key: cord-255378-qgklt8wa authors: huang, yi-ping; cho, chao-cheng; chang, chi-fon; hsu, chun-hua title: nmr assignments of the macro domain from middle east respiratory syndrome coronavirus (mers-cov) date: 2016-03-18 journal: biomol nmr assign doi: 10.1007/s12104-016-9676-9 sha: doc_id: 255378 cord_uid: qgklt8wa the newly emerging human pathogen, middle east respiratory syndrome coronavirus (mers-cov), contains a macro domain in the highly conserved n-terminal region of non-structural protein 3. intense research has shown that macro domains bind adp-ribose and other derivatives, but it still remains intangible about their exact function. in this study we report the preliminary structural analysis through solution nmr spectroscopy of the mers-cov macro domain. the near complete nmr assignments of mers-cov macro domain provide the basis for subsequent structural and biochemical investigation in the context of protein function. dispensable for coronavirus replication (putic et al. 2005) , however it seems to play a role in the pathogenesis of mouse hepatitis virus infection (eriksson et al. 2008 ). in addition, viral macro proteins may act via adp-ribose binding to influence the cellular macro domains-regulated pathways either to promote virus replication or to inhibit host responses directed against the virus (neuvonen and ahola 2009 ). since the biochemical, enzymatic and structural analysis of mers-cov macro domain is not available, here we present the expression, purification, and chemical shift assignments of mers-cov macro domain. the nmr chemical shift assignments serve as the basis for further structural characterization and ligand screening, which provides insight into the conformational properties of this domain in solution and contributes to understanding its function. the dna sequence containing mers-cov macro domain (aa. 1110-1274) was chemical synthesized and cloned into ndei/xhoi site of pet-28a(?) vector system (novagen). bacteria of the e. coli strain bl21(de3) transformed with the pet-28a (?)-macro domain plasmid were grown in 4 liters of lb medium at 37°c until the absorbance at 600 nm reach 1.0. cells were then harvested by centrifugation and re-suspended in one liter of m9 minimal medium supplemented with 1 g/l of 15 nh 4 cl and 2 g/l of 13 c 6 -glucose (cambridge isotope laboratories). isopropylb-d-thiogalactoside (iptg, 1 mm) was added to induce his-tagged protein for 20 h at 16°c. the cell culture was harvested by centrifugation at 6000 rpm. for purification, cell pellets were re-suspended in 50 ml buffer containing 25 mm sodium phosphate and 100 mm nacl at ph 7.0 and then disrupted by sonication for 20 min. the cell extract was clarified by centrifugation at 12,500 rpm for 30 min at 4°c to remove debris. the supernatant was then applied to ni-nta column (ge, healthcare) equilibrated with the same re-suspension buffer, and his-tagged protein was eluted with 200 mm imidazole. the purified his-tagged macro domain was then digested with thrombin for 6 h at 16°c to remove the his-tag. finally, macro domain protein (20 kda, with extra gly, ser, his and met at n-terminus) was purified using size-exclusion superdex75 xk 16/60 column (ge, healthcare). the purified protein was concentrated to 0.1-0.5 mm in 20 mm sodium phosphate (ph 6.5) and 100 mm or 150 mm nacl for nmr structural studies. all nmr experiments were carried out at 293 k on bruker avance 600 mhz nmr or 800 mhz spectrometers equipped with 5 mm triple resonance cryoprobe and z-gradient. the data was acquired and processed using the software topspin2.1 (bruker, germany) and further analyzed using sparky, version 3.114 (t. d. goddard and d. g. kneller, sparky 3, university of california, san francisco), following the procedures as described previously (yang et al. 2010; chen et al. 2014) . 1 h chemical shifts were externally referenced to 0 ppm of 2,2-dimethyl-2-silapentane-5-sulfonate, whereas 13 c and 15 n chemical shifts were indirectly referenced according to iupac recommendations (markley et al. 1998) . protein backbone resonance assignments were based on standard triple resonance experiments (sattler et al. 1999 ): hncacb, cbca(co)nh, hnco and hn(ca)co. aliphatic side-chain assignments were primarily done by hcch-tocsy and hcch-cosy with the help of hcc(co)nh and hbha(co)nh experiments. the backbone resonance assignments were nearly complete. figure 1 illustrates the 2d ( 1 h-15 n) hsqc spectrum and assignments of the amide resonances. except for the first four amino acids on n-terminal (gly -2 , ser -1 , his 0 and met 1 ) and six proline residues (pro 1 , pro 72 , pro 96 , pro 118 , pro 123 , and pro 134 ), amides of all other residues (158 out of 168) have been assigned under the experimental conditions (ph 6.5 at 293 k). among these 158 residues, all other backbone resonances ( 1 ha, 13 ca, 13 cb and 13 c) are 100 % completed. completeness of 1 h resonances assignment, including side-chain, calculated by cyana3.9 (güntert 2004 ) is 86.7 %. secondary structure elements of mers-cov macro domain were identified by calculating the chemical shift deviations of the ca(ddca) and cb(ddcb) from the random coil values and was corroborated by analysis of the chemical shift data using the program talos? (shen et al. 2009 ). positive and negative values of the difference between ddca and ddcb correspond to a-helix and b-sheet secondary structure, respectively and correlated well with talos? index (fig. 2) . six helices and seven b-strands could be deduced for mers-cov moacro domain protein based on the secondary chemical shift analysis, which results in residues 21-27, 47-54. 58-69, 104-115, 134-144 and 156-162 in a-helices and residues 5-10, 14-19, 32-25, 78-81, 89-94, 119-124 and 148-153 forming b-sheets. the resonance assignments have been deposited to the biomagresbank (http://www.bmrb.wisc.edu/) under the accession number 26657. backbone resonance assignments of the a sub-domain of brevibacillus thermoruber lon protease middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group mouse hepatitis virus liver pathology is dependent on adpribose-1 00 -phosphatase, a viral function conserved in the alphalike supergroup coronaviruses: an overview of their replication and pathogenesis automated nmr protein structure calculation with cyana the macro domain protein family: structure, functions, and their potential therapeutic implications bats as reservoirs of severe emerging infectious diseases dissection of aminoterminal functional domains of murine coronavirus nonstructural protein 3 the macro domain is an adpribose binding module recommendations for the presentation of nmr structures of proteins and nucleic acids. iupac-iubmb-iupab inter-union task group on the standardization of data bases of protein and nucleic acid structures determined by nmr spectroscopy differential activities of cellular and viral macro domain proteins in binding of adp-ribose metabolites the hepatitis e virus orf1 'x-domain' residues from a putative macrodomain protein/appr-1 00 -pase catalyticsite, critical for viral rna replication adp-ribose-1 00 -monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture heteronuclear multidimensional nmr experiments for the structure determination of proteins in solution employing pulsed field gradients talos?: a hybrid method for predicting protein backbone torsion angle from nmr chemical shifts resonance assignments of human c35 (c17orf37) protein, a novel tumor biomarker isolation of a novel coronavirus from a man with pneumonia in saudi arabia acknowledgments the nmr spectra were obtained at high-field nuclear magnetic resonance center (hf-nmrc) and grc nmr core facility in academia sinica, taiwan. this work was supported by the ministry of science and technology, taiwan (103-2113-m-002-009-my2), and national taiwan university (ntu-erp-104r8600 and ntu-icrp-104r7560-5) to c.-h. hsu. key: cord-258892-1xmoeoyh authors: thomas, helen lucy; zhao, hongxin; green, helen k.; boddington, nicola l.; carvalho, carlos f.a.; osman, husam k.; sadler, carol; zambon, maria; bermingham, alison; pebody, richard g. title: enhanced mers coronavirus surveillance of travelers from the middle east to england date: 2014-09-17 journal: emerg infect dis doi: 10.3201/eid2009.140817 sha: doc_id: 258892 cord_uid: 1xmoeoyh during the first year of enhanced mers coronavirus surveillance in england, 77 persons traveling from the middle east had acute respiratory illness and were tested for the virus. infection was confirmed in 2 travelers with acute respiratory distress syndrome and 2 of their contacts. patients with less severe manifestations tested negative. during the first year of enhanced mers coronavirus surveillance in england, 77 persons traveling from the middle east had acute respiratory illness and were tested for the virus. infection was confirmed in 2 travelers with acute respiratory distress syndrome and 2 of their contacts. patients with less severe manifestations tested negative. east respiratory syndrome coronavirus (mers-cov) died in june 2012 in the kingdom of saudi arabia and was reported on september 20 (1) . the second case reported globally was in a qatari national patient who had been transferred from qatar to a hospital in england; preliminary data sharing on september 23 indicated that isolates from the second case-patient had 99.5% identity with the virus identified in the first case. (2) . on september 24, 2012, public health england (phe) (formerly the health protection agency [hpa]) established an enhanced surveillance system to rapidly detect and investigate possible cases of mers-cov infection among travelers to england from the middle east. the first 12 months of surveillance in england identified 1 additional case of mers-cov in a traveler returning from the middle east and 2 cases among family contacts of this second case. definitions for possible and confirmed cases were established. possible cases were defined by clinical and epidemiologic criteria. clinical criteria specified acute respiratory syndrome (including fever ≥38°c or history of fever and cough) requiring hospitalization and clinical or radiologic evidence prompting suspicion of lower airway involvement not explained by another etiology. epidemiologic criteria specified travel to or residence in an area where infection with mers-cov could have been acquired during the 10 days before onset of illness. at the time these criteria were initiated, the kingdom of saudi arabia and qatar were the 2 areas indicated. a confirmed case was defined by respiratory samples testing positive for mers-cov by at least 2 specific pcr assays targeting different regions of the mers-cov genome. because mers-cov is an emerging pathogen, case definitions were, and continue to be, revised in response to new information (2), in agreement with world health organization case definitions (3) (4) (5) . substantial revisions included extension of the geographic areas where infection could have occurred to include all countries neighboring those where infection could have been acquired (november 29, 2012), the recommendation to test patients with the appropriate clinical and epidemiologic criteria if they had an alternative etiology which did not fully explain their clinical manifestation (february 12, 2013), and extension of the incubation period to 14 days (june 21, 2013). enhanced surveillance involved the collection of a minimum dataset for each possible case, including demographic data, clinical symptoms, travel and contact history, and results of testing for respiratory pathogens (6) . nose and throat swab specimens and, when possible, lower respiratory tract specimens, were tested at 1 of 4 regional laboratories. although the testing guidelines recommended mers-cov testing after exclusion of alternative etiologies, other tests were conducted in parallel with mers-cov testing for most suspected cases. during the first few days of surveillance, a pan-coronavirus assay conducted at the phe national reference laboratory was used as a screening test; then the viral genome was fully sequenced. after the generation of mers-cov specific assays, a first-line screening assay targeting the viral genomic area upstream of the e gene (7) was conducted, followed by confirmatory testing at the hpa/phe national reference laboratory. results of mers-cov testing are reported regularly in the hpa/phe weekly influenza report (8). a descriptive analysis included the number of persons tested and proportion positive for mers-cov by key demographic, epidemiologic, and clinical characteristics. definition were tested for mers-cov. seventy-five travelers tested negative on the screening assay, and 2 tested positive. positive results on the screening assay were confirmed by positive results at the hpa/phe national reference laboratory. in addition to testing the 77 persons who met all of the possible case criteria, mers-cov testing was conducted on 13 patients who had severe acute respiratory disease but did not meet the travel requirements: 2 had a travel history outside the middle east, 4 had no travel history in the relevant exposure period, and travel histories of the remaining 7 were unknown. mers-cov was not detected in any of these persons. the clinical and epidemiologic characteristics of the 77 persons tested and their mers-cov test results are shown in table 1 . those tested ranged in age from 3 months to 90 years; 34 (44%) had signs of pulmonary parenchymal involvement. the 2 confirmed cases were in male patients, 45 and 60 years of age; both had severe acute respiratory symptoms requiring treatment by extracorporeal membrane oxygenation; both subsequently died. mers-cov pcr testing was conducted on 53 contacts of the 2 confirmed case-patients in england; 2 of these contacts tested mers-cov positive (9,10). the positive predictive value for mers-cov infection of different combinations of signs and symptoms is shown in table 2 . no case-patients who did not have pulmonary parenchymal involvement tested positive for mers-cov, and the positive predictive value of the clinical manifestations increased as the severity of disease increased. of the 77 patients tested, 22 had positive results for alternative respiratory pathogens, including 10 with influenza (7 influenza a and 3 unlike surveillance for established organisms, surveillance for a novel pathogen requires analysis of information collected from all patients tested, even from those that test negative, to build knowledge of the predictive value of different epidemiologic and clinical manifestations. this report on the characteristics of patients traveling to england from the middle east and tested for mers-cov enables a first crude estimation of the positive predictive value of different signs and symptoms during the first year following the emergence of this pathogen. because this study is based on a cohort of 77 suspected case-patients, of whom only 2 laboratory-confirmed cases were identified during the surveillance period, estimates on the basis of identified symptoms are very imprecise ( table 2) . however, in the context of an emerging pathogen, reporting such data progressively helps optimize case detection and surveillance systems. during the 12-month surveillance period, no patients who had respiratory symptoms but no pulmonary parenchymal involvement were positive for mers-cov by pcr, and the positive predictive value of signs and symptoms increased with the severity of clinical manifestation. this suggests that the case definitions that were in use during this period (which recommended mers-cov testing only for patients who met the epidemiologic criteria and had a severe respiratory illness) were appropriate. a range of respiratory pathogens were found in those patients that were mers-cov negative, highlighting the importance of looking for alternative diagnoses. however, the diagnosis of 1 of the mers-cov case-patients was delayed because of an initial diagnosis of influenza. the testing algorithm was subsequently changed to ensure that patients meeting the possible case definition were tested for mers-cov if they had an alternative etiology which did not fully explain their clinical manifestation. the predictive value of the possible case definition depends on the incidence of infection and would be expected to vary across different population groups and change over time, especially in the context of an emerging pathogen. we encourage other countries to similarly report the characteristics of all patients tested for mers-cov to improve understanding of the predictive value of different clinical and epidemiologic manifestations in various populations at different times. this will help inform the evolving international public health response to this novel pathogen. severe respiratory illness caused by a novel coronavirus mers-cov case definitions and algorithms world health organization. interim case definition for case finding severe respiratory disease associated with novel coronavirus as of 25 world health organization. revised interim case definition -novel coronavirus. interim case definition as of 29 world health organization. revised interim case definition for reporting to who -novel coronavirus. interim case definition as of enhanced case and contact protocol v5.0. epidemiological protocols for comprehensive assessment of early middle east respiratory syndrome coronavirus cases and their close contacts in the united kingdom detection of a novel human coronavirus by realtime reverse-transcription polymerase chain reaction evidence of person-to-person transmission within a family cluster of novel coronavirus infections we thank all the clinical, microbiological, and health protection staff who have reported cases to the english enhanced mers-cov surveillance system and collected the relevant clinical, virologic, and epidemiologic data.dr thomas is a consultant epidemiologist at the national centre for infectious disease and control, public health england. her research interests include field epidemiology studies of infectious disease outbreaks and tuberculosis surveillance. key: cord-282293-pdhjl508 authors: park, wan beom; kwon, nak-jung; choe, pyoeng gyun; choi, su-jin; oh, hong sang; lee, sang min; chong, hyonyong; kim, jong-il; song, kyoung-ho; bang, ji hwan; kim, eu suk; kim, hong-bin; park, sang won; kim, nam joong; oh, myoung-don title: isolation of middle east respiratory syndrome coronavirus from a patient of the 2015 korean outbreak date: 2016-01-14 journal: j korean med sci doi: 10.3346/jkms.2016.31.2.315 sha: doc_id: 282293 cord_uid: pdhjl508 during the 2015 outbreak of middle east respiratory syndrome coronavirus (mers-cov) in korea, 186 persons were infected, resulting in 38 fatalities. we isolated mers-cov from the oropharyngeal sample obtained from a patient of the outbreak. cytopathic effects showing detachment and rounding of cells were observed in vero cell cultures 3 days after inoculation of the sample. spherical virus particles were observed by transmission electron microscopy. full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade b of mers-cov. middle east respiratory syndrome coronavirus (mers-cov) is a betacoronavirus causing a severe acute respiratory infection (1, 2) . it was first isolated from the sputum of a patient with severe pneumonia in saudi arabia in 2012 (3) . since then, 26 countries have reported 1,618 laboratory-confirmed cases of infection with mers-cov to the world health organization (who), including 579 fatalities (4) . the korean outbreak of mers-cov was initiated in may 2015 by a business man returning from the middle east (5) . the transmission of mers-cov continued until early july, resulting in 186 cases with 38 deaths. one of the most important characteristics of the korean outbreak was 4 large clusters of cases due to superspreading event at hospitals, accounting for > 80% of the total cases. another characteristic was that many cases of second-and third-generation of transmission occurred (5, 6) . this finding is quite contrast to the previous studies suggesting limited person-to-person transmissibility of mers-cov (7, 8) . to better understand transmissibility and assess epidemic risk, characterization of mers-cov of the korean outbreak would be of paramount importance (9) . here, we report the mers-cov isolated from a patient of the korean outbreak. a 39-year-old healthcare worker was admitted to the hospital because of fever and cough. on may 27, 2015, he was unknowingly exposed to the index case (designated as patient number 14 by korea ministry of health and welfare) of the hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) at emergency department of a hospital (10) . two days later, he developed fever and dry cough. on june 2, he was diagnosed with mers-cov infection as sputum sample was positive on real-time reverse transcriptase polymerase chain reaction (rt-pcr) assay, and admitted to the isolation unit of the mers-designated hospital by the government. he had a history of cough variant asthma, but did not take any regular medication, and otherwise healthy. on admission (june 2, 2015), the physical examination revealed a body temperature of 38.8°c, a respiratory rate of 22 breaths per minute, a pulse of 78 per minute, and a blood pressure of 118/71 mmhg. chest radiography showed patchy consolidation in the upper zone of the left lung. his pneumonia progressed, and on june 8, he developed shortness of breath, his arterial oxygen saturation decreased below 90%, requiring oxygen supplementation, and chest radiography showed multiple consolidations in the both lungs. on june 10, he was intubated and mechanical ventilation was started. his hypoxemia worsened rapidly, and veno-venous extr acorporeal membrane oxygenation support was started since june 11. on july 2 (day 35 of his illness), real-time rt-pcr for mers-cov turned negative, and was removed from the isolation unit. he recovered gradually. the patient's oropharyngeal samples were obtained by using utm tm kit containing 3 ml of viral transport media (copan di-agnostics inc., murrieta, ca, usa). the samples were stored at -70°c until assays. we inoculated m onolayers of vero cells with the samples and cultured the cells at 37°c in a 5% carbon dioxide atmosphere. cytopathic effects consisting of rounding and detachment of cells were observed 3 days after the inoculation of the sample taken on day 11 of his illness ( fig. 1a and b) . the rna titer in the sample was 5.80 × 10 7 copies/ml for upe gene and 4.97 × 10 7 copies/ml for orf1a gene. in order to observe virus particles, vero cell monolayer showing the cytopathic effects was fixed as previously described (11) . it was cut on ultramicrotome (rmc mt-xl) at 65 nm. ultrathin sections were stained with saturated 4% uranyl acetate and 4% lead citrate before examination with a transmission electron microscope (jem-1400; jeol usa inc., peabody, ma, usa) at 60 kv. spherical particles ranging 77 to 131 nm in diameter were observed within the cytoplasm of infected cells (fig. 1c and d) . for full-length genome sequencing of the virus isolate (mers-cov hu/kor/snu1_035/2015), vero cell monolayer showing cytopathic effects was harvested and used for rna extraction. rna was extracted by using qiaamp viral rna mini kit (qiagen, valencia, ca), according to the manufacturer's instructions. the rna was used for cdna synthesis using su-perscript iii reverse transcriptase (invitrogen, ma, usa) by each specific rt primer as described previously (12) . finally, about 2.5 kb pcr products were amplified by each primer pair (table 1) , and the amplicons were sheared by covaris s2 according to the 200 bp target bp condition (covaris, ma, usa). to generate the next generation sequencing (ngs) library, the fragments were ligated with adapter and index (barcode) using truseq nano dna ht library prep kit (illumina, ca, usa), and the library was sequenced by miseq (illumina, ca, usa). the ngs data were aligned to mers-cov, nc_019843, used for binary sequence alignment/map (bam) file generation, and genome assembly. in order to evaluate genetic relationship between this isolate and homo sapiens and camelus dromedaries mers-cov sequences reported from other countries, phylogenetic analyses were conducted using the whole genome, the s gene and the ofr1a gene. the full-length genome sequence (30,119 bp) of the virus isolate was obtained and deposited in the genbank (accession no. ku308549). the genome sequence of the virus had high level of nucleotide identity (97.80%-99.95%) to those of mers-cov reported previously ( fig. 2a) . of note, the closest ones were korea/seoul/035-1-2015 and 035-2-2015 (genbank accession no. , that were directly sequenced from sputum of the same patient as ours (13) . a previous study about s gene of mers-cov reported from korea showed that a culture isolate from patient number 002 contained two nonsynonymous variants (s137r and v530l) (14) . these variants were not found in our isolate and there was no difference in amino acids of s protein between our isolate and directly sequenced ones (kt374054-5). this difference can be explained by cell culture-adaptation in that our culture isolate was obtained before passage whereas one with nonsysnonymous variant was from the third passage in vero cells. phylogenetic analyses of the whole genome showed that this virus closely clustered with those reported from korea (gen-bank accession nos. kt029139, kt374050-kt374057), china (genbank accession no. kt006149.2) and saudi arabia in 2015 (genbank accession no. kt026453-6). phylogenetic analyses based on orf1ab genes revealed that this virus fell into the group 3, but those based on s genes showed that this virus belongs to the group 5 along with other viruses reported from korea ( fig. 2b and c) . these findings are compatible to a previous study (15) . in summary, we isolated mers-cov from a patient with severe pneumonia who had been infected during the korean outbreak in 2015. we also obtained full-length sequence of the the evolutionary history was inferred by using the maximum likelihood method based on the tamura-nei model (16) . evolutionary analyses were conducted in mega6 (17 middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. middle east respiratory syndrome corona saudi arabia: disease outbreak news middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission characteristics of mers and sars in the healthcare setting: a comparative study interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study what needs to be done to control the spread of middle east respiratory syndrome coronavirus? middle east respiratory syndrome coronavirus superspreading event involving 81 persons, korea processing tissue and cells for transmission electron microscopy in diagnostic pathology and research full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus microevolution of outbreak-associated middle east respiratory syndrome coronavirus variations in spike glycoprotein gene of mers-cov origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees mega6: molecular evolutionary genetics analysis version 6.0 we thank ms. myoung im shin (department of pathology, seoul national university hospital) for her technical assistance in electron microscopy. the authors have no potential conflicts of interest to disclose. key: cord-259200-65b267ic authors: harypursat, vijay; chen, yao-kai title: six weeks into the 2019 coronavirus disease outbreak: it is time to consider strategies to impede the emergence of new zoonotic infections date: 2020-05-05 journal: chin med j (engl) doi: 10.1097/cm9.0000000000000760 sha: doc_id: 259200 cord_uid: 65b267ic nan coronaviruses have in the past been known to be the etiologic agents of mild upper respiratory infections in humans, similar to the ubiquitous and relatively benign "common cold"-type upper respiratory illnesses induced by the human rhinoviruses in adults and children. subsequent to the severe acute respiratory syndrome (sars) outbreak in china 2003, and the middle east respiratory syndrome (mers) outbreak in the middle east in 2012, global concerns regarding the pathogenicity and epidemic/pandemic potential of novel human coronaviruses began to emerge, with some experts predicting that novel coronaviruses could likely again cross the species barrier and present humans with future pandemic-potential infections. [1] these concerns have proven prescient with the emergence, late in 2019, of the 2019 coronavirus disease (covid-19) or novel coronavirus pneumonia. a significantly large variety of coronavirus species cause a diverse range of diseases in domesticated and wild mammals and birds, and these animals may also be carriers of and reservoirs for coronaviruses. [2] six coronavirus species had, before the january 8th, 2020, been known to cause disease in humans. four species are endemic in human populations, and cause mild common cold symptoms in immunocompetent humans. the two remaining species, sars-cov and mers-cov, are zoonotic in origin, and their infection of humans may have fatal outcomes. 2019-ncov is the seventh coronavirus species that is now known to infect humans, is also zoonotic in origin, and is the causative organism for the current viral pneumonia epidemic in china. both sars-cov and mers-cov are believed to have originated from bats, with common masked civets and dromedary camels respectively being intermediary hosts. [3] sars-like coronaviruses have been isolated from chinese horseshoe bats, and may attach to and utilize the angiotensin-converting enzyme 2 receptor in human lower respiratory tract cells to gain entry into these cells, thus facilitating transmission to, and initiating infection in, humans. [4] the genomic sequence of 2019-ncov is strikingly similar to that of sars-like coronaviruses found in bats, and phylogenetic data from recent genomic studies on bat-associated coronaviruses and 2019-ncov suggest that bats are the natural reservoir for coronaviruses in general, and 2019-ncov in particular. [5] it has been postulated that the reservoir for 2019-ncov is the chinese horseshoe bat, which is known to host sars-like coronaviruses. it is now hypothesized that one of the reservoir coronavirus species in bats crossed the species barrier to an intermediate mammal host (presumed to be a masked civet) sold at the wet market at the epicenter of the current epidemic, with subsequent mutation and transmission to humans, initiating the present epidemic of covid-19. it has been noted that the two previously known human coronaviruses causing epidemic disease and spread, sars-cov and mers-cov, had a relatively low rate of spread from an individual infected patient (an index referred to as its basic reproductive number-r°). the r°of sars was estimated to be around 3, meaning that on average, each infected patient is presumed to spread the virus to three other individuals. [6] it is currently estimated that the r°for 2019-ncov is between 2.2 and 2.7. [6, 7] however, approximately 10% of individuals infected with sars-cov and mers-cov were associated with a phenomenon referred to as "super spreading," associated with an r°> 10. [8] wide transmission and spread of sars-cov and mers-cov occurred to a large extent by means of super-spreading events. [8] human super spreaders for 2019-ncov have not been identified thus far in limited epidemiological studies conducted in the past 6 weeks of the outbreak. [6] however, clinicians and researchers should be acutely aware of the likelihood for the potential existence of such transmitters of 2019-ncov infection in the general population, and of the means to identify and isolate such individuals expeditiously to prevent a reduction of the current epidemic doubling time of approximately 7 days, and to limit viral transmission and spread. [7] a compelling mathematical modeling study done by researchers at the hong kong university indicates, despite limitations to their study, that these figures may not be a fair representation of the actual scale of the 2019-ncov outbreak in china. they estimated that the basic reproductive number for 2019-ncov was 2.68 (95% confidence interval 2.47-2.86) and the epidemic doubling time was 6.4 days (95% confidence interval 5.8-7.1 days). [7] ominously, a further mathematical model, proposed by tang et al, [9] suggests that the basic reproductive number for 2019-ncov might be as high as 6.47. the putative zoonotic origin of 2019-ncov, and the zoonotic origins of the sars and mers epidemics, brings into sharp focus the existence of unregulated wet markets in china, trading in live wild game, game meat, and game products. zoonotic origins for emerging viral infections are not new, with acquired immunodeficiency syndrome, ebola, influenza viruses, sars, mers, and a multitude of other viral illnesses all crossing the species barrier and causing devastating illness in humans, at enormous economic and human cost. [10] the presence and availability of markets that trade in wild animals for human consumption, and for purchase as pets, greatly increases the potential for viral infections originating from these reservoir animals to jump to human populations. the complete ban on market trading and sale of wild game meat in china on january 26th, 2020 will help prevent zoonotic transmission of 2019-ncov in the current epidemic and, to a certain degree, help prevent emergence of new zoonotic infections. further social and cultural changes regarding wild game trading and consumption is required in china and worldwide, to prevent scenarios where regular emergence of zoonotic infections becomes commonplace, with their inevitably attendant economic and human costs. it is estimated that the sars epidemic cost the global economy approximately $ 54 billion in 2003 alone. the 2015 mers outbreak in the republic of korea resulted in a $ 2.6 billion loss for the south korean tourism industry alone. the 2014 outbreak of ebola in guinea, liberia, and sierra leone cost their already lean economies approximately $ 300 million. the human and economic costs of the 2019-ncov outbreak to the global economy will, without doubt, be scrupulously studied after the present outbreak ends, and the global economic costs will be immense, and the human cost, agonizing. each preventable zoonotic outbreak costs the country of origin and the world vast amounts of money and resources, and an inestimable cost in human lives, and if emerging zoonotic outbreaks can be prevented by severely limiting human exposure to wild animals and their trade, then effective measures to ensure that this occurs should be implemented by regulatory government authorities globally as soon as it is practicable. it is clearly apparent that the work done thus far in the quest to contain the current 2019-ncov outbreak is massive, focused, and resolute. it is also abundantly evident that a large quantum of work remains to be done in order for the current public health effort to be successful in containing the present outbreak. managing this requires international cooperation using traditional and proven public health strategies that ultimately succeeded in the sars epidemic. it is, however, inevitable that new zoonotic infections will emerge in the future. it is, therefore, an urgent priority for local and international health and wildlife regulatory authorities to structure and implement robust control mechanisms that effectively reduce human exposure to wild game meat and their products. in contrast to africa, the consumption of wild game meat in asia is not generally motivated by poverty, hunger, or starvation. the common motivations for the human consumption of wild game meat in asia are for their purported medicinal value, and the supposed health-enhancing effects of certain varieties of wild game meat, or their products. specific rare and exotic asian and other international wild game and their products, are also consumed and offered to guests and influential persons in an effort to project status, prestige, and wealth, depending on the rarity of the animal involved. there is also the existence of wildlife trafficking between asia and other regions of the world, which has created an international supply and demand chain, with savvy wildlife entrepreneurs marketing wild game meat and products as "traditional specialties," in their effort to boost sales. the existence of local and international wildlife trade for meat and animal products needs urgent and decisive change. it is fervently hoped that the steadfast efforts by china, in partnership with the international community, will reap positive results with respect to 2019-ncov control in the future weeks and months. additionally, urgent international attention to and curtailment of the hitherto unregulated and commonplace trade in wild game, meat and products is essential if a repeat of the human and economic loss, and public fear and social disruption wreaked by the current 2019-ncov outbreak is to be avoided in the future. this work was supported by a grant from the chongqing special research project for prevention and control of novel coronavirus pneumonia (no. cstc2020jscx-fyzx0074). efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential the battle against sars and mers coronaviruses: reservoirs and animal models clinical features of patients infected with 2019 novel coronavirus in wuhan, china isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study the role of super-spreaders in infectious disease estimation of the transmission risk of the 2019-ncov and its implication for public health interventions economic impact of the 2015 mers outbreak on the republic of korea's tourism-related industries six weeks into the 2019 coronavirus disease outbreak: it is time to consider strategies to impede the emergence of new zoonotic infections none. key: cord-265279-0zjpqnqp authors: hoteit, rouba; shammaa, dina; mahfouz, rami title: use of the human coronavirus 2012 (mers) genesig kit for mers-cov detection date: 2016-04-16 journal: gene rep doi: 10.1016/j.genrep.2016.04.004 sha: doc_id: 265279 cord_uid: 0zjpqnqp introduction: mortality due to mers-cov infection is common especially among immunocompromised patients. the pathogenesis and the transmission mode of this virus are still not well understood. the name of the virus is derived from the area of its appearance and the genomic sequence that was used in the development of qrt-pcr assays for mers-cov detection was retrieved from the first detected case isolate. the employed assays target various regions including the area upstream of the envelope gene (upe) that is used for screening and the open reading frames (orf) 1a and 1b used for confirmation. aim: this study assesses the use of a mers-cov specific assay for screening of respiratory samples in anticipation of the possible spread of the virus in the region. methods: 46 respiratory specimens were tested using the qualitative one-step qrt-pcr genesig human coronavirus 2012 (mers) kit (primerdesign™). results: out of the 46 tested samples, 45 were negative for mers-cov and one sample was found mers-cov positive. conclusion: the genesig human coronavirus 2012 (mers) kit is very useful for the screening of suspected respiratory cases in the middle east area as well as other regions. the middle east respiratory syndrome (mers) is a novel coronavirus first discovered in september 2012 in a cell-culture from a patient after his death from pneumonia in june 2012 in saudi arabia (assiri et al., 2013) . mortality due to mers-cov infection is common especially among immunocompromised patients (al-abdallat et al., 2014) . coronaviruses are the largest positive single-stranded rna viruses with a genome that can reach up to 32 kb long. the genome entails a 5′-terminal cap structure and is polyadenylated at its 3′ end, and is occupied by two large overlapping open reading frames, orfs 1a and 1b (subissi et al., 2014) . mers-cov is part of the c lineage of the betacoronavirus genus and it is related to the hku4 and hku5 coronavirus detected in bats (omrani et al., 2013) . mers-cov and highly homologous mers-like cov were isolated in dromedary camels and african neoromicia capensis bats, respectively. although mers-cov is known to have zoonotic origins the pathway of transmission from the animal reservoir to humans is still not well understood. it is, however, suggested that a bat to camel to human route could explain the mers-cov 2012 outbreak in humans, due to the emerging camel trade between equatorial africa and saudi arabia during the last 20 years (gralinski and baric, 2015) . mers-cov causes severe lower respiratory tract infection that sometimes requires admission to the intensive care unit. patients usually respond well to alpha interferon or cyclosporine treatment, however, no specific antiviral or vaccine is available for mers-cov (ohnuma et al., 2013) . more than 1540 cases from 21 countries worldwide have been infected and diagnosed with mers-cov since june 2012, and almost a third of those died (saad et al., 2014; who, c 2015) . patients with mers-cov infection tend to have leukocytosis, lymphopenia and some express thrombocytopenia and coagulopathy. moreover, creatinine levels, lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase levels may be elevated and thus implying a liver or kidney disease (van den brand et al., 2015) . males and elderly are more commonly infected with mers-cov than females and younger people, and human-to-human transmission in the community is mainly in healthcare settings where poor infection control measures are implemented especially in intensive care units, and where overcrowding of patients may occur (saad et al., 2014) . other reported cases of mers-cov, especially the ones reported in europe or the us are more likely associated with the travel history to the middle east. the rate transmission of the virus from infected patients to their household contacts is as low as 5% (drosten et al., 2014) . rapid detection and implementation of proper infection control measures are two essential steps to limit and control mers-cov transmission in healthcare settings. this includes applying contact and airborne precautions such as wearing gloves, gowns and surgical masks upon entry to a room of mers-cov infected patient in isolation and removing such personal protective equipment upon leaving the room (zumla and hui, 2014) . protective measures outside healthcare settings include avoiding contact with dromedary camels, the host of mers-cov, and restraining from drinking camel urine or raw milk or eating raw or undercooked camel meat. moreover people working with camels must wear protective clothing and facial masks, and maintain a good personal hygiene (reusken et al., 2014) . ideally, lower respiratory specimens such as sputum, bronchoalveolar lavage, bronchial wash and tracheal aspirates are the most representative and appropriate for mers-cov testing (cdc, june 14, 2013) . the name of the virus is derived from the area of its appearance and the genomic sequence that was used in the development of qrt-pcr assays for mers-cov detection was retrieved from the first detected case isolate (lu et al., 2014) . the employed assays target various regions including the area upstream of the envelope gene (upe) that is used for screening and the open reading frames (orf) 1a and 1b used for confirmation . the aim of this study was to assess the use of a mers-cov specific assay for screening of respiratory samples referred to a major tertiary care center in anticipation of the possible spread of the virus in the region. rna was extracted from 46 respiratory specimens using the qiaamp viral rna mini kit (qiagen, hilden, germany) that does not use phenol/ chloroform extraction or alcohol precipitation protocols. briefly, a lysis buffer was used to isolate non-degraded viral rna and inactivate rnases. the sample was then transferred to the qiaamp mini spin column under optimal conditions that ensured effective binding of the rna to the qiaamp membrane. possible contaminants were removed using two different wash buffers, then, a rnase-free buffer was employed to elute the rna. mers-coronavirus qualitative one-step qrt-pcr testing was done using the genesig human coronavirus 2012 (mers) kit (primerdesign™, london, uk). the hcov primers are designed for the specific and exclusive in vitro detection of the novel coronavirus isolates based on sequences for the emc strain by targeting the orf1ab and the upe regions of orf5. the orf1ab and orf5/e primer and probe sets are designed to be specific to the mers strain regardless of the species of origin and are detected through the fam channel. an internal rna extraction control is available in the kit and was added to the lysis buffer as an external source of rna template, during the extraction step, and co-purified with the sample rna to monitor this process as well as to rule out the presence of inhibitors in the realtime pcr step. for the latter purpose, a separate mix of primers for the internal control, with limited concentrations that allow for multiplexing with the target sequence primers without affecting the ability to detect the target even when present in low concentrations, was used. the detection of the internal control was through the vic channel. moreover, a primer mix for the endogenous control actin beta (actb) gene was used to evaluate the quality of the original biological sample. the detection of actb is through the vic channel, thus a separate reaction mix than that of the target is necessary to test for actb as multiplexing for actb and hcov primers is not possible. for each sample, three reaction mixes were prepared. for the detection of orf1ab and orf5/e two 20 μl reaction tubes were prepared as follows: 10 μl of oasig™ qrt-pcr mastermix, 1 μl of hcov_2012 (orf1ab or orf5/e) primer/probe mix, 1 μl of internal extraction control primer/probe mix, 6 μl of rnase/dnase free water and 2 μl rna material. to detect the endogenous actb control, a separate 20 μl reaction mix was prepared, containing, 10 μl of oasig™ qrt-pcr mastermix, 1 μl of endogenous actb primer/probe mix, 7 μl of rnase/dnase free water and 2 μl rna material. the amplification conditions involved a reverse transcription step at 42°c for 10 min, an enzyme activation step at 95°c for 2 min and 50 cycles of 95°c for 10 s followed by 60°c for 60 s. a positive control for orf1ab and orf5/e, as well as a negative control (rnase/dnase free water) was used with each run. the test requires 65 min hands-on time and the pcr protocol lasts two hours after which the rotorgene-q (qiagen, uk) software is used for analysis. out of the 46 tested samples, 45 were negative for mers-cov and one sample was found mers-cov positive. the positive result was validated by the naval medical research unit (namru) laboratory, located in cairo-egypt, in collaboration with the world health organization (who). all other results were validated with a cap accredited referral laboratory in germany and the concordance rate was 100%. fig. 1 represents a graph of the amplification of the mers-cov rna and the endogenous actb in the mers-cov positive sample. positive and negative controls were used and signal detection was done on the fam channel. the graph in fig. 2 represents the detection of the internal rna extraction control (iec) in the fluorescence channel vic, for the same positive sample. iec was only added to the sample during extraction and not to the positive or negative controls. an acceptable ct value of the iec ranges between 28 and 34. the genesig human coronavirus 2012 (mers) kit is a rapid and useful kit for the screening of suspected respiratory cases in the middle east area as well as other regions. the collaboration of different molecular microbiology laboratories with the world health organization (with viral cultures done as gold standard reference testing) has always been a rewarding and teaching experience. no extensive sensitivity and specificity work-up has been performed from our side on multiple samples due to the presence of a single mers-cov positive case; however, our lower limit of detection is compatible with the 100 copies/ml reported by the manufacturer. the clinicians have advised that this detection limit is acceptable for their management algorithms. to our knowledge, this is the first report that describes the clinical application of the genesig kit in the mers-cov epidemic era. hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide transmission of mers-coronavirus in household contacts molecular pathology of emerging coronavirus infections real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case rna and neutralising antibodies in milk collected according to local customs from dromedary camels clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities middle east respiratory syndrome coronavirus (mers-cov) c who infection control and mers-cov in health-care workers key: cord-271004-gtmo5ixs authors: al-tawfiq, jaffar a.; rabaan, ali a.; hinedi, kareem title: influenza is more common than middle east respiratory syndrome coronavirus (mers-cov) among hospitalized adult saudi patients date: 2017-10-12 journal: travel med infect dis doi: 10.1016/j.tmaid.2017.10.004 sha: doc_id: 271004 cord_uid: gtmo5ixs background: since the initial description of middle east respiratory syndrome coronavirus (mers-cov), we adopted a systematic process of screening patients admitted with community acquired pneumonia. here, we report the result of the surveillance activity in a general hospital in saudi arabia over a four year period. materials and methods: all admitted patients with community acquired pneumonia from 2012 to 2016 were tested for mers-cov. in addition, testing for influenza viruses was carried out starting april 2015. results: during the study period, a total of 2657 patients were screened for mers-cov and only 20 (0.74%) tested positive. from january 2015 to december 2016, a total of 1644 patients were tested for both mers-cov and influenza. none of the patients tested positive for mers-cov and 271 (16.4%) were positive for influenza. the detected influenza viruses were influenza a (107, 6.5%), pandemic 2009 h1n1 (n = 120, 7.3%), and influenza b (n = 44, 2.7%). pandemic h1n1 was the most common influenza in 2015 with a peak in peaked october to december and influenza a other than h1n1 was more common in 2016 with a peak in august and then october to december. conclusions: mers-cov was a rare cause of community acquired pneumonia and other viral causes including influenza were much more common. thus, admitted patients are potentially manageable with oseltamivir or zanamivir therapy. the emergence of the middle east respiratory syndrome coronavirus (mers-cov) in september 2012 had attracted international attention. the virus was initially isolated from a patient with a fatal community acquired pneumonia (cap) in saudi arabia [1] . since then, multiple hospital outbreaks occurred within [2] [3] [4] [5] [6] [7] and outside saudi arabia [8] [9] [10] [11] . as of may 1st, 2017, the world health organization reported 1952 laboratory-confirmed cases worldwide and at least 693 related deaths [12] . a wide-spectrum of mers-cov infection was described and ranges from mild to severe and fulminant infections leading to severe acute respiratory disease [2, [13] [14] [15] . in the kingdom of saudi arabia, the number of mers-cov cases was 1601 as of may 6th, 2017 [16] . since most of the cases of mers-cov in saudi arabia occurred due to intra-and inter-hospital transmissions, there was an increased amplification of the transmission [2] [3] [4] [9] [10] [11] 17] . early detection and isolation of patients with mers-cov infection remains an important factor for the control of mers-cov transmission [18, 19] . one of the goals of the surveillance of emerging respiratory viruses is the rapid and early identification and placement of control measures [20] . following the initial description of the disease [1] , the ministry of health in the kingdom of saudi arabia put in place a surveillance and screening program for patients admitted with respiratory illness [21] . similarly, we adopted universal screening of admitted patients with community acquired pneumonia. here, we report the result of the surveillance activity in a general hospital in saudi arabia over a four year period. the study was conducted at a 350-bed general hospital, which also accepts referred patients. the hospital provides medical care for about 160,000 individuals eligible for medical care. the hospital has 5 intensive care units (cardiac, medical, surgical, pediatric, and neonatal) [22] . all admitted patients with community acquired pneumonia from suspected mers-cov was an acute febrile respiratory illness (fever, cough, or dyspnea) with radiographic evidence of pneumonia [22] . we collected data for all suspected patients using a standard microsoft excel data collection sheet. both electronic and paper medical records were reviewed. we recorded the age and the date of admission and the mers-cov and influenza results. the study was approved by the johns hopkins aramco healthcare institutional review board (irb). suspected patients had either dacron-flocked nasopharyngeal swabs, or sputum testing for mers-cov. the testing was done at the saudi ministry of health mers-cov laboratory and at the main hospital. clinical samples were screened with real-time reverse-transcriptase (rt)epcr as described previously [23] . the test amplified both the upstream e protein (upe gene) and orf1a for mers-cov and if both assays were positive then the diagnosis of mers-cov was made, as described previously [14] . the influenza test was carried out at the johns hopkins aramco healthcare centre, dhahran, using the cepheid ® xpert flu assay multiplex real-time pcr. the tested influenza viruses were pandemic 2009 h1n1, influenza a (other than h1n1), and influenza b. the test was systematically carried out starting april 2015. statistical analysis was done using excel and descriptive analyses were done for demographic, results of the tests and the monthly number of cases. minitab ® (minitab inc. version 17, pa16801, usa; 2017) was used to calculate the mean age ( ± sd) of patients with influenza. during the study period from 2013 to 2016, a total of 2657 patients were screened for mers-cov and only 20 (0.74%) tested positive. during the first two years (april 2013-march 2015), a total of 1013 patients were screened for mers-cov. only 1.8% of them were positive for mers-cov (table 1 ) and unfortunately these were not systematically screened for influenza. there was an increased number of tests in november 2015-march 2016 (fig. 1) . from april 2015 to december 2016, a total of 1644 patients were tested for both mers-cov and influenza. none of the patients tested positive for mers-cov and 271 (16.4%) were positive for influenza. the detected influenza viruses were influenza a (107, 6.5%), pandemic 2009 h1n1 (n = 120, 7.3%), and influenza b (n = 44, 2.7%) ( table 1 and fig. 2 ). it is interesting to note the pattern of the influenza in 2015 and 2016 (fig. 3) . pandemic h1n1 was the most common influenza in 2015 and influenza a other than h1n1 was more common in 2016. the 2015 influenza season peaked october to december and the 2016 season had a peak in august and then october to december (fig. 3 ). there was a significant difference in the mean age ( ± sd; 95% ci) of patients with h1n1 and other influenza (fig. 4) in this study, we presented the surveillance data on mers-cov over a four year period and the surveillance for influenza over a two year period. mers-cov was only detected in 20 (0.75%) from a total of 2657 patients as detailed in previous publication [22, 24] . the earliest surveillance study from saudi arabia was done from 1 october 2012 to 30 september 2013 and tested a total of 5065 samples [21] . in that study, the mers positivity rate was 2% [21] . a second surveillance of mers-cov in saudi arabia was conducted from april 1, 2015 to february 1, 2016 and included a total of 57,363 suspected mers cases [25] . the study showed only 384 (0.7%) mers-cov positive cases [25] . in a study in the united states, two (0.4%) imported cases were detected among 490 patients-under investigation in 2013-2014 [26] . in a surveillance study of 1586 unique persons from the united arab emirates between january 1, 2013and april 17, 2014, 41 (3%) tested positive for mers-cov infection [27] . in the south korea outbreak, 184 (1%) had mers among 16,752 suspected cases [28] . in a small study from saudi arabia, mers-cov was not detected in 182 cases tested november 2013 and january 2014 (winter time) [29] . thus, the overall positivity of mers-cov among a large cohort remains low. there is a need for a better tool to identify patients with high probability of mers-cov. however, a case control study and a large cohort study did not reveal significant predictor of mers-cov infection [22, 30] . the monthly frequency of suspected mers cases that were tested showed variation with an apparent increase in the tested number during november 2015-march 2016. this apparent increase likely represented an increased activity of influenza during that time. there was no relation to the hajj season as it occurred during september 21-26, 2015 (fig. 1) . in addition at that time, there were no known outbreaks in the kingdom of saudi arabia to account for such an increase in the testing. the 2015 outbreaks occurred in al-hasa in may 2015 [31] and in riyadh in august 2015 [7, 32, 33] . previous studies had shown increased testing of patients for mers-cov during outbreaks [4] . in the current study, the 2015 season was predominated by 2009 pandemic h1n1 whereas influenza a was more common during 2016. similarly, in the united states the 2014-2015 season was predominated by pandemic h1n1 and h3n2 was more common during the 2016-2017 season [34, 35] .we found that influenza rather than mers-cov was more common among the tested patients. the findings are also consistent with other studies among travelers and pilgrims where influenza far exceeded mers [36] [37] [38] [39] [40] . similarly, in a small study in saudi arabia, influenza viruses were detected in 16% of 182 patients [29] . similarly, among a small study of 52 suspected mers cases in the united states of america, influenza was the most commonly (35%) identified respiratory agent [41] and another study found influenza a and b in 11% of 296 investigated patients [26] . thus, it is important to test for common respiratory pathogens such as influenza viruses and it should be noted that identification of a respiratory pathogen should not exclude mers-cov testing [42] . one report indicated co-infection with influenza and mers in four patients [43] . however, epidemiologic differences between different countries should remain as an important predictor of the existence of mers-cov infection. the mean age of patients with h1n1 was younger than the other influenza patients of at least 10 years (45.09 vs. 63.70 for influenza a, 55.11 for influenza b, and 61.28 for influenza negative patients (p < 0.0001). the inital cases of pandemic 2009 h1n1 were also younger than the influenza negative patients [44] . in a small study of 196 patients, influenza b patients were younger than other influenza [45] and in another study the mean age was lower for patients with influenza b (16.4 yr) than (h1n1) pdm09 influenza infection. however, these studies included children and thus are not comparable with the present study [46] . similar results were obtained in travelers returning from the middle east. these studies showed the lack of mers-cov among travelers and that influenza was more common among french travelers [47, 48] , austrian returning pilgrims [40] , british travelers [49] , german travelers [50] , and travelers to california, united states [41] . the presence of influenza infection among those travelrs stress the need for influenza vaccination in travelers, notably tfor those going for the hajj and umrah in saudi arabia. in conclusion, mers-cov was a rare cause of community acquired pneumonia (cap) and other viral causes including influenza are much more common. the epidemiology of influenza mirrored the epidemiology of influenza worldwide. the study highlights the importance of the surveillance system to elucidate the epidemiology of respiratory infections in order to formulate appropriate control measures. interhospital and intra-hospital transmission of mers-cov infection is an important element of the transmission of this virus and it is imperative to continue to have early recognition of cases and constant application of infection control measures to abort the hospital transmissions of the virus [18, 19] . isolation of a novel coronavirus from a man with pneumonia in saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia drivers of mers-cov transmission: what do we know? epidemiological findings from a retrospective investigation the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea preliminary epidemiologic assessment of mers-cov outbreak in south korea middle east respiratory syndrome coronavirus (mers-cov). who family cluster of middle east respiratory syndrome coronavirus infections epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study recovery from severe novel coronavirus infection saudi ministry of health c and cc. mers-cov statistics n hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus in healthcare settings middle east respiratory syndrome coronavirus infection control: the missing piece? surveillance for emerging respiratory viruses screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study middle east respiratory syndrome-coronavirus (mers-cov): a case-controlstudy of hospitalized patients assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections. euro fig. 4. interval plot of age and 95% confidence interval of age among influenza patients hematologic, hepatic, and renal function changes in hospitalized patients with middle east respiratory syndrome coronavirus surveillance and testing for middle east respiratory syndrome coronavirus evaluation of patients under investigation for mers-cov infection response to emergence of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications patterns of human respiratory viruses and lack of mers-coronavirus in patients with acute upper respiratory tract infections in southwestern province of saudi arabia predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study influenza activity -united states, 2015-16 season and composition of the 2016-17 influenza vaccine cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during 2012-2015 active screening and surveillance in the united kingdom for middle east respiratory syndrome coronavirus in returning travellers and pilgrims from the middle east: a prospective descriptive study for the period 2013-2015 influenza not mers cov among returning hajj and umrah pilgrims with respiratory illness hajj-associated viral respiratory infections: a systematic review influenza a and b viruses but not mers-cov in hajj pilgrims laboratory testing for middle east respiratory syndrome coronavirus interim guidelines for clinical specimens from pui | cdc n the impact of coinfection of influenza a virus on the severity of middle east respiratory syndrome coronavirus pandemic influenza a (2009 h1n1) in hospitalized patients in a saudi arabian hospital: epidemiology and clinical comparison with h1n1-negative patients differences in clinical features between influenza a h1n1, a h3n2, and b in adult patients clinical differences between influenza a (h1n1) pdm09 & influenza b infections identified through active community surveillance in north india lack of mers coronavirus but prevalence of influenza virus in french pilgrims after infections in symptomatic travelers returning from the arabian peninsula to france: a retrospective cross-sectional study enhanced mers coronavirus surveillance of travelers from the middle east to england acute respiratory infections in travelers returning from mers-cov-affected areas all authors have no conflict of interest to declare. all authors have no funding. key: cord-268943-arjtjy53 authors: reuss, annicka; litterst, annette; drosten, christian; seilmaier, michael; böhmer, merle; graf, petra; gold, hermann; wendtner, clemens-martin; zanuzdana, arina; schaade, lars; haas, walter; buchholz, udo title: contact investigation for imported case of middle east respiratory syndrome, germany date: 2014-04-17 journal: emerg infect dis doi: 10.3201/eid2004.131375 sha: doc_id: 268943 cord_uid: arjtjy53 on march 19, 2013, a patient from united arab emirates who had severe respiratory infection was transferred to a hospital in germany, 11 days after symptom onset. infection with middle east respiratory syndrome coronavirus (mers-cov) was suspected on march 21 and confirmed on march 23; the patient, who had contact with an ill camel shortly before symptom onset, died on march 26. a contact investigation was initiated to identify possible person-to-person transmission and assess infection control measures. of 83 identified contacts, 81 were available for follow-up. ten contacts experienced mild symptoms, but test results for respiratory and serum samples were negative for mers-cov. serologic testing was done for 53 (75%) of 71 nonsymptomatic contacts; all results were negative. among contacts, the use of ffp2/ffp3 face masks during aerosol exposure was more frequent after mers-cov infection was suspected than before. infection control measures may have prevented nosocomial transmission of the virus. 76% also had >1 underlying chronic condition (4). the median age of case-patients was 50 years (range 14 months to 94 years). all cases were directly or indirectly related to countries in the middle east or on the arabian peninsula. mers-cov shows a close genetic relationship with coronaviruses found in bats (1, (5) (6) (7) (8) (9) (10) , but no zoonotic link has been confirmed. person-to-person transmission has been reported in the work environment, among family contacts, or to health care workers (hcws) (11) (12) (13) . although situations involving consecutive human transmission events have been documented (13) , none of the known clusters have led to sustained person-to-person transmission in the general population. in europe, single imported infections have been reported in the united kingdom, germany, france, and italy, and secondary cases have been reported in the united kingdom, france, and italy (12, 14, 15) . because a large proportion of cases are fatal and the virus could acquire the ability to spread more efficiently (as was the case with severe acute respiratory syndrome coronavirus), who has recommended thorough contact investigations for confirmed human cases to identify, quantify, and prevent person-to-person transmission (16) . in germany, mers-cov infection was initially reported in a person from qatar (17) . he was in his third week of illness and was already on mechanical ventilation when he was admitted to a hospital in essen in october 2012. a retrospective contact investigation found no indication of person-to-person transmission to contacts in germany (17) . on march 23, 2013, the institute for virology of the university of bonn reported an imported case of mers-cov infection to the department of health and environment in munich (city health department). a 73-year-old man from abu dhabi, united arab emirates, had been admitted to a hospital in munich and had positive test results for mers-cov infection ( figure 1 ). clinical details and virologic findings have been reported elsewhere (18) . briefly, the patient had underlying multiple myeloma and had received several modes of treatment, including high-dose chemotherapy and autologous stemcell transplantation in 2009. on march 8, 2013, influenzalike illness with fever and cough developed in the patient. after his symptoms worsened, he was hospitalized in his country on march 10 with a diagnosis of pneumonia; he was intubated on march 17 and transferred by flight ambulance services to germany on march 19, eleven days after illness onset, for further intensive care treatment and mechanical ventilation. general infection control guidelines of the munich hospital required that patients from areas such as the middle east, where prevalence of multidrug-resistant pathogens is high, be isolated until colonization or infection with a multidrug-resistant pathogen is ruled out. this rule is particularly enforced when patients have been previously hospitalized in the country of origin. thus, at the time of hospital admission in germany, the patient was isolated from other patients. when mers-cov infection was suspected and included in the differential diagnosis on march 21, standard hygiene measures for hcws were changed to infection control measures as recommended for severe acute respiratory syndrome patients, including the use of ffp2 face masks for usual patient care (19) . mers-cov infection was diagnosed in the patient on march 23; he died on march 26 of multiorgan failure and acute respiratory distress syndrome. after mers-cov infection was diagnosed, the city health department, in cooperation with the state health department, the institute for virology in bonn, and the robert koch institute, initiated an investigation to 1) monitor all contacts of the patient to identify possible person-to-person transmission, 2) assess infection control measures, and 3) explore possible sources for the patient's infection to prevent further cases. for the investigation, the city health department assessed all contact persons (contacts) retrospectively and monitored them prospectively. all contacts received a questionnaire for retrospective documentation and prospective daily self-monitoring of symptoms, exposure to the patient, and infection control measures applied. for every day from march 19 through april 5, information was collected about the contacts' distance from the patient (<2 meters vs. >2 meters); type of contact with the patient (aerosol-producing procedures, non-aerosol-producing procedures, care of patient, handling of urine catheter, handling of respiratory samples in the laboratory, handling of urine samples in the laboratory); type of protection used (surgical mask, ffp1 mask, ffp2 mask, ffp3 mask, gown, gloves, protective glasses); and symptoms experienced by the contacts (cough, fever, temperature, sore throat, diarrhea, shortness of breath). an aerosol-producing procedure was defined as respiratory suction, bronchoalveolar lavage, intubation, or bronchoscopy. on the basis of the self-reported information in the questionnaires and personal interviews with the contacts, we divided contacts into 2 groups. close-distance contacts had face-to-face contact with the patient (<2 meters from the patient) or direct contact with secretions or body fluids of the patient, irrespective of protective measures worn. all other contacts were classified as less-close-distance contacts. according to who recommendations on the duration of follow-up at that time, close-distance contacts were asked to contact the city health department daily for 10 days after the last exposure to the patient. those who failed to do so were contacted by the city health department, supported by the occupational health service of the hospital. less-close-distance contacts were asked to report to the city health department only in case of onset of symptoms. respiratory illnesses in contacts that occurred 1-10 days after exposure to the patient were assessed through the city health department by telephone contact with the contact; a respiratory tract sample was taken from any contact with respiratory illness. in addition, attempts were made to obtain paired serologic samples from all contacts, the first taken immediately after contact and the second >28 days after the last exposure. because the mers-cov patient was on mechanical ventilation and could not be interviewed, family contacts who had accompanied him to germany were interviewed about the onset of his symptoms and possible exposures in the 10 days before disease onset. for the interview, a structured questionnaire was used, and information collected was documented on paper. pcr testing and serologic testing were done as described (17, 20) . serum samples from contacts were tested for mers-cov antibodies if a serum sample was taken >28 days after last exposure. in addition, serum samples were tested for antibodies against influenza a, b, and c; rhinovirus a, b, and c; parainfluenzavirus 1, 2, 3, and 4; respiratory syncytial virus a and b; human metapneumovirus; coronavirus 229e, nl63, oc43, and hku1; and adenovirus. all samples were analyzed at the institute for virology of the university of bonn. data from the city health department's contact monitoring, the contacts' questionnaires, and the laboratory findings were integrated in 1 database. results were validated and analyzed by using stata version 12.0 (statacorp, college station, tx, usa). the city health department identified 83 contacts. of these, 69 (83%) were classified as close-distance contacts and 14 (17%) as less-close-distance contacts (table) . four (5%) of the contacts were members of the patient's family, 16 (19%) §probability that the distribution as indicated occurs by chance given the column and row totals. ¶nonsymptomatic contacts are asymptomatic persons and those who were symptomatic before exposure. of other professional groups. clinical follow-up was available for 81 (98%) contacts. a respiratory symptom or fever developed in 10 (12%) contacts. of these, swab specimens were collected from 9 (90%) and blood samples from 7 (70%). all 9 swab specimens were negative for mers-cov; 1 (11%) was positive for cov nl-63, and 2 (22%) were positive for rhinovirus. all 7 serum samples were negative for mers-cov antibodies. all symptomatic contacts had >1 sample type (respiratory swab or serum) collected for laboratory testing; results of pcr and serologic testing were available from 6 (60%), pcr only from 3 (30%), and serologic testing only from 1 (10%). in addition, serologic test results were available for 53 (75%) of the 71 nonsymptomatic contacts; all were negative for mers-cov antibodies. overall, persons for whom serologic testing results were available were more likely to be close-distance contacts than were persons without available serologic results (p = 0.007; table) . the 4 family members who accompanied the patient were his wife, daughter, son, and son-in-law. their ages were 35-37 years, and none reported symptoms. the patient's children and son-in-law had their last contact with the patient on march 20 and his wife on march 23. because no protection measures had been used until after march 20, the family members were considered at high risk for infection. all 4 provided respiratory swab and serum samples on march 24; all samples had negative results. serum samples taken >28 days after last exposure to the patient were not available. mers-cov infection was added to the differential diagnosis for the patient on march 21. the daily numbers of hcws who had any contact with him (regardless of protection measures) and of those who had aerosol exposure were lower after that date than before ( figure 2 ): 4.4 hcw per illness day vs. 7.5 hcw per illness day (p = 0.05) and 2.8 hcw per illness day vs. 6 hcw per illness day (p = 0.03). among hcws with aerosol exposure, 1 (8%) of 12 daily exposures occurred while ffp2 or ffp3 masks were being used before march 21; after that date, 11 (79%) of 14 daily exposures occurred while ffp2 or ffp3 masks were being used (p<0.01). the patient was a 73-year-old married man from abu dhabi, united arab emirates; he had a medical history of multiple myeloma. at the time of his mers-cov infection, he was receiving corticosteroid therapy. his profession was camel breeding; in the 2 weeks before his onset of illness, 1 of his camels was reported to have had a respiratory illness. in the questionnaire, we did not differentiate between dromedary (camelus dromedaries) and bactrian (c. bactrianus) camels. his neighborhood had palm trees, and bats were known to dwell in the area. the patient had no known contact with other mers-cov patients, had no personal contacts in qatar or jordan, and had no travel history in the 10 days before illness onset. he consumed different types of fruit juices and cooked goat meat, beef, and sheep meat, but no raw meat. he ate dates from his region, but he reportedly did not consume date or palm syrup. other than the camels on his farm, he had no contact with animals; he did not practice falconry and did not visit camel racetracks or animal markets. we describe the case and contact investigation of a confirmed case of mers-cov infection that was imported to germany. we did not identify person-to-person transmission from the patient to any of the contacts. as with the previous imported case in this country, the patient was already on mechanical ventilation when he was transferred to germany. however, whereas the previous case was in the late third week of illness, this patient was in the second week of illness. sample from this patient taken from different body locations and at different times were positive for mers-cov by pcr, and the viral load detected was several logs higher than in samples from the patient with the previous imported case (18, 20) . these results indicate that this patient may have been more infectious than the previous patient. nosocomial transmission from mers patients to hcws has been documented (13, 21, 22) . in our study, the patient was isolated during the first 2 days of his hospital stay (before mers was suspected), although the reason for this intervention was the hospital's policy to isolate every patient from the middle east, irrespective of the assumed diagnosis, because of perceived increased risk of carrying drug-resistant pathogens, rather than any special measures taken because of the patient's respiratory illness. after mers was suspected, hcws used ffp2 masks significantly more frequently than they had before, and fewer hcws had daily contact with the patient. our result suggest that, in the later stages of this disease, the combination of standard protection measures (use of surgical masks for potentially aerosol-generating procedures), cautious handling of the patient (because of his potential to harbor drugresistant bacteria), and possible decreased infectiousness compared with the first week of illness may have prevented transmission to hcws. these findings also underline the importance of following who recommendations on infection prevention and control when managing a patient who may be infected with a pathogen that could lead to nosocomial transmission (23) . regarding possible sources of infection, an extensive interview was conducted with family members because the patient could not be interviewed. the patient's illness was likely a primary case, and possible exposures that might have caused the mers-cov infection were explored. of note were the presence of bats in the neighborhood of his residence, the patient's profession as camel breeder, and his contact with a camel that was reported to have had a respiratory illness before his own illness onset. bats are a likely reservoir for mers-related cov (5, 8) , and serum samples from omani racing camels have shown to have neutralizing antibodies against mers-cov (24). these findings suggest these animals' possible relevance (e.g., as intermediate hosts) for human acquisition of mers-cov. two complementary monitoring instruments for contact persons were used: active follow-up with daily telephone contact and a self-administered monitoring questionnaire. both methods have merits, and a combination of both is likely to ensure the most thorough contact follow-up. advantages of personal interviews on the telephone are immediacy and the possibility for the interviewer to receive intangible information, such as the self-assessment of symptoms, as well as the opportunity to answer questions from the contacts. this process enables a more specific way to judge a person's health status. on the other hand, a daily monitoring questionnaire provides detail in clinical information, exposure, and protection measures that might be used for more in-depth analyses (e.g., when a few contacts have become infected). such a questionnaire could be expanded to include a section for contact persons to fill in the names of persons with whom they had face-to-face contact during each day. this information might become crucial for second-generation contact tracing when contacts under observation become infected. rapid availability of this type of information is essential for efficient investigation of clusters or outbreaks similar to those that have been reported already (13) . in conclusion, we conducted a contact investigation of an imported case of mers-cov infection in germany. laboratory testing of symptomatic and asymptomatic contacts of the index case-patient did not indicate transmission of the virus. furthermore, we documented the change from standard hygiene to infection control measures after mers-cov was suspected, an adaptation that may have prevented nosocomial transmission. exposure to camels as a possible etiologic mechanism for human mers-cov infection requires further evidence from other studies. dr reuss is an epidemiologist at the respiratory infections unit, robert koch institute, berlin, germany. her research interests include emerging infectious respiratory diseases, pandemic preparedness, and influenza vaccination. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. novel coronavirus infection in the united kingdom middle east respiratory syndrome coronavirus (mers-cov)-update state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe coronaviruses in bats from mexico full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus close relative of human middle east respiratory syndrome coronavirus in bat genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans group c betacoronavirus in bat guano fertilizer middle east respiratory syndrome coronavirus (mers-cov)-update health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission investigation of an imported case of middle east respiratory syndrome coronavirus interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus contact investigation of a case of human novel coronavirus infection treated in a german hospital clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection empfehlungen des robert koch-institutes für die hygienemaßnahmen und infektionskontrolle bei patienten mit schwerem akutem respiratorischem syndrom (sars) assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections updated rapid risk assessment: severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus infections in health care workers infection prevention and control of epidemic-and pandemic-prone acute respiratory diseases in health care. who interim guidelines key: cord-263391-18x4ann5 authors: harvey, ruth; mattiuzzo, giada; hassall, mark; sieberg, andrea; müller, marcel a.; drosten, christian; rigsby, peter; oxenford, christopher j. title: comparison of serologic assays for middle east respiratory syndrome coronavirus date: 2019-10-17 journal: emerg infect dis doi: 10.3201/eid2510.190497 sha: doc_id: 263391 cord_uid: 18x4ann5 middle east respiratory syndrome coronavirus (mers-cov) was detected in humans in 2012. since then, sporadic outbreaks with primary transmission through dromedary camels to humans and outbreaks in healthcare settings have shown that mers-cov continues to pose a threat to human health. several serologic assays for mers-cov have been developed globally. we describe a collaborative study to investigate the comparability of serologic assays for mers-cov and assess any benefit associated with the introduction of a standard reference reagent for mers-cov serology. our study findings indicate that, when possible, laboratories should use a testing algorithm including >2 tests to ensure correct diagnosis of mers-cov. we also demonstrate that the use of a reference reagent greatly improves the agreement between assays, enabling more consistent and therefore more meaningful comparisons between results. middle east respiratory syndrome coronavirus (mers-cov) was detected in humans in 2012. since then, sporadic outbreaks with primary transmission through dromedary camels to humans and outbreaks in healthcare settings have shown that mers-cov continues to pose a threat to human health. several serologic assays for mers-cov have been developed globally. we describe a collaborative study to investigate the comparability of serologic assays for mers-cov and assess any benefit associated with the introduction of a standard reference reagent for mers-cov serology. our study findings indicate that, when possible, laboratories should use a testing algorithm including >2 tests to ensure correct diagnosis of mers-cov. we also demonstrate that the use of a reference reagent greatly improves the agreement between assays, enabling more consistent and therefore more meaningful comparisons between results. s ince the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 (1), more than 2,250 laboratory-confirmed cases have been reported to the world health organization (who); approximately one third of these cases were fatal. a large proportion of mers-cov cases have been the result of human-to-human transmission in healthcare settings (2, 3) ; outbreaks have occurred in several countries, with the largest outbreaks seen in saudi arabia, united arab emirates, and south korea (4). dromedary camels are the putative reservoir hosts for mers-cov; they experience no or mild symptoms upon infection (5) . primary infection can occur from dromedary camels to humans, and new cases with evidence of camel contact continue to occur sporadically (6) . mers-cov is 1 of the 10 high-threat pathogens on the who research and development blueprint (7) , a document that sets out a roadmap for research and development of diagnostics, preventive and therapeutic products for prevention, and early detection and response to these high-priority pathogens. the who roadmap for mers-cov lists several priority activities, including improved diagnostics and vaccines for humans and camels as well as basic and translational research (8) . serologic assays are critical for the evaluation of the efficacy of new vaccines and patient treatment, as are diagnostic tools to confirm infections and perform serosurveillance. a variety of serologic assays have been developed globally, both commercially and in-house; however, there is no evidence supporting the quality of performance of these assays and their consistency with one another. participants at the who intercountry meeting on mers-cov in cairo, egypt, june 20-22, 2013, recognized this issue as a public health priority and called for a study to compare currently available serologic assays (9) . therefore, we assembled a panel of human serum or plasma and polyclonal antibodies to compare the performance of serologic assays for mers-cov. we invited participants to use their testing algorithms to diagnose each sample as if it were a real patient sample. the assays were evaluated for sensitivity and specificity. pas et al. described in 2015 the impact that a single international standard would have on reducing interlaboratory variability for mers-cov diagnostics (albeit in this case for nat assays) (10) . to this end, we included 2 samples in the panel as examples of potential who international standard material, and we assessed their effectiveness in harmonization of the data from the participant laboratories. the national institute for biological standards and control (nibsc) human material advisory committee (project 16/005mp) approved this project. the ministry of health, oman; ministry of health, saudi arabia; and korea national institute of health, south korea, donated convalescent serum and plasma samples from pcr-confirmed mers-cov-infected patients. all patient donors gave informed consent for the use of their serum or plasma, and samples were anonymized. we treated all mers-cov convalescent-phase serum and plasma with solvent detergent using a validated method (11) to ensure there was no residual infectious virus. we stored all study samples at -20°c until dispatched on ice packs to participants. we blind coded the study samples provided to the participants (table 1) . we included 5 plasma samples (samples 1, 5, 9, 11, and 12) as individual patient samples. other smaller serum donations were pooled in 3 samples (samples 14, 16, and 18) based on their antibody titer, which we determined using the recombivirus human mers-cov nucleoprotein (np) antibody (igg) elisa kit (alpha diagnostic international, https://4adi.com). we categorized samples into high-, medium-, or low-positive pools ( figure 1 ). we included mers-cov-negative serum with antibodies against other human coronavirus hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1 (samples 3, 6, 7, 8, 13, 15, and 17) to test specificity of the assays ( table 2 ). co-authors a.s., m.a.m., and c.d. precharacterized and donated these samples. purified human mers polyclonal antibodies from transchromosomal (tc) cattle (12) immunized with either recombinant spike protein or whole inactivated virus (samples 2, 4, and 10) were donated by eddie j. sullivan (sab biotherapeutics, inc., sioux falls, sd, usa). we diluted the material in universal buffer (10 mmol/l tris-hcl, ph 7.4, 0.5% human serum albumin, 2% trehalose) at a concentration of 1 mg/ml. the 10 participating laboratories (listed at the end of this article) were located in australia, china (2 mainland, 1 hong kong), germany, south korea, the netherlands, united states, and the united kingdom (2 laboratories). participating organizations included national control laboratories, diagnostic laboratories, and research laboratories. participants tested the sample panel using their routine assays for mers-cov serology. we asked participants to perform 3 independent assays on different days. we provided an excel spreadsheet (microsoft, http://www.microsoft.com) for reporting the raw data for each assay and any interpretation of the results, such as positive or negative diagnosis of the samples. we combined titers and relative potency (relative titer) estimates as unweighted geometric means (gms) for each sample and laboratory and used these laboratory gms to calculate overall unweighted gms for each sample. we expressed variability between laboratories using geometric coefficients of variation (gcv = [10 s − 1] × 100%, where s is the sd of log 10 transformed estimates). to mitigate the effect of any potential outliers, we calculated robust estimates of s using the r package wrs2 (13). we referred to all participating laboratories by code numbers, which were randomly allocated. if a laboratory returned data using different assay methods, we assayed the results separately for each method and referred to them according to their laboratory number and assay code; for example, 04 ppnt (pseudoparticle neutralization test) and 04 tcid 50 (50% tissue culture infectious dose). a total of 27 datasets were returned (table 3 , https://wwwnc. cdc.gov/eid/article/25/10/19-0497-t3.htm). data covered a range of different assay formats: neutralization assays, eli-sa, immunofluorescence tests, and 1 microarray. in general, there was good agreement between all the assays tested in this study. in assays with a quantitative measurement, the limit of detection and titer of samples varied greatly, but overall determination of positive or negative agreed between all assays except for 1 (laboratory 04 tcid 50 mn [microneutralization]), which failed to detect 2 positive samples (samples 9 and 18) that all other tests detected as positive. the panel of negative control samples was deemed to be negative in all quantitative assays. there were 3 instances of laboratories reporting a result above cutoff for samples in 1 assay, but these samples were correctly diagnosed as negative overall by their testing algorithms: laboratory 02 detected samples 3 and 7 as above cutoff at 1:80 dilution in 1 assay only; laboratory 02 detected sample 13 as above cutoff at 1:100 and 1:400 dilutions in 1 assay; and laboratory 03 detected sample 13 as above cutoff at 1 dilution tested. participants detected pool a, the high-titer mers-cov antibody pool (sample 16) in all assays (table 3) . they detected pool b, the medium-titer pool (sample 18), in all but 1 of the quantitative assays, a tcid 50 mn assay from laboratory 04. in all other quantitative assays, participants detected the high pool at a higher titer than the medium pool. in the qualitative assays, 3 assays gave borderline positive or equivocal results for the medium pool; these assays were a euroimmun s1 elisa (https:// www.euroimmun.com) in laboratories 01 and 10 and an in-house immunofluorescence assay in laboratory 07. the low-positive pool (pool c, sample 14) was only detected as positive in a single assay in the study, the alpha diagnostic international mers np elisa performed in laboratory 05. participants scored positive the 2 purified igg samples from mers-cov-immunized transchromosomal bovine samples (samples 4 and 10) in all the qualitative assays, but 2 of the quantitative assays, n elisa from laboratory 02 and the alpha diagnostic international mers np elisa from laboratory 05, were unable to detect these samples. we expected these 2 np assays to fail to detect sample 10 because this antibody was raised against recombinant 1880 emerging infectious diseases • www.cdc.gov/eid • vol. 25, no. 10, october 2019 mers spike protein only; however, it was surprising that the assays did not detect sample 4, which was an antibody raised against whole inactivated virus. for the individual convalescent-phase plasma samples, we saw more variability in the results when compared with the pooled material, but despite this, 3 of the samples (samples 5, 11, and 12) were correctly identified as positive in all tests. sample 1 was correctly diagnosed as positive in all tests but was identified as borderline positive by laboratory 01 in a euroimmun elisa assay. sample 9 was the most difficult individual patient plasma to detect as positive; it was negative in the tcid 50 mn assay in laboratory 04; equivocal/positive in the in-house immunofluorescence assay in laboratory 07; and, in the euroimmun elisa, borderline/negative in lab 01, equivocal in lab 07, and weak positive in lab 10. sample 9 was the weakest positive of the individual samples tested in the panel, as we saw from the titers in the quantitative assays that detected it as positive. we compiled the results of quantitative assays for the 5 individual positive plasma samples ( figure 2 ). to simplify comparison of the assays, we reported results from the different laboratories as relative to either the transchromosomal bovine igg sample raised against whole inactivated virus (sample 4) or the high-positive pooled human serum (pool a, sample 16). when we expressed data as a potency relative to either of the 2 chosen potential reference preparations with an assigned arbitrary value of 1,000, we observed improvement in the agreement between tests (table 4 ; figure 3 ). we saw the greatest reduction in the variation between laboratories (smaller sem in figure 3 and smaller percentage geometric coefficient of variation [gcv] in table 4 ) when we used pooled human serum (sample 16) as standard. the transchromosomal bovine igg raised against whole virus (sample 4) showed a substantial improvement in the agreement between laboratories; however, 2 elisa methods included in this study could not identify this sample as positive. as detection of sporadic cases of mers-cov continues, development of new vaccines to combat the disease remains important, as does serosurveillance to understand exposure to the disease and the severity of illness in persons exposed to the virus (14) . the importance of serologic assays for the diagnosis of mers cases should also be considered; the who guideline for laboratory testing for mers-cov, updated in january 2018, specifically includes sample collection from suspected mers-cov cases for serology (15) . the guideline lists 3 situations in which laboratories should conduct serologic testing for mers-cov: for defining a sporadic mers-cov case if molecular testing, such as nucleic acid amplification methods, is not possible; as part of an investigation of an ongoing outbreak; or serologic surveys, such as retrospective analysis of the extent of an outbreak. in this collaborative study, we evaluated the performance of assays to detect mers-cov antibodies using a panel of serologic samples. all laboratories correctly identified the negative samples in the panel, including those containing antibodies against other coronaviruses, implying good specificity of the assays. laboratories reported the positive results correctly except for sample 14, which tested negative by all but 1 assay; however, these results demonstrated the importance of a testing algorithm. we observed 14 instances of a sample being incorrectly determined as negative or borderline/equivocal in a single test in a single laboratory (table 3) . however, because each laboratory used a testing algorithm involving >1 method of analysis, all the samples with sporadic spurious results were correctly diagnosed as positive or negative; if they had used a single assay type, we would have found a higher proportion of incorrect results. the results for sample 14, which was a pool of serum samples from patients with confirmed mers-cov, highlight a lack of sensitivity in most of the assays in this study; further investigation would be needed to determine why the antibody titers in this pool were below the limit of detection in all but 1 assay targeting n protein. it is important to understand whether there is a specific window of time in which clinical samples for serology should be taken or whether some patients do not mount a detectable antibody response against the spike protein during infection. such information may lead to further updates of who guidelines on laboratory testing for mers-cov. the raw titers reported from the laboratories performing quantitative assays varied greatly, for some samples >1,000-fold, between laboratories ( table 3 ). the use of a reference material in the assays tightened the values from the laboratories for all the samples, enhanced comparability (figure 3) , and reduced the gcv percentage between all laboratories (table 4) , perhaps unsurprisingly, but the magnitude of reduction in gcv percentage was noteworthy. mers-cov is an example of an important emerging pathogen with potential to cause outbreaks; diagnostic tests for emerging pathogens are often developed during outbreaks without proper validation or calibration. this study showed the importance of using a standard reagent to allow better comparison of results from different laboratories or interpretation of results from different studies or clinical trials. as we continue to face emerging pathogens, which pose significant risks to human health, it is important to use the experience gained from studies such as this to improve our response to the next threat. standardizing assays is a key issue when different groups around the world are working to develop and produce novel assays and vaccine products. the need for a standard for mers-cov serology was discussed and was widely supported at the who-international vaccine institute joint symposium for mers-cov vaccine development in seoul, south korea, june 26-27, 2018. several potential vaccines are in development, and the immune response elicited, their efficacy, and correlates of protection must be assessed. the use of a reference such as who international standards (16) will enable worldwide harmonization of assays and comparability of the results from different preclinical and clinical studies. assessing the specificity and sensitivity of methods is crucial to improve our understanding of the use and limitations of serologic assays for emerging diseases for which we have little knowledge of disease progression, antibody profiles, and other key information that is available for other infectious diseases. national institute for viral disease control and prevention department of viroscience high-containment microbiology isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. who mers-cov global summary and assessment of risk scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases annual review of diseases prioritized under the research and development blueprint a roadmap for mers-cov research and product development: report from a world health organization consultation report on the intercountry meeting on the middle east respiratory syndrome coronavirus (merscov) outbreak in the eastern mediterranean region geneva: the organization first international external quality assessment of molecular diagnostics for mers-cov robustness of solvent/detergent treatment of plasma derivatives: a data collection from plasma protein therapeutics association member companies triple immunoglobulin gene knockout transchromosomic cattle: bovine lambda cluster deletion and its effect on fully human polyclonal antibody production wrs2: wilcox robust estimation and testing mers: progress on the global response, remaining challenges and the way forward world health organization. laboratory testing for middle east respiratory syndrome coronavirus interim guidance recommendations for the preparation, characterization, and establishment of international and other biological reference standards (revised we thank the following institutions and organizations: universitätsklinikum, bonn, germany; charité-universitätsmedizin, berlin, germany; korea national institute of health; the central public health laboratory, ministry of health, sultanate of oman; public health department, supreme council of health, qatar; the ministry of health, public health directorate, kingdom of saudi arabia; sab biotherapeutics, south dakota, usa. we also thank humayun asghar from the who eastern mediterranean regional office for assistance obtaining the serum for the study. dr. harvey is a principal scientist at the national institute for biological standards and control, united kingdom. her research interests include influenza virus vaccines and respiratory viruses including mers coronavirus. key: cord-022046-q1exf47s authors: toosy, arshad haroon; o'sullivan, sean title: an overview of middle east respiratory syndrome in the middle east date: 2018-09-28 journal: fowler's zoo and wild animal medicine current therapy, volume 9 doi: 10.1016/b978-0-323-55228-8.00042-4 sha: doc_id: 22046 cord_uid: q1exf47s nan middle east respiratory syndrome (mers) is an emerging infectious zoonotic disease caused by a novel coronavirus (cov). mers was first reported in 2012 in jeddah, kingdom of saudi arabia (ksa), and in jordan, respectively. 1 the disease was considered a potential pandemic threat to public health in the persian gulf region. 2 (see also chapter 19.) most known covs infect and circulate in animals, mainly bats, but a number of covs are known to cause human disease (see also chapter 40). [3] [4] [5] the rapid emergence of mers-cov coupled with its limited geographic distribution has led to the suspicion that this is a zoonotic disease with an animal reservoir, 4 and the evidence supports the hypothesis that dromedary camels (dcs) are the reservoir host. in dcs mers-cov causes a mild, transient upper respiratory tract (urt) infection. [4] [5] [6] a mild or asymptomatic disease has also been reported in humans, but this is not always the case. mers-cov infection in humans often results in a severe, life-threatening disease of the lower respiratory tract (lrt), with high mortality. 1, 2, 5 immunocompromised, elderly people and those with comorbidities, usually with a history of close contact with infected dcs, are particularly susceptible. 7 is a positive-sense enveloped single-stranded rna virus and is the first lineage of 2c betacoronavirus known to infect humans. 2, 8 it is more closely related to bat covs hku4 and hku5 (lineage 2c) than to the severe acute respiratory syndrome cov (sars-cov, (lineage 2b). 2,3 recent genome sequencing analysis reported the genomic evolution rate (1.12 × 10 3 substitutions per site), suggesting that mers-cov diverged from its viral ancestor in march 2012. 12 analysis of human mers-cov sequences has identified several circulating genotypes. these distinct genotypes are phylogenetically classified into clades a, b, and, most recently, c, which correlate with outbreaks of mers among humans. 4, 5, 8, 12 the emergence of divergent mers-cov clades in humans since 2012 is consistent with several independent sporadic introductions into the human population from an animal reservoir, of which the camel was unquestionably the source. 6, 8, 12, 13 pathogenesis host cell entry of mers-cov is mediated by the binding of mers spike (s) proteins to a specific cellular receptor known as dipeptidyl peptidase 4 (dpp4). 11 dpp4 is expressed on the epithelial and endothelial cells of most human organ tissues in ex vivo studies using human tissue culture lines; this may account for the multisystem clinical spectrum of the mers-cov infection. 2, 14 a strain cultured from a fatal human case was experimentally inoculated into three dcs using intratracheal, intranasal, and conjunctival routes. 15 a mild transient disease resulted in submucosal inflammation and necrosis in the urt and lrt, but the alveoli remained unaffected. 15 experimental inoculation of rhesus macaques (macaca mulatta) and common marmosets (callithrix jacchus) resulted in mild to severe lrt disease causing multifocal interstitial pneumonia in the macaques and extensive fatal pneumonia in the marmosets. 2, 14, 16 since at least 1992. 13 several mers-cov serologic surveys confirm that the disease is not present in domesticated livestock (namely, horses, sheep, goats, and cattle) and is enzootic in the dc population across the arabian peninsula as well as in north and east africa. 6, 7, 13, 15, [19] [20] [21] [22] [23] historically, the camel was the mainstay of land-based trade transportation and was used extensively as a food source across the entire region prior to industrialization during the latter part of the 20th century. 5, 10 the free movement of humans and animals across the region supports the widespread prevalence and genetic diversity of mers-cov in the dc populations of arabia and east africa today. 5, 10 the temporal dynamics of mers infection in dcs in al-ahsa, ksa, was examined by collecting nasal swabs and lung tissue during postmortem examination from two independent groups of animals over the course of a year and testing these for mers-cov rna by real-time reverse-transcriptase polymerase chain reaction (rt-rtpcr). 24 positive samples were typically associated with young immunologically naive animals (<4 years of age) rather than adults (>4 years of age). seasonal peaks were detected during the winter months and coincided with the calving season, less extreme environmental conditions, cooler ambient temperatures, and higher relative humidity, for the transmission of infection amongst susceptible individuals. 24 this seasonal peak has also been described in epidemic nosocomial outbreaks in humans that occur more frequently during the winter months. 25, 26 extensive virologic evidence has been accumulated since 2012 supporting the epidemiologic link between dcs and humans in the transmission of mers-cov, although epidemiology mers-cov belongs to a lineage commonly associated with bats, the closest relatives of which lineage were recently identified in vesper bats (i.e., various species of the family vespertilionidae) from europe, asia, and south africa. initial research efforts have focused on establishing an epidemiologic link between bats and humans. 3, 4, 6, 17 there is no conclusive evidence to support the theory that bats are the source of human infection, although there is consensus that bats are the ancestral hosts of the disease. 4,5,17 a related mers-like cov virus, isolated from an african pipistrelle bat (pipistrellus hesperidus) in uganda, has shown high divergence of the s protein nucleotide sequence compared with an index mers-cov s protein sequence (46% amino acid identity divergence). 17 this suggests that the two viruses differ significantly in receptor binding properties, implying that the mers-like cov virus is not a zoonotic threat and supporting the theory of a common ancestry. 17 to date the only direct link between bats and human disease was a single instance when an rna sequence from a fatal human mers index case showed a 100% nucleotide match of a polymerase chain reaction (pcr)-amplified sequence of a fecal pellet from an egyptian tomb bat (taphozous perforates) collected in the same area of bisha, ksa. 18 the human fatality was an owner of four dcs, which also tested positive for the same strain of mers-cov. 18 at present, bat-to-human infection by mers-cov is considered to be purely speculative. 4 surveillance of dcs in ksa has shown that mers-cov clade b has been enzootic in the camel population in arabia genetic deep sequencing methods (i.e., high-throughput sequencing) have been readily available to researchers since the disease was first reported. 12 sequenced data have been used in these cases to successfully construct the phylogenetic tree between related viruses and hosts. 2, 6, 7, 12 direct mers-cov antigen detection is possible but has been rarely performed. 10 immunochromatography assays and monoclonal antibody-based capture elisas targeting the mers-cov nucleocapsid protein have been described. 20 since the virus was first reported in 2012, a range of comprehensive laboratory tests has been developed. 10, 30 to better understand the disease, it has been important to collate sampling methodology data, laboratory results, and analyses in combination with clinical and epidemiologic data. 10 until laboratory assays are fully validated, a combination of molecular and serologic laboratory tests is required to improve confidence in laboratory diagnosis during outbreaks. 30 in cases of mild or asymptomatic infection, full validation of serologic assays is required to rule out false-negative results. 31 validation is also required to successfully apply newly developed diagnostic serology algorithms to inform public health decisions. 10, 30, 31 treatment therapeutic options for mers-cov are limited. supportive treatment is indicated for hospitalized patients, but vigilance for complications is essential. 8 empirical use of antimicrobial agents or steroids has not succeeded in reversing the progression of severe disease. 8, 27, 29 no specific drug or vaccine is currently available to treat mers. indeed, it has been stated that the complexity and time required for the development and registration process of drugs for human use impedes the ability to counter the rapid threat against an emerging infectious agent. 8 for example, there is no vaccine available against sars-cov because of the brevity of the threat to the public health. 2, 8 it is likely that a mers-cov vaccine for human use may not be developed due to a lack of commercial interest, or if the threat posed by mers-cov declines in the meantime. 8 nevertheless, given the prevalence of mers-cov infection in the middle east's dc population and due to the potential for spillover to the human population in direct contact with dcs, the development of a vaccine for use in dcs may be feasible. 4, 5, 32 a recent successful trial of a mers orthopoxvirus vaccine has conferred mucosal immunity in the urts of dcs. 32 eradication of mers-cov from herds may be possible, if vaccines are administered to young, immunologically naive camels prior to exposure. 4, 5, 32 identification of the zoonotic source of mers guides control strategies at the human-animal interface. 3, 30 by preventing spillover of mers-cov from animals to humans, the risk of nosocomial and familial outbreaks in the middle east could be eliminated. 3 strategic serosurveys of humans using samples collected after 2012 have been infrequent. 4, 5, 10 there is a paucity of baseline data to describe the proportion of the potentially infected human population for much of the arabian peninsula and all of east africa, including the horn of africa. 10 the exact mechanism of transmission from camels to human remains uncertain. 10 sustained close contact is most probably necessary for transmission by aerosolized droplets, as mers-cov viral rna has been detected in air samples from a barn housing infected dcs in qatar, and the virus may remain viable in aerosol for up to 45 minutes. 10, [27] [28] [29] the potential public health risk resulting from aerosol-generating activities ranges from contamination of a room occupied by a symptomatic patient to slaughter practices. 8, 24, 30 aerosolized transmission of mers-cov has been attributed to hospital outbreaks in ksa and south korea. 26, 27, 29 mers-cov spreads inefficiently from human to human, but transmission is effective in a hospital environment, where susceptible individuals are concentrated and the risks are amplified by poor infection prevention and control (ipc) protocols. 8 in some reported cases of mers, direct contact with camels was not apparent. 27, 29 camel-to-human transmission through other routes is, however, possible owing to the consumption of unpasteurized camel milk or raw camel meat and in traditional medicine, when camel urine is consumed as a natural remedy for a variety of ailments. 3, 10 a recent survey has found that infected camels may shed mers-cov virus in milk and urine, and the virus has been shown to remain infectious for 3 days in milk stored at 4°c. 3, 10 the transmission risks associated with the handling of camel products, raw milk, urine, and meat during animal slaughter are yet to be fully elucidated. further studies are needed to demonstrate the potential of camel-to-human transmission. 8 serologic methods with high sensitivity and specificity to detect mers-cov antibodies have been developed for use in seroepidemiologic studies. methods include indirect immunofluorescence assays, enzyme-linked immunosorbent assays (elisas), protein microarray technology, and microneutralization (mn) assays. 13, [20] [21] [22] pseudoparticle virus neutralization tests (ppnts) and conventional mn assays have also been used to detect neutralizing antibodies to mers-cov. 18 validated molecular assays have been developed. 10, 13, 19 rt-rtpcr is the preferred diagnostic method for the detection of mers-cov and has been endorsed by the who. 1 confirmation of mers in suspected cases requires the screening of samples targeting a number of genes specific to mers-cov, namely up e, orf 1a, orf 1b, and n genes. 1, 10, 13, 19 its infancy. aside from bats, the role that other wildlife may play in the ecology of mers-cov in east africa and arabia is yet to be elucidated. at present the implementation of intensive ipc measures in human health care is vital, including improving education and awareness among healthcare workers. 1, 8 most human cases have been linked to lapses in ipc, as one-fifth of viral infections have been reported among healthcare workers. 1, 8 stringent precautions while handling suspected mers-cov patients include the use of personal protective equipment (ppe) (i.e., disposable gloves, gowns, respiratory protection, and eye protection). 2, 8, 33 immunocompromised individuals and those with preexisting medical conditions should avoid close contact with dcs. 2, 8 public health authorities should adopt a standardized public health response protocol to include standardized case reporting methodology as defined by the who. 1, 30 standardization of case definitions aids accurate calculation of a case fatality ratio by including mild or asymptomatic cases. 30 the health authority of abu dhabi in the united arab emirates recently implemented a standardized reporting option for mers, successfully incorporating it into existing epidemiologic surveillance systems with the aim of enhancing surveillance, educating healthcare workers, and ensuring laboratory capacity. 25 in countries where mers-cov is enzootic in dcs, mers control at the animal-human interface is unlikely to succeed unless appropriate preventive strategies are implemented. 5 these should include the following: • strict regulation of camel movement with imposition of a requirement for mers clearance prior to the importation and transport of camels, including animals presented for slaughter. • camels with detectable mers-cov rna should be quarantined and tested at regular intervals. • use of appropriate ppes while handling dcs. • increased awareness among camel owners and the general public of the risks of consuming unpasteurized camel milk and urine. this may prove challenging given the depth of customs and beliefs in some areas. • accelerated development of safe and effective mers vaccines for animal and human use. 5, 33 conclusions mers-cov has been observed for only 4 years, and vigilance is vital for the containment of the disease due to the high case fatality rate in humans and possible genetic instability of the virus. 8 continued laboratory testing, genetic sequencing, analysis, timely data sharing, and clear communication are essential if such vigilance is to be effective. 8 nonetheless, despite the potential for a pandemic outbreak at multiple mass gatherings during the islamic calendar (hajj, eid, and umrah) there were no reported outbreaks of mers during or immediately after these events. 10 as such mers-cov is not a virus of pandemic concern. 10 since 2012 our understanding of mers has increased greatly although gaps in knowledge still exist. the understanding of the disease's ecology-especially the interplay between camels, humans, and the environment-is still in who mers-cov global summary and risk assessment middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd who emergencies: mers-cov. available at mers coronavirus: diagnostics, epidemiology and transmission dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia pathogenesis of middle east respiratory syndrome coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels response to emergence of middle east respiratory syndrome coronavirus transmission of middle east respiratory syndrome coronavirus infections in healthcare settings hospital outbreak of middle east respiratory syndrome coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient mers-cov outbreak in jeddah-a link to health care facilities mers coronavirus: data gaps for laboratory preparedness asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels centers for disease control and prevention: interim prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). available at an animal model of mers produced by infection of rhesus macaques with mers coronavirus further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia antibodies against mers coronavirus in dromedaries middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan isolation of mers coronavirus from a dromedary camel mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia the authors wish to thank the following: h. e. ghanim mubarak al hajeri and the senior management of al ain zoo for their encouragement and support, dr. ahsan ul haq of dubai camel hospital for his technical insight, and dr. andrew higgins, fellow of the zoological society of london and honorary editor in chief of the veterinary journal, who reviewed the manuscript. key: cord-002070-8y24j34j authors: adney, danielle r.; bielefeldt-ohmann, helle; hartwig, airn e.; bowen, richard a. title: infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas date: 2016-06-17 journal: emerg infect dis doi: 10.3201/eid2206.160192 sha: doc_id: 2070 cord_uid: 8y24j34j middle east respiratory syndrome coronavirus is a recently emerged pathogen associated with severe human disease. zoonotic spillover from camels appears to play a major role in transmission. because of logistic difficulties in working with dromedaries in containment, a more manageable animal model would be desirable. we report shedding and transmission of this virus in experimentally infected alpacas (n = 3) or those infected by contact (n = 3). infectious virus was detected in all infected animals and in 2 of 3 in-contact animals. all alpacas seroconverted and were rechallenged 70 days after the original infection. experimentally infected animals were protected against reinfection, and those infected by contact were partially protected. necropsy specimens from immunologically naive animals (n = 3) obtained on day 5 postinfection showed virus in the upper respiratory tract. these data demonstrate efficient virus replication and animal-to-animal transmission and indicate that alpacas might be useful surrogates for camels in laboratory studies. middle east respiratory syndrome coronavirus is a recently emerged pathogen associated with severe human disease. zoonotic spillover from camels appears to play a major role in transmission. because of logistic difficulties in working with dromedaries in containment, a more manageable animal model would be desirable. we report shedding and transmission of this virus in experimentally infected alpacas (n = 3) or those infected by contact (n = 3). infectious virus was detected in all infected animals and in 2 of 3 in-contact animals. all alpacas seroconverted and were rechallenged 70 days after the original infection. experimentally infected animals were protected against reinfection, and those infected by contact were partially protected. necropsy specimens from immunologically naive animals (n = 3) obtained on day 5 postinfection showed virus in the upper respiratory tract. these data demonstrate efficient virus replication and animal-to-animal transmission and indicate that alpacas might be useful surrogates for camels in laboratory studies. cov) was first detected in samples from a man in saudi arabia who had severe respiratory disease in 2012 (1) . since its identification, >1,600 cases of infection have been documented, and the case-fatality rate is ≈36% (2) . although efficient human-to-human transmission has been documented, zoonotic spillover probably plays a major role in human infection (3) (4) (5) (6) (7) . dromedary camels were identified early after recognition of the virus as a possible reservoir host for the disease, although not all patients report contact with camels. numerous investigators have reported the presence of mers-cov rna or infectious virus in nasal swab specimens of dromedary camels in saudi arabia (3, 4, (8) (9) (10) , qatar (5, (11) (12) (13) , oman (14) , the united arab emirates (15), nigeria (16) , and egypt (17) . in some areas of the middle east and africa, nearly 100% of animals tested were serologically positive for mers-cov, which suggested widespread circulation among camel populations (9, 18, 19) . historical samples contained specific antibodies against mers-cov as long ago as 1992, which indicated that mers-cov has been circulating much longer than originally believed (19, 20) . young animals appear to be at a greater risk for productive infection, and handling practices, such as weaning or shipping animals, might play a major role in animal-to-animal transmission. many dromedary camels tested had high antibody titers. these results support field data suggesting that young animals become infected, and their immune responses probably are repeatedly boosted by subsequent exposure to the virus (18) . however, it is currently unknown whether these repeated exposures result in productive infection or whether antibodies generated from a previous infection are protective. we have previously demonstrated that dromedary camels can be experimentally infected with mers-cov and found that mild upper respiratory tract disease associated with shedding copious amounts of virus by nasal secretions develops during the first week after infection (21) . however, because of the cost of dromedaries, their size, and the requirement for specialized facilities to conduct such studies, it would be useful to identify alternative animal models that respond similarly to infection with mers-cov. we report characterization of an alpaca model of mers-cov infection in which we evaluated virus shedding and pathology, transmission by contact, and protective immunity 10 weeks after initial infection. results indicate that alpacas might be a useful substitute for dromedary camels in certain types of mers-cov experiments. animal experiments were approved by the animal care and use committee of colorado state university. every effort was made to minimize stress and pain of the animals. animals were infected with a low-passage human isolate of mers-cov (strain hcov-emc/2012). this strain was propagated in vero e6 cells cultured in dulbecco modified eagle medium as described (21) . nine locally bred alpacas were obtained by private sale for use in this study. animals were allowed to acclimate to the facility for 1 week before infection and were fed hay ad libitum. one day before infection, animals were subcutaneously injected with an identification and temperaturesensing transponder (lifechip; destron fearing, dallas/ fort worth airport, tx, usa), and their body temperatures were monitored throughout the study. alpacas a1-a3 were housed together and experimentally infected by intranasal instillation of 10 7 pfu of mers-cov diluted in sterile phosphate-buffered saline (3 ml/nare). two days later, alpacas a4-a6 were introduced into the same room as alpacas a1-a3 and housed together for the duration of the study. nasal swab specimens were collected by inserting and rotating sterile swabs into both nares, immediately placed in virus transport medium, and frozen until assay. blood was collected weekly into serum-separating tubes for detection of neutralizing antibodies. animals a1-a6 were held in the facility for 70 days postinfection, and all 6 animals were then reinfected intranasally with 10 7 pfu of mers-cov. three additional alpacas (a7-a9) were also infected to serve as infection controls and evaluate tissue distribution of virus replication. nasal swab specimens were collected daily from all animals for 5 days, at which time animals a7-a9 were humanely euthanized. tissues collected at necropsy for detection of infectious virus from these 3 animals included nasal turbinates, trachea, larynx, and all 4 lung lobes. these samples plus additional samples, including brain, kidney, liver, skeletal muscle, heart, spleen, bladder, mesenteric lymph node, submandibular lymph node, and mediastinal lymph node, were fixed in formalin for histopathologic and immunohistochemical analysis. nasal swab specimens and serum samples collected from alpacas a1-a6 were sampled for 2 weeks after the second infection, and then these animals were then humanely euthanized. tissues were fixed in 10% neutral-buffered formalin for >7 days and embedded in paraffin. tissue sections were stained with hematoxylin and eosin and evaluated by a veterinary pathologist (h.b.-o.). immunohistochemical analysis was preformed to detect mers-cov antigen by using a rabbit polyclonal antiserum against hcov-emc/2012 antigen (diluted 1:1,000) as a primary antibody as described (22). mers-cov was titrated from nasal swab specimens in virus transport medium and homogenized tissue by plaque assay as described for camels (21) . a 1-ml volume of virus transport medium was considered a 10 -1 dilution, and 10-fold serial dilutions were prepared in ba1 medium. neutralizing antibodies were detected by plaque reduction neutralization test (prnt) as described, and seropositive animals were identified by using a 90% neutralization cutoff (23) . field studies and experimental infections suggest that mild respiratory disease associated with nasal discharge develops in mers-cov-infected camels (21, 24, 25) . similar to dromedaries, none of the alpacas had any appreciable increase in body temperature during challenge or rechallenge ( figure 1 ). unlike dromedary camels, none of the alpacas emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 6, june 2016 had any observable nasal discharge over the course of infection. all alpacas maintained consistent activity level, temperament, and food intake throughout the study. nasal swab specimens were collected from infected animals immediately before challenge, on days 1-5 postinfection, and on day 10 postinfection. all 3 experimentally infected animals (a1-a3) had detectable infectious virus on day 15 postinfection but had stopped shedding virus by day 10 postinfection (figure 2, panels a, b) . the 3 co-housed animals (a4-a6) were placed in the room with the infected animals 2 days after initial virus infection. nasal swab specimens were collected from co-housed animals on days 3-10 after infection of animals a1-a3, and then 3 times/ week through day 19. infectious virus was detected from animal a6 during days 7-14 and from animal a4 only on day 14. we did not isolate infectious virus from animal a5 (figure 2 , panel a). although infectious virus was detected in animal a6 on day 7, infectious virus was not detected in animal a4 until day 14 (figure 2, panel a) . we speculate that animal a4 became infected by contact with animal a6 after animals a1-a3 had cleared their infections, which suggested that transmission is linked to intimate animal contact, rather than to aerosol transmission. to test whether previous infection was protective against subsequent virus challenge, all 6 original study animals (a1-a6) were allowed to clear their infections and rechallenged by intranasal infection on day 70 postinfection. challenge was also performed with 3 immunologically naïve alpacas (a7-a9) (infection controls). the 3 immunologically naive animals became infected and shed virus during days 1-5 postinfection, at which time they were euthanized. the 3 animals that became infected through contact (a4-a6) shed minimal virus between days 1-2 after rechallenge, but not on days 3-5. in contrast, animals that had been experimentally infected were completely protected against rechallenge and did not shed detectable quantities of virus ( figure 2 , panels c, d). serum was collected weekly and tested for neutralizing antibodies against mers-cov. all 3 experimentally infected animals (a1-a3) had detectable levels of antibodies beginning on day 14 (table) . although infectious virus was isolated only from 2 of the 3 co-housed animals, these 3 animals had neutralizing antibodies detected first on day 21 (animals a5 and a6) or day 28 (animal a4) (table) . nasal turbinate, upper trachea, lower trachea, larynx, and all 4 lung lobes were sampled at necropsy from alpacas a7, a8, and a9 and tested for infectious virus by using a plaque assay. virus was detected in the nasal turbinates, larynx, and trachea of the 3 alpacas but not in any of the lung lobes tested (figure 3 ). gross lesions were not observed at necropsy in any of the alpacas. however, microscopic analysis of formaldehydefixed tissue sections from animals a7-a9 showed mild squamous metaplasia of the epithelium of the turbinates in animal a8 (figure 4 , panel a) and rare foci of mucosal erosion accompanied by minimal-to-mild subepithelial infiltration of neutrophils and macrophages and fewer lymphocytes ( figure 4 , panel c). all 3 animals also had follicular hypertrophy and hyperplasia of the draining lymph nodes, which suggested immune activation. immunohistochemical analysis detected rare, scattered, virus antigen-positive cells in respiratory epithelium of turbinates (figure 4, panel b ) and in rare cells interpreted to be intraepithelial leukocytes. virus antigen was not detected in any of the other tissues examined. animals a7 and a9 had histopathologic evidence of mild encephalitis with perivascular infiltrates of lymphocytes and monocytes and mild gliosis (figure 4, panel d) . we did not assay brain tissue for virus, either by isolation or pcr, because of the high potential of contamination from the nasal cavity during extraction. brain tissue was negative for virus by immunohistochemical analysis, but the etiology of the encephalitis observed remains unknown and might have been unrelated to mers-cov infection. many difficulties are associated with high containment experiments involving dromedary camels. thus, additional animal models are necessary for mers-cov research. because of their greater availability in the united states and smaller size, we tested an alpaca model. we report an alpaca model of mers-cov infection in camelids and analysis of animal-to-animal transmission and reinfection dynamics. infected alpacas shed considerable quantities of infectious virus nasally, although at lower concentrations than those reported for dromedary camels (21, 24) . in addition, none of the infected alpacas had a noticeable nasal discharge, which is distinctly different from what has been observed in camels and might explain the relatively low efficiency of contact transmission we observed with alpacas. , antibody titer a1 a2 a3 a4 a5 a6 0 <10 <10 <10 <10 <10 <10 14 40 40 40 <10 <10 <10 21 40 40 40 <10 10 20 28 40 80 80 10 160 20 35 80 160 160 20 80 40 42 160 320 160 20 40 20 49 80 320 80 20 80 80 56 80 640 160 20 80 80 63 80 640 160 40 80 80 70 160 640 80 20 40 80 77 320 640 80 160 320 80 84 320 640 160 320 320 80 *alpacas a1-a3 were experimentally infected, and alpacas a4-a6 were co-housed with infected alpacas. titers were determined by using a 90% cutoff. infectious virus was detected in nasal swab specimens from 2 of 3 alpacas co-housed with experimentally infected animals, and each of the 3 co-housed animals had neutralizing antibodies against mers-cov, which indicated virus transmission. the antibody titers observed approximate those seen for infected dromedaries with the exception of a4, whose antibodies titers remained low until after rechallenge (21) . finally, experimentally infected alpacas were completely protected against subsequent virus rechallenge, and contact-infected alpaca were only partially protected. these results suggest that infection can easily spread among closely grouped camelids infected with mers-cov. camels are frequently moved within the middle east for grazing, camel shows, and races. such movement enables mixing and close mingling of animals and could play a major role in mers-cov transmission among animals and to handlers. khalafalla et al. reported that animals bound for slaughter were held in a livestock market for several days, transferred to an abattoir, and kept for up to 24 hours before slaughter (25) . our data suggest that these handling practices could promote animal-to-animal virus transmission and that at the time of slaughter virus could potentially be transmitted to slaughterhouse workers. a major question related to the pathogenesis of mers-cov infection in camels, and of great relevance to vaccination strategies, is whether animals that have been infected are resistant to reinfection and virus shedding and, if so, for how long. our experimentally infected animals were completely protected against rechallenge 70 days later, which suggests that sterilizing immunity can be achieved. however, the animals that were infected through contact (animals a4-a6) shed infectious virus after reinfection, albeit at much lower levels than infected control animals (animals a7-a9). although not tested in the present study, it might be surmised that the 3 in-contact animals would have acquired sterilizing immunity from the second (booster) infection. these results support field data that suggest that young animals become infected and probably receive booster infections; most older animals have acquired immunity and are not susceptible to infection and virus shedding (26) . this finding also highlights the possibility that widespread vaccination of dromedary camels could result in a major decrease in virus transmission to humans. to date, neutralizing antibodies against mers-cov have not been detected in camelids outside africa or the middle east. however, if virus were to be introduced into immunologically naive camelid populations, it probably would be readily transmitted among animals. many new world camelids are valued for their fiber, and such transmission might devastate fiber-related industries (27) . thus, as travel-associated cases of mers-cov infection continue to be documented, human-to-human virus transmission and possible human-to-animal virus transmission should be monitored. this study had several limitations. each of the 3 experimental groups had only 3 animals, which limited our ability to perform statistical analyses. in addition, we evaluated protective immunity 10 weeks after the original infection, which is a relatively short period and does not fully recapitulate seasonal exposures. thus, further studies are necessary to better understand duration of immunity in camels and alpacas. note added in proof: crameri et al. also report experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus in this issue of emerging infectious diseases (28) . isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) evidence for camel-to-human transmission of mers coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates occupational exposure to dromedaries and risk for mers-cov infection mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through wholegenome analysis and virus cultured from dromedary camels in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels isolation of mers coronavirus from a dromedary camel high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels antibodies against mers coronavirus in dromedary camels antibodies against mers coronavirus in dromedary camels mers coronavirus neutralizing antibodies in camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels infection with mers-cov causes lethal pneumonia in the common marmoset dynamics of passive immunity to west nile virus in domestic chickens an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels mers-cov in upper respiratory tract and lungs of dromedary camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries medicine and surgery of camelids experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus we thank erasmus medical center (rotterdam, the netherlands) for proving virus isolate hcov-emc/2012 and vincent munster and the rocky mountain laboratories for providing the rabbit antiserum against mers-cov. key: cord-259051-6kuh4njb authors: elkholy, amgad a.; grant, rebecca; assiri, abdullah; elhakim, mohamed; malik, mamunur r.; van kerkhove, maria d. title: mers-cov infection among healthcare workers and risk factors for death: retrospective analysis of all laboratory-confirmed cases reported to who from 2012 to 2 june 2018 date: 2019-05-02 journal: j infect public health doi: 10.1016/j.jiph.2019.04.011 sha: doc_id: 259051 cord_uid: 6kuh4njb background: approximately half of the reported laboratory-confirmed infections of middle east respiratory syndrome coronavirus (mers-cov) have occurred in healthcare settings, and healthcare workers constitute over one third of all secondary infections. this study aimed to describe secondary cases of mers-cov infection among healthcare workers and to identify risk factors for death. methods: a retrospective analysis was conducted on epidemiological data of laboratory-confirmed mers-cov cases reported to the world health organization from september 2012 to 2 june 2018. we compared all secondary cases among healthcare workers with secondary cases among non-healthcare workers. multivariable logistic regression identified risk factors for death. results: of the 2223 laboratory-confirmed mers-cov cases reported to who, 415 were healthcare workers and 1783 were non-healthcare workers. compared with non-healthcare workers cases, healthcare workers cases were younger (p < 0.001), more likely to be female (p < 0.001), non-nationals (p < 0.001) and asymptomatic (p < 0.001), and have fewer comorbidities (p < 0.001) and higher rates of survival (p < 0.001). year of infection (2013–2018) and having no comorbidities were independent protective factors against death among secondary healthcare workers cases. conclusion: being able to protect healthcare workers from high threat respiratory pathogens, such as mers-cov is important for being able to reduce secondary transmission of mers-cov in healthcare-associated outbreaks. by extension, reducing infection in healthcare workers improves continuity of care for all patients within healthcare facilities. middle east respiratory syndrome coronavirus (mers-cov) was first detected in a patient living in saudi arabia in september of 2012 [1] . subsequent cases have included human infections across the arabian peninsula, occasional importation of cases outside the arabian peninsula and associated clusters in other regions of the world. outbreaks of non-sustained, human-to-human transmission have occurred primarily in healthcare settings [2] . while mers-cov appears to be inefficient at transmitting between humans in the general community, about half of the reported mers-cov infections have occurred in healthcare settings [2] . healthcare-associated transmission of mers-cov has been reported in france, jordan, saudi arabia, united arab emirates, republic of korea and the united kingdom and has on occasion resulted in large outbreaks [3] [4] [5] [6] [7] [8] [9] . secondary transmission has occurred between patients, from patients to healthcare workers, and from patients to visitors of the hospital. to date, there has been limited evidence of transmission documented between healthcare workers in saudi arabia [10] and anecdotal evidence from healthcare workers to patients in saudi arabia [10] . because hcw have not been consistently tested for mers-cov infection, nor has detailed outbreak investigations occurred within all hospital outbreaks, the role of healthcare workers in onward transmission remains unclear. given the large number of hcws infected to date, it is possible that hcw can propagate an outbreak within a healthcare facility. among reported secondary mers cases in such outbreaks, a substantial proportion have been healthcare workers [3, 4, [9] [10] [11] [12] [13] . the clinical spectrum of mers-cov infection ranges from asymptomatic infection to severe pneumonia with acute respiratory distress syndrome and other life-threatening complications [13] [14] [15] [16] . mild symptoms are non-specific and can include headache, tiredness, fever, mild cough, sore throat and runny nose. some patients may present with gastrointestinal symptoms, including diarrhoea. the non-specificity of mers signs and symptoms poses several challenges, not only for the timely identification and isolation of infected patients, but also for reducing secondary transmission within the healthcare facility, particularly to healthcare workers and between patients. preventing mers-cov infection in healthcare workers is critical because of their role in the clinical management of patients and in ensuring adequate infection prevention and control measures are implemented in healthcare facilities. unnecessarily exposing healthcare workers to mers-cov affects the safety of both the healthcare workers and other patients in the healthcare facility, potentially propagating secondary transmission in healthcare-associated outbreaks. understanding mers-cov infection in healthcare workers to date and the risk factors for adverse outcomes is important for preventing future infection of healthcare workers, for informing and updating infection prevention and control measures in healthcare facilities and for reducing secondary mers-cov transmission within healthcare settings. the international health regulations [17] require all laboratoryconfirmed cases of mers-cov to be reported to the world health organization (who) within 24 h of laboratory confirmation [18] . in this study, we use the epidemiological data of all mers cases reported to date to who to describe secondary cases of mers-cov infection among healthcare workers and to identify the risk factors for death among healthcare workers with secondary infection. a retrospective analysis was conducted on epidemiological data of laboratory-confirmed mers-cov cases reported to who [18] from september 2012 to 2 june 2018. we looked specifically at healthcare workers involved in the delivery of health care to patients within the same healthcare facility as a confirmed or suspected mers-cov case. primary cases were defined as cases with laboratory confirmation of mers-cov infection with no epidemiological link to a suspected or confirmed human mers case. secondary cases were defined as those with laboratory confirmation of mers-cov infection and with a direct epidemiological link with a confirmed or probable mers-cov case. prior to 2015, data from individual cases including occupation, signs/symptoms, exposures and risk factors for infection, etc. was not collected systematically. following the large mers outbreak in jeddah and riyadh in 2014, case report forms and policies related to the investigation of cases and contacts became more systematic from 2015 onwards. given the inconsistencies in the collection and reporting of epidemiologic data from mers-cov cases to who prior to 2015, we performed a secondary analysis with data reported only from 1 january 2015 to 2 june 2018. descriptive analysis was performed for all mers-cov cases reported to who from 2012 to 2018. for all statistical analyses, p < 0.05 was considered statistically significant and all analyses were performed using the epidemiological data display package in r, version 3.2.2.0 (https://cran.r-project.org/ package=epidisplay). we compared all secondary cases in healthcare workers with all secondary cases among non-healthcare workers, using the student t-tests for continuous variables and chi-squared tests for categorical variables. we also aggregated survival outcomes of healthcare workers with mers-cov infection by year of infection. to identify risk factors for adverse outcomes in healthcare workers with secondary infection, we performed multivariable logistic regression analysis on all healthcare worker secondary cases. variables considered were dichotomous (sex, residency, symptomatic clinical presentation, presence of any comorbidities), categorical (year of infection) or continuous (age). the final multivariable model, constructed using the backward, stepwise elimination method, included all variables with an adjusted p < 0.10. our analysis considered all 2223 cases of mers-cov reported to who up to 2 june 2018. among these cases, 462 were primary cases, 989 secondary cases, 380 cases for which the information reported was insufficient to be able to determine whether it was a primary or secondary case, and 392 cases with missing case information. among all cases, 790 (35.5%) deaths occurred. of the 2223 cases, 415 were reported as healthcare workers. the mean age of healthcare workers was 39.3 (interquartile range 30.0-46.0) years and 54.9% were women. five were primary cases, 338 were secondary cases, 54 cases had insufficient information to be able to determine whether it was a primary or secondary case and 18 cases had missing case information. fig. 1 shows the epidemic curve of cases of mers-cov among healthcare workers and non-healthcare workers reported from 2012 to 2 june 2018. table 1 provides further description of the 415 healthcare worker cases, as well as the 178 healthcare worker cases reported to who from 1 january 2015 to 2018. table 2 compares all secondary healthcare worker cases and all secondary non-healthcare worker cases. compared with secondary non-healthcare worker cases, secondary healthcare worker cases were younger (p < 0.001), had a higher proportion of women (p < 0.001), were non-national residents (p < 0.001), and had asymptomatic infection (p < 0.001), fewer comorbidities (p < 0.001) and higher survival (p < 0.001). further comparison between laboratory-confirmed mers-cov cases in healthcare workers and non-healthcare workers are shown in the supplementary material. tables 3a and 3b shows the survival outcomes of all infections by year of infection. tables 3a shows outcomes of all infections by year of infection for healthcare workers, while table 3b shows outcomes of all infections by year of infection for non-healthcare workers. there have been no fatal mers infections among healthcare workers since 2015. table 4 shows the regression coefficients and adjusted odds ratios (95% confidence interval) for the two variables retained in the final multivariable risk model for death in secondary cases among healthcare worker. year of infection and having no comorbid conditions were found to be independent protective factors against death in healthcare workers with secondary mers-cov infection. healthcare workers include, but are not restricted to: doctors, nurses, pharmacists, physiotherapists, radiologists, rehabilitation staff, infection prevention and control staff, intensive care staff, ambulance staff, respiratory therapists, auxiliary healthcare workers, attendants, laboratory, x-ray and ultrasound technicians, and healthcare administrators. a lack of consistency in reporting specific job titles prohibited subgroup analysis by different roles of healthcare worker. healthcare workers continue to constitute a substantial proportion of secondary mers-cov infections. that is, among all cases of mers-cov reported to who as of 2 june 2018, healthcare workers accounted for 18.6% of all mers-cov cases and 34.2% of secondary mers-cov cases. this is similar to other respiratory pathogens: in the 2003 outbreak of severe acute respiratory syndrome, 23% of cases in hong kong, 40% in canada and 41% in singapore were healthcare workers [19] . protecting health care workers from infectious hazards is paramount to ensuring their safety in delivering health care. in addition, being able to protect healthcare workers, constituting the front line response against high-threat respiratory pathogens, such as mers-cov, is important for reducing secondary transmission in healthcare-associated outbreaks. reducing infection in healthcare workers, even if their infection does not cause morbidity or mortality, will improve the continuity of care for all patients in the same healthcare facility. we also found that the demographic and clinical profile of secondary infections in healthcare workers was different from secondary infections among non-healthcare workers. healthcare workers infected with mers-cov were younger, more were female and non-national residents, and had fewer comorbidities, more asymptomatic infections and higher survival. importantly, no deaths have occurred among healthcare workers with secondary mers infection since the end of 2015. these results are similar to the findings in individual outbreaks reports [4, 5, [7] [8] [9] [10] [11] [12] 14] . the highest burden of mers cases to date is in saudi arabia where healthcare workers are more likely to be female, a large proportion of whom are expatriates. the younger age and fewer comorbidities may partly explain the higher survival observed among healthcare workers, as well as the possibility of earlier identification or suspicion of mers among healthcare workers. indeed, the case fatality rate among all healthcare workers was 5.8% compared with 42.8% among all non-healthcare workers. this is the first study to perform multivariable logistic regression to identify risk factors for death among 338 healthcare workers with secondary infections reported to date. the analysis showed that year of infection (2013-2018) and having no comorbidities were independent protective factors against death. the decline in risk of death since 2013 may reflect the substantial improvements in surveillance and the infection prevention and control measures that have been introduced in affected countries in recent years. in saudi arabia, for example, the ministry of health regularly review and update national infection prevention and control guidelines, according to which, healthcare workers who had unprotected "high-risk exposure" (within 1.5 m of the patient) or have suggestive symptoms regardless of exposure type are required to stop performing their duties immediately; to have a nasopharyngeal swab tested for mers-cov; to not resume their duties until cleared by the infection control team and to delay travel until cleared by infection control team [20] . any healthcare worker who tests positive for mers, any healthcare worker who develops mers suggestive symptoms and any healthcare worker who had unprotected high-risk exposure are considered clear and able to resume work if they meet all of the following criteria: asymptomatic for at least 48 h, and the 14-day observation period is over, and they have at least one negative rt-pcr [20] . the enhanced infection prevention and control (ipc) efforts which have been introduced since 2015 include regular training of healthcare workers on ipc, auditing of ipc in healthcare facilities, improved case notification and isolation within emergency departments, and comprehensive contact-tracing and testing of all contacts, including healthcare workers, regardless of the development of symptoms. guidelines on contact tracing were revised after 2015 to include the testing of all contacts of confirmed mers cases (including healthcare workers) for mers-cov. prior to 2015, contacts were only tested if they developed symptoms. this has increased the detection of asymptomatic or mildly symptomatic cases, which in turn, may have decreased the case fatality rate. specific ipc measures which may have contributed to the decreased case fatality rate include more systematic use of appropriate personal protective equipment and increased testing of asymptomatic personnel which effectively increases the detection of asymptomatic healthcare workers who are less likely to die, therefore increasing the denominator of healthcare worker cases and decreasing the case fatality rate. in addition, earlier detection of cases, and more efficient contact tracing have likely identified mers-cov infections in healthcare workers at an earlier stage of infection, enabling more timely treatment and clinical management, and, as a result, a reduction in the case fatality rate. further efforts to prevent and manage emerging respiratory disease infections, including mers among healthcare workers, still need to be made. these include improving identification and rapid diagnosis of mers, further understanding of the mechanisms of transmission in healthcare settings, optimizing the layout of emergency departments for better triage of patients with respiratory symptoms, standardization of infection prevention and control practices and (re)training at facilities with high hospital staff turnover, and auditing of healthcare facilities for adherence to infection prevention and control measures. these will be critical to minimizing transmission in healthcare facilities, particularly until interventions are introduced to stop the virus entering the human population from the dromedary camel reservoir. the role of environmental contamination was evaluated in a number of hospitals following the 2015 mers outbreak in the republic of korea and collaborative, experimental studies were conducted to evaluate the viability and persistence of mers-cov on surfaces and in the air [21] [22] [23] . the role of mild or asymptomatic cases in transmission chains, however, remains unclear [24] [25] [26] [27] . further epidemiologi outcomes cal and environmental studies to determine route(s) of secondary transmission will also be critical. the results of our study are strengthened by the size of the study, which included all cases reported to who since the first case was notified in 2012. our database has cases from all 27 countries reporting cases globally. however, since 2012, some inconsistencies have occurred in the way data have been collected and reported to who, for example in the use of a systematic data collection tool and in reporting the outcome of all cases. challenges also exist in classifying cases based on the available information at the time of reporting. for example, thorough outbreak investigations, which include full genome sequencing to clarify the transmission chains within the outbreak or separate introductions from outside, may find that cases that were initially classified as secondary case are in fact primary cases [4, 11] , but this information is not systematically relayed to who. efforts are currently being made to retrospectively review and update the epidemiological data for all 2223 cases reported to who to date, particularly before 2015. healthcare workers still make up a significant proportion of secondary mers-cov infections. understanding transmission to healthcare workers, preventing infection and improving clinical management of infected healthcare workers will all be critical to further reducing the incidence of secondary infections in healthcare settings. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. who mers-cov global summary and assessment of risk epidemiological findings from a retrospective investigation hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia response to emergence of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission epidemiological investigation of mers-cov spread in a single hospital in south korea middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control molecular epidemiology of hospital outbreak of middle east respiratory syndrome clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study middle east respiratory syndrome a cohort study of patients suspected for mers-cov in a referral hospital in saudi arabia geneva: world health organization middle east respiratory syndrome case definition for reporting to who: interim case definition severe acute respiratory syndrome (sars) and healthcare workers middle east respiratory syndrome coronavirus: guidelines for healthcare professionals -version 5.0 environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers outbreak units stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection the authors would like to thank the many individuals involved in the collection of individual case data and in the care of mers-cov infected patients in the affected countries. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jiph.2019.04. 011. all authors contributed to the study design, and reviewed and edited the manuscript. aae, rg and me performed the statistical analysis. all authors were involved in the interpretation of the findings. no funding sources. none declared. the case-patient data reported to who is anonymized, thus neither informed consent, nor approval from an institutional review board were required by who or countries providing this data to who under the international health regulations (2005). key: cord-276769-th7iou21 authors: khan, suliman; siddique, rabeea; bai, qian; shabana; liu, yang; xue, mengzhou; nabi, ghulam; liu, jianbo title: coronaviruses disease 2019 (covid-19): causative agent, mental health concerns, and potential management options date: 2020-07-25 journal: j infect public health doi: 10.1016/j.jiph.2020.07.010 sha: doc_id: 276769 cord_uid: th7iou21 coronavirus disease-2019 (covid-19) pandemic started from wuhan, china has infected more than 6.7 million individuals and killed more than 3,90000 individuals globally. due to the higher transmissibility and infectiousness, asymptomatic infection, and lack of effective treatment options and vaccine, fatalities and morbidities are increasing day by day globally. despite physical health consequences, covid-19 pandemic has created stress and anxiety, as result there is an increased risk of mental illnesses both in the infected and normal individuals. to eradicate these risks, it is necessary to determine the covid-19 zoonotic source of transmission to humans and clinical manifestations in infected individuals. although, identification or development of the highly effective therapeutic agents is necessary, however, development of protective strategies against the covid-19 by enhancing immune responses will be an asset in the current scenarios of the covid-19 pandemic. in this paper, we discuss the transmission, health consequences, and potential management (therapeutic and preventive) options for covid-19 disease. the new decade of the 21 st century (2020) started with a deadly coronavirus disease 2019 pandemic caused by a novel coronavirus (sars-cov-2) in wuhan, china [1] . . in december 2019, various cases of pneumonia with unknown etiology were reported in wuhan, china. later, on 10 th january 2020, the disease was confirmed as viral pneumonia and found epidemiologically linked with huanan seafood market of wuhan, where wild animals including bats were sold [2, 3] . soon after the chinese authorities declared an emergency situation in wuhan, other countries in the world started reporting covid-19 cases [4] . due to high spread, morbidity, mortality, and infectiousness, who declared covid-19 outbreak as a pandemic [5] . as of june 6, 2020, more than 6.7 millions confirmed cases and more than 3,90000 deaths are reported from j o u r n a l p r e -p r o o f covid-19 pandemic (walker, 2020). to curb the risk of spread and manage the adverse impacts of virus, it is necessary to know the medical consequences associated with covid-19 infection. in this article, we briefly describe the consequences of highly infectous human corona viruses, and elaborate the transmission and health consequences (both physical and mental health) of covid-19 disease. we further discuss the potential therapeutic and preventive strategies, and required research investigations that may help managing and controlling the spread of covid-19 disease. before covid-19, severe acute respiratory syndrome (sars) outbreak caused by sars-cov during 2002 in guangdong, china affected 8098 individuals and killed 774 individuals in 29 different countries [6] . only a decade later (during 2012), the world witnessed the middle east respiratory syndrome (mers) outbreak caused by mers-cov in the middle east [7] . until 2020, mers-cov infected 2468 individuals and caused 851 fatalities worldwide [8] . while researchers were still investigating the mechanisms to develop therapeutic strategies against mers, another member of coronaviruses called sars-cov-2, emerged in wuhan [9] . market civets were thought to be the possible source of sars-cov transmission to human, however, the virus strains in civets were found transmitted from horseshoe bats [10] . mers-cov strains obtained from camels were highly similar to those isolated from humans; with highly prevalent mers-cov-specific antibodies in camels from the middle east, africa, and asia [7, [11] [12] [13] . these observations suggest that mers-cov transmitted from camel to human. however, genome sequencing analysis revealed that mers-cov is phylogenetically related to bat coronaviruses [14] . the zoonotic source of sars-cov-2 is not confirmed, however, its genome sequence exhibited close relatedness with two bat-derived sars-like coronaviruses [15, 16] . thus, it is thought that bats are the possible source of origination for sars-cov-2. like mers-cov and sars-cov, sars-cov-2 belongs to betacoronavirus (subgenus; sarbecovirus) [3, 17] . reportedly, the genome sequencing analysis has revealed over 80% similarity between sars-cov-2 and sars-cov, however, there are some differences at the structural protein levels. for instance, the 8a protein was reported missing while the aminoacids number in 8b and 3c protein were found fluctuating in sars-cov-2 [17] . these differences can help in studying the infectiousness, transmission, and health impacts of covid-19 infection. j o u r n a l p r e -p r o o f although bats are thought to be the source of origin for sars-cov-2, the intermediate animal that caused the transmission of virus to humans, is still unknown [3] . once sars-cov-2 transfers from zoonotic source to human, it can potentially transmit from human-to-human, mainly through respiratory droplets from an infected individual via coughing or sneezing [18] . the virus can also transmit from an asymptomatic infected individual to infect healthy individual [19] . the entry of virus to host cell occurs in several steps including binding to a target host cell via cellular receptors, fusing the envelope with a cellular membrane, and forking over its genetic material inside the cell [20, 21] . this process is highly dependent upon binding specificity to receptors, proteolytic activation, and endocytosis efficiency. this entry process is facilitated by glycosylated spike (s) fusion protein, which is capable of significant structural rearrangement, thus plays an important role in fusing the viral membrane with the host cell membrane [15] . the spike glycoproteins are comprised of two subunits known as s1 and s2, where s2 subunit contains fusion peptide [22] . this fusion process is the key to virus entry into a cell, however, the fusion process is linked with the accessibility of the receptor determined by hinge-like conformational movements of the receptor-binding domain (rbd) of s1. the rbd can transiently hide or expose the determinants of receptor binding [23] . soon after the entrance of the virus to the host cell, transcription of polyprotein 1a/1ab (pp1a/pp1ab) is initiated by the activity of the replicationtranscription complex (rtc) [22] . binding of s protein to the cellular receptor ace2 initiates the life cycle of sars-cov-2 in host cells [22] . studying the infectiousness mechanism further can help to understand the source of origination and transmission. knowing the intermediate sources of transmission is important in disease control. a recently report indicated pangolin as the possible intermediate source that might have transferred the virus to humans after receiving it from bat [24] . the main reasons for the high rate of transmission are not well documented however, the higher affinity of rbd for binding to ace2 receptors is considered to be one of the possibilities for the high rate of infectiousness [17, 25] . the rbd amino acids (l455, f486, q493, s494, n501, and y505) in sars-cov-2 can play a role in the determination of the host range [25] . further research is required to investigate these rbd amino-acids in a wide range of animal species. despite the rbd aminoacids, proteases associated with infectiousness such as furin, can also help to determine host range. this j o u r n a l p r e -p r o o f determination can further be facilitated by studying the higher genetic variation in spike glycoproteins [3, 25] . it has been reported that the mutation found in polybasic cleavage site in sars-cov-2 from humans was dissimilar to that in bat and pangolin viruses [25] , suggesting its association transmission and infection in humans. these observations indicate the need for further research on understanding the impact of polybasic cleavage on transmissibility and pathogenesis. without knowing the zoonotic source of transmission, range of animals hosts, and source of origination, it may be impossible to eradicate the virus. covid-19 disease has caused millions of morbidities and hundred of thousands of mortalities worldwide (walker, 2020). . the clinical manifestations of covid-19 are characterized by fever, cough, dyspnea, and bilateral infiltrates on chest imaging [26] . after infection, the majority of individuals show moderate symptoms whereas approximately, 20% of the infected patients show severe illness of respiratory failure, septic shock [26] , gastrointestinal complications [26, 27] , myalgias, lymphopenia, and parenchymal lung abnormalities [2] . the severity of symptoms and death causing ability of the virus are highly dependent on underlying diseases such as cancer, hypertension, and cardiopulmonary diseases [3, 26] . the infection has been reported to cause high mortalities in older people [28] and individuals with blood group a [29] . moreover, pregnant women with confirmed covid-19 pneumonia can face adverse pregnancy and neonatal impacts [30] . the individuals at higher risk of developing severe disease after contracting the infection should be give the priority for treatment and providing the mangeemtn and health servicesconsidering the importance of covid-19 in the aspects of the asymptomatic spread of the virus and adverse health impacts, it is deemed necessary to investigate the factors associated with the rate of infectiousness and severity of symptoms. covid-19 outbreak is affecting physical health as well as mental health, however, the primary attention is given to physical health [9] . fear of being infected due to close contact with infected patients, prolonged working schedules without proper rest, and disturbed wake and sleep routines have increased the risk of stress and anxiety in the healthcare workers [9] . the consequences can be alarming as mental illnesses have been reported to alter immunity, thus j o u r n a l p r e -p r o o f increasing the vulnerability to diseases [31] . it might be one of the risk factors for higher covidnetwork for epidemics) [32], a large population has no access to this information. therefore, a large number of people rely on electronic and social media, in an attempt to win the race, share forged news also termed as infodemia, thereby, increasing the risk of stress, fear, and anxiety [32, 33] . normal daily routines are inevitable to maintain normal rhythms, however, due to the current restrictions to outdoor activities, millions of people rely on indoor activities. these individuals may experience disturbance in eating routines, exposure to irregular light-dark cycles and disturbed sleep-wake behaviors which may dysregulate circadian rhythms and mood [34] . while, mistimed exposure to light and disrupted sleep perturb the circadian rhythm that may lead to mental illnesses including stress and anxiety [35, 36] . notably, children have no or limited exposure to the open environment and playgrounds, indicating that they may have more access to electronic devices or smartphones. these situations can lead to severe mental illnesses in the future [37, 38] . . nonetheless, the researchers should conduct psychological investigations and identify the mental health concerns if any, not only in children but also in women, elder individuals, and healthcare workers, so that timely treatment and psychological assistance are provided currently, no promising treatment options are available against the covid-19. however, to contain the covid-19, several therapeutic strategies have been evaluated. among the trialed antiviral drugs, remdesivir alone or in combination with chloroquine or interferon beta showed effectiveness against the covid-19 infection [39] . chloroquine efficacy against the covid-19 may be linked with its ability to prevent binding and cellular entry of the sars-cov-2 through interfering with ace2. while remdesivir inhibits the action of viral rna polymerase to prevent viral replication [40] . currently, clinical clinical trials are underway in different regions of the world, while china based preliminary data has demonstrated benefits of chloroquine in patients with pneumonia, as the drug significantly eliminatied the virus and improved recovery from covid-19 disease [41] . thus, it was included in the guidelines for the prevention, diagnosis, and treatment of covid-19 disease, chinese national health commission [41, 42] . however, j o u r n a l p r e -p r o o f chloroquine is also known to have toxicity and side effects to central nervious system [42] . on the other hand, a multi-center trial of remdesivir showed serious side effects in 12 out of the 53 covid-19 patients, although 36 of them were reported clinically recovered [43] . therfore, both remdesivir and chloroquine should further be evaluated for their suitability against covid-19. baricitinib an approved drug for the treatment of rheumatoid arthritis has been considered as the potential candidate to treat covid-19. this drug inhibits the endocytosis regulating enzymes (ap2-associated protein kinase 1) [44] , thus, can inhibit endocytosis of sars-cov-2 into the cell. thus, it could be another potential candidate to treat covid-19 [42] , however, wide range of clinical trials are necessary to evaluate its effectiveness furthermore, ritonavir and lopinavir are promising protease inhibitors, which have been included in the list of anti-covid-19 drugs. although, earlier treatment of covid-19 patients with ritonavir and lopinavir in wuhan, china, did not indicate sufficient benefits of these drugs [45] , however, their combination may successfully inhibit both protease cytochrome p4503a4 which can reduce metabolism and inhibit viral replication [46] . therefore, further clincal trials with combination of these drugs are required to evaluate their effectiveness. recently another antiviral drug known as favipiravir designed for influenza that inhibits rna-dependent rna polymerase, was found effective against the covid-19. however, further studies are required to find its broad term efficacy and associated side effects [47] . moreover, some antiviral drugs in combination with traditional chinese medicines are currently being evaluated in covid-19 patients. it is thought that lower mortality and higher recovery rate from covid-19 disease in china may be linked to the treatment with tradiations chinese medicines in combined with western medicines, as approximately 85% patients in china received these combinations [42, 48] . however, we did not find convincing evidence to conclude that traditional chinese medicines are siginifanclty effective, thereore, further investigations are required to determine their effects against covid-19 disease. despite the afore-mentioned therapeutic options, further studies are necessary to develop promising therapeutic strategies against covid-19 infection. the serious concern with coronaviruses is the ability to suppress counteracting response from the host innate interferons [6] , thus, utilizing interferon inducers or recombinant interferons may help to treat covid-19 pneumonia. surface structural spike protein (s) and ace2 play a critical role in the process of pathogenicity through facilitating sars-cov-2 entry into the host cells [6, 16] . targeting these regions through monoclonal antibodies or fusion j o u r n a l p r e -p r o o f inhibitors [6] may be effective therapeutic options against coronaviruses. therefore, antiviral peptides such as hp2p-m2 that target s protein [6] should be evaluated against the sars-cov-2. effective vaccines are important to prevent and control sporadic epidemics of emerging viruses. although sars-cov was fully controlled during 2003, and mers-cov spreads poorly, yet the sars-cov-2 is spreading efficiently, thus the availability of vaccine is necessary to control further spread of the disease. viral vectors, recombinant protein, viral-like particles, rhesusθdefensin-1, and protein cage-nanoparticles are generally effective in prevention against coronaviruses [6] , therefore such vaccination strategies can be developed for individuals at high risk of covid-19 infection, to control the ongoing pandemics [49] . covid-19 disesae has created an alarming situation worldwide and therefore, it requires serious attention from healthcare authorities and research communities in the aspects of transmission, treatment and prevention. after originating in bats, sars-cov-2 emerged in wuhan, spread all over the world through human to human transmission, and infected millions of individuals. currently, each pasing day, the virus is causing deaths in thousands of covid-19 infected individuals across the globe. although, the virus mainly affects lungs to cause severe to moderate pneumonia, it may adversely impact mental health to develop stress, anxiety and depression. to control the spread of covid-19 infection, there is a need for developing therapeutic options, vaccines and proper management of daily activities and human to human interactions. given the importance of the covid-19 pandemic, further studies are necessary to understand replication, pathogenesis, and biological properties in order to control covid-19 disease and prevent novel emerging diseases in future. the research work should focus on identifying or designing therapeutic agents and developing effective vaccines. testing the drugs for coronaviruses requires suitable animal models prior to their use in humans. however, the j o u r n a l p r e -p r o o f currently established models for sars and mers are not promising except few small animals such as transgenic mice expressing human ace2 and mouse-adapted sars-cov strains [6] . therefore, more animal models from mouse to non-human primates are needed to be developed. neverthelesss, healthcareworkers and medical professionals should focus on helping the public to cope with the mental health concerns. they should further educate the people to understand the spread mechanisms, infection consequences and required management to control the spread. mental health. hum vaccin immunother 2020;00:1-2. novel coronavirus is putting the whole world on alert hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china the emergence of a novel coronavirus (sars-cov-2), their biology and therapeutic options the spread of novel coronavirus has created an alarming situation worldwide covid-19 pandemic ; prevention , treatment coronaviruses-drug discovery and therapeutic options origin and evolution of pathogenic coronaviruses middle east respiratory syndrome coronavirus transmission novel coronavirus: how the things are in wuhan severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae mers coronaviruses from camels in africa exhibit region-dependent genetic diversity genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory sy genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin covid-19 infection: origin, transmission, and characteristics of human coronaviruses importation and human-to-human transmission of a novel coronavirus in vietnam transmission of 2019-ncov infection from an asymptomatic contact in germany dynamics of virus-receptor interactions in virus binding, signaling, and endocytosis fusion of enveloped viruses in endosomes evaluation and treatment coronavirus (covid-19). statpearls [internet cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding on the origin and continuing evolution of sars-cov-2 the proximal origin of sars-cov-2 articles clinical features of patients infected with 2019 novel coronavirus in wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention abo blood group and susceptibility to severe acute respiratory syndrome clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records brain , behavior , and immunity depressive symptoms and immune transcriptional profiles in late adolescents impact of coronavirus outbreak on psychological health 2020 effects of chronic jet lag on the central and peripheral circadian clocks in cba/n mice circadian rhythm disruption and mental health health risks associated with genetic alterations in internal clock system by external factors circadian rhythms have broad implications for understanding brain and behavior the genetics of circadian rhythms, sleep and health remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies covid-19: learning from lessons to guide treatment and prevention interventions compassionate use of remdesivir for patients with severe covid-19 family-wide structural analysis of human numb-associated protein kinases a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 treatment of middle east respiratory syndrome with a combination of lopinavir/ritonavir and interferon-β1b (miracle trial): statistical analysis plan for a recursive two-stage group sequential randomized controlled trial the mechanism of resistance to favipiravir in influenza traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective novel coronavirus, poor quarantine, and the risk of pandemic declaration: the authors of this manuscript declare that there is no conflict of interest key: cord-279503-w4tn03w0 authors: kim, hanbi; park, minseon; hwang, joonki; kim, jin hwa; chung, doo-ryeon; lee, kyu-sung; kang, minhee title: development of label-free colorimetric assay for mers-cov using gold nanoparticles date: 2019-05-07 journal: acs sens doi: 10.1021/acssensors.9b00175 sha: doc_id: 279503 cord_uid: w4tn03w0 [image: see text] worldwide outbreaks of infectious diseases necessitate the development of rapid and accurate diagnostic methods. colorimetric assays are a representative tool to simply identify the target molecules in specimens through color changes of an indicator (e.g., nanosized metallic particle, and dye molecules). the detection method is used to confirm the presence of biomarkers visually and measure absorbance of the colored compounds at a specific wavelength. in this study, we propose a colorimetric assay based on an extended form of double-stranded dna (dsdna) self-assembly shielded gold nanoparticles (aunps) under positive electrolyte (e.g., 0.1 m mgcl(2)) for detection of middle east respiratory syndrome coronavirus (mers-cov). this platform is able to verify the existence of viral molecules through a localized surface plasmon resonance (lspr) shift and color changes of aunps in the uv–vis wavelength range. we designed a pair of thiol-modified probes at either the 5′ end or 3′ end to organize complementary base pairs with upstream of the e protein gene (upe) and open reading frames (orf) 1a on mers-cov. the dsdna of the target and probes forms a disulfide-induced long self-assembled complex, which protects aunps from salt-induced aggregation and transition of optical properties. this colorimetric assay could discriminate down to 1 pmol/μl of 30 bp mers-cov and further be adapted for convenient on-site detection of other infectious diseases, especially in resource-limited settings. t he point-of-care testing (poct) market of infectious disease represents promising and significant growth in the global in vitro diagnostics (ivd) industry. 1 there are several factors that are stimulating the demand for infectious disease poct, including the increasing spread of human immunodeficiency virus (hiv), tuberculosis (tb), and malaria in developing countries, and the threat of emerging and reemerging infectious diseases such as the middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars), zika, a variety of influenza strains, and the west nile virus. 2 infectious diseases pose a significant risk to human health and has led to more than half of the deaths worldwide. 3 additionally, widespread infectious diseases have caused a continuous increase in fatality rates in developing countries. the best way for containment of the epidemic is an early diagnosis, which is difficult using ordinary methods because of costly and large equipment, the necessity of experts, and slow data output. 4 therefore, rapid poct methods are crucial for overcoming these limitations by miniaturizing and reducing the device cost and providing simple, fast, easy-to-use diagnostic tests without specialized training. colorimetric detection originating from gold nanoparticles (aunps) have been intensively studied because of their particular optical properties, i.e., localized surface plasmon resonance (lspr), which represent a color with maximal absorbance wavelength. aunps have been utilized in colorimetric assays for the detection of diverse biological molecules (e.g., proteins 5 and nucleic acids 6 ) in that the change in particle color is generated by sensitive reactivity of nanosized particles to external condition. in addition, the disperse state is adjustably modified from artificial electrostatic force control by ion, ph, biomacromolecules, and so forth. 7, 8 for example, a positive electrolyte (salt) causes metallic nanoparticle aggregation, causing a significant color change as a red-shift in the lspr spectrum. 9,10 based on that, a modified aunp cross-linking method was demonstrated in mirkin's group for dna identification. 11 in the research, the surfaces of aunps were conjugated with two thiolated single-stranded dna (ssdna) via strong au−s interactions. the hybridization of target dnas with the ssdnas on the surface of the aunps induces the formation of double-stranded dna (dsdna)-aunp networks, resulting in aunps aggregation and dramatic color changes. the target dna acts as a crosslinker between two ssdna-functionalized aunps. on the other hand, colorimetric dna sensing methods using functionalized aunps have been demonstrated using noncross-linking dna hybridization with the benefit of the powerful salting-out effect of dsdna compared to ssdna. 12 since the ssdna density on the surface of aunps and the ionic strength of the solution affect the stability of functionalized aunps, control of the aggregated state by modification is quite difficult in practical applications. rothberg et al. suggested a label-free detection method based on the fact that ssdna in the solution could bind to citrate-capped aunps and electrostatically stabilize them at high ionic strength. in such a system, aunps are stabilized in the presence of ssdna, but aggregated with dsdna in a highly concentrated electrolyte solution. this strategy does not need the direct binding of ssdna on the surface of nanosized metallic particles, as the dna amplification step prior to detection is inevitable due to the low sensitivity of this method. 13 since both ssdna and dsdna allow aunps to stabilize at low salt concentration, 14−17 cationic agents are mainly used to identify the target with probes. 18, 19 in this regard, farhad rezaee et al. demonstrated a modified non-cross-linking aunp aggregation using disulfide self-assembly of terminal modified dna. long and flexible sulfur-rich, self-assembled products were well combined on the surface of aunps and effectively inhibit the particles from salt-induced aggregation. 15 inspired by this research, we developed a colorimetric assay that relies on bare aunps that employs a disulfide-induced self-assembly. the bare aunp-based colorimetric method consists of two thiol modified probes at the 3′ or 5′ ends for targeting partial genomic regions (30 bp) of mers-cov along with upstream e protein gene (upe) and encoding open reading frames (orf) 1a. this assay could be highly reliable for mers-cov diagnosis as we have followed who updated recommendations for infectious disease laboratory testing, which targets the two regions on mers-cov considered for potential preclinical screening and high sensitivity 20 the developed assay platform was able to detect the target dna through optical properties of the gold nanoparticles such as color changes with the naked eye and spectral shifts on uv− vis wavelength. the specific thiolated probes form the complementary dsdna with the target and make a disulfideinduced long self-assembled complex due to continuous disulfide bond formation. the extended self-assembled complex can protect bare aunps for stability against saltinduced aggregation since sulfur-group at ends of dsdna are mediated covalent bond with gold surface. the interaction is known to generate stable conjugation within disulfide-induced self-assembled complex and aunps and intermolecular force between au−s attains about 40 to 50 kcal mol −1 . 21, 22 otherwise, probes build a disulfide induced interconnection with each other in the absence of targeting dna. the probe dna terminal coupling is unable to cover the surface of aunps exposed to aggregation, leading to significant color changes as well as a broader red-shift of the lspr peak ( figure 1 ). with this method, we can visually observe the results with the naked eye, not using costly equipment. this colorimetric assay is an important step to use of infectious disease poct across the developing world. moreover, this concept of a disulfide bond based colorimetric assay could be applied for diagnosis of other infectious diseases. preparation of the ideal gold nanoparticle upon a spectral centroid. we developed various sized gold nanoparticles for optimization as a colorimetric indicator. the citrate reduction method, i.e., turkevich-frens, is generally applied to synthesize stabilized gold nanoparticles under electrostatic repulsion. 23 additionally, the ratio of the reducing agent and haucl 4 allow for the preparation of a diverse size of aunps. we changed the volume of trisodium citrate acid from 300 to 1600 μl to acquire different-sized aunps. the morphology of the prepared aunps and dls records are reported in figure 2a . according to the tem images and dls measurements, the average diameter of aunps decreased as the amount of trisodium citrate dehydrate increased. for instance, the addition of reducing agents (300, 500, 1200, 1600 μl) allow us to synthesize 72-, 41-, 26-, and 19-nm-sized gold nanospheres, respectively. this was the result of changed citrate and gold ratio to control the reduction and stabilization of the nanoparticle surface. 24, 25 we optimized an average of 19 nm aunp through reduction with 1600 μl of trisodium citrate and called that formed particle "bare gold", which means "unmodified and inartificial conjugation with any molecules after synthesized process" and "citrate ion capped gold nanoparticle derived from reduction and stabilization effects of sodium citrate" for this experiment. the structural and spectroscopic properties are presented as (iv) in figure 2 . to evaluate the effects of salt on as-prepared aunps, the absorbance spectra of the bare aunps and those with mgcl 2 were measured using a conventional uv−visible spectrometer (figure 2b,c) . since the lspr spectral band of nanosized metallic particle is derived from dielectric factors, particle size and shape, the absorption peak shift indicated a changed environment surrounding aunps such as by conjugation of biomolecules. thus, the addition of mgcl 2 induces a significant change in the absorption spectra. for example, the lspr band has a decrease in intensity and increase in bandwidth, as well as new bands at longer wavelength. these lspr band shifts represent the aggregation of aunps caused by the loss of interparticle repulsive force as salt disrupts the charge interaction surround aunps and promotes the interparticle van der waals attractive forces. 26 as shown in the salt induced results, the solutions of gold nanoparticles, excluding 19 nm aunps, became gradually transparent due to severe aggregation of metal particles and also a broader, flattened spectra appeared in the uv−vis measurement as figure 2c . therefore, we selected the 19-nm-diameter aunps that detected the lspr changes on the surface of the nanosized metallic particles in this experiment. furthermore, a spectral centroid was derived by calculating the centroid wavelength in each experiment spectrum to determine a credible and accurate point of the lspr band. the spectral centroid represents several parameters of the lspr band including the peak wavelength, bandwidth, and intensity that depend on the size, shape, concentration, and interparticle interactions of the aunps. 15, 27 the shift of the centroid can be a comprehensive indicator for the overall transition of the spectral distribution toward shorter or longer wavelengths. for example, 19-nm-diameter aunps possess a spectral centroid at the orange-red wavelength 516 nm, whereas larger 72-nmdiameter aunps exhibit a purple color with a spectral centroid at 576 nm. optimization of disulfide-induced long self-assembly and the salting agent. in this report, bare 19-nmdiameter aunps were used, and we selected a target and control upstream of the e protein gene (upe) and open reading frames (orf) 1a on mers-cov, and the tobacco mosaic virus (tmv), respectively. furthermore, we synthesized forward and reverse thiol modified probes specifically binding the target dna and forming complementary base pairs. each of the thiolated probes were modified at the 5′ site (right: 5′r) and 3′ site (left: 3′l). once two probes simultaneously recognize a specific half of the target, the disulfide bonds at the 3′ and 5′ terminals readily form a sulfurrich self-assembled complex. specifically, target dna samples were mixed with thiolated probes (5′ r, 3′ l) in distilled water (d.w.) and then incubated at 90°c for a few minutes to establish disulfide-induced long self-assembled products. 90 μl of the aunps solution was then add to the above-mentioned mixture (60 μl). the solution was incubated at room temperature for 30 min and various amounts of mgcl 2 solutions were added. each test was evaluated in at least triplicate. we analyzed the color of the resulting solutions with the naked eye and uv−vis spectrometer. disulfide self-assembled products were visualized along with the gel electrophoresis, where the presence of the target (positive reaction) displayed bands with a characteristic ladder due to the formation of long assemblies of dsdna (figure 3a) . polymorphous bands appeared with the formation of disulfide self-assembly between the target and probes on original full-length native polyacrylamide gel electrophoresis (page) analysis image ( figure s1 ). additionally, we confirm the sizes of dna fragments with 4% agarose gel which is commonly used to separate and visualize the amplified dna outcomes depended on number of base pairs ( figure s2 ). furthermore, we confirmed the adsorbed components on the surface of gold nanoparticle via x-ray photoelectron spectroscopy (xps) (figure 3b ). in the case where thiolated probe met the incompatible dna, the s 2p signals mainly indicated the absorbed thiol component at s 2p3/2 photoelectron binding energy (be) of 162.2 ev since a sulfur monolayer was formed on the surface of aunps from the probe solution (s̅ , sh̅ , or sh 2 species) (figure 3b(i) ). 28, 29 meanwhile, xps spectrum can be fitted at 163.37 and 164.55 ev of s 2p3/2 binding energy when target dna and probes compose the disulfide-induced self-assembled multilayers on aunps (figure 3b(ii) ). the observed components at 163−164 ev of be are assigned to some s in multilayer which highly defect to the gold surface or domain boundaries. 29, 30 although be of 163.7 appeared in figure 3b (i), the area in the xps spectrum was lower than thiol species (1:0.64), and in page, the size of dna fragments only appeared close to 20 bp ladder that indicates thiol containing short layers formed, not the disulfide based multilayer, and unbound thiol assigned that be of s 2p 3/2 when target dna is absent. 31 also, the increased area at higher binding energy indicates higher coverage with absorbed polysulfides. 22 based on results mentioned earlier from page and xps analyses, disulfide-induced self-assembled complex composed the multilayers on aunps, but thiolated ssdna probes form monolayer which is hard to cover the surface of gold nanoparticle from salt-induced aggregation. dls measurements also supported the size increasing of dna covered aunps which depended on extended layers ( figure s3 ). dynamic light scattering (dls) was used to monitor the distribution of the hydrodynamic size of the gold nanoparticles after immobilized ssdna or dsdna complex layer on the gold surface, respectively. as shown in figure s3 , the average hydrodynamic size of the gold nanoparticle protected by disulfide-induced self-assembly from the positive reaction was increased by approximately 8.11 nm compared to immobilized ssdna on gold nanoparticle from the negative reaction. the stability of the assay was investigated through the addition of various concentrations of mgcl 2 into the bare aunps solution in the (i) absence or (ii) existence of the target (figure 3c ). mixed products with target dna and probe solutions shielded the particles, which tolerated the salting more than gold nanoparticles conjugated with thiol from disulfide-interconnected probes, since the disulfide-induced self-assembled complex was used to protect bare aunps against salt addition. the spectral peak of the aunps colloid was not quite different with bare aunps when the target dna is in the solution with 0.05 to 0.5 m mgcl 2 , while aunps linked to disulfide-interconnected probes aggregated with any concentration of salt. however, the absorbance declined slightly, corresponding to an increased salt concentration whether extended disulfide assemblies were formed or not. therefore, the spectral centroid shifts depending on salt concentration were plotted and are shown in figure 3d . at all concentrations (0.05 to 0.5 m), the delta centroid of the negative control showed more shift than positive controls due to intergold nanoparticle aggregation, while the centroid shift of the positive control was the smallest at 0.1 m of mgcl 2. as previously mentioned, the absorbance declined conversely according to the increase in salt concentration. hence, 0.1 m of mgcl 2 was chosen as the optimized concentration. verification of the colorimetric assay. to consider the disulfide self-assembled reaction dependence on target dnas and their concentrations, we measured the absorption change of the aunp solution before and after adding 0.1 m mgcl 2 in the presence or absence of each target using a uv−vis spectrometer. when the target was absent, the wavelength was shifted more toward the red side of the spectrum compared to when the target was present. the wavelength peak shifts were 11, 19, and 51 nm after complementary binding reactions between probes and (i) orf1a, (ii) upe, and (iii) tmv, respectively ((i) 522 to 533 nm, (ii) 520 to 541 nm, and (iii) 521 to 572 nm) (figure 4a,b) . due to the broad spectrum of the negative control, delta centroid was estimated as 2.5 times greater than a positive reaction (avg. 29 nm vs avg 12 nm). these changes indicate that positive samples (orf1a and upe) that are a mixture of thiol-modified probes and their target dna that hindered the aggregation of aunps through composition of the long self-assembled structures. negative samples (tmv) were unable to form long self-assembled structures due to mismatched targets. furthermore, we calculated a p-value to prove the reproducibility of this colorimetric platform (figure 4b ). the p-value is the probability that might yield the same results observed in the biological study, if the null hypothesis is true. all data groups were found to pass the null-hypothesis assuming a normal distribution was followed. 32 therefore, the p-value of the delta centroid implies that this assay is considered statistically significant (p-value <0.01). the limit-of-detection (lod) was determined in order to apply to the medical laboratory field. the lod of this colorimetric platform was 1 pmol/μl, which was calculated by the equation mean blank + 3σ blank defined from the iupac standard ( figure 4c ). 33 1 pmol/μl is about 6 × 10 11 copies/ μl, which requires only 26 pcr cycles to diagnose mers patients who release 1.5 × 10 3 to 6.7 × 10 3 copies/μl in the sputum for 10 days and have recovered without specific treatment for mers-cov. this assay can reduce the number of pcr cycles for diagnosis of mers even with a small amount of viral molecules, compared to conventional pcr reactions that require more than about 34 cycles to detect mers-cov. 34−37 ■ conclusions a simple and fast colorimetric assay for detecting infectious disease that can be seen by the naked eye without costly equipment was developed. we proposed a colorimetric assay using disulfide bonds formed by hybridizing with thiolated probes and a target; this method inhibited the aggregation of aunps by salt and limits the color change for diagnosis of mers. this assay can confirm the presence of mers-cov within 10 min without electrophoresis or other procedures. the assay could discriminate mers-cov with a potential detection limit of 1 pmol/μl, which means it could distinguish a lower amount of target with a lower level of amplification, or even without amplification. a follow-up study will be conducted to apply this method to clinical samples with longer targeting regions. we anticipate that this method will provide rapid and accurate diagnostic results in epidemic areas, especially in resource-limited settings and will evolve by state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans emerging infectious diseases: threats to human health and global stability nanobioimaging and sensing of infectious diseases an operationally simple colorimetric assay of hyaluronidase activity using cationic gold nanoparticles electrostatic assembly of nanoparticles and biomacromolecules guided assembly of nanoparticles on electrostatically charged nanocrystalline diamond thin films localized surface plasmon resonance spectroscopy and sensing molecular linker-mediated self-assembly of gold nanoparticles: understanding and controlling the dynamics selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles gold nanoparticles for naked-eye dna detection: smart designs for sensitive assays label-free colorimetric detection of specific sequences in genomic dna amplified by the polymerase chain reaction disulfide-induced self-assembled targets: a novel strategy for the label free colorimetric detection of dnas/rnas via unmodified gold nanoparticles colorimetric detection of dna sequences based on electrostatic interactions with unmodified gold nanoparticles unmodified gold nanoparticles as a colorimetric probe for potassium dna aptamers laboratory testing for middle east respiratory syndrome coronavirus interim guidance thermal stability of dna functionalized gold nanoparticles the chemistry of the sulfur-gold interface: in search of a unified model controlled nucleation for the regulation of the particle size in monodisperse gold suspensions size controlled synthesis of gold nanoparticles using photochemically prepared seed particles turkevich method for gold nanoparticle synthesis revisited design of gold nanoparticle-based colorimetric biosensing assays spontaneously formed sulfur adlayers on gold in electrolyte solutions: adsorbed sulfur or gold sulfide? sulfur-substrate interactions in spontaneously formed sulfur adlayers on au(111) sulfur multilayer formation on au(111): new insights from the study of hexamethyldisilathiane x-ray photoelectron spectroscopy sulfur 2p study of organic thiol and disulfide binding interactions with gold surfaces value of p-value in biomedical research limits of detection in spectroscopy comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity kinetic studies and infection control of respiratory viruses in infectious mers-cov isolated from a mildly ill patient middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands key: cord-259347-3acsko74 authors: cheng, qi; yang, yue; gao, jianqun title: infectivity of human coronavirus in the brain date: 2020-05-28 journal: ebiomedicine doi: 10.1016/j.ebiom.2020.102799 sha: doc_id: 259347 cord_uid: 3acsko74 a new strain of human coronaviruses (hcovs), severe acute respiratory syndrome coronavirus-2 (sars-cov-2), has been identified to be responsible for the current outbreak of the coronavirus disease 2019 (covid-19). though major symptoms are primarily generated from the respiratory system, neurological symptoms are being reported in some of the confirmed cases, raising concerns of its potential for intracranial invasion and neurological manifestations, both in the acute phase and in the long-term. at present, it remains unclear the extent to which sars-cov-2 is present in the brain, and if so, its pathogenic role in the central nervous system (cns). evidence for neuroinvasion and neurovirulence of hcovs has been recognised in animal and human studies. given that sars-cov-2 belongs to the same family and shares characteristics in terms of receptor binding properties, it is worthwhile exploring its potential cns manifestations. this review summarises previous findings from hcovs in relation to the cns, and compares these with the new strain, aiming to provide a better understanding of the effects of sars-cov-2 on the cns. human coronaviruses (hcovs) are a group of enveloped rna viruses that are composed of a single stranded positive sense rna genome (~30 kb). the entire genome of hcovs encodes spike (s) glycoprotein, membrane (m) glycoprotein, envelope (e) glycoprotein, nucleocapsid (n) protein, rna polymerase and genes for numerous accessory proteins that modulate pathogenesis, among which the n protein is highly conserved in most coronaviruses and abundantly expressed during infection [1] , thus acting as a critical antigen for viral detection. the s protein is biologically critical for tropism and modulation [2] , and is considered to be integral in the capability of hcovs to reach the brain [3, 4] . the hcovs include seven strains, three of which, namely sars-cov-2, sars-cov, and middle east respiratory syndrome-related coronavirus (mers-cov), may cause severe respiratory disease with a relatively high morbidity and mortality. in contrast, the other four strains oc43, 229e, nl63 and hku1, are often associated with symptoms akin to the 'common cold' and hence, are relatively harmless. based on genome analysis, the oc43 strain shares a 531% and a 512% identity with sars-cov and hcov-229e, respectively [5] . the new strain, sars-cov-2 (previously called 2019-cov), shares a 79% and 50% identity with sars-cov and mers-cov, respectively [6] . mainly considered as respiratory viruses, a common manifestation from hcovs related acute respiratory disease is hypoxia, which predisposes the brain to edema, disturbed metabolism, and subsequent neurological manifestation. however, hypoxia is not the only way hcovs were related to central nervous system. in fact, hcovs have also been shown to be capable of invading neural cells, and hence manifesting with neurological symptoms and signs ( table 1 ). the potential for significant neurological deficits recently became a concern following the report of a covid-19 patient who demonstrated loss of involuntary control of breathing due to presumed involvement of the inspiratory area in the brainstem, as well as reports of sars-cov-2 infected patients developing ataxia, loss of smell and convulsions [7] . in vitro studies have shown that 229e and oc43 hcov strains can infect a wide range of human neural cell cultures, including neuroblastoma, neuroglioma, astrocytoma, microglial and oligodendrocytic cell lines [8à10]. in addition, animal studies have also revealed the neuroinvasion and neurovirulence of hcov-oc43 [4,5,11à13] . importantly, a significantly higher prevalence of the oc43 strain in terms of viral rna detection has been shown in human brain autopsy samples from multiple sclerosis (ms) patients when compared to other neurological diseases and normal controls, which is consistent with the left lower lobe infiltrates (11) →obvious resolution (28) →progressive bibasilar infiltrations (35) ct: broad encephalic pathological changes of probably ischemia and necrosis and brain edema (33) none eye-ground exam: an exudation around the visual yellow zone (26) death ( recovery [15] all cases had fever and/or flu-like symptoms at the onset. half of the patients (5/10) had headache, dizziness or vomiting as an early sign of neurological manifestation. 2/3 sars patients had seizures, whereas 3/5 mers patients and both oc43-infected cases had facial/limb paralysis. unfortunately, mers-cov has not been detected in csf samples of any of the reported cases. ischaemic changes have been detected in images of half of the patients, while haemorrhagic pathology has been identified on one mers patient. the oc43-related cns-infected patients were younger compared to sars and mers patients. gcs: glasgow coma scale; ncv: nerve conduct velocity; emg: electroneuromyography. capability for neuroinvasion of this hcov [3] . furthermore, oc43 has also been identified in a teenager patient with demyelinating disease, in whom the virus was detected in both csf and nasopharyngeal secretions by pcr technology [14] . the hcov-oc43 has also been associated with a fatal encephalitis in an infant although the underlying circumstances are still unclear [15] . additionally, co-infection with the 229e and oc43 strains has been reported in a young girl who developed an acute flaccid paralysis [16] . sars-cov and mers-cov have also been linked with neurological manifestations. sars-cov has been shown to be capable of infecting human neural cells [17] , and neuroinvasion and neurovirulence have been found in studies involving both sars-cov [18à23] and mers-cov [24, 25] . an association of these two more highly pathogenic viruses with neurological manifestations have also been reported. for instance, sars-cov particles and genomic sequences have been detected from post-mortem brain tissues of sars patients [26à28]. they have also been detected using rt-pcr in csf samples from a 32-year-old pregnant female patient who presented with a brief duration generalized convulsion and accompanying loss of consciousness [29] and within 24 h of a first seizure in a 59-year-old female patient [30] . although there is less of direct evidence of viral presence in the cns, mers patients have also presented with neurological findings, such as altered consciousness, as well as manifested with a wide range of abnormalities on brain mri [31, 32] . regarding the regional distribution of the virus in the cns, data from the post-mortem studies have shown that infection from sars-cov was confined to neurons within selected areas of the brain, including thalamus, cerebrum, brainstem, hypothalamus and cortex [22, 27] . intriguingly, sars-cov has been detected in cerebrum, but not in cerebellum, in both animal [22] and human [28] studies. in animals infected in the cns with mers-cov, the thalamus and brain stem were found to be the highest infected sites [25] . data from multiple hace2 transgenic mouse models has revealed that sars-cov detection in the brain is significantly delayed compared to that within the lung, consistent with the initial establishment of infection within the respiratory system before dissemination to the cns [21à23]. several dissemination routes have been proposed for coronaviruses to gain access to the cns (fig. 1) . the mouse hepatitis virus (mhv), a neurotropic coronavirus, has been shown to enter the cns via a transneuronal route and disseminate within the brain via neuroanatomic pathways [33] . in mice transgenic for hace2, sars-cov seems to enter the brain via the olfactory bulb post intranasal inoculation before disseminating transneuronally to distal connected neurons and causing the dysfunction/ death of these infected neurons [18] . a similar dissemination pattern has also been detected in oc43-inoculated mice [5, 13] . however, different observations were found in another hace2 transgenic mouse model, in which no sars-cov was detected in the olfactory bulb, or preferentially in sites transneuronally connected to the olfactory bulb [22] . in addition, a recent study has revealed that ace2 (angiotensinconverting enzyme 2) and tmprss2 (transmembrane protease, serine 2), two key genes involved in sars-cov-2 entry, are not expressed in olfactory sensory or bulb neurons, but rather in olfactory epithelial cells [63] , suggesting the involvement of other dissemination mechanisms independent of axonal transport. it has also been proposed that hcovs can gain access to the cns via the certain neurotransmitter pathways such as serotoninergic dorsal raphe system [18] , which primarily projects to cerebral cortex, neostriatum, amygdala, substantia nigra, pons, hippocampus, entorhinal cortex and locus coeruleus. however, this hypothesis is speculative and requires to be further tested in vivo models. viremia has been observed following intranasal inoculation with mhv [34] and sars-cov [21] in transgenic mice. in addition, viral rna has been measured in the blood of patients infected with sars-cov and mers-cov [35] and thus, the dissemination of these two viruses to the cns may be mediated by the hematogenous route through a damaged blood-brain barrier (bbb). once crossing the bbb, hcovs-infected cells could potentially invade the brain through perivascular spaces, also known as virchow-robin spaces. these fluid-filled spaces are implicated as sites in which macrophages and lymphocytes interact to initiate immune response in patients with viral encephalitis [36] . for instance, the cryptococcal organisms in aids patients have been shown to disseminate through the virchow-robin spaces before further propagation into other brain regions [37] . whether hcovs could disseminate via the same route remains unclear. the intranasal inoculation of rodents with mhv has also revealed a lymphatic dissemination of coronavirus via cervical and mesenteric lymph nodes in addition to viremia [34] . additional studies are required to determine the role of this potential dissemination route. the infection of hcovs may also result in long-term neurological deficits. the pathologic examination of brain tissue collected from a sars patient revealed necrosis of neurons and broad hyperplasia of gliocytes, indicative of the presence of a chronic progressive encephalitis during the course of viral infection [26] . in addition, scattered red degeneration of neurons, a sign of neuronal hypoxia or ischaemia [27] , as well as oedema around small veins with demyelination of nerve fibres [38] , have been detected in brains of sars patients, which may provide an explanation for an incidence of long-term neurological [39] and/or psychological [40] abnormalities observed in sars survivors. in surviving mice post hcov-oc43 infection, spongiform-like degeneration was a residual finding post acute infection, indicating the possibility of neurodegenerative neuropathological sequelae [4] . further, a decline of locomotor activity and an abnormality in the limb clasping reflex were observed starting several months after viral infection, and neuronal loss especially in the hippocampal region was still detectable one year post infection [41] . although the majority of infectious virus (80%) was cleared in surviving animals, viral rna could persist for months [41] . even for cells that have survived the initial viral infection, little is known about their long-term functional status. to better understand this, cov-infected models that allow the identification and isolation of survival cells, such as the cre reporter mouse model of mhv infection, may be helpful [42] . recovery from acute infection does not necessarily promise complete clearance of the virus, and if an infection were to becomes chronic, it may result in long-term sequelae including chronic neurological diseases. however, given the limited clinical data, the long-term effects of hcovs on the cns post acute infection are unknown and thus, more studies involving the long-term follow-up of survivors are needed. ace2 was reported as a functional cellular receptor for sars-cov due to the ability of the s1 domain of the spike (s) protein to efficiently bind to ace2 for subsequent viral replication in the cytoplasm of sensitive cells [43] . in addition, previous autopsy studies have inferred that only ace2-postive cells were susceptible to sars-cov infection because the s protein and its rna could not be detected in ace2-negative cells [44] . it has been reported that sars-cov-2 might also use ace2 as a cellular receptor, despite a variation at some key residues of the receptor-binding domain compared to that of sars-cov [6] . the lack of ace2 expression in neuronal cells at both transcription and protein level contrasts with the viral susceptibility of these cells and cytopathogenicity during the acute phase of infection in neural cell lines of human and rat origins [17] as well as in humans brains [45] . even in hace2 transgenic mice, when specific brain regions were heavily infected by sars-cov, the expression levels of hace2 in the brain were no more than 01 to 1% of those found in the lungs [18] . furthermore, even in cells highly expressing hace2 in the transgenic animal model, sars-cov infection could be absent, indicating that the expression of hace2 alone might not be sufficient for maintaining viral infection [46] . these findings suggest that other receptor (s) or co-receptor(s) are involved in the viral entry into neural cells. dpp-4 (also known as cd26) was identified as a cellular receptor for mers-cov [47] . two lines of human dpp-4 transgenic mice were found to have high affinity to infection by mers-cov [24, 25] . dpp-4 is expressed in human brains [48] , therefore, when the virus gains access to the brain, cells expressing dpp-4 are potentially available for viral replication within the cns. aminopeptidase n (apn, also known as cd13), a cell-surface metalloprotease, was proposed as a receptor for hcov 229e [49] , as well as sars-cov [50] . human aminopeptidase n was shown, in vitro, to be a cellular receptor for hcov 229e infection of human neuronal, astrocytic and oligodendrocytic cell lines [51] . another glycoprotein, namely cd209l (also called l-sign) has also been identified as an alternative cellular receptor for sars-cov [52] . in addition, the infection of sars-cov on ace2-expressing cells seemed to be dependent on a proteolytic enzyme cathepsin l (ctsl1) in a ph sensitive manner [53, 54] . the expression levels of these receptors in human brain, however, require further elucidation to determine the extent to which they may be responsible for the hcovs infection in the cns. in addition to known and putative receptors, antibody-dependent enhancement (ade) of viral entry using the fusogenic spike protein has also been reported as a pathway to transfer coronavirus (mhv4) from infected cells to non-infected cells [55] . in vitro studies have shown that coronavirus entry could be mediated by special antibodies that bind the virus surface spike protein and facilitate subsequence viral entry of mers-cov [56] and sars-cov [57] in a receptor-like manner. the ade pathway is particularly important for vaccine design and the development of antibody-based drug therapies. 6. is sars-cov-2 likely to be present in the central nervous system? at present, it remains unknown whether sars-cov-2 is present in the cns, possibly due to limited access to brain tissue and csf from patients infected with the virus. autopsies are being increasingly carried out, however, initial biopsies have been taken only from lung, liver and heart for histopathology [58] . given that several strains of hcovs have been shown to be present in the brain, as well as csf [14à16,26à30], with neuroinvasion and neurovirulence shown in cell culture and animal models [4,5,11à13,17à25] , it is reasonable to consider that the sars-cov-2 may also be present in the cns. this is further supported by the fact that some covid-19 patients present with headache, nausea and vomiting, symptoms that potentially indicates neurological involvement [59] . the frequency of neurological manifestations including changes in consciousness and acute cerebrovascular disease increase in parallel with the severity of the disease [60] . one study on nasal epithelium samples taken from a subset of covid-19 patients exhibiting olfactory dysfunction has suggested that the disturbance of smell in these patients is more possibly due to sars-cov-2 infection of non-neuronal cells rather than olfactory neurons [63] . more recently, the report of a cohort of 58 covid-19 patients showed a correlation between acute respiratory distress syndrome (ards) and encephalopathy, mainly agitation, confusion and corticospinal tract signs [61] . however, these findings need to be confirmed by studies on brain samples or csf. the most efficient way to identify involvement of neurological systems remains to be determined. the presence of sars-cov-2 in csf was confirmed by gene sequencing in a 56-year-old patient with covid-19, which was the first direct evidence for the neuroinvasion of this novel coronavirus [62] . brain samples remain the gold standard of confirmation, however, access to such samples will almost certainly remain very limited and it is not a practical or ethical consideration in larger cohorts, especially in mild to moderate cases. several pathways of invasion for hcovs have been proposed, including via the olfactory nerve, neurotransmitter pathways, hematogenous route, virchow-robin space surrounding arterioles and venules, lymphatic systems and receptors, from which alternative parameters and types of samples may offer opportunity for proof of cns involvement. more studies are needed to evaluate and compare these alternative pathways and parameters. in addition, the timing of sampling needs further investigation, as the change of viral load in the human body at different stages of covid-19-related infection and recovery remains unclear. though considered mainly as respiratory viruses, several strains of hcovs have been detected in brains from patients with encephalitis and multiple sclerosis. in addition, the detection of sars-cov in csf of patients with neurological manifestation has also provided direct evidence for the neuroinvasion and neurovirulence of hcovs. however, the role of the virus in the process of the disease in acute phase as well as in the long term still remains elusive. the current strain sars-cov-2 can also potentially infect neuronal cells in the brain. therefore, a better understanding of dissemination characteristics and neuropathogenesis of these hcovs in the cns is urgently needed. catching a viral infection with ensuing cytokine release and tissue lesions may lead to a variety of cns-related manifestations (delirium, headache, vomiting, etc.), which are recognizable for many viruses including hcovs, however, these non-specific clinical symptoms and signs may be constitutional and represent systemic involvement rather than direct cns infection. therefore, we should be cautious when reporting cases with neurological symptoms and/or signs that lack solid evidence for cns infection (e.g. absence of viral detection in csf samples or abnormalities supportive of infection on brain imaging). it is particularly challenging when evaluate the neurological deficits during the advanced stage of covid-19-related disease. the clinical diagnosis and treatment strategy has to be made on the basis of a differential diagnosis which includes hypoxia, respiratory, metabolic acidosis, ards-related encephalopathy, the effect or withdrawal of medications, and viral infection of the cns. pubmed search of articles: "human coronavirus", "sars", "mers", "oc43", "229e", "neurological symptom", "neurological sign", "neurological manifestation", "neuroinvasion", and "neurovirulence". additional articles were selected based on articles in these searches and as suggested by reviewers. we declare no conflicts of interest. identification of an epitope of sars-coronavirus nucleocapsid protein engineering the largest rna virus genome as an infectious bacterial artificial chromosome neuroinvasion by human respiratory coronaviruses vacuolating encephalitis in mice infected by human coronavirus oc43 human respiratory coronavirus oc43: genetic stability and neuroinvasion genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding neurological manifestations in covid-19 caused by sars-cov-2 persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229e acute and persistent infection of human neural cell lines by human coronavirus oc43 infection of primary cultures of human neural cells by human coronaviruses 229e and oc43 murine encephalitis caused by hcov-oc43, a human coronavirus with broad species specificity, is partly immunemediated susceptibility of murine cns to oc43 infection axonal transport enables neuron-to-neuron propagation of human coronavirus oc43 detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis human coronavirus oc43 associated with fatal encephalitis a rare cause of acute flaccid paralysis: human coronaviruses susceptibility of human and rat neural cell lines to infection by sars-coronavirus severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme 2 generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis multiple organ infection and the pathogenesis of sars organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways possible central nervous system infection by sars coronavirus detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) neurological complications of middle east respiratory syndrome coronavirus: a report of two cases and review of the literature effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain viremic dissemination of mouse hepatitis virus-jhm following intranasal inoculation of mice middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease immunological and neuropathological significance of the virchow-robin space dilated virchow-robin spaces in cryptococcal meningitis associated with aids: ct and mr findings the clinical pathology of severe acute respiratory syndrome (sars): a report from china persistence of physical symptoms in and abnormal laboratory findings for survivors of severe acute respiratory syndrome posttraumatic stress, anxiety, and depression in survivors of severe acute respiratory syndrome (sars) human coronavirus oc43 infection induces chronic encephalitis leading to disabilities in balb/c mice murine olfactory bulb interneurons survive infection with a neurotropic coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv human aminopeptidase n is a receptor for human coronavirus 229e aminopeptidase n inhibitors and sars involvement of aminopeptidase n (cd13) in infection of human neural cells by human coronavirus 229e cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells dissemination of mhv4 (strain jhm) infection does not require specific coronavirus receptors molecular mechanism for antibody-dependent enhancement of coronavirus entry antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro pathological findings of covid-19 associated with acute respiratory distress syndrome development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients neurologic features in severe sars-cov-2 infection sars-cov-2: underestimated damage to nervous system non-neuronal expression of sars-cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-associated anosmia we thank a/prof dennis cordato, senior specialist/neurologist, liverpool hospital, sydney, australia, for critical review and grammar check of the manuscript. we received no funding for this review. key: cord-281364-syg0wo77 authors: caì, yíngyún; yú, shuǐqìng; postnikova, elena n.; mazur, steven; bernbaum, john g.; burk, robin; zhāng, téngfēi; radoshitzky, sheli r.; müller, marcel a.; jordan, ingo; bollinger, laura; hensley, lisa e.; jahrling, peter b.; kuhn, jens h. title: cd26/dpp4 cell-surface expression in bat cells correlates with bat cell susceptibility to middle east respiratory syndrome coronavirus (mers-cov) infection and evolution of persistent infection date: 2014-11-19 journal: plos one doi: 10.1371/journal.pone.0112060 sha: doc_id: 281364 cord_uid: syg0wo77 middle east respiratory syndrome coronavirus (mers-cov) is a recently isolated betacoronavirus identified as the etiologic agent of a frequently fatal disease in western asia, middle east respiratory syndrome. attempts to identify the natural reservoirs of mers-cov have focused in part on dromedaries. bats are also suspected to be reservoirs based on frequent detection of other betacoronaviruses in these mammals. for this study, ten distinct cell lines derived from bats of divergent species were exposed to mers-cov. plaque assays, immunofluorescence assays, and transmission electron microscopy confirmed that six bat cell lines can be productively infected. we found that the susceptibility or resistance of these bat cell lines directly correlates with the presence or absence of cell surface-expressed cd26/dpp4, the functional human receptor for mers-cov. human anti-cd26/dpp4 antibodies inhibited infection of susceptible bat cells in a dose-dependent manner. overexpression of human cd26/dpp4 receptor conferred mers-cov susceptibility to resistant bat cell lines. finally, sequential passage of mers-cov in permissive bat cells established persistent infection with concomitant downregulation of cd26/dpp4 surface expression. together, these results imply that bats indeed could be among the mers-cov host spectrum, and that cellular restriction of mers-cov is determined by cd26/dpp4 expression rather than by downstream restriction factors. in 2012, a novel human coronavirus causing frequently fatal disease emerged in western asia [1] and was named ''middle east respiratory syndrome coronavirus (mers-cov)'' [2] . as of june 11, 2014, mers-cov caused 699 laboratory-confirmed human infections in 21 countries, including 209 deaths (proportion of fatal cases <29.9%) [3] . increasing evidence points to dromedaries (camelus dromedarius) as an intermediate reservoir contributing to the emergence of middle east respiratory syndrome (mers) in humans. several seroepidemiology studies have found mers-cov-neutralizing antibodies in dromedaries from egypt, jordan, oman, and saudi arabia [4] [5] [6] [7] [8] [9] . more recently, coronaviral genomes detected in nasal swabs obtained from dromedaries proved to be identical to genomes of human mers-cov isolates [4, 5, 9] . one such genome was detected in a patient who had been caring for a sick dromedary and directly from that animal [10] . in addition, mers-cov was directly isolated from a dromedary in qatar [11] . the source of dromedary mers-cov infection remains to be elucidated, but it is not unlikely that they serve only as intermediary hosts [12] . bats have been proposed as additional mers-cov hosts. this hypothesis is based on the fact that several betacoronaviruses related to mers-cov (e.g., severe acute respiratory syndrome-like coronaviruses, tylonycteris bat coronavirus hku4, pipistrellus bat coronavirus hku5) are known to infect bats in africa, europe, and asia [1, [13] [14] [15] [16] . in addition, mers-cov genome fragments encoding parts of the rnadependent rna polymerase were detected in one egyptian tomb bat (taphozous perforates) living close to a mers-cov-infected patient [14] . finally, a novel coronavirus (neocov) closely related to mers-cov was discovered in cape serotines (neoromicia capensis) in south africa [12] . therefore, bats could possibly maintain mers-cov in nature and may occasionally infect dromedaries and thereby may infect humans similar to the batshorse-human or bats-pig-human transmission cycle observed for henipaviruses [17, 18] . recently, cd26, also known as dipeptidyl peptidase 4 (dpp4) was identified as the human mers-cov cell entry receptor [19] and also as a receptor for tylonycteris bat coronavirus hku4 [20, 21] . cd26/dpp4 receptor is conserved among different mammals (e.g., bats, dromedaries, humans), and the possibly broad species tropism of mers-cov may partly be the result of this conservation [19, 22] . to further evaluate the hypothesis that bats may be implicated in transmission of the virus, we inoculated ten cell lines from phylogenetically diverse bats living in geographically distinct areas with mers-cov. six bat cell lines were productively infected. the susceptibility or resistance of the ten cell lines to mers-cov infection directly correlated with the absence or presence of naturally expressed cd26/dpp4 on the cells surface. anti-human cd26/dpp4 antibodies reduced mers-cov yield in susceptible bat cell cultures in a dose-dependent manner. similar to other studies using mers-cov-resistant (non-bat) cell lines transfected with cd26/dpp4 [19] , expression of human cd26/dpp4 in resistant bat cells rendered these cell lines susceptible to infection. finally, we demonstrate that persistent mers-cov infections can be established in permissive bat cell lines after sequential virus passage, leading to downregulation of natural cd26/dpp4 cellsurface expression. together, our data indicate that the host cell tropism of mers-cov may largely depend on the expression of suitable cd26/dpp4 orthologs, and that bats cannot be excluded as mers-cov reservoirs at this point in time. huh-7 (a kind gift from hideki ebihara, rocky mountain laboratory, hamilton, mt), vero e6 (atcc, #crl-1568, manassas, va), and vero (atcc, #ccl-81) cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum (fbs, sigma-aldrich, st. louis, mo). the bat cell lines used in this study are described in table 1 . r05t, r06e, and hypni/1.1 cell lines were grown in dmem/f-12 (lonza, walkersville, md) supplemented with 10% fbs. all others bat cell lines were maintained in dmem supplemented with 10% fbs. all cells were incubated at 37uc in a humidified 5% co 2 atmosphere. bat cell lines were seeded in collagen-coated 24-well plates (becton dickinson labware, bedford, ma) at 2610 5 cells/well. one day later, media were removed, and cells were washed once with dmem without fbs (0% dmem). cells were then exposed to mers-cov/emc or mers-cov/jor at an moi of 1. after 1 h of incubation at 37uc, viral inocula were removed and cells were washed once with 0% dmem and then supplemented with dmem containing 2% fbs (2% dmem). at 1, 3, or 5 days postexposure, supernatants were harvested and cleared of cellular debris by centrifugation. plaque assay mers-cov particle yields were quantified by plaque assay [23] . briefly, confluent monolayers of vero cells in 6-well plates were exposed to serial dilutions of mers-cov, incubated at 37uc for 1 h under gentle rocking every 15 min, followed by removal of inocula and addition of a 0.8% tragacanth overlay (sigma-aldrich, st. louis, mo). infected cells were then incubated at 37uc for 72 h. the tragacanth overlay was removed, and the cells were stained with 2% crystal violet (sigma-aldrich) in 10% neutral buffered formalin (nbf, fisher scientific, kalamazoo, mi). plaques were enumerated manually. roni/7.1 or huh-7 cells were incubated with different concentrations (0, 1.25, 2.5, 5, 10, 20 mg/ml) of goat anti-human cd26/dpp4 antibody (r&d systems, minneapolis, mn) or control goat igg antibody at 37uc for 1 h. antibody-treated cells were exposed to mers-cov/emc at an moi of 1 at 37uc for 1 h in the presence of antibodies. virus-antibody inocula were then removed, cells were washed in 0% dmem, fresh dmem (2% fbs) was added, supernatants were harvested 24 h postexposure, and viral yields were determined by plaque assay. at the same time, plates were fixed with 10% nbf and then stained with rabbit polyclonal anti-mers-cov spike protein antibody (sino biological, beijing, china) followed by secondary alexa fluor 488conjugated goat anti-rabbit igg antibody (life technologies, carlsbad, ca). hoechst 33342 dye was used to stain nuclei. the percentage of infected cells was measured and analyzed using the operetta high content imaging system (perkinelmer waltham, ma) and analysis software (harmony 3.1). bat cells were washed with phosphate-buffered saline (pbs) and then dissociated with cell dissociation buffer (life technologies). cells were spun down, washed, and resuspended in 4% paraformaldehyde for fixation. cells were stained with goat antihuman cd26/dpp4 antibody followed by alex fluor 488conjugated rabbit anti-goat igg antibody. as a control, the cells were stained with the same concentration of isotype control goat igg antibody followed by the same secondary antibody. samples were collected using an lsr fortessa flow cytometer (bd biosciences, san jose, ca). flowjo software version 9.7.5 (treestar, ashland, or) was used to analyze the data. confluent bat cells were inoculated with mers-cov/emc at an moi of 1 for 1 h at 37uc. after viral inocula were removed, cells were washed once with 0% dmem and then supplemented with 2% dmem. media were removed 24 h later, and electron microscopy grade fixative, 2.5% glutaraldehyde (e.m. sciences, warrington, pa) in millonig's sodium phosphate buffer (tousimis research, rockville, md), was added directly to the dishes. after 10 min, bat cells were scraped off the dishes with a cell scraper, collected into 15-ml tubes, and immediately centrifuged at 5006g for 20 min. to complete fixation, cells were kept in fixative for 24 h at 4uc and were post-fixed in 1% osmium tetroxide (electron microscopy sciences, hatfield, pa). post-fixed cells were stained en bloc with 2% uranyl acetate, dehydrated in a series of graded ethanols, and infiltrated and embedded in spurr plastic resin (electron microscopy sciences). a leica em uc7 ultramicrotome (leica microsystems, buffalo grove, il) was used to section the embedded blocks into ultra-thin sections (60-80 nm). these sections were collected, mounted on 200-mesh copper grids (electron microscopy sciences), and contrasted with reynold's lead citrate. a fei g2 tecnai transmission electron microscope (fei, hillsboro, or), operating at 80 kv, was used to examine and image the grids. fluor 488-conjugated chicken anti-rabbit igg antibody (life technologies). images were acquired using the operetta high content imaging system. 2 flasks were infected with mers-cov/emc or mers-cov/jor at an moi of 1. after 7 days, supernatants were harvested for virus yield analysis by plaque assay, and the cells were subcultured at a 1:10 dilution in new flasks. subsequently, the infected cells were passaged at a 1:10 dilution weekly for a total of nine passages. from each passage, supernatants were harvested, and virus yields were determined by plaque assay. eidni/41.3 cells (non-infected or persistently infected with mers-cov, day 63) were washed with pbs and lysed in cell lysis buffer (cell signaling, danvers, ma) according to the manufacturer's instruction. equivalent amounts of total cellular lysates were resolved in 4% to 12% bis-tris gradient gels (life technologies) and then dry-transferred to polyvinylidene difluoride (pvdf) membranes (life technologies) by using the iblot gel transfer system (life technologies). after blocking in 5% nonfat milk powder in pbs with 0.1% tween (sigma-aldrich), membranes were incubated overnight with goat anti-human cd26/dpp4 antibody (1:500) or anti b-actin antibody (1:500, abcam, cambridge, ma), followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (sigma-aldrich). signals were detected by supersignal west femto chemiluminescent substrate (thermo fisher scientific, rockford, il), and images were acquired using a syngene g: box chemiluminescene imaging system (syngene, frederick, md). six of ten tested bat cell lines are susceptible to mers-cov infection figure 1a, 1b) . these results were also confirmed by immunofluorescence assay (ifa) in cell lines inoculated with mers-cov/emc ( figure 1c ) or mers-cov/jor ( figure 1d ). infected cells were detected by mers-cov spike protein ifa. representative images were picked from those taken on day 1 or day 3 post-exposure, as cytopathic viral effects diminished immunofluorescence in some cell lines (e.g., eidni/41.3, roni/7.1, roni/7.2 cells, figure 1c ). images of bat cells (day 1 post-exposure) inoculated with mers-cov/emc taken by a transmission electron microscope (tem, figure 1e to evaluate whether cd26/dpp4 expression is related to susceptibility of bat cells to mers-cov infection, surface expression of cd26/dpp4 was analyzed in ten bat cell lines by flow cytometry using a polyclonal anti-human cd26/dpp4 antibody. none of the four mers-cov-resistant cell lines tested in this study (pesu-b5l, r05t, r06e, and tb1lu) were recognized by anti-human cd26/dpp4 antibody in this assay, whereas all susceptible bat cells (eidni/41.3, eponi/22.1, hyplu/45.1, hypni/1.1, roni/7.1, and roni/7.2) tested positive for cd26/dpp4 expression (figure 2 ). to confirm the role of cd26/dpp4 in mers-cov bat cell entry, roni/7.1 cells or control human huh-7 cells were incubated with increasing concentrations of a monoclonal antihuman cd26/dpp4 antibody and subsequently exposed to mers-cov/emc. cd26/dpp4 antibody treatment reduced mers-cov particle production in both roni/7.1 cells and huh-7 cells, as evidenced by a dose-dependent reduction in viral yield in plaque assays ( figure 3a ). immunofluorescent images of infected cells stained against mers-cov spike protein confirm these results ( figure 3b ). thus, cd26/dpp4 plays a crucial role in mers-cov cell entry into bat cells. figure 4d ). in vitro studies revealed that mers-cov can infect cell lines derived from nonhuman primates, civets, rabbits, goats, cows, sheep, chickens, and pigs, but not cell lines derived from cats, dogs, hamsters, or mice [22, 24, 25] . in this study, we explored the potential of bats to be a reservoir for mers-cov infection [13] [14] [15] northern africa. in these geographic areas, domestic and wild dromedaries can be found and natural human mers-cov infections are recorded (table 1) . cd26/dpp4 is the cellular receptor for mers-cov [19] . this evolutionary conserved dimeric ectopeptidase is differentially expressed in various tissues but may not be the sole determinant for susceptibility at the organism level [25] . importantly, cellular susceptibility to mers-cov infection not only depends on the expression of cd26/dpp4, but also on its sequence. for instance, five amino-acid variations in the mers-cov-binding domain of hamster, ferret, and mouse cd26/dpp4 compared to human cd26/dpp4 have been linked to the resistance of hamster, ferret, and mouse cell lines to mers-cov infection [22] . our study confirms the role of cd26/dpp4 as receptor for two divergent mers-cov isolates and correlates its presence or absence on the surface of bat cells directly with bat cell susceptibility or resistance to productive mers-cov infection (figures 2 and 3 ). bat cells that tested negative for cd26/dpp4 expression by flow cytometry using the polyclonal anti-human cd26/dpp4 antibody may possibly express a cd26/dpp4 ortholog that is not recognized by this antibody. although the cells were derived from bats of different geographic origins, they could be rendered permissive by ectopic expression of human cd26/dpp4 ( figure 4a-c) . overexpression of human cd26/dpp4 in already mers-cov-susceptible bat cell lines led to an increase in virus yield in some bat cell lines and to a decrease in virus yield in others ( figure 4d ). we hypothesize that this variation may be due to differences in the number of bat cd26/dpp4 molecules on the cell-surface of each bat cell. for instance, overexpression of human cd26/dpp4 in a bat cell line with naturally high bat cd26/ dpp4 surface expression may not have an effect on virion entry efficiency since all virions already find enough binding partners in the untransfected cell. vice versa, if a bat cell line expresses little cd26/dpp4, finding a binding partner would present a bottleneck for mers-cov virions, and overexpression of human cd26/dpp4 might overcome this bottleneck and thereby increase virus yield. second, we hypothesize that overexpression of human cd26/dpp4 may interfere with transport and/or functionality of certain bat cd26/dpp4 orthologs due to heterodimerization and consequent structural changes. our results indicate that, at least on the cellular level, presence or absence of functional cd26/dpp4 with a suitable mers-cov-binding domain is a major determinant for mers-cov cellular tropism in bats. in addition to potential differences in the mers-cov-binding domain of distinct bat cd26/dpp4 orthologs, presence or absence of yet-to-be-identified co-receptors and bat species-specific cellular factors acting downstream of virion adsorption and fusion may further influence to what extent a productive infection can be established. however, these considerations alone are not sufficient to pinpoint bats as epidemiologically relevant mers-cov hosts, as the immune system at the level of the organism may interfere with infection prior to cell entry or lead to rapid viral clearance. one characteristic of natural virus host reservoirs is that hosts frequently are persistently infected with virus in the absence of clinical signs. continuous transmission of viruses from such a reservoir to other animals, including humans, is a hallmark of zoonoses and explains repeated introduction of viruses into susceptible animal populations [26] . a number of zoonotic viruses are known to persistently and subclinically infect rodents (e.g., arenaviruses, hantaviruses) or bats (henipaviruses), which then directly or indirectly infect humans [17, [27] [28] [29] [30] [31] . among the betacoronaviruses, the group of coronaviruses including mers-cov, murine coronavirus and severe acute respiratory syndrome (sars)-related coronaviruses that are related to mers-cov, are known to cause persistent infection in their mammalian hosts [16, 32] . during persistent sars-cov infection in vero e6 cells and also during acute infection in laboratory mice, the sars-cov entry receptor, angiotensin converting enzyme 2 (ace2), is downregulated [33, 34] . ace2 downregulation contributes to the severity of lung pathology of sars [34] . in figure 5 ). the observation that cultures continued to release significant virion amounts on day 63 post-inoculation, but also exhibited low to absent cd26/dpp4 cell-surface expression, is consistent with receptor downregulation as one mechanism for persistent infection with mers-cov in our experiments. virus carryover between cell/virus passages probably did not influence these results as excreted viruses should be unable to re-infect cells in the absence of cd26/dpp4 receptor. the mechanism of cd26/dpp4 downregulation upon persistent mers-cov infection requires further study. western blot-based examination of lysates of persistently infected cells at day 63 indicate that cd26/dpp4 is either not expressed anymore or is very efficiently degraded ( figure 5g ). the reason for the observed differences in establishing persistent infection in these cell lines among the two different mers-cov isolates is unclear, but may have epizootiological significance and needs to be examined in future studies. our results are consistent with the suggested role of bats in mers-cov transmission. if cd26/dpp4-positive cells of bats become infected with mers-cov and if subsequently the receptor surface expression is downregulated as we observe in our experiments, a persistent infection could be established. such animals may provide a reservoir that continuously sheds infectious virus, possibly transmitting the virus to other mammals, such as dromedaries. further evaluation is needed to determine whether downregulation of cd26/dpp4 is a general hallmark of persistent mers-cov infection in animal models of mers and whether the receptor influences pathogenesis in a manner similar to that seen with ace2 downregulation in persistent sars-cov infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov) summary and literature update -as of 11 middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-to-human transmission of mers coronavirus isolation of mers coronavirus from a dromedary camel rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe middle east respiratory syndrome coronavirus in bats, saudi arabia close relative of human middle east respiratory syndrome coronavirus in bat severe acute respiratory syndrome coronavirus persistence in vero cells agricultural intensification, priming for persistence and the emergence of nipah virus: a lethal bat-borne zoonosis urban habituation, ecological connectivity and epidemic dampening: the emergence of hendra virus from flying foxes (pteropus spp.) inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus host species restriction of middle east respiratory syndrome coronavirus through its receptor dipeptidyl peptidase 4 use of gum tragacanth overlay, applied at room temperature, in the plaque assay of fish and other animal viruses differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters how to find natural reservoir hosts from endemic prevalence in a multi-host population: a case study of influenza in waterfowl persistence of lymphocytic choriomeningitis virus in immune animals and its relation to immunity density thresholds for mopeia virus invasion and persistence in its host mastomys natalensis emergence and persistence of hantaviruses hantavirus reservoirs: current status with an emphasis on data from brazil recrudescent infection supports hendra virus persistence in australian flying-fox populations persistent infection promotes cross-species transmissibility of mouse hepatitis virus jnk and pi3k/ akt signaling pathways are required for establishing persistent sars-cov infection in vero e6 cells a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum comparative analysis of ebola virus glycoprotein interactions with human and bat cells evidence supporting a zoonotic origin of human coronavirus strain nl63 cell lines from the egyptian fruit bat are permissive for modified vaccinia ankara differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses we would like to thank eric donaldson (fda) for providing pesu-b5l cells. we are grateful to our colleague, jiro wada (irf-frederick), for assisting us with the preparation of figures. the content of this publication does not necessarily reflect the views or policies of the us department of the army, the us department of defense, the us department of health and human services, or of the institutions and companies affiliated with the authors. key: cord-279976-juz9jnfk authors: xie, mingxuan; chen, qiong title: insight into 2019 novel coronavirus — an updated intrim review and lessons from sars-cov and mers-cov date: 2020-04-01 journal: int j infect dis doi: 10.1016/j.ijid.2020.03.071 sha: doc_id: 279976 cord_uid: juz9jnfk background: the rapid spread of the coronavirus disease 2019 (covid-19), caused by a zoonotic beta-coronavirus entitled 2019 novel coronavirus (2019-ncov), has become a global threat. awareness of the biological features of 2019-ncov should be updated in time and needs to be comprehensively summarized to help optimize control measures and make therapeutic decisions. methods: based on recently published literatures, official documents and selected up-to-date preprint studies, we reviewed the virology and origin, epidemiology, clinical manifestations, pathology and treatment of 2019-ncov infection, in comparison with severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) infection. results: the genome of 2019-ncov partially resembled sars-cov and mers-cov, and indicating a bat origin. the covid-19 generally had a high reproductive number, a long incubation period, a short serial interval and a low case fatality rate (much higher in patients with comorbidities) than sars and mers. clinical presentation and pathology of covid-19 greatly resembled sars and mers, with less upper respiratory and gastrointestinal symptoms, and more exudative lesions in post-mortems. potential treatments included remdesivir, chloroquine, tocilizumab, convalescent plasma and vaccine immunization (when possible). conclusion: the initial experience from the current pandemic and lessons from the previous two pandemics can help improve future preparedness plans and combat disease progression. in late december 2019, a pneumonia outbreak of unknown etiology took place in wuhan, hubei province, china, and spread quickly nationwide. chinese center for disease control and prevention (ccdc) identified a novel beta-coronavirus called 2019-ncov, now officially known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (gorbalenya et al., 2020) , that responsible for the pandemic. this was the third zoonotic coronavirus breakout in the first two decades of 21 st century that allowing human-to-human transmission and raising global health concerns. chinese government had taken immediate, transparent and extraordinary measures, and reached initial achievements to control the outbreak. as of 11 march 2020, the pandemic in pubmed, web of science, embase, cnki, wanfang, vip, preprint biorxiv and medrxiv databases from the earliest available date to 11 march, 2020. initial search terms were "2019-ncov" or "2019 novel coronavirus" or "sars-cov-2" or "covid-19" or "corona virus disease 2019" or "ncp" or "novel coronavirus pneumonia". further search words were above keywords, "sars" or "sars-cov" or "severe acute respiratory syndrome", "mers" or "mers-cov" or "middle east respiratory syndrome", in combinations of with "spike protein" or "genome" or "reproductive number" or "incubation period" or "serial interval" or "fatality rate" or "clinical characteristics" or "pathology" or "autopsy" or "treatment". j o u r n a l p r e -p r o o f moreover, official documents and news released by national health commission of p.r. china, ccdc, cdc(usa) and who were accessed for up-to-date information on covid-19. only the articles in english or chinese were considered. in this review, we highlight the pandemic potential and pathological indications of emerging coronavirus, comprehensively and systematically summarize the up-to-date knowledge of the biological characteristics of 2019-ncov, including virology and origin, epidemiology, clinical manifestations, pathology and treatment. because of its natural structures and biological features to bind receptors on host cells, the spike protein of 2019-ncov may played an essential role in disease spreading. we summarized all of the four available pathology studies of covid-19 biopsy and autopsy, and compared the results with previous two deadly coronavirus diseases. new therapeutic measures are emerging one after another. potential effective treatments were remdesivir, chloroquine, tocilizumab, convalescent plasma and vaccine immunization (when possible). evidence-based medicine should always be advocated to guide our clinical decision. coronavirus belongs to the subfamily orthocoronavirinae in the family of coronaviridae in the order nidovirales, which mainly caused infections in respiratory and gastrointestinal tract. the 2019-ncov is a novel enveloped beta-coronavirus which has a single stranded positive sense rna genome . concerning the origin of the virus, several phylogenetic analysis suggested the bat to be the most probable animal reservoir. based on genome sequencing, 2019-ncov is about 89% identical to bat sars-like-covzxc21, 82% identical to human sars-cov and about 50% to mers-cov (chan et al., 2020; lu et al., 2020) . as both sars-cov and mers-cov were transmitted from bats to palm civets or dromedary camels, and finally to humans, there should be another animal representing as an intermediate host between bat and human. pangolins were suggested as the possible intermediate hosts, because their genome had approximately 85.5%-92.4% similarity to 2019-ncov, representing two sub-lineages of 2019-ncov in the phylogenetic tree, one of which (gd/p1l and gdp2s) was extremely closely related to 2019-ncov (lam et al., 2020) . other research suggested 2019-ncov was the recombinant virus of bat coronavirus and j o u r n a l p r e -p r o o f snake coronavirus, by comparison in conjunction with relative synonymous codon usage bias among different animal species (ji et al., 2020) . the truth is yet to be discovered. the spike surface glycoprotein of coronavirus plays an essential role in binding to receptors on host cells and determines host tropism. spike protein(s-protein) of 2019-ncov is reported to bind with angiotensin-converting enzyme 2 (ace2), the same receptor of sars-cov to invade host cells; whereas mers-cov uses dipeptidyl peptidase 4 (dpp4) as the primary receptor . the amino acid sequence another research team also discovered an "rrar" furin recognition site by an insertion in the s1/s2 protease cleavage site in 2019-ncov, instead of a single arginine in sars-cov. after quantifying the kinetics mediating the interaction via surface plasmon resonance, ace2 is calculated to bind to 2019-ncov ectodomain with ~15 nm affinity, which is approximately 10-to 20-fold higher affinity than ace2 binding to sars-cov (wrapp et al., 2020) . in all, the binding affinity between 2019-ncov s-protein and ace2 is comparable or even stronger than sars-cov s-protein and ace2. this may explain the rapid development and strong ability of human-to-human transmission in covid-19. the pandemic escalated exponentially at the beginning of 2020, which might only be the tip of the iceberg due to delayed case reporting and deficiency in testing kits . the onset of first cluster cases were reported an exposure history to the j o u r n a l p r e -p r o o f huanan seafood(wild animal) wholesale market in wuhan. however, phyloepidemiologic analyses suggested that huanan market was not the origin of 2019-ncov. the virus was imported from elsewhere and boosted in the crowded market (yu et al., 2020) . the proportion of infected cases without an exposure history and in health care workers gradually increased. all of the evidence indicated the human-to-human transmission ability of 2019-ncov, which may already be spread silently between people in wuhan before the cluster of cases from huanan market was discovered in late december. person-to-person transmission may occur mainly through droplet or contact transmission. according to guan's latest pilot study, 2019-ncov was detected positive in the gastrointestinal tract specimens (stool and rectal swabs) as well as in saliva and urine, and even in esophageal erosion and bleeding site of severe peptic ulcer patients . four important epidemiological parameters of 2019-ncov were reviewed in comparison with those of sars-cov and mers-cov(shown in table 1 ). representing the average number of new infections generated by an infectious person in a totally naïve population. for r0˃1, the number of infected is likely to increase; for r0 ˂1, transmission is likely to decline and die out. the reproductive number updated along with the development of the outbreak and interventions. r0 was estimated to be around 3 for sars (bauch et al., 2005) and ˂1 for mers (bauch and oraby, 2013) . the preliminary r0 of 2019-ncov was reported as 2.24-3.58 . several research groups reported estimated r0 of the outbreak depending on distinct estimation methods and the validity of underlying assumptions. liu et al. (2020) reviewed all of the 12 references of an estimated r0 ranged from 1.4 to 6.49, with a mean of 3.28 and a median of 2.79. in clinical studies, a 425-case study by 22 january 2020, reported an r0 of approximately 2.2(95%ci, 1.4-3.9) , while another 4021-case study by 26 january 2020, estimated 3.77(95%ci, 3.51-4.05) . the discrepancy may be due to sample number and different stages of the pandemic. incubation period is defined as the interval from initial exposure to an infectious agent to onset of any symptoms or signs it causes. a long incubation period may lead to a high rate of asymptomatic and subclinical infection. the first prediction of mean incubation period was 5.2 days (95%ci, 4.1-7.0 days), with the 95th percentile of the distribution at 12.5 days, based on 2019-ncov exposure histories of the first 425 cases in wuhan . a 4021-case study reported 4.75 days (interquartile range: j o u r n a l p r e -p r o o f 3.0-7.2 days) . another 88-exported-case study calculated the mean incubation period to be 6.4 days (95%ci, 5.6-7.7 days), using known travel histories to and from wuhan and symptom onset dates (backer et al., 2020) . all these literatures lay the foundation to set 14 days as the medical observation period if any exposure occurred. a latest study collected 1099 cases from 552 hospitals in 31 provinces in china and declared a median incubation period of 3.0 days, ranging from 0 to surprisingly 24.0 days. an adjustments in screening and control policies may be needed. the 2019-ncov generally has a longer incubation time than sars-cov (4.0 days, 95% ci 3.6-4.4 days) (lessler et al., 2009 ) and mers-cov (range 4.5-5.2 days) (park et al., 2018) . serial interval is the interval from illness onset in a primary case to illness onset in the secondary case. the mean serial interval was estimated at 7.5 days(95% ci, 5.3-19days) using contact tracing data from early wuhan cases in 2019-ncov pandemic, which was shorter than the 8.4-day mean serial interval reported for sars (lipsitch et al., 2003) and 12.6-day for mers (cowling et al., 2015) . another estimation of the mean serial interval from 26 infector-infectee pairs was surprisingly 2.6 days, which was shorter than the median incubation period, suggesting a substantial proportion of secondary transmission before illness onset (nishiura et al., 2020) . the cfr in early studies of covid-19 involving relatively small samples of confirmed cases in wuhan, varied from 4.3% to 14.6% huang et al., 2020; , but that may not be able to reflect the truth. the cfr in wuhan was undoubtedly higher than cfr outside of wuhan. the reported cfr ranged 1.4%-3.06% in large nationwide case studies . prognosis factors such as male, elderly patients aged≥ 60 years, underlying disease, severe pneumonia at baseline and a delay from onset to diagnosis >5 days substantially elevated the cfrs . cfrs in patients with cardiovascular disease, diabetes, hypertension and respiratory disorders were as high as 10.5%, 7.3%, 6.0% and 6.3%, respectively. according to who announcement, sars accounted for 8096 cases and 774 death, with a cfr of 9.6% (who, 2004 clinical presentation of covid-19 greatly resembled viral pneumonia such as sars and mers. most cases are mild cases(81%), whose symptoms were usually self-limiting and recovery in two weeks (wu and mcgoogan, 2020) . severe patients progressed rapidly with acute respiratory distress syndrome (ards) and septic shock, eventually ended in multiple organ failure. general information of four inpatient case studies with relatively comprehensive data were summarized in supplementary table 1. the 2019-ncov was more likely to infect elderly men with comorbidities. males were more susceptible to 2019-ncov infection, same as sars-cov and mers-cov studies (badawi and ryoo, 2016) , due to x chromosome and sex hormones' role on innate and adaptive immunity (jaillon et al., 2019) . chronic underlying diseases (mainly hypertension, cardio-cerebrovascular diseases and diabetes) may increase the risk of 2019-ncov infection , which is similar to mers-cov infection (badawi and ryoo, 2016) . smoking may be a negative prognostic indicator for covid-19 guan et al., 2020) . clinical information of the above four selected inpatient case studies were summarized in supplementary table 2. onset of symptoms were usually mild and nonspecific, presenting by fever, dry cough and shortness of breath. very few covid-19 patients had prominent upper respiratory tract and gastrointestinal symptoms (eg, diarrhea) huang et al., 2020) , compared to 20-25% of patients with mers-cov or sars-cov infection developed diarrhea (assiri et al., 2013) . however, only 43.8% of covid-19 patients had an initial presentation of fever, and developed to 87.9% following hospitalization , compared to as high as 99% and 98% frequent in sars-cov and mers-cov infection (badawi and ryoo, 2016) . those patients without fever or even asymptomatic may be left un-quarantined as silent infection source, if the surveillance methods focused heavily j o u r n a l p r e -p r o o f on fever detection. moreover, the onset of symptoms may help physicians identifying patients with poor prognosis. patients admitted to the icu were more likely to report pharyngeal pain, dyspnea, dizziness, abdominal pain and anorexia . in terms of laboratory findings, a substantial decrease in the total number of lymphocytes could be used as an index in the diagnosis of 2019-ncov infection, indicating a consumption of immune cells and an impairment to cellular immune function . non-survivors developed more severe lymphopenia over time . initial proinflammatory plasma cytokine concentrations were higher in covid-19 patients than in healthy adults. icu patients had even higher plasma levels of il2, il7, il10, gscf, ip10, mcp1, mip1a, and tnfα compared to non-icu patients . there were numerous differences in laboratory findings between patients admitted to the icu and those not, including higher white blood cell and neutrophil counts, higher levels of d-dimer, creatine kinase, and creatine in icu patients . typical chest ct manifestation of covid-19 pneumonia were initially small subpleural ground glass opacities that grew larger with crazy-paving pattern and consolidation. after two weeks of growth, the lesions were gradually absorbed leaving extensive opacities and subpleural parenchymal bands in recovery patients. however, guan et al. ( 2020) demonstrated that patients with normal radiologic findings on initial presentation consisted of 23.9% and 5.2% of severe and non-severe cases respectively, which add the complexity to disease control. (nicholls et al., 2003) . thrombi were seen in all six autopsies of sars-cov infected patients, with even huge thrombus formation in part of pulmonary vessels. coagulation function disorders were reported in most of the severe covid-19 patients, by elevated levels of d-dimer and prolonged prothrombin time, some of whom ended in disseminated intravascular coagulation huang et al., 2020; . this may explain some sudden death of clinical recovery patients and serve as an indication for disease severity. in an autopsy study, the only one patient without usage of corticosteroids demonstrated increased cd3+ lymphocyte than five other specimen treated by corticosteroids (pei et al., 2005) . it suggested an inhibition of immune system similarities. the human monoclonal antibody could efficiently neutralize sars-cov and inhibit syncytia formation between s-protein and ace2 expressing cells (sui et al., 2004) . appropriate modification of the monoclonal antibody may be effective for treatment of covid-19. what's more, potential therapies targeting the renin-angiotensin system, to increase ace2 expression and inhibit ace may be there are no effective antiviral treatment for coronavirus infection, even the strong candidates as lopinavir/ritonavir and abidol exhibited no remarkable effect on clinical improvement, day 28 mortality or virus clearance (chen et al., 2020) . expectation and attention were shifted to "remdesivir" which may be the most potential wide-spectrum drug for antiviral treatment of 2019-ncov. remdesivir is an adenosine analogue, which incorporates into novel viral rna chains and results in pre-mature termination. it is currently under clinical development for the treatment of ebola virus infection (mulangu et al., 2019) . wang et al. (2020b) revealed that remdesivir were highly effective and safe in the control of 2019-ncov infection in vero e6 cells and huh-7 cells. a successful appliance of remdesivir on the first 2019-ncov infected case in the united states when the his clinical status was getting worsen, were recently released (holshue et al., 2020) . animal experiments also showed superiority of remdesivir over lopinavir/ritonavir combined with interferon-β, by reducing mers-cov titers of infected mice and improving the lung tissue damage (sheahan et al., 2020) . the effectiveness and safety of remdesivir can be expected by the clinical trial lead by dr bin cao. the 2019-ncov infection is associated with a cytokine storm triggered by over-activated immune system xu et al., 2020b) , similar to sars and mers. the aberrant and excessive immune responses lead to a long-term lung function and structure damage in patients survived from icu. ongoing trials of il-6 antagonist tocilizumab, which shown effective against cytokine release syndrome resulting from car-t cell infusion against b cell acute lymphoblastic leukemia, may be expanded to restore t cell counts and treat severe 2019-ncov infection (le et al., 2018) . the available observational studies and meta-analysis of corticosteroid treatment suggested impaired antibody response, increased mortality and secondary infection rates in influenza, increased viraemia and impaired virus clearance of sars-cov and mers-cov, and complications of corticosteroid therapy in survivors (zumla et al., 2020) . therefore, corticosteroid should not be recommended for treatment of 2019-ncov, or use on severe patient with special caution. a review (nichol et al., 2003) . in conclusion, it still remains a challenging task to fight the 2019-ncov of unknown origin and mysterious biological features, and to control an outbreak of covid-19 with such a high r0, a long incubation period and a short serial interval, by limited treatment and prevention measures. lessons learned from the mers and sars outbreaks can provide valuable insight into how to handle the current pandemic. the successful public health outbreak response tactics of chinese government, such as hand hygiene, wearing masks, isolation, quarantine, social distancing, and community containment, can be copied by other countries according to their national situation. as the pandemic is still ongoing and expanding, experiences and research literatures from china and other countries will increase. the 2019-ncov should be monitored of any possible gene variation of antigenic drift or antigenic conversion, to avoid another round of outbreak. another lessons from this pandemic will be awe for nature and love for life. funding source: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ethical approval: the ethical approval or individual consent was not applicable. all authors declare no conflict of interest. all authors don't have any financial and personal relationships with other people or organizations that could influence our work. epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis dynamically modeling sars and other newly emerging respiratory illnesses: past, present, and future assessing the pandemic potential of mers-cov genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan efficacies of lopinavir/ritonavir and abidol in the treatment of novel coronavirus pneumonia epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study recommendations for influenza and streptococcus pneumoniae vaccination in elderly people in china the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade preliminary epidemiological assessment of mers-cov outbreak in south korea breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies severe acute respiratory syndrome-related coronavirus-the species and its viruses, a statement of the coronavirus study group clinical of coronavirus disease 2019 in china sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically-proven protease inhibitor first case of 2019 novel coronavirus in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan, china sexual dimorphism in innate immunity cross-species transmission of the newly identified coronavirus 2019-ncov identification of 2019-ncov related coronaviruses in malayan pangolins in southern china fda approval summary: tocilizumab for treatment of chimeric antigen receptor t cell-induced severe or life-threatening cytokine release syndrome incubation periods of acute respiratory viral infections: a systematic review early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia transmission dynamics and control of severe acute respiratory syndrome the reproductive number of covid-19 is higher compared to sars coronavirus genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis controlled trial of ebola virus disease therapeutics influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly lung pathology of fatal severe acute respiratory syndrome serial interval of novel coronavirus (2019-ncov) infections. medrxiv(preprint) 2020 mers transmission and risk factors: a systematic review lung pathology and pathogenesis of severe acute respiratory syndrome: a report of six full autopsies a report on the general observation of a 2019 novel coronavirus autopsy comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association evaluation of convalescent plasma for ebola virus disease in guinea chloroquine is a potent inhibitor of sars coronavirus infection and spread clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama 2020a remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro decoding the evolution and transmissions of the novel pneumonia coronavirus (sars-cov-2) using whole genomic data summary of probable sars cases with onset of illness from 1 who . who mers global summary and assessment of risk who. novel coronavirus(2019-ncov) situation report-51 2020 cryo-em structure of the 2019-ncov spike in the prefusion conformation genome composition and divergence of the novel coronavirus (2019-ncov) originating in china characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission pathological findings of covid-19 associated with acute respiratory distress syndrome liqun fang. epidemiological and clinical features of the 2019 novel coronavirus outbreak in china single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov a novel coronavirus from patients with pneumonia in china reducing mortality from 2019-ncov: host-directed therapies should be an option crrt(%) 7 9 1.5 0.8 key: cord-272710-uq2idlca authors: cho, chao-cheng; lin, meng-hsuan; chuang, chien-ying; hsu, chun-hua title: macro domain from middle east respiratory syndrome coronavirus (mers-cov) is an efficient adp-ribose binding module: crystal structure and biochemical studies date: 2016-01-05 journal: journal of biological chemistry doi: 10.1074/jbc.m115.700542 sha: doc_id: 272710 cord_uid: uq2idlca the newly emerging middle east respiratory syndrome coronavirus (mers-cov) encodes the conserved macro domain within non-structural protein 3. however, the precise biochemical function and structure of the macro domain is unclear. using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the mers-cov macro domain as a more efficient adenosine diphosphate (adp)-ribose binding module than macro domains from other covs. furthermore, the crystal structure of the mers-cov macro domain was determined at 1.43-å resolution in complex with adp-ribose. comparison of macro domains from mers-cov and other human covs revealed structural differences in the α1 helix alters how the conserved asp-20 interacts with adp-ribose and may explain the efficient binding of the mers-cov macro domain to adp-ribose. this study provides structural and biophysical bases to further evaluate the role of the mers-cov macro domain in the host response via adp-ribose binding but also as a potential target for drug design. since the severe acute respiratory syndrome (sars) 2 outbreak in 2003 (1, 2), a newly discovered disease, middle east respiratory syndrome (mers), has been spreading from countries in the middle east to america (3) (4) (5) . in the summer of 2015, mers was reported in north east asia (6 -8) . the causative agent of mers was identified as an unknown coronavirus (cov) resembling sars-cov and referred to as middle east respiratory syndrome cov (mers-cov) (9 -12) . mers-cov belongs to the genus betacoronavirus and possesses a positivestrand rna genome that encodes viral proteins essential to the life cycle of the virus (13, 14) . the mortality of mers is 4-fold higher than sars (40% compared with 10%) (15) . since the first case report in saudi arabia, mers has been reported in more than 20 countries and has caused more than 400 deaths worldwide (9) . covs utilize the rna genome to encode structural proteins, including spike glycoprotein (s), membrane protein (m), and nucleocapsid protein (n). they encode a large number of nonstructural proteins (nsps) for rapid replication. a single large replicase gene encodes all proteins involved in viral replication. the replicase gene contains two open reading frames (orfs), orf1a and orf1b, which encode two polyproteins, pp1a and pp1ab; production of pp1ab requires a ribosomal frameshift to transcribe the portion encoded by orf1b (16) . orf1a encodes viral proteases, main protease (m pro , also called 3cl pro ), and papain-like protease (pl pro ), which are responsible for cleavage of the orf1a and orf1b gene products to produce functional nsps. in sars-cov, the largest nsp member, nsp3, is a multidomain protein containing the following domains: n-terminal acidic domain, macro domain, sars-unique domain, pl pro , nucleic acid-binding domain, marker domain (g 2 m), transmembrane domain, and y-domain (17) . the mers-cov genome contains 16 nsps (fig. 1) ; except for 3cl pro and pl pro (18, 19) , most of the functional domains within the nsp3 in mers-cov remain structurally uncharacterized. the macro domain is named after the non-histone motif of the histone variant macroh2a, in which it was originally characterized (20 -22) , a protein module ubiquitous in eukaryotes, bacteria, and archaea. this domain is well known for its affinity to adenosine diphosphate (adp)-ribose (23) (24) (25) . many cellular enzymes bearing macro domains within their structures interact with poly(adp)-ribose (26 -29) . poly(adp)-ribosylation is a post-translational modification linked with dna repair, apoptosis, gene regulation, and protein degradation. thus, macro domain-containing proteins and enzymes may play important roles in regulating various cellular processes (30) . surprisingly, the covs studied so far and a few other viruses such as alphavirus, rubella virus, and hepatitis e virus possess macro domains in their genomes (16) . in addition, some viral macro domains were found to have adp-ribose 1љ-phosphate phosphatase (adrp) activity (31) (32) (33) , which catalyzes the removal of phosphate from adp-ribose 1љ-phosphate (appr1p) to produce adp-ribose. adrp activity has been reported in a yeast protein containing macro domain as well as af1521 protein in archaeoglobus fulgidus (23, 34, 35) . the enzymatic activity of viral macro domains in processing appr1p is low (33, 36 -38) and appears to be dispensable for virus rna synthesis (31) . in addition, the mutant for the cov mouse hepatitis virus a59 (mhv-a59), encoding a single amino acid substitution of a strictly conserved residue for adrp activity, replicated to slightly reduced titers in mouse liver but, strikingly, did not induce liver disease (39) . the mhv macro domain exacerbates mhv-induced liver pathology, most likely by inducing excessive inflammatory cytokine expression. it was also reported that catalytic residues asn-809, his-812, gly-816, and gly-817 for adrp activity in hepatitis e virus macro domain are critical for hepatitis e virus replication (40) . accordingly, the development of drugs targeting the viral macro domain may be a strategy for antiviral therapy. the macro domain of sars-cov nsp3 was previously reported to possess adp-ribose and poly(adp)-ribose binding ability, which suggests that the macro domain may regulate cellular proteins involved in an apoptotic pathway via poly-(adp)-ribosylation to mediate the host response to infection (36) . structural studies of macro domains from covs such as human cov 229e (hcov-229e) and feline cov (fcov) also revealed interactions with adp-ribose (41) (42) (43) and have offered huge advances in our understanding of viral macro domains. the mers-cov genome features a macro domain embedded in nsp3 (fig. 1) . however, we lack structural and functional information regarding the mers-cov macro domain. in the present study, we investigated the mers-cov macro domain as an adp-ribose binding module, with comparison to previously characterized viral macro domains. furthermore, we determined the crystal structure of the mers-cov macro domain in complex with adp-ribose. structural comparison of mers-cov and other human covs revealed divergence in adp-ribose binding by macro domains. our study may shed new light on structurally based design of novel antiviral drugs targeting viral macro domains. protein expression and purification-the dna sequence containing the mers-cov macro domain was synthesized by a local biotechnology company (mdbio, inc.) and cloned into the puc57 plasmid. the macro domain fragment was inserted between the ndei and xhoi sites of the pet28a vector system (novagen). the forward and reverse pcr primers used for amplification were macro-f (5ј-aattcatatgccactga-gcaattttgaaca-3ј) and macro-r (5ј-aattctcgagt-tagatggtcaggctcttatac-3ј). the resulting plasmid with the inserted sequence was transformed into escherichia coli bl21(de3) cells, which were grown at 37°c up to a 600 1.0 with 50 g/ml of kanamycin. the expression of the recombinant mers-cov macro domain with an his tag at the n terminus was induced in cells with 1 mm isopropyl ␤-d-thiogalactoside, followed by growth for 20 h at 16°c. cells were collected by centrifugation and resuspended in lysis buffer (25 mm phosphate buffer, ph 7.0, 100 mm nacl). after 20 min of sonication, the cell extract was clarified by centrifugation at 18,900 ϫ g for 30 min at 4°c to remove debris. the clear supernatant was placed in an open column filled with nickel-nitrilotriacetic acid resin. the resin was washed with 10 times volume of lysis buffer containing 50 and 100 mm imidazole, respectively. the histagged mers-cov macro domain was eluted by lysis buffer containing 300 mm imidazole. the purified mers-cov macro domain was dialyzed against stabilization buffer (25 mm phosphate buffer, ph 7.0, 100 mm nacl, 0.5 mm dithiothreitol). the his tag was removed by using thrombin, which resulted in four additional residues (gshm) at the n terminus. the protein was further purified by gel filtration chromatography with a super-dex75 xk 16/60 column (ge healthcare) in 20 mm tris-hcl buffer (ph 7.0), 100 mm nacl. circular dichroism (cd) spectroscopy-far-uv cd spectra were measured with 10 m protein samples in cd buffer (20 mm phosphate buffer, ph 3.5-8.5) placed into a 1-mm path length cuvette and recorded on a jasco j-810 spectropolarimeter equipped with a peltier temperature control system (jasco international co.). thermal transition of protein samples with or without preincubation of 1 mm adp-ribose were monitored at 220 nm from 25 to 95°c at a scan rate of 1°c/min. baseline subtraction, smoothing, and data normalization involved the use of sigmaplot. the melting temperature (t m ) was calculated with the maximum of the first derivative of the cd signal. differential scanning fluorimetry (dsf)-thermal shift assay with dsf involved use of a cfx48 real-time pcr detection system (bio-rad). in total, a 25-l mixture containing 2 l of sypro orange (sigma), 1.25 l of dialysis buffer (20 mm tris-hcl, and 100 mm nacl, ph 7.0), 10 l of 1 m protein sample, and various concentrations of adp-ribose were mixed on ice in an 8-well pcr tube. fluorescent signals were measured from 25 to 95°c in 0.1°c/30-s steps (excitation, 450 -490 nm; detection, 560 -580 nm). the main measurements were carried out in triplicate. data evaluation and t m determination involved use of the bio-rad cfx manager, and data fitting and dissociation constant (k d ) calculations involved the use of sigmaplot. isothermal titration calorimetry (itc)-binding of adp-ribose to the mers-cov macro domain was measured by itc with the nano isothermal titration calorimeter (ta instruments). aliquots of 3 l of 1.14 mm adp-ribose were titrated by injection into protein (0.057 mm in 0.98 ml) in 20 mm tris-hcl (ph 7.0) and 100 mm nacl. experiments were carried out at 25°c with 250 rpm stirring. background heat from ligand to buffer titrations was subtracted, and the corrected heat from the binding reaction was used to derive values for the stoichiometry of the binding (n), k d , apparent enthalpy of binding (⌬h), and entropy change (⌬s). data were fitted by use of an independent binding model with launch nanoanalyze version 2.3.6. crystallization and data collection-the mers-cov macro domain and adp-ribose were mixed in a molar ratio of 1:15. initial protein crystallization trials were performed at 283 k by the sitting-drop vapor-diffusion method with commercial crystallization screen kits, 96-well intelli-plates (art robbins instruments), and a honeybee 963 robot (genomic solutions). each crystallization drop was prepared by mixing 0.3 l of macro domain/adp-ribose at 10 mg/ml with an equal volume of mother liquor, and the mixture was equilibrated against 100 l of reservoir solution. the crystals for data collection were grown in 1 week at 283 k with the optimal condition of 100 mm phosphate/citrate (ph 4.2), 2.0 m ammonium sulfate, and 10 mm nicotinamide adenine dinucleotide as the additive. for subsequent anomalous phasing, the crystal was soaked for 8 h in 3 mm mercuric(ii) chloride, cryoprotected in mother liquor sup-plemented with 20% glycerol, and flash-frozen in liquid nitrogen at 100 k. the diffraction images were recorded in a 100-k nitrogen gas stream with use of bl13b1 or bl13c1 beamlines (national synchrotron radiation research center, taiwan) and processed by using hkl2000 software (44) . structure determination and refinement-the crystal structure of the mers-cov macro domain in complex with adpribose was solved by the mercury(ii) derivative single-wavelength anomalous dispersion method by using shelxd/ shelxe software (45) . the initial model was refined by the maximum likelihood method implemented in refmac5 (46) as part of the ccp4 suite (47) and rebuilt interactively by inspecting the -weighted electron density maps with coefficients 2mf o ϫ df c and mf o ϫ df c in coot (48) . during the later stages, restrained positional and b-factor refinement involved the program phenix.refine (49) . water molecules were manually added at the final stages. the models were evaluated with use of procheck (50) and molprobity (51) . the data collection and structure refinement statistics are in table 1 . the mers-cov macro domain (pp1a residues 1110 to 1273) was expressed and purified from e. coli. the final purified pro tions of symmetry related reflections of h. c cc 1/2 is a percentage of correlation between intensities from random half-datasets (56) . d cc ano is a percentage of correlation between random half-datasets of anomalous intensity differences. e r work /r free ϭ ⌺͉f obs ϫ f calc ͉/⌺f obs , where f calc is the calculated protein structure factor from the atomic model (r free was calculated with 5% of the reflections selected). tein was a 167-amino acid protein (20 kda), with four additional residues at the n terminus resulting from removal of the hexahistidine tag after thrombin cleavage. cd spectra revealed that the macro domain exhibited a stable ␣/␤-type folding pattern under various ph conditions (fig. 2) . the t m of the macro domain from thermal transition monitored by cd was 43°c. however, the addition of adp-ribose significantly increased the t m to 51°c (fig. 3a) . the significant increase in t m suggests the interaction between the mers-cov macro domain and adp-ribose. to understand the affinity of adp-ribose binding to the mers-cov macro domain, both dsf and itc measurements were used to examine the equilibrium dissociation constant (k d ) of adp-ribose. after fitting dsf data, the k d was determined to be 3.12 ϯ 0.42 m (r 2 ϭ 0.9628) (fig. 3b) , which is similar to the calculated k d of 2.95 m based on itc data (fig. 3c ). in addition, itc data indicated that adp-ribose bound to the mers-cov macro domain with favorable enthalpy change (exothermic, ⌬h ϭ ϫ91.04 kj/mol). the binding reaction was spontaneous at 25°c with exergonic gibbs energy of binding (⌬g ϭ ϫ31.56 kj/mol). the thermodynamic profile (⌬g ͻ 0, ⌬h ͻ 0, and ϫt⌬s ͼ 0) of adp-ribose binding to the mers-cov macro domain suggests that adp-ribose is likely stabilized by hydrogen bond formations (52) . we reviewed the results of previously reported binding assays of adp-ribose binding to cov macro domains ( table 2) . overall structure of mers-cov macro domain in complex with adp-ribose-we determined the crystal structure of adp-ribose-bound mers-cov macro domain for further molecular elucidation. the orthorhombic crystals gave good quality x-ray diffraction and belonged to the space group c222 1 with the following unit cell dimensions: a ϭ 41.798 å, b ϭ 120.807 å, c ϭ 67.659 å, and ␣ ϭ ␤ ϭ ␥ ϭ 90°. the structure of the mers-cov macro domain was solved by mercury singlewavelength anomalous dispersion (see "experimental procedures"). the final protein structure (fig. 4a ) was refined to 1.43-å resolution with r-factor and r-free values of 0.1273 and 0.1619, respectively ( table 1 ). the core of the structure of mers-cov macro domain is a seven-stranded ␤-sheet in the order of ␤1-␤2-␤7-␤6-␤3-␤5-␤4 (fig. 4b) . the central ␤-sheet is sandwiched between six ␣-helices, with ␣1, ␣2, and ␣3 packing onto one face and ␣4, ␣5, and ␣6 onto the other. in the initial refinement cycle, a strong bent electron density (continuous at 1 cutoff) (fig. 4a ) located at the central pocket, was unambiguously identified as an adp-ribose molecule. this molecule is tightly bound in an uncharged crevice located at the c-terminal end of strands ␤3 and ␤6 in the loop regions between ␤3-␣2 and ␤6-␣5 (fig. 4a) . a search of the dali database (53) with the structure of the mers-cov macro domain in complex with adp-ribose used as a model revealed several structural homologs. top-ranked structures were macro domains of covs in complex with adpribose such as those for sars-cov (pdb code 2fav; z score 27.9; r.m.s. deviation 1.3; sequence identity 45%; sequence similarity 65%) (36), hcov-229e (pdb code 3ewr; z score 22.8; r.m.s. deviation 1.8; sequence identity 33%; sequence similarity 56%) (43), fcov (pdb code 3jzt, z score 22.6; r.m.s. deviation 1.8; sequence identity 30%; sequence similarity 53%) (42) , and infectious bronchitis virus (pdb code 3ewp; z score 19.5; r.m.s. deviation 1.9; sequence identity 28%; sequence similarity 47%) (43) . this finding reflects that the viral macro domains are structurally well conserved. however, variability between all these structures arises from the loops connecting the core secondary structure elements, which display great diversity in sequence, length, and conformation and may correspond to different adp-ribose binding ability. domain-to gain insights into the molecular mechanism of adp-ribose binding, we further investigated the binding pocket for adp-ribose in the mers-cov macro domain. the adenine moiety resides in the hydrophobic cavity containing gly-19, ala-21, ile-47, pro-123, leu-124, and val-152 (fig. 5a) . coordination of adp-ribose involves serial hydrogen bond formations and hydrophobic interactions provided by surrounding amino acid residues (fig. 5b) . the side chain of asp-20 contacts the n-6 atom of the pyrimidine ring in adenine moiety via direct hydrogen bonding. this residue is critical for binding specificity of the macro domain af1521 in a. fulgidus (54) . structure-based multiple sequence alignment showed that this aspartic acid is conserved among cov macro domains (fig. 6a ). oxygen atoms of the pyrophosphate in adp-ribose contact surrounding residues via hydrogen bonding with nitrogen atoms in backbone amides of ile-47, ser-126, gly-128, ile-129, and phe-130. the second ribose is stabilized by complex hydrogen bonding with surrounding residues and water molecules (fig. 5a) . the ribose-3љ oxygen atom forms a hydrogen bond with a nitrogen atom in the side chain of asn-38. the ribose-2љ oxygen atom forms hydrogen bonds with the oxygen and nitrogen atoms in the backbone amides of lys-42 and gly-44, respectively. the ribose-1љ oxygen atom forms a hydrogen bond with the nitrogen atom in the backbone amide of gly-46. a water molecule serves as a bridge between the ribose-1љ oxygen atom, asn-38, and his-43. this organization of the terminal ribose and surrounding molecules was also observed in the yeast adrp enzyme (34) , which suggests that asn-38 and his-43 may be critical for the hydrolysis reaction of adp-ribose 1љ-phosphate to adp-ribose. in addition, equivalent residues critical for adrp activity in the sars-cov macro domain (36) hcov-229e macro domains shows that the major structural divergence lies in the ␣1 helices, which participates in stabilipull-down based binding assay tures. as compared with asp-20 in mers-cov and asp-23 in sars-cov, the equivalent residue in hcov-229e is asp-19, which does not contact adp-ribose. instead of forming a hydrogen bond directly with adp-ribose, the side chain of asp-19 in hcov-229e contacts thr-22 in the ␣1 helix via hydrogen bonding with oxygen and nitrogen atoms in the side chain and backbone of thr-22, respectively. hydrogen bonding with thr-22 drags the side chain of asp-19 in the hcov-229e macro domain away from the adenine cavity as compared with the position of asp-20 in the mers-cov macro domain (fig. 6d) . consistent with the previous study, the thermodynamic profile (⌬g ͻ 0, ⌬h ͻ 0, and ϫt⌬s ͻ 0) of adp-ribose binding to the hcov-229e macro domain suggests less contribution of the hydrogen bond to stabilization of adp-ribose (41) ( table 2 ). variations in strength of the hydrogen bond and orientation of the side chain in asp residues may result in differential binding affinities of adp-ribose observed in macro domains of mers-cov (k d 2.95 m), sars-cov (k d 24 m) (36), and hcov-229e (k d 28.9 m) (41) . the relationship between binding affinities of adp-ribose in macro domains and differential pathogenicity of human covs needs further investigation. conclusion-taken together, our biochemical study shows higher binding affinity for adp-ribose in the mers-cov macro domain than macro domains of covs characterized to date. structural analysis revealed that differences in the context of hydrogen bonds formed by the conserved asp with adpribose and residues in ␣1 helices in macro domains of mers-cov, sars-cov, and hcov-229e may result in differential binding affinities for adp-ribose. our studies provide a biochemical basis for further investigating the role of macro domain in mers-cov infection and also the precise structural information for the design of novel antiviral drugs. severe acute respiratory syndrome (sars): epidemiology and clinical features. postgrad the 2003 sars outbreak and its impact on infection control practices laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece middle east respiratory syndrome coronavirus: update for clinicians middle east respiratory syndrome coronavirus in children south korea scrambles to contain mers virus spread of mers to south korea and china the efficacy and safety of prone positioning in adults patients with acute respiratory distress syndrome: a meta-analysis of randomized controlled trials middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps bats as reservoirs of severe emerging infectious diseases the emerging threat of mers isolation of a novel coronavirus from a man with pneumonia in saudi arabia detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction a novel human coronavirus: middle east respiratory syndrome human coronavirus middle east respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3 crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity structural characterization of the histone variant macroh2a splicing regulates nad metabolite binding to histone macroh2a macroh2a, a core histone containing a large nonhistone region the macro domain is an adp-ribose binding module a biochemical genomics approach for identifying genes by the activity of their products differential activities of cellular and viral macro domain proteins in binding of adp-ribose metabolites splitting of the ribose-ribose linkage of poly(adenosine diphosphate-robose) by a calf thymus extract identification and characterization of a mammalian 39-kda poly(adp-ribose) glycohydrolase the structure and catalytic mechanism of a poly(adp-ribose) glycohydrolase poly adp-ribose glycohydrolase from rat liver nuclei, a novel enzyme degrading the polymer new insights into the molecular and cellular functions of poly(adp-ribose) and parps adp-ribose-1љ-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture identification of protease and adp-ribose 1љ-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3 structural basis of severe acute respiratory syndrome coronavirus adp-ribose-1љ-phosphate dephosphorylation by a conserved domain of nsp3 structure and mechanism of adp-ribose-1љ-monophosphatase (appr-1љ-pase), a ubiquitous cellular processing enzyme a highly specific phosphatase that acts on adp-ribose 1љ-phosphate, a metabolite of trna splicing in saccharomyces cerevisiae structural and functional basis for adp-ribose and poly(adp-ribose) binding by viral macro domains the adp-ribose-1љ-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus 229e mediate resistance to antiviral interferon responses adp-ribose-1љ-phosphatase activities of the human coronavirus 229e and sars coronavirus x domains mouse hepatitis virus liver pathology is dependent on adp-ribose-1љ-phosphatase, a viral function conserved in the ␣-like supergroup the hepatitis e virus orf1 "x-domain" residues form a putative macrodomain protein/appr-1љ-pase catalytic-site, critical for viral rna replication crystal structures of the xdomains of a group-1 and a group-3 coronavirus reveal that adp-ribosebinding may not be a conserved property structure of the x (adrp) domain of nsp3 from feline coronavirus crystal structures of two coronavirus adp-ribose-1љ-monophosphatases and their complexes with adp-ribose: a systematic structural analysis of the viral adrp domain processing of x-ray diffraction data collected in oscillation mode experimental phasing with shelxc/d/e: combining chain tracing with density modification refinement of macromolecular structures by the maximum-likelihood method overview of the ccp4 suite and current developments features and development of coot phenix: a comprehensive python-based system for macromolecular structure solution procheck: a program to check the stereochemical quality of protein structures molprobity: all-atom structure validation for macromolecular crystallography thermodynamics of drug-dna interactions dali server: conservation mapping in 3d the crystal structure of af1521 a protein from archaeoglobus fulgidus with homology to the non-histone domain of macroh2a ligplotϩ: multiple ligandprotein interaction diagrams for drug discovery linking crystallographic model and data quality key: cord-284234-9cd2v6bt authors: sebastian, s; gonzalez, h a; peyrin-biroulet, l title: safety of drugs during previous and current coronavirus pandemics: lessons for ibd date: 2020-06-10 journal: j crohns colitis doi: 10.1093/ecco-jcc/jjaa120 sha: doc_id: 284234 cord_uid: 9cd2v6bt the coronavirus 2019 (covid-19) pandemic has posed challenges in the routine care of patients with inflammatory bowel disease. one of the key challenges needing addressing is the quantification of the risks of immunosuppressive and biologic therapies in ibd patients during the pandemic. the similarities and differences between the previous coronavirus outbreaks and the pathobiology of the infections can give useful information in understanding the risks, and perhaps potential beneficial aspects of drugs used in ibd. although clinical, immunological and pharmacological data from the experience with the previous coronavirus outbreaks cannot be automatically translated to predict the safety of ibd therapies during covid-19 pandemic, the signals so far from these outbreaks on ibd patients who are on immunomodulators and biologics are reassuring to patients and clinicians alike. the 21st century has seen the worldwide spread of three previously unrecognized coronaviruses, the severe acute respiratory syndrome coronavirus (sars-cov-1) and middle east respiratory syndrome coronavirus (mers-cov), and more recently sars -cov-2 ( 1) . starting from 2002 in china, the sars-cov-1 produced, then unprecedented, nosocomial transmission resulting in nearly 9000 deaths across 29 countries (2) . exactly a decade later another coronavirus mers-cov emerged with 2254 laboratory positive cases with at least 800 deaths over 27 countries (3) . the current pandemic sars-cov2 originated in hubei province in china, and was declared a pandemic by world health organisation on march 11 2020 (4). understandable concerns have been raised on the safety of steroids, immunosuppressive drugs, and biologics used in patients for a variety of indications including immune mediated inflammatory disease such as inflammatory bowel diseases (ibd), which do increase the risk of opportunistic bacterial, viral and fungal infections (5) . the magnitude of the infection-risk in general with the therapies used in ibd is small and vary based on the patient, disease, and drug characteristics (6) . equally, another aspect needing consideration if patients discontinue their ibd therapies is the potential risk of ibd flares needing hospitalization which will increase the risk of acquiring sars-cov-2. several consensus guidelines recommend continuation of ibd therapies, primarily with the aim of reducing the risk of a flare needing hospitalization or surgery (7, 8, 9) . two studies recently showed that ibd patients are not at increased risk of being infected with covid-19 (10, 11) . by contrast, there is scarce data so far on the risk of sars-cov-2 infection in ibd patients who are on therapies which potentially alter the immune response to pathogens (12) . therefore, continuing concerns remain both from ibd patients and the a c c e p t e d m a n u s c r i p t clinicians managing them, regarding the potential of ibd related drugs causing more frequent infections by sars-cov2, and increased risk of severe complications from covid-19 (13) . in this review we discuss the immune-pathological aspects of previous and ongoing coronaviruses ( 1 ). the s protein is responsible for viral entry and thus plays a pivotal functional role in viral entry to the host cells, and the non-structural proteins are the key players in viral replication (16) . the sars-cov-2 virus enters cells via the same mechanism as sars-cov, which is by binding of the surface spike glycoprotein (s protein) on the surface of the virus to angiotensin-converting enzyme 2 (ace2), a receptor on the cell surface controlling the cleavage of several peptides. ace2 is expressed in type 2 pneumocytes in the lung, blood vessels, oropharyngeal mucosa, small intestine, colon, and kidneys (17, 18) with remarkably high expression in the epithelial cells of the proximal and distal intestines, and this is also a portal for viral entry (figure 2 ). single cell rna sequencing analysis showed that ace2 expression in colon cells was positively correlated with the regulation of viral infection and congenital cellular immunity and was negatively correlated with viral transcription, protein translation, phagocytosis, and complement activation [19] . recently, garg et al. have described differences in the ace2 between inflamed and non-inflamed biopsies in patients a c c e p t e d m a n u s c r i p t with ibd, but the mucosal expression or activity was not associated with the use of therapies in ibd (20) . furthermore, their study indicated that the level of circulating ace is upregulated in patients with ibd. in addition, a more recent elegant study maria abreu and colleagues (21) reported that the expression of ace2 and tmprss2, which are the entry portals for sars-cov-2, are not increased in inflamed colon and ileum of patients with ibd and some medical therapies are associated with lower levels of ace2. therefore, ace2overall, it is becoming increasingly apparent. that the tug of war between coronaviruses and host antiviral defences are at the core of the pathogenic potential of all coronaviruses determining the clinical course and outcome, and this gives insights on the relative risks of drugs and also opens a window for exploring therapeutic options (34) (figure 3 a c c e p t e d m a n u s c r i p t immunosuppressive drugs and severity of coronavirus infections: opportunity in the face of adversity? the impact of drugs, in particular, steroids, immunosuppressive and biologics were brought in to sharp focus during the three coronavirus pandemics despite the wide variation in the severity of the outbreaks and the mortality rates. these drugs remain the cornerstones of therapies in ibd and have revolutionised ibd care. the lack of prospective data specific to the sars-cov-2 infection to date means that our understanding of the genealogy and immunopathology need to be integrated to the pharmacological aspects of these drugs to shed some light into this challenging conundrum. corticosteroids are thought to have a divergent effect on viral infections including sars cov viruses; on one hand they inhibit host immune response acting on migration and chemokines production leading to impaired viral clearance and the resultant prolonged moreover, a prospective, randomized double-blinded, placebo-controlled trial compared early hydrocortisone treatment (before day seven of the illness) with a placebo and found that early hydrocortisone therapy was associated with a higher subsequent plasma viral load (61) . in the setting of mers-cov, use of corticosteroids for critically ill patients did not improve the 90 corticosteroids also impair the induction of anti-viral type-i interferon responses to a range of respiratory viruses (64) . early corticosteroid use did not benefit critically ill patients with ards but was independently associated with higher mortality in the setting of severe influenza pneumonia (65) . overall, the current data does not support the use of corticosteroids in the coronavirus infections (59) and randomised controlled trials are needed before routine use. there is data suggesting that doses above 20 mg of prednisolone are associated with increased risk of bacterial and viral infections in ibd and even with increased hospitalization suggesting a lower dosing strategy (66, 6760) . in a recent italian series of ibd patients with covid-19, a trend towards adverse outcome with concomitant corticosteroids was reported (68) . in a more recent series from new york, the use of steroids appears to predict covid-19 (or 1.38, 95% ci 1.07-1.77) (69) . locally acting steroids such as budesonide and beclomethasone theoretically have some advantages in relation to side effect profile (70, 71) , but no data is available in the setting of coronaviruses. hence overall, while there is no data a c c e p t e d m a n u s c r i p t on whether current use of steroids increases the risk of severe covid-19, it seems prudent to minimise the use of systemic steroids, think of alternatives to steroids, and if used, taper to the lowest possible dose quickly. broad immunosuppression has the potential to increase susceptibility, persistence, and reactivation of viral infections in patients (72) . acute respiratory viruses implicated in causing severe disease in immunocompromised patients include respiratory syncytial virus (rsv), influenza viruses a and b, parainfluenza viruses and adenovirus, and immunocompromised patients are generally considered at increased risk of influenza and at higher risk of complicated infection (73, 74) . there is also risk of opportunistic viral infection with thiopurines, but these are mainly dna viruses such as hepatitis b and c, epstein barr virus and human papilloma virus. (74) partial assessment regarding whether immunosuppression is a relevant risk factor for covid-19 infection and severe course can be guided by the findings of sars-cov-1 and mers-cov outbreaks (75) . on review of large published cohorts of sars-cov1 and mers-cov infections (76, 77, 78, 79, 80) , the risk factors for both infections included advanced age and presence of one or more co-morbidities such as diabetes, heart disease ,hypertension, lung disease and obesity. although there were no specific studies on immunosuppressed individuals including ibd, no fatality was reported in patients undergoing chemotherapy or other immunosuppressive treatments, at any age (76, 78, 79) . although transplant patients were expected to have poorer outcomes following acquisition of sars cov1, at the end of the outbreak no mortality or graft loss had been recorded (75, 77, 79) a c c e p t e d m a n u s c r i p t atypical presentation of mers-cov was reported from korea in 3 patients on immunosuppressants, all of whom made full recovery without the need for invasive ventilation (81) . in another series of 45 patients with serious mers-cov infection, there was a single patient who was on prolonged immunosuppression who also made an uneventful recovery (78) . in a hospital outbreak of mers-cov in jordan, immunosuppressive individuals did not have additional risks (77) . in an analysis of 1253 cases from the epicentre of mers in saudi arabia from 2012-2015, immunosuppressant use was not associated with increased risk for mers-cov infection (80) . in another study, with a cohort of 114 patients, one of the patients was on immunosuppressants for double transplant (kidney and liver) and, while requiring intensive unit care, did not die (76) . in a retrospective cohort study of host susceptibility in south korean mers outbreak, 3 patients of solid organ transplantation were included but none developed mers-cov infection, but 2 out of the 3 patients with autologous cell transplantation did, without any mortality (79) . in an animal model of mers-cov using immunosuppressed rhesus macaques, despite increased viral replication, pathology in the lungs was significantly lower in immunosuppressed animals (82) . intriguingly some immunosuppressive agents used in treatment, including in ibd, have been found to interfere with viral replication (87) . although the doses used were much higher in comparison to that in treatment of immune disorders such as ibd, thiopurines, when used for haematological malignancies, appeared to be specific inhibitors of sars cov-1 virus (88) . (91, 92, 93, 94) . in a monocentric cross-sectional study of 384 patients, 46 of whom ((12%) were on calcineurin inhibitors alone, the immunosuppression with calcineurin inhibitors did not increase the risk of hospitalisation or mortality (95) . cyclosporine was a c c e p t e d m a n u s c r i p t successfully used in a pregnant patient with acute severe ulcerative colitis with covid-19 (96) . these promising signals, and the underlying pharmacological basis, have prompted some to suggest consideration of calcineurin inhibitors for treatment of coronavirus infections (97, 98) . in a study of rheumatoid arthritis patients on methotrexate with resolved hbv infection, the incidence of hbv reactivation was very low at 1.93/100 person-years (99) . the patients on concomitant methotrexate in ibd so far has not reported any additional risks from sars-cov-2 (11, 68, 86) overall, immunosuppressive therapy does neither seem to have a major impact on infection with sars cov-1, mers-cov and sars-cov-2 nor does it seem to lead to a severe disease course in many cases. however, it must be kept in mind that reported case numbers are exceedingly small overall and continued vigilance is needed. furthermore, anti tnf treatment may diminish the degree of protective immunity resulting from vaccination, but levels achieved appear adequate with other viruses (100). paradoxically, it is plausible that the use of biologics may have a beneficial effect in reducing the inflammatory immune responses following covid-19 by reduction of cytokines, including tnf. tnfα has been implicated in the severe immune-based pulmonary injury caused by sars-cov-, suggesting that tnfα inhibitors could be a potential treatment for the acute respiratory disease syndrome caused by coronavirus (101). coronavirus viral spike protein is able to induce a tnf-α-converting enzyme (tace)-dependent shedding of the ace2 ectodomain, and this process, which appears to be strictly coupled to tnfα, is essential for the penetration of the virus into the cell. this means tnf inhibitors might be effective in blocking viral entry and the detrimental effects of exuberant tnf-α (102). anti tnf agents, as an option for therapeutic modulation, were proposed during the sars-cov-1 outbreak although no human studies were performed (103). following this, in a study on 22 piglets to assess the efficacy of an anti-tnfα) therapy for endotoxin respiratory diseases, the investigators observed that tnfα blockade was not associated with decrease in disease severity(104) subsequently, anti tnf for therapy of viral infections caused by respiratory syncytial virus or influenza virus was studied again in animal models and showed that tnf however, it is important to recognise that it is equally plausible that theoretical possibility of increased viral burden resulting from inability of immune response may result in increases severity of inflammation (107) . hence some concerns have been raised by some authors in relation to broad immunosuppression in the presence of an overwhelming infective illness (108) . nevertheless, there is no evidence indicating that tnfα blockade is harmful to patients in the context of severe infections including septic shock (109) . a randomised controlled trial of anti tnf agents with septic shock in intensive care units showed no evidence of increased infections in the anti tnf treated patients (110) . no patients on anti tnf agents have been recorded in any of the series reported during the sars-cov-1 or mers outbreaks, but there is an increasing amount of literature suggesting that tnfα blockade is not harmful to patients in the context of covid-19 (111, 112, 113) . these opinions are supported by the case series of patients on anti tnfs for immune mediated inflammatory disorders such as arthritis and inflammatory bowel diseases (10, 11, 68, 86, 114) . in a report of 320 rheumatology patients on disease modifying drugs, half of whom were on anti-tnf agents at the height of the pandemic in northern italy, monti et al however, the hospitalisation rates, intensive care treatment and mortality appears to be higher in the 106 patients who are on combination therapy with anti tnf and immunomodulators (36%, 5% and 3% respectively), and therefore further data is required. vedolizumab selectively inhibits the interaction of α4β7 with mucosal adhesion molecule -1 preventing the entry of t lymphocytes across the endothelium to the inflamed gastrointestinal mucosa (116) . the receptors are present in gi tract, nasopharyngeal mucosa, and biliary epithelium. the gut-selective mode of action of vedolizumab should theoretically be associated with a lower risk of infections. no increase in viral infections including nasopharyngitis was noted in vedolizumab treated patients in a meta-analysis (117) . higher, but statistically insignificant, rates of enteric infections occurred in vedolizumab-exposed patients (7.4/100 pys; 95% ci: 6.6-8.3) to placebo (6.7 pys; 95% ci: 3.2-10.1) in this metaanalysis (117) . no reactivation of hepatitis b or c was noted in 29 patients with history of hepatitis b or c in a post marketing surveillance study of the licencing trials (118) . in sivof these patients required hospitalisation (10, 11, 86) . the ig-ibd study included 15 patients on vedolizumab, five of whom needed admission but reported no association with risk for covid-19 pneumonia (68) . the secure ibd registry (accessed on 15 th may 2020) has included data from 107 patients on vedolizumab with a 29% hospitalisation rate; (6% in icu) and 4% mortality (115). therapies targeting jaks may interfere with normal anti-viral response including inhibition of ifn-γ activity (121) , and may potentially increase the risk of infection and/or reactivation of several viral infectious diseases including a dose dependent risk for vzv observed for tofacitinib (122). in some studies the use of higher dose of tofacitinib along with corticosteroids was associated with serious infections (123) . jak inhibitors also have anti-viral potential since they lower the pro-inflammatory response mediated by viruses and block many pro-inflammatory cytokines involved in cytokine storm such as il1-, il6, il-8 and tnf (1124). of relevance would be the il-6 or il6_r blockade with jaks or specific anti il-6r antibody tocilizumab (125) . baricitinib, currently not used in ibd but approved for rheumatoid arthritis, blocks viral endocytosis and assembly of virus particles into pneumocytes, and has shown promising results in clinical trials in sars-cov-2 (126, 127) . this potential beneficial effect is not seen with tofacitinib. fedratinib, a selective a c c e p t e d m a n u s c r i p t jak2 inhibitor which inhibits th17 mediated immune hyperstimulation, is also proposed for treatment of severe covid-19 infection (128) . the new york series on imids had 4 patients on tofacitinib, one among them needing hospitalisation but not ventilation (86) . two patients on tofacitinib with covid-19 have been reported in rheumatology literature, both without severe outcomes (114) . in a case report from washington (129), a young patient on tofacitinib continued the treatment uninterrupted following diagnosis of covid-19 and had complete recovery without the need for hospitalisation. seventeen patients on vedolizumab have been reported so far to the secure ibd registry (115), five of whom needed hospitalisation, with one mortality (accessed on 15 th may 2020). hypothetically, blocking il12 and il23 which are involved in cytokine storm may have a beneficial effect in ameliorating the cytokine storm in covid-19 (130, 131) . ustekinumab currently there is no data to indicate that therapies used in ibd will result in more severe outcomes in patients. whether drug-induced immunosuppression will prevent the cytokine storm in patients infected with covid-19 will require further investigation. until we have more data, a risk versus benefit grid based approach ( table 2) covid-19, sars and mers: are they closely related? who guidelines for the global surveillance of severe acute respiratory syndrome (sars) the global burden of premature mortality due to the middle east respiratory syndrome (mers) using standard expected years of life lost comparative risk of serious infections with biologic and/or immunosuppressive therapy in patients with inflammatory bowel diseases: a systematic review and meta-analysis second european evidence-based consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease on behalf of the international organization for the study of inflammatory bowel disease, management of patients with crohn's disease and ulcerative colitis during the covid-19 pandemic: results of an international meeting british society of gastroenterology guidance for management of inflammatory bowel disease during the covid-19 pandemic raga clinical practice update on management of inflammatory bowel disease during the covid-19 pandemic: expert commentary incidence and patterns of covid-19 among inflammatory bowel disease patients from the nancy and milan cohorts novel corona virus disease (covid-19) in patients with inflmaamatory owel disease the risk of sars-cov-2 in immunosuppressed ibd patients. crohn's & on behalf of the ecco-covid task force. inflammatory bowel disease management during the covid-19 outbreak: a survey from the european colitis and crohns organization (ecco) emerging coronaviruses: genome structure, replication, and pathogenesis structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-ncov) and sars coronavirus(sars-cov) characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis the single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to wuhan 2019-ncov infection. fronts of medicine ace2 expression by colonic epithelial cells is associated with viral infection, immunity and energy metabolism imbalance of the renin-angiotensin system may contribute to inflammation and fibrosis in ibd: a novel therapeutic target? expression of sars-cov-2 entry molecules ace2 and tmprss2 in the gut of patients with ibd the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis impact of corticosteroid therapy on outcomes of persons with sars-cov-2, sars-cov, or mers-cov infection: a systematic review and meta-analysis use of corticosteroids in coronavirus disease corticosteroids as adjunctive therapy in the treatment of influenza the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and meta-analysis clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury effect of different doses and time-courses of corticosteroid treatment in patients with acute respiratory distress syndrome: a metaanalysis effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients systemic corticosteroid therapy may delay viral clearance in patients with middle east respiratory syndrome coronavirus infection persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients early corticosteroids in severe influenza a/h1n1 pneumonia and acute respiratory distress syndrome corticosteroid suppression of antiviral immunity increases bacterial loads and mucus production in copd exacerbations the historical role and contemporary use of corticosteroids in inflammatory bowel disease increased risk of influenza and influenza-related complications among 140,480 patients with inflammatory bowel disease inflammatory bowel disease outcomes of covid-19 in 79 patients with ibd in italy: an ig-ibd study covid-19 in immune-mediated inflammatory diseases -case series from new york comparative safety of systemic and lowbioavailability steroids in inflammatory bowel disease: systematic review and network metaanalysis efficacy and safety of oral beclomethasone dipropionate in ulcerative colitis: a systematic review and meta-analysis the natural history of influenza infection in the severely immunocompromised vs non-immunocompromised hosts increased incidence of systemic serious viral infections in patients with inflammatory bowel disease associates with active disease and use of thiopurines risk of serious and opportunistic infections associated with treatment of inflammatory bowel diseases coronaviruses and immunosuppressed patients. the facts during the third epidemic clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study host susceptibility to mers-cov infection, a retrospective cohort study of the 2015 korean mers outbreak outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study atypical presentations of mers-cov infection in immunocompromised hosts pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques clinical characteristics of coronavirus disease 2019 in china coronaviruses and immunosuppressed patients. the facts the third epidemic case report of covid-19 in a kidney transplant recipient: does immunosuppression alter the clinical presentation? characteristics and prognosis of patients with inflammatory bowel disease during the sars-cov-2 pandemic in the basque country (spain) network-based drug repurposing for noval coronavirus 1019-ncov19 sars-cov-2 human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors influences of cyclosporin a and non immunosuppressive derivatives on cellular cyclophilins and viral nucleocapsid protein during human coronavirus 229e replication cyclosporin a inhibits the replication of diverse coronaviruses replication of human coronaviruses sars-cov, hcov-nl63 and hcov-229e is inhibited by the drug fk506 clinical presentation and evolution of covid-19 in immunosuppressed patients. preliminary evaluation in a north italian cohort on calcineurin-inhibitors based therapy management of acute severe ulcerative colitis in a pregnant woman with covid-19 infection: a case report and review of the literature cyclosporine has a potential role in the treatment of sars cyclosporine therapy during the covid-19 pandemic is not a reason for concern incidence of hepatitis b virus reactivation in patients with resolved infection on immunosuppressive therapy for rheumatic disease: a multicentre, prospective, observational study in japan high viral load and respiratory failure in adults hospitalized for respiratory syncytial virus infections immunosuppression for hyperinflammation in covid-19: a double edged sword? anti-tnf-alpha therapy for patients with sepsis: a systematic meta-analysis intersept: an international, multicenter, placebo-controlled trial of monoclonal antibody to human tumor necrosis factor-alpha in patients with sepsis. international sepsis trial study group implications of covid-19 outbreak on immune therapies in multiple sclerosis patients-lessons learned from sars and mers recommendations for coronavirus infection in rheumatic diseases treated with biologic therapy clinical course of covid-19 in a series of patients with chronic arthritis treated with immunosuppressive targeted therapies under research exclusion an overview of the mechanism of action of the systematic review: the safety of vedolizumab for the treatment of inflammatory bowel disease low frequency of opportunistic infections in patients receiving vedolizumab in clinical trials and post-marketing setting sustained virologic control in siv+ macaques after antiretroviral and 4 7 antibody therapy an open-label phase 1 clinical trial of the anti-α4β7 monoclonal antibody vedolizumab in hiv-infected individuals jak inhibitors for the treatment of autoimmune and inflammatory diseases herpes zoster in patients receiving jak inhibitors for ulcerative colitis: mechanism, epidemiology, management, and prevention real-world experience with tofacitinib in ibd at a tertiary center jak-inhibitor tofacitinib suppresses interferon alfa production by plasmacytoid dendritic cells and inhibits arthrogenic and antiviral effects of interferon alfa the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab my be the key to reduce the mortality baricitinib as potential treatment for 2019-ncov acute respiratory disease baricitinib as potential treatment for 2019-ncov acute respiratory disease th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib case report of a sars-cov -2 infection in a patient with ulcerative colitis on tofacitnib il-12, il-23 and il-17 in ibd: immunobiology and therapeutic targeting covid-19 and immunomodulation in ibd a c c e p t e d m a n u s c r i p t 100.cullen g, bader c, korzenik a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-271681-jmoyy8rb authors: assiri, abdullah m.; midgley, claire m.; abedi, glen r.; saeed, abdulaziz bin; almasri, malak m.; lu, xiaoyan; al-abdely, hail m.; abdalla, osman; mohammed, mutaz; algarni, homoud s.; alhakeem, raafat f.; sakthivel, senthilkumar k.; nooh, randa; alshayab, zainab; alessa, mohammad; srinivasamoorthy, ganesh; alqahtani, saeed yahya; kheyami, ali; hajomar, waleed husein; banaser, talib m.; esmaeel, ahmad; hall, aron j.; curns, aaron t.; tamin, azaibi; alsharef, ali abraheem; erdman, dean; watson, john t.; gerber, susan i. title: epidemiology of a novel recombinant middle east respiratory syndrome coronavirus in humans in saudi arabia date: 2016-06-14 journal: journal of infectious diseases doi: 10.1093/infdis/jiw236 sha: doc_id: 271681 cord_uid: jmoyy8rb background: middle east respiratory syndrome coronavirus (mers-cov) causes severe respiratory illness in humans. fundamental questions about circulating viruses and transmission routes remain. methods: we assessed routinely collected epidemiologic data for mers-cov cases reported in saudi arabia during 1 january– 30 june 2015 and conducted a more detailed investigation of cases reported during february 2015. available respiratory specimens were obtained for sequencing. results: during the study period, 216 mers-cov cases were reported. full genome (n = 17) or spike gene sequences (n = 82) were obtained from 99 individuals. most sequences (72 of 99 [73%]) formed a discrete, novel recombinant subclade (nrc-2015), which was detected in 6 regions and became predominant by june 2015. no clinical differences were noted between clades. among 87 cases reported during february 2015, 13 had no recognized risks for secondary acquisition; 12 of these 13 also denied camel contact. most viruses (8 of 9) from these 13 individuals belonged to nrc-2015. discussions: our findings document the spread and eventual predominance of nrc-2015 in humans in saudi arabia during the first half of 2015. our identification of cases without recognized risk factors but with similar virus sequences indicates the need for better understanding of risk factors for mers-cov transmission. middle east respiratory syndrome coronavirus (mers-cov) is known to cause severe respiratory illness in humans, with deaths recorded in 35%-40% of cases reported globally [1] . since the first recognition of mers in 2012, all cases reported to the world health organization have been linked to the arabian peninsula, with >85% of cases reported from saudi arabia [1] . camels (camelus dromedarius) have been suspected as a reservoir for mers-cov, based on case investigations [2] , serologic studies [3] , and the isolation of virus from camels [4] [5] [6] [7] [8] . direct camel contact has also been identified as a risk factor for human illness [9] . secondary human transmission has been demonstrated among close contacts of symptomatic cases, primarily following healthcare-associated exposures [10] [11] [12] and, to a lesser degree, household exposures [13] . there is no definitive evidence of sustained human-to-human transmission in the community [14] . mers-cov infection can exhibit a wide range of clinical manifestations, including mild or limited symptoms among those identified through contact tracing [11] . prolonged viral shedding from the respiratory tract of those without obvious symptoms has been demonstrated [15] , and transmission related to unrecognized cases has been suggested [12, 16] but not documented. mers-cov sequences obtained to date suggest periodic introductions of the virus into human populations, presumably from an animal reservoir, with subsequent limited chains of transmission in households and healthcare settings. the temporal persistence of identified viral clades appears limited, consistent with an r 0 of <1 [17, 18] . intervals between the beginning and end of the circulation of a clade vary, with longer intervals suggesting the existence of undetected human cases [19] . cases and clusters continue to be reported from countries in or near the arabian peninsula, presenting an ongoing threat for broader transmission [20] . to assess the epidemiologic and clinical features of the disease, we investigated all cases reported by the saudi arabia ministry of health (moh) during january-june 2015, and we attempted genetic sequencing on all available specimens. this investigation was part of an emergency public health response and was determined to be nonresearch by the moh and centers for disease control and prevention (cdc) and therefore not subject to institutional review board review. at the time of this investigation, reporting in saudi arabia was required for all patients with clinical or radiologic evidence of mers-cov infection and a positive real-time reverse transcription-polymerase chain reaction (rrt-pcr) test result [21] . all rrt-pcr-positive cases identified at non-moh facilities required confirmation at moh laboratories. we assessed the routinely collected epidemiologic information for all mers-cov cases reported by the moh during 1 january-30 june 2015, to provide a basic epidemiologic description. for this analysis, we included only individuals who met the case definition described above (ie, symptomatic cases). february 2015 was a period of increased reporting. to perform a more in-depth analysis, we collected additional information for all individuals with laboratory-confirmed mers-cov infections during february 2015. this included all cases meeting the case definition as described above, as well as those identified as having a laboratory-confirmed case but no recognized symptoms; individuals not meeting the case definition [21] were typically identified through contact tracing. we reviewed available moh case investigation records and data reported through the moh health electronic surveillance network. we collected demographic information, medical history, outcome information, and treatment location. we assessed the likelihood of acquisition from another person (secondary acquisition) by determining whether a patient (1) was a healthcare professional (hcp), (2) had been admitted to a healthcare facility 2-14 days before illness onset, (3) had visited any healthcare facility in the 14 days before illness onset, or (4) had direct contact with either another documented case of mers-cov infection or with someone with an acute respiratory illness of unknown cause in the 14 days prior to illness. when it was not possible to determine the criteria described above by using available information, we conducted telephone interviews (in arabic) to collect additional exposure information. proxies (a close friend or immediate family member who was familiar with the patient's activities during this period) were interviewed if the case was deceased, still hospitalized, or too ill to participate. among cases without any of the aforementioned risk factors for secondary acquisition (hereafter referred to as sporadic cases), we asked during telephone interviews about the history of exposure to camels [9] . interviewees were prompted to describe examples of camel exposures, including direct contact or visiting a live market, slaughterhouse, or race where camels were present. we also assessed travel history. demographic and clinical characteristics were reported, and differences were assessed for significance by using χ 2 , wilcoxon rank sum, and kruskal-wallis tests, where appropriate. data were analyzed using sas, version 9.3 (sas institute, cary, north carolina). molecular testing was performed on all respiratory specimens available during january-june 2015. specimens and molecular testing at the moh respiratory specimens, including nasopharyngeal and oral pharyngeal swabs, both separate and combined, nasopharyngeal and tracheal aspirates, and sputa collected from suspected mers cases were tested at moh laboratories by upe and orf1a rrt-pcr assays [22] . available specimen aliquots (or rna extracts) that tested positive for mers-cov by both assays were shipped on dry ice to the cdc (atlanta, georgia) for sequencing. sample aliquots (200-300 µl, if available) were extracted on a nuclisens easymag (biomerieux), and 100 µl of total nucleic acid elutes were recovered. the specimen extract were retested by mers-cov n2 and/or n3 rrt-pcr assays [23] , and sequencing was attempted on confirmed positive samples. overlapping nested primer sets were used for amplification and sanger sequencing of the mers-cov spike genes and selected genomes (supplementary table 1 ). amplicon sequencing was performed in both directions, using sequencing and internal amplification primers, with the bigdye terminator v3.1 cycle sequencing kit on a 3730xl genetic analyzer (thermo fisher scientific). sequencher 5.3 software (gene codes) was used for sequence assembly and editing. nucleotide sequences were aligned using clustal x, version 1.83, implemented in bioedit, version 7.2.5. phylogenies were estimated using neighbor-joining and maximum likelihood methods implemented in molecular evolutionary genetics analysis, version 6.0621 [24] , and bayesian inference, using mrbayes v3.2.6 [25] . the neighbor-joining method used maximum composite likelihood distance estimation and maximum likelihood used general time reversible (gtr) model of nucleotide substitution with γ-distributed rate variation and a proportion of invariant sites (gtr + g + i). mrbayes was performed under a gtr model of nucleotide substitution with 4 categories of γ-distributed rate heterogeneity and a proportion of invariant sites (gtr + 4 + i). putative recombination events were identified using recombination detection program software, version 4.70 (rdp4; available at: http://web.cbio.uct.ac.za/~darren/rdp.html), with the default settings [26] . the complete genome sequence of each of the viruses in the nrc-2015 clade was aligned with the genomes outside the clade. the multiple sequence alignment was then imported into the rdp software for detection of recombination. the software uses several algorithms, including gene-conv, bootscan, maxchi, chimaera, siscan, and 3seq, to detect putative recombination events. the potential minor and major parental sequences and the beginning and end breakpoints of the potential recombinant sequences were also defined by rdp4 software. putative recombinant events were considered significant when a p value of ≤ .05 was observed for the same event, using ≥4 algorithms. time estimates to the most recent ancestor were calculated using the bayesian markov chain monte carlo (mcmc) method implemented in beast v1.8.2 [27] . the coding regions (orf1ab, s, orf3, orf4a, orf4b, orf5, e, m, and n) in the genomes grouping within nrc-2015 were concatenated, and the hky+ γ 4 substitution model was used with independent rates for each of the positions in the codon. a lognormal relaxed molecular clock (uncorrelated) was used with gaussian markov random field bayesian skyride coalescent. bayesian mcmc analysis was run for 25 million steps. parameters for tmrca, rate, and trees were sampled every 5000 steps, with the first 10% removed as burn-in. time estimate values thus obtained were also compared with strict and exponential relaxed clock models. during 1 january-30 june 2015, 216 mers-cov cases from 10 of the 13 regions of saudi arabia were reported by the moh; mers-cov-positive individuals with no recognized symptoms, and who therefore did not meet the case definition, were not included. the longest period between case reports was 11 days. among these 216 cases, 214 were hospitalized, and 102 (47%) died. most patients were male (161 [75%]) and of saudi nationality (147 [68%]). median age was 56 years (range, 20-93 years). of the 216 symptomatic cases reported during the study period, 124 had respiratory specimens available for further testing at the cdc; 1 specimen was also available from an individual with no recognized symptoms who did not meet the case definition. of the 125 available respiratory specimens collected during 6 january-3 june 2015, spike gene sequences were obtained from 99 (supplementary table 2 table 3 ). recombination analysis on the newly available genome sequences from nrc-2015 identified 2 possible recombination events involving sequences from outside the clade as potential minor and major parental strains. the first event had a predicted breakpoint at nucleotide position 17 475 (99% confidence interval [ci], 13 502-19 074), located in orf1ab, and the second event had a predicted breakpoint at 23 976 (99% ci, 23 571-24 862), located in the spike gene. recombination analysis was performed using rdp software, and events detected with a p value of ≤ .05 were considered evidence of true recombination (supplementary table 4 ). to date the emergence of nrc-2015, mcmc analysis was performed on the concatenated coding regions of the genomes grouping within nrc-2015, using beast. the most recent common ancestor of the virus was approximately 0.85 years table 5 ). among the 216 cases reported during 1 january-30 june 2015, nrc-2015 was first detected in a case with onset in mid-january 2015 (figure 2a) . during the study period, nrc-2015 viruses were detected in 6 regions of saudi arabia (figure 3) , and the proportion of patients identified with nrc-2015 increased steadily over time ( figure 2b ). nrc-2015 was next compared to past and present subclades within clade b, using sequences available in genbank (figure 4 ). nrc-2015 was more widely distributed geographically than any other identified members of clade b. the duration of circulation of recognized subclades ranged from 16 to 665 days. at the conclusion of our investigation period, nrc-2015 had been circulating for 135 days, which was longer than 7 of 9 other identified subclades. in our analysis, the longest circulating subclade reported was riyadh_kkuh-1_2014, which was first detected in july 2013 and was still circulating as of may 2015. during our investigation period, 10 of 99 sequenced viruses belonged to riyadh_kkuh-1_2014. no viruses belonging to clade a were detected. a comparison of patients infected with nrc-2015 versus other circulating viruses revealed no significant differences in age, sex, rate of mortality, time between onset of symptoms table 6 ). there was also no difference in mean cycle threshold values, a proxy for virus load, with respiratory specimens containing nrc-2015 versus other clades, although these were not adjusted for timing of specimen collection (supplementary table 6 ). for our more detailed analysis of cases reported during 1-28 february 2015, we identified 87 mers-cov-positive patients ( table 1 ). of these, 77 patients (89%) satisfied the case definition for routine reporting and required hospitalization; the remaining 10 individuals (11%) had no recognized symptoms (and did not satisfy the case definition) but are included in this analysis. the 87 patients were reported from 35 different healthcare facilities across 7 regions in saudi arabia; 17 of these facilities reported ≥2 cases within the same 14-day period. of these 87 patients, sequences could be obtained from 34, of which 24 (71%) were associated with nrc-2015. no clinical differences were apparent when comparing nrc-2015 to other circulating viruses ( table 1) . the 87 patients with laboratory-confirmed disease reported during february were also classified according to their reported exposures during the 2 weeks before illness onset. record review and interviews were conducted during 11-25 march 2015. among the 87 cases, 51 were classifiable using information obtained by the initial case investigation. interviews were attempted for the remaining 36 patients. of these, 28 (78%) were interviewed; 1 individual refused to participate, and 7 patients were not available. proxy interviews were conducted for 22 of 28 interviews, including for 18 patients who were deceased and for 4 of 10 patients who survived. among the 87 patients, 13 (15%) were determined to have had household contact with a confirmed mers-cov case, 14 (16%) were hcps, 21 (24%) were inpatients in a healthcare facility, 16 (18%) were hospital visitors, and 10 (11%) were unable to be classified owing to a lack of available information (table 1) . notably, 13 patients (15%) denied exposure to a healthcare facility or to a person with acute respiratory illness in the 2 weeks before illness onset and were classified as sporadic cases (tables 1 and 2); among these, 1 individual reported visiting a camel farm in the 2 weeks before illness onset. among the 13 sporadic cases, 2 were available for interview, and 11 were interviewed by proxy. among the 11 interviewed by proxy, 9 were deceased and 2 were too ill to participate in the interview. sequences were obtained for 9 sporadic cases, and 8 (89%) were nrc-2015, including the individual who had visited the camel farm. in the republic of korea [31, 32] , thailand (accession number kt225476), and china in 2015 [31, 33] . previous documentation of the duration of circulation in humans of 4 different mers-cov clades in saudi arabia during 2012-2013 noted an average detection time of 98 days [19] . in contrast, we demonstrate that nrc-2015 has persisted longer than most previously documented clades. nrc-2015 was found to eventually predominate over the 6-month study period and attain a wide geographic distribution in a comparatively short period. while this apparent emergence and clade displacement is suggestive of greater epidemiologic fitness [34] , we observed no clinical differences between nrc-2015 and other clades; the implications for virus replication and transmission need further study. during preparation of this manuscript, sequences obtained from camels in oman in may 2015 [35] and saudi arabia during july 2014-april 2015 [36] were reported that showed similar recombination features and phylogenetic association with nrc-2015. in camels, nrc-2015 (referred to as lineage 5 [36] ) was first detected in july 2014 and became predominant in saudi arabia during a period that overlaps with our study, corroborating our findings of an increased prevalence in humans relative to other clades. recombination has been documented among covs [37] and has been linked to the emergence of more-pathogenic strains of some animal covs [38] [39] [40] . evidence of intraspecies recombination has also been found with the human covs hku1 [41] , nl63 [42] , oc43 [43] , and, more recently, mers-cov [44] . genome analysis of human mers-cov strains from saudi arabia in 2015 and the recent outbreak in south korea/china [31] [32] [33] and camels as noted above [35, 36] revealed a probable signature recombination event between 2 different parental clade b viruses involving a region of the orf1ab and spike genes. we confirmed this finding and documented an increasing prevalence of this virus in humans among samples collected since january 2015 from geographically distant communities in saudi arabia. similar to recent reports [33] , we estimate that this recombinant virus emerged sometime in mid-to-late 2014. based on recently available sequence data from camels in saudi arabia, nrc-2015 (lineage 5) was predicted to have diverged between december 2013 and june 2014 [36] . in our study, further analysis of 87 patients with laboratoryconfirmed mers-cov reported in february revealed 13 individuals with no recognized risks for secondary acquisition; none of these 13 reported direct camel contact, although 1 individual reported visiting a camel farm. of those sequenced, most were infected with genetically very similar viruses, suggesting a potential for limited transmission from those with unrecognized mers-cov infection. these findings highlight the importance of strengthened epidemiologic and laboratory surveillance. most cases identified in saudi arabia in february had documented exposure to healthcare facilities, a well-demonstrated risk factor for mers-cov infection [10] [11] [12] . seventeen of 35 affected facilities in saudi arabia in february experienced mers-cov infection clusters. moreover, 16 of 87 patients in february (18%) were visitors to healthcare facilities. this is similar to the 2014 jeddah outbreak, where 17% of investigated cases were visitors [11] . recommendations to limit visitation in facilities with ongoing mers-cov transmission should be reinforced to limit these exposures. our investigation, which was performed as part of an emergency public health response, is subject to several limitations. first, specimens were not available for all cases during the study period, meaning that many viruses remained untyped; however, we observed no demographic differences between cases who had specimens sequenced and those who did not. second, since its emergence in 2012, surveillance and sequencing of mers-cov strains has been incomplete; variations in sequence availability and documentation might have influenced the extent of persistence and geographic spread that we have determined for past circulating virus strains. case definitions, testing practices, and testing locations have also changed during this period. third, although we were able to obtain full genome sequences from 12 nrc-2015 samples, all of which possessed the expected recombinant signal, our sequencing was mostly limited to the spike gene alone, which poses the risk of misclassifying recombinant viruses as belonging to nrc-2015. this is illustrated in the recent study by sabir et al [36] , which reported multiple novel recombinant viruses in camels, including recombinants between nrc-2015 (lineage 5) and other virus clades. fourth, because of the high morbidity and mortality of mers-cov infection, interviews with cases were not always possible, necessitating the use of proxies. it is possible that, combined with issues of recall, the quality of the information collected varied. of particular consideration, 11 of 13 sporadic cases were classified on the basis of interviews with proxies, and pre-illness exposures might not have been accurately recognized and reported. fifth, some camel exposures may have gone unrecognized because of disincentives for reporting camel exposures, given their cultural and economic significance in saudi arabia. sixth, given the existing evidence of association between mers-cov illness and pre-illness healthcare exposure or exposure to sick individuals [10, 11, 13] , our risk classification was hierarchical; that is, reported exposure to a setting where secondary acquisition was likely took precedence over reported exposure to camels. as such, we did not assess camel exposures in individuals with recognized risks for secondary acquisition. finally, although we have attempted to link the results of our epidemiologic investigation with mers-cov sequences obtained from investigated cases, we cannot fully assess the possible role of virus introductions from nonhuman sources. recent phylogeny of mers-cov sequences from camels in saudi arabia indicated that the novel recombinant subclade (referred to as nrc-2015 in our manuscript) was also predominant in camels during a period overlapping with our study [36] . as such, our detection of closely related viruses in humans might in part reflect multiple introductions from camels with similar strains. virus introductions from other currently unidentified sources might also be factor. virus transmission dynamics within and between human and nonhuman sources of mers-cov will likely influence transmission routes in ways not yet fully understood. this investigation describes the emergence, persistence, and widespread circulation of a novel recombinant mers-cov in saudi arabia. a lack of clearly defined epidemiologic links in some cases highlights the need for ongoing intensive epidemiologic and laboratory surveillance to better understand mers-cov transmission and to focus infection prevention and control efforts. supplementary materials are available at http://jid.oxfordjournals.org. consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. potential conflicts of interest. all authors: no reported conflicts. all world health organization. summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel mers coronaviruses in dromedary camels human infection with mers coronavirus after exposure to infected camels, saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia family cluster of middle east respiratory syndrome coronavirus infections world health organization. who statement on the ninth meeting of the ihr emergency committee regarding mers-cov a case of long-term excretion and subclinical infection with mers-coronavirus in a health care worker a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus world health organization. who statement on the tenth meeting of the ihr emergency committee regarding mers case definition and management of patients with mers coronavirus in saudi arabia assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus mega6: molecular evolutionary genetics analysis version 6.0 mrbayes: bayesian inference of phylogenetic trees rdp4: detection and analysis of recombination patterns in virus genomes bayesian phylogenetics with beauti and the beast 1.7 preliminary analysis of middle east respiratory syndrome coronavirus (mers-cov) sequences from korea and china evolution patterns of the middle east respiratory syndrome coronavirus (mers-cov) obtained from mers an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china complete genome sequence of middle east respiratory syndrome coronavirus kor/knih/002_05_2015, isolated in south korea origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis viral fitness: definitions, measurement, and current insights asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses emergence of a group 3 coronavirus through recombination importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in china coronavirus diversity, phylogeny and interspecies jumping genomic analysis of 16 colorado human nl63 coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene and evidence of recombination molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination mers-cov recombination: implications about the reservoir and potential for adaptation acknowledgments. we thank shifaq kamili, for logistics of specimen shipment; and laura wright of the centers for disease control and prevention (cdc) geospatial research analysis and services program.disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc.financial support. this work was supported by the saudi arabia ministry of health and the cdc as part of an emergency response. in the 2 weeks before becoming ill, none had known contact with a patient with mers, a person with severe respiratory illness, and anyone mildly ill, and none reported working in or visiting a healthcare facility.abbreviations: g, full genome sequence obtained; s, spike region sequence obtained; ns, not sequenced. a genbank accession number. key: cord-256750-5m7psxri authors: park, hye yoon; park, wan beom; lee, so hee; kim, jeong lan; lee, jung jae; lee, haewoo; shin, hyoung-shik title: posttraumatic stress disorder and depression of survivors 12 months after the outbreak of middle east respiratory syndrome in south korea date: 2020-05-15 journal: bmc public health doi: 10.1186/s12889-020-08726-1 sha: doc_id: 256750 cord_uid: 5m7psxri background: the 2015 outbreak of middle east respiratory syndrome (mers) in the republic of korea is a recent and representative occurrence of nationwide outbreaks of emerging infectious diseases (eids). in addition to physical symptoms, posttraumatic stress disorder (ptsd) and depression are common following outbreaks of eid. methods: the present study investigated the long-term mental health outcomes and related risk factors in survivors of mers. a prospective nationwide cohort study was conducted 12 months after the mers outbreak at multi-centers throughout korea. ptsd and depression as the main mental health outcomes were assessed with the impact of event scale-revised korean version (ies-r-k) and the patient health questionnaire-9 (phq-9) respectively. results: 42.9% of survivors reported ptsd (ies-r-k ≥ 25) and 27.0% reported depression (phq-9 ≥ 10) at 12 months post-mers. a multivariate analysis revealed that anxiety (adjusted odds ratio [aor], 5.76; 95%ci, 1.29–25.58; p = 0.021), and a greater recognition of stigma (aor, 11.09, 95%ci, 2.28–53.90; p = 0.003) during the mers-affected period were independent predictors of ptsd at 12 months after the mers outbreak. having a family member who died from mers predicted the development of depression (aor, 12.08, 95%ci, 1.47–99.19; p = 0.020). conclusion: this finding implies that psychosocial factors, particularly during the outbreak phase, influenced the mental health of patients over a long-term period. mental health support among the infected subjects and efforts to reduce stigma may improve recovery from psychological distress in an eid outbreak. the 2015 outbreak of the middle east respiratory syndrome coronavirus (mers-cov) in the republic of korea had an enormous impact on medical, psychological, and social issues nationwide [1] . the outbreak lasted from may 2015 to dec. 2015 and resulted in 186 infected patients within the initial 2 months, 16,693 officially isolated individuals, and an overall mortality of 38 patients in a total of 50 million population [2] . acute infectious outbreaks of emerging infectious diseases (eids) are known to influence the physical as well as the mental health of affected patients, as observed during similar events such as the severe acute respiratory syndrome (sars) outbreak [3] , which was associated with such issues during the acute phase [4] and the long-term follow-up phase [5, 6] . 35% of 425 survivors expressed anxiety or depressive symptoms at 1-month post-sars in hong kong where 1755 citizens were infected, and its fatality was 17.0% [4] . in ninety survivors' cohort study for 30 months in hong kong, post-sars cumulative incidence of psychiatric disorders was 58.9%. the most common diagnoses were ptsd (25.6%) and depression (13.3%) [5] . few studies have investigated the psychological impact of the 2015 korean mers-cov outbreak, but a survey conducted during this period found that 90% of the general public reported worrying about being infected by mers-cov and that 46% of this population experienced psychological distress [7] . another study reported that 7.6% of 1656 isolated individuals exhibited anxiety symptoms and that 16.6% of this group reported feelings of anger during the isolation period [8] . in contrast, anxiety was present in 47.2% of mers patients [8] , which was more prevalent than the rate of anxiety in isolated people without the mers-cov infection. compared to patients with other diseases, those with eids may experience greater suffering in terms of the physical and psychiatric symptoms of the infectious illness itself [9] ; extreme fear and anxiety due to their unfamiliarity with the disease, which may be lifethreatening [10] ; abrupt isolation from family and society during the illness [8] ; stigma due to the infectious disease [11] ; the unexpected death of a family member; and/or social impairments [12] . given that some factors, such as grief or stigma, may be persistent following the mers illness, the suffering of afflicted individuals may negatively influence recovery in their daily lives during the acute outbreak period as well as the post-mers period. studies of sars survivors in hong kong and china reported persistent psychological burdens, including post-traumatic stress disorder (ptsd), in over 40% of the survivors after 3 years [13] . however, no studies have investigated the mental health status of mers survivors. thus, the present study explored mental health issues and related factors in mers survivors 12 months after the outbreak to determine the long-term psychological outcomes of this population. the present study was part of a prospective nationwide cohort study of mers survivors conducted at multicenters in the republic of korea. for purposes of this study, a mers survivor was defined as a patient who was diagnosed with the mers-cov infection and then completely recovered, as confirmed by the korean government during the 2015 outbreak. of 148 mers survivors who were eligible, 73 consented to participate in the study initially when they were contacted by phone and mail at 6 months post-mers (fig. 1 ). of these participants, 69 survivors completed the 12-month assessment that consisted of medical tests between june 2016 and august 2016. among them, 63 participants provided consent to participate in the psychological assessments in five tertiary hospitals: national medical center, seoul national university hospital, chungnam national university hospital, seoul medical center, and dankook university hospital. all subjects were older than 19 years of age at enrollment, voluntarily participated in the study, and answered the questionnaires independently. written informed consent was obtained from all subjects, and the study was approved by the institutional review board of each study hospital. all subjects responded to self-report questionnaires assessing sociodemographic characteristics, previous history of medical illness or psychiatric visit, illness experiences during the mers-cov infection period, and psychological features. questions about mers-related illness experiences solicited information regarding status during infection, duration of hospitalization, presence of pneumonia, whether a ventilator or extracorporeal membrane oxygenation was applied, and whether a family member died from mers. to determine psychological outcomes, ptsd was assessed with the impact of event scale-revised korean version (ies-r-k) [14, 15] , and depression was evaluated with the patient health questionnaire-9 (phq-9) [16, 17] . the ies-r-k is a 22-item scale that assesses symptoms of intrusion, avoidance and numbing, and hyperarousal related to a particular life-threatening event (i.e., mers-cov infection in the present study). each item is rated on a scale ranging from 0 to 4, and a total score ≥ 25 is considered to be clinically significant [15] . the phq-9 is a nine-item scale that assesses depression based on the symptoms of major depressive disorder included in the diagnostic and statistical manual of mental disorders-fourth edition (dsm-iv) [16] . significant depression is considered to be present when the total score is > 10 [17] . current suicidality was assessed with the suicidality module of the mini-international neuropsychiatric interview (k-mini) [18, 19] , which is composed of six weighted items rated as 'yes' or 'no': wish for death (weight of 1), wish for self-harm (weight of 2), suicidal thoughts (weight of 6), suicide plan (weight of 10), suicide attempt in the past 1 month (weight of 10), and lifetime suicide attempt (weight of 4). the suicidality score is the sum of the weighted score of the six items, and a total score ≥ 6 is considered to be above moderate degree of risk. anxiety was assessed with the generalized anxiety disorder-7 (gad-7) scale, which consists of seven items rated using a four-point likert scoring system [20] . a total score ≥ 10 is considered to be significant. the phq-9 and the gad-7 were administered additionally at two points, before and during infection with mers-cov, based on participant recall. insomnia was evaluated with the korean version of the insomnia severity index (isi-k) [21] , a five-item measure relying on a five-point scale that assesses current sleep problems and interference with daily functioning; clinical insomnia was considered to be present if the total score was ≥15. mers stigma was assessed with an adjusted version of the 40-item berger human immunodeficiency virus (hiv) stigma scale [22] and the 8-item short version of the hiv stigma scale [23] . this questionnaire contains eight items rated on a four-point likert-type scale that ranges from "strongly disagree" to "strongly agree" and assesses the four domains of stigma: personalized stigma, disclosure concerns, negative selfimage, and concerns with public attitudes; the cronbach's α in the present study was 0.919. the present study also included the brief cope, which is a 28-item questionnaire that uses a four-point likert scale to measure three distinctive coping strategies: emotionfocused, problem-focused, and dysfunctional [24] . the social support systems of participants were assessed with the medical outcome study social support survey (mos-sss) [25] , which includes 19 items that are scored on a scale from 0 to 100 and assesses emotional/information support, tangible support, positive social interactions, and affectionate support. to examine the impact of social support on mental health in a regression analysis, poor social support was defined as a mos-sss score lower than that of the 25th percentile for all participants. the sociodemographic characteristics, mers-related clinical characteristics, and mental health status of the participants are presented as both numerical values and percentages. the present study placed a particular focus on ptsd and depression, which were the two most prevalent problems 12 months post-mers in the descriptive analysis. accordingly, the subjects were divided into two groups based on the presence of significant ptsd or depression. independent t-tests were conducted to compare the mental health status between the two groups (p < 0.002, adjusted for multiple comparisons), a stepwise regression analysis was performed to identify independent risk factors for ptsd and depression at 12 months after the mers outbreak, and a univariate analysis was used to identify potential mediating factors associated with ptsd/depression (p < 0.10). subsequently, a backward multivariate logistic regression analysis was performed using variables identified as significant in the univariate analysis (p < 0.05). although depression during mers and current mers stigma were significant in the univariate analysis, these variables were not entered into the multivariate regression analysis due to multicollinearity with anxiety during mers (r = 0.831, p < 0.001) and mers stigma during mers (r = 0.628, p < 0.001), respectively. all data were analyzed with spss for windows version 21.0 (ibm corp.; armonk, ny, usa) except for the multivariate logistic regression analysis, which was performed with stata version 14.0 (stata; college station, tx, usa). the demographic characteristics of the subjects are presented in table 1 . although more male (n = 39, 61.9%) than female subjects were included in the study, the age distribution was relatively even (mean age: 49.2 years, standard deviation [sd]: 12.6). of the 63 subjects, 15.9% had a history of a visit to a psychiatric clinic prior to the mers outbreak. the distribution of respondents at the point of mers-cov infection was as follows: patients, 31.7%; healthcare providers, 23.8%; caregivers, 17.5%; and those visiting the patients in hospitals, 17.5% ( table 2 ). the median length of hospitalization was 21 overall, 54% of the subjects had at least one symptom of ptsd, depression, suicidality, or insomnia that was significantly above the clinical threshold. the mean total score on the ies-r was 25.93 (sd = 20.01), and 42.9% of the subjects had significant ptsd ( table 3 ). the mean score on the phq-9 was 2.49 (sd = 3.53) before infection with mers-cov, 13.54 (sd = 8.80) during the infection, and 6.60 (sd = 6.2) at 12 months after the initial infection. moreover, 27% of the subjects had depression at 12 months post-mers. most subjects had a minimum risk of suicidality, but 22.2% showed at least a moderate degree of suicidal risk. of the survivors, 28% reported significant insomnia at 12 months after the mers outbreak. during mers and 12 months post-mers, all domains of ptsd, anxiety, and depression were more severe, and the quality of life was worse in survivors with current ptsd or depression compared to those without ptsd or depression (p < 0.001) (table s1 ). however, anxiety and depression prior to mers did not significantly differ in either comparison. survivors with ptsd reported higher scores for negative coping strategies compared to those without ptsd (p = 0.001). univariate and multivariate logistic regression analyses were performed to identify risk factors associated with ptsd or depression at 12 months post-mers. the univariate analysis revealed that several factors were significantly associated with ptsd, including previous psychiatry history, having a family member who died from mers, depression and anxiety during the mersaffected period, greater perceived stigma currently and during the illness, and negative coping strategies (table s2) . depression was associated with gender, previous psychiatry history, anxiety before mers, having a family member who died from mers, and depression, anxiety, and greater stigma during the affected phase. neither the severity of mers nor complications, such as the development of pneumonia, use of a ventilator, or extracorporeal membrane oxygenation was associated with ptsd or depression. likewise, not having a spouse, living with a child, and poor social support were not associated with these outcomes. the multivariate logistic regression analysis revealed that previous psychiatric history (adjusted odds ratio [aor]: 9.09, 95% confidence interval [ci]: 1.05-78.67; p = 0.045), anxiety (aor: 5.76, 95% ci: 1.29-25.58; p = 0.021), and greater recognition of stigma (aor: 11.09, 95% ci: 2.28-53.90; p = 0.003) during the mersaffected period were independent predictors of ptsd at 12 months after mers (table 4) . additionally, previous psychiatric history (aor: 9.97, 95% ci: 1.53-65.11; p = 0.016) and having a family member who died from mers (aor: 12.08, 95% ci: 1.47-99.19; p = 0.020) predicted the development of depression at this timepoint. the mers outbreak in 2015 is a noteworthy example of a national disaster that impacted most korean people. its early and rapid dissemination via hospitals concentrated in metropolitan areas [2] , high fatality rate of nearly 20% [2] , and unfamiliarity as a novel infectious disease [26] may have led to high levels of anxiety and fear about being infected among the public and about death among affected people [7] . the present findings confirmed high prevalence of mental health problems in survivors at the recovery phase after the outbreak. the prevalence of ptsd in survivors at 12 months post-mers in the present study was comparable to the rate of 41.7% observed in a study of 63 sars survivors at 3 months post-discharge from a hospital in singapore [27] and higher or comparable to the rates of ptsd in patients with hiv (30-35%), adult survivors of a human-made disaster (30-60%) [28] , and survivors of a stay in an intensive care unit (14-59%) [29, 30] . this indicates that an eid is not only a serious medical illness but also a psychologically traumatic experience for patients that can result in long-term psychological burdens. additionally, the result suggests that mental health adjusting for gender, presence of previous visit to psychiatric clinic, presence of a family member who died from mers, anxiety prior to mers (gad> = 10), anxiety during mers (gad> = 10), mers stigma during mers problems caused by an eid outbreak can continue for a long period. for example, another study showed that 42.5% of sars survivors in hong kong still showed active psychiatric illnesses at 3 years post-sars infection [31] . furthermore, a second study demonstrated that 42% of chinese sars survivors still experienced ptsd at 4 years post-sars [13] . assuming that the experiences of the patients in the mers outbreak are similar in terms of eids, the mental health problems of the mers survivors in the present study may persist for longer than 12 months. therefore, a study on mental health outcomes after 12 months post-mers will be required. of the premorbid characteristics of the subjects, only a history of a visit to a psychiatric clinic was independently related to ptsd and depression at 12 months post-mers, whereas demographic factors, such as gender, age, and level of education were not. on the other hand, high anxiety levels, perceived stigma about mers, and having a family member who died from mers predicted the development of ptsd or depression. these findings indicate that the psychological outcomes associated with an eid are mainly affected by factors during the outbreak period. furthermore, the presence of a physical illness prior to the mers-cov infection and the severity of mers were not associated with ptsd or depression. thus, psychosocial factors, rather than medical factors, may play an important role during mers-cov infection in terms of mental health status. these findings differ from those of a study investigating sars survivors at 30 months post-infection, which found that the risk factors of ptsd included being female, the pre-sars presence of chronic medical illness, and the presence of complications caused by sars treatment [32] . it is possible that the relatively small sample size of the present study was insufficient to statistically identify the influences of demographic characteristics and medical severity on adverse psychological outcomes. however, psychological burdens, such as widespread and extreme fear or feelings of isolation caused by mers [7] , may have outweighed the possible contributions of these other factors. a previous report showing that only a history of mental disease and financial burden are related to anxiety in mers patients [8] supports this assumption. the present findings suggest a need for appropriate psychosocial support during infectious outbreaks to reduce psychological distress in patients [1] . therefore, healthcare professionals who treat these patients should be aware of the risk of developing adverse psychological outcomes during the acute stage of the illness as well as during the follow-up period. in particular, patients with a prior psychiatric history, high levels of psychological distress during the illness, or a negative perception about mers should be given more attention. interestingly, on our univariate analysis, we can assume that negative coping strategy such as denial, substance use, and selfblame may affect the development of ptsd. this relationship between negative coping style and ptsd is consistent with the previous findings in natural disaster and infectious disease [33, 34] . it suggests that providing what is a useful coping strategy should be included in psychosocial support for survivors from eid. similarly, the governmental strategy for the management of eids should include psychosocial support based on group characteristics, risk factors, and severity of distress. the white paper, 'mers 2015,' issued by the korean government proposed that the national policy for eids should include content for "improving ethical problems and strengthening psychological support in eid control." [1] the present findings suggest several considerations in this regard. in general, during the early outbreak phase, it is important that effective risk communication is incorporated into the overall strategy to reduce fear among the general public and quarantined people [35] ; when developing such a strategy for this phase, it is also important to consider the ethical issues related to patients and quarantined people to minimize stigma [36] . more specifically, due to the high prevalence of mental health problems, routine care for eid patients should include effective psychological support that reflects individual risk factors and the current level of distress. in fact, the central and local korean governments provided psychological support for quarantined people, patients, and families who had a member die from mers using designated public mental health care centers and telephone counseling during the outbreak [1] . the core value associated with this program was adequate public accessibility; indeed, rather than rely on the passive provision of information, the program was implemented in a proactive manner [37] . in addition, we should pay attention to stigma as a risk factor amenable to change rather than other psychosocial variables for ptsd in the study. in eid outbreak, the perspective is easily made that an infected patient is regarded as a dangerous vector or perpetrator to spread virus who should be isolated from the society [38] . it can be maintained even after the outbreak [39] . the stigma may produce discrimination and exclusion from a community regardless of medical indications. it would significantly threaten a patient's mental health and social relationship. consequently, their life could be influenced in a variety of domains such as residence, occupation and the use of healthcare for a long time [40] . this study showed that reducing stigma can be an effective strategy to ameliorate psychological consequence after an eid. media and government should respect a patient or quarantined people as a citizen who are suffering and be sensitive to words or actions that might stigmatize a specific person or group. a community and healthcare service need to provide active support for an isolated patient to relieve their burden from the stigma [41] . the present study has several limitations that should be noted. because this study assessed only 43% of the overall mers survivors, the results may not reflect the status of all survivors. however, the distributions of the demographic data on age, gender, and area of residence in the present study were similar to those in the official reports for all mers patients [1] . second, psychological distress and stigma during the pre-mers period and during the mers-cov infection were evaluated based on participant recall and may not accurately represent the actual status of the subjects. additionally, the relatively small sample size may have limited the ability to identify risk factors due to low statistical power. however, given that 47.2% of 34 patients reported anxiety using the same scale in a previous study conducted during the isolation period [8] , it can be assumed that the subjects in the present study were not likely to overestimate their symptoms during recall. finally, we assessed only with self-questionnaire that could be considered less accurate than the ratings of a clinician. our study showed that nearly half the assessed mers survivors experienced significant mental health problems, including ptsd and depression, at 12 months post-mers. mers-specific psychosocial distress may influence long-term psychological sequelae. thus, efforts to control eids should include all levels of government and involve the implementation of effective strategies to reduce fear and stigma among the public; they should also enable the provision of adequate psychological support and hospital care for infected people. supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08726-1. additional file 1 table s1 . comparisons of mental health status and related factors between survivors with and without ptsd/depression in south korea. table s2 . univariate analysis assessing ptsd and depression and related variables 12 months after the mers outbreak, in south korea. the 2015 mers outbreak in the republic of korea: learning from mers middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications outcomes of sars survivors in china: not only physical and psychiatric co-morbidities psychological distress and negative appraisals in survivors of severe acute respiratory syndromes (sars) long-term psychiatric morbidities among sars survivors stress and psychological distress among sars survivors 1 year after the outbreak the emotional distress and fear of contagion related to middle east respiratory syndrome (mers) on general public in korea mental health status of people isolated due to middle east respiratory syndrome psychiatric complications in patients with severe acute respiratory syndrome (sars) during the acute treatment phase: a series of 10 cases posttraumatic stress, anxiety, and depression in survivors of severe acute respiratory syndrome (sars) psychosocial consequences of infectious diseases mental health impact of severe acute respiratory syndrome: a prospective study posttraumatic stress disorder in convalescent severe acute respiratory syndrome patients: a 4-year follow-up study reliability and validity of the korean version of the impact of event scale-revised a study on reliability and validity of the korean version of impact of event scale-revised the phq-9: validity of a brief depression severity measure information c. standardization of the korean version of screening tool for depression (patient health questionnaire-9 validity of korean version of the mini-international neuropsychiatric interview the mini-international neuropsychiatric interview (m.i.n.i.): the development and validation of a structured diagnostic psychiatric interview for dsm-iv and icd-10 a brief measure for assessing generalized anxiety disorder validation of a korean version of the insomnia severity index measuring stigma in people with hiv: psychometric assessment of the hiv stigma scale psychometric properties of a short version of the hiv stigma scale, adapted for children with hiv infection you want to measure coping but your protocol's too long: consider the brief cope the mos social support survey middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications quality of life and psychological status in survivors of severe acute respiratory syndrome at 3 months postdischarge the epidemiology of post-traumatic stress disorder after disasters posttraumatic stress disorder following medical illness and treatment posttraumatic stress disorder and health-related quality of life in long-term survivors of acute respiratory distress syndrome mental morbidities and chronic fatigue in severe acute respiratory syndrome survivors: long-term follow-up risk factors for chronic post-traumatic stress disorder (ptsd) in sars survivors post-traumatic stress disorder and coping in a sample of adult survivors of the italian earthquake post-traumatic stress disorder among recently diagnosed patients with hiv/aids in south africa worry experienced during the middle east respiratory syndrome (mers) pandemic in korea ethical perspectives on the middle east respiratory syndrome coronavirus epidemic in korea system effectiveness of detection, brief intervention and refer to treatment for the people with post-traumatic emotional distress by mers: a case report of community-based proactive intervention in south korea the patient as victim and vector: ethics and infectious disease the sars-associated stigma of sars victims in the post-sars era of hong kong the experience of sarsrelated stigma at amoy gardens stress and health: major findings and policy implications publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to acknowledge all the participants and researchers in the cohort study for mers survivors.authors' contributions shl and hss coordinated the overall study. hyp, shl, jlk, jjl, hl, and hss were involved in the concept and the design of the study. hyp and shl undertook the statistical analysis and drafted the manuscript. hyp, wbp, shl, jlk, jjl, hl, and hss contributed to the acquisition and the interpretation of the data, revised the manuscript and approved the article of its final version. the study was supported by a grant of the korea health technology r&d project through the korea health industry development institute (khidi), funded by the ministry of health and welfare, republic of korea (hi15c3227) and a grant from the korean mental health technology r&d project, ministry of health & welfare, republic of korea (hl19c0007). the funding bodies were not involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. the data obtained from the current study are not publicly available due to the sensitive nature of the study. not applicable. the authors declare that they have no competing interests. key: cord-273182-djb0ozrt authors: díez, josé maría; romero, carolina; vergara-alert, júlia; belló-perez, melissa; rodon, jordi; honrubia, josé manuel; segalés, joaquim; sola, isabel; enjuanes, luis; gajardo, rodrigo title: cross-neutralization activity against sars-cov-2 is present in currently available intravenous immunoglobulins date: 2020-09-09 journal: immunotherapy doi: 10.2217/imt-2020-0220 sha: doc_id: 273182 cord_uid: djb0ozrt background: cross-reactivity against human coronaviruses with flebogamma(®) dif and gamunex(®)-c, two available intravenous immunoglobulins (ivig), has been reported. in this study, these ivig were tested for neutralization activity against severe acute respiratory syndrome coronavirus 2 (sars-cov-2), sars-cov and middle east respiratory syndrome cov (mers-cov). materials & methods: neutralization capacity of lots of ivig manufactured prior to covid-19 pandemic was assessed against these viruses in cell culture. infectivity neutralization was quantified by percent reduction in plaque-forming units and/or cytopathic/cytotoxic methods. results: all ivig preparations showed neutralization of sars-cov-2 isolates. all ivig lots produced neutralization of sars-cov. no ivig preparation showed significant neutralizing activity against mers-cov. conclusion: the tested ivig contain antibodies with significant in vitro cross-neutralization capacity against sars-cov-2 and sars-cov, but not mers-cov. these preparations are currently under evaluation as potential therapies for covid-19. the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which causes the respiratory disease covid-19 was declared a pandemic by the who in march 2020. most infected patients (80%) have mild symptoms. however, about 20% of covid-19 patients can progress to severe pneumonia and to acute respiratory distress syndrome which is associated with multi-organ failure and death [1] . the current critical situation demands an effective and reliable therapy that is immediately available to control the progression of the disease [2] . convalescent plasma or plasma-derived immunoglobulin (ig; either polyvalent ig prepared from healthy donors or hyperimmune ig prepared from donors with high antibody titers against a specific antigen) have been historically used as a readily available therapeutic option in outbreaks of emerging or re-emerging infections [3] . to date, seven human coronaviruses (hcov) have been identified. four of them (hcov-229e, hcov-nl63, hcov-oc43 and hcov-hku1) are globally distributed [4] and are associated with about 15% of common colds, typically causing mild symptoms [5] . in contrast, sars-cov, middle east respiratory syndrome cov (mers-cov), and sars-cov-2 are zoonotic epidemic viruses [6] that can cause severe respiratory infections and fatalities. sars-cov emerged in china in 2002 with the last reported case in 2014. mers-cov emerged in saudi arabia a decade later, in 2012, and led to an outbreak in south korea in 2015. mers-cov still emerges sporadically in humans from its reservoir in camelids [7] [8] [9] . more recently (december 2019), the novel coronavirus sars-cov-2 emerged in china and because of its extraordinary human-to-human transmissibility is currently causing an unprecedented pandemic [10] . coronaviruses share some morphological and functional properties that may be associated with cross-reactive immune responses which may have important therapeutic implications [11] . sars-cov, sars-cov-2 and mers-cov are classified within the family coronaviridae, genus betacoronavirus, subgenera sarbecovirus (sars-cov, sars-cov-2) and merbecovirus (mers-cov). sars-cov-2 has four main structural proteins including spike (s) glycoprotein, small envelope (e) glycoprotein, membrane (m) glycoprotein and nucleocapsid (n) protein [12] . s protein is the main determinant of the coronavirus entry into the host cell and is also the major target of neutralizing antibodies [13, 14] . spikes are formed by trimers of protein s, which is in turn formed by subunit (s1) that mediates the binding to the cell receptor, and a membrane-anchored subunit (s2) that mediates the fusion of the virus with cell membranes [15] . the receptor-binding domain (rbd) is a key functional component within the s1 subunit that is responsible for virus binding to host cell [16] . potent neutralizing antibodies often target rbd. however, the s1 subunit shows a higher variability than s2. antibodies targeting s1 are often virus-specific making s2 a better target for cross-neutralizing antibodies [17, 18] . the amino-acid sequence identity among the s proteins of human betacoronaviruses causing mild (hcov-oc43 and hcov-hku1) and severe (sars-cov, sars-cov-2 and mers-cov) respiratory infections varies between 22 and 33% [14] . however, the s proteins of sars-cov and sars-cov-2 share 77% amino-acid identity [19] . shared protein homologies among coronaviruses can cause cross-reactivity and/or cross-neutralization antigenic responses (i.e., antibodies able to recognize a coronavirus, but that have generated in response to prior infection of other different circulating coronavirues). cross-reactivity has been described among hcovs of the same genus, particularly betacoronaviruses. cross-reactivity between sars-cov, mers-cov and other endemic hcovs has been reported in some studies [20] [21] [22] . however, it is uncertain whether such cross-reacting antibodies among coronaviruses have also the capacity of reducing viral infectivity by a cross-neutralization effect. recently, we reported cross-reactivity in elisa binding assays against antigens of sars-cov, sars-cov-2 and mers-cov with flebogamma r dif 5 and 10% and gamunex r -c, two currently available intravenous igs (ivig) [23] . as a continuation of this study, here we evaluated the neutralization capacity of those same ivig products against these epidemic hcovs. experimental products ivig products used in this study were flebogamma r dif 5% and 10% (instituto grifols s.a., barcelona, spain) and gamunex r -c 10% (grifols therapeutics inc., nc, usa), two highly purified (≥98-99% immunoglobuin g [igg]), unmodified human igs. each product is manufactured from plasma collected from thousands of donors in the usa and/or several european countries. igg concentrations in flebogamma dif products were 50 and 100 mg/ml (5 and 10%) and in gamunex-c, the concentration was 100 mg/ml (10%). to ensure a virus-free product, both ivig manufacturing processes contain dedicated steps with high pathogen clearance capacity, such as solvent/detergent treatment, heat treatment, caprylate treatment and nanofiltration (planova™, asahi kasei, brussels, belgium). the plasma used to manufacture the ivig lots tested was collected from march 2018 to october 2019. six different lots of flebogamma dif and gamunex-c were tested at several dilutions for cross-reactivity against sars-cov, sars-cov-2 and mers-cov by: elisa techniques; and well-established neutralization assays in cell cultures. lots were identified as f1 and f2 for flebogamma 5% dif, f3 and f4 for flebogamma 10% dif and g1 and g2 for gamunex-c. each experiment was performed in duplicate. handling of viruses and cell cultures was carried out at the level 3 biosafety laboratories in the centro nacional de biotecnología -consejo superior de investigaciones científicas (cnb-csic; madrid, spain) and the institut de recerca i tecnologia agroalimentàries -centre de recerca en sanitat animal (irta-cresa; barcelona, spain), following the centers' biohazard safety guidelines and under authorizations #a/es/00/i-8 and #sa-10430-20, respectively. recombinant sars-cov was generated from urbani strain using a previously described reverse genetic technique [24] . two different sars-cov-2 isolates, collected from nasopharyngeal swab from covid-19 patients, were tested: sars-cov-2 mad6 isolated from a 69-year-old male patient from madrid (spain); and sars-cov-2 (accession id epi isl 418268 at gisaid repository: http://gisaid.org) isolated from a an 89-year-old male patient from badalona (spain). both stock viruses were prepared by collecting the supernatant from vero e6 cells, as previously described [25] . recombinant mers-cov was generated using a previously described reverse genetic system [26] from the reference sequence of mers-cov isolated from the index patient emc/2012 (genebank jx869059) [27] . huh7 is a well differentiated human hepatocyte-derived carcinoma cell line, kindly provided by dr l carrasco (centro de biología molecular severo ochoa -consejo superior de investigaciones científicas [cbmso-csic], madrid, spain). huh7 is composed of epithelial-like cells susceptible to infection by mers-cov [28] . vero e6 is a cell line isolated from kidney epithelial cells extracted from an african green monkey. vero e6 is composed of epithelial-like cells susceptible to infection by sars-cov and sars-cov-2 [29] . at cnb-csic, vero e6 cell lines were kindly provided by dr e snjider (university of leiden medical center, the netherlands). both huh7 and vero e6 cell lines were cultured in dulbecco's modified eagle medium (dmem) supplemented with 25 mm hepes buffer, 2 mm l-glutamine (sigma-aldrich, mi, usa), 1% nonessential aminoacids (sigma-aldrich), 10% fetal bovine serum (fbs; biowhittaker, inc., md, usa). in the post infection semisolid medium, the percentage of fbs was reduced to 2%, and diethylaminoethyl (deae)-dextran (sigma-aldrich) was added to a final concentration of 0.08 mg/ml. at irta-cresa, vero e6 cells were obtained from the atcc (atcc crl-1586) and cultured in dmem (lonza, basel, switzerland) supplemented with 5% fbs (euroclone, pero, italy), 100 u/ml penicillin, 100 μg/ml streptomycin and 2 mm glutamine 8 (all from thermofisher scientific, ma, usa). in the post infection medium, the percentage of fbs was 2%. igg elisa testing procedures qualitative determination of igg class antibodies cross-reactivity against antigens of the tested coronaviruses was performed using elisa techniques. ivig samples were serially diluted using the buffer solutions provided in each igg elisa kit. the following kits were used for the qualitative determination of igg class antibodies in the experimental ivig lots: sars coronavirus igg elisa kit (creative diagnostics, ny, usa), against virus lysate; human anti-sars-cov-2 virus spike 1 [s1] igg elisa kit (alpha diagnostic intl. inc., tx, usa), against s1 subunit spike protein; rv-402100-1, human anti-mers-np igg elisa kit (alpha diagnostic intl inc.), against n protein; rv-402400-1, human anti-mers-rbd igg elisa kit (alpha diagnostic intl inc.), against rbd of s1 subunit spike protein (s1/rbd); rv-402300-1, human anti-mers-s2 igg elisa kit (alpha diagnostic intl. inc.), against s2 subunit spike protein; rv-405200 (formerly rv-404100-1). in all cases, the determinations were carried out following the manufacturer's instructions. reactivity was rated as negative if no reaction was observed with neat ivig or positive if the lowest ivig dilution demonstrated reactivity. this neutralization assay was based on the reduction in plaque forming units (pfu) after exposing a given amount of virus to the product to be characterized and comparing with the untreated control. this assay is performed in cell cultured plates with a semisolid ovarlay to allow plaque formation. for this assay, ivig samples were serially diluted (factor 10 dilutions: 1:10 2 , 1:10 3 , 1:10 4 and 1:10 5 ) in dulbecco's phosphate-buffered saline (gibco, thermo fisher scientific, ma, usa). samples of each ivig dilution were incubated for 1 h (37 • c; 5% co 2 ) with 300 pfus of sars-cov, sars-cov-2 or mers-cov. aliquots of 50 μl of each ivig dilution-virus complex were added in duplicate to confluent monolayers of vero e6 cells (for sars-cov and sars-cov-2) or huh7 (for mers-cov), seeded in 12-well plates and incubated for 1 h (37 • c; 5% co 2 ). after this adsorption time, the igg-virus complex inoculum was removed and a semi-solid overlay was added (dmem 2% fbs + 0.6% agarose). cells were incubated for 72 h at 37 • c. the semi-solid medium was removed, the cells were fixed with 10% neutral buffered formaldehyde (sigma-aldrich) for 1 h at room temperature, and stained with 0.2% aqueous gentian violet for 10 min, followed by plaque counting. the sensitivity threshold of the technique was 20 pfu per ml. the neutralization potency of the ivig products was expressed in two ways: percent reduction in pfu calculated from the pfu count after neutralization by ivig relative to initial pfu count inoculated onto the cells; and plaque reduction neutralization test (prnt 50 ) value, calculated as the -log 10 of the reciprocal of the highest ivig dilution to reduce the number of plaques by 50% compared with the number of plaques without ivig. neutralization assay for sars-cov-2 (epi isl 418268 isolate) this neutralization assay measured the cytopathic/cytotoxic virus-induced effect by detecting cellular enzymatic activity after incubation with a given amount of the relevant virus and comparing this with the relevant untreated control. for this assay, a fixed concentration of a sars-cov-2 stock (10 1.8 tcid 50 /ml, a concentration that achieves 50% cytopathic effect) was mixed with decreasing concentrations of the ivig samples (range 1:10 to 1:5120), each mixture was incubated for 1 h at 37 • c, and added to vero e6 cells. to assess potential plasma-induced cytotoxicity, vero e6 cells were also cultured with the same decreasing concentrations of plasma in the absence of sars-cov-2. uninfected cells and untreated virus-infected cells were used as negative and positive infection controls, respectively (see supplementary figure 1 ). plasma from a covid-19 positive patient with a high half-maximal inhibitory concentration (ic 50 ) was included as an active positive control (expressed as the -log 10 of the reciprocal of the dilution). all the cultures were incubated at 37 • c and 5% co 2 for 3 days. cytopathic or cytotoxic effects of the virus or plasma samples were measured at 3 days post-infection, using the cell titer-glo luminescent cell viability assay (promega, wi, usa). luminescence was measured in a fluoroskan ascent fl luminometer (thermo fisher scientific). neutralization curves are shown as nonlinear regressions. ic 50 values were determined from the fitted curves as the plasma dilutions that produced 50% neutralization. details of the technique are available elsewhere [25] . cross-reactivity studies (elisa-binding assays) ivig products showed consistent reactivity to antigens of sars-cov (culture lysate) at 10-100 mg/ml igg, sars-cov-2 (s1 subunit protein) at 100 μg/ml igg and mers-cov (n protein, s1 subunit/rhd protein and s2 subunit protein) at 50 μg/ml igg (table 1) . all the assayed ivig preparations had neutralizing activity against sars-cov ranging from 39 to 61% (figure 1 ). all 10% igg ivig preparations (f3, f4, g1 and g2) showed prnt 50 neutralization titers between 2.0 and 3.3, corresponding to 50-61% pfu reduction ( figure 1b & c) . the highest pfu reductions, 59.3 and 61.9% (prnt 50 neutralization titers of 3.2 and 3.3), were observed with lots f4 and g1, respectively, at 1 and 0.1 mg/ml igg (dilution factors 2 and 3). the f1 and f2 lots, (5% igg) showed a lower neutralization capacity with pfu reductions of 39.5 and 43.3%, respectively ( figure 1a ). for sars-cov-2 mad6 isolate, all ivig lots, except f1 (inconclusive results) showed a significant neutralizing activity and reached prnt 50 titers ranging from 4.5 to >5 (figure 2 ). pfu reductions ranging from 78.2 to 82.5% were observed with lots f2, f3 and f4 at a dilution factor of one. even at the highest dilution factor (5 = 0.5 and 1 μg/ml), the pfu reduction ranged from 38.5 to 50.9% corresponding to prnt 50 titers of 4.5-5.0 (figure 2a & b) . for lots g1 and g2, the pfu reduction was even higher, ranging from 88.5 to 89.5% at a dilution factor of one to 61.7-62.5% at a dilution factor of five with prnt 50 titers greater than five ( figure 2c ). for the sars-cov-2 epi isl 418268 isolate, f4 and g1 lots neutralized 58.4 and 64.7%, respectively, tcid 50 counts at a dilution factor of one (figure 3 ). one replicate of f4 product failed to demonstrate neutralization. no ivig lot showed any significant pfu reduction (i.e., >10%) on mers-cov even at the lowest dilution factor (10 mg/ml igg). the results presented here demonstrate for the first time significant cross-neutralization activity against sars-cov and especially sars-cov-2 in two therapeutic ivig concentrates (flebogamma r dif and gamunex r -c). this neutralizing activity correlates with the cross-reactivity to different coronavirus antigens observed in elisa-binding assays with ivig, as shown in a previous study [23] . the plasma used to manufacture the tested ivig lots was collected prior the detection of sars-cov-2 in europe and the usa. therefore, these results should be ascribed to cross-reactivity against sars-cov-2 by antibodies against endemic hcovs in the human population at large. similar results have been reported for sars-cov and mers-cov [20] [21] [22] . ivig are polyclonal igg antibodies reacting to a broad range of different antigens. antibody titers and specificities may vary slightly among different lots and manufacturers, depending on the plasma donor population [30] . our neutralization studies showed that the studied ivig products contain antibodies with cross-neutralizing capacity against sars-cov (40-60%) and sars-cov-2 (80-90%), but not against mers-cov (<10%). these results suggest that the cross-neutralizing antibodies target antigenic regions more conserved in sars-cov and sars-cov-2 than in mers-cov. no significant differences in the neutralizing capacity were observed among ivig lots regardless the country of origin for the plasma. this reinforces the broad applicability of these results. two different neutralization techniques were used for sars-cov-2 and both techniques showed not only the ivig neutralization capacity, but also the reliability of the results. in addition, results obtained with two different sars-cov-2 isolates confirm that the neutralization capacity is not dependent on the isolate. this was not unexpected since no significant sequence differences have been observed among sars-cov-2 isolates currently circulating throughout the world. the percentage of sars-cov-2 cross-neutralization was higher in the pfu reduction technique than in the cytopathic effect/cytotoxic technique with very low or negative values in some few cases (inconclusive for lot f1 by the pfu study and cytopathic effect in one replicate of lot f4). this suggests that the technique used and/or slight variations in methodology may significantly influence the nature or magnitude of the results. therefore, further evaluation this cross-neutralizing activity should be carried out. cross-neutralization is gaining attention as a protective mechanism against viral infection in the context of the covid-19 health emergency. the results of this study are in agreement with recent studies that describe cross-neutralization of sars-cov-2 by monoclonal antibodies from memory b cells of an individual who was infected with sars-cov [31] . furthermore, sars-cov-2-reactive cd4 + t cells have been detected in around half of unexposed individuals, suggesting that there is cross-reactive t-cell recognition between circulating common cold coronaviruses and sars-cov-2 [32] . however, the levels of cross-neutralizing antibodies against sars-cov-2 in the sera of sars-cov patients can be highly variable [33] . ivig products are prepared using plasma from thousands of different donors, hence containing a broad representation of the state of immunity in the population at that time. this is consistent with the low rate of variability found among the different lots of ivig products tested. nevertheless, greater variability is expected among individuals with respect to infection by a given endemic human coronavirus. therefore, it has been hypothesized that the diversity of symptoms observed in sars-cov-2infected individuals and even the potential for getting infected may depend on pre-existing cross-immunity due to previous exposure to other endemic hcovs. in this regard, a detailed study of the state of immunity in the general population distinguishing those affected and not affected by the sars-cov-2 may be warranted. the higher cross-neutralizing capacity of the tested ivig preparations against sars-cov and sars-cov-2 than mers-cov may be explained by higher sequence identity of the s proteins of circulating mild hcovs (hcov-oc43 and hcov-hku1) with sars-cov and sars-cov-2 compared with mers-cov (32-33% vs [23] [24] [25] [19, 34] . additionally, differences in specific domains of the s protein between sars-cov and sars-cov-2 might explain higher cross-reactivity of the tested ivig against sars-cov-2 compared with sars-cov (80-90% vs 40-60%). the absence of cross-neutralization against mers-cov despite the cross-reactivity observed in elisa assays suggests that these antibodies are not neutralizing. however, this does not necessarily indicate that such antibodies are not functional by another mechanism. for example, these non-neutralizing antibodies could be labeling the virion for identification by immune cells and subsequent destruction [35] . despite the limitations of the in vitro nature of this study, the clinical implications of the findings are encouraging, and the results may support the use of ivig as a therapeutic option for covid19 . in vitro neutralization studies should be deemed as a partial characterization of a more complex response that can take place in vivo where the host's response mechanisms can include antibody dependent cellular phagocytosis, antibody-dependent cellular cytotoxicity [36] , as well as viral mechanisms such as antibody-dependent enhancement [37] . nevertheless, positive results with the administration of ivig (immunomodulatory dose) to counteract hyper inflammation in patients with severe covid-19 [38] have already been reported in case studies [39, 40] . ivig use is being tested in an ongoing clinical trial [41] . further studies looking at the functionality of these antibodies could improve our understanding the human coronavirus acquired immunity. this could pave the way for ivig (and other igg products such as intramuscular or subcutaneous preparations) as a potential therapeutic/prophylactic approach to fight current and future epidemics due to emerging hcovs. under the experimental conditions of this study, flebogamma r dif and gamunex r -c ivig contained antibodies with significant neutralization capacity against sars-cov and sars-cov-2, but not against mers-cov. additional research is warranted to advance ivig toward clinical use for covid-19. • intravenous immunoglobulin products were tested against severe acute respiratory syndrome coronavirus 2 in cell culture neutralization assays. • for plaque forming unit method, viral neutralization ranged from 79 to 89.5%; prnt 50 titers ranged from 4.5 to >5. • for cytopathic method, viral neutralization ranged from 47 to 64.7%; ic 50 was around 1. • there was also neutralization of sars-cov, ranging from 39.5 to 55.1%; prnt 50 ; 2.0-3.3. • results support current trials assessing intravenous immunoglobulin as potential therapy for covid-19. any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no outsourced writing assistance was utilized in the production of this manuscript. this work is licensed under the attribution-noncommercial-noderivatives 4.0 unported license. to view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ strategies for the prevention and management of coronavirus disease 2019 sars-cov-2 causing pneumonia-associated respiratory disorder (covid-19): diagnostic and proposed therapeutic options use of human immunoglobulins as an anti-infective treatment: the experience so far and their possible re-emerging role •• a comprehensive review on the role of intravenous immunoglobulin (ivig) as anti-infective treatment in community and emerging diseases epidemiology, genetic recombination, and pathogenesis of coronaviruses update on human rhinovirus and coronavirus infections the 2019 novel coronavirus disease (covid-19) pandemic: a zoonotic prospective. asian pac sars: prognosis, outcome and sequelae sars-cov-2/covid-19: viral genomics, epidemiology, vaccines, and therapeutic interventions middle east respiratory syndrome european centre for disease prevention and control. covid-19. situation update worldwide global patterns in coronavirus diversity sars-cov-2 envelope and membrane proteins: structural differences linked to virus characteristics? neutralizing antibodies against sars-cov-2 and other human coronaviruses identification of the receptor-binding domain of the spike glycoprotein of human betacoronavirus hku1 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov structure of sars coronavirus spike receptor-binding domain complexed with receptor genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov-2, sars-cov, and mers-cov: a comparative overview phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop an outbreak of human coronavirus oc43 infection and serological cross-reactivity with sars coronavirus. can • a report to understand the key role of antibody cross-reactivity in emerging viral disease cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests • a report to understand the key role of antibody cross-reactivity in emerging viral disease antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 • a report to understand the key role of antibody cross-reactivity in emerging viral disease currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus 2 antigens time report of significant cross-reactivity of currently available ivig products to components of severe acute respiratory syndrome coronavirus 2 (cov-2), severe acute respiratory syndrome cov and middle east respiratory syndrome-cov construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis search for sars-cov-2 inhibitors in currently approved drugs to tackle covid-19 pandemia engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by sars coronavirus and human coronavirus 229e the life cycle of sars coronavirus in vero e6 cells polyclonal intravenous immunoglobulin for the prophylaxis and treatment of infection in critically ill adults. can cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals lack of cross-neutralization by sars patient sera towards sars-cov-2 profiling early humoral response to diagnose novel coronavirus disease (covid-19) protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts antibody-dependent cellular phagocytosis in antiviral immune responses molecular mechanism for antibody-dependent enhancement of coronavirus entry covid-19: consider cytokine storm syndromes and immunosuppression high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease • the first clinical study to evaluate the efficacy of ivig in the treatment of severely ill covid-19 patients effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid-19 key: cord-263508-row2mn17 authors: chan, jasper fuk-woo; lau, susanna kar-pui; woo, patrick chiu-yat title: the emerging novel middle east respiratory syndrome coronavirus: the “knowns” and “unknowns” date: 2013-07-21 journal: j formos med assoc doi: 10.1016/j.jfma.2013.05.010 sha: doc_id: 263508 cord_uid: row2mn17 a novel lineage c betacoronavirus, originally named human coronavirus emc/2012 (hcov-emc) and recently renamed middle east respiratory syndrome coronavirus (mers-cov), that is phylogenetically closely related to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5, which we discovered in 2007 from bats in hong kong, has recently emerged in the middle east to cause a severe acute respiratory syndrome (sars)-like infection in humans. the first laboratory-confirmed case, which involved a 60-year-old man from bisha, the kingdom of saudi arabia (ksa), who died of rapidly progressive community-acquired pneumonia and acute renal failure, was announced by the world health organization (who) on september 23, 2012. since then, a total of 70 cases, including 39 fatalities, have been reported in the middle east and europe. recent clusters involving epidemiologically-linked household contacts and hospital contacts in the middle east, europe, and africa strongly suggested possible human-to-human transmission. clinical and laboratory research data generated in the past few months have provided new insights into the possible animal reservoirs, transmissibility, and virulence of mers-cov, and the optimal laboratory diagnostic options and potential antiviral targets for mers-cov-associated infection. introduction: the "new sars"? ten years after the devastating epidemic of severe acute respiratory syndrome (sars) caused by sars coronavirus (sars-cov), which resulted in a total of 774 deaths among more than 8000 confirmed cases in over 30 countries, the world is facing a new challenge posted by a "sars-like" infection caused by another novel coronavirus emerging from the middle east, which was originally named human coronavirus emc/2012 (hcov-emc) and recently renamed by the coronavirus study group of the international committee for taxonomy of viruses as middle east respiratory syndrome coronavirus (mers-cov). 1e8 the complete genome of the virus was sequenced and released in october 2012 after the isolation of the virus from two patients with severe community-acquired pneumonia in bisha, the kingdom of saudi arabia (ksa), and doha, qatar, first announced by the world health organization (who) on 23 september 2012. 9 as of may 12, 2013, the total number of laboratory-confirmed cases of mers-cov infection has increased to 34 with 20 deaths, including two cases confirmed retrospectively from a jordanian cluster of severe respiratory disease reported by the ministry of health of jordan in april 2012 (table 1) . 6,7,10e14 although the number of laboratory-confirmed cases remains limited, the severe clinical manifestations with an unusually high mortality rate of over 50%, the spread of the infection beyond the geographical confinement in the middle east, and the epidemiological evidence of human-to-human transmission arising from the recent clusters of cases in a family in the united kingdom (cases 10 to 12), and in hospitals in ksa (cases 18 to 30, 32 and 33) and france (cases 31 and 34), have raised significant concerns on the possible emergence of another sars-like epidemic in the near future. in anticipation of the potential spread of this highly pathogenic virus from the middle east and europe to other parts of the world, especially the densely populated southeast asia, an updated review of the latest research findings on mers-cov and their implications on the clinical management of mers-cov infection is essential. viral genomic studies reveal the first lineage c betacoronavirus associated with human infection mers-cov belongs to the genus betacoronavirus in the family coronaviridae under the order nidovirales. coronaviruses are enveloped viruses with positive-sense singlestranded rna genomes. studies in their biodiversity, comparative genomics and phylogeny in the past 10 years have improved our understanding of this family of viruses. 15e30 according to the most recent classification by the coronavirus study group of the international committee for taxonomy of viruses, there are four genera in coronaviridae, namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. 28, 31 the genus alphacoronavirus contains two human coronaviruses, hcov-229e and hcov-nl63, which are associated with the common cold. no human coronavirus has been discovered in gammacoronavirus and deltacoronavirus, which mainly contain avian coronaviruses with just a few mammalian coronaviruses. the genus betacoronavirus is comprised of four lineages (a, b, c and d) which contain four coronaviruses associated with human infections: hcov-oc43 and hcov-hku1 (lineage a), sars-cov (lineage b), and mers-cov (lineage c). 15 mers-cov is the first known lineage c betacoronavirus associated with human infection and is phylogenetically closely related to the other lineage c betacoronaviruses including tylonycteris bat cov hku4 (ty-batcov-hku4) and pipistrellus bat cov hku5 (pi-batcov-hku5), which were discovered in lesser bamboo bats (tylonycteris pachypus) and japanese pipistrelle bats (pipistrellus abramus), respectively, captured in hong kong, china. 9, 22, 32 analysis of the genome of mers-cov revealed that it has a genome size of 30,106 bases with the rdrp and s genes having over 90% and around 70% amino acid identities with those of ty-batcov-hku4 and pi-batcov-hku5. 9,32 molecular clock analysis using the rdrp gene showed that mers-cov might have diverged from the most recent common ancestor of lineage c betacoronaviruses in year w941 ad (529 bc to 1878 ad). 32 it is postulated that the emergence of mers-cov represents another series of interspecies transmission events in coronaviruses, from bats to possibly other animals and then to humans, a scenario similar to the sars epidemic. epidemiology reports and cell line susceptibility studies suggest possible animal reservoirs and human-to-human transmissions epidemiological linkage with animals, including camels, goats, sheep, and farm animals or their caretakers before symptom onset in some of the reported cases supported the hypothesis of mers-cov being a zoonotic agent (table 1) . furthermore, in vitro data from cell line susceptibility studies showed that the virus had a broad species tropism and was able to replicate in bat, primate, porcine, rabbit, and civet cell lines. 33, 34 as in the case of sars-cov, which likely emerged from its natural animal reservoir the horseshoe bats (rhinolophus sp.), and jumped to other mammals during their caging in the wild life markets of south china and then to humans, mers-cov might have also originated from bats to these susceptible animal species before adapting to humans. 16, 35, 36 in addition to pipistrellus bats, which are the natural hosts of the closely related pi-batcov-hku5 and are also found in the middle east, rousettus, rhinolophus, myotis, and carollia bat cell lines are also susceptible to mers-cov infection in vitro. 34 active surveillance of different bat and animal species predominantly found in the middle east would help to delineate the natural bat reservoir and the evolutionary pathway of mers-cov among other susceptible animal species. indeed, a recent study showed that a number of different bat species in ghana and europe are infected with coronaviruses, which also share close homologies with mers-cov. 37 determination of the virus' animal hosts might in turn facilitate the control of the outbreak as in sars, where closure of the wet markets likely contributed to the cessation of the epidemic. human-to-human transmission represents a new stage in the evolution of mers-cov infection. there are, so far, six 12 the seroprevalence and transmissibility of mers-cov remain undetermined, although animal-to-human and human-to-human transmissions both appear to be limited at this stage (fig. 1) . among 2400 persons seeking medical attention in a hospital in jeddah, ksa, none had mers-covspecific igg detectable by indirect immunofluorescence assay, suggesting that the current situation is likely to be different from those of other human coronaviruses which are endemic in humans. 6, 38 the lack of secondary cases among nearly 200 contacts of cases 2 and 5 who did not practice optimal infection control measures before the diagnosis of mers-cov infection was confirmed, implied that this novel virus might be less efficient than sars-cov in human-tohuman transmission. 7, 13, 39 however, the findings of these studies should be interpreted with cautions for two reasons. first, only a relatively small number of subjects (10/64 in case 2 and 85/123 in case 5) were tested by reliable laboratory tests. second, the timing of testing was not clearly described and might be suboptimal for the purpose of excluding the diagnosis. it has been proposed that a second test should be performed in symptomatic patients with initially negative results by reverse transcriptionpolymerase chain reaction (rt-pcr), within the first 10 days of symptom onset, based on the observation in sars where viral load peaked at day 10 of symptom onset. 5,40e42 therefore, the apparently limited spread of mers-cov at present might be an underestimation and ongoing transmission of the virus should be cautiously monitored. unlike its close relatives in bats, mers-cov is highly pathogenic in humans. the most common clinical presentation among the 34 laboratory-confirmed cases of mers-cov infection is acute severe community-acquired pneumonia with acute renal failure following an estimated incubation period of 1e9 days (table 1) . 12, 43 this is unusual among human infections caused by coronaviruses, in which severe pneumonia with respiratory failure is seldom seen, except in sars. the other human coronaviruses, namely hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1, predominantly cause acute self-limited upper respiratory tract infections, and only occasionally cause lower respiratory tract infections in the elderly and immunocompromized populations. only two of 34 patients (cases 12 and 16) had a self-limiting, mild, influenza-like illness not requiring hospitalization. asymptomatic or mild infections have otherwise not been detected among the other contacts of confirmed cases by rt-pcr of upper respiratory tract specimens and/or serological tests, 2400 saudi residents in jeddah by indirect immunofluorescent antibody testing of archived sera, and 169 french hajj pilgrims with upper respiratory symptoms and rt-pcr of prospectively collected nasal swabs. 6, 7, 13, 44 the other 32 (94.1%) patients, many of whom had no underlying medical condition, developed rapid clinical deterioration with lower respiratory tract involvement and respiratory failure requiring ventilator support within a few days to 1 week after initial systemic symptoms of fever, myalgia, and malaise, and upper respiratory tract symptoms such as rhinorrhea and sore throat. this correlated with the finding in our recent cell line susceptibility study, which showed that mers-cov replicated much better in the lower respiratory tract cell lines including calu-3 (polarized airway epithelium), a549 (lung adenocarcinoma), and hfl (embryonic lung fibroblasts), than the upper respiratory tract cell line hep-2 (laryngeal epidermoid carcinoma). 33 another in vitro study also showed that pseudostratified human bronchial epithelium cultures are highly permissive to mers-cov infection. 45 in ex vivo organ cultures, mers-cov productively replicated in both human bronchial and lung tissues, whereas sars-cov only productively replicated in lung tissue. 46 recently, a macaque model showed that mers-cov caused acute, localized to widespread pneumonia, resulting in mild to moderate clinical disease resembling the illness observed in humans. 47 radiologically, pneumonia is evident by focal consolidations involving single or multiple lobes, with progressive involvement of bilateral lung fields, especially the lower zones. in case 1, thoracic computed tomography scans revealed mediastinal hilar lymphadenopathies, airspace opacities with air bronchograms, scattered ground-glass opacities, interstitial septal thickenings, and nodularities in the upper lobes without significant pleural and pericardial effusions. 6 acute renal failure was the other dominant clinical feature in mers-cov infection and is seen in at least six of the 34 reported cases. many of the remaining 28 severe cases probably also developed renal impairment, although their clinical details were not available. this was unusual, even when compared to sars in which 28.8% of the patients had abnormal urinalysis and detectable viral load by quantitative rt-pcr in urine, but only 6.7% developed acute renal failure with histological evidence of acute tubular necrosis and most did not require renal replacement therapy. 48 this clinical presentation correlates with the in vitro finding of efficient replication of mers-cov in kidney cell lines, including hek 293, vero, llc-mk2, and 769p. 33, 34, 49 more importantly, the presence of renal involvement appeared to be a poor prognostic factor, as those with renal failure either died or required renal replacement therapy, while two cases without renal impairment survived ( table 1 ). the lack of extrapulmonary lesions observed in the macaque model of mers-cov infection suggested that acute renal failure was more likely due to hypoxic damage than a direct viral cytopathic effect. 47 other clinical, laboratory and microbiological findings reported in mers-cov infection included pericarditis, disseminated intravascular coagulation, leukocytosis with neutrophilia and lymphopenia, thrombocytopenia, anemia, hyponatremia, hypoalbuminemia, elevated liver enzymes, lactate dehydrogenase, c-reactive protein, and procalcitonin levels, possible secondary bacterial pneumonia caused by klebsiella pneumoniae, staphylococcus aureus, and acinetobacter sp., and coinfection with other respiratory viruses, including influenza a(h1n1)pdm09 and type 2 parainfluenza virus. 6,7,10e13,50 it is interesting to note the absence of watery diarrhea in the acute phase of mers-cov infection, despite the in vitro finding of viral replication in the colonic cell line caco-2 (colorectal adenocarcinoma), in contrast to sars in which around 20% of patients developed enterocolitis. 1, 40 it remains to be seen in future case cohorts whether this is due to under-reporting, or a genuine difference in clinical presentations between the two diseases. together with severe acute respiratory and renal failure, these clinical features underscore the success of the innate immune evasion mechanisms of mers-cov in humans, leading to overwhelming infection and possible cytokine dysregulation, as reflected by the lack of interferon through inhibition of interferon regulatory factor family 3 (irf-3). 45, 49 the discovery of dipeptidyl peptidase 4 (dpp4), a multifunctional 766-amino-acid-long type-ii transmembrane glycoprotein exopeptidase expressed on human non-ciliated bronchial, renal, enteric, hepatic and prostatic epithelial cells, which is important in the regulation of hormone and chemokine bioactivity, glucose metabolism, t-cell activation, chemotaxis modulation, cell adhesion, apoptosis and regulation of tumorigenicity, as a functional receptor for mers-cov, provides further insights into the unique pattern of organ involvement and unusually severe clinical presentation of this emerging infection. 51 clinical utility and practical concerns of published laboratory diagnostic options several laboratory methods are available for establishing a virological diagnosis of mers-cov infection. the most definitive tests are viral culture from respiratory, fecal, urine, or tissue specimens, and/or a fourfold rise in the serum neutralizing antibody titers taken at 14 to 21 days apart. the first clinical isolate of mers-cov was cultured on monkey kidney cells, like vero and llc-mk2, which showed cytopathic effects of syncytium formation, rounding and detachment of cells. 6 subsequent studies have identified various human cell types, including bronchial epithelial, colonic, hepatic, renal, and neuronal cells, monocytes and histiocytes that support the replication of mers-cov. however, the use of viral culture is limited by the requirement of a biosafety level three setting, which is not available in most clinical microbiology laboratories. a serum neutralizing antibody test has the disadvantage of requiring convalescent sera and is therefore mainly used for diagnosis in the convalescent instead of acute phase. 13, 52 furthermore, the sera of sars patients may contain lowtiter cross-reactive neutralizing antibodies against mers-cov, which may lead to the wrong serodiagnosis, especially in countries where the general population had previous exposure. 53 it remains to be seen whether neutralizing antibodies against mers-cov might also crossreact with other closely related lineage c betacoronaviruses, like ty-batcov-hku4 and pi-batcov-hku5. 54 in most of the laboratory-confirmed cases, diagnosis was established by the detection of nucleic acid by rt-pcr of respiratory tract samples, including combined nose and throat swab, sputum, tracheal aspirate, bronchoalveolar lavage, and/or urine taken between day 5 (cases 3 and 11) and day 20 (case 5). 51 two highly sensitive real-time rt-pcr assays targeting regions upstream of the e gene (upe), with sensitivity of up to 3.4 rna copies per reaction and within an open reading frame (orf) 1b, with sensitivity of up to 64 rna copies per reaction, have been proposed for screening and confirmation of mers-cov infection, respectively, and are available in about half of the countries in the who european region. 55, 56 additional testing of other gene targets with partial or whole genome sequence analysis may also be used for confirmation. 55, 56 for example, pan-coronavirus rt-pcr targeting the rdrp gene was used in case 2 and a real-time quantitative rt-pcr assay targeting the n gene has been used in in vitro studies, and may also be useful for clinical diagnosis, although further evaluations are needed. 33 other potential diagnostic options which might be used in areas without rt-pcr are n or s protein-based enzyme-linked immunosorbent assays, which were shown to be highly sensitive and/ or specific for the diagnosis of sars. of note, the detection of other respiratory viruses in the respiratory tract specimen does not preclude the need for specific mers-cov testing in patients with epidemiological risk factors, or unusually severe disease, despite antiviral treatment as exemplified by the presence of coinfections in cases 10e12. the exceptionally high crude mortality rate of 60% in mers-cov infection is partly due to the lack of specific anticoronavirus treatment and effective vaccine. in most of the cases, intensive supportive treatment with extracorporeal membrane oxygenation, renal replacement therapy, and empirical broad-spectrum antibacterial and antiviral agents were used. case 2, who is still in a critical condition nearly 6 months after symptom onset, also received a corticosteroid in the initial phase of treatment, but its efficacy is unknown. its use might be limited by serious side effects and the availability of ecmo for organ support during the critical phase. alternatively, type i interferons appear to be promising therapeutic options, as mers-cov has been shown to be much more sensitive than sars-cov to the antiviral action of interferon in vitro. 49 the replication of mers-cov was shown to be reduced in human lung ex vivo organ cultures treated with type i interferons. 46 a recent study showed that while both interferon-a2b and ribavirin reduced the replication of mers-cov in vero and llc-mk2 cells, the combination of the two drugs achieved the same endpoints at a much lower concentration, which might facilitate their clinical applications. 57 other potential specific antiviral targets include type ii transmembrane serine proteases (tmprss2) and endosomal cathepsins, which are responsible for mers-cov s protein activation required for virus-cell fusion and entry of the virus into host cells. 54 the identification of dpp4, but not angiotensinconverting enzyme 2 (ace2), aminopeptidase n, and carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1), as a functional receptor for mers-cov implies that in vivo manipulation of dpp4 levels and development of inhibitors against the s1 domain-dpp4 interface might have potential therapeutic roles in mers-cov infection, as with ace2 analogues in the case of sars. 51 finally, the recognition of the predicted receptor-binding domain /critical neutralizing domain at residues 377 to 662 in the mers-cov s protein might facilitate vaccine development for this emerging infection. 58 in the past few months, following the who's announcement of the first two cases of mers-cov infection on september 23, 2012, clinicians and scientists worldwide have collaborated closely in an attempt to prevent a sars-like epidemic from happening again. the discovery of certain important characteristics of mers-cov including its genome arrangement, phylogenetic relatedness with other coronaviruses, in vitro tissue and species tropism, and functional receptor, enhanced our understanding of the clinical presentation, pathogenesis, epidemiology, and design of diagnostic and therapeutic options of this emerging novel human coronavirus. however, key questions concerning the definitive animal reservoirs of the virus, evolutionary process, transmissibility, and the prognostic factors and optimal treatment modalities of the infection, remain elusive. more importantly, the increasing number of laboratory-confirmed cases in the middle east, and the recent evidence of human-tohuman transmission in europe are suggestive of continuing viral adaptations in humans, which might precede a largescale epidemic. indeed, as of 23 june 2013, after the acceptance of the article, the total number of laboratoryconfirmed cases have increased to 70 with 39 fatalities. 59 additional clusters of cases involving household and/or hospital contacts were reported in ksa, italy, and tunisia. collaborations between global and local health authorities and their sustained support for further research on mers-cov are crucial to control this "new sars". severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection coronavirus as a possible cause of severe acute respiratory syndrome the severe acute respiratory syndrome relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans latest outbreak news from promed-mail: novel coronavirus e middle east recovery from severe novel coronavirus infection evidence of person-to-person transmission within a family cluster of novel coronavirus infections contact investigation of a case of human novel coronavirus infection treated in a german hospital world health organization. global alert and response: novel coronavirus infectioneupdate. geneva: who coronavirus diversity, phylogeny and interspecies jumping severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia molecular diversity of coronaviruses in bats coronavirus hku1 and other coronavirus infections in hong kong comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome covdb: a comprehensive database for comparative analysis of coronavirus genes and genomes comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events coexistence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku9 belonging to a novel betacoronavirus subgroup discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku14, from domestic rabbits recent transmission of a novel alphacoronavirus, bat coronavirus hku10, from leschenault's rousettes to pomona leafnosed bats: first evidence of interspecies transmission of coronavirus between bats of different suborders taxonomy of viruses. virus taxonomy: 2012 release (current genetic relatedness of the novel human lineage c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications on disease pathogenesis and clinical manifestation human coronavirus emc does not require the sarscoronavirus receptor and maintains broad replicative capability in mammalian cell lines bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia the united kingdom public health response to an imported laboratory confirmed case of a novel corona viral replication in the nasopharynx is associated with diarrhea in patients with severe acute respiratory syndrome viral loads in clinical specimens and sars manifestations clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study incubation period as part of the case definition of severe respiratory illness caused by a novel coronavirus lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential tropism and innate immune responses of the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures pneumonia from human coronavirus in a macaque model acute renal impairment in coronavirus-associated severe acute respiratory syndrome human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus world health organization. global alert and response: novel coronavirus infectione update. geneva: who dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections crossreactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests the spike-protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2 and is targeted by neutralizing antibodies detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction laboratory capability for molecular detection and confirmation of novel coronavirus in europe inhibition of novel b coronavirus replication by a combination of interferon-a2b and ribavirin a predicted receptor-binding and critical neutralizing domain in s protein of the novel human coronavirus hcov-emc global alert and response (gar): middle east respiratory syndrome coronavirus (mers-cov) e update key: cord-253337-xdexrlq3 authors: park, jung wan; lee, keon joo; lee, kang hyoung; lee, sang hyup; cho, jung rae; mo, jin won; choi, soo young; kwon, geun yong; shin, ji-yeon; hong, jee young; kim, jin; yeon, mi-yeon; oh, jong seok; nam, hae-sung title: hospital outbreaks of middle east respiratory syndrome, daejeon, south korea, 2015 date: 2017-06-17 journal: emerg infect dis doi: 10.3201/eid2306.160120 sha: doc_id: 253337 cord_uid: xdexrlq3 from may through july 2015, a total of 26 cases of middle east respiratory syndrome were reported from 2 hospitals in daejeon, south korea, including 1 index case and 25 new cases. we examined the epidemiologic features of these cases and found an estimated median incubation period of 6.1 days (8.8 days in hospital a and 4.6 days in hospital b). the overall attack rate was 3.7% (4.7% in hospital a and 3.0% in hospital b), and the attack rates among inpatients and caregivers in the same ward were 12.3% and 22.5%, respectively. the overall case-fatality rate was 44.0% (28.6% in hospital a and 63.6% in hospital b). the use of cohort quarantine may have played a role in preventing community spread, but additional transmission occurred among members of the hospital cohort quarantined together. caregivers may have contributed in part to the transmission. from may through july 2015, a total of 26 cases of middle east respiratory syndrome were reported from 2 hospitals in daejeon, south korea, including 1 index case and 25 new cases. we examined the epidemiologic features of these cases and found an estimated median incubation period of 6.1 days (8.8 days in hospital a and 4.6 days in hospital b). the overall attack rate was 3.7% (4.7% in hospital a and 3.0% in hospital b), and the attack rates among inpatients and caregivers in the same ward were 12.3% and 22.5%, respectively. the overall case-fatality rate was 44.0% (28.6% in hospital a and 63.6% in hospital b). the use of cohort quarantine may have played a role in preventing community spread, but additional transmission occurred among members of the hospital cohort quarantined together. caregivers may have contributed in part to the transmission. a few respiratory viruses constitute emerging threats to global health security (1); among them are middle east respiratory syndrome (mers) coronavirus (mers-cov), which has caused outbreaks in saudi arabia (2, 3) . the major mers outbreaks that occurred during 2012-2015 have been in or near the arabian peninsula. however, information on the epidemiologic features of mers is insufficient, especially for different environmental and cultural settings. the 2015 mers outbreak in south korea could provide more information about the epidemiology of mers because it was the largest outbreak outside the middle east (4). the first case of mers in south korea was reported on may 20, 2015. the patient had flown among several countries in the middle east (bahrein, the united arab emirates, saudi arabia, and qatar) and became the source of consecutive hospital-to-hospital transmissions after his return to south korea, which led to 186 laboratoryconfirmed cases (5) and 38 deaths. hospital-to-hospital transmission involved 17 hospitals and originated from 1 hospital (hospital p) (5, 6) . this transmission was attributable to "hospital shopping" by some mers patients (4, 5) and was particularly evident in daejeon, which is the fifth largest city in south korea. the index case-patient for nosocomial transmission in daejeon had initially traveled from his home city of daejeon to pyeongtaek, south korea, seeking healthcare at hospital p, after which he returned to daejeon. subsequently, 2 hospitals in daejeon experienced mers cases attributable to this patient. this index case-patient in daejeon was consecutively hospitalized at hospital a in daejeon during may 22-28, 2015, and at hospital b during may 28-30, 2015. thereafter, an additional 25 mers cases (14 in hospital a, 11 in hospital b) were reported. after the south korea government recognized the outbreak of mers in daejeon, cohort quarantine (isolation of persons who had been in contact with patients with confirmed cases in the hospital ward) was applied. this quarantine seems to have played a useful role in preventing the spread of mers-cov to the local community. we describe the mers case-patients, the epidemiologic features of the disease, and the quarantine policy used to prevent additional transmission. hospital a is a 300-bed general hospital in daejeon. the outbreak occurred in ward 51 on the fifth floor, where 13 rooms (5 with 7 beds, 6 with 4 beds, 1 with 2 beds, and 1 with 1 bed) are located. hospital b is an 800-bed university hospital in daejeon. the main outbreak occurred in ward 101 on the ninth floor, where 16 rooms (7 with 6 beds, 1 with 4 beds, 1 with 2 beds, and 7 with 1 bed) are located. epidemiologic investigators of the korea centers for disease control and prevention started their outbreak investigation with face-to-face interviews of the index casepatient in daejeon and the 25 additional case-patients with confirmed mers-cov infection. we collected data on the demographic characteristics and the clinical, contact, and mers-cov exposure histories and thoroughly reviewed the medical records of the case-patients to identify symptoms, underlying concurrent medical conditions, laboratory findings, and clinical courses of illness. clinical outcome was classified as recovery or death, and the ambulation status of the inpatients at the time of admission was clarified. we collected the names of inpatients, their room numbers, medical staff, and caregivers (family members or professionals hired by the family or hospital) exposed to mers-cov in each hospital. the duration and route of exposure were further determined by reviewing recordings from closed-circuit televisions placed in the hospitals. moreover, we used the floor plan of each hospital to estimate the spatial distributions and transmission routes of the virus within the hospitals. these estimates enabled us to identify a possible location of exposure and a transmission route for each confirmed case. when a patient with a confirmed case had experienced several possible exposures, we determined the most probable exposure by author consensus. persons who had had face-to-face contact with patients with confirmed cases were considered the closest contacts. when the data were ambiguous, the following were reviewed independently by the korea centers for disease control and prevention and the daejeon in-depth team: all potential exposures by symptom onset; disease duration; physical distance from a patient with a confirmed case; and infector factors including ambulation status, symptoms (including a productive cough), and sharing of caregivers. when a patient with a confirmed case had been subjected to several potential exposures, the most probable exposure was determined by consensus of the 2 teams. an expert member of the korean society of epidemiology reviewed all decisions. the process was repeated until a final consensus was obtained. sputum samples from the persons suspected of having mers were collected in sterile cups and sent to qualified local or national laboratories for confirmation. as a confirmatory test, a real-time reverse transcription pcr of nucleic acid extracted from sputum specimens was performed (5) . cycle threshold values were also measured to quantify viral loads. for each patient with a confirmed case, the korea centers for disease control and prevention assigned a case number according to the order of confirmation during the 2015 mers outbreak in south korea. for example, the case number of the index case-patient in daejeon was 16. the cases were described in case-series form. attack rates were calculated as the number of cases per number of exposed persons (defined as persons who had experienced face-to-face contact with a symptomatic mers case-patient in either hospital or as persons who had been in the same hospital ward as the symptomatic case-patients). such persons were identified from the outbreak investigation reports and the lists of those undergoing cohort or home quarantine. to assess differences in attack rates and case-fatality rates according to independent variables, we performed χ 2 and fisher exact tests by using sas software version 9.3 (sas institute, inc., cary, nc, usa). comparisons were considered significant at p<0.05 and marginally significant at p<0.1 (both p values were 2-tailed). we defined the incubation period as the time from exposure to onset of mers-associated symptoms, including nonspecific signs and symptoms such as fever, chills, cough, sore throat, sputum production, dyspnea, myalgia, headache, nausea, vomiting, diarrhea, and abdominal discomfort. if the exposure period was >2 days, a single interval-censored estimate of the incubation period was computed by using the earliest and latest dates of exposure and the date of symptom onset for each case-patient (coarsedatatools package in r statistical software version 3.2.2) (7). to construct cumulative fraction curves of all cases by incubation period, we calculated the log-normal density function by fitting the interval-censored data on incubation periods. to do this, we used the maximum-likelihood method and calculated the medians and 5th and 95th percentiles of the incubation periods. the daejeon index case-patient (case-patient 16), a 41-yearold man, lived in daejeon and was a former smoker (10 to 20 pack-years). he had undergone colon surgery in august 2014 at hospital p. the index case-patient of the mers outbreak in south korea (case-patient 1) was in hospital p during may 15-17, 2015. the daejeon index case-patient was admitted to hospital p at the same time (may 15-18, 2015) for a follow-up colonoscopy. after discharge on may 20, the daejeon index case-patient felt feverish and had chills, cough, general weakness, and diarrhea. because of these symptoms, he was hospitalized in hospital a in daejeon during may 22-28; the room was shared by 3 inpatients and 1 caregiver. because his symptoms did not improve, he was transferred to the emergency department at hospital b. after hospitalization in ward 101 in hospital b, he was suspected of having mers and was isolated in a negative-pressure room on may 30. ultimately, he became the 16th confirmed mers patient of 186 total case-patients during the 2015 outbreak. before the daejeon case-patient was isolated, those around him did not use protective equipment. therefore, virus was spread from him during his first 10 days of illness, before mers diagnosis and isolation. when we checked the closed-circuit television recordings from hospital a to estimate how many persons could have been in contact with the daejeon index case-patient, we found that he had been in several sections of the hospital ward, in particular those located on the left side of the nurse station. these sections included his admission room, a restroom, the nurse station, the foyer, and the hall in front of the elevators. the daejeon index case-patient had potentially contacted every inpatient in the same hospital ward. therefore, we classified all patients and caregivers in that ward as possible contacts. a total of 26 cases (including the index case) were confirmed in the 2 hospitals, and 11 case-patients died of mers (4 in hospital a and 7 in hospital b) (online technical appendix, https://wwwnc.cdc.gov/eid/article/ 23/6/16-0120-techapp1.pdf). other than the index case, 14 cases occurred in hospital a and 11 in hospital b. casepatients 30, 38, and 128 were admitted to the same room in hospital a as the daejeon index case-patient. case-patient 85 was a caregiver hired by case-patient 128, so she was in the same hospital room in hospital a during may 22-28. case-patients 23, 24, 31, 36, and 95 were admitted to the same room in hospital b as the daejeon index case-patient. case-patient 82 was the wife of case-patient 36; case-patient 106 was a caregiver hired by case-patient 36; and case-patient 127 was the wife of case-patient 24. therefore, when caregiving, they were in the same room as the daejeon index case-patient. the median age of the case-patients was 71 (interquartile range 38-86) years; 13 (52.0%) were male; 6 (24.0%) were commercial caregivers; and 3 (12.0%) were family caregivers. a total of 18 (72.0%) case-patients had underlying diseases; 7 (28.0%) had pulmonary diseases, such as asthma, chronic obstructive pulmonary disease, idiopathic pulmonary disease, lung cancer, and pulmonary tuberculosis. all patients reported fever. other signs and symptoms included chills (10 patients, 38.5%), cough (8, 30 .8%), sputum (6, 23.1%), myalgia (9, 34.6%), headache (4, 15.4%), dyspnea (6, 23.1%), nausea (3, 11.5%), diarrhea (6, 23.1%), table 1 . quarantine policy to prevent additional transmission of mers, daejeon, south korea* action  the cohort quarantine applied to admitted patients and their caregivers (professional or family) exposed to the mers case-patients.  inpatients admitted to the same hospital room before quarantine were quarantined in the same room because their degree of exposure was probably the same. their caregivers were also quarantined in the same room because of the need for caregiving.  the medical staff (physicians, nurses, and medical technologists) exposed to the mers case-patients were subjected to home quarantine. however, members of the households of medical staff were not subjected to home quarantine until and unless that medical staff member exhibited any symptoms. contact between household members and the medical staff member was severely restricted.  the wards under cohort quarantine were controlled by unexposed medical staff using level d protectors (microguard 2000; 3m, bracknell, uk). each protector included an n95 mask, protective glasses, a whole-body protective gown, gloves, and boots.  the body temperature of persons (including inpatients and caregivers) and medical staff admitted to cohort or home quarantine was checked, and these persons were clinically interviewed twice daily. if they reported any symptoms (including a febrile sensation or chills) or if they were asymptomatic but with a body temperature >37.5c°, they were immediately placed in a quarantined area at each hospital. the kcdc performed laboratory tests at this stage; the results were available 3 d later. if the doctor in charge strongly suspected mers, that patient could be transferred, with careful precautions, to a national isolation hospital within 1 d.  all wards were disinfected by use of sodium hydrosulfite, 80% (vol/vol) alcohol, and 2% (vol/vol) chlorhexidine twice during each shift, thus 6 times/d.  south korea operates a nationwide medical insurance scheme; all costs incurred by mers patients were covered.  persons with confirmed mers were transferred to another quarantine room that had negative-pressure equipment. strategies for caregivers  the infection control team carefully explained the risk for mers and the need for cohort quarantine to all caregivers. some caregivers did not wish to remain in hospital wards with inpatients. they were taken home and placed in in-home quarantine and used the same mers quarantine strategy applicable to medical staff in close contact with the patients.  caregivers attended only noninfected inpatients who required total care. if an inpatient was confirmed to have mers, nursing care was provided by professional nurses wearing protectors.  the infection control team continuously educated caregivers on how mers was transmitted and how to prevent infection. caregivers were told to wear protectors (n95 masks, vinyl gowns, and gloves) and to not touch each other. however, during the first week of quarantine, checks of closed-circuit television footage showed that the protector and contact rules were sometimes not obeyed in hospital a.  hospital a designated 2 rooms for caregivers in the quarantine ward. the caregivers could use these rooms when they were not actively engaged in patient care. to prevent the spread of mers-cov to the local community, on june 1, 2015, the government of south korea ordered cohort quarantine, which hospitals a and b followed (table 1) . persons with a history of exposure to patients with confirmed mers were isolated in the same hospital ward. after the index case-patient in daejeon spread mers-cov in daejeon, the first case occurred on may 30, 2015, and the last on june 15, 2015 (total outbreak duration 17 days) ( figure 1 ). the epidemic curve for hospital a suggested a relatively sporadic pattern compared with that for hospital b. the peak in hospital b comprised mostly patients who shared a hospital room with the index case-patient. most mers cases appeared later in professional or family caregivers rather than in inpatients. the estimated median incubation period for confirmed cases was 6.1 (95% ci 4.7-7.5) days ( figure 2 ). incubation periods were 8.8 (95% ci 7.2-10.4) days for hospital a and 4.6 (95% ci 2.9-6.2) days for hospital b. in hospital a, the index case-patient was admitted to room 5101, in sector a (figure 3) . thereafter, 12 casepatients were in sector a, and 1 was in sector b. however, the case-patient in sector b had a history of contact with case-patient 85, who was transferred to sector b from sector a for quarantine. most case-patients were presumed to have been infected by the daejeon index case-patient case-patient 38 is not included because date of illness onset is unknown. black, weekday; blue, saturday; red, sunday or holiday. (case-patient 16). however, 3 instances of other transmission were noted: case-patient 85 to case-patient 130, casepatient 54 to case-patient 172, and several case-patients to case-patient 129. for this last instance of transmission, we could not identify the most probable source, because many possible exposures were evident (case-patients 54, 84, 86, 87, 107, and 149). in hospital b, the index case-patient was admitted to room 1007, located on the upper side of ward 101 (sector c). eight case-patients were in sector c. case-patient 83 was in room 1013 on the opposite side of ward 101 (sector d). case-patient 45 was in the emergency room and ward 101 with the index case-patient. case-patient 148 was presumed to have been infected by case-patient 36 during performance of cardiopulmonary resuscitation in the intensive care unit. a total of 14 cases developed among 301 persons exposed in hospital a (attack rate 4.7%) and 11 among 371 persons exposed in hospital b (attack rate 3.0%) ( table 2 ). the attack rates for the sectors hosting the index case-patients (sector a of hospital a, sector c of hospital b; figure 3 ) were higher than those for other sectors (sector b of hospital a, sector d of hospital b; figure 3 ) (31.6% vs. 2.4% in hospital a, p<0.05; 18.2% vs. 6.5% in hospital b; table 3 ). the probability of infection for a person admitted to the same rooms as the index case-patient was 75.0%. in both hospitals, attack rates were somewhat higher for caregivers (22.5%) than for inpatients (12.3%), although statistical significance was not attained. the overall case-fatality rate was 44% (table 4 ). this rate was higher for patients in hospital b (63.6%) than for those in hospital a (28.6%), although statistical significance was not attained. during the mers outbreak in south korea, 25 confirmed cases (including 11 deaths) occurred in daejeon, all associated with the same index case-patient. two hospitals were affected. the incubation periods and case-fatality rates for the 2 hospitals differed. under the south korea healthcare system, patients can visit secondary hospitals and the emergency rooms of tertiary hospitals without limitation (5) , which probably facilitated nosocomial transmission of mers-cov. indeed, the outbreak in daejeon accounted for 1 of the 3 major mers-cov outbreaks in south korea. these observations underscored the importance of the outbreak in daejeon, leading the south korea government to focus resources on controlling transmission of the virus. the estimated median incubation period for mers during the outbreak we report was similar to that for outbreaks in the eastern province of saudi arabia in 2013 (1) . incubation period estimates may differ, depending on the method used to select exposure: the most probable exposure versus overlapping exposures. in our study, the incubation periods estimated by using both methods were similar. the incubation period estimated by using the most probable exposure method was 6.1 (95% ci 4.7-7.5) days, and that estimated by using the overlapping exposures method was 5.6 (95% ci 4.2-6.9) days. the overall attack rate among all exposed persons in daejeon was similar to that for pyeongtaek (5) . the case-fatality rate of the outbreak in daejeon was lower than that in the eastern province of saudi arabia in 2013 (1) but similar to that in jeddah, saudi arabia, in 2014 (3). our results indicate various epidemiologic characteristics of mers-cov. all persons acquired infection in a hospital setting, which is consistent with the previous outbreak in saudi arabia, in which nosocomial spread curves indicate estimated cumulative fractions of cases corresponding to the incubation periods, estimated by creating lognormal density functions fitting the observed data. horizontal lines indicate 95% cis for the 5th, 50th, and 95th percentiles of the estimated incubation periods. a) total; estimated median incubation period was 6.1 (95% ci 4.7-7.5) days. b) hospital a; estimated median incubation period was 8.8 (95% ci 7.2-10.4) days. c) hospital b; estimated median incubation period was 4.6 (95% ci 2.9-6.2) days. hospital outbreaks of mers, south korea, 2015 was a major route of mers-cov transmission (1). the characteristics of the specific hospital seemed to affect attack rates and case-fatality rates. the index case-patient in daejeon was consecutively admitted to 2 hospitals. the fifth floor of hospital a specializes in senile patients, most of whom have chronic illnesses, including parkinson's disease, paraplegia attributable to old infarctions, or amyotrophic lateral sclerosis. most beds on the fifth floor are occupied by bedridden patients. the attack rate among caregivers was higher in hospital a than in hospital b. because immobile patients require personal caregiving, their caregivers were required to be in prolonged close contact with patients, which might have resulted in a higher attack rate. hospital b is a university hospital and thus contained more severely ill patients than hospital a. ward 101, to which the daejeon index case-patient was admitted, is the main ward of the pulmonary medicine department. we presumed that the case-fatality rate was higher for hospital b than hospital a because of underlying pulmonary disease, which has been reported to be a risk factor for development of more severe diseases (8) . generally, cohort quarantine may be useful as an infection-control tool to limit virus transmission in hospitals in which susceptible inpatients are gathered or to more effectively detect infected patients (9) . hospitals a and b applied cohort quarantine. in this situation, cohort quarantine had several advantages and disadvantages. the primary purpose was to prevent the spread of mers-cov to the local community. after applying cohort quarantine, no further spread of mers-cov occurred other than within hospitals a and b. this result may have been achieved by quarantining all persons who had been in contact with mers-cov-infected patients and by refusing hospital entry to all susceptible persons. in addition, more cases were diagnosed promptly by active surveillance of the cohort. however, this policy had a limitation. one cohort accommodated inpatients and caregivers in the same hospital room; thus, if 1 person in the cohort was infected by mers-cov, others were exposed, increasing the probability of mers-cov transmission. this practice raises an ethical issue in terms of whether letting persons stay in the same room with potential mers patients is justified by the purpose of preventing transmission of the virus to the community. some caregivers at hospital a may have had difficulty complying with the quarantine policy (the protector and contact rules) because they cared for immobile patients. thus, this practice may have increased transmission within the hospital. we identified several cases with uncommon routes of transmission. case-patient 148 was the head nurse of the intensive care unit to which case-patient 36 was admitted. when case-patient 36 experienced cardiac arrest, that nurse performed cardiopulmonary resuscitation while wearing a level d protector. however, afterward, she may have been exposed to mers-cov when she wiped sweat with her bare arm. case-patient 143 was an employee of hospital a; he worked in information technology. he was employed by hospital a during january-may 30, 2015, and his bedroom was located on the seventh floor. his routine work routes, shown on closedcircuit television, did not reveal any close contact with case-patients; thus, the transmission route was unclear. we presume that he was infected by fomites in an elevator or exposed to a patient in a place lacking closedcircuit television coverage. the transmission route for case-patient 83 was also unidentified. it is possible that some medical staff and caregiver, contaminated with mers-cov after visiting room 1007, may have visited case-patient 83 in room 1013. of note, when case-patient 83 was exposed to mers-cov, the outbreak in daejeon had not yet been recognized and hospital b had not yet implemented infection control strategies (e.g., handwashing; wearing gloves, masks, and vinyl gowns). this study had several limitations. first, we cannot be certain that all chains of infection between case-patients have been identified. we did not perform serologic analyses to seek cases that were potentially missed; such missed cases may have affected the incubation period estimates and case-fatality rate. second, closed-circuit television may not have captured all relevant movements. in conclusion, in 2015, daejeon experienced a hospital-associated outbreak of mers-cov. two hospitals experienced nosocomial outbreaks, and virus transmission was evident among mostly inpatients and caregivers. to prevent the spread of the virus to the local community, we developed a unique and successful cohort quarantine policy. however, ethical issues associated with this policy require thorough discussion by policy makers. middle east respiratory syndrome coronavirus (mers-cov) is a novel cov known to cause severe acute respiratory illness in humans; approximately 40% of confirmed cases have been fatal. human-to-human transmission and multiple outbreaks of respiratory illness have been attributed to mers-cov, and severe respiratory illness caused by this virus continues to be identified. as of february 23, 2014, the world health organization has reported 182 laboratory-confirmed cases of mers-cov infection, including 79 deaths, indicating an ongoing risk for transmission to humans in the arabian peninsula. visit our website to listen: https://www2c.cdc.gov/podcasts/player.asp?f=8631627 ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: epidemiology and disease control measures mers-cov outbreak in jeddah-a link to health care facilities middle east respiratory syndrome coronavirus outbreak in the republic of korea. osong public health and research perspectives mers outbreak in korea: hospital-to-hospital transmission epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital estimating incubation period distributions with coarse data epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study understanding, compliance and psychological impact of the sars quarantine experience address for correspondence: hae-sung nam, department of preventive medicine and public health dr. park is a medical specialist in infectious diseases and an epidemiologist at the korea centers for disease control and prevention, in cheongju, south korea. his research interest is mers-cov infection. key: cord-252600-bvh1o64r authors: galasiti kankanamalage, anushka c.; kim, yunjeong; damalanka, vishnu c.; rathnayake, athri d.; fehr, anthony r.; mehzabeen, nurjahan; battaile, kevin p.; lovell, scott; lushington, gerald h.; perlman, stanley; chang, kyeong-ok; groutas, william c. title: structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element date: 2018-04-25 journal: european journal of medicinal chemistry doi: 10.1016/j.ejmech.2018.03.004 sha: doc_id: 252600 cord_uid: bvh1o64r abstract there are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of middle east respiratory syndrome coronavirus (mers-cov) infections. mers-cov 3cl protease is essential for viral replication; consequently, it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting mers-cov. we describe herein the structure-guided design and evaluation of a novel class of inhibitors of mers-cov 3cl protease that embody a piperidine moiety as a design element that is well-suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and pk properties. the mechanism of action of the compounds and the structural determinants associated with binding were illuminated using x-ray crystallography. proteins, s (spike glycoprotein), e (envelope protein), m (membrane glycoprotein), and n (nucleocapsid protein), which play a critical role in virion-cell receptor binding, replication and virion assembly, are located at the 3 0 end of the genome [1, 12] . coronavirus entry is initiated by the binding of the spike protein (s) to cell receptors, specifically, dipeptidyl peptidase 4 (ddp4) and angiotensin converting enzyme 2 (ace2) for mers-cov and sars-cov, respectively [1e5] . entry into cells requires host proteases for cleavage at two sites in the s protein, in the case of most cov [13, 14] translation of the genomic mrna of orf1a yields polyprotein pp1a, while a second polyprotein (pp1b) is the product of a ribosomal frame shift that joins orf1a together with orf1b. orf1a encodes a papain-like cysteine protease (plpro) and a 3c-like cysteine protease (3clpro). polyproteins pp1a and pp1b are processed by 3clpro (11 cleavage sites) and plpro (3 cleavage sites) resulting in sixteen mature nonstructural proteins, including rna-dependent rna polymerase (rdrp) and helicase, which play important roles in the transcription and replication of coronaviruses (fig. 1 ). both proteases are essential for viral replication, making them attractive targets for drug development [9,10,15e17] . mers-cov 3clpro is a chymotrypsin-like cysteine protease having a catalytic cys148-his41 dyad and an extended binding site [18e21] . the protease displays a stringent primary substrate specificity for a p 1 gln residue [22] and has a strong preference for a p 2 leu residue. the p 3 residue side chain is oriented toward the solvent while the s 4 subsite is shallow, preferring a small hydrophobic p 4 residue (ala). functional and structural studies have delineated the similarities between the 3clpro of coronaviruses that can be exploited in the design of broad-spectrum inhibitors [23] . we have recently reported the first demonstration of clinical efficacy of a coronavirus protease inhibitor (a dipeptidyl aldehyde bisulfite adduct inhibitor designated gc376) [24, 25] . specifically, administration of gc376 to cats infected with fipv, a coronavirus that is 100% fatal in cats, reversed the progression of fatal fip and resulted in clinical remission in a majority of animals (>90%). since fip disease progression is quite rapid and its pathogenesis primarily immune-mediated, features shared by mers-cov, we hypothesized that a viral protease inhibitor could reverse the pathogenesis of mers-cov in affected hosts. interrogation of this hypothesis entailed, as a first step, the design of a new and versatile class of peptidomimetic inhibitors of mers-cov 3cl protease. we describe herein the structure-guided design of inhibitors of mers-cov 3clpro that embody a piperidine moiety as a novel design element, as well as pertinent structural and biochemical studies. these inhibitors were also examined against other coronaviruses, including sars-cov, fipv and mhv to evaluate the spectrum of activity against multiple coronaviruses. the structure-guided design of inhibitor (i) encompassed the following steps: (a) we first determined a high resolution x-ray crystal structure of mers-cov 3clpro in complex with gc376 ( fig. 2/panel a) . examination of the active site of the complex revealed that the aldehyde bisulfite adduct had reverted to the precursor peptidyl aldehyde, which subsequently formed a tetrahedral hemi-thioacetal upon reaction with the active site cys148. notably, the electron density at this stereocenter was consistent with the formation of both r and s enantiomers at the covalent binding site (also observed for the other structures described in the following sections). the structure reveals a network of backbone hydrogen bonds which ensure correct positioning of the inhibitor to the active site, as well as two critical hydrogen bonds with the p 1 gln surrogate [26] side chain. the inhibitor p 2 leu side chain is ensconced in the hydrophobic s 2 subsite of the enzyme. importantly, the structure shows a hydrophobic-driven interaction between the benzyl group of the inhibitor and the g-lactam ring of the gln surrogate side chain; (b) based on the forgoing, we reasoned that extending the "cap" would allow the inhibitor to assume an extended conformation and orient the phenyl ring toward the hydrophobic s 4 pocket of the enzyme. validation of this idea was obtained by synthesizing extended inhibitor gc813 and determining a high resolution x-ray crystal structure of the mers-cov 3clpro:gc813 complex ( fig. 2/panel b) . the m-cl phenethyl side chain is clearly shown to occupy the hydrophobic s 4 subsite. in addition to an array of h-bonds with gln192, glu169, and gln167 and the backbone of the inhibitor, which serve to correctly position the inhibitor at the active site, the inhibitor interacts with the s 1 , s 2 and s 4 subsites, but not the s 3 subsite; (c) we hypothesized that the attachment of a piperidine ring to the peptidyl component would yield a structurally novel peptidomimetic (i) capable of (1) orienting recognition elements r 3 and r 4 in a correct vector relationship for optimal interactions with the s 3 and s 4 subsites, (2) rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor with potentially diminished pk liabilities and, (3) providing a flexible means for the structure-guided parallel optimization of admet/pk and physicochemical properties using diversity sites r 3 and r 4 in inhibitor (i) (fig. 3) . in summary, the piperidine-based design strategy is a hitherto unrecognized effective means of rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor capable of engaging in optimal binding interactions with all four s 1 -s 4 subsites but which, however, is anticipated to display diminished pk liabilities due to its reduced peptidyl character. furthermore, the aforementioned piperidine-based design strategy has wide applicability and can be extended to any protease with an extended binding site. preliminary evidence in support of this approach is provided by the results of enzyme and cell-based screening of derivatives of (i) (tables 1 and 2) , as well as the results of structural studies (vide infra) (see table 3 ). the synthesis of final compounds 9(a-f) and 10(a-f) is outlined in scheme 1. 1-boc-4-piperidinone was reacted with different grignard reagents to yield the corresponding 1-boc-4-piperidinol derivatives (1c and 1e). refluxing (l) leucine methyl ester hydrochloride with trichloromethyl chloroformate yielded the isocyanate which was reacted with (1c and 1e, or commercially-available nsubstituted 4-piperidinol 1a) to form the corresponding carbamate adducts (4a, 4c and 4e) that were hydrolyzed to the corresponding acids (5a, 5c and 5e) with lithium hydroxide in aqueous thf. subsequent coupling with glutamine surrogate methyl ester hydrochloride 11 afforded the desired dipeptidyl esters (6a, 6c and 6e) which were either treated with lithium borohydride directly or were first treated with dry hcl in dioxane followed by reaction with an alkyl sulfonyl chloride or alkyl chloroformate, to yield esters (7b, 7d and 7f) prior to reduction with lithium borohydride, to yield alcohols 8 (a-f). dess-martin oxidation, followed by flash chromatography, yielded pure aldehydes 9(a-f). the enantiomeric purity of the aldehydes was consistently high, with the amount of epimerized aldehyde ranging between 0 and 10%. the corresponding bisulfite adducts 10(a-f) were readily obtained as white solids by stirring the aldehydes with sodium bisulfite in an ethyl acetate/ water mixture. the synthesized compounds are listed in table 1 . the inhibitory activity of the synthesized compounds against 3clpro of mers-cov, sars-cov or fipv, and the antiviral activity of two representative compounds (compounds 10a and 10c) in a cellbased system including mers-cov, fipv and mhv were evaluated as described in the experimental section. the ic 50, ec 50, and cc 50 values, are listed in tables 1 and 2 these are the average of at least two determinations. it is evident that derivatives of (i) function as highly potent inhibitors of all tested coronaviruses in enzyme (table 1) and cell based assays (table 2 and fig. 4 ). more importantly, representative aldehyde bisulfite adduct compounds 10a and 10c display potent inhibition toward mers-cov in both enzyme and cell-based systems, with low cytotoxicity (cc 50 > 100 mm) ( table 2 and fig. 4 ). for example, compound 10a has a selectivity index (si ¼ cc 50 /ec 50 ) of >250. with the exception of compounds 9e-10e, the aldehyde and aldehyde bisulfite adducts were found to have comparable in vitro potency toward mers-cov 3clpro. furthermore, pharmacological activity was found to be dependent on the nature of the r 3 group (compounds 9e-10e are 10-fold less active toward mers-cov 3clpro than compounds 9a-d, 10a-d and 9f-10f). in order to establish the mechanism of action of (i), as well as obtain structural information that can be used to guide the optimization of pharmacological activity, the high resolution x-ray crystal structures of several derivatives of (i) bound to mers-cov 3clpro were determined, including the cocrystal structure of the mers-cov 3clpro:inhibitor 10c complex (fig. 5a) . the formation of a tetrahedral adduct via the reaction of the aldehyde, generated from aldehyde bisulfite adduct 10c under the crystallization conditions used [27, 28] , with the active site cysteine (cys148) is clearly evident, confirming the mechanism of action of (i). inspection of the structure reveals the presence of prominent electron density consistent with the structure of inhibitor 10c; however, the n-bocpiperidinyl moiety was disordered. the position and orientation of the benzyl group suggest that the piperidine ring is likely projecting toward the s 4 subsite. inhibitor 10c is bound to the active site of the enzyme via a network of backbone h-bonds with gln192, gln167, and glu169 (fig. 5b ). additionally, a h-bond with his41 serves to stabilize the hemi-thioacetal tetrahedral adduct. also clearly evident are three critical h-bonds involving the p 1 gln surrogate ring oxygen and nitrogen with glu169, his166 and phe143. the h-bonding interactions are near identical to those of inhibitor gc813 (fig. 2/panel b) . the structural complementarity of inhibitors 10c and gc813 is also evident in the electrostatic surface representation of the enzyme with the two inhibitors nestled in the active site (fig. 6) . the cocrystal structure of the mers-cov 3clpro:aldehyde bisulfite adduct 10e complex also showed that, under the crystallization conditions used, the aldehyde bisulfite adduct reverted to the precursor aldehyde, which subsequently formed a tetrahedral adduct with the active site cysteine (cys148) (fig. 7a ). the piperidinyl moiety was disordered and consequently its precise location could not be discerned. however, inhibitor 10e is engaged in the same h-bonding interactions as inhibitor 10c (fig. 7b ). mers-cov constitutes a global public health concern. there are currently no licensed vaccines or antiviral drugs for the prevention and treatment of coronavirus infections. we disclose herein for the first time the design and utilization of a general class of piperidinebased peptidomimetic inhibitors of coronavirus 3cl proteases. attachment of the piperidine moiety to a dipeptidyl component permits the resultant hybrid inhibitor to engage in favorable binding interactions with the s 3 and s 4 subsites of the enzyme. more importantly, the approach disclosed herein can be extended to other proteases of medical relevance. finally, the disclosed compounds potently inhibit mers-cov, and their mechanism of action and mode of binding to mers-cov 3cl protease have been illuminated using x-ray crystallography. reagents and dry solvents were purchased from various chemical suppliers (aldrich, acros organics, chem-impex, tci america, and bachem) and were used as obtained. silica gel (230e450 mesh) used for flash chromatography was purchased from sorbent technologies (atlanta, ga). thin layer chromatography was performed using analtech silica gel plates. visualization was accomplished dropwise under a n 2 atmosphere to a solution of 1-boc-4piperidinone (10 mmol) in dry thf (15 ml) in an ice bath kept at 0 c. the reaction mixture was stirred for 3 h at room temperature under a n 2 atmosphere while monitoring completion of the reaction by tlc. the reaction mixture was diluted with water (25 ml) and the solution was acidified to ph~3 using 5% hydrochloric acid. the solvent was removed on the rotary evaporator and the residue was extracted with ethyl acetate (75 ml) and the layers separated. the organic layer then washed with brine (40 ml) and dried over anhydrous sodium sulfate, filtered and concentrated to yield a colorless oily product which was purified by flash chromatography to yield 1c and 1e. 4.1.2. synthesis of (l) leucine methyl ester isocyanate 3 (l) leucine methyl ester hydrochloride 2 (100 mmol) was placed in a dry 500-ml rb flask and then dried overnight on the vacuum pump. the flask was flushed with nitrogen and dry dioxane (200 ml) was added followed by trichloromethyl chloroformate (29.67 g, 150 mmol), and the reaction mixture was refluxed for 10 h. the solvent was removed on the rotary evaporator and the residue was vacuum distilled to yield pure isocyanate 3 as a colorless oil [27, 28] . table 3 crystallographic data for mers-cov 3clpro in complex with compounds gc376, gc813, 10c and 10e. mers-cov 3clpro: gc376 mers-cov 3clpro: compound 10c mers-cov 3clpro: compound 10e hkl. c r factor ¼ ʃ hkl jjf obs (hkl) j -jf calc (hkl) jj/ʃ hkl jf obs (hkl)j; rfree is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement. d r meas ¼ redundancy-independent (multiplicity-weighted) r merge. [42, 43] r pim ¼ precision-indicating (multiplicity-weighted) r merge. [44, 45] . e cc 1/2 is the correlation coefficient of the mean intensities between two random half-sets of data [46, 47] . 4.1.3. synthesis of substituted piperidine-derived carbamates 4a, 4c and 4e. general procedure a solution of substituted or unsubstituted 1-boc-4-piperidinol (1c, 1e or 1a) (20 mmol) in dry acetonitrile (15 ml) was treated with triethylamine (4.05 g, 40 mmol) followed by the amino acid methyl ester isocyanate 3 (20 mmol). the resulting solution was refluxed for 2 h and then allowed to cool to room temperature. the solution was concentrated and the residue was taken up in ethyl acetate (75 ml). the organic layer was washed with 5% hcl (2 â 20 ml) and brine (20 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, leaving compounds 4a, 4c and 4e as colorless oils. 4.1.4. synthesis of acids 5a, 5c and 5e. general procedure a solution of ester (4a, 4c or 4e) (20 mmol) in tetrahydrofuran (30 ml) was treated with 1 m lioh (40 ml). the reaction mixture was stirred for 3 h at room temperature and the disappearance of the ester was monitored by tlc. most of the solvent was evaporated off and the residue was diluted with water (25 ml). the solution was acidified to ph~3 using 5% hydrochloride acid (20 ml) and the aqueous layer was extracted with ethyl acetate (3 â 100 ml). the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to yield the corresponding compounds 5a, 5c and 5e as colorless oils. 4.1.5. synthesis of compounds 6a, 6c and 6e. general procedure edci (2.40 g, 12.5 mmol, 1.25 eq) and hobt (1.92 g, 12.5 mmol, 1.25 eq) were added to a solution of compound (5a, 5c or 5e) (10 mmol) in dry dmf (20 ml) and the mixture was stirred for 30 min at room temperature. in a separate flask, a solution of deprotected glutamine surrogate 11 (2.23 g, 10 mmol) in dmf (15 ml) cooled to 0e5 c was treated with diisopropylethylamine (diea) (9.5 g, 40 mmol, 4 eq), stirred for 30 min, and then added to the reaction mixture containing the acid. the reaction mixture was stirred for 12 h while monitoring the reaction by tlc. the solvent was removed and the residue was partitioned between ethyl acetate (100 ml) and 10% citric acid (2 â 40 ml). the layers were separated and the ethyl acetate layer was further washed with saturated aqueous nahco 3 (40 ml), followed by saturated nacl (50 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a yellow-colored oily product. purification by flash chromatography yielded esters 6a, 6c and 6e as white solids. 4.1.6. synthesis of compounds 7b, 7d and 7f. general procedure 4 m hcl in dioxane (8 ml) was added to a solution of compound (6a, 6c and 6e) (10 mmol) in dry dcm (5 ml) and the mixture was stirred for 1 h at room temperature. the solvent was removed and the residue was dried under high vacuum for 2 h before the product was dissolved in dry thf (20 ml). an appropriate alkyl sulfonyl chloride or alkyl chloroformate derivative (11 mmol/1.1 eq) was added to the solution with stirring. the reaction mixture was stirred for 12 h at room temperature and the residue was dissolved in ethyl acetate (50 ml) and washed with 5% hcl (2 â 20 ml). the ethyl acetate layer was further washed with saturated nacl (20 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to yield a crude product. purification by flash chromatography yielded the corresponding esters 7b, 7d and 7f as white solids. lithium borohydride (2 m in thf, 7.5 ml, 15 mmol) was added dropwise to a solution of ester (6 or 7) (5 mmol) in anhydrous thf (30 ml), followed by absolute ethyl alcohol (15 ml) and the reaction mixture was stirred at room temperature overnight. the reaction mixture was then acidified by adding 5% hcl and the ph adjusted tõ 2. removal of the solvent left a residue which was taken up in ethyl acetate (100 ml). the organic layer was washed with brine (25 ml), dried over anhydrous sodium sulfate, filtered, and concentrated to yield compounds 8 (a-f) as white solids. compound 8 (a-f) (5 mmol) was dissolved in anhydrous dichloromethane (50 ml) under a nitrogen atmosphere and cooled to 0 c. dess-martin periodinane reagent (3.18 g, 7.5 mmol, 1.5 eq) was added to the reaction mixture with stirring. the ice bath was removed and the reaction mixture was stirred at room temperature for 3 h (monitoring by tlc indicated complete disappearance of the starting material). a solution of 10% aqueous sodium thiosulfate (20 ml) was added and the solution was stirred for another 15 min. the aqueous layer was removed and the organic layer was washed with 10% aqueous sodium thiosulfate (20 ml), followed by saturated aqueous sodium bicarbonate (2 â 20 ml), water (2 â 20 ml) and brine (20 ml). the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. the yellow residue was purified by flash chromatography (silica gel/methylene chloride/ ethyl acetate/methanol) to yield a white solid 9 (a-f). absolute ethanol (12 ml) was added to a solution of aldehyde 9 (a-f) (5 mmol) in dry ethyl acetate (20 ml) with stirring, followed by a solution of sodium bisulfite (540 mg; 5 mmol) in water (5 ml) and the reaction mixture was stirred for 3 h at 50 c. the reaction mixture was allowed to cool to room temperature and then vacuum filtered. the solid was thoroughly washed with absolute ethanol and the filtrate was dried over anhydrous sodium sulfate, filtered, and concentrated to yield a yellowish oil. the oily product was treated with ethyl ether (2 â 50 ml) to form a white solid. the white solid was stirred with ethyl ether (30 ml) and ethyl acetate (15 ml) for 5 min. careful removal of the solvent using a pipette left the corresponding aldehyde bisulfite adducts 10 (a-f) as white solids. the fret protease assay was performed by preparing stock solutions of the substrate (dabcyl-ktsavlq/sgfrkme-edans derived from the cleavage sites on the viral polyproteins of sars-cov) and inhibitor in dmso and diluting into assay buffer which was comprised of 20 mm hepes buffer, ph 8, containing nacl (200 mm), edta (0.4 mm), glycerol (60%), and 6 mm dithiothreitol (dtt). the expression and purification of the 3clpro of mers-cov, sars-cov or fipv was performed by a standard method described previously by our lab [24, 29] . the protease (3clpro of mers-cov, sars-cov or fipv) was mixed with serial dilutions of each compound or with dmso in 25 ml of assay buffer and incubated at 37 c for 30 min, followed by the addition of 25 ml of assay buffer containing substrate. fluorescence readings were obtained using an excitation wavelength of 360 nm and an emission wavelength of 460 nm on a fluorescence microplate reader (flx800; biotec, winoosk, vt) 1 h following the addition of substrate. relative fluorescence units (rfu) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously [29] . the dosedependent fret inhibition curves were fitted with a variable slope by using graphpad prism software (graphpad, la jolla, ca) in order to determine the ic 50 values of the inhibitors. the effects of compounds 10a and 10c on the replication of mers-cov, fipv or mhv-a59 were examined in vero81, crfk or ccl9.1 cells, respectively [30] . briefly, confluent and semi-confluent cells were infected at an moi of 0.01 pfu/cell. following adsorption, cells were incubated with medium containing dmso (<0.1%) or each compound (up to 100 mm) for 48 h. after incubation, viral titers were determined with a tcid 50 (fipv or mhv) or plaque assay (mers-cov). ec 50 values were determined using graphpadprism software [31]. the cytotoxic dose for 50% cell death (cc 50 ) for compounds 10a and 10c was determined in vero81, crfk or ccl9.1 cells. confluent cells grown in 96-well plates were treated with various concentrations (1e100 mm) of each compound for 72 h. cell cytotoxicity was measured by a cytotox 96 nonradioactive cytotoxicity assay kit (promega, madison, wi). the in vitro therapeutic index was calculated by dividing the cc 50 by the ec 50. purified mers-cov 3clpro, in 100 mm nacl, 20 mm tris ph 8.0, was concentrated to 8 mg/ml (0.5 mm). stock solutions of 100 mm gc376, gc813, compound 10c or compound 10e were prepared in dmso and the complex with mers 3clpro was prepared by mixing the concentrated protein supplemented with 3 mm compound and incubating overnight at 4 c. all crystallization experiments were conducted using compact 300 (rigaku reagents) sitting drop vapor diffusion plates at 20 c using equal volumes of protein and crystallization solution equilibrated against 75 ml of the latter. crystals of mers 3clpro in complex with gc813, compound 10c and compound 10e that displayed a prismatic morphology were obtained from the index ht screen (hampton research) condition g10 (25% (w/v) peg 3350, 100 mm bis-tris ph 5.5, 200 mm mgcl 2 ) in 1e2 days. crystals of the gc376 complex were obtained from the index ht screen (hampton research) condition e6 (30% (v/v) peg 550 mme, 100 mm bis-tris ph 6.5, 50 mm cacl 2 ). samples were transferred to a fresh drop containing 80% crystallant and 20% (v/v) peg 200 before storing in liquid nitrogen. x-ray diffraction data were collected at the advanced photon source beamline 17-id using a dectris pilatus 6 m pixel array detector. intensities were integrated using xds [32, 33] using autoproc [34] and the laue class analysis and data scaling were performed with aimless [35] , which suggested that the highest probability laue class was 2/m and space group c2. structure solution was conducted by molecular replacement with phaser [36] using a previously determined isomorphous structure of mers 3clpro (pdb: 4rsp [37] ) as the search model. structure refinement and manual model building were conducted with phenix [38] and coot [39] , respectively. disordered side chains were truncated to the point for which electron density could be observed. structure validation was conducted with molprobity [40] and figures were prepared using the ccp4mg package [41] . coordinates and structure factors for the mers 3clpro inhibitor complexes were deposited to the worldwide protein data bank (wwpdb) with the accession codes: 5wkj (gc376), 5wkk (gc813), 5wkl (inhibitor 10c) and 5wkm (inhibitor 10e). coronaviridae in field's virology coronaviruses: important emerging human pathogens sars and mers: recent insights into emerging coronaviruses update on human rhinovirus and coronavirus infections middle east respiratory syndrome and severe acute respiratory syndrome pathogenesis of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome coronaviruses: drug discovery and therapeutic options antiviral drugs specific for coronaviruses in preclinical development mers-cov vaccine candidates in development: the current landscape role of the spike glycoprotein of human middle east syndrome coronavirus (mers-cov) in virus and syncytia formation middle east respiratory syndrome infection mediated by the transmembrane serine protease tmprss2 host cell entry of middle east respiratory distress syndrome coronavirus after two-step, furin-mediated activation of the spike protein an overview of severe acute respiratory distress syndrome-coronavirus (sars-cov) 3cl protease inhibitors: peptidomimetics and small molecule chemotherapy the sars-coronavirus papainlike protease: structure, function and inhibition by designed antiviral compounds identification, synthesis, and evaluation of sars-cov and sars-cov 3c-like protease inhibitors structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus inhibitor recognition specificity for mers-cov papain-like protease may differ from that of sars-cov 157e162 where s 1 , s 2 , s 3 , …. s n and s 1 ', s 2 ', s 3 ', …. s n ' correspond to the enzyme subsites on the n-terminus and c-terminus side, respectively, of the scissile bond structure of main protease from human coronavirus nl63: insights for wide spectrum anti-coronavirus drug design reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor efficacy of a 3c-like protease inhibitor in treating various forms of acquired feline infectious peritonitis tripeptide aldehyde inhibitors of human rhinovirus 3c protease: design, synthesis, biological evaluation, and cocrystal structure solution of p1 glutamine isosteric replacements structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3cl protease. structure-activity relationships and biochemical, x-ray crystallographic, cell-based, and in vivo studies inhibition of norovirus 3cl protease by bisulfite adducts of transition state inhibitors broad-spectrum antivirals against 3c or 3c-like proteases of picornaviruses, noroviruses and coronaviruses alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sarscoronavirus infection in a mouse model automatic indexing of rotation diffraction patterns data processing and analysis with the autoproc toolbox an introduction to data reduction: space-group determination, scaling and intensity statistics phaser crystallographic software ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot molprobity: all-atom structure validation for macromolecular crystallography developments in the ccp4 molecular graphics project scaling and assessment of data quality improved r-factors for diffraction data analysis in macromolecular crystallography global indicators of x-ray data quality linking crystallographic model and data quality resolving some old problems in protein crystallography towards automated crystallographic structure refinement with phenix.refine supplementary data related to this article can be found at https://doi.org/10.1016/j.ejmech.2018.03.004. key: cord-263016-28znb322 authors: omrani, a.s.; shalhoub, s. title: middle east respiratory syndrome coronavirus (mers-cov): what lessons can we learn? date: 2015-08-22 journal: j hosp infect doi: 10.1016/j.jhin.2015.08.002 sha: doc_id: 263016 cord_uid: 28znb322 the middle east respiratory coronavirus (mers-cov) was first isolated from a patient who died with severe pneumonia in june 2012. as of 19 june 2015, a total of 1,338 mers-cov infections have been notified to the world health organization (who). clinical illness associated with mers-cov ranges from mild upper respiratory symptoms to rapidly progressive pneumonia and multi-organ failure. a significant proportion of patients present with non-respiratory symptoms such as headache, myalgia, vomiting and diarrhoea. a few potential therapeutic agents have been identified but none have been conclusively shown to be clinically effective. human to human transmission is well documented, but the epidemic potential of mers-cov remains limited at present. healthcare-associated clusters of mers-cov have been responsible for the majority of reported cases. the largest outbreaks have been driven by delayed diagnosis, overcrowding and poor infection control practices. however, chains of mers-cov transmission can be readily interrupted with implementation of appropriate control measures. as with any emerging infectious disease, guidelines for mers-cov case identification and surveillance evolved as new data became available. sound clinical judgment is required to identify unusual presentations and trigger appropriate control precautions. evidence from multiple sources implicates dromedary camels as natural hosts of mers-cov. camel to human transmission has been demonstrated, but the exact mechanism of infection remains uncertain. the ubiquitously available social media have facilitated communication and networking amongst healthcare professionals and eventually proved to be important channels for presenting the public with factual material, timely updates and relevant advice. the middle east respiratory syndrome coronavirus (mers-cov) was first identified in september 2012. as of 19 june 2015, a total of 1,338 mers-cov infections have been reported to the world health organization (who). 1 despite an accumulation of clinical experience and scientific knowledge, new mers-cov infections continue to be reported almost on daily basis. what lessons can we learn after three years of clinical experience and scientific research? lesson one: no substitute for continuous vigilance a 60-year-old man was admitted on june 13th, 2012, to a hospital in jeddah, saudi arabia, with severe pneumonia and multi-organ failure. the patient died after 11 days of hospitalization. 2 indirect immunofluorescence assays and real-time polymerase chain reaction (pcr) for widely occurring respiratory viruses failed to identify an infective aetiology. interestingly, cytopathic changes consistent with viral replication were noted in llc-mk2 and vero cell cultures of the patient's respiratory samples. slides of the infected cell cultures reacted strongly with the patient's serum but not with any of 2400 control sera stored in the same hospital. however, pancoronavirus pcr yielded positive results. the pcr fragments were sequenced at the erasmus medical centre in rotterdam, the netherlands, and phylogenetic analysis showed that the novel coronavirus belonged to lineage c of the genus betacoronavirus. 2, 3 on 20 september 2012, an email was posted on program for monitoring emerging diseases mail (promed-mail) announcing the discovery of a novel human coronavirus. 4 meanwhile, a critically ill 49-year-old qatari man was transferred by air ambulance on september 11th, 2012, to a hospital in england. 5 he had developed respiratory symptoms on september 3rd followed by multi-organ failure. his upper and lower respiratory tract samples were negative for influenza a/b, parainfluenza 1e4, rsv a/b, human metapneumovirus, enterovirus, rhinovirus, adenovirus, human bocavirus, and the human coronaviruses (nl63, 229e, oc43, hku1). on september 21st, 2012, one day after the above promed-mail posting, the patient's respiratory samples tested positive by pancoronavirus pcr. once sequenced, a 250 base-pair fragment from this isolate showed 99.5% homology with the erasmus medical centre's isolate. 5 the third patient was a 45-year-old man who presented to a hospital in riyadh, saudi arabia, on october 10th, 2012, with severe pneumonia and renal failure. mers-cov was detected in samples from the patient's upper and lower respiratory tract. 6 prior to all of this, an outbreak of respiratory illness was reported in april 2012 from an intensive care unit in a hospital in zarqa, jordan. 7 a retrospective epidemiological investigation in november 2012 identified 13 probable cases, two of whom had died. mers-cov was detected by reverse transcription (rt)epcr in stored samples from the two deceased patients. 8 seven more were subsequently confirmed by serological testing. 9 a pattern began to emerge, characterized by severe pneumonia, multi-organ failure, and an epidemiological link to a country in the middle east. in may 2013, the virus, which had been initially known as human coronaviruseerasmus medical centre (hcov-emc), was named the middle east respiratory syndrome coronavirus (mers-cov). 10 notably, phylogenetic analysis of the first five available mers-cov sequences suggested a common ancestor dating back to mid-2011. 11 furthermore, anti-mers-cov antibodies were detected in 15 out of 10,009 serum samples [0.15%; 95% confidence interval (ci): 0.09e0.24%] obtained between december 2012 and december 2013 from 13 provinces in saudi arabia. 12 the authors extrapolated that just fewer than 45,000 individuals (44, 951; 95% ci: 26, 971e71, 922) in saudi arabia could be seropositive for mers-cov. 12 it is therefore reasonable to assume that human mers-cov infections had taken place in the region for some considerable time before it was identified. it is possible that the identification of the virus might have been delayed even more, had it not been for the meticulous investigation by a single virologist, dr a.m. zaki, of the first reported case of mers-cov. 2 the first lesson one has to learn from mers-cov and its discovery is that continuous vigilance and perseverance with diagnostic investigation of undiagnosed infectious diseases are essential to identify emerging pathogens. lesson two: yet again, prevention is better than cure clinically, mers-cov infection may range from an asymptomatic or mild upper respiratory illness to a rapidly progressive and fatal disease. 13e15 the majority of hospitalized patients with mers-cov infection present with fever and respiratory symptoms including cough and shortness of breath with clinical and radiological evidence of pneumonia. 16,17 fatigue, myalgia, headache, and gastrointestinal symptoms such as vomiting and diarrhoea are also frequent. 16, 17 respiratory and renal failure are frequent complications of severe mers-cov infection, in addition to acute liver injury, cardiac dysrhythmias, and coagulopathy. 16e20 overall mortality is around 35.6%, but exceeds 70% in critically ill patients and in those with significant comorbidities. 1,18e22 for reasons yet to be understood, mers-cov infection is rare in children. 23, 24 in-vitro studies have identified numerous agents with anti-mers-cov activity including interferon, ribavirin, mycophenolate, cyclosporine and lopinavir. 25 the combination of interferon and ribavirin showed promising results in experimentally infected macaques. 26 however, in retrospective clinical studies the combination was not associated with significantly improved overall survival. 21,22 treatment of patients with mers-cov infections remains largely dependent on supportive measures. 27 diagnosis is confirmed by detection of mers-cov rna in respiratory samples by real-time pcr targeting the upe and orf 1b genes. 28 samples obtained from the lower respiratory tract have higher viral loads and better diagnostic yield than those obtained from the throat or nasopharynx. 11,29,30 moreover, viral shedding is considerably prolonged in symptomatic and severely ill mers-cov patients compared with asymptomatic infected contacts. 31 interestingly, detection of mers-cov in blood has been associated with worse clinical outcome. 22,32 mers-cov may also be detected in stool for up 16 days and in urine for up to 13 days from disease onset. 11 under certain conditions, mers-cov can survive on plastic and steel surfaces for up to 30 h. 33 in the absence of appropriate precautions, the environment surrounding a symptomatic mers-cov patient can therefore become extensively contaminated with viable, potentially infectious virus. human-to-human transmission of mers-cov has been well documented in family clusters, community settings and more often in healthcare settings. 13e15,17,20,34 common denominators in the largest hospital outbreaks have been overcrowding, especially in emergency departments, and poor adherence to infection control standards. 15,17,35,36 however, mers-cov continues to have relatively limited infectiousness. for example, screening identified secondary mers-cov infections in only 4% of 280 close family contacts and 2% of 5065 healthcare contacts. 37,38 moreover, no secondary cases were identified following extensive epidemiological investigations of imported cases in the uk, germany, france, greece, the netherlands, and the usa. 39e46 it has been phylogenetically demonstrated that mers-cov transmission chains have not extended beyond two to three months and that the virus has remained genetically stable over the past three years. 47, 48 given an effective reproduction number (r 0 ) of less than one, human-to-human mers-cov could be readily interrupted with effective preventive interventions. 49, 50 indeed, even the most explosive hospital outbreaks of mers-cov infection, such those that occurred in jeddah and riyadh in april to may 2014, were brought under control through a strategy based on early case detection and implementation of appropriate infection prevention and control measures; namely contact and droplet precautions for general care in addition to airborne precautions for aerosolgenerating procedures such as intubation and respiratory tract suctioning. 51e53 the poor prognosis associated with mers-cov, especially in patients with multiple comorbidities, and the lack of effective anti-viral therapy make appropriate infection prevention and control all-important. just as is true for most infectious diseases, mers-cov reminds us again that prevention is better than cure. the initial case definitions for mers-cov case finding and reporting focused on patients who are hospitalized, had evidence of acute pulmonary disease with an epidemiological link to confirmed cases or to countries in the middle east. 54, 55 as more clinical experience and epidemiological data became available, updated definitions removed the requirement for hospitalization. 56 the reporting of several community and hospital clusters during the first half of the year 2013, often without identifiable human or animal sources, led to speculation that individuals with no or only mild respiratory symptoms might have a role in mers-cov transmissions. 13, 14, 20 this was reflected in the who revised interim definition published in july 2013 where patients with acute febrile illness of any severity were included; in addition to a recommendation to proactively test asymptomatic close contacts of confirmed mers-cov infections. 57 memish et al. later showed that mers-cov was detectable for up to 12 days in 30% of 12 asymptomatic contacts. 31 in another report, an asymptomatic healthcare worker had detectable mers-cov for more than five weeks. 58 although mers-cov transmission from an asymptomatic individual remains a strong probability, this has never been documented. 37, 38 in the meantime, clinicians were becoming increasingly aware that mers-cov infections were being diagnosed in patients whose clinical presentations did not conform to those definitions, including the absence of fever, lack of respiratory involvement and the predominance of gastrointestinal or nonspecific generalized symptoms. 17, 22, 59 in the aftermath of the surge of mers-cov infection in jeddah and riyadh in april and may 2014, the ministry of health in saudi arabia revised its case definition and surveillance guidance to recommend mers-cov testing in any of four patient categories: e patients with clinical or radiological evidence of community-acquired pneumonia; e patients with clinical or radiological evidence of healthcare-associated pneumonia; e patients with acute febrile illness and myalgia, headache, diarrhoea, nausea, or vomiting, and unexplained leucopenia or thrombocytopenia; e contacts of individuals with confirmed or probable mers-cov infection who develop upper or lower respiratory symptoms within two weeks of exposure. 60 as better understanding of the epidemiology of mers-cov developed, it became obvious that a considerable proportion of cases were probably missed. 12,50 during the steep learning curve of an emerging infectious disease, regularly updated guidelines are important. such guidelines are inevitably based on incomplete evidence and hence may not be comprehensive or applicable in all situations. clinical acumen and heightened medical awareness are essential for early detection of unusual mers-cov cases and to prevent delays in diagnosis and to mitigate additional exposures. 61 a zoonotic origin was suspected soon after the identification of mers-cov. 6 bats are known natural hosts for several coronaviruses and hence were the initial target for investigation. 62, 63 more than 1000 faecal samples were collected from wild bats in the area around where the first mers-cov patient lived. a 190-nucleotide fragment of mers-cov rna was detected in one faecal pellet from an egyptian tomb bat. the sequenced amplification product was genetically identical to the mers-cov sequence obtained from the index human case. 64 more recently, a closely related coronavirus was isolated from bats in south africa, suggesting that mers-cov ancestors might exist in old world bats. 65, 66 to date, no further evidence is available to confirm the role of bats as natural hosts or reservoirs for mers-cov. on the other hand, the evidence implicating dromedary camels in mers-cov epidemiology is more consistent. a role for dromedary camels is supported by the following observations: à neutralizing mers-cov antibodies are highly prevalent in dromedary camels from across the arabian peninsula, north africa, and eastern africa. 67e73 mers-cov antibodies were detected in stored camel sera dating as far back as the early 1990s. 73e75 the prevalence of mers-cov seropositivity is significantly higher in camels aged more than two years than in juvenile camels. 68,74,76 à several groups have reported the detection of mers-cov by rtepcr in nasal and faecal samples from dromedary camels in the arabian peninsula. 72,74,76e79 one study reported mers-cov positivity in more than 60% of lung tissue samples obtained from dromedary camel carcasses. 79 rtepcr was positive in camels that had prior evidence of mers-cov seropositivity, indicating that animal reinfection is possible. 76 interestingly, the prevalence of mers-cov rna is significantly higher in juvenile than in adult camels. 74, 76, 79 furthermore, all mers-cov strains obtained from dromedary camels are phylogenetically clustered within human isolates, supporting possible animalehuman intertransmission. 72 it is important to note, however, that mers-cov seroprevalence studies in individuals with close contact with camels have yielded inconsistent results. a national serosurvey in saudi arabia found prevalence of mers-cov antibodies that was 15 times higher in camel shepherds (p ¼ 0.0004) and 23 times higher in slaughterhouse workers (p < 0.0001), compared with the general population. 12 similarly, mers-cov serology was positive in individuals who had occupational exposure to dromedary camels in qatar but not in those without such exposure. 86 on the other hand, mers-cov antibodies were not detected in sera obtained from individuals who had close contact with camels that had documented mers-cov infection two to three months earlier. 87 likewise, screened slaughterhouse workers and other animal workers in western and southern saudi arabia were all seronegative for mers-cov antibodies. 88, 89 collectively, the available data strongly suggest that mers-cov is highly prevalent in dromedary camels in the arabian peninsula and that transmission of infection from camels to humans, although inefficient, does occur. however, the exact mechanism and route of infection it is still unclear. infections. 30, 94, 95, 97 one pertinent cause for concern has been the potential global spread of mers-cov during the annual hajj pilgrimage when millions of muslims from around the world gather in mecca, saudi arabia. 98e102 though those concerns are well founded, several surveillance studies over the past three years have not identified any mers-cov infections among hajj pilgrims while they are in saudi arabia or after their return to their home countries. 103e108 the situation was entirely different in the recent outbreak in south korea where a single imported case resulted in a total of 186 laboratory-confirmed cases of mers-cov infection, including 36 deaths. 109 the index patient was a 68-year-old man who developed respiratory symptoms seven days after returning to seoul from a two-week visit to bahrain, saudi arabia, united arab emirates, and qatar. 35 he sought medical care in several hospitals before he was diagnosed with mers-cov infection. 35,110 a combination of late recognition, overcrowding in emergency departments and hospital wards, multiple incidents of patient movement between different healthcare facilities, and delayed implementation of adequate infection control precautions culminated in the largest single outbreak of mers-cov infection. 35,36,110e112 the outbreak involved patients, visitors, care-givers and healthcare workers, and spanned across six different hospitals in three south korean cities. 110, 113 notably, phylogenetic analysis of mers-cov strains from south korea revealed no significant biological changes compared to previously sequenced viruses. 114 the outbreak in south korea was eventually controlled through a series of measures including aggressive contact identification, screening and strict isolation, and rigorous infection control precautions. 111, 115, 116 within a few weeks, south korea went from a country with no reported mers-cov cases to one that has the second largest number in the world. 116, 117 with air travel becoming readily accessible and affordable, the south korean experience demonstrates vividly that in the context of an infectious respiratory illness, there is simply no room for complacency. adequate assessment of patients presenting with febrile illness must include their recent travel history to enable early application of proper control measures and to expedite laboratory confirmation and appropriate clinical management. the past decade has witnessed an exponential rise in internet-based social media sites such as facebook, twitter, and youtube. 118 healthcare professionals are increasingly using social media applications to follow medical developments and emerging scientific literature and to share their own research findings, observations, and opinion. 119 the general public often uses these tools as news outlets to seek and share medical and scientific information. 120 however, in the context of mers-cov, social media have been a double-edged sword. for example, social media were at some point rife with inaccurate information that included rumours of hospitals closed due to mers-cov outbreaks and certain social events being nodes for mers-cov transmission. the authors are aware of examples of information and photos shared on social media resulting in patients losing their right to privacy and confidentiality. patients often cancelled their clinic appointments or scheduled surgical procedures for fear of acquiring mers-cov while in hospital. some avoided attending emergency departments despite having acute problems that required medical attention. some individuals posted videos and messages challenging the suggestion that camels may be a source of mers-cov infection. scepticism and mistrust in governmental agencies and the medical community were sometimes promoted and propagated. on the other hand, various government agencies, scientific organizations and healthcare professionals used social media to enhance networking and facilitate communication of epidemiological, medical and scientific developments; in addition to presenting the public with factual material, timely updates, and relevant advice. 121 the saudi ministry of health, for example, posts daily updates on its website and through social media outlining details of current mers-cov cases. the korean ministry of health and welfare did the same during their mers-cov outbreak. such steps are important to gain the public's trust and to remove barriers to appropriate sources of information. taking on board the surging role of social media and using them effectively to disseminate appropriate information turns them into invaluable tools for controlling an emerging infectious disease such as mers-cov. mers-cov is an emerging infectious disease of probable animal origin. sustained human-to-human infection has not occurred and its potential for causing widespread epidemic remains limited. vigilance, early recognition, and institution of appropriate protective measures are the most effective control measures. none declared. middle east respiratory syndrome coronavirus (mers-cov); summary of current situation, literature update and risk assessment 7 isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans novel coronavirus e saudi arabia: human isolate. archive number severe respiratory illness caused by a novel coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) investigations and implications for the prevention of human-to-human transmission a case of imported middle east respiratory syndrome coronavirus infection and public health response middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands lack of transmission among close contacts of patient with case of middle east respiratory syndrome imported into the united states spread, circulation, and evolution of the middle east respiratory syndrome coronavirus an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection; interim guidance infection prevention/control and management guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection world health organization. case definition for case finding severe respiratory disease associated with novel coronavirus. interim case definition as of 25 revised interim case definition e novel coronavirus. interim case definition as of 16 revised interim case definition for reporting to who e middle east respiratory syndrome coronavirus (mers-cov) revised interim case definition for reporting to who e middle east respiratory syndrome coronavirus (mers-cov) a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker successful recovery of mers-cov pneumonia in a patient with acquired immunodeficiency syndrome: a case report case definition and management of patients with mers coronavirus in saudi arabia investigation of cases of human infection with middle east respiratory syndrome coronavirus (mers-cov); interim guidance updated 3 ecology, evolution and classification of bat coronaviruses in the aftermath of sars detection of coronaviruses in bats of various species in italy middle east respiratory syndrome coronavirus in bats, saudi arabia close relative of human middle east respiratory syndrome coronavirus in bat, south africa rooting the phylogenetic tree of mers-coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus antibody reactors among camels in dubai middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity mers coronavirus in dromedary camel herd, saudi arabia mers coronaviruses in dromedary camels isolation of mers coronavirus from a dromedary camel mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia acute middle east respiratory syndrome coronavirus infection in livestock dromedaries tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels evidence for camel-to-human transmission of mers coronavirus occupational exposure to dromedaries and risk for mers-cov infection lack of middle east respiratory syndrome coronavirus transmission from infected camels investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012 middle east respiratory syndrome coronavirus (mers-cov) e thailand. disease outbreak news 20 middle east respiratory syndrome coronavirus (mers-cov) e austria clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus (mers-cov) e the philippines investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in family cluster of middle east respiratory syndrome coronavirus infections laboratoryconfirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response health protection agency uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections a scenario-based evaluation of the middle east respiratory syndrome coronavirus and the hajj middle east respiratory syndrome (mers) coronavirus. what travel health advice should be given to hajj pilgrims preventive measures against mers-cov for hajj pilgrims the hajj pilgrimage and surveillance for middle east respiratory syndrome coronavirus in pilgrims from african countries potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms lack of mers coronavirus but prevalence of influenza virus in french pilgrims after prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj from the hajj: it's the flu, idiot high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage saudi arabia, south korea preliminary epidemiological assessment of mers-cov outbreak in south korea lessons to learn from mers-cov outbreak in south korea spread of mers to south korea and china list of hospitals with known mers exposure preliminary data from sequencing of viruses in the republic of korea and the people's republic of china. mers-cov situation assessment 9 world health organization. managing contacts in the mers-cov outbreak in the republic of korea geneva: who. available at middle east respiratory syndrome coronavirus (mers-cov): summary and risk assessment of current situation in the republic of korea and china e as of 19 korean society for healthcare-associated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea dangers and opportunities for social media in medicine social media and health care professionals: benefits, risks, and best practices social media and clinical care: ethical, professional, and social implications middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd key: cord-278648-hkvurb2k authors: menachery, vineet d.; gralinski, lisa e.; mitchell, hugh d.; dinnon, kenneth h.; leist, sarah r.; yount, boyd l.; graham, rachel l.; mcanarney, eileen t.; stratton, kelly g.; cockrell, adam s.; debbink, kari; sims, amy c.; waters, katrina m.; baric, ralph s. title: middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis date: 2017-11-15 journal: msphere doi: 10.1128/msphere.00346-17 sha: doc_id: 278648 cord_uid: hkvurb2k coronaviruses (covs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. in this study, we focused on highly conserved nonstructural protein 16 (nsp16), a viral 2′o-methyltransferase (2′o-mtase) that encodes critical functions in immune modulation and infection. using reverse genetics, we disrupted a key motif in the conserved kdke motif of middle east respiratory syndrome cov (mers-cov) nsp16 (d130a) and evaluated the effect on viral infection and pathogenesis. while the absence of 2′o-mtase activity had only a marginal impact on propagation and replication in vero cells, dnsp16 mutant mers-cov demonstrated significant attenuation relative to the control both in primary human airway cell cultures and in vivo. further examination indicated that dnsp16 mutant mers-cov had a type i interferon (ifn)-based attenuation and was partially restored in the absence of molecules of ifn-induced proteins with tetratricopeptide repeats. importantly, the robust attenuation permitted the use of dnsp16 mutant mers-cov as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted mers-cov strain. these studies demonstrate the importance of the conserved 2′o-mtase activity for cov pathogenesis and highlight nsp16 as a conserved universal target for rapid live attenuated vaccine design in an expanding cov outbreak setting. importance coronavirus (cov) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome cov (sars-cov), mers-cov, porcine epidemic diarrhea virus, and swine delta cov in the 21st century. these studies describe an approach that effectively targets the highly conserved 2′o-mtase activity of covs for attenuation. with clear understanding of the ifn/ifit (ifn-induced proteins with tetratricopeptide repeats)-based mechanism, nsp16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent cov strains. importantly, other approaches targeting other conserved pan-cov functions have not yet proven effective against mers-cov, illustrating the broad applicability of targeting viral 2′o-mtase function across covs. t he emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 represents the second severe cov to strike the human population since the beginning of the 21st century (1) . similar to its predecessor, severe acute respiratory syndrome cov (sars-cov), mers-cov is characterized by severe lung infection and high mortality rates (2) . associated with elderly patients and nosocomial spread, mers-cov is likely harbored in camel populations with periodic reintroductions into humans, followed by periodic nosocomial outbreaks in hospital settings (3) . importantly, with the continued rate of globalization, mers-cov remains an ongoing threat for future outbreaks both in and outside the middle east, as evidenced by the large outbreak in south korea (4) . together, these factors highlight the importance of examining cov pathogenesis and developing conserved therapeutic targets for the treatment of current and future emergent strains. like all members of the cov family, mers-cov maintains a balance of conserved and novel viral proteins within its genome (5) . it is a member of the group 2c cov family, and a wealth of distinct accessory open reading frames and nonstructural proteins (nsps) have already been established to have important roles in modulation of the host immune response (6) . similarly, a number of viral proteins highly conserved in structure, replication, and fidelity are also maintained in the cov backbone (7) . among these, mers-cov nsp16 provides a potent target for therapeutic development. a 2=omethyltransferase (2=o-mtase), cov nsp16 has been implicated in the capping of viral rna and prevention of its recognition by the intracellular sensor mda5 and antiviral effectors, including members of the ifit (interferon [ifn]-induced proteins with tetratricopeptide repeats) family (8) . generation of mutants with changes in the nsp16 kdke active site resulted in ifn-mediated in vitro and in vivo attenuation of both mouse hepatitis virus (mhv) and sars-cov (9, 10) . therefore, an approach targeting mers-cov nsp16 might be anticipated to result in attenuation and potentially provide a universal platform for cov vaccines against future emergent strains. using reverse genetics to target residues in the highly conserved active site, we evaluated mers-cov infection outcomes in the context of inactive nsp16 (dnsp16). consistent with previous studies of sars-cov (10), the dnsp16 mers-cov mutant maintained no significant attenuation in terms of replication or the initial host immune response. however, both primary human airway epithelial (hae) cells and in vivo studies in a mers-cov mouse model demonstrated robust attenuation of dnsp16 mutant growth and pathogenesis. notably, attenuation was both ifn and ifit1 dependent, providing a clear mechanism for attenuation. importantly, the dnsp16 mutant also provided robust protection against a lethal mers-cov challenge and maintained attenuation in the mouse-adapted backbone. together, the results illustrate the broad conservation and necessity of nsp16 in cov pathogenesis and highlight the targeting of this protein as a rapid-response platform for future emergent cov strains. a combination of structural and biochemical approaches has established a critical role for cov nsp16 in 2=o-mtase activity (fig. 1a) . stabilized by interactions with nsp10 (orange), nsp16 has been identified as a structurally conserved adomet-dependent methyltransferase (11) ; despite divergence in protein sequence across organisms, an invariant kdke motif (red) within the methyltransferase core is required to mediate its activity (12) . this kdke motif is highly conserved within all of the nsp16 sequences examined in the cov family (fig. 1b) . importantly, mutation of any of the kdke residues has been shown to ablate 2=o-mtase activity (11) . in addition, previous alteration of this motif in both group 2b sars-cov (10) and group 2a mhv (9) disrupted 2=o-mtase activity and attenuated various aspects of infection. on the basis of high conservation in the cov family, we hypothesized that disruption of the kdke motif would also attenuate other emerging cov families, including group 2c mers-cov. utilizing a mers-cov reversed genetic system (13), we disrupted the kdke motif by mutating two nucleotides to produce a d130a change (fig. 1a) . the resulting disrupted-nsp16 (dnsp16) mutant had no significant defect noted in stock titer generation (not shown); similarly, infection of both vero cells and calu-3 2b4 cells, a respiratory epithelial cell line, at a low multiplicity of infection (moi) demonstrated only modest attenuation at late time points (fig. 1c and d) . together, these results indicate that nsp16 activity is not required for replication. similar in vitro host responses of sars-cov and mers-cov dnsp16 mutants. having established replication competence in both vero and calu-3 2b4 cells, we next evaluated the induction of host pathways following infection. calu-3 2b4 cells infected at an moi of 5 demonstrated no differences in replication (not shown) and only modest differences in host induction (zero genes with a log 2 change in expression of ͼ1.5-fold), similar to observations with nsp16 mutant sars-cov compared with wild-type (wt) sars-cov (10). however, unlike in studies with sars-cov, a rapid cytopathic effect (cpe) by 24 h limited the analysis to early time points with wt and dnsp16 mutant shown is nsp16 (gray) highlighting the conserved kdke motif (red) required for 2=o-mtase activity. also shown is the nsp10 scaffold required for mers-cov (orange). the inset displays conserved kdke (left) and the d130a mutation (right) that disrupts function. homology models were created with modeler in the max-planck institute bioinformatics toolkit. the known crystal structure of the nsp16-nsp10 complex (3r24 in the rcsb protein data bank) was used as the template structure (38) . homology models were then manipulated with macpymol. (b) heat maps were constructed from a set of representative covs from all four genogroups by using alignment data paired with neighbor-joining phylogenetic trees built in geneious (v.9.1.5) and visualized in evolview david-based analysis compared the network host responses to the mers-cov and sars-cov dnsp16 mutants (fig. 2) . over the first 24 h of infection, both mers-cov and sars-cov dnsp16 mutant infections showed no significant functional enrichment of any categories relative to corresponding wt infections, consistent with the lack of replication attenuation. however, at late times (ͼ24 h postinfection), sars-cov produced robust changes in several host pathways, including cytokine responses, inflammation, and extracellular activity. similarly, changes in apoptosis, transcription repression, and regulation of phosphorylation indicated a host response more hostile to viral infection. while a more rapid cpe following both wt and dnsp16 mutant mers-cov infections precluded an equivalent finding at late time points, the sars-cov results suggest that the absence of nsp16 activity eventually initiates host response changes that contribute to attenuation at late time points. mers-cov dnsp16 mutant attenuated in primary and in vivo models. to further examine the replicative capacity of the dnsp16 mutant, we infected both hae cells and mice expressing human dipeptidyl peptidase 4, the receptor for mers-cov. primary hae cell cultures were challenged with wt and dnsp16 mers-cov at a low moi (fig. 3a ). while robust replication was observed following wt infection, dnsp16 mers-cov had significant attenuation that corresponded well to previous results obtained with dnsp16 mutant sars-cov (10). we next examined dnsp16 mutant mers-cov replication phenotypes in the context of in vivo infection by using an adenovirus balb/c mouse transduction model (14) . while neither infection produced weight loss (not shown), wt mers-cov replicated efficiently at both days 2 and 4 postinfection (fig. 3b ); in contrast, no detectable replication was seen following infection with dnsp16 mutant mers-cov. the lack of replication may be due to residual ifn responses associated with initial adenovirus infection. for greater clarity, we next infected crispr-cas9-targeted mice that include mutations in dpp4 at positions 288 and 330 (288-330 ϩ/ϩ ) conferring efficient wt mers-cov infection and growth in mice but no clinical disease (15) . following infection, no changes were observed in weight loss in either group of mice, consistent with previous findings (data not shown). however, absence of nsp16 activity severely attenuated dnsp16 mutant virus replication at both days 2 and 4 postinfection (fig. 3c ). coupled with data from hae cell cultures and the adenovirus model, the results demonstrate clear attenuation of dnsp16 mutant mers-cov relative to the control. dnsp16 mutant mers-cov attenuation is mediated by ifn and ifit1. having established a deficit in dnsp16 mutant mers-cov replication in relevant in vitro and in vivo models, we next sought to evaluate the mechanism of attenuation. previous work by our lab and others had established increased susceptibility of dnsp16 mutants to type i ifn (9, 10); however, dnsp16 mutant sars-cov had not shown augmented type i ifn stimulation following infection (10) . consistent with this finding, infection with dnsp16 mutant mers-cov produced stimulation of type i ifn transcript similar to that seen following in vivo infection of dpp4 mutant (288-330 ϩ/ϩ ) crispr mice with the wt virus (fig. 4a ). in contrast, while both the wt and mutant viruses were sensitive to ifn treatment, dnsp16 mutant mers-cov had a significant reduction in replication relative to the control virus (fig. 4b) . these attenuation results are consistent with reports of nsp16 mutants of other covs and are in contrast to the equivalent replication observed without pretreatment (fig. 1c) (8) . extending this analysis further, we examined the role of ifit1 and ifit2 gene expression in this attenuation phenotype in previously constructed stable short hairpin rna (shrna) knockdown cell lines (10) . similar to sars-cov, knockdown of ifit1 augmented replication of dnsp16 mutant mers-cov in the context of type i ifn pretreatment (fig. 4c ). in addition, knockdown augmented wt mers-cov infection, suggesting sensitivity to ifit1 activity despite the presence of nsp16. notably, ifit2 knockdown had only a modest, nonsignificant impact on replication, contrasting with results obtained with sars-cov. overall, the data indicate that dnsp16 mutant mers-cov attenuation is driven by sensitivity to type i ifn mediated by the activity of ifit1 rather than augmented ifn responses. nsp16 mutant vaccination protects from a lethal mers-cov challenge. on the basis of ifn and ifit1 attenuation phenotypes, targeting of nsp16 offers a potential platform strategy for live attenuated vaccine generation. while previous work by our group had shown that the dnsp16 mutant sars-cov conferred protection from a lethal challenge, similar phenotypes in other more distant covs are essential for establishing universal principles of attenuation across a virus family. to test this hypothesis, dpp4 288-330 ϩ/ϩ mutant mice were vaccinated with dnsp16 mutant mers-cov and subsequently challenged with a mouse-adapted mers-cov strain (fig. 5) (15) . following the challenge, dnsp16 mutant-vaccinated mice showed only modest weight loss, in significant contrast to the severe disease seen in the control group (fig. 5a ). in addition, both viral replication and lung hemorrhage were significantly reduced in the context of the dnsp16 mutant vaccine (fig. 5b and c) . importantly, serum analysis revealed robust virus neutralization with values similar to those seen in serum from wt virus-infected mice (fig. 5d) . together, the results indicate that dnsp16 mutant mers-cov can function as a vaccine platform that not only induces high levels of neutralizing antibodies but provides compete protection from a lethal mers-cov challenge. nsp16 mutation attenuates mouse-adapted mers-cov. despite conferring protection in the wt mers-cov backbone, it was unclear if the nsp16 mutant would be sufficiently attenuated in a virulent mers-cov backbone. to address this question, we inserted the dnsp16 mutation (d130a) into the mouse-adapted mers-cov backbone (15) . following infection, mouse-adapted mers-cov produced rapid weight loss and death (fig. 6a) . in contrast, the mouse-adapted dnsp16 mutant produced only modest weight loss and 100% survival following infection. in addition, the replication of the dnsp16 mutant was significantly attenuated relative to that of the wt at days 2 and 4 postinfection (fig. 6b) . finally, hemorrhage scoring of the lung revealed minimal disease in dnsp16 mutant-immunized mice relative to control mice at day 4 postinfection (fig. 6c) . overall, the results demonstrate robust attenuation of mers-cov pathogenesis in the context an nsp16 mutation. in the context of the ongoing mers-cov outbreak, the development of universal platform strategies to attenuate emerging and contemporary covs is a significant priority. in this study, we demonstrate the critical importance of nsp16 function to mers-cov pathogenesis in vitro and in vivo. similar to mhv and sars-cov (9, 10), the disruption of 2=o-mtase activity in mers-cov had no significant impact on replication or early host response patterns in vitro. however, dnsp16 mutant mers-cov demonstrated attenuated replication and growth in primary hae cell cultures, as well as reduced disease in vivo, relative to the wt virus. further examination revealed increased sensitivity to type i ifn in an ifit-dependent manner. notably, this ifn/ifitbased attenuation phenotype provided robust protection from a lethal challenge following vaccination with the dnsp16 mutant. importantly, the nsp16 mutant also was fully attenuated in a highly virulent mouse-adapted mers-cov backbone. these results, coupled with previous work with other covs, highlight the viral 2=o-mtase activity as a potential universal platform for therapeutic treatment and vaccine development for current and future emergent human and animal covs. similar to previously viral methyltransferase mutants of both flaviviruses and covs, substitution with the highly conserved kdke motif alters the pathogenicity of mers-cov. like sars-cov mutants (10), dnsp16 mutant mers-cov had normal replication but was attenuated in the context of type i ifn and ifit activity. notably, the absence of 2=o-mtase activity did not result in an increased type i ifn response, suggesting that loss of nsp16 produces a sensitivity to ifit activity not seen in the wt virus. while the mutant rna should induce greater ifn activation through mda5, multiple other cov proteins have been shown to disrupt immune recognition (16) . similarly, viral processes including replication within double membrane vesicles may also limit ifn stimulation. together, the results indicate that even without nsp16, covs are able to control ifn stimulation and replicate as efficiently as the wt virus. however, nsp16 is needed to protect the viruses once the ifn-stimulated gene (isg) effector response is initiated. the attenuation of the mers-cov nsp16 mutant also provides evidence supporting the targeting of viral 2=o-mtase as a global therapeutic strategy against emergent rna viruses. previous work with both flaviviruses and covs has demonstrated robust attenuation by targeting the conserved kdke motif of the viral 2=omtase (8, 17, 18) . importantly, 2=o-mtase activity is the only known function for nsp16 and it appears completely dispensable for cov replication in the absence of a strong ifn response (8) . in contrast, flaviviruses without nsp5-encoded methyltransferase activity are much more sensitive to innate immune responses (19) ; mutants of west nile virus, dengue virus, and japanese encephalitis virus are highly replication attenuated at early time points (20, 21) . while diminished virus yields likely reduce flavivirus vaccine utility in vivo, robust propagation of both mers-cov and sars-cov dnsp16 mutants provides a useful parameter for vaccine generation. for both viral families, drugs targeting 2=o-mtase activity may have great efficacy if paired with stimulation of the ifn-responsive genes, including that for ifit1 (8, 17) . overall, the results indicate that viral 2=o-mtase activity is a critical determinant of pathogenesis and can be leveraged to develop therapeutic treatments. nsp16 also represents the third highly conserved cov protein targeted as the basis of a vaccine platform. previous work by our group demonstrated the importance of cov fidelity for infection and pathogenesis (22) ; disruption of the exonuclease activity of nsp14 rendered attenuated, reversion-proof versions of both mhv and sars-cov (22) (23) (24) . for sars-cov, the disruption served as the basis of a successful live attenuated vaccine (22) . similarly, groups have targeted the envelope protein of sars-cov and defined a key inflammatory role for e protein in pathogenesis. sars mutants targeting e function also conferred protection from a lethal challenge (25, 26) . for both nsp14 and e mutants, fidelity and inflammation induction are partially responsible for attenuation. however, both viral mutants also maintain replication attenuated in terms of kinetics and yields, potentially complicating their use as vaccine platforms, especially in outbred populations (22, 26) . importantly, the nsp14 mutant has not yet been recovered within the context of mers-cov and failure has been reported in group i covs (27) . similarly, disruption of the e protein renders mers-cov, transmissible gastroenteritis virus, and mhv replication deficient without exogenous complementation (28) (29) (30) ; this broad attenuation of the delta e mutant potentially limits its use as a live attenuated vaccine. in contrast, dnsp16 mutant mers-cov maintains robust propagation, as well as ifn/ifit-based attenuation phenotypes. together, these results suggest that targeting ofnsp16 may be the most broadly applicable platform for cov attenuation. despite the success of nsp16 mutants in protection studies with sars-cov and mers-cov, several additional parameters must be considered in the context of cov vaccination. previous work by our group has demonstrated failures of cov subunit vaccines in aged animals and in the context of a heterologous challenge (31) . with the aged representing a population with high mortality rates and marginal vaccination success, the efficacy of the nsp16 mutant in this population is paramount in its pursuit as a platform. similarly, the existence of numerous covs in animal populations raises concerns about heterologous challenges from emergent viruses (32, 33) . prior reports had also demonstrated vaccine-induced disease following heterologous challenges with related sars-like viruses (31, 34) . with this in mind, nsp16-vaccinated mice will need to be examined in the context of a heterologous challenge to determine if vaccine-induced pathology occurs. together, these two factors represent important checkpoints in the pursuit of nsp16 as a universal cov vaccine platform. in addition to aging and heterologous challenges, reversion and baseline pathogenesis also represent important risks that must be evaluated in the context of an nsp16 vaccine. while the nsp14 sars vaccine was absent sterilizing immunity in immunodeficient mice, the lack of reversion over time in vivo indicates safety in the approach (22) . in contrast, passage of the e mutant rendered a novel mutant that transplanted a critical ion channel function from another viral protein and thus restored partial virulence (35) . studies examining the reversion potential of nsp16 are critical prior to its use as a vaccine; these concerns are especially important considering that both the sars-cov and mers-cov dnsp16 mutants have robust replication, permitting additional opportunities for reversion (10) . in addition, equivalent host immune responses during early infection may produce substantial damage prior to ifn/ifit-based attenuations, most notably in aged and immunocompromised mice. therefore, despite the promise of successful attenuation in multiple cov backbones, several additional metrics must be examined prior to the use of nsp16 mutants as a universal cov vaccine platform. overall, the present study demonstrates that targeting of 2=o-mtase activity is a robust strategy to attenuate mers-cov and other emergent covs. in the absence of nsp16 activity, the mers-cov mutant is sensitive to type i ifn in an ifit-dependent manner, providing a clear attenuation mechanism. importantly, unlike other conserved cov platforms, the nsp16 mutant is both viable and robust enough to be used as an effective live attenuated vaccine. while further vaccine characterization is required, the results indicate that disruption of cov nsp16 activity can be the basis of therapeutic strategies for both current and future emergent cov infections in both human and animal populations. cells and viruses. the wt, mutant, and mouse-adapted versions of mers-cov used in this study were previously described (13, 15) and were cultured on vero 81 cells grown in dulbecco's modified eagle's medium or opti-mem (gibco, carlsbad, ca) and 5% fetal bovine serum (hyclone, south logan, ut) along with antibiotic/antimycotic (gibco, carlsbad, ca). growth curves in vero, calu-3 2b4, and hae cells were performed as previously described, with examination of multiple samples (n ն3) at each time point (9, 31) . briefly, cells were washed with phosphate-buffered saline (pbs) and inoculated with virus or mock diluted in pbs for 40 min at 37°c. following inoculation, cells were washed three times and fresh medium was added to signify time zero. samples were harvested at the time points described. for ifn pretreatments, 100 u/ml recombinant human ifn-␤ (pbl laboratories) was added to cells 16 h prior to inoculation, and the cells were infected as described above. stable shrna knockdown vero cell lines for both ifit1 and ifit2 were previously described for previous cov studies and had phenotypic validation (10) . all virus cultivation was performed in a biosafety level 3 laboratory with redundant fans in biosafety cabinets as described previously by our group (32, 33) . all personnel wore powdered air purifying respirators (3m breathe easy) with tyvek suits, aprons, and booties and were double gloved. construction of wt and nsp16 mutant viruses. both wt and mutant viruses were derived from either mers-cov emc or a corresponding mouse-adapted (ma1) infectious clone as previously described (13) . for nsp16 mutant construction, the d130a mutation changed the sequence from the mers-cov e fragment, which was cloned into the psmart vector (lucigen) and used for alanine scanning mutagenesis of conserved residues in nsp16. for the d130a change, a product was generated by pcr with primers against mers-cov nsp16 [fragment 1, emc:e#2(ϩ) (tgaactacctgtagctgtag) and emc:emuc(ϫ) (nnnnnngctcttctcgcggaaataacaagatccacttg); fragment 2, emc:emuc(ϩ) (nnnnnngctcttc cgcgatgtatgatcctactactaag) and emc:e#6(ϫ) (caacctcaatacaagcagac)]. the two resulting products were digested with sapi (in boldface) and ligated overnight with t4 dna ligase. this product was then digested with ppumi and nsii and used to replace the region of the emc e plasmid (puc57) that had been similarly digested. thereafter, plasmids containing wt and mutant mers-cov genome fragments were amplified, excised, ligated, and purified. in vitro transcription reactions were then performed to synthesize full-length genomic rna, which was transfected into vero e6 cells. the medium from transfected cells was harvested and used as seed stock for subsequent experiments. viral mutants were confirmed by sequence analysis prior to use. synthetic construction of nsp16 mutants was approved by the university of north carolina institutional biosafety committee. rna isolation, microarray processing, and identification of de. rna isolation and microarray processing, quality control, and normalization from calu-3 2b4 cells were carried out as previously described (36) . differential expression (de) was determined by comparing virus-infected replicates with time-matched mock-treated replicates. criteria for de in determining the consensus isg list were an absolute log 2 change of ͼ1.5-fold and a false-discovery rate-adjusted p value of ͻ0.05 for a given time point. clustering and functional enrichment. genes identified as differentially expressed were used to generate clustered expression heat maps. hierarchical clustering (using euclidean distance and complete linkage clustering) was used to cluster gene expression according to behavior across experimental conditions. the david online resource (https://david.ncifcrf.gov/) was used to acquire functional enrichment results for the genes in each cluster. david output was manually summarized for each cluster. plots were generated with r. ethics statement. this study was carried out in accordance with the recommendations for the care and use of laboratory animals of the office of laboratory animal welfare (olaw), national institutes of health. the institutional animal care and use committee (iacuc) of the university of north carolina at chapel hill (unc; permit a-3410-01) approved the animal study protocols used in this study (iacuc protocols 15-009 and 13-072). mouse infections and vaccinations. ten-to 20-week-old balb/c (envigo/harlan) or crispr-cas9targeted 288-330 ϩ/ϩ c57bl/6 mice were anesthetized with ketamine and xylazine (in accordance with unc iacuc guidelines) and intranasally inoculated with a 50-l volume containing 10 6 pfu of wt mers-cov, dnsp16 mutant mers-cov, mouse-adapted variants, or pbs (mock inoculation) as indicated in the figure legends. infected animals were monitored for weight loss, morbidity, and clinical signs of disease, and lung virus titers were determined as described previously (37) . in vivo adenovirus transduc-tion with dpp4 were performed as previously described (14) . for vaccination experiments, 10-to 20-week-old 288-330 ϩ/ϩ mice were infected with 10 6 pfu of dnsp16 mutant mers-cov as described above, monitored for clinical symptoms for 7 days, and then challenged 4 weeks postvaccination with 10 6 pfu of mouse-passaged mers-cov ma1. animal housing, care, and experimental protocols were in accordance with unc iacuc guidelines. data availability. raw microarray data for these studies were deposited in publicly available databases at the national center for biotechnology information (ncbi) gene expression omnibus (37) and are accessible through geo series gse65574. middle east respiratory syndrome and severe acute respiratory syndrome middle east respiratory syndrome middle east respiratory syndrome coronavirus vaccines: current status and novel approaches mortality risk factors for middle east respiratory syndrome outbreak jumping species-a mechanism for coronavirus persistence and survival sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon the nonstructural proteins directing coronavirus rna synthesis and processing coronavirus non-structural protein 16: evasion, attenuation, and possible treatments ribose 2=-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2=-o-methyltransferase activity coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2=o)-methyltransferase activity an rna cap (nucleoside-2=-o-)-methyltransferase in the flavivirus rna polymerase ns5: crystal structure and functional characterization reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus a mouse model for mers coronavirus induced acute respiratory distress syndrome coronavirus nonstructural protein 15 mediates evasion of dsrna sensors and limits apoptosis in macrophages the dengue virus ns5 protein as a target for drug discovery polymerases of hepatitis c viruses and flaviviruses: structural and mechanistic insights and drug development evasion of early innate immune response by 2=-o-methylation of dengue genomic rna 2=-o methylation of internal adenosine by flavivirus ns5 methyltransferase a crystal structure of the dengue virus ns5 protein reveals a novel inter-domain interface essential for protein flexibility and virus replication a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics a live attenuated severe acute respiratory syndrome coronavirus is immunogenic and efficacious in golden syrian hamsters immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in e protein protects against lethal respiratory disease mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response engineering a replicationcompetent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome analysis of constructed e gene mutants of mouse hepatitis virus confirms a pivotal role for e protein in coronavirus assembly a doubleinactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine host regulatory network response to infection with highly pathogenic h5n1 avian influenza virus release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-o-methylation by nsp16/nsp10 protein complex this research was supported by grants from the niaid of the nih (u19ai100625 to r.s.b., u19ai106772 to r.s.b., hhsn272201000019i-hhsn27200003 to r.s.b., and k99ag049092 to v.d.m.). support for primary hae cell cultures was obtained from the nih through the unc cystic fibrosis research and translation core center cell models core (nih p30dk065988). the content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the nih. the pacific northwest national laboratory is operated by the battelle memorial institute for the department of energy under contract de-ac05-76rlo1830. key: cord-266313-b518n9dx authors: cao, yu-chen; deng, qi-xin; dai, shi-xue title: remdesivir for severe acute respiratory syndrome coronavirus 2 causing covid-19: an evaluation of the evidence date: 2020-04-02 journal: travel med infect dis doi: 10.1016/j.tmaid.2020.101647 sha: doc_id: 266313 cord_uid: b518n9dx the novel coronavirus infection that initially found at the end of 2019 has attracted great attention. so far, the number of infectious cases has increased globally to more than 100 thousand and the outbreak has been defined as a pandemic situation, but there are still no “specific drug” available. relevant reports have pointed out the novel coronavirus has 80% homology with sars. in the difficulty where new synthesized drug cannot be applied immediately to patients, “conventional drug in new use” becomes a feasible solution. the first medication experience of the recovered patients in the us has led remdesivir to be the “specific drug”. china has also taken immediate action to put remdesivir into clinical trials with the purpose of applying it into clinical therapeutics for corona virus disease 2019 (covid-19). we started from the structure, immunogenicity, and pathogenesis of coronavirus infections of the novel coronavirus. further, we analyzed the pharmacological actions and previous trials of remdesivir to identify the feasibility of conducting experiments on covid-19. the novel coronavirus 2019 (2019-ncov), officially named severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is a newlyemerged human infectious coronavirus. since december 2019, it has spread rapidly in china in a short period of time. as of march 17, 2020, there have been 81116 confirmed cases and 3231 deaths. it has also outbreak in other countries, such as korea, japan, italy, singapore, and iran, with a total of 85296 cases confirmed. due to it is a newlyemerged virus, researchers have taken quick actions to isolate the virus and perform gene sequencing, making identifying treatments possible. even so, it takes time to develop new drugs and vaccines, as well as to explore biotherapeutics, thus it is unlikely to be applied to patients with urgent need. therefore, "conventional drug in new use" becomes a viable solution. the sars-cov-2 is 80% homologous with the acute respiratory syndrome-associated coronavirus (sars-cov), which also broke out in china in 2002, and some enzymes are even more than 90% homologous [1] . consequently, we are expecting to find drugs for the treatment of covid-19 from the experience of sars-cov and middle east respiratory syndrome (mers-cov). some drugs, such as ribavirin, interferon, lopinavir, and corticosteroids, have been used in patients with sars or mers [2] , within the selection range of "conventional drug in new use". through clinical treatment of the covid19 , it has been found that neuraminidase inhibitors (oseltamivir, peramivir, zanamivir), ganciclovir, acyclovir, ribavirin are ineffectual and not recommended for clinical application [3] . when we set our sights on the broad-spectrum antiviral drugs, we found that a drug unlisted, remdesivir, has demonstrated strength in trials related to mers-cov and ebola virus infection. in the united states, the first patient with covid-19 has shown significant improvement in clinical symptoms within 24 h of treatment with remdesivir. this case has convinced the public that remdesivir could become a new "specific drug" for covid-19. this article starts from the structure, immunogenicity, and pathogenesis of infection of the sars-cov-2, and then analyzes the feasibility of conducting trials and putting into clinical use of covid-19 from the pharmacological characteristics and successful cases of remdesivir. different from sars-cov and mers-cov, and becomes the seventh member of the coronavirus family to infect humans [4] . sars-cov-2 shows the typical beta coronavirus organization: 5′ untranslated region (utr), replication enzyme coding region, s gene, e gene, m gene, n gene, 3' utr, and several unidentified nonstructural open reading frames ( fig. 1 ) [4] . the replication enzyme coding region mainly expresses and encodes two large genes: orf1a and orf1b, which encode 16 nonstructural proteins (nsp1~nsp16) that are highly conserved throughout the coronavirus. s gene, m gene, e gene, and n gene respectively encode four main structural proteins: spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins. the s protein is the receptor binding site, which is on the viral surface; the m protein shapes the virions, promotes membrane curvature, and is responsible for the transport of nutrients across cell membranes; the e protein plays a role in the assembly and release of virus, and is involved in viral pathogenesis; the n protein can bind virus rna genome and maintain its stability [5] . among them, s protein plays a key role in virus recognizing and binding to host cell surface receptors, and mediating the fusion of virus envelope and cell membrane [6] . through the analysis of the whole genome sequence of sars-cov-2, it shares 40% sequence similarity with mers-cov and 80% sequence similarity with sars-cov, indicating that sars-cov-2 is more compatible with sars-cov [7] . in addition, by performing systematic structural simulations and immunogenicity scans of the s proteins of all coronaviruses, as well as calculating the immunogenic distance between sars-cov-2 and other coronavirus subtypes, it can be concluded that the immunogenicity of the s protein of sars-cov-2 is closer to that of sars-cov [8] . it is known that sars-cov enters target cells by binding the s protein to the ace2 receptor on the cell surface, which is triggered by the cell serine protease tmprss2 [9] . in view of the 76% amino acid similarity between sars-cov-2 and sars-cov [10] , we can speculate that the novel coronavirus may have a similar function to sars-cov, which has been preliminary proved in bioinformatics prediction methods as well as in vitro tests [9] . previous studies have shown that 4 of the 5 key amino acids of the s protein on the surface of sars-cov-2 that binds to angiotensin-converting enzyme 2 (ace2) receptor on the target cells have changed. it was suspected it may affect the affinity of the s protein to ace2 receptor, and in turn affect the spread of the virus among the public [10] . however, through calculation methods of molecular structure simulation, the interaction between the s protein of sars-cov and the ace2 receptor has perfectly maintained in a holistic manner [11] . at present, it has been proved that the binding affinity between the extracellular domain of the s protein of sars-cov-2 and ace2 receptors is about 10-20 times higher than that of sars-cov, which may facilitate human-to-human transmission of sars-cov-2 [12] . covid-19 is a respiratory syndrome caused by sars-cov-2 infection. in general, covid-19 is an acute resolved disease, and the most common symptoms at onset are fever, dry cough, and fatigue, partly with nausea, diarrhea, or other gastrointestinal symptoms. compared with sars and mers, covid-19 has milder clinical symptoms and lower fatality [13, 14] , but it can also be fatal. severe patients may develop diffuse alveolar injury, progressive respiratory failure, and acute respiratory distress syndrome (ards) and so on. similar to sars-cov, the receptor binding domain (rbd) of s protein on the surface of sars-cov-2 binds to the ace2 receptor on the cell surface to facilitate the virus entering the host cell; then the virus exposes its rna, translates its rna replicase, and forms an rna replicase-transcriptase complex. through transcription and replication, the complex forms rna negative strands that will be translated for the structural proteins of the virus later. then the structural proteins and rna in the cytoplasm assemble into new viral particles, which are released from infected cells by exocytosis to infect other cells (fig. 2 ). each infected cell produces thousands of novel viral particles that spread to bronchi, eventually reach the alveoli, and extrapulmonary organs, causing pneumonia and targeted organic infections. however, the ace2 receptor is not only expressed in the respiratory organs. it has been reported that, by using the rna-seq method to express ace2 receptors in human tissues, the number of ace2 receptors expressed in the gastrointestinal tract (high in esophagus, small intestine, and colon, but low in stomach), kidneys, and testes is nearly 100 times higher than that in the lung [15] , suggesting that these tissues may also be the target organs for sars-cov-2 invasion. it may explain why some patients with covid-19 developed other system injuries clinically besides respiratory system injuries. furthermore, it have been found that sars-cov-2 nucleic acid detection is positive in the feces of some patients, indicating that there may be live virus in the feces, and the digestive system may be a potential route for covid-19 [16] . in covid-19, in addition to the direct damages caused by the virus, the indirect immune injuries caused by the injured tissues also attract great concern, which may be related to the severity and fatality of the disease. previous studies have shown that pulmonary inflammation and extensive lung injury in patients with sars are associated with an increase in proinflammatory cytokines (such as il-1β, il-6, il-12, ifn-γ, ip-10, and mcp-1) in serum [17] . and it has been reported that the mers-cov infection induced elevated proinflammatory cytokine concentrations (such as ifn-γ, tnf-α, il-15, and il-17) in serum [18] . we note that patients with covid-19 also have high levels of il-1β, ifn-γ, ip-10, and mcp-1 in their serum, leading to activation of the th1 cell responses. furthermore, the concentrations of gcsf, ip-10, mcp-1, mip-1a, and tnf-α in icu patients were higher than those in non-icu patients, indicating that cytokine storms were associated with disease severity. apart from this, sars-cov-2 infection also activates the secretion of cytokines (such as il-4 and il-10) in th2 cell responses that suppress inflammation, which is different from sars-cov infection [17] . further researches are needed to investigate the responses of th1 and th2 in sars-cov-2 infection to elucidate the pathogenesis of currently, the pathogenesis of covid-19 is unclear. the first pathologic autopsy of a patient with covid-19 demonstrated that the lungs of the patient reviews diffuse alveolar injury and pulmonary hyaline membrane formation, consistent with ards. the overall pathological manifestations of the lungs were similar to sars and mers. flow cytometry signified that the number of cd4 + and cd8 + t lymphocytes in peripheral blood was greatly reduced, but their state was overactivated. other than this, ccr4 + and ccr6 + th17 lymphocytes with highly proinflammatory effects increased in cd4 + t lymphocytes; cd8 + t lymphocytes had a high concentration of cytotoxic granules, of which 31.6% were perforin positive, 64.2% were particle lysin positive, and 30.5% were both particle lysin and perforin positive. it manifests that the severe immune injury in this patient may be closely linked to the overactivation of t lymphocytes characterized by the increase of th17 lymphocytes and the high cytotoxicity of cd8 + t lymphocytes [19] . we presume that the failure to develop a full adaptive immune response to covid-19 could be due to: the progression of pneumonia was too rapid to allow the available establishment of adaptive immune responses. likewise, the counts of peripheral cd4 + and cd8 + t lymphocytes were substantially reduced, leading to insufficient immune defenses. furthermore, peripheral t lymphocytes are in an over-activated state, manifested by increase of th17 and high cytotoxicity of cd8 + t lymphocytes, accounting for to a certain degree of immune injury in patients. this over activation not only failed to establish an immune response, but also caused tissue injuries, mostly manifested as severe injury in the lungs, and some patients died of multiple organ failure. this situation further accelerates the deterioration and shortens the course of the disease, hampering the establishment of fully adaptive immune response. the immunopathological injuries caused by the over activation also provides us with an idea for treating covid-19, for example, we can probably apply the il-17 inhibitor (secukinwmab) directed against th17 cell activation, but it still need more exploration. also, vaccines are also one of the solutions to make up for the lack of adaptive immune response. the latest study terms that the changes of viral nucleic acid in patients with covid-19 is similar to that in patients with influenza, but different from those with sars. viral load can be detected not only in symptomatic patients but also in asymptomatic patients, pointing out the potential for virus transmission in asymptomatic or mildly symptomatic patients. these findings are coherent with reports evidencing that the virus transmission may have occurred early in infectious processes, illustrating that case detection and isolation may require a different strategy from that required to control sars-cov [20] . remdesivir (gs-5734) is a nucleoside analogues drug (fig. 3b ) with extensive antiviral activity and effective treatment of lethal ebola and nipah virus infections in nonhuman primates [21] . as an rna-dependent rna polymerase (rdrp) inhibitor, it can inhibit the replication of multiple coronaviruses in respiratory epithelial cells. a recent study reported that remdesivir competes with natural counterpart atp. once remdesivir added into the growing chain (i position), it cannot cause an immediate stop. on the contrary, it will continue to extend three more nucleotides down to stop the strand at (i + 3) position (fig. 2) [22] . in the ces1c (−/−) mouse sars model, the preventive treatment trial of remdesivir achieved satisfactory results. administering 1 day after the onset of the disease, lung virus titers decreased significantly, fig. 2 . sars-cov-2 invasion process and how remdesivir works 1 sars-cov-2 enters target cells by binding the s protein to the ace2 receptor on the cell surface; 2 remdeivir, the nucleotide analogues, act as rdrp inhibitors, can provide a scheme for blocking rna replication; 3once remdesivir added into the growing chain (i position), is cannot cause an immediate stop. on the contrary, it will continue to extend three more nucleotides down to stop the strand at (i + 3) position; 4 remdesivir triphosphate cannot be removed by nsp14-exon. the original structure of the drug is derived from drugbank (https://www.drugbank.ca, accessed feb 25). with improvements on pulmonary function. administering 2 days after the onset, the pulmonary virus titer can be obviously reduced, but the survival rate of mice is still relatively low. this study implied that when the pulmonary injuries reach the maximum, simply reducing the virus titer can no longer suppress the strong immune responses in mice, also showing that administering before the peak of virus replication can significantly improve symptoms of the infected mice [23] . in a rhesus monkey model infected with mers-cov, treating with remdesivir 24 h before infection can completely prevent symptoms caused by mers-cov, strongly inhibit viral replications in the respiratory tract, and prevent the formation of pulmonary lesions. administering remdesivir 12 h after infection provides clear clinical benefits, reducing clinical symptoms, lung virus replication, and lung lesions [24] . pharmacokinetic experiments in cynomolgus monkeys showed the first-pass effect of oral remdesivir resulted in a low bioavailability of the drug. intramuscular injection of 3 mg/kg had a 50% survival rate compared with the control group. administering intravenously at a dose of 10 mg/kg, remdesivir rapidly decomposed into the original drug (nucleoside phosphate) in rhesus monkeys. within 2 h, remdesivir quickly distributed in peripheral blood mononuclear cells (pbmcs), and soon afterwards activated to nucleoside triphosphate to reach a peak, with a survival rate of 100% [25] . as for pharmacokinetic studies in vivo, after the intravenous infusion of the remdesivir solution formulation at a single dose of 3-225 mg for 2 h, it showed dose-linear pharmacokinetics. intravenous infusion of 150 mg of a remdesivir solution repeated 1 h per day showed a linear pharmacokinetics over a period of 14 days. after intravenously injecting 75 and 150 mg of remdesivir solution formulations over 2 h, the pharmacokinetic profile was similar to that of a lyophilized formulation. intravenous infusion of 75 mg of drug over 30 min provides similar levels of parent drug exposure to the same dose over 2 h (table 1) . after the intravenous infusion, remdesivir will enter the cellular metabolism to form active gs-443902 (fig. 3c) , but the frequencies of pbmcs exposure of gs-443902 is higher than those of intravenous infusion of remdesivir 150 mg within 2 h. studies in pbmcs show that the half-life of gs-443902 is more than 35 h [26] . in the case of daily administration, the active substance of the drug gs-443902 will accumulate in vivo. as a result, in large-scale clinical trials, after the first dose of 200 mg is administered, the subsequent dose is adjusted to 100 mg to ensure the proper blood concentration in vivo [27] . intravenous infusions in previously phase i clinical trials have good safety and pharmacokinetic properties. also, no cytotoxicity, hepatorenal toxicity, or no serious adverse reactions related to metering have been observed in climbing experiments. subjects were tolerant in studies that repeated 150 mg intravenously daily for 7-14 days. remdesivir did not show any renal injuries in a multi-dose study [26] . phase ii clinical trials were conducted in ebola virus-infected patients. in clinical trials of anti-ebola drugs, the fatality rate of patients in the experimental group using remdesivir was 53%, and the efficacy was significantly worse than that of the two monoclonal antibodies mab114 (fatality rate 35%) and regn-eb3 (fatality rate 33%) [27] . the 53% fatality rate was not significantly different from the average 50% fatality rate of ebola virus infection, and as a result, phase ii clinical trials were stopped. nevertheless, in consideration of ebola's high lethality and monoclonal antibodies with more obvious therapeutic effects, when there are merely 175 patients injected remdesivir, we cannot assume remdesivir of no avail. the small sample size is not enough to deny the effect of remdesivir. moreover, receptors of ebola virus are widely distributed in vivo, not only to the respiratory tract, but also to the digestive tract, urinary tract, and blood system, etc., causing mortally hemorrhagic fever; in addition, ebola virus persists in the eyes and central nervous system for long [28] . once remdesivir entering body, it will be quickly distributed to the testis, epididymis, eyes, and brain, but relatively less in eyes and brain [29] . all these indicate that the wide range of spread of ebola virus in the high lethality tissues make remdesivir control ebola ineffectively. the wuhan virus research institute conducted in vitro experiments on covid-19 of remdesivir and found that remdesivir was the fastest-acting and most powerful antiviral agent. in the primary culture of human airway epithelial cells in vitro, sars-cov's ic 50 = 0.069 μm, mers-cov's ic 50 = 0.074 μm, and the dose-dependent effect on virus inhibition [2] , which is speculatively related to the fact that remdesivir triphosphate cannot be removed by nsp14-exon [30] . it has been conjectured the loss of function of exonuclease may be involved with the three additional nucleotides added after the incorporation of remdesivir into the extended strand [22] . in vitro and animal models, remdesivir has demonstrated activity against both sars and mers that also belong to coronaviruses, and theoretically provides support its effectiveness in treating covid-19. presently, there have been successful cases of remdesivir in the treating covid-19. the new england journal of medicine reported the entire course of rehabilitation of the first patient with covid-19 in the united states. the patient once visited wuhan but was neither directly exposed to wuhan seafood market nor had direct contact with the diagnosed patients. he returned to washington on january 15, 2020. on 19 january, due to cough and fever for four days, he went to the hospital for emergency treatment, and was then diagnosed with covid-19. his condition was stable from the second to the fifth day of admission (the sixth to ninth day of onset). on the evening of the fifth day of admission, the blood oxygen saturation decreased to 90%. the condition continued to worsen, and chest radiographs on the sixth day of admission (tenth day of onset) showed typical characteristics of covid-19. in view of the continuous aggravation of the patient's clinical symptoms, the physicians gave a chartered medication (compassionate use) to remdesivir on the evening of the 6th day of admission, and began to give intravenous to the patient on the evening of the seventh day of admission (the eleventh day of onset), without adverse reactions. vancomycin was discontinued that night and cefepime was discontinued the following day. on the eighth day of admission (the twelfth day of onset), the patient's clinical symptoms were improved, and the oxygen saturation increased to 94%. although the patient was still hospitalized as of january 30, 2020, all symptoms had been resolved except for cough and occasional running nose [31] . it is worth noting that from the data in the article, it can be found the viral load of patients has decreased before remdesivir injection (table 2) , which is not described in detail in the original report. it's known that the viral infection is self-limiting, and the patient is a mild to moderate infectious case with a controlled fever in time, thus it is possible that his recovery is related to the role of self-defense mechanisms and supportive treatment as well. it cannot be inferred that table 1 drug concentrations in plasma and the concentration of pharmacologically active substances in pbmc in healthy people. the improvement of patients' condition after taking the drug is definitely connected to remdesivir. whether there is a link between the improvement of the symptoms and the drug is worth further consideration. clinical symptoms, especially respiratory symptoms, have been improved significantly within 24 h, bringing hope for the treatment of patients with severe covid-19. for covid-19 no specific medication is available, remdesivir is expected to be a "specific drug". however, for the acute infectious diseases, reducing the number of viral copies in the body is the key point. also, the efficacy of the drug should be focused on the pharmacokinetics and kinetics data of covid-19 in the ongoing phase iii clinical trials. the outbreak of sars-cov-2 in wuhan constituted an epidemic threat in china. the world health organization announced it a public health emergency of international concern on january 30, 2020. during the outbreak, the number of confirmed cases in china showed an exponential growth. the people and the government of the country tried their best to fight the epidemic with soaring combat mood. the nation's enthusiasm to fight the epidemic provides the trials on covid-19 a favorable environment. at the same time, article 23 of china's new drug administration law, which came into effect on december 1, 2019, has enabled the "compassionate use" to develop adaptively in china. two clinical trials on remdesivir have passed the most stringent ethical review of the 74 projects. on february 5, 2020 the trial has officially launched with experimental drugs provided by gilead sciences for free in china by professor chen wang, an academician of chinese academy of engineering, an internationally renowned respiratory expert who successfully suggested chinese government building "fang cang" hospitals to cure more than 10 thousand mild or prepatent covid-19 patients [32] . due to the large number of confirmed cases of covid-19 in china with no effective drugs, it is easy to collect clinical samples for trials theoretically. however, the rigor of the included samples hindered recruitment. as public attach more attention on prevention and treatment, fewer patients meet stringent inclusion criteria, resulting in a slow recruitment process. another reason is that there are plenty of drugs in the clinical trials, speeding up the patients' leaving hospital. nevertheless, it has been reported that more severe patients have been recruited, which provides favorable conditions for the trial of the severe group, and as a result, at least, it can be rapidly applied to the clinical treatment of severe patients in the near future. the need of treatment on covid-19 is urgent, so if the results of clinical trials prove it has the potential to benefit the treatment, according to china's "compassionate use", remdesivir will be more immediately used in patients with severe illness. meanwhile, the opening of green channels under special circumstances to speed up the review and approval process of the drug approval center will undoubtedly help save the lives of critical patients and promote the developing of "specific drugs". in the absence of clinical trial results, it is still difficult to put remdesivir into large scale clinical use [33] . with the political support, the rapid development of clinical trials on remdesivir is imperative. a drug, gs-441524 (compound a, fig. 3a ) for treating feline infectious peritonitis (fip) caused by coronavirus infection in cats has been tested in cats. its safety and effectiveness in treating fip have been proven [34] , with fda's approval. it can be seen from the structure that remdesivir is phosphorylated from gs-441524, with identical target rdrp (fig. 2) . it is noteworthy that though coronavirus reproduces more than 20 generations in gs-441524 yields resistance, the resistant virus is still sensitive to high concentrations of remdesivir and the fitness of the resistant virus has reduced to the same level as wild-type mers-cov [35] , which avoids resistant mutant coronaviruses from producing resistant supervirus. at the beginning of developing remdesivir, gilead science selected a large number of nucleosides or their prodrugs to conduct in vitro growth inhibition experiments on ebola-infected human microvascular endothelial cells in the laboratory and found compound a showed inhibitory activity (ec 50 = 0.78 μm), and the compound a was gs-441524. thereafter, on the basis of compound a, after examining the activity and toxicity of compounds surrounding compound a, modifying the prodrug, and optimizing amino acids and acyl groups, the cynomolgus monkey performs a pharmacokinetic test to select a structure such as gs-5734 (fig. 3b) [25] . although in phase ii it was not as effective as competitive drugs and clinical trials were terminated, remdesivir showed good safety and pharmacokinetics in both phases i and ii clinical trials. covid-19 has once again brought remdesivir to the stage of clinical trials. whether the results of phase iii clinical trial will make its comeback to the stage is worthy of expectation. phase ii clinical trials have demonstrated human tolerance to remdesivir. of the 175 patients in the phase ii clinical trial administering remdesivir, 9 were reported to have serious adverse reactions, 8 of whom were considered not related to drugs, and 1 with severe hypotension was thought to be drug-related, but still not confirmed [27] . the gs-441524, a drug used to treat fip, has shown a high degree of safety in feline trials as well. the focal injection site reactions only showed in immediate pain with vocalization, occasional growling, and postural changes lasting for 30-60s. these initial reactions were relieved after the owners became more adept at administering the injection. except for a cat with a slight increase in urea nitrogen and sdma of the third round of treatment, no other symptoms of systemic poisoning were observed [34] . relevant research signified that through a large number of synthesis and structure-activity analysis, the toxicity was greatly reduced after gs-411524 was synthesized into gs-5734 (remdesivir) [36] . the safety of remdesivir in human is further speculated. coronaviruses must replicate nucleic acids to generate new progeny virus after entering human cells. sars-cov-2 is known to be single stranded rna virus, so rdrp must be used to replicate nucleic acids. remdesivir, a nucleotide analogues, act as rdrp inhibitor, can provide a scheme for blocking rna replication. related studies have found that it plays a role in the final stage of entering the cell, which is consistent with its expected mode of action. wuhan virus research institute carried out a vitro inhibition test and found that remdesivir can block virus infection at very low micromolar concentration of vero e6 cells infected with virus, and the cell selectivity is high (ec 50 = 0.77 μm, cc 50 > 100 μm, si > 129.87) [2] . in an anti-ebola infection experiment on cynomolgus monkeys, intravenous injection of 10 mg/kg of remdesivir, the drug can exist in the blood for a long time and can inhibit to ebola virus with a percentage of 100 [25] . wuhan virus research institute's research that applied remdesivir to vero e6 cells with an ec 90 = 1.76 μμ, lower than that of the monkey model, draw to a speculation that it could also play a role in sars-cov-2 infected monkeys. based on the effectiveness in previous researches, although there are many unknowns and limits of remdesivir, the phase iii clinical trials on sars-cov-2 are not only a fight against this epidemic, but also of strategic importance to reserve more effective antiviral drugs for the future. strategic reservation for antiviral drugs will avoid the difficulty of medicine unavailable when an outbreak comes again. remdesivir's situational and political superiority, as well as its previous research results and application effects make it imperative to carry out the clinical trials focusing on the sars-cov-2. given that sars-cov-2 is an rna virus that is easy to mutate, the rapid starting of clinical trials is undoubtedly a right choice to prevent the resistance mutation due to blind medication. it has been covered in the world health organization (who) director-general's opening remarks at the media briefing on covid-19 on february 20, 2020 that the two clinical trials on remdesivir of therapeutics prioritized by the who r&d blueprint are y.-c. cao, et al. travel medicine and infectious disease xxx (xxxx) xxxx expected preliminary results in three weeks. on february 24, the who cast a vote of confidence for gilead sciences' experimental antiviral drug, remdesivir, indicating that remdesivir has great potential and may be the best candidate for the treatment of covid-19. whatever the progress of the clinical trials is, we are expecting that the clinical trials of remdesivir, a starring drug, would bring outstanding breakthroughs to the treatment of covid-19, or more promisingly, other virus infection in the future. all authors contributed to the conception of the review. yc cao and qx deng reviewed the literature and drafted the manuscript. sx dai critically reviewed the manuscript. all authors contributed to the revision of the manuscript. the authors report no conflicts. learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro zhonghua jie he he hu xi za zhi = zhonghua jiehe he huxi zazhi = chin a novel coronavirus from patients with pneumonia in china emerging coronaviruses: genome structure, replication, and pathogenesis the spike protein of sars-cov -a target for vaccine and therapeutic development discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin. biorxiv; 2020 identification of potential cross-protective epitope between 2019-ncov and sars virus the novel coronavirus 2019 (2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells. biorxiv; 2020 evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-em structure of the 2019-ncov spike in the prefusion conformation. biorxiv; 2020 clinical features of patients infected with 2019 novel coronavirus in wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster ace2 expression in kidney and testis may cause kidney and testis damage after 2019-ncov infection. medrxiv; 2020 the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes. biorxiv; 2020 plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile pathological findings of covid-19 associated with acute respiratory distress syndrome sars-cov-2 viral load in upper respiratory specimens of infected patients gs-5734) protects african green monkeys from nipah virus challenge the antiviral compound remdesivir potently inhibits rnadependent rna polymerase from middle east respiratory syndrome coronavirus comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection nucleotide prodrug gs-5734 is a broad-spectrum filovirus inhibitor that provides complete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates summaries of evidence from selected experimental therapeutics a randomized, controlled trial of ebola virus disease therapeutics the pathogenesis of ebola virus disease therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys nucleosides for the treatment of respiratory rna virus infections the first case of 2019 novel coronavirus pneumonia imported into korea from wuhan, china: implication for infection prevention and control measures covid-19 control in china during mass population movements at new year remdesivir as a possible therapeutic option for the covid-19 efficacy and safety of the nucleoside analog gs-441524 for treatment of cats with naturally occurring feline infectious peritonitis coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease synthesis and antiviral activity of a series of 1'-substituted 4-aza-7,9-dideazaadenosine c-nucleosides travel medicine and infectious disease xxx (xxxx) xxxx key: cord-284057-pdjz4z8z authors: choi, jeong sil; kim, kyung mi title: crisis prevention and management by infection control nurses during the middle east respiratory coronavirus outbreak in korea date: 2016-04-01 journal: american journal of infection control doi: 10.1016/j.ajic.2015.10.032 sha: doc_id: 284057 cord_uid: pdjz4z8z a middle east respiratory coronavirus (mers-cov) outbreak occurred in korea between june 20 and july 28, 2015. a total of 186 patients were confirmed as being infected with mers-cov, 36 of whom died. infection control nurses referred to hospital guidelines to address the screening and isolation needs of patients and instigated a variety of infection control activities to prevent mers-cov transmission at the frontlines of patient care. their concerted effort is believed to have been instrumental in ending the outbreak. a middle east respiratory coronavirus (mers-cov) outbreak occurred in korea between june 20 and july 28, 2015. a total of 186 patients were confirmed as being infected with mers-cov, 36 of whom died. infection control nurses referred to hospital guidelines to address the screening and isolation needs of patients and instigated a variety of infection control activities to prevent mers-cov transmission at the frontlines of patient care. their concerted effort is believed to have been instrumental in ending the outbreak. © 2016 association for professionals in infection control and epidemiology, inc. published by elsevier inc. all rights reserved. middle east respiratory syndrome (mers) is an emerging infectious disease caused by a coronavirus that gives rise to respiratory infection. mers was first discovered in saudi arabia in 2012, and cases of this disease have mainly occurred in the middle east region, but the number of countries with reported cases has been increasing. 1 on may 20, 2015, the first case of mers coronavirus (mers-cov) infection was diagnosed in korea. 2 during the period may 20-july 28, 2015, 186 patients were definitely diagnosed with mers-cov infection, 36 (19.4%) of whom died. 3 more than 16,000 people exposed to mers patients were self-isolated or isolated in hospitals. 4 patients with mers were admitted to or visited 98 hospitals across the country, exposing all regions of korea to the risk of mers infection. 5 fifteen hospitals stopped regular medical services and were placed under the government's control for isolation purposes because many of their health care workers and patients had been exposed to patients with mers-cov infection. 4 thirtynine health care workers (21%) were infected with mers-cov, 8 of whom were doctors and 15 of whom were nurses. 3 to date, this outbreak of mers-cov infection was the second largest worldwide, and the largest outbreak of this disease outside of the middle east. 6 unlike in other regions, korea's mers-cov infection outbreak presented no evidence of community transmission, and the epidemiologic pattern was that of health care-associated outbreaks. 7 additionally, mers-cov transmission was accelerated by interhospital infection. 6 the outbreak posed a critical threat to the work of infection control nurses (icns), who played key roles in keeping the disease from spreading further. since icns were first deployed in korea in 1991, 8 the medical service act has ruled that hospitals with 200 or more beds should have infection control committees and infection control departments. 9 however, even large hospitals have only 1-2 icns. 8, 10 although the struggle to stop the transmission of mers-cov infection was beyond the capabilities of these icns, they nonetheless worked diligently to limit the outbreak. a number of difficulties were encountered during the early stages of the mers-cov infection outbreak. the korea centers for disease control and prevention distributed mers-cov response guidelines based on those from the centers for disease control and prevention and the world health organization. unfortunately, the contents of these korea centers for disease control and prevention documents were too general; those working in hospitals needed more detailed guidelines. thus, the korean society for infectious disease, the korean society for healthcare-associated infection control and prevention, and the korean association of infection control nurses (kaicn) jointly released more detailed mers-cov infection control guidelines. however, each hospital's unique environment made the application of these guidelines complicated on an organization level. accordingly, kaicn members who were icns sought answers to urgent questions about infection control using social networking services and built each individual hospital's manual using shared experiences and ideas. based on these communication processes and the guidelines of the korean society for infectious disease, korean society for healthcare-associated infection control and prevention, and kaicn, icns provided information on how to wear personal protective equipment (ppe) and determined the routes of access to the negative pressure rooms. further, icns prepared hospital manuals addressing the screening and isolation of patients, hospital environment cleaning and disinfection, medical waste disposal, laundry management, specimen collection and delivery methods, patient transportation methods, patient admission and discharge, powered air purifying respirator management and cleaning, and the safe disposal of dead patients. moreover, because standard, contact, and airborne precautions had to be applied to patients infected with mers-cov and patients suspected of infection, 11 the use of ppes increased rapidly in hospitals, and the supply of n95 masks was insufficient. in addition to providing education on the proper methods of donning and doffing ppes in hospitals, icns checked their institutions' ppe inventories and were involved in maintaining sufficient ppe quantities via contact with public health centers and suppliers. although it was known that mers-cov had not undergone mutations that would have made it more transmissible, 7 its infectivity was still much stronger than assumed. patients with confirmed mers-cov infection were isolated in negative pressure rooms, and the health care workers attending these patients accessed the rooms in protective whole-body suits (including a full-length gown, goggles, n95 mask, gloves, shoe covers, and other components) that were labeled "level d." because most of the health care workers had no experience using level d ppes, icns provided instruction on how to put on the ppes, monitor the manner in which the health care workers donned and removed the ppes, and provided guidance on how to remain free from infection during the donning and doffing processes. furthermore, icns communicated with local public health centers about tasks related to patients with confirmed mers-cov, delivered the government's mers-cov-related guidelines to hospitals, and implemented the gathering of mers-cov infectionrelated data requested by the government. in addition to the icns' in-hospital activities, some participated in the immediate response task force for mers, which was launched by the korean government in early june of this year. the task force provided mers outbreak hospitals with updated and adapted scientific guidelines for patient care, infection control, and laboratory handling for medium-and small-sized hospitals. the immediate response task force for mers was composed of 17 experts in infection control, including 2 nursing professors who were former icns, and its members made about 300 visits to hospitals with patients with confirmed mers-cov infection. 6 the nursing professors visited small-and medium-sized hospitals that did not have infection control departments, as well as hospitals experiencing large outbreaks. during these visits, they provided instruction on infection control guidelines, such as ppe use and environment decontamination, and offered advice on the practice of infection control. further, they monitored the degree to which infection control guidelines were being observed in the field, participated in tabletop exercises, and provided monitoring and advice when patients were transported. in another important role, the nursing professors informed the government of difficulties and problems related to mers infection control in hospitals, so that these problems would be resolved and there would be support for the necessary resources. on july 28, 2015, the world health organization and the korean government declared the end of the mers-cov infection outbreak. 12 the transmission of an emerging infectious disease like mers-cov brought the entire society of korea to a state of crisis. problems were reported not only in the korean quarantine system, but also in health care delivery and infection control systems. the transmission of mers-cov may have been assisted by the ease of access to the hospital system in korea, as well as by the practice of seeking care at multiple hospitals (so-called doctor shopping). 7 additionally, the extremely crowded emergency rooms and multibed rooms of large metropolitan hospitals in korea led to an unexpectedly major outbreak, in comparison with the outbreak in saudi arabia. 6, 7 many hospitals experienced heavy financial losses due to the outbreak. academic societies related to infection control had earlier suggested establishing persistent infection control infrastructures, activating health care-associated infection surveillance, and constructing an infection control system for small-and mediumsized hospitals. however, the course of this outbreak shows that these suggestions had not been fully implemented. although icns were faced with the first outbreak of mers-cov in a setting with poor infection control infrastructures, they nevertheless wrestled with the disease for more than a month, working both day and night. they undertook this task with a sense of purpose, and their labor is believed to have ended the mers-cov outbreak. public health crisis preparedness and response in korea ministry of health and welfare. briefing for foreign correspondents admission or visiting healthcare facilities korean society for healthcareassociated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea world health organization (who) national survey on the current status of infection control nurses and their activities in general hospitals with more than 300 beds infection control nurse specialist education in korea factors influencing the self-perceived practice levels of professional standard competency among infection control nurses in korea interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) intensified public health measures help control mers-cov outbreak in the republic of korea key: cord-258032-buh1e4tm authors: tang, siming; ma, wanbiao; bai, peifan title: a novel dynamic model describing the spread of the mers-cov and the expression of dipeptidyl peptidase 4 date: 2017-08-15 journal: comput math methods med doi: 10.1155/2017/5285810 sha: doc_id: 258032 cord_uid: buh1e4tm the middle east respiratory syndrome (mers) coronavirus, a newly identified pathogen, causes severe pneumonia in humans. mers is caused by a coronavirus known as mers-cov, which attacks the respiratory system. the recently defined receptor for mers-cov, dipeptidyl peptidase 4 (dpp4), is generally expressed in endothelial and epithelial cells and has been shown to be present on cultured human nonciliated bronchiolar epithelium cells. in this paper, a class of novel four-dimensional dynamic model describing the infection of mers-cov is given, and then global stability of the equilibria of the model is discussed. our results show that the spread of mers-cov can also be controlled by decreasing the expression rate of dpp4. the middle east respiratory syndrome (mers) coronavirus, a newly identified pathogen, causes severe pneumonia in humans, with a mortality of nearly 44%. human-to-human spread has been demonstrated, raising the possibility that the infection could become pandemic [1] . a colorized electron micrograph shows the coronavirus mers-cov acute viral respiratory illness that is characterized primarily by cough, fever, and shortness of breath and is sometimes associated with severe and potentially fatal complications such as pneumonia and kidney failure. the illness was first observed in june 2012 in jiddah, saudi arabia, and soon afterward it was reported in other countries in the middle east, including jordan, qatar, and the united arab emirates (uae). it later was detected in europe, including cases in france, germany, italy, and the united kingdom; in the north african country of tunisia; and in countries more distant from the middle east, including china, malaysia, south korea, and the united states. the largest mers outbreak outside saudi arabia occurred in 2015, when an individual who had recently traveled to the middle east subsequently fell ill in south korea, transmitting the disease to close contacts. the dissemination of the disease by infected travelers leaving the middle east suggested that mers had the potential to escalate into an international public health emergency. the possibility of a pandemic was thought to be impeded, however, by the limited ability of the disease to be passed from one person to another. mers is caused by a coronavirus known as mers-cov, which attacks the respiratory system. the recently defined receptor for mers-cov, dipeptidyl peptidase 4 (dpp4), is generally expressed in endothelial and epithelial cells and has been shown to be present on cultured human nonciliated bronchiolar epithelium cells, providing further information on the respiratory tropism of mers-cov [2] . symptoms of illness appear anytime from 2 to 14 days following infection. cough, fever, and shortness of breath are the primary symptoms, but others such as diarrhea, nausea, vomiting, and myalgia (muscle pain) can also occur. in some persons, infection produces no symptoms or only mild cold-like symptoms, whereas in others, particularly in persons with underlying medical conditions, infection can produce severe illness [3] . it is well-known that dynamic models are still playing important roles in describing the interactions among uninfected cells, free viruses, and immune responses (see, e.g., [4] [5] [6] [7] ). a three-dimensional dynamic model for viral infection is proposed by nowak et al. (see, e.g., [5] [6] [7] ). (1) in model (1), ( ), ( ), and v( ) denote the concentration of uninfected cells, infected cells, and free viruses at time , respectively. the constant > 0 is the rate at which new uninfected cells are generated (from a pool of precursor cells). the constants > 0 and ≥ 0 are the death rate of uninfected cells and the rate constant characterizing infection of the cells, respectively. the constant 1 > 0 is the death rate of the infected cells due to either viruses or immune responses. the infected cells produce new viruses at the rate 1 during their life, on average having the length 1/ 1 , where > 0 is some integer number. the constant > 0 is the rate at which the viruses are cleared, and the average lifetime of a free virus is 1/ . figure 1 shows a interaction procedure between uninfected cells and mers-cov mediated by dpp4 receptors. based on basic dynamic model (1) and figure 1 , we propose the following novel four-dimensional dynamic model which describes the spread of the mers-cov and the expression of dpp4:̇= in model (2), ( ) represents the concentration of dpp4 on the surface of uninfected cells, which can be recognized by surface spike (s) protein of mers-cov (see, e.g., [8] ). infected cells are produced from uninfected cells and free viruses at the rate ( ( ))v( ) ( ). it is assumed that dpp4 is produced from the surface of uninfected cells at the constant rate 1 > 0. dpp4 is destroyed, when free viruses try to infect uninfected cells, at the rate 1 ( ( ))v( ) ( ), and is hydrolyzed at the rate ( ). here, 1 ≥ 0 and > 0 are constants. it is assumed that there is no undestroyed dpp4 on the surface of infected cells. all other parameters in model (2) have similar biological meanings to that in model (1) . the initial condition of model (2) is given as (0) ≥ 0, (0) ≥ 0, v(0) ≥ 0, and (0) ≥ 0. it is not difficult to show that the solution ( ( ), ( ), v( ), ( )) with the initial condition is existent, unique, bounded, and nonnegative for all ≥ 0 (in fact, it also has ( ) > 0 and ( ) > 0 for all > 0). if (0) > 0, (0) > 0, v(0) > 0, and (0) > 0, it is easily proven that the corresponding solution ( ( ), ( ), v( ), ( )) is positive for all ≥ 0. furthermore, it can be easily shown that the set is attractive and positively invariant with respect to model (2), the purpose of the paper is to study local and global stability of the equilibria of model (2) by using roth-hurwitz criterion and constructing suitable lyapunov function (see, e.g., [9] [10] [11] [12] [13] ). the basic reproductive ratio of the virus for model (2) is (2) always has an infection-free equilibrium 0 = ( 0 , 0, 0, 0 ) = ( / , 0, 0, 1 / ). if 0 > 1, model (2) also has unique infected equilibrium * = ( * , * , v * , * ), where, for first, we have the following result. proof. at any equilibrium ( , , v, ), jacobian matrix of model (2) by simple computations, we can get that the characteristic equation at 0 is ( ) = ( + )( + )[ 2 + ( + 1 ) + 1 (1 − 0 )] = 0. clearly, if 0 < 1, all roots of ( ) = 0 have negative real parts. hence, 0 is local asymptotic stability by routh-hurwitz criterion. if 0 = 1, ( ) = 0 has the zero root = 0 and three negative roots. hence, 0 is linearly stable. construct the lyapunov function as follows: it is clear that is continuous on ω 1 and positive definite with respect to 0 and satisfies condition (ii) of definition 1.1 in [14] or lemma 3.1 in [15] on ω = ω \ ω 1 . calculating the derivative of along the solutions of model (2), we have, for ≥ 0, clearly, / ≤ 0 on ω 1 by 0 ≤ 1. define = { / = 0 | ( , , v, ) ∈ ω, ( , , v, ) < +∞}. let be the largest subset in which is invariant with respect to the set model (2) . hence, we have that ⊂ ⊂ {( , , v, ) | ( , , v, ) ∈ ω, = 0 , = 0 }. from the invariance of and model (2), we can easily show that = { 0 }. therefore, it follows from theorem 1.2 in [14] or lemma 3.1 in [15] that 0 is globally attractive. this completes the proof. for local and global stability of the infected equilibrium * , we have the following result. where it is obvious that > 0 ( = 0, 1, 2, 3). furthermore, by using matlab program, it can been shown that δ 3 = 1 2 3 − 2 1 − 2 3 0 has 200 items in which all items are positive. hence, * is local asymptotic stability by routh-hurwitz criterion. construct the lyapunov function as follows: it is clear that is continuous on ω 2 and positive definite with respect to * and satisfies condition (ii) of definition 1.1 in [14] or lemma 3.1 in [15] on ω = ω \ ω 2 . calculating the derivative of along the solutions of model (2), we have, for ≥ 0, since the arithmetical mean is greater than or equal to the geometrical mean, we have that * / + * / + v * / * v * * + v * / * v − 4 ≤ 0, for any , , v, > 0, and that * / + on the other hand, notice the inequality in [16] : we have (2), we can also show that = { * }. hence, it follows from theorem 1.2 in [14] or lemma 3.1 in [15] that * is globally attractive. this completes the proof. let us first give some numerical simulations on the orbits of model (2) . take the following a set of parameters, = 1 = 1, = 0.001, = 1 = 0.05, = 1, = 0.2, 1 = 1, and = 0.11. we can compute the values of the infectionfree equilibrium 0 and the basic reproductive ratio, 0 = (20, 0, 0, 9.0909) and 0 = 0.90909 < 1. figure 2(a) shows the trajectory of model (2) with suitable initial condition, which shows that the infection-free equilibrium 0 is asymptotically stable. let us take = 0.05, and all the other parameters are the same as above. we can also compute the values of the infection-free equilibrium 0 , the infected equilibrium * , and the basic reproductive ratio, 0 = (20, 0, 0, 20), * = (14.142, 5.8579, 1.4645, 14.142), and 0 = 2 > 1. figure 2 (b) shows orbits of model (2) with suitable initial conditions, which shows that the infected equilibrium * is asymptotically stable. we would like to point out here that, based on the numerical simulations, the condition (2 ) 2 ≥ 1 2 1 3 2 may be further weakened or even removed. finally, by using the basic reproductive ratio 0 = 1 / , let us give some simple discussions on the interactions between the protein dpp4 and the virus infection. usually, in the absence of any drug treatment, all the parameters in model (2) and the corresponding basic reproductive ratio 0 can be regarded as relatively fixed constants. if some drug treatment measures are taken, the effectiveness of the treatment can be reflected in the regulation of the parameter . for example, by increasing the value of , the value of the basic reproductive ratio of 0 can be changed from greater than 1 to less than 1. in the numerical simulations in this section, figure 2 (b) shows that the virus infection will be persistent, when = 0.05 and 0 = 2 > 1. if increasing from = 0.05 to = 0.11, figure 2 (a) shows that the virus infection can be controlled, since 0 = 0.9090 < 1. the authors declare that they have no conflicts of interest. rapid generation of a mouse model for middle east respiratory syndrome middle east respiratory syndrome coronavirus (merscov) causes transient lower respiratory tract infection in rhesus macaques merse infectious diseases of humans: dynamics and control population dynamics of immune responses to persistent viruses virus dynamics: mathematics principles of immunology and virology mathematical analysis of hiv-1 dynamics in vivo host cell entry of middle east respiratory syndrome coronavirus after two-step, furinmediated activation of the spike protein global properties of basic virus dynamics models global asymptotic properties of virus dynamics models with dose-dependent parasite reproduction and virulence and non-linear incidence rate complete global stability for an sir epidemic model with delay-distributed or discrete global properties for virus dynamics model with beddington-deangelis functional response stability and hopf bifurcation in a delayed hiv infection model with general incidence rate and immune impairment global dynamics of a mathematical model of competition in the chemostat: general response functions and differential death rates necessary and sufficient conditions for permanence and global stability of a lotka-volterra system with two delays theory of motion stability and their applications the authors are grateful to drs. songbai guo and ke guo for their valuable suggestions. the research is partly supported by srtp of ustb (for s. tang and p. bai) and partly supported by the nnsf of china (11471034) and national key r&d program of china (for w. ma). s. tang and p. bai performed local and global stability analysis and the numerical simulations and wrote the manuscript. w. ma designed the study, developed the methodology of invariance principle, and wrote the manuscript. all authors have read and approved the final manuscript. key: cord-271723-8qoozmgk authors: gelman, ram; bayatra, areej; kessler, asa; schwartz, asaf; ilan, yaron title: targeting sars-cov-2 receptors as a means for reducing infectivity and improving antiviral and immune response: an algorithm-based method for overcoming resistance to antiviral agents date: 2020-06-18 journal: emerging microbes & infections doi: 10.1080/22221751.2020.1776161 sha: doc_id: 271723 cord_uid: 8qoozmgk the ongoing severe acute respiratory syndrome pandemic caused by the novel coronavirus 2 (sars-cov-2) is associated with high morbidity and mortality rates, and it has created a pressing global need for effective antiviral therapies against it. covid-19 disease pathogenesis is characterized by an initial virus-mediated phase, followed by inappropriate hyperactivation of the immune system leading to organ damage. targeting of the sars-cov-2 viral receptors is being explored as a therapeutic option for these patients. in this paper, we summarize several potential receptors associated with the infectivity of sars-cov-2 and discuss their association with the immune-mediated inflammatory response. the potential for the development of resistance towards antiviral drugs is also presented. an algorithm-based platform to improve the efficacy of and overcome resistance to viral receptor blockers through the introduction of personalized variability is described. this method is designed to ensure sustained antiviral effectiveness when using sars-cov-2 receptor blockers. the ongoing severe acute respiratory syndrome pandemic caused by the coronavirus 2 (sars-cov-2), which causes the coronavirus disease , is associated with high morbidity and mortality rates worldwide [1] . two previous coronaviruses have already previously drawn global attention, by causing potentially lethal epidemic outbreaks: the severe acute respiratory syndrome coronavirus (sars-cov), and the middle east respiratory syndrome coronavirus (mers-cov) [2] . currently, there are no specific antiviral drugs or vaccines for treatment of covid-19 [3] . moreover, the potential for resistance to antiviral agents, as is common in numerous viruses, may become a major obstacle for the development of effective therapies against sars-cov-2 [4] . in the present paper, we outline the potential targeting of receptors for sars-cov-2 therapy. the potential effect of blocking these receptors in the modulation of downstream immune responses, both in initial and late phases of the disease, is discussed. an algorithm-based platform to improve the efficacy of and overcome resistance to viral receptor blockers by introducing personalized variability is presented. covid-19 depends on the interactions between the virus and the immune system [6] . coronavirus tropism is predominantly determined by interactions between the viral spike (s) proteins and host receptors [7] . cell entry of coronaviruses requires the binding of the viral s protein to cellular receptors and depends on s protein priming by host cell proteases [8] . the spike protein contributes to host receptor binding, cell tropism, and pathogenesis, and it acts by binding to host receptors on target cells and inducing endocytosis of virions. this is followed by the fusion of host and viral membranes, allowing for the penetration of the viral genome into the host cytoplasm. the s protein is also a target of the host immune system, which adds selective pressures to this biochemical machinery [9] . coronavirus spikes can recognize a broad range of cell-surface molecules in addition to target receptors, thereby augmenting coronavirus cellular attachment and entry [7] . covid-19 disease progression follows a two-step process. in the viral infection phase, cellular infection takes place in various organs via specific receptors [10] . initially, symptoms that present are constitutional such as fever, myalgia, and respiratory symptoms including throat pain, cough, and shortness of breath [11] . the innate immune response is mediated by interferon α (ifnα) secretion and characterized by elevated levels of interleukin 6 (il-6), crp, and neutrophils, with an accompanying decrease in lymphocyte count. the virus can hamper ifn production and downstream signaling, and a dysregulated type i interferon response is part of the pathogenesis of severe infections [12] . the initial viral-infection phase is followed by an inappropriate hyperactivation of the immune response, involving multiple cytokines and immune cells, which induce immune-mediated end-organ damage [12] . in some patients, the disease progresses to a severe form which most commonly manifests as acute respiratory distress syndrome (ards) followed by respiratory failure, acute myocardial injury, cardiac dysfunction, shock, and multiple organ failure [5] . the severe form of the disease is associated with increased cytokine levels (il-6, il-10, and tnfα), lymphopenia (in cd4+ and cd8+ t cells), and decreased ifnγ expression in cd4+ t cells [13] . viral binding to the toll like receptor (tlr) promotes pro-il-1β cleavage by caspase-1, followed by inflammasome activation and an il-1β surge that induces lung inflammation, fever, and fibrosis [14] . the adaptive immune response towards sars-cov-2 is a th1 type response, mediated by cytotoxic t cells (responsible for killing virus-infected cells), and a humoral response comprising antibody production to neutralize the virus and ultimately protect from the disease [15] . a th1 response is associated with stronger levels of t cell activity and neutralizing antibodies, leading to recovery, while a th2 response may be associated with fatal disease [16] . taken together, the data support the presence of virus-immune system interactions, which underlie the pathogenesis of covid-19. the renin-angiotensin system (ras) is essential for the regulation of organ functions including those relating to the cardiovascular system, blood pressure, fluid and electrolyte balance, the kidneys, and the lungs. ras is also known to exert tissue-specific local effects associated with hypertension, myocardial injury, heart failure, diabetes, and inflammatory lung diseases [17] . ras and the angiotensin-converting enzyme (ace) are subjects of interest in determining the pathophysiology of lung inflammation in numerous disease processes [18] . renin secretion from the juxtaglomerular apparatus of the kidneys in response to a variety of stimuli acts on the circulating precursor angiotensinogen to generate angiotensin i. the conversion of angiotensin i, which lacks vasoconstriction properties, to angiotensin ii, a potent vasopressor, is due to the action of ace [19] . the modulation of ras via ace inhibition has been a primary strategy in the treatment of hypertension and heart failure [19] . ace2, a human ace homologue, is found primarily in the heart, kidneys, and testicular tissues in humans [20] . ace2 has a deceptive signaling peptide, a metalloprotease active site, and a transmembrane domain, which share genomic structures with ace, suggesting that the two genes share a common ancestor. ace2 is a negative ras regulator, and ace/ace2 imbalance is an important parameter in several disease processes including lung injuries associated with ards [21, 22] . ace2 is a metallopeptidase that serves as a cellular entry point for sars-cov and is more abundantly expressed on the apical surface than the basolateral surface of polarized airway epithelia, as well as by oral mucosal epithelial cells [23, 24] . ace2 expression positively correlates with the differentiation state of epithelia. undifferentiated cells expressing low ace2 levels are poorly infected by sars-cov, while well-differentiated cells expressing high levels are readily infected [25] . a high affinity between ace2 and the viral s1 protein domain of the sars-cov s protein has been described. viral entry into and replication in host cells depends upon interactions between the viral s protein, and the ace2 receptor on the host cell membrane. this is followed by s protein priming of the transmembrane protease serine 2 (tmprss2) [8, 26] . the ability of anti-ace2 antibodies, but not anti-ace antibodies, to inhibit viral replication in susceptible cells has been shown in a dose-dependent manner, which supports a substantial contribution of ace2 to the efficiency of sars-cov replication [23] . attenuation of ras activity protects the lungs and is dependent on ace2 expression. by deregulation of this lung-protective pathway, the virus is thought to exhibit higher lethality [27] . ace2 is also expressed by gastrointestinal epithelial cells, suggesting that sars-cov-2 can actively infect and replicate in the gastrointestinal tract [28] . there are multiple similarities between sars-cov-2 and sars-cov. the three dimensional structures of the spike protein receptor-binding domain of both viruses are almost identical, with a high degree of homology and 76% amino-acid sequence identity similarity [29] . like other coronaviruses, sars-cov-2 uses the s protein as the main interacting protein with host cell receptors, including the sars-cov receptor ace2 for entry, and the serine protease tmprss2 for s protein priming [16] . a tmprss2 inhibitor inhibited the viral entry and sera from convalescent sars patients cross-neutralized sars-cov-2 s-driven entry [8] . sars-cov, and presumably sars-cov-2, leads to downregulation of the ace2 receptor, but not the ace receptor, via binding of the s protein with ace2. this leads to viral entry and replication, and potentially to severe lung injury [27] . the receptor-binding domain (rbd) of the sars-cov-2 s protein binds to ace2 receptors, and it manifests a higher ace2 binding affinity than that of sars-cov, and it could block the binding and attachment of sars-cov-2 rbd to ace2-expressing cells inhibiting their infection to the host cells [30] . patients with cardiovascular and respiratory diseases such as hypertension, heart failure, and obstructive or inflammatory lung disease are more susceptible to sars-cov-2 infection [31] . this may be related to differences in ras activation and ace2 expression, in addition to upregulation of the receptor in the lungs in response to medical treatment that contributes to ras inhibition [32, 33] . glutamyl aminopeptidase (enpep) is a member of the m1 family of endopeptidases, which are membrane type ii zinc-containing endopeptidases. it is involved in the catabolic pathway of ras-formed angiotensin iii, and it has been proposed as another potential receptor for human coronaviruses [34] . potential therapeutic approaches have been proposed to target various steps in the viral infectious process, including a sars-cov-2 s-protein-based vaccine, a tmprss2 inhibitor aiming to block the priming of the viral s protein, an anti-ace2 antibody to block the surface receptor, and a soluble ace2 analogue which competitively binds with sars-cov-2 to slow viral entry into cells and decrease viral spread [27] . ace2 also plays a role in immune function and may be linked to immune responses associated with covid-19 pathogenesis. ras is associated with inflammation and fibrosis, and the ras axis is composed of ace2 and angiotensin-(1-7), while the mas receptor exerts opposite effects in terms of inflammatory responses and tissue fibrosis. viral respiratory infections such as influenza strains, the respiratory syncytial virus, and sars-cov-2 can mediate lung inflammation and injury via ace2 dysregulation [35, 36] . downregulation of ace2 activity in mice treated with an ace2 inhibitor prior to instilling lipopolysaccharide (lps) was shown. it-facilitated neutrophil infiltration parallel to ace2 reduction, via reduced ace2 inhibition of des-arg9 bradykinin (dabk)/bradykinin receptor b1 (bkb1r) axis signaling. this led to pro-inflammatory chemokine expression, increased neutrophil infiltration, and exaggerated levels of lung inflammation and injury [37] . these effects affect additional inflammatory processes in the lungs, including pulmonary fibrosis, chronic obstructive pulmonary disease (copd), asthma, and acute lung injury due to potentiation of pro-fibrotic and pro-inflammatory cytokines and stimulation of angiotensin ii receptors. these processes are associated with the expression of pro-inflammatory mediators, such as interleukin(il)-8/cytokine-induced neutrophil chemoattractant-3 and il-6 [38] . the anti-inflammatory and anti-fibrogenic effects of the ace2/ang-(1-7)/mas axis are linked to reduced cytokine release and the inhibition of fibrosis-associated signaling pathways in models of atherosclerosis, cerebral ischemia, obesity, chronic kidney disease, liver disease, and asthma [39] . loss of ace2 is associated with increased neutrophil, macrophage, and t-cell infiltration in a model of acute kidney injury. mrna levels relating to pro-inflammatory cytokines, il-1β, il-6, tnfα, the macrophage inflammatory protein 2, and the monocyte chemoattractant protein-1, were increased in ace2 knockout (ko) mice. decreased ace2 expression is associated with increased apoptosis and oxidative stress [40] . amelioration of sepsisinduced-lung injury is mediated via regulation of the ace2/ang-(1-7)/mas axis and inhibition of the mapk/nf-kappab inflammatory signaling pathways [41] . treatment with an angiotensin i receptor blocker prevented angiotensin ii-mediated aortic profilin-1 expression, and inflammation [42] . clinical data suggested that application of ace inhibitors and arb contribute to the clinical outcomes of covid-19 patients with hypertension. patients receiving acei or arb therapy had a lower rate of severe diseases, a trend toward a lower level of il-6 in peripheral blood, and decreased peak viral load [43] . among hospitalized covid-19 patients with hypertension, the use of these drugs was associated with lower risk of all-cause mortality [44] . these data suggest that, under some conditions, worsening of inflammation occurs using ace2 blockers, which may be beneficial in early stages of sars-cov-2 infections. the effects of these blockers during late disease phases, in which the target organ damage is mediated by inflammatory pathways, remain to be shown. receptor, which may also be associated with the antiviral immune response dipeptidyl peptidase 4 (dpp4), also known as cd26, is a 110 kda transmembrane glycoprotein expressed on the surface of a wide variety of epithelial cells and some lymphocytes. it acts as a peptidase, cleaving nterminal dipeptides and degrading numerous substances including hormones, neuropeptides, chemokines, and cytokines. the most notable of these are the incretin hormones, which participate in glucose metabolism [45] . dpp4 is uncommon in surface epithelial cells from the nasal cavities to the conducting airways, with a somewhat increased incidence in the distal airways. it has been detected in mononuclear leukocytes and serous cells of submucosal glands [46] . in the lungs, it is present on the surfaces of non-ciliated bronchial epithelial cells, on type i and ii cells, alveolar macrophages, and on vascular endothelial cells, lymphatics, and pleural mesothelia. patients with chronic lung disease manifest increased dpp4 immunostaining in alveolar epithelia, type i and ii alveolar cells, and alveolar macrophages [46] . dpp4 is a known receptor for mers-cov, and antibodies against it inhibit hcov-emc infection of primary human bronchial epithelial cells and huh-7 cells [47] . a study that examined the sars-cov-2 s protein suggested that its s1 domain, which presumably serves as the viral binding site, interacts with dpp4 [48, 49] . sars-cov-2 is thought to have a zoonotic origin prior to person-to-person transmission, and it has a high genetic similarity to sars-cov and mers-cov, which both originated in bats [50, 51] . the dpp4 protein 's amino acid sequence is highly conserved across different species, including bats. in vitro expression of human and bat dpp4 in previously non-susceptible cells without previous dpp4 expression subsequently enabled infection of those cells by mers-cov. serious adverse outcomes in patients with sars-cov-2 have been described more frequently in patients with copd comorbidity [52] . smokers, and subjects with copd, express higher rates of dpp4 [53] . dpp4 is also expressed on immune cells including t and b lymphocytes, activated natural killer (nk) cells, and myeloid cells. it plays a role in t-cell mediated immune responses, and contributes to t cell development, maturation, differentiation and activation [54] . dpp4 is involved in t-cell co-stimulatory activation via its association with adenosine deaminase (ada), caveolin-1, the caspase recruitment domain-containing membrane-associated guanylate kinase protein-1 (carma-1), cd45, the mannose-6-phosphate/insulin growth factor-ii receptor (m6p/igfii-r), and the c-x-c motif receptor 4 (cxc-r4). dpp4 mediates costimulation in human cd8+ lymphocytes, contributing to acquired immune responses, and its role in tcell immunity is attributed to its peptidase activity and ability to serve as a receptor for numerous molecules, including memory antigens [55] . dpp4 inhibitors modulate tcr signaling, inhibit the proliferation of cd4+ and cd8+ lymphocytes, and suppress antigen-stimulated cd4+ t-cell clone secretion of ifn-γ, tnf-α, and il-4 in a dose-dependent manner [56] . dpp4 is also a positive regulator of b cell activation, and its inhibitors suppress that activation via suppression of dna synthesis in mitogenic active b cells [57] . the interaction of the mers-cov s protein with dpp4 initiates signals that suppress macrophage activation [58] . infection of macrophages with particles pseudotyped with the mers-cov s protein also suppresses macrophage responses, by reducing their capacity to produce tnfα and il-6 in naive and lps-activated thp-1 macrophages, and by augmenting the lps-induced production of the immunosuppressive cytokine il-10. the mers-cov s protein induces the expression of a negative regulator of tlr signaling, irak-m, and of a transcriptional repressor, pparγ [58] . sitagliptin is a dpp4 inhibitor used for treatment of diabetes mellitus. it can inhibit proliferation of phytohemagglutinin-stimulated peripheral blood mononuclear cells (pbmc) from healthy volunteers, decreased cd26 expression, and reduced the proportions of th1, th2, and th17 lymphocytes [59] . clinical treatment with sitagliptin reduced the number of circulating cd4+ t cells, th17 lymphocytes, and regulatory t cells, and increased the number of circulating cd34 cxcr4 cells by approximately a factor of two in patients with type 2 diabetes [60] . inhibition of dpp4 by sirna or sitagliptin diminished the effects of the mers-cov s protein on irak-m, pparγ and il-10, supporting its immunosuppressive effects [58] . the data suggest that blocking dpp4 receptors may have potential benefits in modulation of the covid-19 immune response, both in early and late phases of the disease. sitagliptin has been proposed as a potential therapy for patients infected with sars-cov-2. figure 1a shows a schematic representation of the effects of blocking ace2 and dpp4 receptors on viral infectivity, and on immunomodulation of the inappropriate immune hyperactivation that induces the organ damage during the disease. the glucose-regulated protein 78 (grp78) is an endoplasmic reticulum (er) chaperone that plays a role in viral entry [61, 62] . it is involved in protein folding and assembly, translocation of newly synthesized polypeptides, degradation of misfolded proteins, and the maintenance of er homeostasis [63] . grp78 is a regulator of er stress due to its role in the unfolded protein the introduction of personalized variability to counter resistance to antiviral treatments. regular, fixed dosing using an antiviral agent with robust and rapid effects is expected to lead to viral clearance, while fixed dosing regimens of moderately effective drugs may lead to drug resistance, viral persistence, an augmented hyperactivation of the immune system, and severe disease. the introduction of an algorithm-based dosing method based on quantified variability patterns derived from disease and host parameters improves the response to antiviral drugs. response pathway, and it has also been detected in the mitochondria, nucleus, cytosol, and plasma membrane. it is associated with cell surface regulation of signaling and cellular homeostasis [64] . in the er lumen, it binds to and inactivates three enzymes responsible for cell death or differentiation, including activating transcription factor 6 (atf6), protein kinase rna-like endoplasmic reticulum kinase (perk), and the inositol-requiring enzyme 1 (ire1) [86] . above a certain threshold of accumulated unfolded proteins, grp78 releases and activates atf6, perk, and ire1, leading to the inhibition of protein synthesis and enhancement of protein refolding [64] . overexpression of grp78 is initiated by cellular exposure to stress, which increases the likelihood that it will escape er retention and translocate to the cell membrane. once there, its substratebinding domain (sbd) can be recognized by the virus, which allows viral entry into the cell. grp78 inhibits hepatitis b virus replication by reducing viral production and protein secretion [65] . grp78 is abundantly expressed by epithelial cells of the bronchus, bronchiole, and alveolus. double immunostaining of dpp4 and grp78 demonstrated the colocalization of dpp4 and grp78 in the epithelial cells lining the human airways. grp78 augments viral entry to susceptible cells in the presence of the host cell receptor dpp4 [8] . the co-localization of dpp4 and grp78 to the epithelial cell apical sides further supports the notion that grp78 facilitates mers-cov entry or attachment. grp78 assists in the attraction of virus particles to the cell surface, increasing the likelihood of receptor-mediated mers-cov and bat coronavirus hku9 (bcovhku9) viral entry. mers-cov infection results in an up-regulation of grp78 on the cell surface, which increases viral attachment and enhances viral entry into infected cells [7] . infections by certain coronaviruses, including the infectious bronchitis virus and sars-cov, induce er stress leading to grp78 expression on the cell surface [66, 67] . the sars-cov s protein induces transcriptional activation of grp78, and a substantial amount of s protein accumulates in the er. the expression of the s protein exerts different effects on the three major signaling pathways of the unfolded protein response (upr), inducing grp78 through pkr-like er kinase [68] . a recent study proposed that the sars-cov-2 s protein binds to the grp78 cell-surface receptor [69] . the sars-cov-2 s protein has been modelled using its counterpart, the sars-cov spike protein. pep42 is a cyclic peptide that binds to grp78 at the surface of cancer cells. sequence and structural alignments have shown that four regions of the s protein have sequence and physicochemical similarities to pep42. protein-protein docking studies have shown that the four s protein regions fit tightly in the grp78 substrate binding domain β (sbd β). the docking pose showed an association of the sbd β of grp78 and the receptor-binding domain of the virus s protein in recognition of the host cell receptor. region iv predicted a high binding affinity, and it has been proposed as a potential therapeutic target [69] . similarly to sars-cov and mers-cov, there is no clinically proven specific antiviral agent available to treat sars-cov-2 infections. several potential therapies, including the repurposing of existing antiviral agents, have been proposed [70] . the broad-spectrum antiviral remdesivir and chloroquine have been effective for viral control in vitro. remdesivir is an adenosine analogue which targets the rna-dependent rna polymerase and blocks viral rna synthesis, and it has shown antiviral activity against a wide array of rna viruses including sars/mers-cov. other nucleoside analogues, such as favipiravir, ribavirin and galidesivir have also been suggested as potential treatments [70, 71] . studies supporting some role for broad-spectrum antiviral remdesivir and chloroquine in covid-19 were published [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] . prolonged exposure to antiviral drugs and ongoing viral replication are key factors in the development of drug resistance, which may manifest as persistent or increasing viremia or severe disease resulting from progressive viral infection. in cases where the treatment course is effective and viral fitness is impaired sufficiently, no viral genomes will be successfully replicated. however, in conditions where the treatment is not as effective and some genomes replicate, selective pressures may result in rapid adaptation leading to viral resistance [82] . both host and viral factors are linked to the development of resistance, which involves mechanisms of viral replication, genomic inference, or selective pressures that result in viral adaptation associated with high rates of viral mutations. antiviral resistance is linked to high mutation and recombination rates, demographic histories of transmission, compartmentalization, and selective forces incurred during viral adaptation to drugs [82] . both receptor-level and post-receptor-level resistance may occur. antiviral inhibition of the viral replication cycle can occur at the level of virion inhibition, adsorption, viral penetration, viral uncoating, genome replication, or the release of mature virions [83] . the process of entry into a host cell is important for the life cycle of most viruses, and broad-spectrum antiviral approaches which target host cell proteins and pathways are being used. following the attachment to the cell surface or receptors, many viruses induce changes to environmental conditions, such as ph, interactions with a cellular receptor, or modulate the activity of proteolytic enzymes which leads to conformational changes in key proteins that mediate cell membrane penetration [84] . models have been developed to describe the development of influenza resistance using semi-stochastic simulations which determine the emergence of resistant mutants during infection, in the presence or absence of antiviral agents. a mismatch between surface proteins and internal rna correlated with a reduced likelihood of the appearance of drug-resistant mutants. late occurrence of mutants was thought to be because the mismatch provided an option to prevent the propagation of the mutation. the immune response may also lower the probability of drug-resistant infection [85] . resistance against protease or polymerase inhibitors used for the treatment of hepatitis c viral infections have been linked to a lower genetic barrier to resistance, and they can be achieved through a single or a small number of mutations. combinations such as ledipasvir and sofosbuvir, which have high genetic barriers to resistance, show little cross-resistance between the two drugs. acyclovir and its related analogues used against herpes viruses are nucleoside inhibitors, against which resistance mutations are known that affect either a thymidine kinase required for prodrug activation by phosphorylation, or the dna polymerase. hiv reverse transcriptase is highly error prone, leading to a high rate of nucleotide substitutions, increased population diversity, and frequent resistance mutations and drug resistance. combination therapies reduce the incidence of resistance without known cross-resistance mutations [86] . the overcoming of drug resistance, especially under conditions where the drugs are unable to provide rapid and complete viral clearance, is a major obstacle for improving the morbidity and mortality rates caused by numerous viruses. the use of an integrative antiviral drug repurposing methodology has been proposed to implement a pharmacology-based platform able to shorten the time to develop anti-sars-cov-2 drugs [87] . however, these may not only fail to prevent resistance, but may actually promote it due to the relatively moderate antiviral effects of these agents. 1.6. the introduction of variability into therapy to overcome resistance at the receptor level biological systems are characterized by an inherent variability, which can be viewed as a property of causal processes, contributing to their function. the stochastic behaviour of these systems typifies their dynamic behaviour in response to internal and external triggers. intrinsic stochasticity has been described for intracellular pathways [88] . intra-and inter-subject biological variability has been described at the cellular organelle level, as well as for whole organs [88] . variability also underlies responses to disease-inducing triggers and also to the effect of drugs [89] . individual tolerance to drugs is a random variable that derives from multiple genetic and environmental factors, which suggests that deterministic equations are not suitable for modelling it. variability of the treatment response has been attributed to pharmacogenomic-and pharmacodynamic-based drug metabolism parameters, but the heterogeneity in the responses cannot be attributed solely to these factors [90] . complex intracellular drug-target and drugreceptor interactions have been described. non-specific interactions slow the incorporation kinetics of dnabinding drugs, and have been attributed to irregular drug diffusion in cells. in humans, marked intrapatient variability in drug serum levels between days has been described [91] . the unpredictability of drug effectiveness is partly associated with the dynamicity and continuously changing rules by which these systems function. dosing regimens of drugs, which are based on regular fixed schedules, have been proposed to be incompatible with the physiological variability by which these systems operate, leading to primary or secondary loss of response [92, 93] . chronic anti-tnfs therapy is associated with high rates of drug response loss. dosage escalations and reductions, as well as drug holidays, are used in realworld settings. intermittent dosing with drug holidays has shown clinical benefits while helping to minimize drug exposure and potential adverse effects. in a prospective trial of patients with inflammatory bowel disease, irregular dosing reduced the loss of response compared to regular fixed dosing [94] . antiviral resistance at the receptor or post-receptor levels may be overcome by the implementation of personalized variability patterns in treatment regimens. a patient-tailored approach, which implements personalized variability patterns into treatment algorithms, has been proposed to improve the treatment sustainability. patterns of variability can be quantified based on host and disease-related variability patterns, immune tests, genetic profiling, chronotherapy, heart rate variability, and additional parameters [92, 93] . algorithm-controlled treatment regimens are now being used in several clinical trials for overcoming drug resistance (nct03843697; nct03747705). a similar platform has been proposed to ensure sustained effects of receptor-targeted therapies for sars-cov-2 infections. figure 1b shows a schematic representation of the effect of the introduction of personalized variability as a counter to antiviral drug resistance. regular, fixed dosing using an antiviral agent with robust and rapid effects is expected to aid viral clearance and recovery. on the other hand, fixed dosing with a moderately effective antiviral agent may lead to viral resistance and persistent infection. viral persistence is expected to further augment the immune hyper-activation, leading to worsening of disease severity. the introduction of an algorithm-based variable treatment approach, which quantifies disease and host-related personalized patterns, can overcome drug resistance and improve treatment responses. in summary, the high morbidity and mortality rates in patients with covid-19, and the lack of effective treatments, make them a worldwide challenge. targeting of potential receptors has been proposed to develop receptor-targeted therapies against the virus. these blockers have the potential both to reduce infectivity and to counter the inappropriate over-activation of the immune system that is responsible for end organ damage. as the likelihood of developing a powerful agent, which can rapidly clear the virus, is low, drug resistance is a major obstacle for drug development. administration of antivirals using an algorithm-regulated regimen can improve the efficacy of these therapies, preventing the development of resistance and ensuring sustained effects. future clinical trials will explore the effect of using these personalized algorithms to improve the clinical effects of antiviral agents. yi is the founder of oberon sciences and is a consultant for teva, enzo, protalix, betalin therapeutics, immuron, scim, natural shield, tiziana pharma, plantylight, and exalenz bioscience. orcid yaron ilan http://orcid.org/0000-0003-0802-1220 the covid-19 pandemic in the us: a clinical update a pneumonia outbreak associated with a new coronavirus of probable bat origin pharmacologic treatments for coronavirus disease 2019 (covid-19): a review comparison of antiviral resistance across acute and chronic viral infections clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study molecular immune pathogenesis and diagnosis of covid-19 middle east respiratory syndrome coronavirus and bat coronavirus hku9 both can utilize grp78 for attachment onto host cells sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a potential role for integrins in host cell entry by sars-cov-2 immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region sars-cov-2: virus dynamics and host response immune responses and pathogenesis of sars-cov-2 during an outbreak in iran: comparison with sars and mers induction of proinflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov pulmonary angiotensin-converting enzyme 2 (ace2) and inflammatory lung disease pulmonary epithelial tlr4 activation leads to lung injury in neonatal necrotizing enterocolitis ace revisited: a new target for structure-based drug design a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 angiotensin-converting enzyme 2 protects from severe acute lung failure ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa sars-cov-2 receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target covid-19 and the digestive system evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine potential effects of coronaviruses on the cardiovascular system: a review covid-19 and the cardiovascular system ace2, a new regulator of the renin-angiotensin system single cell rna sequencing of 13 human tissues identify cell types and receptors of human coronaviruses angiotensin-converting enzyme 2 (ace2) mediates influenza h7n9 virusinduced acute lung injury angiotensin-converting enzyme 2 protects from lethal avian influenza a h5n1 infections attenuation of pulmonary ace2 activity impairs inactivation of des-arg(9) bradykinin/bkb1r axis and facilitates lps-induced neutrophil infiltration cross-talk between inflammation and angiotensin ii: studies based on direct transfection of cardiomyocytes with at1r and at2r cdna the anti-inflammatory potential of ace2/angiotensin-(1-7)/mas receptor axis: evidence from basic and clinical research loss of ace2 exacerbates murine renal ischemia-reperfusion injury sini decoction ameliorates sepsis-induced acute lung injury via regulating ace2-ang (1-7)-mas axis and inhibiting the mapk signaling pathway ace2 deficiency enhances angiotensin ii-mediated aortic profilin-1 expression, inflammation and peroxynitrite production renin-angiotensin system inhibitors improve the clinical outcomes of covid-19 patients with hypertension association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 dpp4 in diabetes dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc emerging wuhan (covid-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd26 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond clinical characteristics of coronavirus disease 2019 in china dpp4, the middle east respiratory syndrome coronavirus receptor, is upregulated in lungs of smokers and chronic obstructive pulmonary disease patients. clinical infectious diseases: an official publication of the infectious diseases society of america cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system cd26-mediated co-stimulation in human cd8(+) t cells provokes effector function via pro-inflammatory cytokine production dipeptidyl peptidase-4 inhibitors have adverse effects for the proliferation of human t cells functional role of cd26 on human b lymphocytes middle east respiratory syndrome corona virus spike glycoprotein suppresses macrophage responses via dpp4-mediated induction of irak-m and ppargamma sitagliptin inhibit human lymphocytes proliferation and th1/ th17 differentiation in vitro sitagliptin, a dpp-4 inhibitor, alters the subsets of circulating cd4 + t cells in patients with type 2 diabetes grp78 is an important host factor for japanese encephalitis virus entry and replication in mammalian cells grp78, a coreceptor for coxsackievirus a9, interacts with major histocompatibility complex class i molecules which mediate virus internalization glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential grp78: a cell's response to stress glucose-regulated protein 78 demonstrates antiviral effects but is more suitable for hepatocellular carcinoma prevention in hepatitis b upregulation of chop/gadd153 during coronavirus infectious bronchitis virus infection modulates apoptosis by restricting activation of the extracellular signal-regulated kinase pathway characterization and mechanism of stress-induced translocation of 78-kilodalton glucose-regulated protein (grp78) to the cell surface modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein covid-19 spike-host cell receptor grp78 binding site prediction diagnosis and clinical management of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: an operational recommendation of peking union medical college hospital understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools remdesivir for the treatment of covid-19 -preliminary report remdesivir in covid-19 hopes rise for coronavirus drug remdesivir remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial should clinicians use chloroquine or hydroxychloroquine alone or in combination with azithromycin for the prophylaxis or treatment of covid-19? effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial chloroquine or hydroxychloroquine for prophylaxis of covid-19 potential of chloroquine and hydroxychloroquine to treat covid-19 causes fears of shortages among people with systemic lupus erythematosus use of hydroxychloroquine and chloroquine during the covid-19 pandemic: what every clinician should know remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro antiviral drug resistance as an adaptive process antiviral peptides as promising therapeutic drugs targeting viral entry as a strategy for broad-spectrum antivirals modelling the emergence of influenza drug resistance: the roles of surface proteins, the immune response and antiviral mechanisms the distinct contributions of fitness and genetic barrier to the development of antiviral drug resistance network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 generating randomness: making the most out of disordering a false order into a real one personalized inherent randomness of the immune system is manifested by an individualized response to immune triggers and immunomodulatory therapies: a novel platform for designing personalized immunotherapies determinants of drug-target interactions at the single cell level intrapatient variation in antiepileptic drug plasma concentration after generic substitution vs stable brand-name drug regimens a personalized signature and chronotherapy-based platform for improving the efficacy of sepsis treatment introducing patterns of variability for overcoming compensatory adaptation of the immune system to immunomodulatory agents: a novel method for improving clinical response to anti-tnf therapies dashboard driven vs. conventional dosing of infliximab in inflammatory bowel disease patients: the precision trial key: cord-016451-k8m2xz0e authors: chertow, daniel s.; kindrachuk, jason title: influenza, measles, sars, mers, and smallpox date: 2020-01-03 journal: highly infectious diseases in critical care doi: 10.1007/978-3-030-33803-9_5 sha: doc_id: 16451 cord_uid: k8m2xz0e influenza, measles, sars, mers, and smallpox illnesses are caused by highly infectious viral pathogens that induce critical illness. these biologically diverse viruses enter and replicate within host cells triggering viraland host-mediated damage that results in pneumonia and multiorgan failure in severe cases. early case identification and strict infection control limit healthcare transmission. vaccination allowed smallpox eradication and limits global measles and seasonal influenza mortality. while sars-coronavirus (cov) is no longer circulating, mers-cov and zoonotic influenza viruses, with pandemic potential, remain persistent threats. supportive critical care is the mainstay of treatment for severe disease due to these viral infections. measles virus is a pleomorphic, enveloped, negative-sense, single-stranded rna virus of family paramyxoviridae of approximately 100 nm to 300 nm in diameter [2] . measles virus causes mild to severe illness during seasonal outbreaks in endemic areas and intermittent outbreaks in nonendemic area [10] . measles virus codes for six structural and two nonstructural proteins (fig. 5 .1b) [11] . hemagglutinin (h) and fusion (f) glycoproteins project from the viral surface and facilitate viral binding to cellular receptors and fusion with the host cell membrane, respectively. matrix (m) protein underlies the envelope providing structure. the inner nucleocapsid is composed of rna coated by nucleoprotein (n), bound by the polymerase complex which includes the large (l) polymerase protein, and phosphoprotein (p), a polymerase cofactor. the remaining nonstructural proteins include c and v. coronaviruses are spherical, enveloped, positive-sense, single-stranded rna viruses of family coronaviridae of approximately 120 nm in diameter [12] . coronaviruses are the causative agents of an estimated 30% of upper and lower respiratory tract infections in humans resulting in rhinitis, pharyngitis, sinusitis, bronchiolitis, and pneumonia [13] . while coronaviruses are often associated with mild disease (e.g., hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1), severe acute respiratory syndrome coronavirus (sars-cov), a lineage b betacoronavirus, and middle east respiratory syndrome coronavirus (mers-cov), a lineage c betacoronavirus, are associated with severe and potentially fatal respiratory infection [14, 15] . sars-and mers-cov transcribe 12 and 9 subgenomic rnas, respectively, which encode for the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural proteins (fig. 5 .1c) [14] . s, e, and m are all integrated into the hostderived lipid envelope, and s facilitates host cell attachment to angiotensinconverting enzyme (ace)-2 receptors for sars-cov and dipeptidyl peptidase (dpp)-4 receptors for mers-cov [16, 17] . the n protein encapsidates the viral genome to form the helical nucleocapsid. the viral replicase-transcriptase complex is made up of 16 nonstructural proteins (nsp1-16) including a unique proofreading exoribonuclease that reduces the accumulation of genome mutations [12] . poxviruses are oval-to-brick-shaped double-stranded dna viruses of family poxviridae that range in size from 200 to 400 nm [2] . viruses within genus orthopoxvirus that cause human disease include cowpox virus (cpxv), monkeypox virus (mpxv), vaccinia virus (vacv), and variola virus (varv), the etiologic agent of smallpox [18] . poxviruses contain a biconcave viral core where the dna genome, dnadependent rna polymerase, and enzymes necessary for particle uncoating reside ( fig. 5.1d ) [19] . this nucleosome is surrounded by a core membrane that is flanked by two proteinaceous lateral bodies. a single lipid membrane surrounds the cellassociated form of the mature virion (mv). a second host-derived lipid envelope covers the extracellular virion (ev) [2, 19] . poxvirus genomes are comprised of a large, linear double-stranded viral dna genome that encodes ~200 genes. highly conserved structural genes are predominantly found in the middle of the genome, whereas variable virulence factor genes that function in immune evasion, virulence, and viral pathogenesis are found at the termini of the genome [20] . wild aquatic birds are natural reservoirs for nearly all influenza a virus subtypes, which spread to domestic avian species and mammals, including humans [5] . h17n10 and h18n11 subtypes are exceptions in that they have only been isolated from bats [6, 7] . certain h5 and h7 subtypes are highly pathogenic to domestic poultry when transmitted from wild birds, known as highly pathogenic avian influenza (hpai) viruses [21] . hpai viruses cause spillover infections in humans that may be severe or fatal. examples include outbreaks of h5n1 and h7n9 hpai viruses in asia with high case fatality among humans, although limited human-tohuman transmission [22, 23] has been reported. hpai virus adaptations might lead to sustained human-to-human transmission, and so poultry outbreaks are managed by flock depopulation [24] . influenza a subtypes isolated in swine include h1 to h5, h9, and n1 and n2. subtypes that spillover into humans cause mild to severe illness and are known as swine "variant" viruses [25] . currently circulating seasonal influenza a subtypes h1n1 and h3n2 and influenza b viruses, yamagata or victoria lineage, cause annual epidemics during fall through spring in temperate regions and infections throughout the year in the tropics [26] . antigenic drift of h and n surface glycoproteins drives annual epidemics. from 2017 to 2018, seasonal influenza caused approximately 49 million illnesses, 1 million hospitalizations, and 79,000 deaths in the united states alone [27] . when two or more influenza a viruses infect a common host, such as a bird or pig, individual gene segments may recombine to form a novel virus, known as antigenic shift. influenza pandemics occur when novel viruses emerge into an immunologically naïve population and become adapted for sustained human-to-human spread. the 1918 "spanish" influenza pandemic was the most severe on record, resulting in an estimated 50 million deaths [28] . less severe pandemics occurred in 1957, 1968, and 2009 . in an effort to improve preparedness and response to seasonal, pandemic, and zoonotic influenza, the world health organization (who) conducts global surveillance of influenza a and b isolates (fig. 5 .2a) [29] . measles is pathogenic for humans and nonhuman primates, although sustained transmission occurs only among humans raising potential for global elimination [30] . historically, measles infected an estimated 90% of children by age 5 years, resulting in approximately 2 million global deaths each year [10] . with the introduction of the measles vaccine in 1963 and advances in global vaccination programs, measles cases and mortality have drastically declined (fig. 5.2b ). by 2017, 85% of children worldwide had received at least one dose of the measles vaccine by age 1 year, and during 2000-2017, global measles mortality decreased by 80%, preventing an estimated 21 million deaths [31] . of the 24 known measles genotypes, only five were detected in circulation during 2016-2017. despite these gains, measles remains endemic in many regions of the world including africa, western pacific, south east asia, and europe, and measles has resurged in previously low-incidence areas (e.g., regions within europe and the americas) with epidemics attributable to importation of cases and suboptimal immunization coverage [32] [33] [34] . an estimated 93% population immunity is required to prevent measles transmission within communities, a prerequisite for global elimination [35] . chinese horseshoe bats are the putative reservoir for sars-cov, and dromedary camels are thought to be the reservoir for mers-cov [36] [37] [38] [39] [40] [41] [42] [43] . animal-to-human transmission likely occurs following direct contact with intermediate hosts [38, 44] . during the 2003-2004 sars epidemic, 8096 cases and 774 deaths were reported from 26 countries with no cases reported since ( fig. 5 .2c) [45] . human-to-human transmission of sars-cov occurred primarily in healthcare settings with healthcare workers comprising 22% and >40% of reported cases in china and canada, respectively [45] . mers was first reported in saudi arabia in 2012 with >2000 cases and >800 deaths reported from 27 countries through 2018 [46] . while most cases have been reported from the arabian peninsula, an imported case to south korea in 2015 resulted in a large outbreak in multiple healthcare facilities [47] . mers transmission occurs primarily in healthcare facilities and to a lesser degree within households [48, 49] . while the only known reservoir for varv is humans, it has been postulated that the virus emerged from an ancestral rodent-borne poxvirus more than 10,000 years ago [18, 50] . numerous smallpox epidemics have occurred throughout recorded history including more than 300 million fatalities during the twentieth century alone [51] [52] [53] . smallpox was eventually eradicated following the implementation of the smallpox eradication program by the who from 1966 to 1980 ( fig. 5 .2d) which was facilitated by the absence of a zoonotic reservoir for varv [51] . influenza viruses are transmitted by large respiratory droplets by coughing, sneezing, or talking or through contact with infected surfaces [54] . influenza viruses bind to sugar moieties on the surface of airway epithelial cells where early viral replication, propagation, and shedding occur during an average 1-2 days of incubation period [55] [56] [57] . peak viral replication typically occurs within 4 days of symptom onset and resolves within 7-10 days, lasting longer in children and immunocompromised hosts [58] [59] [60] . on average one person infects -one to two additional people; however, this reproductive number (r 0 ) varies by viral strain and social and environmental factors [61] . viral infection impairs the airway mucosal barrier and disrupts the alveolar-capillary membrane contributing to leakage of fluid and inflammatory cells into the alveolar space which impairs gas exchange resulting in hypoxemia [62, 63] . bacterial coinfection often complicates severe cases contributing to respiratory failure and death, with staphylococcus aureus and streptococcus species as predominant copathogens [64] . seasonal influenza virus infection is largely limited to the respiratory tract; however, h5 and h7 hpai viruses have a polybasic cleave site within the hemagglutinin allowing for replication outside of the respiratory tract [65, 66] . infection with one strain of influenza does not confer complete immunity to other strains or subtypes [67] . measles is among the most highly contagious respiratory infections, spread by exposure to large respiratory droplets through coughing, sneezing, or talking; by indirect contact with infected surfaces; or by small infectious droplets that can remain suspended in air for up to 2 hours [10, 68] . respiratory tract dendritic cells, lymphocytes, and alveolar macrophages are early targets of infection where during an average 8-to 12-day incubation period measles replicates and spreads to local lymphatics and respiratory epithelium and then disseminates in blood via infected lymphocytes to epithelial and endothelial cells in most organs [69] [70] [71] . the infectious period begins with fever onset and extends for several days after rash appears [72] . the estimated r 0 of measles is 9-18 dependent upon host susceptibility and social and environmental factors [73] . measles infects and disrupts tissues throughout the body; however, severe disease is primarily due to lower respiratory tract and neurological complications [72] . natural measles infection confers lifelong immunity, and passive transfer of maternal antibodies protects newborns during the early postnatal period [74] . individuals who recover from measles infection are at increased risk of secondary infection [75, 76] . sars-cov is transmitted by large respiratory droplets and by contact with infected surfaces. epidemiologic data also support small droplet airborne transmission of sars-cov although the estimated r 0 of 0.86-1.83 argues against this being a predominate route of spread [77, 78] . sars-cov binds to angiotensin-converting enzyme (ace)-2 receptors on respiratory epithelial cells, pneumocytes, and alveolar macrophages resulting in diffuse alveolar damage and respiratory failure [79, 80] . sars is a systemic infection with viremia detected in most cases affecting multiple cell types and organs [81, 82] . acute kidney injury is multifactorial with evidence of acute tubule necrosis, vasculitis, and glomerular fibrosis, and central nervous system manifestations are at least in part attributable to direct infection of neurons resulting in edema and degeneration [83] . mers-cov is transmitted by large respiratory droplets and by contact with infected surfaces with an estimated r0 of <1 to >1 outside of versus within healthcare settings, respectively [84] . mers-cov binds dipeptidyl peptidase 4 (dpp4) on respiratory epithelial cells and pneumocytes where it undergoes productive replication during a 2-14 days incubation period [16] . viral shedding from the lower respiratory tract may persist for weeks [85, 86] . viremia, while not documented in all cases, is associated with severe disease and productive infection of dcs, and macrophages is thought to facilitate immune dysregulation [87, 88] . dpp4 is broadly expressed on cells outside of the lung; however, few autopsy data are available to define viral distribution [16, 89] . varv is transmitted primarily by large respiratory droplets and to a lesser degree through contact with contaminated objects such as scabs, bedding, or clothing or by airborne small respiratory droplets [90, 91] . varv is thought to replicate in airway epithelium and spread to regional lymph nodes [92, 93] . varv replicates within lymph nodes and disseminates via the bloodstream seeding distant sights including skin, spleen, bone marrow, liver, kidney, and other organs [94] . fever manifests following an average 12 days incubation, and rash follows fever by 3-4 days, concurrent with high-level viral shedding from oropharyngeal secretions [95, 96] . the estimated r 0 of smallpox is between 3.5 and 6 [97] . high-level viremia is detected more often with hemorrhagic compared with ordinary type smallpox, although exact mechanisms of organ failure observed in fatal case are not well defined [98] [99] [100] [101] . influenza infection manifests as acute onset of fever, chills, malaise, headache, and myalgias following an average 1-2 days asymptomatic incubation period [9] . most infections are self-limited resolving within 1-2 weeks. upper or lower airway complications include otitis media, sinusitis, bronchitis, and pneumonia with or without bacterial coinfection [63, 64, 102] . risk factors for severe infection include age >65 years or <5 years; pregnancy; preexisting respiratory, cardiac, neurologic, or metabolic conditions; immunosuppression; and obesity. progressive lethargy and shortness of breath, typically within 5 days of symptom onset, suggest development of lower respiratory tract complications which may rapidly progress to respiratory failure and death in severe cases [64] . pneumonia due to influenza infection alone versus influenza and bacterial coinfection cannot be reliably distinguished by clinical or radiological grounds, and so a high index of suspicion is needed. influenza complications outside of the respiratory tract include exacerbation of underlying heart disease including ischemic heart disease and heart failure, myocarditis, encephalopathy, and encephalitis [103] . measles infection manifests by acute onset fever, coryza, conjunctivitis, and cough [10] . small white papules, koplik spots, appear on the buccal mucosa within 3 days of fever onset, followed by development of diffuse maculopapular rash 1 or 2 days later. diarrhea commonly begins shortly following rash onset and may result in dehydration. symptoms typically resolve within 7 days of fever onset in self-limited illness. groups at increased risk for measles complications include malnourished infants and those with vitamin a deficiency, adults >20 years old, and immunocompromised individuals [72] . respiratory complications include otitis media, laryngotracheobronchitis (croup), and pneumonia. pneumonia, often complicated by bacterial coinfection, is the most common severe complication of measles contributing to respiratory failure and death [72, 104] . predominant bacterial copathogens include streptococcus pneumonia, staphylococcus aureus, and haemophilus influenzae. three rare but severe neurologic complications occur [105] . acute disseminated encephalomyelitis (adem) is a demyelinating autoimmune process that occurs within weeks of acute illness in approximately 1 in 1000 cases. adem is characterized by fevers, seizures, and neurologic deficits. measles inclusion body encephalitis is a progressive lethal brain infection occurring within months of acute illness primarily among individuals with impaired cellular immunity. subacute sclerosing panencephalitis (sspe) occurs 5-10 years following initial infection resulting in seizures and cognitive and motor decline resulting in death. sspe affects an estimated 1 in 10,000 infants under 1 year of age and is attributed to host responses to defective viral particle production in the brain. following an average 5-day incubation period, sars-cov infection presents with fevers, chills, dry cough, headache, malaise, and dyspnea commonly followed by watery diarrhea [106] [107] [108] . age >60 years and pregnancy are associated with severe disease manifested by progressive respiratory failure within 2 weeks of illness onset [108, 109] . common laboratory features of sars included lymphopenia, thrombocytopenia, abnormal coagulation parameters, and elevated lactate dehydrogenase, alanine aminotransferase, and creatine kinase levels [110] [111] [112] . acute kidney injury and proteinuria were observed in 7% and 84% of patients, respectively [113] . initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of 5 days [114] . gastrointestinal symptoms, including vomiting and diarrhea, occur in onethird of patients [115] [116] [117] [118] . the median times from symptom onset to hospitalization, icu admission, and death are 4, 5, and 12 days, respectively [118] . mers patients present with a rapidly progressing pneumonia requiring mechanical ventilation and additional organ support with the first week of illness [109] . severe disease has been linked to comorbidities including diabetes mellitus (68%), chronic renal disease (49%), hypertension (34%), chronic cardiac disease (28%), chronic pulmonary disease (26%), and obesity (17%) [114] . the median age of those with confirmed mers is 50 years with a male-to-female ratio of 3.3:1 [114] . laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes [114, 115, 117, 119, 120] . high lactate levels and consumptive coagulopathy have also been reported [119, 121] . chest radiographic abnormalities are due to viral pneumonitis with or without secondary bacterial pneumonia, and acute kidney injury occurs in up to 43% of patients [114, 119, 120, [122] [123] [124] . as the smallpox disease course was related to the clinical presentation of disease, rao proposed a clinical classification system [125] that was later adopted by the who in 1972 [51] . ordinary type smallpox was the most common clinical type of smallpox. the incubation period was 7-19 days and was followed by fever onset (38.5-40.5 °c), headaches, backaches, vomiting, and diarrhea [51] . lesions first appeared on mucous membranes (including the tongue, palate, and pharynx) ~1 day prior to macular rash development, where lesions began on the face followed by proximal regions of the extremities, the trunk, and the distal extremities. lesion development followed a centrifugal dispersion pattern, typically most dense on the face, with papules appearing within 2 days of macular rash development. papules became vesicular ~2-4 days later followed by a pustular stage (5-7 days postrash) that peaked ~10 days postrash. pustule resolution quickly followed and was accompanied by lesion flattening, fluid reabsorption, hardening, and scab formation (14-21 days postrash). rao proposed for ordinary type smallpox to be further subdivided based on the macular rash pattern [125] . these included discrete ordinarytype smallpox, characterized by discrete skin lesions; confluent ordinary-type smallpox, where pustular skin lesions were confluent on the face and extremities; and semiconfluent ordinary-type smallpox, where skin lesions were confluent on the face but disparate over the rest of the body. modified-type smallpox, where lesions were less numerous than in ordinary-type smallpox, was primarily associated with vaccinated individuals and had an accelerated nonfatal disease course [125] . flattype and hemorrhagic-type smallpox were the most lethal forms of the disease but were also very rare (~7% and 3% of patients, respectively) [51] . flat-type smallpox had high cfrs in both unvaccinated and vaccinated patients (97% and 67%, respectively). hemorrhagic-type smallpox was nearly 100% fatal in both vaccinated and unvaccinated individuals, and death normally came prior to macular rash development. the clinical symptoms of flat-type smallpox were more severe during the prodromal period and did not subside. skin lesions were flat and often black or dark purple. respiratory complications were common and patients were febrile throughout disease. death typically occurred 8-12 days post-fever onset. hemorrhagic-type smallpox could be divided into early and late hemorrhagic-type smallpox. the early form was characterized by hemorrhage (primarily subconjunctival) early in the disease course. generalized erythema, petechiae, and ecchymosis within 2 days of fever and flat matter lesions formed across the entire body surface. lesions turned purple by day 4 with death by day 6 as a result of cardiac and pulmonary complications. in the late form, hemorrhages occurred following rash development and death followed between 8 and 10 days post-fever onset. in healthcare settings, patients under evaluation for influenza should be isolated, and standard, droplet, and contact precautions should be implemented [126] . traditional antigen-based rapid diagnostic assays (rdas) for influenza lack sensitivity and cannot be relied upon to rule out infection [26] . newer antigen-based rdas that employ a digital scan of the test strip, and molecular assays that employ isothermal amplification technology have improved sensitivity and specificity that more closely approximates highly sensitive and specific reverse transcriptase polymerase chain reaction (rt-pcr)-based assays [127] . acceptable sample types for influenza testing include nasopharyngeal swab or wash and bronchoalveolar lavage specimens. individuals suspected of zoonotic influenza infection should have case evaluation and specimen testing coordinated through local or state public health authorities. measles should be considered in patients without preexisting immunity and a compatible febrile rash illness. travel to a region with ongoing measles transmission or exposure to other individuals with a febrile rash illness should raise suspicion. patients under evaluation for measles require isolation and implementation of standard, airborne, and contact precautions. local or state health authorities should be contacted within 24 hours to assist with confirmatory testing, case finding, and infection control. measles is typically confirmed by measles-specific igm serology or detection of measles rna in a nasopharyngeal, throat, or urine specimen by rt-pcr [10] . a fourfold or greater rise in measles igg titers between acute and convalescent samples tested 2 or more weeks apart can assist with diagnostic uncertainty. virus can also be cultured from respiratory, blood, and urine specimens in appropriate public health laboratories. while sars is no longer circulating, mers should be suspected in individuals with a compatible febrile illness and an epidemiological risk factor [128] . risk factors include travel to the arabian peninsula or contact with a confirmed or suspected case within 14 days of symptom onset. patients under evaluation for mers require isolation and implementation of standard, airborne, and contact precautions. confirmatory testing and infection control should be coordinated through local or state health authorities. mers may be confirmed in designated public health laboratories by rt-pcr testing of lower respiratory tract specimens [129] . multiple other specimen types including upper respiratory tract samples, serum, and stool should also be collected for testing. serologic testing can be used to evaluate for suspected infection among individuals no longer shedding virus [129, 130] . smallpox has not been observed in over 40 years; however, concerns remain for use as a bioweapon. major and minor criteria have been established to assist clinicians in recognition of smallpox [131] . individuals under evaluation should be isolated, and standard, airborne, and contact precautions should be implemented. local or state health authorities should be contacted to assist with confirmatory testing and public health interventions. pcr identification of variola dna or isolation of the virus from a clinical specimen is required to confirm a diagnosis in specialized highcontainment laboratories. annual seasonal influenza vaccination is recommended in the united states for all individuals aged 6 months or older and has been associated with decreased risk of pneumonia and death, particularly among high-risk groups [132] [133] [134] . seasonal influenza vaccination does not provide protection against novel strains. consequently, efforts are underway to develop a vaccine that would protect against most or all influenza strains [135] . three classes of drugs are licensed for the treatment of influenza in the united states [136] . adamantanes, including amantadine and rimantadine, are not currently recommended given resistance of circulating seasonal strains. baloxavir morboxil, a cap-dependent endonuclease inhibitor, was recently approved for the treatment of uncomplicated influenza [137] . neuraminidase inhibitors (nai) include oral oseltamivir, inhaled zanamivir, and intravenous peramivir. prophylactic use of nais is recommended in unvaccinated individuals with risk factors for severe disease and during institutional outbreaks to limit spread. therapeutic use is recommended for individuals with suspected or confirmed influenza that have developed or are at high risk for influenza complications [26] . influenza complications, including respiratory and multiorgan failure, are managed with supportive care. bacterial coinfection should be considered and empirically treated early pending results of microbiologic testing among severe cases. measles can be effectively prevented through vaccination, typically given in combination with vaccines for rubella (mr), mumps (mmr), or varicella (mmr-v). who recommends the first dose of measles vaccine be administered at 9 or 12 months of age in high and low prevalence settings, respectively [138] . a second dose should be administered after a minimum of 4-week interval. nonimmune individuals that have been exposed to measles should receive post-exposure prophylaxis with mmr or immunoglobulin within 72 hours or 6 days, respectively, although not concurrently [139] . clinical management of patients with measles consists of fluid, electrolyte, and nutritional support and early recognition and treatment of bacterial coinfection [10] . two doses of vitamin a in children under 2 years have been associated with reduced risk of pneumonia and death [140] . who recommends administering 200,000 iu of vitamin a daily for 2 days in children aged 1 year and older, with reduced dosing in younger infants [141] . there are currently no licensed therapeutics or vaccines for sars or mers. consequently, supportive care is the mainstay of treatment [142] . renal replacement therapy is frequently required in severe illness [119, 143, 144] . empiric antibiotics are often administered given potential for secondary bacterial infection. ribavirin and pegylated interferon alpha 2b have been administered to mers patients, although effectiveness data is lacking [144] . aerosol-generating procedures including endotracheal intubation are associated with increased risk of healthcare worker infection necessitating strict adherence to infection control measures, including use of eye protection in addition to standard, airborne, and contact precautions [145] . while routine smallpox vaccination ceased at the end of the smallpox eradication program, it is still employed for those at increased risk for exposure. first-generation vaccines comprise a significant proportion of both the us national and global vaccine stockpiles [146] . however, first-generation vaccines carry high risk of adverse events due to use of replication-competent vacv and potential manufacturing contaminants. second-generation smallpox vaccines have reduced concerns for contaminants and are expected to have similar protective efficacy as first-generation vaccines. acam2000® has garnered us food and drug administration licensure for vaccination of those at high risk for orthopoxvirus exposure and is part of the us strategic national stockpile [147] . acam2000® and the lister-derived vaccines rivm and elstree-bn also contribute to the global stockpile. imvamune (mva), a third-generation vaccine, is licensed in europe and canada and is part of the us national stockpile. passive immunization with vig has been employed to treat complications of vaccinations [148, 149] . there has also been increasing interest in the development and licensure of small molecule antivirals for treatment of orthopoxvirus infections. cmx001 (brincidofovir), a dna synthesis inhibitor, has demonstrated protection against lethal varv in nonhuman primates [150] and has been granted ophan drug designation while also being included in the us strategic national stockpile. st-246 (tecovirimat), which inhibits viral egress, has potent (ic50 < 0.010 μm) and selective (cc50 > 40 mm) inhibitory activities against multiple orthopoxvirues [151] , is the only antipoxvirus therapeutic that has been granted approval in the us and has been added to the strategic national stockpile. types of influenza viruses serologic evidence of exposure to influenza d virus among persons with occupational contact with cattle characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family avian influenza virus surveillance and wild birds: past and present a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses a revision of the system of nomenclature for influenza viruses: a who memorandum structure, transcription, and replication of measles virus coronaviruses: an overview of their replication and pathogenesis coronavirus infections in hospitalized pediatric patients with acute respiratory tract disease sars and mers: recent insights into emerging coronaviruses a decade after sars: strategies for controlling emerging coronaviruses dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus an overview of poxviruses discovery of antivirals against smallpox avian influenza: assessing the pandemic threat global epidemiology of avian influenza a h5n1 virus infection in humans, 1997-2015: a systematic review of individual case data epidemiology of human infections with avian influenza a(h7n9) virus in china outbreaks of avian influenza a (h5n2), (h5n8), and (h5n1) among birds--united states influenza a (h3n2) variant virus-related hospitalizations: ohio estimated influenza illnesses, medical visits, hospitalizations, and death in the united states -2017-2018 influenza season influenza: the mother of all pandemics world health organization. global influenza surveillance and response system (gisrs biological feasibility of measles eradication progress toward regional measles elimination -worldwide measles cases in europe a measles outbreak in an underimmunized amish community in ohio progress toward regional measles eliminationworldwide a measles epidemic threshold in a highly vaccinated population middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camel-to-human transmission of mers coronavirus mers coronavirus in dromedary camel herd, saudi arabia isolation of mers coronavirus from a dromedary camel isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor bats are natural reservoirs of sars-like coronaviruses characterization of a novel coronavirus associated with severe acute respiratory syndrome high prevalence of mers-cov infection in camel workers in saudi arabia severe acute respiratory syndrome (sars): a year in review middle east respiratory syndrome coronavirus (mers-cov) mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study transmission characteristics of mers and sars in the healthcare setting: a comparative study transmission of middle east respiratory syndrome coronavirus infections in healthcare settings on the origin of smallpox: correlating variola phylogenics with historical smallpox records smallpox and its eradication. geneva: world health organization smallpox: the fight to eradicate a global scourge jenner and the history of smallpox and vaccination routes of influenza transmission. influenza other respir viruses transmission of influenza: implications for control in health care settings human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals influenza virus receptors in the human airway kinetics of influenza a virus infection in humans the timeline of influenza virus shedding in children and adults in a household transmission study of influenza in managua long-term shedding of influenza virus, parainfluenza virus, respiratory syncytial virus and nosocomial epidemiology in patients with hematological disorders estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature influenza virus-induced lung injury: pathogenesis and implications for treatment the pathology of influenza virus infections bacterial coinfection in influenza: a grand rounds review human infection with highly pathogenic avian influenza a(h7n9) virus, china molecular pathogenesis of h5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif immunobiology of influenza vaccines airborne transmission of measles in a physician's office measles virus host invasion and pathogenesis pathological consequences of systemic measles virus infection predominant infection of cd150+ lymphocytes and dendritic cells during measles virus infection of macaques the clinical significance of measles: a review the basic reproduction number (r0) of measles: a systematic review early waning of maternal measles antibodies in era of measles elimination: longitudinal study measles virus-induced suppression of immune responses long-term measles-induced immunomodulation increases overall childhood infectious disease mortality evidence of airborne transmission of the severe acute respiratory syndrome virus model parameters and outbreak control for sars epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury multiple organ infection and the pathogenesis of sars tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis pathology and pathogenesis of severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov). who mers-cov global summary and risk assessment viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection viral load kinetics of mers coronavirus infection from sars to mers, thrusting coronaviruses into the spotlight viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels an airborne outbreak of smallpox in a german hospital and its significance with respect to other recent outbreaks in europe transmission potential of smallpox: estimates based on detailed data from an outbreak diagnosis and management of smallpox immunopathogenesis of poxvirus infections: forecasting the impending storm the cause of death in smallpox: an examination of the pathology record virus excretion in smallpox. 1. excretion in the throat, urine, and conjunctiva of patients tropical infectious diseases: principles, pathogens and practice transmission potential of smallpox in contemporary populations viraemia in smallpox virus and virus antigen in the blood of smallpox patients; their significance in early diagnosis and prognosis viraemia in haemorrhagic and other forms of smallpox influenza a virus--induced acute otitis media the hidden burden of influenza: a review of the extra-pulmonary complications of influenza infection. influenza other respir viruses coinfection is common in measles-associated pneumonia measles virus and the nervous system coronavirus as a possible cause of severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients severe acute respiratory syndrome vs. the middle east respiratory syndrome severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome (sars): epidemiology, diagnosis and management acute renal impairment in coronavirus-associated severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission epidemiological findings from a retrospective investigation family cluster of middle east respiratory syndrome coronavirus infections state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings middle east respiratory syndrome clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia bombay: the kothari book depot prevention strategies for seasonal influenza in healthcare settings diagnostic accuracy of novel and traditional rapid tests for influenza infection compared with reverse transcriptase polymerase chain reaction: a systematic review and meta-analysis interim patient under investigation (pui) guidance and case definitions respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome epidemiology of coronavirus-associated respiratory tract infections and the role of rapid diagnostic tests: a prospective study evaluating patients for smallpox: acute, generalized vesicular or pustular rash illness protocol effectiveness of influenza vaccination in institutionalized older adults: a systematic review university of nottingham influenza and the immunocompromised (uniic) study group, nguyen-van-tam js. influenza vaccination for immunocompromised patients: systematic review and meta-analysis by etiology prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states the pathway to a universal influenza vaccine influenza antiviral medications: summary for cli baloxavir marboxil investigators g. baloxavir marboxil for uncomplicated influenza in adults and adolescents world health organization. measles vaccines: who position paper measles (rubeola): for healthcare professionals vitamin a for treating measles in children middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease recovery from severe novel coronavirus infection ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review smallpox vaccine and its stockpile in 2005 clinical guidance for smallpox vaccine use in a postevent vaccination program the efficacy of vaccinial immune globulin. a 15-year study experience of anti-vaccinia immunoglobulin in the united kingdom efficacy of tecovirimat (st-246) in nonhuman primates infected with variola virus (smallpox) an orally bioavailable antipoxvirus compound (st-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge key: cord-261421-k1s5iy3u authors: khalafalla, abdelmalik i.; lu, xiaoyan; al-mubarak, abdullah i.a.; dalab, abdul hafeed s.; al-busadah, khalid a.s.; erdman, dean d. title: mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia, 2013–2014 date: 2015-07-17 journal: emerg infect dis doi: 10.3201/eid2107.150070 sha: doc_id: 261421 cord_uid: k1s5iy3u to assess the temporal dynamics of middle east respiratory syndrome coronavirus (mers-cov) infection in dromedary camels, specimens were collected at 1–2 month intervals from 2 independent groups of animals during april 2013–may 2014 in al-ahsa province, saudi arabia, and tested for mers-cov rna by reverse transcription pcr. of 96 live camels, 28 (29.2%) nasal swab samples were positive; of 91 camel carcasses, 56 (61.5%) lung tissue samples were positive. positive samples were more commonly found among young animals (<4 years of age) than adults (>4 years of age). the proportions of positive samples varied by month for both groups; detection peaked during november 2013 and january 2014 and declined in march and may 2014. these findings further our understanding of mers-cov infection in dromedary camels and may help inform intervention strategies to reduce zoonotic infections. m iddle east respiratory syndrome coronavirus (mers-cov) is an emerging pathogen associated with severe respiratory symptoms and renal failure in infected persons (1, 2) . saudi arabia is the country most severely affected by the virus and is where the first recognized case was identified in 2012. the origin of mers-cov remains a mystery. bats seem to be the reservoir host of the virus (3) but are probably not the source of the ongoing mers-cov outbreak because of limited contact with humans in the arabian peninsula. early observations that some mers-cov-infected persons had been exposed to camels suggested a possible role of these animals as intermediate reservoir hosts (2, 4) . serologic surveys subsequently conducted in several countries in the arabian peninsula and africa identified high rates of mers-cov-specific antibodies in dromedary camels (5) (6) (7) (8) (9) (10) (11) (12) . furthermore, mers-cov infection in dromedary camels was definitively proven by the detection of virus and virus sequences in respiratory specimens, feces, and milk collected from camels in qatar (9, 13) , oman (14) , saudi arabia (5, 15, 16) , and egypt (17) . the few published studies that looked for mers-cov in the respiratory tract of naturally infected dromedary camels examined nasal or ocular swab samples but not samples from the lower respiratory tract. moreover, several studies relied on only a few specimens or collected specimens at only 1 time point (9, (13) (14) (15) . to address these limitations and to clarify the dynamics of mers-cov infection in these animals, we conducted a year-round study in which we collected a large number of specimens from the upper respiratory tracts of live dromedary camels and from the lungs of dromedary camel carcasses. this study was approved by the institutional review board of the camel research center, king faisal university, al-ahsa, saudi arabia. respiratory specimens were collected from 2 independent groups of mixed-age dromedary camels (camelus dromedaruis). the first collection was obtained during april 2013-may 2014 at the al omran abattoir, al omran city, in al-ahsa province in the eastern region of saudi arabia. livestock slaughtered at this abattoir include cattle, goats, sheep, and camels originating from al-ahsa and neighboring provinces. animals selected for slaughter were mainly from the livestock market and from herds located around al-ahsa province. at the livestock market in al-ahsa, dromedary camels are housed in small groups (10-15 animals), where they may stay for no more than 4 days. they are then transported in vehicles to the abattoir, where they are kept for no more than 24 hours before slaughter. samples were taken from slaughtered dromedary camels on 8 occasions (every 1-2 months). on each particular collection date, tissue specimens were collected from the lungs of all slaughtered dromedary camels. a total of 91 animal carcasses were sampled; 28 had been young animals (<4 years of age) and 63 had been adults (>4 years of age). lung lobes that showed pulmonary lesions were sampled; if both lobes showed lesions or if no lesions were visible, the left lobe was sampled because of its close proximity to the person collecting the sample. the tissue samples (≈1-2 g) were collected aseptically from inside the lung lobes by using sterile surgical instruments (scalpels, forceps, and scissors). to avoid cross-contamination, lungs were moved to a clean room adjacent to the slaughtering hall and examined on a freshly disinfected table by a person wearing a newly donned gown, face mask, and sterile gloves and using a new set of sterile surgical instruments. collected tissue samples were immediately deposited in labeled sterile plastic bags and placed in a cooler containing ice packs for transport to the laboratory. a second sample was collected from age-matched animals over the same period and consisted of 96 nasal swab specimens (36 young animals and 60 adults), 94 from visually healthy dromedary camels and 2 from camels with nasal and lachrymal discharge. nasal swabs were collected from animals at 3 locations in al ahsa province (al omran abattoir, al ahsa livestock market, and the veterinary hospital of king faisal university). for this procedure, a long sterile flexible swab was inserted into 1 nostril until slight resistance was felt; the swab was then rotated, held in place for 5 seconds, withdrawn, and placed in 1 ml of cold viral transport medium containing antibiotics (this medium was chosen to enable future attempts to isolate the virus). both swab and lung specimens were transported on ice to the laboratory within 1-2 hours of collection and stored at −80°c until testing. collection dates and numbers of samples are listed in table 1 . swab specimens in transport media were mixed and then clarified by centrifugation at 350 × g for 10 minutes; the supernatants were recovered for extraction. lung samples were thawed and homogenized by using a tissueruptor homogenizer (qiagen, hilden, germany), and 20% suspensions were prepared in 5 ml of transport medium. the resulting homogenates were subjected to centrifugation as above, and the supernatants were recovered for extraction. total rna was extracted from 140 μl of each nasal swab or lung sample by using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's instructions. extracted rna was tested by using a gel-based pancoronavirus reverse transcription pcr (rt-pcr) assay according to the protocol of vijgen et al. (18) . realtime rt-pcr (rrt-pcr) was performed by using an assay kit provided by the centers for disease control and prevention (cdc; atlanta, ga, usa). this assay panel targets the mers-cov nucleocapsid protein gene (19) and a region upstream of the envelop protein gene described by corman et al. (20) . all samples were screened by using gel-based rt-pcr and 2 rrt-pcr assays and were considered positive for mers-cov if a positive result was obtained with at least 2 of the 3 tests following world health organization recommendations (http://www.who.int/csr/disease/coronavirus_infections/ who_interim_recommendations_lab_detection_mer-scov_092014.pdf). all rt-pcrs included no-template negative controls and quantified mers-cov transcript as positive control. cdna was prepared from 20 positive samples and shipped to cdc for independent confirmation and sequencing. to assess the genetic variability of mers-cov, we sequenced the spike protein gene coding region (4,062 nt) on the 20 positive samples. sequencing was performed on an applied biosystems 3130xl genetic analyzer (thermo fisher scientific, grand island, ny, usa) by using sequencher version 4.8 software (gene codes, ann arbor, mi, usa) for sequence assembly and editing. sequence alignments were performed by using clustalx version 1.83 implemented in bioedit version 7.2.5 (http://www.mbio. ncsu.edu/bioedit/bioedit.html). phylogenetic analyses were performed by using mega version 6.06 (http://www. megasoftware.net). the neighbor-joining method (tree algorithm inferred with the kimura 2-parameter substitution model of sequence evolution) was used to construct phylogenetic trees, and bootstrap resampling analyses were performed (1,000 replicates) to test tree reliability. during the study, a total of 91 lung tissue samples and 96 nasal swabs were obtained from the 2 groups of camels (table 1) (table 1) . all animals from both groups appeared healthy on visual inspection except for 2. these 8-month-old dromedary camel calves, located outside of the al omran abattoir, exhibited purulent nasal and lachrymal discharge; mers-cov rna was detected in nasal swab specimens from these 2 calves (figure 1 ). mers-cov rna was more often detected in the lung and nasal cavity of young camels than adult camels (table 2) our results confirm previous reports documenting wide circulation of mers-cov in dromedary camel populations in the middle east. in other studies, rt-pcr detection of mers-cov in nasal swab specimens from these animals has ranged from 1.6% to 41.7%. studies conducted in qatar detected mers-cov in 4 (35.7%) of 14 (13) and 5 (41.7%) of 12 (9) animals tested; in saudi arabia, 9 (22%) of 41 (16) and 51 (25%) of 202 (5); in oman, 5 (6.6%) of 76 (14) ; and in egypt, 4 (3.6%) of 110 (17) . a recent large study of 7,803 dromedary camels in the united arab emirates identified mers-cov rna in only 1.6% of animals (22) . of note, these authors found proportionately more positive animals near the border with saudi arabia and detected >5fold more among animals sampled from slaughter houses. overall, we detected mers-cov in the upper respiratory tract of a higher proportion of animals tested in al-ahsa, but this proportion was within the upper range previously reported. in contrast, alagaili et al. (5) , in a comprehensive survey conducted in november and december 2013, sampled 5 regions of saudi arabia (gizan in the south, taif in the west, tabuk in the north, uniza in the center, and hofuf [al-ahsa] in the east) and reported 66% positivity by rrt-pcr in animals from taif versus only 5% from al-ahsa, despite seroprevalence of 92% in the latter. during the same period and in the same region, we detected mers-cov in 38.5% of nasal swab samples. this difference may be because of differences in the numbers and ages of animals sampled, time of specimen collection, or even between geographically proximate dromedary camel herds where rates of mers-cov detection can vary dramatically (9) . of note, detection of mers-cov rna by rt-pcr does not necessarily indicate active virus replication. when 3 dromedary camels were experimentally inoculated, infectious mers-cov was detected in the upper respiratory tract for only 7 days, but rna could be detected by rt-pcr for up to 35 days after inoculation (23). we were unable to perform virus isolation studies because of lack of suitable biosafety infrastructure. we also found that a high proportion of lung tissues from slaughtered dromedary camels at the al omran abattoir were mers-cov positive by rt-pcr. in their experimental inoculation study, adney et al. (23) observed histologic lesions in the epithelium of the upper and lower (trachea, bronchi, and bronchioles) respiratory tract and recovered viable virus from these tissues and from 1 of 4 lung lobes of an animal euthanized 5 days after inoculation; viable virus was not recovered from tissues of 2 other animals at 28 and 42 days after inoculation. although that limited study found infection extending to the lung of 1 animal, the authors found that the upper respiratory tract was the predominant site of virus replication and offered that finding as an explanation for the lack of observed systemic illness among naturally infected dromedary camels. an alternative hypothesis posits that, in the natural setting, subclinical mers-cov infection of the lower respiratory tract also occurs, possibly enhanced by crowding and stress endured during transport and corralling before slaughter. although we did not collect matching premortem nasal swab samples from slaughtered animals to determine how many were also positive for mers-cov in the upper respiratory tract, our findings raise the possibility that testing upper respiratory tract samples alone may underestimate the true number of actively infected animals. in humans, mers-cov was detected in the lower respiratory tract of infected patients for ≈1 month while oronasal swab samples were negative (24) . likewise, mers-cov detection has been found to be enhanced from lower respiratory tract specimens, and therefore these specimens are recommended by the world health organization for diagnosis of mers-cov infection (2, 24, 25) . although great care was taken to avoid contamination with ambient mers-cov present in the abattoir, the possibility that sample contamination occurred cannot be entirely ruled out. further studies that include immunohistologic examination and virus isolation from the lower respiratory tract of naturally infected dromedary camels will be needed to substantiate these findings. our detection of mers-cov rna in 2 camel calves with purulent nasal discharge was consistent with those of hemida et al. (16) , who also observed mild clinical signs characterized by nasal discharge in some naturally infected young dromedary camels, and of adney et al. (23) , who documented appearance of purulent nasal discharge in the 3 experimentally infected adult dromedary camels. we also detected mers-cov rna in a higher proportion of specimens from younger than from older adult dromedary camels, consistent with findings of previous studies that mers-cov infection is more common among young camels (5, 16) . our study also investigated temporal variation in mers-cov infection in dromedary camels. although data interpretation was complicated by discontinuity in the months sampled and sampling from only 1 animal group in some months, a temporal pattern in mers-cov prevalence was apparent. for both animal groups, peak detection occurred during november 2013-january 2014, followed by a steady decline, reaching the lowest point in may 2014. although we observed no clear temporal differences in the geographic origins or ages of dromedary camels brought to slaughter, which might bias these results, our data are nevertheless limited and should not be used to imply a general pattern of mers-cov circulation in dromedary camels in saudi arabia. nevertheless, these findings would not be unexpected. increased circulation of mers-cov among dromedary camels during the cool season is consistent with the prevailing cooler ambient temperatures, which have been shown to enhance coronavirus survivability outside the host (26, 27) , and the cool season is the period of peak circulation of other respiratory viral pathogens of humans in saudi arabia (28) (29) (30) . this period also corresponds with the peak calving season for dromedary camels in saudi arabia (16) ; higher rates of mers-cov infections among a greater proportion of young animals with higher virus loads may increase opportunities for virus spread (5, 16) . whereas the link between dromedary camels and mers-cov infection of humans is well established (15, 31) , the overall contribution of zoonotic infections to community-acquired mers-cov remains unclear. serologic studies of animal handlers in saudi arabia who work 1156 emerging infectious diseases • www.cdc.gov/eid • vol. 21, no. 7, july 2015 in close proximity to dromedary camels have shown limited evidence of mers-cov infection (32) (33) (34) . alghamdi et al. (35) , who examined patterns of mers-cov infections among humans in saudi arabia between june 2013 and may 2014, did not find a concomitant temporal increase in human infections that corresponded with our findings in dromedary camels. those authors observed a slight, temporary increase in cases among humans in june and september 2013 and few cases from october through february, after which cases and deaths sharply increased beginning in april 2014. the authors concluded that lower relative humidity and higher temperatures during these months might have contributed to the dramatic surge in reported cases. however, more recent data from the world health organization (36) show a sharp decline in mers-cov cases among humans in may 2014; low numbers of cases were reported from june through august 2014, when mean temperature was highest and relative humidity was lowest in saudi arabia (34) . moreover, a recent increase in numbers of mers-cov cases in humans from september 2014 through february 2015 corresponds more closely with the temporal pattern we found in dromedary camels the preceding year. further studies conducted over multiple years are needed to better understand the ecology of mers-cov, which might help inform intervention strategies to reduce zoonotic infections. isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus in bats, saudi arabia recovery from severe novel coronavirus infection middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia seroprevalence in domestic livestock in saudi arabia seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralization assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels geographic distribution of mers coronavirus among dromedary camels antibodies against mers coronavirus in dromedary camels seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterization of assay specificity middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels human infection with mers coronavirus after exposure to infected camels, saudi arabia mers coronavirus in dromedary camel herd, saudi arabia mers coronaviruses in dromedary camels a pancoronavirus rt-pcr assay for detection of all known coronaviruses real-time reverse transcription polymerase chain reaction assay panel for middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels mers-cov study group. clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission severe respiratory illness caused by a novel coronavirus stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions t he effects of temperature and relative humidity on the viability of the sars coronavirus viral aetiology and epidemiology of acute respiratory infections in hospitalized saudi children respiratory viruses in children attending a major referral centre in saudi arabia viral agents causing acute lower respiratory tract infections in hospitalized children at a tertiary care center in saudi arabia evidence for camel-to-human transmission of mers coronavirus investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012. influenza other respir viruses lack of middle east respiratory syndrome coronavirus transmission from infected camels the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment we thank isam al jalii and khalid borsais for assistance with sample collection and marzooq m. al eknah for financial support.dr. khalafalla is professor of veterinary virology at king faisal university, al-ahsa, saudi arabia. his research focus is on viral diseases of dromedary camels. key: cord-266260-t02jngq0 authors: ramshaw, rebecca e.; letourneau, ian d.; hong, amy y.; hon, julia; morgan, julia d.; osborne, joshua c. p.; shirude, shreya; van kerkhove, maria d.; hay, simon i.; pigott, david m. title: a database of geopositioned middle east respiratory syndrome coronavirus occurrences date: 2019-12-13 journal: sci data doi: 10.1038/s41597-019-0330-0 sha: doc_id: 266260 cord_uid: t02jngq0 as a world health organization research and development blueprint priority pathogen, there is a need to better understand the geographic distribution of middle east respiratory syndrome coronavirus (mers-cov) and its potential to infect mammals and humans. this database documents cases of mers-cov globally, with specific attention paid to zoonotic transmission. an initial literature search was conducted in pubmed, web of science, and scopus; after screening articles according to the inclusion/exclusion criteria, a total of 208 sources were selected for extraction and geo-positioning. each mers-cov occurrence was assigned one of the following classifications based upon published contextual information: index, unspecified, secondary, mammal, environmental, or imported. in total, this database is comprised of 861 unique geo-positioned mers-cov occurrences. the purpose of this article is to share a collated mers-cov database and extraction protocol that can be utilized in future mapping efforts for both mers-cov and other infectious diseases. more broadly, it may also provide useful data for the development of targeted mers-cov surveillance, which would prove invaluable in preventing future zoonotic spillover. middle east respiratory syndrome coronavirus (mers-cov) emerged as a global health concern in 2012 when the first human case was documented in saudi arabia 1 . now listed as one of the who research and development blueprint priority pathogens, cases have been reported in 27 countries across four continents 2 . imported cases into non-endemic countries such as france, great britain, the united states, and south korea have caused secondary cases [3] [4] [5] , thus highlighting the potential for mers-cov to spread far beyond the countries where index cases originate. reports in animals suggest that viral circulation could be far more widespread than suggested by human cases alone [6] [7] [8] . to help prevent future incidence of mers-cov, public health officials can focus on mitigating zoonotic transfer; however, in order to do this effectively, additional research is needed to determine where spillover could occur between mammals and humans. previous literature reviews have looked at healthcare-associated outbreaks 9 , importation events resulting in secondary cases 10, 11 , occurrences among dromedary camels 12, 13 , or to summarize current knowledge and knowledge gaps of mers-cov 14, 15 . this database seeks fill gaps in literature and build upon existing notification data by enhancing the geographic resolution of mers-cov data and providing occurrences of both mammal and environmental detections in addition to human cases. this information can help inform epidemiological models and targeted disease surveillance, both of which play important roles in strengthening global health security. knowledge of the geographic extent of disease transmission allows stakeholders to develop appropriate emergency response and preparedness activities (https://www.jeealliance.org/ global-health-security-and-ihr-implementation/joint-external-evaluation-jee/), inform policy for livestock trade and quarantine, determine appropriate demand for future vaccines (http://cepi.net/mission) and decide where to deliver them. additionally, targeted disease surveillance will provide healthcare workers with updated lists of the methods and protocols summarized below have been adapted from previously published literature extraction processes [18] [19] [20] [21] [22] , and provide additional context surrounding our systematic data collection from published reports of mers-cov. data collection. we identified published reports of mers-cov by searching pubmed, web of science, and scopus with the following terms: "middle eastern respiratory syndrome", "middle east respiratory syndrome", "merscov", and "mers". the initial search was for all articles published about mers-cov prior to april 30, 2017 , and was subsequently updated to february 22, 2018. these searches were conducted through the university of washington libraries' institutional database subscriptions. we searched the web of science web of science core collection (the subscribed edition includes science citation index expanded, 1900-present; social sciences citation index, 1975-present; arts & humanities citation index, 1975-present; emerging sources citation index, 2015-present). we searched the standard scopus database and the standard, freely available pubmed database; these products have a single version that is consistent across institutional subscriptions or access points. in total, this search returned 7,301 related abstracts, which were collated into a database before a title-abstract screening was manually conducted (fig. 1. flowchart) . articles were removed if they did not contain an occurrence of mers-cov; for example, vaccine development research or coronavirus proteomic analyses. non-english articles were flagged for further review and brought into the full text screening stage. the accompanying supplementary file highlight the title and abstract screening process and the inclusion and exclusion criteria. full text review was conducted on 1,083 sources. to meet the inclusion criteria, articles must have contained both of the following items: 1) a detection of mers-cov from humans, animals, or environmental sources, and 2) mers-cov occurrences tagged with spatial information. additionally, extractors attempted to prospectively manually remove articles containing duplicate occurrences that were already extracted in the dataset. extractors only prospectively manually removed articles if it was clear the articles contained data we were confident had already been extracted and had high-quality data. we excluded 885 sources based on full text review. in addition, we reviewed citations and retroactively added relevant articles to our database if they were not already included. we retroactively added and subsequently marked ten articles for extraction using this process. in total, we extracted 208 peer-reviewed sources reporting detection of mers-cov that included geographic and relevant epidemiological metadata. geo-positioning of data. google maps or arcgis 23 was used to manually extract location information at the highest resolution available from individual articles. we evaluated spatial information as either points or polygons. the geography was defined as a point if the location of transmission was reported to have occurred within a 5 × 5 km area. point data are represented by a specific latitude and longitude. a point references an area smaller than 5 × 5 km in order to be compatible with the typical 5 × 5 km resolution of satellite imagery used for global analyses. the geography was defined as a polygon if the location of transmission was less clear, but known to have occurred in a general area (e.g. a province), or the location of transmission occurred within an area greater than 5 × 5 km (e.g. a large city). we used contextual information to determine location in instances where the author's spelling of a location differed from google maps or arcgis. maps provided by authors were digitized using arcgis. we used three different types of polygons: known administrative boundaries, buffers, and custom polygons. relevant administrative units were sourced from the global administrative unit layers curated by the food and agricultural organization of the un 24 for known administrative boundaries of governorates, districts, or regions, and paired with the occurrence record. buffers were created to encompass areas in cities and regions without corresponding administrative units. to ensure that buffers encompassed the entirety of the area of interest, google maps was used to determine the required radius. in areas with unspecified boundaries (e.g. table mountain national park and the border region between saudi arabia and uae) arcgis was used to generate custom polygons, which were assigned a unique code within a defined shapefile for ease of re-identification. this database is publicly available online 16, 17 . each of the 861 rows represents a unique occurrence of mers-cov. rows containing an index, unspecified, or imported case represent a single case of mers-cov. rows containing mammal and secondary cases may represent more than one case but are still unique geospatial occurrences. table 1 shows an overview of the content available in the publicly available dataset. in addition, online-only table 1 lists occurrences by geography, origin, 405 shape type, and publication and online-only table 2 provides citations of the data. index 34 99 1 0 93 7 234 unspecified 86 50 1 4 35 27 203 mammal 53 56 7 30 43 19 208 import 11 2 0 2 10 9 34 secondary 82 30 1 1 26 8 148 absent 3 8 0 0 7 3 21 environmental www.nature.com/scientificdata www.nature.com/scientificdata/ 15. pathogen: name the pathogen identified (e.g. mers-cov, bat coronaviruses, and other mers-cov-like pathogens). 16. pathogen_note: miscellaneous notes regarding pathogen. 17. patient_type: index, unspecified, na, secondary, import, or absent. • index: any human infection of mers-cov resulting after direct contact with an animal and no reported contact with a confirmed mers-cov case or healthcare setting. • unspecified: cases that lacked sufficient epidemiological evidence to classify them as any other status (e.g. serosurvey studies). • na: non-applicable field; case was not a patient (e.g. mammal) • secondary: defined as any cases resulting from contact with known human infections. cases reported after the index case can be assumed to be secondary cases unless accompanied by specific details of likely independent exposure to an animal reservoir. • import: cases that were brought into a non-endemic country after transmission occurred elsewhere. • absent: suspected case(s) ultimately confirmed negative for mers-cov. 18 . transmission_route: zoonotic, direct, unspecified, or animal-to-animal. • zoonotic: transmission occurred from an animal to a human. • direct: only relevant for human-to-human transmission. • unspecified: lacked sufficient epidemiological evidence to classify a human case as zoonotic or direct. • animal-to-animal: transmission occurred from an animal to another animal. 19 . clinical: describes whether the mers-cov occurrence demonstrated clinical signs of infection. denoted by yes, no, or unknown. • yes: clinical signs of infection were present/reported. clinical signs among humans may range from mild (e.g. fever, cough) to severe (e.g. pneumonia, kidney failure). clinical signs among camels include nasal discharge. • no: clinical signs of infection were not present/reported. • unknown: subject(s) may or may not have been demonstrating clinical signs of infection. for example, some authors did not explicitly mention symptoms, but individuals reportedly sought medical care. another example being when a diagnostic serosurvey was conducted during an ongoing outbreak. the term "unknown" was used when articles lacked sufficient evidence for extractors to definitively label as "yes" or "no". 20. diagnostic: describes the class of diagnostic method that was used. pcr, serology, or reported. 21 . diagnostic_note: more detailed information related to the specific test used (e.g. rk39, igg, or igm serology). 22. serosurvey: describes the context if serological testing was used. • diagnostic: testing of symptomatic patients. • exploratory: historic exposure determined among healthy asymptomatic individuals. 23. country: iso3 code for country in which the case occurred. 24 . origin: open-ended field to provide more details on the specific in-country location of mers-cov case. 25. problem_geography: this field was utilized if the mers-cov case was reported in a location that could cause uncertainty when determining exact geographic occurrence (e.g. hospital, abattoir). 26. lat: latitude measured in decimal degrees. 27. long: longitude measured in decimal degrees. 28. latlong_source: the source from which latitude and longitude were derived. 29. loc_confidence: states the level of confidence that researchers had when assigning a geographic location to the mers-cov case (good or bad). an answer of 'good' meant the article stated clearly that the case occurred in a specific geographic location and no assumptions were required on part of the researcher. an answer of 'bad' meant the article did not clearly state the specific geographic location of the mers-cov case, but the researcher was able to infer the location of occurrence. the field site_notes was utilized to detail the logic behind researchers' decisions when inference was required. 30. shape_type: the geographic shape type assigned to the mers-cov occurrence (point or polygon). 31. poly_type: if the mers-cov occurrence was assigned a shape_type of polygon, was it admin (gaul), custom, or buffer? 32. buffer_radius: if a mers-cov occurrence was assigned a buffer, what is the radius in km? 33. gaul_year_or_custom_shapefile: file path used to reach the necessary shape file in arcgis. users of this dataset can find custom shapefiles created for this dataset at: https://cloud.ihme.washington.edu/index. php/s/dgoykyqnbjg54f2/download 34. poly_id: a standardized and unique identifier assigned to each gaul shapefile. 35 . poly_field: which type of polygon was used to geo-position the occurrence? (e.g. if admin1 polygon was used, enter adm1_code) 36. site_notes: miscellaneous notes regarding the site of occurrence. 37. month_start: month that the occurrence(s) began. if the article provided a specific month of illness onset, the month was assigned a number from 1-12 (1 = january, 2 = february, etc.). if the article did not provide a specific month of illness onset, then researchers assigned a value of 'na' . month that the occurrence(s) ended, defined as the date a patient tested negative for mers-cov. if the article provided a specific month for recovery, the month was assigned a number from 1-12 (1 = january, 2 = february, etc.). if the article did not provide a specific month of symptom onset, then researchers assigned a value of 'na' . 39. year_start: year that the occurrence(s) began. if the year of illness onset was not provided in the article, the ihme standard was used: (year_start = publication year -3). year that the occurrence(s) ended. if the article did not provide a specific year for recovery, the ihme standard was used: (year_end = publication year -1). 41. year_accuracy: if years were reported, this field was assigned a value of '0' . if assumptions were required, this field was assigned a value of '1' . all data extracted from the original search (october 2012 to april 30, 2017) was reviewed independently by a second individual to check for accuracy. challenging extractions from the updated search (may 1, 2017 to february 22, 2018) were selected for group review during bi-weekly team meetings. upon extraction completion, all data were checked to ensure they fell on land and within the correct country. while the protocol implemented above was designed to reduce the amount of subjective decisions made by extractors, total elimination was not possible. wherever a subjective decision had to be made, the extractor utilized the various notes fields in order to document the logic behind decisions. these decisions were subsequently reviewed by other extractors. the techniques described here can be applied to collect and curate datasets for other infectious diseases, as has been previously demonstrated with dengue 20 and leishmaniasis 18 . additionally, since these data were collected independently through published reports of mers-cov occurrence, they may be used to build upon existing notification data 26, 27 . our ability to capture occurrences in this dataset is contingent on the data contained within published literature. therefore, this dataset does not represent a total count of all cases. instead, this dataset's value lies within its geo-precision. data were extracted with a focus on obtaining the highest resolution possible. these data may be merged with other datasets, such as who 26 or oie 27 surveillance records, and are intended to complement, not replace, these resources. together, published reports and notification data can provide a more comprehensive snapshot of current disease extent and at-risk locations. an important consideration, whether using the literature data alone, or in combination with other databases, is the potential for duplication. various pieces of metadata can be used to evaluate where potential duplicates could lie, such as common date fields (month_start, month_end, year_start, year_end) or consistent geographic details (lat, long, poly_id, shape_type) or shared epidemiological tags (patient_type). researchers may wish to consider further steps, such as fuzzy matching of geographic data (e.g. matching a point with an overlapping buffer) or temporal data (e.g. matching a precise month with an overlapping month interval). we acknowledge this duplicate-removal process will not catch all matching records, but it will likely catch several. we recommend occurrences are layered from top to bottom in the following order: index (green), unspecified (orange), mammal (yellow), import (blue), secondary (purple). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this approach because it will allow researchers to remove several duplicates without erroneously deleting any two occurrences that are truly unique (i.e. not duplicates). essentially, we recommend a sensitive approach above a more specific approach, as the latter simply risks culling too many records that aren't actually duplicates. when merging with other databases, consistency in metadata tagging is essential. for the who disease outbreak news data feed 26, 27 for instance, nomenclature for case definitions is slightly different, with who definitions of "community acquired" and "not reported" comparable to "index" and "unspecified" respectively. in addition, it is important to recognize what information is beyond the scope of these additional databases. again, when comparing to the who dataset, it is important to recognize that serologically positive cases do not meet the case definition used in the who database. these adjustments need to be identified on a dataset-to-dataset basis. among cases tagged as index or unspecified. occurrences tagged as index are coloured green, those tagged as unspecified are coloured orange. points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this database can be combined with other covariates (e.g. satellite imagery) to produce environmental suitability models of mers-cov infection risk and potential spillover on both global and regional scales as achieved with other exemplar datasets [28] [29] [30] [31] . this information can be useful in resource allocation aimed at improving disease surveillance and contribute towards a better understanding of the factors facilitating continued emergence of index cases. the addition of sampling techniques and prevalence data may improve this dataset. researchers were ultimately unable to add these data due to inconsistencies in the way literature reported sampling techniques and prevalence date by geography. an attempt to extract these data using the current approach would have led to sporadic inclusion of this information and would not have been comprehensive for the entire dataset. moving forward, we recommend authors report sampling technique and prevalence data at the highest resolution geography possible, as seen in miguel et al. 32 . we encourage continued presentation of paired epidemiological and geographic metadata that would allow for more detailed analysis in the future. this database may also be utilized in clinical settings to provide an evidence-base for diagnoses when used in conjunction with patient travel histories. additionally, it can be used to identify geographies for surveillance, particularly areas where mers-cov has been documented in animals but not humans (e.g. ethiopia and nigeria). identifying locations for surveillance will, in turn, inform global health security. while models will increase the resolution at which these questions can be addressed, datasets such as this provide an initial baseline. a major limitation of this database is the potential for sampling bias, which stems from higher frequency of disease reporting within countries where there exists strong healthcare infrastructure and reporting systems. this database does not attempt to account for such biases, which must be addressed in subsequent modelling activities where such biases are of consequence. similarly, another limitation is potential duplicate documentation of singular occurrences. this can happen when the same occurrence is assigned different geographies (e.g. point, polygon) in multiple publications. even though extractors made efforts to prospectively manually identify duplicate occurrences, this was challenging because the process relied upon papers providing sufficient details for extractors to determine a duplicate occurrence (e.g. geography, patient demographics, dates of occurrence, diagnostic methods, etc.). however, the majority of papers did not report such details for each occurrence. in those instances, it was impossible for extractors to discern whether occurrences may have been duplicates from a previous artic le. even case studies inconsistently reported patient details and demographic information. these are some examples of challenges faced by extractors when we attempted to identify duplicates. without sufficient contextual clues, extractors lacked evidence to determine duplicity and thus likely extracted some unique occurrences more than once. despite efforts to remove duplicate occurrences from the database, it is possible that some remain. geographic uncertainty is similarly problematic for analyses such as this. in some cases, polygons, as opposed to points, are utilised as a geographic frame of reference, reflecting the uncertainty in geotagging in the articles themselves. for some occurrences, there is a strong assumption that the geography listed corresponds to the site of infection. while the use of 5 km × 5 km as the minimum geographical unit allows for some leeway in this precision, it is possible that even with the point data (often corresponding to household clusters) these may not map directly with true infection sites. this must be considered in any subsequent geospatial analysis. finally, this database represents a time-bounded survey of the literature. while all efforts were made to be comprehensive within this period, articles, and therefore data, will continue to be published. efforts to streamline ongoing collection processes are still to be fully realized 33 . regardless, we hope that this dataset provides a solid baseline for further iteration. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission enhanced mers coronavirus surveillance of travelers from the middle east to england control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea molecular evolution of mers coronavirus: dromedaries as a recent intermediate host or long-time animal reservoir? middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from 2015 to 2017 assessing the risk of observing multiple generations of middle east respiratory syndrome (mers) cases given an imported case risk of mers importation and onward transmission: a systematic review and analysis of cases reported to who a systematic review of mers-cov seroprevalence and rna prevalence in dromedary camels: implications for animal vaccination a rapid scoping review of middle east respiratory syndrome coronavirus in animal hosts what have we learned about middle east respiratory syndrome coronavirus emergence in humans? a systematic literature review. vector borne zoonotic dis middle east respiratory syndrome coronavirus (mers-cov) infection: epidemiology, pathogenesis and clinical characteristics geopositioned middle east respiratory syndrome coronavirus occurrences. database 1983-2017. institute for health metrics and evaluation (ihme) database of geopostioned middle east respiratory syndrome coronavirus occurrences global database of leishmaniasis occurrence locations the contemporary distribution of trypanosoma cruzi infection in humans, alternative hosts and vectors a global compendium of human dengue virus occurrence a global compendium of human crimean-congo haemorrhagic fever virus occurrence a comprehensive database of the geographic spread of past human ebola outbreaks food and agricultural organization of the united nations. the global administrative unit layers (gaul): technical aspects global distribution maps of the leishmaniases updates to the zoonotic niche map of ebola virus disease in africa the global distribution of crimean-congo hemorrhagic fever the global distribution and burden of dengue risk factors for mers coronavirus infection in dromedary camels in a supervised learning process to validate online disease reports for use in predictive models critical contribution of laboratories to outbreak response support for middle east respiratory syndrome coronavirus international health regulations (2005) facilitate communication for in-flight contacts of a middle east respiratory syndrome case first confirmed case of middle east respiratory syndrome coronavirus infection in the kingdom of bahrain: in a saudi gentleman after cardiac bypass surgery. case rep event based surveillance of middle east respiratory syndrome coronavirus (mers-cov) in bangladesh among pilgrims and travelers from the middle east: an update for the period middle east respiratory syndrome coronavirus antibodies in dromedary camels middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study definitive diagnosis in suspected middle east respiratory syndrome coronavirus cases characteristics of traveler with middle east respiratory syndrome complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china imported case of mers-cov infection identified in china mers-related betacoronavirus in vespertilio superans bats prevalence and genetic diversity analysis of human coronaviruses among cross-border children no mers-cov but positive influenza viruses in returning hajj pilgrims, china two deletion variants of middle east respiratory syndrome coronavirus found in a patient with characteristic symptoms human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset complete genome sequence of middle east respiratory syndrome coronavirus isolated from a dromedary camel in egypt cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt mers coronavirus neutralizing antibodies in camels mers coronaviruses in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt middle east respiratory syndrome coronavirus: epidemiology and disease control measures geographic distribution of mers coronavirus among dromedary camels first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection a case of imported middle east respiratory syndrome coronavirus infection and public health response laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece cluster of middle east respiratory syndrome coronavirus infections in iran epidemiological and clinical characteristics of patients with middle east respiratory syndrome coronavirus in iran in 2014 health care associated middle east respiratory syndrome (mers): a case from iran influenza virus but not mers coronavirus circulation in iran phylogenetic analysis of merscov in human and camels in middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels the prevalence of middle east respiratory syndrome coronavirus (mers-cov) antibodies in dromedary camels in israel detection of coronaviruses in bats of various species in italy investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov don't forget the migrants": exploring preparedness and response strategies to combat the potential spread of mers-cov virus through migrant workers in sri lanka high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis arabia: an index case investigation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan stillbirth during infection with middle east respiratory syndrome coronavirus antibodies against mers coronavirus in dromedary camels mers-cov antibodies in humans, africa no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response dromedary camels in northern mali have high seropositivity to mers-cov middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels the middle east respiratory syndrome coronavirus (mers-cov) zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae imported case of middle east respiratory syndrome coronavirus (mers-cov) infection from oman to thailand serologic evidence for mers-cov infection in dromedary camels contact tracing the first middle east respiratory syndrome case in the philippines effectiveness of the middle east respiratory syndrome-coronavirus protocol in enhancing the function of an emergency department in qatar high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases isolation of mers coronavirus from a dromedary camel mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels occupational exposure to dromedaries and risk for mers-cov infection risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during 2013-2014: a case-control study mers-cov outbreak in jeddah-a link to health care facilities a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker a comparative study of clinical presentation and risk factors for adverse outcome in patients hospitalised with acute respiratory disease due to mers coronavirus or other causes a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients acute myocarditis associated with novel middle east respiratory syndrome coronavirus an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region, saudi arabia antibody response and disease severity in healthcare worker mers survivors brief report: family cluster of middle east respiratory syndrome coronavirus infections characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a singlecenter experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia descriptive epidemiology and characteristics of confirmed cases of middle east respiratory syndrome coronavirus infection in the makkah region of saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study evidence for camel-to-human transmission of mers coronavirus exposures among mers case-patients first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities hospital outbreak of middle east respiratory syndrome coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome lack of middle east respiratory syndrome coronavirus transmission from infected camels longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia epidemiology of a novel recombinant middle east respiratory syndrome coronavirus in humans in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia mers cov infection in two renal transplant recipients: case report mers-cov in upper respiratory tract and lungs of dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus transmission in extended family middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia outbreak of middle east respiratory syndrome at tertiary care hospital patient characteristics infected with middle east respiratory syndrome coronavirus infection in a tertiary hospital predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients report of middle east respiratory syndrome coronavirus (mers-cov) infection in four patients with hematological malignancies treated at king fahad medical city risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel spontaneous intracranial hemorrhage in a patient with middle east respiratory syndrome corona virus successful recovery of mers cov pneumonia in a patient with acquired immunodeficiency syndrome: a case report surveillance and testing for middle east respiratory syndrome coronavirus, saudi arabia the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study the first case of the 2015 korean middle east respiratory syndrome outbreak travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study the calm before the storm: clinical observations of middle east respiratory syndrome (mers) patients the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection in livestock and temporal relation to locations and seasons assessing the detection of middle east respiratory syndrome coronavirus igg in suspected and proven cases of middle east respiratory syndrome coronavirus infection cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia a cohort-study of patients suspected for mers-cov in a referral hospital in saudi arabia conveyance contact investigation for imported middle east respiratory syndrome cases recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses outbreak of middle east respiratory syndrome-coronavirus causes high fatality after cardiac operations prevalence of antibodies against the middle east respiratory syndrome coronavirus, influenza a and b viruses among blood donors, saudi arabia serological evidence of coronavirus infections in native hamadryas baboons (papio hamadryas hamadryas) of the kingdom of saudi arabia hematologic, hepatic, and renal function changes in hospitalized patients with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus and pulmonary tuberculosis coinfection: implications for infection control outcome of strict implementation of infection prevention control measures during an outbreak of middle east respiratory syndrome outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome coronavirus disease in children close relative of human middle east respiratory syndrome coronavirus in bat rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat mers outbreak in korea: hospital-to-hospital transmission a case report of a middle east respiratory syndrome survivor with kidney biopsy results an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea atypical presentations of mers-cov infection in immunocompromised hosts clinical implications of 5 cases of middle east respiratory syndrome coronavirus infection in a south korean outbreak emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital epidemiological investigation of the 119th confirmed middle east respiratory syndrome coronavirus case with an indefinite mode of transmission during the pyeongtaek outbreak in korea extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus mers epidemiological investigation to detect potential mode of transmission in the 178th mers confirmed case in pyeongtaek mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome in 3 persons, south korea middle east respiratory syndrome-coronavirus infection: a case report of serial computed tomographic findings in a young male patient outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon successful treatment of suspected organizing pneumonia in a patient with middle east respiratory syndrome coronavirus infection: a case report surveillance operation for the 141st confirmed case of middle east respiratory syndrome coronavirus in response to the patient's prior travel to jeju island transmission among healthcare worker contacts with a middle east respiratory syndrome patient in a single korean centre host susceptibility to mers-cov infection, a retrospective cohort study of the 2015 korean mers outbreak a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of korea a middle east respiratory syndrome screening clinic for health care personnel during the 2015 middle east respiratory syndrome outbreak in south korea: a single-center experience evaluation and clinical validation of two field-deployable reverse transcription-insulated isothermal pcr assays for the detection of the middle east respiratory syndrome-coronavirus serologic responses of 42 mers-coronavirus-infected patients according to the disease severity the clinical and virological features of the first imported case causing mers-cov outbreak in south korea mers-cov antibody responses 1 year after symptom onset neurological complications during treatment of middle east respiratory syndrome impact of middle east respiratory syndrome outbreak on the use of emergency medical resources in febrile patients hospital outbreaks of middle east respiratory syndrome zero transmission of middle east respiratory syndrome: lessons learned from thailand family cluster of middle east respiratory syndrome coronavirus infections further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus a phylogenetically distinct middle east respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in dubai with rising seroprevalence with age acute middle east respiratory syndrome coronavirus infection in livestock dromedaries antibodies against mers coronavirus in dromedary camels clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate middle east respiratory syndrome coronavirus antibody reactors among camels in dubai middle east respiratory syndrome coronavirus during pregnancy polyphyletic origin of mers coronaviruses and isolation of a novel clade a strain from dromedary camels in the united arab emirates prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate time course of mers-cov infection and immunity in dromedary camels transmission of middle east respiratory syndrome coronavirus infections in healthcare settings response to emergence of middle east respiratory syndrome coronavirus diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates mers-cov in pregnancy some epidemiological studies on mers coronavirus in dromedaries in the united arab emirates -a short communication emerging and reemerging diseases in the world health organization (who) eastern mediterranean region-progress, challenges, and who initiatives melinda gates foundation opp#1181128 and s.i.h. was supported by opp1132415 curated and catalogued the database. s.s. provided managerial support. all authors participated in interpreting and summarizing the results. r.e.r. wrote the first draft of the manuscript. all other authors critically reviewed the manuscript had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis s.i.h. and d.m.p. are members of the editorial board of scientific data. supplementary information is available for this paper at https://doi.org/10.1038/s41597-019-0330-0.correspondence and requests for materials should be addressed to d.m.p.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.the creative commons public domain dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the metadata files associated with this article. key: cord-255871-dau9tz6u authors: lee, mi-kyung; kim, sinyoung; kim, mi-na; kweon, oh joo; lim, yong kwan; ki, chang-seok; kim, jae-seok; seong, moon-woo; sung, heungsup; yong, dongeun; lee, hyukmin; choi, jong-rak; kim, jeong-ho title: survey of clinical laboratory practices for 2015 middle east respiratory syndrome coronavirus outbreak in the republic of korea date: 2015-12-18 journal: ann lab med doi: 10.3343/alm.2016.36.2.154 sha: doc_id: 255871 cord_uid: dau9tz6u background: it is crucial to understand the current status of clinical laboratory practices for the largest outbreak of middle east respiratory syndrome coronavirus (mers-cov) infections in the republic of korea to be well prepared for future emerging infectious diseases. methods: we conducted a survey of 49 clinical laboratories in medical institutions and referral medical laboratories. a short questionnaire to survey clinical laboratory practices relating to mers-cov diagnostic testing was sent by email to the directors and clinical pathologists in charge of the clinical laboratories performing mers-cov testing. the survey focused on testing volume, reporting of results, resources, and laboratory safety. results: a total of 40 clinical laboratories responded to the survey. a total of 27,009 mers-cov real-time reverse transcription pcr (rrt-pcr) tests were performed. most of the specimens were sputum (73.5%). the median turnaround time (tat) was 5.29 hr (first and third quartile, 4.11 and 7.48 hr) in 26 medical institutions. the median tat of more than a half of the laboratories (57.7%) was less than 6 hr. many laboratories were able to perform tests throughout the whole week. laboratory biosafety preparedness included class ii biosafety cabinets (100%); separated pre-pcr, pcr, and post-pcr rooms (88.6%); negative pressure pretreatment rooms (48.6%); and negative pressure sputum collection rooms (20.0%). conclusions: clinical laboratories were able to quickly expand their diagnostic capacity in response to the 2015 mers-cov outbreak. our results show that clinical laboratories play an important role in the maintenance and enhancement of laboratory response in preparation for future emerging infections. on may 20, 2015, middle east respiratory syndrome coronavirus (mers-cov) was confirmed for the first time in an infected patient in the republic of korea. although the primary case traveled to the united arab emirates and saudi arabia, the patient did not initially report his recent travel in those countries [1] . this case led to transmission of mers-cov both within a hospital and between hospitals and eventually resulted in the largest outbreak of mers-cov infections outside the arabian peninsula. at that time, we had limited information on mers-cov, and only a few clinical laboratories were prepared to perform molecular diagnostic testing for the virus. during the longer than two months of the outbreak, 186 confirmed cases were diagnosed by real-time reverse transcription pcr (rrt-pcr) of mers-cov, and specimens from tens of thousands of suspected cases, including individuals who contacted the confirmed cases, were submitted for this testing. many clinical laboratories were instructed to set up facilities to perform mers-cov rrt-pcr on site in a short time to fight against the transmission of this virus in their own institutes. an earlier outbreak in 2009 of a novel strain of h1n1 influenza virus a (h1n1 influenza) affected laboratories worldwide, with a potentially tremendous impact on the practices of clinical laboratories [2] . the outbreak of h1n1 influenza also had a great influence in the republic of korea. the field of molecular testing for pathogens has been expanded in clinical laboratories, and the molecular testing industry has responded quickly with the production of new molecular test kits. numerous studies, including viral etiology, epidemiology, risk factors, clinical and laboratory characteristics, and diagnostic tests, have been reported [3] . despite the fact that clinical laboratory practice was a critical element in the response to the h1n1 influenza outbreak, there are only a few reports regarding this aspect of testing [2, 4, 5] . the ability of clinical laboratories to respond appropriately to an outbreak is significant in pathogen control. therefore, it is crucial to understand the current status of clinical laboratories in the republic of korea in order to be well prepared for any future emerging infectious diseases. in this article, we present the results of a survey of clinical laboratory practices during the 2015 mers-cov outbreak. the study population consisted of clinical laboratories performing diagnostic testing for mers-cov in medical institutions (hospitals and medical centers) and referral medical laboratories among clinical laboratories accredited by the korean laboratory accreditation program [6] . this survey was an initiative of the mers-cov laboratory response task force of the korean society for laboratory medi-cine. we conducted a survey of 49 clinical laboratories. a short questionnaire to assess clinical laboratory practices related to mers-cov diagnostic testing was sent by email to the directors and the clinical pathologists (laboratory physicians) in charge of the clinical laboratories performing mers-cov tests. the survey focused on the number of tests and the number of positive test results for mers-cov, turnaround time (tat), request process, collection and transportation of specimens, testing and reporting, communication of results, laboratory safety, and reagents and equipment. a total of 40 clinical laboratories (81.6%, 40/49), including 35 medical institutions and 5 referral medical laboratories, responded to the survey. the number of beds in the medical institutions was as follows: < 500 beds, 3 institutions (8.6%); 500-1,000 beds, 20 (57.1%); and > 1,000 beds, 12 (34.3%). all clinical laboratories used rrt-pcr as the detection method for mers-cov. the number of mers-cov rrt-pcr tests performed was collected from 32 medical institutions and five referral medical laboratories. data up to july 15, 2015 were collected according to the day and specimen type. a total of 27,009 mers-cov rrt-pcr tests were performed at 32 medical institutions (n = 11,502) and five referral medical laboratories (n = 15,507) (table 1 and fig. 1 ). the proportion of medical institutions was significantly underestimated because one tertiary care hospital submitted responses for the survey but not the specimen list, and the numbers of mers-cov rrt-pcr tests and positive specimens at this institution would have been predominant in the reporting medical institutions. mers-cov rrt-pcr testing at all medical institutions and referral medical laboratories increased dramatically in early june (fig. 1 ). the number of clinical laboratories that initiated mers-cov testing increased in the first two to three weeks (fig. 2) . daily test volumes peaked on june 24 (1,088 tests) and began to decrease thereafter (fig. 1) . among the 27,009 mers-cov rrt-pcr specimens, 246 (0.9%) and 91 (0.3%) specimens were positive and indeterminate, respectively (153 and 71 specimens at medical institutions, 93 and 20 specimens at referral medical laboratories; table 1 ). most of the specimens for mers-cov rrt-pcr were sputum (73.5%). a total of 74.7% of all specimens tested and 82.9% of positive specimens were specimens from the lower respiratory tract, such as sputum, bronchoscopy specimens, or tracheal aspirates ( table 1 ). all nasopharyngeal aspirates (n = 293) were negative for mers-cov rrt-pcr. the tat is defined as the time from the receipt of specimens in the laboratory to the reporting of the results. unfortunately, 35% of the laboratories (nine medical institutions and five referral medical laboratories) were not able to provide or analyze data on tat. the median tat was 5.29 hr (first and third quartile, 4.11 and 7.48 hr) for 26 medical institutions, and the minimum and maximum median tat (first and third quartile) were 4.00 hr (3.35 and 5.10 hr) and 26.46 hr (19.59 and 31.36 hr), respec30 01 03 05 07 09 11 13 15 17 19 21 23 25 27 29 01 03 05 07 09 11 13 tively. the median tat of more than a half of the laboratories (57.7%) was less than 6 hr. the results were reported within 24 hr in all medical institutions except one ( table 2 ). in the referral medical laboratories, the results were reported in less than 6-9 hr (during weekday daytime) or 9-15 hr (during weekday nights and weekends). table 2 shows the current status of clinical laboratories in medical institutions with respect to their response to the outbreak of mers-cov infections. many laboratories were able to perform testing throughout the week (71.4%, 25/35) and ran tests once or twice per day. specimen was collected mainly by clinicians, and all specimens were directly transported person-to-person. in most cases, clinicians filled out the specimen request information form, and doctors of laboratory medicine completed the test report form. most of the mers-cov rrt-pcr tests were performed only by clinical laboratory technicians of molecular genetics divisions (74.3% of 35 laboratories). the test results were primarily reported by clinical pathologists via electronic processing. laboratory biosafety preparedness in response to the mers-cov outbreak included class ii biosafety cabinets (100%, 35/35); separated pre-pcr, pcr, and post-pcr rooms (88.6%, 31/35); negative pressure pretreatment rooms (48.6%, 17/35); and negative pressure sputum collection rooms (20.0%, 7/35). as shown in table 3 , the current status of clinical laboratories in referral medical laboratories was similar to that of medical institutions in many aspects. specimens were transported twice or three times a day. many laboratories (75%, 30/40) used phosphate-buffered saline for pretreatment of sputum specimens. all reagents for the detection of mers-cov were components of ready-made kits, and the powerchek mers real-time pcr kit (kogenebiotech, seoul, korea) was the most commonly used kit (72.5%, 29/40). most laboratories used cfx96 (bio-rad laboratories, hercules, ca, usa) or abi7500 or 7800 systems (life technologies, carlsbad, ca, usa) for rrt-pcr (85.0%, 34/40) ( table 4 ). the emergence of novel viral pathogens and the evolution of pandemics have presented a new and complex challenge to public health care systems at every level [1, 3, 7, 8] . the outbreak of h1n1 influenza eventually turned out to be relatively mild, despite the fear it engendered as the potential early stage of a pandemic. breban et al. [9] suggested that mers-cov does not yet have pandemic potential. nevertheless a mers-cov outbreak recently occurred in the republic of korea, and its characteristics were very different from those of the outbreak of h1n1 influenza. the mers-cov outbreak was more serious 26 28 30 01 03 05 07 09 11 13 15 17 19 21 23 25 27 29 01 03 05 07 09 11 13 than expected, and numerous problems arose concerning infection control and prevention in hospitals and among the general public. because clinical laboratories are usually on the front lines for the detection of emerging pathogens, the ability of these laboratories to respond to an outbreak is critical for infection control and prevention. the elements of clinical laboratory preparedness and responsiveness include availability of personal protective equipment and its appropriate use, availability and use of appropriate testing supplies, adequacy of staffing, and infrastructure requirements including laboratory space [2] . in 2012 and 2013, the european centre for disease prevention and control (ecdc) and the who regional office for europe conducted a joint survey [10] . although the number of countries that had laboratory capabilities to detect and confirm mers-cov increased in 2013 (55.8%, 29 of 52 countries) compared with 2012 (47.8%, 22 of 46 countries), a half of the countries were still unable to test mers-cov [10] . the present study reveals a snapshot of the current status and capability of clinical laboratories to respond to the mers-cov outbreak in the republic of korea. most clinical laboratories participating in the survey were considered to have sufficient capacity to respond to the outbreak. immediately after the mers-cov rrt-pcr test was approved, many laboratories were able to perform testing seven days a week and the number of tests reported increased dramatically. the median tat of more than a half of the laboratories (57.7%) was shorter than 6 hr, and the minimum median tat was 4.00 hr. the results were reported within 24 hr in all medical institutions except one. one limitation of our study is that even though we obtained responses from the majority of medical institutions and referral medical laboratories, the results may not be fully representative for a number of reasons. data were collected before the end of the mers-cov outbreak and did not include some major medical institutions and public health laboratories such as the korea centers for disease control and prevention. in addition, our results report the number of mers-cov rrt-pcr tests, and not the number of mers-cov cases. previously, sousa et al. [11] suggested that the use of upper respiratory specimens (e.g., nasopharyngeal swabs) for mers-cov diagnosis might not be as sensitive as the use of lower respiratory tract specimens. the laboratory diagnostic guidelines for mers-cov testing of the korean society for laboratory medicine recommended using specimens from the lower respiratory tract. in the present survey, the number of positive results from mers-cov rrt-pcr was significantly higher in specimens obtained from the lower respiratory tract (1.01%, 204/20,183) compared with specimens from the upper respiratory tract (0.6%, 41/6,785) (p = 0.002, chi-square test). therefore, the specimen type is expected to have a significant impact on diagnostic sensitivity and should be considered when testing emerging pathogens. for laboratory biosafety in response to the mers-cov outbreak, class ii biosafety cabinets were adequately supplied but high-end engineering facilities such as negative pressure sputum collection and pretreatment rooms were not readily available. however, no laboratory-acquired infections were reported during the outbreak, thus standard precautions with droplet precautions appeared to be sufficient for the prevention of laboratory-acquired infection of mers-cov. nonetheless, improvements in engineering laboratory biosafety are needed for preparedness to test agents with a higher biosafety level. clinical laboratories have the primary responsibility for testing specimens to support clinical decision-making. although public health laboratories also test specimens to aid clinical decisions, their roles in surveillance, strain identification, and tracking of drug resistance are arguably their main priorities [12] . moreover, clinical laboratories often have resources available that allow for rapid expansion [12] . in conclusion, the results of this survey contribute to the comprehensive view of clinical laboratory response in the republic of korea to the recent outbreak of 2015 mers-cov. on the basis of currently available data, clinical laboratories in korea were able to expand their diagnostic capacity in a short time and achieve a tat of shorter than nine hours with testing seven days per week to response to the recent mers-cov outbreak, although the delay in the early period of the outbreak should be improved. therefore, clinical laboratories should be ready for the maintenance and enhancement of laboratory responses in preparation for future emerging infections. ah ra cho preliminary epidemiological assessment of mers-cov outbreak in south korea ginocchio cc. a survey-based assessment of united states clinical laboratory response to the 2009 h1n1 influenza outbreak h1n1 influenza laboratory surge response to pandemic (h1n1) 2009 outbreak initial response of health care institutions to emergence of h1n1 influenza: experiences, obstacles, and perceived future needs clinical pathology laboratory inspection and accreditation in korea i: development of the system and its trial mers-cov outbreak in jeddah--a link to health care facilities preparedness of institutions around the world for managing patients with ebola virus disease: an infection control readiness checklist interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk laboratory capability and surveillance testing for middle east respiratory syndrome coronavirus infection in the who european region mers coronavirus: data gaps for laboratory preparedness laboratory surge capacity and pandemic influenza no potential conflicts of interest relevant to this article were reported. key: cord-275138-033r259v authors: hayden, frederick g; farrar, jeremy; peiris, j s malik title: towards improving clinical management of middle east respiratory syndrome coronavirus infection date: 2014-07-31 journal: the lancet infectious diseases doi: 10.1016/s1473-3099(14)70793-5 sha: doc_id: 275138 cord_uid: 033r259v nan a decade on from the 2002-03 outbreak of severe acute respiratory syndrome coronavirus (sars-cov) infections, the world is again confronted by the possible international spread of a novel coronavirus, the middle east respiratory syndrome coronavirus (mers-cov), which apparently originated in the arabian peninsula. 1 mers-cov is associated with severe respiratory tract infection, often renal failure, and mortality exceeding 40% in patients admitted to hospital. 2 similar to sars-cov, it is closely related to bat coronaviruses, but mers-cov has diff erent cellular receptor specifi city and a broader species range-camels seem to have a role as a natural host. saudi arabia has been most severely aff ected so far, but imported cases-either recent travellers or those transported for clinical care-have been seen in countries in europe, africa, asia, and north america. major nosocomial outbreaks have happened in the middle east, 3 and non-sustained human-to-human transmission events elsewhere, 4 and many more clinical cases are likely to have occurred. 5 there has been a recent surge in case reporting that could be the result of more human-to-human transmissions due to a change in exposure patterns, expansion of the virus in animal reservoir(s), seasonal variation, ongoing nosocomial clusters, or increased surveillance with reporting of mild or asymptomatic mers-cov detections, or both. 5 severe mers-cov disease has occurred primarily in older adults, particularly men, with comorbidities. 2 however, data on disease pathogenesis, particularly viral replication patterns and clinical manifestations, are scarce at present. despite more than 500 laboratory-confi rmed mers-cov cases so far, only a handful of patients have had systematic virological and biomarker sampling. 4, 6 consequently, there are many unanswered questions regarding sites of infection, pathogen dynamics, innate and adaptive immune responses, and host genetic factors. prolonged viral replication in the lower respiratory tract, extra pulmonary virus detection, severe lung injury with respiratory failure, and often renal failure are notable features, suggesting that an eff ective antiviral regimen, perhaps in combination with immunomodulatory agents, would provide clinical benefi t. although supportive care is central to clinical management of coronavirus infections, appropriate antiviral and immunomodulatory therapy for both sars 7 and mers-cov infections remain uncertain because of a scarcity of quality evidence. an absence of good animal models for mers-cov poses a major challenge. many agents have inhibitory activity in vitro for coronaviruses, including some licensed drugs, 7-9 but it is unclear whether their human pharmacology and tolerability would enable suffi cient doses to be given to exert antiviral eff ects in patients with mers-cov. one available drug that is inhibitory for coronaviruses in vitro at clinically achievable levels is the inosine-5´monophosphate dehydrogenase (impdh) inhibitor mycophenolic acid, 8 but animal data for this eff ect are scarce, and one patient developed infection while receiving mycophenolate mofetil. 4 ribavirin and interferon combinations are associated with modest centre for infections/public health england/science photo library antiviral eff ects in mers-cov inoculated rhesus macaques given high doses. 9 although the clinical relevance of these fi ndings is uncertain, the combined use of antiviral drugs to enhance inhibitory eff ects and reduce the potential for resistance emergence makes sense. at present, the strongest treatment evidence supports the use of convalescent plasma or other preparations that possess neutralising antibodies. 10, 11 convalescent plasma seemed to reduce duration of treatment in hospital and mortality when used early in patients with sars. 7 for mers-cov, low neutralising antibody responses and inability to acquire suffi cient convalescent plasma from survivors with comorbidities might restrict the eff ectiveness of this treatment, although these limitations might not apply to infected health-care workers. additionally, the availability of human neutralising monoclonal antibodies 12 or polyclonal immune globulin produced in transgenic cows or other hosts 13 could overcome these hurdles. the high seroprevalence of high-titre neutralising antibody to mers-cov or a closely related virus in dromedary camels in the region raises the possibility of using camel sera or engineered single domain camel antibodies for therapy. 13, 14 purifi ed immunoglobulins or immunoglobulin fragments (nanobodies) might off er a therapeutic option for severely ill patients until more defi ned, genetically engineered, antibodies become available. for any chosen intervention, we advocate that use must be accompanied by a prospective, protocolbased assessment of safety and eff ectiveness that includes sequential virological, clinical, and biomarker measurements. we wrote about the slow acquisition of such data in the 2009 h1n1 infl uenza pandemic. 15 one outcome from this circumstance was the formation of the international severe acute respiratory and emerging infection consortium (isaric), a global federation of academic clinical research networks. isaric has collaborated with who to develop biological sampling protocols that are applicable for patients with mers-cov. furthermore, working with colleagues in public health england, isaric experts have examined available data and ranked potential therapeutic options with regard to their priority for clinical study; 10 this information will be updated as new data become available. however, no mers-cov patients have yet been enrolled on therapeutic protocols incorporating systematic sampling through isaric or any other organisation. consequently, we are not learning what might benefi t or potentially harm such patients. whatever agent or agents are selected for testing, systematic harmonised data collection involving robust observational studies or, when possible, controlled trials, is needed to assess both disease pathogenesis and candidate therapeutics for mers-cov. clinicians and public health offi cials in aff ected middle eastern countries, particularly in saudi arabia, are uniquely positioned to undertake such studies. with support as needed from international partners like who and isaric, 11 regional governments, and funders, middle eastern colleagues have both the opportunity and the responsibility to undertake studies to advance the understanding of eff ective prevention and treatment strategies for mers-cov and any future novel cov outbreaks. thus far, mers-cov is yet another emerging infection threat for which the clinical research response has been too slow and uncoordinated. new investigative frameworks, possibly incorporating mandates into the international health regulations, are urgently needed. the virus has resurfaced in israel, and might be linked to use of intravenous inactivated polio vaccine (ipv). 5 transnational mobility also contributes to polio's persistence, and circulating vaccine-derived poliovirus (cvdpv) in yemen, mozambique, and madagascar is further complicating eradication eff orts. 6 we know that the spatial distribution of polio (where polio exists in some places, is contained in others, is detected but not virulent, has gained virulence through mutation, or is a threat) is very complex. the distribution is complex because the diff usion of poliovirus is associated with diff erent types of polio, bodies, ecologies, and geopolitical realities. polio can be biomedically engineered polioviruses (ipv), degraded versions of the virus (oral polio vaccine [opv]), mutating viruses, or the so-called wild polio virus and its various strains; bodies can have no poliovirus, wild polio resistance, symptomatic polio, ipv, opv, cvdpv, or be subclinical; ecologies vary across landscapes of built and natural environments; and geopolitical realities create diff erent regulatory structures, biomedical accessibilities, confl icts, migrations, and tensions. this complexity means that eradication of polio in some places for some people with certain forms of a vaccine might not be possible in the immediate future. we thus have to better imagine how diff erent types of viruses, bodies, built and natural ecologies, and geopolitical realities interact to produce the present landscape of infectious disease. 7 as health geographers, we argue that such complexity demands a diff erent spatial imaginary and concomitant vocabulary to understand polio. 8 a set of assumptions in standard epidemiological practice suggest that control can happen through geographical containment in particular places and bodies. 9 polio containment leads to the elimination of wild or mutated viruses in particular places-a process that provides the promise of biomedical science's capacity to eradicate and then extinguish these uncontrolled forms of life. although we are fully supportive of all eff orts to eliminate human suff ering, including vaccination, we also believe in the need to be more realistic about the capacities of the virus; 10 the assumptions embedded in vaccination eff orts do not appreciate the ontological position of viruses circulating through ecosystems. 11, 12 polioviruses are not bound to the humanly produced built and natural ecologies in which they exist nor the political or natural boundaries; the interest of polioviruses is survival, and this depends on their ability to fi nd a human host. polioviruses, therefore, do not rely on the ocularcentric spatial imagination of human beings. people need to see polioviruses to know how to eradicate and control them, including the viruses used in vaccines and laboratory studies. polioviruses know how to negotiate the negative spaces between human vision and the bodies and ecological land scapes that aff ord them their clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection severe acute respiratory syndrome and coronavirus broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques clinical decision making tool for treatment of mers-cov v.1.1 outbreak readiness workshop: clinical management and potential use of convalescent plasma potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein production of human polyclonal antibodies by transgenic animals formatted single-domain antibodies can protect mice against infection with infl uenza virus (h5n2) patient-oriented pandemic infl uenza research key: cord-266031-tlrsco40 authors: haghani, milad; bliemer, michiel c. j. title: covid-19 pandemic and the unprecedented mobilisation of scholarly efforts prompted by a health crisis: scientometric comparisons across sars, mers and 2019-ncov literature date: 2020-09-21 journal: scientometrics doi: 10.1007/s11192-020-03706-z sha: doc_id: 266031 cord_uid: tlrsco40 during the current century, each major coronavirus outbreak has triggered a quick and immediate surge of academic publications on its respective topic. the spike in research publications following the 2019 novel coronavirus (covid-19) outbreak, however, has been like no other. the global crisis caused by the covid-19 pandemic has mobilised scientific efforts at an unprecedented scale. in less than 5 months, more than 12,000 research items and in less than seven months, more than 30,000 items were indexed, while it is projected that the number could exceed 80,000 by the end of 2020, should the current trend continues. with the health crisis affecting all aspects of life, research on covid-19 seems to have become a focal point of interest across many academic disciplines. here, scientometric aspects of the covid-19 literature are analysed and contrasted with those of the two previous major coronavirus diseases, i.e., severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). the focus is on the co-occurrence of key-terms, bibliographic coupling and citation relations of journals and collaborations between countries. interesting recurring patterns across all three literatures were discovered. all three outbreaks have commonly generated three distinct cohorts of studies: (i) studies linked to public health response and epidemic control, (ii) studies on chemical constitution of the virus; and (iii) studies related to treatment, vaccine and clinical care. while studies affiliated with category (i) seem to have been relatively earliest to emerge, they have overall received relatively smaller number of citations compared to publications the two other categories. covid-19 studies seem to have been disseminated across a broader variety of journals and across a more diverse range of subject areas. clear links are observed between the geographical origins of each outbreak as well as the local geographical severity of each outbreak and the magnitude of research originated from regions. covid-19 studies also display the involvement of authors from a broader variety of countries compared to sars and mers. considering the speed at which the covid-19-related literature is accumulating, an interesting dimension that warrants further exploration could be to assess if the quality and rigour of these publications have been affected. on december 31, 2019 an official case of a novel respiratory disease of the category of coronaviruses, named covid-19, was reported in wuhan, china, marking the beginning of what proved to be one of the direst and most devastating viral outbreaks in the modern history (sohrabi et al. 2020; wang et al. 2020 ). this was immediately followed by an unprecedented and swift response of the academic community to address various dimensions of this health crisis and prompted an avalanche of scholarly publications on this topic (golinelli et al. 2020; haghani et al. 2020; kagan et al. 2020) . in less than five months, more than 12,000 publications on this topic have already been indexed by scopus with the number increasing figuratively every day in considerable increments 1 (torres-salinas et al. 2020) . and this figure does not even include many more publications available in various repositories, including cord-19 (colavizza et al. 2020) , in the form of preprints awaiting peer review by their respective journals. such explosion of research on a single topic and the off-the-charts surge in the rate of publications is arguably an unprecedented trend in the history of scholarly publications. an article published by science on may 13, 2020, referred to this phenomenon as one that is "among the biggest explosions of scientific literature ever" (brainard 2020) . it highlighted how, in the face of this phenomenon, it has become extremely challenging for scientists to stay abreast of the latest developments. this has made the importance of research synthesis more tangible than ever and has even resulted in the development of several computational research mining tools for this very topic utilising methods such as artificial intelligence (ai). among such efforts is a research synthesis tool powered by ai algorithms which has harvested datapoints from a large number the cord-19 articles and categorised them (brainard 2020) . though the impact of the covid-19 health crisis has marked it as a rather unique milestone in the history of disease outbreaks, the world, prior to this, was not a stranger with coronavirus disease outbreaks (mcintosh 1974; myint 1994; cavanagh 2005; lim et al. 2016; chen et al. 2020 ). prior to 2020, two major outbreaks of this family of viruses had already been reported with at least one of them carrying the official label of a "global pandemic" ). on november 16, 2002, the first case of the severe acute respiratory syndrome (sars) disease was reported in the guangdong province in southern china, which by 2003, swiftly spread from continent to continent, prompting the world health organisation to declare it as a pandemic. in fact, sars is known to be "the first pandemic of the 21st century" (cherry and krogstad 2004) . nearly ten years later, on june 13, 2012, the first case of the middle eastern respiratory syndrome (mers) disease was discovered in jeddah, saudi arabia. these two constituted the two most major coronavirus outbreaks, until covid-19 came along. similar to covid-19, though at a much smaller scale, each of these previous outbreaks generated a literature of their own (kostoff and morse 2011) . in the face of the flood of scholarly outputs on covid-19, and along with the conventional review and research synthesis studies (chang et al. 2020; chen et al. 2020; cortegiani et al. 2020) , scientometric (colavizza et al. 2020 ) and bibliometric methods (bonilla-aldana et al. 2020; hossain 2020) have also gained traction in documenting and analysing the rapid developments of this literature and discovering emerging patterns (chahrour et al. 2020; dehghanbanadaki et al. 2020; haghani et al. 2020; kumar 2020; le bras et al. 2020 ). here in this work, the literatures of these three major coronavirus diseases are disentangled from one another and analysed in a comparative way and from scientometric perspectives. the aim is to discover possible similarities and discrepancies across these three segments of the coronavirus literature, and to discover whether there are recurring patterns in terms of magnitude, temporal evolution and the structure of these three literatures that were each developed in response to a respiratory disease outbreak. the main focus of the analyses is on keyword co-occurrences, bibliographic coupling and citation relations of sources and collaborations between countries. to compare the scientometric aspects of the studies on sars, mers and covid-19, three separate datasets of publications on these three topics were retrieved from scopus through three separate search strategies. the decision on which general database to use (e.g. web of science (wos) or scopus) was mainly made on the basis of the number of indexed covid-19 studies in particular, as the sector of the coronavirus literature that is currently emerging (compared to the literatures on sars and mers that have already stabilised). at the time of the data retrieval (may 2020), wos had indexed slightly less than 5,000 research items on covid-19, while the number of items in scopus neared 12,000. given the fact that the scopus database was considerably more up to date in that respect, this database was set as the main source of data extraction in this work. therefore, for the sake of consistency, the data for sars and mers were also extracted from scopus. the search strategies were devised in a way to minimise the possible overlap between the datasets on sars, mers and covid-19 and to disentangle the three datasets from one another to the most possible extent. preliminary inspection of the literature on each three topics determined a set of distinct keywords that would return the target literature with reasonable specificity and sensitivity. in each search, key terms associated with the other literatures were combined with the boolean operator "and not" in order to avoid the overlap. the lower bound of the time span for each search was set with consideration of the year when each viral outbreak took place. the query strings associated with the three datasets are as below: sars: ( title-abs-key ( ( ( "severe acute respiratory syndrome" or "sars") and ( coronavirus*)) or ( "sars virus" or "sars disease" or "severe acute respiratory syndrome disease" or "severe acute respiratory syndrome virus" or "sars-cov")) and not title-abs-key ( ( "covid" or "ncov" or "covid-19" or "covid19" or "sars-cov-2" or "severe acute respiratory syndrome-2" or "mers" or "middle east respiratory syndrome"))) and pubyear > 2001. mers: ( title-abs-key ( ( ( "middle east respiratory syndrome" or "mers") and ( coronavirus)) or ( "mers-cov" or "mers virus" or "mers disease" or "middle east respiratory syndrome virus" or "middle east respiratory syndrome disease")) and not titleor "sars-cov-2" or "sars" or "severe acute respiratory syndrome"))) and pubyear > 2011. covid-19: title-abs-key ( "covid-19" or "covid19" or "coronavirus disease 2019" or " 2019-ncov" or "novel coronavirus" or "novel corona virus" or "sars-cov-2") and pubyear > 2018. the search was last time updated on 24 may 2020 where it returned 5,907 items on sars, 1,752 items on mers and 11,859 items on covid-19. figures 1, 2 , 3 respectively show the distribution of the studies on sars, mers and covid-19, across subject areas. figure 4a also shows the composition of the covid-19 literature in terms of the document types, demonstrating that only nearly 50% of the studies on this topic have so far been in the form of full-length articles, while letters, notes, reviews, and other document formats constitute a large portion (i.e. nearly half) of the literature on this topic at the time of this investigation. full records of the three datasets on sars, mers and covid-19 were retrieved in csv excel format from scopus, all on the same day. this included the citation information, bibliographic information, abstract and keywords, funding details and the references. the scopus restriction of maximum 2000 document per export posed challenges for the retrieval of the sars and covid-19 datasets whose size were bigger than 2000 documents. for the sars dataset, the challenge was circumvented by further limiting the search to specific years, in separate bundles, in a way that the size of each bundle was less than 2000 items, therefore allowing us to export the items of each bundle separately. the extraction of the covid-19 dataset, however, posed a further layer of complication. given that nearly all studies of covid-19 have been published in one year, i.e. 2020, the year of publication could no longer be used as a criterion to form a set of mutually exclusive exportable bundles. to decompose the search outcome to non-overlapping bundles of 2000 documents or less, the following strategy was devised. the document type was used to initially limit the search to mutually exclusive (non-overlapping) categories. first, the search was limited to "review or short survey or erratum or conference paper or data paper". this formed a set of 1267 documents which was extracted in one single export (see fig. 4a for details of the number of items within each document type category). subsequently, the search was set back to the original and was limited to notes (1067 items) and then to editorial (1270 items). each of these two subsets, being smaller than 2000 items, were exported separately. there were also 2564 documents of letter type. this set was further decomposed to two mutually exclusive subsets based on the publication stage criterion (1539 article in press, and 1025 final) and was retrieved in two separate exports. for the remaining 5691 article documents, the following strategy was adopted. of the 5691 items, 2944 were article in press and 2747 were final. first, the 2944 article in press items were considered. the list of those studies was sorted as first author (a-z) and the first 2000 items were extracted in one export. then the list was sorted as first author (z-a), i.e. the order was reversed, and the first 944 items were exported. a similar strategy was utilised to extract the remaining 2747 final documents. a supplementary search was also conducted on the general topic of coronaviruses using the string title-abs-key ( "coronavirus*") and pubyear > 1985 which returned 24,620 documents on the same day. only the data related to the number of documents by year was extracted for this search. figure 4b shows the temporal distribution of the studies on the general topic of coronaviruses. the graph clearly shows spikes of publication coinciding with the years when sars, mers and covid-19 outbreaks took place. the first spike is related to the sars outbreak in 2002 which is reflected in an immediate and substantial increase in the number of publications on coronaviruses from 2002 to 2003. the increase continued, though at a slower pace, to 2004 and was then followed by a gradual decline till 2012. the 2012 mers outbreak triggered another spike in the number of publications on coronaviruses, though not as large as that of sars. the intensification of attention to this topic, this time, lasted for about three years till 2015 before another decline began. the spike of coronavirus studies prompted by the covid-19 outbreak, however, seem to have been occurring at a completely different scale which can be deemed unprecedented in the history of coronavirus studies, and perhaps arguably in the history of science. the number of studies emerged in the first five months of 2020 nears an equivalent of the 70% of the total size of the coronavirus literature accumulated during more than 50 years . in fig. 4c , the temporal distribution of the sars, mers and covid-19 studies have been shown separately according to the three datasets explained earlier. note that, the quantities associated with sars and mers are represented by the left vertical axis whereas that of covid-19 is represented by the right vertical axis, while its scale is ten times bigger than the scale of the left axis. the history of previous coronavirus research has suggested that the number of studies will likely keep rising for at least a few years before it peaks. but given the unprecedented magnitude of research and the explosive rate of publications since the begging of 2020, it would be interesting to observe whether this pattern would repeat itself and whether the peak would occur at an earlier or later stage compared to those of the previous outbreaks, a question whose answer will only be determined by time. the co-occurrence of keywords associated with the sars, mers and covid-19 literatures were analysed using vosviewer (van eck and waltman 2010). each analysis was performed on the separate set of data associated with the literature of interest. the maps of keyword co-occurrences associated with sars, mers and covid-19 literatures are provided in figs. 5, 6, 7 respectively. the minimum number of occurrences for the keywords to be included in the map was set to 5 in all three cases. the number was chosen on the basis of the clarity of the resultant clusters. the unit of analysis has also been set fig. 7 with the colour-coding of the average number of citations. given that almost all studies of covid-19 are 2020 items, the colour-coding related to the average publication year was forgone with respect to this literature. maps of term occurrences based on the analysis of the title and abstract of studies on sars, mers and covid-19 have also been presented in the "appendix" in figs. 23, 24, 25 respectively. while the below analysis focuses mainly on the interpretation of the keyword maps, similar patterns are by-and-large observable through analysis of the title and abstract terms of these studies. with respect to each of the three literatures, three distinct clusters of keywords were identifiable. these clusters showed certain patterns of commonality across the three datasets. each map presents a distinct cluster of keywords that seem to be associable to the studies related to public health emergency management and the prevention of epidemic. this cluster has been assigned a yellow colour in all three maps in figs. 5, 6, 7 here, this is referred to as cluster (i). in this cluster, one can observe terms such as those associated with general public health including "wold health organisation", "public health", "public the map of keyword co-occurrences associated with the covid-19 literature health service", "global health", as well as those associated with disease outbreaks including "emergency", "health risk" "epidemics", "pandemic", "outbreak", "viral diseases", "virus infection", "communicable disease", "transmission", "travel". terms representing measures of emergency severity also appear in this cluster including "mortality", "fatality", "morbidity", "infection risk". this cluster also includes terms that are linked to the prediction of disease propagation. these are terms such as "mathematical model", "modelling", "simulation", "statistical model" and "prediction" that have commonly occurred in this cluster. the cluster includes terms affiliated with measures of disease control and spread prevention such as "(social/patient) isolation", "quarantine", "hygiene", "handwashing", "prevention", "infection control", "(population) surveillance", "mass screening", "(face) mask", "contact tracing". the cluster also represents keywords that are attributable to public policy making and social protection such as "health care planning", "health care policy", "health care quality", "leadership", "disaster planning" and "polices". the cluster (i) of keywords also have distinctly and commonly (across all three datasets) represented keywords that are attributable to the studies on mental health impacts of the epidemic. these are keywords such as "mental health (service)", "psychiatry", "psychology", "mental stress", "anxiety", "fear", "mental disease". these studies have often used methods such as "questionnaire(s)" and "survey(s)" that have commonly reflected in this cluster across the three literatures. issues surrounding the safety of medical facilities and medical staff also appear to have been addressed mainly by studies whose keywords are attributable to this cluster. these studies have generated keywords such as "health care personnel", "nurse(s)", "medical staff", "hospital", "health care facility", "personal protective equipment" that are distinctively observable in cluster (i) of keywords across all three datasets. the economic aspects of the epidemics also seem to have been addressed particularly by covid-19 as reflected in cluster (i) of the covid-19 literature. these have been reflected in terms such as "economics", "economic aspect", which have occurred frequently enough in covid-19 studies to appear distinctly on the map. the presence of such cohort of studies is, however, not as clearly identifiable on the sars and mers maps as is it on the covid-19 map. this could be explained by the greater magnitude of the societal impact of covid-19 outbreak compared to sars and mers. the names of the countries and regions have almost invariably appeared in cluster (i) across all three datasets. in certain cases, the country names that have occurred most often are those from which the outbreaks originated or those that suffered most from the impact of the outbreak. for example, "saudi arabia" appears quite distinctly on the cluster (i) of the mers dataset. similarly, the presence of the names of south-east asian countries/regions such as "china", "hong kong", "taiwan", "singapore" on the cluster (i) of the sars map, or the term "wuhan" on the cluster (i) of the covid-19 map are quite pronounced. the occurrence of the names of countries also could be a reflection of the early (case) studies that have addressed the local impacts/spread of the outbreaks within their own society. on the issue of early studies, the terms "letters", "editorial", and "review" (which have intentionally been kept on the maps) seem to also have distinctly occurred in cluster (i) of each literature which is an indication that this cluster includes early studies that appeared at a time where the amount of data and clinical trials were insufficient for full-length articles. an inspection of the figs. 18, 20 does, in fact, confirm this hypothesis at least in association with the sars and mers literature, that the cluster (i) of keywords represent studies that on average emerged earlier during the developments of their respective literatures. figures 19, 21 , 23 that have illustrated the colour-coding of the average number of citations on the maps also show that, although cluster (i) is associated with the early studies that generally preceded studies of the two other clusters and although it represents the largest variety of topics compared to the two other clusters, it is also associated with the studies that, on average, been the recipient of a lesser number of citations when compared to the two other clusters. this pattern appears to have commonly occurred across all three datasets. a second cluster of keywords associated with each of the three literatures were also discovered that is attributable to the studies on the chemistry and physiology of the virus, or viral pathogenesis, or in other words, the chemical constitution of the virus (knight 1954) , a sector of virology that investigates the biological processes and activities of viruses that take place in infected host cells and result in the replication of a virus. this cluster has been assigned the green colour in the maps of figs. 5, 6, 7. according to the maps, the most distinct terms associated with this cohort of virology studies on sars, mers and covid-19 are terms such as "virus protein", "virus entry", "chemistry", "metabolism", "physiology", "pathology", "cell line", "(virus/viral) protein(s)", "molecular model(s)", "virus genome", "virus rna", "virus replication", "mutation", and "enzyme activity". as this sector of studies often use "animal model(s)", terms such as "animal cell", "animal experiment", "controlled study", "mice" and "mouse" have frequently appeared in the representative cluster (ii). in reflection of the fact that these cohort of studies ultimately seek "drug design", in addition to generic common terms such as "drug design/potency/structure/synthesis", the names of the specific potential drugs that have been investigated in relation to each disease have appeared in this cluster. this includes terms such as "hydroxychloroquine" or "remdesivir" on the covid-19 map. an inspection of the maps overlaid with the average year of publications for sars and mers in figs. 18, 20 in the "appendix" suggests that, on average, this cohort of studies are generally the last to appear in the published domain compared to the two other major clusters, but, according to figs. 19, 21, 23, they receive relatively high citations on average. a third and relatively smaller cluster of keywords was commonly identifiable in relation to each three literatures. this cluster has been visualised in blue across all three maps of keyword co-occurrence. the studies represented by this cluster of keywords, here referred to as cluster (iii), appear to have been more closely focused on the developments of antibodies and vaccines. the terms "treatment", "treatment outcome", "disease severity", "antiviral therapy", "prognosis", "drug safety", "prospective/retrospective study", "immunology", "immunotherapy", "innate immunity", "immune response", "virus/viral vaccine(s)", "virus/viral antibody" across studies of this cluster. terms affiliated with studies related to treatments and clinical care of respiratory patients also appear in this cluster. this includes terms such as "artificial ventilation", "intensive care unit", as well as symptom and organ terminologies associated with each disease, terms such as "fever", "headache", "diarrhea", "lung (injury)", "coughing", "liver injury", "kidney". terms affiliated with cohort analysis studies have appeared in this cluster of the maps associated with each literature. this is reflected in terms such as "female", "male", "child", "infant", "young adult", "adult", "age", "middle aged", "pregnant", "pregnancy". bibliographic coupling of the studies on sars, mers and covid-19 were analysed at the level of their sources/journals. figures 8, 9 , 10 show the maps of journal bibliographic coupling associated with sars, mers and covid-19 literatures respectively. the node sizes are proportional to the number of documents published by the corresponding sources and the thickness of the links are proportional to the degree of bibliographic couplings between the pairs of sources connected by each link. the minimum number of documents associated with each node/journal to appear on the map has been set to 10. the number has been set on the basis of maximising clarity of the a first-glance comparison shows that while the maps associated with sars and mers are well connected, connections across the covid-19 map are rather sparse. both the sars and mers maps include three distinct and well-defined clusters of bibliographically coupled journals in addition to one minor and smaller cluster. these clusters show relatively strong degrees of inter-connectivity and coherence, whereas, this feature is not shared by the covid-19 map. the observation is understandable considering the fact that the sars and mers literatures are relatively well established and have stabilised given that they have both been under development over a period of several years, whereas the covid-19 literature is an emerging and evolving field. newly published covid-19 studies do not seem to be sharing many references as of yet, which explains why their coupling connections are relatively weak. the comparison also suggests that the covid-19 studies are generally scattered across a broader variety of journals and subject areas, as opposed to the sars and mers publications that seem to have been concentrated across a smaller set of specialty (mostly medical) journals. this is also consistent with our observations from figs. 1, 2, 3 showing explicitly that studies of covid-19 are scattered across a broader variety of subject areas compared to the sars and mers literatures. though not shown in fig. 3 , due to the respective values being smaller than 1%, journals in the following subject areas (those that are deemed minor areas in relation to covid-19 literature) have each published a relatively considerable number of studies on this topic (a feature that is not necessarily shared where the most active journal has been social anthropology (24 items) covering topics such as "climate change reactions" (bychkova 2020), or "legal voids linked to declared states of emergency" (karaseva 2020)), economics, econometrics and finance (84 items, with economic and political weekly (36 items) being the most active journal of that category, covering topics such as "food supply chains" (reardon et al. 2020) , "economic stimulus packages" (mulchandani 2020) or "reverse migration" (dandekar and ghai 2020)), physics and astronomy (77 items, where chaos solitons and fractals (16 items) has been the most active publication outlet, covering topics such as "mathematical models for forecasting the outbreak" (barmparis and tsironis 2020; bekiros and kouloumpou 2020; boccaletti et al. 2020; ndaã¯rou et al. 2020; postnikov 2020; ribeiro et al. 2020; zhang et al. 2020 )), energy (67 items, with international journal of advanced science and technology (44 items) being the most active journal in that category, covering topics such as "flexible work arrangement in manufacturing" (sedaju et al. 2020) ), material sciences (57 items, with acs nano (10 items) being the most active outlet in that category, covering topics such as "3-d printed protective equipment" (wesemann et al. 2020) ), decision sciences (23 items, with lancet digital health (8 items) and transportation research interdisciplinary perspectives (4 items) being the most active outlets in that category, covering topics such as "the effect of social distancing on travel behaviour" (de vos 2020) or "the implementation of drive-through and walk-through diagnostic testing" (lee and lee 2020)), earth and planetary sciences (22 items, with indonesian journal of science and technology (8 items) being most active in that domain, covering topics such as "the deployment of drones in sending drugs and patient blood samples" (anggraeni et al. 2020) ). a prominent source that seem to have consistently published a substantial portion of studies on sars and mers is journal of virology. this journal, however, has not published a considerable number of studies on covid-19, and with only 8 publications on this topic at the time of writing this article, it does not have a strong representation on the map of covid-19. the lancet and science, however, are two major outlets notably identifiable on all three maps. for covid-19 studies in particular, journals such as journal of medical virology, the bmj, the lancet, journal of infection, science, nature, science of the total environment and medical hypotheses have been most notable outlets of publications so far. some of these outlasts, such as science of the total environment and medical hypotheses, do not have a strong representation on the maps associated with the sars or mers. in terms of the bibliographic coupling of the sources for sars publications, the strong relation between journal of virology and virology, and to lesser extent, with emerging infectious diseases are outstanding. for mers publications, the strong bibliographic coupling of publications between emerging infectious diseases and journal of virology is most outstanding. for covid-19 publications, the one outstanding bibliographic coupling relation is one that exits between journal of medical virology and journal of infection. the analyses of journal citations also showed similar patterns of scatter and relatively cohesive clusters in relation to the covid-19 literature compared to the welldefined clusters of journal citations for sars and mers literatures. consistent with the previous observation with respect to journal bibliographic coupling, the covid-19 literature seems to be also much less cohesive in terms of its journal citation networks, when compared to the sars and mers literatures. as discussed earlier in relation to bibliographic couplings, this could also be partly explained by the fact that covid-19 papers are scattered across a more diverse range of journals and broader variety of subject categories. for the maps of journal citation relations presented in figs. 11, 12, 13 , only the strongest citation relations have been visualised by increasing the minimum strength threshold for the citation links between pairs of journals to show on the map. the nodes have also been overlaid with the average citation colour coding. for sars, the maps present very strong citation relations between publications in journal of virology and those of virology, pnas and science (and to a lesser extent, with emerging infectious diseases, the lancet and new england journal of medicine). for mers in figs. 27, 31 in the "appendix", the nodes of the bibliographic coupling maps have been colour-coded by the average year of publications and the average citations per document associated with the journals that each node represent (except for the covid-19 map that has only been overlaid with the average citations). according to these maps, emerging infectious diseases and the lancet have been a major source of publications for early studies on both sars and mers. this pattern for the lancet seems to have extended to covid-19 studies as well, as this journal has published a substantial portion of the early studies on this topic. for sars, the strong representation of chinese medical journal and chinese journal of microbiology and immunology among the journals that published early studies are notable, a pattern that could be explained by the geographical origin of the sars outbreak. such pattern is to a less obvious extent also observable with respect to the mers literature. collaborations of authors aggregated at the level of the countries were also analysed with respect to the sars, mers and covid-19 literatures. outputs of the analysis are presented in figs. 14, 15, 16, for sars, mers and covid-19 respectively. in each map, the size of nodes, each corresponding with a country, are proportional to the number of published documents with an author affiliated with the institutes of those countries. the links connecting the nodes indicate co-authorships between authors residing in the countries, while the thickness of the links represent the strength (i.e. frequency) of the co-authorships. the colour assigned to each node represents the average number of citations that studies emerged from countries have received. the minimum number of documents for country names to appear on the maps has been set to 5, a number chosen based on the criterion of the quality of the resultant maps. comparison across the three maps of co-authorships shows a pattern of author involvement from the regions where each viral outbreak originated. studies authored by researchers affiliated with chinese institutes are well represented in all three cases, but clearly the map of country co-authorships associated with the covid-19 literature more so with respect to the sars and covid-19 literature, diseases whose first cases were recorded in china. the involvement of chinese authors is relatively less pronounced with respect to the mers studies whose origin was in the middle east. instead, with respect to the mers literature, it appears that authors affiliated with institutes in saudi arabia have been exceptionally overrepresented in the publications. this is also, to a lesser extent, the case with egypt being markedly presented on the mers map of the country co-authorships. on sars studies, chinese authors have most strongly collaborated with authors residing in the united states, followed by authors from germany, taiwan, singapore, japan, france, australia, united kingdom and canada. the sars network of collaborations for the authors affiliated with institutes in the united states has been, by and large, similar to that of china. except, south korea, the netherlands, italy and spain are also strongly represented in the collaborations with the united states. the map of co-authorships associated with the mers literature presents the names of many middle eastern countries including saudi arabia, egypt, lebanon, iran, tunisia, qatar, oman, jordan, and united arab emirates which is a clear indication into the exceptionally strong representation of the authors from this region in these studies. the strongest network of co-authorships on this topic are observed between authors from the united states and those of saudi arabia, followed by china, united kingdom, egypt, south korea, canada and the netherlands. the closest collaborators of chinese authors on this topic, after the united states, have been from saudi arabia, united kingdom and egypt. the closest collaborators of saudi arabia, after the united states, have been egypt, china and united kingdom. the covid-19 map presents a considerably higher number of country names compared to maps of the sars and mers literatures. it clearly shows that authors from a larger number of countries have become involved in studies of covid-19, compared to the research on sars and mers that have apparently engaged a lesser number of countries and institutes. china, on the topic of covid-19, has a very well spread and rather more evenly distributed network of collaborations with countries across the world, when compared with its network of collaboration on sars and mers. while its strongest collaboration has been with the united states, the names of many other countries appear on its network with no particular country standing out distinctly. italy, as a country that was highly affected by the covid-19 outbreak, has been exceptionally well represented on this map with a very strong link of collaboration with the united states, followed by united kingdom at a smaller scale. this pattern of exceptional representation of highly affected countries on the maps has to a lesser extent extended to iran, spain, france and brazil as countries severely affected by the covid-19 outbreak at early stages of the global spread. according to the colour coding of the maps, sars studies in which authors affiliated with the institutes in the netherlands were involved have, on average, received the highest number of citations. this is followed by studies of authors from germany. these are two countries whose authors have published considerable number of documents on sars and received high number of citations at the same time. this pattern was, to some extent, repeated in relation to the mers literature, with studies from the netherlands, germany and united kingdom having received, on average, the highest number of citations. for studies published on covid-19, publications from china have so far stood out in terms of both the magnitude of research activities and the average number of citations. outbreaks of infectious diseases have often shown a pattern of generating a quick surge of publications on their respective topics, such that they often create an entirely new literature over a short period of time (olijnyk 2015; tian and zheng 2015) . by all measures, however, the influx of the research publications that began to emerge following the 2019 novel coronavirus outbreak outsizes those of the previous cases in the history of coronaviruses, and perhaps arguably, in the history of infectious diaereses (tian and zheng 2015) . this has certainly marked a new milestone in the timeline of research on coronaviruses which dates back to 1968 (almeida et al. 1968 ). according to the editor-in-chief of the journal of virology, as quoted in an article of the scientist magazine (jarvis 2020), this surge of research outputs has been to the extent that has inundated established coronavirus researchers and domain experts with peer review requests to an extent that they are unable to cope. parallel to such intensified efforts on the research, peer review and editorial fronts, widespread efforts have also been underway in synthesising, summarising and visualising these rapid developments, a pattern that has also been observed-though at much smaller scales-with respect to the previous epidemics of viral diseases (kostoff and morse 2011) . in line with these endeavours, this work also aimed at quantifying and analysing scientometric aspects of the covid-19 literature in contrast with those of the previous major coronavirus diseases, i.e. sars and mers. the focus for sourcing these literatures has been on peer reviewed and published studies. an analysis of the timeline of the development of publications on coronaviruses made clear that the sars outbreak constitutes the first major milestone in the history of this research, an event that brought a then-unprecedented amount of attention to this topic. while scopus has indexed a total of nearly 4400 studies on coronaviruses from 1968 to 2002, the three immediate years post sars outbreak (i.e. november 2002) have each recorded nearly 1000 coronavirus publications. this means that following the sars outbreak, the then 36 years old literature of coronaviruses almost expanded by 70% within only three years. this trend, however, did not persist and did not extend to the years succeeding 2005, as from 2005 onwards, a gradual decline in the rate of publications on coronaviruses began. this continued until the 2012 mers outbreak, another event that reinvigorated the coronavirus research, though not as substantially as that of the sars epidemic. the pattern of a few years of increase in research activities on coronaviruses followed by a gradual decline, which had occurred in relation to sars, also repeated itself in a similar fashion after the mers outbreak. the outbreak of 2019 novel coronavirus, however, marks a unique milestone in this timeline. the magnitude of scholarly outputs prompted by this novel virus was to the extent that, in less than five months, 12,000 publications was already indexed by scopus, a number that nearly equals 70% of the total amount of literature generated on coronaviruses during the 50 years of this research prior to 2020. in fact, since april 2020, we have made five consistent recordings of the number of scopus-listed publications linked to covid-19 at five cross-sections of time, the last of which representing our scopus search on 21 july 2020 as the revision of this article was being drafted. this set of records may give an approximation for the current rate of accumulation of studies on this topic. according to fig. 17 that visualises these records, the trend seems to be rather linear, although the rate seems to have increased since may 2020. our very latest scopus search on 21 july 2020 showed that the number has reached 30,400 items. if the current trend continues, then by the end of 2020, approximately another 50,000 articles could be expected on this topic, making the total size of this literature exceed 80,000 items. a conservative projection could be obtained using the rate of accumulation associated with the time period between april and may 2020, the earlier stage of the emergence of studies on this topic. this would yield an estimate of approximately 70,000 publications by the end of 2020. by retrieving and disentangling the literatures linked to these three respiratory diseases, we sought to discover similarities and discrepancies of their research landscape from scientometric perspectives. the most interesting pattern was the recurrence of three distinct clusters of studies in each literature as suggested by keyword co-occurrence analyses. it appeared that, following each outbreak, an early cluster of studies first emerged, addressing issues attributable to the public emergency management aspects of a pandemic, such as prediction of disease propagation, measures of outbreak control, public policy making, and concerns related to the protection of medical professionals and mental health. accordingly, as a result of these three outbreaks, three significant cohort of studies related to health emergency management have already emerged, which, if synthesised effectively, could constitute a valuable and evidence-based guide to help government and communities better prepare for and react to possible future disease outbreaks, at least of those of the respiratory nature. citation and bibliographic coupling analysis at the level of journals demonstrated that firstly, covid-19 studies are scattered across a broader variety of sources and subject categories, and secondly, its network of journal relations is still not as cohesive as those of the sars and mers literatures. as the literature on covid-19 further develops, more cohesive patterns of bibliographic coupling or journal citations may form. while journal of virology and emerging infectious diseases seem to have been two major outlets commonly prominent across both sars and mers literatures, their presence in the literature of covid-19 seem to not be distinctly notable. instead, a great portion of covid-19 studies have concentrated across three journals: the lancet, the bmj, fig. 17 the number of studies related to covid-19 indexed by scopus at five different points in time since april 2020, along with a projection of the number at the end of 2020 and journal of medical virology. while major multidisciplinary journals, particularly science, nature, pnas and plos one, have, to varying degrees, been active in publishing studies on all three topics, their influence is most notable in relation to the sars literature where they have published a substantial portion of studies. those studies happen to have been recipients of relatively high number of citations. the involvement of authors from various countries on the publications linked to these three diseases seem to be distinctly correlated with the regions where the outbreaks originated, with authors from china, for example, being much more strongly represented on sars and covid-19 studies, two diseases whose origin of outbreaks were in that country. middle eastern countries, on the other hand, are exceptionally represented in the mers literature. the questions of where the covid-19 literature is headed, how big it will grow in the next coming months/years, at what point in time the rate of publications on this topic is going to slow down and how widely this literature is going to spread across journals and subject categories are just a few examples of questions that will only be determined by time. these scholarly patterns may also be influenced by future success or failure of countries in controlling the extent of the surge and re-surge of outbreaks and by possible medical discoveries in relation to vaccine and treatment. but given the pace at which scholarly outputs are currently emerging, the number of under-review preprints, the extent of studies and clinical trials that have already been conceived around the world; and also considering the seemingly long-lasting and far-reaching consequences of this global emergency that have impacted on aspects of life, it will probably not be so soon before we observe a decline in covid-19 research interest. macro-syntheses of the scholarly literatures generated in response to the three most significant outbreaks of coronaviruses, i.e. sars, mers and covid-19, revealed interesting structural similarities across all three literatures and suggested that they are commonly made up of three major segments. in each case, publications related to public health emergency management have been the earliest to merge, followed by studies on virus chemistry and clinical treatments. however, despite this similarity of the structures and the recurring patterns of publications following each outbreak, the magnitude of the rate of scholarly research prompted by the 2019 novel coronavirus outbreak remains a striking phenomenon, arguably, unique in the history of scientific publication. the main motive of this study was to document this major milestone and the magnitude of these scientific efforts. it is, per se, a heartening observation to note how the 2020 global health crisis has mobilised the efforts of scholars across the globe and across scientific disciplines. a question that naturally follows, however, would be to discover how the rush to combat this global crisis through scientific work has influenced the rigour and quality of the publications. this could warrant further research on this rapidly evolving literature in the form of quality assessment of publications. the deployment of drones in sending drugs and patient blood samples covid-19 estimating the infection horizon of covid-19 in eight countries with a data-driven approach sbdiem: a new mathematical model of infectious disease dynamics modeling and forecasting of epidemic spreading: the case of covid-19 and beyond sars-cov, mers-cov and now the 2019-novel cov: have we investigated enough about coronaviruses?-a bibliometric analysis. travel medicine and infectious disease scientists are drowning in covid-19 papers can new tools keep them afloat? science covid-19 and climate change reactions: sts potential of online research coronaviridae: a review of coronaviruses and toroviruses a bibliometric analysis of covid-19 research activity: a call for increased output 31 the map of bibliographic coupling of sources for covid-19 overlaid with the colour-coding of the average citation per document coronavirus disease 2019: coronaviruses and blood safety emerging coronaviruses: genome structure, replication, and pathogenesis sars: the first pandemic of the 21st century a scientometric overview of cord-19 a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 migration and reverse migration in the age of covid-19 the effect of covid-19 and subsequent social distancing on travel behavior. transportation research interdisciplinary perspectives bibliometric analysis of global scientific research on coronavirus (covid-19) the impact of early scientific literature in response to covid-19: a scientometric perspective the scientific literature on coronaviruses, covid-19 and its associated safety-related research dimensions: a scientometric analysis and scoping review current status of global research on novel coronavirus disease (covid-19): a bibliometric analysis and knowledge mapping journals, peer reviewers cope with surge in covid-19 publications. solana beach: the scientist scientometric trends for coronaviruses and other emerging viral infections the legal void and covid-19 governance the chemical constitution of viruses structure and infrastructure of infectious agent research literature: sars author productivity of covid-19 research output globally: testing lotka's law 2020. visualising covid-19 research. arxiv testing on the move: south korea's rapid response to the covid-19 pandemic human coronaviruses: a review of virus-host interactions current topics in microbiology and immunology ergebnisse der mikrobiologie und immunitã¤tsforschung covid-19 crisis: economic stimulus packages and environmental sustainability human coronaviruses: a brief review mathematical modeling of covid-19 transmission dynamics with a case study of wuhan an algorithmic historiography of the ebola research specialty: mapping the science behind ebola estimation of covid-19 dynamics "on a back-of-envelope": does the simplest sir model provide quantitative parameters and predictions? covid-19's disruption of india's transformed food supply chains short-term forecasting covid-19 cumulative confirmed cases: perspectives for brazil flexible work arrangement in manufacturing during the covid-19 pandemic: an evidence-based study of indonesian employees world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) emerging infectious disease: trends in the literature on sars and h7n9 influenza open access and altmetrics in the pandemic age forescast analysis on covid-19 related literature software survey: vosviewer, a computer program for bibliometric mapping review of the 2019 novel coronavirus (sars-cov-2) based on current evidence 3-d printed protective equipment during covid-19 pandemic predicting turning point, duration and attack rate of covid-19 outbreaks in major western countries see figs. , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ,and 31. key: cord-262673-j2ot35lt authors: ahmed-hassan, hanaa; sisson, brianna; shukla, rajni kant; wijewantha, yasasvi; funderburg, nicholas t.; li, zihai; hayes, don; demberg, thorsten; liyanage, namal p. m. title: innate immune responses to highly pathogenic coronaviruses and other significant respiratory viral infections date: 2020-08-18 journal: front immunol doi: 10.3389/fimmu.2020.01979 sha: doc_id: 262673 cord_uid: j2ot35lt the new pandemic virus sars-cov-2 emerged in china and spread around the world in <3 months, infecting millions of people, and causing countries to shut down public life and businesses. nearly all nations were unprepared for this pandemic with healthcare systems stretched to their limits due to the lack of an effective vaccine and treatment. infection with sars-cov-2 can lead to coronavirus disease 2019 (covid-19). covid-19 is respiratory disease that can result in a cytokine storm with stark differences in morbidity and mortality between younger and older patient populations. details regarding mechanisms of viral entry via the respiratory system and immune system correlates of protection or pathogenesis have not been fully elucidated. here, we provide an overview of the innate immune responses in the lung to the coronaviruses mers-cov, sars-cov, and sars-cov-2. this review provides insight into key innate immune mechanisms that will aid in the development of therapeutics and preventive vaccines for sars-cov-2 infection. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) reportedly emerged at a live animal market in the chinese city of wuhan is currently causing a pandemic and negatively affecting global health (1) (2) (3) . there are more than 11 million confirmed sars-cov-2 infections with an mortality rate that widely varies by country and region (4) . even in industrialized countries, sars-cov-2 led healthcare systems approach the brink of collapse by overwhelming their capacity and straining resources. governments and local leaders ordered the shutdown of cities, regions, countries leading to massive disruptions in the local and global economy. unlike previous coronavirus (cov) infections, the rapid global spread, high transmission rate, longer incubation time, and disease severity of sars-cov-2 requires a better understanding of the epidemiology and immunopathogenesis of this viral outbreak in order to learn from this experience and to manage future pandemics. sars-cov-2 is a highly pathogenic cov (5) (case-fatality rate of 3.6-3.8%) (4, 6) that is related to severe acute respiratory syndrome cov (sars-cov) (case-fatality rate of 14-15%) and the middle east respiratory syndrome cov (mers-cov) (case-fatality rate of 34.4%), see also table 1 (138, 139) . sars-cov, sars-cov-2 and mers-cov target the lower respiratory system, causing respiratory illnesses, including severe pneumonia, acute lung injury (ali) and acute respiratory distress syndrome (ards) (140, 141) . sars-cov-2 infection results in higher hospitalization rates in the elderly (>65) and persons with pre-existing conditions including hypertension, diabetes and obesity compared to rates among younger populations without pre-existing conditions ( table 1 ) (142, 143) . in addition to an age disparity, males with covid-19 appear to have higher risk for worse outcomes and death (143, 144) . epidemiological research of the sars and mers infections also showed that males had a higher mortality rate than females (144) (145) (146) . while sars-cov-2 is a novel coronavirus, several important insights have already been made about its basic mode of transmission. virus particles are inhaled in respiratory droplets expelled from the airways of infected individuals. angiotensinconverting enzyme 2 (ace2), expressed on the ciliated bronchial cells, endothelial cells, and type i and ii alveolar cells, is the host receptor for cell entry into the respiratory tract by both sars-cov-2 and sars-cov ( table 1 ) (147) (148) (149) (150) . the spike protein (s) of cov is responsible for the entry of the virus into the target cell (figure 1 ) (147, 151) . ace2 is a type i transmembrane metallocarboxypeptidase that plays a vital role in the renin-angiotensin system (ras) (152, 153) , which in turn is critical in maintaining blood pressure homeostasis as well as fluid and salt balance in mammals. ace2 is found in vascular endothelial cells, in the renal tubular epithelium, and in leydig cells of the testes (154) . studies have shown that ace2 is expressed in gastrointestinal (gi) tissues, making it a potential site for harboring sars-cov (155) . this may be one of the reasons for gi pathology reported in some patients with covid-19 and viral shedding in stool. in contrast, mers-cov uses dipeptidyl-peptidase 4 (dpp4) as an entry receptor, which is expressed on endothelial cells and some epithelial tissues ( table 1 ) (19, 156) . accumulating data suggest that the innate anti-viral response and adaptive immunity may contribute to a cytokine storm leading to systemic hyper inflammation and exacerbation of the disease in patients with (a) comorbidities (b) older than 65 years of age (c) of the male sex. the exact role of the innate immune system in disease pathogenesis and prevention between the sexes and the impact of age is not fully elucidated. in addition, the potential dysregulation of the innate immune response by sars-cov and sars-cov-2 is not completely understood which warrants further research. the cells of the airway epithelium are the first line of defense, providing a mechanical barrier (mucociliary escalator) that expels particles and pathogens via cilia, mucus, and induced coughing (157, 158) . this barrier includes cells of the pulmonary epithelium and tissue-resident macrophages and dendritic cells (dcs). the macrophages and dcs express pattern recognition receptors (prrs) that can detect molecules from pathogens (pathogen-associated molecular patterns-pamps) or molecules released from damaged cells (damage or danger-associated molecular patterns-damps) (158) (159) (160) . in the lung, there are two populations of macrophages, alveolar and interstitial macrophages (161) . in addition to these macrophages, dcs play a vital role in facilitating the host defenses against respiratory diseases (162) (163) (164) . dcs can be divided into plasmacytoid (pdc) and myeloid types (mdc) (165) (166) (167) . macrophages and the two dc subtypes trigger antiviral responses by generating a substantial amount of type i interferon and these cells play important roles in immune surveillance in the airways and the distal lung (74, (168) (169) (170) (171) (172) . during steady state, the dc density in lung associated tissue declines from the trachea to the alveoli (173) while the representation of cells in macrophage compartment seems more complex (174) . if a virus infects airway epithelial cells, the viral rna would be sensed via intrinsic innate receptors, including rig-1, mda5, nlrp3 inflammasome, and the rna sensing tlrs 7 and 8. in the case of influenza a infection, triggering the prrs causes a strong induction of type 1 interferon (ifn) in epithelial cells (175, 176) . in other viral infections, such as respiratory syncytial virus (rsv), alveolar macrophages are the predominant source of type 1 ifns (161). furthermore, respiratory epithelial cells and lung macrophages are capable of secreting a broad range of chemokines like il-8, macrophage inflammatory protein-1 (mip-1), rantes and cytokines including tnf-α, il-6, il-1β that influence the types of immune cells being recruited to the area in response to acute viral infections (177, 178) . macrophages, depending on their polarization status of either m1 or m2, and in a similar way as airway epithelial cells, can further elicit a th1 or th2 response (158, 161, 178) . in the case of influenza virus infection, the magnitude of epithelial cell response can be proportional to the amount of virus which result in paracrine induction of ifn λ (175) . not only can airway epithelial cells produce a large array of cytokines/chemokines in response to an acute viral infection, but, depending on the magnitude of prr engagement and the combination of pamps and damps triggered, these epithelial cells can modulate the type of chemokines/cytokines produced and influence the influx of innate and adaptive immune cells (158, 160) . the response to different viral infections is generally similar, however, the response can be tailored in timing, magnitude and the induced gene signatures in response to each virus (179) . unlike rsv and mers-cov, which both productively infect alveolar multiple cell types in the lower respiratory tract were found to be infected, including type i alveolar epithelium, macrophages, and putative cd34 + oct-4 + stem/progenitor cells in human lungs (15) (16) (17) ciliated bronchial epithelial cells and type ii pneumocytes (7, 18) un-ciliated bronchial epithelial cells and type ii pneumocytes (19) (20) (21) infect mostly human type i and type ii pneumocytes and alveolar macrophages (22) respiratory, nasal, corneal and intestinal epithelial cells (23) club cells, ciliated cells, type i and type ii alveolar cells (24) ciliated epithelial cells of the upper and lower respiratory tract (25) the ciliated cells of the human airway epithelium are the main target, it also infects basal cells (26) and immune cells, such as macrophages, b cells cd4 + and cd8 + t cells (27) upper and lower airways epithelial cell ( no specific antiviral drugs (134) antiviral drugs may be a treatment option (135) no antiviral agents symptomatic treatment supportive care (136) there are no approved antiviral medications (137) ace2 macrophages (73, 180) , seasonal influenza and sars-cov usually lead to non-productive infections in these cells (181) . in addition, sars-cov infection of primary monocytes yielded little virus, likely due to the suppressive effects of ifn-α (182) . thus, the initial cell type(s) involved in propagating a viral infection intensifies the complexity of the immune response. another key factor that determines the magnitude of the immune response is the induction and rate of cell death. although related, mers-cov induces widespread cell death when compared to sars-cov in human bronchial epithelial cell cultures (183) . however, the sars-cov open reading frame 3a (orf3a) protein can induced necrotic cell death in a variety of cell lines (184) . the same orf3a protein can activate the nlrp3 inflammasome, leading to activation of nf-κb and elevated secretion of active il-1β in cell culture (185) . cytokine (186), with slightly different cytokine/chemokine profiles. this delay in cytokine induction was confirmed in another study using the same epithelial cell lines (187) as well as in human alveolar type ii cells (18) . in both cell lines and primary alveolar type ii cells, sars-cov induced ifn-β, ifn-λ, cxcl10, cxcl11, il-6, ip-10, and tnf-α (18, 187) . mers-cov did not induce ifn-β but induced higher level of il-8 transcript in cell culture. however, no difference in il-8 production was observed between sars-cov and mers-cov at 48 h post-infection (186) . this was confirmed in-vivo using a non-human primate model comparing the immune responses to sars-cov infection between young adult cynomolgus macaques (3-5 yrs) and older macaques (10-19 yrs) (188) . interestingly, the high induction of il-8 was observed on a transcript level in the older animals, while the younger once showed higher levels of ifn-β transcript (188) . in all animals, the expression of ifn-β was inversely correlated with the pathology score, supporting the role of ifn-β in controlling disease severity (188) and introducing a potential area of research to define age disparity observed in patients infected with sars-cov-2. both older age and male sex are important factors associated with high mortality of sars-cov and sars-cov-2 infection (189, 190) . many viruses have evolved to disrupt and subvert the immune responses. a common virus that is well-known to affect the lower airway and counteract the immune response is rsv (178, 191) . the rsv genome encodes non-structural proteins ns1 and ns2 that can block type 1 ifn production and signaling in cell cultures (191) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat1 and stat2 signaling in cell lines (192) , whereas mers-cov m protein also suppresses type 1 ifn by inactivating irf3 (193) , leading to the low expression of ifn-β. in contrast to reports in epithelial cell lines or primary alveolar type ii cell culture and observations in non-human primates, sars-cov nucleocapsid (n) protein and membrane (m) protein, as well as nsp1, can suppress ifn response via various mechanisms in cell lines (194) (195) (196) . to bridge the dichotomy of inhibition of ifn signaling in cell lines, and the ifn expression in vivo, cells recruited by the infection need to be considered as a potential source. as previously discussed, infected epithelial cells via paracrine signaling to neighboring cells and resident macrophages, secrete chemokines and cytokines to attract other immune cells. in general, monocytes/macrophages are recruited by ccl3 (mip-1a), ccl2 (mcp-1), and neutrophils are recruited by il-8 (cxcl8), cxcl2, and cxcl5 chemokines, all of which can be secreted by airway epithelial cells (178, 197, 198) . both monocytes and neutrophils are also recruited by complement fragment (anaphylatoxin) c5a (figure 1) . both influenza and sars virus can induce acute lung injury (ali) which is accompanied by high levels of c5a, leading to the influx and activation of innate immune cells (199) (figure 1) . serum samples and lung tissue of sars patients showed high-level expression of cxcl10 (ip-10), which is also found to be induced by sars-cov in the epithelial cell line calu-3 (200) . significant neutrophil, macrophage and cd8 t-cell infiltration can be found in the lung of sars patients by immunohistochemistry (76, 77, 201) . in addition to post-mortem lung histology analysis in patients with sars-cov, experiments using rhesus macaques infected with sars-cov found monocyte and macrophage recruitment. the accumulation of pathogenic inflammatory monocytemacrophages (imms) was also observed in a sars-cov mouse model. the accumulation of imms resulted in heightened lung inflammatory cytokine/chemokine levels, extensive vascular leakage, and impaired virus-specific t cell responses (202) . a strong infiltration of cd68 and mac387 positive monocytes/macrophages were found in the human and animals lung samples (203, 204) . macrophages further stimulate fibroblasts to deposit extracellular matrix leading to pulmonary fibrosis (205) , which was also observed in patients who recovered from sars (206, 207) . autopsy samples acquired from patients with sars-cov-2 patients contained viral nucleocapsid protein (np) positive cd68 + macrophages in the capillaries of the spleen and in lymph nodes, indicating that sars-cov-2 might migrate into the spleen and lymph nodes through macrophages. this study also found that cd169 + macrophages appear to mediate sars-cov-2 into these tissue sites, contributing to virus dissemination (208) . similar to sars-cov-2, sars viral particles and genomic sequences were detected in monocytes, macrophages as well as within different organs of sars patients (15) . sars-cov was shown to infect both immature and mature human monocyte-derived dcs by electron microscopy and immunofluorescence. the detection of negative strands of sars-cov rna in dcs suggests viral replication, but no increase in viral rna was observed (209) . as mentioned above, there was no perceivable increase to sars-cov replication in primary monocytes (182) . another study looked at sars-cov and mers-cov replication in human macrophages, human monocyte-derived macrophages, and dendritic cells (mddcs) and found that both viruses replicated poorly. mers-cov induced ifn-λ1, cxcl10, and mxa mrnas in both macrophages and mddcs, however, sars-cov was unable to induce such responses (69) . interestingly, depletion of inflammatory monocyte-macrophages in the mouse model partially protected from lethal sars infection (210) . these data suggest that monocytes, macrophages and dendritic cells have essential roles in cov infection. the severity of disease and the response to these viruses seems to be dependent on the induced cytokine/chemokine profiles and the amplification of the immune response by the recruited cells. growing evidence of dysregulation of an innate anti-viral response originates from studies using clinical samples (211) and murine models (202, 212, 213) . in addition to dysregulated cellular responses, the complement system may play an important role in sars-cov-2 infection (figure 1) . evidence comes from sars infected patients who had lower levels of mannan binding lectin (mbl) in serum compared to healthy controls (214) . the sars patients with a higher frequency of mbl gene polymorphisms were associated with lower serum levels or deficiency of mbl (214) . it is still unknown if this is also true for covid-19 patients, which requires further investigation. in cell culture experiments sars-cov was able to bind and activate the complement cascade and block viral infection (214) . preliminary findings in a limited number covid-19 patients found significant deposits of the membrane attack complex (mac), c4d and mbl-associated serine protease (masp)2 in the microvasculature indicating sustained, systemic activation (215) . the sars-cov-2 spike protein was co-localized with c4d and mac (215) . in a non-peer reviewed publication by gao et al., mers-cov, sars-cov and sars-cov-2 n protein are able to bind to masp-2 leading to enhanced complement activation (216) (figure 1) . in a later phase of the infection, the complement system might be also triggered via antibodies bound to the virus (classic activation pathway, figure 1 ). this excessive complement activation might play a role in multi organ damage in severe covid-19 cases (217) . in a mers-cov mouse model the blockade of the c5a-c5ar axis alleviated not only lung damage but also spleen damage (218) . mice treated with a monoclonal antibody to c5a showed reduced lung infiltration of cd68 + cells and significantly lower cytokine levels of il-1 β, tnf-α, inf-γ and il-12 (218) . complement blockade might be an important way to curb part of the immune dysregulation associated with covid-19. overall, we need to look closer at the role of the complement system, the recruited innate immune cells and their combined role in pathogenesis, viral clearance and the eventual resolution of the infection. the most abundant leukocytes, neutrophils, play a critical role in clearing viral infections. neutrophils, attracted by chemokines/cytokines released by tissue-resident macrophages and dcs, swarm to the site of infection. they recognize and release bioactive compounds, including cytokines, chemokines and ros, as well as nos in the very early phase of the infection (219, 220) . neutrophils modulate other innate and adaptive immune responses via cytokine/chemokine release and cell death and, therefore, can ameliorate or exacerbate disease progression. neutrophils infiltrate tissues infected by cov, including sars-cov, rat coronavirus (rcov), and mouse hepatitis virus (mhv). a high neutrophil count in the blood of sars patients at the time of hospital admission is associated with a poor prognosis (221, 222) . gao et al. suggested that patients with sars-cov-2 pneumonia can be stratified by neutrophil to lymphocyte ratio (nlr) and age (216) . patients older than 50 years of age and having an nlr ≥ 3.13 had more severe illness, so rapid access to the intensive care unit is required (79, 223) . experiments in mice showed that sars-cov disease severity in older mice correlated with increased pulmonary inflammation and influx of neutrophils (224, 225) . infection of rats with rcov could lead to neutrophil infiltrating into the respiratory tract early after inoculation, followed by the recruitment of macrophages and lymphocytes (226) . infection of mice with a neurotropic murine cov (mhv-jhm) showed infiltration of neutrophils into the brain as early as the first day after inoculation, which then promoted the recruitment of other types of inflammatory cells into the brain, likely through the loss of the blood-brain barrier integrity (227) . gene expression analysis in experimentally infected rhesus macaques with mers-cov revealed the recruitment of neutrophils into infected lung tissue (228, 229) . angiotensin-converting enzyme inhibitors (ace-is) could serve as a potential risk for fatal covid-19 through the upregulation of ace2 (230) and may provide a direct linkage to neutrophils and disease progression. investigators found that ace2 modulates il-17-mediated neutrophil influx by impacting stat3 activity (231) . animal models used to study the pathogenesis of sars-cov-2 have revealed important roles of neutrophils in infection and confirmed findings observed in patients. a new aspect in sars-cov-2 infection is the potential role of neutrophil extracellular traps (nets). the process of net formation is a specific type of cell death that can be triggered under inflammatory conditions (232, 233) , such as gm-csf+c5a, il-8, ifn-α+c5a or other tlr response mediators; all conditions present in severe sars-cov-2 infection (232, 233) . the net formation has been observed in covid-19 patients and may contribute to thrombotic complications in covid-19 patients (234, 235) . microvascular injury and thrombosis have been reported in covid-19 patients, increasing the likelihood that neutrophil net formation might play a role (215, 236, 237) . net formation was reported to be involved in clot formation and thrombosis and can further increase inflammation (232, 233) . therefore, neutrophils can attract a second wave of immune cells to the site of infection by cytokine/chemokine secretion as well as via netosis (238, 239) , which included monocytes and natural killer cells. on the other hand aggregated nets were reported to limit inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation (240) . despite the presence of neutrophils in sars-cov-2-infected tissues, their role in the clearance and/or immunopathology of the viral infection remains unclear. future studies on the responses of neutrophils to sars-cov-2-infection or infected cells in vitro may elucidate the role of neutrophils in the pathogenesis of respiratory cov infections. natural killer (nk) cells are a heterogenic immune cell subset that acts promptly to combat viral infections. they produce significant amounts of ifn-γ, kill virus-infected cells, provide direct support to other innate immune cells, and aid in the adaptive immune response to counter viruses. nk cells express activating receptors that detect viral antigens, enabling the destruction of infected cells (241) (242) (243) (244) . lectin-like receptor cd94 and killer immunoglobulin-like receptors, such as cd158b, regulate the function of nk cells. a study of 221 patients with sars explored the relationship of the number of nk cells and the expression level of their immunoglobulin-like receptor cd158b in the peripheral blood to the severity of sars (245) . the overall count of nk cells and cd158 + nk cells and the percentage of cd158 + nk cells in patients with sars were significantly lower than counts in healthy subjects (245) . a separate study that analyzed lymphocytes and lymphocyte subsets in a cohort of 38 patients with sars observed reduced nk cell counts in 21 patients (55%) (246) . clinical reports reveal that children appear to have a milder form of sars-cov-2, with peripheral blood lymphocyte levels remaining in the normal range, suggesting less immune dysfunction following the disease (247) . this could be attributed to healthy children expressing lymphocytes, especially nk cells, in a greater quantity compared to healthy adults (248) . interestingly, previous studies found rapid and significant restoration of lymphocyte subsets including, nk cells, in peripheral blood in patients recovering from the initial stages of sars infection (249) . although the primary mechanism for the decrease in nk cells and other subsets during disease onset remains unknown, their contribution to sars-cov-2 needs further study especially from a treatment perspective. innate lymphoid cells (ilcs) are a family of innate immune cells that include ilc1, ilc2 and ilc3. although ilc2 facilitates lung repair after injury, the role of ilcs during respiratory viral infection is not clearly defined (250) . evidence for the potential involvement of ilc2 cells in the lung during viral infection was reported in a murine model (251) . this study found a rapid accumulation of ilc2 cells in the lung after an influenza virus infection, however their initial contribution to exacerbation of the disease was limited (251) . a recent study identified an interaction between ace2-expressing sars-cov-2 target cells and ilcs in the colon (252) . thus, elucidating the role of ilc subsets will be important in understanding the pathogenesis of sars, sars-cov-2 and mers infections. there is distinct evidence indicating an important role of ifns in sars and other cov infections (201, 253) . the sera of patients with sars revealed the presence of high levels of il-1, il-6, infγ, ccl2, cxcl10, and il-8 and products of interferon stimulated genes (254, 255) . high expression levels of isgs such as cd58, ifnar1, and ifngr1 and ifn-stimulated chemokines cxcl10 and ccl2 were observed in another cohort of sars patients and were correlated with the severity of pathogenesis (256) . significant upregulation of cxcl10 gene expression was observed in the severe phase of patients who died from sars. this data is corroborated by studies in patients with mers that found upregulation of cxcl10 in the serum of patients who developed pneumonia (254) . cxcl10 and infα were also correlated with severity of disease (255) . the importance of ifn signaling in response to cov infection has been well-demonstrated in several knockout mouse models. type i, ii, and iii ifn signaling deficient mice have increased susceptibility to mouse-adapted sars-cov strains (257, 258) . studies using mice lacking the ifnar1 and ifnlr1 or stat1 identified higher sars-cov replication in the lungs and delayed virus clearance (259, 260) . another study with mers-cov in mice expressing the human dpp4 receptor showed a role for the ifnar1 in viral clearance and lung inflammation (112) . these mouse models suggest an important role of ifn response for cov clearance. this quick expanding medical literature is very suggestive of an important role of ifn responses for cov control and clearance. many viruses have evolved to disrupt and subvert the immune response. rsv counteracts the immune response (178, 191) ; as discussed earlier, the rsv genome encodes non-structural proteins (ns1 and ns2) that are able to block type 1 ifn production and signaling in cell cultures (191) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat1 and stat2 signaling in cell culture lines (192) . covs have developed several ways to escape from innate immune pressure. mers-cov m protein suppresses type 1 ifn by blocking the irf3 activation (193) , explaining the low expression of ifn-β. in various cell lines, sars-cov nucleocapsid (n) protein, membrane (m) protein, as well as nsp1, were reported to suppress ifn response (194, 196, 261) . the nucleocapsid protein (n) of sars-cov interferes with the function of irf3. although it does not form a complex with rig-i or mda5, rna binding activity at the initial recognition stage of viral rna potentially contributes to immune evasion (261, 262) . aside from the hcov, structural proteins, accessory, and non-structural proteins (nsp) are involved in innate immunity modulation. in both sars-cov and mers-cov, host mrna endonucleolytic cleavage is promoted by nsp1 protein, which modifies capped non-viral rnas (263, 264) . nsp1 in sars-cov prevents host mrna translation by interacting with the 40s subunit of the ribosome; in turn, transcription and translation of viral rna is given preference over the host mrna (263) . another study found that additional sars-cov nsp1 residues interfered with ifn-dependent signaling (265) . ifn production is affected by nsp3 proteins in sars-cov and mers-cov. these proteins have both papain-like protease (plpro) and a plp2 domain, and the plpro domains in both sars-cov and mers-cov downregulate mrna levels of ccl5, infβ, cxcl10, and other pro-inflammatory cytokines (266) . the suppression of ifn responses by sars-cov plpro is due to the inhibition of phosphorylation of ifn-regulatory factor 3 (irf3) and its subsequent translocation to the nucleus where it enhances ifn gene transcription (267) . mers-cov plpro also suppresses rig-i and mda5 and antagonizes ifn induction (266, 268) . in hcov-229e and sars-cov suppression of ifn responses, the key molecule is a adp-ribose-1-monophosphatase macrodomain encoded within nsp3 (269) . accessory proteins are not key in viral replication; however, in human cov, this group of proteins are involved in diverse cellular signaling, including cell proliferation, apoptosis, and ifn signaling (270) . by downregulating phosphorylation and nuclear translocation of irf3, open reading frame orf3b and -6 interfere with ifnβ synthesis and prevent ifnβ-induced activation of ifnstimulated response element (isre) in the promoter of isg in sars-cov (262) . in cells transfected with orf4a, -4b, and -5 of mers-cov, ifnβ promoter-driven luciferase activity is significantly reduced, and it may follow a similar pattern of suppression of irf3 nuclear translocation (141) . therefore, the suppression of signaling events in infected immune and airway epithelial cells, as well as the magnitude of suppression due to elevated expression levels of these accessory proteins, needs to be further elucidated to understand delayed or hyperimmune responses and cytokine storm that occurs in cov infection. in addition to revealing our unpreparedness of handling a worldwide pandemic by a viral infection, covid-19 exposed our lack of understanding of the pathogenesis of diseases as well as the host immunity. the interaction of the host innate immune system and other factors including age, sex, and pre-existing conditions need further investigation regarding disease severity and morbidity of sars/mers and covid-19. disease severity and its related progression are further associated with dysregulation of multiple components of both innate and adaptive immune responses leading to a cytokine storm and severe pathology. for the development of a therapeutic intervention, it is vital to elucidate the interplay among the different layers of the innate immune response and their relation to the clinical factors associated with increased morbidity and mortality in cov infection. investments in basic science research are needed to help elucidate the roles of different immune cells, and their contribution to disease severity; it will pave the way to prevent or abrogate cov outbreaks and potentially new viruses. nl, td, dh, zl, and nf performed the literature search, analyzed the literature, and wrote the manuscript. ha-h, bs, yw, and rs performed the literature search and wrote the manuscript. all authors contributed to the article and approved the submitted version. a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china korean society of epidemiology -nco vtft. an interim review of the epidemiological characteristics of 2019 novel coronavirus cross-country comparison of case fatality rates of covid-19/sars-cov-2. osong public health res perspect understanding of covid-19 based on current evidence current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019. (covid-19) angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structural basis for the recognition of sars-cov-2 by full-length human ace2 the mers-cov receptor dpp4 as a candidate binding target of the sars-cov-2 spike. iscience the sialic acid binding activity of human parainfluenza virus 3 and mumps virus glycoproteins enhances the adherence of group b streptococci to hep-2 cells cx3cr1 as a respiratory syncytial virus receptor in pediatric human lung rhinoviruses and their receptors multiple organ infection and the pathogenesis of sars a novel subset of putative stem/progenitor cd34+oct-4+ cells is the major target for sars coronavirus in human lung time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome-coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc anaesthetic and medico-legal problems in patients intoxicated by alcohol reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes investigating influenza a virus infection: tools to track infection and limit tropism parainfluenza virus infection respiratory syncytial virus can infect basal cells and alter human airway epithelial differentiation respiratory syncytial virus (rsv) infects cd4+ t cells: frequency of circulating cd4+ rsv+ t cells as a marker of disease severity in young children the emerging role of rhinoviruses in lower respiratory tract infections in children -clinical and molecular epidemiological study from croatia summary table of sars cases by country q&a: influenza and covid-19 -similarities and differences epidemiology of acute respiratory infections in children of developing countries 2019 case definition coronavirus as a possible cause of severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia association between age and clinical characteristics and outcomes of covid-19 distribution of influenza virus types by age using case-based global surveillance data from twenty-nine countries human parainfluenza virus 4 outbreak and the role of diagnostic tests age-specific incidence rates and risk factors for respiratory syncytial virusassociated lower respiratory tract illness in cohort children under 5 years old in the philippines. influenza other respir viruses respiratory syncytial virus infection in adults human rhinovirus c: age, season, and lower respiratory illness over the past 3 decades who. consensus document on the epidemiology of severe acute respiratory syndrome (sars) who. contract mers transmission and risk factors: a systematic review estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature transmission of respiratory syncytial virus among children under 5 years in households of rural communities, the philippines lethal respiratory disease associated with human rhinovirus c in wild chimpanzees investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia incubation period and other epidemiological characteristics of 2019. novel coronavirus infections with right truncation: a statistical analysis of publicly available case data incubation periods of acute respiratory viral infections: a systematic review human parainfluenza viruses (hpivs) available online at transmission dynamics and control of severe acute respiratory syndrome q&as on covid-19 and related health topics clinical features and short-term outcomes of 144 patients with sars in the greater toronto area acute renal impairment in coronavirus-associated severe acute respiratory syndrome prevalence of comorbidities in cases of middle east respiratory syndrome coronavirus: a retrospective study respiratory tract cell-mediated immunity clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis hospitalized patients with 2009. h1n1 influenza in the united states severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults respiratory syncytial virus infection in elderly and high-risk adults clinical characteristics and outcomes of human rhinovirus positivity in hospitalized children detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies middle east respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocytederived macrophages and dendritic cells macrophages: a trojan horse in covid-19? depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (piv5) p/v mutant productive infection of isolated human alveolar macrophages by respiratory syncytial virus alveolar macrophage-derived type i interferons orchestrate innate immunity to rsv through recruitment of antiviral monocytes rhinovirus replication in human macrophages induces nf-kappabdependent tumor necrosis factor alpha production sars-cov regulates immune function-related gene expression in human monocytic cells modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells dysregulation of immune response in patients with covid-19 in wuhan, china depressed human monocyte function after influenza infection in vitro infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3 human airway epithelial cells direct significant rhinovirus replication in monocytic cells by enhancing icam1 expression productive replication of middle east respiratory syndrome coronavirus in monocytederived dendritic cells modulates innate immune response heightened innate immune responses in the respiratory tract of covid-19 patients influenza a virus infection of human primary dendritic cells impairs their ability to cross-present antigen to cd8 t cells human parainfluenza virus type 2 vector induces dendritic cell maturation without viral rna replication/transcription innate immune components that regulate the pathogenesis and resolution of hrsv and hmpv infections human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and b7-h1 expression complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract innate immunity to influenza virus infection respiratory syncytial virus (rsv) and its propensity for causing bronchiolitis chemokines and inflammation in the nasal passages of infants with respiratory syncytial virus bronchiolitis elevated cytokine concentrations in the nasopharyngeal and tracheal secretions of children with respiratory syncytial virus disease haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways high levels of virus-specific cd4+ t cells predict severe pandemic influenza a virus infection infection and immunoregulation of t lymphocytes by parainfluenza virus type 3 protective and dysregulated t cell immunity in rsv infection rhinovirus has the unique ability to directly activate human t cells in vitro lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study middle east respiratory syndrome coronavirus (mers-cov): infection, immunological response, and vaccine development breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 a b cell population that dominates the primary response to influenza virus hemagglutinin does not participate in the memory response b-cell fate decisions following influenza virus infection respiratory syncytial virus infection in infants is associated with predominant th-2-like response innate immune signals modulate antiviral and polyreactive antibody responses during severe respiratory syncytial virus infection peripheral blood t and b lymphocyte subpopulations in infants with acute respiratory syncytial virus brochiolitis human rhinoviruses enter and induce proliferation of b lymphocytes neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes rapid generation of a mouse model for middle east respiratory syndrome persistence of antibodies against middle east respiratory syndrome coronavirus neutralizing antibodies against sars-cov-2 and other human coronaviruses protective antibodies against influenza proteins protective effect of antibody to parainfluenza type 1 virus respiratory syncytial virus neutralizing antibodies in cord blood, respiratory syncytial virus hospitalization, and recurrent wheeze the time course of the humoral immune response to rhinovirus infection dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome the relationship between serum interleukins and t-lymphocyte subsets in patients with severe acute respiratory syndrome active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis contribution of cytokines to tissue damage during human respiratory syncytial virus infection induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies cytokines induced during influenza virus infection human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium human rhinovirus induced cytokine/chemokine responses in human airway epithelial and immune cells the deadly coronaviruses: the 2003. sars pandemic and the 2020 novel coronavirus epidemic in china evaluation and treatment coronavirus (covid-19) cdc. influenza vaccines -united states rhinovirus vaccination: the case against review of the clinical characteristics of coronavirus disease 2019. (covid-19) available online at respiratory syncytial virus: diagnosis, treatment and prevention update on human rhinovirus and coronavirus infections coronavirus-associated pneumonia in previously healthy children clinical disease in children associated with newly described coronavirus subtypes identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area gender differences in patients with covid-19: focus on severity and mortality do men have a higher case fatality rate of severe acute respiratory syndrome than women do? the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis structure, function, and evolution of coronavirus spike proteins angiotensin-i-converting enzyme and its relatives receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters a novel coronavirus associated with severe acute respiratory syndrome dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv airway epithelial differentiation and mucociliary clearance respiratory epithelial cells orchestrate pulmonary innate immunity the role of toll-like receptors in the production of cytokines by human lung macrophages lung epithelial cells: therapeutically inducible effectors of antimicrobial defense pulmonary macrophages: key players in the innate defence of the airways the development and function of lungresident macrophages and dendritic cells contribution of dendritic cells in protective immunity against respiratory syncytial virus infection the contributions of lung macrophage and monocyte heterogeneity to influenza pathogenesis plasmacytoid dendritic cells: development, regulation, and function human dendritic cell subsets: an update the multifaceted biology of plasmacytoid dendritic cells role of dendritic cells in sars coronavirus infection plasmacytoid dendritic cells and type i ifn: 50 years of convergent history the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting macrophage polarization in virus-host interactions macrophages: sentinels and regulators of the immune system lung dendritic cells at the innate-adaptive immune interface lung interstitial macrophages: past, present, and future innate immune response to influenza virus at singlecell resolution in human epithelial cells revealed paracrine induction of interferon lambda 1 the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna innate immunity in the respiratory epithelium the airway epithelium: soldier in the fight against respiratory viruses lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses helix 1 tryptophan variants in galleria mellonella apolipophorin iii synergy of il-27 and tnfalpha in regulating cxcl10 expression in lung fibroblasts sars-coronavirus replication in human peripheral monocytes/macrophages middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis molecular mechanisms of cell death: central implication of atp synthase in mitochondrial permeability transition severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection exacerbated innate host response to sars-cov in aged non-human primates analysis on 54 mortality cases of coronavirus disease 2019 in the republic of korea from prevalence of sars-cov antibody in all hong kong patient contacts respiratory syncytial virus measles virus v protein blocks interferon (ifn)-alpha/beta but not ifn-gamma signaling by inhibiting stat1 and stat2 phosphorylation middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination neutrophil recruitment and function in health and inflammation host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells the role of c5a in acute lung injury induced by highly pathogenic viral infections characterization of cytokine/chemokine profiles of severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) spatiotemporal interplay of severe acute respiratory syndrome coronavirus and respiratory mucosal cells drives viral dissemination in rhesus macaques location or origin? what is critical for macrophage propagation of lung fibrosis? thinsection ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience pathogenesis of severe acute respiratory syndrome the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) directly decimates human spleens and lymph nodes. medrxiv chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome the pathogenicity of sars-cov-2 in hace2 transgenic mice pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2 mannosebinding lectin in severe acute respiratory syndrome coronavirus infection complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases highly pathogenic coronavirus n protein aggravates lung injury by masp-2-mediated complement over-activation. medrxiv the case of complement activation in covid-19 multiorgan impact blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov neutrophils in viral infections: current concepts and caveats a role for neutrophils in viral respiratory disease. front immunol severe acute respiratory distress syndrome (sars): a critical care perspective a major outbreak of severe acute respiratory syndrome in hong kong neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019. novel coronavirus in the early stage mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice effect of heterophil depletion by 5-fluorouracil on infectious bronchitis virus infection in chickens neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology neutrophils promote mononuclear cell infiltration during viral-induced encephalitis middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? a dynamic variation of pulmonary ace2 is required to modulate neutrophilic inflammation in response to pseudomonas aeruginosa lung infection in mice neutrophil extracellular traps in immunity and disease neutrophil extracellular traps (nets) -formation and implications targeting potential drivers of covid-19: neutrophil extracellular traps the role of neutrophil extracellular traps in covid-19: only an hypothesis or a potential new field of research? autopsy findings and venous thromboembolism in patients with covid-19 coagulation abnormalities and thrombosis in patients with covid-19 mechanisms underlying neutrophil-mediated monocyte recruitment partners in crime: neutrophils and monocytes/macrophages in inflammation and disease aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines selective reduction of natural killer cells and t cells expressing inhibitory receptors for mhc class i in the livers of patients with hepatic malignancy expression of killer cell inhibitory receptors is restricted to true nk cell lymphomas and a subset of intestinal enteropathy-type t cell lymphomas with a cytotoxic phenotype modulation of expression of the mhc class i-binding natural killer cell receptors, and nk activity in relation to viral load in hiv-infected/aids patients hla-cw7 zygosity affects the size of a subset of cd158b+ natural killer cells the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome sars-cov-2 infection in children: transmission dynamics and clinical characteristics will children reveal their secret? the coronavirus dilemma significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus t cells and ilc2s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice single cell rna sequencing of 13 human tissues identify cell types and receptors of human coronaviruses how the sars coronavirus causes disease: host or organism? comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection mobile group ii introns of yeast mitochondrial dna are novel site-specific retroelements resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists sars coronavirus nsp1 protein induces templatedependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin identification of residues of sars-cov nsp1 that differentially affect inhibition of gene expression and antiviral signaling mers-cov papain-like protease has deisgylating and deubiquitinating activities regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the adp-ribose-1''-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus 229e mediate resistance to antiviral interferon responses accessory proteins of sars-cov and other coronaviruses the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 ahmed-hassan, sisson, shukla, wijewantha, funderburg, li, hayes, demberg and liyanage. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-275404-hv3y4x4g authors: zumla, alimuddin; hui, david s title: infection control and mers-cov in health-care workers date: 2014-05-20 journal: lancet doi: 10.1016/s0140-6736(14)60852-7 sha: doc_id: 275404 cord_uid: hv3y4x4g nan the recent exponential rise in the number of reported cases of middle east respiratory syndrome coronavirus (mers-cov) is of major global concern. the fifth meeting of the international health regulations emergency committee concerning mers-cov was convened on may 13, 2014, by who's director-general and concluded that, although the seriousness of the situation had increased, there was no evidence of sustained human-to-human transmission and that conditions for a public health emergency of international concern have not yet been met. 1 mers-cov was fi rst reported in september, 2012, when a novel β coronavirus was isolated from a saudi arabian patient in jeddah, who had died of severe (a third now exercise for at least 30 min on 5 days per week) would be expected to reduce blood pressure. 8 comparison with data from the usa and canada suggests that the uk has some way to go in terms of cardiovascular disease prevention, but this must be set against the distinctly lukewarm evidence for benefi t from treatment of people with stage 1 hypertension and no additional risk, on which uk guidance is based. 1, 9 furthermore, there were important methodological diff erences between countries, particularly in the canadian data in which multiple measurements of blood pressure will have led to improved results compared with those from england. 10 nevertheless, falaschetti and colleagues' data point to the potential for further improvements in both the detection and treatment of hypertension. people with hypertension and raised cardiovascular risk, albeit based on relatively small numbers in the study, might be undertreated. data from uk primary care suggests that many of these people will be elderly, who might go untreated because of lack of evidence (particularly for decreased targets and in the presence of comorbidities). 11 we have previously shown that patients can self-monitor blood pressure and self-titrate their own medication; these and other new interventions can help to build on the improvements of blood pressure control that have been achieved so far. 12 such improvements are worthwhile because treatment of blood pressure is cost saving. 1 overall, however, physicians in primary care-who provide most hypertension management in the uk-seem to have made substantial advances in their management of hypertension, perhaps encouraged by the uk's pay-forperformance policy within which hypertension care has received a large and sustained proportion of the funding on off er. 13 falaschetti and colleagues' study provides a welcome example of the combined eff ects of individual physicians and policy makers on a simple but important risk factor. after 50 years of treatment, it seems that the drugs are working. 4 3 the large number of mers-cov cases (229 cases) reported between april 11, 2014, and may 4, 2014, by saudi arabia were probably seasonal (related to the camel birthing season), reminiscent of the clusters of hospital cases that were previously confi rmed in a hospital in jordan in april, 2012, 4 which involved haemodialysis units within hospitals in al hasa in april and may, 2013. 5 sequencing of the mers-cov isolates from the jeddah outbreak has shown no substantial genetic changes. 1 the who emergency committee concluded that the increase in cases reported among health-care workers from hospitals in jeddah was amplifi ed due to overcrowding and inadequate infection control measures. 1, 3 acute viral respiratory tract infections, such as severe acute respiratory syndrome (sars) and mers, are predominantly spread by large respiratory droplets (≥10 μm in diameter) during coughing and sneezing, whereas contact with fomite (including hand contamination with subsequent self-inoculation) might be another potential route of transmission. 6, 7 the sars outbreak in 2003 provided good lessons for the evaluation of environmental infl uences on the aerosol transmission of communicable respiratory diseases and the importance of good infection control measures in the prevention of nosocomial infections. one intriguing aspect of the 2003 sars epidemic was the occurrence of super-spreading events, which accounted for 71·1% and 74·8% of sars cases in hong kong and singapore, respectively. 8 during the sars outbreak in 2003, sars-coronavirus (cov) was moderately transmissible, with 2·7 secondary infections for every index case. 8 however, infectivity was substantially increased when coupled with environmental factors: 138 patients, many of whom were health-care workers, were infected within 2 weeks as a result of exposure to one patient with community-acquired pneumonia who was admitted to a general medical ward. 9 this super-spreading event seemed to be related to overcrowding and poor ventilation in the dry air-conditioned hospital ward, together with some contribution by the use of a jet nebuliser for the index case. 10 evidence of airborne transmission of sars-cov was also supported by positive air samples of the virus obtained from a hospital room occupied by a patient with sars in toronto, canada. 11 on the basis of analysis of data in a case-control study that involved 124 medical wards in 26 hospitals in guangzhou, china, and hong kong, the risk factors for super-spreading events of sars-cov in the hospital setting were: close separation between beds of less than 1 m; performance of resuscitation; staff working while experiencing symptoms; and patients requiring oxygen or non-invasive ventilation therapy. 12 this study also showed that the availability of washing or changing facilities for health-care staff was a protective factor. 12 these fi ndings have important clinical implications in the prevention of nosocomial infections of mers-cov in health-care facilities in the middle east. a systematic review of fi ve case-control and fi ve retrospective cohort studies identifi ed tracheal intubation, tracheotomy, and manual ventilation before intubation as procedures associated with risk of transmission of sars-cov to health-care workers. 13 opportunistic airborne transmission might occur through fi ne particle aerosols as an effi cient means of propagation under special environmental conditions, such as with aerosol-generating procedures in a ward science photo library environment with poor ventilation and insuffi cient air changes. 7 the main infection prevention and control measures for managing acute viral respiratory tract infections are simple and well documented: droplet precaution (wearing a surgical mask within 1 m of the patient) and contact precaution (wearing gown and gloves on entering the room and removing them on leaving). 14 droplet precautions should be added to standard precautions for patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confi rmed cases of mers-cov infection. airborne precautions should be applied when performing aerosol-generating procedures. 3, 6, 7, 15 to reduce room contamination in the hospital setting, major health organisations have recommended the application of a minimum room ventilation rate of six air changes per hour in existing facility, whereas a higher ventilation rate of 12 air changes per hour is recommended for new or renovated construction, especially when caring for patients receiving mechanical ventilation and during aerosol-generating procedures. 16, 17 infection source and engineering control, including avoidance of aerosol generation with appropriate airborne precautions, and improvement of ventilation design in hospital wards warrant serious consideration for the prevention of nosocomial outbreaks. the mers-cov outbreak in jeddah, and the increasing number of health-care workers acquiring the infection as a result of poor infection control measures, remind us of the need to go back to the basics of infection control to help prevent mers-cov infection in health-care workers. az and dsh serve on the scientifi c advisory committee of the saudi ministry of health global centre for mass gathering medicine. we declare no competing interests who. who statement on the fifth meeting of the ihr emergency committee concerning mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-update hospital outbreak of middle east respiratory syndrome coronavirus severe acute respiratory syndrome vs the middle east respiratory syndrome infection prevention and control measures for acute respiratory infections in healthcare settings: an update transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions a major outbreak of severe acute respiratory syndrome in hong kong sars: experience at prince of wales hospital, hong kong detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) who guidelines on natural ventilation for infection control in health-care settings. geneva: world health organization guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) key: cord-265666-27ckjl7w authors: kang, hee sun; son, ye dong; chae, sun‐mi; corte, colleen title: working experiences of nurses during the middle east respiratory syndrome outbreak date: 2018-05-30 journal: int j nurs pract doi: 10.1111/ijn.12664 sha: doc_id: 265666 cord_uid: 27ckjl7w aims: to explore working experiences of nurses during middle east respiratory syndrome outbreak. background: since the first case of middle east respiratory syndrome was reported on may 20, 2015 in south korea, 186 people, including health care workers, were infected, and 36 died. design: a qualitative descriptive study. methods: seven focus groups and 3 individual in‐depth interviews were conducted from august to december 2015. content analysis was used. results: the following 4 major themes emerged: “experiencing burnout owing to the heavy workload,” “relying on personal protective equipment for safety,” “being busy with catching up with the new guidelines related to middle east respiratory syndrome,” and “caring for suspected or infected patients with caution.” participants experienced burnout because of the high volume of work and expressed safety concerns about being infected. unclear and frequently changing guidelines were 1 of the common causes of confusion. participants expressed that they need to be supported while caring for suspected or infected patients. conclusion: this study showed that creating a supportive and safe work environment is essential by ensuring adequate nurse staffing, supplying best‐quality personal protective equipment, and improving communication to provide the quality of care during infection outbreak. information on the new guidelines and job-related information via text messages using smartphones was helpful for the nurses. • creating a supportive work environment and providing adequate training for nurses is essential. the implications of this paper: • nurse managers and hospital administrators should establish strategies to prevent nurses from burnout and to ensure their safety during the outbreak of infectious diseases. • clear and consistent practice guidelines and effective communication methods among nurses should be developed. • increasing awareness of health care workers about infectious diseases to enhance emergency preparedness is essential. with mers on may 20, 2015, which was 9 days after his first visit (lee & ki, 2015; yang et al., 2015) . a mers outbreak in korea was caused by hospital-to-hospital transmission because patients were moved to other hospitals without appropriate quarantine (ki, 2015; kim et al., 2016) . it was exacerbated by overcrowding in the emergency room, delayed diagnosis, and lack of self-protection (balkhy, perl, & arabi, 2016; xia, zhang, xue, sun, & jin, 2015) . as more and more mers cases were reported, hospitals restricted the visitors and checked all visitors and employees for the presence of fever. additionally, for the temporary screening of mers-suspected cases, triage was set up to screen the infected or suspicious patients and to block the cross-transmission in and outside hospitals. furthermore, the government adopted the national safe hospital program to control mers infections within hospitals (korea centers for disease control and prevention, 2015 along with high risk of being infected, studies reported that health care personnel experienced occupational risks, distress, and the fear of contacting and transmitting the disease during epidemics of h1n1, severe acute respiratory syndrome (sars), and ebola virus (bukhari et al., 2016; chou et al., 2010; corley, hammond, & fraser, 2010; koh, hegney, & drury, 2012; speroni, seibert, & mallinson, 2015) . nurses also reported positive experiences of becoming more confident, mature, and broad-minded while caring for sars patients (liu & liehr, 2009) and positive feelings about their experience of caring for h1n1 patients (honey & wang, 2013) . however, few studies have been conducted on nurses' working experiences during the mers outbreak. the aim of the study was to explore the working experiences of nurses during the mers outbreak. data were collected using 7 focus group interviews and 3 individual in-depth interviews from august to december 2015 until the data were saturated. focus group questions were developed based on a literature review (chou et al., 2010; corley et al., 2010) . each focus group was comprised of 2 to 5 participants. individual in-depth interviews were conducted for those who were not able to meet in focus groups because of time conflicts. prior to the interview, participants were informed about the reasons for doing this study and the goals of the study. the first author (hsk), who has experience with qualitative research, conducted the focus groups and the individual in-depth interviews. the focus group discussions and individual interviews were conducted in a private room at a site with convenient participant access. each session lasted for 1 to 2 hours. no one was present besides the participants and the researchers during the interviews. a semistructured interview guide was used. we conducted a pilot test with 2 nurses caring for the patients with mers and refined the interview questions. the following questions guided the interviews: • what are your working experiences of caring for suspected or infected patients with mers during the outbreak? • what are the challenges of working during the mers outbreak? to ensure consistency and accuracy of our data, interviews were audio-taped with the participants' permission and transcribed verbatim. the researchers made field notes during and right after the • it's so sweaty and hard to breathe with it. it is hard to work and see clearly while wearing it (protective measures) and i feel dizzy when wearing it for long hours. • (we were) sweating, (find it) hard to breathe; it was difficult to work wearing personal protective equipment. being busy with catching up with the new guidelines for mers frequently changing guidelines • mers guidelines kept changing. at first, (we were told to) do thing this way and this is the guideline. we needed time to understand and practice a new guideline; however, guidelines kept changing without considering our adjustment to a new one. • the most difficult thing was that protocols were changed daily. working along with memorizing new protocols was very difficult. while workload increased, (we) were told this has been changed this way and that has been changed that way in shift change meetings. sharing the new information • we promptly communicated and shared updated information among nurses within the unit, through kakao talk (a free mobile instant messaging application for smartphones with free texting). • we had a notice note summarized about new information on mers. when changing shifts, we read the note and were also told what we have to be cautious because of what has been changed and it helped. it helped because we never had mers before and didn't know how we have to send the specimens and did not know how to cope with it. it worked as basic guidelines. lack of support • why do you have to do it, and what if you are infected? why? why does it have to be you? • when the patients' condition was bad and when we were having a hard time, no one showed appreciation of our hard work. identifying the best way to care for patients • after spending many days in the isolated room, we started using a messenger. we supported each other and shared information. it was very helpful for me to ask my colleagues when i was unsure about patient care. • we made a package for mers patients, a package for mers. at first, we brought water bottles to patients because they cannot come out a negative pressure room and we complained regarding this matter. next thing, we agreed to make a package for the patients in the isolation room. when a patient comes, we give this package that has water, sleeper, and disposable products that patients need. (continues) interviews to help understand the interviews. there were no repeat interviews carried out. the study was approved by the institutional review board (1041078-201506-hrsb-099-01). all participants were informed about the purpose of this study and participants' right to withdraw from the study at any time, without penalty. confidentiality of participants was ensured, and written informed consent was obtained from each participant. the responses from the participants were analysed, using qualitative content analysis (krueger & casey, 2009 ). data collection was conducted concurrently with data analysis and continued until no new information emerged from the responses. the researchers read each verbatim transcript several times to obtain an overall understanding of the content and to gain a sense of the whole. the meaning units (words, sentence, and paragraphs) in the interviews related to nurses' work experiences were identified and coded. the codes were sorted into similar things together and grouped into categories based on similarities and differences. after assessing themes across groups, overarching themes were derived. two of the investigators independently coded each transcript. when discrepancies in coding occurred, the investigators discussed and resolved them by consensus. trustworthiness of the study was maintained following criteria by lincoln and guba (1985) . the study participants included 25 female and 2 male nurses. their mean age was 29.5 (4.7) years, ranging from 20 to 24 years, and their work experience ranged from 3 months to 17 years. the following 4 major themes emerged: "experiencing burnout owing to the heavy workload," "relying on personal protective equip participants reported discomfort in wearing ppe all day on duty. the amount of time of wearing ppe varied according to their work and the severity of the patients' condition. participants said that they preferred a mask that led to less breathing difficulties. one participant said, "i prefer the mask made by a company because it has a space that helps me to breathe easily. many nurses prefer to use it." the patterns of staying in the isolation room wearing a papr differed. nurses from one hospital stated that they stayed in the isolation room for a maximum of 2 hours while wearing their papr and then came out; they stayed in the anteroom (a room in front of the negative pressure isolation room) and went back into the isolation room when needed. contrary to this, nurses from another hospital stayed in the isolation room for their entire shift, except for the lunch hour. meanwhile, nurses who wore a papr said that they felt like wearing a space suit. they had a backache from wearing heavy equipment. "i had put the battery of the papr on a side participants communicated with others by writing on paper. they reported that it was easy to hear the intercom sound in the room, but it was difficult to talk back because the head shield of the papr blocked out sounds. permission to use computers or smartphones in the isolation room varied across hospitals. in one hospital, nurses working in an isolated intensive unit communicated with other nurses in another isolated room using a smartphone messenger application. they said it helped them to know how others in isolation rooms were doing and to ask them when they were unsure about patient care. participants said that they gradually returned to normal life. the hospitals rewarded working with mers patients differently. these included participating in healing programs, receiving financial incentives, eating out in teams, or receiving several days off for resting. a participant said "i enjoyed participating in the healing camp. the post-trauma prevention education was also helpful. i was relieved to hear that we could seek psychiatric counselling if necessary." after completing their volunteered job with the mers patients, participants stated that they had learned on site while caring for infected patients and that it was very rewarding and worthwhile. they expressed that they felt being matured and gained a lot of confidence from these experiences. participants also said that when they returned to their work unit, they often heard "you did a good job" from their peers, and that "it felt supportive and healing." this study explored the nurses' work experience during the mers outbreak. our participants reported that their workload increased with time. this result indicates that, as part of emergency planning, nurse managers and hospital administrators should prepare for the extra workload during the emergency of an infection outbreak and to ensure quality of care. participants reported that restricting unauthorized access of visitors was one of the main issues. restricting visitors was one of the strategies used for controlling further outbreak during the norovirus outbreak (danial et al., 2016) . visiting hospitalized patients in a group is a part of the korean culture, as it is a way of expressing support and wishing for a quick recovery. rather than just restricting the visitors, it would be helpful to suggest alternative ways of expressing support for patients, such as sending a message through a phone or social networking service. participants expressed their concerns about the possibility of being infected. in fact, health care personnel who had close contact with mers patients were at a high risk for infection (alraddadi et al., 2016) . previous studies support our results. during the mers epidemic, health care workers felt fearful about being infected; however, they continued to work during the epidemic as it was their professional and ethical duty (al-dorzi et al., 2016; khalid, khalid, qabajah, barnard, & qushmaq, 2016) . emergency room nurses working during the outbreak of mers also expressed high concerns about being infected, and that they would have like to avoid caring for patients with mers if there was a choice (choi & kim, 2016) . these fears of nurses could be reduced by sharing the correct information about the quality of the protection devices they wear and appropriate ways to use them to prevent the transmission of infection (speroni et al., 2015) . in addition, hospitals experiencing mers epidemic suggested that institutional plans be made in advance to provide personal safety equipment when there is a rapid increase in its demand (al-dorzi et al., 2016; stirling, hatcher, & harmston, 2017) . however, our participants mentioned discomfort in wearing ppe. likewise, a study on a simulation exercise for health care workers wearing ppe in a hospital in the uk reported that they found the ppe uncomfortable, and even basic tasks took longer than usual while wearing it (phin et al., 2009) . thus, feedback from nurses on protection devices would help medical equipment companies design more comfortable medical protection equipment. our participants complained of having to continuously catch up with the frequently updated guidelines. likewise, it was reported that one of challenges for hospitals was the changing and conflicting guidelines and the overwhelming amount of information that required sifting through during the h1n1 influenza pandemic (rebmann, 2010 regarding going back to routines, our participants felt supported when they received positive responses from peers when they went back to their unit after taking care of patients at risk. additionally, hospitals implemented various programs for health care professionals to reward or appreciate their hard work during the outbreak. the common response of participants on these was very positive. in a study, nurses who took care of h1n1 high-risk infected patients and who worked in an isolated area in taiwan said that nurses needed counselling services (honey & wang, 2013) . after any outbreak, it may be important to offer a healing program for nurses, to help them share their experiences and feelings with others. a limitation of this study was that all participants were staff nurses. further research is needed to explore the mers experiences of patients, nurse managers, and other health care workers. because of variations in nurses' work schedule, the focus groups were quite small. the disadvantage of a small group is that it limits generating a rich diversity in views and the total range of experiences, although the advantage of smaller groups is that they are easier to recruit and allow everyone to have a greater opportunity to share experiences. another limitation was that this was a cross-sectional study. longitudinal studies are needed to examine the impact of any changes in hospital regulations or policies on nursing care. these study results suggest that nurse managers and administrative personnel should understand that overload of nurses' work during the outbreak may lead them burned out, which may negatively affect quality of care to patients. furthermore, establishing consistent and solid practice guidelines and efficiently disseminating them and training health care workers to deliver them could lead to less confusion during an infection outbreak. it is important to acknowledge nurses' work as valuable and to create a supportive environment in workplace of nurses. these efforts will empower nurses to work as an expert and will positively influence the quality of care. finally, it is essential to raise awareness about infection control among health care workers and people in general to strengthen emergency preparedness. the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel middle east respiratory syndrome coronavirus: current situation and travel-associated concerns preventing healthcareassociated transmission of the middle east respiratory syndrome (mers): our achilles heel middle east respiratory syndrome: a new global threat middle east respiratory syndrome coronavirus (mers-cov) outbreak perceptions of risk and stress evaluation in nurses factors influencing emergency nurses' ethical problems during the outbreak of mers-cov uniformed service nurses' experiences with the severe acute respiratory syndrome outbreak and response in taiwan the experiences of health care workers employed in an australian intensive care unit during the h1n1 influenza pandemic of 2009: a phenomenological study lessons learned from a prolonged and costly norovirus outbreak at a scottish medicine of the elderly hospital: case study new zealand nurses perceptions of caring for patients with influenza a (h1n1) healthcare workers emotions, perceived stressors and coping strategies during a mers-cov outbreak 2015 mers outbreak in korea: hospital-to-hospital transmission the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea nurses' perceptions of risk from emerging respiratory infectious diseases: a singapore study middle east respiratory syndrome coronavirus outbreak in the republic of korea focus groups: a practical guide for applied research strengthening epidemiologic investigation of infectious diseases in korea: lessons from the middle east respiratory syndrome outbreak naturalistic inquiry instructive messages from chinese nurses' stories of caring for sars patients personal protective equipment in an influenza pandemic: a uk simulation exercise pandemic preparedness: implementation of infection prevention emergency plans worry experienced during the 2015 middle east respiratory syndrome (mers) pandemic in korea nurses' perceptions on ebola care in the united states, part 2: a qualitative analysis communicating the changing role of a nurse in an epidemic: the example of the mers-cov outbreak in saudi arabia consolidated criteria for reporting qualitative research (coreq): a 32-item checklist for interviews and focus groups modeling the transmission of middle east respirator syndrome corona virus in the republic of korea middle east respiratory syndrome in 3 persons, south korea working experiences of nurses during the middle east respiratory syndrome outbreak none. the authors declare no conflict of interest. all listed authors meet the authorship criteria and all authors are in agreement with the content of the manuscript. sun-mi chae http://orcid.org/0000-0002-3010-2265 key: cord-277823-vijh6x1l authors: teramichi, takurou; fukushi, shuetsu; hachiya, yuma; melaku, simenew keskes; oguma, keisuke; sentsui, hiroshi title: evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of middle east respiratory syndrome coronavirus among livestock in ethiopia date: 2019-11-05 journal: j vet med sci doi: 10.1292/jvms.19-0436 sha: doc_id: 277823 cord_uid: vijh6x1l a serological survey of middle east respiratory syndrome coronavirus (mers-cov) was conducted among dromedary camels and herbivorous animals sharing the same pasturage in ethiopia. the pseudotyped vesicular stomatitis virus coated with the spike protein of mers-cov was used in virus neutralization (vn) tests performed in a biosafety level (bsl)-2 laboratory. the results were similar to those obtained from the vn test using live mers-cov and were more sensitive than the elisa performed using synthetic mers s1 fragment as the antigen as well as the competitive elisa performed using a monoclonal antibody against mers-cov. according to the comprehensive results of the four types of serodiagnosis methods, positive antibodies were detected only in dromedary camels and the remaining herbivorous animals were not infected with the virus. moreover, using the present procedure, serological tests for mers-cov can be conducted even in bsl 2 laboratory. pseudovirus vsv-mers/gfp, a recombinant vesicular stomatitis virus pseudotype, integrated with the gfp gene and coated with the spike protein of mers-cov was used in the neutralization test [8] . the infectivity of vsv-mers/gfp was detected based on gfp expression observed using fluorescence microscopy. gfp-expressing cells in photographic images were counted using vh analyzer. the vsv-mers/gfp titers were then expressed as focus forming units (ffu) per ml. cell culture 293t and vero cells were cultivated in dulbecco's modified eagle's medium supplemented with 5% fetal bovine serum (fbs), 100 u/ml of penicillin, and 100 µg/ml of streptomycin at 37°c [8] . 293t cells were used to prepare vsv-mers/gfp and mers-cov recombinant receptor binding domain (rbd) for the celisa antigen. vero cells were used for neutralization tests by live mers-cov and vsv-mers/gfp, respectively. serum samples were collected from dromedary camels (n=38; mean age, 4.3 years; range, 1-13 years), goats (n=25; mean age, 3.9 years; range, 1-8 years), sheep (n=25; mean age, 2.7 years; range, 1-4 years), and cattle (n=15; mean age, 6.7 years; range, 1-11 years) from two herds in bati district, amhara region, and one herd in fafen district, somali region, ethiopia. all animals appeared healthy, shared the same pasturage during the day, and stayed in barns or small grounds specific for each animal species surrounded by fences at night. transportation of the serum samples to japan was conducted with the permission of the japanese government (animal quarantine inspection number nfib070602-011) and followed the rules and regulations of the oie/fao for biological sample transportation. serum from a rabbit immunized with recombinant mers-cov s protein was used as a positive control for neutralization [8] . sera from animals (5 cattle, 5 sheep and 5 goats) reared on the attached farm of nihon university were used as negative controls. the neutralization test using vsv-mers/gfp was performed as previously described [8] . a medium composed of eagle's mem supplemented with 5% fbs was used for virus and serum dilution. serially diluted 0.05 ml of test sera were mixed with equal volumes of 3,000 ffu of vsv-mers/gfp and incubated at 37°c for 1 hr. the mixture was inoculated to vero cells seeded on a 96-well culture plate and incubated at 37°c for 24 hr in a co 2 incubator. gfp-positive cells were then detected using a fluorescence microscope. the number of positive gfp cells was counted as described above. the neutralization titer was determined as the highest serum dilution showing ≤50% of the number of gfp-positive cells compared with the no serum control. neutralization test using live mers-cov was performed as previously described except using vero cells instead of vero-tmprss2 cells [16] . briefly, 0.05 ml of serially diluted test sera was mixed with an equal volume of 100 tcid 50 of mers-cov (emc isolate) in a 96-well culture plate and incubated at 37°c for 1 hr; thereafter,vero cells were added in each well and cultivated at 37°c. cytopathic effects (cpe) on the vero cells were observed at 3 days after infection. the neutralization titer was determined as the highest serum dilution showing at least 50% cpe on the inoculated cells. synthetic s1 fragment of mers-cov was obtained from sino biolobical inc. (beijing, china) and used as the antigen [17] . elisa microplates were coated with 50 µl of 50 ng/ml antigen per well at 4°c overnight, following which the wells were incubated with pbs containing 2% block ace and 0.05% tween 20 for 2 hr at 37°c. following the removal of blocking reagent, diluted serum samples were added and incubated for 1 hr at 37°c. after washing the wells thrice with 0.05% tween 20 in pbs (pbs-t), a peroxidase-labeled protein ag (thermo fisher scientific, waltham, ma, u.s.a.) was added and incubated for 1 hr at 37°c. following further washing thrice with pbs-t, 100 µl of 2,2′-azinobis-3-ethylbenzthiazolinesulfonic acid (abts) substrate solution (roche applied science, penzberg, germany) was added and incubated for 20 min at room temperature. the optical density (o.d.) of each well was measured at 450 nm using a microplate reader, and mean absorbance was determined for each serum sample. one of camel serum that showed a high antibody titer in the neutralization test by live mers-cov was treated as a positive control. the mers-cov rbd was used as the antigen of the celisa. for the preparation of recombinant rbd, the mammalian expression plasmid pcaggs-rbd, which encodes histidine-tagged mers-cov rbd (amino acid 358-588), was transfected to 293t cells. at 2 days after transfection, the recombinant rbd was purified from the supernatant of transfected cells using his-bind purification kit (merck, damastadt, germany). the celisa was performed as described by fukushi et al. [9] . briefly, mers-cov recombinant rbd with pre-determined optimal quantity was coated on a 96-well elisa plate at 4°c for overnight, following which the wells were incubated with pbs containing 2% bovine serum albumin and 0.05% tween 20 (blocking reagent) for 2 hr at 37°c. following the removal of the blocking reagent, 100 µl of a biotin-labeled monoclonal antibody mixed with serially diluted serum samples was added and incubated for 1 hr at 37°c. one of camel serum that showed a high antibody titer in according to the results of the previous study, antibody titers of ≥16 are treated as positive in neutralization test using vsv-mers/gfp. in dromedary camels and cattle, 31 out of 38 and 1 out of 15, respectively, were mers antibody positive. goats and sheep were all mers antibody negative (table l) . moreover, in 15 camels, the antibody titer was 16 or 32; in 7, it was 64 or 128; and in 9 and control positive rabbit serum, it was ≥256. the antibody titer and the positive rate of antibody against mers-cov increased with the age of the camels. the antibody titer in one cow was 64. all sera collected from animals on the attached farm of nihon university were negative. overall, 31 camels and control positive rabbit serum were found to be antibody positive in the neutralization test using mers-cov, and 16 camels were positive in s1-elisa. in the celisa, 26 camels and 1 cow were positive ( table 2 ). the sera collected as negative controls from animals on the attached farm of nihon university were all negative in s1-elisa and celisa. each camel that was antibody positive in s1-elisa or in neutralization tests using mers-cov was found to be positive in neutralization tests using vsv-mers/gfp. cows that were antibody positive in the neutralization test using vsv-mers/gfp or celisa were different animals and both were antibody negative in the neutralization test using mers-cov. a comparison of the antibody titers detected in the neutralization tests using vsv-mers/gfp with those detected using live mers-cov showed a high correlation between both antibody titers, with a correlation coefficient of 0.9753. all camel sera that were positive in any one of the tests-the s1-elisa, celisa, or neutralization tests using live mers-cov-were positive in the neutralization test using vsv-mers/gfp. one cow serum was positive for celisa and another cow was positive in the neutralization test using vsv-mers/gfp, but negative in all other tests. because the neutralization test using live mers-cov is considered the most accurate serological test and because the two positive reactions were observed in only one serological test among the four, these two bovine sera should be considered as nonspecific reactors. the exact reason of nonspecific reaction of these sera remains unclear. the nonspecific reaction may result from differences in immunological conditions of animals or in the degree of hemolysis during the preparation of samples. for example, a nonspecific reaction sometimes appears in cattle after injection with a certain inactivated vaccine in the elisa kit for johne's disease diagnosis in japan. some rhabdovirus cross react with vsv [6] and such a virus might be subclinically infected in ethiopian cattle. there are reports that mers-cov specific antibodies were not detected in sera from cattle, goat and sheep in jordan and saudi arabia, the mers-cov prevalence region [11, 13] . however, they were kept indoor or did not share the pastureland. present results indicate that mers-cov infects only dromedary camels and is unlikely to infect other domestic animals even sharing the pastureland. since the antibody positive rate and antibody titer of mers increased with age, it is considered that mers-cov establishes an infection cycle and inapparently present only among dromedary camels [11] . in present studies, the limited numbers of samples were tested because the import of animal sera from the foot-and-mouth disease endemic region is regulated in japan. further studies using additional samples from other countries would be required to clarify the role of other animal on the ecology and epidemiology of mers-cov. a comparison of the two neutralization tests using vsv-mers/gfp and mers-cov showed a high correlation between both antibody titers. the former showed an antibody titer of ≥256, whereas the latter showed an antibody titer of ≥512. s1-elisa was not sensitive compared to other tests because only 16 serum samples were positive and they required an antibody titer of ≥64 in vsv-mers/gfp. however, the sensitivity of s1-elisa would be improved by examining the cut-off value and dilution of the test serum. in celisa, five serum samples were negative out of the 31 positive samples in the neutralization tests, and an antibody titer of 16 was observed in both neutralization tests. therefore, neutralization test using vsv-mers/gfp exhibits sensitivity similar to the neutralization test using mers-cov and is more sensitive compared with the s1-elisa as well as celisa. the present study shows that the neutralization test using vsv-mers/gfp, s1-elisa, and celisa are as specific to mers-cov infection as the serological tests, although their sensitivities slightly differ. the detection of antibody and epidemiological survey of mers-cov could be possible by appropriately selecting these tests according to the desired purpose, without using a bsl-3 laboratory. middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates ksa mers-cov investigation team 2013. hospital outbreak of middle east respiratory syndrome coronavirus evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group cross-neutralization between vesicular stomatitis virus type indiana and chandipura virus transmission of mers-coronavirus in household contacts inability of rat dpp4 to allow mers-cov infection revealed by using a vsv pseudotype bearing truncated mers-cov spike protein characterization of novel monoclonal antibodies against the mers-coronavirus spike protein and their application in species-independent antibody detection by competitive elisa middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus infection not found in camels in japan characterization of anti-mers-cov antibodies against various recombinant structural antigens of mers-cov in an imported case in china acute middle east respiratory syndrome coronavirus infection in livestock dromedaries isolation of a novel coronavirus from a man with pneumonia in saudi arabia acknowledgment. this work was partly supported by jsps kakenhi (grant number 17h04642), japan agency for medical research and development (grant number jp18fk0108058) and the academic frontier project for private universities (s1491007) from the ministry of education, culture, sports, science and technology of japan. key: cord-272622-2wceu3o9 authors: park, mi hye; kim, hee ryun; choi, duck hwan; sung, ji hee; kim, jong hwa title: emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report date: 2016-06-01 journal: korean j anesthesiol doi: 10.4097/kjae.2016.69.3.287 sha: doc_id: 272622 cord_uid: 2wceu3o9 only a few reports have been published on women with an infectious respiratory viral pathogen, such as middle east respiratory syndrome (mers) coronavirus delivering a baby. a laboratory confirmed case of mers was reported during a mers outbreak in the republic of korea in a woman at gestational week 35 + 4. she recovered, and delivered a healthy baby by emergency cesarean section (c-sec). we present the clinical course and the emergency c-sec in a pregnant woman with mers. surgery on an infectious patient tional-related diabetes, but it was well-controlled by diet without medication. she was admitted to obstetrics ward of our institution on april 19 due to premature labor. during the 6-week admission period, she visited her mother who was hospitalized in the emergency department of the same institution on may 27 and 28, which was the same time that a known mers-cov super-spreader patient was treated there. her mother developed illness with fever on day 7 after contacting the super-spreader and was confirmed positive by mers-cov rt-pcr. our case complained of severe myalgia without fever on june 8 and was admitted to the isolation ward 5 days after discharge. her sputum sample was positive for mers-cov by rt-pcr. creactive protein (crp) was elevated to 1.95 mg/dl and a chest x-ray revealed patchy opacity in both lower lobes, suggesting bronchopneumonia ( fig. 1) . a chest x-ray taken 4 days later was improved and crp had decreased to a value within the normal range (0.15 mg/dl). however, she complained persistently of episodes of dyspnea, sputum, and myalgia without fever. she was provided supportive treatment, but no antiviral agent or steroid because the disease was not progressing. fetal growth was within normal gestational limits, and she recovered gradually from the myalgia and other symptoms. the results of rt-pcr follow-up tests performed on june 19 and 21 were all negative in sputum samples. the division of infectious diseases confirmed a full recovery, and she moved to the obstetrics ward on june 22 where she was expected to have a normal delivery. however, she began sudden bleeding with abdominal pain on the day after recovery was declared. a pelvic examination with a speculum confirmed the vaginal bleeding. however, the transabdominal ultrasonography did not detect any placental problems or hid-den hemorrhaging. baseline fetal heart rate (fhr) was 120-130 bpm with moderate variability. an obstetrician diagnosed placental abruption based on the symptoms and physical examinations. we considered spread of mers-cov because her symptom onset was very recent, and delivery would involve a large volume of contagious body fluids. an emergency c-sec under combined spinal-epidural anesthesia (cse) was scheduled with precautions. the surgery was performed in a designated negative pressure-ventilated isolation operating room. all designated specialized infection control personnel prepared for the surgery and wore enhanced personal protective equipment (ppe) (fig. 2) . a medical worker with ppe transferred the patient on an exclusive elevator. she was very anxious in the operating room, so we provided emotional support but no premedication. she was 167 cm tall and weighed 79 kg (body mass index, 28.33 kg/m 2 ). her baseline blood pressure and heart rate were 120/50 mmhg and 82 beats/min, respectively. spo 2 was 100% on room air and electrocardiograph results showed normal sinus rhythm. the patient was placed in the right lateral decubitus position, and epidural anesthesia was attempted with a 17-gauge tuohy needle via the median approach at l3-4. the epidural space was detected using the loss-of-resistance technique, and a 22-gauge epidural catheter was inserted (fig. 3) . a lumbar puncture was performed with a 25-gauge whitacre spinal needle at the l4-5 interspace, and 6 mg of 0.2% ropivacaine and 20 μg fentanyl were injected intrathecally. levobupivacaine (0.25%, 10 ml) was administered through the epidural catheter. the patient was placed in the supine position with a left lateral tilt to avoid aortocaval compression. anesthesia was assessed bilaterally using cold and light touch, and the spinal injection produced a t4 block after 10 min. the c-sec was performed uneventfully without pain or discomfort. the baby was 3.14 kg, with apgar scores at 1 and 5 min of 9 and 10. umbilical vein ph was 7.363. a 10 × 10 cm blood clot was extracted and approximately 20% of the placenta appeared to be abrupted. total operation time was 150 min, with an estimated blood loss of 700 ml. a total of 2700 ml of crystalloid solution and 1000 ml of colloid were administered during the surgery. an infusion of oxytocin (10 units/1000 ml ringer's lactate solution) and carbetocin (1000 μg/10 ml normal saline) was administered to produce uterine contractions and minimize blood loss, respectively. she was administered in the operating room until a recovery block to t8, then the patient was transferred directly to a single room on the obstetrics ward. the baby was isolated in the intensive care unit, and a mers-cov test was negative. maternal serum and the placenta were mers-cov negative. breastfeeding was allowed by pumping instead of sucking. she was discharged on postoperative day 7. the mother and baby are doing well. we performed an emergency c-sec in a patient with a highly infectious respiratory viral pathogenic disease. sudden placental abruption occurred at midnight, and an immediate c-sec was recommended [5] . our multidisciplinary team had discussed the delivery and had developed emergency scenarios; thus, the c-sec progressed without delay under the regional anesthesia (ra) for several reasons. she was not actively bleeding, and only moderate variability in fhr was detected without deceleration, so we had ample time to perform the cse. although the decision-to-deliver interval for general anesthesia (ga) is faster than that for ra [6] , ra is preferred to ga because it is safer and causes less maternal and neonatal morbidity than those of ga [6, 7] . the other consideration in this case was spread of the infection. although her chest x-ray and laboratory results improved, the patient had pneumonia within 2 weeks. we were not convinced that the lung parenchyma had fully recovered, and tracheal intubation/extubation in a patient with mers could expose the medical staff to a high aerosolized viral load. thus, the c-sec was performed by an experienced senior anesthesiologist. potentially high-risk procedures should be performed by experienced medical staff during an infectious disease outbreak period [8] . mers is thought to be transmitted mainly via respiratory droplets and close human contact, but the mode is not completely defined. although we already had some infection-control protocols applicable for the operating room for known infectious diseases, such as pulmonary tuberculosis, viral hepatitis, human immune deficiency virus, and antibiotics-resistance bacterial infection before the mers outbreak, we developed a new operating room protocol for patients with confirmed or suspected mers because of the high infectivity and unknown transmission mode. the patient's sputum was mers-cov negative based on rt-pcr before delivery, so it is unclear if strict precautions were necessary [9] . however, the patient had been declared recovered for only 1 day, and illness onset was within 2 weeks. in addition, a c-sec frequently involves large volumes of blood, amniotic fluid, and other body fluids, and these could be infection sources. no other case reports are available on delivering a baby for a women with mers, but several cases of labor and delivery in women with severe acute respiratory syndrome (sars) have been published [10, 11] . viral shedding from maternal body secretions, stool, cerebrospinal fluid, and peritoneal fluid has been reported in sars cases, suggesting that mers-cov could be present in any body fluid and transmitted during c-sec, which is why we treated this patient for confirmed mers perioperatively. we followed our institution protocol for patients with mers and referred to the sars obstetrics guidelines. according to the management guidelines for obstetric patients with sars [12] , all deliveries should be managed in a designated negative-pressure isolation room and by designated personnel with specialized infection control ppe. as we have no permanent negative pressure operating rooms at our institution, two operating rooms were converted to negative-pressure [13] . these rooms were connected, but had separate air-conditioning and humidification units with individual atmospheric air inlet and exhaust systems. we used one as the surgery on an infectious patient operating theater and the other as a ppe changing room. the air pressure in these rooms was maintained at −4.7 pa (operating theater) and −1.2 pa (changing room). an airflow visualization (smoke) test was carried out to ensure negative pressure. modifications were made to minimize outflow of contaminated air from the mers-related operating rooms. airflow in the operating room was > 14 air exchanges per hour. all anesthetic and surgical team staff used enhanced ppe comprised of a n95 mask, surgical cap, double gowns, double gloves, shoe covers, and powered air-purifying respirators. after the patient recovered from anesthesia in the operating room, the patient was transferred back to the ward by the same dedicated anesthesiologist who changed into new ppe to minimize mers-cov exposure. an n95 mask was fit on the patient during the transfer and roberge et al. [14] reported that use of an n95 mask by a healthy women during late pregnancy did not change any physiological or subjective findings compared with those of healthy, non-pregnant women during sedentary activities and exercise for 1 hour. if a pregnant woman is infectious, they may be able to use an n95 mask without complications. finally, the mersdesignated spaces were all ventilated for 100 min and thoroughly decontaminated after the case. all medical staff involved in the delivery remained healthy. placental abruption occurred in our case, which is one of most commonly encountered uteroplacental bleeding disorders with high rates of perinatal-maternal morbidity and mortality. several reports have indicated that maternal acute and chronic respiratory diseases during pregnancy are associated with placental abruption [15] . acute and chronic respiratory conditions may trigger an inflammatory and hypoxic process at the mater-nal-fetal interface that may lead to placental abruption. however, placental abruption occurs in only about 1% of pregnancies [15] . only two case reports are available on pregnant women with mers who delivered a baby. a woman in abu dhabi died after giving birth to a baby by c-sec, and the other case was associated with a stillbirth in jordan [4] . thus, we were not convinced that the placental abruption was related with mers. our patient was complicated by a history of premature labor, gestational diabetes, and advanced maternal age. however, the pregnancy had a favorable outcome for the mother and baby. the reasons for the good outcome are unclear but may be due to the late stage of gestation and earlier detection of mers. long-term follow-ups of infants and mothers are needed to fully define the pregnancyrelated risks of mers infection. most human infections result from human-to-human spread, and when particular medical procedures are combined with poor infection control the virus can disseminate within the hospital. thus, protocols and education for medical workers are essential to minimize exposure and protect against transmitting the pathogen during an infectious disease outbreak. surgeries, particularly those performed in an emergency situation, have a high risk for spreading a contagious pathogen. thus, a multidisciplinary team of emergency personnel should be prepared, including surgeons, physicians, nurses, and anesthesiologists. early and strict infection control strategies and clinical guidelines for infectious patients, such as those with mers, should be established. mi hye park, http://orcid.org/0000-0002-3760-1067 contact investigation of a case of human novel coronavirus infection treated in a german hospital middle east respiratory syndrome coronavirus in healthcare settings a balancing act: mechanisms by which the fetus avoids rejection by the maternal immune system stillbirth during infection with middle east respiratory syndrome coronavirus abruptio placentae. diagnosis, management and maternal-fetal prognosis: a retrospective study of 100 cases mode of anaesthetic for category 1 caesarean sections and neonatal outcomes anesthetic practice for caesarean section and factors influencing anesthesiologists' choice of anesthesia: a population-based study infection control measures for operative procedures in severe acute respiratory syndromerelated patients lack of transmission among close contacts of patient with case of middle east respiratory syndrome imported into the united states severe acute respiratory syndrome in pregnancy infants born to mothers with severe acute respiratory syndrome management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) conversion of operating theatre from positive to negative pressure environment n95 respirator use during advanced pregnancy acute and chronic respiratory diseases in pregnancy: associations with placental abruption park et al. key: cord-277337-ij0dn77h authors: cho, hae-wol; chu, chaeshin title: outbreak of middle east respiratory syndrome in korea? date: 2015-08-28 journal: osong public health res perspect doi: 10.1016/j.phrp.2015.08.005 sha: doc_id: 277337 cord_uid: ij0dn77h nan it was a total surprise to everybody! since late may 2015, we have observed an imported pathogen make a huge impact on the general public and economy of the republic of korea, which has one the most advanced medical and public health systems in the world. nobody expected korea to be in second place on the list of countries with the highest incidence of middle east respiratory syndrome (mers), right after saudi arabia. the korea centers for disease control and prevention (kcdc) has been at the center of this unprecedented event. we have seen the incredible commitment of the members of the division of epidemic intelligence service in the kcdc, and of the mers outbreak investigation team. this editorial is a record of what the virus fighters have experienced in the past 2 months. before we review the whole picture, we would like to give our sincere homage to their professional attitude and heroic sacrifices. we would also like to acknowledge the efforts of medical staff in treating infected patients and of public health officials in tracing contacts. the mers-coronavirus (cov) outbreak in korea started with a 68-year-old male case who visited bahrain, saudi arabia, and the united arab emirates from april 24 to may 4, 2015. the symptom onset date of this index case was may 11, and this patient visited two small clinics and two hospitals: pyeongtaek st. mary's hospital in pyeongtaek, samsung medical center (smc) in seoul. whether he also visited saudi arabia from may 1 to may 2 was not known until after laboratory confirmation results were released to smc on may 20. he was immediately transferred to the national medical center for isolation. since august 19, 186 cases, including 1 case exported to china (patient 10), were laboratory-confirmed. of those cases, 140 (75.3%) have recovered, 36 have died (19.4%), and 10, including 3 serious cases, are still being treated. eighty-two (44.1%) were patients in 16 hospitals, 111 (59.7%) were caregivers, and 25 (13.4%) were healthcare workers. the average incubation period was estimated to be 6.83 days [1] . the clinical presentation of those laboratoryconfirmed patients has ranged from mild illness to severe disease and death. their symptoms include fever or chills (74.2%); myalgia (25.3%); cough (17.7%); nausea, vomiting, diarrhea, or gastric discomfort (12.9%); headache (8.6%); sputum (7.5%); and sore throat (4.3%). pneumonia was initially seen in 18.2% of the cases. overall, 59.7% (n z 111) were male, and the median age was 55 years (range, 16e87). fifteen of the 36 death cases had underlying diseases, such as chronic pulmonary diseases, cancer, and cardiovascular diseases. of the 36 death cases, 80.6% were aged >60 years old [1] . the first epi curve ( figure 1 ) shows the progression of the outbreak based on the laboratory-confirmed cases up to june 15, 2015. the horizontal axis represents the date when a person showed symptoms after exposure. the vertical axis is the number of persons who became ill on each date. the colors represent the hospitals, index cases, and deaths with case number. the index cases in red are the four identified clusters in four hospitals. blue, yellow, and green indicate the number of patients from pyeongtaek st. mary's hospital, smc, and gun-yang hospital, respectively. gray squares indicate death cases [1, 2] (see figure 2 ). as of june 16, 2015, 75 smc-related cases have been confirmed. patient 14, who visited the smc emergency department on may 27e29, played a role in exposing many people to mers-cov, however, spread related to this patient is being controlled. among the smc-related cases, 69 have been hospitalized, and 6 are dead. of the 4,075 close or casual contacts related to smc, 558 are hospital staff members, and 2,238 are patients. one hundred and forty-six close contacts are quarantined at the hospital, 1,078 are under home quarantine, 1,570 casual contacts are under active monitoring, and 1,237 have been released from quarantine or monitoring [2] . smc has enhanced the personal hygiene and education of its healthcare workers and the daily cleaning of rooms with confirmed cases. thorough cleaning was conducted following the detection of case 14. the patients and healthcare workers' risks of exposure were grouped by the location and duration of exposure using electronic medical records, telephone interviews, and closedcircuit television (cctv). group-specific risk was highest at zone 2. smc is revising the contact tracing list to identify additional contacts using the data from the health insurance review and assessment service. cctv analysis is ongoing to confirm the contacts of case 137 within the hospital [2] . to detect mers-cov, we are using real-time reversetranscription polymerase chain reaction (rt-pcr) following the protocol recommended by the world health organization (who). after the confirmation of the first mers case in kcdc, we expanded the screening capacity by including provincial-level laboratories and major private diagnostic centers (n z 5) on may 30 and june 5, respectively. all participating laboratories are using the same screening method of real-time pcr, and the laboratories in the network can process at least 2000 samples/d in case of a surge of samples. a total of 6,776 samples have been tested and 154 cases have been confirmed as mers positive since may 19. we have also successfully isolated mers after the confirmation of the mers cases on may 20, we implemented several measures to better control this mers outbreak. case definition for case detection and management protocol for hospitalized patients has been developed and revised for the early detection and better management of cases in alignment with the guidance for mers-cov prevention and control from the kcdc. among the 154 confirmed cases since june 16, 95 confirmed cases are from the close contact list and one from the casual contact list. forty-nine confirmed cases (31.8%) were not included in the contact list. in the early stage of this outbreak (may 20e29), the list of the contacts of confirmed cases was not accurate. from may 30 to june 7, most of the confirmed cases were from the contact list, and the proportion of confirmed cases among nonlisted contacts decreased. however, because of some missing contact information from smc, the percentage of confirmed cases among nonlisted contacts increased on june 8e11. as a result of the strong control measures implemented on june 10, the percentage of missed contacts has decreased again since june 12 [3] . as of june 16, 5:00 am, 10,776 contact persons are under quarantine: 462 close contacts (4.3%) are in health facilities, 5,768 close contacts (53.5%) are under home quarantine, and 2,682 people have been released from quarantine; 4,546 casual contacts are under active monitoring, and 2,850 contacts have been released from monitoring [3] . patients that show no symptom and obtain two consecutive negative pcr results (at a 24-hour interval) are discharged, whereas contacts with two negative pcr results (48-hour interval) within 14 days of monitoring are released from isolation after that period. a travel ban for close contacts was imposed on june 1, and one for casual contacts was added on june 6. however, the prohibition of casual contacts from travel was lifted on june 13. currently, we are monitoring close contacts that travel, at the airport. as of june 16, 7,499 people are under the travel ban. the list of contacts under the travel ban is provided to the ministry of justice, and a short message service (sms) is sent to each person to notify them of their travel ban status [3] . in healthcare facilities, an enhanced infection prevention and control (ipc) strategy against mers has been implemented for case isolation, the management of hospitalized patients, and the monitoring of exposed healthcare personnel. in addition to standard precautions, including hand hygiene, appropriate personal protective equipment, including gloves, gowns, and respiratory and eye protection, is used when seeing confirmed or suspected mers cases. each patient is placed in a negative-pressure room with a high-for contact tracing, contact lists are obtained by interviewing cases, viewing cctv surveillance, and global positioning system (gps) tracking of mobile phones. close contacts with confirmed or suspected cases are quarantined at home or at health facilities and monitored actively twice a day by phone call, checking for fever or any new symptoms for 14 days from the last-exposure date. law enforcement has been in place for noncompliant persons. casual contacts with confirmed or suspected cases are monitored actively twice a day by telephone for fever or new-symptom development for 14 days from the last-contact date. if any suspected case is detected during the quarantine period, he/she is isolated, and respiratory specimens are immediately obtained for laboratory confirmation. editorial efficiency particulate air filter or a minimum of 12 air changes/h. also, aerosol-generating procedures are recommended to be performed in a negative-pressure isolation room by fully protected healthcare personnel. visits to mers-cov patients are restricted, and facilities maintain a record of all visitors. healthcare workers are trained in the ipc guidelines. all healthcare workers are monitored for fever and all respiratory symptoms twice a day for 14 days after the last known contact with a mers-cov patient to minimize exposure to and transmission of mers-cov in hospital settings [3] . since june 5, strict pneumonia surveillance has been implemented in 262 hospitals in four areas with major outbreaksdseoul, gyeonggi province, daejeon city, and chungnam province. as of june 16, 503 severe pneumonia cases have been identified, among which 375 respiratory specimens were tested for mers-cov and two tested positive. the two positive cases were already on the contact list and were isolated in a cohort isolation hospital in pyeongtaek. a cross-sectional nationwide survey of hospitalized pneumonia cases who had ever visited affected hospitals was conducted between june 9 and june 13. as of june 15, six suspected cases among 7,468 pneumonia patients in 2,575 hospitals have been identified. among those cases, three tested negative for mers-cov, and the other three were already under appropriate management [2] . the government has designated 17 referral hospitals for mers-cov patient management, and 35 hospitals are currently caring for mers patients. also, over 30 triage hospitals for suspected cases with a temporary space for safe triage of respiratory-illness patients and isolation of suspect mers cases have been designated nationwide. guidelines for ipc and education, and resources such as personal protective equipment have been provided to the hospitals. financial and human resources support has also been provided [2] . as the whoekorea joint mission team concluded, this outbreak was unexpected, and mers-cov was unfamiliar to all doctors and the general public in korea. although we may need more weeks to control this unexpectedly large and complex outbreak, the number of new cases seems to be declining. as the whoekorea joint mission team summarized, several factors contributed to the spread of mers-cov in korea: (1) suboptimal ipc measures in hospitals; (2) crowded emergency and multibed rooms; (3) the practice of "doctor shopping," which involves visits to multiple hospitals; and (4) the custom of having many family members or other visitors in the patient's room, which leads to the secondary spread of infection among contacts [3] . to date, virus sequencing results show no strong evidence to suggest the change in virus transmissibility and all cases are associated with hospital-related infection. currently, there is no evidence of ongoing community transmission of mers-cov in korea. the role of environmental factors, such as inadequate ventilation in hospitals, and other factors, is being investigated. the decline of new cases indicates that the implementation of much stronger contact tracing and monitoring and quarantine measures might be working. however, we need to be vigilant to end this outbreak because sporadic cases related to smc are still occurring. we also want to invest in strengthening medical capacity and human resources to deal with new infectious diseases. finally, we would like to thank all the international experts who participated in the whoekorea joint mission and other experts with whom we collaborated in the virus sequence analysis. the views and advice of the experts from the joint mission were extremely useful to fully understand the situation and effectively control the outbreak. kcdc is willing to further collaborate with the who, the us centers for disease control and prevention and its saudi arabian and chinese counterparts, and other international partners to share its experiences related to these mers-cov cases and fill gaps in knowledge about mers-cov. the ministry of health and welfare will make full efforts for the early identification of cases, the quarantine/isolation and monitoring of all contacts and suspected cases, the full implementation of ipc measures, and risk communication to the public and national and international partners [4] . middle east respiratory syndrome coronavirus outbreak in the republic of korea ministry of health and welfare, mers portal statement for the 9th meeting of international health regulation emergency committee on mers-cov outbreak from the ministry of health and welfare, the republic of korea intensified public health measures help control mers-cov outbreak in the republic of korea editorial hae-wol cho * osong public health and research perspectives hwcho@eulji.ac.kr chaeshin chu* osong public health and research perspectives key: cord-283966-eln8ljjj authors: meyer, benjamin; müller, marcel a.; corman, victor m.; reusken, chantal b.e.m.; ritz, daniel; godeke, gert-jan; lattwein, erik; kallies, stephan; siemens, artem; van beek, janko; drexler, jan f.; muth, doreen; bosch, berend-jan; wernery, ulrich; koopmans, marion p.g.; wernery, renate; drosten, christian title: antibodies against mers coronavirus in dromedary camels, united arab emirates, 2003 and 2013 date: 2014-04-17 journal: emerg infect dis doi: 10.3201/eid2004.131746 sha: doc_id: 283966 cord_uid: eln8ljjj middle east respiratory syndrome coronavirus (mers-cov) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the arabian peninsula since 2012. dromedary camels have been implicated as possible viral reservoirs. we used serologic assays to analyze 651 dromedary camel serum samples from the united arab emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. recombinant spike protein–specific immunofluorescence and virus neutralization tests enabled clear discrimination between mers-cov and bovine cov infections. most (632/651, 97.1%) camels had antibodies against mers-cov. this result included all 151 serum samples obtained in 2003. most (389/651, 59.8%) serum samples had mers-cov–neutralizing antibody titers >1,280. dromedary camels from the united arab emirates were infected at high rates with mers-cov or a closely related, probably conspecific, virus long before the first human mers cases. cov) is an emerging pathogen associated with severe respiratory symptoms and renal failure in infected patients (1, 2) . globally, 156 laboratory-confirmed cases of infection with mers-cov, including 65 deaths, were reported as of early november 2013. all human cases were linked to the arabian peninsula (saudi arabia, jordan, oman, qatar, kuwait, and the united arab emirates). imported cases were detected in countries in europe and africa (united kingdom, germany, italy, france, and tunisia) (3) . transmission patterns, including the putative zoonotic source of the virus, remain unclear. hypotheses include frequent zoonotic infections with limited subsequent human-to-human transmission chains and existence of a self-sustained epidemic in humans (4) . a recent study found evidence to support the existence of epidemiologically unlinked cases in a large outbreak in the al-hasa region, saudi arabia (5) . it was speculated that zoonotic introductions of mers-cov from an unknown reservoir might occur at high rates, in addition to obvious human-tohuman transmission. coronaviruses (cov) are positive-sense rna viruses. viruses in the genera alphacoronavirus and betacoronavirus are associated with mammals and show a particularly high level of diversification in bats. viruses in the genera gammacoronavirus and deltacoronavirus are mostly avian-associated viruses (6, 7) . mers-cov belongs to betacoronavirus phylogenetic lineage c that, in addition to mers-cov, contains 2 distinct bat-associated cov species (hku4 and hku5) (1, 8) . insectivorous bats of the family vespertilionidae were recently shown to carry viruses that are probably conspecific with mers-cov (9) . however, the limited rate of contact between humans and insectivorous bats makes a continuous and frequent acquisition of mers-cov from bats an unlikely scenario. in a manner similar to observations regarding severe acute respiratory syndrome cov (sars-cov), an intermediate reservoir host might exist from which human infections are acquired. dromedaries from different regions in africa and the arabian peninsula have been shown to have antibodies against mers-cov (10, 11) . animals from the arabian peninsula had high neutralizing serum activities overall and reciprocal antibody titers <320-1,280, which support recent infection with mers-cov or a highly related virus. thus, dromedaries might serve as intermediate hosts. however, detailed serologic studies in countries with actual incidence of mers-cov infections in humans have not been conducted. serologic analysis of covs is challenging because of cross-reactivity between covs infecting the same host and the broad distribution of covs in diverse mammalian species (6, 7, (12) (13) (14) . antibodies directed against some of the major antigens of different covs are known to crossreact in standard serologic assays (15, 16) . potential crossreactivity is a diagnostic challenge because camelids are known to be infected with bovine cov (bcov), a distinct betacoronavirus of phylogenetic lineage a unrelated to the mers-cov (17, 18) . as an additional challenge, camel immunoglobulins lack a light chain peptide, which affects specific physical properties, such as altered size and stability, compared with immunoglobulins of other mammals (19, 20) . the influence of this feature on serologic assays has not been thoroughly investigated. thus, serologic assays should be applied with caution, and different assay formats should be tested concurrently. we reported a 2-staged approach for mers-cov serologic analysis in humans (15, 16) . expanding upon these studies, we used in the present study a recombinant mers-cov spike protein immunofluroescence assay (rifa) augmented by a validated protein microarray (10, 21) , followed by mers-cov-specific neutralization assay, to screen 651 dromedary serum samples from the united arabian emirates. cross-reactivity against clade a betacoronaviruses was assessed by using a immunofluorescence assay (ifa) and a bcov-specific neutralization assay. serum samples obtained in 2003 and 2013 were compared to obtain information for the time in which mers-related cov has been circulating in camels. a total of 651 dromedary (camelus dromedarius) serum samples were systematically sampled in dubai, united arab emirates and the surrounding area in 2003 (collection 4, n = 151) and in 2013 (collections 1a, 1b, 2, and 3; n = 500). the total number of camels in that area was 360,000 in 2010 (22) . fecal samples were also available for collections 1a and 1b (n = 182), all obtained in 2013. animals in collection 1b were born and raised at the dubai central veterinary research laboratory, which tests ≈70,000 camels per year (23) and had no contact with other camels. camels in collection 2 were racing camels (age range 2-8 years), and camels in collection 3 were adult livestock camels originally purchased from saudi arabia, sudan, pakistan and oman. dromedary blood was obtained for routine health screening by jugular vein puncture according to standard veterinary procedures by trained personnel. for most serum samples, animal owners requested sample codes to be anonymous. all samples obtained during 2003 and 2013 were stored at -80°c until further analysis. for comparison, 16 serum samples from c. bactrianus camels in zoologic gardens in germany were included in the study. all serum samples were shipped in agreement with german import regulations. for screening purposes, an rifa was used (15, 24) . in brief, vero b4 cells were transfected with pcg1 eukaryotic expression vector that contained the complete spike sequence of mers-cov or human cov-oc43. cells were fixed 24-h post-transfection with ice-cold acetone/ methanol and stored dry at 4°c. serum samples were applied at a dilution of 1:80 for 1 h at 37°c, which was optimal for reducing nonspecific reactions and maintaining sensitivity. secondary detection was conducted by using a goat anti-llama igg fluorescein isothiocyanate-conjugated antibody. for some negative serum samples, dilutions of 1:20 and 1:40 were also tested. a confirmatory assay based on a protein microarray was performed as described (10, 21) by using the spike s1 subunits of mers-cov, human cov-oc43, and sars-cov. serum samples were used at 1:20 dilutions on microarray chips. relative light units were determined by using secondary cyanine 5-conjugated goat anti-llama igg. a mers-cov ifa with infected vero cells was conducted as described (15) by using commercially available mers-cov ifa slides (euroimmunag, lübeck germany). serum samples were used at dilutions of 1:20-1:5,120. secondary detection was conducted by using goat anti-llama fluorescein isothiocyanate-labeled igg (1:200 dilution; agrisera, vännas, sweden). serum neutralization tests were conducted as described (10) with camel serum diluted in 25 ml serum-free dulbecco minimum essential medium. the starting dilution was 1:40. after incubation for 1 h at 37°c, each well was infected for 1 h at 37°c with a 50 ml virus-serum mixture. supernatants were removed and fresh complete dulbecco minimum essential medium was added. assays were terminated by fixation with 8% paraformaldehyde for 30 min and stained with crystal violet after 3 days. neutralization titers were defined as serum dilutions reducing cytopathic effects in 2 parallel wells. viral rna was extracted from serum and fecal samples by using the magna pure system (roche, basel, switzerland) and an input volume of 100 μl of serum or fecal material suspended 1:10 in phosphate-buffered saline buffer. the elution volume was 100 μl for serum and fecal suspensions. to identify cov-specific nucleic acids, 2 generic cov pcrs were performed as described (25) (26) (27) , followed by subsequent sanger sequencing of the amplified dna. to characterize reactivity of camel serum samples with mers-cov in different assay formats, we chose 11 camel serum samples with weak and strong reactivity predetermined by using a simple ifa. the 11 serum samples were titrated in a 2-fold dilution series in all applied assays. the reactivity pattern of the mers-cov spike protein (mers-s) was compared against that of the human cov-oc43 spike protein (oc43-s). as in our previous study (10) , human cov-oc43 was used instead of bcov in these initial experiments because it is serologically indistinguishable from bcov and is not subject to handling restrictions of german animal diseases protection act (28) . overall titers against mers-s were higher than those against oc43-s, and several serum samples reacted exclusively against 1 of the 2 viruses (table 1) , suggesting the absence of general cross-reactivity between spike proteins of both viruses by ifa. typical patterns of reactivity observed for camel serum samples are shown in the figure, panel a. a previously published microarray-based assay that used the receptor-binding s1 spike subunit of mers-cov (mers-s1), human cov-oc43 (oc43-s1), and sars-cov (sars-s1) was also evaluated. in contrast to our previous studies (10,21) we chose a lower fluorescence intensity cutoff of 4,000 instead of 20,000 relative fluorescence units (rfu) to maximize the sensitivity and thereby challenge the target specificity. all 3 mers ifanegative serum samples had signal intensities <4000 rfu at serum dilutions of 1:20 (table 1 ). all rifa-positive serum samples had saturated signals >65,535 rfu. the oc43-s1 reactivity pattern across the serum panel was comparable with that for the oc43-s rifa. as expected, all serum samples were negative against the sars-s1 control antigen. a comparison of typical reactivity patterns in the microarray with those of the ifa is shown in the figure, panel b. results for the rifa and protein microarray were highly congruent. the panel of camel serum samples was additionally tested in a commercially available ifa that used cells infected with mers-cov (vifa) (euroimmun ag). the use of whole virus provides additional structural and nonstructural protein antigens, including envelope, membrane, nucleocapsid, and diverse replicase proteins. however, because of conserved features of nonstructural proteins among even distantly related covs (7,12) cross-reactivity was possible with this assay (15) . in the tested panel of camel serum samples, vifa titers corresponded well to titers determined by rifa and generally equal to or higher than titers in the rifa (table 1) to confirm results from affinity assays with results from a functional test, we determined endpoint virus neutralization titers by using a microneutralization test against mers-cov and bcov. in most animals mers-cov serum neutralization titers were higher than titers against bcov (serum samples 4-11) ( table 1) . high ifa titers generally corresponded with high neutralization titers, with exceptions for some bcov antibody-positive serum samples. divergence between affinity and neutralization assays can result from waning neutralizing antibody activity for infections that occurred long ago. neutralization assays confirmed the absence of cross-neutralization between mers-cov and bcov antibodies in either direction even at low dilutions, such as 1:40. however, sample no. 1 (table 1) neutralized bcov at a dilution of 1:40 despite showing negative results in all other serologic assays. this finding indicates that nonspecific neutralization activities might be encountered with camel serum samples, suggesting that higher serum dilutions should be used when conducting critical investigations such as viral reservoir studies. on the basis of the validation studies, we investigated 4 collections of serum samples from dromedaries from the united arab emirates that were sampled in 2003 and 2013. for initial screening, we chose the rifa because of its proven sensitivity and decreased chances of generating false-positive results. all 667 camel serum samples from the united arab emirates and germany were initially screened at dilutions of 1:80. a total of 89.0%-100.0% of serum samples in 4 collections showed positive results ( table 2) . seroprevalence was higher for collections from exclusively adult animals (collections 3 and 4) than for a collection from young racing camels (2-8 in the rifa. re-testing at lower dilutions of 1:20 and 1:40 confirmed absence of reactivity in these serum samples. subcollection 1b contained serum samples from 5 animals that were born in, and had never left, a closed animal research facility in dubai; these animals were seronegative. a confirmatory microneutralization test was conducted at dilutions of 1:640 and 1:1,280 for all ifa-reactive serum samples. these high dilutions were chosen on the basis of our observation of high levels of neutralizing serum activity in camels (10) . most (59.8%, 389/651) serum samples had high neutralizing titers >1,280 (table 2 ). in 18.4% (120/651) of all serum samples, neutralization titers ranged from 640 through 1,280, and 21.8% (142/651) of rifa-positive serum samples had neutralizing titers <640. to rule out cross-reactivity and to study additional exposure of mers-cov-positive camels with bcov (17, 18) , all serum samples having mers-cov neutralizing titers >640 were tested by using a bcovspecific microneutralization assay. at a dilution of 1:640, a total of 19.2% (23/120) of mers-cov-neutralizing serum samples had concomitant neutralizing activities against bcov (table 3 ). of serum samples that had mers-cov neutralizing antibody titers >1,280, a total of 24.2% (94/389) had concomitant neutralizing activities against bcov. fecal samples were available for 182 dromedaries in collection 1. all samples were tested by using a subfamily coronavirinae-specific broad-range reverse transcription pcr (rt-pcr) and a highly sensitive rt-pcr specific for genus betacoronavirus phylogenetic lineage c. both assays were specific for the viral rnadependent rna polymerase gene. two positive fecal samples were identified by both assays. sequencing of amplified cdna fragments of 182 nt and 404 nt identified sequences 99% identical with bcov strain mebus (genbank accession nos. kf894801 and u00735.2). to further confirm virus identity, we amplified a region within the spike protein gene (positions 24303-24702 in bcov strain mebus) by using rt-pcr and sequencing it. amplicons from both animals were 97% identical at nucleotide level with bcov strain mebus, indicating the presence of bcov in camels as reported (10) . we tested all serum samples in the same way by rt-pcr and obtained uniformly negative results. we have shown that dromedaries from the united arab emirates, a country with human cases of mers-cov infection, have antibodies that can neutralize mers-cov at high rates. antibodies were detected in serum samples obtained in 2013 and in serum samples obtained >10 years earlier, which indicated the longstanding presence of mers-cov or a closely related virus in dromedaries in that region. our data add to previous studies in which our group and others have reported wide antibody prevalence in camels in various regions, including oman, egypt, and the canary islands (10, 11) . a 10% lower seroprevalence in collection 2, which contained young racing camels, suggests that animals might be infected as juveniles. however, because only limited data were made available by owners, a definite statement awaits confirmation. the absence of antibodies in a control cohort from germany might be explained by the fact that these animals belonged to a different camelid species (c. bactrianus vs. c. dromedarius). however, because mers-cov has a highly conserved receptor structure, we did not assign high priority to the hypothesis that the closely related camel species c. bactrianus, should be less susceptible than c. dromedarius camels to mers-cov (29, 30) . differences in antibody prevalence rates might reflect a restricted geographic distribution of the virus, which corresponds to our previous finding of a relatively lower prevalence of antibodies against mers cov in camels from the canary islands, which have been isolated from their point of origin in africa for many years (10) . therefore, mers-cov-like viruses in camelids might be spreading across a region covering at least the eastern arabian peninsula, including oman, the united arab emirates, egypt, and morocco from where some of the antibody-positive camels described by reusken et al. originated (10) . the high rates of antibody prevalence in contemporary serum samples and samples from 2003 suggest that the virus has spread in camelids for some time. however, recognition of camelids as the bona fide reservoir for mers-cov has to await sequencing of camelid-associated mers-related cov. in this context, only animals infected with conspecific viruses can be regarded as reservoirs for a given virus. although neutralization assays can provide evidence of infection with a virus belonging to the same serotype, no systematic studies have defined whether serotypes correlate with covs species. nevertheless, for several cov clades, serotypes defined by neutralization assay will not include >1 viral species. members of the species betacoronavirus 1, including cov-oc43 and bcov, show cross-neutralization with each other, but the closely related sister species (human cov-hku1) does not show cross-neutralization (31) . feline cov (fcov) comprises 2 subserotypes that show limited cross-reactivity but are considered 1 virus species. transmissible gastroenteritis virus of swine shows more efficient cross-neutralization with 1 of these fcov subserotypes than the other and is classified as 1 species with fcov even though it is carried by a different host (32) . human covs 229e and nl63, which form 2 closely related sister taxa, do not show cross-neutralization and concordantly form 2 different species by genetic criteria (33) . therefore, our finding of high neutralizing antibody titers in camelids is suggestive (but not evidentiary) of the presence of viruses conspecific with mers-cov in camelids. final confirmation will depend on the identification of virus sequences in camelids, which should expectably be closely related to human-specific mers-cov sequences. camels probably acquired mers-cov at some unknown time. potential sources include bats of the family vespertilionidae, in which a virus with a close phylogenetic relationship with mers-cov has been detected (9). this virus, which is carried by vespertilionid bats of the genus neoromicia, has been confirmed to be conspecific with mers-cov. lineage c betacoronaviruses in other bat taxa have also been proposed to be related to mers-cov (34, 35) . however, although these viruses cluster phylogenetically with mers-cov, they are not conspecific with mers-cov on the basis of sequence distance criteria, such as that were proposed by drexler et al. (36) . in vespertilionid bats, including those in the genus neoromicia, virus conspecific with mers-cov differs from human mers-cov, even if formally a member of the same species. the observed degree of sequence divergence between this virus and mers-cov makes any direct and recent transmission from bats to humans seem unlikely. nevertheless, it cannot be excluded from available data that the virus source population in bats has not been detected. for example, a recent investigation of rhinolophus bats in china identified viruses with close relationships to the bona fide ancestor of sars-cov, and viruses described in many studies yielded only conspecific yet less related viruses (37) . in that study, viruses from civet cats, which are deemed to be intermediary hosts in the transition of sars-cov from bats to humans, were still more closely related to human sars-cov than even the closest bat-borne virus. if camelids should function as intermediary hosts in a similar manner, we should expect a virus in camelids that has a closer phylogenetic relationship with any bat-borne cov and thus should be easily detectable with available rt-pcrs. larger studies to confirm the presence of mers-cov in camelids should receive high priority so as to define the animal reservoir of mers-cov and possibly control it by such measures as vaccination or control of animal movement. however, before implementation of any control measures, whether camelids are a continuous source of infection for humans needs to be firmly established. isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov)-update transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus genomic characterization of sars-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt molecular diversity of coronaviruses in bats distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats detection and prevalence patterns of group i coronaviruses in bats, northern germany contact investigation of a case of human novel coronavirus infection treated in a german hospital investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughtethouse workers in jeddah and makkah, saudi arabia, fall 2013 analysis of the genome sequence of an alpaca coronavirus enteric coronavirus infection in a juvenile dromedary (camelus dromedarius) comparison of physical chemical properties of llama vhh antibody fragments and mouse monoclonal antibodies naturally occurring antibodies devoid of light chains specific serology for emerging human coronaviruses by protein microarray cvrl. 26th annual report detection of a novel human coronavirus by realtime reverse-transcription polymerase chain reaction human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray antigenic and biological relationships between human coronavirus oc43 and neonatal calf diarrhoea coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc examination of seroprevalence of coronavirus hku1 infection with s protein-based elisa and neutralization assay against viral spike pseudotyped virus antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry coronaviruses in bats from mexico group c betacoronavirus in bat guano fertilizer genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor we thank tobias bleicker for providing excellent technical assistance.the work was supported by a european research project on emerging diseases detection and response (emperie; www.emperie.eu/emp/) (contract no. 223498) and antigone (contract no. 278976). c.d. has received infrastructural support from the german centre for infection research, the german ministry for research and education, and the german research council (grants 01kio701 and dr 772/3-1).mr meyer is a doctoral student at the bonn institute of virology, bonn, germany. his research interests are spike protein-mediated entry of bat-borne coronaviruses into cells and advancement of specific serologic tests for antibodies against coronaviruses. key: cord-266987-ikt8r2o1 authors: loeffelholz, michael j.; tang, yi-wei title: laboratory diagnosis of emerging human coronavirus infections – the state of the art date: 2020-03-30 journal: emerg microbes infect doi: 10.1080/22221751.2020.1745095 sha: doc_id: 266987 cord_uid: ikt8r2o1 the three unprecedented outbreaks of emerging human coronavirus (hcov) infections at the beginning of the twenty-first century have highlighted the necessity for readily available, accurate and fast diagnostic testing methods. the laboratory diagnostic methods for human coronavirus infections have evolved substantially, with the development of novel assays as well as the availability of updated tests for emerging ones. newer laboratory methods are fast, highly sensitive and specific, and are gradually replacing the conventional gold standards. this presentation reviews the current laboratory methods available for testing coronaviruses by focusing on the coronavirus disease 2019 (covid-19) outbreak going on in wuhan. viral pneumonias typically do not result in the production of purulent sputum. thus, a nasopharyngeal swab is usually the collection method used to obtain a specimen for testing. nasopharyngeal specimens may miss some infections; a deeper specimen may need to be obtained by bronchoscopy. alternatively, repeated testing can be used because over time, the likelihood of the sars-cov-2 being present in the nasopharynx increases. several integrated, random-access, point-of-care molecular devices are currently under development for fast and accurate diagnosis of sars-cov-2 infections. these assays are simple, fast and safe and can be used in the local hospitals and clinics bearing the burden of identifying and treating patients. cov, mers-cov and sars-cov-2). in temperate regions endemic hcovs usually display a winter seasonality, although hcov-229e has been detected sporadically throughout the year [12] . endemic hcovs are globally distributed and are maintained in the human population. the sars-cov pandemic came to an end in 2003 (https://www.who.int/csr/resources/ publications/cds_csr_aro_2004_2.pdf?ua=1. accessed 3 february 2020), less than a year after the first reported case. in contrast, human cases caused by mers-cov continue to be reported at the time of writing, more than seven years after the first reported case. most laboratory-confirmed mers cases have occurred in the eastern mediterranean region, and the majority of those in saudi arabia. unlike the endemic hcovs, sars-cov and mers-cov are maintained in zoonotic reservoirs. the sars and mers outbreaks were driven in part by super-spreading events in which individuals directly infected a disproportionally large number of contacts [13] . the sars-cov-2-caused coronavirus disease 2019 (covid19) epidemic originated in a wuhan, china market that sold exotic animals for consumption. based on genetic relatedness to other betacoronaviruses, sars-cov-2 likely has a zoonotic reservoir. however, the precise source of sars-cov-2 that initially infected humans remains to be confirmed. the sars-cov-2 appears to be substantially more contagious than sars-cov ( table 1 ). the distribution of sars-cov-2 in different mammalian species is unknown. an interesting question is the susceptibility of farm animals and pets, and their role in the epidemiologic cycle as their angiotensin-converting enzyme 2 (ace2) receptor shares similarity with human ace2 [14] . infections caused by endemic hcovs have an incubation period of 2-5 days and are associated with mild upper respiratory symptoms (the "common cold"). endemic hcovs are among the most frequent cause of upper respiratory tract infections. lower respiratory tract infections (bronchiolitis, pneumonia) are rare. following an incubation period of usually 4-5 days, patients infected with sars-cov often present with symptoms of fever, headache, and myalgias. respiratory symptoms including cough and dyspnoea usually develop from several days to a week after illness onset. atypical pneumonia and respiratory deterioration occur in 20-30% of cases. the incubation period and clinical course of mers are similar to that of sars, the exception being a higher proportion of cases progressing to respiratory deterioration and distress. the incubation period and clinical course of sars-cov-2 infection are probably similar to that of sars. li et al. first reported a mean incubation period of 5.2 days [15] . fever and cough are frequently reported early in the course of illness [16, 17] . infections are also characterized by dyspnoea, respiratory distress and positive chest x-ray [10] . lower respiratory symptoms often develop about 1 week from the onset of initial symptoms [16] . globally over 8000 cases and over 900 deaths due to sars-cov were reported, with a case-fatality ratio of approximately 11% (https://www.who.int/csr/sars/en/ whoconsensus.pdf. accessed 3 february 2020). between september 2012 and november 2019, there were 2494 laboratory-confirmed cases of mers, with 858 deaths (https://www.who.int/emergencies/merscov/en/. accessed 4 february 2020). the mers casefatality rate of 34.4% is about triple that of sars, and persons in the 50-59 year age group are at highest risk for primary cases. in the short time from its emergence in december 2019 to 15 march 2020, the sars-cov-2 has been reported in 134 countries. at the time of writing, the situation was evolving rapidly, with over 142,000 confirmed cases reported globally (over 81,000 in china) and 3194 deaths in china (3.9% case-fatality rate) and over 2100 deaths outside of china. of countries and continents outside of china, south korea, iran, and europe (particularly italy) have experienced a high number of covid-19 cases (https://www.who.int/emergencies/diseases/novel-coro navirus-2019/situation-reports/. accessed 15 march 2020). mortality rates vary widely, and depend on the age of patients, underlying risk factors, and the denominator definitionhospitalized cases, all symptomatic cases, only moderate to severe cases, etc. in a study of adult patients (mean age 59.7 y; 40% with chronic illnesses) with sars-cov-2 pneumonia admitted to the intensive care unit (icu), 61.5% died within 28 days [18] . in contrast, a study of hospitalized patients (median age 47.5 years) across beijing showed [8, 20, 22, 59] 18% of cases to be severe and 73% mild, with a fatality rate of 0.9% [19] . mortality is highest in older persons, with a median age of 59-75 years [15, 17] . treatment for all severe hcov infections is supportive although a randomized, double-blinded, control clinical trial has been conducted on a gilead drug remdesivir [20] based on one study focused on children, a total of 28 children aged from 1 month to 17 years have been reported in china. all paediatric cases with laboratory-confirmed sars-cov-2 infection were mild cases with no deaths reported [21] . during the first 2 months of the current outbreak, covid-19 spread rapidly throughout china and caused varying degrees of illness with a death rate of 1.3%. patients often presented without fever, and many did not have abnormal radiologic findings [22] . the chinese centers for disease control and prevention team analysed more than 72,000 patient records, of which 44,672 were laboratory-confirmed cases, 16,186 suspected cases, 10,567 clinically diagnosed cases, and 889 asymptomatic cases. of the confirmed cases, about 14% of the illnesses were severe, which included pneumonia and shortness of breath, and about 5% have the critical disease, marked by respiratory failure, septic shock, and multi-organ failure. the overall case-fatality rate was 2.3%, and of 1023 deaths included in the study, the majority were in people age 60 and older or those with underlying medical conditions http://www.cidrap.umn.edu/news-perspective/ 2020/02/more-outbreak-details-emerge-covid-19-casestop-70000 (accessed 18 february 2020). it must be appreciated that no matter how accurate and fast laboratory testing methods are, the diagnosis of viral pneumonias such as caused by sars-cov-2 involves collecting the correct specimen from the patient at the right time. the endemic hcovs have been detected from a variety of upper and lower respiratory sources including throat, nasal nasopharyngeal (np), sputum, and bronchial fluid [12, 23, 24] . wang et al have just reported that oropharyngeal (op) swabs (n = 398) were used much more frequently than np swabs (n = 8) in china during the covid-19 outbreak; however, the sars-cov-2 rna was detected only in 32% of op swabs, which was significantly lower than that in np swabs (63%) [25] . the us centers for disease control and prevention (cdc) recommends collecting the upper respiratory np swab. collection of an op specimen is a lower priority, and, if collected, should be combined in the same tube as the np swab (https://www.cdc.gov/coronavirus/ 2019-ncov/lab/guidelines-clinical-specimens.html. accessed 16 march 2020). swab specimens should be placed in a universal or viral transport medium. nasopharyngeal aspirates are also suitable specimens for the detection of hcovs. for the most sensitive detection of sars-cov, mers-cov, and sars-cov-2, the collection and testing of both upper and lower respiratory samples [sputum, bronchoalveolar lavage fluid (bal)] is recommended [26] . however, the collection of sputum and particularly bal via bronchoscopy increases biosafety risk to healthcare workers through the creation of aerosol droplets. proper use of personal protective equipment (ppe) by healthcare workers is important. bronchoscopy is a highly technical procedure requiring well-trained staff and may not be available in many parts of the world. upper respiratory specimens are easy to collect, thereby increasing access to testing for patients with mild symptoms, and in the resource limited settings. sars-cov and mers-cov rna are also detected from stool, urine and blood specimens, although generally less reliably than from respiratory specimens [26] [27] [28] ]. an exception is sars-cov rna which is consistently detected in feces at about two weeks after symptom onset [26, 29] . for the most sensitive detection of endemic hcovs, upper respiratory specimens should be collected within the first few days of symptom onset. the dynamics of rna shedding in mers and sars patients may reflect the specimen source, severity of illness, as well as underlying risk factors. among hospitalized patients who did not require ventilator support, mers-cov rna levels in the upper respiratory tract usually peaked in the first week after symptom onset. among eventual fatal cases requiring ventilation, rna levels in lower respiratory tract specimens peaked between weeks 2 and 3 [27] . similar shedding patterns were seen for sars-cov: rna positive rates peaked in upper respiratory tract specimens at 7-10 days after symptom onset and then steadily declined after that, while rna positive rates in lower respiratory tract specimens remained higher throughout 3 weeks after onset of illness [26] . in one study, diabetes was associated with prolonged mers-cov rna shedding in the respiratory tract [27] . viral pneumonias typically do not result in the production of purulent sputum. thus, a nasopharyngeal swab/wash is usually the collection method used to obtain a specimen for testing. nasopharyngeal specimens may miss early infection; a deeper specimen may need to be obtained by bronchoscopy. alternatively, repeated testing can be used because over time, the likelihood of the sars-cov-2 being present in the nasopharynx increases. self-collected saliva specimens were tested positive in 11 of 12 covid-19 patients, suggesting it is a promising non-invasive specimen for diagnosis, monitoring, and infection control in sars-cov-2 infections [30] . at the time of writing there was little data on the performance of upper vs. lower respiratory tract specimens for the detection of sars-cov-2 [16] . serum is another source for the detection of sars-cov-2. however, only 15% of patients hospitalized with pneumonia had detectable rna in serum [16] . specimens collected for laboratory testing of hcovs should be maintained at refrigerated temperature for up to 72 h, or frozen at −70°c or below (https://www.cdc.gov/coronavirus/ 2019-ncov/lab/guidelines-clinical-specimens.html. accessed 15 march 2020). rectal specimens have been reported positive in patients infected with sars-cov-2 [20] . if the patient's travel or exposure history or symptoms suggest possible infection with a high-risk, novel agent, sars-cov, or mers-cov, then the initial handling of the specimen should be performed under biosafety level 3 (bsl-3) conditions until the specimen or an aliquot is rendered noninfectious by lysis or another method. virus isolation should not be routinely performed in this situation (https://www.asm.org/ articles/policy/laboratory-response-network-lrn-sentinel-level-c. accessed 4 february 2020). the u.s. cdc biosafety guidelines state that routine diagnostic testing of specimens from suspected or confirmed sars-cov-2 patients, can be handled in a bsl-2 laboratory using standard precautions (https://www.cdc. gov/coronavirus/2019-ncov/lab/lab-biosafetyguidelines.html. accessed 21 march 2020). isolation of hcovs in cell culture is not routinely performed for diagnostic purposes due to the lack of permissive cell lines, time to results, labour and expertise requirements, and the lack of commercial antisera for culture confirmation (table 2) . sars-cov and mers-cov and sars-cov-2 will grow in primary monkey cells and cell lines such as vero and llc-mk2, but cell culture should not be performed for suspect cases in routine diagnostic laboratories for biosafety reasons [2, 6, 31, 32] . however, virus isolation in cell cultures is critical to obtain isolates for characterization and to support the development of vaccines and therapeutic agents. rapid antigen tests would theoretically provide the advantage of fast time to results and low-cost detection of hcovs but are likely to suffer from poor sensitivity based on the experience with this method for influenza (flu) viruses [33] [34] [35] [36] [37] (table 2 ). in a pre-peer reviewed article, diao et al. reported that a fluorescence immunochromatographic assay is an accurate, rapid, early and simple method for detecting nucleocapsid protein of sars-cov-2 in np swab for diagnosis of covid-19 (https://www.medrxiv.org/content/10.1101/2020.03. 07.20032524v2. accessed 15 march 2020). the incorporation of colloidal gold-labeled immunoglobulin g (igg) as the detection reagent is an approach that may increase the sensitivity of rapid antigen tests for respiratory viruses [38] . monoclonal antibodies specifically against sars-cov-2 have been under preparation. novel approaches to concentrate antigen, or to amplify the detection phase are needed if these methods are to have clinical utility. sona nanotech (halifax, canada) is developing a quick-response lateral-flow test to screen covid-19 patients targeting to produce results in 5-15 min (https://sonanano.com/sona-develops-rapidscreening-test-for-coronavirus/. accessed 15 february 2020). timing of specimen collection, when viral titres are highest, may improve the diagnostic sensitivity of rapid antigen tests for hcovs [39] . serological assays are not routinely used for diagnosis of hcov infections due to the lack of commercial reagents, let alone commercial reagents that have been vetted by clinical trials and the regulatory review process [40, 41] (table 2 ). serological assays, on the other hand, are important for understanding the epidemiology of emerging hcovs, including the burden and role of asymptomatic infections. it has been particularly important for antibody detection in the diagnosis of cases of novel and emerging hcovs, such as sars-cov and mers-cov [2, 3] . in these situations, affected patients may not test positive for viral rna, particularly in the early phase of the disease, but retrospectively can be shown to have developed an immune response. when sars-cov-2 was identified, especially when rapid antigen testing and/or molecular assays are neither available nor stable, serology can be used as a supplementary diagnostic tool. a recent study demonstrated that both igm and igg antibodies were detected 5 days after onset in all 39 patients infected with sars-cov-2 infection. the authors recommended to use serology to facilitate the diagnosis of sars-cov-2 infections when an np swab specimen was collected inappropriately and the molecular assays were performed unsatisfactorily [42] . in china, six serology devices have just received urgent approval from the national medical products administration (nmpa) by 12 march 2020 (table 3) . proper specimen handling and storage are important to maintain the integrity of specimens and the performance of serologic tests. random-amplification deep-sequencing approaches played a critical role in identifying mers-cov and sars-cov-2 [6, 11, [43] [44] [45] [46] [47] . for the clinical diagnostic application, the genetic heterogeneity of hcovs precludes a single "pan-hcov" molecular assay [48] [49] [50] [51] ( table 2 ). some pan-cov assays use degenerate primers [52] , some utilize multiple primer sets [53] , and others employ a single set of nondegenerate primers [54] . current molecular respiratory panels that detect the endemic hcovs (hcov-nl63, hcov-hku1, hcov-oc43, and hcov-229e) require multiple sets of pcr oligonucleotides [12, [55] [56] [57] . sars-cov-2 cases tested negative for endemic hcovs included in molecular respiratory panels [10] . in china, at the time of revising, eleven molecular devices from shanghai zj bio-tech, shanghai geneodx biotech, bgi biotech (wuhan), mgi tech, da an gene, sansure biotech, shanghai biogerm medical biotech capitalbio (chengdu), beijing applied biological technologies, maccura biotechnology, and wuhan easydiagnosis biomedicine have received urgent approval from nmpa and their characteristics are contrasted in table 3 . variable performance has been reported on these devices [47, 58] . in their registration certificates, it was clearly indicated that the certificate was for urgent and supplemental diagnosis of pneumonia caused by sars-cov-2. additional multi-centre clinical trial data are needed for extension after one year. among them, one (mgi tech) uses its ngs technique to detect all pathogens in a given specimen including sars-cov-2 and the other one (innovita) uses its isothermal amplification followed by chip detection. the other nine devices incorporated real-time pcr technique with hydrolysis probes. after nucleic acids get extracted (separated reagents and systems), the extracts are transferred to a real-time pcr thermocycler (e.g. abi 7500 fast dx real-time pcr instrument) for nucleic acid amplification and detection. several rt-pcr protocols for detection of sars-cov-2 rna have been posted by the world health organization at https://www.who.int/emergencies/ diseases/novel-coronavirus-2019/technical-guidance/ laboratory-guidance. (accessed 15 march 2020). three of these protocols are listed below. the us cdc developed developed a rt-pcr diagnostic panel for universal detection of sars-like betacoronaviruses and specific detection of sars-cov-2 [20] . three separate rt-pcr reactions target the n gene. one primer/probe set detects all betacoronaviruses, while two sets are specific for sars-cov-2. all 3 assays must be positive to report presumptive positive for sars-cov-2 (https://www.fda.gov/ media/134922/download. accessed 15 march 2020). specimen types included upper and lower respiratory specimens (such as np or op swabs, sputum, lower respiratory tract aspirates, bal, and nasopharyngeal wash/aspirate or nasal aspirate). it the charité algorithm (berlin, germany) begins with two rt-pcr assays that detect e and rdrp genes of subgenus sarbecovirus (sars-cov, sars-cov-2, and bat-associated betacoronaviruses). both assays must be positive to advance to the next step in the testing algorithm. the second step consists of a [52] [53] [54] naat, monoplex, specific-hcov high sensitivity and specificity for special species, potential quantification 1-8 h diagnosis (detection, differentiation, and limited typing) and research [69, 70] naat, multiplex high sensitivity and specificity, covering other pathogens, filmarray rp ez is clia-waived 1-8 h diagnosis (detection, differentiation, and limited typing) and research [12, [55] [56] [57] naat, poct rapid and safe, good sensitivity and specificity, some are clia-waived diagnosis (detection and limited differentiation) and research [63, 67] note: eia, enzyme immunoassay; ifa, immunofluorescent assay; naat, nucleic acid amplification test; clia, clinical laboratory improvement act. sars-cov-2 specific rt-pcr that targets rdrp [59, 60] . exclusivity testing showed that alphacoronaviruses (cov-nl63 and −229e) and betacoronaviruses hcov-oc43, hcov-hku1 and mers-cov were not detected (https://www.who.int/docs/default-source/ coronaviruse/protocol-v2-1.pdf?sfvrsn=a9ef618c_2. accessed 8 february 2020). the university of hong kong li ka shing faculty of medicine protocol uses two assays (n gene screening assay followed by orf1b assay for confirmation) to detect subgenus sarbecovirus [30, 61] . since sars-cov is not circulating in humans currently, cases that are positive should be considered as sars-cov-2 infected cases. exclusivity testing showed that 229e, oc43 and mers, 229e, hku1, nl63, oc43 yielded negative results (https://www.who.int/docs/defaultsource/coronaviruse/peiris-protocol-16-1-20.pdf? sfvrsn=af1aac73_4. accessed 8 february 2020). all three novel coronaviruses are highly contagious. fast, safe, simple to use diagnostic devices performed at or near the point of care (poc) (figure 1 ) which have been shown to impact patient management and control of infectious disease epidemics [62], are extremely desirable in poc when biosafety facility is limited (table 3) . several manufactures have been spending efforts to generate devices for poc testing (poct) [63] . the id now™ (previously alere i) influenza a & b assay (abbott, san diego, ca) was cleared by the us fda for direct use on np swabs as the first-ever clinical laboratory improvement amendments (clia)-waived nucleic acid-based test in january 2016 [64, 65] . similarly, the xpert® xpress flu/rsv (cepheid, sunnyvale, ca) and cobas® liat® flu a/b & rsv (roche molecular systems, pleasanton, ca) assays are integrated nucleic acid extraction-independent devices that have recently received fda clearance and clia-waiver for simultaneous detection and identification of flua, flub, and rsv in nasopharyngeal swabs [66] . the filmarray® respiratory ez panel (biofire, salt lake city, ut) so far so far is the only clia-waived syndromic panel that covers a set of 14 respiratory viral and bacterial pathogens including classical coronavirus species [67] . considering the increased levels of mortality and infectivity associated with three novel-coronavirus outbreaks, these random-access, safe and simple tests, which offer fast and accurate detection and identification, are likely to have an immediate impact on prompt clinical and epidemiological decisions [7, 63] . lysis buffer can be used to inactivate the infectivity of specimens so the testing can be run at poc when a biosafety cabinet is not available. fast near-patient and poct could help more efficiently triage of suspected cases of novel coronavirus, helping to focus limited resources on enabling appropriate use of quarantine. a handful of diagnostics developers are now striving to bring fast sars-cov-2 tests to market as soon as possible, with hopes of ultimately assisting with the ongoing outbreak in china. molecular diagnostic tests for use at the are in development from cepheid and hibergene (dublin, ireland). cepheid has some advantages in the molecular poct space because it already has instruments placed in china. mobidiag, meanwhile, may offer additional benefits with a multiplex test for coronavirus and flu viruses (https://www. genomeweb.com/pcr/diagnostics-firms-rush-developrapid-point-care-tests-novel-coronavirus#.xkea3sgzy 2x. accessed 15 february 2020). mjl and y-wt are employees of cepheid, the commercial manufacturer of the xpert xpress sars-cov-2 test. figure 1 . evolutions in molecular testing procedures. the point-of-care test (poct) devices incorporate nucleic acid extraction, amplification and detection together into an integrated and sealed cartridge making it simple, rapid and safe. during end-point pcr, dna is detected or measured at the completion of pcr amplification, requiring post-pcr processing. real-time pcr is a closed-tube system in which dna is detected or measured during the exponential phase of amplification. epidemiology, genetic recombination, and pathogenesis of coronaviruses a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle a novel coronavirus from patients with pneumonia in china the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method the role of super-spreaders in infectious disease covid-19: epidemiology, evolution, and cross-disciplinary perspectives early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with 2019 novel coronavirus in wuhan updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study characteristics of covid-19 infection in beijing first case of 2019 novel coronavirus in the united states diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement clinical characteristics of coronavirus disease 2019 in china practical guidance for clinical microbiology laboratories: viruses causing acute respiratory tract infections simple method for combining sputum and nasal samples for virus detection by reverse transcriptase pcr detection of sars-cov-2 in different types of clinical specimens viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome middle east respiratory syndrome coronavirus infection dynamics and antibody responses among clinically diverse patients, saudi arabia kinetics and pattern of viral excretion in biological specimens of two mers-cov cases persistent shedding of viable sars-cov in urine and stool of sars patients during the convalescent phase consistent detection of 2019 novel coronavirus in saliva growth and intracellular development of a new respiratory virus cultivation of a novel type of common-cold virus in organ cultures a highly specific rapid antigen detection assay for on-site diagnosis of mers detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzymelinked immunosorbent assay differentiation between human coronaviruses nl63 and 229e using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies immunofluorescence assay for detection of the nucleocapsid antigen of the severe acute respiratory syndrome (sars)-associated coronavirus in cells derived from throat wash samples of patients with sars comparison of immunofluorescence with monoclonal antibodies and rt-pcr for the detection of human coronaviruses 229e and oc43 in cell culture evaluation of a commercial colloidal gold assay for detection of influenza a and b virus in children's respiratory specimens rapid detection and monitoring of human coronavirus infections examination of seroprevalence of coronavirus hku1 infection with s protein-based elisa and neutralization assay against viral spike pseudotyped virus seroepidemiology of group i human coronaviruses in children molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia rna based mngs approach identifies a novel human coronavirus from two individual pneumonia cases in 2019 wuhan outbreak a new coronavirus associated with human respiratory disease in china identification of a novel coronavirus causing severe pneumonia in human: a descriptive study co-infection with sars-cov-2 and influenza a virus in patient with pneumonia laboratory diagnosis of respiratory tract infections in children -the state of the art molecular diagnosis of severe acute respiratory syndrome: the state of the art emerging molecular assays for detection and characterization of respiratory viruses molecular assays for the detection and characterization of respiratory viruses no novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide codehop-based primers clinical disease in children associated with newly described coronavirus subtypes characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia multicenter evaluation of the eplex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital clinical evaluation of the luminex nxtag respiratory pathogen panel positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in transmission of 2019-ncov infection from an asymptomatic contact in germany detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan establishing ebola virus disease (evd) diagnostics using genexpert technology at a mobile laboratory in liberia: impact on outbreak response, case management and laboratory systems strengthening point-of-care testing for infectious diseases: past, present, and future evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b profile of the alere i influenza a & b assay: a pioneering molecular pointof-care test parallel validation of three molecular devices for simultaneous detection and identification of influenza a and b and respiratory syncytial viruses performance and impact of a clia-waived, point-of-care respiratory pcr panel in a pediatric clinic identification of a new human coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays key: cord-254976-la9g6g5t authors: kim, ji soo; choi, jeong sil title: factors influencing emergency nurses' burnout during an outbreak of middle east respiratory syndrome coronavirus in korea date: 2016-11-09 journal: asian nurs res (korean soc nurs sci) doi: 10.1016/j.anr.2016.10.002 sha: doc_id: 254976 cord_uid: la9g6g5t purpose: emergency department (ed) nurses suffer from persistent stress after experiencing the traumatic event of exposure to middle east respiratory syndrome coronavirus (mers-cov), which can subsequently lead to burnout. this study aimed to assess ed nurses' burnout level during an outbreak of mers-cov and to identify influencing factors in order to provide basic information for lowering and preventing the level of burnout. methods: study participants were ed nurses working in eight hospitals designated for treating mers-cov-infected patients in korea. we performed multiple regression analysis to explore the factors influencing burnout. results: the ed nurses' burnout was affected by job stress (β = 0.59, p < .001), poor hospital resources for the treatment of mers-cov (β = −0.19, p < .001) and poor support from family and friends (β = −0.14, p < .05). these three variables explained 47.3% of the variance in burnout. conclusions: ed nurses taking care of mers-cov-infected patients should be aware that burnout is higher for nurses in their divisions than nurses in other hospital departments and that job stress is the biggest influential factor of burnout. to be ready for the outbreak of emerging contagious diseases such as mers-cov, efforts and preparations should be made to reduce burnout. job stress should be managed and resolved. working conditions for mitigating job stress and systematic stress management programs should be provided, and hospital resources for the treatment of mers-cov need to be reinforced. moreover, promoting support from family and friends is required. the middle east respiratory syndrome coronavirus (mers-cov) is an emerging infectious disease that infects the respiratory system. since its first outbreak in saudi arabia, the number of affected countries has been increasing [1, 2] . korea had its first mers-cov infected patient in may 2015. as of july 30, 2015, the total number of patients in the country diagnosed with the disease was 186. of these, 33 patients have died, and 153 patients have been treated [3] . most mers-cov-infected patients first visit a hospital's emergency department (ed). consequently, ed nurses are the first healthcare professionals to care for patients infected with the novel contagious disease. in fact, there were many cases of exposure to the disease in eds during the mers-cov outbreak [3] . compared to nurses in other areas, ed nurses are faced with hectic, unpredictable, and ever-changing situations [4] . because they deal with various diseases, traumatic events, and urgent situations, they do not have enough time for recovery, putting them under persistent stress. as a result, ed nurses are reported to experience much higher burnout than nurses in other hospital departments [5e7] . burnout is a longterm consequence of prolonged exposure to certain job demands and a reaction that appears when a person can no longer endure the stress they have been undergoing [8] . it is a syndrome of physical, mental, and emotional exhaustion that includes a negative selfconcept and work attitude, and a reduced interest in patients [8] . it has also been strongly associated with working conditions in nursing [5e8], leading it to be the focus of many nursing studies [4e10] . previous studies of ed nurses' burnout have focused on the severity of burnout or its influencing factors [4e7] . according to a systematic review of 17 reports on emergency room (er) nurses' burnout published over the past 25 years, 26.0% of them suffered from burnout [5] . although varying between studies, the factors influencing burnout have been largely divided into individual factors and work-related factors [5, 9] . individual factors reported in previous studies include demographic characteristics such as gender, age, religion, education level [5, 9] , having children, living with family [10] , job stress [7, 11] , personality, coping strategies, and job attitude [5, 9] . major work-related influencing factors are exposure to traumatic events, level of wages, social support, staffing, and lack of material resources [5, 9] . a nationwide outbreak of mers-cov is an unfamiliar traumatic event for ed nurses, so there has been little research into mers-covrelated burnout. similarly, when severe acute respiratory syndrome such as sars spread rapidly, most nurses experienced severe stress, and some nurses refused to care for patients [12, 13] . in addition, the factors influencing nurses' desire to leave their jobs during an outbreak of sars were identified as the perceived risk of fatality from sars, tenure, work stress, and social relationships [13] . when h5n1 avian flu spread rapidly, taiwanese nurses' fear of infection from the disease was a significant factor influencing their willingness to care for patients infected with the avian flu [14] . since an emerging respiratory infectious disease such as mers-cov can happen anywhere in the world, nursing managers need to pay attention to ed nurses' burnout in association with their experiences of a nationwide mers-cov outbreak. however, not even a basic survey has been conducted on the level of burnout experienced by ed nurses, who are in the front line taking care of mers-cov patients, or factors of burnout that consider the specific nature of mers-cov. thus, this study attempted to assess ed nurses' burnout level during an outbreak of mers-cov and to identify influencing factors in order to provide basic information for lowering and preventing the level of burnout. this was a cross-sectional design study conducted to identify the factors influencing mers-cov-related burnout in ed nurses who had experienced an outbreak of mers-cov in korea. during the outbreak of mers-cov in korea, 15 hospitals designated for treating mers-infected patients in seoul, gyeonggi-do, and incheon, korea. the participants were drawn from nurses working in the eds of these hospitals. this study used convenience sampling to select eight eds, all of which gave their consent to the survey. the sample size was estimated using g*power 3.1 [15] . with an input a at .05, a medium effect size of .15, power of .90, and the number of predictors at 18 for a linear multiple regression analysis, the minimum sample size required for this study was 183. however, for an even distribution of the participants among the hospitals selected, this study sampled 30 ed nurses from each institution by simple random sampling, for a total of 240 ed nurses. of all the participants, 223 replied to the survey (response rate 92.9%). after unanswered questionnaires were excluded, 215 questionnaires were used as valid data in this study. approval for conducting this study was obtained from the institutional review board of gachon university in korea (no. 1044396-201504-hr). burnout [16] and job stress [17] were measured with a scale from previous studies, which was translated into korean, validated, and used by choi et al [18] . other scales were developed by the researcher through a literature review [5, 9, 13, 14] and the developed tools were tested for content validity by two infection control nurse practitioners, one infectious disease specialist (doctor), and one nursing professor. the developed tools were translated into korean by two phd-prepared bilingual nursing faculty and then back translated into english. back translation was performed by a separate professional translator, who did not have prior information about the scale. in addition, the scales were tested through a pilot study with 15 ed nurses. mers-cov-related burnout was assessed using the oldenburg burnout inventory (olbi) developed by demerouti et al [16] . in order to limit the study to burnout related to mers-cov, the phrase "caused by mers-cov" was added to each item. the olbi consists of 16 items in two subdomains: emotional exhaustion and disengagement from work. each item is answered on a 5-point scale ranging from 1 (strongly disagree) to 5 (strongly agree), with a high score meaning a high level of burnout. cronbach a of the scale was .78 in the previous study, .78 in the preliminary survey, and .80 in the main survey. mers-cov-related job stress was assessed by measuring the pressure from time and anxiety with a scale developed by parker and decotiis [17] . in order to limit the study to job stress related to mers-cov, the phrase "caused by mers-cov" was added to each item. this scale consists of nine items, with each item answered on a 5-point scale ranging from 1 (strongly disagree) to 5 (strongly agree). a high score means a high level of stress. cronbach a of the scale was .78 in the previous study, .90 in the preliminary survey, and .93 in the main survey. the scale for fear of mers-cov infection was developed by the researcher based on a previous study of nurses' fear during the outbreak of h5n1 avian flu [14] . this scale has just one item, "i am afraid of being infected with mers-cov," which is answered on a 10-point visual analogue scale. a high score means a high fear of mers-cov infection. the scale for measuring hospital resources for the treatment of mers-cov was developed by the researcher based on previous studies reporting material resources as one of the influencing factors of burnout [5, 9, 13, 14] . in this scale, each item is answered on a 4-point scale ranging from 1 (strongly disagree) to 4 (strongly agree), with a high score meaning satisfactory hospital resources for the treatment of mers-cov are available. the three items of this scale are as follows: "my hospital is equipped with facilities sufficient for preventing the spread of mers-cov," "my hospital applies the best infection control guideline for preventing the spread of mers-cov," and "my hospital discusses how to prevent mers-cov regularly." cronbach a of the scale was .78 in the preliminary survey and .81 in the main survey. content validity index was .95. the scale for measuring support from family and friends was developed by the researcher based on previous studies reporting social support as one of the influencing factors of burnout [5, 9, 13, 14] . in this scale, each item is answered on a 4-point scale ranging from 1 (strongly disagree) to 4 (strongly agree), with a high score meaning high support from family and friends. the four items of this scale are as follows: "my friends will avoid me if they find that i have cared for mers patients," "my friends will support me caring for mers patients," "my family will avoid me if they find that i have cared for mers patients," and "my family will support me caring for mers patients." cronbach a of the scale was .76 in the preliminary survey and .80 in the main survey. content validity index was .90. data were collected during the period from july 20, 2015 to july 31, 2015, about 2 months after the outbreak of mers-cov and when the disease had not yet been controlled. the researcher visited the eight convenience-sampled hospitals designated for treating mers-cov patients, explained the purpose of this study, obtained their consent, and delivered the questionnaires. the charge nurse of the relevant department in each hospital explained the purpose of the study to the nurses, obtained their written consent, and then distributed and collected the questionnaires. the whole process of the survey was conducted anonymously, and all personal information was kept confidential. the data were collected and analyzed using spss for windows version 21.0 (ibm corp., armonk, ny, usa), and the normal distribution of the main variables was confirmed before analysis (kolmogorov-smirnov test). the participants' general characteristics, mers-cov-related burnout, job stress, fear of mers infection, available hospital resources for the treatment of mers-cov, and support from family and friends were analyzed with frequencies, percentages, means, and standard deviations. scale reliability was assessed with cronbach a. differences in burnout according to general characteristics were analyzed using independent t tests, analysis of variance, and scheffe's post hoc test. correlation was computed using pearson's correlation test. multiple regression was performed using the enter method with input variables found to be significant in the difference testing and correlation analysis, to explore factors influencing mers-cov-related burnout. the participants' mean age was 28.17 years, and 201 (93.5%) were female. in addition, 172 (80.0%) were unmarried, and 128 (59.5%) had at least a bachelor's degree. the mean length of clinical experience was 2.58 years. most of the participants (183, 85.1%) were working under the three-shift system, and 119 (55.3%) had actual experience in caring for mers-cov-infected or mers-covsuspected patients. the mean number of hospital beds was 857.37. the level of mers-cov-related burnout was found to be significantly higher in nurses who worked a three-shift system, and in those who had nursed mers-cov-infected or mers-covsuspected patients than those who did not (p < .05) ( table 1) . the mean score of mers-cov-related burnout was 3.02 out of 5; mers-cov-related job stress was 3.25 out of 5; fear of mers-cov infection was 6.71 out of 10; hospital resources for the treatment of mers-cov was 2.88 out of 4; support from family and friends was 2.49 out of 4 ( table 2) . mers-cov-related burnout was significantly correlated with mers-cov-related job stress, fear of mers-cov infection, availability of hospital resources for the treatment of mers-cov, and support from family and friends (p < .05). however, it was not significantly correlated with age, number of beds, or length of clinical experience. mers-cov-related job stress was found to be the biggest influencing factor of mers-cov-related burnout (b ¼ .59, p < .001), with the level of mers-cov-related burnout higher when job stress was high. in addition, poor hospital resources for the treatment of mers-cov (b ¼ à0.19, p < .001) and poor support from family and friends (b ¼ à0.14, p < .05) increased mers-cov-related burnout. these three variables explained 47.3% of the variance in mers-covrelated burnout ( table 3) . the regression analysis satisfied the basic assumption of the model. the durbin-watson statistic was 1.75, indicating that there was no autocorrelation. tolerance was .78e.98, which was higher than .10; the variance inflation factor was 1.02e1.55, which was smaller than 10, indicating that there was no multicollinearity problem. this study was meaningful in that it surveyed factors influencing mers-cov-related burnout in ed nurses who experienced the traumatic event of a mers-cov outbreak. the study adapted scales from previous studies to measure fear of mers-cov infection [13, 14] , social support, and lack of material resources [5, 9] . during the outbreak of mers-cov in korea, one super-spreader (a host, an organism infected with a disease that infects disproportionally more secondary contacts than other hosts also infected with the same disease) had contact with 594 people in an emergency department and transmitted mers-cov to 85 of them [3] . as such, it is highly likely that ed nurses who dealt with him or her then were more inclined to experience burnout when other enduring stress situations arise [13] . the burnout level in ed nurses who experienced the mers-cov outbreak was 3.02 on a 5-point scale (60.4 on a 100-point scale). the olbi, a burnout scale suggested by demerouti et al [18] , is an adequate scale for assessing the emotional aspects of exhaustion, including its physical and cognitive elements [16,18e20] . the study of hopper et al [6] , which surveyed er nurses' burnout level using maslach's burnout inventory, reported that 22.4% and 19.2% of ed nurses experienced a high level of burnout, respectively. in particular, a systematic review of 17 studies that surveyed burnout in ed nurses reported that 25.9% of them experienced emotional exhaustion exceeding the cut-off score [5] . another study reported that burnout among ordinary nurses, midwives and nurses with some sort of specialization surveyed using the olbi, was 2.40 on a 5-point scale [21] . accordingly, the burnout level surveyed in this study using the olbi shows that the participants' experience of burnout was above average compared to other nurses dealing with burnout in general. this suggests a need to pay particular attention to burnout in er nurses who are on the front line in treating mers-cov-infected or mers-cov-suspected patients. this also suggests the necessity for programs designed to help manage and control burnout levels in ed nurses during the outbreak of an infectious disease like mers-cov. in the univariate analysis, mers-cov-related burnout was significantly higher in nurses working under the three-shift system and in those with experience in caring for mers-covinfected or mers-cov-suspected patients. in the multiple regression analysis, the three-shift system was not a significantly influential factor of burnout. shift work is a major factor in increasing mers-cov-related burnout not only for er nurses but also for nurses in other departments [22] . however, in some studies, shift work was not a factor influencing burnout [5, 9] . in this study, although shift work did not show a significant relationship with mers-cov-related burnout for ed nurses, it may have an indirect effect on mers-cov-related burnout. therefore, the ed nurses who came into contact with mars-cov-related patients, their burnout need to be monitored by reducing their shift work. previous studies have reported that repeated exposure to traumatic events causes burnout in nurses [23, 24] . it has also been found that when ed nurses have been exposed to traumatic events, they experience higher emotional exhaustion than those without such exposure [25] . consistent with previous reports, the burnout level in this study was significantly higher in ed nurses who cared for mers-cov-infected or mers-cov-suspected patients during the nationwide outbreak of the disease. this finding supports the proposal that the outbreak of mers-cov was a traumatic event for which nurses' burnout needs to be managed. previous studies have reported demographic characteristics such as gender, age, religion, education level [5, 9] , having children, and living with family [10] as factors influencing burnout. in other studies, however, burnout was not significantly correlated with gender, age, experience, shift work, education level, or religion [6] , and financial reward was not an influencing factor for ed nurses' burnout [1] . because previous studies report conflicting results related to gender, age, education level, and experience, replication studies should be continuously conducted [5, 9] . stress is a significant influencing factor that correlates with burnout in previous studies [7, 11] . in this study, job stress induced by the outbreak of mers-cov was also found to be the biggest influencing factor, showing a correlation with burnout, that is, the burnout level was higher when job stress was high. thus, during the outbreak of an emerging infectious disease such as mers-cov, the continuous inflow of patients and the highly infectious nature of the disease increase ed nurses' stress, which in turn, aggravates their burnout [7] . in order to manage ed nurses' burnout, efforts should be made to find the sources of stress and to resolve them. in addition to mers-cov-related job stress, the second and third biggest influencing factors increasing mers-cov-related burnout were poor hospital resources for the treatment of mers-cov and poor support from family and friends. this finding is consistent with the systematic review [5] and literature review [7] , which reported that hospital resources and support from family and friends were major influencing factors for ed nurses' burnout [5, 7] . in particular, a lack of material resources is reported to be correlated with a high level of burnout [22] . these findings suggest that, in preparation for the outbreak of an emerging infectious disease, hospitals should prepare facilities for preventing infection, establish systematic infection control guidelines, and continue discussions about preventive measures [13, 14] . what is more, support from family and friends experienced as more extensive social support, was found to be another influencing factor, as reported in previous studies [5, 9] . in the context of employment, typical sources of social support are coworkers, supervisors, and the organization in general. social support from one's supervisor and colleagues is found to provide a buffering effect that directly or indirectly reduces job stress [5, 9, 12, 14] . accordingly, it is suggested that the aspect of social support from an employee's supervisor and colleagues should be taken into consideration for further research in future studies. if social support is provided in consideration of national and cultural factors, it may reduce burnout in nurses caring for patients in such a national crisis as the outbreak of mers-cov [12, 14] . the principles of mers-cov infection prevention and control strategies associated with healthcare suggest the need for administrative controls and hospital resources [2] . in addition, if the government, with strong leadership, develops stronger and more resilient health systems in preparation against emerging contagious diseases such as mers, ed nurses' mers-cov-related burn out and job stress will decrease. one limitation of this study is that the scope of research was confined to the outbreak of mers-cov in one country. as such, it is necessary to expand the number of study participants for comparison with cases in other countries. furthermore, as most of the survey respondents were female, future studies need to include more male nurses so as to examine gender characteristics. in addition, factors such as personality, coping strategies, and job attitude could be surveyed as additional influencing factors of burnout. lastly, we also suggest that a mixed-method research design combining cross-sectional surveys and in-depth interviews be adopted for an in-depth inquiry into mers-cov-related burnout. during the outbreak of an emerging infectious disease such as mers-cov, nursing managers need to pay attention to burnout in ed nurses, who are the first to treat patients. the participants' mers-cov-related burnout was higher than the average of nurses dealing with burnout in general. the biggest influencing factor of mers-covrelated burnout was mers-cov-related job stress, followed by availability of hospital resources for the treatment of mers-cov and support from family and friends. these three variables explained 47.3% of the variance in mers-cov-related burnout. the outcome of this study is expected to provide basic information related to ed nurses' burnout in connection to an outbreak of an emerging infectious disease such as mers-cov and to contribute to programs and strategies for reducing burnout. in order to lower the level of burnout, nursing managers need to make efforts to reduce job stress, to reinforce hospital resources for the treatment of mers-cov, and to promote support from family and friends. particularly in the area of ed nursing, it is essential that we develop effective and systematic burnout management programs for monitoring and preventing burnout in preparation against possible future outbreaks of infectious diseases. the authors declared no conflict of interest. middle east respiratory syndrome (mers) department of health and human services clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected middle east respiratory syndrome (mers) violence against nurses and its impact on stress and productivity determinants and prevalence of burnout in emergency nurses: a systematic review of 25 years of research compassion satisfaction, burnout, and compassion fatigue among emergency nurses compared with nurses in other selected inpatient specialties to what extent do nurses and physicians working with emergency department experience burnout: a review of the literature job burnout factors that influence the development of compassion fatigue, burnout, and compassion satisfaction in emergency department nurses psychosocial work environment and burnout among emergency medical and nursing staff burnout in relation to specific contributing factors and health outcomes among nurses: a systematic review sars risk perception, knowledge, precautions, and information sources, the netherlands factors predicting nurses' consideration of leaving their job during the sars outbreak nurses' fears and professional obligations concerning possible human-to-human avian flu power 3: a flexible statistical power analysis power analysis program for the social, behavior, and biomedical sciences the convergent validity of two burnout instruments: a multitrait-multimethod analysis organizational determinants of job stress burnout and work engagement: a thorough investigation of the independency of both constructs affecting factors of nurses' burnout in secondary general hospitals linking job demands and resources to employee engagement and burnout: a theoretical extension and metaanalytic test exploring within-and between-gender differences in burnout: 8 different occupational groups the relationship between psychosocial job stress and burnout in emergency department: an exploratory study occupational risk factors in the emergency medical services correspondence the prevalence and impact of post traumatic stress disorder and burnout in nurses ambulance personnel and critical incidents: impact of accident and emergency work on mental health and emotional well-being the authors wish to thank all the ed nurses who so willingly participated in this study. key: cord-268388-kkhuzf3p authors: sharif-yakan, ahmad; kanj, souha s. title: emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives date: 2014-12-04 journal: plos pathog doi: 10.1371/journal.ppat.1004457 sha: doc_id: 268388 cord_uid: kkhuzf3p nan two years have passed since the initial description of the middle east respiratory syndrome coronavirus (mers-cov), yet the epidemic is far from being controlled. the high case fatality rate, the recent steep increase in reported cases, and the potential to cause a global pandemic during the upcoming hajj season are serious concerns. although a wealth of information about the pathophysiology, proposed animal reservoir, and intermediate host has been revealed, many questions remain unanswered. we herein review mers-cov, covering its proposed origins, route of transmission, treatment options, and future perspectives. first reported in 2012 [1] , middle east respiratory syndrome coronavirus (mers-cov) is a novel coronavirus and the first lineage 2c betacoronavirus known to infect humans [2] . with a case fatality rate of 35%, an urgent response is needed to prevent a global pandemic [3] . prior to 2003, coronaviruses were not considered serious human pathogens since they only caused mild upper respiratory tract infections (urtis) [4] . the first zoonotic introduction of a coronavirus into the human population occurred with the severe acute respiratory syndrome coronavirus (sars-cov) in 2002. sars-cov caused a global pandemic, with 8,400 recorded cases and 800 deaths [5] . mers-cov marks the second known zoonotic introduction of a highly pathogenic coronavirus, probably originating from bats. three lines of evidence currently support this theory: (1) the very close phylogenetic similarity with the bat betacoronaviruses: btcov-hku4 and btcov-hku5 [6] ; (2) closely related coronavirus sequences have been recovered from bats in africa, asia, the americas, and eurasia; and (3) mers-cov uses the evolutionary conserved dipeptidyl peptidase-4 (dpp4) protein in pipistrellus pipistrellus bats for cell entry [7] . since human-bat contact is limited, camels have been implicated as probable intermediate hosts. mers-cov appears to have been circulating in dromedary camels for over 20 years [8] . mers-cov uses the dpp4 (cd26) receptor to gain entry and effectively replicate in camel cell lines [9] . neutralizing antibodies for mers-cov have been detected in dromedary camels from oman, canary islands, egypt, jordan, united arab emirates, and saudi arabia [10, 11] . the exact mode of transmission from camels to humans remains to be confirmed [12] . camel milk was investigated as a possible route of transmission, given the common practice of consuming camel milk in the arabian peninsula. the first reported case of mers-cov in yemen occurred in a yemeni pilot who consumed raw camel milk [13] , and reusken et al. reported the finding of mers-cov in camel milk in qatar [14] . however, respiratory transmission is currently considered as the most likely route of transmission [15] . mers-cov has been detected by reverse transcription pcr (rt-pcr) from the nasal swabs of three camels in qatar and was linked to two confirmed human cases with high similarity upon sequencing, suggesting a possible respiratory mode of transmission [16] . several clusters of mers-cov cases have been reported, mainly among household members and health care workers (hcws), suggesting that transmission is through close contact. the largest cluster reported so far has been in 23 hcws in a hospital in al hasa, kingdom of saudi arabia (ksa), while the largest family cluster has been in three infected brothers from riyadh, ksa [17, 18] . the basic reproductive rate for mers-cov has still not been determined with certainty [11] . using two transmission scenarios, breban et al. reported an r 0 of 0.60 and 0.69 [18] . cauchemez et al. reported a similar r 0 at 0.63, but warned that in the absence of infection control measures, r 0 may range from 0.8-1.3 and could lead to a self-sustaining transmission [19] . propensity for the mers-cov to replicate in the lower respiratory tract may account for the observed limited transmission [20] . the united states centers for disease control and prevention (cdc) recommends standard contact and airborne precautions with the use of an n-95 mask when caring for an infected patient [21] . the cdc defines a laboratory-confirmed case of mers-cov as a patient with a positive pcr from a respiratory sample, and a probable case as a patient who had close contact with a confirmed case but inconclusive laboratory evidence [22] . the incubation period for the presentation of mers-cov symptoms is 2-14 days and it remains unknown whether patients are infectious during the incubation period [23] . the average age of presentation is 50 years, with a male predominance [24] . clinically, mers-cov causes symptoms of upper and lower rtis [23] . the severity of symptoms varies widely. most asymptomatic cases have been discovered through screening after contact with a known case [2] . presenting signs and symptoms may include highgrade fever, non-productive cough, dyspnea, headache, myalgia, nausea, vomiting, and diarrhea that may precede the respiratory symptoms [25, 26] . renal failure has been frequently reported, yet no conclusive evidence of a direct viral invasion of renal tissues exists [11, 27, 28] . notably, most patients who developed complications had coexisting medical co-morbidities [11] . laboratory findings on admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels [25] . mers-cov can also cause severe pneumonia with acute respiratory distress syndrome (ards), requiring mechanical ventilation and intensive care admission [24] . to date, there is still a lack of surgical and pathological information from patients infected with mers-cov, which hampers full understanding of the pathogenesis. lastly, coinfection with other respiratory viruses and with community-acquired bacteria has been also reported in mers-cov patients [11, 29, 30] . as of june 26, 2014, who officially reported 707 affected patients in 21 countries in three continents. two-hundred and fifty-two patients have died of mers-cov, setting the case fatality rate at 35% [3] . the cases so far have been acquired either directly through a probable zoonotic source, or as a result of human-human transmission via close contact. retrospective analysis tracked the first outbreak to a hospital in the city of al-zarqa in jordan in april 2012 [31] . an unexplained observation has been the seasonal variation in reported numbers, with a peak between april and june of each year. the number of cases reported during april 2014 alone was alarming, because it was greater than the cumulative number of cases reported since the outbreak began [32] . the recent increase in the number of infected patients may arguably be attributed to better case detection and active surveillance programs. yet other factors may have contributed to the observed surge, including suboptimal infection control practices in affected hospitals in saudi arabia, as documented in a recent report of the who mission to jeddah [33] . another explanation for the seasonal variation may be that it coincides with camel birthing season, and younger camels seem to be more often infected than their older counterparts [34] . the distribution of the total reported cases by country is as follows: 85.8% in the ksa, 8.1% in the united arab emirates, 1.7% in jordan, and 1% in qatar [32] . cases have also been reported in kuwait, yemen, oman, iran, lebanon, tunisia, algeria, bangladesh, malaysia, france, italy, germany, the netherlands, united kingdom, greece, italy, and the united states [32] . furthermore, seropositive camels for mers-cov were detected in egypt, kenya, nigeria, tunisia, and ethiopia, suggesting that there may be mers-cov cases that are undetected in africa [8, 35, 36] . in 2012, 3,161,573 muslims from 188 countries gathered in mecca to perform the annual hajj, the largest gathering of muslims in the world. the identification of mers-cov in saudi arabia has generated international concern of a global pandemic. as a response, the saudi government requested that elderly and chronically ill muslims avoid hajj in 2013 and restricted the number of pilgrims to 2,061,573. consequently, no cases were reported during the pilgrimage of that year [37] . nasopharyngeal specimens collected from 5,235 pilgrims revealed no cases of mers-cov nasal carriage [38] . a prospective cohort study of 129 french pilgrims did not reveal any mers-cov cases [39] . nevertheless, the potential for spreading of mers-cov during the 2014 hajj season (october 2-6) remains possible, especially since documented transmission occurred this year in patients from iran and malaysia after their return from umrah in mecca. it is worth noting that the two most frequently visited cities during the hajj, mecca and medina, have so far reported 32 and 35 cases respectively [32] . mers-cov binds to the dpp4 (cd26) surface receptor using the spike (s) surface protein with subsequent cell entry [40] . the exact mechanism of entry after receptor binding is still unknown. the s surface protein is composed of a core subdomain that shares similarity with that of sars-cov and a receptor binding subdomain (rbsd) that exhibits significant variation from the sars-cov rbsd. the development of vaccines targeting the rbsd of mers-cov is currently under investigation because they are thought to be safer and more effective than vaccines based on inactivated virus, dna, or viral vectors [40] . another potential therapeutic approach is the inhibition of the papain-like and/or 3c-like protease of mers-cov [41] . to date, no effective therapy or prophylaxis for mers-cov exists. supportive therapy remains the cornerstone of management. current treatment is based on previous experience with the sars-cov, in-vitro studies, and case series. various agents have been tried, including those that block virus entry, inhibit viral replication, or interfere with host immune response [42] . the international severe acute respiratory and emerging infection consortium (isaric) suggested therapeutic options for treatment of mers-cov infection with various agents alongside continuous evaluation of efficacy, and in the setting of clinical trials [43] . based on experience with sars-cov, the use of convalescent plasma, hyper-immune globulin, or human monoclonal antibodies that contain neutralizing antibodies may be efficacious and is recommended as first-line treatment when available [43] . ribavirin and interferon alpha-2b both showed promising results, especially when used in combination, both in vitro and in animal studies using rhesus macaques monkeys [44] . however, these positive results did not translate clinically in an observational study in five patients, all of whom succumbed to the infection [45, 46] . repurposing of currently available agents may be an efficient approach. dyall et al. screened various agents with potential therapeutic efficacy [46] . cyclosporin a, mycophenolic acid, interferon-beta, homoharringtonine, cycloheximide, anisomycin, and emetine dihydrochloride hydrate were found to have the most potent in vitro activity against mers-cov. despite the progress in our understanding of mers-cov, many questions remain unanswered. the definitive origin, exact mechanism of transmission, and the reason behind seasonal variability are still unclear. although most cases have been described in countries of the arabian peninsula, the increasing travel to the region and the hajj season in ksa pose a threat of a potential global pandemic. extensive efforts are required to speed up the development of an effective therapy and vaccine. repurposing of currently available pharmaceutical agents is highly desirable for a more rapid drug development. meanwhile, hcws who encounter patients with respiratory symptoms who have lived or traveled to areas with mers-cov should have a low threshold to consider a diagnosis of mers-cov, with testing and immediate implementation of proper infection control practices to prevent further spread. finally, given the important role that camels may play in transmission of the virus, the common practices in the arabian peninsula of herding and consuming unpasteurized camels' milk should be discouraged until conclusive evidence is obtained that such practices do not contribute to infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers: emergence of a novel human coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 26 identification of a novel coronavirus in patients with severe acute respiratory syndrome consensus document on the epidemiology of severe acute respiratory syndrome (sars) genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc antibodies against mers coronavirus in dromedary camels replicative capacity of mers coronavirus in livestock cell lines middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study the emergence of the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? global alert and response: middle east respiratory syndrome coronavirus (mers-cov) -update middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels, qatar middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation hospital outbreak of middle east respiratory syndrome coronavirus interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques interim infection control and prevention recommendations for hospitalized patients with mers-cov mers case definition mers clinical update from the idsa center for disease control and prevention cdc epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission family cluster of middle east respiratory syndrome coronavirus infections a patient with severe respiratory failure caused by novel human coronavirus a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection epidemiological findings from a retrospective investigation who concludes mers-cov mission in saudi arabia mers-cov enigma deepens as reported cases surge geographic distribution of mers coronavirus among dromedary camels mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 20 prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj lack of mers coronavirus but prevalence of influenza virus in french pilgrims after 2013 hajj the receptor binding domain of mers-cov: the dawn of vaccine and treatment development proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease what are our pharmacotherapeutic options for mers-cov? international severe acute respiratory & emerging infection consortium (isaric)-clinical decision making tool for treatment of mers-cov v.1.1 an animal model of mers produced by infection of rhesus macaques with mers coronavirus ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection the authors would like to acknowledge sima l. sharara for editing the manuscript. key: cord-264956-wbi0ird5 authors: ahmed, anwar e.; alshukairi, abeer n.; al‐jahdali, hamdan; alaqeel, mody; siddiq, salma s.; alsaab, hanan a.; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; aloudah, nouf m.; baharoon, salim; alsalamah, majid a.; al johani, sameera; alghamdi, mohammed g. title: development of a risk‐prediction model for middle east respiratory syndrome coronavirus infection in dialysis patients date: 2018-04-14 journal: hemodial int doi: 10.1111/hdi.12661 sha: doc_id: 264956 cord_uid: wbi0ird5 introduction the middle east respiratory syndrome coronavirus (mers‐cov) infection can cause transmission clusters and high mortality in hemodialysis facilities. we attempted to develop a risk‐prediction model to assess the early risk of mers‐cov infection in dialysis patients. methods this two‐center retrospective cohort study included 104 dialysis patients who were suspected of mers‐cov infection and diagnosed with rrt‐pcr between september 2012 and june 2016 at king fahd general hospital in jeddah and king abdulaziz medical city in riyadh. we retrieved data on demographic, clinical, and radiological findings, and laboratory indices of each patient. findings a risk‐prediction model to assess early risk for mers‐cov in dialysis patients has been developed. independent predictors of mers‐cov infection were identified, including chest pain (or = 24.194; p = 0.011), leukopenia (or = 6.080; p = 0.049), and elevated aspartate aminotransferase (ast) (or = 11.179; p = 0.013). the adequacy of this prediction model was good (p = 0.728), with a high predictive utility (area under curve [auc] = 76.99%; 95% ci: 67.05% to 86.38%). the prediction of the model had optimism‐corrected bootstrap resampling auc of 71.79%. the youden index yielded a value of 0.439 or greater as the best cut‐off for high risk of mers infection. discussion this risk‐prediction model in dialysis patients appears to depend markedly on chest pain, leukopenia, and elevated ast. the model accurately predicts the high risk of mers‐cov infection in dialysis patients. this could be clinically useful in applying timely intervention and control measures to prevent clusters of infections in dialysis facilities or other health care settings. the predictive utility of the model warrants further validation in external samples and prospective studies. an important lesson was learned from the world's largest middle east respiratory syndrome coronavirus (mers-cov) outbreaks that occurred in saudi arabia and south korea: that health care-associated infection is a major cause of rapid pathogen spread in health care settings with a high risk of cluster infections. in particular it was discovered that they spread rapidly in hemodialysis, inpatient, emergency, and intensive care facilities. [1] [2] [3] [4] [5] [6] dialysis patients were associated with a high risk of mortality 6 compared to the national mortality estimates in the mers-cov population. 7, 8 assiri et al. were able to track hospitals, units, rooms, beds, symptoms onset, and diagnoses status to map a large cluster of infections between april 1 and may 23, 2013. 1 according to the authors, the clusters developed in the hemodialysis facility, where 1 health careassociated infected patient who underwent long-term hemodialysis transmitted the virus to 7 dialysis patients and the transmission then continued to other hospital settings. 1 in a recent study, assiri et al. reported a high likelihood of transmission in dialysis patients and health care workers within the outpatient dialysis facility. 6 among 186 laboratory-confirmed mers-cov patients in south korea, only 1 dialysis patient was identified, but no cluster viral transmissions were identified in other patients who utilized the same hemodialysis facility. 9 park et al. developed a guideline to control cluster infections and prevent mers outbreaks in hemodialysis facilities. 9 earlier studies on dialysis patients focused on virus transmission and clinical outcomes, while limited by the small number of mers cases. our understanding of early diagnoses of mers and identifying patients at high risk of infection is incomplete, 10 particularly in a hemodialysis facility. a mers-cov risk assessment tool is urgently needed to accurately identify dialysis patients at high risk of infection and apply infection control measures to prevent future cluster transmission in these patients and patients in other health care facilities. exploring an efficient screening system to detect mers-cov infection at an earlier stage may result in immediate isolation 11 and improve clinical outcomes and economic burdens. 12, 13 a valid risk-predictive model for mers-cov infection in dialysis patients may increase the likelihood of early virus detection. the authors attempt to develop an algorithm that combines demographic, clinical, radiological, and laboratory data to assess the early risk of mers-cov infection in dialysis patients who are suspected of having mers-cov infection and were diagnosed by real-time reverse transcription-pcr (rrt-pcr) between september 2012 and june 2016. the authors hypothesized that mers-cov infection in dialysis patients could be predicted by a set of clinical, radiological, and laboratory indices. this two-center retrospective cohort study included 104 dialysis patients who were suspected of having mers-cov, according to the saudi ministry of health guidelines, 14 1. acute respiratory illness and/or chest radiological findings of pneumonia, 2. hospitalized with health care associated-pneumonia, 3. upper or lower respiratory tract illness within 14 days after exposure to a confirmed/probable case of mers-cov infection, and 4. high fever (388c), headache, body aches, nausea/ vomiting, diarrhea, or with or without respiratory symptoms, leucopenia, and thrombocytopenia. 14 data were abstracted into 15 potential predictors of mers, including demographic data (age and gender); clinical presentations (fever, cough, short breath, chest pain, abdominal pain, diarrhea, vomiting, diabetes); radiology findings in chest (abnormal ct scan or x-ray); baseline laboratory measurements (number of white cells) (wbc) 109/l in the blood, blood platelet count 10 9 /l, alanine transaminase (alt) u/l, and aspartate transaminase (ast) u/l). in order to evaluate whether mers-cov infection was associated with a decrease in wbc count, a cut-off of less than 4 (10 9 /l) indicates leukopenia. 14 similarly, platelet count of less than 150 (10 9 /l) indicates thrombocytopenia, 14 alt greater than 55 (u/l) indicates elevated alt, and ast greater than 34 (u/l) indicate elevated ast. data were analyzed using stata (statacorp. 2017. stata statistical software: release 15. college station, tx: stata-corp llc). overall sample summary and subgroup analysis were provided in table 1 . p value of independent samples t test/chi-square test and unadjusted odds ratio (or) were reported to test whether specific characteristics were associated with mers-cov infection in dialysis patients ( table 1 ). the area under the curve (auc) and 95% confidence interval (ci) were used to evaluate the accuracy of each predictor in identifying mers-cov infection ( table 2) . we developed the mers riskprediction model in dialysis patients using the stepwise logistic regression model. fifteen potential predictors of mers-cov were evaluated at a 0.05. the goodness-offit of the final model was evaluated using the hosmer-lemeshow test. a p value of greater than 5% (a > 0.05) indicates the model fit the data well. the discrimination of the model was evaluated by the receiver operator characteristic curve and was compared with the each of the most important predictors (figure 1 ). the risk model was internally validated in 100 bootstrap samples drawn with replacement from the study sample (n 5 104). the model was presented in the form of the predictive probability of mers-cov infection in dialysis, which is a function of the important selected variables, refer to the supplement file. the youden index was used to identify optimal probability cut-off value for the mers risk stratification. of the 104 dialysis patients studied, 76% had respiratory symptoms, and 26.9% had gastrointestinal symptoms at presentation. the sample age was relatively older at 60.3 6 16.7 years and 72.1% were males (table 1) the auc in table 2 shows the predictors of mers infections. it indicates that chest pain, diabetes, abnormal radiology findings, and elevated ast (auc 0.60) were the most powerful predictors of discriminating mers. when controlled for 15 potential predictors (table 3) , the final risk-prediction model retained 3 independent variables (at a 0.05) that increased the risk of mers-cov infection. mers dialysis patients were more likely to have chest pain (or 5 24.194; p 5 0.011), leukopenia (or 5 6.080; p 5 0.049), and elevated ast (or 5 11.179; p 5 0.013). according to the hosmer-lemeshow test, the adequacy of this prediction model was good (p 5 0.728). the model shows high potential for predicting mers (auc 5 76.99%; 95% ci: 67.05% to 86.38%). the prediction of the model had optimism-corrected bootstrap resampling auc of 71.79%. figure 1 shows that the riskprediction model improved the accuracy of risk classification as compared to the individual predictors. the predicted probability of mers can be calculated by: [1 1 exp (2.362 -3.186 3 chest pain -1.805 3 leukopenia -2.414 3 elevated ast)] 21 . table 4 presents cut-off values for risk probability. this is the first study to develop a risk-prediction model in dialysis patients who screened for mers-cov infection by rrt-pcr. the study included data on 104 dialysis stepwise selection significant at a 5 0.05. patients from 2 centers, kfgh-jed and kamc-r. mers-cov infection is common in dialysis patients, 1, 5, 6 and is associated with increased rapid spread, 1 which can be prevented through early detection, isolation, and monitoring individuals at risk. subsequently, a predictive model was developed for mers-cov infection in hemodialysis facilities. the model shows promising accuracy in detecting high-risk dialysis patients with an auc of 76.99%. the model identified the 3 most important clinical and laboratory characteristics that could help in distinguishing mers-cov infection from other respiratory illnesses. dialysis patients with chest pain were associated with a 24-times higher risk of mers-cov infection than dialysis patients without chest pain. earlier studies reported that chest pain was one of the most common symptoms in the mers-cov population. 17, 18 in agreement with a matched case-control study, 19 we found no differences between mers and non-mers groups in regards to fever, shortness of breath, cough, and other gastrointestinal symptoms. dialysis patients with low wbc count or leukopenia was associated with a 6-times higher risk of mers-cov infection as compared to dialysis patients without leukopenia. this finding is in agreement with saudi ministry of health guidelines, as they developed a tool to identify and evaluate individuals for mers-cov infection, 14 and several other reports, 17, 20, 21 where the wbc was found to be lower in patients with mers-cov infection. our findings support the matched case-control study 19 which showed that mers-cov patients are more likely to have leucopenia and transaminitis. in concordance with earlier studies, 17, 19, 22 elevated ast was found to be a feature of mers-cov infection, where dialysis patients with elevated ast were associated with 11-times higher risk of mers-cov infection as compared with dialysis patients with no elevated ast. according to our risk-prediction model, alt has poor predictive utility. this association was also described by ajlan et al., 22 where normal alt levels have been frequently encountered in mers patients. a prospective study is needed to understand further the link between abnormal ast and mers-cov infection in dialysis patients. the model with the 3 mentioned predictors can be useful in clinical decision to identify high-risk dialysis patients for further investigations and interventions. we presented a simple form of a probability prediction model to calculate the potential risk of infection. for instance, a randomly selected dialysis patient who presented with chest pain, leukopenia, and elevated ast has a probability of mers of 0.994. another case, a randomly selected dialysis patient who did not present with chest pain, leukopenia, or elevated ast has a probability of mers of 0.086. the cut-off values of the probabilities that discriminate between the high-risk and low-risk mers were provided in table 4 . according to the youden index, a cut-off value (p 0.439) produces sensitivity and specificity of 0.950 and 0.259, respectively was found optimal to identify high-risk mers infection. diabetes and abnormal radiology were risk factors for mers-cov infection when we presented the unadjusted analysis. however, these 2 factors were not significant after adjustment for other confounding factors. several limitations should be reported that could influence the prediction of the risk model. the model needs to be validated in a prospective mers-cov investigation. the study included two of the largest hospitals in saudi arabia, yet the model may not be generalizable to dialysis patients in other hospitals. although this study is the largest rrt-pcr study on dialysis patients who screened for mers-cov infection, yet it is limited by the small number of cases screened. the authors were not able to include many other potential confounding factors in the analysis because they were not available. we also acknowledge that the small number of dialysis patients and unequal distribution of mers between hospitals limit our report. despite the limitations mentioned, the prediction ability of the model appears to be promising in clinical decision making to identify suspected dialysis patients with mers-cov infection at an early stage of the infection. in summary, this risk-prediction model in dialysis patients appears to depend markedly on chest pain, leukopenia, and elevated ast. the model accurately predicts high-risk of mers-cov infection in dialysis patients. this could be clinically useful in applying timely intervention and control measures to prevent clusters of infections in dialysis facilities or other hospital settings. the predictive utility of the model warrants further validation in an external sample and a prospective study. hospital outbreak of middle east respiratory syndrome coronavirus managing mers-cov in the healthcare setting middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases guidelines for the laboratory diagnosis of middle east respiratory syndrome coronavirus in korea outbreak of middle east respiratory syndrome at tertiary care hospital multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia the predictors of 3-and 30-day mortality in 660 mers-cov patients estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics, and regions: a worldwide study middle east respiratory syndrome clinical practice guideline for hemodialysis facilities diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia identify-isolate-inform: a modified tool for initial detection and management of middle east respiratory syndrome patients in the emergency department accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach available from: www.moh.gov.sa/en/ccc/ regulations/case%20definition.pdf (accessed date mers-cov outbreak in jeddah-a link to health care facilities the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study early identification of pneumonia patients at increased risk of mers-cov infection in saudi arabia health care associated middle east respiratory syndrome (mers): a case from iran predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea epidemiological and clinical characteristics of patients with middle east respiratory syndrome coronavirus in iran in 2014 middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings key: cord-279733-c0w9bw5u authors: lui, pak-yin; wong, lok-yin roy; fung, cheuk-lai; siu, kam-leung; yeung, man-lung; yuen, kit-san; chan, chi-ping; woo, patrick chiu-yat; yuen, kwok-yung; jin, dong-yan title: middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 date: 2016-04-20 journal: emerg microbes infect doi: 10.1038/emi.2016.33 sha: doc_id: 279733 cord_uid: c0w9bw5u middle east respiratory syndrome coronavirus (mers-cov) infection has claimed hundreds of lives and has become a global threat since its emergence in saudi arabia in 2012. the ability of mers-cov to evade the host innate antiviral response may contribute to its severe pathogenesis. many mers-cov-encoded proteins were identified to have interferon (ifn)-antagonizing properties, which correlates well with the reduced ifn levels observed in infected patients and ex vivo models. in this study, we fully characterized the ifn-antagonizing property of the mers-cov m protein. expression of mers-cov m protein suppressed type i ifn expression in response to sendai virus infection or poly(i:c) induction. this suppressive effect was found to be specific for the activation of ifn regulatory factor 3 (irf3) but not nuclear factor-κb. mers-cov m protein interacted with traf3 and disrupted traf3–tbk1 association leading to reduced irf3 activation. m proteins from mers-cov and sars-cov have three highly similar conserved n-terminal transmembrane domains and a c-terminal region. using chimeric and truncation mutants, the n-terminal transmembrane domains of the mers-cov m protein were found to be sufficient for its inhibitory effect on ifn expression, whereas the c-terminal domain was unable to induce this suppression. collectively, our findings suggest a common and conserved mechanism through which highly pathogenic mers-cov and sars-cov harness their m proteins to suppress type i ifn expression at the level of tbk1-dependent phosphorylation and activation of irf3 resulting in evasion of the host innate antiviral response. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in september 2012 as a novel coronavirus that causes severe acute respiratory disease. 1 since then, this virus has caused recurrent outbreaks in the arabian peninsula and has spread, occasionally, to other parts of the world. [2] [3] [4] [5] [6] [7] [8] [9] according to the world health organization, 1626 laboratory-confirmed cases were reported between september 2012 and 7 january 7 2016 with 586 related deaths in 26 countries. 10 in particular, 186 people were infected and 36 were killed in one recent outbreak in south korea. 10 mers-cov is classified into lineage c of betacoronavirus and is most phylogenetically related to two bat coronaviruses, hku4 and hku5, providing insight on its evolutionary origin. 11, 12 mers-cov is a polycistronic positive-sense single-stranded rna virus with a genome of~30 kb in size. the 5′ most two-thirds of mers-cov genome encodes polyproteins 1a and 1ab, which are further cleaved to yield 16 non-structural proteins, whereas the 3′ end of the genome encodes several structural or lineage-specific proteins. 13 upon infection, these proteins are expressed to facilitate viral replication and propagation in the host. 14 mers-cov infection has been widely reported to mildly induce type i interferons (ifns), including ifn-α and -β, in patients as well as in animal and cellular infection models. [15] [16] [17] [18] [19] [20] [21] this has been attributed to the ifnantagonizing property of some mers-cov-encoded proteins, which directly perturb the host ifn production mechanisms, [22] [23] [24] [25] [26] lending support to the notion that mers-cov uses multiple strategies to evade the innate immune response. in non-specialized epithelial cells as well as a subset of specialized immune cells that are susceptible to mers-cov infection, 16, 18, 27 type i ifn production is an important part of the host innate immune response and is initiated by ubiquitously expressed cytoplasmic viral sensors in the retinoic acid-inducible gene-i (rig-i)-like receptor (rlr) family in response to the detection of viral pathogen-associated molecular patterns such as double-stranded rna (dsrna). 28, 29 stimulated rlrs mobilize downstream signal transducers that lead to the activation of the transcription factors ifn regulatory factor 3 (irf3) and nuclear factor-κb (nf-κb) that drive ifn-β expression. 28 the transduction events within this signaling cascade are prone to negative regulation by many mers-cov proteins. in a comparative analysis of mers-cov structural and accessory proteins, it has been shown that m, orf4a, orf4b and orf5 possess ifn-antagonizing properties. 22 we, and others, have characterized the orf4a protein as a dsrna-binding protein that interferes with the activation of rlr by either a dsrna ligand or the protein co-activator pact. 24, 25 however, the molecular mechanisms through which other mers-cov proteins manipulate the rlr signaling pathway to disrupt ifn-β expression have not been elucidated. in this study, we focused on the characterization of the mers-cov m protein in ifn antagonism. coronavirus m protein is a transmembrane glycoprotein localized predominantly to the golgi complex and is required for virion assembly. [30] [31] [32] mers-cov m protein is of particular interest because sars-cov m protein also inhibits ifn production through a mechanism by which the formation of traf3·tank·tbk1/ikk-ε complex is impeded to ablate the activation of irf3 transcription factor. 30 in contrast, m protein encoded by human coronavirus hku1 associated with common cold has no influence on ifn production. 32 here we reported that the mers-cov m protein also specifically inhibited irf3 activation but not nf-κb signaling. mers-cov m protein was capable of interacting with traf3 adapter protein and hampered traf3-tbk1 interaction leading to diminished irf3 activation. using a chimeric protein containing the mers-cov m protein n-terminal transmembrane domains and a dormant sars-cov m protein c-terminal domain, we confirmed that the n-terminal transmembrane domains of mers-cov m protein sufficiently account for its inhibitory effect. although another chimera containing sars-cov m protein n-terminal transmembrane domains and a mers-cov m protein c-terminal domain was fully competent in ifn antagonism, a truncation mutant lacking the functional first transmembrane domain of sars-cov m was not, suggesting that the c-terminal domain of the mers-cov m protein is largely dispensable for its immunosuppressive activity. the ifnβ-luc reporter plasmid and rig-i expression plasmid are gifts from professor takashi fujita (kyoto university, kyoto, japan). 28 the expression plasmids for tbk1, irf3 and traf3 were generous gifts from dr genhong cheng (university of california, los angeles, ca, usa), 33, 34 whereas those for rig-i n, ikk-ε and mavs and iκb-α as well as irf3-luc and κb-luc reporter plasmids have been described elsewhere. 30, [35] [36] [37] viral rna was extracted from mers-cov-infected vero-e6 cells. the m gene was pcr-amplified from complementary dna and cloned into the ecori/xhoi sites of pcagen plasmid with the addition of a c-terminal v5-tag with the following primers: 5′-atg tct aat atg acg caa ctc act ga-3′ (forward) and 5′-agc tcg aag caa tgc aag ttc-3′ (reverse). the sars-cov m protein expression plasmid has been described elsewhere. 30, 32 the expression plasmids for the sn and mn chimeras were constructed by assembly pcr with the following forward primers covering the breakpoints: 5′-agg ctg ttt gct cgt acc cgc tca tgg tgg tca ttc aat cct gag-3′ (sn) and 5′-ccg gct gtt tat gag aac tgg atc aat gtg gtc att caa ccc a-3′ (mn). the reverse primers were complementary to their respective forward primers. the truncation mutant of the sn chimera lacking the first transmembrane domain was constructed using the forward primer: 5′-atg gta aca ctt gct tgt ttt gtg ct-3′. mouse anti-flag (m2) and anti-β-actin antibodies were purchased from sigma-aldrich (st. louis, mo, usa). mouse anti-v5 and anti-ha (y11) antibodies were purchased from life technologies (grand island, ny, usa) and santa cruz biotechnology (dallas, tx, usa), respectively. rabbit anti-irf3 and anti-phospho-irf3 (ser 386) antibodies were purchased from ibl-america (minneapolis, mn, usa). cell culture and sendai virus hek-293 human embryonic kidney cells were maintained in dulbecco's modified eagle's medium with 10% fetal bovine serum (life technologies) at 37°c in a humidified chamber supplemented with 5% carbon dioxide. plasmid transfection was performed with genejuice (merck millipore; billerica, ma, usa). poly(i:c) was purchased from sigma-aldrich and was transfected with lipofectamine 2000 (life technologies). sendai virus (cantell strain) was purchased from american type culture collection (manassas, va, usa). dual-luciferase reporter assay, co-immunoprecipitation and western blotting were performed as previously described. 30, 38 particularly, relative luciferase activity in arbitrary units was calculated by normalizing firefly luciferase activity to renilla luciferase activity recovered from cell lysate. non-denaturing native polyacrylamide gel electrophoresis (page) was performed as previously described. 36, 39, 40 bioinformatic analysis sequence alignment was performed using cluster omega, an online tool based on the hidden markov model, 41 and hosted by the embl-ebi server (http://www.ebi.ac.uk/tools/msa/clustalo/). transmembrane domain prediction was performed using tmfinder, which considers hydrophobicity and helicity of the amino-acid sequence (http://tmfinder.research.sickkids.ca/). 42 to characterize the mers-cov m protein in terms of its ifn antagonism, m protein was ectopically expressed in cultured cells for functional assays ( figure 1a) . a luciferase reporter construct driven by ifn-β promoter was used to reflect ifn-β promoter activity stimulated during infection. sendai virus was used as a model virus to potently induce ifn expression in transfected cells. when increasing doses of m proteins were expressed in advance, a dose-dependent inhibition of ifn-β promoter activity was observed ( figure 1b ; bars 3-5 compared with bar 2). a similar observation was also noted when synthetic dsrna analog poly(i:c) was used as an alternative inducer that specifically stimulates the rlr pathway of ifn production ( figure 1c ; bars 3-5 compared with bar 2). these two pieces of data are generally consistent with a previous report, 22 and they further strengthen the current model of the ifn antagonism of mers-cov m protein. cellular ifn-β expression is under the control of multiple transcription factors, which work cooperatively to form a large enhanceosome complex. 43 in particular, irf3 and nf-κb are two transcription factors that are primarily activated by rlr signaling. 28, 44, 45 mers-cov m protein has previously been shown to have no influence on nf-κb activation induced by sendai virus infection. 22 however, it remains to be seen whether the mers-cov m protein could preferentially inhibit irf3 and nf-κb signaling after rig-i activation. to address this issue, two different luciferase reporter constructs, in which tandem copies of either irf3-or nf-κb-binding elements were inserted into their promoter region, were used. the truncation mutant of rig-i known as rig-i n that contains only the n-terminal card domain was chosen to be the inducer in these assays because it is constitutively active and highly competent to induce these two pathways. 28 the mers-cov m protein was able to suppress the promoter activity of the irf3-driven luciferase construct in a dose-dependent manner (figure 2a ; bars 3-5 compared with bar 2), but no inhibitory effect was observed with the nf-κb-driven construct ( figure 2b ; bars 3-5 compared with bar 2) although the canonical inhibitor iκb-α could efficiently blunt its activation as a positive control ( figure 2b ; bar 6 compared with bar 2). hence, the suppressive effect of the mers-cov m protein is specific for irf3 signaling but not nf-κb activation. to delineate the action point of the mers-cov m protein in ifn antagonism, we tested the ability of the m protein to inhibit the activation signal induced by different signal transducers of the rlr pathway individually. the activation signal will be mostly unaffected if the activation event mediated by that transducer is downstream of the action point where m protein exerts its inhibitory effect. as described above, rig-i n is a constitutively active mutant that resembles (1 μl/ml) in c for 16 h before harvest for dual-luciferase reporter assay. bars represent the mean of three biological replicates (n = 3) and error bars indicate their s.d. the statistical significance between selected samples was evaluated using a two-tailed student's t-test for unpaired samples with equal variance and p-value (p) was indicated. figure 3a ; bars 3-5 compared with bar 2). a similar result was also obtained using mavs as an activator ( figure 3b ; bars 3-5 compared with bar 2), which is a mitochondrial adapter that diverts the activation signal from rig-i to the irf3 and nf-κb pathways. [44] [45] [46] [47] when activators committed to the irf3 pathway were used, greater inhibitory effects were observed, as in the cases of tbk1 ( figure 3c ; bars 3-5 compared with bar 2) and ikk-ε ( figure 3d ; bars 3-5 compared with bar 2), which are kinases which recognize and phosphorylate irf3 as direct substrate. [48] [49] [50] [51] surprisingly, when a constitutively active mutant of irf3 transcription factor (irf3 5d), with five inducible phosphorylation sites at ser/thr residues mutated to asp, 52 was employed, the expression of the m protein no longer quenched the irf3-induced activation of ifn-β promoter ( figure 3e ), suggesting that the inhibitory effect of m protein occurs upstream of irf3 activation. to further analyze the molecular mechanism and consequences through which mers-cov m protein exerts its inhibitory effect, we first investigated what signal transducer molecule might interact with the m protein. several rlr transducers were ectopically expressed with mers-cov m protein in cultured cells for a coimmunoprecipitation experiment. when the transducers were precipitated with an anti-flag antibody, the m protein was only detected in traf3-containing precipitate ( figure 4a ; lane 4 compared with lanes 1-3) even though m protein was abundantly expressed in all samples with other transducers (figure 4a ; lower panel for input), traf3 functions as an adapter that bridges the mitochondrial transducer mavs with the downstream signaling complex containing tbk1 and ikk-ε kinases that are essential for irf3 activation. 34, 53 the physical association of the mers-cov m protein with traf3 ( figure 4a ) prompted us to test whether the adapter function of traf3 would be particularly affected by m protein. we performed another co-immunoprecipitation experiment to explore the possibility that m protein could perturb the interaction of traf3 with the downstream transducer complex. when traf3 and tbk1 were ectopically expressed in cultured cells, the detection of tbk1 in traf3-immunoprecipitate confirmed the specific recruitment of tbk1 to traf3 in the absence of m protein ( figure 4b ; lane 2 compared with lane 1). however, when m protein was added to the system, the interaction between traf3 and tbk1 was significantly disrupted ( figure 4b ; lane 3 compared with lane 2), demonstrating that the physical association of mers-cov m protein with traf3 perturbs traf3-tbk1 interaction. it was observed that mers-cov m protein disrupted traf3-tbk1 interaction ( figure 4b ), which is required for irf3 activation. we then evaluated whether irf3 activation would be affected by the expression of m protein. irf3 dimerization visualized by non-denaturing native page is a sensitive assay for evaluating direct irf3 activation. 39 therefore, we ectopically expressed irf3 and m protein with the inducer rig-i n in cultured cells and subjected cell lysates directly to native page to check for irf3 dimerization. when the inducer rig-i n was exclusively co-expressed with irf3, the detection of an additional slow-migrating band indicated the activation and dimerization of irf3, which would otherwise be entirely in its monomeric form in the absence of any activator ( figure 4c ; lane 2 compared with lane 1). interestingly, when m protein was expressed, the signal reflecting the dimeric form of irf3 molecules was significantly diminished, even though the total irf3 level expressed in all samples was highly comparable as detected by conventional denaturing sds-page ( figure 4c ; lower panel for sds-page), suggesting that irf3 activation was greatly inhibited by the mers-cov m protein. irf3 phosphorylation was also suppressed with the expression of mers m protein in a similar experimental setup ( figure 4d ; lane 3 compared with lane 2). together with other results, mers-cov m protein was thought to interact with traf3 to perturb traf3-tbk1 interaction, which, in turn, affects irf3 phosphorylation and activation. given that the m proteins of both sars-cov and mers-cov were capable of antagonizing ifn production through highly similar innate immunosuppression by mers-cov m protein p-y lui et al mechanisms, 30 it will be of interest to analyze the sequence and domain architecture of the two proteins. sequence alignment of sars-cov and mers-cov m proteins revealed a strikingly high sequence similarity (470%) and the presence of three transmembrane domains at the n-termini ( figure 5a ). according to the prediction results, we have initially constructed two truncation mutants for mers-cov m protein, an n-terminal transmembrane domain-containing mutant and a c-terminal mutant, and tested their inhibitory capacity in suppressing ifn-β expression using a luciferase reporter assay. however, neither exhibited an inhibitory effect (data not shown), possibly due to unstable expression or aberrant localization. to overcome the inactivity of truncation mutants and to define the inhibitory activity of different domains, we decided to create chimeric proteins using domain swapping between mers-cov and sars-cov m proteins. particularly, the sn chimera contains the n-terminal transmembrane domains from sars-cov m protein and the c-terminal domain from mers-cov m protein, whereas the mn chimera contains the n-terminal transmembrane domains from the mers-cov m protein and the c-terminal domain from the sars-cov m protein ( figure 5b ). the breakpoint was designed to occur immediately after the third predicted transmembrane domain at residue 106 and before the conserved ser residue in both proteins at residue 107 ( figure 5b ). we next compared the inhibitory effect of these two chimeras and the full-length m proteins on ifn-β expression using the luciferase reporter assay. our previous study showed that the ifn-antagonizing activity of the sars-cov m protein is mediated by its n-terminal transmembrane domains, but the c-terminal domain has no effect. 32 when we swapped the c-terminal domain of the sars-cov m protein with that of the mers-cov m protein in the sn chimera, a similar suppressive effect on ifn-β promoter activity was observed ( figure 5c ; bar 4 compared with bar 3), consistent with our previous results. 32 likewise, when we swapped the c-terminal domain of mers-cov m with that of sars-cov m protein in mn, the chimera was capable of suppressing ifn-β promoter activity to comparable level ( figure 5c ; bar 8 compared with bar 7). given that the c-terminal domain of sars-cov m protein possesses no suppressive effect, 32 the inhibitory activity of the mn chimera would be predominantly due to the n-terminal domains of mers-cov m protein. a biochemical assay also confirmed that the mn chimera maintained the ability to interact with the traf3 adapter protein in a co-immunoprecipitation experiment ( figure 5d ; lane 2 compared with lane 1). therefore, we concluded that the n-terminal transmembrane domains of the mers-cov m protein are required and sufficient for its innate immunosuppressive activity. to further determine whether the c-terminal domain of the mers m protein also possesses ifn-antagonizing activity, we utilized the knowledge that the first transmembrane domain of sars-cov m protein is fully responsible for its suppression effect 32 in this study, we reported that the mers-cov m protein inhibited irf3 activation, hence ifn expression, by disrupting traf3-tbk1 interaction. this innate immunosuppressive activity of the mers-cov m protein was due to its conserved n-terminal transmembrane domains. our mechanistic study complemented the previous work that showed that mers-cov m protein had ifn-antagonizing activity. 22 both studies demonstrated that mers-cov m protein suppressed irf3 activity but not nf-κb signaling. it is known that the activation of rig-i and mavs results in the activation of both irf3 and nf-κb. 28, [44] [45] [46] our results indicated that the mers-cov m protein was capable of differentially suppressing the rig-i-induced activation of irf3 (figure 2 ). this provides further support to the bifurcation of irf3 and nf-κb signaling subsequent to rig-i and mavs activation. further investigations should elucidate the mechanism by which the mers-cov m protein preferentially modulates irf3 activators such as tbk1, while sparing nf-κb activators such as card9. 47 we provided evidence that the traf3-tbk1 interaction as well as irf3 phosphorylation and dimerization were affected by the mers-cov m protein (figure 4 ). our findings fill the knowledge gap by providing novel mechanistic insight into the innate immunosuppressive activity of mers-cov m protein. in our study, traf3 was also shown to interact with the mers-cov m protein ( figure 4a ). in line with this, the adapter function of traf3 in tbk1 recruitment and subsequent irf3 activation was perturbed by mers-cov m protein ( figures 4b-d) , which plausibly contributed to the ifn antagonism of the mers-cov m protein (figures 1-3) . the mers-cov m protein is a transmembrane protein that was shown to co-localize with markers of the golgi apparatus and endoplasmic reticulum (er)-golgi intermediate compartments in the perinuclear area. 22 although traf3 is known to adapt the mitochondrial transducer mavs, it is not associated with mitochondria but rather with the golgi apparatus and er-golgi intermediate compartments in unstimulated conditions, 30, 54 rendering it susceptible to interaction with the mers-cov m protein. upon stimulation with ligands or viral infection, traf3 appears on membrane-bound fragments originating from golgi. retention of traf3 in er-to-golgi compartments and inability to form golgi fragments rendered ifn-β expression inefficient. 54 therefore, whether traf3-containing golgi fragment formation is affected by mers-cov m protein warrants further analysis. this may serve as a novel mechanism by which virus-encoded proteins counteract host ifn production. mers-cov and sars-cov are two highly pathogenic coronaviruses that have caused hundreds of deaths. on one hand, the development of relevant prophylactic and therapeutic agents has been well under way. [55] [56] [57] on the other hand, the identification of the pathogenic factors in these viruses is also in full swing. the m protein is a pathogenic factor by virtue of its ifn-antagonizing property. both the mers-cov and sars-cov m proteins were found to suppress ifn production with a highly similar mechanism in which the irf3phosphorylating complex of traf3·tank·tbk1/ikk-ε was affected by their n-terminal transmembrane domains. 30, 32 interestingly, in the case of community-acquired human coronavirus hku1, which normally causes common cold in infected individuals, its m protein showed no ifn antagonistic property, 32 further supporting the importance of the m protein in sars-cov and mers-cov pathogenesis. using a side-by-side comparison of sars-cov and mers-cov m proteins, we discovered that the extent by which the mers-cov m protein suppressed ifn-β promoter activity was lower than that by sars-cov m protein. this difference might be explained by the strengths of the interaction of mers-cov m protein with other transducers. whereas the mers-cov m protein was found to be strongly associated with traf3, its interaction with tbk1 or ikk-ε was undetectable (data not shown). this distinguished mers-cov m protein from the sars-cov m protein, which interacts potently with every component of the traf3·tank·tbk1/ikk-ε complex. 30 further investigations are required to shed light on how the interaction of the m protein with traf3 complex might influence mers-cov pathogenesis. coronaviruses encode multiple proteins to counteract the host innate antiviral response. 58-60 mers-cov is no exception. several mers-cov-encoded proteins have been identified to be ifn antagonists. we, and others, have characterized at least three ifn-antagonizing proteins encoded by mers-cov. in addition to the m protein reported in this study, orf4a is a dsrna-binding protein, which directly inhibits rlr activation induced by dsrna and/or the protein co-activator pact. 24, 25 in addition, our unpublished data also revealed that orf4b is a potent ifn antagonist. this is in line with findings by other groups although the mechanistic details of its action have not well documented. 22, 23 one recent report suggested that orf4b might not only interact directly with tbk1/ ikkε in the cytoplasm but also perturb ifn production in the nucleus through an as yet unknown mechanism. 61 nevertheless, how m, orf4a, orf4b and the other ifn-antagonizing proteins of mers-cov coordinate with each other to modulate the host innate antiviral response to facilitate viral replication and propagation remains elusive. severe respiratory illness associated with a novel coronavirus-saudi arabia and qatar severe respiratory illness caused by a novel coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in a case of imported middle east respiratory syndrome coronavirus infection and public health response first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease spread of mers to south korea and china middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses cell type-specific involvement of rig-i in antiviral response severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3·tank·tbk1/ ikkε complex a structural analysis of m protein in coronavirus assembly and morphology suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain irf3 mediates a tlr3/tlr4-specific antiviral gene program modulation of the interferon antiviral response by the tbk1/ikki adapter protein tank mip-t3 is a negative regulator of innate type i ifn response the double-stranded rna-binding protein pact functions as a cellular activator of rig-i to facilitate innate antiviral response suppression of type i and type iii interferon signalling by nss protein of severe fever-with-thrombocytopenia syndrome virus through inhibition of stat1 phosphorylation and activation comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku1 induction of irf-3/-7 kinase and nf-κb in response to double-stranded rna and virus infection: common and unique pathways suppression of pact-induced type i interferon production by herpes simplex virus 1 us11 protein fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega tm finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales virus induction of human ifnβ gene expression requires the assembly of an enhanceosome identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-κb and irf 3 ips-1, an adapter triggering rig-i-and mda5-mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-β signaling recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1β production ikkε and tbk1 are essential components of the irf3 signaling pathway ifnregulatory factor 3-dependent gene expression is defective in tbk1-deficient mouse embryonic fibroblasts ikk-i signals through irf3 and nfkappab to mediate the production of inflammatory cytokines phosphorylation of innate immune adapter proteins mavs, sting, and trif induces irf3 activation virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation regulation of antiviral responses by a direct and specific interaction between traf3 and cardif proteomic profiling of the traf3 interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity yeast-expressed recombinant protein of the receptorbinding domain in sars-cov spike protein with deglycosylated forms as a sars vaccine candidate structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon to sense or not to sense viral rna-essentials of coronavirus innate immune evasion a molecular arms race between host innate antiviral response and emerging human coronaviruses middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets this work is licensed under a creative commons attribution 4.0 international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ key: cord-284374-sqxlnk9e authors: park, jiyeon; yoo, seung yeon; ko, jae-hoon; lee, sangmin m.; chung, yoon joo; lee, jong-hwan; peck, kyong ran; min, jeong jin title: infection prevention measures for surgical procedures during a middle east respiratory syndrome outbreak in a tertiary care hospital in south korea date: 2020-01-15 journal: sci rep doi: 10.1038/s41598-019-57216-x sha: doc_id: 284374 cord_uid: sqxlnk9e in 2015, we experienced the largest in-hospital middle east respiratory syndrome (mers) outbreak outside the arabian peninsula. we share the infection prevention measures for surgical procedures during the unexpected outbreak at our hospital. we reviewed all forms of related documents and collected information through interviews with healthcare workers of our hospital. after the onset of outbreak, a multidisciplinary team devised institutional mers-control guidelines. two standard operating rooms were converted to temporary negative-pressure rooms by physically decreasing the inflow air volume (−4.7 pa in the main room and −1.2 pa in the anteroom). healthcare workers were equipped with standard or enhanced personal protective equipment according to the mers-related patient’s profile and symptoms. six mers-related patients underwent emergency surgery, including four mers-exposed and two mers-confirmed patients. negative conversion of mers-cov polymerase chain reaction tests was noticed for mers-confirmed patients before surgery. mers-exposed patients were also tested twice preoperatively, all of which were negative. all operative procedures in mers-related patients were performed without specific adverse events or perioperative mers transmission. our experience with setting up a temporary negative-pressure operation room and our conservative approach for managing mers-related patients can be referred in cases of future unexpected mers outbreaks in non-endemic countries. high-volume healthcare facility, which is how the previous south korea outbreak occurred 11 . moreover, there may be very few hospitals that are prepared to provide perioperative care for mers patients. therefore, herein, we share our experience of providing infection prevention and control measures for surgeries for mers-related patients in our hospital. during the mers outbreak in our hospital, six mers-related patients underwent surgery including three possibly exposed patients, one directly exposed patient, and two mers-confirmed patients who recovered from the disease. all patients were negative during two preoperative mers screenings using real-time reverse transcription polymerase chain reaction (rrt-pcr) 14 . figure 1 shows the total number of surgeries performed during the outbreak period at our hospital and the distribution of mers-related patients undergoing surgery. temporary set-up of a negative-pressure operating room. the operating rooms in our hospital were generally positive-pressure environments, and we had no permanent negative-pressure operating rooms. because a negative-pressure operating room is the optimal environment to prevent airborne virus spreading to adjacent areas 13 , two of our 25 operating rooms in the main operating suite of the hospital were temporarily converted into negative-pressure operating rooms to perform surgical procedures on mers-related patients. operating rooms no. 16 and 17 were selected because they were connected to each other, but each room had separate atmospheric air inlets and exhaust systems. they also had separate air-conditioning and humidification systems. of the two connected rooms, one was used as the main operating room and the other was used as the anteroom where healthcare workers (hcws) applied and removed the personal protective equipment (ppe). in each room, fresh air was supplied from an inlet duct and discharged outside through the exhaust duct (fig. 2) . because a constant exhausting air volume was maintained through the outlet duct, negative pressure in the operating room was achieved by decreasing the inflow air volume that entered through the inlet duct. first, the blades of the air volume control damper in the inlet duct were closed as much as possible (fig. 2) . however, because the damper was not intended to be air-tight, the inflow volume to the operating room did not decrease sufficiently. second, as an additional measure to decrease the inflow volume, we opened the access hole in the inlet duct, which was originally used for duct inspection purposes (fig. 2) . finally, a smoke test was carried out to ensure negative pressure. the room pressure was maintained at −4.7 pa in the main operating room and at −1.2 pa in the anteroom (fig. 3) ; −4.7 pa is below the negative pressure room standard of −2.5 pa 15 . www.nature.com/scientificreports www.nature.com/scientificreports/ airflow in both rooms reached 14-18 air exchanges per hour, according to airflow velocity measurements with an anemometer (ebt731 balometer; tsi alnor ® , minnesota, united states). in this environment, removing airborne contaminants requires 18 minutes for 99% efficiency and 28 minutes for 99.9% efficiency 16 . therefore, 30 minutes of room ventilation was required after aerosol forming high-risk procedures, such as endotracheal intubation or extubation 12, 17 . the cleanliness level of each room was also measured using a particle counter (tsi 9310; tsi, united states): main operating room = 2,604 and the anteroom = 2,540, which were much lower than the institutional target level of <10,000 for general surgery (fig. 3) . cleanliness level was defined as the number of particles smaller than 0.5 µm in 0.3048 m 3 . equipment preparation and disinfection. all built-in instruments such as computers, telephones, and ventilators were covered with plastic paper. sufficient amounts of drugs, fluids, and other equipment were prepared in the operating room before surgery, and other unnecessary equipment was moved out. additionally, we used disposable equipment, when possible. high efficiency particulate air (hepa) filters were installed in the breathing circuits, both on the inspiratory and expiratory limbs of the ventilators and at the patient's site that connected to endotracheal tube. after operations with mers-exposed patients, 30 minutes of room ventilation was followed by surface disinfection with diluted chlorine bleach (500 ppm) 18, 19 . cleaners wore standard ppe while disinfecting surfaces. for mers-confirmed patients, surface disinfection was performed twice. institutional guidelines for perioperative management of mers-related patients. during the mers outbreak, we set the following principles for perioperative management of mers-related patients: all elective surgeries for mers-confirmed patients were postponed to reduce the risk of potential in-hospital transmission. for mers-exposed patients, surgical procedures were delayed until after the potential incubation period of 14 days 20 . when a mers-related patient required an urgent or emergency operation, mers-cov pcr tests were performed twice with distinct specimens preoperatively, to account for asymptomatic mers patients or delayed positive conversion in symptomatic mers-exposed patients. for patients with ambiguous pcr results or without a pcr test, operations were performed according to the management guidelines for mers-confirmed patients. all the surgical procedures for mers-related patients were performed in the last order of the day as possible. perioperative protection level for hcws. when an operation for a mers-related patient was scheduled, the division of infectious diseases and infection control department confirmed the protection level of the hcws, according to institutional guidelines (table 1 ). in principle, standard ppe was applied to hcws who cared for asymptomatic mers-exposed patients. standard ppe includes surgical gloves, surgical gowns, eye shields, and n95 respirators. while managing mers-confirmed or mers-exposed patients with mers-associated symptoms including fever, myalgia, respiratory symptoms, or diarrhea, hcws implemented enhanced ppe, which included coverall clothes with head cover, shoe covers, goggles, two pairs of surgical gloves, and powered air purifying respirator (papr) or n95 respirators. although we performed preoperative mers-cov pcr screening, enhanced ppe was still recommended when managing symptomatic mers-exposed patients regardless of their pcr results. anesthesiologists were recommended to apply enhanced ppe (including papr from the middle of the outbreak) when managing all mers-related patients because they were most directly exposed to the aerosol-producing high-risk procedures, such as endotracheal intubation and extubation. only minimal numbers of hcws were present in the operating room. institutional education regarding the precise use of ppe was provided to the all associated hcws and they were assisted by skilled nurses in the operating room during the ppe donning and doffing processes. patient transfer for operation. mers-related patients were transferred directly to the negative-pressure main operating room through an exclusive path and elevator by a physician wearing proper ppe. the walls and the floor of the passageways and the elevator were covered with plastic paper. mers-related patients wore a www.nature.com/scientificreports www.nature.com/scientificreports/ surgical mask during transfer. because only anesthesiologists wore enhanced ppe when in proximity to asymptomatic mers-exposed patients, 30 minutes of room ventilation was performed after anesthetic induction, including endotracheal intubation. the surgical team then entered the main operating room through the anteroom. in the cases of symptomatic mers-exposed patients or mers-confirmed cases, all hcws wore enhanced ppe and the 30-minute ventilation time was not required. after completion of operation procedures, patients who were moved to the general ward recovered in the main operating room without going through the post-anesthesia care unit. thirty minutes of room ventilation was performed after tracheal extubation. a physician in the main operating room sent the patient into the corridor, while the other physician outside the main operating room wearing ppe took over and transferred the patient to the general ward directly through the exclusive pathway (fig. 4) . patients moving to the intensive care unit (icu) were transferred while remaining intubated. before transfers, we injected patients with a sufficient amount of intravenous muscle relaxant and sedative drugs to prevent coughing or movement and we applied a portable ventilator or bag-valve mask with a hepa filter to the patient. operations for six mers-related patients. the details of the six mers-related patients undergoing surgery are presented in table 2 . two patients had operations during phase 1 and four patients during phase 2 of the outbreak (fig. 1) . the negative-pressure operating room was set up to be used from phase 2. regarding ppe levels for the hcws attending these six patients, standard ppe was applied during management of patient a (asymptomatic mers-exposed patient), while anesthesiologists wore enhanced ppe for high-risk procedures (tracheal intubation). enhanced ppe was applied to hcws for patient b because the patient was symptomatic and still within the two-week incubation period, even though both pcr results were negative. enhanced ppe, including papr to reduce risk of mers-transmission, was applied for patient c who underwent surgery in the middle of the outbreak (phase 2). papr provides more perfect sealing and protection of the head surface. patients d and e had documented mers-cov infection and their recovery was confirmed with symptom resolution and two negative mers-cov pcr tests. however, enhanced ppe with papr was applied to hcws because the infection risk could not be eliminated during exposure to a large amount of body fluid, especially during cesarean section (patient d). after spinal anesthesia, she recovered in the main operating room and was transferred directly to the general ward. patient f had a history of exposure to a mers patient in the emergency room and was isolated due to a fever. enhanced ppe was applied to the hcws for patient f and she underwent surgery with only one set of negative pcr results because of her emergency condition. mers, as well as sars, is associated with coronaviruses, both of which have high affinity for the lower repiratory tract and easily produce severe pneumonia [21] [22] [23] [24] [25] . although mers has lower human-to-human transmission potential and has resulted in fewer large outbreaks than sars, there may be occasional amplification of clusters in healthcare settings [21] [22] [23] . moreover, mers case fatalities are reported to be much higher than sars (35-45% for mers and 10-15% for sars) 12, 17, 23, 24, 26 . unlike sars, ongoing small and large mers outbreaks in the arabian peninsula foster potential future mers outbreaks in non-endemic countries. however, there is likely to be a very limited number of hospitals that are prepared with negative-pressure operating rooms, except for a few hospitals in hong kong that experienced the 2003 sars outbreak 13 . almost all hospitals generally have positive-pressure operating rooms and they may experience an outbreak without facilities that are prepared for perioperative management of mers patients, as our hospital did in 2015. one of the highlights of our experience during the outbreak was the temporary set-up of a negative-pressure operation room with an adequate pressure gradient (≥2.5 pa) by modifying two connected operating rooms according to us centers for disease control and prevention (cdc) guidelines 15 . continuous negative pressure was maintained in the main operating room (−4.7 pa) and the anteroom (−1.2 pa). this temporary setting was possible because the two adjacent rooms had separate atmospheric air inlets and exhaust systems. although we could not measure the airflow pattern or dispersion of infectious particles directly 27 , the cleanliness levels in both operating rooms were 2,500 particles, well below the institutional target cleanliness for general surgery (<10,000 particles). although the precise route of mers-virus transmission is currently not clearly understood 21 , mers, as well as sars, is known to spread by direct contact with infectious material, such as large respiratory droplets, and also by airborne routes 28, 29 . touching contaminated objects may also be a source of transmission; this is different from tuberculosis, which is transmitted by airborne routes 19, 29 . therefore, when performing procedures that generate aerosols, such as endotracheal intubation, in patients with mers or sars, hcws must wear enhanced ppe, including gloves, a gown, either a face-shield that fully covers the front and sides of the face or goggles, and respiratory protection at least as protective as an n95 filtering face piece respirator 19, 28, 30 . when removing ppe, care should also be taken not to contact contaminated materials. considering potential aerosol generation in operating rooms and the transmission risk of mers-cov while changing ppe, the temporary modification of an operating room to a negative-pressure room with an anteroom should provide suitable protection for hcws participating in operations on mers-related patients 5 . a second highlight of our experience is the highly conservative application of ppe to hcws. at the time of the outbreak, there were no specific guidelines for perioperative management 31 . therefore, we used a conservative approach based on our experience and previous reports. first, although the previous guidelines recommended that asymptomatic mers-exposed patients be managed as general patients undergoing surgery, we applied standard ppe to hcws and we performed mers-cov pcr screening twice. although mers progressed gradually after symptom onset 32 , we could not exclude the possibility that asymptomatic mers-exposed patients had the potential to develop symptomatic disease perioperatively. moreover, we observed development of mers after the known incubation period of 14 days in an immunocompromised host 10, 33 ; thus, the possibility of exceptional cases could be considered. furthermore, a certain proportion of asymptomatic mers-exposed patients could actually be asymptomatic mers-infected patients. approximately 21% of laboratory confirmed mers patients have been classified as asymptomatic or having nonspecific mild symptoms at the time of testing 34 . the potential for transmission from asymptomatic mers-cov pcr-positive person is currently unknown, but there are reports about prolonged viral rna detection in the upper respiratory tract in asymptomatic pcr-positive person 9 . considering these points, it would be reasonable to prepare more conservatively than the existing guidelines call for. www.nature.com/scientificreports www.nature.com/scientificreports/ another point on which our preparations differed from the guidelines was the application of enhanced ppe, which emphasizes full protection of the body surface with a hooded coverall. during the outbreak in our hospital, mers transmission events occurred among hcws who were equipped with standard ppe, including n95 masks. transmission may have occurred after possible contamination of uncovered head or face surfaces 35 . therefore, if a patient with a mers contact history had mers-associated symptoms, applying enhanced ppe (either a n95 respirator or papr) during surgery would be appropriate for hcws because numerous droplets and aerosols may be produced during airway interventions. because the n95 may fit inadequately if worn for a long time or after movement during surgery, wearing papr will be more beneficial. however, unlike hcws dealing with ebola virus, impermeable and fluid-resistant gowns are not required because body fluids are not infectious as with ebola virus diseases 9, 28, 35, 36 . our experience was limited in that, as a mers outbreak outside the endemic country, we did not have an opportunity to perform surgical procedures in actively virus-shedding mers-infected patients. additionally, our infection-prevention protocols would be too conservative to apply in mers-endemic situations. however, considering the potential risk of infected hcws, preventing mers transmission is extremely important in the management of a mers outbreak. importantly, our experience can be generalized to other non-endemic countries for managing potential outbreaks of emerging respiratory diseases. in the era of globalization, a mers outbreak can occur in any country outside the middle east. a very limited number of hospitals are equipped with negative-pressure operating rooms, and therefore, most hospitals are likely to experience a mers or other outbreak in an unprepared circumstance. we hope that this report will help other hospitals in preparing for future mers outbreaks and infection control in unexpected conditions. this study was based on all available data at the samsung medical center from the mers outbreak and on interviews with hcws associated with the outbreak. the study was approved by samsung medical center institutional review board. the documents for review included electronic medical records of mers-related patients who underwent operative procedures and institutional guidelines for perioperative management of mers-related surgical patients. the mers guidelines were prepared through multidisciplinary team discussions that were held by our hospital's infection control department during and after the mers outbreak. the records about the temporary set-up of a negative-pressure operating room were also reviewed. we also collected data through interviews with hcws who participated in surgery and anesthesia for mers-related patients. we defined mers-related patients as those who were possibly or directly exposed to mers or who had a previously confirmed mers diagnosis 10 . in brief, patients who had a potential but unconfirmed close contact history with a mers patient were defined as possibly exposed patients, and they were allowed to continue their normal activities until mers-like symptoms developed. directly exposed patients included those who had close contact with a known mers patient and who did not wear proper ppe; these patients were isolated in their homes or in private negative-pressure rooms at our hospital. because the number of mers-infected patients continuously increased at our hospital, we partially closed the hospital on june 13 10 , at which point outpatient-care clinics were closed and the emergency department was only available for life-threatening emergencies. all elective surgeries were postponed if possible 10 . we defined the early phase of the outbreak (before june 13) as phase 1 and the middle phase of the outbreak (from june 13) as phase 2. for mers-cov pcr tests, either sputum or nasopharyngeal swab samples were collected and sputum samples were preferred if available 14 . sputum was collected directly into a sterile, leak-proof, screw-capped sputum collection sterile container and nasopharyngeal swab was collected with an eswab (482 c, copan diagnostics inc., murrieta, ca, usa). clinical samples were screened by rrt-pcr testing with amplification targeting the upstream e region (upe) and confirmed by subsequent amplification of the open reading frame (orf)1a using powerchek ™ mers real-time pcr kits (kogene biotech, seoul, korea). all rrt-pcr reactions were performed using the 7500 fast real-time pcr system (applied biosystems, foster city, ca, usa). the pcr reaction was performed in a total volume of 20 μl (15 μl pcr reaction mixture and 5 μl template rna). thermocycling conditions included a step at 50 °c for 30 min, followed by 95 °c for 10 min and then 40 cycles of 15 s at 95 °c and 60 s at 60 °c. positive viral template control and no-template control were included in each run. the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was amplified simultaneously as a heterologous internal control to monitor pcr inhibition. a positive test result was defined as a well-defined exponential fluorescence curve that crossed the cycle threshold (ct) < 35 cycles for both upe and orf1a. a sample was considered "equivocal" if the upe result was positive but the ct value for orf1a was >35 and <40. we interpreted the result as "indeterminate" if (1) the upe result was positive but the ct value for orf1a was undetected or if (2) the ct value for upe was >35 and <40. institutional review board statement. the study was performed in accordance with the declaration of helsinki and experimental protocols were revised and approved by irb at samsung medical center. (irb no. smc 2016-08-156-002). informed consent statement. irb at samsung medical center has approved the waiver of patient consent form because of the retrospective nature of this study. patient confidentiality is maintained at all time in accordance with samsung medical center policies. middle east respiratory syndrome: new disease, old lessons isolation of a novel coronavirus from a man with pneumonia in saudi arabia contact investigation for imported case of middle east respiratory syndrome enhanced mers coronavirus surveillance of travelers from the middle east to england laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities family cluster of middle east respiratory syndrome coronavirus infections euro surveillance: bull. europeen sur les. maladies transmissibles = european communicable dis ebola virus disease: an update for anesthesiologists and intensivists control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study infection control measures for operative procedures in severe acute respiratory syndrome-related patients conversion of operating theatre from positive to negative pressure environment importance of specimen type and quality in diagnosing middle east respiratory syndrome guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) guidelines for preventing the transmission of mycobacterium tuberculosis in health-care settings infection control and anesthesia: lessons learned from the toronto sars outbreak effects of environmental disinfection on the isolation of vancomycin-resistant enterococcus after a hospital-associated outbreak of middle east respiratory syndrome middle east respiratory syndrome infection control and prevention guideline for healthcare facilities surveillance for human infection with middle east respiratory syndrome coronavirus (mers-cov), interim guidance severe acute respiratory syndrome vs. the middle east respiratory syndrome mers coronavirus: diagnostics, epidemiology and transmission transmission characteristics of mers and sars in the healthcare setting: a comparative study middle east respiratory syndrome lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore middle east respiratory syndrome coronavirus in healthcare settings infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care, who guidelines guidance on personal protective equipment (ppe) to be used by healthcare workers during management of patients with confirmed ebola or persons under investigation (puis) for ebola who are clinically unstable or have bleeding, vomiting, or diarrhea in u.s. hospitals, including procedures for donning and doffing ppe, page last reviewed on august 30 guidelines for preventing the transmission of mycobacterium tuberculosis in healthcare settings infection prevention and control during health care for probable or confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection, interim guidance predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients atypical presentations of mers-cov infection in immunocompromised hosts management of asymptomatic persons who are rtpcr positive for middle east respiratory syndrome coronavirus (mers-cov), interim guidance serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak surgical protocol for possible or confirmed ebola cases anesthetic implications of ebola patient management: a review of the literature and policies j.p. this author helped with data collection, data analysis, writing of the first draft and revision of the manuscript, and archiving of the study files. s.y.y. this author helped with data analysis, discussion of results, writing and revision of the manuscript, archiving of the study files and approval of final version. j. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.r.p. or j.j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-253238-ptmxkpae authors: kopel, jonathan; perisetti, abhilash; gajendran, mahesh; boregowda, umesha; goyal, hemant title: clinical insights into the gastrointestinal manifestations of covid-19 date: 2020-05-23 journal: dig dis sci doi: 10.1007/s10620-020-06362-8 sha: doc_id: 253238 cord_uid: ptmxkpae the month of december 2019 became a critical part of the time of humanity when the first case of coronavirus disease 2019 (covid-19) was reported in the wuhan, hubei province in china. as of april 13th, 2020, there have been approximately 1.9 million cases and 199,000 deaths across the world, which were associated with covid-19. the covid-19 is the seventh coronavirus to be identified to infect humans. in the past, severe acute respiratory syndrome and middle east respiratory syndrome were the two coronaviruses that infected humans with a high fatality, particularly among the elderly. fatalities due to covid-19 are higher in patients older than 50 years of age or those with multimorbid conditions. the covid-19 is mainly transmitted through respiratory droplets, with the most common symptoms being high fever, cough, myalgia, atypical symptoms included sputum production, headache, hemoptysis and diarrhea. however, the incubation period can range from 2 to 14 days without any symptoms. it is particularly true with gastrointestinal (gi) symptoms in which patients can still shed the virus even after pulmonary symptoms have resolved. given the high percentage of covid-19 patients that present with gi symptoms (e.g., nausea and diarrhea), screening patients for gi symptoms remain essential. recently, cases of fecal–oral transmission of covid-19 have been confirmed in the usa and china, indicating that the virus can replicate in both the respiratory and digestive tract. moreover, the epidemiology, clinical characteristics, diagnostic procedures, treatments and prevention of the gastrointestinal manifestations of covid-19 remain to be elucidated. the covid-19 is a novel coronavirus with the first reported case in wuhan, hubei province of china, in early december 2019. the infected patients presented with pneumonia from an unknown cause, and by december 2019, the world health organization (who) recognized these cases due to a potential virus. in march 2020, covid-19 spread to 166 other countries with a declaration as a pandemic by who [1] . the coronaviruses (covs) belong to a large family of single-stranded, positive-sense rna viruses that infect both humans and animals, causing several respiratory, gastrointestinal (gi), hepatic and neurologic diseases [2] . the covs can be further divided into four major groups: alpha-coronavirus, beta-coronavirus, gamma-coronavirus and deltacoronavirus [3] . however, there were only six human covs that have been identified, including the alpha-covs hcovs-nl63 and hcovs-229e and the beta-covs hcovs-oc43, hcovs-hku1, severe acute respiratory syndrome-cov (sars-cov) and middle east respiratory syndrome-cov (mers-cov) [4] . the covid-19 is the seventh human cov to be identified, which has continued to spread globally. the covid-19-related disease can lead to pneumonia, acute respiratory distress syndrome (ards) and congestive heart failure [2] . although covid-19-related fatality data continue to change rapidly, current estimates from china put the case fatality rate in men and women at 2.8% and 1.7%, respectively. infected patients < 50 years of age have a fatality risk of < 0.5%, while those > 70 years old have a fatality up to 8% [5] . however, current estimates put the cumulative fatality for covid-19 at 5.6% (95% ci 5. 4-5.8) for china and 15.2% (12.5-17.9 ) outside of china [6] . globally, the fatality rate for covid-19 is estimated to be 5.7% (5.5-5.9) [6] . a recent study on the epidemiology of covid-19 found that the daily cumulative index (dci) of covid-19 cases was greatest in china (1320.85 per day), followed by republic of korea (78.78 per day), iran (43.11 per day), italy (30.62 per day), bahrain (9.5 per day), kuwait (9 per day) and japan (5.11 per day) [7] . furthermore, the overall fatality rate of covid-19 was highest in eastern mediterranean regions (6.7%, n = 34), followed by asia (3.4%, n = 2861) and europe (2.1%, n = 23%) [7] . however, these fatality data continue to changes as more cases appear in the usa and abroad. besides, patients with heart and lung diseases, diabetes, decompensated cirrhosis, hiv with low cd4 counts and other immunosuppressed patients are at higher risk for contracting covid-19 and developed severe disease. the diagnosis of covid-19 is performed by the combination of history and physical examination, reverse transcriptasepolymerase chain reaction (rt-pcr) analysis and computerized tomography (ct) scan. however, the ct scans have been found to have the highest specificity for the diagnosis of covid-19, although this may increase healthcare costs and reduces time to start treatment to individuals suspected of covid-19 [8] . currently, prevention of the transmission of covid-19 remains at paramount importance. therefore, the centers of disease control (cdc) has recommended frequent hand washing, social distancing, avoiding crowds and avoidance of touching the mouth, nose and eyes to prevent the spread of covid-19 until a vaccine or novel treatment is approved [9] . covid-19 is the disease caused by sars-cov-2 or covid-19 virus. current evidence indicates that covid-19 virus (or sars-cov-2) belongs to the beta-cov group and shares 80% nucleotide identity at the genomic level with sars-cov [10] . similar to other covs, covid-19 is believed to have originated from bats and transferred to other intermediate hosts before being transmitted to humans [11] . in most cases, covid-19 is transmitted through respiratory droplets [12] . once an individual is infected with covid-19, the most common symptoms are fever (98%), cough (76%), myalgia or fatigue (44%), and sometimes atypical symptoms such as sputum (28%), headache (8%), hemoptysis (5%) and diarrhea (3%) are present [2] . however, a patient can harbor a virus for a period of 2-14 days without any symptoms [2] . this is especially true with gi symptoms in which patients can still shed the virus even after pulmonary symptoms have resolved [13] . given the high percentage of covid-19 patients that present with gi symptoms (diarrhea, nausea, etc.), screening these patients is essential. therefore, pre-screening for covid-19 should extend to asymptomatic individuals while keeping the gi manifestations in mind [9] . the main limitations of our current understanding of covid-19 and its effects on the gi tract are primarily due to poor understanding of the effects of other covs on the gi tract. for sars-and mers-cov, the mechanism of gastrointestinal entry remains unknown; however, covid-19 has shown a mechanism by which covs might infect and proliferate in the gi tract for a long time. although the respiratory symptoms of covs are well-known, the gi symptoms and continual viral shedding in the feces are often overlooked. this review provides a comprehensive list of the gi symptoms, pathophysiology and mechanisms related to those symptoms, including the role of fecal shedding of covid-19. recently, cases of fecal-oral transmission of covid-19 have been confirmed in the united states (us) and china, indicating that the virus can replicate in both respiratory and digestive tracts [14] . furthermore, it is hypothesized that the virus can be re-transmitted by feces through aerosolization of viral-containing droplets; however, this has not been confirmed [14] . interestingly, some patients with positive covid-19 stool samples did not experience any gi symptoms, nor did it correlate with the severity of lung infection [13] . however, testing stool samples for covid-19 may provide an alternative method for diagnosing covid-19 [13] . furthermore, testing stool after a patient has been infected with covid-19 may be necessary to monitor any gi complications, and the potential for fecal-oral transmission after respiratory symptoms has resolved. specifically, 20% of covid-19 patients have viral rna that remains positive even after the negative conversion of viral rna in the respiratory tract [15] . therefore, it might be possible that covid-19 can produce long-term changes to the gi tract anatomy and physiology even after the respiratory infection has passed [15] . it could happen for the gut-microbiome and gut-lung crosstalk with the covid-19 genome, which has been observed with the influenza virus in the past [16] . a few patients with covid-19 showed intestinal microbial dysbiosis with decreased probiotics, such as lactobacillus and bifidobacterium [17] . among covid-19 patients, gi symptoms, such as diarrhea (2-10.1%), and nausea and vomiting (1-3.6%), occur with modest frequency compared to the fever and pulmonary symptoms in most patients (table 1) [18] [19] [20] . a recent study suggests that gi symptoms can be as high as 50% (39.6-50%, nausea (17.3%), diarrhea (12.9%), anorexia (12.2%), abdominal pain (5.8%), belching (5%) and emesis (5%) [9, 21] . this difference in the gi and pulmonary symptoms suggests differences in the covid-19 viral tropism compared to sars-cov, mers-cov and influenza virus [22] [23] [24] . although the pathogenesis is still being investigated, the first step of viral entry into the enterocytes occurs via the angiotensin-converting enzyme 2 (ace2) protein, similar to sars-cov [15, 25, 26] . ace-2 is a type-1 transmembrane metallocarboxypeptidase that helps regulate blood pressure along with the ace through the renin-angiotensin systems (ras) [27] . the ace-2 degrades angiotensin ii to generate angiotensin 1-7, thereby negatively regulating ras [28, 29] . although ace-2 is expressed mostly in the vascular endothelial cells, the renal tubular epithelium and in leydig cells in the testes, ace-2 has also been detected in the lung, kidney and gi tract [30] [31] [32] . the sars and mers-cov are also believed to use the ace-2 and ddp4, respectively, to enter cells for viral replication and release [33, 34] . as shown in fig. 1 , the virus can then spread to other digestive organs, such as the liver, by using the same ace2 enzyme [9] . reports of covid-19 presenting with abnormal liver chemistries were found to be as high as 20-30% [9] . despite the limited information on covid-19 and its gi symptoms, information from sars-cov and mers-cov provides some insights on the symptoms and disease severity from other covs. the mers-cov has shown to infect human primary intestinal epithelial cells, small intestine it is also found to transmit via the fecal-oral route [35] . given the prevalence of both mers-cov and covid-19 in the middle east, the co-infection of both coronaviruses through fecal-oral transmission remains a concern in countries with poor sanitation or healthcare infrastructure [36] . patients infected with the mers-cov show almost a similar frequency of gi symptoms (35%) as covid-19 [22, 37, 38] . specifically, the most common gi symptoms of mers-cov were diarrhea (22%) and vomiting (17%) [37] . another study on mers-cov patients showed the presence of gi symptoms in almost 25% of patients; the study found diarrhea (26%), vomiting (21%) and abdominal pain (17%) being the most common gi symptoms [22] . sars-cov also showed a similar frequency of gi illnesses (38.4%) to mers-cov [32] . furthermore, the frequency of diarrhea (19-50%), nausea and vomiting (19.6%) and abdominal pain (13%) in sars patients are similar to mers-and covid-19 [23, 39, 40] . interestingly, some sars patients (8%) can present with diarrhea and fever without respiratory symptom; in most cases (20.3%), sars patients present with watery diarrhea [32] . the study also found that the sars-cov rna can still be detected in the stool for more than 10 weeks after initial symptoms [32] . therefore, covid-19, like other covs, may continue to shed the virus through stool many weeks after the resolution of pulmonary symptoms. it is of a significant public health concern as poor water and sanitation practices could continue to spread covid-19 in the community. although no clinical studies have examined the release of the cytokine in the gi tract of covid-19 patients. however, th1 and th2 cytokines cause hypocontractility and hypercontractility of inflamed intestinal smooth muscle through downregulating l-type ca 2+ channels and upregulating regulators of g protein-coupled receptors [41] . furthermore, several cytokines such as interleukin-1 (il-1), il-33, il-36, tumor necrosis factor-α, il-6 and tumor necrosis factor-like cytokine 1a all contribute to maintaining the integrity and function of the gi tract [42] . in covid-19 patients with cytokine storm, elevations of il-2, il-7, granulocyte colony-stimulating factor, interferon-γ inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1-α and tumor necrosis factor-α were elevated [43] . although these cases are rare, these cytokines may become elevated in mild or persistent covid-19 cases [44] . for most covid-19 patients, cytokines il1-β, il-1ra, il-7, il-8, il-9, il-10, fgf2, gcsf, gmcsf, ifnγ, ip10, mcp1, mip1α, mip1β, pdgfb, tnfα and vegfa were also elevated [44] . some of these cytokines have been previously shown to be involved in gi health and disease [45] [46] [47] [48] [49] [50] . for example, il-2 is a potent cytokine that binds to lymphocytes and macrophages to maintain and preserve intersinal epithelium after injury from mechanical stresses, infections or viruses [45] . similarly, il-10 tnfα are believed to maintain intestinal epithelium by termination of excess inflammatory responses after infections or cell death and survival mechanisms [49, 50] . the elevation of these cytokines suggest an increased protective response to covid0-19 infection and damage to the gi tact. further studies are needed to indemnity the cytokines elevated in covid-19 patients and how it may influence the health of the gi and respiratory tract through chronic alterations in cytokine expression and secretion. white blood cell (wbc) counts vary in covid-19 patients ranging from leukopenia, leukocytosis and lymphopenia; however, lymphopenia is the most common wbc derangement [5, 19, 51] . elevated lactate dehydrogenase, ferritin and aminotransferase levels have also been observed [5, 19, 51] . as it is a viral infection, patients usually have normal procalcitonin levels; however, higher procalcitonin levels have been seen in patients admitted to the intensive care unit (icu) care [5, 19, 51] . the covid-19 patients typically present with bilateral ground-glass opacification with or without consolidations are consistent with viral pneumonia on chest ct scans [52, 53] . chest ct scans, together with rt-pcr testing, have a high sensitivity of 97% for detecting covid-19 but at the expense of specificity being only 25% [54] . in patients with gi symptoms, the best test of covid-19 diagnosis appears to be the collection and analysis of stool samples [13, 14, 17] . given the susceptibility of the gi tract to sars-cov-2 infection, it is also recommended to measure aminotransferase levels to evaluate the severity of the infection [9] . however, more studies of covid-19 are needed to establish rigorous and reliable markers for evaluating gi injury. there are currently no approved treatment recommendations for gi manifestations of covid-19 except for symptomatic and supportive care. the covid-19 is currently being treated with antiviral agents that are under experimentation for effectiveness. these agents include chloroquine, chloroquine/hydroxychloroquine, remdesivir (novel nucleotide analog), lopinavir-ritonavir (protease inhibitor) and tocilizumab (il-6 inhibitor) [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] . each of these agents was shown to be effective against sars-cov in preliminary in vivo and in vitro studies; however, further human clinical trials are needed to assess the effectiveness of these agents [69, 70] . therefore, the medical community presumes that the same overlap might exist to counteract covid-19 too. as of today, preventive measures (washing hands, social distancing, avoiding crowds, etc.) remain the only effective method for reducing the spread and severity of covid-19. currently, there is no evidence that the gi symptoms of elderly patients infected with covid-19 are different from younger populations. furthermore, given the potential risk of spread of the covid-19 via fecal-oral transmission, all major american gastroenterological societies including american association for the study of liver diseases (aasld), american gastroenterological association (aga), american college of gastroenterology (acg) and american society of gastrointestinal endoscopy (asge) have made recommendations for managing covid-19 in the patients both in outpatient and endoscopy settings [9, 71] . they divided those procedures into "non-urgent/postpone" and "non-urgent/perform" depending on the need for the endoscopy. it has been strongly advised to reschedule nonurgent endoscopic procedures such as cancer evaluations, prosthetic removals and evaluation of significant symptoms [9, 71] . furthermore, physicians are encouraged to screen patients using telemedicine for non-emergent procedures to decrease office visits for at-risk or infected patients and provide needed care to patients who are less willing or unable to travel. besides, telemedicine between patients allows effective triage the urgent and non-urgent procedures, thereby reducing a patient's risk of being exposed to covid-19. deferral of the elective procedures is likely to decrease the risk of transmission of covid-19 as luminal contents are frequently encountered during upper and lower endoscopies. second, all personnel involved with endoscopic procedures should wear appropriate personal protective equipment (ppe) (gloves, mask, eye shield/goggles, face shields and gown) [9] . these protocols and ppe are strongly encouraged by the cdc and who [9] . with a shortage of medical supplies, it is also recommended that physicians extend the use or reuse of surgical masks and eye protection; however, reusing medical equipment may inadvertently increase covid-19 transmission or exposure [9] . further investigation is needed to determine proper protective equipment protocols for covid-19 infections. patients should be pre-screened again at the time of arrival to endoscopy center to determine whether they are at high risk if they report any history of fever or respiratory symptoms, family members or close contacts with similar symptoms, any contact with a confirmed case of covid-19 and recent travel to a high-risk area [9] . high-risk patients with symptoms should be offered covid-19 testing. particular emphasis should be given to check the body temperature of patients arriving at the endoscopy center and avoid bringing patients older than 65 years to medical facilities unless necessary [9] . furthermore, endoscopy units should inform patients at least 24 h before any procedure to discuss their risks of covid-19, risks of endoscopy in viral transmission and appropriate intervention, including the postponement of the procedure. furthermore, the world endoscopy organization (weo) suggests endoscopy centers should be at a biosafety level 2 (includes laboratories or medical facilities that work with agents associated with human diseases that pose a moderate health hazard) for all endoscopies. the biosafety level should be increased to level 3 (includes laboratories or medical facilities that work with microbes of either indigenous or exotic that cause serious or potentially lethal disease through inhalation) for covid-19 patients [72] . as described by the british society of gastroenterology (bsg), the different biosafety levels minimize the transmission of covid-19 and delay or "flatten" the outbreak curve of the infection, which prevents overwhelming the healthcare resources [73] . therefore, the bsg recommends that endoscopic procedures only be performed in patients with acute upper gi bleeding, acute esophageal obstruction, endoscopic vacuum therapy for perforations/leaks, acute cholangitis/jaundice secondary to malignant/benign biliary obstruction, acute biliary pancreatitis, and/or cholangitis with stone and jaundice, infected pancreatic collections, urgent inpatient nutrition support and gi obstruction needing urgent decompression/stenting [73] . it is particularly important since these patients tend to have weaker immune systems and have a higher risk of infection [73] . the covid-19 virus remains a global healthcare emergency as the number of cases and fatality continue to rise. as more information is being gathered, understanding of the virus will most likely improve with better diagnosis, prevention and treatment options for patients exposed or experiencing symptoms from the disease. however, the gi manifestations of covid-19 pose a continuing challenge for the prevention of the virus spread. physicians should monitor for gi symptoms in covid-19-infected patients and examine whether the virus continues to remain in their stools after their respiratory symptoms have resolved. this knowledge may provide greater insight into preventing further infections and long-term complications arising from the covid-19 and might help in policy-making to prevent the community from the virus spread. novel coronavirus infection and gastrointestinal tract the sars-cov-2 outbreak: what we know the structure and functions of coronavirus genomic 3′ and 5′ ends isolation of a novel coronavirus from a man with pneumonia in saudi arabia updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan real estimates of mortality following covid-19 infection global epidemiology of coronavirus disease 2019: disease incidence, daily cumulative index, mortality, and their association with country healthcare resources and economic status performance of radiologists in differentiating covid-19 from viral pneumonia on chest ct covid-19 clinical insights for our community of gastroenterologists and gastroenterology care providers. bethesda: american college of gastroenterology novel coronavirus: where we are and what we know another decade, another coronavirus a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) fecal specimen diagnosis 2019 novel coronavirus-infected pneumonia first case of 2019 novel coronavirus in the united states evidence for gastrointestinal infection of sars-cov-2 microbiotadriven tonic interferon signals in lung stromal cells protect from influenza virus infection management of corona virus disease-19 (covid-19): the zhejiang experience a new coronavirus associated with human respiratory disease in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of pediatric patients with covid-19: a report of two family cluster cases clinical characteristics of 140 patients infected with sars-cov-2 in wuhan epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study a major outbreak of severe acute respiratory syndrome in hong kong factors associated with clinical outcome in 25 patients with avian influenza a (h7n9) infection in guangzhou, china. bmc infect dis a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin-i-converting enzyme and its relatives trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters angiotensin-converting enzyme 2 (ace2) is a key modulator of the renin angiotensin system in health and disease a novel coronavirus associated with severe acute respiratory syndrome quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme enteric involvement of severe acute respiratory syndrome-associated coronavirus infection. gastroenterology differential downregulation of ace2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl63 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus covid-19 in the shadows of mers-cov in the kingdom of saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus family cluster of middle east respiratory syndrome coronavirus infections a cluster of cases of severe acute respiratory syndrome in hong kong sars: experience at prince of wales hospital, hong kong cytokine-induced alterations of gastrointestinal motility in gastrointestinal disorders innate cytokines dictate the fate of acute intestinal inflammation covid-19: consider cytokine storm syndromes and immunosuppression the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak interleukin 2 modulates intestinal epithelial cell functionin vitro granulocyte-macrophage colony stimulating factor and inflammatory bowel disease: establishing a connection the role of granulocyte macrophagecolony-stimulating factor in acute intestinal inflammation interleukin-7 links t lymphocyte and intestinal epithelial cell homeostasis monocyte chemoattractant protein-1 contributes to gut homeostasis and intestinal inflammation by composition of il-10-producing regulatory macrophage subset tumour necrosis factor alpha in intestinal homeostasis and gut related diseases clinical features of patients infected with 2019 novel coronavirus in wuhan radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study relation between chest ct findings and clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter study correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine inhibition of human coronavirus 229e infection in human epithelial lung cells (l132) by chloroquine: involvement of p38 mapk and erk antiviral activity of chloroquine against human coronavirus oc43 infection in newborn mice clinical and virological factors associated with gastrointestinal symptoms in patients with acute respiratory infection: a two-year prospective study in general practice medicine persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229e intussusception in a premature infant simulating neonatal necrotizing enterocolitis detection of human coronaviruses in simultaneously collected stool samples and nasopharyngeal swabs from hospitalized children with acute gastroenteritis detection of human coronaviruses in children with acute gastroenteritis vacuolating encephalitis in mice infected by human coronavirus oc43 detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis human coronavirus oc43 associated with fatal encephalitis pathogenesis of mouse hepatitis virusinduced demyelination long-term human coronavirus-myelin cross-reactive t-cell clones derived from multiple sclerosis patients neuroinvasion by human respiratory coronaviruses current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019 (covid-19) pharmacologic cutrell jb. treatments for coronavirus disease 2019 (covid-19) considerations in performing endoscopy during the covid-19 pandemic suggestions of infection prevention and control in digestive endoscopy during current 2019-ncov pneumonia outbreak in wuhan acknowledgments none. key: cord-259374-m7q1roay authors: agostini, maria l.; pruijssers, andrea j.; chappell, james d.; gribble, jennifer; lu, xiaotao; andres, erica l.; bluemling, gregory r.; lockwood, mark a.; sheahan, timothy p.; sims, amy c.; natchus, michael g.; saindane, manohar; kolykhalov, alexander a.; painter, george r.; baric, ralph s.; denison, mark r. title: small-molecule antiviral β-d-n(4)-hydroxycytidine inhibits a proofreading-intact coronavirus with a high genetic barrier to resistance date: 2019-11-26 journal: j virol doi: 10.1128/jvi.01348-19 sha: doc_id: 259374 cord_uid: m7q1roay coronaviruses (covs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no fda-approved antivirals to treat the infections. one class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. while these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting covs. this has been attributed to the proofreading activity of the viral 3′-5′ exoribonuclease (exon). β-d-n(4)-hydroxycytidine (nhc) (eidd-1931; emory institute for drug development) has recently been reported to inhibit multiple viruses. here, we demonstrate that nhc inhibits both murine hepatitis virus (mhv) (50% effective concentration [ec(50)] = 0.17 μm) and middle east respiratory syndrome cov (mers-cov) (ec(50) = 0.56 μm) with minimal cytotoxicity. nhc inhibited mhv lacking exon proofreading activity similarly to wild-type (wt) mhv, suggesting an ability to evade or overcome exon activity. nhc inhibited mhv only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of g:a and c:u transition mutations present after a single infection. low-level nhc resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both mhv and mers-cov. these results point to a virus-mutagenic mechanism of nhc inhibition in covs and indicate a high genetic barrier to nhc resistance. together, the data support further development of nhc for treatment of covs and suggest a novel mechanism of nhc interaction with the cov replication complex that may shed light on critical aspects of replication. importance the emergence of coronaviruses (covs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. however, no antivirals have been approved to treat these infections. here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-n(4)-hydroxycytidine (nhc), against two divergent covs. viral proofreading activity does not markedly impact sensitivity to nhc inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the cov replicase. further, passage in the presence of nhc generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. together, these data support a mutagenic mechanism of inhibition by nhc and further support the development of nhc for treatment of cov infections. keywords coronavirus, nucleoside analogue, rdrp, rna-dependent rna polymerase, sars-cov, mers-cov, pandemic, antiviral resistance t he emergence of severe acute respiratory syndrome (sars) in 2002 and middle east respiratory syndrome (mers) in 2012 has underscored the ability of coronaviruses (covs) to cause lethal disease in humans (1, 2) . mers-cov continues to infect humans in the middle east, and four additional human covs (hcovs), hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1, continue to circulate globally and cause respiratory disease (3) (4) (5) (6) . the continued circulation in bat populations of sars-and mers-like covs that can replicate efficiently in primary human airway cells further demonstrates the potential for covs to emerge and cause severe disease in the future (7) (8) (9) (10) . while sars-cov and mers-cov outbreaks have been controlled, largely through public health measures (11) (12) (13) , the potential for future outbreaks highlights the need for safe and effective therapeutics to combat cov infections. there are currently no approved therapeutics or vaccines for any human cov infection. previous efforts to treat cov infections with existing antivirals did not conclusively benefit clinical outcomes; thus, the current standard of care remains mostly supportive (14) (15) (16) . several targets for direct-acting antivirals are being investigated to treat cov infections (17) (18) (19) . because the viral replication machinery performs an essential role in genome replication, therapeutics approved to treat multiple different viral infections are aimed at this target (20) . many approved antivirals are classified as nucleoside analogues, compounds that mimic natural nucleosides to inhibit viral replication (21) . inhibition by nucleoside analogues can be accomplished through a variety of mechanisms. common mechanisms of action include incorporation of the analogue by the viral polymerase to induce premature termination of strand synthesis and loss of essential genetic information through mutagenesis (22) (23) (24) (25) . a previous study reported that the nucleoside analogues ribavirin (rbv) and 5-fluorouracil (5-fu) did not potently inhibit covs, and this finding was attributed to the proofreading capabilities of the viral 3=-5= exoribonuclease (exon) (26) . recent reports have demonstrated the inhibition of wild-type (wt) covs by nucleoside analogues such as galidesivir (bcx4430) and remdesivir (gs-5734) (27) (28) (29) . while these compounds have shown efficacy against covs, administration of multiple compounds simultaneously may be required to effectively treat cov infections and control the emergence of drug resistance, as has been demonstrated for other viral infections (30) . ␤-d-n 4 -hydroxycytidine (nhc) (eidd-1931; emory institute for drug development), a cytidine analogue, has recently been shown to inhibit multiple viruses, including chikungunya virus, venezuelan equine encephalitis virus (veev), respiratory syncytial virus (rsv), hepatitis c virus, norovirus, influenza a (iav) and b viruses, and ebola virus (31) (32) (33) (34) (35) (36) . previous reports have demonstrated increased introduction of transition mutations in viral genomes after treatment, as well as a high genetic barrier to resistance (31, 36) . antiviral activity of nhc has also been reported against the human ␣-cov hcov-nl63, as well as the ␤-cov sars-cov (43, 44) . neither the nhc mechanism of action nor nhc resistance has been described for any cov to date. in this study, we investigated nhc inhibition and resistance in two divergent ␤-covs, murine hepatitis virus (mhv) and mers-cov. we show that nhc potently inhibits wt mhv and mers-cov with minimal cytotoxicity. we also demonstrate that mhv exon proofreading activity has a limited but measurable effect on sensitivity to nhc. we observed an nhc inhibition profile consistent with a mutagenic mechanism of action featuring an accumulation of transition mutations, indicative of a high genetic barrier to resistance. experiment with two divergent ␤-covs: the model cov mhv and the epidemically circulating zoonotic cov mers-cov. nhc treatment resulted in a dose-dependent reduction in viral titers for mhv ( fig. 2a ) and mers-cov (fig. 2b ). this inhibition resulted in 50% effective concentrations (ec 50 s) of 0.17 m for mhv (fig. 2c ) and 0.56 m for mers-cov (fig. 2d) . we detected negligible changes in dbt-9 cell viability out to 200 m (fig. 2e ) and 50% cytotoxic concentration (cc 50 ) values above 10 m in vero cells (fig. 2f ). the antiviral activity was not due to cytotoxicity, as the selectivity indexes were ͼ1,000 for mhv and ͼ20 for mers-cov. together, these results confirm potent inhibition of ␤-covs by nhc. the nhc inhibition profile in covs is consistent with mutagenesis. to better understand the mechanism through which nhc inhibits cov replication, we performed a time of drug addition assay to determine at what point in the viral replication cycle nhc acts (40) . we added 16 m (ϳ100 times the ec 50 ) nhc at the indicated times preor postinfection (p.i.) of cells with wt mhv at a multiplicity of infection (moi) of 1 pfu/cell and quantified viral replication after a single infectious cycle. compared to the vehicle (dimethyl sulfoxide [dmso]) control, nhc significantly inhibited mhv replication when added at or before 6 h postinfection (fig. 3a) , suggesting that nhc acts at early stages of the viral replication cycle. we next determined the effect of nhc on mhv rna levels and compared it to the effect on the infectious-virus titer. rna levels were reduced by approximately 10-fold at the highest tested concentration of nhc in both mhv-infected cell monolayers (fig. 3b ) and supernatants (fig. 3c ). in contrast, the viral titer was reduced up to 5,000-fold at this concentration. we therefore calculated the ratio of infectious virus per viral rna genome copy number normalized to the untreated control (specific infectivity) after nhc treatment and found that the specific infectivity of wt mhv was reduced in a dose-dependent manner after treatment with increasing concentrations of nhc (fig. 3d ). together, these data are consistent with a mutagenic mechanism of nhc anti-cov activity. nhc treatment increases transition mutations present across the mhv genome. to directly test the effect of nhc treatment on the mutational burden, we treated wt mhv with increasing concentrations of nhc and performed full-genome next-generation sequencing (ngs) on viral populations released after a single round of infection. our data demonstrated a dose-dependent increase in mutations present at low frequencies (ͻ5% of the viral population) across the genome after treatment with increasing concentrations of nhc ( fig. 4a to c). further analysis of the types of mutations introduced by nhc revealed an increase in the total number of transition mutations with increasing nhc concentrations ( fig. 4d to f). the relative proportions of g:a and c:u transitions among all observed mutations were increased by 13 to 15% in the presence of 2 m nhc and 36 to 40% in the presence of 4 m nhc compared to the vehicle control ( fig. 4g and h) . conversely, the relative proportions of a:g and u:c transitions decreased with increasing nhc concentrations compared to the vehicle control ( fig. 4g and h) . together, these results demonstrate that nhc treatment during a single round of wt mhv infection causes predominantly g:a and c:u transition mutations that are detectable at low frequencies across the genome. these data further support a mutagenic mechanism of action for nhc inhibition of wt mhv. nhc inhibition is modestly enhanced in the absence of exon proofreading. mutagenic nucleoside analogues, such as rbv and 5-fu, have been ineffective at potently inhibiting wt covs due to the exon proofreading activity (26) . a proofreadingdeficient [exon(ϫ)] mhv mutant displays increased sensitivity to previously tested nucleoside analogues, indicating that proofreading dampens inhibition by these compounds (26, 37, 38) . therefore, we tested the sensitivity of exon(ϫ) mhv to nhc inhibition. our results indicate that nhc decreases the titers of both wt and exon(ϫ) mhv in a dose-dependent manner but that exon(ϫ) mhv demonstrates a statistically passage in the presence of nhc yields low-level resistance associated with multiple transition mutations. to better understand the development and impact of nhc resistance in covs, we passaged two lineages of wt mhv 30 times in the presence of increasing concentrations of nhc and tested the sensitivity of passage 30 (p30) mhv populations to nhc inhibition. we found that the lineage 1 (mhv p30.1) viral population showed no change in sensitivity to nhc compared to wt mhv (fig. 6a ). however, lineage 2 (mhv p30.2) showed a decrease in sensitivity to nhc inhibition in a titer reduction assay, especially at higher concentrations of compound. we observed a modest (approximately 2-fold) increase in ec 90 values for mhv nhc passage viruses (wt mhv, ec 90 ϭ 1.53 m; mhv p30.1, ec 90 ϭ 2.61 m; mhv p30.2, ec 90 ϭ 2.41 m) (fig. 6b ). this suggests that mhv passage resulted in minimal resistance to nhc. we next sought to determine if passaging wt mhv in the presence of nhc altered the replication capacities of these viruses. we found that both lineages showed a delay in replication but ultimately reached peak titers similar to that of wt mhv (fig. 6c ). this delay in replication suggests that mhv p30 is less fit than wt mhv. to identify mutations associated with these phenotypes after passage, we sequenced complete genomes of mhv p30.1 and mhv p30.2. both lineages passaged in the presence nhc had accumulated over 100 consensus mutations distributed across the genomes ( fig. 6d and e; see table s1 in the supplemental material). in comparison, a previous study reported that wt mhv accumulated only 23 total mutations after 250 passages in the absence of drug (38) . further analysis of the p30 mhv mutational profile demonstrated that slightly more of the total mutations in both lineages were synonymous changes that did not result in an amino acid change as opposed to nonsynonymous changes, which did alter the amino acid sequence ( fig. 6f ; see table s1 ). additionally, the vast majority of mutations in both lineages were transition mutations resulting in a purine-to-purine or pyrimidine-to-pyrimidine change (fig. 6g ). both lineages contained only two transversion mutations resulting in a purine-topyrimidine or pyrimidine-to-purine change. though all possible transition mutation types were detected in both viral-lineage populations, the majority in both passage lineages were g:a transitions (fig. 6h) , which is consistent with the mhv ngs data (fig. 4) . to determine if the mutational profile at p30 was consistent with an earlier passage, we analyzed the whole genomes of both lineages 1 and 2 at p19. both lineages demonstrated fewer mutations at p19 than at p30, but the profiles of synonymous versus nonsynonymous changes and the transition mutations were similar (see fig. s1 and table s2 in the supplemental material). to determine whether the lack of robust resistance to nhc was broadly applicable across ␤-covs, we assessed the capacity of mers-cov to evolve resistance to nhc. as (fig. 7b ), corresponding to approximately 2-fold resistance. similar to mhv, we observed no substantial shift in the dose-response curve for mers-cov, indicating minimal acquired resistance. nhc p30 viruses replicated similarly to wt p30 mers-cov (fig. 7c) . we sequenced both lineages of the mers-cov p30 population and detected 27 consensus mutations in mers-cov nhc p30.1 (fig. 7d ; see table s3 in the supplemental material) and 41 consensus mutations in mers-cov nhc p30.2 ( fig. 7e ; see table s3 ) that were randomly distributed across the genome. both mers-cov nhc p30.1 and mers-cov nhc p30.2 accumulated nonsynonymous and synonymous mutations in roughly equal proportions (fig. 7f) . as in mhv, the mutations detected in mers-cov p30 lineages were predominantly transition mutations (fig. 7g ). further analysis of these mutations revealed that the predominant type of transition was lineage dependent. the majority of transition mutations in mers-cov nhc p30.1 were g:a transitions, as was observed in both p30 mhv lineages, whereas mers-cov nhc p30.2 contained similar numbers of each type (fig. 7h) . these results indicate that mers-cov can achieve low-level resistance to nhc and that development of resistance is associated with the accumulation of multiple transition mutations. together, our data suggest nhc acts as a mutagen and that it poses a high genetic barrier to resistance for ␤-covs. in this study, we demonstrate that nhc potently inhibits the divergent ␤-covs mhv and mers-cov. our data are consistent with a virus-mutagenic mechanism of action, as evidenced by a decrease in specific infectivity and an increase in g:a and c:u transition mutations present at low frequencies across the genome after treatment with nhc. we also demonstrate that robust resistance to nhc is difficult to achieve in both mhv and mers-cov. both wt mhv and exon(ϫ) mhv are sensitive to nhc inhibition, suggesting that nhc is able to overcome exon-mediated proofreading to inhibit wt covs and that it interacts with covs differently than other previously tested nucleoside analogues. utility of the broad-spectrum antiviral nhc as a pan-cov therapeutic. early work with nhc focused on the mutagenic effects of the compound in multiple bacterial systems (39, 41, 42) . more recently, the antiviral properties of the compound have been reported for multiple rna viruses, including chikungunya virus, venezuelan equine encephalitis virus, respiratory syncytial virus, hepatitis c virus, norovirus, influenza a and b viruses, and ebola virus (31) (32) (33) (34) (35) (36) . nhc has also been shown to potently inhibit sars-cov and hcov-nl63 (43, 44) , suggesting potential utility in treating cov infections (17) . based on previous studies, nhc appears to primarily inhibit viral replication by mutagenesis (31, 34) . serial passaging in the presence of nhc led to low-level resistance for veev, but no detectable resistance for rsv, iav, or bovine viral diarrhea virus, indicating a high barrier to resistance (31, 34, 36) . consistent with the previous studies, we demonstrated that nhc is mutagenic in covs and that serial passaging yields low-level, approximately 2-fold resistance. low-level resistance has also been observed for remdesivir, another nucleoside analogue that potently inhibits covs. approximately 6-fold resistance to remdesivir is conferred by two mutations in the cov rna-dependent rna polymerase (rdrp) (37) . this study further expands the known antiviral spectrum of nhc to include mhv and mers-cov, two genetically divergent ␤-covs, and supports nhc development as a broad-spectrum cov antiviral. nhc inhibition may circumvent exon-mediated proofreading. nhc is the first mutagenic nucleoside analogue demonstrated to potently inhibit proofreading-intact covs. previous studies have demonstrated that viruses lacking exon proofreading activity [exon(ϫ) viruses] are more sensitive to inhibition by nucleoside analogues, especially rbv and 5-fu (26, 37, 38, 45) . this increased sensitivity has been attributed to the inability of exon(ϫ) viruses to efficiently remove incorrect nucleosides (46) . however, we observed a minimal change in nhc sensitivity between wt mhv and exon(ϫ) mhv, especially by ec 90 . this suggests that nhc interacts with the cov replicase differently than other previously tested nucleoside analogues. one explanation is that nhc may evade removal by the proofreading exon. studies investigating nucleosides that inhibit dna viruses have suggested an inability of the viral exonuclease to efficiently excise some nucleoside analogues (47, 48) . further, a previous study suggested that the t4 dna exonuclease activity was incapable of removing nhc (49) . while the sars-cov exon efficiently removes 3=-terminal mismatches regardless of type (46, 50) , the effect of nhc on this activity has not been investigated. interestingly, mismatches readily observed during single-nucleotide elongation by the sars-cov polymerase in the absence of drugs correspond to mismatches that would lead to the g:a and c:u transitions observed after nhc treatment (46) . this suggests that the cov polymerase could be naturally more prone to make these types of errors, which are then magnified by nhc. this could lead to a scenario where exon cannot prevent dipping below the error threshold, ultimately resulting in lethal mutagenesis and similar inhibition of both wt mhv and exon(ϫ) mhv (51) . several nucleosides, including the mutagenic rbv, have multiple demonstrated mechanisms other than direct incorporation into the genome (52, 53) . thus, another explanation for the unique potency of nhc in the presence of an active proofreading exon is that it may inhibit viral replication by additional mechanisms beyond mutagenesis. indeed, previous reports have suggested that nhc may also interfere with the rna secondary structure or virion release to cause inhibition (31, 36) . further, exogenous c or u in the presence of nhc could rescue viral replication in hcv, chikungunya virus, rsv, and influenza a virus (32, 34, 36) , indicating that nhc competes with exogenous nucleosides at some stage prior to viral inhibition. these results raise the possibility that nhc could inhibit a process that results in similar inhibition of these viruses by a mechanism unrelated to exon. thus, future studies will be important to investigate the role of proofreading in nhc inhibition of covs to shed light on the intricacies of nhc inhibition of the cov replication complex. nhc mutagenesis may hinder emergence of robust resistance to nhc. the decrease in specific infectivity, along with the accumulation of transitions across the cov genome, supports a mutagenic mechanism of action for nhc in covs. nhc resistance in covs was modest and difficult to achieve, as we obtained approximately 2-fold resistance after 30 passages. resistance was associated with multiple mutations. interestingly, mers-cov accumulated fewer mutations over 30 passages than mhv. while differences in viral mutation rates could be the driver of this difference, previous studies have suggested that mhv does not have a higher mutation rate than mers-cov (54) (55) (56) . the differences in mutation accumulation between mhv and mers-cov may be a product of different passage conditions. while mhv was passaged with a consistent transfer volume, mers-cov passage volumes were adjusted over time to sustain viral replication under escalating selection for drug resistance. the constant-volume passaging conditions may have more severely bottlenecked mhv populations and fixed more mutations in the genome than the variable-volume passaging conditions applied to mers-cov (57) . alternatively, this difference could also reflect a difference in mutational robustness of the mhv and mers-cov genomes, though this proposition needs to be investigated further (58, 59) . while a portion of the mutations that accumulated during passage likely contribute to nhc resistance, mutations in proteins dispensable for viral replication in cell culture, such as ns2 and nsp2, may be merely tolerated because of their limited effect on viral fitness in the context of our passage conditions (60) (61) (62) . few common mutations arose in both mhv and mers-cov passage series (see tables s1 to s3), suggesting that multiple pathways to low-level nhc resistance exist in covs. interestingly, for both mhv and mers-cov, the p30 lineage that demonstrated a greater change in sensitivity to nhc was the lineage that had fewer overall mutations (fig. 6 and 7) . both mhv passage lineages replicated less well than wt mhv, suggesting that the accumulation of mutations during passage may negatively impact viral fitness and the ability of mhv to evolve robust resistance to nhc. further, the mhv lineage that did not result in changed sensitivity to nhc by p30 (mhv p30.1) had fewer mutations present at consensus by p19 than the other lineage (see fig. s1 ). thus, it is possible that the accumulation of deleterious mutations counteracts the potential benefits of resistance mutations (63) . if this is the case, mutations promoting nhc resistance would need to arise early during passage to help mitigate the accumulation of excess deleterious mutations. alternatively, the inability to evade inhibition by nhc may lead to the accumulation of a greater number of nhc-associated transitions and ultimately a higher mutational burden that may impact viral fitness (64, 65) . together, our results support the hypothesis that establishment of resistance to nhc in covs requires a delicate balance of resistance-promoting mutations, viral fitness, and accumulation of deleterious mutations. thus, defining the roles of individ-ual nhc resistance-associated mutations will be an important goal for future studies. overall, our results support further development of nhc as a broad-spectrum antiviral for treatment of cov infections and contribute new insights into important aspects of cov replication. cell culture. murine astrocytoma delayed brain tumor (dbt) (66) and vero (atcc ccl-81) cells were maintained at 37°c in dulbecco's modified eagle medium (dmem) (gibco) supplemented with 10% fetal bovine serum (fbs) (invitrogen), 1% penicillin and streptomycin (gibco), and 0.1% amphotericin b (corning). viruses. all work with mhv was performed using the recombinant wt strain mhv-a59 (genbank accession number ay910861 [67] ). mers-cov stocks were generated from cdna clones (genbank accession number jx869059 [68] ). compounds and cell viability studies. nhc was synthesized at the emory institute for drug development and prepared as a 20 mm stock solution in dmso. cell viability was assessed using celltiter-glo (promega) in 96-well plates according to the manufacturer's instructions. dbt and vero cells were incubated with the indicated concentrations of compound at 37°c for 24 h (dbt) or 48 h (vero). cell viability was determined using a veritas microplate luminometer (promega) or glomax (promega), with values normalized to those of vehicle-treated cells. nucleoside analogue sensitivity studies and generation of ec 50 curves. subconfluent monolayers of dbt cells were infected with mhv at an moi of 0.01 pfu per cell for 1 h at 37°c. the inoculum was removed and replaced with medium containing the indicated compound concentration. cell supernatants were harvested 24 h postinfection. titers were determined by plaque assay as described previously (69) . subconfluent monolayers of vero cells were infected at an moi of 0.01 pfu/cell of mers-cov. after virus adsorption for 30 min at 37°c, the inoculum was removed. the cells were washed with pbs and incubated with medium containing the indicated concentrations of nhc or dmso (vehicle control). after 48 h, the supernatant was collected and titers were determined by plaque assay as described previously (70) . ec 50 and ec 90 values and curves were generated using the nonlinear regression curve fit in graphpad (la jolla, ca) prism software. time of drug addition assay. subconfluent monolayers of dbt cells were treated with medium containing dmso or 16 m nhc (ϳ100 times the ec 50 ) at the indicated times pre-or postinfection. the cells were infected with wt mhv at an moi of 1 pfu/cell for 1 h at 37°c. the virus inoculum was removed and replaced with fresh medium. culture supernatant was harvested 12 h postinfection, and the viral titer was determined by plaque assay. quantification of viral genomic rna. subconfluent dbt cells were infected with wt mhv at an moi of 0.01 pfu/cell. the inoculum was removed after 1 h of incubation at 37°c, and medium containing the indicated concentration of nhc was added. total rna from cells and supernatant rna were harvested using trizol reagent (invitrogen) after 20 h. both total rna and supernatant rna were extracted by phase separation. total rna was purified by ethanol precipitation, and supernatant rna was purified using a purelink rna minikit (invitrogen) according to the manufacturer's protocol. total rna was reverse transcribed using superscript iii (invitrogen) to generate cdna, which was quantified by quantitative pcr (qpcr) as previously described (26) . data are presented as 2 ϫδδct , where δδc t denotes the change in the threshold cycle for the viral target (nsp10) normalized to the control (glyceraldehyde-3-phosphate dehydrogenase [gapdh]) before and after drug treatment. the supernatant rna was quantified using one-step quantitative reverse transcriptase pcr (qrt-pcr) as previously described (45) . the data are presented as the fold change in genome rna copies normalized to vehicle control. determination of specific infectivity. subconfluent dbt cells were infected with wt mhv at an moi of 0.01 pfu/cell. the inoculum was removed after 1 h of incubation at 37°c, and medium containing the indicated concentration of nhc was added. supernatant rna was harvested using the trizol reagent (invitrogen) after 20 h, followed by extraction and quantification as described above. the viral titer was determined by plaque assay. the specific infectivity was calculated as the number of pfu divided by the supernatant genome rna copy number. this ratio was then normalized to that of the vehicle control. ngs studies. subconfluent dbt cells were infected with wt mhv at an moi of 0.01 pfu/cell and treated with the indicated concentrations of nhc. the supernatant was collected 24 h postinfection. purified viral rna was submitted to genewiz (south plainfield, nj) for library preparation and sequencing. briefly, after quality controls, viral rnas were randomly fragmented using heat. libraries were prepared and sequenced on the illumina hiseq platform. genewiz performed base calling and read demultiplexing. trimmomatic was used to trim adapter contaminants and reads shorter than 36 bp and to filter low-quality bases (q score ͻ 30) (71). the paired-end fastq reads were then aligned with the mhv genome using bowtie2 to generate a sam file (72) . samtools was used to process the resultant alignment file and to calculate the coverage depth at each nucleotide, generating a sorted and indexed bam file. lofreq was used to call substitution variants, including low-frequency variants, and to generate a variant file (73) . the bash shell and excel were used to further process and analyze the resultant vcf file. a frequency of 0.001 was used as a cutoff for variants, consistent with previous reports (74) . absolute numbers of mutations are reported for each nhc treatment. the percentage of the total mutations for each specific mutation type was calculated using these numbers. the difference in percentage for each class of mutation after treatment compared with vehicle control is referred to as the relative proportion of these mutations. mhv population passage in the presence of nhc. wt mhv was passaged in triplicate in increasing concentrations of nhc from 1 m to a maximum of 5 m. infection was initiated for passage 1 at an moi of 0.1 pfu/cell. viral supernatants were harvested from each viral lineage and frozen when the cell monolayer demonstrated 80% cytopathic effect (cpe) or after 24 h. a constant volume of 16 l was used to initiate subsequent passages. all three lineages were maintained until passage 16, when lineage 3 demonstrated no visible cpe upon multiple attempts at varying concentrations. lineages 1 and 2 were maintained until passage 30. after each passage, total rna was harvested from infected cell monolayers using the trizol reagent. viral rna was extracted from passage 19 and passage 30 samples and reverse transcribed using superscript iii, followed by generation of 12 pcr amplicons to cover the whole genome. dideoxy amplicon sequencing was performed by genewiz and analyzed to identify mutations present at greater than 50% of the total using macvector. viral mutation maps depicting the identified mutations were generated using macvector. mers-cov population passage in the presence of nhc. three parallel independent passage series of wt mers-cov were performed on vero cells in the presence of gradually increasing concentrations of nhc up to a maximum concentration of 6.5 m to select for drug-resistant mutant viruses. virus adaptation to nhc-supplemented complete culture medium was assessed by monitoring the progression of characteristic mers-cov cpe. the volumes of transferred culture supernatants were adjusted empirically to balance continuous selective pressure against culture extinction. each of triplicate lineages in the mers-cov passage experiment was sustained through passage 30. however, the third lineage was severely impaired in replication and was excluded from further analysis. total infected-cell mers-cov rna purified from monolayers infected with terminal-passage (p30) culture supernatant was used to generate rt-pcr products for consensus sanger sequencing of the complete viral genome (genewiz). changes in passaged virus nucleotide and deduced amino acid sequences were identified via alignment with the wt parental virus genomic sequence using macvector. virus replication assays. subconfluent monolayers of dbt (mhv) or vero (mers-cov) cells were infected with wt or nhc-passaged viral populations at an moi of 0.01 pfu/cell for 1 h (mhv) or 30 min (mers-cov). inocula were removed, and the cells were washed with pbs before addition of prewarmed medium. supernatants were harvested at the indicated times postinfection, and titers were determined by plaque assay. statistics. statistical tests were performed using graphpad (la jolla, ca) prism 7 software as described in the respective figure legends. supplemental material for this article may be found at https://doi. a novel coronavirus associated with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia global seasonal occurrence of middle east respiratory syndrome coronavirus (mers-cov) infection seroepidemiologic studies of coronavirus infection in adults and children epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emer further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus control measures for severe acute respiratory syndrome (sars) in taiwan description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea clinical management and infection control of sars: lessons learned sars: systematic review of treatment effects ribavirin and interferon therapy for critically ill patients with the middle east respiratory syndrome: a multicenter observational study potential antivirals and antiviral strategies against sars coronavirus infections coronavirusesdrug discovery and therapeutic options antiviral drugs specific for coronaviruses in preclinical development antivirals and antiviral strategies approved antiviral drugs over the past 50 years advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases antimicrobial strategies antiviral nucleoside and nucleotide analogs: a review inhibitors of the hepatitis c virus polymerase; mode of action and resistance coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses antiviral combination therapy for treatment of chronic hepatitis b, hepatitis c, and human immunodeficiency virus infection ␤-d-n(4)-hydroxycytidine is a potent antialphavirus compound that induces high level of mutations in viral genome characterization of ␤-d-n4-hydroxycytidine as a novel inhibitor of chikungunya virus antiviral activity of nucleoside analogues against norovirus orally efficacious broadspectrum ribonucleoside analog inhibitor of influenza and respiratory syncytial viruses identification of a new ribonucleoside inhibitor of ebola virus replication ribonucleoside analogue that blocks replication of bovine viral diarrhea and hepatitis c viruses in culture coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease proofreading-deficient coronaviruses adapt for increased fitness over long-term passage without reversion of exoribonuclease-inactivating mutations n4-hydroxycytidine-a new mutagen of a base analogue type a timeof-drug addition approach to target identification of antiviral compounds mutagenic action of n4-hydroxycytidine on escherichia coli b cytϫ the metabolism of n4-hydroxycytidine-a mutagen for salmonella typhimurium inhibition of human coronavirus nl63 infection at early stages of the replication cycle inhibition of severe acute respiratory syndrome-associated coronavirus (sarscov) by calpain inhibitors and ␤-d-n4-hydroxycytidine homology-based identification of a mutation in the coronavirus rna-dependent rna polymerase that confers resistance to multiple mutagens structural and molecular basis of mismatch correction and ribavirin excision from coronavirus rna inhibition of purified human and herpes simplex virus-induced dna polymerases by 9 cidofovir diphosphate inhibits adenovirus 5 dna polymerase via both nonobligate chain termination and direct inhibition, and polymerase mutations confer cidofovir resistance on intact virus effect of proofreading and dam-instructed mismatch repair systems on n 4 -hydroxycytidineinduced mutagenesis rna 3=-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex theories of lethal mutagenesis: from error catastrophe to lethal defection demethylation therapy as a targeted treatment for human papillomavirus-associated head and neck cancer the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase viral mutation rates mers coronavirus in dromedary camel herd, saudi arabia spread, circulation, and evolution of the middle east respiratory syndrome coronavirus viral quasispecies evolution evolution favors protein mutational robustness in sufficiently large populations the origins of mutational robustness murine coronavirus nonstructural protein ns2 is not essential for virus replication in transformed cells cell-typespecific type i interferon antagonism influences organ tropism of murine coronavirus the nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication pathways to extinction: beyond the error threshold the contribution of epistasis to the architecture of fitness in an rna virus mutation and epistasis in influenza virus evolution molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 engineering infectious cdnas of coronavirus as bacterial artificial chromosomes high fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants growth and quantification of mers-cov infection trimmomatic: a flexible trimmer for illumina sequence data scaling read aligners to hundreds of threads on general-purpose processors lofreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets sequence-specific error profile of illumina sequencers we thank members of the denison laboratory for thoughtful discussions regarding this work. key: cord-262045-r2iqpmmc authors: smits, saskia l.; raj, v. stalin; pas, suzan d.; reusken, chantal b.e.m.; mohran, khaled; farag, elmoubasher a.b.a.; al-romaihi, hamad e.; alhajri, mohd m.; haagmans, bart l.; koopmans, marion p. title: reliable typing of mers-cov variants with a small genome fragment date: 2014-12-15 journal: j clin virol doi: 10.1016/j.jcv.2014.12.006 sha: doc_id: 262045 cord_uid: r2iqpmmc background: middle east respiratory syndrome coronavirus (mers-cov) is an emerging pathogen that causes lower respiratory tract infection in humans. camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. human-to-human transmission occurs sporadically. the wide geographic distribution of mers-cov among dromedary camels and ongoing transmissions to humans provides concern for the evolution of a mers-cov variant with efficient human-to-human transmission capabilities. phylogenetic analysis of mers-cov has occurred by analysis of full-length genomes or multiple concatenated genome fragments, which is time-consuming, costly and limited to high viral load samples. objective: to develop a simple, reliable mers-cov variant typing assay to facilitate monitoring of mers-cov diversity in animals and humans. study design: phylogenetic analysis of presently known full-length mers-cov genomes was performed to identify genomic regions with sufficient phylogenetic content to allow reliable mers-cov variant typing. rt-pcr assays targeting these regions were designed and optimized. results: a reverse-transcription pcr assay for mers-cov targeting a 615 bp spike fragment provides a phylogenetic clustering of mers-cov variants comparable to that of full-length genomes. the detection limit corresponds to a cycle treshold value of ∼35 with standard upe real time pcr assays on rna isolated from mers-cov emc. nasal swabs from rt-pcr positive camels (ct values 12.9–32.2) yielded reliable sequence information in 14 samples. conclusions: we developed a simple, reliable mers-cov variant typing assay which is crucial in monitoring mers-cov circulation in real time with relatively little investment on location. middle east respiratory syndrome coronavirus (mers-cov; family coronaviridae) may cause severe lower respiratory tract infection in humans [1, 2] . camels are considered the likely animal source for zoonotic infection; sporadic human-to-human transmission does occur, but is considered to be inefficient based on currently available data [3] [4] [5] [6] [7] [8] [9] [10] . until recently, new mers-cov infections in humans were reported at a steady low rate reaching ∼200 confirmed human cases early 2014. in march and april 2014, however, a surge of mers-cov infections occurred mainly in hospitals around jeddah, kingdom of saudi arabia (ksa) and united arab emirates (uae). this increased case load was in part attributed to an increase in community cases, but mostly to transmission within hospitals, with no evidence for evolution of a mers-cov variant with more efficient human-to-human transmission capabilities (who;http://www.who.int/csr/disease/coronavirus infections/ mers cov update 09 may 2014.pdf?ua=1). the ongoing occurrence of new cases, however, and the finding that mers-cov is endemic in dromedary camels in a wide geographic region [3, 10, 11] , stresses the need for surveillance of strain diversity, to help unravel the epidemiology of this newly identified pathogen, and to provide a reference for studies into mers-cov evolution. all currently sequenced human and camel mers-cov genomes share >99% nucleotide identity across the ∼30 kb genome. phylogenetic analysis has occurred mainly by analysing full-length genomes or multiple concatenated genome fragments, to provide reliable phylogenetic information [5, [12] [13] [14] . however, full length genome sequencing capacity is not widely available, and requires relatively high viral load, leading to limited success when trying to sequence animal or human samples [5] . an accurate typing of mers-cov variants, preferably with a simple assay encompassing a short region of the mers-cov genome, is crucial in monitoring the mers-cov outbreak in real time with relatively little investments on location. in this study, we describe the development of a mers-cov variant typing assay, which can be used in monitoring mers-cov circulation, especially when more information on virus type is required rapidly from a large number of viruses from animals/humans. full or near full-length mers-cov genomes encompassing nucleotides 215-29770 (numbering corresponding to mers-cov emc genome jx869059) were aligned with mafft version 7 (http://mafft.cbrc.jp/alignment/server/) ( table 1) . to remove the redundancy from the dataset, fastgroupii analysis (http://fastgroup.sdsu.edu/fg tools.htm) was performed grouping all currently available viral genomes based on nucleotide composition, resulting in 15 groups (table 1) . one viral genome from each group was taken as representative in subsequent analyses. a summary of nucleotide positions that vary across the genomes was created using bioedit v7.2.0 [15] and the number of nucleotide positions with variations was plotted over 1000 nucleotide windows. phyml trees were generated using seaview 4 software with the approximate likelihood ratio test based on a shimodaira-hasegawa-like procedure which used general time reversible as substitution model. nearest neighbor interchange, subtree pruning, and regrafting-based tree search algorithms were used to estimate tree topologies, as described previously [5] . total nucleic acids were isolated using an automated mag-napure 96 extraction with the total nucleic acid isolation kit (roche, mannheim, germany) as described previously [13] . the detection limit of the assay was determined on rna isolated from 10x dilutions of cell culture derived mers-cov emc 2012 (jx869059), as described above. virus stocks were prepared as described previously [16] . rna was isolated from serial 10-fold dilutions of mers-cov emc 2012 [13] . serial 10-fold dilutions of this rna were amplified in parallel with the mers-cov variant typing assay described above and the upe and n gene real time pcr assays [17, 18] . the sensitivity of the mers-cov variant typing assay was expressed as cycle threshold value based on the upe real time pcr assay. on may 13 and 15, 2014, the first two mers-cov infected patients in the netherlands who became infected upon travel to saudi arabia were reported to who; throat swabs from these patients were available [19] . in february and april 2014, nasal swabs were taken from dromedary camels of different age and sex from a slaughterhouse in doha, qatar [13] , which were available for this study. to identify genome regions for reliable phylogenetic analysis comparable to that of full-length genome, (near) full-length mers-cov genomes (table 1) were aligned. viral genomes that were 99.9% identical were grouped and one viral genome from each group was taken as representative in the analysis (table 1 ) and a phyml tree was generated (fig. 1a) . was plotted over 1000 nt windows (fig. 1b) . four genome fragments, two located in orf1a, one in s and one in orf4b, showed a relatively high number of snps (fig. 1b) and for this reason already had been used as concatenated genome fragment for phylogenetic analysis [12] . however, phylogenetic trees created for these four fragments separately did not accurately reflect the phylogenetic positions of the currently known full-length mers-cov genomes (data not shown). in a subsequent analysis all identified snps were inspected visually and regions containing snps with phylogenetic information regarding the previously identified four clusters of viruses were identified (fig. 1a) . the s2 domain of the spike protein contains a number of these mutations (fig. 1b) . a fragment of 615 bp, containing three of these snps, provided a phylogenetic tree similar to the one obtained upon full-genome analysis regarding the previously identified four mers-cov clusters (fig. 2) , whereas other mers-cov genome regions did not provide similar results. as observed for the full-length genomes, human, and camel mers-cov genomes shared >99% nucleotide identity across the 615 bp s2 domain fragment. mers-cov genomes that were released recently and not taken along in the variation analysis were typed using the 615 bp s2 domain and clustered similar to their phylogenetic positions as upon full genome analysis ( fig. 2 and table 1 ), thereby validating the assay. a sequencing rt-pcr targeting the identified genome fragment was developed and optimized using rna isolated from cell culturederived mers-cov. in limiting dilution experiments, the mers-cov variant typing assay amplified the 615 bp fragment down to a cycle treshold value of ∼35 as determined by diagnostic upe real time pcr assays [17, 18] . the mers-cov variant typing assay was used to type rt-pcr positive nose swabs from the first two human dutch mers-cov cases in 2014. the results showed grouping consistent with previous findings based on long sequence fragments [19] (fig. 2 ). in addition, the mers-cov variant typing assay was performed on camel samples from a slaughterhouse in qatar [13] and sequences for 14 mers-cov positive animals with cycle threshold values ranging from 12.9 to 32.2 as determined by upe real time rt-pcr [17, 18] were obtained (fig. 2) . five different camel mers-cov variants in clusters b1 and b2 were detected, without the need for full genome sequencing. phylogenetic analysis of representative mers-cov full-length genomes indicated that four regions in the mers-cov genome exist with a substantially higher nucleotide variation across genomes. however, phylogenetic analysis of these genome regions sepa-rately did not provide reliable phylogenetic information, in contrast to an analysis of the concatenated fragments [5, 12, 19] . subsequent analyses revealed a region in the open reading frame that encodes the spike protein with a number of positions in which nucleotide variation occurs between mers-cov variants with a strong phylogenetic signal regarding previously identified clusters of viruses based on full-length mers-cov genomes. the here[19] and from camels from a slaughterhouse in doha, qatar [13] . the observed detection limit of a cycle treshold value of ∼35 allows variant typing in clinical samples obtained from humans and animals with relatively low viral loads. it enables inclusion of samples in phylogenetic analysis that would not have been included when only full length genomes would have been accepted. this mers-cov variant typing assay, targeting a part of the mers-cov spike gene, is a relatively simple rt-pcr sequencing assay that could be performed more widely as initial screening assay in laboratories with basic sequencing capacity. it provides accurate crude mers-cov type information, applicable in monitoring viral variants in real time. new variants identified through this initial screening could then be sent to a reference laboratory for further characterization. the continued occurrence of transmission between humans in health care and family settings is an ongoing concern as stated by the world health organization (http://www.who.int/csr/disease/coronavirus infections/mers cov update 27 march 2014.pdf?ua=1), although the outbreaks appear to be self-limiting or extinguishable with rigorous implementation of appropriate infection control guidelines at present. however, as the primary route of transmission to humans is uninterrupted, human-to-human transmissions will continue to occur. the data obtained from the mers-cov variant typing assay would aid in informing the most effective international preparedness and response, allowing ad hoc risk assessment and implementation of containment strategies if necessary. state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia human infection with mers coronavirus after exposure to infected camels antibodies against mers coronavirus in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands this work was funded by zonmw top project 91213058z. this does not alter our adherence to all the policies on sharing data and materials. all authors contributed to gathering and analysis of the information. saskia smits, bart haagmans, and marion koopmans drafted and revised the manuscript based on all authors contributions. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2014.12.006. key: cord-264267-weat0qs6 authors: kleine-weber, hannah; schroeder, simon; krüger, nadine; prokscha, alexander; naim, hassan y.; müller, marcel a.; drosten, christian; pöhlmann, stefan; hoffmann, markus title: polymorphisms in dipeptidyl peptidase 4 reduce host cell entry of middle east respiratory syndrome coronavirus date: 2020-01-21 journal: emerg microbes infect doi: 10.1080/22221751.2020.1713705 sha: doc_id: 264267 cord_uid: weat0qs6 middle east respiratory syndrome (mers) coronavirus (mers-cov) causes a severe respiratory disease in humans. the mers-cov spike (s) glycoprotein mediates viral entry into target cells. for this, mers-cov s engages the host cell protein dipeptidyl peptidase 4 (dpp4, cd26) and the interface between mers-cov s and dpp4 has been resolved on the atomic level. here, we asked whether naturally-occurring polymorphisms in dpp4, that alter amino acid residues required for mers-cov s binding, influence cellular entry of mers-cov. by screening of public databases, we identified fourteen such polymorphisms. introduction of the respective mutations into dpp4 revealed that all except one (δ346-348) were compatible with robust dpp4 expression. four polymorphisms (k267e, k267n, a291p and δ346-348) strongly reduced binding of mers-cov s to dpp4 and s protein-driven host cell entry, as determined using soluble s protein and s protein bearing rhabdoviral vectors, respectively. two polymorphisms (k267e and a291p) were analyzed in the context of authentic mers-cov and were found to attenuate viral replication. collectively, we identified naturally-occurring polymorphisms in dpp4 that negatively impact cellular entry of mers-cov and might thus modulate mers development in infected patients. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus with a single-stranded rna genome of positive polarity. it belongs to the coronaviridae family (genus betacoronavirus), which is part of the order nidovirales. mers-cov was isolated in 2012 from the sputum of a 60 year old man suffering from acute pneumonia and renal failure in saudi arabia [1] . since its discovery, mers-cov has caused 2,442 human infections of which 842 (34.5%) had a fatal outcome (as of may, 2019) [2] . dromedary camels are reservoir hosts of mers-cov and display only common cold-like symptoms upon infection but constitute the main source of human infections. transmission to humans occurs via close contact to animals or contaminated animal products [3] [4] [5] [6] . human-to-human transmissions seem limited and were mainly observed in health care settings, leading to mers outbreaks in hospitals [7] [8] [9] [10] [11] [12] . finally, differences in the tissue specific expression of the cellular receptor for mers-cov, dpp4, were recently suggested to account for the differences in mers-cov transmission and disease induction in camels and humans, respectively [13, 14] . in order to infect a host (cell) and replicate, mers-cov has to deliver its genome into the cellular cytoplasm for gene translation and genome replication. this process is facilitated by the viral spike (s) glycoprotein, a type-i transmembrane protein embedded in the viral envelope. for host cell entry, the surface unit, s1, of mers-cov s binds to the cellular type-ii transmembrane protein dipeptidyl peptidase 4 (dpp4, cd26) [15] . the structure of the interface between dpp4 and mers-cov-s was resolved on the atomic level and fifteen residues in dpp4 were found to make direct contact with residues in the viral s protein [16] . upon dpp4 engagement, mers-cov s undergoes proteolytic activation through the cellular serine protease tmprss2 or the endosomal cysteine protease cathepsin l [17] [18] [19] , which allows the transmembrane unit, s2, of mers-cov s to fuse the viral membrane with cellular membranes. dpp4 is a prolyl oligopeptidase that is expressed in various tissues [20] and involved in multiple biological processes including t-cell activation [21] , control of the activity of growth factors, chemokines and bioactive peptides [22] [23] [24] , and regulation of the glucose metabolism [25] . mature dpp4 is embedded in the plasma membrane as a homodimer and each monomer consists of an n-terminal cytoplasmic domain, followed by a transmembrane domain and a large ectodomain, which can be further subdivided into a short stalk domain, a glycosylation-rich and a cysteine-rich region as well as the c-terminal catalytic domain (α/ β-hydrolase domain) [26] . polymorphisms in the dpp4 gene were implicated in several diseases and conditions, including diabetes [27, 28] and myocardial infarction [29] but their potential impact on mers-cov infection has not been analyzed. we asked whether naturally-occurring amino acid polymorphisms in dpp4 residues making contact with mers-cov s have an impact on mers-cov entry. we identified fourteen polymorphisms by screening public databases and introduced the respective mutations into a dpp4 expression plasmid. we identified four mutations that reduced mers-cov s binding to dpp4 and mers-cov s-driven host cell entry without affecting dpp4 expression at the cell surface. 293t cells were transfected with expression vectors for wt or mutant dpp4, or empty expression vector (negative control). at 16 h post transfection, the culture medium was replaced and the cells were further incubated for additional 32 h. then, the cells were washed with pbs and mixed with 2x sds-sample buffer (0.03 m tris-hcl, 10% glycerol, 2% sds, 0.2% bromophenol blue, 1 mm edta). cell lysis was achieved by incubating the samples for 10 min at room temperature followed by incubation at 96°c for an additional 10 min. the samples were further loaded on polyacrylamide gels and sds-page (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed. next, the proteins were transferred onto nitrocellulose membranes (hartenstein gmbh) by immunoblotting. the membranes were further blocked by incubation in pbs-t (pbs containing 0.5% tween 20 and 5% skim milk powder) for 30 min at room temperature. afterwards, the membranes were incubated overnight at 4°c with undiluted supernatant of a hybridoma cell line secreting anti-cmyc antibody 9e10 (for dpp4 detection) or pbs-t containing anti-ß-actin (actb) antibody (mouse, 1:1,000, sigma aldrich). following three washing intervals with pbs-t, the membranes were further incubated with pbs-t containing horseradish peroxidaseconjugated anti-mouse antibody (goat, 1:5,000, dianova) for 1 h at room temperature before an in house-prepared enhanced chemiluminescent solution (0.1 m tris-hcl [ph 8.6], 250 µg/ml luminol, 1 mg/ ml para-hydroxycoumaric acid, 0.3% h 2 o 2 ) was added and signals were recorded using the chemocam imaging system and the chemostar professional software (intas science imaging instruments gmbh). in order to quantify the signal intensity of the protein bands, the program imagej (fiji distribution) [30] was used. to account for differences in the total protein content of the samples and variations, we normalized the dpp4 signals against the respective signals of the loading control (actb). 293t (human kidney cells, dsmz no. acc 635), bhk-21 (hamster kidney cells, dsmz no. acc 61) and vero 76 (african green monkey kidney cells, kindly provided by andrea maisner, philipps-university marburg) were cultivated in dulbecco's modified eagle medium (pan-biotech) while caco-2 cells (human colorectal adenocarcinoma cells) were cultivated in minimum essential medium (thermofisher scientific). the media were supplemented with 10% fetal bovine serum (biochrom), 100 u/ml of penicillin and 0.1 mg/ml of streptomycin (pan-biotech). all cell lines were incubated at 37°c and 5% co2 in a humidified atmosphere. for subcultivation and seeding, cells were washed with phosphate-buffered saline (pbs) and detached by incubation with trypsin/ edta solution (pan-biotech) (bhk-21, vero 76 and caco-2) or by resuspending the cells in culture medium (293t). transfection of 293t and bhk-21 cells was carried out by calcium-phosphate precipitation or with the help of icafectin-441 (in-cell-art) or fugene hd (promega). all dpp4 mutants were generated based on a pcdna3.1/zeo(+)-based expression vector in which the coding sequence for human dpp4 (genbank: xm_005246371.3) containing an c-terminal cmyc epitope was inserted into via bamhi/ecori restriction sites. the following aa (amino acid) substitutions were introduced via overlap-extension pcr: k267e, k267n, q286k, t288i, t288s, a289v, a291p, a291v, r317k, y322h, i346t, i346v and k392n . in addition, a deletion mutant was generated that lacks aa residues 346-348 (δ346-348). information on dpp4 polymorphisms was retrieved from the ensembl database (https://www.ensembl.org/index.html) [31] and the single nucleotide polymorphism database (dbsnp) of the national center for biotechnology information (ncbi) (https://www.ncbi.nlm.nih.gov/snp) [32] , and is based on data provided by the gnomad database (genome aggregation database, https://gnomad. broadinstitute.org/), topmed program (trans-omics for precision medicine, https://www.nhlbiwgs.org/), exac consortium (exome aggregation consortium, http://exac.broadinstitute.org/) [33] and the 1000g project (1,000 genomes project, http://www. internationalgenome.org/) [34] (for detailed information see supplementary table 1) . we further utilized pcaggs-based expression vectors for vesicular stomatitis virus (vsv) glycoprotein (g), mers-cov s wildtype (wt) and mers-cov s (d510g) (the latter two either untagged or equipped with a c-terminal v5 epitope) that have been described elsewhere [35] [36] [37] . in addition, a previously described expression vector for angiotensin converting enzyme 2 was employed [38] . similar to the strategy used for the generation of dpp4 mutants, we employed the overlap-extension pcr technique to introduce a single mutation into the mers-cov s open reading frame, thus generating untagged and v5-tagged mers-cov s (d539n). soluble s comprising the s1 subdomain of mers-cov s (aa residues: 1-747) fused to a human igg fc tag was generated by inserting the pcr-amplified s1 sequences into the pcg1fc vector [39] (kindly provided by georg herrler, university of veterinary medicine hannover) making use of the bamhi/sali restriction sites. in addition, we generated an expression vector for the enhanced green fluorescent protein (egfp) by inserting the egfp coding sequence, which was pcr-amplified from the pegfp-c1 vector (clontech), into the pcaggs plasmid using the ecori/xhoi restriction sites. all pcr-amplified sequences were subjected to automated sequence analysis (microsynth seqlab) to verify their integrity. sequences of primers used for cloning of the different constructs are available upon request. analysis of dpp4 surface expression by immunofluorescence analysis bhk-21 cells were grown on coverslips and transfected with the different dpp4 constructs or empty expression vector using icafectin-441 (in-cell-art) at 24 h post seeding according to the manufacturer's instructions. after changing the culture medium at 4 h post transfection, the cells were incubated for additional 20 h. then, the culture medium was aspirated and the cells were washed with pbs, before they were fixed by incubated with pbs containing 4% paraformaldehyde (pbs/pfa) for 15 min at room temperature. subsequently, the cells were washed with 0.1 m glycine/ pbs solution followed by a washing step with pbs. next, the coverslips were incubated with anti-dpp4 antibody (mouse, diluted 1:200 in pbs containing 1% bovine serum albumin [pbs/bsa], abcam) for 1 h at 4°c. for this, the coverslip was put on a drop (20 µl) of antibody solution that was added on a sheet of parafilm inside a humidity chamber (a glass dish in which the parafilm was placed on wet paper tissue). thereafter, the cells were washed 3x with pbs before incubation with alexafluor568-conjugated antimouse antibody (goat, 1:1000, diluted in pbs/bsa, thermofisher scientific) for 30 min at 4°c was performed. subsequently, the cells were washed 3x with pbs. finally, the cells were incubated with dapi (4',6-diamidino-2-phenylindole, carl roth) and mounted in prolong gold antifade mountant (ther-mofisher scientific) before they were analyzed using a zeiss lsm800 (zeiss) confocal laser scanning microscope and the zen imaging software (zeiss). analysis of dpp4 surface expression by flow cytometry bhk-21 cells were transfected with expression vectors for wt or mutant dpp4, or empty expression vector (negative control). at 16 h post transfection, the culture medium was replaced and the cells were further incubated for additional 32 h. then, the cells were washed with pbs, resuspended in pbs/ bsa and pelleted by centrifugation (600 x g, 5 min, 4°c). after aspiration of the supernatant, the cells were resuspended in pbs/bsa containing anti-dpp4 antibody (mouse, diluted 1:100, abcam) and incubated for 1 h at 4°c. next, the cells were pelleted, washed with pbs/bsa, pelleted again, resuspended in pbs/bsa containing alexafluor488-conjugated anti-mouse antibody (donkey, diluted 1:500, thermo-fisher scientific) and incubated for 1 h at 4°c. subsequently, the cells were washed (as described above) and resuspended in pbs/pfa for 2 h at 4°c for fixation. finally, the cells were washed (as described above) and resuspended in pbs/bsa for flow cytometric analysis using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of dpp4 surface expression, the mean fluorescence intensity (mfi) value of the negative control was subtracted from all samples. for normalization of dpp4 surface expression, values obtained for cells expressing dpp4 wt were set as 100% and the relative surface expression of the respective dpp4 mutants was calculated accordingly. in order to generate soluble mers-cov s for binding studies, 293t cells were transfected with an expression vector for the s1 subunit of mers-cov s fused to the fc fragment of human immunoglobulin g (solmers-s1-fc). at 24 h post transfection, the culture medium was exchanged and the cells were further incubated for 24 h before culture supernatants were harvested and freed from cellular debris by centrifugation (4,700 x g, 10 min, 4°c). the clarified supernatants were loaded on vivaspin protein concentrator columns with a molecular weight cut-off of 30 kda (sartorius) and centrifuged at 4,700 x g at 4°c until the sample was 10-fold concentrated. for the binding studies with solmers-s1-fc, a similar protocol was followed as described for the analysis of dpp4 surface expression with the exceptions that sol-mers-s1-fc was used instead of the primary antibody (1:10 dilution in pbs/bsa) and that an alexafluor488conjugated anti-human antibody (goat, 1:500 dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. bhk-21 cells transfected with expression vectors for wt or mutant dpp4, ace2 or empty expression vector (both negative controls) were analyzed by flow cytometry for solmers-s1-fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of solmers-s1-fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of solmers-s1-fc to cells expressing dpp4 wt was set as 100% and the relative binding efficiencies to cells expressing the respective dpp4 mutants or ace2 were calculated accordingly. 293t cells (grown in 6-well plates) were cotransfected with expression plasmids coding for solmers-s1-fc and wt or mutant dpp4. cells transfected with empty expression vector instead of dpp4 or sol-mers-s1-fc (or both) served as controls. at 16 ,400 x g at 4°c, before 400 µl of the supernatant were mixed with 50 µl of protein a-sepharose (1 g protein a-sepharose [sigma-aldrich] in 4 ml pbs) while the residual 100 µl of the cell lysate were mixed with 100 µl 2x sds-sample buffer and incubated for 15 min at 96°c (these samples were later analyzed to confirm comparable total protein levels [via detection of actb] as well as comparable dpp4 and solmers-s1-fc levels before the co-immunoprecipitation [co-ip] step.). following incubation of the lysate/protein a-sepharose mixtures for 2 h at 4°c in an overhead shaker, the samples were centrifuged for 5 min at 16,400 x g at 4°c to pellet the protein a-sepharose/solmers-s1-fc/dpp4-complexes. after aspiration of the supernatant, 500 µl of np40 lysis buffer (without protease inhibitors) were added and the cells were mixed by vortexing, before being centrifuged again. this washing routine was repeated three times, before finally 50 µl of 2x sdssample buffer were added to the pelleted complexes and the samples were further incubated for 15 min at 96°c. thereafter, the samples were subjected to sds-page and western blot analysis (see above). detection of dpp4 (lysate and co-ip samples) and actb (lysate samples) was carried out as described above. solmers-s1-fc was detected (lysate and co-ip samples) by incubation with a peroxidase-conjugated anti-human antibody (goat, 1:5,000, dianova). signal intensities of the protein bands were quantified as described above. further, signals obtained for dpp4 were normalized against the respective signals for solmers-s1-fc in order to account for variations in transfection efficiency and sample processing. for the binding studies with soluble dpp4, a similar protocol was followed as described for the analysis of binding of solmers-s1-fc with the exceptions that a soluble dpp4 fused to the fc region of human igg (soldpp4-fc, acro biosystems) was used instead of solmers-s1-fc (1:200 dilution in pbs/bsa) and that an alexafluor488-conjugated anti-human antibody (goat, 1:500 dilution in pbs/bsa, thermofisher scientific) was employed as the secondary antibody. 293t cells transfected with expression vectors for wt or mutant (d510g and d539n) mers-cov s, or empty expression vector (negative control) were analyzed by flow cytometry for soldpp4-fc binding using an lsr ii flow cytometer and the facs diva software (both bd biosciences). additional data analysis was carried out using the fcs express 4 flow research software (de novo software). for quantification of soldpp4-fc binding, the mfi value obtained for cells transfected with empty expression vector was subtracted from all samples. further, binding of soldpp4-fc to cells expressing mers-cov s wt was set as 100% and the relative binding efficiencies to cells expressing the respective mers-cov s mutants were calculated accordingly. we employed a previously described protocol for the generation of vsv pseudotype particles (vsvpp) that is based on a replication-deficient vsv vector that lacks the genetic information for vsv-g but instead contains the genetic information for egfp and firefly luciferase (fluc) as reporters of transduction efficiency (vsv*δg-fluc, kindly provided by gert zimmer, institute of virology and immunology, mittelhäusern/switzerland) [37, 40] . in brief, 293t cells transfected with expression vectors for mers-cov s, vsv-g (positive control) or empty expression vector (negative control) were inoculated with vsv*δg-fluc for 1 h before being washed with pbs and further incubated for 16 h with culture medium that was supplemented with anti-vsv-g antibody (i1, mouse hybridoma supernatant from crl-2700; atcc) (except for cells expressing vsv-g). the produced vsvpp were inoculated onto bhk-21 cells expressing wt or mutant dpp4, or no dpp4 (empty expression vector, negative control) and incubated for 16-18 h before fluc activity in cell lysates was quantified as an indicator for transduction efficiency using the beetle-juice kit (pjk) and a plate luminometer (hidex) [41] . bhk-21 cells were transfected with expression vectors for wildtype or mutant dpp4 (k267e or a291p), or empty expression vector (negative control) using fugene hd (promega) according to the manufacturer's instructions. at 24 h posttransfection, the cells were infected with mers-cov (human betacoronavirus 2c emc/2012, mers-cov emc-2012, genbank accession number: jx869059) at a multiplicity of infection of 0.01 for 1 h. thereafter, the inoculum was removed and the cells were washed 3x with pbs before fresh medium was added and the first sample (time point 0 h postinfection) was taken. the cells were further incubated and additional samples were taken at 24 and 48 h postinfection. viral titers in the culture supernatant were analyzed by quantitative reversetranscriptase pcr, using the upe assay according to a published protocol [42] . in brief, viral rna was isolated from cell culture supernatant using the nucleospin rna virus kit (macherey-nagel), reversetranscribed into cdna using the superscript iii one step rt-pcr system (thermofisher scientific) and analyzed on a lightcycler 480 qpcr cycler platform (roche) with primers and conditions as specified for the upe assay [42] . in vitro-transcribed standard samples containing defined amounts of mers-cov fragments (10, 100, 1,000 and 10,000 copies) were included for absolute quantification as genome equivalents (ge). the dpp4 protein structure (4pv7) [43] and the structure of the complex formed by the mers-cov s receptor binding domain bound to dpp4 (4l72) [16] were retrieved from the research collaboratory for structural bioinformatics protein database (rscb pdb, https://www.rcsb.org/). structure visualization and colorization was performed using the yasara software (http://www.yasara.org/index.html) [44] and ucsf chimera version 1.14 (developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco) [45] . one-way or two-way analysis of variance (anova) with dunnett's posttest was used to test for statistical significance. only p values of 0.05 or lower were con identification of polymorphisms in dpp4 that alter amino acid residues which make contact with mers-cov s the binding interface between mers-cov s and the cellular receptor dpp4 was resolved by wang and colleagues using crystallography, revealing the interacting amino acid residues for each binding partner [16] : fourteen residues of mers-cov s (y499, n501, k502, l506, d510, r511, e513, d537 g538, d539, y540, r542, w553 and v555) interact with a total of fifteen residues in dpp4 (k267, f269, q286, t288, a289, a291, l294, i295, h298, r317, y322, r336, q344, i346 and k392) [16] , which are distributed over the glycosylation-rich domain and the cysteinerich domain ( figure 1a-c) . in order to identify polymorphic residues in dpp4 that contact mers-cov s, we screened public databases that provide information on polymorphic amino acid residues based on data derived from different bio projects (i.e. gnomad, topmed, exac, 1000g; more information is given in the materials and methods section). by this method we found that nine out of the fifteen dpp4 residues interacting with mers-cov s are polymorphic (k267, q286, t288, a289, a291, r317, y322, i346 and k392) ( figure 1c ). while five of these residues can be replaced by only a single different amino acid residue (q286[k], a289 . circles with sticks represent glycosylation sites, while small numbers indicate the amino acid residues. triangles below the domains highlight the positions of amino acid residues that directly interact with mers-cov s (grey triangles mark residues for which no polymorphism has been reported, while red triangles indicate polymorphic residues). (b) side (left) and top (right) view of homodimeric dpp4 (the dotted line indicates the border between the two monomers and the cellular plasma membrane is schematically depicted below the side view model of dpp4). the protein model was constructed on the published crystal structure (4pv7) deposited in rscb pdb and the binding interface with mers-cov s has been highlighted (green). (c) close-up on the dpp4 residues that directly interact with mers-cov s and for which no polymorphic (yellow) or polymorphic (red) residues have been reported. in addition, the specific residues in dpp4 (regular letters and numbers), including the respective polymorphic residues (letters in brackets), and the corresponding interacting residues in mers-cov s (italicized letters and numbers) are indicated. (d) frequency of polymorphic dpp4 residues in the human population. public databases (see supplementary table 1 and the materials and methods section for detailed information) were screened for the frequency of the polymorphic residues under study (y-axis). error bars indicate standard error of the mean (sem) and refer to polymorphic residues found in more than one database. table 1 ). finally, the frequency of these polymorphisms in the human population is low, ranging roughly from 1:19,000 (a289v) to 1:245,000 (t288i) ( figure 1d and supplementary table 1 ). we next introduced the polymorphisms in a dpp4 expression plasmid. western blot analysis and signal quantification revealed that all resulting dpp4 variants were robustly expressed and total expression levels were comparable (figure 2a-b) . as dpp4 needs to be transported to the plasma membrane to be engaged by mers-cov s for host cell entry, we next investigated whether the presence of the polymorphic dpp4 residues has an impact on dpp4 cell surface localization. for this, we performed flow cytometry and confocal laser scanning microscopy, using transfected bhk-21 cells and an antibody targeting the dpp4 ectodomain. we found that all dpp4 variants but one, a deletion variant lacking amino acid residues 346-348 (δ346-348), displayed comparable cell surface expression levels ( figure 3a-b) . we next investigated whether polymorphic dpp4 residues impact mers-cov host cell entry. for this, we made use of vesicular stomatitis virus (vsv) pseudotypes (vsvpp) bearing mers-cov s or vsv g, which does not bind to dpp4 and served as negative control [46] . as expected, vsvpp harboring vsv g were able to efficiently transduce bhk-21 target cells irrespective of dpp4 expression. in contrast, transduction of bhk-21 cells mediated by mers-cov s critically depended on ectopic expression of human dpp4, in accordance with published findings [47] ( figure 4) . notably, four dpp4 polymorphisms -k267e, k267n, a291p and δ346-348 -severely reduced mers-cov s-driven transduction compared to dpp4 wt (figure 4 ). in order to analyze whether the reduction in mers-cov s-driven host cell entry would translate into attenuated mers-cov replication, we next investigated two dpp4 polymorphisms (k267e and a291p) in the context of infection with authentic mers-cov. when followed over a period of two days post infection it was observed that mers-cov replication in bhk-21 cells expressing human dpp4 was significantly reduced when dpp4 contained either k267e or a291p ( figure 5 ). after the identification of dpp4 polymorphisms that reduce s-driven cellular entry of rhabdoviral vectors as well as mers-cov replication, we next sought to investigate whether the attenuating phenotype was due to reduced binding of mers-cov s to dpp4. for this, we used soluble mers-cov s, produced by fusing the s1 subunit, which contains the dpp4 binding domain, to the fc portion of human immunoglobulin g (solmers-s1-fc). co-immunoprecipitation analysis demonstrated that dpp4 variants harboring polymorphisms k267e, k267n or a291p, which were not compatible with efficient mers-cov s-driven host cell entry, displayed significantly reduced ability to interact with mers-cov s as indicated by weaker dpp4 signals upon protein a-sepharosemediated pull-down of dpp4/solmers-s1-fc (as compared to dpp4 wt, figure 6a-b) . notably, dpp4 variant δ346-348 could be as efficiently coimmunoprecipitated as dpp4 wt, indicating that its inefficient receptor function was solely due to its defect in proper surface transport. the findings obtained by co-ip analysis were confirmed by flow cytometry. it was revealed that polymorphisms that reduced mers-cov s-driven host cell entry (k267e, k267n, a291p and δ346-348) and spread of authentic mers-cov (k267e and a291p) also reduced mers-cov s binding to cells expressing dpp4 on the cell surface ( figure 6c ). in addition, polymorphism a289v, which decreased mers-cov s-driven transduction to a lesser extent than the aforementioned polymorphisms (figure 4) , also reduced mers-cov s binding to dpp4. thus, dpp4 polymorphisms k267e, k267n and a291p reduce mers-cov s-driven host cell entry and mers-cov infection by diminishing mers-cov s binding to dpp4. host cell entry of mers-cov critically depends on the interaction between the viral s protein and the cellular receptor dpp4. a link between obesity or underlying diseases like diabetes mellitus, which both can affect dpp4 expression levels [48] , and the risk of fatal outcome of mers-cov infection has been made [49] . moreover, alanine scanning mutagenesis identified dpp4 residues critical for mers-cov entry, including k267, l294, i295, r317 and r336 [50, 51] . however, the impact of natural-occurring variations on host cell entry of mers-cov has not been addressed so far. we identified dpp4 polymorphisms that reduce s protein-driven host cell entry and replication of authentic mers-cov by lowering the binding efficiency of mers-cov s to dpp4, suggesting that the dpp4 phenotype may impact the course of mers-cov infection. western blot analysis, flow cytometry and confocal laser scanning microscopy revealed that none of the polymorphisms studied, except deletion of amino acids 346-348, had a significant impact on total or cell surface expression of dpp4, at least in the context of dpp4 transfected cells. four polymorphisms located at three different sites in dpp4 (k267e, k267n, a291p and δ346-348) severely reduced s protein-driven host cell entry. as dpp4 δ346-348 was shown to be incompatible with robust cell surface transport but able to interact with mers-cov s in co-ip analysis, we conclude that the reduction in entry efficiency is solely due to insufficient dpp4 surface levels. in contrast, reduction of host cell entry by k267e, k267n and a291p could not be explained by reduced dpp4 expression and these polymorphisms were thus further investigated. mers-cov infection of bhk-21 cells transfected to express dpp4 wt and variants k267e or a291p revealed that k267e or a291p were not compatible with robust mers-cov replication. finally, co-ip analyses and binding studies with soluble mers-cov s showed that these dpp4 polymorphisms reduced s protein binding to dpp4. when looking at the crystal structure of the complex consisting of the mers-cov s receptor binding domain bound to dpp4, these observations do not come as a surprise. dpp4 residue k267 has been reported to contact mers-cov s residues g538 and d539, including a salt bridge interaction with d539 [16] . the exchange of k267 to either glutamate (e) . whole cell lysates (wcl) were prepared and analyzed for dpp4 expression by sds-page under non-reducing conditions and wb using a primary antibody targeting the c-terminal cmyc epitope and a peroxidase-conjugated secondary antibody. further, expression of beta-actin (actb) was analyzed as a loading control. shown are the expression data from a representative experiment. numbers at the left indicate the molecular weight in kilodalton (kda). (b) quantification of total dpp4 expression in wcl. after normalization of dpp4 band intensities with that of the corresponding actb bands. dpp4 wt expression was set as 100% and the relative expression of mutant dpp4 was calculated accordingly. presented are the combined data of three independent experiments with error bars indicating the sem. no statistical significance for differences in total expression between wt and mutant dpp4 was observed by one-way analysis of variance with dunnett's posttest (p > 0.05, not significant [ns]). . surface expressed dpp4 was stained by subsequent incubation of the non-permeabilized cells with a primary antibody that targets the dpp4 ectodomain and an alexafluor488-conjugated secondary antibody. fluorescent signals representing surface-expressed dpp4 were analyzed by flow cytometry and the mean fluorescence intensity (mfi) values for each sample were calculated. for normalization, the mfi value of the negative control was subtracted from all samples. further, surface expression of dpp4 wt was set as 100% and the relative surface expression of the dpp4 mutants was calculated accordingly. shown are the combined data of three experiments with error bars indicating the sem. statistical significance for differences in surface expression between wt and mutant dpp4 was tested by one-way analysis of variance with dunnett's posttest (p > 0.05, not significant; p ≤ 0.05, *). (b) dpp4 surface expression was further analyzed by immunofluorescence analysis. for this, dpp4 wt or dpp4 mutants were expressed in bhk-21 cells grown on coverslips (cells transfected with empty expression vector served as negative control). after fixation of the cells, surface expressed dpp4 was stained by subsequent incubation of non-permeabilized cells with a primary antibody that targets the dpp4 ectodomain and an alexafluor568-conjugated secondary antibody. in addition, cellular nuclei were stained with dapi. finally, images were taken using a confocal laser scanning microscope at a magnification of 80x. or asparagine (n) likely abolishes/decreases the interaction with mers-s due to the different biochemical properties of k267 (positively charged, basic) versus e267 (negatively charged, acidic) and n267 (not charged, acidic) (supplementary figure 1) . for dpp4 residue a291, which has been reported to contact the mers-cov s residue e513, no information on the type of interaction is available [16] . here, we speculate that the bulky and distorted side chain of proline (in comparison to the small side chain of alanine) abolishes/decreases interaction with mers-cov s residue e513 (supplementary figure 1) . in contrast to that, valine contains a small side chain and also has identical biochemical properties as alanine and thus might be efficiently contacted by e513 of mers-cov s, which is why we did not observe any impact of polymorphisms a291v on mers-cov s-driven entry and mers-cov s mers-cov s binding/interaction (supplementary figure 1 ). the observation that certain polymorphisms in dpp4 reduced mers-cov s binding and viral entry triggered the question whether residues in mers-cov s that are in direct contact with the respective dpp4 residues are also polymorphic. indeed, we obtained initial evidence to support such a concept. thus, we found that residue 539 in mers-cov s which contacts dpp4 residue 267 is polymorphic, with certain mers-cov variants harboring an asparagine instead of an . reduced mers-cov s-driven host cell entry is caused by inefficient s protein binding to dpp4 harboring polymorphic amino acid residues. in order to investigate whether reduced mers-cov s-driven host cell entry and mers-cov replication is due to inefficient mers-cov s binding to dpp4 harboring amino acid polymorphisms at the binding interface, we performed co-immunoprecipitation (co-ip) as well as binding experiments with a soluble s protein comprising the s1 subunit of mers-cov s fused to the fc region of human igg. (a) 293t cells were cotransfected with expression plasmids coding for soluble, fc-tagged mers-cov s1 (solmers-s1-fc) and the indicated dpp4 variant containing a c-terminal cmyc-tag. cells that were transfected only with empty expression vector alone, or empty expression vector instead of either solmers-s1-fc or dpp4 served as controls. at 48 h posttransduction, cells were lysed and incubated with protein a sepharose. next, samples were subjected to sds-page and western blot analysis. dpp4 levels were detected via antibodies specific for the cmyc-tag, whereas solmers-s1-fc was detected using a peroxidase-coupled anti-human antibody. similar results were obtained in three individual experiments. analysis of whole cell lysates (wcl) for expression of solmers-s1-fc, dpp4 and ß-actin confirmed comparable ß-actin levels in each sample and comparable expression levels for solmers-s1-fc and dpp4. (b) for quantification of mers-cov s/dpp4 interaction we first normalized the dpp4 signals against the respective solmers-s1-fc signals. then, mers-cov s/dpp4 interaction was set as 100% for wildtype (wt) dpp4 and the relative interaction efficiency for each dpp4 mutant was calculated accordingly. presented are the mean data from three independent experiments. error bars indicate the sem. statistical significance of differences in mers-cov s/dpp4 interaction between wt and mutant dpp4 was analyzed by one-way analysis of variance with dunnett's posttest (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.001, ***). (c) soluble mers-cov s1-fc was incubated with bhk-21 cells expressing wildtype (wt) or mutant dpp4, or cells transfected with empty expression vector or an ace2-expression plasmid (controls). to detect bound s protein, the cells were subsequently incubated with an alexafluor488-conjugated anti-human antibody directed against the fc-tag. fluorescent signals representing bound solmers-s1-fc were analyzed by flow cytometry and mfi values for each sample were calculated. for normalization, the mfi value of the negative control (empty expression vector) was subtracted from all samples. further, binding of sol-mers-s1-fc to cells expressing dpp4 wt was set as 100% and the relative binding to cells expressing the dpp4 mutants was calculated accordingly. shown are the combined data of five independent experiments with error bars indicating the sem. statistical significance of differences in solmers-s1-fc binding to cells expressing wt or mutant dpp4 was analyzed by one-way analysis of variance with dunnett's posttest (p > 0.05, ns; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). aspartate residue at this position. d539n reduced entry into cells expressing relatively low amounts of dpp4 but had no effect on entry into cells expressing high amounts of dpp4 (supplementary figure 2) . moreover, and more interestingly, d539n slightly rescued mers-cov s-driven entry from the negative effect exerted by dpp4 polymorphism k267n (supplementary figure 2) . similarly, residue 510 in mers-cov s, which is known to interact with dpp4 residues 317 and 322, was found to be polymorphic, and previous studies demonstrated that polymorphism d510g reduced dpp4 binding but also increased resistance to neutralizing antibodies [37] . notably, d510g slightly increased entry via dpp4 harboring polymorphism y322h and allowed mers-cov s to use dpp4 with polymorphism r317k with the same efficiency as wt dpp4. it should be stated that none of these effects was statistically significant and that dpp4 and mers-cov s polymorphisms occur with low frequency. although it is unlikely that the dpp4 polymorphisms have emerged as a result of evolutionary pressure from mers-cov infections, our results suggest that certain existing dpp4 polymorphism(s) might foster the emergence of mers-cov variants with altered biological properties. the polymorphisms studied here occur with relatively low frequencies of one per ∼19,000 (a289v) to ∼245,000 (t288i) individuals. however, detailed information on the geographic distribution or incidence in certain ethnical groups is largely missing. thus, dpp4 polymorphisms could contribute to the perplexing absence of mers cases in africa, where the virus circulates in camels [52] [53] [54] [55] [56] [57] . however, recent evidence suggests that sequence variations between african and arabian mers-cov might be a factor [53, 57] . more importantly, it remains to be analyzed how frequent dpp4 polymorphisms that affect s protein binding occur in the middle east and whether they are associated with the clinical course of mers. isolation of a novel coronavirus from a man with pneumonia in saudi arabia replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg infect dis evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation isolation of mers coronavirus from a dromedary camel hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus molecular epidemiology of hospital outbreak of middle east respiratory syndrome middle east respiratory syndrome coronavirus outbreak in the republic of korea mers-cov outbreak in jeddah-a link to health care facilities epidemiological investigation of mers-cov spread in a single hospital in south korea transmission of mers-coronavirus in household contacts differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 physiology and pharmacology of dpp-4 in glucose homeostasis and the treatment of type 2 diabetes revisiting an old acquaintance: cd26 and its molecular mechanisms in t cell function dipeptidyl-peptidase iv (cd26)-role in the inactivation of regulatory peptides cd26/ dipeptidylpeptidase iv-chemokine interactions: double-edged regulation of inflammation and tumor biology inhibitors of dipeptidyl peptidase iv (dp iv, cd26) induces secretion of transforming growth factor-beta 1 (tgf-beta 1) in stimulated mouse splenocytes and thymocytes dpp-4 inhibitors and their potential role in the management of type 2 diabetes cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system association of dpp4 gene polymorphisms with type 2 diabetes mellitus in malaysian subjects association between dpp4 gene polymorphism and serum lipid levels in chinese type 2 diabetes individuals polymorphisms in dipeptidyl peptidase iv gene are associated with the risk of myocardial infarction in patients with atherosclerosis fiji: an open-source platform for biological-image analysis ensembl variation resources. database (oxford) dbsnp: the ncbi database of genetic variation analysis of protein-coding genetic variation in 60,706 humans a global reference for human genetic variation the glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization differential sensitivity of bat cells to infection by enveloped rna viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon the hemagglutinin of bat-associated influenza viruses is activated by tmprss2 for ph-dependent entry into bat but not human cells detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction discovery of dipeptidyl peptidase iv (dpp4) inhibitors based on a novel indole scaffold yasara: a tool to obtain structural guidance in biocatalytic investigations ucsf chimera-a visualization system for exploratory research and analysis ldl receptor and its family members serve as the cellular receptors for vesicular stomatitis virus host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 high circulating plasma dipeptidyl peptidase-4 levels in non-obese asian indians with type 2 diabetes correlate with fasting insulin and ldl-c levels, triceps skinfolds, total intra-abdominal adipose tissue volume and presence of diabetes: a case-control study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study identification of residues on human receptor dpp4 critical for mers-cov binding and entry dipeptidyl peptidase-4 levels are increased and partially related to body fat distribution in patients with familial partial lipodystrophy type 2 middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses from camels in africa exhibit region-dependent genetic diversity antibodies against mers coronavirus in dromedary camels mers coronavirus neutralizing antibodies in camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate emc we are grateful to thank g. herrler, a. maisner and g. zimmer for providing plasmids and reagents. we further thank a.-s. moldenhauer for excellent technical support. no potential conflict of interest was reported by the authors. this work was supported, including the efforts of stefan pöhlmann and christian drosten, by the bundesministerium für bildung und forschung [grant numbers 01ki1723d and 01ki1723a], network project rapid (risikobewertung bei präpandemischen respiratorischen infektionserkrankungen). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-270534-ebkwv4zo authors: bodmer, bianca s.; fiedler, anna h.; hanauer, jan r.h.; prüfer, steffen; mühlebach, michael d. title: live-attenuated bivalent measles virus-derived vaccines targeting middle east respiratory syndrome coronavirus induce robust and multifunctional t cell responses against both viruses in an appropriate mouse model date: 2018-06-11 journal: virology doi: 10.1016/j.virol.2018.05.028 sha: doc_id: 270534 cord_uid: ebkwv4zo cases of middle east respiratory syndrome coronavirus (mers-cov) continue to occur, making it one of the who´s targets for accelerated vaccine development. one vaccine candidate is based on live-attenuated measles virus (mv) vaccine encoding the mers-cov spike glycoprotein (mers-s). mv(vac2)-mers-s(h) induces robust humoral and cellular immunity against mers-s mediating protection. here, the induction and nature of immunity after vaccination with mv(vac2)-mers-s(h) or novel mv(vac2)-mers-n were further characterized. we focused on the necessity for vector replication and the nature of induced t cells, since functional cd8(+) t cells contribute importantly to clearance of mers-cov. while no immunity against mers-cov or mv was detected in mv-susceptible mice after immunization with uv-inactivated virus, replication-competent mv(vac2)-mers-s(h) triggered robust neutralizing antibody titers also in adult mice. furthermore, a significant fraction of mers cov-specific cd8(+) t cells and mv-specific cd4(+) t cells simultaneously expressing ifn-γ and tnf-α were induced, revealing that mv(vac2)-mers-s(h) induces multifunctional cellular immunity. the middle east respiratory syndrome coronavirus (mers-cov) is a member of the coronaviridae family and emerged in 2012 in the kingdom of saudi arabia (zaki et al., 2012) . coronaviruses typically cause mild infections of the upper respiratory tract, but already in 2002, the severe acute respiratory syndrome cov (sars-cov) with a mortality rate of about 10% among infected patients was introduced in the human population. sars-cov spread world-wide and caused more than 8000 diagnosed infections, but was contained within a year after its emergence (http://www.who.int/csr/sars/country/table2004_04_21/ en/). in contrast, infections with mers-cov are ongoing for more than 5 years, with 2103 laboratory-confirmed cases distributed over 27 countries with at least 733 deaths that were reported to the who by november 2017 (http://www.who.int/emergencies/mers-cov/en/). this apparent case-fatality rate of 35% is of grave concern, because epidemic spread as has been observed for sars-cov could result in a disastrous death toll. mers-cov has been introduced zoonotically by transmission from dromedary camels to human patients (alagaili et al., 2014; haagmans et al., 2014; reusken et al., 2013a) and serological studies indicate wide-spread and early distribution among this animal host (alagaili et al., 2014; reusken et al., 2013b) . therefore, a continuous risk of transmission especially to persons in close contact to camels is evident. fortunately, the human to human transmission rate has remained low. aside of individuals with regular contact to camels, only health care workers or relatives of mers-cov patients have a considerable risk of infection (alraddadi et al., 2016; drosten et al., 2014) , but still at a modest level. nonetheless, the high case fatality rate, the recurrent outbreaks of mers-cov infections, and especially the risk of virus adaptation potentially resulting in epidemic or even pandemic spread make the development of an effective vaccine against mers-cov an international priority. the efficacy of several vaccine candidates has been demonstrated in different animal models up to even dromedary camels (reviewed in (okba et al., 2017) ). one of these candidates, mv vac2 -mers-s(h) (malczyk et al., 2015) , is based on the measles virus (mv) vaccine platform technology (mühlebach, 2017) , and encodes the mers-cov spike protein (s) as an additional antigen in the backbone of recombinant mv vac2 (del valle et al., 2007) resembling vaccine strain moraten that is authorized and in use in the us since 1968. this candidate induces both robust humoral and functional cellular immuneresponses against mers-cov. moreover, mers-cov viral load and inflammation of the lung were significantly reduced in challenged mice that had been vaccinated with mv vac2 -mers-s(h), before (malczyk et al., 2015) . while these experiments provided proof of concept for efficacy of this vaccine candidate, further mechanistic insights into the nature of the induced t cell responses remain to be elucidated. these are of special interest, since it has been shown that t cells are essential for clearance of the infection (coleman et al., 2017; zhao et al., 2014) : depletion of cd8 + t cells increased overall inflammation, bronchiolar inflammation, lymphocyte infiltration, and pleuritis at day 7 post-infection in mice (coleman et al., 2017) , while mers cov-susceptible mice depleted of all t cells were unable to clear the virus . as an alternative to the spike glycoprotein, conserved (internal) structural proteins such as the nucleocapsid protein n are of special interest as putative target of anti-viral t cell responses to be triggered by future mers vaccines . therefore, we have also generated and characterized mers-cov n protein-encoding vaccine candidates based on the mv vac2 vaccine platform, in this study. to further characterize the induction of mers cov-specific immune responses, we first analyzed the necessity for viral replication for the induction of mers cov-and mv-specific immune responses using the highly immunogenic mv vac2 -mers-s(h) vaccine candidate. in addition, we characterized the functionality of cd8 + and cd4 + t cell responses in juvenile (6-12 week old) and adult (7 months of age) mice using flow cytometry and functional assays. vero (african green monkey kidney) (atcc# ccl-81) and 293 t (atcc crl-3216) cell lines were purchased from atcc (manassas, va, usa) and cultured in dulbecco's modified eagle's medium (dmem, biowest, nuaillé, france) supplemented with 10% fetal bovine serum (fbs; biochrom, berlin, germany) and 2 mm l-glutamine (l-gln; biochrom). jawsii dendritic cells (atcc crl-11904) were purchased from atcc and cultured in mem-α with ribonucleosides and deoxyribonucleosides (gibco brl, eggenstein, germany) supplemented with 20% fbs, 2 mm l-gln, 1 mm sodium pyruvate (biochrom), and 5 ng/ml murine gm-csf (peprotech, hamburg, germany). dc2.4 murine dendritic cells (shen et al., 1997) were cultured in rpmi containing 10% fbs, 2 mm l-gln, 1% non-essential aminoacids (biochrom), 10 mm hepes (ph 7,4), and 50 μm 2-mercaptoethanol (sigma-aldrich, steinheim, germany). cells were cultured at 37°c in a humidified atmosphere containing 6% co 2 for a maximum of 6 months of culture after thawing of the original stock. the codon-optimized gene encoding mers-cov-n (genebank accession no. jx869059) flanked with aatii/mlui binding sites in plasmid pma-rq-mers-n was obtained by gene synthesis (invitrogen life technology, regensburg, germany). the antigen and the immediate early cytomegalovirus (cmv) promoter (martin et al., 2006) were inserted into plasmids p(+)br-mv vac2 -atu(p) (del valle et al., 2007) or p(+)mv vac2 -gfp(h) via mlui/aatii and sfii/sacii, respectively, to generate p(+)polii-mv vac2 -mers-n(p) or p(+)polii-mv vac2 -mers-n (h). for construction of lentiviral transfervectors encoding mers-cov-n, the orf of mers-n was amplified by pcr with primers encompassing flanking restriction sites nhei/xhoi and template pma-rq-mers-n. details on primers and pcr are available upon request. pcr products were cloned into pcr2.1-topo (invitrogen life technology) and fully sequenced. intact antigen orf was cloned into pcscw2gluc-ires-gfp (hewett et al., 2007) using nhei/xhoi restriction sites to yield pcscw2-mers-n-ires-gfp. lentiviral vectors were produced and used for the generation of antigen-expressing dendritic cell lines as described, before (malczyk et al., 2015) . in short, hiv-1-derived vectors were generated using a standard 3 plasmid system and the transfer vector plasmid pcscw2-mers-n-ires-gfp by pei transfection. subsequent purification after harvest of transfected 293 t cells yielded virus stocks used to transduce dc cell lines, which were single cell-sorted by facs and selected for antigen expression. mers-n encoding vaccine candidates mv vac2 -mers-n(p) and mv vac2 -mers-n(h) were rescued as described (malczyk et al., 2015; martin et al., 2006) . single syncytia were picked and overlaid onto 50% confluent vero cells cultured in 6-well plates and harvested as "passage 0" (p0) by scraping and freeze-thaw cycle of cells at the time of maximal infection. subsequent passages were generated as described for the following viruses. mers-n encoding vaccine viruses in p3 were used for characterization, viruses in p4 for vaccination. mers-s encoding vaccine virus mv vac2 -mers-s(h), and control virus mv vac2 -atu(p) (malczyk et al., 2015) were also used in p4 for vaccination. both as well as mv vac2 -gfp(p) and mers-cov (isolate emc/2012) (zaki et al., 2012) used for neutralization assays were propagated and titrated on vero cells by the method of spearman and kaerber (hubert, 1984; kärber, 1931) . mv vac2 -mers-s(h) was inactivated by uv-irradiation using a cl-1000 uv crosslinker (uvp, cambridge, uk). 100 μl of virus suspension in 48-well-plates on ice were exposed to uv light of 254 nm at 3 cm distance from the uv source of 1,85 × 10 5 μj/cm 2 for 30 min. inactivation of virus was controlled by incubation of vero cells with a control aliquot inactivated, in parallel. all virus stocks were stored in aliquots at −80°c. cells were lysed and immunoblotted as previously described (funke et al., 2008) . a rabbit anti-mers-cov serum (1:1000) was used as primary antibody for mers-cov-n and a rabbit anti-mv-n polyclonal antibody (1:25,000) (abcam) for mv-n detection. a donkey hrp-coupled anti-rabbit igg (h&l) polyclonal antibody (1:10,000) (rockland, gilbertsville, pa) served as secondary antibody for both. peroxidase activity was visualized with an enhanced chemiluminescence detection kit (thermo scientific, bremen, germany) on amersham hyperfilm ecl (ge healthcare, freiburg, germany). all animal experiments were carried out in compliance with the regulations of german animal protection laws and as authorized by the rp darmstadt. six-to 12-week-old or 7 months old ifnar -/--cd46ge mice (mrkic et al., 1998) deficient for type i ifn receptor and transgenically expressing human cd46 were inoculated intraperitoneally (i.p.) with 1 × 10 5 tcid 50 of recombinant viruses or uv-inactivated vaccine preparations on days 0 and either on day 21 or 28. mice were bled on days 0, 28, and 49 post initial infection (p.i.). serum samples were stored at − 20°c. mice were euthanized on days 32, 42, or 49 p.i., and splenocytes were harvested for assessment of cellular immune responses. quantification of vnts was done as described, before (malczyk et al., 2015) . in brief, mouse sera were serially diluted in 2-fold dilution steps in dmem in duplicates. a total of 50 pfu of mv vac2 -gfp(p) or 200 tcid 50 of mers-cov (strain emc/2012) were mixed with diluted sera and incubated at 37°c for 1 h. virus suspensions were added to 1 × 10 4 vero cells seeded 4 h prior to assay in 96-well plates and incubated for 4 days at 37°c. vnts were calculated as the reciprocal of the highest mean dilution that abolished infection. murine gamma interferon (ifn-γ) enzyme-linked immunosorbent spot (elispot) assays (ebioscience, frankfurt, germany) were performed according to the manufacturer's instructions using multiscreen immunoprecipitation (ip) elispot polyvinylidene difluoride (pvdf) 96well plates (merck millipore, darmstadt, germany). 5 × 10 5 isolated medium inoculated mice served as mock control. vnts were calculated as the highest dilution abolishing infectivity. dots represent single animals (n = 6); horizontal lines represent mean per group. the y-axis starts at the detection limit; all mice at the detection limit had no detectable vnt. (g) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes harvested 32 days post prime immunization and after co-culture with jawsii (left) or dc2.4 (middle) dendritic cells transgenic for mers-n (black) or untransduced controls (nc, white). (right) to analyze cellular responses directed against mv, splenocytes were stimulated with 10 μg/ml mv bulk antigen (mv bulk) or left unstimulated (sham). the reactivity of splenocytes was confirmed by cona treatment (10 μg/ml). tcid 50 , tissue culture infectious dose 50; one-way-anova with tukey multiple comparison. *: p < 0,05; **: p < 0,01; ***: p < 0001; ****: p < 0,0001. splenocytes were co-cultured with different stimuli in 200 μl rpmi + 10% fbs, 2 mm l-gln, and 1% penicillin-streptomycin for 36 h. for re-stimulation of mers n-specific t cells, splenocytes were co-cultivated with 5 × 10 4 jawsii, dc2.4 dendritic cells, or clones of either cell line encoding mers-n. on the other hand, splenocytes were stimulated with 10 μg/ml mv bulk antigen (serion immunologics, würzburg, germany), 10 μg/ml mers s-derived peptide s1165 (biosynthesis inc., lewisville, tx, usa, (channappanavar et al., 2014) ), or 10 μg/ml siinfekl control peptide (sin) of ovalbumin (aa 257-264) (invivogen, san diego, ca, usa), as appropriate. for general t cell stimulation, 10 μg/ml concanavalin a (cona, sigma-aldrich, st. louis, mo, usa) was used. as negative control, splenocytes were left untreated. after 36 h, cells were removed from the plates, and plates were incubated with biotin-conjugated anti-ifn-γ antibodies and avidin-hrp according to the manufacturer's instructions. 3-amino-9ethyl-carbazole (aec; sigma-aldrich) substrate solution for development of spots was prepared according to the manufacturer's instructions using aec dissolved in n,n-dimethylformamide (merck millipore) and used for peroxidase-dependent staining, afterwards. spots were counted using an eli.scan elispot scanner (ae.l.vis, hamburg, germany) and elispot analysis software eli.analyse v5.0 (ae.l.vis). for flow cytometry-based determination of cytokine expression by intracellular cytokine staining (ics), splenocytes of vaccinated mice were isolated, and 2 × 10 6 splenocytes per mouse were cultivated in 200 μl rpmi1640 + 10% fbs, 2 mm l-gln, 1 × non-essential amino acids (biochrom), 10 mm hepes, 1% penicillin-streptomycin, 50 μm βmercaptoethanol, 10 μg/ml brefeldin a (sigma-aldrich), and one of the stimuli also used for elispot analysis. for general t cell stimulation, 0.25 μg/ml tetradecanoylphorbol acetate (tpa, sigma aldrich) and 0.5 μg/ml ionomycin (iono, sigma-aldrich) were used as positive control, and only medium was used as negative control. splenocytes were stimulated for 5 h at 37°c. subsequently, cells were stained with fixable viability dye efluor450 (ebioscience), cd4-pe (1:2000) (bd, franklin lakes, nj, usa), cd8-fitc (1:500) (bd), and, after permeabilization with fixation/permeabilization solution (bd) and perm/ wash buffer (bd), stained with ifn-γ-apc (1:500) (bd) and tnf-α-pe-cy7 (1:500) (bd). cells were fixed with ice-cold 1% paraformaldehyde (pfa) in pbs and analyzed via flow cytometry using an lsrii sorp flow cytometer (bd) and fcs express software (de novo software, glendale, ca, usa). since the nucleocapsid protein (n) of cov is quite conserved, it is regarded as an appropriate target to induce anti-viral t cells. therefore, mers-cov n was chosen as an alternative antigen to be expressed by the recombinant mv vaccine platform. full-length mers-n was cloned into two different additional transcription units (atus) either behind p (post p) or h (post h) cassettes of measles vaccine strain mv vac2 genome, and virus clones were successfully rescued and amplified in vero cells with titers of up to 2 × 10 8 tcid 50 /ml. the essential verification of antigen expression by western blot analysis of vero cells infected with the mv vac2 -mers-n vaccines revealed expression of the n antigen with only little impact of the genomic position of the transgene cassette (fig. 1a) , while growth kinetics showed no impairment of virus replication compared to the respective mv vac2 -gfp(p) control virus ( fig. 1 b, c) . to test the efficacy of the mv vac2 -mers-n candidate in vivo, genetically modified ifnar -/--cd46ge mice were chosen, since they are the prime small animal model for analysis of mv-derived vaccines (mrkic et al., 1998) . thus, 6 mice per group were inoculated via the intraperitoneal (i.p.) route on days 0 and 28 with each time 1 × 10 5 tcid 50 of mv vac2 -mers-n(p), mv vac2 -mers-n(h), or empty control virus mv vac2 -atu(p). medium-inoculated mice served as negative controls. 21 days or four days after boost immunization, sera or splenocytes of immunized mice were sampled, respectively (fig. 1d) . as expected, all mice immunized with recombinant mv (including the control virus) developed high mv virus neutralizing titers (vnt) (512-2048 vnt, fig. 1f ). little evidence for induction of neutralizing antibodies against mers-cov was found in all mice, as expected for the intra-particular antigen (fig. 1e) . no vnts against mv or mers-cov were detected in control mice inoculated with medium alone. to analyze splenocytes of animals vaccinated with mv vac2 -mers-n (h) or control animals inoculated with medium or mv vac2 -atu(p) by elispot assay for antigen-specific ifn-γ secretion, the antigen-specific t cells were re-stimulated in vitro by syngeneic murine dc cell lines (jawsii and dc2.4), which had been genetically modified by lentiviral vector transduction to stably express mers-n protein and thereby to present the respective t cell epitopes on mhc. single cell clones were derived by flow cytometric sorting of single gfp-positive cells. antigen expression by transduced dcs was verified by western blot analysis (data not shown). elispot assays using splenocytes of vaccinated animals in co-culture with jawsii-mers-n or dc2.4-mers-n revealed about 200 ifn-γ secreting cells per 1 × 10 6 splenocytes after immunization with mv vac2 -mers-n(h) (fig. 1g) , which was significant over controls. additionally, cellular immune responses targeting mv antigens were detected upon stimulation with mv bulk antigens in vaccinated mice that had received any recombinant virus, as expected. however, mv bulk antigens stimulated about 930-1500 ifn-γ secreting cells per 1 × 10 6 splenocytes of mv vaccinated animals, as described, before (malczyk et al., 2015) . finally, splenocytes of all mice revealed a similar basic reactivity to unspecific t cell stimulation, as confirmed by similar numbers of ifn-γ secreting cells upon cona treatment (fig. 1g) . thus, the generated mv-based vaccine platform expressing mers-n induces significant mers n-specific cellular immune responses, as desired. in any case, humoral and cellular responses induced by vaccine candidate mv vac2 -mers-s had been considerably higher in previous analyses under similar conditions (malczyk et al., 2015) . therefore, further characterization of anti-mers-cov immunity induced by mv vac2 -derived vaccines proceeded with this mers-s encoding vaccine candidate, which yielded approximately 5-fold higher numbers of reactive t cells after vaccination. since the mers vaccine candidate mv vac2 -mers-s(h) induced robust protective humoral and cellular immune responses in ifnar -/--cd46ge mice (malczyk et al., 2015) , we were interested in the necessity of viral replication of this life-attenuated vaccine for the induction of mers cov-specific immunity. for these analyses ifnar -/--cd46ge mice were chosen as the animal model, again, since these mice are the standard animal model for analysis of mv-derived vaccines (mühlebach, 2017) , their genetic composition is compatible with an established mers-cov challenge model , as shown, before (malczyk et al., 2015) , and their size allows housing under regularly available conditions opposed to dromedary camels, the only know natural host of mers-cov, to date. as all morbilliviruses, the mv-based vaccine virions are highly cellassociated, and transfer of antigenic protein within the vaccine preparation cannot be excluded. therefore, we vaccinated these mv-susceptible mice with either 1 × 10 5 tcid 50 of live or of the same formulation and quantity uv-inactivated mv vac2 -mers-s(h) in a primeboost regimen ( fig. 2a) . mv vac2 -atu(p), which does not encode any additional antigen, was included as vector control. blood was drawn from naïve mice on day 0 before vaccination, and on days 28 and 49 post-immunization. serum samples were tested for their ability to neutralize mv vac2 -gfp(p) (fig. 2b , d, f) or mers-cov (fig. 2c, e, g) . sera of naïve mice had no neutralizing antibodies against either virus (fig. 2b, c) . after the first immunization, both live virus preparations induced neutralizing antibodies against mv, with mv vac2 -atu(p) triggering significantly higher titers (1280-1920 vnt) than mv vac2 -mers-s(h) (480-640 vnt). after the second immunization, anti-mv vnts increased to titers of 640-2560 in both cohorts. in contrast, only one out of four animals in the uv-inactivated vaccine group had a borderline neutralizing antibody titer of 20 after the first immunization, and another animal had a titer of 30 after the boost. while mv vac2 -atu(p) and the uv-inactivated mv vac2 -mers-s(h) vaccine did not induce neutralizing antibodies against mers-cov above background levels over the course of the experiment, the group vaccinated with live mv vac2 -mers-s(h) developed titers around 50 after the first immunization and 40-320 (mean of 300) after the boost. taken together, these data reveal that replication of the vaccine is necessary to induce functional antibody responses against mv and the additional antigen mers-s. to assess the capacity of the different mv vac2 -mers-s(h) vaccine preparations to induce mers-cov s-specific cellular immune responses, splenocytes of mice, which had already been tested for humoral responses ( fig. 2a) , were isolated and analyzed 49 days after immunization for antigen(ag)-dependent ifn-γ secretion using elispot assay. the isolated splenocytes were re-stimulated with mers-s immunodominant peptide s1165 (channappanavar et al., 2014) or mv bulk antigen (mv bulk) to analyze mv-specific cellular immune responses. ovalbumin-derived siinfekl-peptide (sin) served as peptide negative control, or cells were left untreated (mock). stimulation with concanavalin a (cona) was used to confirm general t cell reactivity in splenocyte preparations (fig. 2h) . while splenocytes of all mice responded to cona with 1000 to 1500 spots per 1 × 10 6 splenocytes, only those from animals vaccinated with live mv vac2 -mers-s(h) could be stimulated with mers s-specific peptide s1165 reaching mean values of 1040 spots per 1 × 10 6 splenocytes. in contrast, splenocytes of the uv-inactivated group or control virus mv vac2 -atu(p) could not be restimulated to secrete ifn-γ. furthermore, only replication-competent vaccine viruses induced mv-specific cellular immune responses in vaccinated mice. re-stimulation with mv bulk ag induced a mean of 800 and 970 spots per 1 × 10 6 splenocytes for mv vac2 -mers-s(h) or mv vac2 -atu(p) vaccinated mice, respectively. consequently, replication of the vaccine candidate is essential to induce both arms of the immune system with responses against mv as well as the additional mers-s antigen. usually, 6-12 weeks old juvenile mice are used for our mers-cov neutralizing antibodies in sera of (b, c) naїve mice, or in sera of mice after (d, e) prime-or (f, g) boost-immunization. one-way anova with tukey multiple comparison. * : p < 0,05; * *: p < 0,01; ***: p < 0001; ****: p < 0,0001. (h) secretion of ifn-γ after antigen-specific re-stimulation of splenocytes. ifn-γ elispot analysis using splenocytes of mice vaccinated on days 0 and 28 with indicated vaccines isolated 21 days after boost immunization and after incubation with indicated stimuli (mers-s peptide s1165, mv bulk antigen (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control) or untreated (mock). the reactivity of splenocytes was confirmed by concanavalin a (cona) treatment (10 μg/ml). the number of cells per 1 × 10 6 splenocytes represent the amount of cells expressing ifn-γ upon re-stimulation. dots represent individual animals, horizontal bars mean. one-way anova with tukey multiple comparison. ****: p < 0,0001. b.s. bodmer et al. virology 521 (2018) 99-107 immunization studies. to evaluate if there is an age-dependent change in vaccination efficacy, approximately 7 months-old mice were vaccinated with mv vac2 -mers-s(h) in a prime-boost vaccination scheme with 3 weeks between prime and boost vaccination (fig. 3a ). mice were sacrificed at day 42 post-immunization, and splenocytes were re-stimulated with mv-antigens or mers-s peptide s1165. we found that reactive ifn-γ-secreting t cells were also specifically induced in mice of this age (fig. 3b) . a mean of 520 spots per 1 × 10 6 splenocytes was detected upon re-stimulation with mv bulk antigen, whereas 900 spots per 1 × 10 6 splenocytes were induced by re-stimulation with the mers s-derived peptide s1165, illustrating that mv-and mers-cov-specific cellular immune responses are effectively induced in adult mice. to gain more detailed insights in the quality of the observed t cell responses, we further characterized the responsive t cell populations by flow cytometry, determining the expression of cd8 + and cd4 + surface markers as well as ifn-γ and tnf-α upon re-stimulation with s1165 or mv bulk antigen. as a positive stimulus for t cell activation tetradecanoylphorbol-acetate and ionomycin (tpa/iono) were used. exocytosis of cytokines was blocked by addition of brefeldin a (10 μg/ ml) during stimulation. cells were permeabilized, labelled, and fixed for flow cytometry. the gating strategy excluded duplicates (not shown), selected for living cells (fig. 4a, upper panel) , and separated cd8 + and cd4 + t cells (fig. 4a, lower panel) . selected cd8 + t cells were then analyzed for their expression of ifn-γ (fig. 4b left panel) , tnf-α (fig. 4b middle panel) , or both ( fig. 4b right panel) as exemplarily shown for splenocytes re-stimulated with mers-s peptide s1165. likewise, cd4 + t cells expressing ifn-γ (fig. 4c, left panel) , tnf-α (fig. 4c, middle panel) , or both (fig. 4c , right panel) are depicted after re-stimulation with mv bulk antigen. vaccination with mv vac2 -mers-s(h) induced a significant amount of mers s-specific cd8 + t cells expressing either ifn-γ (fig. 4d, left panel) or tnf-α (fig. 4d, middle panel) , with means of 0.6% and 0.4% of total positive cells, respectively. among those, a significant fraction of cells revealed to be multifunctional, with a mean of 0.3% of all cd8 + cells or 75% of the tnf-α − responsive cells being positive for both cytokines (fig. 4d, right panel) . moreover, vaccination induced a significant fraction of vector-specific cd4 + t cells expressing ifn-γ (fig. 4e, left panel) , or tnf-α (fig. 4e , middle panel) upon re-stimulation with mv bulk antigen. among those, multifunctional cd4 + t cells expressing both cytokines were induced with a mean of about 0.1% (fig. 4e, right panel) . to conclude, vaccination with mv vac2 -mers-s(h) induces not only ifn-γ or tnf-α expressing t cells directed against mers-cov and mv, but also a significant fraction of multifunctional cytotoxic t cells specific for mers-s and cd4 + t cells specific for mv antigens, illustrating that a broad and robust mers-covspecific immune response is induced by vaccination with mv vac2 -mers-s(h). in this study, we aimed to understand the induction of immunity and the functionality of induced t cell responses after vaccination with mv vac2 -mers-s(h), a vaccine candidate that induces protective immunity against mers-cov in an appropriate animal model. in parallel, we generated and tested also alternative mv-based vaccine candidates expressing mers-cov n protein as conserved t cell antigen. mv vac2 -mers-n vaccine candidates indeed induced significant antigen-specific cellular immune responses in vaccinated transgenic mice, revealing that also mers n-expression by recombinant mv may have a useful role to combat mers-cov. since the immune responses induced by mers-s expressing candidates had been nevertheless considerably higher, we proceeded with s-expressing vaccine virus to characterize the induction of anti-mers-cov immunity by mv-based vectors. using mv vac2 -mers-s(h), robust anti-mers cov immune responses were induced also in older mice, while replication of the vaccine vector was necessary to induce either arm of adaptive immunity against vector or pathogen. furthermore, vaccination with mv vac2 -mers-s(h) triggered significant numbers of multifunctional mers s-specific cd8 + t cells and mv-specific cd4 + t cells, simultaneously producing ifn-γ and tnf-α upon stimulation with respective antigens. since not only numbers, but also the quality of the induced mers cov-specific t cell responses might be relevant for protection against mers-cov, it is quite encouraging to see that approx. 50% of ifn-γ reactive cd8 + t cells also expressed tnf-α, whereas in reverse 75% of tnf-α-reactive cd8 + t cells co-expressed ifn-γ upon stimulation with the immune-dominant mers-s peptide. this induction of multifunctional t cells is quite in accordance with previous studies, since the potential of mv during natural infection or the recombinant mv to induce multifunctional, antigen-specific t cells has already been demonstrated. infection of macaques with wild-type mv induces polyfunctional t cells specific for mv proteins with increasing numbers of cells secreting il-2, tnf-α, as well as ifn-γ over time (nelson et al., 2017) , and polyfunctional t cells directed against mv-h can be expanded from pbmc of human donors (ndhlovu et al., 2010) . likewise, hiv-vaccine candidates mv1-f4, which encode gag, rt, and nef of an hiv-1 clade b or a clade c strain as foreign antigen, induce antigenspecific multifunctional t cells simultaneously expressing ifn-γ, tnf-α, and il-2 in mice and also macaques (stebbings et al., 2013 (stebbings et al., , 2012 . while the combination of ifn-γ and tnf-α indicates functional t cells fig. 3 ) were re-stimulated and subjected to intracellular staining (ics) for ifn-γ and tnf-α and stained for extracellular t-cell markers cd4 and cd8 for flow cytometry analysis. (a -c) gating strategy for analysis of cd8 + or cd4 + t-cells expressing ifn-γ or tnf-α within splenocytes stimulated with (b) s1165 peptide or (c) mv-bulk ag. duplicates (not shown) and dead cells (a) were excluded from analysis. (b, c) cd8 + and cd4 + cells were separately subjected to analysis for ifn-γ-(left panels), tnf-α-(middle panels) or double-positive cells (right panels). quantification of flow cytometry data of (d) cd8 + -and (e) cd4 +positive cells after incubation with indicated stimuli (mers s-specific peptide s1165, mv bulk ag (mv bulk), immunodominant ovalbumin-derived siinf-ekl-peptide (sin) as a peptide negative control, or untreated cells (mock); reactivity of splenocytes was confirmed by tetradecanoylphorbol-acetate and ionomycin (tpa/iono) treatment (10 μg/ml). dots represent individual animals, horizontal bars mean. repeated-measures one-way anova with tukey multiple comparison. *: p < 0,05. with higher potency, in general, expression of il-2 is a sign of the induction of cd8 + memory t cells (williams et al., 2006) . in our study, the strong correlation of ifn-γ and tnf-α expression thus indicates a high functionality of induced t cell responses. extension of the antibody panel for il-2 detection could yield further insight into the durability of these t cell responses induced by the mv vaccine platform in future studies. such multifunctional cd8 + t cells specific for mers-cov may become especially important, since mouse studies have shown that cd8 + t cells are crucial for clearance of mers-cov infection (coleman et al., 2017; zhao et al., 2014) . noteworthy, for other viral infections such as human immunodeficiency virus (hiv), modified vaccinia virus ankara (mva), or cytomegalovirus (cmv) the amount of just ifn-γ producing t cells does not correlate with ctl killing effectivity, but the multifunctionality of antigen-specific t cells inversely correlated with viral load (betts et al., 2006; lichterfeld et al., 2004; precopio et al., 2007; sandberg et al., 2001) , further underlining the importance of multifunctionality. besides these cellular immune responses, also considerable humoral immunity was induced in vaccinated animals, here. the mean vnt was somewhat lower than expected (malczyk et al., 2015) , but still quite high. an alternative vaccine candidate derived from modified vaccinia virus ankara, mva-mers-s, revealed protection in dromedary camels, the natural host for mers-cov (haagmans et al., 2016) . passive immunotherapy with dromedary immune sera significantly reduced mers-cov titers in lung tissue of challenged mice, starting with a vnt of 160 (zhao et al., 2015) . neutralizing antibody titers in reconvalescent plasma of human patients diagnosed with mers were determined by microneutralization tests in two previous studies, and were on average at 175 (arabi et al., 2016) or 58.3 (zhao et al., 2017) . furthermore, a prnt 50 titer of at least just 50 was required to lower virus titers by more than 0.5 log in mice challenged after transfer of human reconvalescent plasma (zhao et al., 2017) . these titers were exceeded in this study. in addition, mv vac2 -mers-s(h) induced higher anti-mers-s titers in c57/bl6 mice than mva-mers-s in balb/c mice, when comparing studies that used similar virus titers for vaccination (malczyk et al., 2015; volz et al., 2015) , thus indicating an at least comparable efficacy. thus, also vnt determined here indicate efficacy and were anyway not statistically different from those published before for mv vac2 -mers-s(h) (malczyk et al., 2015) . nevertheless, the exact correlates of protection for mers-cov remain to be determined in future studies, since it will be essential to evaluate the efficacy of the different vaccine candidates against this most important benchmark. in contrast, uv-inactivated mv vac2 -mers-s(h) did not induce any antibodies able to neutralize mers-cov or mv. while neutralizing antibodies can in principle also be induced by inactivated vaccines or proteins, e.g. full-length or truncated mers-s protein in combination with adjuvant (wang et al., 2015) . obviously, the amount of mers-s antigen within the mv vac2 -mers-s(h) vaccine formulation or the adjuvant effect of the inactivate were not sufficient during application. therefore, replication of the mv-derived mers vaccine candidate is necessary for the induction of immune responses both against vector and antigen of interest in vaccinated animals. indeed, the induction of cellular immunity is usually more efficient by de novo expression of antigen after immunization. consequently, the application of a replication competent vaccine platform is justified here to robustly induce potent responses of both arms of the adaptive immune system. these powerful immune responses were not only induced in juvenile mice 6-12 weeks of age, but also in adult mice older than half a year of age. this is quite of interest, since adult vaccinees are also the target group for vaccination in response to the mers-cov outbreak, as defined in the target product profile by the who (http://www.who.int/ blueprint/what/research-development/mers_cov_tpp_ 15052017.pdf). remarkably, mv vaccine strain virus encoding chikungunya virus (chikv) antigens was already tested in a phase i clinical trial in adult human vaccinees (18-45 years old) (ramsauer et al., 2015) . these adult test subjects all developed significant humoral immunity against chikv, despite their adult age and most interestingly also independent from measles pre-immunity. taken together, these study shows that mv vac2 -mers-s(h) induces surprisingly high numbers of multifunctional t cells specific for mers-s also in adult test subjects, as a result from replication of the recombinant vector. therefore, high quality cellular immune responses are induced in addition to the robust antibody responses by this vaccine candidate, further qualifying mv vac2 -mers-s(h) for evaluation as vaccine candidate against mers-cov. in parallel, mers-n encoding mv can be a further option to generate protection against mers in future studies and constructs. evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia (e00884-14) risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia hiv nonprogressors preferentially maintain highly functional hiv-specific cd8+ t cells t cell-mediated immune response to respiratory coronaviruses cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a vectored measles virus induces hepatitis b surface antigen antibodies while protecting macaques against measles virus challenge transmission of mers-coronavirus in household contacts targeted cell entry of lentiviral vectors middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels mutant torsina interferes with protein processing through the secretory pathway in dyt1 dystonia cells bioassays: spearman-karber method beitrag zur kollektiven behandlung pharmakologischer reihenversuche hiv-1-specific cytotoxicity is preferentially mediated by a subset of cd8(+) t cells producing both interferon-gamma and tumor necrosis factor-alpha a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform rna polymerase ii-controlled expression of antigenomic rna enhances the rescue efficacies of two different members of the mononegavirales independently of the site of viral genome replication measles virus spread and pathogenesis in genetically modified mice vaccine platform recombinant measles virus dynamic regulation of functionally distinct virus-specific t cells evolution of t cell responses during measles virus infection and rna clearance middle east respiratory syndrome coronavirus vaccines: current status and novel approaches immunization with vaccinia virus induces polyfunctional and phenotypically distinctive cd8(+) t cell responses immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, doubleblind, placebo-controlled, active-comparator, first-in-man trial middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study functional heterogeneity of cytokines and cytolytic effector molecules in human cd8+ t lymphocytes cloned dendritic cells can present exogenous antigens on both mhc class i and class ii molecules immunogenicity of a recombinant measles-hiv-1 clade b candidate vaccine immunogenicity of a recombinant measles hiv-1 subtype c vaccine protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov interleukin-2 signals during priming are required for secondary expansion of cd8+ memory t cells isolation of a novel coronavirus from a man with pneumonia in saudi arabia recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses rapid generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection this work was supported by the german center for infection research (dzif; ttu 01.802). the authors would like to thank vivian scheuplein, jürgen schnotz, and daniela müller for excellent technical assistance. the authors are indebted to ron fouchier for providing mers-cov strain emc/2012, kenneth rock for dc2.4 cells, roberto cattaneo for providing the pb(+)mvvac2 construct, and urs schneider for providing the polii rescue system originally used to generate and to rescue recombinant mv vectors. the authors would further like to thank bakhos tannous for providing pcscw2gluc-ires-gfp. moreover, the authors would like to thank veronika von messling for critically reading the manuscript. key: cord-256537-axbyav1m authors: kimball, ann marie title: emergence of novel human infections: new insights and new challenges date: 2016-10-24 journal: international encyclopedia of public health doi: 10.1016/b978-0-12-803678-5.00153-3 sha: doc_id: 256537 cord_uid: axbyav1m novel human infections have continued to emerge over the past decade. their presentation, epidemiology, and microbiology have shifted the paradigms of traditional science. in particular insights into nongenetic or paragenetic mechanisms (plasmid mediated), modes of infection have challenged biology. in reviewing the new challenges posed by these emergent events, new technologies promise some answers; however, global health security against pandemic threats, particularly given the uneven distribution of global resources for prevention, detection, and response, remains a critical area of challenge. new human infections have continued to come forth over the last decade. this discussion will focus on the period from 2006 to 2016. given the importance of the severe acute respiratory syndrome (sars) as the harbinger of coronaviruses such as the middle east respiratory syndrome (mers), some discussion of sars is also included. the phenomenon of emerging infections has been constant with the majority of human infections reflecting the introduction into humans of zoonotic pathogens. over the last decade, great progress has been made in defining more fully how emergence occurs. in fact the emergence of new infections has expanded the paradigms of microbiology in a number of ways, which will be highlighted here. specifically: (1) it is now well appreciated that influenza can migrate directly from avian sources to humans, and the appreciation of the actual directness of 'species jumping' has moved forward; (2) new infections have also introduced uncertainty in transmission dynamics with emphasis on super-spreader events as well as nosocomial transmission; (3) infectious particles are not confined to those organisms which contain genetic material; (4) a new paradigm such as 'planetary health' may be necessary for defining these trends; and (5) global preparedness and response is not in place for the next pandemic. for further background information, the reader is referred to the original article on this phenomenon (kimball, 2008) . a general discussion of antimicrobial resistance and xenotransplantation is not included in this discussion as they were thoroughly covered in the original article. antimicrobial resistance will be covered in detail in another article of this volume. nonetheless they continue to be important considerations. while it has long been appreciated that the majority of human pathogens arise from zoonotic sources, the directness of the path from animal to human has become clear. ideally detecting pathogens in animals could be seen as a harbinger for human outbreaks. in fact this has been a working hypothesis of major us agency for international development (usaid) funding to their pandemic agents program. however, recent outbreaks of influenza, sars, and mers coronavirus (cov) demonstrate how emerging infections are shifting this longstanding paradigm. they lend further urgency to this area of research. in addition, these outbreaks have challenged our understanding of transmission dynamics. empirical observation has shown that there is not a single transmission force 'number' which completely characterized the spread of infectious diseases. this further complicates efforts to control infection within populations. in 2009, a cluster of high mortality influenza cases was detected in mexico. an observational study of 899 patients hospitalized in mexico between late march and 1 june 2009 showed that pandemic (h1n1) disproportionately affected young people. fifty-eight patients (6.5% of those hospitalized) became critically ill, with complications including severe acute respiratory distress syndrome and shock. among those who became critically ill, the mortality rate was 41% (domínguez-cherit et al., 2009) . the pandemic and its management have been the subject of an in-depth analysis commissioned by the world health organization (who), and that analysis proved prescient in more recent global outbreak events such as ebola (who, 2005) . this cluster heralded a global pandemic and the declaration by who of a public health emergency of international concern (phiec). it occurred in the context of a decade of planning for a potential h5n1 outbreak, which included who updating its pandemic guidance (pandemic influenza preparedness and response, 2009 ). in 1997 a small but lethal cluster of influenza occurred in children in a hong kong nursery. of the 18 cases, 6 died (mounts et al., 1999) . science shifted its understanding of influenza and recognized that flu could come directly from birds to humans. in fact h5n1, which emerged in 2003, acquired the popular moniker of 'bird flu.' the outbreak was curtailed through active surveillance, changes in practice, and poultry culling of live markets in hong kong. however, the outbreak proved a harbinger of more widespread recognition of 'bird flu.' h5n1 was identified through active surveillance of poultry and wild birds; waterfowl were particularly affected. human cases were sporadic but carried a very high mortality rate (estimated at 60%) (webster et al., 2006) . although the virus proved difficult to transmit to humans, case fatality was high. as of february 2011, about 500 laboratory-confirmed human cases had been reported to the who from 15 countries; about 60% of reported cases were fatal. the spread of h5n1 was geographically broad with extension into nigeria and throughout southern and se asia in waterfowl and in domestic fowl. extensive planning for a potential pandemic was put into place given the apparent proximity of the threat and the high mortality in humans. veterinary and human health collaboration was a central precept of this planning in the 'one health' concept (heymann and dixon, 2013) . the h5n1 threat did not materialize despite the high level of circulation in birds. preparedness in se asia in particular included tabletop exercises, culling of birds through rapid response, and at least one joint investigation carried out between laos and thailand. in this setting, h1n1 ran its course, with a milder clinical syndrome (although it is estimated that the global death toll was 284 000). the pandemic of h1n1 precipitated some confusion as well as competition for access to antivirals and vaccines. in the aftermath of these contentious discussions, new policy initiatives were put into place. this included the 'pandemic influenza preparedness' (pip) (who) plan which took some years to negotiate. it balances access to new viral strains for vaccine production with access to these vaccines for poor countries. the fineberg commission to examine an 'after action' performance of the international health regulations (ihr) was central to potential reform as well. this will be more fully discussed below. in 2013 a new influenza virus, h7n9, was isolated from patients in the people's republic of china. isolates were both from birds and from humans. although 571 cases occurred globally with 212 deaths, international spread was limited to two countries. the national response to this epidemic was very strong which is a key to control (discussed below). the 2009 influenza pandemic also followed another global respiratory emergency, sars. the etiology of sars (a new coronavirus) was not known in late 2002 when cases and deaths began to occur in guangdong province of the people's republic of china. initially misdesignated as a chlamydial pneumonia early in the course, public alarm mounted as antibiotics proved futile in treatment. between november 2002 and july 2003, a total of 8427 probable sars cases were reported from 29 countries with 813 deaths for a mortality rate of 9.6% (mmwr, 2003) . sars effectively demonstrated the potential mobility of respiratory pathogens. while sars was declared poorly transmissible, it effectively jumped continents in travelers and infiltrated populations through nosocomial spread. in contrast to influenza, which is highly transmissible, sars still requires direct exposure to bodily fluids for transmission. however, sars shifted the scientific paradigm of zoonotic transmission more fullywith incrimination of wet markets vending civet cats initially thought to be the source. this initial assessment has proved less robust in terms of 'reservoir' for new human pathogens as discussed below. sars also challenged our understanding of transmission dynamics. for reasons that remain unclear, a single infected person who spent a single night in a hotel in kowloon resulted in geographically broad transmission. the concept of a 'superspreader' was popularized (lloyd-smith et al., 2005) . this phenomenon was also described in a second scenario in beijing (shen et al., 2004) . traditionally infection has been characterized by a 'reproductive rate' which is essentially the rate at which an infectious case replaces itself. if the rate is 1, then there will be no spread, as the infection will simply stay the same absolute number as the index case recovers. however, if the rate is higher (i.e., >1), then spread into the population will occur as a single case infects multiple individuals so the number of cases increases. super-spreaders seem to have high infection rates although the overall rate for the pathogen in question may be low. this has, of course, changed our assumptions for modeling disease spread in populations. following the 2009 h1n1 pandemic (and the avian influenza emergence of 1997) and the sars pandemic of 2003, a new virus emerged on the arabian peninsula. mers-cov is a new human pathogen that also causes pneumonia and respiratory distress. the story of mers is less history and more present in terms of defining its epidemic potential. it reinforces the need for 'one health' collaboration between veterinarians, clinicians, and public health specialists, making use of their relative expertise. mers was first reported in 2012 as a case report of a patient in an icu with severe respiratory disease from jordan (hijawi et al., 2013) . at the time of this writing, 1321 cases and 466 deaths have been reported to the who, with an average case fatality ratio of 35%. in one epidemiological analysis, the case fatality ratio for primary cases was 74% (95% ci, 49-91), whereas for secondary cases, it was 20% (95% ci, 7-42) (alsolamy, 2015) . like its coronavirus cousin, sars, mers-cov has demonstrated agile spread within hospitals (oboho et al., 2015) and across continents. in 2015 a large outbreak occurred in south korea (cowling et al., 2015) . that outbreak was characterized by a mortality rate of about 20% among 186 cases, 33 were fatal. it also featured 'superspreading' events. in fact a single case housed in the emergency room with persistent cough was linked to 81 cases in one hospital (kucharski, 2015) . to summarize, the recent episodes of respiratory infectious diseases related to influenza, sars-cov, and mers-cov have demonstrated increasingly direct links between animal and human infections, agile intercontinental geographic spread, and complex transmission dynamics including 'superspreader' events. transmission within health-care settings has also been a prominent feature. these characteristics have challenged traditional assumptions about the pathogenesis and epidemiology of infectious diseases. traditionally microbiology has held that microbes (bacteria, viruses, protozoa, fungi, etc.) are organisms that replicate through genetic mechanisms. that replication is a major factor in the illness in humans these agents cause. recent emerging infectious events have brought an additional complexity into that decades' old assumption. a new form of a human neurodegenerative disease that emerged in britain in the 1990s was linked to the emergence of bovine spongiform encephalitis (bse) in cattle ('mad cow disease'). this link was demonstrated through multiple casecontrol studies. the biological proof of a common etiology came somewhat later (hill et al., 1997) . prions are not microbial life in the traditional sense. they are 'autocatalytic proteins' or proteins that make change. in the instance of 'transmissible spongiform encephalopathies' (tses) of mad cow disease (bse), sheep 'scrapie,' elk chronic wasting disease, as well as in the human diseases of creutzfeldt-jakob disease (cjd), new variant creutzfeldt-jakob disease (vcjd) and kuru, these changes occur in the central nervous system. while the new vcjd epidemic related to ingestion of infected beef from bse-affected cows has waned due to enhanced global surveillance and animal husbandry practices, research into prion disease continues. it appears that despite their lack of genetic material, prions do undergo mutation and strain diversification in response to selective pressures (collinge et al., 2007) . soberingly, it is believed that some humans (estimated 1 in 4000 britons) silently carry pathogenic prions for many years. this risk persists and creates safety concerns for blood transfusions and organ transplantation. the story of bse demonstrated the limitation of the traditional assumption that genetic reproduction of pathogens is necessary for infection as outlined above. the uk beef industry had historically been a relatively stable one when fragmented among many smaller farms across the british isles. to protect this industry, the government maintained a tariff on the imports of competing products from abroad. with the explosion of global trading in beef after world war ii, coincident with refrigerated transport, and the movement toward global free trade, the united kingdom negotiated a timetable under the general agreement on tariffs and trade to scale down tariffs on beef, which heightened competition in the uk beef industry and increased pressure for more efficient and less costly production methods. against this backdrop, innovation in rendering was introduced into the slaughterhouses of the united kingdom. the rendering or processing of carcasses of cows and other animals after the edible and usable bits of flesh and meat have been cut away has been done for centuries. and for decades, uk farmers used the meat and bone meal from rendering as a protein source for beef cattle. historically, the rendering process was similar to pressure cookingapplying very high temperatures for very long times so eventually even the bones broke down into powder. it was an expensive, fuel-consuming, and timeconsuming process. when a new cold vacuum extraction method of rendering was introduced requiring lower temperatures (i.e., less energy) and less time, it seemed a win-win situation considering the increasing pressure on the uk beef industry in the face of global competition. but sometime after the new rendering practice was introduced into the united kingdom, the prion disease known as mad cow disease emerged. the new process, discovered by uk beef industry, was not effectively disinfecting for prions. existence of prion disease was unknown prior to its dramatic emergence, first in cows and then in people. the context is important to appreciate. somehow the streamlining of the rendering process played a role. british scientists tested the new process by deliberately introducing animals with mad cow disease and assaying the resulting meat and bone meal product. they found that the newer rendering process does not remove the infection, whereas the older process did. zika virus emerged in sub-saharan africa in the late 1940s. it is a close cousin of dengue virus and in the same family as yellow fever. all of these viruses are transmitted to humans through the bite of a mosquito. all of these diseases are clinically mild, but occasionally severe causing fever, rash, and joint pain. unfortunately all of these diseases have become globally distributed. zika is the most recent arrival in the americas. it has circulated in asia for some time (chen and hamer, 2016) . while clinically relatively mild, epidemiologic studies in brazil have suggested a link between zika virus and severe birth defects in newborns. viral infection in early pregnancy appears to be associated with microcephaly, which is associated with profound brain injury in newborns. again, the emergence of a new infectious disease is pushing the boundaries of biomedical knowledge. in the absence of certain prevention or treatment options, the government of brazil has gone as far as advising women not to become pregnant (mcneil, 2015) . the emergence of antimicrobial resistance has continued to be a major concern. in 2008 a new enzyme in gram-negative bacteria was detected which caused broad antibiotic resistance. this enzyme was produced by mobile genes that travel on plasmids. plasmids are small circular dna (genetic) packages, which are distinct from the dna of the bacteria itself. these are mobile, with the ability to be taken in across classes of bacteria if and when they confer a selective advantage for survival, as in the case of antimicrobial resistance. the new enzyme ndm-1 (new delhi metalloproteinase-1) was traced to an infection that occurred in patients who had been treated in india. a more complete study of the epidemiology of this enzyme in gram-negative bacterial isolates from the subcontinent suggested a broad range of gram-negatives were affected (kumarasamy et al., 2010) . this new biology, which was first described for extended spectrum beta lactamase (esbl) resistance in the 1990s, has provoked renewed concern in antimicrobial resistance. 2011 was declared by the who as the year to address antimicrobial resistance. the pharmaceutical pipeline is bereft of new antibiotics to address this challenge. chillingly another new panresistant plasmid has been reported from china, which is resistant even to the last line of defense, the polymixin class of antimicrobials (liu et al., 2015) . this report included isolates from pigs at slaughter, retail pig and chicken meat, and humans. again, the specter of intensive agriculture fostering new biological threats calls for careful study. the isolates were largely from areas of intensive porcine agriculture in china. figure 1 indicates sites sampled for polymixin plasmid resistance. the question of where new infections lurk in nature has been an active area of research. as noted above, the initial reservoir for sars coronavirus was thought to be civet cats which are domestically raised for food in china. however, further investigation has suggested that there may be a single host involved in many emergent diseases. in an elegant demonstration of interdisciplinary research, han et al. (2015) have recently published a persuasive article on the role of bats in emerging infections. whether or not the theses of the article prove true with further research, the work provides additional evidence of how extremely powerful interdisciplinary research can be toward solving the puzzles of emergent diseases in humans. in discussing the spillover of viruses from bats to humans, the authors write: factors that contribute to the intrusion of bats into human living environment can be summarized into a 'push' and a 'pull' (brüssow, 2012) . a 'push' refers to the enormous demand for more space and resources brought by the human population explosion, which leads to the destruction of bat habitats and shortage of food. natural environmental changes, such as typhoons and droughts, can also place stresses on bats. a 'pull' involves the living environments built by humans, characterized by urbanization, intensive agriculture and food animal breeding, which attracts bats into human living environments for an abundant of food supply. so coming full circle, this discussion highlights the critical importance of further understanding these events of 'species jumping' or 'spillovers,' given the pressures will only augment rather than abate. infections potentially housed in bats as their natural reservoir (from han and colleagues) is shown in table 1 . it is an impressive array, which brings us to consider how indispensible further understanding in these interactions is to gain insight into emergent human infections. so what explains the apparent increased pace of emergence of new human infections? is it simply that we are more able, with our new technologies, to detect them? or are there forces at work that are fostering this trend? the stories of bse and nvcjd recounted above were outlined because the united kingdom, although a small country, has excellent epidemiologic, statistical, laboratory, and clinical acumen. while cases of bse and nvcjd were not confined to the united kingdom, the origin of emergence was tracked and described relatively efficiently. but, what of influenza? the potential contribution of intensive poultry and swine agriculture to the emergence of influenza a emergence was outlined in my earlier article for this publication. research over the past decade has continued to provide evidence of this risk. as intensive practices have spread to developing countries, the assurance of biosecurity has become less sure as noted in the discussion (above) of polymixin resistance through plasmids in pigs and humans. while many new strains of influenza arise in wild birds (particularly waterfowl), the link to human populations appears to be through domestic poultry (leibler et al., 2009) . however, other anthropogenic mechanisms are also at play. without the global 'mobile' environment of travel and trade, emergence would remain a largely local phenomenon. however, as we saw with the influenza discussion above that is not the case. nor was it the case with sars or with mers-cov. international tourism surpassed 1.1 billion arrivals in 2013 according to the world bank (world bank international tourism). after a slump in global trade during the global recession exports of food in 2014 reached nearly $1.5 trillion in value for selected countries where data were available. this, of course, does not include all of global trade, which has surpassed $3 trillion in value (merchandise trade by product). guarding against transcontinental transmission in food or through human travel is a complex undertaking, with safeguards at the source the most likely answer. however, research, testing, and demonstration of effective measures remain wanting. finally, climate change, largely attributed to human activity, seems to be readjusting the boundaries of mosquito borne diseases. the recent incursion of zika into brazil is attributed to the el niño weather pattern now in force in that geography. while el niño oscillation is not directly attributable to human activity, the shifting of such natural oscillations and their severity may well be. in his landmark 1993 book planetary overload: global environmental change and the health of the human species, anthony j. mcmichael outlines the human-generated stress on natural systems (mcmichael, 1993) . he posits that food will become increasingly scarce for the human community. the macroecologic effects of human activity on climate, water, food, agriculture, pollution, and human health are well described, but the systematic link between the macrolevel (what we can see) and what is occurring on the microlevel remains an important area of research. to address the emergence of new pathogens, we need more precise knowledge about the mechanisms that form the critical pathway to emergence. a follow-up report to the landmark iom report, emerging infections, microbial threats to health: emergence detection and response was published in 2003 (smolinski et al.) (microbial threats to health) . additional factors of emergence were examined in this report: human susceptibility to infection, climate and weather, changing ecosystems, poverty and social inequity, war and famine, the lack of political will, and intent to harm. thus the original 6 factors grew to 13. while enriching the discussion and description of emergence, this proliferation of factors also created overlapping domains within factors; for example, climate and weather are an integral physical science aspect of ecosystems, the failure of political will is integral to the neglect of public health systems, and so forth. from an analytic point of view, the need for in-depth study of factors and how they actually work has become critical for scientific insight into public health protection. mcmichael has suggested the term 'the anthropogenic epoch' to describe our contemporary situation. in other words, human kind is changing the nature of our environment in unprecedented ways. more recently the rockefeller-lancet commission has suggested the new scientific discipline of 'planetary health' as a unifying concept to bring the disparate strands of life sciences, and ecology more closely together to foster interdisciplinary research and insight (the rockefeller foundation, 2015). the key to success will require intense investment in transdisciplinary research which brings in disparate databases and talents into risk analysis. while a full discussion is beyond the scope of this discussion, a number of new tools have come into use that allow more rapid diagnosis and response to newly emergent outbreaks. a few will be highlighted here. in addition to formal disease surveillance reporting a number of informal diseases, surveillance networks have arisen among countries which share common borders or work within a common economic bloc. these networks facilitate the flow of information across borders and allow collaborative investigations, tabletop exercises, and resource sharing on an ongoing basis. in postconflict areas such as the mekong basin, they contribute to health security (gresham et al., 2013) . increasing sophistication of bringing disparate data sets together and creating models to understand possible scenarios has brought additional insight into prevention and control efforts for emerging diseases. prediction of where outbreaks are most likely to occur remains a very imperfect science (jones et al., 2008) . retrospective niche modeling has brought additional insight into how different factors may interact to foster outbreaks (daszak et al., 2013) . laboratory diagnosis of unknown agents has also advanced. during the sars outbreak, the who convened an informal network approach to fully sequence the new agent in 1-month time (david, 2004) . one group has put forward a vision of bringing full genomic sequencing into the laboratories of developing countries (aarestrup et al., 2012) . clearly the ability to quickly diagnose new agents without the necessity of culture would be an important advance. as importantly the integration of informatics linking genomic analyses to phylogenetic metadata would allow global tracking of agents. as the boundaries of microbiology are stretched with new insights from emergent diseases, the ability of the people of the world to protect against pandemics is more important than ever. global traffic and trade continue to grow, confounding national approaches with their international span. in 2005 (following sars) the who adopted the ihr (international health regulations). this is the only law with the force of an international treaty, which is in place to govern the conduct of countries during global security emergencies. the ihr outline 'core capacities' for national-level protection. it is not the optimal level for an emergent pandemic, but it is the minimum considered essential for global health security. the ihr implementation was to have been completed by all member countries by 2012. however, implementation has languished with only one-third of countries implementing and many not disclosing their status of implementation to the who in 2015. after the 2009 influenza pandemic, an independent expert panel was convened to assess how the ihr functioned. that panel, lead by dr harvey fineberg, then president of the institute of medicine, was explicit in outlining the gaps in global health security. the world is ill-prepared to respond to a severe influenza pandemic or to any similarly global, sustained and threatening public-health emergency. beyond implementation of core public-health capacities called for in the ihr, global preparedness can be advanced through research, reliance on a multisectoral approach, strengthened health-care delivery systems, economic development in low and middle-income countries and improved health status. fineberg (2014) . in 2013, ebola (a known infection) emerged in guinea. at the time of its appearance, none of the countries in sub-saharan africa had implemented the core capacities of the ihr (kimball and heymann, 2014) . the outbreak went on to create pandemonium in the three most affected countries (guinea, liberia, and sierra leone) killing over 11 000 people (and afflicting more than 28 000) (ebola-situation). infection was introduced in other countries, but onward transmission was limited. following ebola, the international systems are again under review. initial observations remain distressingly similar to those made in 2009. with reform of the un and who, once again underway, it will be important to follow through. of particular importance as highlighted above is the interdisciplinary (and in the case of the un interagency) nature of prevention, detection, and response to emergent threats. despite the new technological tools, the ecological factors in emergence are ever gathering force. clearly additional efforts are required. emerging infections remain an intersectoral challenge with every indication they will continue to be with us over the coming decades. integrating genome-based informatics to modernize global disease monitoring, information sharing, and response middle east respiratory syndrome: knowledge to date zika virus: rapid spread in the western hemisphere preliminary epidemiological assessment of mers-cov outbreak in south korea interdisciplinary approaches to understanding disease emergence: the past, present, and future drivers of nipah virus emergence the international response to the outbreak of sars in 2003 critically ill patients with 2009 influenza a(h1n1) in mexico pandemic preparedness and responsedlessons from the h1n1 influenza of creating a global dialogue on infectious disease surveillance: connecting organizations for regional disease surveillance (cords). emerg bats as reservoirs of severe emerging infectious diseases the value of the one health approach: shifting from emergency response to prevention of zoonotic disease threats at their source epidemiological findings from a retrospective investigation the same prion strain causes vcjd and bse global trends in emerging infectious diseases ebola, international health regulations and global safety factors influencing the emergence of new (and "old") disease, international encyclopedia of pubic health superspreading in mers emergence of a new antibiotic resistance mechanism in india, pakistan and the uk: a molecular, biological and epidemiological study industrial food animal production and global health risks: exploring the ecosystems and economics of avian influenza emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in china: a microbiological and molecular biological study superspreading and the effect of individual variation on disease emergence planetary overload global environmental change and the health of the human species zika virus, a mosquito-borne infection, may threaten brazil's newborns global health microbial threats to health: emergence, detection, and response by committee on emerging microbial threats to health in the 21st century update: severe acute respiratory syndrome -worldwide u.s case-control study of risk factors for avian influenza a (h5n1) disease, hong kong mers-cov outbreak in jeddah-a link to health care facilities a who guidance document. world health organization the rockefeller foundation-lancet commission on planetary health safeguarding human health in the anthropocene epoch: report of the rockefeller foundation-lancet commission on planetary health h5n1 outbreaks and enzootic influenza report of the review committee on the functioning of the international health regulations the author wishes to thank ms kellie creamer for her assistance in preparing this manuscript.see also: antimicrobial resistance in a one health and one world perspective -mechanisms and solutions; arboviruses; ebola and other viral hemorrhagic fevers; global health law: international law and public health policy; influenza. key: cord-257511-4ftedh1a authors: gunaratne, gihan s.; yang, yang; li, fang; walseth, timothy f.; marchant, jonathan s. title: naadp-dependent ca2+ signaling regulates middle east respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system date: 2018-11-30 journal: cell calcium doi: 10.1016/j.ceca.2018.08.003 sha: doc_id: 257511 cord_uid: 4ftedh1a abstract middle east respiratory syndrome coronavirus (mers-cov) infections are associated with a significant mortality rate, and existing drugs show poor efficacy. identifying novel targets/pathways required for mers infectivity is therefore important for developing novel therapeutics. as an enveloped virus, translocation through the endolysosomal system provides one pathway for cellular entry of mers-cov. in this context, ca2+-permeable channels within the endolysosomal system regulate both the luminal environment and trafficking events, meriting investigation of their role in regulating processing and trafficking of mers-cov. knockdown of endogenous two-pore channels (tpcs), targets for the ca2+ mobilizing second messenger naadp, impaired infectivity in a mers-cov spike pseudovirus particle translocation assay. this effect was selective as knockdown of the lysosomal cation channel mucolipin-1 (trpml1) was without effect. pharmacological inhibition of naadp-evoked ca2+ release using several bisbenzylisoquinoline alkaloids also blocked mers pseudovirus translocation. knockdown of tpc1 (biased endosomally) or tpc2 (biased lysosomally) decreased the activity of furin, a protease which facilitates mers fusion with cellular membranes. pharmacological or genetic inhibition of tpc1 activity also inhibited endosomal motility impairing pseudovirus progression through the endolysosomal system. overall, these data support a selective, spatially autonomous role for tpcs within acidic organelles to support mers-cov translocation. coronaviruses (cov) are enveloped, single strand (+)rna viruses that cause respiratory and enteric infections across a broad range of animal species. several coronaviruses have recently emerged as zoonotic infections that cause life-threatening human disease, exemplified by the severe acute respiratory syndrome (sars-cov) epidemic in 2002/2003 as well as more recent clusters of infections caused by the middle east respiratory syndrome coronavirus (mers-cov). mers-cov is a lineage c beta-coronavirus first isolated in the summer of 2012 from a hospitalized patient in saudi arabia [1] , and to date there have been > 1500 mers cases worldwide. mers-cov infection causes symptoms of high fever and acute, progressive pneumonia in humans, and infection can be associated with a significant mortality rate (∼30-50%) in individuals with comorbidities [2, 3] . as no vaccine exists and trials of drugs and immune response modulators have demonstrated poor efficacy in vivo, there is considerable interest in identifying and optimizing novel therapies to resolve mers-cov infections [3, 4] . therapeutic strategies encompass those targeting viral components as well as host-based processes that support mers-cov infectivity and replication [3, 4] . consequently, resolution of the cell biology of mers-cov infection to illuminate the cellular infrastructure that controls viral entry, organelle passage and transferal into the cytoplasm for replication is of particular interest for evaluating new host targets with promise for development, or repurposing, of mers-cov therapeutics. research into the cell biology of mers has shown that mers-cov particle entry is facilitated by interaction between the viral spike (s) protein and a specific host surface receptor, dipeptidyl peptidase 4 (dpp4, also known as cd26, [5] ). proteolytic priming of the spike protein promotes fusion of the viral envelope with host cell membranes, thus allowing successful translocation of the infectious viral genome into the host cell. recent reports have demonstrated that such proteolytic priming and membrane fusion may occur either at the cell surface via the serine protease tmprss2 [6] , or intracellularly in endocytic compartments via proprotein convertases such as furin (7) . following clathrin-mediated endocytosis, the virus traffics through the endolysosomal system where it is proteolytically activated by host proteases to mediate vesicular fusion and liberation into the cytoplasm [7, 8] . this subcellular translocation pathway affords opportunity for pharmacological intervention as generalized manipulations of endolysosomal function, through inhibition of endocytosis, cytoskeletal dynamics and bulk alkalization of acidic organelles, have been shown to impair mers-cov infectivity [7] [8] [9] [10] . such observations provide justification for pharmacological profiling of targets within acidic organelles to identify novel, more selective opportunities to impair mers-cov translocation through the endolysosomal system. ion channels of the two-pore channel (tpc1, tpc2) and mucolipin family (e.g. trpml1) reside within the endolysosomal system where they regulate endolysosomal microenvironment and trafficking functions [11, 12] . as mers-cov translocation and release into the cytoplasm requires the interplay with the endolysosomal milieu [7, 8] , the ability of these ca 2+ -permeable channels to acutely regulate luminal ionic composition and ph promotes their consideration as potential therapeutic targets. manipulation of endolysosomal ion channel function has been shown to impact endolysosomal morphology and homeostatic trafficking in a variety of cell types [13] [14] [15] [16] [17] [18] [19] [20] . pharmacological manipulation of these channels may therefore permit a defter approach for impairing mers-cov translocation than more generalized perturbations of endolysosomal function. of special relevance is the recent discovery that the natural product tetrandrine acts as a potent blocker of both tpc activity and ebola infectivity, reducing viral titers in the serum of infected mice [21] . the efficacy of tetrandrine related to interference with a late step in ebola virus translocation, possibly by preventing viral-endosome membrane fusion from within tpc2-positive structures [21] , or by interfering with a tpc2-driven late endosome/lysosome maturation process [22] . such data provide impetus for considering tpcs as druggable targets for combating a potentially broad range of infectious pathogens that must traverse, or reside within, the acidic ca 2+ store milieu. here, we use both pharmacological and molecular approaches to address this concept in the context of mers-cov infectivity, as the contribution played by endolysosomal channels in facilitating mers-cov translocation is currently unknown. chemicals were sourced as follows: hernandezine, metocurine, thaligine (isofangchinoline), cycleanine (specs chemistry database), ym201636 (invivogen), gly-phe-b-naphthylamide (gpn), trans-ned-19, (santa cruz biotechnology), arn14988 (echelon biosciences), d-nmapdd and fty720 (cayman chemicals), fumonisin β1 (enzo life sciences), n,n'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[n-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (bapta-am, biotium). all other ligands were purchased from sigma aldrich. naadp was synthesized from nicotinamide adenine dinucleotide phosphate as previously described [23] . pegfp-n3 was from clontech. anti-gfp (rabbit polyclonal), anti-rhodopsin c9 (mouse monoclonal), anti-ha (rabbit polyclonal) and anti-gapdh (rabbit polyclonal) antibodies were from santa cruz. plasmids encoding tpc1-gfp, tpc1[l273 p]-gfp, tpc2-gfp, tpc2pm and rab7a-gfp were from sandip patel (ucl) and have been described previously [14, 24, 25] . pgp-cmv-nes-gcamp6m was from douglas kim (addgene plasmid #40754), tmprss2 was from tom gallaher (loyola) and egfp-rab7a [q67 l] from qing zhong (addgene plasmid #28045 and #28049, respectively). silencer select sirnas targeted against tpcn1, tpcn2, and mcoln1 and non-targeting negative control sirna were purchased from invitrogen. sirna sequences were: tpcn1 sirna#1 -gcgucuucuu caucgugua, tpcn1 sirna#2 -ggcuacuauuaucucaaua; tpcn2 sirna#1 -cgguauuacucgaacguau, tpcn2 sirna#2 -acagaa gugugguuaaaga; mcoln1 sirna#1 -ccuucgccgucgucucaaa, mcoln1 sirna#2 -gaucacguuugacaacaaa; smpd1 sirna#1 -ucacagcacuugugaggaatt, smpd1 sirna#2 -cuaccuacaucgg ccuuaatt; asah1 sirna#1 -ccuugauagauguuaccaatt, asah1 sirna #2 -gcaguuccaugguacaccatt; cers2 sirna#1 -ggcta ttacttcttcaatttt, cers2 sirna#2 -gcattgcctctgatgtcaatt. hek293 (human embryonic kidney) were sourced from atcc. huh7 (human liver) cells and u-2os (human bone osteosarcoma) were gifts from charles m. rice (rockefeller) and eugen brailoiu (temple) respectively. cell lines were maintained in dmem (invitrogen) supplemented with 10% fetal bovine serum (fbs, invitrogen), 100 units/ml penicillin and streptomycin, 292 μg/ml l-glutamine (invitrogen) and cultured at 5% co 2 and 37°c. transfection of plasmid dna was performed in 6-well dishes (nunc) using lipofectamine ® 2000 (invitrogen). cells were transfected using 750 ng of each dna construct, using a 1:3 dna:lipofectamine ® 2000 ratio. complexes were prepared in opti-mem (invitrogen) and added to cells in dmem without fbs or antibiotics. complexes were removed after 6 h and media was exchanged with dmem containing fbs. transfection of sirna was performed in 6well dishes using interferin (polyplus) according to the vendor's protocol. cells were passaged three times to maintain subconfluency over the course of 5 days during which time sirna:interferin complexes were replenished to ensure protein knockdown. knockdown was validated by rt-pcr for positive and negative controls. mers pseudovirus manipulations were carried out as described previously [26, 27] . mers-cov-spike-pseudotyped retroviruses expressing a luciferase reporter gene were prepared by co-transfecting hek293 t cells with a plasmid carrying env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re) and a plasmid encoding mers-cov spike (s) protein. the s protein has previously been shown to be necessary and sufficient to facilitate mers-cov cell entry. m.erspseudovirus particles were harvested from supernatant 72 h after transfection. huh7 cells, which express hsdpp4 endogenously [26] , were used to resolve the effects of drugs on mers-pseudovirus translocation. huh7 cells were seeded into 96-well plates (midwest scientific) at a concentration of 1 × 10 4 cells/well. the following day, cells were pre-incubated with individual drugs (10μm final concentration) for 1 h prior to mers-pseudovirus addition. cells were incubated (5% co 2 /37°c) for an additional 5 h in the presence of drug and pseudovirus. after 6 h, the culture media was replaced with complete dmem and cells were incubated for a further 60 h. cells were then washed 3 times with dpbs (invitrogen) and assayed for luciferase activity. cells were lysed in 80 μl lysis buffer (promega) per well, and 40 μl of lysate was transferred to solid-white 96-well plates (corning) and mixed with 40 μl of luciferase substrate (promega). luminescence (relative luminescence units, rlus) were measured using a glomax-multi detection system (promega). luminescence values are reported relative to levels measured in cells treated with virus alone, background corrected by luminescence values in cells unexposed to virus, except where indicated. for cell viability assays, huh7 cells were lysed 60 h post drug treatment, using cell lysis buffer (promega). lysates were transferred to solid white 96-well plates to be screened in an atp-based viability assay (celltiter-glo 2.0, promega) according to the vendor's protocol. finally, for molecular manipulations, hek293 cells (1.5 × 10 4 cells/ well) were used owing to higher transfectability. extra samples were harvested, for immunoblotting or rna extraction, from the same samples used to study pseudovirus infectivity. for colocalization analyses, huh7 cells or hek293 cells (co-transfeced with hsdpp4-ha) were transfected with plasmids encoding gfptagged proteins of interest. one day after transfection, cells were incubated with mers-pseudovirus (2 h, 4°c) to allow adsorption of the pseudovirus particles to hsdpp4 receptors at the cell surface. after a brief incubation (45 min, 5% co 2 /37°c), cells were then fixed in methanol. samples were blocked with 3% bsa and incubated with primary antibody (1:250 dilution) overnight at 4°c. cells were incubated with an alexa fluor-conjugated secondary antibody (invitrogen) for 1 h at room temperature (5 μg/ml). cells were imaged on an olympus ix81 inverted microscope using a plan-apochromat 60x/1.42 oil-immersion objective, using a spinning disk confocal unit (yogogawa csu-x1). images were captured using a clara interline ccd camera (andor). u2os cells were transfected with plasmid encoding gcamp6m two days prior to microinjection experiments. one day post-transfection, 1 × 10 6 cells were seeded onto collagen coated mattek dishes. for microinjection experiments, dishes were mounted on an olympus ix81 inverted microscope equipped with a piezo nanopositioning stage (prior scientific). cells were perfused with ca 2+ -free hank's balanced salt solution (thermo scientific) at a rate of 0.5 ml/min. isolated u2os cells expressing gcamp6m identified by fluorescence were selected for injections. cell morphology was assessed by acquiring z-stack images and reconstructing three-dimensional models of each cell to be injected. regions that were not relatively close to the nucleus or cell periphery were targeted for injection sites. femptotip (eppendorf) injection pipettes were backfilled with intracellular buffer (110 mm kcl, 10 mm nacl, 20 mm hepes, ph 7.2) containing either vehicle or naadp (100 nm), and positioned using an injectman-4 (eppendorf) micromanipulation system. cells were injected at a z-position approximately 70% of the cell thickness at the site of injection using a femtojet4i (eppendorf). injection parameters were 85 hpa injection pressure, 40 hpa compensation pressure, 0.5 s injection duration, 45°injection angle, and 600um/s injection speed. cells to be injected were imaged (λ ex = 488 nm, λ em = 514 ± 15 nm bandpass) using a plan-apochromat 60x/1.42 objective, and fluorescence changes were monitored using a yokogawa spinning disk confocal (csu-x-m1n), and an andor ixon ultra 888 emccd camera. image acquisition and data collection was done using metamorph version 7.10. cells were lysed at 4°c on a nutating mixer in ice-cold lysis buffer consisting of pbs (invitrogen), 1% triton x-100 (fluka), 1x complete protease inhibitors (roche). protein concentration was determined by bradford assay (pierce), and 25 μg of protein was loaded onto 'any-kd' mini-protean tgx gels (biorad) for sds-page. after electrophoresis, protein was transferred to nitrocellulose membranes using a transblot turbo (biorad) semi-dry transfer machine. membranes were blocked for 1 h at room temperature in 5% milk in pbs supplemented with 0.1% tween-20, prior to addition of primary antibody (1:1000 dilution for anti-gfp and anti-ha antibodies, 1:2000 dilution for anti-gapdh antibody) and overnight incubation at 4°c. the following day, membranes were incubated with irdye secondary antibodies (1:5000 dilution, li-cor) for 1 h at room temperature. signals were detected using a li-cor odyssey imaging system. rna was isolated from hek293 cells after sirna treatment using trizol (invitrogen) according to the vendor's protocol. rna aliquots were frozen at −80°c prior to rt-pcr analysis. rt-pcr evaluation of knockdown of mrna of interest was assessed using the following primers: step rt-pcr system with platinum taq (invitrogen) was used to convert mrna to cdna and amplify samples in a single reaction. semi quantitative rt-pcr reactions (35 cycles) were multiplexed to amplify gapdh and mrna of interest simultaneously. pcr products were separated on a 2% agarose gel. gels were imaged using a myecl (thermo fisher) and quantified by densitometry (imagej). intracellular furin activity was detected using a fluorogenic substrate, boc-arg-val-arg-arg-7-amino-4-methylcoumarin (boc-rvrr-amc, enzo life science). for pharmacological assays, huh7 cells were cultured in the presence of vehicle or drug for 4 h, or were left untreated. for knockdown assays, hek293 cells were treated with the indicated sirnas as described above. cells were washed in pbs, and lysed in cold pbs containing 1% triton x-100 and phosphatase inhibitors (thermo scientific). protein concentration was determined by bradford assay and diluted to 1 μg/μl, 100 μl was dispensed into individual wells in a clear-bottom, black walled 96-well plate (cellstar). furin substrate (10μm final concentration) was added to each well. to test for direct inhibition of furin activity, fangchinoline was added immediately before addition of furin substrate. fluorescence was monitored using a tecan m1000 plate reader at 37°c, λ ex = 360-380 nm, λ em = 440-460 nm. for baseline endosomal motility measurements, huh7 cells were cultured in the presence of 200ug/ml fitc-dextran (sigma) in complete media for 20 min to allow endocytic uptake, before rinsing with pbs and imaging. motility of endosomes expressing wild-type or dominantnegative tpc1 was done by transfecting huh7 cells with the indicated gfp-tagged constructs and imaging gfp fluorescence. assessment of pharmacological inhibition of endosomal motility was performed by treating huh7 cells with the indicated compounds for 1 h at 37c prior to loading with fitc-dextran as described above. cells were imaged on an olympus ix81 microscope equipped with a piezo nanopositioning stage (prior scientific) using a planapochromat 60x/1.42 oil-immersion objective, using a spinning disk confocal unit (yokogawa, csu-x1) and an ixon 888 emccd camera (andor). endosomal structures were tracked by acquiring a series of z-stacks over a time course of 5 min. maximum intensity projections of z-stacks at each time point were generated, and the track particles addon was used in metamorph to assess movement of endosomes and produce trajectory plots. mers pseudovirus entry and subcellular trafficking was monitored using a luciferase assay [26, 27] in which the pseudovirus genome was engineered to encode a luciferase that generates a luminescence signal after release into the cytoplasm, thereby reporting the efficiency of subcellular translocation events (receptor binding, internalization, endolysosomal trafficking, cytoplasmic release; fig. 1a ). to validate this assay in our hands, preliminary experiments were performed following infection in huh7 cells, using drugs previously shown to inhibit specific steps required for mers infectivity. ouabain, a cardiac glycoside inhibitor of early coronavirus internalization events [9] inhibited mers-cov pseudovirus translocation when applied prior to pseudovirus addition (fig. 1b) . similarly, chlorpromazine -an inhibitor of clathrin-mediated endocytosis [28] -resulted in lower luminescence values. inhibition was also observed with several drugs that elevate endolysosomal ph, including chloroquine, the lysomotropic weak base nh 4 cl and bafilomycin a1, an inhibitor of the vacuolar-type h + -atpase, [7, 8, 10, 27, 29] . the cell permeable ca 2+ -chelator bapta-am also inhibited mers-cov pseudovirus translocation (fig. 1b) . bafilomycin was a particularly effective inhibitor of mers-cov pseudovirus trafficking, reducing luminescence signal to background levels ( fig. 1b , [8] ). this inhibitory effect was ablated when bafilomycin application was delayed 5 h after viral addition (fig. 1c) . the inhibitory action of bapta-am was similarly timesensitive ( supplementary fig. 1) , both results demonstrating that mers-cov pseudovirus translocation is rapid and regulated contemporaneously by the endolysosomal microenvironment. endolysosomal ion channels, including the two-pore channel family (tpc1 and tpc2) and the mucolipin family (trpml) of trp channels (fig. 1a) , regulate organellar microenvironment and trafficking dynamics [11, 30, 31] . to examine their impact on mers-cov pseudovirus translocation, we applied both molecular and pharmacological tools to modulate individual channel activity. for rnai analyses, constructs were expressed in hek293 cells to capitalize upon the high transfectability of this cell line. experiments assaying mers infectivity in hek293 cells also necessitated co-expression of the mers-cov entry receptor dipeptidyl peptidase 4 (hsdpp4 [5] ,) owing to low endogenous hsdpp4 expression: luminescence levels were low in the presence of pseudovirus and endogenous dpp4 alone and expression of hsdpp4 resulted in ∼5-fold enhancement of luminescence signal ( fig. 2a , [29] ). co-expression of a gfp control plasmid did not alter infectivity levels ( fig. 2a) . as negative and positive controls, transfection of a dominant negative rab construct (rab5a[s34 n]) which impairs endocytic activity depressed mers-cov pseudovirus infectivity, whereas a constitutively active rab variant (rab7a[q67 l]) potentiated mers-cov pseudovirus infectivity ( fig. 2a) . loss of function analyses were performed using multiple sirnas targeting individual endolysosomal ca 2+ channels. discrete sirnas targeting tpc1, tpc2 and trpml1 were transfected into cells and infectivity assays performed 1 day after the final transfection. whereas two discrete control sirnas, or dual sirnas targeting trpml1 had little effect in this assay, knockdown of endogenous tpc1 or tpc2 markedly inhibited mers translocation ( fig. 2a) . co-transfection of tpc1 and tpc2 sirna did not enhance this inhibitory effect ( fig. 2a) . the penetrance of knockdown attained with these sirnas was evaluated by rt.pcr in the same set of transfections used for the pseudovirus infection assay. representative gels are shown (fig. 2b) together with associated densitometry from all additional experiments evidencing knockdown of individual channels (fig. 2b) . overexpression of tpc isoforms, a manipulation known to perturb endolysosomal trafficking, also impaired mers-cov pseudovirus translocation ( fig. 2a) . overexpression of trpml1 was without effect (fig. 2a) . this inhibitory effect was dependent on appropriate subcellular targeting of the active channel as overexpression of a functional tpc2 channel rerouted from acidic ca 2+ stores to the cell surface (tpc2pm, [25] ) by deletion of the nh 2 -terminal lysosomal targeting motif did not inhibit mers-cov pseudovirus infectivity (fig. 2a) . moreover, the inhibitory action of these manipulations was not caused by alterations in dpp4 expression, as similar dpp4 expression levels were observed across all conditions ( supplementary fig. 2) . fig. 1 . mers-pseudovirus assay validation. a, top, schematic representation of mers-pseudovirus infection pathway, highlighting individual processes during mers translocation (binding, internalization, trafficking, release) that are targets for pharmacotherapy. mers particle association with dpp4 (the host entry receptor) is followed by dpp4-dependent internalization and trafficking through acidic ca 2+ stores and release of luciferase-encoding rna into the cytoplasm of infected cells after fusion with internal membranes. note, the pseudovirus translocation assay used for these experiments is replication defective and reports only virus translocation. bottom, trafficking events through the endolysosomal system are regulated by the activity of ion channels resident within these acidic ca 2+ stores, including members of the trpml (activated by pi(3,5)p 2 ) and tpc family (activated by naadp and pi(3,5)p 2 ). b, effect of drug incubation (spanning 1 h prior to pseudovirus addition and for a 5 h co-incubation after) on mers infectivity measured in terms of luminescence intensity measured 3 days post-infection. drug concentrations were: ouabain (100 nm), chlorpromazine (10 μm), chloroquine (10 μm), nh 4 cl (5 mm), bapta-am (10 μm), bafilomycin (100 nm). pseudovirus infectivity was unaffected by the presence of dmso (0.1%) as drug vehicle. c, delayed exposure to bafilomycin ( †, 6 h exposure, 5 h after pseudovirus addition) attenuated the inhibitory effect of bafilomycin (100 nm) on mers infectivity. data from each experiment are normalized to untreated control samples, and values represent mean ± sem from three or more independent experiments. p-values, ** p < 0.01, * p < 0.05, calculated relative to dmso sample. colocalization between tpc isoforms and the mers-cov spike protein was then examined by immunofluorescence staining against the c9-epitope tagged spike protein. these experiments were performed using huh7 cells owing to the fact that this cell line expresses higher endogenous levels of hsdpp4 (∼18-fold, compared with hek293 cells [7] ). this obviates the need for hsdpp4 transfection and allows examination mers-cov pseudovirus trafficking in a native cell line. while no signal was evident in huh7 cells unexposed to pseudovirus, mers-cov spike protein could be resolved in vesicular structures in cells previously incubated with pseudovirus (2 h, 4°c) and fixed 45 min after a 37°c incubation (fig. 3) . co-transfection with rab5-gfp or rab7-gfp evidenced mers cov spike protein co-localization within a subset of both rab5-gfp and rab7-gfp positive vesicles (supplementary fig. 3) . expression of tpc constructs (tpc1-gfp, tpc2-gfp) evidenced colocalization between the mers cov spike protein with both tpc2 (biased toward lysosomes [24, 32] ) and tpc1 (biased toward endosomes [24, 32] ) in all samples examined (fig. 3) . colocalization between mers-cov spike protein and gfp-trpml1 was also observed (fig. 3) . collectively these data are consistent with mers pseudovirus translocation through tpc positive endolysosomal organelles (fig. 3) , with the properties and/or dynamics of these structures that are permissive for mers-cov pseudovirus translocation being regulated by endogenous tpc activity (fig. 2 ). can tpc modulators attenuate mers trafficking through the endolysosomal system? tpc activity is regulated by the endolysosomal phospholipid pi(3,5)p 2 , as well as the potent ca 2+ releasing second messenger naadp (fig. 1a) . lipid modulators, naadp antagonists and a range of voltage-operated ion channel blockers have all been shown to regulate tpc activity [18, 21, 24, [32] [33] [34] [35] [36] . to examine the effects of tpc regulators on mers-cov pseudovirus translocation, a fixed concentration (10μm) primary drug screen was performed in huh7 cells, with the goal of identifying plasma membrane-permeable compounds with inhibitory activity on mers-cov pseudovirus translocation. the effects of naadp antagonists/pore blockers (fig. 4) and lipid modulators (supplementary fig. 6 ) are described in turn below. tpcs show affinity for a broad range of voltage-operated channel ligands, possibly reflecting their ancient evolutionary pedigree as antecedents of four domain voltage-gated ion channels [33] . consistent with these observations, several na + channel blockers (procaine, benzocaine) and voltage-operated ca 2+ antagonists (verapamil, nicardipine and nimodipine) attenuated mers-cov pseudovirus translocation (fig. 4a) . of particular relevance were the effects of bisbenzylisoquinoline alkaloids, compounds consisting of dual benzylisoquinoline moieties linked together by ether bridge(s) (fig. 4b, supplementary fig. 3 . mers-cov spike protein colocalizes with endolysosomal ion channel positive structures. huh7 cells were transfected with tpc1-gfp (top, green), tpc2-gfp (middle, green) and gfp-trpml1 (bottom, green) and subsequently infected with mers-pseudovirus (red). cells were fixed, immunostained for mers-cov spike protein (spike-af555) and visualized by confocal microscopy. white boxes in overlay panel (right) show enlarged regions to assess colocalization between the red and green channel. scalebars, 10 μm (left column) and 1 μm (inset, right). fig. 4) . these compounds are of interest in light of the recent discovery that tetrandrine, a bisbenzylisoquinoline alkaloid natural product, blocked tpc activity and potently inhibited ebola virus infectivity in vitro and in vivo [21] . therefore, we screened several bisbenzylisoquinoline alkaloids from two different groupings (fig. 4b) tubocurarinelike ligands where the isoquinoline pairs were non-adjacent within the bisbenzylisoquinoline ring structure (abab, 'head-to-tail'), and tetrandrine-like ligands, where the isoquinoline groups are directly conjoined (aabb, 'head-to-head and tail-to-tail'). these two groups are discriminated by shading intensity in fig. 4a&b and structures of individual compounds are provided in supplementary data (supplementary fig. 4) . several structure-activity insights were clear from the screening dataset. first, the tubocurarine-like bisbenzylisoquinolines (tubocurarine, cycleanine, metocurine) were considerably less effective compared to the tetrandrine-like ligands (tetrandrine, thaligine and fangchinoline) which strongly impaired mers-cov pseudovirus translocation (fig. 4a&b) . second, the presence of individual methoxy moieties around the compound ring structure markedly influenced the penetrance of individual ligands, with examples of ring substitutions that preserved (fangchinoline, thaligine) or decreased (berbamine, herandezine) drug effectiveness relative to the anti-ebola prototype tetrandrine. finally, fangchinoline, and the stereoisomer thaligine ('isofangchinoline') were the most effective compounds at inhibiting mers translocation in these assays. compounds from the initial screen were further evaluated in full concentration-response relationships for inhibition of mers-cov pseudovirus infectivity (fig. 4c) . the relative sensitivities to screened compounds fangchinoline (ic 50 = 1.7 ± 0.1 μm) > tetrandrine (ic 50 = 7.0 ± 0.8 μm) > berbamine (ic 50 = 29.2 ± 7.0 μm) were consistent with the primary screen (fixed concentration, 10 μm). cellular viability was measured in parallel and used to calculate a 'selectivity index' for these selected compounds. calculation of the selectivity index (cytotoxic concentration 'cc 50 ' for cellular toxicity / ic 50 for mers inhibition) underscored the improved performance of fangchinoline over tetrandrine (fig. 4c, inset) . these data support an efficacy of specific bisbenzylisoquinoline alkaloids as agents for targeting fig. 4 . pharmacological blockade of mers pseudovirus infectivity. a, single-concentration drug screening (10μm) to identify ion channel modulators that inhibit mers pseudovirus translocation. huh7 cells were treated with indicated drugs for 6 h, starting 1 h prior to incubation with mers pseudovirus (i.e. a 5 h co-incubation). drugs represent known modulators of intracellular trpml (ml-sa1) and tpc channels (ned-19, voltage-operated channel blockers), as well as a series of structurally related bisbenzylisoquinolines encompassing the known tpc blocker tetrandrine. screened bisbenzylisoquinolines comprised two groupings: tubocurarine-like compounds (light dashed) and tetrandrine-like compounds (heavy dashed). p-values, ** p < 0.01, * p < 0.05. b, structures representing the two groups of bisbenzylisoquinolines highlighted in 'a'. 3d confomers were downloaded from pubchem and displayed in pubchem 3d viewer v2.0. c, complete concentration response relationships for inhibition of mers translocation by fangchinoline, tetrandrine, berbamine or ned-19. inset, selectivity index (cc 50 for cellular toxicity/ ic 50 for pseudovirus translocation) for indicated compounds. d, inhibition of ca 2+ signals in u2os cells microinjected with naadp (100 nm pipette concentration) after drug treatment (0.1% dmso, 50μm ned-19, 10μm tetrandrine, 10μm fangchinoline, 10 min preincubation). traces represent average of 3 independent injections, error bars represent s.e.m. to assess the ability of the same compounds to inhibit naadp-evoked ca 2+ release, confocal ca 2+ imaging experiments were performed in human u2os cells microinjected with naadp. preincubation of cells with tetrandrine or fangchinoline (10μm) inhibited ca 2+ signals triggered by naadp microinjection (69 ± 16% and 88 ± 3% inhibition for tetrandrine and fangchinoline, respectively), evidencing fangchinoline as an effective inhibitor of both naadp-evoked ca 2+ release (fig. 4d) and mers-cov pseudovirus translocation (fig. 4a&c) . no antiviral activity was observed following inhibition of ip 3 -or cadpr-evoked ca 2+ release (fig. 1 in [37] ). the inhibition of naadp-evoked ca 2+ release and mers-cov pseudovirus translocation by these compounds was not due to lysosomotropism, as we found no significant decrease in lysosomal ca 2+ content or disruption of lysosomal ph upon addition of these drug (supplemental fig. 5) . finally, the effects of two other cell permeable endolysosomal ion channel modulators were examined: the naadp antagonist ned-19 [38] , and the trpml agonist ml-sa1 [13] . ned-19 showed poor inhibitory activity in the mers translocation assays (fig. 4a) , which was surprising as it is widely employed as a naadp blocker. however, in our hands, trans-ned-19 (up to ∼50μm) also failed to potently inhibit naadp-evoked ca 2+ release in either mammalian cells (fig. 4d ) or sea urchin homogenate (fig. 4 in [37] ), suggesting some caution in interpretation of results obtained with commercially sourced ned-19. the activity of the cell permeable trpml agonist ml-sa1, which did not modulate mers-cov pseudovirus infectivity (fig. 4a) , was confirmed as application to mammalian cell lines elicited clear ca 2+ transients (data not shown). tpc channels are also regulated by bioactive lipids, including pi (3,5)p 2 [18] and sphingosine for tpc1 [39] . addition of the pikfyve inhibitor ym201636 to reduce pi(3,5)p 2 levels, a phosphoinositide which activates tpc channels, decreased mers-cov pseudovirus translocation ( supplementary fig. 6 a&b) . sphingosine is generated through the action of acid ceramidase (ac, asah1) on ceramide, a product of acid sphingomyelinase (asm, smpd1) activity (supplementary fig. 6 ). acid sphingomyelinase is required for ebola infection [40] , although the roles of asm and ac have not been studied in mers-cov pseudovirus infectivity. in huh7 cells exposed to pseudovirus, addition of the antidepressant drugs desipramine or amitriptyline, both of which act as functional inhibitors of asm [41, 42] , inhibited mers-cov pseudovirus translocation (supplementary fig. 6b ). concentration-response curves demonstrated potent inhibition (ic 50 = 796 nm for desipramine, ic 50 = 4.8 μm for amitriptyline) with little deleterious effects on cellular viability over the concentration range studied ( supplementary fig. 6c ). pharmacological inhibition of ac, to reduce sphingosine levels, also reduced luminescence levels ( supplementary fig. 6b ). in contrast, incubation with fumonisin b1, an inhibitor of ceramide synthase enzymes localized in the endoplasmic reticulum, which utilize sphingosine to synthesize ceramide, did not significantly decrease luminescence values. discrete sirnas targeting smpd1 or asah1 inhibited mers translocation ( supplementary fig. 6d) , and successful knockdown of these enzymes was validated ( supplementary fig. 6e) . therefore, inhibition of enzymes upstream, but not downstream, of the tpc1 activator sphingosine impaired mers-cov pseudovirus infectivity. in summary, these data demonstrate dysregulation of known lipid tpc regulators also impeded mers pseudovirus translocation. tpc activity changes both the cytosolic and luminal microenvironment within the acidic ca 2+ stores. local, cytoplasmic ion fluxes potentially regulate vesicular dynamics and fusion events [17, 18] to impact mers-cov pseudovirus progression through the endolysosomal system. tpc triggered changes in luminal ca 2+ and ph (51, 52) may also regulate mers-cov pseudovirus translocation by regulating proprotein convertase activity needed for proteolytic activation of the spike protein and thereby mers-cov fusion and release into the cytoplasm (6, 53) . therefore, we examined the effects of fangchinoline on both these cell biological aspects of mers-cov pseudovirus translocation. first, we assessed the effects of drug incubation on mers-cov pseudovirus progression through tpc-positive structures. treatment of huh7 cells with fangchinoline (10μm) increased mers particle colocalization with both tpc1-gfp and tpc2-gfp labelled structures (fig. 5a) . quantification of colocalization using pearson's correlation coefficient [43] from single cell regions of interest in fixed samples showed that fangchinoline treatment increased levels of pixel-to-pixel covariance across mers and tpc-positive structures (fig. 5a, inset) . analysis of mers-cov spike protein colocalization with tpcs using a different algorithm (manders' coefficient) also demonstrated a similar increase in colocalization after fangchinoline treatment (fig. 5b) . these results are suggestive of a drug-evoked blockade of endolysosomal mers-cov pseudovirus translocation events. in live cell imaging experiments, it was also evident that fangchinoline treatment impacted the mobility of fluorescent dextran labelled endosomal structures. single particle tracking analysis revealed that incubation of cells in fangchinoline, or tetrandrine, decreased the mobility of dextran-labelled endosomal structures (fig. 5c ). impaired movement of tpc1labelled structures was also seen in cells overexpressing the pore-dead mutant tpc1[l273 p]-gfp, but not tpc1-gfp, suggesting that drugevoked inhibition of tpc1 activity underpinned this effect on endosomal motility (fig. 5d) . next, we assessed the effects of drug incubation, or tpc knockdown, on the activity of furin, a ca 2+ -dependent serine endoprotease. for these experiments, the kinetics of furin-evoked cleavage of a fluorogenic substrate (boc-rvrr-amc) was measured in cell lysates under different experimental conditions. in huh7 cells, pretreatment of cells with fangchinoline (10μm, 4 h) markedly reduced the rate of substrate cleavage (19.3 ± 2.5% of control, fig. 6a&b ). in contrast, inhibition was not observed during acute drug treatment, demonstrating fangchinoline did not act as a direct furin inhibitor (fig. 6a&b) . similar assays were repeated in hek293 cells treated with the validated sirnas against tpc1 or tpc2 (fig. 6c) . knockdown of either tpc isoform impaired furin activity, decreasing the initial rate of substrate cleavage by 59.9 ± 3.6% (tpc1) or 54.3 ± 3.0% (tpc1, fig. 6d ). these data show that pharmacological or molecular inhibition of tpc function impaired furin activity. consequently, we tested whether furin overexpression could rescue drug inhibition of mers-cov pseudovirus translocation. overexpression of furin markedly attenuated drugevoked inhibition of mers-cov pseudovirus infectivity in huh7 cells (fig. 6e) . the inhibition observed with tetrandrine and fangchinoline in control experiments (> 80% inhibition) was considerably attenuated by transfection with exogenous furin (< 30% inhibition, fig. 6e ). overexpression of the serine protease tmprss2 to bias mers-cov pseudovirus translocation to direct entry via the plasma membrane also rendered both bisbenzylisoquinoline alkaloids ineffective at blocking mers-cov pseudovirus infectivity (fig. 6f) . the lack of inhibitory action of tetrandrine or fangchinoline after direct cell entry at the plasma membrane rule out the possibility that these compounds act processes downstream of membrane fusion, including transcription or translation. these data confirm that tetrandrine and fangchinoline act by blocking the trafficking and processing of internalized mers-cov pseudovirus particles within the endolysosomal system. mers-cov infections are clinically challenging and are associated with high mortality rates (∼30-50%, [2, 3] ) due to disease severity and lack of effective pharmacotherapy. here, we demonstrate that endolysosomal tpcs may represent a druggable host target for mers-cov antiviral therapy based on data showing that inhibition of endogenous tpc activity via either molecular or pharmacological methods impaired g.s. gunaratne et al. cell calcium 75 (2018) [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] the cellular translocation of a mers-cov pseudovirus. such findings merit consideration of the role of tpcs as host-factors in supporting mers-cov infectivity, and the potential druggability of these ion channels to source novel antivirals. these issues are discussed below. tpcs are evolutionarily ancient ion channels, resident within the endolysosomal system, where they fulfill homeostatic trafficking functions supporting internalized substrate distribution [13] [14] [15] [16] [17] [18] [19] [20] . the mechanistic basis of how these channels regulate endolysosomal trafficking events has been the subject of varied speculation, but it seems clear that perturbing tpc localization, or their activity away from the physiological set-point, disrupts endolysosomal morphology and substrate trafficking. their importance in subcellular transport extends beyond endogenous substrates, as impactfully highlighted in the context of ebola infectivity [21] . a role for tpcs in supporting viral translocation is further evidenced here in the context of mers, another single stranded rna virus. investigation of the role of tpcs in other viral infections and in other paradigms of infectious disease, where pathogens exploit or reside within endolysosomal derived organelles is merited. how do tpcs support mers passage from cell surface to cytoplasm? fig. 5 shows that tpc activity is necessary to support trafficking events and passage of pseudovirus particles through tpc-positive compartments in the endolysosomal trafficking pathway. effects on particle progression and processing are likely interdependent: tpcs also regulate furin activity (fig. 6) . tpc activity is known to alter both endolysosomal ca 2+ content and ph [44, 45] and both factors regulate the activity of proprotein convertases (such as furin) required for proteolytic activation of the spike protein, mers-cov fusion activity and cytoplasmic translocation [7, 46] . dysregulation of the luminal microenvironment (ph, ca 2+ ) owing to changes in tpc activity may then impair mers-cov fusion depending on protease levels, or diversity of protease expression, within a given cell type. the inhibitory and non-additive effects of tpc1 (biased toward endosomes) and tpc2 (biased toward lysosomes) knockdown ( fig. 2a) suggest that mers translocation occurs throughout proximal and distal compartments within the acidic ca 2+ stores. prior investigations of mers infectivity suggest fusion and cytoplasmic translocation occurs from early endosomes [8] , while more recent studies demonstrate inhibition of cathepsin l, a lysosomal cysteine protease, blocks mers infectivity [47] , both studies consistent with the demonstrated role for both tpc1 and tpc2 (fig. 2) . a further conclusion that can be drawn from the current data is that the role of tpcs appears selective: manipulation of trpml1 function, another 'acidic ca 2+ store' ion channel, failed to impair mers-cov pseudovirus infectivity. these data suggest sub-specialization in endolysosomal channel function and/or trafficking pathways, as tpc inhibition may selectively impair only a subset of transported substrates rather than effect a global disruption of endolysosomal functiona feature that may prove critical in advancing the viability of tpcs as selective drug targets. selectivity in drug action against tpcs was also evident through a correlation between the extent of inhibition of naadp-evoked ca 2+ signals and impairment of mers translocation (fig. 4) , a relationship which is evidenced further in the companion paper [37] . further drugs that inhibited mers pseudovirus translocation do not inhibit other intracellular ca 2+ mobilization pathways ( fig. 1 in [37] ), even though the role of tpcs in amplifying ca 2+ signals through other intracellular ca 2+ channels beyond the endolysosomal system is well appreciated. in short, the data support a selective, spatially autonomous role for tpcs within acidic organelles in supporting mers infectivity. are tpcs druggable targets for antiviral development? this is a topical issue given renewed interest in repurposing ca 2+ channel ligands to impair viral infectivity [21, 48, 49] , although most attention to date has been directed toward cell surface targets such as voltage-operated ca 2+ channels (voccs). however many vocc blockers also inhibit naadp-evoked ca 2+ release activity in the high micromolar range [50] , likely due to the pedigree of the tpc structural blueprint as an antecedent for cell surface voltage-gated channel architecture [33] . tpc blockade may therefore constitute a component of previously described antiviral vocc antagonist action. the promiscuous pharmacology of tpcs may nevertheless prove a challenge for discovering selective, high affinity tpc ligands. however, the emerging capacity to (i) interrogate cell biological assays that depend on tpc function (such as explored here), (ii) miniaturize naadp-evoked ca 2+ release screening platforms (see companion paper, [37] ) and (iii) integrate structurebased approaches based upon the recent resolution of tpc crystal structures [51] [52] [53] , will collectively spur opportunities to identify novel leads and optimizing ligand affinity and selectivity for these channels. in this context, clear structure-activity correlations emerge from our study of the bisbenzylisoquinoline alkaloids. these compounds are a well-studied natural product group with hundreds of unique compounds described, some displaying activity against other pathogenic eukaryotes [54, 55] . bisbenzylisoquinoline alkaloids are classified based on the number, type and orientation of ring linkages [56] [57] [58] . while the compounds studied here provide only limited insight into the structural diversity of this series, representing compounds with two diphenyl ether linkages differing in their orientation ('head-to-tail' versus 'headto-head' and 'tail-to-tail'), nevertheless clear insight into structure-activity relationships impacting mers translocation/intracellular ca 2+ release properties was discernable (fig. 4) providing impetus for further structural exploration of the bisbenzylisoquinoline scaffold. this should encompass compounds with differing number of ring linkages (effect of ring flexibility), the position and nature of the ring substitutions, as well as the stereochemistry around the dual asymmetric carbons. we note the potency of tetrandrine against mers was lower than previously reported for tetrandrine against ebola (tens of nm in vitro, [21] ). the improved performance of fangchinoline over tetrandrine in this particular assay supports future optimization of structure activity relationships in this compound series against specific pathogens (fig. 4d) . also noteworthy in our experiments was the efficacy and good selectivity indices seen with the fda-approved serotonin reuptake inhibitors, amitriptyline and desipramine ( supplementary fig. 6 ). further investigation of the interrelationship between the antiviral activity of ssris ( supplementary fig. 6 , [49] ), endolysosomal bioactive lipid content (these compounds act as functional inhibitors of acid sphingomyelinase [41, 42] ) and effects on tpc activity is also warranted. in conclusion, these findings support a unique role for tpcs and naadp-sensitive ca 2+ release in mers infectivity, providing further support for exploration and development of tpc ligands as novel antiviral therapeutics. none. fig. 6 . pharmacological and molecular inhibition of tpcs reduces furin activity. a, huh7 cells were treated with vehicle or fangchinoline (10μm), lysates were harvested from cells, and furin activity was assessed using fluorogenic substrate, boc-rvrr-amc. drug was added either 4 h prior to harvesting (pretreated) or immediately before addition of substrate (acute). representative traces are shown, linear range used to calculate furin substrate cleavage (rfu/min) is shown using dashed lines. b, quantification of cumulative data set of furin activity in pharmacologically treated huh7 lysates. p-values, ** p < 0.01 relative to dmso control c, hek293 cells were treated with the indicated sirnas, lysates were collected and assayed for furin activity. representative traces are shown, with linear range of substrate cleavage denoted using dashed lines. d, quantification of cumulative data set of furin activity using sirna treated hek293 cells, p-values: ** p < 0.01, relative to non-targeting sirna treated samples. e, effect of furin or tmprss2 overexpression on pharmacological blockade of mers-pseudovirus infectivity, pvalues: ** p < 0.01 relative to empty vector transfected dmso treated controls, ## p < 0.01 relative to empty vector transfected samples treated with tetrandrine or fangchinoline. f, effect of tetrandrine or fangchinoline treatment on huh7 cells overexpressing tmprss2. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sarslike disease coronaviruses -drug discovery and therapeutic options dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner atp1a1-mediated src signaling inhibits coronavirus entry into host cells screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture two-pore channels at the intersection of endolysosomal membrane traffic pairing phosphoinositides with calcium ions in endolysosomal dynamics: phosphoinositides control the direction and specificity of membrane trafficking by regulating the activity of calcium channels in the endolysosomes lipid storage disorders block lysosomal trafficking by inhibiting a trp channel and lysosomal calcium release the two-pore channel (tpc) interactome unmasks isoform-specific roles for tpcs in endolysosomal morphology and cell pigmentation purified tpc isoforms form naadp receptors with distinct roles for ca 2+ signaling and endolysosomal trafficking tpc1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of tpc2 high susceptibility to fatty liver disease in two-pore channel 2-deficient mice tpc proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes two pore channel 2 (tpc2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal ph dysregulation of lysosomal morphology by pathogenic lrrk2 is corrected by two-pore channel inhibition ebola virus. two-pore channels control ebola virus host cell entry and are drug targets for disease treatment ebolavirus glycoprotein directs fusion through npc1+ endolysosomes photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (naadp) targets in mammalian cells essential requirement for two-pore channel 1 in naadp-mediated calcium signaling an naadp-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying ca 2+ signals a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus the essential role of clathrin-mediated endocytosis in the infectious entry of human enterovirus 71 role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation regulation of membrane trafficking by signalling on endosomal and lysosomal membranes role of trpml and two-pore channels in endolysosomal cation homeostasis naadp mobilizes calcium from acidic organelles through two-pore channels two-pore channels provide insight into the evolution of voltage-gated ca 2+ and na + channels expression of ca(2)(+)-permeable two-pore channels rescues naadp signalling in tpc-deficient cells convergent regulation of the lysosomal two-pore channel-2 by mg(2)(+), naadp, pi(3,5)p(2) and multiple protein kinases the two-pore channel tpcn2 mediates naadpdependent ca(2+)-release from lysosomal stores a screening campaign in sea urchin egg homogenate as a platform for discovering modulators of naadp-dependent ca 2+ signals in human cells identification of a chemical probe for naadp by virtual screening intracellular sphingosine releases calcium from lysosomes ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs identification of novel functional inhibitors of acid sphingomyelinase a practical guide to evaluating colocalization in biological microscopy fertilization and nicotinic acid adenine dinucleotide phosphate induce ph changes in acidic ca(2+) stores in sea urchin eggs the ecto-enzyme cd38 is a nicotinic acid adenine dinucleotide phosphate (naadp) synthase that couples receptor activation to ca2+ mobilization from lysosomes in pancreatic acinar cells furin at the cutting edge: from protein traffic to embryogenesis and disease glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) the clinically approved drugs amiodarone, dronedarone and verapamil inhibit filovirus cell entry a screen of approved drugs and molecular probes identifies therapeutics with anti-ebola virus activity pharmacological properties of the ca2+-release mechanism sensitive to naadp in the sea urchin egg structure of the voltage-gated two-pore channel tpc1 from arabidopsis thaliana structure, inhibition and regulation of two-pore channel tpc1 from arabidopsis thaliana structural insights into the voltage and phospholipid activation of the mammalian tpc1 channel in vitro antiplasmodial, antiamoebic, and cytotoxic activities of a series of bisbenzylisoquinolgulation of the lysosomal two-pore channel-2 bine alkaloids antimycobacterial activity of bisbenzylisoquinoline alkaloids from tiliacora triandra against multidrug-resistant isolates of mycobacterium tuberculosis the bisbenzylisoquinoline alkaloids bisbenzylisoquinoline alkaloikds -a review supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ceca.2018.08.003. key: cord-252883-1ub01j2x authors: bleibtreu, a.; bertine, m.; bertin, c.; houhou-fidouh, n.; visseaux, b. title: focus on middle east respiratory syndrome coronavirus (mers-cov) date: 2019-11-11 journal: med mal infect doi: 10.1016/j.medmal.2019.10.004 sha: doc_id: 252883 cord_uid: 1ub01j2x since the first case of human infection by the middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia in june 2012, more than 2260 cases of confirmed mers-cov infection and 803 related deaths have been reported since the 16th of october 2018. the vast majority of these cases (71%) were reported in saudi arabia but the epidemic has now spread to 27 countries and has not ceased 6 years later, unlike sars-cov that disappeared a little less than 2 years after emerging. due to the high fatality rate observed in mers-cov infected patients (36%), much effort has been put into understanding the origin and pathophysiology of this novel coronavirus to prevent it from becoming endemic in humans. this review focuses in particular on the origin, epidemiology and clinical manifestations of mers-cov, as well as the diagnosis and treatment of infected patients. the experience gained over recent years on how to manage the different risks related to this kind of epidemic will be key to being prepared for future outbreaks of communicable disease. the first case of infection attributed to middle east respiratory syndrome coronavirus (mers-cov) was detected in saudi arabia in june 2012 [1] . mers-cov then spread to several neighboring countries, mainly jordan and qatar (see fig. 2 ), and imported cases of the disease were reported throughout the world in asia, africa, europe and the americas [2] . by the 16th of october 2018, 2260 con the first two coronaviruses demonstrated to cause respiratory infections in humans, the coronaviruses 229e and oc43, were identified in the 1960s. they were held responsible for respiratory infections of moderate severity in humans. despite these viruses being identified in several reports as causing lower respiratory tract infections, it was generally accepted that coronaviruses were of low pathogenicity until the emergence of sars-cov (severe acute respiratory syndrome coronavirus) in 2002, a virus with a fatality rate estimated at 10%. the sars outbreak that resulted in more than 8400 cases was finally contained two years later, in 2004, and the virus has not been detected again since [4] . there was renewed interest in coronavirus research following the sars epidemic, and two novel endemic human coronaviruses were identified, nl63 and hku1 respectively in 2004 and 2005, but could not be replicated in cell culture. both of these new viruses were responsible for respira-tory infections of moderate seriousness like the coronaviruses 229e and oc43. great effort has been made to identify coronaviruses in animal populations, both before and after the sars outbreak, in order to better understand and control the risk of animal-to-human transmission. this resulted in the discovery of coronaviruses in numerous animal species, with a few exceptions such as sheep and goats, fish and non-human primates [5] . the first case of mers-cov infection was reported in jeddah, saudi arabia, in june 2012 [1] . the patient, a 60-year-old man, died from lung and kidney failure 11 days after being admitted to hospital. very shortly afterwards, in september 2012, a second patient was admitted to hospital in the united kingdom for severe respiratory infection related to a novel coronavirus following travel to the middle east. the new virus was found to replicate in a tissue culture model and was rapidly isolated and identified for both cases [6, 7] . retrospectively, other cases of the disease were found to have occurred before the 2 aforementioned cases: in april 2012, an outbreak at zarqa hospital in jordan affected the staff of the intensive care unit, with two fatal cases. the respiratory samples collected were later confirmed to be positive for mers-cov [8] . these initial cases were rapidly followed by a series of outbreaks in all saudi arabian provinces that were characterized by the infection of health professionals in direct contact with the patients. other similar outbreaks were observed in several neighboring countries: qatar, bahrain, kuwait, jordan and tunisia. health authorities reacted quickly to the reports of these epidemics and the strong resemblance with observed clinical features of sars-cov infections. indeed, although a few patients developed mild infections, the fatality rate for patients infected with mers-cov was over 30% [2] . following the identification of mers-cov, great effort was put into finding which animal species it originated from in order to stop the further spread of the disease to humans. mers-cov was very rapidly determined to be genotypically closely related to the betacoronavirus lineage c viruses identified in bats [9] . based on these findings, and the major role of bats in the genetic diversity and spread of coronaviruses, much of the initial work aiming at finding the natural reservoir of mers-cov focused on bats. however, no conclusive evidence demonstrating that bats were the natural reservoir of mers-cov in the arabian peninsula were found, despite the identification of closely related viruses in bats in sub-saharan africa [10] , far from the existing outbreaks. very strong epidemiological links were identified between the human cases and camels and resulted in the isolation in camels of viruses that were directly related to mers-cov and that could replicate in cultured human cells [11] . the investigation of dromedary camel serum collections, some of which collected as early as 1983, demonstrated that the virus was already widespread (seropositivity rate > 80%) in the east african countries (somalia, sudan and egypt). these countries export dromedary camels to arabian countries, but also in kenya, nigeria, tunisia, ethiopia, burkina faso and morocco [12] [13] [14] . phylogenetic analysis revealed 5 distinct coronavirus lineages in dromedary camels, including one recombinant lineage that led to the mers-cov epidemic in humans [15] . mers-cov is a betacoronavirus belonging to lineage c. it is an enveloped virus with a positive-sense single-stranded rna genome of about 30 kb. under electron microscopy, virions are generally spherical with surface projections (spikes) formed by the surface protein s creating an image reminiscent of a crown or solar corona. the positive-sense single-stranded rna genome acts as messenger rna (mrna) with a 5 cap and a 3 polyadenylated tail. it plays three roles during the host cell cycle: (i) it acts as the initial rna molecule for the infection cycle; (ii) it is the template for replication and transcription; (iii) it is the substrate that is packaged into the newly assembled viral particles [16] . the mers-cov genome is organized in the same way as other coronavirus species. the first two thirds of the mers-cov genome contain two overlapping reading frames (orf1a and orf1b) that translate into the replication-transcription complex including 16 non-structural proteins. the remaining third of the genome encodes the four structural proteins, the spike (s), envelop (e), membrane (m) and nucleocapsid (n) proteins, as well as five accessory proteins (orf3, orf4a, orf4b, orf5 and orf8b) that are not required for genome replication but are probably involved in virulence. the flanking sequences, on both ends of the genome, contain untranslated 5 and 3 regions (utr) (fig. 3 ) [17] . the viral particle can enter the cell in two ways, which probably contribute to the broad tissue tropism of this virus that replicates mainly in respiratory epithelial cells but can also infect many other cell types. via the endosomal pathway, the s1 domain of the mers-cov spike protein (s) binds its receptor, dipeptidyl peptidase 4 (dpp4) [18] , induces endocytosis of the viral particle and a change in the conformation of the s2 subunit of the s protein that then mediates virus-host membrane fusion and uncoating of virus rna. mers-cov can also enter host cells via a non-endosomal mechanism by direct fusion of the virus with the plasma membrane following s protein cleavage by human proteases [19] . following entry into the cytoplasm and uncoating of the virus nucleocapsid, the viral genomic rna is translated to produce two polypeptides, pp1a and pp1b, that form the replicase-transcriptase complex. this initial replicase-transcriptase complex uses the genomic rna to produce 16 non-structural proteins that assemble into the replication complex. the replication complex then replicates the genomic rna and produces other subgenomic rnas that ensure the translation of the structural proteins. virions are assembled at the endoplasmic reticulum membrane as viral proteins and genomic rna are grouped together and then bud into the lumen of the endoplasmic reticulum. the virions are then exported via the secretory pathway of the endoplasmic reticulum into the golgi intermediate compartment and then into the extracellular environment. the m protein drives the packaging process by selecting and organizing the viral envelop components at the assembly sites and interacting with the nucleocapsid to allow budding [20] . several large serology studies suggest that cases of asymptomatic or mild mers-cov infection occur regularly, although infrequently. the importance of such cases is difficult to assess [10] . it is therefore difficult to determine whether these cases are due to or take part in human-to-human transmission. several studies suggest that less than 50% of infected patients transmit the virus to individuals they come into contact with, even at the beginning of an outbreak [10] . the disease therefore seems to spread due to frequent animal-to-human transmission, from camels to humans, with limited subsequent human-to-human transmissions [21] . there are unfortunately exceptions to this observation and local outbreaks caused by human-to-human transmission have been observed on a regular basis, mostly in hospitals. to date, the most poignant example is the outbreak that occurred in south korea in which the index case caused 185 secondary cases, among whom 30 were care providers, leading to 24 fatalities [3] . this outbreak was characterized by the key role of a few "super spreaders", delayed diagnosis, high doctor shopping behavior and the importance of confined spaces (waiting room, hospital room, ambulance). in this example, the resemblance with sars-cov's spreading mechanisms is striking, despite lower degrees of transmission to care providers for mers-cov [22] . these regular cases of imported-mers, the most recent was reported in england in august 2018 [23] , represent a real threat of local epidemics outside saudi arabia and special screening and isolation procedures need to be implemented in units likely to receive patients suspected of mers-cov infections. when possible, the first measure to be taken is to delay departure, in particular for individuals over 65 or with chronic disease, and for pregnant women or children. such measures are nevertheless challenging to maintain today as that the virus is still present 6 years after its apparition. all other preventive measures aim at preventing both animalto-human transmission and human-to-human transmission. it is therefore recommended to avoid any contact with domestic animals (firstly dromedary camels), their secretions, raw milk and insufficiently cooked meat. it is also advised to avoid eating fruit and vegetables that might have been in contact with animal secretions if not washed and peeled by oneself. to avoid human-to-human transmission, the usual recommendations for preventing the spread of any respiratory virus should be applied: hand washing with soapy water or an alcohol-based solution, covering one's nose and mouth when sneezing, refraining from shaking hands and touching one's mouth and nose with one's hands, avoiding contact with people with respiratory symptoms. finally, a last series of recommendations focus on how to behave in case of suspicious symptoms: (i) consult a doctor as soon as symptoms occur during travel and delay the return until symptoms disappear; (ii) if symptoms occur with 14 days of returning home, consult a doctor and tell him/her about the recent travel [24] . pcr-based detection methods are currently the preferred option for detecting the virus in respiratory samples and making a diagnosis of mers-cov infection. serology tests can also be performed and are often used for second-line diagnostic investigation in patients with a high suspicion of mers-cov but negative results by direct pcr testing. various respiratory matrices can be used: nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal), and even in some cases, induced sputum. the deepest samples, tracheal aspirates and bals, show the greatest sensitivity and significantly higher viral loads [25] . the genome amplification and detection methods used (pcr) were initially mostly developed in situ and performed in biosafety level 3 (bsl-3) reference facilities. the time to results is generally relatively long, 24-48 h, due to the usual time required for conventional pcr testing to which must be added the additional preparation and sample neutralization time needed to protect the laboratory staff against this virus. the pcr methods used are generally semi-quantitative and some studies suggest a correlation between the amount of virus detected and the severity of the symptoms [26] . nevertheless, no consensus has been reached yet regarding a threshold level that could actually predicts clinical severity. targeting the envelop gene upe is recommended with confirmatory testing for orf 1a or 1b or the n gene. if results diverge, sequencing is sometimes required to obtain conclusive results [27] . today, an increasing number of commercial tests are becoming available (altona diagnostics, fast track diagnostics, primerdesing ltd.) some even with a time to results of less than 1 hour (biofire-biomérieux). some of these tests are point-of-care, or can be performed in bsl3 facilities or a standard laboratory following sample neutralization in a bsl3 facility. these commercial tests must always be validated before use to check their sensitivity and compare their performance with reference methods. as with any other acute viral infection, antibodies can only generally be detected about 10 days after the onset of symptoms. in some patients, especially those with severe infections, the time interval to antibody detection may be even longer [28] . serological testing is therefore of little help for the initial diagnosis of symptomatic patients, but can be useful for epidemiological investigations. the highly immunogenic s and n viral proteins are widely used targets for serological tests and are found on all coronaviruses. various approaches have been developed: serum neutralization assays [29] , microarrays [30] , or more recently elisa confirmed by a microneutralization test [31] . all methods are technically complex and require a high level of expertise that restrict their use to a few highly specialized facilities. the first cases of infection with mers-cov were reported in 2012 [1] . hospital-acquired mers-cov infections have been described worldwide and represented a third of all cases reported in saudi arabia in the early stages of the epidemic [1, 32, 33] . clustered hospital-acquired infections were frequently observed during the first outbreaks and probably contributed to spreading the disease from the primary site of virus infection to the whole arabian peninsula, the most striking example of hospital-acquired outbreak being the korean outbreak in 2015 [34] . care providers are often affected and represent 15-22% of cases [33] [34] [35] [36] [37] [38] [39] . most of the cases are described in middle east countries, in particular saudi arabia (73%), with a predominance of male patients (66-69% in various studies) and a mean patient age ranging from 40 to 55 years [34, 38, 40] . comorbidities are found in 46-68.6% of patients, in particular diabetes and high blood pressure, followed by other heart conditions and finally obesity [34, 37, 38, 41] . the mean incubation time is 5 to 6.5 days. the generation interval (time between the onset of symptoms of the first case and those of the second case) is 7.6 days, which is identical to that of the respiratory syncytial virus (rsv) but threefold more than the influenza virus [36, [42] [43] [44] . the main challenge of mers-cov infection is the absence of specific clinical features for differential diagnosis with other viral respiratory diseases [37, 45] . this difficulty, combined with precautionary action taken to avoid potential secondary contamination with mers-cov [46] , can result in medical confusion and inappropriate patient management due to prolonged, difficult isolation that makes it impossible to perform the necessary complementary tests while waiting for pcr results [47] . the clinical features of mers-cov infection are extremely variable, ranging from an absence of symptoms (14-80% of cases) to a flu-like syndrome, pneumonia and acute respiratory distress syndrome (ards) [37, 48] . the three most frequent symptoms are: fever (77% [iqr: 59-82]), cough (90% [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] ), and dyspnea (68% ). many other secondary symptoms have been reported, such as sputum production (40%), odynophagia (39%), digestive system signs (20%), hemoptysis (4.3%), myalgia (43%) and headache (20%) [34, 37, 41, 42] . diarrhea is significantly more frequent in patients infected with mers-cov than in patients with another acute, febrile respiratory conditions [45] . severe mers is characterized predominantly by ards, acute kidney failure, and in the most severe cases, by multiple organ failure that can be fatal [49, 50] . one third of patients develop pneumonia and 20% develop ards [51] . the median time to respiratory failure is 12 days after the onset of symptoms. depending on studies, 53 to 89% of hospitalized patients are admitted to an intensive care unit (icu) [43, 52] . since the first mers outbreak, who had documented, in october 2018, 2260 cases of mers-cov infection confirmed by laboratory testing and 803 related deaths in 27 different countries. the retrospective fatality rate varies between outbreaks, ranging from 36.5 to 60% [33, 35, 37, 38, 42] . the mortality rate of 20.4% observed for the korean outbreak is probably the most reliable epidemiologically due to the comprehensive investigations carried out [34] . the death rate is highest among patients admitted to an icu, ranging from 58% to 90% [49, 53] . in the only cohort study performed in saudi arabia, the fatality rate for mers-cov patients was of only 10% (8/80). however, the patients of this cohort were younger, had less symptoms, showed less radiological features and only 17% were admitted to an icu [37] . the findings of the latter study diverge therefore with the situations observed in other hospitals, but are perhaps a better reflection of the infection profile in the general population in which younger subjects are less symptomatic and therefore less frequently admitted to hospital. the time interval between the onset of symptoms and death ranges from 11.5 to 27 days [34, 44, 54] . finally, co-infection with other respiratory viruses, in particular influenza, has been described although the impact of such combined infections have not been evaluated [44, 55] . co-infections with bacteria have also been reported in the patients developing the most severe disease [49, 51] . there are no specific laboratory findings related to mers-cov infection. nevertheless in patients with acute respiratory infection in mers-endemic areas, mers-cov infections have been associated with normal leukocyte and/or polymorphonuclear neutrophil counts but elevated transaminases [37, 45] . moreover, hyperleukocytosis, lymphocytopenia, thrombocytopenia, hypoalbuminemia, elevated serum creatinine, ldh and crp levels, and hypoxemia (pao2/fio2 < 300) have been repeated reported in mers-cov infected patients and are associated with severity and death [34, 45] . imaging (chest x-ray and sometimes chest ct) has revealed infection-related features in 51-75% of cases. the lesions observed are uni-or multi-focal ground glass opacifications, of subpleural and lower lobe predominance, with sometimes bilateral bi-basal involvement or features of organizing pneumonia [34, 37, 42, 45] . mortality is highest in elderly, male patients with comorbidities, especially diabetes [38, 45, 56] . patients from saudi arabia and the middle east have an increased mortality rate compared with patients from korea or other countries [38, 40] . in contrast, being a medical professional significantly reduces the risk of mortality [38, 45] . other factors associated with a higher mortality risk have been described in various studies: digestive symptoms, prolonged delay between the onset of symptoms and admission to hospital, smoking, low blood pressure, impaired gas exchange, leukopenia, anemia, disturbance of liver or kidney function, use of mechanical ventilation and prolonged stay in the icu [42, 57] . for the korean outbreak in 2015, the independent risk factors for mortality were: age > 55 years, dyspnea, diabetes, chronic lung disease, systolic blood pressure at admission < 90 mmhg, hyperleukocytosis at admission (> 10,000/mm 3 ) and the use of mechanical ventilation [34] . positive pcr results for mers-cov in blood at diagnosis are associated with an increased risk of requiring mechanical ventilation, extracorporeal membrane oxygenation (ecmo) or to lead to death [58, 59] . the lack or delayed detection of mers antibodies (elisa igg and iga, or prnt) in the blood or airways is a poor prognostic factor [54, 60] . it should however be noted that no seroconversion is observed in asymptomatic mers-infected patients [54] . finally, the mers-cov viral loads in distal lung samples were higher among deceased patients [60] . in a study including 45 patients in a tertiary referral hospital in south korea: • the predictive factors for pneumonia in mers-cov patients were: age > 45 years, body temperature > 37.5 • c on day 3, platelet counts < 150,000/mm 3 , lymphocytopenia (< 1000/mm 3 ), crp ≥ 20 mg/l and high viral loads (ct value < 28.5); • the predictive factors for respiratory failure were male sex, high blood pressure, thrombocytopenia, lymphocytopenia, hypoalbuminemia < 35 g/l and crp ≥ 40 mg/l. the patients with at least two, one and none of the predictive pneumonia factors developed pneumonia in 100%, 50% and 0% of cases, respectively [61] . several therapeutic options targeting various viral elements are currently available or under development (fig. 4) [62] . the different classes of available treatment are (i) immunotherapy with specific anti-mers-cov antibodies, (ii) molecules with antiviral activity, (iii) symptomatic treatment. few molecules have shown real curative action and the reports in the literature generally describe isolated cases or small series of cases. more studies have focused on associated treatment and supportive care. at this time, preventive therapies are still in preclinical stages. the efficacy and safety of plasma from convalescent patients have not been assessed. three separate reports concluded that such therapeutic approaches were inappropriate [63] . one trial is listed on www.clinicaltrials.gov. two cases of therapy with intravenous polyclonal iggs have been reported. in one of them, the iggs originated from donors in regions negative for mers specific antibodies. several monoclonal antibodies were tested and seemed to show anti-mers-cov activity in vitro [64] . no clinical trials are currently underway. recently, a phase i placebo-controlled, dose escalation study evaluated the efficacy of polyclonal iggs produced by transchromosomal cattle with human immunoglobulin genes immunized with the mers-cov spike (s) protein [65] . the primary outcome of tolerance to a single dose was reached. the secondary pharmacodynamic endpoint (serum neutralization activity) showed efficacy with a dose of 50 mg/kg. no phase ii trials are currently underway. a phase i study has been registered to assess the immunogenicity and tolerance of a combination of two monoclonal anti-mers-cov antibodies. the study has not yet started recruiting patients. infection with mers-cov reduces the host's interferon response. mers-cov is 100 times more sensitive to ifn-␣. treatment with ifn-␣ has been reported for many clinical cases and several retrospective cohort studies have been performed, in combination with ribavirin, lopinavir or mycophenolate mofetil (mmf). none of these studies have demonstrated increased overall survival. one study reported increased survival at d14 but not at d30 for critically ill intubated and ventilated patients [66] . a ifn/mmf combination trial is currently underway (see below). high doses of ribavirin have shown anti-mers-cov activity in vitro. ribavirin has been used to treat patients in saudi arabia as well as in france for the most severe cases managed in icus [67] . no significant effects were demonstrated either on the mortality rate or the time spent in the icu. ritonavir-boosted lopinavir has shown efficacy against mers-cov in vitro. as a result, the fda has extended the indications of lopinavir to patients infected with mers-cov. two case reports (in greece and korea) have described improvement in patients treated with lopinavir, type 1 interferon and ribavirin [68] . a phase ii-iii clinical trial is registered on clinicaltrials.gov. the aim of this study is to evaluate the feasibility, efficacy and safety of the combination lopinavir/ritonavir/recombinant ifn␤-1b vs. a placebo in patients with confirmed mers receiving optimal symptomatic care. chloroquine is among the molecules approved by the fda following in vitro studies. no clinical data or studies support its use in vivo at the present time. in vitro, anti-mers-cov activity has been demonstrated for doses of nitazoxamide that could be reached with two daily oral doses. no clinical data or studies support its use in vivo at the present time [69] . in vitro, anti-mers-cov activity has been demonstrated for doses of mmf that are acceptable for use in humans. mmf seems to show a synergistic effect with ifn-␤1b in vitro [70] . but in a non human primate common marmosets model, animals treated with mmf developed more severe lesions and showed a higher case fatality rate compared with untreated animals [70] . in contrast with animal model, the combination ifn-␤1b/mmf was administered to 8 patients in saudi arabia. all the patients survived but had lower apache ii scores that other patient groups [71] . alisporivir has been shown to provide additive in vitro anti-mers-cov activity when used in combination with ribavirin. no clinical data or studies support its use in vivo at the present time [72] . silvestrol is a molecule of the flavagline family found in plants. it binds to eif4a and enhances the affinity of eif4a for mrna. this blocks helicase activity and inhibits protein translation. a recent in vitro study demonstrated that silvestrol has anti-mers-cov activity [73] . no clinical data or studies support its use in vivo at the present time. corticosteroid therapy is currently the most widely studied therapeutic option. in a retrospective study, arabi et al. [67] compared the outcome of 309 patients with confirmed mers-cov infection managed in an icu setting and treated with (151) or without (159) corticosteroid therapy. the overall fatality rate was 67%. univariate analysis showed that mortality in the icu, during the hospital stay or at 90 days was higher in the corticosteroid group. then, following adjustment using a marginal structural model for causal inference, corticosteroid therapy was shown not to be associated with mortality, but delayed virus clearance. these findings, together with the absence of any description of the adverse effects caused by corticosteroid treatment, argue against the use of corticosteroids. a retrospective study was recently carried out in saudi arabia in mers-cov patients with refractory respiratory failure [74] . the patients were included in the study from 2014 to 2015 in five icus. the study consisted of two patient groups: ecmo versus conventional treatment. the primary endpoint was inhospital mortality. secondary endpoints included the length of stay in the icu and in hospital. thirty-five patients were included: 17 were treated with ecmo and 18 received conventional care. both groups had similar baseline characteristics. inhospital mortality was lower in the ecmo group (65 vs. 100%; p = 0.02) although they stayed longer in the icu (median stay of 25 days vs. 8 days; p < 0.01). the overall time in hospital was similar in both groups (median stay of 41 vs. 31 days; p = 0.421). in addition, patients in the ecmo group showed improved pao2/fio2 values at 7 and 14 days after admission into the icu (124 vs. 63, and 138 vs. 36, respectively; p < 0.05), and lower levels of vasoactive amines at d1 and d14 (29 vs. 80%, and 36 vs. 93%, respectively; p < 0.05). the results of this study support the use of ecmo as salvage treatment for mers patients with respiratory failure, as is the case for other respiratory infections. two trials with candidate vaccines are currently registered at https://clinicaltrials.gov/ct2/home. a phase-i clinical trial on healthy volunteers was set up to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls-5300) that expresses the s protein of mers-cov. this trial was planned to last one year and started in 2016. no results are available yet. a second phase-i trial was started by oxford university in january 2018. it uses a chimpanzee adenovirus vector containing the mers-cov s protein gene [75] . patient inclusion is currently underway. many other candidate vaccines using various different technologies are at a less advanced stage of development. the mers epidemic started in 2012. in contrast with sars-cov that disappeared 2 years after it first appeared, mers-cov continues to persist in the middle east 6 years later. although the disease has not become pandemic, outbreaks have occurred worldwide. today, it is impossible to predict with certainty whether mers-cov will disappear or continue to remain a threat for human populations. efficient vaccine development for host ani-mals and humans could play a key role in tilting the balance from potentially-pandemic to mers-cov elimination. furthermore, the epidemiological and viral determinants of the emergence of mers-cov in the middle east are difficult to comprehend, due to the high seropositivity rate of african dromedary camels but no similar disease in local human populations. the constant increase of transcontinental travel, in particular towards the main focal points of mers outbreaks with religious pilgrimages and mass tourism, raises the problem of the management of patients suspected of mers-cov infection and the absence of efficient treatment options to this date. the main problem in non-epidemic countries is to detect a mers-cov case among a great number of non-mers patients. in france, with the exception of the first 2 cases, no further cases have been detected. the current strategy is to isolate any suspicious cases as rapidly as possible to contain the infection and prevent local outbreaks as seen in south korea. the ability to rapidly test patients suspected to have mers-cov infection is the cornerstone of this strategy. the experience gained over the last few years by the health community will also help deal with any respiratory infections that will emerge in the future. the authors declare that they have no competing interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov). who mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) molecular evolution of human coronavirus genomes genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans severe respiratory illness caused by a novel coronavirus who | background and summary of novel coronavirus infection-as of 21 comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological stud human infection with mers coronavirus after exposure to infected camels, saudi arabia mers coronavirus neutralizing antibodies in camels antibodies against mers coronavirus in dromedary camels risk factors for mers coronavirus infection in dromedary camels in co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov accessory orfs play key role for infection and pathogenesis host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein a structural analysis of m protein in coronavirus assembly and morphology east respiratory syndrome coronavirus (mers-cov)-update. who transmission characteristics of mers and sars in the healthcare setting: a comparative study new case of mers-cov identified in the united kingdom middle east respiratory syndrome coronavirus (mers-cov): prevention in travelers respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome association of higher mers-cov virus load with severe disease and death, saudi arabia a roadmap for mers-cov research and product development: report from a world health organization consultation presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study specific serology for emerging human coronaviruses by protein microarray inclusion of mers-spike protein elisa in algorithm to determine serologic evidence of mers-cov infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from 2012 to 2015 clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea mers-cov outbreak in jeddah-a link to health care facilities mers outbreak in korea: hospital-to-hospital transmission predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia the predictors of 3-and 30-day mortality in 660 mers-cov patients middle east respiratory syndrome coronavirus infections in health care workers estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome a comparative study of clinical presentation and risk factors for adverse outcome in patients hospitalised with acute respiratory disease due to mers coronavirus or other causes rapid risk assessment: severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov), 22nd update clinical management of respiratory syndrome in patients hospitalized for suspected middle east respiratory syndrome coronavirus infection in the paris area from outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus infection: a short note on cases with renal failure problem state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia serologic responses of 42 mers-coronavirus-infected patients according to the disease severity the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea ifn-(2a or ifn-(1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients prevention and treatment of respiratory viral infections: presentations on antivirals, traditional therapies and host-directed interventions at the 5th isirv antiviral group conference feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study a review of treatment modalities for middle east respiratory syndrome corticosteroid therapy for critically ill patients with the middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-(1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model broad-spectrum antiviral activity of the eif4a inhibitor silvestrol against corona-and picornaviruses extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice the chapters on the origin, emergence, structure, transmission mechanisms, prevention and diagnostic methods were mainly written mb, nh, and bv.bv produced the figures. the chapters on clinical presentation, prognosis and available treatment options were mainly written by ab and cb.all authors read, amended and agreed with the entire final manuscript. key: cord-018016-r7tg0s45 authors: john, maya; shaiba, hadil title: shiny framework based visualization and analytics tool for middle east respiratory syndrome date: 2019-12-04 journal: advances in data science, cyber security and it applications doi: 10.1007/978-3-030-36365-9_16 sha: doc_id: 18016 cord_uid: r7tg0s45 people in the middle east have been affected by the middle east respiratory syndrome coronavirus (mers co-v) since 2012. new cases are continuously reported especially in the kingdom of saudi arabia, and the risk of exposure remains an issue. data visualization plays a vital role in effective analysis of the data. in this paper, we introduce an interactive visualization application for mers data collected from the control and command centre, ministry of health website of saudi arabia. the data corresponding to the period from january 1, 2019 to february 28, 2019 was used in the present work. the attributes considered include gender, age, date of reporting, city, region, camel contact, description and status of the patient. the visualization tool has been developed using shiny framework of r programming language. the application presents information in the form of interactive plots, maps and tables. the salient feature of the tool is that users can view and download data corresponding to the period of their choice. this tool can help decision makers in the detailed analysis of data and hence devise measures to prevent the spread of the disease. mers is a viral disease that was first discovered in 2012 when a patient in saudi arabia was diagnosed with critical respiratory distress and kidney trouble [1] . the infection can either be asymptomatic or show symptoms such as cough, fever etc. along with difficulty in breathing [2] . the disease has been brought under control in all middle eastern countries except saudi arabia where each month, new cases are still reported. studies have confirmed that this disease is zoonotic and camels are its significant reservoir [3, 4] . people in close contact with infected camels and health care professionals who care for infected patients have a high risk of acquiring the infection. many cases of community and household acquired infections have been reported from saudi arabia [5] . the ministry of health, saudi arabia has issued guidelines for infection prevention, control and management. to the best of our knowledge, there is a lack of interactive visualization tools for mers data visualization in saudi arabia. this work deals with developing an application where users can interactively view information about the infection in the form of plots, tables and maps. the data used in this study was obtained from the saudi ministry of health website and it includes the cases reported in saudi arabia from january 1, 2019 to february 28, 2019. by viewing the data visualizations, users can analyze mers cases better, find trends, monitor the disease and help authorities set detection and prevention guidelines. the mers data analyzed consists of 82 cases reported during the first two months of the year 2019. the information is present in the form of a pdf file corresponding to the cases reported each week. the cases reported contains details which include the date of reporting and patient information such as personal information, demographic information, camel contact details, description of infection and status of the disease. the latitude and longitude of the regions and cities are stored in separate .csv files. the details of the attributes present in the database are shown in table 1 . shiny is an r package used to create interactive web applications [6] . the shiny framework is a reactive programming model where browser refreshing is not required to instantiate the output changes when the user modifies the input. it can be used to build web applications without using javascript coding. shiny combines the computational power of r programming language with the highly interactive nature of the web. leaflet package is used to create interactive maps in r based on the javascript library leaflet [7] . these interactive maps can be rendered in r markdown, shiny apps, rstudioide and r console as it was developed in association with htmlwidgets. the leaflet( ) function is used to create the map widget. the different types of layers which can be included in the map widget are map tile, markers, lines, polygon, popups, raster images, color legends, layer groups, layer control etc. the maps created can be zoomed or downsized interactively. the googleviz package in r facilitates the use of google chart apis [8] . interactive charts can be incorporated into web pages using google charts. the data stored in r data frames can be visualized in the form of google charts without uploading the information to google. an internet connection is required to view the output rendered by this package. the mers data visualization tool was developed using r programming language. the tool consists of three sections (tabs) namely "different cases analysis", "miscellaneous analysis" and "summary" section. the users can choose the period for which they would like to visualize the mers data. in the case of different cases analysis, the user can view the information as pie charts and maps, or tables. the users can view details about either all the cases together or recovered cases, death cases or hospitalized cases separately. the details regarding the cases can be viewed with respect to all cities, all regions or cities within a region. in the case of the option "cities within a region", the user has to choose a region from the drop down list provided. unlike conventional pie charts, the pie charts created using googleviz package are interactive in nature. by pointing the cursor over a portion in the pie chart, one can understand the actual number of cases in that particular part of the chart. in the map, places are marked based on longitude and latitude of that place. on clicking the marker displayed in the map, the name and number of cases in that place will be displayed as a pop-up. based on the number of cases, markers are assigned colors such as red, orange or yellow. the colors red, orange and yellow represent "large number of cases", "moderate number of cases" and "few number of cases" respectively. the information in figs. 1 and 2 is presented in the form of pie chart and map. the screen shot of the page corresponding to different cases analysis is shown in fig. 1 . the figure depicts the analysis of all mers cases reported for the first two months of the year 2019 with regard to cities. it can be inferred from fig. 1 that during that period 76.8% of the patients recovered from the disease and the maximum number of cases (61%) were reported from the city of wadi aldawasir. the analysis based on all cases reported in "all cities within riyadh region" during january to february 2019 is shown in fig. 2 . it can be observed from the figure that majority of cases in riyadh region were reported from the city of wadi aldawasir. nearly 78% of the infected people in riyadh region recovered from the disease. the application page with table output is shown in fig. 3 . it can be observed from the table that 12 people died due to mers in riyadh region during the first two months of the year 2019. the table which displays the information is interactive in nature. when the user clicks a column name in the table, the records in the table are sorted based on values in the clicked column and displayed accordingly. the table also has provision for searching values and selecting the number application page corresponding to "different cases analysis" tab for cities within a region of records to be displayed in a page. the users can also download the details contained in the table. the different cases analysis will help decision makers in gathering information regarding the cases reported in different places. this type of analysis is essential to identify disease prone areas and hence take measures to curtail the spread of infection. the miscellaneous analysis consists of analysis based on age, camel contact and months. the users can analyze the data for all cases, death cases or recovered cases. depending on the user's choice, the analysis corresponding to all cities, all regions or cities within a region are displayed. analysis based on age. the infected people are divided into three age categories namely below 25 years, 26-60 years and greater than 60 years. in the case of age based analysis, the information is displayed both in the form of pie chart and stacked bar chart. the pie chart represents the number of cases corresponding to each age group. the stacked bar chart depicts the number of cases in each age group corresponding to the location type selected. the screen shot corresponding to age category wise analysis is shown in fig. 4 . the figure corresponds to the analysis corresponding to all cases reported with respect to cities for the first two months of the year 2019. it is evident from the pie chart fig. 4 that the percentage of cases reported is 4.9%, 65.9% and 29.3% for the age group below 25 years, 26-60 years and above 60 years respectively. the age category wise helps in understanding the number of cases reported among people of different age groups. this type of analysis will help in identifying which group has more mortality rate and health authorities may conduct extensive analysis of causes leading to death. analysis based on camel contact. camels have been identified as a carrier of the disease, and hence it is essential to perform analysis of patient cases involving camel contact. depending on the choice of the user, the information is displayed for "all cases", "death cases" or "recovery cases". the analysis is carried out for "all cities", "all regions" or "cities within a region". the details are displayed as pie chart, map and table. the pie chart represents the numbers of cases corresponding to the location type selected by the user. the places where camel contact cases are reported are marked in the map. this can help in analyzing whether the cases pertaining to people with camel contact are clustered in a region or not. the table displays the details of infected people who had contact with camels. the data visualization based on camel contact is shown in fig. 5 . the figure depicts the analysis corresponding to all cases with regard to cities in saudi arabia. it can be observed from fig. 5 that 56% of the camel contact based cases were reported from the city of wadi aldawasir. camel contact based analysis of mers patients is highly essential to identify places where people in contact with camels have been infected with the disease. this will help health authorities in taking more measures to spread awareness about the precautionary methods to be taken when handling camels. the infected camels in such regions may be identified and isolated to prevent the spread of the disease. month-wise comparison of the data can be carried out for "all cases", "death cases" and "recovery cases". the stacked bar chart for cases reported in different months is plotted based on the location type specified by the user. when the cursor is moved to a region in the bar chart, the number of cases in the particular month will be displayed corresponding to the location. the screenshot corresponding to month-wise comparison is shown in fig. 6 . the figure portrays the analysis corresponding to death cases reported in various regions. the maximum number of death cases were reported in riyadh region during february. monthwise analysis is useful in identifying whether the infection is related to climate. summary based visualization gives a graphical summary of the count of the "status of the patients" for different attributes. the information is depicted in the form of stacked bar charts where the charts are plotted based on the frequency of status of patients corresponding to different values of the attributes. this visualization will help the users in getting an overall idea regarding the distribution of data with regard to the status of the patients. figures 7 and 8 depict the screenshots of the visual summary of data for the first two months of the year 2019. figure 7 confirms the earlier findings that riyadh region and specifically wadi aldawasir city has the highest number of mers cases and death cases. figure 8 shows that most mers patients are male and that people below 25 years are less likely to get infected by mers. elderly patients are more prone to die of mers. the majority of patients were not in contact with camels and death rate was low in the case of patients in contact with camels. many patients acquired the infection from healthcare facilities and high death rate is reported among this group. the users can view information corresponding to their period of interest. the interactive feature of the pie chart prevents the chart from being cluttered with description. the use of maps to represent the information will give an idea regarding the spatial distribution of mers cases. these maps will help the health authorities to identify the areas with large number of cases and hence alert the hospitals and the general public regarding that. the interactive nature of the table helps the users in analyzing the data as per their requirement. a salient feature of the application is that the users can easily download details corresponding to the period of their choice. analyzing the data based on camel contact will aid health authorities to identify the areas where many such cases are present and hence intensify the awareness programs to reduce the rate of infection. in this paper, we have created an interactive visualization tool for mers co-v infection cases based on details of cases reported in saudi arabia. the attributes used include the date of reporting, city, region, age, gender, description of the disease, camel contact and status of the patient. the user can view details regarding all cases, recovered cases, death cases or hospitalized cases for all the cities, all regions or cities within a region. our tool provides the flexibility to view information in the form of charts and maps, or downloadable tables. the analysis is also carried out based on age group, camel contact and month-wise cases. by viewing the maps, users can easily differentiate between places based on the number of cases. moreover, the users can view a visual summary of the number of cases for different values of attributes based on the status of the patients. understanding and analyzing the disease related information can help decision makers in setting guidelines for preventing and controlling the spread of disease. location-based analysis of the infection is highly essential in formulating region specific awareness programs to reduce the rate of infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia main factors influencing recovery in mers co-v patients using machine learning mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission key: cord-265128-i0d4lxko authors: gurung, arun bahadur; ali, mohammad ajmal; lee, joongku; farah, mohammad abul; al-anazi, khalid mashay title: unravelling lead antiviral phytochemicals for the inhibition of sars-cov-2 m(pro) enzyme through in silico approach date: 2020-05-22 journal: life sci doi: 10.1016/j.lfs.2020.117831 sha: doc_id: 265128 cord_uid: i0d4lxko a new sars coronavirus (sars-cov-2) belonging to the genus betacoronavirus has caused a pandemic known as covid-19. among coronaviruses, the main protease (m(pro)) is an essential drug target which, along with papain-like proteases catalyzes the processing of polyproteins translated from viral rna and recognizes specific cleavage sites. there are no human proteases with similar cleavage specificity and therefore, inhibitors are highly likely to be nontoxic. therefore, targeting the sars-cov-2 m(pro) enzyme with small molecules can block viral replication. the present study is aimed at the identification of promising lead molecules for sars-cov-2 m(pro) enzyme through virtual screening of antiviral compounds from plants. the binding affinity of selected small drug-like molecules to sars-cov-2 m(pro), sars-cov m(pro) and mers-cov m(pro) were studied using molecular docking. bonducellpin d was identified as the best lead molecule which shows higher binding affinity (−9.28 kcal/mol) as compared to the control (−8.24 kcal/mol). the molecular binding was stabilized through four hydrogen bonds with glu166 and thr190 as well as hydrophobic interactions via eight residues. the sars-cov-2 m(pro) shows identities of 96.08% and 50.65% to that of sars-cov m(pro) and mers-cov m(pro) respectively at the sequence level. at the structural level, the root mean square deviation (rmsd) between sars-cov-2 m(pro) and sars-cov m(pro) was found to be 0.517 å and 0.817 å between sars-cov-2 m(pro) and mers-cov m(pro). bonducellpin d exhibited broad-spectrum inhibition potential against sars-cov m(pro) and mers-cov m(pro) and therefore is a promising drug candidate, which needs further validations through in vitro and in vivo studies. j o u r n a l p r e -p r o o f coronaviruses (covs) are positive-sense rna enveloped viruses which derive their name from the crown-like spikes on their surface and they belong to coronaviridae family. they are classified into four main subgroups-alpha, beta, gamma, and delta depending on their genomic structure [1] . alpha-and beta coronaviruses cause respiratory infections in humans and gastroenteritis in other mammals [2, 3] . likewise, the middle east respiratory syndrome coronavirus (mers-cov) caused a disastrous pandemic in 2012 leading to 37% mortality [1] . all coronaviruses infecting humans usually known to have intermediate hosts such as bats or rodents [4] . previous outbreaks of sars-cov and mers-cov involved civet cats and dromedary camels for their direct transmission to humans [1] . a new coronavirus caused an outbreak of the pulmonary disease in wuhan (the capital of hubei province in china) in december 2019 and has since spread across different parts of the world [5, 6] . since its rna genome shows about 82% identity to that of the sars coronavirus (sars-cov), the new virus has been termed as sars-cov-2 [7] . however, both these viruses belong to the same clade of the genus betacoronavirus [5, 6] . the sars-cov-2 caused a disease known as covid-19. at the initial outbreak, cases were linked to the huanan seafood and animal market in wuhan but active human-to-human transmission caused exponential growth in the number of reported cases. the world health organization (who) confirmed the outbreak a pandemic on march 11, 2020. there have been more than 170,000 cumulative cases worldwide accounting for approximately 3.7% case-fatality rate as of march 15, 2020 [8] . due to the close similarity to sars-cov, the biochemical interactions and the pathogenesis of sars-cov-2 are highly likely to be similar [1] . the virus entry into the host cell is mainly mediated through the binding of the sars spike (s) protein to the angiotensinconverting enzyme 2 (ace-2) receptor on the cell surface [9] . among coronaviruses, the main protease (m pro , also called 3cl pro ) has emerged as the best-described drug target [10] . the j o u r n a l p r e -p r o o f 4 polyproteins that are translated from the viral rna are processed by this enzyme together with the papain-like protease(s) [11] . the m pro recognizes and acts remarkably on eleven cleavage sites typically leu-gln↓(ser,ala,gly) on the large polyprotein 1ab (replicase 1ab) of approximately 790 kda. blocking the activity of this enzyme would help in inhibiting viral replication. there are no reported human proteases with a similar cleavage specificity and therefore, inhibitors against this enzyme are less probable to be toxic [8] . the three dimensional x-ray crystal structure of this enzyme in complex with α-ketoamide inhibitor 13b (o6k) was recently solved by zhang et al. (2020) (pdb id: 6y2f) which offers an opportunity for structure-based drug design against the enzyme target. understanding the relevance of the steady rise in the number of infected and death cases in recent time from covid-19 and lack of effective therapeutic interventions such as drugs and vaccines, computer-aided drug design is an important strategy to be sought after. this rational based drug design will reduce the cost and time incurred in the drug discovery process. structure-based drug design primarily relies on molecular docking to identify lead molecules against the target proteins from chemical libraries [12, 13] . compared to the synthetic inhibitors plant based-drugs have less toxicity and much safer to use. the natural products such as traditional medicines and plant-derived compounds (phytochemicals) are the rich sources of promising antiviral drugs [14] . around 44% of the approved antiviral drugs between 1981 and 2006 were derived from natural products [15] . the plant extracts have been extensively used and screened for drug molecules to evaluate theirs in vitro antiviral activities. few examples of medicinal plants with proven antiviral activities include phyllanthus amarus schum. and thonn which blocks human immunodeficiency virus (hiv) replication both in vitro and in vivo [16] ; azadirachta indica juss. (neem) shows in vitro and in vivo inhibition properties against dengue virus type-2 (denv-2) [17] ; geranium sanguineum l. significantly inhibits the replication of herpes simplex virus type-1 and 2 (hsv-1 and hsv-2) in vitro [18] ; acacia nilotica l. possesses activity against hepatitis c virus (hcv) in vitro etc [19] . in the present study, we have screened small drug-like molecules from a dataset of phytochemicals possessing antiviral activities using drug-like filters and toxicity studies. the the information about a set of thirty-eight phytochemicals from medicinal plants with antiviral activities was retrieved through literature search [14] . the summary of the selected phytochemicals (class, plant source and antiviral activity) is provided in suppl. table 1 the phytochemicals were screened based on physicochemical properties obeying lipinski's rule of five (rof) filters [23] and further tested for in silico toxicity studies such as mutagenicity, tumorigenicity, reproductive effects and irritancy. the physicochemical properties of the phytochemicals were determined using datawarrior program version 4.6.1 (sander et al., 2015) . table 1 . the sequence percentage identity of the sars-cov-2 m pro to sars-cov m pro and mers-cov m pro was determined using a standard protein basic local alignment search (blastp) tool (https://blast.ncbi.nlm.nih.gov/blast.cgi?page=proteins). multiple sequence alignment of the three sequences were performed using clustal w algorithm [26] . pairwise structural clustering of sars-cov-2 m pro , sars-cov m pro and mers-cov m pro was analyzed using ucsf chimera tool [27] . to check the suitability of molecular docking parameters and algorithm to reproduce the native binding poses, a redocking experiment was performed using the co-crystal compound. where ∆g is the binding energy in kcal/mol, r is the universal gas constant (1.987 calk −1 mol −1 ) and t is the temperature (298.15 k) a stable complex is formed between a protein and ligand which exhibits more negative free energy of binding and low k i indicates high potency of an inhibitor [29, 30] . the hydrogen bonds and hydrophobic interactions between the compounds and the target enzymes were studied using a total of 38 bioactive phytochemicals possessing antiviral activities were selected for the study. these compounds were chosen based on the previous reports of their potent antiviral effects against various pathogenic viruses such as adenovirus, influenza virus, respiratory syncytial virus, human cytomegalovirus, herpes simplex virus, poliovirus, varicella-zoster virus etc. (suppl. table 1 ). the compound set consists of different classes of phytochemicals including active flavonoids (n=14), active organic acids (n=5), active alkaloids (n=5), active essential oils (n=3), active stilbenes (n=6) and other phytoconstituents (n=5). the three-dimensional structures the compounds were modelled and optimized. a list of these phytochemicals is enumerated in table 2 . these optimized structures were used further for virtual screening and molecular docking studies. from a total of 38 phytochemicals, 10 compounds (four active flavonoids, two active alkaloids, two active essential oils and two other phytoconstituents) were found to be orally bioactive with j o u r n a l p r e -p r o o f 8 respect to rof criterion (molecular weight (mw) ≤500, clogp (partition coefficient between noctanol and water) ≤5, number of hydrogen bond donors (hbd) ≤5 and number of hydrogen bond acceptors (hba) ≤10 [23] ) without any significant toxicity issues such as being nonmutagenic, non-tumourigenic, non-irritant and no adverse effects on reproductive health (table 3 ). these drug-like compounds were further taken for molecular docking studies. the drugattrition rate in preclinical and clinical trials is quite high due to the poor pharmacokinetic studies and therefore initial screening of these drug-like molecules can increase the chances of passing through the clinics. his163, his164, met165, pro168, asp187, gln189, thr190 and gln192) ( figure 3d ). interestingly, the residues his41 and cys145 which form catalytic dyad residues are also found interacting with the inhibitor. thus all the three lead molecules have better binding affinity to sars-cov-2 m pro compared to the standard inhibitor. (n=17) ( figure 4d ). it also shows good binding to mers-cov m pro which involves seven hydrogen bonds with cys145, ser147, cys148, gln167 and glu169 and hydrophobic interactions via residues met25, thr26, leu27, his41, phe143, leu144, gly146, his166, met168, leu170, ala171, gln192, val193, his194 and gln195 (n=15) ( figure 5d ). the binding energies and inhibition constants of the phytochemicals with the sars-cov-2 m pro enzyme were compared with that of a set of twelve fda approved antiviral drugs-a) viral sars-cov-2 and coronavirus disease 2019: what we know so far, pathogens origin and evolution of pathogenic coronaviruses fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin bat coronaviruses in china others, a pneumonia outbreak associated with a new coronavirus of probable bat origin others, a new coronavirus associated with human respiratory disease in china severe acute respiratory syndrome-related coronavirus--the species and its viruses, a statement of the coronavirus study group crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus--induced lung injury coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design molecular docking: a powerful approach for structure-based drug discovery molecular docking in modern drug discovery: principles and recent applications antiviral properties of phytochemicals natural products as sources of new drugs over the last 25 years concerted inhibitory activities of phyllanthus amarus on hiv replication in vitro and ex vivo inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type-2 replication antiherpes virus activity of extracts from the medicinal plant geranium sanguineum l antiviral activity of acacia nilotica against hepatitis c virus in liver infected cells merck molecular force field. i. basis, form, scope, parameterization, and performance of mmff94 exploring the physicochemical profile and the binding patterns of selected novel anticancer himalayan plant derived active compounds with macromolecular targets pubchem substance and compound databases lead-and drug-like compounds: the rule-of-five revolution datawarrior: an open-source program for chemistry aware data visualization and analysis molecular docking of the anticancer bioactive compound proceraside with macromolecules involved in the cell cycle and dna replication clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice ucsf chimera--a visualization system for exploratory research and analysis autodock4 and autodocktools4: automated docking with selective receptor flexibility insights into protein-ligand interactions: mechanisms, models, and methods molecular docking studies of lonchocarpus cyanescens triterpenoids as inhibitors for malaria ligplot+: multiple ligand-protein interaction diagrams for drug discovery structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design in silico study of fucoxanthin as a tumor cytotoxic agent molecular structures and antiviral activities of naturally occurring and modified cassane furanoditerpenoids and friedelane triterpenoids from caesalpinia minax antibacterial, antifungal, and antiviral activities of some flavonoids list of abbreviations ace-2 :angiotensin converting enzyme 2 blast: basic local alignment search clogp: partition coefficient between n-octanol and water hba: hydrogen bond acceptor hbd: hydrogen bond donor mers-cov: middle east respiratory syndrome coronavirus mpro: main protease mw: molecular weight pdb: protein data bank rmsd: root mean square deviation rof: rule of five sars-cov: severe acute respiratory syndrome coronavirus ligplot analysis for top three lead compounds along with the control against sars 83 å) 91 å) key: cord-280029-g1k3zlax authors: gabutti, giovanni; d’anchera, erica; sandri, federica; savio, marta; stefanati, armando title: coronavirus: update related to the current outbreak of covid-19 date: 2020-04-08 journal: infect dis ther doi: 10.1007/s40121-020-00295-5 sha: doc_id: 280029 cord_uid: g1k3zlax in december 2019, some cases of viral pneumonia were epidemiologically related to a new coronavirus in the province of hubei, china. subsequently, there has been an increase in infections attributable to this virus throughout china and worldwide. the world health organization (who) has officially named the infection coronavirus disease 2019 (covid-19), and the virus has been classified as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this appears to be a virus from rhinolophus bats, but the intermediate host has not yet been identified. the mechanism of infection of sars-cov-2 is not yet known; it appears to have affinity for cells located in the lower airways, where it replicates. the interhuman transmission of coronaviruses mainly occurs through saliva droplets and direct and indirect contact via surfaces. as of march 10, 2020, the number of cases worldwide was 113,702. along with severe acute respiratory syndrome (sars) and middle eastern respiratory syndrome (mers), covid-19 appears to cause a severe clinical picture in humans, ranging from mild malaise to death by sepsis/acute respiratory distress syndrome. the prognosis is worse in elderly patients with comorbidities. to date, there is no specific therapy for covid-19. prevention of sars-cov-2 infection implies strategies that limit the spread of the virus. who and other international and national bodies have developed continuously updated strategic objectives and provisions to contain the spread of the virus and infection. background coronaviruses (covs) are a large family of respiratory viruses that can cause mild to moderate diseases, ranging from the common cold to severe respiratory syndromes [1] . these viruses are common in many animal species, and, in some cases, albeit rarely, they can evolve and infect humans and then spread to the population [2] . most of the numerous human pathogenic coronaviruses are associated with mild clinical symptoms, with two notable exceptions: severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). sars is caused by a virus that emerged in southern china in november 2002 and led to [ 8000 human infections and 774 deaths in 37 countries in the 2002-2003 period [3] ; mers is related to a virus detected for the first time in saudi arabia in 2012, responsible for 2494 laboratoryconfirmed cases of infection and 858 deaths since september 2012 [4] . in december 2019, some cases of viral pneumonia were epidemiologically linked to a new coronavirus in the province of hubei in china. in the following days a possible association with the huanan fish market in wuhan was identified. in fact, most of the initially identified patients had visited, worked at or lived near this market in the month preceding the infection. in early january, a new coronavirus, tentatively called 2019-ncov, was isolated, and interhuman transmission was confirmed [5] . on february 11, 2020 , the world health organization (who) officially named the new coronavirus 2019 infection coronavirus disease 2019 (covid-19) [6] . subsequently the coronavirus study group (csg) of the international taxonomy committee of viruses (ictv) officially classified the virus as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [7] . this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. coronaviruses (covs) are rna viruses belonging to the subfamily of the coronavirinae, coronaviridae family, order nidovirales [8] . their definition is related to the fact that covs are virions with projections resembling a crown. all covs share similarities in organization and genomic expression with 16 nonstructural proteins and at least 4 structural proteins (spike: s; envelope: e; membrane: m; nucleocapsid: n proteins) [9] . they are divided into four genera: alphacov, betacov, gammacov and deltacov [10] . covs can infect different hosts and have a different tissue tropism: generally, alphacovs and betacovs infect mammals while gam-macovs and deltacovs infect other animals such as birds, fishes and only a few mammals [8] ( table 1) . seven human covs are known to date, common worldwide. the first ones were identified in the mid-1960s, others more recently [9] . the human covs are: 229e (alfacov), nl63 (alfacov), oc43 (betacov), hku1 (betacov), mers-cov (betacov, which causes mers), sars-cov (betacov, which causes sars) and 2019-ncov/sars-cov-2 [11] . in the past 20 years, two major human cov epidemics have occurred: sars and mers. both viruses infect the lower airways and cause severe respiratory syndromes in humans. other covs have a global distribution with a seasonality characterized by a peak in winter and in spring and few cases in summer [10] . the sars epidemic began in 2002, starting from the town of foshan in the chinese province of guangdong, and then spread globally, involving 33 countries in 5 different continents. more than 8000 cases were recognized (21% in health workers) with a case fatality rate (cfr) of almost 10% [3] and a basic reproduction number (r 0 ) equal to 2-3 [12] . in humans, the incubation period ranges between 2 and 10 days, and interhuman transmission is very effective, which is why the delay of 3 months between the beginning of the epidemic and the first investigations has allowed a worldwide spread of the virus. the bat was identified as the natural reservoir of sars-cov and the civet as the intermediate host [13] . despite active surveillance, no more sars cases have been recognized since july 2003 [3] . mers was first identified in 2012. from then until january 16, 2020, 2521 cases were reported worldwide. these include 919 deaths, with a cfr of 36% [4] and an estimated r 0 of 0.7 [12] . most cases were found in the arabian peninsula and approximately 84% in saudi arabia [4] . however, the virus was isolated in 27 countries [10] , and all patients outside the arabian peninsula had a travel history to or a contact with someone who had traveled to this region [4] . in humans, the incubation period ranges between 2 and 14 days, and, unlike sars, interhuman transmission is limited. the virus is not easily transmitted from person to person, unless a close contact occurs. the bat was also identified as an animal reservoir for the mers-cov, while the intermediate host was the camel [13] . both sars and mers have a broad spectrum of clinical pictures ranging from flu-like symptoms to acute respiratory distress syndrome (ards). age and comorbidities are prognostic predictors. sars mainly involved healthy young adults while half of mers cases were identified in subjects [ 50 years of age. the clinical picture at hospital admission is characterized by fever, cough, dyspnea and myalgia in both sars and mers. atypical symptoms such as diarrhea and vomiting are common in both syndromes [10] . epidemiology on december 31, 2019, the who chinese office was informed about some cases of pneumonia of unknown origin in the city of wuhan, located in the chinese province of hubei [14] . on january 3, 2020, a new coronavirus was isolated from the bronchial wash samples of a patient from wuhan and was identified as the pathogen responsible for pneumonia [15] . on january 11 and 12, 2020, the who received further details and information from the chinese national health commission regarding the possible association of this epidemic with exposure in a fish market in wuhan, and the chinese authorities shared the genetic sequence of a new coronavirus, subsequently identified as sars-cov-2 [14] . new evidence shows the relation between sars-cov-2 and other covs that are circulating in bats, specifically in rhinolophus bats [16] . these subspecies are abundant in southern china and asia, the middle east, africa and europe. the mode of transmission to humans is still unclear. bats are rare in chinese markets; however, they are captured and sold directly to restaurants as food. the most recent hypothesis is that there is an intermediate animal host that has played a role in the new sars-cov-2 infection [6] . several animals have been considered as possible hosts, including pangolins [17] . identifying the animal source of this new virus would help to prevent other new infections and to understand the initial dynamics of spreading in the wuhan market. in this regard, strengthening the control of food and hygiene within live animal markets is essential to prevent future new zoonosis [6] . the mechanism of sars-cov-2 infection is not yet known. the key to human transmission is the ability of the virus to bind to human cells: covs use the spike protein to bind to cells, and it seems that sars-cov-2, as sars-co virus, uses the same receptor for the conversion enzyme of angiotensin 2 (ace2) [5, 13] . a recent study showed that the binding affinity between the viral receptorbinding domain (rbd) and the host receptor ace2, in the initial viral attachment step, determines the host susceptibility to sars-cov-2. the greater transmissibility of sars-cov-2 than sars-cov is partly explained by the fact that, due to a higher affinity, the binding capacity of viral rbd increases and the number of viruses required to infect a cell is reduced [18] . sars-cov-2 appears to have affinity for cells located in the lower airways, where it replicates, causing radiologic evidence of pneumonia in patients without any particular clinical symptoms [19] . usually, the interhuman transmission of covs occurs through different pathways that include droplets, direct contact and indirect contact through surfaces [13] . the virus has also been isolated in serum, blood, rectal swabs, saliva, urine and stool [18] . fecal-oral transmission has not yet been proven [20, 21] . a recent study indicates that the highest viral load is found immediately after the onset of symptoms and in greater quantities in the nose rather than in the throat. this analysis suggests that the spread of the sars-cov-2 virus resembles that of influenza more than that of sars. the viral load found in asymptomatic subjects was similar to that of symptomatic ones; this suggests the potential transmission role of asymptomatic subjects [22] . despite several uncertainties, the evidence indicates that the cfr for hospitalized covid-19 patients is substantially lower than that for hospitalized sars, mers and 2009 pandemic flu h1n1 patients (4%, 28%, 65% and 9%, respectively). taking into account covid-19 and influenza (pandemic and seasonal), it is important to carefully consider the differences in definition of cases, as these are relevant for cfr estimation [18] . the r 0 has been estimated between 2 and 3 [18] , and the risk assessment calculated by who is very high for the whole world [23] . as of march 10, 2020, there were 113,702 confirmed cases of sars-cov-2 infection in the world, with 4125 new cases in the last 24 h. in china, the country with the highest number of confirmed cases, 80,924 occurred, of which 20 were within 24 h with 3140 total deaths (17 new in the last 24 h) [23] . the first case of sars-cov-2 laboratory-confirmed infection outside china was reported on january 13, 2020, in thailand [14] . as of march 10, 2020, outside china there were 32,778 confirmed cases and 872 deaths, and 109 countries were involved [23] . in the western pacific region, the countries with the highest number of cases were republic of korea (7513), japan (514) and singapore (160) [23] . in the european region, italy counted 9172 cases; thus it was the second country with the most cases after china. france, germany and spain reported 1402, 1139 and 1024 cases, respectively, while another 44 countries reported \ 400 cases [23] . in the eastern mediterranean region, iran was the country with the highest number of cases (7161) [23] . in the usa, 472 cases were reported [23] . of note, 696 cases, including 7 deaths, were reported in subjects on a cruise ship that had been anchored in japanese territorial waters [23] . several clinical pictures have been associated with sars-cov-2 infection; they range from mild malaise to death, which occurs from sepsis and/or ards. early recognition of the clinical picture is essential to promptly start the adequate preventive measures and supportive treatments to avoid spreading of the virus and possible complications in patients [19] (table 2) . it seems that covid-19 presents with three worsening clinical pictures that evolve in few days: at the onset a slight malaise with symptoms of the upper respiratory tract, subsequently a mild pneumonia that can later worsen with a picture of ards [19] . several retrospective studies show that hospitalization generally takes place on the 7th day after the onset of the symptoms (fever, fatigue and dry cough with dyspnea) [1, 15] . other possible symptoms are myalgia, headache, anorexia, pharyngodynia and gastrointestinal complaints with diarrhea. many patients developed multiorgan dysfunction, radiologic signs of bilateral pneumonia with ards and acute renal failure; furthermore, mental confusion may occur [15] . the incubation period lasts up to 14 days (4-5 days as median incubation period) [12, 18] . recently, the chinese center for disease control and prevention (ccdc) has published a work on a series of cases, currently the most numerous in the literature, concerning mainland china (updated on february 11, 2020). healthcare workers represent 3.8% of cases; 14.8% of these have been confirmed as serious or critical cases [24, 25] . as for the distribution by age, 1%, 8% and 87% of cases were registered in young subjects (\ 10 years and between 10 and 19 years of age), young adults (aged between 20 and 29 years) and adults and elderly people (age range 30--79 years), respectively. elderly patients aged c 80 years represented 3% of cases. regarding the spectrum of the disease, 81%, 14% and 5% of cases were affected by a medium intensity, severe and critical form, respectively [24, 25] (table 3) . according to these data, the overall cfr rate is 2.3%. in detail, cfr was equal to 8% and 14.8% in subjects aged between 70 and 79 years and in patients aged c 80 years, respectively. no subject \ 9 years of age died. no deaths have been registered in subjects suffering from mild or medium intensity forms, while cfr was equal to 49% in critical cases [24, 25] (table 3) . notably, cfr was high in subjects with comorbidities; in detail, cfr was equal to 10.5%, 7.3%, 6.3%, 6.0% and 5.6% for cardiovascular diseases, diabetes, chronic respiratory diseases, hypertension and tumors, respectively [24, 25] (table 3 ). in conclusion people [ 60 years of age and patients with comorbidities (most of all cardiovascular diseases) have a greater risk for a more severe clinical picture and for fatal outcome. furthermore, deaths occurred only in critical cases. fortunately, covid-19 seems to be relatively rare in children, whose clinical picture is often mild [18] . according to another chinese study, the median age of patients is 56 years, with a slightly higher percentage in males (54.3% males vs. 45.7% females). intensive care unit (icu) admission was needed in 26% of subjects, and 4.3% of them died of multiorgan failure. once more, the worst clinical pictures refer to elderly subjects with comorbidities such as diabetes, high blood pressure and cardiovascular diseases [15] . inter-human transmission was considered for 41% of patients: the infection seems related to previous exposure to the huanan fish market in 8.7% of cases, family members in 12.3% and healthcare workers in 29% [15] . according to another study, some cases have also been reported in children aged 1--11 months: the clinical picture was less severe than in adults, with fever and mild respiratory infections as prevalent symptoms. the transmission was mainly intra-familiar or through contacts with visitors to the huanan market [26] . a recent study analyzed nine pregnant women with sars-cov-2 pneumonia. their clinical picture was similar to that of nonpregnant women affected by the virus. in all cases, a cesarean delivery was done, and no vertical transmission has been documented. therefore, it can be assumed that there is no evidence of vertical transmission during the third trimester of pregnancy [27] . the diagnosis and confirmation of sars-cov-2 infection are carried out by specific tests recommended by who that are described on a dedicated webpage [28] . the european centre for disease prevention and control (ecdc) also provided a specific webpage on laboratory support by a coronavirus-specialized laboratory in the european union [29] . any person satisfying the criteria of a suspected case ( table 2) should be tested for sars-cov-2, and, when possible, samples from both the lower respiratory tract (bronchoalveolar lavage, endotracheal aspirate, expectorated sputum) and upper respiratory tract (nasopharyngeal swab, oropharyngeal swab, nasopharyngeal aspirate or nasal wash) should be collected [29] . according to a recent chinese study, collecting specimens from different sites could be useful to improve the sensitivity and reduce false-negative test results. this study highlights that bronchoalveolar lavage fluid specimens showed the highest positive rates, followed by sputum, nasal swabs, bronchial biopsy, pharyngeal swabs, feces and blood [30] . to date, there is no specific therapy for covid-19. patients with covid-19 should receive supportive care to help relieve symptoms, and, for severe cases, treatment should include care to support vital organ functions [31, 32] . several randomized clinical trials (rcts) are currently underway, not yet published, which are testing different therapies for covid-19. one of the drugs, normally used in the treatment of rheumatoid arthritis, targets the interleukin 6 receptor and has been included in the covid-19 treatment guidelines issued by the chinese national health commission [33, 34] . in addition, several rcts are underway regarding the use of antiviral drugs for the treatment of covid-19 [32, [35] [36] [37] [38] . finally, a recent study evaluates the use of plasma from convalescent patients with previous sars-cov-2 infection as a potential therapeutic treatment [39] . another study shows that the use of systemic corticosteroids for covid-19 is not recommended [40] . the advent of covid-19 is unquestionably reminiscent of previous sars and mers epidemics. the increase in the number of cases and expansion of geographical areas have revealed issues regarding the future management of the infection: on march 11, 2020, the who declared covid-19 a pandemic [41] . at the beginning, efforts were made by the chinese government to limit the spread of the virus, such as suspension of public transport, closure of airports [42] , cancellation of the lunar new year celebrations and closure of parks and cinemas. a ban on the wild animal trade within china was declared on january 26, 2020 [12] . as the number of cases increased, more drastic containment measures were applied in china up to the suspension of all non-essential activities. these measures appear to have slowed the progression of sars-cov-2 infections in china, as can be seen from the progressive decrease in incidence in wuhan [23] . since the beginning, the who and the emergency committee under international health regulations have stressed china's need to strengthen screening of exits from affected areas. in general, evidence in the peer-reviewed literature does not support entry screening as an effective measure for detecting infected travelers, especially when symptoms of the disease are very common/aspecific and the seasonal flu activity in europe and china is ongoing. however, some imported cases of covid-19 in asian countries have been detected through entry screening procedures at the destination airports [12] . one of the screening methods for covid-19 is the measurement of body temperature, although current evidence shows that this method is not effective in controlling disease transmission [18] . as of march 10, 2020, who believes that restrictive measures for travelers and the exchange of goods must be proportional to the risk to public health, with the minimum duration possible and daily updates accordingly on the available epidemiology [43] . currently, the us centers for disease control and prevention (cdc) has increased the level of traveler health alert and recommends avoiding non-essential travel [44] . other countries are considering implementing restrictions for people traveling from and to the most affected countries. since the beginning of the pandemic, the who has developed a strategy and response plan to contain the impact of covid-19. the main points included in this plan are related to blocking the chain of transmission (working on patients as well as close contacts), identifying and reducing transmission from animal sources, developing correct and scientifically sound risk communication and controlling the social and economic impact of the pandemic as much as possible to minimize it through multisectoral partnerships [6] . the ecdc, taking into account the evolving epidemiologic situation, considers five possible scenarios (from 0 to 4). the objective is to avoid the health system disruption and to limit the impact of the pandemic as much as possible [18] ( table 4) . crucial general preventive measures should include: rigorous hand hygiene, avoiding coughing and/or sneezing without covering the mouth and the use of disposable tissues to mechanically block droplets [18] . the who recommends hand washing with soap and water or use of alcohol-based solutions [45] . the use of surgical face masks can reduce the risk of infection transmission; masks should be used by subjects with respiratory symptoms. there is no evidence of the usefulness of face masks by healthy subjects; besides, their use can be related to an increased risk due to a false sense of safety [18] . isolation of symptomatic subjects can be considered to reduce transmission; patients (suspected or confirmed) should be asked to wear a surgical mask to reduce the spread of table 4 different scenarios and options to limit the impact of the epidemic (modified from [18] ) risk management and options for response localized outbreaks, which start to merge, becoming epidemiologically indistinct reduce burden on the health system and protect the population at risk scenario 4 widespread sustained transmission and health care system over-burdened because of the large demand for services mitigate the impact of dissemination, protect the population at risk and reduce excessive mortality respiratory droplets, considered the most likely route of transmission [18] . currently, many countries are considering or have already implemented relevant social measures such as school closures, smart working and cancelling meetings, sports and cultural events. social distancing is essential. it implies avoiding shaking hands and kissing, use of public and crowded means of transportation, and gatherings of people [18] . strict compliance with all these measures is essential to lower the spread of infection to gain time to identify adequate therapeutic options and to design and hopefully develop a vaccine [18] . italy is now the second country in the world in terms of case numbers [23] . the first cases occurred in some areas of lombardy and the veneto regions and then spread all over the country. as of march 9, 2020, considering that new cases of covid-19 registered in italy had started to increase considerably and national restrictive measures had not yet been put in place, the council of ministers issued a decree law with measures to prohibit access to and exit from the country as well as suspension of all the activities that were not strictly necessary [46, 47] . the current italian situation confirms that a local outbreak, once started, can quickly spread and have a huge impact on the most vulnerable citizens, mainly elderly subjects with comorbidities [18] . although to date no sars-cov-2 bloodborne transmission has been documented, the national blood center of the national institute of health (iss) has activated precautionary measures to prevent the spread of the new coronavirus sars-cov-2 from blood donors [48] . ecdc reports that, as of march 2, 2020, the risk of covid-19 infection in europe is currently moderate to high and can change. it should be considered that the transmission chain has not always been identified and that the epidemiologic situation is constantly evolving [18] . the spread of the sars-cov-2 virus represents a global health emergency involving the health authorities of all countries, especially since the pandemic state was declared. many aspects of the infection have been studied. available epidemiologic, clinical and impact data have made it possible to outline preventive interventions that have been shared internationally. unfortunately, the spread of the virus is ongoing, and the impact of the infection is still growing, despite the application of preventive interventions, which in some contexts are very restrictive. the impact of the infection is evident not only from a clinical but also from an economic point of view. the considerable cost in terms of infected/ dead health workers, who operate on the front line and, as such, are particularly exposed to the risk of infection, should not be underestimated [25] . strict observance of the rules issued by the who and the other international bodies (e.g., cdc, ecdc, etc.) is essential, as we need to understand the dynamics of virus spread in more detail, identify new diagnostic and therapeutic approaches and develop a vaccine reasonably quickly. the international effort is enormous and hopefully will allow preventing further spreading of the virus [42] . epidemiologic daily updated cases can be found on the following web pages: https://www.who. int/emergencies/diseases/novel-coronavirus-2019/ >situation-reports/. https://www.ecdc.europa.eu/ en/novel-coronavirus-china. funding. no funding or sponsorship was received for this study or publication of this article. authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole and have given their approval for this version to be published. disclosures. erica d'anchera, federica sandri, marta savio and armando stefanati declare that they have no conflict of interest. giovanni gabutti declares that he does not have a specific conflict of interest related to this paper; however, he reports grants from sanofi pasteur msd, gsk biologicals sa, pfizer, sanofi pasteur italy, msd italy, emergent biosolutions and seqirus for taking part to advisory boards, expert meetings, for acting as speaker and/or organizer of meetings/congresses and as principal investigator and chief of o.u. in rcts. compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. data availability. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. open access. this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/bync/4.0/. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study focolaio di infezione da un nuovo coronavirus (2019-ncov severe acute respiratory syndrome (sars) risk assessment guidelines for infectious diseases transmitted on aircraft (ragida) middle east respiratory syndrome coronavirus (mers-cov) genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding who. novel coronavirus (2019-ncov) situation report-22 focolaio di infezione da nuovo coronavirus sars-cov-2 emerging coronaviruses: genome structure, replication, and pathogenesis epidemiology, genetic recombination, and pathogenesis of coronaviruses sars and other coronaviruses as causes of pneumonia outbreak of acute respiratory syndrome associated with a novel coronavirus, china: first local transmission in the eu/eea-third update emerging threats from zoonotic coronaviruses-from sars and mers to 2019-ncov who. novel coronavirus (2019-ncov) situation report-1 clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan the 2019-new coronavirus epidemic: evidence for virus evolution angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target covid-19): increased transmission globally-fifth update advisory group for infectious hazards t. comment covid-19: what is next for public health? first case of 2019 novel coronavirus in the united states the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes sars-cov-2 viral load in upper respiratory specimens of infected patients who. situation report-50 situation in numbers total and new cases in last 24 hours characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72,314 cases from the chinese center for disease control and prevention the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china novel coronavirus infection in hospitalized infants under 1 year of age in china articles clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records who. coronavirus disease (covid-19) technical guidance: laboratory testing for 2019-ncov in humans case definition for eu surveillance of covid-19, as of 25 detection of sars-cov-2 in different types of clinical specimens cdc. prevention, treatment of coronavirus disease 2019 (covid-19)|cdc reducing mortality from 2019-ncov: host-directed therapies should be an option coronavirus puts drug repurposing on the fast track avviso sull'emissione di un nuovo piano di diagnosi e trattamento della polmonite da coronavirus infect dis ther 03/46c9294a7dfe4cef80dc7f5912eb1989.shtml. accessed 12 how to conquer coronavirus: top 35 treatments in development efficacy and safety of darunavir and cobicistat for treatment of pneumonia caused by 2019-ncovfull text view-clinicaltrials the efficacy of lopinavir plus ritonavir and arbidol against novel coronavirus infection-full text view-clinicaltrials a randomized, open, controlled clinical study to evaluate the efficacy of asc09f and ritonavir for 2019-ncov pneumonia-full text view-clini-caltrials convalescent plasma as a potential therapy for covid-19 clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury director-general's opening remarks at the media briefing on covid-19 a novel coronavirus outbreak of global health concern updated who recommendations for international traffic in relation to covid-19 outbreak cdc. covid-19 information for travelers|cdc nuovo coronavirus, nuove misure di prevenzione dopo la trasmissione locale|centro nazionale sangue key: cord-272306-92rz2byz authors: morra, mostafa ebraheem; van thanh, le; kamel, mohamed gomaa; ghazy, ahmed abdelmotaleb; altibi, ahmed m.a.; dat, lu minh; thy, tran ngoc xuan; vuong, nguyen lam; mostafa, mostafa reda; ahmed, sarah ibrahim; elabd, sahar samy; fathima, samreen; le huy vu, tran; omrani, ali s.; memish, ziad a.; hirayama, kenji; huy, nguyen tien title: clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta‐analysis date: 2018-04-17 journal: rev med virol doi: 10.1002/rmv.1977 sha: doc_id: 272306 cord_uid: 92rz2byz middle east respiratory syndrome (mers) is a respiratory disease caused by mers coronavirus. because of lack of vaccination, various studies investigated the therapeutic efficacy of antiviral drugs and supportive remedies. a systematic literature search from 10 databases was conducted and screened for relevant articles. studies reporting information about the treatment of mers coronavirus infection were extracted and analyzed. despite receiving treatment with ribavirin plus ifn, the case fatality rate was as high as 71% in the ifn‐treatment group and exactly the same in patients who received supportive treatment only. having chronic renal disease, diabetes mellitus and hypertension increased the risk of mortality (p < .05), and chronic renal disease is the best parameter to predict the mortality. the mean of survival days from onset of illness to death was 46.6 (95% ci, 30.5‐62.6) for the ifn group compared with 18.8 (95% ci, 10.3‐27.4) for the supportive‐only group (p = .001). delay in starting treatment, older age group, and preexisting comorbidities are associated with worse outcomes. in conclusion, there is no difference between ifn treatment and supportive treatment for mers patients in terms of mortality. however, ribavirin and ifn combination might have efficacious effects with timely administration and monitoring of adverse events. large‐scale prospective randomized studies are required to assess the role of antiviral drugs for the treatment of this high mortality infection. tions range from supportive to antiviral therapy. two in vitro studies suggested a possible efficacious effect of interferon alpha-2b and ribavirin in the treatment of mers infection. 10, 11 consequently, the investigators further examined the efficacy of these drugs in an animal study. 12 potential benefits of these antiviral treatments in both in vitro and animal studies persuaded clinicians to question the feasibility and applicability of such an approach in humans. therefore, we aimed to recapitulate the evidence from all human published data about mers clinical management in a systematic review and metaanalysis, to summarize the efficacy and safety of current applied therapeutics and define risk factors associated with outcomes. as a result, we may provide a better approach to more compatible management of fatal consequences of mers infections and the risk imposed by recent outbreaks in densely populated areas. our study was performed according to the recommendations of the preferred reporting items for systematic reviews and meta-analyses. 13 we developed a protocol of methods and registered it in the international prospective register of systematic reviews (pros-pero) (reference, crd42015024819). in june 2015, we conducted a systematic search of 10 after removing duplicates, three trained reviewers were assigned to independently screen the titles and abstracts of all references generated from the aforementioned search strategy on the basis of the following inclusion and exclusion criteria. inclusion criteria were (a) any study that gives information about the treatment of mers-cov infection, (b) all types of study designs were included, and (c) no restriction was made with respect to language, age, and area. exclusion criteria were (a) data that could not be reliably extracted, (b) data sets considered as overlapping, (c) studies published before 1/1/2012, (d) book chapter, thesis, letter, conference paper, poster, or editorial, and (e) animal or in vitro studies. three reviewers compared their screening results and discussed the differences. a consensus was reached through discussion. extracted data included publication year, year of research, country and city of the patients, year of subject recruitment, study design, participant enrollment, data collection method, baseline characteristics before treatment, diagnostic method of mers-cov, time from admission to treatment start, treatment for mers-cov, and outcome survival. this work was conducted by 2 investigators evaluating the references independently, and all disagreements were discussed to reach a consensus from supervisors. the quality of included clinical data was assessed using (care) statement for case reports 14 and the 9 metrics tool for nonrandomized studies. 15 three reviewers were assigned to assess each included reference independently. mortality rates were treated as dichotomous variables with their respective 95% confidential intervals (ci). statistical heterogeneity was assessed using the i 2 statistic 16, 17 and assumed to be influential when i 2 was greater than 50% or p ≤ .1. 18, 19 a fixed-effect model was used because there was no evidence of heterogeneity between studies. meta-analysis was performed using data analysis and statistical software (stata) that was developed by statacorp. fisher exact (or chi-square, as appropriate) and mann-whitney u tests were used for the categorical and continuous variables, respectively. the classification and regression tree (cart) model was used to identify independent variables that predict mortality outcome. 20 invasive ventilation and renal replacement therapy were also chosen as the outcomes in the cart model because of direct correlation with severity and mortality. 20 all possible variables were extracted to build cart (table s4 a total of 1095 references were retrieved. upon screening them regarding inclusion and exclusion criteria from section 2, eleven references were included for data extraction and analysis. [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] additionally, 5 references were identified through manual search. 7-9,32,33 so a total of 16 studies were eligible for selection as shown in figure 1 . of the 16 studies including 116 patients, 10 were case reports, [7] [8] [9] 21, 24, 25, 28, 30, 31, 33 2 were case series, 23,26 and 4 were observational studies. 22, 27, 29, 32 saudi arabia was the country of origin for patients in 13 studies, and the rest were from france, greece, and qatar. detailed characteristics of patients in our included articles are presented in table 1 . eight studies used specific antiviral treatment 21, 22, 25, 26, [28] [29] [30] 32 while 7 studies used supportive treatment (including invasive ventilation, prone position, renal replacement therapy, vasopressors, corticosteroids, immunoglobulins, and oseltamivir). 7-9,23,24,31, 33 omrani et al 27 used both specific antiviral treatment and supportive treatment. the specific antiviral treatments were ifn (alpha-2a, alpha-2b, and beta-1a), ribavirin, and several others including tenofovir, emtricitabine, lopinavir, and ritonavir. among 116 patients recruited, 29 were reported with detailed information regarding baseline characteristics, comorbidities, treatments, outcomes, and some with survival time, which was subsequently used to perform univariable analysis and kaplan-meier survival curves (table s4 , found in the supporting information). hemodialysis dependency appeared in higher frequency in the ifn group than in the supportive-only group (p = .025). conversely, renal replacement therapy and vasopressors were used more often in the supportive-only group than in the ifn group (p = .019) ( table 2 ). the quality of 10 case reports [7] [8] [9] 21, 24, 25, 28, 30, 31, 33 and 2 case series 23, 26 was assessed using the care checklist (table s1 , found in the supporting information). all of them contained an introduction and described the importance of the case in the abstract, main introduction section, demographics and symptoms of the patients, significant the quality of 4 observational studies 22, 27, 29, 32 was assessed using the 9 metrics tool 15 (table s2 , found in the supporting information). three of them had a score 23,27,29 of 5 of 9 as they described study design, characteristics of the patient population, inclusion criteria, method quality, and mers diagnosis. however, none of the 3 studies described data collection method, assignment method of patients, exclusion criteria, and interpretation. treatment with antiviral drugs, other than oseltamivir, was reported in 9 studies. ifns (ifn alpha-2a, alpha-2b, or beta-1a) in combination with ribavirin were the common remedies used in all 9 studies. ifn beta-1a was used in 2 studies (n = 12), both of which used a dose of 44 μg/wk for treatment. 28, 29 ribavirin administration was started with a loading dose followed by subsequent doses in all 9 studies. the loading dose was 2000 mg for all studies while 400 mg in one study of alghamdi et al. 21 the subsequent doses, however, were variable among studies and ranged between 400 and 3600 mg/d. the frequency of administration of ribavirin was 3 doses per day in 2 studies 22, 30 and 2 doses per day in another 3 studies. 21, 28, 29 duration of treatment with ribavirin was also variable and ranged between 5 and 26 days. the subsequent oral ribavirin dose was adjusted according to the calculated creatinine clearance in 3 studies. [25] [26] [27] however, the duration of treatment (8-10 days) and the loading dose (2000 mg) used in the 3 studies were similar, regardless of creatinine clearance (table s3 , found in the supporting information). in addition to treatment with ifn and ribavirin, the treatment regimen in shalhoub et al 28 included treatment with tenofovir/ regarding mortality, studies with more than 2 cases were included in the meta-analysis. the pooled proportion of mortality was 0.714 (0.618-0.795) from 8 studies including 106 mers patients (figure 2 ). in 68 patients received ifn treatment, the mortality rate was high (71%) in spite of receiving treatment with ribavirin plus ifn (alpha-2a, alpha-2b, or beta-1a). likewise, the mortality rate was high in patients who received supportive treatment only (71%, n = 48). there was no statistical significant mortality difference when comparing mortality of both groups (p = 1) ( there was a significant difference between death and survival in patients with chronic renal disease (crd the modeling tool, cart, identified crd as the best parameter to predict mortality ( figure 3a ). the performance of the decision tree tool that classified mortality outcome was at an accuracy of 72.4%, sensitivity of 52.9%, specificity of 100%, ppv of 100%, and npv of 60%. in addition, treatment with inotropes was exhibited as the best parameter to predict renal replacement therapy outcome ( figure 3b ). the performance of the decision tree tool that classified renal replacement therapy outcome was at an accuracy of 86.2%, sensitivity of 66.67%, specificity of 95%, ppv of 85.7%, and npv of 86.4%. no significant results were detected regarding the invasive ventilation outcome. survival time from admission to death was compared for ifn-treated (n = 30) and for non-ifn (supportive care only, n = 14) patients. all 44 cases died within 80 days after hospital admission ( figure 4a ). only one female case treated with ifn was eliminated from analysis because the patient remained intubated when the original study ended. however, she had met death criteria; thus, she was included the difference between the 2 groups was statistically significant (p = .001) ( figure 4b ). the longer survival period from onset to death was simply attributed to the duration between onset and admission. our systematic review highlights the significance of age and period between the illness onset and start of antiviral therapy in mers cases' prognostic assessment. our results revealed that younger age and there is no evidence of any difference between ifn treatment and supportive treatment for mers patients in terms of mortality. the ribavirin and ifn combination might have promising effects where therapy can be started promptly and adverse effects monitored carefully; a randomized controlled trial is required to assess this possibility. concerning prognostic factors, delayed treatment, older age, and accompanying comorbidities such as hypertension, dm, chronic kidney disease, and dialysis dependence are associated with worse outcomes. because of high fatality, the seriousness of this newly emerging disease, and a limited number of available cases, we believe there is an urgent need for large-scale clinical trials on the efficacy of antiviral treatment of mers-cov infections. the authors declare no competing interests. orcid ahmed abdelmotaleb ghazy http://orcid.org/0000-0002-9145-7115 nguyen tien huy http://orcid.org/0000-0002-9543-9440 infectious diseases. mers surges again, but pandemic jitters ease middle east respiratory syndrome coronavirus (mers-cov) screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission family cluster of middle east respiratory syndrome coronavirus infections isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin treatment with interferon-[alpha] 2b and ribavirin improves outcome in mers-cov-infected rhesus macaques the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration case report guidelines the newcastle-ottawa scale (nos) for assessing the quality of nonrandomized studies in meta-analyses meta-analysis in clinical trials measuring inconsistency in meta-analyses meta-analysis of genetic association studies synthesis of genetic association studies for pertinent gene-disease associations requires appropriate methodological and statistical approaches post-dengue acute disseminated encephalomyelitis: a case report and meta-analysis mers cov infection in two renal transplant recipients: case report ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection a patient with severe respiratory failure caused by novel human coronavirus ribavirin and interferon-alpha2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study successful recovery of mers cov pneumonia in a patient with acquired immunodeficiency syndrome: a case report ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome coronavirus in children characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case infection prevention/control and management guidelines for patients with middle east respiratory syndrome coronavirus (mers cov) infection severe acute respiratory syndrome vs. the middle east respiratory syndrome clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta-analysis key: cord-256806-g42n51n9 authors: khudhair, ahmed; killerby, marie e.; al mulla, mariam; abou elkheir, kheir; ternanni, wassim; bandar, zyad; weber, stefan; khoury, mary; donnelly, george; al muhairi, salama; khalafalla, abdelmalik i.; trivedi, suvang; tamin, azaibi; thornburg, natalie j.; watson, john t.; gerber, susan i.; al hosani, farida; hall, aron j. title: risk factors for mers-cov seropositivity among animal market and slaughterhouse workers, abu dhabi, united arab emirates, 2014–2017 date: 2019-05-17 journal: emerg infect dis doi: 10.3201/eid2505.181728 sha: doc_id: 256806 cord_uid: g42n51n9 camel contact is a recognized risk factor for middle east respiratory syndrome coronavirus (mers-cov) infection. because specific camel exposures associated with mers-cov seropositivity are not fully understood, we investigated worker–camel interactions and mers-cov seroprevalence. we assessed worker seroprevalence in 2 slaughterhouses and 1 live-animal market in abu dhabi, united arab emirates, during 2014–2017 and administered an epidemiologic survey in 2016 and 2017. across 3 sampling rounds during 2014–2017, we sampled 100–235 workers, and 6%–19% were seropositive for mers-cov at each sampling round. one (1.4%) of 70 seronegative workers tested at multiple rounds seroconverted. on multivariable analyses, working as a camel salesman, handling live camels or their waste, and having diabetes were associated with seropositivity among all workers, whereas handling live camels and either administering medications or cleaning equipment was associated with seropositivity among market workers. characterization of high-risk exposures is critical for implementation of preventive measures. m iddle east respiratory syndrome (mers) coronavirus (mers-cov) was first identified as a cause of severe respiratory tract infections in saudi arabia in october 2012 (1) . the clinical spectrum of mers ranges from asymptomatic infection to acute respiratory distress syndrome and death (2) . as of april 3, 2019, a total of 2,374 laboratory-confirmed cases of infection have been reported by 27 countries to the world health organization (who); the reported case-fatality rate is 35% (2) . all reported cases have an epidemiologic link to the arabian peninsula, and imported cases have been reported in europe, asia, north america, and africa. the united arab emirates has reported the third-highest number of mers cases since 2012 (3) . mers-cov is a zoonotic virus, and dromedaries (camels) are recognized as a major virus reservoir for spillover to humans (4) . multiple studies have isolated mers-cov or mers-cov rna from camels across the arabian peninsula and africa (5) (6) (7) (8) (9) (10) (11) . serologic studies of camels in the middle east and africa have revealed mers-cov seroprevalence of >90%-97% (8, (11) (12) (13) . in natural infection, camels have been found to shed mers-cov in respiratory secretions and to a lesser extent in stool (14, 15) . evidence of virus rna has also been found in milk collected by traditional milking procedures, which involve calf suckling as a stimulus for milk letdown (15) . epidemiologic links between infected camels and human mers-cov infections have been shown, with identical or nearly identical mers-cov genomes found in human cases and in camels with which they had direct contact (16) (17) (18) . also, a case-control study identified exposure to camels as a risk factor for human mers-cov infection (19) . human seroprevalence studies also support the association between mers-cov infection and camel contact; in saudi arabia mers-cov seroprevalence was found to be 15 times greater in camel shepherds and 23 times greater in slaughterhouse workers compared with the general population (20) . further studies have also shown high seroprevalence in specific occupational groups with various camel exposures (e.g., seropositivity was detected in 6.8% of a cohort of 294 camel workers in qatar [21] and in 53% of a cohort of 30 camel workers in saudi arabia [22] ). although multiple lines of evidence suggest camel exposure is associated with human mers-cov infection, the exact mechanisms of transmission are not fully understood. information on specific risk factors relating to camel interactions are needed to further understand how the virus might be transmitted from camels to humans and to guide interventions to prevent zoonotic transmission, including changes to camel management practices. because mers-cov vaccines are currently in development and have reported success in phase i clinical trials (23), knowledge of groups at risk for mers-cov infection might also be useful when considering future vaccine use. our study aimed to identify risk factors for mers-cov seropositivity among live-animal market and slaughterhouse workers. the study sites consisted of an open-air animal market and 2 slaughterhouses (1 commercial and 1 public). all 3 facilities housed camels, goats, sheep, and cattle ( figure 1 ). typically during the study period, approximately 460 persons worked at the market, 101 at the public slaughterhouse, and 29 at the commercial slaughterhouse. the market investigated in this study was linked to a human mers case in 2015 (24) . prior investigation showed a large diversity of mers-covs circulating among camels at the market; 109 (29%) of 276 screened camels had detectable mers-cov rna in nasal swab specimens in the spring of 2015 (25) . we conducted 3 rounds of worker serum sampling. the first round was conducted during may 11-14, 2014 , and the second round during march 23-april 1 and may 7-13, 2015. during the first 2 rounds of sampling, all available workers at the market and public slaughterhouse were requested to provide a serum sample as part of a public health investigation. we conducted a third round of serum sampling during september 22-october 5, 2016, and march 20-23, 2017 . the third round of sampling included workers at the market, public slaughterhouse, and the newly opened commercial slaughterhouse. all available workers were requested to provide serum samples, although participation was voluntary. some, but not all, workers were repeatedly sampled, when feasible, during multiple rounds. we administered an epidemiologic survey to all workers only during the third round of serum sampling in 2016 and 2017. no surveys were administered in 2014 or 2015. the survey consisted of questions covering worker demographics; occupational history; contact with various animal species; travel history; medical history; consumption of raw camel milk, raw camel meat, and camel urine; specific tasks performed with camels; types of personal protective equipment (ppe) worn; and handwashing practices (appendix 1, https://wwwnc.cdc.gov/eid/article/25/5/18-1728-app1.pdf). separate lists of questions covering specific camel tasks performed were asked of market and slaughterhouse workers because of the different nature of camel tasks among occupational groups. interviews were conducted in arabic by staff from the abu dhabi department of health. human serum samples were tested for mers-cov antibodies at the us centers for disease control and prevention (cdc) by using indirect elisas for nucleocapsid (n) and spike (s) proteins, followed by a confirmatory microneutralization test, as previously described (26) . samples were initially tested by using both n and s elisas as screening assays with serum diluted to 1:400. all serum samples with optical densities above assay cutoff were diluted serially, 4-fold, from 1:100 to 1:6,400, and used for endpoint titer determinations. serum samples that were positive by n or s elisa with titers at 1:400, 1:1600, or 1:6,400, plus 10% of samples negative by n or s elisa at these titers, were tested by using microneutralization with live mers-cov performed in a biosafety level 3 laboratory, as previously described (26) . in addition, we conducted confirmatory microneutralization tests on seronegative samples from any persons who showed a change in seropositivity status over time to confirm changes in seropositivity status. samples were considered positive if positive on n and s elisa or if positive on microneutralization. specimens near the limits of detection but not consistently above or below these limits were considered indeterminate. for the epidemiologic analysis, persons with an indeterminate result were considered seronegative. we used epi info 7 (https://www.cdc.gov/epiinfo) for data entry and r version 3.3.1 (https://cran.r-project.org/ bin/windows/base/old/3.3.1) for data analysis. we performed comparisons between prevalence of work practices by setting (market vs. slaughterhouse) by using the pearson χ 2 square test. we used univariable logistic regression to estimate odds ratios, 95% cis, and p values (wald test) for all associations between potential risk factors and seropositivity. we assessed associations between demographics, occupational history, contact with various animal species, consumption of camel products, travel history, and medical history with seropositivity for all workers. we separately tested associations between specific interactions with camels, types of ppe worn, and handwashing practices with seropositivity for stratified subgroups of market and slaughterhouse workers because of the different nature of work setting and standard practices between these 2 populations. we then performed additional exploratory data description by occupation on the basis of results of univariable analyses. we developed 3 multivariable logistic models to identify associations between risk factors and seropositivity. first, we constructed a model of risk factors common to all workers and then constructed occupationally stratified models (i.e., separate models for market workers and slaughterhouse workers) to model specific interactions with camels, ppe use, and handwashing practices. we combined or eliminated highly correlated variables, which were determined by condition indices and variance decomposition proportions. we reduced categorical variables to binary options if small group size was observed. we performed initial variable selection by using least absolute shrinkage and selection operator (lasso) and then tested person-variable significance by using the likelihood ratio test with a cutoff of p<0.05 within an ordinary logistic regression model. we then included age and number of years worked at current setting as potential confounders in all 3 final models. we excluded persons with missing data at the lasso stage but included them for the final logistic regression model. for the stratified market worker and slaughterhouse models, we also included variables significant in the all workers model but not directly relating to camel interactions (e.g., reported underlying conditions) in the final occupationally stratified models. we did not include significant variables directly relating to camel exposures in the all workers model in the stratified models because more specific camel risk practices were assessed in the stratified models. for market and slaughterhouse models, we tested interactions between significant risk practices and select ppe use and handwashing practices for a protective effect. we sampled 100 workers in round 1 (2014), 151 workers in round 2 (2015), and 235 workers in round 3 (2016 and 2017); overall mers-cov seroprevalence was 6% for round 1, 19% for round 2, and 17% for round 3. twenty-one persons had specimens taken at rounds 1 and 2, twenty-three at rounds 2 and 3, thirteen at rounds 1 and 3, and twenty-two at all 3 rounds ( figure 2 ). of 70 persons who were seronegative at their first sample, only 1 (1.4%, 95% ci 0.1%-8.8%) seroconverted: a 30-year-old man who was a cleaner at the public slaughterhouse tested negative at round 1 and positive at round 2. of 8 persons who were seropositive at their first sample, 1 (13%) was later found to be seronegative: a 28-year old man who was an administrative supervisor at the market was resampled between rounds 1 and 3. this person did not report handling camels or their waste and did not perform any tasks directly relating to camels. one additional person who had a positive serologic result at their first and second samples and an indeterminate result at their third sample was not subsequently evaluated for change in seropositive status. because some study participants might have had different medical record numbers across the 3 sampling rounds, we could not determine all potential seroconversions or losses of seropositivity, although we also performed matching by name and age. we compiled serologic results for all participants who ever tested positive (appendix 2, https://wwwnc.cdc.gov/eid/ article/25/5/18-1728-app2.pdf). in total, 235 persons both completed the epidemiologic survey and were sampled during round 3. one additional person completed the epidemiologic survey but refused serum sampling and was not included in any analyses. all 235 workers were men, and their median age was 35 years (range 19-64 years). the median number of years worked at the current settings was 6 (range 0.2-15 years). we observed no significant effect of age (p = 0.26) or years worked (p = 0.18) on seropositivity on univariable analysis. worker occupations were categorized into animal handlers (n = 16), camel salesmen (n = 37), other animal salesmen (n = 41), animal or waste transporters (n = 27), butchers (n = 65), cleaners (n = 26), veterinarians (n = 9), and other (e.g., supervisor, cashier, and tourist guide) (n = 14). salesmen only worked in the market, and butchers only worked in the slaughterhouses. the remaining occupations were found in both settings, but each person could only work at a slaughterhouse or the market. none of the workers reported working at any other job outside of the market or slaughterhouses, and the only animals reported present at home were poultry and stray cats. overall, 64 (44%) of 145 market workers had daily contact with camels or their waste, compared with 47 (52%) of 90 slaughterhouse workers (p = 0.28). certain ppe use and handwashing were more frequently reported by slaughterhouse workers than market workers. among slaughterhouse workers, 99% reported wearing a dust mask (equivalent to a surgical mask), compared with 21% of market workers (p<0.01). only 37% of slaughterhouse workers reported taking their work clothes home, compared with 97% of market workers (p<0.01). eighty-one percent of slaughterhouse workers reported washing their hands before and after each animal-related task, compared with 21% of market workers (p<0.01). ninety-three percent of slaughterhouse workers reported washing their hands at the beginning and end of the day, compared with only 56% of market workers (p<0.01). rates of seropositivity were higher among market workers (29 [20%] of 145) than among slaughterhouse workers (11 [12%] of 90), although this difference was not statistically significant on univariable analysis (p = 0.17). by occupation, camel salesmen and animal or waste transporters had significantly higher odds of seropositivity than the reference group of other salesmen (table 1) . univariable analyses showed that several characteristics were associated with seropositivity among all workers (table 1) , including handling camels or their waste daily. not all seropositive workers reported handling camels or their waste; 7 workers initially claimed they never handled camels or their waste, although 3 of these later reported that they contacted either camel equipment, viscera, or waste within the slaughterhouse. for the subgroup of market workers, univariable analyses revealed multiple camel exposures to be associated with seropositivity and 2 handwashing practices that were inversely associated with seropositivity (table 2 ). for the subgroup of slaughterhouse workers, no individual risk factors were associated with seropositivity (table 3) . because camel salesmen had the highest odds of mers-cov seropositivity, we summarized their frequency of specific camel exposures separately (figure 3 ). direct observation of camel salesmen in the market showed that most of their time was spent in the camel pens, including while they ate and rested, and direct handling of the animals occurred frequently (data not shown). for the multivariable model evaluating risk factors associated with seropositivity in all workers, the following variables remained in the final logistic regression model: handling camels or their waste daily (adjusted odds ratio [aor] 4.2, 95% ci 1.7-11.8), working as a camel salesman (aor 4.0, 95% ci 1.6-10.1), and self-reported diabetes (aor 6.2, 95% ci 1.2-30.3). all 3 factors significantly increased odds of seropositivity. for market workers, multivariable analysis resulted in a final model in which the following variables were each independently associated with seropositivity: handling live camels (aor 12.2, 95% ci 3.2-62.9), administering medications to camels (aor 3.4, 95% ci 1.1-11.2), and self-reported diabetes (aor 20.9, 95% ci 1.6-341.3). cleaning equipment was also significantly associated with seropositivity (aor 3.3, 95% ci 1.1-10.3); substituted for administering medication to camels, this factor produced a model with a near-identical fit along with the other risk factors. given that administering medications to camels was highly correlated with cleaning equipment, the statistical significance of both factors was lost if both factors were included in the model because of collinearity (ρ = 0.65). none of the select ppe and handwashing practices evaluated as interactions with risk practices showed a significant protective effect. no individual risk factors were significantly associated with slaughterhouse workers by multivariable analysis. our study investigated risk factors for mers-cov seropositivity in animal market and slaughterhouse workers at a site previously associated with zoonotic transmission of mers-cov. given the large number of camels present, including many young camels, and the mixing of camels from multiple sources, this site probably facilitates mers-cov transmission among camels. our results demonstrated a relatively high mers-cov seroprevalence in workers at this site, ranging from 6% to 19% at each round across all occupations. because we did not record occupation and other risk factors during the first 2 sampling rounds, we were unable to further assess reasons for the different seropositivity rates between sampling rounds. we found particularly high seroprevalence in specific occupational groups, namely camel salesmen (49%) and animal or waste transporters (22%). previous studies of workers with occupational exposure to camels have reported either lower seropositivity rates (e.g., 6.8% of 294 workers with occupational camel contact seropositive in qatar [21] and 2.3% of 87 camel shepherds seropositive in saudi arabia [20] ) or comparable seropositivity (e.g., 53% of camel workers positive in saudi arabia [22] ). our rates of seropositivity might underestimate actual exposure to mers-cov. previous studies have demonstrated that examining mers-covspecific t cells from mers patients is more sensitive than examining serum antibodies alone (27) . to examine t-cell responses, peripheral blood mononuclear cells must be collected, which was beyond the scope of our study. on multivariable analysis, we found that contact with camels or their waste, working as a camel salesman, and self-reported diabetes were all independently associated with seropositivity in all workers. because of small stratum size, belonging to other occupational groups could not be meaningfully explored as risk factors. diabetes has previously been shown to be a commonly reported underlying condition in mers cases (28) , has been associated with risk for infection in a case-control study (19) , and has been associated with increased risk for death in mers patients (29) . we found an association between diabetes and mers-cov seropositivity in a cohort with occupational exposure to camels. although persons with diabetes might be at increased risk for mers-cov infection, the association between diabetes, mers-cov infection, and the resulting antibody response is still not fully understood. however, because persons with diabetes are considered at high risk for developing severe disease from mers-cov infection, who recommends these persons take precautions when visiting farms or markets where camels are present, including avoiding contact with camels (3). among market workers, handling live camels and either administering medications to camels or cleaning equipment were practices associated with significantly increased risk for mers-cov seropositivity. given that administering medications to camels was highly correlated with cleaning equipment, neither factor was statistically significant if both were included in the model. the biological importance of these associations might therefore be difficult to interpret, because either or both risk factors could be statistically associated with mers-cov seropositivity and have an undefined strength of association. practices potentially associated with camel calves, such as milking or assisting with camel birth, were not associated with mers-cov seropositivity despite a higher prevalence of viral rna in camels <1 year of age compared with other ages (30) and a previously reported association between milking camels frequently and seropositivity (31) . however, these practices were not commonly reported by market workers in our study, limiting the power to detect an association with seropositivity. no specific work practices were found to be associated with seropositivity among slaughterhouse workers. compared with market workers, slaughterhouse workers had less exposure to live camels and a higher self-reported prevalence of potentially protective practices such as ppe use and frequent handwashing. although our multivariable analysis did not show a significant association between ppe use (e.g., wearing a dust mask and gloves) or handwashing practices and seropositivity, the small sample size might have restricted the power to detect interactions between ppe and camel exposures. because camel-to-human transmission of mers-cov is not fully understood, who recommends broad preventive measures for slaughterhouse and market workers, including wearing facial protection when feasible, washing hands before and after each animal-related task, and washing soiled work clothes and shoes at the work place to avoid exposing family members to soiled work clothing (3) . where feasible, increased use of such measures could be encouraged, particularly in market workers, to decrease risk for infection. because only a single human mers case has been reported in connection with the study site, our reported rates of seroprevalence suggest unrecognized transmission (and potentially unrecognized illness) at this site. however, because the length of time mers-cov antibodies persist is unknown (32), the time and place these infections might have occurred is unknown; transmission potential also exists in the united arab emirates outside of markets and slaughterhouses. whether infections were symptomatic is also unknown. participants were asked whether they had seen a healthcare provider for respiratory illness in the previous 12 months, but such reported illness was not associated with seropositivity, and multiple pathogens other than mers-cov could be responsible for any reported respiratory illness. despite these limitations, mers-cov was detected in camels at the market during our study period (25) , and an interim seroconversion was noted in 1 worker, suggesting active zoonotic transmission. taken collectively, our findings suggest an underestimated prevalence of human mers-cov infection in settings where the virus is circulating among camels, probably resulting from camel-to-human transmission. our study had additional limitations, including the overall sample size and limited number of subjects within specific substrata. concentration of camel interactions within particular occupational groups limited our ability to differentiate risk among specific camel interactions, despite our use of multivariable analysis. furthermore, because most persons reported interactions either daily or never, determining whether increased risk was associated with increased frequency of individual tasks was not possible. also, some mers-cov infections might not result in detectable antibodies, particularly when the infections are asymptomatic or mild (32) . persistence of detectable mers-cov antibodies after infection is not well-defined, limiting the ability of serologic testing to define previous infection. finally, because of incomplete linkage of study participants by medical record numbers across the 3 sampling periods, not all potential seroconversions or losses of seropositivity could be determined. in summary, our study found significantly increased odds of mers-cov seropositivity in persons with exposure to camels, in particular among those who handle live camels. odds of seropositivity were also significantly higher for camel salesmen, suggesting that preventive measures such as ppe use could focus on specific occupational groups, in addition to individual work practices. determining groups at highest risk for zoonotic mers-cov infection could also inform future vaccine trials in geographic regions where mers-cov is known to circulate. middle east respiratory syndrome coronavirus (mers-cov) is a novel cov known to cause severe acute respiratory illness in humans; approximately 40% of confirmed cases have been fatal. human-tohuman transmission and multiple outbreaks of respiratory illness have been attributed to mers-cov, and severe respiratory illness caused by this virus continues to be identified. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) world health organization. who mers-cov global summary and assessment of risk middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction risk factors for mers coronavirus infection in dromedary camels in mers coronaviruses from camels in africa exhibit region-dependent genetic diversity middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels human infection with mers coronavirus after exposure to infected camels, saudi arabia high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study occupational exposure to dromedaries and risk for mers-cov infection high prevalence of mers-cov infection in camel workers in saudi arabia inovio pharmaceuticals i. inovio's mers vaccine generates high levels of antibodies and induces broad-based t cell responses in phase 1 study man in germany dies of complications stemming from mers virus diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing inclusion of mers-spike protein elisa in algorithm to determine serologic evidence of mers-cov infection recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study korean society of infectious diseases. clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea acute middle east respiratory syndrome coronavirus infection in livestock dromedaries risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during 2013-2014: a case-control study mers-cov antibody responses 1 year after symptom onset we thank yassir eltahir for critical input during the conceptualization and implementation of this study. this investigation was considered a public health response by the abu dhabi department of health and cdc. dr. khudair is a senior officer in the communicable disease control and management section of the abu dhabi department of health. his research interests include mers-cov and tuberculosis. dr. killerby is an epidemiologist in the division of viral diseases, national center for immunization and respiratory diseases, cdc. her research interests include respiratory viruses such as mers-cov, human coronaviruses, and adenoviruses. key: cord-103046-w8bm4p44 authors: suarez, david l.; pantin-jackwood, mary j.; swayne, david e.; lee, scott a.; deblois, suzanne m.; spackman, erica title: lack of susceptibility of poultry to sars-cov-2 and mers-cov date: 2020-06-16 journal: biorxiv doi: 10.1101/2020.06.16.154658 sha: doc_id: 103046 cord_uid: w8bm4p44 chickens, turkeys, ducks, quail and geese were challenged with sars-cov-2 or mers-cov. no disease was observed, no virus replication was detected, and antibodies were not detected in serum. neither virus replicated in embryonating chicken’s eggs. poultry are unlikely to serve a role in the maintenance of either virus. which it has 82% identity across the genome (2). sars-cov-2 is highly transmissible among 21 humans and particularly virulent for elderly individuals and those with certain underlying health 22 conditions. multiple studies have examined the susceptibility of domestic animals to cov-2 to establish the risk of zoonotic transmission and two studies have shown chickens and 24 pekin ducks were not susceptible to infection (3, 4) . 25 middle east respiratory syndrome coronavirus (mers-cov), another coronavirus of 26 high concern associated with zoonotic infection, was first detected in patients with severe acute 27 lower respiratory tract disease in saudi arabia in 2012. mers-cov causes lower respiratory 28 disease similar to the sars-covs (5). unlike sars-cov-2, mers-cov transmits poorly to 29 humans and does not exhibit sustained human-to-human transmission; however, it has a high 30 case fatality rate of around 30%. although the mers-cov case count is low, human cases 31 continue to be reported, therefore there is a possibility for the virus to adapt to humans. 32 based on sequence similarity, the closest relatives of sars-cov-2 and mers-cov are 33 believed to be bat beta-coronaviruses (6), but because of the amount of sequence difference 34 between human and bat isolates an intermediary host likely exists. for mers-cov, dromedary 35 camels appear to be the primary natural reservoir of infection to humans, but other domestic 36 animals seem to be susceptible to infection (7, 8) . there is only a single study of mers-cov in 37 chickens that looked for antibodies, but all samples were negative (9). 38 because poultry are so widespread and have close and extended contact with humans, 39 and other mammals in many production systems, including live animal markets, susceptibility 40 were conducted with sars-cov-2 and mers-cov in five common poultry species. 41 additionally, embryonating chicken eggs (ece) have been utilized for the isolation and as a laboratory host system, including use in vaccine production, for diverse avian and mammalian 43 viruses. therefore, ece were tested for their ability to support the replication of both viruses. hosts and sources of endemic human 101 genomic characterization of the 103 2019 novel human-pathogenic coronavirus isolated from a patient with atypical 104 pneumonia after visiting wuhan transmission studies of sars-cov-2 in fruit bats, ferrets, pigs and chickens the 107 lancet susceptibility of ferrets, cats, 109 dogs, and other domesticated animals to sars-coronavirus 2. science mers-cov: a global challenge human coronavirus infections-severe acute 113 middle east respiratory syndrome (mers), and sars-114 reference module in biomedical sciences identification of mers-cov in dromedary camels middle east 118 respiratory syndrome coronavirus infection in non-camelid domestic mammals. emerg 119 microbes infect respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in 122 saudi arabia key: cord-257587-xjoyrdhj authors: gunaratne, gihan s.; johns, malcolm e.; hintz, hallie m.; walseth, timothy f.; marchant, jonathan s. title: a screening campaign in sea urchin egg homogenate as a platform for discovering modulators of naadp-dependent ca2+ signaling in human cells date: 2018-11-30 journal: cell calcium doi: 10.1016/j.ceca.2018.08.002 sha: doc_id: 257587 cord_uid: xjoyrdhj abstract the ca2+ mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (naadp) regulates intracellular trafficking events, including translocation of certain enveloped viruses through the endolysosomal system. targeting naadp-evoked ca2+ signaling may therefore be an effective strategy for discovering novel antivirals as well as therapeutics for other disorders. to aid discovery of novel scaffolds that modulate naadp-evoked ca2+ signaling in human cells, we have investigated the potential of using the sea urchin egg homogenate system for a screening campaign. known pharmacological inhibitors of naadp-evoked ca2+ release (but not cadpror ip3-evoked ca2+ release) in this invertebrate system strongly correlated with inhibition of mers-pseudovirus infectivity in a human cell line. a primary screen of 1534 compounds yielded eighteen ‘hits’ exhibiting >80% inhibition of naadp-evoked ca2+ release. a validation pipeline for these candidates yielded seven drugs that inhibited naadp-evoked ca2+ release without depleting acidic ca2+ stores in a human cell line. these candidates displayed a similar penetrance of inhibition in both the sea urchin system and the human cell line, and the extent of inhibition of naadp-evoked ca2+ signals correlated well with observed inhibition of infectivity of a middle east respiratory syndrome coronavirus (mers-cov) pseudovirus. these experiments support the potential of this simple, homogenate system for screening campaigns to discover modulators of naadp, cadpr and ip3-dependent ca2+ signaling with potential therapeutic value. ca 2+ signals originating from the 'acidic' ca 2+ stores of endosomes and lysosomes regulate a steadily growing list of cellular and developmental processes [1] . one important endolysosomal ca 2+ release pathway is activated by nicotinic acid adenine dinucleotide phosphate (naadp), a potent ca 2+ releasing second messenger in many cells and tissues [1] [2] [3] . naadp mobilizes intracellular ca 2+ stores by engaging the activity of members of the two-pore channel (tpc) family ( [4] [5] [6] [7] [8] [9] , but see [10, 11] ). tpcs are broadly expressed ion channels and evolutionarily ancient members of the voltage-gated ion channel superfamily [12, 13] . their identification and subsequent study has facilitated resolution of many pathophysiology processes dependent upon endolysosomal ca 2+ release [14] [15] [16] . for example, using knockdown or knockout approaches, a role for tpcs has been shown during viral infectivity [17] , parkinson disease [18] , muscle function and development [11, [19] [20] [21] , late-onset obesity [22] and susceptibility to non-alcoholic fatty liver disease [23] . such discoveries prioritize the importance of identifying small molecule modulators of tpc-and naadp-dependent ca 2+ signaling pathways as research tools and perhaps, over the longer term, as a therapeutic avenue. for example, the natural product tetrandrine recently identified as a tpc blocker and inhibitor of naadp-evoked ca 2+ signaling improved survival in mice infected with ebola [17] . modulators of naadpdependent signaling have been shown to be effective against other eukaryotic pathogens [24] and viral infections [17, 25] as well as regulators of neoangiogenesis [26, 27] . current pharmacological tools for inhibiting naadp-evoked ca 2+ signals comprise several groups. first, compounds that generally perturb the mobilizable, organellar ca 2+ pool (e.g. bafilomycin, a vacuolar derivatives [25] . finally, a flotilla of other compounds working through the above mechanisms that have inhibitory actions over a range of tens of micromolar. these include alkyl pyridinium analogs (ic 50 ∼15μm, [33] ) na v blockers (ic 50 > 100μm, [13] ) and naringenin (ic 50 ∼180μm, [27] ). overall, there is scope for identifying an improved pharmacopeia of selective, potent modulators of the naadp-evoked ca 2+ release pathway. one approach for executing an unbiased screening campaign targeting naadp-sensitive ca 2+ signaling is based upon interrogation of ca 2+ release responses in the sea urchin egg homogenate system, the preparation in which the ca 2+ releasing activity of naadp (and cyclic adp ribose, cadpr) was first discovered [34, 35] . the sea urchin egg homogenate system represents a simple, yet robust preparation [36] : it is an easily prepared cell-free system that, within each independent preparation, provides reproducible and robust responsiveness to multiple ca 2+ -releasing second messengers (naadp, cadpr and ip 3 ) within a minaturizable, high signal-to-noise, room temperature assay. homogenate can be prepared in bulk, and stored as frozen aliquots which remain responsive for many years. for all these reasons, the system has long been regarded as the 'gold-standard' for studying naadp action [34, 35] , and has been frequently used to assess the action of molecules eliciting ca 2+ release through each of the discrete, endogenous ca 2+ mobilization pathways [13, 30, 32, 33, [37] [38] [39] [40] [41] [42] . although the system is clearly amenable to high throughput profiling, no screening campaign has, to our knowledge, yet been reported. this is surprising given the many advantages of this preparation, not in the least the intrinsic sensitivity to the three ca 2+ -releasing second messengers that permits counter-screening of small molecule specificity. here, we have performed a pilot screen for novel modulators of naadp-sensitive ca 2+ release in the sea urchin egg homogenate system and assessed tractability of the resulting 'hits' against both endogenous naadp-evoked ca 2+ responses and a pseudotyped mers-cov translocation assay in human cell lines [25] . chemicals were sourced as follows: fluo-4 am and lysotracker red (ltr, thermo scientific); fluo-3 pentapotassium salt (biotium). gly-phe-β-naphthylamide (gpn, santa cruz biotechnology); pf-543, skf 96365 hydrochloride, ly-310,762 hydrochloride, pdmp hydrochloride, and ppads tetrasodium salt (cayman chemical); fluphenazine dihydrochloride, gbr-12935 dihydrochloride, racecadotril, clemastine fumarate, prochlorperazine dimaleate salt, thioridazine hydrochloride, salmeterol xinafoate, oxybutynin chloride, trifluoperazine dihydrochloride, naringenin, hepes, chaps, potassium gluconate, n-methylglucamine, nad, naad, nadp, nicotinamide mononucleotide, nicotinic acid, nicotinamide, atp, and dtt (sigma aldrich); a-315456, 3-(1h-imidazol-4-yl)propyl di(p-fluorophenyl)methyl ether hydrochloride (ipfme), st-148, tmb-8 hydrochloride, and trans-ned-19 (santa cruz biotechnology); dilazep hydrochloride (tocris); complete™ edta-free protease inhibitor cocktail (roche). naadp and cadpr were synthesized in house using previously described methods [40, 43] . the libraries used for screening activities were sourced from sigma (lopac ®1280 , library of pharmacologically active compounds, 1280 compounds) and selleck (gpcr compound library, 254 compounds). for the lopac ®1280 library all compounds were screened in triplicate (n = 3, independent assays). for the smaller gpcr library, compounds were screened in duplicate (n = 2, independent assays), owing to limitations on material. strongylocentrotus purpuratus homogenates (25%) were prepared as previously described [44] and stored at −80°c for subsequent usage. homogenates were loaded with ca 2+ and fluo-3 by incubation at 17°c in an intracellular medium, consisting of 250 mm potassium gluconate, 250 mm n-methyl-d-glucamine, 20 mm hepes, 1 mm mgcl 2 , ph 7.2, supplemented with 0.3 mg/ml creatine kinase, 0.5 mm atp, 4 mm phosphocreatine, and 3μm fluo-3 [45] . homogenate was diluted in a step-wise fashion over the course of 3 h to a final concentration of 1.25% homogenate. fluo-3 fluorescence was monitored using a tecan infinite m1000 pro plate reader (λ ex = 485 ± 5 nm, λ em = 525 ± 5 nm). baseline fluorescence readings from samples in the presence of individual drugs were measured, followed by stimulation with a submaximal concentration of naadp. the screening studies were performed in 96-well assay plates (corning 3590 flat bottom, transparent) and each library was screened at a final concentration of 25μm. an epmotion ® 96 liquid handling workstation (eppendorf) was used to dispense homogenate and naadp into assay plates. fluo-3 fluorescence was monitored using a tecan infinite m1000 pro plate reader. for all screening experiments, fluo-3 fluorescence changes were monitored in the presence of compound for 35 cycles (6 min) prior to the addition of an ec 90 concentration of naadp (167 nm final concentration). for the lopac ®1280 library, 0.25ul of vehicle (dmso) or compound (10 mm) was dispensed into the assay plates using a labcyte ech0550 acoustic nanoliter dispensing system. the assay was started by addition of 99.75 μl of sea urchin egg homogenate. for experiments screening the selleck gpcr compound library, baseline fluo-3 fluorescence of the homogenate (97.5ul) was monitored for 1.5 min prior to the addition of 2.5ul vehicle (dmso) or compound (1 mm) using the epmotion ® 96. z' values were calculated to assess separation of distributions of positive and negative controls, as described elsewhere [45] . 2.4. 32 p-naadp binding and ca 2+ release assays in sea urchin egg homogenate [ 32 p]-naadp was synthesized from [ 32 p]-nad and used for binding studies as previously described [45, 46] . for imaging experiments to assess changes in lysosome properties and ca 2+ content, human u2os cells (bone osteosarcoma) were seeded in optical bottom black walled 96-well plates (thermo scientific) at a density of 6 × 10 5 cells per well. after 4 h at 37°c and 5% co 2 , cells were loaded with lysotracker ® red (ltr) and fluo-4 am according to the vendors' respective protocols. cells were then thoroughly rinsed and media was replaced with hanks balanced salt solution (hbss, thermo scientific). fluorescence of ltr (λ ex = 575 ± 5 nm, λ em = 590 ± 5 nm) and fluo-4 (λ ex = 490 ± 5 nm, λ em = 506 ± 5 nm) were simultaneously monitored using a tecan infinite m1000 pro plate reader at 37°c. baseline fluorescence values were monitored for 10 cycles, followed by addition of either vehicle or drug (final concentration, 30μm) and changes in fluorescence values were monitored for an additional 35 cycles. cells were then treated with gpn (final concentration, 300μm) to stimulate osmotic disruption of lysosomes and ca 2+ release with fluorescence monitored for a further 35 cycles. changes in lysosomal ca 2+ content due to drug treatment were quantified by assessing fluorescence ratios (f/f 0 ) during gpn treatment in control and drug-treated samples, where 'f' represents fluo-4 fluorescence at peak, and f 0 represents fluorescence at time = 0. changes in lysosomal labelling due to drug treatment were quantified by assessing fluorescence ratios (f/f 0 ) of ltr during drug treatment, where again 'f' represents minimum ltr fluorescence ratio after drug addition prior to gpn treatment, and 'f 0 ' represents ltr fluorescence at time = 0. naadp microinjection assays in human u2os cells were performed as described in the companion paper [25] . u2os cells were seeded in white 96-well plates (corning) at a density of 2 × 10 5 cells per well. the following day, cell cultures were supplemented with test compounds or vehicle for 8 h at 37°c and 5% co 2 . viability of the cells was assessed using celltiter-glo 2.0 (promega) according to the vendor's protocol. atp-dependent luciferase activity from celltiter-glo 2.0 reagent was quantified using a plate reader (tecan infinite m1000 pro). mers pseudovirus experiments were performed in huh7 cells (human hepatocyte-derived carcinoma) as described in the companion paper [25] . in brief, mers-cov spike pseudotyped retroviruses expressing a luciferase-encoding reporter gene was generated by transfecting hek293 t cells with plasmid carrying env-defective, luciferaseexpressing hiv-1 genome (pnl4-3.luc.re) and plasmid encoding mers-cov spike protein. following receptor-mediated endocytosis of the mers-pseudovirus, translocation of the viral particle from the lumen of the endolysosomal system to the cytosplasm is detected 72 h post infection by measuring luciferase activity. as an initial feasibility test for the validity of screening sea urchin egg homogenate to discover leads with mammalian activities, we took advantage of our existing compound dataset resulting from the mers pseudovirus bioassay [25] . a set of compounds, known to display various degrees of attenuation of mers pseudovirus infectivity, were screened for inhibition of ca 2+ release in the sea urchin egg homogenate system. a typical experiment is shown in fig. 1a , which resolves ca 2+ release kinetics evoked by naadp, or cadpr or ip 3 in the absence and presence of fangchinoline. fangchinoline, an inhibitor of naadpevoked ca 2+ signals and mers pseudovirus translocation in a human cell line [25] , decreased the magnitude of naadp-evoked ca 2+ release (peak amplitude 47 ± 2% of control response, blue traces in fig. 1a ) with lesser effects on the size of ip 3 or cadpr-evoked ca 2+ transients (fig. 1a) . ca 2+ release assays were performed for ∼20 other ligands shown to be inhibitors in the mers pseudovirus translocation assay, and then the impact of these ligands on ca 2+ signals evoked by naadp-, cadpr-and ip 3 were correlated with effects in the viral assay (fig. 1b) . inspection of regression plots from each dataset revealed that compounds that inhibited naadp-evoked ca 2+ release were associated with blockade of mers pseudovirus translocation, with more effective naadp inhibitors causing greater decreases in infectivity (fig. 1b) . no positive correlation was seen for the identical set of compounds between modulation of either cadpr or ip 3 -evoked signals and mers pseudovirus translocation (fig. 1b) . overall, these data establish that identification of pharmacological inhibitors of naadp-evoked ca 2+ signals in the sea urchin system has potential utility for discovering modulators of naadp dependent processes in human cells, such as mers pseudovirus translocation [25] . this provides rationale for a broader screening campaign against sea urchin egg homogenate to discover novel modulators of naadp-evoked ca 2+ release. a schematic overview of the four-step screening workflow is shown in fig. 2a . the primary screen (fixed concentration of 25μm, 1534 compounds), and secondary validation of potential 'hits' (full concentration response curve analysis), were both performed using sea urchin egg homogenate (steps '1' and '2') in a miniaturized format (96well plate). these activities would be predicted to yield a smaller number of candidates for the subsequent, more laborious validation approaches in a human cell line (u2os). these final activities (steps '3' and '4') encompassed: (i) counter-screening for more generalized actions against acidic ca 2+ stores, for example lysosomotropism [28, 29] , (ii) quantifying effects on naadp-evoked ca 2+ signals evoked by single cell microinjection of naadp, and (iii) correlating effects on ca 2+ release with bioactivities in the mers pseudovirus translocation assay. and are expressed as mean ± sem. b, correlation plot comparing the extent of inhibition of naadp-(blue) ip 3 -(red) or cadprevoked ca 2+ release (green) observed with individual ligands (10μm) correlated with the extent of inhibition of mers-pseudovirus translocation evoked by the same ligands (at the same concentration, 10μm). none of these tested ligands evoked ca 2+ release by themselves. solid (naadp) and dotted lines (ip 3 , cadpr) represent linear regression of datapoints. ligand key: 1 = dmso, 2=cycleanine, 3= tubocurarine, 4=nimodipine, 5=procaine, 6=chondocurine, 7=benzocaine, 8=hernandezine, 9=berbamine, 10=nicardipine, 11=verapamil, 12=tetrandrine, 13=fangchinoline, 14=amitriptyline, 15=loperamide, 16=ned-19, 17=bafilomycin, 18=u18666 a, 19 = ym201636, 20=fluoxetine, 21=citalopram, 22=desipramine, 23=siramesine. mers-pseudovirus infectivity was measured using a luciferase-based cell entry assay, assay methodology and inhibition of naadpevoked ca 2+ shown in this figure are described in detail in the companion paper [25] . the overall pipeline would therefore evaluate the translatability of compounds discovered from urchin screening platform for modulating naadp-evoked ca 2+ signaling in mammalian cells. first, we optimized conditions for executing the miniaturized screen in sea urchin egg homogenate, defining a basic protocol depicted in fig. 2b . compounds were preincubated with homogenate for 6 min (1st addition) during which fluorescence readings were monitored, followed by a single subsequent addition of naadp (167 nm, 2nd addition). the positive control was naadp itself (1st addition, fig. 2c ), known to selfdesensitize the sea urchin naadp-evoked ca 2+ release pathway [46] . the negative control was dual additions of vehicle (fig. 2d ). these positive (naadp) and negative vehicle (dmso) controls were run in parallel for each plate. the robustness of the screening platform was assessed by calculating the z' factor (z'), a widely employed indicator of assay quality in screening applications [47] . z' values over 0.5 are considered a prerequisite for executing reliable higher throughput screens. calculations of z' were therefore made using peak fluorescence values during the naadp-evoked ca 2+ mobilization response after initial preincubation with vehicle control versus dual vehicle additions (fig. 2d) , averaging 8 replicate wells within a 96 well plate. using this protocol, z' = 0.73 ± 0.12, an acceptable value defining assay conditions for subsequent experiments. the primary screen (1534 compounds) was then performed using two libraries (lopac ®1280 , 1280 compounds; and a g protein coupled receptor (gpcr) library, 254 compounds). results of the dual library screens are presented together in fig. 3a which collates the averaged magnitude of the naadp-evoked ca 2+ signal observed following preincubation with each compound. these data were then replotted (fig. 3b ) as a progressive ranking of inhibition from the most penetrant inhibitor (pf-543) through to compounds that showed potentiation versus the control naadp signal (phenytoin). most compounds (∼87% of those screened) fell within a ± 25% range of control values (shaded area, fig. 3b ). reassuringly, two established inhibitors of naadpevoked ca 2+ release and 32 p-naadp binding in the sea urchin egg homogenate systemthe purinergic blockers ppads and ppnds [31] displayed clear inhibitory effects in the primary screen, with naadpevoked ca 2+ release reduced to 40 ± 8% (ppads) and 22 ± 4% (ppnds) of control values (fig. 3c) . as our focus here was on identifying novel, penetrant inhibitors of naadp signaling, an arbitrary cut-off of > 80% inhibition of the control naadp-evoked ca 2+ signal amplitude was used for candidate prioritization (red box, fig. 3d ), a threshold which corralled 20 compounds. compounds that caused changes or elevations of baseline fluorescence values during the preincubation period were also excluded from subsequent analysis, one example being the ionophore a23187 (fig. 3) . another example was the serca inhibitor thapsigargin, which depleted the er ca 2+ content, but did not abrogate naadp responsiveness from the acidic ca 2+ stores (fig. 3c) . also excluded were known modulators of naadp-evoked ca 2+ signaling such as thio-nadp (known to contain contaminating naadp [48] , fig. 3c ). following this pruning, a top cohort of 18 putative inhibitors was prioritized and ranked (#1, pf-543 through #18, trifluoperazine; fig. 3d ). analysis of the known pharmacological activities of the screened ligands, and comparison with the subset of these top eighteen candidate inhibitors, revealed enrichment of the 'neurotransmission' classification and dopaminergic modulators in particular ( supplementary fig. 1 ). table 1 collates the ranking of these 18 candidate hits from the primary screen and subsequent data from other assays in the screening pipeline. three compounds showed > 90% inhibition of naadp-evoked ca 2+ signaling in the primary screen (fig. 3e) . the top two hits were pf-543 (rank #1, 6.0 ± 5.0% of control naadp response) and skf96365 (rank #2, 6.3 ± 4.5% of control naadp response). pf-543 is a cell permeable inhibitor of sphingosine kinase 1 (k i ∼ 4 nm, [49] ), which catalyzes the formation of sphingosine 1-phosphate from sphingosine; skf96365 is a lva t-type ca v blocker, with additional antagonist action at trpc channels and other ca v s [50, 51] . secondary validation of the primary screening hits was then performed (step '2', fig. 2 ). for each of the top hits, full concentration response curves for inhibition of naadp-evoked ca 2+ release in the sea urchin egg homogenate was performed. representative curves are shown in fig. 4a , and ic 50 values for each compound are collated in table 1 . each of the eighteen prioritized candidates elicited a concentration-dependent inhibition of naadp-evoked ca 2+ release, validating the robustness of the primary screen. ic 50 values spanned from low micromolar (e.g. racecadotril, ic 50 = 1.6 ± 0.1μm) to tens of micromolar, a range that compares favorably with data obtained with currently used inhibitors of naadp evoked ca 2+ signals, including ppads (ic 50 = 5.4 ± 0.2μm) and the lower potency of commercially sourced trans-ned-19 in our hands (156 ± 3μm, but compare with [30] ). a recently proposed tpc2 inhibitor -naringenin [27] also displayed little inhibitory activity in this system. the selectivity of inhibition of the naadp pathway was assessed by monitoring effects of the same candidate (30μm) on ip 3 -evoked ca 2+ signals, cadpr-evoked ca 2+ signals and naadp-evoked ca 2+ signals. representative compounds in this assay are shown in fig. 4b . finally, the effects of compound on 32 p-naadp binding was also examined, as one potential mechanism for inhibition of naadp-evoked ca 2+ responses. except for naadp and the positive control ppads, none of the compounds displayed significant inhibition of 32 p-naadp binding in sea urchin egg homogenates (fig. 4c) . these data were also consistent with a failure of the candidates to displace a photoaffinity probe [8, 45, [52] [53] [54] from the naadp receptor binding protein in mammalian u2os cell extracts (data not shown). these sea urchin screening activities generated a group of eighteen compounds that merited assessment for activities against naadpevoked ca 2+ signaling in human cells (steps '3' and '4', fig. 2a ). to generate a priority order for assessing inhibition of responses to numbered 1281-1534) . b, results from both libraries were combined and compounds were ranked by amplitude of response from greatest inhibition (rank #1, left) to potentiation (rank #1534, right). the majority of compounds were in a range ± 25% of control response (shaded box). compounds that exhibited > 80% inhibition of naadp-evoked ca 2+ release were prioritized (red box) and selected for further characterization (table 1) . c, raw data from the primary screen for selected compounds -ppads, ppnds, thapsigargin, a23187, thio-nadp and vehicle control (dmso). d, enlargement of red box from 'b' showing ranking of top eighteen hits after pruning, which displayed > 80% inhibition of naadp-evoked ca 2+ release. pruned compounds were thio-nadp and a23187 (4 th and 5 th top hits), shown in grey. e, traces of naadp-evoked ca 2+ release in the presence of the two top ranked candidates (#1, pf-543; #2, skf96365; coloured lines, 25μm). microinjected naadp in single cells, which is a relatively time-consuming process, we first counter-screened the compounds for deleterious effects on cell viability, or non-specific actions on the acidic ca 2+ stores. the cell viability screen was performed using a luciferase based system to quantify cellular atp levels following incubation of u2os cells (bone osteosarcoma) with each compound. none of the compounds exhibited toxicity over this treatment paradigm compared to control samples ( supplementary fig. 2) . next, the effects of the candidate drugs on lysosomal number and ca 2+ content were assessed by simultaneously monitoring changes in lysotracker ® fluorescence and cytoplasmic ca 2+ following addition of gpn (glycyl-l-phenylalanine-2-naphthylamide). gpn causes lysosomal permeabilization and ca 2+ release, concomitant with loss of lysotracker ® staining intensity [55, 56] . decreased lysosomal ca 2+ content in drug-treated samples relative to controls assessed after gpn addition, or decreases in lysotracker ® signals on initial drug addition ('lysosomotropism' [28, 29] ) were regarded as more generalized actions of the drug candidates on the lysosomal ca 2+ stores distinct from activity against the naadp-evoked ca 2+ release pathway. representative traces showing ratios (f/f o ) of green (fluo-4) and red (lysotracker ® ) fluorescence signals over time are shown in fig. 5a for several of the candidates (examples lacking and displaying effects) and controls (vehicle, no gpn, bafilomycin and a protease inhibitor cocktail to impair gpn action). several of the candidate drugs (for example, prochlorperazine and trifluoperazine in fig. 5a ) caused a rapid decrease in lysotracker ® staining (fig. 5a, bottom) and a decrease in mobilizable lysosomal ca 2+ content on gpn addition (fig. 5a, top) . these effects were related, with a strong observed correlation between loss of lyso-tracker ® staining and gpn-evoked ca 2+ transient amplitude (fig. 5b) . data from the portfolio of all 18 candidates are shown in fig. 5b , identifying three broad groupings -(i) compounds with no effect on lysotracker ® or gpn signal intensity, clustering with negative controls (water, dmso; boxed in fig. 5b ), (ii) compounds with penetrant effects on both lysotracker ® and gpn signal intensity (the positive control bafilomycin, and several phenothiazines: prochlorperazine (rank #9), thioridazine (rank #10) and trifluoperazine (rank #18) and (iii) a group of 8 compounds with a profile intermediate between these groupings (∼30-50% decrease in fluorescence ratio versus controls). only the seven candidates with no effect on gpn-mobilizable ca 2+ or ltr staining -the first grouping, pf-543 (rank #1), skf96365 (rank #2), racecadotril (rank #5), a-315456 (rank #6), ly-310,762 (rank #12), pdmp (rank #15) and salmeterol (rank #16) -were advanced for further validation. the remaining 11 candidates were not pursued further in the context of this study (shaded rows in table 1 ). the effects of the remaining seven candidates on the amplitude of naadp-evoked ca 2+ signals in human u2os cells was examined. these experiments were performed by monitoring ca 2+ release kinetics following microinjection of naadp into single cells (fig. 6) . whereas injection of buffer alone evoked only a small stimulus artefact, injection of naadp evoked a robust ca 2+ transient (peak f/f o = 4.0 ± 0.6, n = 3 injections, fig. 6a ). the action of naadp was then examined in cells preincubated with the candidate inhibitors (10μm, 10 min pretreatment), as well as other compounds of interest. ppadsthe positive control naadp inhibitor from sea urchin assays [31] -decreased the amplitude of the naadp-evoked ca 2+ transient to 24.2 ± 2.1% of control values (fig. 6a) . however, neither ned-19, nor naringenin significantly attenuated ca 2+ signal amplitude following naadp microinjection (fig. 6a) . examination of each candidate inhibitor revealed varying degrees of inhibition of naadp-evoked ca 2+ responses under the preincubation conditions (10μm) with the most effective compounds being skf96365 (12.4 ± 8.9% of control values), pf-543 (14.1 ± 3.0% of control values) and racecadotril (16.6 ± 10% of control values). these compounds were highly ranked in the sea urchin screen (skf96365 (rank #2), pf-543 (rank #1) and racecadotril (rank #5)) and compared well with ppads (24.2 ± 2.1% of control values). pdmp (rank #15) caused the lowest extent of inhibition table 1 summary of compounds from primary screen with > 80% inhibition of naadp-evoked ca 2+ release. compounds were ranked (#1 through #18) in order of maximum average inhibition of naadp-evoked ca 2+ release in the primary screen (fig. 3) . hits were then further assessed in concentration-response and 32 p-naadp binding experiments in sea urchin egg homogenate (fig. 4) . counter-screening activities were performed in u2os cells, with the cut-off threshold for nonpursuit of the candidates being > 30% decrease in fluorescence ratios in either ca 2+ release or ltr assays. validation assays represent extent of inhibition of naadpevoked ca 2+ release signals, and mers pseudovirus translocation by the remaining seven candidates (bold in compound list). ipfme (rank #8): 3-(1h-imidazol-4-yl) propyl di(p-fluorophenyl)methyl ether hydrochloride. (43.7 ± 15.0% of control values) which was none-the-less still a considerable improvement over both ned-19 and naringenin in our hands. finally, in the companion paper [25] , we had established that inhibition of either naadp-sensitive ca 2+ release, or tpc1/tpc2 activity, impaired the translocation of a mers pseudovirus through the endolysosomal system. the unbiased screening approach described here generated an additional panel of inhibitors of naadp-evoked ca 2+ signaling. therefore, the potential effectiveness of these compounds in the mers pseudovirus infectivity assay was assessed. results from this mers pseudovirus bioassay were plotted along with the ca 2+ release inhibition data in fig. 6b . visual inspection of the results from both datasets revealed a strong correlation between results from these independent assays, further supporting the conclusions of the companion paper that ligands targeting the naadp pathway inhibit mers pseudovirus translocation [25] , while highlighting new scaffolds for manipulation of naadp-dependent signaling processes in mammalian cells. here we have performed a 'proof of principle' unbiased screen in the sea urchin egg homogenate system with the goal of using this system as an entry point to a validation pipeline aimed at discovering novel chemical scaffolds to inhibit naadp-evoked ca 2+ release. discovery of modulators of naadp-evoked ca 2+ signaling is important as appreciation grows of the role of this pathway in (dys)regulating cellular processes [11, [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . there is certainly room for improvement in defining ligands with improved selectivity and reliable activity against the naadp signaling pathway. the small pilot screen (1534 compounds) was robust in terms of signal amplitude (fig. 2) , the population spread of inhibitory values (fig. 3b) and most importantly success in identifying known blockers of naadp-evoked ca 2+ release within the screened inventory. examples of such compounds which ranked highly in the primary screen include (i) [ 32 p]-naadp binding inhibitors, ppnds (primary screen rank #27, [31] ) and ppads (rank #62 [31] ), and (ii) previously identified ca v blockers, nicardipine (rank #35, [32] ), diltiazem (rank #113 [32] ), verapamil (rank #120 [32] ), and nifedipine (rank #149 [32] ). other ca v modulators found within the top hundred 'hits' included fpl64176 (rank #34), nitrendipine (rank #85) and methoxyverapamil (rank #95), as well as the highly ranked hit skf96365 (rank #2, discussed below [50, 51] ). these ca v blockers would be predicted to serve as tpc pore blockers, as supported by virtual docking analyses and microinjection studies [13] . table 1 . b, averaged peak ca 2+ release in the presence of drug (30μm, 5 min preincubation time) relative to vehicle controls, in response to ec 70 concentrations of naadp (70 nm), cadpr (100 nm), or ip 3 (200 nm). c, analysis of effects of all 18 candidates on specific 32 p-naadp radioligand binding levels in the presence of drug (30μm), relative to vehicle controls. p-values: ** p < 0.01 relative to dmso controls. here, we prioritized candidates showing ≥80% inhibition of the peak naadp-evoked ca 2+ signal. this comprised eighteen candidates, seven of which progressed through validation in subsequent assays. this 'hit' proportion (∼0.5% from 1534 compounds) is consistent with discovery rates observed in other screens, and is especially agreeable given the low execution cost for the urchin screening platform. prior reticence to use this system for unbiased screening may have related to concern over tractability of structure-activity relationships from the urchin to human pathways. differences in sensitivities between sea urchin and mammalian systems have previously been noted for structural analogs of naadp [42] implying differences in naadp binding protein specificity [45, 52] . however, this relates to finer structure-activity relationships within defined chemical series, rather than discovery of new scaffolds. while obviously data reflects the sensitivities and specificities of an invertebrate ca 2+ release system, the utility of c. elegans and drosophila as drug screening models is noted [57] . in our opinion, the advantages of high assay throughput in the sea urchin system offsets the need for subsequent validation of 'hits' against naadp-evoked responses in human cells. even so a similar ordering of potency was ultimately observed between the sea urchin and human cell bioassays: skf96365, pf-543 and racecadotril were the most penetrant inhibitors in both systems (fig. 4, table 1 ). identification of these three novel hits ( table 1 ) that (i) inhibited ca 2+ release by > 80% in a human cell line, more than seen with tetrandrine under identical conditions [25] , (ii) reduced mers pseudovirus translocation to levels observed with fangchinoline ( fig. 1, [58] ), and (iii) lacked demonstrable action in the counterscreen provides strong support for execution of a higher throughput screening campaign using sea urchin egg homogenate. screening large compound libraries against endogenous naadp-evoked signals mediated via tpcs within their acidic store native environment in mammalian cells would be a much more daunting prospect. screens of tpcs targeted to the cell surface [59] fig. 5 ). numerous compounds and chemical scaffolds have been shown to accumulate within the acidic ca 2+ store lumen where they can modulate (e.g. functional inhibitors of acid sphingomyelinase, fiasmas [63, 64] ) or act as substrates of luminal enzymes [65] to alter the structure and/or function and even integrity of the lysosome (e.g. osmotic lysis by gpn). examples of compounds dropped from the pipeline based on counter-screening assays, that are known to fall within this category [63, 66] are multiple phenothiazine compounds (rank #3, #9, #10, #18), clemastine (rank #7) and dilazep (rank #11). these compounds display higher lipophilicity (average clogp, 4.85) and basic pka (average pka, 8.9) than the remaining dataset. other excluded compounds (gbr-12935, ipfme, st-148, tmb-8 and oxybutynin) are new lysosomotropic suspects. this does not equate to a lack of usefulness as research tools, or even clinical drugs, as many approved therapeutics show marked lysosomotropism, a feature that may actually contribute to their clinical efficacy [63, 67, 68] . this is especially relevant for novel uses of existing clinical agents to target pathogens that traverse the endolysosomal system, where drug accumulation with acidic ca 2+ stores would be a desirable attribute for pathogen targeting [25, 69] . such activities may have good repurposing potential, but for our purposes here, inhibition of naadp-evoked ca 2+ release by these candidates is likely indirect. the seven candidates advanced through the pilot screen pipeline deserve further scrutiny. all inhibited naadp evoked ca 2+ signals responses in the human cell line (fig. 6 ) under conditions less penetrant than the counterscreen where no changes in lysosomal properties were observed (fig. 5) . the three top ranked hits -skf96365, pf-543 and racecadotrilhave not previously been shown to impair naadpevoked ca 2+ signaling. none of these compounds interfered with specific 32 p-naadp binding implying inhibition through other fig. 5 . characterization of candidate inhibitors on lysosomal ca 2+ stores in u2os cells. a, representative traces of gpn induced ca 2+ release (top) and lysosomal disruption (bottom). u2os cells were loaded with fluo-4 am and lysotracker ® red (ltr). baseline fluorescence values were recorded for 15 min before addition of test compounds (30μm). fluorescence was then monitored for 40 min before addition of gpn (300μm) to release lysosomal ca 2+ . measurements were made to quantify gpn evoked ca 2+ release (top) and loss of ltr fluorescence following initial drug addition (bottom) by assessing fluorescence ratio values in the regions highlighted by the green (peak f/f o ratio) and red bars (minimal f/f o ratio). pic, protease inhibitor cocktail; bafa1, bafilomycin a1. b, correlation plot depicting relationship between measurements of gpn induced calcium release (y-axis, green) and drug-induced lysosome disruption (xaxis red). values are replotted from highlighted regions in (a). the cluster of non-lysomotropic compounds is highlighted (box). data represents peak fluo-4 fluorescence ratios (f fluo /f 0 , where 'f fluo ' represents fluo-4 fluorescence at peak, and f 0 represents fluorescence at time = 0) and minimum lysotracker ® fluorescence ratios (f ltr /f 0 , where 'f ltr ' represents minimum lysotracker ® red fluorescence prior to gpn addition, and f 0 represents fluorescence at time = 0). compounds are labeled according to ranking # in table 1 . mechanisms (fig. 4c ). skf96365 is a low voltage-activated t-type ca v blocker, with antagonist action at other ca v s and trpc channels [50, 51] . this polypharmacological profile may now extend to tpcs. direct electrophysiological analysis will be needed to confirm if the observed inhibition of naadp action in ca 2+ -free extracellular media by skf96365 (fig. 6a ) results from tpc pore blocking ability. our data also suggest caution in attribution of the mechanistic basis of effects of skf96365 in studies of ca 2+ signaling [70] . pf-543 is a cell-permeant inhibitor of sphingosine kinase [49] , application of which causes a dose-dependent increases in cellular sphingosine levels. elevated sphingosine levels attenuate acidic ca 2+ store signaling, as evidenced by impaired responses to naadp in patients with niemann-pick type fig. 6 . validation of naadp-inhibitors in mammalian cells. a, ca 2+ traces resolved by fluo-4 fluorescence in response to naadp microinjection (100 nm pipette concentration) in u2os cells treated with indicated drugs (10μm, 10 min pretreatment). individual traces shown in red, averaged response shown in black. b, quantification of peak amplitude of naadp-evoked ca 2+ transients in microinjected u2os cells relative to control (blue bars) following preincubation with indicated drugs as shown in (a). red bars report luciferase levels in a mers-pseudovirus cell translocation assay in huh7 cells relative to controls (h 2 o, dmso) following treatment with the same panel of drugs (10μm for 1 h prior to exposure to mers-pseudovirus for a 5 h period). mers-pseudovirus cell entry was detected 3 days post infection by measuring luciferase activity as described fully in the companion paper [25] . p-values: * p < 0.05, ** p < 0.01 relative to dmso controls. c1 disease [71] . this inhibition may be caused by sphingosine-dependent tpc1 activation [72] , impaired lysosomal ca 2+ uptake and/or lysosomal permeabilization [65, 71] . action of pf-543 through any of these mechanisms would result in the observed inhibition of naadpevoked ca 2+ signals. racecadotril (acetorphan) is a neutral endopeptidase inhibitor (nep), used therapeutically as an antidiarrheal agent by blocking enkephalin-mediated intestinal fluid secretion. it is also a prodrug, being rapidly converted to thiorphan, a low nanomolar nep inhibitor. inhibition of neprilysin (an amyloid β (aβ) peptide degrading enzyme) by infusion of thiorphan in a mouse model of alzheimer's disease is associated with extensive lysosomal accumulation of aβ as well as changes in lysosomal number and size [73] . these data evidence lysosomal alterations which, as seen in other neurodegenerative models [18] , dysregulate naadp action. further experiments will be needed to define mechanistically how these drugs impair naadp action. in conclusion, interrogation of the sea urchin egg homogenate platform provided new leads for inhibiting naadp-dependent processes -naadp-evoked ca 2+ signaling, as well as mers pseudovirus infectivityin human cells. even though only a small number of compounds were profiled in this initial unbiased pilot screen, the effectiveness of the highly ranked compounds was as good as achieved through structure-activity based screening around the known tetrandrine scaffold (see companion study, [25] ). these data provide strong support for execution of a higher throughput screening campaign using the sea urchin egg homogenate system to discover new ligands for manipulation of naadp signaling, as well as for modulators of cadpr and ip 3 action. none. molecular mechanisms of endolysosomal ca 2+ signalling in health and disease nicotinic acid adenine dinucleotide phosphate (naadp)-mediated calcium signaling a primer of naadp-mediated ca(2+) signalling: from sea urchin eggs to mammalian cells essential requirement for two-pore channel 1 in naadp-mediated calcium signaling naadp mobilizes calcium from acidic organelles through two-pore channels the two-pore channel tpcn2 mediates naadpdependent ca(2+)-release from lysosomal stores convergent regulation of the lysosomal two-pore channel-2 by mg(2)(+), naadp, pi(3,5)p(2) and multiple protein kinases expression of ca(2)(+)-permeable two-pore channels rescues naadp signalling in tpc-deficient cells tpc: the naadp discovery channel? tpc proteins are phosphoinositide-activated sodium-selective ion channels in endosomes and lysosomes ren, mtor regulates lysosomal atp-sensitive two-pore na(+) channels to adapt to metabolic state an ancestral deuterostome family of two-pore channels mediates nicotinic acid adenine dinucleotide phosphate-dependent calcium release from acidic organelles two-pore channels provide insight into the evolution of voltage-gated ca 2+ and na + channels two-pore channels: catalyzers of endolysosomal transport and function from mucolipidosis type iv to ebola: trpml and two-pore channels at the crossroads of endo-lysosomal trafficking and disease function and dysfunction of two-pore channels ebola virus. two-pore channels control ebola virus host cell entry and are drug targets for disease treatment dysregulation of lysosomal morphology by pathogenic lrrk2 is corrected by two-pore channel inhibition ca(2+) release via twopore channel type 2 (tpc2) is required for slow muscle cell myofibrillogenesis and myotomal patterning in intact zebrafish embryos two-pore channels (tpc2s) and nicotinic acid adenine dinucleotide phosphate (naadp) at lysosomal-sarcoplasmic reticular junctions contribute to acute and chronic beta-adrenoceptor signaling in the heart lysosomal two-pore channel subtype 2 (tpc2) regulates skeletal muscle autophagic signaling absence of intracellular ion channels tpc1 and tpc2 leads to mature-onset obesity in male mice, due to impaired lipid availability for thermogenesis in brown adipose tissue high susceptibility to fatty liver disease in two-pore channel 2-deficient mice ned-19 inhibition of parasite growth and multiplication suggests a role for naadp mediated signalling in the asexual development of plasmodium falciparum inhibition of two-pore channels blocks middle east respiratory syndrome coronavirus translocation through the endolysosomal system vegf-induced neoangiogenesis is mediated by naadp and two-pore channel-2-dependent ca2+ signaling naringenin impairs two-pore channel 2 activity and inhibits vegf-induced angiogenesis quantitation of the lysosomotropic character of cationic amphiphilic drugs using the fluorescent basic amine red dnd-99 identification of a chemical probe for naadp by virtual screening ppads is a reversible competitive antagonist of the naadp receptor pharmacological properties of the ca2+-release mechanism sensitive to naadp in the sea urchin egg cell-permeant small-molecule modulators of naadp-mediated ca2+ release calcium mobilization by dual receptors during fertilization of sea-urchin eggs a derivative of nadp mobilizes calcium stores insensitive to inositol trisphosphate and cyclic adp-ribose preparation and use of sea urchin egg homogenates for studying naadp-mediated ca(2)(+) release carvedilol inhibits cadpr-and ip3-induced ca(2+) release scaffold hopping with virtual screening from ip3 to a drug-like partial agonist of the inositol trisphosphate receptor triazine dyes are agonists of the naadp receptor nicotinic acid dinucleotide phosphate analogs containing substituted nicotinic acid: effect of modification on ca 2+ release analogues of the nicotinic acid adenine dinucleotide phosphate (naadp) antagonist ned-19 indicate two binding sites on the naadp receptor activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (naadp) analogs in a human cell line: difference in specificity between human and sea urchin naadp receptors synthesis and characterization of antagonists of cyclic-adpribose-induced ca 2+ release pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (naadp)-binding proteins in sea urchin egg activation and inactivation of ca2+ release by naadp+ a simple statistical parameter for use in evaluation and validation of high throughput screening assays thio-nadp is not an antagonist of naadp modulation of cellular s1p levels with a novel, potent and specific inhibitor of sphingosine kinase-1 the transient receptor potential channel antagonist skf96365 is a potent blocker of low-voltage-activated t-type calcium channels sk&f 96365, a novel inhibitor of receptor-mediated calcium entry photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (naadp) targets in mammalian cells nicotinic acid adenine dinucleotide 2'-phosphate binding proteins in t lymphocytes the molecular basis for ca 2+ signalling by naadp: two-pore channels in a complex use of glycyl-l-phenylalanine 2-naphthylamide, a lysosome-disrupting cathepsin c substrate, to distinguish between lysosomes and prelysosomal endocytic vacuoles activity-dependent exocytosis of lysosomes regulates the structural plasticity of dendritic spines drug discovery in fish, flies, and worms a screening campaign in sea urchin egg homogenate as a platform for discovering modulators of naadp-dependent ca 2+ signals in human cells an naadp-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying ca 2+ signals structure of the voltage-gated two-pore channel tpc1 from arabidopsis thaliana structure, inhibition and regulation of two-pore channel tpc1 from arabidopsis thaliana structural insights into the voltage and phospholipid activation of the mammalian tpc1 channel identification of novel functional inhibitors of acid sphingomyelinase functional inhibitors of acid sphingomyelinase (fiasmas): a novel pharmacological group of drugs with broad clinical applications lysosomotropic agents: impact on lysosomal membrane permeabilization and cell death a high content screening assay for identifying lysosomotropic compounds acid sphingomyelinase-ceramide system mediates effects of antidepressant drugs acid sphingomyelinase inhibition prevents development of sepsis sequelae in the murine liver repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection nicotinic acid adenine dinucleotide phosphate plays a critical role in naive and effector murine t cells but not natural regulatory t cells niemann-pick disease type c1 is a sphingosine storage disease that causes deregulation of lysosomal calcium intracellular sphingosine releases calcium from lysosomes activation of the amyloid cascade in apolipoprotein e4 transgenic mice induces lysosomal activation and neurodegeneration resulting in marked cognitive deficits supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.ceca.2018.08.002. key: cord-265282-v3n9ff16 authors: ahn, inkyung; heo, seongman; ji, seunghyun; kim, kyung hyun; kim, taehwan; lee, eun joo; park, jooyoung; sung, keehoon title: investigation of nonlinear epidemiological models for analyzing and controlling the mers outbreak in korea date: 2018-01-21 journal: journal of theoretical biology doi: 10.1016/j.jtbi.2017.10.004 sha: doc_id: 265282 cord_uid: v3n9ff16 abstract much concern has arisen regarding serious epidemics due to the middle east respiratory syndrome (mers) coronavirus. the first mers case of korea was reported on 20 may 2015, and since then, the mers outbreak in korea has resulted in hundreds of confirmed cases and tens of deaths. deadly infectious diseases such as mers have significant direct and indirect social impacts, which include disease-induced mortality and economic losses. also, a delayed response to the outbreak and underestimating its danger can further aggravate the situation. hence, an analysis and establishing efficient strategies for preventing the propagation of mers is a very important and urgent issue. in this paper, we propose a class of nonlinear susceptible-infectious-quarantined (siq) models for analyzing and controlling the mers outbreak in korea. for the siq based ordinary differential equation (ode) model, we perform the task of parameter estimation, and apply optimal control theory to the controlled siq model, with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the proposed siq model can explain the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well, and the number of the mers cases can be controlled reasonably well via the optimal control approach. first confirmed on 20 may 2015, the latest outbreak of middle east respiratory syndrome coronavirus (mers-cov) infections in korea accounted for 186 laboratory-confirmed cases, including 36 deaths, 138 recovered individuals discharged from hospitals, and 12 remaining in hospitals up to 28 july 2015, a de facto end date of the outbreak ( ministry of health and welfare , moh ). it spread remarkably fast in hospitals, which has caused the largest mers outbreak outside the middle east. the case fatality rate of 19.4% is, however, much lower than the reported rate of 37.7% prior to the outbreak in korea, according to the world health organization (who). the mers-cov is a novel betacoronavirus which was first identified from the sputum of a 60-year-old man in fall 2012 ( zaki et al., 2012 ) . clinical features of mers range from mild illness to fatal conditions such as acute respiratory distress syndrome * corresponding authors. e-mail addresses: ahnik@korea.ac.kr (i. ahn), parkj@korea.ac.kr (j. park). and multi-organ failure resulting in death, especially in patients with underlying comorbidities ( zumla et al., 2015 ) . although it is initially known as a zoonotic disease, human-to-human transmission occurs in health care settings and now is linked with significant morbidity ( oboho et al., 2015 ) . this outbreak of the mers-cov infection, including the index cases roommates, their caregiver, and even the healthcare workers, in what is called as a super-spreading event ( kupferschmidt, 2015 ) , raised several important issues for global public health surveillance and raised several issues regarding infection control policies, including the control of nosocomial transmission to avoid a repeated outbreak ( keeling and rohani, 2008 ) . in this study, we carry out an epidemiological assessment of the mers-cov outbreak in korea in order to provide a mathematical framework for understanding the complex dynamics of the pathogen spread and establishing efficient guidelines for implementing quarantine and isolation strategies. more specifically, we propose a class of nonlinear susceptible-infectious-quarantined (siq) models for analyzing and controlling the mers outbreak in korea. the proposed siq model is innovative, in that the mers https://doi.org/10.1016/j.jtbi.2017.10.004 0022-5193/© 2017 elsevier ltd. all rights reserved. transmission probability is time-dependent, monotone decreasing, and squashing-type. more specifically, it is initially almost flat, then decreasing rapidly, and finally gradually reaching a saturation point, which is reasonable because this can reflect the change in individuals' hygienic behaviour with time. recently, there has been much interest in investigating some aspects of the time-dependent nature of the disease transmission probability. in particular, several methods have been considered to model the time-dependence due to the impact of media coverage ( cui et al., 2008; liu et al., 2007; sun et al., 2011; xiao et al., 2015 ) . however, these studies have mostly focused on only a single factor under consideration. in this paper, we consider all relevant factors that can affect the mers transmission probability ( e.g. , media coverage, increased awareness, etc.) collectively, and try to model the resultant time-dependent transmission probability using a sigmoidal function. note that the sigmoidal functions are quite popular in the field of machine learning when one needs to address monotone and squashing-type phenomena. for the proposed siq model, we perform the task of parameter estimation, and apply optimal control theory to the controlled siq model, with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the proposed siq model can explain the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well, and the number of the mers cases can be controlled reasonably well via the optimal control approach. finally in the last two sections of this paper, discussion and concluding remarks are given along with brief descriptions of data treatment. in this section, we will propose a nonlinear susceptibleinfectious-quarantined (siq) model for analyzing the mers outbreak in korea. the siq model is a generalization of an epidemiological population model involving susceptible, infectious, and quarantined compartments ( hethcote et al., 2002; keeling and rohani, 2008; lenhart and workman, 2007; xiao et al., 2015 ) . in the siq model, the four compartment populations are used as the model's state variables: s ( t ), the number of individuals that are susceptible to the mers disease at time t; i ( t ), the number of individuals that are actively infectious at time t; s q ( t ), the number of quarantined susceptible individuals at time t ; and c q ( t ), the number of confirmed cases at time t . note that in the siq model, q stands for the super-compartment comprising s q and c q (see fig. 1 ). also, note that c q ( t ) is a collective sink-type compartment, which includes the number of the mers patients under treatment, the recovered cases, and the dead cases altogether at time t . as shown in fig. 1 , the siq model considers two kinds of non-pharmaceutical interventions: quarantine of the susceptible and infected individuals, and isolation of the infectious individuals following contact tracing. as a result of a contact tracing, a proportion, q , of individuals who are contacted in connection with a mers patient is quarantined. the quarantined individuals can move to compartments c q or s q , depending on whether they are exposed to the mers coronavirus or not. hence, the quarantined individuals, if uninfected, move to the compartment s q at a rate of c(1 − β(t)) q, where c is the contact rate, i.e. , the average number of contacts of the whole population per unit time, and β( t ) is the probability of the mers transmission per contact at time t . note that in the siq model, the mers transmission probability, β( t ), is time-dependent, monotone decreasing, and squashing-type. obviously, using timedependent transmission probability is more reasonable than using the constant one, because it can reflect the change of individuals' hygienic behavior with time. as shown in fig. 1 , if infected, the quarantined individuals move to the compartment c q at a rate of c β( t ) q . also, the remaining proportion ( i.e. , the proportion missed from the contact tracing), 1 − q, can move to compartment i or stay in compartment s , depending on whether they are exposed to the mers coronavirus or not. the transmission dynamics of the siq model is illustrated in fig. 1 , and its state equations are as follows: the first equation of (1) describes the rate of change of the susceptible compartment population, with four terms on its right-hand side. its first term concerns the transition from s to s q due to quarantine of susceptible individuals. this term can be explained in terms of a bilinear incidence law having a contact rate c together with β( t ), the probability of the mers transmission per contact at time t , and q , the probability of quarantine per contact. the second and third terms model the transition from s to c q and the transition from s to i , respectively. the fourth term represents the transition from s q to s , and this transition means the release from quarantine into the wider community. in the second equation of (1) , the rate of change of the infectious compartment population is described by two terms. the first represents the transition from the susceptible state to the infectious state, and the second term denotes the transition from i to c q due to detection and isolation of the infectious patients. the remaining equations of (1) describe the rates of change of s q and c q in the super-compartment q , respectively, and the exact meaning of their terms can be explained similarly. the natural birth and death rates are not considered in the siq model, and this omission allows us to focus on the core theme of the paper. note that consideration of these additional aspects is relatively straightforward, and would lead us to some further related observations. for example, if the natural birth rate of a susceptible population, , and the natural death rate, d , are consid ered, the resultant reduced siq system 1 with a constant transmission probability, β 0 , would have the disease-free equilibrium point as previously mentioned, the mers transmission probability, β( t ), of our siq model, is time-dependent, monotone decreasing, and squashing-type. since many factors ( e.g. , media coverage, increased awareness, etc.) can alter individuals' hygienic behavior, we employ the squashing-type function of fig. 2 for β( t ). 2 more specifically, we utilize where σ ( ·) is the logistic sigmoidal function ( bishop, 2006 ) defined as σ (x ) = 1 / (1 + exp (−x )) ; t β is the inflection point of β( t ); s β is the parameter determining the slope of β( t ) at its inflection point. note that the use of logistic sigmoidal functions is quite popular in the field of machine learning ( bishop, 2006 ) when one needs to represent monotone and squashing-type phenomena. also, note that using the logistic sigmoidal function of (2) lead to the following simplification when we compute its derivative: this property can be utilized effectively in further studies on the qualitative properties of the siq model ( e.g. , study of the global stability of the disease-free and endemic equilibrium points of the siq model). an explanation of the siq parameters is given in table 1 . by fitting the siq model to the reported data for the confirmed cases and the quarantined cases, we obtain the following tant approach that can be addressed along these lines is the event-dependent approach, where the transmission probability could be dependent on the number of the newly-added or accumulated mers cases. parameters: 3 we performed simulations based on the estimated parameters. simulation results ( fig. 4 ) show that the proposed siq model can explain the observed data ( fig. 3 ) for the confirmed cases and the quarantined cases in the mers outbreak very well. our parameter estimation results show that the mers transmission probability, β( t ), is initially almost flat, then decreasing rapidly, and finally gradually reaching a saturation point (see the solid line of fig. 5 ). this has a natural interpretation, in that as information on the mers outbreak becomes more widely known with the passage of time, health authorities' and individuals' effort s against the epidemic intensify accordingly, which results in the mers transmission probability decreasing in the squashing-type fashion, as in fig. 5 . in retrospect, if the initial effort to reduce the mers transmission probability were more effective, the magnitude of β 0 could be decreased further. in order to investigate this aspect, we additionally performed simulations for a scenario with β 0 reduced to 90% of its estimated value (see the dashed line of fig. 5 ). fig. 6 shows that the infectious population could be reduced significantly if the aforementioned effort were successful. in order to clarify why it is important to consider the incidence rate of the type shown in (2) , we also considered the constant beta case, and provided the corresponding simulation results ( fig. 7 ) . comparing figs. 4 and 7 shows the superiority of using the time-dependent β. since quarantine and isolation strategies are the most important and effective measures against the outbreaks of disease when one does not have valid medicines and vaccine (see e.g. , castillo-chavez et al., 2003; day et al., 2006; yan and zou, 2009; yan et al., 2007 ) , one can view the effort s of implementing quarantine and isolation strategies as the actions that control the entire model. in this paper, we utilize optimal control theory ( lenhart and workman, 2007; lewis and syrmos, 1995 ) for the possibility of improving our control effort s. note that recent applications of optimal control theory are increasingly used in communities of biological systems ( e.g. ( buonomo and messina, 2012; joshi et al., 2006; jung et al., 2002; kirschner et al., 1997; de pillis et al., 2007 ) ), and in particular, they have been widely used and discussed in the control of epidemics ( e.g. caetano and yoneyama, 2001; feng et al., 2009; gupta and rink, 1973; joshi et al., 2006; lenhart and workman, 2007 ) . by incorporating the control inputs, q * ( t ) and θ * ( t ) into our siq model (1) , one can obtain the following state equations for the controlled model: note that for the optimal control of (5) , it is enough to consider the first three variables only. hence, we consider the following controlled siq (c-siq) model: from the data fitting based on the siq model, we have already obtained the estimation results for the quarantine probability, q , and the isolation rate, θ . in retrospect, however, control-theoretic investigation is desirable for the purpose of improving our response to the outbreak. in the following, we consider the problem of improving the quarantine probability and isolation rate further with additional effort s ( q * ( t ) and θ * ( t )) by minimizing an objective function in the form of j = t f 0 g(i(t ) , q * (t) , θ * (t)) dt. note that in this problem, the quarantine probability and isolation rate at time t are represented by and the control inputs to be determined via optimal control theory are the additional effort s described by q * ( t ) and θ * ( t ). here g ( · ) should be reasonably chosen to reflect the relative importance of the quarantine and isolation effort s over the infection. more precisely, in order to minimize an objective function comprising the infection cost ( i.e. , the infectious compartment population) and the cost of implementing quarantine and isolation strategies, we consider the following optimal control problem: subject to the c-siq state equations (6) . in the cost rate of this problem, c q and c θ are the trade-off constants defining the relative importance of the implementation costs over the infection cost. note that the cost rate considers quadratic cost terms for the control inputs, which is a commonly used strategy in related control problems dealing with epidemicmodel-based systems ( e.g. , lenhart and workman, 2007 ) . also, note that the existence of an optimal control and corresponding optimal state trajectory comes from the convexity of the integrand of the objective function with respect to the control and the lipschitz property of the state system with respect to the state variables (see, e.g. , fleming and rishel, 1975 ) , and based on the existence, we can now rely on the pontryagin maximum principle (pmp) ( pontryagin et al., 1987 ) for an optimal solution. as is well known, the necessary conditions for an optimal solution of (8) can be obtained via the pontryagin maximum principle. for this, the hamiltonian h of the optimal control problem (8) is defined as and its costate equations can be obtained via 2 0 1 5 -0 5 -2 0 2 0 1 5 -0 5 -2 2 2 0 1 5 -0 5 -2 4 2 0 1 5 -0 5 -2 6 2 0 1 5 -0 5 -2 8 2 0 1 5 -0 5 -3 0 2 0 1 5 -0 6 -0 1 2 0 1 5 -0 6 -0 3 2 0 1 5 -0 6 -0 5 2 0 1 5 -0 6 -0 7 2 0 1 5 -0 6 -0 9 2 0 1 5 -0 6 -1 1 2 0 1 5 -0 6 -1 3 2 0 1 5 -0 6 -1 5 2 0 1 5 -0 6 -1 7 2 0 1 5 -0 6 -1 9 2 0 1 5 -0 6 -2 1 2 0 1 5 -0 6 -2 3 2 0 1 5 -0 6 -2 5 2 0 1 5 -0 6 -2 7 2 0 1 5 -0 6 -2 9 2 0 1 5 -0 7 -0 1 2 0 1 5 -0 7 -0 3 2 0 1 5 -0 7 -0 5 2 0 1 5 -0 7 -0 7 t 0 1000 2000 3000 4000 5000 6000 7000 s q (t) 2 0 1 5 -0 5 -2 0 2 0 1 5 -0 5 -2 2 2 0 1 5 -0 5 -2 4 2 0 1 5 -0 5 -2 6 2 0 1 5 -0 5 -2 8 2 0 1 5 -0 5 -3 0 2 0 1 5 -0 6 -0 1 2 0 1 5 -0 6 -0 3 2 0 1 5 -0 6 -0 5 2 0 1 5 -0 6 -0 7 2 0 1 5 -0 6 -0 9 2 0 1 5 -0 6 -1 1 2 0 1 5 -0 6 -1 3 2 0 1 5 -0 6 -1 5 2 0 1 5 -0 6 -1 7 2 0 1 5 -0 6 -1 9 2 0 1 5 -0 6 -2 1 2 0 1 5 -0 6 -2 3 2 0 1 5 -0 6 -2 5 2 0 1 5 -0 6 -2 7 2 0 1 5 -0 6 -2 9 2 0 1 5 -0 7 -0 1 2 0 1 5 -0 7 -0 3 2 0 1 5 -0 7 -0 5 from the optimality conditions, ∂h ∂ q * = 0 and ∂h ∂ θ * = 0 , we can also obtain the following condition for optimal control: also, if upper bounds for the nonnegative control inputs are forced, then by confining the control input to be nonnegative and subject to a positive upper bound q max and θ max , the optimal control of (8) can be written in the following form: from the above steps, we can conclude that any solution to the optimal control problem (8) must satisfy the following: s(0) = s 0 , by solving this boundary value ordinary differential equation (ode) problem numerically, we can obtain optimal control inputs for problem (8) . in order to illustrate the optimal control policy, we simulated the optimally controlled c-siq system. the parameters considered for the simulations are set to be the same as the estimation results except the control inputs, q * ( t ) and θ * ( t ). for the tradeoff constants of (8) , we used c θ = 10 , 0 0 0 , c q = 1 . the initial conditions for the simulations were also taken from the estimation results, i.e. , s 0 = 16 , 0 0 0 , i 0 = 1 , and s q 0 = 0 . the simulations considered two scenarios. the first scenario does not have bounds on the control inputs, while the second scenario has the bounds of q max = 0 . 007 and θ max = 0 . 05 . with the goal of keeping the infection level low with reasonable control effort s, we solved the boundary value ode problem (13) using bvp4c , the matlab function to solve boundary value problems for ordinary differen-tial equations). figs. 8-10 show the simulation results for the first scenario. figs. 8 and 9 show that under the optimal control of the first scenario, the best method of fighting the infection is to initially enter large amounts of q ( t ) and θ ( t ), and later after 30 days to slowly reduce them to zero. the resultant state trajectory of i ( t ) (the dashed line of fig. 10 ) shows that, with the optimal control strategy, the infectious compartment population can be reduced significantly compared to the original case (the solid line of fig. 10 ) . to consider the robustness of the quarantine and isolation control, we also conducted sensitivity analysis for the following cases: (1) when c is 10% lower and 10% higher; (2) when λ is 50% lower and 50% higher. for each case we simulated the resultant controlled system, and the results on the number of infectious individuals are shown in figs. 11 and 12 , respectively, for cases (1) and (2). comparing figs. 11 and 12 shows that c is more important for the quarantine and isolation control. simulation results for the second scenario, which deals with the bounded input case, are shown in show that under the optimal control of the second scenario, the best method of fighting the infection is to apply the maximum amounts of the control inputs from the start, and then to slowly reduce them after 30 days to zero. the state trajectory of i ( t ) resulting from the optimal control is shown in fig. 15 . note that the infectious compartment population can be reduced significantly even with the bounds on control inputs. from the simulation results ( figs. 8-15 ), we can conclude that the mers disease spread can be properly handled by the optimal control approach, and we can obtain a practical guideline, whereby quarantine and isolation effort s in the early stage are critically important in effectively controlling the mers outbreak. in recent years, global pandemic viral infections, including the 2003 severe acute respiratory syndrome, the 2009 h1n1 influenza, and the 2014 ebola outbreak, have been devastating but provided valuable experience in outbreak responses. for public health control, increased vigilance by health professionals and voluntary compliance by the public are essential in implementing rapid effective response interventions. in this study, we carried out an epidemiological assessment of the mers-cov outbreak in korea in order to provide a mathematical framework for understanding the complex dynamics of the pathogen spread and establishing efficient guidelines for implementing quarantine and isolation strategies. the following have been observed by the assessment: • by fitting the siq model, which employs a squashing-type function of fig. 2 for β( t ) , to the real data on the confirmed cases and the quarantined cases, we obtained reasonable performance, as shown in fig. 4 . also, it turned out that the resultant estimated parameters belonged to plausible ranges. by comparison, the conventional siq model utilizing a constant transmission probability could not explain the observed data well. • our nonlinear epidemiological models showed that the mers transmission probability decreased in the squashing-type fashion and then approached a saturation point in a timedependent manner. as information on the mers outbreak be-came widely known in the nation, effort s against this epidemic, including individuals hygienic behavior, and interventions by health care facilities and by authorities were accordingly strengthened. in our siq-based analysis, the inflection point for transmission probability was found to be t β = 21 . 8 , corresponding to a couple of days after 7 june 2015. interest-ingly, 7 june 2015 was the day when the korean government revealed the names of 24 mers-affected hospitals to the public. after releasing the names of affected medical facilities, the rate of increase in new confirmed cases abated. as a practical guideline to avoid another similar unexpected outbreak, we draw the conclusion that combined effort s in the early stage are critically important, and sharing information including the names of affected hospitals or countries, clinical situations, and prevention methods might be important for global public health control. • we applied optimal control theory to the controlled siq model with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. simulation results show that the number of the mers cases can be controlled reasonably well via the optimal control approach. in conclusion, this paper proposes a nonlinear epidemiological ode for the mers outbreak in korea, the siq model, in which the state variables are defined as the populations of four compartments ( s ( t ), i ( t ), s q ( t ), and c q ( t )), and the mers transmission probability, β( t ), is modelled by the time-dependent sigmoidal function. we performed the task of parameter estimation for the siq model, and the data fitting results explained the observed data for the confirmed cases and the quarantined cases in the mers outbreak very well. we also applied optimal control theory to the controlled siq model with the goal of minimizing the infectious compartment population and the cost of implementing the quarantine and isolation strategies. our simulation results show that mers propagation was controlled reasonably well via the optimal control approach. in future work, we will conduct further simulation studies, with the aim of revealing the strengths and weaknesses of the proposed method, and investigate stability and control issues for its various extensions, including a stochastic differential equation (sde) approach. we retrieved publicly available data ( ministry of health and welfare , moh ) from the centers for disease control and prevention and the ministry of health and welfare in the republic of korea. the data included information on the cumulative number of reported cases. the first mers case in korea was confirmed on 20 may 2015, and the numbers of newly confirmed cases and suspected patients who had been quarantined to prevent possible spread of the mers have reached 186 cases and 16,693 cases, respectively, as of 28 july 2015. the data also included a brief description of each confirmed case with exposure date and onset of symptoms, and they were sufficient to estimate our siq model for the mers outbreak epidemiology. in this model, we assumed mers is unlikely to spread to another region. we implemented the parameter estimation procedure as a matlab program.we used fminsearch , a matlab function for unconstrained nonlinear optimization, along with some changes of variables in order to carry out the data fitting procedure for the siq model (5) with the time-dependent variable β( t ) specified in (2) . the performance index (pi) used in the optimization for parameter estimation was defined as follows: p i = w 1 × ( square d error of c q ) + w 2 × ( square d error of s q ) . the objective function of (14) , pi , was based on the numbers of the confirmed mers cases and the quarantined cases between 20 may and 07 july. the weight values ( w 1 = 10 4 and w 2 = 1 ) were obtained empirically via a tuning process based on the training data. simulation results show that the above set of weight values yielded reasonably good fitting results. finally, note that a more sophisticated markov chain monte carlo (mcmc) algorithm ( e.g. , rasmussen et al., 2011 ) could be used for parameter estimation of the siq model. however, the use of fminsearch , which was simpler and more transparent, was sufficient for the purposes of this paper. pattern recognition and machine learning impact of vaccine arrival on the optimal control of a newly emerging infectious disease: a theoretical study optimal and sub-optimal control in dengue epidemics mathematical models of isolation and quarantine the impact of media on the control of infectious diseases when is quarantine a useful control strategy for emerging infectious diseases? timely identification of optimal control strategies for emerging infectious diseases deterministic and stochastic optimal control optimal control of epidemics effects of quarantine in six endemic models for infectious diseases optimal control methods applied to disease models optimal control of treatments in a two-strain tuberculosis model modeling infectious diseases in humans and animals optimal control of the chemotherapy of hiv amid panic, a chance to learn about mers optimal control applied to biological models optimal control media/psychological impact on multiple outbreaks of emerging infectious diseases ministry of health and welfare (moh) and center for disease control and prevention mers-cov outbreak in jeddah-a link to health care facilities chemotherapy for tumors: an analysis of the dynamics and a study of quadratic and linear optimal controls the mathematical theory of optimal processes inference for nonlinear epidemiological models using genealogies and time series effect of media-induced social distancing on disease transmission in a two patch setting media impact switching surface during an infectious disease outbreak control of epidemics by quarantine and isolation strategies in highly mobile populations optimal quarantine and isolation strategies in epidemics control isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome this work was supported by a korea university grant. key: cord-269437-0pvqvhqs authors: gastañaduy, paul a. title: update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov) — worldwide, 2012–2013 date: 2013-06-14 journal: mmwr morb mortal wkly rep doi: nan sha: doc_id: 269437 cord_uid: 0pvqvhqs cdc continues to work in consultation with the world health organization (who) and other partners to better understand the public health risk posed by the middle east respiratory syndrome coronavirus (mers-cov), formerly known as novel coronavirus, which was first reported to cause human infection in september 2012. the continued reporting of new cases indicates that there is an ongoing risk for transmission to humans in the area of the arabian peninsula. new reports of cases outside the region raise concerns about importation to other geographic areas. nosocomial outbreaks with transmission to health-care personnel highlight the importance of infection control procedures. recent data suggest that mild respiratory illness might be part of the clinical spectrum of mers-cov infection, and presentations might not initially include respiratory symptoms. in addition, patients with comorbidities or immunosuppression might be at increased risk for infection, severe disease, or both. importantly, the incubation period might be longer than previously estimated. finally, lower respiratory tract specimens (e.g., sputum, bronchoalveolar lavage, bronchial wash, or tracheal aspirate) should be collected in addition to nasopharyngeal sampling for evaluation of patients under investigation. an emergency use authorization (eua) was recently issued by the food and drug administration (fda) to allow for expanded availability of diagnostic testing in the united states. on june 7, 2013 , this report was posted as an mmwr early release on the mmwr website (http://www.cdc.gov/mmwr). cdc continues to work in consultation with the world health organization (who) and other partners to better understand the public health risk posed by the middle east respiratory syndrome coronavirus (mers-cov), formerly known as novel coronavirus, which was first reported to cause human infection in september 2012 (1) (2) (3) (4) . the continued reporting of new cases indicates that there is an ongoing risk for transmission to humans in the area of the arabian peninsula. new reports of cases outside the region raise concerns about importation to other geographic areas. nosocomial outbreaks with transmission to health-care personnel highlight the importance of infection control procedures. recent data suggest that mild respiratory illness might be part of the clinical spectrum of mers-cov infection, and presentations might not initially include respiratory symptoms. in addition, patients with comorbidities or immunosuppression might be at increased risk for infection, severe disease, or both. importantly, the incubation period might be longer than previously estimated. finally, lower respiratory tract specimens (e.g., sputum, bronchoalveolar lavage, bronchial wash, or tracheal aspirate) should be collected in addition to nasopharyngeal sampling for evaluation of patients under investigation. an emergency use authorization (eua) was recently issued by the food and drug administration (fda) to allow for expanded availability of diagnostic testing in the united states. as of june 7, 2013, a total of 55 laboratory-confirmed cases have been reported to who. illness onsets have occurred during april 2012 through may 29, 2013 ( figure 1 ). all reported cases were directly or indirectly linked to one of four countries: saudi arabia, qatar, jordan, and the united arab emirates ( figure 2 ). most cases (40) were reported by saudi arabia. four countries, the united kingdom (uk), italy, france, and tunisia, have reported cases in returning travelers and their close contacts (5) (6) (7) (8) . ill patients from qatar and the united arab emirates have been transferred to hospitals in the uk and germany. to date, no cases have been reported in the united states. who and cdc have not issued any travel advisories at this time; updated information for travelers to the arabian peninsula is available at http://wwwnc.cdc.gov/travel/notices/ watch/coronavirus-arabian-peninsula. the median age of patients is 56 years (range: 2-94 years), with a male-to-female ratio of 2.6 to 1.0. all patients were aged ≥24 years, except for two children, one aged 2 years and one aged 14 years. all patients had respiratory symptoms during their illness, with the majority experiencing severe acute respiratory disease requiring hospitalization. thirty-one of the 55 patients are reported to have died (case-fatality rate: 56%) (5) (6) (7) (8) . two cases in tunisia, in siblings whose father's illness was a probable case, and a case from the uk, were in persons with mild respiratory illnesses who were not hospitalized (5, 9) . information was not available for all cases; however, several patients had accompanying gastrointestinal symptoms, including abdominal pain and diarrhea, and many cases occurred among persons with chronic underlying medical conditions or immunosuppression, as reported to who (5, 9) . the original source(s), route(s) of transmission to humans, and the mode(s) of human-to-human transmission have not been determined. eight clusters (42 cases) have been reported by six countries (france, italy, jordan, saudi arabia, tunisia, and the uk) (5) among close contacts or in health-care settings and provide clear evidence of human-to-human transmission of mers-cov. the first documented patient-to-patient nosocomial transmission in europe was confirmed recently in france (10). the first french patient, a man aged 64 years with a history of renal transplantation, became ill on april 22, 2013, within 1 week after returning from dubai. he presented with fever and diarrhea. pneumonia was diagnosed incidentally on radiographic imaging, and he subsequently died with severe respiratory disease. the secondary case is in a man aged 51 years on long-term corticosteroids who shared a room with the index patient during april 26-29 and who remains hospitalized on life support. the incubation period for the secondary case was estimated to be 9-12 days; this is longer than the previously estimated 1-9 days (10). a larger cluster, consisting of 25 cases including 14 deaths, ongoing since april 2013 in the region of al-ahsa in eastern saudi arabia, also has included cases linked to a health-care facility (5) . cases have included health-care personnel and family contacts. an additional five cases, not linked to the cluster in al-ahsa, were reported recently in another region of eastern saudi arabia (5). thus far, no evidence of sustained community transmission beyond the clusters has been reported in any country. in some instances, sampling with nasopharyngeal swabs did not detect mers-cov by polymerase chain reaction (pcr); however, mers-cov was detected by pcr in lower respiratory tract specimens from these same patients. in the two patients reported by france, nasopharyngeal specimens were weakly positive or inconclusive, whereas bronchoalveolar lavage and induced sputum were positive (10). in consultation with who, the period for considering evaluation for mers-cov infection in persons who develop severe acute lower respiratory illness days after traveling from the arabian peninsula or neighboring countries* has been extended from within 10 days to within 14 days of travel. persons who develop severe acute lower respiratory illness within 14 days after traveling from the arabian peninsula or neighboring countries should be evaluated according to current guidelines (available at http://www.cdc.gov/coronavirus/mers/case-def. html). persons whose respiratory illness remains unexplained and who meet criteria for "patient under investigation" should be reported immediately to cdc through state and local health departments. persons who develop severe acute lower respiratory illness who are close contacts † of a symptomatic traveler who developed fever and acute respiratory illness within 14 days of traveling from the arabian peninsula or neighboring countries may be considered for evaluation for mers-cov. in addition, cdc recommends that clusters of severe acute respiratory illness be investigated and, if no obvious etiology is identified, local public health officials be notified and testing for mers-cov conducted, if indicated. to increase the likelihood of detecting mers-cov, cdc recommends collection of specimens from different sites (e.g., a nasopharyngeal swab and a lower respiratory tract specimen, such as sputum, bronchoalveolar lavage, bronchial wash, or tracheal aspirate). specimens should be collected at different times after symptom onset, if possible. lower respiratory tract specimens should be a priority for collection and pcr testing; stool specimens also may be collected. specimens should be collected with appropriate infection control precautions (available at http://www.cdc.gov/coronavirus/mers/case-def.html). testing of specimens for mers-cov currently is being conducted at cdc. fda issued an eua on june 5, 2013, to authorize use of cdc's novel coronavirus 2012 real-time reverse transcription-pcr assay (ncv-2-12 rrt-pcr assay) to test for mers-cov in clinical respiratory, blood, and stool specimens. this eua is needed because, at this time, there are no fda-approved tests that identify mers-cov in clinical specimens. this assay will be deployed to laboratory response * countries considered to be on or neighboring the arabian peninsula include bahrain, iraq, iran, israel, jordan, kuwait, lebanon, oman, palestinian territories, qatar, saudi arabia, syria, the united arab emirates, and yemen. † close contacts are defined as 1) persons who provided care for the patient, including health-care personnel and family members, or who had other similarly close physical contact, or 2) persons who stayed at the same place (e.g., lived with or visited) as the patient while the patient was ill. network (lrn) laboratories in all 50 states over the coming weeks. updated information about laboratories with the capacity to conduct mers testing with the ncv-2-12 rrt-pcr assay will be provided on cdc's mers website (http://www. cdc.gov/coronavirus/mers/case-def.html). in consultation with who, the definition of a probable case of mers-cov infection has been updated to also include persons with severe acute respiratory illness with no known etiology with an epidemiologic link to a confirmed case of mers-cov infection. until the transmission characteristics of mers-cov are better understood, patients under investigation and probable and confirmed cases should be managed in health-care facilities using standard, contact, and airborne precautions. as information becomes available, these recommendations will be reevaluated and updated as needed. recommendations and guidance on case definitions, infection control (including use of personal protective equipment), case investigation, and specimen collection and testing, are available at the cdc mers website (http://www.cdc.gov/ coronavirus/mers/index.html). the mers website contains the most current information and guidance, which is subject to change. state and local health departments with questions should contact the cdc emergency operations center (770-488-7100). cases associated with a cluster cases reported in al-ahsa governorate cases not associated with a cluster travel history associated with cases occuring outside of arabian peninsula and neighboring countries * dots representing the cases are not geographically representative of the exact location of the residence of the patient. severe respiratory illness associated with a novel coronavirus-saudi arabia and qatar novel coronavirus associated with severe respiratory disease: case definition and public health measures isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group global alert and response (gar): novel coronavirus infection -update (middle east respiratory syndrome coronavirus) updated rapid risk assessment: severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) european centre for disease prevention and control. epidemiological update: additional confirmed cases of middle east respiratory syndrome coronavirus (novel coronavirus) in france, saudi arabia, and tunisia world health organization. global alert and response (gar): novel coronavirus summary and literature update family cluster of middle east respiratory syndrome coronavirus infections clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission key: cord-016842-sow7k53m authors: an, jisun; kwak, haewoon title: multidimensional analysis of the news consumption of different demographic groups on a nationwide scale date: 2017-08-02 journal: social informatics doi: 10.1007/978-3-319-67217-5_9 sha: doc_id: 16842 cord_uid: sow7k53m examining 103,133 news articles that are the most popular for different demographic groups in daum news (the second most popular news portal in south korea) during the whole year of 2015, we provided multi-level analyses of gender and age differences in news consumption. we measured such differences in four different levels: (1) by actual news items, (2) by section, (3) by topic, and (4) by subtopic. we characterized the news items at the four levels by using the computational techniques, which are topic modeling and the vector representation of words and news items. we found that differences in news reading behavior across different demographic groups are the most noticeable in subtopic level but neither section nor topic levels. demographics play an important role in news consumption. what women in their fifties read is very different from that of men in their twenties. understanding such differences in news consumption can potentially help journalists to pitch news articles better, help editors to decide which ones to put on the front page, and help computer scientists design new algorithms for recommending articles. that is the reason why news consumption of demographic groups has been actively studied in both the domains of journalism study and computer science [14, 18, 24, 31, 39, 40] . the previous literature regarding news consumption in the study of journalism has mainly focused on the gender differences in the consumption of news genres [18, 24, 40] primarily due to the lack of other detailed data. on the other hand, in computer science, previous studies have mainly focused on developing models for predicting clicks and for news recommendations at an individual level with large-scale data [14, 31, 39] . in this study, we attempt to bridge these two worlds and uncover the differences in news consumption across demographic groups by large-scale news consumption data. specifically, we aim to quantify such differences in four dimensions: actual news items, sections, topics, and subtopics. while the existence of the "differences" is expected, our multidimensional analysis shows how such differences can be differently captured in each dimension. for this study, we collected and analyzed the daily top 30 news items for each gender (male and female) and age group (10s, 20s, 30s, 40s, and 50s) in daum news, the second most popular news portal service in south korea, for the entire year of 2015. the number of the unique news items collected is 103,133. daum news can have the accurate, not self-reported, information on the user's age and gender based on one's social security number. this practice is not common in the western web services. in south korea, to join a website, it is mandatory for identity verification to provide the social security number that contains your birth year and gender. also, daum news has a strong user base that reads news with a logged-in status mainly because daum news offers a wide range of services, which include e-mail, internet community, or messenger, for example, based on the logged-in status. sex-typed media preference has long been investigated for various types of media. regarding movie and tv genres, studies have found that women are more likely to watch tragedies, soaps, dramas, medical serials, and romances; men, on the other hand, tend to prefer horror, sports, and action and adventure content [21, 37, 38, 41, 42] . psychological research suggests that such sex-typed media selections might well be rooted in societal gender stereotypes -men are expected to achieve more, and women are expected to interact more [16] . scholars of the study of journalism have examined how demographics, such as sex or age, relate to news-seeking behavior and news preferences [15] . when attending to news, the sexes typically pursue remarkably different interests -in terms of topics, men tend to follow news on politics, sports, and business and finance, whereas women turn to news about community and health issues [18, 40] . also, women read more about social/interpersonal issues than men, and men read more about achievement/performance than women [24] . scholars suggested that the origin of gender difference in news consumption is not considered from biological differences but from the psychological traits led by the sex-typed socialization [33] . while news preferences have been extensively studied for the news section, little is known about the topic or subtopic preferences of different demographic groups' news consumption. the key reason for this oversight is mainly due to a lack of data. most of the previous work is based on surveys or experiments in the laboratory setting [18, 24, 40] . by contrast, our work relies on the longitudinal data collection where a huge number of korean internet news readers are unobtrusively monitored. since news sites have been publishing online, we now have access to large-scale data of individual news consumption with detailed personal profiles. computer scientists have addressed news-related questions but with different interests and approaches from scholars in journalism or communication studies. news-related research by computer scientists has predominantly focused on modeling news sharing behavior [3] [4] [5] , news diffusion [1, 7, 17, 19, 26, 29, 36, 44, 49] , and modeling the relative prominence of items or topics [2, 6, 8, 13, 25, 28] . computer scientists also have exploited news consumption patterns of individuals mainly for building a better news recommendation system to give readers a personalized experience when reading the news. systems that make recommendations according to demographic classes were initially introduced [39] . more recently, the demographic information has often been used as a feature of those models [14, 31] . however, the demographic differences in news consumption have not been fully uncovered, particularly in different dimensions of news such as topic or subtopic. online news consumption in south korea has increased drastically. about 86% of south korean people access news online at least once a week 1 . given the 92% internet and 85% smartphone penetrations, such a drastic increase makes sense. web portal sites such as naver and daum are especially popular digital news platforms. due to the extreme popularity of these portals, news providers in south korea have been eager to publish their content via portals for years. in 2015, naver and daum formed the committee for the evaluation of news partnership, complete with a set of ethical standards to help decide which providers should be eligible to supply news to portals. as a result, we can reasonably say that news providers and the news readers of daum news are representative of the general south korean news media and population, respectively. daum is the second largest web portal in south korea, followed by naver. daum plays a significant role in providing a place for accessing online news to south korea; 41% of south koreans (24.6 m users) access daum news on a weekly basis. daum news provides different ways to explore news articles, for example, by its recency, by current issues, by regions, or by popularity based on the number of views or the number of comments. a unique feature of daum news is that it provides the top 30 most popular news articles on a particular day for each gender and age group, which are [male or female] and [10s, 20s, 30s, 40s, and 50s and above]. we note that naver does not provide a ranked list of news articles by different demographic groups, and thus, we focus on daum news data even though it is the second largest news portal in south korea. we crawled the top 30 most popular articles for different age and gender groups of each day for a one-year period (01/01/2015−31/12/2015). we carefully designed our crawlers not to degrade the performance of daum's web servers. in our data set, we have 103,133 listed news items with 54,274 unique news titles. for each news item, we have its unique item id, demographic group, rank in the group, title, summary, news source, and published date. we note that news articles about entertainment and sports are not included in the lists due to daum news's policies. we need to determine the section, topic, and subtopic of the news items we collected. next, we will briefly describe the methodology that we adopt for the analysis. defining the operational coding scheme for news has been a central issue for communication studies. in this study, we introduce four dimensions of news (sections, topics, subtopics, and individual news items) by which we investigate news consumption patterns. the diagram of the four dimensions of news is illustrated in fig. 1 . the first dimension is the news section (e.g., society, politics, etc.), which is a category of news often adopted in newsrooms and which has been popularly used for sex-typed news preference studies. next, a news topic refers to a specific happening (e.g., a mers outbreak in south korea or child abuse case in a daycare center). within a topic, we further explore subtopics by distinguishing different aspects of a topic. finally, there is a dimension where all individual news items are aligned. we then can characterize the entire news collection (e.g., a news collection of south korea in 2015) according to the four dimensions. the most common way of categorizing news is perhaps to use a news section as defined by the news media. especially for online news, which news section a news article belongs to can be inferred from the meta information embedded in the news urls. for example, the url http://media.daum.net/society/labor/ newsview?newsid=20160709180100906l is categorized as "society." we parsed all of the collected urls and extracted the section information. figure 2 shows the proportion of news items for each demographic group for each news section. in our dataset, daum news has seven different news sections, which are culture, digital, economic, foreign, life, politics, and society. we note that the entertainment and sports sections are not included in the dataset. we observe that female groups are more alike than male groups in terms of news section preference. for female groups, society and culture were the two most popular news sections. for age10 male and age20 male groups, society, foreign, and politics were more popular than the others. then, for the age30 male and age40 male groups, their interests were digital (i.e., technology news), economics, and politics. we also find one noticeable behavior of the age50 male group: 78% of their top 30 most popular news items over a year are about politics. these news section preferences align well with previous work on sex-typed news consumption observed internationally [18, 40] . the news section category provided by daum news abstractly captures the high-level topic of a news item. in this study, we go beyond a mere news section preference and examine whether the gender-or age-specific topic or subtopic preferences exist. to do that, we need to understand the semantic content of the news articles. for example, news about "violence at daycare" and "a killer of his family" are both categorized under society, but they are two different topics. we automatically discover a topic-specific categorical structure from a set of titles and classify each news article based on it. topic modeling techniques such as probabilistic latent semantic indexing (plsi) [23] , latent dirichlet allocation (lda) [11] , and hierarchical dirichlet processes (hdp) [46] can be employed to induce topics from the set of news titles. we manually compare the three methods in terms of the interpretability of the induced topics and the quality of the clustered news titles for each of the included topics. for two slices of subsets, may and october 2015, we examined the top 20 topics resulting from the three methods with their top 30 words. for our dataset, plsi's top topics were mapped more clearly with news events in the corresponding month than lda's and hdp's topics were. one possible reason is that lda and hdp were penalized more for modeling topics with short titles than plsi was. thus, we use plsi to detect candidate topics. there are also specialized topic models for short texts, such as a biterm topic model (btm) [48] , and we leave it as a future work to improve our topic categorization method. one problem with these topic modeling techniques is that they are not timesensitive. the following two news titles, "anyang killer -a man killed his family" and "wife killer -a man killed his wife but did not show any grief," are likely to be categorized as the same topic even though one event happened in january 2015, and the other happened in october 2015. to handle this problem, we first split our dataset by month, and then we build a plsi model for each of the monthly datasets. each plsi model induces 100 topics, giving us 1,200 candidate topics in total. each topic is represented by word distributions. we then aggregate similar candidate topics by examining the representative keywords for topics (words with the highest probabilities). in our case, we aggregate topics if they share more than three top words. after this, we classify each news title as one of these candidate topics based on the score given by the plsi model. finally, we split every candidate topic further into multiple topics by considering the publication times of news items. only news items published for days in a row are tagged as the same topic, resulting in 2,122 topics. news titles without any matching candidate topic are considered stand-alone topics. altogether, we have 41,452 topics. across all topics, the most popular news event is the middle east respiratory syndrome (mers) outbreak in south korea. we find 2,756 matching news items regarding mers. the most frequent words are mers, a vice prime minister, confirmed patients, infected, hospital, daejeon, tourists, etc. validation of topic categorization. for the purpose of validation, we crossmatched the set of news items extracted by the topic-model-based clustering method with the set of news items extracted by a keyword-based method. we focus on one particular news topic, mers outbreak in south korea. the outbreak lasted for a month and a half starting on may 20th. a total of 186 cases occurred during the outbreak, with a death toll of 36. due to the outbreak, 2,208 schools were temporarily closed, and 16,693 people were quarantined. mers was the most sensational news event in south korea in 2015. we extract news items whose title contained the word "mers", which results in 961 news items. by including more keywords, we may be able to extract more news items. however, we use a single word to be sure that all the retrieved news items are relevant to the news event. we find that 97% of news items about the mers outbreak (929 out of 961 news items) overlap with those by the topic modeling based method. then, to examine the relevance of those articles without the word "mers," we randomly select 100 articles that do not explicitly include the word "mers" and examine what they are about. we find that all are relevant to the mers. this indicates that our method can extract news items even when the title of news does not include a key topic word. however, a set of topic words (not one word) will also be able to retrieve all relevant news items. once we have a set of news articles on a certain topic, we further need to group them by subtopic. to this end, we train our data to represent each news item on a vector space and then cluster news items. semantic vector space models of language represent each word with a real-valued vector. firstly introduced by hinton [22] , the methods have been extended using neural network [9, 45] and applied for the practical uses. in recent years, milkolvo's skip-gram and distributed bag-of-word (dbow) models [34] are popularly used for learning vector representation of words and documents due to its computational efficiency. in the skip-gram model, the objective is to predict a word's context given the word itself, whereas the objective in the dbow model is to predict a word given its context. more recently, the concept of embeddings has been extended beyond words to a number of text segments, including phrases [35] , sentences and paragraphs [27] , and documents. adopting the document representation method, we learn distributed representations of news items in our daum news collection. each of news items is represented as low-dimensional vectors and are jointly learned with distributed vector representations of words using a dbow model explained in [27] . in this vector space, two news items of semantically similar meaning are located nearby. in our news embeddings (henceforth news2vec), every news item is mapped to a unique vector in a matrix that represents news items, and every word is mapped to a unique vector in a matrix that represents words. we denote by n2v the s × f matrix of s which is the sum of n news items (n 1 , n 2 , . . . , n n ) and f dimensions (f 1 , f 2 , . . . , f f ). a great advantage of learning distributed representation vectors for news in this way is that the algorithm is not sensitive to news item length and does not require specific tuning for word weights. the row of the matrix n2v, n i , is a vector of f dimensions representing the i-th news item. the dimension of vector f is set to 200, and the model is trained with 40 epochs. once we have news2vec, we apply the hierarchical clustering method using ward's method [43] to this resulting matrix to cluster news items of the same subtopic. the hierarchical clustering method builds a dendrogram among entities. then, one can cluster entities based on the dendrogram. its main advantage is that the dendrogram is computed only once regardless of the number of clusters of interest. once the dendrogram is built, we can simply choose the number of clusters (k) of our interest. validation for subtopic categorization. to evaluate the news2vec based subtopic clustering method, we prepared a corpus in which each news item is labeled by two authors. we used 961 news items about mers extracted by the keyword-matching method. then, we manually classify news items by subtopics. we conduct a qualitative content analysis to develop a taxonomy of subtopics for mers news. following an open-coding method [47] , we identify the subtopics of mers news in a two-phase process. we first read titles and descriptions of 100 news items to develop an initial coding scheme and then used an affinity diagramming technique [10] to iteratively develop a classification scheme for subtopics until a new subtopic did not emerge. table 1 lists the resulting 10 subtopics. the individual authors manually classified all news items into one of the subtopic categories. using the delphi method [30] , after each researcher independently coded the titles, we then iteratively compared and recoded the news items as necessary until we came to an agreement. the ten subtopics regarding the mers with the number of corresponding news items are listed in table 1 . st1 "reporting new cases" was the most popular subtopic with 254 news articles in our data set, followed by st7 "responsibility of government" with 153 news items and st9 "economical consequences" with 106 news items. we then use this labeled data for the evaluation of our subtopic categorization method. we first learn news2vec using entire daum news data. then, we cluster those 961 news items into ten groups (k = 10) using vectors from the resulting news2vec. we evaluate the resulting subsets of news items with manually tagged clusters. for each detected subset, we find the best matched manual cluster based on the proportion of matching news items. across ten subsets, our news2vec subtopic classification achieved an 82.2% matching rate on average where the maximum matching rate is 92.8% and the minimum is 77.2%. in our method, selecting the k is challenging. here, we propose one possible solution to assist in the k selection procedure. the idea is that the average similarity scores for all pairs of news items within the same subtopic (s within ) should be smaller than across the subtopics (s across ). when news items are in vector representation, one can use any distance measures, such as euclidean distance or cosine similarity, to measure the similarity score between two news items. those two values, once found empirically, can play a role as thresholds for selecting the k for different topics. in our evaluation data set, s within is 0.02 and s across is 0.15 when using cosine similarity. for further evaluation, we use these two values to find the subtopics of another topic, "daycare child abuse." for 342 news items regarding the topic, we find k equals five. the manual inspection reveals the following five subtopics emerged: (1) what the teacher did to a child; (2) how cruel the teacher is; (3) investigation and prosecution; (4) a new regulation on cctv installation at daycare centers; and (5) other cases of child abuse. we now quantify differences in news consumption across demographic groups in four dimensions: (1) by actual news item, (2) by section, (3) by topic, and (4) by subtopic. as a first attempt to compare the news consumption of different groups, we look at actual news items. we measure the similarity among groups based on commonly consumed news items (by their unique news ids) among the top 30 articles consumed by each group over a one-year period. we use jaccard similarity to compute group similarity. for the two sets, a and b, the jaccard similarity is given by : j(a, b) = |a∩b| |a∪b| . in our case, let a and b be sets of news items corresponding to the two groups to be compared. strictly, |a ∩ b| would translate to the count of the news items matched across the sets of a and b. figure 3(a) shows the jaccard similarity among groups as a heatmap. for example, the pair of age10 female and age20 female has a jaccard score of 0.15. this means that, among the union set of all their consumed news items, 15% are common. the higher the similarity score is, the more news items are viewed in common between the two groups. we find that within same-sex groups, the similarity generally increases as the age difference decreases, with female groups have a stronger tendency of it than male groups (the average similarity score among all pairs of female groups is 0.217 while that of male groups is 0.071). however, we observe two exceptions, age10 female and age10 male. they are more similar to the age30 or age40 than the age20 same-sex groups. in fig. 2 , we can see that the age10 groups have more politics and foreign news items in the top lists, indicating their similarity to older groups. we then find strikingly low similarity scores between different sex groups. age40 male and age50 male have almost no news items in common with the female groups, and the same happens for the age40 female and age50 female groups. with these results, we can conclude that the set of popular news items that females consume is very different from what males read. we have shown that there is a striking difference between the popular news items for the male groups and those of the female groups. now, we will examine the news consumption of those groups in terms of sectional interests. this analysis will tell us whether the existing framework of news consumption based on sex or age is also found in korea. for each group g, we created a vector s g = (w 1,g , w 2,g , . . . , w s,g ) in which each dimension corresponded to a predefined section from daum news where s = 7 in our case (see 7 sections in fig. 2) . the weight w s,g was computed by the proportion of the news items in the section s for the group g. we then computed the cosine similarity between two vectors to compare the sectional interests of two groups. the results are shown in fig. 3(b) as a heatmap. for female groups, we find high similarity scores between all pairs (>0.97), showing that the proportion of news items in each section is similar in each group. a similar pattern is also observed for male groups but to a lesser extent, and with one exception, age50 male, which shows a very different sectional interest. the reason is that they exclusively read political news -83% of the top 30 news items for a one-year period are about politics (see fig. 2 ). male groups are further split into two groups, as age10 male and age20 male are more similar to female groups, but age30 male and age40 male have sectional interests distinct from those of female groups. this partly supports the traditional sex-typed news consumption theory -our data set also shows different sectional preferences in different gender groups. however, we find such differences are driven more by age30 male and age40 male and less by age10 male and age20 male. in summary, the sectional interests seem to be alike across all groups except age50 male. considering that news consumption largely depends on current, local issues, this could make sense. however, given the striking differences in common news items, the fact that groups largely share sectional interests is still surprising. we now move onto the similarities in the topics that different groups consume. given that sectional interests are similar among groups, but not the actual news items, it is intuitive to think that even if two groups are visiting the same news section, such as society, they might consume different topics -older people might read more about "baby killer" while young people read more about "violence at school." to investigate such topic-specific differences in news consumption, we map each news item to a specific topic. the topics are identified by the method we described in sect. 4.3. then, we quantify the importance or the level of attention to a specific topic for a group by computing the lifespan. we define the lifespan of a topic as the longest period of time when that topic appeared on the top 30 list for each group. we then measure the similarities between groups based on the importance of the different topics. we select topics that are consumed by at least two groups, resulting in 36,134 topics and compute each topic's lifespan for each group. this gives us a ranked list of topics for each group, and we use spearman's rank correlation coefficient (ρ) to compare the two ranked lists. figure 3(c) shows the results as a heatmap. all pairs of rankings are statistically significantly different (p < 0.05). in this heatmap, we compare pairs of values. for example, a value of 0.9 between age40 female and age40 male is hard to interpret by itself. comparing one similarity score to other entries, one observes that this value is higher to that for the 'age40 male' -'age30 male' pair or 'age40 male' -'age50 male' pair. simply put, one could claim that gender differences lead to more strongly pronounced news consumption than 10 years of age difference. by comparing pairs of values, we observe that age differences play important roles in news consumption -a similar pattern was also found when looking at common news items in sect. 5.1. given that a pair of different sex groups have few common news items consumed, the high similarity between two ranked list of topics (ρ > 0.8) is striking. this means that all users of daum news are interested in similar topics, but what they read is different; less than 10% of news items on average were in common for those pairs of different sex groups while the average ρ is 0.65 for these pairs. we also find that two groups, age10 male and age10 female, are generally more different from other groups, confirming the existence of an age gap between 10-year-olds and others. we also note that while the age50 male group has very different sectional interests, it has similar topic preferences to those of other groups. we firstly observed that demographic groups show such different news consumption patterns at news item level. then, the high similarity scores at section and topic levels tell us that the overall news consumption is largely driven by current issues. however, groups still have distinct news consumption patterns. this suggests that news consumption even for one particular topic may be very different across groups. for this analysis, we use our evaluation data set and focus on the subtopic consumption regarding the mers outbreak. the mers outbreak was a deviant event, and all ten demographic groups have at least one news item about mers. however, the volume of news items about mers is different across groups. news items about mers are more popular in female groups than in male groups-on average, the female group has 427.4 popular news items about mers while that of the male group is 123.8. we then quantify the differences in mers news consumption in terms of the content between two groups. to do this, for each group, we rank the subtopics of mers outbreak in table 1 by the number of news items. then, we test the similarity between two groups by computing the spearman's ranking correlation coefficient. this will tell us which two groups have the most similar consumption of subtopics about mers. figure 3(d) shows the results as a heatmap. all pairs of rankings are statistically significantly different (p < 0.05). from the heatmap, we observe that (1) the popular news is more similar within the same sex groups than within the different sex groups; (2) female groups are more similar to each group than male groups are; and (3) age differences matter, except in the age10 male and age10 female groups. interestingly, all three of these observations are also found in our previous analysis that compares actual news items in sect. 5.1. all these results lead us to conclude that all groups are generally interested in similar news sections or topics; however, for the same topic, they are attracted to different subtopics, leading to the big differences in popular news among groups. to gain insights into how popular news items about mers differ between different demographic groups, we extract the most discriminative words in news titles for each group. we focus on the group-specific words of news titles, specifically on those with a high phi score, the chi-square test statistics [12] , for discriminating between one group and others (e.g., age10 female vs. non-age10 female (all other groups)). table 2 shows the top 20 words ranked by phi. two authors of this work translated korean words to english words. some interesting differences were observed. overall, female groups are likely to check the status of the mers outbreak, such as how many people are infected (the number of patients, death, this week), the symptoms of mers (high fever, cytokine storm), and the protection against mers (mask, gloves). the age30 female group showed an interest in news about pregnant women who had been diagnosed with mers and other women's cases. the age40 female group, in particular, was more interested in the status of closed schools and other education-related topics. on the other hand, the male groups were more interested in the political issues surrounding the mers outbreak, the accusations towards the government's response to the mers outbreak (e.g., ruling and opposition parties, the lack of a proper response, misreporting, false propaganda), and the responsibility of politicians. news2vec offers an opportunity to visualize each news item in the vector space by applying t-sne, a widely-used dimensionality reduction based on manifold learning [32] . figure 4 shows where each news item (colored circle) consumed by each gender (fig. 4(a) ) or age ( fig. 4(b) ) group is located in the vector space. in the figure, closed circles are that news items have similar representations in the vector space and thus fall in similar subtopics. for the clarity of the visualization, we focus on the news items consumed by a single demographic gender or age group. figure 4 (a) shows a better clustered structure than fig. 4(b) , meaning that gender difference is well aligned with the difference of vector representation of news items than age difference. in fig. 4(b) , we can also see some clustered structures, such as groups of green circles (news on patients' deaths read by 30s), blue circles (news on choongbuk-daejeon regions read by 10s), red circles (news on the closing of schools read by 40s), and purple circles (news on the prime minister read by 50s) from the top to the bottom, while colored circles are mostly dispersed over the space mostly. this is another evidence that gender difference is more noticeable than age difference at the subtopic level. to the best of our knowledge, this is the first study to conduct a multidimensional analysis of the news consumption of different demographic groups on a nationwide scale. differences in news consumption between different demographic groups exist among south koreans. we look into news consumption at different levels and find that section and topic preferences are similar across groups, but subtopic preferences are not. this means that only the behavioral differences in the subtopic level can explain the strikingly low numbers of common news items across different demographic groups, whereas the differences in the news section or topic levels cannot. in summary, while different demographic groups are interested in similar topics, they read news articles belonging to different subtopics, indicating that subtopics make the news consumption of the different groups different. for the following studies, our work suggests that the differences between demographic groups should be examined at the appropriate level. one potential limitation is that we analyze a single news portal service, even though it is an extremely popular service in south korea. to the best of our knowledge, daum is the only data source where (1) the user-registered demographic information is credible, and (2) the user base spans all generations and parties. we are willing to extend our approach to new data sources that satisfy above two conditions so that we can find demographic differences in news consumption. we note that all the users are exposed to the same layout and the same items if they visit the website at the same time. thus, the ways to show news, such as news clustering or adaptive layout, cannot selectively influence on a certain user segment. while the differences in news consumption between demographic groups can be explained partly by different interests on subtopics, there could be other latent factors such as sentiments or frames of news. for instance, grabe and kamhawi found that men recognize and respond more to negatively framed messages, while women are more aroused by and engaged with positively framed messages [20] . by adding another dimension to our news dimensions, we can understand human behavior on consuming news in depth. in this work, we examined the most critical aspects of news, which are section, topic, and subtopic, and saved the other dimensions for the future work. our results also bring the practical implications for news organizations. our characterization of user behavior allows them (1) to gain a better understanding of what people consume and (2) to produce more relevant and engaging content for different demographic groups. such demographic-based profiling can help tackle the cold-start problem for new users. by simply knowing the gender and age of a new user, one can provide a better user experience when reading online news. the opposite direction of the inference can also be useful. given the first few sets of news articles, one can infer the demographics of the reader. this will be particularly useful if the demographics of users are not readily available on news sites. tracking information epidemics in blogspace a peek into the future: predicting the evolution of popularity in user generated content icwsm 2011: proceedings of the 5th international aaai conference on weblogs and social media sharing political news: the balancing act of intimacy and socialization in selective exposure partisan sharing: facebook evidence and societal consequences trends in social media: persistence and decay the role of social networks in information diffusion the pulse of news in social media: forecasting popularity a neural probabilistic language model contextual design: defining customer-centered systems latent dirichlet allocation statistical inference can cascades be predicted? personalized recommendation on dynamic content using predictive bilinear models online news consumption: a structural model linking preference, use, and paying intent models of the self: self-construals and gender the anatomy of large facebook cascades news in online and print newspapers: differences in reader consumption and recall the structural virality of online diffusion hard wired for negative news? gender differences in processing broadcast news music and music videos distributed representations probabilistic latent semantic indexing the gender news use divide: americans' sextyped selective exposure to online news topics prediction of retweet cascade size over time what is twitter distributed representations of sentences and documents using a model of social dynamics to predict popularity of news meme-tracking and the dynamics of the news cycle the delphi method: techniques and applications personalized news recommendation based on click behavior visualizing data using t-sne gender as a social category efficient estimation of word representations in vector space distributed representations of words and phrases and their compositionality information diffusion and external influence in networks exploring the paradox of the enjoyment of sad films the respondent gender gap a framework for collaborative, content-based and demographic filtering news audiences increasingly politicized-online news audience larger, more diverse: biennial media consumption sensation seeking, television viewing motives, and home television viewing patterns selective viewing: cognition, personality and television genres clustering methods differences in the mechanics of information diffusion across topics: idioms, political hashtags, and complex contagion on twitter continuous space language models sharing clusters among related groups: hierarchical dirichlet processes qualitative research: analysis types and software tools a biterm topic model for short texts modeling information diffusion in implicit networks key: cord-275313-mfyff9ne authors: modjarrad, kayvon title: treatment strategies for middle east respiratory syndrome coronavirus date: 2016-01-01 journal: journal of virus eradication doi: nan sha: doc_id: 275313 cord_uid: mfyff9ne middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than 1600 people, resulting in almost 600 deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of this novel pathogen have created an urgent need for effective public health countermeasures, including safe and effective treatment strategies. despite the relatively few numbers of cases to date, research and development of mers-cov therapeutic candidates is advancing quickly. this review surveys the landscape of these efforts and assesses their potential for use in affected populations. respiratory tract infections are the leading cause of mortality in resource-limited settings, accounting for more than 4 million deaths each year globally [1] . epidemic-and pandemic-prone respiratory viruses are the aetiological pathogens in many cases, and have caused several of the most prominent infectious disease outbreaks of the past two decades: these include h5n1 influenza in 1997, severe acute respiratory syndrome (sars) in 2003 and pandemic h1n1 influenza in 2009. most recently, middle east respiratory syndrome coronavirus (mers-cov) has emerged as a novel cause of severe acute respiratory illness after first being identified in a saudi arabian patient in 2012 [2] . although initially restricted to the arabian peninsula, this emerging pathogen has respectively infected and killed more than 1600 and 580 people on four continents across 26 countries [3, 4] . phylogenetically related to sars-cov [5] , mers-cov has a similar clinical presentation [6] [7] [8] [9] , albeit with a higher case fatality rate (~40% versus 10%) [3] [4] [5] . dromedary camels serve as the principal animal reservoir for this virus; and zoonotic spillover from dromedaries to humans has, thus far, driven the course of the epidemic [10] [11] [12] [13] [14] [15] [16] [17] [18] . although humanto-human transmission has been documented -particularly in the context of nosocomial outbreaks [19] [20] [21] [22] [23] [24] -the spread of mers-cov is inefficient and unsustained, as reflected in an estimated reproduction rate of no higher than 0.7 [25, 26] . mers-cov is an enveloped, single-stranded, positive-sense rna virus that comprises a 30-kilobase genome that codes for four structural proteins and an rna polymerase [27] , typical of the coronaviridae family ( figure 1 ). the most immunogenic of these proteins is the virus' only surface glycoprotein, spike (s) [28] [29] [30] that mediates viral attachment and fusion via the host cognate receptor, dipeptidyl peptidase 4 (dpp4) [31] . although the broad principles of the virus' life cycle and its mechanisms of pathogenesis are beginning to be understood, this knowledge has not yet translated to a licensed therapy or vaccine. much of the work to develop safe and effective mers-cov countermeasures has centred on vaccines, but the relatively low prevalence of the disease, the sporadic nature of the case clusters and the dearth of detailed knowledge on chains of transmission highlight the need for greater investments into the discovery of effective therapeutic and secondary prophylactic regimens for infected and exposed individuals. efforts to research and develop treatment strategies for mers-cov are accelerating but remain limited in their scope and stage of advancement. there are few novel compounds being studied that are specific for mers-cov molecular targets, as most treatment options, investigational and licensed, are being repurposed from their use for other rna viruses or other non-infectious diseases. the current landscape of mers-cov therapies, therefore, is dominated by an armamentarium of repositioned drugs with in vitro activity against mers-cov replication, but is also speckled with agents that are directed towards and derived from host immunity. the current review surveys the landscape of therapeutic products in each category and assesses their potential for advanced testing and development. respiratory and circulatory support, preservation of renal, hepatic and neurological function, and prevention of secondary infections. beyond implementing basic principles of critical care medicine, immune-based therapies have been used most commonly during both the sars-cov pandemic of 2003 and the current mers-cov epidemic, each time yielding equivocal results. there have been some promising animal data where combination treatment with ribavirin and interferon (ifn)-α2b improved clinical outcomes in mers-cov-infected non-human primates (nhps). however, treatment was initiated very soon after viral challenge (~8 hours), a window that is unlikely to be replicated in a real-world clinical setting [32] . various ifn regimens, in combination with ribavirin, have been intermittently administered to severely ill patients, although typically in an ad hoc manner and in the absence of systematic evaluation [33] [34] [35] [36] [37] . individual case reports and uncontrolled case series not only limit determination of whether an intervention works but if it is safe as well. ribavirin, for example, is a potent nucleoside analogue that has been used with varying measures of success against a range of rna viruses [38] . however, patients can experience significant toxicities when given the drug alone or in combination with an interferon, including but not limited to haemolytic anaemia and metabolic abnormalities. interferons also can elicit systemic adverse effects, psychiatric disturbances and neutropenia [39] . thus, without the benefit of randomised controlled trial data, it becomes difficult to assess whether the treatment is worse than the disease. certain strategies, however, have been shown to worsen clinical outcomes in the setting of a coronavirus infection. for example, studies during the sars pandemic showed that corticosteroids, when used early on sars-cov infected patients, significantly increased viral load, icu admission and mortality [40, 41] . the role for interferon therapies has been less clear in the current mers-cov epidemic, as some data show a positive impact on proximate outcomes, such as oxygenation and inflammation, but no effect on more significant outcomes like hospital stay and long-term survival [35, 36, 42] . rapidly scaled treatments based on naturally occurring neutralising antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have been shown to be relatively safe and potentially effective for reducing mortality from several infections such as sars-cov and influenza [43] [44] [45] , and may hold promise for mers-cov as well. this strategy, however, relies on the rapid identification of cases and contacts and immediate deployment of products to have maximal impact. one study found that convalescent plasma decreased mortality in sars-cov patients only if administered within 14 days of illness [44] . a network for the use of convalescent plasma for case clusters of mers-cov is currently being assembled [43] to test its safety, efficacy and feasibility. however, actualisation of this plan is limited by logistical challenges, local technical capacity and donor supply. unfortunately, no host-derived experimental interventions have yet demonstrated appreciable benefit in acutely ill, mers-covinfected patients in a consistent or controlled manner. this reality, although, has not slowed down the discovery and advancement of passive prophylactic products derived from vaccinated and infected animals and humans. despite intensive efforts to develop a mers-cov vaccine, the prevalence and transmissibility of this emerging pathogen are both relatively low [3, 26] , making it difficult to define a target population for vaccination. mabs, on the other hand, can be administered in the setting of an outbreak without the need to discriminate who might be at greatest risk for infection. they can be used to treat cases early in their natural history and for post-exposure prophylaxis of case contacts. mabs also carry the benefits of higher potency, greater specificity, more extensive pre-licensing evaluation and consequently a more vetted safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic-level detail for the design of better immunogens. they also have been proven as effective therapies in the areas of cancer treatment and autoimmune disease management. although there is only one pathogen, respiratory syncytial virus, for which a mab is licensed for use, there are a number of other infectious disease indications-such as ebola virus disease treatment and human immunodeficiency virus primary and secondary prevention-for which mabs are being tested in advanced phase clinical trials (www.clinicaltrials.gov). despite all of these advantages, the timelines and costs of mab research and development (r&d) are respectively longer and higher than that for polyclonal antibody preparations. in spite of the requirements for greater upfront investments and a more rigorous testing and approval process, several groups have identified highly potent mers-cov mabs and are advancing them through preclinical stages of development (table 1) . some have been isolated from immunised animals (mice/humanised mice/nhps) [46] [47] [48] [49] [50] [51] [52] [53] [54] , while others have been identified from either an antibody human phage library [55] or memory b cells of infected and recovered human survivors [56] . almost all of the published mabs and all of those in development target the s receptor-binding domain (rbd), which contains the most immunogenic epitopes on the virus. many bind to the rbd, expressed both on a recombinant s and on the surface of live virus, with picomolar affinity and neutralise mers-cov at a half maximal inhibitory concentration (ic 50) of 10 ng/μl or less. additionally, several groups have demonstrated new york blood center mersmab1 s1 imunised mouse rbd in vitro [46] nih national cancer institute m336, m337, m338 human antibody library rbd in vitro [52] nih niaid d12, f11, g2, g4 s/s1 immunised mouse rbd, s1, s2 nhp efficacy [51] regeneron regn3048/regn3051 humanised mouse rbd mouse/nhp efficacy [49] tsinghua university mers-4, mers-27 human antibody library rbd in vitro [47] rbd: receptor binding domain; s: spike glycoprotein; s1: spike domain containing rbd; s2: spike domain containing fusion machinery. journal of virus eradication 2016; 2: 1-4 protective efficacy in pre-and post-exposure prophylaxis animal models. because most of the antibodies target the rbd, there is a potential for viral escape from any one mab. thus, there may be a need to develop antibodies against other vulnerable sites on s or to investigate the use of combination mabs to overcome the potential emergence of therapeutic resistance. it is likely that mabs directed at other sites on the s glycoprotein have already been recovered but are not as potent neutralisers, as is the case in one report [51] . a more efficient search for potent neutralising antibodies that target epitopes outside the rbd could be facilitated by a more detailed understanding of the atomic-level structure of the entire s glycoprotein, as has already been resolved for the rbd. the successes thus far in isolating potent and protective mabs, although significant, are likely to be tempered by the challenges of advancing these products to licensing and full-scale production at affordable costs for as of yet undefined populations in a relatively short timeframe. thus, mabs should be advanced along a development pipeline in parallel with a program of rational drug design and discovery. although intensive, supportive care still serves as the primary treatment option for mers-cov and mabs are the focus of the most advanced r&d efforts, antiviral therapies are being actively investigated for use in severely ill patients. there are two main pathways for drug discovery: de novo development and repurposing licensed medications. there are few new antivirals for mers-cov; however, the ebola epidemic has had an unanticipated consequence of facilitating their development. one in particular, gs-5734 developed by gilead sciences, is an adenine analogue that is incorporated into viral rna to disrupt replication [57] . it has shown survival benefit in nhps inoculated with ebola virus and is now advancing through a phase i dose escalation trial. it has been claimed to have in vitro activity against mers-cov as well, but publication of these data is pending. similarly, bcx4430 is a nucleoside analogue that is being developed by biocryst pharmaceuticals for potential treatment of filoviruses, coronaviruses and other rna viruses [58] . additionally, small interfering rna molecules and peptide inhibitors are being investigated for their ability to disrupt mers-cov replication, although these products are still in very early phases of investigation [59, 60] . as the life cycle and genetic sequence of this new coronavirus has become better elucidated, the rational design and development of novel and approved agents with potent antiviral activity have become possible. the advent of high-throughput screens of licensed compounds and small molecules has also allowed researchers to efficiently evaluate large libraries of drugs for their in vitro antiviral activity against novel targets [61] [62] [63] [64] [65] [66] . to date, several dozen licensed drugs have been reported to inhibit mers-cov replication. using slightly different screening technologies, different groups have converged on some common classes of compounds, including nucleoside analogues, antibacterial protein synthesis inhibitors, kinase signalling modifiers, antimetabolites and antiprotozoal agents. mycophenolic acid, an inhibitor of both t an b lymphocytes, has also been found to have strong activity against mers-cov, as it does against other rna viruses such as west nile, hepatitis c and dengue [63] . only one of the drugs to show in vitro activity against mers-cov, lopinavir, however, has been tested in an animal model. mers-cov-challenged marmosets that were treated with this protease inhibitor had better clinical, pathological, virological and radiological outcomes than controls or those treated with mycophenolate mofetil [67] . additionally, two peptides, hr1p and hr2p are being developed as potential fusion inhibitors [59] . by acting on the six-helix bundle core of the mers-cov s protein to prevent protein-mediated cell-to-cell fusion, this class of compounds may hold potential beyond mers-cov towards a long-term objective of a pan-coronavirus antiviral. given some of the common life cycles and pathways of pathogenesis for rna viruses and homologies in protein structures across different coronaviruses, there may be economies of effort and investment in developing antivirals that have activity against more than one virus or family of viruses. irrespective of the breadth of these novel or repurposed compounds, treatment studies should be carried out prospectively according to protocols that plan for the collection of quality controlled data and serial biological sampling to assess viral evolution and biomarkers of favourable clinical outcomes. recent infectious disease outbreaks such as the 2009 h1n1 influenza pandemic, the h7n9 influenza epidemic in china, the ebola crisis in west africa and now the mers-cov outbreak have highlighted the need for better r&d preparedness and improved coordination of clinical testing in the face of the accelerating number of emerging and re-emerging infectious diseases. the ability to have an armamentarium of countermeasures and clinical trial infrastructure in the early phases of an outbreak is critical for mounting an effective public health campaign. for example, the sars-cov pandemic caused more than 8000 cases of severe acute respiratory illness and nearly 900 deaths but few prospective, controlled studies were undertaken to determine the optimal management of the disease. consequently, treatment options for sars-cov were never defined clearly and thus difficult to adapt for mers-cov. although global coordination has resulted in the advancement of some urgently needed, novel countermeasures for mers-cov, they will have to be developed along faster timelines than before, with greater investments earlier in the preclinical development pipeline that can generate products for more timely efficacy testing in affected populations. as the global community takes lessons from the most recent outbreak and prepares for the potential of another regional epidemic or broader pandemic, stakeholders in mers-cov r&d must set out a sound strategy now for where to best target their investments in anticipation of the changing dynamics of the current and future outbreaks. global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) summary and literature update -as of 11 european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov) from sars to mers: 10 years of research on highly pathogenic human coronaviruses clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient evidence for camel-to-human transmission of mers coronavirus epidemiological investigation of mers-cov spread in a single hospital in south korea transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea transmission among healthcare worker contacts with a middle east respiratory syndrome patient in a single korean centre transmission characteristics of mers and sars in the healthcare setting: a comparative study health-care associate transmission of middle east respiratory syndrome corona virus, mers-cov, in the kingdom of saudi arabia assessment of the middle east respiratory syndrome coronavirus (mers-cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach estimation of mers-coronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome: a case report ribavirin and interferon-alpha2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review interferon in oncological practice: review of interferon biology, clinical applications, and toxicities the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis the use of convalescent plasma to treat emerging infectious diseases: focus on ebola virus disease a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for middle east respiratory syndrome coronavirus (mers-cov) evaluation of candidate vaccine approaches for mers-cov exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus nucleotide prodrug gs-5734 is a broad-spectrum filovirus inhibitor that providescomplete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx4430 in a non-human primate model of filovirus infection structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus design of potential rnai (mirna and sirna) molecules for middle east respiratory syndrome coronavirus (mers-cov) gene silencing by computational method antiviral drugs specific for coronaviruses in preclinical development a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors alternative screening approaches for discovery of middle east respiratory syndrome coronavirus inhibitors treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset the opinions expressed herein are those of the authors and should not be construed as official or representing the views of the us department of defense or the department of the army. key: cord-273391-vmtfn78x authors: li, kun; li, zhuo; wohlford-lenane, christine; meyerholz, david k.; channappanavar, rudragouda; an, dong; perlman, stanley; mccray, paul b.; he, biao title: single-dose, intranasal immunization with recombinant parainfluenza virus 5 expressing middle east respiratory syndrome coronavirus (mers-cov) spike protein protects mice from fatal mers-cov infection date: 2020-04-07 journal: mbio doi: 10.1128/mbio.00554-20 sha: doc_id: 273391 cord_uid: vmtfn78x middle east respiratory syndrome coronavirus (mers-cov) can cause severe and fatal acute respiratory disease in humans and remains endemic in the middle east since first being identified in 2012. there are currently no approved vaccines or therapies available for mers-cov. in this study, we evaluated parainfluenza virus 5 (piv5)-based vaccine expressing the mers-cov envelope spike protein (piv5/mers-s) in a human dpp4 knockin c57bl/6 congenic mouse model (hdpp4 ki). following a single-dose intranasal immunization, piv5-mers-s induced neutralizing antibody and robust t cell responses in hdpp4 ki mice. a single intranasal administration of 10(4) pfu piv5-mers-s provided complete protection against a lethal challenge with mouse-adapted mers-cov (mers(ma)6.1.2) and improved virus clearance in the lung. in comparison, single-dose intramuscular immunization with 10(6) pfu uv-inactivated mers(ma)6.1.2 mixed with imject alum provided protection to only 25% of immunized mice. intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated mers-cov, suggestive of a hypersensitivity-type response. overall, our study indicated that piv5-mers-s is a promising effective vaccine candidate against mers-cov infection. m iddle east respiratory syndrome (mers) emerged as a significant illness on the saudi arabian peninsula in mid-2012, and the causative agent was identified as a novel coronavirus (cov), mers-cov (1) . mers has a high mortality rate (ϳ35%) associated with severe lung disease that can advance to acute respiratory distress syndrome (ards). mers-cov, similarly to sars-cov, which caused a similar epidemic in 2003, has been a global cause for concern due to its high fatality rate. epidemiologic studies established that mers-cov is zoonotic in origin, with transmission occurring from dromedary camels on the arabian peninsula (2) (3) (4) . spread from camels to people is documented (5) , as well as person-to-person spread among health care workers in hospital settings (6) . to date, mers-cov has spread to 27 countries and caused 858 deaths in 2,494 confirmed cases (4 february 2020, world health organization [who]), including a large travel-related outbreak in south korea in 2015 (7) . mers-cov is an enveloped positive-stranded rna virus whose entry into target cells is mediated by the viral envelope s protein. the s protein consists of an s1 subunit responsible for binding to the virus receptor, dipeptidyl peptidase 4 (dpp4 or cd26), via a receptor-binding domain (rbd), and an s2 subunit that mediates membrane fusion (8) (9) (10) . thus, the s protein, particularly the rbd, is an important target for mers-cov vaccine development (8, 11, 12) . there is currently no vaccine or antiviral therapeutic against mers-cov. a number of candidate mers-cov vaccines, including those based on recombinant virus, viral vectors (e.g., mva, adenovirus, and measles virus), nanoparticles, dna, and dna/protein, as well as subunit vaccines, are under development (12, 13) . none are approved; thus, the need remains for an effective and broadspectrum vaccine against mers-cov infection (14) . piv5, formerly known as simian virus 5 (sv5), is a nonsegmented, negative-strand, rna virus (nnsv). it is a member of the rubulavirus genus of the family paramyxoviridae, which includes mumps virus (muv) and human parainfluenza virus type 2 (hpiv2) and type 4 (hpiv4) (15) . piv5 encodes eight known viral proteins (15) . nucleocapsid protein (np), phosphoprotein (p), and large rna polymerase (l) protein are important for transcription and replication of the viral rna genome. piv5 is an excellent viral vector candidate for vaccine development; it is safe and infects a large number of mammals without being associated with any diseases, except kennel cough in dogs (16) (17) (18) (19) (20) . because piv5 does not have a dna phase in its life cycle, its use avoids the possible unintended consequences of genetic modifications of host cell dna through recombination or insertion. in comparison to positive-strand rna viruses, the genome structure of piv5 is stable. a recombinant piv5 expressing f of respiratory syncytial virus (rsv) has been generated, and the f gene was maintained for more than 10 generations (21) . piv5 can be grown to 8 ϫ 10 8 pfu/ml, indicating its potential as a costeffective and safe vaccine vector that may be used in mass production. we have discovered that piv5-based influenza, respiratory syncytial virus (rsv), and rabies vaccines are efficacious (22) (23) (24) (25) (26) (27) (28) . in studies of influenza, we previously reported that that a piv5 vector expressing influenza virus na provided sterilizing immunity (no mortality, no morbidity, and no virus detected in the lungs of challenged mice at 4 days postchallenge) and piv5 expressing np protected 100% of mice against lethal influenza virus h1n1 challenge in mice (25) , demonstrating that piv5 is an excellent vector for developing vaccines for respiratory pathogens. here we investigate the utility of a piv5-based vaccine expressing the mers s protein in a robust humanized mouse model of lethal mers-cov infection. construction of a piv5 vector expressing mers-cov spike glycoprotein. previously, we inserted the ha gene of influenza a virus at different locations within the genome of piv5 and found that the insertion at sh and hn generates the best immune responses (24) . thus, we inserted the full-length gene of s of mers at the sh and hn junction. a plasmid containing full-length piv5 cdna with the s gene insertion at sh and hn junction was constructed using standard molecular cloning techniques (fig. 1a) . the plasmid was transfected into bhk cells along with plasmids expressing t7 rna polymerase, np, p, and l of piv5, and infectious virus piv5-mers-s was rescued as described before (24) . the rescued virus was plaque-purified and then expanded to large quantity in mdbk cells for further analysis. the viral genome was sequenced and confirmed to contain the desired input dna sequence. to verify s protein expression in piv5-mers-s-infected cells, the cells were infected at different mois and then lysed for immunoblotting using anti-s antibody. the full-length s and cleaved s2 fragments were observed in piv5-mers-s-infected cells, suggesting that the s protein was properly processed (fig. 1b) . expression of s protein in piv5-mers-s-infected cells was further confirmed by immunofluorescence assay (fig. 1c) . interestingly, piv5-mers-s caused massive syncytium formation in vero cells. piv5-mers-s had a similar growth kinetics as wild-type piv5 (fig. 1d) . to determine whether piv5-mers-s can generate immune responses in mice, c57bl/6 mice were immunized with a single dose of piv5-mers-s or control piv5-gfp virus at 10 4 pfu or 10 6 pfu per mouse via intranasal route. while both doses generated antibody responses, neutralizing titers were modest at 1:64 and 1:128 for the 10 4 and 10 6 doses, respectively ( fig. 2a and b) . it is known that c57bl/6 and balb/c mice generate th1-and th2-dominant immune responses, respectively, following immunization. a single dose of 10 6 pfu of piv5-mers-s resulted in a neutralization antibody titer as high as 1:2,000 in balb/c mice (fig. 2c) , consistent with a th2dominant response in balb/c mice. to assess the primary cd8 t cell response generated by piv5-mers-s immunization, hdpp4-ki mice were intranasally immunized with 10 4 pfu piv5-mers-s. four weeks later, lungs were harvested and examined for mers-cov-specific lung-resident cd8 t cells (fig. 3a) . as shown in fig. 3b to d, we observed a significant increase in the immunization with piv5 expressing mers-cov spike ® percentage and total number of cd8 ϩ -ifn-␥ ϩ cells in the lungs of piv5-mers-simmunized mice in comparison to those infected with piv5-gfp virus, consistent with a mers-s-specific primary cd8 t cell response in the lungs. further, to examine recall response of mers-s-specific cd8 t cells, we challenged piv5-mers-s-and piv5-gfpimmunized mice with 10 4 pfu of mers-cov ma 6.1.2. our results show a significant increase in the recall cd8 t cell response at day 4 p.i. in comparison to piv5-gfpimmunized mice ( fig. 3e to g). we also observed 10-fold increase in mers-s-specific cd8 t cells compared to the primary cd8 t cell response ( fig. 3b to g). collectively, these results indicate that mers-s immunization induces a significantly increased mers-s-specific cd8 t cell response upon piv5-mers-s immunization. to determine the efficacy of piv5-mers-s in preventing or modifying a mers-cov infection, hdpp4 ki mice on the c57bl/6 background were immunized with 10 4 pfu piv5-mers-s via the intranasal route. at 4 weeks after immunization, mice were challenged with a mouseadapted mers-s strain (fig. 4a ). all piv5-mers-s-immunized mice survived this lethal challenge and lost little weight ( fig. 4b and c). in contrast, piv5-gfp-or pbs-immunized mice all died following challenge ( fig. 4b and c), indicating that piv5-mers-s completely protected mice against lethal challenge. while piv5-mers-s-immunized mice had a higher rate of virus clearance from the lungs, this did not provide sterilizing immunity (fig. 4d) . histopathology of lung tissues. histopathology studies of lungs after challenge with mers ma 6.1.2 indicated that piv5-mers-s-immunized mice had significantly less cellular debris present and greater mononuclear infiltrates ( fig. 5a and b ). piv5-mers-s-immunized mice exhibited robust cellular infiltration of leukocytes (mostly mononuclear cells) and less evidence of lesions (edema, hyaline membranes, necrotic cellular debris, etc.) indicative of severe disease (fig. 5) . to investigate the protective responses elicited by piv5-mers-s and the inactivated mers-cov, hdpp4 ki mice were immunized with 10 4 pfu of piv5-mers-s or pbs via i.n. or uv-inactivated mers-cov with adjuvant via i.m. route. while piv5-mers-s provided 100% protection against lethal challenge, inactivated mers-cov protected 25% of mice from mortality (fig. 6 ). it has been reported that mice immunized with inactivated sars-cov and mers-cov developed a hypersensitivity-type response after respective sars-cov and mers-cov challenge, manifested by increased il-4 and il-5 expression and an influx of eosinophils (29) . we examined the lungs of mice that were immunized and then challenged with mers-cov (fig. 7) . we observed more eosinophils in the lungs of mice immunized with inactivated mers-cov than in pbs-or piv5-mers-s-immunized mice following mers-cov challenge. compared to pbs, perivascular eosinophilic infiltration was significantly increased in the inactivated mers-cov group, but no statistical difference was seen many strategies have been considered to develop vaccines for both sars-cov and mers-cov. a live attenuated sars-cov with rationally introduced mutations was efficacious in golden syrian hamsters (30) . however, the development of a live attenuated vaccine for a positive-stranded rna virus like sars-cov has often been hampered by safety concerns. several mers vaccine candidates are under investigation. a dna-based vaccine expressing the full-length s protein is the most advanced to date (31) ; it is well tolerated in humans, as shown in a phase i clinical trial. the prime-boost regimen of mva (modified vaccinia ankara) expressing mers s protein induced neutralizing antibodies and t cell responses in mice and limited viral replication after challenge in mice and camels. however, mva-s did not provide sterilizing immunity, immunization with piv5 expressing mers-cov spike ® and infectious mers-cov and genomic rna were detected after challenge in mice and camels (32, 33) . the prime-boost regimen of measles virus (mv) expressing mers s or soluble s induced both humoral and cellular immune responses. after mers challenge, infectious mers-cov or genomic rna significantly decreased, but these two vaccines did not provide sterilizing immunity, and signs of inflammation were observed in mouse lung tissue (34) . an inactivated rabies virus (rabv) expressing mers s1 provided complete protection from mers challenge in mice but three 10-g doses of vaccine were needed (35) . furthermore, the ad5/hdpp4-transduced mouse model used in these studies has limitations. adenovirus (ad5) expressing mers s or s1 also induced a neutralizing antibody in mice (36) . ad41, an enteric adenovirus, may induce enhanced mucosal immunity when administered via an oral or intragastric (i.g.) route (37, 38) . however, i.g. immunization of both ad41-s and ad5-s failed to generate mucosal immunity. although ad41-s induced humoral immunity in serum, it was significantly less than ad5-s (39). chimpanzee adenovirus-based vector systems have also been used (40) . in our work, we demonstrated that a single dose as low as 10 4 pfu of piv5-mers-s was sufficient to provide 100% protection against lethal mers-cov challenge. the low dose is especially advantageous in a situation where a mass immunization program is needed in a short period of time. to the best of our knowledge, this is the most efficacious mers-cov vaccine tested in a relevant animal model. the protective mechanism of piv5-mers-s vaccine in c57bl/6 mice is likely due to robust cellular immune responses after piv5-mers-s immunization. while neutralizing antibody was generated in c57bl/6 mice after a single-dose immunization with piv5-mers-s, titers were modest at 1:64 and 1:128 with 10 4 pfu and 10 6 pfu of piv5-mers-s, respectively ( fig. 2a and b) . consistent with protective cellular immune responses protecting the mice, a significant influx of cd8 ϩ ifn-␥ ϩ cells was detected in lungs of c57bl/6 mice following piv5-mers-s immunization (fig. 3) . furthermore, the observation that piv5-mers-s-immunized mice had a higher rate of mers virus clearance (fig. 4c ) suggests a role for t cell-based immunity in protecting c57bl/6 mice against lethal challenge. interestingly, in balb/c mice, piv5-mers-s generated neutralizing antibody titers as high as 1:2,000 (fig. 2c) . it is possible that the higher neutralizing antibody titers in balb/c mice may be protective. unfortunately, the only available small animal model is a humanized mouse model on the c57bl/6 background. it is known that the s protein is a major protective antigen for coronaviruses. it may be possible to improve our vaccine efficacy by expressing additional mers-cov proteins such as n and m using piv5 as a vector. however, a parainfluenza virus 3 (piv3)-based sars-cov vaccine candidate expressing n, m, or e without the s protein failed to protect hamsters from sars-cov challenge (41) . the ability of piv5-mers-s to generate cellular and humoral immune responses in mice may be in part attributed to the ability of piv5 to express the mers s protein in its native conformation. as shown in fig. 1c , piv5-mers-s caused massive syncytium formation in vero cells, indicating the s protein was functional in promoting cell-to-cell fusion. thus, we reasoned that the s protein produced in piv5-mers-s-infected cells maintains a native conformation. the mers s protein has 1,353 amino acid residues. the entire insertion of the s gene with proper regulatory sequences is over 4,000 nucleotides in length. this is the longest single gene we have inserted into the piv5 genome. since we inserted this gene between sh and hn, and the sh gene is not essential, it may be possible to remove sh to allow insertion of longer sequences. thus, we speculate that the piv5 genome can accommodate sequences longer than 4,000 nucleotides. it has been reported that inactivated sars-cov-immunized mice generated a hypersensitive-type lung pathology after virus challenge, raising the concern of vaccine-enhanced disease (42, 43) . previously, a formalin-inactivated, whole-virus respiratory syncytial virus (rsv) vaccine caused enhanced disease in vaccinated children, leading to vaccine-related deaths (44) . similarly, inactivated mers-cov has been reported to generate a th2-type immunopathology after mers-cov challenge in mice (29) . in the case of a piv5-based rsv vaccine, extensive studies indicate that piv5-based rsv vaccine does not cause enhanced diseases (45) . thus, as a viral vector, piv5 is not known to cause any enhanced diseases, and in our experiment, we observed no abnormal immune responses in piv5-mers-s-immunized mice after mers-cov challenge, suggesting that piv5-mers-s is unlikely to be associated with enhanced disease. immunization with piv5 expressing mers-cov spike ® lung tissues of mice immunized with inactivated mers-cov had an influx of eosinophils after mers-cov challenge, indicative of a hypersensitivity-type response. this result is consistent with a previous report that inactivated mers-cov immunization caused increased il-4 and il-5 expression and an influx of eosinophils in lungs after challenge (29) . understanding whether immunization with inactivated mers-cov can cause enhanced disease is critical for developing a safe and effective vaccine. while mers-cov has a high morbidity and mortality, it has very a low prevalence in human populations. dromedary camels are considered the intermediate host that transmits mers-cov to humans. thus, it may be possible to control the spread of mers-cov in humans by controlling infection in dromedary camels. perhaps virus transmission from camels to humans can be blocked, with concomitant immunization of high-risk human populations, as proposed by cepi (the coalition for epidemic preparedness innovations) and who. as a vaccine vector, piv5 has been effective in mice, cotton rats, hamsters, guinea pigs, ferrets, dogs, and nonhuman primates (25, (46) (47) (48) (49) . it will be worthwhile to test piv5-mers-s in camels in the future. recently, sars-cov-2 (2019-ncov) was identified in wuhan, china, in late 2019. this is a novel zoonotic cov related to the sars-cov that can cause severe respiratory disease . to date, this virus resulted in a significant disease burden, with more than 465,000 cases reported in 199 countries and an estimated case fatality rate of~2%. the finding that piv5 expressing mers s protected mice against lethal mers-cov challenge at a single low dose of 10 4 pfu suggests its potential use as a vaccine vector for emerging viruses such as sars-cov-2. further studies of using piv5 expressing the s protein from sars-cov-2 as a vaccine candidate are ongoing. cells. vero cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs), 100 iu/ml penicillin, and 100 g/ml streptomycin (1% p/s; mediatech inc., manassas, va, usa). bhk21 cells were maintained in dmem containing 10% tryptose phosphate broth (tpb), 10% fetal bovine serum (fbs), 100 iu/ml penicillin, and 100 g/ml streptomycin. mdbk cells were grown in dmem containing 5% fbs and 1% p/s. cells were prepared 1 day prior to infection, achieving approximately 90% confluence by the following day. viruses. the plasmid containing the cdna clone of piv5 with mers-s inserted between sh and hn was constructed using previously described methods (22, 26, 29) . primer sequences are available upon request. infectious virus was rescued in bhk cells as previously described (26) . recombinant piv5 or piv5-mers-s was propagated in mdbk cells as previously described (26, 29) . piv5 plaque assays were performed as previously described (24) . briefly, 10-fold serial dilutions were prepared in dmem with 1% bsa. one hundred microliters of each dilution was transferred to 6-well plates of bhk21 cells, in a total infection volume of 1 ml. after adsorption for 1 to 2 h at 37°c, 5% co 2 , the inocula were aspirated, and cell monolayers were overlaid with dmem containing 10% tryptose phosphate broth (tpb), 2% fbs, 1% p/s, and 1% low-melting-point agarose. after 5 days, the cells were fixed with 2% formaldehyde, overlays were removed, and the cells were stained with crystal violet to visualize the plaques. to obtain virus titers in lung tissues, lungs of infected mice were removed at the indicated days after challenge and homogenized in pbs using a manual homogenizer. virus titer was determined in vero 81 cells by plaque assay. infected vero 81 cells were fixed in 25% formaldehyde and stained with 0.1% crystal violet to delineate plaques. to determine growth rates of piv5 and piv5-mers-s, mdbk cells were infected with piv5 or piv5-mers-s at an moi of 0.1. after adsorption for 1 to 2 h at 37°c, 5% co 2 , dmem with 2% fbs and 1% p/s was added to the plates. one-hundred-microliter samples of supernatant were collected daily for 5 days and frozen at ϫ80°c. virus titers in the samples were quantified by plaque assay. immunization and infection of mice. specific-pathogen-free 6-week-old c57bl/6 and balb/c mice were purchased from charles river laboratory (cr). specific-pathogen-free human dpp4 knockin (hdpp4 ki) mice were generated on a c57bl/6 background as previously reported (50) . all mice were bred and maintained in the university of iowa animal care facility. all protocols were reviewed and approved by the university of iowa institute animal care and use committee. six-to 8-week-old male and female mice were used for these studies. mouse-adapted mers-cov strain mers ma 6.1.2 was generated as reported earlier (50) . mice were anesthetized with xylazine-ketamine (97.5 mg/kg of body weight ketamine,12.5 mg/kg xylazine) and infected intranasally with 10 4 pfu or 10 6 pfu piv5-mers-s or piv5-gfp in 60 l dmem. the mouse-adapted mers ma 6.1.2 strain was inactivated by exposure to uv light for 1 h using a wattage of 4,016 w/cm 2 . then 10 6 pfu uv-inactivated viruses were 1:1 (vol/vol) mixed with imject alum (thermo, catalog no. 77161) and delivered to mice intramuscularly. four weeks postimmunization, mice were infected intranasally with 10 5 pfu mers ma 6.1.2 in 50 l dmem. for passive immunization, sera were collected from hdpp4 ki mice that received 10 4 pfu piv5-mers-s or piv5-gfp intranasally at 4 weeks postimmunization. two hundred microliters of sera were transferred into hdpp4 ki mice intraperitoneally 1 day before challenge with 10 5 pfu mers ma 6.1.2. infected mice were examined daily, and weights were recorded. all work with mers-cov was performed in the biosafety level 3 (bsl3) laboratory of the university of iowa. immunization with piv5 expressing mers-cov spike ® histology. at the indicated days postchallenge, mice were anesthetized and perfused with pbs by intracardiac injection followed by perfusion with zinc formalin. lungs were removed, fixed in zinc formalin overnight, and paraffin embedded. lung sections (ϳ4-m thickness) were stained with hematoxylin and eosin. tissues were evaluated by board-certified veterinary pathologists and scored using a postexamination masking method (51) . lungs were scored for edema, hyaline membranes, cellular debris, and hemorrhage, with scores of 0, 1, 2, 3, and 4 representing detection in 0%, less than 5%, 6% to 33%, 33% to 66%, and more than 66% of lung fields, respectively. lungs were scored for mononuclear infiltrates, with scores of 0 representing values within normal parameters, 1 representing small aggregates in peribronchial and perivascular areas, 2 representing perivascular and periairway aggregates filling perivascular space, and 3 representing a score of 2 plus expanding sheets of infiltrates into septa and consolidation lesions in regions of the lung, respectively. lungs were scored for granulocytic infiltrates, with scores as follows: 0, within normal parameters; 1, scattered pmns sequestered in septa; 2, a score of 1 plus solitary pmns extravasated in airspaces; 3, a score of 2 plus small aggregates in vessels and airspaces, respectively. lung tissues were evaluated for perivascular eosinophil infiltration. briefly, vessels with cellular infiltration (n ϭ 20/lung) were randomly selected by a masked pathologist, and the number of eosinophils was enumerated and averaged for a final score for each lung (52) . neutralizing antibody assay. four weeks postimmunization, sera from immunized mice were collected. all serum samples were heat inactivated by incubation at 56°c for 30 min. heat-inactivated serum was serially diluted by 2-fold in 96-well plates before the same volume of mers-cov pseudovirus was added and incubated at 37°c for 1 h. the mixture was added into vero 81 cells in 96-well plates and incubated at 37°c for 1 h to allow virus binding. then the mixture was removed, and cells were rinsed with pbs. the next day, the neutralization results were measured by luciferase assay and plotted relative to the value for serum-free wells. immunoblotting and immunofluorescence. vero 81 cells were infected with piv5-mers-s at different mois or mock infected at 37°c for 1 h. cell lysates were collected at 2 days postinfection and applied to western blotting. the expression of mers-cov spike protein was detected by a rabbit anti-mers-cov s2 antibody (sino biological, catalog no. 40070-t60) and colocalized using a mouse anti-␣-tubulin antibody (sigma, catalog no. t9026). indirect immunofluorescence assays were performed to detect expression of the s protein in piv5-mers-s-infected cells. vero cells were infected with piv5 or piv5-mers-s. forty-eight hours later, cells were fixed with 2% formaldehyde in phosphate-buffered saline (pbs) and permeabilized by adding 0.1% saponin to the immunostaining buffers. anti-mers-s from sino biological was used (catalog no. 40070-t60). the cells were imaged using a nikon a1r confocal microscope. analyses of mers-cov-specific cd8 t cell response. hdpp4 ki mice were immunized with 10 4 pfu piv5-gfp or piv5-mers-s via intranasal route. at 4 weeks postimmunization, lung cells were harvested and mers-cov-specific cd8 ϩ t cells were stimulated with 1 m s434 and s1165 peptides as described previously (53) in the presence of golgi-plug (1 l/ml) for 5 h. cells were then washed and stained for cell surface cd45, cd4, and cd8 markers followed by intracellular ifn-gamma staining. to examine the recall cd8 t cell response, hdpp4 ki mice immunized with 10 4 pfu piv5-gfp or piv5-mers-s via intranasal route were challenged with 10 5 pfu mers ma 6.1.2. four days after mers ma 6.1.2 infection, lungs were harvested and cd8 t cells were stimulated with s434 and s1165 peptides in the presence of golgi-plug (1 l/ml) for 5 h. cells were then washed and stained for cell surface cd45, cd4, and cd8 markers followed by intracellular ifn-gamma staining. the following monoclonal antibodies were used: pecy7 anti-cd45 (30-f11), anti-cd4 (rm4-5), and anti-cd8␣ (53-6.7), all from bd bioscience, and ifn-␥ (xmg1.2) from ebioscience. isolation of a novel coronavirus from a man with pneumonia in saudi arabia prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study acute middle east respiratory syndrome coronavirus infection in livestock dromedaries evidence for camel-to-human transmission of mers coronavirus ksa mers-cov investigation team. 2013. hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study current advancements and potential strategies in the development of mers-cov vaccines receptor recognition mechanisms of coronaviruses: a decade of structural studies dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc vaccines for the prevention against the threat of mers-cov middle east respiratory syndrome: current status and future prospects for vaccine development single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against mers-cov infection calling for rapid development of a safe and effective mers vaccine paramyxoviridae: the viruses and their replication, ch 41 viruses recovered from laboratory dogs with respiratory disease studies of canine respiratory viruses. i. experimental infection of dogs with an sv5-like canine parainfluenza agent isolation of parainfluenza virus sv5 from dogs with respiratory disease a study of dogs with kennel cough kennel cough complex: confirmation and analysis of the outbreak in japan genetic stability of parainfluenza virus 5-vectored human respiratory syncytial virus vaccine candidates after in vitro and in vivo passage recombinant parainfluenza virus 5 (piv5) expressing the influenza a virus hemagglutinin provides immunity in mice to influenza a virus challenge recombinant parainfluenza virus 5 vaccine encoding the influenza virus hemagglutinin protects against h5n1 highly pathogenic avian influenza virus infection following intranasal or intramuscular vaccination of balb/c mice recombinant parainfluenza virus 5 expressing hemagglutinin of influenza a virus h5n1 protected mice against lethal highly pathogenic avian influenza virus h5n1 challenge single dose vaccination of a recombinant parainfluenza virus 5 expressing np from h5n1 provides broad immunity against influenza a viruses efficacy of parainfluenza virus 5 mutants expressing ha from h5n1 influenza a virus in mice a respiratory syncytial virus (rsv) vaccine based on parainfluenza virus 5 (piv5) a novel rabies vaccine based on a recombinant parainfluenza virus 5 expressing rabies virus glycoprotein immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a live attenuated severe acute respiratory syndrome coronavirus is immunogenic and efficacious in golden syrian hamsters safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform one-health: a safe, immunization with piv5 expressing mers-cov spike ® efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice mucosal vaccines: the promise and the challenge enhanced induction of intestinal cellular immunity by oral priming with enteric adenovirus 41 vectors systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus humoral immunogenicity and efficacy of a single dose of chadox1 mers vaccine candidate in dromedary camels contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus a doubleinactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge respiratory syncytial virus vaccine development parainfluenza virus 5 expressing wild-type or prefusion respiratory syncytial virus (rsv) fusion protein protects mice and cotton rats from rsv challenge a single-dose recombinant parainfluenza virus 5-vectored vaccine expressing respiratory syncytial virus (rsv) f or g protein protected cotton rats and african green monkeys from rsv challenge efficacy of a parainfluenza virus 5 (piv5)-based h7n9 vaccine in mice and guinea pigs: antibody titer towards ha was not a good indicator for protection evaluating a parainfluenza virus 5-based vaccine in a host with preexisting immunity against parainfluenza virus 5 vaccination with recombinant parainfluenza virus 5 expressing neuraminidase protects against homologous and heterologous influenza virus challenge mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice principles and approaches for reproducible scoring of tissue stains in research nonglycosylated g-protein vaccine protects against homologous and heterologous respiratory syncytial virus (rsv) challenge, while glycosylated g enhances rsv lung pathology and cytokine levels rapid generation of a mouse model for middle east respiratory syndrome we thank the members of the perlman, mccray, and he laboratories for their helpful discussion and technical assistance. key: cord-104500-m0kfom0x authors: kyriakopoulos, anthony m.; papaefthymiou, apostolis; georgilas, nikolaos; doulberis, michael; kountouras, jannis title: the potential role of super spread events in sars-cov-2 pandemic; a narrative review date: 2020-09-21 journal: arch acad emerg med doi: nan sha: doc_id: 104500 cord_uid: m0kfom0x coronaviruses, members of coronaviridae family, cause extensive epidemics of vast diseases like severe acute respiratory syndrome (sars) and coronavirus disease-19 (covid-19) in animals and humans. super spread events (sses) potentiate early outbreak of the disease and its constant spread in later stages. viral recombination events within species and across hosts lead to natural selection based on advanced infectivity and resistance. in this review, the importance of containment of sses was investigated with emphasis on stopping covid-19 spread and its socio-economic consequences. a comprehensive search was conducted among literature available in multiple electronic sources to find articles that addressed the “potential role of sses on severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic” and were published before 20(th) of august 2020. overall, ninety-eight articles were found eligible and reviewed. specific screening strategies within potential super spreading host groups can also help to efficiently manage severe acute respiratory syndrome coronavirus 2 (sars-cov-2) epidemics, in contrast to the partially effective general restriction measures. the effect of sses on previous sars epidemics has been documented in detail. however, the respective potential impact of sses on sars-cov-2 outbreak is composed and presented in the current review, thereby implying the warranted effort required for effective sse preventive strategies, which may lead to overt global community health benefits. this is crucial for sars-cov-2 pandemic containment as the vaccine(s) development process will take considerable time to safely establish its potential usefulness for future clinical usage. severe acute respiratory syndrome (sars) has periodically emerged as epidemics and its natural history could be utilized as a "compass" to comprehend and manage the current pandemic of sars-cov-2. sars-cov-2 the etiologic agent of the novel coronavirus disease 2019 (covid19) , be-although the prediction and subsequently the prevention of sses seems to be complicated, the virus, host, environmental, and mass behaviors determine relative approaches to prevent and control sses; core community health programs can inhibit and decrease the incidence and the effect of sses (9) . nevertheless, horizontal austerity measures, such as recommending or compelling individuals to self-isolate at home, which might cause serious social and psychological burden, and quarantine, also leading to loss of income due to social distancing, are associated with negative psychological and religious effects, which can be long lasting (10) , thereby leading to serious instability of the global society. prolonged social isolation and loneliness are associated with increased mortality (11) . currently, limited piece of information exists regarding the effect of sses on coronavirus epidemics. the aim of this narrative review is to mainly focus on the potential impact of sses on large outbreaks of coronavirus. the development of an emergency sars-cov-2 vaccine has its potential usefulness and/or limitations and may result in severe health outcomes, which prompts better screening for sses in order to control coronavirus pandemics. to avoid, in most respects, literature selection bias (12) , multiple electronic sources: medline/pubmed, scifinder, science direct and goggle scholar as well as researchgate and general (google) were investigated via queries with a nonrestricted time frame reaching the 20th of august 2020. initial investigation of sses and sars, sses and mers, and sses and covid-19, gave narrative results from pubmed. the selected literature, which is included in the study, is presented in table 1. same items were also searched in all other mentioned sources. the scope of the study was not only to investigate the transmission of sars-cov-2 due to sses, its comparison with sars-cov-1 and mers-cov, but also to assess the general global impact due to sses by covid-19. therefore, further literature investigation was performed using the same electronic sources. further investigation was made on: a) the prevention of sses by coronaviruses causing sar-1, mers and covid-19, b) the socio-economic relation of sars-cov-1, mers and covid-19 due to sses, c) the austerity caused by sses of covid-19, and d) the relation of sses containment to future vaccination programs. for further investigation, the following items were searched: "sars, mers and covid-19 epidemic prevention", "sars mers and covid-19 infectivity and pathogenicity", "coronavirus sse prevention", sse coronavirus crisis and socio-economics", "holy cup reli-gion and transmission of pathogens and sses", and "coronavirus immunity and vaccination". studies providing an adequate determination of an sse related to sars, mers and covid-19 were primarily screened and selected by two reviewers (authors) blinded to one another. the results were thereafter cross-matched and duplicates were removed. based on this primary search, the socio-economic impact of coronavirus, produced by sses, was extrapolated by two other reviewers (authors). following this initial selection stage, further screening was performed by all reviewers, using the previously described search items to identify parameters determining the global impact of covid-19 due to sses. identified parameters included the global impact of immunity and vaccination, the holy cup and religion transmission, and the austerity caused by covid-19 and other coronavirus epidemics due to restrictions applied. all search results were cross-matched to remove duplicates and thereafter, exclusion and inclusion criteria were applied. after removing the duplicates, review was conducted on titles and abstracts. also, a decision was made to remove "news press opinions". computational model methodologies producing contradictory results, studies with wrong interpretation of sses, and studies with non-clear-cut results were also removed. studies using the interpretation "a super spreading individual, known as the index case, produces a cluster of sars, mers, and covid-19 secondary infections" were included. a second exclusion criterion was applied. in this stage, peer reviewed literature of recent dates, studies assessing sars, mers, and covid-19 epidemiology measures, studies on covid-19 restriction measures producing social and economic austerity, articles discussing the perspective for future vaccination and population immunity, and finally genetic studies on coronaviruses causing sars, mers, and covid-19. by following the described methodology, on medline/pubmed: a) 23 articles were found on sars and mers and sse, and b) 11 articles were found on covid-19 and sses. out of: a) 13 of the 23 articles on sars and mers and sse, and b) 7 out of the 11 articles on covid-19 and sse were deemed relevant hits. after applying the exclusion criteria, 12 articles from the first category, and 4 from the second category were included in the study. suitable articles found by searching, which were selected and reviewed for each part, are illustrated in figure 1 . further investigation in all other electronic sources described, using the same method(21) original research description of sse importance in covid-19 epidemic ! severe acute respiratory syndrome; ∧ middle east respiratory syndrome; &coronavirus disease-19; *super spread events; **when clearly indicated in article, the type of study is also mentioned. ology, increased the number of the included literature to a) 17 and b) 14, for their respective categories of search. studies included from pubmed in these categories of searches are briefly described and listed in table 1. further, assessing the general global impact of sses related to covid-19, using all the mentioned sources, via the same methodology, led to the inclusion of a) 10 articles related to genetic analysis of sars-cov-1 and mers-cov and sars-cov-2, b) 5 articles related to super spread events, c) 2 articles related to austerity, d) 18 articles related to infectivity and pathogenicity of sars, mers and covid-19, e) 17 articles related to prevention of sses concerning human coronaviruses, f) 9 articles related to socio-economic impact, and g) 9 articles related to immunity and future vaccination. table 2 illustrates the initial numbers of hits using all search items in all sources, and the final number of articles reviewed in each category. the involvement of sses in sars extensive outbreaks (1, 4, 5, (13) (14) (15) (16) (17) , necessitates urgent elucidation as global tranquility is disturbed by covid-19 pandemic. epidemiological research has proposed that the outbreak was related to a seafood market in wuhan (hubei, china), underlining the ongoing risk of viral transmission from animals to induce severe diseases in humans. metagenomic rna sequencing of bronchoalveolar lavage fluid from a patient with pneumonia identified a novel rna virus strain from the coronaviridae family (called sars-cov-2); and phylogenetic analysis (by introducing the widely used in silico protein screening) (18) (19) (20) (21) of the complete viral genome (29,903 nucleotides) disclosed that the virus was most closely connected (89.1% nucleotide similarity) with a group of sars-like coronaviruses (genus betacoronavirus, subgenus sarbecovirus) formerly isolated from bats in china (18) (19) (20) (21) (22) . (1, 5, 13, 15) , that closely related sars-like viral genes were traceable in chinese bat populations. authors claimed that these viruses were capable of infecting humans, by selective adaptations or adjustments, and thereby, causing a new epidemic (23) . enhancement of virulence is also attributed to these adaptations due to acquisition of spike protein via adaptive mutations (24) . continuous viral random mutations are possible through intermediate host transmission, until a deadly virus develops, as illustrated in figure 2 . recent evidence revealed that recombination within intermediate hosts has contributed to development of sars-cov-2 (1, 24). asian outdoor markets could constitute the ideal places for continuous viral mutation exchanges (25) . as presented in table 3 , the best way to circumvent continuous virus production is targeted surveillance; to at least stop the overspreading by sses (2, 3, 22, 26) . this has also been proposed by menachery et al. (23) . sars-cov-2 is accountable for the unprecedented covid-19 pandemic (27) , and the interplaying mechanisms involved in the pathophysiology of covid-19 include sars-cov-2 virulence, host immune response, and complex inflammatory reactions (28) . emerging data, also, imply that the reser-voirs of sars-cov-1 infection may be similar to covid-19 (1, 4, 5, 13, 29) , as remarkable similarities exist between sars and swine acute diarrhea syndrome (sads) in topographical, temporal, environmental and etiological backgrounds. however, the increasing coronavirus variety and spread in bats were recognized as a potential target to diminish future epidemics that might impend livestock, community health, and financial progress (30) . probably, identification of animal and insect vectors that transmit the disease, identification and control of alternative routes of transmission like fecal-oral route, and identification of super spreader patient groups could help minimize the epidemiological extent compared to the one observed for sars-cov-2 infection worldwide. lessons from sars epidemic taught us that the key to control is minimizing the time from the diagnosis of infection to prompt hospital isolation and diminishing the probability table 3 : key clinical and laboratory screening functions to appropriately forecast, prevent, and confront sars! coronavirus 2, and future coronavirus epidemic waves to estimate the probability of a major outbreak, use simulations of stochastic compartmental epidemic models. use of diagnostic tests to detect asymptomatic susceptibility and pre-symptomatic infectivity estimation of super spread events of current and previous coronavirus epidemics introduction of individual reproductive number. integrated and computational analysis of the influence of individual variation by binomial distribution and use of branching process analysis of disease data. genetic characterization of inpatient viral isolates to identify intermediate animal hosts facilitating the infection next generation sequencing of samples and cultured viral isolates to obtain full sequence and phylogenetic analysis application. environmental detection and continuous sewage monitoring rt-qpcr# screening on sewage systems, vectorsùĺ and potential air transmission. autopsies and detection of serology conversion of potential vectors. heptad repeat region screening for positive selection computer simulation models to detect positive selection events e.g. codeml branch-site test coupled with bayes empirical bayes procedure, and mixed effects model of evolution. receptor recognition analysis of ace-ii+ to identify origin of crossspecies and human to human transmissions coronaviruses genetic sequencing and phylogenetic analysis of ace-ii to provide origin and efficiency of cross-species and human to human transmission and identification of intermediate hosts. !severe acute respiratory syndrome; #reverse transcriptase quantitative polymerase chain reaction; +angiotensin-converting enzyme-ii. fecal -oral frequent contact with wild animal reservoirs (including domestic animals) and birds** airborne and fecal -oral construction area workers air particles sewage system workers*** fecal -oral *in both community and hospital environments. **including slaughter houses, pet shops, animal and bird collectors and breeders, cow, and pig farmers. ***including workers coming in contact with environment contamination. !human immunodeficiency virus, #methicillin resistant staphylococcus aureus. of another sse (5). the typically recognized 20-80 rule or the so-called "pareto rule", states that 20% of efforts lead to 80% of results (31) . more specifically, this comprises a principally convenient state when tackling infectious diseases and is applied to investigate infection transmission, and initially among cattle farms. in this regard, woolhouse et al. (17) reported that targeted actions concerning disease control and prevention in 20% of the farms that mainly supplied the basic reproduction number (ro) decreased spread by 80% (32) . focusing on the covid-19 virus, ro is a sign of virus transmissibility, denoting the average figure of novel infections caused by an infectious individual in a totally naive population. for r0 > 1, the number of infected people tends to increase, whereas for r0 < 1, transmission is likely to stop; ro represents a chief model in the epidemics, signifying the risk of an infectious mediator with regard to epidemic spread (33) . recent data indicate that the estimated mean ro for covid-19 is almost 3.28, with a median of 2.79 and the interquartile range (iqr) of 1.16, which is substantially higher than who's estimation of 1.95. however, due to biased methodology, ro for covid-19 is expected to be about 2-3, which is approxi-mately consistent with the who estimate (33) . sses appear to be a main limitation of the ro concept. ro, when calculated as a mean or median value, does not include the heterogeneity of transmission between infected individuals (4); two infective agents with equal r0 estimates might have noticeably diverse patterns of transmission. moreover, the goal of a health care system is to achieve ro <1, which is probably only phenomenally feasible in certain conditions without scheduled prevention, recognition, and response to sses (9) . naturally, epidemics follow the aforementioned 20 / 80 rule (17) . specifically, in human population, due to heterogeneous exposure to infectious agent, the 20% core population may transmit the disease, widely. for sars, the rate might have been even lower than 20% (4). the increased infectious potential of a small population subgroup seems to be related to immunodeficiency, such as in hemodialysis, cancer, immunosuppressive therapies (4, 5, 15, 34) . additionally, facilitation of disease spread and transmission due to vector exposure has been investigated in relation to cockroaches (35) . possible mechanical transportation by rats and cat (13, 36) and air transmission (37) in sars-cov-1 have also been studied. other animals capable of being sars-cov-2 carriers (excluding mice and rats), like pigs, ferrets, cats, and nonhuman primates have recently been introduced (3), and contamination of sewage with sars-cov-2, has probably preceded covid-19 outbreak in france (29) . all these agents may contribute to a minimum of 80% of the total transmission potential (17), maybe even more (4, 5) . table 4 displays possible super spreader groups; thus, indicating screening targets to prevent sses. sars epidemic taught us that control programs were inefficient in controlling the epidemic within a population, and failed to identify and provide a targeted infection diagnosis in groups causing potential sses (5, 17) . on the other hand, sars-cov-2 having the ability to cause a pandemic rather than an epidemic, resulted in an increased number of cases and deaths; albeit having a lower mortality rate than sars coronavirus (2) . sses during covid-19 may involve not only one city, but also a whole country or many countries, requiring investigation of their effects on a national or international level (2, 38, 39). preventing and decreasing covid-19-related sses necessitates the decryption of the mechanism through which sars-cov-2 spreads through super spreader individuals, for example within healthcare facilities (7, 9) . healthcare facilities are essential for prevention and control of sses (9) . sse prevention may enable us to even overcome initial low covid-19 virus infectiveness. the capability of the virus to produce sses troubles the epidemiological attempts to restrict viral spread only by isolating individuals at high risk and performing obsolete isolation at home for the general population as carried out in countries such as greece (5) . during the sars epidemic in china (beijing) and singapore, the vast majority of infected individuals were barely infective and only 6% of the population was highly infectious, in contrast to many published sars models (4, 5) . other ways of potential coronavirus transmission between hosts may provide explanations for enormous outbreaks (16) . it should not be disregarded that coronaviruses cause both respiratory and intestinal infections and share common evolutionary roots with hepatitis viruses (40, 41) . passing the cross-species barrier and genetic adaptation within hosts may promote virulence of coronaviruses in humans (14) . this, prompts to specifically identify potential super spreader groups within populations through targeted diagnosis. some of these groups are listed in table 2 . for this purpose, a usual infection must be distinguished from a super spread infection (4, 5) . during sars epidemic, the coronavirus infectiousness mostly occurred in the late stages of infection (5, 17) , whereas in covid-19, viruses are transmitted even in pre-symptomatic stages (42) . as with influenza a virus subtype h1n1 transmission (43) , accurate diagnosis of covid-19 in potentially asymptomatic super spreaders may help contain the magnitude of large outbreaks (44) . in the case of diamond princess cruise ship, an earlyassessed r0 of 14.8 (âl'ĺ4 times higher than the r0 in the epicenter of the outbreak in wuhan, china) was decreased to an assessed effective ro of 1.78 following on-board isolation and quarantine processes (45) . similarly, in china (wuhan) the application of non-pharmaceutical interventions in the society, including a cordon sanitaire of the town; interruption of community transport, school, and most employment; and termination of all community events decreased the ro from 3.86 to 0.32 over a 5-week period (46) . nevertheless, these strategies could not be maintained. emerging research evidence (29) regarding sewage contamination that preceded paris covid-19 epidemic is pointing to the reports of 2003 from the health department of hong kong (35, 36) , the noble work by ng (13) , and urge for extensive environmental monitoring (29, 37) to prevent future covid-19 relapses. however, the flow of genetic variation may be even more complex as illustrated in figure 2 . therefore, advanced clinical and laboratory monitoring is required to prevent sses and thereafter, new coronavirus epidemics. assembly of key functions and screening techniques of reference centers is presented in table 3 . newer therapeutic agents and protocol applications are promising (47) , although probably carrying the possibility of resistance state (48) . first, these also require specific diagnostic and surveillance strategies to overcome any unknown adverse epidemiology consequences (48) . inhibiting wild meat markets and related consumption of wild meat by creating vivid campaigns could be a critical for interrupting the introduction of coronaviruses crossing from animals to the human population, as was the case for sars (1, 4, 5) and middle east respiratory syndrome (mers) (49) epidemics, and probably now for covid-19 pandemic (1-3) . furthermore, the food production process requires radical reconsideration, concerning the industrial environment of current food production and serious violations of natural ecosystems (50) . current industrial procedures for preparing food increasingly favor conditions where viral evolution produces new mutations and increased rates of mutations (25) , thus raising the probability of new and more infectious viral strains. in sars and mers epidemics, the role of sses in vigorously distributing the epidemics has been substantially proven (51) (52) (53) (54) (55) . the new covid-19 epidemiology evidence also adequately highlights the important role of sses in homeland of china (21, 56, 57) , although surprising evidence from neighboring countries show the unlikely role of sse in the spread of the disease (58). the coronaviridae family is characterized by a positive-sense single-stranded rna genome. mouse hepatitis virus is a representative member of the family (41) . additionally, human hepatitis e virus also has a positive-sense single stranded rna genome and shares a common evolution pathway with coronaviruses (40) . hepatitis-related incidents were described for sars (59) . the genetic recombination of these viruses within arbitrary intermediate hosts produced contagious strains that are extremely pathogenic to humans (40, 60) . in this respect, the relation of sars-cov genetic sequences isolated from human, civets, and bats permitted us to find the reason for such a dangerous epidemic, which affected people on a worldwide scale in 2003 (61) . moreover, the unpredictable epidemic of mers-cov posed a serious risk to the health of communities worldwide. these underscored the necessity for further research of the virus epidemiology and pathophysiology to develop successful therapeutic and preventive medications against mers-cov infection (62) . while sars-cov-2 is genetically and structurally connected with mers-cov, it has its own exclusive structures which are responsible for its quick spread throughout the world (60). specifically, variations in coronavirus pathogenicity within different species (63) make the understanding of sars epidemics even more unclear through their capability to overcome the barrier for cross species transmission, which also alters their infectivity status (14, 64) . as a result, boosting the pathogenic behavior of coronavirus strains, within species (65) , and across species barriers (49) , which is a reflection of their positive adaptation to rapid recombination events (49) . the recent mers epidemic revealed the tendency of the strain to genetically adapt and produce greater outbreaks (49) as occurred in sars epidemic in 2003 (66) . however, mainly for socioeconomic reasons, alarm signals were ignored until recently (67) . a new phylogenetic analysis technique employed on clustered covid-19 strains displayed a geographic variation preference in infectivity and pathogenesis (39) . this is probably due to predominating strain's tendency to cause an sse as an outcome of a multi-factorial epidemic process presented in figure 2 (23, 24) . marked sses for covid-19 have already been fully characterized and warrant urgent investigation (23, 24) . as presented in tables 3 and 4 , each way of transmission should be investigated. heterogeneity of epidemic characteristics across nations (39) implies that in this way we may minimize coronavirus transmission. therefore, salvation of national economic catastrophes will also be achieved in this way (66) . thus, the whole biomedical science machinery needs to perform targeted diagnosis of sses and share the obtained experience. subsequently, central authorities will no longer need excessive non-specific contact measures, which will in turn normalize both societal and economic activities. on the other hand, improper understanding of how covid-19 spreads resulted in societal imbalance due to arbitrary restriction of social and religious life including holy communion cup. it has been consecutively demonstrated by expert research that the holy cup (chalice) and the holy cloth are not sources or pathways, for potential spreading of infectious diseases including human immunodeficiency virus (hiv) (68), hepatitis b virus (hbv) (69) as well as other communicable pathogens (70) . specifically, a review (69), considered other 129 relative studies. in this review, the possibilities that the shared communion cup can act as a vehicle for indirect transmission of human immunodeficiency virus, since it was detected in the saliva of infected individuals, was investigated. it was emphasized that although for bacterial contamination, the alcoholic content of the wine, the material that the cup is made of, or the practice of partially rotating the cup, cannot stop the occasional transmission of microbes, the microbial transmission was considerably reduced by the intervening use of a cloth to swab the lip of the cup between communicants. notably, it was emphasized that transmission means not an obligatory inoculation or infection. furthermore, it was also emphasized that out of the epidemiology of microbes transmitted via saliva, particularly for the transmission of the herpes viruses, the indirect transmission is rare, and indeed transmission is highly possible by other means than by the saliva. it was also emphasized that neither hepatitis b virus nor human immunodeficiency virus infection can be transmitted by saliva, rendering their indi-rect transmission also less likely by inorganic objects. finally, the study concluded that no episode of disease transmission has ever been reported as a result of the shared communion cup use, and that there was not any scientific evidence that the communion cup practice should be abandoned due to the possible risk of spreading of any infection (71, 72) . likewise, kingston et al. (68) , by considering 44 relative papers, also concluded that there is no evidence that the holy communion cup spreads infections. moreover, more recent estimations also demonstrated that no infections have ever been observed as a result of religious rituals including christian common communion chalice practice (70) ; whereas, data of previous studies implied that saliva could play a role in hbv transmission, are likely to be trivial (69) . similarly, recent evidence indicate that, although hbv dna and hcv rna can be discovered in the saliva of infected patients, they seem unlikely to transmit infection (72) . it should be noted that, as in the case of coronavirus (73, 74) , hbv also exists in many body fluids including saliva, nasopharyngeal fluid or tears by measures of qualitative and pcr methods (75) . the detection of hbv dna in saliva motivated our study group to investigate the potential viral transmission through the holy communion cup. two successive retrospective studies were conducted to investigate the role of holy communion as an independent risk factor of hbv dispersion. the first preliminary study included patients from our registry of those with chronic hepatitis b under entecavir (jannis kountouras-personal communication) treatment (76) , and in the next step, the relative registry of another department of the same hospital was incorporated. other parameters studied, the substantial independent categorical variable to evaluate our hypothesis was the patients' occupation, thereby introducing two sub-groups; priests and non-priests. this classification was performed based on a standard active and perpetual exhibition (at least once weekly) of priests to many people's saliva, as a part of the grounded process of the holy communion cup. the control group comprised of the aggregate of orthodox priests in greece (10,338) and the rest general population (10, 680, 866) at that timeframe. approval of the institutional ethics committee was obtained and all predispositions of the helsinki declaration were fulfilled. the reservoir database did not include any personified information (name, id number, etc.) and thus no informed consent was required. pearson's chi-squared test with 1 degree of freedom was performed to evaluate whether there was a statistically significant difference between the frequencies of hbv infection in case and control groups and statistical significance was set at p <0.05. the first single-centre registry included 71 patients and one (1.4%) of them was a priest. chronic hepatitis b was significantly more frequent among non-priests compared to priests (x2 (1, n=71)=12.65, p <0.05). the extended sample (n=429) included the registry of another department and an aggregate of four (0.93%) priests were diagnosed with chronic hepatitis b. likewise, the chi-square test revealed that non-priest subjects were more likely to suffer from chronic hepatitis from hbv infection compared to priests (x2 (1, n=429) = 31, p <0.001). in conclusion, both of our analyses indicated a lower prevalence of hbv chronic hepatitis among priests when compared to other occupations. currently, vaccines for covid-19 are in pre-clinical development, and no final clinical phase has been ended due the recent emergence of the disorder. many global entities have stated their plans to produce a vaccine for covid-19. according to the who, 41 candidate vaccines are being produced for covid-19 as of march 13, 2020 (77) . importantly, for production of highly effective and safe covid-19 vaccines, features such as the possibility of the induction of antigen-dependent enhancement (ade) and additional severe opposing effects previously detected with sars and mers should be considered. ade is a phenomenon that occurs when non-neutralizing antibodies against proteins of a virus increase, also increasing virus infectivity (78) . in this regard, coronaviruses can escape the immunity provided by inactivated or recombinant protein vaccines via fast evolution (79) . the problem with live attenuated vaccines is that the coronavirus can recover its virulence via serial passages in cell culture or in vivo (80) . moreover, vaccination in animals and humans could facilitate, rather than inhibit, the pathogenesis of the targeted viruses. this can be the consequence of an ade phenomenon. this underlines a mechanism by which specific antibodies facilitate infection with the targeted virus, or cell-based augmentation, a process resulting in an allergic inflammatory response induced by immunopathology (81, 82) . many experimental sars-cov-1 vaccines have been formulated from whole inactivated viruses, due to their advantage of large-scale production, multiple epitope presentation and high conformation stability (83) . one such vaccine uses viruses from ay71a217 strain of sars-cov-1, which are double inactivated using formalin and uv irradiation, the socalled double-inactivated virus (div) vaccine (84) . although div had initially been demonstrated to induce neutralizing antibodies and to protect against sars-cov-1 viral replication, both in tissue culture and in young mice, it soon became apparent that older mice suffered from vaccine-induced immune pathologies, including failure to contain viral replication, augmented clinical disease and associated symptoms, and increased inflammatory response and eosinophilic influx (84, 85) . in this respect, there is an overlap between the immunopathologic responses connected with coronavirus disease and vaccination, and the role of t helper (th) 17 cells in immune augmentation and eosinophilic lung immunopathology; host th17 polarized inflammatory reactions portray an important role in the pathophysiology of covid-19 pneumonia and edema (86, 87) . eosinophilic pathology, indicating increased pathogenesis and disease severity in the elderly, has been attributed to the nucleocapsid (n) protein, despite the incorporation of multiple sars-cov-1 antigens in the div (82, 84) . this is on grounds that the n protein is a strong modulator of innate immunity, also acting as an interferon antagonist, and therefore, it has the capability to induce inflammation with subsequent immune pathology in situations of heterologous viral challenge or in immune senescence, where patients fail to mount effective immune responses against the disease (84, 88) . the route of transmission is important to be established for sars cov-2. as seen with other important infectious diseases of a) air borne transmission such as tuberculosis (89), b) orofecal transmission such as hev (90) and c) blood transmission such as hbv and hepatitis d virus (91) , even if efficient vaccination is established, understanding of sses is still important. recent research data on the immune receptors used by coronaviruses, which reflect their ability to propagate in the human population, imply that complex immune reactions are responsible for a cell to cell transmission. in addition to ace-ii receptor, as is the case with sars-cov, mers-cov (92) and possibly for sars-cov-2 (92, 93), viruses use complex receptor recognition systems common to immunopathology damage mechanisms in coronavirusinfected individuals, which clearly define the clinical outcome (94) . therefore, application of vaccines that may interfere with antibody-mediated infection by coronaviruses (95) without true epidemiologic containment of coronaviruses, to restrict genetic adaptation events and inevitably producing an sse, may be a miscellaneous attempt. however, synergy of sse prevention measures with proper vaccination can provide a robust attempt for disease containment. this study aimed to perform a literature review. although effort was made to decrease the risk of bias of results via double-blind screening of literature and employment of multiple electronic search engines, bias cannot be eliminated due to incomplete retrieval of identified research and biased estimations of included literature conclusions and methods used. outcome of the study may also contain biased estimations originating from wrong interpretation of super spreading individuals in literature reviewed for sars, mers, and covid-19 outbreaks. although the importance of sses in covid-19 was recognized by this study, more data from future accumulated epidemiology studies are needed to justify these findings. taken all together, management of sses is mandatory to yield efficient control over sars-cov-2. this is achievable through early diagnosis of pre/asymptomatic infected individuals within potential super spreading groups. prevention of outbreaks is more essential, especially due to the lack of efficient vaccination and therapeutic protocols, which necessitates efficient monitoring, as sars-cov-2 virus follows complex infectious patterns. the sars-cov-2 epidemiological models that do not take sses into consideration seem to lead to confusing results with high uncertainty. sars-cov-2 causes prolonged "pandemics" through complex adaptation routes. currently, in addition to the high technology utilized for diagnosis, clinical observation is indispensable to deeply comprehend sses and prohibit further outspread of covid-19. reference laboratories with efficient and accredited molecular and serological diagnosis must be inter-linked between countries. all these parameters could contribute to avoiding a second blind unjustified response that characterized the first covid-19 pandemic spread. understanding the epidemiology of covid-19 through sses could be preventive for future epidemics. a systematic meta-analysis research methodology, when covid-19 epidemiology data accumulate further, would be advisable to confirm the conclusions of this study. this study did not involve the participation of any humans or animals as it was based only on literature research. ak inspired the conception and drafted the initial manuscript. jk revised substantially the manuscript, intellectual content and provided disclosed data for hbv investigation. ak and jk made the primary double-blind search. ap, jk, ak, mn, and ng, made all other searches. ap contributed to the immunology aspect of manuscript. ap and ng aided in the clinical part and preparing the final version of the manuscript. md contributed to bibliographical search and revision of the manuscript. all authors contributed to the english editing of the manuscript. âȃč genomic and protein structure modelling analysis depicts the origin and infectivity of 2019-ncov covid-19, sars and mers: are they closely related? clinical microbiology and infection receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus superspreading and the effect of individual variation on disease emergence transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission the role of super-spreaders in infectious disease network analysis of mers coronavirus within households, communities, and hospitals to identify most centralized and super-spreading in the arabian peninsula identifying and interrupting superspreading eventsâȃťimplications for control of severe acute respiratory syndrome coronavirus 2. emerging infectious diseases the psychological impact of quarantine and how to reduce it: rapid review of the evidence loneliness and social isolation as risk factors for mortality why systematic review rather than narrative review? psychiatry investigation possible role of an animal vector in the sars outbreak at amoy gardens. the lancet coronaviruses post-sars: update on replication and pathogenesis superspreading sars events soluble receptor potentiates receptor-independent infection by murine coronavirus heterogeneities in the transmission of infectious agents: implications for the design of control programs identification of a novel antibody associated with autoimmune pancreatitis helicobacter pylori and autoimmune pancreatitis: role of carbonic anhydrase via molecular mimicry? challenge in the pathogenesis of autoimmune pancreatitis: potential role of helicobacter pylori infection via molecular mimicry in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus a new coronavirus associated with human respiratory disease in china a sars-like cluster of circulating bat coronaviruses shows potential for human emergence genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. the lancet the population genetics of mutations: good, bad and indifferent the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in china convalescent plasma in covid-19: possible mechanisms of action covid-19 âȃť toward a comprehensive understanding of the disease time course quantitative detection of sars-cov-2 in parisian wastewaters correlates with covid-19 confirmed cases fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin cannabidiol as prophylaxis for sars-cov-2 and covid-19? unfounded claims versus potential risks of medications during the pandemic amplification of the basic reproduction number in cattle farm networks the reproductive number of covid-19 is higher compared to sars coronavirus the current treatment landscape of irritable bowel syndrome in adults in hong kong: consensus statements outbreak of severe acute respiratory syndrome (sars) at amoy gardens, kowloon bay, hong kong main findings of the investigation legislative council select committee to inquire into the handling of the severe acute respiratory syndrome outbreak by the government and the hospital authority of hong kong environmental transmission of sars at amoy gardens sars-cov-2 (covid-19) phylogenetic network analysis of sars-cov-2 genomes evolutionary biology of human hepatitis viruses presymptomatic transmission of sars-cov-2 âȃť singapore transmission during presymptomatic phase, japan. emerging infectious diseases detecting presymptomatic infection is necessary to forecast major epidemics in the earliest stages of infectious disease outbreaks covid-19 outbreak on the diamond princess cruise ship: estimating the epidemic potential and effectiveness of public health countermeasures evolving epidemiology and impact of nonpharmaceutical interventions on the outbreak of coronavirus disease structure of the rna-dependent rna polymerase from covid-19 the mechanism of resistance to favipiravir in influenza the heptad repeat region is a major selection target in mers-cov and related coronaviruses current status and development strategy for community-supported agriculture (csa) in china transmission characteristics of mers and sars in the healthcare setting: a comparative study pathogen cross-transmission via building sanitary plumbing systems in a full scale pilot test-rig sars: future research and vaccine predicting super spreading events during the 2003 severe acute respiratory syndrome epidemics in hong kong and singapore the 2003 sars outbreak and its impact on infection control practices covid-19 super-spreaders: definitional quandaries and implications reconstruction of transmission pairs for novel coronavirus disease 2019 (covid-19) in mainland china: estimation of superspreading events, serial interval, and hazard of infection inferring super-spreading from transmission clusters of covid-19 in hong kong, japan, and singapore sars-associated viral hepatitis caused by a novel coronavirus: report of three cases a review of sars-cov-2 and the ongoing clinical trials molecular epidemiology, evolution and phylogeny of sars coronavirus. infection molecular characteristics, functions, and related pathogenicity coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus genotype and phenotype of covid-19: their roles in pathogenesis bat origin of human coronaviruses the impact of sars epidemic. washington, d.c.: national academies press estimating the global economic costs of sars memorandum on the infections hazards of the common communion cup with especial reference to aids the hazard of infection from the shared communion cup infections associated with religious rituals saliva and viral infections commentary: a review of risk of hepatitis b and c transmission through biting or spitting transmission routes of 2019-ncov and controls in dental practice saliva: potential diagnostic value and transmission of 2019-ncov high levels of hepatitis b virus dna in body fluids from chronic carriers experience of entecavir administration in patients with chronic hepatitis b. annals of gastroenterology the possible of immunotherapy for covid-19: a systematic review introduction to modern virology animal coronavirus vaccines: lessons for sars identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge avoiding pitfalls in the pursuit of a covid-19 vaccine successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome the potential role of th17 immune responses in coronavirus immunopathology and vaccine-induced immune enhancement. microbes and infection th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib specific epitopes of the structural and hypothetical proteins elicit variable humoral responses in sars patients the role of super-spreading events in mycobacterium tuberculosis transmission: evidence from contact tracing laboratorybased surveillance for hepatitis e virus infection hepatitis b and hepatitis d virus infections in the central african republic, twenty-five years after a fulminant hepatitis outbreak, indicate continuing spread in asymptomatic young adults carcinoembryonic antigen-related cell adhesion molecule 5 is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus virus cell-to-cell transmission clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study'. the lancet interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells understanding the super-spreading events of sars in singapore super-spreading events of sars in a hospital setting: who, when, and why super-spreaders in infectious diseases all authors agree to publish this manuscript. all data used for this manuscript are available upon request all authors declare that they have no competing interests. no funding or grant was received for this study. we thank our families for providing moral assistance to accomplish this study. key: cord-280941-ds6x0yym authors: kim, young-seok; son, ahyun; kim, jihoon; kwon, soon bin; kim, myung hee; kim, paul; kim, jieun; byun, young ho; sung, jemin; lee, jinhee; yu, ji eun; park, chan; kim, yeon-sook; cho, nam-hyuk; chang, jun; seong, baik l. title: chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles date: 2018-05-17 journal: front immunol doi: 10.3389/fimmu.2018.01093 sha: doc_id: 280941 cord_uid: ds6x0yym the folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (np) vaccines in a timely and reproducible manner. despite significant advances in in silico design and structure-based assembly, most engineered nps are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. the failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. capitalizing on a novel function of rna as a molecular chaperone (chaperna: chaperone + rna), we provide a robust protein-folding vehicle that may be implemented to np assembly in bacterial hosts. the receptor-binding domain (rbd) of middle east respiratory syndrome-coronavirus (mers-cov) was fused with the rna-interaction domain (rid) and bacterioferritin, and expressed in escherichia coli in a soluble form. site-specific proteolytic removal of the rid prompted the assemblage of monomers into nps, which was confirmed by electron microscopy and dynamic light scattering. the mutations that affected the rna binding to rbd significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of nps of a defined size. this underscored the rna-antigen interactions during np assembly. the sera after mouse immunization effectively interfered with the binding of mers-cov rbd to the cellular receptor hdpp4. the results suggest that rna-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of nps into highly regular and immunologically relevant conformations. the concentration of the ion fe(2+), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the nps. the kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of nps and virus-like particles as recombinant vaccines and for serological detection of viral infections. introduction various types of viral vaccines have been developed over the last century with a wide spectrum of efficacy and safety (1, 2) . the manufacturing of most conventional vaccines-live attenuated, inactivated, or subunit vaccines-invariably require the culturing of infectious viruses in cell substrates (3) . despite dedicated efforts, conventional cell culture often fails to produce sufficient amounts of virus for evaluating the immunogenicity, protective efficacy, and safety of viral vaccines. moreover, some emerging viruses cause high-mortality rates, without options for treatment or prophylaxis, necessitating their manipulation, and manufacture under stringent bio-safety environment (4) . not surprisingly, alternative technologies that circumvent these limitations are a high priority in the areas of vaccine development and production. nanoparticles (nps), virus-like particles (vlps), and assembly of multimeric peptides each provide attractive platforms for vaccine design (5) . virus-like particles and nps structurally resemble infectious virions, but are non-infectious due to the lack of viral genomes. recombinant surface antigens from natural virions are assembled into highly ordered conformations as empty particles devoid of genetic material. antigenic epitopes are presented on the multivalent and highly repetitive outer structure of the nps, which leads to the crosslinking of b-cell receptors and the induction of long-lasting immune responses (6) (7) (8) . by mimicking the morphology of the natural infectious virions, the regularly assembled particles are highly immunogenic, and are amenable to diagnostic and prophylactic exploitation. among the simplest targets are the vlps of non-enveloped viruses, such as hepatitis e virus or human papilloma virus, and are composed purely of viral capsid proteins (9) (10) (11) . in contrast to non-enveloped viruses, where virion assembly is exclusive to capsid proteins, enveloped viruses (e.g., coronavirus or flavivirus), require an additional membrane component for assembly into mature virions. consequently, in enveloped vlps, the assembly of matrix proteins provides a molecular scaffold, and viral antigens are embedded into lipid membranes. different types of glycoproteins may be embedded in the lipid membrane as target antigens for generating immunological responses (12) . however, this process requires multiple proteins (surface antigens and matrix proteins), and the enveloped vlps are not structurally uniform and are difficult to characterize. a promising alternative is to present the target antigens on the surfaces of self-assembled nps, which, in lieu of lipid membranes, serve as the macromolecular scaffold for the presentation of the antigens of interest. in developing np vaccines, consideration should be given regarding the selection of a robust and faithful system for np assembly that enables the cost-effective development and delivery of vaccines in a timely manner. structure-based approaches in silico and their underlying principles are relatively advanced for np assembly (13) (14) (15) . most of the approaches consider the thermodynamic stability of the final assembled nps, without due recognition for the kinetic complexities controlling regular assemblage over random interactions that lead to misfolded aggregations. therefore, it is not surprising that most engineered nps are refractory to soluble expression, which presents practical challenges in production, both at a laboratory-scale and in commercial manufacturing processes. this problem becomes augmented when expressed in bacterial hosts because of a lack of folding assistance in the bacterial cytoplasm for viral antigens. therefore, due to advantages in assisted folding, posttranslational modifications, and the possibility of generating multiple-component nps and vlps, eukaryotic hosts such as yeast, insects, and mammalian cells have been favored over bacterial hosts (16) (17) (18) . however, these systems are significantly more expensive than bacterial systems, are more time-consuming, and the down-stream processes are usually more complex. moreover, the purification of vlps from insect cell systems poses a challenge due to similar physicochemical properties between the vlps and the baculoviruses (1, 16) . bacterial systems, if available, would provide a cost-effective means to develop and deliver vaccines, as well as sero-diagnostic antigen kits used to diagnose-specific infection diseases. middle east respiratory syndrome (mers) was first reported in saudi arabia in 2012 and has caused multiple cases of infection with high mortality in europe and asia (19, 20) . mers is caused by mers-coronavirus (mers-cov), which can be transmitted from camels to humans, and from humans to other humans (21, 22) . worldwide transmission is increasing in direct household and community-wide transmission, as well as in nosocomial settings, as exemplified in a 2015 outbreak in korea (23, 24) . neither effective vaccines nor therapeutic interventions are currently available. because of this, assembly of mers-cov antigens into immunologically relevant conformation as nps would be of interest and may be helpful in developing vaccines, sero-diagnostic tools, and therapeutic monoclonal antibodies. in the current study, we present a novel bacterial np of mers-cov antigen using ferritin as a molecular scaffold for self-assembly. ferritin, which is present in most living organisms, has 24 identical subunits that spontaneously self-assemble and form np complexes with internal and external diameters of 8 and 12 nm, respectively (25, 26) . previous studies show that ferritins of helicobacter pylori from a human isolate can be used as scaffold for hiv and influenza np vaccines, using eukaryotic host cells such as human embryonic kidney cells (hek293f or hek293s) (27, 28) . likewise, bacterioferritin (fr), which self-assembles into nanocages with octahedral symmetry, has also been evaluated as a potential drug delivery system (29) . however, viral antigens of human pathogens are prone to misfolding into aggregates, which necessitates chemical refolding of the insoluble aggregates in order to regain solubility and to allow regular assembly of the antigen (30, 31) . in addition, displaying antigens on the surface of multi-molecularly assembled scaffolds in bacterial hosts remains a daunting challenge. we hypothesized that nps displaying the receptor-binding domain (rbd) of the spike protein from mers-cov could be produced in a bacterial system by harnessing the function of a molecular chaperone. conventionally, protein folding and the prevention of non-functional aggregation have been ascribed to molecular chaperones (32) (33) (34) . recently, it has been shown that rna molecules are able to provide novel functions as molecular chaperones (35) (36) (37) . based on novel findings, the concept of chaperna (chaperone + rna) function was established (38) . in this report, chaperna function was harnessed for the folding and assembly of hybrid ferritin monomers into nps using a bacterial expression system. we also demonstrated that the biophysical properties, including solubility, yield, and stability of mers-cov nps, could be improved by properly controlling the rna-binding affinity, and the concentrations of fe 2+ and salts. the chapernabased np assembly may prove to be a versatile tool for developing and delivering recombinant vaccines and for serological detection of emerging/re-emerging viruses. the expression vector pge-hrid(3) was constructed from the parental vector pge-lysrs (3) (39) . the pge-lysrs(3) vector was enzymatically cut with ndei and kpni. the pcr product of hrid, which carries the tev protease cleavage site and a 6-histidine tag at the c-terminus, was cut using the same restriction enzymes and the digested fragment inserted into the vector to generate pge-hrid(3). fr (genebank accession no. nc_000913.3) dna was synthesized by, and purchased from, cosmo genetech (korea). the dna was cleaved with sali and hindiii, and inserted into pge-hrid(3) to generate hrid(3)-fr. the receptor binding domain (rbd), n-terminal residues 367-606, of the mers-cov s protein (genbank accession no. afs88936.1), was generated by gene synthesis, cut with kpni and sali, and inserted into hrid-fr to generate pge-hrid(3)-rbd-fr. linker ssg or asg was inserted into the c-terminus of the rbd using overlapping pcr, cleaved with kpni and sai, and ligated into hrid-fr, generating pge-hrid(3)-rbd-[ssg]-fr or pge-hrid(3)-rbd-[asg]-fr, respectively. the schematic diagrams of each expression vector are illustrated in figure 1b . the genes of mutant hrid(2 m) (k19a and k23a) and hrid(9 m) (k19a, k23a, r24a, k27a, k30a, k31a, k35a, k38a, and k40a) were generated by gene synthesis, cleaved with ndei and kpni, and inserted into pge-hrid(3)-rbd-fr, generating pge-hrid(2 m)-rbd-fr and pge-hrid(9 m)-rbd-fr, respectively. the mutation sites and amino acid sequences of the mutants are shown in table s1 in supplementary material. the resulting expression vectors were transformed into the escherichia coli strain shuffle ® t7. the cells were grown in 50 ml of lb medium with ampicillin (50 µg/ml) at 30°c overnight. each type of transformant was inoculated into 500 ml of lb medium with ampicillin, grown at 30°c until an optical density (od600) of 0.6-0.8 was reached. protein expression was induced with 1 mm iptg for 12 h. each sample was harvested by centrifugation, lysed by sonication in lysis buffer (50 mm tris-hcl, ph 7.5; 10% glycerol; 2 mm 2-mercaptoethanol; and 0.1% tween-20). the soluble fraction of each lysate was purified on a ni-affinity histrap™ hp column by atka prime (ge healthcare) and concentrated with centriprep™ (merck millipore ltd.). the purified proteins were treated with tev protease to remove the fusion partner hrid. the assembled nps were purified by gel filtration on 10/300 superose™ 6 increase columns (ge healthcare). to examine the size and structure of the purified nps, microscopic evaluations using tem and cryo-em were performed. for tem analysis, a drop of the nps was placed onto a formvar/carboncoated tem grid (spl). the grid was negatively stained with 2% uranyl acetate, dried, and examined using a jem-1011 electron microscope (jeol) at an accelerating voltage of 80 kv. the particle sizes were calculated using camera-megaview iii (soft imaging system-germany) for measuring the nps in random image fields. for cryo-em, the nps were placed onto plasma-treated formvar/ carbon 200 copper grid (ems) and negatively stained with 2% uranyl acetate. the grid was accelerated at 200 kv with an fei cryotecnai f20 cryo-em microscope made available through the korean institute of science and technology. the nps were examined and photographed in high resolution. nanoparticle samples (3 ml) were placed into a dispo-h cell, and analyzed using a zeta-potential & particle size analyzer (els(37, 30, and 18°c ) and the cell lysates were separated into total (t), soluble (s), and insoluble (p) fractions by centrifugation (left panel). the solubility of each protein expressed at 18°c was measured by a gel densitometer and the data were summarized and shown in the right panel (n = 3). statistical significance (**p < 0.01, ***p < 0.001) was indicated for the samples compared with the control using a two-tailed student's t-test. (d) illustration of mers-cov rbd-fr nps using the chaperna-based hrid fusion partner. the hrid facilitated folding of the aggregation-prone rbd-fr through interaction with rna. the monomer of rbd-fr formed a properly folded trimeric structure by cleaving hrid with tev protease. eight trimers assembled and formed into mers-cov-like nps. red triangles indicate the rbd trimer on the fr nps. of the nps was measured twice at 25°c in water as a solvent with the sample accumulation time at 200 s. effect of salt and fe 2+ concentrations on np assembly and stability cultured cells (3 ml) were lysed with lysis buffer in the presence of various concentrations of nacl (0, 50, 100, 150, 200, 225, 250, 275 , and 300 mm) to evaluate the intracellular proteins. all samples were performed in triplicate. the cell lysates were separated into soluble and insoluble fractions by centrifugation, and the protein stabilities analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). thus, the proteins from cell lysates (500 ml culture) were purified using hispur™ ni-nta resin (thermo fisher scientific) in buffer a depending on nacl concentration (0-300 mm). to evaluate the effects on fe 2+ on np formation, cells were cultured in lb media with various concentrations of fe 2+ (0, 200, 500, and 1,000 µm). np formation was examined by size exclusion chromatography (sec), sds-page, tem, and dls at the various concentrations of nacl or fe 2+ . the cells were harvested, sonicated with lysis buffer, and separated into soluble and pellet fractions by centrifugation. target proteins in the soluble fraction were purified using hispur™ ni-nta resin (thermo fisher scientific), following the manufacturer's instruction. t (total lysate), s (soluble fraction), p (pellet fraction), w (wash fraction), and e (the elution fraction were analyzed by sds-page. co-purification of the nucleic acids and proteins in the wash and elute were analyzed on a native agarose gel. the nucleic acids were visualized with ethidium bromide (etbr), and the proteins with coomassie staining. cultured cells (10 ml) were harvested using the same method described above. the cells were lysed with 500 µl of protein extraction reagent b-per™ ii (thermo scientific) and separated into soluble and pellet fractions by centrifuged 12,000 rpm for 10 m. a 200 µl aliquot of each soluble fraction was further treated with 250 µg/ml of rnase a (intron biotechnology) and incubated at 37°c for 15 min. the nuclease treated samples were clarified by centrifugation at 12,000 rpm for 15 min and the soluble supernatants and the pelleted precipitates were analyzed on an sds-page gel followed by western blot analysis. to confirm the proper folding of rbd-fr and its variant (rbd-[ssg]-fr), the binding of the purified proteins with the mers-cov receptor hddp4 was performed by elisa. fr only and phosphate-buffered saline (pbs) were used as negative controls. nunc 96-well microtiter immunoplates (thermo fisher scientific) were coated with 100 ng/well of hdpp4 proteins (abcam) and incubated at 4°c overnight. the plates were washed and blocked with 150 µl/well of blocking buffer (1% bsa) for 1 h at room temperature. rbd (ssg linker, wt, 2 m, or 9 m)-fr (100 ng/ well) were added for 2 h at 37°c. an anti-penta his antibody (100 µl/well; qiagen) was serially diluted (1/100 to 1/12,800) in tbst [50 mm tris-cl (ph 7.4), 0.05% tween-20], added to the wells, and incubated for 1 h at 37°c. a secondary goat anti-mouse igg antibody conjugated with hrp in a 100-µl volume (1:5,000, sigma-aldrich) was added and incubated for 1 h at 37°c. the plates were washed three times with tbst at the end of each step. after washing, 100 µl/well of substrate tmb solution (bd biosciences) were added to the well and the plates were incubated at 37°c for 30 min in the dark. 50 µl of stop solution (2 n h2so4) was added to the well to stop the colorimetric reaction, and the absorbance at 450 nm was measured using an elisa reader, fluostar optima (bmg labtech). . the coating antigens were removed, and the wells were blocked with pbst (5% skim milk in pbs and tween-20) for 1 h at 37°c. after 2 h, the blocking solution was removed. twofold serially diluted sera from four patients (cnnh-0709, 0809, 1009, and 1309) were added to each well and incubated at 37°c for 2 h. the antigencoated wells were incubated with peroxidase-conjugated goat anti-human igg antibody (kpl, seracare life sciences, milford, ma, usa) at 37°c for 1 h. the primary antibody was removed and 3,3′,5,5′-tetramethylbenzidine (tmb; sigma-aldrich) was added to each well as colorimetric substrate. immediately after treatment of the reactions with stopping solution (sigma-aldrich), the od was read at 450 nm. six-week-old female balb/c mice were immunized with 20 µg/ mouse of the rbd-fr, rbd-[ssg]-fr, or rbd protein generated as described above, or with commercially available mers-cov rbd protein (mers-rbd-005p; eenzyme) as antigen in bsl-2 facility in ylarc. antigens were diluted in pbs. for the first group, equal volume of mf59 adjuvant (addavax, cat. no vacadx-10) (43) was mixed by pipetting. for the other group, equal volume of antigens and alum adjuvant (thermo fisher scientific) were mixed by pipetting following the manufacturers' protocol. pbs plus adjuvant and fr were used as negative controls. the immunized mice were boosted twice with intramuscular injections on days 14 and 28. mice were anesthetized on days 27 and 41 for ocular bleeding from the orbital sinus ( figure s10 in supplementary material). immune sera were processed by centrifugation of the collected blood at 12,000 × g for 30 min. the spleen and the balf (bronchoalveolar lavage) were obtained at 7 days after the last immunization from sacrificed mice. balf was taken by washing the airways with 1 ml of pbs. t-cell population from immunized mice were analyzed by flow cytometric analysis (43, 44) . the spleens were taken at 7 days after the last immunization from the sacrificed mice. to obtain single-cell suspensions, the tissues were homogenized and passed through 70 µm cell strainers (spl). after centrifugation, erythrocytes were removed by red blood cell lysing buffer (sigma). the cells were washed and resuspended in iscove's modified dulbecco's media containing 10% fbs. for intracellular cytokine staining, the splenocytes were stimulated with 10 µg/ml rbd protein or phorbol myristate acetate/ionomycin in the presence 10 ng/ml recombinant human il-2 (biolegend) and brefeldin a (1:1,000; ebioscience) at 37°c for 5 h. after stimulation, the cells were blocked with rat anti-mouse cd16/cd32 (bd biosciences) and surface stained with anti-cd8 (fitc, clone 53-6.7; biolegend) and anti-cd4 (pe/cy7, clone gk1.5; biolegend) at 4°c for 30 min. the stained cells were fixed in facs lysing solution (bd biosciences) at room temperature for 20 min, and permeabilized with facs buffer (0.5% fbs, 0.1% nan3 in pbs) containing 0.5% saponin (sigma) at room temperature for 15 min. then, the cells were stained with anti-ifn-γ (pe, clone xmg1.2; biolegend) and anti-tnf-α (apc, clone mp6-xt22; biolegend) at room temperature for 40 min. all data were collected by bd lsr fortessa (bd biosciences) and analyzed with flowjo software (tree star inc., ashland, or, usa). competition elisa was performed to determine whether mers-cov antigen [rbd-[ssg]-fr, rbd-fr, rbd, and fr (negative control)]-immunized mouse serum inhibited binding of rbd protein to hdpp4 receptor (45, 46) . 500 ng/well hdpp4 protein (abcam) was coated on nunc 96-well microtiter immunoplates (thermo fisher scientific) and incubated overnight at 4°c. plates were washed and blocked with 150 µl/well of blocking buffer [5% skim milk in pbs and tween-20 (pbst)] for 1 h at 37°c. at the same time, mouse sera immunized with rbd, rbd-[ssg]-fr, rbd-fr, and fr were serially diluted (1/10 to 1/160) with 500 ng/well rbd protein (mers-rbd-005p; eenzyme) in tbst [50 mm tris-cl (ph 7.4), 0.05% tween-20], added to new wells, and incubated for 1 h at 37°c. 100 µl solution was added to each well at 37°c and incubated for 2 h. after that, 100 µl of anti-6xhis tag antibody conjugated with horseradish peroxidase (1:1,000, thermo fisher scientific) was added to each well and incubated for 1 h at 37°c. plates were washed three times with tbst, and 100 µl/well of substrate tmb solution (bd biosciences) was incubated at 37°c for 30 min in the dark. 50 µl of stop solution (2 n h2so4) was added to the well to stop the color reaction and measure the absorbance at 450 nm using an elisa reader fluostar optima (bmg labtech). the hrid facilitated the solubility of mers-cov rbd-fr the spike glycoprotein (s) of mers-cov was used for the generation of mers-cov-like nps. s protein forms trimers, resulting in large spikes on the virus envelope (47) . it is challenging to express the full-sized s protein (~200 kda) in e. coli. thus, the s1 domain of s protein (~80 kda), which includes the receptor-binding ability, was used. our initial attempt to express the s1 domain, either as s1 or as an s1-fr fusion protein, failed; the expression level and solubility of the protein was below the lower limit of detection by sds-page and western blotting ( figure s1 in supplementary material). we therefore used the rbd (367-606 a.a.) of the s1 protein, which has a pivotal function as illustrated in figure 1b (48, 49) . when expressed alone in e. coli, the rbd is not able to form the trimeric assembly (unpublished observation), due to the lack of the hr2 domain within the s2 domain (50) . to overcome this problem, fr was used as scaffold for the assembly. fr is a spherical np whose subunits form trimers that subsequently result in octahedral structures composed of 24 identical subunits (51) . we therefore performed computational modeling to evaluate the potential of fr as scaffold for trimer formation of the rbd. possible trimer formation was analyzed by computational modeling using modeler (13, 52) and cluspro (41, 42) . various linkers, including ssg, asg, and d6, were introduced between the rbd and fr with a goal to minimize steric hindrance between the two domains so as to enhance trimer and np formation. in silico analysis showed energy-stable trimeric models of rbd-fr, rbd-[ssg]-fr, and rbd-[asg]-fr, whereas rbd-d6-fr failed to form a trimeric structure ( figure 1a) . the rbd-[ssg]-fr was predicted to be the most stable and well-structured compared with rbd-fr and rbd-[asg]-fr. initial testing of the rbd-fr constructs without hrid fusion showed that none of the constructs were solubly expressed, even under low-temperature culture conditions ( figure 1c ) ( figure 1b) . we previously confirmed that by using chaperna, the globular domain of influenza hemagglutinin (ha) is efficiently assembled into a trimeric complex with an immunologically relevant conformation (yang et al., in press) . as shown in figure 1c , the hrid fusion significantly increased the solubility of both rbd-fr (59.1%) and rbd-[ssg]-fr (62.83%), indicating that the chaperna platform effectively increased both the solubility and the folding of its fused target proteins. because of the poor expression level and low solubility of the rbd-[asg]-fr construct (figure 1c) , further experiments were performed using only the rbd-fr and rbd-[ssg]-fr constructs. after purification of the soluble proteins ( figure s2 in supplementary material), we determined the potential effects of using hrid as a fusion partner for the self-assembly of the nps. as shown in figure s3 in supplementary material, hrid-rbd-fr failed to form nps. because of this, we performed tev protease cleavage of the hrid. removal of the hrid domain facilitated the self-assembly of the rbd-fr monomers, and also eliminated the immune response against the non-self hrid domain in balb/c mice ( figure s4 in supplementary material). after hrid cleavage, rbd-fr and rbd-[ssg]-fr were purified using sec (figure 2a) . as expected, rbd-[ssg]-fr assembled into properly formed nps (1,080 kda) more efficiently than did rbd-fr nps, which were mainly detected in the void-volume fractions, suggesting they were irregularly assembled soluble aggregates. the size of the rbd-[ssg]-fr nps was further confirmed by tem. tem images of the rbd-[ssg]-fr np structures showed hollow, spherical particles that were more compact than the rbd-fr nps. the average diameter of the rbd-[ssg]-fr nps was 28-30 nm (figure 2b) . in contrast, dls analysis of the rbd-fr np structure without the ssg linker appeared to be smaller with an average intensity diameter of 26.3 nm, and this compared with rbd-[ssg]-fr that had an average intensity distribution diameter of 30.5 nm (figure 2c) . consistent with the sec analysis, rbd-fr without a fusion partner was mostly produced in a soluble aggregated form. therefore, we identified that the protein folding did not occur properly without hrid, and the formation of nps was confirmed by both sec and sds-page analyses. as shown in figure s5 in supplementary material, the purified nps retained their stability over an extended period of time at various temperatures (25, 4 , and −20°c). thus, these results indicate that the ssg linker allowed the rbd-fr to generate properly assembled nps. it should also be noted that the efficiency of protein folding and nps formation may be further enhance through appropriate linker selection. it has been reported that ionic strength plays an important role in the stability and self-assembly of ferritins (53, 54) . we examined the effect of salt concentration on the formation and stability of the rbd-[ssg]-fr nps at various concentrations (0-300 mm). consistent with the previous studies, the stability of the protein was highly affected by the concentration of nacl in the lysis buffer by sds-page ( figure 3a ) (n = 3). the solubility of the protein significantly decreased as the concentration of nacl increased from 0 to 100 mm, with the solubility being about 8.79-fold lower at 100 mm compared with 0 mm. unlike previous studies, the solubility of the protein was gradually recovered at higher nacl concentrations (>100 mm); the solubility at 300 mm was 1.45-fold higher than at 0 mm. furthermore, the yield of soluble of protein per liter of culture increased in a salt concentrationdependent manner (figure 3b) . to further investigate the effect of salt concentration, the physicochemical and morphological properties of the rbd-[ssg]-fr protein were examined by sec, tem, and dls. in 50 mm nacl, most of the protein was aggregated during the purification process, and the purified protein failed to form spherical structures, but instead, existed predominantly as 45 kda monomers (figures 3c,d; figure s6 in supplementary material). in contrast, the protein that was lysed in 0 mm nacl and purified in 200 mm nacl, developed well-structured nps according to tem and dls analyses (figures 3c,d; figure s6 in supplementary material). however, based on sec analysis, at high-salt concentrations (>250 mm), the protein failed to form stable structures with the proteins being eluted predominantly in the void volume, suggesting they were soluble aggregates under the high-salt concentrations ( figure 3c) . transmission electron microscopy images under the various salt concentrations clearly supported the conclusion, showing that the tendency for aggregation was dependent on the salt concentration ( figure 3d) . taken together, the results underscored the importance of salt concentration on the solubility of monomers and the quality of multimeric assembly of hybrid nps. ferritin has an intrinsic ability to interact with fe 2+ to form ferritin-iron cores (55) . thus, it was worth investigating the effect of fe 2+ on the assembly and stability of rbd-[ssg]-fr nps. cells were grown in lb medium with various concentrations of fe 2+ . as shown in figure 4a , the yield of purified protein was significantly increased from cultures with 500 µm fe 2+ , reflecting a 2.7-fold increase compared with similar cultures 0 µm fe 2+ . the cell growth and purification yield at 1,000 µm fe 2+ were slightly decreased, presumably due to the toxicity of ferric acid. np formation under the various concentrations of fe 2+ was analyzed by sec ( figure 4b) . consistent with the previous results, the proteins were eluted mainly in the fractions expected for the size of assembled nps (1,080 kda). of note, the ratio between nps and soluble aggregates in the sec analysis showed that np formation was facilitated at high concentrations of fe 2+ (figure 4b) . the formation of rbd-[ssg]-fr nps at an fe 2+ concentration of 1,000 µm was confirmed by tem ( figure 4c ) and dls ( figure 4d) . the tem analysis clearly showed that the morphology of the proteins was more compact, and probably highly stable, when assembled at high fe 2+ concentrations (500 µm) than at lower concentrations (0 µm) ( figure 4c ). as shown in figure 4d , the average diameter of nps examined by dls was 25.1 nm at high fe 2+ concentration (500-1,000 µm) and 27.7-32.2 nm at lower concentration (0-200 µm). these results suggest that both fe 2+ and salts concentrations influenced the efficiency and quality of the regular assembly of hybrid ferritin monomers into nps. our previous studies show that an rna-protein interaction is crucial for transducing the chaperone function of rna into the folding of client proteins (38) . consistent with that, our present study showed that rna facilitated the folding of its interacting proteins. the solubility of hrid(wt)-rbd-fr was 5.69-fold higher than rbd-fr without hrid fusion (figure 1) , strongly supporting the previous studies. in addition, the solubility of rbd alone was completely insoluble (figure 5b ; figure s7 in supplementary material). it has been shown that the positively charged residues of lysine moieties in hrid contribute to trna binding (56) . in the current study, the trna binding induced the intrinsically disordered protein (idp) status of hrid to form alpha-helical structures ( figure 5a) . thus, two rna-binding (table s1 in supplementary material). the total e. coli lysate (t) was fractionated into the soluble fraction (s) and the pellet fraction (p) by centrifugation. as expected, both rbd and rbd-fr without fusion to hrid domain, were refractory to being produced as soluble proteins ( figure 5b) . interestingly, the solubility of the rna-binding mutants did not decrease, but actually increased to 75.3% for the 2 m mutant and 93.4% for the 9 m mutant compared with wild-type protein at 60.1% (figure 5b) . considering that hrid is relatively unstructured in the absence of trna binding, the results are consistent with previous reports that the fusion with idps promotes the solubility of target proteins (57) (58) (59) . following purification of wild-type hrid-rbd-fr (hrid(wt)-rbd-fr), electrophoretic mobility shift assays showed that greater amounts of nucleic acids were co-purified with hrid(wt)-rbd-fr protein than with the mutant hrid-rbd-frs (2 and 9 m) under non-denaturing conditions ( figure 5c ). the relative ratio of nucleic acid based on etbr staining and proteins based on coomassie staining in the eluted fraction confirmed the reduced affinity of mutants to nucleic acids. to test if rna had a role in maintaining the stability of the target proteins, the lysates were treated with rnase a to eliminate rna, and the solubility of each protein was analyzed by sds-page and western blotting. the soluble fractions of the lysates (s) were incubated at 37°c in the presence and absence of rnase a and the samples were further separated into soluble fraction (ss′) and insoluble fraction (sp′) by centrifugation. as shown in the left panel of figure 6 , rnase a treatment completely abolished the effect of rna on protein solubility as compared with the control (rnase a−) or with samples prior to rnase treatment. parallel experiments with the 2 and 9 m mutants showed much less rna co-purified with the proteins, confirming the reduced affinity to nucleic acids and the complete depletion of rna by rnase a treatment (figure 6, left panel) . remarkably, the solubility of hrid(wt)-rbd-fr was greatly reduced by depletion of rna as reflected in the ratio of [ss′]/ [sp′] [0.1 and 0.4 for rnase (+) and rnase (−), respectively] by both coomassie staining and western blot analyses (figure 6 , right panel). however, the solubility of the mutants (2 and 9 m), was not significantly affected by rnase a treatment, probably due to their lower affinity to rna (figure 6, right panel) . taken together, the results demonstrate that hrid(wt)-rbd-fr maintained a strong affinity for rna, and that affinity was pivotal for maintaining the solubility of the protein. to further define the rna dependence of solubility of the ferritin hybrids (figure 6) , we investigated if the rna binding had a role in the formation of nps. rbd-fr and the various hrid-rbd-fr (wt, 2, and 9 m) proteins were purified by nickel-affinity chromatography ( figure s2 in supplementary material) and their physicochemical properties analyzed by sec (figure 7a) , tem (figure 7b) , and dls ( figure 7c) . the soluble yields of rbd-fr (hrid fusion) was approximately 1.6 mg/l of culture, representing greater than 1,000-fold higher levels than its hrid (−) counterpart (~15 μg/l culture), again confirming the role of hrid as a robust enhancer for solubility and assembly. it was striking to note that the two mutant proteins, despite high solubility (figure 5b) , were detected at disproportionately higher amounts in the void fractions of sec, indicating that they failed to form nps of a defined size, and existed predominantly as soluble aggregates ( figure 7a) . however, hrid(wt)-rbd-fr predominantly formed nps of a defined size (~1,080 kda). it is also interesting to note that there was a slight shift of the rna-binding mutants (2 and 9 m) in the elution pattern, suggesting a larger size of nps compared with wild-type nps. overall, the ratio between soluble aggregates in the void volume and the nps of defined size clearly showed that rna binding was crucial for assembly of the monomers into nps. as a control, rbd-fr (without hrid fusion) existed predominantly as soluble aggregates ( figure s8 in supplementary material). consistent with these results, em analysis confirmed well-structured nps by hrid(wt)-rbd-fr, compared to largely aggregated structures by the mutant proteins ( figure 7b) . even if multi-molecular structure was formed, the structure becomes unstable, mostly as soluble aggregates. consistently, the intensity distribution diameter of the wild-type protein, as estimated by dls analysis, was 25 nm compared with larger sizes of hrid(2 m) at 34.2, 519.2 nm and hrid(9 m) at 52, 717.7 nm (figure 7c ; figure s9 in supplementary material). it is conceivable that soluble aggregates may shield the exposed 6-histidine tag, resulting in a decreased binding affinity to nickel resins and elution in earlier fractions compared with wt protein ( figure s2 in supplementary material). taken together, the data demonstrate that rna binding prevented aggregation into irregular conformations and guided the self-assembly of the hybrid ferritin monomers into nps of a stable structure. the immunological properties of ferritin nps were analyzed by elisa. the hddp4 (human dpp4) receptor has been previously identified as the receptor for mers-cov human infection (46) . therefore, using hdpp4 as a coating antigen, elisa-binding assays between rbd nps and the receptor were performed (figure 8) . fr without rbd fusion failed to bind, and was similar to the pbs negative control. strikingly, the binding ability increased in the same order as the rna-binding ability (hrid(wt) > 2 m > 9 m), with highest absorbance observed in the wt with the ssg linker (hrid(wt)-rbd-[ssg]-fr). the results show that the conformation of rbd in the wt nps better resembled the protective antigen of mers-cov rbd from 293 cells, compared with the rna-binding mutants 2 and 9 m. again, judicious choice of linker between the ferritin carrier and the antigen was important for receptor binding and was reflected in its importance for np assembly into a stable conformation (figure 2) . finally, the elisa results for np against human patients was investigated using the sera from four mers-cov-infected patients (figure 9) . six different proteins, including five recombinant nps (hrid(wt)-rbd-fr, hrid(2 m)-rbd-fr, hrid(9 m)-rbd-fr, hrid(wt)-rbd-[ssg]-fr, and fr), and mers-cov rbd protein were compared by elisa using them as capture antigens. strong elisa signals were detected for the four recombinant nps and mers-cov rbd from 293 cells (positive control). the wt form consistently showed a higher response than the rnabinding mutants (hrid(wt) > 2 m > 9 m), with hrid(wt)-rbd-[ssg]-fr being the best binder among constructs tested. these results address to the utility of the e. coli assembled mers-cov rbd-fr nps as useful tools for sero-diagnosis of mers-cov infection. taken together, the results confirmed the immunologically relevant conformation of the mers-cov rbd displayed on the hybrid ferritin particles, and the crucial role of rna in controlling the kinetic pathway for the assembly of viral antigen monomers into stable nps. to evaluate the immunogenicity of ferritin-based nps, balb/c mice (n = 5) were immunized with rbd, rbd-fr, and rbd-[ssg]-fr nps antigens. the trnas were found to be removed from the hrid protein during the purification process. before immunization, potential rna contamination in the purified proteins was determined by gel electrophoresis. as shown in figure s11 in supplementary material, rna was below detection level, if any, after several purification steps, compared with the proteins purified in the first step. previously, mf59-adjuvated and alum-adjuvated mers-cov antigen have been reported to increase the antibody and t-cell responses in mice (44, 60) . thus, the first group and second group were immunized twice with 20.0 µg of antigen containing the equal volume of alum figure s2 in supplementary material and size exclusion chromatography was used to explore hdpp4 receptor-binding affinity to the protein. all data are shown as mean ± sd from triplicate samples. fr alone and phosphate-buffered saline were used as negative controls. figure s2 in supplementary material were used as coating antigens. fr alone and infected cell lysates were used as negative and positive controls, respectively. virus-infected sera from four patients were serially diluted from 1:100 (twofold dilution). all data are presented as mean ± sd of duplicate samples. higher than rbd, respectively. the antibody responses by rbd-fr and rbd-[ssg]-fr nps were much stronger than the rbd in all antibody subtypes tested (igg1, igg2a, and igg2b) (figures 10b-d) . as a test of mucosal immune responses, the rbd-specific iga antibody levels from balf were also analyzed by elisa (figure 10e) . mf59 adjuvanted rbd-[ssg]-fr nps presented significantly higher od values than rbd and fr (negative control). these results suggested that rbd-[ssg]-fr nps induces local mucosal immune response stronger than rbd. in addition, it was confirmed that antibody responses of igg, igg1 (th1), igg2a, and igg2b (th2) against mf59-adjuvated antigens were higher than those from alum-adjuvated antigens. in contrast, pbs and fr control groups failed to, or only weakly induce an antibody response against rbd protein. these results suggest that fr-based nps significantly enhance various antibody responses than monomeric antigens. the cellular immune responses were investigated in mice immunized with protein (rbd, rbd-fr, rbd-[ssg]-fr) and fr (negative control). splenocytes of mice (n = 4) were harvested 1 week after the last immunization, stimulated with | nanoparticles-immunized mouse serum inhibited interaction between middle east respiratory syndrome-coronavirus receptor-binding domain (rbd) and hdpp4 receptor. competition enzyme-linked immunosorbent assay showed that anti-rbd mouse sera (1:10, from mice immunized with rbd-[ssg]-fr, rbd-fr, and rbd) blocked binding between rbd (5 µg/ml) and hdpp4 receptor (5 µg/ml). fr-immunized mouse serum (1:10) was used as a negative control. all sera were serially diluted from 1:10 (twofold dilution). all data are presented as mean ± sd (n = 5) and p-values were obtained using student's two-tailed tests (***p < 0.001). figure 10 | immune responses in receptor-binding domain (rbd) nanoparticles (nps) immunized mice (n = 5). endpoint titer of igg (a), igg1 (b), igg2a (c), and igg2b (d) antibody binding to middle east respiratory syndrome-coronavirus rbd were detected using mice serum after two immunizations. rbd-specific antibodies were detected after immunizations of rbd nps, rbd, fr with adjuvant (alum and mf59) using enzyme-linked immunosorbent assay. (e). rbd-specific iga antibodies were detected using balf (diluted 1:8) after immunization of protein with mf59. od, optical density. each endpoint titer was shown by individual. all error bars were shown as mean ± sd (n = 5) and all p-values were obtained using student's two-tailed tests (**p < 0.01, ***p < 0.001). rbd protein, and analyzed for cytokines by flow cytometry. in the rbd-immunized group, ifn-γ and tnf-α-producing cd4 + t-cell responses were detected at low levels. however, ifn-γ and tnf-α-producing cd4 + t cells were significantly increased in rbd-fr and rbd-[ssg]-fr-immunized groups compared with rbd and fr-immunized group ( figure s12 in supplementary material) . these results demonstrated that the rbd nps vaccination induced antigen-specific cd4 + t cells that produced ifn-γ and tnf-α upon antigen stimulation. anti-nps serum effectively blocked rbd protein binding to the hdpp4 receptor middle east respiratory syndrome-coronavirus infection is mediated by the interaction of rbd and the host receptor hdpp4 (45, 46) . as a correlate of protection, a competition elisa was performed to investigate whether antibodies generated from nps immunization were able to interfere with the binding to hdpp4. thus, after incubation of rbd protein with mouse serum (1:10), the binding of serum-mixed samples to hdpp4 protein was measured. as shown in figure 11 , rbd-[ssg]-fr, rbd-fr, and rbd-immunized sera strongly abolished the binding of rbd to hdpp4 receptor (93.3, 82.2, and 75.67%, respectively). interestingly, the relative efficiency of interference correlates with that of np assemblage (figure 11 ). in contrast, the fr-immunized mouse serum (negative control) failed to inhibit the interaction. taking together, these results demonstrate that immunization of nps greatly stimulates mers-covspecific antibody response that effectively interferes with the cellular receptor binding, suggesting its possibility as a vaccine. however, protection efficacy should ultimately be tested in a live virus challenge model. having key immunologic features, like a highly repetitive nanostructure, provides a designing principle for nps in inducing potent and long-lasting antibody responses. for vlps of non-enveloped viruses, assembly is made purely by capsid proteins. for enveloped viruses, however, additional membrane components and matrix proteins are required to display the target antigens on the surface of assembled vlps. a promising alternative is to present target antigen on the surfaces of selfassembled nps, which, in lieu of lipid membranes and matrix proteins, serve as a macromolecular scaffold for the presentation of antigens of interest (61) . ferritins, as a substitute for matrix proteins and membranes, have been used as scaffold for the regular assembly of target antigens. however, ferritin-based nps have been produced only in host cells of mammalian or insect origin (28, 62) . previously, we showed that influenza ha could be assembled in a soluble, trimeric, and immunologically relevant conformation by exploiting chaperna activity (63) . the present study is the first report of using rnas as molecular chaperone for supra-molecular structures. here, we present a novel bacterial system for np assembly of hybrid ferritin displayed surface antigens from mers-cov. the nps reacted strongly with sera derived from mers-cov-infected patients (figure 9 ) confirming their utility in sero-diagnosis of infection. moreover, the antisera, generated from immunization of mice, were able to interfere with the binding to the cellular receptor hdpp4 (figure 8 ), in part of essential protective immune responses. the efficiency of receptor-binding inhibition (figure 11) , as well as the ability for inducing the mucosal responses (figure 10e) , correlated with the regular assembly of nps as examined by dls or em (figure 2) , confirming that presentation of antigenic epitopes on a multivalent and highly repetitive structure is indeed important for the quality of immune responses. overall, the quality of nps and consequent immune responses were governed by the rna-mediated assembly of antigens. we hypothesized that chaperna function could be harnessed for presenting target antigens as highly repetitive nanostructures ( figure 1d) . the hrid is the n-terminal domain of hlysrs and was previously identified as a nucleic acid-binding domain ( figure 5 ) (63) . in this report, the hrid was exploited as a transducer for chaperna function (tcf) by serving as a docking-tag for cellular rna for the folding/assembly of the hybrid fr containing client antigen proteins [rbd of mers-cov (figure 1d) ]. the advantage of using hrid as a tcf could be many fold. first, hrid is small (8.3 kda), monomeric, and was flexible enough to allow the access of site-specific protease for the removal of hrid ( figure s3 in supplementary material). of note, hrid belongs to idps, which switches into stabile α-helixes upon binding with trnas. second, the bound rna, due to its highly negative charge, may resist uncontrolled intermolecular interactions among monomers into amorphous aggregation. finally, even the naked hrid (in the absence of rna binding), due to its intrinsically flexible nature, may not pose physical hindrance to multiple interactions among monomers, enabling assembly into stable super-structures, upon removal of the hrid. thus, the potential "pace-making" function harnessed with the rna molecule, allows a regular assembly of monomers as highly repetitive nanostructures. consequently, in the current study, hybrid fr was produced in soluble forms, could be purified by one-step affinity chromatography, and most remarkably, assembled into nps of defined sizes upon removal of the hrid ( figure s2 in supplementary material). consistent with the principles of design, the loss of rna binding by hrid significantly hampered the regular assembly of the ferritin monomers and increased the amount of non-functional misfolded proteins as soluble aggregates (figure 7) . thus, the overall yield, as well as the quality of nps, were dependent on the chaperna function transduced by the hrid, which in turn was mediated by interaction with cellular rnas (likely to be trnas). the driving and controlling factors for de novo assembly of biomolecules are poorly understood. historically, host factors like groel/s were initially discovered as molecular chaperones for supporting viral growth in e. coli and supporting the assembly of viral capsid proteins (64, 65) . moreover, groel/s also cooperates with rbcx in plant cells for the assembly of multicomponent rubisco, which is the most abundant protein in the biosphere responsible for photosynthesis (66) . therefore, it is intriguing that rna could provide such a robust folding/ assembly of a supra-molecular structure. we recently confirmed that the present strategy could be successfully applied to the assembly of bacterially synthesized monomers of norovirus into vlps composed of 180 monomers (unpublished observation, seong, b.l.). whether rna can substitute for, or collaborate with pre-existing protein-based molecular chaperones remains an exciting avenue for future investigations. it should be noted that the defined versatile functions are being expanded for rna molecules. as an engineered system for harnessing chaperna function, the present report may prove to be the tip of an iceberg for pivotal function of rna molecules as chaperones for the folding and supra-molecular assembly of proteins in living organisms (36, 38) . various factors were identified as important for efficient assembly of mers-cov nps. as an extrinsic factor, the binding affinity of hrid to cellular rnas was crucial for the assembly and the quality of the assembled nps (figure 7) . as intrinsic factors, the concentration of salts and fe 2+ also influenced the assembly and stability of nps (figures 3 and 4) . the ionic strength played an important role in the stability and self-assembly of ferritins, and aggregation increased with increasing concentrations of nacl (54) . the assembly of the hybrid mers-cov nps revealed an interesting change in salt dependence, with 200-225 mm nacl buffer as optimal condition as confirmed by em and dls analyses (figure 3) . the change in salt dependence was probably due to the presence of electrostatic interactions among rbd domains (54, 67) . the dependence on fe 2+ was not surprising considering that ferritin has an intrinsic ability to interact with fe 2+ to form ferritin-iron cores (55) . based on our experience, to enhance the quality of nps, it is advisable to control fe 2+ concentrations, both during the culturing of the bacterial cells and during the purification of the soluble monomer proteins (figure 4) . first, the yield of the purified protein was increased in the presence of 500 µm fe 2+ (figure 4b) , up to 2.7-fold greater compared with the control conditions lacking fe 2+ . second, the ratio between nps and soluble aggregates in sec showed that nps formation was facilitated at high concentrations of fe 2+ , and resulted in a more compact morphology under em (figures 4b,c) . thus, both the overall yield and the quality of nps were governed by their intrinsic ability to interact with fe 2+ . finally, our data show that the presence and the nature of the linker between the ferritin and the rbd antigen was also important to the assembly of nps. it is possible that a linker with flexibility and sufficient length would accommodate the steric requirements for assembly of multimeric nps. however, it is difficult to precisely predict the effect of the linker, and therefore it is advisable to screen multiple constructs during the early stages of testing the assembly of nps displaying antigens of interest. in conclusion, the chaperna-based antigen assembly platform holds promise for the development and delivery of np-based vaccines to enhance rbd-specific antibody responses, and the serological detection of emerging viruses. various types of designing principles have advanced the structure-based approaches to np assembly (61, 68) . however, most of the in silico methods consider the thermodynamic stability of the final assembled nps, but not necessarily the kinetic pathways leading to their successful folding into regular assemblages. consequently, most nps are refractory to soluble expression and fail to assemble as designed, resulting in significant, and practical challenges in the manufacturing process. the chaperna-mediated folding and the "pace-keeping" assembly of monomers into higher ordered structures will enable faithful production of np and vlp-based vaccines against emerging and re-emerging viral infections. this study was carried out in accordance with the recommenda figure 7 | elucidation of rna-mediated nanoparticle (np) formation of receptor-binding domain (rbd)-fr. (a) size exclusion chromatography analysis of rbd-fr nps purified from the tev protease-cleaved hrid(wt, 2, or 9 m)-rbd-fr. the fractions (11-12 ml) estimated as nps were further analyzed by transmission electron microscopy (b) and dynamic light scattering (c) exploiting virus-like particles as innovative vaccines against emerging viral infections traditional and new influenza vaccines vaccine manufacturing: challenges and solutions management of accidental exposure to ebola virus in the biosafety level 4 laboratory vaccine delivery using nanoparticles vaccine delivery: a matter of size, geometry, kinetics and molecular patterns virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development the influence of antigen organization on b cell responsiveness structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding self-assembly of human papillomavirus type 1 capsids by expression of the l1 protein alone or by coexpression of the l1 and l2 capsid proteins how will hpv vaccines affect cervical cancer? virus-like particles as universal influenza vaccines comparative protein structure modeling using modeller structure-based design of peptides that self-assemble into regular polyhedral nanoparticles comparative protein structure modeling of genes and genomes construction and characterization of virus-like particles: a review self-assembling protein nanoparticles in the design of vaccines differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan human infection with mers coronavirus after exposure to infected camels, saudi arabia interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk an outbreak of middle east respiratory syndrome coronavirus infection in south korea mers outbreak in korea: hospital-to-hospital transmission the composition and the structure of bacterioferritin of escherichia coli self-assembly in the ferritin nano-cage protein superfamily presenting native-like trimeric hiv-1 antigens with self-assembling nanoparticles hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection ferritin nanocages: great potential as clinically translatable drug delivery vehicles? refolding of recombinant proteins advances in refolding of proteins produced in e. coli the role of molecular chaperones in protein folding molecular chaperones in the cytosol: from nascent chain to folded protein the hsp70 and hsp60 chaperone machines rna-mediated chaperone type for de novo protein folding m1 rna is important for the in-cell solubility of its cognate c5 protein: implications for rna-mediated protein folding rnas as chaperones the folding competence of hiv-1 tat mediated by interaction with tar rna protein solubility and folding enhancement by interaction with rna modeling of loops in protein structures cluspro: a fully automated algorithm for protein-protein docking the cluspro web server for protein-protein docking universal vaccine against respiratory syncytial virus a and b subtypes optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of rhodobacter capsulatus bacterioferritin and the native protein evaluation of comparative protein structure modeling by modeller-3 salt-dependent aggregation and assembly of e coli-expressed ferritin ferritin protein nanocages use ion channels, catalytic sites, and nucleation channels to manage iron/oxygen chemistry a peptide from the extension of lys-trna synthetase binds to transfer rna and dna sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression intrinsically disordered proteins and intrinsically disordered protein regions intrinsically unstructured proteins and their functions identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus protein/peptide-templated biomimetic synthesis of inorganic nanoparticles for biomedical applications self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies harnessing an rna-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation host participation in bacteriophage lambda head assembly properties of a mutant of escherichia coli defective in bacteriophage lambda head formation (groe). i. initial characterization coupled chaperone action in folding and assembly of hexadecameric rubisco modeling the influence of salt on the hydrophobic effect and protein fold stability rational design of an epstein-barr virus vaccine targeting the receptor-binding site key: cord-009594-0rfbmi0q authors: nan title: news date: 2014-11-26 journal: aust vet j doi: 10.1111/avj.139 sha: doc_id: 9594 cord_uid: 0rfbmi0q nan please email emma.malcolm@ava.com.au a small version of your photo and confirm that you also have a high resolution to send us. final images have to be in crystal-clear focus, at a minimum of 300 dpi and at least 150mm at the longest edge. for us to publish an image on the cover it needs to be at least 210x275mm at 300dpi. we had some lovely photographs that we couldn't publish this year because they were taken on a camera phone and the resolution was too low. as good as phone cameras can be, many still aren't going to be able to take a photograph that is high resolution enough to print at a reasonable size. so get your 'old fashioned' digital camera out! if you would like to try to take a cover shot, keep in mind that we have to be able to add the avj masthead at the top and highlights down one or both sides. it's also important to send us an accurate description of the photo, otherwise we might get eagle-eyed members calling to tell us we've got our cows confused -thank you robert mills. where to on welfare? julia nicholls, president f ootage of australian animals being mistreated during slaughter in kuwait, jordan and gaza prompted our members to respond in various ways to the confronting images and to ask what the ava is doing in response. it was hard not to be moved and horrified by the footage, and members have questioned the currency of our policies and position statements on live animal export and humane slaughter. the details of what ava can do are relevant to the debate; we can only comment and question. these breaches of the exporter supply chain assurance system are being investigated, export of animals to gaza is suspended and the department of agriculture keeps us informed of developments in this regard. enormous efforts have been made to improve the welfare of exported animals and it is remarkable that so much has changed for the better in destination countries given that australia has no jurisdictional control. despite this, i appreciate that there are many of us who would like to see an end to live export and others whose livelihood depends on the trade. our membership reflects the diversity of opinion of australian society and this makes it difficult for the ava to take sides in some circumstances. our membership is diverse, but our opinions are reinforced by a deeper knowledge of animal science and physiology. the ava can and does comment on issues of the day relevant to our expertise, and our profile and influence are steadily increasing. we have invested significantly in our public affairs program, which includes setting goals and tracking our achievements on behalf of members. however, i think many members overestimate the ava's ability to influence public opinion and bring about change. just because we are best qualified to comment doesn't mean that we enjoy the most attentive listeners to those comments! part of the art of advocacy is to understand what you can change, as well as how you can change it. there are many animal welfare issues of concern to both the community and members. what are the most important ones for our members and the long-term benefits for animals? how do we reflect your views and represent you to stakeholders and politicians? is there a common thread or philosophy on animal welfare across all members? can we better articulate a more robust and satisfying basis for the ava's policy and advocacy work on animal welfare issues? these are the questions we are hoping to explore and develop further within our public affairs program over the next year. stay tuned to ava communications for opportunities to be part of the conversation. other veterinary associations are doing a range of activities in relation to animal welfare issues. late last year, the american veterinary medical association held a forum called 'the conversation' , 1 which involved veterinarians, ethicists and animal scientists who presented on the scientific, social, political, market, and legal aspects of how and why animal welfare decisions are made. the papers and discussion are being collated into a resource. the nzva has an animal welfare strategy with a stand-alone vision: "our members, using a science-based and ethically principled approach to the humane treatment of animals in new zealand, are respected and recognised for their leadership and educative role in animal welfare and well-being" and mission: "to enable and promote our membership as having the knowledge, skills and leadership in the field of animal welfare and wellbeing". 2 the strategy also emphasises the need to be proactive in relation to animal welfare. the canadian veterinary medical association (cvma) has an animal welfare committee whose purpose is to advocate and promote animal welfare within the animal industry, to government and the public, and to advise the cvma on animal welfare issues and develop pertinent position statements. animal welfare advocacy is a priority of the cvma. should ava hold a conversation-type event? should we have a stand-alone strategy? should we have a welfare committee? perhaps all of these? i'd be interested to hear your thoughts in the discussion forums or as a comment on this article on the website. finally, i wish each and every one of you a very enjoyable festive season, filled with catching up with friends and family and not too many calls on your work time. this is a time of year when we take stock and reflect on the past while making resolutions for the next year. the ava is no different and we are looking ahead to planning our new 3-year strategy for 2016-2018 in the first months of next year. this will involve input from as many of you as possible, so start thinking about what you expect from your association. i wish you all the best for 2015 in your work and personal lives, may you find that elusive balance and continue to enjoy your chosen profession. financial products and services described in this document are provided by boq specialist bank limited abn 55 071 292 594 (boq specialist). boq specialist is a wholly owned subsidiary of bank of queensland limited abn 32 009 656 740 (boq). boq and boq specialist are both authorised deposit taking institutions in their own right. neither boq nor boq specialist guarantees or otherwise supports the obligations or performance of each other or of each other's products. the issuer of these products is boq specialist afsl and australian credit licence 234975. all finance is subject to our credit assessment criteria. terms and conditions, fees and charges and eligibility criteria apply. equipment and fit-out finance / home loans / commercial property finance / car finance / practice purchase loans / smsf lending and deposits / transactional banking and overdrafts / savings and deposits / foreign exchange stress, insomnia, anxiety. we're expert in handling these conditions. veterinary professionals know just how important it is to put an owner's mind at ease. it takes expertise to understand the problem and the ability to clearly explain the options to patient and owner. at boq specialist, we operate in much the same way. over the last 20 years, we've developed a profound understanding of how the veterinary profession works. you are our area of expertise and we've developed an extensive range of products to meet your banking and finance needs. so, if there's anything on your mind talk to us, and then enjoy a good night's sleep. visit us at boqspecialist.com.au/ava or speak to our financial specialists on 1300 131 141. boqs000004 e ducation is the cornerstone of every profession. the ava has close ties to australia's veterinary schools and we provide input where appropriate to help universities produce the veterinarians of the future. as you know if you read these columns, one of the ava's key strategic priorities is workforce planning. we want to develop and advocate for good evidence-based policies that will provide the right number of veterinarians, with the right skills, in the right places, to meet australia's need for veterinary services into the future. when the commonwealth government announced significant changes to higher education in the may budget, we were concerned that this could affect the veterinary workforce. after more analysis, our modelling showed that the proposed changes had dire implications for the debt accrued by a veterinary student and its repayment. our detailed submission to the senate inquiry into the legislation was followed by an invitation to speak at a public hearing. national strategy manager, dr debbie neutze, spoke passionately and persuasively about the potential effects on the profession and answered a range of questions about the profession. the submission we made to the senate and the communications we've been sending to the media have emphasised that veterinarians provide services that no-one else can provide. these include essential services to biosecurity, public health and food safety. the higher education changes in the proposed bill, we believe, could create unintended and inequitable outcomes for the veterinary profession that threaten those vital services. the changes will affect veterinary students more than other student groups because of the length of study and the high cost of delivering the course combined with the significantly lower average lifetime earnings than graduates of comparable courses. we are already witnessing market distortions that will potentially affect the viability of the veterinary profession and its ability to provide essential services. our view is that that the proposed changes to higher education are almost certain to distort the market further. at the time of writing, the senate had not voted on the bill. we are expecting the vote to happen at the beginning of december -close to the time you'll be reading this article. however, we anticipate at least one favourable outcome -the interest rate increases are unlikely to be passed. because of the attention ava focussed on this aspect, particularly for female graduates, we can claim some of the credit for this amendment. over the past few weeks, ava president julia nicholls and i have been meeting with each veterinary school dean or head to discuss our position on the proposed changes. whatever the outcome of the proposed higher education changes, it is critical that we maintain open and honest dialogue with the universities and educators. our meetings have been very positive and i'd like to thank each of the deans for their time and candour. early next year, we are looking forward to reporting on the workforce modelling we've been doing throughout this year. the stock and flow model of the veterinary workforce currently in preparation will analyse both supply and demand factors for veterinary services. the ultimate aim is to create policies that are relevant to the current political and policy environment to ensure a healthy future for the profession. we want to ensure appropriate levels of supply and demand for both current and future veterinarians, and also ensure student debt doesn't become something that veterinarians have to service for decades in their careers. the number and skills of new entrants to the workforce will be part of this analysis, so we anticipate further cooperation and conversations with the veterinary school deans as we respond to the workforce modelling findings. the only person who is educated is the one who has learned how to learn and change. international veterinary, biomedical and business journals at your fingertips ebola, dogs and a vaccine i n october, a nurse in spain tested positive for ebola virus after caring for an infected patient in hospital. people who were in contact with this nurse were quarantined, and the madrid regional government obtained a court order to euthanase her pet dog against her wishes, on the grounds that available scientific information could not rule out a risk of contagion. wsava is strongly of the view that available technology should allow for testing and quarantine, rather than automatic euthanasia of exposed animals. 1 professor michael day, chairman of the wsava's one health committee, noted: "zoonotic diseases, particularly those transmitted through pets, are concerning to the pet-owning public, but there have been no scientific reports indicating that ebola virus has been isolated from or directly transmitted by dogs." the virus that causes ebola is not airborne and can only be spread through direct contact with the bodily fluids of an infected person who is showing symptoms or who has died. dogs appear to be the first animal species shown to be naturally and asymptomatically infected by ebola virus. during the 2001-2002 ebola outbreak in gabon, one group sampled 439 dogs, following the observation that several dogs were highly exposed to ebola virus by eating infected dead animals. 2 dogs were screened using ebola virus-specific immunoglobulin (ig) g assay, antigen detection, and viral pcr. the serological report showed a significant positive association between seroprevalence and the distances to the ebola virusepidemic area, with seroprevalence up to 31.8% in dogs from villages with both infected animal carcasses and human cases. the authors did not detect any circulating ebola antigens or viral dna sequences (tested by pcr), and were unable to isolate any virus. they suggest that this indicated either old, transient ebola infection of the tested dogs, or antigenic stimulation. none of these highly exposed dogs during the outbreak showed any clinical signs. it is possible that dogs may excrete infectious viral particles in urine, faeces, and saliva for a short period before virus clearance, as observed experimentally in other animals. further work on experimental canine infection is needed to establish the potential human risk of ebola virus-infected dogs, including the mechanisms of viral excretion. the main concern in west africa is infection via fruit bats and non-human primates, and by eating bush meat that may include fruit bats and primates infected with ebola. a house pet that may potentially be exposed in developed countries represents a very different scenario to those in epidemic west africa. early human testing of an investigational ebola vaccine co-developed by the national institute of allergy and infectious diseases (niaid) and glaxosmithkline (gsk) began in early september. 3 initial data on safety and immunogenicity (the capacity to generate an immune response) from clinical trials of the niaid/gsk ebola vaccine are expected by the end of 2014. human testing of a second ebola vaccine candidate is under way at the national institutes of health's clinical center in bethesda, maryland. one group is conducting a trial to evaluate the vaccine, called vsv-zebov, for safety and its ability to generate an immune system response in healthy adults who are given two intramuscular doses. this nih phase 1 placebo-controlled clinical trial of the vsv-zebov vaccine candidate will enrol 39 healthy adults. enrolment at each dosing level is staggered, so interim safety assessments of vaccinated individuals can be conducted. vsv-zebov is based in part on a genetically engineered version of vesicular stomatitis virus (vsv), which primarily affects rodents, cattle, swine and horses. the gene for the outer protein of the vesicular stomatitis virus has been replaced with a segment of the gene for the outer protein of the zaire ebola virus species, and the vaccine cannot cause a vaccinated individual to become infected with ebola. a second group at the walter reed army institute of research is simultaneously testing the vaccine candidate as a single dose, to evaluate in real time the safety profile at different dosages and compare the immune responses induced by one injection versus two injections. initial safety and immune response data on the vsv-zebov vaccine are expected by the end of 2014. this vaccine candidate was developed by researchers at the public health agency of canada's national microbiology laboratory, and has been developed from preclinical to clinical testing stage remarkably quickly. this story can be compared to the extraordinary work of dr deborah middleton's group, which developed the hendra virus vaccine so quickly in australia. medivet animal health, a world leader in veterinary regenerative medicine, has a comprehensive product offering for animals including stem cell therapy, platelet rich plasma and nanofiber technologies, amongst others. â�¢ improves the quality of life of your patients t he ava is seeking nominations for its board. can you join the leadership crew and help to steer the profession? here are some tips and information to help you decide if joining the ava board might be the right thing for you. the ava has nine directors. six are elected directly by the general membership and three are nominated and appointed by the three largest special interest groups (cattle, equine and asava). each director has a 3-year term, with a maximum of two successive terms. the president, vice president and treasurer are elected each year by the board. nominations are invited for two elected positions on the board of directors for a 3-year term to take office in may 2015. these elected positions in 2015 are in addition to a position to be nominated by asava. the first requirement is that candidates have prior experience as an office holder within a special interest group or division committee. "to be a good board member, you need to be a good listener and then you need to take the information given to you and be a good advocate for the profession, " dr gilkerson said. some additional skills and experience that are useful include being a member of community organisations, being a member of other boards and committees, a commitment to animal health and welfare, and the ability to prepare reports for the ava board. with a lot of the work involving the ava's groups and committees, communication and team work are key skills to bring to the ava board. "it helps to be patient, diplomatic and to communicate clearly. directors need to have a broad knowledge of the different groups within the ava and how they work together, " board member dr robert johnson said. "if you are interested in helping our profession, joining the board is a great way to do that. you soon learn what matters to your fellow members, and it is a privilege to be a voice for them. " the board has a charter and a code of conduct that outline the expectations and operational details of the board. all election candidates need to agree to abide by the charter, the code and the ava constitution before their nominations are accepted. directors have legal responsibility for the australian veterinary association limited (acn 008 522 852) under the corporations act 2001 and they undertake mandatory training to help them understand and fulfil their legal responsibilities. these include ensuring that the ava complies with all aspects of the law, risks are managed appropriately and all parts of the organisation are doing what they should be doing for the benefit of members. the board sets the direction for the ava, makes the big strategic decisions and ensures effective risk management. the board appoints the chief executive officer and is responsible for managing his or her performance. the ceo is responsible for managing the ava's employees and ensuring the board's strategy is realised. if you haven't already, you can put some faces to names by viewing a video of the ava board meeting earlier this year: http://ow.ly/e2rmf. t he object of the ava's animal welfare trust (awt) is to provide small grants for research, education or promotion and action programs that lead to improvements in the welfare and wellbeing of animals. in october 2014, awt's grants committee critically evaluated and ranked eight worthy research proposals involving a range of species. awt trustees accepted the committee's recommendations and offered two grants for the following research projects. yu zhang, phd candidate, university of queensland this project focuses on how ammonia accumulation affects sheep's feed intake during simulated sea export by studying their physiology, behaviour and emotional state. as the largest live export industry in the world, livestock shipments from australia mostly involve sheep, especially to the middle east. during a voyage, ammonia is released from their excreta, which accumulates as a pad that the sheep have to lie on. ammonia especially accumulates when there is insufficient ventilation, high temperatures and humidity. this is a recognised welfare problem and causes sheep to stand more, feed and ruminate less, hold their heads high for fresher air and suffer conjunctivitis. the university of queensland recently conducted research on gaseous ammonia accumulation on livestock ships that has been used by rspca to seek the establishment of legal limits. however, it is unknown how sheep's feed intake is affected by ammonia on ships. using a simulation, the research objectives are to: â�¢ understand how high concentrations of gaseous ammonia affect feed intake â�¢ study the sheep's emotional state after exposure to gaseous ammonia. project results will increase our understanding of the welfare implications of long-distance sheep export. in the longer term, they will contribute to modification of sea transport standards to improve sheep welfare and reduce lower returns caused by weight loss. outcomes may also help improve welfare issues associated with ammonia accumulation in intensive farming. effect of shearing in pre-embarkation feedlots on sheep feeding behaviour before export awt will fund an honours project under associate professor anne barnes at murdoch university. inappetence late in feedlotting has been shown to increase the risk of shipboard death and consistent feed intake is the key to preventing shipboard deaths from inanition and salmonellosis. therefore, any interference with consistent intake might have poor health and welfare outcomes. sheep exported live from australia are required to spend time in registered premises before shipping. during this time they are fed pelleted feed similar to that provided on the ship so that they can adapt to it, which may take several days. some sheep may never adapt to the different feed, leading to inappetence, which is associated with salmonellosis and eventual starvation. disruption and moving may delay feed adaptation, increasing the risk of inappetence and disease at the feedlot and/or on board. sheep are often shorn during their pre-embarkation preparation to limit wool cover and enhance heat loss. there are no specific standards for the timing of shearing in sheds in relation to the time of embarkation. previous studies report that short periods of restraint, isolation and shearing are acute stressors for sheep. superimposing these on animals undergoing adaptation to a novel environment and feed could affect their continued intake of feed. the research objective is to determine whether moving and handling sheep for shearing at the pre-embarkation feedlot interferes with their feeding pattern. video footage of the sheep's initial entry to the shed, after shearing and before exit will be analysed using qualitative behavioural assessment (qba). using this, observers score descriptors of how animals behave to create an integrated measure of behaviour for comparison of treatments. qba scores are correlated to physiology and ethology and are a non-invasive means of assessing livestock welfare. project results will provide guidance for the industry on whether and when shearing should take place during pre-embarkation. it will inform best practice in the intensive sheep industry regarding the effects of shearing on feeding behaviour during feedlotting. outcomes of both projects will be disseminated via refereed journals, conference presentations and advice to industry. 1 of the 20 european countries that provided data over this timespan, 18 observed decreases ranging from 0.4% to 49%. "these latest figures, which suggest a positive trend in terms of the responsible use of antibiotics in animals in europe, are highly welcome, " explains david mackay, the head of the ema's division on veterinary medicines. "however, the report also shows that there is scope for further decrease. "measures to promote the rational use of antibiotics in animals need to continue as part of the european commission's action plan against antimicrobial resistance, " he said. public health authorities worldwide are confronted with increasing levels of resistance to antibiotics in humans and animals and are engaged in actions at various levels to fight this issue. the responsible use of antibiotics is a key factor to minimise the risk of resistance. the ava has been working closely with the australian department of agriculture and human health groups to join this global campaign to promote responsible use of antibiotics. â�¢ national programs and campaigns on the responsible use of antimicrobials â�¢ restrictions on the use of certain antimicrobials â�¢ increased awareness of the threat of antimicrobial resistance â�¢ reduction targets for the use of antimicrobials in animal production in certain member states â�¢ fluctuations in size and types of animal populations. while additional analysis is needed to confirm the main reasons for this decline, the reduction in the use of antibiotics is a positive sign. l ife for women was very different when judith went to school. she had dreamed of being a veterinarian while spending her school holidays on her uncle's property in guyra. attending a private girls' school in the 1940s and early 1950s put her at a disadvantage for pursuing a career as a veterinarian, as physics and chemistry were not deemed important areas of study for young girls. luckily, she was able to do biology and latin, which were approved prerequisites for veterinary science at the time. "my school leaving certificate marks were good enough to receive a commonwealth scholarship to study at the university of sydney, " she said. "i accepted it and indicated that i planned to study veterinary science. "i was called for an interview at which it was strongly recommended that i use the scholarship to do an arts degree, particularly as i was so disadvantaged, not having studied physics or chemistry. "i stood firm -i had my scholarship and i had my opportunity, " she said. in her first year at vet school, she was the only woman and for whatever reason, her male counterparts decided to elect her as the year representative. this role gave her valuable contact with staff through vet school meetings and allowed her greater access to the vet school area of the university than other first years who had very limited access because they were 'lumped in' with medicine and science students. "i loved the vet school building; the people, the work, the smells and the lifestyle. "elbert was a good teacher, and would be a boss i enjoyed and respected but to my horror he went on holiday a few days after i started workâ�¦ "how to make a nervous wreck of a new graduate!" "in those early days i remember monitoring a sick heifer in my backyard, which died and whose autopsy revealed one of the early cases of sbe. calvings were chain and rope pull jobs, distemper was rife and haemonchus in lambs was devastating, " she said. after marrying rod, the couple moved to armidale. he had become a jackaroo after deciding not to complete veterinary science. the local veterinarian, dr joe o'brien, had heard that she was coming. "very soon after arrival, joe arrived on my doorstep, announced who he was, checked who i was and asked why the [expletive] was i sitting up reading a book when there was vet work to be done?" "i learnt much from joe, who calls a spade a spade and was an innovative, real-thinking vet and excellent surgeon. we had an excellent ava branch and meeting up with colleagues was a vital way for us all to learn and share information, " she said. however, the erratic hours and unpredictable life of a veterinarian in general practice and marriage were not sitting well and she reluctantly applied for a lecturing position in rural science at the university of new england. everything looked great until the fur started to grow. a siamese with a fluffy hindleg with the fur growing the wrong way made a good talking point," australian veterinary journal volume 92, no 12, december 2014 news n15 "i was told that as a female i was not a likely long-term proposition for the lecturing position in parasitology. prejudice withstanding, i became demonstrator in anatomy, histology and parasitology and enrolled to do my phd, working on ovine brucellosis. "i enjoyed the teaching, but research and the experimental work were not really my thing. i yearned for general practice over those years, and by the time i submitted my thesis, i had four children. "further experimental work was requested from a biochemist examiner of my thesis. so, i decided to call it quits. this was a huge decision as i had invested so much time and my 'no more research' stand represented failure. but i took great delight in being a mother and knew that my priorities would always be with family, " she said. the tb and brucellosis eradication scheme gave dr grieve her next veterinary venture. "i was often working with four children and a surrogate grandmother as baby sitter travelling to remote properties and 'interesting' yards, like one that was constructed of iron bedheads. "over this time i made good friends with many of the land holders and found myself doing more small jobs and bringing the odd cat or dog back with me to return on the next visit having done whatever treatment it required, " she said. dr grieve's next career move was to buy an old building, which she moved to her property and set up a small surgery. "with my children at the local school, i met more of the locals and built up my small practice, and then had another baby. "mixed practice hours didn't work with school hours and i started having to exploit friends and neighbours for school pick-ups and drop-offs. to prioritise my children, it became obvious that i should be keeping close to home (and the school) by doing small animal and 'anything that could be brought to the surgery' work, with limited local large animal cases, " she said. w ith 21 ava special interest groups (sigs), there's one for everyone. you bring your own expertise, skills and experience to join others with similar interests to exchange ideas, inspire and support each other. here's a rundown of the different sig opportunities available to ava members. our largest sigs are for veterinarians working with cattle (acv), horses (eva) and small animals (asava) and in practice management (avapm). "you can really benefit from connecting with your like-minded veterinarians through the ava's sigs, " ava president julia nicholls said. "the big sigs offer outstanding value if you're involved in these areas. they keep you on the cutting-edge of your field by providing you with a range of member benefits. from clinical journals, to tailored continuing professional development and a novel strain of coronavirus that causes a rapid onset of severe respiratory disease in humans was first identified in saudi arabia in 2012. as of 3 november 2014, the world health organization (who) has been notified of 897 laboratoryconfirmed human cases of mers-cov, including at least 325 related deaths. 1 the virus appears to be circulating widely throughout the arabian peninsula, and travel-related cases have been reported in europe, africa, asia and the united states. 2 there have been no cases reported in australia. mers-cov is genetically and biologically distinct from other known coronaviruses such as the virus causing severe acute respiratory syndrome (sars) in humans. it is considered to be a serious public health threat, because the infection can cause severe disease in humans and coronaviruses may adapt to new hosts and become more easily transmittable between humans. 3 the epidemiology of mers-cov infection is not yet fully understood. similar strains of mers-cov have been identified in samples taken from camels and humans in the middle east and in some cases there has been an association between infections in humans and camels, suggesting that camels are a likely primary source of the mers-cov infection in humans. 4 viruses genetically related to mers-cov have also been detected in bat species around the world, and a fragment of viral genetic material matching the mers-cov was found in one bat from saudi arabia. however according to the world organisation for animal health (oie), the current evidence does not indicate a direct link between bats and mers-cov in humans and further research is warranted. 3 other species of animals, including sheep, goats, cattle, water buffalo and wild birds, have tested negative for the presence of antibodies to mers-cov. nevertheless, owing to the relatively small sample sizes, the results of these studies cannot exclude infection in other animal species. 3 the oie, who and fao emphasise that joint human health and animal health investigations are needed to develop a better understanding of the overall epidemiology of mers and the role of camels and potentially other animal species in the maintenance and transmission of mers-cov. 3 clusters of confirmed human cases have occurred in healthcare settings and households, but there is no evidence of sustained human to human transmission in the community. immunocompromised persons and those with diabetes, cancer and chronic lung disease are considered at high risk of severe disease from mers-cov infection. the who recommends that as a general precaution anyone visiting farms or other places where camels are present should practise general hygiene measures, including regular hand washing before and after touching animals, avoiding contact with sick animals and following food hygiene practices. 4 for latest information on mers-cov, visit the who website: www.who.int/ resolutions are generally of two types -special resolutions and ordinary resolutions. any resolution to amend the ava constitution requires a special resolution. in order to be passed special resolutions require the approval of at least 75% of the members entitled to vote on that resolution. ordinary resolutions are for matters relating to the accounts or reports of officers of the ava. in order to be passed an ordinary resolution requires the approval of a simple majority of more than 50% of members entitled to vote on the resolution. the wording of a proposed resolution may require amendment after submission (in consultation with the proposers of the resolution). this is to ensure the resolution is in an acceptable form and provides sufficient clarity, particularly if the resolution involves an amendment to the ava constitution. before submitting a resolution, please contact the company secretary who may assist with preparation of an acceptable form of wording of the resolution. it is also appropriate to discuss any proposed resolution with a wide range of members prior to formally lodging it with the company secretary to ensure that the proposed wording of the resolution clearly conveys your objectives. all proposed resolutions will be considered by the ava board, and if they are of the view they are in the best interests of the ava, put to the annual general meeting. for more information contact john robb, company secretary at ava national office on 02 9431 5040 or corporate@ava.com.au. chief executive officer comment on this article at www.ava.com.au/13325 member communications, information resources and guidelines, client-centred materials and accredited programs, your sig keeps you at the forefront of your field. "the collegiality that comes with joining your sig is invaluable. it provides you with support, and enables you to support your colleagues, inspires new ways of thinking and doing things, and may even provide some much needed light relief from time to time, " she said. not involved with cows, horses, small animals or practice management? then we have 17 boutique sigs to cater for nearly everyone. these sigs have fewer members but no less collegiality or passion for their work. most communicate with members via email or through the ava's discussion forums. like the larger sigs, these smaller ones bring together leading veterinarians in a particular field to exchange knowledge and support each other. "the ava's sigs are representative of the diversity within our profession, " dr nicholls said. "whether these smaller sigs represent your daily working activity or they cover something that you're interested in, it's well worth the nominal investment to connect with your colleagues in these groups, " she said. learn the ins and outs of all the unusual and exotic pets or explore some new territory through the acupuncture or integrative medicine group. the public health vets share important information about government issues, while the animal welfare and ethics group explores the philosophical and practical aspects of welfare. the behaviour and dental groups are growing quickly as practitioners understand the value of broadening their skills and knowledge into new areas to meet the demands of modern practice. pig, sheep, reproductive, alpaca, greyhound and poultry vets are active in creating networks and sharing the latest information within their defined fields of practice. conservation biologists come together to explore the latest in wildlife health, disease and management and species conservation. the education and research sig represents veterinarians working in educational institutions and research bodies, while the history group produces the australian veterinary history record, which chronicles the rich and diverse history of veterinary medicine in australia. you can view the array of sigs on offer at www.ava.com.au/about-us/who-does-what/groups. w ith the christmas holiday season fast approaching, it's a good time to stop and think about the forthcoming social events that will be organised within workplaces for staff, clients and visitors. such celebrations provide a great opportunity to bond with colleagues and clients, as you can get to know them more in an informal setting. it's not an opportunity to lower your conduct standards such that you end up embarrassed to turn up to work on monday. together with employees, employers have a responsibility to take all reasonable steps to ensure the safety and health of all who attend work-related functions. this includes a duty of care under work (occupational) health and safety, a range of antidiscrimination legislation, including provision for dealing with sexual and racial harassment, and the criminal code, which includes assault of either of a physical or verbal nature. all of which promote an environment free of discrimination, harassment, bullying and violence, whether or not the function is held offsite or onsite. we have all heard those embarrassing stories of staff doing things they should not have done at christmas parties, only to find photos that you did not want people to see on social media and the professional and social fallout that comes from such posts. this should be enough to make you stop and think about the photo you would prefer to see of yourself on social media. although there are great advantages and value to holding workrelated functions, there are also risks, which, if not managed appropriately, can quickly turn a joyous and enriching time into a nightmare. as many social functions are provided as an extension of the workplace, it is important that employees are aware of the inappropriate behaviour that can surface, especially when functions are highly charged with alcohol. if you're planning to attend an upcoming work function, be aware that you have a responsibility to take reasonable precautions for your own health and safety and that of others. you are expected to conduct yourself in a respectable manner that leaves the workplace function free from harassment and other offensive behaviour. avoid becoming intoxicated to the point where your behaviour causes risks to health and safety. such behaviour includes unwanted touching and kissing, derogatory comments and humour, violence, inappropriate gifts and send-ups, oversharing and illegal drug taking. strategies to minimise the risk of staff social functions getting out of control may include the following: â�¢ before the function, remind all staff of company policies and standards of behaviour in relation to drugs, smoking and alcohol, dress code, bullying and harassment, particularly sexual harassment â�¢ discourage excessive drinking and remind all staff of the dangers of drink-driving â�¢ take reasonable steps to ensure the consumption of alcohol is limited and make sure non-alcoholic drinks are also available â�¢ ensure staff make arrangements to get home safely â�¢ provide plenty of food, especially when serving alcohol â�¢ have set start and finish times for the function â�¢ have a grievance handling procedure in place to deal with any complaints, should they arise â�¢ remind management and senior staff that they are role models for expected appropriate behaviour â�¢ raise staff awareness of the consequences of breaching of company policy, such as warnings and possible dismissal. the best functions are those that are well thought out and fundamentally underpinned by clear, concise and reasonable policies, procedures and expectations in relation to the acceptable codes of conduct of all who attend. such planning will go a long way to making sure the work function is successful and enjoyed by all. comment on this article at www.ava.com.au/13326 the material contained in this article is general comment and is not intended as advice on any particular matter. no reader should act or fail to act on the basis of any material contained herein. the material contained in this publication should not be relied on as a substitute for legal or professional advice on any particular matter. the veterinary nurse council of australia and hill's pet nutrition presented the award, which recognises qualified veterinary nurses employed within a veterinary clinic, who provide exceptional service and deliver the highest possible standard of patient care. editor in chief t ri-solfen (t-s) was developed in response to animal welfare concerns about the animal husbandry practice of mulesing of lambs intended for wool production. many woolproducing sheep are excessively wrinkly in the breech area, which predisposes them to blowfly strike. removal of this wrinkly skin in the breech area by a process known as 'mulesing' produces a plainer skin profile, which in turn mitigates the occurrence of flystrike. the active ingredients of t-s are the local anaesthetics lignocaine (lig) and bupivacaine (bup), together with adrenaline as a vasoconstrictor and the antiseptic cetrimide. these are formulated together in t-s and the product is used as a post-mulesing dressing in lambs. the recommended age for mulesing lambs is 2-12 weeks, with the maximum age allowed under the present commonwealth government model code of practice for welfare of animals being 12 months. 1 mulesing is only carried out on young sheep that are destined for wool production. in reality, any concerns about residues from the four actives contained in t-s would be essentially zero, as any traces would have disappeared long before the sheep entered the human food chain. the interval between application and slaughter in almost all cases is likely to be many years, with some sheep dying or being used for pet food. to further reduce human health concerns, all four active ingredients were chosen by the developers because they already had widespread and safe use in human and veterinary medicine. three of the active components are used topically in humans, and lig and bup are also administered parenterally. the hazard is known and is essentially zero. the australian medicines handbook (amh) recommends that the maximum single injected dose of lig in humans is 3 mg/kg body weight (bw) when used alone or 7 mg/kg bw when administered with adrenaline. 2 the injected dose for bup with or without adrenaline is recommended at 2 mg/kg bw. the amh also lists a number of human preparations of lig used in the nasopharyngeal area or gastrointestinal tract, which results in oral intake of lig. the amh does not recognise therapeutic doses of lig or bup in humans as a particular hazard. 2 lig and bup do not persist in mammals because they are rapidly metabolised, particularly in the liver. the plasma halflives of lig and bup in all species are very short (range 1-190 minutes), depending on the species, age, dose and route of administration. the metabolic pathways are common to humans and other mammalian species, including sheep, cattle and pigs. excretion of parent compounds and their metabolites is rapid and predominantly in the urine. 3, 4 metabolism results in degradation of the compounds, which is responsible for the loss of local anaesthetic action. the australian office of chemical safety has reviewed the toxicology and occupational safety of t-s 5 and established an acceptable daily intake (adi) for its active ingredients: 0.009 mg/ kg bw/day for lig, 0.001 mg/kg bw/day for bup and 0.01 mg/kg bw/day for cetrimide. an adi for adrenaline was not set because residues are not expected to be measurable above endogenous levels normally found in the tissues. the biotransformation of lig and bup produces 2,6-xylidine as a minor metabolite that has been shown to be a weak mutagenic agent in vitro and to have genotoxic characteristics in vivo. 4 this metabolite produced nasal tumours in a 2-year oral toxicity study in rats receiving daily doses of the metabolite equivalent to 150 mg/kg bw/day. the incidence of these tumours was dosedependent and there was no significant increase in nasal tumours at either of the two lower dose rates of 15 and 50 mg/kg bw/ day. 4 in this rat study it was reported that some of the 2,6-xylidine evaporated from the feed and was inhaled by the rats throughout the study period. the nasopharangeal anatomy of rats is unlike that of humans, which leads to differences in the amount and distribution of inhaled materials. rodents are also obligate nose breathers with a highly efficient nasal filtering capacity. a further complication in rats is spontaneous respiratory infections and their sequelae. rats appear to be uniquely susceptible to chronic inflammation and tumours from insoluble cytotoxic particles. 6, 7 overall, this makes the rat an unsuitable model for predicting the significance of chemicals on the induction of nasal tumours in humans. the unique characteristics of the rat and the volatility of the 2,6-xylidine make it difficult to place any significance on the appearance of nasal tumours in the chronic rat study. the results of the 2-year rat study using 2,6-xylidine have been extensively reviewed and it is accepted that this metabolite of lig and bup does not represent a hazard to human health. 4, 8 the end result is that when meat-producing animals are treated with t-s, the possibility of 2,6-xylidine residues being present and causing problems in humans is not an issue. furthermore, human pharmaco-vigilance studies over a long period have not revealed any reports of carcinogenicity following the widespread therapeutic use of lig or bup. the lower limits for finding or detecting (lod) lig and bup and their metabolites in tissues and bodily fluids using contemporary mass spectrometric based methods are in the range of 1-10 ng/ml or gram. 3 the regulatory methods actually used to measure the amount of residues in meat and offal have limits of quantification (loq) of 0.02 mg/kg for bup, 0.5 mg/kg for lig and 0.1 mg/kg for cetrimide. 9 in the case of t-s, in order to keep residues of lig, bup and cetrimide as low as practicable, the australian pesticides and veterinary medicines authority (apvma) originally used the loqs for these active constituents to set the maximum residue limit (mrl). 9 continued on page n20 australian veterinary journal volume 92, no 12, december 2014 news n20 based on published methods of calculation 10 and data, 5, 9 if the adi for lig of 0.009 mg/kg bw/day is used, the acceptable intake of lig and its metabolites is 0.54 mg/day for a 60-kg human (0.009 x 60 = 0.54). similarly, the acceptable intake of bup plus metabolites is 0.06 mg/day. thus, the total acceptable maximum daily intake of the parent local anaesthetics and their metabolites, expressed as parent drug is 0.6 mg (0.54 + 0.06 = 0.6) for a 60-kg human. the standard intake of meat is a total of 0.5 kg/day, made up of the sum of muscle, liver, kidney and fat. the most conservative mrl set for either of the local anaesthetics is for bup at 0.02 mg/ kg meat. therefore, the highest acceptable daily consumption of meat from treated animals can be estimated by dividing the total acceptable drug intake of 0.6 mg by the conservative maximum amount of drug permitted in 0.5 kg meat of 0.01 mg. this results in a theoretical maximum daily intake of 60 kg of meat, which would be unrealistic. it must also be stressed that adis and the resultant mrls are levels for regulatory action and are not human health levels. in the case of lig and bup for t-s, the adis have been calculated using 1000-fold safety factors. this means that the occasional consumption of meat containing residues that marginally exceed the mrl does not translate into a human health hazard. the management of any risks associated with the use of t-s for mulesing lambs has been achieved by standard regulatory procedures. the risk of residues in food and the associated risk to trade have been addressed by the apvma. 11 the active ingredients are included in table 5 of the australian maximum residue limit standard, where substances for which mrls are not necessary are listed. the entry for t-s in the mrl standard also contains the qualifier "as a component in a post-mulesing treatment of lambs that are kept for wool production". as a further safeguard, a meat withholding period (whp) of 90 days after treatment has been established. at this time, more than 30 million sheep have been treated with t-s and there have been no reported residue violations in australia, or in any other country. the safety of t-s has also been assessed by the australian national drugs and poisons scheduling committee and as a result has recently been placed in schedule 5, 12 which identifies t-s as having "low potential for causing harm, the extent of which can be reduced through the use of appropriate packaging with simple warnings and safety directions on the label". the proposed new australian animal welfare standards include the requirement for pain relief for many routine animal husbandry practices in sheep and cattle. 13 the result of these proposed changes will be to place ongoing pressure to make it a requirement to provide pain relief in sheep and cattle undergoing procedures such as castration, dehorning and tail docking. additional pressures to improve farm animal welfare are also being applied by processors of human food and by consumers of animal products. in order to meet these welfare requirements, there is a need for products, such as t-s, that can provide pain relief and infection control. however, as a consequence of these pressures, there have been reports of the use of t-s for purposes other than mulesing of lambs, which is the only registered use at this time. although it may be possible for a veterinary practitioner to use t-s 'off-label' for individual animals, this usage pattern was never intended to accommodate the mass medication of foodproducing animals. there is currently development work under way to provide data to support the extension of the registration of t-s to support other husbandry procedures in livestock. such work should provide a registered product that will meet the new and additional requirements of the livestock industries, through expanded therapeutic claims and associated appropriate usage patterns. it will also ensure that the risk of violative residues from all registered uses will not be an issue. this outcome will also continue to protect the use of t-s for mulesing of lambs. i came across a book recently by psychologist daniel kahneman, titled thinking fast and slow. for veterinary practitioners, this book makes for interesting reading. thinking fast and slow has become a bestseller and in it the author describes two different modes of thinking and decision-making. system 1 operates automatically, with little or no effort, and no sense of voluntary control. system 2 involves effortful mental activities, including complex computations. a punter may make a selection in a race because he thinks he is a good judge of horse flesh and this is a system 1 or a heuristic selection. alternatively, a punter may spend a week studying form before he makes his selection and this is a system 2 judgement. kahneman argues that although the heuristic process is useful, there are biases and traps that intelligent people and even statisticians fall into. system 2 performs better, if established protocols and formulae can be set up. much of the book is about the biases of intuition. kahneman's stated aim is to improve our ability to identify and understand errors of judgment and choice, in others, and eventually in ourselves, by providing a richer and more precise language to discuss them. consider an anorexic cat presented to a practitioner. practitioner a: palpates pain in the anterior abdomen and quizzes the owner, who advises that the cat is fed a high-fat diet. a lipase test may or may not be performed. acute pancreatitis is diagnosed, which is a heuristic decision, especially if pathology tests are not performed. practitioner b: finds the same clinical signs but orders bloods, radiology and sonography. based on those results, a system 2 decision is made. kahneman describes the traps and biases made by practitioner a as follows. 1. insensitivity to prior probabilities of outcomes. available textbooks indicate that pancreatitis in cats is rare. 2. insensitivity to sample size. even if pancreatitis was correctly diagnosed in two cats last year, that sample is too small to state that it is a common condition. 3. misconceptions of chance. there hasn't been a case of a cat with pancreatitis so far this year, so it is about time one showed up. this is using the gambler's fallacy -three tosses of a coin landing heads does not mean that the next one is any more likely to be tails. 4. availability. the client comes in with a preconceived diagnosis, usually made by an internet search. the client is often wrong, because of not having available experience or data. "i think my dog has ringworm, " for example. practitioner b is a big fan of evidence-based medicine, which seems to be gaining popularity in the veterinary as well as the medical profession. system 1 is continually reinforced by system 2, so a more experienced practitioner will need less evidence-based medicine than a new graduate. in the emergency room, there is often no time for system 2, so an experienced practitioner with welldeveloped system 1 knowledge will be more effective. our clients may not be able to afford evidence-based medicine, in which case, they will favour an intuitive practitioner. some practices may find it more profitable to use evidence-based medicine, particularly those with in-house pathology. fear of litigation may encourage some practitioners to use evidence-based medicine. i recommend this book to practitioners. it's pretty heavy going, but repays persistence. comment on this article at www.ava.com.au/13329 in tasmanian dogs t he paper by david jenkins and others 1 and an associated news article published in the august 2014 edition of the journal remind us of the need for ongoing management of tapeworms in dogs, especially hydatids. i believe, however, that the present situation in tasmania has been misrepresented, especially in the news article, which concluded that "hydatid tapeworm (echinococcus granulosus) is still present in australian rural dogs, most commonly in tasmania, despite 'provisional eradication' having been declared in tasmania in 1996". tasmania ran an effective campaign to control hydatids from the 1960s until provisional freedom was declared in 1996. since then all detections of hydatids in livestock at slaughter have been investigated. although many of these detections can be explained by the animals having been infected on the mainland, there are occasional animals that have only ever resided in tasmania, so must have been infected locally. this indicates a low level of transmission of hydatids in tasmania, but the level of detection in abattoir suggests that transmission is a rare event. continued on page n22 avma seeks to promote intraprofessional dialogue about animal welfare issues ebola virus antibody prevalence in dogs and human risk world health organization (who) frequently asked questions on middle east respiratory syndrome coronavirus (mers-cov questions & answers on middle east respiratory syndrome coronavirus (mers-cov) update on mers-cov transmission from animals to humans, and interim recommendations for at-risk groups model code of practice for the welfare of animals: the sheep. primary industries ministerial council publication no. 89 australian medicines handbook. australian medicines handbook pty ltd local anaesthetics that metabolize to 2,6-xylidine or o-toluidine: final review of toxicological literature opinion of the scientific committee of the norwegian scientific committee for food safety. norwegian scientific committee for food safety australian government department of health and ageing office of chemical safety. health risk assessment for tri-solfen topical anaesthetic and antiseptic solution for pain relief in sheep hayes principles and methods of toxicology toxicology: the basic science of poisons. 5th edn mcgraw-hill the european agency for the evaluation of veterinary medicinal products, veterinary medicines evaluation unit. lidocaine summary report tri-solfen: anaesthetic and antiseptic solution for pain relief in sheep research permit (rp 8660) hayes principles and methods of toxicology national drugs and poisons schedule committee. scheduling proposals, final decisions and reasons australian animal welfare standards and guidelines echinococcus granulosus and other intestinal helminths: current status of prevalence and management in rural dogs of eastern australia publishing asia pty ltd, 155 cremorne street, richmond vic. 3121, australia. tel +61 (0)3 9274 3100. fax +61 (0)3 9274 3101. corporate citizenship wiley's corporate citizenship initiative seeks to address the environmental, social, economic, and ethical challenges faced in our business and which are important to our diverse stakeholder groups. we have made a long-term commitment to standardize and improve our efforts around the world to reduce our carbon footprint. follow our progress at www. wiley.com/go/citizenship journal customer services for ordering information, claims and any enquiry concerning your journal subscription please go to www.wileycustomerhelp.com/ask or contact your nearest office. prices include delivery of print journals to the recipient's address. delivery terms are delivered at place (dap); the recipient is responsible for paying any import duty or taxes. title to all issues transfers fob our shipping point, freight prepaid. we will endeavour to fulfil claims for missing or damaged copies within six months of publication, within our reasonable discretion and subject to availability. ltd. all journals are normally despatched direct from the country in which they are printed by surface air-lifted delivery. copyright â© 2014 australian veterinary association. all rights reserved. no part of this publication may be reproduced, stored or transmitted in any form or by any means without the prior permission in writing from the copyright holder. this consent does not extend to other kinds of copying such as copying for general distribution, for advertising or promotional purposes, for creating new collective works or for resale. authorisation to photocopy items for internal and personal use is granted by the copyright holder for libraries and other users registered with their local reproduction rights organisation (rro), eg. copyright clearance center (ccc), 222 rosewood drive, danvers, ma 01923, usa (www.copyright.com), provided the appropriate fee is paid directly to the rro. this consent does not extend to other kinds of copying such as copying for general distribution, for advertising or promotional purposes, for creating new collective works or for resale. special requests should be addressed to permissionsuk@wiley.com disclaimer the publisher, the australian veterinary association and editors cannot be held responsible for errors or any consequences arising from the use of information contained in this journal; the views and opinions expressed do not necessarily reflect those of the publisher, the australian veterinary association and editors, neither does the publication of advertisements constitute any endorsement by the publisher, the australian veterinary association and editors of the products advertised. submission of photographs for publication will be held to imply that permission for publication has been obtained from the photographer and from the subject(s) of the image.avj.pi.feb13 trademarks ava and the ava logo are registered trademarks of the australian veterinary association limited. there is no evidence of local transmission of hydatids to people in tasmania. the tasmanian department of health and human services advises there has not been any detection of hydatids in people that can be related to infection acquired in tasmania after 1996 and evidence suggests that transmission to people ceased in tasmania in the early 1970s. the tasmanian dogs reported by jenkins et al. were sampled as part of investigations into cattle infections detected at slaughter and as such comprise a sample that is heavily biased towards high-risk dogs. the prevalence detected in these dogs cannot be considered an indication of the broader situation in tasmanian dogs and also cannot be reliably compared with the results from a volunteer sample of dogs tested in other states. it is illegal in tasmania to feed livestock offal to dogs unless it has been cooked to the point of being commercially sterile. unfortunately, the section in the paper dealing with dog feeding practices did not differentiate tasmanian results from other states, so does not provide any indication of the level of compliance with this requirement.we are fortunate in tasmania that, unlike on the mainland, hydatids have never been detected in wildlife. this means that by continuing to educate at-risk groups not to feed livestock offal to dogs we have an effective tool for the control of the disease.tasmania will continue to follow up all detections of hydatids in livestock at slaughter to investigate and try to eliminate sources of infection. we will also continue to provide targeted information to dog and livestock owners about the steps they should take to prevent the disease. comment on this article at www.ava.com.au/13330 vic div hits a century! t he ava victorian division and more than 70 of its members recently celebrated 100 years with a dinner at parliament house. the premier of victoria, dr denis napthine also attended and is pictured below with victorian division president dr trish stewart.anne jackson editor in chief key: cord-261533-73721b24 authors: mok, chris ka pun; zhu, airu; zhao, jingxian; lau, eric h y; wang, junxiang; chen, zhao; zhuang, zhen; wang, yanqun; alshukairi, abeer n; baharoon, salim a; wang, wenling; tan, wenjie; liang, weiwen; oladipo, jamiu o; perera, ranawaka a p m; kuranga, sulyman a; peiris, malik; zhao, jincun title: t-cell responses to mers coronavirus infection in people with occupational exposure to dromedary camels in nigeria: an observational cohort study date: 2020-10-06 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30599-5 sha: doc_id: 261533 cord_uid: 73721b24 background: middle east respiratory syndrome (mers) remains of global public health concern. dromedary camels are the source of zoonotic infection. over 70% of mers coronavirus (mers-cov)-infected dromedaries are found in africa but no zoonotic disease has been reported in africa. we aimed to understand whether individuals with exposure to dromedaries in africa had been infected by mers-cov. methods: workers slaughtering dromedaries in an abattoir in kano, nigeria, were compared with abattoir workers without direct dromedary contact, non-abattoir workers from kano, and controls from guangzhou, china. exposure to dromedaries was ascertained using a questionnaire. serum and peripheral blood mononuclear cells (pbmcs) were tested for mers-cov specific neutralising antibody and t-cell responses. findings: none of the participants from nigeria or guangdong were mers-cov seropositive. 18 (30%) of 61 abattoir workers with exposure to dromedaries, but none of 20 abattoir workers without exposure (p=0·0042), ten non-abattoir workers or 24 controls from guangzhou (p=0·0002) had evidence of mers-cov-specific cd4(+) or cd8(+) t cells in pbmc. t-cell responses to other endemic human coronaviruses (229e, oc43, hku-1, and nl-63) were observed in all groups with no association with dromedary exposure. drinking both unpasteurised camel milk and camel urine was significantly and negatively associated with t-cell positivity (odds ratio 0·07, 95% ci 0·01–0·54). interpretation: zoonotic infection of dromedary-exposed individuals is taking place in nigeria and suggests that the extent of mers-cov infections in africa is underestimated. mers-cov could therefore adapt to human transmission in africa rather than the arabian peninsula, where attention is currently focused. funding: the national science and technology major project, national institutes of health. middle east respiratory syndrome coronavirus (mers-cov) is one of eight emerging pathogens identified in the who research and development blueprint requiring urgent action for development of effective vaccines and antiviral drugs. 1 the emergence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as a pandemic virus emphasises the threat posed by zoonotic coronaviruses. mers-cov causes a zoonotic disease, middle east respiratory syndrome (mers), with out breaks in health-care facilities associated with trans mission between humans. as of november, 2019, 2494 laboratoryconfirmed cases of mers, including 858 associated deaths (case-fatality ratio of 34·4%), were reported globally; the majority of these (2102 cases, including 780 deaths) occurred in saudi arabia. 2 travel-associated outbreaks led to 186 cases and 39 deaths in south korea. 2 dromedary camels are the source of zoonotic mers-cov disease. 3 the majority (>70%) of dromedaries are found in africa. they have comparable seroprevalence and virus shedding to those in the arabian peninsula, 4 but no zoonotic disease has been reported in africa. humans with prolonged close exposure to dromedaries in the arabian peninsula have serological evidence of mers-cov infection, sometimes having seroprevalence as high as 50%, 5, 6 but serological evidence is rare in africa, even in dromedary-exposed individuals. 7, 8 however, virologically confirmed infection, especially if it is asymptomatic or mild, might not lead to a serological response. 9 thus, alternative and more sensitive methods for detection of past human mers-cov infection are needed. specific t-cell responses have been shown to be longlasting in sars-cov and mers-cov infected humans, 10, 11 and persist longer than antibodies in sars. we therefore aimed to test peripheral blood mononuclear cells (pbmc) in workers from an abattoir in kano, nigeria, for mers-cov-specific t-cell responses to understand if the dromedary-exposed individuals in africa have been infected by mers-cov. in this observational cohort study, workers at an abattoir in kano, nigeria, consenting to participate in the cohort study in march 13-26, 2018 , were recruited. nonabattoir workers were also recruited randomly from the city of kano during the same period, and blood donors aged 18-65 years sampled in may 10-aug 31, 2018, at guangzhou blood center, guangzhou, china, were randomly included as healthy controls from a region with no dromedary camel exposure. convalescent blood samples collected from 14 people with symptomatic or asymptomatic virologically confirmed mers-cov infections detected at the king abdulaziz medical city, riyadh, and king faisal specialist hospital, jeddah, saudi arabia, collected as part of a previously reported study 11 were included as positive controls. the clinical, serological and t-cell responses (using only interferon [ifn]-γ as a readout of positive cells) of this patient cohort have been previously reported. 11 pbmcs were collected at 6 months (patients 1-6, 8-9, 11-14 as reported in the previous publication) or 24 months (patients [18] [19] after infection. written informed consent was obtained from all study participants in nigeria and the study was approved by the health research ethics committee of the ministry of health, nigeria (moh/off/797/t.i/630). we obtained institutional review board approval from the health commission of guangdong province to use the anonymised blood donor samples for this study. written informed consent was obtained from all recovered patients with mers to participate in this study and approval obtained from the institutional review boards of the national guard hospital, riyadh, and king faisal specialist hospital, jeddah. 12 procedures 8 ml of blood were collected from each study participant from the abattoir and from donors from guangzhou. pbmcs were isolated from blood using leucosep tubes (greiner, kremsmünster, austria) and ficoll-paque plus (ge healthcare, chicago, il) according to the manu facturer's instructions. pbmcs were stored in liquid nitrogen and plasma at -80°c or lower before and during shipping before analysis. plasma was heat inactivated for 30 min at 56°c before the serology testing. anti-mers-cov antibody titres were determined using plaque reduction neutralisation tests. 9, 13 a set of 20-mer peptides overlapping by ten amino acids en comp assing the four mers-cov (hcov-emc/2012) structural proteins (peptides s1, s2, n, and me encompassing the n-terminal and c-terminal portions of the spike [ evidence before this study middle east respiratory syndrome coronavirus (mers-cov) is recognised as one of eight emerging pathogens of greatest threat to global public health, and dromedary camels are the source of human zoonotic infection. the emergence of sars-cov-2 highlights the pandemic potential of zoonotic coronaviruses. although zoonotic disease has been restricted to the arabian peninsula, the largest number (>70%) of mers-cov infected camels are found in africa. we searched pubmed for articles published between nov 8, 2012, and dec 15, 2019, in english with the search terms "mers" and "coronavirus" and "human" and "africa" and manually screened all retrieved articles. there was one mers outbreak reported in tunisia initiated by a traveller returning from the arabian peninsula but no reports of zoonotic disease in africa. there were six sero-epidemiological studies of camel-exposed or other humans in kenya, egypt, nigeria, and morocco and only two (two of 1122 in kenya and three of 476 tested in morocco) found any evidence of mers-cov infection. because there was evidence that serological assays for mers-cov had suboptimal sensitivity for past infection and because we had previous data showing that t-cell assays for mers-cov are specific and potentially more sensitive than antibody detection, we investigated t-cell responses in dromedary-exposed abattoir workers and controls in nigeria. we found that 18 (30%) of 61 abattoir workers with exposure to dromedaries had mers-cov specific t-cell responses, but of 20 abattoir workers without exposure to dromedaries and ten non-abattoir workers from kano, none had such t-cell responses. no individuals with mers-cov t-cell responses had detectable antibody. by contrast, t-cell responses to endemic human coronaviruses were detected comparably in abattoir workers with and without exposure to dromedaries and control groups. we document that dromedary-exposed individuals in africa are frequently infected with mers-cov without evidence of severe disease. our findings indicate that there is substantial zoonotic transmission of mers-cov to people with dromedary exposure in parts of africa. the contribution of mers-cov to zoonotic respiratory disease remains to be established. our findings have implications for global mers-cov control policy. there is a need to confirm our findings elsewhere in africa and to include molecular testing for mers-cov in the investigation of patients with severe acute respiratory infections in dromedary-exposed populations in africa. orf4b, orf5 and orf8b) were synthesised by sino biological (shanghai, china), and used for stimulation of pbmcs. t-cell responses were measured using intracellular cytokine staining assays for interferon-γ (ifn-γ) and tumour necrosis factor (tnf). structural proteins peptide libraries of hku1-cov, oc43-cov, nl63-cov, and 229e-cov were also synthesised by sino biological to detect viral-specific t-cell responses. to enhance specificity, only cells with dual expression of both ifn-γ and tnf after peptide stimulation were considered as positive. flow cytometry was used to determine the phenotype and function of t cells. the following anti-human monoclonal antibodies were used: bv510-cd3 (hit3a; bd, san jose, ca), percp-cy5. 5 in a previous study of dromedary abattoir workers in saudi arabia, ten of 30 workers sampled had detectable t-cell responses to mers-cov. 14 on the basis of this finding, and the assumption that abattoir workers without dromedary exposure and the other control groups would have no detectable t-cell responses, eight abattoir workers would be the minimal sample size required to detect a positive result with 95% probability, where the detection probability is given by: 1 -(1 -p)n with p equivalent to 10/30 and n being the sample size. we aimed at sampling all abattoir workers who consented to participate, as long as we successfully sampled at least eight dromedary-exposed abattoir workers. association of t-cell responses with different exposure to dromedaries was done using fisher's exact test. in univariate analysis, we estimated the crude odds ratio (or) for each potential epidemiological exposure factor in relation to mers-cov t-cell positivity using a logistic regression model. independent risk factors for t-cell positivity were identified using multivariable logistic regression. we included a-priori variables, such as years of work in abattoir and whether other household members frequently visited camel farms, and other variables with a crude or of more than 2 or less than 0·5 in the univariate analysis. due to small sample size and cross-related practices of drinking camel milk and camel urine, we first fitted a logistic regression model which considered all four combinations of the two practices (eg, drinking camel milk only, drinking camel urine only, drinking both camel milk and urine or not drinking either), adjusted for potential confounding factors (model 1). then we further assessed the effect of drinking camel milk and camel urine separately in two models (models 2 and 3) . missing data were handled using multiple imputation with 50 imputations by predictive mean matching using the aregimpute function in r. all statistical analyses were done using r version 3.5.1. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. we recruited 81 volunteers working in an abattoir in kano, nigeria. dromedaries, sheep, goats, and cattle were slaughtered in different areas of this abattoir (appendix p 4), and workers usually restrict themselves to work with one animal type. 61 (75%) workers had occupational exposure to dromedaries, whereas 20 (25%) were only involved in the slaughtering of sheep, goats, or cattle. ten people residing in kano not involved in abattoir-work and 24 volunteers from guangzhou, china, with no exposure to dromedaries, were also recruited as additional controls. 14 patients with confirmed mers from saudi arabia were included in this study as positive controls. 11 all participants were adults (aged ≥18 years). boots were the main protective equipment used by abattoir workers with (48 [79%] of 61) and without (14 [70%] of 20) exposure to dromedaries, whereas other protection, such as gloves, coveralls, masks, or goggles, were rarely used. there was no significant difference in the demographic characteristics between the three groups recruited in kano (table 1) . none of the sera collected neutralised the nig1657 virus (previously isolated at the same abattoir) at the dilution of 1:10 to levels of greater than 50% of control, the lowest threshold for a positive result (data not shown). pbmcs possessed good viabilities in all groups from which they were collected (appendix p 5) and responded to anti-human cd3 stimulation (appendix p 6). 18 (30%) of 61 samples from workers with exposure to dromedaries contained cd4+ or cd8+ t cells that responded to at least one peptide pool, particularly s1 and s2 pools ( figure 1a, b; appendix p 7) . no mers-cov specific cd4+ or cd8+ t-cell responses were detected in the three groups without exposure to dromedaries ( figure 1c, d) . the proportion of individuals with both cd4+ and cd8+ t-cell responses was significantly larger among dro medaryexposed abattoir workers than in workers without exposure (cd4+ p=0·0014; cd8+ p=0·0009), non-abattoir workers (cd4+ p=0·0038; cd8+ p=0·0018), or the ghuangzhou control group (cd4+ p=0·0005; cd8+ p=0·0003). the magnitude of the cd4+ t-cell responses in abattoir workers with exposure to dromedaries was similar to individuals in the saudi arabian positive control group with a subclinical condition (p=0·094), whereas the cd8+ t-cell responses were comparable to the symptomatic group (p=0·49). for stimulation with peptide pools derived from mers-cov accessory proteins (orf3, orf4a, orf4b, orf5 and orf8b), pbmcs were available from 11 workers with exposure to dromedaries who had t-cell responses to mers-cov structural proteins, from 11 who had negative t-cell responses to mers-cov structural proteins, and from four each from abattoir workers without exposure to dromedaries and non-abattoir workers. eight of the 11 dromedary-exposed workers who had t-cell responses to structural proteins also had t-cell responses to accessory proteins ( figure 1e ). none of the abattoir workers with dromedary exposure who did not have t-cell responses previously, nor those without dromedary exposure and non-abattoir workers had t-cell responses to accessory proteins ( figure 1e ). all the t-cell responses detected to accessory proteins were cd4+ t-cell responses and no cd8+ t-cell responses were detected (data not shown). taken together, of 61 workers with exposure to dromedaries in our cohort, six had both cd4+ and cd8+ t-cell responses against mers-cov structural proteins, four had only cd4+, and eight had only cd8+ t-cell responses. the mers-cov-specific cd4+ and cd8+ t cells were multifunctional with dual expression of two cytokines (ifn-γ and tnf). the majority of mers-cov-specific cd4+ t cells from dromedary-exposed workers were phenotypically effector memory (cd45ra-ccr7-) cells (figure 1f), whereas cd8+ t cells consisted of effector memory (cd45ra-ccr7-) and effector (cd45ra+ ccr7-) cells ( figure 1g, h) , comparable to the temra subset (effector memory t cells expressing cd45ra) described in mers survivors. 11 thus, these multifunctional cells are expected to rapidly and efficiently respond to subsequent mers-cov reinfection. 61 (53%) of the 115 participants had pbmcs available for additional testing for four endemic human coronaviruses (229e, hku1, nl63, and oc43), including 18 dromedary-exposed workers positive and ten negative for a mers-cov t-cell response and 33 from the negative control groups who were all mers-cov t-cell negative. 47 (77%) of 61 were t-cell positive to one or more of the human coronaviruses, with cd4+ t-cell responses being detected in all four groups (figure 2a), whereas cd8+ t-cell responses were found less often (figure 2b). in this group of 61 people, mers-cov t-cell responsiveness was not significantly associated with t-cell responses to any of the other coronaviruses (fisher's exact test; 229e p=0·57, hku1 p=0·58, nl63 p=0·37, and oc43 p=0·40). of the 47 with t-cell response to any of the other coronaviruses, ten (21%) had t-cell responses to mers-cov. by contrast, seven (50%) of 14 with no detectable t-cell response to any other coronavirus had t-cell responses to mers-cov, the negative association being statistically significant (fisher's exact test p=0·047) . human coronaviruses did not differ between the exposure groups and this was in marked contrast with the observations with mers-cov, which was observed exclusively in the dromedary-exposed group. drinking unpasteurised camel milk (or 0·24, 95% ci 0·07-0·83) and drinking camel urine (0·30, 0·09-0·94) were significantly and negatively associated with t-cell positivity (table 2). in the multivariate analysis, drinking both camel milk and urine was significantly negatively associated with t-cell responses (0·07, 95% ci 0·01-0·54; model 1; table 3). similar findings were obtained from a model without adjustment for potential confounders (data not shown). we further assessed the effect of each practice separately (models 2 and 3; table 3) and found that drinking unpasteurised camel milk (0·14, 0·02-0·81) and camel urine (0·19, 0·04-0·84) remained a significant factor for t-cell negativity. the two practices of drinking camel milk and camel urine were closely cross-related; 48 (79%) of 61 dromedary-exposed workers drank camel milk or urine, 15 drank milk without drinking urine, and two drank urine without drinking milk. our results indicated that drinking camel milk or camel urine was associated with a protective effect against mers-cov infection, but we could not separate their independent effects in the analysis. dromedaries in africa have comparable seroprevalence of mers-cov and virus shedding to those in the arabian peninsula, but zoonotic disease has not been reported. 4, 12, 15 even serological evidence of mers-cov infection in dromedary-exposed populations is uncommon. we previously found no serological evidence of mers-cov infection in 261 dromedary-exposed abattoir workers in an abattoir in kano, nigeria, although virus rna was repeatedly detected in the camels slaughtered during the winter months, with a peak of 11% of animals shedding virus in some weeks. 16 the negative serological results in workers from the same abattoir in this study were thus consistent with those of other studies of dromedary-exposed populations in kenya and egypt, which also did not find mers-cov-specific antibodies. 7, 16, 17 one study in kenya 18 found two seropositive individuals among 1010 people tested, and our study 8 in morocco detected three seropositive individuals among 476 people living in dromedary herding areas. because some patients with confirmed mers disease might not manifest neutra lising antibody responses and because such antibody responses can wane over time, serological studies could underestimate the extent of mers-cov infections in africa. 9 furthermore, antibody responses might not be positive in those with mild or asymptomatic infection, 9, [19] [20] [21] and t-cell responses are known to be more sensitive and long-lasting following sars-cov infections. 22 we have therefore previously analysed t-cell responses to mers-cov. 11 in these studies, both mers survivors (symptomatic and asymptomatic) and camel workers one abattoir worker with exposure to dromedaries had missing data for years working in abattoir, one for other household members frequently visited camel farms, two for travel outside kano in the past 6 months, and one for participated in mass gathering. *mean for age was 27·7 years (sd 8·9). †mean for years working in abattoir was 11·4 years (sd 9·8). (asymptomatic) identified in saudi arabia were shown to have mers-cov specific t cells in their blood, and some of those with t-cell responses did not have neutralising antibodies. comparable findings were observed in the korean outbreak; some patients with mild mers did not produce neutralising antibodies but had mers-cov-specific t cells in their peripheral blood. 21 we have shown that mers-cov-specific t cells were present in 18 (30%) of 61 dromedary-exposed workers but not in controls without exposure to dromedaries, and we conclude that mers-cov infections in people with occupational contact with dromedaries is underestimated in nigeria, and probably elsewhere in africa. t-cell responses in these workers recognised the highly variable s1 region and unique accessory proteins found in mers-cov, arguing for the mers-cov specificity of the t-cell responses. by contrast, t-cell responses to human coronaviruses nl63, hku1, 229e, and oc43 were found equally distributed in the dromedary-exposed worker group and the control groups (abattoir workers without dromedary exposure, non-abattoir workers, and ghuangzhou negative control). cross-reactive t-cell responses to other human endemic coronaviruses were not likely to be an explanation for the mers-cov t-cell responses in the dromedary-exposed workers, the association being a negative one. the observation that dromedary-exposed individuals with mers-cov t-cell responses did not have antibody responses is consistent with previous studies on mers and the underlying mechanisms needs further investigation. a question of relevance to public health is why no human zoonotic mers has been documented in africa even though zoonotic infection seems to be taking place as assessed by specific t-cell responses. the perception that mers does not occur in africa might reduce the use of mers-cov diagnostics in patients who have travelled to the arabian peninsula, precluding detection of zoonotic mers in africa. our finding that zoonotic mers-cov infection is occurring in dromedaryexposed populations in africa highlights that mers-cov needs to be considered in the differential diagnosis of patients with severe acute respiratory infections in these regions. an alternative hypothesis is that mers-cov strains in africa differ in pathogenic potential to those circulating in the arabian peninsula-ie, causing infection but less likely to cause severe disease. we have shown that mers-covs identified from africa (clade c), including those isolated in nigeria (clade c1), are phylogenetically distinct from contemporary viruses causing disease in the arabian peninsula (clade b). 12, 23, 24 viruses from the african clade c1-lineage were found to replicate less efficiently in human respiratory epithelial cell lines, in ex-vivo cultures of the human lung and in experimentally infected human dpp4 transgenic mice, possibly suggesting impaired pathogenic potential. 12 the absence of antibodies in individuals with t-cell responses might also be indicative of less severe infections, because patients with mild or asymptomatic mers-cov infections often do not have detectable antibody in both the acute and convalescent stages of infection. 9, 21 irrespective of whether mers-cov in africa is less pathogenic than the virus strains in the arabian peninsula, our findings argue for more intensive investigation of mers-cov in both humans and camels in africa. if repeated unsuspected zoonotic transmission of mers-cov continues to take place in africa as our findings indicate, given the much larger number of mers-cov-infected dromedaries in africa, the possibility of the virus adapting and efficiently transmitting between humans is probably more likely here than in the arabian peninsula where mers control efforts have been focused. the phylogenetic diversity of clade c viruses in africa suggests that these are the precursors that gave rise to the potentially more pathogenic clade b viruses currently enzootic in the arabian peninsula. 12, 25 if so, similar pathogenic mers-cov might independently emerge in africa. overall, our findings suggest that the mers control in the arabian peninsula needs to be extended to africa. occupational contact with camels was found to be a key risk factor for mers-cov infection, as defined by the positive t-cell responses against mers-cov. a univariate analysis of exposure factors associated with mers-cov infection (ie, mers-cov t cell reactivity) in the dromedary-exposed worker group revealed that drinking unpasteurised camel milk and drinking camel urine for medicinal purposes were significantly and negatively associated with infection risk. because the practices of drinking raw camel milk and urine were often associated and because of the small sample size, it was not possible to estimate their independent effects in a multivariate analysis in which both factors were concurrent variables. the finding that drinking unpasteurised camel milk was negatively correlated with infection risk is surprising and requires independent confirmation. camel milk has been previously thought of as a potential risk factor for mers-cov infection because mers-cov virus has sometimes been detected in camel milk. however, camel milk contains high titre antibodies to mers-cov, which is likely to neutralise any infectious virus particles, and viable mers-cov was not isolated from milk samples in which mers-cov rna was detected. 26 thus, mers-cov antibody present in camel milk could provide protection against mers-cov infection. our study had some limitations. exposure and risk factors associated with t-cell positivity were self-reported and the details on frequency or intensity for different modes of contacts with dromedaries were not collected. a small sample size reduced the power of the multivariable logistic regression analysis, although we were still able to identify a large protective effect of drinking unpasteurised camel milk or urine on t-cell positivity. in conclusion, we have shown that detection of virusspecific t-cell responses was a more sensitive method for detecting past infection compared with the serological tests being used hitherto, findings that may be also relevant to assessment of population-based infection attack rates of sars-cov-2 using seroprevalence that are currently under way. our findings suggest that the incidence of mers infections taking place in africa is underestimated. these findings have implications for policies on global mers prevention and control and highlight the need for attention towards camel-herding regions in africa as well as the arabian peninsula. ckpm, jincz, and mp designed the study. ckpm, joo, and sak coordinated and carried out the field work. az, jingz, and jincz designed and performed the experiments. jw, zc, zz, and rapmp participated in the experiments. ckpm, az, jingz, and mp analysed the data. ehyl and wl did the statistical analysis. yw collected pbmc from guangzhong blood donors. ana and sab provided mers patients samples from saudi arabia. ww and wt contributed new reagents. ckpm, az, jincz, and mp drafted the manuscript. all authors critically reviewed and commented on the manuscript. we declare no competing interests. review of emerging infectious diseases requiring urgent research and development efforts middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation risk factors for mers coronavirus infection in dromedary camels in burkina faso presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study occupational exposure to dromedaries and risk for mers-cov infection no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study middle east respiratory syndrome coronavirus (mers-cov) neutralising antibodies in a high-risk human population mers-cov antibody responses 1 year after symptom onset, south korea lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses mers coronaviruses from camels in africa exhibit region-dependent genetic diversity seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt high prevalence of mers-cov infection in camel workers in saudi arabia who mers global summary and assessment of risk. world-health-organization lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria mers coronaviruses in dromedary camels mers-cov antibodies in humans antibody response and disease severity in healthcare worker mers survivors transmission of mers-coronavirus in household contacts immune responses to middle east respiratory syndrome coronavirus during the acute and convalescent phases of human infection memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in africa and middle east middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria enzootic patterns of middle east respiratory syndrome coronavirus in imported african and local arabian dromedary camels: a prospective genomic study middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels key: cord-278238-w1l8h8g8 authors: okba, nisreen ma; raj, v stalin; haagmans, bart l title: middle east respiratory syndrome coronavirus vaccines: current status and novel approaches date: 2017-04-13 journal: curr opin virol doi: 10.1016/j.coviro.2017.03.007 sha: doc_id: 278238 cord_uid: w1l8h8g8 middle east respiratory syndrome coronavirus (mers-cov) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. the re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. ongoing efforts to develop mers-cov vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. extending on our current knowledge of mers, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish mers-cov transmission to humans. in addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next cov outbreak. nisreen ma okba, v stalin raj and bart l haagmans middle east respiratory syndrome coronavirus (mers-cov) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. the re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. ongoing efforts to develop mers-cov vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. extending on our current knowledge of mers, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish mers-cov transmission to humans. in addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next cov outbreak. coronaviruses are the largest positive sense single stranded rna viruses. there are six human coronaviruses (hcov) to date; hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, severe acute respiratory syndrome (sars)-cov, and middle east respiratory syndrome (mers)-cov. prior to the sars-cov epidemic in 2002-2003, covs were known to cause mild respiratory infections in humans. sars-cov, on the other hand, infected around 8000 cases causing severe respiratory disease with a 10% fatality rate [1] . ten years later, mers-cov emerged in the human population also to cause severe respiratory infection [2] . in contrast to the sars-cov epidemic, which was contained within one year, mers-cov still continues to cause outbreaks with increasing geographical distribution, four years after its first identification. as of march 2nd 2017, 1905 cases in 27 countries have been reported to the who with 677 deaths accounting for a 35% case fatality rate (http://www.who.int/emergencies/mers-cov/en/). like sars-cov, mers-cov emerged as a result of zoonotic introduction to the human population. despite its close genome similarity with bat coronavirus hku4 and hku5 [2] , accumulating serological and molecular evidence pointed to dromedary camels as the most probable reservoir for mers-cov [3] [4] [5] . this poses a continuous risk of virus spill-over to people in contact with camels, such as those working in slaughter houses and animal farms, evidenced by the presence of mers-cov antibodies in sera of those individuals [6, 7] . nosocomial transmission, however, accounts for the majority of mers-cov cases reported in outbreaks [8] [9] [10] , although a substantial part of infections that occur result in unrecognized asymptomatic or mild illnesses [11] . thus, in addition to camel contacts, other highly-at-risk groups are healthcare workers and patient household contacts [8, 12, 13] . considering the ongoing mers-cov outbreaks, it is crucial to develop intervention measures among which vaccines play an important role. despite the fact that the emergence of mers-cov and sars-cov has dramatically changed the way we view covs, there is no licensed cov vaccine or therapeutic drug available to date [14, 15] . a cornerstone for rational vaccine design is defining the determinants of immune protection. accumulating data from studies done so far on mers-cov and other coronaviruses revealed that a combination of both virusspecific humoral and cellular immune responses is required for full protection against coronaviruses. especially neutralizing antibodies are considered key players in the protective immunity against covs. neutralizing monoclonal antibodies (mabs) reduced viral loads in mers-cov receptor-transduced mice, rabbits and macaques [16] [17] [18] [19] . similarly, convalescent camel sera increased virus clearance and decreased lung pathological outcomes in mice with an efficacy directly proportional to anti-mers-cov-neutralizing antibody (nab) titers [20] . also polyclonal sera produced in transchromosomic bovines protected mice against mers-cov challenge [21] . evidence for the protective role of antibodies also comes from recent studies analyzing immune responses in patients that survived or succumbed to mers-cov. although neutralizing antibodies were only weakly inversely correlated to viral loads, serum antibody responses were higher in survivors compared to fatal cases but viral rna was not eliminated from the lungs [22, 23] . administration of convalescent sera, however, did not lead to significant reduction in viral loads [22, 24] . the presence of mucosal iga abs, on the other hand, was found to influence infectious virus isolation [25] . besides humoral immunity, cellular immune responses are also considered to play a crucial role in protection against coronaviruses. while b-cell deficient mice were able to clear mers-cov, those lacking t-cells failed to eliminate the virus, pointing out the crucial role of t-cells in viral clearance [26] . this is supported by the observation that t-cells were able to protect aged mice against sars-cov infection [27 ] and the fact that a reduced tcell count was associated with enhanced disease severity in sars patients [28] . along with other studies, these data highlight the importance of t-cells for virus clearance and protection against mers-cov [26, 27 ] and sars-cov [29, 30] . it is also noteworthy to mention that while neither antibodies nor memory b cells were detectable 6-years post-infection [31] , sars-cov-specific memory t-cells, despite being low in frequency, persisted up to 11 years post-recovery [32] . nonetheless, the protective capacity of such memory response is not known. hence, taking into account the waning of virus-specific humoral responses, generating a long-lived memory t cell response through vaccination could be favorable, but as proper b-and t-cell immune responses are required for efficient protection, vaccination should target the induction of both. at the moment we lack information concerning the longevity of anamnestic immune responses following mers-cov infection, except for a recent study showing that antibody responses, albeit reduced, persisted up to 34 months post-infection [33] . the role of immune responses in protection is also in line with the observed increased fatality among the aged population following mers-cov infection. retrospective studies on mers-cov patients from saudia arabia and south korea have found a significant correlation between old age and mortality [8, 13, [34] [35] [36] , a pattern that has been also reported for other respiratory viruses such as sars-cov [1] and influenza virus [37] . this is most likely caused by immunosenescence; a failure to produce protective immune response to new pathogens in elderly due to impaired antigen presentation, altered function of tlrs, and a reduced naïve b and t cell repertoire [38] . this agerelated increase in mortality was also reported in sars-cov laboratory-infected animals, that is, mice and nonhuman primates (nhps) [39, 40] , and was associated with low neutralizing antibodies and poor t-cell responses [41, 42, 43 ] . several factors that play a role in t-cell activation were also found to be dysregulated in an age-related manner. age-related increase in phospholipase a 2 group iid (pla 2 g2d), and prostaglan-dind 2 in the lungs contributed to a diminished t-cell response and severe lung damage through diminishing respiratory dendritic cell (dc) migration [44, 45] . likewise, adoptive transfer of t-cells to mice enhanced viral clearance and survival [29] , highlighting the contribution of a reduced t-cell response in severe disease outcome. these observations also highlight the need for more effective preventive measures for the elderly. in this sense, induction of a potent airway t-cell response may be crucial to protect against covs [27 ] . thus, a promising approach to protect against mers-covinduced fatality is to enhance virus-specific tissue (airway) resident memory t-cell responses through intranasal vaccination. although the mers-cov genome encodes for 16 nonstructural proteins (nsp1-16) and four structural proteins, the spike (s), envelope (e), membrane (m), and nucleocapsid (n) [46] , the viral structural proteins, s and n, show the highest immunogenicity [47] . while both s and n proteins can induce t-cell responses, neutralizing antibodies are almost solely directed against the s protein, with the receptor binding domain (rbd) being the major immunodominant region [48] . thus, current mers-cov vaccine candidates mainly employ the spike protein or (parts of) the gene coding for this glycoprotein. these mers-cov vaccines candidates were developed using a wide variety of platforms, including whole virus vaccines, vectored-virus vaccines, dna vaccines, (table 1) and protein-based vaccines ( table 2) . although live attenuated vaccines produce the most robust immune responses, they pose a risk from reversion to virulence. inactivated virus vaccines may cause harm due to incomplete attenuation or the capacity to induce lung immunopathology [49] . viral-vector-based vaccines, on the other hand, provide a safer alternative and have been developed using modified vaccinia virus ankara (mva) [50, 51, 52 ] , adenovirus (adv) [53, 54] , measles virus (mev) [55] , rabies virus (rabv) [56] , and venezuelan equine encephalitis replicons (vrp) [26, 57] , all expressing mers-s/s1 proteins. additionally, vrp expressing the n protein have also been developed [27 ] . a major hurdle facing these viral-vector-based platforms is preexisting immunity in the host which potentially can impair the vaccine efficacy. however, this can be prevented by using virus strains not circulating in the targeted population or immunization strategies involving heterologous prime-boost immunization, for example, mva and adv. although plasmid dna vaccines are considered to be of low immunogenicty in humans, current versions developed seem to induce potent immune responses. dna-based vaccines directed at inducing anti s responses were also shown to exert protection in nhps [58, 59] . noteworthy to mention is middle east respiratory syndrome coronavirus vaccines: current status and novel approaches okba, raj and haagmans 51 ad/hdpp4-mice, mice transduced with hdpp4 in an adenoviral vector; alum, aluminum hydroxide; e, envelope protein; hdpp4, human dipeptidyl peptidase 4; i.m., intramuscular; i.n., intranasal; m, matrix protein; mers-cov, middle east respiratory syndrome coronavirus; nab, neutralizing antibodies; nd, not done; nhp, non-human primate; rntd, recombinant n-terminal domain; rbd, receptor-binding domain; rrbd, recombinant rbd; rbd-fc, rbd fused to the antibody crystallizable fragment of human igg; s, spike protein; s1, s1 domain of spike protein; s367-606, amino acid residues 367-606 of the s protein; s736-761-klh, peptide s736-761 coupled to keyhole limpet haemocyanin; s.c., subcutaneous; vlps, virus-like particles; a i.m.;alum/cpg odn produced higher neutralizing antibody responses than s.c.; ifa/cpg odn. b s350-588-fc, s358-588-fc, s367-588-fc, s367-606-fc, and s377-588-fc were tested and s377-588-fc had the highest nab titers although some produced equal s1 igg response [66] . c mf59 produced the highest immunogenicity at low doses of antigen compared to s377-588-fc only, or with freund's/alum/mpla-sm/isa51/mf59. [67] 1 mg of antigen with mf59 was sufficient to produce humoral and cellular immune responses similar to higher doses (5 mg or 20 mg) [68] . d i.n. + poly(i:c) vaccination induced stronger systemic cellular responses and higher local immune responses in mice lungs (iga and neutralizing antibody titers) than s.c. + montanide isa51 vaccination. that a combination of dna (s) and recombinant protein (s1) in a heterologous prime-boost immunization strategy induced higher immune response (nab) compared to each component alone [58] . additionally, protein-based vaccines were developed in various platforms as virus-like particles (vlps) [60] , nanoparticles [61] , peptide-based [62] , and subunit vaccines directed against various regions of the spike protein s1 [58] , the n-terminal domain [63] , and the rbd [48, [64] [65] [66] [67] [68] [69] [70] . those vaccines have the highest safety profile among vaccine platforms but confer variable degrees of immunogenicity which need adjustment for the dose, adjuvants, and site of administration to get optimal protective responses. adjuvants influence the type and magnitude of immune response produced by vaccines, and thus the doses used [61, 65, 68] . additionally, the route of administration is a determining factor for the type of vaccine-induced immune response produced in the host. while intranasal (i.n.) vaccination with sars-n produced a protective airway t-cell response against sars-cov in mice, subcutaneous (s.c.) vaccination, inducing systemic t-cell responses, did not [27 ] . likewise, i.n. vaccination with mers-rbd induced a significantly higher neutralizing and iga antibody responses in the mice airways compared to s.c. vaccination [70] . this is important because mucosal immunity and airway memory t-cell responses are crucial players in protection against respiratory viruses, since these areas are the first to encounter the virus. therefore, along with selecting antigens for a vaccine, the route of vaccination and adjuvants are key players that cannot be neglected in vaccine design. because the spike protein and more specifically the s1 domain, is highly divergent among different covs, neutralizing antibodies only provide homotypic protection. thus far, the variability in the amino acid sequence of the spike protein observed among mers-cov strains is low [71] , and circulating mers-cov strains did not show any significant variation in the serological reactivity [25, 59] , implying that the development of a vaccine that is effective against one strain is likely to be protective against all circulating strains. another risk posed from the development of antibody escape variants is still present [72, 73] , although this is not likely to happen as mutations in two epitopes may be required, and mutants that develop may have reduced viral fitness [73, 74] . while the rbd is considered an ideal vaccine candidate for mers-cov, the spike s2 domain and n protein are more conserved, and thus adaptive immune response directed against these proteins can potentially lay the basis for a more broadly acting coronavirus vaccine. however, evidence for cross reactive immune responses against different covs is limited to a few studies. convalescent sars-cov patient sera weakly neutralized mers-cov [75] and sars-s reactive antisera showed low level neutralization of mers-cov [61] . extra-rbd s1 or s2 epitopes could be responsible for this effect, as some neutralizing epitopes have been identified in these regions of the s protein [58, 62] . these may not be as immunodominant as the rbd epitopes but could provide a rationale for the development of a cross protective cov epitope-focused vaccine. a recent study also demonstrated the potential role of adaptive response against n protein in protection against mers-cov infection as this vaccine candidate produced a protective t-cell response against mers-cov challenge which was also partially protective against sars-cov [27 ] . moreover, infection of mice with sars-cov reduced mers-cov titers 5 days p.i. upon challenge suggesting the development of a cross reactive t-cell response [26] . thus, mapping and focusing the immune response towards these critical neutralizing and t-cell epitopes, which could be subdominant, may provide a way to induce immune responses with a broader activity against different covs. immune focusing may also be beneficial for the generation of a robust virus-specific immune response. as during vaccine preparation, some epitopes which are normally hidden in the full length protein structure get exposed. some epitopes could be immunodominant and have a negative contribution on the overall neutralization capacity produced by the vaccine [76 ] . this also holds true for some non-neutralizing immunodominant epitopes, as s1-based vaccines induced slightly higher neutralization than whole s ectodomain-based ones [58, 53] . additionally, the rbd induced higher neutralizing antibodies compared to an s1 subunit vaccine [48] , and shorter regions of rbd induced even higher neutralization responses [66] , indicating that additional regions inducing non-neutralizing antibodies may contribute negatively to the overall neutralization response produced. additionally, antibody-dependent enhancement of the viral infection by non-neutralizing antibodies [77] [78] [79] , despite not being reported so far for mers-cov, needs also to be taken into consideration when developing a coronavirus vaccine. one approach to enhance the efficacy of subunit vaccines is to mask those negativelycontributing epitopes through glycosylation [76 ] . other approaches are immunefocusing and epitope-based vaccines, all aiming at narrowing the immune response to target only critical or beneficial epitopes to produce a stronger protective response. a prerequisite to reach that goal is to map epitopes targeted by the immune system and identify their biological role as being neutralizing, non-neutralizing, infection enhancing, containing a tcell epitope, and so on. this can be achieved by analyzing the activity and fine specificity of convalescent patient sera, infected animal polyclonal sera, monoclonal antibodies, animal and human pbmcs. subsequently the predicted epitopes can also provide a basis for potential vaccine candidates when produced as nanoparticles or vlps. further characterization of the immune responses induced by these vaccine candidates when evaluated in an animal model may be utilized to optimize the vaccines for efficacy (figure 1 ). this epitope-focused vaccine approach may allow for targeting less immunodominant b-and t-cell epitopes having broader protection, avoid eliciting immune responses against epitopes playing no role in protection or having a negative or harmful role. in addition to better targeting of protective immunodominant epitopes, a combination of those epitopes, b-and tcell epitopes targeting different viral proteins, could be used to produce a broader and stronger protective immune response for both strain-specific and universal cov vaccines. next to the choice of the mers-cov vaccine candidate, it is also important to take into account the target population that needs to be protected through vaccination. populations at risk of mers-cov infection include camel contacts, healthcare workers and patient contacts. the latter two groups could benefit from the rapid onset of immunity though passive immunization using mabs or convalescent sera, provided that it is given in time. another alternative strategy for short-term protection is the use of vaccines capable of rapidly inducing high titers of neutralizing antibodies. this will provide a short-term immunity beneficial to protect those highly-at-risk groups when a new case is identified, to prevent outbreaks. to prevent virus infection of primary cases, vaccination of the dromedary camels may also be considered. so far, among the available vaccine candidates, only two have been tested in dromedary camels, pvax1-s and mva-s. pvax1-s, a dna-based vaccine, induced neutralizing antibodies in two of three camels tested so far, but has not been tested for efficacy [59] . the other candidate mva-s, a viral-vector-based vaccine, induced systemic neutralizing antibodies and mucosal immunity which conferred protection against mers-cov challenge and reduced virus shedding in vaccinated camels [52 ] therefore, this vaccine candidate may provide a means to prevent zoonotic transmission of the virus to the human population. for camel contacts and healthcare workers in endemic areas, being at a continuous risk of mers-cov infection either from infected camels or patients, respectively, it would be beneficial to induce a longer-term (mucosal) protection. while these could be rewarding approaches to stop mers-cov outbreaks, it is still worthwhile to develop platforms and vaccines that aim to induce more broad protection against different related covs, that could potentially cause future outbreaks. learning from previous epidemics, the who issued a list of priority pathogens posing a risk to the human population and requiring urgent research and development (r&d) for intervention measures, among which mers-cov and highly pathogenic covs are of high priority. the lack of intervention measures along with the increase in geographical area and ongoing mersepitope-based vaccine design. following a virus infection, potential protective b-and t-cell epitopes are mapped. peptides or proteins containing promising epitopes are produced and formulated using a suitable platform, for example, nanoparticles and tested for immunogenicity and efficacy in animals. follwing several cycles of testing and optimization, a final vaccine suitable for human use may be produced. cov cases, raise worries for the future occurrence of larger epidemics as a result of virus adaptation in the human population and more efficient human-to human transmission. further development of mers-cov and other cov vaccines thus needs proactive collaborative efforts from researchers filling knowledge gaps, and market stakeholders providing funding for this costly process. the latter can be insufficient and/or unsustainable, therefore hindering development of even some promising candidates. in an initiative aiming at accelerating vaccine r&d process by providing sustained funding to be prepared for future epidemics, the world economic forum launched the coalition for epidemic preparedness innovations (cepi) [80] . cepi is an international non-profit association aiming at removing barriers facing vaccine development for epidemic infections and getting ready for future epidemics, including mers-cov. however, we still face a number of challenges despite the fact that various promising mers-cov vaccine candidates are currently available. there is a lack of animal models mimicking the disease in humans in which vaccine platforms can be tested prior to human use. we need to take into account the populations to target with vaccination, with camels and camel handlers being the most relevant ones. the lack of full understanding of the pathogenesis and immune responses to the virus in humans and camels, which is crucial for vaccine development, also needs further investments. in addition, the longevity of immune responses post-vaccination has not been evaluated for vaccine candidates, which is important for the vaccination scheme development and for the choice of the best candidates for further development. lastly, most of the vaccine candidates are developed against the highly variable spike protein and thus may not be able to provide protection against cov strains evolving in the future. a more targeted vaccination approach aiming at conserved epitopes should be considered for the development of a more broadly-acting cov vaccine. given the propensity of covs to jump the species barrier, current efforts to develop a mers-cov vaccine may also be of benefit to prepare for potential novel covs that may emerge in the future. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest the immunobiology of sars* isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study occupational exposure to dromedaries and risk for mers-cov infection presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission characteristics of mers and sars in the healthcare setting: a comparative study unraveling the drivers of mers-cov transmission risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel transmission of mers-coronavirus in household contacts clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia animal models of middle east respiratory syndrome coronavirus infection a comparative review of animal models of middle east respiratory syndrome coronavirus infection 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012. virology prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome airway memory cd4(+) t cells mediate protective immunity against emerging respiratory coronaviruses this study shows the efficacy of an anti-nucleocapsid vaccine for mers-cov, t-cell mediated protection and potential cross protection, and the role of route of administration in protection haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection persistence of antibodies against middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility mortality risk factors for middle east respiratory syndrome outbreak, south korea association of higher mers-cov virus load with severe disease and death mortality associated with influenza and respiratory syncytial virus in the united states grubeck-loebenstein b: vaccines for the elderly mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice exacerbated innate host response to sars-cov in aged non-human primates evasion by stealth: inefficient immune activation underlies poor t cell response and severe disease in sars-cov-infected mice successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus t cell-mediated immune response to respiratory coronaviruses review on the role of t-cells in protection against respiratory coronaviruses age-related increases in pgd(2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus. hum. vaccines immunother middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform one-health: a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus a mouse model for mers coronavirus-induced acute respiratory distress syndrome evaluation of candidate vaccine approaches for mers-cov a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice the amino acids 736-761 of the mers-cov spike protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents the recombinant nterminal domain of spike proteins is a potential vaccine against middle east respiratory syndrome coronavirus (mers-cov) infection recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection intranasal vaccination with recombinant receptorbinding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution recombinant receptor-binding domains of multiple mers-coronaviruses induce cross-neutralizing antibodies against divergent human and camel mers-coronaviruses and antibody-escape mutants cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus sars cov subunit vaccine: antibody-mediated neutralisation and enhancement antibody-dependent sars coronavirus infection is mediated by antibodies against spike proteins vaccine initiative marks bold resolution engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate this work was supported by the zoonoses anticipation and preparedness initiative (zapi project; imi grant agreement no. 115760), with the assistance and financial support of imi and the european commission, inkind contributions from efpia partners. key: cord-283586-o8m6xdra authors: spanakis, nikolaos; tsiodras, sotirios; haagmans, bart l.; raj, v. stalin; pontikis, kostantinos; koutsoukou, antonia; koulouris, nikolaos g.; osterhaus, albert d.m.e.; koopmans, marion p.g.; tsakris, athanassios title: virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen date: 2014-12-31 journal: international journal of antimicrobial agents doi: 10.1016/j.ijantimicag.2014.07.026 sha: doc_id: 283586 cord_uid: o8m6xdra abstract serological, molecular and phylogenetic analyses of a recently imported case of middle east respiratory syndrome coronavirus (mers-cov) in greece are reported. although mers-cov remained detectable in the respiratory tract secretions of the patient until the fourth week of illness, viraemia was last detected 2 days after initiation of triple combination therapy with pegylated interferon, ribavirin and lopinavir/ritonavir, administered from day 13 of illness. phylogenetic analysis of the virus showed close similarity with other human mers-covs from the recent jeddah outbreak in saudi arabia. immunoglobulin g (igg) titres peaked 3 weeks after the onset of illness, whilst igm levels remained constantly elevated during the follow-up period (second to fifth week of illness). serological testing confirmed by virus neutralisation assay detected an additional case that was a close contact of the patient. an upsurge of middle east respiratory syndrome coronavirus (mers-cov) infection has been recently described in countries of the arabian peninsula resulting in exported cases from these countries to the european union [1] . cases of mers-cov infection are associated with a high case fatality rate since there is no available treatment. there is a scarcity of data on specific therapeutic interventions for the disease. published reports propose the use of known antivirals based on extrapolation of data from: (i) the severe acute respiratory syndrome (sars) epidemic that was also associated with the circulation of a novel coronavirus; (ii) in vitro data; (iii) animal experimental infections and therapy data; and (iv) limited clinical data for actual mers-cov infections [2] [3] [4] . however, no clear-cut recommended therapeutic regimen exists and the evidence for grading such interventions is generally low, with the exception of the use of convalescent serum that based on biological effects is given the highest grade [5] . moreover, little is known about the viral kinetics of mers-cov-associated infection, especially when a specific antiviral or other therapeutic intervention is attempted. a case of mers-cov has recently been described in greece in a traveller who had extensive contact with the healthcare environment in jeddah (saudi arabia) [6] . here we describe molecular, serological and phylogenetic analyses of this case as well as evidence for a second case that was a close contact of the first patient. furthermore, we provide evidence of the kinetics and the pattern of viral excretion in biological specimens obtained from the first greek case while the patient was on a triple antiviral regimen. a preliminary report of the first imported, laboratory-confirmed mers-cov case in greece has been described elsewhere [6] . a full description of the course of illness and treatment regimen in http relation to kinetics of virus shedding and immune response was prepared by review of the patient records. in the course of the outbreak investigation, 40 of 75 patient's contacts, including the patient's wife, provided an oropharyngeal sample for pcr testing 1 week after contact with the positive case; 5 additional contacts were included in the serology examination group. all were submitted to personal clinical monitoring for fever and upper respiratory infection symptoms and were advised to call the hellenic centre for disease control and prevention (cdc) command centre immediately in such an instance. in addition, all were offered the chance to provide serum samples on a voluntary basis for specific anti-mers antibody testing at baseline (same time as the oropharyngeal pcr testing) and 3 weeks after exposure. during the patient's stay in the intensive care unit (icu), samples from the oropharynx, trachea, urine and faeces were tested for diagnostic evaluation and to monitor viral shedding. a real-time reverse transcription pcr (rt-pcr) method based on amplification of the upstream envelope gene (upe), the nucleocapsid (n) gene and the open reading frame (orf) 1a of the virus was used for detection of mers-cov according to previously described methodology [7, 8] . immunoglobulin g (igg) and igm antibody titres in serum samples were determined using an anti-mers-cov indirect immunofluorescence assay (euroimmun ag, lübeck, germany). confirmation of the serological findings was performed with a virus neutralisation assay as described previously [9] . samples from the patient's upper respiratory tract underwent conventional or molecular testing for the presence of other respiratory pathogens: thus, cultures applied for bacterial testing, whilst real-time rt-pcr was performed for several respiratory viruses including influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza, adenovirus, enterovirus, bocavirus and human metapneumovirus (hmpv) (m.w.s. r-gene; biomérieux, marcy-l'étoile, france). specific urine antigen testing of urine samples was utilised for legionella pneumophila and streptococcus pneumoniae (binaxnow ® ; alere, orlando, fl). a stool culture was performed due to a history of possible typhoid fever, diagnosed by treating physicians in saudi arabia [6] . nucleotide sequences of 3-kb concatenated sequences of representative mers-covs were analysed and a phylogenetic tree was constructed by the phyml method as described previously [10] . a 69-year-old patient of greek origin who was a permanent resident of jeddah presented to a tertiary care centre a few hours after arriving in athens (greece) on 17 april 2014. his chief complaints included fever since 8 april 2014 and diarrhoea since 10 april 2014. the most likely source of exposure was the hospital environment in jeddah. the patient had no known co-morbidities. at the time of initial evaluation, a fever of 38.3 • c was noted together with low oxygen saturation (92%), although the patient exhibited minimal respiratory symptoms. a chest radiograph depicted bilateral lung infiltrates consistent with viral pneumonia. the patient was immediately placed under isolation because of suspicion of mers-cov infection, and an antimicrobial regimen targeting communityacquired pneumonia was initiated. on 18 april 2014, mers-cov infection was confirmed by means of viral rna detection in a pharyngeal swab at the department of microbiology, university of athens medical school (athens, greece). after laboratory confirmation of mers-cov, the patient was transferred to a specialised respiratory disease unit in the 'sotiria' chest diseases hospital of athens where he was treated in a negative pressure regular room in isolation until 20 april 2014 when, due to deterioration of his respiratory function and development of acute respiratory disease syndrome (ards), he was intubated, ventilated and transferred to a negative pressure room in the icu of the same hospital. an empirical antiviral regimen was initiated on day 13 of illness consisting of oral (p.o.) lopinavir/ritonavir (400/100 mg twice daily), pegylated interferon (180 g subcutaneously once per week for 12 days) and ribavirin (2000 mg p.o. loading dose, followed by 1200 mg p.o. every 8 h for 8 days) based on available evidence [3] [4] [5] 11, 12] (fig. 1) . the patient remained intubated exhibiting hypoxia and occasionally hypercapnia while breathing inspired oxygen in the range of 0.45-0.60. he remained febrile with a plateau temperature of >39 • c and a maximum value of 40.5 • c on day 18 of illness. fever started subsiding below 38 • c on day 22. acute kidney injury was diagnosed on day 16 of illness and rapidly progressed to non-oliguric renal failure that reverted to rifle injury level (i.e. two-fold increase in the serum creatinine, or glomerular filtration rate decrease by 50%, or urine output <0.5 ml/kg/h for 12 h) on day 21. the patient's diarrhoea resolved gradually starting on day 13 and he developed constipation thereafter with normalisation of his bowel movements and gastrointestinal function on day 19. owing to development of jaundice and hyperbilirubinaemia attributed to ribavirin [13] , the drug was discontinued on day 20. during the course of his hospitalisation, the patient was diagnosed with adenocarcinoma of the colon and eventually died from septic shock 2 months and 19 days after the initial diagnosis. cultures and antigen detection were negative for l. pneumophila and s. pneumoniae. virological testing was negative for the presence of any other respiratory virus. no relevant enteric pathogens were identified as a cause of the patient's diarrhoea. rna was detected in several consecutive patient samples from different sites that included faecal material and serum (fig. 1) . shedding of mers-cov in the respiratory secretions of the patient was noted until the fourth week of illness, whereas viraemia was last detected 15 days after onset of illness and 2 days after initiation of the triple combination antiviral regimen. consecutive urine testing did not reveal the presence of mers-cov rna (fig. 1) . serological testing showed a peak igg titre during the third week of illness, whilst during the fourth and fifth weeks igg titres were substantially declining. igm titres were persistently elevated during the whole survey period (day 13 until day 34 of illness) (fig. 1 ). viral neutralisation assays performed at erasmus medical center (rotterdam, the netherlands) confirmed the immunofluorescence testing results. initial and follow-up serological testing were performed on serum samples from 45 patient's contacts. seroconversion was revealed in one of them who developed an igg titre of 1/500 and an igm titre of 1/100 at 21 days after making contact with the patient. this was a 63-year-old man with a past medical history of coronary artery heart disease and diabetes. the presence of specific mers-cov antibodies was confirmed by the virus neutralisation assay. without other symptoms from any other system. no nasopharyngeal pcr testing was performed at the time since he was outside the incubation period of 14 days. he has been well since then and during the time that he was symptomatic he only had contact with his family members (four persons). the initial patient's wife had a brief episode of fever on 30 may 2014. oropharyngeal pcr testing was negative for mers-cov, and all contacts remained seronegative on repeat testing. partial genomic sequencing [14] revealed the close phylogenetic relationship with clinical mers-cov strains associated with severe respiratory infection from patients in jeddah (fig. 2) . in this report, we further characterised serological and virological parameters of the first mers-cov case in greece. rising titres of igg were demonstrated in sequential serum samples, with the peak titres approximately 3 weeks into the course of the disease. this is in accordance with serological testing guidance from the world health organization (who) recommending baseline testing from initial contact with an affected case and repeated serological testing on day 21 [15] . on the other hand, igm titres of the patient remained constantly elevated above the threshold of detection, albeit at a lower level than igg antibodies, for a prolonged period of ≥1 month of follow-up. thus, isolated use of igm testing without concomitant igg determination appears not to be sufficient to reveal a recent infection. it should be noted, however, that in the absence of detailed studies, use of serological testing for mers-cov detection in humans needs to be further evaluated. prolonged shedding of the virus was noted from the respiratory tract of the patient. this finding is consistent with data regarding the sars coronavirus. in a report dealing with patients affected by sars, prolonged shedding of the virus was noted in stool (up to 126 days) and respiratory specimens (up to 52 days) [16] . data regarding the length of mers-cov excretion from different body sites are scarce [17] . excretion of the virus probably depends on the amplitude of replication in different body sites, the underlying immune status and co-morbidities, and appropriate antiviral therapy. the non-detectable viral rna in serum by day 3 after initiation of the antiviral treatment could be explained either by viral clearance in an otherwise immunocompetent person or by effectiveness of the instituted antiviral regimen. literature on appropriate antiviral intervention for mers-cov is very limited and currently no evidence-based therapy exists. the regimen chosen was based on the best available literature as well as evidence from animal and patient data that have been described elsewhere [2] [3] [4] . the role of interferon therapy for mers-cov infection needs to be further elucidated. an attenuated interferon-␤ (ifn-␤) response has been described as a result of mers-cov infection [18] and extensive use of interferon-based regimens alone or in combination with ribavirin has been described for sars [4] . however, interferons appear to have a better antiviral effect on mers-cov compared with sars-cov in in vitro experiments [19] . in vitro, ifn-␤ appears to exhibit the best anti-mers-cov effect [20] . interferon activity has been enhanced by the addition of ribavirin in in vitro experiments [21] . furthermore, this combination has shown promising clinical and radiological effects in rhesus macaques experimentally infected with mers-cov [12] . thus, the clinical team elected to use this combination despite the fact that a prestigious public health agency ranks ribavirin use as not supported by high-quality evidence [5] . in a more recent update published by the same public health agency, the use of interferons and lopinavir is ranked under the recommendation of benefit is likely to exceed risk, whereas the combination of interferon and ribavirin is ranked as data are inadequate for assessment [22] . the frequent side effects of ribavirin limit its use in combination regimens for actual mers-cov-infected patients, as was the experience with the current patient where liver toxicity, although not definitively associated, was mainly attributed to this medication. the renal function deterioration of the patient described here was considered multifactorial and probably also a complication of the virus infection [23] . the possibility that drug toxicity might have contributed in the renal dysfunction could not be excluded, however. no drug levels were measured since the patient was under continuous renal replacement therapy at that time and drug levels would be unreliable. in the actual clinical human setting, the combination of ribavirin and interferon has been tried, with no successful outcome reported among any of the mers-cov-infected recipients [3] . nevertheless, the group studied consisted of severely ill patients who received this combination quite late in the course of their disease [3] . lastly, we added the protease inhibitors lopinavir/ritonavir based on experience accumulated from the sars epidemic where the addition of this agent to ribavirin improved the outcome of infection [24] . expanding the knowledge regarding viral kinetics and the pattern of shedding especially in association with specific therapeutic interventions has important implications for infection control in the healthcare environment, especially as it relates to potential transmission to other patients and healthcare workers [25] . phylogenetic analysis of the greek mers-cov strain showed close similarity with circulating patient viral strains from the recent jeddah outbreak as well as with a strain isolated from a dromedary camel in qatar. this is in accordance with previous genetic studies that have shown identical viral strains between infected humans and dromedary camels and generated the hypothesis that dromedary camels are among the reservoirs of the virus in nature [10, 26] . also, the presence of mers-cov-specific antibodies in camels across a wide geographic area in africa and the arabian peninsula signifies the possibility for zoonotic transmission between camels and humans [27] [28] [29] . the potential for transmission across different individual strains should be further explored. in this patient, the most likely source of exposure was the hospital environment in an endemic area, as his wife was hospitalised in a local hospital in jeddah [6] . it appears that healthcare-associated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic in the recent upsurge [30] . in conclusion, we describe the genetic stability of the mers-cov in a strain from the recent jeddah outbreak. although reassuring, this finding should not limit the level of awareness regarding the increased number of cases in the arabian peninsula reported recently and the potential evolution and more efficient transmission of the virus. a who committee recently concluded that the conditions for a public health emergency of international concern have not yet been met. nevertheless, important gaps in current knowledge about mers-cov exist. more investigations to clarify the natural reservoir and modes of transmission are necessary. persistence of virus shedding in patients' secretions and the effect of immune status and antiviral therapy together with the implementation of appropriate infection control measures are of paramount importance in limiting further spread of this potentially lethal virus. funding: this work was funded by a grant from the netherlands organisation for scientific research (nwo) [no. 40-00812-98-13066]. competing interests: none declared. ethical approval: not required. european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov). ecdc what are our pharmacotherapeutic options for mers-cov? ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy clinical decision making tool for treatment of mers-cov v a case of imported middle east respiratory syndrome coronavirus infection and public health response assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation ribavirin and interferon (ifn)-␣-2b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases treatment with interferon-␣2b and ribavirin improves outcome in mers-covinfected rhesus macaques schering corp product information. rebetol® (ribavirin, usp). kenilworth, nj: schering corp isolation of mers coronavirus from dromedary camel seroepidemiological investigation of contacts of middle east respiratory syndrome coronavirus (mers-cov) patients. geneva: switzerland: who long-term sars coronavirus excretion from patient cohort clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential interferon-␤ and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays inhibition of novel ␤ coronavirus replication by a combination of interferon-␣2b and ribavirin in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 9 the authors would like to acknowledge the department of epidemiological surveillance and response of the hellenic centre for disease control and prevention (cdc), and especially dr georgia spala, theano georgakopoulou and agoritsa baka, for mers-related activities, as well as dr spyros sapounas for contacting the case investigation of the second case. key: cord-260024-yrhlg6wm authors: ha, kyoo-man title: a lesson learned from the mers outbreak in south korea in 2015 date: 2015-10-24 journal: j hosp infect doi: 10.1016/j.jhin.2015.10.004 sha: doc_id: 260024 cord_uid: yrhlg6wm nan middle east respiratory syndrome (mers) viruses can spread rapidly to many people, and thus pose a global pandemic threat. mers viruses broke out in one hospital in pyeongtaek, south korea (hereinafter korea), on may 2015, 2015. thirty-six patients died and 186 people were infected. 1 the koreans experienced a national crisis, contributed to by the poor initial response of the affected hospitals, an inadequate response from the government, the economic depression that followed the outbreak, and the psychological impact of the outbreak on the korean population. to date, the korean government has not taken systematic actions to deal with global pandemics in the future. this article comments on how korea can learn from its response to a mers outbreak to be better prepared to control other epidemic and pandemic infectious diseases. we define the korean reaction (which includes four major stakeholders) as a hospital infection control issue (figure 1 ). the four stakeholders were either directly or indirectly involved in the outbreak of mers in korea. we argue that the nation has to transform its current reaction into an emergency management issue involving all stakeholders. hospitals mainly because of poor ventilation and ineffective disinfection in one hospital, mers viruses began to spread rapidly to patients, visitors, and even to healthcare workers. a few neighbouring hospitals also showed a similar pattern of nosocomial transmission. 2 consequently, healthcare workers made all efforts to improve infection control or hygiene; however, this took time. therefore, hospitals became major stakeholders in preventing the loss of human lives. the korea centers for disease control and prevention (kcdc) under the ministry of health and welfare (mw) insisted on not sharing mers information from the hospitals with the public at the initial stage of the outbreak under the pretext of hospital protection, although in reality this decision may have been based on nepotism. further, the ministry of public safety and security (mpss), which is a single, comprehensive emergency management agency, did not implement any specific action to prevent the loss of human lives. thus far, only the kcdc has tried to improve preparedness against similar pandemics. some koreans were involved in manipulating the facts on the mers outbreak and then spreading rumours through the internet or mobile phones. further, given the korean culture, many people did not realize that it was not advisable for them to visit the infected patients in hospitals and share their drinks or foods. also, a few infected residents attempted to go to public places without permission from the government. however, the majority of residents considered the mers outbreak to be a national emergency and thus paid attention to its progress by exchanging relevant information. other stakeholders took steps to efficiently respond to the outbreak of mers. for instance, business establishments continued to operate but measured the body temperature of their customers and distributed hand sanitizers to them, which nonetheless constituted an incomplete preventive measure. mass media also attempted to trace and reveal the sources of rumours, in the process causing political conflicts. many schools temporarily cancelled classes, although unnecessarily, so that their students could stay at home. the military isolated infected soldiers in remote facilities, but this move came rather late. korea did not give all four stakeholders equal involvement in dealing with the mers outbreak. rather, one stakeholder e healthcare workers in hospitals e played multiple roles in controlling mers viruses. thus, the situation was perceived as almost entirely a hospital infection control issue. considering that the mers outbreak was not only a health issue but also an emergency management issue, the model for controlling similar epidemics or pandemics in the future-oriented model should involve all stakeholders in an early and co-ordinated response. emergency management consists of four phases: emergency prevention/mitigation (legalization, inspection, disease prediction, etc.), emergency preparedness (emergency operation planning, training, etc.), emergency response (infection control, health treatment, etc.), and emergency recovery (insurance, medical evaluation, etc.). 3 appropriate roles and responsibilities in all the phases have to be assigned to each of the four stakeholders in advance. although many stakeholders tried to play their own roles during the mers outbreak in korea, their responses were somewhat late and unco-ordinated, and thus contributed to the national crisis. in fact, their specific roles and responsibilities should have been assigned before the mers outbreak. in this context, nations should implement regular training in and exercise of emergency management measures. pandemics, as a type of emergency, pose three kinds of risk: loss of human lives, economic damages, and psychological impact. in the case of the mers outbreak in korea, local government and hospitals were oriented toward decreasing the number of deaths, mainly because they regarded the outbreak as an infection control issue. they did not realize the need to address the economic damage or the psychological impact on the general population, especially at the initial response stage. without a co-ordinated response, the mers outbreak caused considerable economic damage in korea. almost nobody dared to visit shopping malls for fear of infection. further, because mers scared away foreign tourists, including many chinese and some japanese visitors, the tourism industry suffered considerably. thus, the national economy was significantly depressed, and the economic growth projection fell to about 2%. 4 regarding the psychological impact, most people worried about catching mers and thus wore masks whenever they went out. similarly, many primary and middle schools throughout the peninsula cancelled classes to protect their students from mers, contrary to the recommendation of the world health organization. hospitals played a major role in reducing the loss of human lives during the mers outbreak; however, this does not mean that they have done extraordinary work. after pyeongtaek st mary's hospital failed to screen the first infected patient, mers viruses spread to many parts of korea. further, the samsung medical center in seoul refused to share mers information with the public, which made the situation worse. hence, hospitals need to be more professional in dealing with infection control, in particular by educating quarantine doctors and in following the hippocratic oath. 5 the kcdc and mw will certainly remain the major government institutions that should take charge when a pandemic occurs in korea. however, the mpss must also become proactively involved during such an outbreak. thus far, the mpss officially considers only three types of hazard under its management scope: fires, floods that accompany typhoons, and maritime accidents. as a co-operative or co-ordinating institution in relation to all hazards, the mpss must therefore extend its activities to the kcdc, other departments, and local government, and apply countermeasures against new diseases. contrary to the expectation of the government, the level of emergency awareness of korean residents increased considerably during the mers outbreak, particularly as the death toll rose. these residents should now directly demand that the government and the whole nation take more systematic actions against new pandemics toward achieving efficient emergency management. the public must also be willing to challenge cultural practices, and in particular to co-operate with restrictions on visiting hospitals. other stakeholders, including business establishments, mass media, schools, and the military played their own roles in responding to the outbreak of mers within their areas. however, they should have approached the issue more seriously from the beginning of the emergency response instead of merely acting as outsiders in a national crisis. considering that a pandemic may spread quickly to anyone, these entities should join the major stakeholders from the initial response stage. the mers outbreak in korea in may to july 2015 caused the biggest loss of human lives due to the disease outside the middle east. thus far, however, the korean government has yet to comprehensively improve its national response against pandemics. the key tenet is that korea must not consider the mers outbreak to be a hospital infection control issue. rather, the nation must regard such an outbreak as an emergency management issue involving all stakeholders, particularly in fighting against new pandemics in the international community. middle east respiratory syndrome e advancing the public health and research agenda on mers e lessons from the south korean outbreak federal emergency management agency. introduction to continuity of operations planning for pandemic influenzas mers in south korea and china: a potential outbreak threat? what can we learn from mers outbreak in south korea? none declared. none. key: cord-270258-9vgpphiu authors: ko, jae-hoon; park, ga eun; lee, ji yeon; lee, ji yong; cho, sun young; ha, young eun; kang, cheol-in; kang, ji-man; kim, yae-jean; huh, hee jae; ki, chang-seok; jeong, byeong-ho; park, jinkyeong; chung, chi ryang; chung, doo ryeon; song, jae-hoon; peck, kyong ran title: predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients date: 2016-08-09 journal: j infect doi: 10.1016/j.jinf.2016.08.005 sha: doc_id: 270258 cord_uid: 9vgpphiu background: after the 2015 middle east respiratory syndrome (mers) outbreak in korea, prediction of pneumonia development and progression to respiratory failure was emphasized in control of mers outbreak. methods: mers-cov infected patients who were managed in a tertiary care center during the 2015 korean mers outbreak were reviewed. to analyze predictive factors for pneumonia development and progression to respiratory failure, we evaluated clinical variables measured within three days from symptom onset. results: a total of 45 patients were included in the study: 13 patients (28.9%) did not develop pneumonia, 19 developed pneumonia without respiratory failure (42.2%), and 13 progressed to respiratory failures (28.9%). the identified predictive factors for pneumonia development included age ≥45 years, fever ≥37.5 °c, thrombocytopenia, lymphopenia, crp ≥ 2 mg/dl, and a threshold cycle value of pcr less than 28.5. for respiratory failure, the indicators included male, hypertension, low albumin concentration, thrombocytopenia, lymphopenia, and crp ≥ 4 mg/dl (all p < 0.05). with ≥ two predictive factors for pneumonia development, 100% of patients developed pneumonia. patients lacking the predictive factors did not progress to respiratory failure. conclusion: for successful control of mers outbreak, mers-cov infected patients with ≥ two predictive factors should be intensively managed from the initial presentation. middle east respiratory syndrome (mers) is an emerging lethal respiratory disease caused by a novel betacoronavirus (mers-cov). 1 from may to july 2015, there was a hospital-associated mers outbreak in the republic of korea reporting 186 laboratory-confirmed cases, which is the largest recorded outbreak outside the arabian peninsula. 2e6 the outbreak featured several super-spreading events with unexpectedly high human-to-human transmission rate: 136 of 186 cases (73.1%) were transmitted from only three patients. 5,7e10 as these large transmission clusters were exclusively originated from patients with pneumonia, prediction of pneumonia development has been emphasized in control of mers outbreak. 9 in addition, pneumonia progression to respiratory failure should be anticipated in advance to avoid urgent intubation or cardiopulmonary resuscitation which might break protection of healthcare workers. although several studies analyzed prognostic factors for fatal outcome, 11e16 predictive factors for pneumonia development and progression to respiratory failure have not been reported. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated mers-cov infected patients managed in a tertiary care center during the 2015 mers outbreak in korea. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we reviewed the electronic medical records of who were diagnosed with mers-cov infection and admitted at samsung medical center, a 1950 tertiary care university hospital which managed the largest number of mers-cov infected patients as a single center during the 2015 korean mers outbreak. as it is still unclear whether initially asymptomatic patients would develop pneumonia or not, 17 we included all the mers-cov infected patients managed at our hospital during the outbreak regardless of symptoms presence. to avoid confusion with the case definition of mers which did not included asymptomatic cases, 18 we used the term of 'mers-cov infected patient' rather than 'mers patients' throughout the present paper. mers-cov infections were confirmed on the basis of rrt-pcr assays targeting upstream of the e gene and the open-reading frame gene 1a. 18, 19 disease status of included patients was assessed at six weeks from their symptom onset and patients were divided into three groups depending on pneumonia development and progression to respiratory failure: patients without the development of pneumonia (group 1), patients who developed pneumonia without respiratory failure (group 2), and pneumonia patients who progressed to respiratory failure (group 3). for practical purposes, respiratory failure was defined as the need for mechanical ventilation (mv). the institutional review board of our hospital approved the present study. we retrospectively collected data from electronic medical records and epidemiologic investigation. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated clinical variables measured within three days from symptom onset. during the 2015 korean mers outbreak, fever was defined as body temperature !37.5 c to increase sensitivity of screening and the same definition was used in the present analysis. 9 thrombocytopenia was defined as a platelet count lower than 150 â 10 3 cells/mm 3 , lymphopenia as an absolute lymphocyte count lower than 1,000 cells/mm 3 , and hypoalbuminemia as albumin concentration lower than 3.5 g/dl. lower respiratory tract specimens including sputum and endotracheal aspirates were used for mers-cov rrt-pcr. cycle threshold (ct) values of mers-cov rrt-pcr were used as a surrogate of viral load. pneumonia development of mers-cov infected patient was defined as presence of parenchymal infiltration on chest x-ray with respiratory symptoms. test days or events were counted from the day of symptom onset for each patient: day 1 was defined as the day of symptom onset. for asymptomatic patients, the day of diagnosis of mers-cov infection was considered as day 1. to identify predictive factors for pneumonia development and progression to respiratory failure, clinical variables measured within three days from symptom onset were compared. for evaluation of pneumonia development, patients who developed pneumonia (group 2 and 3) were compared to those who did not (group 1). for factors for respiratory failure, patients who progressed to respiratory failure (group 3) were compared to those who did not (group 1 and 2). student's t-tests or mannewhitney u tests were used to compare continuous variables, and chi-square tests or fisher's exact tests were used to compare categorical variables. statistically significant continuous variables were re-categorized into binary factors using threshold values between mean of each group, which showed lowest p value. statistically significant categorical variables and binary factors re-categorized from continuous variables were defined as predictive factors. for significant predictive factors, as a measure of association, odds ratio (or) and 95% confidence interval (ci) for or were calculated using the woolf procedure. 20 multivariate analysis was not performed due to the limited sample size. all pvalues were two-tailed, and those <0.05 were considered to be statistically significant. ibm spss statistics version 20.0 for windows (ibm, armonk, ny, usa) was used for all statistical analyses. time course of pneumonia development and progression to respiratory failure a total of 45 mers-cov infected patients were hospitalized during the outbreak with 13 patients in group 1 (including 3 asymptomatic patients), 19 patients in group 2, and 13 patients in group 3. the clinical course of symptomatic mers patients progressed serially: patients developed initial symptoms after a median 5-day incubation period (iqr 3.5e7.0), pneumonia after a median of 6 days from symptom onset (iqr 5.0e7.0), and respiratory failure after a median of 12 days from symptom onset (iqr 10.0e13.0). in group 3 patients, it took a median of 2 days from desaturation to respiratory failure (iqr 1e3 days). the development and progression of pneumonia by time sequence is depicted in fig. 1 . no one developed pneumonia before day 4 of symptom onset. to evaluate predictive factors for pneumonia development, demographics, underlying diseases, and clinical variables of patients in group 2 and 3 were compared to those of patients in group 1 (tables 1 and 2 ). identified predictive factors are summarized in table 3 with odd ratios (or). increasing age was significantly associated with pneumonia development as a continuous variable (p z 0.015), and age older than 45 years was a predictive factor for the development of pneumonia (or, 8.04; 95% ci, 1.52e42.43; p z 0.007). although proportion of male also increased with progression of pneumonia (38.5%, 57.9%, and 84.6% for group 1, 2, and 3, respectively), statistically significant association between male sex and the pneumonia development was not identified (p z 0.097). fever over 37.5 c by day 3 were more frequently detected in patients with pneumonia (18.2%, 71.4%, and 77.8% in groups 1, 2, and 3, respectively), and was identified as a predictive factor for the development of pneumonia (or, 12.75; 95% ci, 2.12e76.57; p z 0.002). thrombocytopenia (or, not applicable (na); p z 0.007), lymphopenia (or, 17.50; 95% ci, 1.88e163.02; p z 0.003), elevated c-reactive protein (crp ! 2 mg/dl; or, na; p z 0.018), and high viral load (ct value < 28.5; or, 14.00; 95% ci, 1.14e172.65; p z 0.024) were distinctly observed in pneumonia patients from the initial presentation, and identified as predictive factors for pneumonia development. to evaluate predictive factors for progression to respiratory failure, patients in group 3 were compared to those in group 1 and 2 (tables 1e3). although the mean age of patients in each group tended to increase with progression of pneumonia (37.3, 47.7, and 55.2 years in groups 1, 2, and 3, respectively) and increasing age was significantly associated with respiratory failure as a continuous variable (p z 0.036), there was no statistically significant cut-off value for prediction of respiratory failure. proportion of male also increased with progression of pneumonia, and male sex was a predictive factor for respiratory failure (or, 5.50; 95% ci, 1.05e28.88; p z 0.045). among underlying diseases, hypertension was identified as a predictive factor for respiratory failure (or, 6.04; 95% ci, 1.18e30.88; p z 0.021). initial symptoms including fever were not significantly different between patients who progressed to respiratory failure and those who did not. among initial laboratory test results, thrombocytopenia (or, 6.67; 95% ci, 1.18e37.78; p z 0.023), lymphopenia (or, 14.88; 95% ci, 1.56e142.20; p z 0.006), hypoalbuminemia (or, 14.17; 95% ci, 1.83e109.86; p z 0.005), and elevated crp (crp ! 4 mg/ dl; or, 23.00; 95% ci, 2.01e262.57; p z 0.002) were distinctly observed in group 3 patients, and identified as predictive factors for respiratory failure. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) by number of predictive factors were presented in table 4 . when patients presented with ! two of the predictive factors for pneumonia development, 100% of these patients developed pneumonia (sensitivity 56.3%, specificity 100.0%, ppv 100%, and npv 48.1%). patients lacking the predictive factors for respiratory failure did not progress to respiratory failure. when patients presented with ! two of these predictive factors, 50.0% of these patients progressed to respiratory failure (sensitivity 69.2%, specificity 75.0%, ppv 52.9%, and npv 85.7%). initial rapid propagation of mers-cov during the korean mers outbreak was caused by three super-spreading events responsible for 73.1% of all transmissions. 5, 7, 8 in addition to these super-spreaders, transmission of mers-cov despite application of personal protective equipment (ppe) occurred from patients with progressed pneumonia at our hospital. 9 in this regard, identifying the predictive factors for pneumonia development and progression is not only important in patient care, but also in infection control to prevent further in-hospital transmission. the present analysis of predictive factors for pneumonia development and progression to respiratory failure using variables obtained by day 3 of symptom onset could be conducted owing to the observation of entire clinical course of the disease from the exposure to mers-cov. compared to mers outbreaks in the arabian peninsula where community-acquired infections might simultaneously occur from animals, identifying epidemiologic links, exposure date, and symptom onset were relatively clear for each case. 5, 19 in our observation, the clinical course of symptomatic mers patients progressed serially and no one developed pneumonia before day 4 of symptom onset. this is the reason why we used clinical data obtained by day 3 of symptom onset. there is no other comparable data to which presented time interval from the symptom onset to the development of pneumonia. although there were no ideal cut-off scores of predictive factors with good sensitivity and specificity, it should be noted that 100% of patients with ! two predictive factors for data are expressed as the number (%) of patients or mean ae sd. as missing values were also removed from the population parameter, variables with missing values are expressed with modified population parameters. continuous variables with statistical significance were re-categorized into binary factors which are presented in italics. abbreviations: res., respiratory; wbc, white blood cell; alc, absolute lymphocyte count; ast, aspartate transaminase; alt, alanine transaminase; bun, blood urea nitrogen; crp, c-reactive protein; ld, lactate dehydrogenase; ct, threshold cycle; rrt-pcr, real-time reverse transcriptase polymerase chain reaction. a data are presented as mean value of day 1e3 ae sd. pneumonia actually progressed to pneumonia. thus, careful and intensive management should be implemented for such patients including adequate isolation of patient in an airborne infection isolation room (aiir), minimizing chance for exposure, application of ppe with hooded coverall, and consideration of experimental antiviral treatment. 9,21e24 for patients with ! two predictive factors for respiratory failure, aiirs in intensive care units should be prepared for early elective intubation. although the time interval from symptom onset to mv support was much longer than in previous reports (median 12 days versus 7 days), 1 we also experienced rapid progression of pneumonia from the moment of desaturation: 73% of group 3 patients required mv within 2 days from desaturation (median 2, iqr 1e3 days). to avoid urgent situations which might break protection of healthcare workers, elective intubation should be considered when desaturation begins to progress. in addition, sensitivities of predictive values are relatively low with cut-off value of ! two factors, clinical course of patients with any predictive factors also should be carefully monitored. of note, thrombocytopenia, lymphopenia, and increased crp level were shared predictive factor for the pneumonia development and respiratory failure. they were observed in the very early course of the illness, indicating that inflammation had already been enhanced. lymphopenia and thrombocytopenia presenting from the initial presentation of severe mers-cov infected patients were also observed in the recent report by min et al. 25 although time of measurements were not specifically described, these laboratory abnormalities were previously observed in severe mers cases and other respiratory viral illnesses including severe acute respiratory syndrome (sars) and influenza, which are caused by intense inflammatory response to the viruses. 15,26e33 this is the first report that identified these laboratory findings as predictive factors for pneumonia development and progression to respiratory failure in mers. although other predictive factors for pneumonia development and respiratory failure were different due to discordance of statistical significance, they shared the same spectrum of etiology. age increased according to pneumonia progression and was associated with both pneumonia and respiratory failure as a continuous variable (p z 0.015 and p z 0.036, respectively). these findings correlate with previous data suggesting that old age is associated with poor prognosis. 11,13e15,34,35 similarly, the proportion of males increased according to disease severity, though male sex was only significant for predicting respiratory failure. although the mean age of males was older than that of females (49.7 and 42.6 years, respectively), it was not statistically significant (p z 0.169). previous data also reported that overall proportion of male was higher and was associated with severe infection. 15, 34 it could be meaningful observation that the same finding was observed in the republic of korea where the social activity of females is not restricted, especially among healthcare workers. on the other hand, hypoalbuminemia and hypertension were predictive factors only for respiratory failure, while high viral load was predictive factor for the development of pneumonia. these factors were related with severe disease and poor prognosis of mers in previous reports. 1, 12, 13, 15, 16, 34, 35 our study has several strengths and limitations. due to its retrospective nature, there may be a bias regarding collecting medical information in retrospective manner. however, as all electronic medical records were standardized to record symptoms and signs in the same way from the beginning of the outbreak, bias was minimized. secondly, there were missing values when calculating the sensitivity and specificity of predictive factors, which is another limitation of retrospective study. lastly, the present study did not perform multivariate analysis due to limited sample size and need to be validated. prospective studies with sufficient number of patients are required for validation of the predictive factors identified in the present study. despite these limitations, our data would be suitable for identifying predictive factors because we could observe entire course of the disease from exposure and apply homogenous management to patients. in conclusion, based on 45 cases from a single tertiary care hospital during the largest mers outbreak outside of the arabian peninsula, we identified six predictive factors for the development of pneumonia and progression to respiratory failure, respectively. thrombocytopenia, lymphopenia, and high crp level were shared predictive factors. mers-cov infected patients with ! two predictive factors should be intensively managed from the initial presentation for successful control of mers outbreak. there are no potential conflicts of interest relevant to this article to report. this work was supported by the samsung biomedical research institute (sbri) grant [#smx1161321]. middle east respiratory syndrome summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus (mers-cov) e republic of korea. world health organization an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea preliminary epidemiological assessment of mers-cov outbreak in south korea identifying determinants of heterogeneous transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, 2015: a retrospective epidemiological analysis transmission characteristics of mers and sars in the healthcare setting: a comparative study control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection association of higher mers-cov virus load with severe disease and death, saudi arabia mortality risk factors for middle east respiratory syndrome outbreak, south korea clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia management of asymptomatic persons who are rtpcr positive for middle east respiratory syndrome coronavirus (mers-cov), interim guidance. world health organization case definition and management of patients with mers coronavirus in saudi arabia mers-cov outbreak in jeddah e a link to health care facilities on estimating the relation between blood group and disease treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia treatment strategies for middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov), us cdc comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong white cell differential count and influenza a thrombocytopenia in patients with severe acute respiratory syndrome (review) platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis discovery of t cell infection and apoptosis by mers coronavirus middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates both the extrinsic and intrinsic apoptosis pathways epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease we would like to express our sincerest condolences to the patients and families who suffered from the mers outbreak. we also greatly appreciate the health care personnel and staff members at samsung medical center and all other hospitals who worked together to overcome the mers outbreak. key: cord-260420-4s7akmdp authors: mubareka, samira; groulx, nicolas; savory, eric; cutts, todd; theriault, steven; scott, james a.; roy, chad j.; turgeon, nathalie; bryce, elizabeth; astrakianakis, george; kirychuk, shelley; girard, matthieu; kobinger, gary; zhang, chao; duchaine, caroline title: bioaerosols and transmission, a diverse and growing community of practice date: 2019-02-21 journal: front public health doi: 10.3389/fpubh.2019.00023 sha: doc_id: 260420 cord_uid: 4s7akmdp the transmission of infectious microbes via bioaerosols is of significant concern for both human and animal health. however, gaps in our understanding of respiratory pathogen transmission and methodological heterogeneity persist. new developments have enabled progress in this domain, and one of the major turning points has been the recognition that cross-disciplinary collaborations across spheres of human and animal health, microbiology, biophysics, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. collaborative initiatives support advances in topics such as bioaerosol behavior, dispersion models, risk assessment, risk/exposure effects, and mitigation strategies in clinical, experimental, agricultural, and other field settings. there is a need to enhance the knowledge translation for researchers, stakeholders, and private partners to support a growing network of individuals and agencies to achieve common goals to mitigate interand intra-species pathogen transmission via bioaerosols. the transmission of infectious microbes via bioaerosols is of significant concern for both human and animal health. however, gaps in our understanding of respiratory pathogen transmission and methodological heterogeneity persist. new developments have enabled progress in this domain, and one of the major turning points has been the recognition that cross-disciplinary collaborations across spheres of human and animal health, microbiology, biophysics, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. collaborative initiatives support advances in topics such as bioaerosol behavior, dispersion models, risk assessment, risk/exposure effects, and mitigation strategies in clinical, experimental, agricultural, and other field settings. there is a need to enhance the knowledge translation for researchers, stakeholders, and private partners to support a growing network of individuals and agencies to achieve common goals to mitigate inter-and intra-species pathogen transmission via bioaerosols. keywords: bioaerosols, microbes, virus, infections, viral dissemination, network, caniban, collaborations the importance of infectious bioaerosols in disease transmission has been long-acknowledged, yet poorly understood. paltry data and methodological heterogeneity limit many related studies. effective ventilation and infection prevention and control (ip & c) measures in the form of droplet and airborne isolation in healthcare institutions underscore the contribution of these modes of pathogen dispersion. moreover, recent outbreaks such as the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) outbreaks highlight major gaps in our ability to assess and determine risk and to mitigate patient and healthcare worker (hcw) exposure alike (1, 2) . the sars outbreak was eventually controlled in the absence of an effective vaccine or antiviral, strictly through public health interventions and ip & c measures aimed at controlling, among other things, bioaerosols emitted by infected patients (3) . a decade on from this experience, mers-cov has caused community and healthcare-associated severe acute respiratory infections in the middle east and south korea, spreading by similar mechanism(s) (4) (5) (6) . from an animal health perspective, pathogens such as porcine reproductive and respiratory syndrome virus may be transmitted through the air for significant distances and have significant economic impact on the agricultural sector (7) . efforts to characterize this and other relevant organisms have been undertaken previously, specifically looking at the burden of inoculum related to transmission (8) and the prevention of spread of aerosols through various adequate ventilation strategies (9) (10) (11) . emission of african swine fever virus into the air by infected pigs raises the possibility for droplet and/or airborne transmission of this virus, which has recently produced significant alarm after documented spread in china (12) (13) (14) . this hemorrhagic virus is associated with high mortality and significant loss for producers through depopulation and trade restrictions (15, 16) . in recent years, new developments have enabled progress in bioaerosol research, thus establishing a community of practice in the field. although many developments have been technical, one of the major catalysts has been the recognition that crossdisciplinary collaborations across the various spheres of human and animal health, microbiology, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. a network approach has proven successful in other cross-disciplinary fields, including one health and eco-health whereby wildlife, computational and evolutionary biologists, microbiologists, virologists, epidemiologists, ecologists, environmental scientists, climatologists, and human, animal, and public health practitioners are collaborating to address challenges in zoonotic diseases research and control (17, 18) . this has enhanced surveillance efforts and launched ambitious, large scale projects such as the global virome project, though gaps remain in stakeholder engagement and monitoring activities (19, 20) . translation of bioaerosol research stands to benefit in a similar fashion, provided coordinated and committed efforts. potentially infectious bioaerosols are pertinent to a wide range of pathogens, some of which may be endemic and cause sporadic infections and outbreaks (e.g., mycobacterium tuberculosis and legionella pneumophila, the causative agents of tuberculosis and legionnaires' disease, respectively) and/or have potential to cause epidemics or pandemics (e.g., influenza virus a) (21) (22) (23) (24) . in addition, the importance of potentially infectious bioaerosols across different settings is underscored. these include, but are not limited to, agriculture (both crop and livestock), wastewater treatment plants, environmental reservoirs (soil and water), acute and long-term healthcare institutions, and shared public spaces (transportation hubs, recreational areas, etc.). here, we discuss the state of the science for this nascent field and identify gaps requiring urgent attention. progress in the field has been stimulated by advances in other areas which have been applied to the study of infectious bioaerosols. these include: (a) enhanced detection by molecular methods, principally real-time and quantitative pcr, nextgeneration sequencing, metagenomics, and biosensors (25) (26) (27) (28) (29) (30) , and (b) establishment of both conventional and novel infrastructure, such as small and large-scale wind tunnels, biocontained rotating drums for aging aerosols, and field-ready aerosol samplers (31) (32) (33) . ongoing research has also facilitated the development and dissemination of procedures and protocols for experimental work, including artificial aerosols, as well as animal models of transmission including the ferret model for influenza virus transmission and macaque model for ebola virus transmission (34, 35) . whilst aerosols can be used as a means of delivery of therapeutic agents directly to the respiratory tract, they may also be used for the nefarious dispersion of human or animal pathogens. scientists focused on bioaerosol generation, pathogen survival in air and aerobiological fitness must be acutely cognizant of any potential for dual use and routinely re-assess projects and proposed experiments with this perspective in mind. oversight by institutional biosafety officers and committees may also consider this aspect of bioaerosol research during risk assessments and other internal approval processes. given the role of potentially infectious bioaerosols to transmit infectious agents to human and animal populations, it stands to reason that their detection and characterization will ultimately contribute to mitigating the spread of disease. this is possible through efforts focused on the following themes: unresolved fundamental questions may be answered by studying infectious bioaerosols. areas meriting attention are numerous and a few are briefly discussed here. in the case of agents for which airborne transmission is well-recognized (i.e., m. tuberculosis), associations between particle size and generation, pathogen content and virulence, as well as the deposition within the respiratory tract may be determined. the physical and chemical aspects of aerosols including the effects of relative humidity can affect pathogen viability and understanding these aspects of bioaerosol behavior may be beneficial to understanding conditions for controlling bioaerosol dispersion (36) (37) (38) . moreover, pathogens such as viruses may vary with respect to isoelectric points, possibly imparting different charges to infectious bioaerosols which may affect their behavior (39, 40) . finally, although climate change and pollution have been major issues within our biosphere, little is known about the impact of pollution particulates and gases, such as ozone, on the biology and physical properties of bioaerosols (41) . our understanding of bioaerosol production from expulsion events such as breathing, talking, sneezing, and coughing have generally been extrapolated from models, though more recent empiric evidence has become available, significantly enhancing our understanding of the transmission potential of bioaerosol emissions from naturallyinfected hosts (42, 43) . awareness of factors contributing to particle velocity and penetration into space enables modeling strategies that inform engineering controls in a multitude of settings. for example, in healthcare, understanding the dispersion of potential pathogens in the environment can inform infection prevention and control practices (44) . there is also potential benefit to determining pathogen characteristics. if enhanced infectious bioaerosol survival in air is associated with certain strains, genotypes or mutations, this provides possibilities for follow-on work to (a) determine the mechanism and (b) enhance surveillance. the latter would have implications for public and animal health. studying these factors individually or in combination can optimize air handling and other mechanical, environmental or chemical means of neutralizing bioaerosols prior to host exposure, thus alleviating dependence on personal protective equipment, which is the last means of protection prior to exposure. new understanding of the role of bioaerosols in the propagation of human and animal pathogens could lead to new practices. as it stands, it remains uncertain which bioaerosol particles are predominantly responsible for personto-person transmission. there may also be differences between pathogen populations in the infected host vs. what is emitted into the air. this poses an excellent opportunity to leverage advances in metagenomics with developments in aerobiology to extract more sophisticated data from aerosol samples and, potentially, determine genetic bottlenecks for transmission. this would feed back into more fundamental work on bacterial or viral determinants of transmission, as well as lead to novel means for surveillance and mitigation. similarly, translational studies enhance our understanding of risk and determinants of exposure. for many settings and situations, several components of risk assessments are based on limited evidence. this is of particular concern from an occupational health perspective, in healthcare (e.g., purported aerosol-generating procedures), agricultural, or specific settings such as wastewater treatment plants (45) (46) (47) . developing the capacity to generate empiric data on pathogen content and biology from bioaerosols can lead to tools for outbreak investigation, surveillance, and development of risk assessment strategies and policies. as the technology for biosensors accelerates, the possibilities for rapid point-of-care testing and even remote sampling are highlighted. integration of biosensors with bioaerosol sampling (26) has significant potential for early warning and public safety. as advances are made, capacity is built to optimize and evaluate known and novel means to control the dispersion of infectious bioaerosols. these include the use of germicidal and pulsed ultraviolet light, mechanical air filtration and respiratory protection which have both mechanical and electrostatic filtering systems (48, 49) . in addition, the potential to use ozone to control indoor bioaerosols in urban and rural settings is being examined. ozone is a strong oxidizing agent having high redox potential and a short half-life, dependent on temperature. it has been used extensively in the food industry (specifically water treatment, washing of produce, and food preparation), as well as in domestic restoration to remediate odors and smoke damage. its historical use and proven efficacy demonstrate its potential to remediate contaminated indoor air (50, 51) . this would present a novel application of a known treatment modality. a number of important gaps have been identified (52) and these can be prioritized based on potential benefit: biocontainment of infectious particles is the first consideration when aerosolizing human and/or animal pathogens in an experimental setting. these systems are generally customdesigned and constructed, with few facilities capable of conducting aerosol work with risk group 2 pathogens, and fewer still with the capacity to work with risk group 3 or 4 agents. as challenges in infrastructure are overcome, opportunities to optimize and improve methods and techniques in bioaerosol research must be taken. a limitation of many studies focused on viral bioaerosols is the pervasive use of nucleic acid detection, rather than infectious virus isolation. the latter is a much more accurate indicator of the infectious potential of a bioaerosol but is infrequently performed due to poor sensitivity and other technical issues (53) . the development of sampling devices and techniques optimized to preserve pathogen viability would considerably advance the utility of studying bioaerosols for risk assessment and management. for other applications, a more rapid, field-ready point of care test would be useful and would offer remote sampling possibilities. biosensors have the potential to fill this gap and integration into aerosol sampling devices is under development (26) . finally, the collection of nucleic acid may be leveraged to obtain more sophisticated information than is available by pcr and sanger sequencing. metagenomics on environmental samples has been well-described in other spheres and is currently being explored for air samples (54) (55) (56) (57) . to advance bioaerosol-related data interpretation, an effort must be made to standardize and share protocols and reagents to reduce the degree of data heterogeneity to enable meaningful analyses across studies. this may apply to animal models, artificial aerosolizations, collection strategies in the field and clinical settings, processing of samples, and detection methods. developing or adopting a standardized approach to aerosol sampling is challenging and requires substantial efforts to select the best sampling strategy to minimize sampling biases, optimize sample concentration and organism retrieval whilst preserving integrity (58) . establishing standard approaches also enables training and implementation and eases knowledge translation amongst different groups (figure 1) . multiple investigators have published small studies on the recovery of viral rna emitted by naturally infected humans using different approaches for recruitment, sampling, processing, and detection. unfortunately, the results are difficult to compare and, standing alone, are of limited statistical significance (45, (59) (60) (61) . by standardizing approaches, a more feasible method can be used to compare separate studies allowing for larger multi-center studies that are substantially more conclusive and impactful. additional educational and operational needs, such as training of research personnel, proper use of personal protective equipment and developing decontamination protocols must be addressed. as more highly qualified personnel are properly trained, the greater the capacity for designing and utilizing experimental systems and for completing meaningful clinical and field studies. the need for cross-disciplinary experience is underscored, since robust knowledge of physics, mechanical engineering, and microbiology are required. fostering this expertise requires a collaborative effort as each discipline offers unique insight. currently, there are a limited number of workshops or accredited courses available to cultivate both interest and baseline knowledge. although the profile of bioaerosol research is rising, few trainees are exposed to the field early in their careers. courses such as bioaã©rosols et aã©robiologie (bioaerosols and aerobiology) developed at the universitã© laval in quã©bec may help close this gap further given the opportunity to expand reach. the relevance of bioaerosol data to occupational health and infection prevention and control requires attention. validation of bioaerosol data for risk assessments and risk management is needed. air sampling was conducted during both sars-cov and mers-cov outbreaks, accruing important insight into the environmental distribution and persistence of these coronaviruses (4, 62, 63) . the absence of data for more common, lower consequence pathogens represents missed opportunities to build a contextual and impactful body of knowledge during inter-outbreak and inter-pandemic periods. to develop evidence-based policy and ensure the relevance of this work, early stakeholder engagement is needed. consultation with human and animal public health, infection prevention and control, industrial hygiene, and occupational health stakeholders will ensure that the work is germane to the challenges presented by bioaerosol exposures. this pull also increases the likelihood that there will be data generated for risk assessment, policy development and implementation. part of the chaos that characterized the sars epidemic can be attributed to a lack of knowledge regarding the route(s) of transmission. this remains true for high consequence coronaviruses, as well as for a range of other respiratory pathogens, both novel and established. we propose the following to address the challenges outlined above and to further develop the field of applied bioaerosol research: 1. an open network approach working in isolation, microbiologists, aerobiologists, engineers, and epidemiologists have made only incremental progress. a synergistic and pluralistic approach is required for action-driven research which effects change. as capacity grows, so will opportunities for training and response. a networked approach will lead to context-specific follow-on research to ultimately inform policy for the mitigation of respiratory pathogens spread in healthcare institutions, agricultural settings and public spaces. toward this end, the present authors have established the canadian infectious bioaerosols network (caniban) that brings together members of each of these disciplines with the primary objectives of understanding transmission of pathogens. a network would form a hub around which key resources such as infrastructure, equipment, and operational procedures could be shared, thus also enabling training and underscoring best practices in biosafety and biosecurity. shared resources may encompass wind tunnels, cough chambers and mannequins, animal exposure and other biosafety enclosures, and rotating drums to examine pathogen survival in air, together with instrumentation and analysis tools for fluid flow measurement and biochemical assays. limited awareness and understanding of infectious bioaerosols among potential knowledge users, coupled with equally limited outreach by bioaerosol researchers has restricted knowledge transfer and applications. knowledgeuser engagement in research planning from the outset and ongoing involvement through to dissemination is key for effective projects. potential knowledge users include infection prevention and control practitioners, infectious disease specialists, veterinarians and animal health epidemiologists, industrial hygienists, and public health agencies. increasingly, bioaerosol sampling has been proposed and implemented in outbreak investigations and pathogen surveillance using ad hoc approaches. the development of best practices in these areas is essential to generating valid and actionable data. in summary, a collective path for researchers, stakeholders, and private partners is needed to support a network of individuals and agencies to achieve common goals. as the field of bioaerosol studies grows, applications will diversify, along with novel technologies for remote sampling and point of care testing yielding results in real time. this stands to mitigate the spread of nefarious pathogens and contribute to early warning and response measures, thus ultimately benefiting human and animal health. sm framed the manuscript and contributed content. ng contributed content and generated the figure. es contributed content and supported manuscript organization. tc, st, js, cr, nt, eb, ga, sk, mg, gk, and cz contributed content and expertise and cd helped to frame and contributed content. transmission characteristics of mers and sars in the healthcare setting: a comparative study extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards research gaps in protecting healthcare workers from sars and other respiratory pathogens: an interdisciplinary, multi-stakeholder, evidence-based approach environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study mers coronavirus outbreak: implications for emerging viral infections genetic diversity of prrs virus collected from air samples in four different regions of concentrated swine production during a high incidence season the use of bioaerosol sampling for airborne virus surveillance in swine production facilities: a mini review use of a production region model to assess the efficacy of various air filtration systems for preventing airborne transmission of porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae: results from a 2-year study an evaluation of interventions for reducing the risk of prrsv introduction to filtered farms via retrograde air movement through idle fans epidemiological study of air filtration systems for preventing prrsv infection in large sow herds quantification of airborne african swine fever virus after experimental infection molecular characterization of african swine fever virus, china emergence of african swine fever in china inferring within-herd transmission parameters for african swine fever virus using mortality data from outbreaks in the russian federation african swine fever: an unprecedented disaster and challenge to china climate change and health: transcending silos to find solutions pathways to zoonotic spillover the global virome project the growth and strategic functioning of one health networks: a systematic analysis bioaerosol production by patients with tuberculosis during normal tidal breathing: implications for transmission risk evaluation of legionella air contamination in healthcare facilities by different sampling methods: an italian multicenter study health risks from exposure to legionella in reclaimed water aerosols: toilet flushing, spray irrigation, and cooling towers healthcare personnel exposure in an emergency department during influenza season challenges of studying viral aerosol metagenomics and communities in comparison with bacterial and fungal aerosols biosensors for monitoring airborne pathogens first metagenomic survey of the microbial diversity in bioaerosols emitted in waste sorting plants sampling and detection of airborne influenza virus towards point-of-care applications bioaerosol sampling for respiratory viruses in singapore's mass rapid transit network metagenomic survey of bacterial diversity in the atmosphere of mexico city using different sampling methods airborne influenza virus detection with four aerosol samplers using molecular and infectivity assays: considerations for a new infectious virus aerosol sampler evaluation of physical sampling efficiency for cyclone-based personal bioaerosol samplers in moving air environments susceptibility of monkeypox virus aerosol suspensions in a rotating chamber influenza a virus transmission via respiratory aerosols or droplets as it relates to pandemic potential detection of ebola virus rna through aerosol sampling of animal biosafety level 4 rooms housing challenged nonhuman primates relationship between humidity and influenza a viability in droplets and implications for influenza's seasonality physico-chemical characteristics of evaporating respiratory fluid droplets isoelectric points of viruses nanoscale aerovirology: an efficient yet simple method to analyze the viral distribution of single bioaerosols the pollution particulate concentrator (popcon): a platform to investigate the effects of particulate air pollutants on viral infectivity influenza virus in human exhaled breath: an observational study infectious virus in exhaled breath of symptomatic seasonal influenza cases from a college community the effect of environmental parameters on the survival of airborne infectious agents exposure to influenza virus aerosols during routine patient care influenza aerosols in uk hospitals during the h1n1 (2009) pandemicthe risk of aerosol generation during medical procedures human viral pathogens are pervasive in wastewater treatment center aerosols respiratory performance offered by n95 respirators and surgical masks: human subject evaluation with nacl aerosol representing bacterial and viral particle size range comparison of aerosol and bioaerosol collection on air filters water and air ozone treatment as an alternative sanitizing technology effect of different disinfectants on bacterial aerosol diversity in poultry houses drivers of airborne human-to-human pathogen transmission challenge of liquid stressed protective materials and environmental persistence of ebola virus. sci rep metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses functional metagenomics of spacecraft assembly cleanrooms: presence of virulence factors associated with human pathogens the metagenomics and metadesign of the subways and urban biomes (metasub) international consortium inaugural meeting report. microbiome whole metagenome profiles of particulates collected from the international space station a next generation sequencing approach with a suitable bioinformatics workflow to study fungal diversity in bioaerosols released from two different types of composting plants distribution of airborne influenza virus and respiratory syncytial virus in an urgent care medical clinic influenza virus emitted by naturally-infected hosts in a healthcare setting quantification of influenza virus rna in aerosols in patient rooms detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units airborne severe acute respiratory syndrome coronavirus concentrations in a negative-pressure isolation room we should like to thank the centre de recherche de l'institut universitaire de cardiologie et de pneumologie de quã©bec (criucpq), assek technology, quebec frqs respiratory health network, the groupe de recherche en santã© respiratoire (geser), and tsi for supporting the 2016 symposium on the transmission of respiratory pathogens in quã©bec city, qc, canada, and symposium participants for their contributions during the course of this event. we are also grateful for support from the canadian institutes of health research, the natural sciences and engineering research council of canada and the ontario ministry of labor. key: cord-278182-75u57fw1 authors: goh, gerard kian-meng; dunker, a. keith; foster, james a.; uversky, vladimir n. title: shell disorder analysis predicts greater resilience of the sars-cov-2 (covid-19) outside the body and in body fluids date: 2020-03-31 journal: microb pathog doi: 10.1016/j.micpath.2020.104177 sha: doc_id: 278182 cord_uid: 75u57fw1 the coronavirus (cov) family consists of viruses that infects a variety of animals including humans with various levels of respiratory and fecal-oral transmission levels depending on the behavior of the viruses' natural hosts and optimal viral fitness. a model to classify and predict the levels of respective respiratory and fecal-oral transmission potentials of the various viruses was built before the outbreak of mers-cov using ai and empirically-based molecular tools to predict the disorder level of proteins. using the percentages of intrinsic disorder (pid) of the nucleocapsid (n) and membrane (m) proteins of cov, the model easily clustered the viruses into three groups with the sars-cov (m pid = 8%, n pid = 50%) falling into category b, in which viruses have intermediate levels of both respiratory and fecal-oral transmission potentials. later, mers-cov (m pid = 9%, n pid = 44%) was found to be in category c, which consists of viruses with lower respiratory transmission potential but with higher fecal-oral transmission capabilities. based on the peculiarities of disorder distribution, the sars-cov-2 (m pid = 6%, n pid = 48%) has to be placed in category b. our data show however, that the sars-cov-2 is very strange with one of the hardest protective outer shell, (m pid = 6%) among coronaviruses. this means that it might be expected to be highly resilient in saliva or other body fluids and outside the body. an infected body is likelier to shed greater numbers of viral particles since the latter is more resistant to antimicrobial enzymes in body fluids. these particles are also likelier to remain active longer. these factors could account for the greater contagiousness of the sars-cov-2 and have implications for efforts to prevent its spread. in december of 2019, a mysterious virus that causes pneumonia and produces pneumonia-like symptoms; i.e., dry cough and fever was noticed to be spreading in wuhan, hubei province, china [1] [2] [3] [4] . the culprit was quickly identified as a new respiratory coronavirus (cov). this virus and its disease are currently known as severe acute respiratory syndrome coronavirus-2 (sars-cov-2 and coronavirus infectious disease 2019 (covid-19) respectively [1] [2] [3] [4] . sars-cov-2 is observed to be even more contagious (but fortunately less fatal) than sars-cov that was seen in 2003. in 2011-2012, just before the outbreak of the middle eastern respiratory syndrome (mers-cov), we built an empirically-based model that measures the percentage of intrinsic disorder (pid) of the membrane (m) and nucleocapsid (n) proteins in viruses [5, 6] . the main tool uses ai technology to recognize intrinsic disorder, given the protein sequence. the model involves the listing of shell disorder by coronaviruses that is easily clustered into three groups, which incidentally correlate with the known levels of fecal-oral and respiratory transmission of the various coronaviruses. the model predicts that the 2003 sars-cov (m pid = 8% and n pid = 50%) is included into a category of covs with intermediate levels of both respiratory and fecal-oral transmission potentials. an opportunity to test the validity of this model came when mers-cov first struck the middle east in 2012. upon the availability of the sequences of both mers-cov m and n proteins, the authors were able to confirm that mers-cov falls solidly into a category of viruses that have lesser respiratory transmission potential but have greater fecaloral transmission capabilitie [6, 7] . the categorization of mers-cov within this category was supported by the observations that mers-cov https://doi.org/10.1016/j.micpath.2020.104177 received 17 february 2020; received in revised form 18 march 2020; accepted 27 march 2020 is not efficiently spreading among humans, unlike sars-cov, and requires close contact [8] . furthermore, it is now known that camels are the natural reservoir for mers-cov, and viruses that are associated with farm animals, such as camels, tend to have greater fecal-oral transmission potentials [9] . in this study, we will have the opportunity to once again test the model. this time, it will involve the sars-cov-2. we shall see that not only the model is reliable and consistent, but is able to detect something very odd about this virus that could account for its quick spread. protein intrinsic disorder is found when portions of or an entire protein has no structure. it is also called by other names such as unstructured, natively unfolded [10] [11] [12] . the main tool used to develop the covs shell disorder model involves the use of neural networks that had been trained to recognize ordered and disordered regions of a protein given its sequence. a suite of such artificial intelligence tools (ai) have been named pondr® (http://www.pondr.com). of a particular interest. pondr® vlxt has been found to be accurate for structural proteins with protein-protein and protein-rna/dna interactions [13] [14] [15] [16] [17] . in fact, pondr® vlxt has shown consistency and reproducibility when used for the study of structural viral shells that protect the virion from environmental damage. a wide range of viruses have been studied using pondr® vlxt including equine infectious anemia virus (eiav), human immunodeficiency virus (hiv), herpes simplex virus (hsv), smallpox virus, polio virus, nipah and ebola viruses, sars-cov, mers-cov, influenza virus, hepatitis c virus (hcv), hepatitis a virus (hav), yellow fever virus (yfv), and zika virus [2, [5] [6] [7] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] . the coronavirus virion contains four major structural proteins: the spike (s) protein, nucleocapsid (n) protein, membrane (m) protein, and the envelope (e) protein [5] [6] [7] [28] [29] [30] . these proteins are needed for production of a structurally complete viral particle [30] [31] [32] [33] . the main focus of our intrinsic disorder-based model is at the viral m and n proteins. the m protein of cov was chosen over the e protein because the m protein is a major transmembrane protein that is found in large numbers in the virion, whereas e protein is a minor protein of the envelope. also, in previous studies, membrane or matrix proteins of various viruses were analysed with respect to their intrinsic disorder content and roles in protecting the respective virions. the n protein is also important for our model, as it has been shown that greater disorder in the inner shell is associated with the mode of infection and virulence in other viruses [23] [24] [25] [26] . the protein sequences of the selected cov m and n proteins were downloaded from uniprot (http://www.uniprot.org) the sequences were fed into a mysql database using java [19] . the sequences were fed into the pondr® vlxt, and the corresponding results were archived in the database. pondr® vlxt provides intrinsic disorder predisposition scores for each residue. residues with the scores of 0.5 and above are considered disordered, and, conversely, residues with the scores below 0.5 are taken as ordered. we represented the outputs of these analyses in the form of the percentage of intrinsic disorder (pid), which is defined as the number of residues predicted to be disordered divided by the total number of residues in a query protein. statistical analyses using regression analysis and analysis of variance (anova) were done using r statistical package [34] . regression analyses were done using category as the dependent variable with m and n pids as independent variables or n pid as a sole independent variable. categories were assigned the values of 1, 2 and 3 to the three groups as according to the level of respiratory transmission potential. the amino acid sequences of the sars-cov-2 m and n proteins are available at ncbi (https://www.ncbi.nlm.nih.gov/nuccore/mn908947). as aforementioned, the model predicting the mode of viral transmission was built before the 2012 mers-cov outbreak. this model was based on the analysis of mainly the pids found for n proteins, the viruses were clustered into three groups according to their transmission mode, while the m pids provide an indicator of resilience of the virus in the harsh environment. the three groups are represented in table 1 . a two-way anova (p < 0.001) indicates that the groups are statistically identifiable. the table shows that category a consists of viruses that have greater respiratory transmission capabilities but lower fecaloral transmission potentials. category b involves coronaviruses that have intermediate levels of both respiratory and fecal-oral transmission potentials, whereas category c comprises of viruses with lower respiratory transmission. furthermore, a higher inner shell disorder is often associated with the greater infectivity, especially with regard to respiratory transmission potential since disorder allows greater promiscuity of binding [6, 7] . table 1 shows that sars-cov is grouped into category b along with other viruses that have intermediate respiratory and fecal-oral transmission potentials. mers-cov falls into category c (lower respiratory potential but higher fecal-oral transmission capabilities). these are consistent with what we know about transmission behaviour of sars-cov and mers-cov [5] [6] [7] [8] 35] . it should be noted that category a are viruses with n pid above 53%, whereas category c comprises of those with n pid below 47%. since sars-cov-2 has an n pid of 48%, which is above 46% but below 54%, it must be considered as being in category b. 3.3. sars-cov-2 has one of the hardest outer shell among covs (m pid = 6%) as already mentioned, based on the similar analysis of the sars-cov-2 (m pid = 6%, n pid = 48%), it is clear that this virus has to be placed in category b, along with sars-cov (table 1) . however, our data also pointed to something odd about the sars-cov-2. in fact, table 1 and fig. 1 show that data for the sars-cov-2, the m pid is 6%, which is second lowest in our sample of a fairly diverse selection of covs. this means that the sars cov-2 has one of the hardest outer shells among its counterparts. the model is consistent with the theme that viruses that remain in harsh environments require harder; i.e., less disordered, shells to survive. furthermore, the outer shell is likely to play an even greater role in protecting the virion [20, 22, 27] . this has important implications pertaining to the characteristics and behaviors of the virus as we shall see below. a superficial glance at the model that places sars-cov and sars-cov-2 in the same category, with intermediate levels of respiratory transmission potentials may mislead the reader to believe that the model is not reliable, given the fact that we have now seen that sars-cov-2 is much more contagious than sars-cov. we believe that this is not the case. if sars-cov-2 would be placed in category c, the same category as mers-cov, then the model would be definitely wrong in terms of reproducibility. on the other hand, the placing of sars-cov-2 in category a would be an inconsistency that would be difficult to explain. one would further notice that very few covs fall into category a, with the exception for hcov-229e and infectious bronchitis virus (ibv), which make the chance of any covs including sars-cov-2, to be in category a slim. more importantly, because sars-cov-2 is zoonotic and the only non-human cov in category a is the avian ibv, placing sars-cov-2 in category a could suggest that this virus is likely to be of avian origin, which is inconsistent with what we know about sars-cov-2. phylogenetic study has confirmed that the closest relatives of sars-cov-2 are bat covs [4, 36] . we also noticed that at least three of closest neighbors with similar n pids of 47-48% are those of bat origin (see table 1 ). this suggests that bat covs are likely to have an optimal mix of fecal-oral and respiratory transmission potentials that reflect their n pid range of 47-48%, which provides for greater fitness for the spread of the viruses among bats and that sars-cov-2 is likely to be of bat origin too given its n pid is 48%. there are highly debatable suggestions that snakes or pangolins [3, 37, 38] may be an intermediary host of the virus. even if any of these is later deemed as true, our data imply that the virus had not the chance to evolved much with any intermediary host except bats, unlike the cases of sars-cov and mers-cov, in which the model does suggest that they had evolved with hosts such as civet cats and camels respectively. this suggestion is, however, based on n pid alone. the cov shell disorder model was originally conceived as part of a spin-off from a parent research project that studied relationships among the mode of transmission, shell disorder and the absence of vaccines for certain sexually transmitted viruses such as hiv, hsv and hcv [19, 20, 27] . at that time even after 2003 sars-cov, the wealth of cov knowledge lies in the field of veterinary medicine [5, 6] . using what was then known about behaviors of porcine covs, a successful attempt to correlate m and n pids to modes of transmission was made [5, 6] . the results were similar to those found in table 1 and fig. 1 without the data pertaining to mers-cov and sars-cov-2, of course, since the mers-cov and covid-19 outbreaks had yet to occur then. evidence of a strong correlation of m and n pids to the mode of transmission (r 2 = 0.83, p < 0.001) can be seen in the regression analysis. furthermore, a strong but oddly weaker correlation (r 2 = 0.77, p < 0.001) can also be found when n pid is the sole independent variable even if poor correlation (r 2 = 0.21) is found when m pid is the sole independent variable. this basically means that the model should include both m and n pids as the statistical model seems to suggest that m pid does contribute, even if slightly, to the mode of transmission in conjunction to the major contribution by n pid. .this is consistent with the idea that both m and n play roles in protecting the virion from environmental damage and the need to protect the virion is dependent on mode of transmission. a qualitative inspection of the viruses in the three groups also reveals consistency. for example, as in table 1 , canine cov-respiratory falls into category b, while canine coventeritis is in category c. the porcine covs are also consistent with observation of their behaviors as we will later see. we need to keep in mind, however, that the outer shell is likely to play a greater and more immediate role in protecting the virion from environmental damage as it encases the entire virion, whereas the n protein is likely to protect only the rna. m pid is therefore a more crucial indicator of the "hardness" of the virion. however, because of the higher correlation of n to the mode of transmission, our model is apparently suggesting that some other mechanisms are involved. in fact, studies of inner shell of other viruses such as nipah virus, ebola virus and flaviviruses [23] [24] [25] [26] have indicated strong correlations between inner shell disorder and virulence. in the case of nipah virus, the virulence and inner shell disorder were linked to the modes of transmission [26] . the mechanism in which the virus acquires greater virulence via inner shell disorder arises from the ability of the viral protein to bind promiscuously to the host protein [6, [23] [24] [25] [26] . this ability provides for rapid replication of the viral proteins and particles. this quick replication is also an efficient way to evade the host immune system by reproducing a large number of viral particles before the immune system even recognizes the virus, which then often goes on to overwhelm the body leading to the death of the host [6, [23] [24] [25] [26] . one highly plausible explanation for the link between rapid replication and the modes of transmission is that viral load in the body fluids of the infected host needs to be sufficiently high before the virus can be infectious via the respiratory mode. this therefore explains the fact that rapid viral replication and inner shell disorder were found to be the common link between virulence and respiratory transmission in the case of nipah virus [26] . we don't, however, know if there is correlation between cov virulence and inner shell disorder, even though the authors believe it exists, just like the other viruses. if so, n will make an excellent target for cov vaccine development.the difficulty in the search for such links is that covs infect a large variety of animals and each cov often uses a different receptor [4, 6, 39] . for example, mers-cov has a case-fatality rate of above 30% [8] for humans but is generally harmless to camels [27] . furthermore, covs were generally not known to be virulent before the 2003 sars-cov outbreak and therefore lack adequate data for us to work with. the reason that we can make the necessary above-mentioned extrapolations pertaining to inner shell disorder among unrelated viruses with somewhat greater confidence is that inner shell proteins of different species of viruses often share similar functions and structures [30, 40, 41] . for example, the cov n proteins, like the nucleocapsids in other viruses, are responsible for assembling viral particles by forming complexes with other viral proteins and rna [24, 25, 41] . the n protein also helps with the packaging and budding of viral particles in the host er (endoplasmic reticulum) [23, 30, 40] . such tasks require binding to host proteins, and the promiscuous protein binding capabilities of a more disordered n will certainly help towards rapid replication of the virus. since both sars-cov and sars-cov-2 have intermediate respiratory transmission potentials, how do we account for the greater contagiousness of the latter? we need to understand that the respiratory transmission potential of a single viral particle is just one of several factors that might contribute to the contagiousness of the virus. other factors include how many particles are released by the host and how long does the particle remain in the environment. we mentioned that our model has detected something odd about sars-cov-2: this virus has one of the hardest outer shell within the family as seen in fig. 1 and table 1 (m pid = 6%). since the outer shell plays the greater role in protecting the virion in comparison to the inner shell, the harder outer shell provides virus greater resilience to outside the body environment and to the presence of digestive enzymes found in the saliva, mucus, and other bodily fluids [20, 22, [42] [43] [44] [45] [46] . as a result, the virus with the harder outer shell is able to remain active for a longer time and, therefore a lesser number of viral particles is required for a chance to infect someone. furthermore, because the virus is more resistant to the digestive enzymes in the bodily fluids, an infected body is likely to discharged more infectious particles. evidence of the protective role of the outer shells be seen in a wide variety of viruses. viruses associated with saliva (e.g., yfv, zikv, eiav, rabies) or fecal-oral transmission (e.g., poliovirus), have hard outer shells with low pid, whereas sexually transmitted viruses (e.g., hiv, hsv-2, hcv) have higher outer shell pids [6, [20] [21] [22] [23] [24] [25] [26] [27] . also, viruses that are notorious for lasting in the environment for a long time such, as the smallpox virus, have low outer shell pids. before the sars-cov outbreak, coronaviruses were not considered medically important, as, in the past, they had been primarily associated with cold viruses that cause minor sniffles. this is, however, not true in the field of veterinary medicine, where coronaviruses have posed a menace to livestock. this is especially so in the case of tgev (transmissible gastroenteritis virus) and pedv (porcine epidemic diarrhea virus). tgev could move rapidly among farm pigs to quickly devastate the farming community if not controlled early [5] [6] [7] . our analysis reveals that tgev is of category c (higher fecal-oral, lower respiratory transmission potentials) with m and n pids at 14% and 43% respectively. the reason that it is able to move rapidly is that its high fecaloral transmission potential represents a more efficient mode of transmission among the farm animals. there is an antigenically related cousin, pedv, which has similar characteristics as tgev [29] . like tgev, diarrhea and vomiting are main symptoms of pedv infection. while tgev is highly infectious, pedv is less contagious among farm pigs. a peculiar characteristic has been observed by farmers and veterinarians: pedv can reappear out of nowhere from within the same pen that was previously occupied by infected pigs, even after the disease has passed on several months ago. an inspection of the m and n pids reveals the reason showing that pedv belongs to the category b, along with sars-cov and sars-cov-2, unlike tgev and mers-cov. this means that pedv has a higher respiratory transmission potential than tgev and that implies greater chances of fecal-respiratory transmission for the former. obviously, viral particles in fecal materials that was somehow inadvertently left behind go on to infect a new population of pigs several months later [5] [6] [7] . infectious droplets from mucus, vomit, or feces have actually been seen during the sars-cov outbreak. a stark example happened in a 2003 outbreak at amoy gardens, a housing complex in hong kong, where a huge cluster of infected patients was found and the inefficient sewerage and toilet ventilation systems were responsible for the spread [5, 35, 47] . we need to be reminded that pedv, sars-cov, and sars-cov-2 are all under the same category b, and that sars-cov-2 has the hardest outer shell within the entire cov family.sars-cov-2 is thus likely to be able remain infectious in the environment for the longest time regardless of being in feces, mucus, vomit, sweat, or saliva. a puzzling thing can be observed is when we notice that pedv (m pid = 8%) has a harder outer shell than tgev (m pid = 14%), given that tgev is predicted as having higher fecal-oral transmission potential. we would think that having greater fecal-oral potential requires harder outer shell among covs. apparently, the result is implying that this is not necessarily true, as we have seen, and having higher fecaloral transmission potential can actually provide greater efficiency in the spread among farm animals, as we have seen in tgev, such that the virus does not have to stay long outside the body. the ability to stay longer in the environment could, however, provide advantages to a virus with greater respiratory transmission potential, especially with respect to spread via airborne virus from feces or body fluids. this is likely the case for sars-cov-2. we have presented here a set of data that not only place the sars-cov-2 within the category of covs that have intermediate levels of both respiratory and fecal-oral transmission potentials, alongside sars-cov and pedv, but have also predicted sars-cov-2 to have one of the hardest outer shells among most covs. this peculiarity is likely responsible for its high level of contagiousness, since hardness of its outer shell could provide the virus with the greater resilience to the conditions outside the body and in body fluid, as the harder shell will better protect the virion from damage as a result of the hostile environment and action of the digestive enzymes found in bodily fluids. as a result, it is likely that the infected body can shed more infectious particles that have a greater chance of infecting a person over its lifetime. chances of infections via indirect contact and airborne virus from feces and bodily fluids are therefore greater. the results of our research could have important implications for epidemiologists and public health officials. gkmg. conceived the idea, collected and analyzed the data and wrote the first draft. vnu. helped with the collection and analysis of literature data, reviewed and revised the draft. akd and jaf reviewed the manuscript and provided the resources necessary for the research. gkmg. is an independent researcher and the owner of goh's biocomputing, singapore. gkmg. has also written a book ("viral shapeshifters: strange behaviors of hiv and other viruses") on a related subject. the authors have no other potential conflict of interests. who, novel coronavirus rigidity of outer shell predicted by protein disorder model sheds light on covid-19(wuhan-2019-ncov) infectivity evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin understanding viral transmission behavior via protein intrinsic disorder prediction: coronaviruses viral shapeshifters: strange behavoirs of hiv and other viruses, simplicity research institute prediction of intrinsic disorder in mers-cov/ hcov-emc supports a high oral-fecal transmission who, middle eastern respiratory syndrome coronavirus (mers-cov) identification of mers-cov in dromedary camels intrinsically unstructured proteins why are "natively unfolded" proteins unstructured under the physiological conditions? intrinsically unstructured proteins: re-assessing the protein structure-paradigm predicting protein disorder for n-, c-, and internal regions predicting binding regions within disordered proteins sequence complexity of disordered protein mining alpha-helix-forming molecular recognition features with cross species sequence alignments coupled folding and binding with alpha-helix-forming molecular recognition elements viral disorder or disordered viruses: do viral proteins possess unique features? protein intrinsic disorder toolbox for comparative analysis of viral proteins a comparative analysis of viral matrix proteins using disorder predictors protein intrinsic disorder and influenza virulence: the 1918 h1n1 and h5n1 viruses shell disorder, immune evasion and transmission behaviors among human and animal retroviruses detection of links between ebola nucleocapsid and virulence using disorder analysis correlating flavivirus virulence and levels of intrinsic disorder in shell proteins: protective roles vs. immune evasion zika and flavivirus disorder: virulence and fetal morbidity nipah shell disorder, mode of transmission and virulence hiv vaccine mystery and viral shell disorder fundamentals of molecular virology antigenic relationships among procine epidemic diarrhea virus and transmissible gastroenteritis virus strains fielding bc the coronavirus nucleocapsid is a multifunctional protein the molecular biology of coronaviruses efficient assembly and release of sars coronavirus-like particles by a heterologous expression system mers-cov virus-like particles produced in insect cells induce specific humoural and cellular immunity in rhesus macaques r: a language and environment for statistical computing twenty-first century plague: the story of sars gemomic characterisation and epidemiology of 2019 novel-coronavirus repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus (2019-ncov) related coronavirus model cross-species transmission of the newly identified coronavirus 2019-ncov bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (mers-cov) after experimental infection characterization of filovirus protein-protein interactions in mammalian cells using bimolecular complementation specific interaction of capsid protein and importin alpha/beta influences west nile virus production antiviral activities in saliva lysozyme in body fluids from bacterial killing to immune modulation: recent insights into functions of lysozyme antimicrobial peptides and skin defense immune system innate antimicrobial activity of nasa secretions evidence of airborne transmission of the severe acute respiratory syndrome virus key: cord-266253-oyid5haj authors: al-abaidani, i.s.; al-maani, a.s.; al-kindi, h.s.; al-jardani, a.k.; abdel-hady, d.m.; zayed, b.e.; al-harthy, k.s.; al-shaqsi, k.h.; al-abri, s.s. title: overview of preparedness and response for middle east respiratory syndrome coronavirus (mers-cov) in oman date: 2014-10-29 journal: int j infect dis doi: 10.1016/j.ijid.2014.10.003 sha: doc_id: 266253 cord_uid: oyid5haj several countries in the middle east and around 22 countries worldwide have reported cases of human infection with the middle east respiratory syndrome coronavirus (mers-cov). the exceptionally high fatality rate resulting from mers-cov infection in conjunction with the paucity of knowledge about this emerging virus has led to major public and international concern. within the framework of the national acute respiratory illness surveillance, the ministry of health in the sultanate of oman has announced two confirmed cases of mers-cov to date. the aim of this report is to describe the epidemiological aspects of these two cases and to highlight the importance of public health preparedness and response. the absence of secondary cases among contacts of the reported cases can be seen as evidence of the effectiveness of infection prevention and control precautions as an important pillar of the national preparedness and response plan applied in the health care institutions in oman. several countries worldwide have reported cases of human infection with the middle east respiratory syndrome coronavirus (mers-cov). the exceptionally high fatality rate resulting from mers-cov infection in conjunction with the paucity of knowledge about this emerging virus has led to major public and international concern. 1 the ministry of health in the sultanate of oman has announced two confirmed cases of mers-cov. the aim of this report is to describe the epidemiological aspects of the reported mers-cov cases in oman and to highlight the public health response and the activities done to face any future resurgence of mers-cov in the country. based on the world health organization (who) interim case definition for mers-cov as of july 3, 2013, 2 the first laboratory-confirmed case of mers-cov in oman was diagnosed on october 29, 2013. a 68-year-old omani man from dakhliyah governorate complained of fever and cough of 2-day duration. he then developed right lower lobe pneumonia and multi-organ failure and died on november 10, 2013. he had a history of type 2 diabetes mellitus and uncontrolled hypertension, and he had previously undergone coronary artery bypass grafting. he did not have a history of travel outside the country or contact with animals. the second case was a 59-year-old omani man from north batinah governorate who presented on december 22, 2013 with a high-grade temperature and cough of 6-day duration. he later developed severe right upper lobe pneumonia and died on december 30, 2013. he was a heavy smoker, but had no known medical comorbidities. the patient had attended a camel race in abu dhabi, uae, 4 weeks before the onset of his symptoms. the ministry of health implemented a national mers-cov preparedness and response plan. this plan was based on the exceptionally high fatality rate resulting from mers-cov infection in conjunction with the paucity of knowledge about this emerging virus has led to major public and international concern. within the framework of the national acute respiratory illness surveillance, the ministry of health in the sultanate of oman has announced two confirmed cases of mers-cov to date. the aim of this report is to describe the epidemiological aspects of these two cases and to highlight the importance of public health preparedness and response. the absence of secondary cases among contacts of the reported cases can be seen as evidence of the effectiveness of infection prevention and control precautions as an important pillar of the national preparedness and response plan applied in the health care institutions in oman. strengthening five pillars of action, including public health surveillance and contact management, building laboratory capacity, infection prevention and control, case management, and risk communication. algorithms were developed describing response actions in the event of a suspected mers-cov case. checklists for the preparedness of health care facilities were developed and action plans were later developed to rectify the deficiencies. field visits were conducted immediately after confirmation of cases by the regional and national rapid response teams from the ministry of health, and contact surveillance and monitoring was conducted for 14 days after the last exposure. laboratory surveillance for mers-cov started by building laboratory diagnostic capacity with the availability of the primers for mers-cov testing, and with the training of laboratory personnel countrywide on the triple-packing and shipment of samples. training on how to collect nasopharyngeal swabs for testing for mers-cov was conducted for emergency room physicians, internists, and intensivists in all district hospitals. national infection prevention and control guidelines were developed for dealing with suspected or confirmed cases of mers-cov. mask-fit testing was done for all healthcare workers who could be involved in taking care of patients with mers-cov. a project was initiated for triaging of patients presenting to emergency rooms or health centers with an acute respiratory illness. in 2013, post hajj surveillance for mers-cov was done using nasopharyngeal swabs for people returning from hajj and presenting with respiratory symptoms. three hundred and fifty samples were tested by real-time pcr and all were negative for mers-cov. the surveillance system for severe acute respiratory infections (sari) was implemented in oman in january 2008 in four regional hospitals as sentinel sites; sari aims to determine the epidemiology of severe respiratory infections and the contribution of influenza and other etiological agents to severe respiratory infections in the country. 3 it also aims to detect emergent influenza strains with pandemic potential or any other respiratory infections, and to detect any unusual morbidity or mortality due to acute respiratory illness. in 2012, sari sentinel sites were used as a platform to test 10% of cases for mers-cov at the central public health laboratory; 2000 samples were tested and all were negative. in conclusion, we have described the epidemiological aspects of the two reported cases of mers-cov in oman and the preparedness efforts made by the ministry of health. strengthened infection control practices and having a powerful active surveillance program for acute respiratory illnesses is key to the rapid and prompt response for emerging respiratory infections. conflict of interest: none. middle east respiratory syndrome coronavirus (mers-cov): summary and literature update revised interim case definition for reporting to who-middle east respiratory syndrome coronavirus (mers-cov) world health organization. interim global epidemiological surveillance standards for influenza key: cord-258323-vdeffy4l authors: jiang, yuting; li, junfeng; teng, yue; sun, hong; tian, guang; he, lei; li, pei; chen, yuehong; guo, yan; li, jiangfan; zhao, guangyu; zhou, yusen; sun, shihui title: complement receptor c5ar1 inhibition reduces pyroptosis in hdpp4-transgenic mice infected with mers-cov date: 2019-01-09 journal: viruses doi: 10.3390/v11010039 sha: doc_id: 258323 cord_uid: vdeffy4l middle east respiratory syndrome coronavirus (mers-cov) is a highly pathogenic virus with a crude mortality rate of ~35%. previously, we established a human dpp4 transgenic (hdpp4-tg) mouse model in which we studied complement overactivation-induced immunopathogenesis. here, to better understand the pathogenesis of mers-cov, we studied the role of pyroptosis in thp-1 cells and hdpp4 tg mice with mers-cov infection. we found that mers-cov infection induced pyroptosis and over-activation of complement in human macrophages. the hdpp4-tg mice infected with mers-cov overexpressed caspase-1 in the spleen and showed high il-1β levels in serum, suggesting that pyroptosis occurred after infection. however, when the c5a-c5ar1 axis was blocked by an anti-c5ar1 antibody (ab), expression of caspase-1 and il-1β fell. these data indicate that mers-cov infection induces overactivation of complement, which may contribute to pyroptosis and inflammation. pyroptosis and inflammation were suppressed by inhibiting c5ar1. these results will further our understanding of the pathogenesis of mers-cov infection. middle east respiratory syndrome coronavirus (mers-cov), the second highly pathogenic coronavirus to emerge after severe acute respiratory syndrome coronavirus (sars-cov), causes severe acute respiratory failure and extra-pulmonary multi-organ damage accompanied by severe systemic inflammation [1] [2] [3] . however, the pathogenesis of mers-cov still needs to be explored. complement activation and pyroptosis are two proteolytic cascades that defend the host against dangerous pathogens. they are important parts of the innate immune system and have some similar characteristics, including pore-formation and proinflammatory characteristics. pyroptosis is a lytic and inflammatory mode of regulated cell death catalyzed by the caspase family [4] . activation of caspase-1 relies on assembly of inflammasome complexes, which contain nlrp1b, nlrc4, nlrp3, and aim2. different inflammasomes are activated by different pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps) via particular pattern recognition receptors (prrs). the best-characterized inflammasome is the nlrp3 inflammasome, which responds to a variety of bacterial, viral, and fungal agents [5, 6] , damps (e.g., atp, monosodium urate crystals, and amyloid-β aggregates) [7, 8] , and even environmental and industrial particles such as silica and asbestos [9] . the nlrp3 inflammasome comprises the nlrp3 scaffold, the asc (pycard) adaptor, and pro-caspase-1. the activated nlrp3 inflammasome promotes transformation of pro-caspase-1 to its active form, which proteolytically cleaves gasdermin d, pro-il-1β, and pro-il-18 to yield their bioactive forms. the n-terminal domain of cleaved gasdermin d perforates the cell membrane, resulting in osmotic lysis [10] , whereas mature il-1β and il-18 act as proinflammatory cytokines [11] . the complement system is an ancient molecular cascade; indeed, homologs have been found in sea urchin [12] and mosquitoes [13] . complement is activated via three pathways: the classical, lectin, and alternative pathways. during the process of activation, an enzyme named c3 convertase cleaves c3 to c3a and c3b, which are recruited to the c3 convertase to form the c5 convertase. c5 convertase catalyzes cleavage of c5 to c5a and c5b to initiate the terminal complement pathway, resulting in formation of the membrane attack complex, which has pore-forming properties. during this process, two split products, c3a and c5a (known as anaphylatoxins), promote inflammation or serve as chemoattractants by engaging their cognate receptors [14, 15] . in a previous study we demonstrated that aberrant complement activation contributes to severe outcomes in hdpp4 transgenic mice infected with mers-cov, and that preventing over-activation of the complement system may be an effective clinical therapy for mers [16] . here, we examined the role of pyroptosis in the pathogenesis of mers, along with the relationship between pyroptosis and complement. the results may help us to better understand the mechanism underlying severe outcomes after mers-cov infection. all animal experiments were approved by the institutional animal care and use committee (iacuc) of the beijing institute of microbiology and epidemiology (iacuc permit no: bime 2017-0011; permit date: 8 march 2017). animal studies were carried out in strict accordance with the recommendations set out in the guide for the care and use of laboratory animals. human monocytic cells (thp-1) were purchased from the american type culture collection (manassas, va, usa, atcc number: tib-202) and cultured in rpmi 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (fbs), 100 u/ml penicillin, 100 µg/ml streptomycin sulfate, 1× glutamax-i (l-glutamine alternate), and 0.05 mm 2-mercaptoethanol. thp-1 (differentiated) macrophages were obtained by exposing the cells to 60 nm phorbol-12-myristate-13-acetate (pma) for 12 h, followed by culture for a further 24 h in complete growth medium without pma. mers-cov (hcov-emc/2012 strain) was propagated and titrated on vero cells in an approved biosafety level 3 laboratory. thp-1 differentiated macrophages cultured in 75-cm 2 flasks were infected with mers-cov at a multiplicity of infection of 0.1. the virus was adsorbed at 37 • c for 1 h and unbound virus was washed away. hdpp4-tg mice (6 weeks old, female) [17] were maintained in a pathogen-free facility and housed in cages containing sterilized feed and drinking water. following intraperitoneal anesthetization with sodium pentobarbital (5 mg/kg body weight), mice were inoculated intranasally with mers-cov (103.3 50% tissue culture infectious dose (tcid50)) in 20 µl dulbecco's modified eagle's medium (dmem). mice in the sham group received the same volume of dmem. for the experiments of c5ar1 inhibition, mice were received an intravenous (i.v.) injection (600 µg/kg) of a monoclonal ab (mab) specific for mouse c5ar1 (hycult biotech, uden, the netherlands) to block the interaction of c5a to c5ar1 or an injection of phosphate-buffered saline (pbs) (sham treatment control) at the same time as mers-cov inoculation. all infectious experiments related to mers-cov were performed in an approved biosafety level 3 facility. thp-1 differentiated macrophages were lysed in trizol™ reagent (life technologies, carlsbad, ca, usa) at 24 h post-infection with mers-cov. total rna and proteins were isolated according to the reagent user guide. mice were euthanized by overdose inhalation of carbon dioxide at different time points after infection with mers-cov. lungs were harvested and total rna was extracted and purified using an rneasy extraction kit (qiagen, hilden, germany). to detect expression of inflammasomes and complement components in mers-cov-infected thp-1 differentiated macrophages and hdpp4-tg mice, 2 µg of total rna from cells or the lung of mice we used as template for first-strand cdna synthesis. the resulting cdna was subjected to quantitative pcr using power sybr ® green pcr master mix (life technologies, carlsbad, ca, usa) to determine the relative abundance of inflammasome and complement components. the forward and reverse primers used for each component are listed in the table 1 . the relative amount of each gene was obtained by normalization against an endogenous control gene (gapdh) and calculated using the comparative 2 −∆∆ct method. sham-infected monocytes and sham-infected hdpp4-tg mice were used as respective calibrators. an identical amplification reaction comprising (i) polymerase activation and dna denaturation at 95 • c for 10 min, (ii) 40 cycles each of the denaturation at 95 • c for 10 s, and (iii) an annealing/extension step at 60 • c for 30 s, was used for each gene analyzed. proteins isolated from thp-1 monocytic cells and thp-1 differentiated macrophages were electrophoresed in a 12% sds-page gel and transferred to a pvdf membrane (ge healthcare, dassel, germany). pvdf membranes were then blocked for 1 h at room temperature in 5% non-fat cytokines in mouse serum were measured using a milliplex mouse cytokine/chemokine magnetic panel kit (merck millipore, burlington, ma, usa). a panel of inflammatory cytokines (il-1β, il-6, tnf-α, and ifn-γ) was detected according to the manufacturer's protocol. sections of paraffin-embedded spleen and lung tissues (4 µm thick) were prepared and stained to detect antigen expression. briefly, retrieved sections were incubated overnight at 4 • c with the following antibodies: mouse anti-caspase-1 mab (adipogen, san diego, ca, usa), polyclonal rabbit anti-cd68 (abcam, cambridge, ma, usa), and polyclonal anti-ifn-γrα (santa cruz biotechnology). biotinylated immunoglobulin g was then added, followed by an avidin-biotin-peroxidase conjugate (beijing zhongshan biotechnology co., ltd., beijing, china). immunoreactivity was detected using 3,3 diamino benzidine (dab). slides were counter-stained with hematoxylin. statistical analyses were performed using graphpad prism software, version 5.01 (graphpad software, san diego, ca, usa). student's t test was used to compare two groups with respect to relative expression of mrna and cytokine levels in serum. p values < 0.05 were considered significant. unlike abortive infection of sars-cov in human macrophages, mers-cov can establish a productive infection in macrophages and induce production of proinflammatory cytokines and chemokines [18] . many rna viruses, such as ev71, h1n1, h7n9 influenza a virus, and zika virus, can infect macrophages and trigger il-1β secretion via the nlrp3 inflammasome [19] [20] [21] [22] . to evaluate the response of macrophages to mers-cov infection, we inoculated thp-1 monocytic cells and thp-1 differentiated macrophages with mers-cov or rpmi 1640 medium (sham-infection). we then examined expression of nlrp3, pro-caspase-1, and pro-il-1β 24 h later by rt-qpcr. as shown in figure 1 , mers-cov infection induced relatively higher expression of pro-caspase-1 ( figure 1a ) and pro-il-1β ( figure 1b ), but not nlrp3 ( figure 1c ), in both thp-1 monocytes and macrophages. expression of pro-il-1β in monocytes increased by 170-fold, whereas that in macrophages increased by 26-fold (on average). we verified expression of caspase-1, il-1β, and mers nucleocapsid protein (np) by western blotting ( figure 1d ). mers-cov-infected thp-1 macrophages expressed higher levels of pro-caspase-1, pro-il-1β, and activated il-1β (p17) than sham-infected thp-1 macrophages or mers-cov-infected thp-1 monocytes. mers np was detected in both mers-cov-infected thp-1 monocytes and macrophages. these results indicate that mers-cov infection induces high levels of proinflammatory il-1β secretion and thp-1 macrophage pyroptosis. to determine whether mers-cov infection induces pyroptosis in mice, we used rt-qpcr to detect mrna encoding nlrp3, pro-caspase-1, and pro-il-1β in lung tissue from hdpp4 transgenic mice at day 3 post-mers-cov infection. although there was no significant difference in expression of nlrp3 and pro-caspase-1 between the sham-infected and mers-cov-infected groups ( figure 2a ,b), expression of pro-il-1β mrna was significantly higher after mers-cov infection ( figure 2c ). in addition, we measured the concentration of il-1β in serum. the results showed that mers-cov infection induced production of il-1β ( figure 2d ). furthermore, we examined expression of caspase-1 in the lung and spleen at day 7 post-mers-cov infection by ihc. in line with the mrna results, there was no significant difference in expression of caspase-1 in the lung of sham-infected and mers-cov-infected mice. however, the spleens of mice infected with mers-cov showed higher expression of caspase-1 than those of mice in the sham group ( figure 2e ). the results indicated that mers-cov infection could induce pyroptosis in mice. (d) samples of total protein were subjected to western blotting to detect pro-caspase-1, pro-il-1β, activated il-1β, and mers np. to determine whether mers-cov infection induces pyroptosis in mice, we used rt-qpcr to detect mrna encoding nlrp3, pro-caspase-1, and pro-il-1β in lung tissue from hdpp4 transgenic mice at day 3 post-mers-cov infection. although there was no significant difference in expression of nlrp3 and pro-caspase-1 between the sham-infected and mers-cov-infected groups (figure 2a,b) , expression of pro-il-1β mrna was significantly higher after mers-cov infection ( figure 2c ). in addition, we measured the concentration of il-1β in serum. the results showed that mers-cov infection induced production of il-1β ( figure 2d ). furthermore, we examined expression of caspase-1 in the lung and spleen at day 7 post-mers-cov infection by ihc. in line with the mrna results, there was no significant difference in expression of caspase-1 in the lung of sham-infected and mers-cov-infected mice. however, the spleens of mice infected with mers-cov showed higher expression of caspase-1 than those of mice in the sham group ( figure 2e ). the results indicated that mers-cov infection could induce pyroptosis in mice. il-1β plays an important role in mediating autoinflammatory diseases and in generating inflammatory responses to infection [23] . therefore, to assess the inflammatory responses in mice, we measured tnf-α, ifn-γ, and il-6 in serum at day 3 post-mers-cov infection. as shown in figure 3a -c, serum from mice in the mers-cov-infected group contained more tnf-α, ifn-γ, and il-6 than that from sham-infected mice. ihc examination of cd68 and ifn-γ receptor expression also suggested greater macrophage infiltration and activation in the lung and spleen of mice at 7 days post-mers-cov infection ( figure 3d ). these results indicate that mers-cov infection causes systemic inflammation, as reported in clinical mers patients and mers-cov infected animal models [17, 24] . il-1β plays an important role in mediating autoinflammatory diseases and in generating inflammatory responses to infection [23] . therefore, to assess the inflammatory responses in mice, we measured tnf-α, ifn-γ, and il-6 in serum at day 3 post-mers-cov infection. as shown in figure 3a -c, serum from mice in the mers-cov-infected group contained more tnf-α, ifn-γ, and il-6 than that from sham-infected mice. ihc examination of cd68 and ifn-γ receptor expression also suggested greater macrophage infiltration and activation in the lung and spleen of mice at 7 days post-mers-cov infection ( figure 3d ). these results indicate that mers-cov infection causes systemic inflammation, as reported in clinical mers patients and mers-cov infected animal models [17, 24] . the complement system links activation of toll-like receptors to transcription of il-1β mrna [25, 26] . it has been studied that intracellular c3 is converted to biologically active c3a and c3b by the protease cathepsin l [27] , and c3a activates nlrp3 and triggers il-1β production in human monocytes by regulating efflux of atp [28] . in addition, c5a is believed to induce a proinflammatory or anti-inflammatory response when ligated to c5ar1 or c5ar2 respectively [29] [30] [31] . thus, we used rt-qpcr to examine expression of complement components and their receptors c3ar, c5ar1, and c5ar2. as shown in figure 4 , c3 and c3ar expression by both thp-1 monocytes and macrophages was highly upregulated (by 5-20-fold) after mers-cov infection. c5ar1 was upregulated, whereas c5ar2 was downregulated, after mers-cov infection. the complement system links activation of toll-like receptors to transcription of il-1β mrna [25, 26] . it has been studied that intracellular c3 is converted to biologically active c3a and c3b by the protease cathepsin l [27] , and c3a activates nlrp3 and triggers il-1β production in human monocytes by regulating efflux of atp [28] . in addition, c5a is believed to induce a proinflammatory or anti-inflammatory response when ligated to c5ar1 or c5ar2 respectively [29] [30] [31] . thus, we used rt-qpcr to examine expression of complement components and their receptors c3ar, c5ar1, and c5ar2. as shown in figure 4 , c3 and c3ar expression by both thp-1 monocytes and macrophages was highly upregulated (by 5-20-fold) after mers-cov infection. c5ar1 was upregulated, whereas c5ar2 was downregulated, after mers-cov infection. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [17] and inhibiting c5ar1 alleviates mers-cov infection-induced tissue damage by regulating host immune responses [16] . here, we used an anti-c5ar1 ab to block the c5a-c5ar1 axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase-1 in the spleen and il-1β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c5ar1 ab expressed less caspase-1 in the spleen at day 7 post-mers-cov infection ( figure 5a ). although there was no significant difference between the two groups with respect to pro-il-1β mrna expression ( figure 5b ), serum levels of il-1β were lower in the anti-c5ar1 ab-treated group than in the pbs-treated group at day 1 ( figure 5c ) and day 3 [16] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il-1β and caspase-1, in mice infected with mers-cov. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [17] and inhibiting c5ar1 alleviates mers-cov infection-induced tissue damage by regulating host immune responses [16] . here, we used an anti-c5ar1 ab to block the c5a-c5ar1 axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase-1 in the spleen and il-1β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c5ar1 ab expressed less caspase-1 in the spleen at day 7 post-mers-cov infection ( figure 5a ). although there was no significant difference between the two groups with respect to pro-il-1β mrna expression ( figure 5b ), serum levels of il-1β were lower in the anti-c5ar1 ab-treated group than in the pbs-treated group at day 1 ( figure 5c ) and day 3 [16] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il-1β and caspase-1, in mice infected with mers-cov. our previous study demonstrated that mers-cov infection results in dysregulated host immune responses and severe tissue damage [17] and inhibiting c5ar1 alleviates mers-cov infection-induced tissue damage by regulating host immune responses [16] . here, we used an anti-c5ar1 ab to block the c5a-c5ar1 axis. the antibody was administered at the same time as mers-cov infection. we then measured expression of caspase-1 in the spleen and il-1β in the lung and serum. compared with the pbs-treated group, mice receiving the anti-c5ar1 ab expressed less caspase-1 in the spleen at day 7 post-mers-cov infection ( figure 5a ). although there was no significant difference between the two groups with respect to pro-il-1β mrna expression ( figure 5b ), serum levels of il-1β were lower in the anti-c5ar1 ab-treated group than in the pbs-treated group at day 1 ( figure 5c ) and day 3 [16] post-mers-cov infection. these results suggest that complement inhibition decreased the expression of pyroptosis indicators, il-1β and caspase-1, in mice infected with mers-cov. at 1 day after mers-cov infection, we measured proinflammatory cytokines (ifn-γ, tnf-α, and il-6) in serum. ifn-γ levels in mice treated with the anti-c5ar1 ab were much lower than those in the pbs-treated group ( figure 6a ). to further evaluate the effect of complement inhibition on the local inflammation at later time after mers-cov infection, we examined expression of cd68 and ifn-γ receptor in lung and spleen at 7 days post-mers-cov infection. ihc revealed that macrophage infiltration and activation were lower in the anti-c5ar1 ab-treated group ( figure 6d ). taken together, these results suggest that inhibiting complement dampens the over-activated inflammatory response in mice infected with mers-cov. at 1 day after mers-cov infection, we measured proinflammatory cytokines (ifn-γ, tnf-α, and il-6) in serum. ifn-γ levels in mice treated with the anti-c5ar1 ab were much lower than those in the pbs-treated group ( figure 6a ). to further evaluate the effect of complement inhibition on the local inflammation at later time after mers-cov infection, we examined expression of cd68 and ifn-γ receptor in lung and spleen at 7 days post-mers-cov infection. ihc revealed that macrophage infiltration and activation were lower in the anti-c5ar1 ab-treated group ( figure 6d ). taken together, these results suggest that inhibiting complement dampens the over-activated inflammatory response in mice infected with mers-cov. macrophages play important roles in host defense by clearing dead cells, ingesting and destroying microbes, and presenting antigens to t lymphocytes. in addition, macrophages produce the full array of complement components [32] and prrs, which are closely associated with inflammasome activation and pyroptosis. the accumulated studies indicate that macrophages play an important role in the pathogenesis of sars and mers [18, 33] . macrophages infected with mers-cov secrete proinflammatory cytokines and chemokines [18] . widespread distribution of these macrophages throughout many organs is one of the reasons underlying multi-organ damage and systemic inflammation after virus infection. pyroptosis or inflammasome activation plays an important role in virus-mediated pathogenesis. for example, abortive hiv-1 infection in quiescent macrophages play important roles in host defense by clearing dead cells, ingesting and destroying microbes, and presenting antigens to t lymphocytes. in addition, macrophages produce the full array of complement components [32] and prrs, which are closely associated with inflammasome activation and pyroptosis. the accumulated studies indicate that macrophages play an important role in the pathogenesis of sars and mers [18, 33] . macrophages infected with mers-cov secrete proinflammatory cytokines and chemokines [18] . widespread distribution of these macrophages throughout many organs is one of the reasons underlying multi-organ damage and systemic inflammation after virus infection. pyroptosis or inflammasome activation plays an important role in virus-mediated pathogenesis. for example, abortive hiv-1 infection in quiescent lymphoid cd4 t cells leads to cd4 t cell pyroptosis independent of the nlrp3 inflammasome [34] . ev71 3d and zikv ns5 activate the nlrp3 inflammasome by interacting with nacht and the lrr domain of nlrp3 [19, 22] . the pb1-f2 protein of avian influenza a virus h1n1 or h7n9 induces inflammation by activating the nlrp3 inflammasome [35, 36] , and deficiency of nlrp3 or caspase-1 protects mice against h7n9 infection-associated morbidity and mortality [21] . here, we demonstrate that the dysregulated macrophage-mediated immune responses after mers-cov infection may contribute to a severe outcome. several studies demonstrate relationships between complement and inflammasomes. for example, c3-/-mice display reduced inflammasome activation in an intracerebral hemorrhage (ich) model [37] . engagement of c5a and c5ar1 on cd4+ t cells generates reactive oxygen species, which are a classical damp, thereby triggering inflammasome assembly [38] . in monocytes, c3a alters metabolic programming and increases atp efflux, leading to nlrp3 activation and il-1β generation via the receptor p2 × 7 [28] . thus, the complement system is considered to be an essential regulatory component of the cellular alarm system that controls inflammasome activation [39] . here, we inhibit mers-cov infection-induced inflammation and pyroptosis by blocking the c5a-c5ar1 axis with an anti-c5ar1 antibody. in our study, we first demonstrated that virus infection leads to pyroptosis by measuring activation of caspase-1 and il-1β in human macrophages infected with mers-cov ( figure 1 ). next, we found that mers-cov infection induced transcription of pro-il-1β in the lung and expression of caspase-1 in the spleen. activated caspase-1 could be released into the extracellular space and delivered to the lung via exosomes [40] or the systemic circulation; it then cleaves pro-il-1β into its bio-activated form, which is secreted into the serum ( figure 2d ). we cannot exclude the possibility that pyroptosis occurs in the spleen because we did not detect expression of pro-il-1β. meanwhile, examination of cd68 and ifn-γ receptor expression revealed macrophage infiltration and activation in the lungs and spleen ( figure 3d ). thus, pyroptosis could occur in both organs. in our previous study, we showed that inhibiting c5ar1 increased splenic cell regeneration and decreased splenic cell apoptosis, thereby alleviating mers-cov infection-induced tissue damage [16] . the spleen happens to be a large reservoir for myeloid lineage cells such as macrophages, dendritic cells and cd4+ t cells in which pyroptosis mainly occurred [4, 34, 41] . here, we studied and demonstrated that inhibiting c5ar1 suppressed caspase-1 activation ( figure 5a ) and macrophage infiltration and activation in the spleen ( figure 6d ), and thereby reduced the secretion of il-1β into the serum ( figure 5c ) and the systemic inflammation ( figure 6 ). however, in the lung tissue, there was no significant difference of pro-il-1β transcription between the ab and sham-treated groups ( figure 5b ), which may due to the limited macrophages, the main cell type in which pyroptosis occurred, when compared to that in spleen. although many studies have focused on the link between complement and pyroptosis, the pathways that link them remain unclear. here, our results showed that mers-cov infection induces pro-il-1β transcription, and complement activation, which leads to pyroptosis in macrophages. induction of pyroptosis was related with complement activation and may be promoted by ligation of c5a and c5ar1, which was confirmed by the blockade of anti-c5ar1 antibody (figure 7) . in summary, these data indicate that mers-cov infection induces overactivation of complement, which may contribute to pyroptosis and inflammation. pyroptosis and inflammation were suppressed by inhibiting c5ar1. these research will further our understanding of the pathogenesis of mers-cov infection. occurred, when compared to that in spleen. although many studies have focused on the link between complement and pyroptosis, the pathways that link them remain unclear. here, our results showed that mers-cov infection induces pro-il-1β transcription, and complement activation, which leads to pyroptosis in macrophages. induction of pyroptosis was related with complement activation and may be promoted by ligation of c5a and c5ar1, which was confirmed by the blockade of anti-c5ar1 antibody (figure 7) . clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description critical role for cryopyrin/nalp3 in activation of caspase-1 in response to viral infection and double-stranded rna fungal pathogen recognition by the nlrp3 inflammasome cryopyrin activates the inflammasome in response to toxins and atp brief report: granulocyte-macrophage colony-stimulating factor drives monosodium urate monohydrate crystal-induced inflammatory macrophage differentiation and nlrp3 inflammasome up-regulation in an in vivo mouse model innate immune activation through nalp3 inflammasome sensing of asbestos and silica pore-forming activity and structural autoinhibition of the gasdermin family the inflammasomes sea urchin coelomocytes specifically express a homologue of the complement component c3 conserved role of a complement-like protein in phagocytosis revealed by dsrna knockout in cultured cells of the mosquito the role of anaphylatoxins c3a and c5a in regulating innate and adaptive immune responses the role of the complement anaphylatoxins in the recruitment of eosinophils blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis ev71 3d protein binds with nlrp3 and enhances the assembly of inflammasome complex the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the h7n9 influenza a virus infection results in lethal inflammation in the mammalian host via the nlrp3-caspase-1 inflammasome zika virus infection induces host inflammatory responses by facilitating nlrp3 inflammasome assembly and interleukin-1beta secretion immunological and inflammatory functions of the interleukin-1 family clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission crosstalk pathways between toll-like receptors and the complement system cholesterol crystals induce complement-dependent inflammasome activation and cytokine release intracellular complement activation sustains t cell homeostasis and mediates effector differentiation c3a modulates il-1beta secretion in human monocytes by regulating atp efflux and subsequent nlrp3 inflammasome activation the chemotactic receptor for human c5a anaphylatoxin evidence for a functional role of the second c5a receptor c5l2 an anti-inflammatory function for the complement anaphylatoxin c5a-binding protein, c5l2 extrahepatic complement biosynthesis: where, when and why? multiple organ infection and the pathogenesis of sars cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection activation of the nlrp3 inflammasome by iav virulence protein pb1-f2 contributes to severe pathophysiology and disease pb1-f2 peptide derived from avian influenza a virus h7n9 induces inflammation via activation of the nlrp3 inflammasome nlrp3 is required for complement-mediated caspase-1 and il-1beta activation in ich intracellular complement activation-an alarm raising mechanism? inflammasome-derived exosomes activate nf-kappab signaling in macrophages structure and function of the spleen key: cord-256086-8qfeoayb authors: lin, leesa; mccloud, rachel f.; bigman, cabral a.; viswanath, kasisomayajula title: tuning in and catching on? examining the relationship between pandemic communication and awareness and knowledge of mers in the usa date: 2016-04-15 journal: j public health (oxf) doi: 10.1093/pubmed/fdw028 sha: doc_id: 256086 cord_uid: 8qfeoayb background: large-scale influenza outbreaks over the last decade, such as sars and h1n1, have brought to global attention the importance of emergency risk communication and prompted the international community to develop communication responses. since pandemic outbreaks are relatively infrequent, there is a dearth of evidence addressing the following questions: (i) have the resources invested in strategic and routine communication for past pandemic outbreaks yielded public health preparedness benefits? (ii) have past efforts sensitized people to pay attention to new pandemic threats? the middle east respiratory syndrome (mers) that was followed closely by major media outlets in the usa provides an opportunity to examine the relationship between exposure to public communication about epidemics and public awareness and knowledge about new risks. methods: in december, 2013, we surveyed a nationally representative sample of 627 american adults and examined the associations between people's awareness to prior pandemics and their awareness of and knowledge about mers. results: awareness of prior pandemics was significantly associated with awareness and knowledge of mers. the most common sources from which people first heard about mers were also identified. conclusions: communication inequalities were observed between racial/ethnic and socioeconomic positions, suggesting a need for more effective pandemic communication. public health practitioners face unique challenges when developing and implementing risk communication in times of emergencies, as there is limited information on the nature of the threat (including limited data regarding mortality and morbidity, transmission modes, and prevention measures), limited response time, the potential for severe health and economic consequences, media hype and public concern. all of these factors coalesce and intertwine with the diverse social and individual characteristics of the audience when developing emergency risk communication strategy. 1 -5 the need for effective communication plans that enable coherent, credible and timely communication and community engagement during public health emergencies is increasingly being seen as integral to emergency response and planning. 6, 7 one area where emergency response and planning is key is with large-scale disease outbreaks. over the last decade, there has been a succession of large-scale outbreaks of influenza, including the sars, avian flu (h5n1), new bird flu (h7n9) and h1n1 pandemics. these outbreaks raised fears among both scientists and laypeople that an emerging influenza outbreak could repeat the devastation of the spanish flu of 1918. governments and public health agencies recognize the importance of emergency risk communication and have invested significant resources in the development and implementation of public health and communication responses to these outbreaks. the stakes of conducting emergency risk communication are even higher during the early stages of an outbreak, as a treatment and/or vaccine is unlikely to be available for at least several weeks or months after the start of a pandemic. emergency risk communication, such as raising awareness of the disease and promoting health prevention behaviors like hand washing, social distancing and cautioning vigilance among others, plays a vital role in controlling disease transmission. 2, 6, 7 one key importance is to study the association between social and individual factors and communication inequalities-differences among people from different socioeconomic positions (seps), racial, ethnic and geographical backgrounds, to understand how individuals access, interpret and act on messages they have received 1,5,8 -13 and to identify the best ways to quickly and effectively reach diverse populations with important preventive information. for example, low sep individuals have been found to have lower levels of awareness and knowledge regarding pandemics, leading to poorer behavioral responses when dealing with an outbreak. 2,14 -17 however, as pandemic outbreaks are relatively infrequent, there has been a lack of evidence assessing whether the efforts and resources invested in the strategic risk communication during past pandemic outbreaks yielded public health benefits that improved preparedness. gaining an understanding of whether or not the messages emphasized during the response to previous pandemics helped the public, particularly members of low sep populations, become more aware and better prepared is invaluable. therefore, the question that remains to be answered is: does an awareness of past epidemic risk communication help people become more health aware of an emerging epidemic, or, on the contrary, have the past few pandemic communication experiences created a 'boy cries wolf ' effect, making people less attentive to information provided concerning pandemic outbreaks? 18, 19 in late 2012, the middle east respiratory syndrome (mers), a viral illness caused by a coronavirus, was first reported in saudi arabia. although this particular virus had a very low probability of impacting the usa, major media outlets followed it closely, providing the american general public with an opportunity to become familiar with the outbreak. in this study, we assessed people's awareness of previous pandemic outbreaks and how that awareness affected their awareness and knowledge of mers. we identified other predictors of mers awareness and knowledge and investigated the information sources from which people who had heard of mers first learned about it. we also identified a subgroup of people who had have not heard of most or all risk communication messages regarding five pandemics (i.e. sars, avian flu (h5n1), new bird flu (h7n9), h1n1, and mers) which have erupted in the past decade. the analyses in this paper will help inform and calibrate strategic risk communication during a future pandemic. the data for this study, collected from 17 to 31 december 2013, were drawn from a nationally representative sample of us adults aged 18 and older. the survey instrument was adapted from previously tested communication surveys we developed based on focus groups, cognitive testing results and the national cancer institute health information national trends survey. 20 -22 respondents participated in knowledge networks' knowledgepanel w and were recruited using a dual sampling frame, which is a combination of random digit dial and address-based sampling, thus allowing for sampling of individuals with no telephone landlines. once recruited into the study, participants completed an internetbased survey in their home including questions about demographics, pandemic awareness and mers-specific topics. households were provided with internet access and necessary hardware if needed. post-stratification weights were used to adjust for non-coverage and non-responder bias. the survey included an online field experiment that is not the focus of this analysis, including the experimental conditions as a covariate did not materially alter the pattern of findings and therefore they are not reported in this analysis. independent variables † awareness of previous pandemic outbreaks was assessed by asking the participants: 'have you heard of [1] sars, [2] h1n1 or swine flu, [3] bird flu or avian flu (h5n1), [4] new bird flu or influenza a(h7n9) and [5] mers (also called mers-cov, middle east respiratory syndrome, novel coronavirus, or ncov), in the past 10 years?' two awareness variables were created based on respondents' answers: † awareness of pandemic outbreaks prior to mers: respondents were categorized into three groups based on their response to diseases (1) to (4): low (heard of 2 outbreaks), medium (heard of three outbreaks) and high (heard of all four outbreaks). † low pandemic awareness: including mers and the four prior pandemics listed above, those who have heard of only one or none of any of them were labeled to be having low pandemic awareness. † age and gender. † race/ethnicity: non-hispanic white, non-hispanic black and hispanic † sep was measured by their household income ($50 000, $30 000 -49 999, $15 000 -29 999, $14 999) and education (bachelor's degree or higher, some college, high school, less than high school). for the purpose of this study, to measure respondents' knowledge about mers and to ensure its accuracy and equal accessibility to all, we referred to centers for disease control and to account for randomly guessed responses, correct answers are discounted if the respondents also selected incorrect answers. a score of 2 was given if the following correct statements were checked: [someone can get mers from] 'being in close contact with someone who has mers (within arm's length of someone)' and 'no, there is not a vaccine against mers' and none of the following wrong options were checked: [someone can get mers from] 'eating chicken', 'coming in contact with chicken', 'eating pigs', 'coming in contact with pigs' or 'none of the above.' a score of 1 was given if either one of the two correct statements and none of the wrong ones were checked. a score of 0 was given to any other combination of responses. † source of initial mers information: participants were asked to report the source where they first learned about mers. a descriptive analysis was conducted to explore the characteristics of the surveyed sample (table 1 ). in table 2 , logistic and ordered logistic regressions, respectively, were conducted to evaluate the associations between awareness of previous pandemic outbreaks in the past 10 years, socio-demographic factors and (i) awareness of mers (model 1) and (ii) knowledge levels about mers (model 2). using cross-tabulations and x 2 , we identified the associations between respondents' socio-demographic characteristics and the sources from which they first received the information about mers. lastly, we ran logistic regression to determine the predictors of having very little awareness of previous major pandemic outbreaks (including mers) in the past decade. the associations between low awareness of previous pandemic outbreaks and socio-demographic factors were examined, and the results are presented in table 3 . stata w version 11 was used for all analyses. there were 627 respondents who participated in the study, reflecting a response rate of 31.5%. these respondents had a high awareness of the four pandemic outbreaks that affected international communities prior to mers. the prior pandemics were sars, h1n1, avian flu (h5n1) and the new bird flu (h7n9). more than half of the sample (52%) had heard of all four of them and about ninety percent (89%) were aware of at least two outbreaks. specifically, the recent h1n1 pandemics are the best known outbreaks among the sample population, 91% (n ¼ 551) of them have heard of h1n1, followed by avian flu (h5n1) (87%, n ¼ 495), sars (75%, n ¼ 405) and the new bird flu (h7n9) (59%, n ¼ 327). only one-third of the respondents had heard of mers (33%, n ¼ 144); among those who have heard of mers, more than half (52%) received a knowledge score of 2, one quarter had a knowledge score of 1, and the rest (23%), those who had no or incorrect knowledge about how mers spreads, received a score of 0. more information on sample characteristics is presented in table 1 . to inform future pandemics risk communication strategies for future pandemics, we further investigated the following: (i) among those who had heard of mers, what were the sources they used to first learn about it? (ii) among those who have low awareness of pandemics, who are they and what are their background characteristics? our data showed that national news network (18%) and local news television stations (14%), family and friends (8%) and internet-based search engine such as google or bing (6%) are the most commonly used information sources from which the respondents first learned about mers. social media such as facebook, twitter, googleþ, etc. had only minimal contributions as sources of pandemic information (,5%). forty percent of those who had heard of mers said that they could not recall where they first learned about the virus. people with low awareness of pandemics in the past decade among the surveyed population, 8% (n ¼ 64) had never heard of any of the pandemic outbreaks which had occurred in the past decade, including mers and the four pandemics prior to it, as discussed above, and 2% (n ¼ 37) had only heard of one outbreak, of which most respondents reported hearing of h1n1. the logistic regression analysis, seen in what is already known on this topic? the need for effective communication plans that enable coherent, credible and timely communication and community engagement during public health emergencies is increasingly being seen as integral to emergency response and planning. 6, 7 taking population diversity into consideration when developing risk communication plans has been shown to improve responding agencies' risk communication capabilities and, ultimately, the effectiveness of the response, especially in communities with limited local capacity. 24, 25 this lesson was reinforced by the experience of recent international pandemic outbreaks of diseases and viruses such as the sars, avian flu and h1n1 when the constructs of strategic risk communication such as public awareness, media exposure and knowledge about specific threats were further identified and assessed. 1,7,13,19,26 -29 studies confirmed that awareness of media reporting about current threats, general news exposure, people's attitudes and beliefs and people's knowledge about a specific threat are positively associated with a person's knowledge about a specific threat and their adoption of recommended prevention behaviors. 2,9,10,30 -37 main finding of this study in this study, awareness of prior pandemics was significantly associated with both awareness of a new threat, mers, and higher knowledge levels regarding it; racial disparities were found in awareness and knowledge levels of mers. there was no evidence that having heard about pandemics that occurred prior to mers had a 'boy cries wolf ' effect, in which people tuned out information about mers. however, we found that individuals who were younger, had lower income or had less than a bachelor's degree were more likely to report having no awareness of previous pandemics compared with their counterparts. national and local tv networks were the most commonly used information sources from which people first heard about mers. this finding is consistent with previous studies, in which national news networks and/or local news television stations were found to be the most effective channels through which to convey public health messages, while the impact of social media was found to be surprisingly small. 4 what this study adds? increasing awareness alone may not be enough to prompt preventive action, particularly among diverse groups. pandemic communication need to contain clear, comprehensible information about the pandemic offered through trusted, commonly accessed media channels, such as national and local tv networks. customizing messages about risk to one's intended audience and communicating these messages to them via appropriate information channels are instrumental to running an effective communication campaign. 38 -44 it is notable that minority participants had both lower awareness of and less correct knowledge about mers and that individuals with lower education and lower income were less likely to have an awareness of any pandemic, indicating the presence of communication inequalities in pandemic awareness among these subgroups. more research is needed on the awareness and knowledge of future pandemics in a diverse low sep sample to best understand the impact of communication inequalities and how to address them through targeted campaigns. the current findings indicate a need to pay attention to segments that may not be actively seeking out information and to deliver it via channels that they use. given the fact that few people reported that they had first learned about mers through social media, our data suggested that national media such as tv are still important and social media, at least in times of pandemics, appear to be less effective. emergency risk communication has to be strategic, evidence-based and must take into account potential communication inequality. the data for this study are cross-sectional in nature and thus limit us from drawing a causal relationship between the independent and dependent variables. nevertheless, this study finds a link between having heard of prior pandemics and knowledge and awareness of a subsequent pandemic (i.e. mers) that should be further investigated. future studies using experimental, longitudinal or case -control designs could help provide evidence for causal relationships. although the data rely on self-reporting, our survey items were adopted from widely tested national surveys and validated by cognitive testing. the response rate for the survey was 31.5%. poststratification weights were used to adjust for non-coverage and non-responder bias. in the case of the 2009/2010 h1n1 flu pandemics, mexicans and other latinos living in the usa were more likely to be stigmatized by non-hispanic americans as carriers of the virus, partly because of news reports on the outbreak's alleged origin in mexican pig farms. 3 hispanic americans also reported higher levels of risk perceptions of the flu. 45 therefore, in light of the origin of mers, it could be useful for emergency risk communication scientists to further investigate the possible association between knowledge and awareness levels of the mers virus and subsets of populations in the usa with potential personal or family ties to the outbreak regions (e.g. middle eastern migrants). this study found that awareness of past pandemics was associated with higher awareness of and correct knowledge about the 2012/2013 mers outbreak. despite these associations, the overall level of awareness of this new threat was low and communication inequalities were observed between racial/ ethnic and low sep groups. results suggest that awareness of past pandemics might indicate that an individual is more likely to have heard about a new threat, and that more research is needed to discover barriers to awareness that may be present in lower sep samples. emergency risk communication has to be strategic, evidence based and must take into account potential communication inequality. this project was funded by the harvard school of public health preparedness and emergency response center (harvard perrc)-linking assessment and measurement to performance in phep systems (lamps), cdc grant number: 5po1tp000307-05. the content of this publication as well as the views and discussions expressed in this paper are solely those of the authors and do not necessarily represent the views of any partner organizations, the cdc or the us department of health and human services nor does mention of trade names, commercial practices or organizations imply endorsement by the us government. race, ethnicity, language, social class, and health communication inequalities: a nationally-representative cross-sectional study media use and communication inequalities in a public health emergency: a case study of 2009 -2010 pandemic influenza a virus subtype h1n1 the h1n1 pandemic: media frames, stigmatization and coping implementation of nonpharmaceutical interventions by new york city public schools to prevent 2009 influenza a communication inequalities during public health disasters: katrina's wake communications in public health emergency preparedness: a systematic review of the literature what have we learned about communication inequalities during the h1n1 pandemic: a systematic review of the literature preparing racially and ethnically diverse communities for public health emergencies individual differences in behavioral reactions to h1n1 during a later stage of the epidemic statement to the press: world now at the start of 2009 influenza pandemic. geneva: world health organization socioeconomic status, demographics, beliefs and a(h1n1) vaccine uptake in the united states socioeconomic status and health communication inequalities in japan: a nationwide crosssectional survey communications under uncertainty: communication behaviors of diverse audiences during the a(h1n1) incidence of spring and summer getting vaccinated or not getting vaccinated? different reasons for getting vaccinated against seasonal or pandemic influenza early responses to h7n9 in southern mainland china awareness, anxiety, compliance: community perceptions and response to the threat and reality of an influenza pandemic chinese urban-rural disparity in pandemic (h1n1) 2009 vaccination coverage rate and associated determinants: a cross-sectional telephone survey public health communications and alert fatigue implications of public understanding of avian influenza for fostering effective risk communication health information national trends survey (hints) 2007: final report. bethesda, md: national cancer institute hints 2005): final report. bethesda, md: national cancer institute health information national trends survey middle east respiratory syndrome (mers) effective health risk communication about pandemic influenza for vulnerable populations disaster planning and risk communication with vulnerable communities: lessons from hurricane katrina infectious diseases and governance of global risks through public communication and participation perceived threat, risk perception, and efficacy beliefs related to sars and other (emerging) infectious diseases: results of an international survey mass media and population health: a macrosocial view early response to the emergence of influenza a(h7n9) virus in humans in china: the central role of prompt information sharing and public communication the impact of communications about swine flu (influenza a hiniv) on public responses to the outbreak: results from 36 national telephone surveys in the uk situational awareness and health protective responses to pandemic influenza a (h1n1) in hong kong: a cross-sectional study community psychological and behavioral responses through the first wave of the 2009 influenza a(h1n1) pandemic in hong kong knowledge and practices towards influenza a (h1n1) among adults in three residential areas in tampin negeri sembilan: a cross sectional survey surveillance of perceptions, knowledge, attitudes and behaviors of the italian adult population (18-69 years) during the 2009 -2010 a/h1n1 influenza pandemic preventive behaviors, beliefs, and anxieties in relation to the swine flu outbreak among college students aged 18 -24 years knowledge, attitudes, and behaviors of low-income women considered high priority for receiving the novel influenza a (h1n1) vaccine. matern child health h1n1 preventive health behaviors in a university setting socioecological and message framing factors influencing maternal influenza immunization among minority women message framing strategies to increase influenza immunization uptake among pregnant african american women patient knowledge and recall of health information following exposure to 'facts and myths' message format variations message formats and their influence on perceived risks of tobacco use: a pilot formative research project in india providing health messages to hispanics/latinos: understanding the importance of language, trust in health information sources, and media use did h1n1 influenza prevention messages reach the vulnerable population along the mississippi gulf coast? trust influences response to public health messages during a bioterrorist event public risk perceptions and preventive behaviors during the 2009 h1n1 influenza pandemic key: cord-261041-nrmj1qre authors: algaissi, abdullah; hashem, anwar m. title: evaluation of mers-cov neutralizing antibodies in sera using live virus microneutralization assay date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_9 sha: doc_id: 261041 cord_uid: nrmj1qre the microneutralization (mn) assay is a standard and important technique in virology, immunology, and epidemiology. it is a highly specific and sensitive assay for evaluating virus-specific neutralizing antibodies (nabs) in human and animal sera. it provides the most precise answer to whether or not an individual or animal has antibodies that can neutralize or inhibit the infectivity of a specific virus strain. however, using live virus-based mn assay might require working under high containment facilities especially when dealing with high-risk pathogens such as the middle east respiratory syndrome-coronavirus (mers-cov). in this chapter, we describe the isolation, amplification, and titration of mers-cov, as well as detailed mn assay to measure nab levels in sera from different mammalian species. the middle east respiratory syndrome-coronavirus (mers-cov) is a novel zoonotic β-coronavirus that was first identified in saudi arabia in 2012 [1] . epidemiological evidence suggests that dromedary camels are the main zoonotic source of mers-cov [2, 3] . mers-cov causes a wide range of manifestations ranging from asymptomatic infections to mild or severe respiratory disease. detection of anti-mers-cov antibodies (abs) in humans and/or animals represents a valuable tool in diagnostics as well as epidemiological, virological, and immunological studies including evaluation of vaccine immunogenicity [4] [5] [6] [7] [8] . several serological assays have been developed and used for mers-cov, including elisabased assays, immunofluorescence assays, protein microarrays, and pseudovirus-based neutralization assays [9] [10] [11] [12] [13] [14] [15] [16] [17] . however, most of these assays pose several drawbacks and limitations such as low specificity and sensitivity, need for expensive and special equipment and reagents, and/or highly trained technical staff, which could limit their use. on the other hand, live virus-based microneutralization (mn) assay is a highly sensitive and specific technique used for the quantitation of virus-specific neutralizing antibodies (nabs) to a given virus in mammalian sera as well as the evaluation of antiviral activities of small molecules and biologics. the assay has several advantages in detecting nabs against mers-cov. it can precisely detect virus-specific nabs in human and animal sera without the need for specific reagents or equipment, it can be carried out readily once the virus is isolated, and it can overcome strain-specific antigenic changes. the protocol presented in this chapter consists of four major steps, including mers-cov isolation, amplification, titration, and neutralization. the mn assay described here is suitable to quantitatively measure the titer of nabs in sera from different mammalian species. proper aseptic techniques should be used, and all equipment and solutions to be used with cells must be sterile. all cell culture incubations should be performed in a humidified 37 c incubator with 5% co 2 , and all solutions should be pre-warmed to room temperature or 37 c before use with cells. 1. remove a cryovial of frozen vero e6 cells from liquid nitrogen storage and quickly thaw the cells for <1 min by gently swirling in a 37 c water bath until there is just a small bit of ice left in the vial (see note 1). 2. transfer the vial into a laminar flow biosafety cabinet and quickly disinfect the outside of the vial with 70% ethanol. 3. open the vial and transfer all the volume to a sterile 15 ml falcon tube containing 10 ml of pre-warmed m-10 (see note 2). 4. centrifuge the cell suspension at approximately 200-500 â g for 5 min at room temperature. 5. aseptically decant the supernatant without disturbing the cell pellet (see note 3). 6. gently re-suspend the cells in 10 ml of pre-warmed m-10 (see note 4). 7. transfer the cell suspension to t75 tissue culture flask using sterile 10 ml serological pipette. 8. incubate the flask in 37 c incubator with 5% co 2 . 10. when cells reach >90-95% confluency, passage cells into new tissue culture flasks at 1:5 to 1:10 split ratio (see notes 6 and 7). 11. maintain cells in continuous culture and passage them as needed for at least 2-3 passages after removal from long-term storage and before use. amplification of mers-cov 1. harvest confluent vero e6 cells from a t75 tissue culture flask using standard trypsinization procedure (see note 7). 2. count cells using cell counter or hemocytometer and prepare a cell suspension of 5 â 10 5 cells/ml in pre-warmed m-10. 3. seed 5 ml (~2.5 â 10 6 cells) or 20 ml (~1 â 10 7 cells) of the cell suspension into a t25 or t175 tissue culture flask, respectively, so that they are 90-95% confluent the next day (see note 8). 4. incubate the flasks in 37 c incubator with 5% co 2 for overnight. 5. next day, change the media by removing old media and add 2 ml or 5 ml of fresh pre-warmed m-2 into a t25 or t175 tissue culture flask, respectively. 6. if using positive mers-cov sample, filter sterilize samples using sterile 0.22 μm γ-irradiated syringe filters before inoculation onto vero e6 cells (see note 9). 7. add 0.5-1 ml of isolated mers-cov or filtered positive sample to the cells (see note 10). 8. distribute the virus evenly over the cells and incubate for 1 h in 37 c incubator with 5% co 2 . 9. make up the final volume of media to 5 ml or 20 ml in t25 or t175 tissue culture flasks, respectively. 10. incubate the flask in 37 c incubator with 5% co 2 for 2-3 days or until significant cytopathic effect (cpe) is observed (fig. 1 ). 11. check the flask daily post-infection (see note 11). 12. when cpe is >50%, collect supernatant from the flask and centrifuge at 500 â g for 5 min to remove cellular debris (fig. 1 ). 13. aliquot collected clarified supernatant in 100 μl or 1 ml aliquots in sterile 1.5 ml tubes and store at à80 c (see note 12). culture infective dose 50 (tcid 50 ) 1. harvest confluent vero e6 cells from the t75 tissue culture flask using standard trypsinization procedure (see note 7). 2. count the cells using cell counter or hemocytometer and prepare a cell suspension of 1 â 10 5 cells/ml in pre-warmed m-10. re-suspend 1 â 10 6 cells in 10 ml per 96-well plate. 3. seed 1 â 10 4 vero e6 cells (100 μl) per well into sterile 96-well tissue culture plate so that they are 90-95% confluent the next day (see note 8). 4. incubate the plate in 37 c incubator with 5% co 2 for overnight. 5. next day, in a new sterile u-shaped 96-well plate, add 135 μl pre-warmed m-2 to all wells (fig. 2 ). 6. add 15 μl of mers-cov per well in all wells of column 1 to have 1:10 dilution (fig. 2) . 7. perform tenfold serial (log10) dilution by transferring 15 μl progressively from column to column (fig. 2) 14. calculate tcid 50 using reed-muench formula [18] . 2. count the cells using cell counter or hemocytometer and prepare a cell suspension of 1 â 10 5 cells/ml in pre-warmed m-10. re-suspend 1 â 10 6 cells in 10 ml per 96-well plate. 3. seed 1 â 10 4 vero e6 cells (100 μl) per well into sterile 96-well tissue culture plate so that they are 90-95% confluent the next day (see note 8). 4. incubate the plate in 37 c incubator with 5% co 2 overnight. 5. next day, heat-inactivate test sera to be used for virus microneutralization by incubation for 30 min at 56 c. 6 . in a new sterile u-shaped 96-well plate, add 60 μl pre-warmed m-2 to all wells (fig. 3) . 7. add an additional 48 μl pre-warmed m-2 to wells a1-a10 in row a (fig. 3 ). 8. add 12 μl heat-inactivated serum per well in wells a1-a10 in row a to have 1:10 dilution (fig. 3) . do not add serum to a11 and a12 (see note 17) . . perform twofold serial dilutions on added serum samples by transferring 60 μl progressively from row to row (i.e., a1 to b1; b1 to c1; etc. up to g1 to h1) using a multichannel pipette (fig. 3) . 10 . during each dilution step mix well by pipetting eight times up and down (see note 14) . 11. discard the final 60 μl after row h. 12. prepare virus suspension in pre-warmed m-2 so that 60 μl contains 120 tcid 50 (i.e., 2 â 10 3 tcid 50 /ml). approximately 6 ml/plate is needed (see note 18). 13. add 60 μl diluted virus to all wells except wells in columns 12 (cc wells). 14. add 60 μl pre-warmed m-2 to all cc wells (i.e., columns 12). 15. incubate the serum-virus mixtures for 1 h in 37 c incubator with 5% co 2 . 16. remove the 96-well tissue culture plate containing confluent vero e6 cells and aspirate the media (see note 15) . 18. incubate the 96-well tissue culture plate in 37 c incubator with 5% co 2 for 3 days (see note 11) . 19. calculate mn 50 or mn 100 titers of each serum sample as the highest serum dilution that completely protect the cells from cpe in half or all wells, respectively. 1. the water bath is a potential source of contamination. to reduce the risk of contamination, keep the o-ring and cap of the cryovial out of the water. 2. frozen cell stocks contain dimethyl sulfoxide (dmso), which is harmful to the cells, and it should be diluted and removed after thawing the cells and before transferring the cells to tissue culture flasks. 3. after centrifugation, check the clarity of the supernatant and visibility of a complete pellet. 4. different volumes and culture vessels could be used. it is better to initiate vero e6 cells culture in a t25 tissue culture flask. if using a t25 tissue culture flask, re-suspend the cells in 5 ml media, and if using t75 tissue culture flask, re-suspend the cells in 10 ml media. 5. vero e6 cells recover slowly after freezing and may take more than a week before they are ready to be passaged. it may take 2-3 passages before the vero e6 cells reach their normal growth rate. 6. it is important to monitor vero e6 cells and to subculture them once confluent. depending on the number of seeded cells and the size of the used flask, vero e6 cells usually need to be passaged 2-3 times per week. to harvest or maintain vero e6 cells, remove media from the flask, wash the cell monolayer gently with 3-5 ml of sterile pre-warmed dpbs without calcium or magnesium, and discard the used washing solution. add 2-5 ml pre-warmed 1â trypsin-edta in dpbs without calcium or magnesium to the cell monolayer and incubate for 5-10 min at 37 c, 5% co 2 to detach cells (incubation may vary, so check the cells every 2-3 min). after cells are detached, add 5 ml pre-warmed m-10 to the flask to inactivate trypsin activity, and collect detached cells in 15 ml sterile falcon tube. make sure to centrifuge the collected cells and discard the supernatant. then, add new 1-2 ml pre-warmed m-10 and re-suspend the cells by pipetting up and down using 1 ml pipette to make a homogenous cell suspension. 11. cpe could be strain specific, and it depends on the strain and starting titer of the seed virus. 12. each tube should be used once only to avoid freezing and thawing as this can significantly decrease the virus titer. use 1 ml aliquots tubes for virus amplification. 13. other dilutions such as ½ log10 dilution could be used. 14. change pipette tips between wells. 15. avoid cell drying by minimizing the time between media aspiration and adding the virus inoculum or the serum-virus mixtures. 16. alternatively, remove media from cells and fix cells with 100 μl ice-cold 4% paraformaldehyde for 5 min at room temperature. remove fixative and stain cells with 100 μl crystal violet (0.05% w/v) in 20% methanol for 30 min at room temperature, and wash cells in tap water. score wells as positive for mers-cov (i.e., no crystal violet) or negative for mers-cov (i.e., cells are stained with crystal violet). 17. for each serum sample, 12 μl are needed per single test; however, sera should be tested in at least duplicates, so more volume is needed. different plates should be used when testing neutralization against different virus strains. 18. set up back virus titration to ensure working virus concentration is accurate. starting with the working virus dilution (2 â 10 3 tcid 50 /ml), prepare twofold serial dilution in pre-warmed m-2 in a final volume of 60 μl (4 replicates per dilution). after dilution, add 60 μl of pre-warmed m-2 to each well for a final volume of 120 μl and incubate for 1 h in 37 c incubator with 5% co 2 . then, transfer 100 μl to vero e6 cells in 96-well tissue culture plate and incubate for 3 days in 37 c incubator with 5% co 2 . after incubation, examine the plate for cpe and calculate tcid 50 using reed-muench formula. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-to-human transmission of mers coronavirus kinetics and pattern of viral excretion in biological specimens of two mers-cov cases presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses immunogenicity of candidate mers-cov dna vaccines based on the spike protein seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt specific serology for emerging human coronaviruses by protein microarray hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity inability of rat dpp4 to allow mers-cov infection revealed by using a vsv pseudotype bearing truncated mers-cov spike protein comparison of serological assays in human middle east respiratory syndrome (mers)-coronavirus infection characterization of novel monoclonal antibodies against the mers-coronavirus spike protein and their application in speciesindependent antibody detection by competitive elisa inclusion of mers-spike protein elisa in algorithm to determine serologic evidence of mers-cov infection development and validation of different indirect elisas for mers-cov serological testing a simple method of estimating fifty per cent endpoints this work was supported by king abdulaziz city for science and technology (kacst) through the mers-cov research grant program (number 09-1 to amh), which is a part of the targeted research program. key: cord-284286-qfl6hehj authors: woo, patrick c. y.; lau, susanna k. p.; fan, rachel y. y.; lau, candy c. y.; wong, emily y. m.; joseph, sunitha; tsang, alan k. l.; wernery, renate; yip, cyril c. y.; tsang, chi-ching; wernery, ulrich; yuen, kwok-yung title: isolation and characterization of dromedary camel coronavirus uae-hku23 from dromedaries of the middle east: minimal serological cross-reactivity between mers coronavirus and dromedary camel coronavirus uae-hku23 date: 2016-05-07 journal: int j mol sci doi: 10.3390/ijms17050691 sha: doc_id: 284286 cord_uid: qfl6hehj recently, we reported the discovery of a dromedary camel coronavirus uae-hku23 (dccov uae-hku23) from dromedaries in the middle east. in this study, dccov uae-hku23 was successfully isolated in two of the 14 dromedary fecal samples using hrt-18g cells, with cytopathic effects observed five days after inoculation. northern blot analysis revealed at least seven distinct rna species, corresponding to predicted subgenomic mrnas and confirming the core sequence of transcription regulatory sequence motifs as 5′-ucuaaac-3′ as we predicted previously. antibodies against dccov uae-hku23 were detected in 58 (98.3%) and 59 (100%) of the 59 dromedary sera by immunofluorescence and neutralization antibody tests, respectively. there was significant correlation between the antibody titers determined by immunofluorescence and neutralization assays (pearson coefficient = 0.525, p < 0.0001). immunization of mice using recombinant n proteins of dccov uae-hku23 and middle east respiratory syndrome coronavirus (mers-cov), respectively, and heat-inactivated dccov uae-hku23 showed minimal cross-antigenicity between dccov uae-hku23 and mers-cov by western blot and neutralization antibody assays. codon usage and genetic distance analysis of rdrp, s and n genes showed that the 14 strains of dccov uae-hku23 formed a distinct cluster, separated from those of other closely related members of betacoronavirus 1, including alpaca cov, confirming that dccov uae-hku23 is a novel member of betacoronavirus 1. coronaviruses (covs) are found in a wide variety of animals [1] in which they can cause respiratory, enteric, hepatic, and neurological diseases. based on genotypic and serological characterization, covs were traditionally classified into three distinct groups [2] [3] [4] . recently, the coronavirus study group of the international committee for taxonomy of viruses has replaced the traditional groups 1, 2, and 3 covs with three genera, alphacoronavirus, betacoronavirus, and gammacoronavirus, respectively [5] . as a result of their unique mechanism of viral replication, covs have a high frequency of recombination [2, 6] . their tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches [7] [8] [9] [10] . in 2003, the severe acute respiratory syndrome (sars) epidemic, discovery of sars-cov, and identification of sars-cov-like viruses from himalayan palm civets from wild live markets in china boosted interest in the discovery of novel covs in humans and animals [11] [12] [13] [14] [15] [16] . a novel human cov (hcov) of the genus alphacoronavirus, human cov nl63 (hcov-nl63), was reported in 2004 [17] [18] [19] . in 2005, we also described the discovery, complete genome sequence, and molecular epidemiology of another novel hcov, human cov hku1 (hcov-hku1), in the genus betacoronavirus [20] [21] [22] . as for animal covs, we and others have described the discovery of sars-cov-like viruses in chinese horseshoe bats in hong kong and other horseshoe bats in other provinces of china [23, 24] . recently, the discovery of sars-cov-like viruses in chinese horseshoe bats in yunnan has further highlighted the importance for hunting the animal origin of human infections [25] . in addition, we have also discovered 21 other animal covs, which include two novel lineages in betacoronavirus and a novel genus deltacoronavirus [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] . from our studies it was shown that bats are the gene source for alphacoronavirus and betacoronavirus and birds are the gene source for gammacoronavirus and deltacoronavirus to fuel cov evolution and dissemination [33] . in 2012, a novel cov, named middle east respiratory syndrome coronavirus (mers-cov), closely related to tylonycteris bat cov hku4 (ty-batcov hku4) and pipistrellus bat cov hku5 (pi-batcov hku5), has emerged as a cause of severe respiratory infections associated with high mortalities [37] [38] [39] [40] . it has been shown that dromedaries in the middle east possessed neutralizing antibodies against mers-cov [41, 42] . furthermore, mers-cov was also detected in the nasal swabs of dromedaries in qatar, saudi arabia, egypt, and the united arab emirates [43] [44] [45] [46] [47] . the recent emergence of mers and the discovery of mers-cov in dromedaries have boosted interest in the search for other novel viruses in dromedaries [48, 49] . in a recent molecular epidemiology study, we discovered a novel betacoronavirus, named dromedary camel cov uae-hku23 (dccov uae-hku23), from fecal samples of dromedaries from dubai [50] . in this study, we report the isolation of dccov uae-hku23 from the fecal sample of a dromedary and its characterization. of the seven cell lines inoculated with dromedary fecal samples positive for dccov uae-hku23, viral replication was detected by rt-pcr in the supernatants of hrt-18g in two of the 14 dromedary fecal samples at day 7, with viral loads of 2.6ˆ10 10 and 9.7ˆ10 6 copies/ml in hrt-18g cells in the presence of trypsin. cytopathic effects (cpe), mainly in the form of rounded, fused and granulated giant cells rapidly detaching from the monolayer, were also observed in infected hrt-18g cells five days after inoculation (figure 1a) , which showed viral nucleocapsid expression by immunofluorescence in 20% of cells. electron microscopy of ultracentrifuged cell culture extracts from infected hrt-18g cells showed the presence of cov-like particles around 70-100 nm in diameter with typical club-shaped surface projections (figure 1b ). covs are characterized by a unique mechanism of discontinuous transcription with the synthesis of a nested set of subgenomic mrnas [51, 52] . to assess the number and size of dccov uae-hku23 subgenomic mrna species, northern blot analysis with a probe specific to the nucleocapsid sequence was performed. at least seven distinct rna species were identified, with the sizes corresponding to predicted subgenomic mrnas of ns2 (~9600 bp), hemagglutinin-esterase (he) (~8760 bp), spike (s) (~7460 bp), ns5 (~3040 bp), envelope (e) (~2800 bp), membrane (m) (~2410 bp) and nucleocapsid (n) (~1710 bp) (figure 2a ). covs are characterized by a unique mechanism of discontinuous transcription with the synthesis of a nested set of subgenomic mrnas [51, 52] . to assess the number and size of dccov uae-hku23 subgenomic mrna species, northern blot analysis with a probe specific to the nucleocapsid sequence was performed. at least seven distinct rna species were identified, with the sizes corresponding to predicted subgenomic mrnas of ns2 (~9600 bp), hemagglutinin-esterase (he) (~8760 bp), spike (s) (~7460 bp), ns5 (~3040 bp), envelope (e) (~2800 bp), membrane (m) (~2410 bp) and nucleocapsid (n) (~1710 bp) (figure 2a) . covs are characterized by a unique mechanism of discontinuous transcription with the synthesis of a nested set of subgenomic mrnas [51, 52] . to assess the number and size of dccov uae-hku23 subgenomic mrna species, northern blot analysis with a probe specific to the nucleocapsid sequence was performed. at least seven distinct rna species were identified, with the sizes corresponding to predicted subgenomic mrnas of ns2 (~9600 bp), hemagglutinin-esterase (he) (~8760 bp), spike (s) (~7460 bp), ns5 (~3040 bp), envelope (e) (~2800 bp), membrane (m) (~2410 bp) and nucleocapsid (n) (~1710 bp) (figure 2a ). by determining the leader-body junction sequences of subgenomic mrnas from dccov uae-hku23-infected cell cultures, the subgenomic mrna sequences were aligned to the leader sequence which confirmed the core sequence of the transcription regulatory sequences (trs) motifs as 5′-ucuaaac-3′ (figure 2b ), as in other betacoronavirus lineage a covs [53] [54] [55] [56] . the leader trs and subgenomic mrna of ns2, he, s, and n exactly matched each other, whereas there was one base mismatch for ns5, e, and m. these results are in line with our previous prediction on the trs [50] . the dccov uae-hku23 common leader on subgenomic mrnas was confirmed as the first 65 nucleotides of the dccov uae-hku23 genome. fifty-nine serum samples from dromedaries were subject to immunofluorescence and neutralization antibody assays for dccov uae-hku23. results were positive in 58 (98.3%) and 59 (100%) of the 59 samples as detected by immunofluorescence and the neutralization antibody test, respectively (table 1 ; figure 1c ,d). in addition, there was significant correlation between the titers determined by the immunofluorescence antibody test and the neutralization antibody assay (pearson coefficient = 0.525, p < 0.0001) (figure 3 ). by determining the leader-body junction sequences of subgenomic mrnas from dccov uae-hku23-infected cell cultures, the subgenomic mrna sequences were aligned to the leader sequence which confirmed the core sequence of the transcription regulatory sequences (trs) motifs as 5 1 -ucuaaac-3 1 (figure 2b ), as in other betacoronavirus lineage a covs [53] [54] [55] [56] . the leader trs and subgenomic mrna of ns2, he, s, and n exactly matched each other, whereas there was one base mismatch for ns5, e, and m. these results are in line with our previous prediction on the trs [50] . the dccov uae-hku23 common leader on subgenomic mrnas was confirmed as the first 65 nucleotides of the dccov uae-hku23 genome. fifty-nine serum samples from dromedaries were subject to immunofluorescence and neutralization antibody assays for dccov uae-hku23. results were positive in 58 (98.3%) and 59 (100%) of the 59 samples as detected by immunofluorescence and the neutralization antibody test, respectively (table 1 ; figure 1c ,d). in addition, there was significant correlation between the titers determined by the immunofluorescence antibody test and the neutralization antibody assay (pearson coefficient = 0.525, p < 0.0001) ( figure 3 ). to examine possible cross-antigenicity between the dccov uae-hku23 and mers-cov n proteins, recombinant n proteins of dccov uae-hku23 and mers-cov were cloned and purified and tested against serum samples of balb/c mice immunized with the n proteins of dccov uae-hku23 and mers-cov, respectively. the highest dilutions of the serum, obtained from the mouse immunized with the dccov uae-hku23 n protein, which generated immunoreactive bands in the western blot assay, were 1:64,000 and 1:2000, respectively, when the dccov uae-hku23 n to examine possible cross-antigenicity between the dccov uae-hku23 and mers-cov n proteins, recombinant n proteins of dccov uae-hku23 and mers-cov were cloned and purified and tested against serum samples of balb/c mice immunized with the n proteins of dccov uae-hku23 and mers-cov, respectively. the highest dilutions of the serum, obtained from the mouse immunized with the dccov uae-hku23 n protein, which generated immunoreactive bands in the western blot assay, were 1:64,000 and 1:2000, respectively, when the dccov uae-hku23 n protein and mers-cov n protein were used as the antigens, respectively (figure 4 ). the highest dilutions of the serum, obtained from the mouse immunized with the mers-cov n protein, which generated immunoreactive bands in the western blot assay, were 1:512,000 and 1:8000, respectively, when the mers-cov n protein and dccov uae-hku23 n protein were used as the antigens, respectively ( figure 4 ). protein and mers-cov n protein were used as the antigens, respectively (figure 4 ). the highest dilutions of the serum, obtained from the mouse immunized with the mers-cov n protein, which generated immunoreactive bands in the western blot assay, were 1:512,000 and 1:8000, respectively, when the mers-cov n protein and dccov uae-hku23 n protein were used as the antigens, respectively ( figure 4 ). to further examine possible cross-antigenicity between dccov uae-hku23 and mers-cov, heat-inactivated dccov uae-hku23 was used for immunization of balb/c mice and the serum obtained was tested for neutralization antibodies against dccov uae-hku23 and mers-cov, respectively. neutralization antibody assays showed that the neutralization antibody titer of the pooled serum sample was 1:160 for dccov uae-hku23 but <1:20 for mers-cov. to determine the number of genotypes/circulating strains of dccov uae-hku23, the complete rna-dependent rna polymerase (rdrp), s, and n genes of 11 additional dccov uae-hku23 strains from dromedary fecal samples positive for dccov uae-hku23 were sequenced. for the rdrp and n genes, all these 11 dccov uae-hku23 strains together with those of the three dccov uae-hku23 strains with complete genome sequences [50] possessed identical amino acid sequences, although there were 0-2 nucleotide differences among the strains for both genes ( figure 5 ). as for the s gene, phylogenetic analysis using either nucleotide or amino acid sequences revealed two clusters ( figure 5 ). there were 10 nucleotide differences among the two groups. five were synonymous substitutions, and the other five resulted in amino acid changes (v105a, s436a, l845v, s967p, and p1188s). by corresponding analysis (ca) on relative synonymous codon usage (rscu) values, patterns in codon usage were observed that allowed different groups in betacoronavirus 1 to be distinguished. the rdrp, s, and n genes of the 14 strains of dccov uae-hku23 formed a distinct cluster, separated to further examine possible cross-antigenicity between dccov uae-hku23 and mers-cov, heat-inactivated dccov uae-hku23 was used for immunization of balb/c mice and the serum obtained was tested for neutralization antibodies against dccov uae-hku23 and mers-cov, respectively. neutralization antibody assays showed that the neutralization antibody titer of the pooled serum sample was 1:160 for dccov uae-hku23 but <1:20 for mers-cov. to determine the number of genotypes/circulating strains of dccov uae-hku23, the complete rna-dependent rna polymerase (rdrp), s, and n genes of 11 additional dccov uae-hku23 strains from dromedary fecal samples positive for dccov uae-hku23 were sequenced. for the rdrp and n genes, all these 11 dccov uae-hku23 strains together with those of the three dccov uae-hku23 strains with complete genome sequences [50] possessed identical amino acid sequences, although there were 0-2 nucleotide differences among the strains for both genes ( figure 5) . as for the s gene, phylogenetic analysis using either nucleotide or amino acid sequences revealed two clusters ( figure 5 ). there were 10 nucleotide differences among the two groups. five were synonymous substitutions, and the other five resulted in amino acid changes (v105a, s436a, l845v, s967p, and p1188s). by corresponding analysis (ca) on relative synonymous codon usage (rscu) values, patterns in codon usage were observed that allowed different groups in betacoronavirus 1 to be distinguished. the rdrp, s, and n genes of the 14 strains of dccov uae-hku23 formed a distinct cluster, separated from those of other closely related members of betacoronavirus 1, including alpaca cov (dq915164) (figure 6a ) [57] . analysis of the nucleotide sequence corresponding to the rdrp, s, and n genes showed that alpaca cov has a lower genetic distance to bovine cov (bcov) and other recently isolated wild ruminant covs than to dccov uae-hku23 (figure 6b) . ( figure 6a ) [57] . analysis of the nucleotide sequence corresponding to the rdrp, s, and n genes showed that alpaca cov has a lower genetic distance to bovine cov (bcov) and other recently isolated wild ruminant covs than to dccov uae-hku23 (figure 6b ). in this study, we have successfully isolated dccov uae-hku23 using the hrt-18g cell line. with only a few exceptions, such as sars-cov and mers-cov, covs are notoriously difficult to isolate in cell lines [58, 59] . for the hcovs oc43 and 229e, they induce only subtle cpes, even if isolated. in our previous report on the discovery of dccov uae-hku23 from fecal samples of dromedaries by rt-pcr, no positive culture results were obtained from any of the three samples used for complete genome sequencing [50] . in the present study, after inoculation of all 14 fecal samples that were rt-pcr-positive for dccov uae-hku23, the virus was successfully isolated from two of the 14 samples using hrt-18g cells, with rounding and fusion to giant cells rapidly detaching from the monolayer. northern blot experiments and determination of leader-body junction sequences of subgenomic mrnas confirmed our previous predictions on the open reading frames (orfs) and trs of dccov uae-hku23 by comparison to other covs of betacoronavirus 1 [50] . although sequencing of the rdrp, s, and n genes showed that there were two or more strains circulating in the dromedaries, no obvious genotypes were observed for the 14 strains of dccov uae-hku23 in this study. since hcov-oc43, bcov [60] , equine cov, alpaca cov, the murine hepatitis virus-h2 variant, our recently discovered rabbit cov (rbcov) hku14 [34] , and dccov uae-hku23 can all replicate in hrt-18g cells, it suggests that these covs that belonged to betacoronavirus lineage a (the traditional "group 2 covs") may share similar cellular tropisms. the hrt-18g cell line could be used to isolate other members of betacoronavirus lineage a for further characterization of these covs. although dccov uae-hku23 and alpaca cov are both isolated from camelids, they represent two distinct covs. camelids (family camelidae) are classified into the old world camels (genus camelus), including dromedaries and bactrians, and the new world camels (genera lama and vicugna), including llama, guanaco, alpaca, and vicuna. since the alpaca cov, which infects alpaca, and dccov uae-hku23, which infects dromedaries, are both members of betacoronavirus 1, it raised the question whether alpaca cov and dccov uae-hku23 are the same or different covs. we therefore analyzed the codon usage bias in the rdrp, s, and n genes of these two viruses. results showed distinct codon usage bias and genetic distance for alpaca cov and dccov uae-hku23. in fact, alpaca cov showed much more similar codon usage bias and lower genetic distance to bcov and other wild ruminant covs including water buffalo cov, giraffe cov, himalayan tahr cov, nyala cov, sable antelope cov, sambar deer cov, sitatunga cov, waterbuck cov, wisent cov, and white-tailed deer cov than to dccov uae-hku23 in the rdrp, s, and n genes ( figure 6 ) [10, [61] [62] [63] [64] . in addition to codon usage and genetic distance, the s protein of dccov uae-hku23 contains one additional potential n-glycosylation site at amino acid position 492 compared to alpaca cov (data not shown). this potential n-glycosylation site is also present in the s proteins of canine respiratory coronavirus and hcov-oc43, but not in those of other wild ruminant covs. all these indicated that alpaca cov and dccov uae-hku23 are two different covs adapting to two different hosts residing in two different geographical locations (alpaca in south america and dromedaries in the middle east and north africa) in the world. minimal cross-antigenicity was observed between the n proteins of dccov uae-hku23 and mers-cov. in our study on genotyping and serological characterization of rousettus bat cov hku9 (ro-batcov hku9), we have shown that there is minimal serological cross-reactivity among the four lineages of betacoronaviruses using human serum samples positive for antibodies against the n protein of hcov-hku1 (lineage a), chinese horseshoe bat serum samples positive for antibodies against the n protein of sarsr-rh-batcov (lineage b), and leschenault's rousette bat serum samples positive for antibodies against the n protein of ro-batcov hku9 (lineage d), and against recombinant n proteins of hcov-hku1 (lineage a), sarsr-rh-batcov (lineage b), ty-batcov hku4 (lineage c), pi-batcov hku5 (lineage c), and ro-batcov hku9 (lineage d) [31] . in our recent study on the discovery of dccov uae-hku23, the serological data further showed little cross-reactivity between dccov uae-hku23 (lineage a) and sarsr-rh-batcov (lineage b), pi-batcov hku5 (lineage c), and ro-batcov hku9 (lineage d), respectively, using dromedary serum samples positive for antibodies against the n protein of dccov uae-hku23 [50] . in the present study, we showed that the antibody titer of the serum obtained from the mouse immunized with the recombinant n protein of mers-cov for the anti-n of mers-cov was 64 folds higher than that for the anti-n of dccov uae-hku23. similarly, the antibody titer of the serum obtained from the mouse immunized with the recombinant n protein of dccov uae-hku23 for the anti-n of dccov uae-hku23 was 32 folds higher than that for the anti-n of mers-cov. furthermore, for the mice immunized with heat-inactivated dccov uae-hku23, the neutralization antibody titer against dccov uae-hku23 was at least 16 folds higher than that against mers-cov. all these results confirmed that there is minimal serological cross-reactivity among covs of different lineages in betacoronavirus, supporting their classification as separate lineages under betacoronavirus. a high seroprevalence for dccov uae-hku23 was observed in dromedaries. since dromedaries are the natural reservoirs of both dccov uae-hku23 and mers-cov, the seroprevalence of these covs can only be ascertained if there is minimal serological cross-reactivity between these two viruses. based on the minimal serological cross-reactivity between dccov uae-hku23 and mers-cov shown in the present study, we confirmed a high seroprevalence for dccov uae-hku23 in dromedaries as determined by immunofluorescence antibody tests and neutralization antibody assays. the relatively lower seropositivity as determined by western blot in our previous study as compared to the very high seropositivity as determined by immunofluorescence antibody tests and neutralization antibody assays in the present study is because only prominent immunoreactive bands were considered unambiguously positive in western blot analysis and hence might have underestimated the true seroprevalence [50] . original fecal samples from all the 14 dromedaries tested positive for dccov uae-hku23 by rt-pcr [50] were subject to virus isolation in hrt-18g (human rectal tumor epithelial), rk13 (rabbit kidney), mdck (canine kidney), mdbk (bovine kidney), dubca (dubai camel fetus skin fibroblast), caki-3-r (camel kidney), and bsc-1 (african green monkey renal epithelial) cells as described previously [65] . cell lines were prepared in culture plates and inoculated with 200 µl of fecal samples diluted at 1:10. non-attached viruses were removed by washing the cells twice in phosphate-buffered saline. the monolayer cells were maintained in serum-free minimal essential medium (mem, invitrogen, carlsbad, ca, usa) with or without supplementation by tosylsulfonyl phenylalanyl chloromethyl ketone (tpck)-treated trypsin (1 µg/ml) (sigma, st. louis, mo, usa). all infected cell lines were incubated at 37˝c for seven days. cpes were examined at day 1, 3, 5, and 7 by inverted light microscopy. the assay was performed using a real-time one-step qrt-pcr with dccov uae-hku23 primers 5 1 -atagcggctacacgtggtgtt-3 1 and 5 1 -tcccagccgccataaaact-3 1 and probe 5 1 -(fam) ctgttgttataggcaccact (bhq1)-3 1 . to generate calibration curves, we prepared a series of six log 10 dilutions equivalent to 10 1 -10 6 copies per reaction mixture and ran them in parallel with the test samples. negative contrast electron microscopy was performed as described previously [66, 67] . tissue culture cell extracts infected with dccov uae-hku23 were centrifuged at 19,000ˆg at 4˝c, after which the pellet was resuspended in phosphate-buffered saline and stained with 2% phosphotungstic acid. samples were examined with a philips em208s electron microscope (royal philips, amsterdam, the netherlands). total rna was extracted from dccov uae-hku23-infected hrt-18g cells using trizol reagent (invitrogen, carlsbad, ca, usa). rna was separated on 1% agarose gel with 7% formaldehyde at 100 v in 1ˆ3-(n-morpholino)propanesulfonic acid (mops) buffer (20 mm mops, 5 mm sodium acetate, 1 mm edta), transferred to a positively charged nylon membrane (roche diagnostics, basel, switzerland) with transfer buffer (ambion, foster city, ca, usa) by means of capillary force for 2 h, cross-linked to the membrane by ultraviolet light (120 mj/cm 2 ) and baked at 80˝c for 1 h. the blot was prehybridized with ultrahyb-oligo hybridization buffer (ambion, usa) and probed with a dccov uae-hku23 nucleocapsid-specific oligodeoxynucleotide probe 5 1 -ccagaacgatttccagaggacgctctact-3 1 , which was labeled with digoxigenin (dig) at 3 1 end. the blot was hybridized at 42˝c overnight and washed with low-and high-stringency buffers as recommended by the manufacturer (ambion). detection of dig-labeled probe on the blot was performed using dig luminescent detection kit according to manufacturer's protocol (roche diagnostics). to determine the location of the leader and body trss used for dccov uae-hku23 mrna synthesis, the leader-body junction sites and flanking sequences of all dccov uae-hku23 subgenomic mrnas were determined using rt-pcr as described previously [56, 68] . briefly, intracellular rna was extracted from dccov uae-hku23-infected hrt-18g cells using trizol reagent (invitrogen). reverse transcription was performed using random hexamers and the superscript iii kit (invitrogen). cdna was pcr amplified with a forward primer located in the leader sequence and a reverse primer located in the body of each mrna ( table 2 ). the pcr mixture (25 µl) contained cdna, pcr buffer (10 mm tris-hcl ph 8.3, 50 mm kcl, 2 mm mgcl 2 , and 0.01% gelatin), 200 µm of each dntps, and 1.0 u taq polymerase (applied biosystems, foster city, ca, usa). the mixtures were amplified in 40 cycles of 94˝c for 1 min, 50˝c for 1 min, and 72˝c for 1 min, and a final extension at 72˝c for 10 min in an automated thermal cycler (applied biosystems). rt-pcr products corresponding to each subgenomic mrna could be distinguished by size differences on agarose gel electrophoresis. pcr products were gel-purified using qiaquick gel extraction kit (qiagen, hilden, germany) and sequenced to obtain the leader-body junction sequences for each subgenomic mrna. hrt-18g cells infected with dccov uae-hku23 were fixed in chilled acetone at´20˝c for 10 min. the fixed cells were incubated with four-fold dilutions of serum from 1:10 to 1:10,240 from 59 dromedaries, followed by 1:50 diluted novex fitc-goat anti-llama igg (invitrogen). cells were then examined under a fluorescence microscope. uninfected cells were used as negative control. neutralization antibody assays for dccov uae-hku23 were carried out according to previously described protocols with modifications [11, 23] . heat inactivated serum samples from dromedaries were serially diluted from 1:10 and then mixed with 100 50% tissue culture infective dose (tcid 50 ) of dccov uae-hku23 isolate 263f. available sera from 59 dromedaries were included. after incubation for 2 h at 37˝c, the mixture was inoculated in duplicate on to 96-well plates of hrt-18g cell cultures. results were recorded after six days incubation at 37˝c. to prepare antibodies specific against the n proteins of dccov uae-hku23 and mers-cov respectively, 30 µg of purified (his) 6 -tagged recombinant n proteins of dccov uae-hku23 or mers-cov was mixed with an equal part of complete freund's adjuvant and injected subcutaneously into balb/c (h-2 d ) mice (six to eight weeks old, 18 to 22 g). incomplete freund's adjuvant was used in subsequent injections. serum samples were taken two weeks after the third injection for western blot analysis. western blot analysis was performed according to our published protocol [15] , with 1.5 µg purified (his) 6 -tagged recombinant n proteins of dccov uae-hku23 and mers-cov, respectively. serum samples of mice were titrated with serial two-fold dilutions, beginning at 1:2000. antigen-antibody interaction was detected with 1:4000 diluted horseradish peroxidase-conjugated goat anti-mouse igg (novex, san diego, ca, usa) and ecl fluorescence system (ge healthcare, buckinghamshire, uk). dccov uae-hku23 was cultured in hrt-18g cells using serum-free mem for four days and was heat-inactivated at 60˝c for 1 h. the cells were frozen and thawed once. the cell debris was removed by centrifugation and the supernatant was titrated and six balb/c mice were inoculated intraperitoneally using 1ˆ10 5 tcid 50 of heat-inactivated dccov uae-hku23. three boosters were administered on days 10, 20, and 30, respectively, with the heat inactivation of dccov uae-hku23 carried out on the day of injection. serum samples were obtained 14 days after the fourth injection. the sera from the six mice were pooled and neutralization antibody assay for dccov uae-hku23 was performed as described above and that for mers-cov was performed as we described previously [69] . viral rna was extracted from the fecal samples using ez1 virus mini kit v2.0 (qiagen). the rna was eluted in 60 µl of ave buffer (qiagen) and was used as the template for rt-pcr. the complete rdrp, s, and n genes of dccov uae-hku23 strains from 11 additional dromedaries were amplified and sequenced using primers designed by multiple alignments of the three complete genome sequences of dccov uae-hku23 [50] . reverse transcription was performed using the superscript iii kit. the pcr mixture (25 µl) contained dna, iproof high-fidelity pcr buffer (bio-rad, hercules, ca, usa), 200 µm of each dntp, and 0.5 u iproof high-fidelity dna polymerase (bio-rad). the mixtures were amplified by using 40 cycles of 98˝c for 10 s, 55˝c for 30 s, and 72˝c for 50 s, with a final extension at 72˝c for 10 min. standard precautions were taken to avoid pcr contamination and no false-positive was observed in negative controls. the pcr products were gel-purified using the qiaquick gel extraction kit (qiagen). both strands of the pcr products were sequenced twice with an abi prism 3700 dna analyzer (applied biosystems), using the pcr primers. the sequences of the pcr products were assembled manually. the phylogenetic trees for rdrp, s, and n genes were constructed by the maximum likelihood method using phyml. the optimal model for the nucleotide sequences were determined using modelgenerator v85. the trees for rdrp and n genes were inferred by the gtr + i substitution model and the tree for s gene was inferred by the gtr + i + g model. the codon usage bias of the rdrp, s, and n genes of the members in betacoronavirus 1 and rbcov hku14 was estimated using rscu. rscu values were calculated for the 59 relevant codons (excluding methionine, tryptophan, and stop codons) by sse [70] . ca was performed on the rscu values using the r statistical software, version 2.15.1 [71] and the function "corresp" from the modern applied statistics with s (mass) library. the simplot program (version 3.5.1) was used to analyze the genetic distance of the rdrp, s, and n genes of dccov uae-hku23 and other wild ruminant covs in reference to the rdrp, s, and n genes of alpaca cov, and this genetic distance was plotted versus nucleotide positions. the similarity plot was configured with window size: 200 nucleotides; step: 20; f84 (maximum likelihood). dccov uae-hku23 was successfully isolated in dromedary fecal samples using hrt-18g cells. there was minimal serological cross-antigenicity between dccov uae-hku23 and mers-cov. dccov uae-hku23 is a novel member of betacoronavirus 1. the authors declare no conflict of interest. coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives the molecular biology of coronaviruses molecular biology of severe acute respiratory syndrome coronavirus coronavirus genome structure and replication virus taxonomy: ninth report of the international committee on taxonomy of viruses subgenomic messenger rna amplification in coronaviruses feline coronavirus type ii strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type i and canine coronavirus comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 tracing the transmission of bovine coronavirus infections in cattle herds based on s gene diversity biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (bubalus bubalis) calves isolation and characterization of viruses related to the sars coronavirus from animals in southern china the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection a previously undescribed coronavirus associated with respiratory disease in humans identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia coronavirus hku1 and other coronavirus infections in hong kong severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor molecular diversity of coronaviruses in bats comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events coexistence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku9 belonging to a novel betacoronavirus subgroup recent transmission of a novel alphacoronavirus, bat coronavirus hku10, from leschenault's rousettes to pomona leaf-nosed bats: first evidence of interspecies transmission of coronavirus between bats of different suborders discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku14, from domestic rabbits discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus discovery of a novel coronavirus, china rattus coronavirus hku24, from norway rats supports murine origin of betacoronavirus 1 with implications on the ancestor of betacoronavirus lineage a isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia acute middle east respiratory syndrome coronavirus infection in livestock dromedaries metagenomic analysis of viromes of dromedary camel fecal samples reveals large number and high diversity of circoviruses and picobirnaviruses a contemporary view of coronavirus transcription studies into the mechanism for mhv transcription coronavirus transcription mediated by sequences flanking the transcription consensus sequence molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination bovine coronavirus 5 1 -proximal genomic acceptor hotspot for discontinuous transcription is 65 nucleotides wide genomic characterization of equine coronavirus analysis of the genome sequence of an alpaca coronavirus a new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay first detection of equine coronavirus (ecov) in europe inter-and intra-variant genetic heterogeneity of human coronavirus oc43 strains in france bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences detection and characterization of bovine-like coronaviruses from four species of zoo ruminants biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe characterisation of bubaline coronavirus strains associated with gastroenteritis in water buffalo (bubalus bubalis) calves differential susceptibility of different cell lines to swine-origin influenza a h1n1, seasonal human influenza a h1n1, and avian influenza a h5n1 viruses newly discovered coronavirus as the primary cause of severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome genome structure and transcriptional regulation of human coronavirus nl63 cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests sse: a nucleotide and amino acid sequence analysis platform the r foundation. the r project for statistical computing key: cord-260518-mswb3q67 authors: zumla, alimuddin; dar, osman; kock, richard; muturi, matthew; ntoumi, francine; kaleebu, pontiano; eusebio, macete; mfinanga, sayoki; bates, matthew; mwaba, peter; ansumana, rashid; khan, mishal; alagaili, abdulaziz n.; cotten, matthew; azhar, esam i.; maeurer, markus; ippolito, giuseppe; petersen, eskild title: taking forward a ‘one health’ approach for turning the tide against the middle east respiratory syndrome coronavirus and other zoonotic pathogens with epidemic potential date: 2016-06-15 journal: int j infect dis doi: 10.1016/j.ijid.2016.06.012 sha: doc_id: 260518 cord_uid: mswb3q67 the appearance of novel pathogens of humans with epidemic potential and high mortality rates have threatened global health security for centuries. over the past few decades new zoonotic infectious diseases of humans caused by pathogens arising from animal reservoirs have included west nile virus, yellow fever virus, ebola virus, nipah virus, lassa fever virus, hanta virus, dengue fever virus, rift valley fever virus, crimean-congo haemorrhagic fever virus, severe acute respiratory syndrome coronavirus, highly pathogenic avian influenza viruses, middle east respiratory syndrome coronavirus, and zika virus. the recent ebola virus disease epidemic in west africa and the ongoing zika virus outbreak in south america highlight the urgent need for local, regional and international public health systems to be be more coordinated and better prepared. the one health concept focuses on the relationship and interconnectedness between humans, animals and the environment, and recognizes that the health and wellbeing of humans is intimately connected to the health of animals and their environment (and vice versa). critical to the establishment of a one health platform is the creation of a multidisciplinary team with a range of expertise including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral phylogeneticists, and researchers to co-operate, collaborate to learn more about zoonotic spread between animals, humans and the environment and to monitor, respond to and prevent major outbreaks. we discuss the unique opportunities for middle eastern and african stakeholders to take leadership in building equitable and effective partnerships with all stakeholders involved in human and health systems to take forward a ‘one health’ approach to control such zoonotic pathogens with epidemic potential. benefit the large majority of affected people. some foreign aid workers and researchers were not familiar with local cultural and medical services norms and aroused local anxieties. 10 the evd epidemic highlighted the need for developing more comprehensive local, national, international, and global surveillance, as well as epidemic and outbreak preparedness response infrastructures. multiple animal, human, and environmental factors are obviously playing a critical role in the evolution, transmission, and pathogenesis of zoonotic pathogens, and these require urgent definition to enable appropriate interventions to be developed for optimal surveillance, detection, management, laboratory analysis, prevention, and control in both human and animal populations. an important need exists for establishing long-term, sustainable, trusting and meaningful and equitable collaborations between the animal, human, ecosystem, and environmental health sectors at the local, national, and international levels. these should include sustainable political and funder support for developing human and laboratory capacity and training that enables effective human-animal health cooperation leading to proactive surveillance, early detection of potential pandemic pathogens, and rapid initiation of public health prevention and control guidelines and interventions. whilst a long list of pathogens with epidemic potential are on the radar of the world health organization (who), 2 ideally 'prevention is better than cure' and new pathogens should be dealt with at the animal source, tackling the drivers and triggers of pathogen evolution and emergence. this requires close cooperation between human and animal health systems and an appreciation of human impacts on the environment at all levels and easy access to adequate laboratory facilities. on december 10, 2015 an expert panel convened by who prioritized a list of emerging pathogens ''considered likely to cause severe outbreaks in the near future, and for which no, or insufficient, preventive and curative solutions exist''. 11, 12 the list of the top 10 includes the new viral zoonotic pathogen of humans mers-cov, 13, 14 which was first isolated from a patient who died of a severe respiratory illness in a hospital in jeddah, saudi arabia in june 2012. 15 the emergence of mers-cov in 2012 15 was the second time (after sars-cov 16 ) that a highly pathogenic coronavirus of humans emerged in the 21 st century. 17 a strong link between human cases of mers-cov and dromedary camels has been established through several studies. [18] [19] [20] [21] [22] [23] [24] [25] [26] mers-cov is endemic in the camel populations of east africa and the middle east 21, 25, 26 and presents a constant threat to human health in both regions. retrospective studies using stored serum from different geographical locations have indicated that mers-cov has been circulating for several decades. 25 as of may 1, 2016, there have been 1733 laboratoryconfirmed cases of mers reported to the who, 27 with a mortality of 34% (628 cases died). whilst most mers cases have been reported from the middle east (a large proportion from saudi arabia), mers cases have been reported from 27 countries in all continents. 27 the who has held nine meetings of the emergency committee (ec) for mers-cov. 28 since evidence of sustained human-to-human transmission of mers-cov in the community is lacking, the who currently does not recommend travel restrictions to the middle east. however, mers-cov remains a major global public health threat with continuing reports of new human mers cases in saudi arabia, where millions of pilgrims from over 184 countries travel throughout the year. 29 furthermore, a more intensive farm-based camel livestock system has emerged and there is a large, wellestablished trade in camels between countries at the horn of africa and countries in the middle east. this has increased significantly, particularly following the lifting of the ban on live animal imports from somalia by saudi arabia in 2009/2010. somalia now exports some five million live animals every year to the gulf arab states (including 77 000 camels), making it the single biggest exporter of live animals in the world. the positive experience of reviving somalia's livestock export industry through increased investment in animal disease prevention and control strategies highlights how effective the 'one health' approach can be. most of the african countries do not have the resources, expertise, or capacity, including laboratory facilities, to have active surveillance for mers-cov in place. in light of this, the need for increased vigilance and watchful surveillance for mers-cov in sub-saharan africa has been highlighted previously. 30 such an initiative could be supported through investments by countries that import large numbers of camels and other livestock from the region. the epidemic potential of mers-cov was recently illustrated by a large outbreak in hospitals in seoul, the republic korea, in mid-2015: mers-cov was imported by a traveller to the middle east (an agriculture businessman), resulting in 184 mers cases with 33 deaths. 31 the first case was reported on may 20, 2015 and over the ensuing 3 weeks, the number of secondary, tertiary, and perhaps quaternary cases of mers from this single patient rose rapidly, resulting in the largest mers case cluster occurring outside the middle east. the unprecedented outbreak was attributed to poor infection control measures at the hospitals. 30 sequencing studies of the mers-cov isolate showed genetic recombination of mers-cov in the case exported from korea to china. 32 however, recombination is a frequent event in mers-cov and the korean outbreak is unlikely to represent a special form of the virus. nonetheless, the potential evolution of mers-cov into a more virulent form needs to be monitored closely. research on sequencing seems to have stagnated and there have been no further sequences published from new human mers cases reported from the middle east. furthermore, the genetic evolution of mers-cov strains infecting humans over the past year remains unknown. there is an urgent need for more sequencing studies on mers-cov evolution in camels and humans, with the development of appropriate local capacity for these studies. the kingdom of saudi arabia has kept proactive watchful mers-cov surveillance with regular reports to the who of mers-cov cases. 33 the who and ministries of health of middle eastern countries continue watchful surveillance of the mers-cov situation, and the watchful anticipation is that mers-cov may disappear with time like sars-cov. however, with the continuing, regular reports of community cases of mers-cov from saudi arabia, 27 there are no signs of this happening in the near future and lessons must be learnt from the korean outbreak. 34 whilst there is a growing camel livestock industry in the region, elimination of the virus is unlikely in the short term. several animal, human, and environmental factors are obviously playing a critical role in the repeated movement of mers-cov from camels to humans. the disease ecology remains largely unknown. urgent definition is required to enable appropriate interventions to be developed for optimal surveillance, laboratory detection, management, prevention, and control in both human and animal populations. whilst several ad hoc research studies have been conducted and findings published over the past 4 years, more comprehensive investments in tackling mers-cov have not been forthcoming. there remain huge knowledge gaps on mers-cov. much of the information that we have about the source of mers-cov infections is based on small local studies and it is difficult to develop general country-wide policies without a clear understanding of the zoonotic problem. questions remain, for example are new local mers outbreaks in saudi arabia always seeded by the same type of human exposure to camels? are there particular regions of africa that provide infected camels to saudi arabia? or is there a general risk from all regions? is there a way to efficiently control the entry of infected camels? are animal vaccination strategies economically viable given the large number of imported animals and the frequency of the infection? a clear policy in which full virus genome sequences are generated from every outbreak in the country and in which virus from subsets of imported camels is routinely screened and sequenced after 2 years, would provide incredibly useful information about the transmission patterns of the virus and how to stop it. certainly the resources and expertise to perform this sequence monitoring are available and only governmental support is needed to run such a survey. the cost of such a survey would be far less than the management costs and grief associated with a single hospital outbreak. numerous priority research questions regarding mers-cov (basic science, epidemiology, management, and development of new diagnostics, biomarkers, treatments, and vaccines) in both humans and camels, highlighted 2 years ago by the who mers expert groups 35 and by others, 36 remain unanswered. these have again been raised recently, highlighted by calls from saudi arabian health care staff and scientists 37, 38 and by yet another who mers expert group, which has defined a ''roadmap for research and product development against mers-cov''. 39 in 2000 the who set up the global outbreak alert and response network (goarn) 40 for better coordination of surveillance efforts across the globe. it networks 150 institutions and partner agencies, with cooperation with other agencies such as public health england and the us centers for disease control and prevention (cdc) and consortia such as the international severe acute respiratory and emerging infection consortium (isaric). 41 recent consortia such as glopid-r aim to bring together research funding organizations on a global scale to facilitate an effective research response within 48 h of a significant outbreak of a new or re-emerging infectious disease with pandemic potential. 42 the past 4 years has seen outbreaks of ebola virus, zkv, and mers-cov, [2] [3] [4] 13 which indicate that the global community needs to seriously reflect on what is critically missing from current political, scientific, and public health agendas, and how to delineate what is required at the national, regional, and global levels to prevent future epidemics. the factors and operating conditions that promote the emergence and geographical spread of zoonoses are complex and may be related to a single event or chain of multiple events influenced by the genetic evolution of the pathogen, environmental and climate changes, anthropological and demographic changes, and movement and behaviour of humans, animals, and vectors. with animal, human, and environmental factors playing a critical role in its evolution, mers-cov requires more close collaboration between human and animal health systems and university academics to reduce the risk of pandemic spread. 43 moreover, a better understanding of the agricultural dynamics involved in its persistence and spread in camels and studies on interactions between hosts in the environment are urgently needed. the intermittent detection and reporting of mers cases in the community and sporadic nosocomial mers-cov outbreaks will require a more coordinated response plan to study clinical cases, conduct translational basic science and clinical trials research, and perform longitudinal sequencing studies from human and camel mers-cov isolates. a more collaborative mers-cov response plan is required to better define mers-cov epidemiology, transmission dynamics, molecular evolution, laboratory capacity, optimal treatment and prevention measures, and development of vaccines for humans and camels. 44 a better understanding of the prevailing disease ecology and investigations into the dynamics of infectious agents in wildlife could act as a better means of preventing outbreaks in livestock and people at source. the 'one health' concept is an important concept that focuses on the relationship and interconnections between humans, animals, and the environment, and recognizes that the health and wellbeing of humans is intimately linked to the health of animals and their environment (and vice versa). [45] [46] [47] [48] [49] a balanced ecological approach improves understanding of the true threat of novel pathogens and helps to avoid costly, poor, and inappropriate responses to new diseases. in many cases, solutions can be found through altered development pathways and are not inevitably requiring of costly, unsustainable technical and pharmaceutical interventions. thus it is ideally suited to the mers-cov situation in which camels, humans, and environmental factors are central to its persistence and evolution. since the kingdom of saudi arabia is host to millions of pilgrims each year travelling from all continents, 29 tackling the threat of mers and other infectious diseases with epidemic potential will require enhanced closer cooperation between those who provide human health, animal health, and environmental health services, locally, nationally, regionally, and internationally: the middle eastern, european, african, asian, and american governments, veterinary groups, the who, the food and agriculture organization (fao), the african union, the united nations international children's emergency fund (unicef), the world bank, office international des epizooties (oie), cdc, public health england, the newly formed africa cdc, and funding agencies among others. they should now demonstrate increased commitment towards local, national, and global multidisciplinary collaborative efforts to secure optimal health for people, animals, and the environment. global efforts need to be focused on establishing the capability for and strengthening of surveillance systems in developing countries, particularly in africa where emerging and re-emerging zoonoses are a recurrent problem. a prime emphasis should be on developing awareness and response capacity in all countries and on promoting interdisciplinary collaboration and coordination. critical to the establishment of a well-functioning 'one health' platform is the creation of a multidisciplinary team with a range of expertise, including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral geneticists, and researchers, with easy access to adequate laboratory facilities, who will collaborate in order to learn more about zoonotic spread between animals, humans, and the environment and to monitor, respond to, and prevent major outbreaks. there is an urgent and critical need to build a sustainable public health programme and rapid response capability for outbreaks of zoonotic pathogens in the middle east and in low-income countries, especially in africa. importantly there is a need for capacity development programmes designed to strengthen research training and build career pathways for the best and brightest post-doctoral researchers, including phd and masters students working at the interface of humans, animals, and environment. these should include national or regional laboratory facilities, as surveillance requires laboratory support to be meaningful. the development of human and animal health research leaders will create a critical mass of local research capacity and the development of self-funding research environments in african universities and research institutes. this capacity growth could be facilitated through the further development and support of a geographical network of equitable and enduring south-south and north-south partnerships. 9. need for more effective political and scientific engagement to eradicate the threat of mers-cov and other zoonotic diseases the persistence of mers-cov 4 years since its first discovery has created major opportunities for each of the middle eastern and african countries to take leadership of the 'one health' approach with a view to bringing this under regional and global umbrellas, to tackle new emerging and re-emerging infectious diseases with epidemic potential. this will also devolve current dominance of the global health agenda by western groups and consortia and allow equitable partnerships to be established with long-term sustainability. the past year has seen some progress in research into mers-cov, but there remains a need for a more effective, coordinated, and multidisciplinary 'one health' consortium to take forward mers-cov research on priority areas already defined by saudi scientists 37, 38 and the who mers committee. 39 the establishment of regional 'one health' centres of excellence in the middle east (under the league of arab states) and at specific geographical locations in west, central, east, and southern africa could make an important difference in mitigating the risks and factors that pose a risk to both human and animal health. furthermore, any operational plan developed will contribute to strengthening the sentinel surveillance systems in sub-saharan africa in the preparedness and response to potential outbreaks. regional centres should be sufficiently empowered to manage the spectrum of 'one health' approaches to zoonotic disease control in humans and animals, from behaviour change and social interventions for prevention to surveillance of infections and antimicrobial resistance, and preparedness and response to outbreaks. a model for the major syndromes (respiratory, neurological, haemorrhagic, gastro-enteric, and sepsis-like presentations) should be developed so that clinical protocols may be adapted rapidly for any major outbreak during mass gatherings. this should include the development and introduction of innovative and smart platforms for data sourcing, sample collection, and analysis, in order to give clinicians and public health workers continuously updated information on which clinical decisions may be based. there is a pressing need to develop and strengthen the national ethics and medicines regulatory frameworks in sub-saharan africa in order to strike a balance between the public health interest, the interests of the pharmaceutical industry, and ethical values. parallel initiatives across africa and the tropics could be harmonized to create regional networks that can serve as a repository for expert 'one health' advice on agriculture, sustainable livestock, and the links to human development. there are several ongoing important initiatives on developing 'rapid response' and broader 'one health' capacity development groups in europe, asia, and the americas to assist in the surveillance and response to emerging infectious disease threats. the public health systems of west african countries failed with the ebola epidemic, and the response from the who and the international community was very slow and uncoordinated. this led to thousands of people, including over 500 health care workers, losing their lives. the factors governing the appearance and disappearance of new coronaviruses affecting humans are complex and it has been over 4 years since the first patient died of mers-cov. mers cases continue to be reported throughout the year from the middle east. there is a large mers-cov camel reservoir and there is no specific treatment or vaccine. the precise pathway from infected camel to the recurring mers hospital outbreaks needs to be understood in order to devise effective control measures. with 10 million people visiting saudi arabia every year for umrah and/or hajj and the increasing importation of live animals from sub-saharan africa, the potential risk of global spread will be everpresent, especially if mutations or recombinations in mers-cov occur. a major 'one health' initiative to tackle mers-cov at source in animal populations is thus required. 50 middle eastern and african governments should now work more closely together and increase collaborative efforts with international partners and global public health authorities if we are to prevent yet another global zoonotic pandemic. conflict of interest: all authors have a specific interest in 'one health'. the authors declare no conflicts of interest. there was no financial support. emerging and re-emerging infectious threats in the 21st century world health organization. ebola virus disease outbreak world health organization. zika virus be prepared: europe needs ebola outbreak consortium ethics for pandemics beyond influenza: ebola, drug-resistant tuberculosis, and anticipating future ethical challenges in pandemic preparedness and response ebola: missed opportunities for europe-africa research rapid spread of zika virus in the americas-implications for public health preparedness for mass gatherings at the 2016 brazil olympic games lessons from the ebola outbreak: action items for emerging infectious disease preparedness and response challenges in controlling the ebola outbreak in two prefectures in guinea: why did communities continue to resist? world health organization. who publishes list of top emerging diseases likely to cause major epidemics world health organization. a research and development blueprint for action to prevent epidemics middle east respiratory syndrome coronavirus (mers-cov) state of the art seminar: middle east respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia the severe acute respiratory syndrome emerging respiratory tract viral infections evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia deciphering mers-cov evolution in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov) world health organization. ihr emergency committee concerning middle east respiratory syndrome coronavirus hajj: infectious disease surveillance and control middle east respiratory syndrome-need for increased vigilance and watchful surveillance for mers-cov in sub-saharan africa middle east respiratory syndrome coronavirus (mers-cov)-update. disease outbreak news origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis saudi ministry of health. weekly mers-cov monitor middle east respiratory syndromeadvancing the public health and research agenda on mers-lessons from the south korea outbreak state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans advancing priority research on the middle east respiratory syndrome coronavirus in riyadh, ksa knowledge gaps in therapeutic and non-therapeutic research on the middle east respiratory syndrome world health organization. a roadmap for research and product development against middle east respiratory syndrome-coronavirus (mers-cov) international severe acute respiratory and emerging infection consortium. the website for the international severe acute respiratory and emerging infection consortium (isaric) global research collaboration for infectious disease preparedness website emerging infectious diseases and pandemic potential: status quo and reducing risk of global spread development of medical countermeasures to middle east respiratory syndrome coronavirus what is one health? one health global network one health: a new professional imperative. one health initiative task force one world, one health. oie-world organisation for animal health sharing responsibilities and coordinating global activities to address health risks at the animal-human-ecosystems interfaces. a tripartite concept note international organization for standardization. who develops iso standards. geneva: who towards a one health approach to controlling zoonotic diseases key: cord-261876-7rsc803x authors: kaslow, david c. title: certainty of success: three critical parameters in coronavirus vaccine development date: 2020-05-25 journal: npj vaccines doi: 10.1038/s41541-020-0193-6 sha: doc_id: 261876 cord_uid: 7rsc803x vaccines for 17 viral pathogens have been licensed for use in humans. previously, two critical biological parameters of the pathogen and the host–pathogen interaction—incubation period and broadly protective, relative immunogenicity—were proposed to account for much of the past successes in vaccine development, and to be useful in estimating the “certainty of success” of developing an effective vaccine for viral pathogens for which a vaccine currently does not exist. in considering the “certainty of success” in development of human coronavirus vaccines, particularly sars-cov-2, a third, related critical parameter is proposed—infectious inoculum intensity, at an individual-level, and force of infection, at a population-level. reducing the infectious inoculum intensity (and force of infection, at a population-level) is predicted to lengthen the incubation period, which in turn is predicted to reduce the severity of illness, and increase the opportunity for an anamnestic response upon exposure to the circulating virus. similarly, successfully implementing individualand population-based behaviors that reduce the infectious inoculum intensity and force of infection, respectively, while testing and deploying covid-19 vaccines is predicted to increase the “certainty of success” of demonstrating vaccine efficacy and controlling sars-cov-2 infection, disease, death, and the pandemic itself. in the absence of an existing, safe, effective vaccine for a pathogen (i.e., absent proof of clinical efficacy and safety), the risks and uncertainties in developing a new vaccine can be broadly divided into two categories: "biologic uncertainty"-the inherent biological ability of the candidate vaccine to elicit a protective immune response in humans with a safety profile that results in a positive benefit-risk balance; and, "execution uncertainties and risks"-the successful performance of literally thousands of tasks required to develop the vaccine. the subfactors that determine "biologic uncertainty" are perhaps the most difficult to overcome because most are largely immutable. two dominant parameters that underlie "biologic uncertainty" are the safety/tolerability and the efficacy of a candidate vaccine. unlike execution risks, such as the underlying failure rate of a production run under a given set of operational conditions, biologic uncertainties have a relatively binary outcome-the candidate vaccine does or does not have a favorable benefit-risk profile-that remains largely unchanged under a given set of epidemiologic conditions. relatively because the outcome measures, such as efficacy and effectiveness, have inherent variability around the point estimate. this article revisits previously described key parameters of biologic feasibility proposed to determine the "certainty-of-success" (also referred to as the "probability of success") in developing prophylactic vaccines 1 , but now in the context of coronaviruses. oftentimes safety and efficacy are inversely related, the socalled double-edged sword of attenuation 2 : an improvement in one resulting in a loss of performance in the other. of the two, safety is often given priority over efficacy in regulatory review because the target populations of prophylactic vaccines are usually healthy. the risk tolerance for safety in the midst of an outbreak for a pathogen with a high r 0 and a high case fatality rates may be higher 3 than for vaccine use in routine immunization for relatively low-prevalence endemic diseases, particularly those with a low case fatality rate. despite the paramount importance of safety, this article will focus on estimating the "certainty of success" from the efficacy/effectiveness side of the benefit-risk balance. as noted above, the importance (and difficulty) of accurately estimating the performance of a candidate vaccine relative to the efficacy threshold in humans has been reviewed previously 1 . herein the previously proposed paradigm that effective vaccines have been developed mainly for pathogens with lengthy incubation periods is re-explored as it pertains to active prophylactic immunization for coronaviruses, particularly sars-cov-2. whereas in the original analysis, discussion of populationbased effects of immunization (e.g., herd effects) were considered, here the focus is mainly at the level of the individual vaccinee, with the notable exception of the population-based effect of force of infection (see glossary of terms), and the continued circulation of virus in the population on the durability of individual immunity. several simplifying assumptions are made, including that vaccinees are immunocompetent and share similar underlying condition profiles, that the kinetics of an effective acquired and anamnestic immune response is similar for different vaccine modalities, and that the immune responses elicited by vaccination prior to any previous pathogen exposure is at least similar to that acquired during natural infection. the previously proposed paradigm specifically considered, as part of the "certainty-of-success" analysis of biological feasibility, two critical properties of the many inherent biological properties of viral pathogens and the dynamic interaction with the human host: incubation period; and, broadly protective, relative immunogenicity. the combination of these properties appeared to have accounted for much of the successes so far achieved in vaccine development for viral pathogens (see fig. 1a ) 1 . in the original analysis conducted more than a dozen years ago, an effective, durable vaccine against sars-cov was predicted to be on the cusp of biological feasibility when applying the 5-day incubation period rule. in the current specific analysis for sars-cov-2 vaccine development, infectious inoculum intensity is elevated to a critical parameter because of its putative implications in assessing the "certainty of success" of developing an efficacious covid-19 vaccine for use in an outbreak setting and in the design, conduct, and interpretation of covid-19 vaccine efficacy and effectiveness studies. incubation period and infectious inoculum intensity incubation period, defined as the time between exposure to the pathogen and onset of signs and/or symptoms of clinically apparent disease (see box 1. glossary of key terms), incorporates, in an empirically derived unit of time: (1) multiple inherent biological properties of the pathogen; (2) the dynamic interaction between the virus and the host; and (3) real world conditions of transmission (see force of infection below). factors that determine the incubation period include the amount of infectious virus in a typical inoculum, the infectivity of the viral pathogen, the rate of viral replication, the rate of viral clearance by a variety of host mechanisms, including innate and adaptive immunity, the impact of viral immune evasion tactics, and the viral load that results in signs or symptoms of disease. the clinical signs and/or symptoms that define the endpoint of the incubation period also impact the reported value. not surprisingly, the incubation period can be quite variable, and retrospectively, difficult to precisely and accurately measure. in its simplest iteration, the incubation period can be viewed as a race between: (1) the immune system's ability to generate a sufficient and appropriate innate and/or adaptive response; and (2) the replication of the pathogen to a viral load that results in symptoms. as noted previously, an important inflection point occurs around 3 days when considering incubation periods for viral pathogens. the "certainty of success" for viruses, such as influenza (median incubation period 2 days, with a range of 1-4 days), that have short incubation periods do not benefit from the opportunity of an anamnestic response (see box 1. glossary of key terms). in addition, for vaccines against viral pathogens, particularly those with a short incubation period, protection against mild symptoms is often a much more difficult endpoint to achieve than protection against severe disease. in the absence of vaccine-induced, persistent, high-level immune effector function (e.g., circulating high-titer neutralizing antibodies and/or cytotoxic t-cell lymphocytes), early protection against lower level viral replication (i.e., early mild disease) may be more difficult to achieve than an anamnestic response (e.g., newly activated memory b-and t-cell responses) to protect against higher-level viral loads (i.e., more severe disease) that occur much later during infection. this model of being able to elicit high-level protection against severe disease, but not mild clinical symptoms (e.g., observed for rotavirus vaccines), will likely apply to sars-cov-2. infectious inoculum intensity, at an individual-level, and force of infection, at a population-level, are factors that may inversely contribute to the length of the incubation period, the latent period, fig. 1 "certainty of success" of vaccine development as a function of incubation period and broadly protective, relative immunogenicity. a two-dimensional analysis of 29 major human viral pathogens based on incubation period (x-axis; time, in days or weeks, from exposure to clinical signs or symptoms) and broad, relative immunogenicity (y-axis; high, moderate or low-see reference 1 for definition). the 17 viral pathogens for which vaccine efficacy have been established are depicted in boxes with gray backgrounds; those for which vaccine efficacy has yet to be established are depicted in ellipses with white backgrounds. the area of graph associated with higher "certainty-of-success" for vaccine development (light gray) and lower "certainty-of-success" (dark gray) are separated by a thick black line. (reprinted from an open-access article licensed under a creative commons attribution-noncommercial 3.0 unported license 1 . b effect of low and high infectious inoculum intensity on the assessment of the "certainty-of-success" (cos) of sars-cov-2 vaccines. interval bar (white) reflect the uncertainty in the inherent broadly protective, relative immunogenicity (see glossary of key terms) associated with sars-cov-2 natural infection. double-headed arrow (white with black outline) reflects the effect of infectious inoculum intensity higher (light gray) and lower (dark gray) "certainty-of-success" for vaccine development. anamnestic response: a secondary or subsequent immune response that yields a faster, greater, and longer lasting immune response upon re-exposure to an immunogen than that induced during the preceding primary immune response. broadly protective, relative immunogenicity: a semi-quantitative term that captures the genetic diversity of the virus, and two aspects of the host immune responses: (1) the quality of the immune response that is elicited during and immediately after a primary infection; and (2) the ability and duration of that elicited immune response to protect against subsequent symptomatic reinfection. certainty of success: an estimate of the confidence in successfully demonstrating biological activity of a candidate vaccine based on pre-defined endpoints of efficacy/effectiveness. force of infection: rate at which susceptible individuals in a population acquire an infectious disease in that population. incubation period: time interval between infectious agent exposure moment in an individual and appearance of first sign or symptom of disease in that individual. infectious inoculum intensity: magnitude (or area under the curve) in an individual of the infectious agent exposure at the exposure moment(s) associated with the duration of the incubation period in that individual. latent period: time interval between infectious agent exposure moment in an individual and onset of period of infectious transmissibility to others in the population, which may be shorter or longer than the incubation period. and directly contribute the severity of symptoms associated with infection 4, 5 . equally, if not more relevant to "certainty of success" for vaccine development is the relationship between infecting dose and severity of disease 6 , as demonstrated by influenza inoculum dose-related rate of mild-to-moderate disease in a controlled human infection model 7 . with respect to coronaviruses, an inverse correlation between the length of the incubation period and the severity of disease was recently evaluated from data collected during the 2003 sars outbreak in hong kong. comparing the length of the incubation period between fatal cases and non-fatal cases suggested a correlation between shorter incubation and greater severity, allowing for potential confounding by age, sex and occupation 8 . a similar finding was observed between the estimated incubation period of middle east respiratory syndrome cov (mers-cov) cases and mortality during the 2015 mers outbreak in south koreapatients who died had a shorter incubation period than patients who survived 9 . with respect to sars-cov-2, jiang et al. 10 and amodio et al. 11 have both noted that a longer incubation time may lead to a high rate of asymptomatic and sub-clinical infection among immunocompetent individuals; however, an inverse relationship between incubation period and severity of disease has yet to be demonstrated. while the relationship between incubation period and perhaps more importantly, the infectious inoculum intensity of srs-cov-2 and severity of covid-19 requires further validation, data consistent with an inverse relationship was highlighted by sanche et al. 12 when noting that a potential caveat of their estimation of a shorter incubation period [4.2 days (95% ci 3.5-5.1 days)] for sars-cov-2 than most other published reports is because most of their case reports were collected from the first few persons detected in each province, which may have biased case detection toward patients with more severe symptoms. a noteworthy exception to the inverse relationship between incubation period and severity of disease comes from the observation that a longer incubation period among human influenza h7n9 cases was associated with a greater risk of death. virlogeux et al. 13 noted that h7n9 virus infection differs from h5n1, sars, and mers coronaviruses in several respects, including tropism limited to the human upper airways, the absence of a cytokine storm, and the stronger association of severe h7n9 disease with exacerbation of other underlying diseases, while h5n1, sars, and mers coronaviruses cause severe disease in otherwise healthy persons. as such, it is proposed here that the exception to the inverse relationship between incubation period and severity of disease is unlikely for sars-cov-2. admittedly confounded by multiple other factors, the population-based force of infection appears, in at least two recent examples, to be inversely associated with vaccine efficacy/ effectiveness (ve/vef). in the case of rotavirus vef, a review of the first decade of post-licensure data from 24 countries showed a gradient of median vef of 84%, 75%, and 57% in countries with low, medium, and high child mortality, respectively, for the monovalent vaccine based on a single human rotavirus strain, and a vef difference of 90% and 45% in countries with low and high child mortality, respectively, for a pentavalent vaccine based on five bovine-human reassortant rotavirus strains 14 . prelicensure rotavirus vaccine ve data demonstrate a similar gradient 15 , and, when analyzed by the pre-existing rotavirus disease burden (mortality) 16 as an indicator of the force of infection, are consistent with an inverse association with ve, as suggested by feiken, et al. 17 this inverse association was also recently suggested during the regulatory evaluation of the malaria vaccine, rts,s/as01 e . the european medicines agency (ema) noted that "ve tends to be lower in high transmission areas" 18 when analyzing the pivotal mal-055 efficacy trial conducted in eleven research centers in seven sub-saharan african countries, where the force of infection (categorized by annual mean p. falciparum parasite rate, age-standardized in 2 to 10-year olds 19 ) differed by two orders of magnitude. lastly, this notion of an association between force of infection and severity of disease is consistent with multi-year observations of seroconversion rates for the four human endemic coronavirus and frequency of virus in hospitalized children 20 . dijkman et al. reported that the frequency of infection observed via seroconversion in the study population, presumed to be associated with the relative force of infection in that population, had the same rank order of hcov-oc43 ≥ hcov-nl63 > hcov-hku1 ≥ hcov-229e as the frequency of virus in hospitalized children, presumed to be associated with the severity of disease. given the extensive spread of sars-cov-2, it should be possible to determine if a similar association between force of infection and severity of disease is observed during the current pandemic. broadly protective, relative immunogenicity a composite of additional biological features has been incorporated into a single semi-quantitative term, broadly protective, relative immunogenicity (see box 1. glossary of key terms; nominally categorized as high, moderate or low; also see table 1 ), to provide a needed second dimension to refine the estimate of "certainty of success". specifically broadly protective, relative immunogenicity incorporates both genetic diversity of the virus, and two aspects of the host immune response: (1) the quality of the immune response that is elicited during and immediately after a primary infection; and (2) the ability and duration of that elicited immune response to protect against subsequent symptomatic reinfection. factors relevant to coronaviruses, particularly sars-cov-2, that could contribute to categorizing broadly protective, relative immunogenicity might include the number of circulating coronavirus strains that cross-react or cross-protect against other coronaviruses 20 , the propensity for coronaviruses to propagate mutants during the incubation, latent, disease and recovery periods, the frequency of asymptomatic infections due to viral clearance by an adaptive immune response during the primary infection and during reinfection, and the durability of the protective immune response. as to genetic diversity, coronaviruses have the largest positivesense, single-stranded rna (+ss rna) genomes known to cause disease in humans, from 27 up to 32 kilobases (kb), with the genus betacoronavirus sars-cov-2 at 29.9 kb (see table 1 ). the long coronavirus genome displays a high degree of plasticity, particularly the spike or s protein (see below), which can adapt with relative ease to exploit different cellular receptors, likely underlying the propensity of the four genera of animal coronaviruses to jump hosts 21 . while the bat-and rodentderived alpha-and beta-coronaviruses have likely attempted to jump into humans frequently, three cross-species transmission events, over the past dozen and a half years, have resulted in outbreaks of sars-cov, mers-cov, and sars-cov-2 in the human population. four well adapted "common cold"-type coronaviruses also widely circulate in humans (i.e., hcov-229e, -nl63, -oc43, and -hku1) 22 . of the nine open-reading frames encoded in the coronavirus genome and the four structural proteins, the spike or s protein, particularly the receptor-binding domain (rbd) in the s1 subunit and the s2 subunit, is of specific interest because this essential structural protein, expressed in multiple copies on the lipid bilayer envelope of the virus, determines in part the host range through its role in host cell attachment, fusion, and entry 23-26 . in the case of sars-cov-2, evolution within the modular structure of the s protein, the main target of protective immunity, may be driven by the known error rates of coronavirus rna-dependent rna polymerases, and the marked capacity of coronaviruses to employ homologous recombination in the context of coinfections. while not nearly as genetically diverse as the +ss rna hepatitis c virus nor as stable as the +ss rna hepatitis a virus, sars-cov-2 is likely susceptible to moderate genetic diversity, despite the sars-cov-2 rbd already significantly higher binding affinity to the human angiotensin-converting enzyme 2 (ace2) receptor than sars-cov rbd 27 . so, although sars-cov-2 outbreak appeared after sars-cov, phylogenetically, sars-cov-2 appears to be an "older" virus more closely related to the progenitor bat cov than sars-cov 26 . one surrogate for the level of broadly protective, relative immunogenicity is the frequency of reinfection. reinfection with the four circulating human "common cold"-type coronaviruses appears to be a frequent event. examples from the two human coronaviruses studied since the 1960s, include a 4-year study of hcov-oc43 infection in tecumseh, michigan following an hcov-229e outbreak in 34% of the study population 28 . the incidence of hcov-oc43 infection in children <5 years of age was high, yet subsequent symptomatic reinfection, albeit mild except chronic bronchitis in some, was quite frequent in older children and in adults. when analyzed immunologically, >80% of these subsequent symptomatic infections occurred despite prior neutralizing antibodies, calling into question the protective value of circulating neutralizing antibody (or the assays used) 29 . reinfections were also frequently observed, commonly associated with respiratory symptoms, for these two human coronaviruses in young children 30 , as well as in a longitudinal study of working adults 31 . similar but less robust epidemiological findings have been reported from the more recently described endemic human coronaviruses, hcov-nl63 (first described in 2004 32 ) and hcov-hku1 (first described in 2005 33 ) (see also table 1 for references). controlled human infection model (chim) studies 34 provide another source of evidence to inform categorization of broadly protective, relative immunogenicity. in the case of hcov-229e, chim studies in adults document susceptibility to symptomatic reinfection despite the presence of detectable antibodies, although homologous re-challenge a year later led to only asymptomatic reinfection 35 . as noted by callow et al. 35 the human challenge data are consistent with the notion that adults have human coronavirus infections on a 2-3 year cyclic pattern and that "protective amounts of antibody may have disappeared by 2 years, and that if we had been able to reinoculate the volunteers after a further year, the reinfection rate would have been even higher". the totality of the findings from natural and controlled challenge infections, in conjunction with a moderate degree of genetic diversity in these four endemic human coronaviruses, led to a "low" broadly protective, relative immunogenicity categorization in table 1 . similar to the four endemic coronaviruses, the quality and the durability of the protective immune response after natural infection with the three human epidemic coronaviruses appear to be "low" or at best "moderate", the difference being that severe disease has been observed more frequently for sars-cov, mers-cov, and sars-cov-2 than the common cold human coronaviruses. several longitudinal sero-epidemiology studies after the sars-cov outbreak reported a high post-convalescent seroconversion rate with igg peaking at 2-4 months in patient serum samples. in a small sample size study evaluating neutralizing activity in serial serum samples from patients with sars, >85% contained neutralizing antibodies (nab) against sars-cov and most of the nab activities could be attributed to immunoglobulin g (igg) 36 . however, the duration of circulating igg appeared relatively short-lived as the longest longitudinal study reported that at 3 years, the igg positivity had declined to 55.56% 37 . if the incubation period of sars-cov is sufficiently long to allow a protective anamnestic response, then subsequent re-exposure would likely lead to an asymptomatic infection. similar seroconversion and nab rates have been published for mers-cov, with several studies suggesting that antibody levels and longevity following mers-cov infection are correlated with disease severity [38] [39] [40] . okba et al. 41 reported that all fifteen severe mers-cov incubation period: median (range); italicized are published but unconfirmed incubation periods. route of transmission: dc direct non-genital contact of secretions, fo fecal-oral, r respiratory droplets. rna genome: +ssr positive single-stranded rna, -ssr negative single-stranded rna, α alphacoronavirus, β betacoronavirus. broadly protective, relative immunogenicity: using a delphi-type approach, incorporates in a single, semi-quantitative term [high; moderate (mod); low; or to be determined (tbd)]: (a) the genetic diversity of the virus (see column labeled "genetic stability"); and (b) two aspects of the host immune response: (1) the quality of the immune response that is elicited during and immediately after a primary infection (see first term in column labeled "immune response"); and (2) the ability and duration of that elicited immune response to protect against symptomatic reinfection (see second term in column labeled response"). cases tested positive in all tested platforms up to 1 year after disease onset, indicating a robust immune response of high antibody titers in severe cases; however, low or undetectable seroconversion rates and undetectable neutralizing antibodies were observed after most asymptomatic and some mild mers-cov infections. early data from the current sars-cov-2 pandemic suggest a similar pattern of immune responses in severe and mildly symptomatic sars-cov-2 patients 42 . given these data, it is tempting to speculate that a lower infectious inoculum intensity leads not only to a longer incubation period and less severe disease, but also to a less robust broadly protective, relative immunogenicity after natural infection. active immunization that optimally balances efficacy with reactogenicity/tolerability may represent the best of both worlds-robust broadly protective, relative immunogenicity without the severity of disease by administration of a high inoculum intensity without infectiousness. while it is too soon to have significant empiric data on the durability of sars-cov-2 immune responses to protect against symptomatic reinfection, the durability after natural infection as well as the long-term efficacy of active immunization may be influenced by the extent to which sars-cov-2 and/or related cross-reacting human coronaviruses continue to circulate in the population or "herd". under conditions in which insufficient herd immunity exists to curtail or even eliminate circulation of these viruses in the "herd", repeated sub-clinical infections may serve to maintain a protective immune response in an individual of the "herd". as the prevalence of these viruses diminishes in the "herd" as a result of adequate vaccine coverage and/or naturally acquired immunity, likely so will durability of protective immunity in the individuals in the "herd" as subsequent sub-clinical infections no longer occur and no longer serve to naturally "boost" an adequate protective immune response. in such situations where circulation of relevant coronaviruses significantly diminish, re-vaccination must be considered if a high "certainty-of-success" for long-term protection against future outbreaks is sought. a limitation of this rudimentary approach taken herein to assign a qualitative value to this composite biological feature of pathogens (fig. 1a) and sars-cov-2 (fig. 1b) is that it did not employ nor benefit from more powerful tools such as system biology analyses or mathematical models, which have been shown to provide important non-intuitive insights into host-virus interactions. such tools would need to be brought to bear on this topic for a more rigorous evaluation and for a more accurate and precise placement in the broad categories of high, moderate, and low levels of broadly protective, relative immunogenicity depicted in fig. 1a, b. estimating certainty-of-success by simultaneously considering the incubation timeline with the genetic diversity and the quality and durability of the host immune response, an approach to estimating the "certainty of success" based on biological feasibility emerges ( fig. 1 ) 1 . the model predicts an inverse relationship between the length of the incubation period and the level of the broadly protective, relative immunogenicity needed to achieve an equivalent "certainty of success". that is, for those pathogens that have a short incubation period and less opportunity for protection through an anamnestic response, a higher broadly protective, relative immunogenicity is needed to have a high "certainty-of-success"; likewise, for those pathogens having a long incubation period that benefit from protection through an anamnestic response, a lower broadly protective, relative immunogenicity is needed to have a high "certainty-ofsuccess". the association between incubation period and "certainty-ofsuccess" is just that-an association. although the proposed model may well accommodate the dataset presented in fig. 1a , cause and effect clearly has not been demonstrated. in fact, many of the viral pathogens that have short incubation periods also cause hit-andrun, local mucosal infections. these pathogens (e.g., rhinovirus, influenza, rsv, piv, and mpv) cause relatively brief illnesses and have limited tropism. whether the latter is the key parameter of biologic feasibility that determines "certainty-of-success" for developing prophylactic vaccines for these pathogens remains to be determined. as described in detail previously 1 , given its short incubation period and low broadly protective, relative immunogenicity (see table 1 ), influenza is a particular outlier in estimating "certainty of success" because of a relatively predictable transmission season, and the availability of annual immunization with an updated vaccine just prior to exposure in high-resource settings. in many low-resource settings, the latter is not an option and a fit-forpurpose influenza vaccine that does not require annual updating and annual administration has yet to be developed. predictions for the biological feasibility of developing effective covid-19 vaccines in the end, "predictions ought to count more than accommodations, because of the risk of 'fudging' that accommodations run and predictions avoid 43 ". with this mind, four guiding principles and three implications on the design, conduct and interpretation of vaccine clinical trials (see box 2) are offered for pressure-testing the three factors identified herein (see fig. 1b) , as sars-cov-2 candidate vaccines advance into proof-of-efficacy studies. while many other factors that are not easily controlled will affect the robustness of the principles and implications proposed, some box 2 proposed guiding principles and implications for covid-19 vaccine clinical trials proposed guiding principles in determining "certainty of success" 1. reducing the infectious inoculum intensity will: a. lengthen the incubation period. b. lengthen the latent period. c. increase vaccine efficacy. 2. lengthening the incubation period (see "note" below) will: a. reduce the risk of severe disease. b. increase the opportunity for anamnestic responses upon subsequent infectious inoculum exposure. 3. lengthening the latent period will: a. increase the herd effect of naturally acquired immunity and/or vaccine-induced protective immunity. 4. increasing the opportunity for anamnestic responses will: a. increase vaccine efficacy beyond that predicted by circulating antibody levels. b. increase durability of protective immunity while the pathogen still circulates in the population. implication for vaccine trial design, conduct, and interpretation: 5. assessing/estimating the incubation period during vaccine efficacy trials could provide insights into the infectious inoculum intensity and could provide insights into benefit-risk assessments for different use cases (e.g., high-risk first responders and healthcare workers with high-level exposure vs. general use to protect against low-level exposure during reopening after mitigation vs. routine use during interpandemic period). 6. determining vaccine efficacy for specific use cases and comparing vaccine efficacy of different vaccines should account for the infectious inoculum intensity in that specific use case setting and in the vaccine efficacy trial setting, respectively. 7. evaluating the correlates of protection (particularly in infectious inoculum intensity settings in which the incubation period is ≥5 days) should include measures of anamnestic responses, in addition to circulating functional antibody levels. note: by reducing the infectious inoculum intensity [through individual measures (e.g., handwashing, other hygiene practices, and personal protective equipment) and/or population-based measures to reduce the force of infection] or by naturally acquired or vaccine-induced protective immunity. consideration to the factors under human control would seem prudent. the one factor that emerges for consideration in sars-cov-2 vaccine development and implementation is reducing the infectious inoculum intensity (and force of infection, at a populationlevel) to lengthen the incubation period, reduce the severity of illness, and increase the opportunity for an anamnestic response upon exposure to the circulating virus. successfully implementing individual-and population-based behaviors that reduce the infectious inoculum intensity and force of infection, respectively, while testing and deploying covid-19 vaccines may be a critical human-controlled factor in assuring the "certainty of success" through immunization in controlling and eliminating sars-cov-2 infection, disease, death, and the pandemic itself. received: 27 april 2020; accepted: 7 may 2020; biological feasibility of developing prophylactic vaccines for viral pathogens: incubation period as a critical parameter the double-edged sword: how evolution can make or break a liveattenuated virus vaccine extraordinary diseases require extraordinary solutions sars-cov-2 viral load and the severity of covid-19 inoculum size, incubation period and severity of malaria. analysis of data from malaria therapy records the relationship between infecting dose and severity of disease in reported outbreaks of salmonella infections validation of the wild-type influenza a human challenge model h1n1pdmist: an a(h1n1)pdm09 dose-finding investigational new drug study brief report: incubation period duration and severity of clinical disease following severe acute respiratory syndrome coronavirus infection association between severity of mers-cov infection and incubation period does sars-cov-2 has a longer incubation period than sars and mers? outbreak of novel coronavirus (sars-cov-2): first evidences from international scientific literature and pending questions high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2. emerg association between the severity of influenza a(h7n9) virus infections and length of the incubation period effectiveness of rotavirus vaccination: a systematic review of the first decade of global postlicensure data a systematic review of the effect of rotavirus vaccination on diarrhea outcomes among children younger than 5 years global, regional, and national estimates of rotavirus mortality in children <5 years of age use of vaccines as probes to define disease burden mosquirix: public assessment report. mosquirix h-w-2300 a world malaria map: plasmodium falciparum endemicity in 2007 the dominance of human coronavirus oc43 and nl63 infections in infants molecular evolution of human coronavirus genomes common human coronaviruses | cdc recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission molecular pathogenesis of middle east respiratory syndrome (mers) coronavirus adaptive evolution of mers-cov to species variation in dpp4 zoonotic origins of human coronaviruses characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine community-wide outbreak of infection with a 229e-like coronavirus in tecumseh, michigan the tecumseh study of respiratory illness. vi. frequency of and relationship between outbreaks of coronavims infection epidemiology of coronavirus respiratory infections coronavirus infections in working adults. eight-year study with 229 e and oc 43 identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia controlled human infection model (chim) studies: summary of a workshop the time course of the immune response to experimental coronavirus infection of man neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection duration of antibody responses after severe acute respiratory syndrome antibody response and disease severity in healthcare worker mers survivors transmission of mers-coronavirus in household contacts mers-cov antibody responses 1 year after symptom onset sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections severe acute respiratory syndrome coronavirus 2−specific antibody responses in coronavirus disease 2019 patients testing hypotheses: prediction and prejudice an outbreak of influenza aboard a commercial airliner the experimental production of combination forms of virus realities and enigmas of human viral influenza: pathogenesis, epidemiology and control complete genome sequence of the first h5n1 avian influenza virus isolated from chickens in lebanon in 2016 h5 influenza, a global update kinetics of neutralizing antibodies in patients naturally infected by h5n1 virus adaptive evolution of human-isolated h5nx avian influenza a viruses probable person-to-person transmission of avian influenza a (h5n1) probable limited person-to-person transmission of highly pathogenic avian influenza a (h5n1) virus in china who influenza fact sheet (avian-and-other-zoonotic human cases of avian influenza a (h5n1 past, present, and possible future human infection with influenza virus a subtype h7 specificity, kinetics and longevity of antibody responses to avian influenza a(h7n9) virus infection in humans specific memory b cell response in humans upon infection with highly pathogenic h7n7 avian influenza virus comparison of the first three waves of avian influenza a(h7n9) virus circulation in the mainland of the people's republic of china signs and symptoms in common colds human coronaviruses 229e and nl63: close yet still so far effects of a 'new' human respiratory virus in volunteers human coronavirus nl63: a clinically important virus? human coronavirus-nl63 infections in korean children new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia human coronavirus nl63 infection in canada a previously undescribed coronavirus associated with respiratory disease in humans human coronavirus nl63 understanding human coronavirus hcov-nl63 identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection epidemiologic linkage and public health implication of a cluster of severe acute respiratory syndrome in an extended family lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study duration of serum neutralizing antibodies for sars-cov-2: lessons from sars-cov infection sars incubation and quarantine times: when is an exposed individual known to be disease free? multiple contact dates and sars incubation periods molecular evolution of the sars coronavirus during the course of the sars epidemic in china severe acute respiratory syndrome coronavirus 2 isolate wuhan-hu-1, complete genome the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application european centre for disease prevention and control. outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2): increased transmission beyond china -fourth update 14 incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan recovery in tracheal organ cultures of novel viruses from patients with respiratory disease an outbreak of coronavirus oc43 respiratory infection in normandy, france epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method prevalence and clinical characteristics of human cov-hku1 in children with acute respiratory tract infections in china clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 coronavirus hku1 and other coronavirus infections in hong kong hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans preliminary epidemiological assessment of mers-cov outbreak in south korea middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide d.c.k. conceived, wrote, reviewed, approved, and is accountable for this paper. d.c.k. is an employee of path (a not-for-profit organization), has no financial interest in any for-profit organization, and declares no competing interests. correspondence and requests for materials should be addressed to d.c.k. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-007828-c7jxj74b authors: memish, ziad a.; al-tawfiq, jaffar a. title: middle east respiratory syndrome coronavirus infection control: the missing piece? date: 2014-11-25 journal: am j infect control doi: 10.1016/j.ajic.2014.08.003 sha: doc_id: 7828 cord_uid: c7jxj74b nan since the initial occurrence of middle east respiratory syndrome coronavirus (mers-cov) in 2012, 1,2 the disease had caused 837 cases, with a case fatality rate of 34.7%. 3 as with any emerging infectious diseases of pandemic potential there is a concern of the global spread of the disease. it is therefore the first priority of the global public health community to develop and implement the required infection control practices to prevent the dissemination of these emerging organisms within health care facilities (hcfs) and worldwide based on the best available evidence and previous experience with similar or related groups of pathogens. the world health organization (who) through its expert technical committees was prompt in developing its first infection control guidelines based on available knowledge on the new emerging virus, but it mostly drew on experience from a similar virus, severe acute respiratory syndrome coronavirus (sars). these guidelines were updated based on accumulating experience from reports of mers-cov cases and clusters in the community and hcfs in affected countries and regions. this became particularly important with the third and largest wave of hcf-associated mers-cov cases reported from the kingdom of saudi arabia and united arab emirates in april-may 2014. 4 the initial cases were reported in hospital a in jeddah with subsequent appearance of cases in 3 other hospitals. during the initial jeddah outbreak, there were 5 involved hospitals: a-e. hospital a had 3 cases: a community case and 2 health care workers (hcws) assumed to have been infected from the first case. hospital b had 2 community-acquired infections. hospital c reported 1 community-acquired case. hospital d had 1 fatal case in an hcw. the source of infection was believed to be the community; however, hospital-acquired infection could not be ruled out. hospital e was where the main nosocomial cluster occurred. hospital e is a large and busy referral teaching hospital where staff share accommodation. the emergency room is busy with an occupancy rate of 578% for the 76 emergency beds. there were initially 4 community cases reported. extensive screening of their contacts among hcws and family members identified 26 laboratory-confirmed cases among hcws (19 were asymptomatic), 1 family member, and 6 patient contacts. careful review of the recent increase in the number of cases revealed that about 25% were among hcws. 4 of the initial 128 recent mers-cov infected patients in jeddah, kingdom of saudi arabia, most (60%) were infected in the health care setting. of those, 39 were hcws. 5, 6 these 128 cases occurred between february 17, 2014, and april 26, 2014, 6 and were treated in 14 hospitals in jeddah. most hospitals had 1 or 2 patients, and hospital e had 45 cases. 6 of these cases, 33% were primary cases. 6 of the 128 cases, 60% (including 39 hcws) were from health careeacquired infection. 6 an extensive screening of contacts showed that 7 of 554 household contacts (1.3%) were polymerase chain reaction positive for mers-cov 6 compared with 3.03% of 462 family contacts screened during 2012-2013. 7 because the largest percentage of secondary human-to-human transmission occurs in hcfs, 8 the critical question remains of whether the recent large multi-hcf clustering in 2014 was caused by failure of the evidence-based recommended infection control measures outlined by the who or failure of its strict application in the affected facilities. the who continues to stress 3 different approaches to infection control: contact precautions, droplet precautions, and airborne isolation. 9 the use of contact precautions is thought to be needed because of the presence of diarrhea and vomiting in approximately 30% of cases. 2, 10, 11 although, there was no documented transmission of mers-cov through this route, the virus was isolated from stool in a few patients. 12 in addition, studies investigating environmental stability of mers-cov have revealed that mers-cov was stable at different temperature and humidity conditions and could still be recovered after 48 hours, which supports the potential of mers-cov to be transmitted via contact or fomite transmission because of prolonged environmental presence. 13 drawing on data from similar viruses, the potential transmission of viral respiratory infections by contacts was highlighted previously. 14 because mers-cov is similar to sars in many aspects, patients with sars had high virus concentrations and prolonged virus excretion in stools. [15] [16] [17] assessment of infection control practices applied in the affected hcfs during the peak of the outbreak conducted by local and international agencies concluded that the jeddah outbreak was related to poor compliance with the recommended basic infection control measures. 18 the finding is supported by similar observations from sars. during the sars outbreak, the infection in health care settings was further exaggerated by overcrowding, short distance between patient beds, inadequate ventilation, use of aerosol-generating techniques, and during cardiopulmonary resuscitation. 19, 20 the who report on infection control measures for severe acute respiratory infections also elucidated the major risks associated with high-risk aerosol-generating procedures. 9 the recommendations to use droplet precautions for all patients admitted with confirmed or suspected mers-cov, except in aerosol-generating procedures, come from the understanding of the al-hasa outbreak. 2 during that outbreak, a total of 23 patients were recorded across 4 health care settings. the outbreak was controlled with implementation of the basic infection control measures without airborne isolation. 2, 21 the recommendation also relies on the fact that viral respiratory tract infections (eg, sars) spread by large (10 mm in diameter) respiratory droplets. 22 there is a clear seasonal disease activity because the zarqa, jordan, outbreak was in in april 2012, the al-hasa outbreak was in april-may 2013, and the jeddah and united arab emirates outbreak was in april-may 2014 (fig 1) . therefore, exposure of hcws is more likely during these months because of increased community cases. 23, 24 airborne infection isolation (aii) precautions should be applied during any aerosol-generating procedures as recommended by the who. 21, 25 during the sars outbreak, aerosol-generating procedures associated with increased risk of transmission of sars were intubation, tracheotomy, and manual ventilation. 26 indeed, in the only study addressing the risk of mers-cov transmission among hcws, the reported staff were involved in at least 1 of the following highrisk procedures: intubation, airway suctioning, and sputum induction. 27 the centers for disease control and prevention in the united states and european centre for disease prevention and control continue to recommend the application of aii precautions when caring for patients with mers-cov. 28 these recommendations and the recommendations from other experts rely on the fear of the disease and high case fatality rate. 29 in a recently published debate, the presented evidence supported the use of droplet precautions, not aerosol-generating procedures. 29 a recent study showed that mers-cov rna was isolated from the barn of camels linked to a human mers-cov case suggesting possible aerosolization of mers-cov. 30 the study had several limitations, including the following: it was only 1 positive sample; there was a lack of internationally acceptable sampling strategies of the air, and the sequences are all 100% identical to all other sequences from the patient, camel, and laboratory, which suggests contamination; there was a lack of proof of the causation of aerosol dissemination of the virus; and finally, the viral load in the air sample was higher than the viral load in the camel's nose. 30 in addition, there are no established methods for sampling airborne exposures. 31 for the transmission of sars, and this is likely true with mers-cov, multiple factors play a role in the propagation of infection in a health care setting. these factors include the following: lax basic infection control procedures, aerosol-generating procedures, improper use of personal protective equipment, and mouth exposure to patients' body fluids and excretions. [32] [33] [34] [35] [36] [37] [38] teasing out the most important factors contributing to hcw infection of sars, mers-cov, or emerging respiratory viruses is of paramount importance. in addition, the utilization of maximum respiratory protection is easily applied when there are a few cases, but this strategy puts a burden on any health care system when the number of cases increases substantially. when resources are available, using aii precautions in conjunction with contact precautions would provide the best protection for hcws. isolation of a novel coronavirus from a man with pneumonia insaudi arabia hospital outbreak of middle east respiratory syndrome coronavirus global alert and response (gar): middle east respiratory syndrome coronavirus (mers-cov) e update world health organization. global alert and response (gar): coronavirus infections european centre for disease prevention and control. epidemiological update middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov) summary and literature updateeas of 9 screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study assessment of potential risk factors of infection of middle east respiratory syndrome coronavirus (mers-cov) among health care personnel in a health care setting global alert and response (gar): infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions infection prevention and control measures for acute respiratory infections in healthcare settings: an update identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome the severe acute respiratory syndrome world health organization. who concludes mers-cov mission in saudi arabia sars: experience at prince of wales hospital, hong kong detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units hospital-associated middle east respiratory syndrome coronavirus infections infection control and mers-cov in health-care workers travel implications of emerging coronaviruses: sars and mers-cov coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers global alert and response (gar): middle east respiratory syndrome coronavirus (mers-cov) e update aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review middle east respiratory syndrome coronavirus infections in health care workers european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov) debate on mers-cov respiratory precautions: surgical mask or n95 respirators detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient commentary: protecting health workers from airborne mers-covdlearning from sars which preventive measures might protect health care workers from sars? clinical management and infection control of sars: lessons learned cluster of cases of severe acute respiratory syndrome among toronto healthcare workers after implementation of infection control precautions: a case series transmission of severe acute respiratory syndrome during intubation and mechanical ventilation illness in intensive care staff after brief exposure to severe acute respiratory syndrome sars transmission among hospital workers in hong kong risk factors for sars transmission from patients requiring intubation: a multicenter investigation in toronto key: cord-267090-jc1k3fki authors: gardner, emma g.; kelton, david; poljak, zvonimir; van kerkhove, maria; von dobschuetz, sophie; greer, amy l. title: a case-crossover analysis of the impact of weather on primary cases of middle east respiratory syndrome date: 2019-02-04 journal: bmc infect dis doi: 10.1186/s12879-019-3729-5 sha: doc_id: 267090 cord_uid: jc1k3fki background: middle east respiratory syndrome coronavirus (mers-cov) is endemic in dromedary camels in the arabian peninsula, and zoonotic transmission to people is a sporadic event. in the absence of epidemiological data on the reservoir species, patterns of zoonotic transmission have largely been approximated from primary human cases. this study aimed to identify meteorological factors that may increase the risk of primary mers infections in humans. methods: a case-crossover design was used to identify associations between primary mers cases and preceding weather conditions within the 2-week incubation period in saudi arabia using univariable conditional logistic regression. cases with symptom onset between january 2015 – december 2017 were obtained from a publicly available line list of human mers cases maintained by the world health organization. the complete case dataset (n = 1191) was reduced to approximate the cases most likely to represent spillover transmission from camels (n = 446). data from meteorological stations closest to the largest city in each province were used to calculate the daily mean, minimum, and maximum temperature ((ο)c), relative humidity (%), wind speed (m/s), and visibility (m). weather variables were categorized according to strata; temperature and humidity into tertiles, and visibility and wind speed into halves. results: lowest temperature (odds ratio = 1.27; 95% confidence interval = 1.04–1.56) and humidity (or = 1.35; 95% ci = 1.10–1.65) were associated with increased cases 8–10 days later. high visibility was associated with an increased number of cases 7 days later (or = 1.26; 95% ci = 1.01–1.57), while wind speed also showed statistically significant associations with cases 5–6 days later. conclusions: results suggest that primary mers human cases in saudi arabia are more likely to occur when conditions are relatively cold and dry. this is similar to seasonal patterns that have been described for other respiratory diseases in temperate climates. it was hypothesized that low visibility would be positively associated with primary cases of mers, however the opposite relationship was seen. this may reflect behavioural changes in different weather conditions. this analysis provides key initial evidence of an environmental component contributing to the development of primary mers-cov infections. middle east respiratory syndrome coronavirus (mers--cov) is an emerging zoonotic agent that was first isolated in 2012 from a patient hospitalized in saudi arabia [1] , and has since infected over 2200 people with a 36% case fatality ratio [2] . after an incubation period of 2-14 days [3] , the virus causes a disease (middle east respiratory syndrome, or mers) characterized by fever, cough, and shortness of breath, which commonly leads to pneumonia and respiratory failure [4] . the virus circulates silently in dromedary camels, the only known reservoir species and zoonotic source of spillover to humans [5] . however, not all primary human cases have documented exposure to dromedaries or their products, such as milk and meat. although human-to-human community-acquired infections have not been documented, there is evidence that asymptomatic infections of mers-cov exist and could be a source of community transmission [6] . zoonotic spillover from dromedary camels to humans has been documented in the arabian peninsula [7] . subsequent secondary cases can occur after unprotected contact with family members and within healthcare facilities once the primary case seeks medical assistance [8] . while the sizes of mers-cov outbreaks have decreased thanks to improved infection control in healthcare settings in affected countries, cases continue to be reported regularly, especially in saudi arabia, where surveillance is strong [9] . in order to further reduce cases and prevent human outbreaks, a better understanding of zoonotic transmission of mers-cov is needed. a deeper understanding of the epidemiology of primary human cases can inform evidence-based interventions at the level of the community at the animal-human interface. zoonotic modes of mers-cov transmission have not yet been definitively determined. mers-cov in dromedary camels causes a mild upper respiratory infection with no documented viremia [10] , and therefore droplet or aerosol transmission by close camel contact is most likely. however, transmission through contaminated milk, meat, and urine is possible, although the contribution of camel products cannot currently be estimated due to a lack of scientific evidence. the effects of weather and environmental conditions on respiratory diseases with similar modes of transmission (direct contact or droplet), such as influenza and respiratory syncytial virus, have been documented. temperature and humidity are associated with transmissibility of influenza virus [11] , and the seasonality of both influenza and respiratory syncytial virus is linked to these two factors [12] . air quality is also associated with respiratory infections. air pollution has been linked to pneumonia and acute lower respiratory infections [13, 14] , while dust storms are associated with infectious respiratory disease by acting both as a carrier of pathogens and increasing airway susceptibility to infection [15] . the risk of acquiring primary mers may be influenced by changes in weather conditions in two ways. first, weather conditions may affect the viability and persistence of the virus in the environment and therefore its transmissibility [11, 16] . secondly, weather influences behaviour, and it is plausible that the likelihood of people contacting camels depends on environmental conditions. seasonal or meteorological patterns of primary mers-cov infections have yet to be explored. this study examined whether meteorological conditions were associated with the development of known primary mers-cov infections using a case-crossover study design. case-crossover studies are designed so that exposures during a period of interest before a case are compared to exposures during control periods before or after the case. in this regard, case-crossover studies answer the question "why now?" as opposed to "why these subjects?" [17] . the design is well suited for rare diseases with short incubation periods such as mers-cov. the effect period, that is, the period of time after the proposed "trigger", typically has a degree of uncertainty [17] , leading to exposure windows with intervals of biological relevance to the outcome of interest. for infectious diseases, this would equate to the incubation period [18] . furthermore, with appropriate selection of referent windows, the case-crossover design controls for confounding effects of temporal fluctuations such as climatic and livestock-associated seasons (e.g. the dromedary breeding cycle) [19] . by comparing weather conditions immediately before mers cases to weather conditions at other times, this study aimed to identify environmental factors that are associated with primary human mers in saudi arabia. the world health organization (who) maintains a list of all human laboratory confirmed cases of mers-cov. publicly available case data from january 2015-december 2017 were obtained. case data prior to 2015 were excluded due to a lack of standardized data collection prior to 2015 [20] . a mers case was defined throughout the study period as "a person with laboratory confirmation of mers-cov infection irrespective of clinical signs and symptoms" [21] . of the 1191 confirmed cases with onset dates between january 2015 -december 2017, 298 cases were removed where exposure to camels and camel products were known not to have occurred. geographically, cases were restricted to those reported from saudi arabia, where the province of exposure was provided (n = 651). cases that were likely primary cases were retained by excluding healthcare workers and cases with documented contact with known mers cases (n = 128). cases were further removed where symptom onset date was after hospitalization date (n = 44). of the remaining cases (n = 479), 10 (2.1%) had missing symptom onset dates. to retain these ten cases, the median time between symptom onset date and lab confirmation date was calculated (6 days) and subtracted from the lab confirmation date to obtain an estimate of the symptom onset date. visual inspection of the timeline of retained cases identified a spike from riyadh province around august 2015, which corresponds to a documented mers-cov outbreak in the city of riyadh from july-september 2015. data from a published report of the outbreak contained weekly counts of primary and secondary cases [22] . these weekly counts were compared with the case list for this analysis and thirty-two secondary cases associated with the riyadh outbreak were removed. the final number of retained cases fitting the primary case definition was 446 ( fig. 1 ). for the purposes of the descriptive results, age groups were chosen for ease of reading while still providing a visualization of the distribution, and according to age categories provided by the statistical yearbook of the general authority for statistics of the kingdom of saudi arabia, which was used for standardization. meteorological stations closest to the largest city in each province were identified by a numeric identifier and location using google earth [23] (fig. 2) . meteorological data were obtained from the noaa global hourly index [24] . the daily mean, minimum, and maximum temperature, wind speed, and visibility were calculated. relative humidity was calculated using temperature and dew point data [25] . a case-crossover design was used to explore the associations between primary mers cases and preceding meteorological conditions [17, 26] . each case's exposure status on individual days before disease onset (the exposure window) was compared to the exposure status on different days during a control period. under the assumption that weather effects on virus transmission were immediate, the exposure window, that is, the time lag between weather events and disease onset, was set to be equal to the mers incubation period of 2-14 days [3] . univariable conditional logistic regression was used to assess statistical associations between cases and weather variables on each day within the case and control exposure windows. associations with p < 0.05 were considered statistically significant. a time-stratified design was used, with a 28-day strata length with random bi-directional controls matched by day of the week. using a 28-day time window provides at least three control days for each case exposure day while minimizing bias introduced from seasonal changes [27] . temperature and humidity variables were categorized into tertiles calculated within each time stratum. wind speed and horizontal ground visibility were categorized into two groups within each stratum with the median as cutoff. therefore, there is no single threshold for each weather variable, but rather "low", "medium" and "high" are determined according to the measurements in each stratum. statistical analyses were conducted using stata 15.0 (stata corporation, college station, tx). four hundred and forty-six cases of mers-cov in saudi arabia with symptom onset dates between january 2015 -december 2017 were included in the analysis. table 1 presents the case counts as well as crude and age-and sex-standardized rates by province, sex, and age group. all 13 provinces in saudi arabia reported cases during this 3-year period. riyadh province had the highest count of reported cases with 185 cases (42%), although qasseem had the highest cumulative incidence (3.8 cases per 100,000 people), followed by riyadh (2.54 cases per 100,000 people). the median age of cases was 58 years (range, 15-98), and 79% of cases were male. age and sex proportions are similar to figures reported for primary cases in previously published literature [28] . figure 3 presents the case count by month from 2015 to 2017 for the entire country. cases were reported in every month of the year, although no clear seasonality is apparent. temperature and humidity conditions were associated with case occurrence 8-11 days later. the odds of a mers case 9 days after low minimum temperatures was 1.29 (95% confidence interval [ci], 1.06-1.58) higher than after control days, while low mean daily temperature was similarly associated with cases at 9 (or, 1.25; 95% ci, 1.02-1.54) and 10 day lags (or, 1.27; 95% ci, 1.04-1.56) (fig. 4) . conversely, high minimum, maximum, and mean temperatures were protective at similar lag days. for example, the odds ratios of mers cases for the high mean daily temperature was 0.77 (95% ci, 0.61-0.96) with a 9-day lag, and 0.73 (95% ci, 0.58-0.92) with a 10-day lag. humidity followed a similar pattern to temperature. when maximum daily humidity was low 8 days earlier, the odds ratio for a mers cases was 1.35 (95% ci, 1.10-1.65). high humidity was associated with fewer cases across all three daily measurements (fig. 5) . for example, the odds ratios of cases for high maximum daily humidity was 0.68 (95% ci, 0.53-0.86) and 0.75 (95% ci, 0.60-0.95) at 9-and 10-day lags, respectively. high visibility was positively associated with occurrence of a mers case 7 days later, whereas low visibility demonstrated protective effects for risk of mers (fig. 6) . the odds of a mers case 7 days after both minimum and mean daily visibility were high was 1.27 and 1.26 times higher than after control days (95% ci, 1.01-1.60 and 1.01-1.57). conversely, when minimum and mean wind speed results were conflicting, with low minimum daily wind speed and high maximum wind speed both positively associated with cases at similar time lags (fig. 6) . the odds of a mers case was 1.40 times higher 6 days after low minimum wind speed (95% ci, 1.05-1.87), while the odds ratio of cases for when minimum wind speed was relatively high was 0.72 (95% ci, 0.54-0.96). conversely, the odds of a case when maximum wind speed was relatively high was 1.23 (95% ci, 1.003-1.50) (not shown in figure) . mers is a global public health threat that causes severe respiratory disease with a high case fatality ratio, identified by who as a priority pathogen for research and development in public health emergency contexts [29] . it is primarily characterized by healthcare-associated outbreaks triggered by index cases who acquire infection from dromedary camels and possibly from unidentified asymptomatic human carriers. improving our understanding of the epidemiology and risks of primary cases of mers is vital for designing effective interventions that aim to reduce these index cases and prevent subsequent outbreaks in humans. the list of cases maintained by the who was restricted to a subset of primary cases based on explicit inclusion and exclusion criteria and was used to analyze the effect of weather on case occurrence using a case-crossover design. all four weather variables demonstrated statistically significant correlations within the incubation period for mers in humans. the statistically significant time lags for each variable do not match up perfectly, which is to be expected and could be due to a number of reasons including natural variability in incubation periods, variable impact of weather on transmission, the interaction of unmeasured cofactors on weather variables as well the direct effect of unmeasured factors on transmission, and stochasticity in general. acute weather events as well as general seasonal patterns may affect disease transmission rates by altering pathogen viability and persistence in the environment as well as by influencing human behaviour and contact patterns. this study found that mers-cov, although a zoonotic disease, follows similar environmental transmission patterns to other non-zoonotic respiratory diseases with analogous modes of transmission such as influenza and respiratory syncytial virus. tamerius et al. [30] have shown that global trends of influenza broadly follow either a "cold-dry" or "humid-rainy" pattern, corresponding to temperate and tropical climates. additionally, temperate climates tend to have a single annual peak and tropical climates have semi-annual peaks. they further demonstrate that for countries with an annual influenza peak such as saudi arabia, temperature and humidity can be predictive of those peaks, even at latitudes close to the equator. respiratory syncytial virus also follows similar environmental conditions, with peak timing in the arabian peninsula from december to february, following the distribution of cases in the temperate northern hemisphere [12] . the influence of weather is further supported by experimental evidence, which has demonstrated that lower temperatures and lower relative humidity each favour influenza transmission [11] . furthermore, coronaviruses have been shown to exhibit strong seasonal variation in natural hosts, and the theory that these fluctuations may increase risk of zoonotic transmission at certain times of the year has been discussed [16] . the results here demonstrate that colder, drier conditions may increase the risk of zoonotic transmission of mers from dromedaries to humans. sandstorms, dust storms, and air pollution in saudi arabia and elsewhere have been associated with increased morbidity and mortality, including from respiratory disease [31, 32] . a case-crossover study in the united states demonstrated the short-term effects of air pollution on acute lower respiratory infections [14] , while another study demonstrated increased numbers of pneumonia admissions following acute dust storm events in taiwan [33] . dust storms can act as a pathogen carrier and also induce inflammatory reactions, potentially increasing both exposure and susceptibility to disease agents [15] . horizontal ground visibility and wind speed were used as proxies for the occurrence of sandstorms and acute air pollution events. visibility can be reduced to 5000 m for an average of 3.5 h during a sandstorm [34] . summarizing the weather data used in this study, the mean daily visibility by province ranged from 82 m to over 10,000 m, although the median value was over 9000 m in all but one province. the distribution of visibility indicates that fig. 4 daily mean and minimum temperature and risk of primary mers by province in saudi arabia. odds ratio (solid line) and 95% confidence limits (dashed lines) are plotted on the y-axis, while time lags preceding case occurrence are plotted on the x-axis. the odds of primary mers is increased with low temperature at 9 and 10 day lags (a &b), while the odds of primary mers are decreased with high temperatures at 10 and 11 day lags (c & d). asterisks indicate statistically significant odds ratios on corresponding days anything less than full clarity was categorized as low visibility, and that according to the measurements in [34] , could indicate the presence of a sandstorm. it was hypothesized that primary mers infections are more likely to increase following sandstorms or other severe events of air pollution that affect visibility. however, results indicate that the risk of primary mers infection increased following high visibility days, and decreased following low visibility days. this may be due to behaviour, if people are more likely to stay inside during acute weather events, and less likely to engage in activities such as interacting with camels. it was further hypothesized that higher wind speeds would be associated with more cases of mers. while a positive association was found between cases and high maximum wind speeds 5 days prior, there were also similar results to those of visibility. low minimum wind speed was positively correlated with cases, and conversely, when minimum wind speed was relatively high there were statistically fewer cases of mers. results suggest that further investigation of wind speed as a factor for primary mers is warranted. there are several limitations and potential sources of bias in this study. the major cities in saudi arabia are severely polluted and exceed who guidelines, as measured by particulate matter (pm) [35, 36] . sand and dust storms as well as other sources of air pollution such as industrial activities, fuel combustion, and traffic emissions contribute to elevated levels of pm in the country [35] , all of which contribute to reduced visibility [37] [38] [39] . this study did not differentiate between sandstorms and other acute events that reduce visibility, and discerning between different forms of air pollution may provide insights about the risk of mers-cov transmission under different environmental conditions. fifty-two cases (12.3%) in the subset of primary cases had no known exposure history (no information on camel exposure, contact with a known case, nor healthcare worker status). the subset of primary cases investigated likely also include secondary cases, and is a source of selection bias. furthermore, given that mers is an emerging disease, case reporting and data collection standardization may have improved over the 3-year period included here. geographical case data were available only at the provincial level, while exposure data from the weather station closest to the largest metropolitan city in each province were used. while camel raising in the middle east is moving from extensive to intensive production systems and concentrating around cities [40] , human spillover cases would be scattered throughout the provinces to an unknown degree. therefore, if environmental conditions differ significantly within a province, this could be a source of misclassification bias. the risk of primary human cases of mers was associated with a decrease in temperature and humidity, and an increase in ground visibility. the temperature and humidity findings are consistent with associations between the environment and other respiratory diseases. further study of weather and seasonal risk factors may strengthen the evidence for an environmental component of mers-cov transmission. a better understanding of virus viability in different environmental conditions is also a key research need. evidence of environmental risk factors for mers could be utilized by public or one fig. 6 daily visibility and wind speed variables and risk of primary mers by province in saudi arabia. odds ratio (solid line) and 95% confidence limits (dashed lines) are plotted on the y-axis, while time lags preceding case occurrence are plotted on the x-axis. the odds of primary mers is increased with high visibility and decreased with low visibility after 7 days (a & c), while the odds of primary mers are increased with low wind speed and decreased when wind speed is high at 6-day lags (b & d). when maximum wind speed was high, the odds of a mers case were increased with a 5-day lag (not shown). asterisks indicate statistically significant odds ratios on corresponding days health practitioners for targeted interventions during higher-risk periods. the risk of mers acquired from zoonotic transmission, or from asymptomatic carriers in the community, appears to be sensitive to weather conditions, providing key initial evidence of an environmental component for the development of primary mers-cov infections. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. who | middle east respiratory syndrome coronavirus (mers-cov). who world health organization. investigation of cases of human infection with middle east respiratory syndrome coronavirus (mers-cov) interim guidance middle east respiratory syndrome coronavirus (mers-cov) fact sheet mers-cov technical working group. mers-cov: progress in global response to epidemic threat, remaining challenges and way forward: report from the fao-oie-who global technical meeting on mers-cov presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome world health organization. mers situation update replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels influenza virus transmission is dependent on relative humidity and temperature latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review international approach to environmental and lung health a perspective from the fogarty international center short-term elevation of fine particulate matter air pollution and acute lower respiratory infection lung health in era of climate change and dust storms seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us should we use a case-crossover design? a comparison of case-crossover and case-control designs in a study of risk factors for hemorrhagic fever with renal syndrome bias in the case-crossover design:implications for studies of air pollution middle east respiratory syndrome coronavirus (mers-cov) disease outbreak news world health organization. who | middle east respiratory syndrome coronavirus: case definition for reporting to notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital national centers for environmental information weathermetrics: functions to convert between weather metrics the case-crossover design: a method for studying transient effects on the risk of acute events referent selection in case-crossover analyses of acute health effects of air pollution reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases world health organization. who | list of blueprint priority pathogens environmental predictors of seasonal influenza epidemics across temperate and tropical climates the pulmonary consequences of sandstorms in saudi arabia: a comprehensive review and update the effect of sandstorms and air pollution on causespecific hospital admissions in taipei asian dust storm events are associated with an acute increase in pneumonia hospitalization predicting the development of weather phenomena that influence aviation at abu dhabi international airport. pretoria: university of pretoria outdoor particulate matter ( pm ) and associated cardiovascular diseases in the middle east world health organization. who air quality guidelines for particulate matter, ozone, nitrogen dioxide and sulfur dioxide: global update 2005: summary of risk assessment the influence of meteorological conditions and atmospheric circulation types on pm 10 and visibility in tel aviv estimation of particulate matter from visibility in bangkok fine particulate matter characteristics and its impact on visibility impairment at two urban sites in korea: seoul and incheon human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection the authors would like to thank all of the many individuals who investigated and collected information from mers patients in saudi arabia. availability of data and materials all laboratory confirmed human cases of mers included this publication can be found on the who disease outbreak news website, at the following website: http://www.who.int/csr/don/archive/disease/mers-cov/en/ authors' contributions eg designed the study, analyzed and interpreted the data and wrote the manuscript. ag and dk provided significant guidance in all aspects of the research. ag, dk, svd, mvk and zp substantially contributed to the conception of the study and interpretation of the results, critically reviewed the manuscript and provided final approval for publication.ethics approval and consent to participate not applicable: all data used were publicly available. not applicable. publicly available, non-individually identifying data were used in this publication. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-258281-gxwk8jq9 authors: wenling, yao; junchao, qiu; xiao, zhirong; ouyang, shi title: pregnancy and covid-19: management and challenges date: 2020-08-31 journal: revista do instituto de medicina tropical de sao paulo doi: 10.1590/s1678-9946202062062 sha: doc_id: 258281 cord_uid: gxwk8jq9 the consequences of covid-19 infecting pregnant women and the potential risks of vertical transmission have become a major issue. since little is currently known about covid-19 in pregnancy, the understanding of covid-19 in this particular group will be updated in time, and a comprehensive review will be useful to evaluate the impact of covid-19 in pregnancy. based on recently published literature and official documents, this review provides an introduction to the pathogenesis, pathology, and clinical features of covid-19 and has focused on the current researches on clinical features, pregnancy outcomes and placental histopathological analysis from pregnant women infected with sars-cov-2 in comparison with sars-cov and mers-cov. these viruses trigger a cytokine storm in the body, produce a series of immune responses, and cause changes in peripheral leukocytes and immune system cells leading to pregnancy complications that may be associated with viral infections. the expression of ace2 receptors in the vascular endothelium may explain the histological changes of placentas from pregnant women infected by sars-cov-2. pregnant women with covid-19 pneumonia show similar clinical characteristics compared with non-pregnant counterparts. although there is no unequivocal evidence to support the fetal infection by intrauterine vertical transmission of sars, mers and sars-cov-2 so far, more and more articles began to report maternal deaths due to covid-19. in particular, from february 26, 2020 (date of the first covid-19 case reported in brazil) until june 18, 2020, brazil reported 124 maternal deaths. therefore, pregnant women and neonates require special attention regarding the prevention, diagnosis and management of covid-19. a series of a new coronavirus pneumonia (ncp), whose pathogen is the novel 2019 coronavirus (2019 -ncov) was officially recognized by the world health organization on february 11, 2020 and the disease was called covid-19. meanwhile, the international committee on taxonomy of viruses named the 2019 novel coronavirus as sars-cov-2. at 10:00 a.m. on march 30, 2020, a total of 82,447 confirmed cases and 3,310 deaths had been reported in china, with a case fatality rate of 4.0%, while there were 693,224 confirmed cases and 33,106 deaths worldwide, with a case fatality rate of about 4.8% 1 . furthermore, the epidemic had a tendency to spread around the world. therefore, the who has raised the global risk of the covid-19 epidemic to the highest level (very high). together with the other two highly pathogenic coronaviruses, the sars-cov and mers-cov, sars-cov-2 poses a global threat to public health. pregnant women are particularly vulnerable to respiratory pathogens and severe pneumonia due to physiological and immunological changes, such as altered t lymphocyte immunity, increased oxygen consumption, decreased functional residual capacity and decreased chest compliance, which may result in higher maternal and fetal morbidity and mortality 2 . furthermore, pregnant women with pneumonia have a significantly higher risk of giving birth to low birth weight, as well as preterm and, small for gestational age infants, with low apgar scores, born by cesarean section, whose mothers presented with preeclampsia/ eclampsia more often than uninfected women 3 . however, there is still insufficient data to evaluate the impact of covid-19 on pregnant women. considering that sars-cov-2, sars-cov, and mers-cov are all β-coronaviruses, and their genomes, pathogenesis and clinical symptoms have certain similarities, this article draws lessons from previous studies on sars-cov and mers-cov infecting pregnant women to predict the impact of sars-cov-2 on pregnant women and fetuses and make some suggestions. the functional receptor for sars-cov is the angiotensin-converting enzyme 2 (ace2) 4 , which is abundantly present in lung alveolar epithelial cells and enterocytes of the small intestine, as well as in arterial and venous endothelial cells and arterial smooth muscle cells in virtually all of the organs 5 . in contrast, mers-cov uses the dipeptidyl peptidase 4 (dpp4) as its receptor 6 . dpp4 is widely expressed on epithelial cells in the kidney, alveoli, small intestine, liver, prostate and activated leukocytes 7 . sars-cov-2 is a novel β-coronavirus from the subgenus sarbecovirus, genetically similar to sars-cov (about 79%) and mers-cov (about 50%) 8 . like in sars-cov infections, ace2 is the cell receptor for sars-cov-2 9 . the spike proteins of these viruses bind with cellular receptors of sensitive cells to mediate the infection of their target cells, after which viral replication begins in the cell cytoplasm. sars-cov and mers-cov were both found to be able to show extremely high and early replication rates, infecting dendritic cells, macrophages and t cells 10-13 , using several strategies to avoid the host innate immune response 11, 14 , and resulting in a robust and sustained production of proinflammatory cytokines and chemokines 11, 12, 15, 16 17 . il6 (interleukin 6) levels in patients with severe covid-19 were also significantly higher than in patients with milder infections 18 . these results suggest that the cytokine storm may also play a relevant role in the development of covid-19. the pathological features of covid-19 greatly resemble those seen in sars and mers coronavirus infections [19] [20] [21] [22] [23] . diffuse alveolar damage is the predominant pulmonary histological pattern. other changes included hyaline membrane formation, alveolar hemorrhage, desquamation of pneumocytes, extensive infiltration of neutrophils and macrophages in the interstice and alveoli. an interesting report showed the pathological characteristics of a patient who died from a severe infection by sars-cov-2 and underwent postmortem biopsies 23 . both lungs showed diffuse alveolar damage with cellular fibromyxoid exudates and interstitial mononuclear inflammatory infiltrates, with predominance of lymphocytes. the right lung showed evident desquamation of pneumocytes and hyaline membrane formation and the left lung showed a pulmonary edema with hyaline membrane formation, related to the acute respiratory distress syndrome (ards). a recent study reported that the lung histopathology in the early phase of covid-19 pneumonia include edema, proteinaceous exudate, focal reactive hyperplasia of pneumocytes with patchy inflammatory cellular infiltration, and multinucleated giant cells 24 . diffuse alveolar damage with hyaline membranes and pulmonary edema play important roles in ards. pregnant women with ards are more prone to hypoxia, their oxygen consumption is increased by 20% and their functional residual pulmonary capacity is decreased during pregnancy, rendering the woman intolerant to hypoxia. severe pneumonia is characterized by hypoxemia, which subsequently leads to placental hypoxia. the hypoxic placenta releases antiangiogenic and proinflammatory factors that converge to the maternal endothelium, inducing endothelial dysfunction, hypertension, and organ damage 25 . women with pneumonia during pregnancy have a significantly higher risk of adverse pregnancy outcomes, such as preterm delivery, pre-eclampsia, low birth weight and small-for-gestational-age infants 2,3,26 . therefore, severe maternal respiratory distress syndrome may affect the fetal oxygen supply and endanger the fetus. in a study of the placental histopathology of mothers with sars 27 , a total of seven placentas were studied. two placentas from women recovering from sars in the first trimester were normal. three placentas from women that gave birth during the acute stage of sars showed an increase in intervillous and subchorionic fibrin, and these findings may be related to disturbances in maternal placental blood flow due to hypoxia. two placentas from women recovering from sars in the third trimester of pregnancy were found to have an extensive thrombotic vasculopathy on the fetal side (ftv). the etiology of ftv may be related to the tendency to thrombosis due to sars and/ or placental hypoxia. these two pregnancies were also accompanied by intrauterine growth restriction, oligohydramnios and small for gestation newborns. a recent study analyzed the placental histopathology of three pregnant women who were infected by sars-cov-2 in the third trimester of pregnancy, and all of them presented with mild disease 28 . in one of the placentas a chorioangioma was evidenced, and another placenta showed multifocal infarctions. all three cases had varying degrees of increment of intervillous or subchorionic fibrin associated with increased syncytial nodules. no villitis or chorioamnionitis were found. whether these changes were caused by placental ischemia due to 2019-ncov infection still needs to be further investigated by enlarging the sample size. in humans, maternal viral infections caused by ah1n1, dengue and hiv have been associated with impaired maternal and fetal hemodynamics and abnormal placental villous architecture. a healthy functioning placenta relies on a proper vascularization and perfusion of the placenta. early studies indicated that systemic maternal infection and consequent inflammation can disrupt the placental vasculogenesis and angiogenesis and the alterations in placental hemodynamics may contribute to adverse pregnancy outcomes including preeclampsia, preterm delivery, small-for-gestational-age, low birth weight infants and stillbirths. in addition, it is believed that placental ischemia/ hypoxia can trigger an increased production of inflammatory biomarkers, such as il6 and tnfα, which contribute to endothelial dysfunction in preeclampsia 29 . the clinical features of sars-cov and mers-cov infections are similar but patients with mers have a higher incidence of acute respiratory distress syndrome, which may explain why the fatality rate of sars is about 10%, while in mers it is about 36%. the major clinical features of sars-cov infections include persistent fever, chills or rigor, myalgia, dry cough, headache, malaise and dyspnea. sore throat, rhinorrhea, sputum production, nausea, vomiting and dizziness were less common. in contrast, sars and mers-cov infections usually start with fever, cough, chills, sore throat, myalgia and arthralgia, progressing with dyspnea and a rapid development of pneumonia within the first week, usually requiring respiratory support and ventilation, as well as other supportive measures 30 . in comparison with sars, patients with mers are older, with male predominance, a higher incidence of comorbidities and relatively lower human-to-human transmission potential 31 . pregnant women infected with mers may develop severe disease with fatal consequences, including stillbirths [32] [33] [34] [35] . the transmission route of covid-19 is similar to the ones of sars and mers, mainly spread by respiratory droplets and direct contact. it is unclear if covid-19 can be transmitted by the fecal-oral route, given that sars-cov-2 has been identified in stool specimens 36 . sars-cov-2 can also infect the lower respiratory tract and cause pneumonia, but its symptoms seem to be milder than in sars and mers 37 . among all covid-19 cases, severe manifestations accounted for 18.1% 38 . data suggest an incubation period of about 5 days (range 2-14 days). fever, cough, myalgia, fatigue and dyspnea were the most common clinical manifestations, whereas diarrhea, hemoptysis, headache, sore throat and shock only occur in a small number of patients 17,38-41 . bilateral ground-glass or patchy opacities were the most common signs of radiological abnormalities 17,38-41 . lymphopenia and eosinopenia were observed in most patients 17, [40] [41] [42] . the viral load of 2019-ncov detected from the patients' respiratory tract was positively correlated with the lung disease severity 42 . complications of covid-19 included the acute respiratory distress syndrome, anemia, acute cardiac injury, and exuberant secondary infections 17 . the majority of patients were older than 50 years 39-41 . sars-cov-2 is more likely to affect elderly men suffering from chronic comorbidities that may lead to serious and even fatal respiratory failure 41, 43 . the case fatality rate of patients with 2019-ncov infection is lower than those of sars and mers. twelve pregnant women were diagnosed with sars in hong kong between february 1 and july 31, 2003 44 . all patients had high fever (>38 °c) and most presented with chills, rigor, malaise, myalgia and lymphopenia. only 33% of the pregnant patients presented with shortness of breathe. six (50%) were admitted to intensive care units because of hypoxemia. four (33%) required mechanical ventilation, three of whom, died from respiratory failure or nosocomial infection. among seven pregnant women in the first trimester, four had spontaneous miscarriages, two underwent termination of pregnancy due to social reasons and one had an uncomplicated ongoing pregnancy. all five newborns in the second and third trimester groups survived and four of them were delivered by cesarean section. four newborns (80%) were preterm and three of them were delivered by emergency cesarean sections due to the deterioration of maternal respiratory conditions. among the live newborn infants, none had clinical or laboratory evidence of sars-cov2 infection. zhang et al. 45 reported five primigravidas with sars (including 2 twin pregnancies) from guangzhou, china. two were infected in the 2 nd trimester while the other three in the 3 rd trimester. two presented with hospital-acquired infections and the other three had community-acquired infections. all five pregnant women had fever and abnormal chest x-ray. other symptoms included cough (n=4), hypoalbuminemia(n=4), chills or rigor (n=3), elevated alanine aminotransferase (n=3), decreased lymphocytes (n=2) and decreased platelet count (n=2). all five pregnant women were cured with one of them required intensive care hospitalization. in a twin-pregnancy, one of the fetuses evolved to intrauterine death. the five neonates were followed-up and none had evidence of sars. there were no cases of vertical transmission identified among pregnant women infected with sars 44-49 so far, but sars during pregnancy is associated with high incidences of spontaneous miscarriage, preterm delivery, intrauterine growth restriction, endotracheal intubation and admission to the neonatal intensive care unit [44] [45] [46] . there are limited data on the clinical features of mers-cov during pregnancy. only 11 cases of mers-cov in pregnancy have been documented [32] [33] [34] [35] 50, 51 . the clinical presentations of pregnant women with mers were variable and ranged from asymptomatic presentations to shortness of breathe, fever, cough, myalgia and even fatal cases. seven (63.6%) patients required icu hospitalization and three (27%) patients died during the hospital stay. regarding the infants, three (27%) infants died. only one case resulted in both maternal and fetal death: the infant died four hours after birth and the mother died of severe refractory hypoxia and cardiac arrest 24 days after delivery. there were other two cases of intrauterine fetal demise, one maternal death for septic shock eight days after delivery, and one maternal death due to multiple organs failure 19 days after delivery. few studies documented mers-cov testing in infants, except one report of an infant whose blood sample did not contain any igg, igm, or iga antibodies raised to mers-cov 52 . although data are limited, they also indicate that mers infections may cause unfavorable clinical outcomes in pregnancy. chen et al. 53 performed a retrospective review of medical records from nine pregnant women with covid-19 pneumonia in the zhongnan hospital, wuhan university, from january 20 to 31, 2020. none of the nine patients developed severe pneumonia requiring mechanical ventilation or died of covid-19 pneumonia (february 4, 2020). seven of the nine patients presented with fever but none had high fever (> 39°c). other symptoms, including cough (n=4), myalgia (n=3), sore throat (n=2), malaise (n=2), lymphopenia (n=5), and elevated crp (n=6) were also observed. all nine patients had cesarean sections during the third trimester and nine live births were recorded. one infant had a slightly increase in myocardial enzymes but without any clinical symptoms nine days after birth. none of the newborns needed special pediatric treatment. neonatal throat swabs, amniotic fluid, cord blood and breast milk from six patients were tested, all of them were negative for sars-cov-2. zhu et al. 54 retrospectively analyzed the clinical features and outcomes of 10 neonates, including two twins, whose mothers had covid-19 and were hospitalized in five hospitals of hubei, from january 20 to february 5, 2020. among these nine pregnant women, the initial symptoms were fever and cough, one patient also had diarrhea. there were some prenatal complications including prematurity (5 to 7 hours before the onset of the true labor), intrauterine distress (n=6), abnormal amniotic fluid (n=2), rupture of membranes (n=3), abnormal umbilical cord (n=2), and abnormal placenta (placenta previa) (n=1). among these 10 newborns only four were full-term infants and the other six were preterm infants; and one was a large-forgestational-age (lga) infant, while two were small-forgestational-age (sga). the newborns' symptoms were mainly short of breathe (n=6), digestive tract symptoms (n=4), fever (n=2), abnormal liver enzymes accompanied by thrombocytopenia (n=2), neonatal respiratory distress syndrome (nrds) (n=2), increased heart rate (n=1) and vomiting (n=1). until this article was published, five neonates had been cured and discharged from hospital, four neonates were still hospitalized in stable conditions and one died. pharyngeal swab specimens were collected 1-9 days after birth, from nine of the 10 neonates, and none of them were positive to sras-cov-2. on february 5, 2020, one newborn whose mother had confirmed covid-19, was admitted at the wuhan children's hospital of hubei province presenting with stable vital signs, no fever or cough, normal liver function, absence of respiratory symptoms and normal chest x-ray. throat swabs collected from the newborn 30 hours after birth were positive to sars-cov-2 but there was no direct evidence of intrauterine-acquired infection. unfortunately, amniotic fluid, umbilical cord blood and placenta were not tested, thus one cannot conclude if this is a case of vertical transmission or a perinatal transmission by close contact with the infected mother. in the same hospital, another newborn had confirmed covid-19 infection 17 days after birth and a history of close contact with two confirmed cases 55 , the infant's mother and the maternity matron. in this case, there is no reliable evidence to support the vertical transmission of covid-19. one mother tested positive for sars-cov-2 two days after delivery 56 . she was admitted to the hospital due to elevation of her liver enzymes and had no fever or digestive tract symptoms when admitted to the hospital. covid-19 was confirmed after delivery, and the prenatal infection cannot be ruled out. no sars-cov-2 was detected in the blood, urine, breast milk and in the throat swab of the newborn. there was no neonatal asphyxia, but the cardiac myoglobin and ck-mb enzymes were increased in the newborn, suggesting that a myocardial injury might exist. liu et al. 57 reported 13 pregnant patients with laboratoryconfirmed sars-cov-2 infection admitted to hospitals outside of wuhan between december 8, 2019, and february 25, 2020. ten patients (77%) presented with fever (range 37.3-39.0 °c), mostly accompanied by fatigue. three patients (23%) complained of dyspnea and one patient (7.6%) developed severe pneumonia with multiple organs dysfunction syndrome requiring icu hospitalization in the third trimester. three patients (23%) improved after treatment and were discharged with an ongoing pregnancy. the other 10 patients (77%) underwent caesarean sections. five out of 10 (50%) patients had pregnancy complications including fetal distress (n=3), premature rupture of the membranes (n=1) and a stillbirth (n=1) and six patients (60%) had preterm infants. no severe neonatal asphyxia was observed in the nine live births and no vertical transmission was found. yu et al. 58 reported seven pregnant women with covid-19 admitted to tongji hospital in wuhan, china. six patients (86%) had fever, one patient (14%) had cough, one patient (14%) had shortness of breathe and one patient (14%) had diarrhea. chest tomography (ct) revealed that six patients (86%) had bilateral pneumonia and one patient (14%) had unilateral pneumonia. no one needed icu hospitalization and the outcomes of these pregnant women were favorable. among three infants tested for sars-cov-2, one was positive to sars-cov-2 in a throat swab 36 h after birth. this infant had a mild pulmonary infection and mild shortness of breathe, no fever or cough. in addition, the placenta and cord blood of this infant were negative for sars-cov-2 by rt-pcr so that intrauterine vertical transmission may not have occurred. since ahmed et al. 59 reported the first maternal death of a 29-year-old pakistani woman was reported in the heart zone hospital in birmingham, uk on april 8, 2020, more and more articles began to report maternal deaths due to covid-19. in particular, from february 26, 2020 (date of the first covid-19 reported case in brazil) until june 18, 2020, brazil had already reported 124 maternal deaths, in the same period mexico had reported seven maternal deaths 60 . brazil's elevated covid-19 mortality rate in pregnancy might have several explanations. firstly, a possible shortage of healthcare providers and lack of intensive care resources are some of the chronic challenges in brazilian health care. secondly, brazil has a higher cesarean section rate than most countries firstly hit by covid-19 60 . a comparison of pregnant women from the above described studies is shown in table 1 . this is a review on pregnant women infected by sars-cov-2, sars, and mers, including their pathogenesis, clinical manifestations and pregnancy outcomes. these viruses mainly spread through the respiratory mucosa and infect other target cells, triggering a cytokine storm in the body, producing a series of immune responses and causing changes in peripheral leukocytes and immune system cells such as lymphocytes, which might be an important pathological pathway that inhibits the body's cellular immune function, leading to the deterioration of the patient's condition. the angiotensin-converting enzyme 2 (ace2) has been identified as the functional receptor for sars-cov-2 and sars-cov. the fact that ace2 is abundantly present in the epithelia of the lungs and small intestine, provides a possible explanation for the pathological lung and gastrointestinal symptoms. the abundant expression of ace2 in alveolar cells may cause a rapid viral expansion and destruction of the alveolar wall, resulting in a rapid progression of extensive pulmonary consolidations and diffuse alveolar damage with hyaline membrane formation. its presence in the vascular endothelium might also provide a step forward in understanding the histological changes of placentas from pregnant women infected by sars-cov-2. the clinical manifestations of covid-19 infection also show great similarities with sars and mers. however, covid-19 has affected more people in a shorter period of time compared to sars and mers, although with a lower fatality rate than sars and mers. pregnant women with covid-19 pneumonia showed a similar pattern in comparison with non-pregnant counterparts, including fever, cough, myalgia, fatigue, shortness of breathe or asymptomatic presentation. it is worth noting that there is currently no evidence that pregnant women with covid-19 are at higher risk of severe illness. however, sars and mers were found to be greatly associated with severe maternal illness, spontaneous abortion and even maternal death and intrauterine fetal demise. some pregnancy complications have occurred in pregnant women with covid-19, such as fetal distress, premature rupture of membranes, preterm deliveries and stillbirths. furthermore, these pregnancy complications might be closely related to the cytokine storm, lung injury and placental ischemia/ hypoxia caused by sars-cov-2 infections. although there is currently no evidence to support the fetal infection by intrauterine vertical transmission of sars, mers, and covid-19, more and more articles began to report maternal deaths due to covid-19. in particular, from february 26, 2020 (date of the first covid-19 case in brazil) until june 18, 2020, brazil had already reported 124 maternal deaths. thus, we should be alert that these diseases may follow the same trend of greater severity and poorer prognosis in pregnant women. therefore, pregnant women and newborns require special attention in the prevention, diagnosis and management of covid-19. the maternal physiological and immune function changes in pregnancy make pregnant women more susceptible to covid-19. furthermore, considering that pregnant women with covid-19 may not have typical symptoms such as fever, we suggest that pregnant women with any symptoms suggestive of covid-19 should undergo careful examination to prevent adverse pregnancy outcomes. covid-19 infection itself is not an indication for cesarean section deliveries. the timing and mode of delivery should be individualized based on the disease severity, pre-existing maternal comorbidities, obstetric history, gestational age and fetal conditions. newborns from women with suspected or confirmed covid-19 should undergo a careful examination, and have the body temperature, respiratory rate and heart rate closely monitored, as well as digestive tract symptoms. so far, breast milk samples were negative for sars-cov-2 and this virus is mainly transmitted by respiratory droplets and close contact. furthermore, the protective effect of breastfeeding on newborns is particularly strong. precautions should be taken to enable infected mothers to breastfeed, including respiratory hygiene, hand hygiene and disinfection, the use of n-95 masks by the mother while breastfeeding in cases in which mothers or newborns are suspected of or have confirmed covid-19. personal protection must be taken in order to minimize the risk of contracting the virus. future researches should cover different pregnant stages of covid-19 as much as possible. it is recommended that the placenta and other tissues of pregnant women with 2019-ncov infection should be evaluated by histopathological examinations, to provide more detailed pathological analyses. the epidemic situation of covid-19 is still spreading, and this review has limitations. thus, further investigation is needed to elucidate how covid-19 affects pregnant women and fetuses, as well as the exact impact of covid-19 on pregnant women themself and on pregnancy outcomes. yao w, qiu j and xiao z had the idea for the article and performed the literature search and data analysis. the first draft of the manuscript was written by yao w and all authors critically revised the previous versions of the manuscript. all authors read and approved the final manuscript. world health organization. coronavirus disease 2019 (covid-19): situation report -70 characteristics and pregnancy outcomes of patients with severe pneumonia complicating pregnancy: a retrospective study of 12 cases and a literature review pneumonia and pregnancy outcomes: a nationwide population-based study angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways sars and mers: recent insights into emerging coronaviruses plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients clinical features of patients infected with 2019 novel coronavirus in wuhan characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia(ncp) the clinical pathology of severe acute respiratory syndrome (sars): a report from multiple organ infection and the pathogenesis of sars lung pathology of fatal severe acute respiratory syndrome clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates pathological findings of covid-19 associated with acute respiratory distress syndrome pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer hypertension in pregnancy: taking cues from pathophysiology for clinical practice pneumonia during pregnancy: radiological characteristics, predisposing factors and pregnancy outcomes the placentas of patients with severe acute respiratory syndrome: a pathophysiological evaluation pregnant women with new coronavirus infection: a clinical characteristics and placental pathological analysis of three cases pathophysiology of hypertension during preeclampsia: linking placental ischemia with endothelial dysfunction epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study severe acute respiratory syndrome vs. the middle east respiratory syndrome middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus during pregnancy stillbirth during infection with middle east respiratory syndrome coronavirus first case of 2019 novel coronavirus in the united states emerging coronaviruses: genome structure, replication, and pathogenesis clinical characteristics of hospitalized patients with sars-cov-2 infection: a single arm meta-analysis epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 140 patients infected with sars-cov-2 in wuhan, china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia pregnancy and perinatal outcomes of women with severe acute respiratory syndrome clinical analysis of pregnancy in second and third trimesters complicated severe acute respiratory syndrome sars during pregnancy, united states 225-management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) sars and pregnancy: a case report middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report e62 pregnancy and covid-19: management and challenges page mers-cov infection in a pregnant woman in korea clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia neonatal coronavirus expert confirmed at 30 hours of birth: vertical transmission from mother to infant perinatal covid-19: a case report clinical manifestations and outcome of sars-cov-2 infection during pregnancy clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study first covid-19 maternal mortality in the uk associated with thrombotic complications in brazil: 124 maternal deaths and counting we declare no conflict of interests. this work was supported by the national natural science foundation of china, grant nº 81803884 key: cord-281802-9k6klcno authors: german, matthew; olsha, romy; kristjanson, erik; marchand-austin, alex; peci, adriana; winter, anne-luise; gubbay, jonathan b. title: acute respiratory infections in travelers returning from mers-cov–affected areas date: 2015-09-17 journal: emerg infect dis doi: 10.3201/eid2109.150472 sha: doc_id: 281802 cord_uid: 9k6klcno we examined which respiratory pathogens were identified during screening for middle east respiratory syndrome coronavirus in 177 symptomatic travelers returning to ontario, canada, from regions affected by the virus. influenza a and b viruses (23.1%) and rhinovirus (19.8%) were the most common pathogens identified among these travelers. peak travel periods to saudi arabia (e.g., ramadan, umrah, or the hajj) are of particular concern, although after the 2012 and 2013 hajj, no mers-cov cases were identified in persons returning to france (7) . high incidences of other respiratory diseases in pilgrims varied by year. in this study, we aimed to explore the array of respiratory pathogens in travelers with ari returning to ontario from mers-cov-affected areas or in their close symptomatic contacts. during november 2012-june 2014, a total of 177 international travelers returning to ontario were considered persons under investigation (puis) for mers-cov, according to the guidelines of the ontario ministry of health and long-term care (6) . puis were recommended to be isolated and screened for mers-cov and other respiratory pathogens (6) . nasopharyngeal swab samples and, for persons on ventilators, bronchoalveolar lavage specimens were collected from patients and submitted to public health ontario laboratories (phol), the provincial reference laboratory for mers-cov testing (6) . fecal specimens were collected when patients had diarrhea, and urine was collected during early phases of the outbreak when appropriate specimens were ill-defined (6) . mers-cov real-time reverse transcription pcr (rrt-pcr) targeted regions upstream of the e gene and within open reading frame 1b, as recommended by who (8) . influenza rrt-pcrs targeted the influenza a matrix gene and influenza b nonstructural 1 gene using centers for disease control and prevention (cdc; atlanta, ga, usa) protocols. if the rrt-pcr was positive for influenza a virus, we conducted subtyping for seasonal influenza a(h3n2) virus hemagglutinin gene (cdc assay) and influenza a(h1n1) pdm09 virus neuraminidase gene (in-house assay) (9) . respiratory specimens were further tested by using seeplex rv15 ace multiplex respiratory viral assay (seegene inc., seoul, south korea). targets included human rhinovirus, enterovirus, influenza a and b viruses, parainfluenza viruses 1-4, respiratory syncytial virus a and b, adenovirus, bocavirus, human metapneumovirus, human coronavirus oc43, and human coronavirus 229e/nl63. mycoplasma pneumoniae and chlamydophila pneumophila testing was conducted by using propneumo-1 multiplex assay (gen-probe inc., san diego, ca, usa). pcr was conducted for legionella species by using a protocol developed by cdc (10); binaxnow legionella urinary antigen test (binax inc., portland, me, usa) was also conducted to test for l. pneumophila serogroup 1. of 177 puis (mean age 48.1 years, range <1-88 years; 56% male), 54.8% returned from saudi arabia or uae. identification of puis peaked after the october 2013 hajj and after the first 2 mers-cov cases were imported into the united states in may 2014 ( figure) . all puis had ari; of the 85 puis for whom data were available, 47 (55%) and 74 (87%) had respiratory specimens collected within 5 and 14 days (median 4 days) from symptom onset, respectively. specimens collected were as follows: 185 upper respiratory, 10 lower respiratory, 98 urine, 97 blood, 11 fecal, and 1 pleural fluid. symptom onset varied from 17 days before return to 10 days after return (median <1 day after return) for the 20 puis for whom this information was supplied. one patient was excluded from the time-to-collection analysis because the specimen was collected under extenuating circumstances: testing was conducted because of worsening respiratory symptoms beginning 57 days before the patient returned from overseas. at least 1 respiratory pathogen (bacterial or viral) was detected in 89 (50.3%) puis; however, for most (87 [98%] of 89) patients, only viral pathogens were identified (table) . influenza was the most common virus identified: 27 (15.3%) persons tested positive for influenza a, 14 (7.9%) for a(h3n2) and 13 (7.3%) for a(h1n1)pdm09; †among the 177 returned travelers, no respiratory pathogen was found for 88 (49.7%). among the remaining 89 (50.3%) returned travelers, at least 1 respiratory pathogen was found; 12 (6.8%) of these persons had viral co-infections. among the 12 co-infections were 8 rhinovirus co-infections (4 persons with influenza a and 1 each with influenza b, enterovirus, cov oc43, parainfluenza); 1 influenza a-cov oc43 co-infection; 1 influenza b-respiratory syncytial virus; 1 cov 229e/nl63-adenovirus; and 1 cov 229e/nl63-human metapneumovirus co-infection. ‡comprises all reported infections, including viruses that were involved in co-infections. 14 (7.9%) tested positive for influenza b. rhinovirus was also common, detected in 35 (19.8%) persons, with a peak in the fall, in keeping with its seasonality in canada (figure; table) . similarly, influenza a(h3n2) peaked in the fall, whereas influenza b and a(h1n1)pdm09 peaked in late spring. no specimen submitted to the phol tested positive for mers-cov. given the relatively low volume of travelers arriving to canada and ontario from mers-cov-affected areas (0.6% of total global travel from mers-cov-affected areas entered canada during june-november, 2012, and <50,000 nonresident travelers entered ontario from affected countries in 2012 [11, 12] ) and lower rates of humanto-human transmission, risk of importation to ontario and subsequent local spread is likely low (1, 13) . although the risk for mers-cov importation is low, respiratory virus infections acquired abroad or locally after returning to canada might be relatively high and consistent, occurring in 87 (49.2%) of 177 puis during the study period. most influenza b cases were detected shortly after the 2014 ontario peak (phol, unpub. data). furthermore, 75% of puis with influenza b reported symptom onset within 4 days after their return, possibly indicating local acquisition. similarly, puis with enterovirus or rhinovirus detected probably acquired disease in canada, given the short incubation period (mean 1.9 days) of rhinovirus (14) . because limited information about clinical severity or outcomes was reported to phol, we were unable to report on the clinical spectrum of pui presentation. furthermore, pathogens were not identified for all samples, possibly because of delays between symptom onset and specimen collection, sampling technique, or other factors. the number of puis with influenza (41 [23.2%]), whether acquired locally or abroad, is of particular concern. unnecessary identification of puis might have been avoided with more comprehensive vaccination coverage. influenza vaccination should be a priority for all persons and should be recommended by health care practitioners who advise travelers. in addition, surveillance should continue for other respiratory pathogens so that their effects on health systems, when they co-circulate with emerging pathogens with similar clinical presentation, can be better understood. emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives world health organization. global alert and response (gar) middle east respiratory syndrome coronavirus update on mers-cov transmission from animals to humans, and interim recommendations for at-risk groups the kingdom of saudi arabia general authority of civil aviation. statistical yearbook tools for preparedness: triage, screening and patient management for middle east respiratory syndrome coronavirus (mers-cov) infections in acute care settings respiratory viruses and bacteria among pilgrims during the detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction analytical and clinical validation of novel real-time reverse transcriptase-polymerase chain reaction assays for the clinical detection of swine-origin h1n1 influenza viruses dual detection of legionella pneumophila and legionella species by real-time pcr targeting the 23s-5s rrna gene spacer region potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia table 427-0006. number of non resident travellers entering canada, by selected country of residence, excluding the united states, seasonally adjusted monthly (persons transmission of mers-coronavirus in household contacts incubation periods of acute respiratory viral infections: a systematic review we are thankful to public health ontario and phol technical staff for support and help with data collection. mr. german is an infectious disease epidemiologist at st. michael's hospital in toronto, ontario. his research interests include global migration and emerging and re-emerging infectious disease. key: cord-273893-3nd6ptrg authors: lu, guangwen; hu, yawei; wang, qihui; qi, jianxun; gao, feng; li, yan; zhang, yanfang; zhang, wei; yuan, yuan; bao, jinku; zhang, buchang; shi, yi; yan, jinghua; gao, george f. title: molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 date: 2013-07-07 journal: nature doi: 10.1038/nature12328 sha: doc_id: 273893 cord_uid: 3nd6ptrg the newly emergent middle east respiratory syndrome coronavirus (mers-cov) can cause severe pulmonary disease in humans(1,2), representing the second example of a highly pathogenic coronavirus, the first being sars-cov(3). cd26 (also known as dipeptidyl peptidase 4, dpp4) was recently identified as the cellular receptor for mers-cov(4). the engagement of the mers-cov spike protein with cd26 mediates viral attachment to host cells and virus–cell fusion, thereby initiating infection. here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (rbd) of the mers-cov spike protein and its complex with cd26. furthermore, binding between the rbd and cd26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nm. the viral rbd is composed of a core subdomain homologous to that of the sars-cov spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades iv and v of the cd26 β-propeller. the atomic details at the interface between the two binding entities reveal a surprising protein–protein contact mediated mainly by hydrophilic residues. sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the mers-cov rbd core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition. supplementary information: the online version of this article (doi:10.1038/nature12328) contains supplementary material, which is available to authorized users. the newly emergent middle east respiratory syndrome coronavirus (mers-cov) can cause severe pulmonary disease in humans 1,2 , representing the second example of a highly pathogenic coronavirus, the first being sars-cov 3 . cd26 (also known as dipeptidyl peptidase 4, dpp4) was recently identified as the cellular receptor for mers-cov 4 . the engagement of the mers-cov spike protein with cd26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (rbd) of the mers-cov spike protein and its complex with cd26. furthermore, binding between the rbd and cd26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nm. the viral rbd is composed of a core subdomain homologous to that of the sars-cov spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades iv and v of the cd26 b-propeller. the atomic details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues. sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the mers-cov rbd core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition. the recent identification of a novel coronavirus, mers-covwhich, as of may 15th 2013, had infected 40 patients with a total of 20 fatalities-has drawn worldwide attention as a potential cause of a future pandemic 5 . unlike most coronaviruses circulating in humans that only cause mild respiratory illness 6 , mers-cov possibly represents a second reported coronavirus of severely high virulence after sars-cov, which caused over 8,000 infection cases globally in 2003, with more than 800 deaths 3 . the clinical manifestations of mers-cov infection include fever, cough, acute respiratory distress syndrome and, in some cases, accompanying renal failure 1,2 , and are very similar to those caused by sars-cov. however, the novel coronavirus diverges from sars-cov in genomic sequence, and is much more closely related to the bat-derived hku4 and hku5 coronaviruses 7, 8 . consistent with phylogenetic analysis, mers-cov does not use the sars-cov receptor, angiotensin converting enzyme 2 (ace2), as its entry receptor 9 ; rather, a recent study showed that it uses human cd26 for this purpose 4 . cd26 is the third peptidase to be identified as a functional coronavirus receptor, the others being aminopeptidase n (anpep, also known as apn and cd13) 10, 11 and ace2 (ref. 12) . the recognition of cd26 by mers-cov is mediated by virus surface spike (s) protein 4 . as with other coronaviruses, the mers-cov s protein would be cleaved in host cells into s1 and s2 subunits (fig. 1a) . s1 engages the receptor 4 whereas s2, with typical sequence motifs homologous to those identified as the heptad repeats in class i enveloped viruses [13] [14] [15] , should mediate membrane fusion. the exploitation of the virus-receptor interaction and thus of the intervention strategies requires an atomic delineation of the receptor-binding properties of s1. on the basis of previous studies, the receptor attachment sites of coronavirus s1 subunits might locate to either the amino-terminal (such as in murine hepatitis virus 16 ) or the carboxy-terminal (such as in, for example, sars-cov 17 and human coronavirus nl63 (ref. 18)) domain. we therefore tested individually the binding of mers-cov s1 and its n-and c-terminal-domain proteins to cell-surface-expressed cd26 molecules. the receptor-binding capacity was attributed to the c-terminal amino acids 367-606 of mers-cov s1 (fig. 1b) . we hereby referred to this domain as rbd. the potent interaction between mers-cov rbd and cd26 was further demonstrated by surface plasmon resonance assays, in which cd26 binds to mers-cov rbd with a dissociation constant (k d ) of about 16.7 nm (k on , 1.793 10 5 m 21 s 21 ; k off , 2.99 3 10 23 s 21 ), but does not bind to the rbd of sars-cov (fig. 1c) . we crystallized mers-cov rbd and solved its structure at a resolution of 2.5 å (supplementary table 1 ). two molecules of essentially the same structure are present in the asymmetric unit. each molecule contains 208 consecutive density-traceable amino acids from v381 to l588. a dali 19 search within the protein data bank (pdb) revealed clear structural homology between mers-cov rbd and sars-cov rbd (pdb code, 2dd8; z score, 15.1). we therefore divided the mers-cov rbd structure into two subdomains: a core and an external b-sheet, using the structure of sars-cov rbd as a reference. the core subdomain reveals a five-stranded antiparallel b-sheet (b1, b3, b4, b5 and b10) in the centre. the connecting helices (four a-helices: a1-4 and two 3 10 -helices: g1 and g2) and two small b-strands (b2 and b11) further decorate the sheet on both sides, together forming a globular fold. three disulphide bonds, connecting c383 to c407, c425 to c478, and c437 to c585, respectively, stabilize the core-domain structure from the interior. at the solvent-exposed side, the rbd termini are clinched adjacent to each other (fig. 2a, b) . this subdomain fold is very similar to that of the sars-cov rbd core (a root mean squared deviation of 2.79 å for 76 ca pairs). superimposition of the two structures reveals a well-aligned centre sheet and homologous peripheral helices and strands, although several intervening loops are observed to exhibit large conformational variance (fig. 2c) . the external subdomain of mers-cov rbd is mainly a b-sheet structure with three large (b6, b8 and b9) and one small (b7) strand arranged in an antiparallel manner. it is anchored to the rbd core through the b5/6, b7/8 and b9/10 intervening loops, which touch the core subdomain like a clamp at both the top and bottom positions. two small 3 10 helices (g3 and g4) and most of the connecting loops in this subdomain locate on the interior side of the sheet, hence exposing a flat exterior sheet-face to the solvent. residues c503 and c526 form the fourth disulphide bond, linking the g3 helix to strand b6 (fig. 2a, b) . with no observable structure homology (fig. 2c) , the external subdomains of mers-cov and sars-cov rbds are topological equivalents, both being present as an 'insertion' between the equivalent core-strands (strands b5 and b10 in mers-cov, and b6 and b9 in sars-cov) (supplementary fig. 1 ). to elucidate the structural basis of the virus-receptor engagement, we further prepared the rbd-cd26 complex by in vitro mixture of the two proteins and then purification on a gel filtration column. consistent with the high binding affinity between mers-cov rbd and cd26, the complex is easily obtainable and stable ( supplementary fig. 2 ). the complex structure was solved at 2.7 å resolution (supplementary table 1 ) with one rbd binding to a single cd26 molecule in the asymmetric unit. the receptor, as shown in previous reports 20,21 , is composed of an eight-bladed b-propeller domain and an a/b hydrolase domain. mers-cov rbd binds to the side-surface of the cd26 bpropeller, recognizing blades iv and v and a small bulged helix in the blade-linker. as for the viral ligand, the entire receptor binding site locates in the external subdomain and to the solvent-exposed sheetface, qualifying the subdomain as the receptor binding motif (rbm) (fig. 3a) . overall, engagement of the receptor does not induce obvious conformational changes in rbm, although small structural variance could be observed for the tip-loops. the g2-a4 loop in the rbd core, however, unexpectedly exhibits a large conformational difference between the free and the bounded structures ( supplementary fig. 3) . we believe this is due to a crystal contact present in the free rbd structure, which is interrupted in the complex crystal by the engaging receptor. cd26 is a type ii transmembrane protein. it is present as a homodimer on the cell surface 20-22 . the dimerization of the peptidase relies on broad intermolecule contacts contributed by the hydrolase domain and the extended strands in blade iv of the b-propeller 20,21 . a lateral binding of mers-cov rbd to cd26 would therefore not disrupt cd26 dimerization. accordingly, a similar u-shaped cd26 dimer could be generated by symmetry operations of the complex structure. the viral ligand locates at the membrane-distal tip of the dimer, corresponding well to a trans interaction between the virus and the receptor (fig. 3b) . considering that the rbd n and c termini are on the same side distant from cd26, it is unlikely that the remaining s domains would contact the receptor molecule. the binding mode revealed by the complex structure is also in good accordance with a previous study showing that the virus-receptor interaction is independent of the peptidase activity of cd26 (ref. 4) . the bound rbd is far away from interfering with either the substrate/product accessing tunnels or the catalytic centre 20,21 (fig. 3b) . overall, a surface area of 1203.4 and 1113.4 å 2 in cd26 and mers-cov rbd, respectively, is buried by complex formation (fig. 4a) . scrutinization of the binding interface reveals a group of hydrophilic residues at the site, forming a polar-contact (h-bond and salt-bridge) network. these interactions are predominantly mediated by the residue side chains (including rbd y499 with cd26 r336, n501 with q286, k502 with t288, d510 with r317, e513 with q344, and d539 with k267), although cd26 l294 and rbd d510 are observed to contact rbd r542 and cd26 y322, respectively, through the mainchain oxygen atom (fig. 4b) . in addition, the bulged helix in cd26 properly positions three hydrophobic residues a291, l294 and i295 into close proximity with the rbd amino acids y540, w553 and v555, forming a hydrophobic centre at the interface (fig. 4c) . further virusreceptor contacts include v341 and i346 of cd26 packing against p515 and the apolar carbon atoms of r511 and e513 in rbd (fig. 4d) , and a cd26 n229-linked carbohydrate moiety interacting with rbd amino acids w535 and e536 (fig. 4e) . overall, the virusreceptor engagement is dominated by the polar contacts mediated by the hydrophilic residues, and mutations of those in rbd (six alanine substitutions and one y499f mutation of the cd26-interacting amino acids) completely abrogated its interaction with cd26 ( supplementary fig. 4 ). the features of these residue interactions are very similar to those mediating the interaction between adenosine deaminase (ada) and cd26 (ref. 23). by a pairwise comparison, we unexpectedly found that all those cd26 residues identified in the virus-receptor interface are also involved in ada binding, indicating a competition between ada and the virus for cd26 receptor. as the ada-cd26 interaction is shown to induce co-stimulatory signals in t cells 22 , this may indicate a possible manipulation of the host immune system by mers-cov through competition for the ada-recognition site. it is also noteworthy that those cd26 residues involved in rbd binding are highly conserved between human and bat, with only two variations (i295t and r317q), explaining the capability of mers-cov using bat cd26 for cell entry 4 ( supplementary fig. 5 ). coronaviruses can be categorized into three main genera or groups (group 1 (alpha), group 2 (beta) and group 3 (gamma) coronaviruses) 24 . both mers-cov and sars-cov belong to the betacoronavirus genus, but are classified into different lineage subgroups (subgroup 2b for dimer observed in the complex crystal. the two-fold axis is shown as an upright arrow. the transmembrane topology of cd26 is indicated with a modelled lipid-bilayer membrane. in cd26, the propeller and side openings indicated as the substrate entrance/exit tunnels are marked with arrows, and the catalytic triad residues are highlighted as spheres. colour selections are the same as in a, and the cd26 a/b hydrolase domain is shown in orange. the n and c termini are labelled. to facilitate comparison, the secondary-structure elements of sars-cov rbd (pdb code, 2dd8) are marked with spiral (helices) and arrow (strands) lines below the sequence. the cysteine residues that form disulphide bonds are labelled as in a, and residue n410 with a star. c, a structural alignment between mers-cov (magenta for core and cyan for external subdomains) and sars-cov (green) rbds. sars-cov and 2c for mers-cov) 8 . we noted that the spike sequences are of low identity among different subgroup members. for example, mers-cov and sars-cov s proteins show a sequence identity of less than 28%. nevertheless, rbds of the two coronaviruses are homologous for the core subdomain. notably, the three interior disulphide bonds in the core are well-aligned for the steric positions in the two rbd structures and well-conserved in sequence among betacoronaviruses. conversely, the external rbm region is highly variable in both length and residue composition ( supplementary fig. 6 ). consistently, no structural homology in this subdomain is observed between mers-cov and sars-cov. yet it is this subdomain that engages cellular receptors. we therefore assume that betacoronaviruses probably have a similar core-domain fold in the s protein to present the external amino acids with divergent structures for viral pathogenesis, such as receptor recognition. our work presents the fifth structure of virus s protein-receptor complexes in the coronaviridae family [16] [17] [18] 25 . taking into account both the rbd structure and the binding mode with receptors, mers-cov is related to sars-cov 17 (a single insertion functioning as rbm) but differs from porcine respiratory coronavirus 25 and nl63 (ref. 18 ) of alphacoronaviruses (multiple discontinuous rbms) ( supplementary fig. 7) . nevertheless, related structural topologies can still be observed in rbds of these coronaviruses 26 . we noted that in the rbd-receptor complex structures of both mers-cov and porcine respiratory coronavirus the binding interfaces involve a receptor n-glycan. this might represent another cross-genus similarity in the coronaviridae family, which supports a proposed common evolutionary origin of coronavirus s proteins 26 . it would therefore be interesting to investigate the contribution of the sugar moiety to the virus-receptor interaction for mers-cov in the future. vaccination remains the most useful measure to combat viral infection and transmission. a large number of antibodies show neutralization activity by targeting the rbd and thereby disrupting the virus-receptor engagement. therefore, a properly folded rbd could be an ideal immunogen for vaccination, as demonstrated for sars-cov 27 . a recent report indeed shows the presence of s-specific neutralizing antibodies in mers-cov-infected patients 28 . it may be worth attempting to test the immunization effect of mers-cov rbd in the future. protein expression, purification, crystallization and structure determination. both his-tagged cd26 and mers-cov rbd proteins were expressed in insect high five cells using the bac-to-bac baculovirus expression system (invitrogen). the recombinant proteins were then purified via nickel-chelated affinity chromatography and gel filtration. crystals were obtained by initial screening with the commercially available kits followed by optimization. the rbd structure was solved by single-wavelength anomalous diffraction and the complex structure by molecular replacement. full methods and any associated references are available in the online version of the paper. supplementary information is available in the online version of the paper. isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc world health organization. novel coronavirus infection -update coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans sars-like virus in the middle east: a truly bat-related coronavirus causing human diseases human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human aminopeptidase n is a receptor for human coronavirus 229e aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus combating the threat of pandemic influenza: drug discovery approaches coiled coils in both intracellular vesicle and viral membrane fusion following the rule: formation of the 6-helix bundle of the fusion core from severe acute respiratory syndrome coronavirus spike protein and identification of potent peptide inhibitors crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor structure of sars coronavirus spike receptorbinding domain complexed with receptor crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor dali server: conservation mapping in 3d crystal structure of the swine-origin a (h1n1)-2009 influenza a virus hemagglutinin (ha) reveals similar antigenicity to that of the 1918 pandemic virus processing of x-ray diffraction data collected in oscillation mode collaborative computing project number 4. the ccp4 suite: programs for protein crystallography advances in direct methods for protein crystallography pushing the boundaries of molecular replacement with maximum likelihood density modification for macromolecular phase improvement phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics procheck: a program to check the stereochemical quality of protein structures espript: analysis of multiple sequence alignments in postscript acknowledgements this work was supported by the ministry of science and technology of china (most) 973 project (grant no. 2011cb504703) and the national natural science foundation of china (nsfc, grant no. 81290342). assistance by the staff at the shanghai synchrotron radiation facility (ssrf) of china and the high energy accelerator research organization (kek) of japan is acknowledged. we thank z. fan and t. zhao for their technical assistance. g.f.g. is a leading principal investigator of the nsfc innovative research group (grant no. 81021003). we thank m. yang from tsinghua university for his help with data collection. author contributions g.f.g. designed and coordinated the study. g.l., y.h., q.w. and y.s. conducted the experiments. j.q. and f.g. collected the data sets and solved the structures. y.l., y.z., w.z., y.y. and j.y. assisted with the cell maintenance and protein preparations. g.l. and g.f.g. wrote the manuscript and j.y., j.b. and b.z. participated in the manuscript editing and discussion.author information the coordinates and related structure factors have been deposited into the protein data bank pdb under accession numbers 4kqz for the free mers-cov rbd structure and 4kr0 for the rbd-cd26 complex structure. reprints and permissions information is available at www.nature.com/reprints. the authors declare no competing financial interests. readers are welcome to comment on the online version of the paper. correspondence and requests for materials should be addressed to g.f.g. (gaof@im.ac.cn). protein expression and purification. the proteins used for crystallization and surface plasmon resonance experiments were prepared with the bac-to-bac baculovirus expression system (invitrogen). the coding sequences for mers-cov rbd (genbank accession number jx869059, spike residues 367-606), sars-cov rbd (accession number nc_004718, spike residues 306-527), human cd26 (accession number np_001926, residues 39-766) and human ace2 (accession number baj21180, residues 19-615) were individually cloned into the pfastbac1 vector. for each construct, a previously described gp67 signal peptide sequence 29 was added to the protein n terminus for protein secretion, and a hexa-his tag was added to the c terminus to facilitate further purification processes. transfection and virus amplification were conducted with sf9 cells, and the recombinant proteins were produced in high five cells. the cell culture was collected 48 h after infection and passed through a 5-ml histrap hp column (ge healthcare). after removal of most of the impurities, the recovered proteins were then pooled and further purified on a superdex 200 column (ge healthcare). finally, each collected protein was prepared in a buffer consisting of 20 mm tris-hcl (ph 8.0) and 150 mm nacl and concentrated to about 10 mg ml 21 for further use.to obtain the complex of mers-cov rbd bound to cd26, the individual proteins were in vitro mixed at a molar ratio of 1:1 and incubated at 4 uc for about 2 h. the complex was then further purified on a superdex 200 column, and concentrated to about 15 mg ml 21 for crystallization experiments.to prepare the fc chimaeric proteins, the fragment encoding mers-cov s1 (residues 1-751) or ntd (residues 1-353) or rbd (adding the s residues 1-17 of the signal peptide to its n terminus to facilitate protein secretion) was fused 59terminally to a fragment coding for the fc domain of mouse igg and ligated into the pcaggs expression vector. a mutant rbd-fc protein-expressing plasmid was also constructed by site-directed mutagenesis, for which the identified hydrophilic residues involved in cd26 binding were mutated simultaneously (y499f; n501a, k502a, d510a, e513a, d539a and r542a). the expression plasmids were then transfected into hek293t cells. the cell culture was collected 48 h after transfection and directly used in the flow cytometric assay. analytical gel filtration. mers-cov rbd, cd26 and their protein complex were individually prepared and adjusted to the same volume. the samples were then loaded onto a calibrated superdex 200 column (ge healthcare). the chromatographs were recorded and overlaid onto each other. the pooled proteins were analysed on a 12% sds-page gel and stained with coomassie blue. surface plasmon resonance assay. the biacore experiments were carried out at room temperature (25 uc) using a biacore 3000 machine with cm5 chips (ge healthcare). for all the measurements, an hbs-ep buffer consisting of 10 mm hepes, ph 7.5, 150 mm nacl, 3 mm edta and 0.005% (v/v) tween-20 was used, and all proteins were exchanged to the same buffer in advance via gel filtration. the mers-cov rbd and sars-cov rbd proteins were immobilized on the chip at about 500 response units. gradient concentrations of human cd26 (0, 5, 10, 20, 40, 80, 160, 320, 640 and 1,280 nm) or human ace2 (0, 10, 20, 40, 80, 160, 320, 640 and 1,280 nm) were then used to flow over the chip surface. after each cycle, the sensor surface was regenerated via a short treatment using 10 mm naoh. the binding kinetics were analysed with the software biaevaluation version 4.1 using the 1:1 langmuir binding model. flow cytometric assay. for the surface expression of cd26, the full-length coding sequence was cloned into the pegfp-c1 vector which yields a plasmid encoding a recombinant cd26 protein with an egfp-tag fused to its n terminus. the plasmid was transfected into the cd26-negative bhk cells using lipo2000 (invitrogen) according to the manufacturer's instructions. the cells were collected 48 h after transfection.for staining, the mock-transfected bhk cells or the cells transfected with the cd26-expressing plasmid were suspended in pbs and incubated with the individual fc-fusion protein culture or goat anti-cd26 igg (r&d systems) at room temperature for 1 h. the cells were then washed and further incubated at room temperature for about 0.5 h with anti-mouse or anti-goat secondary igg antibodies (r&d systems). after washing, the cells were analysed by flow cytometry with a bd facscalibur machine. the cells incubated only with the secondary antibodies were used as the negative controls. crystallization. all the crystals were obtained by vapour-diffusion sitting-drop method with 1 ml protein mixing with 1 ml reservoir solution and then equilibrating against 100 ml reservoir solution at 18 uc. the initial crystallization screenings were carried out using the commercially available kits. the conditions that yield crystals were then optimized. diffractable crystals of the free rbd protein were finally obtained in a condition consisting of 0.1 m ammonium tartrate dibasic, ph 7.0, and 12% peg 3,350 with a protein concentration of 10 mg ml 21 . derivative crystals were obtained by soaking rbd crystals for 24 h in mother liquor containing 2 mm kaucl 4 n2h 2 o. the complex crystals were grown in 6% (v/v) 2-propanol, 0.1 m sodium acetate ph 4.5 and 26% peg550 with a protein concentration of 15 mg ml 21 . data collection, integration and structure determination. for data collection, all crystals were flash-cooled in liquid nitrogen after a brief soaking in reservoir solution with the addition of 20% (v/v) glycerol. the native rbd data set was collected at the high energy accelerator research organization (kek) bl1a (wavelength, 1.03818 å ), whereas the diffraction data for the au derivative crystal (wavelength, 1.0382 å ) and the complex crystal (wavelength, 0.97930 å ) were collected at the shanghai synchrotron radiation facility (ssrf) bl17u. all data were processed with hkl2000 (ref. 30) . additional processing was performed with programs from the ccp4 suite 31 .the structure of rbd was determined by the single-wavelength anomalous diffraction (sad) method. the au sites were first located by shelxd 32 for the au-sad data. the identified position were then refined and the phases were calculated with sad experimental phasing module of phaser 33 . the real space constraints were further applied to the electron density map in dm 34 . the initial model was built with autobuild in phenix package 35 . additional missing residues were added manually in coot 36 . the final model was refined with phenix.refine in the phenix 35 with energy minimization, isotropic adp refinement, and bulk solvent modelling. the complex structure was solved by molecular replacement module of phaser 33 , with the solved rbd structure and previously reported cd26 structure (pdb code, 2bgr) as the search models. the atomic model was completed with coot 36 and refined with phenix.refine 35 . the stereochemical qualities of the final models were assessed with procheck 37 . the ramachandran plot distributions for the residues in the free rbd structure were 86.8, 11.8 and 1.4% for the most favoured, additionally and generously allowed regions, respectively. these values were 86.5, 13.1 and 0.5% for the rbd-cd26 complex structure. data collection and refinement statistics are summarized in supplementary table 1 . all structural figures were generated using pymol (http://www.pymol.org). secondary-structure determination. the secondary structure determination was based on the espript 38 algorithm. key: cord-267001-csgmc155 authors: george, parakkal jovvian; tai, wanbo; du, lanying; lustigman, sara title: the potency of an anti-mers coronavirus subunit vaccine depends on a unique combinatorial adjuvant formulation date: 2020-05-27 journal: vaccines (basel) doi: 10.3390/vaccines8020251 sha: doc_id: 267001 cord_uid: csgmc155 vaccination is one of the most successful strategies to prevent human infectious diseases. combinatorial adjuvants have gained increasing interest as they can stimulate multiple immune pathways and enhance the vaccine efficacy of subunit vaccines. we investigated the adjuvanticity of aluminum (alum) in combination with rasp-1, a protein adjuvant, using the middle east respiratory syndrome coronavirus mers-cov receptor-binding-domain (rbd) vaccine antigen. a highly enhanced anti-mers-cov neutralizing antibody response was induced when mice were immunized with rasp-1 and the alum-adjuvanted rbd vaccine in two separate injection sites as compared to mice immunized with rbd + rasp-1 + alum formulated into a single inoculum. the antibodies produced also significantly inhibited the binding of rbd to its cell-associated receptor. moreover, immunization with rasp-1 co-administered with the alum-adjuvanted rbd vaccine in separate sites resulted in an enhanced frequency of tfh and gc b cells within the draining lymph nodes, both of which were positively associated with the titers of the neutralizing antibody response related to anti-mers-cov protective immunity. our findings not only indicate that this unique combinatorial adjuvanted rbd vaccine regimen improved the immunogenicity of rbd, but also point to the importance of utilizing combinatorial adjuvants for the induction of synergistic protective immune responses. vaccination is one of the most successful strategy to prevent infectious diseases in the human population, including those caused by emerging viruses [1] . among various vaccine types, such as inactivated virus, live attenuated virus, and viral vector-based vaccines, subunit vaccines using proteins or peptides are believed to be much safer as they do not contain any live virus components and/or cause undesirable severe side effects [2] . however, unlike attenuated vaccines (composed of a virus or bacterium that replicates within the host) or inactivated vaccines (composed of either heat or chemically-inactivated parts of the pathogen), subunit vaccines (that are derived from known pathogen target antigens) are generally much less immunogenic, which can be improved with the addition of appropriate adjuvant(s) to the vaccine [1] . adjuvants play an important role in enhancing the potency of subunit vaccines by improving humoral and/or cellular immune responses to the various subunit protein vaccines, decreasing the antigen dosages, and/or reducing immunization regimens [3, 4] . aluminum salts (hereinafter alum) c57bl/6 mice were immunized intramuscularly (i.m.) twice, three weeks apart, with mers-rbd-fd (5 µg; hereinafter rbd) formulated with or without alum (alhydrogel ® 250 µg) (invivogen, san diego, ca, usa), rasp-1 (25 µg), or with distinct adjuvant combinations. inoculums were prepared in a final volume of 100 µl per mouse and 50 µl was injected in the caudal thigh muscle of each hind leg in the appropriate site as outlined in table 1 . control mice were injected with pbs in 0.1% sds, the buffer solution of rasp-1 and is referred to as naive mice hereafter. complete adsorption of the rbd and rasp-1 proteins by alum was confirmed by sds-gel electrophoresis of the unbound protein samples after absorption with alum for 30 min on a rotator at rt. sera samples were collected 7 days post-2nd immunization for analyses of anti-mers-cov neutralizing antibody titers, inhibition of mers-cov rbd-dpp4 receptor binding, and mers-cov rbd-specific antibody responses using mers-cov s1 as a target. the draining lymph nodes from each hind leg per mouse were also recovered at day 7 post-2nd immunization for analyses of the various cell profiles within. the neutralizing activity of sera from the immunized mice against mers-cov infection in vitro was carried out using our established pseudovirus neutralization assay [17, 20] . briefly, 293t cells were co-transfected with a plasmid encoding the s protein of mers-cov (strain emc2012) and a plasmid encoding env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re). the supernatants containing mers-cov s expressing pseudovirus collected 72 h post-transfection were incubated with serially diluted mouse sera at 37 • c for 1 h. the virus-serum mixtures were then added into huh-7 cells expressing the mers-cov receptor dpp4. the cells were refed with fresh medium 24 h later, and after 72 h, lysed using cell lysis buffer (promega, madison, wi, usa) before the supernatants were transferred into 96-well luminometer plates. after addition of luciferase substrate (promega, madison, wi, usa), the plates were measured for relative luciferase activity using infinite 200 pro luminator (tecan, männedorf, switzerland). neutralizing activity was calculated using the calcusyn computer program [25] and is expressed as 50% pseudovirus neutralizing antibody titers (nt 50 ). sera from immunized mice were tested for their ability to inhibit the binding of the recombinant mers-rbd-fc protein to the cell-associated hdpp4 receptor in huh-7 cells using flow cytometry analysis [26] . briefly, cells were incubated with the mers-rbd-fc protein (5 µg/ml) in the presence or absence of diluted mouse sera (1:50) for 30 min at room temperature. after three washes and staining with fitc-labeled goat anti-human igg fc secondary antibody (1:500, thermo fisher scientific, waltham, ma, usa) for 30 min at room temperature, the cells were measured for fluorescence in a flow cytometer (bd lsrfortessa 4 system). mean fluorescence intensity (mfi) values of the fitc channel from cells incubated with mers-rbd-fc protein in the absence of diluted sera were treated as 100 percentage binding. inhibition of binding was calculated as the percentage of reduced binding to hdpp4 receptor in the presence of diluted sera from the different immunization groups versus the maximum binding observed in the absence of sera. elisa to measure mers-cov rbd-specific antibody responses in immunized mouse sera was performed with mers-cov s1 as the target antigen and as previously described with some modifications [17, 20] . the mers-cov s1 subunit of the mers-cov s protein contains the rbd region of the virus. briefly, 96-well elisa plates were coated with mers-cov s1 (1 µg/ml) overnight at 4 • c, blocked with 2% fat-free milk in pbs containing tween-20 (pbst) at 37 • c for 2 h, and then washed with pbst 3 times. the plates were subsequently incubated at 37 • c for 1 h with serially diluted mouse sera, and horseradish peroxidase (hrp)-conjugated anti-mouse igg (1:5000), igg1 (1:5000), or igg2c (1:2000) antibodies (thermo fisher scientific, waltham, ma, usa). the substrate 3,3 ,5,5 -tetramethylbenzidine (sigma-aldrich, st. louis, mo, usa) was added to the plates after additional washes, and the reaction was stopped by the addition of 1 n h 2 so 4 . absorbance at 450 nm was measured using an elisa plate reader (tecan, männedorf, switzerland). endpoint titers were calculated as the reciprocal of the highest dilution of sera giving an optical density greater than the mean ± 3 times the standard deviation of sera from naïve mice. draining lymph nodes from each hind leg per mouse were harvested at day 7 post-2nd immunization. the lymph nodes were dissociated into single cell suspensions using a syringe plunger, then passed through a 70 µm cell strainer and resuspended in complete rpmi 1640 media containing 10% fetal bovine serum (r10). subsequently, 2.5 × 10 6 cells were washed and resuspended in fresh r10 media in a 96-well cell culture plate for flow cytometry staining. 1.5 × 10 6 cells were stained with a labeled antibodies: cd45-af700, cd11b-pe-cy5, cd11c-bv711, ly6c-percp, cd40-apc, ccr7-pe, cd80-bv650, cd86-bv421, ly6g-pe-cy7, and b220-bv605. while 1 × 10 6 cells were stained with a cocktail of the following fluorescently labeled antibodies: cd45-af700, cd4-pe-cy7, cxcr5-bv605, pd-1-bv421, b220-bv650 and gl-7-af647 (all from biolegend, san diego, ca, usa) , and cd95-bv510 (bd biosciences, dublin, ireland), in a brilliant violet cell stain buffer (bd biosciences, dublin, ireland) for 20 min in the dark at room temperature. cells were then washed, resuspended in cell stain buffer (biolegend, san diego, ca, usa), and the number of stained cells was acquired using bd lsrfortessa cell analyzer (bd biosciences, dublin, ireland). the data were analyzed using flowjo software (tree star, ashland, or, usa). cd45 + cd11c -ly6c + cells were identified as monocytes, cd45 + cd11c -ly6c + cd40 + cells were identified as activated monocytes, cd45 + cd11c -ly6c + ccr7 + cells were identified as migratory monocytes, cd45 + cd4 + cells were identified as cd4 + t cells, cd45 + cd4 + cxcr5 + pd-1 + cells were identified as tfh cells, cd45 + b220 + cells were identified as b cells and cd45 + b220 + cd95 + gl-7 + cells were identified as gc b cells. one-way anova test with tukey's multiple comparison was used for statistical analysis using graphpad prism v6 (graphpad, san diego, ca, usa). spearman correlation was performed to determine the association of the fold increase in the frequency of tfh and gc b cells with neutralizing antibody titers using graphpad prism v6 (graphpad, san diego, ca, usa). p < 0.05: *, p < 0.01: **, p < 0.001: ***, p < 0.0001: ****. nd: not detectable. to investigate whether rasp-1 in combination with alum enhances the humoral immune responses induced by the mres-rbd-fd (herein after rbd) vaccine, we immunized c57bl/6 mice twice. three weeks apart, using a formulation where rasp-1 and the rbd vaccine proteins were completely adsorbed to alum and then administered as a single inoculum (table 1 ; group 5). this adjuvanted vaccine was compared to that in which rasp-1 was co-administered with the alum-adjuvanted rbd vaccine as two inoculums and in two separate sites of the caudal thigh muscle (table 1 ; group 6). rbd formulated with either rasp-1 or alum alone, rbd alone, and pbs alone were included as controls. table 1 . immunization of mice using different combinations and/or formulations of the vaccines and the site of injection: c57bl/6 mice were immunized intramuscularly (i.m.) with mers-rbd-fd (rbd) formulated with or without alum and/or rasp-1 alone or together in different combinations and/or formulations. mice were immunized twice, 3 weeks apart according to the various g1-g6 experimental groups either at the front (a: 50 µl of inoculum) and/or the back (b: 50 µl of inoculum) of the caudal thigh muscle in each hind leg. injection immunization of mice with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6, figure 1 ) significantly resulted in the highest neutralizing antibody titers against mers-cov infection in vitro, nt 50 = 17,657. it was approximately four-fold higher than in mice that were vaccinated with rbd + rasp-1 + alum in a single inoculum (g5 − nt 50 = 4453; figure 1 ),~10-,~3-, and 85-fold higher when compared to rasp-1-adjuvanted rbd vaccine, alum-adjuvanted rbd vaccine, and rbd only, respectively (g3 − nt 50 = 1839, g4 − nt 50 = 6528, g2 − nt 50 = 207, respectively; figure 1 ). it appears that this unique rasp-1 and alum combinatorial adjuvants promoted synergy in the functional humoral response produced vs. the vaccines that utilized the other formulations and/or regimens, including the rasp-1-adjuvanted rbd vaccine. ). it appears that this unique rasp-1 and alum combinatorial adjuvants promoted synergy in the functional humoral response produced vs. the vaccines that utilized the other formulations and/or regimens, including the rasp-1-adjuvanted rbd vaccine. (table 1 and x-axis legend). sera samples were collected 7 days post-2nd immunization and analyzed for neutralization of the pseudotyped mers-cov. the data represents the mean and standard error (sem) of nt50 titers from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.001: ***, p < 0.0001: ****, nd: not detectable. when the total igg response to the mers-rbd antigen was studied using the mers-cov s1 protein as the target protein, we found that although the rbd-specific total igg antibody titers were ~8 times higher in mice that were vaccinated by co-administrating rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6-142,525 end point titer), they were not significantly different from those elicited by immunization with rbd + rasp-1 + alum administered in a single inoculum (g5-18,149 end point titer), or with the alum-adjuvanted rbd vaccine (g4-53,104 end point titer; figure s1a ). nevertheless, the co-administration of rasp-1 and the alum-adjuvanted rbd vaccine in separate sites significantly increased the rbd-specific total igg antibody titers by ~20-fold compared to rasp-1-adjuvanted mers-rbd vaccine or ~160-fold compared to the rbd vaccine alone (g6-142,525, g3-6700 and g2-872 end point titers respectively; figure s1a ); clearly showing that immunization using the unique rasp-1 and alum combinational adjuvants had a beneficiary effect in comparison to the rasp-1 (~20 fold) or the alum (~3 fold) adjuvanted vaccines. to elucidate the igg subtypes induced in the different immunization groups, we also analyzed the rbd-specific igg1 and igg2c antibody titers. we observed that the highest titer of igg1 antibodies was induced when the rbd vaccine was formulated with alum alone or with rasp-1 + alum in one inoculum (g4-250,951 and g5-369,000 end point titers respectively; figure s1b ), with the titers being (table 1 and x-axis legend). sera samples were collected 7 days post-2nd immunization and analyzed for neutralization of the pseudotyped mers-cov. the data represents the mean and standard error (sem) of nt 50 titers from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.001: ***, p < 0.0001: ****, nd: not detectable. when the total igg response to the mers-rbd antigen was studied using the mers-cov s1 protein as the target protein, we found that although the rbd-specific total igg antibody titers werẽ 8 times higher in mice that were vaccinated by co-administrating rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6-142,525 end point titer), they were not significantly different from those elicited by immunization with rbd + rasp-1 + alum administered in a single inoculum (g5-18,149 end point titer), or with the alum-adjuvanted rbd vaccine (g4-53,104 end point titer; figure s1a ). nevertheless, the co-administration of rasp-1 and the alum-adjuvanted rbd vaccine in separate sites significantly increased the rbd-specific total igg antibody titers by~20-fold compared to rasp-1-adjuvanted mers-rbd vaccine or~160-fold compared to the rbd vaccine alone (g6-142,525, g3-6700 and g2-872 end point titers respectively; figure s1a ); clearly showing that immunization using the unique rasp-1 and alum combinational adjuvants had a beneficiary effect in comparison to the rasp-1 (~20 fold) or the alum (~3 fold) adjuvanted vaccines. to elucidate the igg subtypes induced in the different immunization groups, we also analyzed the rbd-specific igg1 and igg2c antibody titers. we observed that the highest titer of igg1 antibodies was induced when the rbd vaccine was formulated with alum alone or with rasp-1 + alum in one inoculum (g4-250,951 and g5-369,000 end point titers respectively; figure s1b ), with the titers being significantly higher when the vaccine was with both adjuvants and in one inoculum. the rbd-specific igg1 titers were significantly decreased when rasp-1 was used as an adjuvant in aqueous formulations; when the rasp-1 and the alum-adjuvanted rbd were co-administered in separate sites (~7-fold decrease; g6-55,945 end point titers) or with the rasp-1-adjuvanted rbd vaccine (~40-fold decrease; g3-9165 end point titers) when it was compared to the administration of rbd + rasp-1 + alum in a single inoculum (g5-369,000 end point titers; figure s1b ). rasp-1 is known as an igg2 (th1)-biased adjuvant [8] . notably, igg2c responses were only elevated when rasp-1 was also administered as an adjuvant (g3-254, g5-1064, and g6-726 end point titers), with the combinatorial adjuvanted vaccines performing similarly and best (g5 and g6; figure s1c ). these data suggest that when rasp-1 is adsorbed to alum in a vaccine formulation (g5), the two adjuvants work in synergy not only to elicit a stronger igg1 response than the alum-adjuvanted vaccine formulation, but also for inducing the igg2c antibody response as compared to alum-adjuvanted vaccine formulation alone (~14-fold increase), suggesting that the combination of rasp-1 and alum in a vaccine with the rbd antigen works in synergy to elicit a more balanced igg1-igg2c antibody response (igg1/igg2c ratio of 1626 in g5 vs. 4403 in g4; figure s1d ). the reduced igg1/igg2c ratio is more pronounced in a vaccine formulation where rasp-1 is not adsorbed to alum but co-administered separately (igg1/igg2c ratio of 601 in g6; figure s1d ). sera samples from day 7 post-2nd immunization were also tested for their ability to inhibit the binding of mers-rbd-fc protein to the hdpp4 receptor-expressing huh-7 cells by flow cytometry. similarly to the enhanced induction of neutralizing antibodies, immunization with rasp-1 and the alum-adjuvanted rbd vaccine in separate injection sites also increased the ability of the generated antibodies to inhibit the binding of the rbd protein to its receptor by~2-fold (g6-50%; figure 2 ) as compared to rbd + rasp-1 + alum adjuvanted vaccine in a single inoculum or with alum-adjuvanted rbd vaccine (g5-25% and g4-35%; figure 2 ), and by >3-fold as compared to the rasp-1-adjuvanted rbd vaccine (g3-16%; figure 2 ). when group g6 was compared to groups g3 and g4, a synergy of the combinatorial adjuvants, though when rasp-1 is not adsorbed to alum, was evident. however, when rasp-1 was adsorbed to alum (g5), the functionality of the antibodies elicited by the vaccine is much reduced. the inhibitory activity in group g5 was reduced by~30%, albeit not significantly, as compared to g4. (table 1 and x-axis legend). sera samples were collected on day 7 post-2nd immunization and assayed for inhibition of the binding of mers-cov rbd-fc to huh-7 cells expressing mers-cov receptor dpp4. the data represents the mean and standard error (sem) of percentage inhibition of binding from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.05: *, p < 0.0001: ****. nd: not detectable. the draining lymph nodes (ln) were harvested from each leg 7 days post-2nd immunization and analyzed for the number of monocytes as well as their activation and migratory status. migration of innate cells from the site of injection to the lns are required to initiate an effective adaptive immune response [27] . the analyses demonstrated that there was no significant difference in the total number of monocytes (cd45 + cd11c -ly6c + ; figures s2a and s3a) recruited into the ln per mouse between the various immunization groups. however, the number of activated monocytes (c45 + cd11c -ly6c + cd40 + ) in the ln were significantly higher in mice where rasp-1 and the alumadjuvanted rbd vaccine were co-administered in separate sites than in mice immunized with rbd + rasp-1 + alum in a single inoculum, or with alum-adjuvanted rbd vaccine (g6 vs. g5 and g4, respectively; figure 3b ). notably, the number of activated monocytes in the ln of mice that received only the rasp-1-adjuvanted rbd vaccine (g3) were similar to those present in mice where rasp-1 was administered without being adsorbed to alum (g6). it appears that alum does not add to the activation of the monocytes in any of the formulations tested (g4, g5 or g6; figure 3b ). (table 1 and x-axis legend). sera samples were collected on day 7 post-2nd immunization and assayed for inhibition of the binding of mers-cov rbd-fc to huh-7 cells expressing mers-cov receptor dpp4. the data represents the mean and standard error (sem) of percentage inhibition of binding from at least two independent experiments with 3 to 5 mice per group. "+" indicates the presence and "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.05: *, p < 0.0001: ****. nd: not detectable. the draining lymph nodes (ln) were harvested from each leg 7 days post-2nd immunization and analyzed for the number of monocytes as well as their activation and migratory status. migration of innate cells from the site of injection to the lns are required to initiate an effective adaptive immune response [27] . the analyses demonstrated that there was no significant difference in the total number of monocytes (cd45 + cd11c -ly6c + ; figures s2a and s3a ) recruited into the ln per mouse between the various immunization groups. however, the number of activated monocytes (c45 + cd11c -ly6c + cd40 + ) in the ln were significantly higher in mice where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites than in mice immunized with rbd + rasp-1 + alum in a single inoculum, or with alum-adjuvanted rbd vaccine (g6 vs. g5 and g4, respectively; figure 3b) . notably, the number of activated monocytes in the ln of mice that received only the rasp-1-adjuvanted rbd vaccine (g3) were similar to those present in mice where rasp-1 was administered without being adsorbed to alum (g6). it appears that alum does not add to the activation of the monocytes in any of the formulations tested (g4, g5 or g6; figure 3b ). nevertheless, a synergistic effect was observed when rasp-1 was added to the rbd vaccine with or without alum with respect to ccr7 + (migratory) monocyte subset. the number of migratory monocytes (cd45 + cd11c -ly6c + ccr7 + ) in the ln were significantly higher in mice where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites, rbd + rasp-1 + alum was administered in a single inoculum or the rasp-1-adjuvanted rbd vaccine vs. the alum-adjuvanted rbd vaccine (g6, g5 and g3 vs. g4; figure 3c ). the number of migratory monocytes in the ln of mice that received the alum-adjuvanted rbd vaccine were actually similar to those in the control group that received only rbd (g4 vs. g2). as observed with the number of activated monocytes, migratory monocytes in the lns of mice that received rasp-1 and the alum-adjuvanted rbd vaccine co-administered in separate sites were significantly higher in mice than in mice received rbd + rasp-1 + alum administered in a single inoculum (g6 vs g5; figure 3c ). nevertheless, a synergistic effect was observed when rasp-1 was added to the rbd vaccine with or without alum with respect to ccr7 + (migratory) monocyte subset. the number of migratory monocytes (cd45 + cd11c -ly6c + ccr7 + ) in the ln were significantly higher in mice where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites, rbd + rasp-1 + alum was administered in a single inoculum or the rasp-1-adjuvanted rbd vaccine vs. the alum-adjuvanted rbd vaccine (g6, g5 and g3 vs. g4; figure 3c ). the number of migratory monocytes in the ln of mice that received the alum-adjuvanted rbd vaccine were actually similar to those in the control group that received only rbd (g4 vs. g2). as observed with the number of activated monocytes, migratory monocytes in the lns of mice that received rasp-1 and the alum-adjuvanted rbd vaccine co-administered in separate sites were significantly higher in mice than in mice received rbd + rasp-1 + alum administered in a single inoculum (g6 vs g5; figure 3c ). to investigate the possible contribution of tfh and gc b cells to the robust functional antibody responses induced in mice administered with rasp-1 and the alum-adjuvanted rbd vaccine in separate injection sites, ln from 7 days post-2nd immunization were analyzed for the tfh ( figure 4a ) and gc b ( figure 5a ) cell frequencies. although the frequency of total cd4 + t cells within the ln was not significantly different between the various immunization groups (figure s4b ), the frequency of the tfh (cd4 + cxcr5 + pd-1 + ) cells within the ln of mice administered with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6) was 2.3-fold higher than in mice administered with the rbd + rasp-1 + alum vaccine in a single inoculum, and 5.5-and 2.1-fold higher versus rasp-1-and alum-adjuvanted rbd vaccines (g6 vs. g3 and g4, respectively; figure 4b) . importantly, the fold increase in the tfh cells was positively and significantly associated with the neutralizing antibody titers against the pseudotyped mers-cov in two immunization groups, namely mice that were immunized with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6; r = 0.535, p = 0.0150) and mice immunized with rbd + rasp-1 + alum vaccine in a single inoculum (g5; r = 0.593, p = 0.0058) ( figure 4c) . notably, the frequency of b cells (b220 + ) in the ln was also significantly higher in mice administered with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites than in mice administered with rbd + rasp-1 + alum in a single inoculum, or with the alum-adjuvanted rbd vaccine or with the rbd vaccine alone (g6-33% vs. g5-25%, g4-25%, and g2-24% respectively; figure 5b ). additionally, the frequency of b cells in mice immunized with rasp-1-adjuvanted rbd was also significantly higher as compared to mice immunized with rbd + rasp-1 + alum in a single inoculum (g3-29% vs. g5-25% respectively; figure 5b ). formulating the vaccine with rasp-1 in an aqueous formulation (not adsorbed to alum) might have been critical for the increased number of b cells in these two vaccine formulations (g6 and g3). when the frequency of gc b cells (b220 + cd95 + gl-7 + ) in the draining ln ( figure 5c ) were analyzed, it appeared that immunization of mice with rasp-1 and the alum-adjuvanted rbd vaccine in separate sites (g6) induced~2-fold increase in the frequency of the gc b cells versus immunization with rbd + rasp-1 + alum in a single inoculum and the alum-adjuvanted rbd vaccine (g5 and g4), and~4-fold increase in gc b cells was induced versus immunization with the rasp-1-adjuvanted rbd vaccine (g3; figure 5c ). interestingly, the alum-adjuvanted vaccines (g5 and g4) had >2-fold increase in gc b cells in the ln compared to rasp-1-adjuvanted rbd vaccine (g3; figure 5c ). importantly, only when rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites (g6) was the fold increase in the gc b cells positively and significantly associated with the neutralizing antibody titers against the pseudotyped mers-cov (r = 0.550, p = 0.029). no significant association was observed in mice when the rbd + rasp-1 + alum vaccine was administered (g5) as a single inoculum (r = −0.051, p = 0.829; figure 5d ). "−" indicates the absence of the protein or adjuvants in the formulation. statistics was performed using one-way anova with tukey's multiple comparison. p < 0.05: *, p < 0.001: ***. spearman correlation was performed to determine the association of tfh cells with neutralizing antibody titers. adjuvants are essential components in both prophylactic and therapeutic vaccines since they ameliorate antigen-specific protective immune responses [28] . however, choosing the appropriate adjuvant that can be employed safely and that enhances vaccine efficacy is still elusive and needs to be optimized experimentally first [29] . besides a handful of adjuvants such as cpg, poly i:c, mpla and mf59, aluminum-based (alum) adjuvants are being used in most of the adjuvanted vaccines for humans globally even today since its inception 85 years ago [30] . although beneficial effects of alum as an adjuvant were observed with the dtap, hepb, and hepa vaccines, a biased th2-type immune response, absence of strong cellular responses, and the induction of adverse reactions were some of the limitations found with the various alum-adjuvanted vaccines [30] . therefore, the utilization of a combination of adjuvants in vaccines that can improve the safety and efficacy of vaccines against emerging pathogens is being actively pursued by the research community [31] . the use of combinatorial adjuvant system is beneficial since they can be tailored to target varied pattern-recognition receptors (prrs) with each being able to enhance antigen-specific responses (cellular and humoral) in a complementary or synergistic outcome [32] . for instance, intranasal vaccination with emulsified fine particles like pelc in combination with ld-indolicidin enhanced protective influenza-specific serological immunity in mice [33] . mpl and cpg combination adjuvants promoted homologous and heterosubtypic cross protection when used with the inactivated split influenza virus vaccine [34] . the co-administration of alum and a tlr-7 adjuvant enhanced memory b cell response to lymphocytic choriomeningitis virus (lcmv) antigen [35] . alum in combination with mpla-ha-adjuvanted hbsag increased both the magnitude and the persistence of hbsag-specific immune responses against hepatitis b virus infection [36] . the aim of the present study was to explore the synergistic potential of combining the o. volvulus-derived protein adjuvant, rasp-1 with alum as a novel combinatorial adjuvant system using mers-rbd-fd as the model vaccine antigen. we have previously shown that rasp-1 enhances the immune response when co-administered in an aqueous formulation with several bystander vaccine antigens [9] [10] [11] . moreover, we have also reported that rasp-1-adjuvanted trivalent influenza vaccine (iiv3) elicits a balanced igg1/igg2c response to iiv3 and protects mice following h1n1 virus challenge, potentially via myd88-independent tlr4 signaling [8, 12] . in this study, we have shown that mice immunized with rbd + rasp-1 + alum in a single inoculum elicited neutralizing antibody titers against pseudotyped mers-cov that were not significantly different from mice that received either rasp-1-adjuvanted rbd vaccine or alum-adjuvanted rbd vaccine alone ( figure 1) . notably, when rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites the vaccine ameliorated the production of neutralizing antibody titers by~4-fold as compared to the combinatorial adjuvant system administered in a single inoculum (figure 1) . we also observed that mice that received two immunizations at three-week intervals of the combinatorial adjuvant system where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered separately elicited neutralizing antibody titers against pseudotyped mers-cov infection that were similar to, or slightly lower than, those elicited in mice that received three immunizations of the montanide isa51-adjuvanted, or two immunizations of the mf59-adjuvanted mers cov-rbd-fc or mers-rbd-fd vaccines [20] . another noteworthy observation is that the heightened neutralizing antibody titers induced by the unique experimental combinatorial adjuvant system was achieved using 5 µg of the mers-rbd-fd vaccine protein, compared to 10 µg of mers-rbd-fc or mers-rbd-fd proteins used in previous studies [20] . this suggests that the rasp-1 in this unique combinatorial adjuvant system enabled also rbd dose sparing and with two immunizations. we have previously reported that rasp-1 also facilitates iiv3 antigen dose sparing up to a 10-or 40-fold decrease, and with a single immunization of the rasp-1-adjuvanted iiv3, mice were still protected from a lethal h1n1 influenza virus challenge [12] . importantly, the antibodies elicited by the different combinatorial rbd-adjuvanted vaccines were also functional in their ability to inhibit the binding of mers-rbd to the human dpp4 receptor. mice that received rasp-1 and the alum-adjuvanted rbd vaccine separately inhibited the binding bỹ 2-fold more as compared to mice administered with rbd rasp-1 + alum in a single inoculum (figure 2 ). although mice that received alum-adjuvanted rbd vaccine significantly inhibited binding (35% ± 2.3) compared to rasp-1-adjuvanted rbd vaccine, they were not significantly different compared to the combinatorial adjuvant system administered in a single inoculum (figure 2 ). the inhibition of binding, however, was only enhanced in the combinatorial adjuvant system where rasp-1 and alum-adjuvanted rbd vaccine are co-administered separately. these data collectively suggest that adsorption of rasp-1 to alum in a combinatorial adjuvant system does not enhance the functional antibody responses elicited by alum-adjuvanted rbd vaccine. while rasp-1 when not adsorbed to alum in a combinatorial adjuvant system was able to ameliorate the functional antibody responses. an important concern raised when anti-viral vaccine are developed, especially with the ongoing covid-19 crisis, is that some vaccine approaches may induce unwantedly adverse side effects due to antibody dependent enhancement (ade) and thus more severe pathology [37] . since ade is generally correlated to the neutralizing antibody titers, studies have also shown that high neutralizing antibody titer may eliminate the potential induction of ade [38, 39] . [18] [19] [20] 40] . in our study, we show immunization of mice with rasp-1 and the alum-adjuvanted mers-cov rbd vaccine in separate sites have induced nt 50 neutralizing antibody titers greater than 1:15,000 against pseudotyped mers-cov infection. we expect that such high-titer neutralizing may prevent mers-cov infection in vivo without causing any adverse effects. however, this will have to be proven experimentally in the future. in this study, we also observed that immunization with the alum-adjuvanted rbd vaccine elicits an rbd-specific igg1-biased response, while the rasp-1-adjuvanted rbd vaccine elicits a balanced rbd specific igg1-igg2c response ( figure s1b,c) . notably, in the combinatorial adjuvant system where rasp-1 and the alum-adjuvanted rbd vaccine are co-administered separately the balanced igg1/igg2c response ( figure s1d ) was preserved, while mice that received the combinatorial adjuvant system in a single inoculum elicited an igg1-biased response ( figure s1b,d) . these results suggest that the presence of rasp-1 in an aqueous formulation shifts the dominant igg1 response elicited by the alum-adjuvanted rbd vaccine to a balanced igg1-igg2c response and this was more pronounced when rasp-1 was not adsorbed to alum. interestingly, the administration of the combinatorial adjuvant system showed differences in the ly6c + activated monocyte but not in cd11c + activated dc subsets ( figure s3 ). this may likely be due to the presence of rasp-1 in the vaccine, since we have previously shown that intra-muscular injection rasp-1 alone or the rasp-1-adjuvanted trivalent influenza (iiv3) vaccine elicited an increased recruitment of monocytes than dcs at the site of injection (24 h after injection) as compared to pbs control group or iiv3 alone [12] . in the present study, the administration of the combinatorial adjuvant system where rasp-1 was completely adsorbed to alum (single inoculum immunization group) significantly reduced the number of cd40 + (activated) monocytes and ccr7 + (migratory) monocytes to the draining ln compared to the administration of the combinatorial adjuvant system where rasp-1 was not adsorbed to alum ( figure 3b,c) . interestingly, the number of cd80 + monocytes in the ln was similar whether rasp-1 + alum + rbd were administered as a single inoculum or as a co-administered vaccine in two separate sites. however, both of these vaccine formulations as well as the rasp-1 adjuvanted-mers-rbd vaccine resulted in significantly higher number of recruited cd80 + monocytes than the alum-adjuvanted rbd vaccine, suggesting that the presence of alum did not significantly alter the number of cd80 + monocytes recruited by the combinatorial rasp-1 and alum adjuvanted-mers-rbd vaccines ( figure s3b ). moreover, there was a 2-fold increase in the number of the activated monocytes and migratory monocytes recruited to the draining ln in mice that received the rasp-1-adjuvanted rbd vaccine when compared to the alum-adjuvanted rbd vaccine ( figure 3b ,c). the number of migratory monocytes doubled in the draining ln of mice immunized with the combinatorial adjuvant system where rbd + rasp-1 + alum was administered in a single inoculum as compared to the alum-adjuvanted rbd vaccine alone. the number of migratory monocytes further increased in mice that received the combinatorial adjuvant system where rasp-1 and the alum-adjuvanted rbd vaccine were co-administered separately ( figure 3c ). there were no significant differences observed in the number of activated and migratory dcs across all the immunization groups. collectively, these data suggest that the rasp-1 in the combinatorial adjuvant system may play a significant role in the enhanced recruitment of monocyte subsets. this is supported with the data where the administration of the rasp-1-adjuvanted rbd vaccine also significantly increased the number of activated (cd40 + ) and migratory (ccr7 + ) monocytes in the draining ln compared to alum-adjuvanted rbd vaccine ( figure 3b,c) . also, rasp-1 and alum may work in synergy to improve the number of migratory monocytes in the draining ln compared to what alum could do alone. one of the important events in the generation of an adaptive cellular response is the effective migration of innate cells to the lymph nodes to encounter naïve t cells, a process in which ccr7, a chemokine receptor, is known to play a dominant role [41] . in addition, the absence of ccr7 has been shown to affect the magnitude of protective responses against viral infections in mouse models [42, 43] . therefore, we suggest that rasp-1, when not adsorbed to alum in a vaccine formulation, may improve the effective recruitment of innate cells that lead to the induction of effector adaptive cellular responses. tfh cells can determine humoral immunity that is also derived from gc b cells, and therefore both of these cell types have become an important aspect for rational designs of more effective vaccines, in particular those depending on functional antibodies for their efficacy [44, 45] . to better understand what contributed to the improved elicitation of functional anti-mers-cov neutralizing antibodies, the frequencies of tfh (cd4 + cxcr5 + pd-1 + ) cells and gc b (b220 + cd95 + gl-7 + ) cells in the draining ln of immunized mice were analyzed. a two-fold increase in both tfh and gc b frequencies were induced when rasp-1 and the alum-adjuvanted rbd vaccine were co-administered in separate sites as compared to the combinatorial adjuvant system where rbd + rasp-1 + alum were administered in a single inoculum (figures 4b and 5c ). while no significant difference was observed in the fold increase of the frequency of gc b cells in the ln of mice that were immunized with rasp-1 and the alum-adjuvanted rbd vaccine co-administered separately as compared to the alum-adjuvanted rbd vaccine, a six-fold increase was observed when this was compared to rasp-1-adjuvanted rbd vaccine alone ( figure 5c ). these data suggest that the complete adsorption of rasp-1 to alum diminished not only the ability to induce migratory monocyte, but also the development of cells that are important for mounting an effective humoral response. importantly, we found a significant and positive correlation between the neutralizing antibody titers in sera of mice vaccinated with rasp-1 and the alum adjuvanted rbd vaccine separately and the fold increase in the frequency of tfh and gc b cells recruited in the draining ln ( figures 4c and 5d) . interestingly, the fold increase in the frequency of tfh cells was also significantly and positively associated with the titers of neutralizing antibodies in mice that were immunized with the combinatorial adjuvant system administered in a single inoculum (rbd + rasp-1 + alum; figure 4b ), suggesting that the rasp-1 and alum may work in synergy. our study demonstrates that a unique combination of rasp-1 (a helminth-derived protein) protein adjuvant with alum and the mers-rbd-fd using the model vaccine antigen enhanced the protective immune responses to mers-cov, despite the fact that adjuvants have to be co-administered separately (where rasp-1 was not adsorbed to alum). also, for the first time, we were able to determine that the tfh and gc b cells in the lns in mice immunized with combinatorial adjuvanted-mers-rbd vaccine were significantly and positively associated with the essential functional protective immune responses to mers-cov neutralizing antibodies. further studies will be necessary, however, to elucidate the precise underlining mechanisms of this unique adjuvant combination of rasp-1 and alum. in our study, it appeared that the adsorption of rasp-1 to alum reduced the immunopotentiating activities of either rasp-1 or alum. as the potency of rasp-1 is highest when it is in an aqueous formulation, a better understanding of the targets of multiple immune pathways that are induced may also help us utilize the rasp-1 protein adjuvant in combination with other prr agonists that can be used in aqueous formulations as adjuvants in novel combinatorial formulations. such combinatorial adjuvants may be more advantageous with subunit vaccine models that generally are known to induce suboptimal protective immune responses alone and/or induce vaccine enhanced disease (ved) when used with the alum adjuvant [46] . the following are available online at http://www.mdpi.com/2076-393x/8/2/251/s1, figure s1 : induction of mers-cov-rbd specific igg subtypes in sera of immunized mice, figure s2 : representative flow cytometry plot determining the gating strategy of the immune cells recruited to the draining lymph nodes (lns) of immunized mice, figure s3 : number of monocyte and dc subsets recruited into the lymph nodes (lns) of immunized mice, figure s4 : frequency of cd4 + t cells in the lymph nodes (lns) of immunized mice. funding: this research was funded by nih grants u01ai124260 and r01ai139092. recent advances of vaccine adjuvants for infectious diseases the latest advancements in zika virus vaccine development augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant from 1920 to 2015 and beyond. vaccines (basel) aluminium adjuvants-in retrospect and prospect optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want old and new adjuvants -1, a th1-biased protein adjuvant derived from the helminth onchocerca volvulus, can directly bind and activate antigen-presenting cells the adjuvanticity of an o. volvulus-derived rov-asp-1 protein in mice using sequential vaccinations and in non-human primates asp-1, a recombinant secreted protein of the helminth onchocercavolvulus, is a potent adjuvant for inducing antibodies to ovalbumin, hiv-1 polypeptide and sars-cov peptide antigens enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant, rov-asp-1. vaccine the parasite-derived rov-asp-1 is an effective antigen-sparing cd4(+) t cell-dependent adjuvant for the trivalent inactivated influenza vaccine, and functions in the absence of myd88 pathway isolation of a novel coronavirus from a man with pneumonia in saudi arabia crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc advances in mers-cov vaccines and therapeutics based on the receptor-binding domain recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection combinatorial delivery of dual and triple tlr agonists via polymeric pathogen-like particles synergistically enhances innate and adaptive immune responses the novel complex combination of alum, cpg odn and hh2 as adjuvant in cancer vaccine effectively suppresses tumor growth in vivo combination of adjuvants: the future of vaccine design hookworm burden reductions in balb/c mice vaccinated with recombinant ancylostoma secreted proteins (asps) from ancylostoma duodenale, ancylostoma caninum and necator americanus theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development dendritic cell migration to peripheral lymph nodes vaccine safety evaluation: practical aspects in assessing benefits and risks efficacy and safety of immunological adjuvants. where is the cut-off? advances in aluminum hydroxide-based adjuvant research and its mechanism from discovery to licensure, the adjuvant system story triggering intracellular receptors for vaccine adjuvantation mucosal delivery of a combination adjuvant comprising emulsified fine particles and ld-indolicidin enhances serological immunity to inactivated influenza virus mpl and cpg combination adjuvants promote homologous and heterosubtypic cross protection of inactivated split influenza virus vaccine alum/toll-like receptor 7 adjuvant enhances the expansion of memory b cell compartment within the draining lymph node evaluation of hyaluronic acid-based combination adjuvant containing monophosphoryl lipid a and aluminum salt for hepatitis b vaccine the potential danger of suboptimal antibody responses in covid-19 cross-reactivity, and function of antibodies elicited by zika virus infection engineering a stable cho cell line for the expression of a mers-coronavirus vaccine antigen rot, a. ccr7 and its ligands: balancing immunity and tolerance impact of ccr7 on priming and distribution of antiviral effector and memory ctl antiviral immune responses in the absence of organized lymphoid t cell zones in plt/plt mice the adjuvant gla-se promotes human tfh cell expansion and emergence of public tcrbeta clonotypes can follicular helper t cells be targeted to improve vaccine efficacy? a unique combination adjuvant modulates immune responses preventing vaccine-enhanced pulmonary histopathology after a single dose vaccination with fusion protein and challenge with respiratory syncytial virus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we gratefully acknowledge kathy tang, head of the lars facility at nybc for providing animal and veterinary care. the authors also thank mihaela barbu-stevanovic, head of the flowcytometry core facility at nybc. the authors also acknowledge maria elena bottazzi and bin zhan from baylor college of medicine, texas children's hospital center for vaccine development, houston, texas for the production of the recombinant ov-asp-1 protein. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-271244-6m8sbbi1 authors: bonilla-aldana, d. katterine; quintero-rada, keidenis; montoya-posada, juan pablo; ramírez-ocampo, sebastian; paniz-mondolfi, alberto; rabaan, ali a.; sah, ranjit; rodríguez-morales, alfonso j. title: sars-cov, mers-cov and now the 2019-novel cov: have we investigated enough about coronaviruses? – a bibliometric analysis date: 2020-02-29 journal: travel medicine and infectious disease doi: 10.1016/j.tmaid.2020.101566 sha: doc_id: 271244 cord_uid: 6m8sbbi1 nan we read with interest the recent systematic review of momattin et al. [1] , which clearly reflects that although there have been concerns about emerging pathogenic coronaviruses, still there is a lack of enough research on many of their clinical, epidemiological, diagnostic and therapeutic aspects. then, we would like to discuss how much has been investigated about coronaviruses (cov), based on a bibliometric analysis in three bibliographic databases. coronaviruses cause respiratory and intestinal infections in animals and humans. they were not considered to be highly pathogenic to humans until the outbreaks of severe acute respiratory syndrome corthe coronaviruses that circulated before that time in humans mostly caused mild infections in immunocompetent people [2] . in 2018, the world health organization (who) held its annual review of the blueprint list of priority diseases, where coronaviruses were considered and included. these diseases, given their potential to cause public health emergencies of international concern (pheic) and the absence of efficacious drugs and vaccines, are considered to need accelerated research and development [3] . we conducted a bibliometric analysis using available information from major biomedical journals-indexing databases in order to assess the current state of cov-related literature worldwide. we used science citation index (sci), scopus, and pubmed. our search strategy involved collected data in indexed articles from the databases using the term "coronavirus" as main operator, from january 1951-january 2020. the scopus search identified 18,158 articles (31.3% from usa, china 13.6% and united kingdom 7.4%) followed by pubmed with 14,455 (20.1% usa, china 18.6% and germany 4.2%), and sci with 11,775 articles (34.9% from usa, 22.4% china and 6.8% germany) (fig. 1 ). in addition, 75.0%, 71.4% and 91.2% of the articles respectively were published after january 2002 (fig. 1) . from the total, 13.7%, 29.5%, 19.2%, respectively corresponded to sars-cov, and 8.3%, 13.3% and 12.9% to mers-cov, in such databases (fig. 1) . the results of this study show that usa and china have primary roles in cov research, with usa leading the scientific production with nearly a third of the articles (fig. 1) . from the directly affected countries only china has significant article production with 22% of the total articles from sci. in asia, hong kong and south korea are among the top ten, and they have been also affected by sars-cov and mers-cov [4, 5] . in the case of countries in the middle east, affected by mers-cov, such as saudi arabia, this area only contributed 3.6% of the publications in sci and 2.5% at scopus. with more than 7700 cases and 170 deaths, up to jan. 30th, 2020, in 20 countries (14 of which are in asia), research from asia can be expected to increase significantly due to the current spread of the 2019-ncov outbreak [6] , but also from countries in other continents, especially where already cases have been confirmed, as is the case of australia (7), usa (5), canada (3), france (5), germany (4) and finland (1), among others. there are still no licensed vaccines for the prevention of any cov and the treatment options are limited. as momattin et al., indicated, there are a few promising therapeutic agents on the horizon for infections such as mers-cov, including the combination of lopinavir/ritonavir and interferon-beta-1β, and ribavirin and interferon [1] . thus, most preventive measures are aimed to reduce the risk of infection. however, it must be noted that the 2019-2020 epidemic has set an unprecedented milestone in virology research by the way open science is tackling with exceptional speediness this outbreak. the availability of genome sequences, phylogenetic tracing analysis, and development of predictive models to better understand the biology of this virus has been generated almost in real-time, allowing a more rapid monitoring and response when compared to previous coronavirus outbreaks. in conclusion, it is time to translate research findings into more effective measures, as with other priority diseases [7] , such as a vaccine or effective therapeutic options, aimed at controlling viruses with clear epidemic potential, and to prioritize those interventions, to reduce and control the negative impact of diseases such as those caused by cov, including the new emerging 2019-ncov. a systematic review of therapeutic agents for the treatment of the middle east respiratory syndrome coronavirus origin and evolution of pathogenic coronaviruses list of blueprint priority diseases sars-cov antibody prevalence in all hong kong patient contacts lessons to learn from mers-cov outbreak in south korea a novel coronavirus from patients with pneumonia in china severe fever with thrombocytopenia syndrome -a bibliometric analysis of an emerging priority disease ramírez-ocampo public health and infection research and incubator group, faculty of health sciences instituto de investigaciones biomedicas idb / incubadora venezolana de la ciencia key: cord-273626-zy8qjaai authors: gong, shu‐ran; bao, lin‐lin title: the battle against sars and mers coronaviruses: reservoirs and animal models date: 2018-07-28 journal: animal model exp med doi: 10.1002/ame2.12017 sha: doc_id: 273626 cord_uid: zy8qjaai in humans, infection with the coronavirus, especially the severe acute respiratory syndrome coronavirus (sars‐cov) and the emerging middle east respiratory syndrome coronavirus (mers‐cov), induces acute respiratory failure, resulting in high mortality. irregular coronavirus related epidemics indicate that the evolutionary origins of these two pathogens need to be identified urgently and there are still questions related to suitable laboratory animal models. thus, in this review we aim to highlight key discoveries concerning the animal origin of the virus and summarize and compare current animal models. in the aftermath of the sars outbreak, its high morbidity and mortality made the identification of natural reservoirs and an appropriate animal model necessary in order to ascertain the interspecies transmission chain, to develop procedures for protecting public health, to promote research on the sars-cov mechanism, and to establish animal models for use in developing antivirals and vaccines. although the sars outbreak occurred over 10 years ago, another member of the coronaviridae family of viruses has since caused illness in the middle east. this illness has been named middle east respiratory syndrome and the pathogen (mers-cov) has been shown to be a type of coronavirus that is highly related to sars-cov. infection with mers-cov results in higher mortality and new symptoms such as renal failure. thus, failure to fully resolve the initial sars pathogen has been followed by a more virulent infection with a related virus, mers-cov. therefore, we need urgently to identify the origins of the viruses and find ways to deal with them. to make a useful comparison, this review will investigate the yuan ky et al 1 assayed 13 different species of bats, 5 different species of rodents and 20 rhesus macaques. the results proved that sars-cov in bats was the most closely related to that in humans. in this assay, the sars-like cov in chinese horseshoe bats has 88%-92% sequence homology with human sars-cov. moreover, the s2 m motif in its 3ʹutrs and the phylogenetic analyses of four fully characterized genomes of sars-like cov indicated that the horseshoe bats have great potential to be one of the natural reservoirs. 2, 3 given that, the same research suggested that, even though there are no available migration patterns, it is known that bats can migrate approximately 30 miles for hibernation, and the distance between their wild habitats and markets in shenzheng and hong kong is only 17 miles, which means that geographically widespread infections of sars-like cov can be explained by transmission via bats. recently, a five year study provided futher evidence that bats in the yunnan province of china are more likely to be the prime reservoir than those detected elsewhere. the study compared the nonstructural protein genes orf1a and 1b of the virus and concluded that sarsr-cov strains from a cave in yunnan village were more closely related to human sars-cov and cell entry studies demonstrated that three newly identified sarsr-covs with different s protein sequences are all able to use human ace2 as the receptor. 4 just as horseshoe bats were postulated to be the primary sars host, van boheeman et al. 5 indicated that two kinds of bats carry similar mers coronaviruses. then, susanna k. p. lau et al. 6 used sequences of rna polymerase (rdrp), spike (s), and nucleocapsid (n) genes to determine that human mers-cov rdrp is more closely related to the pipistrelle bat cov hku5 (92.1%-92.3% amino acid identity) and the s and n genes are more closely related to the tylonycteris bat cov hku4 (respectively, 66.8%-67.4% and 71.9%-72.3% amino acid identity), indicating that these three viruses may share the same ancestor. however, these results did not definitively prove bat cov to be the ancestor of the human cov. subsequently, victor max corman et al 7 isolated a virus named "neo cov" from south african neoromicia capensis bats, and this virus has 85% similarity with mers-cov, with even higher rates for some specific viral rna segments. additionally, mers-cov has been found to grow better in bat cells than in human, bovine, cat, and swine cells. 8 interestingly, within this article, it is noted that the great horn of africa, where neo cov was discovered, is also a place where camels are transported and traded. therefore, the original transmission from bats to camels may have occurred in sub-saharan africa. based on the above evidence, there is a strong possibility that bats are the initial mers-cov host. a paper by hana fakhoury et al analyzed the possible viral distribution route that mers-cov traveled in the spring of 2014, leading the authors to speculate that hibernation might be the reason for seasonal outbreaks. 9 after hibernation, bats wake up in the warm, food-abundant spring weather and eat palm or other seeds. those seeds may carry bat feces and then drop to ground, where they might be eaten by camels. if mers-cov is found in cereal plantations near where bats congregate, this view will be confirmed. during the sars outbreak, masked palm civet cats (paguma larvata) and raccoon dogs (nyctereutes procyonoides) were found to carry sars-like viruses, even before the virus was discovered in lesser bamboo bats (tylonycteris pachypusa and pipistrellus). 10 at the same time, wild animal sellers were found to possess higher than average relevant neutralizing antibodies. 11 initial phylogenetic analyses reveal that the sars-covs in civets and humans actually come from two distant branches, but the sars-cov from civets during the incipient phase of the epidemic had 99.8% sequence similarity to the human sars-cov. 12 until recently, it was thought that civets were the immediate zoonotic source of sars-cov in the guangdong sars outbreak. regarding how the civets gain the cov, janies et al used dynamic homology phylogenetic analyses to investigate other species besides civets and the results further supported the idea of civets as the immediate reservoir. it is worth mentioning that the "other species" even included humans. however, some phylogenetic analyses including humans have shown a limited numbers of infectious civets in the relevant areas. 13, 14 based on these data, it is likely that civets may be only a "bypass" reservoir that adapted transiently before the epidemic. whatever the truth is, we can at least be sure that the civet cat is one of the intermediate hosts. in ruminants, almost all evidence indicates that camels are the most additionally, an investigation in saudi arabia revealed that younger camels are prone to infection by mers-cov. 18 with regard to the transmission route, there are three possible ways for the virus to be conveyed to humans from camels: organs, flesh, and discharges such as feces and camel milk. mers-cov rna was found in a camel lymph node in qatar. 19 this finding indicates that mers-cov may be maintained in camel organs or muscles. if that speculation proves true, the convention in the middle east of cooking and trading camel meat and organs might be the interspecies transmission route. mers-cov can also survive longer in camel milk than in other ruminant milk. in addition to the above two routes, mers-cov rna can be detected in 59% of nasal discharge and 15% of feces in camels. thus, there is supportive evidence for the three postulated transmission routes, but further verification is needed to confirm them. it should also be noted that mers is a type of infectious respiratory disease, and therefore, in addition to the above three possible transmission routes, infection via aerosols produced by camels should be considered. research in qatar in 2015 showed that mers-cov was found in alpacas centrally housed with camels. 20 although mers-cov has so far not been found in camelids other than dromedaries outside of the arabian peninsula, the increasing export of alpacas increases the risk of an outbreak, since they have the potential to be the interspecies host. while it is not clear that alpacas have the same function as the camels in the 2012 outbreak, their ability to spread the virus exists. currently it is very popular to trade and keep ornamental alpacas, so strict quarantine is necessary. reusken et al 19 infected swine and chickens with sars-cov and determined that they are not susceptible to becoming hosts. in contrast, dromedary camels have been found to be capable of sars-cov infection. a similar capability to transmit mers-cov makes camels a focal link in the coronavirus transmission chain. as for other mers-cov domestic reservoirs, between 2010 and 2013, some investigators tested serum samples from sheep, goat, cattle and chickens, which pervade saudi arabia and europe. 16 on the other hand, another study 22 that found mers-cov rna, but no neutralizing antibodies, in 6 lambs. to some extent, in the context of methodical investigations, sheep could be another virus carrier in addition to camels. thus, it would be prudent to conduct field research and take necessary precautionary measures to preclude the possibility of transmission of coronaviruses from sheep. coronaviruses can be found in many kinds of birds. 23 within hong kong alone, cov-hku11 has been found in nightingales, cov-hku12 in thrushes, cov-hku13 in munias, cov-hku16 in whiteeyes, cov-hku17 in sparrows, and cov-hku18 in magpies. fortunately, avian coronaviruses are not that closely related to sars-cov. additionally, these are not migratory birds and therefore do not expand the range of the pathogen. moreover, unlike bats, these birds were accessible enough to sterilize. however, in light of the findings above, randomly hunting them for food or for pets is unwise. compared to other symptom-limited models, non-human primary models are better-established models that have close psychological and physical similarities with humans 24 (table 1 ). 29 the animal's age is the key factor affecting these results, but this is hard to identify in wild-caught monkeys. another suitable and well-established model is the common marmoset (saguinus mystax), which can show more severe clinical signs than rhesus macaques when infected with mers-cov. [30] [31] [32] when administered through a combination of intraoral, intranasal and intravascular inoculation, with doses ranging from 5 9 10 6 tcid 50 to 5 9 10 7 pfu, mild to moderate respiratory disease was observed, and interstitial pneumonia was observed clinically and microscopically. when infected with sars-cov, common marmosets exhibit fever, diarrhea, multifocal pneumonitis and hepatis. 33 research using this model is progressing. the common marmoset is a potential nonhuman primate model for sars-cov infection and deserves more attention. to date, mers-cov only has two mature models. this section will deal with additional non-human prime models for sars-cov. rhesus, cynomolgus (macaca fasicularis), and african green (chlorocebus aethiops sabaeus or cercopithecu aethiops sabaeus) monkeys have been used to investigate vaccine immunogenicity or efficacy against sars-cov. squirrel monkeys (saimiri sciureus) and mustached tamarins (saguinus mystax) have been shown to be incapable of being infected. in the case of cynomolgus monkeys, clinical evidence, such as lethargy, temporary skin rash or respiratory distress, has not been reported. however, 4 to 6 days post-inoculation (dpi), there was diffuse alveolar damage and extensive loss of epithelium from alveolar and bronchiolar walls. 28, 34 regarding african green monkeys, clearance of the virus takes approximately 4 dpi, and the infection niduses are patchy. respiratory secretions cannot accurately reflect the viral titers. however, there is a report that showed that the titer is higher and the residence time is longer in african green monkeys than in two other kinds of old world monkeys (cynomolgus and rhesus). in contrast, young balb/c mice aged 4-6 months and 12-to 14month-old balb/c mice displayed clinical syndromes such as weight loss, dehydration, and ruffled fur at 3-6 dpi, along with interstitial pneumonia, 36 cov, reaching the viral replication peak at 2 or 3 dpi and viral shedding by 10 dpi. there was histochemical evidence of pneumonia but no clear clinical symptoms. in addition, recovered hamsters produced high levels of neutralizing antibodies to resist subsequent infection. 39 unlike mice, the hamsters developed temporary viremia and viral replication in the liver and spleen. however, no inflammation was observed in those organs. this experiment proved that hamsters develop a more severe syndrome than mice. martine be et al 40 three alpacas were experimentally intranasally inoculated with mers-cov. 43 all of them shed viruses and antibodies were found in their serum. additionally, infected alpacas spread the virus to two other healthy alpacas housed in the same space, indicating the alpacas' own natural transmission capability and their potential as natural hosts. the authors also noted that mers-cov may be able to infect alpaca kidney cells. 44 as with camels, alpacas never showed symptoms such as fever. however, unlike camels, alpacas did not demonstrate observable nasal secretion. hagamans bl et al 44 regarding susceptibility to infection, goats are more susceptible than sheep. though the mers-cov neutralizing antibody has not been found in horses, three of the four experimentally infected horses had detectable viral replication in their nasal secretions starting at 3 dpi. determining the evolutionary pathway leading to the ability to transfer between species may be helpful in predicting the next epidemic outbreak. regarding models for mers-cov, camels do not develop the same clinical signs as humans, although they are natural hosts. in addition, they require too much space to house and thus are not the first choice for a laboratory model. rabbits can be infected by mers-cov, but the histology is unstable, which would limit routine observation of disease. compared to those animal models, other than dpp4 transgenic mice, rhesus macaques and marmosets are currently the best mers animal models. since their immune system is like that of humans in terms of physiology and anatomy, they can be used to study the pathogenesis mechanism and the efficacy of vaccines and antivirals. however, research using rhesus macaques requires bsl-3 laboratories and high investments. moreover, they are smart, fast, and strong, which means precautions must be taken against them escaping. therefore, the need for more user-friendly models still exists, but to date non-human primate models are still the best option. however, there is still a possibility of establishing new models. chi wai yip et al 47 phylogenetically analyzed the dpp4 receptor in various species and determined that humans, rhesus macaques, horses and rabbits belong to one family. cattle and swine are not in the same group, but their receptor is close to the human dpp4 receptor. small animals (including ferrets and mice) have dpp4 receptors far more distantly related to that of humans. this analysis could provide a reference point for potential animal models. in summary, non-human primate models are still the best choice of model. comparatively speaking, there is a greater variety of sars-cov animal models than mers-cov animal models. regarding priorities for research on vaccines and antivirals for both coronaviruses, suitable mers-cov models should be considered first. 12m-006), the chinese national major s & t project wild animal surveillance for coronavirus hku1 and potential variants of other coronaviruses prevalence and genetic diversity of coronaviruses in bats from china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats discovery of a rich gene pool of bat sars related coronaviruses provides new insights into the origin of sars coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syn rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat re-emerging middle east respiratory syndrome coronavirus: the hibernating bat hypothesis possible role of an animal vector in the sars outbreak at amoy gardens prevalence of igg antibody to sars-associated coronavirus in animal traders civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates comments to the predecessor of human sars coronavirus in 2003-2004 epidemic cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques pneumonia from human coronavirus in a macaque model treatment with interferon-a2b and ribavirin improves outcome in mers-cov-infected rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus macaque model for severe acute respiratory syndrome an animal model of sars produced by infection of macaca mulatta with sars coronavirus treatment with lopinavir/ritonavir or interferon-b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset infection with mers-cov causes lethal pneumonia in the common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease pneumonitis and multiorgan system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans glycosylation of mouse dpp4 plays a role in inhibiting middle east respiratory syndrome coronavirus infection severe acute respiratory syndrome coronavirus infection of golden syrian hamsters therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters pathology of experimental sars coronavirus infection in cats and ferrets animal models for sars studies of severe acute respiratory syndrome coronavirus pathology in human cases and animal models infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas replicative capacity of mers coronavirus in livestock cell lines asymptomatic middle east respiratory syndrome coronavirus infection in rabbits inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding bats as animal reservoirs for the sars coronavirus: hypothesis proved after 10 years of virus hunting phylogenetic perspectives on the epidemiology and origins of sars and sars-like coronaviruses how to cite this article: gong sr, bao ll. the battle against sars and mers coronaviruses: reservoirs and animal models key: cord-270077-mfl0iagr authors: chefer, svetlana; thomasson, david; seidel, jurgen; reba, richard c.; bohannon, j. kyle; lackemeyer, mathew g.; bartos, chris; sayre, philip j.; bollinger, laura; hensley, lisa e.; jahrling, peter b.; johnson, reed f. title: modeling [(18)f]-fdg lymphoid tissue kinetics to characterize nonhuman primate immune response to middle east respiratory syndrome-coronavirus aerosol challenge date: 2015-11-16 journal: ejnmmi res doi: 10.1186/s13550-015-0143-x sha: doc_id: 270077 cord_uid: mfl0iagr background: the pathogenesis and immune response to middle east respiratory syndrome (mers) caused by a recently discovered coronavirus, mers-cov, have not been fully characterized because a suitable animal model is currently not available. (18)f-fluorodeoxyglucose ([(18)f]-fdg)-positron emission tomography/computed tomography (pet/ct) as a longitudinal noninvasive approach can be beneficial in providing biomarkers for host immune response. [(18)f]-fdg uptake is increased in activated immune cells in response to virus entry and can be localized by pet imaging. we used [(18)f]-fdg-pet/ct to investigate the host response developing in nonhuman primates after mers-cov exposure and applied kinetic modeling to monitor the influx rate constant (k(i)) in responsive lymphoid tissue. methods: multiple [(18)f]-fdg-pet and ct images were acquired on a pet/ct clinical scanner modified to operate in a biosafety level 4 environment prior to and up to 29 days after mers-cov aerosol exposure. time activity curves of various lymphoid tissues were reconstructed to follow the [(18)f]-fdg uptake for approximately 60 min (3,600 s). image-derived input function was used to calculate k(i) for lymphoid tissues by patlak plot. results: two-way repeated measures analysis of variance revealed alterations in k(i) that was associated with the time point (p < 0.001) after virus exposure and the location of lymphoid tissue (p = 0.0004). as revealed by a statistically significant interaction (p < 0.0001) between these two factors, the pattern of k(i) changes over time differed between three locations but not between subjects. a distinguished pattern of statistically significant elevation in k(i) was observed in mediastinal lymph nodes (lns) that correlated to k(i) changes in axillary lns. changes in lns k(i) were concurrent with elevations of monocytes in peripheral blood. conclusions: [(18)f]-fdg-pet is able to detect subtle changes in host immune response to contain a subclinical virus infection. full quantitative analysis is the preferred approach rather than semiquantitative analysis using standardized uptake value for detection of the immune response to the virus. electronic supplementary material: the online version of this article (doi:10.1186/s13550-015-0143-x) contains supplementary material, which is available to authorized users. the pathogenesis and immune response to middle east respiratory syndrome (mers) caused by a recently discovered coronavirus, mers-cov, has not been fully characterized, in part, because a suitable animal model that mimics human mers is currently not available. nonhuman primates (nhps), such as rhesus monkeys (macaca mulatta) or common marmosets (callithrix jacchus) inoculated with mers-cov via combined intratracheal, intranasal, oral, and ocular routes, develop transient respiratory disease with little or no viremia although lethal disease was observed in a small number of marmosets [1] [2] [3] [4] . 18 f-fluorodeoxyglucose ([ 18 f]-fdg) pet/ct as a real-time noninvasive approach can be beneficial in providing biomarkers for host immune response and disease progression. [ 18 f]-fdg-pet/ct has been used to track host immune response during monkeypox virus and human immunodeficiency virus-1 infections [5] [6] [7] . as [ 18 f]-fdg uptake is increased in activated macrophages, lymphocytes, and granulocytes during inflammation, the immune response can be localized by pet imaging [8] . tracking the host response noninvasively is especially useful when animal species studied is limited and/or expensive to obtain or when animals do not develop overt clinical signs of disease. we applied [ 18 f]-fdg-pet/ct imaging to monitor infection development in rhesus macaques after mers-cov inhalation. compared to standardized uptake value (suv), we increased the accuracy of measurement of [ 18 f]-fdg uptake by applying kinetic modeling and patlak graphical analysis. we assessed the net [ 18 f]-fdg uptake rate constant (k i ) in primary lymphoid tissues engaged in the host response to mers-cov exposure. this study is the first application of the methodology to an acute infectious disease process. rhesus macaques were housed in a biosafety level 4 containment facility accredited by the association for assessment and accreditation of laboratory animal care international. experimental procedures were approved by the national institute of allergy and infectious diseases (niaid), division of clinical research (dcr), animal care and use committee and were in compliance with the animal welfare act regulations, public health service policy, and the guide for the care and use of laboratory animals recommendations. for aerosol inhalation, mers-cov-hu/jordan-n3/2012 strain (genbank accession no. kc776174.1) [9] was grown in eagle's minimum essential medium (lonza, md, usa) on vero e6 cells. prior to aerosol challenge, four rhesus macaques, two males and two females, 3-5 years old, weighing 3-5 kg each, were anesthetized by intramuscular ketamine (10-15 mg/kg) injection. head-out plethysmography (buxco-data sciences international, mn, usa) was used to calculate an average respiratory minute volume (ml/min) by multiplying the respiration rate by the tidal volume. aerosol concentrations derived from a skc biosampler (skc inc., pa, usa) were used to calculate the presented dose [10] . within a negative-pressure (−24.9 pa), head-only aerosol exposure chamber, macaques were exposed to a small-particle (0.5-3 μm aerodynamic diameter targeting lung alveoli) aerosol challenge (inhaled dose = log 10 -4.64 plaque-forming units). imaging data were acquired with gemini pet/ct clinical scanner (philips healthcare, andover, ma, usa) [5, 11] . with an axial field-of-view (fov) of 180 mm of the pet scanner, the entire nhp thorax is imaged in a single bed position. use of the scanner's brain protocol resulted in a transverse field of view of 256 mm and led to cubic 2mm-wide voxels in the reconstructed images. low-dose ct images of the thorax for pet attenuation purposes were acquired at 120 kvp, 3-mm slice thickness, and 1.5-mm spacing. no contrast was given, and the subjects were freely breathing during the scan. pet image acquisition was initiated immediately after the low-dose ct scans and 1 min prior to intravenous injection of [ 18 f]-fdg (9-10 mbq/kg) into the saphenous vein and continued for up to 60 min (3600 s). nine imaging sessions per animal were conducted on pre-inoculation days −14 or −13 and −11 or −10 and post-inoculation days +1 or +2, +3 or +4, +7 or +8, +9 or +10, +15 or +16, +21 or +22, and +28 or +29 with mers-cov. suv pet images were reconstructed iteratively using the manufacturer supplied 3d line-of-response (lor)-based row-action maximum-likelihood algorithm [12] . methods for scatter, decay, random, and attenuation correction were applied during the image reconstruction process. both scatter and attenuation corrections [13] were based on the low-dose ct images acquired prior to the pet scans. the list mode data were sorted into 46 dynamic frames during creation of the histograms. to extract the early tracer dynamic distribution in the arterial blood, the initial data set (up to 720 s or 12 min) was comprised of 39 frames with the following time sequence: 15 frames × 2 s, 6 frames × 5 s, 5 frames × 10 s, 5 frames × 20 s, 4 frames × 40 s, and 4 frames × 120 s. this sequence was followed by 3 frames × 240 s and 4 frames × 480 s to capture the late slow phase of dynamic distribution of the tracer in both the blood and the tissues. pet images were reconstructed iteratively using 3d ordered-subset expectationmaximization algorithm with two iterations and nine subsets followed by 18 iterations of maximum a posteriori reconstruction [14] . maximum a posteriori parameters were adjusted to provide a uniform spatial resolution of 4.8 mm (full-width half-maximum = 4.8 mm) in all three directions. methods for scatter, decay, random, and attenuation correction were applied during the process of pet image reconstruction. reconstructed suv pet images were analyzed without any post-reconstruction smoothing using pmod version 3.5 (pmod technologies, zurich, ch). to extract an image-derived input function (idif), voi (2-mm spheres) were placed on the left ventricles and arch of the aorta using frames over the first 6 min (360 s) after [ 18 f]-fdg injection (fig. 1a, b) . averaged data from two vois were used to generate the idif (fig. 1c, d) . two-ml spheres were placed on axillary and mediastinal lns and lumbar spine bone marrow as described previously [6] , and 5-mm spheres were placed on right and left sides of the lungs to obtain the tissue time activity curves (tacs). the last time point of the tacs was used to generate the suv data. using the standard two-tissue compartment kinetic model with irreversible tracer metabolism (k 4 = 0, fig. 1e ), the [15] . the blood volume fraction (v b ) was included in the modeling. patlak linear regression method was applied for parameter estimation utilizing the idif, [ 18 f]-fdg tissue tacs [16] , and pmod version 3.5 (pmod technologies). tissue tacs were fitted to the models by use of the nonlinear least-squares method with the levenberg-marquardt algorithm, which minimizes the weighted sum of squared errors between pet measurement and model solutions. a plot of the ratio c tis (t)/c bl (t) against the ratio of cumulative to instantaneous blood activity concentration ("normalized time") became linear in the late phase after the tracer injection when the concentration of free (i.e., unmetabolized) [ 18 f]-fdg in the blood had equilibrated with that of free tracer in extravascular volume of distribution. this linear part of the plot was fitted by eq. (1) to identify the k i as a slope of a regression line: in which c tis (t) and c bl (t) represent the radioactivity concentration in the region of interest and the arterial blood assessed from the pet images at different time points after an [ 18 f]-fdg injection, respectively, and v dist is an initial distribution volume. a criterion for maximum error was set to 5 % to derive the model parameter values. for the [ 18 f]-fdg model described in fig. 1 , the slope equals k 1 × k 3 ÷ (k 2 + k 3 ). complete blood cell counts were determined on pet-scan days [5] . body temperature or body weight were monitored once daily or once every other day, respectively. two-way repeated measures analysis of variance (anova) with post hoc bonferroni multiple comparison test used k i obtained pre-inoculation and post-inoculation with mers-cov and voi location as independent variables to characterize the host immune response. for two-way repeated measures anova, we used k i at different time points pre-inoculation with mers-cov and voi location as within and between two factors, respectively. the correlations between k i values in mediastinal and axillary lns and bone marrow and between monocyte fraction in the blood and mediastinal and axillary lns were calculated using the pearson product moment correlation coefficient (r). the d'agostino and pearson test [17] was applied to confirm that the data followed a gaussian distribution. graphpad prizm 6.01 (graphpad software inc., la jolla, ca, usa) was used for all statistical analyses. analysis of lung data revealed no pathology on ct images and no changes in [ 18 f]-fdg uptake up to day 30 after mers-cov inhalation (data not shown). no changes in body temperature, body weight, and blood glucose concentrations (69.9 ± 7.4 mg/dl prior to exposure and 63.9 ± 10.4 mg/dl after exposure) were observed. however, [ 18 f]-fdg uptake as indicated by suv increases in mediastinal and axillary lns post-inhalation (fig. 2 , additional file 1: movie s1). analysis of complete blood cell counts revealed a slight increase (within normal range) in circulating monocytes only that peaked on day 5 or 6 post-inhalation and remained elevated through the remainder of the study (fig. 3a) . images of the first 37 time frames comprised of 2-120 s each caught the fast kinetics of [ 18 f]-fdg distribution in the arterial blood (fig. 1c, d) . the rest of six time frames, 4-8 min in duration, covered the slow distribution and accumulation of [ 18 f]-fdg in the tissues at later time points (16-60 min, slow phase) (additional file 2: movie s2, fig. 4 ). suv tacs for axillary lns plateaued 15 min (900 s) after fdg injection and were similar throughout the study duration of 1.5 months (fig. 4b) . analogously, bone marrow suv tacs did not show significant variation during the study. however, compared with the axillary lns, the tacs for bone marrow continued to rise at 60 min (3,600 s) after [ 18 f]-fdg injection (fig. 4b, c) suggesting a longer time after [ 18 f]-fdg injection for the tissue with high cell glycolytic activity to reach a steady state. in mediastinal lns, tacs rise was pronounced on days 5-9 post-inhalation only, as indicated by tacs (fig. 4a) . representative patlak plots for bone marrow and mediastinal and axillary lns are shown in fig. 5 . k i obtained 2 days prior to virus exposure remained unchanged for each lymphoid tissue (fig. 3a, b) . the mean baseline k i prior to mers-cov exposure was similar in axillary and mediastinal lns (0.0062 ± 0.002 sd and 0.008 ± 0.004 sd, respectively). greater elevation in mean k i values (up to almost six-fold increase from pre-exposure scan) in mediastinal lns was observed within the first week after mers-cov exposure compared to mean k i values (<two-fold increase from pre-exposure scan) in axillary lns (0.04 ± 0.01 sd and 0.01 ± 0.002 sd for mediastinal and axillary lns on day 5/6 post-exposure), respectively, figs. 3a, b and 6b, e) . the [ 18 f]-fdg mean k i in bone marrow prior to mers-cov exposure was five-fold higher (0.03 ± 0.006 sd) compared to that observed in the lns but did not follow the pattern of ln changes observed after virus challenge (fig. 3b) . statistical analysis using two-way repeated measures anova revealed that alteration in k i values was associated with the time point (p < 0.001) after virus exposure and the location of lymphoid tissue (p = 0.0004). as revealed by a statistically significant interaction (p < 0.0001) between these two factors, the pattern of changes in k i over time differed between three locations but not between subjects. post hoc analysis by bonferroni's multiple comparison test specified a statistically significant increase in k i in mediastinal lns post-exposure compared with the k i values prior to virus challenge (p < 0.015, fig. 3a ). correlation between k i changes in axillary and mediastinal lns was statistically significant, r = 0.758, p = 0.0179 (data not shown). in addition, changes in [ 18 in peripheral blood, r = 0.85, p = 0.0035 and r = 0.70, p = 0.04, respectively (fig. 3c, d) . for comparison with the results obtained with k i , we evaluated the host response to mers-cov exposure using suvs from 49 to 57 min (2940 to 3420 s) after [ 18 f]-fdg injection (additional file 3: fig. s1c ), the last time point from the tacs (fig. 4a, b) . the main difference between the findings using k i and suvs is that the correlations between the changes in mediastinal and axillary lns (data not shown) and between monocytes fraction and the suvs in axillary lns were lost (additional file 3: fig. s1c ). in addition, the first pronounced increase in [ 18 f]-fdg uptake in axillary ln observed on day 5/6 post-exposure with k i (fig. 6e) was not detected when analyzed by suvs (fig. 6d) . furthermore, the magnitude of changes in axillary lns associated with virus exposure was greater with k i compared with suvs, (fig. 6c, f) . for example, average percent change for k i in axillary lns was 52.9 ± 19.4 % sd, while an increase in [ 18 f]-fdg uptake after mers-cov exposure determined by suvs was about two-fold less, 25.1 ± 13.9 % sd. we monitored 18 f-fdg uptake primarily in lymphoid tissues at different locations in response to aerosol mers-cov challenge in nhps. a group of mediastinal lns showed a specific pattern of changes in k i up to 29 days post-virus exposure that correlates to k i changes in axillary lymph nodes. as the mediastinal lns are the lung-draining lns, these lns had the greatest increase in k i values compared to other lymphoid tissues. the increase in k i in axillary lns was 1/3 of that observed in mediastinal lns and was not statistically significant. nevertheless, changes in k i in axillary and mediastinal lns were concurrent with elevated percentage of monocytes in peripheral blood. as shown by previously published studies, ln activation during viral infection generates an [ 18 f]-fdg signal that can be easily discerned from background activity [7, 18] . activated lymphocytes increase glucose uptake by approximately 20-fold over 24 h [19] . moreover, results from a few longitudinal studies in rhesus macaques challenged with simian-human immunodeficiency virus or humans withdrawing from antiretroviral therapy indicate a b c d fig. 3 changes in [ 18 f]-fdg uptake and monocytes fraction in the blood after mers-cov aerosol challenge. a k i in mediastinal lns (y-axis on the left) and monocyte fraction in the blood (y-axis on the right) at different days prior to and after mers-cov exposure. b k i in bone marrow and axillary lns at different days prior to and after mers-cov exposure. each column represents mean ± sd (n = 4). post hoc analysis by bonferroni's multiple comparison test specified a statistically significant increase in k i in mediastinal lns up to days +15 or +16 post-exposure compared with the k i values prior to virus challenge (adjusted p = 0.0152 on day +3 or +4, p < 0.0001 on days +5 or +6, +7 or +8, and +9 or +10, respectively, and p = 0.0004 on day +15 or +16). c, d pearson product moment correlation between elevation in [ 18 f]-fdg uptake in mediastinal (c) and axillary (d) lns and changes in blood monocyte fraction. abbreviations: fdg, fluorodeoxyglucose; k i , [ 18 f]-fdg uptake rate constant; lns, lymph nodes that increased ln tissue [ 18 f]-fdg uptake preceded fulminant virus replication. systemic factors such as viral proteins or cytokines could be engaged in lymphoid tissue response [7, 18] . as monocytes are part of the immune response to virus infection (e.g., cytokine release, phagocytosis), correlation between an increase in monocyte fraction and [ 18 f]-fdg uptake in the lns is not surprising. we compared kinetic modeling using k i to analysis using suv to quantify [ 18 f]-fdg uptake. by definition, suv represents the radioactivity concentration in the region of interest adjusted for the injected dose and the body weight. therefore, the suv is influenced by several parameters such as (1) body composition, (2) blood glucose concentration, and (3) time interval from [ 18 f]-fdg injection to image acquisition. as noted in tacs of bone marrow throughout the study and of mediastinal lns on days 5-9 post-exposure, suvs did not reach a plateau within 60 min (3600 s) after [ 18 f]-fdg injection. thus, high metabolic activity of proliferating cells in bone marrow requires longer time for [ 18 f]-fdg to reach steady state in this tissue even under physiological conditions. as the ln biopsies have not been performed in the current study, future studies sampling the tissue from mediastinal lns at peak response will be able to identify the specific cell type associated with increase in [ 18 f]-fdg uptake in the lns. notably, by applying kinetic modeling analysis, we were able to determine a steady state parameter that characterizes the system based on two processes: [ 18 f]-fdg delivery from the blood to the tissue of interest and the state of cell metabolic activity. as multiple time points from pet-acquired data are used in the kinetic modeling analysis, the accuracy of k i computation for small regions of interest is higher compared with suvs even in the presence of high noise [16] . in this study, kinetic modeling increases the signal-to-noise ratio resulting in an improvement of the accuracy of measurement and method sensitivity over analysis with suv. if the effect is mild, then a semiquantitative analysis by suv will not be able to detect it. in this study, we attempted to develop an aerosol nhp studies in rhesus macaques is hampered by the different dose units and route(s) of administration. doses given in previous studies were expressed as tissue culture infectious dose 50 % (tcid 50 ) and was given by a combination of intratracheal, ocular, oral, and intranasal routes [2, 3] or by intratracheal route only [4] . nevertheless, as [ 18 f]-fdg-pet quantitated the host response to virus challenge while ct revealed no pathology on the images, the sensitivity of ct for detection of host response is lower than [ 18 f]-fdg-pet [20] . similarly, results from a mers-cov exposure in rabbits demonstrated that mers-cov replicates in the lung tissue without the development of overt clinical signs [21] . as subclinical mers-cov infection has been reported more often in patients without comorbidities than in patients with comorbidities [22] [23] [24] , this animal model can be used to study the host response to mers-cov and to test the intervention strategies aimed at inhibition of mers-cov replication [21] . however, suppression of immune responses to mers-cov exposure in these animals may be needed to reflect the severity of mers observed in the majority of patients. other limitations of this feasibility study are the small number of subjects and preexisting health status. as this study used young, previously healthy animals with an apparently intact immune response, translation of these findings to older mers patients with numerous comorbidities should be done with caution. since [ 18 f]-fdg-pet identifies the location and the pattern of host response to virus challenge, future studies with a higher challenge dose will investigate changes in [ 18 f]-fdg uptake along with results of histopathological and immunological analysis of responsive lymphoid tissues. [ 18 f]-fdg is generally considered to be a nonspecific tracer for inflammation/immune activation, and the cells involved in the host response cannot be specified by [ 18 f]-fdg-pet. however depending on the virus and the disease stage, certain populations of immune cells will be involved in the response. therefore, the location and pattern of [ 18 f]-fdg changes in the body during infection progression will be virus specific. compared with clinical application in which [ 18 f]-fdg-pet is mostly used for diagnostic purposes, preclinical studies are performed under controlled conditions using within subject design. the metabolic map of the body determined prior to virus exposure is used to identify the location of the tissues with elevated glycolytic activity after the exposure. the specific pattern of [ 18 f]-fdg changes in the foci identified by pet at pre-and post-exposure states are monitored day-by-day. when the pattern and the location of [ 18 f]-fdg changes are identified following virus exposure, this pattern can be used to monitor the effects of treatment. in our previous study [6] , we this study extends the use of kinetic modeling of [ 18 f]-fdg uptake during lung inflammation to host immune response after mers-cov exposure in rhesus macaques. compared with suvs, k i computation increases signalto-noise ratio of pet data that results in improved accuracy of measurement and method sensitivity. with application of kinetic modeling of [ 18 f]-fdg uptake in infected animals, researchers will be able to pinpoint sites of immune response and quantify anti-inflammatory effects of potential antiviral treatments. host response assessed by patlak graphical analysis and semiquantitative analysis using suv. suvs (a) and k i (b) in mediastinal lns. suvs (d) and k i (e) in axillary lns. c, f percent increase in suv (black columns) and in k i (grey columns) in mediastinal (c) and axillary (f) lns was calculated as (k i or suv post-exposure) − (k i or suv pre-exposure) ÷ (k i or suv pre-exposure) × 100 % infection with mers-cov causes lethal pneumonia in the common marmoset pneumonia from human coronavirus in a macaque model middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus evaluation of monkeypox disease progression by molecular imaging the effect of volume of interest definition on quantification of lymph node immune response to a monkeypox virus infection assessed by 18 f-fdg-pet fluorodeoxyglucose imaging in healthy subjects with hiv infection: impact of disease stage and therapy on pattern of nodal activation assessment of lung inflammation with 18f-fdg pet during acute lung injury middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group the automated bioaerosol exposure system: preclinical platform development and a respiratory dosimetry application with nonhuman primates performance of philips gemini tf pet/ct scanner with special consideration for its time-of-flight imaging capabilities a row-action alternative to the em algorithm for maximizing likelihood in emission tomography optimization of a fully 3d single scatter simulation algorithm for 3d pet high-resolution 3d bayesian image reconstruction using the micropet small-animal scanner tomographic measurement of local cerebral glucose metabolic rate in humans with (f-18)2-fluoro-2-deoxy-d-glucose: validation of method analysis of 2-[fluorine-18]-fluoro-2-deoxy-d-glucose uptake kinetics in pet studies of pulmonary inflammation numerical recipes in c: the art of scientific computing whole body positron emission tomography imaging of activated lymphoid tissues during acute simian-human immunodeficiency virus 89.6pd infection in rhesus macaques expression of glycolytic isoenzymes in activated human peripheral lymphocytes: cell cycle analysis using flow cytometry accuracy of fdg-pet-ct in the diagnostic work-up of vascular prosthetic graft infection asymptomatic middle east respiratory syndrome coronavirus infection in rabbits mers-cov outbreak in jeddah-a link to health care facilities community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans we thank niaid comparative medicine branch and comparative medicine veterinarians for successful implementation of pet-ct scanning protocols in the biosafety level 4 environment. we thank jiro wada for outstanding assistance in figure preparation of this manuscript on behalf of battelle memorial institute. the authors declare that they have no conflicts of interest.authors' contributions lb and mgl performed this work as employees of battelle memorial institute. subcontractors to battelle memorial institute who performed this work are as follows: sc and dt, as employees of tunnell government services, inc.; jkb, as an employee of lovelace respiratory research institute; and cb and ps, as employees of mri global. key: cord-252397-qlu7dilh authors: johnson, reed f.; via, laura e.; kumar, mia r.; cornish, joseph p.; yellayi, srikanth; huzella, louis; postnikova, elena; oberlander, nicholas; bartos, christopher; ork, britini l.; mazur, steven; allan, cindy; holbrook, michael r.; solomon, jeffrey; johnson, joshua c.; pickel, james; hensley, lisa e.; jahrling, peter b. title: intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease date: 2015-11-01 journal: virology doi: 10.1016/j.virol.2015.07.013 sha: doc_id: 252397 cord_uid: qlu7dilh middle east respiratory syndrome coronavirus (mers-cov) continues to be a threat to human health in the middle east. development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. a recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. based on these data we sought to compare two isolates of mers-cov. we followed disease progression in common marmosets after intratracheal exposure with: mers-cov-emc/2012, mers-cov-jordan-n3/2012, media, or inactivated virus. our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (ct), with limited other clinical signs. based on ct data, clinical data, and virological data, mers-cov inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication. infection with middle east respiratory syndrome coronavirus (mers-cov) has been associated with middle east respiratory syndrome commonly known as mers, a respiratory syndrome with acute severe hypoxic respiratory failure often accompanied by renal failure (arabi et al., 2014; assiri et al., 2013; zaki et al., 2012) . as of march, 2015 there have been approximately 1000 laboratory confirmed cases reported with a 36% case fatality rate (http://www.who.int/csr/disease/coronavirus_infections/). considering the geographical location of mers and the hajj pilgrimage which draws an estimated 2.5 million visitors, roughly 1.8 million of whom travel internationally (http://www.cdsi.gov.sa/english/ index.php?option=com_docman&itemid=173) mers-cov represents a global health risk. common signs and symptoms of mers include fever, cough, shortness of breath, and myalgia. gastrointestinal signs are also frequently observed which include vomiting, diarrhea, and abdominal pain (assiri et al., 2013) . often, mers patients also have underlying comorbidities such as diabetes, hypertension, and chronic cardiac or renal disease (assiri et al., 2013) . to guide development of mers countermeasures, an appropriate animal model must be identified and characterized. ideally, a laboratory animal model would demonstrate clinical signs consistent with all aspects of human disease. as with most infectious diseases, mice have been evaluated as a potential mers model for pathogenesis and countermeasure screening. balb/c and stat-1 knockout mice did not develop signs of disease, such as weight loss, nor could infectious virus be recovered from lung homogenates (coleman et al., 2014) . zhao et al. developed a murine model for mers by transduction of the respiratory tract with the putative mers-cov receptor, human dipeptidyl peptidase 4 (dpp4 or cd26), using an adenovirus construct (zhao et al., 2014) . infected mice developed limited clinical signs including a small degree of weight loss. histopathological analysis found peribronchial and perivascular lymphoid infiltrates which later progressed to an interstitial pneumonia. nearly 6 log 10 plaque forming units (pfu)/g of infectious virus could be detected in the lungs of infected dpp4 transduced mice (zhao et al., 2014) . the dpp4 transduced mouse model has been used to evaluate countermeasures and pathogenesis (channappanavar et al., 2014a (channappanavar et al., , 2014b . nonhuman primate (nhp) models are considered to be essential to understanding pathogenesis and evaluating countermeasures. results from several challenge studies of mers-cov in rhesus monkeys (macaca mulatta) have varied between laboratories. the first published nhp model used rhesus monkeys inoculated via multiple routes and evaluated for virological, immunological, and histopathological changes up to 6 days postinoculation . nhps demonstrated signs of pneumonia and virus could be detected in tissues and mucous membranes by quantitative reverse transcriptase polymerase chain reaction (qrt-pcr), but attempts to determine the load of infectious virus was not reported. a follow up study demonstrated that administration of interferon-alpha2b and ribavirin reduced viral burden and lessened disease . results from a natural history study of mers-cov-infected rhesus monkeys indicated that intratracheal inoculation induced a non-lethal disease with limited pathology observed in recovering animals at 28 days post-inoculation and infectious virus could be recovered from lung but not other tissues assayed (yao et al., 2014) . standard radiological examination revealed lung infiltrates at days 3 and 5 post-inoculation, suggesting virus-induced lung disease. more recently, falzarano et al. described multiple route inoculation of the common marmoset (callithrix jacchus) in which transcriptional changes indicating induction of immune, inflammatory, and repair pathways were cataloged and partial lethality was observed (falzarano et al., 2014) . here we characterize intratracheal (it) inoculation of mers-cov into common marmosets as a model for mers and to determine differences between two common isolates of mers-cov, mers-cov-jordan-n3/2012 virus and mers-cov-emc/2012 virus. we inoculated 4 groups of marmosets and followed disease progression by periodic physical exams that included computed tomography (ct). previously, positron emission tomography with computed tomography (pet)/ct has been used to evaluate tuberculosis therapies in common marmosets (via et al., 2013) , disease progression in monkeypox-virus-inoculated cynomolgus monkeys (dyall et al., 2011) , and influenza-a-virus-inoculated ferrets (jonsson et al., 2012) . ct has two distinct advantages when compared to standard x-ray radiography 1) ct provides three dimensional data, and 2) ct data can be quantified, which allows unbiased comparisons (elke et al., 2013; li et al., 2013; romanova et al., 2014) . an ex-vivo experiment with marmoset lung and kidney primary cultures found that these tissues could support mers-cov replication; this suggested that common marmosets might be developed as a suitable animal model for mers (fig. 1) . therefore, we sought to determine if intratracheal inoculation of marmosets would result in disease presentation similar to human disease. the experimental design is shown in fig. 2 . two pre-inoculation baseline ct's were performed to establish normal lung volumes and any pre-existing anomalies. mers-cov-jordan-n3/2012 virus (mers-jor, genbank kc776174) and mers-cov-emc/2012 virus (mers-emc, genbank jx869059) were obtained and propagated as described in materials and methods. to ensure that no gross cross-contamination of the mers-cov stocks used for the marmoset experiments occurred during preparation, the spike protein of each stock was sequenced and compared to reference sequences (cotten et al., 2013; frey et al., 2014) . the spike region was chosen for comparison due to high diversity associated with viral glycoproteins. strain-specific differences found in mers-jor and mers-emc reference sequences were maintained in our stocks (table 1) , indicating that no gross cross-contamination occurred. three single nucleotide polymorphisms (snps) were seen in our stocks, two in mers-emc and one in mers-jor. blastx alignments indicate that two of these snps lead to changes in the s2 protein sequence. the mers-jor stock had a t to c change at position 2636 which induced an i839t change in s2. two changes were observed in mers-emc c to t at position 2604 that did not alter amino acid sequence and a c to a change at 3044 which resulted in a n1016t change in s2. changes in the s2 region seen here are likely a result of serial passage in cell culture (frey et al., 2014) . body temperature, peripheral oxygen saturation, respiratory rate, and overall condition were evaluated at each physical exam. no increases in body temperatures above normal ranges were observed; subjects maintained peripheral oxygenation throughout the study, and respiratory rates increased above normal range sporadically throughout the study (fig. 3a) . tremors were noted on daily observations including and between days 3 and 9 postinoculation, but were not consistently observed (data not shown). subjects underwent blood withdrawal on days 0, 4 or 5, and at necropsy to determine if hematological parameters indicated changes consistent with disease. day 4 or 5 was chosen based on our data from mers-jor inoculated rhesus monkeys which demonstrated a peak in lung disease at day 5 post-inoculation by ct (manuscript in preparation). subjects did not develop clinically significant changes in total white blood cell count, lymphocyte number, monocyte number, or neutrophil number on day 4 or 5 post-inoculation and remained within the normal range, as indicated by shaded gray area (fig. 3b ). together, these data indicate that subjects did not develop systemic clinical disease. to determine if virus was disseminating or shedding, whole blood, oropharyngeal, rectal swabs, and fecal samples from mock and inactivated virus subjects, 2 subjects per group, were collected on days à 21, à8, 1, 3, 5, 7, 14 or 15, and 25. mers jordan subjects ex-vivo analysis of primary cells. cells were isolated from lung, kidney, and bronchoalveolar lavage (bal), and one-step growth kinetics were performed as described in materials and methods. lung, kidney and bal demonstrate that mers-cov is able to replicate to at least 4 log 10 pfu/ml. were swabbed on days à 21, à8, 1 (n ¼1), 2 (n ¼2), 3(n ¼1), 4 (n ¼2), 5(n¼ 1), 6 (n ¼2), 7(n¼ 1), 8(n ¼2) 14(n ¼3) and 32 and mers-emc exposed subjects were swabbed on days à 21(n¼ 3), -8 (n ¼3), 1(n ¼3), 3 (n ¼3), 5(n¼ 3), 7(n ¼3), 14(n ¼3), and 25 (n ¼3) as outlined in fig. 2 . these samples were assayed by qrt-pcr as described in materials and methods. all samples were negative for mers-cov, suggesting that virus did not disseminate nor was shed. furthermore, plaque assays were performed on necropsy tissues and no infectious virus could be detected. virus isolation was attempted using lung homogenates for two sequential passages on vero cells; no infectious virus could be detected. fluorescent reduction neutralizing assays were performed to determine if marmosets developed an antibody response to mers-cov (fig. 4 ). subjects inoculated with mers-jor developed a low antibody titer to mers-cov with an average titer of 1:100 across the group at study end, day 25-32 post-inoculation. animals inoculated with mers-emc developed an antibody titer of 1:80 for all 3 subjects. media only, and inactivated virus did not develop detectable neutralizing antibody responses to mers-cov. qualitative assessment of the imaging data indicates that mers-cov inoculated marmosets developed lung disease that mainly affected the medial and caudal regions of the lung. lung abnormalities could be observed in media-only and the inactivated virus inoculation groups, with the inactivated virus stimulating increased lung abnormalities when compared to the media only mock inoculated subjects (fig. 5a ). lung infiltrates presenting as air bronchograms did not completely clear by end of study (fig. 5b ). more importantly, quantitative assessment of abnormal lung volume data supported the qualitative assessment. the quantitative assessment suggests that mock inoculated subjects developed a short lived response that was observed one day post-inoculation but returned to near baseline by the day 7 ct and remained near that volume throughout the course of the study. subjects inoculated with inactivated virus demonstrated increased abnormal lung volumes when compared to the media only group, but not as elevated as the groups that received live virus. mers-cov inoculated groups demonstrated increasingly abnormal lung volumes beginning day 1 post-inoculation in an individual dependent manner that did not completely resolve by study end (fig. 5c and d). comparison of the mean peak values of diseased lung volume and percent relative change of abnormal lung volume for each group indicated no statistically significant difference (unpaired t-test) between the virus isolates ( fig. 5e and f). when the peak diseased lung volume of the infectious mers-emc receiving group is compared to the mock infected the pvalue ¼0.026 and when compared to the γ-irradiated virus receiving group it is p ¼0.548. comparison of the mers-jor group to the control groups indicates a similar pattern p ¼ 0.057 and p ¼0.1245, respectively. comparison of the % fold change in diseased lung volume between the mers-emc and mock group gave p ¼0.0049 and the γ-irradiated virus receiving group was 0.0195. comparison of the % fold change in diseased lung volume between the mers-jor and mock group gave p ¼0.2102 and p ¼0.1591. the data indicate that, in common marmosets, the genetic sequence differences between the isolates do not impact disease progression as indicated by changes in diseased lung volume and that the mers virion itself can induce an inflammatory response. however, a significant difference was observed between the mers-emc group and the mock exposed group when compared by fold change of diseased lung volume, which likely reflects the small group sizes. differences in temporal progression were observed between the individual subjects that did not segregate by virus isolate. one subject in the mers-emc inoculated group appeared to develop a secondary infection observed by ct that increased to study end, day 25 post-exposure. however, no infectious virus could be recovered, suggesting an opportunistic infection either due to repeated manipulations or effects of mers-cov. at necropsy, lung lesions included multifocal to coalescing interstitial pneumonia with consolidation of the dorsocaudal lung lobe in the mers-cov inoculated animals (fig. 6 ). gross pathology of mers-cov infected subjects revealed no to moderate changes. these changes include interstitial multifocal to coalescing moderate pneumonia as shown in fig. 6a . histopathological examination of select lymphoid tissues revealed mild lymphoid hyperplasia, with medullary histiocytosis of the mandibular and tracheobronchial lymph nodes in several of the mers-cov inoculated subjects, while the control animals showed minimal changes. examination of representative lung tissue samples showed interstitial lymphohistiocytic pneumonia with type ii pneumocyte and bronchial associated lymphoid tissue (balt) hyperplasia and a few syncytia were observed in the mers-cov inoculated animals ( fig. 6b and d) . in addition, the lung lesions observed in the mers-cov inoculated subjects are consistent with a chronic respiratory disease. in general, the gross and histopathological findings in the lung tissue did somewhat correspond with the ct findings. examination of representative lung tissue from a single subject that received mers-jor and demonstrated the greatest increase in diseased lung volume as seen by ct did not differ grossly or histologically from the other subjects. no signal was detected for the presence of mers-cov antigen by immunohistochemistry (ihc) performed on the lung tissue sections of the infected subjects. ideally, an animal model for mers will recapitulate the key features of the human disease in the severe form. therefore, an ideal mers model might result in lethal disease with severe bronchopneumonia and extrapulmonary complications such as renal failure. with the use of ct, we observed that it inoculation of common marmosets with mers-jor or mers-emc isolates resulted in a non-lethal disease characterized by limited clinical signs and moderate consolidative lung pathology that did not completely resolve by study end. no difference in clinical signs was observed between the two isolates or control subjects, nor could we detect mers-cov genome using a genome specific qrt-pcr assay with a sensitivity of 100 genome copies of mers-cov. detection of neutralizing antibody in the infectious virus receiving groups and not the γ-irradiated virus receiving group does suggest that an infectious process did take place which allowed for antigen processing for development of neutralizing antibodies. artifacts introduced by manipulation of the subjects should be accounted for by use of control subjects. control marmosets were inoculated with media or inactivated virus which also resulted in limited lung pathology. in the present study, examining the percent change in diseased lung volume determined a significant difference was observed between the mers-emc group and the mock group, but not between the mers-emc and γ-irradiated group, which suggests that some viral-induced disease process did occur. however, statistical significance was not found when the total diseased lung volumes were compared between any of the groups. across all groups, pathology findings were predominantly confined to the lungs showing chronic and resolving changes. the absence of viral antigen signal in the lung tissue sections by ihc might be a result of probing for the viral antigen only at a late stage of disease, when there was likely considerable clearance of the virus. falzarano et al. observed inconsistent lethality highlighted by lung pathology that shared some characteristics of mers following mers-emc exposure of common marmosets. three notable differences between the present study and falzarano et al. are 1) our use of control subjects receiving media or inactivated virus, 2) single it route vs. multiple inoculation routes (oral, intranasal, ocular, and it), and 3) the volume of lung inoculum (0.2 ml vs. 0.5 ml). similar to their rhesus experiment, their marmoset experiment was designed as a serial euthanasia study. six subjects were euthanized at early timepoints post-exposure, before the disease course could resolve. however, two subjects met moribund endpoint criteria on day 4, and 3 survived to day 20 post-inoculation. it is unknown what the true outcome of the subjects that were serially euthanized would have been and if the subjects that met endpoint criteria did so due to virus induced disease or subject manipulations, thus skewing the interpretation of disease severity. adding to the difficulty in data interpretation, qrt-pcr data is not supported by immunohistochemistry, em, or virus isolation. furthermore, the pattern of infectious virus isolation shown by falzarano et al. demonstrates that initially only the lungs contain infectious virus in 11/15 tissues (trachea and lung lobes) examined, by day 4 postinoculation virus can be isolated from fewer lung lobes (8/10 tissues), but virus can be isolated from the trachea. by day 6 postinoculation virus is detected in 1/8 of lung lobes and all tracheas examined. no quantification is provided, therefore it is difficult to determine if propagated infectious virus was recovered or merely the inoculum. based on our data and falzarano et al.'s data, it is possible that intratracheal inoculation results in mechanical damage to the respiratory epithelium, followed by inflammation and repair, which leads to restoration of mucociliary motion that clears the liquid (and virus) from the lung. in the absence of a lethal model, other quantitative measures become more valuable for demonstrating pathogenesis. in the present study, we demonstrated that ct is effective for evaluating disease progression and regression following mers cov exposure in common marmosets. ct data can be quantified, which provides an opportunity to effectively evaluate countermeasures in sub-optimal models and strengthen data gained in well-established models. in the case of mers, an optimal animal model must still be developed. staged euthanasia studies similar to the adenovirus transduced mouse mers model could be performed in nhps, but such studies are undesirable due to the ethical concerns of nonhuman primate use. vero cells (atcc catalog number ccl81) were maintained in dulbecco's modified eagle's medium (dmem) (hyclone, logan, ut) and supplemented with 10% fetal bovine serum (fbs) (sigma st. louis mo) at 37 1c with 5% co 2 . mers-cov isolates (jordan-n3/ 2012 and emc/2012) were propagated at a multiplicity of infection of 0.1 on vero cells in 5% fbs and 5% co 2 for 3-4 days postinoculation until cytopathic effect encompassed 75-80% of the vero cell monolayer. virus was concentrated by ultracentrifugation and pelleted through a 20% sucrose cushion at 221,000 â g for 2 h at 4 1c. the pellet was re-suspended in dmem 5% fbs, frozen and titered by limiting dilution plaque assay (see below). inactivated virus for the control groups was generated by treating virus with 60,000 gy of gamma irradiation from a 60 cobalt irradiator (jl shepherd 484-r2), followed by confirmation of inactivation. mers-cov-jordan-n3/2012 was a kind gift of armed forces health surveillance center, division of global emerging infections surveillance and response system and mers-cov-emc/2012 was kindly provided by vincent munster, laboratory of virology, national institute of allergy and infectious diseases (niaid). marmoset lung tissue was ground in a sterile petri dish using a plunger from a sterile syringe, digested with 100 u/ml of collagenase (life technologies, ny usa), and 10% fetal calf serum (fcs, hyclone) 37 1c for 30 min. the reaction was quenched, and the homogenate was passed through a 70-mm filter, washed, and centrifuged (500 â g, 5 min, 4 1c). after the final wash, cells were re-suspended in 5 ml of ack lysing buffer (life technologies) for 15 min and washed twice with 25 ml pbs/5 mm edta/0.5% bsa. washed and pelleted cells were re-suspended in rpmi (lonza, switzerland) supplemented with 10% fcs (hyclone, ut, usa) and 1% penicillin/streptomycin (ps) and plated. media was changed after 24 h. kidneys were processed similar to the lung but strained through 70 mm and 40 mm filters prior to the ack lysis step. bronchoalveolar lavage samples were centrifuged at 1000 â g for 10 min at 4 1c, re-suspended in rpmi 10% fcs, and 1% ps (life technologies) before plating. multi-step growth curves were performed on isolated primary cells infected with mers-jor at a multiplicity of infection of 0.1. supernatants were harvested at 0, 24, 48, and 72 h and stored at à80 1c until plaque assays were performed (described below). mers-cov isolates were sequenced using the primers in table 2 . spike genomic sequences were generated by aligning sanger to generate a comparison between study stocks and references, spike sequences obtained from sequencing and associated reference sequences were aligned using clustal omega with default settings (goujon et al., 2010; mcwilliam et al., 2013; sievers et al., 2011) . the resulting clustal alignment was filtered for alignment positions with at least one variant base using a custom python script. potential coding changes were assessed by blastx alignments (altschul et al., 1990) . ten common marmosets, ranging in weight from 250 g to 475 g and age from 3 to 9 years were divided into 4 groups. mock inoculated subjects (n ¼2) received dmem supplemented with 5% fbs by it inoculation. two subjects received inactivated virus by it inoculation; one subject received inactivated mers-jor virus isolate, and one subject received inactivated mers-emc virus isolate. three marmosets received 5 â 10 7 pfu of mers-jor and 3 others received 5 â 10 7 pfu of mers-emc by it inoculation. prior to handling, marmosets were anesthetized with isoflurane to effect. it inoculation was performed by placement of a 20 gauge â 1 in. catheter into the trachea followed by installation of the virus inoculum in a 0.2 ml volume followed by a 0.2 ml air flush of the syringe and catheter. all animal procedures were approved by the national institute of allergy and infectious diseases (niaid) animal care and use committee, and adhered to national institutes of health (nih) policies. the experiments were carried out at the niaid integrated research facility, an aaalac and aalas accredited facility. prior to and after inoculation, ct scans, physical exams, including temperature and weight measurements were performed, oropharyngeal and rectal swabs, and stool samples were collected (fig. 2) . nhps were monitored at least twice daily. a preestablished scale was used to evaluate subject health and disease progression. these criteria included: (1) overall clinical appearance, (2) labored breathing, (3) activity and behavior, (4), responsiveness, and (5) core body temperatures. no subjects met moribund clinical endpoint criteria by study end. eight of 10 marmosets were humanely euthanized for histopathological and virological analysis. blood withdrawal was performed pre-inoculation, either day 4 or 5 post-inoculation, and at necropsy. we acquired high resolution chest ct scans, without contrast, using a hybrid philips gemini 16 slice pet/ct scanner specifically designed to function in a biological safety level 4 environment. due to the rapid respiratory rate of marmosets, a breath hold could not be performed. the ct parameters were as follows: helical scan, 140 kvp, 300 ma s, 90 mm field of view, 0.5 mm/rotation table speed, high-resolution collimation of 16 â 0.75 mm 2 , 0.8 mm slice thickness, and 0.4 mm increment covering the whole lung. the images were acquired using a standard algorithm and reconstructed using a y-detail algorithm. images were analyzed as previously described (via et al., 2013) . unpaired t-test of peak abnormal lung volume was performed with graphpad prism 6.0 (graphpad software, ca, usa). complete blood cell differential count was determined from blood samples collected in edta-coated blood tubes and analyzed using a sysmex xt2000v ™ hematology analyzer (sysmex america). viral load in samples was determined by quantitative pcr using the following primer probe set: forward primer: 5ʹtggcc gtggtggttatcact 3ʹ, reverse primer: 5ʹctcaaaatcgtccatcca ctca3ʹ, and probe 5ʹ6-fam/caccccattccactatgagcgagacaac/ 36-tamsp/3ʹ. cycling conditions were 48 1c for 30 min and 95 1c for 10 min for the rt-step followed by 95 1c for 15 s and 60 1c for 60 s for 40 cycles. the taqman one-step rt-pcr master mix was used (life technologies, ny, usa). samples were extracted with trizol and screened for the presence of mers-cov using specific primers on an applied biosystems 7900ht fast real time pcr system (life technologies). the limit of detection was 100 gene copies. virus stock and tissue samples were excised at necropsy, flash frozen, and stored at à 80 1c. for tissues, a w/v homogenate between 10% and 30% was generated. serial 10-fold dilutions were made for virus stocks, growth curve samples, and tissues and incubated on confluent veroe6 cells using 0.8% tragacanth in emem 2% fbs, and 1% ps overlay and incubated for 5 days. following incubation, tragacanth overlays were removed, the monolayers were stained with crystal violet (0.1% crystal violet, 20% ethanol 10% formalin v/v), and plaques were enumerated. serum samples taken at necropsy or experiment end were heat inactivated at 56 1c for 1 h then serial diluted and mixed with 120 pfu (final moi¼1.0) of either mers-jor or mers-emc and incubated for 1 h. after incubation virus and serum mixture was added to veroe6 cells (atcc, manassas va) for 1 h at 37 1c, 5% co 2 on 96 well operetta (perkin-elmer) compatible plates. negative control (no virus and no serum) samples and positive control (virus þ rabbit polyclonal antibody to the mers spike protein (sino biological)) samples were also included. cells were washed and incubated for 24 h followed by fixation with 20% nbf. mers-cov infection was visualized by fluorescence using a mers-cov anti-spike antibody (sino biological) on an operetta high content imager (perkin-elmer). the dilution at which 50% inhibition of relative fluorescence intensity was observed was reported as the frna 50 . forty-one tissues from all major organ systems were collected and fixed in 10% neutral buffered formalin (nbf) for 30 days. following fixation in 10% nbf tissues were processed following standard procedures. hematoxylin and eosin (he) stain was applied using the leica automated staining system (leica, microsystems). stained slides were then examined via standard light microscopy. to detect mers-cov antigen, ihc was performed using a rabbit polyclonal antiserum against mers-cov (sino biological p.r. china) (1:1000). tissues from an uninfected control animal were used to validate all ihc procedures. he and ihc sections were examined by light microscopy by the veterinary pathologists (sy and lh). in this experiment, we sought to determine if there were virus specific differences in disease progression following intratracheal inoculation of common marmosets with middle eastern respiratory syndrome coronavirus, commonly known as mers-cov, with two common laboratory viral isolates (mers-emc and mers-jordan). in contrast to previous results, we observed a non-lethal disease and few, if any, signs of virus-specific pathology. in addition, we were unable to isolate infectious virus from tissues. computed tomography was used to evaluate disease progression and provide quantitative data for comparisons between mockexposed, inactivated virus-exposed, and virus-exposed subjects. the data indicate that marmosets do not faithfully replicate human mers pathogenesis and that alternate models must be developed to efficacy test medical countermeasures. basic local alignment search tool clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study virusspecific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection t cell-mediated immune response to respiratory coronaviruses wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques evaluation of monkeypox disease progression by molecular imaging quantification of ventilation distribution in regional lung injury by electrical impedance tomography and xenon computed tomography infection with mers-cov causes lethal pneumonia in the common marmoset treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques full-genome sequence of human betacoronavirus 2c jordan-n3/2012 after serial passage in mammalian cells a new bioinformatics analysis tools framework at embl-ebi molecular imaging reveals a progressive pulmonary inflammation in lower airways in ferrets infected with 2009 h1n1 pandemic influenza virus growth pattern analysis of murine lung neoplasms by advanced semiautomated quantification of micro-ct images analysis tool web services from the embl-ebi magnetic resonance imaging versus computed tomography for identification and quantification of intraventricular hemorrhage fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega differential virulence and disease progression following mycobacterium tuberculosis complex infection of the common marmoset (callithrix jacchus) an animal model of mers produced by infection of rhesus macaques with mers coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome this work was supported by the national institute of allergy and infectious disease, division of intramural research. we are grateful to marisa st. claire, russell byrum, dan ragland, and the entire evps and irf team for their contributions to these studies. we key: cord-277781-v9hw1cdi authors: ejima, keisuke; aihara, kazuyuki; nishiura, hiroshi title: probabilistic differential diagnosis of middle east respiratory syndrome (mers) using the time from immigration to illness onset among imported cases date: 2014-04-07 journal: journal of theoretical biology doi: 10.1016/j.jtbi.2013.12.024 sha: doc_id: 277781 cord_uid: v9hw1cdi abstract middle east respiratory syndrome (mers) has spread worldwide since 2012. as the clinical symptoms of mers tend to be non-specific, the incubation period has been shown to complement differential diagnosis, especially to rule out influenza. however, because an infection event is seldom directly observable, the present study aims to construct a diagnostic model that predicts the probability of mers diagnosis given the time from immigration to illness onset among imported cases which are suspected of mers. addressing censoring by considering the transmission dynamics in an exporting country, we demonstrate that the illness onset within 2 days from immigration is suggestive of influenza. two exceptions to suspect mers even for those with illness onset within 2 days since immigration are (i) when we observe substantial community transmissions of mers and (ii) when the cases are at high risk of mers (e.g. cases with close contact in hospital or household). it is vital to collect the information of the incubation period upon emergence of a novel infectious disease, and moreover, in our model, the fundamental transmission dynamics including the initial growth rate has to be explored to differentiate the disease diagnoses with non-specific symptoms. middle east respiratory syndrome (mers), caused by the novel mers coronavirus (mers-cov), has been reported since march 2012 (cauchemez et al., 2014) . mers-cov has not caused substantial human-to-human transmissions yet, but the extent of geographic distribution of this disease has gradually expanded from middle east countries (e.g. jordan and saudi arabia) to other countries, including europe. recent studies have suggested that mildly symptomatic cases are common (cauchemez et al., 2014; fisman and tuite, 2014; assiri et al., 2013a) , while hospitalized cases tended to exhibit severe respiratory symptoms (assiri et al., 2013b; guery et al., 2013) . in october 2012, a saudi boy was suspected of mers in hong kong and was admitted to a hospital which is equipped with an isolation ward (south china morning post, 2012) . two days in advance, his father had developed fever and coughing, and the boy followed similar symptoms to his father. one day after admission to the hospital in hong kong, the boy was tested positive to influenza a (h1n1) virus, while testing negative to mers-cov. the similar suspected cases caused by influenza have been also contents lists available at sciencedirect journal homepage: www.elsevier.com/locate/yjtbi reported from other countries (nishiura et al., 2012) . not only influenza but also many other respiratory viruses induce only nonspecific clinical signs and symptoms. thus, it is difficult to selectively detect and differentially diagnose only mers among imported cases with upper respiratory symptoms (e.g. by screening febrile individuals at an international border (nishiura and kamiya, 2011) ). laboratory diagnosis such as pcr takes time and cost, and there would be a substantial number of suspected imported cases to be tested and isolated in hospital if we intend to test and intervene all suspected febrile individuals arriving from affected countries. as complimentary information to partly resolve this problem, a probabilistic model has been proposed to help differential diagnosis based on a known incubation period (nishiura et al., 2012) . employing a bayesian approach, and assuming that the incubation period distribution and the prior probability (or the population risks) of all suspected respiratory viruses are known, the model has permitted us to calculate the probability of mers given a certain length of the incubation period. nevertheless, it has been recognized as a core issue of infectious disease epidemiology that an infection event of non-sexual directly transmitted diseases is seldom directly observable (clancy and o'neill, 2008) . thus, the exact length of the incubation period is seldom known for each individual case. nevertheless, it is frequently the case that the time of illness onset is remembered among cases, and the time from immigration to illness onset among imported cases is readily available and can be useful for demonstrating the practical usefulness of the probabilistic model to assist clinical diagnosis. the present study aims to construct a statistical model that predicts the probability of mers diagnosis given a certain length of the time from immigration to illness onset among imported cases. through this exercise, we also aim to assess practical and theoretical importance of the proposed model and identify associated data gaps in epidemiological observations. further to the present study, nishiura and inaba (2011) proposed an estimation framework of the incubation period based on the time from immigration to illness onset among imported cases of influenza a (h1n1-2009) in japan. while that study aimed to estimate the incubation period distribution, the present study extends the model structure in the earlier study to predict the probability of mers diagnosis. in particular, the present study focuses on the distinction between influenza and mers. during the early stages of the epidemic, the epidemiological parameters of mers had yet to be estimated based on the empirical data. thus, we also examine the corresponding estimates of the severe acute respiratory syndrome (sars) as a substitute for mers during the early stages. let t be the time from immigration to illness onset in an imported case which developed a disease in country b at time tz 0 (where t¼0 stands for the time of immigration; fig. 1 ). suppose that he or she traveled to country a (where mers has spread) for k days. in the case of resident of country a, we may drop the data or assume that k-1. we consider two different patterns of spread in country a: (i) an endemic state (i.e. the risk of infection is in a stationary state) and (ii) an epidemic state. in the case of the latter, we assume that the epidemic of novel coronavirus is in an early stage with an approximately exponential growth of infections. the frequency of exposure among imported cases during their travel is assumed as proportional to the incidence in country a. the exponential growth rate of incidence is known to be characterized by the basic reproduction number r 0 and the mean generation time t g (wallinga and lipsitch, 2007) . if the generation time is exponentially distributed, the growth rate r is calculated as r ¼ (r 0 à1)/t g , while we have r ¼ln(r 0 )/t g for a constant generation time (wallinga and lipsitch, 2007) . given identical values of r 0 and t g , the exponential distribution is known to yield the largest value of r among all the possible distributions of the generation time, while a constant t g gives the smallest r (roberts and heesterbeek, 2007) . here we briefly describe the time from immigration to illness onset among imported cases (nishiura and inaba, 2011) . let i(t,τ) be the number of incubating individuals at time t after immigration and at infection-age τ (i.e. the time since infection). supposing that the rate of illness onset at infection-age τ is γ(τ), the dynamics of the density of incubating individuals are described by ∂iðt; τþ ∂t þ ∂iðt; τþ ∂τ ¼ àγðτþiðt; τþ; ð1þ with boundary conditions iðt; 0þ ¼ 0 for t 4 0; ið0; τþ ¼ jðτþ: here j(τ)¼i(0,τ) represents the density of incubating population at an infection-age τ at the time of immigration t ¼0 (i.e. the initial age distribution). i(t,τ) can be integrated as for τ àt 40, where l(τ) represents the survival probability of incubating individuals at infection-age τ. the survival probability is calculated by using the rate of illness onset at infection-age τ, γ (τ) as follows: and, based on survival analysis, the probability density of the incubation period f(τ) is given as note that l(τ) is also written as 1 à f(τ) where f(τ) is the cumulative distribution of the incubation period. let c(t) be the number of new symptomatic cases (illness onsets) at time t after immigration. supposing that the duration of travel is k days, then the mechanism and timing of importation: spending k days for travel, an exposure occurs in country a with a risk proportional to the incidence. entering country b, the exposed individual develops illness at t days since immigration. since an infection event is not directly observable, the exact length of the incubation period has to be inferred by addressing censoring and using an explicitly infection-age structured model. which can be rearranged as the frequency of illness onset is obtained by normalizing c(t) over t. hereafter, we focus on differential diagnosis of two specific diseases, influenza and mers. suppose that both diseases are growing in a similar manner (i.e., both in an endemic state or both in an exponential growth phase with different growth rates due to different r 0 and t g ). as was discussed by nishiura and inaba (2011) , the frequency of exposure in an epidemic case is written as for τ 4k. in the endemic case, we have r ¼0, and thus, (8) is simplified as let i represent a label for disease i and θ i be the population parameter of the incubation period of disease i. the probability density of observing the illness onset at t days from immigration, g i (t), is written as the function g i (t) is the normalized version of c(t) in eq. (7). let q i be the prior probability of disease i in country a that may be derived from cause-specific prevalence such as those based on viral etiological study. as was discussed by nishiura et al. (2012) , a bayesian approach is employed, and the present study uses the following formula for the differential diagnosis: where i¼0 denotes mers. in practical instances, the exact time of illness onset may not be precisely known for all suspected cases due to coarsely recorded data, and we may only know that the illness onset occurred at time t from immigration where 0 rt rt m , where t m is the possible maximum incubation period. in such an instance, we use a doubly interval censored likelihood (reich et al., 2009) which we use to compute the following crude model for prediction: it should be noted that eq. (13) investigates the cumulative probability of mers from time 0 to t m (and thus, the interpretation is different from (11)), but the consistent discrete version of (11) can be obtained by alternatively integrating time from (t m à 1) to t m in (12). for illustration of the proposed predictive model, we consider the differential diagnosis between two diseases, using influenza and mers as the case study. in addition, we consider sars and compare it against influenza, because sars share many clinical, virological and epidemiological features in common with mers and the empirical data of the incubation period and epidemiological parameters were available during the early stages of mers outbreaks (wallinga and lipsitch, 2007; donnelly et al., 2003) . let r 0 and t g of influenza be 1.5 and 3.0 days, respectively. according to recent studies, r 0 and t g of mers are assumed as 0.6 and 10.7 days, respectively (cauchemez et al., 2014; breban et al., 2013) . similarly, r 0 and t g of sars are assumed as 3.0 and 7.0 days, respectively (donnelly et al., 2003) . the incubation period was assumed to follow a lognormal distribution with the scale parameter (or the median incubation period), exp(μ) ¼1.6, 5.0 and 4.0 days and shape parameter s 2 ¼0.21, 0.19 and 0.37 for influenza, mers and sars, respectively (cauchemez et al., 2014; nishiura and inaba, 2011; reich et al., 2009; donnelly et al., 2003) . for illustration, we assumed that the length of travel was k ¼5 days (which is consistent with the empirical best estimate (cauchemez et al., 2014) ) and also that a prior probability for each disease was 0.50. to compare against influenza, a common disease, the equal prior yields a conservative result when the diagnosis involves a novel infectious disease without known q i (nishiura et al., 2012) . for the exposition of our proposed method, we first compare mers against influenza, using both continuous and discrete models (i.e. eqs. (11) and (13)), and examine the possible time from immigration to illness onset as ranging from 0 to 10 days. as mentioned above, as an alternative to mers during the early stages of pandemic, we also compare influenza against sars. in both comparisons, we assume endemic and epidemic scenarios, and the latter is restricted to the early exponential growth phase. in the epidemic scenario, we use two different exponential growth rates, i.e., one assuming that the generation time is a constant, and the other assumes that the generation time follows an exponential distribution. as the sensitivity analysis, we compute the probability of influenza (in comparison with mers) given the time from immigration to illness onset, by varying the length of travel (from 0 to 20 days), prior probability of influenza (from 0.1 to 0.9), and r 0 of mers (from 0.6 to 3.0) (cauchemez et al., 2013) . fig. 2a shows the probability of influenza given the exact time from immigration to illness onset (computed by eq. (11)). one minus the probability of influenza gives the posterior probability of mers. since the median incubation of influenza is 3.4 days shorter than that of the mers, the probability of influenza is high if the cases develop the disease shortly after immigration. however, as time goes by since immigration, the probability of influenza lowers 50% at day 2, and the probability of mers exceeds that of influenza thereafter. exponential growth of cases during the travel yielded higher probability of influenza than the case of uniformly distributed risk, although the difference was hardly visible on day 3 since immigration and later. if the growth rate was much greater, the probability of infection shortly before immigration would be elevated, increasing the probability of influenza. a similar qualitative pattern was seen in the comparison between influenza and sars (fig. 2b) . in this comparison, the posterior probability of influenza again appeared to be greater than 50% given that a case develops illness within 2 days since immigration. fig. 2c and d shows the average probability of influenza, as compared with mers and sars, respectively, given that the case developed a disease between day 0 and day t since immigration (computed by eq. (13)). although the prediction becomes crude as compared to those based on the exact length of time in fig. 2a and b, it clearly indicates that the average posterior probability of influenza is greater than 70% as long as the case develops the disease within 2 days since immigration. the probability is gradually lowered thereafter eventually reaching to 50%, i.e., the prior probability. again, comparison between influenza and sars yielded very similar results to that between influenza and mers. fig. 3a -c examines the sensitivity of the posterior probability of influenza to the duration of travel, the prior probability of influenza and r 0 of mers, respectively. fig. 3d -f shows the corresponding results of the average probability of influenza using eq. (13). as the duration of travel was extended, it appeared that the posterior probability of influenza decreased for the early days from immigration ( fig. 3a and d) . since the incubation period of influenza is short (on the order of a few days at most), the long travel indirectly increased the likelihood of mers. short travel (e.g. 1 day) led us to have the high probability of influenza for those developing illness within 2 days since immigration, because the distribution of time from immigration to illness onset becomes closer to the incubation period distribution. the posterior probability of influenza appeared to be very sensitive to prior probability of influenza ( fig. 3b and e) . especially, if the prior probability of mers was high (e.g., 90% due to widespread community transmission of mers or if we have to examine cases at high risk such as those following close contact), the posterior probability of influenza was apparently smaller than a and b) the posterior probability of influenza given illness onset at t days since immigration (as compared to middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars)), calculated by using eq. (11). we assumed that the travelers stay in country a for 5 days with two different rates of exponential growth (where exponential 1 corresponds to exponentially distributed generation time, while exponential 2 corresponds a constant generation time) or the uniformly distributed risk over time. the prior probability of influenza was assumed as 0.50. r 0 of mers was assumed to be 0.63. (c and d) the average probability of influenza given illness onset from 0 to t days since immigration (as compared to middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars)), calculated by using eq. (13). only the results that assumed exponential growth with exponentially distributed generation time are shown, but other assumption yielded quantitatively similar estimates. other parameters are identical to those adopted in panels a and b. 50% for those developing illness within 2 days since immigration. in the realistic situation with much greater endemicity of influenza than mers for general travelers, the illness onset within 2 days from immigration was clearly suggestive of influenza. r 0 of mers had little impact on the prediction of influenza diagnosis (fig. 3c and f), which is in line with the limited sensitivity of the posterior probability to the growth rate of infection in fig. 2 . the findings are summarized in table 1 . it appeared that the posterior probability of influenza for those developing illness within 2 days since immigration is strongly influenced by the prior probability of influenza. this indicates that the high probability of influenza would not be the case even for those developing illness in 2 days if mers transmission was more widespread than influenza or if the cases were at high risk of mers (e.g. with suspected exposure to camels or other mers cases in hospital) (assiri et al., 2013b; reusken et al., 2013a reusken et al., , 2013b . the present study proposed a probabilistic model that permits us to estimate the posterior probability of a specific infectious disease given the time from immigration to illness onset among imported cases. the estimation requires us to assume that we know not only the incubation period and the prior probability but also the length of travel and the transmission dynamics of exporting country. we have shown that the illness onset within 2 days from immigration is suggestive of influenza rather than unless varied in the corresponding univariate analysis, the duration of travel was 5 days, the prior probability of influenza was assumed as 0.50, and r 0 of mers was assumed to be 0.63. only the results that assumed exponential growth with exponentially distributed generation time are shown, but other assumption yielded quantitatively similar estimates. sensitivity of differential diagnosis to four different variables. baseline assumption sensitivity of diagnosis to the increase in assumed value how sensitive within assumed parameter range? growth rate of infection exponential growth or endemic steady state mers or sars, which is consistent with a simpler model based on the known exact length of the incubation period (nishiura et al., 2012) . the results of using sars data were similar to those obtained using mers data, which were consistent with similar viral etiology and common clinical characteristics between two diseases (assiri et al., 2013a; guery et al., 2013; cotten et al., 2013; de wit et al., 2013) . moreover, we have demonstrated that our approach to doubly interval censored data can correspond to common practical settings in which the exact length from immigration to illness onset is not known. assuming that the risk of mers is likely much smaller than 0.50, the illness onset within 2 days can be said to be strongly suggestive of influenza as compared to novel coronavirus infection. nevertheless, this predictive statement is not applicable to suspected cases at high risk of mers-cov infection, such as those previously exposed to camels or other confirmed cases in household or hospital (assiri et al., 2013b; reusken et al., 2013a reusken et al., , 2013b ); a high weight should be given to the prior probability of mers among these cases. to the best of our knowledge, the present study is the first to explicitly relate the observable time length (i.e. the time from immigration to illness onset) to the differential diagnosis of infectious diseases, examining the sensitivity of the prediction model to key model variables. by doing so, we have theoretically shown that accounting for such delay mechanism is critical not only for quantifying the natural history (nishiura and inaba, 2011) , but also for utilizing and interpreting the observable information among imported cases. moreover, the applicability of the model is not limited to imported cases. the proposed statistical framework is widely applicable to other settings in interpreting the observable epidemiological data: the most typical application may be the distinction between nosocomial and community infections in hospitals by using the time from hospitalization to illness onset among hospitalized cases with an infectious disease (lessler et al., 2007 (lessler et al., , 2010 ejima et al., 2013) . the formulation on this subject is our ongoing research. in practical terms, our proposed model not only predicts the posterior probability of influenza but also suggests that various data gaps have to be filled in empirical observation. as discussed in an earlier study (nishiura et al., 2012) , it is vital to collect the information of the incubation period upon emergence of a novel infectious disease. it should also be emphasized that the viral etiological study is valuable to directly quantify q i based on empirical data, although such data may be only applicable to general travelers (and not the travelers with close contact with other mers cases or animals (nishiura et al., 2014) ). in addition, the present study identified that addressing censored information of exposure at an exporting country requires us to understand the transmission dynamics at a global scale (and not at the country level) (lam et al., 2011) . namely, at least, either the exponential growth rate of infection, or a combination of the estimates of r 0 and t g has to be derived from epidemiological data nishiura, 2010) . if we have a disease with similar etiology and characteristics (e.g. sars as a substitute of mers), r 0 , t g and the incubation period of the substitute disease could complement the uncertainty by the time these estimates become available for the novel disease. two specific limitations have to be noted. first, our assumed exposure rests on a homogeneous population model, and the heterogeneous transmissions as well as the heterogeneous incubation period have yet to be explored extensively. second, if the transmission dynamics is dependent on the illness onset mechanism klinkenberg and nishiura, 2011) , the dependence structure has to be addressed within the model system, requiring us to account for this matter explicitly in the process of model building. since the proposed clinical prediction solely relied on the incubation period and assumed transmission dynamics, improvements have to be made with a broader scope. our ongoing future study includes an explicit assessment of the diagnostic performance of the proposed prediction system (i.e. assessment of validity and reliability), and also an inclusion of additional exposure variables other than the incubation period in the model (e.g. the presence of risky contact behavior during travel). despite the need to drastically improve the model structure to fully achieve the practical modeling exercise, we believe that the present study successfully improved the applicability of our modeling approach to empirically observable dataset, identifying data gaps and modeling needs to be addressed in the future. epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study investigation team. hospital outbreak of middle east respiratory syndrome coronavirus interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility bayesian estimation of the basic reproduction number in stochastic epidemic models transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong the impact of model building on the transmission dynamics under vaccination: observable (symptom-based) versus unobservable (contagiousness-dependent) approaches the epidemiology of mers-cov clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission the correlation between infectivity and incubation period of measles, estimated from households with two cases the feasibility of age-specific travel restrictions during influenza pandemics an evaluation of classification rules based on date of symptom onset to identify health-care associated infections identifying the probable timing and setting of respiratory virus infections time variations in the generation time of an infectious disease: implications for sampling to appropriately quantify transmission potential estimation of the incubation period of influenza a (h1n1-2009) among imported cases: addressing censoring using outbreak data at the origin of importation incubation period as part of the case definition of severe respiratory illness caused by a novel coronavirus how to interpret the transmissibility of novel influenza a(h7n9): an analysis of initial epidemiological data of human cases from china missing information in animal surveillance of mers-cov estimating incubation period distributions with coarse data middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study model-consistent estimation of the basic reproduction number from the incidence of an emerging infection saudi boy in hong kong has flu, not sars-like virus: south china morning post how generation intervals shape the relationship between growth rates and reproductive numbers hn received funding support from takahashi industrial and economic research foundation and the japan science and technology agency (jst) presto program. ke received scholarship support from the japan society for promotion of science (jsps). ka received funding support from the aihara project, the first program from jsps, initiated by cstp. the funding bodies were not involved in the collection, analysis and interpretation of data, the writing of the manuscript or the decision to submit for publication. key: cord-258611-uzzs8w1j authors: ma, xuezheng; liu, fang; liu, lijuan; zhang, liping; lu, mingzhu; abudukadeer, abuduzhayier; wang, lingbing; tian, feng; zhen, wei; yang, pengfei; hu, kongxin title: no mers-cov but positive influenza viruses in returning hajj pilgrims, china, 2013–2015 date: 2017-11-10 journal: bmc infect dis doi: 10.1186/s12879-017-2791-0 sha: doc_id: 258611 cord_uid: uzzs8w1j background: there is global health concern that the mass movement of pilgrims to and from mecca annually could contribute to the international spread of middle east respiratory syndrome coronavirus (mers-cov). in china, about 11,000 muslim pilgrims participate in the hajj gathering in mecca annually. this is the first report of mers-cov and respiratory virus molecular screening of returning pilgrims at points of entry in china from 2013 to 2015. methods and results: a total of 847 returning hajj pilgrims participated in this study. the test results indicated that of the travelers, 34 tested positive for influenza a virus, 14 for influenza b virus, 4 for metapneumo virus, 2 for respiratory syncytial virus, and 3 for human coronavirus. there was a significant difference in the rates of positive and negative influenza virus tests between hajj pilgrims with symptoms and those without. the detection rates of influenza virus were not significantly different among the three years studied, at 5.3, 6.0 and 6.3% for 2013, 2014 and 2015, respectively. discussion and conclusion: the mers-cov and respiratory viruses detection results at points of entry in china from 2013 to 2015 indicated that there were no mers-cov infection but a 5.7% positive influenza viruses in returning chinese pilgrims. as of november 2015, there had been 1618 laboratoryconfirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection reported to the world health organization, and at least 579 cases had died [1, 2] . most cases of mers-cov infection were reported from the kingdom of saudi arabia. annually, more than 2 million muslim pilgrims from 184 countries attend the hajj pilgrimage in mecca, saudi arabia [3] . this mass gathering of pilgrims presents a global health risk due to the potential spread of infectious diseases, and respiratory infections are the most common infections transmitted between hajj pilgrims [4, 5] . the viruses most commonly isolated from symptomatic patients during the hajj pilgrimage were influenza virus and coronaviruses [4] [5] [6] [7] . there is global concern that travelers returning from pilgrimage could contribute to the international spread of mers-cov. the international health regulations (ihr) emergency committee suggested that all countries perform surveillance for mers-cov among pilgrims during and after hajj [8] . in china, about 11,000 muslim pilgrims participate in the hajj gathering in mecca annually [9] . this is the first report of the molecular screening for mers-cov and respiratory viruses among returning pilgrims at points of entry in china, carried out from 2013 to 2015. the participants in this study were adult hajj pilgrims who traveled in groups to mecca, saudi arabia, and stayed there for 35-40 days from september to october, 2013-2015. in china, the government arranged charter flights for hajj pilgrims to visit mecca. infectious disease monitoring and surveillance of foreigners travelers coming from other countries is the responsibility of aqsiq (general administration quality supervision inspection and quarantine of the people's republic of china). aqsiq supervises 283 entry-exit ports in china, and operates on behalf of the national government. for all chinese hajj pilgrims, personal and flight information was recorded and a medical examination was conducted, including vaccination by a local aqsiq office, before the trip to mecca. xinjiang and gansu province have the highest number of hajj pilgrims visiting mecca each year. in this study, our institute, the chinese academy of inspection and quarantine, cooperated with the xinjiang and gansu entry-exit inspection and quarantine bureau. we randomly selected 847 returning pilgrims arriving at xinjiang and gansu airports, and asked for their consent to participate in this study. in china, every entry-exit airport has an infrared radiation thermometer, installed by aqsiq, to screen travelers' body temperatures. among 847 returning pilgrims, 20 returning pilgrims triggered the alarm on passing through the infrared radiation thermometer installed at the airport to monitor travelers' body temperature. sixteen of these travelers were confirmed by using a clinical thermometer to have the onset of fever (>37.5°c), and also reported a sore throat or cough on the returning flight. the remaining 831 returning pilgrims did not have a fever or other symptoms. the numbers of travelers with fever in each year were 7 (2013), 4 (2014), and 5 (2015). the mean age of all participants was 62.24 years old (sd = 6.19). in this study, 351 were females and 496 were males, and they were all moslem. all pilgrims were asked to undergo a health examination and were vaccinated against influenza a and b in a local travel health center a week prior to departure. all participants included in this study were voluntary and signed consent forms. for the detection of viral infection, samples included lower respiratory tract sputum, washes, and upper respiratory tract oropharyngeal swab specimens. lower respiratory tract sputum samples were used to test for respiratory viruses during this 3-year period. all pilgrims were tested for influenza and mers, but only those with fever were tested for the other viruses. all specimens were collected immediately when returning pilgrims arrived at each point of entry, and nucleic acid was isolated and immediately screened by real time rt-pcr for the upe and orf1a genes of mers-cov provided by the world health organization [10, 11] . all real time pcr protocols for influenza a and b followed those used by a previous study [12] . samples from travelers displaying a fever were also tested by real time rt-pcr for human metapneumo virus (hmpv), human respiratory syncytial virus (hrsv), and human coronaviruses hku1, 229e, and oc43 [12] . according to the infection control and health quarantine rules at airports, the time taken between specimen collection and the reporting of results was within 4 h. all real time rt-pcr results for mers-cov were negative. a total of 34 influenza a and 14 influenza b virus positive samples were detected from 2013 to 2015 (table 1) . of these, the test results from participants with a fever indicated that 7 samples were positive for influenza a, 4 were hmpv positive, 2 were hrsv positive, and 1 participant was positive for each of hku1, 229e, and oc43. in addition, 27 influenza a and 14 influenza b positive samples were detected from nonsymptomatic travelers. no dual infections were detected. two hypotheses were tested: (1) there is a significant difference in the positive and negative rates of influenza virus detection between hajj pilgrims with symptoms and those without. pearson's chi-square analysis indicated that there was a significant difference in the influenza virus detection rates between travelers with fever and those without symptoms (χ 2 = 44.24, p = 0.00). it is of interest, although of unclear significance, that none of the influenza b positive subjects were symptomatic. (2) there is a significant difference in the rates of influenza (a and b) virus detection among the years 2013, 2014, and 2015. the rates of influenza virus detection for the years 2013, 2014, and 2015 were 5.3, 6.0, and 6.3%, respectively, and statistical analysis revealed that there was no significant difference in the rates of influenza virus detection among these three years (χ 2 = 0.37, p = 0.83). all participants with fever were followed up, and none of these individuals were admitted to hospital after 15 days. in this study, we did not detect any cases of mers-cov infection but respiratory virus infections including influenza a and b, hmpv, hrsv, and human coronavirus were detected among hajj pilgrims returning to china. this result was consistent with the outcomes of similar studies of respiratory virus detection in hajj pilgrims in france, north india, egypt, ghana, saudi arabia, and the uk [12] [13] [14] [15] [16] [17] . regarding the detection of influenza viruses, these studies reported detection rates of 7.8% in france (no vaccination) [15] , 11% in north india (72% vaccination rate) [14] , 14% in egypt (20% vaccination rate) [13] , 1.3% in ghana (vaccination rate unknown) [5] , and 7% in the uk (37% vaccination rate) [17] . in our study, all participants had been vaccinated against influenza virus, but 5.7% tested positive for influenza virus infection. we are unable to measure the direct impact of influenza vaccination on the resistance of hajj pilgrims to influenza infection and further studies are required to understand the efficacy of the influenza vaccine among this population. however, increasing the rate of vaccination will help protect individuals, particularly those travelers that are most vulnerable to infection such as older adults and those that may be immunocompromised. a combination of vaccination and rapid antiviral treatment of symptomatic individuals currently offer the best strategy for the prevention and treatment of infections among hajj pilgrims. in this study, mers-cov was not detected in any of the upper respiratory swabs or sputum specimens tested. however, limiting the time taken for sample collection, the type of samples collected and the selection of participants can all affect the rates of positive detection. in previous studies, most samples were nasal swabs collected from strongly suspected symptomatic participants after they were under investigation in hospital [13] [14] [15] [16] [17] . however, in our study, swabs were collected from both suspected and asymptomatic returning pilgrims immediately after their arrival at airports. our sampling design would therefore include some healthy pilgrims, thereby decreasing the rate of detection of respiratory virus infections. in addition, upper respiratory samples (nasopharyngeal swabs and sputum) have been demonstrated to have a lower mers-cov genome load than lower respiratory specimens such as tracheal aspirates and bronchoalveolar lavage specimens [18] . this may also have limited the detection of mers-cov in our study. the findings from our study demonstrate the risk of influenza infection among travelers during mass gatherings, and confirming the need for effective surveillance of imported infectious diseases at entry points into china. the hajj pilgrimage provides a unique opportunity to test the effectiveness of different infectious disease preventive and detective measures that require a large sample size. continued annual monitoring of mers-cov, influenza viruses, and other respiratory viruses (such as human rhinovirus), is needed to increase our understanding of the epidemic patterns of respiratory virus infections among hajj pilgrims in china. world health organization. global alert and response (gar) middle east respiratory syndrome middle east respiratory syndrome hajj-associated viral respiratory infections: a systematic review high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana prevention of influenza at hajj: applications for mass gatherings the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus world health organization statement on the tenth meeting of the ihr emergency committee concerning mers-cov characteristics of traveler with middle east respiratory syndrome assays for laboratory confirmation of noval human coronavirus (hcov-emc) infection detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction epidemiology of 11 respiratory rna viruses in a cohort of hospitalized children in riyadh, saudi arabia cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during 2012-2015. influenza other respir viruses influenza not mers cov among returning hajj and umrah pilgrims with respiratory illness lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms lack of mers coronavirus but prevalence of influenza virus in french pilgrims after viral respiratory infections at the hajj: comparison between uk and saudi pilgrims respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome we wish to thank dr. dexin li for his extensive support and assistance with the study. all data generated or analyzed during this study are included in this published article.authors' contributions xm and fl carried out sample collection and drafted the manuscript. ll, lz, and lm extracted rna and collected clinical samples. aa, lw, wz, and py recorded the experimental data and collated the results tables. kh designed the study, edited the manuscript, and supervised the experiments. all authors read and approved the final manuscript. the study was conducted according to the protocol approved by the human research ethics committee, chinese academy of inspection and quarantine, in compliance with the provisions for human research in the helsinki declaration (es-0823696/2015/384hq). written informed consent was obtained from all participants. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord-018438-1tkevj8v authors: edholm, christina j.; emerenini, blessing o.; murillo, anarina l.; saucedo, omar; shakiba, nika; wang, xueying; allen, linda j. s.; peace, angela title: searching for superspreaders: identifying epidemic patterns associated with superspreading events in stochastic models date: 2018-10-25 journal: understanding complex biological systems with mathematics doi: 10.1007/978-3-319-98083-6_1 sha: doc_id: 18438 cord_uid: 1tkevj8v the importance of host transmissibility in disease emergence has been demonstrated in historical and recent pandemics that involve infectious individuals, known as superspreaders, who are capable of transmitting the infection to a large number of susceptible individuals. to investigate the impact of superspreaders on epidemic dynamics, we formulate deterministic and stochastic models that incorporate differences in superspreaders versus nonsuperspreaders. in particular, continuous-time markov chain models are used to investigate epidemic features associated with the presence of superspreaders in a population. we parameterize the models for two case studies, middle east respiratory syndrome (mers) and ebola. through mathematical analysis and numerical simulations, we find that the probability of outbreaks increases and time to outbreaks decreases as the prevalence of superspreaders increases in the population. in particular, as disease outbreaks occur more rapidly and more frequently when initiated by superspreaders, our results emphasize the need for expeditious public health interventions. ebola outbreak to date [38] . this spread might have been increased due to infected health-care workers' close contact with susceptible individuals. additionally, burial ceremonies may increase contact with infectious deceased bodies that contain the virus. the incubation period, defined as the time of infection to onset of symptoms, ranges from 2 to 21 days [38] . individuals can recover from ebola; however, mortality rates range from 25 to 90%. in 2016, the who announced that the first vaccine trial implemented in guinea was 100% effective [17, 37] . the recent preventive measures announced by the centers for disease control and prevention (cdc) include: reducing contacts with infected animals or bodily fluids of infected individuals, isolating infected and deceased individuals, early detection of infected individuals, and maintaining a clean environment [8] . mers was first identified in 2012 from an outbreak that occurred in saudi arabia [40] . the source of infection was identified as dromedary camels. however, most cases are not due to camel-to-human infections. mers outbreaks among humans arise from human-to-human interactions, where many cases occur in healthcare settings with poor health prevention and control practices. in 2015, an outbreak of mers in south korea was driven by three ss, initiated with one ss contracting mers during international travel. the first ss was responsible for 29 secondary infections through various clinical visits. two subsequently infected individuals were responsible for 106 tertiary infections [39, 40] . individuals infected with mers can be asymptomatic, while others may experience the following symptoms: fever, coughs, shortness of breath, diarrhea, and pneumonia. nearly, 35% of mers cases resulted in death. while no vaccine or treatments are available, individuals are advised to maintain good hygiene when coming into contact with animals, particularly camels, such as washing hands and avoiding contact with sick animals. additional prevention strategies include consuming thoroughly cooked and prepared animal products [39] . mathematical models formulated for recent outbreaks of mers and ebola have applied the compartmental setting with various disease stages such as susceptible, exposed, infectious, and recovered (seir) or performed statistical analyses to identify important parameters in spread of the disease ( [11, 23, 24] mers and [4, 10, 16, 22] ebola). additional classes for asymptomatic, hospitalized, or isolated individuals were also included in mers models [11, 23] . time-dependent transmission parameters accounted for superspreading events (e.g., [22] [23] [24] ). superspreading events have also been investigated with multitype branching processes by including individual heterogeneity in offspring generating functions [18, 26] . all of these models have contributed to a better understanding of the role of superspreaders in disease outbreaks. our models incorporate the compartmental framework and apply stochastic simulations with theory from branching processes to further elucidate the role of superspreaders in disease dynamics. in this investigation, we develop a mathematical modeling framework that incorporates the heterogeneity of hosts through differences in transmission rates to assess the role of ss in disease spread at the population level. specifically, we aim to study the disease dynamics in a heterogeneous population consisting of ss and ns individuals, and develop a deterministic model based on ordinary differential equations (odes) which is expanded to a stochastic model that is implemented as a continuous-time markov chain (ctmc) system and approximated by a multitype branching process [1, 2] . we incorporate estimated parameter values from published data of prior mers and ebola epidemics into our models. next, we compute the basic reproduction number for the ode model, and perform sensitivity analysis using latin hypercube sampling and partial rank correlation. by varying the initial size of ss and model parameters of the ctmc model, we derive and verify analytical estimates obtained using multitype branching process approximations with model simulations to predict the probability of an epidemic outbreak. in further numerical simulations of the ctmc model, we compute sample paths, probability of outbreak, number of deaths, time to outbreak, time to peak infection, and peak number of infectious individuals. our analyses and numerical simulations reveal how ss influence the dynamics of epidemic outbreaks, which may provide useful insight for public health interventions. we formulate a simple modeling framework for host heterogeneity due to differences in individuals that account for either ss or ns. in particular, ss or ns may differ in transmission, transitions between disease stages, deaths, recovery, or population size. the ss and ns mix homogeneously, such as in a hospital setting (mers) or at a large gathering such as a funeral (ebola). our basic modeling framework is a system of odes with five disease stages for ss and ns as described by the compartmental diagram in fig. 1 and by the differential equations in (2.1), where i = 1 is ns and i = 2 is ss. the description of the model variables are summarized in table 1 . such types of models have been used in metapopulation settings and are referred to as multigroup models (e.g., [25, 34] ). for the ode model, we assume that the disease duration is short and, therefore, we do not include birth or natural death rates. in addition, we make the simplifying assumption that ns cannot become ss and vice versa. we make this assumption due to the short duration of the epidemic period and the fact that no control measures are applied (which could change the transmission patterns). in the model, the number of susceptible ns and ss are denoted by s 1 and s 2 , respectively. susceptible individuals transition into their respective exposed classes, e 1 and e 2 , at a rate of where β 1 is the transmission rate of the ns asymptomatic a 1 and infective i 1 classes with n 1 as the total number of ns and similarly for ss variables. the total number of individuals is . from the exposed class, individuals transition to the asymptomatic class, a 1 and a 2 , at a rate of α 1 or α 2 . in the asymptomatic class, there is diseaseinduced mortality with rates μ a1 or μ a2 , respectively. asymptomatic individuals do not display symptoms but are infectious. individuals transition into the infective class at a rate of δ 1 or δ 2 , where the disease-induced mortality rates are μ i 1 or μ i 2 . [10] (9.9, 100) × 10 −5 μ ji disease-induced death rate 0.08 [7] (0.02, 0.14) 0.09 [9] (0.075, 0.125) γ i recovery rate 0.075 [19] (0.05, 0.1) 0.05 [31, 35] table 2 . for ebola parameter values, we used the outbreak in sierra leon in 2014 [7, 12, 19, 31] and for the mers 2015 outbreak in south korea [9, 10, 31, 35] . note that these parameter values are taken from a single outbreak of mers and ebola, which means that they vary from other outbreaks and may present some constraints when asserting conclusions for outbreaks of the same infectious disease [14, 20] . however, the parameter values used from the two outbreaks provide an excellent baseline for our model simulations. we compute the reproduction number for the ode system (2.1) using the nextgeneration matrix [34] . the basic reproduction number, r 0 , is defined as the number of secondary cases produced by the introduction of a single infected individual into a fully susceptible population. if r 0 > 1, an outbreak occurs in the ode model. we start by defining two matrices, f and v , where the f matrix represents the newly infected rates in the system, and v represents the remaining rates in the infected compartments, eqs. (1) and (2) respectively. the matrix f −v is the jacobian matrix of the infected compartments evaluated at the disease-free equilibrium (dfe), wherē s i = n i (0) and e i = a i = i i = r i = 0, i = 1, 2. we find the spectral radius of the matrix f v −1 (appendix 1), which equals the basic reproduction number, (2. 2) the basic reproduction number has the form typical of a multigroup/stage progression model [34] . it is the sum of two basic reproduction numbers, one for each group, ns when i = 1 and ss when i = 2. in particular, the two terms in the preceding expression represent new infections resulting from either the asymptotic stage a i or from the infectious stage i i , i = 1, 2. in addition for group i, the term β i (n i /n ) is the number of successful transmissions from an individual in stage a i (first term) or from an individual in stage i i (second term) that result in exposed individuals. the term 1/(δ i + μ a i ) is the average length of the asymptotic stage while 1/(γ i + μ i i ) is the average length of the infectious stage, and δ i /(δ i + μ a i ) is the probability of transitioning from a i to i i . for parameter values in table 2 and for equal proportion of ss and ns, n 1 /n = 0.5 = n 2 /n, the basic reproduction number for mers is r 0 = 2.36 and for ebola it is r 0 = 3.75. if the number of hosts/pathogens is sufficiently small, an ode model is not appropriate. to that end, we utilize a continuous-time markov chain (ctmc) model, which is continuous in time and discrete in the state space, to study the variability at the initiation of an outbreak, in time to outbreak, and in the peak level of infection. for simplicity, we use the same notation for the state variables as in the ode model. in particular, time t ∈ [0, ∞) and the states are discrete random variables, e.g., the markov property implies that the future states of the stochastic process only depend on the current states. in particular, there is an exponential waiting time between events. to formulate a ctmc, it is necessary to define the infinitesimal transition probabilities corresponding to each change (event) in the state variables. the ctmc model consists of 12 distinct events, six events for each of the groups, ns and ss. the changes and the corresponding infinitesimal transition rates are summarized in table 3 . the theory of multitype (galton-watson) branching processes has a long history (e.g., [2, 13, 36] and references therein). it has been used to approximate the dynamics of the ctmc model near the dfe and the stochastic threshold for a disease outbreak [1] [2] [3] 36] . in fact, the stochastic threshold (i.e., probability of a disease outbreak) is directly related to the basic reproduction number as defined in table 3 state transitions and rates for the ctmc model with poisson rates the corresponding deterministic model (2.1) (see [3, 36] ). more specifically, if the basic reproduction is less than unity, then disease extinction occurs with probability one. in this case, the branching process is called subcritical. however, if the basic reproduction number is greater than unity, the probability of disease extinction is less than one (probability of outbreak is greater than zero) and the process is referred to as supercritical. in what follows, we will apply a multitype branching process approximation of the ctmc model at the dfe to estimate disease extinction probability. first, let us define the offspring probability generating function (pgf) for the exposed, asymptomatic, and infectious individuals in ns and ss. let x = (x 1 , . . . , x 6 ) := (e 1 , a 1 , i 1 , e 2 , a 2 , i 2 ) be a vector of integer-valued random variables and δ ij denote the kronecker delta (i.e., δ ij = 1 if i = j and zero otherwise). in general, the offspring pgf for type i given x j (0) = δ ij is a function from [0, 1] 6 to [0, 1], and it takes the form: here, p i (k 1 , k 2 , . . . , k 6 ) is the probability that the individual of type i gives "birth" to k j individuals of type j for j = 1, 2, . . . , 6. in particular, the pgfs f i : 6) are given by: according to the theory of multitype branching processes [5, 13] , the fixed points of the offspring pgfs give an estimate of the disease extinction probability. let (q 1 , q 2 , q 3 , q 4 , q 5 , q 6 ) be the minimal fixed points of pgfs; that is, f i (q 1 , . . . , q 6 ) = q i for i = 1, . . . , 6. then, an estimate of the extinction probability given and hence the probability of an outbreak is however, due to the simplicity of f 1 and f 4 (no deaths during stage e i ), the pgfs can be simplified. that is, x 1 = x 2 and x 4 = x 5 . therefore, we only solve for q 2 , q 3 , q 5 , and q 6 . the expectation matrix m = (m ij ) can be shown to be directly related to the basic reproduction number [3] with m ij = ∂f j ∂x i | x=1 . we include this calculation in appendix 2. it is known that the spectral radius of m, denoted as ρ(m), determines whether the disease extinction probability is equal to or less than the unity [2, 3, 13] . specifically, if ρ(m) < 1, q 1 = · · · = q 6 = 1, then the extinction probability is one; if ρ(m) > 1, then there exists a unique fixed point (q 1 , · · · , q 6 ) ∈ (0, 1) 6 , and hence the extinction probability is strictly less than one. by the threshold theorem of reference [3] , it follows that the spectral radius of the matrix m is strictly less than one if and only if the basic reproduction number is strictly less than one. analogous statements hold whenever the spectral radius of m is equal to one or is strictly greater than one. we perform a sensitivity analysis on the parameters ranges given in table 2 for the ode models for mers and ebola using a uniform distribution for the values. latin hypercube sampling (lhs), first developed by mckay et al. [29] , with the statistical sensitivity measure partial rank correlation coefficient (prcc), performs a sensitivity analysis that explores a defined parameter space of the model. the parameter space considered is defined by the parameter intervals depicted in table 2 . rather than simply exploring one parameter at a time with other parameters held fixed at baseline values, the lhs/prcc sensitivity analysis method globally explores multidimensional parameter space. lhs is a stratified monte carlo sampling without replacement technique that allows an unbiased estimate of the average model output with limited samples. the prcc sensitivity analysis technique works well for parameters that have a nonlinear and monotonic relationship with the output measure. prcc shows how the output measure is influenced by changes in a specific parameter value when the linear effects of other parameter values are removed. the prcc values were calculated as spearman (rank) partial correlations using the partialcorr function in matlab 2016. their significances, uncorrelated p-values, were also determined. the prcc values vary between −1 and 1, where negative values indicate that the parameter is inversely proportional to the output measure. following marino et al. [27] , we performed a z-test on transformed prcc values to rank significant model parameters in terms of relative sensitivity. according to the z-test, parameters with larger magnitude prcc values had a stronger effect on the output measures. we start by verifying the monotonicity of the output measures. monotonicity was observed for all parameters except μ i 2 with total ss deaths, which exhibited two monotonic ranges [0.02, 0.0278] and [0.0278, 0.14]. for non-monotonic trends, alternative methods based on decomposition of model output variances such as efast (extended fourier amplitude sensitivity test) can be used instead of prcc [27] ; however, since all other parameters were monotonic, we use prcc and just consider the two monotonic ranges of μ i 2 separately. prcc analysis of these two ranges produces similar results. for an analysis of the monotonicity, refer to appendix 3. once meeting the monotonicity requirements, we proceed to utilize lhs with prcc for both mers and ebola parameters. for each disease, we calculate the prcc for the following output measures: total ns cases, total ss cases, total ns deaths, and total ss deaths. the number of total cases refers to the total number of transmission events where susceptible individuals become exposed (latently infected) individuals. for the outputs of ns/ss cases, the prcc results were similar in both ebola and mers. according to the prcc values, the β 2 and μ i 2 are significant in the model for mers. meanwhile, in the ebola model, both transmission parameters are significant in the model (see fig. 2 ). note that β 2 is calculated from r 0 , which we will vary later in simulations. for the ctmc model, we numerically simulate sample paths to compute the probability of an outbreak, number of deaths, time to outbreak, time to peak infection, and peak number of infectious individuals. for sample paths and probability of outbreak, we compare our results with the deterministic model. in the remainder of this analysis, we assume that the initial total population size is n(0) = 2000. reference to infected individuals will imply the variables i 1 and i 2 , unless stated otherwise. for example, peak number of infectious individuals refers to the maximum value of the number of total cases refers to the total number of transmission events where susceptible individuals become exposed (latently infected) individuals. p -values that are greater than 0.05 are labeled as not significant (n.s.) i 1 + i 2 and the time to peak infection refers to the time t at which this maximum occurs. however, an outbreak means that the total number in classes e i , a i , and i i for both ns and ss has reached at least 50, i.e., (e i + a i + i i ) ≥ 50. in addition, we note that for the ctmc model, an outcome measure (e.g., peak values, time to peak, and number of deaths) is defined by a corresponding probability distribution and a sample path yields one outcome from the distribution. an example of the sample paths resulting from our ctmc model is shown in fig. 3 , for both mers and ebola cases. these sample paths are generally well aligned with the population average response that is captured by our ode model (shown with a black line). however, the sample paths of the ctmc model illustrate the potential variability in timing of the peak level of infection and the peak number of infectious individuals. note that some sample paths are not shown because in those simulations the disease becomes extinct. also, note that the a class is not shown for ebola (fig. 3b) given that the asymptomatic stage is extremely short for this disease. next, in order to do a comprehensive comparison of the stochastic simulation and ode model results, we probe the relationship between two model parameters-the value of r 0 (fig. 4) as well as the fraction of the susceptible population that are in the ss class (fig. 4 ) and a key model output: the probability of outbreak. probability of outbreak is defined by monitoring the number of people in the e, a, and i classes and an outbreak is declared when the cumulative size of these compartments reaches the threshold value of 50. although the value of 50 appears relatively large, it is reasonable given that we are counting the cumulative number in all three classes for a relatively large population size of 2000. for these simulations, we vary β 2 given the significant effect of this parameter on the model outputs as confirmed by the lhs analysis. we note a negative correlation between the proportion of ss in the s class and the probability of outbreak (fig. 4) and attribute this to the fact that the value of β 2 is varied in order to maintain a constant value of r 0 (mers, r 0 = 2.5 and ebola, r 0 = 2.39). in other words, as the fraction of ss susceptible individuals is increased, the value of β 2 decreases and results in a reduction in the probability of outbreak (1 − q 6 ). results in fig. 4 are shown only for q 3 and q 6 since these outputs are similar to q 1 and q 4 , respectively. we also note that q 1 = q 2 and q 4 = q 5 and therefore exclude those plots as well. as expected, the probability of an outbreak is dependent on the initial fraction of the population that is infected, with an increasing chance of an outbreak (fig. 4) . furthermore, the probability of outbreak is significantly enhanced when the initially infected population is composed of ss rather than ns individuals. we also find a strong agreement between the probability of outbreak predicted by stochastic simulations of the ctmc model and the associated branching process approximations for all of these analyses (fig. 4a-f ). utilizing our stochastic model of mers and ebola dynamics within a population of individuals, we next sought to investigate whether the presence of ss individuals within the population could be reflected in key metrics that capture the severity of disease outbreak: the number of deaths, time to disease outbreak, probability of outbreak, time to peak number of infections, and the peak number of infectious individuals. we first assess the impact of ss individuals on the number of deaths that accumulate over a 150-day time frame following disease initiation. we observe a modest increase in the frequency of deaths as the size of the susceptible ss class of individuals is increased from 5 to 50% of the total population for both mers and ebola disease simulations (not shown). we note a higher frequency of epidemics for all subsequent simulations, we initialize the population consisting of 1000 ss and 1000 ns susceptible individuals. most notably, there is a ten-fold increase in the frequency of deaths expected when the initial infected individual (for both mers and ebola) is an ss rather than an ns (fig. 5) . the statistical significance of the difference between ns-and ss-initiated epidemics is confirmed with a kolmogorov-smirnov test (p < 0.001) [28] . it is clear that the distributions are bimodal. this is due to the fact that there may be only a minor outbreak (with probability q 3 or q 6 ) with none or a few deaths or a major outbreak (with probability 1 − q 3 or 1 − q 6 ) with a significant number of deaths. we next explore the relationship between the number of deaths and the number of initially infected individuals. for each fraction of the population initially infected, there are 1000 points, one point from each of the 1000 sample paths, representing the total number of deaths over a 150-day time period. as expected, we find that as the number of initially infected ns individuals increases, the expected number of deaths increases as well (fig. 6) . interestingly, we find a threshold response as the fraction of initially infected ss individuals increases. as the fraction of initially infected ss individuals increases beyond 0.005 for mers (fig. 6b) and 0.0075 for ebola (fig. 6d) , we find that the simulation always gives rise to an outbreak, resulting in a maximal number of around 1000 deaths over a 150-day simulated period. we also note that there is a decrease in the variability in the number of deaths when the outbreak is initiated by an ss rather than an ns, which contributes to this threshold response. the seemingly binary response in the number of deaths resulting from a mers or ebola epidemic initiated by infected ss individuals who only contribute to 0.5-0.75% of the starting population is a good indication that by tracking the number of deaths in an epidemic, the presence of an ss may be predicted. thus, while the observation that an outbreak has occurred does not necessarily suggest the existence of ss individuals in the population, the severity of the outbreak in terms of lives lost may be more suggestive of the presence of an ss, especially when the number of known initial infections is low. similarly, we find that the time to outbreak-where an outbreak is defined as 50 or more people in all of the e, a, and i classes-is reduced when the initial infected individual in a simulated mers or ebola disease situation is an ss rather than an ns (fig. 7a, c) . we confirmed that this reduction is, indeed, statistically significant ( fig. 8a-b) . these results also illustrate that as the fraction of susceptible ss increases the time to outbreak increases as well, which we attribute to the fact that β 2 values decrease (detailed in figs. 7 and 8) . in fig. 7 , each distribution is based on 10,000 sample paths, whereas in fig. 8 , for each fraction initially infected, the time points are based on 1000 sample paths. we also find a clear separation between the time to outbreak of an epidemic initiated by a fraction of ss versus ns infected individuals. mean differences were significantly distinct for each percentage in (fig. 8a-b) , p < 0.001. in fact, if a mers or ebola outbreak is initiated by 1.5% or more of the initial population size and these individuals are ss, then the time to outbreak is predicted to be no more than 20 days where an outbreak is defined as 2.5% of the population becoming infected (fig. 8c-f ). the fraction of susceptible ss is increased for comparison. all results were statistically significant when p < 0.05 from t-test (two-tailed). comparisons that are not statistically significant were denoted n.s. given that the time to outbreak shows a significant difference between epidemics initiated by ss versus ns individuals, we next asked whether ss-initiated epidemics will also reach peak infection in a shorter time. to investigate this, we calculated mean (± sd) of time to peak infection (in days) for mers and ebola, where the percent of ss varied in the susceptible population (see fig. 9a-b) . mean differences between the introduction of 1 infected ns (black) compared to 1 infected ss (white) were assessed separately as the percent of ss varied (e.g., 5%, 25%, and 50%) using t-tests where statistical significance was accepted when p < 0.05. for mers, time to peak infection was slightly significantly lower for ss when 5% and 25% of the susceptible population was ss, but not significant when 50% of the population was ss (fig. 9a) . for ebola, time to peak infection was significantly lower for ss regardless of changes in the percent of ss in the susceptible population (fig. 9b) . hence, while the differences in mean time to peak infection between ss and ns-initiated epidemics are only modestly different, we find their difference to be statistically significant (fig. 9a-b) . thus, this confirmed that epidemics initiated by infected ss individuals reaches its peak value more quickly. we repeated the same analysis to assess mean differences in the peak number of infections. surprisingly, we did not find a significant difference between the peak number of infections for epidemics initiated from a single infected ns versus ss individual for ebola (fig. 9d) . however, significant differences were observed when 5% or 50% of the susceptible population was ss for mers. in this investigation, we capture the dynamics of mers and ebola epidemics by applying both deterministic and stochastic modeling strategies. to investigate the role of ss on the epidemic dynamics and to compare our results, we keep the r 0 constant for both mers and ebola while varying β 2 , the transmission rate of ss. parameter sensitivity analysis, using latin hypercube sampling and partial rank correlation coefficient, shows that β 2 has a significant effect on all the output measures (fig. 2) . from fig. 4 , we can conclude that the stochastic model simulations agree with the branching process analytical results. as the value of r 0 increases, we observe that the probability of an outbreak increases for both diseases. this result is expected since more individuals in the population are infected. the probability of an outbreak is greater for ebola than mers, which is due to the transmission parameters for ebola being larger than mers. furthermore, these results show that if the outbreak is initiated by an ss, then the probability of an outbreak is significantly higher. additionally, fewer ss individuals than ns individuals are sufficient to cause an outbreak irrespective of the disease (mers or ebola). as an outbreak initiated by ss has a greater probability of occurrence and peaks earlier than with ns, the accumulated number of deaths is more severe in an epidemic initiated with the same proportion of ss than ns (figs. 5 and 6). disease severity (number of deaths) for both mers and ebola occurs earlier with ss than ns. our findings agree with prior epidemiological studies on superspreading events [15, 32, 40] . for example, the 2003 outbreak of the respiratory infection sars in beijing found that ss had higher mortality rates, higher attack rates, and greater number of contacts in comparison to ns [40] . from a public health perspective, as ss events will be observed more frequently, intervention/prevention methods must have rapid response to reduce disease severity. for example, wong et al. [40] suggested that several community-based efforts could have been made to reduce the number of mers and ebola cases in guinea and sierra leone, such as tracking contacts, earlier diagnosis, treatment strategies, and community education. effective responses to control superspreading events and reduce disease transmission in mers and ebola outbreaks included: "early discovery, diagnosis, intervention, and quarantine of confirmed cases." [40] . other epidemics that are more likely to occur in hospital settings, e.g., sars, could be controlled through hospital administrative strategies, such as reducing contact between the infected patient and healthcare workers, visitors, or other patients whose immune system may be comprised due to other infections [32] . thus, a rapid response is needed to reduce disease severity of ss events. evident in figs. 7 and 8 , when an outbreak is initiated by an ss rather than an ns, the time to outbreak is shorter and has less variability. therefore, if the number of disease cases rises rapidly, there may be ss in the community. in this scenario, healthcare managers should search for potential ss. similar results apply for time to peak infection, fig. 9 . if peak infection occurs quickly, it is more likely that there is an ss in the population. interestingly, varying the percentage of ss in the population has little influence on the peak number of infections (fig. 9c, d) . this is likely due to the fact that the r 0 values are held constant. we have formulated, analyzed, and numerically simulated deterministic and stochastic epidemic models that include heterogeneity in transmission for ns and ss. we applied our models to emerging and re-emerging infectious diseases, mers and ebola, where the models were parameterized with data from the literature but with a fixed initial population size of 2000. there are a number of extensions and generalizations that we will consider in the future work. we assumed homogeneous mixing and only two types of classifications of individuals (ns/ss) for the entire population. generalizing this model to include heterogeneous mixing and spatial components are key features that can provide insight on how a superspreaders can be classified. in our model, we considered inter-host variability, which naturally leads to constructing a model with intra-host variability utilizing stochastic differential equations or other types of models. in addition, variability of the pathogen on epidemic dynamics can be explored. additionally, we will validate our models' findings against time series data, test our models' abilities to detect the presence of ss, and interpret the results for public health implementation. finding answers to these problems will lead to our ultimate goal of constructing novel ways to quantify, characterize, and identify an ss during the initiation of an outbreak. women through research-focused networks" (nsf-hrd 1500481), society for mathematical biology, and microsoft research. we give special thanks to the wamb organizers: ami radunskaya, rebecca segal, and blerta shtylla. anarina l. murillo acknowledges that this work has been supported in part by the grant t32dk062710 from the national institute of diabetes and digestive and kidney diseases and grant t32hl072757 from the national heart, lung, and blood institute. omar saucedo acknowledges that this research has been supported in part by the mbi and the grant nsf-dms 1440386. nika shakiba is the recipient of the nserc vanier canada graduate scholarship. this work was partially supported by a grant from the simons foundation (#317047 to xueying wang). in addition, we thank texas tech university for hosting our second wamb group meeting and the paul whitfield horn professorship of linda js allen for providing financial support. we thank the two anonymous reviewers for their helpful suggestions on the original manuscript. in the calculations below, we denote n i (0) as n i and n(0) = n 1 (0) + n 2 (0) as n : and the next-generation matrix is for each pgf, we take partial derivatives with respect to x 1 , . . . , x 6 (jacobian matrix of the functions f 1 , . . . , f 6 ), then evaluate at x 1 , . . . , x 6 = 1 and take the transpose of the matrix. the result is the expectation matrix, m, where eq. (3) reduces to (4) . in addition, we create the matrix w , eq. (5), a diagonal matrix. and finally, we check that for the mers parameters, the graph of the output measure with μ i 2 parameter had a concave curvature implying that another sensitivity analysis maybe implemented. however, the range of the output measure is small enough that we can ignore the monotonicity. the remaining graphs for mers (fig. 10 ) and all the graphs for ebola (fig. 11 ) are all monotonic which means that we can trust the sensitivity analysis and proceed to the prcc analysis (fig. 12 , tables 4, 5 and 6). stochastic population and epidemic models extinction thresholds in deterministic and stochastic epidemic models relations between deterministic and stochastic thresholds for disease extinction in continuous-and discrete-time infectious disease models estimating the reproduction number of ebola virus (ebov) during the 2014 outbreak in west africa branching processes dynamically modeling sars and other newly emerging respiratory illnesses: past, present, and future mortality risk factors for middle east respiratory syndrome outbreak ebola virus disease) prevention ebola outbreak in west africa-case counts transmission dynamics and control of ebola virus disease (evd): a review synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission preliminary epidemiologic assessment of mers-cov outbreak in south korea in the garden of branching processes a pandemic risk assessment of middle east respiratory syndrome coronavirus in saudi arabia epidemiology: dimensions of superspreading assessing the international spreading risk associated with the 2014 west african ebola outbreak efficacy and effectiveness of an rvsvvectored vaccine expressing ebola surface glycoprotein: interim results from the guinea ring vaccination cluster-randomised trial an event-based model of superspreading in epidemics the daily computed weighted averaging basic reproduction number for mers-cov in south korea estimating the basic reproductive ratio for ebola outbreak in liberia and sierra leone a deterministic model for gonorrhea in a nonhomogeneous population spatial and temporal dynamics of superspreading events in the 2014-2015 west africa ebola epidemic a dynamic compartmental model for the middle east respiratory syndrome outbreak in the republic of korea: a retrospective analysis on control interventions and superspreading events statistical inference in a stochastic epidemic seir model with control intervention: ebola as a case study spatial heterogeneity in epidemic models superspreading and the effect of individual variation on disease emergence a methodology for performing global uncertainty and sensitivity analysis in systems biology the kolmogorov-smirnov test for goodness of fit comparison of three methods for selecting values of input variables in the analysis of output from a computer code compartmental disease models with heterogeneous populations: a survey modeling the impact of interventions on an epidemic of ebola in super-spreaders in infectious diseases reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission time from infection to disease and infectiousness for ebola virus disease, a systematic review the outcome of a stochastic epidemic-a note on bailey's paper who, final trial results confirm ebola vaccine provides high protection against disease ebola virus disease fact sheet east respiratory syndrome coronavirus (mers-cov) fact sheet the role of super-spreaders in infectious disease heterogeneities in the transmission of infectious agents: implications for the design of control programs key: cord-271504-t3y1w9ef authors: luo, zichao; ang, melgious jin yan; chan, siew yin; yi, zhigao; goh, yi yiing; yan, shuangqian; tao, jun; liu, kai; li, xiaosong; zhang, hongjie; huang, wei; liu, xiaogang title: combating the coronavirus pandemic: early detection, medical treatment, and a concerted effort by the global community date: 2020-06-16 journal: research (wash d c) doi: 10.34133/2020/6925296 sha: doc_id: 271504 cord_uid: t3y1w9ef the world health organization (who) has declared the outbreak of 2019 novel coronavirus, known as 2019-ncov, a pandemic, as the coronavirus has now infected over 2.6 million people globally and caused more than 185,000 fatalities as of april 23, 2020. coronavirus disease 2019 (covid-19) causes a respiratory illness with symptoms such as dry cough, fever, sudden loss of smell, and, in more severe cases, difficulty breathing. to date, there is no specific vaccine or treatment proven effective against this viral disease. early and accurate diagnosis of covid-19 is thus critical to curbing its spread and improving health outcomes. reverse transcription-polymerase chain reaction (rt-pcr) is commonly used to detect the presence of covid-19. other techniques, such as recombinase polymerase amplification (rpa), loop-mediated isothermal amplification (lamp), clustered regularly interspaced short palindromic repeats (crispr), and microfluidics, have allowed better disease diagnosis. here, as part of the effort to expand screening capacity, we review advances and challenges in the rapid detection of covid-19 by targeting nucleic acids, antigens, or antibodies. we also summarize potential treatments and vaccines against covid-19 and discuss ongoing clinical trials of interventions to reduce viral progression. the recent global outbreak of covid-19 has led to a public health emergency. as of april 23, 2020, over 2.6 million confirmed cases were reported to who from 213 countries and territories [1] . on january 30, 2020, who declared the covid-19 outbreak as the sixth public health emergency of international concern, following h1n1 (2009), polio (2014), ebola in west africa (2014), zika (2016), and ebola (2019) [2] . the rapid global expansion and rising fatalities have raised grave concerns on the viral spread across the globe. with the rapid increase in the number of confirmed cases, who classified the global covid-19 outbreak as a pandemic on march 11, 2020 [3] . covid-19 can spread from person-to-person and animal, and transmission of infection may occur with exposure to symptomatic patients or asymptomatic individuals. coronaviruses (covs) (corona: crown-like shape) are enveloped, single-stranded rna viruses that belong to the order nidovirales in the subfamily coronaviridae. covs are divided into four genera: alpha (α), beta (β), gamma (γ), and delta (δ) (figure 1(a) ) [4] . alpha-and beta-covs infect mammals, while gamma-and delta-covs primarily infect birds [5] . before december 2019, six types of covs had infected humans, including two α-covs (hcov-229e and hcov-nl63) and four β-covs (hcov-oc43, hcov-hku1, sars-cov, and mers-cov). the first two β-covs (hcov-oc43 and hcov-hku1) mainly cause self-limiting upper respiratory infections, while the other two β-covs (sars-cov and mers-cov) are mostly associated with severe respiratory illness [6, 7] . full-genome sequence analysis of 2019-ncov confirms that it is a β-cov, distinct from sars-cov and mers-cov [8] . investigations reveal that 2019-ncov shares~80% sequence identity with sars-cov while maintaining~89% nucleotide identity to the sars-like covs (zc45 and zxc21) from bats [9] . a recent report suggests that a bat cov (ratg13) is 96% identical to 2019-ncov [10] . a typical cov genome is a single-stranded, positivesense rna (+ssrna) (~30 kb) enclosed by a 5′-cap and 3′ -poly-a tail [11] . the genome size of 2019-ncov is 29,891 nucleotides, encoding 9860 amino acids, with a g+c content of 38% [12] . the 2019-ncov genome contains two flanking untranslated regions (utrs) on 5 ′ -and 3 ′ -terminals, one single long open reading frame 1ab (orf1ab) encoding a polyprotein and at least five other orfs encoding structural proteins, and eight accessory proteins (figure 1(c) ). the first orf (orf1a/b) is about two-thirds of the whole-genome length and encodes the 16 nonstructural proteins (nsp116) . the other one-third of the genome contains four orfs encoding the spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, whereas other orfs encode accessory proteins (figure 1(b) and (c)). most of the nonstructural proteins are essential for 2019-ncov replication, while structural proteins are responsible for virion assembly and viral infection [12, 13] . the m and e proteins are required in viral assembly, while the n protein involves rna genome assembly. the s protein, a surface-located trimeric glycoprotein of covs, is the primary determinant of cov tropism, as it binds to the membrane receptor on host cells, mediating viral and cellular membrane fusion [14] . the s protein of 2019-ncov reportedly binds to angiotensin-converting enzyme 2 (ace2), a homolog of ace on host cell membranes, contributing to 2019-ncov cell invasion [15] . moreover, this particular s protein shows a higher binding affinity to ace2 than the s protein of sars-cov, enabling 2019-ncov to invade host cells more effectively [16, 17] . recently, a transmembrane glycoprotein, cd147, also known as basigin or emmprin, has been confirmed as another receptor for binding of the 2019-ncov s protein, thereby mediating viral invasion [18] . the e protein is an integral membrane protein that regulates viral life cycles, including pathogenesis, envelope formation, assembly, and budding [19] [20] [21] . among the four structural proteins, protein e appears to have the highest antigenicity and the most significant potential as an immunogenic target, highlighting the possibility of developing protein e-derived peptides as a 2019-ncov vaccine [22] . systemic studies of proteins s and e have inspired scientists to take creative approaches to design anti-covid-19 drugs. although some covid-19 patients show no symptoms, most patients have some common symptoms such as fever, cough, fatigue, sputum production, shortness of breath, sore throat, and headache. in some severe cases, infections can cause pneumonia, severe acute respiratory syndrome, kidney failure, and death. according to the who-china joint report [23] , on average, people infected with 2019-ncov develop mild respiratory symptoms and fever, 5-6 days after infection (mean incubation period, 5-6 days; range, 1-14 days). people over 60 years of age and those with hypertension, diabetes, or cardiovascular diseases are at high risk for severe illness and death. in comparison, children under 19 years appear to be infected minimally by 2019-ncov (around 2.4% of all reported cases). based on the chinese center for disease control and prevention (china cdc) report (from 72,314 patient records, dated 11 february 2020), among the confirmed cases, 86 .6% of patients are 30-79 years of age, 80.9% of patients have mild-to-moderate disease, 13.8% have a severe illness, and 6.1% are critically ill [24] . notably, the mortality rate of children under 19 years is 0.2%, while people aged over 80 years have the highest mortality rate of 14.8%. currently, there are no effective antiviral drugs or specific vaccines against covid-19. thus, there is an urgent need for rapid detection to prevent further spread, to reduce the intensity of the pandemic, and to slow the increase in cases. recently, several new technologies, including lamp-lfa, rpa-lfa, rpa-crispr, and other nanomaterial-based igg/igm kits, have been adopted for 2019-ncov detection. a significant number of drug candidates, including chemical drugs, biological drugs, nutritional interventions, and traditional chinese medicine (tcm), have been proposed for clinical trials after the 2019-ncov outbreak. in this review, we concentrate on the most significant developments in 2019-ncov detection and provide an overview of medical treatments and vaccines currently in development to combat and contain the disease. properties of the virus and biomarkers that hosts exhibit after infection. these biomarkers include viral proteins and nucleic acids, as well as antibodies induced in response to viral infection. the most common 2019-ncov detection methods include viral nucleic acid detection and serum antibody (igg or igm) detection. a confirmed case should have at least one of the following criteria: (i) a positive result for 2019-ncov nucleic acid, using real-time pcr tests from respiratory or blood samples; (ii) a high homogeneity between viral gene sequencing from respiratory or blood samples and known 2019-ncov; and (iii) serum samples positive for igm or igg to 2019-ncov, or seroconversion in igg, or a fourfold or more significant increase in igg antibody titer to 2019-ncov in the recovery phase than in the acute phase [25] . 2.1.1. high-throughput sequencing (2 nd -generation sequencing). high-throughput sequencing (hts) technology contains various strategies that depend on a combination of library preparation, sequencing and mapping, genome alignment, and data analysis [26] (figure 2(a) ). unlike the 1977 sanger sequencing method (1 st -generation sequencing) [27] , 2 nd -generation sequencing has been widely applied in genome sequencing, transcriptional profiling (rna-seq) disease mapping, and population genetic studies. the wholegenome nucleotide sequence of 2019-ncov was identified and compared with the full-length genome sequence of coronavirus from bats [10] through hts. hts-based technology is also applied to detect 2019-ncov. for example, wang et al. developed a hts method based on nanopore target sequencing (nts) by harnessing the benefits of target amplification and long-reads for real-time nanopore sequencing [28] . this nts strategy detects 2019-ncov with higher sensitivity (100-fold) than standard qpcr, simultaneously with other respiratory viruses within 6-10 h. moreover, all targeted regions can be identified by nts in higher copies of samples (1000-3000 copies/ml) within 10 min, indicating the potential for rapid detection of an outbreak in the clinic. for 1 h sequencing data, reads mapped to 2019-ncov differed remarkably from those of negative controls in all targeted regions at concentrations ranging from 10 to 3000 copies/ml. importantly, nts can identify mutated nucleic acids. however, the nts platform cannot readily detect highly degraded nucleic acid fragments that are less than 200 base pairs in length [29] . moreover, the strategy requires tedious sample preparation and lengthy turnaround time. although hts technology provides fast, low-cost dna sequencing, it is not suitable for detection in clinics. on the other hand, the hts strategy may be suitable for amplicon sequencing or de novo sequencing of a whole genome [30] . reaction (rt-pcr). rt-pcr is considered the gold standard to detect nucleic acids extracted from 2019-ncov specimens qualitatively. positive results indicate infection with 2019-ncov. rt-pcr is an advanced technique for coronavirus detection because of its optimized sensitivity, specificity, and simplicity for quantitative assay [31, 32] . it provides accurate and reliable identification for confirmed and suspected cases. there are many commercial 2019-ncov detecting kits with oligonucleotide primers and probes (sybr green or taqman chemistries) for detecting double genes of 2019-ncov (nucleocapsid n gene and orf1ab/e/orf1b/s gene). this strategy usually requires four steps: sample collection (respiratory swabs), sample preparation (rna isolation), one-step qrt-pcr, and data analysis ( figure 2 (b)) [33] . real-time rt-pcr has been adopted as the gold standard diagnostic approach for 2019-ncov worldwide. however, rt-pcr is time-consuming (4-6 h) and requires wellequipped laboratories and skilled technicians, thereby limiting full deployment in developing countries. 2.1.3. reverse transcription-isothermal amplification (rt-iamp)-based detection. isothermal amplification technology has been developed to eliminate the need for a high-precision instrument in rt-pcr assays. this approach can amplify dna at isothermal conditions without a thermocycler [35] . there are mainly four isothermal amplification technologies for nucleic acid detection: lamp, rpa, nucleic acid sequence-based amplification (nasba), and transcriptionmediated amplification (tma) [36] . in nasba and tma assays, input rna is converted to a double-stranded dna intermediate with a promoter region. detection of rna using dna polymerase-based amplification requires a reverse transcriptase step. lamp and rpa do not require thermal or chemical melting with the aid of enzymes. combined with a visual detection platform, such as a lateral flow assay (lfa) or organic dyes, lamp and rpa have been widely employed in viral detection kits. lamp is a rapid, one-step amplification technique that amplifies nucleic acids with high sensitivity and specificity at an optimal temperature of 65°c [37] . lamp processing comprises three steps: an initial step, a cycling amplification step, and an elongation step (figure 3(a) ). lamp employs six primers to amplify targeted genes by creating stem-loop structures that promote new dna synthesis using a dna polymerase with strand displacement activity. the two inner primers (fip, bip) and two outer primers (f3, b3), along with loop structures (lf, lb), create multiple initiating sites in the growing dna products, enabling rapid amplification. lamp is also highly specific since the amplification reaction occurs only when the primers correctly recognize all six regions. a reverse transcriptase step is included in the lamp reaction to allow rna targets to be detected [38] . rt-lamp offers improved sensitivity and specificity in screening sars-cov, hcov-nl63, and mers-cov compared to conventional real-time rt-pcr [39] [40] [41] . recently, yu et al. used a commercial lamp kit to amplify fragmented orf1ab genes of 2019-ncov (figure 3 (b) and (c)) [42] . they optimized the lamp system through incubation at 65°c for different periods using a 2019-ncov-positive rna sample as the template. results require a 15 min reaction time at 65°c, and detection sensitivity is comparable to that of the taqman-based qpcr approach (10 copies). rt-lamp employs two additional protocols for 2019-ncov rna detection. park et al. performed rt-lamp at 65°c for 40 min to identify the nsp3, s, and n genes of 2019-ncov using colorimetric detection [43] . the sensitivity of this rt-lamp assay was 100 copies of 2019-ncov rna. the other rt-lamp protocol was conducted at 63°c for 30 min to detect the orf1ab, e, and n genes simultaneously [44] . the results confirmed the specific nature of orf1ab and the high sensitivity of the n gene. based on an analysis of 208 clinical specimens, the sensitivity of this rt-lamp was similar to conventional rt-pcr, and the specificity was 99%. interestingly, ei-tholoth et al. designed a two-stage isothermal amplification procedure by combining rpa (37°c) with lamp (63°c) to detect synthesized dna fragments of 2019-ncov [45] . the test was performed in closed tubes within 1 h using either fluorescence or colorimetric detection. this method has a sensitivity of 100 times better than conventional lamp and rt-pcr, suggesting a rapid, sensitive, point-of-care test for use at home. rpa is an isothermal dna amplification method that utilizes a specific combination of enzymes and proteins (recombinase, single-strand binding (ssb) protein, and strand-displacing dna polymerase) to amplify target genes step 2: cycling amplification step 3: elongation step 1: initiation step 2: ssb binds to displaced strand step 3: bsu initiates polymerization step 4: parental strand is displaced and elongation continues step 1: recombinase binds primers step 2: ssb ds to displaced strand step 3: bsu tiates polymerization step 4: tal strand is displaced elongation continues step 1: recombinase binds primers rapidly at a constant low temperature between 25 and 42°c in as little as 15 min [46] . rpa usually requires four steps to achieve dna amplification: formation of a recombinaseprimer complex, strand invasion, d-loop formation (stabilized by ssb, dna polymerization through the use of strand-displacing dna polymerase), and dna amplification ( figure 3 (d)) [47] . results of rpa can be detected by agarose gel electrophoresis, quantitatively measured using twis-tamp ™ probes, or simply applied in lateral flow assays. apart from dna target amplification, rpa formats have been developed for the detection of rna targets (rt-rpa) by adding a reverse transcriptase enzyme to reaction mixtures [48] . because rpa-(rt-rpa-) based detection achieves more rapid and sensitive results and operates efficiently, it has been widely adopted to detect animal and human pathogens, such as hand, foot, and mouth disease (hfmd) virus, human immunodeficiency virus (hiv), bovine coronavirus, or mers-cov [49] [50] [51] [52] . currently, rpa has been applied to detect 2019-ncov, in combination with other technologies, such as crispr or microfluidic technology. repeat-(crispr-) based detection. the crispr-associated protein 9 (cas9) system (crispr/cas9) is a revolutionary gene-editing toolbox that can modify target genes with high precision and that can control various types of genetic diseases in preclinical studies [53] [54] [55] [56] . due to the collateral nucleic acid cleavage activity of cas effectors, crispr/cas systems have also been widely used in nucleic acid detection with fluorescent and colorimetric signals [56] . there are mainly two kinds of crispr/cas systems for diagnostics, based on the cutting activity of cas protein on nucleic acids outside of the grna target site: the crispr/cas13a and crispr/cas12a systems. the crispr/cas13a system (specific high-sensitivity enzymatic reporter unlocking (sherlock)) was developed by zhang's group, based on the collateral effect of an rnaguided and rna-targeting crispr effector, cas13a (figure 4 (a)) [57, 58] . the detection system is highly sensitive and specific because it is capable of single-molecule nucleic acid detection. subsequently, they developed an enhanced sherlock version 2 (sherlockv2) detection system with a 3.5-fold improvement in detection sensitivity and lateral flow readout. sherlockv2 has been used to detect dengue and zika virus single-stranded rna or mutations in clinical samples, showing great potential for multiplexable, portable, rapid detection of nucleic acids [59] . recently, they combined rt-rpa technology with the sherlock system (namely crispr diagnostics) to detect the s and orf1ab genes of 2019-ncov (figure 4(b) and (c)) [ diagnostics-based test can be conducted in 1 hour and can be read using a dipstick. the analysis is performed at 37°c and 42°c, and its detection sensitivity is ten copies per microliter of input, exhibiting unique advantages, such as high sensitivity, specificity, speed, and suitability for point-of-care testing. however, this approach needs to be validated using real patient samples. unlike the crispr/cas13a system, the crispr/cas12a system is based on the collateral effect of cas12a on singlestranded dna (ssdna). chen and colleagues combined cas12a ssdnase activation with rpa technology to create a new approach, named dna endonuclease-targeted crispr trans reporter (detectr), with attomolar sensitivity for dna detection (figure 4 (a)) [61] . detectr was also validated with clinical samples, showing the capacity for rapid, specific detection of human papillomavirus (hpv) [61] . recently, detectr was investigated to identify the nucleic acid of 2019-ncov. lucia et al. applied the detectr (crispr-cas12a and rt-rpa) to detect the rnadependent rna polymerase (rdrp), orf1b, and orf1ab genes of 2019-ncov using synthetic rna fragments as samples [62] . remarkably, all steps of the test were completed in 1 h, and results were visible to the unaided eye. the limit of detection for orf1ab was ten copies/μl. the advantages of this method are its portability and low cost (~us$2 per reaction). but this proposed approach also needs to be validated with clinical samples before commercialization. another detectr-based 2019-ncov detection strategy was developed by chiu's lab [63] . they employed lamp, crispr/-cas12, and lateral flow assay to detect the e and n genes of 2019-ncov in clinical samples. this protocol supplied rapid (~30 min), low-cost, and accurate (100% specific vs. 90% specific for qrt-pcr) detection of 2019-ncov in respiratory swab samples. realistically, crispr/cas-based 2019-ncov detection technology is highly specific, rapid, and low cost, but the detection strategy also needs to be validated using clinical samples. microfluidic-based detection. the abovementioned methods are based on relative quantification, because they require external calibration with genetic standards or inner reference dna templates, resulting in unavoidable errors and other uncertainties. on the contrary, methods that do not need standard curves can provide a quantitative analysis of nucleic acids using absolute quantification of genetic copies. recently, digital pcr and digital lamp have been achieved with microelectromechanical and microfluidic technologies [64, 65] . microfluidic or lab-on-a-chip techniques use microsized channels to process or manipulate fluids. microfluidics has been widely utilized in various fields, including drug screening, tissue engineering, disease diagnostics, and nucleic acid detection [66, 67] . based on its portability and ultralow sample consumption, microfluidics shows significant promises in clinical applications [68] . regarding nucleic acid analyses, microfluidic devices aliquot diluted nucleic acid samples into hundreds to millions of discrete nanoliter chambers. the isolated chambers contain only one or zero target molecule according to a poisson distribution. consequently, the abso-lute copy number of target nucleic acid can be calculated from the number of positive and negative reactions, based on the poisson distribution formulas [69, 70] . both digital pcr and digital lamp have employed microfluidics for pathogen analysis, which is suitable for covid-19 detection. for instance, ottesen and colleagues used digital pcr to amplify and analyze multiple genes on a microfluidic chip [71] . this chip consisted of parallel chambers and micromechanical valves. the micromechanical valves segmented chambers into independent pcr reactors after the sample flowed into chambers through connection channels. the chip was able to detect several kinds of genes with parallel sample panels. digital pcr can also be conducted with droplets generated by the microfluidic chip. however, the detection of fluorescent signals in droplets requires special instruments, such as flow cytometers, which may limit its application in point-of-care testing. additionally, the high temperature in pcr amplification tends to evaporate the reaction liquid (nanoliter or even femtoliter), leading to unacceptable errors. using airtight devices or high pressure delays liquid evaporation but complicates the devices and increases testing costs. digital lamp is more compatible with microfluidics than digital pcr because it is executed at a moderate temperature. this simplifies microfluidic devices and reduces testing costs. many microfluidic devices have been reported for nucleic acid detection using digital lamp, such as self-digitization chips, self-priming compartmentalization chips, and dropletgeneration chips [72] [73] [74] . as an example, xia et al. designed a mathematical model using the monte carlo method according to the theories of poisson statistics and chemometrics [70] . the mathematical model illustrated influential factors of the digital lamp assay, guiding the design and analysis of digital lamp devices. based on the established mathematical model, they fabricated a spiral chip with 1200 chambers (9.6 nl) for pathogen detection ( figure 5 (a)-(c)). this spiral chip operated at 65°c without visible liquid evaporation and achieved a quantitative analysis of nucleic acids over four orders of magnitude in concentration with a detection limit of 87 copies per ml. this portable gadget shows significant promise in future point-of-care testing. microfluidics, combined with enzyme-dna nanostructures, is also applied to detecting 2019-ncov. ho et al. developed a modular detection platform (termed envision) consisting of an integrated circuit of enzyme-dna nanostructures for direct and versatile detection of pathogen nucleic acids from infected cells [75] . built-in enzymatic cascades in the envision microfluidic system supply a rapid color readout for detecting hpv. the assay is fast (<2 h), sensitive (limit of detection < 10 attmol), and readily quantified with smartphones. recently, they adopted the envision microfluidic system to detect 2019-ncov [76] . preliminary results showed that the envision platform is sensitive, accurate, fast (within 0.5-1 h), and inexpensive (less than $1 per test kit). this novel platform works at room temperature and does not require a heater or special pumps, and it uses a minimal amount of samples, making it highly portable. however, this platform needs to be further validated with real clinical samples. antigen and antibody. as mentioned above, the primary diagnostic methods are virological detection involving viral nucleic acids. another approach to detection is with serological assays that measure antigens or antibodies present in the host. such testing provides vital information about host exposure to 2019-ncov and is useful for detection and surveillance purposes. for instance, this method greatly helps medical professionals to determine whether some recovered patients have a higher risk of reinfection. however, the disadvantage is that one should be cautioned that in the early stages of covid-19 infection, the host's antibodies are often not within the detectable range of serological test kits. besides, there was no proven evidence on the duration of igm or igg antibodies circulating in the host after recovery. it could be merely a short time frame for detection. as such, serological tests should not be solely used for covid-19 diagnosis. early diagnosis of 2019-ncov infection is of utmost importance both for medical teams to manage patients effectively and for policymakers to curb the viral spread. presently, elisa in cell culture extracts has proven to be the working "gold standard" for laboratory diagnosis of 2019-ncov [77] . elisa is a plate-based assessment method for detecting and quantifying biomolecules, including peptides, proteins, antibodies, and hormones. elisa techniques depend on specific antibodies to bind target antigens and a detection system to indicate the presence of antigen binding. in an elisa, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. detection is accomplished by assessing the conjugated enzyme activity after incubation with a substrate to produce a measurable product [78] . recently, coronavirus proteins have been widely used in elisa to diagnose sars-cov or other viruses within the coronavirus family [79] . in a bold, novel approach, a team of infectious disease experts in singapore utilized an elisa against 2019-ncov to ascertain that suspected subjects were infected with covid-19 and discovered the connection between two covid-19 clusters in the local community [80] . using blood samples taken from alleged covid-19 patients, the researchers detected antibodies targeting the spike protein that prevented the virus from killing cells in laboratory tests. they verified that a couple allegedly infected with covid-19 had the disease because they had exceedingly elevated levels of virus-specific antibodies in their blood. interestingly, pcr tests on the couple yielded negative results. because the couple had recovered from the 2019-ncov infection, they had no viral genetic materials in their bodies, but the antibodies persisted. there were also other reports of using elisa to diagnose 2019-ncov infection [81, 82] . each study confirmed the high reproducibility and specificity of elisa in diagnosing covid-19 patients accurately in clinics. research has established that the presence of immunoglobulin m (igm) indicates a primary defense against viral infections. this igm defense occurs before the production of high-affinity and adaptive immunoglobulin g (igg) that is critical for prolonged immunity and immunological memory [83] . from a previous study on sars infections, both igm and igg antibodies could be detected in the patient blood after 3-6 days and beyond 8 days, respectively [84] . given that 2019-ncov belongs to the same family of coronaviruses including mers and sars, 2019-ncov should also generate igm and igg antibodies in infected humans. therefore, the detection of igm and igg antibodies may provide epidemiologists with crucial information on viral infection of test subjects, allowing them to adjust policies to combat the pandemic more effectively. point-of-care lateral flow immunoassays are performed qualitatively to quickly determine the presence of 2019-ncov by detecting anti-2019-ncov igm and anti-2019-ncov igg antibodies in human plasma, serum, or whole blood. a typical device is shown in figure 6 (a). reddishpurple lines in the readout indicate the presence of 2019-ncov igm and igg antibodies in the sample. lfa is based on the lateral chromatographic flow of reagents that bind and interact with the sample. as the sample flows through the test device, starting at the sample pad region, the anti-2019-ncov igm and igg antibodies, if present, bind tightly to 2019-ncov antigen-labeled gold nanoparticles, located on the conjugated pad. when conjugated products in the sample continue to move up the strip, anti-2019-ncov igm antibodies and anti-2019-ncov igg antibodies bind to anti-human igm (m line) or anti-human igg (g line), respectively. no visible lines can be seen when the specimen does not contain anti-2019-ncov antibodies because no labeled complexes bind at the test zone. igg-labeled gold colorimetric nanoparticles serve as the control when they bind to anti-rabbit igg antibodies at the control line (c) (figure 6(b) ). lfa has proven useful in detecting 2019-nco-vigm/igg antibodies in clinical studies, demonstrating 88.66% test sensitivity and 90.63% specificity in human blood, serum, and plasma samples. results from six patients are shown in figure 6 (c). common 2019-ncov detection methods are summarized in table 1 . in clinics, medical imaging tools are indispensable and form an essential component of viral diagnosis, as well as for monitoring viral progression [85] . they have also been used for follow-up in outpatient settings for coronavirus-related pulmonary disorders. just like both sars and mers, pulmonary complications in covid-19 patients have been observed. learning from the well-documented sars and mers studies, ct imaging results in the acute and chronic periods of covid-19 are invariant, but not always present [86] [87] [88] [89] . evidence is found in previous studies on sars and mers. the glass opacities observed are not always found in covid-19 patients. crucially, preliminary imaging discoveries indicate that covid-19 yields nonspecific results as well [90] [91] [92] . radiologists are presently striving to any characteristics specific to covid-19, although present medical information remains limited. given the precarious situation, there is a pressing need for alternative, complementary diagnostics. ct is one example. covid-19 patients often develop "ground glass" lung opacities [93] . as such, a ct imaging scan can readily identify lung abnormalities in human subjects, thereby enabling early treatment against covid-19. ct has demonstrated some common imaging characteristics in covid-19 patients. these features include bilateral, multifocal, ground glass opacities, with a peripheral distribution (figure 7 (a)) [93] . crucially, more than half of 90 patients under study presented multilobar involvement and lesions more prominently in the lower lobes of their lungs. given its feasibility and ease of use, ct has become an essential tool for the 2019-ncov infection diagnosis. from a radiological perspective, the advantages of using ct imaging could expedite the rate of diagnosis. it also supports the current shortage and heavily reliant on technical knowhow during rt-pcr testings. nonetheless, one limitation is that it should be cautiously utilized as a diagnostic approach because there are no proven, evidence-based clinical benefits of using ct. it could also cause false securities if results are negative. other limitations include requirements of relatively high-dose ct scans and long-term, continuous usage, which can altogether be logistically challenging and deplete additional medical resources. transmission electron microscopy (tem) has been used for many years and has had a profound impact on our understanding of illnesses, including viral infections. the thousandfold enhanced resolution provided by tem enables investigators to visualize viral morphology and to classify viruses into families [94] . mechanistically, tem operates based on interactions between electrons emitted from a source and materials under examination. in the present context, it is usually 2019-ncov in a cellular sample [95] . the detector collects a multitude of signals from transmitted electrons, before processing them to reveal viral morphology and location within cells [96] . typical specimen preparation for tem includes sample fixation, embedding, sectioning, staining, and loading onto the tem copper grids [94, 97, 98] . 2019-ncov sampling typically uses supernatants from patient airway epithelial cells. infected cells are fixed and dehydrated before embedding in resin. a negatively stained, film-coated grid for examination is similarly prepared for contrast enhancement. 2019-ncov virus particles seen with tem are shown [97] (figure 7(b) ). tem enables microbiologists to rapidly diagnose patients with a single examination of a single tissue sample. there are three general approaches to develop potential antiviral treatments of the human coronavirus. firstly, standard assays may be used to evaluate existing broadspectrum antiviral drugs. secondly, chemical libraries containing existing compounds or databases may be screened. thirdly, specific, new medications based on the genome and biophysical understanding of 2019-ncov can be designed and optimized. therefore, this section will discuss some of the potential 2019-ncov therapeutics obtained through these general approaches. besides chemical and biologic drugs commonly used in antiviral therapies, we further elaborate on how nanomaterials, nutritional interventions, traditional chinese medicine, and stem cell therapy can be potentially used for treatment or as an adjuvant to reduce the mortality and morbidity rate of 2019-ncov patients. finally, to end this section, we highlight vaccines as a key therapeutic option to eradicate covid-19 through herd immunity without getting the disease. 3.1. chemical drugs. there are currently no approved antiviral drugs to treat covid-19, and patients must depend upon their immune systems to combat the infection. a full-fledged treatment plan has yet to emerge, and both academia and pharmaceutical companies are racing to develop new treatments and vaccines to address covid-19. research into the cellular and molecular pathogenesis of 2019-ncov has provided essential insights with the hope of developing viable therapies. while researchers are working on cures or preventive measures for covid-19 [99] , a more robust, efficient, and economical way to tackle the disease is to repurpose existing drugs into a viable therapeutic strategy. drug repurposing, also termed drug repositioning, refers to the process of discovering new therapeutic applications for existing drugs. it offers various advantages over traditional de novo drug discovery, i.e., reduced cost and drug development time, established drug characteristics, and, most importantly, established safe dosages for human use [100] . repurposed drugs often negate the need for phase 1 clinical trials and can be used immediately [100, 101] . at present, repurposed drugs are the only option available at treatment centers for covid-19 patients. as covid-19 is a viral infection, the most obvious choices for repurposed drugs come from known antiviral drugs [102] . antiviral creation strategies focus on two approaches: targeting viruses or targeting host cell factors. in this section, we will review antiviral drugs prescribed or proposed against covid-19 based on the antiviral drug creation strategies. when an infection occurs, the virus gains entry into a host cell by attaching itself to the host cell surface (figure 8 ) [103] . this relies on numerous interactions between the virion surface and the specific proteins on the cell membrane. in general, these surface proteins have other functions but are serendipitously recognized by the virus as entry receptors [103] . molecules that prevent such recognition, either by competitive binding or by downregulating the receptors, are known as viral entry inhibitors (figure 9(a) ). these inhibitors are valuable as therapeutics since blocking infection early in the life cycle reduces cellular and tissue damage associated with viral replication and production of viral progeny. as mentioned above, coronavirus particles comprise four structural proteins: the s, e, m, and n proteins [104] [105] [106] [107] . the s protein is the most crucial in viral attachment, fusion, and entry [108] . it comprises two subunits. s1 facilitates attachment to the host cell receptor, while s2 mediates membrane fusion of the virion and the host cell. as mentioned above, viruses have specific attachment sites. sars-cov recognizes ace2 as its host receptor, while mers-cov recognizes dipeptidyl peptidase 4 [109, 110] . like sars-cov, 2019-ncov also targets host ace2 [111] [112] [113] . biophysical and structural analysis indicates that the 2019-ncov s protein binds ace2 with higher affinity than the sars-cov s protein [16] . therefore, it is vital to target ace2 for the development of viral entry inhibitors. to the best of our knowledge, not much is known about ace2-specific inhibitors that are commercially available or under commercial development [114] . however, ace2 stimulators have been used in the treatment of hypertension, cardiac diseases, and diabetes mellitus to regulate the reninangiotensin system [115, 116] . there are also ace inhibitors known for treating the diseases mentioned, but these lack inhibitory activity toward ace2 due to their distinct substrate-binding pockets [116] [117] [118] [119] . in brief, there are concerns that both ace2 stimulators and ace inhibitors can increase the expression of ace2, which in turn may increase susceptibility to viral host cell entry [120, 121] . much work needs to be done on ace2-targeting drugs, and controversial issues that lie beyond the treatment pathway need to be addressed soon. a small antiviral molecule, umifenovir, has entry inhibitory effects on the influenza virus. umifenovir targets hemagglutinin for its anti-influenza virus effect [122] [123] [124] . hemagglutinin, a viral cell surface protein, facilitates infection by undergoing a conformational change when the virus binds to host cells [122] . umifenovir interacts with hemagglutinin to stabilize it against low ph-induced conformational change via the formation of an extensive network of noncovalent interactions that prevent hemagglutininmediated membrane fusion [122, 124] . it also interacts with phospholipids by altering membrane fluidity [125] , which is vital for the fusion process. this is most likely due to umifenovir's molecular interactions (bearing both the h donor and acceptor groups) with the interfacial region of the lipid bilayer by competing for the hydrogen bonding of phospholipid c=o groups with water molecules [126] . this renders lipid bilayers of host cells less prone to viral fusion [125] . no studies have shown that umifenovir is effective in inhibiting sars-cov or 2019-ncov. wang et al. reported that 4 patients with mild/severe covid-19 recovered after prescription of combined lopinavir/ritonavir, arbidol (umifenovir), and shufeng jiedu capsule (a traditional chinese medicine) [127] . on the other hand, dong et al. found in an in vitro study that arbidol may effectively inhibit 2019-ncov infection at a concentration of 10-30 μm [128] . chloroquine, also a small molecule, is a quinine analog used to prevent and treat malaria. similar to umifenovir, chloroquine exhibits its inhibitory effect on influenza by ph stabilization. chloroquine is a weak base and becomes protonated intracellularly in a manner described by the henderson-hasselbalch law [129] . it can raise lysosomal ph to facilitate autophagy intracellularly [130] [131] [132] . chloroquine also alters the signaling pathway of enzymes, causing enzyme glycosylation, ultimately inhibiting viral replication in host cells [133, 134] . liu et al. claimed that chloroquine could inhibit sars-cov entry by changing glycosylation of the ace2 receptor and s protein [135] . chloroquine's effective inhibition of sars-cov was demonstrated in vitro on primate cells and human rectal cells [136, 137] . hydroxychloroquine is a derivative of chloroquine with an additional hydroxyl group. these two chloroquines share similar struc-tures and mechanisms. both have shown in vitro antiviral activities toward 2019-ncov [138] [139] [140] . hydroxychloroquine was more effective than chloroquine in inhibiting 2019-ncov in vitro on primate cells [141] . until now, chloroquine has shown apparent efficiency and safety against 2019-ncov in clinical trials conducted in china [139] . currently, chloroquine or hydroxychloroquine has been administered to hospitalized 2019-ncov patients on an uncontrolled basis in various countries, including china and the usa [42] . however, it must be noted that chloroquine and hydroxychloroquine cause ocular toxicity [142] . hydroxychloroquine is reportedly less toxic than chloroquine, making it more attractive as a prescription drug [135, 143] . nevertheless, more investigation and clinical trials are needed to evaluate further their efficacy and safety in treating 2019-ncov. proteases are essential enzymes that regulate cell life processes such as cell growth and death, blood clotting, inflammation, fertilization, and infection [103] . viral entry into host cells requires s protein priming by host proteases, which subsequently enables the fusion of viral and cellular membranes [113] . membrane fusion enables the release of the viral genome into the host cytoplasm, initiating rna translation into protein. most viruses also encode their proteases to protect viral proteins by modulating host cell responses. while proteases are vital for cell life processes, they have become promising targets for antiviral therapeutic agents. protease inhibitors prevent viral replication by binding selectively to viral proteases or blocking proteolytic cleavage of protein precursors necessary for the production of infectious particles [144] . it is noteworthy that protease inhibitors were a major therapeutic breakthrough of antiviral drug design in the mid-1990s for the treatment of hiv. most hiv protease inhibitors have found prominent clinical use (figure 9(b) ). coronavirus s proteins can be primed by a multitude of proteases [145] . hoffmann et al. demonstrated that the s protein of 2019-ncov could be primed by serine protease tmrpss2 [113] . similarly, both sars-cov and mers-cov can be activated by other tmprss family members [145] . tmprss family proteases are widely expressed in the respiratory tract [145, 146] , which is likely the reason that coronaviruses cause acute respiratory distress syndrome. upon successful priming, the viral genome encoding rna and several nonstructural proteins, including coronavirus main protease (3clpro), papain-like protease (plpro), and rdrp, are released [147] [148] [149] . the single-stranded positive rna is translated into viral polyproteins by ribosomes in the host cell cytoplasm. the polyproteins are then cleaved into effector proteins by viral proteases: 3clpro and plpro. plpro also acts as a deubiquitinase that may remove specific host cell proteins (e.g., interferon regulatory factor 3 (irf3) and nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb)), thus weakening the immune system [147, 149, 150] . both host and viral proteases are essential therapeutic targets in the case of covid-19. camostat mesylate is a small molecule that has shown an excellent therapeutic effect for chronic pancreatitis treatment by targeting proteases [113, 151, 152] . camostat mesylate primarily inhibits enzymatic autodigestion of the pancreas [153] . in vivo studies on rats with pancreatic fibrosis showed that camostat mesylate inhibits inflammation, cytokine expression, and fibrosis in the pancreas [154] . it has an additional clinical benefit for pancreatic pain by preventing enzyme-evoked activation of pain receptors [155] . as mentioned above, the tmprss family, especially tmrpss2, is most likely the protease targeted by a coronavirus. camostat mesylate inhibits tmprss2 activity on primate cells in vitro, completely blocking membrane fusion between the host cell and the viral mers-cov particle [156] . zhou et al. claimed that camostat mesylate displays an inhibitory effect in mice for sars-cov infection [152] . recent research by hoffmann et al. showed a promising in vitro inhibitory effect of this serine protease inhibitor in sars-cov and 2019-ncov on human lung cells, showing potential as a viable option for covid-19 treatment [113] . unfortunately, in vitro and in vivo data for camostat mesylate against coronaviruses are limited. more investigation is required to evaluate camostat mesylate as a potential therapeutic against covid-19. lopinavir-ritonavir is a coformulated antiretroviral drug with excellent efficacy against hiv-1. the lopinavir has a core molecular structure identical to ritonavir. the 5thiazolyl end group and 2-isopropylthiazolyl group in ritonavir are replaced by the phenoxyacetyl group and a modified valine, respectively, in which the amino terminus has six-membered cyclic urea attached. in brief, lopinavir is a potent protease inhibitor developed from ritonavir with high specificity for hiv-1 protease [103] . it represents a higher proportion of the coformulation. lopinavir contains a hydroxyethylene scaffold mimicking a standard peptide bond cleavable by hiv-1 protease [157] . this results in the production of noncontagious viral particles. on the other hand, ritonavir binds to hiv-1 protease, interrupting the maturation and production of viral particles [158] . a clinical study from hong kong has shown that the combination of lopinavir-ritonavir and ribavirin treatment for 152 patients against sars-cov had an overall favourable clinical response [159] . it has been demonstrated that lopinavirritonavir targets 3clpro of 2019-ncov and further indicated that 3clpro might also be the targets of protease inhibitors for other coronaviruses [160] . regrettably, a recent clinical trial using lopinavir-ritonavir in wuhan, china, reported that 199 hospitalized adult patients infected with 2019-ncov did not benefit from the treatment [161] . given such conflicting clinical data, physicians must carefully weigh lopinavir-ritonavir as a covid-19 treatment. darunavir is another antiretroviral protease inhibitor drug effective against hiv-1. darunavir is designed for multidrug-resistant hiv-1 protease variants, due to its molecular structure, which introduces more hydrogen bonds compared to conventional antiretroviral medicines. in general, changes in van der waals and hydrogen bonding interactions between inhibitors and proteases affect the potency of antiretroviral drugs [162] . aside from enzymatic inhibition, darunavir inhibits protease dimerization [163] . the dimerization of hiv protease is essential for the acquisition of its proteolytic activity for the maturation of viral particles [163] . lin et al. claimed that darunavir inhibits 2019-ncov. the group has used molecular modeling to evaluate darunavir binding to 3clpro and plpro proteases and found targeted activity against the latter [164] . nevertheless, the therapeutic effect of darunavir in covid-19 clinical cases remains untested [164] . this may be in part due to potential side effects, such as liver damage and severe skin rashes [103, 165] . these contraindications must be carefully evaluated if darunavir is to be considered a potential therapeutic agent for covid-19. polymerases are enzymes essential for viral replication to produce viral progeny. viral dna and rna polymerases are responsible for duplicating the viral genome and facilitating transcription and replication [103] . replication inhibitors (figure 9 (c)) interfere with the production of viral particles by blocking enzymatic activity, ultimately causing chain termination during viral dna or rna replication [166] . there are four types of viral polymerases in viruses: rna-dependent rna polymerases, rdrp, dna-dependent rna polymerases, and dna-dependent dna polymerases. in the section protease inhibitors, we mentioned that rdrp is released upon successful priming. rdrp is a necessary polymerase that catalyzes the replication of rna from an rna template for coronaviruses [167] . release of rdrp from the virus initiates the synthesis of a full negativestrand rna template to be used by rdrp to replicate more viral genomic rna, which eventually turns host cells into virus factories [147] . therefore, rdrp is an attractive therapeutic target to prevent host cells from producing viruses. ribavirin is a synthetic guanosine nucleoside analog that mimics purines, including inosine and adenosine, and ribavirin has been used in the treatment of respiratory syncytial virus [168] . it has only one ring at the heterocyclic base, compared with guanine's two rings. notably, ribavirin has a ribose sugar moiety with a hydroxyl group at the 2 ′ -carbon position, enabling preferential activity in rna-related metabolism [168, 169] . ribavirin inhibits cellular enzyme and inosine monophosphate dehydrogenase involved in purine nucleotide biosynthesis [170, 171] . ribavirin is also known for its inhibitory effect on viruses by forcing viral genome replication to become catastrophically error-prone. it is likely that as a nucleoside analog, ribavirin is incorporated by rdrp into the newly synthesized viral genome, where it induces mutagenesis [170, 172] . although ribavirin has proven effective against viral infections, its mechanism of action has not been firmly established, and there are several proposed mechanisms of action that require further validation [168, 173] . ribavirin was initially used in treating sars; however, ribavirin treatment lacked an in vitro antiviral effect and caused adverse side effects including anemia, hypoxemia, and decreased hemoglobin levels [174] . however, ribavirin was used as the primary treatment during the mers outbreak [175] . in general, clinical studies of ribavirin treatment for sars and mers did not show strong evidence of efficacy against these coronaviruses [176] [177] [178] . there have been no studies of ribavirin's efficacy against covid-19. therefore, the use of ribavirin remains controversial and requires more investigation for a better understanding of its mechanism of action, efficacy, and toxicity, even though it is a widely available drug. favipiravir is a synthetic guanine analog frequently used for influenza treatment [179] . structurally, favipiravir is closely related to ribavirin, in which it shares the same carboxamide moiety [180] . while ribavirin interacts with the viral polymerase directly, favipiravir must be phosphoribosylated by cellular enzymes to its active form, favipiravir-ribofuranosyl-5 ′ -triphosphate (rtp) [181, 182] . the viral polymerase mistakenly recognizes favipiravir-rtp for a purine nucleotide, thereby disrupting viral genome replication [181, 182] . favipiravir has not been used against sars and mers previously, but interestingly, it has been shown to reduce viral infection of 2019-ncov [138, 183] . in a clinical study involving 80 patients infected with 2019-ncov, conducted in shenzhen, china, favipiravir showed better efficacy than lopinavir-ritonavir in terms of disease progression and viral clearance [183] . another clinical study involving 240 patients with covid-19 conducted in hubei province, china, also demonstrated that those treated with favipiravir had a higher recovery rate compared to those treated with umifenovir (preprint) [184] . more clinical data are needed to validate favipiravir's efficacy and safety in 2019-ncov treatment. remdesivir is a trial synthetic adenosine analog that has not yet been clinically approved [185] . it was synthesized and developed by gilead science in 2017 for ebola virus infection [186] . remdesivir needs to be metabolized into its active form, gs-441524, to initiate its activity. the active form of remdesivir inhibits viral rna polymerase and evades proofreading by viral exonuclease, causing an interruption in viral rna production [138, 185, 186] . it has been demonstrated that remdesivir is effective against mers-cov infection in vivo and 2019-ncov in vitro [138, 187] , showing great potential as a therapeutic agent for 2019-ncov. the drug is currently being validated in clinical trials [188] . given that antiviral drugs have previously demonstrated reasonable inhibition of coronaviruses and therapeutic efficacy against coronavirus outbreaks, umifenovir, chloroquine, hydroxychloroquine, lopinavir-ritonavir, and ribavirin have been recommended in the latest guidelines for diagnosis and treatment of covid-19, updated on 17 february 2020 [189] . recent studies also demonstrated that some antibiotics potentially inhibit 2019-ncov replication. anderson et al. (preprint) recently developed the first bat genome-wide rna interference (rnai) and crispr libraries and identified mthfdi as the critical host factor for viral infections [190] . mthfdi is a trifunctional enzyme involved in the one-carbon (c1) metabolic pathway, participating in the cellular production of purine, dtmp, and methyl groups [191] . anderson et al. demonstrated that purine synthesis activity of mthfdi is an essential activity for viral replication, making mthfdi a potential target for developing antiviral drugs [190] . they further explained that an mthfd1 inhibitor, carolacton, restricts replication of influenza virus, mumps virus, melaka virus, zika virus, and, most importantly, 2019-ncov [190] . carolacton is a secondary metabolite derived from the mycobacterium sorangium cellulosum. it is a macrolide ketocarbonic acid. carolacton has been studied as an antibacterial compound against biofilms of pathogenic streptococcus mutans and growth of pathogen streptococcus pneumoniae [192, 193] . it has no toxic effect against eukaryotic cells [194] . it has recently been identified as a potent inhibitor of mthfdi, and its mechanism of action is presumably due to the ability of carolacton to bind with mthfdi [194] . more research is needed to validate the mechanism of action, efficacy, and safety of carolacton as a possible treatment for covid-19. on the other hand, ivermectin is originally a medication used to treat parasite infestation. it comprises different analogs of avermectin: 22,23-dihydroavermectin b1a and 22,23-dihydroavermectin b1b, at a ratio of 4 : 1 [195] . they are macrolide antibiotics isolated from the fungus streptomyces avermitilis. it has reportedly stopped hiv-i proliferation by inhibiting interaction of the retroviral integrase protein with adapter protein (importin), responsible for the nuclear protein import cycle [196] . caly et al. reported that ivermectin successfully inhibited 2019-ncov in vitro but the mechanism of action is unclear [197] . since ivermectin is an approved drug, it shows great potential as a therapeutic agent for covid-19. in vivo work or clinical trials need to be done to confirm its efficacy and safety for treatment against covid-19. potential drugs for covid-19 are summarized in table 2 . 3.2. nanodrug delivery system. nanomaterials have recently been utilized for the treatment of diseases such as cancer [198] [199] [200] and various types of infections [201, 202] . the ease of modification of surface properties, large surface area [203] , and multivalent interactions with targets [204] imbues nanomaterials with massive potential as highly efficacious covid-19 therapeutic options. however, to the best of our knowledge, no nanoparticle treatment option has been applied to covid-19. nonetheless, results obtained from nanoparticle research against other viruses have shown promising potential. for example, fujimori et al. utilized a cui nanoparticle to treat h1n1 influenza through the generation of reactive oxygen species (ros) that inactivate the virus [205] . silver nanoparticles also show much promise in treating covid-19 with their broad antiviral properties against a multitude of viruses, including hiv, hepatitis b virus, herpes simplex virus, respiratory syncytial virus, and monkeypox virus [206] . the broad antiviral properties of silver nanoparticles and the generality of ros inactivation suggest that these nanoparticles can be utilized therapeutically without any modifications. nanoparticles could also be used for drug delivery. recently, herold and sander demonstrated the use of pulmonary surfactant-biomimetic nanoparticles to encapsulate a stimulator of interferon gene (sting) agonist, 2 ′ ,3 ′ -cyclic guanosine monophosphate-adenosine monophosphate, as an adjuvant in a variety of influenza vaccines [207] . using nanoparticles as a delivery agent, immune cells were activated without excessive inflammation in the lung. this could provide a considerable benefit for use in covid-19 vaccines in the future, but as the field is still relatively new, especially in medicinal applications, safety should remain a key consideration in the adoption of nanoparticles in humans. in addition to chemical medicines, another vital form of therapy for covid-19 may be the use of biologics. currently, interferon-α2b nebulization of 100,000 to 400,000 iu/kg twice a day for 5 to 7 days is one of the main treatments for covid-19 in children, and it has demonstrated efficacy in reducing the viral load during early stages of infection [208, 209] . another promising biologic drug is convalescent plasma, the plasma of patients who have recovered from covid-19 [210, 211] . antibodies in the donated plasma could confer temporary, passive immunity against covid-19, allowing patients time to develop active immunity. clinical trials are currently ongoing [212, 213] , and preliminary results announced from the chinese hospitals have been promising. on the other hand, human monoclonal antibodies or their fragments developed in the lab have shown encouraging results as well. tian et al. confirmed the binding of a human monoclonal antibody cr3022 to the receptor-binding domain (rbd) of 2019-ncov with high affinity [214] , highlighting the therapeutic potential of cr3022 toward covid-19, though further in vitro and in vivo studies are required before it could be used clinically. another supportive treatment for covid-19 involves dietary interventions. various research studies have shown supplementation of multiple vitamins and minerals such as vitamins a, c, and d and zinc can reduce the severity of respiratory infections [215] [216] [217] [218] [219] [220] [221] . however, most of these studies targeted children below the age of 5 who were suffering from malnourishment or preexisting diseases. therefore, vitamin and mineral supplementation may offer more significant benefits to covid-19 patients in developing countries. moreover, aggressive supplementation of calories and protein in nutritionally at-risk patients has shown significant benefits in reducing mortality [222] . using a modified nutrition risk in critically ill (mnutric) score, kalaiselvan and coworkers demonstrated that 42.5% of mechanically ventilated patients have high nutritional risks (mnutric score ≥ 5), accompanied by long intensive care unit (icu) stays and high mortality rates [223] . an estimated 5% of covid-19 patients require icu care, and of these critically ill patients, most need mechanical ventilation [224, 225] . therefore, nutritional intervention using aggressive calorie and protein supplementation may provide substantial benefits to a significant number of critically ill patients. evidence of such benefits may be provided by the clinical trial (nct04274322) that is expected to end in july 2020. 3.5. traditional chinese medicine. traditional chinese medicine (tcm) is considered a prospective supplementary treatment of covid-19, due to its impressive performance in treating sars in 2003 [226] . first, tcm shows a generalized antiviral effect through direct inhibition of viruses and control of inflammation. for example, weng et al. reported that the smabucus formosana nakai (a traditional medicinal herb) ethanol stem extract displayed strong anti-hcov-nl63 activity [227] . moreover, tcm can alleviate damage induced by inflammatory reactions and immune responses initiated by viral infections. single and combined chinese medicines could mitigate the cytokine storm by clearing the heat and toxicity in the body. for instance, tcm approaches were adopted to prevent and treat sars in 2003 and h1n1 influenza in 2009 [228] . as of february 17, 2020, over 85.2% of total confirmed cases (over 60,000 cases) had been treated with tcm, showing that tcm yields excellent outcomes. notably, in a trial of 102 cases with mild symptoms, tcm achieved remarkable therapeutic effects, demonstrated [192, 193] in vitro antiviral activities against bat kidney cells [190] not known none ivermectin genome replication antiparasitic drug (broad-spectrum). in vitro antiviral activities against 2019-ncov on primate cells [197] not known none n/a: not available. note: "coronaviruses" only target sars-cov and mers-cov. by faster clinical symptom disappearance and reduction of fever, shorter disease course, higher cure rate (by 33%), and lower rate of moderate-to-severe cases [229] . to date, the national health commission (nhc) of the people's republic of china has published seven editions of the guidelines for diagnosis and treatment of covid-19 [25] . since the fourth version, a list of tcm prescriptions (including tcm soup and tcm capsules) has been recommended for patients based on the stage of the disease and their symptoms [230] . according to the 7 th edition of the guidelines, there are three kinds of tcm prescriptions recommended for different stages of patients: the medical observation period, the clinical treatment period, and critical condition (details in table 3 ) [25] . among tcm recipes, the qing fei pai du decoction is strongly recommended for treatment of covid-19 by the nhc of the people's republic of china, because it gave a cure rate of over 90% of covid-19 patients in a clinical trial involving 701 confirmed cases [231] . another tcm recipe, xue bi jing injection is specifically recommended for treating critically ill covid-19 patients, because it suppresses severe sepsis, according to the china food and drug administration. it also promoted significant improvement in cases of severe community-acquired pneumonia (cap) [232] . therefore, tcm could be an alternative prophylactic approach to covid-19 and a supplementary treatment in combination with western medicine to cure covid-19. 3.6. stem cell therapy. stem cell therapy is a promising treatment strategy for degenerative diseases, including huntington's disease, parkinson's and alzheimer's diseases, and chronic diseases such as cardiac failure and diabetes [233] . a clinical study showed that transplantation of mesenchymal stem cells (mscs) significantly lowered the mortality of patients with h7n9-induced acute respiratory distress syndrome (ards), with no harmful effects [234] . as h7n9 and 2019-ncov share similar genome structures and corresponding infection mechanisms, as well as related clinical symptoms (lung failure), msc-based therapy could be a possible alternative for treating covid-19. currently, stem cellbased therapy for covid-19 is being conducted by different hospitals in china. doctors from baoshan hospital (yunnan province, china) used human umbilical cord mesenchymal stem cells (hucmscs) to treat a 65-year-old critically ill woman with covid-19. two days after the 3rd injections of stem cells, the woman recovered, and the throat swab test for covid-19 turned negative [235] . another clinical trial involving stem cell therapy was conducted in seven confirmed covid-19 patients in different clinical stages in beijing youan hospital (beijing, china). two to four days after intravenous transplantation of ce2 -mscs, all symptoms such as high fever, weakness, and shortness of breath disappeared in all seven patients without observed adverse effects, indicating that mscs can cure or significantly improve functional outcomes [236] . there are at least 12 other trials using stem cells to treat covid-19 in china, according to the who report. vaccines are another promising treatment to prevent or cure specific viruses. currently, there is no effective vaccine against 2019-ncov. fortunately, two covid-19 vaccines are undergoing clinical trials. the first is moderna's mrna-1273, an mrna vaccine, which started at kpwhri in seattle, usa, on march 16, 2020 [237] . it targets the spike protein of 2019-ncov. the other vaccine, ad5-ncov (a recombinant novel coronavirus disease vaccine (adenovirus type 5 vector)), was conducted at tongji hospital in wuhan, china, on march 16, 2020 [238] . the trial was jointly developed and administered by cansino biologics inc. and the academy of military medical sciences. ad5-ncov, a genetically engineered vaccine, expresses the 2019-ncov s protein using replication-defective adenovirus type 5 as an expression vector, thereby inducing a virus-specific immune response to prevent covid-19. there are also several other types of covid-19 vaccines, including deoptimized live attenuated vaccines, protein vaccine, dna vaccine, rna vaccine, and subunit vaccine, all of which are in the preclinical stage (table 4 ) [239] . notably, there is a new microneedle array (mna) approach based on delivering coronavirus s1 subunit vaccines against covid-19. expedited by prior experience in developing vaccines against mers, this approach was developed within 4 weeks and enabled long-term induction of potent virus-specific antibody responses. significantly, the mna work can be extended to other emerging infectious diseases. however, these will require further clinical studies for efficacy and safety, which requires more time. according to the 7 th edition of the diagnostic criteria [25] , patients severely or critically ill with covid-19 should receive comprehensive antiviral treatment, including lopinavir/ritonavir, arbidol, or shufeng jiedu capsule. meanwhile, they also need additional treatments, according to their symptoms, including respiratory support (oxygen therapy, high-flow nasal cannulas, or noninvasive ventilation, invasive mechanical ventilation, or extracorporeal membrane oxygenation-(ecmo-) based therapy), circulatory support, or continuous renal replacement therapy. the main therapeutic approaches proposed for covid-19 are summarized in table 5 . as the most recent pandemic, covid-19 induces much fear. it is highly infectious and is transmitted asymptomatically. as such, our best options to slow and prevent transmission are to understand the origin of 2019-ncov, its transmission route, and associated disease pathways and systems. generally, a pathogen must remain viable outside the host to allow for environmental spread [240] . collective effects of many biotic and abiotic factors determine the period that the pathogen can survive [240] . as of now, covid-19 is thought to be transmitted directly from person-to-person through liquid (droplets) and, more importantly, transmitted indirectly via contact with contaminated surfaces. 2019-ncov remains viable for a fairly long period outside the human body (up to 72 hours) and is more stable on plastic and stainless steel than on copper and cardboard [241] . therefore, aerosol and fomite transmission of 2019-ncov is possible, as the virus lingers among particles or fibers, in airborne liquid droplets, and on surfaces, in some cases for days [241] . although there are currently insufficient data on the inactivation of environmental 2019-ncov, data from other coronaviruses can be used as a reference. however, it should be noted that biocidal agents may only limit the survival of coronavirus in critical environments and have no efficacy for infected patients. given the high transmissibility of covid-19, its propensity for asymptomatic transmission, and its persistent nature, confirmed patients could only be quarantined and treated in adequately equipped facilities. this also applies to anyone who has come into contact with these patients. as such, contact tracing is still a mainstay for disease control. confirmation can be achieved only when specific diagnostic methods have been employed. chest ct imaging is useful as an initial evaluation for covid-19, as ct confirmation is often possible even before symptoms appear; therefore, it is recommended for suspected covid-19 cases [242] . once the primary diagnosis reveals abnormal chest ct findings, a nucleic acid test should be performed to confirm whether a patient is infected. once a person is confirmed positive, tests such as c-reactive protein (crp), complete blood count, urinalysis, biochemical indicators (i.e., liver enzymes, myocardial enzymes, and renal function), blood coagulation function, arterial blood gas analysis, and cytokine levels should be performed to monitor the patient's condition [189] . chest ct should be performed as a follow-up to treatment as well [242] . the currently adopted procedure in identifying potential covid-19 cases in china is summarized in figure 10 . on a community scale and beyond, strict controls over human traffic are essential to limiting disease transmission. by establishing lockdowns, china has been able to bring the crisis under control. other nations are now following the chinese's approach in restricting movement of residents within their borders. as evidenced globally, social distancing is essential to halt the spread of covid-19. on the other hand, individuals have the responsibilities to follow the guidelines given by the authorities, to practice good hygiene, and to behave responsibly. covid-19, like the past epidemics, does not recognize political boundaries, ethnicity, or gender. the disease has challenged the economic and medical infrastructure of the entire globe. as evidenced by events of the past few months, the impact of the outbreak depends upon how well we are prepared to face such a challenge. only with time will we be able to fully evaluate the measures that are being taken against covid-19 today. previous coronavirus epidemics like sars and mers have expedited the process of finding useful diagnostic and therapies against 2019-ncov. it is of paramount importance for all countries to share essential information about 2019-ncov to mitigate its spread. because of this strategic approach, research has been mobilized to rapidly develop diagnostic methods and worldwide implementation to minimize the impact of the pandemic. practical diagnostic tests have aided management and contact tracing of covid-19 cases in hotspot areas. in this regard, molecular virological techniques have assisted the scientific community in characterizing infectious agents for years. these include qrt-pcr, isothermal amplification, and crispr technology. on the other hand, serological assays for antibodies and antigens present essential tools to obtain valuable information about prior exposure to 2019-ncov and the prevalence of infection. these include elisa and lfa technologies. serological screening also enables novel vaccines to be assessed and supports the design of functional vaccine approaches. other approaches, including chest computed tomography (ct) and transmission electron microscopy (tem), can boost existing detection approaches. notably, there has been a marked increase in the use of both ct and tem to detect 2019-ncov and other coronaviruses. these complimentary tools reveal the progression of suspected infection, which cannot be accomplished by conventional diagnostic means. nonetheless, there is a pressing need for continuous development of rapid, accurate diagnostic devices and strategies to characterize unknown respiratory pathogens. despite these signs of progress, the present data suggest that current public health policies and improved diagnostic measures alone may not be sufficient to eradicate covid-19 in the short term. efficacious and novel treatments are desperately required. presently, large numbers of ongoing clinical trials of various drugs may succeed in minimizing morbidity and mortality. we have highlighted several of them in this review. some are highly promising, while others may require more time to demonstrate usefulness. while some drug candidates appear promising and have been used in treating covid-19 patients in desperation, it does not necessarily mean that they are proven safe and efficacious in the long run. as such, stringent criteria must be established by health regulatory agencies. however, in the long term, vaccines and prophylactics may be required to curb the spread of 2019-ncov. the authors declare no conflicts of interest. covid-19) outbreak situation who, 2019-ncov outbreak is an emergency of international concern who-director-general-s-openingremarks-at-the-media-briefing-on-covid more and more coronaviruses: human coronavirus hku1 discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus from patients with pneumonia in china return of the coronavirus: 2019-ncov a pneumonia outbreak associated with a new coronavirus of probable bat origin genome composition and divergence of the novel coronavirus (2019-ncov) originating in china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerging coronaviruses: genome structure, replication, and pathogenesis ace2 expression by colonic epithelial cells is associated with viral infection, immunity and energy metabolism, medrxiv singlecell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection cryo-em structure of the 2019-ncov spike in the prefusion conformation structural basis for the recognition of sars-cov-2 by full-length human ace2 sars-cov-2 invades host cells via a novel route: cd147-spike protein, biorxiv coronavirus envelope (e) protein remains at the site of assembly subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein a single polar residue and distinct membrane topologies impact the function of the infectious bronchitis coronavirus e protein design of multi epitope-based peptide vaccine against e protein of human 2019-ncov: an immunoinformatics approach report of the who-china joint mission on coronavirus disease 2019 (covid-19) the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china, 2020 national health committee of the people's republic of china, diagnosis and treatment guidelines for covid-19april 2020 high-throughput determination of rna structures dna sequencing with chain-terminating inhibitors nanopore target sequencing for accurate and comprehensive detection of sars-cov-2 and other respiratory viruses rapid multiplex small dna sequencing on the minion nanopore sequencing platform mycobiome diversity: highthroughput sequencing and identification of fungi recent advances and perspectives of nucleic acid detection for coronavirus real-time rt-pcr in covid-19 detection: issues affecting the results quantification of mrna using real-time rt-pcr sars-cov-2 test to detect novel coronavirus receives fda emergency use authorization and is available in markets accepting the ce mark nucleic acid isothermal amplification technologies-a review isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases development and application of reverse transcription loop-mediated isothermal amplification for detecting live shewanella putrefaciens in preserved fish sample a simple and multiplex loop-mediated isothermal amplification (lamp) assay for rapid detection of sars-cov a rapid and specific assay for the detection of mers-cov development of loopmediated isothermal amplification assay for detection of human coronavirus-nl63 rapid colorimetric detection of covid-19 coronavirus using a reverse tran-scriptional loopmediated isothermal amplification (rt-lamp) diagnostic plat-form: ilaco development of reverse transcription loop-mediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus 2 rapid detection of sars-cov-2 using reverse transcription rt-lamp method a single and two-stage, closed-tube, molecular test for the 2019 novel coronavirus (covid-19) at home, clinic, and points of entry recombinase polymerase amplification applied to plant virus detection and potential implications recombinase polymerase amplification: basics, applications and recent advances a portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus rapid detection of hiv-1 proviral dna for early infant diagnosis using recombinase polymerase amplification a new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus development of a reverse transcription recombinase-aided amplification assay for the detection of coxsackievirus a10 and coxsackievirus a6 rna multiplex genome engineering using crispr/cas systems development and applications of crispr-cas9 for genome engineering crispr gene-editing tested in a person for the first time harnessing crispr/cas9 technology in cardiovascular disease nucleic acid detection with crispr-cas13a/c2c2 sherlock: nucleic acid detection with crispr nucleases multiplexed and portable nucleic acid detection platform with cas13, cas12a, and csm6 sherlock, a protocol for detection of covid-19 using crispr diagnostics crispr-cas12a target binding unleashes indiscriminate single-stranded dnase activity an ultrasensitive, rapid, and portable coronavirus sars-cov-2 sequence detection method based on crispr-cas12 crispr-cas12-based detection of sars-cov-2 digital pcr research highlights: digital assays on chip lab-on-a-chip: microfluidics in drug discovery the origins and the future of microfluidics the present and future role of microfluidics in biomedical research theoretical design and analysis of multivolume digital assays with wide dynamic range validated experimentally with microfluidic digital pcr monte carlo modeling-based digital loop-mediated isothermal amplification on a spiral chip for absolute quantification of nucleic acids microfluidic digital pcr enables multigene analysis of individual environmental bacteria digital lamp in a sample self-digitization (sd) chip self-priming compartmentalization digital lamp for point-of-care microfluidic continuous flow digital loop-mediated isothermal amplification (lamp) visual and modular detection of pathogen nucleic acids with enzyme-dna molecular complexes nus scientists work on covid-19 vaccine trial and rapid test kits detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay differential sensitivities of severe acute respiratory syndrome (sars) coronavirus spike polypeptide enzyme-linked immunosorbent assay (elisa) and sars coronavirus nucleocapsid protein elisa for serodiagnosis of sars coronavirus pneumonia singapore claims first use of antibody test to track coronavirus infections a human monoclonal antibody blocking sars-cov-2 infection molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes igm in microbial infections: taken for granted? development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis radiology perspective of coronavirus disease 2019 (covid-19): lessons from severe acute respiratory syndrome and middle east respiratory syndrome radiology of severe acute respiratory syndrome (sars): the emerging pathologic-radiologic correlates of an emerging disease middle east respiratory syndrome coronavirus: what does a radiologist need to know? follow-up chest radiographic findings in patients with mers-cov after recovery chest radiograph scores as potential prognostic indicators in severe acute respiratory syndrome (sars) ct imaging features of 2019 novel coronavirus (2019-ncov) a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster 2019 novel coronavirus (2019-ncov) pneumonia what happens to people's lungs when they get coronavirus? application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future probing the nature of upconversion nanocrystals: instrumentation matters unravelling biological macromolecules with cryo-electron microscopy here are the first images of how coronavirus replicates in cells preparation of viral samples within biocontainment for ultrastructural analysis: utilization of an innovative processing capsule for negative staining novel and potent inhibitors targeting dhodh, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against rna viruses including newly emerged coronavirus sars-cov-2 innate immune cells and bacterial infection in zebrafish drug repurposing from an academic perspective drug repurposing approaches for the treatment of influenza viral infection: reviving old drugs to fight against a long-lived enemy chapter 34 -antiviral drugs characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine subunit vaccines against emerging pathogenic human coronaviruses vaccines for the prevention against the threat of mers-cov prospects for a mers-cov spike vaccine mechanisms of coronavirus cell entry mediated by the viral spike protein angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure analysis of the receptor binding of 2019-ncov functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structure-based discovery of a novel angiotensinconverting enzyme 2 inhibitor the potential actions of angiotensin converting enzyme ii (ace2) activator diminazene aceturate (dize) in various diseases angiotensin-converting enzyme 2 as a therapeutic target for heart failure diabetes and cvd risk during angiotensin-converting enzyme inhibitor or angiotensin ii receptor blocker treatment in hypertension: a study of 15 990 patients ace inhibitor use and major cardiovascular outcomes in type 2 diabetes treated with the dpp-4 inhibitor alogliptin evaluation of angiotensin-converting enzyme (ace), its homologue ace2 and neprilysin in angiotensin peptide metabolism covid-19 and angiotensinconverting enzyme inhibitors and angiotensin receptor blockers are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? structural basis of influenza virus fusion inhibition by the antiviral drug arbidol new small-molecule drug design strategies for fighting resistant influenza a characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol membranotropic effects of arbidol, a broad antiviral molecule, on phospholipid model membranes clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment discovering drugs to treat coronavirus disease 2019 (covid-19) effects of chloroquine on viral infections: an old drug against today's diseases effect of weak bases on the intralysosomal ph in mouse peritoneal macrophages mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology chloroquine inhibits autophagic flux by decreasing autophagosomelysosome fusion new insights into the antiviral effects of chloroquine anti-hiv effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies chloroquine and hydroxychloroquine as available weapons to fight covid-19 in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) effects of chronic exposure to hydroxychloroquine or chloroquine on inner retinal structures animal toxicity and pharmacokinetics of hydroxychloroquine sulfate retroviral proteases and their roles in virion maturation host cell proteases: critical determinants of coronavirus tropism and pathogenesis influenza and sars-coronavirus activating proteases tmprss2 and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases virusencoded proteinases and proteolytic processing in the nidovirales the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds crystal structures of the main peptidase from the sars coronavirus inhibited by a substrate-like aza-peptide epoxide simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry protease inhibitors targeting coronavirus and filovirus entry efficacy of camostat mesilate compared with famotidine for treatment of functional dyspepsia: is camostat mesilate effective? camostat mesilate attenuates pancreatic fibrosis via inhibition of monocytes and pancreatic stellate cells activity the proteinase inhibitor camostat mesilate suppresses pancreatic pain in rodents middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 anti-hiv drugs: 25 compounds approved within 25 years after the discovery of hiv ritonavir-boosted protease inhibitors in hiv therapy role of lopinavir/-ritonavir in the treatment of sars: initial virological and clinical findings potential inhibitors against 2019-ncov coronavirus m protease from clinically approved medicines a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 revealing origin of decrease in potency of darunavir and amprenavir against hiv-2 relative to hiv-1 protease by molecular dynamics simulations dimerization of hiv-1 protease occurs through two steps relating to the mechanism of protease dimerization inhibition by darunavir molecular modeling evaluation of the binding effect of ritonavir, lopinavir and darunavir to severe acute respiratory syndrome coronavirus 2 proteases, biorxiv drugs for hiv infection inhibitors of virus replication: recent developments and prospects the potential chemical structure of anti-sars-cov-2 rna-dependent rna polymerase the application and mechanism of action of ribavirin in therapy of hepatitis c the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen signatures of nucleotide analog incorporation by an rna-dependent rna polymerase revealed using high-throughput magnetic tweezers ribavirin in the treatment of sars: a new trick for an old drug? proceedings of the national academy of sciences of the united states of america mechanism of action of ribavirin in the treatment of chronic hepatitis c pharmacologic treatment of sars: current knowledge and recommendations middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) -possible lessons from a systematic review of sars-cov therapy antiviral treatment guidelines for middle east respiratory syndrome understanding the mechanism of the broad-spectrum antiviral activity of favipiravir (t-705): key role of the f1 motif of the viral polymerase a focus on ebola virus polymerase favipiravir (t-705), a novel viral rna polymerase inhibitor favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase experimental treatment with favipiravir for covid-19: an open-label control study favipiravir versus arbidol for covid-19: a randomized clinical trial, medrxiv therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys remdesivir as a possible therapeutic option for the covid-19 comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov centers for disease control and prevention, information for clinicians on therapeutic options for covid-19 patients chinese centre for disease control and prevention, covid-19 prevention and control: diagnosis and treatment orthogonal genome-wide screenings in bat cells identify mthfd1 as a target of broad antiviral therapy, biorxiv one-carbon metabolism in health and disease dental composite materials containing carolacton inhibit biofilm growth of streptococcus mutans the biofilm inhibitor carolacton enters gram-negative cells: studies using a tolcdeficient strain of escherichia coli damage of streptococcus mutans biofilms by carolacton, a secondary metabolite from the myxobacterium sorangium cellulosum targeting human onchocerciasis: recent advances beyond ivermectin ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of hiv-1 and dengue virus the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro cancer cell membrane-coated adjuvant nanoparticles with mannose modification for effective anticancer vaccination light-activatable red blood cell membrane-camouflaged dimeric prodrug nanoparticles for synergistic photodynamic/chemotherapy near-infrared upconversion mesoporous cerium oxide hollow biophotocatalyst for concurrent ph-/h 2 o 2 -responsive o 2 -evolving synergetic cancer therapy near-infrared light-controllable on-demand antibiotics release using thermosensitive hydrogel-based drug reservoir for combating bacterial infection induction of potent neutralizing antibody responses by a designed protein nanoparticle vaccine for respiratory syncytial virus metal nanoparticles: the protective nanoshield against virus infection nano-oncology: drug delivery, imaging, and sensing novel antiviral characteristics of nanosized copper(i) iodide particles showing inactivation activity against 2009 pandemic h1n1 influenza virus silver nanoparticles as potential antiviral agents toward a universal flu vaccine diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus the convalescent sera option for containing covid-19 convalescent plasma as a potential therapy for covid-19 more than 80 clinical trials launch to test coronavirus treatments takeda initiates development of a plasmaderived therapy for covid-19 potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody vitamin a supplements ameliorate the adverse effect of hiv-1, malaria, and diarrheal infections on child growth vitamin a supplementation in infectious diseases: a meta-analysis vitamin c and sars coronavirus vitamin c intake and susceptibility to pneumonia vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data zn 2+ inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture zinc supplementation for the prevention of pneumonia in children aged 2 months to 59 months the relationship between nutritional intake and clinical outcomes in critically ill patients: results of an international multicenter observational study use of nutrition risk in critically ill (nutric) score to assess nutritional risk in mechanically ventilated patients: a prospective observational study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china in vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, cimicifuga rhizoma, meliae cortex, coptidis rhizoma, and phellodendron cortex antiviral activity of sambucus formosananakai ethanol extract and related phenolic acid constituents against human coronavirus nl63 antiviral activity of isatidis radix derived glucosinolate isomers and their breakdown products against influenza a in vitro/ovo and mechanism of action traditional chinese medicine for covid-19 treatment application of integrative medicine protocols on treatment of coronavirus disease 2019 publicity department of the people's republic of china, press conference of the joint prevention and control mechanism of state council on xuebijing injection versus placebo for critically ill patients with severe communityacquired pneumonia: a randomized controlled trial welcome to progress in stem cell clinical study of mesenchymal stem cell treatment for acute respiratory distress syndrome induced by epidemic influenza a (h7n9) infection: a hint for covid-19 treatment clinical remission of a critically ill covid-19 patient treated by human umbilical cord mesenchymal stem cells transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia nih clinical trial of investigational vaccine for covid-19 begins china approves first homegrown covid-19 vaccine to enter clinical trials draft landscape of covid-19 candidate vaccines environmental survival and microbicide inactivation of coronaviruses aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 coronavirus disease 2019 (covid-19): a systematic review of imaging findings in 919 patients key export products for covid-19 diagnosis & treatment researchers develop new test kit to detect coronavirus in 15 mins china approves 29-minute testing kit for new coronavirus s'pore researchers invent covid-19 test that can tell if someone's infected in 5 minutes a protocol for rapid detection of the 2019 novel coronavirus sars-cov-2 using crispr diagnostics: sars-cov-2 detectr new covid-19 test kits used to screen swab samples collected at singapore checkpoints the development of beihang university on detecting and preventing of 2019-ncov preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies efficacy and mechanism of lianhua qingwen capsules(lhqw) on chemotaxis of macrophages in acute lung injury (ali) animal model anti-sepsis protection of xuebijing injection is mediated by differential regulation of pro-and anti-inflammatory th17 and t regulatory cells in a murine model of polymicrobial sepsis re-du-ninginhalation solution exerts suppressive effect on the secretion of inflammatory mediatorsviainhibiting ikkα/β/iκbα/nf-κb, mapks/ap-1, and tbk1/irf3 signaling pathways in lipopolysaccharide stimulated raw 264.7 macrophages andrographolide sulfonate ameliorates lipopolysaccharide-induced acute lung injury in mice by down-regulating mapk and nf-κb pathways tanreqing injection attenuates lipopolysaccharide-induced airway inflammation through mapk/nf-κb signaling pathways in rats model action mechanism of shenfu injection by computational system biology analysis key: cord-259703-9ef3u2mz authors: alsolamy, sami; arabi, yaseen m title: infection with middle east respiratory syndrome coronavirus. date: 2015 journal: can j respir ther doi: nan sha: doc_id: 259703 cord_uid: 9ef3u2mz nan t he middle east respiratory syndrome coronavirus (mers-cov) was first recognized as a new febrile respiratory illness in saudi arabia in june 2012. as of september 21, 2015, the who reported 1569 laboratory-confirmed cases, including at least 554 related deaths. cases have been reported in 26 countries; however, the majority of cases have occurred in saudi arabia (79%) and south korea (13%) (1) . mers-cov infection has been documented in dromedary camels; evidence suggests that they are the most common source of animal-to-human transmission (2) . although the exact mode of transmission from camels is unknown, the presence of high viral loads in the upper respiratory system of infected camels suggests that transmission occurs through close contact (2) . humanto-human transmission of mers-cov has been demonstrated among close household contacts (3). however, sustained community transmission has not been observed. in a cross-sectional serosurveillance study involving 10,009 individuals in saudi arabia, positive serology was documented in only 0.15% of individuals sampled (4) . transmission within health care settings has been a predominant feature of mers-cov infection, and has been attributed to breaches of infection prevention and control practices (5) . coronaviruses are a family of single-stranded rna viruses. mers-cov is the sixth coronavirus and the first lineage c betacoronavirus known to infect humans. severe acute respiratory syndrome coronavirus is of lineage b (6). mers-cov enters cells via a common receptor, the dipeptidyl peptidase-4, and it infects type i and type ii alveolar cells (7) . the virus has primarily been detectable in respiratory secretions, with the highest viral loads in the lower respiratory tract (8) . the median incubation period of mers-cov infection is 5.2 days, but it can be as long as 14 days (9) . the most severe cases of mers-cov infection have been reported in adult patients with underlying comorbidities, including diabetes mellitus, ischemic heart disease, end-stage kidney disease or immunosuppression (9, 10) . however, severe infection may also occur among younger patients, especially health care workers. the disease spectrum ranges from asymptomatic infection to rapidly progressive multiorgan failure. the most common clinical features in severe cases are fever (71%), cough (68%), dyspnea (66%) and gastrointestinal symptoms (32%) (9) . laboratory abnormalities commonly associated with severe mers-cov infection include leukopenia, lymphocytopenia and thrombocytopenia, in addition to elevated serum levels of creatinine, lactate dehydrogenase and liver enzymes (9) . initial chest radiographs are abnormal in the majority of symptomatic patients. findings range from minimal abnormality to extensive bilateral infiltrate consistent with acute respiratory distress syndrome patterns (9) . respiratory samples in suspected patients should be tested using real-time reverse transcriptase-polymerase chain reaction. lower respiratory tract specimens have been found to be more sensitive than upper respiratory tract specimens for the detection of mers-cov (11) . rapid progression to hypoxemic respiratory failure requiring mechanical ventilation usually occurs within the first week and it is notable that it has been associated with acute kidney failure (12) . the management of patients with mers-cov infection entails early case recognition, appropriate patient isolation, strict implementation of infection prevention and control measures, and supportive treatment. the who has issued interim guidance for the management of suspected and confirmed mers-cov infection (13) . early supportive management includes supplemental oxygen to all patients with signs of hypoxemia or respiratory distress, conservative fluid management, early endotracheal intubation in patients with laboured breathing or persistent hypoxemia, and a lung-protective ventilation strategy (13) . other adjunctive hypoxemic rescue therapies, such as early prone positioning and neuromuscular blockade, may be considered in patients with moderate-to-severe acute respiratory distress syndrome (14) . in addition, systematic corticosteroids should generally be avoided unless there is another indication. high-flow oxygen and noninvasive ventilation should be used with caution because of the potential to generate aerosols (15) . a systematic review to assess the risk for transmission of respiratory pathogens to health care workers through aerosol-generating procedures found that the following procedures were associated with an increased risk for pathogen transmission: endotracheal intubation, noninvasive ventilation, tracheotomy and manual ventilation (15) . to date, there are no clinical trials involving humans for virusspecific therapies for mers-cov infection. data regarding ribavirin, interferon and convalescent plasma are limited (9) . other medications, such as mycophenolic acid, chloroquine, chlorpromazine, loperamide and lopinavir, have shown an inhibitory effect on mers-cov replication in vitro; however, in the absence of clinical data, these drugs are not recommended for clinical use outside clinical trials (16) . there is ongoing work investigating monoclonal or polyclonal antibodies against mers-cov, but no clinical data to date (17) . the overall case-fatality rate of mers-cov infection is 35%, but the mortality rate of mechanically ventilated patients reaches 60% to 70% (12) . given the potential for transmission in the health care setting, compliance with infection control measures is critical. according to who guidelines, droplet precautions should be added to the standard precautions when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of mers-cov infection, and airborne precautions should be applied when performing aerosol-generating procedures with mers-cov patients (13) . the centers for disease control and prevention (georgia, usa), on the other hand, recommends using airborne precautions with mers-cov patients at all times (19) . hospitals should develop an infectious disease emergencies response plan. after more than three years since the first mers-cov patient was identified, this virus continues to be a significant global threat because of its high fatality rate and the gaps in our knowledge about the disease. this open-access article is distributed under the terms of the creative commons attribution non-commercial license (cc by-nc) (http:// creativecommons.org/licenses/by-nc/4.0/), which permits reuse, distribution and reproduction of the article, provided that the original work is properly cited and the reuse is restricted to noncommercial purposes. for commercial reuse, contact reprints@pulsus.com middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia: disease outbreak news evidence for camelto-human transmission of mers coronavirus family cluster of middle east respiratory syndrome coronavirus infections presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study mers-cov outbreak in jeddah -a link to health care facilities genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome: knowledge to date association of higher mers-cov virus load with severe disease and death, saudi arabia an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected -interim guidance hypoxaemic rescue therapies in acute respiratory distress syndrome: why, when, what and which one? aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). cdcgov <www.cdc.gov/coronavirus/mers/infection-prevention-control.html> key: cord-274007-zndtddty authors: rasmussen, sonja a.; smulian, john c.; lednicky, john a.; wen, tony s.; jamieson, denise j. title: coronavirus disease 2019 (covid-19) and pregnancy: what obstetricians need to know date: 2020-02-24 journal: am j obstet gynecol doi: 10.1016/j.ajog.2020.02.017 sha: doc_id: 274007 cord_uid: zndtddty coronavirus disease 2019 is an emerging disease with a rapid increase in cases and deaths since its first identification in wuhan, china, in december 2019. limited data are available about coronavirus disease 2019 during pregnancy; however, information on illnesses associated with other highly pathogenic coronaviruses (ie, severe acute respiratory syndrome and the middle east respiratory syndrome) might provide insights into coronavirus disease 2019’s effects during pregnancy. coronaviruses cause illness ranging in severity from the common cold to severe respiratory illness and death. currently the primary epidemiologic risk factors for coronavirus disease 2019 include travel from mainland china (especially hubei province) or close contact with infected individuals within 14 days of symptom onset. data suggest an incubation period of ∼5 days (range, 2–14 days). average age of hospitalized patients has been 49–56 years, with a third to half with an underlying illness. children have been rarely reported. men were more frequent among hospitalized cases (54–73%). frequent manifestations include fever, cough, myalgia, headache, and diarrhea. abnormal testing includes abnormalities on chest radiographic imaging, lymphopenia, leukopenia, and thrombocytopenia. initial reports suggest that acute respiratory distress syndrome develops in 17–29% of hospitalized patients. overall case fatality rate appears to be ∼1%; however, early data may overestimate this rate. in 2 reports describing 18 pregnancies with coronavirus disease 2019, all were infected in the third trimester, and clinical findings were similar to those in nonpregnant adults. fetal distress and preterm delivery were seen in some cases. all but 2 pregnancies were cesarean deliveries and no evidence of in utero transmission was seen. data on severe acute respiratory syndrome and middle east respiratory syndrome in pregnancy are sparse. for severe acute respiratory syndrome, the largest series of 12 pregnancies had a case-fatality rate of 25%. complications included acute respiratory distress syndrome in 4, disseminated intravascular coagulopathy in 3, renal failure in 3, secondary bacterial pneumonia in 2, and sepsis in 2 patients. mechanical ventilation was 3 times more likely among pregnant compared with nonpregnant women. among 7 first-trimester infections, 4 ended in spontaneous abortion. four of 5 women with severe acute respiratory syndrome after 24 weeks’ gestation delivered preterm. for middle east respiratory syndrome, there were 13 case reports in pregnant women, of which 2 were asymptomatic, identified as part of a contact investigation; 3 patients (23%) died. two pregnancies ended in fetal demise and 2 were born preterm. no evidence of in utero transmission was seen in severe acute respiratory syndrome or middle east respiratory syndrome. currently no coronavirus-specific treatments have been approved by the us food and drug administration. because coronavirus disease 2019 might increase the risk for pregnancy complications, management should optimally be in a health care facility with close maternal and fetal monitoring. principles of management of coronavirus disease 2019 in pregnancy include early isolation, aggressive infection control procedures, oxygen therapy, avoidance of fluid overload, consideration of empiric antibiotics (secondary to bacterial infection risk), laboratory testing for the virus and coinfection, fetal and uterine contraction monitoring, early mechanical ventilation for progressive respiratory failure, individualized delivery planning, and a team-based approach with multispecialty consultations. information on coronavirus disease 2019 is increasing rapidly. clinicians should continue to follow the centers for disease control and prevention website to stay up to date with the latest information (https://www.cdc.gov/coronavirus/2019-ncov/hcp/index.html). e merging infections have been shown to have an important impact on pregnant women and their fetuses, 1 with the increased risk of complications in pregnant women with the 2009 pandemic h1n1 influenza virus 2 and the severe fetal effects of zika virus as recent examples. 3, 4 the emergence of a coronavirus not previously seen in humans, first reported in wuhan, china, on dec. 31, 2019, has attracted much interest throughout the world. since then, the number of reported cases has increased rapidly, with more than 51,800 laboratory-confirmed cases and 1600 deaths as of feb. 16, 2020. in addition to china, cases have spread to 25 other countries ( figure 1 ) including 15 cases in the united states. initial outbreak data from china show a near exponential growth of reported cases. 5 reported numbers are likely underestimates of the true numbers because milder cases are less likely to be reported. on jan. 30, 2020, the world health organization declared the outbreak as a public health emergency of international concern; on jan. 31, 2020, the united states declared a public health emergency, and the centers for disease control and prevention issued a federal quarantine for 195 americans who traveled from wuhan, china, its first federal quarantine in more than 50 years. on feb. 11, the new coronavirus disease (previously referred to as 2019 novel coronavirus (2019-ncov)) received an official name from the world health organization (who), coronavirus disease 19 (covid-19) (figure 2 ). 6 the international committee on taxonomy of viruses has proposed severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as the name of the virus that causes covid-19. 7 coronaviruses are single-stranded rna, nonsegmented, enveloped viruses, which cause illness ranging in severity from the common cold to severe and fatal illness. the term coronavirus derives from the latin word corona, which means crown or halo; that designation arises from the appearance of coronavirus virions viewed by electron microscopy, in which the virus particles display a crown-like fringe typically referred to as spikes ( figure 3 ). in the past 2 decades, 2 other coronaviruses that cause severe respiratory illness in humans have emerged: severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). with the emergence of sars-cov-2, a third coronavirus that can cause severe respiratory illness has been identified. in a short period of time, this novel coronavirus has caused more cases of illness than were reported for mers and sars combined. here we summarize what is currently known about covid-19 and what this means for practicing obstetricians and their pregnant patients. because so little is currently known about covid-19 in pregnancy, we also review available information on the effects of sars and mers during pregnancy to inform care of pregnant women with covid-19 until additional data on pregnant women and their fetuses become available. severe acute respiratory syndrome (sars) is caused by the sars-cov. reports of the emergence of sars-cov appeared in february 2003, with the first cases observed in guangdong province in china. the virus spread to nearly 30 countries throughout the world, resulting in more than 8000 cases and 770 deaths. 8 the outbreak was brought under control after public health control measures to reduce contact with infected persons were put into place, and no cases have been seen since 2004. manifestations of sars consist of fever, chills, headache, malaise, and myalgia. diarrhea was seen in some patients. pneumonia was nearly always seen in patients diagnosed with sars, with mechanical ventilation being required in 10e20% of cases. case fatality rate was estimated at 9e10% (table) . the natural reservoir for sars-cov is believed to be bats; however, some evidence supported civet cats or raccoon dogs as possible intermediate sources of these illnesses. 8 sars is transmitted by close person-to-person contact through contact of the mucus membranes of the respiratory tract with respiratory droplets formed when an infected person coughs or sneezes. fecal-oral transmission and transmission via fomites have also been reported. 8 airborne spread because of inhalation of small particle aerosols may also be possible. transmission in health care settings was frequently seen during the 2003 outbreak, with superspreading (when a single patient transmits infection to a disproportionate number of contacts) reported. 9 the incubation period was estimated at a mean of 4.6 days, with a range of 2e14 days. transmission appeared to occur most often during the second week of illness when viral excretion is highest; there is no evidence that a person with sars is contagious before symptom onset. the largest case series of pregnant women with sars was from the 2003 outbreak in hong kong, in which 12 pregnant women were identified. 10 the case-fatality rate was 25% (3 deaths). clinical and laboratory findings were similar to those seen in the nonpregnant population. pneumonia on chest radiograph or computed tomography was seen in all patients. major medical complications included adult respiratory distress syndrome in 4, disseminated intravascular coagulopathy (dic) in 3, renal failure in 3, secondary bacterial pneumonia in 2, and sepsis in 2 patients. pregnancy outcomes varied by trimester of presentation. 10 among the 7 women who became ill in the first trimester, 4 had a spontaneous abortion, 2 had pregnancy terminations for social reasons after recovery from sars, and 1 delivered a full-term healthy infant. among the 5 women who presented after 24 weeks' gestation, 4 delivered preterm. three women delivered by cesarean delivery because of deteriorating maternal condition from their sars illness at 26, 28, and 32 weeks' gestation. 11 these babies had birthweights appropriate for gestational age. two of the infants had respiratory distress syndrome requiring surfactant (born at 26 and 28 weeks' gestation), with one later developing bronchopulmonary dysplasia. gastrointestinal complications were observed in 2 infants, including a jejunal perforation in an infant delivered at 26 weeks and necrotizing enterocolitis with ileal perforation in an infant delivered at 28 weeks' gestation. whether these gastrointestinal complications were related to complications from sars or its treatment or whether they were secondary to preterm delivery is unknown. 11 the two infants who were delivered after their mothers' recovery from sars had intrauterine growth restriction. no clinical, radiologic, or laboratory evidence for transmission from mother to fetus was observed, despite laboratory testing of different specimens. 12, 13 a matched case-control study 14 compared 10 of the 12 pregnant women noted in the previous text (2 were excluded because they were unable to be matched) with 40 nonpregnant women with sars. women were matched on sex, age, timing of contracting sars, health care worker status, underlying illness, and whether the woman resided in a housing area in which there was a large outbreak. pregnancy appeared to have no effect on clinical symptoms or time to presentation after symptom onset. however, complications and adverse outcomes were more common among pregnant women: women who were pregnant had a longer hospital stay, were statistically significantly more likely to develop renal failure, sepsis, and dic, and were more likely to require intensive care unit admission. forty percent of pregnant women required mechanical ventilation, compared with 13% of nonpregnant patients (p ¼.07). pregnant women were also significantly more likely to die (p ¼ .01). we identified 5 reports of additional cases of sars during pregnancy treated in hong kong (n ¼ 2), the united states (n ¼ 2), and canada (n ¼ 1). 15e19 two of the 5 women required mechanical ventilation, 1 required hemodialysis for acute renal failure, and 1 had seizures and positive cerebrospinal fluid for sars-cov, suggestive of a central nervous system infection. all patients recovered from their illness. in 1 case, the pregnancy was terminated at the mother's request; the remaining pregnancies ended in liveborn infants (2 at term and 2 preterm). testing of neonatal specimens for sars-cov rna was negative. several hospitals in toronto and hong kong reported measures instituted on obstetrics services during the sars outbreak to decrease transmission to pregnant women, their families, community members, and health care workers. 20, 21 for example, all hospital staff, patients, and visitors were screened for symptoms at the hospital entrance and wore n95 respirators. visitors were limited to 1 per patient on labor and delivery, with no visitors allowed in the postpartum ward. global map of confirmed covid-19 cases (as of feb. 14, 2020) (from https://www.cdc.gov/ coronavirus/2019-ncov/locations-confirmed-cases.html). rasmussen. 2019 novel coronavirus and pregnancy. am j obstet gynecol 2020. postpartum stays were reduced in length with a postpartum nurse home visit added. postpartum patients were asked to observe a 10 day home quarantine. health care workers were asked to observe a work quarantine in which they were asked to go directly from home to work and vice versa to minimize interaction in the community. obstetric services considered to be nonessential such as routine ultrasound and prenatal diagnosis were suspended. although the impact of these interventions was not evaluated, there may be some relevant lessons learned from these experiences during sars that could help inform the approach to covid-19. middle east respiratory syndrome (mers) is a respiratory illness caused by mers-cov. the illness was first identified in saudi arabia in 2012, with spread to other countries in the arabian peninsula and eventually to countries outside the arabian peninsula, including the united states. 22, 23 the largest outbreak outside the arabian peninsula was in the republic of korea in 2015. nearly 2500 cases of mers-cov illness and more than 860 deaths have been reported with continuing reports into the present. the manifestations of mers include severe respiratory illness characterized by fever, cough, and shortness of breath. some patients also have diarrhea. the case fatality rate is estimated to be 35e40%. patients who developed mers were more likely to be older (median age is 50 years) with about two thirds of patients being male. patients with mers were also more likely to have an underlying illness. some patients with mers-cov infection have been asymptomatic (identified through contact investigations). the mean incubation period is 5.2 days, with a range of 2e13 days. as with sars, mers is mainly spread person to person through close contact, with transmission in health care settings, and superspreading events have been observed. however, since 2016, the number of cases of mers-cov has been dramatically reduced after public health efforts to prevent mers-cov transmission were put into place. 24 information on mers among pregnant women is limited. we identified reports of 13 cases of pregnant women with mers from several countries, including saudi arabia (n ¼ 8), korea (n ¼ 2), jordan (n ¼ 1), united arab emirates (n ¼ 1), and philippines (n ¼ 1). 13,25e31 two women were asymptomatic, identified as part of a contact investigation. among the 11 symptomatic women, manifestations were similar to those seen in nonpregnant patients with mers. timeline showing key events in the covid-19 outbreak expert review ajog.org seven of 13 patients were admitted to an intensive care unit for respiratory deterioration or acute respiratory distress syndrome, 5 required ventilator support, 3 died, and 8 recovered. among the 3 deaths, the mothers died 8e25 days after delivery. both babies born to asymptomatic women were born healthy at term; among those who were symptomatic, there was 1 intrauterine fetal demise, 1 stillbirth, 1 baby delivered at 25 weeks who died 4 hours after birth, 2 healthy preterm infants, and 5 healthy term infants (infant status was not mentioned for 1). early data suggested an association between the huanan seafood wholesale market and covid-19 with 27 of 41 cases in 1 report 32 and 26 of 47 in another report 33 with epidemiologic links to the market, leading to closure of the market on jan. 1, 2020. given that the earliest case reported (illness onset on dec. 1, 2019) 32 did not have exposure to the market raises the possibility that the initial emergence into humans occurred elsewhere. however, sampling of the market's environment supports the market's importance in early transmission of the virus. later cases were much less likely to have visited the market, supporting the role of personto-person transmission in later cases. the sars-cov-2 is a betacoronavirus similar to sars-cov and mers-cov (table) . sequencing data show that the sars-cov-2 is most closely related to coronaviruses found in bats, with more than 85% nucleotide identity with a bat sars-like cov. 34, 35 the virus has 79% nucleotide identity to sars-cov and about 50% to mers-cov. 35 bats appear to be the natural reservoirs of both sars-cov and mers-cov. the emergence of these viruses in humans has been attributed to host switching: the virus jumped from an intermediary host species (eg, civet cats for sars-cov and dromedary camels for mers-cov) to humans. an intermediary host species is thought to be likely for sars-cov-2, 35 although it has been yet to be identified. sequence data show a high degree (>99.98%) of similarity of the virus among different patients, suggesting a recent emergence in humans. clinical manifestations of covid-19 are similar to those with sars and mers (table) . studies of hospitalized patients with covid-19 show that patients commonly develop severe pneumonia, with 23e32% admitted to the intensive care unit and 17e29% of cases progressing to acute respiratory distress syndrome. 32, 36, 37 among hospitalized patients, 4e15% have died. 32, 36, 37 overall case fatality ratio estimates (including asymptomatic and symptomatic infections) appear to be in the range of 1% (95% confidence interval, 0.5e4%), 38 although these estimates should be considered preliminary. average age of hospitalized patients was 49e56 years, with 32e51% having an underlying illness. most patients (54e73%) were men. children with covid-19 appear to be rarely identified, with only 28 children reported as of jan. 30, 2020 (<1% of total), and most of those identified had mild symptoms. 39 no pregnant women were reported in any of these initial cohorts. common manifestations among hospitalized patients were fever (83e100%), cough (59e82%), myalgia (11e35%), headache (7e8%), and diarrhea (2e10%). all patients had abnormalities on radiographic imaging of the chest. person-to-person transmission of sars-cov-2 is thought to be similar to transmission of influenza and other respiratory pathogens; respiratory droplets are formed when an infected person coughs or sneezes and these droplets are inhaled by close contacts, generally within 6 feet. it is unclear whether infection can be transmitted from fomites. fecal-oral transmission might be possible, given that sars-cov-2 has been identified in stool specimens 40 and sars-cov might have been transmitted in this manner. 41 the basic reproduction number, r0 (the average number of people who will become infected from a single infected person in a population in which all persons are susceptible) is affected by factors such as the duration of infectivity, the transmissibility of the pathogen, and the number of susceptible contacts. measles, which is highly infective, has an r0 of 12e18, while 2009 h1n1 influenza and sars have an r0 of 1.2e1.6 and 2e5, respectively. 42 current estimates of r0 for sars-cov-2 places it at 2.2 (95% confidence interval, 1.4e3.9) 33 as with sars and mers, nosocomial transmission is playing a key role in transmission, presumed to be responsible for infection of 29% of affected health professionals and 12% of hospitalized patients in a recent study. 37 in the midst of a rapidly evolving outbreak that could have significant effects on our public health and medical infrastructure, the unique needs of pregnant women should be included in preparedness and response plans. in previous outbreaks, clinicians have at times been reluctant to treat or vaccinate pregnant women because of concerns for fetal safety. 43 it is critical that pregnant women not be denied potentially lifesaving interventions in the context of a serious infectious disease threat unless there is a compelling reason to exclude them. as with all decisions regarding treatment during pregnancy, carefully weighing of the benefits of interventions for the mother and fetus with potential risks is necessary. as surveillance systems 32, 33, 36, 37 this observed gender difference could be due to differences in reporting, susceptibility, exposure, or recognition and diagnosis of infection. there are no data to inform whether pregnancy increases susceptibility to covid-19. previous data on sars and mers suggest that clinical findings during pregnancy can range from no symptoms to severe disease and death. the most common symptoms of covid-19 are fever and cough, with more than 80% of hospitalized patients presenting with these symptoms. 36 in a recent study by chen et al, 44 9 women diagnosed with covid-19 during the third trimester of pregnancy were reported. in this small series, clinical presentation was similar to that seen in nonpregnant adults, with fever in 7, cough in 4, myalgia in 3, and sore throat and malaise each in 2 women. five had lymphopenia. all had pneumonia, but none required mechanical ventilation, and none died. all women had a cesarean delivery, and apgar scores were 8e9 at 1 minute and 9e10 at 5 minutes. in a second series of 9 pregnancies with 10 infants (1 set of twins) reported by zhu et al., 45 symptom onset was before delivery (1e6 days) in 4, on the day of delivery in 2, and after delivery (1e3 days) in 3 cases. clinical presentation of covid-19 was similar to that seen in nonpregnant patients. among the 9 pregnancies, intrauterine fetal distress was noted in 6, 7 were cesarean deliveries, and 6 infants were born preterm. based on these limited reports and the available data from other respiratory pathogens such as sars and influenza, it is unknown whether pregnant women with covid-19 will experience more severe disease. travel guidance for pregnant women. travel recommendations have been instituted to limit exposure to persons in the united states. all persons, including pregnant women, should not travel to china. on feb. 2, 2020, the us state department upgraded their travel advisory to level 4, the highest level of travel advisory. obstetric providers should obtain a detailed travel history for all patients and should specifically ask about travel in the past 14 days to areas experiencing widespread transmission of sars-cov-2. currently this is limited to china, but this situation is rapidly evolving and obstetricians should stay alert to the global situation by consulting the centers for disease control and prevention website and following media coverage. there is currently no vaccine to prevent covid-19. since posting of a sars-cov-2 virus genetic sequence online on jan. 10, 2020, multiple organizations, including the national institutes of health, have been working to rapidly develop a covid-19 vaccine. development of this vaccine builds on and benefits from work on sars and mers vaccines. 46 however, it is not known how quickly a safe and effective vaccine may be readily available. infection control measures and diagnostic testing. all patients, including pregnant women, should be evaluated for fever and signs and symptoms of a respiratory infection. ideally, screening procedures begin before arrival on a labor and delivery unit or prenatal care clinic. for example, when those patients with respiratory symptoms should be separated from other waiting patients and a facemask should be placed on them. patients who meet criteria for a person under investigation (box 1) should be immediately placed in an airborne infection isolation room (single-patient rooms at negative pressure). once in isolation, the patient's facemask may be removed. health care personnel should adhere to standard, contact, and airborne precautions. infection control personnel and local/ state health departments should be notified immediately; local/state health departments can help to arrange testing of relevant specimens (upper and lower respiratory specimens and serum are currently recommended; other specimens [stool and urine] may also be sent). general principles regarding management of covid-10 during pregnancy include early isolation, aggressive infection control procedures, testing for sars-cov-2 and coinfection, oxygen therapy as needed, avoidance of fluid overload, empiric antibiotics (because of secondary bacterial infection risk), fetal and uterine contraction monitoring, early mechanical ventilation for progressive respiratory failure, individualized delivery planning, and a team-based approach with multispecialty consultations (box 2). team-based management is recommended for pregnancies managed in a health care facility and should include a determination of the optimal clinical unit on which to provide care. ability to provide surveillance for early detection of a worsening maternal course of illness, as well as an ability to monitor for evidence of obstetric complications (eg, preterm labor or fetal compromise), are needed. changes in fetal heart rate pattern may be an early indicator of maternal respiratory deterioration. based on experience with sars and mers, severe respiratory failure might occur in box 2 principles for management of pregnant women with confirmed or suspected covid-19 48,53,54,a patients with respiratory symptoms should adhere to respiratory hygiene, cough etiquette, and hand hygiene. ensure rapid triage of pregnant patients with respiratory symptoms. patients with respiratory symptoms should wear a facemask and wait in a separate, wellventilated waiting area at least 6 feet from other people. confirmed and suspected cases of covid-19 should be isolated as soon as possible in an aiir. if an aiir is not available, consider transfer to a hospital with an aiir. implement cdc infection prevention and control procedures for health care providers including standard, contact, and airborne precautions. eye protection and properly fitted n95 respirators should be used. provide additional staff training in correct use of personal protective equipment including correct donning, doffing, and disposal of personal protective equipment. contact hospital infection personnel. in coordination with local/state health department, collect and send relevant specimens for diagnostic sars-cov-2 testing. limit visitor and health care personnel access to patient rooms with a confirmed or suspected case. pregnancy should be considered a potentially increased risk condition and monitored closely including fetal heart rate and contraction monitoring. consider early oxygen therapy (target o 2 saturations 95% and/or po 2 70 mm hg). consider early mechanical ventilation with evidence of advancing respiratory failure. noninvasive ventilation techniques may have a small increased risk of aspiration in pregnancy. use intravenous fluids conservatively unless cardiovascular instability is present. screen for other viral respiratory infections and bacterial infections (because of risk of coinfections). consider empiric antimicrobial therapy (because of risk for superimposed bacterial infections). consider empiric treatment for influenza, pending diagnostic testing. do not routinely use corticosteroids. use of steroids to promote fetal maturity with anticipated preterm delivery can be considered on individual basis. if septic shock is suspected, institute prompt, targeted management. delivery and pregnancy termination decisions should be based on gestational age, maternal condition, and fetal stability, and maternal wishes. consult with specialists in obstetrics, maternal-fetal medicine, neonatology, intensive care, anesthesia, and nursing. communicate with patients and families regarding diagnosis, clinical status, and management wishes. ajog.org expert review pregnant women, and in the most severe cases, mechanical ventilation might not be sufficient to support adequate oxygenation. if that occurs, limited literature suggests a potential role of extracorporeal membrane oxygenation in pregnancy; use should be considered only in centers that have experience with this technique. 47 whether delivery provides benefit to a critically ill mother is unknown; decisions regarding delivery should consider the gestational age of the fetus and should be made in conjunction with the neonatologist. 48 there are currently no antiviral medications approved by the us food and drug administration for treatment of covid-19, although broad-spectrum antivirals used in animal models of mers are being evaluated for activity against sars-cov-2. 46 corticosteroids for the treatment of coronavirusassociated pneumonia should be avoided unless other indications are present because they were not shown to be beneficial in mers and could lead to delayed mers-cov clearance. 49 therefore, decisions about the use of corticosteroids for fetal lung maturity should be made in consultation with infectious disease specialists and maternal-fetal medicine consultants. all guidance should be considered subject to revision as additional data on pregnant women with covid-19 become available. care of infants born to mothers with covid-19. although the limited experience with newborn evaluations after delivery with sars and mers has not identified cases of maternalto-fetal transmission, reports have appeared in the media of a 30 hour infant who was diagnosed with covid-19, suggesting the possibility of in utero transmission. 50 however, insufficient information is included in media reports to rule out perinatal or postnatal modes of transmission. data from the recent case series published by chen et al 44 and zhu et al 45 of 18 women (19 infants) infected in the third trimester of pregnancy with sars-cov-2 identified no laboratory evidence of vertical transmission. testing of amniotic fluid, cord blood, and neonatal throat swab samples was negative for sars-cov-2 in the 6 patients reported by chen et al. 44 in the report by zhu et al., 45 some infants were symptomatic (shortness of breath in 6, cyanosis in 3, gastric bleeding in 2, and 1 baby died of multiple organ failure and dic); however, throat swab testing of all infants was negative for sars-cov-2 , suggesting that these neonatal complications might not be related intrauterine transmission. thus, at this time, it is unknown whether sars-cov-2 can be transmitted from mother-to-fetus. given the current lack of information, it seems reasonable to assume that a newborn born to a mother with covid-19 at delivery could possibly be infected, either in utero or perinatally, and thus should be placed in isolation to avoid exposure to other newborns. although the ideal setting for a healthy infant is within a healthy mother's room, temporary separation of an ill mother and her infant, as was recommended during pandemic h1n1, 51 seems prudent. whether covid-19 can be transmitted through breastmilk is unknown. we are aware of a single report of sars-cov testing of breastmilk in a mother who had recovered from sars and no viral rna was detected; however, the specimen was collected w130 days after illness onset. 15 sars-cov antibodies were seen in breastmilk of that patient 15 but not in another patient who was infected at 7 weeks' gestation with breastmilk tested at postpartum days 12 and 30. 16 breastmilk was tested for sars-cov-2 in 6 of the mothers reported by chen et al 44 ; all specimens were negative. until additional data are available, mothers who intend to breastfeed and are well enough to express breastmilk should be encouraged to do so; breastfeeding can be instituted after she is no longer considered infectious. no data are available to guide length of separation and will need to be decided on a case-by-case basis after discussion between infection control experts and neonatologists. the covid-19 outbreak is rapidly increasing in the number of cases, deaths, and countries affected. much is unknown about the virus and its effects, including its modes of transmission, the basic reproduction number, risk factors for illness, and case fatality rate. although cases are primarily in china, it is highly likely that there will be additional global spread of the virus. at the present time, limited data are available on pregnant women with covid-19 on which to base recommendations for pregnancy-specific care; however, early reports and lessons from sars, mers, and other respiratory infections suggest that pregnant women could have a severe clinical course. surveillance systems for cases of covid-19 need to include information on pregnancy status as well as maternal and fetal outcomes. it is important to be vigilant about the spread of the disease and be able to provide rapid implementation of outbreak control and management measures once the virus reaches a community. standard interventions to manage any severe respiratory infection is the foundation of care for any pregnant woman with covid-19 and should be implemented aggressively in a team-based care model. public health approach to emerging infections among pregnant women pandemic 2009 influenza a (h1n1) virus illness among pregnant women in the united states characterizing the pattern of anomalies in congenital zika syndrome for pediatric clinicians zika vrus and birth defectsreviewing the evidence for causality preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak world health organization. coronavirus disease (covid-19) outbreak. available at: https:// expert review ajog.org www.who.int/emergencies/diseases/novelcoronavirus-2019 severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group severe acute respiratory syndrome: historical, epidemiologic, and clinical features the role of superspreaders in infectious disease pregnancy and perinatal outcomes of women with severe acute respiratory syndrome infants born to mothers with severe acute respiratory syndrome sars in newborns and children emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report a casecontrolled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome sars and pregnancy: a case report sars during pregnancy, united states severe acute respiratory syndrome in pregnancy specific immunoglobulin g antibody detected in umbilical blood and amniotic fluid from a pregnant woman infected by the coronavirus associated with severe acute respiratory syndrome possible central nervous system infection by sars coronavirus the effect of severe acute respiratory syndrome on a hospital obstetrics and gynaecology service managing obstetrical patients during severe acute respiratory syndrome outbreak first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities middle east respiratory syndrome (mers) worldwide reduction in mers cases and deaths since 2016 middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases and review of the literature impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia middle east respiratory syndrome coronavirus during pregnancy stillbirth during infection with middle east respiratory syndrome coronavirus contact tracing the first middle east respiratory syndrome case in the philippines mers-cov infection in a pregnant woman in korea clinical features of patients infected with 2019 novel coronavirus in wuhan, china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china who collaborating centre for infectious disease modelling, mrc centre for global infectious disease analysis diagnosis and treatment of 2019 novel coronavirus infection in children: a pressing issue first case of 2019 novel coronavirus in the united states evidence of airborne transmission of the severe acute respiratory syndrome virus pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses pregnant women and the ebola crisis clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia coronavirus infections-more than just the common cold extracorporeal membrane oxygenation (ecmo) during pregnancy and postpartum management of acute respiratory failure in pregnancy corticosteroid therapy for critically ill patients with middle east respiratory syndrome can coronavirus pass from mother to baby? maybe, but experts need more research preparing for influenza after 2009 h1n1: special considerations for pregnant women and newborns receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars. j virol, in press. for disease control and prevention. interim infection prevention and control recommendations for patients with confirmed 2019 novel coronavirus (2019-ncov) or patients under investigation for 2019-ncov in healthcare settings centers for disease control and prevention. interim guidance for implementing home care of people not requiring hospitalization for 2019 novel coronavirus (2019-ncov) ajog.org key: cord-259658-rgrt6e6r authors: yan, bingpeng; chu, hin; yang, dong; sze, kong-hung; lai, pok-man; yuan, shuofeng; shuai, huiping; wang, yixin; kao, richard yi-tsun; chan, jasper fuk-woo; yuen, kwok-yung title: characterization of the lipidomic profile of human coronavirus-infected cells: implications for lipid metabolism remodeling upon coronavirus replication date: 2019-01-16 journal: viruses doi: 10.3390/v11010073 sha: doc_id: 259658 cord_uid: rgrt6e6r lipids play numerous indispensable cellular functions and are involved in multiple steps in the replication cycle of viruses. infections by human-pathogenic coronaviruses result in diverse clinical outcomes, ranging from self-limiting flu-like symptoms to severe pneumonia with extrapulmonary manifestations. understanding how cellular lipids may modulate the pathogenicity of human-pathogenic coronaviruses remains poor. to this end, we utilized the human coronavirus 229e (hcov-229e) as a model coronavirus to comprehensively characterize the host cell lipid response upon coronavirus infection with an ultra-high performance liquid chromatography-mass spectrometry (uplc–ms)-based lipidomics approach. our results revealed that glycerophospholipids and fatty acids (fas) were significantly elevated in the hcov-229e-infected cells and the linoleic acid (la) to arachidonic acid (aa) metabolism axis was markedly perturbed upon hcov-229e infection. interestingly, exogenous supplement of la or aa in hcov-229e-infected cells significantly suppressed hcov-229e virus replication. importantly, the inhibitory effect of la and aa on virus replication was also conserved for the highly pathogenic middle east respiratory syndrome coronavirus (mers-cov). taken together, our study demonstrated that host lipid metabolic remodeling was significantly associated with human-pathogenic coronavirus propagation. our data further suggested that lipid metabolism regulation would be a common and druggable target for coronavirus infections. coronaviruses are enveloped viruses with a large single-strand, positive-sense rna genome [1, 2] . as of today, there are a total of six coronaviruses that are known to infect humans, including human coronavirus oc43 (hcov-oc43), human coronavirus 229e (hcov-229e), severe acute respiratory syndrome coronavirus (sars-cov), human coronavirus hku1 (hcov-hku1), human coronavirus nl63 (hcov-nl63), and the middle east respiratory syndrome coronavirus (mers-cov) [3] . these human-pathogenic coronaviruses cause a broad range of clinical manifestations. hcov-oc43, hcov-229e, hcov-hku1, and hcov-nl63 cause mild, self-limiting upper respiratory tract infections. in contrast, sars-cov and the recently emerged mers-cov may cause severe pneumonia with acute respiratory distress syndrome, multi-organ failure, and death in both immunocompetent and immunocompromised hosts [4] [5] [6] [7] . lipids play crucial roles at various stages in the virus life cycle. first, lipids can serve as the direct receptors or entry co-factors for enveloped and non-enveloped viruses at the cell surface or the endosomes [8, 9] . second, lipids and lipid synthesis play important roles in the formation and function of the viral replication complex [10, 11] . third, lipid metabolism can generate the required energy for efficient viral replication [12] . moreover, lipids can dictate the proper cellular distribution of viral proteins, as well as the trafficking, assembly, and release of virus particles [13, 14] . in this regard, the host lipid biogenesis pathways play indispensable roles in modulating virus propagation. as in other viruses, lipids play key roles in the life cycle of coronaviruses. coronaviruses confiscate intracellular membranes of the host cells to generate new compartments known as double membrane vesicles (dmvs) for the amplification of the viral genome. dmvs are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors, which collectively orchestrate a unique lipid micro-environment optimal for coronavirus replication [15] . a recent study indicated that a key lipid processing enzyme, cytosolic phospholipase a2α enzyme (cpla2α) that belongs to the phospholipase a2 (pla2) superfamily, was closely associated with dmvs' formation and coronaviruses' replication [16] . the viral protein and rna accumulation, as well as the production of infectious virus progeny, were significantly diminished in the presence of cpla2α inhibitor [16] . at the same time, phospholipase a2 group iid (pla2g2d), an enzyme that predominantly contributes to anti-inflammatory/pro-resolving lipid mediator expression, contributed to worsened outcomes in mice infected with sars-cov by modulating the immune response [17] . however, to date, the change and modulating effects of the specific lipids involved in lipid rearrangement upon coronavirus infection remains largely unexplored. to obtain a comprehensive and unbiased profile of perturbed lipids upon coronavirus infection, we performed mass spectrometry (ms)-based lipidomics profiling on coronavirus-infected cells using hcov-229e as a model virus. specific lipids including glycerophospholipids and fatty acids (fas) upon virus infection were identified, which represented the lipid species that were rearranged by hcov-229e infection. further pathway analysis revealed that the linoleic acid (la) and arachidonic acid (aa) metabolism axis was the most perturbed pathway upon hcov-229e infection. importantly, supplement of additional la and aa to coronavirus-infected cells significantly inhibited virus replication of both hcov-229e and the highly virulent mers-cov, suggesting that the la-aa metabolism axis is a common and essential pathway that could modulate coronavirus replication. in this regard, temporal modulation of the host lipid profile is a potential novel strategy to combat emerging human coronaviruses. high performance liquid chromatography (hplc)-grade methanol, acetonitrile, chloroform and 2-propanol were purchased from merck (darmstadt, germany). hplc-grade water was prepared using a milli-q water purification system (millipore, burlington, ma, usa). analytical grade acetic viruses 2019, 11, 73 3 of 16 acid and commercial standards used for biomarker identification were purchased from sigma-aldrich (st. louis, mo, usa). internal standards (is) including arachidonic acid-d8, 15(s)-hete-d8, leukotriene-b4-d4 and platelet-activating factor c-16-d4 (paf c-16-d4) were purchased from cayman chemical (ann arbor, mi, usa) [18] . huh-7 and veroe6 cells were maintained in dulbecco's modified eagle medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum (fbs), 100 u/ml penicillin, and 100 g/ml streptomycin (5% co 2 at 37 • c). mers-cov (emc/2012 strain) was kindly provided by professor ron fouchier (erasmus medical center, rotterdam, the netherlands). mers-cov and hcov-229e were cultured in veroe6 cells in serum-free dmem supplemented with 100 u/ml penicillin and 100 g/ml streptomycin as we described previously [19] [20] [21] . the supernatants were harvested when cytopathic effects (cpe) were observed and centrifuged to generate the viral stocks. the viral stocks were titrated by plaque assay on veroe6 cells and stored at −80 • c as previously described [22, 23] . briefly, confluent veroe6 cells were infected with 10-fold serial viral dilutions. the cells were incubated with diluted viruses at 37 • c for 1 h and subsequently overlaid with 1% low-melting-point agarose (promega, madison, wi, usa). the cells were fixed with 4% formaldehyde as the plaques were observed and then stained with 0.2% crystal violet. all experiments involving live mers-cov followed the approved standard operating procedures of the biosafety level 3 facility as previously described [24] [25] [26] [27] . huh-7 cells were seeded into 24-well plate to reach 90% confluency and infected with mers-cov or hcov-229e at multiplicity of infection (moi) of 0.005 or 1, respectively. after 1 h of inoculation, the cells were washed with phosphate-buffered saline (pbs) and maintained in lipids-supplemented medium at the indicated concentrations for 24 h. aa, la, oleic acid (oa), and palmitic acid (pa) were dissolved in ethanol and ethanol was used as a negative control. the lipids were purchased from cayman chemical (ann arbor, mi, usa). the supernatants and cell lysates were collected at 24 h post-infection. the viral genome copy numbers were determined by reverse-transcription quantitative polymerase chain reaction (rt-qpcr) as previously described [28] [29] [30] . confluent huh-7 cells were mock infected or infected with hcov-229e at moi of 1 and incubated in dmem medium. at 24 hpi, cells were collected for cellular lipid extraction. the lipid extraction was performed for liquid chromatography-mass spectrometry (lc-ms) analysis according to a previously described protocol with slight modifications [31, 32] . inactivation of virus infectivity was confirmed before further processing as we previously described with some modifications [33] . briefly, 500 µl of ice-cold 150 mm ammonium bicarbonate solution was added to dissociate cells. two millilitres of chloroform/methanol (v/v 2:1) containing is were added, followed by vortexing and centrifugation at 4500 rpm for 10 min at 4 • c. the bottom phase was transferred to glass vials and dried using a vacuum concentrator for storage at −80 • c. the dried samples were reconstituted in 250 µl solvent mixture containing methanol/2-propanol/water (v/v/v 5:4:1) for lc-ms analysis. after centrifugation at 14,000 rpm for 10 min at 4 • c, supernatants were transferred to lc vials for lc-ms analysis. the lipid extract was analyzed using an acquity uplc system coupled to a synapt g2-si high definition mass spectrometry (hdms) system (waters corp., milford, ma, usa). the chromatography was performed on a waters acquity beh c18 column (1.7 µm, 2.1 × 100 mm, i.d., 1.7 mm, waters corp., milford, ma, usa). the mobile phase consisted of (a) 0.1% acetic acid in water and (b) acetonitrile. gradient elution applied for ultra-high performance liquid chromatography-mass spectrometry (uplc-ms) analysis was described in table s1 . the column and autosampler temperature were maintained at 45 • c and 4 • c, respectively. the injection volume was 5 µl [34] . the mass spectral data were acquired in both positive and negative modes. the capillary voltage, sampling cone voltage and source offset were maintained at 2.5 kv, 60 v, and 60 v, respectively. nitrogen was used as desolvation gas at a flow rate of 800 l/h. the source and desolvation temperatures were maintained at 120 • c and 400 • c, respectively. mass spectra were acquired over the m/z range of 50 to 1200. the synapt g2-si hdms system was calibrated using sodium formate clusters and operated in sensitivity mode. leucine enkephalin was used as a lock mass for all experiments. ms/ms acquisition was operated in the same parameters as ms acquisition. collision energy was applied at the range from 20 to 40 ev for fragmentation to allow putative identification and structural elucidation of the significant lipids. acquisition of the raw data was performed using masslynx software version 4.1 (waters corp., milford, ma, usa) and raw data were converted to the common data format (netcdf) files using conversion software databridge (waters corp., milford, ma, usa). the netcdf data were subsequently deconvolved into a usable data matrix using the xcms software (http://metlin.scripps. edu/download/) [35] and the grouping of features was performed using the camera r package [36] . preprocessed data were then exported as a .csv file for further data statistical analysis. metaboanalyst 3.0 (http://www.metaboanalyst.ca) and simca-p v12.0 (umetrics, umeå, sweden) were used for univariate and multivariate statistical analysis, respectively [37] . for univariate analysis, statistical significance of features was determined between the mock and hcov-229e infected group using the student's t-test and fold change. the p-value < 0.05 and fold change > 2 were used as criteria for significant features selection. for multivariate analysis, the features were subjected to pareto scaling firstly then orthogonal partial least squares discriminant analysis (opls-da) was performed as a supervised method to find important variables with discriminative power. the opls-da model was evaluated with the relevant r2 and q2. the variable importance in projection (vip), which reflects both the loading weights for each component and the variability of the response explained by this component, was used for feature selection [38] . ms/ms fragmentation was performed on the significant features with high abundances. the significant features identification were carried out by searching accurate ms and ms/ms fragmentation pattern data in the metlin database (metabolomics database, http://metlin.scripps. edu/), human metabolome database (http://www.hmdb.ca/), and lipd maps (lipidomics gateway, http://www.lipidmaps.org/). for confirmation of lipid identity using authentic chemical standard, ms/ms fragmentation pattern of the chemical standard was compared with that of candidate lipid under the same lc-ms condition to reveal any matching [18, 39] . to investigate how coronavirus perturbs host lipid metabolism, we performed lipidomics analysis on hcov-229e-infected huh7 cells and compared the results with those of the mock-infected cells. the preliminary features list included precursor ions, adducts and isotope ions, which were imported into the metaboanalyst and simca-p software for further analysis. the r2x/ r2y, represented the x/y variables explanation rate of the opls-da model, were 83.0% and 98.8%, respectively. the predicted component, as estimated by cross-validation, was 0.97 (q2). these cross-validated parameters were satisfactory for opls-da mode (supplementary figure s1a) . at the same time, the permutation test (100 times) also indicated that the validated model was satisfied (supplementary figure s1b) . overall, our results demonstrated that these significant lipid features could be selected by the validated statistical model for subsequent identification. a total of 206 (positive mode) and 100 (negative mode) ion features were selected according to the omics-based statistical analysis method. these ion features were significantly discriminative between hcov-229e-infected and mock-infected cells. to observe the discrimination trend in more detail, a hierarchical clustering analysis was performed based on the degree of similarity of lipid abundance profiles to show the overview trend of all significant ion features. as indicated in figure 1 , most of the significant features from both negative mode ( figure 1a ) and positive mode ( figure 1b ) expressed an up-regulation trend after hcov-229e infection compared with the mock infection controls. furthermore, to identify lipids specific to hcov-229e infection, these significant features were grouped and annotated using the camera software, and the potential precursor ions were used to perform further ms/ms experiments for obtaining their fragmentation patterns. finally, a total of 24 lipids were identified, which could be classified into three lipid classes, including lysophosphatidylcholine (lysopc), lysophosphatidylethanolamine (lysope) and fatty acid (fa). the chromatogram peak heights of these identified lipids were generated by lc-ms raw data and the ratio between infected and mock-infected cells was determined. as demonstrated in figure 2 , we found a consistent up-regulation trend of the identified lipids in hcov-229e-infected cells. in particular, lysopc was the predominant lipid class of all identified, accounting for approximately 60% of all identified lipids with significant elevation (figure 2a ). at the same time, arachidonic acid (aa), which belongs to the fa class, showed the highest increase in fold-change among all identified lipids with a maximum of 7.1-fold increase ( figure 2b ). in addition, the level of lysopes ( figure 2c ) was also up-regulated with a maximum fold change of 2.93, which was comparatively less than that of the lysopcs and fas. the identities of lysopc (16:0/0:0), platelet-activating factor c-16 (paf c-16), lysope (16:0/0:0), aa, la, pa and oa were confirmed by matching the retention time (rt) and ms/ms fragmentation patterns of the authentic chemical standards that distinguish between hcov-229e-infected cases and non-infected controls ( figure 3 ). the detailed information of the 24 identified lipids was listed in table 1 based on the list of significantly up-regulated lipids after hcov-229e-infection, metaboanalyst (http://www.metaboanalyst.ca) was applied to investigate which pathway might be markedly perturbed. the result of the pathway analysis was graphically presented in figure 4 . from the enrichment analysis results, the la metabolism pathway and fa biosynthesis pathway had a statistically significant raw p-value (raw p < 0.05, as shown in the y-axis). pathway impact results indicated that the la metabolism and aa metabolism pathways presented higher impact than the other pathways, as indicated in the x-axis value. combining the above two analysis results, we postulated that the la metabolism pathway to be a markedly perturbed pathway that correlated with the lipid rearrangement process induced by hcov-229e infection. from the enrichment analysis results, the la metabolism pathway and fa biosynthesis pathway had a statistically significant raw p-value (raw p < 0.05, as shown in the y-axis). pathway impact results indicated that the la metabolism and aa metabolism pathways presented higher impact than the other pathways, as indicated in the x-axis value. combining the above two analysis results, we postulated that the la metabolism pathway to be a markedly perturbed pathway that correlated with the lipid rearrangement process induced by hcov-229e infection. to better understand the current pathway analysis results and the cellular lipid signaling response upon hcov-229e infection, we constructed a global la pathway map based on the pathway information in the kyoto encyclopedia of genes and genomes (kegg) database (https://www.genome.jp/kegg/) and literature mining ( figure 5 ). upon hcov-229e infection, the glycerophospholipids, as main components of the cell membrane, were metabolized to lysophospholipids and fas after cpla2 enzyme activation. lysophospholipids such as lysopcs and lysopes were correspondingly increased after hcov-229e infection. moreover, lysopcs could be further metabolized to platelet-activating factor. fas were also released from glycerophospholipids but only la and aa could initiate downstream pathways to generate corresponding metabolites. the up-regulation of both lysophospholipids and fas were partially confirmed by authentic standards. furthermore, to investigate the downstream pathways trend of fas, the authentic standards were also applied in lc-ms method to confirm whether these downstream lipids were changed correspondingly. as illustrated in figure 5 , aa is a downstream lipid of la and the origin lipid of aa metabolism pathway. the identity of aa was confirmed by authentic standard (supplementary figure s2d) , which was found to be significantly up-regulated. therefore, combining pathway analysis and the authentic standards verification results, our data to better understand the current pathway analysis results and the cellular lipid signaling response upon hcov-229e infection, we constructed a global la pathway map based on the pathway information in the kyoto encyclopedia of genes and genomes (kegg) database (https://www.genome.jp/kegg/) and literature mining ( figure 5 ). upon hcov-229e infection, the glycerophospholipids, as main components of the cell membrane, were metabolized to lysophospholipids and fas after cpla2 enzyme activation. lysophospholipids such as lysopcs and lysopes were correspondingly increased after hcov-229e infection. moreover, lysopcs could be further metabolized to platelet-activating factor. fas were also released from glycerophospholipids but only la and aa could initiate downstream pathways to generate corresponding metabolites. the up-regulation of both lysophospholipids and fas were partially confirmed by authentic standards. furthermore, to investigate the downstream pathways trend of fas, the authentic standards were also applied in lc-ms method to confirm whether these downstream lipids were changed correspondingly. as illustrated in figure 5 , aa is a downstream lipid of la and the origin lipid of aa metabolism pathway. the identity of aa was confirmed by authentic standard (supplementary figure s2d) , which was found to be significantly up-regulated. therefore, combining pathway analysis and the authentic standards verification results, our data suggested that the la-aa metabolism axis was the most significantly perturbed pathway and might be associated with lipids rearrangement or other processes in hcov-229e infection. suggested that the la-aa metabolism axis was the most significantly perturbed pathway and might be associated with lipids rearrangement or other processes in hcov-229e infection. to investigate the potential implication of the perturbed la-aa metabolism axis in hcov-229e infection, we treated hcov-229e-infected huh7 cells with la and aa and included pa and oa for comparison. the la and aa were mapped and played a vital role in the perturbed la-aa metabolism axis ( figure 5 ). in contrast, pa and oa were not mapped in the perturbed pathway and may only be produced from glycerophospholipids due to cpla2 enzyme activation. huh-7 cells were infected with hcov-229e and treated with aa, la, pa, or oa. the cell lysates and culture supernatants were harvested at 24 h post-infection to determine the viral genome copy number by rt-qpcr. as shown in figure 6 , la and aa consistently inhibited the replication of hcov-229e as evidenced by the decrease in virus genome copies in both cell lysate ( figure 6a ,c) and supernatant samples ( figure 6b-d) . in contrast, pa inhibited hcov-229e replication only when supplied at high concentration while hcov-229e replication was largely independent of oa ( figure 6a-d) . to further investigate if the modulatory effects of la and aa were conserved among other human-pathogenic coronaviruses, we evaluated the effects of these lipids on the replication of the recently emerged and highly virulent mers-cov. our data demonstrated that la and aa potently suppressed mers-cov replication in a similar manner as hcov-229e ( figure 6e,f) . overall, our results demonstrated that exogenously supplied la and aa could interfere with the optimal to investigate the potential implication of the perturbed la-aa metabolism axis in hcov-229e infection, we treated hcov-229e-infected huh7 cells with la and aa and included pa and oa for comparison. the la and aa were mapped and played a vital role in the perturbed la-aa metabolism axis ( figure 5 ). in contrast, pa and oa were not mapped in the perturbed pathway and may only be produced from glycerophospholipids due to cpla2 enzyme activation. huh-7 cells were infected with hcov-229e and treated with aa, la, pa, or oa. the cell lysates and culture supernatants were harvested at 24 h post-infection to determine the viral genome copy number by rt-qpcr. as shown in figure 6 , la and aa consistently inhibited the replication of hcov-229e as evidenced by the decrease in virus genome copies in both cell lysate ( figure 6a ,c) and supernatant samples ( figure 6b-d) . in contrast, pa inhibited hcov-229e replication only when supplied at high concentration while hcov-229e replication was largely independent of oa ( figure 6a-d) . replication of human-pathogenic coronaviruses, which suggested that the la-aa metabolism axis was significantly involved in the propagation of these viruses. to further investigate if the modulatory effects of la and aa were conserved among other human-pathogenic coronaviruses, we evaluated the effects of these lipids on the replication of the recently emerged and highly virulent mers-cov. our data demonstrated that la and aa potently suppressed mers-cov replication in a similar manner as hcov-229e ( figure 6e,f) . overall, our results demonstrated that exogenously supplied la and aa could interfere with the optimal replication of human-pathogenic coronaviruses, which suggested that the la-aa metabolism axis was significantly involved in the propagation of these viruses. in this study, a ms-based lipidomics approach was established to characterize the host cell lipid changes upon coronavirus infection. univariate and multivariate statistical analyses were applied in data processing for the selection of significant lipid features. a total of 24 lipids including lysophospholipids and fas were identified and were consistently up-regulated in hcov-229e-infected cells. seven representative lipids were confirmed by authentic standards, including lysopc (16:0/0:0), paf c-16, lysope (16:0/0:0), aa, la, pa and oa. subsequent pathway analysis indicated that the la-aa metabolism axis, consisting of la and aa as important precursor lipids, was substantially perturbed after hcov-229e infection. moreover, we demonstrated that exogenously supplied la and aa were capable of inhibiting the replication of hcov-229e and the highly pathogenic mers-cov, which suggested the la-aa metabolism axis to be a conserved and essential pathway in the propagation of human coronaviruses. a total of 24 lipids including lysopcs, lysopes and unsaturated/saturated fas were identified to be significantly upregulated after hcov-229e infection. twenty of these 24 (83.3%) lipids were lysopc and lysope. lysopc is the most abundant lysophospholipid in humans, with a high plasma concentration of several hundred micromoles. in addition, lysopc was a potent inhibitor and could reversely arrest pore expansion during syncytium formation mediated by diverse viral fusogens [40] . another lysophospholipid, lysope, is present at low concentrations in vivo but they induce various cellular responses such as activation of mitogen-activated protein kinase (mapk) and neuronal differentiation when applied to cells in vitro [41] . among the identified fas, the la and aa both belong to polyunsaturated omega-6 fatty acid and are essential fatty acids. in addition, la is the metabolic precursor of aa, both of which are key components of the cell membrane. la and aa also play fundamental roles in the biological function of many tissues by modulating enzymes, ion channels, receptors, as well as inflammation [42] . coronavirus replication is associated with intracellular membrane rearrangement and depends on the formation of double membrane vesicles (dmvs) and other membranous structures as replicative organelles [16] . the cell membrane components consist mainly of glycerophospholipid components such as phosphatidylcholine (pc), phosphatidylethanolamine (pe), lysophosphatidylcholine (lysopc), and lysophosphatidylethanolamine (lysope). a specific phospholipids composition is required by different viruses to form the optimal replicative organelles best suited for their replication [43] . moreover, the lysopc/pe was produced from pc/pe by cpla2 activation, which simultaneously generated corresponding fatty acid moiety. in this regard, cpla2 activation is commonly believed to be beneficial for virus replication [16, 17] . in our study, we found that a number of lysophospholipids and fas downstream of cpla2 activation, were upregulated upon hcov-229e infection. the upregulation of these lipid species including la and aa were believed to promote efficient coronavirus replication. however, when we evaluate this hypothesis by exogenously supplementing additional la and aa to hcov-229e-or mers-cov-infected cells, we noticed a significant reduction in virus replication. taken together, our data suggested that coronavirus infection did not randomly perturb the cellular lipid compositions. instead, we speculate that coronaviruses precisely modulate and rearrange the host lipid profile to reach an intricate homeostasis optimized for its replication. any exogenous manipulation that disrupts the equilibrium may interfere with the optimal replication of the viruses. alternatively, supplementing la and aa might disturb the la-aa metabolism axis and result in feedback reversion of lysophospholipids into phospholipids through land's cycle [44] , thus limiting virus replication. in addition, la and aa are polyunsaturated fatty acids that are biological signaling precursors. they can be metabolized to important eicosanoids and metabolites, which play multiple roles in the host immune response and the pathogenesis of viral infections [45] [46] [47] . however, previous study had suggested that arachidonic acid (aa) downstream metabolites show no evidence of anti-coronaviral activity as observed through special inhibitors of cyclooxygenases (cox) 1/2 and 5-lipoxygenase (lox), which are two key enzymes requiring aa as a precursor. the results indicated aa downstream products may not have a significant effect on coronaviruses replication, at least in vitro [16, 48] . in this regard, the function of the downstream metabolites of la and aa may play key roles in the pathogenesis of coronaviruses in vivo. in the present study, we revealed that the cellular lipid profile was rearranged upon hcov-229e infection. a total of 24 lipids including lysopcs, lysopes and fas were upregulated. among them, la and aa, which were mapped into the la-aa metabolism axis, demonstrated strong modulatory effects on the replication of hcov-229e and the highly pathogenic mers-cov. in this regard, our data suggested that optimal coronavirus replication required a specific composition of cellular lipids and any disruption could decrease the efficiency of coronavirus replication. thus, the ms-based lipidomics strategy could be used to monitor virus-specific lipid requirement, to discover the perturbed pathways and identify novel lipids to interfere with virus replication. in further studies, combining lipidomics data with biological and immunological data may help to elucidate specific pathogenic mechanisms and identify novel treatment strategies for virus infections. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/11/1/73/s1, table s1 : gradient elution program applied for uplc-ms analysis, figure s1 : opls-da model validation and permutation test. the funding sources had no role in study design, data collection, analysis or interpretation or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? interspecies transmission and emergence of novel viruses: lessons from bats and birds the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection coronavirus as a possible cause of severe acute respiratory syndrome coronaviruses-drug discovery and therapeutic options glycosphingolipids as receptors for non-enveloped viruses virus entry, assembly, budding, and membrane rafts. microbiol building viral replication organelles: close encounters of the membrane types viral reorganization of the secretory pathway generates distinct organelles for rna replication temporal proteome and lipidome profiles reveal hepatitis c virus-associated reprogramming of hepatocellular metabolism and bioenergetics phosphatidylinositol (4,5) bisphosphate regulates hiv-1 gag targeting to the plasma membrane influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum inhibition of cytosolic phospholipase a2alpha impairs an early step of coronavirus replication in cell culture critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection uhplc-ltq-orbitrap ms combined with spike-in method for plasma metabonomics analysis of acute myocardial ischemia rats and pretreatment effect of danqi tongmai tablet differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment middle east respiratory syndrome coronavirus and bat coronavirus hku9 both can utilize grp78 for attachment onto host cells carcinoembryonic antigen-related cell adhesion molecule 5 is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by severe acute respiratory syndrome coronavirus and human coronavirus 229e a method for simultaneous pathogen inactivation and extraction of samples for multi-omics profiling a robust and universal protocol for single-sample integrative proteomic, metabolomic, and lipidomic analyses talaromyces marneffei mp1p is a virulence factor that binds and sequesters a key proinflammatory lipid to dampen host innate immune response srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target xcms: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification camera: an integrated strategy for compound spectra extraction and annotation of liquid chromatography/mass spectrometry data sets metaboanalyst 3.0-making metabolomics more meaningful variable influence on projection (vip) for orthogonal projections to latent structures (opls) colon-derived uremic biomarkers induced by the acute toxicity of kansui radix: a metabolomics study of rat plasma and intestinal contents by uplc-qtof-ms(e) lysophosphatidylcholine reversibly arrests pore expansion during syncytium formation mediated by diverse viral fusogens novel lysophosphoplipid receptors: their structure and function arachidonic acid: physiological roles and potential health benefits-a review rna virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes metabolic interactions between the lands cycle and the kennedy pathway of glycerolipid synthesis in arabidopsis developing seeds the arachidonic acid metabolome serves as a conserved regulator of cholesterol metabolism the role of arachidonic acid in the regulation of nitric oxide synthase isoforms by hiv gp120 protein in astroglial cells. free radic an insight into the role of arachidonic acid derived lipid mediators in virus associated pathogenesis and malignancies l-lysine acetylsalicylate + glycine impairs coronavirus replication key: cord-009476-4emc4o6n authors: madani, tariq a title: case definition and management of patients with mers coronavirus in saudi arabia date: 2014-09-22 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70918-1 sha: doc_id: 9476 cord_uid: 4emc4o6n nan the threat to global health security from emerging and re-merging respiratory tract infections will be ever present because of the genetic adaptability of microbes, and their ability to resist clinical interventions and public health measures aimed at their elimination. although much has been learned from previous outbreaks, present surveillance systems have their inherent weaknesses, and recent experiences with mers-cov 14 show that pandemic preparedness still faces major political and scientifi c challenges. 15 an important priority for control of infectious disease is to ensure that scientifi c and technological advances in molecular diagnostics and bioinformatics are well integrated into public health. more eff ective and wider partnerships based on equity and best ethical practice, across governments, health care, academia, industry, and with the public, are essential to eff ectively galvanise economic, political and scientifi c measures required to develop core capacities, including legislation, national focal points, and pandemic planning to reduce risk of global spread and reduce the burden of respiratory tract infectious diseases. an urgent need exists to establish trusting and eff ective meaningful collaborations between countries to tackle new emerging microbial threats. this will facilitate early and rapid detection of potential pandemic infectious diseases through public health actions within the framework of the international health regulations. 16 outbreak and prevent human-to-human and animalto-human transmission; an appropriate management algorithm, including best-practice guidelines for accurate diagnosis, infection control, intensive care, emergency medicine, and treatment; prioritise research related to the mers-cov outbreak such as case-control and cohort studies, seroprevalence studies, and clinical trials; and to eff ectively monitor outbreak control activities. a continously operating command and control centre was established in the minister's office. in addition to the advisory council, nine further platforms were established: interministerial to coordinate efforts between the ministry of health (moh) and other concerned ministries; capacitybuilding to recruit and mobilise qualified staff to work in hospitals treating patients with mers-cov, increase the number of beds in intensive care units, and provide state-of-the-art machines such as extracorporeal membrane oxygenation to treat patients with respiratory failure refractory to conventional ventilation; public relations to communicate relevant information to the public, health-care workers, and local and international media; clinical operation to coordinate management of patients and transfers between hospitals; public health to collect data related to patients and their contacts; data analysis to enter and analyse data; epidemiological to provide consultations on data analysis and interpretation; laboratory to ensure fast and reliable diagnostic testing; and, infection control to oversee infection control practice and staff training activities. a mers referral hospital run by well trained staff was designated in riyadh, jeddah, and dammam to receive and manage all patients infected with mers-cov. the moh enforced strict infection prevention as new clinical information became available, a revision of the mers-cov case defi nition seemed appropriate. 2 the new case defi nition (appendix) was developed based on reported health-care-associated mers-cov pneumonia (added as category 2 in the new case defi nition) and non-respiratory characteristics of patients with confi rmed infection who fi rst presented with acute febrile dengue-like illness with body aches, leucopenia, and thrombocytopenia (added as category 3). the new case defi nition added a fourth category for contacts of people with mers-cov who present with not only lower respiratory tract but also isolated upper respiratory tract features. this defi nition classifi ed the status of patients into three categories of suspect, probable, or confi rmed infection. the new mers-cov case defi nition was revised and approved by the advisory council after seeking external cdc expert opinion. an algorithm for mers-cov case management was developed (fi gure). according to this algorithm, patients with confi rmed mers-cov who have no evidence of pneumonia or who recover from pneu monia but remain positive for mers-cov, can be isolated at home after careful assessment of the home situation and suitability for isolation by the treating physician, highly trained social workers, or other health-care professionals by telephone or home visits. the cdc has released recommendations on how to assess the home situation and the advice to be given to patients on home isolation and his or her caregivers and household members, 3 and also released guidance for the public, clinicians, and public-health authorities in the usa on control of the mers-cov infection. 4 ministry of health, riyadh, saudi arabia; and department of medicine, faculty of medicine, king abdulaziz university, jeddah, saudi arabia tmadani@kau.edu.sa tim uyeki, and ray arthur for their critical review of the case defi nition and the mers-cov case management algorithm, and fadwa mushtaq for her secretarial assistance world health organization. who experts probe middle-eastern respiratory syndrome coronavirus (mers-cov world health organization. who revised interim case defi nition for reporting to who-middle east respiratory syndrome coronavirus (mers-cov): as of us centers for disease control and prevention. interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus first confi rmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities health-care-associated bacterial infections are an important cause of morbidity and mortality in critically ill patients, especially patients needing mechanical ventilation. decolonisation with topical antibiotics, such as selective digestive tract decontamination (sdd) or selective oropharyngeal decontamination (sod), eradicates potentially pathogenic bacteria, preventing ventilator-associated pneumonia and bacteraemia. in two dutch studies, 1,2 sdd and sod reduced mortality, intensive-care unit (icu) length of stay, icu-acquired bacteraemia, and carriage with antibiotic-resistant bacteria. accordingly, both measures were deemed cost eff ective. 3 only studies that assessed the unit-wide implementation of sdd or sod provide evidence of a key: cord-256300-emsvxxs5 authors: tortorici, m. alejandra; veesler, david title: structural insights into coronavirus entry date: 2019-08-22 journal: adv virus res doi: 10.1016/bs.aivir.2019.08.002 sha: doc_id: 256300 cord_uid: emsvxxs5 coronaviruses (covs) have caused outbreaks of deadly pneumonia in humans since the beginning of the 21st century. the severe acute respiratory syndrome coronavirus (sars-cov) emerged in 2002 and was responsible for an epidemic that spread to five continents with a fatality rate of 10% before being contained in 2003 (with additional cases reported in 2004). the middle-east respiratory syndrome coronavirus (mers-cov) emerged in the arabian peninsula in 2012 and has caused recurrent outbreaks in humans with a fatality rate of 35%. sars-cov and mers-cov are zoonotic viruses that crossed the species barrier using bats/palm civets and dromedary camels, respectively. no specific treatments or vaccines have been approved against any of the six human coronaviruses, highlighting the need to investigate the principles governing viral entry and cross-species transmission as well as to prepare for zoonotic outbreaks which are likely to occur due to the large reservoir of covs found in mammals and birds. here, we review our understanding of the infection mechanism used by coronaviruses derived from recent structural and biochemical studies. three dimensional 9-o-ac-sia 5-n-acetyl-9-o-acetyl-sialosides ace2 angiotensin-converting enzyme 2 apn amino-peptidase n cov coronavirus cryoem cryo-electron microscopy dpp4 dipeptidyl peptidase 4 fab antigen-binding fragment of an antibody mers-cov middle-east respiratory syndrome coronavirus mhv mouse hepatitis virus pdcov porcine δ-cov pedv porcine epidemic diarrhea virus sars-cov severe acute respiratory syndrome coronavirus tgev transmissible gastroenteritis virus coronaviruses (covs) are enveloped viruses with a positive sense rna genome, that belong to the subfamily coronavirinae within the family coronaviridae, which is part of the nidovirales order. they are classified in four genera (α, β, γ, and δ) and four lineages are recognized whithin the β-cov genus (a, b, c and d). covs cause a variety of respiratory and enteric diseases in mammalian and avian species. until recently, covs were considered to be pathogens with a largely veterinary relevance but with limited impact on human health. however, outbreaks of severe acute respiratory syndrome (sars) in 2002-2004 and of middle-east respiratory syndrome (mers) starting in 2012, with fatality rates of 10% and 35%, respectively, led covs to be recognized as zoonotic threats with pandemic potential. four other covs are endemic in the human population and cause up to 30% of mild respiratory tract infections as well as occasional severe disease in young children, the elderly or immunocompromised individuals (isaacs et al., 1983; su et al., 2016) . these viruses are hcov-nl63 and hcov-229e (α-covs) as well as hcov-oc43 and hcov-hku1 (β-covs). numerous sars-cov and mers-cov-like viruses currently circulate in bats and dromedaries making outbreaks of highly pathogenic human covs a global health threat (ge et al., 2013; haagmans et al., 2014; hu et al., 2017; menachery et al., 2015 menachery et al., , 2016 sabir et al., 2016) . the cov virion contains at least four structural proteins: spike (s), envelope (e), membrane (m) and nucleocapsid (n) . in contrast to other β-cov lineages, lineage a covs also encode a hemagglutinin-esterase which serves as receptor-destroying enzyme to facilitate release of viral progeny from infected cells and escape from attachment to non-permissive host cells or decoys (bakkers et al., 2017 (bakkers et al., , 2016 . s is a class 1 viral fusion protein that promotes host attachment and fusion of the viral and cellular membranes during entry (bosch et al., 2003) . as a consequence, s determines host range and cell tropism. s is also the main target of neutralizing antibodies elicited during infection and the focus of vaccine design. s is trimeric and each protomer is synthesized as a single polypeptide chain of 1100-1600 residues, depending on the cov species. for many covs, s is processed by host proteases to generate two functional subunits, designated s 1 and s 2 , which remain non-covalently bound in the prefusion conformation (bosch et al., 2003) . the s 1 subunit comprises the apex of the s trimer, including the receptor-binding domains, and stabilizes the prefusion state of the s 2 fusion machinery, which is anchored in the viral membrane. for all covs, s is further cleaved by host proteases at the so-called s 2 ' site located immediately upstream of the fusion peptide. this cleavage has been proposed to activate the protein for membrane fusion via large-scale, irreversible conformational changes (heald-sargent and gallagher, 2012; millet and whittaker, 2015) . although several class 1 viral fusion proteins have been extensively studied, cov s proteins have proven reluctant to structural characterization until recently. structural studies were largely limited to x-ray crystallographic analysis of isolated receptor-binding domains in complex with viral receptor ectodomains or neutralizing antibodies (li et al., 2005a; lu et al., 2013; peng et al., 2011; prabakaran et al., 2006; reguera et al., 2012; wang et al., 2013; wong et al., 2017; wu et al., 2009; yu et al., 2015) and of the s 2 postfusion core (duquerroy et al., 2005; gao et al., 2013; supekar et al., 2004; xu et al., 2004a, b; zheng et al., 2006) with the exception of two low-resolution electron microscopy reports (beniac et al., 2006 (beniac et al., , 2007 . in the past few years, however, technical advances in single-particle cryo-electron microscopy (cryoem) (bai et al., 2013; brilot et al., 2012; campbell et al., 2012 campbell et al., , 2015 li et al., 2013; punjani et al., 2017; scheres, 2012) together with the implementation of strategies for the stabilization of cov s proteins in prefusion conformation (pallesen et al., 2017; walls et al., 2017a) led to a surge of structural data for multiple s ectodomain trimers. we review here our current understanding of the mechanism used by covs to infect host cells based on recent structural and biochemical studies of s glycoprotein ectodomains in prefusion and postfusion states as well as complexes with known receptors or neutralizing antibodies. cryoem studies of the s glycoproteins of mouse hepatitis virus (mhv) and hku1 led to the first structures at high-enough resolution to obtain an atomic model of the prefusion state (kirchdoerfer et al., 2016; walls et al., 2016a) . these structures revealed that prefusion s ectodomains are 160 å -long trimers with a triangular cross-section ( fig. 1a and b) . the s 1 subunit adopts a "v" shaped architecture for β and γ covs (gui et al., 2017; kirchdoerfer et al., 2016; shang et al., 2018a; tortorici et al., 2019; walls et al., 2016a; yuan et al., 2017) (fig. 1c) , or a squareshaped organization for αand δ-covs (shang et al., 2018b; walls et al., 2016b; xiong et al., 2018) . the s 1 subunit folds as β-rich domains designated a, b, c, d. several α-covs harbor a likely duplication of their domain a at the n-terminus of the s glycoprotein walls et al., 2016b) . this additional domain, designated domain 0, was visualized in the nl63 s structure and hypothesized to interact with heparan sulfate present at the host cell surface during viral entry (milewska et al., 2014; walls et al., 2016b) . domain a and domain 0 adopt a galectin-like β-sandwich fold conserved across all cov genera (kirchdoerfer et al., 2016; peng et al., 2011 peng et al., , 2012 walls et al., 2016a) (fig. 1d) . domain b, which shows the highest sequence variability within cov s 1 subunits, has a markedly different architecture between α-, β-, γand δ-covs. b domains of β-covs contain a β-sheet core subdomain decorated with a highly variable external subdomain mediating receptor engagement kirchdoerfer et al., 2016; li et al., 2005b; lu et al., 2013; tortorici et al., 2019; walls et al., 2016a; wang et al., 2013) (fig. 1e) . b domains of α-, γand δ-cov form a β-sandwich decorated with loops mediating receptor attachment (reguera et al., 2012; shang et al., 2018a, b; walls et al., 2016b; wong et al., 2017; xiong et al., 2018) . in the context of the s trimer, β/γ cov b domains interact with the a and b domains of another protomer, whereas they pack against the a domain of the same protomer in α/δ-covs (shang et al., 2018a; walls et al., 2016a, b; xiong et al., 2018) . the s 2 subunit, which is more conserved than s 1 , comprises the fusion machinery and connects to the viral membrane. it is assembled from a large number of α-helices, an antiparallel core β-sheet, a β-rich connector domain and a stem helix leading to the heptad-repeat 2 (hr2) and the transmembrane region (fig. 1f ) (gui et al., 2017; kirchdoerfer et al., 2016 kirchdoerfer et al., , 2018 pallesen et al., 2017; shang et al., 2018a, b; tortorici et al., 2019; walls et al., 2016a walls et al., , b, 2017b xiong et al., 2018; yuan et al., 2017) . key s 2 features facilitating virus-cell fusion include the fusion peptide, two heptad repeat regions (named hr1 and hr2) and the transmembrane domain. in the prefusion s conformation, a central helix stretches along the threefold axis, perpendicular to the viral membrane, and is located downstream the hr1 motif, which folds as four consecutive α-helices (kirchdoerfer et al., 2016; walls et al., 2016a) . moreover, an upstream helix runs parallel to and is zipped against the central helix via hydrophobic contacts (fig. 1f ). the cov s 2 subunit shares similarity with the pneumovirus/paramyxovirus f proteins-including a comparable 3d organization of the core β-sheet, the upstream helix and the central helix-suggesting an evolutionary relatedness between the viral fusion proteins of these different viruses and a conservation of their fusion mechanism (mclellan et al., 2013; walls et al., 2016a walls et al., , 2017b wong et al., 2016; xu et al., 2015; yin et al., 2006) . a conserved tryptophan-rich segment (y(v/i)kwpw(y/w)vwl) directly preceding the cov s transmembrane region is crucial for proper trimerization. this segment is also required functionally for formation of a fusion pore (schroth-diez et al., 2000) . furthermore, transmembrane domain interactions within and possibly between s trimers have been proposed to be essential to complete the membrane fusion process (schroth-diez et al., 2000) . the transmembrane domain is followed by an intraviral/cytoplasmic tail of variable length (36-46 residues) depending on the coronavirus species, which contains a palmitoylated cysteine-rich region (of about 18-24 residues with 7-10 cysteines) and a variable c-terminal end (thorp et al., 2006) . the cytoplasmic tail is involved in assembly, intracellular transport, cell-surface expression and cell-cell fusion (bos et al., 1995; bosch et al., 2005; chang et al., 2000; lontok et al., 2004; petit et al., 2005; ye et al., 2004; youn et al., 2005) . currently, no structural information is available for any cov full-length s, hindering our understanding of the influence of the transmembrane and cytoplasmic domains on the conformation of exposed antigenic sites, as previously studied for hiv-1 envelope (chen et al., 2015; dev et al., 2016) . cov entry into susceptible cells is a complex process that requires the concerted action of receptor-binding and proteolytic processing of the s protein to promote virus-cell fusion (heald-sargent and gallagher, 2012; millet and whittaker, 2015) . domain 0, domain a and/or domain b can act as receptor-binding domains and both attachment and entry receptors have been described, depending on the cov species. lineage a β-covs attach via their s domain a to 5-n-acetyl-9-oacetyl-sialosides (9-o-ac-sia) found on glycoproteins and glycolipids at the host cell surface to promote entry into susceptible cells (vlasak et al., 1988) . these include human covs oc43 and hku1, bovine cov (bcov) and porcine hemagglutinating encephalomyelitis virus. we recently identified and visualized by cryoem the hcov-oc43 s sialoside-binding site, which is located in a groove at the surface of domain a ( fig. 2a ) (hulswit et al., 2019; tortorici et al., 2019) . this site is conserved in all other covs known to attach to 9-o-ac-sia (β-covs, lineage a) and shares architectural similarity with the ligand-binding pockets of cov hemagglutinin-esterases and influenza virus c/d hemagglutinin-esterase-fusion glycoproteins, highlighting common structural principles of recognition (bakkers et al., 2017 (bakkers et al., , 2016 hulswit et al., 2019; rosenthal et al., 1998; tortorici et al., 2019) . the current consensus in the field is that hcov-oc43 only utilizes 9-o-ac-sialosides as host receptors. in line with this statement, ligand-interacting residues were shown to be essential for s-mediated viral entry (hulswit et al., 2019; tortorici et al., 2019) and 9-o-ac-sia depletion from target cells resulted in severe decrease in virus infectivity (krempl et al., 1995; vlasak et al., 1988) . free 9-o-ac-sia, however, did not trigger s conformational changes associated with membrane fusion . this observation contrasts with data for sars-cov s, for which addition of the human angiotensin-converting enzyme 2 (ace2) ectodomain (the proteinaceous receptor) promoted s refolding to the postfusion state (song et al., 2018; walls et al., 2019) . these findings suggested that either 9-o-ac-sia-containing receptors differ from proteinaceous receptors in their mode of action, or that an interaction with a yet unidentified proteinaceous receptor is required before or after virus internalization for hcov-oc43 entry into target cells. the sialoside-binding site identified in hcov-oc43 s is not conserved among covs which are also known to interact with sialoglycans to initiate host cell infection but are outside of the lineage a of β-covs, such as mers-cov (β-cov, lineage c) or infectious bronchitis virus (ibv, δ-cov) wickramasinghe et al., 2011) . some α-covs such as transmissible gastroenteritis virus (tgev) and porcine epidemic diarrhea virus (pedv) use domain 0 to attach to sialoglycans, presumably to increase virus concentration at the cell surface and enhance subsequent attachment to proteinaceous receptors schwegmann-wessels et al., 2003) . carbohydrate binding via this domain has been proposed to be a determinant of the tgev enteric tropism since loss of domain 0 appears to correlate with a loss of enteric tropism for porcine respiratory coronavirus (prcov), the latter virus being a naturally occurring tgev variant (krempl et al., 1997) . covs exploit a limited variety of proteinaceous receptors compared with the large number and diversity of viral species. all covs known to engage proteinaceous receptors do so using domain b with the exception of mhv, which binds ceacam1a using domain a (dveksler et al., 1991; peng et al., 2011; williams et al., 1991) . remarkably, viruses from different genera, such as hcov-nl63 (α-cov) and sars-cov (β-cov), can recognize the same region of ace2 (entry receptor) using structurally distinct b domains (hofmann et al., 2005; li et al., 2005a li et al., , 2003 wu et al., 2009 ). many α-covs, including hcov-229e, tgev and prcv, as well as porcine δ-cov (pdcov) utilize aminopeptidase n (apn) as entry receptor (delmas et al., 1992; delmas et al., 1993; li et al., 2018; reguera et al., 2012; wong et al., 2017; yeager et al., 1992) whereas mers-cov uses dipeptidyl peptidase 4 (dpp4) raj et al., 2013; wang et al., 2013) . crystal structures of sars-cov, hcov-nl63, mers-cov, hcov-229e b domains in complex with their cognate receptors provided atomic details of the interacting-interface and identified key residues for cross-species transmission and infection (fig. 2) (li et al., 2005a; lu et al., 2013; wang et al., 2013; wong et al., 2017; wu et al., 2009) . this information will be useful to guide the development of therapeutics and vaccines against human covs. recent cryoem studies revealed that mers-cov s and sars-cov s can adopt open and closed conformations in which the receptor binding site of domain b is exposed and occluded, respectively (gui et al., 2017; kirchdoerfer et al., 2018; pallesen et al., 2017; song et al., 2018; walls et al., 2019; yuan et al., 2017) . in contrast, the mhv, hcov-nl63, hcov-hku1, pdcov, ibv and hcov-oc43 s glycoproteins appear to only adopt a closed conformation (kirchdoerfer et al., 2016; shang et al., 2018a, b; tortorici et al., 2019; walls et al., 2016a, b; xiong et al., 2018) and unknown trigger(s), besides proteolytic activation, might be necessary for these viruses to expose their receptor-binding motifs for recognition to occur. these findings suggest that covs have evolved a fine-tuned mechanism to balance masking of the receptor-binding motifs, putatively to avoid neutralization by the host humoral immune response, and their necessary exposure to enable receptor recognition and infection of host cells (walls et al., 2016b wong et al., 2017) . upon host recognition, covs are internalized via receptor-mediated clathrin-dependent, caveolin-dependent or other uptake pathways (burkard et al., 2014; eifart et al., 2007; inoue et al., 2007; nomura et al., 2004) . for instance, both clathrin-dependent and clathrin/caveolae-independent entry pathways have been reported for sars-cov (inoue et al., 2007; wang et al., 2008) . feline infectious peritonitis virus was suggested to enter host cells via a clathrin/caveolin-independent internalization route (regan et al., 2008; van hamme et al., 2008) whereas a caveolin-dependent endocytic uptake has been suggested for hcov-229e and hcov-oc43 (nomura et al., 2004; owczarek et al., 2018) . several reports have demonstrated the key role of proteolytic processing of cov s for cell-cell fusion activity and/or virus entry into host cells using experiments of inhibition of intracellular proteases (burkard et al., 2014; frana et al., 1985; simmons et al., 2005; yamada and liu, 2009 ) and/or substitutions of residues at the s 1 /s 2 or s 2 ' cleavage sites (belouzard et al., 2009; millet and whittaker, 2014; wicht et al., 2014; yang et al., 2015) . prior to and/or after uptake of the virion by a host cell, the s protein is proteolytically processed by host proteases at one or two cleavage sites and both receptor-binding and proteolytic processing act in synergy to induce large-scale s conformational changes promoting cov entry. one of the cleavage sites is located at the boundary between the s 1 and s 2 subunits (s 1 /s 2 cleavage site), whereas the other is located immediately upstream of the fusion peptide (s 2 ' cleavage site), reviewed in (millet and whittaker, 2015) . cleavage at the s 1 /s 2 site can occur upon viral egress, such as for mhv (frana et al., 1985) , or upon encounter with a target cell, such as for sars-cov (belouzard et al., 2009; bosch et al., 2008; shulla et al., 2011) , to yield two non-covalently associated subunits. this first cleavage event, along with binding to the host receptor, promotes further cleavage at the s 2 ' site for sars-cov s (belouzard et al., 2009 ) and mers-cov s (millet and whittaker, 2014; park et al., 2016) . proteolysis at the conserved s 2 ' site is essential for fusion activation of all characterized cov s proteins, and it can occur at the host membrane or in internal cellular compartments of the target cell (belouzard et al., 2009; burkard et al., 2014; millet and whittaker, 2015; park et al., 2016) . cleavage at the mers-cov s 1 /s 2 site by furin during viral egress enables subsequent exposure of the s 2 ' site upon binding to the host receptor and a second cleavage step by serine proteases anchored in the membrane of the target cells, eventually leading to fusion at the cytoplasmic membrane (early entry) . conversely, mers-cov budding with uncleaved s glycoproteins traffic to the endosomes of target cells where cathepsin l or other proteases promote membrane fusion (late entry) . the former mechanism has been proposed to be the route of mers-cov entry into cell types relevant to lung infection, and therefore a significant determinant of mers-cov virulence . moreover, tetraspanin cd9 has been implicated in clustering dpp4 and transmembrane serine proteases to promote early entry of mers-cov (earnest et al., 2017 (earnest et al., , 2015 . pedv, which replicates in the epithelial cells of the small intestine, undergoes s proteolytic activation by trypsin, which is highly abundant in the intestinal lumen . the critical importance of cleavage at the s 1 /s 2 site was also exemplified in studies with the mers-cov-related bat coronavirus hku4. although hku4 s recognizes human dpp4, in vitro infectivity assays revealed that entry into human cells required addition of exogenous trypsin, suggesting proteolytic activation of this bat virus did not occur in human cells (wang et al., 2014) . in line with these findings, various dpp4 mammalian orthologues, with variable binding affinities for the mers-cov s receptor-binding domain, were shown to support virus or pseudovirus entry into target cells in the presence of an activating protease (barlan et al., 2014) . these results collectively illustrate how specific s proteolytic cleavage participates in determining the intracellular site of fusion and also viral tropism and pathogenesis of covs. therefore, the zoonotic potential of covs is not only determined by receptor engagement, but also by proteolytic processing of the s protein required for fusion activation. we showed that in vitro trypsin cleavage of mhv, sars-cov and mers-cov s, under limited proteolysis conditions, recapitulated fusion activation by inducing the pre-to postfusion transition (walls et al., 2017b) . the cryoem structure of the mhv s 2 subunit ectodomain trimer revealed that membrane fusion involves large-scale s conformational changes that are reminiscent of the ones described for other class 1 fusion proteins, including the pneumovirus/paramyxovirus f glycoproteins (fig. 3) (mclellan et al., 2011; swanson et al., 2010 swanson et al., , 2011 walls et al., 2016b; yin et al., 2005) . these experiments also demonstrated that (i) the s 1 subunits stabilize the s 2 fusion machinery in the spring-loaded, metastable prefusion state before initiation of infection; and (ii) postfusion s is the ground state of the fusion reaction. similarly to the organization of influenza virus hemagglutinins (xiong et al., 2013) , domain b interacts with the hr1-central helix hairpin in prefusion closed s structures likely to stabilize s 2 in the spring-loaded prefusion state. this interaction appears to coordinate receptor engagement with fusion. upon receptor binding and proteolytic cleavage at the s 1 /s 2 and s 2 0 sites, the s 1 crown is likely shed (as observed for mers-cov s by yuan et al., 2017) to facilitate a conformational change of s 2 , which involves projection of the fusion peptide to a distance of 100 å and its insertion into the target membrane (fig. 3) (walls et al., 2016a (walls et al., , 2017b . the free energy released upon s 2 refolding from the prefusion to the postfusion state is believed to bring the viral and host membranes in close proximity and promote membrane merger (harrison, 2008) . recent structural work comparing recombinant s proteins from sars-cov and mers-cov in isolation and in complex with their cognate receptors or neutralizing antibodies suggested an activation mechanism for coronavirus fusion (gui et al., 2017; kirchdoerfer et al., 2018; song et al., 2018; walls et al., 2019; yuan et al., 2017) . specifically, sars-cov and mers-cov s structures in complex with neutralizing antibodies isolated from survivors showed both antibodies competitively blocked receptor interaction, in agreement with previous surface plasmon resonance data (corti et al., 2015; rockx et al., 2008; traggiai et al., 2004; walls et al., 2019) . the anti-sars-cov s230 antibody, however, functionally mimicked the receptor by promoting s fusogenic conformational rearrangements through a molecular ratcheting mechanism (fig. 4) . these observations suggested that upon receptor recognition, bound b domains are locked in the open state, thereby releasing the constraints imposed on the hr1-central helix hairpin, allowing refolding of the s 2 fusion machinery and membrane fusion to occur (pallesen et al., 2017; song et al., 2018; walls et al., 2019; yuan et al., 2017) (fig. 4) . proteolytic activation is likely required to ensure that s glycoproteins will work in synergy, with proper spatial and temporal coordination, to drive fusion of the viral and host membranes. a deep knowledge of the organization and chemical composition of carbohydrates obstructing the surface of cov s glycoproteins is key for understanding accessibility to neutralizing antibodies and for guiding the rational development of subunit vaccines and therapeutics. s glycoproteins feature 20-35 predicted n-linked oligosaccharides per protomer. a cryoem structure of the hcov-nl63 s ectodomain allowed to visualize for the first time the extensive n-linked glycans covering the surface of a cov s trimer (walls et al., 2016b) (fig. 5) . a subsequent study revealed that numerous glycosylation sites are strictly or topologically conserved between pdcov s and hcov-nl63 s although the two glycoproteins share only 43% amino acid sequence identity and the two viruses belong to different genera infecting different hosts (xiong et al., 2018) . this observation suggested that all covs face similar immune pressure in their respective hosts, and that the areas that are masked by the conserved glycans might be key to the function of s. based on the information gained from the hcov-nl63 s structure, in which a glycan participates to masking the receptor-binding loops, it was proposed that the s glycan shield is involved in immune evasion, similarly to the well-characterized hiv-1 envelope trimer (walls et al., 2016b) . comparison of the n-linked oligosaccharides of full-length mers-cov s derived from virions produced in african green monkey veroe6 cells, or of a purified mers-cov s ectodomain recombinantly produced in hek293f cells, revealed an extensive overlap of glycan composition, including the presence of hybrid and complex glycans . processed oligosaccharides were also observed decorating s trimers at the surface of authentic sars-cov virions (krokhin et al., 2003; ritchie et al., 2010) . these data indicated that at least a fraction of the mers-cov and sars-cov virions produced in a cell are exposed to the glycan-processing enzymes residing in the golgi apparatus during assembly and budding, in contrast with previous models of cov budding (ng et al., 2003; stertz et al., 2007) . a common feature observed in the glycosylation patterns of s glycoproteins is the presence of less densely glycosylated regions surrounding the s 1 /s 2 cleavage site and the conserved fusion peptide, near the s 2 ' cleavage site, probably to allow access to activating host proteases and for membrane fusion to take place (walls et al., 2016b; walls et al., 2019) (fig. 5) . these "glycan holes" could be targeted for epitope-focused immunogen design or new therapeutic development against cov, as supported by the identification of a neutralization epitope within a comparable breach of the hiv-1 envelope glycan shield (mccoy et al., 2016) . recent structural and functional characterization of cov s glycoproteins provided insights into the mechanism used by these viruses to infect host cells and suggested possible strategies for rational design of vaccines and therapeutics. introducing stabilizing mutations, which prevent the prefusion to postfusion s transition, led to the elicitation of improved neutralization titers in mice and will be a key tool for the design of subunit vaccines against covs pallesen et al., 2017) . furthermore, the exposure of the fusion peptide at the surface of prefusion s trimers (walls et al., 2016a) and its conservation among covs indicate it might be an attractive target for broad inhibition of cov entry. major antigenic determinants of mhv and sars-cov s overlap with the fusion peptide region (daniel et al., 1993; zhang et al., 2004) and binding of neutralizing antibodies to this site could putatively prevent fusogenic conformational changes, as proposed for influenza virus hemagglutinin or hiv envelope (corti et al., 2011; kong et al., 2016; lang et al., 2017) . finally, masking strain-specific antigenic regions via engineering of additional n-linked glycosylation sites, as implemented for the mers-cov domain b (du et al., 2016) , bears the promise of focusing the immune response on highly conserved epitopes and eliciting broadly neutralizing antibodies against covs. ribosome structures to near-atomic resolution from thirty thousand cryo-em particles betacoronavirus adaptation to humans involved progressive loss of hemagglutinin-esterase lectin activity coronavirus receptor switch explained from the stereochemistry of protein-carbohydrate interactions and a single mutation receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites architecture of the sars coronavirus prefusion spike conformational reorganization of the sars coronavirus spike following receptor binding: implications for membrane fusion site directed mutagenesis of the murine coronavirus spike protein. effects on fusion cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex beam-induced motion of vitrified specimen on holey carbon film coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysisdependent manner movies of iceembedded particles enhance resolution in electron cryo-microscopy 2.8 å resolution reconstruction of the thermoplasma acidophilum 20s proteasome using cryo-electron microscopy coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein hiv-1 envelope. effect of the cytoplasmic domain on antigenic characteristics of hiv-1 envelope glycoprotein crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus identification of an immunodominant linear neutralization domain on the s2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev further characterization of aminopeptidase-n as a receptor for coronaviruses structural basis for membrane anchoring of hiv-1 envelope spike introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the sars coronavirus spike glycoprotein cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv the tetraspanin cd9 facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains role of endocytosis and low ph in murine hepatitis virus strain a59 cell entry proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation viral membrane fusion ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus coronavirus spike protein and tropism changes human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted epidemiology of coronavirus respiratory infections pre-fusion structure of a human coronavirus spike protein stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis fusion peptide of hiv-1 as a site of vulnerability to neutralizing antibody analysis of cellular receptors for human coronavirus oc43 point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus mass spectrometric characterization of proteins from the sars virus: a preliminary report antibody 27f3 broadly targets influenza a group 1 and 2 hemagglutinins through a further variation in vh1-69 antibody orientation on the ha stem structure of sars coronavirus spike receptor-binding domain complexed with receptor broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a single point mutation creating a furin cleavage site in the spike protein renders porcine epidemic diarrhea coronavirus trypsin independent for cell entry and fusion receptor and viral determinants of sars-coronavirus adaptation to human ace2 electron counting and beam-induced motion correction enable nearatomic-resolution single-particle cryo-em receptor usage and cell entry of porcine epidemic diarrhea coronavirus intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 holes in the glycan shield of the native hiv envelope are a target of trimer-elicited neutralizing antibodies structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence human coronavirus nl63 utilizes heparan sulfate proteoglycans for attachment to target cells host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein host cell proteases: critical determinants of coronavirus tropism and pathogenesis proliferative growth of sars coronavirus in vero e6 cells human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor crystal structure of bovine coronavirus spike protein lectin domain genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody cryosparc: algorithms for rapid unsupervised cryo-em structure determination dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc differential role for low ph and cathepsin-mediated cleavage of the viral spike protein during entry of serotype ii feline coronaviruses structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia relion: implementation of a bayesian approach to cryo-em structure determination the role of the transmembrane and of the intraviral domain of glycoproteins in membrane fusion of enveloped viruses binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins cryo-electron microscopy structure of porcine deltacoronavirus spike protein in the prefusion state a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 the intracellular sites of early replication and budding of sars-coronavirus epidemiology, genetic recombination, and pathogenesis of coronaviruses structure of a proteolytically resistant core from the severe acute respiratory syndrome coronavirus s2 fusion protein structure of the newcastle disease virus f protein in the post-fusion conformation structural basis for immunization with postfusion respiratory syncytial virus fusion f glycoprotein (rsv f) to elicit high neutralizing antibody titers palmitoylations on murine coronavirus spike proteins are essential for virion assembly and infectivity an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus clathrin-and caveolae-independent entry of feline infectious peritonitis virus in monocytes depends on dynamin human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo-electron microscopy cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion unexpected receptor functional mimicry elucidates activation of coronavirus fusion structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 endocytosis of the receptor-binding domain of sars-cov spike protein together with virus receptor ace2 proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins receptor-binding loops in alphacoronavirus adaptation and evolution structure and stabilization of the hendra virus f glycoprotein in its prefusion form crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor receptor binding by a ferret-transmissible h5 avian influenza virus glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections crystal structure of the pre-fusion nipah virus fusion glycoprotein reveals a novel hexamer-of-trimers assembly structural basis for coronavirus-mediated membrane fusion. crystal structure of mouse hepatitis virus spike protein fusion core crystallization and preliminary crystallographic analysis of the heptad-repeat complex of sars coronavirus spike protein proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for chargerich and cysteine-rich regions of the endodomain human aminopeptidase n is a receptor for human coronavirus 229e structure of the uncleaved ectodomain of the paramyxovirus (hpiv3) fusion protein structure of the parainfluenza virus 5 f protein in its metastable, prefusion conformation contribution of trafficking signals in the cytoplasmic tail of the infectious bronchitis virus spike protein to virus infection structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains identification of an antigenic determinant on the s2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies core structure of s2 from the human coronavirus nl63 spike glycoprotein we acknowledge support from the national institute of general medical sciences key: cord-282560-tofppr3b authors: henderson, jack a.; verma, neha; harris, robert c.; liu, ruibin; shen, jana title: assessment of proton-coupled conformational dynamics of sars and mers coronavirus papain-like proteases: implication for designing broad-spectrum antiviral inhibitors date: 2020-09-21 journal: j chem phys doi: 10.1063/5.0020458 sha: doc_id: 282560 cord_uid: tofppr3b broad-spectrum antiviral drugs are urgently needed to stop the coronavirus disease 2019 pandemic and prevent future ones. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is related to the sars-cov and middle east respiratory syndrome coronavirus (mers-cov), which have caused the previous outbreaks. the papain-like protease (plpro) is an attractive drug target due to its essential roles in the viral life cycle. as a cysteine protease, plpro is rich in cysteines and histidines, and their protonation/deprotonation modulates catalysis and conformational plasticity. here, we report the pk(a) calculations and assessment of the proton-coupled conformational dynamics of sars-cov-2 in comparison to sars-cov and mers-cov plpros using the recently developed graphical processing unit (gpu)-accelerated implicit-solvent continuous constant ph molecular dynamics method with a new asynchronous replica-exchange scheme, which allows computation on a single gpu card. the calculated pk(a)’s support the catalytic roles of the cys–his–asp triad. we also found that several residues can switch protonation states at physiological ph among which is c270/271 located on the flexible blocking loop 2 (bl2) of sars-cov-2/cov plpro. simulations revealed that the bl2 can open and close depending on the protonation state of c271/270, consistent with the most recent crystal structure evidence. interestingly, despite the lack of an analogous cysteine, bl2 in mers-cov plpro is also very flexible, challenging a current hypothesis. these findings are supported by the all-atom fixed-charge simulations and provide a starting point for more detailed studies to assist the structure-based design of broad-spectrum inhibitors against cov plpros. over the last two decades, three coronaviruses have caused deadly epidemics, threatening the global human population. the severe acute respiratory syndrome coronavirus (sars-cov) caused an outbreak in 2003, and a related middle east respiratory syndrome coronavirus (mers-cov) caused an outbreak in 2012. today, the world is facing the pandemic of the coronavirus disease 2019 (covid-19) caused by a novel coronavirus sars-cov-2, which shares about 82% genome sequence identity with the original sars-cov. 1 all three viruses are thought to have originated from animal reservoirs, and zoonotic transmission into the human population has led to the outbreaks. 2 currently, no effective treatment exists for any of the three coronavirus diseases; thus, there is an urgent need to understand the potential therapeutic targets and develop inhibition strategies. following the release of the coronavirus genome from the acidic endosome, the replicase polyproteins are translated and site is solvent exposed and flanked by a flexible β-hairpin loop called the blocking loop 2 or blocking loop 2 (bl2) (g266-g271). the fingers' subdomain contains a zinc finger coordinated by four cysteines, which upholds the structural integrity and is essential for the plpro activity. 12 the structure of sars-cov-2 plpro is nearly identical to that of sars-cov plpro, 13 as expected from the highly similar sequences [96% similarity and 83% identity, fig. 1(c) ]. in contrast, although the structure of mers-cov plpro overlays well with the sars-cov plpro structures, small differences are visible [ fig. 1(a) ], as expected from the larger sequence differences [66% similarity and 30% identity with sars-cov plpro, fig. 1(c) ]. all three cov plpros are rich in cys and his residues. sars-cov/cov-2 plpro contains 8/11 cys and 11/9 his, while mers-cov plpro contains 14 cys and 10 his residues [ fig. 1(c) ]. among them, five cys and two his residues are conserved in all three plpros, including the catalytic cys and his. cys and his have model pka's of 8.6 and 6.5, respectively; thus, in model compounds or peptides at physiological ph 7.4, they are both predominantly neutral, i.e., cys is protonated and his is singly protonated. however, in the protein environment, a small pka downshift for cys or upshift for his may occur, leading to a significant population of or a complete switch to the alternative protonation state, i.e., negatively charged, deprotonated cys and positively charged, doubly protonated his. a protonation state switch is an important energy transduction mechanism to enable functionally required conformational changes in biology. for example, the coronavirus spike protein makes use of protonation state switches to induce large conformational changes required for membrane fusion. 14, 15 our previous work employing the hybrid-solvent based continuous constant ph molecular dynamics (cphmd) simulations 16 demonstrated that the elucidation of proton-coupled conformational dynamics offers a deeper understanding of the structure-dynamics-function relationships 17 and inhibition mechanisms 18-20 of aspartyl proteases. toward understanding the (possibly) proton-coupled structurefunction relationship and assisting broad-spectrum inhibitor design, here, we report the pka calculations and preliminary assessment of the proton-coupled conformational dynamics of sars-cov-2 plpro in comparison to sars-cov and mers-cov plpros. this work employed the recently developed graphical processing unit (gpu)-accelerated gb-neck2 implicit-solvent based cphmd method 21 with a new asynchronous implementation of the ph replica exchange sampling protocol. to confirm our findings, the conventional all-atom fixed-charged md in amber18 22 was also applied. the simulations allowed us to determine the protonation states of all titratable sites, including the catalytic cys-his-asp triad, offering a timely knowledge to facilitate the md studies of plpros in the community. importantly, we tested a hypothesis regarding the proton-coupled conformational plasticity of the bl2 loop, which modulates substrate and inhibitor binding. the contrasting features among the three cov plpros have implications for designing broadspectrum antiviral inhibitors. for ease of discussion, the acronyms used in this work are listed in table i . the coordinates were retrieved from the protein data bank (pdb): sars-cov plpro (pdb 2fe8 13 ), sars-cov-2 plpro (pdb 6w9c), and mers-cov plpro (pdb 4rna 23 ). if multiple chains were available in the x-ray crystal structure, only the first chain was used. any small molecules or solvent were removed. for each structure, the acetylated n-terminus and amidated c-terminus along with all missing hydrogens were added using the charmm program (c36b2). 24 in the crystal structure of sars-cov-2 plpro (pdb 6w9c), a disulfide bond is present in the fingers' subdomain in place of a zinc ion. considering that the zinc ion cannot be represented in implicit-solvent simulations, we removed the zinc ion and added an analogous disulfide linkage between the closest non-adjacent cysteine pairs in all other structures to maintain the integrity of the fingers' subdomain. the disulfide linkage should not affect the pka's of the discussed residues, as they are located far away in other subdomains [ fig. 1(a) ]. following the addition of hydrogens and disulfide bridge, the structure was subject to a 20-step energy minimization with the heavy atoms restrained and a 20-step energy minimization with all atoms restrained but the disulfide bonded cysteine pairs. the minimization used ten steps of steepest decent and ten steps newton-raphson methods. from there, the force field parameters and coordinate files were constructed from the charmm output with the leap utility in amber. 22 the ff14sb force field 25 was used to represent the protein, and the gb-neck2 (igb = 8) implicit-solvent model 26 was used to represent solvent. the mbondi3 intrinsic born radii were modified for improving the titration simulations of his 21 and cys 27 side chains. the structure was then energy minimized and equilibrated in gb-neck2 implicit solvent, 26 following our previous protocol. 28 the energy minimization was performed using the steepest decent algorithm for 5000 steps and the conjugate-gradient algorithm for 1000 steps. the equilibration was performed at ph 7 in four stages, each having 2000 md steps with gradually decreased restraining force constants of 5 kcal/mol/å 2 , 2 kcal/mol/å 2 , 1 kcal/mol/å 2 , and 0 kcal/mol/å 2 . the final structure was used for cphmd titration simulations. the titration simulations were performed using the recently implemented gpu-accelerated gbneck2-cphmd method 28 in the pmemd engine of amber18. 22 the implementation is built upon the cpu version of the gbneck2-cphmd module 21 and the gpu version of the gbneck2 module 26, [29] [30] [31] in amber18. 22 the gbneck2-cphmd method has its origin in the gbsw-cphmd method implemented in charmm. [32] [33] [34] here, we implemented an asynchronous version of the ph replica-exchange protocol 16 to allow amber replica-exchange simulations to be performed on a single gpu or several gpus with a total number smaller than the number of replicas. a similar implementation that allows replicaexchange molecular dynamics on cpus to progress without centralized synchronization steps and the need for direct communication between processors was developed by gallicchio, levy et al. in the past. 35 the python script of our asynchronous ph replica-exchange algorithm is freely available at https://gitlab.com/shenlab-ambercphmd/async_ph_replica_exchange. the conventional way of running replica exchange is to use one gpu (or one cpu core) per replica. under this scheme, all replicas are running at the same time, and periodically, an attempt is made to exchange ph values (or configurations) between replicas according to the metropolis criterion. this method is not feasible if the number of replicas is larger than the number of available gpus. instead, in the asynchronous method, the replicas are consecutively run on each available gpu, starting from the lowest ph condition. as soon as two replicas that are supposed to exchange at that exchange step are completed, the exchange is attempted, not waiting for other replicas to finish. as soon as a gpu finishes a replica, that gpu is assigned the next available ph value and begins a new single-ph simulation. if all replicas at a single exchange step are being run, the gpu will be assigned the first replica from the next exchange step to avoid idling gpus. in the current amber implementation of ph replica exchange, 22 the ph conditions are swapped, but to simplify the arrangement of replicas, the asynchronous method instead swaps configurations and keeps a constant arrangement in ph space. this also eliminates a post-processing step in which the replica trajectories are sorted and stitched together according to their ph conditions. our previous work showed that ph replica-exchange enhances both protonation and conformational state sampling, allowing pka's to rapidly converge. 16, 28, 36 in the protocol, nine ph replicas were placed at ph values ranging from ph 4.5 to 8.5 at an interval of 0.5 ph units. an exchange of two adjacent ph conditions was attempted every 1000 md steps (or 2 ps). each replica was run for 55 ns, resulting in an aggregate time of 495 ns for each. the λ values were recorded after each exchange attempt. all side chains of asp, glu, his, cys, and lys were allowed to titrate, with their titration model parameters taken from our previous work. 21, 27, 28 an ionic strength of 0.15m was used to represent the physiological salt condition. simulations were run at a temperature of 300 k and an effectively infinite cutoff (999 å) for nonbonded interactions. shake was used to constrain bonds involving hydrogens to allow for a 2-fs time step. to support the findings from the gb-cphmd simulations, two all-atom md simulations were carried out for sars-cov-2 plpro (pdb 6w9c) using the predicted protonation states for asp, glu, his, cys, and lys at ph 8.5. note, since several residues may switch protonation states at physiological ph according to the cphmd predictions, ph 8.5 was used to avoid ambiguity in choosing protonation states. two additional runs were carried out using the protonated form of cys270. all simulations were performed with amber18. 22 the protein and water were represented by the ff14sb 25 and tip3p 37 force fields. the initial structure was placed in a truncated octahedron box of water molecules. longrange electrostatic interactions were handled by using the particle mesh ewald method. 38 a non-bonded cutoff of 8 å was used with a time step of 2 fs. the starting structure underwent energy minimization by applying 5000 steps of steepest descent, followed by 5000 steps of conjugate gradient minimization with a force constant of 25 kcal/mol/å 2 applied to the solute heavy atoms. the force constant was reduced to 5 kcal/mol/å 2 , and the system was heated from 100 k to 300 k for 50 ps. following heating, solvent was equilibrated in the npt ensemble for 250 ps using the isotropic berendsen barostat 39 and with the same force constant. subsequently, the restraints were removed, and the system was further relaxed for 100 ps in the npt ensemble. finally, two production runs of 1 μs each were performed for each system starting from a different random initial velocity seed. all analysis was performed with the amber module cpptraj. 40 the first 300 ns from each trajectory was discarded. we performed ph replica-exchange cphmd simulations to estimate the pka values of asp/glu/his/cys/lys side chains and assess possible proton-coupled dynamics in sars-cov, sars-cov-2, and mers-cov plpros. the titration simulations were conducted in the ph range of 4.5-8.5 and lasted 55 ns per replica (aggregate simulation time of 495 ns for each protein). the protonation states were well converged. consistent with our previous work, 36 we found that the protonation states of his residues converge rapidly within 10 ns per replica, and those of cys converge more slowly due to the formation of hydrogen bonds that are not present in the crystal structure (see later discussion). the convergence analysis of protonation state sampling and replica walks along the ph ladder are given in figs. s1-s6 of the supplementary material. for the ease of discussion, we refer to the "standard" protonation states as the default settings in the md programs, i.e., deprotonated asp/glu (negatively charged), deprotonated his (neutral), with one proton on either δ or ε nitrogen, protonated cys (neutral), and protonated lys (positively charged). our simulations showed that several cys, his residues and one asp are in the "non-standard" protonation state or can switch to this state at physiological ph in all three plpros (table ii) . a complete list of the calculated pka's is given in table 1 of the supplementary material. we first consider the catalytic triad in sars-cov-2 plpro. the catalytic cys111 is located in the thumb, his272 is located in the foothill of the palm adjacent to the flexible loop bl2, and the journal of chemical physics asp286 is located at the end of β18 [ fig. 1(b) ]. currently, no measured pka data are available. biochemical experiments of sars-cov plpro suggested that the cys serves as a nucleophile, while the his functions as a general acid with the assistance of a negatively charged asp; 12 however, it is unclear whether the reactive nucleophile is the thiolate of the cys⋯his ion pair or the neutral thiol, which becomes deprotonated upon binding of the substrate. 4 the calculated pka's of cys111 and asp286 are <4.5, whereas the pka of his272 is >8.5, indicating that the catalytic triad residues are all in the charged state at physiological ph. thus, our data support the mechanism in which the reactive nucleophile is the thiolate ion, rather than the neutral thiol that needs to be first activated by the substrate. 4 the cphmd simulations showed that the cys-his-asp triad maintains a catalytic geometry through several hydrogen bonds, which supports their protonation states. the catalytic his272 forms a hydrogen bond simultaneously with cys111 and asp286 in the entire ph range of 4.5-8.5, stabilizing his272 in the doubly protonated state and asp286 and cys111 in the deprotonated states [figs. 2(a) and 2(d)]. the doubly protonated form allows his272 to perform its role as a general acid. 4 the catalytic cys111 forms a hydrogen bond not only with his272 but also with the indole nitrogen of trp106, which provides further stabilization for the thiolate form and explains the significant downshifted pka relative to the solution value of 8.5 (table ii) . the cys111⋯trp106 hydrogen bond is important, as it maintains the position of trp106; the analogous residue in sars-cov plpro has been hypothesized as the oxyanion hole residue that donates a hydrogen bond to stabilize the negatively charged tetrahedral intermediate developed in the course of peptide hydrolysis. 13 the three hydrogen bond interactions in sars-cov-2 plpro are consistent with those in sars-cov plpro [ fig. 2(b) ]. in mers-cov plpro, trp106 is replaced with a leu, which is incapable of forming a hydrogen bond with the catalytic cys or with the negatively charged intermediate. this has been hypothesized as a cause for the significantly lower catalytic activity of mers-cov as compared to sars-cov plpro. 41 in addition to the missing cys⋯trp hydrogen bond, cphmd simulations of mers-cov plpro showed that the hydrogen bond between the catalytic his and asp is nearly abolished [fig. 2(c) ]. the loss of hydrogen bond interactions involving the catalytic his and cys appears to provide less stabilization to the respective charged states in mers-cov plpro, as suggested by the partial titration under the highest and lowest ph conditions, respectively (see fig. s3 ). to test whether the loss of hydrogen bond network affects the flexibility of the regions near the catalytic triad, we calculated the root-meansquare fluctuations (rmsfs) of the cα atoms of the catalytic triad and nearby five residues (fig. 3) . interestingly, the rmsfs of the loop residues that are sequence neighbors of the catalytic triad in mers-cov plpro are increased as compared to those in sars-cov-2 and sars-cov plpros, which are similar except for the flexible bl2 loop (267-271) region. the loop (106-116) adjacent to α4, which harbors the catalytic cys, the bl2 loop next to the catalytic his, and the β-hairpin loop next to the catalytic asp all display enhanced mobility [ figs. 3(a) and 3(b) ]. the largest increase in rmsf is seen for the bl2 loop, whereby the rmsf in mers-cov plpro is nearly doubled relative to sars-cov-2 plpro, which shows a somewhat higher mobility than sars-cov plpro. the extremely high flexibility of the bl2 loop in mers-as compared to sars-cov plpro is consistent with the lack of electron density for the region in the first x-ray structure of the apo mers-cov plpro. 41 the bl2 loop is perhaps the most prominent feature of the substrate binding site in sars-cov plpro, as its movement modulates the substrate and inhibitor binding. 4 crystal structures show that bl2 is open in the unbound sars-cov plpro and it closes by about 1.5 å-2 å in the bound form, which allows hydrogen bonds to form between tyr269/gln270 and the inhibitor. 4 upon inspection of the x-ray structures of sars-cov-2 plpro, we noticed that the bl2 loop is open in the ph 7.5 structure (pdb 6w9c) and a zinc ion is found within the binding distance of cys270; however, in the structure determined at ph 4.5 (pdb 6wrh), the bl2 loop closes in by 1.9 å (cα distance between tyr268 and asp164) and the zinc ion is absent. thus, we hypothesized that the bl2 loop dynamics is coupled to the titration of c270. cphmd titrations gave the pka's of 6.7 and 6.9 for cys270 in sars-cov-2 plpro and the equivalent cys271 in sars-cov plpro, respectively (table ii) . thus, cys270/c271 samples both protonated and deprotonated states at physiological ph. the pka that downshifts relative to the model cys pka of 8.5 is due to the formation of local hydrogen bonds, which favors the thiolate state. in the crystal structure of sars-cov-2 plpro, cys270 does not interact with thr265. the titration simulations showed that the distance between cys270 and thr265 varies widely between 5 and 15 å, when cys270 is protonated (λ value close to 0); however, when cys270 is deprotonated (λ value close to 1), the distance is locked to 1.5 å-2.5 å, indicating the formation of a hydrogen bond [ fig. 4(a) ]. in addition to the hydroxyl group of thr265, the deprotonated cys270 can also accept a hydrogen bond from the backbone amide groups of his272 and gly271, stabilizing the charged state [ fig. 4(b) ]. the same hydrogen bonds were also formed in the simulations of sars-cov plpro, which explains the similarly downshifted pka of the equivalent cys271. to test the hypothesis that protonation/deprotonation of cys270 modulates the bl2 dynamics, we examined the cα distance between tyr268 on the bl2 loop and the conserved asp164 next to α7, which represents the width of the s3 subpocket [figs. 4(c) and 4(d)]. the equivalent tyr269 in sars-cov plpro is an important residue, as it forms a hydrogen bond with the inhibitors that bind to the s3 pocket. 4 when cys270 is protonated, the probability distribution of the tyr268-asp164 distance covers a broad range of 8 å -20 å with a peak at around 12 å; however, when cys270 is deprotonated, the distribution samples a narrower range of 14 å-20 å with a peak at around 16 å [ fig. 4(c) ]. thus, the cphmd data suggest that the deprotonated cys270 is correlated with the bl2 conformations that are more open and rigid, which might be attributed to the aforementioned hydrogen bond formation between the deprotonated cys270 and the surrounding residues. turning to sars-cov plpro, fig. 4(c) shows that the bl2 movement is coupled to the protonation/deprotonation of the analogous cys271. however, in sars-cov plpro, it appears that the bl2 with a deprotonated cys270 can sample a wider range of 10 å-20 å as compared to sars-cov-2 plpro, although the peak remains around 16 å. the wider range of bl2 movement may be attributed to the somewhat weaker hydrogen bonds involving the deprotonated cys271. the movement of the bl2 loop is very similar between sars-cov and sars-cov-2 plpros when cys270 is protonated. for broad-spectrum inhibitor design, it is important to understand the difference in the bl2 conformation across the three plpros. the distributions of the tyr269/268-asp165/164 distance for sars-cov/cov-2 and the equivalent thr274-asp164 distance for mers-cov plpro at physiological ph [figs. 5(a) and 4(d)] show that the widest position of the bl2 loop is about the same across the three plpros; however, the bl2 in sars-cov-2 plpro samples the narrowest range, followed by sars-cov plpro and mers-cov plpro, which samples the widest range between 5 å and 22 å. the enhanced flexibility of the bl2 in mers-cov plpro may be attributed to the lack of a cys equivalent to cys271/270 in sars-cov/cov-2 plpro, which can form hydrogen bonds with neighboring residues to restrict the loop motion, and perhaps also the loosening of the nearby catalytic his, which no longer forms double hydrogen bonds as in sars-cov/cov-2 plpro. in agreement with the bl2 dynamics described with the protonated and deprotonated states of cys270 [ fig. 4(c) ] and the x-ray structures determined at ph 7.5 (pdb 6w9c) and ph 4.5 (pdb 6wrh), a significant ph dependence is observed with the bl2 dynamics in the simulations of sars-cov-2 plpro (fig. 5) structure [ fig. 5(c) ]. in the simulation at ph 4.5, the bl2 loop assumes a wide range of dynamics, allowing it to sample a state that leaves the s3/4 subpocket more closed, similar to the low ph crystal structure [ fig. 5(c) ]. as expected, nearly all asp/glu residues adopt standard protonation states (i.e., charged) at physiological ph; however, asp12 in sars-cov-2 plpro has a pka abnormally upshifted from its model pka of 4.0 to 6.7 (table ii) , making it possible to occasionally sample the protonated state at ph 7.4. trajectory analysis suggested that this upshift is, in part, due to the protonated asp12 acting as a hydrogen bond donor to either (deprotonated) glu67 or asn15 [figs. 6(a) and 6(c)], which stabilizes the protonated state. the two hydrogen bonds are mutually exclusive such that asp12 is a hydrogen bond donor 82%-96% of the time when it is in the protonated state [ fig. 6(a) ]. in addition to hydrogen bonding, asp12 is buried in a hydrophobic pocket with a very low solvent accessible surface area, which increases as asp12 becomes deprotonated at higher ph [ fig. 6(b) ]. in sars-cov plpro, the analogous asp13 experiences a similar degree of hydrogen bonding and solvent sequestration, resulting in a upshifted pka of 5.9. in mers-cov plpro, the analogous asp11 primarily donates a hydrogen bond to asn15, as the analogous residue to glu67 is missing (fig. s7 ). compared to asp12/13 in sars-cov-2/cov plpro, asp11 is more solvent exposed in the lower ph range (fig. s7) , which may contribute to a smaller degree of pka upshift of asp11 in mers-cov plpro as compared to sars-cov/cov-2 plpro. cphmd titrations revealed that three histidines unique to sars-cov/cov-2, h74/73, h90/89, and h176/175 (fig. 7) , have pka's around 7 (table ii) fig. s8 ), which stabilizes the charged state. his90/89 located on the c-terminal end of α3 in sars-cov/cov-2 has a pka of 7.0/6.9. analysis showed that a small pka upshift relative to the model pka is due to the stabilization of the charged state by the occasional hydrogen bonding with the backbone carbonyl of ser86/85 or transient salt-bridge interaction with asp107/108 located near the oxyanion hole of the cov plpro (fig. s9 ). his176/175 is located on the c-terminal end of α7 and opposite to his74/73. the increased pka of 7.4/7.3 can also be attributed to local hydrogen bonding either with his172 in sars-cov-1 or tyr171 in sars-cov-2 plpro (fig. s10) . to provide support for the protonation states determined by gb-cphmd titrations and test the proton-coupled dynamics of the bl2 loop, we performed conventional all-atom fixed-charge md simulations of sars-cov-2 plpro with the catalytic side chains fixed in the charged states and cys270 fixed in the protonated or deprotonated state. all other residues were fixed in the standard protonation states. two 1-μs trajectories were obtained with each cys270 protonation state. consistent with the gb-cphmd simulations at physiological ph, the catalytic triad remained very stable, with the hydrogen bond between his272 and cys111 being the strongest, followed by the his272⋯asp286 and cys111⋯trp106 hydrogen bonds, as shown in the hydrogen bond occupancy plots [ fig. 8(a) ]. interestingly, while the effect of cys270 protonation/deprotonation appears negligible for the latter two hydrogen bonds (occupancy change is below 5%), protonation of cys270 weakens the his272⋯cys111 hydrogen bond (occupancy decreases by over 20%). this decrease is consistent with the gb-cphmd data, which shows that the catalytic his⋯cys hydrogen bond is significantly weakened below ph 6 as cys270 becomes fully protonated in sars-cov-2 plpro [ fig. 2(a) ]. to test the effect of cys270 titration on the bl2 conformation, we calculated the probability distribution of the cα distance between tyr268 and asp164. for the trajectories with protonated cys270, the distribution displays a single peak at about 11 å; however, for the trajectories with deprotonated cys270, a second peak appears at about 17 å [ fig. 8(b) ]. these data indicate that deprotonated cys270 is correlated with the more open bl2 loop conformations, consistent with the findings from the gb-cphmd simulations [ fig. 4(c) ]. however, due to the slow transition between the open and closed bl2 loop conformations in explicit solvent, more trajectories or longer simulations are needed to solidify the conclusion. we compare the cphmd-predicted pka's of sars-cov-2/cov and mers-cov plpros with those from the delphipka server, 42 which performs continuum poisson-boltzmann (pb) calculations with a smooth gaussian dielectric function. 43 note, cys pka calculation is a newly added functionality in delphipka. 44 the default setting with a protein internal dielectric constant of 8 was used. for the purpose of this work, we focus on the pka's of his, cys, and the abnormal asp13/12/11 (fig. s11 ). there appears to be a good correlation for his and cys pka's in the pka range of 5-7.5 between the two methods; however, there is a large disagreement for the pka's that are predicted to be highly down-or upshifted relative to the model values by the cphmd method. close inspection suggests that the disagreement is related to the small pka ranges from the delphipka calculations: 6-8 for his and 5.5-7 for cys (i.e., all cysteines are thiolates). specifically, cysteines that have cphmd predicted pka's above 8.5 have pka's below 7 according to del-phipka calculations. similarly, histidines that have cphmd predicted pka's below 4.5 have pka's of about 6 according to delphipka calculations. another significant disagreement is for asp13/12/11, which have significantly upshifted pka's according to cphmd (5.2-6.7, see table ii ) but have downshifted pka's based on delphipka (pka 2-3, fig. s11) . a possible explanation is that in the crystal structure, one of the carboxylate oxygens of asp13/12/11 accepts a hydrogen bond from the side chain amino group of asn15/14/13, thereby stabilizing the deprotonated form. by contrast, in the cphmd simulations, asn15/14/13 rotated such that its carbonyl group accepts a hydrogen bond from asp13/12/11, which additionally donates a hydrogen bond to glu67 in sars-cov-2/cov-2 plpro (fig. 6) . experimental measurements and future community efforts such as the 2009 blind pka prediction exercise 45 are needed to assess and promote the further development of various pka calculation approaches. the protonation states and possible proton-coupled conformational dynamics of sars-cov-2 plpro were investigated in comparison to sars-cov and mers-cov plpros using the gpuaccelerated gbneck2-cphmd titration simulations with a new asynchronous ph replica-exchange scheme as well as conventional all-atom md. the simulations showed that the catalytic cys, his, and asp are charged in the entire simulation ph range of 4.5-8.5 for all three plpros, which supports the mechanism in which the reactive nucleophile is the thiolate ion and the catalytic his serves as a general acid stabilized by the catalytic aspartate. the catalytic the journal of chemical physics article scitation.org/journal/jcp triad in sars-cov-2/cov plpro forms a hydrogen bond network among themselves and with a nearby trp, which serves as an oxyanion hole residue to stabilize the tetrahedral intermediate developed in the peptide hydrolysis. in contrast, the hydrogen bond with trp is missing and the hydrogen bond between the catalytic his and asp is nearly abolished in mers-cov plpro, consistent with the significantly lower catalytic activity compared to sars-cov plpro. 41 interestingly, the lack of a hydrogen bond network for the catalytic triad in mers-cov plpro is correlated with the increased mobility of nearby loop residues, in particular the bl2 loop. the simulations revealed that several titratable residues have shifted pka values such that they switch between two protonation states at physiological ph. these include three his and one cys residues unique to sars-cov-2/cov and one asp residue common to all three plpros (table ii) . of particular interest is cys270/271 on the flexible bl2 loop of sars-cov-2/cov, which has a pka of 6.7/6.9 and samples both the standard thiol and charged thiolate forms at neutral ph. cphmd simulations showed that the bl2 loop samples an open or a closed conformational ensemble with deprotonated or protonated cys270/271, respectively, consistent with two crystal structures of sars-cov-2 plpro determined at neutral and low ph conditions and the conventional all-atom md trajectories of sars-cov-2 plpro with either deprotonated or protonated cys270. thus, the simulation data and experiment together support our hypothesis that the bl2 loop conformation is coupled to the titration of c270/271 in sars-cov-2/cov plpro. an induced fit mechanism, by which bl2 closes in to form hydrogen bonds with the inhibitor, has been proposed in designing potent inhibitors targeting the s3/s4 pocket of sars-cov plpro. 4 our finding suggests that in the absence of a ligand, protonation of c270/271 induces the closure of bl2 in sars-cov-2/cov plpro, which raises the possibility that a conformational selection mechanism may be operative, in which inhibitor binding shifts the bl2 conformational population to the closed form, perhaps by favoring the protonation of c270/271. while the unliganded crystal structures show that bl2 in mers-cov plpro is more open than in sars-cov-2/cov plpro, cphmd simulations suggest that bl2 has an increased flexibility in mers-cov plpro and it can sample closed conformations. this finding challenges a current hypothesis, according to which sars-cov plpro inhibitors do not bind to mers-cov plpro due to the more open bl2 loop as a result of the sequence difference and one extra residue. 23, 46 a main caveat of our work is the use of the gb-neck2 implicit-solvent model. 26 although it has been demonstrated in the accurate de novo folding simulations of nearly two dozen small proteins with α and β topologies, 31 inherent issues such as the lack of solvent granularity may limit the accuracy of detailed conformational representation. nonetheless, our work provides a starting point for further mechanistic investigations using higher-level approaches such as the all-atom cphmd 47 and more extensive conformational sampling to assist the structure-based drug design targeting the coronavirus plpros. see the supplementary material for convergence analysis, a list of pka estimates for all titratable residues, and additional hydrogen bond analysis. α-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds sars and mers: recent insights into emerging coronaviruses nsp3 of coronaviruses: structures and functions of a large multi-domain protein mers-cov papain-like protease has deisgylating and deubiquitinating activities rna-virus proteases counteracting host innate immunity deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus papain-like protease 2 (plp2) from severe acute respiratory syndrome coronavirus (sars-cov): expression, purification, characterization, and inhibition the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structure, function, and evolution of coronavirus spike proteins sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor continuous constant ph molecular dynamics in explicit solvent with ph-based replica exchange ph-dependent population shift regulates bace1 activity and inhibition constant ph molecular dynamics reveals ph-modulated binding of two small-molecule bace1 inhibitors proton-coupled conformational allostery modulates the inhibitor selectivity for β-secretase how ligand protonation state controls water in protein-ligand binding generalized born based continuous constant ph molecular dynamics in amber: implementation, benchmarking and analysis inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov charmm: the biomolecular simulation program ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb improved generalized born solvent model parameters for protein simulations assessing lysine and cysteine reactivities for designing targeted covalent kinase inhibitors gpu-accelerated implementation of continuous constant ph molecular dynamics in amber: pk a predictions with single-ph simulations generalized born model with a simple, robust molecular volume correction routine microsecond molecular dynamics simulations with amber on gpus. 1. generalized born folding simulations for proteins with diverse topologies are accessible in days with a physicsbased force field and implicit solvent constant-ph molecular dynamics using continuous titration coordinates constant ph molecular dynamics with proton tautomerism toward the accurate first-principles prediction of ionization equilibria in proteins asynchronous replica exchange software for grid and heterogeneous computing predicting reactive cysteines with implicitsolvent-based continuous constant ph molecular dynamics in amber comparison of simple potential functions for simulating liquid water particle mesh ewald: an n log(n) method for ewald sums in large systems molecular dynamics with coupling to an external bath ptraj and cpptraj: software for processing and analysis of molecular dynamics trajectory data crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features delphipka web server: predicting pk a of proteins, rnas and dnas pk a predictions for proteins, rnas, and dnas with the gaussian dielectric function using delphi pk a delphipka: including salt in the calculations and enabling polar residues to titrate progress in the prediction of pk a values in proteins x-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases all-atom continuous constant ph molecular dynamics with particle mesh ewald and titratable water we acknowledge financial support from the national institutes of health (grant no. gm098818). the python script to enable the asynchronous replica-exchange protocol for amber simulations is freely available at https://gitlab. com/shenlab-amber-cphmd/async_ph_replica_exchange. the data that support the findings of this study and raw trajectory files are available from the corresponding author upon reasonable request. key: cord-263042-qdmunb9l authors: zhao, yongkun; wang, chong; qiu, boning; li, chufang; wang, hualei; jin, hongli; gai, weiwei; zheng, xuexing; wang, tiecheng; sun, weiyang; yan, feihu; gao, yuwei; wang, qian; yan, jinghua; chen, ling; perlman, stanley; zhong, nanshan; zhao, jincun; yang, songtao; xia, xianzhu title: passive immunotherapy for middle east respiratory syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model date: 2016-11-24 journal: antiviral res doi: 10.1016/j.antiviral.2016.11.016 sha: doc_id: 263042 cord_uid: qdmunb9l middle east respiratory syndrome (mers) is a highly lethal pulmonary infection caused by a coronavirus (cov), mers-cov. with the continuing spread of mers-cov, prophylactic and therapeutic treatments are urgently needed. in this study, we prepared purified equine f(ab’)(2) from horses immunized with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins. both igg and f(ab’)(2) efficiently neutralized mers-cov replication in tissue culture. passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of mers-cov infected mice. our data show that horses immunized with mers-cov vlps can serve as a primary source of protective f(ab’)(2) for potential use in the prophylactic or therapeutic treatment of exposed or infected patients. middle east respiratory syndrome (mers)-cov is an emerging pathogen that causes severe pneumonia in humans in the arabian peninsula and in travelers from this region (assiri et al., 2013a; zaki et al., 2012b; zumla et al., 2015) . human-to-human spread has been documented (assiri et al., 2013b) . while infections of immunocompetent patients generally present with only mild symptoms, the elderly and patients with pre-existing illnesses such as diabetes or renal failure are likely to develop more severe disease (assiri et al., 2013a) . as of september 21, 2016, 1806 cases with 643 deaths (35.6% mortality) had been reported to the world health organization, although the actual number of infections could be much larger since mild, asymptomatic or undiagnosed cases are likely to be common (drosten et al., 2014) . as yet there are neither licensed vaccines nor any prophylactic or therapeutic treatments effective against mers-cov. given the ability of coronaviruses to rapidly adapt to new hosts, a major public health concern is that mers-cov will further adapt to replication in humans, triggering a global severe acute respiratory syndrome (sars)-like pandemic (peiris et al., 2004; zaki et al., 2012a) . as of now, the most promising treatment is the passive administration of anti-mers-cov neutralizing antibodies. several research groups have developed and produced anti-mers patientderived or humanized monoclonal neutralizing antibodies in vitro that were able to protect mers-cov infected mice (corti et al., 2015; li et al., 2015; zhao et al., 2014) . however, since these antibodies react with a single epitope on the mers-cov spike (s) protein and since coronaviruses are prone to mutate, this approach has raised concerns about possible antibody escape (corti et al., 2015; sabir et al., 2016) . recently, we showed that sera from middle east dromedary camels contained high levels of anti-mers-cov neutralizing antibodies. passive immunotherapy with sera from these animals significantly reduced virus loads and accelerated virus clearance from the lungs of mers-cov infected mice . this provides proof of concept that immune animal sera are potentially useful in the treatment of patients with mers (hayden et al., 2014) . passive immunotherapy with animal sera or antibodies has been successfully used to prevent rabies and to neutralize snake venom (both et al., 2012; gutierrez et al., 2014) . convalescent plasma used to treat patients with sars has been found safe and has demonstrated some efficacy in a study with a small number of patients (mair-jenkins et al., 2015) . however, neutralizing antibody titers in mers patients are generally low and the limited number of mers survivors makes this approach impractical (drosten et al., 2013) . here, we show that immunization of healthy horses with mers-cov virus-like particles (vlps) expressing mers-cov s, m and e proteins induces strong polyclonal neutralizing antibodies against mers-cov. since administration of whole antibodies can induce allergic responses in some humans, we further tested f(ab') 2 fragments prepared by digestion of antibody with pepsin. prophylactic or therapeutic treatment of mers-cov infected mice with either igg or f(ab') 2 significantly decreased the virus load in their lungs. mers-cov vlps were produced and purified as previously described . in brief, army worm sf9 cells were infected with a single recombinant baculoviruses co-expressing mers-cov structural protein genes s, m, and e, at a multiplicity of infection (moi) of 0.5. culture supernatants were harvested at 96 h post-infection and centrifuged at 2000 g for 30 min to remove cell debris. following centrifugation of the clarified supernatants at 100,000 g for 1 h at 4 c the resulting vlp pellets were resuspended in pbs and loaded onto a 30e40e50% discontinuous sucrose gradient. after an additional centrifugation at 100,000 g for 1.5 h at 4 c, bands between 30 and 40% sucrose containing mers-cov vlp were collected. four 4-year-old healthy horses received multi-point intramuscular injections of 0.5, 1.5, 2, 3, and 5 mg mers-cov vlps in 4 ml pbs at weeks 0, 2, 4, 6, and 8, respectively. freund's complete adjuvant (sigma) was included in the first dose, and incomplete adjuvant in the remaining ones. sera were collected from the jugular vein 2 weeks after each injection, and stored at à20 c before further analysis. mers-cov specific antibodies in the sera were measured by an indirect enzyme-linked immunosorbent assay (elisa) using purified mers-cov receptor-binding domain (rbd) protein (i.e., s protein residues 358e662 cloned into the pet-30a expression vector and purified by ni-nta affinity chromatograph column). briefly, 96-well microtitration plates (corning costar, usa) were pre-coated with 100 ml purified rbd antigen diluted in 0.05 mol/l carbonate sodium buffer (ph 9.6) to a final concentration of 1 mg/ ml and incubated at 4 c overnight. after blocking with skimmed milk for 2 h at 37 c, 100 ml twofold serially diluted serum samples were added to the wells, and incubated at 37 c for 1 h. the plates were washed three times with pbs containing 0.05% tween-20 (pbst), before addition of 100 ml hrp-labeled rabbit antibody against horse igg (bioss, china; 1:20,000) and incubation at 37 c for 1 h. after washing with pbst, 100 ml 3, 3 0 , 3, 5'-tetramethylbenzidine (tmb) (sigma, usa) as substrate was added to each well and incubated for 30 min. the reaction was stopped with 50 ml 2 m h 2 so 4 . optical densities at 450 nm were measured in an elisa plate reader (bio-rad, usa). horse antiserum was diluted with 2 vol of normal saline (0.9% nacl) and a half volume of saturated ammonium sulfate was then added and mixed gently at room temperature for 30 min before centrifugation at 5000 g for 20 min. the resulting sediment was redissolved in saline and mixed with a one-third volume of saturated ammonium sulfate. after incubation at ambient temperature for 30 min and centrifugation at 5000 g for 20 min, the second sediments were dissolved in normal saline and dialyzed against normal saline to remove any remaining ammonium salt. immunoaffinity resins were prepared by coupling 10 mg rbd protein to 0.02 m sodium periodate-activated sepharose 4b (4 g), and then incubating with 150 ml sodium borohydride for 30 min. after reaction with 1 m tris (ph 7.5) for 30 min, a purified igg sample was diluted 9-fold with pbs and incubated with the rbd resin overnight at 4 c with constant rotation. the flowthroughs (anti-rbd depleted) were collected, and then the flowthroughs were tested against the rbd protein by elisa to ensure rbdspecific igg all bound with the rbd sepharose 4b. after washing with pbs, the bound antibodies (anti-rbd) were eluted in 0.2 m glycine-hcl buffer (ph 2.7). the eluates were neutralized with 1 m tris buffer (ph 9.0), and then dialyzed against pbs. all samples were adjusted to the same protein concentration and sterilized by passage through microspin filters (0.2 mm pore size; millipore). neutralizing activity of the igg, rbd-specific igg, and flowthroughs were tested. the ph of the horse antiserum was adjusted to 3.3 with 1 mol/l hcl. following incubation with pepsin (10000 iu/ml) at 30 c for 2.5 h, the reaction was stopped by adjusting the ph to 7.2 with 1 mol/l naoh. the solution was then applied to protein-a and protein-g columns sequentially to remove whole immunoglobulins. the purity of the resulting f(ab') 2 protein was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by coomassie blue staining and the target fraction in the gel was analyzed in a thin layer chromatography scanner (transmission, zigzag scan, dual wavelength, swing width:8 mm, delta y: 0.1 mm) (cs-9301, shimadzu). specific pathogen-free 6 week old balb/c mice were purchased from charles river laboratories international and maintained in the animal care facility, university of iowa. briefly, all mice were housed in thoren individually ventilated cages. caging and bedding were autoclaved. irradiated diet was fed. filtered water (0.2 mm filter) was provided with edstrom automatic watering system. hepa-filtered cage changing stations were used. all persons entering animal rooms worn autoclaved gowns, gloves, hair bonnets, face masks, and shoe covers. serum samples, purified igg or f(ab') 2 were serially diluted in dmem and mixed with an equal volume of mers-cov containing 80 pfu. following incubation at 37 c for 1 h, aliquots were added to cultures of vero 81 cells in 48 well plates and incubated at 37 c in 5% co 2 for 1 h with gentle rocking every 15 min. plates were then overlaid with 1.2% agarose/dmem/2% calf serum. after further incubation for 3 days, agarose plugs were removed using a small spatula, and the remaining plaques were visualized by staining with 0.1% crystal violet. six-week-old female balb/c mice were lightly anesthetized with isoflurane and transduced intranasally with 2.5 â 10 8 pfu of ad5-hdpp4 in 75 ml dmem as described elsewhere (zhao et al., 2014) . five days post transduction, mice were infected intranasally with mers-cov (1 â 10 5 pfu) in a total volume of 50 ml dmem. mice were monitored daily for morbidity (weight loss) and mortality. all work with mers-cov was conducted in the university of iowa biosafety level 3 (bsl-3) laboratory. separate groups were injected with 200 ml horse antiserum or 500 mg igg or f(ab') 2 intraperitoneally (ip) 1 day before or after intranasal infection with 1 â 10 5 pfu mers-cov. control mice were given an equal volume of normal horse serum (sigma). to obtain virus titers, lungs were harvested from subgroups of 3 animals at the indicated time points (see results) and homogenized into 3 ml of phosphate buffered saline (pbs), using a manual homogenizer. lung homogenates were aliquoted into micro tubes and kept in à80 c. virus was titered on vero 81 cells. cells were fixed with 10% formaldehyde and stained with crystal violet three days post-infection (p.i.). viral titers are expressed as pfu/g tissue for mers-cov (zhao et al., 2014) . due to the biosafety risk, mers-cov must be handled in a bsl-3 laboratory, whereas vlps can be rapidly generated under bsl-2 conditions as an immunogen inducing high antibody titers. in addition, the horse provides little risk to humans and produces high antibody yields, making these animals an effective source for production of hyperimmune sera (zheng et al., 2016) . rbd-specific igg titers in the sera were all above 1:20,480 after five immunizations (fig. 1) as assessed by elisa. rbd contains the major neutralizing epitopes of the s protein, as shown by the observation that absorption of sars patient convalescent sera with sars-cov rbd removes the majority of neutralizing antibodies (he et al., 2005) . independent research groups have also shown more directly that the mers-cov rbd sequence contains the major antigenic determinants for inducing neutralizing antibodies, and that neutralizing epitopes within mers-cov s1 are also localized primarily in the rbd region (du et al., 2013; mou et al., 2013) . here, we have demonstrated that anti-rbd antibodies function as major components of neutralizing antibodies. we found that rbd-specific igg neutralized mers-cov infection with half maximal inhibitory concentration of 15.74 mg/ml, and 2.612 â 10 3 mg/ml for flowthroughs (fig. 2) , suggesting that the rbd of s protein act as an important neutralization determinant of mers-cov. our results demonstrate that equine antibodies are polyclonal and recognize more antigen determinants in mers-cov s protein than single mabs, which could potentially prevent antibody escape. the integrity of igg and f(ab') 2 fragments was evaluated using an sds-page gel (fig. 3a) . the purity of the f(ab') 2 fragments after protein-a/g chromatography was >91% after gel electrophoresis (fig. 3b ). passive transfer of blood products from other humans poses a safety concern, with possible contamination with agents of blood-borne diseases (e.g., hiv, hepatitis). heterologous antibody carries a potential risk of allergic reaction, but generation of f(ab') 2 fragments, results in antibodies being less immunoreactive and safer for use in humans. while we successfully generated equine antibodies against mers-cov vlps, their protective effect against authentic mersfig. 1 . robust mers-cov rbd-specific antibody in immunized horse sera. horses (n ¼ 4) were injected intramuscularly with mers-cov vlps and boosted every two weeks an additional 4 times. sera were collected 2 weeks after each immunization. rbd-specific antibodies in immunized horse sera were detected using elisa. cov infection remained untested. using a plaque reduction neutralizing assay, we confirmed that immune sera significantly neutralized mers-cov infection in vitro, with a half effective maximal dilution of 1: 20,900 (fig. 4a, b) . further, we found that equine igg and f(ab') 2 also neutralized mers-cov infection with half effective maximal concentrations (ec 50 ) of 2.16 mg/ml and 2.60 mg/ml for igg and f(ab') 2 , respectively (fig. 4c, d) . collectively, these results show that equine antibody products exhibit highly potent neutralizing activity against mers-cov. next we asked if adoptive transfer of equine antibodies could protect mice from mers-cov infection prophylactically and therapeutically. by using a mouse model we previously generated (zhao et al., 2014) , we injected animals with immune serum (fig. 5a, b) , purified igg (fig. 5c, d) or f(ab') 2 (fig. 5e, f) i.p. 1 day before (fig. 5a , c, e) or after (fig. 5b, d, f) mers-cov challenge. in both prophylactic and therapeutic settings, passive transfer of equine immune antibodies resulted in a 2e4 log reduction of virus titers in the lungs of mers-cov infected mice, and accelerated virus clearance in the serum treated group (fig. 5a, b) . we did not observe any difference in body weight loss and pathologic changes on the exterior surface of the lungs in treated and untreated mice after fig. 2 . neutralizing activity of the rbd-specific antibodies in igg. in vitro neutralization tests of total igg, rbd-specific igg, and flowthroughs, were determined in a series of 2-fold dilutions and 50% neutralization was calculated using graphpad prism. infeciton, since in this model, mice only develope mild lung disease. rapid virus replication and inflammatory cell infiltration in the infected lungs are the major parameters to measure (zhao et al., 2014) . since the half-life of f(ab') 2 in vivo is relatively short and mers-cov is cleared within 6 days in this model (zhao et al., 2014) , we did not inject f(ab') 2 antibodies before day à1 or after day 1 p.i. of note, the purified igg seemed to have lower protective potency than that of the immune serum in vivo (fig. 5) . the concentration of igg in serum is > 10 mg/ml. we used 200 ml of immune serum (equal to 2 mg igg) per mouse which is much higher than the immune igg we used (500 mg/mice). the other reason could be we purified immune igg using saturated ammonium sulfate precipitation method, which needed to be performed under room temperature. we speculated that some iggs were degraded or misfolded, and unable to bind to mers-cov spike protein under this circumstance. while, immune sera were properly stored at à20 c and contained high concentration of bsa and other proteins, which made the antiserum more stable. to date, there are several anti-mers-cov antibodies developed from different origins. each antibody contains its own advantages and disadvantages. for monoclonal antibodies, mouse-derived monoclonal antibody needs to be humanized before human use (li et al., 2015) ; a human neutralizing antibody derived from a convalescent mers patient can be produced in large amount from cho cells (corti et al., 2015) . however, the single clone antibody raises the concern of viral escape mutant when applied to human. administration of transchromosomic bovine human immunoglobulins (luke et al., 2016) or dromedary immune serum resulted in rapidly viral clearance in infected mouse lungs. the disadvantage of these antibodies is that these animals are not readily available. compared to the antibodies described above, the administration of equine igg-derived f(ab') 2 fragment proved to be a versatile and feasible method (lu et al., 2006; zhou et al., 2007) . it provides a useful platform to produce therapeutics against emerging infectious diseases. in summary, by immunizing healthy horses with mers-cov vlps, we have successfully developed the first equine igg-derived f(ab') 2 fragment that neutralizes mers-cov in vitro and in vivo. both prophylactic and therapeutic treatments decreased virus loads and accelerated virus clearance in the lungs of mers-cov-infected mice. therefore, horses immunized with mers-cov vlps can serve as a useful initial source for developing protective f(ab') 2 fragments, for the purpose of preparedness and to serve as a strategic reserve for a potential mers epidemic and other emergent pathogens. the authors declare no competing interests. epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus transmission of mers-coronavirus in household contacts clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development immunological profile of antivenoms: preclinical analysis of the efficacy of a polyspecific antivenom through antivenomics and neutralization assays towards improving clinical management of middle east respiratory syndrome coronavirus infection identification of a critical neutralization determinant of severe acute respiratory syndrome (sars)-associated coronavirus: importance for designing sars vaccines a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein passive immunotherapy for influenza a h5n1 virus infection with equine hyperimmune globulin f(ab')2 in mice human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies severe acute respiratory syndrome co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of a novel coronavirus from a man with pneumonia in saudi arabia rapid generation of a mouse model for middle east respiratory syndrome passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from ebola virus infection inhibition of infection caused by severe acute respiratory syndrome-associated coronavirus by equine neutralizing antibody in aged mice middle east respiratory syndrome ad5-hdpp4 transduced balb/c mice (6wks, female) were injected intraperitoneally with 200 ml horse serum key: cord-259443-5sv3dwbs authors: banik, gouri rani; alqahtani, amani salem; booy, robert; rashid, harunor title: risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia date: 2016-01-25 journal: virol sin doi: 10.1007/s12250-015-3679-z sha: doc_id: 259443 cord_uid: 5sv3dwbs [image: see text] however, these studies were relatively small and the risk factors for disease severity were rarely explored (majumdar et al., 2015; korea cdc, 2015; saad et al., 2014) . to this end, we have explored the key risk factors for mortality and severity of mers-cov from a larger dataset of saudi arabian patients. data for this analysis were obtained from the command and control center (ccc) of saudi arabian ministry of health (moh). this center has been publicly reporting the mers-cov cases in saudi arabia since may 2013 on their website (http://www.moh.gov.sa/en/ccc/ pressreleases/pages/default.aspx). the site contains data on > 80% of the saudi arabian mers cases. initially, only limited information such as patients' age, sex, nationality, address, date of diagnosis, presenting symptoms, and presence of any pre-existing condition were made publicly available; however, since 24 th september 2014, additional information on likely exposure to animals and other suspected mers cases were added, and it was recorded whether the exposure likely occurred at health care settings or in community settings. two authors (asa and grb) extracted the data from the saudi moh website and added it into a data extraction spread sheet; the first author (grb) double checked the entries, any disagreement was resolved through verifying and ensuring data integrity. the following data were abstracted in the extraction sheet: date of confirmation of the diagnosis, age and sex of the patient, nationality, city or region of the patient's residence, symptoms, whether the patient required intensive care unit (icu) admission, status of the patient at the time of data entry (recovered, being cared in the hospital or icu, or died) and whether the patient was a health care worker (hcw). it was also noted if the patient had any pre-existing medical condition and, if so, what those medical conditions were. additional information (e.g., history of exposure to animal or contact with suspected cases in health care and/or community settings) were collated in a separate column irrespective of whether or not the information was used in this analysis. the proportion of patients with a particular risk factor, such as pre-existing illness or age ≥ 65 years, was calculated separately for those who died and for those who survived. for this analysis, a case of mers was considered 'severe' if he/she needed admission to icu at any time point during the course of the illness, all other cases were considered 'not severe', irrespective of whether they required hospitalization or not. the proportion of patients with a particular risk factor such as the presence of a named pre-existing medical condition was calculated separately for 'severe' and 'not severe' cases. odds ratios (ors) for mortality and severity in the presence of a potential risk factor were calculated with 95% confidence interval (95%ci) by using an authentic online calculator (http://vassarstats.net/odds2x2.html). a p value of < 0.05 was considered statistically significant. as of 11 october 2015, data on 1060 mers-cov cases aged 9 months to 109 years (median 52 years) were published in the saudi moh website; 65% were male (m/f ratio: 1.5). among them, 81% (846/1049) were saudi residents; the non-saudis (19%, 203/1049) included expatriates and immigrants mainly from the philippines, palestine, pakistan, and egypt. riyadh had the highest number of cases (45.5%, 482/1058), followed by jeddah (18%, 192/1058) ( figure 1 ). one hundred and forty one (14%) of all mers cases were hcws. of 379 patients for whom such data were available, 84 (22%) had a history of exposure to health care settings, and of 595 patients for whom such data were available, 250 (42%) gave a history of exposure to suspected cases in the community. of 350 patients for whom the data were available, 17% (60) gave a history of exposure to anim-als: 3% to camels and 14% to other unspecified animals. the presence or absence of a co-morbidity was recorded in two-thirds of cases (n = 685); of these, 496 (72.4%) were considered as being at risk, either because they were aged ≥ 65 years or had one or more pre-existing medical conditions. of 496, 208 (41.9%) were aged 65 years or older, and apart from seven (3%), all had preexisting medical conditions; in 108 (22%) cases, the conditions were specified: diabetes mellitus (50%, 54/108), hypertension (45%, 49/108), and renal (30%, 32/108), cardiac (19%, 21/108), pulmonary (17%, 18/108), malignant (13%, 14/108), and neurological diseases (5%, 5/108). another 16% (17/108) had other chronic diseases in various combinations. over half (52%) of the patients had multiple comorbidities. of the total 1060 cases, 384 (36%) died by the time the data were analyzed, of which 270 (70%) were male. diabetes, hypertension, renal disease, malignancy, and several other conditions hitherto termed as miscellaneous conditions (e.g., anemia, obesity and congenital abnormalities, diseases of the liver and gall bladder, or steroid use) were significant risk factors for mortality from mers-cov (table 1) . male sex was also a significant risk factor for mortality but the proportions of diabetes, hypertension, renal disease, malignancy, and miscellaneous conditions in men were not significantly different from that in women. of 676 mers patients who survived, 130 (19.2%) required icu admission, therefore were considered to have a severe form of the disease. of those who survived, 428 (63%) had pre-existing conditions, of which diabetes and neurological conditions were significant risk factors for severity (table 2) . these results corroborate the findings of other smaller studies from saudi arabia and south korea that showed that older age and presence of co-morbid conditions were associated with higher mortality (saad et al., 2014; feikin et al., 2015; majumder et al., 2015; korea cdc, 2015) . our study has specifically identified that diabetes, hypertension, renal disease, malignancy, miscellaneous conditions, and male sex were the main risk factors for mortality. another small study also demonstrated that diabetes was significantly associated with mortality (or = 15.7; 95%ci 2.5-100.7) (shalhoub et al., 2015) . in contrast, among south korean patients, underlying respiratory disease and older age were the key risk factors for mortality (korea cdc, 2015) . in our study, presence of a respiratory disease was not a significant risk factor and we did not explore the association of older age with mortality, because essentially all patients aged ≥ 65 years in our cohort had a pre-existing disease, but age itself could be an independent risk factor, as other studies from saudi arabia and south korea demonstrated that age > 60 years (in some studies ≥ 65 years) was significantly associated with mortality (feikin et al., 2015; majumder et al., 2015; saad et al., 2014) . additionally, majumder et al. (2015) demonstrated that for every one year of increase in age, the odds of fatality increased by 12% (or 1.1, 95% ci 1.1-1.2). the risk factors might vary geographically; additionally, the relatively small sample sizes in other studies could have missed other significant risk factors. our study also demonstrates that men had higher odds of dying from mers-cov, which is unsurprising given the fact that the disease predominantly affects men. another saudi study also demonstrated that the case fatality rate was higher for men (52%) than for women (23%) (alghamdi et al., 2014) . in the south korean cohort, even though being male increased the odds of mortality, the association was not significant (majumder et al., 2015) , likely because of small size. in our cohort, 65% of the patients were men, while in some other series, 97% of the cases were men, which is explained by the fact that men more often come in close contact with camels than women in arabian countries (alraddadi et al., 2015) . mers-cov patients with diabetes and neurological conditions were more likely to require icu admission than those without these conditions, which are unique findings. in contrast, a study involving 70 saudi arabian mers patients showed that concomitant infections (or 14.1, 95% ci 1.6-126.1, p = 0.02) and low albumin levels (or 6.3, 95% ci 1.2-31.9; p = 0.03) were independent risk factors for icu admission (saad et al. 2014) . approximately 14% of the mers-cov patients in our cohort were hcws, and exposure to healthcare setting was an important ground for transmission of the virus (over 20%), which is supported by other available data (oboho et al., 2015; petersen et al., 2014; saad et al., 2014) , and mathematical modelling suggests that nosocomial transmission is over four times higher than community transmission (chowell et al., 2014) . considering that pre-existing conditions such as diabetes, hypertension, renal, malignant, neurological, and miscellaneous conditions are risk factors for severe outcomes of mers-cov, due attention should be paid to optimum control of these conditions. co-infection with mers-cov and influenza plus other bacterial and viral infections have been reported (zumla et al., 2015) , and co-infection is seen to increase the severity of the disease (saad et al., 2014) . therefore, vaccinations against influenza and pneumococcal disease should be considered for the high-risk individuals (those with pre-existing disease and/or aged ≥ 65 years). these vaccines could prevent co-infections by influenza and streptococcus pneumoniae as well as the fatal outcomes of mers-cov. they may even reduce the need for hospital visits or even icu admission. the limitations of this study are: data could not be validated from the original source and multivariate analysis was not considered. despite these limitations, the study provides valuable information on risk factors for severity and mortality in mers patients from a relatively large dataset, and has actually ascertained some key risk factors. we hope this study would inspire further research based on even larger datasets. middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia middle east respiratory syndrome key: cord-261163-n9tp9nx7 authors: ko, jae-hoon; müller, marcel a.; seok, hyeri; park, ga eun; lee, ji yeon; cho, sun young; ha, young eun; baek, jin yang; kim, so hyun; kang, ji-man; kim, yae-jean; jo, ik joon; chung, chi ryang; hahn, myong-joon; drosten, christian; kang, cheol-in; chung, doo ryeon; song, jae-hoon; kang, eun-suk; peck, kyong ran title: serologic responses of 42 mers-coronavirus-infected patients according to the disease severity date: 2017-10-31 journal: diagnostic microbiology and infectious disease doi: 10.1016/j.diagmicrobio.2017.07.006 sha: doc_id: 261163 cord_uid: n9tp9nx7 abstract we evaluated serologic response of 42 middle east respiratory syndrome coronavirus (mers-cov)-infected patients according to 4 severity groups: asymptomatic infection (group 0), symptomatic infection without pneumonia (group 1), pneumonia without respiratory failure (group 2), and pneumonia progressing to respiratory failure (group 3). none of the group 0 patients showed seroconversion, while the seroconversion rate gradually increased with increasing disease severity (0.0%, 60.0%, 93.8%, and 100% in group 0, 1, 2, 3, respectively; p = 0.001). group 3 patients showed delayed increment of antibody titers during the fourth week, while group 2 patients showed robust increment of antibody titer during the third week. among patients having pneumonia, 75% of deceased patients did not show seroconversion by the third week, while 100% of the survived patients were seroconverted (p = 0.003). since the first reported case of middle east respiratory syndrome coronavirus (mers-cov) in 2012 (zaki et al., 2012) , small and large outbreaks have occurred, resulting in 1917 mers-cov infections and 677 related deaths to date (who, 2017) . to understand this fatal respiratory viral infection, several serologic investigations have been conducted (corman et al., 2016; min et al., 2016; park et al., 2015; payne et al., 2016) . however, practical analysis of serodiagnostic parameters for clinical usage was limited in previous studies, due to insufficient sample size or clinical information. we managed 45 mers-cov-infected patients, which is the largest number of patients as a single center during the 2015 korean mers outbreak (total 186 patients identified) kim et al., 2016; park et al., 2016) , and reported that mers-cov-infected patients experienced 4 distinct clinical courses, ranging from asymptomatic infection to severe pneumonia requiring mechanical ventilation . based on these findings, we evaluated serologic response of 42 mers-cov-infected patients according to the disease severity to investigate potential role of serodiagnostic parameters as prognostic markers. among 45 mers-cov-infected patients who were admitted to samsung medical center, a 1950-bed tertiary care university hospital, during the 2015 korean mers outbreak , we obtained sera from 42 patients. mers-cov infections were confirmed on the diagnostic microbiology and infectious disease 89 (2017) [106] [107] [108] [109] [110] [111] basis of real-time reverse transcriptase polymerase chain reaction (rrt-pcr) assays targeting upstream of the e gene (upe) and the openreading frame gene 1a (orf1a) (corman et al., 2012a; madani, 2014) . epidemiologic investigation data and electronic medical records were reviewed to obtain exact exposure date, symptom onset, clinical course, and outcome data for the patients. one or 2 residual serum samples per week of illness were used for serologic testing during hospitalization periods. follow-up serum samples obtained at outpatient clinics were also tested up to 6 months from symptom onset. the institutional review board of samsung medical center approved the present study. the clinical course of mers-cov-infected patients was assessed 6 weeks after symptom onset and patients were divided into 4 disease severity groups: asymptomatic infection (group 0), symptomatic infection without pneumonia (group 1), pneumonia without respiratory failure (group 2), and pneumonia progressing to respiratory failure (group 3) . for practical purposes, respiratory failure was defined as the need for mechanical ventilation. only patients in group 3 experienced fatal outcomes (5/13, 38.5%), and interval from symptom onset to death was 27 days in median ). proportion of underlying immunocompromising conditions including diabetes, solid cancer, or hematologic malignancies was not different between groups . the distinct clinical presentation of the 4 severity groups are presented in supplementary figs. 1 and 2, and supplementary table 1, in addition to the previous report . seroconversion status was determined based on neutralization activity: if none of the serum samples from a mers-cov-infected patient, necessarily including sera obtained after the third week of illness, showed neutralization activity, the patient was considered to have negative seroconversion; if none of the serum samples obtained by the end of the third week of illness showed neutralization activity and no samples were available for neutralization tests thereafter, the patient was considered to have an indeterminate response (i.e. interpretation not applicable); if any serum showed neutralization activity, the patient was considered to have positive seroconversion. patients with an indeterminate response were excluded from calculation of the seroconversion rate. this definition is based on the premise that no patients had previous exposure to mers-cov, as this was the first mers outbreak in korea as a non-endemic country. during the outbreak, mers-cov exposure dates and symptom onsets were clearly identified in most patients, owing to thorough contact investigation and monitoring of exposed individuals park et al., 2016) . mers-related symptoms included fever, myalgia, cough, sputum, and diarrhea. to provide a common point of reference, we used 'days post onset of illness (dpoi)' to evaluate mers-covinfected patients. for asymptomatic patients, the day of diagnosis of mers-cov infection was considered as day of symptom onset . anti-mers-cov elisa igg and iga (euroimmun, lübeck, germany) were based on soluble mers-cov spike protein s1 domain expressed in hek-293 t cells (muller et al., , 2015 muth et al., 2015; raj et al., 2013) . sera were tested according to the manufacturer's instructions with 1:100 dilutions. secondary detection was done with peroxidase-labeled anti-human igg and iga. cutoff values of od ratio 0.4 for elisa igg and 0.2 for elisa iga were applied in the present study, as these values exhibited optimal performance in predicting neutralization activity . anti-mers-cov ifa igm (euroimmun) was performed with slides carrying vero cells infected with full mers-cov (corman et al., 2012b; meyer et al., 2014; muller et al., 2014 muller et al., , 2015 . sera were tested according to the manufacturer's instructions with 1:10 dilutions. weekly positive ifa intensity was considered cutoff intensity value of ifa igm, which exhibited optimal performance in predicting neutralization activity . mers-cov prnt was performed as previously described muller et al., 2014 muller et al., , 2015 . pre-dilution before setting up the log2dilution series was 1:10, defining 1:20 as the lowest possible significant titer for categorizing a sample as positive . for comparison of clinical variables between groups, one-way analysis of variance (anova) or kruskal-wallis test was used for continuous variables, and chi-square or fisher's exact test was used for categorical variables. six-week survival probability was calculated using the kaplan-meier method. the cox proportional hazard model and logrank test were used to examine the association of seroconversion status with the 6-week mortality of mers patients having pneumonia. all pvalues were 2-tailed, and those b0.05 were considered to be statistically significant. r-3.3.1 for windows (rstudio, boston, ma, usa) was used for all statistical analyses. seroconversion status of 42 mers-cov-infected patients is summarized in table 1 . none of the group 0 patients showed seroconversion, and the seroconversion rate gradually increased with increasing disease severity (0.0%, 60.0%, 93.8%, and 100% in groups 0, 1, 2, and 3, respectively; p = 0.001). seroconversion was observed from 14 to 24 dpoi (18 dpoi in median), mostly during the third week of illness (88.0% of seroconverted patients with a known timeline). group 3 patients showed slightly delayed timing of seroconversion compared to group 2 patients (18.5 and 17.5 dpoi in median, respectively, without statistical significance), and seroconversion during the fourth week of illness was exclusively observed in group 3. serologic responses of seroconverted patients are depicted according to the severity groups with 7-day intervals in fig. 1 . serologic response occurred from the third week of illness, and antibody response is weaker in patients with mild symptomatic patients (group 1) than patients with pneumonia (groups 2 and 3). group 2 patients showed robust increment of antibody titer during the third week (compared to the 2nd week, the median od ratios of elisa igg and iga increased more than 3-fold, and ifa igm and prnt increased from negative to 2+ and 1:80, respectively), and the titers did not significantly increase thereafter (in comparison of the median values of third week and fourth week, no statistical significance was observed). meanwhile, group 3 patients showed delayed and continuous increment of antibody titers from the third week: the median values of each serologic test were significantly higher during the fourth week compared to those of the third week in group 3 (all p b 0.05). in comparison between groups 2 and 3, antibody titers of group 3 patients during the third week were numerically lower than those of group 2, although only elisa igg showed statistically significant difference (p = 0.016). the antibody titers of group 3 patients continuously increased, showing numerically higher titers compared to those of group 2 patients during the fourth week (without statistical significance). detailed serologic test results for each patient are presented according to timeline and severity groups in supplementary tables 2 to 5. as seroconversion rates were low in mild severity groups (0% in group 0 and 60% in group 1), outcome analysis was performed in patients having pneumonia (groups 2 and 3). only 25% of deceased patients showed seroconversion by the end of the third week of illness, while 100% of survived patients seroconverted (p = 0.003, table 2 ). this difference also could be discriminated by elisa igg (with od ratio cutoff value of 0.4, p = 0.003) and elisa iga (with od ratio cutoff value of 0.2, p = 0.010). ifa igm response was not significantly different between survivors and non-survivors (with intensity cutoff value of weakly positive, p = 0.135). in a kaplan-meier analysis comparing seroconverted patients and non-converted patients by the third week of illness, seroconverted patients showed significantly higher survival probability compared to patients with negative seroconversion (fig. 2 , p b 0.001 by log-rank test). negative seroconversion in pneumonia patients by the third week of illness showed a hazard ratio of 27.83 (95% ci 2.76-280.21, p = 0.005, by the cox proportional hazard model) in predicting 6-week mortality. since previous hospital-associated outbreaks of mers occurred in endemic countries, where primary infections flow from community into hospitals, detailed clinical data of each patient were hard to obtain (corman et al., 2016) . however, during the 2015 korean mers outbreak, the first outbreak in a non-endemic country, epidemiologic links and entire clinical course of each patients could be clearly identified park et al., 2016) . owing to the detailed epidemiologic and clinical information about patients, we could find out different serologic response depending on disease severity and outcome. although different seroconversion rates depending on disease severity can be inferred from previous serologic investigation (min et al., 2016) , the number of evaluated mers patients was limited to 14 and neutralization testing was not performed. in that study, a robust increment of elisa igg titer with a 3-fold increase in od ratio was exclusively observed among patients with severe pneumonia, while mild infections exhibited a modest increment in od ratio, if any. likewise, we noted that asymptomatic mers-cov-infected cases did not show serologic response including prnt within 6 months, and the seroconversion rate increased with the disease severity. although the number of asymptomatic patients was limited to 3 in the present analysis, it is less likely that asymptomatic patients will experience seroconversion considering that even group 1 patients with obvious mers-related symptoms showed low seroconversion rate of 60%. this finding correlates with another serologic study that evaluated 11 rrt-pcr-confirmed mers patients (choe et al., 2017) . in that study, antibody titers in 4 of 6 patients with mild illness were undetectable. in addition, most contact surveys of mers-cov could not detect additional rrt-pcr-negative prntpositive mers-cov infections (breakwell et al., 2015; buchholz et al., 2013; choi et al., 2016; . these findings imply that serologic surveys to detect subclinical infections among asymptomatic individuals would not be effective. serologic response was delayed in group 3 patients, and negative seroconversion by the third week of illness was associated with fatal outcome among patients with mers pneumonia (hr 27.83, 95% ci 2.76-280.21, p = 0.005). delayed commencement of serologic response in severe disease was also suggested by previous report by park et al. (park et al., 2015) . although seroconversion timing was not statistically significantly delayed in group 3 patients in the present study, delayed increment of igg, iga, and igm titers after the third week was demonstrated in group 3. however, the delayed serologic response in group 3 could not be used as predictor for respiratory failure, as respiratory failure progressed during the 2nd week of illness (12 dpoi in median). meanwhile, negative seroconversion in mers pneumonia by the third week of illness was associated with fatal outcome in the present analysis. impaired serologic response in deceased patient was also noted in the paper of corman et al., but insufficient clinical information, especially day of symptom onset, hampered more detailed analysis in association with timeline (corman et al., 2016) . in this study, we could obtain exact clinical information including day of symptom onset, and figured it out that seroconversion status by the third week of illness (by 21 dpoi) can serve as a prognostic marker. another important point is that mers-cov-infected patients in the present analysis died later than previous reports, probably owing to antiviral therapy or aggressive critical care including extracorporeal membrane oxygenation (ecmo). the median interval from symptom onset to death was 27 days in the present study, which is much longer than 11.5 days in previous reports (zumla et al., 2015) . although rapidly deteriorating mers cases would die before the third week of illness, there certainly is a population that benefit from prognosis prediction by serologic response. aggressive managements including ecmo should be considered for pneumonia patients without seroconversion by the third week of illness. although seroconversion status can be confirmed by neutralization tests, it cannot be readily performed worldwide (corman et al., table 1 seroconversion status of mers-cov-infected patients according to the disease severity group. classification by the disease severity group 0 asymptomatic (n = 3) group 1 symptomatic (n = 10) group 2 pneumonia (n = 18) group 3 resp. failure (n = 11) 2016). in the present analysis, seroconversion status of the third week assessed by elisa igg and iga was similar with that by prnt. these elisa tests can be practically used for predicting poor prognosis of mers pneumonia in the field of patient management. as a retrospective study, serum samples of each patient could not be collected with same interval. however, we applied strict criteria for seroconversion, excluding patients who did not have follow-up samples after the third week of illness as indeterminate response. in the previous report with the same patient population, we also suggest predictive factors for disease progression using clinical variables within 3 days from symptom onset . together with the present paper, these factors could be used complementarily in managing mers-cov-infected patients. in addition, although we identified that seroconversion status by the third week was associated with fatal outcomes of mers pneumonia, we could not perform multivariate analysis due to limited sample size. this finding need to be further evaluated with enough patient numbers of mers pneumonia. in conclusion, in a serologic investigation of 42 mers-cov-infected patients, mild cases showed low seroconversion rates, while fatal cases showed impaired serologic responses. this work was supported by a samsung biomedical research institute (sbri) grant [#smx1161321]. cd reports funding by eu grants antigone (ga no. 278976) and prepare (ga no. 602525). there are no potential conflicts of interest relevant to this article to report. lack of transmission among close contacts of patient with case of middle east respiratory syndrome imported into the united states contact investigation of a case of human novel coronavirus infection treated in a german hospital mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study mers-cov antibody responses 1 year after symptom onset clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak suggested new breakpoints of anti-mers-cov antibody elisa titers: performance analysis of serologic tests. suggested new breakpoints of anti-mers-cov antibody elisa titers: performance analysis of serologic tests predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients case definition and management of patients with mers coronavirus in saudi arabia antibodies against mers coronavirus in dromedary camels comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity mers coronavirus neutralizing antibodies in camels, eastern africa survival probability according to the seroconversion status was evaluated in mers-cov-infected patients having pneumonia, whose seroconversion status during the third week of illness is identifiable. seroconverted patients showed significantly higher survival probability compared to patients with negative seroconversion (p b 0.001 by log-rank test) presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea kinetics of serologic responses to mers coronavirus infection in humans persistence of antibodies against middle east respiratory syndrome coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc who, middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome we would like to express our sincerest condolences to the patients and families who suffered from the mers outbreak. we also greatly appreciate the hcp and staff members at samsung medical center and all other hospitals who worked together to overcome the mers outbreak. finally, we would like to thank jinseob kim for statistical advice and figure development, as well as mingu kang for ifa testing. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2017.07.006. key: cord-255815-5d9bqji0 authors: malik, ajamaluddin; alsenaidy, mohammad a. title: mers‐cov papain-like protease (pl(pro)): expression, purification, and spectroscopic/thermodynamic characterization date: 2017-05-30 journal: 3 biotech doi: 10.1007/s13205-017-0744-3 sha: doc_id: 255815 cord_uid: 5d9bqji0 within a decade, mers-cov emerged with nearly four times higher case fatality rate than an earlier outbreak of sars-cov and spread out in 27 countries in short span of time. as an emerging virus, combating it requires an in-depth understanding of its molecular machinery. therefore, conformational characterization studies of coronavirus proteins are necessary to advance our knowledge of the matter for the development of antiviral therapies. in this study, mers-cov papain-like protease (pl(pro)) was recombinantly expressed and purified. thermal folding pathway and thermodynamic properties were characterized using dynamic multimode spectroscopy (dms) and thermal shift assay. dms study showed that the pl(pro) undergoes a single thermal transition and follows a pathway of two-state folding with t (m) and van’t hoff enthalpy values of 54.4 ± 0.1 °c and 317.1 ± 3.9 kj/mol, respectively. an orthogonal technique based on intrinsic tryptophan fluorescence also showed that mers-cov pl(pro) undergoes a single thermal transition and unfolds via a pathway of two-state folding with a t (m) value of 51.4 °c. our findings provide significant understandings of the thermodynamic and structural properties of mers-cov pl(pro). frequent fatal coronavirus outbreaks in humans and animals have caused serious concerns in the healthcare sector, scientific community, and animal husbandry. first human outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in 2002 caused life-threatening atypical pneumonia in more than 8000 people in 26 countries with a case fatality rate (cfr) of 10% (pillaiyar et al. 2015; al-tawfiq et al. 2014; who 2016b) . another lethal coronavirus outbreak emerged in the arab peninsula countries in 2012 which was caused by what is now known as the middle east respiratory syndrome coronavirus (mers-cov) . from september 2012 to december 2016, 1864 laboratory-confirmed mers-cov cases of infection in 27 countries with 659 mortalities (nearly four times higher cfr than sars-cov) have been reported (al-tawfiq et al. 2016; who 2016a) . coronavirus survivors after acute infections suffer from many health issues and require long-term medical assistance (han et al. 2003; ong et al. 2005; chan et al. 2003; leow et al. 2005; siu 2016; cha et al. 2016) . in addition to sars-cov and mers-cov, at least four other pathological coronaviruses (hcov-oc43, hcov-229e, hcov-hku1, and hcov-nl63) are continuously circulating in humans causing relatively mild respiratory conditions that may in some instances escalate to severe pathological illnesses (mackay et al. 2012; carbajo-lozoya et al. 2012; simon et al. 2007) . coronaviruses have also caused deadly diseases in animals, leading to huge economic losses in the animal husbandry sector (vlasova et al. 2014; lee and lee 2014; sun et al. 2016) . moreover, high mutation and recombination rates in coronaviruses allow them to cross species barriers and adapt to new hosts more easily (denison et al. 2011; lau and chan 2015) . viral proteases are essential for pathogenesis and virulence. like all coronaviruses, mers-cov contains two cysteine proteases (main protease and papain-like protease) which processes viral nonstructural polypeptides (kilianski et al. 2013; hilgenfeld 2014) . mers-cov main protease (m pro , also called the 3c-like protease, 3cl pro ) cleaves at eleven sites, while mers-cov papain-like protease (pl pro ) cuts at three sites on the nonstructural polypeptides and releases mature nonstructural proteins (hilgenfeld 2014) . thus, mers-cov proteases make up a suitable target for antiviral therapies. mers-cov open-reading frame 1 (orf1) encodes two large polyproteins (pp1a and pp1b). mers-cov pl pro domain is encoded on the pp1a proteins (residue 1484-1800) (yang et al. 2014; hilgenfeld 2014; kilianski et al. 2013) . like other coronaviruses, mers-cov pl pro contains a catalytic triad and exhibits similar proteolytic, deubiquitination, and isg15-linked isgylation properties (lin et al. 2014; chen et al. 2007; clementz et al. 2010; yang et al. 2014; zheng et al. 2008) . in this study, we expressed and purified mers-cov pl pro . thermal stability was studied by thermal shift assay using intrinsic fluorescence and dynamic multimode spectroscopy (dms). mers-cov pl pro was found to unfold via a single thermal transition and follows a pathway of two-state folding. this study will not only help in the understanding of the folding and stability of mers-cov pl pro but also could help shed some light on other deubiquitinating enzymes with the similar folding scaffold. the orf of mers-cov pl pro (1484-1800 polyprotein residues, genbank accession number nc_019843.2) was cloned into pet28a plasmid under t7 promoter as published before (lin et al. 2014) . the codon was optimized (genscript, usa) and cloned between ncoi and xhoi sites which was in frame of c-terminal his tag present on the vector. e. coli bl21 (de3) plyss was used for the expression of recombinant protein. low-molecular weight protein markers, prepacked ni-nta, and superdex 75 columns were from amersham biosciences (united kingdom). chicken egg lysozyme was from usb corporation, usa. benzonase, ans, and kanamycin from sigma. iptg was purchased from bio basic, canada. all other chemicals used in this study were of reagent grade. cary 60 spectrometer and cary eclipse spectrofluorometer were from agilent technologies, usa. akta purification system was from amersham biosciences (united kingdom) and sds-page assembly from bio-rad (usa). thermomixer and benchtop cooling centrifuge were from eppendorf, germany. innova 44r shaking incubator was from new brunswick, germany. chirascan-plus spectropolarimeter was from applied photophysics, united kingdom. expression and purification of mers-cov pl pro in e. coli bl21 (de3) plyss e. coli bl21 (de3) plyss harboring pet28a-mpl pro was used for expression of mers-cov pl pro . protein expression and soluble protein extraction were performed as described in lin et al. (2014) . purification of mers-cov pl pro was performed with minor modification of an earlier published protocol (lin et al. 2014) . briefly, 1 mm dtt was used throughout unless described. cleared crude lysate was passed through a 1-ml ni-nta column pre-equilibrated with 20 mm tris, ph 8.5, 500 mm nacl, 10 mm imidazole, and 1 mm dtt and washed with 20 cv equilibration buffer. bound protein was eluted with a linear gradient of 0-50% buffer b (equilibration buffer containing 500 mm imidazole) at 1 ml/min flow rate on akta purification system. the purity of eluted fractions was analyzed on sds-page. the prepacked superdex 75 equilibrated with 20 mm tris, ph 8.5, 100 mm nacl, and 1 mm dtt was calibrated with proteins of known molecular weight. subsequently, ni-nta-purified mers-cov pl pro was further purified using superdex 75 column. the purity of eluted fractions was analyzed on sds-page. highly pure fractions were pooled, aliquoted, and stored at -80°c. before analysis, the frozen aliquots were thawed and centrifuged at 13,000 rpm for 15 min at 4°c. protein concentration was determined spectrophotometrically at 280 nm using a molar extinction coefficient of 42,400 m -1 cm -1 . tryptophan fluorescence spectra (50 lg/ml) of mers-cov pl pro were recorded using a cary eclipse fluorescence spectrophotometer in a 10-mm-path length cuvette. to measure tryptophan fluorescence, mers-cov pl pro sample was excited at 295 nm and emission spectra were collected between 305 and 400 nm (5 nm excitation and 5 nm emission bandwidth). temperature melting studies of mers-cov pl pro were performed at 10°c increments as well as a linear increase of 1°c/min. mers-cov pl pro incubated at different temperatures from 20 to 90°c in peltier-controlled cary eclipse fluorometer. the temperature of the protein samples was monitored using an internal temperature probe. when the desired temperature was reached, the sample was allowed to equilibrate for 2 min before tryptophan fluorescence spectra were measured. the maximum fluorescence intensity (i max ) and maximum fluorescence wavelength (k max ) were plotted with respect to temperature. in a similar experiment, mers-cov pl pro was gradually heated from 20 to 80°c at a rate of 1°c/min during which tryptophan fluorescence was measured by exciting at 295 nm and collecting at 330 and 350 nm to obtain the temperature melting curve. to study the secondary structure of mers-cov pl pro in terms of conformational and thermal stability, dynamic multimode spectroscopy was applied. the measurement was performed using chirascan-plus spectrophotometer, calibrated with (1s)-(?)-10-camphorsulfonic acid. in this study, 0.2 mg/ml of mers-cov pl pro was gradually heated from 20 to 94°c at 1°c/ml rate. internal thermal probe was inserted in the 0.1-cm-path length cuvette to precisely monitor the actual temperature of the samples. far-uv cd spectra from 200 to 250 nm were recorded at each temperature. thermal transition data were processed using manufacturer's global 3 software. the steady-state kinetics of mers-cov pl pro was measured as described in lin et al. (2014) , with slight modification. briefly, 50 lm fluorogenic peptidyl substrate, dabcyl-frlkggapikgv-edans (genscript), was mixed with different concentrations of mers-cov pl pro (6.8-0.1 lm, in twofold serial dilution) using 50 mm phosphate at ph 6.5 as a buffer at room temperature. the fluorescence signal was measured for 30 min at 60-s intervals in a hidex chameleon plate reader using 340 nm (excitation) and 535 nm (emission filter) with 20% gain. expression and purification of recombinant mers-cov pl pro in this study, mers-cov pl pro was overexpressed in e. coli bl21 (de3) plyss. mers-cov pl pro was purified in two-step chromatography as described in lin et al. (2014) . ni-nta elute contained minor impurities (data not shown). when ni-nta elute passed through gel filtration column, one major symmetrical sharp peak was obtained (fig. 1a) . sds-page analysis of the pooled fractions showed the yield of highly pure mers-cov pl pro (fig. 1b) . we obtained nearly 10 mg of mers-cov pl pro from a 1-l shake flask culture. in an earlier study, the yield of soluble mers-cov pl pro was strain dependent and the best yield (*52 mg purified protein from 1 l shake flask culture) was obtained with e. coli bl21 (de3) star strain (lin et al. 2014) . the difference in the yield of mers-cov pl pro was due to the different strain's genetic background. intrinsic fluorescence spectroscopy is a highly sensitive tool, which provides information about the microenvironment of trp and tyr residues within proteins. tyrosine emission maximum is less sensitive to its local environment compared to tryptophan. indole ring in the tryptophan residues undergoes two isoenergetic transitions which causes polarity sensitivity, while tyrosine undergoes through a single electronic state (ghisaidoobe and chung 2014) . during the course of protein unfolding, the polarity of fluorophore microenvironment changes, which in turn leads to changes in the maximum fluorescence intensity (i max ) as well as maximum fluorescence wavelength (k max ). therefore, intrinsic tryptophan fluorescence emission is sensitive to the tertiary structure and detects subtle protein conformational changes in solution. it has been frequently used for the characterization of protein's conformational changes under different stress conditions (kumar et al. 2005; xiao et al. 2015) . a previous study analyzing the quaternary structure of mers-cov pl pro using analytical ultracentrifugation technique has shown that pl pro is found in the monomeric state (lin et al. 2014) . mers-cov pl pro contains ten tyrosine and five tryptophan residues (fig. 2a) . mers-cov pl pro consists of two domains: n-terminal ubiquitinlike (ubl) domain and a catalytic core domain. the n-terminal ubl domain consists of 62 residues and contained one a-helix, one 3 10 -helix and five b-strands. the substrate-binding region is solvent exposed and comprised of the right-hand scaffold (lei et al. 2014) . three tryptophan residues (w93, 243 and 303) are buried, while w187 and w190 are surface exposed (fig. 2b) . figure 3a shows the decrease in intrinsic fluorescence of mers-cov pl pro with increasing temperature. at low temperature (20°c), highest fluorescent intensity (i max ) was observed with maximum fluorescence wavelength (k max ) at 343 nm, indicating an overall localization of all five tryptophans in the partially hydrophobic environment. as the temperature increases gradually from 20 to 90°c, i max decreased with major transitions occurring between 50 and 60°c (fig. 3a, b) . initially, k max increased from 343 to 344 nm when the temperature was increased from 20 to 30°c. as the temperature was increased further, a blue-shift in k max with increasing temperature was observed, indicating conformational rearrangements in different temperature regimes. the major blue-shift in k max was found between 50 and 60°c (fig. 3c) . the 14-nm blue-shift of tryptophan fluorescence spectrum indicated that the microenvironment of the tryptophan residues is becoming more hydrophobic during thermal denaturation. mers-covpl pro thermal unfolding is different from most other proteins. commonly, protein unfolding fluorescence spectra are characterized by a long wavelength shift ''red-shift.'' but some proteins, fig. 2 a sequence of c-terminal his-tagged mers-cov pl pro showing ten tyr and five trp residues, which are highlighted in green and blue, respectively. potential internal protease site is highlighted in red and his-tagged residues are underlined. b three-dimensional structure of mers-cov pl pro (pdb id: 4r3d) with the side chains of the five trp residues being numbered and colored in blue including mers-cov pl pro , exhibit blue-shift upon denaturation (slutskaya et al. 2015; duy and fitter 2006; pattanaik et al. 2003) . pig pancreatic a-amylase showed red-shifted fluorescence spectra when chemically unfolded and showed blue-shifted spectra during thermal unfolding (duy and fitter 2006) . equine lysozyme first exhibits a blue-shift transition at lower temperature and red-shift above 50°c (morozova et al. 1991) . to obtain temperature melting curve, mers-covpl pro was gradually heated from 20 to 80°c at 1°c/min and the ratio of 330/350 nm tryptophan fluorescence was plotted with respect to temperature (fig. 4) . data were fitted according to the equation f = y 0 ? a/(1 ? exp(-(xx 0 )/ b)) with an r 2 value of 0.9910. our results showed that mers-cov pl pro was moderately stable and unfolds via a single transition with a t m value of 51.4°c. since mers-cov pl pro is a protease, it may undergo autolysis during thermal shift assay. it contains one potential autolysis site (lkgg) as shown in fig. 2a . to evaluate the extent of autolysis as well as the extent of irreversible thermal unfolding and aggregation, mers-cov pl pro (0.2 mg/ml) was incubated on a thermomixer from 20 to 70°c with 10°c intervals. six samples were gradually heated and equilibrated at the respective temperatures for 3 min. when the desired temperatures were attained, the samples were removed, kept on ice, and centrifuged at 13,000 rpm for 15 min to remove any forming aggregates. equal volumes of the supernatant were analyzed (fig. 5) . if mers-cov pl pro would undergo autolysis during the course of thermal incubation, we would expect to see the appearance of two or more bands of mers-cov pl pro fragments on sds-page gels or at least a decrease in the band intensity of mers-cov pl pro . we found that the intensity of mers-cov pl pro band was apparently unchanged, indicating that autolysis was not occurring during the thermal shift assays applied in this study. in another scenario, irreversible unfolding aggregates may form during thermal shift assay. if this is the case, then aggregated protein will be pelleted down upon centrifugation and band intensity would have decreased in the supernatant sample. our result showed that the band intensity of the supernatant samples incubated from 20 to 70°c was apparently unchanged (fig. 5) , indicating fig. 3 thermally induced structural changes in mers-cov pl pro as monitored by the intrinsic tryptophan fluorescence spectroscopy. a to monitor tryptophan fluorescence at different temperatures, mers-cov pl pro was slowly heated and allowed to equilibrate for 2 min at the respective temperatures. the sample was excited at 295 nm and the emission spectra were collected from 305 to 400 nm (5 nm excitation and 5 nm emission bandwidth). b effect of temperature on the tryptophan emission intensity showing the decrease of i max with increasing temperature. major transition occurred between 50 and 60°c. c effect of temperature on the tryptophan maximum emission wavelength (k max ). blue-shift was observed during thermal unfolding of mers-cov pl pro . initially, red-shift was observed when the temperature was increased from 20 to 30°c. further increase in temperature leads to blue-shift with major changes occurring between 50 and 60°c no or insignificant aggregation occurring during thermal shift assays. information about thermal stability, unfolding pathway, and secondary structure of mers-cov pl pro was obtained using an orthogonal method. we employed dms, a newly developed information-rich experimental technique, to obtain spectroscopic and thermodynamic data of melting temperature (t m ) and van't hoff enthalpy (dh vh ) (john and weeks 2000; greenfield 2006; al-ahmady et al. 2012) . temperature-induced secondary structural changes in mers-cov pl pro in the far-uv region were monitored to study thermodynamic parameters. moreover, dms also characterized thermal unfolding pathway and identified the number of folding intermediate species and their relative concentrations during the unfolding process (malik et al. 2015 (malik et al. , 2016 . mers-cov pl pro was gradually heated from 20 to 94°c at 1°c/min increments and far-uv cd spectra were recorded from 200 to 250 nm. far-uv cd spectra at several wavelengths were plotted as a function of temperature (fig. 6a) . mers-cov pl pro underwent a single thermal transition as a function of temperature, suggesting two-state folding. similar transition was also observed in the thermal shift assay using fluorescence spectroscopy. in an earlier study, secondary structure content was calculated and it was found that b-sheet structure (31%) is dominant in mers-cov pl pro (lin et al. 2014) . similarly, our results showed that mers-cov pl pro presented a single negative minimum at *218 nm suggesting a predominant b-sheet structure as well. when mers-cov pl pro was gradually heated, secondary structure was lost and became irregularly disorder structure (fig. 6b) . the relative concentrations of folded and unfolded species as a function of temperature are shown in fig. 6c . the thermal melting point (t m ) and van't hoff enthalpy (dh vh ) of mers-cov pl pro calculated using global 3 analysis software were 54.4 ± 0.1°c and 317.1 ± 3.9 kj/mol, respectively. the thermal melting points (t m ) of mers-cov pl pro calculated by thermal shift assay and dms were found to be in close proximity, reflecting tertiary-secondary structure unfolding events, respectively. in a recent study, papainlike protease of murine coronavirus also underwent a single thermal transition with a t m value of *46°c (mielech et al. 2015) . a three-dimensional model of the thermal transitions of mers-cov pl pro was generated using global 3 analysis software (fig. 6d) . a single transition is clearly evident at lower wavelengths in the far-uv spectra region. mers-cov pl pro was expressed in soluble state in e. coli and purified to homogeneity in two-step chromatography. two orthogonal techniques were used for studying unfolding pathway that include the utilization of dms and thermal shift assay. the results showed that mers-cov pl pro unfolds via a single thermal transition and follows a two-state unfolding pathway. thermal shift assay calculated a t m value of 51.4°c and dms method calculated a t m value of 54.4 ± 0.1°c, in agreement with sequential unfolding events of tertiary and secondary structures, respectively. similar folding behavior and thermal melting point were also observed in papain-like protease of murine lipid-peptide vesicle nanoscale hybrids for triggered drug release by mild hyperthermia in vitro and in vivo travel implications of emerging coronaviruses: sars and mers-cov middle east respiratory syndrome coronavirus: current situation and travelassociated concerns replication of human coronaviruses sars-cov, hcov-nl63 and hcov-229e is inhibited by the drug fk506 a case report of a middle east respiratory syndrome survivor with kidney biopsy results sars: prognosis, outcome and sequelae proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl63 deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases coronaviruses: an rna proofreading machine regulates replication fidelity and diversity how aggregation and conformational scrambling of unfolded states govern fluorescence emission spectra intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on forster resonance energy transfer techniques using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions a follow-up study of 69 discharged sars patients from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design van't hoff enthalpies without baselines assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferasebased biosensors second derivative tryptophan fluorescence spectroscopy as a tool to characterize partially unfolded intermediates of proteins coronaviruses: emerging and re-emerging pathogens in humans and animals outbreak-related porcine epidemic diarrhea virus strains similar to us strains crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features hypocortisolism in survivors of severe acute respiratory syndrome (sars) structural and functional characterization of mers coronavirus papain-like protease co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 spectroscopic and thermodynamic properties of recombinant heat shock protein a6 from camelus dromedarius structural and thermodynamic properties of kappa class glutathione transferase from camelus dromedarius murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis stability of equine lysozyme. i. thermal unfolding behaviour 1-year pulmonary function and health status in survivors of severe acute respiratory syndrome unusual fluorescence of w168 in plasmodium falciparum triosephosphate isomerase, probed by single-tryptophan mutants middle east respiratory syndrome-coronavirus (mers-cov): an updated overview and pharmacotherapeutics acute life threatening event (alte) in an infant with human coronavirus hcov-229e infection coping with future epidemics: tai chi practice as an overcoming strategy used by survivors of severe acute respiratory syndrome (sars) in post-sars hong kong heat-induced conformational changes of tet peptidase from crenarchaeon desulfurococcus kamchatkensis epidemiology and vaccine of porcine epidemic diarrhea virus in china: a minireview distinct characteristics and complex evolution of pedv strains middle east respiratory syndrome coronavirus who (2016b) sars (severe acute respiratory syndrome heat-induced unfolding of apo-cp43 studied by fluorescence spectroscopy and cd spectroscopy proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production acknowledgements the authors extend their appreciation to the deanship of scientific research at king saud university for funding this work through the research project no r5-16-02-05. conflict of interest to the best of our knowledge, no conflict of interest, financial or others, exists. all authors are fully aware of this submission. key: cord-267540-9p4rky4c authors: joseph, iype title: middle east respiratory syndrome corona virus (mers cov): the next steps date: 2015-03-26 journal: j public health policy doi: 10.1057/jphp.2015.9 sha: doc_id: 267540 cord_uid: 9p4rky4c developing countries are at risk of importing middle east respiratory syndrome corona virus (mers cov) from the middle east. hospitals in the middle east currently reporting the disease are staffed by immigrants. in the current hot spots for mers cov a sizeable portion of the population is from other countries, but many of these countries have yet to detect any importation of mers cov. to assess the disease transmission in these countries, supplemental surveillance strategies are urgently needed beyond the currently recommended measures. a few strategies to address the situation are: (i) improving preparedness with enhanced surveillance in particular regions; (ii) targeting certain sentinel groups for surveillance in hot spots; and (iii) limited use of serosurveillance. recovered, immune patients can be employed to give patient care during outbreaks. saudi arabia reported the first case of middle east respiratory syndrome corona virus (mers-cov) in september, 2012. 1 also caused by a corona virus, it is not unlike sars. world over, a total of 965 laboratory-confirmed cases of infection, including at least 357 related deaths have been reported to the world health organization (who) as of 3 february 2015. 2 in addition to outbreaks in the arabian peninsula, sporadic cases have been imported to europe, africa, asia, and north america in returning travellers. no sustained transmission has been reported outside the arabian peninsula. evidence point to a reservoir for the virus among both bats 3 and camels, 4 but the issue is not yet settled. 5 who coordinates international surveillance. deaths, sick persons who recover, and asymptomatic carriers continue to be found. cases arise from unknown sources in the community and from within hospitals in the middle east region that have cared for laboratory-proven cases. surveillance systems in parts of the world outside the arabian peninsula remain deliberately inactive. how will the situation evolve? will the disease be extinguished at its source? will the current state of geographically limited transmission persist indefinitely? will it spread far and wide, immediately or after a delay? 6 exportation is being closely watched, but might the virus find another niche environment where it can successfully establish sustained transmission? clinically, 7 mers cov presents with symptoms of lower respiratory tract disease (fever, cough, dyspnoea, and chest pain), sore throat, myalgia, malaise, and gastro-intestinal symptoms, such as diarrhoea, vomiting and abdominal pain. complications described in fatal cases are hyperkalaemia with associated ventricular tachycardia, disseminated intravascular coagulation leading to cardiac arrest, pericarditis and multi-organ failure. a large proportion of the severely ill patients require mechanical ventilation. fatalities are more in those with co-morbid conditions and an age over 60 years. preparedness to face the mers cov threat in tropical developing countries is limited. experience of managing pandemic h1n1 (gathered from the prior threat of sars) might help prepare countries. [8] [9] [10] despite persisting knowledge gaps about transmission dynamics rational preparedness can begin. preparedness can begin in priority countries and in geographical locations with increased potential for importation. mers outbreaks linked to hospitals and to health care centre visits have been documented many times in the middle east. hospitals in the middle-east employ immigrant health staff, especially from india 11 and the philippines. 11, 12 in addition, much of the population includes labour immigrants 13 from pakistan, sri lanka, egypt, bangladesh, and indonesia. a look at where immigrants come from and their travel back to their native lands in the past 28 months would demarcate the geographical areas with potential for importation. a useful technique has already used hajj pilgrim data 14 (from the population of muslims who come from all over the world to mecca each year). the data on hospital employees can be extracted from the immigration departments of the affected middle-east countries. who might collate this important kind of data. health-care workers 15 are a high risk group to acquire mers cov. ten studies that addressed this issue have found that, among those infected, 24.4 per cent (90/369) were health-care workers. 16 the role of the asymptomatic health-care workers in the incubation period and of minimally sick health-care workers who continue to offer clinical services thus propagating the disease transmission have also been documented. 16 in addition, the hajj pilgrims have spread the disease. among the travellers, currently symptomatic individuals who satisfy the who case definition are being targeted for virological surveillance. travelling health-care workers, irrespective of whether they are symptomatic can be selected for virological testing. what is happening with mers cov transmission in countries with high likelihood of importation? this has not yet been adequately assessed. using the current strategy of virological testing of patients fulfilling the who case definition, india, pakistan, egypt, bangladesh, philippines, sri lanka, and indonesia 13 (countries having sizable numbers of health workers employed in the middle-east) have not yet detected more than one case each. is this because of the absence of importation or to surveillance systems in these countries? perhaps an alternate strategy is needed to detect arrival of the virus into these countries. as virological testing is not readily available in less developed countries they might use serological methods. even with the limits of serological tests, results from multiple tests may yield important information. 17 indigenously acquired sero-positivity against mers cov would currently be a good indicator of importation. overcrowded hospitals 18 have been shown to provoke nosocomial outbreaks of airborne diseases, like sars. particular characteristics enhance transmission, possibly through aerosol and contact: distance between beds of ⩽1m; staff continuing work while symptomatic; and host patient requiring oxygen therapy. such hospital environments are quite common in developing countries. mers cov also shares many symptoms with other respiratory diseases, consequently misdiagnosis is likely. thus the clinical staff of hospitals likely to receive returnees seeking treatment for their illnesses can act as 'sentinels' for mers cov surveillance. serious respiratory illness in the clinical staff of hospitals, especially those with icus, might be targeted for virological testing. serological testing of other staff may detect those who had asymptomatic illness. these tests would identify importation and help assess the gravity of the situation. these supplemental, limited, and targeted serological investigations would require some new financial inputs. hospital-based serum collection from selected staff is generally inexpensive and countries can manage this by themselves. storage and transportation can be tricky, but both are amenable to centralized management. health departments of developing countries can step forward to carry out this under international coordination from who. the serological tests for mers (currently available) do require specialized skill and expertise. 17 to start, it would be ideal if these tests were done under direct supervision by the labs that initially developed the tests. these labs would have to be empowered to test a large number of samples from many countries. later, the original labs might limit themselves to quality control. new funds would be required and the countries where the original labs are situated should, perhaps, come forward to support their labs to help people in the developing countries who face mers. how long will this surveillance be needed? perhaps not for long. the relevance of this surveillance strategy would end when either the outbreak extinguishes itself at the original hot spots or when clear evidence of indigenous transmission in many other sites becomes evident. the effort to optimize these in-country surveillance protocols can be managed by epidemiologists and biostatisticians of each developing country. hospital staff who are found to be immune either from asymptomatic infections or having recovered from symptomatic infection are likely to become the greatest resource when the next patient with suspected mers cov arrives. when all care givers of a patient acutely ill with mers cov are immune, further transmission within the hospital is unlikely. this way to block transmission seems a reasonable option as no vaccine is available. (in fact, this strategy can be used to manage propagated outbreaks of diseases that leave those who recover immune, like chickenpox or ebola virus disease.) outbreak management should include enhanced recruitment of recovered persons after their convalescent period for the care of new patients. the duration of immunity should be monitored as the outbreak progresses. when care givers are immune, they may provide better clinical care, as they are not afraid of contact with the patient. this approach would be a good addition to the currently recommended hospital infection control measures. 19 mers is a challenge. let us face it. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization (who) close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan missing information in animal surveillance of mers-cov lancet infect dis improving the evidence base for decision making during a pandemic: the example of 2009 influenza a/h1n1 pathogenesis of middle east respiratory syndrome coronavirus 10 years on, the world still learns from sars from sars in 2003 to h1n1 in 2009: lessons learned from taiwan in preparation for the next pandemic from sars to h7n9: the mechanism of responding to emerging communicable diseases has made great progress in china nursing shortage in india with special reference to international migration of nurses international organization for migration (iom) philippines united nations expert group meeting on international migration and development in the arab region un/pop/ egm potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome coronavirus: implications for health care facilities laboratory testing for middle east respiratory syndrome coronavirus interim recommendations (revised why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? infection prevention and control during health care for probable or conformed cases of novel coronavirus (ncov) infection iype joseph, mb.bs, mph is a research scientist i (medical) in the pathogen biology group at the rajiv gandhi centre for biotechnology, kerala, india. key: cord-283709-y59h5bw8 authors: chan, renee w y; hemida, maged g; kayali, ghazi; chu, daniel k w; poon, leo l m; alnaeem, abdelmohsen; ali, mohamed a; tao, kin p; ng, hoi y; chan, michael c w; guan, yi; nicholls, john m; peiris, j s malik title: tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study date: 2014-08-28 journal: lancet respir med doi: 10.1016/s2213-2600(14)70158-4 sha: doc_id: 283709 cord_uid: y59h5bw8 background: middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic infection causing severe viral pneumonia, with index cases having resided in or recently travelled to the arabian peninsula, and is a global concern for public health. limited human-to-human transmission, leading to some case clusters, has been reported. mers-cov has been reported in dromedary camels but phenotypic characterisation of such viruses is limited. we aimed to compare mers-cov isolates from dromedaries in saudi arabia and egypt with a prototype human mers-cov to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung. methods: we characterised mers-cov viruses from dromedaries in saudi arabia and egypt and compared them with a human mers-cov reference strain. we assessed viral replication kinetics and competence in vero-e6 cells (rhesus monkey), tissue tropism in cultures of ex-vivo human bronchial and lung tissues, and cytokine and chemokine induction, gene expression, and quantification of viral rna in calu-3 cells (human respiratory tract). we used mock-infected tissue as negative controls for ex-vivo experiments and influenza a h5n1 as a positive control for cytokine and chemokine induction experiments in calu-3 cells. findings: we isolated three dromedary strains, two from saudi arabia (dromedary/al-hasa-kfu-hku13/2013 [ah13] and dromedary/al-hasa-kfu-hku19d/2013 [ah19d]), and one from egypt (dromedary/egypt-nrce-hku270/2013 [nrce-hku270]). the human and dromedary mers-cov strains had similar viral replication competence in vero-e6 cells and respiratory tropism in ex-vivo cultures of the human respiratory tract, and had similar ability to evade interferon responses in the human-respiratory-tract-derived cell line calu-3. interpretation: the similarity of virus tropism and replication competence of human and dromedary mers-cov from the arabian peninsula, and genetically diverse dromedary viruses from egypt, in ex-vivo cultures of the human respiratory tract suggests that dromedary viruses from saudi arabia and egypt are probably infectious to human beings. exposure to zoonotic mers-cov is probably occurring in a wider geographical region beyond the arabian peninsula. funding: king faisal university, egyptian national research centre, hong kong food and health bureau, national institute of allergy and infectious diseases, and european community seventh framework program. middle east respiratory syndrome coronavirus (mers-cov) is a disease of public health concern globally, with 837 laboratory-confi rmed cases and 291 deaths reported to who as of july 23, 2014. 1 index-case patients have all resided in or recently travelled to the arabian peninsula or adjacent countries. travel-associated cases or secondary transmission arising from such cases have been reported in europe, north america, africa, and asia. 2 some case clusters have been associated with limited human-to-human transmission occurring within family or health-care settings, and the remaining sporadic cases are presumed to be zoonotic in origin. [3] [4] a recent increase in reported cases from saudi arabia associated with health-care facilities is a particular cause of concern. 3 establishment of the proximate zoonotic source of human infection and modes of transmission from animals to human beings remains crucial for public health. mers-cov has been detected in dromedary camels (camelus dromedarius), in association with and preceding human infection, and in dromedary slaughterhouses and farms. [5] [6] [7] [8] [9] viruses from most of the recent human cases from the arabian peninsula phylogenetically cluster within clade b, but the earliest human mers-cov strains from 2012 are genetically distinct and are denoted as clade a. 10 dromedary mers-cov strains detected in the arabian peninsula in 2012-13 were clade b viruses, phylogenetically related to viruses from human cases from the same regions, but genetically diverse (non-clade a/b) viruses were reported from egypt. 11 researchers have also reported serological evidence collected between 2009 and 2013 of mers-cov infection in dromedary camel serum samples from tunisia, nigeria, ethiopia, and egypt, suggesting that mers-cov or antigenically related viruses might be geographically more widespread than is appreciated at present. 6, 12 findings from two studies of 226 people in saudi arabia and 179 people in egypt working in dromedary abattoirs showed that all remained seronegative for mers-cov, suggesting that trans mission to humans is uncommon 11, 13 and raising the question of whether dromedary mers-cov strains have the capacity to infect human beings. full genome sequences of dromedary viruses suggest that these viruses are similar to viruses in humans. 11, 14 there were some aminoacid diff erences between the human and dromedary viruses, including in the genetic sequence of the spike protein (a major determinant of host specifi city), but they did not aff ect the receptor binding interface with the cell receptor dpp4. 11, 14 however, because a single aminoacid change can profoundly aff ect host range and because the host range determinants of mers-cov are not well defi ned, dromedary viruses need to be phenotypically characterised in a physiologically relevant manner. 15 so far, few virus isolates from dromedaries have been available for biological characterisation and direct evidence for replication competence of dromedary mers-cov in human respiratory tissues has been lacking. the only biological characterisation of a dromedary mers-cov virus so far was the fi nding of dpp4-receptor-dependent virus replication in huh-7 cells, 16 a transformed human hepatoma cell line physiologically unrelated to the human respiratory tract. we aimed to compare mers-cov isolates from dromedaries in saudi arabia and egypt with the prototype human mers-cov emc strain to assess virus replication competence and cell tropism in ex-vivo cultures of human bronchus and lung. we used a human mers-cov strain emc (hmers-cov) provided by r a m fouchier (erasmus university medical center, rotterdam, netherlands). we prepared the emc virus stock as previously described. 17 three dromedary camel strains, two from saudi arabia (dromedary/al-hasa-kfu-hku13/2013 [ah13] and dromedary/al-hasa-kfu-hku19d/2013 [ah19d]) and one from egypt (dromedary/egypt-nrce-hku270/2013 [nrce-hku270]), were prepared. the dromedary camel mers-cov isolates ah13 and ah19d had been isolated in vero-e6 cells (atcc, crl-1586, manassas, va, usa) from nasal (ah13) and faecal (ah19d) swabs, collected from dromedaries in al-hasa, saudi arabia, as previously described. 14 the detection of dromedary camel mers-cov nrce-hku270 from a nasal swab of a dromedary in an abattoir in egypt has been previously reported. 11 for this study, we successfully isolated the virus from this specimen in vero-e6 cells as previously described. 14 viruses were grown, aliquoted, and stored at -80°c until used. all the dromedary mers-cov strains used in this study were passage 3 after isolation in vero-e6 cells. we had previously compared the full genome sequence of the ah13 virus in the original nasal swab with the vero-e6 passaged virus used in this accession numbers of virus genomes retrieved for this analysis are jx869059, kc776174, kc164505, kc667074, kf192507, kj156881, kj156949, kj156944, kj556336, kj156952, kf600613, kf186564, kf600627, kf186567, kf600651, kj156866, kf600634, kf600632, kf600644, kf186565, kf186566, kf600645, kf600647, kf600630, kf600652, kf745068, kj156869, kj650297, kj650296, kj650295, kf600628, kf961221, kf961222, kj156874, kj156910, kj156934, methods for viral titrations are described in the appendix. the methods we used for ex-vivo organ cultures and infection, quantifi cation of viral rna and host cytokine and chemokine mrnas, and quantifi cation of protein and immunohistochemistry have been previously described 17 and are also detailed in the appendix. we did experiments with live mers-cov in a biosafety level 3 biocontainment facility at the university of hong kong. this study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster (uw-13-104). we cultured vero-e6 and calu-3 cells using dulbecco's modifi ed eagle's medium. cells were seeded at 1 × 10⁵ cells per well in 24-well tissue-culture plates and infected with the mers-cov strains at a multiplicity of infection of 0·01 or 2 as indicated. after 1 h of virus adsorption at 37°c, we removed the virus inoculum, washed the cells with phosphate-buff ered saline (pbs) three times to remove unbound virus, and replenished with fresh culture medium. we measured virus replication kinetics by titrating infectious virus in infected culture supernatants. the human-respiratory-tract-derived cell line calu-3 (atcc htb-55, manassas, va, usa) was cultured and infected with virus, as for the vero-e6 cells. rna from the infected cells was also collected at 6, 24, and 30 h post infection for analysis of gene expression and quantifi cation of viral rna. we harvested 1 ml of culture supernatants from calu-3 cells at 30 h post infection to measure cytokine and chemokine protein concentrations using elisa (r&d systems, minneapolis, mn, usa) and cytometic bead array (bd bioscience, san jose, ca, usa), using methods specifi ed by the manufacturer. as a positive control for assessment of cytokine and chemokine induction in calu-3, we used a highly pathogenic avian infl uenza h5n1 virus, a/hong kong/483/1997. infl uenza virus was prepared in madin-darby canine kidney cells as previously described. we created thermal inactivation curves of the coronaviruses at 37°c, using culture wells inoculated with virus dilutions in the absence of permissive cells, or with an ex-vivo culture of mouse lung prepared from c57bl/6n mice. the mouse lung culture was prepared similar to the ex-vivo organ culture of human samples. we added 1 ml of virus dilution into 24-well plates with diff erent starting concentrations (10⁴, 10³, and 10² tcid 50 /ml). we collected 130 μl of supernatant at 1, 24, 48, and 72 h post incubation to measure viral titration. to assess tissue tropism in ex vivo experiments, we infected bronchial and lung tissues with 1 ml of each mers-cov virus dilution at a titre of 10⁶ 50% tissue culture infective dose per ml (tcid 50 /ml). after incubation for 1 h at 37°c, we washed the cultures three times with 5 ml of warm pbs to remove unbound virus. mock-inoculated tissue served as controls (1 ml of dulbecco's modifi ed eagle's medium without virus). we collected culture supernatants from the infected cultures at 1, 24, 48, and 72 h post infection and titrated them in parallel for infectious virus using the tcid 50 assay. samples for experiments on the ex-vivo cultures of human bronchus and lung were provided by fi ve independent donors. we assessed the extent of the overall viral replication with area-under-curve analysis using the trapezoid rule. we used the area calculated from 24 to 72 h post infection above the limit of detection of infectious virus titre for this analysis. the results of the four viruses were compared with the non-parametric friedman test. we did the experiments with vero-e6 and calu-3 cells with three biological replicates of each cell line and calculated mean and standard error of the mean (sem). we compared diff erences in log 10 transformed viral titres by a two-way anova followed by a bonferroni multiple-comparison test. we compared the quantitative cytokine and chemokine mrnas between viruses and over time with a two-way anova followed by a bonferroni multiple-comparison test. the protein concentrations of the cytokines and chemokines between viruses at 30 h post infection were compared by a oneway anova followed by a bonferroni multiplecomparison test. see online for appendix we used p less than 0·05 to indicate statistical significance. statistical analyses were done with graphpad prism version 6.0 for mac. the funder of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. figure 1 shows the phylogeny of mers cov strains used in this study within the global context of mers-cov. only mers-cov with full genome sequences were included in this analysis. most viruses cluster within clade b mers-cov, including the dromedary camel virus isolates from saudi arabia used in this study (ah13 and ah19d). the prototype human mers-cov virus (emc) is a clade a virus. the dromedary mers-cov isolates, nrce-hku270 and dromedary/egypt-nrce-hku205/2013, have a long branch in the phylogenetic tree separating them from clade a and clade b viruses and also from each other, suggesting that they are genetically diverse from previously described mers-cov. we used the dromedary mers-cov isolate nrce-hku270 in this study. the nucleotide and aminoacid similarity between these human and dromedary camel viruses is provided the appendix. all four mers-cov strains replicated effi ciently in vero-e6 cells at multiplicities of infection of 0·01 and 2 (fi gure 2, table). egyptian dromedary mers-cov nrce-hku270 replicated at least as well as did human mers-cov emc, and reached peak titres even faster than human mers-cov at 48 h post infection when infected at low multiplicity of infection. although the two saudi dromedary mers-cov strains (ah13 and ah19d) replicated less effi ciently than did human mers-cov during the fi rst 24 h post infection, the viral titre of ah13 was similar by 72 h post infection. replication of dromedary mers-cov nrce-hku270 was signifi cantly higher than that of the other two dromedary mers-cov strains at 24 h and 48 h post infection at multiplicity of infection of 0·01. immunohistochemistry of ex-vivo cultures of human bronchus and lung infected with human and dromedary mers-cov strains suggested that all four viruses could infect these tissues (fi gure 3). because of their threedimensional nature, infection in ex-vivo cultures was non-uniform throughout the histological specimen (appendix). figure 4 shows viral replication kinetics of the viruses in the ex-vivo cultures of human bronchus and lung. in the absence of permissive cells, the thermal inactivation curves of each of these mers-cov strains showed virus infectivity largely decreasing to undetectable levels by 24 h post infection (fi gure 4a). we noted similar thermal inactivation kinetics when we substituted mouse lung tissue (non-permissive to mers-cov) for the human ex-vivo tissues (data not shown). although the extent of virus replication varied from donor to donor, comparison with the thermal inactivation curves provided convincing evidence of virus replication of all four viruses in the ex-vivo human bronchial (fi gure 4b) and lung (fi gure 4c) cultures from each donor over the 72 h experiment period. two replicates of ex-vivo cultures per donor from three donors, infected with the same virus (human mers-cov emc) on diff erent occasions, showed that the change of area-under-curve from the same tissue donor in bronchus ranged between 0 to 1·36-fold and in lung ranged between 0 to 1·46-fold, providing an indication of the range of technical variation in these ex-vivo assays (appendix). by contrast, the diff erence of area-undercurve for human mers-cov emc between donor 3 and donor 1 was 5·3-fold for the bronchus (area under curve above detection limit 112·8 for donor 1 patchy nature of the infection, the variation between donors, and lack of adequate statistical power, statistical comparisons between viruses have to be interpreted with caution. within these limitations, we noted no signifi cant diff erence in the replication competence of the four viruses in either the bronchus (p=0·151) or the lung (p=0·270) as assessed by the area-under-the-curve using the friedman test (appendix). immunohistochemical analysis of virus-infected bronchial tissues showed that the types of cells infected by human and dromedary mers-cov were very similar (fi gure 5). co-staining of viral antigen with the goblet cell marker muc5ac (fi gure 5a) and ciliated cell marker β-tubulin (fi gure 5b) lent support to the notion that mers-cov infects non-ciliated bronchial epithelium other than goblet cells. co-staining of viral antigen and alveolar epithelial cell marker ae1/3 showed that all four viruses infected alveolar epithelial cells (fi gure 5c), and co-staining with the alveolar type ii epithelial cell marker prosurfactant protein c showed that type ii alveolar epithelium was infected (fi gure 5d). we noted no evidence of infection of alveolar macrophages (fi gure 5e). all four viruses infected lung endothelium (appendix). thus, overall, the respiratory tropism of the human and dromedary mers-cov was similar. all three mers-cov strains (emc, ah13, nrce-hku270) replicated effi ciently in calu-3 cells (ah19d was not included because it is in the same clade as ah13). to quantify the viral rna and cytokine and chemokine gene induction and expression, the humanrespiratory-tract-derived cell line calu-3 was cultured and infected with virus as for the vero-e6 cells. rna from the infected cells was also collected at 6, 24, and 30 h post infection for analysis of gene expression and quantifi cation of viral rna. we harvested 1 ml of culture supernatants from calu-3 cells at 30 h post infection to measure cytokine and chemokine protein concentrations using elisa (r&d systems, minneapolis, mn, usa) and cytometric bead array (bd bioscience, san jose, ca, usa), using methods specifi ed by the manufacturer. as a positive control for assessment of cytokine and chemokine induction in calu-3, we used a highly pathogenic avian infl uenza h5n1 virus, a/hong kong/483/1997. infl uenza virus was prepared in madin-darby canine kidney cells as previously described. 17 despite effi cient replication, none of the mers-cov strains induced signifi cant gene expression or protein secretion of tumour necrosis factor α (tnfα), cxcl10, or interferon β (fi gure 6). we used infl uenza a (h5n1) virus, a potent inducer of interferon β and cxcl10 in epithelial cells, for comparison. we noted evidence of interleukin 6 induction by all three mers-cov strains; although ah13 and nrce-hku270 induced greater production of interleukin 6 mrna than the human emc strain, the human emc strain induced higher concentrations of interleukin 6 protein than did either dromedary strain (fi gure 6f). we compared the viral replication competence and respiratory tropism of three mers-cov isolates from dromedary camels with the prototype human clade a mers-cov isolate emc (panel). the two dromedary mers-cov isolates from al-hasa, ah13 and ah19d, are phylogenetically closely related to recent clade b human viruses from saudi arabia. 18 as shown by the long-branch in the unrooted phylogenetic tree (fi gure 1) and genetic similarity tables (appendix), egyptian dromedary mers-cov nrce-hku270 and dromedary mers-cov nrce-hku205 are genetically divergent from all other mers-cov detected to date, and are also divergent from each other, suggesting that the global diversity of mers-cov is wider than previously appreciated. the lack of a representative human clade b mers-cov isolate is a limitation of this study. comparison of viral genetic data can provide clues, but is not of itself fi nal evidence for the competence of an animal virus to infect human beings. with sars coronavirus, two aminoacid changes in the spike protein strikingly altered the ability of that virus to transmit effi ciently in human beings. 15 because all the crucial determinants of host tropism with mers-cov are not fully understood, especially those outside of the spike-receptor binding domain, phenotyping of such viruses provides essential complementary information to the genetic analysis. the three mers-cov isolates from dromedary camels (ah13, ah19d, and nrce-hku270) were similar to the human mers-cov emc virus in replication competence in the rhesus monkey cell line vero-e6. all four viruses replicated in ex-vivo cultures of the human bronchus and lung and infected the same cell types in the bronchus (non-ciliated bronchial epithelium) and lung (alveolar epithelium, including type ii alveolar epithelial cells). we noted infection of lung endothelial cells with all four viruses, suggesting that potential for dissemination beyond the respiratory tract was also comparable. overall, the tropism of human mers-cov emc for the human respiratory tract was similar to that previously reported. 17 phenotypically, human and dromedary mers-cov have much the same tropism and replication competence in human respiratory ex-vivo cultures. to our knowledge, this report provides the fi rst comparative data from living human respiratory tissue in situ. therefore, our study provides new and crucial information to the emerging understanding of a disease with global importance for public health. the heterogeneity of virus replication between individual donors in our ex-vivo infection experiments might result from diff erences in host susceptibility, which are physiologically relevant to the reported epidemiology of human mers and could imply that individual variations might be an important determinant of disease susceptibility and severity. however, this question needs more systematic investigation. thus, overall, our data support and complement the viral genetic sequence data of the spike proteins of the dromedary viruses, which seem similar to the spike proteins of human mers-cov strains, 11, 13, 16 and strongly suggest that dromedary mers-cov strains have the capacity to infect human beings. infection of ex-vivo cultures provided useful information of virus cell tropism in the absence of autopsy data for mers. ex-vivo organ cultures provided information about virus tropism at early stages of the infection, which is not usually available in autopsies or from patients with late-stage disease (often after lengthy mechanical ventilation). thus, our fi ndings emphasise the usefulness of ex-vivo cultures for risk assessment of animal viruses for human health. mers-cov is reported to be a weak inducer of type i and type iii interferons. 17, 19 immune evasion mechanisms are relevant to pathogenesis and interspecies transmission, and diff erences might aff ect virus replication competence in new hosts. we therefore investigated whether human mers-cov and dromedary mers-cov diff ered in their ability to evade eliciting type i interferon and other innate immune responses. because the multiplicity of infection cannot be accurately controlled in experiments with ex-vivo cultures of the human bronchus and lung, we used calu-3, an epithelial cell line derived from human lung adenocarcinoma that has been used as a cell line to study viral infections of the human respiratory tract. 20 human mers-cov emc, dromedary mers-cov ah13, and dromedary mers-cov nrce-hku270 replicated effi ciently in these cells, although the dromedary mers-cov strains replicated signifi cantly more effi ciently in these cells (appendix). despite this effi cient virus replication, no virus elicited any tnfα, cxcl10, or interferon β mrna or protein responses after virus infection (fi gure 6). by contrast, infl uenza a h5n1 virus was a potent inducer of interferon β and cxcl10. we noted some induction of interleukin 6 by dromedary mers-cov strains ah13 and nrce-hku270 and human mers-cov at late timepoints post infection (30 h; fi gure 6e and 6f). importantly, the human and dromedary mers-cov behaved similarly by not eliciting interferon and cxcl10 responses, suggesting that these viruses do not diff er in evasion of type i interferon responses. in view of the high prevalence of infection in dromedaries [5] [6] [7] [8] 10 and implied frequent human exposure, the rarity of human infection and disease remains a puzzle and is reminiscent of the epidemiology of avian infl uenza a h5n1. 21 the exact mode of transmission of mers-cov from dromedaries to people remains unclear and whether unusual modes of exposure or host susceptibility have a role should be investigated. serological and virological evidence of mers-cov in dromedaries has been reported from countries in east and north africa outside of the arabian peninsula, 11,12 but zoonotically acquired mers has not yet been diagnosed in human beings in the african continent. our fi nding that the genetically divergent dromedary mers-cov isolated in egypt is phenotypically similar to human mers-cov emc in explant cultures from human respiratory tissue underscores the possibility that zoonotic mers might be occurring, unrecognised, in northeast africa and beyond. at present, many countries only test for mers-cov in patients with a recent travel history to the arabian peninsula, and locally acquired mers could be missed. alternatively, diff erences in cultural practices of camel husbandry, modes of animal contact, and consumption of animal products (eg, fresh camel's milk) 22 might account for diff erences in occurrence of zoonotic disease in diff erent geographical regions. in conclusion, our fi ndings suggest that dromedary mers-cov has competence to infect the human respiratory tract and does not diff er phenotypically from human mers-cov in this respect. this evidence strengthens the contention that dromedary camels are the proximate source of infection for human beings. our fi ndings also emphasise the need to consider mers in the diff erential diagnosis of viral pneumonia in a much wider geographical region in northeast africa and beyond. rwyc planned and did the experiments with ex-vivo cultures, analysed the data, and contributed to drafting of the report. mgh, gk, aa, and maa did the fi eld studies and detection of virus in dromedary specimens, and planned the experiments. dkwc, llmp, and yg did the genetic sequencing and phylogenetic analysis. kpt did the in-vitro culture and quantitative pcr assays. hyn isolated the mers-cov strains. mcwc was involved in study design and obtained research funding. jmn did and interpreted the histology and immunohistochemistry studies. jsmp obtained research funding, planned and coordinated the study, analysed the data, and wrote the report. all authors contributed to the data interpretation, analysis, and provided critical comments on the draft report. we declare no competing interests. to assess the infection potential of dromedary camel middle east respiratory syndrome coronavirus (mers-cov) strains for humans, genetic analysis should be complemented with phenotypic characterisation in physiologically relevant invitro cell cultures. we searched pubmed for reports published up to july 5, 2014, with the terms "mers coronavirus" and "dromedary" and "culture" or "isolate". we applied no date or language restrictions. three papers were retrieved, only two of them related to mers-cov isolates from dromedary camels. one other recent paper not yet cited in pubmed was identifi ed through search of the literature. only one of these studies pertained to the phenotypic characterisation of dromedary mers-cov in cell cultures in vitro. 16 this study used a transformed human hepatoma cell line, not physiologically relevant to the human respiratory tract. our fi ndings show similar virus tropism and replication competence of human and dromedary mers-cov in ex-vivo cultures of the human respiratory tract. these fi ndings suggest that dromedary mers-cov from the arabian peninsula, in addition to genetically diverse dromedary viruses from egypt, can be infectious to human beings. exposure to zoonotic mers-cov might be happening in a wider geographical region beyond the arabian peninsula. virological evidence of mers-cov needs to be sought in patients with severe unexplained viral pneumonia in africa in addition to the middle east. middle east respiratory syndrome coronavirus (mers-cov)-update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update who. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: quantifi cation of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-tohuman transmission of mers coronavirus spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers coronaviruses in dromedary camels geographic distribution of mers coronavirus among dromedary camels investigation of antimiddle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 mers coronavirus in dromedary camel herd, saudi arabia structural biology: adaptation of sars coronavirus to humans isolation of mers coronavirus from dromedary camel tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia effi cient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential evaluation of the calu-3 cell line as a model of in vitro respiratory syncytial virus infection avian infl uenza virus (h5n1): a threat to human health stability of middle east respiratory syndrome coronavirus in milk key: cord-271211-frkk6w0a authors: han, yu; yang, hailan title: the transmission and diagnosis of 2019 novel coronavirus infection disease (covid‐19): a chinese perspective date: 2020-03-12 journal: j med virol doi: 10.1002/jmv.25749 sha: doc_id: 271211 cord_uid: frkk6w0a 2019 novel coronavirus (sars‐cov‐2), which originated in wuhan, china, has attracted the world's attention over the last month. the chinese government has taken emergency measures to control the outbreak and has undertaken initial steps in the diagnosis and treatment of 2019 novel coronavirus infection disease (covid‐19). however, sars‐cov‐2 possesses powerful pathogenicity as well as transmissibility and still holds many mysteries that are yet to be solved, such as whether the virus can be transmitted by asymptomatic patients or by mothers to their infants. our research presents selected available cases of covid‐19 in china to better understand the transmission and diagnosis regarding this infectious disease. both of them did not wear masks. 5 sars-cov-2 possesses powerful pathogenicity and transmissibility, being more infectious than sars-cov and mers-cov. 6 encountering a confirmed patient and being close for 15 or 50 seconds is not the only route of infection, though it is the most likely route in the above two cases. moreover, being infected within a very short exposure time is possible in the absence of masks. 7 the powerful infectivity of sars-cov-2 may be explained by the latest findings reported by wrapp d et al, 8 which showed that the sars-cov-2 s binds to angiotensin-converting enzyme 2 (ace2) receptors with a higher affinity than sars-cov s. another underlying reason was reported by zou l et al 9 in that the shedding pattern of viral nucleic acid in patients infected with sars-cov-2 is similar to that in patients with influenza and appears to be different from that in patients infected with sars-cov. sars-cov-2 often causes cluster transmission, especially within family clusters. in some cities, cases involving cluster transmission accounted for 50% to 80% of all confirmed cases of covid-19. 10 human-to-human transmission of sars-cov and mers-cov occurred mainly through nosocomial transmission, and transmission between family members only occurred in 13% to 21% of mers cases and 22% to 39% of sars cases. 11 sars-cov-2 can be transmitted by droplets and contact. a study in south korea showed that many environmental surfaces of patients with mers were contaminated by mers-cov, and virus rna was detected from environmental surfaces within 5 days after the last positive pcr of patients' respiratory samples. 12 guangzhou cdc also found sars-cov-2 in the house of a confirmed patient, 13 which serves as evidence of contact transmission. moreover, live viruses have been found in the feces of patients with covid-19, 14 however, the fecal-oral transmission of the virus has not been shown. studies have shown that sars-cov may be detected in the feces of most sars patients, 15 and the virus within feces could survive at room temperature for at least 1 to 2 days. 16 it is possible but infrequent for sars-cov to spread via the fecal-oral route. 17 in patients with mers, feces and urine samples also yielded viral rna. 18 given the evidence of fecal contamination of sars-cov and mers-cov as well as their ability to survive in feces, it is possible that sars-cov-2 may also be transmitted through the fecal-oral route. 19 additionally, in regard to the expression of ace2 in the intestine and kidney, sars-cov-2 may infect these tissues and enter the feces, allowing its potential spread via fecal-oral route. 20 the lancet also reminded doctors not to ignore sars-cov-2 transmission via ocular surfaces as infected droplets and bodily fluids may easily contaminate the human conjunctival epithelium. 21 guangfa wang, a member of the national health commission of the people's republic of china (nhc) expert panel on pneumonia, was exposed to a fever clinic in wuhan with only his eyes unprotected. he then demonstrated symptoms of conjunctivitis in his left lower eyelid 2 days before the onset of covid-19. 22 on february 19, the nhc published the 6th edition of guideline on diagnosis and treatment of covid-19 (the 6th guideline for covid -19) . 23 this document asserted that the transmission of sars-cov-2 mainly occurs via large droplets and contact. additionally, the virus may spread in an unventilated environment with high levels of viral aerosols. in addition, researchers claimed the spread of sars-cov-2 could be characterized by super-spreading events. 24 however, there is no evidence of super-spreading events in any medical institution that treats patients suffering from covid-19. 6 and a study in south korea showed that for transmission of mers-cov nonisolated in-hospital days was the only factor which tended to be higher in super-spreaders than usual-spreaders. 25 therefore, whether there are super-spreaders or not, early isolation is necessary to reduce the size of the outbreak of sars-cov-2. over the past few days, asymptomatic patients were found in many chinese cities. table 1 outlines its summary. t a b l e 1 summary of asymptomatic patients wuhan were confirmed to have covid-19 after returning to shenzhen, with their asymptomatic child presenting with no fever, respiratory tract symptoms or diarrhoea but had ground-glass lung opacities seen on radiography. 26 subsequently, asymptomatic patients were discovered in many chinese cities with most of them having an epidemiological history. asymptomatic infections may occur due to weakened immune responses and subclinical manifestations, or because the virus is waiting for opportunities to reproduce and invade. to understand its mechanism requires additional investigation of asymptomatic patients as well as blood tests pointing to signs of an immune response, which can help detect asymptomatic or presymptomatic cases. 27 a study showed that during the outbreak of sars-cov, of all exposed health care workers, 7.5% were asymptomatic sarspositive cases. asymptomatic sars was associated with lower sars antibody titers and a higher use of masks compared to that of pneumonic sars. 28 another study showed that of 255 patients with laboratory-confirmed mers-cov, a total of 64 patients (25.1%) were reported to be asymptomatic. however, 33 (52%) of the 64 patients were interviewed with 26 (79%) of them having reported at least one respiratory symptom. 29 this phenomenon indicates whether asymptomatic patients were actually infected without showing symptoms. whether asymptomatic people can transmit sars-cov-2 to others is unclear. the 6th guide for covid-19 noted that asymptomatic patients may serve as a source of infection. an article in nejm first reported a german to be confirmed with covid-19 after contact with an asymptomatic chinese patient. 30 however, it turned out that the chinese patient had a fever in germany and took antipyretics. a recent study in nejm reported that a viral load detected in an asymptomatic patient was similar to that detected in symptomatic patients, indicating the potential for transmission in asymptomatic patients. 9 table 1 indicates that these asymptomatic patients may infect others or develop symptoms later, but the number of patients involved is small. we remind readers to take this into account when interpreting the research results and conclusions as some of the above observations may be accidental. another uncertainty is whether those who are asymptomatic can cause large-scale infections. a study in south korea showed that during the mers outbreak in korea, an asymptomatic patient with mers was discovered, and none of the 82 persons exposed to that patient without protection was infected. 31 most of the asymptomatic patients had close contact with confirmed patients, hence, they may be isolated in a timely manner when tracking close contacts. moreover, the number of asymptomatic patients was very small; according to epidemiological data in mainland china, only 1.2% of patients with covid-19 are asymptomatic. 6 due to the above reasons, such patients will generally not cause large-scale transmissions of sars-cov-2. 32 february 2, was delivered by a mother who was confirmed to have covid-19. the cord blood and placental tissue were collected immediately and tested negative for sars-cov-2, but nasopharyngeal swab samples collected 36 hours after birth were positive. 33 sars-cov-2 is a novel virus and shares a 79.0% nucleotide identity with the sequence of sars-cov and a 51.8% identity with that of mers-cov. 34 the alveolar lavage test is the best way to confirm sars-cov-2, however, the detection of alveolar lavage fluid is mostly used in severe patients using an invasive ventilator. sputum is considered the second choice, though the coughs of many patients with covid-19 are unproductive. 46 therefore, pharyngeal swabs are the most common sampling method, however, they may occasionally cause missed diagnoses for smaller levels of sars-cov-2 residing in the pharynx. in addition, pcr detection takes up a lot of time, hampering the control of infectious diseases. therefore, the revised version of the 5th edition added clinical confirmed standards for the hubei province. 47 genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a man was infected by contacting with a confirmed patient for 15 seconds without a mask a man was infected by contacting with a confirmed patient for 50 seconds without a mask novel coronavirus pneumonia emergency response epidemiology team central people's government of the people's republic of china. 14 key questions and answers cryo-em structure of the 2019-ncov spike in the prefusion conformation sars-cov-2 viral load in upper respiratory specimens of infected patients special expert group for control of the epidemic of novel coronavirus pneumonia of the chinese preventive medicine association sars and other coronaviruses as causes of pneumonia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea nanfang metropolis daily. 2019-ncov was found in the house of the confirmed patient information office people's government of guangdong province. the 19th news conference on epidemic prevention and control of guangdong province clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study world health organization. first data on stability and resistance of sars coronavirus compiled by members of who laboratory network critical care lessons from severe acute respiratory syndrome viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? structure analysis of the receptor binding of 2019-ncov -ncov transmission through the ocular surface must not be ignored. the lancet peking university hospital wang guangfa disclosed how he was infected national health commission of the people's republic of china. the notice of launching guideline on diagnosis and treatment of covid-19 return of the coronavirus: 2019-ncov clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster scientists are racing to model the next moves of a coronavirus that's still hard to predict asymptomatic sars coronavirus infection among healthcare workers mers-cov outbreak in jeddah-a link to health care facilities transmission of 2019-ncov infection from an asymptomatic contact in germany infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection zhang wenhong, leader of shanghai medical treatment expert panel: asymptomatic infected patients will not become superspreaders chinese academy of engineering academicians, member of the national medical team of hubei province identification of a novel coronavirus causing severe pneumonia in human: a descriptive study pregnancy and perinatal outcomes of women with severe acute respiratory syndrome mers-cov infection in a pregnant woman in korea novel coronavirus infection in hospitalized infants under 1 year of age in china what are the risks of covid-19 infection in pregnant women? the lancet clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records a confirmed patient had real-time rt-pcr detection before admission for three times, all negative for interview with peng zhiyong, director of critical medicine department bulletin of 2019 novel coronavirus pneumonia from wuhan municipal health commission bulletin of 2019 novel coronavirus pneumonia from wuhan municipal health commission national health commission of the people's republic of china. the notice of launching guideline on diagnosis and treatment of the novel coronavirus pneumonia (ncp) tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis national health commission of the people's republic of china. the notice of launching guideline on diagnosis and treatment of the novel coronavirus pneumonia (ncp) key: cord-279979-3ecnbqom authors: anthony, s. j.; gilardi, k.; menachery, v. d.; goldstein, t.; ssebide, b.; mbabazi, r.; navarrete-macias, i.; liang, e.; wells, h.; hicks, a.; petrosov, a.; byarugaba, d. k.; debbink, k.; dinnon, k. h.; scobey, t.; randell, s. h.; yount, b. l.; cranfield, m.; johnson, c. k.; baric, r. s.; lipkin, w. i.; mazet, j. a. k. title: further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus date: 2017-04-04 journal: mbio doi: 10.1128/mbio.00373-17 sha: doc_id: 279979 cord_uid: 3ecnbqom the evolutionary origins of middle east respiratory syndrome (mers) coronavirus (mers-cov) are unknown. current evidence suggests that insectivorous bats are likely to be the original source, as several 2c covs have been described from various species in the family vespertilionidae. here, we describe a mers-like cov identified from a pipistrellus cf. hesperidus bat sampled in uganda (strain predict/pdf-2180), further supporting the hypothesis that bats are the evolutionary source of mers-cov. phylogenetic analysis showed that predict/pdf-2180 is closely related to mers-cov across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. indeed, several amino acid substitutions were identified in key binding residues that were predicted to block predict/pdf-2180 from attaching to the mers-cov dpp4 receptor. to experimentally test this hypothesis, an infectious mers-cov clone expressing the predict/pdf-2180 spike protein was generated. recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in vero cells or primary human airway epithelial cells. our findings suggest that predict/pdf-2180 is unlikely to pose a zoonotic threat. recombination in the s1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of mers-cov in humans. express the functional receptor for mers-cov, we further show that recombination was likely to be the critical step that allowed mers to emerge in humans. keywords bat, mers coronavirus, spike, uganda, zoonoses i n 2012, middle east respiratory syndrome (mers) emerged in saudi arabia. clusters of fatal pneumonia in adults were determined to be caused by a novel lineage c betacoronavirus (2c cov), termed mers-cov (1) . this was the first 2c cov known to cause disease in humans and at the time of its discovery was most closely related to two known bat coronaviruses (2) , raising the possibility that bats were a reservoir and source for the virus. concurrently, epidemiologists identified an association between mers infections in patients and their contact with dromedary camels (3, 4) . mers-cov was subsequently detected in camels at a farm linked to two human cases in qatar (5) and in camels in egypt (6) , followed by surveys that demonstrated widespread exposure to the virus in the middle east and in north and east africa as early as the 1980s (7) (8) (9) (10) . it is now clear that camels play an important role in the transmission of mers-cov to people (11) , with seroprevalence highest among those who have had contact with camels (12) . while camels are thought to be important for the transmission of mers-cov, bats are widely considered to be the evolutionary source of the virus. several 2c covs have now been described in bats, including hku4 from tylonycteris pachypus (13) , hku5 from pipistrellus abramus (13) , and the recently identified neocov from neoromicia capensis (14) . neocov is the closest relative yet discovered (85% identical to mers) and shares sufficient genetic similarity in the replicase genes to be considered part of the same viral species (15) ; however, despite being closely related across much of the genome, the s1 subunit of the spike gene is highly divergent as a result of a prior recombination event. recombination in the spike gene is particularly significant because the derived protein is responsible for host receptor recognition and membrane fusion (16) and thus is central in determining host specificity. the s1 subunit contains the receptor binding domain and therefore has a specific role in defining host tropism (17) . other processes are also important, such as the activation of the spike protein by host proteases (18) , but the ability of s1 to bind with a host receptor is a critical step in the emergence pathway-and it can be quickly altered by a single recombination event. the sequence variation in the s1 region of mers-cov and neocov could therefore indicate differences in host binding preferences. predicting the interactions of virus binding domains with a particular host receptor (for example, the human mers-cov receptor dpp4) is possible through the use of structural modeling and the generation of infectious clones. protein-protein interactions can be modeled using a related homologous complex (19, 20) while reverse genetic strategies can test the permissiveness of human or other primate cells for infectious clones expressing the novel receptor binding domains or complete spike glycoprotein (21) (22) (23) (24) . pseudotyped lentivirus systems have also been used, for example, to show that dpp4 is the receptor for hku4 but not for the closely related hku5 (25, 26) . and while pseudotypes are not always accurate predictors of spike glycoprotein function (23) , these findings indicate that multiple cell-entry strategies could exist for 2c viruses and that not all mers-like covs pose an equal risk of zoonotic emergence. here, we investigated the receptor binding properties of a new strain of mers-like cov found in a bat from uganda. this virus (predict/pdf-2180) shares the same putative s1 subunit recombination that was observed in neocov, allowing us to also consider whether the spike recombination was critical for the emergence of mers-cov in humans. sampling and site characterization. a bat (identifier [id] otba03-20130220) was trapped on 20 february 2013 in the nkuringo area of kisoro district, in southwestern uganda (latitude ϫ1.12, longitude 29.68) (fig. 1) . this area is an established settlement of villages comprising approximately 15,000 inhabitants adjacent to the southwestern boundary of bwindi impenetrable national park. communities include subsistence farmers growing small crops, with some members working inside the national park or supporting tourism-related businesses. livestock, including cattle, pigs, sheep, goats, and poultry, are present in the village and are raised on a small scale primarily for local consumption. the sampled bat weighed 3.0 g and had a forearm length of 25 mm (fig. 1 ). it was identified as pipistrellus cf. hesperidus based on 95% sequence identity in the cytochrome b (cytb) gene. the cytochrome oxidase subunit 1 (co1) was also sequenced, but no corresponding co1 sequences for p. hesperidus were available in genbank for comparison. we therefore relied on the cytb sequence for species identification. discovery and sequence characterization. the oral swab, rectal swab, and whole blood of bat otba03-20130220 were assayed for the presence of coronavirus by consensus pcr (cpcr). two separate assays were used, each targeting a different region of the orf1b rna-dependent rna polymerase (rdrp). bands of the expected size were amplified from the rectal swab (pdf-2180) by both assays and confirmed to represent viral products by traditional sanger dideoxy sequencing. both fragments showed ͼ98% amino acid sequence identity to mers-cov, prompting further characterization of the virus. the oral swab and blood were negative. the near-full-length genome (identified as predict/pdf-2180) was assembled from 100-nucleotide (nt) illumina single-end reads at an average depth of 26ϫ. only the 5= and 3= noncoding regions were left incomplete. the order of all predicted open reading frames (orfs) was consistent with mers-cov and with the recently described neocov (kc869678) identified in a bat from south africa. similarly, the hexanucleotide transcription regulatory sequence (aacgaa) was conserved and found in the same position as both mers and neocov upstream of each predicted orf. across the full genome, the sequence had 86.5% amino acid identity to mers-cov and 91% to neocov; however, considerable variation was observed in different genes. uganda mers-like virus in bats ® amino acid identity could be as high as 97% to both mers-cov and neocov in orf1b or as low as 45% to mers-cov in subunit 1 of the spike protein. for the full spike protein, identity was 94% to neocov and 63% to mers-cov. percent sequence identity of the spike protein (subunits 1 and 2) to other 2c viruses is shown in tables 1 and 2, respectively. based on the current criteria for species demarcation established by the international committee for the taxonomy of viruses (ͼ90% amino acid sequence identity in the replicase proteins), predict/pdf-2180 shares sufficient genetic identity to mers-cov to be considered a member of the mers-like coronavirus species. phylogenetic analysis. maximum likelihood phylogenetic reconstructions showed that predict/pdf-2180 is most closely related to neocov (fig. 2) . the two viruses were basal or formed sister clades to mers-cov in all genes except subunit 1 of the spike. the full-genome alignment was scanned for recombination using seven different algorithms (rdp, geneconv, bootscan, maxchi, chimaera, siscan, and 3seq) implemented in rdp v4.46. a single recombination event was detected within the spike gene by rdp, bootscan, maxchi, chimaera, siscan, and 3seq (bonferroni-corrected p of ͻͻ0.001), suggesting that the incongruent phylogenies observed between spike subunit 1 and the rest of the genome are the result of recombination. attempts to date the divergence of these two viruses to estimate the "minimum" number of years since this recombination were prevented by evidence of strong negative (purifying) selection across the genome (fig. 2) . given that purifying selection can confound true phylogenetic depth, we felt that attempts to estimate the number of years to common ancestry were inappropriate and would result in artificially "recent" dates. zoonotic potential of predict/pdf-2180. the high genetic variability in subunit 1 suggests that human and bat strains of mers have different receptor binding properties. to investigate this, we modeled the specific affinity of the predict/pdf-2180 spike protein for the human mers-cov receptor dpp4 (27) . we utilized the crystal structure of the mers-cov spike binding domain in complex with dpp4 to create a homology model for the comparable region of the predict/pdf-2180 spike (fig. 3) . previous work has demonstrated 11 specific amino acid residues in mers-cov that (28) . of these residues, only one is conserved for predict/pdf-2180. to determine whether the binding interactions may be conserved between dpp4 and predict/pdf-2180 regardless of the differences in amino acid residues at these positions, we analyzed the predicted interactions between predict/pdf-2180 and dpp4, compared to mers-cov and dpp4. overall, we found a global reduction in predicted hydrogen bonding interactions in the dpp4-predict/ pdf-2180 binding interface compared with dpp4-mers-cov (fig. 3) . while the interactions in conserved residue y499 were maintained, dpp4 interactions with predict/ pdf-2180 residues 501, 502, 510, 511, 513, 539, and 542 were disrupted. the interaction between dpp4 y322 and mers d510 is abolished in the predict/pdf-2180 prediction, where d510 is replaced by k510. this is a charge change from negative to positive. interestingly, a change from r511 in mers to d511 in predict/pdf-2180 facilitates a potential interaction with y322 to replace the hydrogen bond lost with k510. regardless, due to the predicted loss of the majority of the dpp4 binding interactions, the model predicts that predict/pdf-2180 will not bind to dpp4. to confirm these results in vitro, a recombinant mers-cov cdna clone was constructed containing the predict/pdf-2180 spike gene in the context of the full-length mers-cov backbone. the chimeric virus maintains the entire ectodomain of the predict/pdf-2180 spike with the exception of the first 20 amino acids of the 5= end, which were taken from wild-type mers-cov. similarly, the transmembrane domains (tmds) and cytoplasmic tail of the chimeric virus used the wild-type mers-cov sequence in order to minimize incompatibility in virion formation. following transfection into vero cells, pcr amplification of leader-containing transcripts for all of the expected nested subgenomic (sg) mrnas (including the sg spike mrna) confirmed replication of the recombinant virus (fig. 4) . however, subsequent passages by supernatant transfer to uninfected monolayers failed to reproduce the infection, suggesting that the predict/pdf-2180 spike protein is unable to mediate cell entry in vero cells as seen with wild-type mers-cov (fig. 4) . (fig. 5a) . similarly, viral rna expression analysis indicated no evidence of replication following infection with the predict/pdf-2180 chimeric virus (fig. 5b) . in contrast, wild-type the discovery of predict/pdf-2180 in uganda adds to the growing number of group c betacoronaviruses that have now been identified in bats. these include neocov from south africa (15), mex_cov-9 from mexico (29), batcov/kw2e from thailand (30), p.pipi/vm314 from the netherlands (31), h.sav/206645-40 from italy (32), and betacov/sc2013, hku4, and hku5, all from china (33) . collectively, these examples demonstrate that the mers-related covs are highly associated with bats and are geographically widespread. the group 2c viruses appear to have a particular, though not exclusive, association with vespertilionid bats, which form a highly diverse and widely distributed family within the microchiroptera. neocov, sc2013, hku4, hku5, h.sav/206645-40, p.pipi/ vm314, and predict/pdf-2180 were all found in species belonging to this family. if the full diversity of 2c viruses reflects the number of vespertilionid species described (n ϭ 475 species), there is potential for a substantial diversity of mers-related viruses to be circulating in bats. our data suggest that predict/pdf-2180 cannot infect humans and is not likely to pose a threat to human health, at least in its current form. the spike protein of this virus is distinct from the mers-cov spike, sharing only 46% amino acid identity, and it appears unable to enter cells that express the functional receptor used by mers-cov (dpp4)-or any other receptor expressed by either primate vero cells or human airway epithelial cells. importantly, failure to assemble and release viral particles from the initial infection could also explain our results; however, we suggest that receptor incompatibility is more likely given the steps taken to minimize particle disruption (see materials and methods). these results suggest that adaptation of the spike would be required to permit predict/pdf-2180 replication in human airways. while we did not examine the specific binding properties of the related virus neocov, the high amino acid sequence identity with predict/pdf-2180 indicates that it shares a similar phenotype and is most likely also refractory for human infections. our data suggest that rna recombination is the mechanism that underlies the observed difference in receptor binding. recombination can occur at high frequency during mixed coronavirus infection, allowing different viral lineages to exchange specific functional motifs or even entire genes (22, 34, 35) . phylogenetic incongruence was noted in subunit 1 of the spike protein, and breakpoints were observed in this same region by multiple recombination detection algorithms. it is also parsimonious with the high purifying selection observed across the genome of 2c viruses (which argues against receptor adaptation via drift or selection) and with previous reports citing recombination in association with host switching for other coronaviruses (36) (37) (38) . given that the recombination is observed in both predict/pdf-2180 and neocov, we support the previous suggestion by corman et al. (15) that it was the mers-cov that acquired a new spike. given also that the predict/pdf-2180 spike does not use dpp4 and is seemingly not competent for human infection, we further suggest that the recombination event was the critical factor driving the emergence of mers-cov. what is less clear is whether this recombination occurred in bats or an intermediate host. lineage 2c strains that use dpp4 have been reported in bats (25, 26) , and there is also evidence of positive selection in the bat dpp4 that would indicate the existence of a large diversity of (as-yet-unknown) dpp4-competent strains (39) . just as detailed metagenomics studies have revealed the presence of several severe acute respiratory syndrome (sars)-like bat covs that can use the human angiotensin converting enzyme 2 receptor and/or replicate efficiently in human cells (23, 24, (40) (41) (42) , it seems likely that subsets of diverse mers-cov-like bat coronaviruses will also exist which are preprogrammed to efficiently use the human dpp4 receptor. this would support the hypothesis that the recombination occurred in bats; however, the mers-cov spike seems to have adapted and acquired a preference for human dpp4 over the bat homologue (26, 43) making it difficult to conclude with certainty that the mers-cov spike has bat origins. increased surveillance will be required to understand the full diversity of spike phenotypes circulating in bats or in intermediate hosts such as camels. in recent years, global surveillance efforts such as the usaid emerging pandemic threats predict program have advanced our understanding of the viral diversity that exists in wildlife (44) . while this knowledge can be useful for proving the existence of novel viruses (29, 30, (45) (46) (47) (48) (49) , quantifying overall viral diversity (45, 46) , and measuring infection prevalence within a population, it does not provide information on their specific zoonotic threat. given that no single correlate of pathogenicity or virulence has been determined for any viral family (50, 51) and that it is not possible to determine risk through phylogenetic data alone (51), the approach used here is an important tool in characterizing the zoonotic potential of viral sequences detected in wildlife. doing so on a large scale (for example, as part of projects like usaid predict) will also provide critical information on host and geographic variation in key viral traits, like potential host tropism, which are currently missing from most risk-based models forecasting hot spots of disease emergence. a bat (id otba03-20130220) was trapped on 20 february 2013 in the nkuringo area of kisoro district in southwestern uganda. the bat was caught with a mist net (3.8-mm mesh; avinet, inc.) according to established protocols and was released unharmed postsampling. standard morphometric measurements (weight and forearm length) and photographs were obtained to aid species identification, which was confirmed by dna barcoding of the cytochrome b (cytb) and cytochrome oxidase subunit 1 (co1) mitochondrial dna genes (52) . approximately 200 l of whole blood was collected into edta. oral and rectal swabs were also collected in duplicate (one into viral transport medium and one dry). specimens were stored temporarily on gel packs and frozen in liquid nitrogen in the field within 4 h of collection and then transferred to ϫ80°c for storage until testing. samples were transferred to the center for infection and immunity at columbia university for viral discovery and characterization. coronavirus discovery by consensus pcr. total nucleic acid (tna) was extracted using the roche magna pure 96 platform according to the manufacturer's instructions. tna was reverse transcribed into cdna using superscript iii (invitrogen) according to the manufacturer's instructions. two broadly reactive consensus pcr assays targeting partial and nonoverlapping regions of the coronavirus orf1b (containing the rdrp) were performed (53, 54) . bands of the expected size were excised from 1% agarose, cloned into strataclone pcr cloning vector, and sequenced to confirm detection. sequencing and bioinformatic processing. total rna extract was dnase treated (dnase i; ambion, life technologies, inc.) and reverse transcribed using superscript iii (invitrogen, life technologies, inc.) with random hexamer primers. the cdna was rnase h treated before second-strand synthesis with klenow fragment (3= to 5= exonuclease) (new england biolabs). the resulting double-stranded cdna was sheared to 200-bp (average) fragments using a covaris focused ultrasonicator e210, according to the manufacturer's standard settings, and used for library construction using the kapa hyper library preparation kit (kapa biosystems, roche), again according to the manufacturer's instructions. the final library was quantified using an agilent bioanalyzer 2100 and pooled to allocate 20 million reads on the illumina hiseq 2500 platform. the q30-filtered fastq files were used to generate quality control reports using prinseq software (v0.20.2) (55) and were further filtered and trimmed. host background levels were determined by mapping the filtered reads against a bat reference database using bowtie2 mapper (v2.0.6, http://bowtie -bio.sourceforge.net) (56) . the host-subtracted reads were de novo assembled using mira assembler (v4.0) (57) . contigs and unique singletons were subjected to homology search using megablast against the genbank nucleotide database. sequences that showed poor or no homology at the nucleotide level were screened by blastx against the viral genbank protein database. viral sequences from blastx analysis were subjected to another round of blastx homology search against the entire genbank protein database to correct for biased e values and taxonomic misassignments. the genome of predict/pdf-2180 was mapped with bowtie2 against the filtered data set to visualize depth and coverage in integrated genomics viewer. genetic and phylogenetic analyses. sequences were analyzed and edited using geneious (version 6.0.3). full genome and individual gene sequences were aligned with clustalw, and maximum likelihood phylogenetic trees were constructed in paup* (500 bootstraps). models of nucleotide substitution were selected using jmodeltest. nucleotide sequence similarity between mers-like viruses was assessed using simplot v3.5.1 (58) with a sliding window size of 500 bp, a step size of 50 nucleotides, and 1,000 bootstrap replicates using gap-stripped alignments and the f84 (maximum likelihood) distance model. the full-genome alignment was scanned for recombination using seven different algorithms (rdp, geneconv, bootscan, maxchi, chimaera, siscan, and 3seq) implemented in rdp (v4.46) (59). structural modeling. predicted binding differences between dpp4 and either mers or uganda were determined by structural analysis. the crystal structure demonstrating the interactions between dpp4 and mers spike binding domain has previously been reported (28) , and the crystal structure is pdb id 4kr0. we created a homology model of the region of the uganda spike protein homologous to the mers spike binding domain based on the 4kr0 structure in association with dpp4. we first aligned the amino acid sequences for 4kr0 (28) and the uganda spike using clustal omega (60). we then used modeller (61) to create predicted structural coordinates for the uganda spike based on the coordinates of 4kr0. because modeller requires the two sequences to be the same length, we introduced gaps in the sequences where appropriate to maintain the best sequence identity between the 2 amino acid sequences. numbering is based on mers-cov amino acid residues. we then imported the predicted crystal structure for uganda and the known dpp4-mers structure into pymol (62) for visualization and comparative analysis. hydrogen bonding interactions were predicted by selecting the known dpp4 and 4kr0 or the homologous dpp4 and uganda interaction sites and using the "find polar interactions" function within pymol. generation of a mers-cov recombinant virus. previously, we reported the isolation of recombinant mers-cov that was derived from a cdna clone (63) . to reconstitute a mers genome expressing the predict/pdf-2180 cov spike, new e and f plasmids were ordered synthetically (bio-basic) to contain the predict/pdf-2180 spike ectodomain; these plasmids were named mers-uganda e and f. mers orf1 and orf2 overlap, so to maintain a functional replicase sequence and signal sequence for spike, the first 20 amino acids of the mers spike were retained and the predict/pdf-2180 sequence was fused in frame downstream of the mers-cov s glycoprotein signal peptidase domain beginning at its 24th amino acid. in short, the sequence of the mers spike coding for amino acids 21 to 1306 was replaced with the sequence of the predict/pdf-2180 spike coding for amino acids 24 to 1298, so that following processing, an intact spike glycoprotein was expressed during virus infection. the e and f plasmids were sequence verified prior to the assembly of full-length recombinant dnas. the mers a through f inserts (containing the uganda s gene) were restriction digested, resolved on 0.8% agarose gels, visualized, excised, and purified using a qiaquick gel extraction kit (qiagen). the mers a to f inserts were mixed and ligated overnight at 4°c, phenol-chloroform extracted, and precipitated under isopropyl alcohol. full-length t7 transcripts were generated in vitro as described by the manufacturer (ambion; mmessage mmachine) with certain modifications (63) . for mers-cov n transcripts, 1 g of plasmid dna containing the n gene (amplified using forward primer 5=-atttaggtgacactat agatggcatcccctgctgcacc-3= and reverse primer 5=-ttttttttttttttttttttttctaatcagtgttaaca tcaatcattgg-3=) was transcribed by sp6 rna polymerase with a 4:1 ratio of cap analog to gtp. rna transcripts were added to 800 l of vero cell suspension (8.0 ϫ 10 6 cells) in an electroporation cuvette, and four electrical pulses of 450 v at 50 f were delivered with a gene pulser ii electroporator (bio-rad). the transfected vero cells were allowed to recover for 10 min at room temperature and then incubated at 37°c for 2 to 4 days in a 75-cm 2 flask. virus progeny were then passaged several times in vero cells or primary human airway epithelial cells for 48 h to detect viable viruses. all viruses were maintained under biosafety level 3 (bsl3) conditions with redundant fans, and personnel used powered air-purifying respirators (paprs) and tyvek suits. to detect leader-containing rnas, intracellular rna from wild type and recombinant mers-cov-uganda (rmers-cov-uganda) was reverse transcribed with a primer at the 3= end of the genome and cdna was isolated for pcr using a reverse primer located in orf5 and a forward primer located in the leader rna sequence at the 5= end of the genome (5=-ctatctcacttcccctcgttctc-3=). leadercontaining amplicons were sequenced as previously described (64) . the cdna products were separated and visualized in 0.8% agarose gels. viruses, cells, and infection. wild-type and chimeric covs were cultured on vero e6 cells, grown in dulbecco modified eagle medium (dmem) (gibco, carlsbad, ca) and 5% fetal clone serum (hyclone, south logan, ut) along with antibiotic-antimycotic (anti-anti; gibco, carlsbad, ca). growth curves in vero and primary human airway epithelial cells were performed as previously described (65, 66) . human lungs were procured under university of north carolina at chapel hill institutional review board-approved protocols. biosafety and biosecurity. reported studies were initiated after the nih and the university of north carolina institutional biosafety committee approved the experimental protocol (project title, generating infectious clones of bat sars-like covs; lab safety plan id, 20167715; schedule g id, 19982). accession number(s). the near-complete genome sequence for predict/pdf-2180 has been deposited in genbank under accession number kx574227. isolation of a novel coronavirus from a man with pneumonia in saudi arabia genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation uganda mers-like virus in bats ® mers coronaviruses in dromedary camels geographic distribution of mers coronavirus among dromedary camels antibodies against mers coronavirus in dromedary camels mers coronavirus neutralizing antibodies in camels, eastern africa middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study molecular diversity of coronaviruses in bats close relative of human middle east respiratory syndrome coronavirus in bat rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat coronavirus spike proteins in viral entry and pathogenesis bat-to-human: spike features determining "host jump" of coronaviruses sars-cov, mers-cov, and beyond host cell proteases: critical determinants of coronavirus tropism and pathogenesis comparative protein structure modeling of genes and genomes template-based structure modeling of protein-protein interactions synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 coronaviruses in bats from mexico group c betacoronavirus in bat guano fertilizer circulation of group 2 coronaviruses in a bat species common to urban areas in western europe detection of coronaviruses in bats of various species in italy comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features establishing a genetic recombination map for murine coronavirus strain a59 complementation groups a clustering of rna recombination sites adjacent to a hypervariable region of the peplomer gene of murine coronavirus genetic diversity of coronaviruses in miniopterus fuliginosus bats severe acute respiratory syndrome (sars) coronavirus orf8 protein is acquired from sarsrelated coronavirus from greater horseshoe bats through recombination coronavirus diversity, phylogeny and interspecies jumping adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus bat severe acute respiratory syndrome-like coronavirus wiv1 encodes an extra accessory protein, orfx, involved in modulation of the host immune response isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection reducing pandemic risk, promoting global health. one health institute a strategy to estimate unknown viral diversity in mammals non-random patterns in viral diversity diversity of coronavirus in bats from eastern thailand adenovirus and herpesvirus diversity in free-ranging great apes in the sangha region of the republic of congo detection of diverse novel astroviruses from small mammals in china the search for meaning in virus discovery prediction and prevention of the next pandemic zoonosis identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit i and cytochrome b gene sequences identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria bat coronaviruses and experimental infection of bats, the philippines quality control and preprocessing of metagenomic datasets fast gapped-read alignment with bowtie 2 genome sequence assembly using trace signals and additional sequence information full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination rdp4: detection and analysis of recombination patterns in virus genomes fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega comparative protein structure modeling using modeller the pymol molecular graphics system, version 1.8. schrödinger llc reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus rewiring the severe acute respiratory syndrome coronavirus (sars-cov) transcription circuit: engineering a recombination-resistant genome release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus we thank nancy simmons (american museum of natural history) and carlos zambrana (ecohealth alliance) for help with species identification. we also thank komal jain for bioinformatic assistance (columbia university) and bernard erima, titus tugume, and raymond mayanja (all from makerere university) for processing samples.this study was made possible by the support of the american people through the united states agency for international development (usaid) emerging pandemic threats predict project (cooperative agreement number ghn-a-oo-09-00010-00) and by support from the national institutes of allergy and infectious disease (ai109761 and ai110700) and aging (k99ag049092). human airway epithelial cell cultures were supported by the national institute of diabetes and digestive and kidney diseases (dk065988). key: cord-269885-r8molh8c authors: jeong, soo young; sung, se in; sung, ji-hee; ahn, so yoon; kang, eun-suk; chang, yun sil; park, won soon; kim, jong-hwa title: mers-cov infection in a pregnant woman in korea date: 2017-08-08 journal: j korean med sci doi: 10.3346/jkms.2017.32.10.1717 sha: doc_id: 269885 cord_uid: r8molh8c middle east respiratory syndrome (mers) is a lethal respiratory disease — caused by mers-coronavirus (mers-cov) which was first identified in 2012. especially, pregnant women can be expected as highly vulnerable candidates for this viral infection. in may 2015, this virus was spread in korea and a pregnant woman was confirmed with positive result of mers-cov polymerase chain reaction (pcr). her condition was improved only with conservative treatment. after a full recovery of mers, the patient manifested abrupt vaginal bleeding with rupture of membrane. under an impression of placenta abruption, an emergent cesarean section was performed. our team performed many laboratory tests related to mers-cov and all results were negative. we report the first case of mers-cov infection during pregnancy occurred outside of the middle east. also, this case showed relatively benign maternal course which resulted in full recovery with subsequent healthy full-term delivery without mers-cov transmission. middle east respiratory syndrome (mers) is a lethal respiratory disease caused by mers-coronavirus (mers-cov) and occurs mostly in the middle east, initially by camel-to-human transmission, and then by human-to-human transmission. however, the disease was spread to other continents, probably by an index case, with subsequent pandemic outbreaks through human-to-human transmission through droplets and contact. during these respiratory viral outbreaks, pregnant women can be expected as highly vulnerable candidates for infection (1) . a mers outbreak occurred in korea in 2015 with 186 infections, including 38 deaths (2, 3) . we experienced a case of a korean pregnant woman who was confirmed for a mers-cov infection via a polymerase chain reaction (pcr) test. this is the first case of a mers-positive pregnancy reported outside the middle east and is also the first case of having been exposed and confirmed on 3rd trimester of pregnancy with full-recovery and successful full-term delivery. on may 27, 2015, the patient's mother was exposed to the 14th mers patient, had a fever starting from june 3 and was diagnosed with mers on june 7. while febrile, she had been in close contact with her daughter, a 39-year-old pregnant woman (gravida 2 para 1). on june 8 (35 weeks and 4 days of gestational age [ga]), this pregnant woman visited the emergency room complaining of mild myalgia. based on this contact history with a mers patient and her symptoms, a mers-cov pcr test was performed and the result was found to be positive on june 9. starting from june 9, the patient developed dyspnea and sputum production. although chest auscultation was normal, the oxygen saturation (spo2) was 95% in room air and chest radiography showed diffuse opacity in the left lower lung area compared to a previously obtained radiographic image. the laboratory findings included a leukocyte count of 5,570/mm 3 (normal range 4,000-10,000/mm 3 ), with a differential of 71.4% segmented neutrophils, 20.5% lymphocytes, and 7.9% monocytes; and c-reactive protein level of 1.95 mg/ dl (normal range 0-0.3 mg/dl). she was given supplemental oxygen for hypoxia and conservative treatment, with hydration and pain control. the antiviral agents used in other severe mers-cov patients were not used in this patient, because her symptoms and laboratory findings were not severe. also, there was no evidence of any potential harm to the fetus and pregnant woman related to those drugs. after several days, her dyspnea and myalgia improved. the spo2 was 98% in room air and chest radiography showed interval improvement. on june 19 and 21, mers-cov pcr was performed and the results were negative. she had no symptoms related to mers. on june 23, the patient manifested abrupt vaginal bleeding with rupture of membranes. a fist-sized blood clot was found through speculum examination and she had abdominal pain. fetal cardiotocography showed no deceleration, but a variability of fetal heart rate changed from moderate to minimal. with an impression of placental abruption, her obstetrical team decided on emergent cesarean delivery. a 3,140 g male newborn was delivered at 37 weeks and 5 days of gestation. apgar scores at 1 and 5 minutes were 9 and 9, respectively. as expected, about 10% placental abruption was found (fig. 1) . after delivery, the baby was immediately moved to the airborne infection isolation room (aiir) and received an initial care with all health care personnel (hcp) completely protected according to the centers for disease control and prevention (cdc) guidelines (4). mers-cov pcr tests and antibody tests were performed with umbilical cord blood and placenta, and all results were negative. a systematic testing procedure for coronavirus infection, including chest radiograph and serial reverse transcription (rt)-pcr assays with peripheral blood and nasopharyngeal swab, did not demonstrate the presence of mers-cov in the newborn. mers-cov antibody tests were performed with mother and newborn sera on june 16 and june 28, respectively (5). in the mother's serum, immunoglobulin g (igg) was detected, albeit weakly, (0.302) via enzyme-linked immunosorbent assay (elisa; euroimmun ag, luebeck, germany), and via indirect immunofluorescence test (iift; euroimmun ag) with a titer of 1:100. igm and iga were not detected through elisa and the plaque reduction neutralization test (prnt) result was below the cutoff value. however, mers antibodies for igg, igm, and iga were not detected in the newborn's blood samples ( table 1) . the patient and her newborn baby were discharged in stable condition on june 30 with no clinical abnormalities on followup at the outpatient clinic. mers-cov was first isolated from a patient who died from a severe respiratory illness in jeddah, saudi arabia in june 2012 (6) . since then, more than 1,698 confirmed cases were reported to https://doi.org/10.3346/jkms.2017.32. 10.1717 the world health organization (who). clinical features of mers are variable, and infected patients can be asymptomatic or have an acute febrile illness, upper respiratory tract disease, or even multiple organ failure resulting in death (7) (8) (9) (10) (11) . however, there are limited data about the clinical features of mers-cov infection during pregnancy and the perinatal outcome of patients diagnosed with mers-cov infection. to our best knowledge, there have been 9 reported cases in which pregnant patients had positive laboratory results for mers-cov including this case (12) (13) (14) (15) (table 2) . unlike other cases, this case is not only the first mers-cov infection during pregnancy occurred outside of the middle east, but also the first case of mers confirmed on 3rd trimester of pregnancy showing good outcome of both mother and baby. currently, an exposure time to this virus during pregnancy and a severity of maternal disease could be expected to affect the perinatal outcome. however, there is limited knowledge about the clinical implications of mers-cov infection on the maternal, fetal, and placental aspects of pregnancy. from the maternal aspect, there is no epidemiologic data regarding whether pregnant women are more susceptible to mers. also, it is unknown whether mers-cov infected pregnant women have a more severe disease course compared with the non-pregnant population. in our case, she showed a mild disease course. she had low level of igg antibody by elisa and iift but not detectable neutralization activity by prnt. it has been suggested that neutralizing antibodies are produced at low levels and are potentially short-lived after mild or asymptomatic mers-cov infection (16, 17) . from the fetal aspect, it is unclear whether mers was a causative factor in the stillbirth or preterm birth. fetal specimen and/or placenta were not available for evaluation in the previous cases. as pregnancy alters maternal pulmonary function and consumes more oxygen, severe respiratory illness during pregnancy results in maternal hypoxemia. maternal hypoxemia can be associated with poor fetal oxygenation, which eventually could lead to preterm birth or stillbirth. also, altered immune responses during pregnancy could affect the fetal outcome (13) . from the placental aspect, there have been no reports of mers causing pathology of the placenta including infarction, insufficiency, or villus placentitis. our case showed placenta abruption clinically, which can be caused by maternal infection. there is no evidence of a relationship between mers-cov and placenta disorder. however, the possibility that this virus may be a cause of placenta abruption should be of concern. lastly, the remaining question was whether the virus could cross the placenta causing significant infection in the fetus, and whether mers could cause vertical transmission. camel-tohuman transmission, and human-to-human transmission via contact, droplet, and possibly airborne routes are the known modes of transmission (18, 19) . however, there are no data about perinatal transmission of mers-cov. moreover, if the mother mounts an appropriate immune response to produce enough neutralizing antibodies without serious conditions, passive antibodies transferred from mother to fetus may have a protective effect on the fetus. there is only one case reporting the mother's serologic data previously (15) , in which stillbirth occurred at approximately 5 months of gestation, although the mother had mers-cov antibody by elisa (titer 1:1,600) , immunofluorescent antibody (ifa), and microneutralization titer assay (titer 1:80). in our case, although the mother had igg antibody (titer 1:100 by iift), antibody was not detected in neonatal serum. this finding may provoke different interpretations in regard to the role of maternal antibodies in the fetus or to transmission of maternal antibodies, necessitating more data in the future. to know whether prenatal transmission of mers-cov can occur, collection of samples including amniotic fluid, placenta, and umbilical cord is needed from an infected pregnant patient. further studies with a larger sample size will help in understanding of the pathophysiology and perinatal outcome of mers during pregnancy and the optimal mode of delivery. the effect of asian influenza on the outcome of pregnancy mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome coronavirus (mers-cov) nosocomial outbreak in south korea: insights from modeling interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak isolation of a novel coronavirus from a man with pneumonia in saudi arabia characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea mers outbreak in korea: hospital-to-hospital transmission better understanding on mers corona virus outbreak in korea case definition and management of patients with mers coronavirus in saudi arabia impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia middle east respiratory syndrome coronavirus during pregnancy stillbirth during infection with middle east respiratory syndrome coronavirus transmission of mers-coronavirus in household contacts persistence of antibodies against middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control the emergence of the middle east respiratory syndrome coronavirus we thank dr. christian drosten and dr. marcel a. muller in institute of virology, university of bonn medical center for performing immunoglobulin a (iga) enzyme-linked immunosorbent assay (elisa) and plaque reduction neutralization test (prnt). we also thank prof. kyong ran peck in division of infectious disease, department of medicine, samsung medical center, sungkyunkwan university school of medicine for providing the reagent and practical help for us to obtain the antibody test results. the authors have no potential conflicts of interest to disclose. jong-hwa kim https://orcid.org/0000-0003-4356-0561 key: cord-256020-wrui3i2l authors: fadaka, adewale oluwaseun; sibuyi, nicole remaliah samantha; adewale, olusola bolaji; bakare, olalekan olanrewaju; akanbi, musa oyebowale; klein, ashwil; madiehe, abram madimabe; meyer, mervin title: understanding the epidemiology, pathophysiology, diagnosis and management of sars-cov-2 date: 2020-08-26 journal: j int med res doi: 10.1177/0300060520949077 sha: doc_id: 256020 cord_uid: wrui3i2l the emergence of coronavirus disease 2019 (covid-19) in december 2019 has resulted in over 20 million cases and 741,808 deaths globally, affecting more than 200 countries. covid-19 was declared a pandemic on 11 march 2020 by the world health organization. the disease is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2). there is limited information on covid-19, and treatment has so far focused on supportive care and use of repurposed drugs. covid-19 can be transmitted via person-to-person contact through droplet spread. some of the recommended precautionary measures to reduce the rate of disease spread include social distancing, good hygiene practices, and avoidance of crowded areas. these measures are effective because the droplets are heavy and can only travel approximately 1 meter in the air, settling quickly on fixed surfaces. promising strategies to combat sars-cov-2 include discovery of therapeutic targets/drugs and vaccines. in this review, we summarize the epidemiology, pathophysiology, and diagnosis of covid-19. we also address the mechanisms of action of approved repurposed drugs for therapeutic management of the disease. covid-19 has become one of the most dangerous pandemics in recent history. the pandemic has claimed more than 740,000 lives, with more than 20 million reported cases since the original outbreak. the disease is caused by sars-cov-2, a zoonotic pathogen that acquired mutations as it crossed the species barrier from bat to pangolin enabling it to infect humans. 1 sars-cov-2 was confirmed as a novel coronavirus using molecular methods and initially named 2019 novel coronavirus (2019-ncov). 2 the disease caused by this virus was later renamed covid-19 by the world health organization (who). 3 sars-cov-2 is highly infectious and has spread to more than 200 countries in all continents. hence, the virus was declared a global threat (pandemic) by the who. sars-cov-2 has a single-stranded positive sense rna genome (þssrna) approximately 30 kb long. the sars-cov-2 genome is organized similarly to those of sars-cov-1 and middle east respiratory syndrome (mers)-cov. 4 using phylogenetic analysis, the sars-cov-2 genome was demonstrated to share 96% sequence similarity with a bat cov genome. sars-cov-2 belongs to the b-coronavirus genus that includes sars-cov-1 and mers-cov. 5 the clinical symptoms of covid-19 include fever, cough, and pneumonia, which makes the disease enormously dangerous with a high case fatality rate. 6, 7 in contrast to other b-coronaviruses, many sars-cov-2 deaths have resulted from multiple organ dysfunction syndrome (msof) rather than respiratory failure. 8 this could be attributed to the widespread distribution of angiotensin converting enzyme-2 (ace-2), the primary receptor for sars coronaviruses, in multiple organs. 9,10 ace-2 is expressed as a cellsurface molecule in the respiratory tract (epithelium, arterial and venous endothelium), the small intestinal epithelium, and arterial smooth muscle cells. 11 sars-cov-2 is morphologically spherical and is characterized by presence of a spike protein, lower pathogenicity and higher transmissibility between humans. 12 some of the primary measures taken to reduce the number of infections and prevent community transmission are to avoid crowds and gatherings and to practice good hygiene. interestingly, countries with the highest reported prevalence and mortality such as the united states, spain, italy, the united kingdom, russia, germany, brazil, france, turkey, iran and china, are more concerned with flattening the curve through early case detection, isolation, and development of therapeutic drugs and vaccines. because of the novel nature of this disease, there is limited information regarding risk factors for severe outcomes. specific factors such as the serial interval, viral lifespan, incubation period, pathogenic mechanisms, clinical features and optimal disease management remain unclear. therefore, this review aimed to summarize the epidemiology and pathophysiology of sars-cov-2 as well as the use of repurposed food and drug administration (fda) approved drugs for therapy. the entry of this virus into host cells and possible downstream complications deserve closer attention. covs are members of the subfamily othocoronavirinae of the family coronaviridae. the subfamily consist of four genera: alpha, beta, gamma and delta covs. 13 both alpha-and beta-covs can infect mammals, including humans, while the gamma-and delta-covs only infect birds. 14 about seven covs have been isolated from humans ( figure 1 ). these include two alpha-covs, human coronavirus 229e (hcov-229e) and human coronavirus nl63 (hcov-nl63), and five beta-covs: human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), sars-cov-1, mers-cov, and sars-cov-2. sars-cov-1, mers-cov, and sars-cov-2 are pathogenic and cause severe infections in humans following contact with the respective intermediate hosts (bats) (figure 2 ). however, hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1 do not appear to cause severe infections in humans. 14 covs are enveloped viruses with þssrna genomes. they have the largest genomes (approximately 26-33 kb) among rna viruses. all covs possess nonsegmented genomes with similar organization. 15 generally, about two-thirds of the genome consists of two large and overlapping open reading frames (orf1a and orf1b), which are translated into polyproteins pp1a and pp1ab and subsequently processed to yield 16 non-structural proteins (nsp1 to nsp16). 16 the remaining one-third of the genome consists of orfs encoding structural proteins including the spike (s) glycoprotein embedded in the envelope and the envelope (e), matrix (m), and nucleoproteins (n). 17 there are short untranslated regions at both the 3 0 and 5 0 ends. the s protein plays a role in receptor-binding and entry of virus into host cells and is thus considered a major therapeutic target. the m and e proteins are important for viral assembly, while the n protein is necessary for synthesis of rna. 18 overview of the sars-cov family sars-covs belongs to a global family of viruses causing respiratory disease and influenza-like symptoms such as fever, muscle pain, sore throat, headache, and cough. 19 figure 3 highlights the onset and progression of sars-cov-1, mers-cov, and sars-cov-2 infection. the first case of sars-cov-1, reported in china, resulted in an outbreak that caused hundreds of deaths and thousands of infected cases in the early 2000s. 20 a pneumonia-like syndrome (mers) was first discovered in saudi arabia and then spread to several countries, where it incurred a mortality rate of about 3% to 6%. marra et al (21) observed that the mers mortality rate increased with age and was as high as 43% to 55% in people older than 60 years. in december 2019, sars-cov-2, caused an outbreak in china and then spread worldwide. the resulting disease (covid-19) is a serious threat mostly in people with compromised immune systems or underlying conditions such as lung disease, diabetes mellitus, and human immunodeficiency virus infection. 22 sars-cov-1. sars-cov-1 causes a viral respiratory disease and belongs to lineage b of the beta-covs. 11 of uncooked meat, milk or urine, as shown in figure 2 . human-to-human infection also occurred through nosocomial transmission. 11 symptoms of human sars-cov-1 infections include headache, fever and respiratory complications such as cough, dyspnea, and pneumonia. 23 the sars-cov-1 genome is 29,727 nucleotides in length. the 5 0 end of the genome contains orf1a and orf1b. the polyproteins encoded by these orf are auto-catalytically processed to yield a number of viral proteases as well as the rna-dependent rna polymerase (rdrp). the remainder of the genome encodes the viral structural proteins (s, e, m and n) as well as several accessory proteins. 18 the receptor for sars-covs is ace-2, a surface molecule found on cells of the respiratory tract, the small intestinal epithelium, and smooth muscle. in the respiratory tract, ace-2 is expressed on epithelial cells of the alveoli, bronchi, trachea, and bronchial serous glands, as well as on alveolar monocytes and macrophages. the ace-2 enzyme plays an important role in protection against lung failure. 11 mers-cov. mers-cov causes a viral respiratory disease and belongs to lineage c of the beta-covs. 11 the first cases of human infection by mers-cov were reported in saudi arabia in 2012. cases were subsequently reported in other countries including qatar, egypt, and the republic of korea following contact with infected camels (figure 3b ). cases of mers-cov were identified in about 27 countries between 2012 and 2018. 18 mers-cov rna in camels showed more than 99% genomic sequence similarity to human mers-cov. 23 bats are the natural hosts of mers-cov (figure 2) , and a high prevalence of mers-cov infections in dromedary camels (intermediate hosts) was confirmed in the middle east, spain, and africa. mers-cov infections were transmitted to humans following contact with camels infected with the virus. 23 the mers-cov genome is about 30,119 nucleotides in length and has a 5 0 terminal cap structure and a poly (a) tail at the 3 0 end. the genome encodes 16 non-structural proteins (nsp1-nsp16) at the 5 0 end, four structural proteins (s, e, m, and n) at the 3 0 end, 24 and five accessory proteins in orf3, orf4a, orf4b, orf5, and orf8. 18 risk factors for severe mers-cov include age and the presence of underlying conditions such as diabetes, obesity, hypertension, chronic renal disease, and lung diseases. 25 the receptor for mers-cov is dipeptidyl peptidase 4 (dpp4 or cd26). dpp4 is a multifunctional cell surface protein and is expressed on the epithelial cells of the respiratory tract, liver, kidney, small intestine, and prostate, as well as on activated white blood cells. dpp4 also plays a vital role in activation of t cells and providing co-stimulatory signals for immune responses of t cells. 11, 18 consequently, mers-cov causes acute pneumonia and renal dysfunction with associated clinical symptoms such as cough, chest pain, sore throat, fever, diarrhea, vomiting, and abdominal pain. mers-cov can infect human dendritic cells and macrophages in vitro, thereby contributing to dysregulation of the immune system. 18 sars-cov-2. sars-cov-2, a newly discovered coronavirus, has a þssrna genome and a spherical morphology when observed under the electron microscope. 26,27 sars-cov-2 encodes a richly glycosylated spike protein responsible for binding to the ace-2 receptor. 28 the virion's shape and the ability of spike proteins to form a crownlike structure gave coronaviruses their name. 29, 30 the sars-cov-2 genetic material is surrounded by a lipid-bilayer envelope. other structural components include the nucleocapsids, membrane, envelope, and hemagglutinin. unlike the envelope, the membrane exists in higher quantities within the virions. 31 among other functions, the envelope serves to release viral particles from the host cells. 32 the nucleocapsid assist in rna packaging during virion assembly. 33, 34 hemagglutinin enhances the entry and pathogenesis of coronaviruses. 33 some of the characteristics of sars-cov-2 are summarized in table 1 . many of these features are shared with sars-cov-1. more information on the epidemiology and general characteristics of sars-cov-1 can be found later in the review. other important human covs include alpha-covs (hcov-229e and hcov-nl63) and two beta-covs (hcov-oc43 and hcov-hku1). the receptor for hcov-229e is human aminopeptidase n (cd13), a cell-surface metalloprotease present on cells of the kidney and lung, as well as on epithelial and intestinal cells. the receptor for hcov-nl63 is also ace-2. the receptor for hcov-oc43 is 9-o-acetylated sialic acid, while no receptor has been identified for hcov-hku1. 42, 43 generally, the most common diagnostic tools for human covs are molecular detection techniques such as reverse transcription-polymerase chain reaction (rt-pcr) using rna extracted from respiratory tract samples as template. other methods include serological tests and viral cultures. 11 although several agents against covs, including antibodies, antiviral peptides, and cell or viral protease inhibitors, have been shown to be effective both in vitro and/or in vivo, clinical trial outcomes have not been reported. 11 therefore, clinical treatments for covs are still lacking. nevertheless, supportive and symptomatic therapy are used for at present, 215 countries and territories are affected by the sars-cov-2 outbreak, including the united states, italy, germany, south africa, and nigeria. the distribution of sars-cov-2 confirmed cases, including mortality and recovery, is shown in figure 4 . the united states, china, and some european countries have high case numbers and mortality rates. recovery rates are increasing worldwide with higher numbers reported in china. the cfr is defined as the percentage of deaths recorded among confirmed cases. as of 14 april 2020, the number of deaths among confirmed cases was estimated at 126,066 to 1,992,189, resulting in a cfr of 6.33%. this differs from the cfr of 3.70% calculated on 15 march 2020. however, several factors can prevent the accurate determination of the cfr. we compared the global cfr to the african cfr. the highest cfr was observed in italy, followed by spain and the netherlands (figure 5a ). in africa, countries such as egypt, algeria, burkina faso, and morocco had cfrs above 5% (figure 5b) . a major challenge in the accurate calculation of the cfr is the denominator (the number of confirmed cases). asymptomatic cases of covid-19, patients with mild symptoms, or individuals who are misdiagnosed may be left out of the denominator, leading to underestimation or overestimation of the cfr. 45 high cfrs reflect limited access to health care for the most vulnerable patients and limitations in health-care systems, including limited capacity of surveillance systems to trigger a timely response (who, 2020). to date, over 20 million cases of covid-19 have been reported globally. it is important to estimate the reproductive number for this virus to enable accurate predictions. two primary factors, the reproductive ratio (r o ) and the serial interval (s i ), are essential to estimate the rate of transmission of this disease. serial interval (s i ). to understand the case turnover and transmissibility of covid-19, the serial interval (s i ) from the onset of illness in a primary case to the onset of illness in a secondary case is important. 49 recent studies estimated the average s i for covid-19 as 3.77 (2.23-4.82) days. 49-51 a shorter s i makes covid-19 harder to contain and more likely to rapidly transmit within populations ( figure 6 ). taking the r 0 and s i of covid-19 into consideration, it can be inferred that approximately 4 days are required for an infected person to transmit covid-19. it is highly likely that the individual can infect approximately two or three other persons, making the spread of covid-19 extremely rapid and dangerous ( figure 7a ). the extent of spread is totally dependent on the r o value (figure 7b ). if r 0 < 1, each existing infection causes less than one new infection. in this case, the disease will decline and eventually die out. if r o ¼ 1, each existing infection causes one new infection. the disease will remain stable in the population, but will not result in epidemic spread. if r 0 > 1, each existing infection causes more than one new infection. the disease will spread between individuals and eventually lead to an outbreak. covid-19 was regarded as an outbreak (30 january 2020) with a r 0 > 1 (figure 7b) . importantly, the r 0 value of a disease only applies when everyone in a population is completely susceptible. this means that no one has been vaccinated or had the disease previously, and there is no way to control the spread of the disease using interventions such as drugs or vaccines. there are currently two known modes of covid-19 transmission: the fecal-oral route and respiratory droplets. 6, [52] [53] [54] [55] [56] droplets have potential to come into contact and infect a healthy person within 3 to 6 feet (1 meter). droplets that stick to surfaces can survive for more than 24 hours, remaining infectious. the virus can remain airborne for about 3 hours, long enough to permit transmission. upon infection with sars-cov-2, the virus infects type ii pneumocytes of the alveoli. 57 these pneumocytes are responsible for surfactant production. surfactant decreases the surface tension within alveoli and reduces the collapsing pressure. the spike protein of the virus binds to ace-2 on the pneumocytes (figure 8 ) permitting virion entry into the host cell. 58 the virus hijacks the host cell's machinery (ribosomes) to enable translation of its þ ssrna genome into different protein molecules. the virus can also use its rdrp to produce additional copies of its þ ssrna genome (figure 9 ). the translated polyproteins are further processed into different individual components within the host cell. these processes give rise to multiple virions, which are then released upon pneumocyte damage. in response to this process, type ii pneumocytes releases specific inflammatory mediators that instruct macrophages to secrete interleukins 1 and 6 (il-1 and il-6) and tumor necrosis factor-alpha. these cytokines cause the endothelial cells lining blood vessels to dilate, leading to increased capillary permeability. in response, fluids accumulate in the alveoli leading to edema. 59 as surface tension increases, the collapsing pressure of the alveoli increases. a decrease in gas exchange is also observed through this process, which in turn leads to hypoxia and difficulty breathing (dyspnea). this can progress to a critical condition such as acute respiratory distress syndrome (ards). inflammatory mediators further stimulate neutrophils, which release reactive oxygen species and proteases. this process damages the alveoli (both type 1 and 2), leading to consolidation and alveolar collapse. high levels of il-1 and il-6 travel through the blood to the central nervous system, instructing the hypothalamus to release prostaglandins and causing fever. severe lung inflammation leads to systemic inflammatory respiratory syndrome. progression can lead to increased capillary permeability. the overall blood volume decreases, and through a series of processes involving hypotension and decreased perfusion of multiple different organs, multiple system organ failure (msof) can occur. during msof, elevated levels of blood urea, nitrogen, and creatinine accrue in the kidney. the liver also releases specific inflammatory and acute phase response biomolecules (aspartate transaminase, alanine transaminase, bilirubin, c-reactive protein [crp], fibrinogen and il-6) that can serve as biomarkers for patients with covid-19. there is continued research by multiple groups into the mechanism of transmission of sars-cov-2 through the eyes. investigations were necessary because health care providers, including a perking university physician, may have contracted the virus while not wearing eye protection when treating patients. 61 some researchers have asserted that avoidance of touching the eyes, nose or mouth with unwashed or unsterilized hands can reduce covid-19 transmission. the mucous membranes that line various cavities in the body are generally most susceptible to viral transmission. 62 ocular symptoms such as viral conjunctivitis can result from sars-cov2 upper respiratory tract infections. 63 this was confirmed in 9 of 1,099 patients in china. 64 other research also found that 1 of 30 patients hospitalized with covid-19 were diagnosed with conjunctivitis. 65 in a study conducted by the american optometric association, covid-19 was linked to ocular signs and symptoms including photophobia, irritation, conjunctival infection and ocular discharge. 66 thus, the superficial blood vessels of the conjunctiva are an alleged route of exposure and transmission of sars-cov-2. the clinical manifestations of sars-cov-2 are uncertain and change frequently. some infections are asymptomatic. symptoms can include respiratory distress syndrome, pneumonia of different levels of severity, 67 and sometimes death. according to the who, the most common symptoms of covid-19 are fever, fatigue, dry continuous cough, and shortness of breath. 68, 69 some patients may have a runny nose, sore throat, nasal congestion, aches and pains, and diarrhea. 70 some patients report a loss of sense of smell and taste. 71 in some cases, symptoms are mild and similar to those of the common cold; in such patients, recovery can occur without any treatment. 72 the least commonly observed symptoms include nausea or vomiting, coughing up blood or bloody mucus, and viral conjunctivitis causing red eyes, watery discharge from the eyes, swollen eyelids and light sensitivity. 3 occasional upper respiratory and gastrointestinal symptoms, accompanied by changes vital signs such as increased respiration (heart rate) and low blood pressure may also be observed, especially in the elderly and among individuals suffering from heart disease, chronic respiratory conditions and diabetes. 73 additionally, patients critically ill with covid-19 may present with increased venous thromboembolism including thrombocytopenia, elevated d-dimer, prolonged prothrombin time and disseminated intravascular coagulation (dic). these coagulation abnormalities are associated with a systemic inflammatory response and an imbalance between pro-coagulant and anticoagulants homeostasis mechanisms. and increase the risk of mortality. 74 some of these clinical features are also observed in cases of dic observed in septic patients. these features are very distinct in covid-19 patients as their levels are higher than the standards for sepsis. 75, 76 diagnosis, prognosis, treatment, and management of sars-cov-2 sars-cov-2 causes various complications ranging from fever, dry cough, and pneumonia to decreased organ perfusion leading to msof. early symptoms are similar to those of influenza, and the first step to differentiate covid-19 from flu and pneumonia is a nasopharyngeal swab test (viral testing for influenza a/b). 77 quantitative polymerase chain reaction-based (qpcr) methods are the major diagnostic tests for sars-cov-2 using nasal swab, aspirate, sputum, or blood as samples. these method have some limitations as they are time consuming and have variable sensitivity (30%-80%). [78] [79] [80] another diagnostic method is the newly approved nucleic acid test, which is carried out based on the principle of fluorescence pcr. 81 the main goal of sars-cov-2 diagnosis is to accurately detect the virus and to minimize further transmissions by timely isolation and treatment of infected patients. other tests (not specific for sars-cov-2) used in conjunction with the methods above are based on clinical manifestations. these include blood tests such as complete blood count, comprehensive metabolic panels, basic metabolic panels, and assessment of liver/kidney markers and procalcitonin levels (for bacterial infections). inflammatory markers may also be assessed including crp, erythrocyte sedimentation rate, il-6, lactate dehydrogenase, d-dimer, ferritin, troponin and creatine kinase-mb. imaging investigations are typically computed tomography scans: in covid-19 patients these often show glass opacities, areas of consolidation, and paving patterns in cases of severe and progressive disease. ground glass opacities can also be observed on chest x-ray. finally, ultrasound can show b-lines, pleural line thickening, and lung consolidation. air bronchograms can also be used for assessment. these tests are non-specific but helpful to determine patients' health status. covid-19 patients with severe ards could potentially present with pneumonia. pneumonia may be severe, leading to ards and msof. it is therefore imperative to mechanically ventilate the lungs to avoid ards and msof. although the risk factors for covid-19 remain unclear, some risk factors put patients at a significantly higher risk of mortality, especially in individuals with underlying conditions. 6, 35, 40, 82 some medical disorders are correlated with higher risk of mortality in covid-19 patients. these include, cardiovascular diseases mortality risk 10.6%), 41 lung disease (mortality risk 7.3%), type 1 and 2 diabetes (mortality risk 6.3%), immunosuppression (e.g., cancer patients; mortality risk 5.6%), [83] [84] [85] [86] and age ( figure 10 ). 87 covid-19 mortality rates increase with advancing age, and are especially high in those aged >60 years. elevated inflammatory markers in response to tissue damage (elevated levels of d-dimer, ferritin, creatine kinase-mb, and troponin) have been associated with higher mortality rates. the first line of treatment for patients presented with the symptoms of covid-19 (fever, dry cough, and shortness of breath) is self-quarantine for at least 14 days. cases are monitored for progression of symptoms such as increased fever (>40 c), significant difficulty breathing or shortness of breath, mouth breaks, constant coughing, and dehydration. if there is no significant improvement in symptoms, it is advisable to consult a clinician for confirmation of the diagnosis and to avoid further spread of the virus. the main treatment currently available is supportive care. although there is limited information on covid-19, it has been linked to ards. for patients with high fevers that could potentially lead to dehydration, intravenous fluids such as normal saline or lactated ringer's fluid can be administered sparingly to prevent lung overload. to reduce fever, antipyretic drugs such as paracetamol or acetaminophen can be administered. drugs such as remdesivir, chloroquine, ritonavir, tocilizumab, corticosteroids have been repurposed for the treatment of covid-19 (figure 11 ), although their clinical effectiveness has not yet been confirmed. [88] [89] [90] [91] [92] [93] unfortunately, the use of chloroquine and derivatives such as hydroxychloroquine, alone or in combination with other drugs, resulted in cardiac toxicity. investigations of these drugs were recently suspended by the who. 94 mechanism of action of selected repurposed drugs for treatment of covid-19. the elucidation of potential targets could lead to covid-19specific treatments (figure 11 ). approaches to anti-sars-cov-2 drug development include (a) inhibition of sars-cov-2 entry, (b) disruption of sars-cov-2 þ ssrna synthesis after entry, (c) inflammatory response suppression, and (d) disruption of sars-cov-2 translation. an fda-approved immunosuppressive and anti-malarial parasite drug (chloroquine) can inhibit the entry of covid-19 into the endosome after binding to ace-2 receptors, thereby preventing the release of þ ssrna for translation. 89, 93, 95, 96 studies also showed that hydroxychloroquine, an analog of chloroquine, was more potent and could be used in place of chloroquine. 97 remdesivir, a novel antiviral drug and nucleotide analog used to treat ebola virus infection, is currently under clinical development. the mechanism of the drug is reported to be at the post viral entry stage. 89 its mode of action is to inhibit the rdrp, preventing synthesis of the viral þ ssrna (figure 11 (2)). this drug is currently in phase 2 clinical trials. the protein inhibitor ritonavir has also been proposed for the treatment of covid-19. this drug interferes with the protease enzymes (proteinases) by inhibiting the conversion of polyproteins into the mature components (spike proteins, nucleocapsids) needed by the virus for multiplication (figure 11 (3)). another immunosuppressive drug, tocilizumab, has also been repurposed for covid-19 because of its ability to block il-6 and inhibit inflammatory responses. corticosteroids can also decrease inflammation by inhibiting phospholipases such as phospholipase a 2 , and thus suppress the excessive production of prostaglandins (figure 11 (4) ). sars-cov-2 appears unfamiliar to the human innate immune system. coupled with its emergence and its spread globally via human-to-human transmission, the development of vaccines for prevention is no longer a debate but a necessity. several vaccine platforms are being developed and some have entered clinical trials. however, approval by regulatory agencies such as the fda and manufacturing may take 12 to 18 months. 98 studies of the antiviral activity of host-directed drugs and compounds have identified two classes of molecules (protein biogenesis inhibitors and ligands of the sigma1 and sigma2 receptors) as effective inhibitors of viral infectivity. molecules that target the sigma1 and sigma2 receptors perturb the virus through different mechanisms from translation inhibitors, potentially including modulation of cellular stress responses. 99 the ligands haloperidol, pb28, pd-144418, and hydroxychloroquine are currently undergoing clinical trials in covid-19 patients. 100 these molecules exert their antiviral effects during viral replication by inhibiting nucleoprotein expression after viral entry has occurred. the lack of selectivity of chloroquine and hydroxychloroquine, including off-target effects on the human ether-a`-go-go-related gene (herg) and other molecules, may be related to the adverse cardiac reactions that have limited their use. 101 a recent study by gordon et al identified 332 highconfidence sars-cov-2-human protein interactions connected to multiple biological processes, including protein trafficking, translation, transcription and ubiquitination regulation. 102 the study identified 69 ligands, including fda approved drugs, compounds in clinical trials, and preclinical compounds that might theoretically have therapeutic effects as host-directed interventions against covid-19. to date, no known antiviral drugs nor vaccines against sars-cov-2 with proven clinical efficacy have been identified. part of the reason for the absence of such agents is limited information regarding the molecular details of the infection. to develop therapeutic interventions against covid-19, it is crucial to understand how the virus interactions with the host during infection. ace-2, a potential target for covid-19 treatment. ace-2 has been identified as the functional receptor for sars-cov-2. it is an outer membrane enzyme expressed in vascular endothelial cells, the renal tubular epithelium, and leydig's cells of the testes, lungs, heart, kidney, and gastrointestinal tract. 59, 103 it is a type-ii transmembrane metallocarboxypeptidase with its enzymatically active domain exposed at the cell surface. 104 ace-2 is a key player in the renin-angiotensin system (ras) and a target for treatment of hypertension. 105 ace-2 catalyzes the cleavage of angiotensin ii, a vasoconstrictor, into angiotensin 1-7, a vasodilator, thereby lowering blood pressure by negatively regulating ras. 106 ace-2 is a promising drug target for treating cov infection as well as other cardiovascular diseases. 5 ace-2 confers a protective effect against lung injury induced by viruses by increasing the production of angiotensin 1-7. the virus presumably causes lung damage by reducing ace-2 levels on cells through the process of degradation and internalization. 107, 108 because studies have shown that ace inhibitors and angiotensin ii receptor blockers could be used to up-regulate the expression and activity of ace-2 in hypertensive patients, similar strategies might reduce the severity of covid-19. 109, 110 recent studies have shown that the expression of human ace-2 is associated with sars-cov infection and that genetic variations of this receptor may contribute to susceptibility and/or resistance against infection. 111 for instance, a single-cell rna sequencing analysis of ace-2 revealed that type ii alveolar cells had higher expression of ace-2 in eight individual lung tissues obtained from normal donors. ace-2 expression was higher in the two male samples compared with the six female samples. additionally, ace-2 expression was higher in the only asian male in the study compared with caucasian and black americans, 59 which might explain why the four german cases showed mild clinical symptoms with no severe illness. 112 this implies that variation in ace-2 expression in covid-19 patients is likely to affect susceptibility, symptoms and intervention outcomes following sars-cov-2 infection. the degree of spread of covid-19 is currently about 5.3% and could potentially increase if precautionary measures are not considered. global prevention of the spread of covid-19 is therefore a crucial and urgent goal. to prevent further spread of this disease, detection and isolation of individuals with covid-19 is of the utmost priority. examples of measures to curb spread include self-quarantine, isolation of infected individuals, social distancing, good personal hygiene (frequent hand washing with soap and water/alcohol-based sanitizers and avoiding touching the eyes, nose and the mouth), and use of personal protective equipment. certain classes of compounds, called surfactants, are contained in soap and have the ability to neutralize microbes such as sars cov-2. 113 this is because soap can assemble into bubblelike structures called micelles that trap viral matter and other biomaterials. 114 surfactants in soap lather have their hydrophilic parts pointing outwards and interacting with solvent and their hydrophobic heads pointing inwards. this opens the coronavirus outer membrane and encapsulates viral molecules within micelles, thus making insoluble viral molecules easily soluble in water and effectively removing them from hands, surfaces or other areas after about 20 s. 115 therefore surfactants in soap can disrupt and sequester viruses and other contaminants while sanitizer and disinfectants are designed to kill sars-cov-2. 116 the outbreak of sars-cov-2 has become a global threat. however, information regarding this virus remains limited. the available information is inconsistent and there are constant data updates, which may contribute to variation between study results. for more consistent and accurate results, well-annotated data from clinical patients and subclinical subjects in normal populations could help to better understand the pandemic. the information provided in this review is based on data provided by the center for systems science and engineering (csse) at johns hopkins university during specific date ranges. key insights into the prevalence, pathophysiology, diagnosis, and potential treatment of sars-cov-2 are herein summarized. research efforts are being intensified to address the current challenges in the quest for adequate treatments, diagnostics, and vaccines. clinical studies into the genetic variation of receptors such as ace-2 in tissues and across populations will remain an active area of research until relevant targets and therapies are found. while the development of adequate treatments and vaccines for covid-19 is underway, it is advisable that good hygiene practices including washing of hands and social distancing should be practiced, and government guidance/guidelines should be followed. this will help to reduce the spread of the disease. we hope that the insights gained from this review will enable researchers to help patients develop accommodating lifestyles and improve the efficiency of health care practitioners. data are freely available at the following sources. centers for disease control and prevention (https://www.cdc.gov/). center for systems science and engineering (csse) at johns hopkins university (https://coronavirus.jhu. edu/map.html). worldometers (https://www. worldometers.info/coronavirus/?). zoonotic origins of human coronaviruses detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in wuhan, china a novel coronavirus emerging in china-key questions for impact assessment recent insights into the biology of pancreatic cancer paracrine and cell autonomous signalling in pancreatic cancer progression and metastasis current and emerging therapies for patients with advanced pancreatic ductal adenocarcinoma: a bright future sars and other coronaviruses as causes of pneumonia isolation and characterization of sars-cov-2 from the first us covid-19 patient potential maternal and infant outcomes from coronavirus 2019-ncov (sars-cov-2) infecting pregnant women: lessons from sars, mers, and other human coronavirus infections subunit vaccines against emerging pathogenic human coronaviruses genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding viral and cellular mrna translation in coronavirus-infected cells molecular evolution of human coronavirus genomes from sars to mers, thrusting coronaviruses into the spotlight a complete sequence and comparative analysis of a sars-associated virus (isolate bj01) ultrastructural characterization of sars coronavirus the genome sequence of the sars-associated coronavirus asymptomatic cases in a family cluster with sars-cov-2 infection bat origin of human coronaviruses isolation and growth characterization of novel full length and deletion mutant human mers-cov strains from clinical specimens collected during middle east respiratory syndrome: emergence of a pathogenic human coronavirus cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the proteolytic regulation of virus cell entry by furin and other proprotein convertases an overview of their replication and pathogenesis differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza c virus and bovine coronavirus identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study oxford research encyclopedia of classics review of a modern contagion: imperialism and public health in iran's age of cholera. middle east quarterly disasters and community resilience: spanish flu and the formation of retail cooperatives in norway does sars-cov-2 has a longer incubation period than sars and mers clinical characteristics of 2019 novel coronavirus infection in china prevalence of comorbidities and its effects in patients infected with sars-cov2: a systematic review and meta-analysis human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a human coronavirus hku1 spike protein uses 9-o-acetylated sialic acid as an attachment receptor determinant and employs hemagglutinin-esterase protein as a receptor-destroying enzyme ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study the many estimates of the covid-19 case fatality rate estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis early dynamics of transmission and control of covid-19: a mathematical modelling study feasibility of controlling covid-19 outbreaks by isolation of cases and contacts the serial interval of covid-19 from publicly reported confirmed cases epidemiological and clinical features of the 2019 novel coronavirus outbreak in china community transmission of severe acute respiratory syndrome coronavirus 2 a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster pandemic potential of 2019-ncov active monitoring of persons exposed to patients with confirmed covid-19-united states modes of transmission of virus causing covid-19: implications for ipc precaution recommendations: scientific brief quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses single-cell rna expression profiling of ace2, the putative receptor of wuhan critical care crisis and some recommendations during the covid-19 epidemic in china novel coronavirus disease 2019 (covid-19): the importance of recognising early possible ocular manifestation and using protective eyewear can the coronavirus disease 2019 (covid-19) affect the eyes? a review of coronaviruses and ocular implications in humans and animals clinical characteristics of coronavirus disease 2019 in china evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection ophthalmologists are more than eye doctors-in memoriam li wenliang asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths world health organization the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records presumed asymptomatic carrier transmission of covid-19 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention the covid-19 epidemic changes in blood coagulation in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis coagulation abnormalities and thrombosis in patients with covid-19 prominent changes in blood coagulation of patients with sars-cov-2 infection the utility of preemptive mass influenza vaccination in controlling a sars outbreak during flu season characterization of a new member of alphacoronavirus with unique genomic features in rhinolophus bats real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus development and evaluation of novel realtime reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china nosocomial infections in patients with cancer selective depletion of regulatory t cell subsets by docetaxel treatment in patients with nonsmall cell lung cancer features of postoperative immune suppression are reversible with interferon gamma and independent of interleukin-6 pathways myeloid suppressor cells in cancer and autoimmunity severe outcomes among patients with coronavirus disease 2019 (covid-19)-united states drug treatment options for the 2019-new coronavirus (2019-ncov) remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel coronavirus in the united states arguments in favour of remdesivir for treating sars-cov-2 infections chloroquine for the 2019 novel coronavirus sars-cov-2 role of chloroquine and hydroxychloroquine in the treatment of covid-19 infection -a systematic literature review hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid-19: a multinational registry analysis fda-approved drug, prevents zika virus infection and its associated congenital microcephaly in mice efficacy of chloroquine versus lopinavir/ritonavir in mild/general covid-19 infection: a prospective, open-label, multicenter, randomized controlled trial in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus timely development of vaccines against sars-cov-2 sigma-1rs are upregulated via perk/ eif2a/atf4 pathway and execute protective function in er stress outcomes related to covid-19 treated with hydroxychloroquine among in-patients with symptomatic disease cardiotoxicity of antimalarial drugs a sars-cov-2 protein interaction map reveals targets for drug repurposing angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target substernal electrical stimulation system compensation of ace2 function for possible clinical management of 2019-ncov-induced acute lung injury potent neutralization of 2019 novel coronavirus by recombinant ace2-ig evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin receptor blockers as tentative sars-cov-2 therapeutics structural variations in human ace2 may influence its binding with sars-cov-2 spike protein comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations cationic gemini-surfactants based on waste cooking oil as new 'green' inhibitors for n80-steel corrosion in sulphuric acid: a combined empirical and theoretical approaches the effect of surfactant concentration, salinity, temperature, and ph on surfactant adsorption for chemical enhanced oil recovery: a review the micelle thermodynamics and mixed properties of sulfobetaine-type zwitterionic gemini surfactant with nonionic and anionic surfactants managing neonates with respiratory failure due to sars-cov-2-authors' reply the authors declare that there is no conflict of interest. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. adewale oluwaseun fadaka https://orcid. org/0000-0002-3952-2098 nicole remaliah samantha sibuyi https:// orcid.org/0000-0001-7175-5388 ashwil klein https://orcid.org/0000-0002-5606-886x key: cord-274480-aywdmj6o authors: song, wenfei; wang, ying; wang, nianshuang; wang, dongli; guo, jianying; fu, lili; shi, xuanling title: identification of residues on human receptor dpp4 critical for mers-cov binding and entry date: 2014-10-21 journal: virology doi: 10.1016/j.virol.2014.10.006 sha: doc_id: 274480 cord_uid: aywdmj6o middle east respiratory syndrome coronavirus (mers-cov) infects host cells through binding the receptor binding domain (rbd) on its spike glycoprotein to human receptor dipeptidyl peptidase 4 (hdpp4). here, we report identification of critical residues on hdpp4 for rbd binding and virus entry through analysis of a panel of hdpp4 mutants. based on the rbd–hdpp4 crystal structure we reported, the mutated residues were located at the interface between rbd and hdpp4, which potentially changed the polarity, hydrophobic or hydrophilic properties of hdpp4, thereby interfering or disrupting their interaction with rbd. using surface plasmon resonance (spr) binding analysis and pseudovirus infection assay, we showed that several residues in hdpp4–rbd binding interface were important on hdpp4–rbd binding and viral entry. these results provide atomic insights into the features of interactions between hdpp4 and mers-cov rbd, and also provide potential explanation for cellular and species tropism of mers-cov infection. middle east respiratory syndrome (mers), a novel coronavirus which causes severe respiratory illness, was first reported in a patient from saudi arabia in 2012 (de groot et al., 2013) . to date, individual cases as well as small clusters and large outbreaks have been reported in several countries and the mortality rate is estimated at 30% among laboratory-confirmed cases (organization, 2014) . phylogenetic analysis demonstrates that the mers coronavirus (mers-cov) is genetically closest to clade 2c betacoronavirus found in camels and insectivorous bats (ithete et al., 2013) although the true viral reservoir remains uncertain. the clinical symptoms caused by mers-cov are similar to those caused by severe acute respiratory syndrome coronavirus (sars-cov) although the two viruses use two distinct receptors; mers-cov uses dipeptidyl peptidase 4 (dpp4) while sars-cov uses angiotensin-converting enzyme 2 (ace2). other coronaviruses use other receptors and perhaps this provides partial explanation for their cellular and species tropism. mers-cov can replicate in a range of cell lines derived from human, non-human primate, porcine, and bat (de wit et al., 2013) . traditional small laboratory animals, such as mice (coleman et al., 2014) , hamsters (de wit et al., 2013) , and ferrets (raj et al., 2014) , were shown to resist mers-cov infection. the finite host range of mers-cov has seriously restricted the development of appropriate animal models to study the pathogenesis of this virus and to assess the efficacy of potential therapeutic strategies. raj et al. (2014) demonstrated that human receptor dpp4 (hdpp4) domain (residues 246 to 505) could confer the susceptibility of ferret dpp4 to mers-cov infection. zhao et al. (2014) are the first to describe a method of developing a small-animal model for mers-cov in which an adenovirus expressing hdpp4 was utilized to transiently transduce mouse airway cells and make mice susceptible to mers-cov infection. recently van doremalen et al. (2014) showed that dpp4 played an important role in the observed species tropism of mers-cov infection and identified residues in dpp4 responsible for this restriction. these results indicate that the insusceptibility to infection is primarily determined by the inability of mers-cov binding to dpp4 of a non-permissive cell line. previous findings have shown that hdpp4 extracellular domain consists of a variable n-terminal eight-blade β-propeller domain and a conserved c-terminal α/β-hydrolase domain (engel et al., 2003; rasmussen et al., 2003) . however, our understanding of critical residues of hdpp4 on mers-cov interaction and entry is quite limited. we and others have previously characterized rbd-hdpp4 crystal structure lu et al., 2013; wang et al., 2013) . the rbd-hdpp4 crystal structure showed that the viral rbd recognized blades iv and v of the dpp4 β-propeller domain. the atomic interaction details of the binding interface revealed that the rbd receptor recognition was predominantly mediated by several amino-acid residue interactions, including rbd residue d539 with dpp4 residue k267, rbd y499 with dpp4 r336, rbd residues d510 and e513 with dpp4 residues r317 and q344, rbd l506, w553 and v555 with dpp4 l294 and i295. previously, we have generated a panel of mers-cov mutant rbd proteins at the residues d539, y499, d510, e513, l506, w553 and v555 to characterize their impacts on binding activity to hdpp4 and the entry efficiency into target cells. however, the impacts of the corresponding residues on hdpp4 have not been well characterized. here, through structure-guided mutagenesis, we identified several key residues in hdpp4 that were critical for rbd binding measured by both real-time surface plasmon resonance (spr) and pseudovirus entry. these residues included k267 and r336 on binding patch 1, and l294, i295, r317 and q344 on binding patch 2. the mutations of three positively charged residues k267, r336 and r317 perhaps interfere with the interaction of the negatively charged residues on the surface of rbd; the mutations of l284, i295 and q344 may lead to the change of hydrophobic or hydrophilic properties of hdpp4 at the interface with rbd. our previous findings have shown that the binding interface between hdpp4 and mers-cov rbd is mainly composed of two binding patches, patch 1 and patch 2 (fig. 1a) . the patch 1 interface is characterized by interactions between c-terminal end of the long linker connecting the rbd β6/β7 strands and the hdpp4 blade 4. the contact in patch 1 is critically determined by the polar interactions among a group of hydrophilic amino-acid residues, including rbd e536, d537, d539 and y499 and hdpp4 k267 and r336. in this patch, dpp4 residue k267 interacts with rbd d539 by salt bridge (fig. 1b) , while dpp4 residue r336 forms hydrogen bond with rbd residue y499 (fig. 1c) . patch 2 has a hydrophobic core surrounded by a hydrophilic periphery. in the hydrophobic core, rbd and hdpp4 contacts are critically dependent on a few 'hot spot' residues including rbd l506, w553 and v555, and dpp4 l294 and i295. however, the surrounding hydrophilic surface consists of rbd residues d510, e513 and y540, and dpp4 residues h298, r317 and q344. among these hydrophilic residues, the salt bridge and hydrogen bond between d510 and r317, e513 and q344 contribute to the maintenance of rbd-receptor contact (fig. 1d) . to study the impacts of the substitutions of the critical residues on hdpp4 described above on the interaction between mers-cov rbd and hddp4, we determined the binding efficiency between these two proteins by employing spr technique. first, we constructed a series of hdpp4 mutants guided by the rbd-hdpp4 complex crystal structure information . the wide-type and mutant hdpp4 were introduced into baculovirus expression system. all wide-type and mutant forms of hdpp4 were expressed efficiently (data not shown). second, the binding efficiency was measured by spr. as shown in fig. 2 and table 1 , mutations at several hdpp4 residues, in individual or combination, resulted in a significant attenuation in binding to mers-cov rbd. in patch 1, residue k267 mutation (k267a and k267e) presumably damaged the salt-bridge interaction, completely abrogated the binding between hdpp4 and rbd, while r336a reduced rbd and hdpp4 binding about 100 fold. in patch 2, double mutations at l294 and i295 (l294a þ i295a and l294d þi295d) completely eliminated the binding between rbd and hdpp4, presumably by disrupting hydrophobic interactions with rbd l506, w553 and v555. in contrast, the single-residue substitution of r317a and q344a in the hydrophilic surface of patch 2 had negligible effect on binding efficiency. to further study the importance of the critical residues on hdpp4 on viral entry, we measured the entry efficiency of pseudovirus into cos7 cells expressing the wide-type and mutant forms of hdpp4. the expression levels of the wide-type and mutant hdpp4 were analyzed by fluorescence-activated cell sorting (facs) using goat anti-hdpp4 polyclonal antibody. all of the wide-type and mutant hdpp4 proteins could be expressed on the surface of cos7 cells with the similar expression efficiency (fig. 3a) . forty-eight hours later, these cells were exposed to pseudovirus infection and their entry efficiency was measured by luciferase activity 48 h later. as showed in fig. 3b , the residue mutations located at patch 1 (k267a, k267e and r336a) and hydrophobic region of patch 2 (l294a þi295a and l294aþ i295d) fig. 1 . the amino-acid residue interaction details at the binding interface. (a) two patches of the binding interface. patch 1 interface is characterized by interactions between the c-terminal end of the long linker connecting the rbd β6/β7 strands (light magenta) and the hdpp4 blade 4 (cyan). in patch 2, a gently concaved outer surface in rbd (light magenta) accommodates a linker containing a short α helix between hdpp4 blades 4 and 5 (cyan). (b) and (c) hydrophilic residues of rbd and hdpp4 interact through polar contacts in patch 1. rbd d539 has salt-bridge interaction with hdpp4 residue k267 (b). dpp4 residue r336 forms hydrogen bond with rbd residue y499 (c). the polar contacts (salt-bridge and hydrogen bond) are drawn as black dashed sticks. (d) hot spot residues in the hydrophobic core and hydrophilic periphery of patch 2. resulted in significantly reduction in viral entry. this is consistent with the binding results described previously. in the hydrophilic region of patch 2, residue substitution r317 led to partial loss of viral infection (41.4%), while the mutation q344 modestly increased viral infection (22.8%). in summary, we have identified several key residues in hdpp4 critical for viral binding and entry into target cells. these residues include positively charged residues of patch 1 (k267 and r336) and hydrophobic zone of patch 2 (l294 and i295). in contrast, the mutations at hydrophilic zone of patch 2 (r317 and q344) had little influence on binding and virus entry efficiency. these results showed that the positively charged residues at the outer surface of blade 4 and the hydrophobic regions of blade 5 may play an important role in mediating viral binding and entry into the target cells, while the impact of mutations at hydrophilic region of patch 2 was barely detectable. this is consistent with our earlier findings where residue mutations at the corresponding negatively charged and hydrophobic core positions on rbd of mers-cov could significantly reduce both binding and viral entry efficiency. sequence analysis of dpp4 from multiple animal species (fig. 4 ) showed that mers-cov susceptible animals, such as macaque, camel and bat, shared the same sequence with hdpp4 at blades iv and v. in contrast, those mers-cov resistant animals, such as mouse, rat and ferret, have residues at l294, i295 and r366 that are all different from hdpp4. raj et al. (2014) reported that when these sites of hdpp4 were changed to the residues of ferret, the binding and viral infection efficiency could also be decreased. van doremalen et al. (2014) found 5 residues involved in the hdpp4-rbd interaction which were important to determine the susceptibility to mers-cov infection, in which i295 and r336 were included. these results are consistent with our findings and suggest these residues play an important role in rbd binding and viral entry, and determining the tropism to mers-cov infection. mers-cov rbd (residues 367-606) and the extracellular domain of hdpp4 (residues 39-766) were expressed using a bacto-bacs baculovirus expression system (invitrogen). in brief, the dna encoding rbd and hdpp4 were respectively cloned into the pfastbac™ dual vector (invitrogen) incorporating an n-terminal gp67 signal peptide to facilitate secretion and a c-terminal hexa histidine-tag for purification. the constructed dna was then transformed into the bacterial dh10bac competent cells and the recombined bacmid dna was extracted and transfected into sf9 cells using cellfectin ii reagent (invitrogen). after 5-7 days of incubation at 300 k, the low-titer viruses were harvested and then amplified. the amplified high-titer viruses were then used to infect sf9 cells and the cell culture supernatant containing target protein was harvested 60 h after infection, concentrated, loaded to nickel (ni)-charged resin (ge healthcare), and eluted with 0.5 m imidazole and further purified using the superdex™ 200 highperformance column (ge healthcare) pre-equilibrated with tris buffer (50 mm tris, ph 8.8, 40 mm nacl). fractions containing the purified protein were collected and applied directly to a preequilibrated resource™ q column (ge healthcare) and then eluted with a 0.05-1 m nacl gradient in 40 mm tris buffer (ph 8.8). fractions containing protein were finally purified using super-dex™ 200 column pre-equilibrated with hbs (10 mm hepes, ph 7.2, 150 mm nacl) and centrifuged to 1 mg/ml. mutants of the extracellular domain of hdpp4 were constructed using a standard pcr-based cloning strategy. and the mutant proteins were expressed and purified in the same way. the spr analyses were carried out using a biacore t200 instrument (ge healthcare) equipped with a research-grade cm5 sensor chip. to measure the affinity binding between rbd and wide-type or mutant hdpp4, the rbd was immobilized on the sensor chip by standard amine coupling procedure. the flow cell 1 was left blank to serve as a reference. purified rbd at a concentration of 5 μg/ml in sodium acetate buffer (10 mm, ph 5.0) was immobilized to a density of 300-400 response units on the flow cell 2. for the collection of binding data, hdpp4 or its mutants in a buffer of 10 mm hepes, ph 7.2, 150 mm nacl, and 0.005% (v/v) tween-20 were injected over the two flow cells at a series of concentration at a 30 μl/min flow rate and 298 k. the rbd-hdpp4 complex was allowed to associate for 60 s and dissociated for 60 s. the surfaces were regenerated with an injection of 5 mm naoh between each cycle if needed. the data was analyzed with the biacore t200 evaluation software by fitting to a 1:1 langmuir binding model. mers-cov pseudovirus was generated by co-transfection of human immunodeficiency virus (hiv) backbone expressing firefly luciferase (pnl43r-e-luciferase) and mers-cov spike glycoprotein expression vector (pcdna3.1 þ , invitrogen) into the 293 t cells. viral supernatants were harvested 48 h later, normalized by p24 elisa kit (beijing quantobio biotechnology co., ltd, china) before infecting the target cos7 cells transiently expressing wide-type or mutant hdpp4. the wide-type and mutant hdpp4 expressing cos7 cells were incubated with goat anti-hdpp4 polyclonal antibody (r&d) followed by incubation with fluorescein phycoerythrin (pe)labeled rabbit anti-goat igg antibody (santa cruz). the expression levels of wide-type and mutant hdpp4 were measured by flow cytometer (bd aria ii) and the mean fluorescence intensity (mfi) was analyzed. the cos7 cells infected by mers-cov pseudovirus were lysed at 48 h post infection and viral entry efficiency was quantified by comparing the luciferase activity between pseudoviruses-infected cos7 cells expressing wide-type and those infected cos7 cells expressing mutant hdpp4. the entry efficiency (%) of pseudovirus was calculated on the basis of luciferase activity. and the percentages of pseudovirus entry efficiency shown for mutant hdpp4 were luciferase activity values versus that of the wide-type hdpp4, as the entry efficiency for wide-type hdpp4 was defined as 100%. data shown were corrected for the expression of different hdpp4 constructs by the parameter of mfi. error bars represent standard errors of the means of three independent experiments. student's t-test; n po 0.05; nn po 0.01. crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus wild-wide-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the crystal structure of dipeptidyl peptidase iv (cd26) reveals its functional regulation and enzymatic mechanism close relative of human middle east respiratory syndrome coronavirus in bat molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 middle east respiratory syndrome coronavirus (mers-cov) -update. world health organization adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus crystal structure of human dipeptidyl peptidase iv/cd26 in complex with a substrate analog host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 rapid generation of a mouse model for middle east respiratory syndrome we thank drs linqi zhang and xinquan wang for their kind support and helpful suggestions. this work was supported by national natural science fund 81101236, 81471929, ministry of science and technology of china (2014cb542500), the national science and technology major projects (2012zx10001-004). key: cord-265380-2gs34xcw authors: leist, sarah r.; cockrell, adam s. title: genetically engineering a susceptible mouse model for mers-cov-induced acute respiratory distress syndrome date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_12 sha: doc_id: 265380 cord_uid: 2gs34xcw since 2012, monthly cases of middle east respiratory syndrome coronavirus (mers-cov) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. the ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with mers-cov in human infections. the inability of mers-cov to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. identification of human dipeptidyl peptidase iv (hdpp4) as the receptor for mers-cov infection opened the door for genetic engineering of mice. precise molecular engineering of mouse dpp4 (mdpp4) with clustered regularly interspaced short palindromic repeats (crispr)/cas9 technology maintained inherent expression profiles, and limited mers-cov susceptibility to tissues that naturally express mdpp4, notably the lower respiratory tract wherein mers-cov elicits severe pulmonary pathology. here, we describe the generation of the 288–330(+/+) mers-cov mouse model in which mice were made susceptible to mers-cov by modifying two amino acids on mdpp4 (a288 and t330), and the use of adaptive evolution to generate novel mers-cov isolates that cause fatal respiratory disease. the 288–330(+/+) mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for mers-cov. the chapter starts with a historical perspective on the emergence of mers-cov and animal models evaluated for mers-cov pathogenesis, and then outlines the development of the 288–330(+/+) mouse model, assays for assessing a mers-cov pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice. in february of 2018 mers-cov was listed as a priority on the r&d blueprint for the global strategy and preparedness plan outlined by the world health organization (who) [1] . the r&d blueprint includes viruses that pose a global public health risk, and for which there are no available therapeutic countermeasures [1] . twenty-seven countries have reported cases of mers-cov with most cases confined to the arabian peninsula. diagnosed cases of mers-cov in countries outside the arabian peninsula are primarily traveler associated. the potential for global spread of mers-cov was realized in 2015 when a single traveler returning to south korea initiated an outbreak that infected 186 people resulting in 20% fatality and caused widespread fear that crippled the economy for nearly 6 months [2] [3] [4] . human-to-human transmission is often associated with close contact in the health care setting, but can also occur between family members within a household [5] . asymptomatic individuals pose a particular risk of transmission due to their unknown carrier status as demonstrated in the health care setting [6] . despite the high percent of fatalities associated with mers-cov outbreaks on the arabian peninsula most epidemiological studies suggest r 0 values <1, indicative of a low risk of sustainable human-to-human transmission, whereas epidemiological studies from the south korean outbreak describe r 0 values (>1) akin to more sustainable human-to-human transmission [7] . recurring spillover events from dromedary camels (zoonotic reservoir for mers-cov on the arabian peninsula) likely contribute to newly diagnosed cases in humans [8] [9] [10] . the potential for continuous reintroduction to humans increases the risk of mers-cov adapting in humans to acquire enhanced human-tohuman transmission profiles, a scenario suspected to have initiated the sars-cov pandemic in 2002-2003 [11] . effective public health measures and culling of civet cats, the zoonotic host for sars-cov, brought the sars-cov pandemic to a rapid end [11] . eliminating mers-cov through culling of infected camel herds is not a practical solution. furthermore, detection of pre-emergent mers-cov-like, and sars-cov-like, strains circulating in bat species indicate that the natural environment is ripe for future human exposures to potentially pathogenic coronaviruses [12] [13] [14] . therefore, the development of therapeutic countermeasures that can interfere with mers-cov pathogenesis is critical to break zoonotic-to-human and human-to-human transmission cycles that may instigate global spread. evaluating the toxicity and efficacy of novel mers-cov therapeutics require the availability of animal models that effectively recapitulate mers-cov pathogenesis during fatal cases of human infections. therefore, the first question in generating a mers-cov animal model would be: what are the pathological features of a human infection? limited histopathological findings from human autopsies indicate that fatal cases of mers-cov results from pneumonia initiated by infection of bronchiolar and alveolar epithelia of the lower respiratory tract (lrt) [15, 16] . pneumonia in the lrt is also the prominent finding on radiographs from x-rays and cts of diagnosed human cases [17] . high viral loads in tracheal aspirates from patients are also associated with severe pulmonary disease [18] , which is indicative of actively replicating mers-cov in the lrt. initial evaluation of the human mers-cov emc/2012 isolate in rhesus macaques demonstrated replication in the lrt with mild pneumonia-like disease ( fig. 1 ) [28] . achieving respiratory pathology reflecting a lethal human disease proved to be more complicated in nonhuman primates. severe respiratory disease in the marmoset produced clinical endpoints consistent with fatal disease that required euthanasia ( fig. 1) [29, 30] . evaluation of two human isolates, jordan and emc/2012, and a tissue cultureadapted mers-cov strain (mers-0) in nonhuman primates resulted in mild disease in rhesus macaques or marmosets ( fig. 1 ) [31] [32] [33] , confounding the reproducibility of near-lethal disease in nhps. nonhuman primates are central to late-stage preclinical evaluation of therapeutic countermeasures, but may be impractical for initial preclinical studies. a small animal model may be applicable if there is limited therapeutic available for toxicity and efficacy testing, especially if large animal numbers are needed to determine confidence and reproducibility. early studies in mouse, hamster, and ferret revealed that conventional small animal models were fully resistant to mers-cov infection and replication (fig. 1) [19, 20, 36] . a seminal study identifying the mers-cov receptor as human dipeptidyl peptidase iv (hdpp4) [49] , and publication of the crystal structure of hdpp4 interacting with the receptor binding domain (rbd) of the mers-cov spike protein [50] , exposed tropism determinants critical for susceptibility. dipeptidyl peptidase iv contact amino acids at the hdpp4/rbd interface are highly conserved among mers-cov-susceptible mammalian species (human, camel, and specific events since the emergence of mers-cov in 2012 are emphasized above the timeline. references to mammalian models evaluated for mers-cov pathogenesis comprise hamster [19] , ferret [20] , rabbit [21] [22] [23] [24] , camel [25] [26] [27] , nonhuman primates [28] [29] [30] [31] [32] [33] [34] [35] , and mouse [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] bat) (fig. 2) [51] . although mouse, hamster, ferret, and guinea pig dpp4 orthologs exhibit high overall similarity to hdpp4, specific amino acid differences at the dpp4/rbd interface account for the inability of these species to support infection [51] [52] [53] [54] [55] [56] . overexpression of a mouse dpp4 (mdpp4) with changes in the contact residues at the dpp4/rbd altered cellular profiles from resistant to susceptible to mers-cov infection [52, 53, 56] . the dependence on dpp4-specific contact was further substantiated by similar studies evaluating modified dpp4 orthologs from the hamster, ferret, and guinea pig [55] . dipeptidyl peptidase iv was identified as the major determinant of mers-cov tropism. researchers rapidly leveraged knowledge of the dpp4 receptor to generate susceptible small animal models ( fig. 1 ) [12] . zhao et al. utilized a unique approach for producing susceptible mice that could replicate human isolates of mers-cov in the lungs by infecting mouse lungs with an adenovirus that constitutively expresses the full-length hdpp4 gene ( fig. 1) [37] . transient expression of hdpp4 supported infection and replication with human strains of mers-cov in the lungs and indicated that this technology may be an effective rapid response platform for initial (c) zoomed-in view of the human dpp4 structure (light gray) with highlighted mers-cov rbd contact residues (dark gray). species-specific contact residues that differ from human are highlighted in red evaluation of emergent and pre-emergent viruses. however, pathology associated with a fatal mers-cov infection was not observed in the ad-hdpp4 model [37] , which limited the capacity to evaluate the efficacy of therapeutic countermeasures. genetic engineering of mice would be necessary to develop preclinical mers-cov mouse models with respiratory phenotypes that reflected clinical outcomes in patients. knock-in of full-length hdpp4 rendered mice susceptible to human isolates of mers-cov at low infection doses ( fig. 1 ) [38] [39] [40] . knock-in mice exhibited severe pulmonary pathology and increased mortality; however, widespread constitutive expression of full-length hdpp4 resulted in high levels of mers-cov infection and replication in extrapulmonary tissues [38] [39] [40] . in some studies, higher viral loads could be detected in the brain compared to the lungs [39, 40] . mice with infections of the central nervous system (cns) exhibited encephalitis that corresponded with the kinetics of mortality [39] . currently, there is no evidence to support a cns component associated with mers-cov pathogenesis in humans. attempts to restrict hdpp4 expression to epithelial cells of the lungs using constitutive tissue specific promoters (e.g., cytokeratin k18) yielded outcomes similar to those observed with sars-cov mouse models, wherein high levels of mers-cov infection/replication were detected in the brains (fig. 1 ) [39] . to circumvent confounding problems associated with global bio-distribution of overexpressed hdpp4 receptor, researchers engineered mouse models using sophisticated molecular approaches. pascal et al. employed regeneron's velocigene technology to replace sequences encoding nearly the entire mdpp4 genomic region with those encoding the exons/introns from the hdpp4 genetic region ( fig. 1 ) [41] . retaining the mdpp4 5 0 and 3 0 genetic elements that regulate expression maintained inherent expression profiles of full-length hdpp4 in mice [41] . importantly, mers-cov infection/replication was readily detected in the lungs with little involvement of extrapulmonary tissues [41] . infection with human isolates of mers-cov caused moderate respiratory pathology with mortality determined by euthanasia of mice at 20% weight loss [41] . unfortunately, commercial restrictions limit the availability and use of this model to the broader scientific community. in addition to the concerns raised above, the first generation of mouse models was developed with the full-length hdpp4, which may alter the inherent physiological properties of the mouse. the multifaceted involvement of dpp4 in maintaining immune homeostasis is of significant importance regarding susceptibility to infectious disease [57] . dpp4 exists in two forms: (1) a membrane anchored form on the surface of multiple cells types (e.g., b cells, t cells, nk cells, and epithelial cells to mention a few) and (2) a secreted form that can be identified in human serum [57] . dpp4 interacts with and modifies heterologous protein molecules involved in nociception, neuroendocrine function, metabolism, cardiovascular function, immune regulation, and infection [57] . modification of heterologous protein function can proceed through cleavage of n-terminal amino acids through the enzymatic activity of the α/b-hydroxylase domain, or allosteric interaction/ signal transduction [57] . the species specificity of dpp4 is exemplified by the interaction of hdpp4 with adenosine deaminase (ada), a well-recognized binding partner of hdpp4, which modulates downstream t cell functions [58] [59] [60] . the hdpp4/ada interaction evolved in higher mammalian species (human, nhp, bovine, rabbit), but not in mouse or rat [58] [59] [60] . interestingly, in one study ada was demonstrated to block infection of mers-cov in tissue culture [20] , indicating that the binding site on hdpp4 for ada, and the mers-cov rbd, may overlap. consequently, introducing full-length hdpp4 into mice may skew innate immune mechanisms that could influence responses to therapeutic countermeasures. in the second generation of mers-cov-susceptible mouse models amino acid residues predicted to function at the mdpp4/ mers-cov rbd interface were modified to avoid the introduction of full-length hdpp4 ( fig. 1) [42, 43] . li et al. recently developed a mouse model wherein the mdpp4 genomic region encompassing exons 10-12 were replaced with the respective genomic region from hdpp4, referred to as an hdpp4 knock-in model (hdpp4-ki) [43]. exons 10-12 encode contact amino acids at the hdpp4/mers-cov rbd interface that were able to support replication of human mers-cov isolates in the lungs, but did not elicit a mortality phenotype [43] . adaptive evolution of human mers-cov in the hdpp4-ki mouse resulted in mouseadapted viruses that evoked a lethal respiratory phenotype with little involvement of extrapulmonary tissues. the lethal respiratory phenotype is a consequence of novel mutations acquired during adaptive evolution. a combination of mutations in both the s1 and s2 regions of the mers-cov spike protein facilitated a lethal respiratory phenotype [43] . results in the hdpp4-ki model substantiate an earlier mouse model referred to as the 288-330 +/+ model, which was designed with only two amino acid changes in mdpp4 to generate mers-cov susceptible mice. genetic engineering and implementation of the 288-330 +/+ mouse model, combined with mers-cov adaptive evolution, is the subject of this chapter. initial studies in tissue culture revealed that human and rodent cell types were resistant to mers-cov infection upon overexpression of mdpp4; however, overexpression of hdpp4 conferred permissivity to infection/replication [53] . comparative structural modeling of hdpp4 and mdpp4 revealed putative contact residues in mdpp4 amenable to modification at the dpp4/rbd interface. modification of two amino acids (a288l and t330r) was sufficient to endow mdpp4 with the capacity to mediate mers-cov infection/replication [53] . shortly after the emergence of mers-cov into humans in 2012, the crispr/cas9 genome editing technology became available for applications to modify mammalian genomes in vitro and in vivo [61] [62] [63] . recognizing our unique situation, we designed crispr/cas9 targets to modify the mouse genome encoding amino acids a288 and t330 in exons 10 and 11 of the mdpp4 gene (fig. 3) [12, 42] . concomitant with mouse development, in vitro studies were initiated to adapt mers-cov to the modified mdpp4 [42] . tissue culture adaption resulted in mers-0 virus, which contained an rmr insertion and s885l mutation in the s2 region of the mers-cov spike protein [42] . a mers-0 molecular clone exhibited enhanced replication kinetics and higher titers compared to human mers-cov isolates. additionally, the mers-0 virus replicated to higher levels in the lungs of 288-330 +/+ mice, compared to human and camel mers-cov isolates [42] . based on these data the mers-0 virus was used to initiate passaging in mice heterozygous for mdpp4 with a288l and t330r mutations, 288-330 +/à (fig. 4) . we reasoned that adaptation around one expressed copy of the mdpp4 with 288-330 mutations, and a wild-type mdpp4 expressed copy, might cultivate generation of a mouse-adapted mers-cov that could utilize wild-type mdpp4 as the primary receptor. after 15 passages we obtained a mouse-adapted mers-cov (mers15c2) exhibiting a lethal respiratory phenotype in the 288-330 +/+ mice [42] . our mers-cov reverse genetic system was used to generate an infectious clone of the mouse-adapted virus, icmersma1 [42] . lethal respiratory pathology with icmersma1 required high infectious doses (5 â 10 6 pfu). an additional 20 passages of icmersma1 in 288-330 +/à mice bore a novel mouse-adapted mers-cov that produced lethal respiratory disease at doses of 5 â 10 5 pfu, and lung pathology associated with severe respiratory disease at 5 â 10 4 to 5 â 10 5 pfu [44] (fig. 1) . this mers-cov model system (288-330 +/+ mice and mouseadapted mers-cov viruses) is now being employed to: (1) understand complex virus-host interactions [12, 31, 42, [64] [65] [66] [67] , (2) evaluate antibody-based therapeutics [42] , (3) evaluate drug-based therapeutic countermeasures [68] , and (4) evaluate anti-mers-cov vaccines [42, 66] . the goal of this chapter is to provide an outline of how to rationally design a mouse with altered susceptibility to mers-cov. for additional information there are a number of detailed reviews and book chapters describing the design and utilization of the crispr/cas9 technology for generating mouse models [69, 70] . . agarose gel separation based on size allows for discrimination between target dna, cas9 digested targets, and guide rnas. (b) schematic utilizing crispr/cas9 technology to genetically engineer mice. fertilized c57bl/6 j zygotes are collected and injected with rna encoding cas9, dpp4 single guide rna, and oligos to facilitate homology-directed repair (hdr). microinjected zygotes are implanted into pseudopregnant recipient female c57bl/6 j mice. offspring are screened by sequencing for the intended change at positions 288 and 330. mice identified as having the appropriate changes are backcrossed to c57bl/6 j mice to maintain the pure c57bl/6 j background, or may be crossed to any desired strain (e.g., balb/cj or 129s1/svimj). (c) table describing sequences of cas9 guide rnas and oligos for hdr to genetically engineer amino acid changes at position 288 (ala to leu) and 330 (thr to arg). (d) sequencing chromatograms highlighting how the f0 offspring from embryo implantation can be a mosaic of insertion/ deletions (indel's) generated by random non-homologous end joining from cas9 cutting at the genomic alleles, and the hdr repair that incorporates the intended changes encoding amino acids at positions 288 and 330. pure homozygous 288-330 +/+ lines were obtained by backcrossing onto c57bl/6 j mice. the highlighted mutations caa (ttg in the reverse orientation) and aga encode the novel 288 l and 330r amino acids 9. after a predetermined number of passages the region encoding the spike protein of mers-cov is sequenced using rt-pcr to amplify the region of interest followed by standard sanger sequencing (see note 6). 10. after 10 passages viruses were plaque purified by diluting the heterogenous stock of virus 10 à1 to 10 à6 , and infecting a monolayer of vero ccl81 cells similar to a standard plaque assay. 11. single plaques are isolated using a pipet tip and the virus expanded on a freshly seeded monolayer of vero ccl81 cells. 12. virus stocks are generated, viral rna is isolated using standard trizol purification, and the region encoding the mers-cov spike protein is amplified by standard rt-pcr techniques and sequenced using standard sanger sequencing. 13 . mutations identified by sequencing must be confirmed using a reverse genetic system to generate an infectious clone encoding the identified mutations [72] . cockrell (fig. 3) the details for generating and using the crispr/cas9 system to generate mutations are outlined in the materials and methods by cockrell et al. [42] . notably, the 288-330 +/+ mice were initially generated in the animal models core facility at the university of north carolina at chapel hill. the extensive technical expertise required for genetic engineering of mice is the subject of many expert reviews and book chapters that will not be covered here. nevertheless, we provide a conceptual overview of the steps to generate the 288-330 +/+ mice. 1. design guide rnas to target each of the a288 and t330 alleles. cockrell et al. designed the guide rnas to direct the cas9 to cut as near the mutation site as possible (fig. 3 ) (see note 7 for helpful resources to design and genetically engineer mouse knockouts). 2. test guide rnas in vitro for the capacity to cut a target sequence (fig. 3) . (a) generate double-stranded odns or a plasmid containing the target sequence with the correct pam site. (b) assemble ribonucleoprotein (rnp) complexes according to manufacturer's instructions (see note 8). (c) subject double-stranded dna with target sequence to rnp complexes and assess digestion pattern on an agarose gel (fig. 3 ). 3. two separate oligos are also designed to introduce the novel mutations on exons 10 (a288l) and 11 (t330r) of mdpp4, through homology-directed repair (fig. 3 ). 4. fertilized zygotes are collected from c57bl/6j female mice that were superovulated and mated to male c57bl/6j mice. cas9 endonuclease, combined with odns encoding the replacement alleles for 288 and 330 in mdpp4, were all pronuclear injected into the fertilized zygote [42] (fig. 3) . the fertilized zygotes were from c57bl/6j mice. 6. the injected embryos were implanted into pseudopregnant recipient females. 7. newly born pups were screened for the presence of the correct change at the 288 and 330 alleles by standard pcr amplification and sanger sequencing (fig. 3 ) (see note 9). 3. 288-330 +/+ mice are brought into the bsl3 laboratory 7 days before start of the experiment to allow for environmental acclimation. 4. mice are anesthetized via intraperitoneal injection of 50-100 μl of a ketamine (50 mg/kg)/xylazine (15 mg/kg) mixture (see note 10). level of anesthesia is assessed by pedal reflex. 6. measure initial weight (day 0 weight) while waiting for mouse to be anesthetized (see note 11). 7. once a pedal reflex is no longer triggered, mice should be immediately infected by the intranasal route. holding the animal vertically, apply 50 μl of virus solution by pipetting onto their nostrils and allow them to inhale. to ensure that all of the 50 μl reach the lower respiratory tract hold the mouse upright for an additional 30 s (see note 12). 8. note any inconsistencies during infection, including: (1) presence of bubbles of inoculum from nasal cavity, (2) occurrence of inoculum in mouth, or (3) failure to inhale entire dose of inoculum. notes will help to explain potential inconsistencies in readout parameters and may be used as exclusion criteria for inefficient infections. 9. mice are put back into the cage and placed on their back to ensure virus solution will stay in the lungs. note: cages are returned to cage rack, but the respiration of mice is continuously monitored by observing breathing. 10. place mice next to each other to keep body temperature as close to normal as possible. 11. check cage after 30-45 min to confirm that all mice wake up from anesthesia and infection. adaptation of mers-0 in 288-330 +/à mice (fig. 4) 1. mouse adaptation was initiated in heterozygous 288-330 +/à mice by infecting with 50 μl of the mers-0 infectious clone (see note 13). 2. at 3 days post-infection the mouse is euthanized by extended exposure to isoflurane.~2 ml of isoflurane is added to the bottom of a jar that can be firmly sealed (see note 14). a thoracotomy is performed to expose the lungs. 4. lungs are removed and placed in a 2 ml gasket sealed skirted screw cap tubes. tubes are previously prepared with 1 ml of 1â pbs containing~5-10 mm of glass beads. lungs are homogenized for 60 s in a bead homogenizer. 6. lung lysates are centrifuged in a microcentrifuge for 5 min at max speed to pellet debris. 7. this is considered passage 1 (p1) and 50 μl of lung homogenate is used to infect a naïve 288-330 +/à mouse. 8. the process is repeated for a desired number of cycles. 1. after infection mice are monitored daily for weight loss for the entire duration of the experiment. 2. to record daily weights, pick up mice by the tail, identify by ear notch, and place into cup on a scale. record weight and calculate percentage of starting body weight (see note 15). 3. mice can also be monitored to determine if they are moribund using a clinical scoring scale whereby: 0 ¼ no clinical signs; 1 ¼ ruffled fur; 2 ¼ ruffled fur with hunched posture (only slight with no signs of dehydration); 3 ¼ as defined in number 2 with more severe signs of dehydration and some loss of body strength; 4 ¼ pronounced dehydration and prominent loss of mobility; 5 ¼ unresponsive to stimuli and prominent eye squinting. 4. it is important to note that weight loss might not always be the most appropriate parameter and animals should be euthanized at the discretion of the researcher even if animals have not reached 80% of their starting weight. mice that approach 80% of their starting body weight (20% weight loss) are euthanized via isoflurane overdose followed by a secondary euthanasia method (thoracotomy or cervical dislocation). depending on the experimental circumstances an institutionally approved exception may be implemented to allow continuation of the experiment (increasing the frequency with which the mice are monitored will likely need to be implemented) (see note 16). 3. respiratory function can be performed every day over the entire course of infection, or on single selected days. investigating a novel respiratory virus may require the investigator to perform a time course to determine the most effective time points for measurement. the largest differences between groups typically correlate with peak viral replication. 4. at each time point measured the mice need to be randomized into different chambers to avoid technical artifacts (e.g., a mouse measured at day 1 in chamber 1 should be evaluated in a different chamber on day 2 measurement). practical considerations dictate that 8-12 animals can be measured at any one time, and each group of 8-12 mice may take an hour for a proficient technician. therefore, experiments should be carefully planned to limit the number of mice to be evaluated. 5 . mice that are difficult to handle can be slightly anesthetized by applying isoflurane to the chamber in order to remove them from the chamber and return to their cage (see note 18). 6. open thorax, paying attention not to cut into lung tissue. 7. assess lung tissue for reddish discoloration and record severity by applying a number 0 (no hemorrhaging) to 4 (severe hemorrhaging in all lobes of the lungs). 8. harvest lung tissue and place in tubes prefilled with sample type specific solution. 9. put scissors and forceps first into cidecon to remove any residual blood and then into 70% ethanol in order to not crosscontaminate samples. 10. whole lung can be used for one assay or different lobes can be used for different assays. steps 1-6 from subheading 3.7. 2. cut into superior vena cava and collect blood with a pipette. 3. blood is typically transferred to a serum/plasma separation tube that allows for separation of serum/plasma from cells. 4. in the event that it is necessary to harvest cells for flow cytometry analysis or vetscan hm5 analysis, the blood sample can be transferred to a tube containing edta. note: vetscan hm5 is a veterinary diagnostic machine that analyzes basic immune cell counts and additional hematological parameters within 2 min. 5. all vetscan assays are performed under bsl3 conditions. removal of samples for downstream analysis outside of bsl3 conditions require specific inactivation procedures that must be pre-approved by the institutional biosafety committee. 3. this can also be achieved using a readily transfectable human embryonic fibroblast cell line such as hek293t cells and selecting for stably transfected cells. 4. cells can be counted by seeding an extra well, but it is safe to assume that the cell number is approximately doubled after 24 h. 5. this can also be achieved using a microscope to assess plaque size if plaques can be readily detected. 6. since the major determinant of mers-cov tropism is the spike protein, it would be anticipated that mutations having the most significant impact on infection might occur within the gene encoding the spike protein. 7. at the time that these mice were being generated in early 2014, crispr/cas9 reagents were not readily available. additionally, there were few bioinformatics tools available to facilitate guide rna design and off-target potential. in the current research environment crispr/cas9 reagents can be sourced from multiple commercial entities and there are a number of bioinformatics tools to assist with design. addgene is a nonprofit plasmid repository where crispr reagents and resources can be readily obtained (https://www.addgene. org/). additional guidance for generating mice using crispr/cas9 technology can be found in more comprehensive protocols [69, 70] . 8. all relevant reagents and protocols can now be obtained from commercial sources as readily synthesized rnas and purified proteins (e.g., integrated dna technologies). 9 . although a number of pups may be identified to have the correct mutation, many will likely be mosaic for random mutations including insertions/deletions due to the higher efficiency of non-homologous end joining (nhej) after cas9 digestion compared to the desired hdr employed to mediate allele modification. 10. the administered dose will depend on the weight of the animal which should be predetermined the day prior to initiating the experiment. 11. it is not necessary to anesthetize mice for measuring daily weights. 12. it is important that the inoculum reaches the lower respiratory tract for a successful mers-cov infection. 13 . mouse adaptation can be initiated with any mers-cov strain that exhibits some pulmonary replication. 14. mice should never come into contact with the isoflurane. to prevent direct contact, a layer of aluminum foil is placed at the bottom of the jar and this is covered with two additional layers of paper towel. 15 . the percent body weight is typically calculated after leaving the bsl3 environment. therefore, the weight sheets should have the anticipated weights of each animal at 20% weight loss. this will provide a real-time indication of when the mice are approaching the criteria established for humane euthanasia. 16. institutional approval is required for animals to be placed under exception. it cannot be emphasized enough that all animal work should be pre-approved by appropriate university iacuc and ibc committees and should be in accordance with the recommendations for the care and use of animals by the office of laboratory animal welfare at nih. 17. assessing respiratory function using plethysmography under bsl3 conditions requires costly equipment and extensive training prior to use. 18. anesthesia should be avoided prior to measuring lung function to prevent interference with lung function measurements. the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from economic impact of the 2015 mers outbreak on the republic of korea's tourism-related industries costly lessons from the 2015 middle east respiratory syndrome coronavirus outbreak in korea middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control mers transmission and risk factors: a systematic review high prevalence of mers-cov infection in camel workers in saudi arabia reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) severe acute respiratory syndrome modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice origin and evolution of pathogenic coronaviruses jumping species-a mechanism for coronavirus persistence and survival histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates middle east respiratory syndrome viral load kinetics of mers coronavirus infection the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus asymptomatic middle east respiratory syndrome coronavirus infection in rabbits enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection lack of middle east respiratory syndrome coronavirus transmission in rabbits bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (mers-cov) after experimental infection replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels pneumonia from human coronavirus in a macaque model treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset infection with mers-cov causes lethal pneumonia in the common marmoset a spike-modified middle east respiratory syndrome coronavirus (mers-cov) infectious clone elicits mild respiratory disease in infected rhesus macaques 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease elevated human dipeptidyl peptidase 4 expression reduces the susceptibility of hdpp4 transgenic mice to middle east respiratory syndrome coronavirus infection and disease cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with middle east respiratory syndrome coronavirus haagmans bl (2013) dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptorbinding domain complexed with human receptor dpp4 coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection glycosylation of mouse dpp4 plays a role in inhibiting middle east respiratory syndrome coronavirus infection permissivity of dipeptidyl peptidase 4 orthologs to middle east respiratory syndrome coronavirus is governed by glycosylation and other complex determinants host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system direct association of adenosine deaminase with a t cell activation antigen revisiting an old acquaintance: cd26 and its molecular mechanisms in t cell function crystal structure of cd26/ dipeptidyl-peptidase iv in complex with adenosine deaminase reveals a highly amphiphilic interface multiplex genome engineering using crispr/cas systems rna-guided human genome engineering via cas9 one-step generation of mice carrying reporter and conditional alleles by crispr/cas-mediated genome engineering the human sodium iodide symporter as a reporter gene for studying middle east respiratory syndrome coronavirus pathogenesis giving the genes a shuffle: using natural variation to understand host genetic contributions to viral infections middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis mers-cov accessory orfs play key role for infection and pathogenesis broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses generating genetically modified mice: a decision guide genome editing in mouse embryos with crispr/cas9 reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses new metrics for evaluating viral respiratory pathogenesis key: cord-014546-arw4saeh authors: janies, daniel a.; ford, colby; damodaran, lambodhar; witter, zachary title: spread of middle east respiratory coronavirus: genetic versus epidemiological data date: 2017-05-01 journal: online j public health inform doi: 10.5210/ojphi.v9i1.7581 sha: doc_id: 14546 cord_uid: arw4saeh nan mers-cov was discovered in 2012 in the middle east and human cases around the world have been carefully reported by the who. mers-cov virus is a novel betacoronavirus closely related to a virus (neocov) hosted by a bat, neoromicia capensis. mers-cov infects humans and camels. in 2015, mers-cov spread from the middle east to south korea which sustained an outbreak. thus, it is clear that the virus can spread among humans in areas in which camels are not husbanded. we calculated a phylogenetic tree from 100 genomic sequences of mers-cov hosted by humans and camels using neocov as the outgroup. in order to evaluate the relative order and significance of geographic places in spread of the virus, we generated a transmission graph (figure 1) based on methods described in 1. the graph indicates places as nodes and transmission events as edges. transmission direction and frequency are depicted with directed and weighted edges. betweenness centrality, represented by node size, measures the number of shortest paths from all nodes to others that pass through the corresponding node. places with high betweenness represent key hubs for the spread of the disease. in contrast, smaller nodes at the periphery of the network are less important for the spread of the disease. web scraping and mapping due to the journalistic style of the who data, it had to be structured such that mapping software can ingest the data. we used import.io to build the api. we provided the software a sample page, selected the data that is pertinent, then provided a list of all urls for the software. we used tableau to map the information both geographically and temporally. most important among the places in the mers-cov epidemic is saudi arabia as measured by the betweenness metric applied to a changes in place mapped to a phylogenetic tree. in figure 1 , the circle representing saudi arabia is slightly larger compared to other location indicating its high importance in the epidemic. saudi arabia is the source of virus for jordan, england, qatar, south korea, uae, indiana, and egypt. the united arab emirates has a bidirectional connection with saudi arabia indicating the virus has spread between the two countries. the united arab emirates also has high betweenness. the united arab emirates is between saudi arabia and oman and between saudi arabia and france. south korea, and qatar have mild betweeness. south korea is between saudi arabia and china. qatar is between saudi arabia and florida. other locations (jordan, england, indiana, and egypt) have low betweenness as they have no outbound connections. certain articles include the infected individuals' countries of origin. ln constrast, many reports are in a lean format that includes a single paragraph that only summarizes the total number of cases for that country. if we build the api in a manner that recognizes features in the detailed reports, we can generate a map that draws lines from origin to reporting country and create visualizations. however, since only some of the articles contain this extra information, mapping in this manner will miss many of the cases that are reported in the lean format. our goal is to develop methods for understanding syndromic and pathogen genetic data on the spread of diseases. drawing parallels between the transmissions events in the who data and the genetic data has shown to be challenging. analyses of the genetic information can be used to imply a transmission pathway but it is hard to find epidemiological data in the public domain to corroborate the transmission pathway. there are rare cases in the who data that include travel history (e.g. "the patient is from riyadh and flew to the uk"). we conclude that epidemiological data combined with genetic data and metadata have strong potential to understand the geographic progression of an infectious disease. however, reporting standards need to be improved where travel history does not impinge on privacy. a transmission graph for mers-cov based on viral genomes and place of isolation metadata. the direction of transmission is represented by the arrow. the frequency of transmission is indicated by the number. the size of the nodes indicates betweenness. isds annual conference proceedings 2017. this is an open access article distributed under the terms of the creative commons attribution-noncommercial 3.0 unported license (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. phylogenetics; transmission graph; mers-cov; syndromic; genetic phylogenetic visualization of the spread of h7 influenza a viruses janies e-mail: djanies@uncc research reported in this publication was supported by a unc research opportunities initiative grant to unc charlotte, nc state university, and unc-chapel hill. the data used in this report are publically available in genbank and the who. we gratefully acknowledge all the researchers and institutions who submitted prepublication data to the public domain. key: cord-271512-owidim7o authors: thabet, farah; chehab, may; bafaqih, hind; almohaimeed, sulaiman title: middle east respiratory syndrome coronavirus in children date: 2015 journal: saudi med j doi: 10.15537/smj.2015.4.10243 sha: doc_id: 271512 cord_uid: owidim7o the middle east respiratory syndrome (mers) is a new human disease caused by a novel coronavirus (cov). the disease is reported mainly in adults. data in children are scarce. the disease caused by mers-cov in children presents with a wide range of clinical manifestations, and it is associated with a lower mortality rate compared with adults. poor outcome is observed mainly in admitted patients with medical comorbidities. we report a new case of mers-cov infection in a 9-month-old child complicated by severe respiratory symptoms, multi-organ dysfunction, and death. we reviewed the literature in an attempt to characterize the mode of presentation, the risk factors, and outcome of mers-cov infection in the pediatric population. t he middle east respiratory syndrome (mers) is a new human disease caused by a novel coronavirus (cov) first reported in the kingdom of saudi arabia (ksa) in september 2012. 1 the mers-cov is characterized by severe respiratory illness and highcase-fatality rates. 2, 3 as per the june 2014 update, 701 laboratory-confirmed cases of infection with mers-cov including at least 249 related deaths have officially been reported to the world health organization. most of the affected patients are adults. 4 data in children including presentation, risk factors, and outcome is poorly documented in the literature. here, we report a new case of mers-cov infection in a 9-month-old child with a literature review in an attempt to characterize the mers-cov infection in the pediatric population. case report. a 9-month-old child recently diagnosed to have infantile nephrotic syndrome and started on oral prednisolone was transferred to the pediatric intensive care unit (picu) after 5 days of ward admission due to streptococcus pneumonia sepsis. he was started on vancomycin and meropenem, and required mechanical ventilation and inotropic support. blood gas showed metabolic acidosis, and the chest x-ray was normal. he gradually improved. on day 4 of picu admission, he was completely weaned from inotropes, and repeated blood culture was negative. he remained on low-ventilatory support because of ascites and chest wall edema. on day 8, his condition deteriorated. he had increased respiratory effort and increased oxygen requirement. blood gases showed compensated respiratory acidosis. chest x-ray showed diffuse bilateral haziness (figure 1 ). at the same time, a decrease in urine output, and worsening in renal function was noted. disclosure. authors have no conflict of interests, and the work was not supported or funded by any drug company. echocardiography showed mild tricuspid regurgitation with normal cardiac function. he was switched to highfrequency oscillatory ventilation. he was afebrile, and laboratory investigations showed a c-reactive protein of 24 mg/l (normal value [nv] -0-6 mg/l), white blood cell count of 9.600/mm 3 (nv -5-14.5/mm 3 ), hemoglobin of 10.9 g/dl (nv -11.5-15.5 g/dl), and platelet of 32000/mm 3 (nv -150-450/mm 3 ). his coagulation profile was initially within the normal range but later become prolonged he continued to receive supportive treatment and was started on continuous renal replacement therapy due to decreased urine output and worsening renal failure, but he continued to deteriorate, and died 4 days later. no other explanation for the sudden deterioration of the patient could be identified. we could not determine the source of infection in our patient despite extensive investigations, including screening of the healthcare providers who came in contact with the patient in the emergency department, in the pediatric ward, and the picu, as well as, all the family contact members. discussion. since the emergence of mers-cov, the prince sultan military medical city in riyadh has taken extensive measures (the "no risk" policy) to prevent further spread of the disease. apart from extensive infection control measures, a vast education campaign for the staff and the community including parents and their children was initiated. in compliance with the saudi arabian ministry of health recommendations, screening with pcr for mers-cov was carried out for every patient that presented or developed fever, and respiratory symptoms with new chest infiltration documented on the chest x-ray. despite all this extensive screening, and in contrast to what was observed by our colleagues dealing with adult cases in the hospital, our pediatric department could identify only one case of mers-cov in a 9-month-old child known to have nephrotic syndrome. furthermore, out of 701 patients confirmed mers-cov reported, so far worldwide, only 14 were pediatric cases (2%). 4 does this mean that mers-cov is mainly an adult disease? or alternatively, does mers-cov in children have a different clinical picture, including a less severe disease, or an atypical presentation, or even an asymptomatic carrier state? what are the risk factors, the comorbidities and the outcome? these questions are still unanswered in the pediatric literature. the 14 children with confirmed mers-cov have a mean age of 99 months, and they were 8 females and 6 males ( table 1) . nine patients were asymptomatic and were detected during screening of family contact; all these 9 patients were healthy without underlying diseases. 5 three patients developed mild respiratory symptoms, 5-7 while the last 2 patients developed severe respiratory symptoms. 5 these 2 patients have associated comorbidities. the outcome was good in 12 patients, while the 2 patients with severe respiratory symptoms and comorbidities developed multi-organs dysfunction and died (14.2%). it is interesting to note that similar to adults, pediatric patients with poorer outcome develop multi-organ impairment, and mainly renal failure. 8 from this small series, compared to adult patients, mers-cov in children is less frequent and seems to be associated with less mortality unless the patient has underlying comorbidities. this impression of low mortality in children with mers-cov was also reported during the acute respiratory syndrome coronavirus (sars-cov) infections in children, where symptoms were milder, and resulted in no mortality, and few hospitalizations. 9 the low incidence of mers-cov infection in children is corroborated by a jordanian prospective year-round viral surveillance study in children less than 2 years of age admitted with acute respiratory symptoms and/ or fever from march to september 2010 tested for mers-cov, all 474 tested patients were negative. 10 we were, unfortunately, unable to identify how our patient got infected. three patterns of transmission of the disease are suggested by the literature: 11 the first pattern is the occurrence of sporadic cases in communities. the true incidence of the disease in the community is not known. the second pattern is transmission within families. the rate of intrafamilial transmission is not known, but it is estimated to be approximately 3.6%. 10 the third transmission pattern is nosocomial transmission from patient to patient, or from a patient to healthcare provider, or vice versa the study by memish et al 11 reported a positivity rate of mers-cov by pcr of only 1.12% of all tested health care workers. these data suggest that the recommended infection control measures are adequate to reduce the risk of transmission within healthcare facilities. during our investigations, we focused on the last 2 patterns of transmission, and we searched for any link between our patient and adult patients admitted during the same period with mers-cov in our hospital, however we failed to demonstrate any positive contacts. in conclusion, these data indicate that although few cases of mers-cov in children have been detected, it remains mainly a disease of adults. the disease caused by mers-cov in children presents with a wide range of clinical manifestations, and it is associated with a lower mortality rate compared with adults. poor outcome is observed mainly in admitted patients with medical comorbidities. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) update world health organization middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus (mers-cov) -update world health organization middle east respiratory syndrome coronavirus (mers-cov) -update world health organization clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study key: cord-103899-6tqm99g1 authors: mirzaei, rasoul; mahdavi, farzad; badrzadeh, fariba; reza hosseini-fard, seyed; heidary, maryam; salimi jeda, ali; mohammadi, tayeb; roshani, mahdane; yousefimashouf, rasoul; keyvani, hossein; darvishmotevalli, mohammad; zarei sani, melika; karampoor, sajad title: the emerging role of micrornas in the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection date: 2020-11-13 journal: nan doi: 10.1016/j.intimp.2020.107204 sha: doc_id: 103899 cord_uid: 6tqm99g1 the novel coronavirus disease 2019 (covid-19) pandemic has imposed significant public health problems for the human populations worldwide after the 1918 influenza a virus (iva) (h1n1) pandemic. although numerous efforts have been made to unravel the mechanisms underlying the coronavirus, a notable gap remains in our perception of the covid-19 pathogenesis. the innate and adaptive immune systems have a pivotal role in the fate of viral infections, such as covid-19 pandemic. micrornas (mirnas) are known as short noncoding rna molecules and appear as indispensable governors of almost any cellular means. several lines of evidence demonstrate that mirnas participate in essential mechanisms of cell biology, regulation of the immune system, and the onset and progression of numerous types of disorders. the immune responses to viral respiratory infections (vris), including influenza virus (iv), respiratory syncytial virus (rsv), and rhinovirus (rv), are correlated with the ectopic expression of mirnas. alterations of the mirna expression in epithelial cells may contribute to the pathogenesis of chronic and acute airway infections. hence, analyzing the role of these types of nucleotides in antiviral immune responses and the characterization of mirna target genes might contribute to understanding the mechanisms of the interplay between the host and viruses, and in the future, potentially result in discovering therapeutic strategies for the prevention and treatment of acute covid-19 infection. in this article, we present a general review of current studies concerning the function of mirnas in different vris, particularly in coronavirus infection, and address all available therapeutic prospects to mitigate the burden of viral infections. for centuries, infectious diseases have caused a significant damage to human health and devastated the entire organisms [1] . currently, there is no particular medication or prophylactic vaccine for the eradication of all types of coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), severe acute respiratory syndrome coronavirus 1 (sars-cov-1), and middle east respiratory syndrome coronavirus (mers-cov) [2] [3] [4] [5] [6] . the novel human coronavirus, named sars-cov-2, is the causative agent of the coronavirus disease 2019 (covid19) pandemic, initially identified in wuhan, hubei province, china, which has now affected millions of individuals over the world [7, 8] . the sars-cov-2 is a beta coronavirus (a positivesense, enveloped, and single-stranded rna) belonging to the coronaviridae family [9, 10] . human coronaviruses (hcovs) cause respiratory tract infections (rtis). some of these infectious agents, such as human coronavirus nl63 (hcov-nl63), human coronavirus oc43 (hcov-oc43), and human coronavirus hku1 (hcov-hku1), may be benign and cause the common cold. nonetheless, the science of virology has characterized a group of highly pathogenic human coronaviruses, including sars-cov, over the past two decades, which caused infectious diseases in about 8,000 people in the world with a fatality rate of ~10% or mers-cov in 2012 that infected 2,500 cases with a mortality rate of 37% [9] . numerous studies indicated that these pathogenic coronaviruses led to the emergence of acute respiratory distress syndrome (ards), leading to extensive pulmonary inflammation, dyspnea, and even death. compared with mers and sars-cov-1, sars-cov-2 tends to spread more quickly than other types of coronaviruses; nevertheless, it has a lower mortality rate of 2-3% [11] . by november 3, 2020, more than 46, 591, 622 million cases have been diagnosed in 219 countries, and more than 1, 201, 200 deaths have been reported [12] . micro-rnas (mirnas) are classified as small molecules with a length of 17-24 nucleotides [13, 14] . they are noncoding single-stranded rnas that can suppress the translation of mature messenger rnas (mrnas) [13] . micrornas are significant regulators of the gene expression process and play a decisive function in different biological phenomena, including cell propagation, differentiation, growth, and apoptosis [15] . recently, it has been known that mirnas contribute to the regulation of both innate and adaptive immune systems by controlling the expansion and activation of immune cells [15] . as mentioned earlier, there is no definite therapy or vaccine to treat and prevent highly pathogenic coronaviruses. therefore, seeking new biological strategies for the therapy of viral diseases is highly recommended. data imply that mirnas have important functions in modulating the immune system toward respiratory viruses, such as hcovs that include sars-cov-1, mers, sars-cov-2, and other types of hcovs, human metapneumovirus (hmpv), human rhinovirus (hrv), iv, and rsv. also, changes in the expression of mirnas in epithelial cells may participate in the pathogenesis of both severe and chronic respiratory infections [16] . this review will summarize the recent discoveries associated with mirnas in various respiratory infections caused by viruses, especially coronavirus, and address all feasible therapeutic options to mitigate the burden of vris. the humoral immunity is immunologically categorized as an acquired immune response in which t helper cells collaborate with b cells to differentiate these types of cells to plasma cells [17] [18] [19] . plasma cells can synthesize specific antibodies (abs) against a particular viral antigen (ag) [17] . secreted abs are capable of blocking viruses to prevent them from entering the host cells. henceforth, they play an influential defensive role in preventing infection and its recurrence [17] . the capability of humoral immune responses as well as b and t cell epitopes against sars-cov have been broadly investigated and mapped to analyze the envelope as well as spike (s), nucleocapsid (n), membrane (m), and envelope (e) proteins (structural proteins) [20] . angiotensin-converting enzyme 2 (ace2) acts as a receptor for the entrance of sars-cov-2 when infecting the epithelial cells of the lungs. it has been discovered that the s proteins of 2019-ncov and sars-cov-1 have similar structural dominant t cell epitopes [21] . for the generation of compensating abs, two proteins are needed to support b cell epitopes in the form of receptor binding domains (rbd) [17] . a potent t cell response is a prerequisite for generating a higher concentration of neutralizing abs [17] . it has been demonstrated that a specific site is not required for t cell epitopes compared with b cell epitopes; thus, they are abundantly expressed in viral infections [17] . t helper cells participate in the switching of isotypes, stimulating b cells to secrete immunoglobulin m (igm) and immunoglobulin g (igg) abs against sars-cov-1. also, seroconversion may occur, which could be mediated by the production of t helper cells [22] . following this phase, at the end of week 12, igm starts to disappear. simultaneously, igg can persist for a more extended period, implying that igg abs may be potent protectors during infection [23] . current evidence strongly suggests that the t helper 1 (th1) response is a crucial factor for successful controlling of mers-cov and sars-cov-1, and it might be the same for sars-cov-2 [24] . cellular immunity is immunologically named as acquired immunity [17] . in contradiction of the humoral immune responses, cellular immunity can kill infected cells mediated by t cells [17] . t helper cells are responsible for the overall direction of adaptive immune responses. in contrast, cytotoxic t cells are implicated in killing and eliminating cells infected with viral agents [17] . for the construction of potent vaccines, cellular immunity granted by t cells would be indispensable, as shown by murine sars-cov and mers-cov models [24] . it has been demonstrated that t cell deficiency results in failure of viral clearance in infected mice, suggesting the significance of these cells in viral clearance [25] . pointing back to mers-cov and sars-cov-1, it has been indicated that cd8+ (interferon (ifn)-γ and tumor necrosis factor (tnf)-α) and cd4+ (tnf-α, ifn-β, and interleukin 2 (il-2)) memory t cells can be persevered in patients recovered from sars-cov for up to four years while having a functionality by the propagation of t cells, generation of ifn-γ, and delayed-type hypersensitivity (dth) responses [26] . currently, the study results show a distinct memory t cell response to the s library of peptides belonging to sars-cov years after the onset of infection [20] . different cd8+ t cells were detected through the clearance of mers-cov in a murine model [27] . these data could be analyzed in the case of sars-cov-2 [17] . nevertheless, the current study demonstrated that the numbers of cd8+ and cd4+ t cells are reduced in individuals infected with sars-cov-2, resulting in the compromised generation of memory t cells in patients recovered from sars-cov-2 [17] . several reports have studied the diverse lymphocyte populations in subjects with sars-cov-2 infection. in a study by qin et al. [28] lymphocytes were evaluated in 44 subjects and according to their results, the total amount of b, t, and nk cells was significantly diminished in the infected group as nk and t cells were below the usual rate, while b lymphocytes were within the lower range of the reference rates [28, 29] . t lymphocytes are usually the most changed by the viral infection, nevertheless, the numbers of cd8+ t, cd4+ t and nk cells were within the normal range in qinʼs study [28] . moreover, studying several subsets of t lymphocytes showed that both cytotoxic t (cd3+cd8+) and helper (cd3+cd4+) lymphocytes were below the normal rate in subjects with covid-19, and the t helper/suppressor ratio within the normal rates [28] . additionally, covid-19 subjects showed lower numbers of regulatory t lymphocytes (treg) (cd3+cd4+cd25+cd127low+), and this decrease was particularly documented in critical covid-19 cases [28] . also, a reduction in stimulated (cd45ro+cd3+cd4+cd25+cd127low+) regulatory t and naïve (cd45ra+cd3+cd4+cd25+ cd127low+) lymphocytes was documented in critical covid-19 subjects [28] . cytokine storm syndrome (css) occurs; meanwhile, massive amounts of proinflammatory cytokines are secreted by immune cells due to the overreaction of the human immune system to the external signals [17, 30] . one study showed css as one of the complications of covid-19 [27] . this study was in line with the data on death-related risk factors of sars-cov-2 infection [17] . ards is highly correlated with css as the immune effector cells secrete high amounts of proinflammatory cytokines and chemokines during ards, leading to the uncontrolled systemic inflammatory responses [31] . transforming growth factor beta (tgf-β) [17] . in the case of mers-cov, increased expression of proinflammatory cytokines, such as ifn-α, il-6, as well as chemokines, including ccl-5, cxcl-8, and cxcl-10, have been reported [32] . in a study, xu et al. found that peripheral blood of covid-19 subjects had an extremely high rate of ccr6 t-helper 17 (th17) lymphocytes, sustaining a th17-type css in this infection [29, 33] . cytokine storm-induced ards has destructive effects on the immune system, resulting in multiple organ failure and subsequent death [34] . this deleterious effect has caused similar mortality in the sars-cov-2 outbreak as in sars-cov-1 and mers-cov infections [17] . it is now known that drugs that target il-1, il-6, il-18, and ifn-γ are useful for attenuating cytokine storm syndrome in other viral infections. this casts a doubt on the effectiveness of these therapeutic agents for mitigating the severity of covid-19 [35] . recently, one of the specific inhibitors of il-6 was demonstrated to be effective in reducing covid-19 severity in a few cases of patients in china [36] . css is a crucial factor that determines the intensity of signs, the mortality rate, and the onset of extrapulmonary signs during covid-19 [29] . severe infections show features suggestive of a series of pathologies associated with css, in which multi-organ failure and hyper inflammation are demonstrated following excessive cytokines produced from an enhanced immune stimulation [29] . hence, studies with css are promising and could urge further pathogenetic studies on identification of effective therapeutic biomarkers. the innate immunity is regarded as the primary defense line against most infectious agents, such as bacteria, viruses, and other pathogens, posing a hazard to the host [16] . it has been reported that mirnas contribute to regulating the function of dendritic cells, epithelial cells, monocytes, granulocytes, nk cells, and macrophages that participate in innate immune responses [16] . activation of the intracellular signaling pathway triggers the innate immune response and stimulates the coupling of pathogen-associated molecular patterns (pamps) with pattern recognition receptors (prps), which are specialized transmembrane proteins. this family of proteins includes nod-like receptors, toll-like receptors (tlrs), and rig-i-like receptors (rlrs), which have a fundamental function in antimicrobial protection [37] . since the primary target of vris is the epithelial cells, these types of cells are involved in viral-induced immune responses. for instance, stimulating the epithelial cells by tlr agonists incites the expression and synthesis of chemokines, ifns, and cytokines [16] . the most effective stimulating response is achieved by treating epithelial cells with tlr3 ligands, which detect double-strand rnas (dsrnas) produced as intermediate products during the replication of viral genomes [38] . recently, it has been shown that mirnas effectively participate in signal transduction of tlrs [16] . studies have indicated the enhanced expression of mir-146a and mir-146b in response to lipopolysaccharide (lps) when exposed to the thp-1 cell line. this finding was in agreement with other related studies, indicating that similar responses occur upon the activation of the tlr signaling pathway in myeloid cells in response to fungal, viral, and bacterial agonists of tlrs [39, 40] . interestingly, no significant rise was seen in the expression of mir-146a after the stimulation of tlr-3, tlr-7, and tlr-9 in response to bacterial and viral nucleic acids [16] . these results were in contrast to findings obtained from the expression of mir-155, which showed increased levels following the activation of tlr-3 and tlr-9 in response to poly i: c and cpg, respectively [16] . several lines of evidence showed that mir-155 is a definite regulator of the pro-inflammatory cytokines, as demonstrated by research carried out on the transgenic mouse model [40, 41] . also, reports indicated that mir-146a could modulate the tlr signaling pathway by targeting traf6 and irak1, which are introduced as protein complexes involved in the activation of il-1βr, tlr2, tlr4, and tlr5 signal transduction [42, 43] . studies showed that mir-9 is overexpressed upon tlr4 activation in human monocytes and neutrophils [44, 45] . remarkably, it has been reported that the stimulation of tlr3, a signal transduction activator dependent on myd88, did not influence the expression of mir-9, denoting that mir-9 induction is linked with signal transduction dependent on myd88 activation in monocytes. current evidence indicated that the activation of tlr2, tlr4, and tlr7/8 promotes the activation of the myd88 signaling pathway, leading to an improvement in the expression of mir-9 in human monocytes and neutrophils [16] . following the exposure of murine macrophages to tlr2, tlr3, and tlr4 agonists and lipopolysaccharide (lps), the expression of mir-147 is activated [38, 46] . a recent study showed that mir-147 has anti-inflammatory properties and attenuates excessive inflammatory responses [16] . mirnas control the expression of some cytokines involved in the activation of innate immune responses [16] . the generation of some cytokines, such as tnf-α, il-6, and il-12 are lowered after the induction of mir-152 and mir-148, associated with tlr3, tlr4, and tlr9 activation [42] . except for ifn-γ, proinflammatory cytokines, such as il-1β, induce mir-9 expression [38] . other receptors belonging to the prps family, such as rlrs, consist of melanoma differentiationassociated gene 5 (mda5) and retinoic acid-inducible gene (rig-i) [16] . rlrs are surrounded in the cytoplasm and contribute to the identification of viral components [16] . viral infections caused by negative-and positive-sense rna viruses, such as iv and rsv, can lead to rig-1 activation, while mda5 can identify picornaviruses, such as rvs [47] . rv can induce antiviral responses by triggering rig-i, tlr3, and mda5 activation [16] . furthermore, the stimulation of hela cells infected with rv led to the enhanced levels of mir-23b, implying that the expression of this mirna is regulated by ifn-and rig-mediated signaling pathways [48] . adaptive immunity is a member of the antigen-specific defense system, mediated by t and b lymphocytes [16] . recently, it has been indicated that the mir-17-92 cluster is the primary regulator of t cell proliferation involved in t cell expansion, leading to the increased endurance of these cells by the impairment of the expression of pro-apoptotic protein-encoding mrnas, such as phosphatase, tensin homolog, and bcl-2 [49] . the expression of some sets of mirnas contributes to the polarization of t helper cells. for instance, mir-155 enhanced the differentiation of th-2 cells, whereas mir-326 promotes the development of th-17 cells [16] . besides, mirnas are responsible for the regulation of mechanisms underlying the central-adaptive immune responses, such as presenting ags, engaging mir-155, and promoting the development of b and t cells in the presence of mir-181a [49, 50] . li and colleagues [51] observed the expression of mir-181a in immature t cells resident in the thymus. this mirna can also modulate t-cell receptor signal transduction by suppressing the activation of various phosphatase enzymes [51] . in contrast to human naive t cells, mir-146a is highly expressed in human t cm [50, 52] . studies indicated that mir-146a interferes with apoptosis initiation and targets the fas-associated death domain [16] . correspondingly, the engagement of mir-146a in the acquired immunity is more pronounced when the overproduction of mir-146a leads to the disturbed production of il-2 and action of activator protein-1 [53] . several studies showed that the memory and effector cd8+ t cell-mediated antiviral responses recruit mir-155 for their activation [16] . cd8+ t cells lacking mir-155 exhibit decreased effector responses, and they are unable to be differentiated into memory cells [54] . another study conducted on mice knock-out for mir-155 and infected with iv demonstrated the impaired clearance of viral infection in these genetically-manipulated animals [55] . besides, mir-155-deficient cd8+ t cells are more resistant to the anti-proliferative properties of ifns and show increased ifn-i signaling [16] . a study conducted by lind et al. [55] showed that immunity to the lymphocytic choriomeningitis virus is compromised in mir-155-deficient mice. remarkably, mir-155 is overexpressed in resting primary human b cells infected with ebv that cause the increased survival of virus within the lymphoblastic cells. this implies the importance of mir-155 for ebv latency. therefore, mir-155 seems to be a vital component of antiviral responses. the immune responses against vris, such as iv, hrv, human coronavirus (hcov), hmpv, and rsv, are correlated with the aberrant expression of several mirnas in epithelial cells and participate in the pathogenesis of chronic and acute forms of respiratory disorders (table 1 ) [16] . in this section, the role of mirnas in vris is comprehensively reviewed. rv causes upper respiratory tract infections (urtis) in adults and children. the epithelial cells resident in the respiratory tract is the main target of rv [56] . rv is a single-stranded-rna (ssrna) virus that possesses icosahedral capsids and belongs to the picornaviridae family [57, 58] . the dsrna is generated during viral replication and is identified by rig-i and tlr3 [47, 59] . bioinformatics software have recently enabled us to efficiently predict whether the natural or designed mirnas can target some viruses to act as therapeutic agents [60] . several studies showed that mir-155 and mir-128 could regulate innate immune responses against rv-1b due to targeting the genetic components of rv [61] . it has been shown that the knock-down of these two mirnas can stimulate rv replication [62] . mir-23b contributes to the immune response against rv, leading to the decreased expression of the transmembrane region of vldlr and lpr5 [16] . these two receptors are hijacked by twelve rv types, including rv23, rv25, rv29, rv30, rv31, rv1a, rv1b, rv2, rv44, rv47, rv49, and rv62 [48] . the genome of rsv is a negative-sense ssrna, encoding 11 proteins (ns1, ns2, m, p, n, sh, m2-1, m2-2, g, f, and l), which is related to the paramyxoviridae family [63] [64] [65] . rsv is a prevalent human pathogenic respiratory virus that attacks the lower respiratory tract (lrt) and causes similar clinical symptoms to those of the common cold in children and adults. the virus is classified as a respiratory virus regularly isolated from hospitalized children with bronchitis [56] . primary infection typically causes acute illness, whereas secondary infection can provoke obstructive bronchitis [66] [67] [68] . in hbepc, rsv can reduce the production of mir-221, while the degree of let-7i and mir-30b production is increased after 48 h of infection [56] . increased expression of let-7i and mir-30b has been reported in rsv-infected hbepc lacking ns1 and ns2 proteins, implying that these proteins have antagonistic properties against mir-30b and let-7i; thus, suppressing the generation of type i ifn [56] . among mirnas aberrantly expressed during the infection period of rsv-a2, mir-221, mir-27a, mir-339-5p, mir-574, mir-453, and mir-744 are more affected compared with others. except for the mir-744, the expression levels of all the aforementioned mirnas are increased [69] . a case-control study explained that patients infected with rsv had a marked decrease in the expression of mir-34c, mir-34b, mir-29c, mir-125b, mir-125a, mir-27b, and mir-429 when compared with healthy individuals. in contrast, the expression levels of mir-31, mir-155, let-7d, mir-203a, and mir-16 were elevated [56] . in this case-control study, subjects were allocated to three groups based on the severity of the disease: mild, moderate, and severe. the results demonstrated that mir-125a and mir-429 expression decreased in the moderate group [70] . some investigations indicated that rsv could provoke different expression levels of mirnas in at least two various ways. first, the induction of let-7b and let-7i is dependent on the expression of ifn-β in hbepc and monocyte-derived dendritic cells (mddcs) [71] . second, mir-30b is not induced by ifn-β while being reliant on the expression of nf-κb in hbepc [71] . evidence also demonstrates that rsv can diminish mir-221 expression in hbepc [71] . rsv infection can lower the expression of some mirnas, such as mir-337-3p, let-7f, mir-24, mir-26b, mir-520a-5p, mir-595, and mir-198 [72] . all of these mirnas have related victims, such as genes encoding the cell cycle proteins (ccnd1, dyrk2, and elf4), suppressor of cytokine signaling 3 gene (socs3), and chemokines (ccl7) [72] . moreover, the rsv g-protein can increase the expression of let-7f, having an antagonistic activity against dyrk2 and ccnd1. these proteins contribute to cell cycle check in the g1 phase and enhance viral propagation [72] . it has been demonstrated that mir-let-7 is vital for inducing the host genes during viral infections [72] . hmpv is a recently identified prominent member of the paramyxoviridae family [46] , including the human parainfluenza virus [73, 74] . ns1 and ns2 are non-structural genes, which are not expressed in the genome of hmpv with eight open-reading frames: 3′-n-p-m-f-m2-sh-g-l-5′ [75] . some clinical trials demonstrated that hmpv is responsible for developing pediatric lrti [76] [77] [78] . previous works have indicated that hmpv causes some alterations in the expression of a collection of mirnas, such as mir-4552, let-7f, mir-16, mir-30a, mir-192, and mir-374a* which are expressed in epithelial cells resident in the respiratory tract [76] [77] [78] . it has been reported that hmpv is responsible for regulating the expression of 174 mirnas in a549 cells. among these mirnas highly expressed in a549 cells is let-7f, which can target the rna polymerase enzyme of hmpv to control the viral replication [79] . the influenza virus (iv) is an ssrna virus related to the orthomyxoviridae family, consisting of three influenza viruses: a, b, and c [56] . type a influenza virus (iva) is sub-categorized based on two proteins expressed on the exterior surface of the virus: neuraminidase and hemagglutinin (n and h proteins, respectively) [56] . to date, 16 types of h proteins, along with 9 types of n enzymes, have been characterized [80] . influenza a (h1n1 or h3n2) and b have currently the highest incidence rate in the united states [81] . iv is a highly contagious respiratory disorder, characterized by the emergence of featured signs, including cephalea, fever, coryza, myalgia, sore throat, and coughing [81] . the iv preferably infects the upper respiratory tract (urt); nevertheless, in severe cases, the lower respiratory tract (lrt) (bronchioles) might also be infected with the virus [82] . the mirnas expression during iv infection might be dysregulated [56] . many mirnas, including mir-491, mir-323, and mir-654, can hinder the replication of iva h1n1, resulting in the reduced iva gene expression in infected cells [83] . the mrna degeneration of the pb1 protein (contributing to the viral propagation of iva) by the host mir-491, mir-323, and mir-654 is one of the exemplary mechanistic roles of mirnas [84] . suppression of the m1 protein expression by let-7c regulates the replication of the iva in a549 cells [84, 85] . studies indicated a substantial reduction in the production of mir-221 and mir-17-3p in human alveolar basal epithelial cells during the iv infection [86] . coronaviruses are responsible for a broad range of respiratory infectious diseases, from mild urtis to critical and life-threatening forms of the disease [87] . up to now, the role of mirnas in the infectivity of coronaviruses has not been examined in in-vivo studies. a limited number of invitro studies were carried out on the oc43 virus, and some in-silico analyses have been conducted on sars-cov and mers-cov [88] . the coronavirus oc43 causes the common cold [89] . the n protein of coronavirus is requisite for viral replication and binding the genomic rna to produce helical capsids. the oc43 n protein triggers nf-kb activation by attaching to its negative regulator called mir-9 [88] . whether the activation of nf-kb contributes to viral replication directly or acts as a bystander to limit the viral replication has not been so far revealed [88] . compared with other pathogenic coronaviruses, a lower rate of oc43 virulence, along with minor symptoms, may affect the interaction between infected and non-infected subjects and consequently increase the distribution of the virus within a community [90] . this innovative mechanism to boost gene expression may explain how rna viruses suppress the host immune scheme or induce pathological alterations in humans [88] . sars-cov-1 is a newly-identified coronavirus that and e proteins, serve as targets for host mirnas. [91] . the abolition of viral replication may support the viral evasion from the immune system surveillance until the infection of other cells [88] . these outcomes describe how sars-cov-1 can cause some changes in the host mirna expression pattern to its authority. regarding the sars-cov-1, it is thought that this virus cannot encode its mirnas [91] . however, in due progress of evolution, the virus may evolve some highly sophisticated mechanisms to take advantage of the host cellular biosynthetic system and evade from the cellular defense mechanisms [91] . under specific conditions, sars-cov-1 employs cellular mirna transcripts to promote its own progress [91] . in a pioneer work, mallick et al. [91] assessed mirna-interfered host-sars-cov-1 interplays in bronchoalveolar stem cells (bascs) at the early stage of the disease. they found that bascs could be categorized as a subset of oct-4 + ace2 + cells and major targets for sars-cov infection [91] . recently, it has been discovered that sars-cov-1 could enter the host cells, contaminate pulmonary bascs, and regulate its developmental steps through molecular controls-mirnas [91] . the interaction between the host mirnas and viruses exhibits a planned scenario to control the viral replication to prevent immune elimination until the infection of other cells [91] . afterwards, the virus undergoes rapid mutations to optimize target-mirna mismatches and promote its propagation until cells reach an entirely differentiated status [91] . mallick et al. [91] indicated that bascs are the primary target cells for sars-cov. they revealed that the mirnas-17*, -574-5p, and -214 are upregulated following sars-cov infection in bascs to suppress the viral replication and escape from the immune system [91] . it has also been noted that n and s proteins downregulate mir-223 and mir-98 in bascs to manage several steps of the differentiation process in these cells and activate inflammatory chemokines to downregulate the activity of the ace2 enzyme [91] . these processes effectively account for a fruitful viral transference and reproduction within bascs, leading to the ongoing destruction of lung cells and loss of repair capacity [91] . generally, this study revealed the different modes of exploiting cellular mirna tools by a virus to its benefit. [92] . an in-silico study recognized some mirnas ( table 2 ) that possess remarkable sequence identity to hairpin constructions in the mers-cov genome. these mirnas may accordingly downregulate the viral gene expression to hinder its replication [93] . in a recent study, zhanga et al. [94] , investigated the impact of host cerna (competitive endogenous rna) alternations along with the biological circrnas (circular rnas) on calu-3 (human lung adenocarcinoma epithelial) infected with in mirna expression (table 2 ) [94] . these phenomena pave the way to better understand hostvirus interactions and design novel antiviral agents against mers-cov, a highly fatal respiratory disorder. the host mirna expression has a fundamental effect on viral pathogenesis control through interfering with t cells and immune reactions to viral infections [95] . the mir-32 is the primary described example of a cellular mirna that targets the viral rna genome that decreases viral replication within the cells [96] . besides, it has been demonstrated that two proteins (l and p) of vsv are targets of mir-24 and mir-93, while mir-29a targets the nef protein of hiv to inhibit its replication. moreover, some mirnas, including mir-1, mir-30, mir-128, mir-196, mir-296, mir-351, mir-431, and mir-448, target the hcv proteins such as c and ns5a proteins to suppress the translation/replication by provoking ifn signaling [97] [98] [99] . hence, mirna can provide a featured concept for elucidating the pathogenesis and infectivity of the new pandemic sars-cov-2. while the sars-cov-1 is far related to sars-cov-2, some relationships have been found in their manifestations. however, there are marked discrepancies between the two disorders [100] . currently, numerous countries deal with sars-cov-2, and such a prevalence makes the virus prone to undergoing some mutations and developing variations in different populations [101] . several lines of evidence showed that viral pathogens could evade the immune system surveillance by utilizing the host mirnas [102, 103] . khan et al. [101] analyzed the host mirnas profile and the epigenetic regulators of the different expression levels of mirnas among sars-cov and sars-cov-2. they applied computational procedures to prognosticate possible host and viral mirnas and their potential tasks in various operative pathways [101] . they also distinguished multiple presumed host mirnas (with antiviral function) capable of targeting sars viruses and their mirnas to target the host genes [101] . the contrast among the host mirna profiles associated with distinct sars-cov-2 genomes among 24 distinct nations with corresponding normalized deaths elucidated some mirna clusters correlated with raised mortality rates [101] . in this context, khan and colleagues [80] detected that the provoked cellular mirnas could act as a double-edged sword for host protection, as they have significant functions in compensating the viral infection. additionally, mirnas can function as proviral determinants. khan et al. [101] indicated that both sars-cov-1 and sars-cov-2 viral mirnas can target a wide range of signaling pathways associated with the immune system, and some sars-cov-2 mirnas can target a set of immuneassociated signaling pathways, including autophagy and ifn-i signaling, suggesting their immuneevasion tools for continued latency inside hosts [101] . besides, various crucial cellular pathways can be modulated by sars-cov-2, which may result in extended aberrations in cases with comorbidities such as cardiovascular disorders, diabetes, and respiratory difficulties [101] . this inflammation-linked micrornas, such as mirna-155, following infection with these viruses [104] . infection of the calu-3 with sars-cov-2 approximately causes a 2-fold increase in the expression of the ifn-stimulated response element genes and the expression of cytokines, such as cxcl10 or il6 compared with sars-cov-1 [104] . they showed the significant increased expression of mir-155 in cells infected with sars-cov-2 [104] . this mirna has been correlated with numerous viral diseases [105] [106] [107] , and has been recognized as a regulator of immune cell differentiation, especially t-cells [108, 109] . the involvement of this mirna in directing the innate immune responses has further been addressed elsewhere [110] . recently, mir-155-5p induction was determined to be provoked in a murine model infected with the iva [111] . significantly, in the study of wyler and colleagues, the pulmonary damage caused by ards was reduced by the elimination of mir-155, denoting that this mirna could be a possible curative target for the treatment of covid-19 [104] . additionally, in bioinformatics studies by another team, some mirnas, such as mir-218-5p, let-7g-5p, mir-506-3p, mir-133b, mir-124-3p, mir-22-3p, and mir-133a-3p have been identified in sars-cov-2 disease [112] . in another bioinformatics work by sardar et al. [113] , a unified sequence-based examination of sars-cov-2 genome from diverse geographic places was carried out to recognize notable genomic characteristics of sars-cov-2 by integrated analysis. their analysis revealed nine host mirnas that can conceivably target sars-cov-2 genes (table 2 ) [113] . interestingly, they found that nine mirnas did not have targets in another related viral genomes, including those of sars-cov-1 and mers [113] . liu et al. [114] used computational methods to investigate the sars-cov-2 genome for alleged mirnas and detected the viral mirna targets on the virus, and human genome simultaneously and also detected the human mirnas that can target the viral genome. besides, they examined mirnas implicated in dysregulation prompted by a viral disease that involved the immunity and cytoskeleton structure, which are the common critical biological means modulated by the mirnas, induced by infection [114] . notably, they observed that hsa-mir-4661-3p could target the s gene of sars-cov-2, and that mir147-3p (virus-encoded mirna) enhanced the induction of tmprss2 by increasing the virulence of sars-cov-2 in the intestine. ivashchenko et al. [5] studied how mirnas can protect humans from covid-19 using bioinformatics tools. they found that among 2565 mirnas, three mirnas, including mir-6864-5p, mir-5197-3p, and mir 4778-3p, were recognized as the common significant mirnas communicating with the guide rna of sars-cov-1, mers-cov, and sars-cov-2 [5] . using the genome sequences of several mirnas, including mir-6864-5p, mir-4778-3p, and mir-5197-3p, they identified the cc-mir (complete complementary mirna) in the guide rna of coronaviruses [5] . the indicated that the binding sites of these cc-mirs did not create intramolecular networks in the 2d construction of the guide rna of sars-cov-1, mers-cov, and sars-cov-2 [5] . hence, the cc-mirs can join guide rna via these sites out of the race [5] . it has been revealed that in these coronaviruses, cc-mirs are devoid of target genes among 17508 subjects encoding genes with a δg/δgm of higher than 85%, implying that these cc-mirs do not affect the translation of the mrna in humans [5] . it has been shown that cc-mirs could be employed as remedial molecules by being fused with exosomes or additional biological vesicles to be transferred within the lung through inhalation [5] . the transfer of cc-mir within the blood can hinder the propagation of the virus in the bloodstream and the entire organs it can penetrate [5] . the recommended approach of the hindrance of coronaviruses replication can further be employed for other viruses. in another study, tak-sum chow and colleagues predicted and examined the micrornas in the human lung epithelium (hle) that could target sars-cov-2 [115] . they have recognized 128 micrornas (from human) with the ability of targeting the sars-cov-2 genome [115] . this study indicated that six of these micrornas are differentially expressed simultaneously in in-vitro sars-cov-2 infection. besides, 28 microrna targets (for sars-cov-1) and 23 microrna targets (for mers-cov) have been found [115] . furthermore, 48 and 32 micrornas have been typically distinguished in two other investigations. more investigations into distinguishing authentic micrornas that target the coronavirus will help understand the importance of micrornas as a cellular security mechanism toward pathogenic coronavirus diseases [115] . sabbatinelli et al. [116] investigated the levels of mir-21-5p, mir-146a-5p, and mir-126-3p in the serum of 29 subjects with covid-19 receiving tocilizumab along with sex and age-matched healthy control. the study results indicated that the serum levels of mir-146a-5p were significantly lowered in cases who did not respond to tocilizumab [116] . they also demonstrated that among non-responders, those with the lowest serum levels of mir-146a-5p experienced the most adverse outcomes [116] . finally, they proposed that the biomarkers in blood, such as mir-146a-5p, can present a molecular connection between inflammation and the covid-19 clinical course, thus expanding the drug arsenal toward this global health threat [116] . the authors also demonstrated that between non-responders, those with the lowermost levels of mir-146a-5p in serum experienced a majority of unfavorable consequences. it has been found that ace2 helps in maintaining the blood pressure as well as electrolyte balance and it also decreases the level of angiotensin ii in the circulation by suppressing the reninangiotensin-aldosterone system (raas) conferring anti-hypertensive influences [117] . currently, it has also been found that ace2 acts as a receptor for the s protein of sars-cov-2 that plays a pivotal activity in covid-19 [118] . of note, in severe covid-19 patients, various health complications, such as hypertension, cardio-vascular disorders, and diabetes have been reported [119] . in particular, it has been found that ace2 is produced in cardiomyocytes and raised in individuals with cardiac disorders [120] . in an outstanding study by lu et al. [120] , several mirnas with the ability of modulating ace2 which can be exploited to control the sars-cov-2 receptor were investigated. their findings showed that both ace2 mrna and ace2 protein rates are suppressed by mir-200c in animal cardiomyocytes and more importantly, in cardiomyocytes derived from humans [120] . lu et al. [120] found the first mirna candidate that could target ace2 in cardiomyocytes that could be used as a preventive approach for cardiovascular complications in covid-19 patients [120] . ace2 is one of the crucial factors by which sars-cov-2 accesses the human cells, and it has been noted that internalization of the sars-cov-2 requires not only binding to ace2 but also the tmprss2 (transmembrane protease serine 2) [121] . of note, the potential involvement of tmprss2 and targeting it in covid-19 as a novel therapeutic method has not been fully evaluated. in a study by matarese et al. [121] the micrornas that specifically target tmprss2 were evaluated by an in-silico approach. they found mir-98-5p as a proper candidate and mechanistically validated it as a modulator of tmprss2 transcription in two different umbilical vein endothelial cells derived from the human lung [121] . overall, these data show that tmprss2 can be a promising target by specific non-coding-rnas in the treatment of covid-19 patients. to date, there is no available drug to increase/ inhibit any mirnas in vris; nevertheless, progress has been made in other disorders. the primary inhibitory medication designed for mir-122 was constructed in 2010, and it is currently in a phase ii trial for hepatitis c therapy [56] . mrx34 is the primary artificial mirna (2013) used to treat advanced hepatocellular carcinoma [122] . in further investigations, artificial mirnas have been produced to transfect mononuclear cells of peripheral blood with liposomal carriers [56] . these procedures boost particular proinflammatory cytokines, such as tnf-α, and promote innate immunity [123] . the most current purpose of these mirnas was to establish modern vaccines by attenuated viruses filled with an expression cassette (ec) encoding an artificial mirna to target necessary viral proteins [56] . the viral pr8-amir-93np was produced by inserting an ec for mir-93 in attenuated iv within viral genes (genes encoding non-structural proteins). the target of this mirna is the nucleoproteins of iv [56] . this vaccine is given by intranasal injection, protecting against various dissimilar viral strains [124] . plants also generate mirnas that can control the replication of viruses [56] . for example, the expression of pb2 and ns1 proteins in ivas (h1n1 and h7n9) was repressed by mir-2911 (a mirna in honeysuckle) [125] . the absence of an in-vivo delivery system is a fundamental challenge for the advancement of mirna-based therapeutics. up to now, aerosolization is the most traditional and valuable tool for passing small rnas in the respiratory tract with a micro sprayer [126, 127] . this method presents the possibility of mirna transmission for potential applications in ris. concerning covid-19, the viral glycoprotein called the s protein mediates viral entry to the target cells via binding to the ace2 expressed on cell surface [128] . the s glycoprotein is activated through proteolytic cleavage by a host protease called furin [128] . adam17 (a disintegrin and metalloprotease 17 controls the ace2 expression on the plasma membrane, promoting the protein shedding [128] . the notch signaling elevates furin expression, while repressing adam17 via mirna-145. hence, blocking the initiation of notch signaling pathway may serve as an approach to hinder the entry of virus into the cells by diminishing furin and increasing adam17 shedding (such as γ-secretase inhibitor) [128] . besides, approaches that can increase the expression of adam17, such as the application of antagomir to mirna-145, must be considered [128] . commercializing a novel medicine is time-consuming and expensive owing to the administrative manners correlated to pre-clinical and clinical analyses. notwithstanding, we believe that various molecules previously investigated and expanded after the former sars-cov-1 disease in 2002 can be used as a platform for developing active molecules for the current sars-cov-2 disease. one of the exciting facets of these molecules is the feasibility of changing the functionality of the viral replication machinery and disrupting the secondary construction of the central 5'utr genomic area [129] . major molecules reported in recent works are natural rna molecules which have exhibited no side effects in in-vivo and can penetrate the cells without the need for transmission vehicles or transfecting tools. this is a beneficial characteristic as, usually, rna molecules are not much alive per se. they demand a means to enter the cells; for example, mrna-1273 is transported via lipid nanoparticles to covid-19 patients (phase-i clinical trial) [130] . additionally, antagomir has been characterized to act on mirnas turnover [131] . antagomir is a new type of engineered oligonucleotide, which can specifically silence the endogenous mirnas [131] . in this regard, such approaches can act with a good efficacy on covid-19 patients with severe conditions due to the css [132] . antagomirs -easy to be produced in the laboratory, and cheaper than traditional biological approaches, especially when produced in a large scale -could be customized upon the immunological hallmarks of a given condition to block the activation of a range of compounds, effectively tailoring the whole cytokine storm to prevent the patient from having serious, life-threatening complications associated with the covid-19. taken together, antagomir can be used to specifically tailor the action of key mirnas that contribute to the inflammatory process in covid-19 patients, and can improve the clinical symptoms of these patients. using the administration of mirnas, the armory that can operate to combat sars-cov-2 is encouraging and conceivably very efficacious. as postulated, a few of these curative possibilities could mitigate covid-19 signs or diminish viral replication. we firmly propose that none of these opportunities should be dropped unexamined, as identifying an efficacious vaccine could be an extraordinarily long and unpredictable way. this work did not receive any particular grant from funding agencies in the commercial and public sectors. none declared. [135] the mir-let-7c expression was remarkably raised in iv-infected a549 cells. [136] let-7f human metapneumovirus following rsv infection, the let-7f expression was significantly increased. [137] mir-185-5p human metapneumovirus the hmpv provokes mir-185-5p expression. [138] mir-16 human metapneumovirus during hmpv infection, has-mir-185-5p expression was increased. hmpv (wild type) infection does not provoke mir-30a and mir-16 expression, but the virus deficient the m2-2 gene enhancing mir-16 and mir-30a. [139] mir-374a, mir-192 human metapneumovirus the mir-374a* was diminished by hmpv infection. [79] mir-221 respiratory syncytial virus the rsv infection significantly diminished the expression of mir-221. [140] mir-30, let-7i respiratory syncytial virus the expression of let-7i was observed in healthy hbepc cultures infected with an rsv that deficient ns1 and ns2 proteins. [71, 141] let-7 respiratory syncytial virus the increase in the expression of let-7b was observed in hbepc cultures infected with an rsv. [56, 71] mir-339-5p respiratory syncytial virus following the rsv infection, mir-339-5p targets the trkb and apaf1 genes and result in trkb-brain-derived neurotrophic factor binding as well as apoptosis. [140, 142] mir-453 respiratory syncytial virus during the rsv disease, the low-affinity p75 ntr receptor gene was declined via mir-453. [140, 142] mir-34 respiratory syncytial virus in patients infected with rsv, the expression levels of mir-34b and mir-34c were reduced. [56] mir-125 respiratory syncytial virus among the mir-125 family, in patients infected with rsv, the expression of mir-125a and mir-125c were reduced. [56, 143] mir-29 respiratory syncytial virus among the human mir-29 family, the expression levels of mir-29c were declined in rsv infected patients contrasted to the control group. [ 56, 144] the host cerna and circrnas analysis in human lung adenocarcinoma epithelial cells contaminated with the highly pathogenic mers-cov indicated that mers-cov infection could impact on host gene expression. [94] mir628-5p, mir-6804-3p, mir-4289, mir-208a-3p, mir-510-3p, mir-18a-3p, mir-329-3p, mir-548ax, mir-3934-5p, mir-4474-5p, mir-7974, mir-6865-5p, and mir-342-3p the computational approach provided an exciting hypothesis that those mirnas involved in mers-cov pathogenesis, and this approach may help to understand host-pathogen interplay better and promote new antiviral treatment toward mers-cov. [152] mir-146a-5p, -21-5p, and -126-3p in patients with covid-19 that not respond to the tocilizumab treatment, the levels of mir-146a-5p were decreased in the serum. also, this study's data suggest mirs in the blood, such as mir-146a-5p, can act as biomarkers and provide a molecular link between inflammation and the covid-19 clinical course. [116] mir-200c sars-cov-2 i a study investigated several identified mirnas which could regulate ace2 which may be exploited to regulate the sars-cov-2 receptor. their data reveal that both ace2 mrna and ace2 protein levels are inhibited by mir-200c in rat primary cardiomyocytes and importantly, in human-derived cardiomyocytes [120] mir-98-5p sars-cov-2 in a study evaluated the micrornas that specifically target tmprss2. through a bioinformatic approach, they identified mir-98-5p as a suitable candidate and they mechanistically validated mir-98-5p as a regulator of tmprss2 transcription in two different human endothelial cell types, derived from the lung and from the umbilical vein [121] global trends in epidemiology of coronavirus disease 2019 (covid-19) virus against virus: a potential treatment for 2019-ncov (sars-cov-2) and other rna viruses a realistic two-strain model for mers-cov infection uncovers the high risk for epidemic propagation development of genetic diagnostic methods for novel coronavirus 2019 (ncov-2019) in japan how mirnas can protect humans from coronaviruses covid-19 overview of the current promising approaches for the development of an effective severe acute respiratory syndrome coronavirus 2 (sars-cov-2) vaccine clinical features of patients infected with 2019 novel coronavirus in coronavirus disease 2019 (covid-19): immunological approaches and emerging pharmacologic treatments a novel coronavirus outbreak of global health concern bacterial co-infections with sars-cov-2 covid-19, sars and mers: are they closely related? coronavirus disease (covid-19) pandemic exosome and exosomal microrna: trafficking, sorting, and function role of micrornas in staphylococcus aureus infection: potential biomarkers and mechanism role of micrornas in the immune system, inflammation and cancer micrornas and the immune response to respiratory virus infections host immune response and immunobiology of human sars-cov-2 infection the human immune system against staphylococcus epidermidis prevalence of atle, ica, meca, and mupa genes in staphylococcus epidermidis isolates t cell-mediated immune response to respiratory coronaviruses structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-ncov) and sars coronavirus (sars-cov) t cell responses to whole sars coronavirus in humans profile of specific antibodies to the sars-associated coronavirus recent advances in the vaccine development against middle east respiratory syndrome-coronavirus vaccine-elicited cd8+ t cells protect against respiratory syncytial virus strain a2-line19f-induced pathogenesis in balb/c mice interferon interplay helps tissue cells to cope with sars-coronavirus infection cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome dysregulation of immune response in patients with covid-19 in immunopathology of sars-cov-2 infection: immune cells and mediators, prognostic factors, and immune-therapeutic implications a contemporary review on pathogenesis and immunity of covid-19 infection in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients pathological findings of covid-19 associated with acute respiratory distress syndrome cytokine storm induced by sars-cov-2 interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome covid-19: consider cytokine storm syndromes and immunosuppression pattern recognition receptors and inflammation activation of airway epithelial cells by toll-like receptor agonists nf-κb-dependent induction of microrna mir-146, an inhibitor targeted to signaling proteins of innate immune responses advances in micrornas: implications for immunity and inflammatory diseases expression and function of micro rnas in immune cells during normal or disease state integrating micrornas into a system biology approach to acute lung injury mir-146a is critical for endotoxin-induced tolerance implication in innate immunity micrornas in inflammatory lung disease-master regulators or target practice? induction and regulatory function of mir-9 in human monocytes and neutrophils exposed to proinflammatory signals mir-147, a microrna that is induced upon toll-like receptor stimulation, regulates murine macrophage inflammatory responses co-ordinated role of tlr3, rig-i and mda5 in the innate response to rhinovirus in bronchial epithelium retinoic acidinducible gene i-inducible mir-23b inhibits infections by minor group rhinoviruses through downregulation of the very low density lipoprotein receptor micrornas: small rnas with big effects micrornas and immunity: novel players in the regulation of normal immune function and inflammation, seminars in cancer biology skare, mir-181a is an intrinsic modulator of t cell sensitivity and selection expression profiling of human immune cell subsets identifies mirna-mrna regulatory relationships correlated with cell type specific expression an emerging player in the adaptive immune response: microrna-146a is a modulator of il-2 expression and activation-induced cell death in t lymphocytes microrna mir-155 affects antiviral effector and effector memory cd8 t cell differentiation micro-rna 155 is required for optimal cd8+ t cell responses to acute viral and intracellular bacterial challenges rosas-taraco, micrornas in viral acute respiratory infections: immune regulation, biomarkers, therapy, and vaccines a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany structure of a human common cold virus and functional relationship to other picornaviruses rhinovirus and dsrna induce rig-i-like receptors and expression of interferon β and λ1 in human bronchial smooth muscle cells in-silico algorithms for the screening of possible microrna binding sites and their interactions o20-human rhinovirus replication-dependent induction of micro-rnas in human bronchial epithelial cells identification of host mirnas that may limit human rhinovirus replication respiratory syncytial virus (rsv) nonstructural (ns) proteins as host range determinants: a chimeric bovine rsv with ns genes from human rsv is attenuated in interferoncompetent bovine cells human respiratory syncytial virus genome and gene products he, function of the respiratory syncytial virus small hydrophobic protein respiratory syncytial virus infection in elderly and high-risk adults risk of primary infection and reinfection with respiratory syncytial virus respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine respiratory syncytial virus infection of airway cells: role of micrornas nasal mucosal microrna expression in children with respiratory syncytial virus infection respiratory syncytial virus regulates human micrornas by using mechanisms involving beta interferon and nf-κb respiratory syncytial virus modifies micrornas regulating host genes that affect virus replication significant differences in nucleocapsid morphology within the paramyxoviridae analysis of the genomic sequence of a human metapneumovirus effects of viral respiratory infections on lung development and childhood asthma heterogeneity of the association between lower respiratory illness in infancy and subsequent asthma human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children human metapneumovirus infection induces significant changes in small noncoding rna expression in airway epithelial cells update on avian influenza a (h5n1) virus infection in humans influenza: lessons from past pandemics, warnings from current incidents influenza, an existing public health problem cellular micrornas inhibit replication of the h1n1 influenza a virus in infected cells harnessing the power of mirnas in influenza a virus research cellular micro rna let-7c inhibits m1 protein expression of the h1n1 influenza a virus in infected human lung epithelial cells influenza a virus-induced expression of a galnac transferase, galnt3, via micrornas is required for enhanced viral replication molecular pathology of emerging coronavirus infections the role of micrornas in respiratory viral infection: friend or foe? genetic drift of human coronavirus oc43 spike gene during adaptive evolution human coronavirus oc43 nucleocapsid protein binds microrna 9 and potentiates nf-κb activation micrornome analysis unravels the molecular basis of sars infection in bronchoalveolar stem cells middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps a computational approach for predicting role of human micrornas in mers-cov genome competing endogenous rna network profiling reveals novel host dependency factors required for mers-cov propagation microrna-155 enhances t cell trafficking and antiviral effector function in a model of coronavirus-induced neurologic disease a cellular microrna mediates antiviral defense in human cells hypersusceptibility to vesicular stomatitis virus infection in dicer1-deficient mice is due to impaired mir24 and mir93 expression human cellular microrna hsa-mir-29a interferes with viral nef protein expression and hiv-1 replication interferon modulation of cellular micrornas as an antiviral mechanism systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov epigenetic regulator mirna pattern differences among sars-cov, sars-cov-2 and sars-cov-2 world-wide isolates delineated the mystery behind the epic pathogenicity and distinct clinical characteristics of pandemic covid-19 in silico analysis revealed zika virus mirnas associated with viral pathogenesis through alteration of host genes involved in immune response and neurological functions the interplay between viral-derived mirnas and host immunity during infection bulk and single-cell gene expression profiling of sars-cov-2 infected human cell lines identifies molecular targets for therapeutic intervention environmental pollutants modulate rna and dna virusactivated mirna-155 expression and innate immune system responses: insights into new immunomodulative mechanisms microrna 155 and viral-induced neuroinflammation roles of micrornas in the life cycles of mammalian viruses micrornas as regulatory elements in immune system logic regulation of the germinal center response by microrna-155 and its star-form partner mir-155* cooperatively regulate type i interferon production by human plasmacytoid dendritic cells increased expression of microrna-155-5p by alveolar type ii cells contributes to development of lethal ards in h1n1 influenza a virus-infected mice interplay of host regulatory network on sars-cov-2 binding and replication machinery comparative analyses of sar-cov2 genomes from different geographical locations and other coronavirus family genomes reveals unique features potentially consequential to host-virus interaction and pathogenesis implications of the virusencoded mirna and host mirna in the pathogenicity of sars-cov-2 prediction and analysis of sars-cov-2-targeting microrna in human lung epithelium decreased serum levels of inflammaging marker mir-146a are associated with clinical response to tocilizumab in covid-19 patients the renin-angiotensin-aldosterone system and its suppression sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china, zhonghua liu xing bing xue za zhi= micrornas targeting the sars-cov-2 entry receptor ace2 in cardiomyocytes mir-98 regulates tmprss2 expression in human endothelial cells: key implications for covid-19 first microrna mimic enters clinic exosome-derived mirnas and cellular mirnas activate innate immunity generation of a safe and effective live viral vaccine by virus self-attenuation using species-specific artificial microrna honeysuckle-encoded atypical microrna2911 directly targets influenza a viruses intrapulmonary delivery of xcl1-targeting small interfering rna in mice chronically infected with mycobacterium tuberculosis local pulmonary immunotherapy with sirna targeting tgfβ1 enhances antimicrobial capacity in mycobacterium tuberculosis infected mice covid-19 in the heart and the lungs: could we "notch" the inflammatory storm? non-coding rnas and innovative therapeutic strategies to target the 5'utr of sars-cov oxygen-ozone as adjuvant treatment in early control of covid-19 progression and modulation of the gut microbial flora (probiozovid) effects of antagomirs on different lung diseases in human, cellular, and animal models antagomirs: a novel therapeutic strategy for challenging covid-19 cytokine storm identification of host mirnas that may limit human rhinovirus replication retinoic acidinducible gene i-inducible mir-23b inhibits infections by minor group rhinoviruses through downregulation of the very low density lipoprotein receptor influenza a virus-induced expression of a galnac transferase, galnt3, via micrornas is required for enhanced viral replication cellular microrna let-7c inhibits m1 protein expression of the h1n1 influenza a virus in infected human lung epithelial cells human metapneumovirus infection induces significant changes in small noncoding rna expression in airway epithelial cells comparative analysis of mirna profile in human dendritic cells infected with respiratory syncytial virus and human metapneumovirus non-coding rnas and their role in respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) infections microrna-221 modulates rsv replication in human bronchial epithelium by targeting ngf expression comparative analysis of mirna profile in human dendritic cells infected with respiratory syncytial virus and human metapneumovirus mirpower: a webtool to validate survival-associated mirnas utilizing expression data from 2178 breast cancer patients diverse functions of mir-125 family in different cell contexts the mir-29 family: genomics, cell biology, and relevance to renal and cardiovascular injury mir-429 regulates the proliferation and apoptosis of nephroblastoma cells through targeting c-myc micrornas in lipid metabolism a role of mir-27 in the regulation of adipogenesis mir-27b targets ksrp to coordinate tlr4-mediated epithelial defense against cryptosporidium parvum infection microrna as type i interferon-regulated transcripts and modulators of the innate immune response micrornas and their target gene networks in breast cancer the role of micrornas in respiratory viral infection: friend or foe? a computational approach for predicting role of human micrornas in mers-cov genome this work was supported by the hamadan university of medical sciences, hamadan, iran. key: cord-274122-n9jnu2ah authors: mielech, anna m.; kilianski, andy; baez-santos, yahira m.; mesecar, andrew d.; baker, susan c. title: mers-cov papain-like protease has deisgylating and deubiquitinating activities date: 2014-02-01 journal: virology doi: 10.1016/j.virol.2013.11.040 sha: doc_id: 274122 cord_uid: n9jnu2ah coronaviruses encode papain-like proteases (plpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (dub)/deisgylating activity, which is hypothesized to modify the innate immune response to infection. here, we investigate the predicted dub activity of the plpro domain of the recently described middle east respiratory syndrome coronavirus (mers-cov). we found that expression of mers-cov plpro reduces the levels of ubiquitinated and isgylated host cell proteins; consistent with multifunctional plpro activity. further, we compared the ability of mers-cov plpro and severe acute respiratory syndrome coronavirus (sars-cov) plpro to block innate immune signaling of proinflammatory cytokines. we show that expression of sars-cov and mers-cov plpros blocks upregulation of cytokines ccl5, ifn-β and cxcl10 in stimulated cells. overall these results indicate that the plpro domains of mers-cov and sars-cov have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis. middle east respiratory syndrome coronavirus (mers-cov) is a recently described coronavirus with high mortality. as of november 18, 2013, there have been 157 confirmed cases and 66 deaths (http://www.who.int/csr/don/2013_11_18/en/index.html). mers disease is characterized primarily by respiratory symptoms but several patients also developed renal failure drosten et al., 2013) . in most cases reported thus far, immunosuppression or other types of medical disorders have been associated with more severe disease (assiri et al., 2013) . the sequence of the rna genome of mers-cov is most similar to bat coronaviruses hku4 and hku5 ; however, the origin of mers-cov is not known. a recent report showed that dromedary camels have high levels of neutralizing serum antibodies against mers-cov, suggesting a possible zoonotic source (reusken et al., 2013) . in addition, analysis of fecal samples from bats identified an egyptian tomb bat as a potential source of infection (memish et al., 2013) , but more work is needed to identify the animal reservoir(s) for mers-cov. limited humanto-human transmission of mers-cov has been reported, which considering the high mortality, raises a concern that the virus has a potential to become a threat to public health (assiri et al., 2013; guery et al., 2013) similar to severe acute respiratory syndrome coronavirus (sars-cov). the sars-cov pandemic from 2002-2003 was controlled by public health measures of identification and isolation of infected, symptomatic individuals and their contacts which broke the chain of human-to-human transmission. a sars-cov-like virus is endemic in chinese horseshoe bats, but changes in the sequence of the spike glycoprotein are required for this virus to efficiently infect humans (lau et al., 2005; rockx et al., 2007) . for mers-cov, it is unclear if the virus can jump directly from bats to humans, if there are any mutations in the viral genome that facilitate infection or disease in humans, and if there are both symptomatic and asymptomatic cases, which would make any potential epidemic more difficult to control by public health measures alone. our goal was to apply the knowledge gained from the study of sars-cov to identify and characterize the mers-cov papain-like protease domain as an innate immune antagonist and as a potential target for therapeutics. mers-cov, similar to other coronaviruses, is a positive-strand rna virus that upon entry into cells is translated to produce a viral replicase polyprotein. the replicase polyprotein is processed by viral proteases to generate a membrane-associated viral replication complex (snijder et al., 2006; perlman and netland, 2009 ). sequence analysis of mers-cov indicates that the canonical papain-like protease (plpro) and 3c-like proteinase (3clpro) are likely responsible for processing the polyprotein to generate 16 nonstructural proteins that assemble to form the replication complex. the majority of coronavirus papain-like proteases (plps), including sars-cov plpro, have been shown thus far to act as deubiquitinases and interferon antagonists (clementz et al., 2010; frieman et al., 2009; ratia et al., 2006; zheng et al., 2008; xing et al., 2013; barretto et al., 2005; sulea et al., 2005; chen et al., 2007) . the ubiquitin pathway is important for regulating a number of innate immune pathways and the ability of a viral protein to cleave ubiquitin from host cell proteins can contribute to virus pathogenesis. in addition to ubiquitination, modification of cellular proteins with interferon-stimulated gene 15 (isg15) is known to have a broad-spectrum antiviral activity. isg15 is ubiquitin-like protein that can be conjugated to cellular targets via a mechanism called isgylation, regulating innate immune responses. coronavirus plps are known to have the ability to remove isg15 conjugates from cellular substrates (clementz et al., 2010; lindner et al., 2005 lindner et al., , 2007 . in this study, we demonstrate the deisgylating and deubiquitinating (dub) activities of the papain-like protease from mers-cov, and provide new information on the potential role of coronavirus protease/dubs to inhibit the innate immune response. mers-cov plpro is encoded within nonstructural protein 3 (nsp3) of the replicase polyprotein (fig. 1a) . to gain insight into the potential of mers-cov plpro to recognize and cleave ubiquitin and isg15 from proteins, we used the high-resolution x-ray structure of sars-cov plpro in apo-enzyme form (pdb:2fe8, chain c) to generate a homology model of mers-cov plpro. we threaded the mers-cov primary amino acid sequence into the sars-cov structure and then energy minimized the structure. the homology model displays several conserved structural features between mers-cov and sars-cov plpro; including the ubiquitinlike domain (ubl), a catalytic triad consisting of c1594-h1761-d1776 and the ubiquitin-binding domain at the zinc finger. to model ubiquitin (ub) into the zinc finger and palm domains of mers-cov plpro, we used the x-ray structure and associated electron density of sars-cov plpro in complex with ub aldehyde (ubal) (pdb:4mm3) for refinement and energy minimization of the model in complex with ub. the resulting mers-cov-ubal model displays a nearly ideal fit of the ub moiety within the palm and the zinc finger regions of the enzyme with the c-terminal extension of ubiquitin oriented properly towards the mers-cov substrate subsites and catalytic triad (fig. 1b) . from this model, we hypothesize that the plpro domain from mers-cov, like sars-cov is a multifunctional enzyme with protease, deubiquitinating and likely deisgylating activity. we recently described expression and protease activity of mers-cov plpro in cell culture (kilianski et al., 2013) . to determine the deisgylating activity of mers-cov plpro, we transfected hek293t cells with c-myc-isg15 plasmid, isg15 conjugation machinery, and increasing amounts of plasmids expressing mers-cov plpro wild-type and catalytic mutant c1594a (plproca). cysteine 1594 is predicted to be the active site cysteine nucleophile that attacks the substrate peptide bond and mutation to alanine should significantly reduce or abolish enzymatic activity (fig. 1 ). in addition, we transfected cells with plasmids expressing sars-cov plpro wild-type or catalytic mutant (c1651a). we harvested cell lysates at 20 h posttransfection to evaluate the presence of isgylated proteins. we found that both mers-cov and sars-cov plpro can deconjugate isg15 from multiple cellular substrates in a dose-dependent manner. in contrast, plpro catalytic mutants did not deconjugate isg15, indicating that catalytic activity of plpro is required for its deisgylating activity ( fig. 2a) . thus, mers-cov plpro like sars-cov plpro (lindner et al., 2007) has deisgylating activity. to assess the dub activity of mers-cov plpro, we transfected hek293t cells with plasmid expressing flag-ub and increasing amounts of wild-type plpro or plproca. we determined that plpro can deubiquitinate multiple cellular substrates, and that plpro catalytic activity is required for dub activity (fig. 2b ). this dub activity is also observed with expression of sars-cov plpro, consistent with previous reports (frieman et al., 2009; ratia et al., 2006; barretto et al., 2005; lindner et al., 2005 lindner et al., , 2007 . in these experiments, we noticed the difference in the expression levels of sars-cov and mers-cov plpros in transfected cells, which may be due to differences in codon optimization in the mers-cov plpro construct. further in vitro studies using purified enzymes are needed to determine the relative kinetics of sars-cov and mers-cov plpro dub and deisgylating activities. taken together, our data indicate that mers-cov plpro is a potent deisgylating enzyme that also exhibits dub activity and that both activities require cysteine 1594 for catalysis, likely in the context of the predicted catalytic triad (fig. 1b) . coronavirus plps have been shown to block interferon β (ifnβ) induction in transfected cells (clementz et al., 2010; frieman et al., 2009; devaraj et al., 2007) . in addition, the deubiquitinase function of an arterivirus papain-like protease has been shown to have a role in interferon antagonism during virus infection (van kasteren et al., 2013) . therefore, we assessed the ability of mers-cov plpro to antagonize interferon production. first, we addressed if mers-cov plpro can inhibit mda5 induced ifnβ reporter, since mda5 has been implicated in recognition of coronaviruses during virus infection (zust et al., 2011) . we transfected hek293t cells with plasmids expressing ifn-β-luciferase, renilla luciferase, pef-bos-mda5 (rothenfusser et al., 2005) and increasing amounts of wild-type plpro or plproca. at 16 h posttransfection we assessed luciferase reporter activity. we determined that mers-cov plpro can potently inhibit mda5 mediated induction of ifnβ in a dose-dependent manner and that catalytic activity of mers-cov plpro is required for ifnβ antagonism (fig. 3a ). using overexpression of an active form of rig-i, we determined that mers-cov plpro can also inhibit n-rig-i induced ifnβ reporter. similarly to the experiment with mda5 stimulation, the catalytic activity of mers-cov plpro is necessary for ifnβ antagonism upon n-rig-i stimulation (fig. 3b) . upon recognition of viral rna by pattern recognition receptors (prrs) such as mda5 or rig-i the signal is transmitted downstream via mitochondrial antiviral signaling protein (mavs). thus, we tested if plpro is able to inhibit mavs induced ifnβ reporter. to stimulate the ifnβ reporter, we overexpressed pef-bos-mavs (rothenfusser et al., 2005) in hek293t cells, co-expressed reporters, and either the wild-type plpro or plproca. we found that plpro, but not plproca inhibits mavs induced ifnβ reporter (fig. 3c ). finally, we tested the ability of mers-cov plpro to inhibit nf-κb reporter activity as observed with sars-cov plpro. we transfected cells with plasmids expressing nf-κb luciferase, renilla luciferase, and mers-cov wild-type plpro or plproca, treated cells with tnfα to activate the nf-κb pathway, and harvested cell lysates at 4 h post-treatment to assess luciferase activity. we determined that wild-type plpro can reduce induction of nf-κb reporter in a dose-dependent manner and that the catalytic cysteine residue is required for this activity (fig. 3d) . taken together these results indicate that mers-cov plpro is an interferon antagonist and that catalytic activity is required for the antagonism. in addition, plpro can reduce tnfα-mediated induction of nf-κb reporter activity and catalytic activity is also required. to further investigate the role of coronavirus plpros in inhibiting innate immune responses we tested the effect of mers-cov plpro on the expression of endogenous cytokines. first, using the human innate and adaptive immune responses pcr array (sabiosciences) we determined that in hek293t cells ccl5 (rantes), ifnβ, and cxcl10 (ip-10) mrna levels are upregulated more than 20-fold upon mda5 stimulation (data not shown) and therefore selected these genes for further analysis. to determine the effect mers-cov plpro and sars-cov plpro on cytokine expression, we performed qrt-pcr to measure mrna encoding ccl5, ifnβ, and cxcl10 levels in the presence of cov plpros. hek293t cells were transfected with pef-bos-mda5, and wild-type or catalytic mutants of mers-cov or sars-cov plpros. at 18 h posttransfection the total rna was extracted and qrt-pcr was performed. we found that both mers-cov and sars-cov plpro can potently inhibit (over 3-fold reduction) expression of ccl5 upon mda5 stimulation and that catalytic activity is required for this inhibition (fig. 4a ). in agreement with the results from luciferase reporter assays, we observed that expression of ifnβ in mda5 stimulated cells is inhibited in the presence of wild-type mers-cov plpro and sars-cov plpro (fig. 4b ). cxcl10 mrna levels were also significantly reduced (po0.0005) when wild-type, but not catalytic mutant versions of mers-cov plpro and sars-cov plpro were expressed (fig. 4c) . to our knowledge, this is the first report showing that both mers-cov plpro and sars-cov plpro can reduce induction of endogenous proinflammatory cytokines in cells, and that the mechanism requires catalytic activity. viruses must "do more with less" because of the compact nature of their genomes. one example of this is the multifunctional plp domain encoded in all members of the order nidovirales. nidoviruses, including those in the coronavirus and arterivirus families, encode one or more plp domain. these plps are critical for proteolytic processing of the viral replicase polyprotein. in addition to protease activity, many of these plps have also been shown to act as viral deubiquitinating enzymes (dubs), able to deconjugate ubiquitin and isg15 from cellular substrates. coronavirus dub activity was first proposed by molecular modeling of the sars-cov plpro domain which predicted that the protease may be multifunctional . indeed, analysis of the dub activity of purified cov plps and the x-ray crystal structure of sars-cov plpro fully support the initial prediction of viral dub activity (ratia et al., 2006; barretto et al., 2005; lindner et al., 2005; ratia et al., 2008) . analysis of plps from coronaviruses and arteriviruses have revealed conserved dub activity; although the enzymes in the coronavirus family fall into the ubiquitin specific protease (usp) family whereas the arterivirus plps are in the ovarian tumor (otu) domain family of enzymes. the identification of a newly emerged coronavirus mers-cov provides an opportunity to evaluate plpro enzymatic activity and develop new hypotheses about how this protease/dub may contribute to viral pathogenesis. our modeling of the mers-cov plpro domain onto the structure of sars-cov led to the prediction of viral dub/deisgylating activity. although the enzymes are only $ 30% identical to sars-cov plpro at the amino acid level, we show that deisgylating, dub and interferon antagonism activities are conserved. importantly, we show that coronavirus plpro activity can modulate the innate immune response. coronaviruses have been shown to modulate immune responses upon infection, however the mechanisms involved in the regulation are not yet clear (totura and baric, 2012) . cytokine and chemokine responses to sars-cov in non-lymphatic cells and in infected patients results in low levels of several cytokines, including ccl5 and ifnβ (wong et al., 2004; spiegel and weber, 2006) . in addition, cxcl10, ccl5, and ifnβ among others, are not induced in cloned bronchial epithelial cell line and human alveolar type ii cells infected with sars-cov early post infection (yoshikawa et al., 2010; qian et al., 2013) . the innate immune response to mers-cov is also intriguing. microarray analysis of mers-cov infection of calu-3 cells results in distinct immune response compared to sars-cov infection. expression of multiple genes involved in activation of adaptive immune responses, such as mhc class i and ii, are downregulated in mers-cov infected cells (josset et al., 2013) . the ability of sars-cov and mers-cov to modulate early immune responses is likely due to multiple proteins encoded within virus genomes that may act as interferon antagonists. previous reports showed that several coronavirus proteins can block the activation of innate immune responses, particularly production and induction of ifnβ response (reviewed in (totura and baric, 2012) ). structural proteins, such as sars-cov nucleocapsid (n) and membrane protein (m), in addition to being critical elements of the viral particles, have been shown to block the ifn response. several accessory proteins (sars-cov orf3b, orf6, and mouse hepatitis virus ns2) are known to act as antagonists of innate immunity (kopecky-bromberg et al., 2007; zhao et al., 2012) . indeed, mers-cov accessory protein 4a has been reported to block induction of ifn (niemeyer et al., 2013) . in addition, nonstructural proteins including nsp1, nsp7, nsp15 have been implicated as ifn antagonists (frieman et al., 2009; kamitani et al., 2009) . importantly, plps encoded within nsp3 have been shown to block ifnβ induction. sars-cov plpro and hcov-nl63 plp2 are interferon antagonists and catalytic activity is important for plpro antagonism (clementz et al., 2010; frieman et al., 2009; devaraj et al., 2007) . sars-cov plpro is the only plp in the sars-cov genome and it is both a deisgylating and a deubiquitinating enzyme (ratia et al., 2006; lindner et al., 2005; lindner et al., 2007) . on the other hand, hcov-nl63 encodes two papain-like proteases, plp1 and plp2, in the genome but only plp2, which has 22% homology to sars-cov plpro, is an ifn antagonist with deisgylating and dub activities (clementz et al., 2010) . hcov-nl63 plp1 is devoid of these activities . in addition, mouse hepatitis virus plp2 and porcine epidemic diarrhea virus plp have dub activity and act as ifn antagonists (zheng et al., 2008; xing et al., 2013) . plps from arteriviruses are also known to block ifn responses. the n terminal region of nsp2 encodes the papain-like protease in porcine reproductive and respiratory syndrome virus (prrsv). this plp has been characterized as an otu with deubiquitinating and deisgylating ability (frias-staheli et al., 2007) . in addition, sun et al. (2010) , showed that prrsv plp domain can block sendai virus induced ifnβ, and can also inhibit nf-κb by preventing iκbα degradation by its deubiquitination. a more recent report showed that prrsv plp has also deisgylating activity which suggests multiple roles of prrsv papain-like protease in antagonism of innate immunity (sun et al., 2012) . nsp2 of another member of the arteriviridae, equine arteritis virus (eav), has deubiquitinating and deisgylating activities as well (frias-staheli et al., 2007) . the deubiquitinating ability of eav plp can block rig-i induced ifn by inhibiting ubiquitination of rig-i, which is required for its activation . co-crystal structure of eav plp with ubiquitin reveled potential interaction sites between those molecules, and mutagenesis studies showed that plp dub activity is required for inhibition of innate immunity in infected cells (van kasteren et al., 2013) . specific deubiquitinating and deisgylating activities have been shown for crimean-congo hemorrhagic fever virus (cchfv) which is a highly pathogenic, negative-strand rna virus belonging to the family bunyaviridae. the l protease of cchfv contains otu domain with the ability to cleave isg15 modification. l protease can remove isg15-meidated immune protection in type i ifn receptor knock-out mice and make them highly susceptible to sindbis virus infection (frias-staheli et al., 2007) . overall, the results from multiple laboratories studying a variety of coronavirus, arterivirus, and bunyavirus proteases indicate that deubiquitinating and deisgylating activity of viral proteases have an important role for inhibition of innate immune responses and possibly virus pathogenesis. here, we characterized the papain-like protease from mers-cov revealing the deisgylating and deubiquitinating activities, and that it can act as an interferon antagonist. further, we showed for the first time that sars-cov plpro and mers-cov plpro can block induction of several endogenous proinflammatory cytokines. our data suggest that antagonism of innate immune responses mediated by mers-cov and sars-cov plpros is not limited to ifnβ, but may affect expression of many cellular cytokines. our results suggest that plpro might contribute to the modulation of innate immune responses upon sars-cov and mers-cov infection, however, the exact mechanism and the role of coronavirus plps and their associated dub and deisgylating activities in these processes remains to be determined. the crystal structure sars-cov plpro (pdb:2fe8) was used as the template structure to generate a homology model of mers-cov plpro using the automated web-based homology modeling server 3d-jigsaw (bimolecular modeling laboratory, cancer research uk, england). ccp4 program suite 6.2.0 and coot version 0.6.2 were used for final refinement, energy minimization and modeling of ub into the zinc finger and palm regions of mers-cov plpro by using the electron density of sars-cov plpro in complex with ubiquitin-aldehyde (pdb:4mm3). hek293t cells were cultured in dulbecco's modified eagle medium (dmem) with 10% fetal calf serum (fcs) and 2% glutamine. transfections were performed with 70% confluent hek293t cells in cell bind plates (corning) using transit-lt1 reagent (mirus) according to manufacturer's protocol. the mers-cov plpro (pcdna-mers-plpro) expression plasmid and generation of catalytic mutant were described previously (kilianski et al., 2013) . pcdna-sars-plpro wild-type and catalytic mutant expression plasmids were described elsewhere (barretto et al., 2005) . for the luciferase assay experiments we used ifnβ-luc deisgylating activity assay hek293t cells in 12-well plates were transfected with 10, 25, 50, 100 ng of pcdna-mers-plpro wild-type or catalytic mutant, and 250 ng pisg15-myc, 125 ng pubch8, 125 ng pube1l, and 125 ng pherc5. at 20 h post-transfection, cells were lysed with lysis buffer (20 mm tris (ph 7.5), 150 mm nacl, 1 mm egta, 1 mm edta, 1% triton x-100, 2.5 mm na pyrophosphate, 1 mm betaglycerophosphate, 1 mm na ortho-vanadate, 1 mg/ml leupeptin). proteins were separated by sds-page, and transferred to pvdf membrane using a semi-dry transfer apparatus (biorad). following transfer, the membrane was blocked using 5% dried skim milk in tbst buffer (0.9% nacl, 10 mm tris-hcl, ph ¼7.5, 0.1% tween 20) overnight at 4 1c. the membrane was incubated with mouse anti-myc antibody (mbl) at the dilution of 1:2500. the membrane was washed 3 times for 15 min in tbst buffer. following the membrane was incubated with secondary goat-anti-mouse-hrp antibody at the dilution 1:5000 (amersham). then the membrane was washed 3 times for 15 min in tbst buffer. the detection was performed using western lighting chemiluminescence reagent plus (perkinelmer) and visualized using fluorocheme imager (protein simple). to verify expression of the plpro the membrane was probed with mouse anti-v5 antibody (invitrogen) at the dilution 1:5000. mouse anti-calnexin antibody (cell signal) at the dilution 1:2000 was used to determine loading standard. to assess dub activity, hek293t cells in 12-well plates were transfected with 400 ng pcdna3.1-flag-ub and 0.25, 0.5, or 1 mg pcdna-mers-plpro wild-type or catalytic mutant. at 18 h posttransfection, cells were lysed with 100 ml of lysis buffer. proteins were separated by sds-page and transferred to pvdf membrane as described above. membrane probing was performed using mouse anti-flag m2 antibody (sigma) at the dilution of 1:2000. hek293t cells in 24-well plates were transfected with 50 ng renilla-luciferase, 100 ng ifn-β-luc, and 25, 50, and 100 ng pcdna-mers-plpro wild-type or catalytic mutant expression plasmids. as a stimulation 150 ng pef-bos mda5, or 50 ng pef-bos mavs, or 50 ng n-rig-i per well was transfected. empty pcdna3.1-v5/his-b vector plasmid was used to standardize the total amount of dna used for transfection. at 16 h post-transfection cells were lysed using 1x passive lysis buffer (promega). alternatively, the cells were transfected with 50 ng pgl4 32 [luc2 nf-κb-re hyrgo], 100 ng ifnβ-luc and pcdna-mers-plprov5 wild-type or catalytic mutant for 12 h and then treated with 10 ng/ml tnfα (roche) for 4 h, and lysed. for all experiments firefly and renilla luciferase were measured using dual luciferase reporter assay system (promega) and luminometer (veritas). results were normalized to renilla luciferase expression control. experiments were performed in triplicate. remaining lysates were incubated with lysis buffer a (0.9% nacl, 10 mm tris-hcl, ph 7.5, 0.1% tween-20) and analyzed by sds-page as described above. hek293t cells in 12-well plates were transfected with 300 ng pef-bos mda5 and 200 ng pcdna-mers-plpro wild-type or catalytic mutant expression plasmids, or 200 ng pcdna-sars-plpro wild-type or catalytic mutant expression plasmids. empty vector plasmid pcdna3.1-v5/his-b vector was used to standardize the total amount of dna in each sample. the cells were lysed 18 h posttransfection with buffer rlt (qiagen) and rna was extracted using rneasy mini (qiagen). reverse transcription was performed using 1 mg of total rna and the rt 2 first strand kit (qiagen) according to manufacturer's protocol. 1 ml of cdna was used to set up qrt-pcr reaction according to the manufacturer's protocol using single primer assay for ifnβ, cxcl10, and ccl5 (sabiosciences). c t values were normalized to housekeeping gene (rpl13). hospital outbreak of middle east respiratory syndrome coronavirus the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity deubiquitinating and interferon antagonism activities of coronavirus papainlike proteases proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus nl63 clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling ovarian tumor domain-containing viral proteases evade ubiquitin-and isg15-dependent innate immune responses clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus assessing activity and inhibition of mers-cov papain-like and 3c-like proteases using luciferase-based biosensors severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists a twopronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme selectivity in isg15 and ubiquitin recognition by the sars coronavirus papain-like protease middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist coronaviruses post-sars: update on replication and pathogenesis innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome-coronavirus middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme the rna helicase lgp2 inhibits tlr-independent sensing of viral replication by retinoic acid-inducible gene-i a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15 sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells arterivirus and nairovirus ovarian tumor domaincontaining deubiquitinases target activated rig-i to control innate immune signaling plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology we thank dr. xufang deng for helpful discussions and review of the manuscript. this work was supported by the nih grant r01 ai085089 (to scb and adm). amm was supported by arthur j. schmitt dissertation fellowship from loyola university chicago. ak was supported by the nih training grant in experimental immunology (nih t32 ai512795). key: cord-255488-nvgz53su authors: li, kun; mccray, paul b. title: development of a mouse-adapted mers coronavirus date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_13 sha: doc_id: 255488 cord_uid: nvgz53su first identified in 2012, middle east respiratory syndrome coronavirus (mers-cov) is a novel virus that can cause acute respiratory distress syndrome (ards), multiorgan failure, and death, with a case fatality rate of ~35%. an animal model that supports mers-cov infection and causes severe lung disease is useful to study pathogenesis and evaluate therapies and vaccines. the murine dipeptidyl peptidase 4 (dpp4) protein is not a functional receptor for mers-cov; thus, mice are resistant to mers-cov infection. we generated human dpp4 knock-in (hdpp4 ki) mice by replacing exons 10–12 at the mouse dpp4 locus with exons 10–12 from the human dpp4 gene. the resultant human dpp4 ki mice are permissive to mers-cov (hcov-emc/2012 strain) infection but develop no disease. to generate a mouse model with associated morbidity and mortality from respiratory disease, we serially passaged hcov-emc/2012 strain in the lungs of young hdpp4 ki mice. after 30 in vivo passages, an adapted virus clone was isolated and designated mers(ma)6.1.2. this virus clone produced significantly higher titers than the parental clone in the lungs of hdpp4 ki mice and caused diffuse lung injury and a fatal respiratory infection. in this chapter, we will describe in detail the procedures used to mouse adapt mers-cov by serial passage of the virus in lungs. we also describe the methods used to isolate virus clones and characterize virus infection. middle east respiratory syndrome (mers) is a fatal respiratory illness that first appeared on the saudi arabian peninsula in mid-2012. it is caused by a novel betacoronavirus, mers coronavirus (mers-cov) [1] . shortly after the identification of the virus, its receptor dipeptidyl peptidase 4 (dpp4) was discovered [2] . as of september 2019, the world health organization (who) has reported 2468 laboratory-confirmed cases of mers in 27 countries, including 851 associated deaths (fatality rate: 35%). mers-cov does not currently have pandemic potential [3] [4] [5] . however, mers-cov is still epidemic in the middle east and remains a cause for significant concern due to the potential spread by global travel as demonstrated by the outbreak in south korea in 2015 [6] [7] [8] . to date, there are only two published reports on autopsy findings from subjects who died from mers [9, 10] and our understanding of mers-cov pathogenesis in humans is still limited. animal models are useful for the study of viral diseases and play an important role in the investigation of pathogenesis and evaluation of antiviral therapies and vaccines. an ideal animal model should be permissive to the viral infection and develop disease and pathology with similarities to that observed in humans. mers-cov infection has been evaluated in two nonhuman primate (nhp) models, the rhesus macaque and common marmoset [11] [12] [13] [14] [15] . both species are susceptible to mers-cov infection. however, mers-cov caused only a transient lower respiratory tract infection without mortality in rhesus macaques [11] [12] [13] . in common marmosets, the consequences of mers-cov infection are controversial. falzarano et al. reported the common marmoset reproduced several features of mers-cov infection in humans including progressive severe pneumonia [15] , while another group observed only mild to moderate nonlethal respiratory disease following mers-cov infection [14] . thus, the common marmoset is potentially a model to study pathogenesis and evaluate antiviral therapies and vaccines. however, nhps are expensive, their availability limited, and their use may raise ethical concerns. in contrast, small animal models provide advantages over nhps, including reduced cost, availability in large numbers, ease of handling, and species-specific reagents, especially for the studies of highly pathogenic viruses like mers-cov in the biosafety level 3 (bsl-3) laboratory. unfortunately, common small laboratory animals like mice [16, 17] , ferrets [18] , guinea pigs [19] , and hamsters [20] are not susceptible to mers-cov infection because their homologous dpp4 cannot be bound and utilized by mers-cov as a host receptor for entry [21, 22] . haagmans et al. detected mers-cov rna in the respiratory tract of new zealand white rabbits following inoculation but found no clinical signs of disease [23] . several strategies have been used to overcome this receptor incompatibility and develop mouse models of mers-cov infection. in 2014, we developed the first mouse model of mers-cov infection [24] . we delivered a recombinant adenovirus 5 encoding human dpp4 (hdpp4) to the lungs of mice. transient expression of hdpp4 by adenovirus transduction made the mice temporarily permissive for mers-cov infection, but animals developed only mild lung disease. generation of transgenic mice expressing a virus receptor is a common strategy to make mice permissive to infection. several groups in addition to ours developed mice with transgenic expression of hdpp4 using different promoters [25] [26] [27] [28] . the disease severity following mers-cov infection in transgenic mice correlated with the cellular distribution and expression level of hdpp4. in transgenic mice expressing hdpp4 driven by the cytokeratin 18 promoter [26] or a ubiquitous promoter [25, 27, 28] , mers-cov replicates and causes respiratory disease and mortality. however, the lethality was found to be secondary to overwhelming central nervous system (cns) disease or multiorgan damage. this was also observed in mice transgenic for human ace2 driven by the cytokeratin 18 promoter and infected with sars-cov [29] . in transgenic mice expressing hdpp4 under the human surfactant protein c (spc) promoter, which restricts expression to bronchiolar and alveolar epithelia, mers-cov infection caused only mild disease [26] . thus, these transgenic mice do not reproduce a severe lung disease phenotype that resembles mers. alternative strategies for the creation of mouse models of mers-cov infection are generation of dpp4 humanized mice and adaptation of the virus to the animals. pascal et al. reported a model in which all of the mouse dpp4 exons had been humanized and also generated humanized monoclonal antibodies against the mers-cov s protein using a novel strategy [30] . mers-cov infection in this model caused pulmonary edema, vascular cuffing, and alveolar septal thickening with an associated~20% weight loss, necessitating euthanasia [31] . another mers mouse model was engineered by changing two amino acids in the mouse dpp4 locus using crispr-cas9 technology [32] . this model supported mers-cov replication without severe disease. similarly, our human dpp4 knock-in mouse model supported mers-cov replication but did not lead to a severe lung disease phenotype [33] . two mouse-adapted (ma) strains of mers-cov were subsequently developed independently by serial passage of the hcov-emc/2012 strain [1] in the lungs of the two humanized mouse models [32, 33] . the resultant mers-15 and mers ma 6.1.2 mouse-adapted mers-cov strains replicated to high titers in the lungs of the crispr-cas9 genetically engineered mouse model and the hdpp4 knock-in mouse model, respectively. the respiratory disease that developed in both mouse models and the associated mortality shared similarities with severe cases of mers [32, 33] . mouse adaptation was also successfully used to generate several sars-cov strains capable of modeling severe sars-cov lung disease in mice [34] [35] [36] . thus, the adaption of the virus to enhance virulence in the mouse is a very useful approach to generate mouse models for coronavirus-associated lung disease. these materials can be altered to fit the requirements for other viruses of interest. 1. hcov-emc/2012 strain. 2. hdpp4 knock-in mouse (c57bl/6 strain with mouse dpp4 exons 10-12 replaced with the human codons). all procedures are performed under bsl-3 laboratory conditions and must follow the standard operating protocol of a bsl-3 facility and regulatory agencies. all manipulations of infectious specimens, samples, and mice must be performed within a biosafety cabinet or within a contained device such as a centrifuge. all tissue culture media and waste must be bleached and autoclaved prior to disposal. all instruments must be disinfected with virex plus or 10% bleach. 5. insert surgical scissors under the sternum and cut the diaphragm following the costal arch. remove the rib cage using scissors and forceps, exposing the lungs and heart. 6. fill a 10 ml syringe with cold sterile dpbs using a 25 g â 5/8 inch needle. insert the needle into the apex of the left ventricle and make a small incision in the right atrium. slowly perfuse !5 ml cold sterile dpbs into the left ventricle. next, insert the needle into the apex of the right ventricle and perfuse !5 ml cold sterile dpbs (see note 10). 7. remove the lungs and heart from the thoracic cavity. remove the liver, kidney, spleen, and small intestine. place organs in a small polystyrene weighing dish. remove remaining connective tissue. 8. to harvest the brain, turn the mouse over and wet the fur of the head with 70% ethanol. grasp the ears with forceps and cut off the skin and fur to expose the skull. remove remaining skin at the base of the neck to further expose the skull. immobilize the mouse and make an incision along the sagittal suture of the skull using a single edge razor blade (see note 11) . wedge one prong of the curved serrated forceps into the now opened sagittal suture. slowly pry up the skull, grasp the piece of skull with forceps and peel outward to remove. repeat on the other side. use curved forceps to lift the brain from the skull. place brain tissue in the small polystyrene weighing dish. 9 . carefully place each organ into a 50 ml disposable tissue grinder filled with 2 ml sterile cold dpbs for homogenization or into 2 ml of trizol for rna extraction (see note 12). 10. grind the lung tissue and transfer the homogenates or rna samples into 2.0 ml sterile screw-top tubes with o-ring cap (see note 13) . store samples at à80 c. thaw and spin down the cell debris in lung homogenates before use. the virulence of the virus should be evaluated in groups of mice by weight loss and survival after every 5-10 in vivo passages. after the virulence of the virus has been significantly enhanced, single plaques of the adapted virus should be purified and evaluated. 1. plate vero81 cells in d10 media in 6-well plates one day before infection. 2. rapidly thaw lung homogenates from the selected passage. mix the lung homogenates from two mice in a 2.0 ml sterile screwtop tube with o-ring cap on ice. 3. serially dilute the mixed lung homogenates tenfold in 2.0 ml sterile screw-top tubes with o-ring caps, using ice-cold serumfree dmem (see note 15) . keep the dilutions on ice. 4. remove the medium from each well and add diluted samples (in a volume of 400 μl) to each well. 5. place the plates in the 37 c incubator for 1 h and rotate gently every 15 min. 6. melt 2% low melting point agarose and maintain in a 65 c water bath. 7. mix overlay media and 2% low melting point agarose at a volume ratio of 1:1. rotate the tube several times to fully mix. overlay cells with 1.5 ml of mixed media using 10 ml stripettes. 8. let the plates sit in the hood for~5 min at rt or until the agarose overlay turns solid. add 0.5 ml d2 medium on the top of solidified agarose. 9 . place the plates in the 37 c incubator. 10 . after 3 days, plaques should be visible. remove the liquid on the top of the agarose. circle the visible plaques on the underside of the plates using a permanent marker (see note 16). 11. vertically penetrate the agarose and pipette the circled plaque several times with a 1 ml graduated transfer pipette. the agarose above the plaque will be pulled into the pipette. 12. transfer the agarose above the plaque into a 15 ml conical tube filled with 500 μl dmem by pipetting up and down several times. 13. transfer the 500 μl dmem containing the agarose into a 2.0 ml sterile screw-top tube with o-ring cap. 14. repeat the procedure and pick six single plaques. store the tubes at à80 c. 8. animal euthanasia should follow the institution's animal care and use guidelines. there may be differences in institutional requirements regarding when euthanasia is required based on weight loss. 9 . use the lid of an insulated foam shipping box. cut absorbent bench underpad (42 â 58 cm) into small pieces that fit the lid. 10 . carefully keep the tip of the needle in the lumen of the ventricle. be sure to puncture the right atrium as this will help to drain blood during the perfusion. the lung will turn white after perfusion. 11. make sure that the incision does not exceed the thickness of the skull; avoid cutting into the brain tissue. 12. the individual organs can be divided into pieces and transferred to grinders with pbs or trizol separately. 13. lung homogenates are immediately transferred to tubes on ice. rna samples are transferred to a tube and incubated for at least 15 min at room temperature per our bsl-3 specific standard operating procedures. 14. at 2 days post infection with 10 5 pfu/mouse, hcov-emc/ 2012 strain replicates in the lung of the hdpp4 ki mice to a titer of around 4 â 10 6 pfu/ml, which equals 2 â 10 5 pfu in 50 μl. we chose the 10 5 pfu/mouse inoculum to begin because we wanted to use a similar dose range during in vivo serial passage while skipping the titration step. 15. titrate the homogenates of the passage of interest and select a virus dilution that produces 10 plaques in a well. this allows identification of individual clear single plaques. 16 . only circle the unambiguous clear single plaques. 17. compared to vero 81 cells, we found that the mers-cov rna genome is more stable when propagated in huh7 cells (less likely to introduce genomic deletions, insertions, or point mutations). isolation of a novel coronavirus from a man with pneumonia in saudi arabia dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk person-toperson spread of the mers coronavirus-an evolving picture the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission spread of mers to south korea and china infectious diseases epidemic threats and mass gatherings: refocusing global attention on the continuing spread of the middle east respiratory syndrome coronavirus (mers-cov) comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease infection with mers-cov causes lethal pneumonia in the common marmoset mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus domestic pig unlikely reservoir for mers-cov the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection asymptomatic middle east respiratory syndrome coronavirus infection in rabbits rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a mouse model for mers coronavirus-induced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease we thank chris wohlford-lenane and jennifer bartlett for careful review of the manuscript. this work is supported by the national institutes of health p01 ai060699. we also acknowledge the support of the cell morphology core and pathology core, partially supported by the center for gene therapy for cystic fibrosis (national institutes of health p30 dk-54759) and the cystic fibrosis foundation. p.b.m. is supported by the roy j. carver charitable trust. key: cord-280350-ay4cnzn5 authors: chan, jasper f.w.; chan, kwok-hung; kao, richard y.t.; to, kelvin k.w.; zheng, bo-jian; li, clara p.y.; li, patrick t.w.; dai, jun; mok, florence k.y.; chen, honglin; hayden, frederick g.; yuen, kwok-yung title: broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus date: 2013-10-03 journal: j infect doi: 10.1016/j.jinf.2013.09.029 sha: doc_id: 280350 cord_uid: ay4cnzn5 objectives: middle east respiratory syndrome coronavirus (mers-cov) has emerged to cause fatal infections in patients in the middle east and traveler-associated secondary cases in europe and africa. person-to-person transmission is evident in outbreaks involving household and hospital contacts. effective antivirals are urgently needed. methods: we used small compound-based forward chemical genetics to screen a chemical library of 1280 known drugs against influenza a virus in biosafety level-2 laboratory. we then assessed the anti-mers-cov activities of the identified compounds and of interferons, nelfinavir, and lopinavir because of their reported anti-coronavirus activities in terms of cytopathic effect inhibition, viral yield reduction, and plaque reduction assays in biosafety level-3 laboratory. results: ten compounds were identified as primary hits in high-throughput screening. only mycophenolic acid exhibited low ec(50) and high selectivity index. additionally, ribavirin and interferons also exhibited in-vitro anti-mers-cov activity. the serum concentrations achievable at therapeutic doses of mycophenolic acid and interferon-β1b were 60–300 and 3–4 times higher than the concentrations at which in-vitro anti-mers-cov activities were demonstrated, whereas that of ribavirin was ∼2 times lower. combination of mycophenolic acid and interferon-β1b lowered the ec(50) of each drug by 1–3 times. conclusions: interferon-β1b with mycophenolic acid should be considered in treatment trials of mers. a novel lineage c betacoronavirus, previously known as human coronavirus emc/2012 and later renamed as middle east respiratory syndrome coronavirus (mers-cov), has emerged in the arabian peninsula since april 2012 to cause a "severe acute respiratory syndrome (sars)-like" disease in 136 laboratory-confirmed cases with 58 fatalities in 9 countries in the middle east, europe, and north africa as of 4 october 2013. 1e5 animal-to-human transmission has been suspected in view of mers-cov's close phylogenetic relatedness to other lineage c betacoronaviruses found in bats in hong kong, mexico, europe, and africa, 6e13 and its broad species tropism in various animal cell lines including those of bats, primates, pigs, civets, and rabbits. 14, 15 recently, a serological study of major livestock suggested dromedary camels to be a possible host based on the high prevalence of mers-cov neutralizing antibodies in dromedary camels from oman. 16 however, targeted studies are needed to confirm this finding and its possible relevance to human cases of mers-cov infection as most cases did not have contact with camels and the virus has not been isolated in animals yet. the epidemic continues to evolve with recent outbreaks occurring among epidemiologically-linked household contacts in the kingdom of saudi arabia, the united kingdom, italy, and tunisia, and hospital contacts in the kingdom of saudi arabia, jordan, the united kingdom, and france providing evidence for mers-cov's potential for person-to-person transmission. 17e23 unlike most other human coronavirus infections which are generally mild, most patients with mers have suffered from rapidly progressive pneumonia with some also developing acute renal failure, hepatic dysfunction, gastrointestinal upset, pericarditis, disseminated intravascular coagulation, and/or cytopenias. 2, 24 the resulting crude mortality rate of nearly 50% in documented cases far exceeded those seen in all other human coronavirus infections including sars despite aggressive supportive treatment including extracorporeal membrane oxygenation in some of the mers cases. while mild and asymptomatic cases have been recognized, 2,19,24 these recent case clusters signify a global health threat especially in view of the unusual clinical severity of mers, travel of infected persons to other countries and influx of religious pilgrims to the kingdom of saudi arabia, and the lack of proven effective specific antiviral treatment. after our initial success in applying chemical genetics in probing novel targets and compounds for antiviral development, 25 we started looking for broad-spectrum antiviral compounds that may be active against both influenza a viruses and coronaviruses, the two viral pathogens responsible for causing the recent 2009 pandemic and largescale epidemics. 9 while neuraminidase inhibitors such as oseltamivir and zanamivir remain effective against most seasonal and avian influenza a viruses, 26e30 proven antiviral therapeutic options for coronavirus infections is lacking. given the limited time available to develop novel anti-mers-cov agents in this evolving epidemic, we attempted to provide an alternative solution by identifying potential broad-spectrum antiviral agents against mers-cov and influenza a viruses by a small compound-based forward chemical genetics approach using chemical libraries consisting of 1280 drug compounds already marketed or having reached clinical trials in the united states, europe, or asia (microsource discovery systems, usa). 25 we then assessed the anti-mers-cov activities of the identified drug compounds in cell culture by cytopathic effect (cpe) inhibition, viral yield reduction, and plaque reduction assay (pra) assays, as well as drug cytotoxicity. a clinical isolate of mers-cov was kindly provided by r. fouchier, a. zaki, and colleagues. 3 the isolate was amplified by one additional passage in vero cells to make working stocks of the virus (4 â 10 5 tcid 50 /ml). all experimental protocol involving live mers-cov isolate followed the standard operating procedures of the approved biosafety level-3 facility as we previously described. 31 the influenza a/ wsn/1933 (h1n1) virus was expanded in chick embryo as we previously described. 25 chemical reagents and high-throughput screening (hts) a total of 1280 pre-existing drug compounds (microsource discovery systems) were screened against influenza a/ wsn/1933 (h1n1) virus. high-throughput screening (hts) was carried out in a fully automated beckman coulter core system (beckman coulter, usa) integrated with a kendro robotics co 2 incubator (thermo fisher scientific) at chemical genetics unit, department of microbiology, research center of infection and immunology, li kashing faculty of medicine, the university of hong kong as we previously described with modifications. 25 briefly, compounds were added in 96-well microtitre plates (tpp) in duplicate with a final concentration of 10 mm or 100 mm and 20,000 madinedarby canine kidney (mdck) cells per well in 100 ml complete eagle's minimal essential medium (emem) supplemented with 1% heat-inactivated fbs. cells were then inoculated at an moi of 0.01 with influenza a/ wsn/1933 (h1n1) virus for detection of broad-spectrum antivirals. after infection, the plates were incubated at 37 c with 5% co 2 and monitored daily using a leica dm inverted light microscope for virus-induced cpe. drugs that gave full protection of mdck cells (no cpe) were selected for further evaluation with mers-cov in a biosafety level-3 laboratory. the cytotoxicity of selected drug (ribavirin: 1600e0.1 mg/ml; introna 75,000e4.58 iu/ml; avonex: 75,000e4.58 iu/ml; rebif: 250,000e15.26 iu/ml; betaferon: 50,000e3.05 iu/ml; mmf: 32e0.25 mg/ml) was determined by thiazolyl blue tetrazolium bromide (mtt) assay according to manufacturer's instructions. the endpoint was the 50% effective cytotoxic concentration (tc 50 ). the drug compounds identified as primary hits showing a ec 50 of less than or equal to 50 um and a selectivity index of more than 100 were diluted with serum free mem and added to confluent vero cells in 96-well culture plates in triplicate for 2 h at 37 c. after incubation, the drugcontaining media was removed, and mers-cov at 0.0001 moi was added together with fresh drug-compound media to each well containing approximately 60,000 cells. following 1 h adsorption at 37 c, the virus-compound mixture was removed and the cells were washed 2 times with mem to remove unbound virus. subsequently, media with antiviral compounds were added to the cells for further incubation for 72 h at 37 c in a 5% co 2 humidified environment. cpe was examined by inverted light microscopy, and 50 ml of supernatant was collected for virus quantification, as we previously described with modifications. 14 thereafter, 50 ml of serum free mem and 10 ml of 5 mg/ml mtt solution (prepared in 1 â pbs, filtered) were added to the wells. the monolayers were incubated as above for 4 h (away from light). finally, 100 ml of 10% sds with 0.01 m hcl was added and further incubated at 37 c with 5% co 2 overnight. the activity was read at od 570 with reference wavelength at od 640 . the interferon and non-interferon drug compound with the lowest 50% effective inhibitory concentration (ec 50 ) and highest selectivity index were selected for combination studies using the cpe inhibition assay. for the drug compounds with antiviral activity in the mtt assay, further evaluation by quantitative virus yield reduction and plaque reduction assays (pra) was performed. virus yield quantification was performed by quantitative rt-pcr using total nucleic acid extracted from culture supernatants of the vero cells infected by mers-cov on day 3 post-infection as we previously described. 14 pra was performed as we previously described with modifications. 32 briefly, it was performed in duplicate in 24well tissue culture plates (tpp). the vero cells were seeded at 1 â 10 5 cells/well in mem (invitrogen) with 10% fbs on the day before carrying out the assay. after 16e24 h incubation, 70e100 plaque-forming units (pfu) of mers-cov virus were added to the cell monolayer with or without the addition of drug compounds and the plates further incubated for 2 h at 37 c in 5% co 2 atmosphere before removal of unbound viral particles by aspiration of the media and washing once with mem. monolayers were then overlaid with media containing 1% low melting agarose (cambrex) in mem and appropriate concentrations of drug compounds and incubated as above for 72 h. next, the wells were fixed with 10% formaldehyde (bdh) overnight. after removal of the agarose plugs, the monolayers were stained with 0.7% crystal violet (bdh) and the plaques counted. the percentage of plaque inhibition relative to the control (without the addition of compound) plates was determined for each drug compound concentration. the ec 50 and the 50% cellular cytotoxicity concentration (cc 50 ) were calculated using sigma plot (spss) in an excel add-in ed50v10. the pra were carried out in triplicate and repeated twice for confirmation. high-throughput screening (hts) ten drugs compounds, namely mycophenolic acid, flufenamic acid, tolfenamic acid, meclofenamate sodium, mefenamic acid, ribavirin, mercaptopurine, pyrimethamine, emetine, and estradiol were identified as primary hits with protective results in chemical library screening against influenza a/wsn/1933 (h1n1) virus (table 1) . neuraminidase inhibitors were not identified because they were not included in the chemical library. amantadine was not identified because the virus strain had an m2 gene mutation (s31n) conferring drug resistance. using both ec 50 and tc 50 as the hit selection criteria, only mycophenolic acid exhibited a low ec 50 of <10 mm with a high selectivity index of >100. mercaptopurine, which is a competitive, selective, and reversible inhibitor of the sars-cov papain-like protease, 33 in addition to mycophenolic acid (sigmaealdrich, usa), ribavirin (tianxin pharmaceutical, china), intron a (recombinant interferon-a2b, schering-plough, usa), avonex (recombinant interferon-b1a, biogen idec, denmark), rebif (recombinant interferon-b1a, merck serono, italy), betaferon (recombinant interferon-b1b, bayer schering pharma, germany), imukin (recombinant interferon-g1b, boehringer ingelheim, germany), nelfinavir mesylate hydrate (agouron pharmaceuticals, usa), and lopinavir (abbott, usa) were also tested in the mtt assays because of their documented in vitro anti-sars-cov activities in previous reports. 34e37 among them, only mycophenolic acid, ribavirin, intron a, avonex, rebif, and betaferon showed anti-mers-cov activity at the tested concentrations ( table 2) . cpe was completely absent in vero cells infected with mers-cov on day 3 post-infection at concentrations of !0.063 mg/ml for mycophenolic acid and !100 mg/ml for ribavirin, and was decreased but not absent in the tested concentrations of intron a, avonex, rebif, or betaferon (table 3) . combination studies showed that the ec 50 of mycophenolic acid was lowered by 1.7e2.8 times in the presence of 6.25e12.5 iu/ml of betaferon, and that the ec 50 of betaferon was lowered by 1.1e1.8 times in the presence of 0.016e0.063 mg/ml of mycophenolic acid (table 2) . the mean baseline viral load in the cell culture supernatants without drugs was 12.110 ae 0.003 log 10 copies/ml. there was a 50% reduction in viral load as compared to the baseline in cell culture supernatants inoculated with each of the six drugs (fig. 1 ). there was a >2-log reduction in viral load in cell culture supernatants inoculated with mycophenolic acid, ribavirin, rebif, and betaferon. there was >1-log reduction in the viral load in cell culture supernatants at 40 iu/ml of betaferon and >3-log reduction at the highest concentration of 50,000 iu/ml (fig. 1c) . the largest reduction in viral load at clinically relevant drug levels was a nearly 4-log reduction at 16 mg/ml of mycophenolic acid. mycophenolic acid, ribavirin, and rebif achieved 100% plaque reduction at concentrations of 6.4 mg/ml, 400 mg/ml, and 62,500 iu/ml respectively (figs. 2 and 3) . the maximum percentages of plaque reduction achieved by intron a, avonex, and betaferon were 76.2% at 70,000 iu/ml, 70.2% at 5000 iu/ml, and 66.6% at 400 iu/ml respectively (fig. 3) . in pra, betaferon achieved 40e50% plaque reduction at 40 iu/ml (fig. 3c ). novel antiviral targets for sars coronavirus and influenza a virus have been identified previously using small compoundbased forward chemical genetics approaches similar to ours. 25, 38, 39 in this study, we identified ten compounds among approved drugs with as primary hits in chemical library screening that possess antiviral activities. some may offer potential therapies in the evolving mers-cov epidemic. influenza a/wsn/1933 (h1n1) virus, instead of mers-cov, was used for initial screening because its manipulation did not require a biosafetly level iii laboratory. other human betacoronaviruses such as hcov-oc43 and hcov-hku1 were not used because of their slow replication and low viral titres in cell culture. 14 among the 10 identified drug compounds, only mycophenolic acid exhibited an ec 50 of <10 mm, which is a common cut-off value for lead compound detection, and a high selective index of >100. additionally, we tested other agents reported to have in vitro activities against sars-cov and/or mers-cov. 32, 34, 40 imukin (interferon-g1b) and the hiv protease inhibitors, nelfinavir mesylate hydrate and lopinavir, showed suboptimal ec 50 in the initial cpe inhibition assay and were therefore not further evaluated. together with mycophenolic acid, four other drug compounds in five preparations, namely ribavirin, intron a, avonex, rebif, and betaferon, showed in vitro anti-mers-cov activity of varying magnitude across four assays. mycophenolic acid is a selective, non-competitive, and reversible inhibitor of inosine-5 0 -monophosphate dehydrogenase (impdh). it inhibits the proliferation of t and b lymphocytes and production of immunoglobulins by depletion of the lymphocyte guansine and deoxyguanosine nucleotide pools. 41 its major clinical indication is prevention of graft rejection in solid organ and haematopoeitic stem cell transplantations. in addition to potent immunosuppressive activity, mycophenolic acid also has broad activity in vitro and/or in animal models against different viruses including west nile, 42 japanese encephalitis, 43 yellow fever, 44 dengue, 45 chikungunya, 46 and possibly hepatitis b viruses. 47 furthermore, it inhibited the in vitro and in vivo replication of hepatitis c virus by augmentation of interferon-stimulated gene expression and depletion of guanosine. 48, 49 combination treatment with interferon-a showed additive effects on interferon-stimulated gene expression and enhanced interferon-induced luciferase reporter activity. 48 as for coronaviruses, mycophenolic acid test 1 test 2 test 3 12,500.000 t t t 50,000.000 t t t remarks: -, negative; 1þ is defined as 1%e25% involvement; 2þ is defined as >25%e50% involvement; 3þ is defined as >50%e 75% involvement; 4þ is defined as >75% involvement; t, druginduced toxic effects in vero cells. was found to be ineffective against sars-cov in an animal model, although it did not significantly increase the viral load in the lungs of sars-infected balb/c mice as ribavirin did. 50 we are unaware of data on its activity against other human coronaviruses. our study is the first to demonstrate the anti-coronavirus activity of mycophenolic acid against the novel mers-cov. in addition to mycophenolic acid, our in vitro findings indicated that ribavirin, interferon-a, and interferon-b had anti-mers-cov activities in vitro. in the case of sars-cov, their antiviral activities in in vitro susceptibility tests had been conflicting. 34 none of them were tested systemically in large-scale randomized controlled trials and the results from clinical trials involving their use in sars were often confounded with the concomitant use of corticosteroids. 51, 52 although their clinical use in mers-cov infection has not been described, a recent study found that ribavirin had in vitro anti-mers-cov activity at very high concentrations which was potentiated when given together with interferon-a2b. 40 another study showed that mers-cov is 50e100 times more sensitive to pegylated interferon-a than sars-cov in vero cells, which is possibly related to the lineage-specific genetic differences between the two coronaviruses with mers-cov lacking the homolog of the sars-cov orf6 protein responsible for the blockade of interferon-induced nuclear translocation of phosphorylated transcription factor stat1. 53 furthermore, the delayed and aberrant induction of inflammatory cytokines and chemokines by mers-cov might support the use of adjunctive immuno-modulatory treatment combined with antivirals in patients with mers. 54, 55 among the four preparations of interferons tested, betaferon exhibited the lowest ec 50 of 17.64 iu/ml, which was below the mean peak serum concentration of 40 iu/ml after a subcutaneous dose of 16 million iu or an intravenous dose of 0.2 million to 64 million iu. 56 although the other preparations of interferons also demonstrated in vitro anti-mers-cov activities, their ec 50 were generally above the peak serum concentrations achievable with usual therapeutic dosing. combination treatment consisting of mycophenolic acid and betaferon resulted in a 1.7e2.8-fold reduction in the ec 50 of mycophenolic acid in vero cells with 6.25e12.5 iu/ml of betaferon, and 1.1e1.8-fold reduction in the ec 50 of betaferon in vero cells with 0.016e0.063 mg/ml of mycophenolic acid. our finding may provide the basis for combinational mycophenolic acid and betaferon in future clinical trials. compared with ribavirin and interferons, mycophenolic acid exhibits a number of attributes that support its practical use in mers-cov infection. it is commonly available in two forms, the prodrug mycophenolate mofetil and the salt mycophenolate sodium, and could be given orally or parenterally. the serum concentration of mycophenolic acid peaks at around 10e50 mg/ml after a 1000 mg oral dose of mycophenolate mofetil or 26.1 mg/ml after a 720 mg oral dose of mycophenolate sodium. these far exceeds its ec 50 of 0.17 mg/ml and is 60e300 times higher than the concentrations at which the replication of mers-cov is inhibited in cell culture and pra. 57 with average plasma elimination half-lives of 17.9 h and 16.6 h after a 1000 mg oral dose and 1500 mg intravenous dose of mycophenolate mofetil respectively, 41 the usual regimens consisting of 1000 mg twice daily oral or 1500 mg twice daily intravenous mycophenolate mofetil would be sufficient to achieve levels well above the ec 50 throughout the dosing interval. in contrast, the ec 50 of ribavirin for mers-cov between 9.99 and 41.45 mg/ml is just marginally effective in some cell lines and greatly exceeds the drug's serum concentration with usual oral doses. peak concentrations with high intravenous doses may reach approximately 24 mg/ml in humans, but steady-state requires at least 4 weeks to achieve. 40, 58 furthermore, the use of ribavirin, and hence also its combination with interferon-a2b, may be limited in the clinical setting, because a significant proportion of patients with mers-cov infection have developed acute renal failure often requiring renal replacement therapy. 2, 24 it has been suggested that systemic ribavirin should best be avoided in patients with a creatinine clearance of <50 ml/min because of the increased risk of haemolytic anaemia. 59 although mycophenolic acid may also be associated with acute renal impairment, the dosage adjustment in such a setting is generally well established. 41 the potent in vitro anti-mers-cov activity of mycophenolic acid may allow it to be used as a monotherapy if concomitant interferon is not available or tolerated by the patient. finally, drug level monitoring for mycophenolate mofetil is generally available in most tertiary hospitals which are the usual referral centers for cases of severe mers-cov infections requiring intensive care facilities such as extracorporeal membrane oxygenation. however, the risk of immunosuppression at the onset of adaptive immune responses or polarization towards a deleterious th1 response by mycophenolic acid needs to be considered. 60 one possible approach is a short course of mycophenolate mofetil combined with an interferon, particularly interferon-b1b, to provide synergistic antiviral and immune-enhancing effects against mers-cov. these options should be considered for study in randomized control clinical trials for this highly fatal disease. there are a number of limitations in our study. firstly, the cytotoxicity assay likely underestimated more subtle effects of candidate compounds on host cell growth and metabolism. for example, ribavirin inhibits replication of uninfected mdck cells at concentrations of 10 mg/ml and above but does not cause overt cytotoxicity until much higher concentrations are reached. 61, 62 secondly, we used vero cells alone to study the antiviral activity of ribavirin. vero cells have been described as being comparatively resistant to ribavirin due to their inefficient conversion of the drug into its mono-and tri-phosphate forms. 63 however, we decided not to perform the experiment using another cell line as this has been done in another recent report using vero and llc-mk2 cell lines which also demonstrated anti-mers-cov activity of high ribavirin concentrations similar to our findings. 40 it would be important to extend these in vitro studies to human respiratory epithelial cell systems and explants. to optimize treatment options for mers-cov infection, further studies on the anti-mers-cov activities of other potential anti-coronavirus agents which have been previously identified for sars-cov should be undertaken. replication of many coronaviruses including sars-cov and mers-cov requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3clpro) and a papain-like protease (plpro). however, the protease inhibitors such as nelfinavir and lopinavir were not found to be active in our in vitro study. helicase inhibitors are another group of agents with in vitro anti-sars-cov activities but their anti-mers-cov activities remain undetermined. 39, 64 inhalational nitric oxide was used as rescue therapy for sars and might be useful for treating mers-cov infection if organic nitric oxide donors such as s-nitro-n-acetylpenicillamine also show anti-mers-cov activity. 65, 66 antiviral peptides or neutralizing antibodies designed against heptad repeat region 2 of s2 which may inhibit membrane fusion and cell entry of sars-cov could theoretically be harnessed for mers-cov since the s2 region shared significant homology amongst betacoronaviruses. 67e70 other agents with in vitro anti-sars-cov activities such as glycyrrhizin, baicalin, reserpine, aescin, valinomycin, niclosamide, aurintricarboxylic acid, mizoribine, indomethacin, chloroquine, and experimental agents like small interfering rna (sirna) and inhibitors targeting the binding interface between the s1 domian and receptor in vivo, should also be evaluated. 34, 35, 71 we did not test these agents in this study because most of them have the problems of either not being commercially available or having therapeutic levels that are not easily achievable clinically. recently, cyclophilin inhibitors, such as cyclosporine which is available commercially, have also been reported to exhibit anti-mers-cov and anti-coronavirus activity in cell culture and viral load studies. 53, 72 further evaluation of its potential therapeutic effects of these commercially available agents with in vitro activity should be conducted in randomized clinical trials as good animal models for mers are not widely available at this stage. 73 is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group global alert and response: middle east respiratory syndrome coronavirus (mers-cov) e update e as of 4 october comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features genetic relatedness of the novel human lineage c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus interspecies transmission and emergence of novel viruses: lessons from bats and birds genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe coronaviruses in bats from mexico middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications on disease pathogenesis and clinical manifestation human coronavirus emc does not require the sarscoronavirus receptor and maintains broad replicative capability in mammalian cell lines middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov) e worldwide clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study identification of influenza a nucleoprotein as an antiviral target two years after pandemic influenza a/2009/h1n1: what have we learned? avian influenza a h5n1 virus: a continuous threat to humans human infections with the emerging avian influenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome the emergence of influenza a (h7n9) sixteen years after influenza a(h5n1) in humans: a tale of two cities clinical, virological, and histopathological manifestations of fatal human infections by avian influenza a(h7n9) virus delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds treatment of sars with human interferons interferon-gamma and interleukin-4 downregulate expression of the sars coronavirus receptor ace2 in vero e6 cells characterization of sars-cov main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics inhibition of novel b coronavirus replication by a combination of interferon-a2b and ribavirin mycophenolate mofetil: an update identification of active antiviral compounds against a new york isolate of west nile virus mycophenolic acid inhibits replication of japanese encephalitis virus the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase mycophenolic acid inhibits dengue virus infection by preventing replication of viral rna cellular impdh enzyme activity is a potential target for the inhibition of chikungunya virus replication and virus induced apoptosis in cultured mammalian cells antiviral or proviral action of mycophenolic acid in hepatitis b infection? mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis c virus infection in vitro and in vivo mycophenolate mofetil inhibits hepatitis c virus replication in human hepatic cells enhancement of the infectivity of sars-cov in balb/c mice by imp dehydrogenase inhibitors, including ribavirin the management of coronavirus infections with particular reference to sars medical treatment of viral pneumonia including sars in immunocompetent adult mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment active mers-cov replication and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis product information: betaferon ò single use pack drug (aust r 83309) bioequivalence of enteric-coated mycophenolate sodium and mycophenolate mofetil: a metaanalysis of three studies in stable renal transplant recipients ribavirin in the treatment of sars: a new trick for an old bug? ribavirin monitoring in chronic hepatitis c therapy: anaemia versus efficacy mycophenolate mofetil and its mechanisms of action plaque inhibition assay for drug susceptibility testing of influenza viruses enhancement of activity against influenza viruses by combinations of antiviral agents cell type mediated resistance of vesicular stomatitis virus and sendai virus to ribavirin severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase inhalation of nitric oxide in the treatment of severe acute respiratory syndrome: a rescue trial in beijing nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests a predicted receptor-binding and critical neutralizing domain in s protein of the novel human coronavirus hcov-emc identification of a receptor-binding domain in the s protine of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development synthetic peptides outside the spike protein heptad repeat regions as potent inhibitors of sars-associated coronavirus suppression of coronavirus replication by cyclophilin inhibitors pneumonia from human coronavirus in a macaque model the authors have no financial or any other conflicts of interest regarding the contents of the investigations. key: cord-282554-hlcgutzf authors: yoo, jin-hong title: the fight against the 2019-ncov outbreak: an arduous march has just begun date: 2020-01-30 journal: j korean med sci doi: 10.3346/jkms.2020.35.e56 sha: doc_id: 282554 cord_uid: hlcgutzf nan this virus is a positive sense single stranded rna virus belonging to the family coronaviridae. most coronaviruses cause only mild upper respiratory infections, but sometimes they cause fatal respiratory disease and outbreaks, as experienced in cases of sars-cov or mers-cov. this disaster has been warned until recently that new mutants of coronavirus can occur anytime. 5, 6 the emergence of these mutants is caused by species jumping between human and other animals. therefore, it is likely to occur in an environment where human and animals are in close contact. the current outbreak is also suspected of being caused by mutants from species spillover in wuhan's wild animal market. the 2019-ncov is rapidly spreading throughout china and around the world in a relatively short period of time. as of 29 january 2020, a total of 6,140 cases were confirmed in 19 countries, including korea, of which 132 died (in china only) and had a mortality of 2.1%. 7 as much as the 2015 mers-cov outbreak, we are also learning a lot of lesson from this disaster. because epidemic is a national disaster, not only medical institutions but also governments have to be active. hence, honesty and transparency are, above all, the virtues that governments should have. our country is excellent at coping with this disaster, thanks to the experiences that we have gained during the 2015 mers-cov outbreak. 8 our defense system is at least more solid and faster than it used to be. what do you expect this ncov outbreak to be in the future? based on the sars epidemic precedence, the outbreak is expected to last at least three months. and this outbreak is expected to have a greater amount of transmission than the 2015 mers-cov. recently, the possibility of transmission by asymptomatic infected people has also been raised carefully although its evidence is unclear. therefore, we should also prepare for the spread to communities by asymptomatic infections. 9,10 after the mers-cov outbreak in 2015, there was a self-tormenting expression in the medical profession society: 'patients die, hospitals die, and civil servants are praised.' but the bad memories of the past should never be repeated. in fact, at present, cooperation between community health centers and private hospitals is not always harmonious. the government needs to be more active, not just to leave everything to the medical staffs. after all, the responsibility for the controlling nationwide epidemics lies with health authorities of the government. the korea center for disease control and prevention (kcdc) must be the control tower of the present disaster in korea and all other central or local governmental organizations must cooperate with the kcdc. toward the end of the 2019-ncov outbreak we must go on a march of arduousness. health workers, the government, and the people will need to unite to overcome this disaster. a novel coronavirus from patients with pneumonia in china clinical features of patients infected with 2019 novel coronavirus in wuhan world health organization. who issues best practices for naming new human infectious diseases surveillance case definitions for human infection with novel coronavirus (ncov) a sars-like cluster of circulating bat coronaviruses shows potential for human emergence origin and evolution of pathogenic coronaviruses tracking coronavirus: map, data and timeline clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea key: cord-264653-ms6zrrnd authors: bhatnagar, tarun; murhekar, manoj v.; soneja, manish; gupta, nivedita; giri, sidhartha; wig, naveet; gangakhedkar, raman title: lopinavir/ritonavir combination therapy amongst symptomatic coronavirus disease 2019 patients in india: protocol for restricted public health emergency use date: 2020-04-28 journal: indian j med res doi: 10.4103/ijmr.ijmr_502_20 sha: doc_id: 264653 cord_uid: ms6zrrnd as of february 29, 2020, more than 85,000 cases of coronavirus disease 2019 (covid-19) have been reported from china and 53 other countries with 2,924 deaths. on january 30, 2020, the first laboratory-confirmed case of covid was reported from kerala, india. in view of the earlier evidence about effectiveness of repurposed lopinavir/ritonavir against severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronavirus (cov), as well as preliminary docking studies conducted by the icmr-national institute of virology, pune, the central drugs standard control organization approved the restricted public health use of lopinavir/ritonavir combination amongst symptomatic covid-19 patients detected in the country. hospitalized adult patients with laboratory-confirmed sars-cov-2 infection with any one of the following criteria will be eligible to receive lopinavir/ritonavir for 14 days after obtaining written informed consent: (i) respiratory distress with respiratory rate ≥22/min or spo(2) of <94 per cent; (ii) lung parenchymal infiltrates on chest x-ray; (iii) hypotension defined as systolic blood pressure <90 mmhg or need for vasopressor/inotropic medication; (iv) new-onset organ dysfunction; and (v) high-risk groups age >60 yr, diabetes mellitus, renal failure, chronic lung disease and immunocompromised persons. patients will be monitored to document clinical (hospital length of stay and mortality at 14, 28 and 90 days), laboratory (presence of viral rna in serial throat swab samples) and safety (adverse events and serious adverse events) outcomes. treatment outcomes amongst initial cases would be useful in providing guidance about the clinical management of patients with covid-19. if found useful in managing initial sars-cov-2-infected patients, further evaluation using a randomized control trial design is warranted to guide future therapeutic use of this combination. wuhan, hubei, china, with clinical presentations greatly resembling viral pneumonia. deep-sequencing analysis from lower respiratory tract samples indicated a novel cov (ncov) 2 , which was named severe acute respiratory syndrome (sars)-cov-2 3 . since december 31, 2019 and as of february 29, 2020, a total of 85,403 cases of cov disease 2019 (covid -19) have been reported, including 2,924 deaths. of the total deaths reported, 2,838 were in people's republic of china (prc). other than china, confirmed cases have been reported from 53 countries 4 . as per the statement of the who emergency committee, covid-19 had a case-fatality ratio (cfr) of four per cent; however, recent reports suggested it to be between 1 and 2 per cent [5] [6] [7] . the published studies from china indicated that most cases with sars-cov-2-infected pneumonia were aged above 50 yr (median age: 55-59 yr), predominantly men (54-68%) and had chronic medical conditions (46.4-51%). the common symptoms included fever, fatigue, dry cough, myalgia, dyspnoea, expectoration and diarrhoea [8] [9] [10] [11] . the common laboratory abnormalities amongst 138 patients were lymphopenia [lymphocyte count, 0.8×10 9 /l (interquartile range iqr, 0.6-1.1), 70.3%], prolonged prothrombin time [pt, 13.0 sec (iqr, 12.3-13.7), 58%] and elevated lactate dehydrogenase [261 u/l (iqr, 182-403), 39.9%] 10 . unilateral (25%) or bilateral (75%) pneumonia and multiple mottling and ground-glass opacities (14%) were the common findings on chest x-ray/computed tomography (ct) scan 9, 10 . patients were treated with antivirals including oseltamivir, ganciclovir and lopinavir and ritonavir, antibiotics and glucocorticosteroids 8, 10 . no antiviral treatment for sars-cov-2 infection has been proven to be effective. a few historical control studies or case reports indicate the effectiveness of combination of lopinavir/ritonavir against sars-cov and mers-cov infections. ritonavir-boosted lopinavir was approved for use amongst hiv-infected individuals in september 2000 by the u.s. food and drugs administration 12 . the drug has been used for over 15 years in india. heat stable version of the medicine that is based on malt-extrusion technology meltrex™ technology was launched in india in 2007 13 . lopinavir is metabolized by cytochrome p4503a (cyp3a) isoenzyme in the liver. lopinavir is always used with ritonavir to reduce the dose of lopinavir and increase the plasma levels of lopinavir as ritonavir inhibits cyp3a isoenzyme. lopinavir and ritonavir are antiretroviral protease inhibitors used in combination as a second-line drug for the treatment of hiv-1 infection in children and adults and have limited side effects 14, 15 . as per the naco (national aids control organization) guidelines, lopinavir/ritonavir is used as a second-line drug in the treatment of hiv in combination with nucleoside reverse transcriptase inhibitors (nrtis). it is also recommended by naco in post-exposure prophylaxis in hiv for 28 days. it is also part of the first-line regimen for patients with hiv-2 infection 16 . the drug has been used extensively in the management of paediatric hiv infection, especially amongst infants. thus, there is a long period of experience of its use in india. a systematic review suggested no safety or efficacy concerns for use of standard-dose lopinavir/ritonavir amongst pregnant women 17 . boosted lopinavir has been used to prevent mother-to-child transmission of hiv-1 and hiv-2 infection 16 . the main viral protease has been regarded as a suitable target for drug design against cov infection due to its vital role in polyproteins processing necessary for cov reproduction. lopinavir/ritonavir has been shown to have the highest inhibitory potency against cov amongst several anti-hiv-1 protease inhibitors 18 . in a historical control study, lopinavir/ritonavir with ribavirin amongst sars-cov patients was associated with substantial clinical benefit. the adverse clinical outcome (ards or death) was significantly lower in the treatment group than in the controls who received only ribavirin (2.4 vs. 28.8%, p<0.001) at day 21 after the onset of symptoms. a reduction in steroid usage and nosocomial infections was seen in patients initially treated with lopinavir/ritonavir, and these patients had a decreasing viral load and rising peripheral lymphocyte count 19 . findings from in vitro and clinical studies, together with the availability and safety profiles of lopinavir/ritonavir and interferon beta-1b (ifn-β1b) suggest that the combination of these agents has potential efficacy for the treatment of patients with mers-cov. oral treatment with lopinavir/ritonavir in the marmoset model of mers-cov infection resulted in modest improvements in mers disease signs, including decreased pulmonary infiltrates identified by chest x-ray, decreased interstitial pneumonia and decreased weight loss 20 . studies on mers patients with treatment regimens including lopinavir-ritonavir reported positive disease outcomes including defervescence, viral clearance from serum and sputum and survival [21] [22] [23] [24] . arabi et al 25 in india, the first laboratory-confirmed case of covid-19 was reported from kerala on january 30, 2020 (https://pib.gov.in/pressrelesedetail. aspx?prid=1601095). subsequently, two more cases were reported from kerala. all cases had recently returned from wuhan, pr china, had mild illness and were managed symptomatically. more such cases can be expected amongst individuals travelling from china, and wuhan in particular, and amongst their close contacts. as covid-19 is an emerging virus, an effective treatment has not been developed for disease resulting from this virus. in view of the earlier evidence about the effectiveness of lopinavir/ritonavir against sars and mers-cov, the indian council of medical research (icmr) has suggested off-label emergency use of lopinavir/ritonavir combination for symptomatic covid-19 patients detected in the country. use of ifn-β1b and ribavirin was not considered due to their reported toxicity, whereas oseltamivir was not considered due to its unproven efficacy against covs. this article describes the protocol for the administration of lopinavir/ritonavir to such patients and their clinical monitoring. this protocol is to be implemented along with the who guidelines on clinical management of severe acute respiratory infection when ncov infection is suspected: interim guidance 26 . inclusion criteria: include (1) adult over 18 yr of age; (2) laboratory confirmation of covid-19 infection by real-time reverse transcription-polymerase chain reaction (qrt-pcr) from the recommended sample (3) symptomatic patients with any one of the following: (i) respiratory distress with respiratory rate ≥22/min or spo 2 of <94 per cent, (ii) lung parenchymal infiltrates on chest x-ray or ct scan, (iii) hypotension defined as systolic blood pressure <90 mmhg or need for vasopressor/inotropic medication, (iv) new-onset organ dysfunction (one or more of the following): (a) increase in creatinine by 50 per cent from baseline, glomerular filtration rate (gfr) reduction by >25 per cent from baseline or urine output of <0.5 ml/kg for six hours, (b) reduction of glasgow coma scale (gcs) score by two or more, and (c) any other organ dysfunction; (v) high-risk groups with age >60 yr, and those with hypertension, diabetes mellitus, renal failure, chronic lung disease and immunocompromised persons; (vi) informed consent from patient and caretaker. consent from legally authorized representative in case the patient is not able to provide the same due to his/her medical condition. exclusion criteria: (i) a patient with hepatic impairment [child pugh c or alanine aminotransferase (alt) over 5x the upper limit of normal]; (ii) use of medications that are contraindicated with lopinavir/ritonavir and that cannot be replaced or stopped, e.g., rifampicin, benzodiazepines, simvastatin, voriconazole and sildenafil; and (iii) known hiv-infected individual receiving other protease inhibitors containing regimens that cannot be replaced by lopinavir/ritonavir. 200 mg/50 mg -two tablets every 12 h for 14 days or for seven days after becoming asymptomatic, whichever is earlier; and (ii) for patients who are unable to take medications by mouth, 400 mg lopinavir /100 mg ritonavir 5 ml suspension every 12 h for 14 days or seven days after becoming asymptomatic whichever is earlier, via a nasogastric tube. all samples would be stored for future-related tests. should be monitored daily until discharge from the hospital and followed up till 90 days; and (ii) patient should be discharged on clinical recovery and after obtaining two consecutive negative rt-pcr results at least 24 h apart from oropharyngeal swabs (to demonstrate viral clearance). clinical outcomes: (i) hospital length of stay; (ii) intensive care unit (icu)-free days; (iii) requiring use of ventilator; (iv) mortality in the icu; (v) mortality in the hospital; and (vi) mortality at 14, 28 and 90 days. outcomes: (i) acute pancreatitis (defined as having: (a) abdominal pain radiating to the back; (b) serum amylase at least three times greater than the upper limit of normal; (c) radiological evidence, such as contrast ct/magnetic resonance imaging/ultrasonography, of acute pancreatitis); (ii) elevation of alt to more than five-fold upper normal limit; (iii) anaphylaxis; and (iv) adverse events and serious adverse events. laboratory outcomes: (i) viral rna loads and cycle threshold values in serial samples of nasopharyngeal and oropharyngeal swabs and blood, collected every third day (to document viral replication kinetics). the schedule of key investigations is given in the table. the complete clinical picture of covid-19 is not fully understood. the clinical manifestations in infected patients could range from mild illness to severe disease requiring icu admission and ventilatory support. the cfr of covid-19 is lower than that of sars (cfr: 14-15%) and mers (34%) 27, 28 . till now, no effective treatment has been recommended for covid-19, except meticulous supportive care 26 . the icmr has suggested lopinavir/ritonavir combination therapy for laboratory-confirmed covid-19 patients based on the observational studies of clinical benefit amongst patients with sars-cov and mers-cov [19] [20] [21] , as well as the docking studies conducted by the national institute of virology, pune 29 . the indian regulatory authority, central drugs standard control organization, has accorded approval for restricted public health emergency use of this treatment protocol. the initial treatment protocol was for administering the combination treatment to all laboratory-confirmed patients. however, the first three laboratory-confirmed patients from kerala had mild symptoms on diagnosis and had a stable course of illness. hence, lopinavir/ritonavir treatment was not administered in these patients. it is however, crucial to initiate the treatment before patient develops features of severe hbv and hcv elisa  @ hb%, total leucocyte count and differential wbc -neutrophils, lymphocytes, eosinophils, monocytes and basophils, rbc count, platelet count; # renal function test -bun, creatinine; * liver function test -albumin, bilirubin, alt, ast, alkaline phosphatase. ast, aspartate transaminase; alt, alanine aminotransferase; rbc, red blood cell; wbc, white blood cell; bun, blood urea nitrogen; hbv, hepatitis b virus; hcv, hepatitis c virus; ecg, electrocardiogram; sars-cov-2, severe acute respiratory syndrome coronavirus 2; qrt-pcr, real-time reverse transcription-polymerase chain reaction; pt, prothrombin time; inr, international normalized ratio; hba 1c , haemoglobin a 1c ; hb, haemoglobin illness. in view of this, the treatment protocol was subsequently amended to include additional criteria of severity as well as organ damage for initiating the combination treatment. the inclusion criteria also include high-risk group patients associated with higher risk of mortality (age >60 yr, hypertension, diabetes mellitus, renal failure, chronic lung disease and immunocompromised persons) for initiating the combination therapy. the treatment protocol also emphasizes the need for obtaining written informed consent and patients to be enrolled into this protocol on case-to-case basis. it is also equally important to monitor covid-19 patients closely to generate reliable data about clinical, laboratory, as well as safety outcomes. this treatment protocol has a limitation. the combination treatment is approved for emergency public health use, only amongst laboratory-confirmed patients with moderate degree of severity and not designed as a controlled clinical trial. however, the treatment outcomes amongst the first few cases would be useful in providing guidance about clinical management of covid-19 cases in future. if found useful in managing initial covid-19 infected patients, further evaluation using a randomized control trial design is warranted to guide future therapeutic use of this combination. clinical virology, 4 world health organization. novel coronavirus -china. who; 2020 world health organization. who director-general's remarks at the media briefing on 2019-ncov on 11 novel coronavirus (2019-ncov) situation as of 17 statement-on-the-meetingof-the-international-health-regulations-(2005)-emergencycommittee-regarding-the-outbreak-of-novel-coronavirus sars to novel coronavirus -old lessons and new lessons european centre for disease prevention and control. severe acute respiratory syndrome -annual epidemiological report epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china a major outbreak of severe acute respiratory syndrome in hong kong drugs@fda: fda-approved drugs product literature of ritonavir and lopinavir, conclusion of overview of literature and approvals relating to selected fixed-dose combinations arvs (such as ritonavir and lopinavir etc) efficacy and biological safety of lopinavir/ritonavir based anti-retroviral therapy in hiv-1-infected patients: a meta-analysis of randomized controlled trials safety and efficacy of lopinavir/ritonavir during pregnancy: a systematic review lopinavir: a potent drug against coronavirus infection: insight from molecular docking study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-α for middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars). who middle east respiratory syndrome coronavirus (mers-cov). who; 2020 perspectives for repurposing drugs for the coronavirus disease 2019 (covid-19) none. key: cord-266464-wuf3s8m0 authors: kim, so yeon; park, sun jae; cho, sook young; cha, ran-hui; jee, hyeon-gun; kim, gayeon; shin, hyoung-shik; kim, yeonjae; jung, yu mi; yang, jeong-sun; kim, sung soon; cho, sung im; kim, man jin; lee, jee-soo; lee, seung jun; seo, soo hyun; park, sung sup; seong, moon-woo title: viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection date: 2016-10-17 journal: emerg infect dis doi: 10.3201/eid2210.160218 sha: doc_id: 266464 cord_uid: wuf3s8m0 we evaluated the diagnostic and clinical usefulness of blood specimens to detect middle east respiratory syndrome coronavirus infection in 21 patients from the 2015 outbreak in south korea. viral rna was detected in blood from 33% of patients at initial diagnosis, and the detection preceded a worse clinical course. m iddle east respiratory syndrome coronavirus (mers-cov) is a zoonotic, betacoronavirus lineage c rna virus that was first identified in saudi arabia in 2012 (1) . mers-cov causes respiratory and renal illness in humans, and infection often progresses to severe pneumonia, acute respiratory distress syndrome, renal failure, or death in a subset of patients (2) . risk factors, including patient age, preexisting health conditions, and high viral load in upper respiratory specimens, have been suggested to be related to disease severity and death (3, 4) . however, pathogenesis and clinical characteristics promoting recovery from infection or progression to serious organ failure have not been well elucidated. respiratory specimens are preferred for viral rna detection and confirmatory diagnosis of mers-cov infection in humans (5) . mers-cov has broad tissue tropism, including the kidney, intestinal tract, liver, histiocytes, macrophages, and t lymphocytes, but viral rna has been found inconsistently in blood, urine, and fecal specimens (6-10). reports have described small numbers of cases with extrapulmonary virus; therefore, it remains unclear whether extrapulmonary specimens have any diagnostic usefulness in determining infection or whether extrapulmonary viral detection has clinical implications in disease management. a large mers-cov outbreak occurred in 2015 in south korea. this outbreak comprised the first imported case and subsequent infection of 185 patients (11) . our study aimed to evaluate the diagnostic utility of blood specimens for mers-cov infection by using large numbers of patients with a single viral origin and to determine the relationship between blood viral detection and clinical characteristics. we collected 21 pairs of edta whole blood and serum specimens from 21 patients with mers-cov after admission to the national medical center in seoul, south korea. mers-cov infection initially was diagnosed by the korea centers for disease control and prevention using respiratory specimens (11) . after admission, each patient was reassessed for epidemiologic information and clinically managed with monitoring. specimens were stored at −80°c before analyses. viral rna was extracted and eluted with a magna pure lc 2.0 automated nucleic acid extractor and magna pure lc total nucleic acid isolation kit (both from roche diagnostics, mannheim, germany) according to the manufacturer's instructions. specimen volumes were 100 μl edta whole blood and 200 μl serum, and elution volumes were 100 μl for edta whole blood and 50 μl for serum. one-step, real-time reverse transcription pcr (rrt-pcr) was performed for the 3 mers-cov gene regions (upstream envelope [upe] , open reading frame [orf] 1a, and nucleocapsid) with an agpath-id one-step rt-pcr kit (applied biosystems, foster city, ca, usa) and an abi7500 real-time pcr system (applied biosystems). human ribonuclease (rnase) p was amplified in parallel for sample quality control (5, 12, 13) . in each test, the viral rna was considered detected when amplification before cutoff was observed from at least 2 different targets in mers-cov with pass of sample quality control. the robustness of rrt-pcr was demonstrated for qualitative concordance of positivity or negativity by using different types of specimens (online technical appendix table 1 , http://wwwnc.cdc.gov/eid/article/22/10/16-0218-techapp1.pdf). we calculated the viral copy concentration in the blood using standard curves constructed from the cycle threshold (c t ) of serially diluted 10 5 copies/μl of upe rna (provided by the university of bonn medical center, bonn, germany). author the results of the blood viral rna analyses did not affect clinical management. we assessed the relationships among clinical and molecular diagnostic factors with the ibm spss statistics program 22.0 (spss inc., chicago, il, usa). in each test, p<0.05 was considered statistically significant. the institutional review board of the national medical center approved this study (h-1508-057-002). we assessed patient demographics, their clinical features, and disease outcomes (tables 1, 2; online technical appendix table 2 ). the time difference was an average of 1.5 days (median 1, range 0-5 days) between when the initial diagnostic respiratory specimens and blood specimens were obtained. at admission, viral rna was detected in 6 (29%) of 21 edta whole blood and 6 (29%) of 21 serum samples from infected patients. two patients showed viral positivity in either specimen subtype of edta whole blood or serum; therefore, the overall detection rate for mers-cov was 33% (7/21) in blood. the concordance rate of viral assay was 90% (19/21) between edta whole blood and serum specimens. blood virus concentration was 3,130 copies/ml edta whole blood (range 2,080-17,400 copies/ml), equivalent to a median upe c t of 37.55, and 1,300 copies/ml serum (range 490-10,200 copies/ml, median upe c t 36.88). blood viral rna positivity at admission was associated with fever >37.5°c on the sampling date (p = 0.007), requirement for mechanical ventilation during the following clinical course (p = 0.003) and extracorporeal membrane oxygenation (p = 0.025), and patient death (p = 0.025, all by 2-tailed fisher exact test; figure) . blood viral rna positivity was not associated with viral c t in the initial diagnostic lower respiratory specimens, or requirement of oxygen supplementation during the following clinical course. between the blood viral rna-positive and -negative groups, we found no differences in age, duration from symptom onset to diagnosis of mers-cov infection, or an invasive procedure before the specimens were obtained (online technical appendix table 3 ). viral loads in the lower respiratory specimens at the initial confirmatory diagnosis showed no effect on patient survival (figure) . patient death was not associated with length of time from symptom onset to diagnosis of mers-cov infection (online technical appendix table 3 ). our results showed that the detection rate of blood viral rna was low in the early phase of infection in patients with a confirmed diagnosis, similar to results from a previous study (14) . these findings contrasted with those of severe acute respiratory syndrome coronavirus infection (15) . therefore, in the case of mers-cov infection, blood does not have the highest diagnostic yield for the initial confirmatory diagnosis. the viral load in blood was low, even in detected cases. a proportion of mers-cov isolates in the 2015 korea outbreak harbored a c→t substitution in the third nucleotide of the orf1a primer binding site (genbank accession nos. kt374052-374055). this mismatch may partially contribute to the insensitivity of orf1a assay observed in this study. an alternative sensitive target replacing orf1a might be useful in studies using blood specimens. blood viral rna has been detected in a few case reports of mers-cov fatalities (8) (9) (10) . our data of 42 specimens from cross-sectional time points focusing on early viremia showed that blood viral rna was present in a subpopulation of patients and that these patients had significantly poorer prognoses, as demonstrated by the need for more frequent mechanical ventilation and the increased risk for death. further large studies using serial daily specimens that are collected throughout the admission period-, both upper and lower respiratory specimens and paired measurement of viral rna and antibody in blood-might help overcome the limitation of the current study, which included relatively small numbers of deceased patients (24% [5/21]). our data showed a detection rate of 33% for viral rna in blood at initial diagnosis, which was insufficient for initial confirmatory diagnosis. blood viral rna at the early phase was related to a worse clinical course in infected patients and might be a good prognostic indicator of severe outcome. measuring blood viral rna at hospital admission might be useful. middle east respiratory syndrome coronavirus-infected patients, south korea, 2015 . a, b) survival difference between the blood viral rna-positive (solid line) and -negative (broken line) groups. survival was defined as the time from initial confirmatory diagnosis to death before hospital discharge (a) (kaplan-meier survival analysis, log rank p = 0.009; breslow p = 0.006) and as the time from symptom onset to death (b) (kaplan-meier survival analysis, log rank p = 0.017; breslow p = 0.015). c, d) survival difference between the high respiratory viral load (solid line) and low respiratory viral load (broken line) groups. viral loads were classified into 2 groups: patients who harbored viral loads above the median load of patients and patients who harbored below. survival was defined as time from initial confirmatory diagnosis to death. cycle threshold (c t ) values were calculated for real-time reverse transcription pcrs targeting the upstream of envelope region (c) and open reading frame 1a region (d) (kaplan-meier survival analysis, log rank p = 0.739; breslow p = 0.630). tick marks along data lines indicate data-censored time points. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome mortality risk factors for middle east respiratory syndrome outbreak, south korea association of higher mers-cov virus load with severe disease and death, saudi arabia world health organization. laboratory testing for middle east respiratory syndrome coronavirus. interim guidance (revised) differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways mers-cov study group. clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection mers-cov biology group. kinetics and pattern of viral excretion in biological specimens of two mers-cov cases middle east respiratory syndrome coronavirus outbreak in the republic of erratum in: osong public health res perspect detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome we thank christian drosten for providing the in vitro transcribed rnas of mers-cov genes. the public health image library (phil) key: cord-264199-8skyagsz authors: fragaszy, ellen; hayward, andrew title: emerging respiratory infections: influenza, mers-cov, and extensively drug-resistant tuberculosis date: 2014-12-31 journal: the lancet respiratory medicine doi: 10.1016/s2213-2600(14)70250-4 sha: doc_id: 264199 cord_uid: 8skyagsz nan reverted to their baseline on the apnoea-hypopnoea index. the high response rate to this intervention (apnoea-hypopnoea index fell to less than 50% and number of events per h fell below 20 in 66% of patients) seems at least partly due to a careful selection process at enrolment, excluding those with concentric collapse of the airway by endoscopic airway examination during drug-induced sleep, the very obese (body-mass index >32 kg/m 2 ), and those with tonsillar enlargement. further work will be needed to clarify the determinants of treatment response and the short-term and longterm rates of adverse eff ects. however, an advantage of such a treatment is a single surgical procedure to treat the disorder as opposed to the requirement for nightly administration of a mechanical treatment. cpap is still a mainstay of osa treatment, but clinicians clearly now have a wider range of therapeutic options that one day might well be predicted by careful patient phenotyping. clinical decision making might become more complex, but chronic care of patients with osa will be enhanced by recognition that one treatment does not necessarily have to fi t all. the importance of the animal-human interface has also been shown through the emergence of middle east respiratory syndrome coronavirus (mers-cov). meyer and colleagues 4 reported that antibodies against mers-cov were common in samples taken from dromedary camels in 2003 (100% of 151 camel serum samples had detectable antibodies to mers-cov with 57% having high neutralising titres), well before the fi rst human cases were identifi ed in 2012. azhar and colleagues 5 reported identical full genome sequences from mers-cov isolates taken from a patient who died of mers-cov and the symptomatically infected dromedary camel belonging to the patient. this provided strong evidence that mers-cov could be transmitted from camels to humans and establishes camels as a potentially important source of infection, although other species such as bats might also be implicated. fortunately, unlike the 2009 infl uenza pandemic strain (h1n1pdm09), neither mers-cov nor infl uenza a h7n9 (or any other infl uenza subtypes recently identifi ed to infect humans) have evolved the ability to spread effi ciently between humans. national preparedness plans for infl uenza pandemic involved widespread stockpiling of antivirals, and the 2009 pandemic provided an opportunity to study their eff ectiveness. new research on the eff ectiveness of neuraminidase inhibitors to reduce mortality in patients admitted to hospital with infl uenza a h1n1pdm09 has been encouraging. 6 although the eff ectiveness of antivirals to reduce mortality would ideally be derived from randomised controlled trials, a recent meta-analysis of trials was only able to collect data for fi ve reported deaths, only one of which was due to a respiratory cause. 7 the comparative rarity of such events highlights the need for high quality observational research to inform policy and practice. muthuri and colleagues 6 collected individual-level data from around the world on patients admitted to hospital with infl uenza a h1n1pdm09. after adjustment for treatment propensity and potential confounders, the results of their meta-analysis indicate that compared with no treatment, neuraminidase inhibitor treatment at any time during illness was associated with a reduction in mortality risk (adjusted odds ratio [or] 0·81). 6 this association was strongest in adults (or 0·75), but weaker and not signifi cant in children. the timing of treatment was also important because early treatment (within 2 days of symptom onset) was associated with a reduction in mortality (or 0·48) compared with later treatment-treatment given later than 2 days and up to 5 days after symptom onset was not associated with reductions in mortality compared with no treatment except in severely ill adult patients admitted to critical care (adjusted or 0·65). questions remain about the most eff ective and costeff ective means to ensure that those most likely to benefi t are treated early. by contrast with acute respiratory infections, which have the potential to emerge and spread rapidly, the inexorable emergence of drug resistant, multi-drug resistant (mdr), and now extensively drug-resistant (xdr) tuberculosis, has played out during the past seven decades since the discovery of streptomycin in 1944. using whole genome sequencing of 1000 prospectively collected tuberculosis isolates from patients in russia, casali and colleagues 8 provided evidence against the theory that acquiring drug resistance typically comes with a fi tness cost and reduced transmissibility. their fi ndings show the crucial need to strengthen tuberculosis control to prevent the emergence and spread of mdr and xdr tuberculosis. further evidence from south africa shows the long infectious periods and alarmingly high levels of mortality in patients with xdr tuberculosis: at 60 months of follow-up, 78 (73%) patients with xdr had died and a further 11 (10%) had failed treatment. 9 there has been a long-standing and continuing need to develop new drugs and treatment regimens for tuberculosis, which is underscored by this research. emerging respiratory infections highlight the complex interplay between organisms, the environment, animal and human host biology and behaviour and challenges in clinical management. interdisciplinary approaches are needed to address these multifaceted issues and to ensure that research fi ndings help to inform prevention, management, and control strategies. epidemiology of human infections with avian infl uenza a (h7n9) virus in china eff ect of closure of live poultry markets on poultry-to-person transmission of avian infl uenza a h7n9 virus: an ecological study can closure of live poultry markets halt the spread of h7n9? antibodies against mers coronavirus in dromedary camels evidence for camel-to-human transmission of mers coronavirus eff ectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with infl uenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data oseltamivir for infl uenza in adults and children: systematic review of clinical study reports and summary of regulatory comments evolution and transmission of drug-resistant tuberculosis in a russian population long-term outcomes of patients with extensively drug-resistant tuberculosis in south africa: a cohort study key: cord-267333-8b7hvorz authors: watson, john t.; hall, aron j.; erdman, dean d.; swerdlow, david l; gerber, susan i. title: unraveling the mysteries of middle east respiratory syndrome coronavirus date: 2014-06-17 journal: emerg infect dis doi: 10.3201/eid2006.140322 sha: doc_id: 267333 cord_uid: 8b7hvorz nan cov) is a novel cov known to cause severe acute respiratory illness in humans; ≈40% of confirmed cases have been fatal. human-to-human transmission and multiple outbreaks of respiratory illness have been attributed to mers-cov, and severe respiratory illness caused by this virus continues to be identified. mers-cov was first reported in september 2012, and subsequent investigations documented illness onsets as early as april 2012 (1) . as of february 23, 2014, the world health organization has reported 182 laboratory-confirmed cases of mers-cov infection, including 79 deaths, indicating an ongoing risk for transmission to humans in the arabian peninsula (2). the median age of reported case-patients is 52 years (range 2-94 years); most cases are in males (3). most index casepatients have at least reported 1 chronic comorbid condition (4) and have resided in, or recently traveled to, jordan, qatar, united arab emirates, oman, kuwait, or saudi arabia (3). in france, germany, italy, united kingdom, and tunisia confirmed cases of mers-cov have been identified in travelers returning from these countries (3) . although a zoonotic reservoir of mers-cov has been speculated, very little is known about the specific exposures that result in primary human cases. mers-cov infection causes severe acute hypoxemic respiratory failure, extrapulmonary organ dysfunction, and high rates of death; however, the spectrum of illness and clinical course are not fully defined (5) . evidence suggests that mers-cov is capable of limited human-to-human transmission, which results in outbreaks in family and health care settings (5, 6) . the 182 reported cases include multiple distinct spatiotemporal clusters and 32 identified infections in health care workers (3) . modeling performed to assess the extent of human infection and the transmission potential of mers-cov (as of august 2013) estimated that most symptomatic case-patients had not been detected but that chains of transmission were not self-sustaining when infection control was implemented (7) . despite evidence of human-to-human transmission, the number of contacts infected by persons with confirmed infections appears to be limited; sustained transmission in the community has not been documented (3) . the hajj, the annual religious pilgrimage to saudi arabia, involved 1.37 million pilgrims from 188 countries in 2013 but resulted in no reports of confirmed cases in the weeks after the pilgrimage (3). little is known about the pathogenic potential and transmission dynamics of mers-cov. although multiple health care-associated clusters have been identified (4), further investigation is needed of the specific risk factors for transmission within health care facilities. basic information about the temporal and causal patterns of viral shedding and their relationships to clinical outcomes is critical to further understand the virus and to shape prevention and control measures needed to limit transmission. standard, contact, and airborne precautions appear to be effective in limiting transmission and are recommended by the centers for disease control and prevention to manage known or suspected mers-cov infection in hospitalized patients as a primary means of preventing and controlling transmission (8) . potential animal reservoirs and mechanism(s) of transmission of mers-cov to humans remain unclear. of the minority of case-patients for whom information is available about exposure to animals, few have reported owning or visiting a farm with camels, goats, sheep, chickens, ducks, or other animals (4). a zoonotic origin for mers-cov was initially suggested by its high genetic similarity to bat covs (9) and the identification of closely related viruses in bats (10). recent reports have described additional data from camels. these include real-time reverse transcription pcr detection and limited sequencing of mers-cov from 3 camels from a farm in qatar linked author to 2 infections in humans in october 2013 (11) and, more recently, in camels in saudi arabia (12) , and antibodies against mers-cov in camel serum from the arabian peninsula, including serum from the united arab emirates drawn in 2003 (13) (14) (15) (16) (17) . however, more epidemiologic data linking cases to infected animals are needed to determine whether a particular animal species is a host for the virus, a source of human infection, or both. this month's issue of emerging infectious diseases presents results of a study that provides evidence of mers-cov in dromedary camels in egypt (18) . only 3 other reports of mers-cov detection in animals have been published: 1 in a bat and 2 in camels (11, 12, 19) . however, these reports were based on limited genetic information. in contrast, on the basis of their sequence analysis of nearly the entire viral genome showing >99% nt sequence identity with human mers-cov, chu et al. provide the most compelling evidence thus far of mers-cov infection in dromedary camels (18) . although the authors also found neutralizing antibodies to mers-cov (or a mers-like cov) in most of the camels, they did not find serologic evidence of infection in the abattoir workers who had contact with the infected animals. this finding leaves key questions about zoonotic transmission unanswered. most notably, it remains unclear whether zoonotic transmission of mers-cov occurs between camels and humans and, if so, what the directionality and risk factors are for such transmission. these lingering gaps in knowledge about mers-cov emphasize the need for more epidemiologic study to determine risk factors for human infection, more population-level data on the prevalence of mers-cov in camels, risk factors for infection and shedding in camels, and continued vigilance for other possible sources of infection. also, the camels tested were in egypt and were locally reared or imported from sudan or ethiopia, countries in which no cases have been identified in humans. thus, the geographic area for surveillance should be widened beyond the arabian peninsula and include eastern africa, which is a source for importation of dromedary camels. this study emphasizes the need to further define exposure information for all mers-cov cases regarding camels and other animals, as well as exposure to ill humans who might have undetected mers-cov infections. understanding the role of dromedary camels and possibly other animals in transmission of mers-cov to humans remains a priority for future investigation to enable development of targeted control measures and prevent future cases and deaths from this emerging pathogen. dr watson is a medical officer with the division of viral diseases, centers for disease control and prevention, in atlanta, georgia, usa. his research interests include the epidemiology and control of viral respiratory diseases. latest outbreak news from promed-mail: novel coronavirus-middle east middle east respiratory syndrome coronavirus (mers-cov)-update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 20 state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels mers coronaviruses in dromedary camels, egypt. emerg infect dis middle east respiratory syndrome coronavirus in bats, saudi arabia key: cord-256784-wfaqim7d authors: modjarrad, kayvon title: mers-cov vaccine candidates in development: the current landscape date: 2016-06-03 journal: vaccine doi: 10.1016/j.vaccine.2016.03.104 sha: doc_id: 256784 cord_uid: wfaqim7d middle east respiratory syndrome coronavirus (mers-cov), an emerging infectious disease of growing global importance, has caused severe acute respiratory disease in more than 1600 people, resulting in more than 600 deaths. the high case fatality rate, growing geographic distribution and vaguely defined epidemiology of mers-cov have created an urgent need for effective public health countermeasures, paramount of which is an effective means of prevention through a vaccine or antibody prophylaxis. despite the relatively few number of cases to-date, research and development of mers-cov vaccine candidates is advancing quickly. this review surveys the landscape of these efforts across multiple groups in academia, government and industry. middle east respiratory syndrome (mers-cov) was first isolated in september 2012 from a patient in saudi arabia who presented two months earlier with severe acute respiratory infection and acute renal failure [1] . retrospective testing of samples in jordan identified earlier cases from a nosocomial outbreak in april 2012 [2] . although the majority of mers-cov cases (∼75%) have occurred in saudi arabia, 25 other countries have confirmed imported or autochthonously transmitted cases ( fig. 1) [3, 4] . the most recent and largest outbreak outside of saudi arabia occurred in south korea in may 2015 [5] , raising concern for an eruption of regional outbreaks or accelerated global spread, similar to the phylogenetically related severe acute respiratory syndrome coronavirus (sars-cov) that killed nearly a thousand people a decade earlier [6] . although the definitive host for mers-cov has not yet been established, closely related coronaviruses have been isolated from bats across wide geographic areas [7] [8] [9] . mounting evidence has strongly implicated dromedary camels as the intermediate animal reservoir, as serological surveys throughout the middle east and north africa have demonstrated them to have a high prevalence of mers-cov binding or neutralizing abs [10] [11] [12] [13] . additionally, outbreak investigations have suggested epidemiologic linkage between farm camels and human cases [14] . mers-cov is a spherical, enveloped, single-stranded, positive sense rna beta-coronavirus [1, 15] . its genome contains a replicase * tel.: +13015003600. locus at the 5 end and codes for structural proteins toward the 3 end. the most immunogenic of the viral proteins is spike (s), a trimeric, envelope-anchored, type i fusion glycoprotein that interfaces with its human host cognate receptor, dipeptidyl peptidase 4 (dpp4), to mediate viral entry [16, 17] . s comprises two subunits: s1, which contains the receptor-binding domain and determines cell tropism; and s2, the location of the cell fusion machinery. although dpp4 has a broad tissue distribution, most of the clinical manifestations of mers-cov can be attributed to its localization to the lower respiratory tract [18, 19] . much like other coronaviruses, mers-cov can also cause significant dysfunction of the gastrointestinal, cardiovascular, renal, and neurologic systems. mers-cov is distinct, though, in its tendency to cause greatest harm to older individuals with concurrent comorbidities of one or more of these organ-systems [20, 21] . despite past efforts to develop coronavirus countermeasures in response to the sars-cov pandemic, there are currently no prophylactic or therapeutic interventions of proven efficacy for mers-cov or any other coronavirus infection. although combination treatment with ribavirin and interferons were shown to improve clinical outcomes in mers-cov-infected non-human primates (nhps), treatment was initiated very soon after viral challenge (∼8 h) and results have not been replicated in humans [22] . in fact, no experimental interventions have demonstrated appreciable benefit in acutely ill patients in a consistent or controlled manner. rapidly scaled treatments based on naturally occurring neutralizing antibodies such as convalescent plasma or hyperimmune globulin, on the other hand, have demonstrated mortality reductions for other respiratory infections and may hold promise for mers-cov as well [23] . their development, however, is limited by logistical challenges, local technical capacity, and donor supply. supportive management, adapted from guidelines developed for sars-cov, has thus far been the mainstay of mers-cov treatment. the global will to develop a coronavirus vaccine faded in the aftermath of sars-cov pandemic but has since gained renewed momentum in the face of the current mers-cov outbreak. previous approaches to coronavirus vaccine development were broad and included whole-inactivated and live-attenuated viruses, recombinant vectors and protein subunits, as well as dna and rna based platforms [6] . most developers based their immunogen designs on the s surface glycoprotein, the primary target for neutralizing antibodies during any natural coronavirus infection. a number of preclinical and clinical studies showed that the sars-cov s1 protein subunit, and specifically the rbd at its core, could serve as a dominant target for neutralizing antibodies in mice, non-human primates, and humans [24] . s1, therefore, became the basis for a number of promising sars-cov vaccine candidates. the s1 protein subunit and rbd have also been the basis for several mers-cov vaccine candidates (fig. 2 ) [25] [26] [27] [28] [29] . resolution of rbd crystal structures alone or in complex with the dpp4 receptor [25, 30, 31] have informed the design of immunogens that have been expressed either as recombinant protein fragments or conjugates to the fragment crystallizable (fc) region of human antibodies. both types of constructs, in formulation with aluminum salt or oil-in-water adjuvants, have elicited neutralizing antibodies of high potency across multiple viral strains. despite their demonstrated immunogenicity in animal models and anticipated safety in humans, rbd or s1-subunit based vaccine candidates are limited in their epitope breadth. although the coronavirus genomes are not as variable as other rna viruses, the rbd is the most mutable region, containing mutation sites that define antibody escape variants [25, 32] . thus, vaccine candidates that elicit a more diverse antibody repertoire as well as a robust cellular immune response may offer the advantage of broader and more durable protection. full-length s used as an immunogen could at least increase the breadth of the antibody response; however, it has been difficult to express, and may require additional work to produce a stable soluble trimer of the s ectodomain. investigators at the university of maryland, in collaboration with novavax, inc., have overcome this problem through the development of s rosettes that are stable and immunogenic in murine models [33] . vaccines that mimic natural infection, such as live-attenuated viruses or recombinant viral vectors, may elicit even more robust immunity. live attenuated viruses have historically been among the most immunogenic platforms available, as they have the capacity to present multiple antigens across the viral life cycle in their native conformations. although a live-attenuated mers-cov has yet to be tested, one has been constructed and has the potential to be protective [34] . however, manufacturing live-attenuated viruses requires containment in a biosafety level 3 or 4 facility. additionally, live-attenuated viruses carry the hazards of inadequate attenuation or reversion to wild type form and causing disseminated disease, particularly in immunocompromised hosts. given that moderately immunocompromised adults with co-morbidities such as diabetes mellitus and chronic kidney disease have suffered the most severe mers-cov disease, these individuals may comprise a target population for immunization, thus making a live-attenuated virus vaccine a less viable option. replication competent viral vectors could pose a similar threat for disseminated disease in the immunosuppressed. replication deficient vectors, however, avoid that risk while maintaining the advantages of native antigen presentation, elicitation of t cell immunity and the ability to express multiple antigens. to date, two recombinant vector platforms-modified although replication deficient vectors are relatively safe and immunogenic, their ability to deliver genetic material for expression could be impeded by pre-existing or developing immunity to the vector itself. one way to overcome this limitation is by administering different vectors in a so-called prime-boost immunization regimen. as this strategy has been effective for other pathogens, it is likely that the same success could be recapitulated for mers-cov. the use of more than one type of platform or antigen in a single vaccine also increases the likelihood of inducing a broad repertoire of antibodies with diverse mechanisms of viral neutralization. one vaccine regimen developed at the us national institutes of health is based on full-length s dna and a truncated s1 subunit glycoprotein and has elicited neutralizing antibodies in mice directed at both the s1-within and outside the rbd-and s2 subunits. immunization with these constructs also protected nhps from severe lung disease after intra-tracheal challenge with mers-cov [25] . a dnaonly vaccine, expressing multiple antigens, has also been developed by inovio pharmaceuticals and geneone life science inc. and has been advanced to a phase i first-in-human trial. each of the vaccine candidates that have been mentioned is being developed for prophylactic use. however, as the total number of cases (>1600) [3, 4] and reproductive rate (∼0.7) of mers-cov are both relatively low [39] , it will be difficult to define the target populations for vaccination that would support the investment in manufacturing and advanced product development. also, with such low incidence and lack of robust animal models, it would be difficult to achieve a vaccine efficacy result that would be sufficient to support licensure. human mabs, on the other hand, could be used without as much discrimination in an outbreak setting for post-exposure prophylaxis and early treatment. the advantages of mabs over polyclonal antibodies (administered through convalescent plasma or hyperimmune globulin) are their higher potency, greater specificity, more extensive pre-licensing evaluation and consequently improved safety profile. additionally, mabs can help define immunogenic epitopes through crystallographic analysis, thereby providing atomic level detail for the design of better immunogens. however, the timeline for mab development may be longer and potentially cost more than some vaccines. despite requirements for greater upfront investment, several groups have developed highly potent mabs that are currently being advanced through pre-clinical stages of testing (fig. 3) . some have been isolated from immunized animals (mice/humanized mice/nhps) [25, 40] , while others have been identified from either an antibody human phage library [41] [42] [43] [44] or memory b cells of infected and recovered human survivors [45] . almost all of the mabs that have been reported target the spike rbd. it is likely that mabs directed at other sites on the spike glycoprotein have been recovered but are not as potent neutralizers. most of those that have been published bind to recombinant spike with picomolar affinity and neutralize mers-cov pseudovirus at a half maximal inhibitory concentration (ic 50 ) of 0.1 mcg/l or less. additionally, some have demonstrated protective efficacy in pre-and post-exposure prophylaxis animal models [44, 45] . the successes thus far in isolating potent and protective mabs may prove useful for therapeutic development where the target population is well defined. it may prove more challenging to advancing these products to licensure and fullscale production at affordable costs for the purpose of prophylaxis in as of yet undefined populations. the vaguely defined epidemiology of mers-cov has complicated the design and implementation of appropriate public health countermeasures. most transmission events have occurred either in the setting of household clusters or nosocomial outbreaks [46] [47] [48] [49] [50] [51] . it is also likely that the virus has been introduced multiple times into human populations from a large zoonotic reservoir, i.e. dromedary camels. given the broad distribution and ownership of camels in the arabian peninsula where most cases have occurred, a targeted vaccine campaign may prove difficult. as the outbreak in the republic of korea revealed, patients and workers in the same healthcare facility as an infected patient are at high risk for secondary acquisition. an optimal strategy may be to use vaccines in conjunction with stringent infection control practices in hospitals where mers-cov cases are being treated. the epidemiologic link of mers-cov between bats, camels, and humans presents an opportunity for a veterinary vaccine to interrupt the transmission cycle. a successful precedent for this so-called "onehealth" approach toward mitigating human disease with a veterinary vaccine exists in the example of equivac ® , a hendra virus vaccine developed solely for horses [52] . although hendra virus is even more rare than mers-cov, it is highly fatal with no treatment other than intensive supportive management. in 2012, a protein subunit vaccine was licensed and rolled-out in australia, where all outbreaks of the virus occurred. since that time, the incidence in horses has fallen precipitously and no human cases have been detected [53] . a similar strategy may be applicable to mers-cov; however, a veterinary vaccination in this context would be deployed solely for the sake of protecting humans, as the virus causes only mild upper respiratory illness in camels. safety and reduction in viral shedding would have to be demonstrated in immunization, challenge and transmission studies of camel or camelid populations, one of which has shown efficacy [54] . one of the primary challenges to developing countermeasures to mers-cov is the lack of an appropriate animal model that recapitulates the natural history of human disease. much of the difficulty originates from the absence of the virus's cognate dpp4 receptor. one group approached this problem by successfully transducing mice with an adenoviral vector expressing human dpp4 [55] . although more relevant than a standard murine model, transient transduction of the desired protein may result in inconsistent tissue expression of adenovirus antigens. agrawal et al. made an important advance with the development of a transgenic mouse model that demonstrated productive, disseminated mers-cov infection [56] . although rhesus macaques do not manifest full clinical disease, they develop a transient lower respiratory infection that can be quantified and evaluated by computed tomography. investigators at the nih rocky mountain laboratories (rml) and integrated research facility (irf) have also independently been developing potentially lethal marmoset models that could be used for the evaluation of vaccines, mabs and therapeutics [57] . as mers-cov vaccines-both active and passive-are developed and tested, not only will more relevant animal models be required, but there will also be a need for a more detailed understanding of the epidemiology, immunology, and pathogenesis of the virus. in the aftermath of the west african ebola virus epidemic and in the face of the current zika virus outbreak, the global health community has coalesced around the realization that a multi-faceted plan is required to quickly and efficiently respond to global public health emergencies. the world health organization is currently developing a blueprint by which that preparation and response can follow, with mers-cov highlighted as a case study. although mers-cov still causes relatively few cases in a limited geographic distribution, its high case fatality and sudden outbreak in in korea have proven it to be a pathogen of public health concern. the concentration of the epidemic to saudi arabia also raises the specter of international spread every year during hajj, one of the largest mass gathering events in the world. ultimately, the development of a safe and effective vaccine for mers-cov may not yield its greatest benefit for the current epidemic but for the knowledge gained in creating a platform for combating coronaviruses as a whole. the opinions expressed herein are those of the authors and should not be construed as official or representing the views of the us department of defense or the department of the army. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological findings from a retrospective investigation east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 11 update: middle east respiratory syndrome coronavirus (mers-cov) probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea a decade after sars: strategies for controlling emerging coronaviruses detection of coronaviruses in bats of various species in italy diversity of coronavirus in bats from eastern thailand mers-related betacoronavirus in vespertilio superans bats middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus antibody reactors among camels in dubai antibodies against mers coronavirus in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia evidence for camel-to-human transmission of mers coronavirus mers: emergence of a novel human coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques middle east respiratory syndrome novel corona mers-cov infection. epidemiology and outcome update epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis anti-sars coronavirus agents: a patent review (2008-present) evaluation of candidate vaccine approaches for mers-cov a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus receptor-binding domain-based subunit vaccines against mers-cov crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice estimation of mers-coronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution preand postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus family cluster of middle east respiratory syndrome coronavirus infections hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections hendra virus vaccine, a one health approach to protecting horse, human, and environmental health hendra virus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection key: cord-252456-971d0sir authors: hemida, maged gomaa; ba abduallah, mohammed m. title: the sars-cov-2 outbreak from a one health perspective date: 2020-03-16 journal: one health doi: 10.1016/j.onehlt.2020.100127 sha: doc_id: 252456 cord_uid: 971d0sir the sars-cov-2 is a new human coronavirus candidate recently detected in china that is now reported in people on inhabited continents. the virus shares a high level of identity with some bat coronaviruses and is recognised as a potentially zoonotic virus. we are utilizing the one health concept to understand the emergence of the virus, as well as to point to some possible control strategies that might reduce the spread of the virus across the globe; thus, containment of such virus would be possible. the severe acute respiratory syndrome-2 was named the coronavirus infectious diseses-19 . however, it was called the novel coronavirus at the beginning of this outbreak. this virus was provisionally named 2019-ncov at the very beginning of its emergence in china, after emerging in china in late 2019 [1] . since that time, there has been an escalation in the number of confirmed cases, notably from china, but also in more than 113 countries and territories around the globe. historically four coronaviruses candidates (hcov-229e, hcov-oc43, hcov-nl63, and hcov-uku1), causing common cold or flu-like symptoms were known. patients infected by these viruses usually recover spontaneously in a short time [2] . the sars-cov-2 is the seventh coronavirus known to infect humans, appearing in less than ten years since the emergence of middle east respiratory syndrome coronavirus (mers-cov) in late 2012 and sars-cov in 2003 [3, 4] . this virus belongs to the family coronaviridae; a large group of viruses that have the potential to infect and cause diseases to a large number of mammals, birds as well as humans. members of this family cause a wide variety of clinical signs in their affected hosts, including respiratory, enteric, nervous, and systemic health problems. the viral genomes of coronaviruses have many unique features that make them prone to frequent coding changes, thus generating new candidates in a short period [5] . the reasons behind this rapid mutational frequency are the poor proofreading capability of the viral rna polymerase and the possibility of recombination between various members of this family [6] . recent studies showing the (angiotensin-converting enzyme a, (ace-2)), ace-2 is the primary receptors for the sars-cov-2 and other candidates of the b lineage of the beta coronaviruses [7] . high expression levels of some potential viral receptors in many hosts from mammals, birds in addition to humans make them susceptible to j o u r n a l p r e -p r o o f the virus infection. in recent studies, it is apparent that sars-cov-2 uses the mechanism that sars-cov used for cell entry. the mechanism of the virus entry to the cell resulting from the interaction between the virus receptors and the spike glycoprotein, after priming of that viral protein by the serine protease tmprss2 [8] . further, inhibition of tmprss2, blocked cell entry by sars-cov-2, indicating similar or identical pathways for cell invasion and possible strategies for therapeutics [8] . the who declared the virus as a global health emergency on jan 31, 2020 [9] . as of 10 am cet 03 march 2020, there are 128920 laboratory-confirmed cases reported to the who from 113 countries and territories across the globe with a total fatality of 4747 [10] . currently, the case fatality rate is relatively low (⁓3.6%) compared to infections with severe acute respiratory syndrome coronavirus (sars-cov, (10%) and mers-cov (32%) [11] . the numbers of infected and dead persons are much higher than the latter viruses. however, these numbers will be subject to changes with the newly reported cases and the outcomes of the ongoing active human cases. during mid-february 2020, a new criterion for the patient identification has been launched considering the chest computed tomography (ct) scans to identify patients suffering from respiratory distresses. [12] the reasons behind the switch from the detection of the viral nucleic acids by real-time pcr to ct scanning at that time for patient identification are (i) to give a chance to all patients to get the proper care at the right time (ii) the variation in the incubation period of the viral infection among various people (iii) the possibility of super-spreader of the virus who shed the virus to the environment in large numbers and poses a great risk of infection to the close contact people. this phenomenon have been reported previously in ebola, virus, sars-cov, mers-cov, and recently in sars-cov-2 [13] [14] [15] . using some mathematical tools models late feb, 2020, the basic reproduction (r0) to the humanto-human transmission was around 3.58. however, the same study found the r0 for the bat-to-j o u r n a l p r e -p r o o f human was 2.30 [16] . based on these findings, the r0 of sars-cov-2 was almost close to sars-cov but higher than mers-cov, with the exception of the mers-cov outbreak in south korea [16] . the mers-cov and the sars-cov-2 are both considered airborne pathogens [17] with a tendency to transmit through the aerosol of infected patients. the human-to-human nosocomial transmission was recently reported, which is contributing substantially to the spreading and sustainability of the virus. some family clusters were also recently reported inside china [18] . the incubation period of the diseases ranges from 2-14 days post-infection [19, 20] . the asymptomatic individuals may play essential roles in the spread of the virus within a specific community [21] . however, there are ongoing debates about the possibility of infected persons to shed the virus early during the course of an infection with this virus even before the appearance of the clinical signs [20, 22] . the emergence of the virus was believed to be linked with some human cases who suffered from severe pneumonia, which have a relation with a wet animal huanan seafood wholesale market in wuhan, china (1). these observations, along with the sequence identity of the virus, have drawn attention to the likely zoonotic origin of the virus. the sars-cov-2 infection is potentially another important example of the one health concept, after sars-cov, mers-cov, and ebola virus, in which there is an excellent overlapping in human, animal, and environmental health [23] . the full-length genome of the virus was recently made available to the scientific community [20] . this sequence will have a significant impact on the development of novel diagnostic assays, antiviral drugs, and vaccines against the virus soon. interestingly, the virus genome and its spike glycoprotein showing ‫9‬ 6.11 % and 92.86 identities to the rhinolophus affinis bat coronavirus, respectively (table 1) , from wuhan, china (ratg13, j o u r n a l p r e -p r o o f journal pre-proof accession number mn996532.1) recently deposited in the genbank [24] . bats are representing almost one-fourth of the mammalian population [25] . they are widely distributed all over the world in all continents except antarctica [25] . bats are classified under the order chiroptera that are found more than 50 million years ago. this order includes more than 1300 species of bats [25] . several studies suggested that bats are the common reservoir for the sars-cov were the sequences for viruses isolated from chinese horseshoe bats shared a high degree of identity with sars-cov [26] . metagenomics paved the way for the discovery of a large number of viruses in different species, including bats. there are more than 200 coronaviruses have been identified in various species of bats [27] . the main bat reservoirs of the sars-cov was identified in 2003 [28] . meanwhile, several species of bats including, taphozous perforatus, rhinopoma hardwickii, and pipistrellus kuhlii were believed to be the ancestors for the mers-cov [29] . based on the previous emergence history of sars-cov, the presence of a large number of mammals and birds overcrowded in one place may give a chance for pathogens, particularly those with rna genomes such as coronaviruses and influenza viruses, to emerge. however, it remains uncertain whether a similar emergence for sars-cov-2 can be postulated. one potential reservoir of sars-cov was the palm civet cat (paguma larvata), a member of the viverridae, a group of mammals found in asia [30] . sars-cov was able to infect and replicated efficiently in these animals [31] . serosurveillance for the antibodies against sars-cov in workers from one wild animal market in guangzhou was conducted in 2006 [32] . this study showed the presence of specific igg antibodies against sars-cov in these workers where the civet cats were available [32] . this same study confirmed the hypothesis of the implication of civet cats in the emergence and sustainability of sars-cov at that time. the identification of this reservoir was one of the milestones in control and containment of sars-cov (9), and j o u r n a l p r e -p r o o f subsequent banning of the trade of civet cats in the wild animal market was a significant step toward the containing of sars-cov [33] . this approach highlights the roles of one health in controlling such zoonotic pathogens. based on the evidence gained from studies of sars-cov, and from the genetic identity of sars-cov-2, it could be postulated that the virus responsible for the recent outbreak is transmit ted from bats to humans either directly or indirectly through adaptation in an unidentified host [34, 35] . the cone health concept could be the most logical approach in case of fighting and control some zoonotic pathogens, especially those who do not have available medication or vaccines during an epidemic or outbreak. there are many aspects by which the one health concept may contribute substantially to the control of the current sars-cov-2 outbreak. adoption of some of one health-based control strategies was of great success in controlling mers-cov, contributing at least in part to the decline in the case fatality rates from 52% in 2012 to 32% in 2020 in case of mers-cov [23, 36] . similar approaches were also successful in containing some of the highly pathogenic avian influenza viruses [37] . in the following sections, we are suggesting some one health-based control strategies for the containment of sars-cov-2 ( figure 2 ). coronaviruses are enveloped viruses like influenza, hiv, rabies etc, their envelope composed of a lipid bilayer, which the virus acquires from the host cell membrane while releasing from the infected cells [38] . using lipid solvents and detergents was proved to inactivate sars-cov through permeant damage of the lipid components of the viral envelope [39] . this study showed j o u r n a l p r e -p r o o f a drastic drop in the viral titer after one-minute treatment [39] . this raises the mandate of hand as well as personal hygiene as one of the gold standard control measures for not only coronaviruses but also form most enveloped viruses. coronaviruses are small, approximately 100-120 nm in diameter. this is making them able to pass the pores of the surgical masks very easily. thus, wearing special masks with minute pore sizes like n95 could be one of the preventive measures against respiratory pathogens, including influenza and coronaviruses, in comparison to another type of masks [40, 41] . however, the who already indicated certain rules for wearing the n95 masks, particularly for the health care workers and people in close contact with active cases [42] . the genome of coronaviruses is composed of single-strand positive-sense rna molecules. the virus genome act as a mrna and is infectious. the ultraviolet light has harmful effects on the sars-cov genome when used at 254 nm wavelength for 60 minutes [43, 44] . meanwhile, the effect of temperature on the virus infectivity/survival has great impact on the control of this class of respiratory viruses. sars-cov may live in the environment at average room temperature about 22 c for 2hrs without losing its infectivity. however, the virus infectivity is greatly affected by exposure to 56ºc for at least 30 min [44] . these parameters have great roles in the virus control as we discuss below. as mentioned above, the corona viral genomes are prone to frequent changes due to many factors; a new study highlighted the presence of two variants (l and s) of the sars-cov-2. this study confirmed that variant l (more severe) was dominant during the early outbreak in china, which was responsible for at least 70% of the cases at that time. however, the implementation of drastic control measures against the virus posed some selective pressure on this variant then the s variant becomes the predominant at this time [45] . thus, frequent monitoring of the virus on the genomic level is highly encouraged. meanwhile, j o u r n a l p r e -p r o o f the preparation of vaccine and antiviral drugs should be done from the most recent generation and seeds of the circulating strains and variants of the virus to ensure high efficacy rates. meanwhile, there is an urgent need for the development of novel diagnostic assays that enable the early detection of the virus, even in low copy numbers in the patient specimens. detection of cases during the early stage of the infection could be a crucial step in the early identification of the infected person; thus, proper treatment and control should be in place as early as possible. furthermore, the development of multiplex diagnostic assays that enable the detection of the most common respiratory pathogen in one reaction is highly recommended. it would be of high value to develop novel diagnostic tests that distinguish the seven human coronavir uses in one reaction per sample. this unique approach may help in screening a large number of people and animals and will have a significant impact on the fast track testing of travelers from at-risk regions. both sars-cov and mers-cov have zoonotic origins in which the civet cats and dromedary camels played important roles in the sustainability and transmission of these viruses in the community as well as spell over to the human [30, 46] . to identify the animal reservoirs in the context of sars-cov and mers-cov, scientist screened a large number of animals and birds to identify the main reservoir for those two viruses [47, 48] . sas-cov-2 was believed to be originated in people who came in close contact with the live wet market in wuhan, china, late 2019 [22] . in the case of sars-cov-2, recent studies used the informational spectrum tools to identify and predict the actual reservoir, the virus receptors as well as potential therapeutic and vaccine targets [49] . the wuhan wet market was hosting a large number of wild animals, birds, reptiles, and amphibians which never exist in one location in nature [50] . simply, there is a close j o u r n a l p r e -p r o o f human-animal contact in the supply chain of these wild animal markets starting from hunting, transporting, selling, processing, including slaughtering, and cooking. this is in addition to the exposure of people to their blood, secretions, and execrations. this chain poses a great risk for the contact between human and these wild animals, which may put the handlers at risk of infection not only for coronaviruses but also for other pathogens that these animals and birds may harbor. in the light of the one health concept, control of this emerging virus requires the reduction of the viral shedding to the environment from the main reservoirs, for animal-person transmission and from humans for person-person transmission [35] . based on the previous experience from the other emerging diseases, particularly sars-cov and influenza viruses, avoiding the mixing of various species of animals, birds, and mammals, is highly suggested [51, 65, 66] . this strategy will minimize the possibility of recombination among not only these pathogens but also for other common pathogens in the near future. a large number of bats may also be available in this particular market, including the rhinolophus affinis. the viral genome of the bat coronavirus from this particular species was close to the sars-cov-2 (table 1) . this result is suggesting these bats are the main ancestor of this virus. about 49% of the first reported human cases had a various levels of contact with this market [22] . a recent study showed that second, if seroconversion is proved in any of these animals or birds, testing specimens and tissues from these animals or birds must be carried out to detect the virus in these animals. the live poultry markets were responsible for the transmission of many viral pathogens to humans, especially various types of avian influenza viruses [37, 51, 52] . several studies reported that banning the storage of live poultry in live markets at least for a short period of time for overnight drastically reduced the ability to isolates the avian influenza viruses by 84% compared with the standard procedures [51] . another study showed a drastic decrease in the number of reported human cases of avian influenza h7n9 by almost 97% after the closure of live poultry markets in four major provinces in china [53] . thus, banning the live wild animal trading could have substantial effects on the control strategies not only for coronaviruses but also for other pathogens such as avian influenza viruses. regular monitoring of the dynamic changes of coronavirus in different species of bats is highly recommended. the environment serves as the intermediate vessels between the animals and humans. the environmental factors that may contribute to the spread of the virus include air, water, soil, etc. coronaviruses are airborne viruses that produce nosocomial infections [54] . they are mainly transmitted through droplet infection [55] . when the virus released from the animals or humans, it passes through the air and may drop on some services or objects. some respiratory pathogens with pandemic potential such as h1n1, sars-cov, and mers-cov may remain viable in the environment for a longer time up to months on these objects until picked up with another person to contaminate the mucus membranes through touching the nose, mouse or eyes [56] . there are several factors that control the virus's survival in the environment on these objects, such as the j o u r n a l p r e -p r o o f journal pre-proof temperature, the relative humidity, the strain of the virus [56] . some coronaviruses such as hcov-229e and mers-cov were detected on some environmental surfaces during some outbreaks [57, 58] . one of the critical control measures in the case of airborne infection is to develop novel assays that enable us to detect and estimate the virus concentration in the air. this approach will have great implications, especially in the health care settings. the process of decontamination of the virus-contaminated surfaces by the appropriate disinfectants or virucidal agents was successful in case of other respiratory viruses such as sars-cov and avian influenza [59] . the hydrogen peroxide vapor showed great success in the decontamination of surfaces contaminated with sars-cov and avian influenza viruses [59] . the who suggested using two the significant burden in controlling the respiratory viruses is how to manage the person-toperson transmission. this could be adopted through many strategies. first, personal and hand hygiene are the gold standard approach toward minimizing the possibility of infection for j o u r n a l p r e -p r o o f individuals. this can be achieved by washing the hands with the antiseptics and or soaps and water for at least 30 seconds as per the who guidelines [61] . this approach will reduce the virus load in the contaminated hands from virus droplets, which might touch the nose or mouse of persons. second, practice extreme caution by applying a social distance of at least one meter between you and anyone who is coughing or sneezing [61] . third, educate the public on how to avoid the frequent touching of the mucous membranes, especially those of nose, mouse, and eyes [61] . fourth, respiratory protection and hygiene through wearing the most appropriate personal protective equipment, as suggested earlier, according to the who's standards [61] . fifth, vomiting and diarrhea were reported in many sars-cov-2 patients [62] . this is suggesting the possibility of the fecal oral route as a mode of transmission of the virus [62] . this stresses out the importance of hand hygiene to avoid any possible transmission of the virus through the fecal oral route. sixth, one of the main pillars in the control of virus spreads is the identification of the infected personnel. it is one of the crucial steps toward reducing the virus spread in a community. the earlier the identification and detection of the positive cases, the better the control of these cases. thus, they will receive the better health care treatment and avoid virus spread particular ly during the very early stage of the infection. seventh, special attention should be paid to how to protect the health care workers dealing with active cases. they should provide by the standard originated from bats [25, 65] . this highlights the mandate for continuous monitoring of the coronaviruses population in bats as an alarm to the emergence of new novel coronaviruses in the future. second, there is ongoing research to develop antiviral therapy that might help in the treatment of the active human cases of the sars-cov-2. each approach mainly depends on a unique strategy to halt the virus replication or disrupt it at a certain point; thus, the virus cannot complete its replication cycle in the host. therefore, it reduces the possibility of virus shedding from the infected patients to its close contacts. a recent study used some serine protease tmprss2 inhibitors to prevent the virus from entry to the cell. these compounds are clinically approved and potentially will reduce the virus replication [8] . some fda approved anthelminthic compounds such as niclosamide have potent antiviral effects not only for sars-cov-2 but also for other viruses, including sars-cov, mers-cov, zika virus, and hcv [63] . a recent study showed the great values of the remdesivir (gs-5734) for prophylactic as well as treatment for the mers-cov in the rhesus macaque model [64] . this approach could be a promising trend for the prevention and treatment of sars-cov-2; however, further studies are required to confirm these findings. another method was to use the nucleotide analog inhibitors, such as the remdesivir, which targets the viral polymerase (rna dependant rna polymerase). this approach diminished the ability of the virus to copy its genome; thus, the replication cycle j o u r n a l p r e -p r o o f may be interrupted. this compound gave promising results in the cell culture model, which requires further testing in laboratory animals before the clinical trials [65] . although sars-cov-2 is rapidly transmitting across the globe, it may be contained if sincere containment measures are implemented. the drastic drop in the number of reported cases in china, along with a reduction in the reported deaths could be an indicator of an early containment of the virus. implementing the one health concept from all aspects involving the animal, environment, and humans could contribute substantially to the control of sars-cov-2 in the near future. the authors declare there is no conflict of interest. we wish to thank king abdul-aziz city for science and technology (kacst) for their generous funding through the mers-cov research grant program (number 20-0004), which is a part of targeted research program (trp). aetiology: koch's postulates fulfilled for sars virus isolation of a novel coronavirus from a man with pneumonia in saudi arabia another decade, another coronavirus molecular evolution and emergence of avian gammacoronaviruses functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor who, novel coronavirus(2019-ncov) situation report -11. available at global surveillance for human infection with novel coronavirus (2019-ncov) epidemic and emerging coronaviruses (severe acute respiratory syndrome and middle east respiratory syndrome) novel coronavirus (covid-19) pneumonia: serial computed tomography findings clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea agent-based modeling for super-spreading events: a case study of mers-cov transmission dynamics in the republic of korea the role of super-spreaders in infectious disease a mathematical model for simulating the phase-based transmissibility of a novel coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient. mbio a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data a novel coronavirus from patients with pneumonia in china epidemiologic characteristics of early cases with 2019 novel coronavirus (2019-ncov) disease in republic of korea epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study middle east respiratory syndrome coronavirus and the one health concept full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event implications for the evolution of echolocation in bats bat coronaviruses in china. viruses dbatvir: the database of bat-associated viruses. database (oxford) isolation and characterization of viruses related to the sars coronavirus from animals in southern china receptor usage of a novel bat lineage c betacoronavirus reveals evolution of middle east respiratory syndrome-related coronavirus spike proteins for human dipeptidyl peptidase 4 binding sars-cov infection in a restaurant from palm civet pathological changes in masked palm civets experimentally infected by severe acute respiratory syndrome (sars) coronavirus study on the dynamic prevalence of serum antibody against severe acute respiratory syndrome coronavirus in employees from wild animal who queries culling of civet cats coronaviruses: a paradigm of new emerging zoonotic diseases. pathog dis novel coronavirus takes flight from bats? some one health based control strategies for the middle east respiratory syndrome coronavirus. one health one health insights to prevent the next hxny viral outbreak: learning from the epidemiology of h7n9 physiological and molecular triggers for sars-cov membrane fusion and entry into host cells sars-coronavirus (sars-cov) and the safety of a solvent/detergent (s/d) treated immunoglobulin preparation n-95 face mask for prevention of bird flu virus: an appraisal of nanostructure and implication for infectious control mers-cov, surgical mask and n95 respirators who, coronavirus disease (covid-19) advice for the public: myth busters. avialble at inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation on the origin and continuing evolution of sars-cov-2 mers coronavirus in dromedary camel herd, saudi arabia. emerg infect dis the aetiology, origins, and diagnosis of severe acute respiratory syndrome middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study use of the informational spectrum methodology for rapid biological analysis of the novel coronavirus 2019-ncov: prediction of potential receptor, natural reservoir, tropism and therapeutic/vaccine target the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak avian influenza and ban on overnight poultry storage in live poultry markets, hong kong. emerg infect dis avian influenza virus detection rates in poultry and environment at live poultry markets effect of closure of live poultry markets on poultry-to-person transmission of avian influenza a h7n9 virus: an ecological study expert consensus on preventing nosocomial transmission during respiratory care for critically ill patients infected by controversy around airborne versus droplet transmission of respiratory viruses: implication for infection prevention transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination isolation and identification of human coronavirus 229e from frequently touched environmental surfaces of a university classroom that is cleaned daily extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards evaluating the virucidal efficacy of hydrogen peroxide vapour virucidal activity of world health organization-recommended formulations against enveloped viruses, including zika, ebola, and emerging coronaviruses covid-19) advice for the public clinical features of patients infected with 2019 novel coronavirus in wuhan broad spectrum antiviral agent niclosamide and its therapeutic potential prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus key: cord-276193-cngz535o authors: volz, a.; sutter, g. title: modified vaccinia virus ankara: history, value in basic research, and current perspectives for vaccine development date: 2016-08-01 journal: adv virus res doi: 10.1016/bs.aivir.2016.07.001 sha: doc_id: 276193 cord_uid: cngz535o safety tested modified vaccinia virus ankara (mva) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. historically, mva was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. adapted to growth in avian cells mva lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. as a biologically well-characterized mutant virus, mva facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. as extremely safe viral vectors mva vaccines have been found immunogenic and protective in various preclinical infection models. multiple recombinant mva currently undergo clinical testing for vaccination against human immunodeficiency viruses, mycobacterium tuberculosis or plasmodium falciparum. the versatility of the mva vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the middle east respiratory syndrome. recent advances include promising results from the clinical testing of recombinant mva-producing antigens of highly pathogenic avian influenza virus h5n1 or ebola virus. this review summarizes our current knowledge about mva as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology. host (cell) environment. as a biologically well-characterized mutant virus, mva facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. as extremely safe viral vectors mva vaccines have been found immunogenic and protective in various preclinical infection models. multiple recombinant mva currently undergo clinical testing for vaccination against human immunodeficiency viruses, mycobacterium tuberculosis or plasmodium falciparum. the versatility of the mva vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the middle east respiratory syndrome. recent advances include promising results from the clinical testing of recombinant mva-producing antigens of highly pathogenic avian influenza virus h5n1 or ebola virus. this review summarizes our current knowledge about mva as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology. poxviruses engineered to express foreign gene products are established tools for the development of novel vaccines and therapeutics in biomedical research. large packaging capacity for heterologous dna, strict virusspecific control of recombinant gene expression, lack of virus persistence in the host, immunogenicity and efficacy as vaccine, and ease of vector and vaccine production were important contributors to this success story. concerns about the safety of conventional vaccinia viruses as smallpox vaccine have been addressed by the study of replication defective viruses unable to produce infectious progeny in human cells. today, the highly attenuated vaccinia virus strain mva can be considered as one of the vaccine viruses of choice in preclinical and clinical research. mva is replication-deficient in cells of mammalian origin and fails to produce many of the virulence factors encoded by conventional vaccinia virus. because of its safety for the general environment mva can be handled under conditions of biosafety level 1 (bsl-1). nonreplicating mva can enter any target cell and activate its molecular life cycle to express all classes of viral and recombinant genes. therefore, recombinant mva have been established as an extremely safe and efficient viral vector system for basic research and for the development of vaccines suitable for industrial scale production. here, we review the development of mva as product from serial tissue culture passage in chicken embryo fibroblasts (cef), its key biological properties, and recent accomplishments in vaccine research using recombinant mva. modified vaccinia virus ankara (mva) was developed by serial passage in chicken fibroblast tissue culture to serve as safer vaccine during the last years of the who smallpox eradication campaign. its ancestor virus is the vaccinia virus strain ankara which was originally propagated on the skin of calves and donkeys at the turkish vaccine institute in ankara for smallpox vaccine production. in 1953, the vaccinia virus strain ankara was brought to munich and added to the strain collection of the institute for infectious diseases and tropical medicine at the university of munich. herrlich and mayr cultivated the virus on the chorioallantois membranes (cam) of embryonated chicken eggs and therefore named it as chorioallantois vaccinia virus ankara (cva) (herrlich and mayr, 1954) . at the bayerische landesimpfanstalt m€ unchen (bavarian state institute for vaccines), cva was grown on the skin of calves to manufacture smallpox vaccine for the vaccination campaigns in munich in 1954 munich in /1955 . in addition, at the university of munich, cva was tested in passage experiments in various tissue cultures to study the genetic stability and the evolution of orthopoxviruses. mayr and munz (1964) reported that 371 passages of cva in primary cef had resulted in the development of an infection phenotype with restricted host (cell) tropism and it was discussed that similar biological properties were known from poxviruses that are highly adapted to specific hosts, e.g., variola virus (varv) to humans or fowlpox virus to chicken. successive passage of vaccinia virus in minced chicken embryo tissue had been described as successful strategy for in vitro amplification of the smallpox vaccine virus in a culture system ward, 1931, 1933) . the serial passage of cva in cef was further continued by anton mayr and colleagues and, in 1968 after the 516th passage on cef, the virus was renamed modifiziertes vakziniavirus ankara (mva) and provided to the bavarian state institute for vaccines to test its suitability for smallpox vaccine production (stickl and hochstein-mintzel, 1971 ). phenotypic changes relating to the repeated passage of the cva virus in cef cultures were first observed upon infection of the embryonated egg. for many years, cam inoculations were the gold-standard experimental system for the phenotypic study of various poxviruses (goodpasture et al., 1931 (goodpasture et al., , 1932 mayr et al., 1955) . mva infection is characterized by the formation of small proliferative lesions on the cam. in contrast, considerably larger cam lesions with variable size areas of central necrosis are typically found with cva or other conventional vaccinia viruses (herrlich and mayr, 1954) . interestingly, the cam lesions of mva were noted to closely resemble those induced by variola or fowlpox viruses following egg inoculation (mayr and munz, 1964; stickl and hochstein-mintzel, 1971 ). in addition, it was observed early on that mva had lost the capacity of vaccinia virus to cause prominent cytopathic effects and/ or to form plaques in first-generation tissue cultures such as cef, primary bovine, or porcine kidney cells, or human hela cells (mayr and munz, 1964) . the most characteristic changes in the in vivo behavior of the virus were reported from experimental inoculations of rabbits (german great white) (mayr et al., 1975; stickl and hochstein-mintzel, 1971 ). intradermal infections or cutaneous infections by scarification with conventional vaccinia virus (vacv) result in the formation of typical skin lesions. such lesions were totally absent following inoculation with mva suggesting a substantial loss of virulence upon in vivo infection. these data were confirmed by the finding that newborn (strain nmri; 1-3 days old) or adult mice (12-15 g) survived intracerebral inoculations of mva at doses that resulted in 100% mortality following cva infection (mayr et al., 1975; stickl and hochstein-mintzel, 1971 ). the inability of mva to induce primary reactions with pock lesions forming upon intradermal or cutaneous inoculation was also confirmed in the cynomolgus monkey model (macacus fascicularis). macaques tolerated intracranial inoculations with mva without obvious adverse effects, whereas animals injected with cva developed severe systemic disease. moreover, intradermal or intramuscular vaccination with about 2 â 10 5 infectious units (iu) of mva vaccine protected macaques from severe disease following intravenous challenge with varv strain madras 1965 (mayr et al., 1975; stickl and hochstein-mintzel, 1971 ). these data from early preclinical characterization in laboratory animals already suggested that mva had maintained immunogenicity as vaccine but demonstrated a dramatic loss of virulence. upon the 516th cef passage the virus was renamed mva and transferred to the bavarian state vaccine institute in munich for evaluation as safer smallpox vaccine in clinical trials. the first mva vaccine preparation to be tested in humans was produced in cef cultures and contained 10 6 iu mva/ml (stickl and hochstein-mintzel, 1971 ). in first attempts, the use of this vaccine by scarification failed to induce any kind of skin reactions and the application by intracutaneous inoculation (0.2 ml vaccine suspension containing 2 â 10 5 iu mva) was chosen for first clinical testing and primary vaccination of 107 individuals aged 2-38 years (stickl and hochstein-mintzel, 1971 ). after 4-6 days, minor local reactions developed at the site of injection with redness and swelling of the skin of the forearm. however, the investigators observed no pock lesions or other symptoms normally associated with smallpox vaccination and none of the 107 individuals developed fever (body temperature !38°c). interestingly, it was noted that the mva application failed to induce circulation antibodies that inhibited the hemagglutination by vacv. thus, the efficacy of immunization was tested with secondary vaccination by scarification using vacv strain elstree in 64 of the 107 individuals that had received mva for primary vaccination. skin reactions typical for follow-up smallpox vaccinations were noted in 62 of 64 patients and suggested a successful primary immunization with the mva vaccine. these data supported further clinical development of mva as smallpox vaccine and, following the testing in more than 7000 patients (stickl et al., 1974) , the bavarian state vaccine institute in munich obtained the first marketing authorization for mva as primary prevaccine against smallpox in germany in 1977 (paul-ehrlich-institut, 31.01.1977) . until 1980, the mva smallpox vaccine was given to more than 120,000 humans without documentation of severe adverse events otherwise associated with the use of conventional vacv vaccines (mahnel and mayr, 1994) . immunizations with this first licensed mva vaccine stopped with the end of the smallpox vaccination program in germany. only a few years later, targeted genetic modification of the vaccinia virus genome became possible and the concept to generate recombinant viruses for gene expression or vaccination also restored interest in mva. restriction mapping of the mva genome was based on the clonal virus isolate f6 from the 572nd cef passage of mva and revealed alterations in the mva genome that are the likely genetic basis for attenuation and growth restriction (meyer et al., 1991) . comparison to the genome maps of cva ancestor viruses revealed that the mva genome harbors large deletions and mutations affecting many genes with functions in virus-host interaction (fig. 1) . first important observations included (i) the failure to rescue productive mva growth in cells of human origin by restoring the vacv host range gene k1l despite presence of the second vacv host range gene c7l with the mva genome; (ii) the absence of the gene for the major vacv a-type inclusion body protein and the lack of this protein among the polypeptides made in mva-infected cells; (iii) the demonstration that the coding sequences of the vacv hemagglutinin (ha) started right adjacent to deletion iii within the mva genome which a few years later allowed to identify the truncation of the ha promoter sequence explaining for the ha-negative phenotype of mva and the inability to detect ha-specific antibodies upon mva immunization (antoine et al., 1996) . notably, these early findings were further complemented by the elucidation of the fulllength sequence of the mva genome (antoine et al., 1998) and the discovery that mva lacks important immunomodulatory genes . the highly attenuated phenotype of the virus also encouraged the evaluation of mva as an expression vector. yet, mva cannot productively replicate in most cells of mammalian origin and high-level expression of recombinant genes was doubtful because other host range mutants of vacv are inhibited already early in their life cycle (drillien et al., 1978 (drillien et al., , 1981 . the coding sequences for the escherichia coli enzymes β-galactosidase and guaninephosphoribosyl-transferase served as the first heterologous genes to be inserted and expressed at the site of deletion iii in the mva genome (sutter and moss, 1992) . surprisingly, the production of early and late viral proteins turned out to be unimpaired in mva-infected human cells (fig. 2) . indeed, the unique ability to efficiently express viral and recombinant genes supported its general application as exceptionally safe viral vector. to ascertain whether mva vectors would be applicable for immunization experiments with recombinant antigens, a first mva vector vaccine was constructed and tested that simultaneously expressed the ha and nucleoprotein (np) genes of influenza a virus (sutter et al., 1994) . the recombinant mva was found to be immunogenic, since vaccination of mice by various routes resulted in high levels of serum antibodies that inhibited hemagglutination by influenza a virus. moreover, the vector vaccine also elicited strong cytotoxic t cell responses directed to both influenza virus proteins. importantly, animals could be completely protected against a lethal respiratory tract influenza challenge following single inoculations with relatively low doses of the recombinant mva vaccine. to generate these first mva vectors, the foreign gene sequences were targeted precisely to the site of the naturally existing deletion iii in the mva genome. this strategy in designing the vector was to avoid unnecessary changes in the genotype and phenotype of the resulting recombinant mva. the development of other recombinant mva vaccines with heterologous genes from simian immunodeficiency virus (siv) or parainfluenza virus 3 inserted in deletion iii rapidly followed (hirsch et al., 1996; wyatt et al., 1996) . also, the natural deletion ii within the mva genome was successfully used as insertion site to express recombinant bacteriophage t7 rna polymerase (sutter et al., 1995) . in addition, the thymidine kinase locus, a well-exploited insertion site in replication-competent vacv vectors, likewise, served to generate recombinant mva delivering antigens of hiv-1 or plasmodium berghei schneider et al., 1998) . poxviruses including the prototype orthopoxvirus vacv are excellent models to study virus-host interactions. vacv can efficiently antagonize the activity of interferons (ifns), cytokines, chemokines, and innate immune signaling (for review, see smith et al., 2013) . hereby, vacv exploits the expression of soluble-binding proteins and receptor antagonists. other viral immune evasion proteins work intracellularly to inhibit apoptosis or to interfere with host signaling pathways activating antiviral immune mechanisms. notably, of the many vacv genes involved in immune evasion, most are inactivated or truncated in the mva genome (antoine et al., 1998) . interestingly, early studies by mayr and coworkers had suggested distinct immune stimulatory activities associated with experimental mva inoculations (mayr et al., 1975) . e.g., intraperitoneal application of virus to mice enhanced the in vivo clearance of carbon marker particles from the blood of the animals suggesting an increased phagocytic activity of immune cells 2 days after mva inoculation. in addition, intranasal delivery of mva to rabbits resulted within hours in the induction of "serum interferons" that efficiently inhibited sindbis virus replication in an in vitro infection assay. in the more recent past, various studies further elucidated the mechanisms of mva-mediated induction of type i ifns following in vitro and in vivo infections dai et al., 2014; delaloye et al., 2009; ishii et al., 2006; waibler et al., 2007 waibler et al., , 2009 . indeed, the lack of many immune evasion factors and the failure of mva infection to interfere early with key host (cell) defence mechanisms may explain the severe growth restriction of mva, its high attenuation upon in vivo infection and its immunogenicity when used as vaccine. moreover, due to the extensive loss of genetic information mva may serve as a particularly useful tool to study vacv host-regulatory genes. obviously, there are several pathways of the host (cell) defence that are targeted by multiple vacv proteins and the phenotype of a vacv single gene mutant may be masked by the complementary function of (an)other viral gene(s) (dobson and tscharke, 2015) . there are three obvious strategies using mva to elucidate on functions of selected vacv host-interaction factors: (i) the few remaining regulatory genes in the mva genome can be targeted for inactivation, (ii) candidate vacv genes missing in mva are reinserted in the genome to rescue a host-interaction phenotype, or (iii) a combination of these latter approaches may serve to investigate putatively complementary gene functions. in the following we describe some principles learnt by mva research that may help to better understand the regulation of virus-host interactions and likely influence safety and immunogenicity of vacv-based vaccines and therapeutics (fig. 3 ). one most striking feature of mva is its inability to replicate in most cells of mammalian origin (carroll and moss, 1997; drexler et al., 1998; meyer et al., 1991) . in contrast, wild-type vacv has a broad cellular host range and productively infects various cell substrates (drillien et al., 1978) . it is noteworthy that poxvirus infections do not rely on the availability of specific cellular receptors but the viruses can efficiently bind to and enter many different cells from diverse animal species. after entry, however, the success of vacv replication depends on the functional activity of a subset of viral genes, the so-called "host range" genes (for review, see bratke et al., 2013; haller et al., 2014; mcfadden, 2005) . these viral genes encode regulatory proteins that control the intracellular host defence, e.g. by manipulation of the sensing of viral pathogen associated molecular patterns, the signal transduction, the cell cycle, or the onset of programmed cell death. some eminent viral regulators of intracellular host tropism are still functional in the mva genome and include the proteins c7, k3, e3, f1, and b18 (68k) (following the nomenclature as established for the genome of vacv strain copenhagen; goebel et al., 1990) . the vacv genes k1l and c7l (encoding the vacv proteins k1 and c7) are known to control virus replication in mammalian cells and both gene functions need to be inactivated to restrict the growth of wild-type vacv (gillard et al., 1986; perkus et al., 1990) . already early work by drillien and coworkers suggested that the replication defect of a vacv host range mutant (in the absence of k1l and c7l) is established at the level of viral gene expression (drillien et al., 1981) . however, the severe growth defect of the highly attenuated mva is not rescued when its genome is engineered to contain functional copies of both k1l and c7l suggesting that additional viral factor(s) await discovery (backes et al., 2010; meyer et al., 1991; sutter et al., 1994) . however, a c7l-deleted mva lacks late gene expression in human and murine cells and induces phosphorylation of eukaryotic translation initiation factor 2α (eif2α). the deficiency of late gene expression and the phosphorylation of eif2α in the absence of c7 can be prevented by k1 as shown by the reinsertion of the k1l gene into the genome of c7l-deleted mva (backes et al., 2010) .the complementary function of k1l and c7l is intriguing because the two genes and encoded proteins are unrelated in sequence. interestingly, recent studies identified the human genes samd9 (sterile alpha motif domain-containing 9) and wdr6 (tryptophan-aspartic acid repeat 6) as host restriction factors for poxviruses and as targets of c7 and k1 (liu and mcfadden, 2015; sivan et al., 2015) . to date, rather little is known about the molecular functions of samd9 and wdr6, but it can be assumed that they act in (an) innate antiviral defense pathway(s) awaiting further elucidation. the viral proteins k3 and e3 (encoded by k3l and e3l genes) are wellknown intracellular inhibitors of ifn-induced antiviral activities of the host cell (chang et al., 1992; davies et al., 1992) . k3 has homology to the alpha subunit of eif2α and serves as a pseudo-substrate for double-stranded rna (dsrna)-activated protein kinase (pkr) to prevent virus-induced phosphorylation of eif2α by pkr. in turn, e3 can sequester dsrna to inhibit the stimulation of pkr and to prevent activation of 2 0 -5 0 oligoadenylate synthase and the endoribonuclease rnasel. in addition, e3 has been described to independently inhibit the phosphorylation of the transcription factors irf-3 and irf-7 and thus to block the production of type i ifns xiang et al., 2002) . these ifns evasion mechanisms provided by k3 and e3 substantially contribute to the broad cellular host range of vacv and numerous host range phenotypes of vacv mutants lacking e3l or k3l genes have been found (werden et al., 2008) . interestingly, cef-adapted mva has maintained fully active e3l and k3l sequences. indeed, a functional e3l gene is required for mva replication in cef where e3 functions as an inhibitor of apoptosis and/or ifn induction to allow for unimpaired late protein synthesis (hornemann et al., 2003) . in human hela, hacat or 293t cells studies with an e3l-deleted mva mutant (mva-δe3l) revealed that e3 is also essential to secure viral intermediate and late protein synthesis by counteracting host cell-specific activation levels of antiviral pathways pkr or rnasel . mva-δe3l also increased the type i ifn and/or chemokine production when compared to wild-type mva in infections of primary murine fibroblasts or murine bone marrow-derived dendritic cells which is in agreement with the known activation of transcription factors irf-3 and irf-7 in the absence of e3 (dai et al., 2014; ishii et al., 2006) . likewise, apoptosis induction in mva-δe3l infected murine fibroblasts required the proapoptotic cellular bh3-only protein noxa which is well in line with the irf-3/ifn-beta-mediated activation of noxa (delaloye et al., 2009; fischer et al., 2005) . cell death by apoptosis is an important defence mechanism to protect the host against viral infection. in turn, poxviruses have evolved specific viral proteins to inhibit the onset of programmed cell death and these regulatory proteins are relevant determinants of virus tropism at the cellular level (taylor and barry, 2006) . the vacv f1 protein (encoding gene f1l) is an antiapoptotic protein with bcl-2-like structure proposed to block apoptosis by binding to the proapoptotic family protein bak (postigo et al., 2006; wasilenko et al., 2003 wasilenko et al., , 2005 . f1l is also conserved in the mva genome and f1l-deficient mva (mva-δf1l) induces enhanced apoptosis in hela cells and in mouse embryonic fibroblasts (fischer et al., 2005) . additional work with mva-δf1l demonstrated that triggering of apoptosis predominantly requires the induction of the pro-apoptotic bh3-only protein noxa which then activates proapoptotic bak (ferrer et al., 2011) . hereby, activation of noxa was linked to the recognition of viral rna and the upregulation of type i ifn signaling. thus, the noxa-dependent induction of apoptosis observed upon infections with mva-δe3l (fischer et al., 2005) may be explained by the failure to sequester viral rna in the absence of e3 resulting in strong activation of pro-apoptotic noxa (fig. 4) . ankyrin repeat (ank) motifs are found in many poxvirus regulatory proteins that determine the cellular host tropism and/or counteract antiviral host responses controlled by nf-kb signaling (herbert et al., 2015) . otherwise ank motifs are described important for many proteinprotein interactions and cellular ank proteins are involved in diverse regulatory tasks including cellular transcription, cell cycle control, or cellular differentiation (mosavi et al., 2004) . the only ank-containing protein encoded by the mva genome is the 68-kda ankyrin-like protein (68kank; homologue to the vacv cop b18r gene product). in addition to anks, 68k-ank contains an f-box-like pranc (pox protein repeats of ankyrin c-terminal) domain (mercer et al., 2005) . mva 68k-ank binds to cellular skp1a and forms a cullin-1-based scf ubiquitin ligase complex in an f-box-dependent manner (sperling et al., 2008) . a 68k-ank-deficient mva mutant shows reduced transcription of intermediate and late viral genes and suffers from a drastically impaired late protein synthesis in human and murine cells (sperling et al., 2009 ). thus, 68k-ank is essential for the completion of the mva molecular life cycle. interestingly, the f-box domain of 68k-ank is dispensable for rescue of the 68k-ank mutant phenotype suggesting multiple activities of this host-regulatory mva protein. mva is intensely used as vector vaccine being tested against a variety of infectious diseases (see section 4) and some cancers. however, we still know very little about the influence on mva vaccine immunogenicity and vaccine efficacy with regard to these mva proteins regulating the host cell tropism. indeed, modulating the functional activity of these proteins could result in quite different outcomes. in one scenario, the inactivation of these regulators may further enhance mva-mediated activation of the innate immune system and may lead to improved protective immunity; another possible scenario is reduced mva vaccine efficacy because fig. 4 role of the mva regulatory proteins e3 and f1 within the intracellular virus life cycle: e3 can efficiently sequester dsrna produced during infection to counteract the activation of the antiviral host enzymes oas (2 0 -5 0 oligoadenylate synthetase)/rnasel (ribonuclease l) or pkr (protein kinase rna-activated). additional functions of the e3 protein are to prevent the activation of the host proapoptotic protein noxa and to block the phosphorylation of the transcription factors irf-3 and irf-7 inhibiting the production of type i interferons. the mva protein f1 can bind or prevent the activation of proapoptotic host proteins bax/bak or noxa and acts as efficient inhibitor of cytochrome c release (cyto c) and apoptosis induction at the level of host cell mitochondria. the deletion of some of these genes (e.g., e3l, c7l, 68k-ank) may drastically impair viral replication, the levels of gene expression and mvamediated antigen synthesis in addition to regulatory genes targeting the host tropism, the mva genome also contains a number of genes with known immunomodulatory functions. one interesting example is gene b16r encoding the vacv interleukin-1β receptor protein (il-1βr). this viral cytokine-binding protein has highspecific affinity for il-1β (alcami and smith, 1992; spriggs et al., 1992) and is thought to play an important role in the regulation of inflammatory responses following vacv infection (alcamí and smith, 1996) . synthesis of il-1βr could be demonstrated in various mva-infected cultured cells zimmerling et al., 2013) . interestingly, primary murine myeloid dendritic cells are important il-1β producer cells upon mva infection but free il-1β can be detected only in the absence of il-1βr using the deletion mutant mva-δil-1βr. immunizations with this mva deletion mutant led to significantly enhanced virus-specific cd8+ t-cell responses and increased protective capacity against lethal challenge infection with virulent vacv strain western reserve (wr) . in addition, the gene sequence encoding the vacv interleukin-18 (il-18) binding protein (il-18bp; c12l gene) is also functionally retained in the mva genome . vacv il-18bp binds soluble il-18 and prevents it to reach its cellular receptor targeting the proinflammatory and antiviral function(s) of this cytokine (born et al., 2000; calderara et al., 2001; symons et al., 2002) . the possibility to enhance vaccine immunogenicity by inactivation of the il-18bp gene in mva was investigated in two previous vaccination studies in mice. cottingham and coworkers found no significant difference in vacv-specific t cell responses when comparing vaccines based on mva mutated in c12l or wild-type mva (cottingham et al., 2008) . a second study demonstrated functional activity of the mva il-18bp and immunizations with the mva deletion mutant increased vacv epitope-specific cd8 + and cd4+ t-cell responses and protective capacity against a vacv challenge infection (falivene et al., 2012) . to diminish the inflammatory host response, another strategy of vacv is the production of secreted viral chemokine receptor proteins which prevent the chemokine-mediated recruitment of leukocytes ( fig. 5 ) (alcamí et al., 1998; graham et al., 1997; ng et al., 2003) . interestingly, mva has lost much of this vacv immune evasion activity. mva efficiently induces chemotaxis and triggers the rapid immigration of leukocytes to the site of in vivo inoculation (lehmann et al., 2009 ). yet, mva still produces the secreted protein a41 which binds chemokines with relatively low affinity suggesting another functional mechanism than just blocking the binding of chemokines to their cellular receptors (bahar et al., 2008) . moreover, deletion of the a41l gene in mva improved the cd8+ t-cell immunogenicity and the protective capacity of vaccination . vacv encodes an impressive variety of intracellular virus proteins that can inhibit the host signaling pathways for nf-kb and irf-3 (for review, see smith et al., 2013) . together these proteins serve to dampen the innate host response by blocking the induction of type i ifns, chemokines, and proinflammatory cytokines. mva fails to functionally produce a substantial number of these inhibitory proteins including a52, b14, c4, c16, k1, m2, and n1. indeed, mva infection induces the activation of nf-kb (oie and pickup, 2001) and the reinsertion of the original vacv gene sequences into the mva genome allowed to further elucidate the function of k1 and m2 as nf-kb inhibitors (hinthong et al., 2008; shisler and jin, 2004) . other inhibitory genes, such as c6l, k7r, a46r, and a49r, are fully conserved in mva. interestingly, the removal of c6l, k7r, or a46r from the mva genome is reported to contribute to higher frequencies of antigen-specific cd8 + and cd4+ t cells, enhanced polyfunctionality of t cells and higher antigen-specific antibody titers when tested in recombinant mvaproducing hiv candidate antigens (garber et al., 2009; garcía-arriaza et al., 2011 . a similar improved immunogenicity is also described for a recombinant mva-hiv virus deleted in the n2l gene (garcía-arriaza et al., 2014) . these data suggest the functional activity of mva n2 despite the fact that the mva gene harbors an in-frame deletion causing the loss of five amino acids (aa 31-35). vacv n2 is an interesting regulatory protein because it acts as inhibitor of irf-3 activity within the nucleus of infected cells (fig. 6 ) . shortly after the eradication of smallpox, vacv acquired a new mission as eukaryotic cloning vector for the expression of heterologous genes. homologous recombination that frequently occurs between vacv genomes within an infected cell was discovered to allow for efficient insertion of foreign dna into the vacv genome (mackett et al., 1982; panicali and paoletti, 1982; . this technology enabled the development of new recombinant vaccines that could be beneficial to decrease the impact of various infectious diseases. over the years replication-competent recombinant vacv has been successfully introduced for use in veterinary medicine as accomplished with the recombinant vacv vaccines against rabies (blancou et al., 1986; esposito et al., 1987; kieny et al., 1984) . the application of recombinant vacv for vaccination of humans was lagging behind restricted due to the well-known side effects of vacv smallpox vaccines. in these circumstances the availability of replication-deficient vacv such as nyvac or mva has spurred the establishment of exceptionally safe next-generation poxviral vectors (sutter and moss, 1992; tartaglia, 1992) . the technology for recombinant mva has been adapted from vacv. the still most frequently and effectively practiced strategy to generate recombinant poxviruses employs homologous dna recombination in infected cells, a relatively frequent event during vacv genome replication (0.1%) (mackett et al., 1984; nakano et al., 1982) . recombination is typically directed by a gene transfer plasmid. for transcriptional control of foreign genes, various virus-specific promoters are used to achieve moderate to strong expression during the whole virus replication cycle (baldick et al., 1992; chakrabarti et al., 1985; davison and moss, 1989; di pilato et al., 2013 , 2015 wennier et al., 2013; wyatt et al., 1996) . usually, the plasmid carries the specific expression unit with a virus-specific promoter next to the multiple cloning site for insertion of various foreign gene sequences and selectable markers to facilitate the clonal isolation of recombinant mva (mackett et al., 1984; staib et al., 2004) . the foreign gene sequences and the marker gene are flanked on both sides by genomic mva sequences that direct the recombination of the expression cassette to favored loci in nonessential regions of the mva genome. in mva, the sites of major deletions are suited for the insertion of foreign gene sequences without affecting essential regions in the mva genome (sutter and moss, 1992; sutter et al., 1995) . until today, deletion site iii serves as one of the most frequently used insertion loci song et al., 2013; volz et al., 2016) . in addition, standard insertion sites used in conventional vacv, the thymidine kinase gene locus ( j2r) and the ha gene locus (a56r), were readily established for the construction of recombinant mva (antoine et al., 1996; schneider et al., 1998) . moreover, due to the high fidelity of homologous recombination also small nonessential regions between mva genes can be used to introduce heterologous dna for vector construction (wyatt et al., 2009 ). multiple insertion sites may allow for integration of various expression units for foreign genes in the same mva genome when opting for multivalent recombinant vaccines. for the generation of recombinant mva, cells are infected with mva and simultaneously transfected with the respective mva transfer plasmid to allow for homologous recombination (kremer et al., 2012b; staib et al., 2004) . recombinant mva viruses are clonally isolated in repetitive cell culture passages screening for specific selection markers. different protocols have been established to allow differentiation between wild-type and recombinant mva. very first approaches took advantage of specific enzymes that allow for color discrimination. here, the transfer vector contains an antibiotic selection marker or a reporter gene allowing the screening due to a change in phenotype such as coexpression of the e. coli β-galactosidase and β-glucuronidase (carroll and moss, 1995; chakrabarti et al., 1985) . among the coexpressed antibiotic resistance markers, the e. coli gpt gene encoding the enzyme xanthine-guanine-phosphoribosyltransferase is frequently used for purification of recombinant viruses by dominant positive selection for resistance against mycophenolic acid (falkner and moss, 1988; isaacs et al., 1990) . staining procedures require additional time of tissue culture, supplementation of agar overlays, and the use of chromogenic substrates and antibiotics. complementation of a defect in virus production is a faster and more convenient method to obtain recombinant mva viruses. a first growth selection protocol was initiated using the vacv host range gene k1l to rescue recombinant mva replication in rabbit kidney rk-13 cells (staib et al., 2000) . blasco and moss had introduced selection for vacv plaque formation through coinsertion of the f13l gene which was adapted to isolate mva vector viruses (blasco and moss, 1995; sánchez-puig and blasco, 2005) . moreover, mva mutant virus and a matching complementing cell line enables for growth selection based on the essential d4r gene function (ricci et al., 2011) . up to now, the generation of recombinant mva viruses is based on well-established techniques for isolation of clonal viruses that include the use of serum-free media and marker-free recombinant viruses. specific regulatory guidelines help to supervise the generation of recombinant vector vaccines suitable for applications in humans (ema, 2010 ). yet, in consequence, generation of recombinant mva is to a certain extent more limited to the use of some preferred methods (kremer et al., 2012b) . e.g., one preferred scheme is the isolation of recombinant mva through screening for transient cosynthesis of marker proteins without enzyme activity and not related to an antibiotic or chemotherapeutic resistance phenotype. fluorescence proteins such as green or red fluorescent proteins are conveniently used as well-characterized inert marker proteins. alternative procedures to engineer poxvirus genomes have been pioneered more recently. the entire vacv and mva genomes were cloned as bacterial artificial chromosomes (bac), which can be engineered in e. coli by homologous recombination with bacteriophage lambda-derived enzymes (cottingham et al., 2008; moss, 2002, 2005) . the modified bac clones can be used to produce pure recombinant poxvirus in mammalian cells with the initial assistance of a helper virus but without further requirements for plaque purification. another up-to-date method, yet to be adapted to the construction of recombinant mva, utilizes the crispr-cas9 system to insert alternative gene sequences into the vacv genome (yuan et al., 2015) . importantly, independent of the methodology used for generation of a recombinant mva virus any new vector virus has to be thoroughly quality controlled for genetic identity, purity, genetic stability, recombinant gene expression, and growth characteristics (kremer et al., 2012b) . typically, the in vitro characterization already starts during the process of clonal isolation of mva vector viruses by plaque purification. a collection of straightforward methodologies has been established to confirm genomic identity and the correct insertion of the target gene sequence within the mva genome using finger-print pcrs to assess the insertion sites and the six naturally occurring deletion sites. levels and kinetics of recombinant protein synthesis, the stability, or the posttranslational modification of the foreign target proteins are typically monitored using standardized in vitro infection experiments and antigen-specific immune detection assays (e.g., western blotting). in this context, it is also important to analyze the growth capacities of recombinant mva. the well-known replication deficiency of mva in mammalian cells is a key biological characteristic of mva and allows the genetically modified virus to be handled in germany under conditions of bsl-1 with minimal potential biohazard to laboratory personnel, clinicians, patients, and the general environment (bundesamt f€ ur vebraucherschutz und lebensmittelsicherheit, 2002) . therefore, new recombinant mva are routinely tested for their growth characteristics in cells of human origin, preferably in cells with normal differentiation such as hacat cells (boukamp et al., 1988 ( jordan et al., 2011; lohr et al., 2009) . moreover, it is encouraging that some of these designer cell lines have already been used to manufacture first candidate vaccines being approved for clinical evaluation (genzel, 2015) . in medical research and development ongoing efforts focus on the study of candidate vaccines against infectious diseases that are more "complicated" to prevent, e.g., those caused by newly emerging pathogens such as severe acute respiratory syndrome coronavirus, west nile virus (wnv), or avian influenza virus. for decades, the availability of a safe and efficient vaccine against different influenza viruses has been one of the biggest demands of public health systems. until today, infection with seasonal influenza virus kills about 250,000-500,000 people every year. here, the main risk groups comprise the elderly, immunocompromised and children, with the highest incidence of fatal outcome (loubet et al., 2016) . moreover, in addition to the circulating seasonal influenza viruses, there is a continuous threat of new pandemic influenza viruses that might arise from pigs or birds herfst et al., 2012) . during the last century, there have been four pandemics that together caused more than 50 million deaths (russell et al., 2012) . hence, it is not surprising that influenza was the target of the first recombinant mva vaccine. this vector virus was designed to codeliver the influenza virus a/pr/8/34 (h1n1) antigens ha, and np (mva-ha-np) (sutter et al., 1994) . balb/c mice immunized with mva-ha-np mounted efficient levels of ha-specific antibodies as well ha-and np-specific cd8+ t cells. a single intramuscular immunization was sufficient to protect these mice against a lethal respiratory challenge with influenza virus a/pr/8/34. subsequently, mva-ha-np was shown to induce mucosal immunity following oral immunization and to allow for partial protection against a heterologous influenza virus subtype h3n2 challenge infection (bender et al., 1996) . after these results from testing a first recombinant mva-expressing influenza virus antigens, it took sometime to encourage a multitude of other experiments targeting influenza in veterinary as well as human medicine. breathnach and coworkers characterized a recombinant mva-producing ha or np from equine influenza a virus h3n8. vaccination of ponies with mva-ha induced robust levels of antibodies and protected against challenge with influenza virus h3n8 a/equine/kentucky/1/81. also mva-np-vaccinated ponies did not show severe clinical symptoms upon influenza virus challenge infection suggesting the induction of cellular immune responses with protective capacity (breathnach et al., 2006) . because of the zoonotic transmission and the pandemic potential of various highly pathogenic avian influenza viruses of the h5 subtype, the development of h5-specific vaccines for humans is considered a priority since 1997 (de jong et al., 1997; yang et al., 2015) . vaccine development is complicated by antigenically different clades of influenza viruses within the h5 subtype. in this context, kreijtz (kreijtz et al., 2007 (kreijtz et al., , 2009a . vaccination with either of the recombinant mva efficiently induced antibody responses against the homologous influenza virus. moreover, immunization with mva-ha-vn/04 (clade 1) was able to activate antibodies not only against the homologous virus but also to a/hk/156/97 (clade 0) and to a lesser extent to a/indonesia/5/05 (clade 2.1). this potent activation of influenza virus-neutralizing h5-specific antibodies correlated with protective efficacy against homologous and heterologous challenge infection (kreijtz et al., 2007) . interestingly, the exceptional immunogenicity of the ha antigen of influenza a/vietnam/2004 was confirmed in another study comparing recombinant mva vaccines expressing ha antigens from various influenza a viruses h5n1 (clades 0, 1, 2.1, 2.2, 2.3) (hessel et al., 2011) . mva-ha-vn/04 also proved immunogenic and protective when tested as single-shot vaccine in chickens against challenge with the highly pathogenic avian influenza virus a/duck/vietnam/tg24-01/05 (veits et al., 2008) . in cynomolgus macaques, prime-boost immunization with mva-ha-vn/04 was able to protect the animals against homologous virus a/ vietnam/1194/04 and infection with clade 2.1 influenza virus a/ indonesia/5/05. the absence of virus infected cells in the lungs and the lack of fever and severe interstitial pneumonia highlighted the induction of solid protective immunity in vaccinated animals (kreijtz et al., 2009b) . results from further evaluation in mice also demonstrated the efficacy of mva-ha-vn/04 vaccines in dose-sparing and single-shot immunizations. here, minimal requirements for induction of protection against homologous and heterologous influenza virus challenge infections include a single immunization with 10 6 pfu (plaque-forming units) mva-ha-vn/04 or primeboost immunizations with 10 4 pfu mva-ha-vn/04. of note, in some experiments, protection was observed in the absence of detectable ha-specific antibodies. these data look very encouraging with regard to pandemic risks where a vaccine has to protect rapidly and at minimal doses (kreijtz et al., 2009a) . in this context recombinant mva expressing the ha from pandemic h1n1 a/california/04/2009 turned out to be highly immunogenic and protective when tested in the mouse model and ferrets (hessel et al., 2010; kreijtz et al., 2010) . in ferrets, prime-boost vaccination with mva-ha-ca/09 efficiently activated virus-specific antibodies, reduced the clinical signs after challenge infection with a/netherlands/ 602/2009, and protected against severe histopathological changes in the lungs. two immunizations also significantly lowered the presence of infectious virus in the upper and lower respiratory tract (kreijtz et al., 2010) . due to these promising results from in vivo preclinical evaluation, a first-in-man phase i/iia clinical study of mva-ha-vn/04 has been facilitated . the vaccine was safely tolerated and serious side effects were not observed. furthermore, this candidate vaccine proved to be immunogenic in all individuals enrolled in this study. mva-ha-vn/04 efficiently activated antibodies cross-reacting with homologous and heterologous h5 subtype influenza viruses and with the recently emerging highly pathogenic avian influenza virus subtype h5n8 (de vries et al., 2015) . of note, prime-boost applications via the intramuscular route induced levels of influenza h5n1-specific antibodies that raise expectations for protective capacity. interestingly, booster vaccinations allowed for a remarkable enhancement of h5n1 specific antibodies when given 12 months after primary immunization. these results firstly describe a major benefit of booster vaccinations with recombinant mva using an extended time period between primary and secondary immunization . future clinical studies using similar regimens might help to develop new vaccination strategies. overall, the induction of robust levels of influenza ha-specific antibodies can be expected to afford cross-protective immunity to viruses of the same influenza virus subtypes. in addition, mva-mediated delivery or codelivery of influenza virus t cell antigens is being considered to possibly confer broader protection against various subtypes (for review, see altenburg et al., 2014) . probably, most extensively tested as t cell vaccine is a recombinant mva vaccine expressing the influenza virus np and matrix 1 (m1) proteins (mva-np+m1; lambe et al., 2013; . this candidate vaccine was also shown to induce influenzaspecific cd8+ t cells in phase i/iia clinical studies and to protect humans from an experimental influenza challenge infection (berthoud et al., 2011; lillie et al., 2012) . finally, aiming on broadly protective "universal" influenza virus vaccines kamlangdee and coworkers tested an innovative recombinant mva expressing an in silico generated synthetic h5 antigen representing a mosaic sequence of more than 2100 h5n1 viruses (kamlangdee et al., 2014) . interestingly, this candidate vaccine protected mice against h5n1 viruses from all clades but also against infection with influenza virus a/pr/8/34 (h1n1). taken together these highly encouraging data support the clinical evaluation of existing mva candidate vaccines and the further development of novel recombinant mva against influenza. a safe and effective human immunodeficiency virus (hiv) vaccine is urgently needed to control the worldwide hiv epidemic. however, the development of a vaccine against aids represents a substantial scientific challenge related to hiv antigenic variability, the lacking understanding of immune correlates for protection, limitations of available animal models, and the enormous constraints associated with the probable need for multiple large-scale clinical trials in different parts of the world (for review, see excler et al., 2014) . moreover, the fragile immune system of hiv-infected individuals sets high standards to candidate vaccine safety. in the recent past, highly attenuated poxviruses continued to play a major role in the international search for an aids vaccine also taking advantage of established technologies for vector vaccine production at industrial scale. different recombinant mvaexpressing hiv proteins have undergone preclinical and clinical testing for the activation of protective immune responses against aids often in combination with dna-based and/or adenoviral vector vaccines (for review, see iyer and amara, 2014; ondondo, 2014) . recombinant mva vaccines targeting different hiv-1 subtypes continued to prove safe and immunogenic in additional clinical studies (goepfert et al., 2014; joachim et al., 2015; munseri et al., 2015; nilsson et al., 2015) . important new findings included data for the induction of high levels of antibody-dependent cellular cytotoxicity-mediating antibodies and months durability of the vaccine-induced hiv env-specific antibody responses. in this context, a necessary target antigen comprises the immunodeficiency virus envelope (env) protein, as it could be shown to elicit antibody responses with enhanced protective capacity in nonhuman primate chimeric simian/human immunodeficiency virus (shiv) or siv challenge infection models (barouch and michael, 2014; barouch and picker, 2014; roederer et al., 2014) . recent approaches are working on the induction of broadly neutralizing antibodies at the mucosal site of viral entry based on env-specific vaccines. other important hiv immunogens delivered by mva candidate vaccines include gag, pol, and nef proteins targeting the induction of hiv-specific cd4+ and cd8+ t cells . in addition, new synthetic hiv-specific "mosaic" immunogens are under evaluation as improved antigens for induction of cd8 + t cells (ondondo et al., 2016) . the large number of different hiv candidate vaccine necessitates the development of preclinical model systems to evaluate and select the most promising vaccine candidates. in many preclinical experiments, varying degree of protection against homologous immunodeficiency virus infection has been found, predominantly depending on the challenge virus and/or the animal model used for evaluation. however, hiv has an extraordinary genetic diversity and the "holy grail" aids vaccine would have to cross-protect against different hiv clades. a major scientific challenge is now to find appropriate antigens or epitopes that elicit a cross-protective immune response. for sometime, induction of cellular immunity was the primary focus of hiv vaccine development but the generation of broadly neutralizing antibodies is also believed to be indispensable (douek et al., 2006) . previous data from two studies in the macaque model showed that booster vaccinations with oligomeric or native env proteins enhance env-binding and virus-neutralizing antibody responses primed by recombinant mva vaccines, and suggest that such antibodies are indeed likely to play a role in vaccine-induced protection (earl et al., 2002) . currently, siv or shiv challenge infections in different nonhuman primates are considered to be the most appropriate animal models to test for immunogenicity and efficacy. these models very closely mimic the pathogenesis of hiv infections in humans concerning viremia, progressive depletion of cd4+ t cells, and the clinical manifestation of aids (van rompay, 2012). the thorough characterization of recombinant mva candidate vaccines in the siv model continues to further elucidate on the protective capacity of siv antigenspecific immune responses (chamcha et al., 2016; iyer et al., 2015; kwa et al., 2015; valentin et al., 2014) . thus, these nonhuman primate models might allow for the generation of proof-of-concept data on antiviral immunity that effectively inhibits immunodeficiency virus replication and disease development. in addition, mva vector vaccines have proven to be excellent candidates for vaccine development against other infectious diseases with global impact such as tuberculosis and malaria (for review mcshane and williams, 2014) . the incidence of disease caused by mycobacterium tuberculosis is steadily increasing often on the basis of poverty-impaired health services, widespread hiv infection, or the emergence of resistant m. tuberculosis. in recent efforts to elicit more potent antimycobacterial immunity, mva vector viruses served to identify new promising target antigens and resulted in the development of first subunit vaccines entering clinical testing (mcshane et al., 2004; sheehan et al., 2015) . here, the conserved mycobacterial antigen 85a turned out to be a promising immunogen for induction of antigen-specific t cells against m. tuberculosis. a recombinant mva candidate vaccine-expressing 85a under transcriptional control of the vacv early/late promoter p7.5 (mva85a) has been extensively tested in preclinical models and phase i to phase iib clinical studies. vaccination of balb/c mice with mva85a induced both cd4+ and cd8+ t-cell responses and conferred protection against challenge with m. tuberculosis (mcshane et al., 2002) . importantly, in an approach to efficiently trigger activation of 85a-specific cellular immune responses by vaccination, recombinant mva85a has been successfully evaluated for boosting the immunogenic effects of a mycobacterium bovis bcg primary immunization. preclinical studies in different animal models, including mice, guinea pigs, nonhuman primates, and cattle, confirmed the protective efficacy of the heterologous bcg prime-mva85a boost vaccination. in these experiments protection was associated with a reduction of bacterial loads in the lungs rather than with sterilizing immunity (goonetilleke et al., 2003; williams et al., 2005a,b) . unfortunately, when testing bcg-mva85a immunizations in a large phase iib clinical trial in children in africa there was no improvement compared to the conventional bcg only immunization schedule (tameris et al., 2013) . mva85a was well tolerated and immunogenic as shown by the induction of 85a antigen-specific cd4+ t cells. however, the immunizations demonstrated no significant efficacy against tuberculosis or m. tuberculosis infection. nevertheless, the study provides an important and encouraging pool of safety data for a recombinant mva candidate vaccine tested in more than 1300 infants. overall, the results obtained from the clinical testing of mva85a support the development of improved recombinant mva85a candidate vaccines against tuberculosis but also including additional antigens of m. tuberculosis. in 2013, there were more than 500,000 estimated deaths and about 200 million clinical illnesses due to malaria, the majority in central and southern africa. thus, an effective vaccine against malaria is urgently required (hoffman et al., 2015) . a variety of antigens from plasmodium falciparum has been expressed and tested with recombinant vacv. the pathogenesis of malaria involves a complex life cycle including different blood and nonblood life stages of the parasites in the human host as well as in the mosquito host. a major challenge is the choice of optimal target antigens mediating protective immune responses against the multiple phases of malaria. desirable targets should result in an efficient activation of humoral immunity as well as malaria-specific cd8 + t cells. in a landmark study, schneider and coworkers had evaluated recombinant mva vaccines in a mouse malaria model using p. berghei. the study demonstrated efficient induction of malaria-specific t cells that could be further enhanced by using dna prime in combination with recombinant mva as boosting agent. of note, this study demonstrated a need for antigen-specific cd8 + t cells to induce protection against p. berghei sporozoite challenge infection . first clinical trials have been initiated using recombinant mva expressing the important malaria liver stage antigens trap/csp alone or in a heterologous prime-boost schedule in combination with adenoviral vectors (gilbert et al., 2002) . further improvement of this antigen has been achieved by fusion of trap with a peptide sequence encompassing a plasmodium-specific multiple epitope (me-trap) (gilbert et al., 1997) . in detail, moorthy and coworkers generated a single polypeptide me-trap of 789 amino acids which combines a multiple epitope string (me) consisting of 14 cd8+ t-cell epitopes with three cd4+ t-cell epitopes from tetanus toxin, bcg, and pf-circumsporozoite protein (pfcsp) and two b-cell epitopes within the established trap antigen. here, dna prime and recombinant mva boost schedules have been evaluated for safety and immunogenicity in a phase i/ii clinical trial in humans. no severe adverse effects were observed after application of mva-me-trap alone or in combination with dna prime vaccination (moorthy et al., 2003) . in following clinical evaluations, immunogenicity and protection has been assessed in african adults in a malaria-endemic area. heterologous regimens using dna prime and mva boosting proved to be more immunogenic compared to homologous application of either vaccine used alone. however, despite measurable immunogenicity, robust protection against challenge with p. falciparum could not be detected (moorthy et al., 2003 (moorthy et al., , 2004 . another advancement was based on studies that tested different combinations of primary immunizations with recombinant fowlpox and recombinant mva vaccines . next steps included the clinical evaluation of these malaria vaccine candidates in heterologous prime-boost vaccinations. here, the booster immunizations resulted in an efficient activation of malaria-specific cd8 + t cells in adults and children webster et al., 2005) . however, efficacy testing in field studies in endemic areas failed to robustly protect against malaria infection at various stages (bejon et al., 2006) . another innovative approach of heterologous prime-boost vaccination has been conducted by rodriguez and colleagues in the murine p. berghei infection model. the alternative usage of porcine parvovirus-like particles delivering cs protein peptide sequences resulted in efficient priming of protective cd8 + t-cell responses following booster vaccination with recombinant mva expressing the cs antigen . more recently another heterologous prime-boost vaccination schedule has been thoroughly evaluated using chimpanzee-adenovirus vectors for priming and recombinant mva for booster vaccinations (for review, see sebastian and gilbert, 2016) . this combination of vector immunization induced high frequencies of me-trap-specific t-cell responses in humans as a promising approach to robustly protect against p. falciparum (ewer et al., 2013) . moreover, a recent study suggested the feasibility of developing malaria transmissionblocking vector vaccines to target p. falciparum within the mosquito. antibodies with high-level transmission-blocking activity could be elicited upon prime-boost immunizations of mice with recombinant chimpanzee adenovirus and mva-expressing candidate malaria antigens pfs230-c and pfs25 (kapulu et al., 2015) . thus, future approaches in the development of mva vector vaccines against malaria aim at the induction of a more balanced immunity based on both efficient humoral and cellular immune responses. yet, complex clinical phase ii/iii studies in humans in endemic areas will be needed to evaluate the efficacy of these new promising approaches. newly emerging pathogens represent another global public health risk as they can suddenly and unexpectedly arise from a previously unknown ecological niche, in most cases an animal reservoir. thus, major concerns are zoonotic infections that are transmitted from animals to humans, which, when sufficiently adapted to the human host, may rapidly spread in the human population (kuiken et al., 2011; reusken et al., 2016; steffen et al., 2012) . during the past 20 years, public health systems had been confronted with a multitude of new pathogens each demonstrating a different scenario of emergence. in this context, the year 1999 with the sudden occurrence of west nile fever in the western hemisphere (new york city, usa) is often marked as the beginning of a new era of epidemics (for review, see suthar et al., 2013) . wnv is a member of the genus flaviviruses and, as an arbovirus, continuously circulates between different mosquito species and birds as the major animal virus reservoirs. through the bite of an infected mosquito, wnv can be transmitted to mammalian hosts, primarily to humans or horses, causing the so-called west nile fever sometimes resulting in neuroinvasive disease with the potential for severe outcomes especially in elderly and immunocompromised humans. the virus had first been isolated in 1937 from a febrile woman in the west nile district in uganda (goldblum et al., 1954) . since then it was observed to be or become endemic in regions of europe, africa, and asia periodically causing wnv outbreaks in humans and/or horses. in 1999, the virus was introduced into the new york city district of queens, supposedly by an infected mosquito or bird (lanciotti et al., 1999; murray et al., 2010; nash et al., 2001) . thus, wnv had relocated to a new, naïve population facilitating its impressive spread across the north american continent leading to a total of about 41,762 human infections and 1765 deaths between 1999 and 2014 (http://www.cdc.gov/westnile/statsmaps/). concurrently, the virus also increasingly emerges in european countries, resulting in outbreaks of severe disease in horses and humans. thus, a safe and effective wnv vaccine for humans is urgently needed in particular to protect at-risk populations. a recent study tested different recombinant mva vaccines delivering the wnv envelope protein (wnv-e) and fulfilling all the principal requirements to proceed to clinical testing in humans. vaccine immunogenicity, induction of neutralizing antibodies and e-specific cd8 + t-cell responses, and the capacity to protect against lethal challenge infections were evaluated in different mouse models. the mva-wnv candidate vaccines allowed to compare the performance of wnv-e antigens expressed in four different conformations. in mva-prm/me, wnv-e is produced together with the wnv membrane protein to induce the synthesis and the release of virus like particles (vlps) upon mva infection. mva-e sol produces a soluble version of wnv-e that is secreted from infected cells. mva-e tmv and mva-e tmc prominently expose wnv-e antigens on the cell surface by providing heterologous transmembrane domains derived from either vacv (tmv, transmembrane domain vaccinia virus) or chikungunya virus (tmc, transmembrane domain chikungunya virus). upon prime-boost vaccinations in balb/c mice, all four mva-wnv candidate vaccines elicited circulating serum antibodies binding to recombinant wnv-e protein and neutralizing wnv in tissue culture infections in addition, immunizations in hla-a2.1-/hla-dr1-transgenic h-2 class i-/class ii-knockout mice efficiently induced wnv-e-specific cd8+ t-cell responses. finally, the mva-wnv candidate vaccines protected c57bl/6 mice against challenge infections with lineage 1 and lineage 2 wnv and activated crossneutralizing antibodies. thus, further studies are warranted to evaluate these recombinant mva-wnv vaccines in other preclinical models in an effort to select and develop an mva-wnv candidate vaccine for clinical testing in humans . recently, the family coronaviridae provided different new pathogens suddenly arising from an ecological niche. since 2003 two novel beta coronaviruses have been introduced into human populations causing acute atypical necrotizing pneumonia, the so-called severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) . in early 2003, the sars epidemic initially occurred in southern china and rapidly spread to more than 37 countries causing 8096 infections and 774 deaths. bat species and civet cats were identified as animal reservoirs transmitting the virus to humans and/or other animals. the abrupt emergence of the sars-cov in china in november 2002 and its worldwide distribution until the end of 2003 is representative for the scenario of a pandemic public health emergency. luckily, further spread of the disease could be prevented and no further infections with sars-cov were detected since early 2004. the successful containment of sars was likely due to multiple causes, e.g., the relative ease of clinical case isolation and the persistence of the pathogen in an animal reservoir rarely enabling transmission to humans. however, the lessons learned from sars include the need for the timely development of specific vaccines to counteract such an outbreak scenario with high morbidity and mortality rates and the lack of any specific treatment option. the most effective approach to deal with emerging pathogens is vaccination. mva with its excellent safety profile and well-established vector production platform holds great potential to rapidly develop new vaccines against such emerging pathogens ready to use in an immediate public health response. in an ideal scenario, mva emergency vaccines against selected pathogens of risk are being developed and undergo preclinical and phase i/ii clinical evaluations already in preepidemic times. in the case of a disease outbreak, these recombinant mva candidate vaccines could be immediately tested in efficacy studies when used to vaccinate people of special risk in endemic areas. this concept already spurred the development of a first set of recombinant mva candidate vaccines against highly pathogenic avian influenza viruses and the recently emerging mers-cov as current examples of new zoonotic agents. mers-cov was first described in september 2012. in contrast to experiences with sars in the epidemic of 2002/2003, the mers-cov continues to cause disease in humans in the fifth year after its first appearance in 2012. at present, who reports a total of 1782 laboratory confirmed cases including 634 deaths (http://www.who.int/emergencies/mers-cov/en/). the epidemiology of human mers-cov infections centers in the middle east, e.g., in qatar, saudi arabia, jordan, and united arab emirates. sporadically, mers-cov infections are also spread to other countries in europe, north-america, and asia due to transmission by travelers infected in the middle east. raj and coworkers identified the human cell surface amino peptidase dipeptidyl 4 or cd26 as functional receptor of mers-cov-mediating entry into the cell (raj et al., 2013) . by now dromedary camels are known and generally accepted to be the critical animal reservoir responsible for spreading the virus to humans memish et al., 2014; meyer et al., 2014; raj et al., 2014) . these primary zoonotic infections can result in interfamilial or health care related secondary transmissions. the elderly and immunocompromised persons are among the people of risk to suffer most from severe and lethal mers-cov infections. other individuals at risk are health care workers and people with close contact to camels. all these groups are considered relevant target populations for potential mers-cov vaccines. so far, there is no licensed vaccine against mers-cov or sars-cov available. different approaches have been undertaken to develop effective means to prevent or cure these new infectious diseases. here, mva has been tested as viral vector vaccine against both beta coronaviruses. the envelope spike (s) protein has been proven to be a major target of sars-cov-neutralizing antibodies (he et al., 2004; sui et al., 2004) . indeed, a recombinant mva producing the sars-cov s antigen was evaluated in different animal models and demonstrated the induction of sars-cov-specific immune responses, including sars-cov-neutralizing antibodies and s-antigen-specific t cells in mice, rabbits, and rhesus macaques (bisht et al., 2004; chen et al., 2005) . prime boost immunization effectively inhibited sars-cov replication in cynomolgous monkeys after respiratory sars-cov challenge infection suggesting the induction of protective immunity. in the case of the mers-cov, a recombinant mva expressing the s protein of mers-cov (mva-mers-s) was generated rapidly after the discovery of mers in 2012 (song et al., 2013) . here, the mers-cov-s-encoding sequences were introduced into the deletion site iii of the mva genome. preclinical evaluation of mva-mers-s candidate vaccine confirmed the induction of mers-covneutralizing antibodies and mers-s epitope-specific cd8+ t cells in balb/c mice comparing different dosages and application routes (volz et al., 2015) . moreover, preclinical testing in a special mouse model allowed for the first demonstration of the protective capacity of this mers candidate vaccine. after adenovirus-mediated transduction with the human dipeptidylpeptidase 4 receptor, balb/c mice are susceptible for respiratory mers-cov challenge infections and the monitoring of virus loads allows to determine the efficacy of experimental immunization (zhao et al., 2014) . as the virus is assumed to persist in dromedary camels, further preclinical analysis in dromedary camels demonstrated immunogenicity and protective efficacy of mva-mers-s . simultaneous immunizations by the intranasal and intramuscular routes resulted in efficient induction of virus-neutralizing antibodies in serum and nasal secretions of vaccinated animals. contrary to the mock vaccinated control animals, camels vaccinated with mva-mers-s revealed a significant reduction of excreted infectious virus and viral rna transcripts after mers-cov challenge infection. in addition, vaccination with mva-mers-s also induced the activation of orthopoxvirus-specific antibodies that readily cross-neutralized camelpox virus. camelpox virus causes severe systemic disease in camels with case fatality rates as high as 28%. clinical disease is characterized by papules and pustules that initially appear at the primary site of infection. this is then followed by the development of generalized rash and fever between day 9 and 11 postinfection. young camels are more susceptible to severe clinical disease. the activation of camelpox virusneutralizing antibodies suggested the potential dual use of this candidate mers-cov vaccine in dromedaries to efficiently protect against mers-cov and camelpox virus infection . these data further support the general safety and efficacy of the mva-mers-s candidate vaccine and introduce the possibility for application as veterinary vaccine with important implications to the one health concept. vaccination of dromedary camels in areas endemic for mers-cov could reduce the burden of virus excretions from the animal reservoir and thus inhibit the transmission of mers-cov to human populations. in addition, it is important to characterize the mva-mers-s candidate vaccine in humans and efforts are ongoing to start a first-in-man clinical evaluation of mva-mers-s as soon as possible. in this first clinical phase i/ii testing, safety and immunogenicity of the mva-mers-s candidate vaccine will be analyzed in healthy volunteers in germany. results from these phase i/ii clinical evaluations are prerequisite for testing the vaccine in larger phase ii studies in endemic countries. in addition, the availability of an investigational drug batch of mva-mers-s might allow for application of as emergency vaccine in the case of a suddenly occurring outbreak scenario, when the virus is rapidly transmitted and spread throughout a new geographic area. exemplary for this was the introduction of mers-cov to south korea on 20 may 2015 (min et al., 2016) . a 68-year-old man returning from the middle east had been diagnosed with mers 9 days after he initially visited the hospital for medical help. in the hospital further spread of mers-cov occurred by transmission to several health care workers and other patients. in this outbreak, a total of 186 individuals have been infected, with a total of 36 deaths. in this context, the government of the republic of korea began to implement intense case and contact management activities that in the end stopped the epidemic of mers-cov in a nonendemic region. however, the sudden and rapid spread of mers-cov in south korea again highlighted the global risks of newly emerging pathogens that might unexpectedly threaten a naïve population ( jeong-sun et al., 2015; kim et al., 2015) . another very recent example for the sudden reemergence of a highly infectious pathogen is the unprecedented ebola virus epidemic in west africa starting in 2013 and continuing for over 2 years. during this devastating and most widespread ebola virus outbreak, the virus had caused more than 28,000 disease cases and 11,325 deaths mainly geographically linked to the africa but some cases were also diagnosed in travelers from africa to the united states, germany, france, spain, and great britain (de la vega et al., 2015; quaglio et al., 2016) . initially, this large epidemic began in the village of meliandou, gu eck edou prefecture, guinea in the end of 2013. most likely bats transmitted the virus to humans followed by massive spread of the disease to other villages. moreover, the virus causing this outbreak was identified to be the most notorious member of the genus ebolavirus and the species zaire ebolavirus (ebov) (baize et al., 2014) . in consequence, the epidemic in west africa was associated with high morbidity and mortality rates that enabled an undamped transmission and perpetuation of the virus in the population for a rather long time period (gire et al., 2014) . this had not been observed for ebolavirus outbreaks in the past. so far, there are no efficient therapeutics available (choi and croyle, 2013) and there are also no vaccines licensed to protect against ebolavirus. however, since this very recent epidemic, research activities to develop and evaluate candidate vaccines against ebolavirus have been intensified. most promising candidates already advanced to clinical evaluation in humans, and include a recombinant vesicular stomatitis virus (vsv) delivering the ebolavirus zaire glycoprotein (vsv-ebov) and a chimpanzee-adenovirus (chad3)-zaire ebola virus (zebov) also expressing the ebolavirus zaire glycoprotein as target antigen. with regard to the chad3-zaire, the strategy is to use a heterologous prime-boost immunization schedule, boosting with an appropriate recombinant mvaexpressing ebolavirus zaire glycoprotein (ewer et al., 2016; tapia et al., 2016) . this strategy was supported by data from the preclinical evaluation of a chad3-ebov candidate vaccine in nonhuman primates. here, primeboost vaccination with the chad3-ebov vaccine alone did not confer protective immunity over a period of several months. however, robust protection against lethal challenge with ebov could be obtained following booster vaccination with a recombinant mva-ebov candidate vaccine coexpressing the ebolavirus zaire and sudan glycoproteins under the control of the p7.5 vaccinia virus early promoter (stanley et al., 2014) . the approach of using a chad3-ebov prime and mva-ebov boost vaccination has been further developed by the engineering of a multivalent recombinant mva coproducing the zebov and sudan ebola virus glycoproteins and other filovirus antigens (mva-bn-filo). a first phase ib clinical study demonstrated safety, tolerability, and immunogenicity of the chad3-ebov prime and mva-bn-filo booster immunizations in adults in the united states and in mali. without any observation of severe side effects the combined chad3-ebov and mva-bn-filo vaccinations proved to be highly immunogenic as it was shown by the activation of ebov-specific antibodies and cd4+ and cd8+ t-cell responses (tapia et al., 2016) . ewer and colleagues investigated another heterologous prime-boost vaccination schedule in 60 healthy adult volunteers in oxford, united kingdom, using a chad3 vector vaccine and a monovalent recombinant mva encoding the surface glycoprotein of zebov. again, chad3 immunization boosted with mva-elicited b-cell and t-cell immune responses to zebov that were clearly superior to those induced by the chad3 vector vaccine alone (ewer et al., 2016) . nevertheless, a heterologous vaccination strategy with two different viral vector vaccines is a complex regimen to be applied in large field studies or in the case of emergency vaccination when whole populations have to be protected rapidly. thus, it should be interesting to evaluate the efficacy of a prime-boost vaccination scheme based on mva-ebov only. in summary, the highly versatile and safe mva vector platform should be particularly useful to effectively control newly emerging or reemerging infectious diseases. the vector system may be readily exploited in a plugand-play generic approach for the rapid generation of vaccine candidates suitable for rapid emergency immunization and, simultaneously, the clinical-stage development of a new licensable product. eradication of human smallpox has been achieved by prophylactic use of vacv to immunize humans, the only host reservoir of the causative agent varv. however, other members of the genus orthopoxvirus can cause zoonotic infections (kroon et al., 2011; mccollum and damon, 2014; vora et al., 2015) . moreover, recent fears that monkeypox virus (mpxv) or varv could be used as biological weapons have renewed the interest in safe vaccines against varv or other zoonotic orthopoxviruses (moss, 2011) . mva holds great promise for worldwide use as third-generation smallpox vaccine due to its well-established characteristics concerning safety and immunogenicity jones et al., 2016; knitlova et al., 2014; meseda et al., 2016; tree et al., 2016; wyatt et al., 2004) . in 2013, an mva vaccine has been licensed in europe for active immunization against smallpox in adults and for use in situations where it is considered necessary to protect against smallpox in accordance with official recommendations (european medicines agency, 2013). data from multiple phase ii clinical studies have confirmed the safety and immunogenicity of the mva vaccine in patient populations considered at risk for conventional smallpox vaccination including individuals with atopic dermatitis or hiv infection (greenberg et al., 2015; overton et al., 2015; von sonnenburg et al., 2014) . in addition, mva has been successfully tested for cardiac safety in a large phase ii clinical trial (zitzmann-roth et al., 2015) . this is important because vaccines based on replication-competent vacv strains are associated with a high incidence of myo-/pericarditis, a severe cardiac complication. additional recent data from clinical studies addressed the meta-analysis of mva-induced immune responses in various patient populations and the comparative evaluation of inoculation routes or improved formulations of the mva vaccine troy et al., 2015) . today, with the eradication of smallpox, we lack an established human disease representing the pathogenesis of a systemic orthopoxvirus infection in humans. therefore, analyzing the protective efficacy of orthopoxvirus-specific vaccines is not straightforward. preclinical evaluations in animal models are required to test vaccine-mediated protection in relation to antigen-specific responses, and animal models must mimic the target poxvirus diseases in humans as closely as possible. varv infection in cynomolgus macaques can result in a lethal disease with similarities to smallpox wahl-jensen et al., 2011) . however, varv is a biosafety level 4 agent and the handling with this virus is highly restricted. thus, several other animal models using the orthopoxviruses cowpox virus (cpxv), mpxv, ectromelia virus (ectv), and vacv have been developed (chapman et al., 2010; esteban and buller, 2005) . mva, as safe smallpox vaccine, has been tested in these different infection models. mva vaccination by the intramuscular or subcutaneous route protected mice against severe respiratory challenge infection with cpxv or vacv-wr (coulibaly et al., 2005; drexler et al., 2003) . of note, mva could also protect mice lacking several components of the immune system, resembling high-risk groups in the population that would require an alternative to the standard smallpox vaccine . in nonhuman primates, the challenge infection with mpxv is the most appropriate model to evaluate the preclinical efficacy of new candidate smallpox vaccines. here, a standard dosage of 10 8 pfu mva robustly protected cynomolgous monkeys against intravenous or intratracheal mpxv challenges stittelaar et al., 2005) . in this context edghill-smith and coworkers demonstrated the essential need of antibodies for protection against fatal monkeypox disease (edghill-smith et al., 2005) . one major limitation in these orthopoxvirus infection models, using vacv, mpxv, or cpxv to test protective capacity of vaccination, is the high amounts of infectious virus (>10 6 pfu) required to induce lethal disease. in contrast, very few infectious particles of varv could generate fatal smallpox disease in naive humans. to overcome this shortcoming, the ectv infection of mice can serve as an additional highly appropriate model for the reassessment of the correlates of protective immunity (for review sigal, 2016) . ectv, the causative agent of mousepox, is a natural mouse pathogen causing a classical systemic poxvirus disease (esteban and buller, 2005) . very low amounts of ectv are sufficient for infection and induction of lethal mousepox disease in susceptible mouse strains. after an asymptomatic incubation period of 6-8 days following intranasal infection, mousepox disease starts in the respiratory tract followed by a systemic virus spread to internal organs such as liver and spleen (paran et al., 2009; parker et al., 2009) . specific signs of illness are characterized by severe bronchopneumonia and hepatitis. mice that survive this systemic phase of severe disease develop a characteristic pustular rash on the skin very reminiscent to that seen in human smallpox. the ectvmouse model offers the opportunity to study an orthopoxvirus pathogen in an experimentally easily accessible natural host. ectv is highly adapted to the mouse immune system and this allows to thoroughly analyze the mechanisms of viral immune evasion and vaccine-induced immune protection. several studies evaluated mva vaccine-mediated protection against lethal mousepox disease in more detail. coulibaly and coworkers demonstrated the usefulness of respiratory ectv challenge infections as improved model system for efficacy testing. here, intramuscular immunization with a single dose of 10 6 pfu mva prevented severe disease and death in mice challenged 3 weeks after vaccination. a single dose of 10 7 robustly protected against any signs of illness and disease after the respiratory mousepox challenge. however, after vaccination with 10 6 or 10 7 pfu vacv wyeth, all mice suffered from severe lethal disease (coulibaly et al., 2005) . in the cpxv challenge model, these different candidate vaccines were equally protective underlining the impact of choosing a specific challenge virus. this study demonstrates the need for data from various preclinical models as key component in developing next-generation orthopoxvirus-specific vaccines for application in humans. in the case of an emergency with newly arising highly pathogenic orthopoxviruses, e.g., because of unintentional or intentional release of varv, vaccination protocols for rapid induction of protective immunity are urgently needed (reardon, 2014; sasse and gelderblom, 2015) . for smallpox vaccination there are historical reports that vacv application in a time window of up to 4 days after exposure with varv may be protective. analysis of mva as emergency vaccine confirmed short-term protective capacity of vaccination in mice, latest when applied at the day of the lethal respiratory challenge with vacv strain wr. postexposure prophylaxis could not be achieved in the vacv-wr model, independent of dosage and application route (staib et al., 2006) . however, when testing emergency vaccination in the ectv-mouse model, a standard dosage of 10 8 pfu mva robustly protected against lethal respiratory mousepox infection up to 2 days before the challenge. even more, in this natural virus-host system, 10 8 pfu mva also prevented death and severe disease when applied up to 4 days after the lethal challenge. however, postexposure vaccination could not inhibit the onset of sign of disease including respiratory symptoms and body weight loss (paran et al., 2009) . in a follow-up study, kremer and colleagues analyzed the immune components mediating this rapid protection in more detail (kremer et al., 2012a) . analysis of mva-induced protection in mice with defined deficiency in the innate or adaptive immunity identified cd4 + t cells to be essentially required to allow for mva-induced cd8 + t-cell expansion. interestingly, selected components of the innate immune system and b cellmediated responses were fully dispensable for prevention of fatal disease by immunization given 2 days before challenge. analyzing protective capacity of mva immunization in ragà/à mice that lack t cells and b cells, these mice could not be protected against the lethal ectv challenge infection. these results underlined the prerequisite of adaptive cellular immunity for mediating a rapid protection with perforin-mediated cytotoxicity proven to be a key immune mechanism. in the case of emergency, when rapid induction of protective immunity by vaccination is desirable for prevention of morbidity and mortality, the instant activation of protective virus-specific immunity by single-shot vaccination, would be ideal. moreover, the possibilities of dose-sparing immunization regimens would increase the numbers of people that can be vaccinated. volz and coworkers assessed the minimal requirements for the induction of protective immunity against lethal ectv challenge infection 2 days after immunization with mva . c57bl/6 mice had been intramuscularly vaccinated with tenfold increasing doses of mva starting with 10 3 up to 10 8 pfu, the mva standard dosage. interestingly, a minimal amount of 10 5 pfu fully protected the mice against the lethal respiratory challenge infection with ectv. moreover, inoculations with 10 4 pfu mva were still sufficient to prevent death of all vaccinated animals but could not protect against the induction of mousepox disease. analysis of immune responses again identified cd8+ t cells as the key components mediating the rapid protection in the low dose immunization model. moreover, mva immunization at low dosage also protected ifnarà/à mice, indicating efficient activation of cellular immunity even in the absence of type i ifn signaling. when monitoring for virus-specific cd8 + t-cell responses in mice vaccinated with the minimal protective dose of mva, we found significantly enhanced levels of antigen-specific t cells in animals that were mva vaccinated and ectv challenged compared to mice that were only vaccinated. the initial priming of naïve cd8 + t cells by mva immunization appears to be highly efficient and, even at low doses, mediates a rapid in vivo burst of pathogen-specific cd8+ t cells upon challenge. these findings define striking requirements for protective emergency immunization against severe systemic infections with orthopoxviruses. these data are of important practical relevance for public health, as producing sufficient amounts of vaccine is expected to be a major challenge should an outbreak occur. moreover, prevention of other infections may require similar immune mechanisms to elicit rapid protective immunity; hence, mva could be an extremely useful vaccine for delivering heterologous t cell antigens, particularly for infectious diseases that fit a scenario of emergency vaccination. thus, studies evaluating recombinant mva candidate vaccines as emergency vaccines might be promising for the development of new prophylactic or therapeutic approaches. today, almost 40 years after its first licensing in germany, mva is well established as safety-tested, immunogenic, and efficacious thirdgeneration smallpox vaccine. in 2013, the european medicines agency and canada health granted the marketing authorization of an mva vaccine to immunize against varv infection, in the absence of human smallpox or naturally occurring varv (european medicines agency, 2013). thus, efficacy data needed to be generated from animal models of orthopoxvirus infections considered to be representative for human smallpox. a similar licensing process by the us food and drug administration is ongoing and appears to be at an advanced stage. despite the eradication of varv more than three decades ago, these efforts are still important mostly due to the threat of varv being-accidently or intentionally-released into unprotected human populations. moreover, the use of a licensed mva vaccine should be ideal to protect individuals at risk-including laboratory workers-against other zoonotic orthopoxviruses such as cpxv and mpxv that continue to cycle in rodent reservoirs and can cause disease in humans. in addition, during 25 years, mva has been continuously improved as an extremely safe and efficient viral vector system for the synthesis of high levels of foreign proteins in nonpermissive human cells. at the moment, recombinant mva viruses expressing various heterologous antigens are among the most promising vector candidates to develop innovative vaccination strategies to protect against complex infections such as aids, tuberculosis, or malaria, or against rare but threatening emerging diseases. desirable common characteristics of mva as viral vector vaccine include the genetic stability, the well-established production procedures and the general safety for the environment. results coming from clinical testing of various recombinant mva vaccines further emphasize its excellent safety record and its immunogenicity as vaccine in humans. in the context of recent clinical findings, it is noteworthy that repeated vaccinations with the same recombinant mva resulted in substantial booster induction of antigen-specific antibodies even in the presence of high levels of mva-specific immunity. these results together with other promising data gained from the combined application of mva with various other viral vector platforms greatly enhance the general acceptance of viral vectors as next-generation candidate vaccines. in addition to its promising characteristics concerning immunogenicity, the nonreplicating recombinant mva vaccines are also well positioned to satisfy very stringent requirements for a broad safety in various settings. mva vaccines appear highly suitable for use in immunocompromised individuals and in the elderly representing important target populations in ever-ageing populations in the industrialized world. at the same time, recombinant mva should be also safe to immunize persons with severe comorbidities, e.g., hiv infection, tuberculosis, or malaria, as it is often seen in developing nations. new vaccines that rapidly protect against threatening emerging pathogens are urgently needed. this it is further highlighted by the who-list of the "top most wanted" emerging diseases likely to cause major epidemics. here, the longstanding experience with the mva vector vaccine platform in preclinical and clinical research should lead to important contributions to the development of protective vaccination strategies against newly emerging pathogens. finally, the exciting results from ongoing fundamental research on the biology of mva spur the possibility to even improve the efficacy of future mva vaccines. here, an exemplary area of research targets the still unknown functions of host-regulatory genes remaining conserved in the mva genome. indeed, modulating the functional activity of these regulatory mva proteins could be beneficial in enhancing the immunogenicity of mva vaccines and activate innate and/or adaptive immune responses to heterologous antigens. a soluble receptor for interleukin-1β encoded by vaccinia virus: a novel mechanism of virus modulation of the host response to infection soluble interferon-γ receptors encoded by poxviruses blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus modified vaccinia virus ankara (mva) as production platform for vaccines against influenza and other viral respiratory diseases enhanced cd8 + t cell immune responses and protection elicited against plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus characterization of the vaccinia mva hemagglutinin gene locus and its evaluation as an insertion site for foreign genes the complete genomic sequence of the modified vaccinia ankara strain: comparison with other orthopoxviruses viral host-range factor c7 or k1 is essential for modified vaccinia virus ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase r-mediated phosphorylation of eukaryotic translation initiation factor 2α structure and function of a41, a vaccinia virus chemokine binding protein emergence of zaire ebola virus disease in guinea mutational analysis of the core, spacer, and initiator regions of vaccinia virus intermediate-class promoters novel vaccine vectors for hiv-1 a phase 2b randomised trial of the candidate malaria vaccines fp9 me-trap and mva me-trap among children in kenya oral immunization with a replication-deficient recombinant vaccinia virus protects mice against influenza potent cd8 (+) t-cell immunogenicity in humans of a novel heterosubtypic influenza a vaccine, mvaànp+m1 severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice modified vaccinia virus ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine oral vaccination of the fox against rabies using a live recombinant vaccinia virus selection of recombinant vaccinia viruses on the basis of plaque formation identification and characterization of two members of a novel class of the interleukin-1 receptor (il-ir) family: delineation of a new class of il-1r-related proteins based on signaling normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line a survey of host range genes in poxvirus genomes immunization with recombinant modified vaccinia ankara (rmva) constructs encoding the ha or np gene protects ponies from equine influenza virus challenge. vaccine 24, 1180. bundesamt f€ ur vebraucherschutz und lebensmittelsicherheit orthopoxvirus il-18 binding proteins: affinities and antagonist activities e. coli beta-glucuronidase (gus) as a marker for recombinant vaccinia viruses host range and cytopathogenicity of the highly attenuated mva strain of vaccinia virus: propagation and generation of recombinant viruses in a nonhuman mammalian cell line vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques strong, but age-dependent, protection elicited by a deoxyribonucleic acid/ modified vaccinia ankara simian immunodeficiency virus vaccine the e3l gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded rna-dependent protein kinase animal models of orthopoxvirus infection recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region emerging targets and novel approaches to ebola virus prophylaxis and treatment deletion of gene a41l enhances vaccinia virus immunogenicity and vaccine efficacy recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus ankara (mva) the nonreplicating smallpox candidate vaccines defective vaccinia lister (dvv-l) and modified vaccinia ankara (mva) elicit robust long-term protection modified vaccinia virus ankara triggers type i ifn production in murine conventional dendritic cells via a cgas/ sting-mediated cytosolic dna-sensing pathway the vaccinia virus k3l gene product potentiates translation by inhibiting double-stranded-rna-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor 2 structure of vaccinia virus late promoters a pandemic warning? ebolavirus evolution: past and present induction of influenza (h5n8) antibodies by modified vaccinia virus ankara h5n1 vaccine innate immune sensing of modified vaccinia virus ankara (mva) is mediated by tlr2-tlr6, mda-5 and the nalp3 inflammasome new vaccinia virus promoter as a potential candidate for future vaccines modification of promoter spacer length in vaccinia virus as a strategy to control the antigen expression redundancy complicates the definition of essential genes for vaccinia virus cloning the vaccinia virus genome as a bacterial artificial chromosome in escherichia coli and recovery of infectious virus in mammalian cells engineering of a vaccinia virus bacterial artificial chromosome in escherichia coli by bacteriophage [lambda]-based recombination the rational design of an aids vaccine highly attenuated modified vaccinia virus ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells identification of vaccinia virus epitope-specific hla-a*0201-restricted t cells and comparative analysis of smallpox vaccines host range restriction of vaccinia virus in chinese hamster ovary cells: relationship to shutoff of protein synthesis host range deletion mutant of vaccinia virus defective in human cells comparison of vaccine strategies using recombinant env-gag-pol mva with or without an oligomeric env protein boost in the shiv rhesus macaque model smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus guideline on quality, non-clinical and clinical aspects of live recombinant viral vectored vaccines vaccinia virus recombinants expressing rabiesvirus glycoprotein protect against rabies ectromelia virus: the causative agent of mousepox imvanex: modifiziertes vacciniavirus ankara lebend protective cd8(+) t-cell immunity to human malaria induced by chimpanzee adenovirus-mva immunisation hiv-1 vaccines improving the mva vaccine potential by deleting the viral gene coding for the il-18 binding protein escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors vaccinia virus protein n2 is a nuclear irf3 inhibitor that promotes virulence recombinant mva expressing secreted glycoprotein d of bohv-1 induces systemic and mucosal immunity in animal models modified vaccinia virus ankara protein f1l is a novel bh3-domain-binding protein and acts together with the early viral protein e3l to block virus-associated apoptosis h5n1 virus: transmission studies resume for avian flu comparison of lyophilized versus liquid modified vaccinia ankara (mva) formulations and subcutaneous versus intradermal routes of administration in healthy vaccinia-naïve subjects expanding the repertoire of modified vaccinia ankara-based vaccine vectors via genetic complementation strategies a candidate hiv/aids vaccine (mva-b) lacking vaccinia virus gene c6l enhances memory hiv-1-specific t-cell responses improving adaptive and memory immune responses of an hiv/aids vaccine candidate mva-b by deletion of vaccinia virus genes (c6l and k7r) blocking interferon signaling pathways deletion of the vaccinia virus n2l gene encoding an inhibitor of irf3 improves the immunogenicity of modified vaccinia virus ankara expressing hiv-1 antigens designing cell lines for viral vaccine production: where do we stand? a protein particle vaccine containing multiple malaria epitopes enhanced cd8 t cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunisation regimes localization and sequence of a vaccinia virus gene required for multiplication in human cells specificity and 6-month durability of immune responses induced by dna and recombinant modified vaccinia ankara vaccines expressing hiv-1 virus-like particles systems analysis of mva-c induced immune response reveals its significance as a vaccine candidate against hiv/aids of clade c enhanced immunogenicity and protective efficacy against mycobacterium tuberculosis of bacille calmette-gu erin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus ankara the t1/35 kda family of poxvirus-secreted proteins bind chemokines and modulate leukocyte influx into virusinfected tissues a decade after sars: strategies for controlling emerging coronaviruses a multicenter, open-label, controlled phase ii study to evaluate safety and immunogenicity of mva smallpox vaccine (imvamune) in 18-40 year old subjects with diagnosed atopic dermatitis middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels poxviruses and the evolution of host range and virulence immunogenicities of intravenous and intramuscular administrations of modified vaccinia virus ankara-based multi-ctl epitope vaccine for human immunodeficiency virus type 1 in mice airborne transmission of influenza a/h5n1 virus between ferrets comparative experimental works on cow pox virus vaccines a pandemic influenza h1n1 live vaccine based on modified vaccinia ankara is highly immunogenic and protects mice in active and passive immunizations vectors based on modified vaccinia ankara expressing influenza h5n1 hemagglutinin induce substantial cross-clade protective immunity characterization of wild-type and mutant vaccinia virus m2l proteins' abilities to localize to the endoplasmic reticulum and to inhibit nf-kappab activation during infection patterns of viral replication correlate with outcome in simian immunodeficiency virus (siv)-infected macaques: effect of prior immunization with a trivalent siv vaccine in modified vaccinia virus ankara the march toward malaria vaccines replication of modified vaccinia virus ankara in primary chicken embryo fibroblasts requires expression of the interferon resistance gene e3l reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses a toll-like receptor-independent antiviral response induced by double-stranded b-form dna dna/mva vaccines for hiv codelivery of envelope protein in alum with mva vaccine induces cxcr3-biased cxcr5 + and cxcr5à cd4 t cell responses in rhesus macaques exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox middle east respiratory syndrome in 3 persons potent functional antibody responses elicited by hiv-i dna priming and boosting with heterologous hiv-1 recombinant mva in healthy tanzanian adults mva vaccination provides long-term protection against nasal rabbitpox virus challenge a chemically defined production process for highly attenuated poxviruses broad protection against avian influenza virus by using a modified vaccinia ankara virus expressing a mosaic hemagglutinin gene comparative assessment of transmission-blocking vaccine candidates against plasmodium falciparum expression of rabies virus glycoprotein from a recombinant vaccinia virus middle east respiratory syndrome infection control and prevention guideline for healthcare facilities development of eczema vaccinatum in atopic mouse models and efficacy of mva vaccination against lethal poxviral infection recombinant modified vaccinia virus ankarabased vaccine induces protective immunity in mice against infection with influenza virus h5n1 mva-based h5n1 vaccine affords cross-clade protection in mice against influenza a/h5n1 viruses at low doses and after single immunization recombinant modified vaccinia virus ankara expressing the hemagglutinin gene confers protection against homologous and heterologous h5n1 influenza virus infections in macaques evaluation of a modified vaccinia virus ankara (mva)-based candidate pandemic influenza a/h1n1 vaccine in the ferret model ankara-based influenza a h5n1 vaccine: a randomised, double-blind phase 1/2a clinical trial critical role of perforin-dependent cd8+ t cell immunity for rapid protective vaccination in a murine model for human smallpox easy and efficient protocols for working with recombinant vaccinia virus mva zoonotic brazilian vaccinia virus: from field to therapy pigs, poultry, and pandemic influenza: how zoonotic pathogens threaten human health cd40l-adjuvanted dna/modified vaccinia virus ankara simian immunodeficiency virus (siv) vaccine enhances protection against neutralization-resistant mucosal siv infection immunity against heterosubtypic influenza virus induced by adenovirus and mva expressing nucleoprotein and matrix protein-1 modified vaccinia virus ankara triggers chemotaxis of monocytes and early respiratory immigration of leukocytes by induction of ccl2 expression preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+m1, in humans samd9 is an innate antiviral host factor with stress response properties that can be antagonized by poxviruses new avian suspension cell lines provide production of influenza virus and mva in serum-free media: studies on growth, metabolism and virus propagation factors associated with poor outcomes among adults hospitalized for influenza in france: a three-year prospective multicenter study role of viral factor e3l in modified vaccinia virus ankara infection of human hela cells: regulation of the virus life cycle and identification of differentially expressed host genes doublestranded rna-binding protein e3 controls translation of viral intermediate rna, marking an essential step in the life cycle of modified vaccinia virus ankara vaccinia virus: a selectable eukaryotic cloning and expression vector general method for production and selection of infectious vaccinia virus recombinants expressing foreign genes experiences with immunization against orthopox viruses of humans and animals using vaccine strain mva ver€ anderung von vaccinevirus durch dauerpassagen in h€ uhnerembryofibroblastenkulturen experimental research on the s-antigen in the viruses of animal smallpox abstammung, eigenschaften und verwendung des attenuierten vaccinia-stammes mva poxvirus tropism a review of preclinical animal models utilised for tb vaccine evaluation in the context of recent human efficacy data protective immunity against mycobacterium tuberculosis induced by dendritic cells pulsed with both cd8+-and cd4+-t-cell epitopes from antigen 85a recombinant modified vaccinia virus ankara expressing antigen 85a boosts bcg-primed and naturally acquired antimycobacterial immunity in humans human infection with mers coronavirus after exposure to infected camels f-box-like domains are present in most poxvirus ankyrin repeat proteins percutaneous vaccination as an effective method of delivery of mva and mva-vectored vaccines mapping of deletions in the genome of the highly attenuated vaccinia virus mva and their influence on virulence antibodies against mers coronavirus in dromedary camels comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity safety and immunogenicity of dna/modified vaccinia virus ankara malaria vaccination in african adults malaria vaccine developments the ankyrin repeat as molecular architecture for protein recognition improved adjuvanting of seasonal influenza vaccines: preclinical studies of mva-np+m1 coadministration with inactivated influenza vaccine priming with a simplified intradermal hiv-1 dna vaccine regimen followed by boosting with recombinant hiv-1 mva vaccine is safe and immunogenic: a phase iia randomized clinical trial west nile virus and its emergence in the united states of america molecular genetics of vaccinia virus: demonstration of marker rescue the outbreak of west nile virus infection in the new york city area in 1999 equine vaccine for west nile virus hiv-dna given with or without intradermal electroporation is safe and highly immunogenic in healthy swedish hiv-1 dna/mva vaccinees: a phase i randomized trial cowpox virus and other members of the orthopoxvirus genus interfere with the regulation of nf-kb activation the influence of delivery vectors on hiv vaccine efficacy novel conserved-region t-cell mosaic vaccine with high global hiv-1 coverage is recognized by protective responses in untreated infection safety and immunogenicity of modified vaccinia ankara-bavarian nordic smallpox vaccine in vaccinia-naive and experienced human immunodeficiency virus-infected individuals: an open-label construction of poxviruses as cloning vectors: insertion of the thymidine kinase gene from herpes simplex virus into the dna of infectious vaccinia virus postexposure immunization with modified vaccinia virus ankara or conventional lister vaccine provides solid protection in a murine model of human smallpox mousepox in the c57bl/6 strain provides an improved model for evaluating anti-poxvirus therapies vaccinia virus host range genes interaction of f1l with the bh3 domain of bak is responsible for inhibiting vaccinia-induced apoptosis ebola: lessons learned and future challenges for europe isolation of mers coronavirus from a dromedary camel cross host transmission in the emergence of mers coronavirus selection of recombinant mva by rescue of the essential d4r gene cultivation of vaccine virus for jennerian prophylaxis in man further observations on the cultivation of vaccine virus for jennerian prophylaxis in man vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing cs of plasmodium immunological and virological mechanisms of vaccine-mediated protection against siv and hiv the potential for respiratory droplet-transmissible a/h5n1 influenza virus to evolve in a mammalian host isolation of vaccinia mva recombinants using the viral f13l gene as the selective marker lessons learnt from the german smallpox outbreaks after world war ii enhanced immunogenicity for cd8+ t cell induction and complete protective efficacy of malaria dna vaccination by boosting with modified vaccinia virus ankara recombinant modified vaccinia virus ankara-based malaria vaccines a phase i, open-label trial, evaluating the safety and immunogenicity of candidate tuberculosis vaccines aeras-402 and mva85a, administered by primeboost regime in bcg-vaccinated healthy adults the vaccinia virus k1l gene product inhibits host nf-kappab activation by preventing ikappabalpha degradation chapter six-the pathogenesis and immunobiology of mousepox identification of restriction factors by human genome-wide rna interference screening of viral host range mutants exemplified by discovery of samd9 and wdr6 as inhibitors of the vaccinia virus k1l(à)c7l(à) mutant ectromelia, vaccinia and cowpox viruses encode secreted interleukin-18-binding proteins irf3 and irf7 phosphorylation in virus-infected cells does not require double-stranded rna-dependent protein kinase r or ikappa b kinase but is blocked by vaccinia virus e3l protein vaccinia virus immune evasion: mechanisms, virulence and immunogenicity middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies the highly conserved orthopoxvirus 68k ankyrin-like protein is part of a cellular scf ubiquitin ligase complex the orthopoxvirus 68-kilodalton ankyrin-like protein is essential for dna replication and complete gene expression of modified vaccinia virus ankara in nonpermissive human and murine cells vaccinia and cowpox viruses encode a novel secreted interleukin-1-binding protein transient host range selection for genetic engineering of modified vaccinia virus ankara construction and isolation of recombinant mva vaccinia virus and poxvirology inactivation of the viral interleukin 1β receptor improves cd8 + t-cell memory responses elicited upon immunization with modified vaccinia virus ankara short-term, but not postexposure, protection against lethal orthopoxvirus challenge after immunization with modified vaccinia virus ankara chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebola virus challenge henipavirus mediated membrane fusion, virus entry and targeted therapeutics intracutaneous smallpox vaccination with a weak pathogenic vaccinia virus mva-stufenimpfung gegen pocken modified vaccinia virus ankara protects macaques against respiratory challenge with monkeypox virus potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association west nile virus infection and immunity nonreplicating vaccinia vector efficiently expresses recombinant genes a recombinant vector derived from the host range-restricted and highly attenuated mva strain of vaccinia virus stimulates protective immunity in mice to influenza virus non-replicating vaccinia vector efficiently expresses bacteriophage t7 rna polymerase the vaccinia virus c12l protein inhibits mouse il-18 and promotes virus virulence in the murine intranasal model safety and efficacy of mva85a, a new tuberculosis vaccine use of chad3-ebo-z ebola virus vaccine in malian and us adults, and boosting of malian adults with mva-bn-filo: a phase 1, single-blind, randomised trial, a phase 1b, openlabel and double-blind, dose-escalation trial near death experiences: poxvirus regulation of apoptotic death repeated high-dose (5 â 108tcid50) toxicity study, of a third generation smallpox vaccine (imvamune), in new zealand white rabbits sex difference in immune response to vaccination: a participant-level meta-analysis of randomized trials of imvamune ® smallpox vaccine comparative analysis of siv-specific cellular immune responses induced by different vaccine platforms in rhesus macaques protective efficacy of several vaccines against highly pathogenic h5n1 avian influenza virus under experimental conditions rapid expansion of cd8+ t cells in wild-type and type i interferon receptor-deficient mice correlates with protection after low-dose emergency immunization with modified vaccinia virus ankara protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein immunogenicity and protective efficacy of recombinant modified vaccinia virus ankara candidate vaccines delivering west nile virus envelope antigens human infection with a zoonotic orthopoxvirus in the country of georgia differential immunogenicity of various heterologous prime-boost vaccine regimens using dan and viral vectors in healthy volunteers progression of pathogenic events in cynomolgus macaques infected with variola virus modified vaccinia virus ankara induces toll-like receptor-independent type i interferon responses vaccinia virus-mediated inhibition of type i interferon responses is a multifactorial process involving the soluble type i interferon receptor b18 and intracellular components vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis the vaccinia virus f1l protein interacts with the proapoptotic protein bak and inhibits bak activation enhanced t cell-mediated protection against malaria in human challenges by using the recombinant poxviruses fp9 and modified vaccinia virus ankara a novel naturally occurring tandem promoter in modified vaccinia virus ankara drives very early gene expression and potent immune responses chapter 3 poxvirus host range genes boosting with poxviruses enhances mycobacterium bovis bcg efficacy against tuberculosis in guinea pigs an assay to compare the infectivity of mycobacterium tuberculosis isolates based on aerosol infection of guinea pigs and assessment of bacteriology tandem repeats within the inverted terminal repetition of vaccinia virus dna expression of the vaccinia virus genome: analysis and mapping of mrnas encoded within the inverted terminal repetition development of a replicationdeficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge elucidating and minimizing the loss by recombinant vaccinia virus of human immunodeficiency virus gene expression resulting from spontaneous mutations and positive selection blockade of interferon induction and action by the e3l double-stranded rna binding proteins of vaccinia virus human infection with a novel avian influenza a(h5n6) virus efficiently editing the vaccinia virus genome by using the crispr-cas9 system rapid generation of a mouse model for middle east respiratory syndrome interleukin-1β receptor expressed by modified vaccinia virus ankara interferes with interleukin-1β activity produced in various virus-infected antigen-presenting cells key: cord-274506-fzcuu4ma authors: jo, seri; kim, hyojin; kim, suwon; shin, dong hae; kim, mi‐sun title: characteristics of flavonoids as potent mers‐cov 3c‐like protease inhibitors date: 2019-09-12 journal: chem biol drug des doi: 10.1111/cbdd.13604 sha: doc_id: 274506 cord_uid: fzcuu4ma middle east respiratory syndrome‐coronavirus (mers‐cov) is a zoonotic virus transmitted between animals and human beings. it causes mers with high mortality rate. however, no vaccine or specific treatment is currently available. since antiviral activity of some flavonoids is known, we applied a flavonoid library to probe inhibitory compounds against mers‐cov 3c‐like protease (3clpro). herbacetin, isobavachalcone, quercetin 3‐β‐d‐glucoside and helichrysetin were found to block the enzymatic activity of mers‐cov 3clpro. the binding of the four flavonoids was also confirmed independently using a tryptophan‐based fluorescence method. the systematic comparison of the binding affinity of flavonoids made it possible to infer their scaffolds and functional groups required to bind with mers‐cov 3clpro. an induced‐fit docking analysis revealed that s1 and s2 sites play a role in interaction with flavonoids. the experimental and computational study showed that flavonol and chalcone are favourite scaffolds to bind with the catalytic site of mers‐cov 3clpro. it was also deduced that some flavonoid derivatives with hydrophobic or carbohydrate attached to their core structures have a good inhibitory effect. therefore, we suggest that flavonoids with these characteristics can be used as templates to develop potent mers‐cov 3clpro inhibitors. coronaviruses (covs) are a positive sense, single-stranded rna virus coated with viral particles (fehr & perlman, 2015) . together with arteriviridae and roniviridae, covs belong to the coronaviridae family in the order nidovirales. these covs can infect a wide variety of hosts, including avian, swine and humans. human coronaviruses (hcovs) represent a major group of covs associated with various respiratory diseases from common cold to serious pneumonia and bronchitis (mesel-lemoine et al., 2012) . today, hcovs are recognized as one of the most rapidly evolving viruses originated from their characteristic high genomic nucleotide substitution rates and recombination. in human, their infection is known to cause approximately one-third of common cold. severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) covs are highly pathogenic and caused a fatal first major outbreak in 2003 and 2012 , respectively (de wit, doremalen, falzarano, & munster, 2016 . sars-and mers-covs genomes contain two open reading frames orf1a and orf1b translated to two respective viral polyproteins pp1a and pp1ab by host ribosomes. orf1a encodes two cysteine proteases, a papain-like protease (plpro) and a 3c-like protease (3clpro). while plpro cuts the first three cleavage sites of its polyprotein, 3clpro is responsible for cleavage of the remaining eleven locations resulting in release of a total of 16 non-structural proteins (nsp) in both sars-and mers-covs. the homodimeric form of 3clpro is active in the presence of substrates. the crystal structure of 3clpro showed that each monomer is composed of three structural domains: domains i and ii form a chymotrypsinlike architecture with catalytic cysteine and are connected to a third c-terminal domain via a long loop (neddle, lountos, & waugh, 2015) . in the proteolytic site, all 3clpros prefer glutamine at p1 position and leucine, basic residues, small hydrophobic residues at p2, p3 and p4 positions, respectively (chuck, chow, wan, & wong, 2011) . at p1′ and p2′ positions, small residues are required. since the autocleavage process is essential for viral propagation, 3clpro is a good drug target for anti-coronaviral infection. in this study, we used a proteolytic method to probe mers-cov 3clpro inhibitory compounds with a synthetic peptide labelled with the edans-dabcyl fret (fluorescence resonance energy transfer) pair (liu et al., 2005) . since emission wavelengths of edans are widely overlapped with absorbance wavelengths of dabcyl, the energy emitted from edans will be quenched by dabcyl in a close proximity (10-100 å). therefore, an increment of fluorescence can be a sign to judge whether a substrate is cleaved or not in this design. hence from the fluorescence intensity change, the proteolytic activity of protease could be detected. with a synthetic peptide with the fret pair, a flavonoid library was screened to search mers-cov 3clpro inhibitory compounds. recent studies showed that some flavonoids have antiviral activity in some viruses (frabasile et al., 2017; jucá et al.,2018; kiat et al., 2006; yang, lin, zhou, liu, & zhu, 2017; zakaryan, arabyan, oo, & zandi, 2017) . therefore, we assayed flavonoids and tried to induce their structural property crucial to bind with mers-cov 3clpro. the coding sequence of mers-cov nsp5, a 3c-like protease (ncbi ref. seq. nc_019843.3) was synthesized chemically by bioneer and cloned into a bacteriophage t7-based expression vector. the plasmid dna was transformed into e. coli bl21 (de3) for protein expression. e. coli bl21 (de3) cells were grown on luria-bertani (lb) agar plates containing 150 μg/ml ampicillin. several colonies were picked and grown in capped test-tubes with 10 ml lb broth containing 150 μg/ml ampicillin. a cell stock composed of 0.85 ml culture and 0.15 ml glycerol was prepared and frozen at 193 k for use in a large culture. the frozen cell stock was grown in 5 ml lb medium and diluted into 2,000 ml fresh lb medium. the culture was incubated at 310 k with shaking until an od 600 of 0.6-0.8 was reached. at this point, expression of mers-cov 3clpro was induced using isopropyl-β-d-1-thiogalactopyranoside (iptg) at a final concentration of 1 mm. the culture was further grown at 310 k for 3 hr in a shaking incubator. cells were harvested by centrifugation at 7,650 g (6,500 rev min −1 ) for 10 min in a high-speed refrigerated centrifuge at 277 k. the cultured cell paste was resuspended in 25 ml of a buffer consisting of 50 mm tris-hcl ph 8.0, 100 mm nacl, 10 mm imidazole, 1 mm phenylmethylsulfonyl fluoride (pmsf) and 10 μg/ml dnase i. the cell suspension was disrupted using an ultrasonic cell disruptor (digital sonifier 450; branson). cell debris was pelleted by centrifugation at 24,900 g (15,000 rev min −1 ) for 30 min in a highspeed refrigerated ultra-centrifuge at 277 k. the protein was purified by affinity chromatography using a 5 ml hi-trap q column (ge healthcare) followed by a 5 ml hi-trap blue column (ge healthcare). the custom-synthesized fluorogenic substrate, dabcyl-ktsavlqsgfrkme-edans (anygen), was used as a substrate for the proteolytic assay using mers-cov 3clpro (kuo, chi, hsu, & liang, 2004) . this substrate contains the nsp4/nsp5 cleavage sequence, gvlq↓sg (wua et al., 2015) , and works as a generic peptide substrate for many coronavirus including mers-cov 3clpro. the peptide was dissolved in distilled water and incubated with each protease. a spectramax i3x multi-mode microplate reader (molecular devices) was used to measure spectral-based fluorescence. the proteolytic activity was determined at 310 k by following the increase in fluorescence (λ excitation = 340 nm, λ emission = 490 nm, bandwidths = 9, 15 nm, respectively) of edans upon peptide hydrolysis as a function of time. assays were conducted in black, 96-well plates (nunc) in 350 μl assay buffers containing protease and substrate as follows: for the mers-cov 3clpro assay, 1.84 μl of 0.19 mm protease containing 20 mm tris ph 8.0 was incubated with 8.75 μl of 0.1 mm substrate at 310 k for 2 hr before measuring relative fluorescence unit (rfu). before the assay, the emission spectra of 40 flavonoids were surveyed after illuminating at 340 nm to avoid the overlapping with the emission spectrum of edans. every compound was suitable to be tested. the final concentration of the protease, peptide and chemical used at the assay was 1, 2.5 and 20 μm each. at first, mers-cov 3clpro and chemical were mixed and preincubated at room temperature for 1 hr. the reaction was initiated by the addition of the substrate, and each well was incubated at 310 k for 2 hr. after 2 hr, we measured the fluorescence of the mixture on the black 96-well plate using the end-point mode of spectramax i3x where the excitation wavelength was fixed to 340 nm and the emission wavelength was set to 490 nm using 9, 15 nm bandwidth, respectively. all reactions were carried out in triplicate. among the first forty flavonoids (table s1 ), four of them were picked up to further assay at a concentration range of 2 μm-320 μm. ic50 value which is the value causing 50% inhibition of catalytic activity of mers-cov 3clpro was calculated by non-linear regression analysis using graphpad prism 7.03 (graphpad software). the proteolytic assay using mers-cov 3clpro in the presence of triton x-100 has been performed to differentiate artificial inhibitory activity of chemicals through non-specific binding with proteases by forming aggregate or complexation. the concentration used in this study was 0.01%. to confirm the feasibility of the assay method independently, the fluorescence spectra from tryptophans of mers-cov 3clpro with candidate inhibitors were investigated (lin, lan, guan, sheng, & zhang, 2009 ). the fluorescence measurements were recorded with a spectramax i3x multi-mode microplate reader (molecular devices) at excitation and emission wavelengths of 295 nm and 300-500 nm, respectively. the optimal excitation and emission wavelengths were determined by softmax pro. five tryptophans of mers-cov 3clpro showed a strong fluorescence emission with a peak at 340 nm at the excitation wavelength of 295 nm. in contrast, the flavonoids were almost non-fluorescent under the same experiment condition. each 40 μm chemical was incubated with 1 μm mers-cov 3clpro for 1 hr, and the fluorescence intensity of mers-cov 3clpro was measured. all the docking and scoring calculations were performed using the schrödinger suite of software (maestro, version 11.5.011). the compounds were extracted from the pubchem database in sdf format and were combined in one file. the file was then imported into maestro and prepared for docking using ligprep. the atomic co-ordinates of the crystal structure of mers-cov 3clpro (5wkj) were retrieved from the protein data bank and prepared by removing all solvent and adding hydrogens and minimal minimization in the presence of bound ligand using protein preparation wizard. ionizer was used to generate ionized state of all compounds at target ph 7.0 ± 2.0. this prepared low-energy conformers of the ligand were taken as the input for docking analysis. grids for molecular docking were built using receptor grid generation. compounds were docked using ligand docking mode with postdocking minimization including 5 poses per ligand. the docked poses were then refined using an xp (extra precision) option with the threshold value for rejecting minimized pose of 0.5 kcal/mol. the energies were calculated using the opls3 force field. the induced-fit docking protocol (sherman, day, jacobson, friesner, & farid, 2006) was run from the graphical user interface accessible within maestro 11.5.011. receptor sampling and refinement were performed on residues within 5.0 å of each ligand for each of the 20 ligand-protein complexes. with prime (jacobson et al., 2004) , a side-chain sampling and prediction module, the side-chains, as well as the backbone of the target protein, were energy minimized. a total of 20 induced-fit receptor conformations were generated for each of the eight test ligands. re-docking the test ligands into their respective 20 structures within 30.0 kcal/mol of their lowest energy structure. finally, the ligand poses were scored using a combination of prime and glidescore scoring functions . the amount of cell harvested for purification of mers-cov 3clpro was 3.01 g per 2,000 ml of e. coli culture. the protein was purified by ion chromatography using a 5 ml hi-trap q column (ge healthcare). the column was equilibrated with a buffer consisting of 20 mm bis-tris ph 7.0, and the pooled fractions were loaded. the column was eluted using a linear nacl gradient to 1.0 m nacl, and the protein was eluted at 0.16 m nacl. the pooled fractions were loaded on a 5 ml hi-trap blue column (ge healthcare) equilibrated with a buffer consisting of 10 mm sodium phosphate ph 7.0. the target protein was detected in unbound fractions. sds-page showed one band around 33(33,737.77da) kda, corresponding to the molecular weight of mers-cov 3clpro. the protein was concentrated to 31.14 mg/ml for protease assays in a buffer consisting of 10 mm sodium phosphate ph 7.0. the purified mers-cov 3clpro was different from the previous method (kumar et al., 2016) where the native form was obtained after removing of a his 6 -tag wih factor-xa. in our experiment, the enzyme activity was 10-fold decreased when the his 6 -tag protein was used (data not shown). that is due to the location of the his 6 -tag connected to the n-terminus of mers-cov 3clpro. the published crystal structure showed that the active site pocket might be easily hindered by the flexible n-terminal his 6 -tag. a flavonoid library consisting of ten different scaffolds was also built (figure 1 ). it contains three isoflavones, one isoflavane, six flavones, nine flavonols, four flavanols, five flavanones, one flavanonol, one prenylflavonoid, eight chalcones and two unclassified flavonoids (table s1 ). we applied the library to assay mers-cov 3clpro. since flavonoids are known to aggregate through a complexation and thus non-specifically inhibit various proteases, the assay in the presence of triton x-100 was performed. before assay, we tested the influence of triton x-100 on the catalytic activity of mers-cov 3clpro. as shown in the figure s1 , only a slight increment of the catalytic activity was observed even up to 0.1% triton x-100. therefore, we performed the experiment at the concentration of 0.01% triton x-100 where no significant interference was detected. using forty flavonoids, an inhibitory effect of each compound at 20 μm was tested. among them, herbacetin (3,4′,5,7,8-pentahydroxyflavone), isobavachalcone (2′,4,4′-trihydroxy-3′-(3-methyl-2-butenyl)chalcone), quercetin 3-β-d-glucoside (3,3′,4′,5,7-pentahydroxyflavone 3-β-d-glucoside) and helichrysetin (4,2′,4′-trihydroxy-6′methoxychalcone) were found to have prominent inhibitory activity (figure 2 ). the four compounds showed the severely reduced fluorescent intensity and thus represented their mers-cov 3clpro inhibitory activity. the experimental data were plotted as log inhibitor concentration versus per cent fluorescence inhibition (figure 2) . ic50 values obtained from the dose-dependent inhibitory curves of herbacetin, isobavachalcone, quercetin 3-β-d-glucoside and helichrysetin are 40.59, 35.85, 37.03 and 67.04 μm, respectively. in order to confirm the inhibitory activity of the flavonoids independently, a general tryptophan-based assay method was employed. tryptophan was well-known to emit its own fluorescence. therefore, if tryptophan is positioned adequately in proteins, the change in fluorescence intensity can reflect the binding state of chemicals and be used to judge interaction between proteins and chemicals. mers-cov 3clpro contains five tryptophan residues and thus displays a high fluorescence peak at 340 nm at the tryptophan excitation wavelength of 295 nm. we monitored the change in the fluorescence intensities of mers-cov 3clpro depending on the presence or absence of four chemicals. since each compound in the flavonoid library was almost non-fluorescent under the experiment condition, a change in fluorescence intensity reflects interactions between mers-cov 3clpro and chemicals. only the four inhibitory compounds obviously reduced the fluorescence intensity of mers-cov 3clpro (figure 3) . the decreased emission intensity confirmed the complex formation between mers-cov 3clpro and inhibitory compounds. in order to deduce the binding modes of the inhibitory flavonoids, an in-depth theoretical investigation through an induced-fit docking study was carried out. the interactions between mers-cov 3clpro and the inhibitory flavonoids were analysed to predict their binding affinities. top ranked structures (according to the glide scores) from the induced-fit docking results for herbacetin (−10.246), isobavachalcone (−9.364), quercetin 3-β-d-glucoside (−9.751) and helichrysetin (−9.953) were selected and hypothesized to be biological complexes. in order to compare binding affinities to their closest homologues ( figure s2 figure s3 . it is very obvious that the s1 site and hydrophobic s2 site of mers-cov 3clpro play a key role in predicted complexes. flavonoids are natural compounds with multiple pharmacological characteristics such as antioxidant, anti-inflammatory, analgesic, anti-carcinogenic, antibacterial infection, antifungal and antiviral properties (frabasile et al., 2017) . naringenin has therapeutic effects on various neurological, cardiovascular, gastrointestinal, rheumatological, metabolic and malignant disorders (rani et al., 2016) . it also represents antiviral function (zakaryan et al., 2017) . fisetin is a commercially available nutraceutical with anti-inflammatory, antioxidant, anti-tumorigenic, anti-invasive, anti-angiogenic, anti-diabetic, neuroprotective and cardioprotective effects (pal, pearlman, & afaq, 2016) . interestingly, fisetin has an anti-noroviral activity by inducing cytokines (seo & choi, 2017) . although some of flavonoids show an antiviral effect, a molecular mechanism of their antiviral activity was rarely known. in this study, we assayed the inhibitory activity of various flavonoids against mers-cov 3clpro. for the trial, a flavonoid library composed of nine different scaffolds plus one undefined group was constructed. they are classified based on a c6-c3-c6 skeleton and differ in the overall hydroxylation patterns and in the saturation of the heteroatomic ring c together with the position of the attached aromatic ring b (at the positions c-2 or c-3 of ring c) (jucá et al., 2018) . the flavonoid library was tested, and thus, a systematic analysis was possible. among the ten groups, isoflavones, isoflavane, flavanols and flavanones did not show any inhibitory activity over mers-cov 3clpro. the other groups contains some flavonoids displaying moderate inhibitory activity. however, four flavonoids, herbacetin, isobavachalcone, quercetin 3-β-d-glucoside and helichrysetin exhibited prominent inhibitory activity. the immediate inference of the primary scaffold required for binding with mers-cov 3clpro was as follows: first, the orientation of the aromatic ring b at the position c-2 of ring c is essential as shown in isoflavones and isoflavane. second, the saturated heteroatomic ring c is not preferred as shown in flavanols and flavanones. third, chalcone and flavonol scaffolds show a promising binding property. the more detailed structural comparison also provided valuable structural information required for the binding affinity of each flavonoid. helichrysetin is a chalcone derivative (van puyvelde et al., 1989) . therefore, we compared its inhibitory activity with a chalcone, cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) which is the structurally identical homologue except one hydroxyl group at the 4-position of the benzyl moiety of chalcone. the inhibitory activity of cardamonin at the concentration of 40 μm was lower than that of helichrysetin ( figure 4 ). it implies that the 4-hydroxyl group of helichrysetin is functionally important to bind with mers-cov 3clpro. the structural comparison of cardamonin with isobavachalcone also indicated the modification effect of acetophenone ring of chalcone: the hydrophobic modification at the 3′-position of the acetophenone ring moiety of isobavachalcone improves its binding affinity to mers-cov 3clpro. intriguingly, the docking study shows that the 4-hydroxyl group of helichrysetin forms a hydrogen bond with the hydroxyl group of tyr54 of mers-cov 3clpro. since tyr54 is located deep inside of the hydrophobic s2 site, helichrysetin is inserted into deeper than cardamonin ( figure s3a ). as a result, helichrysetin seems to have a better affinity and thus represents a better inhibitory activity. in the flavonoid library, there are quite similar homologues of herbacetin, isobavachalcone and quercetin 3-β-d-glucoside. they are kaempferol (3,4′,5,7-tetrahydroxyflavone), 2,2′,4′-trihydroxychalcone and quercitrin (3,3′,4′,5,7-pentahydroxyflavone-3-l-rhamnoside), respectively ( figure s2 ). their mers-cov 3clpro inhibitory activity is lower than their corresponding compounds at 40 μm. herbacetin is a kaempferol derivative where one more hydroxide is attached at 8-position of kaempferol. the docking study indicates kaempferol derivatives occupy the s1 and s2 sites of mers-cov 3clpro ( figure s3b ). the hydroxyl group at 7-position looks important to bind at the s1 binding site. a f i g u r e 4 comparison of inhibitory activity between homologue flavonoids. the two homologue flavonoids are compared side by side. each of two bars represents the effect of two homologue inhibitory compounds at 40 μm against mers-cov 3clpro compared to the control. each bar is expressed as the mean ± standard error of the mean (n = 3). rfu = relative fluorescence units bit better inhibitory activity of herbacetin indicates the hydroxide at 8-position is favourable for its binding affinity to mers-cov 3clpro. in the predicted complex, the hydroxyl group at 8-position of herbacetin induces a hydrogen bond with his41 at the s1 site. the improved activity of isobavachalcone compared with 2,2′,4′-trihydroxychalcone again points out the importance of its hydrophobic substituent. coincided with the experimental result, the prenyl moiety of isobavachalcone makes hydrophobic interactions with met25 and leu49 at the hydrophobic s2 site ( figure s3c) . quercetin 3-β-d-glucoside is a homologue of quercitrin where rhamnoside is substituted for glucoside. the better inhibitory activity of quercetin 3-β-d-glucoside means that hydroxymethyl is preferred to hydroxide in this position of the glucoside to interact with mers-cov 3clpro. the docking study represents that the hydroxymethyl group of the glucoside moiety makes a hydrogen bond with glu169 and thus contributes its tighter binding to the s1 site than the rhamnoside moiety of quercitrin ( figure s3d ). helichrysetin and isobavachalcone inhibiting the activity of mers-cov 3clpro are chalcone derivatives. chalcone has immunomodulation, antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, anticancer and anti-diabetic activities (yadav, prasad, sung, & aggarwal, 2011) . recently, the non-competitive inhibition of cardamonin against the dengue virus protease has been reported (kiat et al., 2006) . as discussed above, cardamonin is a strong homologue of helichrysetin. the analysis of the crystal structures of three viral proteases, mers-cov 3clpro, dengue virus ns2b/ns3 protease and norovirus 3clpro were performed, and their active sites were compared ( figure s4 ) (erbel et al., 2006; nakamura et al., 2005) . despite their sequential divergence, their overall architecture resembles each other. furthermore, the spatial positions of their catalytic residues were also highly conserved. the observation implies that chalcone and flavonol can be used as templates to develop a potential antiviral agent by targeting these viral proteases. in this study, we showed that the antiviral effects of some flavonoids may be directly from their inhibition of main viral proteases and thus nullify a process of virus peptides. therefore, one of antiviral mechanisms of flavonoids may be originated from their direct binding capability to viral proteases. consequently, flavonoid derivatives can be used as a template to design not only for broad-spectrum but also for virus-specific antiviral agents. a further study is going on to develop better inhibitory lead compounds derived from this study. we built a flavonoid library to systematically search mers-cov 3clpro inhibitory compounds with a fret method. we found four flavonoids effectively reducing the proteolytic activity of mers-cov 3clpro. the binding of the flavonoids was independently proven by a tryptophanbased fluorescence method. the assay in the presence of triton x-100 also eliminated the chance of the false-positive result from the aggregation of flavonoids. the analysis of the four compounds with their homologs using an induced-fit docking study provided an insight of flavonoid scaffolds required to bind with mers-cov 3clpro. the prominent activity of helichrysetin and isobavachalcone indicates that the flexibility of the chalcone scaffold is good to bind with mers-cov 3clpro. in contrast, the comparable activity of flavones with a rigid γ-pyrone ring indicates that their spatial rearrangement together with various functional groups may be an alternative strategy to interact with mers-cov 3clpro. therefore, this study suggests that an antiviral effect of some flavonoids is directly from their structural characteristics binding to viral proteases. profiling of substrate specificities of 3c-like proteases from group 1, 2a, 2b, and 3 coronaviruses sars and mers: recent insights into emerging coronaviruses structural basis for the activation of flaviviral ns3 proteases from dengue and west nile virus coronaviruses: an overview of their replication and pathogenesis the citrus flavanone naringenin impairs dengue virus replication in human cells extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes a hierarchical approach to allatom protein loop prediction flavonoids: biological activities and therapeutic potential inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, boesenbergia rotunda (l.), towards dengue-2 virus ns3 protease identification, synthesis and evaluation of sars-cov and mers-cov 3c-like protease inhibitors characterization of sars main protease and inhibitor assay using a fluorogenic substrate spectroscopic investigation of interaction between mangiferin and bovine serum albumin screening of drugs by fret analysis identifies inhibitors of sars-cov 3cl protease a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes a norovirus protease structure provides insights into active and substrate binding site integrity structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity fisetin and its role in chronic diseases pharmacological properties and therapeutic potential of naringenin: a citrus flavonoid of pharmaceutical promise inhibitory mechanism of five natural flavonoids against murine norovirus novel procedure for modeling ligand/receptor induced fit effects isolation of flavonoids and a chalcone from helichrysum odoratissimum and synthesis of helichrysetin prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus the role of chalcones in suppression of nf-κb-mediated inflammation and cancer activity of compounds from taxillus sutchuenensis as inhibitors of hcv ns3 serine protease flavonoids: promising natural compounds against viral infections the authors declare no conflict of interest. the data that support the findings of this study are available from the corresponding author upon reasonable request. https://orcid.org/0000-0002-2205-1453mi-sun kim https://orcid.org/0000-0002-4092-4203 key: cord-275216-dnt88ycw authors: zhang, xue-yan; huang, hao-jie; zhuang, dong-lin; nasser, moussa ide; yang, ming-hua; zhu, ping; zhao, ming-yi title: biological, clinical and epidemiological features of covid-19, sars and mers and autodock simulation of ace2 date: 2020-07-20 journal: infect dis poverty doi: 10.1186/s40249-020-00691-6 sha: doc_id: 275216 cord_uid: dnt88ycw background: the outbreak of coronavirus disease 2019 (covid-19) has caused a public catastrophe and global concern. the main symptoms of covid-19 are fever, cough, myalgia, fatigue and lower respiratory tract infection signs. almost all populations are susceptible to the virus, and the basic reproduction number (r(0)) is 2.8–3.9. the fight against covid-19 should have two aspects: one is the treatment of infected patients, and the other is the mobilization of the society to avoid the spread of the virus. the treatment of patients includes supportive treatment, antiviral treatment, and oxygen therapy. for patients with severe acute respiratory distress syndrome (ards), extracorporeal membrane oxygenation (ecmo) and circulatory support are recommended. plasma therapy and traditional chinese medicine have also achieved good outcomes. this review is intended to summarize the research on this new coronavirus, to analyze the similarities and differences between covid-19 and previous outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) and to provide guidance regarding new methods of prevention, diagnosis and clinical treatment based on autodock simulations. methods: this review compares the multifaceted characteristics of the three coronaviruses including covid-19, sars and mers. our researchers take the covid-19, sars, and mers as key words and search literatures in the pubmed database. we compare them horizontally and vertically which respectively means concluding the individual characteristics of each coronavirus and comparing the similarities and differences between the three coronaviruses. results: we searched for studies on each outbreak and their solutions and found that the main biological differences among sars-cov-2, sars-cov and mers-cov are in orf1a and the sequence of gene spike coding protein-s. we also found that the types and severity of clinical symptoms vary, which means that the diagnosis and nursing measures also require differentiation. in addition to the common route of transmission including airborne transmission, these three viruses have their own unique routes of transmission such as fecal-oral route of transmission covid-19. conclusions: in evolutionary history, these three coronaviruses have some similar biological features as well as some different mutational characteristics. their receptors and routes of transmission are not all the same, which makes them different in clinical features and treatments. we discovered through the autodock simulations that met124 plays a key role in the efficiency of drugs targeting ace2, such as remdesivir, chloroquine, ciclesonide and niclosamide, and may be a potential target in covid-19. the outbreak of coronavirus disease 2019 (covid19) caused by a novel coronavirus has become a public health emergency of international concern (pheic). the virus can cause severe respiratory disease and can spread from person to person. the virus has been named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) based on its appearance under electron microscopy [1] . covid-19 has been designated a public health emergency of international concern (phiec) [2] . the chinese center for disease control and prevention (china cdc) determined that sars-cov-2 infection is the cause of the outbreak that started in in wuhan city (cdc, 2020) [3] . the virus can cause severe respiratory illness, and human to human transmission has been confirmed [4] . isolated viruses were observed under electron microscopy and named sars-cov-2 [5] . as of march 23, 2020, 187 countries and regions reported 348 667 covid-19 confirmed cases worldwide. although the covid-19 situation has been suppressed recently in china, we still need to strengthen the prevention and control of the epidemic, improve people's awareness of protective measures, and minimize the loss caused by the virus to prevent it from becoming a severe global pandemic. this is the third time in human history, following sars and mers, that a coronavirus has caused a widespread epidemic. given that the epidemic is still spreading and the evidence that there are similarities among the three coronaviruses in terms of their biological, clinical and epidemiological features, a comparison among the three is very helpful to guide the improvement of treatment and prevention measures, and the similarities and differences among the three are likely to provide the key to addressing the covid-19 epidemic. coronaviruses are large, enveloped, positive-strand rna viruses that can be divided into four genera: α, β, γ and δ. there are six coronaviruses that cause disease in humans, including 229e and nl63 of the α genus and oc43, hku1, sars-cov and mers-cov of the β genus [1, [6] [7] [8] . human coronavirus (hcov) infection is mainly related to the respiratory, intestinal and nervous systems. in 2002-2003, sars-cov caused an epidemic of severe acute respiratory diseases in china; mers-cov was found in the middle east in 2012 [9, 10] . both of these coronaviruses are zoonotic pathogens that can cause severe respiratory disease in humans, which can progress to severe acute respiratory distress syndrome. according to the experiment by peng et al. [5] , covid-19 uses ace2 as a cellular entry receptor; ace2 is distributed on the epithelial cells of the alveoli, trachea, bronchi, serous bronchial glands, alveolar monocytes and macrophages in the respiratory tract. ace2 is also widely expressed on the mucosa, such as the eyelid, nasal cavity, lip, and oral cavity. the pathogenic mechanism of covid-19 is clear, as shown in fig. 1 . the virus enters the target cells via ace2 and then releases single-strand rna (ssrna), which combines with the ribosome in the target cell and is translated to rna replicase. the rna replicas copy the ssrna to produce negative-strand rna, positive-strand rna and rna fragments, which combine with the ribosome to produce a protein shell. the protein shell and the positive-strand rna form the new sars-cov-2 virions, which are released to infect more target cells (fig. 1) . this review aims to summarize the biological, clinical and epidemiological characteristics of covid-19 and to elucidate the current clinical diagnosis and treatment. the comparison of the similarities and differences among the three viruses might be useful for further research on the treatment of covid-19. learning from past experience with fighting epidemics, it is very important to understand the epidemiological and clinical characteristics of the virus to control the epidemic, and this review summarizes the existing research and shows a targeted comparative analysis of the characteristics of the three related viruses to provide the scientific basis for later epidemic prevention and control measures. based on the studies published in the last few weeks, this review shows the characteristics of the spread, diagnosis and treatment of the disease. recently, many scientists have reported that the pathogenic mechanism of covid-19 is mainly related to ace2. the binding affinities of remdesivir, chloroquine, ciclenosid, niclosamide and lopinavirus to ace2 were determined and compared. we searched the published journal article and website information for covid-19 in the pubmed database and web of science database within one year, including laboratory studies, hospital case reports, and clinical case analysis. we also searched sars as a key word in the pubmed database and narrow the range according to the published journal article and governmental policy, speech and report, and read the retrieved literature. the latest version of treatment guidelines for covid-19, sars and mers were also searched in the pubmed database. the comparison of features among covid-19, sars-cov and mers-cov is summarized in table 1 . with high-throughput sequencing, researchers announced the sequencing of sars-cov-2. the genome of sars-cov-2 consists of 6 major orfs that are common to coronaviruses, and the sequence of sars-cov-2 has almost 70% similarity to that of sars-cov and nearly 40% similarity to that of mers-cov [5, 6, 11, 12] . the main differences among sars-cov-2, sars-cov and mers-cov are in orf1a and the sequence of gene spike coding protein-s [5] , which was identified as a key protein that interacts with target cells. in terms of electron microscopic morphology, sars-cov-2 virions are generally spherical, but some are polygonal. the diameter is between 60 and 140 nm. the virus particles have prominent spines that are approximately 9 to 12 nm, which cause the virus to have a coronal shape. according to the virus morphology observed under the microscope, the virus is consistent with other in the coronavirus family, including sars-cov and mers-cov [5, 13] . the receptor on the target cells is the factor determining how the virus enters the cell and which tissues are susceptible, and the spike protein initiates the merging of the viral envelope with the host cell cytomembrane. existing experimental studies have shown that ace2 is likely to be the cell receptor of sars-cov-2, and sars-cov-2 does not use other coronavirus receptors. the main receptors of sars-cov and mers-cov are ace2 and hdpp4 (human dipeptidyl peptidase 4 or cd26), respectively [1, 5, 14] . symptoms although the study of covid-19 is still in progress, our summary and comparison of coronaviruses [1, 4, [15] [16] [17] . however, those who are infected with sars-cov-2 rarely have significant upper respiratory signs and symptoms, including nosebleed, sneezing or sore throat, which indicates that the target cell may exist in the lower respiratory tract. this is consistent with the autopsy reports of patients with covid-19 that show that sars-cov-2 infection mainly causes deep airway inflammatory reactions and alveolar damage. some patients may also have headache, hemoptysis, diarrhea, dyspnea and lymphocytopenia, but patients are less likely to have gastrointestinal symptoms [4] . complications include acute respiratory distress syndrome, acute heart injury, and secondary infections. covid-19 patients can be divided into those with asymptomatic, mild and severe cases. for most patients, the incubation period of the virus is generally 7-14 days. typically, covid-19 gradually progresses and worsens. thus, each patient's condition becomes more serious in the second week. covid-19, sars, and mers have different mortality rates. among them, mers had the highest fatality rate, and covid-19 has the lowest fatality rate. it is worth noting that watery diarrhea is common in almost 60% of patients who suffer from sars, and there is a typical biphasic clinical course [10, 18, 19] . in mers, most patients have symptoms that include dry cough fever, malaise, myalgia, sore throat, headache, nausea, vomiting, and diarrhea, which are similar to the symptoms of sars, but mers has an unpredictable and erratic clinical course [19] [20] [21] [22] . fibrosis and consolidation in covid-19 are less serious than the lesions caused by sars, revealing that in covid-19, the chest lesions are not primarily serous inflammation but rather are exudative reactions. whether damage to the brain, myocardium, epicardium, kidneys, spleen and digestive organs is associated with viral infection needs further research. identification and diagnosis next-generation sequencing (ngs) and electron microscopy technology play critical roles in the early diagnosis of covid-19, but their diagnostic values have been weakened by the use of specific nucleic acid detection technology [11, 23] . at present, clinically confirmed patients are usually diagnosed by collecting throat swabs and then detecting the nucleic acid of sars-cov-2. diagnosis based on clinical manifestations can be an early and rapid screening method. patients with mild symptoms may not present positive signs. patients in severe condition may have shortness of breath, moist rales in lungs, weakened breath sounds, dullness on percussion, and changes in voice, and the physical examination can help identify these symptoms. in addition, ct imaging plays an important role in the diagnosis. the imaging features of lesions show characteristic (1) distribution (mainly critically ill patients died of severe pneumonia, septic shock, respiratory failure and multiple organ failure (mof). the authors reached a speculative conclusion that sars-cov-2 is more likely to infect older adult males with chronic comorbidities as a result of their weaker immune systems. in patients with severe coinfections, immune function is important in addition to the virulence of the pathogens. old age, obesity, and the presence of comorbidities might be associated with increased mortality. in addition, a substantial decrease in the total number of lymphocytes indicates that sars-cov-2 consumes many immune cells and inhibits the body's cellular immune function; therefore, a low absolute value of lymphocytes could be used as a reference index in the diagnosis of new sars-cov-2 infections in the clinic [29] . it is essential to analyze the infection source, transmission route, susceptible population and replication rate, especially the intermediate host and the exact route of transmission, to find the best measures to prevent the further spread of covid-19. transmission route viruses can directly infect people but can also infect one or more kinds of animals. although these animals themselves do not cause disease, they can act as vectors for the virus and transmit it to humans; during this process, some viruses may mutate and evolve new characteristics. according to the experimental results of peng et al. [5] , sars-cov-2 can be transmitted through respiratory droplets and direct contact, confirming that while the main transmission route of sars-cov-2 is aerosols, other routes of transmission may exist. moreover, a recent experiment conducted with recovering patients found that sars-cov-2 can also exist in the patient's stool, suggesting that the fecaloral route may be a route of transmisson [36] . li et al. investigated cases of sars and found that sars was spread mainly by respiratory droplets [19] . by analyzing case data, hui et al. also found that direct person-toperson transmission through close contact can also spread sars-cov [18] . mers-cov was mainly transmitted through close contact with infected family members or infected individuals in the hospital. xiao et al. identified seven hypothesized transmission modes based on the three main transmission routes (long-range airborne, close contact, and fomite), and the infection risks associated with each hypothesis were estimated using the multiagent modeling framework. this showed that transmission occurred via both the long-range airborne and close contact routes [22] . based on the available data, all three coronaviruses can be transmitted by breathing respiratory droplets that contain virions, which indicates that wearing masks is an effective means of protecting susceptible people. all three coronaviruses are transmitted from animals to humans and from humans to humans. susceptible population there is no evidence that people with certain characteristics are not susceptible to covid-19. the available data suggest that people of all ages who have close contact with patients can be infected by sars-cov-2 [36] [37] [38] . the general public is susceptible, and the data are still being updated daily. the elderly population and patients with basic diseases are more susceptible to severe illness after infection, and children and infants can also be infected by sars-cov-2 [39] . sars-cov had a tendency to affect healthier and younger persons, with a mean patient age of 39.9 years (range 1-91), and the male to female ratio was 1∶1.3, with a slight female predominance. mers-cov had a tendency to affect the elderly and frail populations, especially males, with a mean age of 56 years (range , and the male to female ratio was 3.3∶1 with a male predominance [8, 10, 40] . a commonly used measure of infectivity is the basic reproduction number (r 0 ), which is the average number of people infected who pass the virus on to others without intervention. in other words, the value is equivalent to how many people can be infected by an average patient. the larger the r 0 is, the harder it is to control the epidemic. researchers have estimated the r 0 to be in the range of 2.8-3.9, assuming extreme cases, which means that on average a covid-19 patient passes the virus on to 2.8-3.9 healthy persons [28, 41] . in comparison, the r 0 of mers has been reported to be less than 1, and the r 0 of sars is estimated to be 3. considering that the disease is now widespread around the world, the r 0 of covid-19 may change and could be higher than those of sars and mers. mers caused 800 deaths, with a mortality rate of approximately 35%. sars was characterized by superspreading events, while covid-19 is unique for its indiscriminate transmission among the general public. however, mers seemed to be less aggressive [8, 10, 42] . epidemiological changes have been monitored, taking into account potential routes of transmission and subclinical infections. the official platform updates the public daily on the number of newly diagnosed cases, deaths and cures in each administrative region based on data from the centers for disease control and prevention and hospitals at all levels. since the outbreak, many emergency measures have been taken to reduce personto-person transmission of sars-cov-2. for example, public services and facilities provide disinfectants on a routine basis to encourage appropriate hand hygiene, and physical contact with wet and contaminated objects is considered when dealing with the virus, especially fecal and urine samples that can potentially serve as an alternative route of transmission. china and other countries have implemented major prevention and control measures, including screening travelers, to control further spread of the virus [43] . there are many people donating money, vegetables, medical supplies, etc. to the areas affected by the epidemic. in wuhan, two hospitals, vulcan mountain hospital and raytheon mountain hospital, were built within 10 days, which can contain 1000 and 1300 patients, respectively. according to the people's daily, the national health and fitness commission reported that there are more than 11 000 critical care workers and more than 2000 intensive care unit nurses, and there will be more pooling of medical resources in places where they are most needed. the chinese government has shut down schools and closed businesses to reduce transmission [44] . the outbreak has also caused widespread public concern. husnayain et al. studied the potential to use google trends (gt) to monitor public restlessness regarding the covid-19 epidemic, and they found that searches related to covid-19 and face masks increased rapidly [45] . with the advent of 5g and the rapid development of the information age, it may be more convenient for the masses to obtain the latest news from the internet; thus, internet-based risk communication is becoming an appropriate strategy. there are many disease control organizations and medical institutions that have played an official role in this outbreak and provided accurate and reliable information to the public in a timely manner. for example, laboratory confirmation of covid-19 was performed in five different institutions, namely, the china cdc, chinese academy of medical science, wuhan institute of virology, and academy of military medical sciences, and chinese academy of sciences [29] . according to the cctv news, with scientific progress has enabled the use of advanced technologies to control this epidemic. in addition, the health code divides the public into three health situations, namely, green, red and yellow. this provides an effective method of facilitating crowd tracking and monitoring. furthermore, the geographic information system (gis), which has long been used by many health professionals when tracking and combating contagion, also plays an important role in the geographical tracking and mapping of epidemics. a range of practical online/mobile gis and mapping dashboards and applications have come into use for tracking the covid-19 epidemic [46] . some treatments have been adopted in clinical practice, and a few have been successful [24, 47] . according to prashant pradhan, the first case cured in seven days in the united states showed that the antiviral medication remdesivir may become one of the specific medicines for covid-19; however, this remains to be verified through clinical trials [16] . according to the research by wang, xf, et al. about the clinical manifestations and epidemiology in children with covid-19 treated with lopinavir and ritonavir and without glucocorticoids and immunoglobulin, all 20 patients improved and were discharged from hospital. this may lead to the conclusion that children's clinical symptoms of covid-19 are nonspecific and milder than those in adults, which has significant clinical value [48] . future research priorities may be focused on biological research on sars-cov-2 and clinical research on covid-19 diagnosis and treatment. according to pradhan et al., there are four unique insertions, which have similarity to hiv, in the s-protein in covid-19, which may explain its contagiousness. the gene binding site may become a new target of therapeutics to prevent transmission of the virus [49] . specifically, virus particles are found in the feces, which suggests that there may exist other routes of transmission, such as fecal-oral transmission. previously, we focused on cutting off transmission routes mainly by limiting contact and preventing respiratory droplet transmission. this finding emphasizes the significance of dealing with the feces of the patient. therefore, for patients who already have covid-19, careful disposal of their feces is an important concern with regard to reducing viral transmission [36] . on the basis of the research by hongzhou lu, lopinavir/ritonavir, nucleoside analogs, neuraminidase inhibitors, remdesivir, peptide (ek1), arbidol, rna synthesis inhibitors (such as tdf, 3tc), anti-inflammatory drugs (such as hormones and other molecules), chinese traditional medicine and so on could be therapies for covid-19, but the effects and safety remain to be tested in clinical trials [27] . docking system test 3d structures of remdesivir, chloroquine, ciclesonide, niclosamide, and lopinavirus were obtained from ncbi pubchem. the crystal structure of ace2 (pdb code: 6 m17) was obtained from the protein data bank. the ligands within the crystal structure complex were extracted by pymol software (san carlos, ca, usa). autodock 4.2 was used for the docking system test. autodock tools initialized the ligands by adding gasteiger charges, merging nonpolar hydrogen bonds, and setting rotatable bonds. the ligands were rewritten into pdbqt format, which can be read by autodock software (autodock 4.2, san carlos, ca, usa). autodock tools were used to add polar hydrogen to the entire receptor. the grid box was set to contain the entire receptor region. the receptor output was also saved in pdbqt format. autodock vina was set with the macromolecule held fixed and the ligands flexible. affinity maps for all the atom types present, as well as an electrostatic map, were computed, with a grid spacing of 0.375 å. the structural models were collected from the lowest-energy docking solution of each cluster of autodocks. it is evident from the findings of fig. 2 and table 2 that combinations of antiviral agents are more successful than a single drug. the outbreak of sars renewed interest in this family of viruses and resulted in the development of new drugs, among which remdesivir, chloroquine, ciclesonide, niclosamide, and lopinavirus are the most promising [50] [51] [52] . in addition, as mentioned above, ace2 plays a vital role in the development of covid-19 [53] . with regard to testing the effectiveness of previous medicines used by scientists for the treatment of diseases caused by coronaviruses, autodock calculations have been performed to classify specific binding amino acids and thus to determine the likely common cure targets for ace2. as shown in table 2 and fig. 2 , we found that chloroquine and ciclesonide share similar binding amino acid residues (met124, leu127, ile472 and val589). likewise, remdesivir and niclosamide also possess met124. taken together, we might therefore hypothesize that met124 plays a key role in the efficiency of these drugs targeting ace2. met24 appears to be a potential target for covid-19. however, there is no similar amino acid for lopinavir, suggesting that further studies are needed to elucidate the molecular mechanism of lopinavir treatment of covid-19. the sars-cov-2 has a certain degree of homology with sars-cov and mers-cov. compared to the previous sars and mers outbreaks, we found that covid-19 has a few similarities with regard to the infection source and clinical symptoms, but it also some differences. for example, covid-19 shares some routes of transmission in common with sars and mers, such as respiratory droplets, but covid-19 can also be transmitted by the fecal-oral route [54] [55] [56] . it is important to consider all transmission routes when seeking to control the epidemic and protect medical staff and the general public. the epidemiological research on the source of infection and the route of transmission could help prevent the further spread of the epidemic. different clinical symptoms could be used for the differential diagnosis of patients infected by coronaviruses. the medicines or therapeutic regimens that proved to be effective for sars or mers offer new approaches to the clinical treatment of covid-19, which is essential in the prevention and control of the covid-19 epidemic. the chinese government's control strategies in wuhan and the active cooperation of the people have substantially contributed to china's efforts to control the outbreak, which is important to the global efforts to combat the epidemic. testing medications that have been shown to have antiviral effects against sars-cov or mers-cov coronavirus infections-more than just the common cold emerging understandings of 2019-ncov outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle clinical features of patients infected with 2019 novel coronavirus in wuhan discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin emerging coronaviruses: genome structure, replication, and pathogenesis recent advances in the detection of respiratory virus infection in humans from sars to mers, thrusting coronaviruses into the spotlight,viruses transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination from sars to mers: evidence and speculation detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr note from the editors: novel coronavirus (2019-ncov) medical journals and the 2019-ncov outbreak genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a novel coronavirus from patients with pneumonia in china first case of 2019 novel coronavirus in the united states ct imaging of the 2019 novel coronavirus (2019-ncov) pneumonia severe acute respiratory syndrome: historical, epidemiologic, and clinical features emerging infections -implications for dental care middle east respiratory syndrome virus pathogenesis middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of korea potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review recent advances in the vaccine development against middle east respiratory syndrome-coronavirus safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial drug treatment options for the 2019-new coronavirus (2019-ncov) early estimation of the case fatality rate of covid-19 in mainland china: a data-driven analysis epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost crossspecies transmission from snake to human novel bat alphacoronaviruses in southern china support chinese horseshoe bats as an important reservoir for potential novel coronaviruses epidemic and emerging coronaviruses (severe acute respiratory syndrome and middle east respiratory syndrome) travel implications of emerging coronaviruses: sars and mers-cov integrated and automated, sample-in to result-out, system for nanotechnology-based detection of infectious pathogens absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia potential of large 'first generation' human-tohuman transmission of 2019-ncov the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan, china sars and mers: recent insights into emerging coronaviruses preliminary prediction of the basic reproduction number of the wuhan novel coronavirus 2019-ncov the battle against sars and mers coronaviruses: reservoirs and animal models the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak feasibility of controlling covid-19 outbreaks by isolation of cases and contacts applications of google search trends for risk communication in infectious disease management: a case study of covid-19 outbreak in taiwan geographical tracking and mapping of coronavirus disease covid-19/severe acute respiratory syndrome coronavirus 2 (sars-cov-2) epidemic and associated events around the world: how 21st century gis technologies are supporting the global fight against outbreaks and epidemics clinical trial analysis of 2019-ncov therapy registered in china retracted: clinical and epidemiological characteristics of 34 children with 2019 novel coronavirus infection in shenzhen uncanny similarity of unique inserts in the 2019-ncov spike protein to hiv-1 gp120 and gag broad spectrum antiviral agent niclosamide and its therapeutic potential remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro patients of covid-19 may benefit from sustained lopinavir-combined regimen and the increase of eosinophil may predict the outcome of covid-19 progression structural basis for the recognition of the sars-cov-2 by full-length human ace2 covid-19: gastrointestinal manifestations and potential fecal-oral transmission review article: gastrointestinal features in covid-19 and the possibility of faecal transmission molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes acknowledgments aje edited the manuscript for grammar, punctuation and spelling.authors' contributions xz and hh conceived of and designed the study, revised the manuscript for important intellectual content, performed the literature search and performed the comparison. xz performed the comparison shown in table 1 . key: cord-280624-7v8xuicg authors: ba abduallah, mohamed m.; hemida, maged gomaa title: comparative analysis of the genome structure and organization of the middle east respiratory syndrome coronavirus (mers‐cov) 2012 to 2019 revealing evidence for virus strain barcoding, zoonotic transmission, and selection pressure date: 2020-08-17 journal: rev med virol doi: 10.1002/rmv.2150 sha: doc_id: 280624 cord_uid: 7v8xuicg the middle east respiratory syndrome coronavirus (mers‐cov) emerged in late 2012 in saudi arabia. for this study, we conducted a large‐scale comparative genome study of mers‐cov from both human and dromedary camels from 2012 to 2019 to map any genetic changes that emerged in the past 8 years. we downloaded 1309 submissions, including 308 full‐length genome sequences of mers‐cov available in genbank from 2012 to 2019. we used bioinformatics tools to describe the genome structure and organization of the virus and to map the most important motifs within various regions/genes throughout the genome over the past 8 years. we also monitored variations/mutations among these sequences since its emergence. our phylogenetic analyses suggest that the cluster within african camels is derived by s gene. we identified some prominent motifs within the orf1ab, s gene and orf‐5, which may be used for barcoding the african camel lineages of mers‐cov. furthermore, we mapped some sequence patterns that support the zoonotic origin of the virus from dromedary camels. other sequences identified selection pressures, particularly within the n gene and the 5′ utr. further studies are required for careful monitoring of the mers‐cov genome to identify any potential significant mutations in the future. belong to the betacoronaviruses. 3 the sars-cov emerged in 2003; the mers-cov was discovered in 2012, and the sars-cov-2 was reported in late 2019. the time gap between sars-cov and mers-cov is around 9 years, and between the emergence of mers-cov and sars-cov-2 is 7 years. the continuous emergence of new coronaviruses candidates may be attributed to the features of their genomes. this may be due to several factors, including the low fidelity of their rna-dependant rna polymerases (rdrp), the possibility of recombination, and the high level of expression of their receptors in many mammalian and avian species. 4 thus, there is an urgent need for the regular monitoring of the genome sequences of coronaviruses from various species of bats, animals, and birds. [5] [6] [7] [8] [9] [10] [11] [12] the main goal of the current study was to do a comincludes the majority of viral isolates from human and some from camel origins. 14 clade-c includes viruses of camel origin isolated from various countries in africa, including egypt, morocco, nigeria, burkina faso, and kenya. 13, 15 interestingly, results from the virus neutralization assay revealed that all three clades are closely related, which supports the notion that one vaccine may be able to protect against all three clades. 13 it is believed that mers-cov originated in bats and spilled over to humans via an intermediate host, dromedary camels. 16, 17 dromedary camels are the main known reservoir until now. 18 genome analysis of mers-cov isolates from human and dromedary camel origins revealed a close relationship between each other, suggesting the zoonotic origin of the virus. 14 like other coronaviruses, mers-cov continues to show some changes at the genome level. thus, new virus clades and sub-clades are recently reported of both human and dromedary camel origins. 19 regular monitoring of the genetic makeup of the virus is very important to track down any potential mutation or recombination. the main goal of this study was to do a deep bioinformatic analysis of the most available mers-cov genome sequences in genbank during 2012 to 2019 to understand the evolution of the virus and map any potential changes across the viral genome. therefore, the development of potential diagnostic assays, vaccines, or therapy should cope with any potential changes over the viral genome. this monitoring may also contribute substantially to the control of the virus by knowing the currently circulating clades in a certain community. an earlier study showed the circulation of three different genotypes of the virus in some patients during 2013 in ksa. 20 one year later, the same group reported the presence of four mers-cov clades during the early emergence of mers-cov. later, during 2014 three clades were no longer contributing to the reported human cases suggesting their extinction. 19 the main reason behind these changes was the dynamic changes among the s gene of the virus. 19, 20 studies on mers-cov isolates from various countries in northern and central africa revealed that the circulating strains of the virus in dromedary camels from these countries belong to lineage-c. this lineage is different from the other two lineages reported earlier in the arabian peninsula. 13 although the three lineages have some genetic variations, their antigenic properties remain identical, as shown by the virus neutralization test. 13 isolates from dromedary camels collected from nigeria, burkina faso, and morocco clustered together into a new sublineage-c1 due to shared genetic signatures, including deletions in orf4b. 13 thus, there is an urgent need for continued study not only of mers-cov but also other coronaviruses in the context of the human-animal interface and to understand the biological diversity of coronaviruses. mers-cov submissions were considered as a full-length genome only if they meet three parameters. first, the length of the sequence must be greater than or equal to 30-kilobases. second, the submission must have the full 5 0 utr sequence. third, the submission must have a poly-a tail even represented by one nucleotide of adenine (a). multiple sequence alignment was conducted using the mafft tool (http://ma.cbrc.jp/alignment/soware/). single nucleotide polymorphism (snp) density (excluding indels) was counted from the multiplesequence alignment of 544 orf1ab and 744 s gene sequences by use of an in-house script written in python. for data visualization, geneious (version 7.1.8) was used. to avoid losing open reading frames (orfs) within different sequences, orfs were collected by retrieving regions flanked by conserved sequences, as shown in (table 1) . conserved sequences were obtained from multiple sequence alignment of 308 complete whole mers-cov genomes. different lengths of orf4b and orf8b were calculated at its minimum possible size (300 nt), allowing any start codon (atg, ttg, or ctg) to initiate the orf. mapping the cleavage sites of the nsps among gene-1 was carried out as previously described. 21 phylogenetic trees were constructed using mega x software 22 using multiple sequence alignment of the whole genome, orf1ab, s gene, orf4b, orf5, e gene, m gene, n gene and orf8b sequences. the trees were constructed using maximum likelihood methods and the tamura-nei model. bootstrap analysis (100 pseudo-replicates) was conducted to evaluate the statistical significance of the inferred trees, and only values greater than 50 were displayed. since most isolated sequences did not meet the parameters used in this study for the whole-genome sequences, we conducted the analysis on individual genes. for the comparative study of phylogenetic trees, we initially considered 493 sequences greater than 30 kb since most of the isolated sequences (especially isolates from african camels) did not meet the parameters used in this study for the whole-genome sequences. then, to make the tree much simpler and easily readable, we narrowed we used 308 full-length genome sequences after applying filteration parameters. all relevant data of these sequences are summarized in table s1 . (table 4) . orf1ab consists of two overlapping orfs (orf-1a and orf-1b). the orf-1b is produced by ribosomal frameshifting in which the ribosome steps back one nucleotide and continues reading and producing orf-1b ( figure 1 ). we analyzed 544 sequences of orf1ab (table s1 ). orf1ab is 21 236 nt in length, and the position of the ribosomal frameshifting is located at 13 433 nt of jx869059. distribution of polymorphisms (snps found in more than one strain in a 50-bp sliding window) showed that snp density in orf1ab varies from 0 to 11 ( figure s1a ). from multiple sequence alignment, we identified eight variants seem to be restricted to mers-cov isolated from african camels. all these variants exited in studied african camels only (table s2 ). table 3 ). details about the nsps, their locations, as well as sizes, are shown in table 2 . we analyzed 744 of the s-gene sequences (table s1 ). the full-length of the s gene is about 4062 nt. the organization of the mers-cov-s gene is described in figure 1b (table 3) . we conducted an snp density analysis to test whether the three observed variants of the s gene in african camel samples were not in a highly variable region. the distribution of polymorphisms (snps found in more than one strain in a 50-bp sliding window) showed that snp density in s gene varies from 0 to 12 ( figure s2 ). the variant v26a is located in a region with a snp density of five, while both r1020q and a1158s/l substitutions are located in a region of low snp density (snp density = 2). the total number of different nucleotides between samples in the phylogenetic tree of the s gene sequences is shown in table s2 . the phylogenetic tree of the 57 mers-cov of s gene sequences is presented in figure 2c . compared to the phylogenetic tree of mers-cov whole-genome and orf1ab sequences (figure 2a (table s1 ). there was no sample observed with the two mutations (i529t and d510g) at the same time. in the case of orf-3, we analyzed 566 sequences (table s1 ). the length of orf3 gene is 312-nt. however, in some submissions, we found truncated versions of the encoded protein of this orf due to deletions varying from 9-nt to 41-nt (table 3) . two variants were the orf-4 mainly consists of two overlapping orfs (orf-4a and 4b). in the case of orf-4a, we analyzed 568 sequences (table s1 ). the orf4a gene is 330-nt in length, and there is 89-nt overlapping between orf4a and orf4b genes. the orf4a is highly conserved, but snp density increases in the last 45 nt (data not shown). we analyzed 575 sequences of the orf-4b gene ( figure 4 revealed that all models of the defective gene orf4b and the wild type gene orf4b could produce an identical potential orf of 90 a.a. in length. we analyzed 569 mers-cov sequences of orf5 (table s1 ). the fulllength of gene orf5 is 675 nt, which encodes a protein of 224 aa in length. we found two strains encoding a truncated orf5 protein due to deletions causing a frameshift and stop codon ( (figure 2 and figure s4 ). we analyzed 579 sequences of e gene and 575 sequences of m gene (table s1 ). the lengths for e protein and m protein are 82-a.a. and 219-a.a., respectively. most of the e and m gene sequences were quite conserved and had no significant observation either at the nucleotide or amino acid level. however, we considered observed variants not significant as they either existed in only one sample or in sequences generated by the same group. the number of retrieved and analyzed sequences of n gene was 565 (table s1 ). the full-length of gene n is 1242 nt, which encodes a table 3 ). we analyzed 598 sequences of gene orf-8b (table s1 ). the fulllength of the wild type gene orf8 is 339 nt, which encodes a protein of 112 aa in length. this orf is located within the n gene (from 28 762 to 29 100 of the jx869059 genome sequencing; figure 1 ). (table s1 ). on the other hand, 13 strains have truncated orf8 protein (34-aa shorter than the wild type) due to a mutation at position 28 996 of jx869059, changing tca to a tga (stop codon). one of these sequences was isolated from a human in qatar during 2013 while the others were of human origin from ksa in 2014 (one of them was sequenced in indiana-kj813439; table s1 ). all studied african camels (25 samples mers-cov isolated from african camels is shown to be phylogenetically distinct from those circulating in the arabian peninsula. 13, 26, 27 in this study, we showed that the cluster of mers-cov african camels into (table 3) . we tested the possibility of a defective version of the orf8 gene that can produce orf8 protein with a length close to the wild type by starting from different frames rather than frame-1, all possible orfs were tested. there was no defective form of gene orf8 that produced orf8 protein with a length close to the wild type ( figure s6 ). however, the biological consequence behind the variation of orf8 protein length is still unknown. comparative genome sequence analysis of the mers-cov of both dromedary camels and human origins revealed significant evidence for potential barcoding of the african clades based on the s gene sequences. it also provided evidence for the zoonotic origins of the virus from dromedary camels to humans and highlighted the role of selection pressure and compensatory mechanisms in virus genome evolution. isolation of a novel coronavirus from a man with pneumonia in saudi arabia ninth report of the international committee on taxonomy of viruses origin and evolution of pathogenic coronaviruses middle east respiratory syndrome coronavirus and the one health concept genomic variance of the 2019-ncov coronavirus full genome characterization of two novel alpha-coronavirus species from italian bats full-length genome analysis of canine coronavirus type i molecular investigation of a fulllength genome of a q1-like ibv strain isolated in italy in 2013 a metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in neoromicia bats within south africa molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards complete genome analysis of equine coronavirus isolated in japan full-length genome sequences of porcine epidemic diarrhoea virus strain cv777; use of ngs to analyse genomic and sub-genomic rnas mers coronaviruses from camels in africa exhibit region-dependent genetic diversity genomics and zoonotic infections: middle east respiratory syndrome detection of distinct mers-coronavirus strains in dromedary camels from kenya dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus transmission spread, circulation, and evolution of the middle east respiratory syndrome coronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets variations in spike glycoprotein gene of mers-cov the severe acute respiratory syndrome a novel coronavirus from patients with pneumonia in china middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in africa and middle east serological evidence of mers-cov and hku8-related cov co-infection in kenyan camels the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination we wish to thank the king abdul-aziz city for science and technology (kacst), saudi arabia, for their generous funding through the mers-cov research grant program (number 20-0004), which is a part of the targeted research program (trp). the authors have no competing interest. https://orcid.org/0000-0002-5986-7237 additional supporting information may be found online in the supporting information section at the end of this article. key: cord-281529-2rec51xg authors: haagmans, bart l; al dhahiry, said h s; reusken, chantal b e m; raj, v stalin; galiano, monica; myers, richard; godeke, gert-jan; jonges, marcel; farag, elmoubasher; diab, ayman; ghobashy, hazem; alhajri, farhoud; al-thani, mohamed; al-marri, salih a; al romaihi, hamad e; al khal, abdullatif; bermingham, alison; osterhaus, albert d m e; alhajri, mohd m; koopmans, marion p g title: middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation date: 2013-12-17 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70690-x sha: doc_id: 281529 cord_uid: 2rec51xg background: middle east respiratory syndrome coronavirus (mers-cov) causes severe lower respiratory tract infection in people. previous studies suggested dromedary camels were a reservoir for this virus. we tested for the presence of mers-cov in dromedary camels from a farm in qatar linked to two human cases of the infection in october, 2013. methods: we took nose swabs, rectal swabs, and blood samples from all camels on the qatari farm. we tested swabs with rt-pcr, with amplification targeting the e gene (upe), nucleocapsid (n) gene, and open reading frame (orf) 1a. pcr positive samples were tested by different mers-cov specific pcrs and obtained sequences were used for phylogentic analysis together with sequences from the linked human cases and other human cases. we tested serum samples from the camels for igg immunofluorescence assay, protein microarray, and virus neutralisation assay. findings: we obtained samples from 14 camels on oct 17, 2013. we detected mers-cov in nose swabs from three camels by three independent rt-pcrs and sequencing. the nucleotide sequence of an orf1a fragment (940 nucleotides) and a 4·2 kb concatenated fragment were very similar to the mers-cov from two human cases on the same farm and a mers-cov isolate from hafr-al-batin. eight additional camel nose swabs were positive on one or more rt-pcrs, but could not be confirmed by sequencing. all camels had mers-cov spike-binding antibodies that correlated well with the presence of neutralising antibodies to mers-cov. interpretation: our study provides virological confirmation of mers-cov in camels and suggests a recent outbreak affecting both human beings and camels. we cannot conclude whether the people on the farm were infected by the camels or vice versa, or if a third source was responsible. funding: european union projects emperie (contract number 223498), antigone (contract number 278976), and the virgo consortium. an emerging betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-causes illness characterised predominantly by mild-to-severe respiratory complaints, with most patients requiring admission to hospital because of pneumonitis or acute respiratory distress syndrome. old age and the presence of comorbidities or immunosuppression seem to increase the risk of infection and are associated with severe forms of the disease. however, some patients can remain asymptomatic or mildly symptomatic and atypical presentations such as gastroenteritis have occurred. 1 as of dec 2, 2013, 163 laboratory-confi rmed cases, including 71 deaths, have been reported to who. 2 all of these cases were directly or indirectly linked to the middle east region, including saudi arabia, qatar, united arab emirates, kuwait, oman, and jordan. coronaviruses reside in many animal hosts and can adapt to diff erent species, including people. mers-cov belongs to the lineage c betacoronaviruses, which are associated with bats 3 and the closest relatives of mers-cov have been identifi ed in vesper bats from europe, asia, and south africa. [4] [5] [6] notably, this coronovirus is able to replicate in various bat cell lines. 7 molecular clock dating of epidemiologically unlinked human mers-cov isolates estimated their divergence from a common ancestor in mid-2011, with a cluster of isolates from the eastern parts of the arabian peninsula diverging in late 2012. these fi ndings suggest that the reported mers-cov diversity in human beings is the result of several independent, geographically structured, zoonotic events from an unknown reservoir in the middle east. 8, 9 human-to-human transmission has been noted, especially in health-care settings and households, but is thought to be relatively ineffi cient. 1 to determine how circulation of mers-cov in human beings is maintained and to mitigate the chain of transmission, identifi cation of possible animal reservoirs and modes of transmission is needed. 10 limited available exposure history of patients suggest that contact with livestock, including dromedary camels, might have a role. 1, 9, 11, 12 in addition, our recent investigations have provided evidence for the circulation of mers-cov or a related virus in dromedary camels. 13 both mers-cov spike protein binding antibodies and virus neutralising antibodies were reported in dromedary camels from diff erent regions, including oman and egypt, but no virus shedding could be detected and, therefore, the signifi cance of these observations remained an issue of debate. 13, 14 in this study we aimed to assess presence of mers-cov in dromedary camels from a farm in qatar that was linked to two human cases of mers-cov. in october, 2013, the qatar supreme council of health recorded two cases of laboratory confi rmed mers-cov. case 1 involved a 61-year-old man who was the owner of a farm that he visited regularly. the patient had substantial contact with animals including camels, sheep, pigeons, and hens, and no history of travel outside of qatar in the 2 weeks before becoming ill. mers-cov infection was diagnosed by e gene (upe) pcr on a sputum sample collected on oct 13, 2013, in the national virology laboratory of qatar. case 2 involved a 23-year-old male employee of the farm owned by case 1 and was identifi ed in the close-contact investigation of that case. he was reported on oct 17, 2013, and had no underlying medical conditions and no recent travel history, but had regular contact with the animals on the farm. mers-cov infection was diagnosed in a throat swab taken on oct 17, 2013, by the national virology laboratory of qatar. diagnosis for both cases was confi rmed (orf1b and n gene pcr) by public health england. 15, 16 qatari authorities, with support from who, did an epidemiological investigation at the farm within 1 week of diagnosis of the fi rst case, which included the collection of various clinical samples from the farm's dromedary camels. full details of the outbreak investigation will be described elsewhere. we collected serum, rectal swabs, and nasal swabs from all camels present on the premises where the two men had been in contact with the animals. after an experienced animal handler fi xated the camel by nose pinching, samples were obtained through swabbing with fl ocked swabs (floqswabs, copan improve diagnostics, brescia, italy) that were inserted deep into one of the nostrils of the camel. after sampling, swabs were put into tubes containing viral transport medium-utm (copan diagnostics, brescia, italy). in addition, we obtained fi ve stool samples from three diff erent cages. the sample collection was done jointly by the communicable disease control outbreaks rapid response team of the public health department and the animal health team, which used full personal-protective equipment including n95 masks, goggles, disposable gowns, gloves, and head covers during the handling of the animals, and during the environmental and human sampling. serum samples were stored at -20°c and all other samples were stored at -80°c and were shipped to the netherlands on dry ice. we tested nose swabs with two independent rt-pcr targets specifi c for mers-cov (upe and nucleocapsid [n gene]) in one laboratory (department of viroscience, erasmus medical center, rotterdam, netherlands), and did rt-pcr testing with a third target (orf1a) in another laboratory (national institute of public health and the environment, bilthoven, netherlands). we inoculated vero and huh-7 cells for 1 h with cellculture medium containing samples from camel nose or rectal swabs, or with mers-cov (emc isolate) as a positive control. after washing, the cells were incubated with medium containing 1% fetal bovine serum at 37°c. we stained formaldehyde-fi xed huh-7 cells infected with mers-cov by use of heat-inactivated (at 56°c for 30 min) camel sera or a mers-cov patient's serum according to standard protocols with fl uorescein isothiocyanateconjugated antibodies as a second step. we isolated rna from 140 μl of swab medium or culture supernatant with the qiaamp viral rna minikit (qiagen, hilden, germany) and eluted it in 50 μl. camel mers-cov rna was quantifi ed on the abi prism 7700 with the taqman fast virus 1-step master mix (applied biosystems, bleiswijk, netherlands), with 10 μl isolated rna, one taqman mix, 0·5 u of uracil-n-glycosylase, primer, and probes targeting the n gene, upe, or orf1a as described elsewhere. 17, 18 we used rna dilutions isolated from a mers-cov isolate emc stock as a standard control. 10 μl of rna was reverse transcribed with the superscript iii fi rst strand synthesis system (invitrogen, bleiswijk, netherlands) with random hexamers. cdna was used to amplify six diff erent camel mers-cov fragments, including a partial spike gene by use of pfu ultra ii fusion hs dna polymerase (agilent technologies, santa clara, ca, usa). we did the pcr as follows: one initial denaturation step of 95°c for 5 min, followed by 39 cycles of 95°c for 20 s, 50°c for 20 s, 72°c for 2 min, and a fi nal extension of 72°c for 5 min. the amplifi ed camel mers-cov fragments were sequenced directly on both strands with the bigdye terminator version 3.1 cycle sequencing kit on an abi prism 3100 genetic analyser (applied biosystems). we generated phyml phylogenetic trees with seaview 4 software with the approximate likelihood-ratio test based on a shimodaira-hasegawa-like procedure, which used a general time reversible as substitution model. we used nearest neighbour interchange and subtree pruning and regrafting-based tree search algorithms to estimate the tree topologies. 19 rna from human samples (sputum for qatar 3 case and throat swab for qatar 4 case) was extracted with the nuclisens easymag system (biomérieux, basingstoke, uk). viral sequences from the two human cases were isolated (with the same techniques as for camel cases) in the reference department, public health england, london, uk. we used primers designed against sequences of mers-cov jx869059_emc/2012 and bat coronaviruses hku4 and hku5 to amplify the entire genomes from human cases for sequencing on an illumina miseq sequencer, subsequently indicated as qatar_3_2013 (case 1) and qatar_4_2013 (case 2). the sequences obtained in this study were deposited in genbank under the following accession numbers: camel_mers-cov/qatar_1_2013_orf1a_1_ partial, kf933380; camel_mers-cov/qatar_1_2013_ orf1a_3_partial, kf933381; camel_mers-cov/ qatar_1_2013_orf1b_partial, kf933382; camel_ mers-cov/qatar_1_2013_spike_partial, kf933383; camel_mers-cov/qatar_1_2013_orf4b_full, kf933384; camel_mers-cov/qatar_1_2013_orf1a_2_ partial, kf933385; qatar_3_2013, kf961221; and qatar_4_2013, kf961222. we tested all sera for the presence of igg antibodies reactive with mers-cov (residues 1-747), severe acute respiratory syndrome coronavirus (sars-cov; residues 1-676), and human coronavirus oc43 (residues 1-760) s1 antigens as described previously. 13, 20 we report results as the relative mean fl uorescent intensity (rfu) for each set of quadruplicate spots per antigen. we did igg antibody testing only because anti-camel igm conjugates were not available. for virus neutralisation, serum samples were heatinactivated by incubation for 30 min at 56°c. we prepared twofold serial dilutions with 96 well plates, starting dilution at 1/10. mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with penicillin, streptomycin, and 1% fetal bovine serum, to a dilution of 2000 tcid 50 per ml. we then added 50 μl of virus suspension to the plates and the plates were incubated at 37°c for 1 h. next, we incubated virus-serum mixtures on 96 well plates containing vero cells for 1 h, and then washed them with phosphate buff er saline and incubated them with imdm and 1% fetal bovine serum for 5 days. the sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. samples from 14 camels were obtained on oct 17, 2013 (table, fi gure 1). nose swab specimens from fi ve camels were positive for all three assays (upe, n gene, and orf1a), one sample reacted in two assays, and fi ve camels tested positive in only one rt-pcr assay. subsequently, all samples were subjected to a pcr assay targeting the spike gene of mers-cov (nucleotides 22449-22806) to obtain a fragment for sequencing as defi nitive confi rmation. such sequencing confi rmed the presence of sequences specifi c to mers-cov in three camels. alignment of these partial spike gene sequences with known human mers-cov sequences, including those determined from the two related human cases and some other human mers-cov isolates such as hafr-al-batin_1_2013 (kf600628) and riyadh_3_2013 (kf600613), showed 100% identity, but the sequences diff ered from emc mers-cov, which is used routinely in the erasmus mc laboratory, by one nucleotide. the three camel mers-cov sequences obtained by this approach were identical. virus culture attempts were not successful, although one sample tested for camel 7 yielded a positive rt-pcr of the supernatant (table) and staining of the fi xed virus-infected cells (data not shown). further passage of the virus on vero cells, however, was not successful. in addition, serum from camel 9 showed low levels of mers-cov as determined by rt-pcr, and all other camel sera tested negative (data not shown). rectal swabs and faecal materials were negative for mers-cov when tested for reactivity in the upe and n gene rt-pcrs. further sequencing of the virus from camel 5 with primers specifi c to mers-cov rendered six diff erent fragments covering 4·2 kb across the mers-cov genome (fi gure 1). fragments 1 (nucleotides 6708-7459), 2 (8095-9034), and 3 (10073-10658) were located in orf1a, fragment 4 (18792-19584) in orf1b, fragment 5 (22449-22806) in the spike gene, and fragment 6 (26074-26846) in orf4b. the sequences obtained from the human cases from the same farm diff ered by one nucleotide diff erence in orf1a and one in orf4b. the mers-cov emc isolate diff ers by eight nucleotides from the camel mers-cov in orf1a. alignment of the divergent 940 nucleotide orf1a fragment (data not shown) and a 4·2 kb concatenated sequence fragment was used for phyml phylogenetic analysis. the camel mers-cov clustered with viral sequences obtained from the two human cases related to the farm and with a sequence from hafr-al-batin as the next closest relative (fi gure 1). less stringent methods of analysis do show limited bootstrap support (data not shown), in line with relatively limited sequence variation within this fragment. all camel sera were positive in the immunofl uorescence assay when tested in a 1/200 dilution (fi gure 2). when serum samples from camels were tested at diff erent dilutions on the microarray, we noted reactivity with mers-cov antigen, but not the sars-cov antigen. reactivity to human coronavirus oc43 antigen, as a proxy to betacoronavirus, varied between negative (less than the cutoff value of 4000) and saturating signals (data not shown). consistent with the array data, all serum samples had mers-cov neutralising capacity, with titres varying between 160 and 5120 (fi gure 2). we also noted a strong correlation between microarray titres and virus neutralising antibody titres (fi gure 2). the two camels with the highest mers-cov load in the swabs as shown by taqman assay (camels 5 and 7) had a relatively low serum neutralisation titre of 640. we present, to our knowledge, the fi rst virological confi rmation of mers-cov in dromedary camels (panel). we and others previously reported mers-cov neutralising antibodies in dromedary camels from the canary islands, oman, and egypt, suggesting circulation of mers-cov or a mers-cov-like virus in camels. 13, 14 high prevalences and antibody titres were reported in the camels from oman and egypt suggesting widespread circulation. however, virological testing was unable to detect mers-cov viral sequences in camels, probably because only faecal and serum samples were analysed. in addition, shedding kinetics of mers-cov in camels (and human beings) are unknown, and therefore, inter pretation of negative rt-pcr results from screening is diffi cult because positive and negative predictive values remain to be determined. 22 in the outbreak reported, we showed evidence for presence of the virus in six camels according to internationally recommended criteria of two independent rt-pcr targets. furthermore, we used sequence confi rmation as an additional test to increase the level of certainty-during an emerging disease outbreak such as this, options for validation of assays for clinical testing are limited. 22 discrepancies between assays can be explained by diff erences in detection limits, or by diff erences in specifi city of the primer sets. in our study, n gene rt-pcr yielded four additional weak positive results that could not be confi rmed by any other method, and an individual orf1a rt-pcr identifi cation was made for only one animal. these cases could have resulted from mispriming, or represent presence of levels of virus around the detection limit, as can be expected at the end of the infection cycle. nevertheless, our results suggest a widespread and recent outbreak in this herd, coinciding with infection in two people. our data should not be taken as evidence for infection of people from the camels. comparison of sequence data from the orf1a gene and a concatenated 4·2 kb sequence from the human and camel viruses showed that these viruses are very similar, but distinct. notably, samples from camels and people were analysed in diff erent laboratories in the netherlands and the uk. the data provided show proof that camels can be infected with mers-cov. the sequence diff erence between human and animal viruses from the farm, based on the available overlapping sequence, is so small that we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels were infected from a third as yet unknown source. although additional sequencing might provide improved resolution, it probably will not provide conclusive evidence. the most important unknown is the exact timing of infections, both in the infected people and camels. if the virus entered the farm through either the people or the camels, the infections observed would probably have resulted from a chain of transmissions that introduced mutations. because we did not identify any seronegative animals that were pcr positive, and because all animals had mers-cov neutralising antibodies, our sampling probably took place relatively late in the outbreak on this farm. a more detailed analysis of the outbreak, including testing of additional animals and environmental samples is ongoing, as are attempts to obtain full mers-cov genomes of the human and animal specimens. we cannot exclude the possibility that other common livestock species including cattle, sheep, and goats, or other animals including wild species were involved in the spread of mers-cov. while confi rmation of the source is awaited, we recommend that a detailed case history is taken of any cases of mers-cov, including review of any animal exposures (including animal products), and targeted (prospective) serosurveys to determine what risk factors are associated with human infection. we searched pubmed for articles published in english up to dec 1, 2013, with the search terms "novel coronavirus emc" or "mers-cov". we identifi ed 121 reports linked to the middle east respiratory syndrome coronavirus (mers-cov). one report 21 described the detection in one bat specimen of a single 181 bp fragment in a highly conserved area of the coronavirus genome, identical to the human mers-cov laboratory isolate emc. our report describes the fi rst detection of mers-cov in dromedary camels on a farm in qatar that had been linked to human cases of the disease. our study provides proof that mers-cov has infected dromedary camels in qatar. our results suggest the simultaneous occurrence of a mers-cov outbreak in people and camels. on the basis of the present data, we cannot conclude whether the people on the farm were infected by the camels or vice versa. another possibility is that people and camels could have been infected from a third as yet unknown source. we recommend that detailed case histories be taken, including review of any animal exposures including animal products, and targeted (prospective) serosurveys to determine what risk factors-other than contact with an individual with the infection-are associated with human infection. blh wrote the report, drew the fi gures, and generated, analysed, and interpreted data. shsad, mma, and mpgk coordinated the study, wrote the report, and interpreted data. cbemr wrote the report, drew the fi gures, did the literature search, and analysed and interpreted data. vsr drew the fi gures, and generated, analysed, and interpreted data. mg, rm, and ab generated and interpreted data. gjg generated, analysed, and interpreted data. mj generated and analysed data. ef led the fi eld investigation, obtained samples, and collected and interpreted data. ad supervised fi eld outbreak management and collected and interpreted data. hg and fa supervised animal health fi eld investigations. ma-t coordinated the study and interpreted data. saa-m coordinated the study. hear led outbreak management and collected and interpreted data. aak coordinated the study and interpreted data. admeo wrote the report and interpreted data. blh, admeo, and vsr hold a pending patent for mers-cov. admeo is scientifi c adviser of viroclinics biosciences. all other authors declare that they have no confl icts of interest. doi:10.1371/ currents.outbreaks.0bf719e352e7478f8ad85fa30127ddb8. 2 who. global alert and response (gar): novel coronavirus infection-update ecology, evolution and classifi cation of bat coronaviruses in the aftermath of sars close relative of human middle east respiratory syndrome coronavirus in bat human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt alert and response (gar): novel coronavirus infection-update alert and response (gar): novel coronavirus infection-update detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building specifi c serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus: data gaps for laboratory preparedness we thank who and the food and agriculture organization of the united nations for their generous help in organisation of the study, theo bestebroer for providing middle east respiratory syndrome coronavirus specifi c primer sets, berend jan bosch for providing antigens for the microarray, and jeroen cremer for technical support. contributions to the study were funded through the european union fp7 projects emperie (contract number 223498; to blh and admeo) and antigone (contract number 278976; to mpgk and admeo). key: cord-292092-o6s5nw49 authors: furuse, yuki; okamoto, michiko; oshitani, hitoshi title: conservation of nucleotide sequences for molecular diagnosis of middle east respiratory syndrome coronavirus, 2015 date: 2015-09-30 journal: int j infect dis doi: 10.1016/j.ijid.2015.09.018 sha: doc_id: 292092 cord_uid: o6s5nw49 infection due to the middle east respiratory syndrome coronavirus (mers-cov) is widespread. the present study was performed to assess the protocols used for the molecular diagnosis of mers-cov by analyzing the nucleotide sequences of viruses detected between 2012 and 2015, including sequences from the large outbreak in eastern asia in 2015. although the diagnostic protocols were established only 2 years ago, mismatches between the sequences of primers/probes and viruses were found for several of the assays. such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. a slight modification in the primer design is suggested. protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as mers-cov. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus with a positive-sense rna genome. infection with the virus causes severe respiratory symptoms in humans, with a case fatality rate as high as 37%. 1 camels may be a source of infection to humans. 2 human-to-human transmission is also possible, but this requires close contact, such as health care-related contact without proper measures for infection control and prevention. 3 the earliest case of mers was reported in jordan, and mers-cov was subsequently isolated from cases in saudi arabia only a short time later. 4 since then, infections have been endemic mainly in the middle east. however, mers-cov has spread sporadically to other areas, including europe, north america, africa, and southeast and east asia, by travelers from the middle east. 5 the laboratory diagnosis of mers-cov infection is mainly performed using real-time reverse transcription pcr (rt-pcr) to detect viral rna in specimens. interim recommendations from the world health organization (who) in 2015 for the laboratory testing of mers-cov included protocols for rt-pcr that were developed by the university hospital bonn and the us centers for disease control and prevention. [6] [7] [8] [9] this document included seven assays: (1) the upe assay, which is considered highly sensitive and is recommended for screening, 7 (2) the orf1a assay, which is considered equally as sensitive as the upe assay, 6 (3) the orf1b assay, which is considered less sensitive than the orf1a assay, 6, 7 and the (4) n2 and (5) n3 assays, which can complement upe and orf1a assays for screening and confirmation. 8, 9 to date, these assays have shown no cross-reactivity with other human coronaviruses. [6] [7] [8] sequencing protocols for further confirmation, namely the (6) rdrpseq and (7) nseq assays, were also developed. 6 because mers-cov is an rna virus that can evolve rapidly, there remains concern that these protocols may not be suitable for the detection of current mers-cov because of a mismatch among sequences in the primer/probe regions. this study was performed to analyze recent viral genomic nucleic acid sequences and to discuss the efficacy of the rt-pcr protocols for the molecular diagnosis of mers-cov infections. 1 data for these 386 sequences, including complete as well as partial genome sequences, were obtained and analyzed. sequence data were aligned with clustalw to assess genetic changes in the nucleotide sequences of the primer and probe regions of the assays described above. the numbers of viral sequences that matched the primer/ probe sequences perfectly were counted. as mentioned in the introduction above, the upe, orf1a, n2, and n3 assays can be used for screening because of their high sensitivity. [6] [7] [8] [9] among these, only the primer and probe designs of the orf1a assay showed 100% conservation of all sequence data available today (table 1) . minor mismatches were found for the upe assay (one nucleotide substitution in two sequences) and n2 assay (one nucleotide substitution in one sequence), and significant mismatches were found for the n3 assay. the primer/probe regions were found to be well conserved, except for the n3 assay. in addition, mismatches were not found in the 3 0 end region of primers for the upe and n2 assays ( table 1 ). the sensitivity of the assays may not be greatly affected. no mismatches were found for the orf1b assay. with regard to the sequencing assays, no sequence data that matched the sequence of the reverse primer for the rdrpseq assay was found. however, a single common mismatch in all sequence data was found. when the mismatched nucleotide was corrected, the rdrpseq assay matched all the sequence data perfectly ('corrected reverse primer', table 1 ). in addition, viral sequences of the reverse primer region for the nseq assay were not highly conserved; the sequence matched only 49% of strains. based on these results, the use of a modified reverse primer for the assay is suggested, in order to reduce the possibility of a mismatch ('modified reverse primer', table 1 ). several mismatches among viral sequences in the primer/probe regions for molecular diagnosis were identified in this study. such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. the mismatched sequence data could have been generated by errors in pcr or sequencing during viral nucleotide sequence analysis because of the incorporation of the wrong nucleotide. 10 however, it is more likely that the rna virus has evolved and that this has accidentally resulted in the induction of mutation/s in the region targeted by the primer/probe for rt-pcr, only 2 years after the establishment of the protocols. fortunately, no or few mismatches were found for most of the mers-cov screening assays. nevertheless, protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as mers-cov. middle east respiratory syndrome coronavirus (mers-cov) evidence for camel-to-human transmission of mers coronavirus first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): situation update and cases reported in the netherlands. who assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus world health organization. laboratory testing for middle east respiratory syndrome coronavirus-interim guidance (revised). who fidelity of dna polymerases in dna amplification this research was supported by the japan initiative for global research network on infectious diseases (j-grid) from the japan agency for medical research and development, amed. the funding source had no involvement in the study design, in the collection, analysis, and interpretation of the data, in the writing of the manuscript, or in the decision to submit the manuscript for publication.conflict of interest: all authors declare no conflicts of interest. table 1 conservation of the primer and probe region sequences of the who-recommended assays for the molecular diagnosis of mers-cov key: cord-287527-ep6ug9c3 authors: algaissi, abdullah; agrawal, anurodh s; han, song; peng, bi-hung; luo, chuming; li, fang; chan, teh-sheng; couch, robert b; tseng, chien-te k title: elevated human dipeptidyl peptidase 4 expression reduces the susceptibility of hdpp4 transgenic mice to middle east respiratory syndrome coronavirus infection and disease date: 2018-09-26 journal: the journal of infectious diseases doi: 10.1093/infdis/jiy574 sha: doc_id: 287527 cord_uid: ep6ug9c3 background: the ongoing middle east respiratory syndrome coronavirus (mers-cov) infections pose threats to public health worldwide, making an understanding of mers pathogenesis and development of effective medical countermeasures (mcms) urgent. methods: we used homozygous (+/+) and heterozygous (+/−) human dipeptidyl peptidase 4 (hdpp4) transgenic mice to study the effect of hdpp4 on mers-cov infection. specifically, we determined values of 50% lethal dose (ld(50)) of mers-cov for the 2 strains of mice, compared and correlated their levels of soluble (s)hdpp4 expression to susceptibility, and explored recombinant (r)shdpp4 as an effective mcm for mers infection. results: hdpp4(+/+) mice were unexpectedly more resistant than hdpp4(+/−) mice to mers-cov infection, as judged by increased ld(50), reduced lung viral infection, attenuated morbidity and mortality, and reduced histopathology. additionally, the resistance to mers-cov infection directly correlated with increased serum shdpp4 and serum virus neutralizing activity. finally, administration of rshdpp4 led to reduced lung virus titer and histopathology. conclusions: our studies suggest that the serum shdpp4 levels play a role in mers pathogenesis and demonstrate a potential of rshdpp4 as a treatment option for mers. additionally, it offers a validated pair of tg mice strains for characterizing the effect of shdpp4 on mers pathogenesis. middle east respiratory syndrome (mers) is an emerging infectious disease caused by a coronavirus (mers-cov), first identified in saudi arabia in 2012, that has since spread to 27 mostly surrounding countries, resulting in more than 2229 laboratory-confirmed cases of infection and 791 deaths (approximately 36%), as of june 2018 [1] . the pandemic potential of this infection calls for a better understanding of mers pathogenesis and the development of effective medical countermeasures (mcms) for humans. like other human covs, mers-cov uses an exoaminopeptidase, human dipeptidyl peptidase 4 (dpp4), as the entry receptor for infection of permissive cells [2] . dpp4, also known as cd26, is involved in many physiological functions via its ubiquitous expression in a variety of tissues, its propensity to interact with adenosine deaminase and other important regulatory molecules of the immune system, and its intrinsic proteolytic activity that cleaves many biologically active peptides or proteins that contain proline or alanine at the penultimate position [3, 4] . not only is dpp4 expressed as a type ii transmembrane glycoprotein, primarily on endothelial and epithelial cells and subsets of immune cells, but it is also present in a functionally intact soluble form (sdpp4) in the circulation and other body fluids [3, 4] . because wild type mice are not susceptible to mers-cov, we established a heterozygous (+/−) transgenic (tg) mouse model globally expressing hdpp4 for studies of mers pathogenesis and development of mcms against mers-cov infection [5, 6] . to ensure a steady and cost-effective supply of the animals, a tg mouse model with homozygous expression of hdpp4, designated hdpp4 +/+ tg mice, was developed through mating of hdpp4 +/− mice and used as breeders for generating offspring of both genotypes of hdpp4 tg mice. because hdpp4 is the functional mers-cov receptor, the doubling of encoded hdpp4 gene in hdpp4 +/+ tg mice could render them more susceptible than their hdpp4 +/− counterparts to mers-cov infection and disease. to our surprise, we found that hdpp4 +/+ mice are more resistant than their hdpp4 +/− counterparts to mers-cov infection. we subsequently found that an increased expression of functionally intact soluble hdpp4 (shdpp4) in the circulation of hdpp4 +/+ tg mice, relative to that of hdpp4 +/− mice, was associated with this increased resistance and might be, at least in part, accountable for the seemingly counterintuitive findings on susceptibility to mers-cov infection. this notion was supported by studies showing that elevated shdpp4 levels, brought about by administration of recombinant shdpp4 (rshdpp4), resulted in increased resistance of recipient hdpp4 +/− mice to mers-cov. together, our results indicate that manipulation of shdpp4 might serve as a strategy for counteracting mers-cov infection and disease in humans. hdpp4 +/− transgenic mice were established, as previously reported [5, 6] . hdpp4 +/+ breeder mice were derived by mating 2 parental hdpp4 +/− mice. the homozygosity was determined by quantitative polymerase chain reaction analysis of tail dna (data not shown) and verified by their subsequent mating with wildtype (wt) mice. only those mice uniformly yielding heterozygous offspring were selected as hdpp4 +/+ breeders. interbreeding between hdpp4 +/+ mice produced additional hdpp4 +/+ mice, whereas backcrossing them to wt mice generated hdpp4 +/− mice. all of the in vitro and animal studies involving infectious mers-cov were conducted at the biosafety level 3 (bsl3) laboratory and animal bsl3 facilities at the galveston national laboratory in accordance with approved protocols and the guidelines and regulations of the national institutes of health (nih) and association for assessment and accreditation of laboratory animal care (aaalac). detailed methodologies for viral infection, isolation from infected lungs and brain, and determination of infectious viral loads have been established and routinely used in our laboratory [5, 6] . the original stock of mers-cov emc-2012 strain, a gift of heinz feldmann (nih, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, netherlands), was expanded in vero e6 cells 3 times consecutively. passage 3 containing a titer of approximately 5 × 10 6 50% cell culture infectious dose (tcid 50 )/ml of virus was used throughout the study. the 50% lethal dose (ld 50 ) values for hdpp4 +/+ and hdpp4 +/− mice was determined by using traditional virus dilution assays and the reed-muench method, as we previously described [6] . briefly, groups of 4 young (6-8 weeks) or old (7-10 months) hdpp4 +/+ and hdpp4 +/− mice were inoculated, via intranasal route, with dosages of emc-2012 mers-cov in 10-fold decrements from 10 2 to 10 -1 tcid 50 in a volume of 60 µl. mice were monitored daily for clinical manifestations (weight loss) and mortality for at least 21 days postinfection (dpi). ld 50 values for each strain of mice were estimated based on the ratio of the surviving mice to the total inoculated mice, as previously described [6] . those surviving for more than 21 days were also evaluated for specific antibody responses to mers-cov receptor binding domain (rbd) protein by enzymelinked immunosorbent assay (elisa) [6] . only those showing specific antibody to rbd were considered as "mers-cov infected. " to quantify the circulating shdpp4 in the sera of naive dpp4 +/+ , dpp4 +/− , and dpp4 −/− mice, a commercial elisa-based assay was used, following the manufacturer's instructions (ebioscience catalog no. bms235). absorbance at 450 nm in 96-well plates was read in an elisa plate reader (molecular device). elisa-based and vero e6 cell-based microneutralization assays, previously described [7] , were used to determine the titers of mers-cov rbd-specific serum igg and neutralizing antibodies in hdpp4 tg mice in response to mers-cov infection. purified insect cell-derived human dpp4 ectodomain (residues 39-766; genbank accession no. np_001926.2) containing an n-terminal human cd5 signal peptide and a c-terminal his6 tag, as we previously described and characterized [7, 8] , was prepared and used for treatment studies. testing binding specificity of the rshdpp4 to the rbd proteins of mers-cov and severe acute respiratory syndrome coronavirus (sars-cov; both rbds were generous gifts of drs du and jiang at new york blood center, ny) was determined in elisa-based assays [7, 9] . for determining the capacity of rshdpp4 to inhibit mers-cov infection in vitro we initially used our standard microneutralization procedure with cytopathic effect (cpe) inhibition as the endpoint. these assays revealed a dose-dependent reduction of cpe at 72 hours, ranging from less than 5% for 100, 50, and 25 μg/ml and gradually increased to approximately 30% for 12.5 μg/ml of rshdpp4. in addition to standard microneutralization assay with a cpe endpoint, we measured the antiviral effect of rshdpp4 using virus yield of each rshdpp4 dilution from 100 to 0.8 μg/ml, expressed as log 10 tcid 50 /ml. the effect of rshdpp4 for inhibiting mers-cov infection in tg mice was determined using hdpp4 +/− mice in 2 pilot studies with 2 different batches of rshdpp4 showing similar, but not identical, binding capacity to mers-cov rbd and in vitro neutralizing activity. briefly, groups of hdpp4 +/− mice (n = 3 per group) were treated twice with either 100 µg or 400 µg of rshdpp4 or phosphate-buffered saline (pbs) as control, via the intraperitoneal route 2 hours before (−2 hours) and 24 hours after (+24 hours) infection (intranasal) with 10 3 tcid 50 of mers-cov. mice were sacrificed at 3 dpi to assess infectious viral loads and histopathology in the lungs. inflated lung specimens and brain tissues were fixed in 10% neutral buffered formalin for 48 hours before paraffin embedding and processing for routine hematoxylin and eosin stain (h&e) to assess the histopathology, as we previously described [5, 6] . statistical analyses were performed using graphpad prism software. neutralizing antibody titers and virus titers were averaged for each group of mice and compared using students t test, 1-way anova, or others as indicated. for the initial comparison of the susceptibility of hdpp4 +/+ and hdpp4 +/− mice to mers-cov, we determined the ld 50 values for mice 7-10 months of age, as we previously described [6] . because hdpp4 is the functional receptor of mers-cov, we anticipated that dpp4 +/+ mice might be more, or at least equally, permissive as dpp4 +/− mice to mers-cov infection. to our surprise, we found that hdpp4 +/+ mice were more resistant than hdpp4 +/− mice as indicated by ld 50 values of 4.3 and 32.4 tcid 50 of mers-cov for hdpp4 +/− and hdpp4 +/+ mice, respectively. to confirm this seemingly counterintuitive finding and rule out any potential effect of age and gender, we repeated the study using age-(6-8 weeks old) and sex-matched tg mice of both genotypes. shown in figure 1a is a representative of 2 independently performed experiments that confirmed the ld50 difference; values for the hdpp4 +/− and hdpp4 +/+ mice were 7.7 and 70.0 tcid 50 of mers-cov, respectively, indicating that hdpp4 +/+ mice are more resistant than their age-and sex-matched hdpp4 +/− counterparts to mers-cov infection. using sera collected 21 dpi from each strain of tg mice that survived the lower challenge dosages, we quantified mers-cov rbd-specific igg antibodies by elisa. we found that infection had occurred in tg mice of both strains. infection rates for those given 100 tcid 50 were similar (1/1 for dpp4 +/− mice and 2/2 for dpp4 +/+ mice) and 10 tcid 50 (2/2 for each strain) but were greater for dpp4 +/− mice (3/3) than dpp4 +/+ mice (1/4) for 1 tcid 50 . the difference in infection rates is consistent with the increased resistance of dpp4 +/+ mice described earlier. to further verify the difference in susceptibility to mers-cov infection, we infected (intranasal) hdpp4 +/+ (n = 9) and hdpp4 +/− (n = 11) tg mice with an equal dose of mers-cov (10 3 tcid 50 /per mouse) and monitored them daily for morbidity (weight loss) and mortality. three mice of each strain, unless indicated otherwise, were euthanized at 3, 5, and 7 dpi to assess infectious viral titers and the histopathology of lungs and brains. in contrast to dpp4 +/− mice, which exhibited marked weight loss, starting at 3-4 dpi, and 2 deaths at 6 dpi (data not shown), infected hdpp4 +/+ mice exhibited minimal weight changes and uniformly survived through 7 dpi when the experiment was terminated ( figure 1b) . when the viral loads were measured at 3 dpi, we readily recovered infectious virus from the lungs, but not the brains, of all 3 hdpp4 +/− mice examined, but from the lung of only 1 of 3 hdpp4 +/+ mice. although we usually recover virus from lungs of some mice, efforts to recover infectious virus from both lung and brain specimens of both strains of tg mice at 5 dpi were unsuccessful (data not shown); however, we were able to retrieve infectious virus from the brain (but not lungs) of the sole hdpp4 +/− survivor and from all 3 hdpp4 +/+ mice that survived to 7 dpi. the virus titer in the brain was 10 6.2 /g for the single hdpp4 +/− mouse, a titer significantly higher than the average of 10 3.7 /g for 3 hdpp4 +/+ mice ( figure 1c ). this ability to recover infectious virus from the lungs approximately 2-3 days earlier than from the brains is consistent with the pattern, kinetics, and tissue distribution of mers-cov infection in dpp4 tg mice we have previously reported [6] . we also compared the histopathology of lungs and brains, 2 of the prime targets of mers-cov infection of hdpp4 tg mice [5] . although infected dpp4 +/− mice elicited mild-to-moderate histopathological changes within the lungs at 3 dpi after a dose of 10 3 tcid 50 of mers-cov infection, as in our earlier study [6] , infected dpp4 +/+ mice exhibited reduced or no lung histopathology (data not shown). brain histopathology at 7 dpi for the sole hdpp4 +/− survivor had infiltrations of mononuclear cells in the meninges ( figure 1d , below), perivascular cuffing ( figure 1d , above and below), microglial nodules ( figure 1d , below), microhemorrhage ( figure 1d , above) and cell death at the junctions of gray and white matter ( figure 1d , above) but not in dpp4 +/+ mice. collectively, the significant differences between these 2 strains of mice in their ld 50 values, seroconversion rates, viral loads, weight loss, and histopathology support the notion that dpp4 +/+ mice are more resistant than dpp4 +/− mice to mers-cov infection. . sera of naive human dipeptidyl peptidase 4 (hdpp4 +/+ ) mice contain significantly higher levels of soluble hdpp4 (shdpp4) that exhibit higher levels of neutralizing antibody-like activity than those of naive hdpp4 +/− mice. a, groups of at least 10 age-and sex-matched hdpp4 +/+ , hdpp4 +/− , and their transgene-negative (hdpp4 −/− ) littermates were subjected to retroorbital bleeding to assess the contents of shdpp4, using commercially available elisa kits that quantify specific hdpp4 (hdpp4 +/+ vs hdpp4 +/− , p < .001 t test). b, sera obtained from naive hdpp4 +/+ and hdpp4 +/− mice (n = 10 of each) and hdpp4 −/− mice (n = 6) were subjected to the standard vero e6-based microneutralization tests to determine their potential to neutralize mers-cov. data are presented as the geometric mean neutralization titers (gmt log 2 ) of 50% neutralization titer (nt 50 ). a 1-10 dilution of sera is approximately 3.4 log 2 . (* p = .026, t test and mann-whitney rank sum test, when compared to those of hdpp4 +/− mice). the gmt titers of hdpp4 −/− mice were uniformly below the limit of detection. abbreviations: elisa, enzyme-linked immunosorbent assay; mers-cov, middle east respiratory syndrome-associated coronavirus. dotted line, limit of detection. tg mice could be different, thereby contributing to their difference in susceptibility to mers-cov infection. using a commercial elisa-based analysis, we were unable to detect shdpp4 in age-and sex-matched hdpp4-negative (hdpp4 −/− ) littermates. however, as shown in figure 2a , a representative of 2 independently performed studies, an average of 8.1 ± 0.2 μg/ml (mean ± sd) and 6.2 ± 0.4 μg/ml of shdpp4 was detected in hdpp4 +/+ (n = 16) and dpp4 +/− mice (n = 10), respectively. as shdpp4 retains its binding specificity to mers-cov rbd protein [7] , the significantly different expression of shdpp4 in the sera of these 2 strains of tg mice (p < .001) prompted us to examine if elevated shdpp4 expression might relate to the higher resistance of dpp4 +/+ to mers-cov infection by possibly acting like a decoy that binds mers-cov rbd and prevents virus infection. using microneutralization tests, we noted that the 50% neutralization titers (nt 50 ), expressed as the geometric mean titers (gmt), were 3.9 ± 0.3 and 3.1 ± 0.1 for hdpp4 +/+ and hdpp4 +/− mice, respectively (p = .026), compared to those of hdpp4 −/− mice which were uniformly below the limit of detection (i.e., ~3.0) ( figure 2b ). because the significantly higher expression of shdpp4 with better neutralizing activity might contribute to the increased resistance of hdpp4 +/+ mice to mers-cov infection, we explored whether an increased shdpp4 expression might increase resistance of naive hdpp4 +/− mice to mers-cov infection. using our limited amount of insect cell-derived rshdpp4, known to specifically bind to rbd of mers-cov but not sars-cov, and to neutralize mers-cov in vitro in a dose-dependent manner ( figure 3a and 3b), we administered 100 μg of rshdpp4, via the intraperitoneal route, into each of 3 dpp4 +/− mice 2 hours before (−2 hours) and 24 hours after (+ 24 hours) infection with 10 3 tcid 50 of mers-cov. the effect of the rshdpp4 against mers-cov infection was assessed at 3 dpi by using the titers of infectious virus within the lungs as the end point for this pilot study. while all of 3 pbs-treated mice exhibited moderate titers of live virus, we were unable to recover infectious virus from any of 3 rshdpp4-treated mice ( table 1 , experiment 1). encouraged by the preliminary data, we generated another batch of rshdpp4 shown to inhibit mers-cov infection in a dose-dependent manner ( figure 3b ) to repeat the experiment. we gave groups of 3 dpp4 +/− mice either 100 or 400 μg of rsh-dpp4, or pbs (as control) 2 hours before and 24 hours after virus infection as before. to determine if administration of rshdpp4 would increase the circulating levels of shdpp4, serum levels of shdpp4 in mice prior to and 2 hours after the first administration and before challenge with mers-cov were measured. we found that titers at 0 and 2 hours of tg mice given 100 μg (6.6 ± 0.4 versus 6.6 ± 0.9 μg/ml), were similar to those of pbs-treated mice (7.5 ± 0.8 versus 7.3 ± 1.0 μg/ml). however, mice treated with 400 μg of rshdspp4 showed significantly increased titers 2 hours after treatment (6.6 ± 0.6 increased to 13.7 ± 0.8 μg/ml; p = .002, t test). these results suggest that an increased serum level of shdpp4 can be achieved by administration of rshdpp4 in a dose-dependent manner. in addition to the viral loads within the lungs at 3 dpi, the pulmonary histopathology was examined to investigate the effect of rshdpp4 on mers-cov infection. unlike the first study in which treatment with 2 doses of 100 μg (at −2 hours and +24 hours) of rshdpp4 fully protected against mers-cov infection, we were able to recover reduced titers of infectious virus from each of 3 mice given 100 μg of the second batch of rshdpp4 when compared to those of pbs-treated controls (p = .077, elisa 96-well plates, precoated with rbd protein of mers-cov or sars-cov, were used to determine the binding specificity of rshdpp4 by using the standard elisa-based assays. absorbance was measured at a wavelength of 450 nm. b, the dose-dependent neutralizing activities of 2 different batches of rshdpp4 (rshdpp4-1 and -2) against mers-cov. the modified vero e6-based microneutralization test (virus yield) was used to quantify the neutralizing antibody-like capacity of rshdpp4, as described earlier [12] and in the methods. *p < .05; **p < .01. abbreviations: elisa, enzyme-linked immunosorbent assay; od, optical density; tcid 50 , 50% cell culture infectious dose. t test). however, as shown in table 1 , experiment 2, the titers of infectious virus in mice treated with 400 μg of rshdpp4 were significantly reduced from an average of 4.3 ± 0.3 (mean ± se) in control mice to 2.6 ± 0.1 tcid 50 /g (p = .011, t test). this finding is consistent with the reduced potency of batch 2 of rshdpp4, as shown in figure 3b . however, the histopathology in mice treated with the high dose of rshdpp4 (ie, 400 μg) was reduced as well when compared to that of the pbs controls (data not shown). in this study we found that hdpp4 +/+ mice were more resistant than hdpp4 +/− mice to mers-cov infection, as evidenced by approximately 10-fold increases of ld 50 , reduced infectious viral yields, and seroconversion rates, as well as less weight loss and lower mortality than their age-and sex-matched hdpp4 +/− counterparts ( figure 1 ). we also found that hdpp4 +/+ mice had significantly higher levels of shdpp4 in their circulation than did hdpp4 +/− mice ( figure 2 ). moreover, these higher serum levels of shdpp4 exhibited higher titers of neutralizing activity against mers-cov in vero e6 cell-based assays. finally, we showed that administration with functionally active rhsdpp4 proteins (figure 3 ) enabled hdpp4 +/− mice to better resist mers-cov infection in a dose-dependent manner (table 1) , a finding in accordance with increased levels of shdpp4 in their circulation. taken together, these results support the notion that increasing the levels of shdpp4 is a potential option for counteracting mers-cov infection and disease in humans. dpp4, ubiquitously expressed on many types of cells and tissues, has been well characterized as critically involved in regulating many important physiological functions, in part through its intrinsic enzymatic activity and propensity to interact with other key regulatory molecules of the immune system [3, 13] . as the functional receptor that mediates entry of mers-cov to permissive host cells, the membrane-associated hdpp4 plays a pivotal role in mers-cov infection and disease. however, specific role(s) that shdpp4 might have in mers pathogenesis remain much less understood. while the levels of sdpp4 vary significantly, even among healthy individuals, it has been shown that the intensities of sdpp4 expression in the circulation, along with its intrinsic enzymatic activity, could be a factor in dictating the severity of many human diseases, including malignancies, autoimmune and inflammatory diseases, diabetes mellitus and other metabolic syndromes, and chronic infectious disease such as aids and hepatitis c [4, 10, 11, 14, 15] . it has been recently reported that serum levels of sdpp4 expression in confirmed mers patients were significantly reduced when compared to those of healthy individuals [16] ; however, the suggestion that these reduced levels could serve as biomarkers for susceptibility requires knowledge regarding the levels in mers cases before onset of infection and disease. in addition, further studies of the therapeutic value of shdpp4 as either a significant resistance factor or a potential countermeasure for mers-cov in humans is warranted. of note, the soluble forms of the viral receptors for several other viruses, including those caused by sars-cov, rhinovirus, and hiv, have been proposed as potentially effective antiviral therapeutics [17] [18] [19] . we showed in this study that sera derived from naive hdpp4 tg mice of either strain, especially hdpp4 +/+ , possess detectable neutralizing antibody-like activity against mers-cov ( figure 2b ). whether the significantly higher shdpp4 expression of hdpp4 +/+ mice could be solely accountable for its greater resistance to mers-cov infection through functioning as receptor decoys seems unlikely, because it took at least 12.5 μg of rsh-dpp4, a level higher that the approximately 8 μg/ml in sera of hdpp4 +/+ mice, to significantly inhibit mers-cov infection in vero e6 cells ( figure 3b ). additional studies are needed to better understand the shdpp4-related protective mechanisms against mers-cov, especially those of the immune system. however, the validated direct correlation between the level of shdpp4 and the susceptibility to mers-cov infection, as shown in this study, may provide a possible genetic basis for the observed wide spectrum of diseases, ranging from asymptomatic, mild-to-moderate, to severe infection and death, in mers patients [20, 21] . with the limited supplies of rshdpp4, we have shown in 2 independently performed proof-of-principle studies that administration of exogenous rshdpp4 might be a treatment a mice (n = 3 each group) were given either 100 μl of pbs or pbs containing 100 or 400 μg of rshdpp4/per mouse by the intraperitoneal route 2 hours before and 24 hours after intranasal challenge with 100 ld 50 (approximately 10 3 tcid 50 ) of mers-cov. lung infectious viral titers were quantified at 3 dpi using a standard vero e6 cell-based infectivity assay. b mean ± se. c none detected (limit of detection was 2.5 log 10 tcid 50 /g). d p = .038 (t test) using 2.4 as the value for nondetectable samples. e p = .011, 1-way anova. option for mers-cov infection (table 1) . additional studies are required to determine if increasing shdpp4 levels by rsh-dpp4 treatment could be a useful treatment option for human mers. the study presented in this report demonstrates the usefulness of this homozygous and heterozygous pair of hdpp4 tg mice to fully explore the interactions between hdpp4 and mers-cov infection and disease, studies that could lead to identification of novel molecular and cellular targets for mcms against mers-cov infection and disease in humans. financial support. this work was supported by the national institute of allergy and infectious diseases, national institutes of health (grant numbers r21ai113206 to c.-t. k. t. and r01ai110700 to f.l). potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. world health organization regional office for the eastern mediterranean dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system unravelling the immunological roles of dipeptidyl peptidase 4 (dpp4) activity and/or structure homologue (dash) proteins generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 imunomodulatory activity of dpp4 dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus the structure and function of cd26 in the t-cell immune response a dipeptidyl peptidase-4 inhibitor directly suppresses inflammation and foam cell formation in monocytes/macrophages beyond incretins dipeptidyl peptidase-4: a key player in chronic liver disease reduction of soluble dipeptidyl peptidase 4 levels in plasma of patients infected with middle east respiratory syndrome coronavirus susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor prevention of rhinovirus infection in chimpanzees by soluble intercellular adhesion molecule-1 aavexpressed ecd4-ig provides durable protection from multiple shiv challenges clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome key: cord-264901-w285on4x authors: ahmadzadeh, jamal; mobaraki, kazhal; mousavi, seyed jalil; aghazadeh-attari, javad; mirza-aghazadeh-attari, mohammad; mohebbi, iraj title: the risk factors associated with mers-cov patient fatality: a global survey date: 2019-07-31 journal: diagn microbiol infect dis doi: 10.1016/j.diagmicrobio.2019.114876 sha: doc_id: 264901 cord_uid: w285on4x risk factors associated with middle east respiratory syndrome coronavirus (mers-cov) infection outcome were established by analyses of who data from september 23, 2012 to 18 june 2018. of the 2220 reported cases, 1408 cases, including 451 mers-cov deaths, were analyzed. the case fatality rate was 32% (95% ci: 29.4–34.5). compared to mers patients ≤30 years old, those with >30 years had the adjusted odds ratio estimate for death of 2.38 [95% ci: 1.75–3.22]. this index was 1.43 [95% ci: 1.06–1.92] for saudi patients in comparison to non-saudi; 1.76 [95% ci: 1.39–2.22] for patient with comorbidity in comparison to those without comorbidity; 0.58 [95% ci: 0.44–0.75] for those who had close contact to a camel in the past 14 days and 0.42 [95% ci: 0.31–0.57] for patients with >14 days with onset of signs and hospital admission compared to patients with ≤14 days. the middle east respiratory syndrome (mers) is a relatively new viral respiratory infection caused by a novel coronavirus, the middle east respiratory syndrome coronavirus, or mers-cov (alshahrani et al. 2018) . coronaviruses are single-stranded positive-sense rna viruses (zumla et al. 2015) , with human pathogenic strains resulting in a common cold to a severe acute respiratory syndrome (castaño-rodriguez et al. 2018 ). mers-cov, an emerging zoonotic virus (alamoudi et al. 2018; cong et al. 2018 ) is pathogenic in human, resulting in shortness of breath, cough, fever, diarrhea, and frequently pneumonia (cong et al. 2018; sherbini et al. 2017) . the exact animal origin of mers-cov is not fully understood, but the transmission pattern and the evidence from virologic studies suggest that it may have originated in bats and was transmitted to camels sometime in the distant past (hu et al. 2015 , wang et al. 2014 . several published studies have assessed the epidemiological status of mers-cov infection. most of these epidemiological studies were derived from specific cohorts with a small sample size, or carried out in a single medical center (alraddadi et al. 2016; assiri et al. 2013; harriman et al. 2013; memish et al. 2013; mobaraki and ahmadzadeh 2019b; park et al. 2017) . however, numerous questions about the epidemiological status and associated risk factors of mers-cov at the global level remain unanswered. for this study, we used the publicly available world health organization (who) mers global epidemiologic data (world health organization 2019) to assess characteristics, clinical information, global distribution status, and probable risk factors associated with mers-cov patient mortality. in this worldwide comprehensive survey, were analyzed publicly available data from the who website:(http://www.who.int/csr/don/ archive/disease/coronavirus_infections/en/) related to laboratory-confirmed mers-cov cases from september 23, 2012 until june 18, 2018. data for the analyses was downloaded on june 20, 2018. this who database is periodically updated by the who. epidemiological characteristics of each patient were retrieved including: age, gender, travel history to endemic countries, nationality, country/city of origin. also retrieved was clinical data on the symptomatic mers patients including day/ month of the onset of symptom, day/month of the admission to the hospital, and comorbidities (diabetes mellitus, hypertension, and ischemic heart disease). exposure to hazardous contacts was also collected, including healthcare workers (who worked in the hospital), camels, consumption of raw camel products, and exposure to mers-cov morbid patients at home or hospital within 14 days prior to the onset of symptomology. all the collected information was checked for missing or invalid data. of a total input of 2220 mers patient cases, 1408 with a complete data set were included in the analysis. in order to avoid measurement bias and miscategorization of cases, 812 of the 2220 patients with incomplete data were not included. statistical analysis was conducted using the spss, version 21 (ibm inc., armonk, ny, usa). quantitative measurement is expressed by the mean and standard deviation. qualitative variables are presented as absolute frequency and percentage. logistic regression was used to calculate the adjusted odds ratio (aor) and unadjusted odds ratio (uor) with a 95% confidence interval to assess the probable relationship between the risk factors and the final outcome (dead/survived) of laboratory-confirmed mers-cov cases. any p-value given was two-sided and was considered statistically significant at 0.05. for this study, a total of 1408 sporadic/clusters patients with confirmed mers-cov infection and complete data were used in the analysis. the characteristics and the number of mers cases per year can be found in . most of the mers cases were male and male-specific % cfr was approximately 33% (332/1008). the male vs. female ratio in the fatal and nonfatal cases was 2.9 (332/119) vs. 2.4 (676/281), respectively (see table 2 ). table 3 illustrates unadjusted and adjusted or for mortality in the laboratory-confirmed mers-cov cases in the survey period. the adjusted analysis showed that the age groups n30 years (2.38; 95% ci: 1.75-3.22), saudi nationality (1.43; 95% ci: 1.06-1.92), and comorbidity (1.76; 95% ci: 1.39-2.22) were independently associated with higher chances of mortality. additionally, in comparison to mers patients who had ≤14 days from onset of clinical signs to hospital admission, adjusted or estimates of the mortality was 0.42; 95% ci: 0.31-0.57 for those who had n14 days from the onset of clinical signs to hospital admission. the adjusted or estimates of mortality was 0.58; 95% ci: 0.44-0.75 for mers patients who were exposed to a camel in the last 14 days compared to those who were not exposed. other probable risk factors such as gender, exposure to a morbid case of mers in the last 14 days, healthcare worker, and admission in negative pressure isolate room or icu had no significant association with higher mortality (p n 0.05 for all). 2014). mers-cov started in saudi arabia by sporadic infections in mid-2012 and later its outbreak progressed to other countries (alamoudi et al. 2018 ). due to the occurrence of a large number of mers-cov cases and its high worldwide mortality rate, this infection must be considered a public health threat (lessler et al. 2016) . the current study focuses on the epidemiological trend of mers-cov infection and mortality rate analysis of its worldwide cases in the aforementioned dates. the findings of this study may have important implications for the infection control practice and also help to ensure global health security. based on the analysis, the overall global %cfr of mers was 32.0% [451/1408], which is substantially lower than the %cfr in the mers-cov endemic region (table 2) . for example, hunter et al. found an overall cfr of 67% in the abu dhabi (hunter et al. 2016 ) and petersen et al. found an overall cfr of 40% in the kingdom of saudi arabia (petersen et al. 2014 ) however, our estimates were higher than the largest mers outbreak in south korea (cfr of 21%) (cowling et al. 2015) . our analyses of the who data was approximately similar to the cfr of 34% reported by memish et al. (memish et al. 2014) , and also cfr of 30.5% declared by mobaraki et al. (mobaraki and ahmadzadeh 2019a). the regional variation of cfr from previously conducted studies may be skewed due to severity of disease and smaller sample sizes than have been investigated previously. on the whole, the cfr of 32% related to the mers-cov infection in the present study should be considered as a major health concern at the global scale. thus, the characteristics of this disease and the potential risk factors associated with patient fatality should be studied comprehensively. our findings confirm that the mortality pattern of the mers in saudi arabia is different from the observed countries in the middle east and affected countries beyond. by far, the greatest burden of this disease in terms of mortality and morbidity rates is located in four countries including saudi arabia, south korea, united arab emirates, and jordan. in this regards, differences in the virus and the genetic background of the population affected can play a role. other reasons can include a difference in the availability or ability to implement patient isolation procedures as well as differences in overall medical technology among involved countries. consistent with the previous reports, age range n30 years is associated with death in cases of mers-cov infection (alzeer 2009; gautret et al. 2013; memish et al. 2014 ). this finding is in line with a saudi arabian case report series. it showed that the age range of the individuals (n30 years) had a greater association with mortality and per every 1year increase in age, the odds of mortality increased by 12% (majumder et al. 2015) . the reason for the higher fatality rates in this age range is unclear, but they may have underlying diseases and impaired immune functions that exacerbate the symptom of mers-cov infection and increase the chances of death. also, calculating the or (table 3) suggested that having saudi nationality, comorbidity, the interval time of onset sign and admission to the hospital n14 days are other potential risk factors for the disease progression and mortality related to mers-cov infection. although camels are a suspected reservoir, this study could not find a risk relationship in the mortality of patients who had contact with camels in the 14 days prior to clinical signs. meanwhile, global concern rests on the ability of mers-cov to cause major illnesses in direct and indirect contact with camels and its products, namely drinking unpasteurized camel milk (conzade et al. 2018; harrath and abu duhier 2018; kamau et al. 2018) . details as to the specific mechanism of zoonotic transmission from dromedaries to humans remain unclear, and further epidemiological studies are required in this regard. in line with the findings of alghamdi et al. (alghamdi et al. 2014 ), we observed a higher rate of mers-cov incidence in males than females (table 1) . however, based on our findings (table 3) this gender difference in mortality rates related to mers-cov was not statistically significant (aor = 1.24; 95% ci: 0.96-1.60), p = 0.098. the current study suffered from some limitations. of the total worldwide cases (2220 laboratory-confirmed cases of mers-cov), only 1408 cases with complete data were investigated in the current study. it should be noted that from 186 mers-cov cases in south korea, only details related to 57 cases were published in the disease outbreak news on the who website. the lack of complete data for all mers cases potentially increases the occurrence of selection and measurement biases in the result. therefore, it might be more appropriate to conduct further large-scale epidemiological studies with complete data related to all morbid cases of mers to obtain a better understanding of mers-cov emergence in humans and also associated risk factors related of this infection. in the future, we may closely monitor the mers-cov infections globally to better understand the risks of this new infection for public health and to provide helpful recommendations for controlling and preventing it. recommendations might change and be updated as additional data becomes available. indeed, despite the above limitations, such studies might be useful to implement educational programs, and access health care for early diagnosis and prevention of modifiable factors to reduce high mortality rates associated with mers-cov. based on our analyses of the who data, 7 years after the emergence of the mers-cov incidence; saudi arabia still has the highest rate of infection. this study estimated a global 32% cfr (95% ci: 29.4-34.5) for mers patients. the results demonstrated a link between mortality and some risk factors such as age n30 years old, saudi nationality, comorbidities, the interval time of onset sign and the admission to the hospital n14 days. unlinked data. all authors express their satisfaction with the publication of this paper. this project was funded by the urmia university of medical sciences (grant no. ir.umsu.rec.1398.84). the funding bodies had no role in study design, data collection, analysis, preparation of the manuscript, or the decision to publish. the data used for the analysis can be obtained from the study authors. we thanks to the urmia university of medical sciences for supporting this study (grant no. 1397-06-44-1834) . we also appreciate all reporting countries with the confirmed mers cases for investigations, data collection and sending of it to the who. the authors would like to acknowledge the miss gazhal akhavan masoumi for editing this paper. preliminary epidemiologic assessment of mers-cov outbreak in south korea no molecular evidence of mers-cov circulation in jeddah, saudi arabia between 2010-2012: a single-center retrospective study the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus respiratory tract infection during hajj hospital outbreak of middle east respiratory syndrome coronavirus role of severe acute respiratory syndrome coronavirus viroporins e, 3a, and 8a in replication and pathogenesis mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia hospital-associated middle east respiratory syndrome coronavirus infections bat origin of human coronaviruses transmission of middle east respiratory syndrome coronavirus infections in healthcare settings knowledge and practices regarding middle east respiratory syndrome coronavirus among camel handlers in a slaughterhouse an update to middle east respiratory syndrome coronavirus and risk of a pandemic in 2019 estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia mortality risk factors for middle east respiratory syndrome outbreak, south korea family cluster of middle east respiratory syndrome coronavirus infections etiology of severe community-acquired pneumonia during the 2013 hajj-part of the mers-cov surveillance program current epidemiological status of middle east respiratory syndrome coronavirus in the world from 1.1. 2017 to 17.1. 2018: a cross-sectional study an update to middle east respiratory syndrome coronavirus and risk of a pandemic in 2019 hospital outbreaks of middle east respiratory syndrome health-care associate transmission of middle east respiratory syndrome corona virus, mers-cov, in the kingdom of saudi arabia middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 world health organization. emergencies preparedness, response:mers-cov.disease outbreak news middle east respiratory syndrome the authors declare that they have no competing interests. key: cord-268483-joiajgs4 authors: shah, vibhuti kumar; firmal, priyanka; alam, aftab; ganguly, dipyaman; chattopadhyay, samit title: overview of immune response during sars-cov-2 infection: lessons from the past date: 2020-08-07 journal: front immunol doi: 10.3389/fimmu.2020.01949 sha: doc_id: 268483 cord_uid: joiajgs4 after the 1918 flu pandemic, the world is again facing a similar situation. however, the advancement in medical science has made it possible to identify that the novel infectious agent is from the coronavirus family. rapid genome sequencing by various groups helped in identifying the structure and function of the virus, its immunogenicity in diverse populations, and potential preventive measures. coronavirus attacks the respiratory system, causing pneumonia and lymphopenia in infected individuals. viral components like spike and nucleocapsid proteins trigger an immune response in the host to eliminate the virus. these viral antigens can be either recognized by the b cells or presented by mhc complexes to the t cells, resulting in antibody production, increased cytokine secretion, and cytolytic activity in the acute phase of infection. genetic polymorphism in mhc enables it to present some of the t cell epitopes very well over the other mhc alleles. the association of mhc alleles and its downregulated expression has been correlated with disease severity against influenza and coronaviruses. studies have reported that infected individuals can, after recovery, induce strong protective responses by generating a memory t-cell pool against sars-cov and mers-cov. these memory t cells were not persistent in the long term and, upon reactivation, caused local damage due to cross-reactivity. so far, the reports suggest that sars-cov-2, which is highly contagious, shows related symptoms in three different stages and develops an exhaustive t-cell pool at higher loads of viral infection. as there are no specific treatments available for this novel coronavirus, numerous small molecular drugs that are being used for the treatment of diseases like sars, mers, hiv, ebola, malaria, and tuberculosis are being given to covid-19 patients, and clinical trials for many such drugs have already begun. a classical immunotherapy of convalescent plasma transfusion from recovered patients has also been initiated for the neutralization of viremia in terminally ill covid-19 patients. due to the limitations of plasma transfusion, researchers are now focusing on developing neutralizing antibodies against virus particles along with immuno-modulation of cytokines like il-6, type i interferons (ifns), and tnf-α that could help in combating the infection. this review highlights the similarities of the coronaviruses that caused sars and mers to the novel sars-cov-2 in relation to their pathogenicity and immunogenicity and also focuses on various treatment strategies that could be employed for curing covid-19. the whole world is currently confronting a crisis situation that first appeared in late december 2019 as merely a few cases of pneumonia in wuhan, china. the patients were exhibiting common symptoms like fever, dry cough, sore throat, breathlessness, and fatigue. sample swabs from the oral cavity and anal region were collected along with the blood and bronchoalveolar lavage fluid (balf) from all seven of the patients, irrespective of their age and gender, which were then sent to the wuhan institute of virology for further examination. as the outbreak initiated at the seafood market with the onset of winter, similar to that of the previous severe acute respiratory syndrome (sars) infection, the scientists first screened the samples using pan-cov qpcr primers. surprisingly, five samples were reported positive for coronavirus. thorough investigation employing next-generation sequencing and phylogenetic analysis led to the identification of the causative agent of this respiratory disease, a novel coronavirus (2019-ncov) (1) . as more cases started to appear around the world, on february 11, 2020, the world health organization assigned a name, corona virus disease 2019 or covid-19, to the disease and declared it a pandemic on march 11, 2020. the virus was renamed from 2019-ncov to sars-cov-2 by the international committee on taxonomy of viruses on the basis of its genetic similarity to a previously known coronavirus, severe acute respiratory syndrome coronavirus (sars-cov) (2) . transmission of sars-cov-2 occurs when a healthy individual inhales or comes into contact with respiratory droplets from an infected person. the average incubation period before patients exhibit disease symptoms ranges from 2 to 14 days (3) . before the spread of covid-19, sars emerged as an epidemic in 2003, followed by middle east respiratory syndrome (mers) in 2012, both caused by a novel coronavirus of zoonotic origin and assigned to the genus betacoronavirus (4) . the worldwide outbreak of sars-cov-2 has put life on hold, having a major impact on the world's economy, and has claimed ∼436,167 lives globally as of june 15, 2020 (5, 6) . unlike previous episodes of coronavirus spread, where it took months to identify the cause of infection and perform genome sequencing (7) , advancement in science and technology made it possible to identify the causative organism swiftly. within a few weeks of the outbreak, different laboratories across the world had sequenced the whole viral genome and had also provided structural and functional insights into the essential proteins required by the virus for its survival. these immediate scientific inputs helped with developing diagnostic kits and defining treatment strategies for effective prognosis and prevention (8) (9) (10) . in this review, we are emphasizing the immunological aspect of sars-cov-2 pathogenesis by taking into consideration the previous experimental and clinical knowledge obtained from the coronaviruses that were responsible for causing sars and mers. this approach will assist in utilizing immunotherapies, repurposing the previously approved antiviral drugs, and developing therapeutic vaccines specific to novel coronavirus more effectively. initial genome sequencing and phylogenetic analysis of novel coronavirus sars-cov-2 has shown that it is genetically similar to previously known coronavirus sars-cov and hence is placed under the family coronaviridae. coronavirus contains positivesense single-stranded rna (+ve ssrna) as its genetic material, which can be about 30 kb in length and is mostly protected by an outer fatty layer of an envelope that also helps the virus to evade host immune response and assists its entry inside the host cell (11, 12) . the subfamily coronavirinae is further subdivided into four genera, namely alpha-, beta-, gamma-, and delta-coronavirus (α-cov, β-cov, γ-cov, and δ-cov). viruses having the potential to infect humans are placed under the genus α-cov and β-cov (sars-cov & mers-cov), whereas viruses of γ-cov and δ-cov genera are mostly known to infect avians and pigs (13) . the novel coronavirus, sars-cov-2 falls under the genus β-cov, as it shares 88% sequence identity with sars-cov-like coronaviruses (derived from bat) but is only 79% identical to sars-cov and 50% identical to mers-cov (3) . thus, it can be deduced by its genome identity that the immediate host of this virus could be a bat, which then transmitted it to some unknown intermediate host that acted as a source for the transmission of the virus to humans. like those of sars-cov and mers-cov, the sars-cov-2 genome comprises of 12 open reading frames (orfs) in number. at the 5 ′ end of the viral genome, overlapping orfs 1a and 1b are present that encode the rna polymerase and other nonstructural proteins of the virus and occupy approximately twothirds of the genome. genes encoding structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n), are present in the remaining one-third of its genome spanning from the 5 ′ to the 3 ′ terminal, along with several genes encoding non-structural proteins (nsps) and accessory proteins scattered in between, as shown in figure 1 . despite being in the same serogroup, there is a slight difference in the nucleotide number, sequence, gene order, and expression method among previously known coronaviruses and the novel sars-cov-2 (1, 14, 15) . recent reports highlight that a few amino acid substitutions have occurred in the novel coronavirus genes encoding the s protein, nsp2, nsp3, and receptor-binding domain (rbd). these mutations in the nsp2 & nsp3 are also believed to impart the enhanced infection abilities of the novel coronavirus (16, 17) . rna viruses are prone to acquiring genetic mutations that eventually help them to escape the host immune system and develop drug resistance. researchers have also found minor mutations in sars-cov-2 genotype in different covid-19 patients (18) . one such hotspot of mutation in the sars-cov-2 genome is the rna-dependent rna polymerase gene. on analyzing 220 sequences across the globe, eight repetitive novel point mutations were observed. viral genetic sequences accessed from europe exhibited five mutation hotspots, whereas the remaining three point mutations were solely present in the sequences from north america. these unique mutations suggest that the viral strains are continuously evolving across the globe figure 1 | schematic representation of the coronavirus structure and genomic comparison of coronaviruses. (a) representation of coronavirus showing different components of the particle, which is 100-160 nm in diameter. the single-stranded rna (ssrna) genome, covered with the envelope and membrane proteins, gains access into the host cell and hijacks the replication machinery. (b) the ssrna of sars-cov-2 is about 30 kb and has similarities with the genomes of sars-cov and mers-cov. translation of this ssrna results in the formation of two polyproteins, namely pp1a and pp1ab, that are further sliced to generate numerous non-structural proteins (nsps). the remaining orfs encode for various structural and accessory proteins that help in assembly of the viral particle and evading immune response. and that the strains from europe, north america, and asia might have co-existed the whole time (19) . another similar report analyzed 7,666 global viral genomic sequences and found 198 unique mutation sites on sars-cov-2 genome that encodes nsps and s protein, suggesting that the virus is trying to adapt to its new host (20) . as numerous drugs are currently being designed to target the proteins that are essential for the survival of the virus, rapid genetic mutation occurring in these proteins might not prove to be a potential candidate for drug design. therefore, the invariable region of the virus could be a better target to avoid drug failures. interestingly, sars-cov-2, similar to sars-cov, exploits the angiotensin-converting enzyme 2 (ace2) receptor to gain access inside human cells, whereas mers-cov binds specifically to dipeptidyl peptidase 4 (dpp4) receptor (21, 22) . binding of the virus particle to the specific receptor on the host cell plays a key role in governing its pathogenicity. functional evaluation was carried out to reveal the potential receptors for different betacoronaviruses (β-cov) including sars-cov-2, and it was found out that the entry of the virus particle was enhanced in human cells expressing ace2 receptor instead of dpp4 or aminopeptidase n (apn) in the case of the novel coronavirus (23) . recent structural insights provided by cryo-em studies of s protein in prefusion conformation highlighted that the binding efficiency of ace2 and s protein of sars-cov-2 is 10-20 times greater than for the previously known sars-cov (24, 25) . the latest reports suggest that the trimeric s protein of sars-cov-2 is sliced by transmembrane protease serine 2 (tmprss2), similar to sars-cov (26, 27) . hence, profound knowledge of the potential receptors to which the virus particle can bind and its associated proteases will help us in designing specific antiviral drugs and neutralizing antibodies and will lead us to foresee whether particular coronaviruses of zoonotic origin could be able to adapt and infect humans. all coronaviruses initiate entry inside the target cell by engaging the host receptor with the s glycoprotein present on their surface so as to gain entry inside the target cell. the region of s protein containing the rbd is present on the s1 subunit. in a few coronaviruses, rbd is present at the n-terminus region of s1, whereas in sars-cov, it is situated at the c-terminus region (28, 29) . the fusogenic activity of virus-cell membrane is governed by two tandem domains, heptad repeats (hr1,2) that are present on the s2 region of s protein (30, 31) . initially, it was believed that sars-cov enters the target cell merely by virtue of cell membrane integration of virus particle and host cell membrane (32) . later, it was discovered that an essential proteolytic cleavage event takes place in the s protein at the s2 position of sars-cov that results in membrane fusion and facilitates virus entry inside the cell (33) . once the coronavirus is inside the host cell via membrane fusion, it releases its +ve ssrna genome into the cytoplasmic compartment, where the translation of orf-1a and orf-1b begins resulting in the formation of two large polyproteins (pp1a and pp1ab). three functional proteases then cleave the polyproteins into 16 non-structural proteins (nsp1-16), which eventually create the viral rna polymerase and other accessory proteins for virus assembly (34) (35) (36) . an uninterrupted replication-transcription event results in the formation of various nested sets of subgenomic (sg) mrnas that eventually translate into numerous structural and accessory proteins (37) . the e glycoproteins after synthesis are incorporated into the rough endoplasmic reticulum or golgi membrane. the +ve ssrna combines with capsid protein to form the nucleocapsid, followed by budding of assembled virus particles in the er-golgi intermediate compartment (ergic) (38) . lastly, the virus particle-loaded vesicles are fused with the cell membrane for effective shedding of the virus (4). these new virions are now accessible to infect the neighboring healthy cells and are also released into the surrounding environment via respiratory droplets that are highly contagious and hence potentially spread the disease to healthy individuals. the path followed by sars-cov-2 to reach the lungs is via the naso-oral cavity. once the virus is inhaled, it enters the epithelial cells of the nasal cavity by engagement of ace2 receptor with the viral rbd and initiates its replication (27, 39, 40) . this initial asymptomatic phase lasts for about 1-2 days, during which the virus multiplies in the upper respiratory tract, where no major hindrance is caused by the innate immune cells. within 2-14 days of initial encounter, the common symptoms of covid-19 start to appear, which are similar to those of sars and mers, i.e., fever, dry cough, pharyngitis, shortness of breath, joint pain, and tiredness. numerous problems arise during this phase of the disease, including nosocomial and fomite transmission of infection, which enhances the chances of community spread (41) . soon, the virus begins to move toward the lower respiratory tract via airways, and this triggers a strong innate immune response. patients at this stage start exhibiting enhanced pro-inflammatory response that leads to viral sepsis accompanied by other complications, including pulmonary edema, acute respiratory distress syndrome (ards), different organ failures, and death in the worst scenarios (42) . the infected individuals rarely show the intestinal symptoms like diarrhea that were evident in other coronavirus infections. patients are recommended to be quarantined to prevent community spread of this pandemic virus (43) . the severity of covid-19 has been found to be greater in aged individuals and in people with a health history, such as those immune-compromised by hiv infection or by chemotherapy for cancer. diabetic and asthma patients, along with individuals with hypertension, obesity, or heart, kidney, or liver disorders, are also at higher risk if they acquire the disease (44) . autopsy reports of individuals who died due to sars show multi-organ dysfunction, with the highest viral titers in the lungs and immune cells in circulation, thus damaging the pulmonary and immune system (45, 46) . as opposed to adults, only a very small population of children has been infected with sars-cov-2. in one study, the symptoms displayed by children above 15 years were found to be milder as compared to those of younger children, who showed severe symptoms but with rare deaths and better prognosis (47) . the study speculated two major possibilities related to covid-19 severity in children among different age groups. one of these rests on the finding that ace2 activity is higher in children aged 4-13 years; after this age, it starts to decline until adolescence. this could be one of the reasons why lung fibrosis is observed mainly in younger children. secondly, differential cd4 + and cd8 + t cell populations have been seen in children as compared to adults (48, 49) . a large number of clinical and epidemiological criteria were defined to assess probable pediatric cases of covid-19 (50) . a preliminary report from a cross-sectional study of children admitted to us and canadian pediatric intensive care units (picus) during march 14-april 3, 2020, revealed that the 48 children were admitted in the usa whereas no covid-19 cases were reported in canadian picus. the study revealed that there are fewer covid-19 cases in children as compared to adults and that there is a median picu time of 5 days (51). a recent preprint from paris reports that 11 children (age 3.7-16.6) were admitted experiencing symptoms similar to kawasaki disease (kd) along with gastrointestinal issues and elevated inflammatory markers. further investigation suggested that they were also sars-cov-2-positive, speculating that this could be the reason for kd shock syndrome (52) . similar cases have been observed in new york, where four otherwise healthy sars-cov-2-positive children started displaying symptoms similar to kd and toxic shock syndrome, thereby needing intensive care (53) . therefore, medical practitioners should be prepared to tackle such sudden post-infection complications to avoid the associated risks. once the virus gains access inside the target cell, the host immune system recognizes the whole virus or its surface epitopes, eliciting the innate or adaptive immune response (figure 2 ). pathogen recognition receptors (prrs) present on immune cells, mainly toll-like receptors 3, 7, and 8, are the first to identify the virus, which leads to enhanced interferon (ifn) production. the function of host innate immune cells is impaired during sars-cov and mers-cov infection by their non-structural proteins, which affects the overall cytokine production (54) (55) (56) . humoral response against sars-cov-2 has been found to be similar to that against other coronavirus infections, involving the characteristic igg and igm production. at the onset of sars-cov infection, b cells elicit an early response against the n protein, while antibodies against s protein could be detected after 4-8 days from the appearance of initial symptoms (57, 58) . although n protein is smaller than s protein, it is highly immunogenic, and the absence of glycosylation sites on it results in n-specific neutralizing antibody production at an early stage of acute infection (59) . sars-cov-specific iga, igg, and igm antibodies were detected after the onset of symptoms at different time points in infected patients. a persistent level of igg was detected for a longer period, whereas igm levels started to decline after 3 months (60, 61) . in an observational case study of 16 sars-cov-2 patients, anti-s-rbd igg was detected in all of the subjects, whereas anti-n igg and anti-s-rbd igm were detected in 15 patients and anti-n igm in 14 patients (62) . an elisa-based time kinetics study to detect the covid-19 specific humoral immune response showed that the patients produced igm and igg antibodies that did not cross-react with other human coronaviruses except sars-cov. igm and iga antibodies were detected 5 days after the onset of initial symptoms, whereas igg was detected after 14 days (63) . another kinetic study of viral shedding and antibody detection was published in a preprint and reported the presence of higher igg and igm antibody titers in severe patients. they also observed that weak responders for igg antibody had higher viral clearance than strong responders. this observation suggests that robust antibody response leads to disease severity while feeble response is associated with the elimination of virus (64) . a case study on pediatric patients reports that 5 out of 6 children showed a protective humoral response, with neutralizing igg and igm antibodies targeting the n and s-rbd proteins of sars-cov-2 (65) . these studies propose that igm-based elisa can be used for early diagnosis of patients along with qpcr techniques to improve the sensitivity and specificity of the technique. in addition to neutralizing antibodies, which are defensive and useful, there are numerous non-neutralizing antibodies in the system that aid the infection of immune cells and apcs. previously existing sars-cov antibodies may promote the viral infection in fcr-expressing cells (66) . this ace2-independent pathway of viral entry does not result in viral replication; rather, viral shedding by macrophages enhances inflammation and tissue injury by myeloid cell activation. this mechanism of viral entry through non-neutralizing antibody that results in aberrant activation of immune cells is called ade (antibody-dependent enhancement) (66, 67) . ade has been observed in a number of viral infections, including sars and mers. in the case of sars, anti-s antibodies were observed to be involved in ade to gain entry into fcr-expressing cells (68) , while in mers, a neutralizing mab (mersmab1) targeting rbd aided in mers pseudo-virus entry via the dpp4 pathway (69) . although there is no clear evidence regarding ade in sars-cov-2 infection, it is still necessary to consider all of the odds in the pursuit of developing vaccines and treatment regimens involving antibodies (70) . during viral infection, t cells also recognize the viral antigens presented by mhc class i [mhc; human leukocyte antigen (hla) in humans], which in turn promotes the cytokine release and cytotoxic activity of cd8 + t cells (71) . but in some other cases, mhc class ii is also found to present sars-cov peptides to cd4 + t cells. due to the genetic polymorphism of hla, some haplotypes, like hla-b * 07, hla-b * 46, hla-drb1 * 12 (72) , and hla-cw * 08 (73) , are found to be more susceptible to coronavirus infection, whereas the hla-drb1 * 03, hla-a * 02, and hla-cw * 15 haplotypes are protected from sars-cov infection (74) . similarly, hla-drb1 * 11 and hla-dqb1 * 02 were found to be vulnerable to mers-cov infection (75) . additionally, mhc expression is also found to be reduced during the infection due to epigenetic modifications of downstream molecules (76, 77) . so far, hla association is not very wellidentified for sars-cov-2 infection, and this could be crucial for the prevention and treatment of covid-19. however, in a recent report, blood plasma from covid-19 patients was able to block the expression of hla-dr on cd14 + monocytes, which was restored effectively on inhibiting il-6, suggesting that decreased hla-dr expression in sars-cov-2 patients is due to the buildup of hyper-inflammatory conditions (78) . decrease in mhc expression is also evident in cancer cells, which is a mechanism by which they evade the immune response by epigenetically modifying calnexin promoter. but infection with influenza virus in these cancer cells results in enhanced mhc-i presentation due to the increased expression of chromatin remodeling proteins, which stabilizes p53 expression and hence augments the immune surveillance of cancer cells (79) . therefore, molecules that can upregulate chromatin regulators and increase the mhc-i expression could potentially be used for covid-19. most of the t-cell epitopes presented by mhc complex are derived from structural proteins such as the s and n proteins of the coronavirus in both humans and animal models, while the nsps have regulatory effects on the signaling cascade (80, 81) . t cells can be stimulated by 14 epitopes, most of which are observed to be located on orf3 and the s protein in sars patients (61) . in a large cohort study during sars-cov infection, s protein was the only immuno-dominant epitope for cd8 + t-cell activation (61) , whereas, in mers, cd8 + response was against the s and n proteins along with some of the m/e epitopes (82) . these t-cell epitopes have been tested in animal models by assessing the lung pathology and t-cell response upon infection in balb/c and c57bl/6 mice (80, 83). the sequence of sars-cov-2 being more similar to sars-cov than to mers-cov, with no mutation in 19 epitopes, provides a prospective subunit vaccine for stimulating a strong t-cell response in covid 19 patients (84) . in a recent study, samples from 20 convalescing covid-19 patients were analyzed to check the development of adaptive immune response during infection. the results highlighted that helper t cells were eliciting a robust immune response against s, m, and n protein. the effect of adaptive immune response on humoral immunity was also compared, where a strong cd4 + t-cell response against sars-cov-2 eventually resulted in an increase in anti-s-rbdspecific igg and iga antibody titer. along with cd4 + t cells, immunogenic epitopes on s, m, and n proteins were also able to activate cd8 + t cells. however, such t-cell response was not specific to recovered patients only but was also present in 40-60% of the individuals who were not exposed to sars-cov-2. further analysis showed that they had pre-existing cross-reactive cd4 + t cells, which might have been generated in response to some previous coronavirus infection. hence, these t-cells could impart protective immunity in such individuals against sars-cov-2 to some extent (85) . these epitopes could be a promising factor in developing immunotherapy by small molecules that can increase the presentation of viral epitopes. a rapid and coordinated immune response during viral infection leads to enhanced secretion of various cytokines, which acts as a defense mechanism against the virus. numerous reports suggest that individuals affected with sars-cov or mers-cov have dysregulated cytokine production from both innate and adaptive immune cells. in the case of sars, infected hematopoietic cell, monocyte-macrophages, and other immune cells trigger enhanced secretion of pro-inflammatory cytokines like tnf-α, il-6, and ifn-α/-γ, with reduced anti-inflammatory cytokines (86) (87) (88) . similarly, mers-cov infection leads to delayed but increased production of ifn-α and pro-inflammatory cytokines like il-6, il-8, and il-1β (89) (90) (91) . such elevated levels of cytokines were associated with multi-organ dysfunctional syndrome (mods) and ards due to the accumulation of numerous immune cells like macrophages, neutrophils, and dendritic cells in the lungs causing alveolar damage and edema (56, 92, 93) . similarly, in covid-19 patients, secretion of cytokines and chemokines, which attract the immune cells to the lungs, was increased, hence causing ards, which is fatal to critically ill individuals (94, 95) . signature cytokines in severely ill covid-19 patients were consistent with those in sars and mers, i.e., enhanced expression of il-6, tnf-α, macrophage inflammatory protein 1-α (mip-1α), mcp3, gm-csf, il-2, and ip-10 along with elevated chemokines (ip-10, ccl2/mcp1, cxcl1, cxcl5) were also detected in sars-cov-2 infection (96) (97) (98) (99) . in children, the increased inflammatory markers include il-6, il-1, and c-reactive protein along with procalcitonin in serum (52) . in a case study, a 14-year-old child with cytokine storm was treated with anakinra (il-1 receptor antagonist) in order to stabilize the respiratory illness and other clinical symptoms (100) . transcriptomic analysis of pbmc and balf showed that a number of immune regulators were upregulated, particularly cxcl10, with respect to balf. this study also reported that several apoptotic genes and p53 signaling molecules were upregulated, suggesting a possible reason for lymphopenia in these patients (101) . therapeutic measures to control such cytokines involve neutralizing antibodies or small molecular drugs that can stop the signaling cascade for cytokine production. the most potent antiviral machinery acquired by immune cells is the secretion of interferons that act as secondary messengers stimulating the neighboring cells. most innate immune cells are efficient in producing ifns that are involved in obstructing cell proliferation, apoptosis, and immunomodulation (54, 102) . as an escape mechanism, sars-cov or mers-cov uses several ways to overcome the host immune response, one of which is by severe leukopenia and lymphopenia (103) (104) (105) . after gaining entry to the cell, these viruses encode different proteins that interact with downstream signaling molecules of tlrs and the jak-stat pathway. mers-cov encoded matrix protein, accessory proteins from orf 4a, 4b, and 5, which directly inhibits the ifn promoter and nuclear localization of irf3 (106) . plpro, encoded by sars-cov and mers-cov, prevents the dissociation of nf-κb from iκbα, whereas nonstructural proteins of sars-cov, i.e., plpro and orf3b, inhibit irf3 phosphorylation and hence its translocation to the nucleus (4, 107, 108). these viral accessory proteins also inhibit the jak-stat pathway, resulting in inhibition of genes by isre promoters (109) (110) (111) (figure 3) . a new investigation revealed that sars-cov-2 infection leads to an overall decrease in the transcription of antiviral genes because of the lower production of type i and iii interferons with sufficient isg expression, along with elevated chemokine secretion. results obtained from in-vivo and ex-vivo covid-19 experiments were in tune with the in-vitro findings. therefore, a decrease in the innate antiviral response, along with hyper-inflammation, could be one of the causes of covid-19 severity (112) . in addition to reduction in t cells, sars-cov-2 infection also enhances the exhaustion of effector t cells, decreasing the immune response against the virus (94, 113) . exhaustion and loss in function of effector t cells is the result of increased expression of inhibitory receptors like pd-1, tim-3, and tigit on its surface as a result of cytokines like il-6, il-10, and tnf-α or by decreasing the regulatory t-cell population (114, 115) . following viral/antigen clearance, most of the effector t cell undergoes apoptosis in the contraction phase. subsequently, a pool of memory t cells are generated that are programmed to fight against re-infection. cd4 + memory t cells, upon restimulation, trigger b cells and other immune cells by cytokine production, while cytotoxic memory t cells help in destroying the infected cells during subsequent infection (116, 117). case studies in recovered sars patients showed that both cd4 + and cd8 + memory t cells were efficient in eliciting immune response from 3 months to 6 years without the presence of any antigens (118) . in a case study of 23 recovered sars-cov patients, the patients showed very low frequencies of memory b cells, while memory t cells elicited a response against the s protein in 60% of recovered individuals (119) . considering the memory t-cell subset, n-specific helper t cells had more of central memory markers (cd45ra − , ccr7 + , cd62l − ) while the cd8 + t cell population had the effector memory (cd45ra + , ccr7 − , cd62l − ) phenotype in a steady-state manner (120) . the study suggests that an effective vaccine or t cell epitopes could be used to target a particular population for rapid viral clearance. in recent reports, covid-19 subjects have shown reduced regulatory t cell populations and memory t cells, which may aggravate the inflammatory response leading to cytokine storm and hence enhance the tissue damage and organ failure (114) . in a mouse model, the use of cd4 + memory t cells as a vaccine by the intranasal, but not the subcutaneous, route imparted a protective response against the human coronavirus. the infused cd4 + memory t cell, upon re-stimulation, produces ifn-γ and recruits cd8 + t cells for rapid clearance in response to sars-s366 peptide (121) . recently, a human ace-2-expressing mouse model has been developed by crispr/cas9 technology that recapitulates the human symptoms upon infection with sars-cov-2 through the intra-nasal route. this tool will be beneficial for evaluating the efficacy of vaccines for covid-19 and also to study its transmission and pathogenesis (122) . just like sars and mers, there are no specific clinically approved drugs available for covid-19 as of june 15, 2020 (123) . currently, the treatment regime focuses mainly on providing intensive care in order to alleviate the symptoms and discomfort associated with covid-19. conservative fluid therapy accompanied by broad-spectrum antibiotics are also given to the patients as a protective measure to avoid opportunistic bacterial infections. however, ventilator support for respiration is provided to the patient under extreme conditions (124) . numerous fda-approved antiviral drugs, vaccines, and immunotherapies that are already being used to treat other diseases have also been considered as a possible approach for treating covid-19 ( table 1) . but this approach may reduce the availability of these drugs and vaccines for the intended diseases and for the patients with the greatest need. the molecular, structural, and functional relationships of lopinavir viral protease involved in immature, noninfectious hiv virus particle, and inhibits plpro or 3clpro in sars-cov-2. favilavir viral rna polymerase purine analog blocking viral rna synthesis. remdesivir (141) ribavirin guanosine nucleoside binds to nucleoside binding pocket of the enzyme. (133, 140, 142) galidesivir adenosine analog, effective against ebola, zika, and other rna viruses. chloroquine/hydroxychloroquine heme polymerase and ace2 increases endosomal ph and terminal glycosylation of ace2, inhibiting sars-cov-2 entry. (144, 145) nitazoxanide glutathione-s-transferase alters ph and inhibits viral maturation. reported against tb, helminthic, and protozoan infection. umifenovir/arbidol n/a interacts with aromatic residues of viral glycoproteins. is being trialed for prophylactic action against covid-19. sars-cov-2 with sars-cov might define the use of existing anti-viral drugs against covid-19 (147, 148) , considering the total time it takes to perform clinical trials and get fda approval for the use of novel drugs and vaccines. the increasing knowledge of the genetic, immunological, and molecular mechanisms behind its enhanced pathogenicity might help in developing specific treatment approaches for covid-19 in the future. considering the studies on the molecular mechanism of coronavirus infection (147) , several antiviral drugs could be repurposed for the treatment of covid-19. remdesivir is a nucleotide analog that acts as an antiviral agent for a wide variety of viruses and has been tested widely against previous epidemics of coronavirus infections in both in-vitro and in-vivo models (138, (149) (150) (151) . this adenosine analog gets incorporated into the newly synthesized viral rna, which inhibits the addition of further nucleotides by viral rnadependent rna polymerase and hence terminates the ongoing transcription. administration of intravenous remdesivir was found to be effective in treating the first known patient of covid-19 in the usa (152) . a randomized double-blinded clinical trial on 1,059 adult hospitalized covid-19 patients was sponsored by the national institute of allergy and infectious diseases, usa, to further test the potency of intravenously administered remdesivir. the preliminary outcomes of the trial reported that remdesivir treatment decreased the median recovery time in the treatment group (11 days) as compared to the placebo group (15 days). the mortality rate was also less in the treatment group (7.1%) in contrast to the placebo group (11.9%) (153) . numerous clinical studies, similar to this, are required so as to validate the proposed drugs for covid-19. favipiravir, ribavirin, and galidesivir are also potential nucleoside analogs that might be useful against novel coronavirus infection (154) . the combinatorial therapy approach of using remdesivir along with chloroquine, a well-known anti-malarial drug, has also been tested in vitro so as to study its effectiveness against sars-cov-2 (141, 155) . it has been reported that chloroquine immuno-modulates the host microenvironment and also interferes with the replication of the virus and its interaction with the receptor (156, 157) . in a randomized clinical trial (nct04308668) involving 821 asymptomatic individuals across the us and canada who had come into close contact with potential covid-19 patients, the individuals were given either hydroxychloroquine or placebo as a prophylactic measure. the results revealed that hydroxychloroquine treatment had the same effect as did the placebo group. the usage of hydroxychloroquine resulted in minor side effects (40.1%) as compared to the placebo treatment (6.8%). however, no cardiovascular disorder or treatment-related major complications were observed (158) . based on the putative function of hydroxychloroquine on the endosomal acidification, whereby it is presumed to hinder viral uncapping, it can be observed that it has a great potential for prophylaxis, not to prevent infection but to reduce effective viral load in patients and thus lead to milder disease. numerous clinical trials to further explore the usage of hydroxychloroquine in different combinations are in the pipeline and will finally provide a better understanding of the efficacy of this drug for covid-19. a few anti-hiv drugs, such as lopinavir/ritonavir in combination with interferon beta (ifn-β), have been tested in vivo for treating coronavirus infections (sars-cov, mers-cov) and have also been used in the case of covid-19 (138, 139, 159) . various complementary therapies could also be employed as a preventive measure against viral infections. many essential proteases, such as chymotrypsin (3c-like protease) and plpro, which are required by coronavirus for completing the replication process, can also be targeted using drugs. cinanserin, flavonoids, and some small molecules are known to inhibit 3clpro, whereas diarylheptanoids are used to inhibit plpro (160) (161) (162) . in a recent study, 16 potential anti-hcov drugs were identified through a systems biology-based approach, such as melatonin, mercaptopurine, sirolimus, dactiomycin, and toremifene, which are to be tested further for their potency (163) . in the absence of any dependable vaccine or drugs with tested efficacy and when the pandemic onslaught is ongoing, a worthy therapeutic approach is passive immunization using purified antibodies. the source of such antibodies could be the sera of convalescing individuals, mabs, or genetically modified antibodies from an animal host, which can efficiently neutralize the virus. this is an age-old practice, with pioneering work having been done by the nobel laureate, emil behring, who applied this approach for diphtheria, and has been used whenever there are sudden outbreaks of viral diseases like sars, mers, h1n1, h5n1, ebola, and many others (61, 164, 165) . as opposed to active vaccination, plasma therapy is the only means to provide immediate immunity for viral clearance, as in the case of sars-cov-2. as in other epidemic diseases, convalescent sera are currently being employed for covid-19 in a number of countries (166, 167) . although a randomized controlled trial is yet to be reported, limited studies in 10 patients have been documented with no remission of severe respiratory afflictions on receiving neutralizing antibodies from 39 convalesced donors with antibody titers of 1:160, along with drugs and oxygen support (168) . a report from hong kong suggested that this therapy had poor outcome in sars patients, with a number of limitations in their study (169) . as with transfusion of any blood products, precautionary screening of infectious agent is warranted in plasma transfusion. recently, the fda in the usa has approved trials of convalescent plasma therapy in covid-19 under specific guidelines; plasma donation is advised 3 weeks after a patient becomes virus-negative on pcr. the major challenge in this therapy is obtaining donors with similar blood antigens with a high antibody titer of sars-cov-2 (170) . another potential adverse effect of this approach is ade of infection, which is common in so many other viruses. but, to date, the incidence of ade has not been reported in the case of sars-cov-2. another major point of contention is the selection of patients for this therapeutic approach. in most clinical trials, patients with severe diseases are being recruited, while the presumed mechanism of action of convalescent plasma, based on its content of virus-neutralizing antibodies, rather points to plausible favorable outcomes in earlier phases of the disease because in the later, more severe phases, the hyperimmune response, rather than the viral load, becomes the more critical pathology. finally, there are no available data on the heterogeneity of response to convalescent plasma transfusion, which may further illustrate the importance of careful evidencebased patient selection, as heterogeneity of response may result from both virus and host-intrinsic factors which are, to date, not revealed. researchers around the world are working hard to develop a potential vaccine candidate so as to stop the deadly pandemic caused by sars-cov-2. however, vaccine development is not an easy task, as a number of successful clinical trials are required before approval for patients. different approaches are being utilized for designing a specific vaccine targeting either the structural proteins or viral replication process, which eventually results in the inhibition of viral growth and its further transmission. the common strategies involve the use of live attenuated vaccine (lav), inactivated virus, subunit vaccines, monoclonal antibody vaccine, virus vectors, protein vaccines, and dna/rna-based vaccines (171) (172) (173) (174) . there are numerous subunit vaccines targeting all or a part of s protein that have already been tested for sars and mers in animal models (175) and could be potential candidates for testing against sars-cov-2. a recent pilot study with a purified inactivated sars-cov-2 virus vaccine displayed very promising outcomes in different animal models. the neutralizing antibodies generated after vaccination were able to effectively target 10 different strains of sars-cov-2 without developing any ade of infection (176) . various randomized controlled trials (nct04327206, nct04328441) are also underway to evaluate the effectiveness of the bcg vaccine against sars-cov-2 for healthcare professionals. an adenovirus vector-based vaccine candidate, chadox1 (presently azd1222), developed by oxford university (licensed to astrazeneca) for use against sars-cov-2 has been reported to activate both the humoral and cell-mediated immune response when tested in rhesus monkey (177) . the phase i clinical trial to confirm its potency is also in progress (nct04324606). another group has followed a similar approach by using a recombinant adenovirus type 5 (ad5-ncov) vectorbased vaccine for covid-19. the full report from the phase i clinical trial (nct04313127) of ad5-ncov shows that it is very effective in generating both humoral and rapid t-cell response post immunization. the group is now ready for the next clinical trial phase to further strengthen the effectiveness of the ad5-ncov vaccine (178) . it should be noted that there are potential risks associated with the usage of live attenuated viruses, for example, complications resulting in lung damage by infiltrating eosinophils, as seen in in vivo models (179, 180) . however, eosinophil immunopathology due to sars-cov vaccine could be reduced by using tlr4 agonist as an adjuvant (181) . viral neutralizing antibodies specifically targeting various regions of s, i.e., s1-rbd, s1-ntd, or the s2 region, and blocking the interaction of virus with the receptor are well-known for sars and mers (182) . these neutralizing antibodies could prove to be the best and potential candidate for cross-neutralization of sars-cov-2. despite being structurally related, some of the sars-cov neutralizing monoclonal antibodies failed to interact with the s-protein of sars-cov-2, which could be attributable to the substantial differences in their rbd (183) . a recent study reported the presence of high titres of neutralizing anti-s-rbd igg antibodies, but no antibodies were detected against the n protein in recovered covid-19 patients, suggesting that anti-s igg persists longer than does anti-n igg. along with the humoral immune response, they also observed an s protein-specific t cell-population producing ifn-γ, which further contributes to conferring protective immunity against sars-cov-2 infection (184). recently, a monoclonal antibody (47d11) has been identified from 51 sars-spike hybridomas that targets the conserved s-rbd region (residue 338-506) and therefore can very effectively neutralize sars-cov-2 along with sars-cov (185). on similar lines, a group has isolated a single-domain antibody from a phage display library targeting the s-rbd region of sars-cov-2. the fully humanized singledomain antibody was able to neutralize the virus by interacting with a cryptic epitope in s protein (186) . these mab and singledomain antibodies could be used to treat as well as to design quick diagnostic kits for covid-19. the new technology of the microneedle array (mna) has been employed for delivering sars-cov-2 s1 subunit vaccine, which could be really helpful in the treatment of the emerging covid-19 outbreak (187) . the transfer of s1 subunit by mna elicited a strong virus specific-antibody response in sars-cov-2 (187) . a novel encapsulated mrna vaccine candidate developed by modernatx, inc. that encodes full length s protein of sars-cov-2, is also under clinical trial (nct04283461). there is an urgent need to develop more such specific vaccines that could neutralize the novel coronavirus effectively (188) . the host innate immune system encounters upcoming infections, and this results in elevated production of various cytokines and type i interferons (ifns). in the case of prolonged infection, hyperactivation of the immune system may also result in the development of a pro-inflammatory microenvironment, leading to adverse outcomes and even death. the induction of numerous lymphokines, such as il-6, il-1β, tnf-α, and ccl2, that are pro-inflammatory in nature has also been observed in the case of covid-19 (189) (190) (191) . a previous study in a mers animal model showed that treatment with recombinant type-1 ifn (rifn) decreased the viral rna level in lungs with a decrease in ifn-stimulating gene expression. early treatment with rifn resulted in a dampening of cytokine and chemokine release that lowered the migration of neutrophils and other cells in lung (91) . an allogenic mesenchymal stem cell-based (remestemcel-l) therapy developed by mesoblast, which has been previously used for inflammatory conditions and graft vs. host disease in children and adults, is now being assessed for covid-19 (192) (193) (194) . in this therapy, bone marrow-derived mscs from the donor are grown in vitro and are then transfused to the recipient patients. upon infusion, these cells exhibit antiinflammatory activity by reducing pro-inflammatory cytokine production via the recruitment of anti-inflammatory cells in the affected tissue (195) . currently, a randomized placebo-controlled trial (nct04371393) with 300 patients is ongoing for treating ards caused by covid-19. treatment with rifn, inhibitors of the pro-inflammatory pathway, cytokine inhibitors such as tocilizumab, lenzilumab, and many others are still to be used in combination with other drugs for treating covid-19. so far, there is not much evidence from clinical trials of such inhibitors with which to predict the outcome of these anticytokine therapies. considering the current situation of more than 8 million people being infected, with ∼436,167 deaths as of june 15, 2020, there is an urgent need to control the sars-cov-2 pandemic. the fatality rate of sars-cov-2 in lower than those of other coronaviruses that caused catastrophes in the past, but the higher infectivity rate makes it worse. raising awareness of this contagious virus is one of the many ways by which its spread can be prevented. the governing authorities concerned in every country have approved guidelines and taken necessary action to quarantine infected people and break the chain of community spread. antibodies, vaccines, and drugs developed for previously emerged coronaviruses could potentially be used for treating sars-cov-2. the combination of various neutralizing antibodies against s protein could enhance the effectiveness of viral clearance. among various antivirals and other small molecules that are fda approved, chloroquine/hydroxychloroquine has shown better positive outcome in covid-19 patients. in clinical trials, some of the combinational antiviral drugs like lopinavir + ritonavir and blockers like angiotensin receptor blocker that were thought to be effective, have failed in curing the disease (139, 196) . cytokine storm being one of the symptoms of infected individuals, anticytokine therapy for tnf and il-6 should be attempted to determine the efficacy of these antibodies in the treatment of sars-cov-2 infection. clinical trial chictr2000029765 with tocilizumab, a monoclonal humanized antibody against il-6 receptor, has shown some efficacy, but this still needs to be tested in a larger cohort. with the increasing number of deaths, there is an immense need to accelerate the development of rapid and sensitive diagnostic kits and to commence clinical trials of the readily available and safe drugs to reduce the rising infections and covid-19-related deaths so as to bring life back on track. vs and pf contributed equally in writing the review. conception of idea was done by sc, vs, and pf. manuscript writing and editing was done by all the authors. we are thankful to csir-indian institute of chemical biology, jadavpur, kolkata and national centre for cell science (nccs), pune for providing infrastructure facilities. we would also like to acknowledge department of biotechnology-systems medicine cluster (dbt-symec) grant and j c bose fellowship-serb (science and engineering research board) to sc, for the financial support. a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient sars and mers : recent insights into emerging coronaviruses estimates of the severity of coronavirus disease 2019 : a model-based analysis united nation, department of economic and social affairs (2020) the genome sequence of the sars-associated coronavirus a new coronavirus associated with human respiratory disease in china crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors structural basis for the recognition of the sars-cov-2 by full-length human ace2 a novel coronavirus from patients with pneumonia in china coronavirus envelope protein: current knowledge human coronaviruses: a review of virus-host interactions origin and evolution of pathogenic coronaviruses from sars to mers, thrusting coronaviruses into the spotlight genome composition and divergence of the novel coronavirus (2019-ncov) originating in china covid-2019: the role of the nsp2 and nsp3 in its pathogenesis origin and evolution of the 2019 novel coronavirus emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant emergence of genomic diversity and recurrent mutations in sars-cov-2 angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus wenhui dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 cryo-em structure of the 2019-ncov spike in the prefusion conformation host cell proteases: critical determinants of coronavirus tropism and pathogenesis sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor localization of neutralizing epitopes and receptor-binding site in murine coronavirus spike protein viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome current advancements and potential strategies in the development of mers-cov vaccines fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus rna-dependent rna polymerase coronavirus transcription: a perspective host factors in coronavirus replication identification of novel subgenomic rnas and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus the c-terminal domain of the mers coronavirus m protein contains a trans-golgi network localization signal structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes available online at infection control: severe acute respiratory syndrome coronavirus 2 (sars-cov-2). (2020) immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic people who are at higher risk for severe illness | coronavirus | covid-19 severe acute respiratory syndrome-associated coronavirus in lung tissue multiple organ infection and the pathogenesis of sars sars-cov-2 infection in children vulnerability of children with covid-19 infection and ace2 profiles in lungs possible causes for decreased susceptibility of children to coronavirus diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus characteristics and outcomes of children with coronavirus disease 2019 (covid-19) infection admitted to us and canadian pediatric intensive care units outbreak of kawasaki disease in children during covid-19 pandemic: a prospective observational features of covid-19 post-infectious cytokine release syndrome in children presenting to the emergency department links between innate and adaptive immunity via type i interferon interferon and cytokine responses to sarscoronavirus infection sars-cov regulates immune function-related gene expression in human monocytic cells profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers early detection of antibodies against various structural proteins of the sarsassociated coronavirus in sars patients serological assays for emerging coronaviruses : challenges and pitfalls profile of specific antibodies to the sars-associated coronavirus t cell responses to whole sars coronavirus in humans temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2 : an observational cohort study profiling early humoral response to diagnose novel coronavirus disease (covid-19) viral kinetics and antibody responses in patients with covid-19 protective humoral immunity in sars-cov-2 infected pediatric patients the potential danger of suboptimal antibody responses in covid-19 medical countermeasures analysis of 2019-ncov and vaccine risks for antibody-dependent enhancement (ade) antibody-dependent enhancement of sars coronavirus infection and its role in the pathogenesis of sars molecular mechanism for antibody-dependent enhancement of coronavirus entry avoiding pitfalls in the pursuit of a covid-19 vaccine novel immunodominant peptide presentation strategy: a featured hla-a * 2402-restricted cytotoxic t-lymphocyte epitope stabilized by intrachain hydrogen bonds from severe acute respiratory syndrome coronavirus nucleocapsid protein association of human leukocyte antigen class ii alleles with severe acute respiratory syndrome in the vietnamese population epidemiological and genetic correlates of severe acute respiratory syndrome coronavirus infection in the hospital with the highest nosocomial infection rate in taiwan in 2003 humanleukocyte antigen class i cw 1502 and class ii dr 0301 genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection association of human leukocyte antigen class ii alleles with severe middle east respiratory syndrome-coronavirus infection cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus mers-cov and h5n1 influenza virus antagonize antigen presentation by altering the epigenetic landscape complex immune dysregulation in covid-19 patients with severe respiratory failure smar1 favors immunosurveillance of cancer cells by modulating calnexin and mhc i expression t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice priming with sars cov s dna and boosting with sars cov s epitopes specific for cd4+ and cd8+ t cells promote cellular immune responses recovery from the middle east respiratory syndrome is associated with antibody and t cell responses sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals human immunopathogenesis of severe acute respiratory syndrome (sars) plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) pathological findings of covid-19 associated with acute respiratory distress syndrome covid-19 : consider cytokine storm syndromes and immunosuppression pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients anti-inflammation treatment of severe coronavirus disease 2019 (covid-19): from the perspective of clinical immunologists from china the cytokine storm in covid-19: an overview of the involvement of the chemokine/chemokine-receptor system novel paediatric presentation of covid-19 with ards and cytokine storm syndrome without respiratory symptoms transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients the interferon in tlr signaling: more than just antiviral haematological manifestations in patients with severe acute respiratory syndrome:retrospective analysis significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses imbalanced host response to sars-cov-2 drives development of covid-19 elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients dysregulation of immune response in patients with covid-19 in wuhan, china lees jr, farber dl. generation, persistence and plasticity of cd4 t-cell memories characterization of sars-cov-specific memory t cells from recovered individuals 4 years after infection lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study longlived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients airway memory cd4+ t cells mediate protective immunity against emerging respiratory coronaviruses a mouse model of sars-cov-2 infection and pathogenesis coronaviruses-drug discovery and therapeutic options a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality preventing covid-19-induced pneumonia with anticytokine therapy fda approves emergency ind use of humanigen's lenzilumab for compassionate use in covid-19 patients altasciences completes phase i study on gimsilumab for ards in covid-19 type 1 interferons as a potential treatment against covid-19 covid-19 and emerging viral infections: the case for interferon lambda learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases small molecule inhibitors of the sars-cov nsp15 endoribonuclease an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice baricitinib as potential treatment for 2019-ncov acute respiratory disease comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a trial of lopinavirritonavir in adults hospitalized with severe covid-19 old weapon for new enemy: drug repurposing for treatment of newly emerging viral diseases remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology the efficacy of lopinavir plus ritonavir and arbidol against novel coronavirus infection molecular mechanisms of severe acute respiratory syndrome (sars) receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses. sci rep prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection gs-5734) protects african green monkeys from nipah virus challenge first case of 2019 novel coronavirus in the united states remdesivir for the treatment of covid-19 -preliminary report new nucleoside analogues for the treatment of hemorrhagic fever virus infections chloroquine analogs as antimalarial candidates with potent in vitro and in vivo activity chloroquine is a potent inhibitor of sars coronavirus infection and spread effects of chloroquine on viral infections: an old drug against today's diseases? a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial cinanserin is an inhibitor of the 3c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro inhibition of sars-cov 3cl protease by flavonoids diarylheptanoids from alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia treatment with convalescent plasma for influenza a (h5n1) infection the convalescent sera option for containing covid-19 breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients use of convalescent plasma therapy in sars patients in hong kong available online at sars vaccines: where are we? neutralizing antibody against severe acute respiratory syndrome (sars)-coronavirus spike is highly effective for the protection of mice in the murine sars model sars corona virus peptides recognized by antibodies in the sera of convalescent cases severe acute respiratory syndrome (sars) coronavirus: application of monoclonal antibodies and development of an effective vaccine subunit vaccines against emerging pathogenic human coronaviruses development of an inactivated vaccine candidate for sars-cov-2 chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine neutralizing antibodies against sars-cov-2 and other human coronaviruses potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals a human monoclonal antibody blocking sars-cov-2 infection identification of human single-domain antibodies against sars-cov-2 microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development sars-cov-2 vaccines: status report induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies mast cells contribute to coronavirus-induced inflammation: new anti-inflammatory strategy clinical features of patients infected with 2019 novel coronavirus in wuhan a phase 3, single-arm, prospective study of remestemcel-l, ex vivo culture-expanded adult human mesenchymal stromal cells for the treatment of pediatric patients who failed to respond to steroid treatment for acute graft-versus-host disease remestemcel-l: human mesenchymal stem cells as an emerging therapy for crohn's disease transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia mesoblast to evaluate anti-inflammatory cell therapy remestemcel-l for treatment of covid-19 lung disease are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? key: cord-261566-fn08b0y2 authors: mudgal, rajat; nehul, sanketkumar; tomar, shailly title: prospects for mucosal vaccine: shutting the door on sars-cov-2 date: 2020-09-15 journal: human vaccines & immunotherapeutics doi: 10.1080/21645515.2020.1805992 sha: doc_id: 261566 cord_uid: fn08b0y2 the sudden emergence of a highly transmissible and pathogenic coronavirus sars-cov-2 in december 2019 from china and its rapid global spread has posed an international health emergency. the rapid development of an effective vaccine is imperative to control the spread of sars-cov-2. a number of concurrent efforts to find an effective therapeutic agent or vaccine for covid-19 (coronavirus disease 2019) are being undertaken globally. oral and nasal mucosal surfaces serve as the primary portal of entry for pathogens like coronaviruses in the human body. as evidenced by studies on similar coronaviruses (sars-cov and mers-cov), mucosal vaccination can provide a safe and effective means for the induction of long-lasting systemic and mucosal immunity to confer protection against sars-cov-2. this article summarizes the approaches to an effective mucosal vaccine formulation which can be a rewarding approach to combat the unprecedented threat posed by this emerging global pandemic. in the 21st century, we have seen a worldwide spread of three previously unknown coronaviruses. the first outbreak of severe acute respiratory syndrome (sars) occurred in november 2002 in the guangdong province, china. the causative agent of the 2002 sars outbreak was identified as sars coronavirus (sars-cov). 1 another coronavirus, middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in 2012. 2 on december 31, 2019, several cases of pneumonia were reported in wuhan, china. 3 the etiological agent of the 2019 outbreak was later identified as sars coronavirus 2 (sars-cov-2) because the genomic sequence was closely related to that of the sars-cov from 2003. 4, 5 on february 12, 2020, the world health organization (who) named the disease caused by the novel coronavirus (sars-cov-2) as coronavirus disease 2019 . coronaviruses belong to the coronaviridae family. the family members are enveloped, positive-stranded rna viruses which appear as crown-like entities under the electron microscope due to spikes of glycoproteins protruding from their viral envelopes, thus exhibiting a corona-like appearance. 6 the coronaviridae family is divided into two subfamilies: letovirinae and orthocoronavirinae. the latter consists of the genera alphacoronvirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the pathogenic coronaviruses: sars-cov, mers-cov, and sars-cov-2 are all betacoronaviruses. 6 among known rna viruses, coronaviruses have the largest genomes size in the range of 26 to 32 kb in length. 7 two-third of the viral genome at 5ˊ end encodes up to 16 nonstructural replicase proteins which are translated as two polyproteins: pp1a and pp1ab. the genes encoding structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins are present at 3ˊ end of genomic rna. 7, 8 the s protein of coronaviruses is one of the most important targets for the development of sars vaccines and therapeutics because it is involved in receptor recognition, as well as virus attachment and entry. the s protein is made up of s1 and s2 subunits. s1 subunit has the receptor-binding domain (rbd) which binds with host receptor and then the s2 subunit mediates the fusion of viral and host membranes. 9 host receptor for sars-cov is angiotensinconverting enzyme 2 (ace2), whereas mers-cov recognizes dipeptidyl peptidase 4 (dpp4) as its receptor. 10, 11 the common symptoms of coronavirus infection are fever, cough, and sore throat. clinically, patients with sars suffer from atypical pneumonia. 12, 13 clinical presentation of sars-cov-2 patients is similar to patients infected with sars-cov. covid-19 manifests itself with symptoms including fever, dry cough, fatigue, and acute respiratory distress syndrome. 3, 14 these clinical features are a direct consequence of massive alveolar epithelial cell and vascular endothelial cell damage which is also accompanied by an exuberant release of proinflammatory cytokines and chemokines. 15 the disease severity and lung damage in the case of sars-cov-2 infection can be directly correlated with the dysregulated immune response at 7-10 days after symptom onset and is characterized by exuberant production of cytokines including il-2, il-7, il-10, mip-1a, ip-10, and tnf-α. 3, 15 this 'cytokine storm' was also reported in animal studies with sars-cov infection and was responsible for the dampening of adaptive immunity of patients in the later phase of infection. 16 as of now, the spread of sars-cov-2 is being quelled by strict policy measures such as travel restrictions, social distancing, patient isolation, and nationwide lockdown in several parts of the world. there are no approved vaccines available against any of the coronaviruses. this emerging global pandemic has instigated an unprecedented search for an effective prophylactic or therapeutic intervention against covid-19. [17] [18] [19] [20] in this review, we have discussed the potential and challenges for the development of a successful mucosal vaccine against sars-cov-2. vaccines have been one of the major contributors in the eradication of most of the infectious diseases in the last century. vaccination of a population interrupts the transmission chain of a communicable pathogen by not only protecting the immunized subjects but also by halting the transmission of the virus by 'breaking the chain' within a population by induction of herd immunity in the vaccine recipients. vaccines can be administered by either intramuscular or subcutaneous injection to introduce them into the systemic circulation. vaccination through systemic routes elicits strong systemic immune response but is not effective in generating efficient mucosal immunity. 21 mucosal vaccines are advantageous not just in evoking strong immune response at both mucosal sites and systemic circulation but also offer greater practicality in terms of cost and administration. 22 mucosal vaccines can be produced at considerably low cost for mass immunization, the administration is needle-free and convenient compared to systemic vaccines. 23 majority of mucosal vaccines are administered through oral or intranasal routes while rectal, vaginal, ocular, and sublingual routes can also be used. the selection of administration route depends on the nature of the antigen and the desired site for induction of immune response. upon oral immunization, immune responses are induced strongly in gastrointestinal (gi) tract, mammary glands, and salivary glands while intranasal vaccination induces marked antigen-specific immune response in respiratory, gi, and genital tracts. the primary mode of transmission of sars-cov is through mucosal membranes of the eyes, nose, or mouth. 24 studies with s protein of sars-cov-2 have revealed that it also recognizes human ace2 (hace2) receptor on the host cells similar to sars-cov. [25] [26] [27] ace2 is abundantly expressed in nasal and oral mucosa rendering them the primary targets for the viral entry and dissemination of sars-cov-2. 28 concomitant gastrointestinal symptoms are reported in confirmed covid-19 patients along with pulmonary pathology, characteristic of sars-cov-2 infection. the occurrence of acute hemorrhagic colitis in a few cases and the presence of sars-cov-2 rna in fecal samples of covid-19 patients indicate enteric involvement in covid-19 reasserting the similarity in tissue tropism with sars-cov. [29] [30] [31] considering the prominent role of nasal and gastric mucosa in the transmission and the clinical progression of sars-cov-2, mucosal immunization using oral or intranasal vaccine could be an effective strategy for immunoprophylaxis against sars-cov-2. vaccination at the entry site such as intranasal or oral immunization induces a strong local immune response in the case of sars-cov. 32, 33 consequently, vaccination at the mucosal sites reduces the risk of antibodydependent disease enhancement (ade) by blocking the virus at the entry site. administration of mucosal vaccine has also been shown to elicit strong systemic humoral immunity thereby neutralizing any virus particle that evades the primary immune response at the mucosal site. currently, several strategies are being examined for the development of a mucosal vaccine against sars-cov-2. 34 a unique mucosal system exists independent of the systemic immune system to protect exposed mucosal surfaces against environmental antigens and downregulation of systemic immune responses. it acts as the first line of defense against most of the antigens and prevents them from evoking systemic immune system. 35 the mucosal system serves as the most common portal for pathogen entry in the human body. the mucosal immune system consists of a complex network of tissues, non-lymphoid /lymphoid cells, and effector molecules including cytokines, chemokines, and antibodies. 36 the mucosal immune system is compartmentalized into immune inductive sites-where antigen sampling from mucosal surface occurs and then priming of b cells and t cells takes place, and immune effector site-where the activated immune effector cells move after extravasation and secrete cytokines to promote iga class-switch recombination. 37, 38 inductive site for the mucosal immune system is constituted by mucosa-associated lymphoid tissue (malt). malt comprises of highly organized structures such as appendix and peyer's patches in the intestine and tonsils in the upper airway. the effector sites of the mucosal immune system include lamina propria of various mucosae, surface epithelia, and exocrine glands. gut-associated lymphoid tissue (galt) and nasopharyngeal-associated lymphoid tissue (nalt) represent the major mucosal inductive sites. waldeyer's ring and oropharyngeal lymphoid tissues (paired palatine tonsils) in humans are considered anatomical equivalent of murine nalt. 39 larynx-associated lymphoid tissue (lalt), salivary duct-associated lymphoid tissue (sdalt), lacrimal ductassociated lymphoid tissue (ldalt), and conjunctiva-associated lymphoid tissue (calt) are also considered part of human malt. 40 these mucosal inductive sites are covered with a layer of enterocytes and microfold cells (m cells). m cells are specialized thin epithelial cells that move soluble antigen from the gut lumen to the underlying lymphoid tissues via transcytosis. exogenous antigens can activate t cells directly or are handed off to dendritic cells (dcs) that can act as antigen-presenting cells (apcs). 41, 42 these primed t cells move to the germinal centers and secrete cytokines to promote b-cell isotype switching to iga production. 43 secretory iga (siga) antibodies are the most essential effector molecule in the mucosa. it is recognized as the first line of protection against foreign toxins, pathogens, and overgrowth of commensal microbes. it is secreted as a dimer joined together by a joining chain and is actively transported exclusively across the mucosal surface via a polymeric iga receptor. 44 the activated t and b cells retain immunological memory and contribute to long-lasting protective immunity against the pathogen in systemic and mucosal systems. siga neutralizes toxins or pathogens in the mucosal environment using three mechanisms: immune exclusion, antigen excretion, and intracellular neutralization ( figure 1 ). 45 in addition to siga, transudated iga and igg, which are generated in response to both systemic and mucosal vaccination, also contribute to local surface defense in the genitourinary mucosa and in the lower respiratory tract which are more permeable to serum-derived antibodies than intestine. these antibodies employ a diverse range of effector functions to protect against pathogens. they neutralize toxins; can mediate opsonization and facilitate internalization of invading pathogens by phagocytes. transudated igg exert its immunopathological effect when siga-dependent elimination of pathogen is unsuccessful. 46 serum igg antibodies protect against viremia and are crucial for virus clearance from systemic circulation. a mucosal vaccine can be developed based on one of the several vaccine platforms including viral vectors, virus-like particle (vlp)-based, dnas, subunit, inactivated whole virus, or live-attenuated vaccine. 6, 47, 48 each of these platforms has its own advantages and disadvantages. vlp-based vaccines are composed of viral structural proteins capable of selfassembly. dna vaccine comprises viral immunogens encoded by a recombinant plasmid that is delivered to the site of administration where the plasmid expresses itself and elicits the desired immune response. 49 both these safe vaccine platforms preserve the inherent antigenic structure of viral immunogens and are noninfectious but often suffer from weak immunogenicity. viral vectors encode the antigenic protein and are delivered to the host cell where the antigen is expressed and is presented on the surface of apcs to induce a cellular and humoral immune response. 50 these vaccines are highly efficient but preexisting immunity against the viral vector might cause a harmful immune response in some recipients. live-attenuated vaccines or inactivated vaccines are based on pathogens that have been attenuated or inactivated by heat or chemical treatment. 46 as of now, all the mucosal vaccines licensed for human use are based on liveattenuated approach including oral polio vaccine (opv) and intranasal influenza vaccine (flumist®). safety concerns are often associated with attenuated vaccines due to the possibility of incomplete inactivation of harmful pathogens. subunit vaccines consist of specific antigenic fragments of virus capable of eliciting strong antibody-mediated and cellular immune response. 51 inactivated whole virus vaccines and subunit vaccines are relatively inexpensive, inert, and nontoxic but the possibility of alteration of immunogenicity of antigens needs to be confirmed consistently. few sars-cov and mers-cov vaccines based on the different platforms are summarized in table 1 . s protein is the main antigenic component of sars-cov, sars-cov-2, and mers-cov among four structural proteins (s, e, m, and n proteins). vaccines using s protein elicit a potent immune response and inhibit viral infection. 9, 53, 64 but the use of fulllength s protein as an antigen for vaccine design has raised few safety issues due to ade of viral infection in vaccinated subjects. a study on the chinese macaque models for sars-cov showed greater lung damage in vaccinated animals (vaccinated with fulllength s proteins) relative to unvaccinated subjects upon virus challenge. 65 anti-s protein igg antibodies have been implicated in this acute alveolar damage on exposure to the virus. similar symptoms have been observed in critical patients with sars-cov infection. 66 it is speculated that ade of viral infection is mediated through the binding of virus-neutralizing antibody complex to the fc receptors on the monocytes/macrophages leading to increased production of pro-inflammatory cytokines. additionally, this virus-antibody complex might activate the classical pathway of complement system or antibody-mediated cytotoxicity leading to cellular damage. 67 taking a cue from studies with sars-cov and mers-cov, caution must be exercised in selecting an antigen target that minimize ade while inducing a potent immune response against future exposure to sars-cov-2. the use of rbd of s protein as an antigen in the case of sars-cov or mers-cov has been extensively explored. 9,68 rbd of s protein is highly immunogenic and confers neutralizing capability against multiple strains of sars-cov and mers-cov. rbd-based vaccine minimizes the risk of ade upon exposure to virus in vaccinated individuals as it lacks the non-neutralizing immunodominant region of s protein. 69 other fragments of s protein of sars-cov and mers-cov that have been used for vaccine design include s1 and s2 fragments ( figure 2 ). 71,72 a mucosal vaccine based on n protein of sars-cov has shown the induction of both cellular and humoral immunity in mice. 61 several immunogenic domains of n protein are highly conserved in coronaviruses. 73 n protein of sars-cov-2 shares high sequence identity (~91%) with n protein of sars-cov and hence can be considered for the development of a broad spectrum coronavirus vaccine. 74 a challenging alternative that can warrant protection against future outbreaks of similar coronaviruses is to identify a universal epitope in the whole virus family or genera. this strategy is being undertaken for influenza viruses and can be extended to coronaviruses as well. 75 immunoinformatics can also be a powerful tool in prediction of epitopes on the viral surface proteins. 76 traditionally, mucosal immune induction needs a higher dose of antigen in comparison to parenteral immunization as antigenic preparation may get diluted in mucus in the nasal cavity or get expelled by mucus and ciliary movement in the respiratory tract. 77 effective intranasal vaccination requires the antigen to reach mucosal sites, cross the mucus layer, and induce local iga production. a vaccine administered through the oral route has to endure the low ph environment in the upper gi tract and a variety of nucleases and proteases present in the digestive tract before it can reach the immunological sites. mucosal antigens when administered alone lead to a weak induction of immune response. to overcome these physical and biochemical obstacles in priming mucosal immune cells, vaccines delivered through oral or nasal routes are often administered complemented with an adjuvant. 78 adjuvants are the supplementary materials (natural or synthetic) in a vaccine formulation that potentiates the immune-induction capacity of a vaccine. 79 adjuvant in a vaccine formulation plays a critical role in maintaining the structural integrity of the antigen, augmenting antigen bioavailability, and enhancing antigenic stimulation. adjuvants are broadly divided into two classes: adjuvants that can serve as carrier systems to facilitate antigen delivery to immune induction sites and adjuvants that can act as immunostimulators through enhanced internalization, presentation, and processing of the antigen in the apcs. 80 a number of polymers including chitosan and poly lacticco-glycolic acid (plga) have been used as carriers in various vaccine formulations for immunostimulation due to their high affinity toward mucosal surfaces. 81, 82 emulsions and liposomes are the other carriers routinely tested for mucosal vaccine. 48 dc and m cells are a major determinant for induction of mucosal immune response at immune inductive sites and thus represent an ideal site for antigen presentation. surface markers at the surface of epithelial cells and dcs can be strategically targeted for antigen delivery with specialized molecules such as lectins or nanoparticles for increased antigen uptake by apcs. 83, 84 pathogen recognition receptor agonists such as synthetic poly-(i:c), or toll like receptor agonists such as cpg oligodeoxynucleotides, engage these receptors present on the surface of apcs to potentiate the immunogenicity of the vaccine. 85, 86 other commonly used adjuvants including cholera enterotoxin (ct) and heat-labile enterotoxin (lt) from e. coli exert their adjuvanticity by interacting with gm1 gangliosides on the surface of follicular dendritic cells and in turn increase the induction of b-cell clones. 87 the use of immune-stimulating complexes (iscoms) has also proven to be highly effective for administration with mucosal vaccines. 88 one of the major factors influencing the rational design of a mucosal vaccine is immunotolerance at the mucosal sites. immunotolerance is the ingenious modulation of the mucosal microenvironment to avoid unnecessary induction of host immune cells against foreign antigens or commensal microbes. mucosal surfaces are exposed continuously to environmental, food, or self-antigens which lead to the development of a tolerogenic microenvironment, especially in gastric mucosa. 89 some vaccination strategies fail to develop effective immunity and can induce immunotolerance. this suppression of immune response is affected mainly by vaccine formulation, antigenic dose, and frequency of administration. administration of antigen at low doses for a long time leads to low-dose immunotolerance. 48 contrastingly, high dose of antigen administered at a low rate induces high-dose tolerance instead of immunostimulation. this hypo-responsiveness at the mucosal induction sites can be overcome by strategically determining the antigen dose, vaccine formulation, and timing of vaccine delivery. the design of a novel mucosal vaccine also aims at mimicking the kinetics of pathogen infection for increasing the clinical efficacy of the vaccine. optimal release timing of antigen at the inductive site helps in dodging the mucosal immunotolerance. to this end, the antigen is conjugated with adjuvants like tlr ligands or cd-40 specific antibodies. 90, 91 immunosenescence several issues are needed to be addressed for the design and development of an efficacious mucosal vaccine against sars-cov-2. it has been observed in human challenge studies that immunity acquired with coronavirus infection is often shortlived and in some cases, re-infection with the same virus was possible after an extended period. 92 it was reported in some cases that immunity acquired with sars-cov or mers-cov also declines considerably 2-3 years after viral infection. 93, 94 another concern for designing a vaccine against sars-cov-2 is that the viral infection is associated with severe pathology specifically in patients of higher age group (typically >50 years). 14 people in this age group do not respond very well to vaccination in terms of neutralizing antibody titers and require a higher amount of antigen to produce sufficient immunogenicity. specialized dose-regime in terms of the amount of antigen, use of specific adjuvants, or the immunization effects of follow-up doses after a single prime dose can be investigated in vulnerable age groups and immunocompromised people before extending vaccination to them. for most parenterally administered vaccines and mucosal vaccines, correlates and precise mechanism of their efficacy remain poorly defined. most of the licensed vaccines rely on the measurement of a single parameter that is statistically correlated with protection afforded by the vaccine. there is no absolute method of sampling, and the choice of sampling method varies according to the parameter to be evaluated. conventionally, quantification and qualification of secreted antibodies is performed using seroimmunoassays in body secretions such as saliva, tears, nasal, blood samples or genital secretions, and gut or organ lavages. 95 cellular correlates of immunity are analyzed using assays such as enzyme-linked immunospot assays (elispots), reverse-transcriptase pcr (rt-pcr) or cell-sorting techniques. 96 a modern system biology approach can also be used that utilizes functional genomics to identify molecular signatures that correlate well with traditional biological markers for evaluating vaccine efficacy. 97 correlates of protection differ greatly based on whether the objective of vaccination is to prevent a mucosal or a systemic infection. 96 several animal models including mice, ferrets, macaques, hamsters, and non-human primates have been used for evaluating safety and efficacy of sars-cov and mers-cov vaccines. 53, [98] [99] [100] [101] [102] ferrets are a suitable animal model for sars-cov vaccine evaluation as they support viral replication in the respiratory tract, develop similar disease symptoms, and display severe lung pathology. 103, 104 smaller animal models like rabbits and mice are a preferred choice for vaccine evaluation in animals because of inexpensive maintenance, ease of genetic manipulation, and standardized methods of testing. wild type mice are nonpermissive to sars-cov-2 viral replication. a transgenic mice model expressing hace2 has recently been developed by genetic manipulation to make the mice susceptible to sars-cov-2 infection. 105 this mice model mirrors the pathological features of sars-cov-2 infection in human patients albeit at a moderate level as compared to sars-cov. moreover, a young animal/mice model can effectively exhibit excellent neutralizing efficiency induced by a vaccine candidate but it cannot effectively mimic the clinical manifestations of covid-19 in an elderly population. hence, robust lethalchallenged mice models using senescent mice that can recapitulate the clinical disease in the aged human population are needed to be developed for assessing the efficacy of a covid-19 vaccine candidate. following animal modelbased preclinical studies, good manufacturing practices are employed for the scale-up of the vaccine production. currently, mucosal vaccines form a small proportion of licensed vaccines for humans. limited choice of adjuvants for human mucosal vaccines, immunotolerance, and differential degree of vaccine-induced immune response in different populations are some of the impediments associated with the development of mucosal vaccines. efficacies of different mucosal vaccines depend on myriad of factors such as age, environment, host genetics, the microbiome of the recipient, and the regimen of immunization. it ranges from 70% to 90% for rotavirus vaccines to 85%-to 90% for oral vaccines against influenza and polio viruses. 22 cholera, polio, and rotavirus oral vaccines are found to be less efficacious in developing countries. this weak induction of immune response can be attributed to nutritional deficiencies such as vitamin a or zinc, concomitant bacterial, helminth, or viral infections, and the presence of high levels of maternal antibodies in the breast milk. 106, 107 intranasal immunization using live attenuated or live vectors is also associated with the risk of the harmful antigens and adjuvants (such as ct and lt) accessing the central nervous system through the cribiform plate. 108 a number of infectious diseases have been successfully controlled with the use of parenterally administered vaccine that may or may not lead to the induction of mucosal immune response. administration of the vaccine through systemic circulation might not necessarily recapitulate the immune response generated by mucosal administration but is adequate for evoking immune response against mucosal pathogens such as human papilloma virus (hpv), influenza virus, or polio virus. all three licensed vlp-based hpv vaccines are administered intramuscularly and induce a high level of durable igg antibody response against the virus. 109, 110 these antibodies are also detectable at mucosal sites of infection such as oral cavity and cervix after vaccination and serve as the primary effectors of protection against hpv. 111, 112 currently, there are two types of licensed influenza vaccines: parenterally administered inactivated influenza vaccine and live attenuated influenza virus vaccine (laiv) which is delivered intranasally. inactivated influenza vaccine elicits some degree of local iga antibodies and high levels of systemic igg antibodies to mediate protection by diffusing into the mucosal sites. laiv, in turn, mimics natural infection and induce highly cross-reactive serum igg, mucosal iga, and cellular immunity in the recipients. 113, 114 yearly influenza vaccines are adjusted according to the prevalent strains in the coming flu season. in clinical settings, inactivated vaccine is found to be more effective than laiv in preventing symptomatic disease in healthy adults while laiv offers greater protection against influenza disease in children. [115] [116] [117] similarly, two types of vaccines are available for polio virus: an injectable polio vaccine (ipv) and opv. sabin opv, with its enormous impact in disease control worldwide, is considered to be the prototype oral vaccine. superiority of opv over ipv is attributed to the induction of high titers of mucosal iga antibodies. 118, 119 despite the generation of higher serum antibody titers, ipv provides limited intestinal immunity. 118 due to the risk of rare cases of vaccine associated paralytic polio, most developed countries have switched to either ipv or a sequential combination of ipv and opv. 120, 121 based on the substantial experience in control of mucosal pathogens using parenterally administered vaccines, several parenteral vaccines are being developed or evaluated against sars-cov-2. notably, dna-based vaccine by inovio pharmaceuticals (ino-4800), mrna-based vaccine by moderna (mrna-1273), adenovirus-vectored vaccine (chadox1), and an inactivated virus vaccine (picovacc) are currently under investigation in human clinical trials. [122] [123] [124] [125] rapid licensure and production of a mucosal vaccine time is the most valuable asset in the rapidly emerging covid-19 pandemic, especially in high-mortality areas. rapid development and testing of a viable vaccine can be achieved by adopting a number of novel strategies. instead of developing a novel vaccine development platform, utilizing an existing vaccine platform will accelerate the design and production of vaccines against sars-cov-2 and will enable a quicker future response against newly emerging viruses. since the outbreaks of sars-cov and mers-cov, several vaccine candidates were developed using different production platforms. these established platforms are being used for the development of vaccine candidate against sars-cov-2. 122, 124, 126 to expedite the process for licensure and use of the vaccine against sars-cov-2 in the wake of the current pandemic, eyal et al. have suggested an alternative model for accelerated rollout of an effective sars-cov-2 vaccine by skipping phase 3 clinical trials. 127 this study proposes a controlled human challenge model wherein nonsusceptible adult volunteers from a healthy group will be challenged with an increasing dose of live virus to determine a dose that induces clinical symptoms similar to the natural infection in the individuals of similar age (preparatory phase). after this dose for controlled human challenge model has been optimized, a randomized cohort of volunteers will receive the candidate vaccine or placebo before being challenged with the controlled dose of the live virus (phase 1). the successful vaccine candidates can then be administered to a large cohort of individuals for evaluation of shortterm safety prior to vaccine rollout (phase 2). this accelerated licensure of the vaccine can be followed up with post-approval surveillance to monitor the occurrence of rare side effects and long-term efficacy for future regulatory approvals. the overwhelming demand for a sars-cov-2 vaccine in the current pandemic by far exceeds the existing production capacity. to deliver required quantities of the successful vaccine, production process will have to be ramped up to an enormous scale. the necessary infrastructure can be developed simultaneously with the continued production on existing capacity. alternatively, rapid manufacturing of a successful vaccine can be facilitated by adopting a new pandemic paradigm, with a fast start and many steps of vaccine development and production executed in parallel before confirming a successful outcome of another step. 128 in recent years, rapid production of live attenuated vaccines is being facilitated by utilizing reverse genetics process that involves the precise deletion of specific genes of viral genome. 129 deletion of pathogenic components of the pathogen is advantageous over traditional methods of viral attenuation in terms of safety and efficacy of a vaccine. the current covid-19 pandemic has virtually brought most world economies to a halt and severely impacted the lives of a large proportion of the world population. development of a viable sars-cov vaccine is imperative to reduce mortality and morbidity associated with this novel virus outbreak. mucosal vaccines offer better patient compliance in terms of the physical and psychological comfort due to absence of needlestick injury and thus are highly compatible for mass immunization in a pandemic scenario. vaccine administration using injection involves the cost of injection device, it's safe disposal, and the employment of trained medical staff which adds a considerable cost for mass vaccination especially in developing countries. mucosally administered vaccines also abrogate the risk of transmission of infections by injection devices. despite our detailed understanding of mucosal system in mice, the complex cellular and molecular interplay of different component of innate immune response to mucosal vaccination in humans is still poorly deciphered. additionally, the emergence and rapid global spread of sars-cov-2 has provided a very small window for basic and translational studies that propel the development and evaluation of vaccine against a pathogen. while the knowledge gained from previous studies on sars-cov and mers-cov can be used for sars-cov-2 vaccine development, it is yet uncertain as to what extent it will work for sars-cov-2 or whether correlates of protection used will faithfully predict protective efficacy. potential ade and waning of vaccine-induced immune response represent other obstacles in the development of a mucosal vaccine against sars-cov-2. the currently licensed mucosal vaccines against viral pathogens are exclusively live-attenuated. the development of a safe and immunogenic live-attenuated vaccine is an attractive possibility for mucosal vaccine against sars-cov-2. live attenuated vaccines, by definition establish a mild infection at the immunization site and exhibit some level of in-built adjuvanticity thereby ensuring the delivery of a higher antigen dose and better targeting of mucosal inductive sites for immunostimulation. this candidate vaccine can be easily administered orally as encapsulated antigens or can be delivered through the intranasal route via droplets and aerosol spray. covid-19 pandemic demands for adopting nonconventional approaches for a viable mucosal vaccine development, hastening the vaccine licensure process, utilizing existing vaccine development and manufacturing platforms, and ensuring global distribution of the licensed vaccine in time to minimize the impact of covid-19 across the globe. it will also need an unprecedented scale-up of the manufacturing process and concerted efforts of the supply chains to make the vaccine available before this pandemic is over. learning from the covid-19 pandemic, sustained investments in vaccine development and production infrastructure should be made to save human lives and curtail the economic impact associated with a looming viral pandemic. moreover, continued global surveillance and vigilance in the post-pandemic scenario should be practiced to help the world counter a second wave of covid-19 or other possible future coronavirus outbreaks. the authors declare no potential conflict of interest. the compilation of this review was financially supported by department of science and technology, india science and engineering research board (serb) govt of india [ipa/2020/000054]. epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features of patients infected with 2019 novel coronavirus in wuhan the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 vaccines: status report origin and evolution of pathogenic coronaviruses coronaviruses: an overview of their replication and pathogenesis the spike protein of sars-cov-a target for vaccine and therapeutic development dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a cluster of cases of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada clinical and immunologic features in severe and moderate coronavirus disease zhang w viral dynamics in mild and severe cases of covid-19. the lancet infectious diseases dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice covid-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics in silico guided drug repurposing to combat sars-cov-2 by targeting mpro, the key virus specific protease tomar s identification of sars-cov-2 cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach sars-cov-2 e protein is a potential ion channel that can be inhibited by gliclazide and memantine immune response of sows vaccinated with attenuated transmissible gastroenteritis virus (tgev) and recombinant tgev spike protein vaccines and protection of their suckling pigs against virulent tgev challenge exposure recent progress in mucosal vaccine development: potential and limitations mucosal vaccines: strategies and challenges the severe acute respiratory syndrome functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses structural basis of receptor recognition by sars-cov-2. nature. 2020 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis prolonged presence of sars-cov-2 viral rna in faecal samples detection of sars-cov-2 in different types of clinical specimens enteric involvement of severe acute respiratory syndrome-associated coronavirus infection intranasal immunization with inactivated sars-cov (sars-associated coronavirus) induced local and serum antibodies in mice intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines the mucosal immune system: from fundamental concepts to vaccine development immunity against mucosal pathogens mucosal dendritic cells antiviral immune responses in the genital tract: clues for vaccines normal structure, function, and histology of mucosa-associated lymphoid tissue terminology: nomenclature of mucosa-associated lymphoid tissue interactions of the invasive pathogenssalmonella typhimurium, listeria monocytogenes, and shigella flexneri with m cells and murine peyer's patches intestinal villous m cells: an antigen entry site in the mucosal epithelium induction of gut iga production through t cell-dependent and t cell-independent pathways new concepts in the generation and functions of iga the role of secretory antibodies in infection immunity mucosal immunity and vaccines recent advances in the vaccine development against middle east respiratory syndrome-coronavirus mucosal vaccine design and delivery dna vaccines: a key for inducing long-term cellular immunity recombinant viruses as tools to induce protective cellular immunity against infectious diseases modern subunit vaccines: development, components, and research opportunities comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (sars-cov) spike protein induces strong mucosal immune responses and provides long-term protection against sars-cov infection singledose, intranasal immunization with recombinant parainfluenza virus 5 expressing middle east respiratory syndrome coronavirus (mers-cov) spike protein protects mice from fatal mers-cov infection single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against mers-cov infection superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length spike protein of middle east respiratory syndrome coronavirus a recombinant vsv-vectored mers-cov vaccine induces neutralizing antibody and t cell responses in rhesus monkeys after single dose immunization expression of sars-coronavirus nucleocapsid protein in escherichia coli and lactococcus lactis for serodiagnosis and mucosal vaccination intranasal immunization with plasmid dna encoding spike protein of sars-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses dendritic cell targeted chitosan nanoparticles for nasal dna immunization against sars cov nucleocapsid protein effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice combination attenuation offers strategy for live attenuated coronavirus vaccines immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools. virol sin. 2020 receptorbinding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor immunogenicity and protection efficacy of monomeric and trimeric recombinant sars coronavirus spike protein subunit vaccine candidates elicitation of immunity in mice after immunization with the s2 subunit of the severe acute respiratory syndrome coronavirus the coronavirus nucleocapsid is a multifunctional protein structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov mucosal vaccines: the promise and the challenge vaccine delivery: a matter of size, geometry, kinetics and molecular patterns 2020 vision for vaccines against hiv, tuberculosis and malaria the development of mucosal vaccines for both mucosal and systemic immune induction and the roles played by adjuvants enhanced mucosal and systemic immune response with intranasal immunization of mice with hiv peptides entrapped in plg microparticles in combination with ulex europaeus-i lectin as m cell target the basics and underlying mechanisms of mucoadhesion differential expression of lectin-binding sites defines mouse intestinal m-cells pre-clinical evaluation of a novel nanoemulsion-based hepatitis b mucosal vaccine vaccine adjuvants: putting innate immunity to work cpg dna as a vaccine adjuvant heat-labile enterotoxins as adjuvants or anti-inflammatory agents dendritic cells and immune responses to orally administered antigens oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens in vivo targeting of antigens to maturing dendritic cells via the dec-205 receptor improves t cell vaccination triggering tlr signaling in vaccination the time course of the immune response to experimental coronavirus infection of man two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome duration of antibody responses after severe acute respiratory syndrome evaluation of events occurring at mucosal surfaces: techniques used to collect and analyze mucosal secretions and cells correlates of protection induced by vaccination systems biological approaches for mucosal vaccine development middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques pneumonia from human coronavirus in a macaque model sars virus infection of cats and ferrets prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice severe acute respiratory syndrome coronavirus infection of golden syrian hamsters severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques the pathogenicity of sars-cov-2 in hace2 transgenic mice enhanced immunogenicity of an oral inactivated cholera vaccine in infants in bangladesh obtained by zinc supplementation and by temporary withholding breast-feeding factors affecting the immunogenicity of oral poliovirus vaccine in developing countries mucosal immunization: a realistic alternative hpv-16 l1 vlp vaccine elicits a broad-spectrum of cytokine responses in whole blood evaluation of the long-term anti-human papillomavirus 6 (hpv6), 11, 16, and 18 immune responses generated by the quadrivalent hpv vaccine quadrivalent human papillomavirus (hpv) vaccine induces hpv-specific antibodies in the oral cavity: results from the mid-adult male vaccine trial long-term persistence of systemic and mucosal immune response to hpv-16/18 as04-adjuvanted vaccine in preteen/adolescent girls and young women live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse t-cell responses in young children development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza a virus vaccine comparative efficacy of inactivated and live attenuated influenza vaccines live attenuated versus inactivated influenza vaccine in infants and young children current status of live attenuated influenza vaccine in the united states for seasonal and pandemic influenza mucosal immunity induced by enhanced-potency inactivated and oral polio vaccines immunoglobulin response in serum and secretions after immunization with live and inactivated poliovaccine and natural infection vaccine policy changes and epidemiology of poliomyelitis in the united states polio vaccination: past, present and future immunogenicity of a dna vaccine candidate for covid-19 an mrna vaccine against sars-cov-2-preliminary report chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques development of an inactivated vaccine candidate for sars-cov-2. science. 2020 microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development human challenge studies to accelerate coronavirus vaccine licensure developing covid-19 vaccines at pandemic speed an improved reverse genetics system for influenza a virus generation and its implications for vaccine production key: cord-253077-61fmul8c authors: vabret, nicolas; britton, graham j.; gruber, conor; hegde, samarth; kim, joel; kuksin, maria; levantovsky, rachel; malle, louise; moreira, alvaro; park, matthew d.; pia, luisanna; risson, emma; saffern, miriam; salomé, bérengère; selvan, myvizhi esai; spindler, matthew p.; tan, jessica; van der heide, verena; gregory, jill k.; alexandropoulos, konstantina; bhardwaj, nina; brown, brian d.; greenbaum, benjamin; gümüş, zeynep h.; homann, dirk; horowitz, amir; kamphorst, alice o.; curotto de lafaille, maria a.; mehandru, saurabh; merad, miriam; samstein, robert m. title: immunology of covid-19: current state of the science date: 2020-05-06 journal: immunity doi: 10.1016/j.immuni.2020.05.002 sha: doc_id: 253077 cord_uid: 61fmul8c abstract the coronavirus disease 2019 (covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has affected millions of people worldwide, igniting an unprecedented effort from the scientific community to understand the biological underpinning of covid19 pathophysiology. in this review, we summarize the current state of knowledge of innate and adaptive immune responses elicited by sars-cov-2 infection and the immunological pathways that likely contribute to disease severity and death. we also discuss the rationale and clinical outcome of current therapeutic strategies as well as prospective clinical trials to prevent or treat sars-cov-2 infection. the recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the resulting coronavirus disease 2019 (covid-19) poses an unprecedented health crisis that was declared a pandemic by the world health organization (who) on march 11, 2020. the origin of sars-cov-2 was traced to the city of wuhan in hubei province, china, where a cluster of viral pneumonia cases was first detected, many in connection with the huanan seafood wholesale market. china reported this outbreak to the who on december 31 st , 2019 and soon after identified the causative pathogen as a betacoronavirus with high sequence homology to bat coronaviruses using angiotensin-converting enzyme 2 (ace2) receptor as the dominant mechanism of cell entry wan et al., 2020b) . following a likely zoonotic spillover, human-to-human transmission events were confirmed with clinical presentations ranging from no symptoms; to mild fever, cough, and dyspnea; to cytokine storm, respiratory failure, and death. sars-cov-2 is also closely related to sars (retrospectively named sars-cov-1) and mers (middle eastern respiratory syndrome) coronaviruses, causing local outbreaks and zoonotic epidemics in 2003 , respectively (de wit et al., 2016 . while sars-cov-2 is not as lethal as sars-cov-1 or mers-cov (fauci et al., 2020) , the considerable spread of the current pandemic has brought tremendous pressure and disastrous consequences for public health and medical systems worldwide. the scientific response to the crisis has been extraordinary with a plethora of covid-19 studies posted in preprint servers, in an attempt to rapidly unravel the pathogenesis of covid-19 and potential therapeutic strategies. in response, trainees and faculty members of the precision immunology institute at the icahn school of medicine at mount sinai (priism) have initiated an institutional effort to critically review the preprint literature (vabret et al., 2020) , together with peer-reviewed articles published in traditional journals, and summarize the current state of science on the fast evolving field of covid-19 immunology. we thematically focus on the innate and adaptive immune responses to sars-cov-2 and related coronaviruses, clinical studies and prognostic laboratory correlates, current therapeutic strategies, prospective clinical trials, and vaccine approaches. (bouvet et al., 2010; chen et al., 2009; ivanov et al., 2004) , thereby resembling host mrna to promote translation, prevent degradation, and evade rlr sensing. finally, covs also encode an endoribonuclease, nsp15, that cleaves 5' polyuridines formed during viral replication, which would otherwise be detected by mda5 (deng et al., 2017; hackbart et al., 2020) . coronaviruses have evolved additional strategies to impede activation of prrs. sars-cov-1 n-protein prevents trim25 activation of rig-i . likewise, mers-cov ns4a, which itself binds dsrna, impedes pkr activation (comar et al., 2019; rabouw et al., 2016) and inhibits pact, an activator of rlrs (niemeyer et al., 2013; siu et al., 2014) . additionally, mers-cov ns4b antagonizes rnasel, another activator of rlrs (thornbrough et al., 2016) . the role of other prrs remains unclear. for example, sars-cov-1 papain-like-protease (plp) antagonizes sting, suggesting that self-dna may also represent an important trigger (sun et al., 2012) . the extent to which sars-cov-2 homologs overlap in these functions is currently unknown. following activation, rlr and tlrs induce signaling cascades leading to the phosphorylation of transcription factors, such as nf-kb and the interferon-regulatory factor family (irf), ultimately leading to transcription of ifn and proinflammatory cytokines. although no experimental studies have delineated the precise functions of sars-cov-2 proteins, proteomic studies have demonstrated interactions between viral proteins and prr signaling cascades. sars-cov-2 orf9b indirectly interacts with the signaling adaptor mavs via its association with tom70 (gordon et al., 2020) , consistent with prior reports that sars-cov-1 orf9b suppresses mavs signaling (shi et al., 2014) . furthermore, sars-cov-2 nsp13 interacts with signaling intermediate tbk1, and nsp15 is associated with rnf41/nrdp1, an activator of tbk1 and irf3 (gordon et al., 2020) . similarly, sars-cov-1 m protein is known to inhibit the tbk1 signaling complex (siu et al., 2009) , as does mers-cov orf4b (yang et al., 2015) . other proteins, including sars-cov-1 plp, n, orf3b and orf6, block irf3 phosphorylation and nuclear translocation (devaraj et al., 2007; . nf-kb is also inhibited by covs proteins. these include sars-cov-1 plp (frieman et al., 2009 ) and mers-cov orf4b and orf5 (canton et al., 2018; menachery et al., 2017) . finally, sars-cov-1 nsp1 (huang et al., 2011a; kamitani et al., 2009 ) and mers-cov nsp1 (lokugamage et al., 2015) initiate general inhibition of host transcription and translation, thus limiting antiviral defenses nonspecifically. to prevent signaling downstream of ifn release, cov proteins inhibit several steps of the signal transduction pathway that bridge the receptor subunits (ifnar1 and ifnar2) to the stat proteins that activate transcription. for sars-cov-1, these mechanisms include ifnar1 degradation by orf3a (minakshi et al., 2009) , decreased stat1 phosphorylation by nsp1 (wathelet et al., 2007) , and antagonism of stat1 nuclear translocation by orf6 kopecky-bromberg et al., 2007) . however, sars-cov-2 orf6 shares only 69% sequence homology with sars-cov-1, suggesting this function may not be conserved. in support of this notion, sars-cov-2 infection fails to limit stat1 phosphorylation, unlike in sars-cov-1 infection (lokugamage et al., 2020) . mucosal immune responses to infectious agents are orchestrated and regulated by myeloid cells with specialized functions, which include conventional dcs (cdcs), monocyte-derived dcs (modcs), plasmacytoid dcs (pdcs), and macrophages (guilliams et al., 2013) . a growing body of evidence points to dysregulated myeloid responses that potentially drive the covid-19 hallmark syndromes such as acute respiratory distress syndrome (ards), cytokine release syndrome (crs) and lymphopenia (mehta et al., 2020) . flow cytometric analyses of pbmcs from symptomatic covid-19 patients have shown a significant influx of gm-csf-producing, activated cd4 + t cells and cd14 + hla-dr lo inflammatory monocytes (im) (giamarellos-bourboulis et al., 2020; zhou et al., 2020b) . this matches single-cell transcriptomic (scrnaseq) data demonstrating cd14 + il-1β + monocytic expansion wen et al., 2020) , interferon-mapk driven adaptive immune responses , and il-1βassociated inflammasome signatures (ong et al., 2020) in peripheral blood of covid-19 patients, although systemic levels of il-1β detected are conspicuously low (gnjatic et al., unpublished) . importantly, these immune signatures track with progression of clinical disease. scrnaseq studies performed on pulmonary tissues of patients with severe covid-19 disease have revealed an expansion of ims and ficolin-1 + monocyte-derived macrophages, at the expense of tissue-resident reparative alveolar macrophages (am) . the aforementioned study also observed signatures of ifn-signaling and monocyte recruitment that likely contribute to the rapid decline in alveolar patency and promote ards. although most of the clinical focus has been on pulmonary damage and mononuclear phagocyte (mnp) dysfunction therein, it is increasingly clear that covid-19 likely presents systemic challenges in other organ sites such as the ileum and kidneys. understanding the role of non-pulmonary myeloid cells in tissue-specific pathology associated with covid-19 will be important. while data on covid-19 patients continues to rapidly emerge, studies of myeloid cell dysfunction in sars-cov-1 and mers-cov can provide an important roadmap to understanding covid-19 pathogenesis ( figure 2 ). sars-cov-1 infection in mouse models results in an aberrant am phenotype that limits dc trafficking and t cell activation (zhao et al., 2009) . additionally, ym1 + fizz1 + alternative macrophages can increase airway hypersensitivity, thus exacerbating sars-associated fibrosis (page et al., 2012) . further, as described above, murine sars-cov-1 studies have demonstrated that delayed ifn-i signaling and inflammatory monocytes-macrophages promote lung cytokine and chemokine levels, vascular leakage, and impaired antigenspecific t cell responses, culminating in lethal disease (channappanavar et al., 2016) . the role played by prominent ifn-producing pdcs in sars-cov-2 control or pathogenesis warrants investigation, as they have been shown to be critical in murine coronavirus (mhv) control (cervantes-barragan et al., 2007) . longitudinal studies in sars-cov-2 models are awaited, but initial phenotypic studies in humanized hace2 mice have shown the characteristic alveolar interstitial pneumonia, with infiltration of lymphocytes and monocytes and accumulation of macrophages in the alveolar lumen (bao et al., 2020a) , which recapitulates patient findings . lastly, nonhuman primate (nhp) studies and patient data on sars-cov-1 have also shown that virus spike-specific igg responses can exacerbate acute lung injury due to repolarization of alveolar macrophages into pro-inflammatory phenotypes and enhanced recruitment of inflammatory monocyte via ccl2 and il-8 (clay et al., 2012; liu et al., 2019) . however, the extent to which the antibody response contributes to disease pathophysiology remains to be confirmed. the initial mode of viral pathogen-associated signal (pamp) recognition by innate cells has a major impact on downstream myeloid signaling and cytokine secretion (de marcken et al., 2019) . while macrophages are somewhat susceptible to mers-cov and sars-cov-1 infection (perlman and dandekar, 2005; zhou et al., 2014) , data do not suggest that they are infected by sars-cov-2, although one study reported ace2 and sars-cov-2 nucleocapsid protein is expressed in lymph nodes and spleenassociated cd169 + macrophages of covid-19 patients producing il-6 . significantly elevated systemic levels of pro-inflammatory cytokine il-6 have been reported in several covid-19 patient cohorts and shown to correlate with disease severity (mehta et al., 2020) . increased il-6 can also be associated with higher levels of il-2, il-7, ifn-ɣ and gm-csf as seen in secondary hemophagocytic lymphohistiocytosis. in response to viral infections, mnps drive interleukin, and ifn-i and ifn-iii production, resulting in inflammasome activation, induction of pathogenic th1 and th17 cell responses, recruitment of effector immune cells and crs pathology (prokunina-olsson et al., 2020; tanaka et al., 2016) . independently, in vitro studies have demonstrated sars-cov-1 infection can induce intracellular stress pathways resulting in nlrp3-dependent inflammasome activation and macrophage pyroptosis shi et al., 2019) . functional studies are required to implicate these myeloid inflammasome pathways in covid-19 lung pathology and to assess other immunogenic pathways such as ripk1/3-dependent necroptosis (nailwal and chan, 2019) . in conclusion, the strength and duration of myeloid interferon-stimulated gene (isg) signaling potentially dictate covid-19 disease severity, but rigorous studies are warranted to confirm this. lastly, more work is needed to ascertain the mechanistic role played by lung-resident and recruited granulocytes in sars-cov-2 control and pathogenesis (camp and jonsson, 2017; flores-torres et al., 2019) . in contrast to their early protective role, neutrophil netosis and macrophage crosstalk can drive later-stage inflammatory cascades (barnes et al., 2020) , underscoring the overall pathogenic nature of damagesensing host responses ( figure 2) . collectively, the current knowledge of coronaviruses and sars-cov-2 infection, in particular, points to an inadvertent collusion involving myeloid cells in covid-19 pathogenesis, despite their critical role in early sensing and antiviral responses. innate lymphoid cells (ilcs) are innate immune effector cells that lack the expression of rearranged antigen receptors (tcr, bcr). the ilc family is divided into two main groups: the cytotoxic natural killer (nk) cells and the non-cytotoxic helper ilcs, which include ilc1, ilc2 and ilc3 (vivier et al., 2018) . conventional nk cells include cd56 bright cd16 -nk cells and cd56 dim cd16 + cells, that are specialized in cytokine production or cytotoxicity, respectively. multiple studies have reported reduced numbers of nk cells in the peripheral blood of covid-19 patients, which is associated with severity of the disease wang et al., 2020f; yu et al., 2020; zheng et al., 2020b) . a recent scrnaseq analysis revealed a transcriptomic signature for nk cells that was equally represented in lungs from patients and healthy donors . the majority of lung nk cells are non-resident (gasteiger et al., 2015; marquardt et al., 2017) , and cxcr3 has been shown to mediate nk cell infiltration upon influenza infection (carlin et al., 2018) . in vitro, cxcr3 ligands (cxcl9-11) are increased in sars-cov-2-infected human lung tissue (chu et al., 2020) , and cxcr3 ligand-producing monocytes are expanded in the lungs of covid-19 patients . this suggests that the cxcr3 pathway might facilitate nk cell recruitment from the peripheral blood to the lungs in covid-19 patients ( figure 2 ). nk cells express inhibitory and activating receptors that regulate their cytotoxicity. they are therefore able to induce the lysis of virus-infected cells that upregulate virus-derived proteins, as well as stress-inducible ligands, which are then recognized by nk cellactivating receptors, such as nkp46 (cerwenka and lanier, 2001; draghi et al., 2007; duev-cohen et al., 2016; glasner et al., 2012) . future studies should investigate the expression of nk receptor ligands on sars-cov-2-infected cells, in order to better understand the mechanisms underlying nk cell activation in covid-19 disease. further, secretion of igg1 and igg3 antibodies during sars-cov-2 infection (amanat et al., 2020) may induce cd56 dim cd16 + nk cell activation through fc receptor recognition of antibodies, either bound to surface antigens expressed on infected cells or to extracellular virions as immune complexes ( figure 2 ). this interaction might trigger both cytokine production by nk cells and lysis of infected cells through antibodymediated cellular cytotoxicity (adcc), as shown in influenza infection (von holle and moody, 2019) . emerging data highlight the capacity for nk-mediated adcc in response to naturally isolated sars-cov-1 anti-s igg that cross-reacts with sars-cov-2 s glycoprotein when transfected into chinese hamster ovary (cho) cells (pinto et al., 2020) . these findings suggest that triggering nk cell activation may not only contribute to the resolution of infection, but also contribute to the cytokine storm in ards. ex vivo nk cells from peripheral blood of covid-19 patients have reduced intracellular expression of cd107a, ksp37, granzyme b and granulysin, suggesting an impaired cytotoxicity, as well as an impaired production of chemokines, ifn-ɣ and tnf-α (wilk et al., 2020; zheng et al., 2020b) . several pathways may contribute to the dysregulation of nk cells. while influenza virus infects nk cells and induces apoptosis (mao et al., 2009) , lung nk cells do not express the entry receptor for sars-cov-2, ace2, and are therefore unlikely to be directly infected by sars-cov-2 (travaglini et al., 2020) . the majority of nk cells found in human lung display a mature cd16 + kir + cd56 dim phenotype and are able to induce cell cytotoxicity in response to loss of hla class i or through fc receptor signaling, although to a lower extent than their peripheral blood counterpart (marquardt et al., 2017) . killer-immunoglobulin receptors (kirs) are acquired during nk cell development alongside cd16 (fcrγiiia) and are essential for nk cell licensing and subsequent capacity for cytolytic function (sivori et al., 2019) . frequencies of nk cells expressing cd16 and/or kir are decreased in the blood following sars-cov-2 and sars-cov-1 infection, respectively (xia et al., 2004; wang et al., 2020d) . collectively, the data suggest either an impaired maturation of the nk compartment or migration of the mature, circulating nk cells into the lungs or other peripheral tissues of sars-cov-2-infected patients. the immune checkpoint nkg2a is increased on nk cells and cd8 t cells from covid-19 patients . nkg2a inhibits cell cytotoxicity by binding the nonclassical hla-e molecule (braud et al., 1998; brooks et al., 1997) , and this interaction is strongly correlated with poor control of hiv-1 infection (ramsuran et al., 2018) . genes encoding the inhibitory receptors lag3 and tim3 are also upregulated in nk cells from covid-19 patients (wilk et al., 2020; hadjadj et al., 2020) . thus, increased immune checkpoints on nk cells might contribute to viral escape. additionally, covid-19 patients have higher plasma concentrations of il-6 , which significantly correlate with lower nk cell numbers . in vitro stimulation by il-6 and soluble il-6 receptor has previously revealed impaired cytolytic functions (perforin and granzyme b production) by healthy donor nk cells, which can be restored following addition of tocilizumab (il-6r-blockade) (cifaldi et al., 2015) . tnf-α is also upregulated in the plasma of covid-19 patients , and ligand-receptor interaction analysis of peripheral blood scrnaseq data suggests that monocyte-secreted tnf-α might bind to its receptors on nk cells . tnf-α is known to contribute to nk cell differentiation (lee et al., 2009) , which includes downregulation of nkp46 (ivagnes et al., 2018) , though no effect of tnf-α or il-6 on nk cell-mediated adcc has been reported so far. collectively, these data suggest that cross-talk with monocytes might impair nk cell recognition and killing of sars-cov-2infected cells, and antibodies targeting il-6 and tnf-signaling may benefit enhanced nk cell functions in covid-19 patients ( figure 2 ). no studies, to date, have reported ilc1, ilc2, or ilc3 functions in sars-cov-2 infection. all three subsets are present in healthy lung (de grove et al., 2016; yudanin et al., 2019) . ilc2s are essential for the improvement of lung function following influenza infection in mice, through amphiregulin-mediated restoration of the airway epithelium and oxygen saturation (monticelli et al., 2011) . however, ilc2s also produce il-13, contributing to the recruitment of macrophages to the lung and influenza-induced airway hyperreactivity (chang et al., 2011) . indeed, ilcs are involved in the polarization of alveolar macrophages, either towards a m1-like phenotype (ilc1 and ilc3) or a m2like phenotype (ilc2) (kim et al., 2018) . given the increased il-13 concentrations and the dysregulation of the macrophage compartment observed in covid-19 patients, the role played by ilcs in sars-cov-2 infection warrants further investigation. t cells play a fundamental role in viral infections: cd4 t cells provide b cell-help for antibody production and orchestrate the response of other immune cells, whereas cd8 t cells kill infected cells to reduce the viral burden. however, dysregulated t cell responses can result in immunopathology. to better understand the role of t cell responses in sars-cov-2 infection, the pursuit of two major questions is imperative: (1) what is the contribution of t cells to initial virus control and tissue damage in the context of covid-19, and (2) how do memory t cells established thereafter contribute to protective immunity upon re-infection? some tentative answers are beginning to emerge. similar to earlier observations about sars-cov-1 infection , several current reports emphasize the occurrence of lymphopenia with drastically reduced numbers of both cd4 and cd8 t cells in moderate and severe covid-19 cases ( figure 3 ) nie et al., 2020b; wang et al., 2020d; zeng et al., 2020; zheng et al., 2020b) . the extent of lymphopenia -most striking for cd8 t cells in patients admitted to icu -seemingly correlates with covid-19-associated disease severity and mortality diao et al., 2020; liu et al., 2020b liu et al., , 2020c tan et al., 2020a; wang et al., 2020d zeng et al., 2020; zhou et al., 2020c) . patients with mild symptoms, however, typically present with normal or slightly higher t cell counts thevarajan et al., 2020) . the cause of peripheral t cell loss in moderate to severe covid-19, though a phenomenon also observed in other viral infections, remains elusive, and direct viral infection of t cells, in contrast to mers-cov (chu et al., 2016) , has not been reported. several mechanisms likely contribute to the reduced number of t cells in the blood, including effects from the inflammatory cytokine milieu. indeed, lymphopenia seems to correlate with serum il-6, il-10, and tnf-α (diao et al., 2020; wan et al., 2020a) , while convalescent patients were found to have restored bulk t cell frequencies paired with overall lower pro-inflammatory cytokine levels diao et al., 2020; liu et al., 2020a liu et al., , 2020b zheng et al., 2020b) . cytokines such as ifn-i and tnf-α may inhibit t cell recirculation in blood by promoting retention in lymphoid organs and attachment to endothelium (kamphuis et al., 2006; shiow et al., 2006) . however, in an autopsy study examining the spleens and hilar lymph nodes of six patients who succumbed to covid-19, chen et al. observed extensive cell death of lymphocytes and suggested potential roles for il-6 as well as fas-fasl interactions . in support of this hypothesis, the il-6 receptor antagonist tocilizumab was found to increase the number of circulating lymphocytes (giamarellos-bourboulis et al., 2020) . t cell recruitment to sites of infection may also reduce their presence in the peripheral blood compartment. scrnaseq analysis of bronchoalveolar lavage (bal) fluid of covid-19 patients revealed an increase in cd8 t cell infiltrate with clonal expansion . likewise, post-mortem examination of a patient who succumbed to ards following sars-cov-2 infection showed extensive lymphocyte infiltration in the lungs . however, another study that examined post-mortem biopsies from four covid-19 patients only found neutrophilic infiltration (tian et al., 2020a) . further studies are therefore needed to better determine the cause and impact of the commonly observed lymphopenia in covid-19 patients. available information about sars-cov-1-specific t cell immunity may serve as an orientation for further understanding of sars-cov-2 infection. immunogenic t cell epitopes are distributed across several sars-cov-1 proteins (s, n, m as well as orf3), although cd4 t cell responses were more restricted to the s protein (li et al., 2008) . in sars-cov-1 survivors, the magnitude and frequency of specific cd8 memory t cells exceeded that of cd4 memory t cells and virus-specific t cells persisted for at least 6-11 years suggesting that t cells may confer long-term immunity (ng et al., 2016; tang et al., 2011) . limited data from viremic sars patients further indicated that virusspecific cd4 t cell populations might be associated with a more severe disease course, since lethal outcomes correlated with elevated th2 cell (il-4, il-5, il-10) serum cytokines (li et al., 2008) . however, the quality of cd4 t cell responses needs to be further characterized to understand associations with disease severity. few studies have thus far characterized specific t cell immunity in sars-cov-2 infection. in 12 patients recovering from mild covid-19, robust t cell responses specific for viral n, m and s proteins were detected by ifn-γ elispot, weakly correlated with neutralizing antibody concentrations (similar to convalescent sars-cov-1 patients (li et al., 2008) , and subsequently contracted with only n-specific t cells detectable in about one third of the cases post recovery . in a second study, pbmcs from covid-19 patients with moderate to severe ards were analyzed by flow cytometry, approximately 2 weeks after icu admission (weiskopf et al., 2020) . both virus-specific cd4 and cd8 t cells were detected in all patients at average frequencies of 1.4% and 1.3%, respectively, and very limited phenotyping according to cd45ra and ccr7 expression status characterized these cells predominantly as either cd4 tcm (central memory) or cd8 tem (effector memory) and temra (effector memory ra) cells. this study is notable for the use of large complementary peptide pools comprising 1,095 sars-cov-2 epitopes (overlapping 15-mers for s protein as well as computationally predicted hla-i-and -ii-restricted epitopes for all other viral proteins) as antigenspecific stimuli that revealed a preferential specificity of both cd4 and cd8 t cells for s protein epitopes, with the former population modestly increasing over ~10-30 days after initial onset of symptoms. a caveat, however, pertains to the identification of specific t cells by induced cd69 and cd137 co-expression, since upregulation of cd137 by cd4 t cells, in contrast to cd154, may preferentially capture regulatory t cells (treg) (bacher et al., 2016) . further analyses of s protein-specific t cells by elisa demonstrated robust induction of ifn-γ, tnf-α and il-2 concomitant with lower levels of il-5, il-13, il-9, il-10 and il-22. a third report focused on s-specific cd4 t cell responses in 18 patients with mild, severe or critical covid-19 using overlapping peptide pools and induced cd154 and cd137 co-expression as a readout for antiviral cd4 t cells. such cells were present in 83% of cases and presented with enhanced cd38, hla-dr and ki-67 expression indicative of recent in vivo activation (braun et al., 2020) . of note, the authors also detected low frequencies of s-reactive cd4 t cells in 34% of sars-cov-2 seronegative healthy control donors. however, these cd4 t cells lacked phenotypic markers of activation and were specific for c-terminal s protein epitopes that are highly similar to endemic human coronaviruses, suggesting that crossreactive cd4 memory t cells in some populations (e.g., children and younger patients that experience a higher incidence of hcov infections) may be recruited into an amplified primary sars-cov-2-specific response (braun et al., 2020) . similarly, endemic cov-specific cd4 t cells were previously shown to recognize sars-cov1 determinants (gioia et al., 2005) . how previous infections with endemic coronavirus may affect immune responses to sars-cov-2 will need to be further investigated. finally, in general accordance with the above findings on the induction of sars-cov-2specific t cells, using tcrseq, huang et al. and liao et al. reported greater tcr clonality of peripheral blood as well as bal t cells in patients with mild vs. severe covid-19. moving forward, a comprehensive identification of immunogenic sars-cov-2 epitopes recognized by t cells (campbell et al., 2020) , as well as further studies on convalescent patients who recovered from mild and severe disease, will be particularly important. while the induction of robust t cell immunity is likely essential for efficient virus control, dysregulated t cell responses may cause immunopathology and contribute to disease severity in covid-19 patients (figure 3 ). this is suggested in a study by zhou et al. which reported a significantly increased pbmc frequency of polyclonal gm-csf + cd4 t cells capable of prodigious ex vivo il-6 and ifn-γ production only in critically ill covid-19 patients . of note, gm-csf + cd4 t cells have been previously implicated in inflammatory autoimmune diseases such as multiple sclerosis or juvenile rheumatoid arthritis, and high levels of circulating gm-csf + cd4 t cells were found to be associated with poor outcomes in sepsis . additionally, two studies observed reduced frequencies of treg cells in severe covid-19 cases qin et al., 2020) . since treg cells have been shown to help resolve ards inflammation in mouse models (walter et al., 2018) , a loss of tregs might facilitate the development of covid-19 lung immunopathology. similarly, a reduction of γδ-t cells, a subset of t cells with apparent protective antiviral function in influenza pneumonia (dong et al., 2018; zheng et al., 2013) , has been reported in severely sick covid-19 patients lei et al., 2020b) . currently, little is known about specific phenotypical and/or functional t cell changes associated with covid-19. in the majority of preprints and peer-reviewed studies, there are reports of increased presence of activated t cells ( figure 3 ) characterized by expression of hla-dr, cd38, cd69, cd25, cd44 and ki-67 (braun et al., 2020; dong et al., 2020; guo et al., 2020; liao et al., 2020; thevarajan et al., 2020; yang et al., 2020a; zheng et al., 2020a) . generally, independent of covid-19 disease severity, cd8 t cells seem to be more activated than cd4 t cells thevarajan et al., 2020; yang et al., 2020a) , a finding that echoes stronger cd8, than cd4, t cell responses during sars-cov-1 (li et al., 2008) . furthermore, in a case study of 10 covid-19 patients, diao et al. showed that levels of pd-1 increased from prodromal to symptomatic stages of the disease (diao et al., 2020) . pd-1 expression is commonly associated with t cell exhaustion, but it is important to emphasize that pd-1 is primarily induced by tcr signaling; it is thus also expressed by activated effector t cells (ahn et al., 2018) . in addition, several studies reported higher expression of various co-stimulatory and inhibitory molecules such as ox-40 and cd137 , ctla-4 and tigit (zheng et al., 2020a) , and nkg2a . reduced numbers of cd28 + cd8 t cells as well as larger frequencies of pd-1 + /tim3 + cd8 t cells in icu patients were also reported . expression of most of these markers was found to be higher in cd8 than in cd4 t cells, and levels tended to increase in severe vs. non-severe cases, which may be due to differences in viral load. cellular functionality was shown to be impaired in cd4 and cd8 t cells of critically ill patients, with reduced frequencies of polyfunctional t cells (producing more than one cytokine) as well as generally lower ifn-γ and tnf-α production following re-stimulation with pma and ionomycin zheng et al., 2020a zheng et al., , 2020b . similarly, zheng et al. reported that cd8 t cells in severe covid-19 appear less cytotoxic and effector-like with reduced cd107a degranulation and granzyme b (gzmb) production . in contrast, a different study found that both gzmb and perforin were increased in cd8 t cells of severely sick patients (zheng et al., 2020a) . in accordance with the latter observation, when compared to a moderate disease group, convalescent patients with resolved severe sars-cov-1 infection had significantly higher frequencies of polyfunctional t cells, with cd4 t cells producing more ifn-γ, tnf-α and il-2, and cd8 t cells producing more ifn-γ, tnf-α and cd107a, respectively (li et al., 2008) . however, given the vigorous dynamics of acute t cell responses and potential differences in sample timing throughout disease course, these observations are not necessarily mutually exclusive. accordingly, rnaseq data by liao et al. showed that cd8 t cells in the bal fluid of severe covid-19 patients express cytotoxic genes such as gzma, gzmb, and gzmk at higher levels, while klrc1 and xcl1 are enriched in mild cases . in summary, t cells in severe covid-19 seem to be more activated and may exhibit a trend towards exhaustion, based on continuous expression of inhibitory markers such as pd-1 and tim-3 as well as overall reduced polyfunctionality and cytotoxicity. conversely, recovering patients were shown to have an increase in follicular helper cd4 t cells (t fh ) as well as decreasing levels of inhibitory markers along with enhanced levels of effector molecules such as gzm a, gzmb and perforin (thevarajan et al., yang et al., 2020a; zheng et al., 2020b) . collectively, these studies provide a first glimpse into t cell dynamics in acute sars-cov-2 infection, but any conclusions have to be tempered at this stage on account of significant limitations in many of the current investigations. the humoral immune response is critical for the clearance of cytopathic viruses and is a major part of the memory response that prevents reinfection. sars-cov-2 elicits a robust b cell response, as evidenced by the rapid and near-universal detection of virusspecific igm, igg and iga, and neutralizing igg antibodies (nabs) in the days following infection. the kinetics of the antibody response to sars-cov-2 are now reasonably well described . similar to sars-cov-1 infection (hsueh et al., 2004) , seroconversion occurs in most covid-19 patients between 7 and 14 days after the onset of symptoms, and antibody titers persist in the weeks following virus clearance (figure 4) , (haveri et al., 2020; lou et al., 2020; okba et al., 2020; wölfel et al., 2020; wu et al., 2020b; zhao et al., 2020a) . antibodies binding the sars-cov-2 internal n protein and the external s glycoprotein are commonly detected (amanat et al., 2020; ju et al., 2020; . the receptor binding domain (rbd) of the s protein is highly immunogenic and antibodies binding this domain can be potently neutralizing, blocking virus interactions with the host entry receptor, ace2 (ju et al., 2020; wu et al., 2020b) . anti-rbd nabs are detected in most tested patients (ju et al., 2020; . although cross-reactivity to sars-cov-1 s and n proteins and to mers-cov s protein was detected in plasma from covid-19 patients, no cross-reactivity was found to the rbd from sars-cov-1 or mers-cov. in addition, plasma from covid-19 patients did not neutralize sars-cov-1 or mers-cov (ju et al., 2020) . rbd-specific cd19 + igg + memory b cells were single-cell sorted from a cohort of 8 covid-19 donors between days 9-28 after the onset of symptoms (ju et al., 2020) . from their antibody gene sequences, 209 sars-cov-2 specific monoclonal antibodies were produced. the monoclonal antibodies had a diverse repertoire, relatively low or no somatic mutations, and variable binding reactivity, with dissociation constants reaching 10 -8 to 10 -9 , similar to antibodies isolated during acute infections. two potent neutralizing sars-cov-2 rbd-specific monoclonal antibodies were characterized that did not cross-react with the rbd of sars-cov-1 or mers-cov (ju et al., 2020) . together, these results demonstrate that antibody mediated neutralization is virusspecific and likely driven by binding of epitopes within the rbd. the b cell response to a virus serves not only to protect from the initial challenge, but also to offer extended immunity against reinfection. following resolution of an infection, plasma cells formed during the acute and convalescent phases continue to secrete antibodies, giving rise to serological memory. memory b cells that are also formed during the primary infection constitute the second arm of b cell memory. memory b cells can quickly respond to a reinfection by generating new high affinity plasma cells. longterm protection is achieved through the induction of long-lived plasma cells and memory b cells. there is great interest in understanding the life-span of b cell memory responses to sars-cov-2. protection from reinfection has direct medical and social consequences as the world works to develop vaccination strategies and resume normal activities. in covid-19 patients, evidence of near universal seroconversion and the lack of substantial descriptions of reinfection point to a robust antibody response, which, along with the t cell memory response, would offer protection to reinfection. indeed, a case study of a single patient described induction of cd38 hi cd27 hi antibody secreting cells (ascs), concomitant with an increase in circulating follicular t helper cells (tfh) cells (thevarajan et al., 2020) , and a scrnaseq study of pbmc from critically ill and recently recovered individuals revealed a plasma cell population . in addition, igg memory cells specific to the rbd have been identified in the blood of covid-19 patients (ju et al., 2020) . consistent with the development of immunity after covid-19 infection, a recent study of sars-cov-2 infection in rhesus macaques found that two macaques that had resolved the primary infection were resistant to reinfection 28 days later (bao et al., 2020b) . due to the timing of this outbreak, it is not yet possible to know the nature and extent of long-term memory responses, but lessons may again be learned from other human coronaviruses. in the case of the human coronavirus 229e, specific igg and nabs are rapidly induced but wane in some individuals around a year after infection with some residual protection to reinfection (callow et al., 1990; reed, 1984) . the life span of the humoral response following sars-cov-1 infection is also relatively short, with the initial specific igg and nab response to sars-cov-1 diminishing 2-3 years after infection and nearly undetectable in up to 25% of individuals (cao et al., 2007; liu et al., 2006) . a long-term study following 34 sars-cov-1 infected healthcare workers over a 13 years period also found that virus-specific igg declined after several years, but the authors observed detectable virus-specific igg 12 years after infection . in the case of mers-cov, antibodies were detected in 6 of 7 volunteers tested 3 years after infection (payne et al., 2016) . igg specific to sars-cov-2 trimeric spike protein was detectable in serum up to 60 days after symptom onset, but igg titers began decreasing by 8 weeks post-symptom onset (adams et al., 2020) . long term protection from reinfection may also be mediated by reactive memory b cells. a study that analyzed sars-cov-1 s protein-specific igg memory cells at 2, 4, 6 and 8 months post-infection found that s-specific igg memory b cells decreased progressively about 90% from 2 to 8 months after infection (traggiai et al., 2004) . a further retrospective study of 23 individuals found no evidence of circulating sars-cov-1-specific igg + memory b cells 6 years after infection (tang et al., 2011) . this is in contrast to the memory t cell response, which was robustly detected based on induced ifn-γ production (tang et al., 2011) . studies of common coronaviruses, sars-cov-1 and mers-cov indicate that virus specific antibody responses wane over time, and, in the case of common coronaviruses, result in only partial protection from reinfection. these data suggest that immunity to sars-cov-2 may diminish following a primary infection and further studies will be required to determine the degree of long-term protection (figure 4) . several studies have demonstrated that high virus-specific antibody titers to sars-cov-2 are correlated with greater neutralization of virus in vitro and are inversely correlated with viral load in patients ( figure 4 ) wölfel et al., 2020; zhao et al., 2020a) . despite these indications of a successful neutralizing response in the majority of individuals, higher titers are also associated with more severe clinical cases okba et al., 2020; zhao et al., 2020a; zhou et al., 2020a) , suggesting that a robust antibody response alone is insufficient to avoid severe disease ( figure 4 ). this was also observed in the previous sars-cov-1 epidemic, where neutralizing titers were found to be significantly higher in deceased patients compared to patients who had recovered . this has led to concerns that antibody responses to these viruses may contribute to pulmonary pathology, via antibody-dependent enhancement (ade) (figure 4 ). this phenomenon is observed, when non-neutralizing virus-specific igg facilitate entry of virus particles into fc-receptor (fcr) expressing cells, particularly macrophages and monocytes, leading to inflammatory activation of these cells (taylor et al., 2015) . a study in sars-cov-1-infected rhesus macaques found that anti-s-igg contributed to severe acute lung injury (ali) and massive accumulation of monocytes/macrophages in the lung . furthermore, serum containing anti-s ig from sars-cov-1 patients enhanced the infection of sars-cov-1 in human monocyte-derived macrophages in vitro (yip et al., 2014) . ade was also reported with a monoclonal antibody isolated from a patient with mers-cov . somewhat reassuringly, there was no evidence of ade mediated by sera from rats vaccinated with sars-cov-2 rbd in vitro (quinlan et al., 2020) , nor in macaque immunized with an inactivated sars-cov-2 vaccine candidate (gao et al. 2020) . as of now, there is no evidence that naturally developed antibodies towards sars-cov-2 contribute to the pathological features observed in covid-19. however, this possibility should be considered when it comes to experimental design and development of therapeutic strategies. importantly, in all of the descriptions of ade as it relates to coronavirus, the fcr was necessary to trigger the antibody-mediated pathology. high-dose intravenous immunoglobulin (ivig), which may blunt ade, has been trialed in covid-19 patients shao et al., 2020) , but further studies are needed to determine the extent to which ivig is safe or beneficial in sars-cov-2 infection. vaccine trials will need to consider the possibility of antibody-driven pathology upon antigen re-challenge; strategies using f(ab) fragments or engineered fc monoclonal antibodies may prove particularly beneficial in this setting (amanat and krammer, 2020) . with the rapidly growing number of cases in the first few months, numerous reports on predictors of covid-19 severity with small cohorts were released. these offered clinicians and immunologists the first understanding of the clinical course and pathological processes that are associated with the novel sars-cov-2 infection. this section highlights key findings from those studies, with a major focus on the immune factors associated with disease risk or severity. there are currently limited known risk factors for susceptibility to covid-19, although this has been evaluated in several studies. zhao et al. compared the abo blood group distribution in a cohort of 2173 covid-19 patients to that of healthy controls from the corresponding regions . they found blood group a to be associated with a higher risk for acquiring covid-19, when compared to non-a blood groups; blood group o had the lowest risk for the infection. another study demonstrated an identical association (zietz and tatonetti, 2020) , and similar results have been previously described for other viruses (lindesmith et al., 2003) , including for sars-cov-1 (cheng et al., 2005a) . several large collaborative efforts are currently underway to generate, share and analyze genetic data to understand the links between human genetic variation and covid-19 susceptibility and severity, the most prominent of which is the covid-19 host genetics initiative (covid19hg.org). these studies are supported by previous observations on sars-cov-1 that followed the 2003 outbreak, which have identified significant associations between genetic variants and immune phenotypes (chan et al., 2007; wang et al., 2011; zhao et al., 2011) . although identifying such polymorphisms and their associated genes and pathways for sars-cov-2 will require large cohorts, several studies have already highlighted genetic polymorphisms that may potentially impact susceptibility, which remain to be tested in clinical trials. these studies have focused on genetic variants that may impact the expression or function of genes important in viral entry, namely ace2 (sars-cov-2 receptor) and tmprss2 (spike protein activator) (asselta et al., 2020; cao et al., 2020c; renieri et al., 2020; stawiski et al., 2020) . cao et al. identified variants that are potentially expression quantitative trait loci (eqtl) of ace2 (i.e. they may potentially alter ace2 gene expression) and analyzed their frequencies in different populations (cao et al., 2020c) . stawiski et al. listed variants that may be critical in ace2 binding and thereby its function, and compared the frequencies of these variants within different populations (stawiski et al., 2020) . while there are several limitations to these studies, the major question is whether the utility of these biomarkers is replicable in large populations with covid-19 clinical outcomes data and in targeted or large-scale genomic analyses that are currently underway. in addition, these studies will reveal the potential associations between genetic variants and susceptibility in a gene or loci agnostic fashion. several routine blood and serological parameters have been suggested to stratify patients who might be at higher risk for complications to aid in allocation of healthcare resources in the pandemic (table 1 ). serologic markers from routine blood work were reported, by comparing patients with mild/moderate symptoms to those with severe symptoms. this includes different acute phase proteins, such as saa (serum amyloid protein) and c-reactive protein (crp) . interestingly, elevations in crp appear to be unique to covid-19 patients, when compared to other viral infections. other consistently reported markers in non-survivors are increased procalcitonin (pct) and il-6 levels , as well as increased serum urea, creatinine, cystatin c, direct bilirubin, and cholinesterase . overall, inflammatory markers are common in severe cases of covid-19 and appear to correlate with the severity of the symptoms and clinical outcome. moreover, the extensive damage that occurs in specific organs of severe covid-19 patients is possibly related to differences in the expression of ace2 ( figure 5 ) . lymphopenia is the most frequently described prognostic marker in covid-19 (table 1) , and it appears to predict morbidity and mortality even at early stages (fei et al., 2020) . tan et al. proposed a prognostic model based on lymphocyte counts at two time points: patients with less than 20% lymphocytes at days 10-12 from the onset of symptoms and less than 5% at days 17-19 had the worst outcomes in this study (tan et al., 2020a) . wynants et al. compared predictors of disease severity across 7 studies (>1330 patients), highlighting crp, neutrophil-to-lymphocyte ratio (n/l) and lactate dehydrogenase (ldh) as the most significant predictive biomarkers . furthermore, a meta-analysis of 30 covid-19 studies with a total of 53000 patients also attempted to identify early-stage patients with poor prognosis . the most consistent findings across the different studies were elevated levels of crp, ldh and d-dimer, as well as decreased blood platelet and lymphocyte counts zhou et al., 2020d) . systemic and pulmonary thrombi have been reported with activation of the extrinsic coagulation cascade, involving dysfunctional endothelium and monocytic infiltration (poor et al., 2020; varga et al., 2020) ; thrombocytopenia and elevated d-dimer levels may be indicative of these coagulopathies in covid-19 patients with important therapeutic implications (fogarty et al., 2020; poor et al., 2020) . immunological biomarkers are particularly important, as immunopathology has been suggested as a primary driver of morbidity and mortality with covid-19. several cytokines and other immunologic parameters have been correlated with covid-19 severity (table 1) . most notably, elevated il-6 levels were detected in hospitalized patients, especially critically ill patients, in several studies, and are associated with icu admission, respiratory failure, and poor prognosis (chen et al., 2020f; huang et al., 2020b; liu et al., 2020f) . increased il-2r, il-8, il-10, and gm-csf have been associated with disease severity as well, but studies are limited and further studies with larger cohorts of patients are needed to indicate predictive power zhou et al., 2020b) . conflicting results regarding il-1β and il-4 have been reported gong et al., 2020; wen et al., 2020) . although elevated cytokine concentrations have been widely described in covid-19 patients, the vast majority (including il-6, il-10, il-18, ctack, ifn-γ) do not seem to have prognostic value, because they do not always differentiate moderate cases from severe cases (yang et al., 2020b) . this stratification was possible with ip-10, mcp-3, and il-1ra. while there are reports that levels of il-6 at first assessment might predict respiratory failure (herold et al., 2020) , other publications with longitudinal analyses demonstrated that il-6 increases fairly late during the disease's course, consequently compromising its prognostic value at earlier stages . liu et al. developed a web-based tool using k-means clustering to predict prognosis in terms of death or hospital discharge of covid-19 patients using age, comorbidities (binary), and baseline log helper t cell count (th), log suppressor t cell count (ts), and log th/ts ratio . total t cell, helper t cell, and suppressor t cell counts were significantly lower, and the th/ts ratio was significantly higher in patients who died from infection, as compared to patients who were discharged. importantly, most serological and immunological changes observed in severe cases are associated with disease severity, but can not necessarily serve as predictive factors, as they may not have utility in early identification of patients at higher risk. discovery of truly predictive biomarkers and potential drivers of hyper-inflammatory processes requires comprehensive profiling of asymptomatic and mild cases and longitudinal studies which are limited to date. confounding variables including age, gender, comorbidities may dramatically affect associations observed. in addition, direct correlation with patient viral load will be important to provide a greater understanding of underlying causes of morbidity and mortality in covid-19 and the contribution of viral infectivity, hyperinflammation and host tolerance (medzhitov et al., 2012) . in summary, lymphopenia, increases in proinflammatory markers and cytokines and potential blood hypercoagulability characterize severe covid-19 cases with features reminiscent of cytokine release syndromes. this correlates with a diverse clinical spectrum ranging from asymptomatic to severe and critical cases. during the incubation period and early phase of the disease, leukocyte and lymphocyte counts are normal or slightly reduced. after sars-cov-2 binds to ace2 overexpressing organs, such as the gastrointestinal tracts and kidneys, increases in non-specific inflammation markers are observed. in more severe cases, a marked systemic release of inflammatory mediators and cytokines occurs, with corresponding worsening of lymphopenia and potential atrophy of lymphoid organs, impairing lymphocyte turnover (terpos et al., 2020). antivirals are a class of small molecules that function as inhibitors of one or more stages of a virus life cycle. because of similarities between different virus replication mechanisms, some antivirals can be repurposed against various viral infections. currently, most of the available antiviral drugs tested against sars-cov-2 are smallmolecules previously developed against sars-cov-1, mers-cov, or other rna and dna viruses. a number of small molecules with known antiviral activity against other human rna viruses are being evaluated for efficacy in treating sars-cov-2. the ribonucleoside analog β-d-n4-hydroxycytidine (nhc) reduced viral titers and lung injury in mice infected with sars-cov-2 via introduction of mutations in viral rna (sheahan et al., 2017) . further, an inhibitor of host dhodh, a rate-limiting enzyme in pyrimidine synthesis, was able to inhibit sars-cov-2 growth in vitro with greater efficacy than remdesivir or chloroquine xiong et al., 2020) . merimepodib, a non-competitive inhibitor of the enzyme inosine-5′-monophosphate dehydrogenase (impdh), involved in host guanosine biosynthesis, is able to suppress sars-cov-2 replication in vitro . finally, n-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (htcc), which was previously shown to efficiently reduce infection by the less pathogenic human coronavirus hcov-nl63, was also found to inhibit mers-cov and pseudotyped sars-cov-2 in human airway epithelial cells (milewska et al., 2020) . much of the antiviral computational and experimental data currently available for sars-cov-2 focus on targeting the 3cl or main protease (mpro). two prominent drug candidates targeting the sars-cov-2 mpro were designed and synthesized, by analyzing the substrate binding pocket of mpro (dai et al., 2020) . the x-ray crystal structures of the novel inhibitors in complex with sars-cov-2 mpro were resolved at 1.5 å. both compounds showed good pharmacokinetic activity in vitro, and one exhibited limited toxicity in vivo (dai et al., 2020) . multiple studies also aimed to repurpose protease inhibitors to reduce sars-cov-2 titers. nine existing hiv protease inhibitors (nelfinavir, lopinavir, ritonavir, saquinavir, atazanavir, tipranavir, amprenavir, darunavir, and indinavir) were evaluated for their antiviral activity in vero cells infected with sars-cov-2 (yamamoto et al., 2020) and nelfinavir was the most potent at inhibiting viral replication. the coronavirus rna-dependent rna polymerase (rdrp) catalyzes the synthesis of viral rna making it essential for viral replication and a prime target for antiviral inhibitors. remdesivir, an adenosine triphosphate analog, inhibits rdrp by binding to rna strands and preventing additional nucleotides from being added, thereby terminating viral rna transcription ( figure 6a ) (agostini et al., 2018) . remdesivir has been previously shown to be effective against mers-cov and sars-cov-1 infections in animal models (sheahan et al., 2017; de wit et al., 2020) . similarly, a study investigated the efficacy of remdesivir treatments on 12 rhesus macaques with sars-cov-2 infections (williamson et al., 2020) . macaques treated with remdesivir showed a reduction in lung viral loads and pneumonia symptoms, but no reduction in virus shedding. this study does provide evidence that if administered early enough, remdesivir may be effective at treating sars-cov-2 infections. a large number of clinical trials using experimental antiviral drugs are currently underway. a small proportion of them are aimed at repurposing existing antivirals including: arbidol (umifenovir), a broad-spectrum antiviral that blocks viral fusion; lopinavir/ritonavir (lpv/r), a combination of anti-hiv protease inhibitors; favipiravir, an rdrp inhibitor used to treat severe influenza infections (hayden and shindo, 2019) ; and remdesivir ( figure 6a ). chen et al. conducted a multicenter, randomized priority trial on 240 patients with confirmed covid-19 infection to test favipiravir or arbidol . favipiravir was suggested to significantly improve symptom relief. however, the interpretation of this study is limited by a short clinical recovery window of 7 days, only 100 of 236 patients with confirmed covid-19, and the lack of a control group. lpv/r has previously shown efficacy in treating sars-cov-1 (chu et al., 2004) , prompting an early sars-cov-2 clinical trial . 44 patients were enrolled in a trial investigating the efficacy and safety of lpv/r (n=21 patients), arbidol (n=16), or control (n=7) as treatment for mild-to-moderate covid-19. at day 14 of treatment, 76.2%, 62.4% and 71.4% of patients had a positive to negative conversion in the lpv/r, arbidol, and control groups, respectively, with no statistical significance between groups. a randomized controlled trial (rct) with 200 severe covid-19 patients did not observe a significant benefit of lpv/r either (cao et al., 2020a) . however, a study that looked at the impact of earlier administration of (lpv/r) treatment showed that when treatment of lpv/r was started within 10 days of symptom onset, a shorter duration of virus shedding was observed. thus, timing of lpv/r administration may be critical to its efficacy . in a multicenter clinical study assessing the compassionate use of remdesivir in severe covid-19 patients, 53 patients across several countries were treated with remdesivir for 10 days (grein et al., 2020) . 68% of the 53 patients who received remdesivir showed clinical improvement assessed through improved oxygen-support/extubations. without a proper control group, limited conclusions can be drawn with regards to the efficacy of remdesivir from this study. the measured 68% clinical improvement may be in line with average clinical improvement across patients treated with standard of care . a small rct in china with 237 severe covid-19 patients randomized 2:1 to remdesivir vs. placebo demonstrated no significant benefit in time to clinical improvement . almost simultaneously, preliminary results from a larger niaid rct with more than 1000 patients were announced with remdesevir to be associated with quicker time to recovery: 11 days compared with 15 days (ledford, 2020) . a non-significant benefit in mortality was also noted and the trial was stopped early to allow access to remdesivir in the placebo arm. complete safety data and full publication are awaited but this study offers encouraging results and have resulted in an fda emergency use authorization for remdesivir in hospitalized covid-19 patients. chloroquine (cq) and its derivative hydroxychloroquine (hcq) have gained traction as possible therapeutics for covid-19. both drugs are used as antimalarial agents and as immunomodulatory therapies for rheumatologic diseases. however, the application of cq and hcq to covid-19 stems for their past use as antivirals (savarino et al., 2003) , including for sars-cov-1 (keyaerts et al., 2004; vincent et al., 2005) . cq and hcq interfere with lysosomal activity and have been reported to have immuno-modulatory effects. cq augments antigen processing for mhc class i and ii presentation, directly inhibits endosomal tlr7 and tlr9, and enhances the activity of regulatory t cells (garulli et al., 2008; lo et al., 2015; schrezenmeier and dörner, 2020; thomé et al., 2013a thomé et al., , 2013b . early studies involving in vitro infection of host cells with sars-cov-2 demonstrated that both cq and hcq significantly impact endosomal maturation, resulting in increased sequestration of virion particles within endolysosomes. however, there has been conflicting evidence whether cq is more potent than hcq in reducing viral load yao et al., 2020a) . notably, one group reported that treatment of infected cells with hcq before and during infection significantly reduced viral load, suggesting that combined prophylactic and therapeutic hcq use yields maximum efficacy (clementi et al., 2020) . to better understand host immune responses to treatment, one group compared bulk transcriptomic changes in primary pbmcs treated with hcq for 24 hours to pbmcs from confirmed sars-cov-2 positive patients and controls, followed by a comparison of hcq treated primary macrophages to bal and postmortem lung biopsies from covid-19 patients (corley et al., 2020) . across all comparisons, there was minimal overlap between host differential gene expression and genes altered by in vitro hcq treatment. thus, the potential mechanistic action of hcq in the context of sars-cov-2 remains poorly defined. despite the apparent widespread use of hcq and cq to treat covid-19 ( figure 6b ), few controlled clinical trials have been performed so far and thus the potential benefits of these drugs for covid-19 remains controversial. one of the earliest trials (2020-000890-25 ) was a single-arm, open label trial of 600mg daily hcq in 20 covid-19 patients. they reported that hcq alone, or in combination with the antibiotic azithromycin (az), reduced viral load by day 6 (gautret et al., 2020a) . a follow up trial in 80 patients treated with hcq + az reported that 93% of patients had a negative pcr result on day 8 of treatment, and 81.3% were discharged within 10 days of treatment. however, it is important to note that both trials had no control arms (gautret et al., 2020b) . rigorous statistical analyses by others that accounted for the patients excluded from the original analysis found limited evidence for hcq monotherapy (hulme et al., 2020; lover, 2020) . a double blind rrct assessed hcq monotherapy in the treatment of mild covid-19 (chictr2000029559) . a total of 62 patients were enrolled; the treatment arm received 400 mg hcq daily over 5 days. by day 6, patients who received hcq had clinical resolution on average one day earlier than controls; no patients progressed to severe disease compared to 4 patients in the control arm. in a smaller rct treated 30 patients with mild covid-19 (nct04261517) with 400 mg hcq for 7 days, there were no significant differences in the number of patients with negative pcr results on day 7 (all but one positive), median duration of hospitalization, time to fever resolution, or progression of disease on chest ct . the largest rct to date enrolled 150 patients with mild covid-19 across 16 centers in an open label trial of hcq + standard of care (chictr2000029868). there were no significant differences between groups in conversion to negative sars-cov-2 rt-pcr result on day 28 or rate of symptom resolution; there were significantly more adverse events in the hcq arm, though largely non-serious; they reported some evidence for faster normalization of c-reactive protein in the patients who received hcq plus standard of care, but this finding was not significant (tang et al., 2020b) . a metaanalysis including most of the studies described here found no clinical benefits to patients receiving standard of care plus an hcq regimen (shamshirian et al., 2020) . two studies have assessed hcq efficacy in severe covid-19. in a prospective study of 11 patients who had received 600 mg hcq over 10 days with az on days 1-5, there were several patients with worsening clinical status and one death; 8/10 patients had a positive pcr result on day 10 ( molina et al., 2020) . an ongoing double blind rct of patients with severe covid-19 (nct04323527) randomized 81 patients into high dose hcq (600 mg 2x/d for 10 days) or low dose (450 mg/day for 5 days) treatment groups (borba et al., 2020) . recruitment into the high dose arm was halted prematurely due to poor safety outcomes. there was no significant difference in negative pcr results on day 4 or need for mechanical ventilation on day 6. taken together, the clinical trials performed thus far to evaluate the efficacy of hcq ± az for covid-19 have not demonstrated clear evidence of clinical benefit in patients with severe disease. a search of clinicaltrials.gov on april 27, 2020 found 140 clinical trials investigating hcq. this number is rapidly growing, indicating the heightened interest in this therapeutic and pressing need for evidence-based recommendations. because of their anti-inflammatory activity, corticosteroids (cs) are an adjuvant therapy for ards and cytokine storm. however, the broad immunosuppression mediated by cs does raise the possibility that treatment could interfere with the development of a proper immune response against the virus. a meta-analysis of 5,270 patients with mers-cov, sars-cov-1, or sars-cov-2 infection found that cs treatment was associated with higher mortality . a more recent meta-analysis of only sars-cov-2 infection assessed 2,636 patients and found no mortality difference associated with cs treatment, including in a subset of patients with ards (gangopadhyay et al., 2020) . other studies have reported associations with delayed viral clearance and increased complications in sars and mers patients (sanders et al., 2020) . in fact, the interim guidelines updated by the who on march 13, 2020 advise against giving systemic corticosteroids for covid-19 (world health organization, 2020a) . yet, new data from covid-19 are conflicting. one group reported no significant difference in time to viral clearance between patients who received methylprednisolone orally (mild disease) or iv (severe) and those who did not . retrospective studies from groups in china report that patients who were transferred to the icu were less likely to have received cs and that patients with ards who received methylprednisolone had reduced mortality risk . in contrast, another retrospective analysis found that patients who received cs were more likely to have either been admitted to the icu or perished, although the cs treated group also had significantly more comorbidities . a smaller observational study of 31 patients found no association between corticosteroid treatment and time to viral clearance, length of hospital stay, or symptom duration (zha et al., 2020) . a larger study of adjuvant cs in 244 patients with critical covid-19 found no association with 28-day mortality; subgroup analysis of patients with ards found no association between treatment with cs and clinical outcomes . they also found that increased dosage was significantly associated with increased mortality risk. a retrospective review of 46 patients, of whom 26 received iv methylprednisolone, found that early, low-dose administration significantly improved spo2 and chest ct, time to fever resolution, and time on supplemental oxygen therapy (wang et al., 2020h) . others have published perspectives in support of early and short-term, low dose administration based on anecdotal evidence, but not clinical trials. most of the current data on cs use in covid-19 are from observational studies, and support either modest clinical benefit or no meaningful effects. larger rcts are necessary to understand the risks and benefits of cs for these patients; there are 22 trials evaluating various corticosteroids registered on clinicaltrials.gov as of april 27, 2020. one of the first defenses of the human body against rna viruses like sars-cov-2 is the release of type i and iii ifns. it is important to note that type i ifn (ifnα/β) receptors are ubiquitously expressed, so ifnα/β signaling can result in not only antiviral effects, but also in the activation of immune cells that potentially exacerbate pathogenesis. in contrast, type iii ifn (also known as ifnλ) signals mainly in epithelial cells, as well as in a restricted pool of immune cells. because type iii ifns have immunomodulatory functions, subsequent signaling could induce a potent antiviral effect without enhancing pathogenic inflammation (andreakos et al., 2017; prokunina-olsson et al., 2020) . recently, there has been a growing interest in the potential therapeutic impact of modulating the ifn response to disable covid-19 pathogenesis. before the current pandemic, groups have studied the role of ifns in other betacoronavirus infections. one study of 40 patients with sars-cov-1 infection described unresolved elevated type i ifns and ifn-stimulated genes (isgs) in those with poor outcomes (cameron et al., 2007) . others report that exogenous type i ifn does not improve outcomes when given with ribavirin in patients with mers-cov infection (arabi et al., 2020) , suggesting that the role of ifn as a therapeutic or prophylactic option may be strain-or species-specific (sheahan et al., 2020) . interestingly, a recent study by mount sinai virology groups revealed that type i ifn signaling is impaired in the early response to sars-cov-2; in vitro, sars-cov-2 may be more susceptible to type i ifn than sars-cov-1 (blanco-melo et al., 2020) . based on additional evidence that ifn responses to betacoronaviruses are altered as compared to other respiratory viruses (blanco-melo et al., 2020; channappanavar et al., 2016; okabayashi et al., 2006) , trials of ifn-i/iii administration have been initiated (nct04343976, nct04331899). hyperinflammatory responses and elevated levels of inflammatory cytokines, including interleukins (il)-6, 8, and 10, have been shown to correlate with covid-19 severity diao et al., 2020; gong et al., 2020; moore and june, 2020; wan et al., 2020a; xu et al., 2020b) . the drivers of this cytokine storm remain to be established, but they are likely triggered initially by a combination of viral pamps and host danger signals. the heterogeneous response between patients suggests other factors are involved, possibly including the sars-cov-2 receptor, ace2 (hirano and murakami, 2020) . several studies have begun to report the cellular programs that may contribute to the cytokine storm detected in covid-19 patients. one group reported that in the context of generalized lymphopenia, certain subsets of cd4 t cells that express gm-csf and il-6 are more abundant in severe covid-19 patients than in covid-19 patients who do not require intensive care . reports that other major proinflammatory cytokines (tnf-α, ifn-ɣ, il-2) and chemokines (ccl2, ccl3, ccl4) are elevated underscore a potentially pathogenic t h 1/2 program in covid-19 (diao et al., 2020; giamarellos-bourboulis et al., 2020) . histological and single-cell analyses identified monocytes/macrophages as other potent sources of inflammatory cytokines in covid-19 cytokine storm giamarellos-bourboulis et al., 2020; law et al., 2005; moore and june, 2020; zhou et al., 2020b) . studies of other betacoronavirus infections, including sars-cov-1 and mers-cov, have also identified similar hyperactivation of monocytes, macrophages, and dendritic cells as a driver of cytokinemediated immunopathology in humans chien et al., 2006; huang et al., 2020c; konig et al., 2020; wang et al., 2005; wong et al., 2004; xu et al., 2020b; zhou et al., 2020b) . following preliminary reports of il-6 as a critical cytokine in covid-19-associated cytokine release syndrome (crs), monoclonal antibodies that target the il-6 signaling pathway have been proposed as therapeutic candidates (moore and june, 2020) ( figure 6c ). the commercial anti-il-6r antibodies tocilizumab (actemra) and sarilumab (kevzara), and the anti-il-6 antibody siltuximab (sylvant), are now being tested for efficacy in managing covid-19 crs and pneumonia in 13 ongoing clinical trials (table 2 ). to date, only one group has reported preliminary results from a cohort of 20 covid-19 patients treated with a single administration of tocilizumab (400 mg, iv), along with lopinavir, methylprednisolone, and oxygen therapy (chictr2000029765) . the single observation study found recuperated lymphocyte counts in 10/19 patients and resolution of lung opacities in 19/20 patients on chest ct; 19/20 patients were discharged. all patients experienced an improvement in symptoms, and no subsequent pulmonary infections were reported. a second report described an association between use of tocilizumab and reduced likelihood of icu admission and mechanical ventilation. still, in 30 declining patients with severe covid-19 pneumonia, this retrospective study did not report significant improvement in mortality on weighted analysis (roumier et al., 2020) . nevertheless, these studies are encouraging but like other treatment approaches, larger rcts are needed. in addition to the il-6 signaling pathway, other cytokine-/chemokine-associated elements, including il-1r, gm-csf and the chemokine receptor ccr5, have been proposed as potential targets for blockade to manage covid-19 crs ( figure 6c ). finally, complement activation was shown to be over-activated in lungs of covid-19 patients. although results from the randomized trial are not yet published, anti-c5a monoclonal antibody therapy showed benefits in two critically-ill covid-19 patients . while vaccines are being developed to educate a person's immune system to make their own nab against sars-cov-2, there is interest in using adoptive transfer of nab as a therapeutic approach ( figure 6d ). this strategy has already proven to be effective against sars-cov-1 (cao et al., 2010; ho et al., 2005; ter meulen et al., 2004; sui et al., 2004; zhu et al., 2007) and mers-cov (forni et al., 2015; jia et al., 2019; ying et al., 2015) . in the case of sars-cov-2, these efforts are primarily centered on identifying nab made during natural infections or generating nab through animal vaccination approaches. patients who have recovered from sars-cov-2 infection are one potential source of nabs (ju et al., 2020; walls et al., 2020; wölfel et al., 2020; ye et al., 2020; yuan et al., 2020) . in an effort to obtain these nabs, scientists sorted rbd specific memory b cells and cloned their heavy and light variable region to express recombinant forms of the corresponding antibodies (ju et al., 2020; ye et al., 2020) . four of the antibodies produced in these studies (31b5, 32d4, p2c-2f6 p2c-1f11) showed high neutralizing potential in vitro, and all inhibited ace2/rbd binding. successful antibody-mediated neutralization of sars-cov-2 seems to be dependent on the inhibition of ace2/rbd binding. however, ye et al. 2020 showed that nearly all antibodies derived from serum of 26 recovered patients bound to s1 and rbd, with only 3 actually inhibiting ace2/rbd binding . of note, a sars-cov-1 derived neutralizing antibody (47d11) and a single chain antibody against sars-cov-2 (n3130) have also been shown to neutralize sars-cov-2 without inhibiting ace2/rbd binding. thus, blocking this interaction may not be a prerequisite for an effective sars-cov-2 nab. the generation of a hybridoma producing a monoclonal nab against sars-cov-2 provides the potential for a therapeutic ab that can be directly administered to patients to block ongoing infection and potentially even as a prophylactic ( figure 6d ). sars-cov-1 and sars-cov-2 consensus sequences share about 80% identity (tai et al., 2020) . thus, a wide range of sars-cov-1 nabs have been tested for crossreactivity with sars-cov-2, as they could help speed up the development of potential covid-19 treatments. in a recent study, antibodies were isolated from the memory b cells of an individual who recovered from sars-cov-1 infection. while 8 out of 25 isolated antibodies could bind sars-cov-2 s protein, one of them (s309, see table 3 ) also neutralizes sars-cov-2 (pinto et al., 2020) . the combination of s309 with a weakly neutralizing antibody that could bind another rbd epitope led to enhanced neutralization potency. in addition, cr3022 (table 3 ) was found to bind sars-cov-2 rbd , but this antibody did not neutralize sars-cov-2 . computational simulations identified 3 amino-acids that could be modified on cr3022 to enhance its binding affinity with sars-cov-2 rbd (corrêa giron et al., 2020) , potentially augmenting its neutralization potential. animal models represent another tool to generate nabs against sars-cov-2 (table 3) . in one study, the authors developed a protocol to synthetize human nanobodies, smaller antibodies that only contain a heavy variable (vh) chain as first described in camelids (figure 6d ). another antibody isolated from camelids immunized with sars-cov-1 and mers-cov s proteins then fused to a human fc fragment showed neutralization potential against sars-cov-2 (vhh-72-fc) (wrapp et al., 2020) . genetically modified mice with humanized antibody genes can also be used to generate therapeutic monoclonal antibodies, as successfully experimented against ebola virus (levine, 2019) . similar studies are now focused on the use of sars-cov-2 or derivatives to generate highly effective nab in animal models, which can be directly given to infected patients, and efforts are already underway with estimates of clinical trials of pooled antibody cocktails beginning in early summer by regeneron. finally, another approach to nab development is to fuse ace2 protein and the fc part of antibodies as they would bind rbd and potentially be cross reactive among other coronaviruses ( figure 6d ). indeed, an ace2-fc as well as an rbd-fc have been shown to neutralize both sars-cov-1 and sars-cov-2 in vitro. although recombinant nabs could provide an effective treatment, they will require a significant time investment to develop, test, and bring production to scale before becoming widely available to patients. a faster strategy consists of transferring convalescent plasma (cp) from previously infected individuals that have developed high titer nabs that target sars-cov-2 ( figure 6d ). despite the current lack of appropriately controlled trials, cp therapy has been previously used and shown to be beneficial in several infectious diseases such as the 1918 influenza pandemic (luke et al., 2006) , h1n1 influenza (hung et al., 2011) , and sars-cov-1 (arabi et al., 2016) . thanks to the development of serological tests (amanat et al., 2020; cai et al., 2020; xiang et al., 2020b; zhang et al., 2020d) , recovered covid-19 patients can be screened to select plasma with high antibody titers. some studies and case reports on cp therapy for covid-19 have evaluated the safety and the potential effectiveness of cp therapy in patients with severe disease (ahn et al., 2020; duan et al., 2020; pei et al., 2020; shen et al., 2020; zhang et al., 2020b) (table 4 ). these studies were neither controlled nor randomized, but they suggest that cp therapy is safe and can have a beneficial effect on the clinical course of disease. further controlled trials are needed to determine the optimal timing and indication for cp therapy. cp therapy has also been proposed for prophylactic use in at-risk individuals, such as those with underlying health conditions or health care workers exposed to covid-19 patients. the fda has approved the use of cp to treat critically ill patients (tanne, 2020) . determining when to administer the cp is also of great importance, as a study in sars-cov-1 patients showed that cp was much more efficient when given to patients before day 14 day of illness (cheng et al., 2005b) , as previously shown in influenza (luke et al., 2006) . this study also showed that cp therapy was more efficient in pcr positive, seronegative patients. the amount of plasma and number of transfusions needed requires further investigation (table 4) . overall, cp therapy seems to be associated with improved outcomes, and appears to be safe, but randomized clinical trials are needed to confirm this. several clinical trials are currently in progress worldwide (belhadi et al., 2020) the devastating effects of the pandemic spread of sars-cov-2 in a globally naïve population has resulted in unprecedented efforts to rapidly develop, test, and disseminate a vaccine to protect against covid-19 or to mitigate the effects of sars-cov-2 infection. although vaccination has a long and successful history as an effective global health strategy, there are currently no approved vaccines to protect humans against coronaviruses (andré, 2003) . previous work after the sars-cov-1 and mers-cov epidemics has provided a foundation on which many current efforts are currently building upon, including the importance of the s protein as a potential vaccine. diverse vaccine platforms and preclinical animal models have been adapted to sars-cov-2, facilitating fast-moving and robust progress in creating and testing sars-cov-2 vaccine candidates. a number of vaccine candidates are already being tested in clinical trials and more are continuing to progress towards clinical testing. since sars-cov-1 first emerged, the s protein has been favored as the most promising target for vaccine development to protect against coronavirus infection. this particular viral protein has important roles in viral entry and in stimulating the immune response during natural infection and in vaccination studies of both sars-cov-1 and mers-cov (du et al., 2009; song et al., 2019; zhou et al., 2018) , which has also been confirmed for sars-cov-2 . the s protein has been found to induce robust and protective humoral and cellular immunity, including the development of nabs and t cell-mediated immunity (du et al., 2009) . in animal models, correlates of protection against sars-cov-1 infection appear to be induction of nabs against the s protein, although antibodies to other proteins have been detected such as those against nucleoprotein (n) and orf3a (qiu et al., 2005; sui et al., 2005) . nabs are also believed to protect against infection by blocking receptor binding and viral entry, which has been shown with pseudovirus-based neutralization assays nie et al., 2020a) . studies of sars-cov-1 indicate that t cell responses, which were targeted to the s protein after natural infection, included cd8+ t cell responses against the membrane (m) and n proteins, may also be a correlate of protection and that memory t cell responses can persist even 11 years after infection (li et al., 2008; ng et al., 2016) . rbd-specific antiviral t cell responses have also been detected in people who have recovered from covid-19, further validating its promise as a vaccine target (braun et al., 2020; dong et al., 2020) . although the antibodies targeting the rbd of the s protein have greater potential for providing cross-protective immunity, other fragments of the s protein and additional viral proteins have been investigated as target epitopes, especially for t cells. researchers have taken advantage of the genetic similarity between sars-cov-2 and sars-cov-1 and mers-cov and bioinformatics approaches to rapidly identify b and t cell potential epitopes in the s and other proteins, with many studies providing data regarding antigen presentation and antibody binding properties and one study looking into the predicted evolution of epitopes (ahmed et al., 2020; baruah and bose, 2020; bhattacharya et al., 2020; fast et al., 2020; grifoni et al., 2020; lon et al., 2020; zheng and song, 2020) . while the s protein has been found to be the most immunodominant protein in sars-cov-2, the m and n proteins also contain b and t cell epitopes, including some with high conservation with sars-cov-1 epitopes . for sars-cov-1 and mers-cov, animal studies and phase i clinical trials of potential vaccines targeting the s protein had encouraging results, with evidence of nab induction and induction of cellular immunity martin et al., 2008; modjarrad et al., 2019) . these findings are being translated into sars-cov-2 vaccine development efforts, hastening the progress drastically. the who provided a report earlier in april that reported sixty-three vaccine candidates in preclinical testing and three in clinical testing (world health organization, 2020b) . a recent search on may 1, 2020, on clinicaltrials.gov revealed ten registered vaccine candidates (table 5 ). the university of pittsburgh is also looking to move their microneedle array vaccine candidate containing a codon-optimized s1 subunit protein into clinical trials . sanofi and glaxosmithkline (gsk) have recently reported their intent to collaborate and bring together sanofi's baculovirus expression system, which is used to produce the influenza virus vaccine, flublok, to create an s protein vaccine adjuvanted with gsk's as03. the purified inactivated sars-cov-2 virus vaccine candidate (picovacc) of sinovac biotech ltd. will also be starting a clinical trial in china after finding that their candidate protected rhesus macaques from viral challenge without signs of detectable immunopathology . although some of these vaccine candidates are based on platforms that have been used or tested for other purposes, there remain questions regarding their safety and immunogenicity, including the longevity of any induced responses, that will require continual evaluation. although the development of a vaccine to protect against sars-cov-2 infection has progressed at an unprecedented rate and produced an impressive volume of candidates for testing, many challenges lie ahead. the prior knowledge gained after sars-cov-1 was first discovered in 2003 and the subsequent emergence of mers-cov in 2012 provided a significant jumpstart, but the progress of sars-cov-2 vaccine development has already far outstripped the point of the blueprint created before covid-19 became a pandemic. while a variety of platforms are simultaneously being innovated or adapted, they each have strengths and limitations, many of which relate to the delicate balance between safety and immunogenicity. many shortcuts have been taken and will continue to be taken due to the urgency of the ongoing covid-19 pandemic, but significant concerns need to be addressed. one such concern involves the accumulating data supporting the initial assessment that covid-19 is disproportionately severe in older adults. in conjunction with the large body of work related to immune-senescence, these findings indicate that vaccine design should take into consideration the impact of aging on vaccine efficacy (nikolich-žugich, 2018). furthermore, questions remain regarding the possibility of antibody-dependent enhancement of covid-19, with in vitro experiments, animal studies, and two studies of covid-19 patients supporting this possibility (cao, 2020; tetro, 2020; zhao et al., 2020a) . assuming vaccine candidates are identified that can safely induce protective immune responses, additional major hurdles will be the production and dissemination of a vaccine. for some types of vaccines, large-scale production will not be as much of an issue and infrastructure already in place to produce current good manufacturing practice (cgmp)-quality biologics can be repurposed, but this will only be applicable to a subset of the candidates (thanh le et al., 2020) . in order to address the urgent need and stem the covid-19 pandemic, regulatory agencies need to continue to support rapid testing and progression of vaccine candidates, companies need to disseminate important findings directly and openly, and researchers need to investigate correlates of protection using in-depth immune monitoring of patients with a broad range of clinical presentations and clinical trial participants. the newly announced accelerating covid-19 therapeutic interventions and vaccines (activ) is designed to bring together numerous governmental and industry entities to help address this need. the rapid spread of sars-cov-2 and the unprecedented nature of covid-19 has demanded an urgency in both basic science and clinical research, and the scientific community has met that call with remarkable productivity. within months, there has been a significant generation of scientific knowledge that has shed some light on the immunology of sars-cov-2 infections. studies of past coronavirus outbreaks, involving sars-cov-1 and mers-cov, have provided a foundation for our understanding. the pathology of severe cases of covid-19 do indeed resemble certain immunopathologies seen in sars-cov-1 and mers-cov infections, like crs. however, in many other ways, immune responses to sars-cov-2 are distinct from those seen with other coronavirus infections. the emerging epidemiological observation that significant proportions of individuals are asymptomatic despite infection, not only reflects our current understanding that sars-cov-2 has a longer incubation period and higher rate of transmission than other coronaviruses, but also speaks to significant differences in the host immune response. therefore, it is imperative that immune responses against sars-cov-2 and mechanisms of hyperinflammation-driven pathology are further elucidated to better define therapeutic strategies for covid-19. here, we reviewed the recent literature and highlighted hypotheses that interrogate mechanisms for viral escape from innate sensing; for hyper-inflammation associated with crs and inflammatory myeloid subpopulations; for lymphopenia marked by t cell and nk cell dysfunction; and for correlates of protection and their duration, among others. still, additional studies are needed to address how these immune differences across patients or between different types of coronavirus infections dictate who succumbs to disease and who remains asymptomatic. existing studies of sars-cov-1 and mers-cov and ongoing studies of sars-cov-2 will likely provide a robust framework to fulfill that unmet need. overview of innate immune sensing (left) and interferon signaling (right), annotated with the known mechanisms by which sars-cov-1 and mers-cov antagonize the pathways (red). [based on data from preliminary covid-19 studies and earlier studies in related coronaviruses] il-6, il-1β and ifn-i/iii from infected pulmonary epithelia can induce inflammatory programs in resident (alternate) macrophages while recruiting inflammatory monocytes as well as granulocytes and lymphocytes from circulation. sustained il-6, and tnf-ɑ by incoming monocytes can drive several hyperinflammation cascades. inflammatory monocyte-derived macrophages can amplify dysfunctional responses in various ways (listed in top left corner) . the systemic crs-and shlh-like inflammatory response can induce neutrophilic netosis and microthrombosis, aggravating covid-19 severity. other myeloid cells such as pdcs are purported to have an ifn-dependent role in viral control. monocyte-derived cxcl9/10/11 might recruit nk cells from blood. preliminary data suggest that the antiviral function of these nk cells might be regulated through cross-talk with sars-infected cells and inflammatory monocytes. a decrease in peripheral blood t cells associated with disease severity and inflammation is now well documented in covid-19. several studies report increased numbers of activated cd4 and cd8 t cells which display a trend towards an exhausted phenotype in persistent covid-19, based on continuous and upregulated expression of inhibitory markers as well as potential reduced polyfunctionality and cytotoxicity. in severe disease, production of specific inflammatory cytokines by cd4 t cells has also been reported. this working model needs to be confirmed and expanded on in future studies to assess virus-specific t cell responses both in peripheral blood and in tissues. in addition, larger and more defined patient cohorts with longitudinal data are required to define the relationship between disease severity and t cell phenotype. abbreviations: il, interleukin; ifn, interferon; tnf, tumor necrosis factor; gm-csf, granulocyte-macrophage colony-stimulating factor; gzma/b, granzyme a/granzyme b; prf1, perforin. virus-specific igm and igg are detectable in serum between 7 and 14 days after the onset of symptoms. viral rna is inversely correlated with neutralizing antibody titers. higher titers have been observed in critically ill patients, but it is unknown whether antibody responses somehow contribute to pulmonary pathology. the sars-cov-1 humoral response is relatively short lived, and memory b cells may disappear altogether, suggesting that immunity with sars-cov-2 may wane 1-2 years after primary infection. the gastrointestinal tract, kidneys and testis have the highest ace2 expressions. in some organs, different cell types have remarkably distinct expressions, e.g. in the lungs, alveolar epithelial cells have higher ace2 expression levels than bronchial epithelial cells; in the liver, ace2 is not expressed in hepatocytes, kupffer cells nor endothelial cells, but is detected in cholangiocytes, which can explain liver injury to some extent. furthermore, ace2 expression is enriched on enterocytes of the small intestine compared to the colon. ace2, angiotensin-converting enzyme 2; bnp, b-type natriuretic peptide; crp, creactive protein; il, interleukin; n/l, neutrophil-to-lymphocyte ratio; pt, prothrombin time; aptt, activated partial thromboplastin time. a. rdrp inhibitors (remdesivir, favipiravir), protease inhibitors (lopinavir/ritonavir), and anti-fusion inhibitors (arbidol) are currently being investigated in their efficacy in controlling sars-cov-2 infections. b. cq and hcq increase the ph within lysosomes, impairing viral transit through the endolysosomal pathway. reduced proteolytic function within lysosomes augments antigen processing for presentation on mhc complexes and increases ctla4 expression on tregs. c. antagonism of il-6 signaling pathway and of other cytokine-/chemokine-associated targets has been proposed to control covid-19 crs. these include secreted factors like gm-csf that contribute to the recruitment of inflammatory monocytes and macrophages. d. several potential sources of sars-cov-2 neutralizing antibodies are currently under investigation, including monoclonal antibodies, polyclonal antibodies, and convalescent plasma from recovered covid-19 patients. abbreviations: gm-csf, granulocyte-macrophage colony-stimulating factor; cq, chloroquine; hcq, hydroxychloroquine; rdrp, rna-dependent rna polymerase. tai, w., he, l., zhang, x., pu, j., voronin, d., jiang, s., zhou, y., and du, l. (2020) . characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine. cell. mol. immunol. tan, l., wang, q., zhang, d., ding, j., huang, q., tang, y.-q., wang, q., and miao, h. (2020a) . lymphopenia predicts disease severity of covid-19: a descriptive and predictive study. signal transduct target ther 5, 33. tan, w., lu, y., zhang, j., wang, j., dan, y., tan, z., he, x., qian, c., sun, q., hu, q., et al. (2020b) . viral kinetics and antibody responses in patients with covid-19. medrxiv 2020.03.24.20042382 . tanaka, t., narazaki, m., and kishimoto, t. (2016) . immunotherapeutic implications of il-6 blockade for cytokine storm. immunotherapy 8, 959-970. tian, x., li, c., huang, a., xia, s., lu, s., shi, z., lu, l., jiang, s., yang, z., wu, y., et al. (2020b) . potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody. emerg. microbes infect. 9, 382-385. evaluation of antibody testing for sars-cov-2 using elisa and lateral flow immunoassays coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies role of pd-1 during effector cd8 t cell differentiation use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice sars-cov-2 vaccines: status report. immunity a serological assay to detect sars-cov-2 seroconversion in humans vaccinology: past achievements, present roadblocks and future promises interferon-λs: front-line guardians of immunity and homeostasis in the respiratory tract clinical assessment of a novel recombinant simian adenovirus chadox1 as a vectored vaccine expressing conserved influenza a antigens feasibility of a randomized controlled trial to assess treatment of middle east respiratory syndrome coronavirus (mers-cov) infection in saudi arabia: a survey of physicians ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study ace2 and tmprss2 variants and expression as candidates to sex and country differences in covid-19 severity in italy regulatory t cell specificity directs tolerance versus allergy against aeroantigens in humans. cell the pathogenicity of sars-cov-2 in hace2 transgenic mice reinfection could not occur in sars-cov-2 infected rhesus macaques targeting potential drivers of covid-19: neutrophil extracellular traps immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov a brief review of antiviral drugs evaluated in registered clinical trials for covid-19 development of epitope-based peptide vaccine against novel coronavirus 2019 (sars-cov-2): immunoinformatics approach chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (sars-cov-2) infection: preliminary safety results of a randomized, doubleblinded in vitro reconstitution of sars-coronavirus mrna cap methylation hla-e binds to natural killer cell receptors cd94/nkg2a, b and c presence of sars-cov-2 reactive t cells in covid-19 patients and healthy donors nkg2a complexed with cd94 defines a novel inhibitory natural killer cell receptor the impdh inhibitor merimepodib suppresses sars-cov-2 replication in vitro a peptide-based magnetic chemiluminescence enzyme immunoassay for serological diagnosis of corona virus disease the time course of the immune response to experimental coronavirus infection of man interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome lack of innate interferon responses during sars coronavirus infection in a vaccination and reinfection ferret model a role for neutrophils in viral respiratory disease prediction of sars-cov-2 epitopes across 9360 hla class i alleles mers-cov 4b protein interferes with the nf-κb-dependent innate immune response during infection covid-19: immunopathology and its implications for therapy a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease disappearance of antibodies to sars-associated coronavirus after recovery comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations potent and persistent antibody responses against the receptor-binding domain of sars-cov spike protein in recovered patients natural killer cell recruitment to the lung during influenza a virus infection is dependent on cxcr3, ccr5, and virus exposure dose control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon natural killer cells, viruses and cancer association of icam3 genetic variant with severe acute respiratory syndrome innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes developing a new class of engineered live bacterial therapeutics to treat human diseases favipiravir versus arbidol for covid-19: a randomized clinical trial clinical and immunological features of severe and moderate coronavirus disease 2019 severe acute respiratory syndrome coronavirus viroporin 3a activates the nlrp3 inflammasome a pilot study of hydroxychloroquine in treatment of patients restoration of leukomonocyte counts is associated with viral clearance in covid-19 hospitalized patients detectable serum sars-cov-2 viral load (rnaaemia) is closely associated with drastically elevated interleukin 6 (il-6) functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) directly decimates human spleens and lymph nodes (medrxiv) efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial abo blood group and susceptibility to severe acute respiratory syndrome use of convalescent plasma therapy in sars patients in hong kong cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 inhibition of natural killer cell cytotoxicity by interleukin-6: implications for the pathogenesis of macrophage activation syndrome primary severe acute respiratory syndrome coronavirus infection limits replication but not lung inflammation upon homologous rechallenge combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-sars-cov-2 effects in vitro antagonism of dsrna-induced innate immune pathways by ns4a and ns4b accessory proteins during mers coronavirus infection comparative in vitro transcriptomic analyses of covid-19 candidate therapy hydroxychloroquine suggest limited immunomodulatory evidence of sars-cov-2 host response genes on the interactions of the receptorbinding domain of sars-cov-1 and sars-cov-2 spike proteins with monoclonal antibodies and the receptor structure-based design, synthesis and biological evaluation of peptidomimetic aldehydes as a novel series of antiviral drug candidates targeting the sars-cov characterization and quantification of innate lymphoid cell subsets in human lung coronavirus nonstructural protein 15 mediates evasion of dsrna sensors and limits apoptosis in macrophages regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus reduction and functional exhaustion of t cells in patients with coronavirus disease currently available intravenous immunoglobulin (gamunex ® -c and flebogamma ® dif) contains antibodies reacting against sars-cov-2 antigens characterization of anti-viral immunity in recovered individuals infected by sars-cov-2 gammadelta t cells provide protective function in highly pathogenic avian h5n1 influenza a virus infection. front. immunol. 9 nkp46 and nkg2d recognition of infected dendritic cells is necessary for nk cell activation in the human response to influenza infection the spike protein of sars-cov -a target for vaccine and therapeutic development multi-omics evaluation of gastrointestinal and other clinical characteristics of sars-cov-2 and covid-19 the feasibility of convalescent plasma therapy in severe covid-19 patients: a pilot study the human 2b4 and ntb-a receptors bind the influenza viral hemagglutinin and co-stimulate nk cell cytotoxicity low-dose corticosteroid therapy does not delay viral clearance in patients with covid-19 potential t-cell and b-cell epitopes of covid-19 -navigating the uncharted reduction of lymphocyte at early stage elevates severity and death risk of covid-19 patients: a hospitalbased case-cohort study early prediction of disease progression in 2019 novel coronavirus pneumonia patients outside wuhan with ct and clinical characteristics eosinophils and respiratory viruses covid-19 coagulopathy in caucasian patients the heptad repeat region is a major selection target in mers-cov and related coronaviruses severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-b signaling virologic and clinical characteristics for prognosis of severe covid-19: a retrospective observational study in wuhan, china. medrxiv the role of corticosteroids in the management of critically ill patients with coronavirus disease 2019 (covid-19): a meta-analysis machine intelligence design of 2019-ncov drugs prognostic value of nt highly pathogenic coronavirus n protein aggravates lung injury by masp-2-mediated complement overactivation primary cd8+ t-cell response to soluble ovalbumin is improved by chloroquine treatment in vivo tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study complex immune dysregulation in covid-19 patients with severe respiratory failure t-cell response profiling to biological threat agents including the sars coronavirus elucidating the mechanisms of influenza virus recognition by ncr1 correlation analysis between disease severity and inflammation-related parameters in patients with covid-19 pneumonia (medrxiv) a sars-cov-2 protein interaction map reveals targets for drug repurposing compassionate use of remdesivir for patients with severe covid-19 a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov-2 division of labor between lung dendritic cells and macrophages in the defense against pulmonary infections tocilizumab treatment in severe covid-19 patients attenuates the inflammatory storm incited by monocyte centric immune interactions revealed by single-cell analysis coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors impaired type i interferon activity and exacerbated inflammatory responses in severe covid-19 patients serological and molecular findings during sars-cov-2 infection: the first case study in finland influenza virus polymerase inhibitors in clinical development effects of severe acute respiratory syndrome (sars) coronavirus infection on peripheral blood lymphocytes and their subsets level of il-6 predicts respiratory failure in hospitalized symptomatic covid-19 patients covid-19: a new virus, but an old cytokine release syndrome neutralizing antibody response and sars severity chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage clinical features of patients infected with 2019 novel coronavirus in wuhan high levels of circulating gm-csf(+)cd4(+) t cells are predictive of poor outcomes in sepsis patients: a prospective cohort study distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus blood single cell immune profiling reveals the interferon-mapk pathway mediated adaptive immune response for clinical characteristics of 36 non-survivors with covid-19 in wuhan convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection tnfr2/birc3-traf1 signaling pathway as a novel nk cell immune checkpoint in cancer multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase analysis clinical features of covid-19 infection in secondary epidemic area and report potential biomarkers in evaluation single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against mers-cov infection multi-antigenic human cytomegalovirus mrna vaccines that elicit potent humoral and cell-mediated immunity potent human neutralizing antibodies elicited by sars-cov-2 infection a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein type i interferons directly regulate lymphocyte recirculation and cause transient blood lymphopenia in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development. ebiomedicine 000 innate immune crosstalk in asthmatic airways: innate lymphoid cells coordinate polarization of lung macrophages sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum targeting the catecholamine-cytokine axis to prevent sars-cov-2 cytokine storm syndrome severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells hopes rise on coronavirus drug remdesivir tumor necrosis factor-alpha enhances il-15-induced natural killer cell differentiation early preemptive immunomodulators (corticosteroids) for severe pneumonia patients infected with sars-cov-2 potent neutralization of 2019 novel coronavirus by recombinant ace2-ig the phenotypic changes of γδ t cells in covid-19 patients monoclonal antibody therapy for ebola virus disease t cell responses to whole sars coronavirus in humans virushost interactome and proteomic survey of pmbcs from covid-19 patients reveal potential virulence factors influencing sars-cov-2 pathogenesis radiographic findings and other predictors in adults with covid-19. medrxiv an exploratory randomized controlled study on the efficacy and safety of lopinavir/ritonavir or arbidol treating adult patients hospitalized with mild/moderate covid-19 (elacoi) potential host range of multiple sars-like coronaviruses and an improved ace2-fc variant that is potent against both sars-cov-2 and sars the landscape of lung bronchoalveolar immune cells in covid-19 revealed by single-cell rna sequencing safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine human susceptibility and resistance to norwalk virus infection persistent sars-cov-2 presence is companied with defects in adaptive immune system in nonsevere covid-19 patients longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019 novel coronavirus in the early stage. medrxiv hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection prediction of the clinical outcome of covid-19 patients using t lymphocyte subsets with 340 cases from wuhan the potential role of il-6 in monitoring severe case of coronavirus disease two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome clinical features and outcomes of 2019 novel coronavirus-infected patients with cardiac injury association between platelet parameters and mortality in coronavirus disease 2019: retrospective cohort study patients with lrba deficiency show ctla4 loss and immune dysregulation responsive to abatacept therapy middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin sars-cov-2 is sensitive to type i interferon pretreatment prediction and evolution of b cell epitopes of surface protein in sars-cov-2 serology characteristics of sars-cov-2 infection since the exposure and post symptoms onset quantifying treatment effects of hydroxychloroquine and azithromycin for covid-19: a secondary analysis of an open label non-randomized clinical trial genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding adjuvant corticosteroid therapy for critically ill patients with covid-19 (medrxiv) meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? potent antiviral activities of type i interferons to sars-cov-2 infection influenza virus directly infects human natural killer cells and induces cell apoptosis tlr7 and tlr8 activate distinct pathways in monocytes during rna virus infection human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional cd69(-)cd56(dim) cells a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial disease tolerance as a defense strategy covid-19: consider cytokine storm syndromes and immunosuppression mers-cov accessory orfs play key role for infection and pathogenesis human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets htcc as a highly effective polymeric inhibitor of sars-cov-2 and mers-cov the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus cytokine release syndrome in severe covid-19 necroptosis in anti-viral inflammation memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection establishment and validation of a pseudovirus neutralization assay for sars-cov-2 metabolic disturbances and inflammatory dysfunction predict severity of coronavirus disease 2019 (covid-19): a retrospective study middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp3 inflammasome the twilight of immunity: emerging concepts in aging of the immune system review-article cytokine regulation in sars coronavirus infection compared to other respiratory virus infections severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease a dynamic immune response shapes covid-19 progression induction of alternatively activated macrophages enhances pathogenesis during severe acute respiratory syndrome coronavirus infection immunoglobulin fragment f(ab') 2 against rbd potently neutralizes sars-cov-2 in vitro zika virus protection by a single low-dose nucleosidemodified mrna vaccination spike protein binding prediction with neutralizing antibodies of sars-cov-2 persistence of antibodies against middle east respiratory syndrome coronavirus convalescent plasma to treat covid-19: chinese strategy and experiences immunopathogenesis of coronavirus infections: implications for sars ly6e impairs coronavirus fusion and confers immune control of viral disease structural and functional analysis of a potent sarbecovirus neutralizing antibody covid-19 critical illness pathophysiology driven by diffuse pulmonary thrombi and covid-19 and emerging viral infections: the case for interferon lambda dysregulation of immune response in patients with covid-19 in wuhan, china antibody responses to individual proteins of sars coronavirus and their neutralization activities platelet-to-lymphocyte ratio is associated with prognosis in patients with coronavirus disease-19 the sars-cov-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement middle east respiratory coronavirus accessory protein 4a inhibits pkr-mediated antiviral stress responses elevated hla-a expression impairs hiv control through inhibition of nkg2a-expressing cells the behaviour of recent isolates of human respiratory coronavirus in vitro and in volunteers: evidence of heterogeneity among 229e-related strains ace2 variants underlie interindividual variability and susceptibility to covid-19 in italian population. medrxiv interleukin-6 blockade for severe covid-19 pharmacologic treatments for coronavirus disease 2019 (covid-19): a review effects of chloroquine on viral infections: an old drug against today's diseases? mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology hydroxychloroquine versus covid-19: a rapid systematic review and meta-analysis on the use of corticosteroids for 2019-ncov pneumonia clinical efficacy of intravenous immunoglobulin therapy in critical patients with covid-19: a multicenter retrospective cohort study broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov treatment of 5 critically ill patients with covid-19 with convalescent plasma open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes association of cardiac injury with mortality in hospitalized patients with covid-19 in wuhan, china cd69 acts downstream of interferon-alpha/beta to inhibit s1p1 and lymphocyte egress from lymphoid organs severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3·tank·tbk1/ikkε complex middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc human nk cells: surface receptors, inhibitory checkpoints, and translational applications covid-19 early warning score: a multi-parameter screening tool to identify highly suspected patients critical role of type iii interferon in controlling sars-cov-2 infection, replication and spread in primary human intestinal epithelial cells human ace2 receptor polymorphisms predict sars-cov-2 susceptibility. biorxiv potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association evaluation of human monoclonal antibody 80r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants sars-cov-2 and sars-cov spike-rbd structure and receptor binding comparison and potential implications on neutralizing antibody and vaccine development coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus a molecular cell atlas of the human lung from single cell rna sequencing safety, efficacy, and immunogenicity of vgx-3100, a therapeutic synthetic dna vaccine targeting human papillomavirus 16 and 18 e6 and e7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial advancing scientific knowledge in times of pandemics endothelial cell infection and endotheliitis in covid-19 chloroquine is a potent inhibitor of sars coronavirus infection and spread innate lymphoid cells: 10 years on influenza and antibody-dependent cellular cytotoxicity structure, function, and antigenicity of the sars-cov-2 spike glycoprotein multidimensional assessment of alveolar t cells in critically ill patients characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus molecular mechanism for antibody-dependent enhancement of coronavirus entry c-reactive protein levels in the early stage of covid-19 a human monoclonal antibody blocking sars-cov-2 infection persistence of lung inflammation and lung cytokines with high-resolution ct abnormalities during recovery from sars clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in no clear benefit to the use of corticosteroid as treatment in adult patients with coronavirus disease 2019 : a retrospective cohort study characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro human-leukocyte antigen class i cw 1502 and class ii dr 0301 genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection the definition and risks of cytokine release syndrome-like in 11 covid-19-infected pneumonia critically ill patients: disease characteristics and retrospective analysis remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial early, lowdose and short-term application of corticosteroid treatment in patients with severe covid-19 pneumonia: single-center experience from wuhan severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain phenotype of sars-cov-2-specific t-cells in covid-19 patients with acute respiratory distress syndrome immune cell profiling of covid-19 patients in the recovery stage by single-cell sequencing clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 sars and mers: recent insights into emerging coronaviruses prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection virological assessment of hospitalized patients with covid-2019 plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected: interim guidance (world health organization) draft of the landscape of covid-19 candidate vaccines structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications fully human single-domain antibodies against systematic review and critical appraisal of prediction models for diagnosis and prognosis of covid-19 infection the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome potential biochemical markers to identify severe cases among covid-19 patients evaluation of enzyme-linked immunoassay and colloidal gold-immunochromatographic assay kit for detection of novel coronavirus (sars-cov-2) causing an outbreak of pneumonia novel and potent inhibitors targeting dhodh, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against rna viruses including newly emerged coronavirus sars-cov-2 acute myocardial injury of patients with coronavirus disease effective treatment of severe covid-19 patients with tocilizumab pathological findings of covid-19 associated with acute respiratory distress syndrome nelfinavir inhibits replication of severe acute respiratory syndrome coronavirus 2 in vitro factors associated with prolonged viral shedding and impact of lopinavir/ritonavir treatment in patients with sars-cov-2 infection prediction of criticality in patients with severe covid-19 infection using three clinical features: a machine learning-based prognostic model with clinical data in wuhan analysis of adaptive immune cell populations and phenotypes in the patients infected by sars-cov-2 middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets exuberant elevation of ip-10, mcp-3 and il-1ra during sars-cov-2 infection is associated with disease severity and fatal outcome the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) human monoclonal antibodies block the binding of sars-cov-2 spike protein to angiotensin converting enzyme 2 receptor development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus immunodepletion with hypoxemia: a potential high risk subtype of coronavirus disease a highly conserved cryptic epitope in the receptor-binding domains of sars-cov-2 and sars-cov spatial and temporal mapping of human innate lymphoid cells reveals elements of tissue specificity mortality of covid-19 is associated with cellular immune function compared to immune function in chinese han population corticosteroid treatment of patients with coronavirus disease 2019 (covid-19) immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with treatment with convalescent plasma for critically ill patients with sars-cov-2 infection covid-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of which correlate with patient outcome. medrxiv serological detection of 2019-ncov respond to the epidemic: a useful complement to nucleic acid testing antibody responses against sars coronavirus are correlated with disease outcome of infected individuals evasion by stealth: inefficient immune activation underlies poor t cell response and severe disease in sars-cov-infected mice antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 relationship between the abo blood group and the covid-19 susceptibility. medrxiv the immune responses of hla-a*0201 restricted sars-cov s peptide-specific cd8(+) t cells are augmented in varying degrees by cpg odn, polyi:c and r848 interferon induction of ifitm proteins promotes infection by human coronavirus oc43 identification of residues controlling restriction versus enhancing activities of ifitm proteins on entry of human coronaviruses ly6e restricts the entry of human coronaviruses, including the currently pandemic sars-cov incidence, clinical characteristics and prognostic factor of patients with covid-19: a systematic review and metaanalysis novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients gammadelta-t cells: an unpolished sword in human antiinfection immunity functional exhaustion of antiviral lymphocytes in covid-19 patients clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis prospects for a mers-cov spike vaccine hhs public access aberrant pathogenic gm-csf+ t cells and inflammatory cd14+cd16+ pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients a new predictor of disease severity in patients with covid-19 in wuhan safety and immunogenicity of a novel recombinant adenovirus type-5 vector-based ebola vaccine in healthy adults in china: preliminary report of a randomised, double-blind safety and immunogenicity of a recombinant adenovirus type-5 vector-based ebola vaccine in healthy adults in sierra leone: a single-centre, randomised, double-blind potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is enriched in specific cell subsets across tissues testing the association between blood type and covid-19 infection, intubation, and death. medrxiv. lymphocyte count predicted the disease severity and the outcomes of hospitalized patients prognostic value was confirmed in numerous studies decreased continuously in non-surviving patients 13 were reported to be more likely to develop severe illness and to require intensive care unit (icu) admission on admission a risk factor for short-term progression of patients with moderate pneumonia to severe pneumonia confirmed to be of prognostic value in covid-19 in several studies even in early stages, crp levels were positively correlated with lung lesions and reflected disease severity confirmed in numerous studies predicted the risk of acute myocardial injury ldh (lactate dehydrogenase) higher in severe cases than in mild cases we apologize to all authors whose work we could not cite due to space limitations. trainee (phd and md/phd or postdocs) and faculty contributing authors are listed in alphabetical order. illustrations by jill k gregory and used with permission of ©mount sinai health system. we would like to acknowledge funding sources including fastgrant (mm), nci cancer center support grant supplement (mm, rms) burroughs wellcome fund (rms), nih director's early independence award (rms) and nih r01ai081848 (nv, nb, bdg) purpose ro uti ne bl oo dw or k predicted severity independently of other variables . elevated levels and disseminated intravascular coagulation are found in non-survivors . identified patients at risk for acute cardiac injury . other coagulation parameters such as fibrin degradation product levels, longer prothrombin time and activated partial thromboplastin time, were also associated with poor prognosis (tang et al., 2020a) . saa (serum amyloid protein) saa was proposed to be used as an auxiliary index for diagnosis as it was elevated in 80% of the patients in a small cohort . nt-probnp (n terminal pro b type natriuretic peptide) nt-probnp was an independent risk factor of in-hospital death in patients with severe covid-19 . high platelet-to-lymphocyte ratio is associated with worse outcome (qu et al., 2020) . thrombocytopenia is associated with poor outcome and with incidence of myocardial injury in covid-19 (liu et al., 2020h; shi et al., 2020) . im mu nol ogi cal cd4+, cd8+ and nk cell counts lower cd4+, cd8+ and nk cells in pbmc correlated with severity of covid-19 (nie et al., 2020b) . validated by several studies zheng et al., 2020b) . pd-1 and tim-3 expression on t cells increasing pd-1 and tim-3 expression on t cells could be detected as patients progressed from prodromal to overtly symptomatic stages (diao et al., 2020) .expression was higher in infected patients versus healthy controls and in icu versus non-icu patients in both cd4 and cd8 t cells . phenotypic changes in peripheral blood monocytes the presence of a distinct population of monocytes with high forward scatter (cd11b+, cd14+, cd16+, cd68+, cd80+, cd163+, cd206+ which secrete il-6, il-10 and tnf-alpha) was identified in patients requiring prolonged hospitalization and icu admission . cd14+cd16+il-6+ monocytes are increased in icu patients . ip-10, mcp-3, and il-1ra ip-10, mcp-3, and il-1ra were, among 48 examined cytokines, the only ones that closely associated with disease severity and outcome of covid-19 in a study by yang et al. (yang et al., 2020b) . associated with disease severity (hospitalization and icu admission) and poor prognosis (chen et al., 2020f; huang et al., 2020b; liu et al., 2020b . increase levels were associated with higher risk of respiratory failure (yao et al., 2020b) . il-8positively correlated with disease severity (chen et al., 2020d; gong et al., 2020) , with severe cases showing the highest il-8 levels. increased in severe or critical patients as compared to mild patients zhou et al., 2020d ) without a statistically significant difference between severe and critical cases . associated with disease severity in a study that, amongst other cytokines, also associated ferroprotein levels, pct levels, and eosinophil counts with covid-19 severity . il-1β cd14+il-1β+ monocytes are abundant in early recovery patients as shown in a single-cell rna-seq analysis and thought to be associated with cytokine storm . il-1β did not correlate with disease severity in a cross-sectional study with mild, severe and critical patients . il-4 il-4 was associated with impaired lung lesions , but some reports point to a potential mediator effect . in modeling immune cell interaction between dc and b cells in late recovery covid-19 patients, il-18 was found to be important in b cell production of antibodies, which suggests its importance in recovery . gm-csf (granulocytemacrophage colony-stimulating factor) gm-csf+ifn-γ+ t cells are higher in icu than non-icu patients, cd14+cd16+gm-csf+ monocytes are higher in covid-19 patients as compared to healthy controls .il-2 and ifn-γ il-2 and ifn-γ levels were shown to be increased in severe cases . anti-sars-cov-2 antibody levels prolonged sars-cov-2 igm positivity could be utilized as a predictive factor for poor recovery . higher anti-sars-cov-2 igg levels and higher n/l were more commonly found in severe cases . between 10 and 22 days after hospital admission.-body temperature normalized within 3 days in 4 of 5 patients -clinical improvement -viral loads became negative within 12d after the transfusion -neutralizing antibody titers increased 10 severe patients (34-78yo)median 16.5 dpo.-disappearance of clinical symptoms after 3d -chest ct improved -elevation of lymphocyte counts in patients with lymphocytopenia.-increase in sao2 in all patients -resolution of sars-cov-2 viremia in 7 patients increase in neutralizing antibody titers in 5 patients (ahn et al., 2020) key: cord-285900-3rr0j5tk authors: du, lanying; tai, wanbo; yang, yang; zhao, guangyu; zhu, qing; sun, shihui; liu, chang; tao, xinrong; tseng, chien-te k.; perlman, stanley; jiang, shibo; zhou, yusen; li, fang title: introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines date: 2016-11-22 journal: nat commun doi: 10.1038/ncomms13473 sha: doc_id: 285900 cord_uid: 3rr0j5tk viral subunit vaccines often contain immunodominant non-neutralizing epitopes that divert host immune responses. these epitopes should be eliminated in vaccine design, but there is no reliable method for evaluating an epitope's capacity to elicit neutralizing immune responses. here we introduce a new concept ‘neutralizing immunogenicity index' (nii) to evaluate an epitope's neutralizing immunogenicity. to determine the nii, we mask the epitope with a glycan probe and then assess the epitope's contribution to the vaccine's overall neutralizing immunogenicity. as proof-of-concept, we measure the nii for different epitopes on an immunogen comprised of the receptor-binding domain from mers coronavirus (mers-cov). further, we design a variant form of this vaccine by masking an epitope that has a negative nii score. this engineered vaccine demonstrates significantly enhanced efficacy in protecting transgenic mice from lethal mers-cov challenge. our study may guide the rational design of highly effective subunit vaccines to combat mers-cov and other life-threatening viruses. a major goal of viral subunit vaccine development is to rationally design immunogens that can elicit strong neutralizing immune responses in hosts [1] [2] [3] [4] . the receptorbinding domains (rbds) of virus surface spike proteins are the prime candidates for subunit vaccine design because they contain epitopes that can trigger strong immune responses 5 . in addition, viral rbds play essential roles in viral infection cycles by binding to their host receptor for viral attachment 6 . thus, part of the host immune responses elicited by viral rbds can target the receptorbinding region and thereby neutralize viral entry into host cells. however, two problems potentially hinder the development of viral rbds as subunit vaccines. first, viruses can evade the host immune responses elicited by their own spikes or rbd-based vaccines. one of the immune evasion mechanisms by viruses is to use immunodominant non-neutralizing epitopes on their rbds to divert host immune responses, which has been thoroughly illustrated in the case of the hiv receptor-binding subunit gp120 (refs 1,3) . second, when taken out of the context of the full-length spike proteins, recombinant viral rbd vaccines expose large areas of previously buried surfaces that likely contain immunodominant non-neutralizing epitopes. whether an outcome of viral evolution or vaccine design, these immunodominant non-neutralizing epitopes on viral rbds can outcompete other epitopes in triggering host immune responses, so that the resulting immune responses target these non-neutralizing epitopes while neglecting neutralizing epitopes on viral rbds (refs 7-10) . rational design of viral subunit vaccines aims to focus the immune responses on neutralizing epitopes through masking or deletion of immunodominant non-neutralizing epitopes [11] [12] [13] . a critical gap in subunit vaccine design is the lack of an effective way to evaluate an epitope's neutralizing immunogenicity (that is, its capacity to elicit neutralizing immune responses). there have been extensive efforts to predict epitopes' immunogenicity based on the physical and chemical properties of the epitopes 14 . however, these methods are not designed to predict epitopes' 'neutralizing' immunogenicity, which holds the key for subunit vaccine design. although some experimental methods are available to measure the neutralizing immunogenicity of linear epitopes by taking linear peptides out of the context of proteins 15, 16 , these methods do not work for conformational epitopes, which are prevalent on rbd-based viral vaccines 5 . finding a way to measure the neutralizing immunogenicity of different conformational epitopes on viral rbds will tremendously help rational design of viral subunit vaccines. rbd-based coronavirus vaccines have been extensively pursued due to the threat that coronaviruses pose to human health. coronaviruses are enveloped and positive-stranded rna viruses. in 2002-2003, sars coronavirus (sars-cov) infected over 8,000 people with b10% fatality rate 17, 18 . since 2012, mers coronavirus (mers-cov) has infected about 1700 people with b36% fatality rate 19, 20 . the rbds from sars-cov and mers-cov both contain a core structure and a receptor-binding motif (rbm). their core structures are highly similar, but their rbms are markedly different [21] [22] [23] [24] , leading to different receptor specificity: sars-cov recognizes angiotensin-converting enzyme 2 (ace2), whereas mers-cov recognizes dipeptidyl peptidase 4 (dpp4) 6, 25, 26 . both sars-cov and mers-cov rbds are capable of eliciting strong neutralizing antibody responses 5, [27] [28] [29] [30] . on the one hand, because of the enriched neutralizing epitopes in their rbm and their high-yield expression as recombinant proteins, coronavirus rbds are promising subunit vaccine candidates. moreover, because of their relatively simple structures compared with the intact spike proteins, coronavirus rbds provide an excellent model system for structure-based subunit vaccine design. on the other hand, recently determined cryo-em structures of coronavirus spike proteins revealed that whereas the rbm of coronavirus rbds is accessible, large surface areas of the rbd core structure are buried in the full-length spike proteins (supplementary fig. 1) 31, 32 . thus, when these previously buried areas on the surface of the rbd core become exposed in recombinant rbd vaccines, they likely contain immunodominant non-neutralizing epitopes that divert host immune responses. therefore, coronavirus rbds both hold promises and present challenges for vaccine development. it is critical to evaluate the neutralizing immunogenicity of different epitopes on coronavirus rbds, such that immunodominant neutralizing and non-neutralizing epitopes can be preserved and eliminated, respectively. in this study we introduce a novel concept 'neutralizing immunogenicity index' (nii) to evaluate the neutralizing immunogenicity of different epitopes on viral subunit vaccines. as proof-of-concept, we used nii as a tool to identify epitopes with different neutralizing immunogenicity on a mers-cov-rbdbased vaccine. furthermore, we successfully applied this tool and significantly enhanced the efficacy of the mers-cov rbd vaccine in protecting human-dpp4-transgenic mice from lethal mers-cov challenge. our study fills in a critical gap in subunit vaccine design, and can facilitate rational design of subunit vaccines against mers-cov and other life-threatening viruses. to evaluate the neutralizing immunogenicity of a specific epitope on viral rbd vaccines, we can either delete or mask the epitope and then measure the corresponding changes in the vaccine's capacity to elicit neutralizing immune responses. alanine scanning of vaccine-surface residues likely leads to changes in the vaccine's overall immunogenicity that are too subtle to be measurable using currently available experimental methods, while deletion of a whole epitope may disturb the tertiary structure of the viral rbd. instead, in this study we chose to mask the epitope of interest using a host-cell-derived glycan probe. this approach is effective and convenient because the glycan probe can impose steric interference for the access of antibodies and immune cells to the epitope, and also because the glycan probe is unlikely to interfere with the folding and solubility of the rbd. to place the glycan probe on an epitope, we introduced the n-linked glycosylation motif, asparagine-x-threonine (where x is any amino acid other than proline) 33 , onto different epitopes on viral rbd vaccines using site-directed mutagenesis. as proof-of-concept, we chose to study several epitopes on the mers-cov rbd vaccine. the fc-tagged rbd fragment containing residues from 377 to 588 was selected in this study because we previously showed that this fragment is a stable and effective vaccine candidate 34 . four distinct epitopes on this mers-cov rbd fragment were selected based on their location on the rbd surface and their possible functional role in receptor binding: (i) arg511 (located on a protruding loop and in the receptor-binding motif (rbm) region); (ii) ala562 (located on a b-strand and in the rbm region); (iii) val403 (located on a b-strand and in the core region); (iv) thr579 (located on a protruding loop and in the core region) (fig. 1a,b) . on the basis of three-dimensional protrusion index map ( supplementary fig. 2) 35 , the epitopes containing arg511 and thr579 both have a high protrusion index, whereas the epitopes containing ala562 and val403 both have a low protrusion index. we introduced a glycan probe onto each of the above four epitopes on mers-cov rbd. to this end, we introduced single mutations v403n, t579n and a562n to pair with the already existent thr405, thr581 and thr564, respectively, to generate three n-linked glycosylation sites. we also introduced double mutations r511n/e513t to generate the fourth n-linked glycosylation site. each of these glycosylation sites was located in an individual mers-cov rbd fragment. we expressed and purified each of the four mutant rbds in mammalian cells ( supplementary fig. 3a ). to test whether each of the above four epitopes on mers-cov rbd was actually glycosylated, we performed both sds gel electrophoresis and mass spectrometry. compared with the wild type rbd, each of the mutant rbds exhibited a slower electrophoretic mobility on the gel, consistent with additional glycosylation (supplementary fig. 3a ). mass spectrometry revealed that the molecular weights of the mutant rbds were b1-2 kda larger than that of the wild type rbd, which was also consistent with an introduced glycan probe in each of the mutant rbds ( supplementary fig. 3b-f) . for each of the purified mutant rbd samples, there was no visible presence of unglycosylated rbd on the sds gel or the mass spectrometry spectrum ( supplementary fig. 3) . thus, each of the four epitopes on mers-cov rbd had been successfully glycosylated. to understand the correlation between the epitopes' role in receptor binding and their potential to be recognized by immune responses, we examined whether these engineered glycan probes on mers-cov rbd interfered with receptor binding. to this end, we used two alternative approaches. one approach was an alphascreen assay, which analysed the interaction between recombinant rbds and recombinant human dpp4 in solution (fig. 1c) , and the other approach was fluorescence-activated cell sorting (facs), which examined the interaction between recombinant rbds and human dpp4 expressed on the huh-7 cell-surface (fig. 1d) . the results from both assays revealed that the glycan probe located at residue 562 reduced the binding of the rbd to dpp4, the glycan probe located at residue 511 reduced the binding of the rbd to dpp4 even more, and the ones located at residues 403 and 579 had no impact on dpp4 binding. structural analysis of the rbd/dpp4 interactions suggests that a glycan probe located at residue 511 would have serious steric clash with dpp4 binding, whereas a glycan probe located at residue 562 would have partial steric interference with dpp4 binding (fig. 1b) . glycan probes located at residues 403 and 579 would be too far away from the receptor-binding region to have any impact on dpp4 binding. hence, both the biochemical and structural analyses similarly elucidated the role of each of the glycan probes in the binding of the rbd to dpp4. to understand the epitopes' potential to interact with neutralizing monoclonal antibodies (mabs), we analysed how the engineered glycan probes interfered with the binding of the rbd to different neutralizing mabs. we had access to four humanized mabs (hms-1, m336-fab, m337-fab, and m338-fab). all of these mabs were previously shown to be highly potent in neutralizing mers-cov infection of human cells [36] [37] [38] [39] . elisa between each of the rbds and each of the mabs demonstrated that the glycan probe located at residue 511 abolished the binding of the rbd to hms-1 (fig. 2a) , reduced the binding of the rbd to m336-fab and m337-fab (fig. 2b,c) , and had no significant impact on the binding of the rbd to m338-fab (fig. 2d ). in contrast, the glycan probes located at the other three residues, 403, 562 and 579, did not interfere with the binding of the rbd to any of the mabs. the binding sites on the rbd for each of the mabs were previously characterized through mutagenesis and/or structural studies [36] [37] [38] [39] . three of the four mabs, hms-1, m336-fab and m337-fab, bind at or near the epitope containing arg511, whereas all of the mabs bind away from the epitopes containing ala562, val403, and thr579 ( fig. 2e) . overall, among the four selected epitopes, the epitope containing arg511 played the most important role in the binding of neutralizing mabs, and consequently the glycan probe covering this epitope interfered most with the binding of neutralizing mabs. this study thus far has characterized the structural features, receptor binding, and neutralizing mab binding for four selected rbd epitopes using a glycan probe strategy. each of the glycan probes introduced to one of the rbd epitopes only interfered with the binding of dpp4 or mabs that interact with this specific epitope, but had no impact on the binding of dpp4 or mabs to distant epitopes. this observation suggests that each of the glycan probes only shielded the epitope where the glycan probe was attached to, but did not affect the structures of other antigenic sites. it is consistent with findings obtained in studies on another viral spike protein, respiratory syncytial (rsv) virus f protein 40 . to evaluate how the glycan probes altered the neutralizing immunogenicity (that is, the capacity to induce neutralizing immune responses) of mers-cov rbds, we immunized balb/c mice with each of the four rbds containing one of the glycan probes. the immunization schedule is shown in supplementary fig. 4a . sera were collected from mice immunized with each of the rbds, and tested for mers-cov-neutralizing antibodies. compared to the wild type rbd vaccine, the rbds containing a glycan probe at residues 579 and 511 induced significantly higher and lower neutralizing antibody titres, respectively, in mouse sera, whereas the rbds containing a glycan probe at residues 403 and 562 failed to induce significant changes in neutralizing antibody titres in mouse sera ( fig. 3a ; supplementary table 1) . thus, masking the epitope containing arg511 led to reduced neutralizing antibody titres in the immunized mice, demonstrating that this epitope made a positive contribution to the vaccine's overall neutralizing immunogenicity. based on the same rationale, the epitope containing thr579 made a negative contribution and the epitopes containing val403 and ala562 made insignificant contributions to the vaccine's overall neutralizing immunogenicity. the experiments were further repeated twice and similar results were obtained. these results provided a qualitative evaluation of the neutralizing immunogenicity for each of these epitopes. how can we quantitatively evaluate the epitopes' neutralizing immunogenicity? here we introduce a novel concept nii to describe an epitope's neutralizing immunogenicity. nii is defined as the contribution of an epitope to the vaccine's overall neutralizing immunogenicity. it can be determined by masking the epitope with a glycan probe and then measuring the relative change of the vaccine's overall capacity to elicit neutralizing antibody titres (fig. 3b) . based on this definition, we calculated the nii for each of the four epitopes on the rbd ( fig. 3c ; supplementary table 1 ). the epitope containing thr579 had an nii of à 3.0. the negative sign of the nii suggests a negative contribution from this epitope to the vaccine's overall neutralizing immunogenicity, and the value of the nii implicates that masking this epitope using a glycan probe increased the vaccine's overall neutralizing immunogenicity by three fold. conversely, the epitope containing arg511 had an nii of 0.6, suggesting that this epitope made a positive contribution to the vaccine's overall neutralizing immunogenicity and that masking this epitope using a glycan probe reduced the vaccine's overall neutralizing immunogenicity to 60% of that of the wild type vaccine. therefore, the nii can serve as an effective tool to quantitatively evaluate the neutralizing immunogenicity of any epitope on the mers-cov rbd vaccine. to investigate why masking a negative epitope led to enhanced neutralizing immunogenicity of the mers-cov rbd vaccine, we performed a competition assay between neutralizing mabs and mutant-rbd-induced mouse serum for the binding of wild type mers-cov rbd. more specifically, elisa was carried out between a neutralizing mab and mers-cov rbd in the presence of mouse serum induced by the 579-glycosylated mers-cov rbd (fig. 4a,b) . as a comparison, the mouse serum induced by the wild type mers-cov rbd was also included. two different mabs were used in the competition binding assay: hms-1, which binds to the rbm epitope containing arg511 (refs 36,39) , and m336-fab, which binds to the rbm epitope surrounding glu536-asp539 (refs 37,38) . the result showed that the serum induced by the 579-glycosylated rbd inhibited the mab-rbd binding significantly better than the serum induced by the wild type rbd, revealing enhanced neutralizing capability of the mouse serum due to the glycosylation at the 579 position. moreover, the mouse serum induced by the 579-glycosylated rbd demonstrated enhanced binding for at least two separate neutralizing epitopes on the rbm, one surrounding arg511 and the other glu536-asp539. thus, masking an epitope on the rbd core structure with a high negative nii refocuses the host immune response on neutralizing epitopes on the rbm, leading to enhanced neutralizing immunogenicity of the rbd vaccine. rational design of rbd vaccine with enhanced efficacy. to prove that highly effective mers-cov rbd vaccines can be rationally designed based on epitopes' neutralizing immunogenicity, we investigated the efficacy of two engineered mers-cov rbd vaccines using virus challenge studies. these engineered rbd vaccines have a negative epitope (that is, the epitope containing thr579 and with an nii of à 3.0) and a positive epitope (that is, the epitope containing arg511 and with an nii of 0.6) masked, respectively, by a glycan probe. we chose to mask the epitopes rather than deleting them or mutating all of their residues to alanines because introducing a glycan is more convenient in practice and less disruptive to the immunogen's tertiary structure. the wild type rbd vaccine was used as a control. the animal model for vaccine testing was the lethal transgenic mouse model expressing human dpp4 (hdpp4-tg mice) 41, 42 . these mice were chosen for analysis because they are very susceptible to mers-cov and also because preventing disease in these mice is a stringent test of efficacy. the immunization schedule is shown in supplementary fig. 4b . briefly, hdpp4-tg mice were immunized with each of the rbd vaccines and challenged with mers-cov, and the survival rate and weight changes of the mice were recorded. the efficacies of the rbd vaccines were evaluated based on the morbidity and mortality of the immunized and challenged mice. first, hdpp4-tg mice immunized with the negative-epitopemasked rbd vaccine (that is, rbd containing t579n mutation) all survived mers-cov challenge (100% survival rate), whereas hdpp4-tg mice immunized with the wild type rbd vaccine and with the positive-epitope-masked rbd vaccine (that is, rbd containing r511n/e513t mutations) demonstrated survival rates of 67 and 17%, respectively, after mers-cov challenge (fig. 5a) . second, mers-cov challenge did not cause any weight loss in hdpp4-tg mice immunized with the negative-epitope-masked rbd vaccine, but led to significant weight loss in hdpp4-tg mice immunized with either the wild type rbd vaccine or the positive-epitope-masked rbd vaccine (fig. 5b) . the experiments were further repeated twice and similar results were obtained. these results revealed the enhanced efficacy of the hms-1 mab current vaccine design lacks an effective approach to evaluate the neutralizing immunogenicity of epitopes on viral subunit vaccines. in this study, we have developed a novel approach to measure vaccine epitopes' neutralizing immunogenicity. using the mers-cov rbd as a model, we singly mask selected epitopes using host-derived glycan probes, and then measure the corresponding changes in the vaccine's overall neutralizing immunogenicity. we have also developed a method for calculating the nii for the selected epitopes. an epitope's neutralizing immunogenicity contains two parts: the neutralization capacity and immunogenicity. on the one hand, an epitope's neutralizing capacity is determined by the physical overlap of the epitope with the receptor-binding region and the potential role of the epitope in receptor binding. on the other hand, an epitope's immunogenicity is determined by its immune selfness (that is, how similar or dissimilar the viral epitope is to a host-originated epitope), protrusion, and other physical and chemical properties of the epitope. logically, an epitope's nii is correlated with a combination of factors such as immune selfness, protrusion, potential overlap with receptor-binding region, and more. because of the complex nature of nii, it is unlikely that the nii can be reliably predicted by software; instead, this study demonstrates that nii can be experimentally measured using the glycan probe approach. as proof-of-concept, we measured the nii for four distinct epitopes on the mers-cov rbd vaccine, and also characterized the protrusion index, receptor binding, and monoclonal antibody binding of the rbds each with an epitope masked by a glycan probe. the results revealed that the epitopes with a high and low protrusion index tend to have an nii with a high and low absolute value, respectively. in addition, epitopes within the receptor-binding region tend to have a positive nii, and the epitopes located outside the receptor-binding region tend to have a negative nii. we cannot correlate the immune selfness of epitopes with nii because there is no good method to evaluate the immune selfness of conformational epitopes. overall, in rational design of viral subunit vaccines, the epitopes with a high positive nii should be preserved and exposed, while those with a high negative nii should be eliminated via deletion or masking. indeed, our study has identified an epitope containing thr579 as one with a high negative nii on mers-cov rbd. thr579 is located on a protruding loop and away from the receptor-binding region, both of which contribute to its high negative nii. importantly, thr579 is buried inside the full-length coronavirus spike proteins, and only becomes exposed on the surface of the recombinant mers-cov rbd vaccine as an outcome of subunit vaccine design ( supplementary fig. 1 ). to overcome this limitation of subunit vaccine design, the newly exposed epitopes with a high negative nii need to be masked or deleted. to apply the nii strategy to vaccine design, we successfully enhanced the efficacy of the mers-cov rbd vaccine in virus challenge studies by masking its strong negative epitope (that is, the epitope containing thr579, with an nii of à 3.0) with a glycan probe. this engineered vaccine effectively protected hdpp4-transgenic mice from a lethal mers-cov infection. compared with the wild type rbd vaccine, mice immunized with the engineered rbd vaccine showed increased neutralizing antibody responses in their sera; when challenged by mers-cov, they also demonstrated higher survival rate and less weight loss. these results prove that negative epitopes should be eliminated in vaccine design. in contrast, another engineered vaccine with a positive epitope masked (that is, the epitope containing arg511, with an nii of 0.6) showed reduced efficacy in virus challenge studies, confirming that positive epitopes should be preserved and exposed in vaccine design. taken altogether, we validated both the significance and feasibility of the nii strategy in vaccine design by successfully engineering a variant form of the mers-cov rbd vaccine with significantly enhanced efficacy. overall, our study contributes to viral subunit vaccine design in the following ways. first, our study introduces a new concept nii for the evaluation of how individual epitopes contribute to the overall neutralizing immunogenicity of subunit vaccines. previous studies could not evaluate the neutralizing immunogenicity of conformational b-cell epitopes that dominate coronavirus rbd vaccines. second, using the nii strategy our study identified an immunodominant non-neutralizing epitope on the surface of the mers-cov rbd core structure. this result shows that exposure of previously buried epitopes on viral subunit vaccines poses a challenge for subunit vaccine design. this concept, although needing further investigations, may be critical for the development of many viral rbd-based vaccines. third, our study demonstrates that masking an immunodominant non-neutralizing epitope with a negative nii value on the surface of the mers-cov rbd core structure can shift host immune responses towards the neutralizing epitopes in the rbm region, providing means to overcome the limitation of viral subunit vaccines from vaccine design. previous studies showed that hypervariable regions on hiv gp120 divert host immune responses and that masking these regions can shift host immune responses towards conserved neutralizing epitopes 11, 12 , providing means to overcome the limitation of viral subunit vaccines from viral evolution. fourth, although the nii strategy was used in the current study to improve the efficacy of viral subunit vaccines, it can also be potentially helpful in other epitope-based vaccine research. for example, previous studies masked or resurfaced non-neutralizing epitopes on viral immunogens, and used the engineered immunogens as baits to screen from neutralizing sera for monoclonal antibodies that bind to conserved neutralizing epitopes [43] [44] [45] [46] . it is conceivable that the nii strategy can help identify immunodominant non-neutralizing epitopes on immunogens, allowing more targeted epitope modifications for efficient antibody screening. finally, our study suggests that a three-dimensional 'neutralizing immunogenicity map' (nim) can be drawn to describe the distribution of epitopes with different neutralizing immunogenicity on the surface of viral subunit vaccines. such an nim can guide targeted masking of multiple strong negative epitopes, further enhancing the efficacy of viral subunit vaccines. we believe that our approach can facilitate the rational subunit vaccine design not only for coronaviruses such as mers-cov and sars-cov, but also for other life-threatening viruses such as hiv, influenza virus, and ebola virus. cell lines. hek293t (human embryonic kidney) and vero e6 (monkey kidney) cells were obtained from american type culture collection. huh-7 (human hepatoma) cells were kindly provided by dr charles m. rice at rockefeller university. these cell lines were cultured in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs), 2 mm l-glutamine, 100 units ml à 1 penicillin, and 100 mg ml à 1 streptomycin (life technologies inc.). sf9 insect cells were purchased from life technologies inc., and cultured in sf-900 iii sfm medium supplemented with 100 units ml à 1 penicillin and 100 mg ml à 1 streptomycin (life technologies inc.) expression and purification of recombinant proteins. the expression and purification of recombinant mers-cov rbd was carried out as previously described 34 . briefly, wild type (wt) rbd (residues 377-588; genbank accession number: afs88936.1) containing a c-terminal human igg 1 fc tag was expressed in hek293t cells, secreted into the cell culture supernatant, and purified by protein a affinity chromatography (ge healthcare). mutant rbd fragments containing engineered glycan probes were constructed via site-directed mutagenesis, and expressed and purified in the same way as the wild type rbd. the expression and purification of recombinant human dpp4 was carried out as previously described 47 . briefly, human dpp4 ectodomain (residues 39-766; genbank accession no. np_001926.2) containing an n-terminal human cd5 signal peptide and a c-terminal his 6 tag was expressed in insect sf9 cells using the bac-to-bac expression system (life technologies inc.), secreted to cell culture medium, and purified sequentially on hitrap nickel chelating hp column and superdex 200 gel filtration column (ge healthcare). sds gel electrophoresis. 5 mg wild type or mutant mers-cov rbds were subjected to sds gel electrophoresis under denatured condition. protein bands were stained using coomassie brilliant blue r (sigma-aldrich), and image captured using myecl imager (life technologies inc.). mass spectrometry. wild type or mutant mers-cov rbds at 100 mm concentration in 20 mm tris-cl, ph 7.4, 200 mm nacl was ultrafiltrated with deionized water five times using an amicon ultra centrifugal filter with a 10 kda molecular weight cutoff. the desalted protein samples were subjected to maldi-tof mass spectrometry at tufts university core facility. mass spectrometry was performed in linear mode for molecular weight screening. alphascreen protein-protein binding assay. binding between recombinant mers-cov rbds and recombinant human dpp4 was measured using an alphascreen assay as previously described 34, 36 . briefly, 3 nm wild type or mutant mers-cov rbd with a c-terminal fc tag was incubated with 300 nm human dpp4 with a c-terminal his 6 tag at room temperature for 1 h. alphascreen protein a acceptor beads and nickel chelate donor beads (perkinelmer life sciences) were added to the mixture at a final concentration of 5 mg ml à 1 each. after incubation at room temperature for 1 h, the alphascreen signal was measured using an enspire plate reader (perkinelmer life sciences), reflecting the binding affinity between the two proteins. facs. the binding between recombinant mers-cov rbds and human dpp4 expressed on the huh-7 cell-surface was measured using fluorescence-activated cell sorting (facs) as previously described 28, 36 . briefly, huh-7 cells were incubated with wild type or mutant mers-cov rbd (1.25 mg ml à 1 ) at room temperature for 30 min, followed by addition of fitc-conjugated anti-human-igg-fc polyclonal antibody (1:50 dilution) (sigma-aldrich) for 30 min. the amounts of rbd-bound huh-7 cells were measured using flow cytometry, and the binding affinity between rbd and cell-surface dpp4 was characterized as median fluorescence intensity. animal immunization and sample collection. animal immunization and sample collection were carried out as previously described 34 . briefly, balb/c mice were subcutaneously immunized with wild type or mutant mers-cov rbd (10 mg per mouse) in the presence of montanide isa51 adjuvant 34, 48 . pbs plus montanide isa51 was included as a negative control. immunized mice were boosted twice with the same immunogen and adjuvant at a 3-week interval, and sera were collected 10 days after the last immunization for detection of neutralizing antibodies. elisa. the binding between recombinant mers-cov rbd and neutralizing mabs was measured using elisa as previously described 36 . briefly, elisa plates were pre-coated with the same amount of wild type or mutant rbd (1 mg ml à 1 ) overnight at 4°c. after blocking with 2% non-fat milk at 37°c for 2 h, serially diluted mabs were added to the plates and incubated at 37°c for 1 h. after washes, the plates were incubated at 37°c for 1 h with horseradish-peroxidaseconjugated anti-human-igg-fab polyclonal antibody (1:5,000 dilution) (sigma-aldrich). enzymatic reaction was carried out using substrate 3,3 0 ,5,5 0tetramethylbenzidine (life technologies inc.) and stopped with 1 n h 2 so 4 . absorbance at 450 nm (a 450 ) was measured using elisa plate reader (tecan group ltd.). the competition between neutralizing mabs and mutant-rbd-induced mouse serum for the binding of wild type mers-cov rbd was carried out using elisa as described above, except that the binding between wild type rbd and the neutralizing mab (hms-1 or m336-fab at 5 mg ml à 1 concentration) was performed in the presence of serially diluted mouse serum (t579n-rbdinduced, wild-type-rbd-induced, or pbs-induced). the rbd-mab binding was detected by addition of horseradish-peroxidase-conjugated anti-human-igg-fab polyclonal antibody (1:5,000 dilution) and subsequent enzymatic reaction. live mers-cov neutralization assay. a micro-neutralization assay was carried out to test neutralizing antibodies against live mers-cov as previously described 36 . briefly, serially diluted mouse sera were incubated at room temperature for 1 h with b100 infectious mers-cov virions (emc-2012 strain), and were then incubated with vero e6 cells at 37°c for 72 h. the neutralizing capability of the mouse sera was measured by determining the presence or absence of virus-induced cytopathic effect (cpe). neutralizing antibody titres were expressed as the reciprocal of the highest dilution of sera that completely inhibited virus-induced cpe in at least 50% of the wells (nt 50 ). mers-cov challenge studies. mers-cov challenge studies were carried out using human-dpp4-transgenic mice as previously described 41 . briefly, mice were intramuscularly immunized with wild type or mutant mers-cov rbd (5 mg per mouse) in the presence of aluminium adjuvant 49 , and boosted once 4 weeks after the initial immunization. 12 weeks after the second immunization, mice were challenged with mers-cov (emc-2012 strain, 10 4 tcid 50 ), and observed for 21 days for detection of survival rate and weight changes. statistical analyses. in fig. 1c -d, comparisons between wt rbd and each of the mutant rbds in their binding to recombinant dpp4 by alphascreen (fig. 1c) or to cell-surface dpp4 by facs (fig. 1d) were done using two-tailed t-test (***, po0.001; 3 measurements for each rbd in figs 1c and 4 measurements for each rbd in fig. 1d) . in figs 2a-d, nonlinear regression was performed using a log(inhibitor) versus normalized response-variable slope model. r 2 of curve fit is larger than 0.97 for all curves in fig. 2athe extra sum-of-squares f test (***, po0.001; 12 different dilutions of each mab, 4 measurements at each dilution for each mab). in fig. 3a , comparisons between wt rbd and each of the mutant rbds in their capacity to induce neutralizing serum in mice were done using two-tailed t-test (*, po0.05; 4 measurements for each rbd). in fig. 4 , nonlinear regression was performed using a log(inhibitor) versus normalized response-variable slope model. r 2 of curve fit is larger than 0.98 for all curves in fig. 4 . comparisons between wt-rbd-induced serum and t579n-rbd-induced serum in their inhibition of rbd/mab binding by elisa were done using the extra sum-of-squares f test (***, po0.001; 4 different dilutions of each serum, 4 measurements at each dilution for each serum). all statistical analyses were performed using graphpad prism 6 software. data availability. all relevant data are available from the authors. rational design of vaccines to elicit broadly neutralizing antibodies to hiv-1. cold spring harb structure-based antigen design: a strategy for next generation vaccines advances in structure-based vaccine design structural vaccinology starts to deliver the spike protein of sars-cov-a target for vaccine and therapeutic development receptor recognition mechanisms of coronaviruses: a decade of structural studies immunodominance of cd4 t cells to foreign antigens is peptide intrinsic and independent of molecular context: implications for vaccine design immunodominance: a pivotal principle in host response to viral infections immunodominance with progenitor b cell diversity in the neutralizing antibody repertoire to influenza infection immunodominance of conformation-dependent b-cell epitopes of protein antigens refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope immune focusing and enhanced neutralization induced by hiv-1 gp140 chemical cross-linking designing immunogens to elicit broadly neutralizing antibodies to the hiv-1 envelope glycoprotein prediction of immunogenicity for therapeutic proteins: state of the art bcr-abl fusion regions as a source of multiple leukemiaspecific cd8 þ t-cell epitopes ctl responses of hla-a2.1-transgenic mice specific for hepatitis c viral peptides predict epitopes for ctl of humans carrying hla-a2.1 a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structure of sars coronavirus spike receptor-binding domain complexed with receptor crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development receptor-binding domain of sars-cov spike protein induces long-term protective immunity in an animal model current advancements and potential strategies in the development of mers-cov vaccines pre-fusion structure of a human coronavirus spike protein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer assembly of asparagine-linked oligosaccharides searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design an algorithm that identifies protruding atoms in proteins a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection prefusion f-specific antibodies determine the magnitude of rsv neutralizing activity in human sera multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease rational design of envelope identifies broadly neutralizing human monoclonal antibodies to hiv-1 a combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor high throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface defining a protective epitope on factor h binding protein, a key meningococcal virulence factor and vaccine antigen receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus 720 and 51: a new generation of water in oil emulsions as adjuvants for human vaccines current adjuvants and new perspectives in vaccine formulation we thank drs. dimiter s. dimitrov and tianlei ying at the national institutes of health for providing m336, m337 and m338 mabs this work was supported by nih grants r01ai089728 and r01ai110700 (f.l.), nih grant po1ai060699 (s.p.), nih grants r01ai098775, u01ai124260, r21ai109094 and the intramural fund of new york blood center nyb000348 (l.d. and s.j.), nih grant r21ai113206-01 and pilot grants from the center for biodefense and emerging infectious diseases and from the galveston national laboratory (c.k.t), china national l.d., s.j., y.z., f.l. designed the experiments; l.d., w.t., y.y., g.z., q.z., s.s., c.l. and x.t. performed the experiments; l.d., c.k.t., s.p., s.j., y.z., f.l. analysed the data; l.d., s.p., s.j., y.z. and f.l. wrote the paper. key: cord-018504-qqsmn72u authors: caron, rosemary m. title: public health lessons: practicing and teaching public health date: 2014-09-23 journal: preparing the public health workforce doi: 10.1007/978-3-319-07290-6_4 sha: doc_id: 18504 cord_uid: qqsmn72u the following four cases represent events that actually occurred at the local, statewide, national, and international levels. a general, succinct overview is provided of each case with references listed should the reader want to access additional resource materials. the concise format of these cases is intended to generate questions. following the general overview of each case, i examine the lessons learned from the practitioner and educator perspective and i list the skills necessary to address the issues in the case. the reader will note that there are skills that are essential for the public health practitioner to master, whether one is in an internship, entry-level position, or the director of a public health organization and so these skills are consistently listed. i encourage the reader to regularly keep abreast of the news locally and abroad and to set aside time before a staff meeting or supervisory group meeting, or use the first few minutes of a class to discuss these issues. ask your workforce or students, “are we ready to handle such an event if it were to occur here?”; “what resources would we need to have accessible?”; “have we partnered with the correct agencies in the community?”; “do we have an established, trusted presence in the community?”; “who else do we need on our team?”; “do we need training in a specialty area, e.g., emergency preparedness?”; “what skills have we mastered and what skills do we need to obtain?” the discussion-based questions are endless but one runs the risk of not being prepared, either individually, or in their agency, should they not discuss how public health events are occurring around us daily. i encourage you to adapt these selected cases to use in your organization and/or classroom. discussing these issues and reviewing the lessons learned will only help us to be better prepared public health practitioners and educators of public health students. prior to the 1978 federal ban on lead paint and the housing in the center of this city is of very poor quality (mhd 2013a) . manchester is the most racially and ethnically diverse community in the state. the city's designation as a refugee resettlement community contributes to this richness in diversity. manchester experiences disparity in socioeconomic status and health, similar to other larger urban communities: manchester, new hampshire represents an urban microcosm of the childhood lead poisoning problem. one-third of all childhood lead poisoning cases occur predominantly in the center of this urban community (mhd 2013a; nhdhhs 2006) . in 2006, 2.7 % of children in manchester who had been screened for lead poisoning had eblls, as compared to 1.3 % of the new hampshire total (nhdhhs 2006) . in 2007, approximately 25 % of the leadpoisoned children in the local health department's caseload were refugees or children of refugees. (mhd 2013b) sargent et al. (1995) previously examined clp in urban, suburban, and rural communities in massachusetts and reported that "…those children living in communities with high rates of poverty, single-parent families, and pre-1950s housing and low rates of home ownership were 7-10 times more likely to have lead poisoning" (p. 531). the center city of manchester reflects similar demographics and is a community at risk for clp. pediatric fatality: although fatalities due to clp are rare, the first pediatric fatality to occur in over a decade in the usa occurred in this community of manchester, nh. the fatality occurred in a 2-year-old sudanese refugee child who had resettled in 2000 to this community with her mother and siblings from a refugee camp in egypt. the family resided in an apartment in a tenement building that was constructed in the 1920s. approximately 8 weeks following resettlement, this child acquired an ebll of 391 micrograms of lead per deciliter of blood. the cdc's action level in 2000 was 10 micrograms per deciliter of blood. hence, this child's ebll was 39 times above cdc's action level at that time. an environmental and epidemiological investigation determined that due to the child's exposure to lead paint dust and chips in the apartment she lived in with her family and her underlying conditions of pica (a craving for nonfood substances) and malnutrition resulted in her acquiring an ebll in a short period of time. the child died as a result of complications triggered by the ebll (caron et al. 2001) . furthermore, despite the existence of federal regulations developed by the environmental protection agency (epa) that require property owners and managers to provide families with information about lead poisoning and any lead hazards in the home before its sale or lease, the investigation into this case revealed that this information was not communicated in a manner that was understood by the mother of this child (caron et al. 2001) . lessons learned: this tragic event underscored the need for attention to be paid to those public health problems that persist in the environment, i.e., those issues that the community may live with because there is no feasible solution to completely eliminate the risk. due to the older housing stock in the community that contains lead paint, the cdc named the community and its surrounding towns as a universal screening site. this means that every child at 1 and 2 years of age must be screened for exposure to lead ). this is a form of secondary prevention. the gold standard is primary prevention where exposure to lead would not occur in the first place, thus the risk is removed from the environment. to achieve primary prevention of clp, lead paint would need to be abated from every apartment unit in the city. however, this is a costly process that the municipality or property owners/managers are unable to afford. yet, there are many families with lead-poisoned children who would argue that the benefits of primary prevention outweigh the costs. this case also highlights the complexity of persistent public health problems, such as clp. for instance, this particular family, not unlike other african refugee families, was illiterate in english as well as their own language. in addition, the refugee resettlement process is designed in a declining model of support where the refugees are placed in available housing, which is often of poor quality, and offered health benefits for a limited period of time, and employment is the benchmark of resettlement success not acculturation, good health, or community engagement (caron and tshabangu-soko 2012) . this community was fortunate in that it already had a functional community coalition that was addressing the problem via policy development, distribution of resources, surveillance, and testing of at-risk children. yet, it is important to consider the multifactorial issues affecting this persistent public health problem in this particular community. selected issues are included below ): • non-english speaking, at-risk population. • public health system that views the problem as complex due to the continuing influx of refugees and the number of agencies involved in refugee resettlement. • multiple stakeholders who view the problem differently and who offer varied, uncoordinated solutions. • intersect of socioeconomic factors, housing policies, cultural practices, english proficiency, and native language literacy. • clp exemplifies the failure of policy development and implementation in the community. • competing demands for food, shelter, clothing, employment for at-risk populations. • exposure results in health effects that are not visible until an ebll is acquired. • providing education in a culturally competent manner. • distrust of community organizations by the at-risk african refugee population. • often, persistent public health problems "…possess no definitive resolutions…" so "…remediation must focus on how to best manage them" (caron and serrell 2009, p. 195) . if this tragic event occurred in your community, what questions would you ask? i offer the following questions for you to consider from a practitioner and educator perspective: 1. how could we prevent children from being poisoned by lead in our community considering that practical solutions are difficult to implement due to the high cost of lead-abatement measures? 2. are there primary and secondary prevention tools we could implement and evaluate in our community? how will we provide lead prevention education for families for whom english is not their first language? there are over 70 different languages spoken in the manchester, nh, school system (mhd 2013a). it is not feasible to provide translation services for every dialect. how would you educate about a serious public health issue, such as clp, for which there are no visible signs or symptoms until there is an ebll? 3. does the community have a plan to address this public health issue? has the community, who lives with the issue (i.e., refugees, "working poor"), been invited to participate with public health practitioners? is there a community coalition formed to work on monitoring the issue and connecting families with testing services? how would you establish such a community group if one does not exist? 4. how would you partner with an academic institution with public health expertise to assist with providing knowledge, expertise, and resources? 5. how would you partner with the local health-care system (i.e., community health centers, hospitals, physician practices) to assure that they are following cdc testing guidelines and to assist with consistent outreach and prevention education efforts? 6. are there refugee resettlement services developed by resettled refugees who can assist with contacting an often hard-to-reach population to offer peer education? how would you engage this social service agency? 7. what data should you be collecting? how will you access these data? who is the "keeper" of the data? how will you conduct surveillance of the public health issue? 8. what stakeholders in the public health system should be invited to address the problem? if a stakeholder refuses to come to the "table" to work on the issue because they believe the issue is either not under their purview or is too complex to address, how would you engage this key partner? 9. policies pertaining to lead paint in housing and occupancy vary from state to state. how would you amend the current (if any) lead housing policies in your community or state? would public health enforcement laws be necessary (i.e., citations for property owners who do not comply with the developed policy)? whom would you work with to develop and enact such policies? 10. this case demonstrates a very tragic, albeit rare, event. with so many competing demands on the public health system, and the fact that clp is a persistent public health problem that the community has lived with for generations, and the costly abatement measures-should clp be in the "top ten" of issues for communities, similar to manchester, nh, to be concerned about? why or why not? 11. if we addressed clp in the community, what other public health issues could potentially be lessened or mitigated? 12. how does the refugee resettlement process exacerbate clp? should the refugee resettlement process be redesigned? if so, how? 13. should communities with refugee children poisoned by lead request a moratorium for refugee resettlement until the community can provide quality housing that does not pose a health risk? what are the implications of a moratorium for the resettled refugees and the community? 14. how would you engage the refugee resettlement agencies, the social service agencies developed by refugees, and the refugees themselves in a coordinated effort to reduce clp? 15. how would you know what the newly resettled refugee concerns are and how they compare to their counterparts who have been living in the community for a period of time? the answers to many of these questions may include more resources, more expertise, and more community support. i agree with this assessment. however, often, the public health principles that guide us are challenging to implement "on the ground." clp is a very real issue for this community. the number of refugees affected by this public health problem is influenced by the type of refugee who is resettled in the community. for instance, refugee children of parents who speak english and have a secondary and/or postsecondary education tend to not experience an ebll. this community is not able to request from which country the "newcomers" will arrive. box 4.1 highlights selected public health tools that should be utilized by a competent public health workforce addressing clp among a refugee population in their community. these skills are not meant to be exhaustive but are important for public health practitioners and educators of the public health workforce to consider when working on this type of public health problem. • engage the community in the public health issue being addressed. community-based participatory research (cbpr) is one approach to involve the community in addressing the public health issue that they live with on a daily basis. cbpr "…in public health focuses on social, structural, and physical environmental inequities through active involvement of community members, organizational representatives, and researchers in all aspects of the research process. partners contribute their expertise to enhance understanding of a given phenomenon and to integrate the knowledge gained with action to benefit the community involved". (israel et al. 1998, p. 173) serrell et al. (2009) previously identified four core values that were important to progress when building community capacity to address clp: "…adaptability, consistency, shared authority, and trust as core values for such partnerships" (p. 58). the type of public health professional required to address this specific public health issue includes, but is not limited to, the following: • public health director • environmental health specialist • nurse case manager • build academic-community partnerships based on cbpr principles (see above). these partnerships do not require the presence of a local academic institution but could operate via distance technology so the correct expertise for the specific public health issue is accessed. it is important to note that it can take time to build operational partnerships. • collect data from screening facilities (e.g., local health department, primary care physicians, community health centers). these data may be centralized in a state clp and prevention program. • analyze the data for descriptive purposes to know the demographics of the affected population and the at-risk population. • implement primary prevention via culturally and linguistically appropriate educational methods. • implement secondary prevention via blood screening. assure screening is being conducted by communicating with screening facilities and engaging in medical record audits. • develop policy that will be protective of the resident and places the burden of care on the property owner/manager to abate lead from the dwelling. • consider the community's ecology (i.e., its social, cultural, economic, and political composition) and social context of risk. caron et al. (2013) proposed the following: …that communities are important determinants in health-related problems for refugee populations. each community has its own environment and public health system that interacts with each other to influence health risks and risk perceptions of its populations. (p. 660) • partner with others in the public health system (e.g., housing development, refugee resettlement agencies, property managers, etc.) and learn their barriers to the problem, as well as their perception of the public health issue so a feasible and equitable solution or management strategy may be developed. • evaluate progress by reviewing the data to determine whether or not there is a decrease in the number of children poisoned by lead. based on the data, which will tell the story, targeted or tailored approaches for the affected population may be warranted. for example, peer education efforts may be implemented, temporary removal of a family from a home with lead paint until the lead can be removed or covered to meet housing code approval, visual aids for education, nurse case management, environmental inspection of the dwelling, etc. background: "hepatitis c is an infection caused by a virus that attacks the liver and may cause liver damage, liver failure, and even cancer" (nhdhhs 2013, p. 8). specifically, hepatitis c arises as a result of a blood-borne infection. for the majority of those infected, the acute phase of the infection is asymptomatic. in addition, for some infected individuals, their immune system will clear the infection. however, there is a risk that many people infected with the hepatitis c virus (hcv) will develop an active, chronic infection and without therapy some will develop liver cirrhosis, liver disease, liver failure, and/or liver cancer (nhdhhs 2013). the cdc estimates that there are approximately 4.1 million people who have been infected with hcv and 3.2 million people with active infection in the usa (cdc 2013): risk factors for acquiring hepatitis c include injection drug use, tattoos with contaminated supplies, use of infected blood products or occupational needlestick injury, transmission during pregnancy, and sexual transmission (which is usually very uncommon). the risk of acquiring hcv from a needlestick injury with blood from an hcv-infected patient is approximately 1-2 %, but it depends on the level of virus in the blood and the nature of the injury. (cdc 2013) hcv can be treated with an antiviral drug regimen that is administered for a period of several months and is quite costly (nhdhhs 2013). for those who are eligible for therapy and have not been treated in the past, the likelihood of cure is very good in acute infection (80-90 %). with newer available agents, the response rate is very good in chronic infection as well (60-80 %). (nhdhhs 2013, p. 10) specific to the transmission of hcv in health-care settings, risk factors include the following: 1. reuse of syringes for more than one patient or to access medication containers used for more than one patient; 2. sharing of contaminated equipment, like point of care or podiatry equipment; and/or 3. drug diversion by an infected healthcare worker (hcw). transmission can occur when the infected hcw self-administers an injectable narcotic, intended for patient administration, fills the syringe with saline, and places the used syringe back into the circulation for patient administration. (nhdhhs 2013, p. 10) reportable diseases are those that "…hospitals, laboratories, healthcare providers, childcare centers, schools, and local boards of health are required to report diagnosis of certain infectious diseases to dphs" (division of public health services; nhdhhs 2013, p. 10): in new hampshire, hcv infection is not in and of itself a reportable disease. however, any suspected outbreak, i.e., the occurrence of illness or disease in a community at a rate clearly in excess of what is normally expected, is reportable to dphs under the mandatory reporting law, part he-p 301 communicable diseases. (nh general court 2008; nhdhhs 2013, pp. 10-11) reported infections are investigated by public health nurses and epidemiologists at the new hampshire dphs. the purpose of the investigation is to prevent additional illness in the population, which may be accomplished through a variety of methods, depending on the specific disease. some examples of how public health works to prevent additional illness include identifying close contacts to the infected person and recommending prophylaxis medication to prevent them from becoming ill (antibiotics, antivirals, vaccine, etc.), providing disease prevention recommendations (washing hands, covering cough, etc.), recognizing outbreaks, and identifying and controlling their source (healthcare-associated outbreaks, foodborne outbreaks, etc.). (nhdhhs 2013, pp. 10-11) investigation overview an outbreak of hcv was identified at exeter hospital in exeter, new hampshire, in 2012. of the initial four patients diagnosed with hcv, one of the individuals was a traveling medical technician in the cardiac catheterization laboratory of the hospital. further investigation by the new hampshire department of health and human services (nhdhhs) revealed that the cause of the outbreak was drug diversion ("…the stealing of narcotic pain medication intended for patients for self use"; nhdhhs 2013, p. 6) by the infected medical technician. the testing of potential patients was conducted based on the hospital units to which the medical technician had access, i.e., patients seen in the cardiac catheterization laboratory and those who were patients in the operating room and the intensive care unit. for these areas, 1200 patients who had procedures in the cardiac catheterization laboratory during a time period that overlapped the medical technician's time of employment were tested for hcv: of the 1,074 who were tested, 32 patients were identified with active hcv infection with the nh hcv outbreak strain. 27 additional patients had evidence of past hcv infection (and their virus could not be tested) and 9 of them were categorized as probable cases (n = 4) and suspect cases (n = 5) based on epidemiological information. (nhdhhs 2013, p. 6) to contact those who were patients in the operating room or intensive care unit during this same time period, nhdhhs partnered with local health departments and clinics to conduct rapid hcv testing on site "…for the first time in an outbreak setting" (nhdhhs 2013, p. 6). …2,679 patients were tested and…no additional cases of active hcv infection matching the outbreak strain were identified. additional investigation of other units in 4. the medical technician worked for a staffing agency that assigned him to 18 different hospitals in seven other states (arizona, georgia, kansas, maryland, michigan, new york, and pennsylvania) over a decade (seelye 2012 ). in addition, he had been fired four times over this time span for allegations of drug use and theft (associated press 2013). thus, the potential for exposure of patients in other states existed and resulted in a multistate outbreak investigation that was conducted by the cdc. "as of may 2013, 13 other cases of the nh hcv outbreak strain were identified and confirmed in two other states (kansas and maryland)" (nhdhhs 2013, p. 6). the traveling medical technician pled guilty to "…obtaining controlled substances by fraud… [and] tampering with a consumer product" (fbi 2013): …he devised a scheme to divert and steal the controlled substance fentanyl for personal use and abuse. fentanyl is a powerful anesthetic intended for patients undergoing medical procedures, among other uses. [he] admitted that he would surreptitiously take syringes of fentanyl prepared for patients, inject himself with the drug, and refill the syringes with saline, causing the syringes to become tainted with his infected blood. he then replaced the tainted syringes for use on unsuspecting patients. consequently, instead of receiving the prescribed dose of fentanyl together with its intended anesthetic effect, patients actually received saline that was tainted with the same strain of hepatitis c carried by [the medical technician]. (fbi 2013) at the conclusion of the investigation, the nhdhhs (2013) recommended the following action areas: • "increase regulation and improve information sharing regarding allied healthcare workers." • "strengthen healthcare systems to promote prevention and early detection of drug diversion." • "assure optimal response to healthcare associated outbreaks to protect patient safety." (p. 63) lastly, as of september 2013, the nhdhhs had partnered with the national association of drug diversion investigators (naddi) in maryland and honoreform, hepatitis outbreaks national organization for reform, a patient advocacy group based in nebraska to influence national policy regarding the regulation of medical technicians (associated press 2013). any criminal act involving a prescription drug. (national association of drug diversion investigators) inciardi et al. (2007) define prescription drug diversion as the following: …the unlawful channeling of regulated pharmaceuticals from legal sources to the illicit marketplace, and can occur along all points in the drug delivery process, from the original manufacturing site to the wholesale distributor, the physician's office, the retail pharmacy, or the patient. (p. 171) in 2012, the cdc declared that the overdose on prescription drugs had reached an epidemic status (cdc 2012a). to further illustrate this point: "in 2007, approximately 27,000 unintentional drug overdose deaths occurred in the united states, one death every 19 minutes" (cdc 2012a, p. 10). opioid analgesics are responsible for the increase in overdose-related deaths (cdc 2012a). regarding the demographics of the abuse of and deaths from opioid analgesic use, it is …highest among men, persons aged 20-64 years, non-hispanic whites, and poor and rural populations. persons who have mental illness are overrepresented among both those who are prescribed opioids and those who overdose on them. (cdc 2012a, p. 774) of those who are prescribed opioid analgesics, the populations of greatest concern are those who seek care from multiple physicians and potentially take advantage of the physician's sensitivity to the patient's pain management (cdc 2012a). it is this population that is estimated to not only comprise approximately 40 % of overdose cases on opioid analgesics but also are diverting drugs for self-use or providing them to others (cdc 2012a). thus, the cdc recommends that prevention efforts should focus on addressing the following target populations: patients who consume opioid analgesics in high doses and those who seek care from multiple physicians and receive high doses of opioid analgesics. this latter group is likely to be involved in drug diversion (cdc 2012a). inciardi et al. (2009) report that the primary populations involved in drug diversion include "…drug dealers, friends and relatives, smugglers, pain patients, and the elderly, but these vary by the population being targeted" (p. 332). due to the complexity of the issue, several comprehensive prevention strategies have been proposed by the cdc and the american medical association: • restrict the number of reimbursement claims for opioid analgesic prescriptions written by a physician and filled by a pharmacy. this restriction is important for low-income populations on public health insurance, such as medicaid, since this population presents as high risk for drug abuse (cdc 2012a). • monitor that the type and prescribed usage of the opioid medication aligns with the diagnoses (cdc 2012a). • develop and enforce legislation that prohibits "doctor shopping" for those physicians who will prescribe opioid analgesics in high doses; elimination of "pill mills" where controlled pain medicine is distributed with little to no medical oversight; and the requirement of a physical examination prior to receiving a prescription for an opioid (cdc 2012a). • provide medical education via evidence-based practice for general and specialist physicians regarding opioid use and risks, thus holding them accountable for their prescribing practice (cdc 2012a). • fund, at the national level, the national all schedules prescription electronic reporting act (nasper). nasper provides …physicians with up-to-date, patient-specific information at the point of care in order to support appropriate prescribing and to identify those patients who were abusing or diverting prescription drugs. (ama 2013, p. 1) nasper was intended to fund prescription drug monitoring programs at the state level (ama 2013). • develop locations that will take back unused or expired medications (ama 2013). • expand access to addiction treatment and recovery centers (ama 2013). • support naddi: …a non-profit organization that facilitates cooperation between law enforcement, healthcare professionals, state regulatory agencies and pharmaceutical manufacturers in the prevention and investigation of prescription drug diversion. (naddi 2013) lessons learned: if this unfortunate event occurred in your hospital, what questions would you ask? i offer the following questions for you to consider from a practitioner and educator perspective: • how could a medical technician with a suspect record be passed from hospital to hospital? why did the staffing agency not disclose the issues with this employee? did the hospital conduct a thorough background check? • what are our hiring processes? how can we see "red flags" before the individual of concern is hired? who should be involved in the hiring process? • is there a system in place for employees to report suspicious behavior to senior management and human resources? should there be incentives to report employees observed in negligent behavior? • do we have a policy to prevent drug diversion in the workplace? if so, how can we improve the policy? • should we implement mandatory, unannounced drug testing for all hospital employees who engage in patient contact? should termination of employment be implemented if an employee refuses to cooperate with this policy? • is there a reporting system in place so that other hospitals across the country could be notified about the infected individual's reason for termination? • should the penalty for engaging in drug diversion be suspension or removal of one's license or certification to practice their skill in a health-care setting? • what other partners in the public health system should be involved in this issue? how can we partner more effectively with law enforcement and drug rehabilitation centers, for example? • should a public registry for those health-care workers found guilty of drug diversion be created at the national level? should access to such a registry be limited to health-care hiring agencies? should the public also have access to this registry? • how can we do a better job in protecting our patients? • how is drug diversion a public health problem, as well as a health-care problem? box 4.2 highlights selected public health tools that should be utilized by a competent public health workforce addressing a hcv outbreak in their community due to drug diversion. these skills are not meant to be exhaustive but are important for public health practitioners and educators of the public health workforce to consider when working on this type of public health problem. • conduct an outbreak investigation. − confirm that there are more cases than expected. − consider whether there is ongoing transmission. − define an outbreak-related case. − confirm existing number of outbreak-related cases. − investigate existing number of outbreak-related cases by reviewing all available data (e.g., medical records, laboratory results, interviews). − determine the infectious period for the outbreak. − determine potential sites of contact in a facility and potential family and others who could be exposed. − determine the exposed cohort of people at each site who may have been present during the case's infectious period. − define the screening action plan (including eligibility, implementation, and follow-up). − create a media plan. − develop and implement recommendations to prevent future outbreaks for particular populations or settings. − evaluate the outbreak response including whether implementations were effective in stopping transmission. − identify lessons learned to prevent future outbreaks (cdc 2012b). • communicate with the affected patients, their families, and the public as soon as the act of negligence is realized. • improve communication between the public health system and the healthcare system professionals. • develop a policy that would serve as safety measures to protect patient populations from health-care workers engaged in drug diversion. examples of such policies could include the establishment of a public registry of health-care workers found to be guilty of drug diversion; mandatory, unannounced drug testing of health-care workers whose employment involves patient contact; coordination of care so the number of physicians prescribing pain medications is limited; continued reporting of mandatory conditions. • collaborate with public health system partners, such as local health departments and law enforcement to assist with drug diversion education initiatives, drug and disease testing, and drug diversion investigations. • support national initiatives, such as nasper and honoreform. • engage in ongoing surveillance of drug diversion in the health-care setting. • educate health-care employees on proper reporting of such adverse events. the type of public health professional required to address this specific public health issue includes, but is not limited to, the following: antibiotic resistance is rising for many different pathogens that are threats to health. if we don't act now, our medicine cabinet will be empty and we won't have the antibiotics we need to save lives. (dr. thomas frieden, director, cdc) overview of public health threat antibiotic use arises from the inappropriate use of antibiotics in humans and animals. for example, with humans, physicians often prescribe an antibiotic when one is not needed and/or the patient does not complete the entire course of antibiotic treatment. thus, "…up to 50 % of all antibiotics prescribed for people are not needed or are not optimally effective as prescribed" (cdc 2013a, p. 11). antibiotic resistance can occur both within and outside of health-care facilities, yet deaths related to antibiotic resistance are most common in the healthcare setting (cdc 2013a). furthermore, antibiotics are also commonly used in food animals to prevent, control, and treat disease, and to promote the growth of food-producing animals. the use of antibiotics for promoting growth is not necessary, and the practice should be phased out. (cdc 2013a, p. 11) antibiotic resistance is not only a public health problem in the usa but it also presents as a major public health problem on a global scale. the statistics that demonstrate the magnitude of this public health issue on a national scale are staggering: • "each year in the united states, at least 2 million people acquire serious infections with bacteria that are resistant to one or more of the antibiotics designed to treat those infections." • "at least 23,000 people die each year as a direct result of these antibiotic-resistant infections." • "many more die from other conditions that were complicated by an antibioticresistant infection." (cdc 2013a, p. 11) the cdc states that these figures most likely underestimate the magnitude of the problem since …the distinction between an antibiotic-resistant infection leading directly to death, an antibiotic-resistant infection contributing to a death, and an antibiotic-resistant infection related to, but not directly contributing to a death are usually determined subjectively, especially in the preponderance of cases where patients are hospitalized and have complicated clinical presentations. (cdc 2013a, p. 18) thus, these statistics could be significantly higher. moreover, the health-care burden this preventable public health issue creates is multifaceted and can include the following cost-related issues for the health-care system: …prolonged and/or costlier treatments, extend hospital stays, necessitate additional doctor visits and healthcare use, and result in greater disability and death compared with infections that are easily treatable with antibiotics. (cdc 2013a, p. 11) these health-care costs are estimated to be in excess of us$20 billion and societal costs due to a loss of productivity are estimated to be us$35 billion a year (roberts et al. 2009) . a further complication of antibiotic resistance is seen in those populations who have underlying disease, such as diabetes, asthma, and rheumatoid arthritis. these groups, in addition to those patients who may undergo chemotherapy, organ and bone marrow transplant surgery, joint replacement surgery, or end-stage renal disease are significantly dependent on antibiotic use to fight off infections (cdc 2013a). these subgroups represent a susceptible population to infection especially if antibiotics that are heavily relied upon do not work optimally for these patients. the cdc readily acknowledges the following significant areas of improvement in the body of knowledge regarding antibiotic resistance: • "limited national, state, and federal capacity to detect and respond to urgent and emerging antibiotic resistance threats….we do not have a complete picture of the domestic incidence, prevalence, mortality, and cost of resistance." • "currently, there is no systematic international surveillance of antibiotic resistance threats. today, the international identification of antibiotic resistance threats occurs through domestic importation of novel antibiotic resistance threats or through identification of overseas outbreaks." • "data on antibiotic use in human healthcare and in agriculture are not systematically collected. routine systems of reporting and benchmarking antibiotic use wherever it occurs need to be piloted and scaled nationwide." • "programs to improve antibiotic prescribing are not widely used in the united states. these inpatient and outpatient programs hold great promise for reducing antibiotic resistance threats, improving patient outcomes, and saving healthcare dollars." • "advancing technologies can identify threats much faster than current practice. advanced molecular detection (amd) technologies, which can identify ar [antibiotic resistance] threats much faster than current practice, are not being used as widely as necessary in the united states." (cdc 2013a, p. 27) chen et al. (2011) propose that rather than identify population groups at risk for ca-mrsa, diagnostic and preventive approaches should focus on addressing risk factors for ca-mrsa, including "…poor personal hygiene, transmission through contaminated environmental services, and care of non-intact skin" (p. 444). ca-mrsa infections typically occur in otherwise healthy people with no recent stay in a health-care facility. in contrast, hospital-acquired mrsa (ha-mrsa) is contracted by patients in a health-care facility and has been attributed to invasive surgical procedures and poor infection control practices (niaid 2013) . health-care providers are concerned about those ha-mrsa infections that are potentially brought into the community once the patient is discharged (johnson 2013) . the cdc's report titled antibiotic resistance threats in the united states, 2013, is an excellent resource on this topic and provides a comprehensive overview of specific, ranked antimicrobial resistance threats, including prevention measures. an abbreviated outline of prevention measures for ca-mrsa and ha-mrsa are presented here. the reader is encouraged to review the cdc's report on this topic for more extensive information. at the state and community level, it is important to: • "know resistance trends in your region." • "coordinate local and regional infection tracking and control efforts." • "require facilities to alert each other when transferring patients with any infection." (cdc 2013a) the north carolina department of public health proposes the following core activities for public health professionals to engage in when managing ca-mrsa as a public health threat: • "recognize outbreaks" − for example, "an isolated case on a wrestling team; several cases within the same prison unit in a month; more than one case in a child care classroom in a month" (ncdph 2013). • "react to community concerns" − "consider the risk factors for transmission; the 5 cs" − "contact (skin-to-skin)" − "contaminated items and surfaces (wrestling mats, weight room equipment)" − "comprised skin integrity (cuts and abrasions)" − "crowding (locker rooms)" − "cleanliness (absence)" (ncdph 2013) • "respond with public health control measures" − "active surveillance to determine scope of problem in specific setting" − "assure specific control measures for wound care and containment of drainage" − "stop any sharing of personal items and promote enhanced personal hygiene" − "consider exclusion from contact activities, especially with actively draining or packed wounds" − "achieve and maintain a clean environment" (ncdph 2013) selected examples of actions health-care administrators and providers can take include the following: • "require and strictly enforce cdc guidance for infection detection, prevention, tracking, and reporting." • "make sure your lab can accurately identify infections and alert clinical and infection prevention staff when these bacteria are present." • "prescribe antibiotics wisely." • "remove temporary medical devices such as catheters and ventilators as soon as no longer needed." (cdc 2013a) patients and their family members should: • "ask everyone, including doctors, nurses, other medical staff, and visitors, to wash their hands before touching the patient." • "take antibiotics exactly and only as prescribed." (cdc 2013a) carbapenem-resistant enterobacteriaceae (cre) is a hospital-associated infection that is difficult to treat because the bacteria, normally found in the gut, have become resistant to all antibiotics, including carbapenem, which is often considered a last resort type of antibiotic (cdc 2013c). according to the cdc (2013c), …cre infections most commonly occur among patients who are receiving treatment for other conditions. patients whose care requires devices like ventilators (breathing machines), urinary (bladder) catheters, or intravenous (vein) catheters, and patients who are taking long courses of certain antibiotics are most at risk for cre infections. additional risk factors for cre infections include a patient's functional status and a stay in the hospital's intensive care unit (schwaber et al. 2008) . research conducted by perez et al. (2010) suggests that acute care health facilities could be significant reservoirs for the transmission of cre infections. furthermore, cre infections "…can contribute to death in up to 50 % of patients who become infected" (cdc 2013c). approximately 9300 cre infections occur in health-care facilities in the usa. "each year, approximately 600 deaths result from infections caused by the two most common types of cre, carbapenem-resistant klebsiella spp. and carbapenem-resistant e. coli" (cdc 2013a). the incidence of cre infections is on the rise, increasing sevenfold over the past decade (mckinney 2013). the cdc reports that "about 4 % of u.s. short-stay hospitals had at least one patient with a serious cre infection during the first half of 2012. about 18 % of long-term acute care hospitals had one" (cdc 2013a). the cdc has a comprehensive "detect and protect" program for cre infections. the reader is referred to the following website which provides information about this program (http://www.cdc.gov/hai/pdfs/cre/cdc_ detect protect.pdf). an abbreviated outline of prevention measures for cre infections is presented here: state and local health departments are well positioned to lead cre control efforts because of their expertise in surveillance and prevention and their ability to interact among all the health-care facilities in their jurisdiction. (jacob et al. 2013, p. 167) thus, at the state and community level it is important to: • "know cre trends in your region"; • "coordinate regional cre tracking and control efforts in areas with cre. areas not yet affected by cre infections can be proactive in cre prevention efforts"; • "require facilities to alert each other when transferring patients with any infection"; • "consider including cre infections on your state's notifiable diseases list". (cdc 2013a) selected examples of actions health-care administrators and providers can take include the following: • "require and strictly enforce cdc guidance for cre detection, prevention, tracking, and reporting"; • "make sure your lab can accurately identify cre and alert clinical and infection prevention staff when these bacteria are present"; • "know if patients with cre are hospitalized at your facility, and stay aware of cre infection risks. ask if your patients have received medical care somewhere else, including another country"; • "follow infection control recommendations with every patient, using contact precautions for patients with cre. whenever possible, dedicate rooms, equipment, and staff to cre patients"; • "prescribe antibiotics wisely"; • "remove temporary medical devices as soon as possible." (cdc 2013a) • "tell your doctor if you have been hospitalized in another facility or country"; • "take antibiotics only as prescribed"; • "insist that everyone wash their hands before touching you." (cdc 2013a). [to address antibiotic resistance] "…will require expanded and coordinated action from clinicians, facility administrators, and public health officials." (jacob 2013) guh et al. (2013) reported that of 11 state health departments surveyed, all perceived emerging infections, such as cre, as a public health priority for prevention. yet, the extent to which these states can engage in prevention-oriented activities depends upon available resources and existing partnerships among their agencies, hospital administrators, and others in the public health and health-care systems. the cdc has developed core actions to help prevent the development of antibiotic resistance: • "preventing infections, preventing the spread of resistance"; • "tracking"; • "improving antibiotic prescribing/stewardship"; • "developing new drugs and diagnostic tests." (cdc 2013a, p. 31) lessons learned the main question is how do we, as public health practitioners and educators, work collaboratively with our partners in the health-care system to prevent antibiotic resistance in the health-care setting and the community? building upon the public health action plan set forth by the cdc, box 4.3 highlights selected approaches and tools to prevent infections, broaden our surveillance approach, and improve antibiotic stewardship. these skills are not meant to be exhaustive but are important for public health practitioners and educators of the public health workforce to consider when working on this type of public health problem. • cdc has several surveillance programs to monitor antibiotic resistance trends in the community: • "cdc's national healthcare safety network (nhsn) is used by healthcare facilities to electronically report infections, antibiotic use, and resistance" (cdc 2013, p. 32). the more hospitals that report to this database will enable cdc to track the level of antibiotic resistance in all bacteria, as well as track antibiotic usage. "this information will allow facilities to target areas of concern, to make needed improvements and to track the success of their efforts" (cdc 2013a). • "cdc manages the get smart program [http://www.cdc.gov/getsmart], a national campaign to improve antibiotic prescribing and use in both outpatient and inpatient settings" (cdc 2013a, p. 33). "one core activity is the development and implementation of the antibiotic stewardship drivers the type of public health professional required to address this specific public health issue includes, but is not limited to, the following: , a tool that provides healthcare facilities with a menu of interventions they can select from to improve antibiotic use" (cdc 2013a, p. 33). • "stewardship is a commitment to always use antibiotics only when they are necessary to treat, and in some cases prevent disease; to choose the right antibiotics; and to administer them in the right way in every case. effective stewardship ensures that every patient gets the maximum benefit from the antibiotics, avoids unnecessary harm from allergic reactions and side effects, and helps preserve the life-saving potential of these drugs for the future." (cdc 2013a, p. 41) • "…new antibiotics will always be needed to keep up with resistant bacteria as well as new diagnostic tests to track the development of resistance". (cdc 2013a, p. 44) and is believed to be spread via direct transmission. the case fatality rate is high in that approximately half of the people with the mers-cov infection have died. "however, the virus has not shown to spread in a sustained way in communities. the situation is still evolving" (cdc 2013 the severe acute respiratory syndrome (sars) pandemic was short lived but certainly tested the preparedness of our public health and health-care systems for a never-before-seen virus that was transmissible from animals to humans. mers-cov possesses some similarities to sars in that both are believed to be evolved from the bat coronavirus, affect the lower respiratory system, and are transmitted via an airborne route (breban et al. 2013 ). however, recent research has also indicated significant differences between these two coronaviruses. for example, assiri et al. (2013) reported that patients diagnosed with mers-cov tended to be older men with underlying chronic medical conditions, including diabetes, heart disease, and renal disease. in addition, these researchers noted that the progression to respiratory failure occurred faster compared to sars (zumla 2013) . furthermore, these authors observed, in contrast to sars, which was much more infectious especially in healthcare settings and affected the healthier and the younger age group, mers appears to be more deadly with 60 % of patients with co-existing chronic illnesses dying, compared with the 1 % toll of sars. (zumla 2013) lastly, the authors note that it is possible we are only detecting the most serious of the mers-cov cases, and there are milder cases going undetected in the community (zumla 2013) . it is these milder cases that also require a case definition: ultimately the key will be to identify the source of mers infection, predisposing factors for susceptibility to infection, and the predictive factors for poor outcome. meanwhile infection control measures within hospitals seem to work. (zumla 2013) public health emergency? although this is a new virus with a high case fatality rate and is of great concern to the public health and health-care communities, the world health organization (who)'s emergency committee of the international health regulations [unanimously decided in july 2013] …that with the information now available, and using a risk-assessment approach, the conditions for a public health emergency of international concern (pheic) have not at present been met. (who 2013) "while not considering the events currently to constitute a pheic, members of the committee did offer technical advice for consideration by who and member states on a broad range of issues, including the following: • improvements in surveillance, lab capacity, contact tracing and serological investigation • infection prevention and control and clinical management • travel-related guidance • risk communications • research studies (epidemiological, clinical and animal) • improved data collection and the need to ensure full and timely reporting of all confirmed and probable cases of mers-cov to who…." (who 2013) furthermore, there are no current travel bans to countries that have reported mers-cov cases. cdc's …travel notice is a watch (level 1) which advises travelers to countries in or near the arabian peninsula to follow standard precautions, such as hand washing and avoiding contact with people who are ill. (cdc 2013) similarly, who does not currently propose any travel or trade restrictions or special screening activities at points of entry into countries (hopp 2013) . public health preparedness cdc is actively monitoring the outbreak of mers-cov cases and working with international public health partners. to date, cdc has engaged in public health preparedness for this new virus in the following ways: • "…developed molecular diagnostics that will allow scientists to accurately identify mers cases." • "…providing mers-cov testing kits to state health departments." • "…developed interim guidance for preventing mers-cov from spreading in homes and communities to help protect people if there is ever a case of mers in the u.s." • "…offering recommendations to travelers when needed. cdc is also helping to assess ill travelers returning from affected areas." • "…provide advice and laboratory diagnostic support to countries in the arabian peninsula and surrounding region." (cdc 2013) research by breban et al. (2013) examined the transmissibility of mers-cov between humans which allowed them to estimate the potential for mers-cov to attain a pandemic status. the authors concluded "…that mers cov does not yet have pandemic potential" (breban et al. 2013, p. 694) . the authors recommend the following public health actions: "…enhanced surveillance, active contact tracing, and vigorous searches for the mers-cov animal hosts and transmission routes to human beings" (breban et al. 2013, p. 694) . knowledge gaps since this outbreak is still evolving, there are many gaps in our knowledge about the epidemiology of the infection, its clinical course, best diagnostic tools, patient management, and infection control. assiri et al. (2013) did an outstanding job in formulating the questions the public health and health-care communities should be addressing. i have highlighted a few of these questions here for discussion purposes. the reader is referred to the descriptive study of mers-cov in saudi arabia that was conducted by assiri et al. (2013) for further probing questions. • "what is the natural reservoir of mers-cov?" • "what is the source of exposure to mers-cov outside of the healthcare facility (e.g., animals, water, sewage, food)?" the type of public health professional required to address this specific public health issue includes, but is not limited to, the following: asymptomatic, mild, severe infection)?" • "what is the infection rate in the community?" • "what are the protective immune system mechanisms against mers-cov?" • "what is the excretion pattern of the virus?" • "what is the best clinical management of mers-cov?" • "is there a role for antiviral agents?" • "how stable is mers-cov under different environmental conditions (e.g., dry surface, in vomit, sputum or diarrhea)?" • "how can we efficiently disinfect against mers-cov?" • "is there a role for herd immunity against mers-cov? public health skills to address a novel disease outbreak • collaborate with public health partners at the local, state, federal, and international levels in the case of mers-cov, public health and health-care professionals and researchers are reviewing the similarities and differences between sars and mers-cov. reviewing how similar outbreaks were managed can help steer a similar • participate in videoconferences and conference calls sponsored by the cdc and who regarding the latest information and best practices pertaining to the epidemiology, prevention guidelines, clinical management engage in diligent surveillance activities to help develop prevention methods specific to your local community • evaluate these prevention efforts and adapt as necessary. • document the approaches implemented and their effectiveness as this may inform evidence-based practice for future disease outbreaks • be prepared, to the extent possible, with sufficient material and personnel resources to plan, respond, and evaluate prevention efforts inform and educate the public about their risk and prevention efforts via media outlets outbreaks of novel diseases can be unpredictable as the virus evolves. be prepared for changes in transmission, the target population, and disease management references local public health case: pediatric fatality in a refugee resettlement community agency for toxic substances disease registry case studies in environmental medicine: lead toxicity community ecology and capacity: keys to progressing the environmental communication of wicked problems environmental inequality: childhood lead poisoning as an inadvertent consequence of the refugee resettlement process fatal pediatric lead poisoning childhood lead poisoning in a somali refugee resettlement community in new hampshire accessed 18 sept. city of manchester, new hampshire health department (mhd) lead poisoning among refugee children resettled in massachusetts review of community-based research: assessing partnership approaches to improve public health new hampshire childhood lead poisoning prevention program: 2002-2006 blood lead level screening data childhood lead poisoning in massachusetts communities: its association with sociodemographic and housing characteristics an academic-community outreach partnership: building relationships and capacity to address childhood lead poisoning combating rx diversion, overdose and death-comprehensive public health strategies needed after hepatitis c probe, nh, groups push for better drug diversion prevention, detection core elements of an outbreak investigation former employee of exeter hospital pleads guilty to charges related to multi-state hepatitis c outbreak mechanisms of prescription drug diversion among drug-involved club-and street-based populations national association of drug diversion investigators new hampshire code of administrative rules new hampshire division of public health services, department of health and human services national public health case: antibiotic resistance centers of disease control and prevention community-acquired methicillin-resistant staphylococcus aureus skin and soft tissue infections: management and prevention. current infectious disease reporting assessment of public health perspectives on responding to an emerging pathogen: carbapenem-resistant enterobacteriaceae vital signs: carbapenem-resistant enterobacteriaceae hospital mrsa infections fall by more than 50 %, report shows superbug a 'triple threat' but cdc issues warning early to prevent spread north carolina public health management of ca-mrsa carbapenem-resistant acinetobacter baumannii and klebsiella pneumoniae across a hospital system: impact of post-acute care facilities on dissemination hospital and societal costs of antimicrobialresistant infections in a chicago teaching hospital: implications for antibiotic stewardship predictors of carbapenem-resistant klebsiella pneumoniae acquisition among hospitalized adults and effect of acquisition on mortality international public health case: middle east respiratory syndrome-coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study. the lancet infectious diseases interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus (mers-cov) who statement on the second meeting of the ihr emergency committee concerning mers-cov fullest clinical report of saudi mers points to important differences with sars cases to date key: cord-293966-5c466xvz authors: fehr, anthony r. title: bacterial artificial chromosome-based lambda red recombination with the i-scei homing endonuclease for genetic alteration of mers-cov date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_5 sha: doc_id: 293966 cord_uid: 5c466xvz over the past two decades, several coronavirus (cov) infectious clones have been engineered, allowing for the manipulation of their large viral genomes (~30 kb) using unique reverse genetic systems. these reverse genetic systems include targeted recombination, in vitro ligation, vaccinia virus vectors, and bacterial artificial chromosomes (bacs). quickly after the identification of middle east respiratory syndrome-cov (mers-cov), both in vitro ligation and bac-based reverse genetic technologies were engineered for mers-cov to study its basic biological properties, develop live-attenuated vaccines, and test antiviral drugs. here, i will describe how lambda red recombination can be used with the mers-cov bac to quickly and efficiently introduce virtually any type of genetic modification (point mutations, insertions, deletions) into the mers-cov genome and recover recombinant virus. coronaviruses are large, enveloped, single-stranded positive-sense rna viruses that cause both significant human and veterinary disease. prior to the severe acute respiratory syndrome-cov (sars-cov) outbreak in 2003, human covs were only known to cause mild, self-limiting upper respiratory diseases. approximately 10 years after the emergence of sars-cov in 2012, middle east respiratory syndrome (mers)-cov emerged in the middle east where it then spread to 27 different countries, and to date (december 2018, who) there have been 2278 laboratoryconfirmed cases and 806 associated deaths for a case fatality rate of 35%. most of these cases have occurred in the middle east, aside from an outbreak of~200 infected individuals in south korea in 2015 [1] . infectious clones are highly valuable research tools that enable modification of viral genomes to better understand their fundamental biology, develop novel vaccine candidates, and test antiviral therapeutics. soon after identifying mers-cov as the causative agent of mers, two distinct infectious clones were reported for mers-cov [2, 3] . these infectious clones were engineered using in vitro ligation or bacterial artificial chromosomes (bacs), each of which had been used previously for covs [4] [5] [6] . in vitro ligation uses unique type ii restriction endonucleases that cleave several bases away from their recognition site, allowing for the reassembly of authentic cov genomes from smaller fragments. each fragment is separately maintained in its own small plasmid for efficient genetic modification using traditional molecular cloning methods. separating specific nucleotide sequences in orf1a helped to eliminate the problem of these sequences being toxic for bacteria. a t7 promoter is inserted at the 5 0 end of the genome, allowing for in vitro transcription of the viral rna and subsequent transfection into mammalian cells for virus production. in contrast, bacs allow for the stable propagation of full-length cov cdna in bacteria, due to the ability to restrict their copy number to 1 or 2 plasmids per cell. different restriction fragments of these bacs can be sub-cloned into smaller vectors for efficient modification, or the full-length genome can be modified using lambda red recombination, which will be discussed here. cov bac plasmids contain a cmv promoter 5 0 of the viral genome, allowing for transcription of the viral genome following transfection of bac dna into mammalian cells. in addition, the cov bacs contain a polya tail, a hepatitis d virus (hdv) ribozyme, and bovine growth hormone (bgh) termination and polyadenylation signals to create genomic rna with an authentic 3 0 end. the full-length nature of bac dna and the cmv promoter subvert the need for in vitro ligation or transcription to recover infectious virus. bacs were initially developed in the early 1990s, and by the mid-late 1990s they were utilized by herpes virologists for modification of these large dna viruses, which revolutionized the field. a few years later a bac for a cov, transmissible gastroenteritis virus (tgev), was engineered, and since then bacs have been successfully developed for several covs including feline infectious peritonitis virus (fipv), oc-43, sars-cov, mers-cov, murine hepatitis virus strain jhm (mhv-jhm), porcine epidemic diarrhea virus (pedv), and the sars-like cov wiv-1 [2, 4, [7] [8] [9] [10] [11] [12] . thus, it is likely that bac-based reverse genetics could be useful for any novel or emergent cov. lambda red recombination utilizes bacteriophage enzymes exo, beta, and gam (red proteins) to mediate homologous recombination near the ends of linear double-stranded dna [13, 14] . pcr products containing positive selection markers are suitable substrates for these enzymes, so long as they bear extensions of 40-50 bases that are homologous to the target sequence. a major advancement in this technique came with the development of an e. coli strain, dy380, where the red proteins were placed under the control of a temperature-inducible promoter [15] . several methods for removing the positive selection markers from the viral genomes have been developed, including flanking sequences with frt or loxp sites [16] , or utilizing positive and negative selection markers on a single gene cassette, such as the galactose kinase (galk)-kan r gene cassette [17] . these methods both have certain downfalls, including the retention of small frt or loxp sites following removal of the marker, or the unintended removal of negative selection markers by repeat sequences in the bac plasmid. to improve the efficiency of removing the positive selection marker, a unique method utilizing the i-scei homing endonuclease under an arabinose-inducible promoter was developed ( fig. 1 ) [18] . i-scei is an endonuclease with an 18 bp recognition site that is not present in the e. coli genome, making it safe to express in e. coli. in the method described here, this recognition site is engineered on a plasmid (pep-kans) just outside of the positive selection marker, and its cleavage with the i-scei enzyme allows for the removal of the positive selection marker by intramolecular red recombination utilizing sequence duplication introduced in the original pcr primers. this method can be utilized to introduce any type of modification into the bac dna, including mutations, deletions, and insertions. here i will outline the procedure for this highly efficient method to engineer markerless modifications, focusing on single point mutations in the full-length mers-cov bac. [2] . the full protocol for creating this bac was subsequently published by the same group in a previous methods in molecular biology book [19] . this plasmid contains the para, parb, and parc genes derived from the e. coli f-factor to prevent more than one or two bacs from coexisting in the same cell. it also contains genes involved in the initiation and orientation of dna replication and the chloramphenicol resistance gene (cml r ). 2. pep-kans. this plasmid contains the aphai-i-scei cassette containing a kanamycin resistance marker (kan r ) and an i-scei restriction site [18] . this plasmid also contains an ampicillin resistance marker. 3. e. coli strains dh10b (see note1) and gs1783 (see note 2) cells. forward 6. aliquot 25 ml into each 50 ml conical tube on ice. centrifuge at 1800 â g for 10 min at 4 c in a tabletop centrifuge. 7. pour off supernatant with one quick motion. while pouring, position the bacterial pellet away from the liquid to limit the amount of bacteria lost. add 5 ml ice-cold sterile ddh 2 o and resuspend pellet by swirling and tapping the tube to the bottom of the ice-cold autoclave bin. once resuspended, add an additional 20 ml ice-cold sterile ddh 2 o and centrifuge at 1800 â g for 10 min at 4 c. repeat 1â. 8. following the second water wash, resuspend each pellet with 10% glycerol, first in 5 ml, then add an additional 15 ml. centrifuge at 1800 â g for 10 min at 4 c. 9. pour off supernatant as described above. following the pour, resuspend the pellet in the remaining 10% glycerol (~500 μl). if the combined amount of cells and 10% glycerol is greater than 550 μl, transfer the suspension to a cold eppendorf tube and pellet the cells for 2 min at 5000 â g and 4 c. after spinning the cells, remove an appropriate amount of supernatant such that~500 μl of cell suspension remains. resuspend the cells to a homogenous solution and aliquot 50 μl to prefrozen eppendorf tubes and flash-freeze tubes in liquid nitrogen or a dry ice-methanol bath. use immediately or store at à80 c. cells are typically good for 6-12 months, but may be useful even after several years. kan r -i-scei primers with 60 bp homology to the region of interest flanking the desired site to be modified (fig. 1a) . to design simple point mutations, start by developing a 60 bp flanking sequence, calling each 20 base pair section as a, b, and c. then incorporate the desired mutation at the end of section b (40th base pair, or the 39th and 40th base pair if two changes are required). finally, attach this sequence to the 22 bp kan r -i-scei sequence below to create your forward recombination primer. to create the reverse recombination primer, create a new block of 20 bp we will call section d 0 that is the reverse complement of the sequence immediately downstream from section c. these 20 bp will be followed on your primer by sections c 0 and b 0 , the reverse complements to sections b and c, with b 0 containing the desired mutations. finally, add the 23 bp kan r -i-scei sequence to finish the reverse primer. during negative selection, sections b/c will recombine with b 0 /c 0 leading to the loss of the kan r -i-scei cassette (fig. 1g) . for deletion mutants, leave out the desired sequence from your primers. for instance, to delete sections d/e/f, simply create the forward primer with sections a/b/ c, and the reverse primer with sections g 0 /c 0 /b 0 . insertions of small sequences can be achieved by adding the entire insertion sequence at the 3 0 end of the forward primer, and at least 50 bp of reverse complement sequence at the 3 0 end of the reverse primer (fig. 2) . larger insertions may require the development of a full plasmid, or potentially the use of nested pcrs. for additional details, see ref. [18] . while designing recombination primers (indicated below), remember to also order short primers about 100-200 bp outside of the insertion site to check for the proper insertion of the gene cassette by pcr. 2. set up pcr reaction and perform reaction according to manufacturer's protocol with following modifications (fig. 1b) . fig. 2 model of the pcr product used for inserting specific sequences into bacs using lambda red recombination. the full sequence for insertion is incorporated at the 5 0 end of the kan r -i-scei cassette while at least 50 nt of sequence homologous to the 3 0 end of the insertion sequence is incorporated at the 3 0 end of this cassette. surrounding these sequences are 50 nt of sequence homologous to the viral sequence where the sequence is to be inserted 3. for a pcr template, use~50 ng of the pep-kans plasmid. 4 . use 1 μl high-fidelity polymerase (see note 10). procedure lowering the annealing temperature by 1 c starting at 68 c and continuing the pcr reaction for 25 cycles. using this method we rarely see spurious pcr products. 6. analyze the pcr product on an agarose gel with ethidium bromide and image it using a uv-gel box and gel-imaging software. pcr product should be~1.2 kb. 7. purify the pcr product using the purelink pcr purification kit (see note 5). use binding buffer b3 according to manufacturer's protocol to remove primer dimers from the mixture. elute dna into 44 μl of water. 8. dpni digest the pep-kans plasmid in the purified pcr product (fig. 1b) . dpni specifically cleaves methylated dna and is needed to digest the pep-kans plasmid used in the pcr reaction. it has a 4 bp recognition site so it should cleave dna approximately every 250 bp. without this digestion all of your transformants will maintain the pep-kans plasmid as its transformation is much more efficient than the recombination of the pcr product. incubate for 1-3 h at 37 c. 4. carefully transfer the mixture into the groove of the electroporation cuvette on ice. 5. wipe any ice water from outside of cuvette and pulse at 25 μf, 6. recover by immediately adding 0.5-1 ml soc to the cuvette, and then transfer the mixture to a 14 ml culture tube. incubate at 32 c and 220 rpm for 3-5 h. this is when the recombination occurs (fig. 1d) fig. 3 replica plate grids. these grids allow for the easy identification of identical colonies that have been plated on each plate. using a toothpick, dot a single colony in the same spot on each plate. for both positive and negative selection, mers-bac clones that have successfully undergone recombination will grow on the plate on the right, but not on the plate on the left day 4 3. following o/n incubation, create a freezer stock of the bacteria, then purify the bac dna using a standard miniprep kit. using 1 μl of the bac dna, use the external primers located 100-200 bp outside the region of homology you previously designed to test for the insertion of kan r -i-scei by pcr. if the insertion was successful, the dna band from the pcr should be~1 kb larger than the band from the mers-cov wild-type bac (see note 12) . to speed this process up, colonies may be collected off the plate on day 3 and directly tested by pcr. colonies that pass the pcr screen can then progress to the negative selection protocol (3.5) (see note 13). 3. after 2 h, add 2 ml of warm lb-cml with 2% arabinose to the culture tube for a final arabinose concentration of 1% (fig. 1) . incubate at 32 c and 220 rpm for 2 h. warm water bath shaker to 42 c. 4. transfer the culture tubes to the water bath shaker at 42 c and 200 rpm and incubate for 30 min (fig. 1f ). 5. transfer the culture tubes back to 32 c and incubate for 3-4 h (fig. 1g ). 6. after the incubation, perform tenfold serial dilutions of the bacteria in lb. plate 100 μl of 10 à4 and 10 à5 dilutions of original culture on pre-warmed lb-cml plates containing 1% arabinose. incubate the plates at 30-32 c. day 3 7. pick 50 colonies and replica plate on lb-cml/kan and lb-cml plates. colonies that underwent correct recombination should grow on lb-cml but not on lb-cml/kan. incubate o/n at 32 c. efficiency is generally anywhere from 5 to 50% (see note 14) . day 4 8. pick 3 separate cml + kan à colonies and culture each one in 100 ml lb-cml at 32 c and 220 rpm o/n. 2. all procedures from here involve working with mers-cov which requires a bsl-3 containment laboratory. 3. replace medium from cells with 2 ml dmem +10% fbs immediately before proceeding to step4. 6. cytopathic effect (cpe) will start being visible at 3-4 days after transfection. collect cells and supernatant when >50% of well has cpe (the more the better). to collect, scrape any remaining cells off the well with a pipette tip or cell scraper and transfer the media and cell debris in a 2 ml microcentrifuge tube and freeze-thaw the sample. then, centrifuge the sample at~5000 â g to spin out the cell debris and transfer the supernatant to a new tube. we term this passage 0 (p0) virus. 3. for creating long oligos, we prefer invitrogen as a supplier, as they can provide oligos up to 100 nt at their standard price per base. many companies do not make oligos longer than 60 nt at their standard rates, and thus will charge a significant amount more for the 80-90 nt oligos required for this protocol. 4. while many companies sell bac-prep kits which will work for these purposes, these columns are often at least 3â the cost of the nucleobond xtra midi kit. we have tested the xtra midi kit side by side with a bac-prep kit and found little to no difference in cov bac dna yield. 5. for purifying pcr products, we prefer the invitrogen pure-link pcr purification kit because it includes a buffer that allows dna products of <300 bp to go through the column. this helps remove primer dimers from the pcr reaction that could interfere with recombination. 6 . a 42 c shaking water bath is essential for this procedure. the bacterial cultures need to heat up to 42 c quickly to properly induce the red enzymes. a shaking water bath is significantly better than a regular shaking incubator at quickly transferring heat to the bacterial culture. 7. we have tried several different transfection reagents, and lipofectamine 2000 has worked the best for us. that does not mean other transfection reagents won't work, feel free to try whichever reagent you prefer. 8. it is important to always culture gs1783 cells at 32 c or lower due to the potential for leaky expression of the red enzymes at temperatures below the induction temperature of 42 c. 9. some shaking water baths cannot shake at 200 rpm; if this is the case, simply shake at a reasonable speed for the shaker. 10. we have occasionally had problems obtaining a pcr product when using the manufacturer recommended 0.5 μl of highfidelity polymerase. using 1 μl of polymerase provides more consistent results. 11. unlike a typical 1 h recovery following electroporation, it is important to incubate these cultures for several hours in soc to allow time for recombination to occur. a recovery time of 5 h or greater is preferred, with a minimal recovery time being 3 h. 12. occasionally, we find that following pcr some clones have bands that correspond to both the wt bac and the desired kan r -i-scei insert bac. it is likely due to at least two copies of bac dna being present in the same cell. unless the molar ratio of the kan r -i-scei insert to the wt bac is very large, these clones should be avoided. 13 . after positive selection, we do not check the bac by restriction digest, because (a) we find that in many cases the amount of bac dna from a miniprep is insufficient for a readable digest, and (b) we find that the colonies that pass the replica plating and pcr tests rarely if ever have any significant problems concerning removing or duplicating regions of the mers-cov bac dna. 14. first, colonies may take over 1 day before they are visible. second, while the efficiency of the negative selection can be very low, any colonies that have grown on lb-cml plates but do not grow on lb-cml/kan plates are very likely correct. therefore, we go directly to starting large-scale cultures for these clones. that way we can do the final diagnostic tests and we can prepare for the bac transfection and viral recovery at the same time. 15 . a kpni digest of pbac-mers-cov results in dna fragments of 19.1, 13.8, 3.9, and 1.5 kb. we have found this is the best digest for diagnostic evaluation of the mers-cov bac; however other enzymes or enzyme combinations will work as well. be sure to use a low-percentage agarose gel to effectively separate the large dna molecules. 16 . other laboratories use bhk-21 cells for the initial transfection since these cells are highly transfectable. the transfected cells can be overlaid on the vero-81 or huh-7 cells for further outgrowth of the recombinant virus. 17. it is feasible to wait o/n to change the media of the transfected vero 81 cells, but this does result in an increase in the cytotoxicity induced by lipofectamine. this is not feasible if you are using huh-7 cells. 18. mutant viruses that do not replicate as well as wt virus may require increased amounts of p0 virus. 19. it is also beneficial to check the integrity of the entire mers-cov genome by rt-pcr after several passages as mers-cov tends to occasionally delete sections of the accessory proteins. middle east respiratory syndrome: emergence of a pathogenic human coronavirus engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus engineering the largest rna virus genome as an infectious bacterial artificial chromosome systematic assembly of a full-length infectious cdna of mouse hepatitis virus strain a59 strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis molecular characterization of feline infectious peritonitis virus strain df-2 and studies of the role of orf3abc in viral cell tropism the nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis recovery of a neurovirulent human coronavirus oc43 from an infectious cdna clone development of the full-length cdna clones of two porcine epidemic diarrhea disease virus isolates with different virulence bat severe acute respiratory syndrome-like coronavirus wiv1 encodes an extra accessory protein, orfx, involved in modulation of the host immune response use of bacteriophage lambda recombination functions to promote gene replacement in escherichia coli in vivo recombineering of bacteriophage lambda by pcr fragments and single-strand oligonucleotides a highly efficient escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of bac dna a new logic for dna engineering using recombination in escherichia coli simple and highly efficient bac recombineering using galk selection two-step red-mediated recombination for versatile high-efficiency markerless dna manipulation in escherichia coli engineering infectious cdnas of coronavirus as bacterial artificial chromosomes en passant mutagenesis: a two step markerless red recombination system acknowledgments i thank jeremiah athmer and andrea pruijssers for their substantial modifications and improvements to this protocol over the years. i also thank catherine kerr, ethan doerger, andrea pruijssers, isabel sola, and luis enjuanes for critical reading of this manuscript. this work was supported in part by nih grants cobre p20 gm113117-02 and k22 ai134993-01, and start-up funds from the university of kansas. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-103739-mmkrwj8t authors: snijder, eric j.; limpens, ronald w.a.l.; de wilde, adriaan h.; de jong, anja w. m.; zevenhoven-dobbe, jessika c.; maier, helena j.; faas, frank f.g.a.; koster, abraham j.; bárcena, montserrat title: a unifying structural and functional model of the coronavirus replication organelle: tracking down rna synthesis date: 2020-03-24 journal: biorxiv doi: 10.1101/2020.03.24.005298 sha: doc_id: 103739 cord_uid: mmkrwj8t zoonotic coronavirus (cov) infections, like those responsible for the current sars-cov-2 epidemic, cause grave international public health concern. in infected cells, the cov rna-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (ro). while double-membrane vesicles (dmvs) appear to be a pan-coronavirus ro element, studies to date describe an assortment of additional coronavirus-induced membrane structures. despite much speculation, it remains unclear which ro element(s) accommodate viral rna synthesis. here we provide detailed 2d and 3d analyses of cov ros and show that diverse covs essentially induce the same membrane modifications, including the small open double-membrane spherules (dmss) previously thought to be restricted to gammaand delta-cov infections and proposed as sites of replication. metabolic labelling of newly-synthesized viral rna followed by quantitative em autoradiography revealed abundant viral rna synthesis associated with dmvs in cells infected with the beta-covs mers-cov and sars-cov, and the gamma-cov infectious bronchitis virus. rna synthesis could not be linked to dmss or any other cellular or virus-induced structure. our results provide a unifying model of the cov ro and clearly establish dmvs as the central hub for viral rna synthesis and a potential drug target in coronavirus infection. epidemic, cause grave international public health concern. in infected cells, the cov rna-23 synthesizing machinery associates with modified endoplasmic reticulum membranes that are 24 transformed into the viral replication organelle (ro). while double-membrane vesicles 25 (dmvs) appear to be a pan-coronavirus ro element, studies to date describe an assortment ro [15, 20, 21] , was entirely possible and started to attract 71 attention. notably, dmvs can be also formed in the absence of vrna synthesis by 72 expression of key transmembrane nsps [22] [23] [24] [25] [26] . moreover, several studies suggested a lack of 73 direct correlation between the number of dmvs and the level of cov replication in the 74 infected cell [27, 28] . 75 the interpretation of the cov ro structure and function was further compounded by the 76 discovery of different ro 106 double-membrane spherules (dmss) 107 we first set out to analyse the ultrastructure of mers-cov-infected huh7 cells under sample 108 preparation conditions favourable for autoradiography (see materials and methods) (fig 1, s1 109 video). strikingly, in addition to the dmvs and cm that are well established hallmarks of 110 beta-cov infections, the presence of small spherules, occasionally in large numbers, was 111 readily apparent (fig 1a and 1b) . these spherules were notably similar to the dmss 112 previously described for the gamma-cov ibv [29] . their remarkably regular size of ~80 nm 113 (average diameter 79.8 ± 2.5 nm, n=58), a delimiting double membrane and their electron-114 dense content, made these spherules clearly distinct from other structures, including progeny 115 virions, which had comparable diameter (fig 1c and 1d ). the double-membrane spherules 116 (dmss) generated during ibv infection were previously described as invaginations of the 117 zippered er that remain open to the cytosol [29] . in mers-cov-infected cells, the dmss 118 were connected to the cm from which they seemed to derive ( fig 1e) . clear openings to the 119 cytosol could not be detected for the large majority (~80%, n=54) of the fully reconstructed 120 dmss, which suggests that the original invagination may eventually transform into a sealed 121 compartment. this type of apparently closed dmss were also present, though in a lower 122 proportion (~50%, n=39), in ibv-infected cell samples processed in an identical manner (s1 the 3d architecture of mers-cov-induced ro aligned with previous observations for other 156 cov [15, 29] . no clear openings connecting the interior of the dmvs and the cytosol could 157 be detected. all three types of mers-cov-induced membrane modifications appeared to be 158 interconnected, either directly or indirectly through the er. while dmss were connected to 159 cm, and cm to er, er membranes were often continuous with dmvs (fig 1f, arrowheads) . 160 therefore, like other covs, mers-cov infection appears to induce a network of largely 161 interconnected modified er membranes that, as a whole, can be considered the cov ro. quantification of the autoradiography signal per subcellular structure (see also s1 table) . labelling 214 densities and relative labelling indexes (rli) in different subcellular regions of (c) vero e6 cells 215 infected with sars-cov (moi 10) or (d) huh7 cells infected with mers-cov (moi 5). 216 radioactively-labelled uridine was provided for the indicated periods of time immediately before 217 fixation at 7 hpi and 12 hpi, respectively. control mock-infected cells are excluded from the rli 218 plots, as rli comparisons between conditions require the same number of classes (subcellular 219 regions) and these cells lack ros and virions. budding vesicles (fig 6a-c) .the m and s proteins also localized to the golgi complex, 348 aligning with previous observations for other covs [20, 44, 48, 49] . the mers-cov n 349 protein was found in regions with cm and dmss, though the distribution of signal was 350 homogenous and dmss were not particularly densely labelled (fig 6d) . the presence of the assembly, or the s protein (fig 6e and 6f ). 356 previously, the cm induced by sars-cov and mhv were shown by iem to accumulate 357 viral nsps, while dsrna signal was primarily found inside the dmvs [15, 20] . similarly, 358 nsp3 mapped to the cm induced in mers-cov infection, but also to the dmss to a 359 comparable extent (fig 6g) . our attempts to combine dsrna antibody labelling with thawed 360 cryo-sections were unsuccessful, which made us resort to hpf-fs samples. in these, 361 however, while dmvs were easily detected, the morphology of cm and dmss was less 362 clearly defined. nevertheless, dsrna signal was clearly associated with dmvs, while the 363 dark membranous regions between dmvs that we interpreted as cm and dmss clusters 364 appeared devoid of signal (fig 6h and 6i ). 365 in summary, for the antibodies tested (recognizing n, m, s, nsp3, and dsrna), the labelling 366 pattern in mers-cov-induced dmss closely resembled that of the cm, from which they 367 seem to derive. the absence of labelling for key proteins in virus assembly, like the m and s 368 proteins, strongly suggest that dmss do not represent (spurious) virus assembly events. the comprehensive analysis presented here demonstrates that viruses across different cov 371 genera induce essentially the same type of membrane structures. after somewhat disparate 372 observations [15, 20, 21, 29, 30, 47] , the unifying model that emerges from our study is that our results add to studies that, in the last years and after much speculation, have started to 419 provide experimental evidence that the dmvs induced by +rna viruses are active sites of 420 vrna synthesis [11, [58] [59] [60] . however, it is not clear that dmvs always play the primary role 421 in virus replication that we demonstrate here for cov. for picornaviruses, for example, viruswere freeze-substituted in a leica afs2 system with 0.1% (wt/vol) uranyl acetate as 576 previously described [71] , with the only modification that acetone was replaced by ethanol 577 from the last washing step before lowycril infiltration onwards. cell sections (75 nm thick) 578 were incubated with the primary mouse antibody, then with a bridging rabbit anti-mouse-igg 579 antibody (dako cytomation), and finally with protein a coupled to 15-nm gold particles. 580 after immunolabelling, samples were additionally stained with 7% uranyl acetate and 581 reynold's lead citrate. large mosaic em maps containing dozens of cell profiles were used for the quantitative 608 analysis of the newly-synthesized rna autoradiography signal (see s1 table) . for each 609 cov, different conditions (infected and mock-infected cells, plus different labelling times) 610 were compared using only samples developed after the same period of time. the analysis of 611 the signal in different subcellular regions was carried out using home-built software. areas of 612 4 µm 2 were randomly selected from the mosaic em maps and the autoradiography grains 613 present in those areas were manually assigned to the underlying cellular structures. the 614 abundance of the different types of subcellular structures was estimated through virtual points 615 in a 5×5 lattice superimposed to each selected area, which were also assigned to the different 616 subcellular classes. regularly along the process, the annotated data per condition was split 617 into two random groups and the kendall and spearman coefficients, which measure the 618 concordance between two data sets [73], were calculated. new random regions were added 619 until the average kendall and spearman coefficients resulting from 10 random splits were 620 higher than 0.8 and 0.9, respectively (maximum value, 1). labelling densities and relative 621 labelling indexes (rli) were then calculated from the annotated points [39] . 622 for the analysis of the association of vrna synthesis with each of the different ros motifs, 623 the specific dmvs, dmss and cm included in the analysis were carefully selected. only 624 individual dmvs that were at least one micron away from any other virus-induced membrane 625 modification were included in the analysis. for every grain present in an area of 750 nm 626 radius around each dmv, the distance to the dmv centre was measured. in the case of 627 dmss, which were always part of clusters of virus-induced membrane structures, only dmss 628 in the periphery of these clusters were selected. the quantified signal was limited to sub-629 areas devoid of other ro motifs, which were defined by circular arcs (typically 30 o to 100 o , 630 radius 500 nm) opposite to the ro clusters. cms are irregular structures that appear partially 631 28 or totally surrounded by dmvs. only large cm (> 0.6 µm across) were selected in order to 632 make more apparent (if present) any decay of the autoradiography signal as the distance to 633 the surrounding dmvs increased. for each autoradiography grain, both the distance to the 634 closest cm boundary (d 1 ) and the distance to the opposite cm edge (d 2 ) were measured. the 635 relative distance to the cm edge was then calculated as d 1 /(d 1 +d 2 ) and expressed in 636 percentages. all the measurements in different dmvs, dmss and cm were made using 637 aperio imagescope software (leica) and pooled together into three single data sets. autoradiography is a classic technique that allows the em visualization of a radioactive 3 marker, usually targeting a certain process, and thus reveals the subcellular localization of 4 that process [1, 2] . tritiated uridine, for example, can be used to locate active rna synthesis 5 [3] [4] [5] , as shown also in this study. a clear advantage over the use of alternatives for metabolic 6 labelling of newly-synthesized rna (e.g. br-uridine, br-utp, 5-ethynil uridine) is that the 7 radioactive precursor is chemically identical to the natural substrate. 8 after labelling, the samples are immediately fixed and processed for em. the location of the 9 radioactive marker can then be made apparent by applying a highly-sensitive photographic 10 emulsion (a nuclear emulsion) on top of the cell sections and exposing it for several weeks to 11 months. the beta particles that are emitted as a result of tritium disintegrations generate 12 electrons that get trapped in the silver halide emulsion and create a "latent image". when the 13 emulsion is developed, these negative charges promote the reduction to metallic silver, 14 generating electron-dense grains that are visible by em. in principle, given enough time to 15 accumulate enough radioactive disintegrations, even low levels of the radioactive marker 16 could be detected. in practice, other factors (e.g. background radiation, emulsion aging) set 17 some limits to autoradiography, which is nonetheless a very sensitive technique. 18 the resolution of em autoradiography is limited by the fact that radioactive disintegrations 19 generate beta particles that are emitted in random directions. importantly, the probability of 20 giving rise to signal degreases with the distance from the radioactive source; however, some 21 beta particles may travel up to a few hundred nanometers before striking the photographic 22 emulsion [2] . therefore, it is important to keep in mind that the silver grains may not directly 23 overlay the structure containing the radioactive source. quantitative analyses of the signal 24 2 that take this factor into account, like those presented in this study, become indispensable to 25 maximize the information that autoradiography can provide. as for mer-cov-infected cells (fig 1) . tomographic slices through two regions containing 32 membranous replication factories induced by plus-646 strand rna viruses ultrastructure of the replication sites of positive-strand rna 649 viruses building viral replication organelles close encounters of the membrane types interaction of the innate immune system with positive-656 strand rna virus replication organelles rna virus replication complex parallels form and function of retrovirus capsids. molecular 660 cell three-dimensional 662 analysis of a viral rna replication complex reveals a virus-induced mini-organelle. plos 663 biology composition and three-dimensional architecture of the dengue virus replication and assembly 667 sites template rna 670 length determines the size of replication complex spherules for semliki forest virus three-674 dimensional imaging of the intracellular assembly of a functional viral rna replicase 675 complex the transformation of enterovirus replication structures: a three-dimensional study 679 of single-and double-membrane compartments complex dynamic 682 development of poliovirus membranous replication complexes membrane alterations induced by nonstructural proteins of human norovirus three-690 dimensional architecture and biogenesis of membrane structures associated with hepatitis c 691 virus replication rna replication of 694 mouse hepatitis virus takes place at double-membrane vesicles sars-coronavirus replication is supported by a reticulovesicular network of 698 modified endoplasmic reticulum ultrastructural characterization of arterivirus replication structures: reshaping the 703 endoplasmic reticulum to accommodate viral rna synthesis extensive 707 coronavirus-induced membrane rearrangements are not a determinant of pathogenicity an integrated analysis of 711 membrane remodeling during porcine reproductive and respiratory syndrome virus 712 replication and assembly double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable 717 amounts by negative-strand rna viruses qualitative and quantitative 721 ultrastructural analysis of the membrane rearrangements induced by coronavirus. cellular 722 microbiology mers-coronavirus replication induces severe in vitro cytopathology and is 726 strongly inhibited by cyclosporin a or interferon-alpha treatment severe acute 730 respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane 731 vesicles mutations across murine 738 hepatitis virus nsp4 alter virus fitness and membrane modifications expression 742 and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral 744 rna replication bronchitis virus nonstructural protein 4 alone induces membrane pairing competitive 751 fitness in coronaviruses is not correlated with size or number of double-membrane vesicles 752 under reduced-temperature growth conditions targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse 757 coronaviruses including the middle east respiratory syndrome virus infectious bronchitis virus generates spherules from zippered endoplasmic reticulum 762 membranes replication organelle comprises double-membrane vesicles and zippered endoplasmic 766 does form meet function in the 769 coronavirus replicative organelle? trends in microbiology isolation of 772 a novel coronavirus from a man with pneumonia in saudi arabia. the new england journal 773 of medicine genomic characterization of a newly discovered coronavirus associated with acute 777 respiratory distress syndrome in humans the new england journal of medicine genomic characterisation and 783 epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding techniques and applications of autoradiography in the light and electron 787 microscope electron microscopy: 789 principles and techniques for biologists virtual nanoscopy: generation of ultra-large high resolution electron microscopy maps relative labelling index: a novel stereological 796 approach to test for non-random immunogold labelling of organelles and membranes on 797 transmission electron microscopy thin sections replication of coronavirus mhv-a59 in sac-cells: 800 determination of the first site of budding of progeny virions ultrastructural characterization of sars coronavirus the intracellular sites of early replication and budding of sars-coronavirus novel 811 contrasting and labeling procedures for correlative microscopy of thawed cryosections coronavirus m proteins accumulate in the golgi complex beyond the site of virion budding proliferative growth of sars 820 coronavirus in vero e6 cells morphogenesis of avian infectious 823 bronchitis virus and a related human virus (strain 229e) ultrastructural 826 characterization of membrane rearrangements induced by porcine epidemic diarrhea virus 827 the e1 glycoprotein of an 830 avian coronavirus is targeted to the cis golgi complex the 838 putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase 839 gene polyprotein and localizes in complexes that are active in viral rna synthesis localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a 844 role for late endosomes in viral replication mouse hepatitis virus replicase proteins 847 associate with two distinct populations of intracellular membranes determination of host proteins composing the microenvironment of coronavirus replicase 852 complexes by proximity-labeling. elife the coronavirus nucleocapsid is a 855 multifunctional protein visualizing 859 coronavirus rna synthesis in time by using click chemistry do viruses subvert cholesterol 863 homeostasis to induce host cubic membranes? trends in cell biology cubic membranes: a legend beyond the flatland* of cell membrane organization morphological and 870 biochemical characterization of the membranous hepatitis c virus replication compartment epub 2013/07/26 escaping host factor pi4kb inhibition: enterovirus genomic rna replication in the 875 absence of replication organelles 878 the origin, dynamic morphology, and pi4p-independent formation of 879 encephalomyocarditis virus replication organelles. mbio sars-coronavirus replication/transcription complexes are membrane-protected 884 and need a host factor for activity in vitro early 888 endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication mechanisms and 892 enzymes involved in sars coronavirus genome expression a new virus isolated from the human respiratory tract reverse genetics system for 898 the avian coronavirus infectious bronchitis virus the coronavirus spike protein is a 902 class i virus fusion protein: structural and functional characterization of the fusion core 903 complex ultrastructure and origin of membrane vesicles associated with the severe 907 acute respiratory syndrome coronavirus replication complex monoclonal 911 antibodies to double-stranded rna as probes of rna structure in crude nucleic acid extracts towards a solution to mers: protective human monoclonal antibodies targeting 916 different domains and functions of the mers-coronavirus spike glycoprotein resolution of a gold latensification-elon 920 ascorbic acid developer for ilford l4 emulsion correlated 923 fluorescence and 3d electron microscopy with high sensitivity and spatial precision a rapid 930 method for assessing the distribution of gold labeling on thin sections dmss (white 33 arrowheads) and zippered er (white arrows). most zippered er consists of long stretches of 34 though branching zippered er, closer to the cm 35 described for beta-cov, was also present (b) virus particles (black arrowheads) budding into 36 the er membranes were often observed. scale bars analysis 39 of previously described samples of cov-infected cells, prepared for em either by hpf(a) or 40 cryo-plunging (b). a targeted search revealed the presence of dmss (white arrowheads) in 41 close association with cm. in comparison with the chemically fixed samples used in this 42 study, the superior ultrastructural preservation of cryo-fixation results in less distorted 43 membranes, but also in a denser cytoplasm and darker cm that makes dms less apparent. 44 (a) example from a mers-cov-infected huh7 cell sars-cov-infected cell (8 hpi) metabolic labelling of newly-synthesized vrna in ibv-infected cells and 48 analysis of the autoradiography signal. vero cells infected with ibv were pre-treated with 49 actinomycin d for 1 hour, then labelled for 30 or 60 min with tritiated uridine a) overview of 51 an ibv-infected vero cell (60 min labelling). the areas containing dmvs and zippered er 52 are outlined in yellow and blue, respectively, and other subcellular structures annotated (n, 53 nucleus; m, mitochondria; au, autophagosome; vcr, virion-containing regions). the 54 autoradiography signal accumulates in areas of virus-induced membrane modifications that 55 often only contain dmvs close-up of the area boxed in black in (a), which contains dmvs the contrast between the densely labelled dmvs and the 58 zippered er and dmss largely lacking signal is apparent and suggests that the 59 autoradiography grains sometimes present on the latter structures arose from radioactive 60 disintegrations in the surrounding active dmvs. (c) in agreement with this possibility, most 61 of the dmss (96%) were devoid of signal, and most of those that contained label where close 62 to an active dmv (n dms = 178). (d) furthermore, the distribution of autoradiography grains 63 around dmss resembled that of a random distribution dmvs proved that these structures are associated with vrna synthesis, as the signal reaches 66 maximum values in the proximity of the dmvs (n dmvs = 51). ((c, d) see materials and 67 methods for the selection criteria and details) electron tomography of the membrane structures induced in mers-cov infection animation illustrating the tomography reconstruction and model presented in fig 1b. the 72 video first shows the tomographic slices (1.2 nm thick) through the reconstructed volume, 73 and then surface-rendered models of the different structures segmented from the tomogram cm (blue) and dmvs (yellow and lilac, outer and inner membranes), er 75 4 (green), and a vesicle (silver) containing virions (pink). the movie highlights the dms 76 association with cm techniques and applications of autoradiography in the light and electron 84 microscope autoradiography & radioautography. electron microscopy: 86 principles and techniques for biologists association of polioviral proteins of the p2 89 genomic region with the viral replication complex and virus-induced membrane synthesis as 90 visualized by electron microscopic immunocytochemistry and autoradiography escaping host factor pi4kb inhibition: enterovirus genomic rna replication in the 94 absence of replication organelles 97 the origin, dynamic morphology, and pi4p-independent formation of 98 encephalomyocarditis virus replication organelles. mbio mers-coronavirus replication induces severe in vitro cytopathology and is 103 strongly inhibited by cyclosporin a or interferon-alpha treatment sars-coronavirus replication is supported by a reticulovesicular network of 108 modified endoplasmic reticulum table 79 data sets and sampling for the quantifications of the autoradiography signal presented 80 in fig 3c and key: cord-291590-24psoaer authors: ogando, natacha s.; zevenhoven-dobbe, jessika c.; posthuma, clara c.; snijder, eric j. title: the enzymatic activity of the nsp14 exoribonuclease is critical for replication of middle east respiratory syndrome-coronavirus date: 2020-06-20 journal: biorxiv doi: 10.1101/2020.06.19.162529 sha: doc_id: 291590 cord_uid: 24psoaer coronaviruses (covs) stand out for their large rna genome and complex rna-synthesizing machinery comprising 16 nonstructural proteins (nsps). the bifunctional nsp14 contains an n-terminal 3’-to-5’ exoribonuclease (exon) and a c-terminal n7-methyltransferase (n7-mtase) domain. while the latter presumably operates during viral mrna capping, exon is thought to mediate proofreading during genome replication. in line with such a role, exon-knockout mutants of mouse hepatitis virus (mhv) and severe acute respiratory syndrome coronavirus (sars-cov) were previously found to have a crippled but viable hypermutation phenotype. remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding exon-knockout mutants of another betacoronavirus, middle east respiratory syndrome coronavirus (mers-cov), to be non-viable. this is in agreement with observations previously made for alphaand gammacoronaviruses. only a single mers-cov exon active site mutant could be recovered, likely because the introduced d191e substitution is highly conservative in nature. for 11 other mers-cov exon active site mutants, not a trace of rna synthesis could be detected, unless – in some cases – reversion had first occurred. subsequently, we expressed and purified recombinant mers-cov nsp14 and established in vitro assays for both its exon and n7-mtase activities. all exon knockout mutations that were lethal when tested via reverse genetics were found to severely decrease exon activity, while not affecting n7-mtase activity. our study thus reveals an additional function for mers-cov nsp14 exon, which apparently is critical for primary viral rna synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in mhv and sars-cov. importance the bifunctional nsp14 subunit of the coronavirus replicase contains 3’-to-5’ exoribonuclease (exon) and n7-methyltransferase (n7-mtase) domains. for the betacoronaviruses mhv and sars-cov, the exon domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. for these viruses, exon knockout mutants are alive while displaying an increased mutation frequency. strikingly, we now established that the equivalent knockout mutants of mers-cov exon are non-viable and completely deficient in rna synthesis, thus revealing an additional and more critical function of exon in coronavirus replication. both enzymatic activities of (recombinant) mers-cov nsp14 were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development. the bifunctional nsp14 subunit of the coronavirus replicase contains 3'-to-5' 58 exoribonuclease (exon) and n7-methyltransferase (n7-mtase) domains. for the 59 betacoronaviruses mhv and sars-cov, the exon domain was reported to promote 60 the fidelity of genome replication, presumably by mediating some form of proofreading. for these viruses, exon knockout mutants are alive while displaying an increased 62 mutation frequency. strikingly, we now established that the equivalent knockout 63 mutants of mers-cov exon are non-viable and completely deficient in rna 64 synthesis, thus revealing an additional and more critical function of exon in coronavirus interestingly, a quite different phenotype was described for the corresponding exon-122 knockout mutants of two betacoronaviruses, mouse hepatitis virus (mhv) and cov. while exon inactivation decreased replication fidelity in these viruses, conferring 124 a 'mutator phenotype', the mutants were viable, both in cell culture (22, 23) and in 125 animal models (24). these findings suggested that exon may indeed be part of an 126 error correction mechanism. subsequently, the ability of exon to excise 3'-terminal 127 mismatched nucleotides from a double-stranded (ds) rna substrate was 128 demonstrated in vitro using recombinant sars-cov nsp14 (25). furthermore, this 129 activity was shown to be strongly enhanced (up to 35-fold) by the presence of nsp10, 130 a small upstream subunit of the cov replicase (26). the two subunits were proposed 131 to operate, together with the nsp12-rdrp, in repairing mismatches that may be 132 introduced during cov rna synthesis (21, 27) . in cell culture, mhv and sars-cov 133 mutants lacking exon activity exhibit increased sensitivity to mutagenic agents like 5-134 fluoracil (5-fu), compounds to which the wild-type virus is relatively resistant (28, 29) . 135 recently, exon activity was also implicated in cov rna recombination, as an mhv 136 exon knockout mutant exhibited altered recombination patterns, possibly reflecting its 137 involvement in other activities than error correction during cov replication and 138 subgenomic mrna synthesis (30). outside the order nidovirales, arenaviruses are the 139 only other rna viruses known to employ an exon domain, which is part of the 140 arenavirus nucleoprotein and has been implicated in fidelity control (31) and/or immune 141 evasion, the latter by degrading viral dsrna (32, 33) . based on results obtained with 142 tgev and mhv exon knockout mutants, also the cov exon activity was suggested 143 to counteract innate responses (34, 35) . 144 in the meantime, cov nsp14 was proven to be a bifunctional protein by the discovery 145 of an (n7-guanine)-methyltransferase (n7-mtase) activity in its c-terminal domain 146 (36) (fig. 1 ). this enzymatic activity was further corroborated in vitro, using biochemical 147 assays with purified recombinant sars-cov nsp14. the enzyme was found capable 148 of methylating cap analogues or gtp substrates, in the presence of s-adenosyl 149 methionine (sam) as methyl donor (36, 37) . the n7-mtase was postulated to be a 150 key factor for equipping cov mrnas with a functional 5'-terminal cap structure, as n7-151 methylation is essential for cap recognition by the cellular translation machinery (25). 152 although, the characterization of the nsp14 n7-mtase active site and reaction 153 mechanism was not completed, alanine scanning mutagenesis and in vitro assays with 154 nsp14 highlighted several key residues ( fig. 1) (36, 38, 39) . moreover, crystal 155 structures of sars-cov nsp14 in complex with its nsp10 co-factor (pdb entries 5c8u 156 and 5nfy) revealed several unique structural and functional features (ma et al., 2015; 157 ferron et al., 2018) . these combined structural and biochemical studies confirmed that 158 the two enzymatic domains of nsp14 are functionally distinct (36) and physically 159 independent (20, 21). still, the two activities are structurally intertwined, as it seems 160 that the n7-mtase activity depends on the integrity of the n-terminal exon domain, 161 whereas the flexibility of the protein is modulated by a hinge region connecting the two 162 domains (21). 163 coronaviruses are abundantly present in mammalian reservoir species, including 164 bats, and pose a continuous zoonotic threat (40) (41) (42) (43) . to date, seven covs that can 165 infect humans have been identified, and among these the severe acute respiratory continues to circulate and cause serious human disease, primarily in the arabian peninsula (45) . occasional spread to other countries has also occurred, including an 172 outbreak with 186 confirmed cases in south korea in 2015 (46-48 viable, while displaying a 15-to 20-fold increased mutation rate (eckerle, lu et al. 2007 , 203 eckerle, becker et al. 2010 ). an alignment of cov nsp14 amino acid sequences is 204 presented in fig. 1 , including sars-cov-2, which emerged during the course of this 205 project. it highlights the key motifs/residues of the two enzymatic domains of nsp14, 206 as well as other structural elements, like the nsp10 binding site, the hinge region 207 connecting the exon and n7-mtase domains, and three previously identified zinc 208 finger domains (20, 21) . the alignment also illustrates the generally high degree of 209 nsp14 sequence conservation across different cov (sub)genera. in the present study, we targeted all five predicted active site residues of the merscov exon domain (d90, e92, e191, d273, and h268) by replacing them with alanine 212 as well as more conservative substitutions (d to e or q; e to d or q) . this yielded a 213 total of 14 exon active site mutants ( fig. 2a) transcripts were electroporated into bhk-21 cells, which lack the dpp4 receptor 220 required for natural mers-cov infection (chan, chan et al. 2013 , raj, mou et al. 2013 but are commonly used to launch engineered cov mutants because of their excellent 222 survival of the electroporation procedure (19, 22, 23, 34, 51, 54) . as bhk-21 cells have 223 a severely compromised innate immune response (55), they would seem an 224 appropriate cell line to launch exon knockout mutants also in case the enzyme would 225 be needed to counter innate immunity (becares, pascual-iglesias et al. 2016 , case, li 226 et al. 2018 . to amplify any progeny virus released, transfected bhk-21 cells were 227 mixed with either innate immune-deficient (vero) or -competent (huh7) cells, which 228 both are naturally susceptible to mers-cov infection. in stark contrast to what was previously described for mhv and sars-cov, 230 mutagenesis of exon active site residues was found to fully abrogate mers-cov 231 replication. when transfected cell cultures were analyzed using immunofluorescence 232 microscopy at 2 days post transfection (d p.t.), abundant signal was always observed 233 for wild-type mers-cov, but no sign of virus replication was observed for 13 out of 14 234 mutants tested (fig. 2 ), regardless whether vero or huh7 cells were used for 235 propagation of recombinant virus. furthermore, infectious progeny was not detected 236 when transfected cell culture supernatants were analyzed in plaque assays ( fig. 2 and 237 data not shown). the single exception was the mutant carrying the conservative e191d 238 replacement in exon motif ii (fig. 1) , which was alive but somewhat crippled, as will 239 be discussed in more detail below. these results were consistent across a large the lack of mers-cov-specific rna synthesis was further analyzed using rt-pcr 259 assays specifically detecting genomic rna or subgenomic mrna3. rna specifying an a1235d substitution in the betacoronavirus-specific marker (βsm) 287 domain of nsp3, which has been predicted to be a non-enzymatic domain (58) and is 288 absent in alpha-and delta-coronaviruses (59, 60). thus, we assumed that any 289 changes in viral replication were likely caused by the e191d mutation in nsp14 exon. the same virus stock was used to assess growth kinetics in huh7 cells (fig. 4b ) and 291 vero cells (fig. 4a ), which were found to be very similar for wt and mutant virus. still, 292 the e191d mutant was found to be somewhat crippled, yielding smaller plaque sizes 293 and somewhat lower progeny titers in huh7 cells (fig. 4 b-c), but not in vero cells 294 (fig. 4a ). 295 we next examined the sensitivity of e191d and wt virus to the mutagenic agent 5-296 fu, which intracellularly is converted into a nucleoside analogue that is incorporated 297 into viral rna (61, 62). previously, mhv and sars-cov exon knockout mutants were 298 found to exhibit increased sensitivity to 5-fu treatment, in particular in multi-cycle 299 experiments, which was attributed to a higher mutation frequency in the absence of 300 exon-driven error correction (28). we employed this same assay to assess the 301 phenotype of the e191d mutant in more detail, by performing plaque assays in huh7 previously, the exon activity of sars-cov nsp14 was found to be dramatically 366 stimulated by the addition of nsp10 as co-factor (26). consequently, we also expressed 367 and purified mers-cov nsp10 and optimized the exon assay by testing different 368 molar ratios between nsp14 and nsp10 (fig. 6a , left-hand side), different nsp14 369 concentrations (fig. 6b , left-hand side), and by different incubation times (fig. 7 , left-370 hand side). mers-cov nsp14 exon activity was found to be stimulated by nsp10 in a 371 dose-dependent manner (fig. 6a) , while nsp10 did not exhibit any nuclease activity by 372 itself (fig. 6b, nsp10 lane) . the full-length substrate is more completely degraded 373 when a fourfold (or higher) excess of nsp10 over nsp14 was used compared to the 374 effect of merely increasing the nsp14 concentration in the assay (fig. 6b ). similar (bouvet, imbert et al. 2012 , ma, wu et al. 2015 . using a 4:1 ratio of nsp10 versus nsp14, mers-cov exon activity was analyzed in 382 a time-course experiment, (fig. 7) . over time, the full-length substrate was 383 progressively converted to a set of degradation products in the size range of 6-18 nt. 384 we anticipated that the structure of the h4 rna substrate would change from a when the two proteins were combined in the same reaction, a strong increase of exon 409 activity was observed for both nsp14-nsp10 pairs, with the sars-cov pair appearing 410 to be somewhat more processive than the mers-cov pair (fig. 8, lanes 2 and 9) . (fig. 2) . 434 we also evaluated the impact of the h229c zf1 mutation, which -despite its 435 conservative nature -yielded a crippled mutant virus (fig. 2) and of two n7-mtase 436 mutations (discussed below). the n7-mtase mutants displayed wt nsp14-like exon 437 activities (fig. 9) , suggesting that -as in sars-cov nsp14 -exon and n7-mtase 438 activities are functionally separated (36). analyzing the substrate degradation pattern 439 of the h229c mutant (fig. 7) , the enzyme seems to be somewhat crippled when 440 compared to wt nsp14. this suggests that this mutation alters exon activity in vitro, 441 potentially by affecting the structure of the exon domain, as zf1 is in close proximity of the nsp10 interaction surface (20). however, a similar reduction of exon activity was 443 observed for the e191d mutant (fig. 7) , which was much more viable than the h229c recombinant mers-cov nsp14 was found to methylate gpppa, but not m7gpppa 464 (fig. 10a) , which yielded a signal that was similar to the background signal in assays 465 lacking nsp14 or substrate (data not shown). methylation increased with time until 466 reaching a plateau after 120 min (fig. 10b) . the n7-mtase activity of the various 467 nsp14 mutants was compared with that of wt nsp14 after reaction times of 30 and 120 468 min (fig. 10c) . while the r310a and d331a control mutations fully inactivated the n7exon catalytic residues abolished all detectable viral rna synthesis (fig. 3 ) and the 484 release of viral progeny (fig. 2) . the only exception was the conservative e191d 485 mutant, which was found to exhibit near-wild-type levels of exon activity ( fig. 7 and 486 9). based on nsp14 conservation (fig. 1 ) and the viable phenotype of sars-cov and 487 mhv exon-knockout mutants, mers-cov was expected to tolerate exon inactivation, 488 in particular since the enzyme was proposed to improve the fidelity of cov replication 489 without being essential for rna synthesis per se (10, 14, 21-23, 26, 29) . this notion is in the only viable mers-cov exon active site mutant obtained, e191d, the catalytic 545 motif was changed from deedh to the deddh that is characteristic of all members of 546 the exonuclease family that exon belongs to (14, 15, 75) . the phenotype of the e191d 547 virus mutant was comparable to that of wt virus (fig. 4) . biochemical assays revealed 548 that e191d-exon enzyme is able to hydrolyze a dsrna substrate with an activity level 549 approaching that of the wt protein (fig. 9) . additionally, the e191d mutant behaved in this study, we developed an in vitro assay to evaluate mers-cov exon activity 554 using a largely double-stranded rna substrate ( fig. 6 and 7) . as previously observed is interchangeable between cov subgenera in its role as co-factor for the nsp16 2′-o-565 methyltransferase, which was attributed to the high level of conservation of the nsp10-566 nsp16 interaction surface (77). as nsp14 and nsp16 share a common interaction 567 surface on nsp10 (21, 26, 66), we explored whether a similar co-factor exchange was 568 possible in the context of nsp14's exon activity, which was indeed found to be the case 569 (fig. 8) . structurally, nsp14 interacts with nsp10 figuratively similar to a "hand (nsp14) 570 over fist (nsp10)" conformation (21). in the formation of this complex, nsp10 induces 571 conformational changes in the n-terminal region of exon that adjusts the distance 572 between the catalytic residues in the back of the nsp14 palm and, consequently, impact 573 exon activity (21). the exchange of the nsp10 co-factor between the two beta-covs 9), indicating that each of these residues is important for catalysis. our study suggests that, in addition to the active site residues, also other motifs in 589 mers-cov exon are important for virus viability, specifically the two zf motifs that 590 were probed using two point mutations each (fig. 2a) . in previous zf1 studies, a 591 mutation equivalent to h229a created solubility issues during expression of recombinant sars-cov nsp14 (20) and resulted in a partially active exon in the case 593 of white bream virus, a torovirus that also belongs to the nidovirus order (79). it was 594 suggested that zf1 contributes to the structural stability of exon, as it is close to the 595 surface that interacts with nsp10 (20). here, we demonstrate that the more 596 conservative h229c replacement, which converts zf1 from a non-classical ccch 597 type zf motif into a classical cccc type (63), was tolerated during recombinant protein 598 expression and yielded an exon that is quite active in vitro (fig. 7) . this likely 599 contributed to the fact that the h229c virus mutant retained a low level of viability ( fig. 600 2), although its overall crippled phenotype and the non-viable phenotype of mutant 601 c201h clearly highlight the general importance of zf1 for virus replication. in contrast, 602 the corresponding tgev mutant (zf-c) was not strongly affected and could be stably quasispecies diversity 879 determines pathogenesis through cooperative interactions in a viral population the hypercycle. a principle of natural self-organization. part a: 882 emergence of the hypercycle viral quasispecies evolution mutational fitness effects in rna and single-stranded dna viruses: common 886 patterns revealed by site-directed mutagenesis studies viral mutation rates lack of evidence for proofreading mechanisms 891 associated with an rna virus polymerase mutation rates among rna viruses error catastrophe and antiviral strategy selforganization of matter and the evolution of biological macromolecules discovery of the first 899 insect nidovirus, a missing evolutionary link in the emergence of the largest rna virus 900 genomes the 902 footprint of genome architecture in the largest genome expansion in rna viruses a planarian 905 nidovirus expands the limits of rna genome size description and 907 initial characterization of metatranscriptomic nidovirus-like genomes from the proposed new 908 family abyssoviridae, and from a sister group to the coronavirinae, the proposed genus 909 alphaletovirus unique and conserved features of genome and proteome of sars-912 coronavirus, an early split-off from the coronavirus group 2 lineage exoribonuclease superfamilies: structural analysis and 914 phylogenetic distribution purification and characterization of escherichia coli rnase 916 t structural basis for the 3'-5' exonuclease activity of escherichia coli 918 dna polymerase i: a two metal ion mechanism a general two-metal-ion mechanism for catalytic rna 922 discovery of an rna virus 3'->5' exoribonuclease that is critically involved in coronavirus rna 923 synthesis structural basis 925 and functional analysis of the sars coronavirus nsp14-nsp10 complex structural and molecular basis of mismatch correction 929 and ribavirin excision from coronavirus rna high fidelity of murine hepatitis virus 931 replication is decreased in nsp14 exoribonuclease mutants infidelity of sars-cov nsp14-exonuclease mutant 934 virus replication is revealed by complete genome sequencing a live, impaired-936 fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal 937 disease in vitro 939 reconstitution of sars-coronavirus mrna cap methylation rna 3'-end mismatch 941 excision by the severe acute respiratory syndrome coronavirus nonstructural protein 942 nsp10/nsp14 exoribonuclease complex one severe acute respiratory syndrome coronavirus protein 945 complex integrates processive rna polymerase and exonuclease activities coronaviruses lacking 948 exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading 949 and potential therapeutics coronaviruses adapt for increased fitness over long-term passage without reversion of 952 exoribonuclease-inactivating mutations routh 954 al, denison mr. 2020. the coronavirus proofreading exoribonuclease mediates extensive 955 viral recombination arenaviridae exoribonuclease presents genomic rna edition capacity structure of the lassa 960 virus nucleoprotein reveals a dsrna-specific 3' to 5' exonuclease activity essential for 961 immune suppression the exonuclease domain of lassa virus 963 nucleoprotein is involved in antigen-presenting-cell-mediated nk cell responses coronavirus nsp14 reveals its potential role in modulation of the innate immune response murine hepatitis virus nsp14 exoribonuclease activity is required for resistance to innate 970 immunity functional screen reveals sars 972 coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase 975 characterization of the guanine-n7 methyltransferase activity of coronavirus nsp14 on 976 nucleotide gtp structure-978 function analysis of severe acute respiratory syndrome coronavirus rna cap guanine-n7-979 methyltransferase methionine-binding residues in coronavirus nsp14 n7-methyltransferase demonstrates 982 differing requirements for genome translation and resistance to innate immunity bat origin of a new human coronavirus: there and back 985 again identification of new human coronaviruses a novel coronavirus 989 emerging in china -key questions for impact assessment a pneumonia outbreak 993 associated with a new coronavirus of probable bat origin isolation of a 995 novel coronavirus from a man with pneumonia in saudi arabia spread, circulation, and evolution of the 999 middle east respiratory syndrome coronavirus. mbio 5 dynamics of scientific publications 1001 on the mers-cov outbreaks in saudi arabia drivers of mers-cov emergence in 1004 qatar drivers of mers-cov transmission: what do we know? additional changes to taxonomy ratified in a special vote by the international committee on 1011 taxonomy of viruses coronaviridae study group of the international committee on taxonomy of v. 2020. the 1013 species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and 1014 naming it sars-cov-2 engineering a replication-competent, propagation-defective middle east 1017 respiratory syndrome coronavirus as a vaccine candidate accessory protein 4a inhibits pkr-mediated antiviral stress responses genomic characterization of a 1024 newly discovered coronavirus associated with acute respiratory distress syndrome in 1025 humans effects of mutagenesis of 1027 murine hepatitis virus nsp1 and nsp14 on replication in culture t7 rna polymerase-dependent and -1029 independent systems for cdna-based rescue of rift valley fever virus a conserved 3'----5' exonuclease 1032 active site in prokaryotic and eukaryotic dna polymerases the curious case 1034 of the nidovirus exoribonuclease: its role in rna synthesis and replication fidelity proteomics analysis 1038 unravels the functional repertoire of coronavirus nonstructural protein 3 bioinformatics and functional analyses of coronavirus nonstructural 1040 proteins involved in the formation of replicative organelles nsp3 of coronaviruses: structures and functions of a large 1042 multi-domain protein 5-fluorouracil incorporation into rna and dna in relation to thymidylate 1045 synthase inhibition of human colorectal cancers effect of 5-fluorouracil combination therapy on rna 1047 processing in human colonic carcinoma cells zinc finger domains as therapeutic targets for metal-based compounds 1049 -an update zn(ii) binding 1051 and dna binding properties of ligand-substituted cxhh-type zinc finger proteins biochemical characterization of 1054 exoribonuclease encoded by sars coronavirus coronavirus nsp10, a critical co-factor for activation of multiple replicative enzymes the crystal 1061 structure of nsp10-nsp16 heterodimer from sars-cov-2 in complex with s-1062 adenosylmethionine alisporivir inhibits mers-and sars-1065 coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model mers-coronavirus replication induces severe in vitro cytopathology and is strongly 1070 inhibited by cyclosporin a or interferon-alpha treatment sars-coronavirus-2 1073 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology fitness barriers limit 1076 reversion of a proofreading-deficient coronavirus structure of the sars-cov nsp12 polymerase bound to 1078 nsp7 and nsp8 co-factors 2020. 1081 structural basis for inhibition of the rna-dependent rna polymerase from sars-cov-2 by 1082 remdesivir structure of 1084 replicating sars-cov-2 polymerase the 3'-5' exonuclease site of dna 1086 polymerase iii from gram-positive bacteria: definition of a novel motif structure genome-wide analysis of protein-protein interactions and involvement of viral proteins in 1090 sars-cov replication coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in 1093 vitro and in vivo to reduce replication and pathogenesis the 3'-5' exonuclease of dna polymerase i of 1095 escherichia coli: contribution of each amino acid at the active site to the reaction characterization of a 1098 bafinivirus exoribonuclease activity to sense or not to sense viral rna--essentials of coronavirus innate 1100 immune evasion sars-coronavirus replication is supported by a reticulovesicular 1103 network of modified endoplasmic reticulum 2020. a unifying structural and functional model of the coronavirus 1106 replication organelle: tracking down rna synthesis characterization of bafinivirus main protease 1108 autoprocessing activities the nonstructural proteins directing coronavirus rna 1110 synthesis and processing arterivirus nsp1 modulates the 1112 accumulation of minus-strand templates to control the relative abundance of viral mrnas en passant mutagenesis: a two step markerless 1115 red recombination system double-stranded rna is 1117 produced by positive-strand rna viruses and dna viruses but not in detectable amounts by 1118 negative-strand rna viruses ultrastructure and origin of membrane vesicles associated with the 1121 severe acute respiratory syndrome coronavirus replication complex reverse genetics of sars-related 1124 coronavirus using vaccinia virus-based recombination new low-viscosity overlay 1126 medium for viral plaque assays cov forms an ion channel: experiments and molecular dynamics simulations toward the identification of viral 1132 cap-methyltransferase inhibitors by fluorescence screening assay fast, scalable generation of high-quality protein 1135 multiple sequence alignments using clustal omega jalview version 2--a 1137 multiple sequence alignment editor and analysis workbench key: cord-289520-i6pv90s9 authors: harris, carlyn; carson, gail; baillie, j kenneth; horby, peter; nair, harish title: an evidence-based framework for priority clinical research questions for covid-19 date: 2020-03-31 journal: journal of global health doi: 10.7189/jogh.10-011001 sha: doc_id: 289520 cord_uid: i6pv90s9 background: on 31 december, 2019, the world health organization china country office was informed of cases of pneumonia of unknown aetiology. since then, there have been over 75 000 cases globally of the 2019 novel coronavirus (covid-19), 2000 deaths, and over 14 000 cases recovered. outbreaks of novel agents represent opportunities for clinical research to inform real-time public health action. in 2018, we conducted a systematic review to identify priority research questions for severe acute respiratory syndrome-related coronavirus (sars-cov) and middle east respiratory syndrome-related coronavirus (mers-cov). here, we review information available on covid-19 and provide an evidenced-based framework for priority clinical research in the current outbreak. methods: three bibliographic databases were searched to identify clinical studies published on sars-cov and mers-cov in the outbreak setting. studies were grouped thematically according to clinical research questions addressed. in february 2020, available information on covid19 was reviewed and compared to the results of the sars-cov and mers-cov systematic review. results: from the research objectives for sars-cov and mers-cov, ten themes in the literature were identified: clinical characterisation, prognosis, diagnosis, clinical management, viral pathogenesis, epidemiological characterisation, infection prevention and control/transmission, susceptibility, psychosocial, and aetiology. for covid19, some information on clinical presentation, diagnostic testing, and aetiology is available but many clinical research gaps have yet to be filled. conclusions: based on a systematic review of other severe coronaviruses, we summarise the state of clinical research for covid-19, highlight the research gaps, and provide recommendations for the implementation of standardised protocols. data based on internationally standardised protocols will inform clinical practice real-time. viewpoints research theme 6: outbreaks, especially of novel agents, create a pressing need to collect data on clinical characterization, treatment, and validation of new diagnostics to inform rapid public health response. in 2018, we conducted a systematic review to identify the most common clinical research questions asked during outbreaks of sars-cov and mers-cov. we identified ten major clinical questions and provided recommendations for standardised protocol study designs that should be designed in the case of a new outbreak of a novel respiratory pathogen. here, we review the currently available information on covid-19 to determine which clinical questions from the systematic review findings have already been addressed, what information is lacking, and compare covid-19 to sars-cov and mers-cov. we included any original study included conducted in a clinical setting during an acute outbreak of mers and sars but limited the sars studies to those published during the sars epidemic and (16 november 2002 through 5 july 2003) and 18 months thereafter to identify clinical questions relevant during the acute phases of the outbreak. therefore, for sars studies, the last publication date considered for full text review was 31 december 2004. there was no publication date restriction for mers studies as outbreaks were ongoing. search terms to capture observational study designs such as cohort studies, cross-sectional studies, case-control studies, and case series were adapted from the scottish intercollegiate guidelines network (sign) search filters [4] . search terms on diagnosis and prognosis were adapted from pubmed clinical query search filters provided in the pubmed help manual [5] . to capture studies that were conducted in the epidemic or outbreak setting, subject headings such as "disease outbreaks", "epidemics", "pandemics", etc. were included. we applied our inclusion and exclusion criteria ( table 1) for both title and abstract screening and subsequently full text screening. following data extraction, the objectives of the included studies were grouped thematically. within each theme, articles with objectives that represented similar research questions were summarised. studies must be: 1) the main objective was not the study of sars-cov or mers-cov -focused sars-cov or mers-cov 2) the study was not conducted primarily in a clinical setting (ie, population epidemiology studies, in-vitro studies, surveillance studies were excluded) -conducted in a clinical setting 3) the study was not conducted in an outbreak setting or did not analyse data that was collected in an outbreak setting -conducted on humans 4) non-human studies study designs considered: observational studies (prospective cohort, retrospective cohort, case-control, case-series) and cross-sectional studies. 5) the sample size was less than 4 6) the study was not original as this outbreak is in its initial stages, we reviewed resources such as: the world health organization disease outbreak news, ministry of health websites from affected countries, the center for disease control' s morbidity and mortality weekly report (mmwr), promed, and publications found on pubmed. for pubmed searches, the search term "novel and coronavirus" was used, with a date range starting at 1 january 2020. the search was conducted on 20 february 2020. references of articles found through these searches were also reviewed. only studies conducted in the clinical settings were included and single case reports were excluded. we compared our findings to the 2018 systematic review on sars and mers to determine which questions have already been addressed, what information is lacking, and provide recommendations for data sharing and clinical study designs to be conducted during the current outbreak. we grouped the results thematically from reviewing available resources on the novel coronavirus based on their relevance to the previously identified ten clinical questions. viewpoints research theme 6: covid-19 pandemic no funding body had any role in study design, in the collection, analysis, and interpretation of data; in the writing of the report; nor in the decision to submit the paper for publication. a total of 124 studies (71% on sars) were included for the final review and data extraction. all were conducted in a hospital setting. after thematically coding the objectives of the 124 studies, ten key themes emerged: clinical characterisation, prognosis, diagnosis, clinical management, viral pathogenesis, epidemiological characterisation, infection prevention and control/transmission, susceptibility, psychosocial, and aetiology. originally, infection prevention and control and transmission were grouped separately. however, they were combined as most of the papers in the "transmission" category were concerned with transmission to hospital workers. of note, only 16% of sars articles were published before the end of the outbreak. table 2 defines the key clinical research questions identified from each theme. this table was modelled after a publication describing harmonisation of zika virus protocols [6] . themes appear in order based on how many sars and mers articles attempted to address a question within that theme. determine effect of illness, treatment, and isolation procedures on the psychological and social well-being of those infected current state of knowledge for 2019-covid-19 here, we answer what clinical information is currently known about the novel coronavirus, using the above questions as a framework. what is the clinical presentation and spectrum of disease? the first aggregated patient data comes from a publication by chaolin huang and colleagues, who described clinical features of 41 admitted hospital patients, 6 of whom died. they collected data using an adaptation of international severe acute respiratory and emerging infection consortium' s (isaric) / world health organisation (who) internationally standardised data collection forms, updated for use with the novel coronavirus (https://isaric.tghn.org/novel-coronavirus/). they report that the novel coro-viewpoints research theme 6: navirus presentation resembles that of sars: a viral pneumonia with fever, cough, dyspnoea, and fatigue. they also found a high concentration of cytokines in critically ill patients, compared to less severe cases [7] . chen one report of pregnant patients demonstrated similar symptomology to non-pregnant patients. they also established that there was no evidence of vertical transmission to the child [11] . in the only study of infants with covid-19, wei et al. reported fever and upper respiratory symptoms common among nine infants. all infants were infected via family clusters [12] . finally, radiological studies attempted to characterise the course of disease in chest imaging. pan et al. report peak lung involvement at 10 days on ct studies [13] . of note is a case series by xie et al. that reported ground glass opacity in five patients with initial negative rt-pcr findings but suspected covid-19. they were later confirmed to be infected [14] . overall, clinical characterisation studies commonly found bilateral involvement with ground glass opacity, though a range of presentations were reported [8] [9] [10] 15] . what are the risk factors for death or severe illness? while risk factors for death and severe illness cannot be firmly established without large groups of patient data and multi-variable adjusted methods, some studies report similar risk factors among cohorts. [17] . it includes indexes such as multi-lobular infiltration, lymphopenia, bacterial co-infection, smoking, hypertension, and age. in the publication by chaolin huang and colleagues, antibiotics and oseltamivir, and oxygen support were administered to some patients. corticosteroids were used if patients were diagnosed with severe community acquired pneumonia [7] . chen et al. reported antiviral, antibiotic, and non-invasive mechanical ventilation use but did not provide comparisons or suggestions for guidelines [8] . kui et al. determined that use of systemic corticosteroids in their cohort did not show benefits, but early respiratory support improved outcomes [10] . in addition to a randomised controlled trial of lopinavir/ritonavir in adults hospitalised with covid-19 (trial registration number: chictr2000029308) [18] , over 80 clinical trials are expected to determine therapeutic options [19] . several diagnostic tests for the novel coronavirus have been developed. of note are those by corman and colleagues [20] and by hong kong university school of medicine [21] . the world health organization has provided preliminary guidance on specimen collection and shipment as well as reporting [22] . all information on diagnostic testing can be found at the who' s technical guidance site. zou et al. obtained upper respiratory specimens from 18 patients in zhuhai, china. highest viral loads were detected soon after symptom onset, with higher loads in the nose and throat. they suggest that the shedding pattern of sars-cov-2 resembles patients with influenza. asymptomatic patients were also found to have nasopharyngeal viral loads similar to symptomatic patients [23] . additionally, y. zhang et al. found live virus in stool samples [24] . zhou the world health organization has provided a case definition in their interim surveillance guidance for the novel coronavirus [28] . the definition includes patients with a severe acute respiratory infection (sari) with relevant travel history within 14 days of illness or a health care worker caring for those with sari. currently, there are no data on what clinical activities are associated with an increased risk to health care workers. as of 14 february 2020, over 1700 health care workers have been infected [29] . the world health organization has provided interim guidelines for infection prevention and control [30] . currently, it is unclear whether certain demographics of the population are more susceptible to infection with the novel coronavirus. most initial cases had contact with wuhan' s huanan seafood market, the suspected source of the outbreak, or had contact with those who had visited the market [1] . what is the causative agent of disease? sequencing analysis from lower respiratory tract samples from bronchoalveolar lavage identified the novel coronavirus. genomes were released on gisaid.org. due to similarities with related viruses, a bat reservoir is suspected [25] . currently, there are no data on the psychosocial impacts of infection, hospitalization, or quarantine among affected patients and health care workers. we summarise the available clinical information on covid-19, using results from a systematic review on sars and mers as a framework. as highlighted by david heymann in a recent lancet commentary, rapid action by clinicians and scientists to share data has made it possible to identify a causative agent, design and implement a diagnostic test, as well as begin to understand patient presentation [31] . as outlined above, there is much work to be done on understanding the clinical picture of the novel coronavirus. the world health organization r&d research blueprint and the global research collaboration for infectious disease preparedness (glopid-r) have identified similar research priorities as those we identified from our systematic review. overlapping priorities include understanding viral pathogenesis, clinical characterisation studies, infection prevention and control, and candidate therapeutics [32] . to facilitate answering these key clinical questions, we suggest the following study designs ( table 3) . notably, the world health organization released a cases and contacts investigation protocol called "the first few x (ffx)". it involves prospective case finding and follow-up to gain an early understanding of key clinical, epidemiological, and virologic characteristics of the first cases of covid-19 in a given country. they have also released a protocol to assess risk factors for infection among health care workers. in terms of clinical presentation, early recognition of symptoms and disease progression also allows for rapid isolation, early clinical care and limits onward transmission. who and isaric have released an updated version of their case report form (crf) specifically for covid-19. this can be used to collect anonymised, standardised clinical data to start to inform our understanding of the presentation and natural history of covid-19. this crf is being rolled out as a tool for public health and may or may not require ethical approval according to local regulations. a more in-depth characterisation, with the collection of serial biological samples through a research protocol and informed consent can be obtained through the isaric/who clinical characterization protocol. this a standardised, prospective, observational study for the rapid investigation of patients with severe acute infections. the protocol was designed to characterise host and pathogen features, triage and treatment of disease [33] . the who ffx protocol, described above, may be used for the earliest cases to identify key clinical characteristics in real-time. the current clinical characterisation articles are a start to our understanding of the clinical presentation and spectrum viewpoints research theme 6: covid-19 pandemic of disease but much larger cohorts are needed for greater precision around estimates and to undertake prognostic and risk factor analyses. based on the sars and mers systematic review, further clinical research in this area should include if and how symptomology, laboratory findings, and imaging studies differ between demographic groups (ie. adults vs children, immunosuppressed patients). what are risk factors for death or severe illness? based on recent data, male gender, advancing age and co morbidities seem to be associated with death and severe illness [16] . understanding prognostic factors for death or severe illness helps hospitals and public health authorities determine resource allocation [34] [35] [36] . during the sars epidemic, risk factors for mortality were advanced age, co-morbidities, and initial high inflammatory laboratory markers [37, 38] . similarly, for mers, advanced age and co-morbidities are predictors of severe illness and death [39] . many studies identified from the mers and sars systematic review were retrospective cohort studies and poor outcomes were assessed 21-30 days from symptom onset. in designing protocols for mortality or severe illness risk factors for covid-19, case-control studies or prospective cohort studies should be used with an end-point at 90 days, so as not to miss late deaths. whatever the optimal end-point for assessing outcome, standardising this outcome measure across studies will allow researchers to contribute to core data sets. while there is some preliminary descriptive data on clinical management, randomized controlled trials are needed to determine the best treatment options for covid-19. for now, the world health organization has issued interim guidelines for clinical management, adapted from their guidance for mers-cov. it includes recommendations for early recognition, early supportive therapy (oxygen, fluids, empirical antimicrobials) and against routine use of systemic corticosteroids unless indicated for another reason. they also provide guidelines for cases of septic shock [30] . clinical trials to test therapeutic efficacy are ongoing, such as a trial of lopinavir/ritonavir (https://www.pharmaceutical-technology.com/analysis/coronavirus-mers-cov-drugs/). more studies are expected soon [19] . most of studies identified in the sars and mers review were descriptive treatment studies. these observational studies are practical in the fast-paced outbreak setting, as they are easier than randomised controlled the first few x (ffx) who protocol https://www.who.int/publications-detail/the-first-few-x-(ffx)-cases-and-contact-investigation-protocol-for-2019-novel-coronavirus-(2019-ncov)-infection) what are the risk factors for death or severe illness? case-control study or prospective cohort with outcome of death or another defined poor outcome what treatments are effective? randomised controlled trials or adaptive trial designs what is the role of antivirals in treatment? who master protocol: https://www.who.int/blueprint/priority-diseases/key-action/multicenter-adaptive-rct-of-investigational-therapeutics-for-covid-19.pdf?ua=1 what is the role of steroids in treatment? what is the optimal diagnostic test for detecting the virus? what is the causative agent of disease? laboratory based study with clinical specimens, fulfilling koch' s postulates viewpoints research theme 6: covid-19 pandemic trials (rcts) to design and require less administrative effort. however, when it comes to treatment, rcts provide the best primary evidence for medical practice [40] . among these studies, there was great heterogenicity for testing efficacy of specific treatments. this reflects the lack of global research coordination in delivering medical countermeasures during the mers outbreaks and the sars epidemic. this should be kept in mind when designing treatment protocols for covid-19 and is being addressed by who. the rct on ebola therapeutics in the democratic republic of the congo is evidence that conducting clinical therapeutics research is possible in the context of an outbreak [41] . the who has released a master protocol for a multi-centre, adaptive, randomized, double-blind placebo-controlled clinical trial to evaluate safety and efficacy of therapeutic agents for the treatment of hospitalized patients with covid-19. using a master protocol across international sites can speed the implementation of clinical trials and quickly inform treatment options (https://www.who.int/blueprint/ priority-diseases/key-action/multicenter-adaptive-rct-of-investigational-therapeutics-for-covid-19.pdf?ua=1). what is the optimal diagnostic test? the rapid development of a diagnostic test for covid-19 was a critical development and the result of international collaboration. as the outbreak progresses, it is important to continue monitoring diagnostic validity, such as sensitivity, specificity, positive predictive value, and negative predictive value. during an outbreak or epidemic, it is important to also validate diagnostic tests in low prevalence areas, as predictive values may change [42] . what is the duration of viral shedding? understanding the duration of viral shedding and the shedding profile from different anatomical sites are key for both diagnosis and instituting infection prevention and control measures [43] . current data suggest that sars-cov-2 viral loads are high at the beginning of symptom onset, are found in upper respiratory specimens and stool specimens, and are detectable in asymptomatic patients at levels similar to symptomatic patients [23] . one sars study revealed that viral detection peaked at 2 weeks after onset for respiratory specimens and 2-3 weeks for stool and rectal specimens. the shedding peak in urine occurred around weeks 3-4. rarely did patients shed virus 6 weeks after onset, however it was documented in a few stool specimens [44] . to evaluate the shedding profile during the covid-19 outbreak, the isaric/who clinical characterization protocol can be adapted to prospectively and systematically collect serial samples from patients with suspected infection [33, 45] . as with studies on sars and mers, serial samples should be collected from multiple body sites, including urine, faecal, and nasopharyngeal samples. what characteristics define a "case"? in an outbreak, if multiple sites adopt a standardised protocol such as the isaric/who clinical characterization protocol to describe cases, case definitions could be created rapidly to inform accurate reports on incidence and prevalence. developing criteria for confirmed cases is usually based on laboratory diagnosis. the world health organization has provided an interim case definition, and it will likely evolve as more data are shared on patient presentation. what risk factors pre-dispose health care workers to infection or transmission? over 1700 health care workers in wuhan have been diagnosed with covid-19 and there is no research available as to how this happened. both mers and sars viruses showed nosocomial transmission amplification in the health care setting [46] . during the sars epidemic, 21% of the infected patients were health care workers, and in some countries, this rate was as high as 50% [47] . risk of sars infection was associated with inconsistent use of personal protective equipment, and less than 2 hours of infection control training [48] . during the 2015 south korea mers outbreak, 44% of the 186 cases were patients that had been exposed to nosocomial transmission in hospitals, and 83% of total transmission events were due to five super spreading events in hospitals [49] . the recently released who protocol for evaluating risks to health care workers should be implemented as soon as possible to prevent future health care worker infections. viewpoints research theme 6: what are the risk factors for infection (in patient population)? the suspected major risk factors for covid-19 are visiting the wuhan market or being in contact with someone who had visited the market. the above-mentioned who ffx protocol can be used globally to assess risk factors for infection. in the 2018 sars and mers systematic review, only three studies with a psychosocial focus were identified. however, integrating social science research into clinical and epidemiological research during an outbreak can help inform the need for psychologists, psychiatrists, and social workers. especially in diseases with human-to-human transmission, the effect of stigma and quarantine on mental health cannot be underestimated. psychosocial manifestations can be explored with mixed methods studies. what is the causative agent of disease? because of rapid data sharing and laboratory protocols by the chinese health officials and the world health organization, the causative agent of the novel coronavirus was rapidly identified. our systematic review summarises the questions that are answered in the context of new outbreaks, but this methodology cannot tell us what questions should be answered. however, we propose this as a proxy for ensuring key clinical questions are addressed early in an outbreak. we have summarised key knowledge gaps for covid19, but we do not intend to suggest that this is an exhaustive list. many of the most important discoveries hinge on the creativity and innovation to identify new questions. in this new outbreak, we need new ideas to build on the foundations that we have comprehensively summarised in this article. the suggested study designs above can be used to inform standardised research protocols and define data sets that should be collected by hospitals around the world, if they are affected by covid-19. as in any outbreak setting, priorities of local clinicians and public health authorities should be considered, especially in countries that integrate traditional medicine with western medicine. researchers may consider adopting a tiered approach as isaric has with the clinical characterisation protocols. the tiered approach allows sites to determine how much data or samples they can collect given their (limited) resources. this allows health care centres in low and middle-income countries to be represented in the data. global solidarity is needed in the clinical research community as we may face the next pandemic of the 21st century. we all benefit from the data collected and shared. who have launched a clinical data collection platform for covid-19 via the international health regulations and hope that member states will share their data. this uses an isaric who co-created crf for covid 19. thanks to lessons learned from sars and mers, the international public health and research community has been able to rapidly respond to the emergence of this novel coronavirus. based on a 2018 systematic review of sars and mers common clinical research questions, we provide a summary of the state of current clinical knowledge for the covid-19, demonstrate what clinical research gaps still need to be filled, and provide recommendations on study designs. many of the identified gaps, such as viral pathogenesis, clinical characterisation, infection prevention, and candidate therapeutics overlap with gaps identified the who' s r&d blueprint research roadmap. if health care facilities around the world collect standardised patient data and quickly share it, it is likely that these core clinical research questions can be answered in real-time to inform clinical practices for covid-19. world health organization. novel coronavirus (2019-ncov) situation report-1 a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster scottish intercollegiate guidelines network harmonisation of zika virus research protocols to address key public health concerns clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records novel coronavirus infection in hospitalized infants under 1 year of age in china initial ct findings and temporal changes in patients with the novel coronavirus pneumonia (2019-ncov): a study of 63 patients in wuhan, china chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan, china the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) -china clinical features predicting mortality risk in patients with viral pneumonia: the mulbsta score a novel coronavirus outbreak of global health concern more than 80 clinical trials launch to test coronavirus treatments diagnostic detection of 2019-ncov by real-time rt-pcr detection of 2019 novel coronavirus (2019-ncov) in suspected human cases by world health organization. laboratory testing for 2019 novel coronavirus (2019-ncov) in suspected human cases interim guidance sars-cov-2 viral load in upper respiratory specimens of infected patients notes from the field isolation of 2019-ncov from a stool specimen of a laboratory-confirmed case of the coronavirus disease 2019 (covid-19) discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes consistent detection of 2019 novel coronavirus in saliva surveillance case definitions for human infection with novel coronavirus (ncov) interim guidance v1 clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected data sharing and outbreaks: best practice exemplified covid 19 public health emergency of international concern (pheic) global research and innovation forum: towards a research roadmap open source clinical science for emerging infections critically ill patients with severe acute respiratory syndrome risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel severe acute respiratory syndrome: clinical outcome and prognostic correlates world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) clinical manifestations, laboratory findings, and treatment outcomes of sars patients mers transmission and risk factors: a systematic review finding the evidence: a key step in the information process a randomized, controlled trial of ebola virus disease therapeutics understanding and using sensitivity, specificity and predictive values viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome laboratory diagnosis of sars clinical characterization protocol transmission characteristics of mers and sars in the healthcare setting: a comparative study guide to infection control in the hospital. international society for infectious diseases sars transmission among hospital workers in hong kong middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea we extend our deepest thanks to emily phipps and jecko thachil of oxford university. with gail carson in 2012, they first proposed the idea of identifying key clinical questions asked in respiratory outbreaks as a means of epidemic preparedness and to emphasize the importance of data sharing. this work would not have been possible without them. authorship contributions: gc, ph, hn, and ch contributed the idea for the review. ch designed the study, conducted the review, and wrote the report. gc, ph, hn, and kb contributed to the critical review of the report. all authors reviewed and approved the final version.competing interests: all authors declare no competing interests. gc and ph are involved in the operation of isaric. kb is the isaric ccp technical lead. the authors have completed the icmje unified competing interest form and declare no further competing interests. key: cord-284581-fl2nt4ak authors: kleine-weber, hannah; pöhlmann, stefan; hoffmann, markus title: spike proteins of novel mers-coronavirus isolates from northand west-african dromedary camels mediate robust viral entry into human target cells date: 2019-07-19 journal: virology doi: 10.1016/j.virol.2019.07.016 sha: doc_id: 284581 cord_uid: fl2nt4ak the highly pathogenic middle east respiratory syndrome (mers)-related coronavirus (cov) is transmitted from dromedary camels, the natural reservoir, to humans. for at present unclear reasons, mers cases have so far only been observed in the arabian peninsula, although mers-cov also circulates in african dromedary camels. a recent study showed that mers-cov found in north/west(morocco) and west-african (burkina faso and nigeria) dromedary camels are genetically distinct from arabian viruses and have reduced replicative capacity in human cells, potentially due to amino acid changes in one or more viral proteins. here, we show that the spike (s) proteins of the prototypic arabian mers-cov strain, human betacoronavirus 2c emc/2012, and the above stated african mers-cov variants do not appreciably differ in expression, dpp4 binding and ability to drive entry into target cells. thus, virus-host-interactions at the entry stage may not limit spread of northand west-african mers-cov in human cells. the middle east respiratory syndrome-related coronavirus (mers-cov) causes the severe lung disease mers (zaki et al., 2012) , which takes a fatal course in roughly~35% of infected patients (who, 2019) . mers-cov is endemic in the middle east, where the virus is transmitted from dromedary camels, the natural reservoir, to humans (perera et al., 2013; reusken et al., 2013) . human-to-human transmission is inefficient but resulted in several hospital outbreaks of mers harriman et al., 2013; memish et al., 2013) , and there is concern that the virus may adapt to humans and cause a pandemic. infection of dromedary camels with mers-cov is not limited to the middle east. african camels are frequently infected with mers-cov (ali et al., 2017a (ali et al., , 2017b chu et al., 2014 chu et al., , 2015 chu et al., , 2018 corman et al., 2014; deem et al., 2015; kiambi et al., 2018; miguel et al., 2017; ommeh et al., 2018; perera et al., 2013; reusken et al., 2013 reusken et al., , 2014 van doremalen et al., 2017) and the responsible viruses are genetically distinct from those circulating in the middle east kiambi et al., 2018; ommeh et al., 2018) . moreover, viruses isolated from animals in morocco, nigeria and burkina faso form a distinct phylogenetic subclade, c1, and exhibit reduced ability to replicate in human respiratory cells . in addition, mers-cov transmission from camels to humans has not been observed in northand west-africa (munyua et al., 2017; so et al., 2018) , although two livestock handlers in kenya were shown to harbor antibodies against mers-cov (liljander et al., 2016) , moreover, no mers cases were documented in africa. at present, the barrier(s) impeding efficient spread of african mers-cov in human cells and camel-human transmission of these viruses remain to be identified. the mers-cov spike protein (s) is incorporated into the viral envelope and facilitates viral entry into target cells (li, 2016) . for this, the s protein binds to the cellular receptor dipeptidyl peptidase 4 (dpp4, cd26) via its surface unit, s1, and fuses the viral membrane with a target cell membrane via its transmembrane unit, s2 (li, 2016) . binding of mers-s to dpp4 is essential for mers-cov infection of cells and dpp4 expression and the s protein/dpp4 interface are major determinants of mers-cov cell and species tropism van doremalen et al., 2014) . the s proteins of north-and west-african mers-cov of the c1 clade harbor 6-9 amino acid substitutions relative to mers-cov (fig. 1a , table 1 ) and these substitutions might reduce s protein-driven entry into target cells. however, this possibility has not been examined so far. we employed a previously described vesicular stomatitis virus (vsv)-based pseudotyping system to study mers-s-driven host cell entry (kleine-weber et al., 2018 known to adequately model key aspects of the coronavirus entry process. in order to study host cell entry driven by s proteins from the c1 subclade, we employed pcr-based mutagenesis to generate expression constructs for the s proteins of mers-cov from morocco (camel/morocco/cirad-hku213/2015, mo), nigeria (camel/nigeria/nv1657/ 2016, ni) and burkina faso (camel/burkina faso/cirad-hku785/ 2015, bf), using a published expression construct for mers-cov emc s protein as template (kleine-weber et al., 2018 . moreover, expression constructs for all s proteins were generated that encoded a cterminal v5 antigenic tag. western blot analysis of cells transfected to express the s proteins under study revealed that mers-s emc, mo, ni and bf were expressed and proteolytically processed to comparable levels ( fig. 1b) . moreover, these s proteins were incorporated into vsv particles with similar efficiency (fig. 1c) . these results suggest that mutations present in north-and west-african mers-s of the c1 subclade do not reduce s protein expression and proteolytic processing in human cells. we next asked whether dpp4 binding of north-and west-african mers-s was altered. for this, 293t cells transfected to express the s proteins under study were incubated with soluble dpp4 fused to the fc portion of human immunoglobulin and binding was quantified by flow cytometry, as described previously (kleine-weber et al., 2019). the results showed that mers-s emc, mo, ni, and bf bound to dpp4 robustly and with comparable efficiency while dpp4 binding to cells expressing no s protein was within the background range (fig. 2 ). finally, we tested whether the robust binding to dpp4 translated into efficient s protein-driven entry. for this, cell lines were selected that were shown to express low levels (293t), intermediate levels (vero 76) or high levels of dpp4 (caco-2, 293t + dpp4) (kleine-weber et al., 2019). mers-s mo, ni and bf mediated entry into all cell lines with at least the same efficiency as mers-s emc (fig. 3) . moreover, under conditions of low or medium dpp4 expression, entry mediated by mers-s mo and bf was even more efficient than entry mediated by mers-s emc (fig. 3 ), although these differences were not statistically significant. our results show that amino acid substitutions present in north-and west-african mers-s proteins relative to mers-s emc do not compromise s protein expression in human cells, at least when transfected cells are examined. similarly, proteolytic processing of the s proteins in the constitutive secretory pathway, which is known to be carried out by furin (gierer et al., 2015; millet and whittaker, 2014) , was not the indicated s proteins were transiently expressed in 293t cells, whole cell lysates (wcl) were prepared at 48 h posttransfection and s protein expression was analyzed via western blot, using an antibody targeting the c-terminal v5-tag. cells expressing no s protein were used as negative control and detection of β-actin (actb) served as loading control. similar results were obtained in two separate experiments. (c) rhabdoviral transduction vectors (vsvpp) harboring the indicated s proteins were concentrated by centrifugation and, following lysis, analyzed by western blot for s protein incorporation, using an antibody targeting the c-terminal v5-tag. transduction vectors harboring no s protein were used as negative controls and detection of vesicular stomatitis virus matrix protein (vsv-m) served as loading control. similar results were obtained in a separate experiment. numbers on the left side of each blot indicate the molecular weight in kilodalton (kda). further, bands representing the precursor s protein (s0, black circle) and the s2 subunit of proteolytically processed s protein (grey circle) are indicated. appreciably altered. moreover, binding of north-and west-african s proteins to dpp4 was not diminished as compared to mers-s emc, despite the presence of at least one substitution in the receptor binding domain (rbd) in each s protein tested. this finding might not be unexpected since the substituted amino acid residues do not make direct contact with residues in dpp4 (lu et al., 2013) . in keeping with these observations, all african s proteins mediated robust viral entry into non-human primate (vero 76) and human cell lines (293t, caco-2) expressing different levels of dpp4 (kleine-weber et al., 2019) . in fact, mers-s mo-and bf-driven entry into cell lines expressing low or intermediate levels of dpp4 was augmented as compared to mers-s emc, in keeping with these s proteins showing slightly enhanced dpp4 binding as compared to mers-s emc. finally, it is noteworthy that mers-s activation in caco-2 cells mainly depends on the cellular serine protease tmprss2 while activation in 293t and vero 76 cells is mediated by the cellular cysteine protease cathepsin l (kleine-weber et al., 2018, 2019). thus, north-and west-african mers-s proteins seem to be able to use both pathways available for s protein activation in human cells. confirmation of our findings with authentic viruses is pending and we cannot exclude that, for instance, the s protein modulates recognition of the virus by sensors of the interferon system, which cannot be measured with the assays available to us. moreover, we note that a recent study examining two mers-s sequences (c2 subclade) from camels in ethiopia demonstrated that these sequences, when inserted into mers-cov emc, reduced viral entry and replication and increased sensitivity to antibody-mediated neutralization (shirato et al., 2019) . the reduction in entry was observed for vero and to a lesser degree for vero-tmprss2 cells and was generally modest. nevertheless, these results suggest that s proteins from viruses circulating in ethiopia might harbor mutations that diminish entry into human cells and that are not present in the mers-s proteins studied here. amino acid residues i139, l515, e851 and s1302 in the spike protein are unique to ethiopian mers-cov and warrant further analysis. collectively, our results suggest that amino acid substitutions present in the s proteins of north-and west-african mers-cov do not compromise the ability of these viruses to enter human cells. thus, future efforts to understand why north-and west-african mers-cov isolates show reduced replicative potential in human cells should be focused on other aspects of the mers-cov lifecycle than s proteinmediated host cell entry. expression plasmids, based on the vector pcaggs, for vsv-g and mers-s emc were previously described (kleine-weber et al., 2018 . the mers-s emc plasmid was used as template for pcr-based mutagenesis to introduce the mutations found in mers-s mo (morocco, camel/morocco/cirad-hku213/2015, genbank: mg923469.1), ni (nigeria, camel/nigeria/nv1657/2016, genbank: mg923475.1) and bf (burkina faso, camel/burkina faso/cirad-hku785/2015, gen-bank: mg923471.1) ( table 1 ). in addition, pcr-based mutagenesis was used to equip the constructs with a c-terminal v5 antigenic tag. the integrity of all sequences was verified using automated sequence analysis. 293t (human embryonal kidney) and vero 76 (african green monkey kidney) cells were cultivated in dulbecco's modified eagle's medium (dmem; pan biotech). the human colorectal adenocarcinoma cell line caco-2 was grown in minimum essential media (mem, life technologies). all media were supplemented with 10% fetal bovine serum (fbs, pan biotech) and 1x penicillin and streptomycin from a 100x stock solution (pan biotech). the cells were incubated under humid conditions at 37°c and 5% co 2 . for transfection of 293t cells the calcium-phosphate precipitation method was used. genbank: mg923469.1 v26a s1 / n/a a89s s1 / n/a t424i s1 / rbd s856y s2 / n/a r884l s2 / ps(s2') a1158s s2 / n/a v1209l s2 / n/a mers-s ni camel/nigeria/nv1657/2016 genbank: mg923475.1 v26a s1 / n/a h167y s1 / n/a h194y s1 / n/a l495f s1 / rbd l588f s1 / rbd s856y s2 / n/a a1158l s2 / n/a l1200f s2 / n/a mers-s bf camel/burkina faso/cirad-hku785/ 2015 genbank: mg923471.1 v26a s1 / n/a a89s s1 / n/a h194y s1 / n/a t424i s1 / rbd s856y s2 / n/a a1158s s2 / n/a a amino acid position (numbering according to mers-s emc). b subunit / functional domain (if applicable); abbreviations: s1 = s1 subunit; s2 = s2 subunit; rbd = receptor binding domain, ps(s2') = priming site at the s2' position (884-rsar-887), n/a = not applicable. fig. 2 . s proteins of north/west-and west-african mers-cov isolates from dromedary camels efficiently bind to dpp4. 293t cells expressing the indicated s proteins or no s protein at all (control) were successively incubated with soluble dpp4 containing a c-terminal fc tag (sol-dpp4-fc) and alexafluor488conjugated anti-human antibody, before dpp4 binding to the respective s protein was analyzed by flow cytometry. presented are the combined data of three independent experiments for which sol-dpp4-fc binding to mers-s emc was set as 100%. error bars indicate the standard error of the mean (sem). statistical significance was tested by one-way analysis of variance with sidak's posttest (p > 0.05, not significant, ns; p ≤ 0.01, **). for western blot analysis, anti-v5 (mouse, 1:2,500; thermofisher scientific), anti-β-actin (mouse, 1:2,500; sigma-aldrich), anti-vsv-m (mouse, 1:2,500; kerafast) were used as primary antibodies and antimouse hrp (horse radish peroxidase) conjugated antibody (goat, 1:2,500; dianova) was used as secondary antibody. antibodies were diluted in phosphate buffered saline [pbs] containing 0.5% tween 20 [pbs-t] supplemented with 5% skim milk powder. for flow cytometry, a recombinant fusion protein of the ectodomain of dpp4 fused to the fc fragment of human immunoglobulin (sol-dpp4-fc, 1:200, acrobiosystems) and an alexaflour488-conjugated anti-human antibody (goat, 1:500; thermofisher scientific) were used (ligand and antibody were diluted in pbs containing 1% bovine serum albumin). for analysis of s protein expression, 293t cells were transfected with expression plasmid for mers-s proteins harboring a c-terminal v5 tag, as described (kleine-weber et al., 2018, 2019). to investigate mers-s incorporation into vsvpp, equal volumes of supernatants containing vsvpp bearing s proteins with v5 tag were centrifuged through a 20% sucrose cushion at 25.000 g for 120 min. subsequently, cells and vsvpp pellets were lysed and analyzed by immunoblot, following an established protocol (kleine-weber et al., 2018 . dpp4 binding was analyzed as described (kleine-weber et al., 2019) . in brief, 293t cells were transfected with expression plasmids for mers-s proteins and empty plasmid as negative control. at 48 h posttransfection, the cells were washed with pbs, pelleted and resuspended in pbs containing 1% bsa and soluble human dpp4-fc fusion protein at a final dilution of 1:200. after incubation for 1 h at 4°c, the cells were washed and incubated with alexafluor488-conjugated anti-mouse antibody at a dilution of 1:500. finally, the cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry using an lsr ii flow cytometer and the facs diva software (both bd biosciences). fig. 3 . host cell entry driven by the s proteins of north/west-and west-african mers-cov isolates from dromedary camels is robust. 293t, 293t transfected to express dpp4, vero 76 and caco-2 cells were inoculated with equal volumes of rhabdoviral transduction vectors harboring the indicated s proteins or no s protein (control). at 18 h posttransduction, the activity of the virus-encoded luciferase, which served as an indicator for transduction efficiency, was measured in cell lysates. presented are the combined data of three independent experiments for which transduction mediated by mers-s emc was set as 100%. error bars indicate sem. statistical significance was tested by one-way analysis of variance (anova) with sidak's posttest (p > 0.05, ns; p ≤ 0.01, **; p ≤ 0.005, ***). transduction vectors based on a replication-deficient vsv (berger rentsch and zimmer, 2011) and pseudotyped with the indicated viral glycoproteins (vsvpp) were generated according to a published protocol (kleine-weber et al., 2018 . target cells were transduced with equal volumes of supernatants containing vsvpp and transduction efficiency was quantified at 16 h posttransduction by measuring the activity of virus-encoded firefly luciferase in cell lysates as previously described (kleine-weber et al., 2018 . cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt hospital outbreak of middle east respiratory syndrome coronavirus a vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type i interferon middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels mers coronaviruses from camels in africa exhibit region-dependent genetic diversity antibodies against mers coronavirus in dromedary camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity hospital-associated middle east respiratory syndrome coronavirus infections detection of distinct mers-coronavirus strains in dromedary camels from kenya mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization structure, function, and evolution of coronavirus spike proteins mers-cov antibodies in humans molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 hospital-associated middle east respiratory syndrome coronavirus infections risk factors for mers coronavirus infection in dromedary host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study genetic evidence of middle east respiratory syndrome coronavirus (mers-cov) and widespread seroprevalence among camels in kenya seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 middle east respiratory syndrome coronavirus (mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors thank gert zimmer and andrea maisner for providing the replication-deficient vsv vector for pseudotyping and the vero 76 cell line, respectively. this work was supported, including the efforts of stefan pöhlmann, by the bundesministerium für bildung und forschung within the network project rapid (risikobewertung bei präpandemischen respiratorischen infektionserkrankungen; 01ki1723d). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. key: cord-296517-414grqif authors: wong, gary; liu, wenjun; liu, yingxia; zhou, boping; bi, yuhai; gao, george f. title: mers, sars, and ebola: the role of super-spreaders in infectious disease date: 2015-10-14 journal: cell host & microbe doi: 10.1016/j.chom.2015.09.013 sha: doc_id: 296517 cord_uid: 414grqif super-spreading occurs when a single patient infects a disproportionate number of contacts. the 2015 mers-cov, 2003 sars-cov, and to a lesser extent 2014–15 ebola virus outbreaks were driven by super-spreaders. we summarize documented super-spreading in these outbreaks, explore contributing factors, and suggest studies to better understand super-spreading. a number of recent virus outbreaks have resulted in rapid virus spread, placing demands on the affected health infrastructures and sparking global concern. in september 2012, middle east respiratory syndrome coronavirus (mers-cov) emerged as a novel virus that can result in severe respiratory disease with renal failure, with a case fatality rate of up to 38%. primary infections typically occur in countries located in the middle east, where dromedary camels have been identified as one of the species harboring the virus. however, travel has contributed to several cases in other countries. notably, between may and july 2015, an outbreak of mers-cov centered in south korea killed 36 people out of 186 confirmed cases (promedmail.org, 2015) , with thousands quarantined as health authorities attempted to control virus spread. severe acute respiratory syndrome coronavirus (sars-cov), a close relative of mers-cov, was the etiological agent responsible for an outbreak centered around guangdong province in the southeast of china. the sars-cov outbreak killed 774 and infected 8,096 people between november 2002 and july 2003, for a case fatality rate of 9.6%, in which the virus was first traced to palm civets, and then bats. over 80% of the total cases and deaths were from mainland china and hong kong. more recently, a devastating ebola virus (ebov) outbreak has swept through western africa. ebov is a filovirus that causes severe hemorrhagic fever in hu-mans with up to a 90% case fatality rate if untreated. outbreaks of ebov are sporadic, unpredictable, and localized to sub-saharan africa. prior to the 2014-15 outbreak, a total of 1,093 deaths out of 1,393 infections were documented dating back to 1976, when ebov first emerged. the natural reservoir of ebov is currently unknown, but fruit bats are suspected to be the animal species harboring the virus. in addition to their significant impact, these outbreaks were all perpetuated by super-spreaders who disproportionately infect a high number of contacts and likely contribute to the speed and degree of the outbreak. as discussed in detail below, these outbreaks can be traced to one or more individuals who largely contributed to subsequent infections. the 2015 mers-cov outbreak in south korea began from an imported case, a 68-year-old male with a recent travel history to several middle eastern countries, including bahrain, the united arab emirates, saudi arabia, and qatar. the latter three countries also had reported human cases of mers-cov during the same period of time. after the patient started exhibiting disease symptoms, he sought medical assistance at several clinics before being admitted to a hospital, where he was confirmed to be infected with mers-cov (who, 2015a). twentynine secondary infections have since been directly traced to this index patient. additionally, two of these secondary cases were shown to be responsible for 106 subsequent infections, out of 166 known cases at the time (cowling et al., 2015) . transmission was mostly within nosocomial settings, and fourth generation infections have been documented for the first time since the virus was identified in 2012. thus, the mers-cov outbreak in south korea was driven primarily by three infected individuals, and approximately 75% of cases can be traced back to three super-spreaders who have each infected a disproportionately high number of contacts ( figure 1a ). this mers-cov outbreak is the second largest on record and the largest outside of the middle east. the mers-cov outbreak currently appears to be under control, with no additional infections reported in south korea since july 4, 2015. the sars-cov outbreak retrospectively, several super-spreading events were also documented during the sars-cov outbreak in 2003. the index patient of the hong kong epidemic was treated at prince of wales hospital and was associated with at least 125 secondary cases (riley et al., 2003) . subsequent super-spreader events occurred at the hotel metropole (13 cases) and the amoy gardens housing complex (over 180 cases) in hong kong and aboard an air china flight traveling from hong kong to beijing (22 cases) (braden et al., 2013) . notably, cases from hotel metropole were responsible for the spread of sars-cov to canada, vietnam, and singapore through travel ( figure 1b) , and the imported case to canada resulted in 128 sars-cov cases at a toronto hospital (braden et al., 2013) . have super-spreaders played a role in other outbreaks? to a lesser extent, similar events were observed with the 2014-15 ebov outbreak, centered in the western african countries of guinea, sierra leone, and liberia. epidemiological work has linked five infections with a 2-year-old toddler in the remote village of meliandou, guinea. one of the contacts, a midwife, infected at least three others, including a hospital worker at gué cké dou hospital. this worker then infected several family members in the gué cké dou farako district, as well as 15 others at macenta hospital (baize et al., 2014) . one of the subsequent cases, a doctor, was known to have infected several others who traveled to kissidougou and nzé ré koré , further propagating virus transmission (baize et al., 2014) . the outbreak eventually spilled over to neighboring liberia and sierra leone. in sierra leone, the funeral of a traditional healer that died from ebov was shown to have directly infected 13 others (gire et al., 2014) and was eventually linked to more than 300 cases (who, 2015b). the ebov outbreak has killed 11,284 people, with 27,741 infected as of july 19 2015, en route to becoming the largest filovirus outbreak ever documented. in addition to ebov, superspreading has also been documented in outbreaks with other pathogens, including measles (paunio et al., 1998) and mycobacterium tuberculosis (kline et al., 1995) . the initial stages of the outbreaks mentioned above involved at least one super-spreading event. the ability of the pathogen to infect subsequent superspreaders who may export the disease through travel is likely the difference between an infection cluster and an outbreak or epidemic ( figure 1c ). differences in the initial response to mers-cov is why south korea reported clusters of infections before bringing the virus under control and why china, thailand, and the philippines have not reported any additional cases despite imported mers-cov infections. it was why sars-cov was so devastating in mainland china and hong kong from 2002 to 2003. it was also why the 2014-15 ebov outbreak spiraled out of control in western africa, with hundreds of new cases reported weekly, before a coordinated international effort (including contact tracing, isolation, diagnosis, and treatment of infected patients, as well as community education) brought the number down to a more manageable 20-30 weekly infections, with most of the cases diagnosed in guinea and sierra leone. early discovery, diagnosis, intervention, and quarantine of confirmed cases is crucial for preventing further disease transmission in humans, especially via super-spreaders. as evidenced by the decreased numbers of new cases in the mers-cov and ebov outbreaks, current responses are effective, provided that the measures are implemented properly and followed strictly. vigilance and patience will be necessary until the ongoing mers-cov and ebov outbreaks are officially at an end. currently, super-spreaders are only retrospectively categorized after epidemiological tracing. given their high propensity to spread infection, super-spreaders should be able to shed higher levels of pathogen and/or for a longer period of time after infection. this would increase the probability that the pathogen contacts and subsequently infects a naive host. to address whether there are factors or parameters that promote or indicate enhanced virus shedding in a laboratory setting, an animal species that closely recapitulates symptoms of the human disease is required, such that any findings can be translated to humans. animals could be experimentally infected with different virus strains/variants at varying doses via different challenge routes, in which virus shedding from the oral, nasal, and rectal cavities should be correlated with different parameters, including viremia or virus titers in the organs. however, these studies are currently challenging for sars-cov as there is currently not an appropriate animal model that replicates the severe disease seen in humans; infected nonhuman primates exhibit variable, but at most mild to moderate, respiratory disease (mcauliffe et al., 2004) (lawler et al., 2006) . nonetheless, these types of studies are feasible for mers-cov. a model of this infection was developed in the common marmoset that results in severe respiratory disease and partial lethality, in which live virus can be detected in nasal and throat swabs of infected animals (falzarano et al., 2014) . thus, the studies proposed above can be investigated with mers-cov in this animal model. for ebov, the cynomolgus and rhesus macaque animal models are well characterized and have been used over the past 20+ years. ebov shedding is known to occur from the oral, nasal, and/or rectal cavities of nonhuman primates during the advanced stages of disease, but factors that influence virus shedding from these animals have not yet been investigated. interestingly, using a guinea pig model, it was shown that animals infected intranasally (i.n.) with guinea pig-adapted ebov were more contagious to naive contact animals than those that were infected intraperitoneally (i.p.) with the same dose . i.n. infected animals shed virus from their nasal cavity earlier than their i.p. counterparts, and i.p. challenged animals died earlier (and thus were removed from contact with naive animals) compared to i.n. inoculated guinea pigs. it was therefore concluded that the route of infection in addition to the duration of contact time with an infectious host may be factors that influence the efficiency of virus transmission. it will take time to elucidate all possible host and viral factors that contribute to virus shedding, which remains an understudied topic to date. therefore, it is not currently possible to predict with any level of confidence or statistical significance whether a person will become a super-spreader of a certain disease. large-scale genome-wide association studies (gwas) of super-spreaders might provide some clues about the genetic backgrounds of super-spreaders, but the feasibility and robustness of these analyses will depend on the number of super-spreaders in a given outbreak. the large amount of patient data and samples available from the recent mers-cov and ebov outbreaks provide examples from mers-cov, sars-cov, and ebov virus mutation: the virus may acquire mutations to replicate more efficiently and become highly pathogenic, which may result in higher levels of virus shedding for a longer period of time. duration of contact with an infectious host and route of infection: in a guinea pig model of ebov infection, animals that were infected mucosally were more infectious than those that were infected systemically. additionally, prolonged contact with a contagious animal resulted in a higher rate of virus transmission. genetic susceptibility: ebov is known to be more pathogenic in cynomolgus macaques compared to other nonhuman primate species, such as rhesus macaques. different nonhuman primate species also demonstrate variable susceptibility to sars-cov infection. hosts that are more susceptible to disease may exhibit higher levels of virus shedding. underlying medical conditions: certain conditions may decrease immunity and increase susceptibility to pathogens, leading to enhanced virus shedding. many fatal mers-cov patients in the recent outbreak in south korea had other pre-existing medical conditions, including pneumonia and kidney disease. air re-circulation in enclosed spaces: this would increase chances of pathogen encounter, especially those that are airborne. during the sars-cov outbreak, this was shown to be a factor behind super-spreading events at the hotel metropole, amoy gardens housing complex, and a flight between china and canada. it is likely a contributing factor to the mers-cov outbreak since super-spreading events occurred at a hospital or medical clinic. population density: increased numbers of people equally increases the chances to infect a naive host through inadvertent direct or indirect contact. traditional customs and beliefs conducive to infectious disease spread: the culture of ''doctor shopping,'' in which patients seek medical attention from multiple doctors at different clinics/hospitals allowed mers-cov to spread rapidly in south korea. unsafe burials and traditional funerals, which involve touching and washing infectious bodies, played a role in ebov spread in western africa. travel and trade: the global nature of today's world allows infectious diseases to easily bypass geographical barriers. for instance, mers-cov originated from the middle east, but imported cases were reported in north america, europe, and asia. sars-cov originated in asia, and imported cases were reported in north america, europe, and other parts of asia. ebov originated from africa, and imported cases were reported in north america and europe. knowledge and adherence to public health advice: during the mers-cov outbreak, a case was imported to china because a south korean patient did not follow the recommendations of health authorities and traveled despite being a symptomatic, high-risk contact. however, china handled the imported case properly, and the imported korean patient did not become a super-spreader. during the ebov outbreak, some patients were able to escape quarantine, thereby increasing the likelihood of infecting others. possible opportunities to investigate factors that may correlate with an increased risk for becoming a super-spreader. using the mers-cov outbreak as an example, detailed records were kept for the majority of patients who were confirmed to be infected with mers-cov. since the identities of the three super-spreaders are known as patients #1, #14, and #16, studies can be performed to investigate whether these three patients have common traits (i.e., age, gender, pre-existing medical conditions or underlying co-morbidities, levels of virus shedding, etc.). any common characteristics can then be compared with non-super-spreader patients to provide insight into possible risk factors behind enhanced spreading of infectious diseases. another approach would be to isolate mers-cov from the three known superspreaders, perform sequence analysis of the viral genomes, and determine whether there are any shared mutations between these isolates. any mutations that are identified and are absent from the viral genomes of other patients could be indicators of enhanced virus shedding, allowing for super-spreading to occur. a complex combination of factors likely plays a role in the number of subsequent infections initiating from a single superspreader. in addition to host and virus factors, other important factors impacting the spread of infectious diseases are the environment and behavior of individuals. environmental factors include the close proximity of other susceptible hosts and the airflow dynamics within an enclosed area. a high population density represents a greater number of susceptible hosts for direct/indirect contact with an infected person, whereas air recirculation would especially facilitate the transmission of airborne viruses, such as mers-cov and sars-cov. hospitals, enclosed housing complexes, and mass transportation, such as airplanes, are documented sites of super-spreader events, especially during the mers-cov and sars-cov outbreaks (table 1) . individual behaviors may also enhance disease spread. these include ''doctor shopping''-going to multiple hospitals to treat the same ailments or traveling to other countries after the appearance of disease symptoms, which was observed with the mers-cov outbreak (su et al., 2015) . traditional customs, such as unsafe funerals/burials in africa, which involve direct contact with infectious bodily fluids of patients that passed away from ebov disease, is another major reason why the outbreak persisted in guinea and sierra leone (table 1) . a thorough study of host, virus, and environmental dynamics will be important to delineate the relative contribution of each factor to the phenomenon of super-spreading. findings from these studies, combined with changes in behavior conducive to the spread of disease, will be important for the effective management of and preparation for future outbreaks. emergence of zaire ebola virus disease in guinea progress in global surveillance and response capacity 10 years after severe acute respiratory syndrome preliminary epidemiological assessment of mers-cov outbreak in south korea infection with mers-cov causes lethal pneumonia in the common marmoset genomic surveillance elucidates ebola virus origin and transmission during the 2014 outbreak outbreak of tuberculosis among regular patrons of a neighborhood bar cynomolgus macaque as an animal model for severe acute respiratory syndrome replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys explosive school-based measles outbreak: intense exposure may have resulted in high risk, even among revaccinees mers-cov (97): south korea, saudi arabia. international society for infectious diseases transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions mers in south korea and china: a potential outbreak threat middle east respiratory syndrome coronavirus (mers-cov) -republic of korea. disease outbreak news sierra leone: a traditional healer and a funeral ebola virus transmission in guinea pigs key: cord-292709-4hn55wui authors: nor, mohd basri mat; richards, guy a.; mcgloughlin, steve; amin, pravin r. title: pneumonia in the tropics: report from the task force on tropical diseases by the world federation of societies of intensive and critical care medicine date: 2017-12-31 journal: journal of critical care doi: 10.1016/j.jcrc.2017.11.004 sha: doc_id: 292709 cord_uid: 4hn55wui abstract the aetiology of community acquired pneumonia varies according to the region in which it is acquired. this review discusses those causes of cap that occur in the tropics and might not be readily recognizable when transplanted to other sites. various forms of pneumonia including the viral causes such as influenza (seasonal and avian varieties), the coronaviruses and the hantavirus as well as bacterial causes, specifically the pneumonic form of yersinia pestis and melioidosis are discussed. in general, community acquired pneumonia (cap) is caused by pathogens that are common to all geographical areas; s. pneumoniae, viruses, chlamydia, mycoplasma, legionella and less commonly s. aureus and k. pneumoniae. however some organisms are endemic to specific regions and early recognition and awareness of these is critical to the diagnosis and to a favourable outcome. this is no less the case in residents of or travellers to tropical regions. this review discusses those that are most prevalent and which can cause potentially lethal infections. in adults, respiratory viruses account for 10% to 40% of cap and the most common of these are influenza, parainfluenza, adenovirus and respiratory syncytial viruses (rsv) [1, 2] . influenza viruses which are classified by their core proteins (i.e. a, b or c) and belong to the family orthomyxoviridae, cause predominantly respiratory disease in humans. influenza type a and b account for n 50% of viral pneumonia in adults whereas influenza c infections generally cause mild respiratory disease and are not thought to cause epidemics [3] . the close contact between humans and animals in tropical areas may enhance the genetic reassortment of influenza viruses which when disseminated into the human population may result in pandemics [4] . other important respiratory viruses in the tropics that can cause severe pneumonia are influenza a h1n1, avian influenza viruses (h5n1, h7n9), severe acute respiratory syndrome associated coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) [5, 6] . standard, droplet and contact precautions are recommended for these selected acute respiratory infections and whenever possible patients should be placed in airborne infection isolation rooms. influenza viruses have been associated with annual epidemics and intermittent pandemics throughout the world. despite the absence of a winter season in the tropics, consistent seasons of infection have nevertheless been observed. the composition of the antigenic surface glycoproteins of the influenza virus, hemagglutinin (h) and neuraminidase (n) are used for subtyping, resulting in names like h3n2 and h1n1. antigenic drift represents the minor changes of h and n side chains and is responsible for seasonal epidemics. influenza pandemics occur less frequently and these result from major changes in antigenic structure in the envelope glycoproteins (antigenic shift) resulting from reassortment of various viruses such as swine, equine and human varieties. contemporary geographic distributions show that east, south and southeast asia influence unduly the evolution of seasonal influenza a (h3n2), exporting most of the evolutionarily strains that ultimately spread globally. the obvious role of asia in h3n2's evolution has been ascribed to the seasonal nature of influenza in temperate climates [7] . seasonal influenza is an acute respiratory illness caused by influenza a or b viruses. given that these infections including the pandemic variety h1n1 now occur in all parts of the world they will not be discussed in any detail in this treatise. avian influenza viruses (e.g. h5n1 and h7n9) have emerged relatively recently and cause disease in humans and currently remain a potential threat, particularly in the southeast asia [5, 8] . the first association of avian influenza h5n1 with clinical respiratory disease was in 1997 in hong kong, as a human infection transmitted from birds. later h5n1 re-emerged in humans in 2003 as a highly pathogenic virus resulting from antigenic drift to which a larger number of species were vulnerable and conferring resistance to adamantine antivirals [9] . as yet, human to human transmission is rare and the vast majority of cases are related to contact with birds. travellers who have a history of recent exposure to birds in affected areas and who present with otherwise unexplained ards should be screened. h5n1 has been reported from 16 countries and is currently most prevalent in egypt [9] . as of august 2017, from 859 laboratory confirmed cases of influenza a h5n1, 453 (53%) patients have died [10] . 3.1.1.1. clinical. following exposure, the incubation period is seven days or less. clinical characteristics include fever, respiratory illness, pneumonia, diarrhoea and encephalopathy. laboratory abnormalities may include leukopenia, lymphopenia, thrombocytopenia and elevated serum aminotransferases. complications include multi-organ failure, pulmonary haemorrhage, pneumothorax and pancytopenia. radiographic findings include diffuse or patchy infiltrates and segmental or lobar consolidation. progression to respiratory failure is associated with diffuse bilateral ground-glass infiltrates. 3.1.1.2. diagnosis. a comprehensive travel and epidemiological history is critical in suspected cases. patients who meet clinical and epidemiological criteria should be tested for h5n1 avian influenza infection. diagnosis can be established by rrt-pcr or viral culture of respiratory specimens. serological testing is not helpful in the acute setting but useful for retrospective diagnosis [11] . 3.1.2.1. introduction. another avian influenza virus, h7n9, derived from reassortment of at least four avian influenza viruses, has caused severe pneumonia in some patients. it emerged in 2013 and originated from eastern china [12] . additional cases have been detected in mainland china, hong kong, macao, taiwan and malaysia. similar to h5n1 this virus occurs primarily in bird handlers or following recent exposure to live poultry or potentially contaminated environments. to date, there is no evidence of sustained human-to-human transmission. the incubation period has been estimated to be from 3 to 7 days, but can be as long as 10 days. presenting signs and symptoms may include fever, cough, dyspnoea, headache, myalgia and malaise. patients present with lrti which may progress rapidly to pneumonia and potentially acute respiratory failure, ards, septic shock, multi-organ failure, rhabdomyolysis and encephalopathy. severe illness and fatal outcome have been frequently observed in pregnant women, older persons and those with chronic illnesses [13, 14] . as of mid-august 2017, a total of 1557 laboratory-confirmed cases have been reported to who including 605 (39%) deaths [10] . real-time reverse-transcriptase polymerase reaction (rrt-pcr) for avian influenza a h7n9 is the preferred diagnostic test, since rapid antigen tests may be insensitive for novel or avian influenza strains. nasopharyngeal swabs or aspirates should be obtained for testing [11] . vaccination is the best method of protection during a pandemic however if not available or if a patient contracts influenza then antiviral therapy is indicated in certain circumstances. currently two classes of antiviral drugs are available for the treatment and prevention of influenza: the neuraminidase inhibitors (oseltamivir, zanamivir and peramivir) and the adamantines (amantadine and rimantadine) [15] . the adamantines block m2 protein channels and inhibit the uncoating of the influenza virus after it enters the host cells. they are only active against influenza a not b viruses and due to a marked increase in resistant isolates, many countries do not recommend that they be used except in combination with neuraminidase inhibitors in selected case. therefore, adamantines are not recommended for treatment of novel influenza a virus infections [16] . neuraminidase inhibitors are recommended to treat patients hospitalised with seasonal influenza, inclusive of h1n1, and avian influenza (h5n1 and h7n9) infections. in adults, the dose of oseltamivir is 75 mg orally twice daily; inhalational zanamivir is 10 mg (two inhalations) twice daily; and peramivir is 600 mg iv once daily. in patients with severe influenza, there are insufficient data to recommend the use of inhaled zanamivir or iv peramivir as empirical therapy. as such empirical treatment with oseltamivir should be started as soon as possible for all hospitalised cases associated with severe disease, and for confirmed and probable outpatient cases. early therapy (especially within 48 h of illness onset) can shorten the duration of the symptoms in high-risk patients and reduce secondary complications associated with influenza. the recommended duration of antiviral therapy is five days for uncomplicated disease. in severely ill hospitalised or immunocompromised patients, longer courses of treatment (e.g. 10 days) should be considered. in these circumstances, a higher dose of oseltamivir has been recommended by some experts (150 mg twice daily) in adults with normal renal function. however, oral oseltamivir has been reported to be adequately absorbed in critically ill patients and higher dosing may not provide additional clinical benefit. in obese patients, studies have demonstrated adequate exposure to oseltamivir carboxylate (active metabolite of oseltamivir) with the 75 mg twice daily dosing regimen. limited data suggest that oseltamivir administered by oro/naso-gastric tube is well absorbed in critically ill patients, including in those on continuous renal replacement therapy and ecmo [16] . for patients who cannot absorb or tolerate oral oseltamivir, the use of iv peramivir or investigational iv zanamivir should be considered. it is possible that novel influenza a viruses may become resistant to oseltamivir and peramivir during treatment but remain susceptible to zanamivir. in resistant infection, zanamivir is the preferred agent and can be delivered by inhalation or by the intravenous route. corticosteroid treatment is controversial with some reports showing benefit and others harm. in all cases of severe influenza whatever the type bacterial coinfection is possible and as such, appropriate antimicrobial treatment directed toward those organisms causing bacterial acute community acquired pneumonia, and mechanical ventilation as required can reduce the mortality rate. extracorporeal membrane oxygenation (ecmo) has been utilised in some patients with ards and one study showed a possible mortality benefit in patients who were referred to a specialised ecmo centre [17] . ongoing monitoring for antiviral resistance among avian influenza a viruses is crucial. some evidence of antiviral resistance has been reported in highly pathogenic avian influenza (hpai) "asian h5n1 viruses" and "asian h7n9 viruses". future treatment strategies may make use of new generation broadly reactive monoclonal antibodies. in addition, a viral rna polymerase inhibitor, favipiravir is registered for the treatment of pandemic influenza in japan [3] . potentially life-threatening coronavirus infections are caused by the severe acute respiratory syndrome associated coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) [6] . cases of sars were first noted in guangdong province, china in november 2002. the illness spread to 30 countries in north america, south america, europe and asia before the outbreak was contained. sars-cov is a novel coronavirus that jumped the species barrier from civet to man. based upon clusters of cases in hong kong and canada, sars-cov is transmitted by means of close person-to-person contact via droplets or fomites. it has been suggested that airborne and faecal-oral transmissions is also possible [18] . the incubation period is two to seven days and approximately 95% will develop symptoms by 10 days. this is followed by an influenza like (ili) prodrome (stage 1) of high fever (n 38°c), malaise, headache, myalgia and chills without upper respiratory tract symptoms. about 10% to 20% of patients have diarrhoea. the prodrome may last for three to seven days after which involvement of the lrt (stage 2) occurs and this begins with a non-productive cough and dyspnoea. this may progress to acute hypoxaemia with radiologic progression to pneumonia. about 20% of patients progress to stage 3 and develop features of ards that may require mechanical ventilation. the chest radiograph shows diffuse interstitial infiltrates characteristics of ards. risk factors for poor outcomes include older age, underlying comorbidities such as diabetes, atypical symptoms and elevated lactate dehydrogenase on admission [18, 19] . the diagnosis is based on clinical, epidemiological and laboratory criteria and case definitions have been developed by the centers for disease control and prevention. the reverse transcriptase polymerase chain reaction (rt-pcr) and serum antibodies as measured by enzyme-linked immunosorbent assay (elisa) testing, are two reliable tests for diagnosis. however serologic testing during the acute illness has limited value since antibodies typically develop several weeks into the illness. there is no specific treatment recommended except for supportive care. the benefit or otherwise of antiviral drugs or glucocorticoids has not been established for treatment of sars [20] . mers is a newly recognized highly lethal respiratory illness caused by mers-cov. mers-cov is a zoonotic virus and studies have shown that humans are infected through contact with infected dromedary camels or infected people. the disease was first reported in saudi arabia in june 2012 [21] . so far, all cases have been linked through travel to, or residence in, countries in and near the arabian peninsula. outside the arabian peninsula, the largest known outbreak of mers occurred in the republic of korea in 2015 which was also linked to a traveller returning from the arabian peninsula. a total of 186 confirmed patients with mers-cov infection across 16 hospitals were identified. eightytwo (44.1%) of the cases were patients exposed in hospitals, 61 (32.8%) were caregivers, and 25 (13.4%) were hospital staff [22] . a total of 83.2% of the cases were linked to five super-spreaders, all of whom presented with pneumonia and were in contact with hundreds of people. since 2012, although 27 countries have reported cases of mers-cov, 80% have been reported by saudi arabia. mers-cov can infect anyone and patients have ranged in age from b1 to 99 years old. the incubation period for mers is usually about 5 to 6 days, but can range from 2 to 14 days. most patients present with a severe acute respiratory illness manifested by shortness of breath but preceded by fever, cough, myalgia and arthralgia. rapid progression to pneumonia may occur within the first week, often requiring mechanical ventilation and other organ support such as renal dialysis [23] . patients in the korean outbreak had fever and chills as the most common symptoms but gastrointestinal symptoms including diarrhoea and nausea/vomiting have also been reported. as of august 2017, 35% of laboratory-confirmed mers-cov cases reported to who have died. studies have shown that older age and pre-existing comorbidities are important risk factors for death [23, 24] . those with mild symptoms (such as cold-like symptoms) or no symptoms at all usually recover. case definitions have been developed by who, the centers for disease control and prevention (usa) and the ministry of health of saudi arabia. confirmation of the diagnosis is by rrt-pcr performed on respiratory secretions. currently, there is no vaccine to prevent mers-cov infection and no proven antiviral therapy. management includes preventive daily hygiene measures and travel precautions. thereafter symptomatic and supportive therapy is required [25] . the hantavirus is a member of the bunyaviridae family and the genus includes n 50 different viruses that manifest with a wide spectrum of clinical illnesses [26] . the pathogenesis of hantavirus is related to the targeting of vascular endothelial cells, alveolar macrophages and follicular dendritic cells, as well as the renal tubular epithelium [27] . the induction of vascular or capillary leak is a key component of the pathogenicity [28] . transmission to humans is via contact with, or inhalation of aerosolised urine, saliva or faeces of the rodent host (family muridae). as it is predominately a rural disease, risk factors include farming, land development and camping, however due to the method of inoculation, it is usually acquired indoors [27] . person to person transmission has been documented previously in some subtypes so respiratory precautions are recommended for health care workers. it is estimated that over 30,000 cases of hantavirus occur globally each year, with the majority in asia [29] . it causes two major clinical syndromes namely hantavirus pulmonary syndrome (hps) and haemorrhagic fever with renal syndrome (hfrs) with the former occurring predominately in the americas [26] . reports have indicated that there is an increasing incidence of hps in south america [30] . the case definition of the hps as per the centers for disease control and prevention (cdc) is: 'a febrile illness with bilateral diffuse interstitial edema that may resemble the acute respiratory distress syndrome, with respiratory compromise requiring supplemental oxygen developing within 72 hours of hospitalisation, in a previously healthy person' [31] . the incubation period is two to three weeks followed by a number of phases that include [29] ; a prodrome with nonspecific viral symptoms however also with thrombocytopenia, lasting for 1 to 5 days; and the pulmonary phase in which symptoms of respiratory failure predominate progressing extremely rapidly (over 8 to 24 h), to shock, coagulopathy, pulmonary oedema, bronchorrhea, arrhythmia and death. lastly in the recovery phase patients often have a period of significant diuresis as endothelial function recovers and fluid redistribution occurs, with urine outputs of 300 to 500 ml per hour for 24 h [27] ; thereafter there is a convalescent phase which can be prolonged over weeks to months [28] . hantavirus is diagnosed by serology, with hantavirus igm positive by the time symptoms are present. if an appropriate history of exposure is obtained, the presence of thrombocytopenia, hemoconcentration, n10% immunoblasts and a lack of toxic granulation has a high sensitivity and specificity for hantavirus [28] . chest radiograph abnormalities consistent with interstitial oedema are usually present on admission which worsen as respiratory function declines [27] . advanced respiratory and haemodynamic monitoring and support, may be necessary, inclusive of mechanical ventilation and extracorporeal membrane oxygenation (ecmo) which is often a challenge in lics [28] . it is recommended that intravenous fluids are minimised with the early institution of vasopressor and inotropic support as aggressive fluid resuscitation has been shown to increase the risk of respiratory failure [28] . unfortunately, no specific antiviral therapy has been demonstrated to be effective. burkholderia pseudomallei is a gram negative pathogen endemic to south east asia, northern australia, india, south china and taiwan and cases have been documented in brazil and elsewhere in south america, papua new guinea, fiji, and new caledonia [32] . despite being the most common cause of fatal community acquired bacteremic pneumonia in north east thailand and darwin, it is seldom recognized when imported to areas where it is not endemic [33] . the prevalence varies in affected regions and may be related to climactic conditions, being particularly influenced by rainfall, having reached record rates (50.2 cases per 100,000 people) after heavier than usual precipitation in 2009-2010 in northern australia [33] . global mortality estimates are extremely high, similar to deaths from measles and much higher than leptospirosis and dengue, infections that receive considerably more attention. in fact the mortality may be even higher, as diagnosis is low in endemic areas primarily due to the low resource settings in which it occurs, as laboratory facilities are generally poor to non-existent [34] . infection occurs from inoculation from contaminated soil or water, through abrasions, ingestion or inhalation with subsequent haematogenous spread. those with chronic diseases such as diabetes, renal failure and cystic fibrosis or those on immunosuppressive agents appear to be more susceptible [35, 36] . most of the infections in endemic areas are asymptomatic with a significant number having antibodies but no history of disease [37] . however at least 50% of cases present with pneumonia of varying severity ranging from a nodular infiltrate with septic shock which has a mortality of up to 90%, to a lobar consolidation presenting with less florid features. the former often has little in the way of respiratory symptoms, presenting with fever, hypotension and organ dysfunction [35] . the more indolent form may mimic tuberculosis radiologically, most commonly involving the upper lobes, however it may also occur in the lower lobes in which case pleural effusions and empyema may occur [32, 37] . pulmonary and diffuse metastatic abscesses (involving spleen, kidney, prostate and liver), osteomyelitis, and arthritis may also occur [35] . the diagnosis is dependent on obtaining a positive culture. melioidosis must be considered in the differential of all febrile patients that have visited endemic regions, as antibiotics used routinely for community acquired pneumonia are not effective, and inappropriate therapy in severe disease increases mortality. although b. pseudomallei grows readily, laboratories may misidentify the organism as a pseudomonas which may lead to confusion and potentially incorrect therapy prior to the availability of the antibiogram. certain specific culture media may enhance growth however these are not always available outside of endemic areas [38] . serological tests are available and a positive indirect hemagglutination test (iha) or enzyme-linked immunosorbent assay (elisa) in a traveller may raise the suspicion of melioidosis but definitive diagnosis is based on culture positivity [33, 39] . treatment for severe disease is administered as an initial intensive intravenous phase with combinations of ceftazidime or meropenem/ imipenem with cotrimoxazole for at least 10 days as recommended in thailand or 14 days as recommended in australia. thereafter an oral eradication phase consisting of cotrimoxazole or amoxicillin/ clavulanate is administered for 12-20 weeks (thailand) or 3 months (australia) [40] . yersinia pestis is a gram negative coccobacillus and is notorious historically as the cause of 'the black plague'. y. pestis is likely to have caused three major pandemics which led to significant loss of life. in the first in the 6th century ad it is thought that in some regions, between 50 and 60% of the population may have perished [41] . according to the who, currently there are high rates of plague in the democratic republic of congo, madagascar and peru. the disease has three clinical syndromes: bubonic, pneumonic or septicaemic [41] . pneumonic plague leads to purulent, exudative bronchopneumonia that is usually fatal if untreated [42] . the yersinioses are zoonotic infections in which humans are accidental hosts via contact with an infected animal, usually with the flea as the vector. the infection can be acquired via a bite from a flea, scratches or bites from infected animals or rodents or inhalation of infected respiratory secretions. the infecting organisms are carried via lymphatics to the local lymph node which and this is followed by a severe inflammatory reaction [43] . there were 3248 cases of plague reported to the who between 2010 and 2015 with 584 deaths with 95% of cases from africa. areas affected by plague include north america, the former soviet union, africa, asia and south america [44] . the majority of cases are of the bubonic form (80 to 95%) which manifests with fever, chills, malaise, dizziness and associated lymphadenitis causing intense pain and swelling in a lymph node region. if untreated, dissemination occurs to the lungs or meninges in 50% of cases causing pneumonia or meningitis. septicaemic plague can occur without the bubonic illness in 10 to 20% of cases and is a severe illness that can lead to profound multi-organ failure [43] . pneumonic plague can either be primary or secondary. the former develops from aerosol exposure to y. pestis, with symptoms developing 1 to 6 days after inoculation [45] . this can be caused by person to person spread via exposure to respiratory droplets and is the cause of outbreaks in extended families. secondary pneumonic plague is due to dissemination of y. pestis into the lungs during either the bubonic or septicaemic phases and occurs in approximately 10% of patients [41] . recent data suggested that pneumonic forms are increasing in incidence accounting for 23% of global plague cases [44] . a high index of suspicion is important in order to make the diagnosis, which is then confirmed by serology or culture or by more recently developed rapid diagnostic tests [46, 47] . a high percentage of blood cultures are positive in septicaemic plague and gram stain and culture of sputum or lymph node aspirate is the usual means of diagnosis demonstrating small gram negative coccobacilli [48] . the mortality rate of plague if untreated is between 50 and 90% with pneumonic plague having a mortality of 100% if untreated and 50% if treated. previously streptomycin was the preferred antibiotic however in most cases gentamicin is now preferred. alternative agents include doxycycline and tetracycline. fluoroquinolones are the preferred agents for post-exposure prophylaxis [45] . cap is usually managed according to national guidelines that are directed toward those organisms seen most frequently in each region. the conditions described above may not be seen frequently if at all in other parts of the world and as such may not be treated appropriately and may also result in considerable spread. all intensivists should be aware of these conditions and make sure that a good travel history is obtained in each and every case. management of infections in critically ill returning travellers in the intensive care unit-i: considerations on infection control and transmission of resistance viral infection in community-acquired pneumonia: a systematic review and meta-analysis influenza-a model of an emerging virus disease community acquired pneumonia in the tropics community-acquired pneumonia explaining the geographical origins of seasonal influenza a (h3n2) community-acquired pneumonia cumulative number of confirmed human cases of avian influenza a(h5n1) reported to who monthly risk assessment summary. influenza at the human animal interface interim guidance for specimen collection, processing, and testing for patients with suspected infection with novel influenza a viruses associated with severe disease in humans human infection with a novel avianorigin influenza a (h7n9) virus human infection with avian influenza a h7n9 virus: an assessment of clinical severity clinical findings in 111 cases of influenza a (h7n9) virus infection optimizing antiviral therapy for influenza: understanding the evidence interim guidance on the use of antiviral medications for treatment of human infections with novel influenza a viruses associated with severe human disease extracorporeal membrane oxygenation for ards in adults severe acute respiratory syndrome severe acute respiratory syndrome sars: systematic review of treatment effects centres for disease control and prevention. centres for disease control and prevention. middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus outbreak in the republic of korea epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study mortality risk factors for middle east respiratory syndrome outbreak, south korea middle east respiratory syndrome hantavirus infection: a global zoonotic challenge hantavirus pulmonary syndrome hantavirus cardiopulmonary syndrome hantavirus infections for the clinician: from case presentation to diagnosis and treatment hantaviruses and cardiopulmonary syndrome in south america case definitions for infectious conditions under public health surveillance centers for disease control and prevention burkholderia pseudomallei and burkholderia mallei: melioidosis and glanders melioidosis: a review predicted global distribution of burkholderia pseudomallei and burden of melioidosis the epidemiology and clinical spectrum of melioidosis: 540 cases from the 20 year darwin prospective study management of infections in critically ill returning travellers in the intensive care unit-ii: clinical syndromes and special considerations in immunocompromised patients development of antibodies to burkholderia pseudomallei during childhood in melioidosis-endemic northeast thailand comparison of ashdown's medium, burkholderia cepacia medium, and burkholderia pseudomallei selective agar for clinical isolation of burkholderia pseudomallei workshop on treatment of and postexposure prophylaxis for burkholderia pseudomallei and b. mallei infection pneumonic plague: the darker side of yersinia pestis early emergence of yersinia pestis as a severe respiratory pathogen clinical problem-solving. sick as a dog development and testing of a rapid diagnostic test for bubonic and pneumonic plague fast and simple detection of yersinia pestis applicable to field investigation of plague foci practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the infectious diseases society of america none. none declared. key: cord-255339-oudj079q authors: al-tayib, omar a. title: an overview of the most significant zoonotic viral pathogens transmitted from animal to human in saudi arabia date: 2019-02-22 journal: pathogens doi: 10.3390/pathogens8010025 sha: doc_id: 255339 cord_uid: oudj079q currently, there has been an increasing socioeconomic impact of zoonotic pathogens transmitted from animals to humans worldwide. recently, in the arabian peninsula, including in saudi arabia, epidemiological data indicated an actual increase in the number of emerging and/or reemerging cases of several viral zoonotic diseases. data presented in this review are very relevant because saudi arabia is considered the largest country in the peninsula. we believe that zoonotic pathogens in saudi arabia remain an important public health problem; however, more than 10 million muslim pilgrims from around 184 islamic countries arrive yearly at makkah for the hajj season and/or for the umrah. therefore, for health reasons, several countries recommend vaccinations for various zoonotic diseases among preventive protocols that should be complied with before traveling to saudi arabia. however, there is a shortage of epidemiological data focusing on the emerging and reemerging of zoonotic pathogens transmitted from animal to humans in different densely populated cities and/or localities in saudi arabia. therefore, further efforts might be needed to control the increasing impacts of zoonotic viral disease. also, there is a need for a high collaboration to enhance the detection and determination of the prevalence, diagnosis, control, and prevention as well as intervention and reduction in outbreaks of these diseases in saudi arabia, particularly those from other countries. persons in the health field including physicians and veterinarians, pet owners, pet store owners, exporters, border guards, and people involved in businesses related to animal products have adopted various preventive strategies. some of these measures might pave the way to highly successful prevention and control results on the different transmission routes of these viral zoonotic diseases from or to saudi arabia. moreover, the prevention of these viral pathogens depends on socioeconomic impacts, available data, improved diagnosis, and highly effective therapeutics or prophylaxis. rudolf virchow , one of the foremost 19th century german leaders in medicine and pathology [1] , noted a relationship between human diseases and animals and then introduced the term "zoonosis" (plural: zoonoses) in 1880 [2] . later, the world health organization (who) in 1959 specified that "zoonoses are those diseases and infections which are naturally transmitted between vertebrate animals and man" [3] . venkatesan and co-authors reported that the term zoonosis is derived from the greek word "zoon" = animal and "noso" = disease [4] . zoonotic pathogens causing different kinds of diseases are of major public health issues worldwide [5] . these zoonotic diseases include frequent mixing of different animal species in the markets in densely populated areas, and the human intrusions into the natural habitats of animals, have facilitated the emergence of novel viruses. the most important zoonotic viral diseases of which eight were diagnosed (in dead or diseased animals or through antibody detection) on the arabian peninsula over the last years include rabies, middle east respiratory syndrome (mers-cov), influenza virus (ifv), alkhurma hemorrhagic fever, crimean-congo hemorrhagic fever (cchf), rift valley fever (rvf), west nile fever (wnv), and dengue fever virus. among these eight zoonotic viral diseases, two (alkhurma and mers-cov) were first reported in a patient in 1994 and 2012, respectively in saudi arabia [33, 34] . these two were transmitted later to several other countries, not only in the middle east but also to africa, asia, and europe. rabies is an almost invariably fatal zoonotic disease, which belongs to the genus lyssavirus of the rna family rhabdoviridae. rabies virus is considered an endemic viral infectious disease in animals in saudi arabia. recent scientific data on rabies cases reported in camels at al-qassim region (one of the thirteen administrative regions of saudi arabia) showed that there is an increasing number of this fatal virus disease [35] . however, the most significant animal bites which have been recorded in saudi arabia were caused by different species of animals including dogs, cats, rodents, and foxes [36] . later, al-dubaib reported rabies in dromedaries in saudi arabia in 2007 and suggested an incidence of about 0.2% for rabies that was reported among 48 camel herdsmen looking after more than 4000 animals [35] . interestingly, another survey was conducted between 1997 and 2006 in the al-qassim region of central saudi arabia among 4124 camels and showed that about 0.2% of clinical rabies incidence is caused by dogs (may be cause it highly used as a perfect guard for camels), followed by foxes; furthermore, the diagnosis of viral rabies in that region was confirmed among 26 dogs, 10 foxes, 8 camels, and 7 cats [35] . lately, the relevant government authorities (the moh and ministry of agriculture in saudi arabia) in an updated report between 2007 and 2009 showed that there were a total of 11,069 animal bites to humans in saudi arabia [36] . furthermore, most cases of animal bites were caused by dogs (49.5%) and cats (26.6%), followed by mice and rats, camels, foxes, monkeys, and wolves [36] . moreover, dogs, particularly feral dogs and foxes, are considered the most important host for rabies virus; however, bats are also considered as reservoirs of this disease. humans can become rabid by direct contact with animal mucosal surfaces via bites. according to the moh and ministry of agriculture data in saudi arabia, pets are responsible for most animal bites in humans [36] , and it is well-known that insufficient vaccination coverage of pets are among the most common hallmarks of the endemic status of rabies worldwide [37] . more recently, many saudi and expatriate families are keeping pets; however, there are limited number of specialized veterinary clinics (~5) within the kingdom of saudi arabia that have fully licensed veterinary laboratories with state of the art technologies and veterinary staff. globally, almost 95% of all human deaths caused by rabies occur in africa and asia [38] . however, saudi arabia, as one of the asian countries, has scarce publications and epidemic data on rabies status [38, 39] . moreover, memish et al., between 2005 and in saudi arabia, reported the histologic detection of the virus by identifying negri bodies in the brain samples of 40 animal rabies cases. the study showed that among the 40 suspected rabies cases, 37 (~92.5% of all cases) were found to be positive; thus confirming rabies cases among 11 dogs, 6 foxes, 6 sheep, 5 camels, 4 goats, 3 wolves, and 2 cows [36] . furthermore, more recent data confirmed the transmission of rabies virus in saudi arabia by feral dogs [23] . in spite of these facts, there are very few studies available, and no case of human rabies has been reported in recent decades from saudi arabia [40] . however, in march 2018, a scientific work was reported as the first confirmed case of human rabies in saudi arabia from makkah city, which has now been published [41] . indeed, several previous global epidemiological data confirmed that rabies accounted for 24,000 to 60,000 human deaths per year [42] , and more than 40% of these cases occur in children < 15 years of age [43] . in september 2016, a 60-year-old saudi man, presented with different clinical features-such as nausea, vomiting, and epigastric pain, with significant features suggestive of gastritis-at makkah hospital. his past medical history was significant for hypertension and diabetes type 2. during the clinical diagnostic procedure of this case, he developed respiratory distress and tachycardia, for which he was transferred to the intensive care unit [41] . because, his case worsened with chest pain and ventricular tachycardia he was referred to the king abdullah medical city in makkah for further management. the written diagnostic report indicated that he had acute anteroseptal myocardial infarction, had coronary angiogram which suggested that two-vessels were diseased with left main involvement, and surgical intervention was planned. after the decision for surgery, he was found to have leukocytosis and severe retching while attempting to drink water (hydrophobic behavior), which necessitated further review by the infectious disease consultants based on the patient's clinical symptoms. the consultant team discovered the history of an unprovoked scratch on the patient's face by a dog in morocco a month prior to the admission at the hospital. also, the patient stated that he only received tetanus vaccine. all diagnostic tests including neurologic examination were unremarkable and his saliva polymerase chain reaction (pcr) test confirmed rabies virus. he was administered verorab rabies vaccine and human hyperimmune rabies immunoglobulin (20 iu/kg) intramuscularly (im) [41] . in addition, he had troponin i (4.65 ng/ml), creatine kinase isoenzyme mb (ckmb) was found (30.08 ng/ml), and serum glucose (200 mg/dl). on the fifth day of hospital, he had recurrent episodes of ventricular tachycardia, progressively worsening of hemodynamic parameters, and he succumbed to his infection on that day. there is no vaccine against rabies recommended for travelers from/to saudi arabia, and no rabies treatment is offered to pet dogs. however, vaccination is given to dogs before they are infected; otherwise they are euthanized if infected. according to a previous study, most patient injuries from animal bites in saudi arabia showed some variations due to the monthly incidence and/or, according to the animal species [36] . bites by dogs and cats were reported frequently throughout the year, with a decrease in april and between august and october. however, bites by foxes increase between august and september while camel bites were more frequent between december and march of the subsequent year. the same previous study suggest that these seasonal variations of injuries might be due to the saudi population habits, with people going to the desert for leisure activities during good weather periods. laboratory diagnosis of rabies viral disease occur with the use of the rabies virus direct fluorescent antibody test (dfat) on brain samples and hippocampal tissue [44] . while rabies is considered nearly 100% fatal, it is also 100% preventable, and thus vaccination to pets is the key element to prevent the risk of rabies zoonotic infection [45] . reports of the epidemiology of rabies virus worldwide, and particularly in saudi arabia, suggest that it is on the increase, thus the implication of this virus' potential to spread across borders from high to low prevalence countries was highlighted [23] . the mers-cov infection is considered to be a new respiratory disease with a dire global concern [46] . mers-cov infections are caused by a newly emerging coronavirus (cov), belonging to the designated lineage c of betacoronavirus of the rna family coronaviridae. with respect to viral origin and transmission, bats are thought to be the reservoir host of betacoronaviruses, and the african neoromicia bats in particular are the natural reservoir of mers-cov [47, 48] . since its emergence in 2012 in saudi arabia, when an elderly patient (60 years old) with respiratory illness died after admission to a hospital in jeddah [34] , the disease was subsequently reported to have been transmitted to several countries worldwide, and has affected more than 1000 patients with over 35% fatality [46, [49] [50] [51] . moreover, a 60-year-old saudi man was admitted to a private hospital in jeddah, saudi arabia in june 2012 with a history of fever, severe acute respiratory syndrome with cough, expectoration, and shortness of breath. he did not smoke; and for the disease, which was suggested to be due to an animal transmission of coronaviruses, he was treated with oseltamivir, levofloxacin, and piperacillin-tazobactam. on day 11, he died [34] . after this, a 61-year-old saudi male with hypertension and diabetes with no history of smoking, reported for surgery. at the time of admission, he was asymptomatic. he was initially screened using nasopharyngeal swab, endotracheal aspirate, and serum sample for mers-cov per protocol with the mers rrt-pcr assay. the results confirmed mers-cov infection. he died three days after admission. it was discovered that the patient owned a dromedary camel barn in saudi arabia, and had a history of close contact with camels, as well as a habit of raw milk consumption of an unknown duration [51] . two studies have suggested a relationship between the infection and contact with dromedary camels [52, 53] . in addition to this, serological diagnostic methods have been used to confirm mers-cov infections in dromedary camels for at least 2-3 decades and has thus confirmed camels as an intermediate host for this virus [54, 55] . thus, in 2012, a novel coronavirus (mers-cov) was isolated from two fatal human cases in saudi arabia and qatar; and since then, more than 1400 clinical cases of mers-cov have been identified, and the great majority of the cases were from saudi arabia [56] . this previous report author raised a thoughtful comment related to the emerging viral diseases "why we need to worry about bats, camels, and airplanes" [56] . moreover, another study suggested that mers-cov infection is usually transmitted from human's direct contact with dromedary camels, especially when people drink the milk or use camel's urine for medicinal purposes [57] . more recently, a metagenomics sequencing analysis of nasopharyngeal swab samples from 108 mers-cov-positive live dromedary camels marketed in abu dhabi, united arab emirates, showed at least two recently identified camel coronaviruses, which were detected in 92.6% of the camels in that study [58] . however, limited human-to-human infections have been reported. the prevalence of mers-cov infections worldwide still remains unclear. in addition to this, the who reported about 1797 cases of these infections since june 2012, with about 687 deaths in 27 different countries, worldwide. recently, a study was conducted from june 2012 to july 2016, during which samples were collected from mers-cov infected individuals, from the national guard hospital in riyadh (the saudi arabian capital city), the moh in saudi arabia, and other gulf corporation council countries, to determine the prevalence of mers-cov [59] . the epidemiologic data that were collected, showed that the highest number of cases (about 1441 of 1797 patients) were reported from saudi arabia (~93%). among the 1441 mers-cov cases from saudi arabia, riyadh was the worst-hit area with 756 infected cases (52.4%), followed by the western region of makkah where 298 cases (20.6%) were reported [59] . furthermore, this study also showed that the incidence of mers-cov infections was highest among elderly people aged ≥60 years [34, 59] ; with speculation that there might be certain conditions or factors involved. it is considered that mers-cov infection might have a peculiar gender predisposition [60] . recent data examined the mortality in patients with mers-cov and the gender relationships, looking at the survival of cases among females and males. it was suggested that males have a higher risk of death [61, 62] ; however, this was contradicted by the findings from two other studies which suggested that males have a low risk of death [63] ; while another survey which examined the influence of gender on 3-day and 30-day survival, found a low risk of death especially in the older age group [64] . on the other hand, badawi et al., suggested that mers-cov infections could be mild and may only result in death among patients suffering from any kind of immune system disorder and/or any chronic disease [46] . more recently, data regarding the mortality in patients with mers-cov have been published. according to saudi arabia's moh daily statements, dated from february 26 through march 3, laboratory-confirmed new cases of mers-cov and 2 deaths occurred [65] . recently, on february 26, patients infected while hospitalized at riyadh included two men (23 and 59 years old) in stable condition, who were not healthcare workers. according to a february 27 update, a new case involved a 71-year-old man from the city of buraydah who later died. meanwhile, on march 1, another mers-cov infection in a riyadh hospital patient, a 64-year-old man who was listed in critical condition and who likewise had contact with camels, as the other two patients, was reported. thus, the moh stated that the spillover from camels is thought to be the main source of mers-cov in saudi arabia, since all these patients were exposed to the animals before reporting ill [65] . furthermore, an 83-year-old patient from riyadh, and other two patients who had camel contacts from hail city in the north central part of saudi arabia were listed in critical condition. the illness in these patients was reported on march 1. according to a march 3 statement, another patient, a 74-year-old man from najran located in southern saudi arabia, was reported. the man was listed in a stable condition. of these new cases, only one death, involving the 83-year-old man from riyadh, according to the march 3 moh statement, was reported [65] . still, much work is needed to detect the mers-cov infection risk in saudi arabia, because data showed increasing number of cases exist among the eight countries including saudi arabia. thus, the emergence of mers-cov in the region and its continuing transmission from 2012-2017, currently poses one of the biggest threats to global health security [66] . most cases (over 85%) reported to date have been from countries in the region (e.g., egypt) notably from saudi arabia, with 1527 cases including 624 deaths [67] . influenza viruses are considered to be important infectious viral diseases, which is caused by three virus types (a, b, and c) [68] . due to their zoonotic spread, influenza type a infects both humans and animals, and causes moderate to severe illness, with more likelihood of fatalities in young children and the elderly [69, 70] . other types of influenza, including type b and c, infect only humans [71] . furthermore, influenza a viruses, members of the rna family orthomyxoviridae, are further classified into human, swine, and avian influenza viruses. however, during the 1918 influenza pandemic, swine influenza virus infected one-third of the world's population (an estimated 500 million people) and caused approximately 50 million deaths [72] . since 2006, several infections with this virus have been recorded from various areas worldwide, including saudi arabia [73] . at the end of april 2009, an outbreak of a new type of influenza, a/h1n1, started in mexico and the usa [69] . the who declared the pandemic influenza a (h1n1) as a "public health emergency of international concern" following the first few initial cases in mexico, and subsequently in the usa [69, 74] . in saudi arabia, the epidemiological data for influenza virus were collected using a predesigned questionnaire with the first 114 confirmed pandemics influenza a (h1n1) cases identified by the infectious diseases department from the moh, and the database during the period covered from 1 june to 3 july 2009 [72] . however, according to the saudi moh data, the number of laboratory-confirmed cases of the virus in saudi arabia as at 30 december 2009 was 15,850, with 124 deaths [72] . the virus later spread worldwide, causing a pandemic, and the most recorded cases then, as reported by the who in the middle east, were in saudi arabia with 14,500 cases, followed by kuwait [69] , egypt, and oman; with less number of infected patients [68, 75] . nevertheless, between 1979 and 1980, a serosurveillance outcome of swine influenza virus from egypt provided evidence of laboratory diagnosis and very early confirmation of the virus in human patients [76] . in saudi arabia, the influenza surveillance system has been established since 2004. moreover, among people with certain chronic medical diseases or conditions, a trivalent influenza vaccine (tiv), which contains inactivated antigens for two different subtypes of influenza viruses (types a and b), became available in saudi arabia [72] . indeed, h1n1 is now in the post-pandemic period and has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude in saudi arabia [77] . a previous data was collected using a predesigned questionnaire for the first 100 cases of pandemic influenza a (h1n1) from different hospitals in saudi arabia. the age of patients enlisted in the data ranged from 1 to 56 years. the age groups with the highest percentage of cases were between: 20 and 30 years (35%), and 1 and 10 years (22%). there were 45 males and 55 females, and 53% patients had some contacts with infected persons within saudi arabia while about 47% had history of travels into saudi arabia and/or the philippines [72] . these facts are similar to the previous relationship noted between the occurrence of zoonotic viral diseases and the gender of patients and/or their ages, as reported for another viral (e.g., mers-cov) infection; provided certain conditions are met [60] [61] [62] [63] . interestingly, among elderly patients, influenza cases were higher in females than males. this relationship with viral infection occurring particularly with respiratory viral diseases, might pave the way and play a big role of more significant importance in the detection of these diseases, taking into account the influence of climate change and the different environmental factors [69, 77, 78] . nevertheless, between september 2013 and october 2014, about 406 samples were taken from several patients presenting with respiratory symptoms to king abdulaziz university hospital, jeddah, saudi arabia. however, during this study conducted to detect the susceptibility to the influenza viruses circulating in the western part of saudi arabia, out of all the tested samples, 25 (6.2%) respiratory samples were positive for influenza h1n1 virus, 1 (0.25%) was positive for influenza h3n2 virus, and 7 (1.7%) were positive for influenza b virus [78] . furthermore, h1n1, now in the post-pandemic period, has become a seasonal influenza virus that continues to circulate with localized outbreaks of varying magnitude [71] . interestingly, the presentation of influenza virus infections in humans usually vary from mild, self-limiting respiratory-like illness, to severe cases that may result in death [70, 79] . nevertheless, a recent study has shown that subclinical infection in human exists, as revealed by the serological surveillance [76, 80] . therefore, the epidemiological surveillance of influenza in saudi arabia is highly important especially with the fact that influenza cases have also been highly reported can spread globally [78] . thus, geographic influences on influenza virus infection in saudi arabia must be of concern [78] . this is relevant because a remarkably high number of egyptian muslims visit saudi arabia yearly to participate in the umrah and/or the hajj pilgrimage; in addition, the impact of the poultry industry in egypt is also worth considering, with an estimated 1 billion birds and several millions of engaged laborers with/without surveillance [81] . it is well-known that the influenza drugs, antiviral agents, and the current seasonal influenza vaccines are effective in reducing the incidence and severity of the disease, sickness, and/or complications. however, the important strategy for influenza management includes the provision of prophylaxis and treatment [78] . however, it is possible for widespread drug resistance against antiviral agents or vaccines to emerge in patients who extensively abused the drugs, in addition to those who have never received such treatment, globally [82] [83] [84] . furthermore, influenza viruses pose a challenge to vaccine developers and manufacturers due to the fact that these viruses are continually changing in nature, including hemagglutinin and neuraminidase [78, 85] . moreover, while resistance to neuraminidase inhibitors (e.g., oseltamivir and zanamivir) have been reported to sporadically occur, the resistance to oseltamivir has been widely reported since 2007, with a worldwide spread [86] . this highlights why there is an urgent need for the public health system to monitor continuously via globally active influenza surveillance programs. furthermore, there is need to monitor the circulating influenza viruses strains, as well as the occurrence of any resistance, using appropriate diagnostic methods. this is considered highly essential in saudi arabia. interestingly, survey data has shown an increasing report of the viral infection from egypt, since hajj egyptians has ranked in the top 10 list of countries with the highest number of mecca pilgrims in the last 10 years [81] . influenza is highly susceptible to antiviral drugs such as oseltamivir, according to a more recent epidemiological study [87] . although millions of muslims, globally, travel annually to saudi arabia to perform hajj and/or umrah in the holy places including both makkah and al-madinah for very limited period (~10 days), this gathering could play a major role in the introduction of new influenza viruses, not only to saudi arabia but also to the rest of the world [83, 87] . unfortunately, there is no such influenza surveillance program in saudi arabia, thus this pose a serious public health concern. recently, in a study of 1600 pilgrims screened on arrival at the 2010 hajj season in saudi arabia, 120 (7.5%) had influenza a virus (9 out of the 120 had h1n1 virus) [88] . additionally, the epidemiological data showed that the pilgrims had the potential not only to introduce these viruses to saudi arabia, but also to export the influenza virus back to their home countries [78, 89] . this can occur in saudi arabia, despite the availability of a tiv containing inactivated antigens for influenza virus types a and b, which can protect against the influenza virus infection [72] . importation of resistant and highly pathogenic viruses including influenza viruses can occur worldwide. despite this, there is lack of studies and data on drug susceptibility, and a very limited number of studies and reports on viral isolates, except for one study conducted in jeddah, according to the best of our knowledge [78] . most importantly, this highlights the importance of circulating influenza viruses in saudi arabia, hence there is need to ensure effective use of antivirals for prophylaxis and treatment of influenza. furthermore, the rate of vaccination against influenza is very low among pilgrims and healthcare workers [78, 90] . moreover, studies are needed to provide a clear picture on the impact of drug resistance on saudi arabia's endemic pathogens, including the influenza viruses. in a recent communique, the ministry of environment, water, and agriculture for saudi arabia reported two cases of h5n8 avian influenza in the kharj governorate [91] . the latest update by the ministry revealed that the number of samples collected from saudi regions since the start of the influenza outbreak had reached 12,829. positive results from samples and laboratory tests indicate 171 positive cases, and the saudi authorities have taken action by culling as many as 254,050 birds within a 24-h period [91] . in contrast, several epidemic zoonotic cases of influenza h5n1 have been reported in domestic cats in several countries in asia, europe, the usa, and italy [87] . moreover, the epidemic of influenza in dogs might be related to a serious public health issue and could be shown to have resulted from zoonotic diseases from pets, similar to the avian influenza h3n2 outbreak reported in pet dogs in south korea in 2007 [92] . nevertheless, a recent study has shown that the role of pets, particularly cats and dogs in the epidemic of influenza as a source of human infection seems limited. however, cats were shown to be fully susceptible to experimental infection, and infected cats were able to infect naive cats [87] . in 2009, pandemic h1n1 infection in a domestic cat in the usa from iowa was diagnosed by a novel pcr assay; thus, human-to-cat transmission was presumed [93] . despite this prior evidence, the role of pets including cats and dogs seem even more limited in the dispersal of avian influenza to humans. rather, humans may be the source of pet infection, as suggested for influenza h1n1 and/or h3n2 virus infections [87, [92] [93] [94] . most importantly, epidemic zoonotic cases of influenza among pets has highlighted the importance of circulating influenza viruses globally; especially, to ensure the effective use of antivirals for the prophylaxis and treatment of influenza, in particular, with the increase in the number of pets stores in saudi arabia, especially in riyadh [78, 90] . surprisingly, previous data focused on the occurrence of zoonotic infection of different influenza virus types, and particularly, the transmission of avian influenza virus h3n2 to domestic dogs [92] . several studies have examined and confirmed the occurrence of zoonotic infection of the influenza a virus h1n1 pandemic, especially in domestic cats [93, 94] . nevertheless, epidemiological studies on different zoonotic infections among the pets in saudi arabia including cats, dogs, and/or baboons are very rare. however, a previous case report confirmed a relationship between some zoonotic diseases causing respiratory symptoms, such as influenza, among pets [95] . this study suggests that severe lung infection with dry cough and severe anemia should lead to the suspicion of a secondary infection with zoonotic balantidiasis, which infected a hamadryas baboon from saudi arabia in a research center for pets in riyadh [95] . furthermore, two other epidemiological zoonotic study on balantidium coli protozoan zoonotic infection in camel was reported from riyadh [31] . in addition, another previous data confirmed the occurrence of toxocara canis zoonotic infection based on respiratory symptoms reported at the pet clinics in saudi arabia (and also in riyadh where the symptoms occurred in dogs) [30] . still, more such studies are needed to highlight the important issues and/or provide clearer pictures of the zoonotic pathogens among pets in saudi arabia; however, pet ownership has been growing rapidly as well as the number of pet stores among the saudi population. alkhurma hemorrhagic fever virus (ahfv) in humans was discovered in 1994 [33] . the first case reported in a butcher from the city of alkhurma, a district south of jeddah in saudi arabia, died of hemorrhagic fever after slaughtering a sheep. the viral infection has a reported fatality rate of up to 25% [96] . interestingly, one of the previous reports regarding this disease showed a misunderstanding of the real name of this infection, called alkhurma, not alkhumra [97, 98] . because subsequent cases were diagnosed in patients from the small town known as alkhurma in jeddah from where the virus got its scientific name; the name was accepted by the international committee on taxonomy of viruses [99] . thus, based on evidence, the first case was confirmed to be the butcher, following the slaughtered sheep [100] . therefore, a study was conducted among affected patients to address this disease as a public health issue. blood samples were collected from household contacts of patients with laboratory-confirmed virus for follow-up testing by enzyme-linked immunosorbent serologic assay (elisa) for ahfv-specific immunoglobulin (ig) g. samples from persons seeking medical care were tested by elisa for ahfv-specific igm and igg using ahfv antigen. viral-specific sequence was performed by reverse transcription pcr (tibmolbiol, lightmix kit; roche applied science, basel, switzerland). a total of 11 cases were identified through persons seeking medical care, whose illnesses met the case definition for ahfv, and another 17 cases were identified through follow-up testing of household contacts [100] . subsequently, the virus was isolated from six other butchers of different ages (between 24 and 39 years) from the city of jeddah, with two deaths. the diagnosis was established from their blood sample tests. the serological tests later confirmed four other patients with the disease [101] . from 2001 to 2003, the study on the virus initial identification in the city of alkhurma again identified 37 other suspected cases; with laboratory confirmation of the disease in 20 (~55%) of them. among the 20, 11 (55%) had hemorrhagic manifestations and 5 (25%) died [102] . the virus was later identified in three other locations: from the western province of saudi arabia (ornithodoros savignyi and hyalomma dromedarii were found by reverse transcription in ticks) and from samples collected from camels in najran [103, 104] . ahfv virus was considered as one of the zoonotic diseases; however, the mode of transmission is not yet clear. recently, it was suggested that the disease reservoir hosts may include both camels and sheep. the virus might also be transmitted as a result of skin wounds contaminated with the blood or body fluids of an infected sheep; through the bite of an infected tick, and through drinking of unpasteurized or contaminated milk from camels [101, 105] . in humans, this zoonotic disease may present with clinical features ranging from subclinical or asymptomatic features to severe complications. it is related to kyasanur forest disease virus, which is localized in karnataka, india [106, 107] . however, epidemiologic findings suggest another wider geographic location for the disease in western (including jeddah and makkah) and southern (najran) parts of saudi arabia, and the virus infections mostly occur in humans [96, 101, 102] . a study was conducted by alzahrani et al. in the southern part of saudi arabia particularly in the city of najran (with populations of~250,000), an agricultural city in saudi arabia, where domestic animals are reared at the backyard of owners. after the initial virus identification, from january 2006 through april 2009, 28 persons with positive serologic test results were identified. infections were suspected if a patient had an acute febrile illness for at least two days; when all other causes of fever have been ruled out [101] . additionally, data analysis indicated that patients infected with the virus were either in contact with their domestic animals, involved in slaughtering of the animals, handling of meat products, drinking of unpasteurized milk, and/or were bitten by ticks or mosquitoes. symptoms consistent with ahfv infection-including fever, bleeding, rash, urine, color change of the feces, gum bleeding, or neurologic signs-then develop [95] . fortunately, infected patients responded to supportive care (including intravenous fluid administration and antimicrobial drugs when indicated), with no fatal cases. in summary, ahfv is a zoonotic disease with clinical features ranging from subclinical or asymptomatic features to severe complications. another study highlighted different characteristics of the exposure to the blood or tissue of infected animals in the transmission of ahfv to humans. of the 233 patients confirmed with infections, 42% were butchers, shepherds, and abattoir workers, or were involved in the livestock industry [108] . more recently, a study on infection using c57bl/6j mice cells showed that the clinical symptoms of the disease were similar to the presentations in humans [109] . however, alkhurma disease resulted in meningoencephalitis and death in wistar rats, when high titers to the infection occurred [98] . in addition, exposures to mosquito bites are regarded as potential sources of transmissions of the infection; however, very few available data support this [97] . although, available data shows that alkhurma virus has been isolated following mosquito bites [102] . however, another study suggested that mosquitoes may play a role only as a vector in the transmission of the disease [100]. cchf is a zoonotic viral disease from the bunyaviridae family, and the principal vector for the disease is ticks of the genus hyalomma. it is most commonly endemic in africa, middle east, asia, and eastern europe [110, 111] . it is an acute, highly-contagious, and life-threatening vector-borne disease responsible for severe hemorrhagic fever during outbreaks, and a fatality rate of up to 40% [112, 113] . the infectious disease was recognized first in the crimean peninsula in 1944, and it was named crimean hemorrhagic fever virus because the virus was isolated for the first time from a febrile child in 1956 from stanleyville (now kisangani), democratic republic of congo [114] . currently, the virus infects both humans and animals following tick bites [115] . however, a human can be infected by the animal through contact with the blood or tissues of the infected animal, in particular, exposures at the abattoirs are common. therefore, workers in contact with animals (e.g., veterinarians, farmers' and workers in slaughterhouses) form a high percentage of those affected [87] . also, different species of infected animals-such as camels, cattle, sheep, goats, and ostriches-might be infected with no clinical signs [83] . in addition, human-to-human transmission is also documented, mostly through a form of nosocomial or in-house infection [113, [116] [117] [118] . lately, antibodies to the virus have been detected in different animal species, as reported in 1976, in egyptians animals' sera [119] . the preliminary seroepidemiological survey detected antibodies to the virus in 8.8% of camels' sera and 23.1% of sheep sera, but no antibody was detected against the virus in the sera of other animals such as donkeys, horses and mules, pigs, cows, and buffaloes [119] . the epidemiology and distribution of cchf in saudi arabia are unclear, but there are reports of cchf as a result of the trading and importing of infected livestock from neighboring countries to saudi arabia [120] . in 1990, the cchf virus (cchfv) caused an outbreak involving seven individuals in makkah, although the virus had not been reported previously in saudi arabia. therefore, a study on the epidemiology of this virus was carried out in makkah, jeddah, and taif from 1991-1993. about 10 out of 13 different species of ticks that were capable of transmitting the disease were collected from camels, cattle, sheep, and goats, but camels had the highest rate of tick infestation (97%), and h. dromedarii was the commonest tick (70%). an investigation in makkah between 1989 and 1990, which included a serological survey of abattoir workers in contact with sheep blood or tissue, identified 40 human cases of confirmed or suspected cchf with 12 fatalities [120] . the report from the investigation stated that the virus might have been introduced to saudi arabia through the jeddah seaport via infected ticks on imported sheep; since then, it has been endemic in the western province of saudi [120, 121] . in addition, another previous study confirmed that the highest seropositivity rate of the virus in saudi arabia localities was associated with animals imported from sudan [121] . furthermore, the who reported 22 countries with cchf including saudi arabia; however, all the remaining countries are either close to saudi arabia or are islamic countries with high numbers of muslims who travel annually to saudi arabia for hajj pilgrimage. the same who epidemiological data suggest that in these 22 countries including saudi arabia, in recent years, there has been report of steadily increasing number of sporadic human cases, incidence, and outbreaks of the virus [122] . furthermore, another study by who investigating cchfv in the eastern mediterranean region (emr) stated that cchf is a clear and growing health threat in the who emr. cases are being reported in new areas, showing a geographical extension of the disease that is probably linked to the livestock trade and the spread of infected ticks by migratory birds. according to ecological models, the increase in temperature and decreased rainfall in the who emr could have resulted in the sharp increase in distribution of suitable habitats for hyalomma ticks and the subsequent drive of cchfv infection northwards [123] . jazan province, the red sea port city on saudi arabia's southern border with yemen, serves as the east-west portal from sub-saharan africa at djibouti and the south-north route across the yemeni frontier. it is a heavily traveled corridor for humans and animals entering saudi arabia, particularly during the annual hajj pilgrimage. in november 2009, a total of 197 (19%) enrolled soldiers reported symptomatic illness during deployment, 49 (25%) of whom were hospitalized. reported signs and symptoms included fever (n = 81), rash (n = 50), and musculoskeletal complaints (n = 128). a surveillance study was conducted to detect the causes of the several outbreaks through that area, which was reported as endemic over a wide geographic range. from the surveillance, serologic testing for cchfv, ahfv, denv, and rvf was completed for 1024 saudi military units from several saudi arabian provinces. these units were previously stationed in other parts of the country, and were deployed to jazan province; the initial screening for igg of each of these viruses was conducted by igm testing for all igg-reactive samples. among the samples from all military forces, the study identified 40 reactive serum samples with a combined seroprevalence of 3.9 cases/100 soldiers tested. a confirmed serologic status of 1024 soldiers who were evaluated for igg and igm elisa reactivity against cchfv, rvf, ahfv, and denv infections were positive for 6, 20, 13, and 1 sample, respectively [124] . rvf is a common arbovirus zoonotic disease caused by the rvf virus. the virus belongs to the genus phlebovirus and family bunyaviridae. it is most common in domestic animals, and causes mild to life-threatening infections in humans. the name of the disease was derived from the great rift valley of kenya, when the disease was described for the first time in 1912 [125] . epidemiological tests have since been described after a highly fatal epizootic occurred there in 1930 [126] . rvf is a viral zoonosis with evidence of widespread occurrence in humans and animals in africa and the arabian peninsula. the epidemiology of this virus in saudi arabia might be closely related to the ecological factors that are prevalent, as shown from another area, along the great rift valley, which traverses ethiopia and kenya to northern tanzania with the drainage ecosystems [127] . saudi arabia has many of the world's mosquito vectors of parasitic and arboviral diseases. however, few studies have addressed their geographic distribution and larval habitat characteristics [128] . there are complex interactions between these factors that significantly impact mosquitoes ecological fitness and vectorial capacity for disease transmission, with important implications for vector management and control at the local and regional levels [129, 130] . therefore, studying these factors for different mosquito fauna will help in monitoring potential modifications of larval habitats due to rains, global climate change, or man-made activities. previous studies on the ecology, distribution, and abundance of mosquito species in kingdom of saudi arabia are generally few and sporadic; and most of these studies were conducted in the western and southern regions. these studies were conducted in the asir province in 1993-1995 and 1999-2001 [131,132] [142, 143] . these studies reported the presence of many species from many genera, the most important of which are anopheles, aedes, and culex. among these studies, only a few provided the description of habitats of the larvae of these vectors. even fewer studies provided evidence on the active role of some species on disease transmission; the existing ones were mainly for anopheles vectors of malaria [138, 144, 145] , as well as aedes and culex vectors of arboviruses such as sindbis and dengue fever [141, 146, 147] . rvf is not considered a major type in the arboviruses family, which mostly are adapted to a narrow range of vectors; however, among this family, the rvf infection has a very wide range of vector including mosquitoes such as aedes and culex, flies, and often, ticks [148] . interestingly, for different rvf species, rvf vectors have special roles about how they sustain the transmission of the disease ecologically to humans [149] . in some cases, the impact of rainfall, soil type, water, the persistence of breeding, and often wind, have significant effect on vector distribution [150] . epizootics studies indicate that rvf disease follows unusually severe rainy seasons, a situation that may likely favor the breeding of a very large insect population, needed as a vector prerequisite. globally, rvf epidemiology was first reported in africa with the 1989 rvf epizootics in kenya when laboratory test reports confirmed virus isolation [151] [152] [153] . in 2000, the disease, for the first time, affected humans and livestock outside africa, with the larger rvf disease incidence following outbreaks, reported in saudi arabia [154] and yemen. lately, rvf infections have been associated with minimal genetic diversity, epidemiologically; which has lately been considered to be a newly introduced single lineage of rvf viral disease [155] . epidemiological reports from both saudi arabia and yemen showed that the outbreak, which occurred in 2000, resulted in about 2171 human infections, and 245 deaths [156] . furthermore, the fatality rate reported in southern saudi arabia then, reached 14%, and was considered the most severe epidemic in that area ever since [157] . moreover, the disease outbreak was thought to have been transmitted in countries such as saudi arabia by infected imported ruminants from east africa via the port of djibouti and probably from kenya and/or sudan [121] . however, the fact remains that the rvf epidemic has been around for more than 70 years, with infections occurring at prolonged intervals in eastern and southern africa [158, 159] . consistent with this, another report showed that the same virus strain was implicated in the 1997-1998 rvf outbreaks in kenya and the 2000 outbreaks in saudi arabia and yemen [130] . the outbreaks in kenya later resulted in about 89,000 human infected with about 478 patients deaths [127, 160] . surprisingly, in 2000, jup et al. found the mosquito species that was identified as a potential vector, which led to the assumption that the zoonotic viral disease in saudi arabia was transmitted by culex tritaeniorhynchus [161] . other species of mosquitoes were implicated in the transmission of this viral disease in other countries closer to saudi arabia [162] [163] [164] . furthermore, another study reported the unexplained rvf virus infection among people from saudi arabia, with isolation and genetic virus characterization associated with illness in livestock, along the southwestern border of saudi arabia in september 2000 [164] . the study reported that vertical transmission of the virus in the epidemic mosquito vector occurred in saudi arabia. in addition, the study stated that the most abundant culicine mosquitoes collected were aedes vexans arabiensis, culex pipiens complex, and culex tritaeniorhynchus, which were considered to be the most important epidemic and epizootic vectors of rvf virus in saudi arabia [164, 165] . however, the same study, focusing on a very important issue which occurred during the rainy seasons; suggested that aedes vexans arabiensis has the potential to be an important epidemic and epizootic vector because of the tremendous numbers of individual mosquitoes produced after a flood [164] . characteristically, once the virus is introduced into permissive ecologies, it becomes zoonotic; thus, they are able to enhance vulnerability of the area to periodic outbreaks, with the potential to spread further into non-endemic environments with favorable conditions [166, 167] . saudi arabia is considered a region where rvf virus has circulated actively. noticeable data regarding zoonotic infection from animal to human from the arabian peninsula including saudi arabia has recently showed that it may be due to the consumption of unpasteurized camel milk [32, 159, 168] . wernery reported camelus dromedarius as the animal host and/or reservoir of rvf zoonotic infection, which was diagnosed in the arabian peninsula [23] . due to the scientific data regarding rvf disease, it is quite clear that globalization of trade and altered weather patterns are a concern for the future spread of more infections, since the causative agent of this viral disease is capable of utilizing a wide range of vectors for its transmission. thus, this poses a significant challenge to outbreak prediction, with inherently complex methods of infection control; therefore, mitigation and management of the virus will require concerted efforts [121, 169, 170] . dengue hemorrhagic fever (dhf) viral disease is a serious global mosquito-borne infection. the clinical manifestation ranges from mild febrile illness to severe sickness which may include dengue shock syndrome [171] . the dhf virus belongs to the genus flavivirus in the flaviviridae family, which can usually be spread by mosquitoes of the genus aedes aegypti, but less often through the genus aedes albopictus [172, 173] . also, this virus is a single-stranded positive-sense rna virus that exists as four different serotypes (den-1, den-2, den-3, and den-4) [174] . in saudi arabia, the disease is limited to the western and southwestern regions, such as jeddah and makkah where aedes aegypti exists. however, all dhf cases in saudi arabia presented as a mild disease [171, 175] . in fact, the first experience of dhf virus isolation from saudi arabia was recorded during an outbreak of the virus in 1994 [176] , where the 289 confirmed cases reported in jeddah were caused by denv-2 [176] [177] [178] . however, during this first outbreak, in both summer and rainy season, at the end of the year, both denv-2 and denv-1 were isolated. in 1997, during the rainy season in jeddah, there was an emergence of the denv-3 virus [179] . in subsequent years, from 1997-2004; the emergence of dhf occurred with the three identified serotypes (denv-1, denv-2, and denv-3) isolated in jeddah [171] . khan [171, 181] . however, egger suggested that the reemergence of the disease in saudi arabia might be explained by the growing levels of urbanization, international trade, and travels [182] . in keeping with the findings of most previous studies, the epidemiological occurrence of dhf infection using the saudi's national data indicated that the majority (68%) of patients with dengue virus infection were saudi nationals [183] . on the contrary, from the epidemiological report based on saudi's national data in previous publications, an estimated 15% of patients with dhf presented in jeddah [184] . kholedi [186] . in yet another recent study, the virus was reported as 38% in saudi patients [187] . all of these saudi studies were conducted in jeddah. from makkah city, the reported epidemiological study identified 63.4% of dhf infection cases among saudi nationals [188] . similarly, a later study puts the estimate at more than 70% of saudi nationals [189] . these previously published studies suggest that differences in proportions may exist between saudi nationals infected with dhf virus in jeddah and makkah city. contrary to previous data from jeddah, in makkah, it was clear that the majority of patients presenting with clinically significant dhf were saudi nationals. therefore, these results emphasized the fact that saudi nationals are at greater risk of dhf infection. the awareness of these results is considered a cornerstone to enhancing the ability of healthcare professionals' identification of the disease; and this might play an important role in the development of effective eradication strategies for the disease in saudi arabia localities. furthermore, the first cases of the virus, confirmed in al-madinah in 2008, showed that the isolated virus serotypes were denv-1 and denv-2 [190] . in 2009, the moh in saudi arabia reported a total of 3350 cases of the dhf infection, with an estimated case fatality rate of about 4.6 per thousand in saudi arabia [171] . in august 2017, several countries in asia, including malaysia, singapore, and pakistan reported about 60,000, 1877, and 738 dengue cases including deaths, respectively. in the same period (2017), saudi arabia reported 39 confirmed dengue cases in makkah, 19 of which occurred in august 2017, 60 suspected cases, and 15 cases pending laboratory confirmations. from these epidemic data indicating the reemergence of dhf infection in saudi arabia; jeddah, makkah, and al-madinah were shown to be the more susceptible areas, for this infectious disease, and this could be due to the fact that these cities are the sites of both the annual hajj pilgrimage and/or the minor umrah pilgrimage, which draw millions of muslims to saudi arabia [171, 190] . currently, there are few epidemiological studies on dhf virus infection in saudi arabia. a study by al-azraqi et al. was conducted in 30 hospitals and 387 primary healthcare centers in two cities in the southern province of saudi arabia, particularly in jizan, and aseer. the study, which was limited to the seroprevalence among clinically suspected hospital-based patients, detected about 31.7% positive cases of dengue virus igg among 965 randomly selected patients attending the outpatient clinics for any reason. the associated risk factors were male gender, younger age (15-29 years) , lack of electricity, and having water basins in the house [191] . the authors suggested that the virus may occur in sporadic cases in jizan, due to the nature of the city. jizan is relatively flat and located at sea level [191] ; thus the likelihood of the formation of small stagnant water following the rainfall in the city is high [171] . interestingly, a retrospective cross-sectional study, which compared the clinical findings and/or the diagnostic laboratory results in uncomplicated patients, and patients who developed dhf, was conducted at dr. soliman fakeeh hospital in jeddah, between january 2010 and june 2014. about 567 patients with a discharge diagnosis of dhf or dengue shock syndrome were identified [183] . of these, 482 (85%) were adult patients within the age range 14-73 years, and 15% were children with age ranging from 2 months to 13 years. however, among all these patients, 67% of the adults and 63% of the pediatric cases were males. the clinical data from the hospital showed that in the adult patients, about 98% made a full recovery without complications while two patients died [183] . more recently in 28 january 2018, the moh began an intensive campaign to eradicate the dhf virus from saudi arabian cities, to enhance public health awareness, and facilitate a change in hygiene behavior of citizens and residents. this resulted in a 50.7% reduction in the number of dhf infection among inpatient cases in jeddah when compared to the same period in the previous year. however, the overall drop in dhf cases reached 38% in 2017, compared to the previous year [192] . furthermore, recently, it is well-known that in saudi arabia, the dhf infection has been limited to the western and southwestern regions such as jeddah and makkah where aedes aegypti exists. however, all dhf cases in jeddah, saudi arabia, were mostly mild cases [171, 175, 192] and the prospect of dengue virus control lies with vector control, health education, and possibly vaccine use. west nile fever is one of the emerging zoonotic infections, which is caused by an arthropod-borne virus belonging to the genus flavivirus, of the rna family flaviviridae. the virus' main reservoir, which is responsible for the transmission of the disease, is the genus culex mosquitoes [193, 194] . the west nile virus (wnv) derived the name from the site where the first case was isolated in 1937, from the blood of a woman with mild febrile illness living in the west nile district of uganda [195] . the first outbreak, in 1951-1952, was reported in israel [196] . this constituted a turning point in the epidemiology of the virus, because it was thought to have originated from israel following the introduction from africa, and later introduced to the usa in 1999 [197, 198] . subsequently, the infection was documented across the globe [199] , with the exception of antarctica [194] , in various species of vertebrates, including humans, mammals, non-human primates, birds, rodents, reptiles, and amphibians [200] . however, birds are considered as one of the main reservoirs of the virus [201] . saudi arabia is geographically close to several of the countries where wnv had circulated actively or had been reported; thus, there is a high risk of the disease being introduced into saudi arabia. wnv is known to cause neurological disease in both humans and horses. however, the clinical manifestations of the disease in horses include ataxia, paralysis of the limbs, recumbency, hyperexcitability, and hyperesthesia. in al-ahsa, saudi arabia, a study was performed on 63 horses to test the incidence of the virus using the clinical examination and serologic elisa test. however, from this previous study, while clinical examination for neurologic signs detected no significant findings, wnv antibodies were positively identified at serology among 33.3% of the tested population [202] . in 1999, lanciotti et al. found this virus to be responsible for an outbreak of encephalitis in two fatal human cases from northeastern usa in late summer; and suggest a closely relation between this outbreak in the usa to a wnv infection which was isolated from a dead goose in israel in 1998 [197] . the first cases of wnv in horses was identified in egypt and france in the 1960s [203] ; ever since, wnv has had significant public health impact worldwide due to its resurgence and dynamic epidemiologic features in humans and animals. between 2008 and 2009, a study in iran identified wnv antibodies in horses, and the results confirmed the highest activity of the virus reported in the western and southern provinces with seroprevalences of up to 88% in some areas of iran [204] . although human cases and/or animal infections with wnv including horses have also been reported in jordan and lebanon (direct and close neighbors of saudi arabia) between 2000 and 2014 [205] [206] [207] ; however, the reported wnv in patients or horses in these areas might have circulated in natural transmission cycles with close relationship to the wnv isolated from human and horses in jordan, lebanon, and iran in 2000, 2010, and 2014, respectively. humans and horses (incidental hosts), are unable to develop sufficient viremia to infect mosquitoes, hence, they are not included in the wnv lifecycle [203] . more recently, in 2016, using standard procedures, the central veterinary research laboratory in dubai, the united arab emirates, described the first wnv isolation in a dromedary calf; and this supports the conclusion that wnv is present in the country [208] . the wnv zoonotic infection was probably transmitted through the human-animal interface; that is through the well-known contact with infected arabian camels in saudi arabia. interestingly, dromedary are exported from the united arab emirates to saudi arabia and vice versa; due to the closely related wnvs genes and their circulation through the natural transmission cycles worldwide, a complete genome sequencing for more wnvs strains, as well as comparative genomic and phylogenetic studies in saudi arabia, are needed to ascertain whether the dromedary infection with wnv exists in the country or not. however, the same facts have been suggested recently (2017), when it was suggested that wnv infection was introduced into turkey at the time of the outbreaks in saudi arabia and yemen. it was further suggested that the virus may have been introduced via unlawful entrance of viremic domestic or wild animals through the borders or through vectors that carry the virus into turkey [209] . camels play an important role in public health issues regarding zoonosis and they have been involved in most of the zoonotic infections which occurred in saudi arabia in the last three decades. they are reported as sources of infections-including rabies, mers-cov, alkhurma virus, cchfv, and rvf virus [52, 94, 101, 108, 120, 124, 210] -via direct physical contacts with camels and/or indirectly by having camels within or near the household in saudi arabia. however, some zoonotic infections among camels are sometimes asymptomatic; thus, they play a vital role in the mechanism of transmission of various diseases [211] . furthermore, wernery et al. reported that wnv can be transmitted by mosquito bites in different species including to humans, horses, camelids, and many other mammalian species as well as reptiles and birds [159, 200, 201] . to the best of our knowledge, there is still no extensive surveillance data regarding this disease among wildlife animals in saudi arabia. strikingly, several of the human zoonotic cases that involve camels-which included different viral, bacterial, and parasitic infections on the arabian peninsula-have recently been highlighted as being caused by the consumption of unpasteurized camel milk [168] . currently, in this review, some aspects of the most common viral diseases of zoonotic importance in saudi arabia were summarized; these are presented in table 1 . however, data regarding emerging and reemerging zoonotic viral diseases are reported as they occur from time to time from the same, new, and/or different localities from saudi arabia. while other viral zoonotic infections occur in other countries, which are considered to be close to saudi arabia, some infections spread to some localities within saudi arabia because of the geographical proximity as shown in figure 1 . interestingly, some of these zoonotic viral pathogens were first exotic to saudi arabia (e.g., mers-cov and ahfv) and should be of more concern when reported in prevalence studies, and whenever they are detected by saudi authorities. epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for saudi arabia (e.g., from yemen, egypt, gulf areas, and sudan). fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in saudi arabia. however, the control measures of these viral zoonotic pathogens will not only benefit saudi arabia or arabian peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. prevention, control, and management of several zoonotic diseases usually require several important measures including the following. having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [212, 213] . because an intensive livestock trade exists between saudi arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [214, 215] . this is supported by several previous studies concerned with the route of livestock trade between saudi arabia and the neighboring countries (e.g., rabies through yemen and/or oman [36, 216] interestingly, some of these zoonotic viral pathogens were first exotic to saudi arabia (e.g., mers-cov and ahfv) and should be of more concern when reported in prevalence studies, and whenever they are detected by saudi authorities. epidemiological data should be focused more on both the trade routes and wildlife migration across the region, since these are potential risks for saudi arabia (e.g., from yemen, egypt, gulf areas, and sudan). fortunately, there are many ways and/or approaches to improve the control of such different zoonotic pathogens in animals and humans in saudi arabia. however, the control measures of these viral zoonotic pathogens will not only benefit saudi arabia or arabian peninsula but will also be of high benefit to other countries, especially those with low prevalence, by stopping or controlling the spread of the epidemic worldwide. prevention, control, and management of several zoonotic diseases usually require several important measures including the following. having vaccination protocols for all suspected animal species by the use of up to date vaccines and compliance with the standards needed for all animals. taking into account the highly needed and important investigation for these zoonotic viral diseases vectors, including vector breeding control (including vectors, hosts, and arthropods), and control of the animals (livestock) movements, with respect to trade and export [212, 213] . because an intensive livestock trade exists between saudi arabia and its neighboring countries, there may be increased risk of reemerging viral diseases of all kinds [214, 215] . this is supported by several previous studies concerned with the route of livestock trade between saudi arabia and the neighboring countries (e.g., rabies through yemen and/or oman [36, 216] ; rvf through kenya, djibouti, and/or egypt [127, 149, 212] ; cchf through sudan [121] ; influenza through oman and egypt [71, 87, 121, [217] [218] [219] ; wnv through emirates, egypt, jordan, and israel [196, 197, 199, 203, 208] ; and dhfv through egypt [190] ; as well as mers-cov and ahfv viral infections, which originated and are transmitted globally from saudi arabia) [34, 52, 53, 97, 98] . therefore, it is clear that a huge gap still exists in the sharing of published data about the acknowledged epidemiology of zoonotic diseases in saudi arabia, which rigorously prohibits speculations about the health burden of people. currently, there are surveillance activities for some viral diseases-such as rabies, mers-cov, and influenza-but these are still being weakly addressed or neglected, especially at the human-animal interface. the important role of vaccination both in the prevention and control of animal diseases and the need to check the human sources in food or water must not be neglected. also, management of animals, both outdoors and indoors must be taken seriously. however, owners of pets clinics and pets stores should be held responsible in ensuring that they keep their pets' vaccination protocols up to date, and prevent any kind of animal behavior that might result in zoonotic risks to humans through bites or scratches by pets. therefore, pet clinics and/or pets stores should be always considered a serious public health issue and vaccination should be obligatory. therefore, the importance of the annual vaccination routine programs for all stray dogs against rabies, and regular investigation of other animals, should be considered. in addition to this, pet clinics and stores should monitor pets' health records, and their owners should be held fully responsible in ensuring that their animals remain healthy and fully vaccinated. this will guarantee for them and their neighbors a zoonotic disease-free environment (e.g., against rabies virus particularly in dogs). this is particularly important in view of the case of human rabies reported in march 2018 from a makkah hospital. this involved a 60-year-old saudi man who was admitted to the hospital with a history of an unprovoked scratch on his face by a dog. a month after his admission, his saliva pcr test confirmed rabies virus [41] . nevertheless, rabies is endemic in animals in the arabian peninsula, with increasing numbers of reported cases form certain countries in the area including saudi arabia, yemen, and oman [36, 41] . kuwait, qatar, and the united arab emirates are considered to be rabies-free, whereas there is no available information about bahrain [216, 220] . furthermore, animal rabies cycle and cases reported in these endemic countries including saudi arabia are characterized by different animal species such as camels, cattle, goats, and sheep; however, the majority of cases are reported in feral dogs [36, 216] . fortunately, studies about pets with different zoonotic infections from pet clinics and/or pet stores in saudi arabia have been rarely detected among cats, dogs, and baboons. however, there was a previous study, which reported the occurrence of toxocara canis infection in pets (dogs) in riyadh, saudi arabia [30] . there were also two previous reports regarding a protozoan zoonotic infection of some pets with clinical manifestation, particularly in papio hamadryas baboon in riyadh [95, 221] . in addition to this, another report highlighted the protozoan zoonotic infection in camels, in riyadh [31] ; however, more of these kind of studies are needed, because, they provide important opportunities to present a clear picture about indoor and outdoor animals and zoonotic pathogens such as viral, bacterial, fungal, etc. which involved, in saudi arabia. by enhancing biosecurity and management in animal farms, the risk of reemerging pathogens particularly responsible for zoonotic diseases caused by viruses, can be reduced. this is a matter of economic importance; in view of the large livestock trade existing or that existed between countries in the indian ocean and eastern africa countries where several zoonotic diseases are endemic. however, a phylogenetic study strongly suggests that some zoonotic infections have been introduced into saudi arabia through ruminant trade [212, 213] . furthermore, following the adoption of the recommended guidelines of the world organization for animal health through its office international des epizooties (oie) code, if such policies regarding the exportation and/or importation of animals are exactly followed, these would greatly limit the extent of this risk [214] . furthermore, an emphasis should be made on surveillance to detect any sign of zoonotic disease that might occur in any animal kept directly in a quarantine station in any country of origin for 30 days prior to shipment to another country to ensure no clinical sign develops during that period. in addition, the longer quarantine periods or restriction of imported animals-particularly pets (e.g., dogs, cats, rodents, and monkeys) or goats, sheep, and camels-from endemic countries may be effective in reducing the introduction of zoonotic viruses. of such measures, the control of vectors (e.g., ticks and mosquitoes), particularly the intermediate hosts and animal reservoirs, should be key components in the intervention strategy for zoonoses in saudi arabia. while the improving, enhancing, providing, and upgrading of laboratory techniques and/or testing in both veterinary and human medicines are fundamental to early detection and containing of any zoonotic disease or transmitted infection. indeed, epidemiologic evidence should be linked with the seasonal time during the year for different zoonoses, and/or with any symptoms related to zoonotic infections that occur on the mainland a few years earlier. up to date ecological factors on evolutionary issues, social movements, economic, and epidemiological mechanisms affecting zoonotic pathogens' or their persistence and emergence, are not yet well understood. however, studies on the ecological, socioeconomic, and health issues are needed to assess the sustainability and acceptability of measures by breeders, as well as information that ensures appropriate slaughtering or consumption practices, which will decrease the risk of infection to humans [200] . due to these facts about the ecological cascade and evolutionary perspectives, authorities can provide valuable insights into pathogen ecology and can inform zoonotic disease control programs; and thus evaluate their global effect in terms of actual disease and its socioeconomic correlations. enhancing biosecurity and management in the treatments of various zoonotic infections may result in appropriate use of vaccinations, drugs, and antibiotics, however, the overuse of these agents result in various types of resistance. furthermore, regardless of the influenza virus resistance level to treatment, according to a serosurveillance, the enzootic influenza virus h5n1 in egypt is endemic [87] . the same result to oseltamivir-resistant influenza viruses are reported globally, with a high susceptibility to these antiviral drugs among all reported cases of the virus from egypt. resistance was also found in most infected viral cases that are usually acquired in humans through intensive contacts, particularly with backyard birds, among women and children [82, 83, 86, 87] . therefore, drug regimens in saudi must include vaccines against this virus during hajj and umrah seasons, for egyptians. most importantly, epidemic zoonotic cases of influenza among pets has highlighted the importance of circulating influenza viruses globally, and the importance of ensuring the effective use of antivirals for the prophylaxis and treatment of influenza, especially because of the increased number of new pets stores in saudi arabia, particularly in riyadh [74, 86] . thus, studies on drug resistance are considered to be of a high public health importance, although, this might demonstrate the best scenario of how drug resistance in saudi arabia can pave its own way and/or role into the reemerging of different zoonotic pathogens. on the other hand, few studies have been done in this area to identify the relationship between different gatherings and the occurrence of signs and clinical symptoms of viral infections, especially among humans of different ages and gender. however, there are several suggestions and information regarding zoonoses (e.g., influenza and mers-cov infections) in saudi arabia among the elderly, based on age and gender [63, 222] . more recently, increased availability of limited public health data on the prevalence of some zoonotic diseases and associated risk factors or data that identifies the relationship between different zoonotic pathogen antibodies in pregnant women, are of importance [223, 224] . central to the profound worldwide changes in religious beliefs and activities is the birth of a new era of both emerging and reemerging diseases that could be arranged under the umbrella of social movements, along with its own role in the spread of zoonotic diseases. thus, any prevention and/or control strategies against any zoonotic pathogen have to take this point of view into account. furthermore, annually, saudi arabia hosts the largest international gathering of hajj where many millions gather in a small geographical area. this puts saudi arabia in the front line of threats of pandemic diseases [215] . thus, saudi arabia must keep a high level of alertness in monitoring the situation of these pathogens, particularly in view of the potential for global spread of pandemic viruses especially during winter and around the hajj season (e.g., mers-cov infections, ahfv, and influenza viruses). therefore, there is need to prevent further spread of the virus locally, regionally, and internationally. interestingly, with wnv outbreaks, the israeli-like wnv that was isolated in white storks in egypt in 1997-2000 suggests that migrating birds do play a crucial role in the geographical spread of the virus [225] . recently, the same fact was again suggested in 2017, when the same infection by this virus was introduced into turkey at the time of the outbreaks in saudi arabia and yemen; it was stated that the wnv virus might have been introduced via unlawful entry of the viremic domestic or wild animals through the borders, or by vectors carrying the virus to turkey [209] . more recently, epidemiological data of zoonotic viral pathogens from saudi arabia and/or from other neighboring countries after it was confirmed through laboratory test isolation from dromedaries (e.g., rabies, mers-cov, rvfv, and wnv) may enhance a high interest in the search for other novel zoonotic viruses in dromedaries [36, 52, 208, 211, 226, 227] . furthermore, the habits of ingestion off unpasteurized milk from camels as a rare delicacy by saudi people need to be checked. moreover, viral pathogens such as rvfv are acquired through the importation of camels, while the remaining pathogens (e.g., rabies and influenza viruses) are endemic worldwide. of these (e.g., influenza virus), there is need for a highly preventive zoonotic control in saudi, due to fact that the isolation and genetic characterization of h5n1 was reported in 2017 among vaccinated meat-turkeys flock in egypt, a neighboring country, that was previously reported to have more than 100,000 travelers to saudi arabia during hajj pilgrimage seasons, annually [219] . this might be considered as one of such important risk factor of possible introduction or spread of influenza pathogen in saudi arabia [218, 219] . lastly, increased zoonotic pathogens surveillance, particularly influenza, during the hajj season, increased infection control interventions, screening, and quarantine of suspected cases, provision of adequate medical treatment, sustainable awareness, increased education and training of target groups at high risk (e.g., doctors, nurses, veterinarians, and animal workers such as farmers and abattoir workers, etc.) are of great importance to reduce the burden of zoonoses among saudi arabian localities. fortunately, in collaboration with three organizations-including the moh in saudi arabia, the usa centers for disease control and prevention, and the who-a successful preparedness plan during the hajj season was put in place to vaccinate all pilgrims before leaving their home countries [68] . altogether, there is an urgent need for collaborative surveillance and intervention plans for the control of zoonotic pathogens in saudi arabia. with saudi arabia, the focal point of the ongoing zoonotic pathogens outbreak could be due to the large number of religious pilgrims congregating annually particularly in makkah, jeddah, and al-madinah, the main three cities for hajj and umrah, which drastically increased the potential for uncontrolled global spread of zoonotic infections [168] . a zoonotic pathogen outbreak could be dramatically decreased among the annual saudi pilgrims if we take into account the fact that: jeddah governorate, the main seaport in saudi arabia is considered to be the main entry point for over 2 million pilgrims coming for hajj or umrah annually. all these numbers of pilgrims arrive through the jeddah islamic port before going on to makkah, for the start of their umrah and/or hajj. surprisingly, the current review showed that during an outbreak, each of these eight most zoonotic viruses (rabies, mers-cov, influenza, ahfv, cchfv, rvfv, dhfv, and wnv) which occurred and/or cases confirmed in saudi arabia particularly from (jeddah and/or makkah) areas with at least one or all of these eight zoonotic viral pathogenic diseases [33, 44, 46, 78, [96] [97] [98] [99] 121, 130, 156, 171] . the spread could also have been due to the fact that jeddah is the main port for animal importation to saudi arabia. at the same time, it is the closest area to several countries where some zoonotic outbreaks were reported. to enhance this spread, the role of the active circulation of zoonotic viruses, during their natural transmission cycle, has been reported, however, an importation might increase risk of disease introduction to saudi arabia. • almost annually, from the more than 7 million pilgrims who come to makkah and madinah from different countries worldwide during hajj and umrah, the kingdom's revenue in 2012 was put at more than 62 billion saudi riyals (~about 16.5 billion us dollars), 10% up from the 2011 figures. this hajj revenue accounted for 3% of the gross domestic product for the kingdom of saudi arabia. to avert all that number of health hazards from zoonotic diseases in view of economic facts, the global community and particularly the pilgrims need more gift items made in saudi arabia to control and prevent the spread of zoonotic diseases which could be transmitted among hajj and umrah pilgrims. therefore, the following recommendations are suggested in order to improve public awareness and/or health education of zoonotic viral diseases in saudi arabia: based on findings of previous studies, health education strategies could enhance the awareness of the saudi population regarding viral zoonotic diseases through health education program experiences of other countries, particularly during hajj and umrah seasons. this response can draw on the availability of several studies on how to improve, control, and prevent the spread of several zoonoses in both animals and humans, worldwide [78, [96] [97] [98] [99] . public health authorities must highlight the importance of promoting health education and facilitate the outcomes of studies for reducing patient cases in saudi arabia. the saudi authorities and government bodies such as the moh should also launch different programs and workshops to increase public awareness about these zoonotic infections. this should involve the cooperation of the saudi regime, and the private and public sectors. different activities may be needed in saudi arabia-such as the practice of self-protection against these diseases, adult control strategies, control activities, and regular workshops-to achieve control and prevention. enhancing of self-awareness among people through health education programs or other strategies for the prevention of viral zoonotic diseases, which require vectors (such as mosquitoes, ticks, and fleas) for their transmission; are important issues on which the saudi population should be educated. they should also be educated about the adverse effects of arbitrary application of insecticides without prior knowledge on dose, resistance, and side effects. increasing the knowledge about the biology and ecology of the animal vectors in society is also crucial. furthermore, the saudi ministry of culture and information should establish intensive health education programs on television channels, radio, and newspapers to increase public awareness and to maintain hygiene conditions within the kingdom and in saudi houses. the saudi ministry of agriculture could play a big role by regularly controlling the application of vaccinations and/or antibiotics on animals which used in the veterinary sector, and also accounting the misuse of such agents following other developed and developing countries on controlling and/or accounting drug strategies [87, 228, 229] . thus, veterinary regulations of animal antibiotics-including overuse of drugs and their application-must be enforced to alleviate the serious public health problems. funding: this research received no external funding. the authors thank the dental oral rehabilitation (dor) research center at king saud university, college of dentistry, kingdom of saudi arabia, riyadh for their support. also the authors wish to appreciate all the researchers whose articles were used in the present research. the authors declare no conflict of interest. the life and work of rudolf virchow 1821-1902: cell theory, thrombosis and the sausage duel joint who/fao expert committee on zoonoses viral zoonosis: a comprehensive review zoonotic disease programs for enhancing global health security risk factors for human disease emergence anthropogenic environmental change and the emergence of infectious diseases in wildlife host range and emerging and reemerging pathogens emerging of new viral zoonoses emerging pathogens: the epidemiology and evolution of species jumps global trends in emerging infectious diseases perspectives on emerging zoonotic disease research and capacity building in canada. can emerging zoonotic diseases: can it be the case of bioterrorism zoonotic diseases and one health approach world health organization. zoonoses and veterinary public health (vph) prevalence and control of zoonotic diseases: collaboration between public health workers and veterinarians in burkina faso initial identification and characterization of an emerging zoonotic influenza virus prior to pandemic spread emerging infections: microbial threats to health in the united states. washington. in infectious disease surveillance animal-associated opportunistic infections among persons infected with the human immunodeficiency virus emerging foodborne diseases: an evolving public health challenge clinical practice and public health control zoonoses in the arabian peninsula trend of diseases among iranian pilgrims during five consecutive years based on a syndromic surveillance system in hajj reimagining the hajj travel epidemiology: the saudi perspective food poisoning in saudi arabia, potential for prevention arab news: one million animals sacrificed over three days arab news: 800 vets to oversee slaughter of over 1 m animals during hajj a puppy with toxocara canis in pets shop from saudi arabia zoonotic balantidiasis in camel from saudi arabia. sch. acad having domesticated animals at home on the rise division of vector-borne infectious disease isolation of a novel coronavirus from a man with pneumonia in saudi arabia rabies in camels at qassim region of central saudi arabia rabies in saudi arabia: a need for epidemiological data 30 years of rabies vaccination with rabipur: a summary of clinical data and global experience global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the global burden of disease study imported human rabies cases worldwide first confirmed case of human rabies in saudi arabia estimating the global burden of endemic canine rabies re-evaluating the burden of rabies in africa and asia rabies among animals in saudi arabia report on the first consultation on wildlife rabies in the arabian peninsula prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis mers coronavirus has probably been present in bats for many years, research shows middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease first confirmed case of middle east respiratory syndrome coronavirus infection in the kingdom of bahrain: in a saudi gentleman after cardiac bypass surgery middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia infection control and mers-cov in health-care workers human infection with mers coronavirus after exposure to infected camels, saudi arabia emerging viral diseases: why we need to worry about bats, camels, and airplanes evidence for zoonotic origins of middle east respiratory syndrome coronavirus identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update middle east respiratory syndrome coronavirus: a review of the current situation in the world risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia middle east respiratory syndrome coronavirus in medinah city, saudi arabia: demographic, clinical and survival data treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia the predictors of 3-and 30-day mortality in 660 mers-cov patients east respiratory syndrome coronavirus-mers-cov|dynamed middle east respiratory syndrome coronavirus: current knowledge and future considerations emerging and reemerging diseases in the world health organization (who) eastern mediterranean region-progress, challenges, and who initiatives. front. public health influenza research in the eastern mediterranean region: the current state and the way forward. influenza other respir swine influenza a (h1n1) infection in two children-southern california avian influenza a viruses: evolution and zoonotic infection influenza research in the eastern mediterranean region: a review pandemic influenza a (h1n1) in saudi arabia: description of the first one hundred cases highly pathogenic avian influenza (h5n1 hpai) spread in the middle east: risk assessment stirring up "swine flu" hysteria human infection with highly pathogenic h5n1 influenza virus epidemiology, ecology and gene pool of influenza a virus in egypt: will egypt be the epicenter of the next influenza pandemic? virulence susceptibility of influenza viruses circulating in western saudi arabia to neuraminidase inhibitors pigs, poultry, and pandemic influenza: how zoonotic pathogens threaten human health an overview of the epidemic of highly pathogenic h5n1 avian influenza virus in egypt: epidemiology and control challenges possible avian influenza (h5n1) from migratory bird characteristics of patients with oseltamivir-resistant pandemic (h1n1) 2009, united states continued emergence and changing epidemiology of oseltamivir-resistant influenza a(h1n1) 2009 virus effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data recent developments in bioinformatics analyses of influenza a virus surface glycoproteins and their biological relevance dual resistance to adamantanes and oseltamivir among seasonal influenza a (h1n1) viruses: 2008-2010 a comprehensive review of common bacterial, parasitic and viral zoonoses at the human-animal interface in egypt demographic distribution and transmission potential of influenza a and 2009 pandemic influenza a h1n1 in pilgrims respiratory viruses and bacteria among pilgrims during the acceptance of h1n1 vaccine among healthcare workers at primary healthcare centers in abha avian flu hits saudi arabia with two new cases transmission of avian influenza virus (h3n2) to dogs acute bronchointerstitial pneumonia in two indoor cats exposed to the h1n1 influenza virus influenza a pandemic (h1n1) 2009 virus infection in domestic cat lung infection and severe anemia secondary to balantidiasis in hamadryas baboons isolation of a flavivirus related to the tick-borne encephalitis complex from human cases in saudi arabia alkhumra, not alkhurma, is the correct name of the new hemorrhagic fever flavivirus identified in saudi arabia propagation and titration of alkhumra hemorrhagic fever virus in the brains of newborn wistar rats virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses alkhurma hemorrhagic fever virus alkhumra virus infection, a new viral hemorrhagic fever in saudi arabia alkhurma hemorrhagic fever virus in ornithodoros savignyi ticks kyasanur forest disease virus alkhurma subtype in ticks, najran province, saudi arabia the alkhurma virus (family flaviviridae, genus flavivirus): an emerging pathogen responsible for hemorrhage fever in the middle east complete coding sequence of the alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in saudi arabia low diversity of alkhurma hemorrhagic fever virus, saudi arabia is the epidemiology of alkhurma hemorrhagic fever changing: a three-year overview in saudi arabia kyasanur forest disease virus infection in mice is associated with higher morbidity and mortality than infection with the closely related alkhurma hemorrhagic fever virus a preliminary report on cchf in turkey world health organization. crimean-congo haemorrhagic fever crimean-congo haemorrhagic fever: an overview antigenic similarity between the virus causing crimean hemorrhagic fever and congo virus recent progress in the treatment of crimean-congo hemorrhagic fever and future perspectives crimean-congo hemorrhagic fever: history, epidemiology, pathogenesis, clinical syndrome and genetic diversity crimean-congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase ski-1 crimean-congo hemorrhagic fever virus fields virology results of a preliminary seroepidemiological survey for crimean-congo hemorrhagic fever virus in egypt crimean-congo haemorrhagic fever virus infection in the western province of saudi arabia detection of crimean-congo haemorrhagic fever virus antibodies in humans and imported livestock in saudi arabia outbreak of crimean-congo haemorrhagic fever in pakistan climate niches of tick species in the mediterranean region: modeling of occurrence data, distributional constraints, and impact of climate change seroprevalence of alkhurma and other hemorrhagic fever viruses, saudi arabia annual report department of agriculture enzootic hepatitis or rift valley fever. an undescribed virus disease of sheep cattle and man from east africa has rift valley fever virus evolved with increasing severity in human populations in east africa? ecology and habitat characterization of mosquitoes in saudi arabia interactions between mosquito larvae and species that share the same trophic level species interactions among larval mosquitoes: context dependence across habitat gradients distribution and ecology of the mosquito fauna in the southwestern saudi arabia prevalence and distribution of anopheline mosquitoes in malaria endemic areas of asir region, saudi arabia studies of identification and population dynamics of anopheline mosquitoes from jeddah province of saudi arabia mosquito fauna (diptera: culicidae) and seasonal activity in makka al mukarramah region, saudi arabia distribution and seasonal activity of mosquitoes in al madinah al munwwrah, saudi arabia prevalence and seasonal distribution of dengue mosquito aedes aegypti (diptera: culicidae) in al-madinah al-munawara, saudi arabia breeding habitats characterization of anopheles mosquito (diptera: culicidae) in najran province, saudi arabia larval habitat, ecology, seasonal abundance and vectorial role in malaria transmission of anopheles arabiensis in jazan region of saudi arabia household survey of containerbreeding mosquitoes and climatic factors influencing the prevalence of aedes aegypti (diptera: culicidae) in makkah city, saudi arabia. asian pac sindbis virus isolations from saudi arabian mosquitoes mosquito vectors survey in the al-ahsaa district of eastern saudi arabia seasonal activity of some haematophagous insects in the riyadh region, saudi arabia distribution of habitats of mosquito larvae (diptera: culicidae) in riyadh region, saudi arabia the mosquitoes of arabia vector bionomics in the epidemiology and control of malaria, part i, the who african region and the southern who eastern mediterranean region, section iii: south-western arabia isolation and genetic characterization of rift valley fever virus from aedes vexans arabiensis, kingdom of saudi arabia integrated vector management: strategic framework for the eastern. the mediterranean region 2004-2010; the who regional office for the eastern mediterranean: cairo rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention population genetics of two key mosquito vectors of rift valley fever virus reveals new insights into the changing disease outbreak patterns in kenya climatic and geographic influences on arboviral infections and vectors observations on the epidemiology of rift valley fever in kenya isolation of arboviruses in kenya rift valley fever virus activity in east africa in 1989 rift valley fever in southwestern saudi arabia: a seroepidemiological study seven years after the outbreak of complete genome analysis of 33 ecologically and biologically diverse rift valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry genetic analysis of viruses associated with emergence of rift valley fever in saudi arabia and yemen rift valley fever: an uninvited zoonosis in the arabian peninsula relevance of rift valley fever to public health in the european union camelid infectious disorders world health organization hemorrhagic fever task force. an outbreak of rift valley fever in northeastern kenya, 1997-98 the 2000 epidemic of rift valley fever in saudi arabia: mosquito vector studies emergence of rift valley fever genetic reassortment of rift valley fever in nature first record of aedes (stegomyia) unilineatus in the kingdom of saudi arabia a review of mosquitoes associated with rift valley fever virus in madagascar rift valley fever in kenya: history of epizootics and identification of vulnerable districts spatial and temporal pattern of rift valley fever outbreaks in tanzania camel milk-associated infection risk perception and knowledge in french hajj pilgrims. vector borne zoonotic dis first field evidence for natural vertical transmission of west nile virus in culex univittatus complex mosquitoes from rift valley province rift valley fever and a new paradigm of research and development for zoonotic disease control the epidemiology of dengue fever in saudi arabia: a systematic review aedes albopictus, an arbovirus vector: from the darkness to the light the global distribution and burden of dengue antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera dengue hemorrhagic fever virus in saudi arabia: a review. vect. borne zoonotic dis update on the global spread of dengue virologic and serologic surveillance for dengue fever in jeddah, saudi arabia, 1994-1999 clinical profile and outcome of hospitalized patients during first outbreak of dengue in makkah, saudi arabia department of communicable diseases, ministry of health (moh) saudi arabia ministry of health: riyadh, saudi arabia phylogeny of dengue viruses circulating in jeddah reconstructing historical changes in the force of infection of dengue fever in singapore: implications for surveillance and control dengue fever clinical features of 567 consecutive patients admitted to a tertiary care center in saudi arabia characteristics of dengue fever in a large public hospital factors associated with the spread of dengue fever in jeddah governorate, saudi arabia. east mediterr geographical distribution and spatiotemporal patterns of dengue cases in jeddah governorate from an update on the incidence of dengue gaining strength in saudi arabia and current control approaches for its vector mosquito clinico-epidemiological features of dengue fever in saudi arabia. asian pac dengue fever in makkah, kingdom of saudi arabia molecular and seroprevalence of imported dengue virus infection in al-madinah, saudi arabia seroprevalence of dengue virus infection in aseer and jizan regions, southwestern saudi arabia fever cases in jeddah drop by 38%-saudi gazette west nile virus review of west nile virus circulation and outbreak risk in madagascar: entomological and ornithological perspectives a neurotropic virus isolated from the blood of a native of uganda isolation of west nile virus in israel origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states nile encephalitis in israel, 1999: the new york connection west nile virus surveillance in romania le virus du nil occidental culex pipiens: species versus species complex taxonomic history and perspective incidence of west nile virus in al-ahsa, saudi arabia incidence history of west nile virus in africa and middle east spread of west nile virus in iran: a cross-sectional serosurvey in equines seroprevalence of west nile virus in blood donors at hotel dieu de france west nile virus infection in horses in jordan: clinical cases, seroprevalence and risk factors first isolation of west nile virus from a dromedary camel the first serological evidence for rift valley fever infection in the camel, goitered gazelle and anatolian water buffaloes in turkey an outbreak of dermatophytosis in camels (camelus dromedaríus) at qassim region, central of saudi arabia a survey of rift valley fever and associated risk factors among the one-humped camel (camelus dromedaries) in sudan. irish an epizootic of rift valley fever in egypt in 1997 genetic evidence for rift valley fever outbreaks in madagascar resulting from virus introductions from the east african mainland rather than enzootic maintenance the role of the world organisation for animal health (oie) to facilitate the international trade in animals and animal products public health pandemic h1n1 of the 2009 hajj spatio-temporal pattern of sylvatic rabies in the sultanate of oman introduction and enzootic of a/h5n1 in egypt: virus evolution, pathogenicity and vaccine efficacy ten years on isolation and genetic characterization of a novel 2.2.1.2a h5n1 virus from a vaccinated meat-turkeys flock in egypt pandemic (h1n1) 2009 and hajj pilgrims who received predeparture vaccination rabies. cdc health information for international travel balantidium coli infection in hamadryas baboon (papio hymadryas) in saudi arabia: a case report middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission hepatitis c virus antibody titers associated with cognitive dysfunction in an asymptomatic community-based sample report on influenza a and b viruses: their coinfection in a saudi leukemia patient the role of birds in the ecology of west nile virus in europe and africa phylogenetically distinct middle east respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in dubai with rising seroprevalence with age seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt challenges of drug resistance in the developing world the threat of antimicrobial resistance in developing countries: causes and control strategies key: cord-279557-hk77e3pp authors: drosten, christian; seilmaier, michael; corman, victor m; hartmann, wulf; scheible, gregor; sack, stefan; guggemos, wolfgang; kallies, rene; muth, doreen; junglen, sandra; müller, marcel a; haas, walter; guberina, hana; röhnisch, tim; schmid-wendtner, monika; aldabbagh, souhaib; dittmer, ulf; gold, hermann; graf, petra; bonin, frank; rambaut, andrew; wendtner, clemens-martin title: clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection date: 2013-06-17 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70154-3 sha: doc_id: 279557 cord_uid: hk77e3pp background: the middle east respiratory syndrome coronavirus (mers-cov) is an emerging virus involved in cases and case clusters of severe acute respiratory infection in the arabian peninsula, tunisia, morocco, france, italy, germany, and the uk. we provide a full description of a fatal case of mers-cov infection and associated phylogenetic analyses. methods: we report data for a patient who was admitted to the klinikum schwabing (munich, germany) for severe acute respiratory infection. we did diagnostic rt-pcr and indirect immunofluorescence. from time of diagnosis, respiratory, faecal, and urine samples were obtained for virus quantification. we constructed a maximum likelihood tree of the five available complete mers-cov genomes. findings: a 73-year-old man from abu dhabi, united arab emirates, was transferred to klinikum schwabing on march 19, 2013, on day 11 of illness. he had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment. the patient died on day 18, due to septic shock. mers-cov was detected in two samples of bronchoalveolar fluid. viral loads were highest in samples from the lower respiratory tract (up to 1·2 × 10(6) copies per ml). maximum virus concentration in urine samples was 2691 rna copies per ml on day 13; the virus was not present in the urine after renal failure on day 14. stool samples obtained on days 12 and 16 contained the virus, with up to 1031 rna copies per g (close to the lowest detection limit of the assay). one of two oronasal swabs obtained on day 16 were positive, but yielded little viral rna (5370 copies per ml). no virus was detected in blood. the full virus genome was combined with four other available full genome sequences in a maximum likelihood phylogeny, correlating branch lengths with dates of isolation. the time of the common ancestor was halfway through 2011. addition of novel genome data from an unlinked case treated 6 months previously in essen, germany, showed a clustering of viruses derived from qatar and the united arab emirates. interpretation: we have provided the first complete viral load profile in a case of mers-cov infection. mers-cov might have shedding patterns that are different from those of severe acute respiratory syndrome and so might need alternative diagnostic approaches. funding: european union; german centre for infection research; german research council; and german ministry for education and research. in june, 2012, a coronavirus belonging to a group of viruses that had previously only been detected in bats was cultured from respiratory secretions of a patient who had died from severe acute respiratory infection. 1 the same agent was retrospectively detected in clinical samples from a hospital outbreak of severe acute respiratory infection that occurred in jordan in april, 2012, marking the fi rst known occurrence of the virus in people. 2 the agent has been named middle east respiratory syndrome coronavirus (mers-cov). 3 as of june 10, 2013, 55 laboratory-confi rmed cases had been reported in jordan, saudi arabia, the uk, france, italy, germany, and tunisia. 3 31 individuals with laboratory-confi rmed infection had died. few virological data have become available for mers-cov cases, and there is no information about the viral genome sequence, which could identify important epidemiological characteristics. [4] [5] [6] here, we provide a full description of a fatal case of mers-cov infection imported to munich, germany, from abu dhabi, including a chronological profi le of virus concentrations in diverse body compartments. we fully sequenced the mers-cov genome, and therefore could do a chronologically calibrated phylogenetic analysis with all available mers-cov genome sequences. these data were complemented by novel sequence data from an unlinked case treated in germany in 2012. 7, 8 we report data for a patient who was admitted to the klinikum schwabing (munich, germany) in march, 2013. investigation was done as part of a public health intervention according to the german infection protection act. written consent for scientifi c assessment was obtained from the patient's spouse as part of the patient treatment contract. we did diagnostic rt-pcr and indirect immunofl uorescence, following who recom mendations. 7, 9 for serum neutralisation tests, we grew vero b4 cells to subconfl uence in 24-well plates. pre-incubation reactions contained 25 plaque-forming units of mers-cov (emc strain) in 100 μl of medium, mixed one-to-one with serum samples from the patient prediluted in medium. the starting dilution was a tenth. after 1 h incubation at 37°c, each well was infected for 1 h at 37°c with the total 200 μl pre-incubation reaction. supernatants were removed and overlaid with avicell resin as described by herzog and colleagues. 10 assays were terminated and stained after 3 days. we defi ned neutralisation titres as the serum dilution reducing the number of plaques in four parallel wells in summary by greater than 90%. antibodies were tested by immunofl uorescence assay. 7 all clinical materials stored in the ward and laboratories were gathered and submitted for virological diagnostic tests. from the time of laboratory diagnosis, respiratory, faecal, and urine samples were obtained. we designed two diff erent sets of primers generating overlapping amplicons (available on request). the fi rst set consisted of 70 amplicons, 386-800 bp in length, with all primers containing two strong watson-crick bps at their 3 ends, so as to bind the template with high affi nity. the second set consisted of 68 amplicons, 415-761 bp in length, with primers that had no more than two strong bps in their fi ve 3 terminal nucleotides and no strong pairings in the two 3 positions. this method of primer design can decrease sensitivity, but it prevents mispriming within the product, which can improve the success of amplifi cation. after rt-pcr, we sequenced all fragments on a roche 454 junior instrument (roche, penzberg, germany) and assembled in geneious (version 6.1.2). virus quantifi cation was done with standard calibration curves that were based on quantifi ed in-vitro transcribed rna for the upe target gene. 9 we constructed a maximum likelihood tree of the fi ve available complete mers-cov genomes with phyml 11 and the gtr+gamma model of molecular evolution; we assessed phylogenetic support with 1000 bootstrap replicates. we inferred a timescale by linear regression of genetic divergence from the root against time of collection of the samples. the root was placed such that the correlation coeffi cient was maximised. a phylogenetic tree based on all available mers-cov sequences was calculated with phyml 11 on a concatenated 4012 bp dataset with the hky substitution model. reduction of the dataset was determined by the small number of sequence fragments that could be retrieved from a stored clinical sample containing a small amount of the virus, derived from a patient treated in essen, germany. the sponsors of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and fi nal responsibility to submit for publication. on march 8 (day 0), the patient-a 73-year-old man from abu dhabi, united arab emirates-abruptly developed fl u-like symptoms, with fever and non-productive cough. he was admitted to mafraq hospital (abu dhabi) on day 2 (fi gure 1), and was diagnosed with pneumonia. he was intubated on day 9 because of progressive hypoxia and acute respiratory distress syndrome (fraction of inspired oxygen 60%; positive end-expiratory pressure 10 cm h 2 o). the patient had received intensive antimicrobial treatment with meropenem, levofl oxacin, vancomycin, caspofungin, aciclovir, and oseltamivir during his stay in an intensive care unit in abu dhabi, without major improvement in his pulmonary function. the patient was transferred to klinikum schwabing (munich, germany) on march 19, 2013 (fi gure 1). the patient had been diagnosed with multiple myeloma in 2008, and had received several lines of treatment in the previous few years, such as high-dose chemotherapy with autologous stem-cell transplantation in 2009. at relapse of his multiple myeloma in november, 2012, he was given lenalidomide plus dexamethasone. relatives reported that the patient owned camels, and had taken care of a diseased animal shortly before onset of symptoms. no animal samples, or further details about potential sources or exposures could be retrieved. during his stay in munich, we recorded thrombocytopenia (table 1) . thrombocytopenia was also reported in the fi rst described case of mers-cov infection, 1 in two of four patients from a family cluster in saudi arabia, 12 and in the two cases reported from france. 5 the patient developed renal insuffi ciency on day 14, and required dialysis. despite continuous invasive ventilation and antibiotic treatment, the patient's health status deteriorated. death occurred on day 18 and was due to septic shock, with signs of haemolysis and acute coagulation disorder (fi gure 1, table 1). after hospital admission in munich, infection with mers-cov was suspected on the basis of treatment-refractory acute respiratory distress syndrome, combined with the geographical origin of the patient. bronchoalveolar fl uid was obtained on march 20 and 22 (days 12 and 14). mers-cov was detected in both samples by rt-pcr. we also detected herpes simplex virus type 1 dna 13 (6·4 × 10⁴ to 1·9 × 10⁷ copies per ml) and rhinovirus rna 14 (3·7 × 10⁵ to 2·1 × 10⁹ copies per ml) by (rt-)pcr in both samples. mers-cov rna concentrations in respiratory samples ranged from 933 to 1·2 × 10⁶ genome copies per ml. virus concentrations seemed to be higher in samples taken earlier in the course than in those obtained later (fi gure 2). concentrations were more variable in tracheobronchial samples than in bronchoalveolar lavage samples (fi gure 2), which was ascribed to variation in volumes of saline solution applied during removal of tracheobronchial samples. notably, suction catheters without opening at point of care and stored for as long as 5 days at 8°c in a refrigerator in the intensive care unit tested consistently positive but yielded up to roughly 3·5log 10 lower rna concentrations than did those in fresh tracheobronchial aspirates taken on the same days (fi gure 2). immunofl uorescence assays yielded endpoint titres on day 16 of infection (table 2 ). an igm-specifi c immunofl uorescence assay confi rmed recent infection in the same serum sample (table 2) . plaque-reduction neutralisation test confi rmed mers-cov specifi city of detected antibody titres (table 2) . these titres were somewhat lower than those recorded for serum samples from an unlinked non-fatal case of mers-cov treated in germany in 2012. 8 serum samples from this patient had been taken later than they were for our patient (table 2) . we tested two urine samples on day 12, one on day 13, and one on day 16. one of the two samples on day 12, and the sample from day 13 were positive, meaning that the virus was not present in urine after renal failure (day 14), with a maximum virus concentration of 2691 rna copies per ml on day 13. both stool samples obtained on day 12 and the fi ve on day 16 were positive, with up to 1031 rna copies per g, which is a concentration close to the lowest detection limit of the assay. we recorded a low virus concentration in one of two oronasal aspirate samples taken from the intubated patient on day 16 (5370 copies per ml). one dialysate sample and two serum samples on day 16 , and one serum sample on day 18 were negative. although we obtained several isolates for the herpes simplex virus type 1, repeated attempts to isolate mers-cov were unsuccessful. herpes simplex virus is a frequent bystander infection in intubated patients, and is known to not aff ect the cardiorespiratory prognosis and outcome. 15 we sequenced the full mers-cov genome directly from respiratory samples (genbank accession number kf192507). we subjected all available mers-cov genome sequences to phylogenetic analysis, including a correlation and regression analysis of known dates of virus isolation versus tree branch lengths (fi gure 3). we estimated the rate of evolution as 1·6 × 10 -³ substitutions per site per year. the time of the common ancestor of all fi ve viruses for which genomes are available was halfway through 2011 (fi gure 3). the virus in our patient clustered with a sequence from a virus imported into the uk from qatar. 16 to compare this sequence with that of another virus from the same region, we reanalysed a stored clinical sample from another case of mers-cov infection imported into germany in october, 2012. this sample contained low concentrations of rna, so the genome of the virus had not been successfully sequenced previously. 8 after many attempts to recover rt-pcr fragments from the available bronchoalveolar lavage sample, we could sequence 12 fragments, covering 4012 nucleotides of the mers-cov genome (genbank accession number kc875821). a concatenated alignment of homologous sequence portions of all available mers-cov sequences was subjected to phylogenetic analyses, confi rming a clustering of sequences from qatar and the united arab emirates (fi gure 3). a sequence from a patient with a history of travel to pakistan and saudi arabia branched next to this cluster. during and up to 10 days after the course of treatment, 14 health-care workers who had direct contact with our patient or patient-derived materials reported mild respiratory symptoms. samples were taken from the upper respiratory tract and tested by two diff erent rt-pcr assays for mers-cov. none yielded positive results. by contrast, one patient who had had direct contact with the patient with mers-cov was infected with hcov-nl63, a common human coronavirus, and four patients were infected with rhinoviruses. these rhinoviruses were not all mutually related, and none was related to the rhinovirus detected in the patient with mers-cov (appendix). follow-up of all contact patients, including investigation for subclinical infections, is in progress. we have outlined the chronological follow-up of a patient with mers-cov, in which we used quantitative virological diagnostic tests (panel). viral loads were highest in the lower-respiratory tract. the viral sequence from this patient clustered with sequences from nearby qatar. laboratory data are crucial for diagnostic recommendations, to make projections about prognosis, and to estimate infection risks. without quantitative laboratory data from well documented cases of mers-cov infection, most considerations had been made on the basis of an assumed analogy to severe acute respiratory syndrome (sars). 12, [18] [19] [20] however, elementary traits of the virus, such as its receptor usage and sensitivity to type i and type iii interferon, diff er substantially from that of the sars coronavirus, suggesting that diff erences in disease patterns (eg, in organ tropism or in virus shedding) might exist. [21] [22] [23] [24] [25] we focused on these aspects with quantitative virus testing in all relevant body compartments, including viral loads in non-respiratory samples. 5, 12 however, our patient-like most other cases anecdotally reported so far-had an underlying disease that could aff ect virus shedding patterns. only analysis of a large number of patients can yield general fi gures about qualitative virus data. faecal shedding was of particular interest, because patients with sars regularly showed high virus concentrations and prolonged virus excretion in stools that led to the use of stool samples, even for routine sars diagnostic tests. 18, 20, 26 diarrhoea was reported in two descriptions of mers-cov clusters, and it was speculated that faecal virus shedding might have occurred. 5, 12 however, no laboratory data for virus in stool samples were provided. our patient had low faecal virus concentrations that were close to the lowest detection limit on days 12 and 16 of illness. in the only other description so far, one stool sample from a patient with mers-cov had a negative result. 16 stool samples from many patients, including those with early stages of disease, should be tested to assess whether faecal sources could have a role in transmission, or whether mers-cov diff ers from sars in this aspect. another important fi nding was that we recorded low concentrations of virus in urine samples. this fi nding is surprising, because early kidney failure during the course of mers-cov infection has been reported, and kidney cells in laboratory models are highly permissive for mers-cov replication. 5, 12, 23 the fact that the virus was present in urine but not in the blood suggests autonomous virus replication in the kidneys, potentially without active secretion of virus into the urine. however, renal failure due to specifi c viral infection or immunopathogenesis is not necessarily indicated, because the patient had received several doses of potentially nephrotoxic antimicrobial agents in a setting of underlying multiple myeloma. post-mortem examinations are urgently needed to clarify whether kidney failure in mers-cov infection is a primary and preventable result of viral infection, or a secondary complication of severe systemic disease. 27, 28 quantitative virus data are needed to orient diagnostics and hospital infection control measures. the recorded viral load profi le, with highest rna concentrations in bronchoalveolar lavage and tracheobronchial aspirates, confi rms suggestions made in another report about the preferential use of lower-respiratory-tract samples for virus diagnostic tests. 5 notably, the reported overall stability of detectable virus rna in closed suction catheters indicates a straightforward and non-contagious way to collect diagnostic samples even from non-intubated patients. oronasal swabs should not be preferentially submitted for testing, especially in patients presenting late in their disease course with substantial lower respiratory involvement. 5 our data for stool, urine, and blood samples suggest a fairly low infection risk during non-respiratory care procedures. the absence of detectable virus in blood matches reports made in an earlier case of mers-cov infection. 16 however, guery and colleagues 5 reported low semi-quantitative virus measurements in the blood of one of their patients. moreover, initial experimental studies suggested that mers-cov infected vascular endothelial cells. 25 however, quantitative data suggest a low risk from general laboratory procedures involving blood. by contrast, the low virus concentrations and failure to isolate infectious virus from respiratory secretions should not be taken as a general indication of airway-associated infection risks. virological monitoring of the patient started only late in the disease course, at a time when the infectiousness of the virus could already have been reduced (as suggested by the occurrence of neutralising antibodies). the fact that we could not isolate mers-cov could have been due to the concomitant presence of herpes simplex virus type 1, which overgrew some of the diagnostic cell cultures. we searched pubmed for reports published in english at any time before june 7, 2013. we used the search term "mers-cov or hcov-emc". we identifi ed 29 reports linked to middle east respiratory syndrome coronavirus (mers-cov), starting with zaki and colleagues' initial report, 1 in which a previously unknown coronavirus isolated from the sputum of a 60-year-old man is described. when we used the search term "mers-cov", we identifi ed four reports, 2,5,12,17 none of which provided quantitative viral load profi les of infected patients. our report provides the fi rst complete viral load profi le in a case of mers-cov infection. the distribution of viral loads in the respiratory tract suggests lower-respiratory-tract samples should be taken preferentially. low concentrations of the virus in stool, urine, and blood samples suggests little virus excretion-at least in our patient-from body compartments other than the respiratory tract. furthermore, coronaviruses that infect people are generally diffi cult to isolate, particularly in late phases of disease. only two successful isolations of mers-cov have been reported worldwide so far. 1, 5 these isolations were done on day 4 of disease, 5 and day 7 of disease. 1 our samples were taken on days 12 and 14, and the sample from day 12 was stored for 3 days before cell culture. isolation success cannot provide any information about infectiousness of the patient. even a highly concentrated rhinovirus does not seem to have been transmitted from the patient, suggesting that eff ective protective measures were in place during treatment of the intubated patient in the intensive care unit. the sequence data from this and another patient treated in germany enable an extended analysis of phylogeny, hinting at a geographical structure of the mers-cov tree. specifi cally, viral sequences from the eastern part of the arabian peninsula cluster together and stem from one common ancestor whose date of existence is projected to be after that of viruses from jeddah and jordan. date estimates will probably be refi ned when more sequences become available. moreover, whether the reported geographical structure represents repeated transfer from a geographically structured viral reservoir population and limiting chains of person-to-person transmission or multiple sustained lineages of human infections is unclear. with only fi ve complete genome sequences available as yet, genetic data are urgently needed to establish the spatial and temporal distribution of cases, estimate the number of independent human chains of transmission, and thus better assess the threat that mers-cov poses to world health. four sequences from a continuing hospital outbreak in al-hasa, saudi arabia, have now been deposited in genbank. preliminary phylogenetic analyses confi rm the clustering of viruses from the eastern part of the arabian peninsula (qatar: strains england2 and essen; abu dhabi: strain munich; al-hasa: genbank accession numbers kf186564-kf186567). cd designed the virological studies, phylogenetic analysis, and virological data analysis. ms, whar, gs, ss, wg, hgu, tr, ms-w, fb, and c-mw contributed to clinical data generation and analysis. ms, vmc, rk, dm, sj, and ar contributed to fi gures. vmc, rk, dm, sj, mam, sa, and ud generated and analysed virological data. ar did phylogenetic analysis. cd, whaa, hgo, and c-mw interpreted data. cd, ms, vmc, fb, ar, and c-mw wrote the report. whaa advised the public health intervention. hgo and pg coordinated the public health intervention. we declare that we have no confl icts of interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov)-update severe respiratory illness caused by a novel coronavirus clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections contact investigation of a case of human novel coronavirus infection treated in a german hospital detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction plaque assay for human coronavirus nl63 using human colon carcinoma cells a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood family cluster of middle east respiratory syndrome coronavirus infections detection of herpes simplex virus dna by real-time pcr real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses respiratory herpes simplex virus type 1 infection/colonisation in the critically ill: marker or mediator? severe respiratory illness caused by a novel coronavirus genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus the severe acute respiratory syndrome the aetiology, origins, and diagnosis of severe acute respiratory syndrome identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome effi cient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures severe acute respiratory syndrome multiple organ infection and the pathogenesis of sars acute renal impairment in coronavirus-associated severe acute respiratory syndrome this work was supported by a european research project on emerging diseases detection and response (emperie; contract no 223498). cd has received infrastructural support from the german centre for infection research, which included full funding of the position of vc. virological analyses were partly support by the german ministry for key: cord-291199-nazl2e97 authors: kleine-weber, hannah; elzayat, mahmoud tarek; wang, lingshu; graham, barney s.; müller, marcel a.; drosten, christian; pöhlmann, stefan; hoffmann, markus title: mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization date: 2018-11-07 journal: journal of virology doi: 10.1128/jvi.01381-18 sha: doc_id: 291199 cord_uid: nazl2e97 middle east respiratory syndrome coronavirus (mers-cov) poses a threat to public health. the virus is endemic in the middle east but can be transmitted to other countries by travel activity. the introduction of mers-cov into the republic of korea by an infected traveler resulted in a hospital outbreak of mers that entailed 186 cases and 38 deaths. the mers-cov spike (s) protein binds to the cellular protein dpp4 via its receptor binding domain (rbd) and mediates viral entry into target cells. during the mers outbreak in korea, emergence and spread of viral variants that harbored mutations in the rbd, d510g and i529t, was observed. counterintuitively, these mutations were found to reduce dpp4 binding and viral entry into target cells. in this study, we investigated whether they also exerted proviral effects. we confirm that changes d510g and i529t reduce s protein binding to dpp4 but show that this reduction only translates into diminished viral entry when expression of dpp4 on target cells is low. neither mutation modulated s protein binding to sialic acids, s protein activation by host cell proteases, or inhibition of s protein-driven entry by interferon-induced transmembrane proteins. in contrast, changes d510g and i529t increased resistance of s protein-driven entry to neutralization by monoclonal antibodies and sera from mers patients. these findings indicate that mers-cov variants with reduced neutralization sensitivity were transmitted during the korean outbreak and that the responsible mutations were compatible with robust infection of cells expressing high levels of dpp4. importance mers-cov has pandemic potential, and it is important to identify mutations in viral proteins that might augment viral spread. in the course of a large hospital outbreak of mers in the republic of korea in 2015, the spread of a viral variant that contained mutations in the viral spike protein was observed. these mutations were found to reduce receptor binding and viral infectivity. however, it remained unclear whether they also exerted proviral effects. we demonstrate that these mutations reduce sensitivity to antibody-mediated neutralization and are compatible with robust infection of target cells expressing large amounts of the viral receptor dpp4. mild respiratory disease. in addition, the betacoronaviruses severe acute respiratory syndrome (sars)-and middle east respiratory syndrome (mers)-cov can be zoonotically transmitted from animals to humans (1) . camels serve as a natural reservoir for mers-cov, and infected animals may exhibit mild respiratory symptoms (2, 3) . in contrast, transmission of mers-cov to humans induces fatal disease in about 36% of the afflicted patients (4) . most mers cases have been documented in the middle east, but the virus has been introduced into several other countries due to international travel. at present, human-to-human transmission of mers-cov is inefficient. however, massive mers outbreaks have been observed in hospital settings (5) . for instance, the introduction of mers-cov into the republic of korea by a single infected traveler in 2015 resulted in 186 infections, including secondary, tertiary, and quaternary cases, and 38 deaths (6, 7) . whether the virus responsible for the korean outbreak harbored mutations that promoted human to human spread is incompletely understood. the infectious entry of mers-cov into target cells is mediated by the viral spike glycoprotein (mers-s), which is incorporated into the viral envelope. mers-s contains a surface unit, s1, and a transmembrane unit, s2. the s1 subunit binds to the main receptor, dpp4/cd26 (8) , and the secondary receptor, sialic acids (9) , while the s2 subunit facilitates fusion of the viral envelope with a cellular membrane. membrane fusion depends on prior proteolytic cleavage (activation) of the inactive s protein precursor, s0, by host cell proteases. specifically, the endosomal cysteine protease cathepsin l (catl) and the type ii transmembrane serine protease, tmprss2, located at the plasma membrane can activate mers-s (10) (11) (12) . sequence analysis revealed that mers-cov variants observed during the korean outbreak contained polymorphisms d510g and i529t and that the respective viral variants were transmitted to other patients (13, 14) . the d510g and i529t polymorphisms are located in the receptor binding domain (rbd) (fig. 1) , a portion of s1 that is required for binding to dpp4, and one can speculate that they might increase viral fitness and/or transmissibility. however, counterintuitively, both d510g and i529t were shown to decrease binding to dpp4 and i529t was shown to decrease mers-s-driven entry into cells (14) , and it is at present unknown whether the virus benefits from these changes. here we show that several parameters controlling efficiency of mers-s-driven entry, including sialic acid engagement and blockade by interferon (ifn)-induced transmembrane proteins (ifitms), are not modulated by d510g and i529t. moreover, we confirm that d510g and i529t reduce dpp4 binding but show that this translates into reduced entry only when small amounts of dpp4 are expressed. finally, we demonstrate that d510g and i529t reduce sensitivity to antibody-mediated neutralization, suggesting that these mutations might not alter viral transmissibility but might increase viral spread in infected patients in the presence of an antibody response. d510g and i529t do not alter sialic acid dependency of viral entry. a recent study showed that mers-s can bind to sialic acids, glycans presented on plasma membrane proteins and lipids, and that sialic acid engagement promotes s proteindriven entry (9) . we enzymatically removed sialic acids by neuraminidase (na) treatment to investigate whether the korean polymorphisms or the polymorphisms found in the middle east impact sialic acid dependence. neuraminidase treatment markedly reduced influenza a virus hemagglutinin (ha)-driven entry but had no effect on entry driven by vsv-g (fig. 4) , as expected. moreover, entry driven by mers-s wt and s protein variants was comparably inhibited by neuraminidase treatment (fig. 4) , indicating that none of the amino acid changes in mers-s altered sialic acid dependence of viral entry. d510g and i529t do not alter the choice of mers-s activating host cell protease. catl and tmprss2 activate mers-s in cell culture (10, 24) , and the activity of tmprss2 or related enzymes is required for sars-cov spread in the host (26) . therefore, we investigated whether the polymorphisms under study modulated the capacity of mers-s to employ catl and tmprss2 for activation. for this, we first employed 293t cells, which express endogenous catl but not tmprss2 and allow mers-s-driven entry in a catl-dependent fashion (10, 24) . the cells were transfected with dpp4 plasmid and either tmprss2 encoding or empty plasmid (control), incubated with the catl inhibitor mdl28170, and then analyzed for s protein-driven transduction. incubation of control cells with mdl28170 reduced entry driven by mers-s wt and s protein variants, and this effect was fully rescued by directed expression of tmprss2 (fig. 5a ). next, we fig 2 all mers-s variants analyzed were robustly expressed and incorporated into rhabdoviral particles. (a, top) expression plasmids for the indicated s proteins or empty plasmid (control) were transfected into 293t cells. whole-cell lysates (wcl) were prepared from transfected cells, and s protein expression was analyzed by sds-page and immunoblotting using anti-v5 antibody reactive against the v5 epitope at the c terminus of the s proteins. detection of ␤-actin (actb) served as a negative control. similar results were obtained in three separate experiments. (b, top) expression plasmids for the indicated s proteins or empty plasmid (control) were transfected into 293t cells and the cells were then used to produce rhabdoviral particles. the particles were subsequently pelleted by centrifugation through a sucrose cushion and analyzed for s protein expression by sds-page and immunoblotting. detection of vesicular stomatitis virus matrix protein (vsv-m) served as a loading control. (a and b, bottom) immunoblots conducted for panels a (n ϭ 3) and b (n ϭ 6) were subjected to quantitative analysis using imagej software. s protein signals were normalized against the corresponding signals for actb or vsv-m, and expression and particle incorporation, respectively, of mers-s wt were set as 100%. for s proteins that yielded two bands (s0 and s2), the two signals were combined before normalization. mean values are shown; error bars indicate sems. statistical significance of differences in particle expression or incorporation efficiency between mers-s wt and variants was analyzed by paired two-tailed student t tests. investigated s protein activation in caco-2 cells, which express endogenous tmprss2 (27) and afforded the opportunity to visualize a potentially more subtle impact of the s protein polymorphisms on activation by tmprss2. however, incubation of caco-2 cells with rising concentrations of camostat mesylate, a tmprss2 inhibitor, comparably reduced entry driven by mers-s wt and s protein variants and had no effect on vsv-g-mediated entry (fig. 5b) , as expected. these findings suggest that the polymorphisms under study did not compromise s protein activation by catl and tmprss2 and did not endow the s protein with the ability to use low levels of tmprss2 activity with increased efficiency for s protein activation. d510g and i529t do not modulate ifitm sensitivity. expression of ifitm proteins is ifn inducible and inhibits mers-s-driven entry into target cells (28) , most likely at the stage of membrane fusion. we investigated whether the s protein polymorphisms under study modulated susceptibility of mers-s-driven entry to blockade by ifitm proteins. for this, we employed 293t cells stably expressing ifitm1, ifitm2, and ifitm3. expression of ifitm proteins had little effect on entry driven by the machupo virus glycoprotein (macv-gpc), while both ifitm2 and ifitm3 markedly reduced entry by the influenza a virus hemagglutinin (fig. 6 ), in accordance with published data (28) . moreover, expression of ifitm2 but not ifitm1 and ifitm3 diminished mers-s wtmediated host cell entry, as expected, and similar effects were observed for the s protein variants analyzed (fig. 6 ). finally, comparable results were obtained with 293t cells transiently transfected with escalating amounts of iftim2 plasmid, although variant f473s showed a slightly reduced ifitm2 sensitivity under these conditions (data not shown). in sum, the rbd polymorphisms observed in korean patients and mers-cov from the middle east did not markedly alter ifitm sensitivity of viral entry. d510g and i529t increase resistance to antibody-mediated neutralization. the antibody response significantly contributes to control of mers-cov infection in the host. therefore, we asked whether the polymorphisms under study impact antibodymediated neutralization. we focused our analysis on d510g and i529t since these changes modulated the efficiency of host cell entry. first, we tested whether d510g and i529t alter inhibition of s protein-driven entry by the murine antibody f11, which binds to an epitope encompassing amino acid 509, and macaque antibody jc57-14, which recognizes an epitope including amino acid 535 (29) . indeed, exchange d510g rendered mers-s-driven entry insensitive to inhibition by f11, while i529t reduced table 1 ). one serum sample was of sufficient quantity for detailed analysis (css53), while the others could be tested only at a single dilution (css16 and css23). remarkably, mers-s harboring polymorphism d510g showed a significantly increased resistance against neutralization by one serum sample (css16), and polymorphism i529t diminished neutralization by two sera (css16 and css53 [ fig. 7b and c]). finally, neither mutation had any impact on neutralization by serum sample css23. however, the overall neutralizing activity of this particular serum sample was reduced compared to those of the other sera tested and might have been too low to allow for differential neutralization of mers-s wt and mutants d510g and i529t. in summary, our results suggest that increased resistance against humoral immune responses might have driven the emergence of the d510g and i529t variants. the large mers outbreak within hospitals in the republic of korea in 2015 raised the question of whether viral variants with increased transmissibility might have been responsible. indeed, the majority of patients in korea harbored viruses with at least one unique mutation in the rbd, d510g and/or i529t (13, 14) . unexpectedly, these mutations reduced dpp4 binding and viral entry (14) , raising the question of whether they were beneficial to the virus. we found that d510g and i529t were compatible with robust s protein-driven entry into cells expressing large amounts of dpp4 and conferred resistance against antibody-mediated neutralization. these findings support the previously proposed concept that escape from antibody responses might shape mers-cov evolution and might come at the cost of reduced dpp4 binding (30) . however, they also suggest that diminished dpp4 binding may not compromise the ability of mers-cov to spread in target cells expressing robust levels of dpp4. the mers outbreak in the republic of korea was initiated by a single infected traveler, comprised 186 cases and 38 deaths, and encompassed primary, secondary, tertiary, and quaternary infections (6, 7). the case-fatality rate was roughly 20% and was thus lower than that reported for mers patients in the middle east. this may have various reasons, including differences in patient care and virulence of the mers-cov strains responsible for the outbreaks (14) . the i529t mutation has been detected in 72% of patients in korea examined by one study and might have already been present in the index patient (14) . similarly, the s protein sequences from 26 out of 38 patients deposited in the virus variation database (https://www.ncbi.nlm.nih.gov/genome/ viruses/variation/) contain the i529t change. moreover, viruses harboring this mutation established secondary, tertiary, and quaternary infections (13, 14) . in contrast, the d510g mutation was present in only 8% of patients analyzed in a previous study, and 6 out of 38 s protein sequences (16%) deposited in the virus variation database contained this exchange. kim and colleagues reported that d510g and particularly i529t diminish binding of mers-s to dpp4 and reduce entry into 293t cells transfected to express dpp4 (14) . moreover, structural evidence that these mutations disturb the interface between dpp4 and mers-s was presented (14) . however, it remained unclear whether these mutations may also aid virus spread under certain conditions. binding of mers-s to dpp4 is essential for infectious host cell entry (8) , and dpp4/s protein interactions are an important determinant of mers-cov species tropism (31) . moreover, patients with chronic lung disease were shown to express increased levels of dpp4 (32, 33) and are known to be at augmented risk of developing fatal mers (34), suggesting that dpp4 levels expressed under physiologic conditions might limit viral spread in patients. therefore, we investigated whether the previously reported negative effect of d510g and i529t on mers-s-driven entry was dependent on dpp4 expression levels. our results show that this is indeed the case: both mutations markedly reduced entry into 293t cells, which expressed low levels of endogenous dpp4 mrna and protein, but had little effect on entry into caco-2 cells, which express large amounts of dpp4. robust expression of dpp4 has been detected in viral target cells in the human respiratory epithelium, type i and type ii pneumocytes and alveolar macrophages (8, 32) , and it is thus conceivable that wt virus and virus bearing d510g or i529t infect these cells with comparable efficiencies in the lung of mers patients. finally, one can speculate that engagement of attachment-promoting factors ex-pressed in viral target cells in the human respiratory tract but not the cell lines studied here might allow mutants d510g and i529t to compensate for reduced dpp4 binding and to enter cells with the same efficiency as wt virus. the proteolytic activation of mers-s by host cell proteases is essential for mers-sdriven entry into target cells (35) . therefore, we addressed whether d510g and i529t might augment viral spread by promoting s protein activation or by changing the choice of s protein-activating protease. two interconnected routes have been described for mers-s activation. upon binding to dpp4, either the virus may be transported into host cell endosomes, where the s protein is activated by cathepsin l, or the s protein may be activated at the cell surface by tmprss2 (10, 11, 24) . notably, the choice between catl and tmprss2 might be determined by s protein processing in the infected cell: s protein cleavage by furin in infected cells seems to be indispensable for subsequent dpp4-induced conformational changes in the s protein that are in turn essential for activation by tmprss2 (36) . the present study demonstrates that d510g and i529t impact neither s protein cleavage by furin nor s protein activation by tmprss2 or catl. this finding may not be unexpected since both residues are located distant to the two sites believed to be important for s protein processing and activation, r751 (s1/s2 site) and r887 (s2= site). several cellular factors can modulate the efficiency of mers-s-driven entry into cells, raising the question of whether d510g and i529t might alter their effects in a way that promotes viral spread. the dpp4 ligand adenosine deaminase, which is highly expressed in lymphoid tissue and constitutes a component of the purine salvage pathway (37) , has been shown to compete with mers-s for dpp4 binding and to block mers-s-driven entry into target cells (31) . moreover, the ifn-induced transmembrane proteins ifitm2 and, to a lesser degree, ifitm3 were demonstrated to block mers-s-driven entry, at least upon overexpression (28) . finally, mers-s has been shown to bind to a coreceptor, sialic acid, which promotes viral entry into target cells (9) . our results indicate that d510g and i529t do not impact the above-named processes, suggesting that both mutations might affect neither viral attachment to cells nor the membrane fusion reaction. the spike protein is the key target of neutralizing antibodies, and such antibodies are detected with high frequency in convalescent mers patients who have survived severe disease (18, (38) (39) (40) . neutralizing antibodies can be directed against the rbd and interfere with viral entry by blocking dpp4/s protein interactions. a previous study conducted with cell culture systems found that mers-cov can acquire mutations that allow escape from rbd-targeting neutralizing antibodies but also provided evidence that such mutations might come at the cost of decreased dpp4 binding and viral fitness (30) . such a scenario generally matches our findings that d510g and i529t render the s protein at least partially resistant against neutralization by monoclonal antibodies and sera from mers patients and reduce dpp4 binding. however, our results also allow the speculation that reduced dpp4 binding might not necessarily translate into reduced viral spread in host cells expressing high levels of dpp4. this possibility awaits confirmation with infectious mers-cov and primary target cells. nevertheless, it would be in keeping with the observation that viruses harboring the i529t change were robustly transmitted between patients during the mers outbreak in the republic of korea (14) , although the i529t change did not provide the virus with an advantage in the absence of humoral responses. finally, our finding that single point mutations in mers-s can increase resistance to antibody-mediated neutralization highlights the need to develop vaccines and therapeutics that simultaneously target multiple epitopes in mers-s. collectively, our findings and previous work suggest that mers-cov variants with at least partial resistance to antibody-mediated neutralization can retain high infectivity for cells expressing robust amounts of dpp4 and can spread between humans. (43) , and wt mers-s (10), which have been described elsewhere. in addition, we employed overlap extension pcr to generate mers-s mutants harboring single (l411f, f473s, d510g, and i529t) mutations in the rbd (primer sequences and detailed information on the cloning procedure are available upon request). for all s proteins, we also generated versions containing a c-terminal v5 epitope for detection by immunoblotting. expression plasmids for tmprss2 n-terminally fused to a cmyc epitope and dpp4 n-terminally fused to dsred (eu827527.1 and dsred-dpp4) were constructed by amplifying the genetic information from existing expression plasmids and cloning the respective open reading frame (orf) into the pqcxip plasmid (kindly provided by a. l. brass). for the tmprss2 expression vector, we further exchanged the selection marker for puromycin resistance by that for blasticidin resistance, which was taken from plasmid pcdna6/tr vector (8, 44) . all pcr-amplified sequences were subjected to automated sequence analysis (microsynth seqlab) to verify their integrity. cell culture. 293t (human) and vero e6 (african green monkey) cells were cultivated in dulbecco's modified eagle's medium (dmem; pan-biotech). in addition, the human colorectal adenocarcinoma cell line caco-2 was grown in minimum essential medium (mem; thermo fisher scientific). 293t cells stably expressing ifitms or chloramphenicol acetyltransferase (cat), 293t-ifitm1, 293t-ifitm2, 293t-ifitm3, and 293t-cat (45), were cultivated as for the parental 293t cell line with the exception that they additionally received 0.5 g/ml of puromycin (biomol). all media were supplemented with 10% fetal bovine serum (fbs; pan-biotech) as well as 1ϫ penicillin and streptomycin from a 100ϫ stock solution (pan-biotech). all cell lines were cultivated in a humidified atmosphere at 37°c and 5% co 2 . transfection of 293t cells was performed by calcium phosphate precipitation. protease inhibitors. we employed inhibitors targeting cathepsin l and related proteases (mdl28170; sigma-aldrich) or tmprss2 and related proteases (camostat mesylate; sigma-aldrich). target cells for transduction experiments were treated with the respective inhibitor (specific concentrations are indicated in the legends of the respective figures) for 2 h prior to inoculation with rhabdoviral transduction vectors. neuraminidase treatment of target cells. to remove terminal sialic acids from macromolecules on the cell surface, target cells were preincubated with 100 mu of recombinant neuraminidase (sigma-aldrich) for 2 h before being inoculated with rhabdoviral transduction vectors. we employed rhabdoviral transduction vectors (pseudoparticles/pseudotypes) based on a replication-deficient vesicular stomatitis virus that lacks the genetic information for vsv-g but contains orfs for enhanced green fluorescent protein (egfp) and firefly luciferase (fluc), vsv*δg-fluc (46) . for production of vsv-based pseudotypes (vsvpp), 293t cells were transfected with expression plasmids for mers-s wt, mutant mers-s harboring rbd polymorphisms, macv-gpc, h1n1(wsn)-ha/na, vsv-g (positive control) or empty expression vector (negative control). at 24 h posttransfection, cells were inoculated with vsv-gtranscomplemented vsv*δg-fluc (indiana strain, kindly provided by g. zimmer) at a multiplicity of infection of 3 and incubated for 1 h at 37°c and 5% co 2 . next the inoculum was removed, cells were washed with phosphate-buffered saline (pbs), and standard culture medium which contained anti-vsv-g antibody (i1, mouse hybridoma supernatant from crl-2700; atcc) was added to neutralize residual input virus (cells transfected with vsv-g expression plasmid received culture medium without anti-vsv-g antibody). the cells were further incubated for 24 h before the supernatant was harvested, freed from cellular debris by centrifugation (3,000 ϫ g for 10 min), and either stored at ϫ80°c or used for transduction experiments. for the latter, target cells were grown in 96-well plates. if necessary, target cells were previously transfected with expression plasmids for dsred-dpp4 and/or tmprss2 (24 h in advance) or were pretreated with protease inhibitors or neuraminidase (2 h in advance). for transduction, the culture medium was aspirated and the rhabdoviral transduction vectors were added to the cells. transduction efficiency was quantified by measuring the virus-encoded fluc activity in cell lysates using commercial kits (beetle juice; pjk) and a plate luminometer (hidex, turku, finland) as described elsewhere (47) . 293t cells were transfected with expression plasmids for c-terminally v5-tagged versions of the mers-s wt or mers-s mutants or were control transfected with empty plasmid (negative control). at 48 h posttransfection, the culture supernatant was removed, cells were washed with pbs, and whole cells lysates (wcl) were prepared as follows. first, 2ϫsds sample buffer (0.03 m tris-hcl, 10% glycerol, 2% sds, 5% beta-mercaptoethanol, 0.2% bromophenol blue, 1 mm edta) was added to the cells, which were then incubated for 10 min before the sample was transferred to 1.5-ml reaction tubes and heated to 95°c for additional 10 min. for analysis of s protein incorporation into vsv particles, the respective culture supernatants were pelleted by high-speed centrifugation (25,000 ϫ g, 120 min, and 4°c) through a 20% (wt/vol) sucrose cushion, mixed with 2ϫ sds sample buffer, and heated to 95°c for additional 10 min. following sds-page, proteins were transferred onto nitrocellulose membranes (hartenstein gmbh) by immunoblotting. the membranes were then blocked by incubation in 5% skim milk in pbs-0.5% tween 20 (pbs-t) for 30 min at room temperature (rt) and then incubated with primary antibody solution (anti-v5, anti-actb, or anti-vsv-m) overnight at 4°c. next membranes were washed with pbs-t and further incubated with secondary antibody solution (anti-mouse-horseradish peroxidase [hrp]) for 1 h at rt. after additional washing steps, the membranes were finally developed using an in house-made chemiluminescence reagent in combination with the chemocam imaging system and chemostar professional software (intas science imaging instruments gmbh). for quantification of the signal intensity, the program imagej (fiji distribution) (48) was used. s protein signals detected in the wcl and supernatants were normalized against the respective signals of the loading control, either actb or vsv-m. antibodies for protein detection in immunoblot analysis. the following antibodies were used as primary antibodies: anti-v5 (mouse, 1:1,000; thermo fisher scientific), anti-␤-actin (actb, mouse, 1:1,000; sigma-aldrich), and anti-vsv-m (mouse, 1:1,000; kerafast). as a secondary antibody, an hrp-coupled anti-mouse antibody was used (goat, 1:5,000; dianova). all antibodies were diluted in pbs-t (carl roth) and 5% skim milk (carl roth). for the detection of mers-s/dpp4 interaction, 293t cells were transfected with expression plasmids for mers-s wt, mers-s mutants, or vsv-g or empty plasmid (both negative controls). at 48 h posttransfection, the cells were washed with pbs and then resuspended in 1% bovine serum albumin (bsa)-pbs. next, the cells were pelleted by centrifugation (5 min at 600 ϫ g at 4°c) and resuspended in 1% bsa-pbs containing soluble dpp4 equipped with a c-terminal human fc tag (soldpp4-fc, 1:200; acrobiosystems). after incubation for 1 h at 4°c, the cells were washed with 1% bsa-pbs and then resuspended in 1% bsa-pbs containing an alexa fluor 488-conjugated anti-human antibody (1:500; thermo fisher scientific). after another incubation period (as described above), cells were washed with 1% bsa-pbs, fixed with 4% paraformaldehyde solution, and analyzed by flow cytometry as described below. for the analysis of dpp4 surface expression by flow cytometry, 293t (untransfected or transfected with expression plasmid for dpp4), caco-2, and vero e6 cells were detached by resuspension in 1% bsa-pbs (293t cells) or by incubation in citric saline (pbs containing 0.135 m potassium chloride and 0.015 m sodium citrate; caco-2 and vero e6 cells). cells were then pelleted and washed as described above, successively incubated with anti-dpp4 (mouse, 1:200; abcam) and alexa fluor 488-conjugated anti-mouse (goat, 1:500; thermo fisher scientific) antibodies, and fixed. finally, all samples were analyzed using an lsr ii flow cytometer and the facs diva software (both from bd biosciences). for further data analysis, fcs express 4 flow research software (de novo software) was employed. quantification of dpp4-specific transcript levels by qpcr. total cellular rna was extracted from 293t, caco-2, and vero e6 cells using the rneasy minikit (qiagen) according to the manufacturer's instructions. then 1 g of rna was treated with dnase (new england biolabs) and reverse transcribed into cdna using the superscript iii first-strand synthesis system (thermo fisher scientific), both following the manufacturers' specifications. subsequently, 1 l of each sample was subjected to quantitative pcr (qpcr) using the quantitect sybr green pcr kit (qiagen) and a rotor-gene q platform (qiagen). all samples were analyzed for ␤-actin (housekeeping gene control) and dpp4 (gene of interest) transcripts in triplicates. finally, data were analyzed based on the threshold cycle (2 ϫδδct ) method (49) using 293t cells as a reference. neutralization experiments. rhabdoviral transduction vectors harboring mers-s wt, mers-s mutants, or vsv-g were normalized for comparable transduction of caco-2 cells (ϳ10 5 luminescent counts/s), incubated for 30 min at rt with increasing concentrations of monoclonal antibodies targeting distinct epitopes within the mers-s rbd, jc57-14 (targeting an epitope around amino acid residue 535 in mers-s, isolated from vaccinated nonhuman primates [pdb code 6c6y]) and f11 (targeting an epitope around amino acid residue 509 in mers-s, isolated from vaccinated mice) (29) or with different dilutions of serum from a mers patient who traveled from the arabian peninsula to germany. furthermore, sera from two additional mers patients, css16 and css23, were tested at a dilution of 1:200 (since the amount of those sera was limited, they were tested at a fixed dilution). please see table 1 for further information on the sera and the patient histories. afterwards, the mixtures of vsvpp and antibodies/ serum were inoculated onto caco-2 cells that were further incubated at 37°c and 5% co 2 . at 18 h posttransduction, transduction efficiency was quantified (as described above). for normalization, transduction efficiency of rhabdoviral transduction vectors that were incubated in the absence of antibodies/ serum was set as 100%. statistical analysis. if not stated otherwise, unpaired or paired, two-tailed student t tests were performed to test statistical significance of data originating from single representative or multiple combined experiments, respectively. in figures, statistical significance is represented as follows: *, p յ 0.05; **, p յ 0.01; ***, p յ 0.001; and ns, not significant. fields virology middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia ksa mers-cov investigation team. 2013. hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation clinical isolates of human coronavirus 229e bypass the endosome for cell entry variations in spike glycoprotein gene of mers-cov spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia the glycoproteins of all filovirus species use the same host factors for entry into bat and human cells but entry efficiency is species dependent inability of rat dpp4 to allow mers-cov infection revealed by using a vsv pseudotype bearing truncated mers-cov spike protein inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus protease inhibitors targeting coronavirus and filovirus entry tmprss2 activates the human coronavirus 229e for cathepsinindependent host cell entry and is expressed in viral target cells in the respiratory epithelium ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome dpp4, the middle east respiratory syndrome coronavirus receptor, is upregulated in lungs of smokers and chronic obstructive pulmonary disease patients middle east respiratory syndrome priming time: how cellular proteases arm coronavirus spike proteins proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism moonlighting adenosine deaminase: a target protein for drug development mers-cov antibody responses 1 year after symptom onset, south korea feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses the glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells transferrin receptor 1 is a cellular receptor for new world haemorrhagic fever arenaviruses proteolytic activation of the 1918 influenza virus hemagglutinin tmprss2 and tmprss4 facilitate trypsin-independent spread of influenza virus in caco-2 cells virion background and efficiency of virion incorporation determine susceptibility of simian immunodeficiency virus env-driven viral entry to inhibition by ifitm proteins a vesicular stomatitis virus repliconbased bioassay for the rapid and sensitive determination of multispecies type i interferon the hemagglutinin of bat-associated influenza viruses is activated by tmprss2 for ph-dependent entry into bat but not human cells fiji: an opensource platform for biological-image analysis analysis of relative gene expression data using real-time quantitative pcr and the 2(ϫdelta delta c(t)) method a patient with severe respiratory failure caused by novel human coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection saudi arabia, germany ex united arab emirates, request for information we are grateful to m. farzan, a. l. brass, and g. zimmer for providing expression plasmids and reagents. key: cord-284289-8emvca57 authors: schuster, jennifer e.; williams, john v. title: emerging respiratory viruses in children date: 2018-03-31 journal: infectious disease clinics of north america doi: 10.1016/j.idc.2017.10.001 sha: doc_id: 284289 cord_uid: 8emvca57 respiratory viral infections are a leading cause of pediatric disease. emerging respiratory viruses can cause outbreaks with significant morbidity and mortality or circulate routinely. the rapid identification of pathogens, epidemiologic tracing, description of symptoms, and development of preventative and therapeutic measures are crucial to limiting the spread of these viruses. some emerging viruses, such as rhinovirus c and influenza c, circulate yearly but were previously undetected due to limited diagnostic methods. although some pathogens have a geographic focus, globalization dictates that providers be aware of all emerging diseases in order to recognize outbreaks and diagnose and treat patients. respiratory viruses are a leading cause of pediatric morbidity and mortality worldwide. in the last 15 years, molecular detection and sequencing have led to increased pathogen identification in common respiratory illnesses as well as identification of pathogens during outbreak scenarios. heightened awareness for these and other emerging viruses is necessary to provide the best care for pediatric patients and to alert public health officials of novel diseases. seasonal influenza a generally causes a yearly epidemic with variable prevalence based on vaccine efficacy and antigenic drift. antigenic drift occurs in seasonal influenza due to minor mutations in the viral hemagglutinin (h) and neuraminidase (na) genes. antigenic shift occurs because of the ability of the virus to infect multiple animals, especially birds, and the ability of the segmented genome to undergo reassortment, mixing different proteins from different viral strains (table 1) . novel influenza strains are antigenically distinct because of complete exchange of gene segments encoding the h or na proteins, introducing h and na variants that have not previously circulated in humans; thus, reassortant viruses have the ability to cause pandemics because of minimal preexisting population immunity. avian influenza viruses can be passed to humans directly through close contact, or indirectly through another animal host. because these viruses are adapted to birds, they usually have limited ability to replicate in humans, restricting person-to-person transmission. however, this can be circumvented by either mutation or reassortment with a human virus, as in the pandemic 2009 h1n1, which was a reassortment between avian, human, and swine influenza viruses. 1,2 initially, reports of novel influenza a viruses centered on cases of h5n1. h5n1 human cases have occurred predominantly in southeast asia, the indian subcontinent, and the middle east; however, h5n1 has been detected in birds across eurasia, indonesia, and north africa. newly emerging avian influenza strains, including h7n9 and h10n8, have been identified in patients with poultry exposure in china. seasonal outbreaks of h7n9 in humans have occurred since 2013, with a recent spike in cases in china. 3 most cases have been reported in adults. 4 influenza a h5n1 causes more severe disease and higher mortality in children, whereas personto-person transmission is more common with h7n9, with nearly half of pediatric cases occurring in secondary clusters. 5 these novel influenza strains retain their preference for avian receptors and are not well adapted for human-to-human transmission. 6 thus, pediatric cases are less common, likely because of lower rates of poultry exposure in children. nonetheless, these viruses have been adapted in a controlled setting to be transmissible among mammals 7 ; therefore, sustained human-to-human transmission could be possible, placing children at risk. the clinical symptoms associated with novel influenza a strains are similar to yearly epidemic strains; however, symptoms are often more severe due to lack of preexisting immunity. since its reemergence in 2003, fatal reports of h5n1 pediatric cases have been described, including symptoms consistent with acute encephalitis. 8 compared with seasonal h3n2 and h1n1, h5n1-infected patients had a higher viral load and more exuberant cytokine response, 9 and mortalities are approximately 60%. 10 any cytopenia and/or liver involvement is associated with more severe disease. 11, 12 h7n9 infection in humans was first reported in china in 2013. three patients suffered from rapidly progressive, fatal acute respiratory distress syndrome with multiorgan system failure; 2 were known to have recent poultry exposure. fever and cough are common symptoms, 13 and similar to h5n1, laboratory findings included cytopenia, elevated liver function tests, and elevated creatinine kinase. 13, 14 in 2013, a fatal case of novel h10n8 was reported in a patient with recent poultry market exposure in china. 15 retrospective testing demonstrated that this was a newly emerged virus with no prior evidence of infection in poultry workers. 16 travel history is important for persons with acute respiratory illnesses, because most novel influenza a viruses occur in southeast asia. although influenza can be detected in cell culture, molecular diagnostics are crucial for the rapid identification of novel influenza viruses. reverse transcriptase polymerase chain reaction (rt-pcr) can identify a broad range of influenza a strains with subsequent genome sequencing for complete identification of novel strains. alternatively, rt-pcr primers and probes specific for avian h and na genes are available. 17 a whole-virus influenza h5n1 vaccine was found to be safe in a pediatric population with good antibody responses to both h and na components. 18 inactivated h7n9 vaccines are undergoing clinical trials, 19 and viruslike particle vaccine candidates are effective in small animal models. 20 oseltamivir is recommended for persons infected with novel influenza a viruses, although reports of resistance have been described. 21 chemoprophylaxis with oseltamivir can be considered based on exposure risk. oseltamivir chemoprophylaxis is recommended in the highest-risk exposure groups (ie, household or close family member contacts of a confirmed or probable case of novel influenza a) and can be considered in moderate-risk exposure groups (ie, health care personnel with unprotected close contact with a confirmed or probable case). 22 institution of appropriate isolation precautions is important ( table 2) . 23 although initially identified in 1950, influenza c, a member of the orthomyxoviridae family, is less well described than influenza a and b viruses. influenza c has 9 viral proteins in contrast with the 10 and 11 viral proteins of influenza a and b, respectively. unlike influenza a and b, it does not contain the na outer membrane protein. this difference contributes to unique disease characteristics and important considerations table 2 isolation for emerging respiratory viruses emerging respiratory viruses in children with respect to antiviral treatment. similar to influenza b, influenza c does not undergo reassortment and antigenic shift, which is in contrast to influenza a. influenza c does exhibit antigenic drift, and multiple variants can co-circulate (see table 1 ). data suggest that the virus circulates globally, and like other respiratory viruses, infection occurs early in life. 1 most children infected are less than 6 years old. 24 influenza c has been rarely identified as a cause of medically attended illness in adults. 25 the prevalence of influenza c is typically less than influenza a, but it can approach influenza b for some years. 26 although overall rates are low, less than 1% of all respiratory specimens in one study, 24 epidemics do occur in conjunction with replacement of the dominant antigenic group. 27,28 influenza c circulates primarily during the winter to early summer. 24 recent studies using molecular detection have suggested that influenza c is more frequent in children than previously recognized. 24,26,28-31 clinical symptoms influenza c symptoms are indistinguishable from influenza a and b. in one cohort, almost one-fifth of children with influenza c, predominantly those less than 2 years old, were hospitalized primarily due to pneumonia and bronchiolitis. in the ambulatory setting, upper respiratory tract infections were common. fever and cough were common symptoms in both groups, 24 and influenza c has been identified as a cause of hospitalized pediatric community-acquired pneumonia. 31 influenza c has been associated with fewer febrile days and less health care utilization than influenza a or b, suggesting a milder pathogen. 29 like other influenza types, influenza c has been associated with encephalopathy. 32 rt-pcr methods have been used to identify influenza c. 30,33 viral culture is difficult because of limited cell culture methods. furthermore, the virus does not display an easily distinguishable strong cytopathic effect like influenza a and b. treatment of influenza c is not well described. neuraminidase inhibitors are ineffective because of the lack of na glycoprotein on the outer membrane of the virus. in vitro data suggest that amantadine has activity against influenza c 34 ; however, the adamantanes have broad toxicities and are not routinely recommended because of high rates of resistance by influenza a. 35 currently licensed seasonal influenza vaccines do not contain antigens to influenza c and are not protective. supportive care is recommended. droplet isolation should be used for children hospitalized with influenza c infection (see table 2 ). coronaviruses (cov) are a common cause of pediatric respiratory tract disease, accounting for about 15% of common colds. viruses are classified into different genera (alpha-, beta-, gamma-, and deltacoronavirus). covs can infect multiple species, and crossover from animals to humans can lead to outbreaks. 1 epidemic covs have been reported, most notably severe acute respiratory syndrome-associated coronavirus (sars-cov) in 2002 to 2003. 36 in 2012, a 60-year-old saudi arabian presented with pneumonia and respiratory failure. a novel betacoronavirus, subsequently termed middle east respiratory syndrome (mers-cov), was identified. although most of the cases were detected in saudi arabia and united arab emirates, imported cases to the united states were described. 37 the virus was closely related to bat covs. 38 however, camels were later identified as an intermediary host when zoonotic transmissions occurred, 39 and mers-cov seroprevalence was significantly higher among persons with camel exposure. 40 person-to-person transmission did occur, which led to secondary hospital outbreaks. 41 although initial reports primarily occurred in adults, likely related to zoonotic and occupational exposures, pediatric cases developed in household contacts. 42 case descriptions of patients with mers-cov are primarily in adults and in patients hospitalized with sars-cov. in one case series, fever was present in 62% of symptomatic patients, and cough was present in 50%. upper respiratory tract symptoms were less common with only 19% of subjects having rhinorrhea. 43 gastrointestinal symptoms, including diarrhea, have been reported. although initial case fatality rates exceeded 50%, 44 a large number (25%) of patients with laboratory-confirmed infection were asymptomatic in other studies. 43 data are sparse in children. in one case series of 11 saudi children with confirmed mers-cov, the median age was 13 years (range 2-16 years), older than most respiratory viruses. only 2 patients had symptoms, and both had underlying medical diseases, a 2-year-old with cystic fibrosis and a 14-year-old with down syndrome and cardiopulmonary disease. although the younger child died of respiratory failure, the older child had a relatively uncomplicated hospital course. pulmonary imaging for both children demonstrated bilateral diffuse infiltrates. 45 in one cohort of household contacts of mers-cov-infected subjects, the secondary attack rate was relatively low at 5%. the virus did not have an increased predilection for children, and none of the infected children developed symptoms, suggesting that disease may be worse with primary transmission and/or in adults. 42 as with novel influenza a viruses, travel and exposure history are important. real-time rt-pcr is available for the diagnosis of mers-cov, and serologic testing also exists. 46 viral culture can be performed but requires proper specimen handling and biosafety at level-3 facilities. 47 no mers-cov-specific treatment exists. in a small retrospective study of adults with severe mers-cov, patients treated with oral ribavirin and subcutaneous pegylated interferon alpha-2a had improved survival at 14 days (70% vs 29%). however, 28-day survival was not significantly different (30% vs 17%). 48 candidate vaccines are being studied; however, most are in the preclinical stage. 49 airborne and contact precautions are recommended to prevent person-to-person transmission (see table 2 ). 50 human rhinoviruses (rv), members of the picornaviridae family, are leading causes of respiratory illness in children. a new rv species, distinct from species a and b, was identified in 2004. 51, 52 sequencing of the viral protein 4 (vp4) region from specimens of children hospitalized with respiratory illness corroborated the discovery of this new species, rhinovirus c (rv-c). most children in whom the isolate was identified were hospitalized with asthma or febrile wheeze. 53 sixty genotypes of rv-c have been identified. the global burden of rv-c is significant with approximately onequarter of rv infections attributed to rv-c. rates of rv-c are generally higher emerging respiratory viruses in children than rv-b and comparable to rv-a in some studies. 54 rv-c is detected yearround. 55, 56 clinical symptoms although rvs are detected in both symptomatic and asymptomatic children, rv-c is more commonly associated with episodes of clinical illness. 57 only 3% of rv-c is detected in healthy children. 58 rv-c can cause severe respiratory disease, particularly in asthmatics, and was associated with asthma exacerbations in children in a casecontrol study. 59 children with rv-c are more likely to require supplemental oxygen and to have wheezing than children with rv-a. 60 however, in other studies, rv-a and rv-c produce similar clinical symptoms. 55, 56 in one case series, 40% of rv-cinfected hospitalized children required supplemental oxygen and 95% wheezed. 56 rv-c-infected children in the outpatient and emergency department settings were more likely to have radiographically confirmed/clinically diagnosed lower respiratory tract illness (eg, bronchiolitis, pneumonia, croup, and asthma) compared with children with other rvs. 61, 62 in addition, children with rv-c-related wheezing were more likely to be hospitalized for respiratory problems compared with non-rv respiratory viruses. 63 diagnosis rv is typically detected from nasopharyngeal specimens using rt-pcr. broad range pcr allows for detection of a variety of rvs by using primers targeting a conserved region. a subsequent step, including seminested pcr or sequencing, can identify the specific species and serotype. rv-c has also been detected in stool 64 as well as in blood, in which higher rates of viremia occurred with rv-c compared with other rv types. 65 rv can be detected using cell culture; however, this method is highly variable and depends on optimal temperatures (33 c-34 c), motion, and time (10-14 days to see cytopathic effect). 1 furthermore, rv-c is extremely difficult to culture and requires highly specialized methods. 66 antigen and antibody detection is hampered by the presence of numerous rv serotypes. no licensed treatment of rv-c exists. experimental antivirals have had some in vitro efficacy against rv-c, although notably, pleconaril did not have activity. 67 rv vaccine development has been complicated by the numerous viral serotypes and lack of cross-serotype protection. 68 droplet isolation should be used for children hospitalized with clinically apparent rv-c infection. respiratory viruses remain a leading cause of childhood disease. the identification and emergence of novel respiratory viruses are important for pediatricians and infectious diseases clinicians. viruses that were previously difficult to culture can now be rapidly identified using molecular diagnostics and sequencing, and these techniques are highly useful for detecting outbreaks. in addition, the ongoing evolution of viruses and ability to mutate allows for species-to-species transmission of novel viruses. practitioners should remain on high alert for emerging viruses in cases whereby a cause cannot be identified for a clinical syndrome or if the clinical syndrome is more severe than expected for the identified pathogen. travel and animal exposure history are important to maintain a high index of suspicion, and rapid institution of appropriate isolation precautions is crucial to prevent person-to-person transmission (see table 2 ). mandell, douglas, and bennett's principles and practice of infectious diseases antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans sudden increase in human infection with avian influenza a(h7n9) virus in china probable person to person transmission of novel avian influenza a (h7n9) virus in eastern china, 2013: epidemiological investigation differences in the epidemiology of childhood infections with avian influenza a h7n9 and h5n1 viruses a human-infecting h10n8 influenza virus retains a strong preference for avian-type receptors experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia highly pathogenic asian avian influenza a (h5n1) in people risk parameters of fulminant acute respiratory distress syndrome and avian influenza (h5n1) infection in vietnamese children clinical factors associated with severity in hospitalized children infected with avian influenza (h5n1) epidemiological, clinical and viral characteristics of fatal cases of human avian influenza a (h7n9) virus in zhejiang province human infection with a novel avian-origin influenza a (h7n9) virus clinical and epidemiological characteristics of a fatal case of avian influenza a h10n8 virus infection: a descriptive study no evidence h10n8 avian influenza virus infections among poultry workers in guangdong province before 2013 development and validation of a one-step real-time pcr assay for simultaneous detection of subtype h5, h7, and h9 avian influenza viruses antiviral dosage. guidance on the use of influenza antiviral agents a novel coronavirus associated with severe acute respiratory syndrome first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for camel-to-human transmission of mers coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study hospital outbreak of middle east respiratory syndrome coronavirus transmission of mers-coronavirus in household contacts mers-cov outbreak in jeddah-a link to health care facilities update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide middle east respiratory syndrome coronavirus disease in children assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections interim laboratory biosafety guidelines for handling and processing specimens associated with middle east respiratory syndrome coronavirus (mers-cov) -version 2 ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study mers-cov vaccine candidates in development: the current landscape interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during emerging respiratory viruses in children frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children patient characteristics and severity of human rhinovirus infections in children community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy australian preschool-aged children during a 12-month period human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children clinical spectrum of human rhinovirus infections in hospitalized hong kong children role of rhinovirus c respiratory infections in sick and healthy children in spain novel human rhinoviruses and exacerbation of asthma in children human rhinovirus c associated with wheezing in hospitalised children in the middle east human rhinovirus c: age, season, and lower respiratory illness over the past 3 decades molecular epidemiology of human rhinovirus infections in the pediatric emergency department human rhinovirus species c infection in young children with acute wheeze is associated with increased acute respiratory hospital admissions high detection frequency and viral loads of human rhinovirus species a to c in fecal samples; diagnostic and clinical implications detection of human rhinovirus c viral genome in blood among children with severe respiratory infections in the philippines molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c multiple classes of antiviral agents exhibit in vitro activity against human rhinovirus type c developing a vaccine for human rhinoviruses guideline for isolation precautions: preventing transmission of infectious agents in health care settings key: cord-284845-on97zu6w authors: falcinelli, shane d.; chertow, daniel s.; kindrachuk, jason title: integration of global analyses of host molecular responses with clinical data to evaluate pathogenesis and advance therapies for emerging and re-emerging viral infections date: 2016-07-29 journal: acs infectious diseases doi: 10.1021/acsinfecdis.6b00104 sha: doc_id: 284845 cord_uid: on97zu6w [image: see text] outbreaks associated with emerging and re-emerging viral pathogens continue to increase in frequency and are associated with an increasing burden to global health. in light of this, there is a need to integrate basic and clinical research for investigating the connections between molecular and clinical pathogenesis and for therapeutic development strategies. here, we will discuss this approach with a focus on the emerging viral pathogens middle east respiratory syndrome coronavirus (mers-cov), ebola virus (ebov), and monkeypox virus (mpxv) from the context of clinical presentation, immunological and molecular features of the diseases, and omics-based analyses of pathogenesis. furthermore, we will highlight the role of global investigations of host kinases, the kinome, for investigating emerging and re-emerging viral pathogens from the context of characterizing cellular responses and identifying novel therapeutic targets. lastly, we will address how increased integration of clinical and basic research will assist treatment and prevention efforts for emerging pathogens. e merging and re-emerging viruses pose a significant threat to public health and global economies. moreover, outbreaks caused by emerging and re-emerging viruses continue to increase in frequency as a result of changing socio-economic, environmental, and ecological factors. 1 notably, the zoonotic viral pathogens, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), ebola virus, chikungunya virus, and zika virus, have emerged on a global scale in recent years; although less widely publicized, other emerging viral pathogens such as monkeypox virus and andes virus have led to smaller recurrent outbreaks. a critical challenge for combating these outbreaks is often the discordant relationship between the economic status of outbreak "hotspots" and resource distribution or control capacity within these regions. in addition, the development and delivery of therapeutics for combating such outbreaks have been complicated by both the associated costs in design and development for novel antiinfective therapeutics and the requirements for regulatory approval and licensure. 2 importantly, emerging infectious diseases present the additional inherent challenge that they are only "emerging", and thus limited resources are made available for research until they present a significant risk. for many emerging viral pathogens, the requirement for highcontainment facilities has further impeded widespread research. from the perspective of drug development, the limited knowledge and understanding of molecular pathogenesis for these agents is a daunting challenge to overcome when outbreaks emerge. in the face of an increasing burden of emerging and reemerging pathogens, it is necessary to overcome the barriers imposed by a paucity of information regarding molecular pathogenesis for these agents. omics-based approaches present a mechanism to rapidly generate large amounts of data in regard to host responses and assist in target identification for drug development efforts. in addition, omics-based analyses allow for the characterization of molecular events that mitigate cellular responses to viral pathogens from a global perspective across multiple levels of cellular complexity (individual cell types < tissues < organs). high-throughput global analyses of host gene expression, including microarrays and rna-seq, provide important information regarding transcriptional responses during infection. although these validated approaches are among the most widespread of the omics-based technologies for infectious disease investigations, they do not provide a direct measure of the activation status of the cell signaling pathways that regulate underlying cellular responses. in contrast, global investigations of cellular kinase activities (the kinome) are able to provide insight into the activation status of cell signaling networks (including those that mediate pathogen recognition and innate immune activation, cell cycle activities, metabolic status, wound healing and repair, and cell death) at the level of individual kinase-mediated phosphorylation events. in addition, kinome investigations allow for potential identification of kinase drug targets. kinases are currently one of the top targets for drug design and development, and there is potential for repurposing of kinase inhibitors with existing regulatory approval. as emerging viral infections often result in severe illness including respiratory failure [severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and influenza] and multiorgan failure [ebola virus disease (evd)], understanding complex pathogenesis of these infections is required for effective vaccine and therapeutic design and for improved patient care. healthcare providers caring for patients with severe emerging viral infections are generally focused on clinical care and biosafety as compared to the complex molecular events that underlie pathogenesis. in contrast, basic researchers typically focus on discrete aspects of pathogenesis through a variety of in vitro and in vivo analyses rather than the complex interplay between these events and the clinical, physiologic, and pathologic abnormalities observed by the clinician. integrating basic and clinical research is needed to accelerate the translation of knowledge for emerging infections toward vaccine development and therapeutic discovery. specifically, detailed natural history studies merging multiple data streams including omics approaches (high-throughput gene expression and kinomics) and focused translational investigations utilizing relevant models that can be validated to human disease are needed to clarify disease pathogenesis, advance therapeutic discovery, and facilitate regulatory approval. although an integrated approach between basic and clinical research is ideal for investigating the connections between molecular and clinical pathogenesis, there has been a paucity of investigations for which this has been undertaken. here, we will discuss emerging pathogens for which there is available information regarding the clinical course of disease, host immune responses during natural infection, and molecular information regarding the global cellular responses to infection, with particular attention on host kinome investigations. in this regard we will focus on the emerging viral pathogens mers-cov, ebola virus (ebov), and monkeypox virus (mpxv) (figure 1 ). we will review the clinical presentation and immunological and molecular features of the diseases and summarize available omics data informing pathogenesis of these pathogens. lastly, we will discuss the benefit of improved integration of available clinical knowledge or data regarding the pathologic manifestations of disease with basic research investigations to advance treatment and prevention of severe emerging viral infections. global gene expression investigations have provided information regarding host response to emerging and re-emerging review pathogens at the level of individual genes or gene clusters. however, there is a paucity of information regarding the relationship of cell signaling networks, and in particular their activation status, with the biological/pathological events that occur throughout infection. it has been well established that many biological processes can be regulated independent of transcriptional or translational changes through post-translational modification (ptm) events. indeed, kinase-mediated phosphorylation of proteins, in which kinases catalyze the transfer of the γ phosphate group from atp to the hydroxyl group of a specific ser, thr, tyr residue, is figure 2 . generation of kinome peptide array targets and kinome peptide arrays: (1) species-specific proteomic or genomic information from a diverse range of species can be used to identify kinase recognition motifs that are composed of a central phosphorylation target and the surrounding amino acids (normally +4 and −4 amino acids from the central phosphorylated residue); (2) peptides that comprise the kinase recognition motifs identified in (1) are synthesized and covalently linked to a glass surface. peptide targets are spotted in replicates of three to nine spots on each array to account for intra-array variability. individual amino acids of the peptides are represented by orange, red, purple, and green spheres. (4) biological samples are processed to generate cell lysates that are activated with atp and applied to the kinome peptide array. (5) following the application of the cell lysate, activated kinases in the cell lysate will recognize their respective kinase recognition motifs and phosphorylate the central phosphorylated residue of the peptide. (6) kinome peptide arrays are subsequently stained with a phospho-specific fluorescent stain and imaged followed by comparative bioinformatics analyses. tissue and cell images were derived and/or modified from servier medical arts under a creative commons attribution 3.0 unported license. among the most thoroughly characterized ptm. virtually all cell signal transduction events are regulated by kinases independent of biological complexity of the host (i.e., prokaryotes and eukaryotes). 3 lending further credence to the biological importance of kinases, >500 kinases have been identified in the human genome, and ∼30% of the human proteome is modulated by kinase-mediated phosphorylation events. 4, 5 thus, considering the central role of kinases in a broad range of cellular processes (including growth and development, metabolism, and immune responses), it has been postulated that the activities of individual kinases may represent more reliable predictors of cellular phenotypes than transcriptional or translational changes. 6 indeed, transcriptional or translational-based omics approaches are often unable to account for regulatory events including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/ substrate subcellular sequestration, or protein activation/ repression ptms. 7 given the central role of kinases in the regulation of biological processes, kinases are a logical drug target. as a testament to this, 33 kinase inhibitors have been granted licensure by the u.s. food and drug administration (fda) for a broad range of malignancies, and there are a continually increasing number of kinase inhibitors that are in various stages of preclinical trials. furthermore, kinases are the second most frequently targeted gene class in cancer therapy after the g protein coupled receptors. 8, 9 the recent prioritization for the repurposing of approved therapeutics for alternative malignancies by the national institutes of health center for advancing translational sciences (ncats) 10, 11 also provides considerable impetus for the investigation of licensed kinase inhibitors as infectious disease therapeutics. concerns exist regarding the therapeutic application of kinase inhibitors as novel therapeutics for infectious disease, in particular to the potential immunosuppressive effects following prolonged treatment. however, it should be appreciated that the application of kinase inhibitors in such cases would need to be targeted in terms of timing and dose, with appropriate molecular biomarkers guiding initiation and cessation. it should also be appreciated that the clinical symptoms associated with many emerging and re-emerging pathogens have been associated with dysregulated host immune responses (in particular pro-inflammatory responses). thus, the global analysis of the activation state of host kinases (the kinome) can provide critical insight into the specific activation state of individual kinases, cell signaling pathways, or larger biological networks. in addition, kinome investigations may offer important, and predictive, insight into the cellular mechanisms that regulate phenotypic changes within cells. 7 as many kinases recognize a particular phosphorylation motif composed of the central phosphoacceptor site and the amino acids +4 and −4 residues from the central phosphorylation site, 12 peptides representing this kinase target motif can be synthesized with relatively high efficiency and low expense. indeed, kinase target motif peptides have been shown to be appropriate substrates for their respective kinases with v max and k m values approaching those of the fulllength protein. 13 thus, peptide kinome arrays can be constructed in an analogous manner to traditional dna microarrays where kinase target motif peptides are spotted onto a glass slide representing hundreds to thousands of unique peptide targets for kinases ( figure 2 ). following this, samples in the form of cellular lysates from whole organs, tissues, or individual cell types can be applied to the kinome peptide arrays, allowing for the phosphorylation of specific peptide targets by kinases within the lysate (figure 3 ). the development of kinome-specific bioinformatics analysis software, including the platform for intelligent, integrated kinome analysis (piika), has provided a mechanism to identify the complex patterns of kinase-mediated phosphorylation events and quantitate the differences between compared conditions. 14 clinical findings during mers-cov infection. syndromic case-definition for mers infection requires a compatible clinical syndrome and an epidemiologic risk factor including travel to an affected region or contact with a known or suspected case. initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of 5 days, which can range from 2 to 14 days. 23 mildly symptomatic or possibly asymptomatic infections have been reported, and progression to severe disease is associated with pre-existing medical conditions including cardiopulmonary disease, obesity, and diabetes. most (98%) of reported mers cases are among adults, with a median age of 50 years. in severe cases respiratory failure requiring mechanical ventilation typically occurs within 7 days of symptom onset. 22 laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes. 23−27 high lactate levels and consumptive coagulopathy have also been reported. 24, 28 chest radiographic abnormalities are observed in most cases consistent with viral pneumonitis, secondary bacterial pneumonia, or acute respiratory distress syndrome. 22 cytokine levels in serum and bronchoalveolar lavage (bal) from two mers patients, one with fatal disease and one who survived. 30 higher levels of retinoic acid-inducible gene 1 (rig-1), melanoma differentiation-associated protein 5 (mda5), interferon regulatory factor (irf)-3 and -7, interleukin (il) 17a, and il-23 and lower levels of il-12 and ifnγ were observed in the fatal case compared with the survivor. more recently, min et al. performed a temporal analysis of cytokine, chemokine, and growth factor blood levels from 14 patients during the recent outbreak of mers in south korea. 31 the patients were subcategorized into four groups on the basis of disease severity: group i patients developed fever and recovered. group ii patients developed mild pneumonia without hypoxemia. group iii patients had prolonged and severe pneumonia. group iv patients had severe pneumonia and acute respiratory distress syndrome. group iv patients included five fatal cases of mers; all patients in groups i−iii fully recovered from illness. ifnα was elevated in all groups and largely peaked during the second week of illness. granulocyte-colony stimulating factor (g-csf) and granulocyte macrophage (gm)-csf were similarly elevated across all patient groups; however, patients with fatal disease had reduced gm-csf responses following antiviral treatment as compared to patients that recovered. patients with pneumonia had relative elevations of il-1, tumor necrosis factor (tnf)-α, il-6, and il-10 during the second and third weeks of illness. elevated il-6 and il-10 appeared to trend positively with the severity of illness. a robust induction of multiple chemokines was found in most patients. notably, eotaxin and regulated on activation, normal t expressed and secreted (rantes) was elevated in all patients. in contrast, il-8, monocyte chemotactic protein (mcp)-3 and macrophage inflammatory protein (mip)-1β were more prominent in groups ii and iii as compared to groups i and iv. furthermore, elevated interferon gamma induced protein (ip)-10 correlated with the development of pneumonia (groups i−iii). multiple growth factors, including epidermal growth factor (egf), fibroblast growth factor (fgf)-2, vascular endothelial growth factor (vegf), and tgf-α, were significantly elevated across all patients; however, egf was significantly higher in patients that recovered from disease as compared to the fatal cases. further evaluations are needed to characterize the natural history of immune response during acute mers-cov infection and recovery. transcriptome analyses of mers-cov. in an effort to better characterize mers-cov pathogenesis in the absence of available samples from human patients and, in particular, address pathologic changes associated with infection, multiple animal species have been employed in mers-cov investigations. while multiple small animals are not susceptible to mers-cov infection, 32 rhesus macaques and marmosets develop mild to severe lung pathology following experimental infection. transcriptome analysis in mers-cov-infected rhesus macaques revealed that genes related to antiviral immunity, chemotaxis, and inflammation were overexpressed in lesional versus grossly normal lung tissue at 3 days postinfection. 33 a significantly smaller number of differentially expressed genes was found on day 6 postinfection with no obvious trends following pathway enrichment analysis. significant changes in the transcriptome profiles of peripheral blood mononuclear cells (pbmcs) were observed at only day 1 postinfection. this global analysis suggests a key role for an initial rapid innate immune and inflammatory response (through pattern recognition receptors) followed by rapid resolution. 33 a study of mers-cov-infected marmosets evaluated lung lesions by rnaseq at days 3−6 postinfection. 34 pathway analyses demonstrated that chemotaxis and cell migration, cell cycle progression, and cell proliferation and fibrogenesis were highly over-represented relative to uninfected controls. to a lesser degree, pathways associated with inflammation, vascularization, endothelial activation, proliferation of smooth muscle, and tissue repair were also overrepresented in infected animals. differences were most significant on days 4 and 6 postinfection during illness progression relative to day 3. recently, menachery and colleagues examined the interaction between mers-cov emc/2012 and the host ifnstimulated gene (isg) response by transcriptomics. 35 isg responses in mers-cov infected calu3 cells, a lung adenocarcinoma cell line, had no discernible induction initially upon infection but were up-regulated by 12 h postinfection. 35 down-regulation of a subset of isgs resulted in altered histone modifications, a potential epigenetic contributor to early impairment of antiviral cellular defenses. in a separate analysis genetically distinct mers-cov strains, mers-cov sa1 and mers-cov eng1, produced distinct gene expression profiles in calu-3 cells. 36 these analyses may better inform early host-cell antiviral responses and the impact of viral evolution on these and other complex biological responses. proteomics analysis corroborated these transcriptional data with induction of isgs observed 18 h postinfection. significantly reduced levels of stat1 and pkr compared with uninfected controls were also noted. differential host transcriptome responses to mers-cov sa1 and mers-cov eng1 highlight both the propensity of emerging viral pathogens to evolve rapidly and the importance of additional host response analyses for augmenting and clarifying such complex biological responses. kinome analyses of mers-cov infection. host responses to mers-cov infection through kinome analysis were recently assessed using huh-7 cells, an immortalized human hepatocyte cell line, that are highly permissive to mers-cov infection. 37 temporal analysis of kinome responses by peptide arrays revealed selective modulation of extracellular signal-regulated kinases (erk)/mitogen-activated protein kinases (mapk) and phosphatidylinositol-3-kinases (pi3k)/akt (also known as protein kinase b)/mechanistic target of rapamycin (mtor) signaling responses. over-representation analysis (ora) revealed erk/mapk and pi3k/akt/mtor signaling responses were consistently up-regulated during infection. multiple erk/mapk family members formed central components of functional networks and signaling pathways throughout infection. similar results were observed for intermediates of the pi3k/akt/mtor signaling pathway at 1 and 24 h postinfection, suggesting that modulation of erk/ mapk and pi3k/akt/mtor signaling may be important for productive mers-cov infection. downstream analysis of the phosphorylation patterns of pathway intermediates from the erk/mapk and pi3k/akt/ mtor signaling supported observations from the kinome analysis. both investigations demonstrated that nuclear factor kappa-light-chain-enhancer of activated b cells (nfκb)regulated family members were important mediators of mers-cov infection. 38 il8-and ifn-mediated signalings were also modulated during mers-cov infection, consistent with prior analyses. 39, 40 these results were also in agreement with in vitro transcriptional analysis of mers-cov infection. 38 prophylactic or therapeutic addition of fda-licensed kinase review inhibitors targeting activated kinases in mers-cov infection impaired viral replication. these hypothesis-generating data may inform directed investigations into mers-cov pathogenesis and, importantly, demonstrate the potential to identify novel host-centric therapeutic targets. ebolaviruses. the filoviridae family of viruses consists of three genera: ebolavirus, marburgvirus, and the newly identified cuevavirus. structurally, filoviruses have a pleomorphic enveloped, filamentous virion particle that encapsulates a negative-sense single-stranded rna genome. ebolaviruses were first described in 1976 following disease outbreaks in the democratic republic of congo and sudan and are composed of five viral species, including ebola virus (ebov), sudan virus (sudv), bundibugyo virus (bdbv), taı̈forest virus (tafv), and reston virus (restv). sporadic outbreaks of ebov, sudv, bdbv, and tafv have occurred throughout central africa for more than three decades, resulting in thousands of infections. case fatality rates during these outbreaks have routinely exceeded 50%. 41 isolated outbreaks of restv have occurred outside africa in nonhuman primate facilities in the united states, italy, and the phillipines, and infection results in high morbidity and mortality in nonhuman primates; however, restv has only been associated with asymptomatic infections in humans. 42 although ebolaviruses have been historically associated with isolated outbreaks involving small cohorts of infected patients (<500), an outbreak of evd in west africa beginning in 2014 has resulted in 28,616 cases and 11,310 deaths (40% cfr) as of june 2016 (http://www.who.int/csr/ disease/ebola/en/). although virus transmission has greatly decreased in west africa, surveillance for sporadic infections continues. clinical findings in evd. ebov transmission occurs through exposure of infected body fluids or tissues to mucous membranes or nonintact skin. 43 the mean incubation period is 6−10 days, ranging from 2 to 21 days. 43 initial signs and symptoms are nonspecific including fever, myalgia, and malaise and cannot be reliably distinguished from other endemic illnesses in africa including malaria and enteric infections. 43 whereas mild illness has been described, most patients develop severe disease within days of symptom onset. massive gastrointestinal fluid losses of up to 5−10 l per day due to vomiting and watery diarrhea may result in progressive dehydration and hypovolemic shock. even in the setting of adequate fluid and electrolyte replacement, sequential multiorgan failure may occur. ebov infects multiple organs and cell types throughout the body with the notable exception of lymphocytes that are indirectly depleted early during infection. organ injury due to direct viral or indirect host-mediated responses results in severe complications including meningoencephalitis, uveitis, respiratory failure, secretory diarrhea, disordered coagulation, renal failure, hepatic necrosis, and myositis. the clinical presentation, laboratory values, viral kinetics, and clinical management of evd patients in west africa, europe, and the united states during the 2014−2015 outbreak have been recently well-characterized. 44 soluble immune mediators associated with ebov infections. there is a paucity of information regarding ebov pathogenesis in humans primarily due to the limited frequency of evd outbreaks prior to 2014 and limitations presented by sample acquisition from infected patients in the field as well as the overall size of patient cohorts. largely contradictory findings regarding the immune responses in those who survive or succumb to evd have further confounded the under-standing of ebov pathogenesis in human patients. for example, villinger et al. reported that serum cytokine concentrations (including ifnα, ifnγ, tnf-α, il-2, and il-4) were elevated in patients with fatal infections in comparison to survivors. 45 in contrast, additional studies have suggested that fatal infections were instead related to general immunosuppression including ifnγ, il-2, and il-4. 46−49 an investigation of sudv infection in humans by sanchez et al. demonstrated limited changes in the expression levels of cytokines, fas antigen, and fas ligand in pbmcs from infected patients relative to those found for uninfected patients. 50 furthermore, an investigation of 42 fatally infected evd patients by wauquier et al. has further confounded the role of host immune responses in fatal evd as hypersecretion of multiple cytokines and growth factors and decreased secretion of t lymphocyte-derived cytokines were associated with fatal disease. 51 transcriptome analyses of ebola virus infection. to date, no investigations of host gene expression in ebov-infected patients have been reported, although limited data are available from animal models of infection or from in vitro investigations. in a study of pbmcs from ebov-infected crab-eating macaques, rubins et al. found few notable changes in the early stages of infection (1−2 days); however, broad changes were observed over days 4−6 post-infection. pro-inflammatory cytokines (il-1β, il-6, il-8, and tnf-α) and chemokines (mip-1α and mcp1−4) were up-regulated at days 4−6 postinfection relative to healthy controls. 52 multiple genes related to apoptosis including bcl-2 family members, multiple caspases, fas-associated death domain protein, and tnf superfamily member 10 were also up-regulated at late time points. ifn-regulated genes were up-regulated by day 2 postinfection and remained so through study day 6. yan et al. investigated pbmc gene expression in ebovinfected rhesus macaques with or without anticoagulant administration. untreated animals displayed up-regulation of immune response genes, b cell receptor signaling intermediates, nk cell mediated cytotoxicity, leukocyte activation, and lymphocyte activation compared with anticoagulant-treated animals during the early stages of infection. the expression levels of these gene clusters fell to pre-infection levels at the late-stage of infection. in contrast, genes related to defense responses, apoptosis, wounding, inflammation, coagulation, and leukocyte activation remained elevated during early-and latestage infection. following the isolation of restv from pigs, 53 subsequent investigations have demonstrated that pigs were susceptible to both restv and ebov infection with preferential targeting of macrophages in the lungs. recently, nfon et al. demonstrated that ebov infection in pigs resulted in up-regulation of chemokine expression beginning on day 3 postinfection as compared to mock-infected pigs. 54 the most pronounced changes in gene expression were found on days 5 and 7 postinfection and included the up-regulation of a broad set of cytokines (il-5, il-6, il-8, il-10, il-22, il-26, il-27, resistin), chemokines (ccl2, ccl10, ccl19, ccl20, amcf-ii, ccl3l1, ccl4), cell adhesion protein (selectin), antimicrobial protein, palate, lung, and nasal epithelium clone proteins, and pro-apoptotic molecules (multiple caspases, caspase recruitment domain-containing protein 6 (card), apoptosisassociated tyrosine kinase (aatk), fas, fas-associated protein with death domain (fadd), tnf receptor-associated factor 3 (traf3), tnfα-induced protein 3-interacting protein 1 review (tnip1)). in addition, expression of multiple genes related to microbial sensing (pattern recognition receptors) or antiviral responses (isgs) was up-regulated in the lungs of infected animals. although the localization of the cytokine response of pigs and humans or nhps differs during the course of ebov infection (localized responses in the lungs of pigs versus a predominantly systemic response in humans and nhps), the cytokine profiles of pigs, humans, and nhps were quite similar. for example, comparison of nhp 52 and porcine responses 54 during ebov infection demonstrated multiple gene expression similarities between the two species (i.e., il-6, il-8, caspase family members). it is also likely that direct comparison of both data sets would likely yield many common gene signatures that are conserved in their identity as well as their directionality (upregulation vs down-regulation). macrophages are an early target of ebov infection and support high-level viral replication. ebov attachment and entry into human macrophages in vitro induces pro-inflammatory mediators including il-6, il-8, and tnf-α as early as 1 h postinfection. 55 noncardiogenic pulmonary edema is a recognized complication of evd, and human autopsy data support that alveolar macrophages are a target of ebov infection. ebov infection of alveolar macrophages in vitro resulted in an early, transient increase in cytokine and chemokine expression, 56 supporting that paracrine-soluble mediators of inflammation may contribute to vascular leakage in the lungs. gene expression responses of ebov-and marvinfected huh7 cells resulted in the global suppression of antiviral responses, including toll-like receptor (tlr), irf3, and protein kinase r (pkr)-mediated pathways. 57 however, signal transducers and activators of transcription (stat) phosphorylation in ebov-and marv-infected cells were differentially modulated. ebov-mediated ifn inhibition has been well characterized and is thought to be attributable to ebov proteins vp24 and vp35. 58 interestingly, restv infection, which does not induce clinical illness in humans, resulted in the activation of >20% of the ifn-stimulated genes (isgs). kinome analysis of ebola virus. hepatocytes are an early target of ebov infection, directly contributing to diffuse hepatic necrosis observed in fatal cases. ebov infection of huh7 cells has been evaluated by kinome analyses, shedding light on liver pathogenesis in evd. 59 ebov infection of huh-7 cells resulted in temporal modulation of the tgf-β signaling pathway as compared to mock-infected cells. pathway ora demonstrated that multiple tgf-β-mediated signaling pathways were up-regulated at 1 and 24 h post ebov infection. furthermore, these responses were associated with changes in the expression patterns of multiple cellular proteins associated with a mesenchyme-like transition. these included the upregulation of matrix metalloproteinase 9, n-cadherin, and fibronectin and down-regulation of e-cadhering and claudin 1. in this process cells lose polarity and cell-to-cell adhesion transforming into mesenchymal stem cells that contribute to wound healing or organ fibrosis; however, the role of these events in ebov infection remains to be elucidated. additional analysis demonstrated that inhibition of pi3k/akt, erk/ mapk, or pkc pathways with kinase inhibitors reduced ebov replication when administered prophylactically or therapeutically. supporting this observation, a subset of kinase inhibitors administered to ebov-infected mice reduced lethality. defining mechanisms by which kinase inhibitors show benefit in these models will better clarify their role as potential therapeutics. monkeypox virus. mpxv, a member of the genus orthopoxvirus, causes zoonotic infections with a case fatality rate of ∼11%. 60 mpxv, vaccinia virus (vacv), cowpox virus (cpxv), ectromelia virus, and variola virus (varv), the etiologic agent of human smallpox, comprise the orthopoxviridae family of viruses. mpxv was first isolated in 1958 from cynomolgus macaques in denmark; however, human mpxv infections were not recognized until 1970 following the isolation of the virus from a suspected case of smallpox infection in the democratic republic of congo. 61 mpxv is composed of two distinct clades that are genetically, clinically, and geographically distinct. the congo basin mpxv (central african mpxv) clade is considered to have both higher lethality and morbidity than the west african mpxv clade as demonstrated from comparative infection models in various animal species (including nonhuman primates, mice, prairie dogs, and ground squirrels) and as well natural infection in humans. 61 64 although human mpxv infections have been recorded in west africa, the majority of human mpxv infections have occurred in the congo basin region of central africa, largely in the democratic republic of congo. 60 clinical findings in mpxv infections. clinical and epidemiological information regarding human mpxv disease has been derived from enhanced surveillance campaigns in the congo basin. 61 from this work, it has been demonstrated that human mpxv infection and illness largely mirror those of discrete, ordinary smallpox. 61 the incubation period for both viruses (varv and mpxv) is 7−17 days with an initial febrile prodromal period of 1−4 days. this prodromal period is normally accompanied by fever, headache, backache, malaise, and prostration. 61 the rash period for both smallpox and mpxv (including lesion appearance and desquamation) normally occurs 14−28 days postinfection with highly similar appearance, distribution, and progression of lesions. 60, 61 as with smallpox, mpxv-associated rash progresses through macular, papular, vesicular, and pustular phases. a second febrile period occurs when the lesions become pustular and is often associated with deteriorating conditions in the patient. lymphadenopathy (maxillay, cervical, or inguinal) is often associated with mpxv infections prior to, or concomitant with, rash development but is absent in varv infections. it has been postulated that this reflects the effective generation of host immune responses during mpxv infection as compared to varv; however, this has yet to be validated. 60, 61 severe complications have been noted late in the course of mpxv infection, including pulmonary distress or bronchopneumonia, corneal scarring and permanent vision loss, and encephalitis. 60 severe dehydration due to excessive vomiting or diarrhea may also occur. long-term sequelae in survivors are most commonly associated with pitted scarring. soluble immune mediators associated with mpxv infections. although mpxv infections in humans have been recorded for over four decades, there has been little information review regarding host immune responses during the course of natural infection. as disease presentation is highly similar during mpxv and varv infections, it has been postulated that immune responses would likely be highly conserved. recently, johnston et al. provided the first empirical evidence for a relationship between cytokine responses and disease severity during mpxv infection. 65 serum cytokines were analyzed from 19 patients with confirmed mpxv infections ranging from mild to severe as assessed by the who smallpox lesion scoring system. 66, 67 serum concentrations of il-1β, il-1ra, il-2r, il-4, il-5, il-6, il-8, il-13, il-15, il-17, mcp-1, and rantes were elevated in all disease groups (mild to severe) as compared to normal serum concentrations. il-10 concentrations were also elevated in all disease groups and were proportional to disease severity. however, patients with serious mpxv disease had significantly higher concentrations of il-10 compared to all other disease groups. mpxv infection resulted in elevated mip-1α and mip-1β; mild cases had significantly elevated levels above the moderate or severe disease groups. serum concentrations of il-2r were elevated across all disease groups; however, patients with serious disease had significantly higher il-2r serum levels than those with mild to severe mpxv disease. gm-csf levels were significantly elevated only in those with serious mpxv disease as compared to normal serum ranges. on the basis of these observations, mpxv infection resulted in prominent t helper 2 (th2) and dampened th1 responses. transciptome analyses in mpxv infection. transcriptome analyses have largely been employed for the in vitro investigation of the molecular pathogenesis of mpxv infection. alkhalil et al. investigated the host transcriptome responses to mpxv infection during the first cycle of viral replication (3 and 7 h postinfection) in rhesus macaque kidney epithelial cells. 68 interestingly, mpxv infection resulted in a strong downregulation of host transcriptional responses. of the transcripts that met the authors' criteria for significance, 89% of the transcripts were found to be down-regulated at both postinfection time points. comparative functional analysis from both time points suggested that the primary biological functions associated with these down-regulated transcriptional responses were largely related to cell morphology, cell development, metabolic responses, and post-translational modifications. canonical pathway analysis demonstrated a general conservation in the identities of over-represented pathways at both time points including multiple growth factor signaling pathways, p53 signaling, and cell cycle-related pathways. more recently, bourquain et al. investigated host transcriptome responses in mpxv-infected hela cells, a cervical epithelial cell line. 69 at 6 h post-mpxv infection, only 1.1% of the transcripts analyzed were found to have >2fold changes in gene expression. in contrast to alkhalil et al., the majority of these transcripts (∼68%) were found to be upregulated as compared to mock-infected controls. functional analysis of all transcripts with >2-fold changes in gene expression demonstrated a strong over-representation of genes involved in the negative regulation of mapk signaling and the intracellular protein cascade. positive regulation of pathways related to toll-like receptor signaling, chemotaxis, and regulation of leukocyte migration was also predicted from the data. an investigation by rubins et al. compared the temporal host transcriptome response to mpxv in multiple human cells targeted by mpxv including primary macrophages, primary fibroblasts, and hela cells. 70 the trasncriptome of mpxv-infected fibroblasts was found to have the most significant changes where mpxv infection resulted in the depletion of ∼2000 genes by a factor of ≥3. interestingly, mpxv infection resulted in the broad repression of many transcripts related to innate immune responses in all cell types tested. in contrast, inactivated mpxv resulted in strong upregulation of innate immune responses in all of the cell types. it was also noted that mpxv infection resulted in strong cytopathic effects across all of the cell types in contrast to an almost universal repression of innate immune responses. kinome analyses in mpxv infection. human mpxv infections and infection models of mpxv in various animal species have demonstrated that the congo basin mpxv clade is more virulent than the west african mpxv clade. however, there has been a paucity of information regarding the underlying molecular mechanisms mitigating these virulence differences. furthermore, previous investigations focusing on gene expression or proteomic changes during mpxv infection have focused solely on congo basin mpxv. to address this, host kinome analysis was performed on congo basin and west african mpxv-infected human monocytes, a host cell targeted by orthopoxviruses. 71 as the genomes of both mpxv clades demonstrate considerable diversity in the regions coding host response modifier proteins, and in particular in genes associated with anti-apoptotic activities, it was postulated that the virulence differences of the two mpxv clades may be related to differential modulation of host cellular responses. hierarchical clustering of the kinome data sets suggested limited similarities at the level of host kinase modulation between the two mpxv clades. the congo basin mpxv kinome data set clustered most strongly with the kinome data set from cpxv-infected monocytes and moderately with the vacv-infected monocyte data set. both cpxv and vacv can cause serious disease in humans. the pathway ora of the kinome data demonstrated that congo basin mpxv infection resulted in strong down-regulation of a large proportion of host cell responses, most notably apoptosis, in comparison to west african mpxv. biological validation through fluorescenceactivated cell sorting (facs) and caspase 3 activity analyses confirmed this phenomenon. from the perspective of individual phosphorylation events, the kinome data also suggested that akt phosphorylation at ser473 was increased in congo basin mpxv-infected cells as compared to west african mpxv-infected cells. pharmacologic inhibition of akt phosphorylation at ser473 resulted in a >250-fold inhibition of congo basin mpxv virus yields, whereas those for west african mpxv were unaffected. prior investigations with cpxv and vacv demonstrated that pharmacological inhibition of akt resulted in decreased viral yields for both viruses. 72 overall, this investigation provided significant insight into the host cellular response differences between the two mpxv clades. emerging and re-emerging pathogens are a continual threat to global health. in recent years, disease outbreaks associated with sars and the 2009 influenza pandemic have also demonstrated that these pathogens can have considerable effects on local, national, and international economies. as a consequence, regional outbreaks of emerging and re-emerging pathogens can have deleterious effects on global stability. thus, it is prudent that a concerted effort is employed to assimilate data that bridge both clinical and molecular information in investigations review of these pathogens. these efforts will not only provide considerable context in regard to the molecular events that potentiate clinical manifestations of pathogenesis but also better inform the design and implementation of novel therapeutics. to this end, global analyses of host molecular responses can provide considerable insight into the complex molecular events that underlie cellular responses. indeed, transcriptome analyses have provided important information regarding host transcriptional responses during emerging and re-emerging pathogen infection. these investigations often provide critical insight into the kinetics of host immune responses during the course of infection as well as mechanistic information regarding the cellular intermediates involved in these processes. however, the role of ptms in the regulation of these events cannot be captured by traditional transcriptome technologies. in particular, the role of kinase-mediated regulation of cell signaling pathways has remained poorly understood. given the central role of kinases in the regulation of cellular processes (e.g., homeostasis, metabolism, proliferation, and stress responses), it is of inherent importance that future investigations also address the role of the kinome in the cellular response to pathogen insult. furthermore, kinomics also provides a mechanism for the identification of novel therapeutic targets based on the direct assessment of the activation state of cell signaling pathways. for example, proinflammatory responses during early stages of infection, and in particular the dysregulation of specific cytokines or cell signaling events that contribute to these, may represent potential therapeutic targets in the early stages of highconsequence viral pathogen infection. however, the selection of immunomodulatory therapeutics that target these dysregulated host responses is complicated by the regulatory events (i.e., kinase-mediated cell signaling events) that occur upstream of changes in gene expression. in addition, mrna is subject to a variety of regulatory processes (including gene silencing, mrna stability, translational efficiencies, protein turnover, enzyme/substrate subcellular sequestration, and/or protein activation/repression ptms). thus, from the standpoint of therapeutic discovery, the sole reliance on technologies for the global investigations of host responses that do not account for these regulatory processes or the role of ptms in the modulation of cellular responses could impede the identification of efficacious therapeutics. to this end, kinome analysis may also facilitate the identification of immunomodulatory therapeutics that have gained licensure through analysis of a quantifiable biological event (kinase-mediated phosphorylation) or for identifying novel host therapeutic targets for which therapeutics could be designed/developed. furthermore, kinase inhibitors may serve as primary or adjunctive therapies for emerging infectious diseases. in addition, preclinical data and the increasing number of kinase inhibitors that have gained regulatory approval for cancer and other maladies suggest this approach is feasible and efficacious. from the perspective of this review, kinome investigations have identified several therapeutic targets and licensed kinase inhibitors that have impaired viral replication in vitro and reduced the severity of disease in vivo (table 1) . for example, it has been demonstrated that the erk/mapk and pi3k/akt/mtor signaling pathways have a role in viral propagation during mers-cov infection. 37 indeed, licensed kinase inhibitors that targeted these pathways (i.e., everolimus, selumetinib, and trametinib) resulted in decreased viral replication in vitro when added prior to, or following, infection. furthermore, the pharmacologic inhibition of pi3k and pkc following ebov infection provided partial protection in a lethal model of evd in mice. 59 it should be noted that although the modulation of an individual kinase may have suppressive effects on infection (i.e., viral replication), this might not provide the level of inhibition required to completely negate viral escape. in addition, given the ability of many cell signaling pathways to signal through both canonical and noncanonical mechanisms, inhibition at a single intermediary point within a pathway may not provide the overall level of inhibition required to negate a deleterious response (i.e., viral replication, changes in cellular phenotypes, etc.). thus, although previous investigations have demonstrated that individual kinases or cell signaling pathways may represent novel targets for anti-infective therapies, it is prudent that future investigations also examine combinations of inhibitors for efficacy and anti-infective activities. furthermore, the targeting of cell signaling pathways at or near the origin point for the cell signaling cascade should also be examined as these likely represent stronger inhibitory targets given the generally reduced branching of cell signaling networks at or near the cell receptor. in addition to host-directed therapeutic targeting, kinomics also confers the ability to identify novel inhibitors of pathogens through detailed characterization of the viral life cycle. hostmediated ptms, and in particular kinase-mediated phosphorylation, have been implicated in the viral life cycle and pathogenesis for several members of the order mononegavirales, including ebov. 73−75 thus, therapeutic targeting of kinases may represent a novel therapeutic strategy that can be employed to modulate host-centric or pathogen-centric molecular events during infection. for example, in silico prediction of viral protein phosphorylation sites provides a mechanism for the construction and, ultimately, the annotation of viral protein ptms that are critical to the viral life cycle. furthermore, the use of kinome peptide arrays has extended beyond the human kinome and now extends to a variety of animal species. 76−78 it has been suggested that the interspecies phenotypic variability may reflect differences in phosphorylation sites found within the proteome. 78 thus, the development of species-specific kinome peptide arrays provides additional utility for kinome analysis as peptide arrays representing traditional laboratory animal species (mouse, guinea pig, nonhuman primate) can be employed to detail the species-specific host response. the results from such analyses, and the overlap between these and those described previously from the analysis of human infections, may inform review the selection of appropriate animal models that meet regulatory approval through the fda animal efficacy rule. 29 taken together, it is of inherent importance that future investigations of emerging and re-emerging pathogens address the complex nature of biological responses. thus, molecular investigations of pathogenesis should be guided by available knowledge regarding the clinical and pathologic manifestations of disease. indeed, technologies that provide further granularity into the precise molecular events that potentiate cellular responses during the course of infection will assist investigations of emerging and re-emerging pathogens and the identification of novel therapeutic targets. to this end, kinomics-based analyses of host responses provide a mechanism to directly address the cellular events at the level of specific cell signaling phenomena that underlie the biological responses and, ultimately, the clinical presentation of disease for emerging infectious pathogens. global trends in emerging infectious diseases a new antibiotic and the evolution of resistance signaling − 2000 and beyond protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling the protein kinase complement of the human genome sample preparation and profiling: probing the kinome for biomarkers and therapeutic targets: peptide arrays for global phosphorylation mediated signal transduction peptide arrays for kinome analysis: new opportunities and remaining challenges protein kinases − the major drug targets of the twenty-first century? the druggable genome ncats launches drug repurposing program overcoming drug development bottlenecks with repurposing: old drugs learn new tricks statistical analysis of protein kinase specificity determinants analysis of yeast protein kinases using protein chips a systematic approach for analysis of peptide array kinome data piika 2: an expanded, web-based platform for analysis of kinome microarray data two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease human coronavirus oc43 causes influenza-like illness in residents and staff of aged-care facilities in melbourne spectrum of clinical illness in hospitalized patients with "common cold" virus infections an outbreak of coronavirus oc43 respiratory infection in normandy molecular evolution of the sars coronavirus during the course of the sars epidemic in china middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission family cluster of middle east respiratory syndrome coronavirus infections isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory acs infectious diseases middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferonstimulated gene responses. mbio 5 cytokine systems approach demonstrates differences in innate and pro-inflammatory host responses between genetically distinct mers-cov isolates antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study ebola and marburg hemorrhagic fever reston ebolavirus in humans and animals in the philippines: a review clinical features and pathobiology of ebolavirus infection and working group of the u.s.− euopean clinical network on clinical management of ebola virus diseases patients in the markedly elevated levels of interferon (ifn)-gamma, ifn-alpha, interleukin (il)-2, il-10, and tumor necrosis factor-alpha associated with fatal ebola virus infection inflammatory responses in ebola virus-infected patients defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in ebola virus-infected patients early immune responses accompanying human asymptomatic ebola infections human asymptomatic ebola infection and strong inflammatory response analysis of human peripheral blood samples from fatal and nonfatal cases of ebola (sudan) hemorrhagic fever: cellular responses, virus load, and nitric oxide levels human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis the temporal program of peripheral blood gene expression in the response of nonhuman primates to ebola hemorrhagic fever discovery of swine as a host for the reston ebolavirus immunopathogenesis of severe acute respiratory disease in zaire ebolavirus-infected pigs ebola virion attachment and entry into human macrophages profoundly effects early cellular gene expression viral replication and host gene expression in alveolar acs infectious diseases review macrophages infected with ebola virus (zaire strain) global suppression of the host antiviral response by ebolaand marburgviruses: increased antagonism of the type i interferon response is associated with enhanced virulence assessing the contribution of interferon antagonism to the virulence of west african ebola viruses ebola virus modulates transforming growth factor beta signaling and cellular markers of mesenchyme-like transition in hepatocytes human monkeypox status of human monkeypox: clinical disease, epidemiology and research a tale of two clades: monkeypox viruses virulence and pathophysiology of the congo basin and west african strains of monkeypox virus in non-human primates clinical manifestations of human monkeypox influenced by route of infection cytokine modulation correlates with severity of monkeypox disease in humans smallpox dna vaccine protects nonhuman primates against lethal monkeypox genomic variability of monkeypox virus among humans comparison of host cell gene expression in cowpox, monkeypox or vaccinia virus-infected cells reveals viral-specific regulation of immune responses stunned silence: gene expression programs in human cells infected with monkeypox or vaccinia virus systems kinomics demonstrates congo basin monkeypox virus infection selectively modulates host cell signaling responses as compared to west african monkeypox virus activation of the pi3k/akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication dynamic phosphorylation of vp30 is essential for ebola virus life cycle structural phosphoprotein m2-1 of the human respiratory syncytial virus is an rna binding protein phosphorylation status of the phosphoprotein p of rinderpest virus modulates transcription and replication of the genome a comparison of the chicken and turkey proteomes and phosphoproteomes in the development of poultry-specific immuno-metabolism kinome peptide arrays characterization of the host response to pichinde virus infection in the syrian golden hamster by speciesspecific kinome analysis computational analysis of the predicted evolutionary conservation of human phosphorylation sites repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection identification of novel cellular targets for therapeutic intervention against ebola virus infection by sirna screening inhibition of lassa virus and ebola virus infection in host cells treated with the kinase inhibitors genistein and tyrphostin productive replication of ebola virus is regulated by the c-abl1 tyrosine kinase pyridinyl imidazole inhibitors of p38 map kinase impair viral entry and reduce cytokine induction by zaire ebolavirus in human dendritic cells variola and monkeypox viruses utilize conserved mechanisms of virion motility and release that depend on abl and src family tyrosine kinases the authors declare no competing financial interest. review key: cord-292836-1o2ynvy3 authors: ogimi, chikara; kim, yae jean; martin, emily t; huh, hee jae; chiu, cheng-hsun; englund, janet a title: what’s new with the old coronaviruses? date: 2020-04-21 journal: j pediatric infect dis soc doi: 10.1093/jpids/piaa037 sha: doc_id: 292836 cord_uid: 1o2ynvy3 coronaviruses contribute to the burden of respiratory diseases in children, frequently manifesting in upper respiratory symptoms considered to be part of the “common cold.” recent epidemics of novel coronaviruses recognized in the 21st century have highlighted issues of zoonotic origins of transmissible respiratory viruses and potential transmission, disease, and mortality related to these viruses. in this review, we discuss what is known about the virology, epidemiology, and disease associated with pediatric infection with the common community-acquired human coronaviruses, including species 229e, oc43, nl63, and hku1, and the coronaviruses responsible for past world-wide epidemics due to severe acute respiratory syndrome and middle east respiratory syndrome coronavirus. understanding the history and epidemiology of the common community human coronaviruses (hcovs) and those responsible for recent past epidemics is crucial to the control and treatment of novel coronaviruses. the epidemiology of previously described community coronaviruses in children is relatively well known from surveillance studies. however, important clinical and laboratory details of these viruses, such as duration of shedding, transmission rates, viral load over time, immunity, and morbidity in high-risk populations, are not well characterized. such information becomes more important and clinically relevant as we consider world-wide pandemics such as the ongoing severe acute respiratory syndrome coronavirus 2 (sars-cov-2) outbreak. coronaviruses are already known to be ubiquitous viruses and recognized as pathogens in both humans and animals. a coronavirus was first isolated as a causative agent of bronchitis in birds in 1937 [1] and was originally discovered in humans during studies that evaluated the common cold. the history of the discovery of hcovs is shown in table 1 [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . the common human coronaviruses known today include the species 229e, oc43, nl63, and hku1. these 4 viruses are primarily viewed as relatively benign respiratory pathogens in humans, typically causing upper respiratory tract disease and common cold symptoms. by contrast sars-cov and middle east respiratory syndrome coronavirus (mers-cov) are highly pathogenic in humans, with high rates of severe pneumonia and fatal outcomes [21] . currently, the novel coronavirus sars-cov-2 is spreading worldwide, causing anxiety, disease, and mortality [22, 23] . despite the heightened interest, limited pediatric data are available regarding either the community hcovs or the newer pathogenic species in children. here, we review what is known about hcov infections in children in the precoronavirus disease 2019 (covid-19) era. coronaviruses belong to the coronaviridae family in the nidovirales order. the coronavirus subfamily is further classified into 4 genera known as the alpha, beta, gamma, and delta coronaviruses. these viruses cause a wide variety of generally species-specific illness in mammals and birds, including chickens, turkeys, bats, rats, dogs, cats, piglets, and whales [21] . coronaviruses were named for their characteristic crown-like surface projections seen in electron micrographs that correspond to large surface spike proteins. these viruses are enveloped, nonsegmented, positive-sense rna viruses and have the largest identified viral rna genomes, with an approximate length of 30 kilobases [24] . only alpha and beta coronaviruses are known to infect humans. both hcovs 229e and nl63 are alpha coronaviruses and hcov-hku1, hcov-oc43, mers-cov, and sars-cov are beta coronaviruses (table 1) [24, 25] . phylogenetic analysis has revealed that sars-cov-2 also falls within the beta coronavirus in the same subgenus as the sars-cov, but in a different clade [26] . all 7 coronaviruses that infect humans are believed to have originated in bats [27, 28] . based on modeling, it is speculated that hcov-oc43 was transmitted to humans around 1890 [29] . bats also appear to be the original host of sars-cov-2, and it is hypothesized that currently unknown wild animal(s) sold at the huanan seafood market in wuhan, china, might have played a role as an intermediate host to humans [26] . hcovs oc43 and 229e were isolated from nasal cavities of people with the common cold in the 1960s. in the 1970s, studies that used serology and viral culture linked hcovs 229e and oc43 with 8% of cases of lower respiratory tract infection (lrti) in hospitalized infants [30, 31] . poor replication in tissue culture and a lack of cytopathic effect in early attempts at culture were major obstacles in making further progress in the field. the development and widespread use of molecular diagnostics as well as the emergence of sars-cov in 2003 significantly accelerated coronavirus research. subsequently, new hcovs nl63 and hku1 were identified and are frequently seen in children. additional pathogenic coronavirus species and outbreaks with mers-cov and, recently, sars-cov-2 have led to increased interest, research, and concern [21, 23] . community-acquired hcovs species oc43, nl63, hku1, and 229e are found worldwide in temperate and tropical countries, as well as low-, middle-, and high-income countries [32, 33] . hcov infection can occur at any time of the year, with unpredictable year-to-year patterns, and outbreaks are reported in some years and in certain parts of the world. all 4 species may circulate yearly and even simultaneously [34] , with the highest rates of hcov infection seen in winter and spring months in temperate climates [21] . almost all surveillance data indicate hcov-oc43 is the most common species. a large prospective surveillance study conducted in norway from 2006 to 2015 that enrolled all hospitalized children aged ≤16 years with respiratory tract infections revealed that hcovs oc43 and nl63 were detected most frequently and were epidemic every second winter [35] . hcov-hku1 was prevalent every second winter during the year when detection rates for hcovs oc43 and nl63 were low; hcov-229e was the least common. seroprevalence data suggest that exposure is common in early childhood [36] . more than 90% of adults are seropositive for at least 1 hcov species [37] . a recent prospective active surveillance study of young infants in nepal that used multiplex polymerase chain reaction (pcr) showed that hcovs are less common in neonates compared with older infants [32] . outbreaks of variable sizes have been reported sporadically in medical students, young asthmatic children, and neonatal intensive care units [38] [39] [40] [41] . for example, an outbreak of hcov-229e was reported in a neonatal intensive care unit where 92% of infected preterm neonates developed symptoms, including worsening respiratory conditions and bradycardia [42] . potential nosocomial transmission by staff members was speculated. large surveillance studies of children and adults to evaluate the prevalence of all major respiratory viruses using multiplex pcr have been conducted in many settings, showing that hcov infections are the fourth or sixth most common virus detected overall and across all age groups [33, 43] . coinfections are relatively common, especially in children aged ≤5 years. a prospective surveillance study in norway demonstrated that annual hospitalization rates of children with lrti associated with hcov detection were 1.5 and 2.8 per 1000 children aged <5 years and aged <1 years, respectively [35] . the mode(s) of hcov transmission is not known [21, 25] . it is assumed that transmission occurs via a combination of droplet and direct/indirect contact, similar to other respiratory viruses. hcovs oc43 and 229e appear to be primarily transmitted during the first few days of illness when symptoms and viral load in the respiratory tract are highest. the incubation period of hcov-229e was 2 to 5 days (median, 3 days) in adults in challenge studies and has not yet been clearly defined for other hcovs or in children [25, 44] . the transmission of hcov in community epidemics is impacted by the characteristics of hcov infection and shedding in children. children have higher attack rates, as high as 78% within households, compared with adults, as shown in an active surveillance study in kenya [45] . these data complement community studies in the 1960s that showed a higher frequency of hcov-229e in families with children aged <15 years [36] , a higher rate of hcov-oc43 infection in children aged <5 years [46] , and frequent clustering of infections within families [46] . the attack rate of hcov in childcare settings has also been shown to be higher with younger age [47] . children may shed hcovs for extended periods of time after infection, potentially leading to additional transmissions within close-contact settings. there are very limited data regarding duration of shedding in children. in prospective childcare studies, 34% of children with hcov had detectable virus at 1 week or more following symptom onset, with shedding documented for up to 18 days ( figure 1 ) [47, 48] . a longitudinal study of weekly nasal swabs taken from symptomatic and asymptomatic adults and children similarly found that viral detection extended beyond 1 week [49] . children aged <5 years with hcov detection were frequently asymptomatic, especially with hcov-229e. these findings reinforce serologic-based findings of asymptomatic infection in 7% of children with hcov-229e in the 1960s [50] . clinical symptoms associated with the 4 common hcovs generally appear to be indistinguishable from cold symptoms or influenza-like illness (rhinorrhea, sore throat, cough, wheezing, and fever) associated with other respiratory viruses [21, 25, 51] and generally are similar in children and adults. hcovs 229e and oc43 were shown to be pathogenic in adult human volunteer studies. cold symptoms (eg, sore throat, rhinorrhea, cough) are very similar to those caused by other respiratory viruses [39, 44] . it is assumed that hcovs nl63 and hku1 have similar pathogenicity, although this has not been proven in challenge studies. the frequent detection of hcovs in asymptomatic patients as well as coinfection with other respiratory viruses make the interpretation of hcov pathogenesis and clinical findings challenging. a multicenter, prospective surveillance study conducted in the united states evaluated the prevalence of hcovs among hospitalized children with acute respiratory illness and demonstrated that the prevalence of hcovs was similar in both asymptomatic and symptomatic groups of children [52] . in contrast, a prospective community surveillance study of respiratory viruses in utah showed hcov detection was often associated with symptoms in all age groups, suggesting hcov infection might have been previously underestimated as an etiology of respiratory tract infection [49] . an analysis of new and old data reveals, not surprisingly, that viral coinfections are less common in children with influenza virus (9% of influenza in seattle, washington; 27% in seoul, south korea) than in children infected with a community hcovs (43% of hcov infections in seattle and 42% in seoul; table 2 ). a norwegian prospective surveillance study compared children hospitalized with respiratory tract [47, 48] . children attending group childcare were tested prospectively for respiratory viruses at each acute respiratory illness (ari). swabs were collected weekly from ari onset until symptoms were nonworsening and swabs were negative by real-time-polymerase chain reaction [47] . human coronavirus (hcov)-positive swabs (for all species) are represented by filled circles, and negative swabs are represented by open circles. weeks on x-axis were calculated as weeks since symptom onset with an hcov (+) acute respiratory illness. the lower limit of positivity was set at 1000 copies/ml for this analysis. sequencing of hcov isolates was not performed. infections vs asymptomatic children admitted for elective surgery [35] . multivariable analyses suggest that higher viral load (cycle threshold <28 by pcr), codetection of generally symptomatic viruses (respiratory syncytial virus [rsv], human metapneumovirus, influenza, and parainfluenza virus), younger age (<2 years), being female, and high-risk underlying conditions (lung, heart, or neurologic disorder) were associated with symptomatic children. children with only hcov detected were more likely to have a higher viral load compared with those with viral coinfection. associations of hcovs with lrti or asthma exacerbation in children have been reported [21, 31, 34, 38, 53, 54] . clearly, respiratory distress and pneumonia have been well documented in children with only hcov detected as a potential pathogen [34, 55] . specific hcov species and associated risk factors for disease severity have been evaluated, although some studies did not address potential confounders, such as the presence of coinfection [56] . one retrospective study of 212 hospitalized children with hcov (54 with and 158 without viral coinfection) showed that similar numbers of children received respiratory support and intensive care [57] . bivariate analyses showed that younger age (<2 years) and chronic complex medical conditions (cardiovascular, respiratory, genetic, or congenital) were associated with increased disease severity. no specific species were associated with severity of illness. although the presence of viral coinfection was not associated with increased disease severity, the analysis did not assess the impact of specific types of coinfections. a retrospective analysis of 1237 children who presented to seattle children's hospital (washington) for acute care with detected hcovs showed that disease severity did not vary by hcov species [58] . younger age, presence of underlying pulmonary disorder, and presence of coinfection, particularly rsv, were associated with increased likelihood of lrti in multivariable models. the impact of rsv coinfection with hcov has been described in other studies [59] . like other respiratory viruses, hcovs have been detected in middle ear effusions and nasal secretions in children with otitis media, and its possible etiology of acute otitis media has been implicated [60, 61] . the significant association between hcov-nl63 and croup has been reported, with evidence suggesting that hcov-nl63 is the second most common etiology of croup following parainfluenza virus type 1 [62, 63] . hcov has been described as a possible etiology of severe pneumonia in immunocompromised hosts [64] [65] [66] [67] . data are limited for this high-risk population and particularly are lacking in pediatric hematopoietic cell transplantation (hct) recipients [68] . among 404 children aged <18 years who underwent allogeneic hct from april 2008 to september 2018 at seattle children's hospital, hcovs were the third most common respiratory viruses detected post-hct following rhinovirus and parainfluenza virus (preliminary data). the cumulative incidence of hcovs in children during the first 365 days following hct are shown in figure 2 . hcovs were detected in bronchoalveolar lavage (bal) specimens from 2 young children in this cohort. the detection of hcov in bal specimens among hct recipients and patients with hematologic malignancy was also evaluated, but only 1 of 35 patients was a child [69] . among 16 patients in that study (15 adults, 1 child) with hcov and without other coinfections identified by bal, 10 required oxygen support, suggesting a role of hcov as a significant respiratory pathogen. in a retrospective study of immunocompromised and nonimmunocompromised children with hcov detected in nasal specimens in seattle, the prevalence of lrti that required oxygen supplementation (severe lrti) was 15% (13/85) and 11% (122/1152), respectively [58] . multivariable models showed that immunocompromised state, presence of rsv, and underlying pulmonary disorder were associated with increased risk of severe lrti. a prospective surveillance study with weekly nasal sampling in 215 allogeneic hct recipients of all ages demonstrated that the median shedding duration of hcovs was 3 weeks (range, 0-22 weeks) [70] . our follow-up study suggests that high viral load, high-dose steroids, and myeloablative conditioning are associated with prolonged shedding (≥21 days) of hcov in allogeneic hct recipients (2 pediatric and 42 adult patients) [71] . using available nasal samples from patients with prolonged shedding, we performed whole-genome sequencing, which revealed only small and slow genomic changes, consistent with previously estimated evolution rates. this is in contrast to more rapid genomic changes associated with influenza infections [72] . in addition, an 18-year-old pediatric patient had 3 hcov species (oc43, hku1, 229e) detected sequentially over a 5-month period without an associated poor outcome. the role of hcovs as enteric pathogens in humans has been debated, perhaps in part because of the known enteric disease associated with coronavirus in animals, including dogs. risku et al investigated the presence of hcovs in stool of children with and without gastroenteritis. all 4 species were found in 2.5% of stool samples (22/878) from children with gastroenteritis, whereas 1.7 % of stool samples (2/112) from controls were positive for hcovs. however, other known enteric pathogens, such as rotavirus or norovirus, were also found in 18 of 22 stool samples in children with gastroenteritis. among 4 patients with only hcov detected in stool, 3 had respiratory symptoms [73] . another study examined both stool and nasopharyngeal swabs in hospitalized children with acute gastroenteritis and in controls [74] . hcovs were more frequently detected in patients with gastroenteritis than in controls (23/260, 8.8% vs 4/151, 2.6%, respectively). interestingly, in patients with gastroenteritis, more than half (13/23) had respiratory symptoms and hcovs were more frequently found in nasopharyngeal samples than in stool samples (22/256, 8 .6%, vs 6/260, 2.3%, respectively). based on these studies, the significance of hcovs as enteric pathogens appears minor. hcov-oc43 has been detected in the central nervous system of children with acute disseminated encephalomyelitis or fatal encephalitis [75, 76] . a prospective study investigated associations between hcovs detection and various clinical manifestations [77] . this latter study proposed an etiological role of hcovs in febrile seizures given that children with febrile seizures had higher rates of hcovs detection with higher viral load in nasopharyngeal swabs than those with bronchiolitis or gastroenteritis and healthy controls. however, an etiologic connection between hcovs and neurologic diseases remains unproven [78] . carefully conducted epidemiological studies have not demonstrated an association between hcovs and kawasaki disease [21, 25] . an outbreak of sars-cov occurred in east and southeast asia in early spring of 2003. this international outbreak began in a hotel in hong kong and ultimately spread to more than 20 countries. the etiologic agent of sars-cov is a novel coronavirus identified by multiple investigators [10] [11] [12] 79] . the virus was classified as a beta-coronavirus, lineage b. this virus uses angiotensin-converting enzyme 2 as a functional cellular receptor. the virus also binds to the c-type lectin cd209l (also known as l-sign) and dc-sign [80] [81] [82] . during the outbreak, approximately 8098 cases occurred with 774 deaths, resulting in an overall mortality rate of 9% [24] . in hong kong, about 5% of the cases were children and adolescents [83] . young children appeared to have a milder form of the disease [84] [85] [86] [87] . among laboratory-confirmed and probable pediatric sars-cov cases, the most common symptoms included fever (98%), cough (60%), nausea or vomiting (41%), and constitutional symptoms such as myalgia (29%), chills (28%), and headache (28%) [84] . clinical manifestations of sars-cov in children are nonspecific, and it can be very difficult to differentiate sars-cov from other respiratory tract infections without laboratory testing in the outbreak setting. however, certain features may provide a clue. a comparison of 15 pediatric patients with laboratory-confirmed sars-cov and 15 age-and sexmatched patients with culture-confirmed influenza in taiwan revealed that rates of fever, cough, and constitutional symptoms such as chills and myalgia were similar between the 2 groups, but patients with sars-cov had significantly less rhinorrhea, sputum production, and sore throat than those with influenza [88] . in general, respiratory and constitutional symptoms are similar in the beginning of the illness in children and adults. however, a much higher proportion of adult patients progress to severe pneumonia, even acute respiratory distress syndrome. in fact, despite a high mortality rate in adults (9.6%-16.7%) [83] , there were no fatalities documented in the pediatric population [84] [85] [86] [87] . a study reported outcomes of 5 pregnant women infected with sars-cov [89] ; the gestational ages of their infants ranged from 26 weeks to 32 weeks. in 3 of these 5 pregnant women, cesarean section was needed because of maternal conditions, including hypotension and worsening pulmonary function. however, a systematic search for perinatal transmission of the sars-cov did not detect the virus in any of the 5 babies. the first case of mers-cov was reported in a man hospitalized in jeddah, saudi arabia, in june 2012. he died of severe pneumonia and renal failure, and a novel coronavirus was isolated from his sputum [14] . this new beta-coronavirus was named mers-cov [13] . it belongs to lineage c and is closely related to tylonycteris bat coronavirus hku4 (ty-batcov hku4) and pipistrellus bat coronavirus hku5 (pi-batcov hku5) [13] . the dromedary camel is known to be the intermediate host of mers-cov. the widely expressed cell-surface protease dipeptidyl-peptidase 4 (also known as cd26) was identified as a functional receptor for host cell entry [90] . as of november 2019, the world health organization reported 2494 laboratory-confirmed cases of mers-cov infection in 27 countries with 858 deaths globally, resulting in an approximate 34% mortality rate [91] . all known mers-cov infections can be traced to countries in the middle east, primarily saudi arabia. most cases have been in adults with underlying chronic diseases or immunosuppression who live or travel in the arabian peninsula. among 1351 confirmed cases between 2012 and 2019, cases aged <18 years were less than 5% of all confirmed cases [92] . mers-cov infection in adults usually occurs as sporadic cases, healthcare-associated infection, or transmission within families [93] . however, most confirmed pediatric cases were secondary cases after exposures to others within the same family [94] . the proportion of children among those infected with mers-cov has consistently been reported as being relatively low compared with the general population. from june 2012 to april 2016, the proportion of pediatric patients was 1.6% (9 of 552) of all positive cases in saudi arabia [94] . among patients hospitalized in riyadh, saudi arabia, from april 2014 to november 2016, the proportion of 295 confirmed pediatric patients (aged <18 years) was 2.4%, with age ranging from 9 months to 17 years (supplementary table 1 ). clinical manifestations in pediatric patients have not been systematically described. among the 31 pediatric patients with mers-cov infection documented from june 2012 to april 19, 2016, 13 patients (42%) were asymptomatic [94] . in another study, among 7 pediatric patients identified from april 2014 to november 2016, 3 were asymptomatic; fever, cough, shortness of breath, diarrhea, and vomiting were observed in 4 patients [95] . although pediatric patients typically have mild disease, high-risk children with underlying conditions, including cystic fibrosis, nephrotic syndrome, and unidentified underlying conditions, died of mers-cov infection, with a fatality rate of 12% (4/33) (see supplementary table 1 ) [96, 97] . a recent study summarized mers-cov infection in 11 pregnant women [98] . the gestational ages ranged from 6 weeks to 38 weeks. among those 11 pregnant women, 7 (64%) were admitted to intensive care units and 3 (27%) died. of the 11 births, 3 (27%) died, 2 had documented intrauterine death at 34 weeks and 5 months of gestation, and 1 was delivered at 24 weeks but did not survive. recognition of the importance of community coronavirus disease due to 4 established hcovs has increased over the past 20 years, with widespread availability of molecular diagnostic methods. however, detailed information on pathogenesis, immunity, and viral characteristics of disease in children remains limited. recent and ongoing epidemics of novel coronaviruses in the 21st century have highlighted issues of zoonotic origins of transmissible respiratory viruses and potential transmission, disease, and mortality related to these viruses. the role of children in the spread of disease with these novel viruses remains unclear. as the current pandemic with sars-cov-2 unfolds, more information regarding the role of children in viral transmission and their clinical presentation and outcome will become more evident. further study of coronaviruses in humans is imperative. supplementary materials are available at journal of the pediatric infectious diseases society online. discovery, diversity and evolution of novel coronaviruses sampled from rodents in china cultivation of a novel type of common-cold virus in organ cultures a new virus isolated from the human respiratory tract identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans growth in suckling-mouse brain of "ibvlike" viruses from patients with upper respiratory tract disease some characteristics of hemagglutination of certain strains of "ibv-like" virus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome sars working group. a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus genome identified in a cluster of pneumonia cases-wuhan chinese researchers reveal draft genome of virus implicated in wuhan pneumonia outbreak molecular basis of binding between middle east respiratory syndrome coronavirus and cd26 from seven bat species viral infections of humans epidemiology and control molecular evolution of the sars coronavirus during the course of the sars epidemic in china sars-cov infection in a restaurant from palm civet human coronaviruses, including middle east respiratory syndrome coronavirus. in: feigin and cherry's textbook of pediatric infectious diseases first case of 2019 novel coronavirus in the united states a novel coronavirus from patients with pneumonia in china coronaviruses: an overview of their replication and pathogenesis human coronaviruses. in: principles and practice of pediatric infectious diseases genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding tracing the sars-coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event coronavirus infection in acute lower respiratory tract disease of infants coronavirus infection in acute lower respiratory tract disease of infants burden and risk factors for coronavirus infections in infants in rural nepal respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multicountry population sample clinical disease in children associated with newly described coronavirus subtypes human coronavirus in hospitalized children with respiratory tract infections: a 9-year population-based study from norway community-wide outbreak of infection with a 229e-like coronavirus in tecumseh, michigan development of a nucleocapsid-based human coronavirus immunoassay and estimates of individuals exposed to coronavirus in a u.s. metropolitan population the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance neonatal nosocomial respiratory infection with coronavirus: a prospective study in a neonatal intensive care unit coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study outbreaks of human coronavirus in a pediatric and neonatal intensive care unit extensive multiplex pcr diagnostics reveal new insights into the epidemiology of viral respiratory infections effects of a "new" human respiratory virus in volunteers viral etiology of severe pneumonia among kenyan infants and children the tecumseh study of respiratory illness. vi. frequency of and relationship between outbreaks of coronavirus infection epidemiology of viral respiratory tract infections in a prospective cohort of infants and toddlers attending daycare epidemiology of multiple respiratory viruses in childcare attendees community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study seroepidemiologic survey of coronavirus (strain 229e) infections in a population of children severity and outcome associated with human coronavirus oc43 infections among children new vaccine surveillance network. human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls community study of role of viral infections in exacerbations of asthma in 9-11 year old children the pediatric burden of human coronaviruses evaluated for twenty years coronavirus-associated pneumonia in previously healthy children epidemiology and clinical characteristics of human coronaviruses oc43, 229e, nl63, and hku1: a study of hospitalized children with acute respiratory tract infection in guangzhou, china epidemiology and clinical features of human coronaviruses in the pediatric population characteristics and outcomes of coronavirus infection in children: the role of viral factors and an immunocompromised state genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction viral upper respiratory tract infection and otitis media complication in young children croup is associated with the novel coronavirus nl63 role of human coronavirus nl63 in hospitalized children with croup human coronavirus oc43 pneumonia in a pediatric cancer patient with down syndrome and acute lymphoblastic leukemia fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency coronavirus 229e-related pneumonia in immunocompromised patients fatal lower respiratory tract disease with human corona virus nl63 in an adult haematopoietic cell transplant recipient a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients clinical significance of human coronavirus in bronchoalveolar lavage samples from hematopoietic cell transplant recipients and patients with hematologic malignancies human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution parallel evolution of influenza across multiple spatiotemporal scales detection of human coronaviruses in children with acute gastroenteritis detection of human coronaviruses in simultaneously collected stool samples and nasopharyngeal swabs from hospitalized children with acute gastroenteritis human coronavirus oc43 associated with fatal encephalitis detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis the role of human coronaviruses in children hospitalized for acute bronchiolitis, acute gastroenteritis, and febrile seizures: a 2-year prospective study human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? canadian severe acute respiratory syndrome study team. identification of severe acute respiratory syndrome in canada angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus pathogenesis of severe acute respiratory syndrome human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry probable secondary infections in households of sars patients in hong kong severe acute respiratory syndrome in children other members of the hospital for sick children sars investigation team. children hospitalized with severe acute respiratory syndrome-related illness in toronto severe acute respiratory syndrome in children: experience in a regional hospital in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children childhood severe acute respiratory syndrome in taiwan and how to differentiate it from childhood influenza infection infants born to mothers with severe acute respiratory syndrome dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus (mers-cov) mers situation update managing mers-cov in the healthcare setting middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia middle east respiratory syndrome coronavirus in pediatrics: a report of seven cases from saudi arabia middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature key: cord-271648-m2c5bvuj authors: ashour, hossam m.; elkhatib, walid f.; rahman, md. masudur; elshabrawy, hatem a. title: insights into the recent 2019 novel coronavirus (sars-cov-2) in light of past human coronavirus outbreaks date: 2020-03-04 journal: pathogens doi: 10.3390/pathogens9030186 sha: doc_id: 271648 cord_uid: m2c5bvuj coronaviruses (covs) are rna viruses that have become a major public health concern since the severe acute respiratory syndrome-cov (sars-cov) outbreak in 2002. the continuous evolution of coronaviruses was further highlighted with the emergence of the middle east respiratory syndrome-cov (mers-cov) outbreak in 2012. currently, the world is concerned about the 2019 novel cov (sars-cov-2) that was initially identified in the city of wuhan, china in december 2019. patients presented with severe viral pneumonia and respiratory illness. the number of cases has been mounting since then. as of late february 2020, tens of thousands of cases and several thousand deaths have been reported in china alone, in addition to thousands of cases in other countries. although the fatality rate of sars-cov-2 is currently lower than sars-cov, the virus seems to be highly contagious based on the number of infected cases to date. in this review, we discuss structure, genome organization, entry of covs into target cells, and provide insights into past and present outbreaks. the future of human cov outbreaks will not only depend on how the viruses will evolve, but will also depend on how we develop efficient prevention and treatment strategies to deal with this continuous threat. coronaviruses (covs) were discovered in the 1960s and they were classified under family coronaviridae, which is the largest family within the order nidovirales ( figure 1 ) [1] . family coronaviridae encompasses two subfamilies: subfamily orthocoronavirinae and subfamily torovirinae ( figure 1 ) [1] . subfamily orthocoronavirinae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus ( figure 1 ) [1] . covs are typically harbored in mammals and birds and are common in camels, cattle, cats, bats, and other animals [2] . alpha and betacoronaviruses circulate in mammals, including bats ( figure 1 ) [2] . gammacoronaviruses mostly infect avian species the first discovered covs were ibv that causes respiratory disease in chickens and the human covs, human cov-229e (hcov-229e) and human cov-oc43 (hcov-oc43), which cause the common cold in humans [8, 9] . since the emergence of hcov-229e and hcov-oc43, several other hcovs were discovered, such as severe acute respiratory syndrome-cov (sars-cov) in 2002, hcov-nl63 in 2004, hcov-hku1 in 2005, middle east respiratory syndrome-cov (mers-cov) in 2012 [10] . starting december 2019, there were reports of patients presenting with severe viral pneumonia in the city of wuhan, china [11] . sequencing of the virus from these patients has identified a novel cov as the causative agent of this respiratory disease [11] . the 2019 novel cov virus (2019-ncov) was recently named sars-cov-2 by the world health organization (who). the disease caused by sars-cov-2 has been named covid-19. prior to 2002, covs were treated as nuisances but never as serious viruses. things changed after the emergence of sars-cov, which caused serious illnesses and deaths in 2002-2003 [12] . unlike all human covs that cause mild respiratory symptoms, sars-cov, mers-cov, and sars-cov-2 are associated with serious respiratory diseases [12, 13] . since its emergence, the sars-cov-2 has drawn well-deserved attention from the world. efforts are underway in an attempt to control this new cov outbreak. covs, including the newly discovered sars-cov-2, are spherical positive single-stranded rna viruses that are characterized by spike proteins projecting from the virion surface [14, 15] . the spherical morphology of the viral particle together with the spike projections led to the name coronavirus from the latin word corona meaning crown, due to the appearance of the virus as a royal crown under the electron microscope [14, 15] . covs are enveloped viruses (envelope is a lipid bilayer derived from the host cell membrane) with the viral structure formed primarily of structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, and hemagglutininfigure 1 . classification of different types of coronaviruses within the family coronaviridae, subfamily orthocoronavirinae, and the respective genera: alpha-, beta-, gamma-, and deltacoronaviruses. the sars-cov-2 is classified as a betacoronavirus. the first discovered covs were ibv that causes respiratory disease in chickens and the human covs, human cov-229e (hcov-229e) and human cov-oc43 (hcov-oc43), which cause the common cold in humans [8, 9] . since the emergence of hcov-229e and hcov-oc43, several other hcovs were discovered, such as severe acute respiratory syndrome-cov (sars-cov) in 2002, hcov-nl63 in 2004, hcov-hku1 in 2005, middle east respiratory syndrome-cov (mers-cov) in 2012 [10] . starting december 2019, there were reports of patients presenting with severe viral pneumonia in the city of wuhan, china [11] . sequencing of the virus from these patients has identified a novel cov as the causative agent of this respiratory disease [11] . the 2019 novel cov virus (2019-ncov) was recently named sars-cov-2 by the world health organization (who). the disease caused by sars-cov-2 has been named covid-19. prior to 2002, covs were treated as nuisances but never as serious viruses. things changed after the emergence of sars-cov, which caused serious illnesses and deaths in 2002-2003 [12] . unlike all human covs that cause mild respiratory symptoms, sars-cov, mers-cov, and sars-cov-2 are associated with serious respiratory diseases [12, 13] . since its emergence, the sars-cov-2 has drawn well-deserved attention from the world. efforts are underway in an attempt to control this new cov outbreak. covs, including the newly discovered sars-cov-2, are spherical positive single-stranded rna viruses that are characterized by spike proteins projecting from the virion surface [14, 15] . the spherical morphology of the viral particle together with the spike projections led to the name coronavirus from the latin word corona meaning crown, due to the appearance of the virus as a royal crown under the electron microscope [14, 15] . covs are enveloped viruses (envelope is a lipid bilayer derived from the host cell membrane) with the viral structure formed primarily of structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins, and hemagglutinin-esterase (he) protein in some betacoronaviruses [16] . the s, m, and e proteins are all embedded in the viral envelope; pathogens 2020, 9, 186 3 of 15 however, n protein interacts with the viral rna and is located in the core of the viral particle, forming the nucleocapsid [16] . the s protein is a heavily glycosylated protein that forms homotrimeric spikes on the surface of the viral particle and mediates viral entry into host cells [17] . in some covs, each monomer of the homotimeric s protein exists as two subunits (s1 and s2) on the viral particle due to cleavage of s protein by host furin-like proteases during viral replication [17, 18] . however, in other covs including sars-cov, s protein forms s1 and s2 domains, remains intact on viral particles, and only gets cleaved inside endocytic vesicles during viral entry [19, 20] . the m protein is one of the most important proteins in the virion structure. it exists in higher quantities than any other protein in the viral particle, in contrast to the e protein which is found in small quantities within the virion [21] . the difference in abundancy may due to the fact that m protein gives the virus its shape and is critical together with e protein in orchestrating the assembly of the virus and in forming mature viral envelopes [22] . the e protein also functions in release of viral particles from host cells, in addition to other functions [22] . the n protein binds the viral rna and is required for packaging of viral rna into the viral particle during viral assembly [23, 24] . as mentioned previously, he is present on the surface of some betacoronaviruses. it is a hemagglutinin similar to influenza virus hemagglutinin (binds sialic acid on host cell-surface glycoproteins) and possesses acetyl-esterase activity [25] . he characteristics may enhance entry and pathogenesis of coronaviruses that contain such protein in their viral structure. the rna genome of covs is the second largest of all rna viruses, ranging from 26 to 32 kilobases (kb) in size [26] . the largest genome of all rna viruses is that of the recently described planarian secretory cell nidovirus, pscnv (41.1 kb genome size) [27] . viral rna codes for structural and nonstructural proteins [28] . the structural proteins together with a few nonstructural proteins, with different functions, are coded within the 3 end of the viral genome [28] . however, the 5' two-thirds of the genome codes for nonstructural proteins that are important in viral replication, including the rna-dependent rna polymerase (rdrp) [28] . once the viral genome is inside the host cell cytoplasm following viral entry, translation of the 5 end of viral rna produces the rdrp, which uses viral rna as a template to generate virus-specific mrnas (subgenomic mrnas) from subgenomic negative strand intermediates [29, 30] . subgenomic mrnas share the same 3 ends and the same leader sequence of 70-90 nucleotides at their 5 ends [28, 31] . translation of subgenomic mrnas leads to production of structural and nonstructural viral proteins [28] . once sufficient structural proteins and genomic viral rna are formed, viral rna is then assembled with viral structural proteins into virions. viral assembly and budding occur in smooth-walled vesicles in the endoplasmic reticulum-golgi intermediate compartment (ergic) [28] . s protein is the viral protein that mediates the entry of covs into host cells [32] . receptor-binding domain (rbd) within the s1 domain mediates binding to the cognate host cell receptor; however, the s2 domain mediates the fusion events, between viral membrane and host cell membrane, that are required for entry of covs into host cells [33, 34] . the rbd is located at n-terminus of the s1 subunit as in mouse hepatitis virus (mhv) or at the c-terminus as in sars-cov and mers-cov [34, 35] . the tissue tropism of covs is determined by the s protein interaction with the receptors on host cells. several cellular receptors were described as receptors for covs. for example, aminopeptidase n (apn) was identified as the receptor for several alphacoronaviruses [36] , angiotensin-converting enzyme 2 (ace2) as the receptor for sars-cov [37] , hcov-nl63 [38] and possibly for the newly discovered sars-cov-2 [39] , ceacam1 as the receptor for mhv [40] , and dipeptidyl-peptidase 4 (dpp4 which is also known as cd26) as the receptor for mers-cov [41] . a study on the rbd of sars-cov-2 s protein showed its similarity in structure to that of sars-cov with some key amino acid differences [42] . the previous finding suggests that sars-cov-2 could employ human ace2 as its cellular receptor. a recent study published in science showed that sars-cov-2 s protein has higher affinity to ace2 than sars-cov s protein [43] . however, the receptor usage and the cellular tropism of sars-cov-2 need further investigation. the trimeric cov s protein is cleaved by host cell proteases during infection to expose the fusion peptide of the s2 domain, which induces the fusion of viral and cellular membranes [18, 20, 44, 45] . fusion of viral envelope with host cell membrane results in the release of the viral genome into the cytoplasm [33, 34] . cleavage of s protein occurs at different sites that were identified, in different coronaviruses, to be between the s1 and s2 domains (s1/s2 site) and within the s2 domain proximal to the fusion peptide (s2 site) [18, 20, 44, 45] . it is believed that cleavage at both sites is required for viral entry [46] . different proteases have been identified to cleave the s1/s2 site depending on its amino acid sequence in different viruses. for example, the s1/s2 site of mers-cov s protein (rsvr↓sv) is cleaved by furin after its biosynthesis during viral replication [47] . sars-cov-2 has an s1/s2 site (ayt↓m) that is identical to the one in sars-cov [48] . this sars-cov s1/s2 site has been shown to be cleaved by cathepsin l following receptor binding and during viral entry in late endosomes [20] . we believe that the sars-cov-2 s1/s2 site may be cleaved by cathepsin l similar to sars-cov. other proteases such as trypsin, elastase, and tmprss2 have been shown to cleave sars-cov s protein at other sites between s1 and s2 domains [44, 46, 49] . unlike sars-cov, sars-cov-2 has an additional furin-like protease cleavage site (rrar↓sv) that is n-terminus to the s1/s2 site (ayt↓m), and is absent in sars-cov [48] . the presence of this furin-like cleavage site in sars-cov-2 suggests its cleavage by furin during viral egress. in addition to the previous s1/s2 sites, sars-cov-2 has a furin-like protease cleavage s2 site (kr↓sf) that is identical to that in sars-cov [48] . however, there is no evidence that sars-cov s protein is cleaved by furin-like proteases at the s2 site during viral egress [20] . similar to s1/s2 site, we believe that sars-covs protein is cleaved at the s2 site by cathepsin l or tmprss2 in different cellular locations during viral entry. the previous notion is supported by several studies which showed that cathepsin l and tmprss2 promote sars-cov entry while their inhibition suppressed infection of permissive cells [20, 44, 50] . given that sars-cov-2 has the same s2 site as sars-cov, we believe that processing of sars-cov-2 s protein at s2 site is similar to sars-cov. similarly, mers-cov has an s2 site (rxxr↓sa) that is less efficiently cleaved by furin and most probably cleaved by tmprss2 or cathepsin l during viral entry [51] . despite the identification of putative protease cleavage sites in sars-cov-2 s protein, their relative importance for sars-cov-2 s protein activation, viral pathogenesis, and cellular tropism needs further investigation. antiviral small molecules that inhibited cathepsin l were able to inhibit sars-cov infections in vitro and that of other viruses that depend on cathepsin l for entry, such as ebola, hendra, and nipah viruses [50] . the presence of a cathepsin l cleavage site in sars-cov-2 s protein (s1/s2 site) suggests that cathepsin l inhibitors may be valuable in inhibiting sars-cov-2 infections [48] . antibodies against rbd and s2 domain of sars-cov and mers-cov s proteins have been found effective in neutralizing infections of permissive cell lines in vitro [52] [53] [54] [55] . in addition, neutralizing antibodies were capable of treating infections in experimental animals and in infected patients during these major outbreaks [56] [57] [58] [59] . in one study, several sars-cov rbd-specific monoclonal antibodies did not bind to sars-cov-2 s protein [43] . another study showed that the sars-cov-specific monoclonal antibody, cr3022, bound with high affinity to sars-cov-2 rbd [60] . the previous studies suggest that there are differences between the two rbds which impact the cross reactivity of many neutralizing antibodies. however, neutralizing antibodies against sars-cov-2, once developed, could be promising in controlling the current sars-cov-2 outbreak. around 8098 cases that were infected over nine months (around 10% fatality) ( table 1 ) [12] . sars-cov was found to infect unciliated bronchial epithelial cells and type ii pneumocytes and cause fever, cough, shortness of breath, and severe complications such as pneumonia and kidney failure [12, 37] . the incubation period for sars-cov was estimated to range from 2 to 10 days, and up to 14 days (table 1 ) [61] . studies have shown that bats harbor covs that are ancestral to sars-cov (table 1 ) [62] . civets and raccoon dogs, of chinese local markets, were shown to harbor sars-like covs (table 1 ) [63] . the detection of sars-related covs (sarsr-covs) in bats and small animals in retail markets may indicate an interspecies transmission from bats to small animals and finally to humans (figure 2 ). studies in bats from different regions of china have identified several sarsr-covs [64] . the previous finding indicates that sars-cov has been circulating in bats for a long time before genetically changing and jumping to humans. since ace2 was identified as the receptor for sars-cov, it is not surprising that sars-cov has adapted itself to bind human ace2 and efficiently infect human cells [65] . that sort of adaptation required a set of amino acid changes in the rbd of s protein of sars viruses that were circulating in bats [65] . therefore, we conclude that the human-to-human transmission that was seen during the sars-cov outbreak is attributed to the ability of sars-cov to adapt its s protein (particularly rbd) to efficiently bind to human ace2 and infect airway epithelia ( figure 2 ). [62] . civets and raccoon dogs, of chinese local markets, were shown to harbor sarslike covs (table 1 ) [63] . the detection of sars-related covs (sarsr-covs) in bats and small animals in retail markets may indicate an interspecies transmission from bats to small animals and finally to humans (figure 2 ). studies in bats from different regions of china have identified several sarsr-covs [64] . the previous finding indicates that sars-cov has been circulating in bats for a long time before genetically changing and jumping to humans. since ace2 was identified as the receptor for sars-cov, it is not surprising that sars-cov has adapted itself to bind human ace2 and efficiently infect human cells [65] . that sort of adaptation required a set of amino acid changes in the rbd of s protein of sars viruses that were circulating in bats [65] . therefore, we conclude that the human-tohuman transmission that was seen during the sars-cov outbreak is attributed to the ability of sars-cov to adapt its s protein (particularly rbd) to efficiently bind to human ace2 and infect airway epithelia ( figure 2 ). mers-cov was first described in 2012 as a new cov that causes a severe respiratory disease in saudi arabia [13] . similar to sars-cov, mers-cov infects unciliated bronchial epithelial cells and mers-cov was first described in 2012 as a new cov that causes a severe respiratory disease in saudi arabia [13] . similar to sars-cov, mers-cov infects unciliated bronchial epithelial cells and type ii pneumocytes and causes severe illness of the respiratory tract, which is characterized by fever, cough, shortness of breath, and severe complications such as pneumonia and kidney failure [13] . the incubation period of mers-cov is quite similar to sars-cov and ranges from 2 to 14 days (table 1 ) [66] . as of january 2020 and since 2012, 862 of 2506 infected cases in 27 countries have died (≈35% fatality), which is more than three times the fatality seen in sars-cov infections (table 1 ) [67] . however, unlike sars-cov, human-to-human transmission of mers-cov is not easy and has not been confirmed except in cases of very close contact with infected patients in health care settings [67] . mers-related covs (mersr-covs) were detected in bats, suggesting a potential bat origin (table 1) [68, 69] . mers-cov was transmitted to humans from dromedary camels ( table 1 ) [70] . studies have also shown that camel mers-cov strains are almost identical to human mers-cov strains [71] . it was postulated that mers-cov existed in camels at least 30 years ago since antibodies to mers-cov were detected in samples that were collected from camels in 1983 [72] . sequence analyses have shown that mersr-covs' rbds share only 60-70% sequence identity with that of human and camel mers-covs [73] . similar to the adaptation of sars-cov to human host, mersr-covs that are circulating in bats had to undergo several amino acid changes in rbd of s protein to become capable of infecting camels and humans ( figure 2 ) [74] . we believe that the amino acid changes in mersr-covs' rbd led to the emergence of mers-cov strains that are capable of binding to human dpp4 with high affinity, infecting humans, and causing the 2012 outbreak ( figure 2) . as mentioned previously, the sars-cov-2 was isolated and sequenced from patients that showed symptoms of respiratory illness and pneumonia in wuhan, china during december 2019. sars-cov-2 is the third identified human cov that causes severe respiratory illness with symptoms and incubation period resembling that of sars-cov and mers-cov infections (table 1) [11, 75] . since december 2019, sars-cov-2 infection rates have been rising in china and worldwide [42] . similar to sars-cov and unlike mers-cov, human-to-human transmission has been confirmed [42] . initial cases of sars-cov-2 infections were somehow connected to the huanan seafood market in wuhan, in the hubei province of china [11] . in this market, a number of nonaquatic animals were on sale, such as birds, snakes, marmots, bats, and rabbits [11] . genetic analyses of viral samples from patients with sars-cov-2 infections revealed that the sars-cov-2 is a betacoronavirus that has 88% sequence identity to two bat sarsr-cov: 79% identity to sars-cov and only 50% identity to mers-cov [42] . the previous findings suggest that sars-cov-2 is a new virus that is distinct from sars-cov and mers-cov but most probably originated in bats, similar to sars-cov and mers-cov [42] . another recent study confirmed that sars-cov-2 significantly clustered with a sequence from the bat sars-like cov that was isolated in 2015 [76] . however, the existence of an intermediate host for sars-cov-2 is still not verified (figure 2 ). to date, the fatality of sars-cov-2 appears to be less than that observed in sars-cov and mers-cov infections. however, since new cases are confirmed everyday as we write this review, the fatality of this virus may keep changing and will not be accurately calculated until after the end of this outbreak. the virus appears to be more fatal in elderly patients or patients with comorbidities [77] . however, it is important to note that there could be cases that went undetected, which makes it hard to accurately calculate the fatality of this new virus. our lessons learned from sars-cov and mers-cov outbreaks include the high mutation rates that characterize all rna viruses [78] , the evolving nature of covs [6, 7] , and the ease of transmission from one species to another [6, 7] . as mentioned previously, it appears that sars-and mers-covs arose at sometime from ancestral covs harbored by bats (figure 2 ). whereas animals served as intermediate hosts, humans served as terminal hosts (figure 2 ). sars-cov was transmitted to civets and raccoon dogs, and to camels in the case of mers-cov (figure 2 ). they were then transmitted pathogens 2020, 9, 186 7 of 15 from these intermediate animal hosts to humans (figure 2 ). the practice of eating raw meat and the close contact between humans and animals are both risk factors for the initiation of a new human cov outbreak. this is due to the constant exposure of humans in these cultures to the ever-changing mutant covs. the first cases of sars-cov-2 infections were reported in the chinese city of wuhan during december 2019 [11] . although other options have not been completely ruled out yet, it is believed that the sars-cov-2 stemmed from a large seafood and animal market in wuhan, the capital of hubei province, china [11] . as for other wet markets in china, live animals are sold, mostly for food or medicine. the attributed medicinal and/or magical uses of wildlife and rare animal parts (such as pangolin scales and tiger paws) are mainly based on traditional chinese medicine (tcm), which has been widely promoted by the current chinese government. however, it is important to note that most of these folk remedies are never prescribed in reputable tcm hospitals. the wuhan market is known to have a lot of exotic animals and exotic animal parts [42] . thus, sars-cov-2 disease can be considered a zoonotic disease (like sars) that has initially spread from animals to humans. however, human-to-human transmission has also been confirmed [79] . it is not well understood why outbreaks of cov infections are mostly occurring in china. we speculate that those viruses may be predominantly circulating in animals in china rather than other animals in different parts of the world. one of the reasons for these sudden outbreaks could be the close interactions with live and wild animals that are consumed as food in wholesale food markets in china. in a very recent study, the genomes of covs isolated from nine patients having viral pneumonia in wuhan were analyzed [42] . the study showed that the genomes of these viruses differed by less than 0.1 percent (more than 99.98% of sequence identity), which indicates that the virus has only recently emerged in humans and has been detected rapidly after its emergence [42] . as the virus keeps spreading to more individuals, more mutations may arise which can potentially make the virus more virulent and thus constant surveillance will be necessary. the u.s. center for disease control and prevention (cdc) reported that sars-cov-2 causes a respiratory illness that is characterized by fever, cough, and shortness of breath. radiographs of some sars-cov-2 patients demonstrated invasive lesions in both lungs [80] . the cdc also reports that the elderly, individuals with underlying health problems, and people with compromised immune systems are at a particularly higher risk of developing severe pneumonia from the virus [77] . we believe that it is too early to assess the impact of the new virus on children. sars-cov infections were significantly less common among children than adults, and kids younger than 12 reported much less severe symptoms than patients who were more than 12 [81] . this may have to do with children being exposed to more cov in school and the outdoors than adults or because of the better overall health status of children as compared to adults. also, children tend to be more up-to-date with vaccinations, which may protect them from secondary infections that are often triggered by the main infections. during the sars-cov outbreak, most schools and factories in china remained open. following the sars-cov-2 outbreak and confirmation of many infected cases, china responded by locking down residents of wuhan city, banning wildlife trade until the epidemic is over, and attempting to build two new hospitals in the city of wuhan to specifically handle the new outbreak. it is currently unclear if these two hospitals will be able to handle a major outbreak in a city of 11 million residents. there is no definite information about the exact time of the start of the outbreak. that is why it is hard to assess how contagious the virus is (the rate of sustained spread) at the present time. however, it seems likely and plausible that it is highly contagious, based on the mounting data about human-to-human transmission outside china [42] . in order to be able to precisely assess the rate of sustained spread, information about numbers of cases and deaths (the overall number of patients) will need to be divided by the overall number of people at risk of acquiring the disease (the number of individuals who have been in contact with the patients). once this is determined, the overall risk can be assessed. underreporting or misdiagnosis of cases can negatively impact the calculations and thus delay the ability of public health officials to truly assess the situation. in an outbreak of this magnitude, the most important number that public health experts will be looking for is the basic reproduction number, also known as the r0 [82] . this number measures the disease's potential and represents the average number of people who will catch the disease from one infected person in a population that has never had the disease in the past [82] . in one study, the mean estimate of r0 was calculated to be between 2.24 and 3.58 [83] . another study estimated the r0 to have a high average value of 2.5 [84] , which is consistent with other groups that reported values from 2 to 3 [83, [85] [86] [87] [88] . these r0 estimates for the sars-cov-2 are consistent with r0 estimates for sars-and mers-covs (from 2 to 5) (table 1 ) [89, 90] . the virus is less deadly than sars, which killed about 10% of the infected patients. because r0 represents an average, outliers (carriers who infect so many people or carriers who infect nobody) can have a huge impact on the final value of the r0. in other words, a bigger r0 does not necessarily mean more infections. for example, the r0 for the seasonal flu typically ranges from 1.2 to 1.4 [91] , but it still infected many more people than the number of people who got infected with sars-cov. at any rate, any r0 above 1 should be taken seriously. the goal will be to reduce the r0 to a value that is below 1. finally, r0 estimates can be higher than the "true" r0 values because of two main reasons: infected people who did not show symptoms and infected people who did not report their symptoms. since r0 is not an intrinsic property of the virus itself, r0 estimates tend to be lower in places where there are sound infection control methods and vice versa. typically, r0 estimates are highest at the beginning of an outbreak and then subside gradually once countries become aware of the outbreak and manage to put in effective control measures to prevent the spread of the virus. the lockdown strategy that china is implementing works best during the early stages of an infection, which is not the case in wuhan, as there are several million people who already left the city before the restrictions were imposed. if we are beyond the early stages, then there can be disadvantages associated with such lockdown on wuhan. among the disadvantages are people evading care to avoid any restrictions on their life. given that the incubation period can be up to 11 days or more, a two-week federal quarantine was ordered for 195 u.s. citizens who were flown back from china [92] . the action was described by the cdc as precautionary and preventive. for comparison, there were no quarantines ordered in the u.s. for the recent sars-cov or mers-cov outbreaks. the last federal quarantine in the u.s. was ordered back in the 1963 to prevent the spread of smallpox from sweden to the u.s. during a smallpox outbreak in sweden [93] . as shown in table 1 , the number of people infected by the virus has exceeded the global total infected with sars-cov (8098 individuals) in a nine-month period that extended from 2002 until 2003. it will be difficult to control the disease without a level of disruption of air travel. we believe that limiting travel to and from china will be a necessary measure. respiratory viruses spread through respiratory droplets that are produced when an infected person coughs or sneezes. the exact modes of transmission of sars-cov-2 are not entirely clear at this early stage, but reports of healthcare professionals in china who contracted the disease suggest a highly contagious virus [77] . prevention of the sars-cov-2 infections entail precautions that are common to other respiratory viruses. the most obvious measure is to avoid contact with people who are sick. this is especially important due to the contagious nature of the virus. if someone is sick, they should stay home. once they recover, they may consider using disposable face masks (while frequently changing them) and avoiding close contact with coworkers. however, the value of wearing face masks is controversial, to say the least [94] . surgical masks do not fully protect against airborne viruses as they do not fully seal the nose and the mouth. thus, small droplets, which can travel farther than large droplets and in more unpredictable patterns, can be inhaled around the sides of the masks. the n95 masks offer a better protection as long as they fit properly. it is worth noting that n95 masks are not suitable for people with facial hair [95] . being an enveloped virus, washing hands with water and soap for at least 30 s would be beneficial in deactivating the sars-cov-2 [96] . hand sanitizers can also be used if water and soap are not readily available, while touching eyes, nose, and mouth should be prevented [97] . disinfection of different environmental surfaces, tools, and objects is crucial in limiting the spread of the virus [97] . the more you use one of these objects or touch one of these surfaces, the more pressing cleaning and disinfection become. public health officials, local health departments, hospitals, doctors, and cdc personnel should work closely with universities and other workplaces to educate and provide needed supplies to contain the spread of the virus. the world health organization (who) declared sars-cov-2 as a public health emergency of international concern (pheic) on 30 january 2020 [98] . a pheic is an atypical event that constitutes a public health risk and potential for the disease to spread to other countries, thus requiring a coordinated international response [99] . this designation could help mobilize more resources to the impacted areas. the current outbreak represents the sixth time the who has declared a global emergency since it gained the power to declare an international emergency in 2005 [100] . the previous five times were the 2009 h1n1 swine flu, the 2013 ebola outbreak in west africa, the 2014 polio outbreak, the 2016 zika outbreak, and 2019 ebola outbreak in the democratic republic of congo [100] . none of these previous emergencies led a worldwide pandemic. however, we predict that the current outbreak is more likely to become a pandemic. the current diagnostic test for the virus is pcr-based [101] . since this test typically takes 48 h, a new quicker diagnostic test needs to be developed. it will not be practical to isolate (quarantine) a large number of individuals until results of the pcr-based diagnostic test become available. this can also overwhelm healthcare facilities, already overwhelmed by the increasing number of cases that are discovered every day in china and elsewhere in the world. containing the outbreak before it can spread is the best way to prevent pandemics. border closures and screening at airports and checkpoints are classical measures that were previously implemented in the 2009 h1n1 flu pandemic [102] . this can reduce the spread of the virus but will not be a fool-proof strategy. the reason is that the incubation period of the virus is believed to be as long as 14 days, as was the case with mers-cov [103, 104] . this means that carriers of the virus can show up at the border with no apparent symptoms and readily pass through security without raising any red flags. compared to the sars-cov outbreak in 2003, the current increase in air traffic in china and worldwide has likely contributed to the more rapid spread of sars-cov-2 in 2020. without a quick diagnostic test, there are not many good options that can completely stop the transmission of the virus. once a test is available, cases can be identified and isolated. based on previous genetic experience with sars-cov, scientists will need to quickly develop a vaccine for sars-cov-2. the world is hoping to succeed in containing this virus as soon as possible. even after success, there needs to be follow-up with patients who are cured and declared virus-free. this is a lesson we learned from the ebola outbreak, in which some patients who walked out from the hospital "virus-free" were found later to harbor the ebola virus that was carefully hiding itself in other parts of the body, such as the immune-privileged eye [105] [106] [107] . although this hidden ebola virus was no longer transmissible to other humans, it rendered the label "virus-free" incorrect with these individuals. natural disasters bring people together but epidemics and outbreaks split them apart. the sars-cov-2 is another cov that may lead to a pandemic, if not timely controlled. our current knowledge of this virus suggests an intermediate host; however, human-to-human transmission is confirmed and is of concern. the number of infected cases to date indicates a very rapid and efficient human-to-human transmission. this necessitates quick development of therapeutics that can inhibit coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin infectious bronchitis viruses with a novel genomic organization epidemiology, genetic recombination, and pathogenesis of coronaviruses molecular evolution of human coronavirus genomes coronaviruses in avian species-review with focus on epidemiology and diagnosis in wild birds human coronaviruses: a review of virus-host interactions human coronaviruses: what do they cause? a novel coronavirus from patients with pneumonia in china identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy an overview of their replication and pathogenesis the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex the proteolytic regulation of virus cell entry by furin and other proprotein convertases the sars-cov s glycoprotein: expression and functional characterization cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles modular organization of sars coronavirus nucleocapsid protein identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza c virus and bovine coronavirus coronavirus: organization, replication and expression of genome a planarian nidovirus expands the limits of rna genome size the molecular biology of coronaviruses subgenomic negative-strand rna function during mouse hepatitis virus infection coronavirus transcription: subgenomic mouse hepatitis virus replicative intermediates function in rna synthesis further characterization of mouse hepatitis virus rna-dependent rna polymerases s protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry identification and characterization of novel neutralizing epitopes in the receptor-binding domain of sars-cov spike protein: revealing the critical antigenic determinants in inactivated sars-cov vaccine localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein structural bases of coronavirus attachment to host aminopeptidase n and its inhibition by neutralizing antibodies angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars the n-terminal domain of the murine coronavirus spike glycoprotein determines the ceacam1 receptor specificity of the virus strain dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding cryo-em structure of the 2019-ncov spike in the prefusion conformation efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 host cell proteases: critical determinants of coronavirus tropism and pathogenesis elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the s2 domain host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay functional analysis of potential cleavage sites in the mers-coronavirus spike protein generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against sars coronavirus using xenomouse identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution identification of an antigenic determinant on the s2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody bats are natural reservoirs of sars-like coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats receptor and viral determinants of sars-coronavirus adaptation to human ace2 middle east respiratory syndrome middle east respiratory syndrome coronavirus transmission genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation receptor usage of a novel bat lineage c betacoronavirus reveals evolution of middle east respiratory syndrome-related coronavirus spike proteins for human dipeptidyl peptidase 4 binding evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission transmission of 2019-ncov infection from an asymptomatic contact in germany the 2019-new coronavirus epidemic: evidence for virus evolution return of the coronavirus: 2019-ncov. viruses mechanisms and concepts in rna virus population dynamics and evolution a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster identification of a novel coronavirus causing severe pneumonia in human: a descriptive study complexity of the basic reproduction number (r0) preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak novel coronavirus 2019-ncov: early estimation of epidemiological parameters and epidemic predictions early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia pattern of early human-to-human transmission of wuhan nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study transmission dynamics and control of severe acute respiratory syndrome modeling the spread of middle east respiratory syndrome coronavirus in saudi arabia approximate bayesian algorithm to estimate the basic reproduction number in an influenza pandemic using arrival times of imported cases the incubation period of 2019-ncov infections among travellers from wuhan, china. medrxiv 2020 comparing mask fit and usability of traditional and nanofibre n95 filtering facepiece respirators before and after nursing procedures a close shave? performance of p2/n95 respirators in health care workers with facial hair: results of the beards (adequate respiratory defences) study evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation efficacy of ethanol against viruses in hand disinfection china coronavirus: who declares international emergency as death toll exceeds 200 ebola outbreak declared a pheic, world waits for next steps global health concern stirred by emerging viral infections first case of 2019 novel coronavirus in the united states transmission dynamics, border entry screening, and school holidays during the 2009 influenza a (h1n1) pandemic the novel coronavirus originating in wuhan, china: challenges for global health governance comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia persistence of ebola virus in various body fluids during convalescence: evidence and implications for disease transmission and control retinal pigment epithelial cells are a potential reservoir for ebola virus in the human eye immune tolerance elicited via unique ocular and oral routes key: cord-288167-976qxja2 authors: park, wan beom; poon, leo l.m.; choi, su-jin; choe, pyoeng gyun; song, kyoung-ho; bang, ji hwan; kim, eu suk; kim, hong bin; park, sang won; kim, nam joong; peiris, malik; oh, myoung-don title: replicative virus shedding in the respiratory tract of patients with middle east respiratory syndrome coronavirus infection date: 2018-05-09 journal: int j infect dis doi: 10.1016/j.ijid.2018.05.003 sha: doc_id: 288167 cord_uid: 976qxja2 background: information on the duration of replicative middle east respiratory syndrome coronavirus (mers-cov) shedding is important for infection control. the detection of mers-cov sub-genomic mrnas indicates that the virus is replicative. this study examined the duration for detecting mers-cov sub-genomic mrna compared with genomic rna in diverse respiratory specimens. methods: upper and lower respiratory samples were obtained from 17 mers-cov-infected patients. mers-cov sub-genomic mrna was detected by reverse transcription pcr (rt-pcr) and mers-cov genomic rna by real-time rt-pcr. results: in sputum and transtracheal aspirate, sub-genomic mrna was detected for up to 4 weeks after symptoms developed, which correlated with the detection of genomic rna. in oropharyngeal and nasopharyngeal swab specimens, the detection of sub-genomic mrna and genomic rna did not correlate. conclusions: these findings suggest that mers-cov does not replicate well in the upper respiratory tract. middle east respiratory syndrome coronavirus (mers-cov) genomic rna can persist for more than 1 month in respiratory specimens (memish et al., 2014) . however, the detection of mers-cov rna may overestimate the duration of shedding of replicative virus. coronaviruses have a unique mechanism of discontinuous transcription with the synthesis of sub-genomic mrnas (sawicki et al., 2007) . the mers-cov has at least seven distinct sub-genomic mrna species and the detection of these indicates that the virus is replicative (woo et al., 2016) . the objectives of this study were to examine the duration for detecting mers-cov sub-genomic mrna vs. genomic rna in different respiratory specimens. respiratory samples were collected from 17 patients admitted to three seoul national university (snu) affiliated hospitals during the 2015 mers outbreak in korea. the patients were categorized into severe (a-i) or mild (j-q) groups depending on their oxygen supplementation requirements. the severe group required oxygen supplementation to maintain arterial saturation above 90%. patients a-e received ventilator therapy, while patients f-i did not (park et al., 2015) . the institutional review board at snu hospital provided study approval and waived the requirement for written consent. oropharyngeal and nasopharyngeal swabs were collected using a utm kit containing viral transport medium (copan diagnostics inc., murrieta, ca, usa). the rna was extracted from respiratory samples using a qiaamp viral rna mini kit (qiagen, valencia, ca, usa). to detect the mers-cov genomic rna, multiplex quantitative real-time reverse transcription (rrt)-pcr was performed using the powerchek mers (upe & orf1a) real-time pcr kit (kogenebiotech, seoul, south korea) and all assays were performed using a viia7 real-time pcr system (applied biosystems, grand island, ny, usa). the results of genomic rna titers have been presented in part in a previous publication (oh et al., 2016) . the mers-cov sub-genomic mrna was detected using accu-power rt-pcr premix (binder inc., alameda, ca, usa). pcr primers were designed to detect sub-genomic mrna that codes for the spike (s) (433 bp) and nucleocapsid (n) (662 bp) proteins (table 1) . forward primer was elaborated from the leader sequence and backward primers of 5 0 untranslated region (utr)-s and 5 0 utr-n were from gene sequences coding for proteins s and n, respectively. the pcr reactions were performed as follows: initial denaturation at 94 c for 5 min and 40 cycles of denaturation at 94 c for 20 s, annealing at 55 c for 30 s, and extension at 72 c for 1 min. sub-genomic mrna was sequenced using a dna engine tetrad 2 peltier thermal cycler (bio-rad) and the abi bigdye terminator v3.1 cycle sequencing kit (applied biosystems, grand island, ny, usa). if the sequences included the leader sequence and were consistent with the mers-cov genome by 98% using basic local alignment search tool (blast) software, they were confirmed as sub-genomic mrna. in sputum and transtracheal aspirates, the detection of mers-cov sub-genomic mrna was more frequent in the severe group than in the mild group ( figure 1a ). sub-genomic mrna was detected 28 days after the illness onset. in the severe group, the period for detecting sub-genomic mrna strongly correlated with the duration for detecting mers-cov genomic rna (pearson correlation coefficient = 0.803, p = 0.009). the mers-cov genomic rna titer was significantly higher in the specimens with subgenomic mrna detection than in those where sub-genomic mrna was not detected (p = 0.0007) (figure 2 ). in oropharyngeal swab specimens, sub-genomic mrna was detected only in the severe group for up to 11 days after the illness onset ( figure 1b) , and the period for detecting sub-genomic mrna was not significantly correlated with that for mers-cov genomic rna (pearson correlation coefficient = 0.335, p = 0.378). no subgenomic mrna was detected in nasopharyngeal swab specimens ( figure 1c ). in the present study, replicative mers-cov was detected in sputum or transtracheal aspirate for up to 4 weeks after symptom development in mers-cov-infected patients with severe pneumonia. this result is consistent with the findings of previous studies that have tested mers-cov genomic rna (memish et al., 2014; corman et al., 2016; min et al., 2016) . on the basis of these results, infection prevention and control precautions should be thoroughly applied for at least 1 month after symptom onset if the patient with mers-cov infection has severe pneumonia. the differences in the detection of replicative viruses between upper and lower respiratory tract specimens may have originated from differences in viral titers. several studies have demonstrated that the viral titer of mers-cov rna in upper respiratory tract specimens is lower than that in lower respiratory tract specimens (oh et al., 2016; corman et al., 2016) . in the present study, subgenomic mrna was not detected in any of the nasopharyngeal specimens. the current guidelines recommend that isolation should continue until two consecutive upper respiratory tract specimens taken at least 24 h apart test negative by rt-pcr (who, 2018) . however, the present study suggests that, if possible, lower respiratory tract specimens should be used to determine the duration of isolation and that nasopharyngeal swab specimens should be avoided. this study has a few limitations. first, differences in sensitivity between the real-time rt-pcr used to detect genomic rna and the conventional rt-pcr for sub-genomic mrna may have affected the results. second, rt-pcr methods for sub-genomic mrna have not been validated elsewhere. other methods, such as detecting live virus, should be performed to validate the methods used in this study. in conclusion, replicative mers-cov was detected in lower respiratory tract specimens for up to 4 weeks after symptom development, which was well correlated with the detection of genomic rna. in upper respiratory tract specimens, the detection of sub-genomic mrna and genomic rna did not correlate. these findings suggest that mers-cov does not replicate well in the upper respiratory tract. viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity kinetics of serologic responses to mers coronavirus infection in humans a contemporary view of coronavirus transcription management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov). interim guidance isolation and characterization of dromedary camel coronavirus uae-hku 23 from dromedaries of the middle east: minimal serological cross-reactivity between mers coronavirus and dromedary camel coronavirus uae-hku 23 middle east respiratory syndrome coronavirus (mers-cov) genomic rna (upe) titers in sputum and transtracheal aspirates with vs. without sub-genomic mrna detection. solid lines indicate the mean and standard error of the mean none. key: cord-297691-w4cdfwv0 authors: nikaeen, ghazal; abbaszadeh, sepideh; yousefinejad, saeed title: application of nanomaterials in treatment, anti-infection and detection of coronaviruses date: 2020-05-07 journal: nanomedicine doi: 10.2217/nnm-2020-0117 sha: doc_id: 297691 cord_uid: w4cdfwv0 nanotechnology and nanomedicine have excellent potential in dealing with a range of different health problems, including viruses, which are considered to be a serious challenge in the medical field. application of nanobiotechnology could represent a new avenue for the treatment or disinfection of viruses. there is increasing concern regarding the control of coronaviruses, among these, middle east respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus-2 are well known and dangerous examples. this article aims to provide an overview of recent studies on the effectiveness of nanoparticles as diagnostic or antiviral tools against coronaviruses. the possibilities of effectively using nanomaterials as vaccines and nanosensors in this field are also presented. protective role, preventing the encapsulated drug or anti-infection agent from degradation because of the shielding properties of these nano-sized materials [15, 16] . viral infections, due to their problematic wide spreading and their ability to evolve by genetic mutation can pose a great threat to human health. in the last few decades, the high number of deaths caused by some viral infections has been also a challenge for healthcare systems [17] . the coronaviridae family, along with three other virus families, the roniviridae, arteriviridae and mesoniviridae, are categorized in the nidovirales order because of their similarity in their distinctive replication strategy and function in hosts [18, 19] . the coronaviridae family, commonly named as coronaviruses (covs), is composed of two subfamilies: coronavirinae and torovirinae. infection with a member of the coronavirinae subfamily can lead to any of reproductive disease, pneumonia, polyserositis, enteritis, hepatitis, encephalomyelitis, nephritis or sialo dacryoadenitis in mammals and birds. coronavirus and coronavirus-like infections have been reported in various wild and livestock animals such as swine, horses, cattle, cats, dogs, camels, birds and bats [18] . in humans, coronaviruses include a wide range of viruses contributing to the common cold as well as to more severe respiratory diseases such as middle east respiratory syndrome (mers), severe acute respiratory syndrome (sars) and novel coronavirus. since coronaviruses can be transmitted between animals and people, their effect is known as zoonotic diseases. previous studies have found that mers and sars coronaviruses jumped to humans from dromedary camels and civet cats, respectively, before being transmitted to humans. there are various known coronaviruses which can infect animals that have not been seen in humans yet [20] . all recent epidemics of coronavirus have emerged unexpectedly and can spread easily. they not only threaten human health but also may lead to catastrophic consequences [21] . in 2003, the sars-cov epidemic occurred. more than 8000 people were infected, leading to about 900 deaths worldwide [22] . mers-cov was first identified in 2012 and according to a report from june 2019, 2494 individuals have been infected with mers-cov since 2012, with a death rate of 34.4% [23] . in late 2019 and early 2020, a number of human cases of new coronavirus infection were reported. it has been suggested that the virus arose in relation with the huanan seafood wholesale market in wuhan, china; however, at the time of writing, there has been no confirmation of where the virus originated. the novel coronavirus was confirmed by china on 7 january 2020 and was later named as 'sars-cov-2', which causes the novel coronavirus disease. on 11 march 2020, the who declared the novel coronavirus outbreak as a pandemic. on 23 april 2020, a total of 2,510,177 cases had been confirmed in worldwide and 172,241 deaths reported [24] . depending on the seriousness of the disease and the mode of transmission, appropriate methods are required to minimize the transmission of infectious diseases [25] . for example, some diseases are entirely preventable by vaccination (e.g., measles and polio) or by access to improved sanitation (e.g., diarrheal and parasitic diseases); and others are treatable when detected in a timely manner (e.g., tuberculosis and malaria). to date, there is no specific treatment or vaccine found to be successful in the treatment of individuals infected with sars-cov-2 [26] . due to high rate of mortality and rapid global transmission of coronaviruses, this article aims to review the use of nano-sized materials for treatment, detection and antiviral activity against previous known coronaviruses. these may present a way to proceed for application of nanotechnology against new coronaviruses. in this special report, different strategies of using nanoparticles in dealing with coronaviruses are discussed in three parts: applications in nano-based vaccines, antiviral activity and development of diagnostic sensors. vaccination has been known as one of the most effective medical intervention used to stimulate immune response against infectious disease [27] . meanwhile, as nanoparticles have been proven to have immunostimulatory effects [28] , a great deal of attention has been given to development of nano-based therapeutic agent or vaccines against different types of coronaviruses. for example, in 2011, staroverov et al. evaluated the protective immune response stimulated by the administration of gold nanoparticles (au nps) conjugated with a type of coronavirus known as swine transmissible gastroenteritis virus (tgev) in immunized mice and rabbits [29] . tgev-conjugated colloidal gold was found to elicit higher concentrations of ifn-γ and superior titers of neutralizing antibody in vaccinated animals. immunization with the antigen-colloidal gold complex increased propagation of t cells tenfold in comparison with the response to free antigen and the authors of the study also reported that administration of the complex resulted in subsequent activation of macrophage respiratory activity and higher protective immunity against tgev. thus, aunps conjugated to a virus could be considered as a potential antiviral candidate for vaccine application. kim et al. proposed a ferritin-based np assembly mediated by rna as a novel molecular chaperone and demonstrated that using their np-based vaccine against mers-cov can induce cd4 + t cells, which in turn leads to the generation of ifn-γ and tnf-α upon antigen stimulation [30] . additionally, jung et al. attempted to develop an immunogenic vaccine against mers-cov using a heterologous prime-boost strategy involving a recombinant adenovirus serotype 5 encoding the mers-cov spike gene (ad5/mers) and spike protein nanoparticles [31] . groups of female balb/c mice were immunized three times with the prime-boost vaccination. it was shown that the homologous spike protein nps successfully induced higher antibody titers compared with the ad5/mers only group. however, a th1 immune response was not observed to be provided by spike protein nps themselves and only a th2 immune response involving induction of neutralizing antibodies was elicited. therefore, in order to provide much more durable immunogenicity and a suitable balance of th1/th2 responses, a heterologous onestage ad5/mers prime and two-stage spike protein nps boost seemed to be more effective than the homologous prime-boost regimen using either ad5/mers or spike protein nanoparticles alone. in 2019 lin et al. developed a novel viromimetic nanoparticle-based vaccine coupled with an immunologic stimulator of interferon genes agonist adjuvant against mers-cov [32] . as shown in figure 1 , a hollow polymeric nanocarriers coated with receptor binding domain (rbd) antigens were prepared followed by loading with cyclic diguanylate monophosphate as an emerging class of stimulator of interferon genes agonist adjuvant. c57bl/6 mice were then immunized with the developed vaccine. lin cellular and humoral immune responses. a strong and constant humoral and cd4 + t-cell response was also detected in the studied mice immunized with the virus-like np vaccine compared with free rbd antigen admixed with either free cyclic diguanylate monophosphate or mf59 (addavax), an adjuvant for influenza vaccines that has been utilized clinically (see figure 1b -c). in contrast to mf59, this viromimetic np vaccine induced higher levels of humoral responses. furthermore, mice immunized with the proposed np vaccine produced high levels of rbd-specific igg2a antibodies without induction of lung eosinophilic immunopathology after the infection (see figure 1d ). in a recent study performed by sekimukai et al., the efficacy of two types of adjuvants (aunps and toll-like receptor agonists) with recombinant s protein were evaluated against sars-cov infection in mice [33] . results indicated that vaccination with aunp-adjuvanted protein elicited strong igg response but, in contrast to a toll-like receptor agonist-adjuvanted vaccine, did not result in induction of protective antibodies and decreasing eosinophilic infiltration. thus, owing to their immunogenic properties, various types of nps, including gold nps, spike protein nps and hollow polymeric nps have all been reported to have considerable potency to induce an immune response against coronaviruses in animal models and in vitro. different application of nps-based vaccine candidates with some details regarding function and mechanism are summarized in table 1 . nanomaterials have regularly been applied in as antiviral agents [34, 35] or as delivery platforms for antiviral compounds [36] . this section assesses the application of nanomaterials in developing antiviral agents against certain coronaviruses. in a patent invented in 2014 by cho et al., a mixture comprising silver colloid, titanium dioxide (tio 2 ) nps, a dispersion stabilizer, a binder and water showed its antibacterial, antifungal and antiviral activities (us 8,673,331 b2) [37] . results of antiviral tests showed that at 100-fold dilution of the composition concentration showed an antiviral activity against porcine epidemic diarrhea virus (pedv) and tgev at a rate of 99.99% or higher. on the other hand, when the composition concentration was 1000-fold diluted, the inhibited growth of the viruses was at a rate of 99.9% for pedv and 93.0% inhibition for tgev. the antiviral activity of the nanomaterial proposed cho et al. was therefore dependent on composition concentration meaning dosage should be adjusted to have desired inhibition [37] . in 2014, lv et al. compared the strength of immune responses induced by four different silver nanomaterials, including silver nps, silver nanowires of 60 and 400 nm and silver colloids, on tgev in infected swine testicle cells [38] . at a concentration of 3.125-12.5 μg/ml, the percentage reduction in virus titer was evaluated in different silver nanomaterials. it was shown that of the different types of silver nanomaterials, only ag nps and the two types of ag nanowires induced protection against tgev. the silver colloids were not reported to restrain cellular entry of the virus. the ag nps and silver nanowires were capable of reducing the number of apoptotic cells elicited by tgev. in the year 2017, hu et al. developed a promising treatment approach based on the nanoformulation of diphyllin for the treatment of feline infectious peritonitis (fip), which is caused by feline coronavirus [39] . diphyllin is a vacuolar atpase that has been demonstrated to inhibit endosomal acidification in fcwf-4 cells, a necessary process for virus uncoating and cellular entry. it was shown that poly(ethylene glycol)-block-poly(lactide-coglycolide), which was used as the diphyllin nanocarrier, enhanced the inhibitory activity of diphyllin against fip and also improved the safety profile. the antiviral activity of diphyllin nanoparticles was also investigated. it should be noted that administration of high doses of the nanoparticles were found to be tolerable in mice. therefore, diphyllin nanoparticles proved to have prominent antiviral effect against fip. while not studied as a vaccine candidate, this study still demonstrates that nanoformulations can be effective against coronaviruses and this specific example could be a potential treatment candidate. a novel therapeutic strategy based on ag nanomaterials against the alphacoronavirus pedv was first introduced in 2018 by du et al. [40] . they demonstrated that ag 2 s nanoclusters (ncs) could restrain pedv proliferation in treated vero cells. as suggested by authors, this may be attributed to the fact that treatment with ag 2 s ncs inhibited the viral budding and the synthesis of viral negative-strand rna. further, the ag 2 s ncs were found to positively regulate the proliferation of ifn-stimulating genes and the expression of pro-inflammation cytokines, leading to protection against pedv infection, making them a good option to be used in further studies as a treatment tool. the antiviral activity of graphene oxide-silver (go-ag) nanocomposites was reported against non-enveloped and enveloped viruses by chen et al. in 2016 [41] . to evaluate the antiviral activity of go-ag, go-ag solutions with different dilution orders were incubated with serially diluted solution of feline coronavirus. after removing the composite particles, the supernatant was tested using a virus inhibition assay. they demonstrated that 0.1 mg/ml of go-ag can inhibits 24.8% of feline coronavirus infection. go-ag showed higher efficacy when compared with treatment with go alone. as illustrated in figure 2 , during the process of restraining viral infection, go sheets, which were negatively charged, interact with the positively charged lipid membranes and silver particles bind to the sulfur groups of the viral proteins. as a result, it has been shown that go could only have an inhibitory effect on enveloped viruses at noncytotoxic concentrations while go sheets containing agnps are capable of impeding the infection caused by both nonenveloped and enveloped viruses. to tackle human coronavirus nl63, ciejka et al. developed a biopolymeric material to form nano/microspheres (ns/ms) which had good potential for adsorbing coronaviruses [41] . the investigation demonstrated that after addition of n-(2-hydroxypropyl)-3-trimethyl chitosan (h-htcc)-ns/ms to viral suspensions there was a decrease in the copy number of viral rna, this showed good correlation with the amount of added h-htcc-ns/ms. their study showed that 2.5 mg/500 μl h-htcc-ns/ms could cause a decrease equal to 99.60%. when 10 mg/500 μl of hhtcc-ns/ms was added, there was a decrease of 99.92%. therefore, nanomaterials can have antiviral applications against a range of coronaviruses. however, more focus needs to be placed in investigating antiviral nanomedicines against sars-cov, mers-cov and sars-cov-2. a summary about the applied nps-based antiviral agents for disinfection of coronaviruses with some details on their function and inhibition efficiency are represented in table 2 . detecting variations in dna sequences has been found to have a key role in diagnosing and subsequently treating genetic-related conditions at early stages. among the various types of genetic alteration diseases, sequence-specific mismatch is of significant importance; however, detection of this variation is very difficult and this problem is more serious in case of single-nucleotide polymorphism [43] . it is noteworthy that sequence-specific detection is an important and interesting topic in different medical fields among which pathogen response studies, inherited diseases diagnosis and bacterial/viral detection can be highlighted [44] . in the year 2017, teengam et al. developed a multiplex colorimetric paper-based analytical device using ag nps as a colorimetric reagent for the detection of dna associated with viral infection such as mers-cov. in their study, under an optimum condition, a limit of detection of 1.53 nm was achieved [45] . another study, performed by layqah and eissa in 2019, described an electrochemical immunosensor utilizing an array of carbon electrodes which were modified with au nps which enabled detection of human coronavirus (hcov) and mers-cov proteins in spiked nasal samples [46] . the method was conducted with lowest detection limit of 1 and 0.4 pgml -1 for future science group www.futuremedicine.com reproduced with permission from [48] , licensed with cc by-nc-nd 4.0. with a detection limit of 47.91 eid/50 ml egg infective dose (eid) [47] . in another study, ahmed and coworkers reported an approach for ibv detection by using zirconium quantum dots (zrqds) and magnetoplasmonic (mp) nps [48] . as indicated in figure 3 , after preparation of zrqds and mp nps, the ibv antibodies were conjugated future science group www.futuremedicine.com table 3 . recent applications of nanoparticles in developing coronaviruses sensors based on different analytical techniques and related limit of detections. detection way lod ref. with both nanomaterials. at this step, no attraction or conjugation was observed between the utilized nanomaterials (i.e., zrqds and mp) and they were apart from each other till the time of adding the target virus. by addition of a virus, a magnetoplasmonic-fluorescent nanohybrid structure was formed and then separated using an external magnet. eventually, the concentration of the virus was measured through photoluminescence intensity of the nanohybrids with a detection limit of 79.15 eid/50 ml. in a study by weng and neethirajan, an immuno biosensor based on antibody-functionalized mos 2 for rapid detection of ibv with a limit of detection equal to 4.6 × 10 2 eid 50 per ml was developed [49] . in contrast to traditional tests, their immunosensor provided remarkable advantages such as higher sensitivity and lower analysis time, as well as acceptable linearity and validation using the elisa technique. in another study, wang et al. developed a nano-nest pcr assay based on aunps for discrimination of the variant and classical strains of pedv [50] . a total of 78 clinical samples collected from different areas in china were assessed by using both nano-nest and common reverse transcription polymerasechain reaction (rt-pcr). results indicated that the nano-nest pcr assay was more powerful, with 100-fold sensitivity, than common pcr. in order to determine the specificity of this nano-based assay, five other viruses; classical swine fever virus, porcine rotavirus, pseudorabies virus, tgev and porcine reproductive respiratory syndrome virus were also evaluated. interestingly, it was shown that this assay did not amplify the dna or cdna of the other viruses or even of the classical pedv strain, leading to the fact that the nano-nest pcr assay can be successfully applied for specific detection of variant pedv [50] . in a study by liu et al., an immunochromatographic strip (ics) test was developed based on five ibv-specific monoclonal antibodies against the target antigens, spike (s) glycoprotein and nucleocapsid proteins (n), for the detection of ibv in infected chickens. during preparation of the ics, monoclonal antibody-colloidal gold conjugate was used as a tracer in conjugation pad of the strip [51] . according to the results, the best detection limit of 10 4.4 eid 50 was achieved for ibvs. moreover, with rt-pcr as a reference, the assembled ics were identified to be specific to ibv antigens only, compared with other respiratory pathogens including infections laryngotracheitis virus, avian influenza virus and newcastle disease virus. there was good agreement between the results obtained by the ics and rt-pcr. therefore, given the fact that the rt-pcr is an expensive, technically demanding approach, the gold nanoparticles-based ics technique appeared to have the potential of on-farm rapid detection of various ibv strains in chickens. the above studies on the recent applications of nps in developing sensors based on different analytical techniques for coronaviruses and their related limit of detections are compared in table 3 . as it was discussed above agnps, mos 2 nanosheets, qd-mp nps, zr nps and au nps had been applied in detection of a range of coronaviruses. thus, coupling nanomaterials with colorimetric sensing, electrochemiluminescence, immunosensing, photoluminescence and chiroimmunosensing are potential techniques to detect coronaviruses. in addition, electrochemical devices can also be a good options for detection of new kinds coronaviruses in future because of their good ability for coupling with nanomaterials. the advantages of using nanomaterials in this aspect can decrease the analysis time and also increasing the sensitivity, which can open a new door for designing better approaches and higher performance in future. today, there is increasing concern on how to battle coronaviruses as they have been changing the way we live with novel coronavirus disease being a particularly problematic example. despite considerable efforts which have been put in to developing an effective therapeutic strategy against different types of coronaviruses, no specific approach has yet been identified. several studies have been conducted on the application of nanomaterials in the treatment, anti-infection and detection of some types of coronavirus, including those discussed in this report, as well as others [52] [53] [54] [55] . thus, an overview of studies regarding the effectiveness of nanoparticles for diagnostic and therapeutic purposes was presented envisioning the possibility of applying nanomaterials in development of a highly effective vaccine against coronaviruses. up to now, gold, silver, silver sulfide, titanium oxide, zirconium, graphene and some biopolymeric compounds have been the most applicable nanomaterials for this goal. it has been suggested that nanoparticle-based vaccines have a great potential to induce higher protective immunity response in contrast to conventional antigen-based vaccines. moreover, results have indicated that nano-assays hold promise for providing superior specificity and sensitivity, compared with the current techniques, when applied for rapid detection of viral infection at early stages. the quest for finding a reliable, safe and effective vaccine against the sars-cov-2 is still underway. several candidates have been developed ever since its appearance. but at the time of writing this article, there is still no licensed vaccine that can fight it off. according to the previous data obtained from other types of coronaviruses, nano-based vaccines have proven to evoke a more potent immune response. therefore, further research is recommended to address the administration of nanoparticles toward the study of sars-cov-2 in order to create an experimental nano-based-vaccine in a suitable animal model hopefully to provide long-term immunization. application of nano assays for detection of sars-cov and sars-cov-2 is also a recommended topic for researchers in this area. colorimetric sensing, electrochemiluminescence, immunosensing, photoluminescence and chiroimmunosensing and electrochemical sensors are potential techniques to detect coronaviruses in future with the aid of. • the field of nanomedicine could be successfully applied for development of the most promising candidates against the coronaviruses. antiviral activity • ag nanoparticles and their compositions can be promising candidate for research on antiviral agents against coronaviruses. • most suggested responses in antiviral activity against coronaviruses are concentration dependent. nanosensors for diagnosing coronaviruses • using nanomaterials in detection of coronaviruses has some advantages such as improving sensitivity, detection limit, and the analysis time. • nanoparticles can be used for colorimetric detection of coronavirus which is a simple, rapid and low-cost method. • gold nanoparticles are good options for immunochromatographic strip test to use in future studies on detection of coronaviruses. • electrochemical devices can also be a good options for detection of new kinds coronaviruses in future because of their good ability for coupling with nanomaterials. the support of shiraz university of medical sciences (98-01-106-22094) is acknowledged. special thanks to reviewers and editorial office of nanomedicine for their useful comments which improved the quality of this article. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. no writing assistance was utilized in the production of this manuscript. future science group www.futuremedicine.com nanotechnology in sustainable agriculture: recent developments, challenges, and perspectives nantotechniques and approaches in biotechnology functionalized-chitosan/quantum dot nano-hybrids for nanomedicine applications: towards biolabeling and biosorbing phosphate metabolites fluorescent carbon-dots thin film for fungal detection and bio-labeling applications recent advances in nanomaterials for gene delivery -a review nano based drug delivery systems: recent developments and future prospects gold nanoparticles: emerging paradigm for targeted drug delivery system quantum dot bioconjugates for ultrasensitive nonisotopic detection nanosurfaces and nanostructures for artificial orthopedic implants gold-based hybrid nanomaterials for biosensing and molecular diagnostic applications nanostructured and nanoscale devices, sensors and detectors cancer nanotechnology: application of nanotechnology in cancer therapy nanoscale covalent organic polymers as a biodegradable nanomedicine for chemotherapy-enhanced photodynamic therapy of cancer the laboratory diagnosis of dengue virus infection, a review fenner's veterinary virology recent discovery and development of inhibitors targeting coronaviruses summarizes essential information for working on a vaccine for coronaviruses van der hoek l. identification of new human coronaviruses coronavirus infections and immune responses lessons from the past: perspectives on severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) world health organization. coronavirus disease (covid-19) pandemic world health organization. international health regulations characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention emerging viruses and current strategies for vaccine intervention nanovaccines and their mode of action immunostimulatory effect of gold nanoparticles conjugated with transmissible gastroenteritis virus chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles heterologous prime-boost vaccination with adenoviral vector and protein nanoparticles induces both th1 and th2 responses against middle east respiratory syndrome coronavirus viromimetic sting agonist-loaded hollow polymeric nanoparticles for safe and effective vaccination against middle east respiratory syndrome coronavirus •• describes an effective nano-viromimetic approch against middle east respiratory syndrome-coronavirus gold nanoparticle-adjuvanted s protein induces a strong antigen-specific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs nano-sized zinc oxide and silver, but not titanium dioxide, induce innate and adaptive immunity and antiviral response in differentiated thp-1 cells photoinduced antiviral carbon nanohorns nano and microparticulate chitosan-based systems for antiviral topical delivery composition with sterilizing activity against bacteria, fungus and viruses, application thereof and method for preparation thereof inhibitory effect of silver nanomaterials on transmissible virus-induced host cell infections compares the strength of immune responses induced by four different silver nanomaterials on transmissible gastroenteritis nanoparticulate vacuolar atpase blocker exhibits potent host-targeted antiviral activity against feline coronavirus dong nglutathione-capped ag2s nanoclusters inhibit coronavirus proliferation through blockage of viral rna synthesis and budding describes a novel therapeutic strategy based on ag nanomaterials against the alphacoronavirus porcine epidemic diarrhea virus antiviral activity of graphene-silver nanocomposites against non-enveloped and enveloped viruses biopolymeric nano/microspheres for selective and reversible adsorption of coronaviruses pcr-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings dna diagnostics: nanotechnology-enhanced electrochemical detection of nucleic acids multiplex paper-based colorimetric dna sensor using pyrrolidinyl peptide nucleic acid-induced agnps aggregation for detecting mers-cov, mtb, and hpv oligonucleotides • proposes simple paper-based colorimetric dna sensor for detection of some different corona viruses an electrochemical immunosensor for the corona virus associated with the middle east respiratory syndrome using an array of gold nanoparticle-modified carbon electrodes self-assembled star-shaped chiroplasmonic gold nanoparticles for an ultrasensitive chiro-immunosensor for viruses chiral zirconium quantum dots: a new class of nanocrystals for optical detection of coronavirus immunosensor based on antibody-functionalized mos 2 for rapid detection of avian coronavirus on cotton thread rapid detection of variant and classical porcine epidemic diarrhea virus by nano-nest pcr. pak a novel immunochromatographic strip for antigen detection of avian infectious bronchitis virus novel gold nanorod-based hr1 peptide inhibitor for middle east respiratory syndrome coronavirus functional carbon quantum dots as medical countermeasures to human coronavirus preparation of virus-like particle mimetic nanovesicles displaying the s protein of middle east respiratory syndrome coronavirus using insect cells label-free, electrical detection of the sars virus n-protein with nanowire biosensors utilizing antibody mimics as capture probes this work was a review article and does not contain any studies with human participants or animals performed by any of the authors. this article does not contain any studies involving human participants. key: cord-295971-jtv1jj2z authors: cho, sun young; kang, ji-man; ha, young eun; park, ga eun; lee, ji yeon; ko, jae-hoon; lee, ji yong; kim, jong min; kang, cheol-in; jo, ik joon; ryu, jae geum; choi, jong rim; kim, seonwoo; huh, hee jae; ki, chang-seok; kang, eun-suk; peck, kyong ran; dhong, hun-jong; song, jae-hoon; chung, doo ryeon; kim, yae-jean title: mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study date: 2016-07-09 journal: lancet doi: 10.1016/s0140-6736(16)30623-7 sha: doc_id: 295971 cord_uid: jtv1jj2z background: in 2015, a large outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection occurred following a single patient exposure in an emergency room at the samsung medical center, a tertiary-care hospital in seoul, south korea. we aimed to investigate the epidemiology of mers-cov outbreak in our hospital. methods: we identified all patients and health-care workers who had been in the emergency room with the index case between may 27 and may 29, 2015. patients were categorised on the basis of their exposure in the emergency room: in the same zone as the index case (group a), in different zones except for overlap at the registration area or the radiology suite (group b), and in different zones (group c). we documented cases of mers-cov infection, confirmed by real-time pcr testing of sputum samples. we analysed attack rates, incubation periods of the virus, and risk factors for transmission. findings: 675 patients and 218 health-care workers were identified as contacts. mers-cov infection was confirmed in 82 individuals (33 patients, eight health-care workers, and 41 visitors). the attack rate was highest in group a (20% [23/117] vs 5% [3/58] in group b vs 1% [4/500] in group c; p<0·0001), and was 2% (5/218) in health-care workers. after excluding nine cases (because of inability to determine the date of symptom onset in six cases and lack of data from three visitors), the median incubation period was 7 days (range 2–17, iqr 5–10). the median incubation period was significantly shorter in group a than in group c (5 days [iqr 4–8] vs 11 days [6–12]; p<0·0001). there were no confirmed cases in patients and visitors who visited the emergency room on may 29 and who were exposed only to potentially contaminated environment without direct contact with the index case. the main risk factor for transmission of mers-cov was the location of exposure. interpretation: our results showed increased transmission potential of mers-cov from a single patient in an overcrowded emergency room and provide compelling evidence that health-care facilities worldwide need to be prepared for emerging infectious diseases. funding: none. since the fi rst identifi cation of middle east respiratory syndrome coronavirus (mers-cov) infection in 2012, 1 most patients infected with the virus have been exposed in the middle east. as of march 23, 2016, 1698 laboratoryconfi rmed cases have been reported to who. 2 on the basis of previous epidemiological fi ndings, 3 the potential of mers-cov to spread to large numbers of people has been considered low, by contrast with severe acute respiratory syndrome coronavirus (sars-cov). the basic reproductive number of mers-cov was estimated to be less than 1·0, suggesting low transmissibility. 4, 5 however, a 2013 outbreak of mers-cov infection in al hasa, saudi arabia, where one patient infected seven other patients in dialysis and intensive care units, 6 raised concerns about potential so-called super-spreaders 7 that were reported during the sars epidemic. 8, 9 from may to july, 2015, a large outbreak of mers-cov infection occurred in south korea from a traveller returning from the middle east, which led to 186 confi rmed cases (patient 1 to patient 186) in the country. 10 patient 1 was diagnosed at our hospital (samsung medical center, seoul, south korea) after transmitting the virus at several health-care facilities before he came to our hospital. patient 14 was exposed to patient 1 outside the hospital and sought additional care at our hospital without knowing he was infected with mers-cov. therefore, we experienced both south korea's fi rst mers-cov case and the case of highest transmission of mers-cov following a single patient exposure in an emergency room. we aimed to investigate the epidemiology of mers-cov infection in a crowded emergency room outside of the middle east and the presence of multiple super-spreaders. in may, 2015, two patients with mers-cov infection (patient 1 and patient 14) sought care in our emergency room at the samsung medical center without knowing they were infected with mers-cov. while these patients were in the emergency room, a large number of patients, visitors, and health-care workers were exposed during both events. when mers-cov infection was suspected in patient 1 and patient 14, contact investigation was immediately initiated. since no one developed mers among contacts who were exposed to patient 1, only contacts of patient 14 are reported here. we identifi ed, from electronic medical record review and security video footage, all patients who had been in the emergency room with patient 14 as contacts, regardless of the location and duration of exposure. we categorised patient contacts into three groups on the basis of their maximum exposure: patients who were in the same zone in the emergency room (group a; considered close contacts), those who were in diff erent zones but had time overlap with patient 14 in the registration area or radiology suite (30 min before and 2 h after; group b), and those who were in diff erent zones (group c). patients who were admitted to hospital for treatment of their primary illness after exposure in the emergency room were quarantined in private rooms for 14 days from the last exposure or discharged home after treatment was fi nished and continued isolation at home. patients and their family members who were already discharged home were reached by telephone, informed about possible mers-cov exposure, and provided with hotline numbers for any inquiries. health-care workers who were exposed were identifi ed through interviews and review of employees' duty schedules, electronic signature on medical records of patient 14 and patient contacts, security video footage, and self-report. health-care workers who provided direct care to patient 14 were initially considered close contacts and were placed into quarantine at home for 14 days from the last day of exposure. other health-care workers who worked in the emergency room during the same time period continued to work with monitoring and were removed immediately from duty if symptoms developed. a confi rmed case was defi ned as a person with laboratory confi rmation of mers-cov infection from sputum samples, initially by real-time rt-pcr testing with amplifi cation targeting the upstream e region (upe) and then confi rmed by subsequent amplifi cation of open reading frame 1a (orf1a) using powerchek mers realtime pcr kits (kogene biotech, seoul, korea). patients' demographic information, underlying disease, dates of emergency room visit, duration of stay with exact arrival and departure times, and location within the emergency room were collected. if radiographic examinations were done, the time of examination was collected. for healthcare workers, age, sex, occupation, history of patient assignments and working or visiting zone, and dates and time of duty or emergency room visits were collected. the attack rate was calculated by dividing the number of confi rmed cases by the total number of exposed individuals in the emergency room in each group. because the total list of visitors was unavailable, we estimated the number of visitors who were in the emergency room by assuming that one patient had at least one visitor during their stay; we also simulated the scenarios of two and four visitors per patient. to avoid underestimation, we chose the assumption of one visitor per patient, which would give the highest attack rate among the scenarios. the incubation period was defi ned as the time of fi rst exposure to the onset of clinical symptoms of mers-cov infections. categorical variables were presented with frequency (percentage) and continuous variables were summarised with median (range, iqr). we calculated overall comparison of attack rates across the groups with χ² test and across zones with fisher's exact test. incubation evidence before this study little information on nosocomial outbreaks caused by middle east respiratory syndrome coronavirus (mers-cov) outside the middle east had been available before the large mers-cov outbreak in south korea in 2015, for which global alert was issued. we searched pubmed for reports published in english from may 1, 2015, to dec 31, 2015, using the terms "mers-cov" and "korea". we identifi ed 38 reports, none of which provided detailed description for the contact investigation of massive transmission of mers-cov from a super-spreader in an overcrowded emergency room setting. to our knowledge, this study is the fi rst to categorise exposed patients into groups according to the type of exposure and to document group-specifi c incubation periods and attack rates. furthermore, this study provides detailed epidemiological data, including a fl oor plan of the emergency room, to understand how mers-cov spread by a single super-spreader through several modes of transmission. results from our contact investigation showed increased transmission potential of mers-cov from a single spreader, as has been documented in the severe acute respiratory syndrome epidemic. the potential for similar outbreaks anywhere in the world should be noted, as long as mers-cov transmission continues in the middle east. our study provides evidence that hospitals, laboratories, and governmental agencies should be prepared for mers-cov infection. period and exposure time were compared among groups with kruskal-wallis test, followed by tukey's test using ranks for multiple comparisons. to assess the risk factors for mers-cov infection among all patient contacts, we did a multiple logistic regression analysis based on likelihood ratio, by regressing on age, sex, underlying disease, and groups. in a subgroup analysis of patients in group a, the length of stay in the same zone and location were included. for these analyses, odds ratios and 95% cis were reported. p values and 95% cis were adjusted with bonferroni's correction for multiple comparisons if necessary. two-sided p values of less than 0·05 were considered signifi cant. we used sas version 9.4 and graphpad prism version 6.04 for statistical analyses. samsung medical center is a modern 1982-bed university-affi liated tertiary hospital providing referral care in south korea (total population roughly 50 million), with roughly 9000 staff , including more than 1400 physicians and 2600 nurses. the emergency room entrance is located on the ground fl oor near the south gate of the main hospital building. more than 200 patients are seen in the emergency room each day; the average duration of stay in the emergency room was 15 h before the mers-cov outbreak (see appendix p 2 for details on emergency room overcrowding index). the emergency room has seven patient care areas, including zones i to iv for see online for appendix adults, a trauma zone, a resuscitation room, and a paediatric zone (fi gure 1). the paediatric zone and zone iv are separated from the rest of the main areas. two negative-pressure rooms are located in the paediatric zone and two are in zone iv. the emergency room has its own radiology suite for emergency room patients only. the sizes of each zone were as follows: zone i 121·7 m², zone ii 64·2 m², zone iii 168·3 m², and zone iv 223·9 m². zones i and ii included seating areas (50 chairs in zone i and 26 chairs in zone ii), where stable patients received treatment and waited for test results. seriously ill patients, who required close observation and needed a designated bed, were moved to zone iii (17 beds) or zone iv (23 beds). zone iv was used for patients being admitted. beds in zones iii and iv were spaced roughly 1·8 m apart, with curtains in between. nurses were assigned to work in designated zones, whereas physicians and transfer agents took care of patients in several zones. all zones in the emergency room were covered by the same air handling units. there was no funding source for this study. the corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. upon arrival at our emergency room, he denied having travel history to the middle east and any possible exposure to people infected with mers-cov. he was treated for possible bacterial pneumonia on the basis of partial improvement from previous antibiotic treatment and increased c-reactive protein concentration of 13·0 mg/dl (normal <0·3 mg/dl; appendix p 4). during his stay in the emergency room, he was provided with a mask but frequently could not hold it because of severe respiratory symptoms. he was not isolated in a separate room; a negative-pressure room was not considered at that time. as his dyspnoea aggravated on may 28, supplemental oxygen was administered at 2 l per min via a nasal cannula (up to 5 l per min). however, no aerosol-producing procedures, including nebuliser treatments, were given. on the night of may 29, he received a notifi cation call from the health authorities about possible exposure to patient 1, notifi ed our hospital, and was immediately transferred from the emergency room to isolation in a negativepressure isolation room. mers-cov infection was confi rmed on may 30, and he was transferred to the nationally designated health-care facility. from may 27 to may 29, he stayed in three zones in our emergency room: zone ii for roughly 10 h on may 27, zone iii for 19 h from may 27 to may 28, and zone iv for 25 h from may 28 to may 29 (fi gure 1). additionally, from may 27 to may 29, he went to the radiology suites four times. on may 27, he walked around and outside the emergency room and went to the toilet several times because of diarrhoea. between may 27 and may 29, 2015, the average ventilation rate in the emergency room was maintained at three air changes per h, taking 2 h to remove airborne contaminant with a 99·9% effi ciency. 11 the median temperature was 23·8°c (range 14·4-32·2), and the median relative humidity was 32·9% (range 27·1-36·8). 675 patients (117 in group a, 58 in group b, and 500 in group c), an estimated 683 visitors, and 218 health-care workers were identifi ed as contacts of patient 14 (table 1). we assumed that each patient had one visitor and added eight extra visitors (fi ve to group a and three to group c) the epidemic curve of this emergency room-associated outbreak is shown in fi gure 2. the incubation period was determined from 73 confi rmed mers-cov cases: six cases were excluded because we could not determine the date of symptom onset, and data were not available from three visitors. the median incubation period was 7 days (range 2-17, iqr 5-10). among 59 patients and visitors in groups a-c (excluding six who were not initially identifi ed as contacts), the median incubation period was signifi cantly shorter in group a than in group c (fi gure 2). excluding three patients with confi rmed mers-cov infection who were not identifi ed in the initial patient contact investigation (appendix p 5), the overall attack rate for patients in the emergency room was 4% (30 of 675). patients in group a had the highest attack rate (20% [23 of 117]), compared with 5% (three of 58) in group b and 1% (four of 500) in group c (fi gure 3). after adjusting for age, sex, underlying disease, and groups, patients in group a had the highest risk for mers-cov infection (table 2). in group b, all three patients who had mers-cov infection had time overlap in the radiology suite with patient 14. the median exposure time for patients in group a to patient 14 was 3·0 h in zone ii (range 0·5-10·3, iqr 1·9-4·5), 13·9 h in zone iii (0·6-18·9, 6·3-18·4), and 17·4 h in zone iv (0·2-23·2, 9·2-21·4). the attack rates were 23% (13 of 57) in zone ii, 32% (seven of 22) in zone iii, and 8% (three of 38) in zone iv (fi gure 3). after adjusting for age, sex, underlying disease, and exposure time, staying in zone ii was associated with a signifi cantly higher risk for mers-cov infection than staying in zone iv (table 2) . mers-cov transmission occurred in zone iii, despite the fact that the distance from patient 14's bed to the beds of other patients were as far as 6 m (fi gure 4). in zone iv, patient 14 moved from bed 12 to bed 23, and six additional cases were documented in patients and visitors occupying beds in the middle of this zone, which were not adjacent to patient 14's bed. no mers-cov infection was reported in patients and visitors who had been in the emergency room on may 29 during the time period when they were exposed only to zones ii (n=81) or iii (n=15), while patient 14 was confi ned to zone iv. these patients were exposed to areas that were potentially environmentally contaminated but not to patient 14 himself (fi gure 4). under the assumption of one visitor per patient and excluding three visitors with confi rmed mers-cov infection who were not identifi ed in the initial visitor contact investigation (appendix p 5), the overall attack rate for visitors was 6% (38 of 683). all patient contacts (n=675) any underlying disease 1·97 (0·79-5·41) 0·12 error bars represent 95% ci. mers-cov=middle east respiratory syndrome coronavirus. the attack rates for patients and visitors were 20% (47 of 239) in group a, 5% (six of 116) in group b, and 2% (15 of 1003) in group c. under the assumptions of two visitors per patient and four visitors per patient, the overall attack rates for visitors were 3% and 1%, respectively. 218 health-care worker contacts were identifi ed, and fi ve (2%) developed mers-cov infection. three healthcare workers who were not initially identifi ed as contacts (one security guard, one physician, and one patient transfer agent) developed mers-cov infection. although they were not involved in the direct care for patient 14, they visited the emergency room between may 27 and may 29. only close contacts were furloughed and other health-care workers were isolated when they developed symptoms. there were no secondary cases from health-care workers among contacts during the their duty hours. we did a contact investigation of the mers outbreak at the samsung medical center by grouping exposed individuals on the basis of the extent of exposure to patients 1 and 14. to our knowledge, we are the fi rst to document group-specifi c incubation periods and attack rates. our results showed the increased transmission potential of mers-cov from a single patient in an overcrowded emergency room setting. overcrowding is an important issue for this outbreak and is also a common feature of modern medicine. this study is unique because the index exposure occurred in a large emergency room in a tertiary-care centre, with electronic medical record information available to track the location and duration of exposure, thus enabling near-complete tracing of exposed contacts. the classic defi nitions of close contact as being within roughly 6 feet (1·8 m) or within the same room or care area for a prolonged period of time were diffi cult to apply to an emergency room setting with high patient volumes, ongoing traffi c within the emergency room and to and from the radiology suite, and large numbers of visitors and family members. we considered all patients who visited the emergency room during the stay of patients 1 and 14 as exposed contacts, developed criteria for close contacts by expanding on the defi nitions of the us centers for disease control and prevention (cdc), 12 and categorised patients into diff erent groups. therefore, we could establish group-specifi c viral incubation periods and attack rates during the outbreak. in close contacts who stayed in the same zone, the incubation period was shorter and attack rate was higher than patients who stayed in diff erent zones. additionally, in zones iii and iv, patients were infected even when they were separated by curtains most of the time and were apart as far as 6 m (for beds on either side of the nurse's station; fi gure 4). similar to the sars outbreak, we observed so-called superspreaders among patients with mers-cov infection, and these super-spreaders can cause large outbreaks through several modes of transmission similar to those in the sars outbreak. 13 among patients who stayed in various locations, those who overlapped with patient 14 at the radiology suite or registration area had higher attack rates (5%) than the rest of the patients (1%), suggesting that transmission might occur by even brief exposures to recently contaminated objects or encounters with individuals carrying a super-spreader. comparisons of environmental exposure and patient exposure also revealed unique fi ndings. no patient developed mers-cov infection after exposure on may 29 only to the environment that had been potentially contaminated on may 27 (zone ii) and may 28 (zone iii) while patient 14 was confi ned to zone iv on may 29. it is plausible that even if the environment was heavily contaminated by a super-spreader, the virus might not persist long enough in the environment to be capable of causing any new infection. although patient exposure is clearly the most important factor in the spread of mers-cov, more research is needed to address the potential of environmental spread. 14 increased viral load and larger amounts of respiratory secretions have been suggested as the factors for sars-cov super-spreaders. 15, 16 in this mers outbreak, frequent ambulation of the index case could be considered as one factor related to high levels of viral transmission, in addition to large amounts of respiratory secretions and high viral load (cycle thresholds 18·6 for upe and 19·3 for orf1a from patient 1's sputum, 17 and 16·2 for upe and 19·9 for orf1a from patient 14's sputum). of note, patient 1 infected 28 patients in another hospital but caused no confi rmed secondary cases in our hospital, whereas patient 14 caused an additional 82 cases in our hospital. the diff erence of transmissibility between these two individuals could be caused by a combination of factors such as the time from onset of disease, clinical symptoms, duration of contact exposure, pattern of behaviour inside and near the emergency room, and kinetics of viral shedding. we showed that obtaining a travel history from patients is an important element of history taking by all physicians, and not only those specialising in infectious diseases or those working in infection control. suspicion for unusual infections should be maintained if patients do not or cannot report accurate histories. readiness of laboratory support is essential for initial investigation and for control of outbreaks, and overly rigorous requirements for laboratory testing have the potential to delay diagnosis and further spread disease. hospital leadership needs to lead in preparedness for disaster management of high-risk communicable infectious diseases. emergency preparedness at a national level and communication and support from government agencies are imperative to prevent and control any serious outbreak. the results of this study need to be interpreted with caution because the study was not suffi ciently powered to study risk factors for transmission. some of our data were collected retrospectively. analysis on visitors was limited because we did not have detailed data. serological tests were not done simultaneously and attack rates were calculated on the basis of results from real-time rt-pcr of mainly symptomatic individuals. the potential transmission of mers-cov by asymptomatic carriers is under investigation. in conclusion, we report a large nosocomial mers outbreak that occurred outside the middle east. the potential for similar outbreaks anywhere in the world from a single traveller should be noted, as long as mers-cov transmission continues in the middle east. emergency preparedness and vigilance are crucial to the prevention of further large outbreaks in the future. our report serves as an international alarm that preparedness in hospitals, laboratories, and governmental agencies is the key not only for mers-cov infections but also for other new emerging infectious diseases. syc and j-mk designed the study and data collection methods, did the initial data analyses, drafted the manuscript, and approved the fi nal manuscript as submitted. yeh, who suspected and diagnosed the fi rst case of mers-cov infection in south korea, engaged in the management of mers-cov outbreak control at the samsung medical center as an infectious disease specialist and reviewed the manuscript. gep, jyel, j-hk, jyol, jmk, jgr, and jrc coordinated data collection, engaged in the management of mers-cov outbreak control at the samsung medical center, and reviewed the manuscript. sk supervised data collection and analysis, analysed the data, and reviewed the manuscript. hjh, c-sk, and e-sk did laboratory tests for mers-cov detection, coordinated laboratory data collection, and reviewed the manuscript. c-ik, ijj, krp, h-jd, and j-hs supervised data collection and reviewed the manuscript. y-jk and drc conceptualised and designed the study, and critically reviewed and revised the manuscript. all authors approved the fi nal submitted manuscript. we declare no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia. geneva: world health organization middle east respiratory syndrome coronavirus: quantifi cation of the extent of the epidemic, surveillance biases, and transmissibility interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission hospital outbreak of middle east respiratory syndrome coronavirus superspreading and the eff ect of individual variation on disease emergence transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions superspreading sars events middle east respiratory syndrome coronavirus outbreak in the republic of korea guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) middle east respiratory syndrome coronavirus (mers) evidence of airborne transmission of the severe acute respiratory syndrome virus stability of middle east respiratory syndrome coronavirus (mers-cov) under diff erent environmental conditions severe acute respiratory syndrome-singapore severe acute respiratory syndrome middle east respiratory syndrome in 3 persons, south korea we express our sincere consolation for the patients and their families who had mers-cov infection. we greatly appreciate the eff orts of all the hospital employees and their families at the samsung medical center, who worked tirelessly during this outbreak. we also appreciate the cooperation of all other hospitals in south korea that worked together to overcome the nationwide outbreak. we acknowledge the consultation and support of the korea centers for disease control and prevention, the mers rapid response team of the public-private joint mers task force, who, and the us centers for disease control and prevention. we also sincerely appreciate the discussion and critical feedback from michael t osterholm (university of minnesota, minneapolis, mn, usa) and janet a englund (seattle children's hospital, seattle, wa, usa). key: cord-299608-wqa98m4v authors: al-turaiki, isra; alshahrani, mona; almutairi, tahani title: building predictive models for mers-cov infections using data mining techniques date: 2016-09-15 journal: j infect public health doi: 10.1016/j.jiph.2016.09.007 sha: doc_id: 299608 cord_uid: wqa98m4v background: recently, the outbreak of mers-cov infections caused worldwide attention to saudi arabia. the novel virus belongs to the coronaviruses family, which is responsible for causing mild to moderate colds. the control and command center of saudi ministry of health issues a daily report on mers-cov infection cases. the infection with mers-cov can lead to fatal complications, however little information is known about this novel virus. in this paper, we apply two data mining techniques in order to better understand the stability and the possibility of recovery from mers-cov infections. method: the naive bayes classifier and j48 decision tree algorithm were used to build our models. the dataset used consists of 1082 records of cases reported between 2013 and 2015. in order to build our prediction models, we split the dataset into two groups. the first group combined recovery and death records. a new attribute was created to indicate the record type, such that the dataset can be used to predict the recovery from mers-cov. the second group contained the new case records to be used to predict the stability of the infection based on the current status attribute. results: the resulting recovery models indicate that healthcare workers are more likely to survive. this could be due to the vaccinations that healthcare workers are required to get on regular basis. as for the stability models using j48, two attributes were found to be important for predicting stability: symptomatic and age. old patients are at high risk of developing mers-cov complications. finally, the performance of all the models was evaluated using three measures: accuracy, precision, and recall. in general, the accuracy of the models is between 53.6% and 71.58%. conclusion: we believe that the performance of the prediction models can be enhanced with the use of more patient data. as future work, we plan to directly contact hospitals in riyadh in order to collect more information related to patients with mers-cov infections. in 2012, saudi arabia witnessed the outbreak of a virus called middle east respiratory syndrome coronavirus (mers-cov). the novel virus belongs to the coronaviruses family which is responsible for causing mild to moderate colds. mers-co is blamed for causing severe acute respiratory illness that lead to death in many cases. according to [1] , mers-cov symptoms include: cough, fever, nose congestion, breath shortness, and sometimes diarrhea. the virus began spreading rapidly in saudi arabia in 2013. since then, the control and command center of saudi ministry of health in saudi arabia started recording and reporting the cases. the ministry website provides daily statistics on new confirmed mers-cov cases, recoveries, and deaths. infection with mers-cov can lead to fatal complications. unfortunately, there is little information about how the virus spreads and how patients are affected. data mining is the exploration of large datasets to extract hidden and previously unknown patterns and relationships [2] . in healthcare, data mining techniques have been widely applied in different applications including: modeling health outcomes and predicting patient outcomes, evaluation of treatment effectiveness, hospital ranking, and infection control [3] . in this paper, we build several models to predict the stability of the case and the possibility of recovery from mers-cov infection. the goal is to better understand which factors contribute to complications of this infection. the models are built by applying data mining techniques to the data provided by the control and command center of saudi ministry of health website [1] . the rest of the paper is organized as follows. in literature review section, we review related work in the applications of data mining in healthcare. methodology section describes the dataset, pre-processing steps, data mining techniques, and our experimental results. finally, conclusion section concludes the paper with findings. in this section, we highlight some of the related work in data mining applications in healthcare. data mining has been widely used for the prognosis and diagnoses of many diseases. ferreira et al. [4] used data mining to improve the diagnosis of neonatal jaundice in newborns. in their experiment, the dataset consisted of 70 variable collected for 227 healthy newborns. many data mining techniques were applied, including: j48, cart, naive bayes classifier, multilayer perceptron, smo, and simple logistic. the best predictive models were obtained by using naive bayes, multilayer perceptron, and simple logistic. for heart disease diagnoses, venkatalakshmi and shivsankar [5] compared the performance of decision tree algorithm and naive bayes. the experimental results using a dataset of 294 records with 13 attributes showed that the performance of the two algorithms is comparable. fp-growth, association rule mining, and decision trees were used for the diagnosis and prognosis of breast cancer [6] . the classification models were built using a dataset of 699 records and 9 attributes and the best accuracy was achieved using decision trees induction algorithms. in terms of survivability predicting, bellaachia et al. [7] used naive bayes, back-propagated neural network, and the c4.5 decision tree algorithm to predict the survivability of breast cancer patients. the dataset used in the study was obtained from the surveillance epidemiology and end results (seer). experimental results indicated that the c4.5 algorithm outperformed the other two techniques. recently, several predictive models for breast cancer survival were developed [8] . the models were based on a dataset of 657,712 records and 72 variables, also obtained from seer. three different data mining techniques were used: support vector machine (svm), bayes networks, and chi-squared automatic interaction detection (chaid). results showed that the best survival prediction model was obtained using svm. the authors in [8] presented a study of predictive models for breast cancer survival. the main goal was to discover important attributes that contribute to breast cancer survival. three data mining techniques were used: support vector machine (svm), bayes net, and chi-squared automatic interaction detection (chaid). experiments on a dataset obtained from seer showed that the svm model outperformed other models in terms of accuracy, sensitivity, and specificity. svm was able to identify ten attributes that are important indicators of breast cancer survivability. sandhu et al. [9] proposed a cloud-based mers-cov prediction system. the system is based on bayesian belief networks (bbn) for initial classification of patients. a geographic positioning system is utilized to represent patients on google maps. patients classified as infected were tracked using gps from their mobile phones. the proposed system is useful to citizens since it allows them to avoid infected areas. in addition, healthcare authorities can manage the infection problem more effectively. the bbn achieved an accuracy of 83.1% on synthetic data. as mentioned earlier, our dataset was obtained from the website of the control and command center of saudi ministry of health [1]. we used the data on mers-cov infections reported between 2013 and 2015. the data was published in three separate categories: new cases, recoveries, and deaths. for all the categories, the following patient information was provided: gender, age, nationality, city, and whether the patient is a healthcare personnel or not. in addition, there was more specific information for each category. the additional information is as follows: • new cases: symptomatic, current status, and whether the patient had any contact with suspected or confirmed mers-cov infection case. • recoveries and deaths: does the patient have pre-existing diseases. we collected 633 new case records, 231 recovery records, and 218 death records, for a total of 1082 records. a sample of the original dataset is shown in fig. 1 . the dataset was published in different formats. some of the records were found as text files. other records were provided in image format. thus, our first step was to prepare the data in a unified format appropriate for data mining. information in image format was manually extracted. records with missing and inconsistent values (ex. gender is adult) were excluded. the age attribute was converted to discrete values. finally, all records were prepared in .csv format. in order to build our prediction models, we split the dataset into two groups. the first group consisted of recovery and death records. a new attribute was created to indicate the record type, such that the dataset can be used to predict the recovery from mers-cov. the second group contained the new case records to be used to predict the stability of the infection based on the current status attribute. classification is a widely used technique in healthcare. here, we build several classification models to predict the stability and recovery of mers-cov infection. we apply naive bayes and j48 [10] decision tree algorithm. here, we briefly describe these algorithms. • naive bayes classifier: is a probabilistic model based on bayes theorem. it assumes class conditional independence, where the dependencies between class attributes are ignored. research has shown that naive bayes classifiers have comparable performance to other classification algorithms such as decision trees and neural networks. in addition, they produce highly accurate models and can deal with large datasets [2] . • j48 decision tree algorithm: is an implementation by the weka project team of the well-known tree induction algorithm c 4.5 [10] . it follows a greedy iterative approach in building the decision tree. the algorithm partitions the dataset based on the best informative attribute. at each iteration, the attribute with maximum gain ratio is selected as the splitting attribute. decision tree classification models have many advantages. they are easy to interpret and are known to have comparable accuracy to other classification models. the weka platform [11] was used in our experiment. it is a well-known data mining software that supports a wide range of data mining algorithms with a friendly user graphical user interface. all models were built using 10-fold cross validation. here, we discuss the obtained prediction models. in the j48 decision tree recovery model, shown in fig. 2 , the attribute healthcare personnel appears as the first splitting attribute. this indicates the importance of this information. the model can be interpreted as follows: if the patient is a healthcare personnel, the model predicts recovery. however, if the patient is not a healthcare personnel then the model examines whether he has any pre-existing disease. if the patient suffers from other diseases, the model predicts death, otherwise recovery is predicted. according to this model, healthcare personnel are more likely to survive mers-cov infections. this could be due to the vaccinations that healthcare workers are required to get on regular basis. fig. 3 shows the stability model using j48 decision tree algorithm. this model shows that the two important attributes for predicting stability are symptomatic and age. the model first checks symptoms, if symptoms exist, then the age of the patient is examined. the status of patients between the ages of 66-87 are predicted as critical. from this model, we conclude that old patients are at high risk of developing mers-cov complications. in order to evaluate the accuracy of the obtained models, three performance measures were used: accuracy, precision, and recall. accuracy refers to the percentage of correctly classified records. precision is the percentage of records that the model correctly classified as positives out of all positive predictions. recall measures the true positives recognition rate. these measures are calculated as follows: where p is the number of positive records. n is the number of negative records. tp is the number of records that were correctly classified as positive. tn is the number of records that were correctly classified as negative. fn is the number of records that were misclassified as negative. tables 1 and 2 summarizes the evaluation measures for the obtained recovery and stability models, respectively. the naive bayes recovery model performs better in terms of overall accuracy. in addition, it shows high recognition rate for the recovery class. however, the j48 has better recognition rate for class death. as for stability models, the results indicate that j48 has better overall accuracy. however, the recognition rate of class critical is very low. in general, we observe that the performance measures of the recovery models are higher than the stability models. in this work, we use a real dataset with two classification algorithms known to produce highly accurate models. however, the performance of the all obtained models is not satisfactory for application in real world. the main limitation lies in the size of the training dataset. we believe that there is a need to increase the size of the dataset in order to improve predictions. in addition, more patient information (ex. medical history) can be included. in this paper, we built several models to predict the stability and recovery of mers-cov infections. our models were built using naive bayes and j48 decision tree classification algorithms. the decision tree recovery model indicated that patients who are healthcare personnel are more likely to survive. the age attribute was found to be important in predicting the stability of the patient. old patients with ages between 66 and 87 are more likely to suffer from critical complications. the performance of all the models was evaluated and compared. in general, the accuracy of the models is between 53.6% and 71.58%. we believe that the performance of the prediction models can be enhanced with the use of more patient data. as future work, we plan to directly contact hospitals in riyadh in order to collect more information related to patients with mers-cov infections. data mining: concepts and techniques, 3rd edition the morgan kaufmann series in data management systems practical applications of data mining applying data mining techniques to improve diagnosis in neonatal jaundice heart disease diagnosis using predictive data mining data mining techniques for diagnosis and prognosis of cancer predicting breast cancer survivability using data mining techniques prediction of breast cancer survival through knowledge discovery in databases an intelligent system for predicting and preventing mers-cov infection outbreak 5: programs for machine learning witten ih. the weka data mining software: an update the project was scientifically supported by king saud university, deanship of scientific research, research chairs and the research chair of health informatics and promotion. no funding sources. none declared. not required.available online at www.sciencedirect.com key: cord-252049-rgdynmla authors: tomar, sakshi; johnston, melanie l.; st. john, sarah e.; osswald, heather l.; nyalapatla, prasanth r.; paul, lake n.; ghosh, arun k.; denison, mark r.; mesecar, andrew d. title: ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3cl(pro)): implications for nsp5 regulation and the development of antivirals date: 2015-06-08 journal: journal of biological chemistry doi: 10.1074/jbc.m115.651463 sha: doc_id: 252049 cord_uid: rgdynmla all coronaviruses, including the recently emerged middle east respiratory syndrome coronavirus (mers-cov) from the β-cov subgroup, require the proteolytic activity of the nsp5 protease (also known as 3c-like protease, 3cl(pro)) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. kinetic studies indicate that in contrast to 3cl(pro) from other β-cov 2c members, including hku4 and hku5, mers-cov 3cl(pro) is less efficient at processing a peptide substrate due to mers-cov 3cl(pro) being a weakly associated dimer. conversely, hku4, hku5, and sars-cov 3cl(pro) enzymes are tightly associated dimers. analytical ultracentrifugation studies support that mers-cov 3cl(pro) is a weakly associated dimer (k(d) ∼52 μm) with a slow off-rate. peptidomimetic inhibitors of mers-cov 3cl(pro) were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that mers-cov 3cl(pro) undergoes significant ligand-induced dimerization. kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. primary sequence comparisons and x-ray structural analyses of two mers-cov 3clpro and inhibitor complexes, determined to 1.6 å, reveal remarkable structural similarity of the dimer interface with 3cl(pro) from hku4-cov and hku5-cov. despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control mers-cov 3cl(pro) dimerization. activation of mers-cov 3cl(pro) through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of mers-cov 3cl(pro) inhibitors as antiviral agents. states (23) , there is an urgent need to study and characterize the properties of important drug targets of mers-cov for the development of effective therapeutics. coronaviruses express a ͼ800-kda replicase polyprotein, which is processed by viral 3cl pro protease (or nsp5) at 11 distinct cleavage sites to yield intermediate and mature nonstructural proteins (nsp) responsible for many aspects of virus replication (3, 24 -26) . because of its indispensable role in the virus life cycle, 3cl pro is an important target for therapeutic intervention against coronavirus infections (27) (28) (29) (30) (31) (32) (33) . a number of kinetic, biophysical, and x-ray structural studies have demonstrated that sars-cov 3cl pro is only active in vitro as a tightly associated dimer with a dimer dissociation constant (k d ) in the low nanomolar range (34 -38) . the addition or deletion of amino acids, e.g. his 6 affinity tags, at either the n or c terminus drastically reduces the enzymatic rate and decreases the ability of sars-cov 3cl pro to dimerize (37) . although cellular evidence for the auto-cleavage mechanism (cis versus trans) of 3cl pro is lacking, models for how 3cl pro cleaves itself from the polyprotein to form the mature dimer have been proposed based on in vitro studies using purified 3cl pro (34, 39, 40) . a current model posits that two inactive 3cl pro molecules within two separate polyproteins recognize each other and form an immature dimer capable of cleaving the nsp42nsp5 and nsp52nsp6 sites in trans, followed by formation of an active and mature dimer that can then rapidly process other cleavage sites and multiple polyproteins. it has also been proposed that substrate-induced dimerization regulates the enzymatic activity of sars-cov 3cl pro during virus replication; however, no experimental evidence of this has ever been demonstrated in infected cells (40) . although our knowledge of sars-cov 3cl pro is extensive, the dimerization properties of 3cl pro from mers-cov and other coronaviruses, as well as the factors regulating their enzymatic activity, remain largely unknown. to understand the properties of mers-cov 3cl pro , we conducted a series of kinetic, biophysical and x-ray structural studies. here, we report a detailed kinetic and biophysical analysis of mers-cov 3cl pro activity and dimerization. these kinetic and biophysical studies provide evidence for a weakly associated mers-cov 3cl pro dimer. in addition, we utilized our previous knowledge on the design of potent sars-cov 3cl pro peptidic inhibitors to design a series of inhibitors of mers-cov 3cl pro that exhibit low micromolar potency. we demonstrate that mers-cov 3cl pro requires the binding of a ligand for dimer formation, indicating that ligand-induced dimerization is likely a key mechanism in the regulation of mers-cov 3cl pro activity during virus infection. construct design and expression of mers-cov 3cl pro -the gene encoding 3cl pro protease of mers-cov (amino acid residues 3248 -3553 in the replicase polyprotein, genbank tm accession number ahc74086.1) was codon-optimized for optimal expression in e. coli (biobasic inc). the gene was subcloned into pet-11a expression vector with an n-terminal his 6 tag followed by the nsp42nsp5 auto-cleavage site using the forward primer 5ј-atatacatatgcaccaccaccac-caccacagcggtgttctgcagtctggtc-3ј and the reverse primer 5ј-gacggatccttactgcatcacaa-cacccatgatctgc-3ј. the construct was verified by dna sequencing at the purdue university genomics core facility. this construct results in the expression of mers-cov 3cl pro without any n-or c-terminal extensions. mers-cov 3cl pro was expressed through auto-induction in escherichia coli bl21-de3 cells in the presence of 100 g/ml carbenicillin as described previously (41) . cells were harvested by centrifugation at 5000 ϫ g for 20 min at 4°c, and the pellets were stored at ϫ80°c until further use. mers-cov 3cl pro purification-frozen pellets from 4 liters of bacterial cell culture were thawed on ice and resuspended in 250 ml of buffer a (20 mm tris, ph 7.5, 0.05 mm edta, 10% glycerol, and 5 mm ␤-mercaptoethanol (bme)), containing 500 g of lysozyme and a small amount of dnase. cells were then lysed using a single pass through a french press at 1200 p.s.i., and cell debris was removed from the cleared lysate by centrifuging at 29,000 ϫ g for 30 min. solid ammonium sulfate was added to the cleared lysate to a final concentration of 1 m through gradual mixing on ice. hydrophobic interaction chromatography-the cleared lysate, mixed with ammonium sulfate, was loaded at a flow rate of 3 ml/min onto a 60-ml phenyl-sepharose 6 fast-flow highsub column (amersham biosciences) equilibrated with buffer b (50 mm tris, ph 7.5, 1 m ammonium sulfate, 0.05 mm edta, 10% glycerol, and 5 mm bme). the column was then washed with 5ϫ column volume (300 ml) of buffer b at a flow rate of 4 ml/min. protein was eluted using a 5ϫ column volume (300 ml) linear gradient to 100% buffer a. fractions (12 ml) were collected, and those containing mers-cov 3cl pro , as judged through sds-page analysis and specific activity measurements, were pooled (120 ml) and exchanged into 2 liters of buffer a via overnight dialysis in a 10,000 molecular weight cutoff snakeskin dialysis tubing (thermo scientific). deae anion-exchange chromatography-dialyzed sample from the previous step was loaded at a flow rate of 3 ml/min onto a 120-ml deae anion-exchange column (amersham biosciences) equilibrated with buffer a. the column was then washed with 2ϫ column volume (240 ml) of buffer a at a flow rate of 4 ml/min. a linear gradient (total volume 480 ml) to 40% buffer c (50 mm tris, ph 7.5, 1 m nacl, 0.05 mm edta, 10% glycerol, and 5 mm bme) was used to elute the protein. fractions (6 ml) were collected, and those containing mers-cov 3cl pro were pooled (66 ml) and dialyzed for 4 h in 4 liters of buffer d (20 mm mes, ph 5.5, 0.05 mm edta, 10% glycerol, and 5 mm bme). mono s cation-exchange chromatography-following dialysis, the ph of the sample was manually adjusted to 5.5 using 1 m solution of mes, ph 5.5, and any precipitated protein was removed by filtering through a 0.22-m pore size millex-gp filter (millipore). the filtered sample was then loaded at a flow rate of 2 ml/min onto an 8-ml mono s 10/100 column (amersham biosciences) equilibrated in buffer d. the column was then washed with 5ϫ column volume (40 ml) of buffer d at a flow rate of 2 ml/min. protein was eluted using a 25ϫ column volume (200 ml) and a linear gradient to 50% buffer e (50 mm mes, ph 5.5, 1 m nacl, 0.05 mm edta, 10% glycerol, and 5 mm bme). fractions (2 ml) were collected, and those containing mers-cov 3cl pro were pooled (22 ml) and concentrated to ϳ5 mg/ml. gel filtration chromatography-as the final purification step, the concentrated protein sample was loaded onto the preparation grade superdex 75 26/60 gel filtration column (amersham biosciences) equilibrated with buffer f (25 mm hepes, ph 7.5, 10% glycerol, 2.5 mm dithiothreitol (dtt)). protein was eluted isocratically at a flow rate of 1 ml/min with buffer f. fractions (2 ml) containing mers-cov 3cl pro were pooled (total volume of 34 ml) and concentrated to ϳ5 mg/ml. for final storage of the purified mers-cov 3cl pro enzyme, 300-l protein aliquots were placed into 1-ml screw-cap vials, flash-frozen under liquid nitrogen, and then stored at ϫ80°c until further use. purification of sars-cov, hku4-cov, and hku5-cov 3cl pro -sars-cov 3cl pro and hku5-cov 3cl pro with authentic n and c termini were expressed and purified as described previously (37, 42) . hku4-cov 3cl pro was purified utilizing a modified protocol from ref. 42 . final protein yield was calculated based on the measurement of total activity units (m product/min), specific activity (units/mg), and milligrams of protein obtained (bio-rad protein assay) after each chromatographic step. synthesis of compounds 1-11-the peptidomimetic compounds with michael acceptor groups (compounds 1-9, table 3 ) were synthesized via very similar methods to those published previously (30, 43) . synthesis of noncovalent peptidomimetic compounds 10 and 11 (table 3 ) has been described previously (33) . fluorescence-based kinetic assays-the enzymatic activity of 3cl pro was measured using the following custom-synthesized peptide: hilytefluor tm -488-esatlqsglrkak-(qxl tm -520)-nh 2 (anaspec, inc.). the hilytefluor tm -488 fluorescence group was internally quenched by qxl tm -520 dye. this substrate works as a generic peptide substrate for 3cl pro enzymes and was designed based on the nsp42nsp5 cleavage sequence for many coronavirus 3cl pro enzymes. the rate of enzymatic activity was determined at 25°c by following the increase in fluorescence ( excitation ϭ 485 nm, emission ϭ 528 nm, bandwidths ϭ 20 nm) of hilyte fluor tm -488 upon peptide hydrolysis by the enzyme as a function of time. assays were conducted in black, half-area, 96-well plates (corning glass) in assay buffer (50 mm hepes, ph 7.5, 0.1 mg/ml bsa, 0.01% triton x-100, and 2 mm dtt) using a final reaction volume of 100 l. the resulting florescence was monitored using a biotek synergy h1 plate reader. the rate of the reaction in arbitrary fluorescence units/s (afu/s) was determined by measuring the initial slope of the progress curves, which were then converted to units of micromolars of product produced per min (m/min) using experimentally determined values of fluorescence "extinction coefficient" as described previously (37) . all reactions were carried out in triplicate. determination of enzymatic efficiency-the apparent enzymatic efficiency for each of the 3cl pro enzymes was determined by measuring the rate of enzymatic activity as a function of varying substrate concentration in 100-l reactions. reactions were initiated by the addition of enzyme to the wells of an assay plate containing varying concentrations of substrate. the final substrate concentrations varied over a range from 0 to 2 m. the final enzyme concentrations for each 3cl pro studied were as follows: mers-cov 3cl pro at 1 m, sars-cov 3cl pro at 100 nm, hku5-cov 3cl pro at 250 nm, and hku4-cov 3cl pro at 200 nm. because 3cl pro enzymes cannot be saturated with this substrate at a substrate concentration that would still allow accurate fluorescent measurements without the inner filter effect, only the apparent k cat /k m values can be determined from the slope of the line that results from a plot of the enzymatic activity (y axis), normalized for the total enzyme concentration, against the substrate concentration (x axis). influence of dimerization on the activity of 3cl pro enzymes-the dependence of the enzymatic activity on the total enzyme concentration was determined using the fret-based assay described above. the final enzyme concentrations were varied over a concentration range from 2 m to 100 nm for mers-cov 3cl pro , 500 to 10 nm for sars-cov 3cl pro , 250 to 0.6 nm for hku5-cov 3cl pro , and 200 to 10 nm for hku4-cov 3cl pro . reactions were initiated by the addition of substrate, at a final concentration of 2 m, to the assay plates containing varying enzyme concentrations in the assay buffer. initial rates were determined from the initial slopes of the progress curves at each enzyme concentration. the rates of the 3cl pro -catalyzed reactions measured over a range of enzyme concentrations can be fit to either equation 1 or 2 to determine the values of the dissociation constant for the monomer-dimer equilibrium as well as the turnover numbers. nonlinear regression and the program tablecurve 2d version 4.0 were used to fit the data to either equation 1 or 2 below (44) . in equation 1, v max is the rate of the enzymatic activity calculated at each enzyme concentration (c t ); k d is the monomerdimer equilibrium dissociation constant, and k cat, m and k cat, d are the turnover numbers for the monomer and the dimer, respectively. in equation 2, v max , c t , and k d have been described previously, and k cat is the turnover number for the dimer only. inhibition assays-to determine the percent inhibition for compounds 1-9, the total concentration of the substrate was fixed at 1.0 m, and the enzymes were fixed at 250 nm for sars-cov 3cl pro , hku5-cov 3cl pro , hku4-cov 3cl pro , and at 500 nm for mers-cov 3cl pro . dmso stocks (100ϫ) of the compounds were diluted a hundred-fold to a final concentration of 50 m in 80 l of the enzyme solution and incubated for 20 min. after 20 min, the enzymatic activity was measured as initial slope of the progress curve, obtained by initiating the reaction with 20 l of 5 m substrate. % inhibition was calculated using equation 3 . in equation 3, rate sample is the initial slope of the progress curve in afu/s measured in the presence of the compound; rate pos is the initial slope measured in the absence of any compound, and rate neg is the baseline substrate hydrolysis calculated in the absence of enzyme. all the reactions were carried out in triplicate and contained a final dmso concentration of 1%. for compounds displaying more than 50% inhibition, a more extensive characterization of the inactivation kinetics was performed through progress curve analysis. to the reaction well, 20 l of 5 m substrate was added to a final concentration of 1 m, and the total inhibitor concentration [i] total was varied from 0 to 50 m. the reaction was initiated with the addition of 80 l of mers-cov 3cl pro to a final concentration of 500 nm. fluorescence intensity was then measured over time as afu t for a period of 70 min. equation 4 describes the resulting time course of reaction. in equation 4 , v i is the initial velocity of the reaction; k obs is the observed first-order rate constant for the reaction in the absence and presence of inhibitor; t is the time in minutes; [p] t is the concentration of product produced at time t, and [p] i is the initial product concentration, which is zero. product concentrations were calculated from the values of afu t , using the experimentally determined fluorescence extinction coefficient. the resulting values of [p] t were then plotted against time t, and the data were fit to equation 4 with [p] i ϭ 0 using the nonlinear regression program tablecurve 2d to derive the fitted parameters v i and k obs and their associated errors ⌬v i and ⌬k obs . values for each k obs were then plotted against [i] total and the data were fit to equation 5 . in equation 5, k inact defines the maximum rate of inactivation at infinite inhibitor concentration, and k i defines the concentration of inhibitor that yields a rate of inactivation equal to 1 ⁄ 2k inact . the half-life of inactivation at infinite inhibitor concentration, which is a measure of inactivation efficiency, is defined as t 1 ⁄2 ∞ ϭ 0.693/k inact . auc analysis-to determine the oligomeric state of mers-cov 3cl pro , sedimentation velocity experiments were performed at 20°c on the beckman-coulter xla ultracentrifuge using varying concentrations of mers-cov 3cl pro (4 -23 m) in 25 mm hepes, ph 7.5, 50 mm nacl, and 1 mm tris(2-carboxyethyl)phosphine at 50,000 rpm. to characterize the effect of the ligand on the monomer-dimer equilibrium of mers-cov 3cl pro , sedimentation velocity experiments were conducted on the beckman-coulter xli instrument using different stoichiometric ratios of mers-cov 3cl pro with compounds 6 and 10. samples were prepared by mixing 25 m mers-cov 3cl pro with 25, 50, and 100 m compound 6 or 10 and incubating the mixture overnight at 4°c before performing the experiments. absorbance optics (280 nm) and interference optics were utilized for protein detection. solvent density, viscosity, and partial specific volumes were calculated using sednterp. sedphat was used to fit the data to the monomer-dimer self-association model to estimate the sedimentation coefficients (s), apparent molecular weights, and k d and k off values from size distribution analysis. to obtain exact molecular weights, sedimentation equilibrium experiments were performed at concentrations of 3 and 17 m mers-cov 3cl pro . the experiments were done at 20°c utilizing a twochannel centerpiece and run at multiple speeds (8100, 13,800 and 24,000 rpm) in a an-60 ti rotor. the rate sample , rate pos , and rate neg are as described above for equation 3 . mers-cov 3cl pro crystallization, x-ray data collection, and structure determination-purified mers-cov 3cl pro was concentrated to 1.6 mg/ml in 25 mm hepes, ph 7.5, and 2.5 mm dtt. inhibitor complexes of mers-cov 3cl pro with compounds 6 and 11 were formed by incubating mers-cov 3cl pro with the compounds in a 1:3 stoichiometric ratio at 4°c overnight. after iterative rounds of optimization of the crystallization conditions based on the initial hits obtained from high throughput screening of qiagen nextel screens, crystals of mers-cov 3cl pro inhibitor complexes suitable for x-ray diffraction were grown by the hanging-drop, vapor diffusion method at 20°c in 0.2 m sodium acetate, 0.1 m bistris, ph 7.0, and 20% peg-3350 for the mers-cov 3cl pro and 6 complex, and 0.2 m ammonium acetate, 0.1 m bistris, ph 5.5, 12% peg-3350 for the mers-cov 3cl pro and 11 complex. for x-ray data collection, crystals were flash-cooled in liquid nitrogen after dragging the crystals through a cryo-solution that contained the crystallization solution supplemented with 15% 2-methyl-2,4-pentanediol. x-ray diffraction data were collected for mers-cov 3cl pro and 6 and mers-cov 3cl pro and 11 complexes at the lilly research laboratories collaborative access team (lrl-cat) sector 31 and the life sciences collaborative access team (ls-cat) sector 21 at the advanced photon source, argonne national laboratory, respectively. data were processed and scaled using mosflm version 7.0.5 (45) and hkl2000 version 706 (46) . the method of molecular replacement was used to obtain initial phases using the program phaser-mr in phenix suite version 1.8.4 (47) . for mers-cov 3cl pro and 6 complex, the x-ray structure of sars-cov 3cl pro (pdb code 3v3m) was used as a phasing model (32) . the final mers-cov 3cl pro and 6 complex structure was then used to calculate the initial phases for the mers-cov 3cl pro and 11 complex model. automated model building using autobuild in phenix was initially used to build a preliminary model of the mers-cov 3cl pro and 6 inhibitor complex. each structure was then refined using iterative cycles of refinement using phenix refine coupled to manual model building using coot (48) based on f o ϫ f c and 2f o ϫ f c maps. coordinates and molecular library files for inhibitor molecules were built using the program elbow in the phenix suite. water molecules were added to peaks in residual (f o ϫ f c ) density maps that were greater than 3 using the "find water" function in coot. molprobity was used to assess structural quality of the final model (49) . the measured structure factor amplitudes and the atomic coordinates for the final structures were deposited in the protein data bank with accession codes 4rsp (mers-cov 3cl pro and 6 complex) and 4ylu (mers-cov 3cl pro and 11 complex), respectively. structural superposition was performed using the method of least squares fitting of c-␣ atoms in coot. pymol was used to generate figures of all the structures (50) . termini-insertion of the nsp42nsp5 cleavage site between the n-terminal his 6 tag and the coding region for mers-cov 3cl pro results in autoprocessing of the his tag and overexpres-sion of mers-cov 3cl pro without any n-terminal extension in e. coli bl21-de3 cells. mers-cov 3cl pro was purified to high purity and an overall yield of 10% using four sequential chromatographic steps. a summary of the percent enzyme yield, total activity units, and the fold-purification after each chromatographic step is summarized in table 1 . approximately 12 mg of highly pure mers-cov 3cl pro can be obtained per liter of bacterial cell culture. to verify the production of the enzyme with correct n and c termini, the molecular mass of purified mers-cov 3cl pro was determined by maldi to be 33.4 kda, which is close to the theoretical molecular mass of 33.3 kda for the authentic/mature mers-cov 3cl pro monomer. western blot analysis of purified mers-cov 3cl pro using an anti-his 6 antibody also confirmed the absence of the n terminus his 6 tag associated with the expression plasmid (data not shown). these results demonstrate that the n-terminal his 6 tag is auto-catalytically removed by mers-cov 3cl pro during its expression in e. coli, indicating mers-cov 3cl pro is enzymatically active when expressed in e. coli. fret-based peptide substrate was used to measure the enzymatic activity of mers-cov 3cl pro as a function of substrate concentration over a substrate concentration range from 0 to 2.0 m (fig. 1a) . we observed that mers-cov 3cl pro cannot be saturated by the substrate over this concentration range, which is typical for other coronavirus 3cl pro enzymes because the k m values for peptide substrates approach 1 mm (51) (52) (53) (54) . therefore, the slope of the kinetic response of mers-cov 3cl pro to increasing substrate concentration was determined to derive an apparent (k cat /k m ) value, which is a measure of enzymatic efficiency. we also determined and compared the apparent (k cat /k m ) values for 3cl pro enzymes from sars-cov, hku5-cov, and hku4-cov under similar experimental conditions (fig. 1b) . mers-cov 3cl pro is able to hydrolyze the peptide substrate; however, the enzymatic efficiency of mersis noticeably lower than other 3cl pro enzymes tested. specifically, mers-cov 3cl pro was 5-fold less efficient at processing the peptide substrate when compared with sars-cov 3cl pro . even among the ␤-covs from the same 2c genogroup (mers, hku5, and hku4), mers-cov 3cl pro was the least efficient enzyme. mers-cov 3cl pro is a weakly associated dimer-because a dimer has consistently been shown to be the catalytically active form of all 3cl pro enzymes studied to date, we tested the hypothesis that the lower enzymatic efficiency of mers-cov 3cl pro is a result of the reduction in its ability to dimerize. therefore, we determined the dependence of the enzymatic activity of mers-cov 3cl pro on the total enzyme concentration and compared it with other 3cl pro enzymes from hku4, hku5, and sars coronaviruses (fig. 2) . it is immediately apparent from the data plotted in fig. 2 that the response of mers-cov 3cl pro enzymatic activity to an increasing enzyme concentration is nonlinear. the strong curvature suggests that a dimer is either the most active form or the only active form of mers-cov 3cl pro . to determine the mechanism of dimerization, the data in fig. 2 were first fit to equation 1 (see "experimental procedures"), which describes a model where both the monomer and the dimer are active. a fit of the data to equation 1 yielded a negative turnover value for the monomer (k cat, m ), suggesting the monomer is inactive and that the dimer is the only active form of the enzyme. therefore, the data were fit to equation 2 (see "experimental procedures"), which considers only the dimer as the active form of the enzyme. the kinetic data for all four 3cl pro enzymes, mers-cov, hku4-cov, hku5-cov, and sars-cov, fit well to this model, and the resulting values for the monomer-dimer equilibrium dissociation constant, k d , and apparent turnover number, k cat , for each enzyme are provided in table 2 . the lower k cat value for mers-cov 3cl pro , when compared with other coronavirus 3cl pro enzymes, indicates a moderate reduction (2-4-fold) in its ability to turn over the substrate, which is consistent with the observed lower apparent (k cat /k m ) value. in contrast, there is a substantial reduction in the ability of mers-cov 3cl pro to dimerize compared with the other 3cl pro enzymes. based on the k d values, the capacity of mers-cov 3cl pro to dimerize is ϳ78 -130-fold weaker than the other enzymes ( table 2 ). these results indicate that the mers-cov 3cl pro dimer is much more weakly associated than the other coronavirus 3cl pro enzymes studied, and these results raise questions as to the structural and mechanistic differences among the 3cl pro enzymes that ultimately regulate protease activity during coronavirus replication. mers-cov 3cl pro inhibition by designed peptidomimetic compounds-in an effort to develop potent inhibitors of mers-cov 3cl pro , we designed and synthesized nine peptidomimetic compounds containing a michael acceptor group, i.e. an ␣,␤-unsaturated carbonyl, capable of irreversibly reacting with the active site cysteine of mers-cov 3cl pro (table 3) . these compounds were designed and synthesized based on our understanding and knowledge of the interactions of similar inhibitor molecules with sars-cov 3cl pro (30, 31) . at a concentration of 50 m, compounds 6 -9 displayed more than 50% inhibition of mers-cov 3cl pro and were further evaluated for their ability to inactivate the enzyme in a time-and concentration-dependent manner (fig. 3) . data from the kinetic progress curve for compound 6 (fig. 3) , as well as for table 2 . final enzyme concentrations varied over the concentration ranges of 2 m to 100 nm for mers-cov 3cl pro , 500 to 10 nm for sars-cov 3cl pro , 250 to 0.6 nm for hku5-cov 3cl pro , and 200 to 10 nm for hku4-cov 3cl pro . final substrate concentration was fixed at 2 m. experiments were done in triplicate. error bars represent the standard deviation for triplicate data. shaded box represents the data that are plotted in b. b, enlarged view of the fitted data at low total enzyme concentrations, marked in shaded box in a, illustrating the nonlinear dependence of enzymatic activity on the total concentrations of 3cl pro from sars-cov, hku5-cov, and hku4-cov. the michael acceptor group for compound 1 is shaded to highlight this group for all the compounds. the stereochemistry at the benzyl stereocenter of compound 5 is a 1:1 mixture of enantiomers (racemic); therefore, the compound was tested as a mixture of diastereomers. * % inhibition was measured as the % loss in enzymatic activity after 20 min of incubation of 500 nm mers-cov 3cl pro with 50 m of the compound. a as compounds 1-5 showed ͻ50% inhibition of mers-cov 3cl pro , values of k inact , t 1/2 ∞ and k i were not determined (nd) for these compounds. 50 compounds 7-9 (data not shown), were fit to the appropriate equations (see under "experimental procedures") to obtain the kinetic parameters, k inact , t 1 ⁄2 ∞ , and k i , and the resulting values are provided in table 3 . we identified four compounds, 6 -9, as micromolar inhibitors of mers-cov 3cl pro with k i values less than 10 m ( table 3) . analysis of structure-activity relationships of these compounds suggests that the s 2 subsite pocket of mers-cov 3cl pro is small and can only accommodate a smaller p 2 -isobutyl substituent (compounds 6-9) but not bigger substituents such as p 2 -benzyl or p 2 -isobutylenyl (compounds 1-5). it was also observed that replacing the p 4 -ethoxy (compound 6) with p 4 -isopropoxy (compounds 7 and 8) had no effect on the inhibitory activity of the compounds. finally, these compounds provide an excellent chemical scaffold to study the molecular details of interactions of substrate-like compounds with the enzyme and to develop more potent inhibitors of mers-cov 3cl pro for therapeutic intervention. to evaluate broad spectrum specificity of these compounds, we also calculated % inhibition of sars-cov 3cl pro , hku5-cov 3cl pro , and hku4-cov 3cl pro after 20 min of incubation in the presence of 50 m compounds 6 -9. except for compound 9, which inhibited sars-cov 3cl pro by 76%, we observed 100% inhibition of all other enzymes in the presence of compounds 6-9. furthermore, we performed progress curve analysis of hku5-cov 3cl pro and hku4-cov 3cl pro in the presence of varying concentrations of compounds 6 -9. the k i values of compounds 6 -9 for hku5-cov 3cl pro are 0.49 ϯ 0.16, 0.60 ϯ 0.21, 1.30 ϯ 0.53, and 0.47 ϯ 0.06 m, respectively. the k i values of compounds 6 -9 for hku4-cov 3cl pro are 0.39 ϯ 0.14, 0.50 ϯ 0.17, 0.85 ϯ 0.33, and 0.64 ϯ 0.25 m, respectively. these data suggest that peptidomimetic compounds 6 -9 have the potential to be developed as coronavirus 3cl pro inhibitors with broad spectrum specificity. weak association of the mers-cov 3cl pro dimer is supported by auc studies-to further explore the mechanism of mers-cov 3cl pro dimerization, we performed analytical ultracentrifugation sedimentation velocity (auc-sv) studies at varying concentrations of mers-cov 3cl pro (fig. 4a) . unlike enzyme kinetics, auc allows determination of the monomer-dimer equilibrium constant (k d ) in the absence of substrate. mers-cov 3cl pro displayed a continuous size distribution at different protein concentrations. two distinct peaks corresponding to monomer (2.9 s) and dimer (3.9 s) species are observed, with the dimer peak becoming more pronounced at higher enzyme concentrations (fig. 4a) . we fit the auc data to a monomer-dimer equilibrium model to determine the values for k d and k off , where k d is the equilibrium dissociation constant for a monomer from the dimer, and k off is the rate constant for dissociation of the monomer from the dimer. the resulting best fit value for k d is 52 ϯ 5 m and that for k off is 10 ϫ4 s ϫ1 . the k d value of 52 m for mers 3cl pro is dramatically different from sars-cov 3cl pro , which has reported k d values ranging from low nanomolar up to 10 m depending on the enzyme construct used and the experimental conditions and methods utilized to determine the dissociation constant (37) . the dimer affinity of mers-cov 3cl pro is substantially weaker than that for sars-cov 3cl pro , when comparing the same enzyme construct, i.e. the enzyme without any n-or c-terminal modifications. the auc-sv calculated k d value for mers-cov 3cl pro is ϳ150,000 times higher than the value of 0.35 nm determined for sars-cov 3cl pro (34) . the auc results (fig. 4a) show that the monomer peak at ϳ2.9s does not gradually shift peak position toward the dimer peak at ϳ3.9s with increasing concentrations of mers-cov 3cl pro ; rather, the two peaks change in area, which is indicative of very slow monomer-dimer exchange rate (k off ϳ10 ϫ4 s ϫ1 ) and the formation of hydrodynamically stable monomer and dimer species (55) . this k off value is 1000 times slower than the k off value (10 ϫ1 s ϫ1 ) reported for sars-cov 3cl pro indicating that the sars-cov enzyme has a significantly more rapid monomer-dimer exchange rate (56) . these observations support a model whereby the mers-cov 3cl pro dimer is weakly associated, suggesting the enzyme exists mainly as a monomer in solution. mers-cov 3cl pro undergoes extensive ligand-induced dimerization-the weak association of mers-cov 3cl pro monomers engenders the following questions. "are higher levels of expression of 3cl pro in mers-cov-infected cells necessary to allow formation of active dimer?" "are other mechanisms such as substrate-or ligand-induced dimerizations involved in activating 3cl pro ?" to explore the latter question of ligand-induced dimerization of mers-cov 3cl pro , we performed auc experiments in the presence of compound 6, which acts as a substrate mimetic and mechanism-based inhibitor, also known as a suicide substrate. peptidomimetic compounds such as compound 6, which contains a michael acceptor group, interact and react with the active site cysteine of cysteine proteases to covalently modify them. we utilized compound 6 to form a covalent mers-cov 3cl pro and inhibitor 6 complex that is stable over long periods of time, making it amenable to analysis by auc-sv experiments. in contrast, incubation of a normal peptide substrate with the enzyme would lead to immediate hydrolysis of the substrate and dissociation of the products from the enzyme, confounding auc experiments and subsequent data analysis. mers-cov 3cl pro was incubated with varying concentrations of compound 6 in stoichiometric ratios of 1:1, 1:2, and 1:4. the modified enzyme was then subjected to auc studies to determine the influence of compound 6 on the monomer-dimer equilibrium (fig. 4b) . a significant shift in the area under 2.9s peak (monomer) to 4.1s peak (dimer) is detected upon addition of increasing concentrations of compound 6. we obtained similar results when auc studies were performed utilizing a complex of mers-cov 3cl pro with a noncovalent peptidomimetic inhibitor (compound 10, figs. 4c). the transition of mers-cov 3cl pro from monomer to dimer in the presence of compounds 6 and 10 suggests that the enzyme undergoes extensive dimerization upon substrate binding. the observed ligand-induced dimerization of mers-cov 3cl pro , as demonstrated through auc studies, prompted us to investigate whether or not the enzymatic activity of mers-cov 3cl pro could be increased at low concentrations of a compound via ligand-induced dimerization. to do so, we chose to use a noncovalent peptidomimetic compound (compound 10, fig. 5a ) that we previously identified as an inhibitor of sars-cov 3cl pro . because of the time-dependent, irreversible nature of the reaction between compound 6 and mers-cov 3cl pro , use of compound 6 was not ideal for these kinetic studies as it would further complicate kinetic data analysis. the kinetic response of mers-cov 3cl pro to increasing concentrations of compound 10 was first measured at a single enzyme concentration of 1.0 m (fig. 5a) . interestingly, an increase in the activity of mers-cov 3cl pro , as high as 195%, was observed in the presence of low inhibitor concentrations (0.1 to 20 m). inhibition of enzymatic activity was observed only at higher inhibitor concentrations (40 m or greater). these results suggest that at low concentrations, compound 10 binds to a monomer and induces the formation of a dimer. the resulting dimer then has one free active site that is capable of processing the substrate. at higher concentrations of inhibitor, the substrate and inhibitor directly compete for the free active site. the model of activation and inhibition suggested by the data at 1 m enzyme would predict that at higher enzyme concentrations less activation by a compound would be observed at lower inhibitor concentrations, and the inhibition of activity would be detected at lower inhibitor concentrations because the equilibrium would be pushed toward dimer formation. in contrast, lower enzyme concentrations would result in higher activation by compounds, and inhibition by the compound would occur at significantly higher compound concentrations. therefore, we further measured the activity of mers-cov 3cl pro at two additional enzyme concentrations (0.5 and 2.0 m) in the presence of varying concentrations of compound 10. remarkably, we observed that the activation effect was most pronounced at the lowest mers-cov 3cl pro concentration tested (0.5 m), and the effect decreased as the enzyme concentration was increased (1.0 and 2.0 m) (fig. 5a ). moreover, inhibition by compound 10 occurred at lower compound concentrations when higher concentrations of enzyme were used. these observations further support a model whereby enzyme activation can occur through ligand-induced dimerization. the activation and inhibition of mers-cov 3cl pro by compound 10 can be explained by a simple kinetic model depicted in fig. 5b . the mers-cov 3cl pro monomer exists in equilibrium with the dimer, and their relative concentrations depend on the total enzyme concentration. in the absence of substrate or compound, the k d value is 52 m, and the equilibrium is represented by the gray spheres (blue box) in fig. 5b . the monomer is unable to hydrolyze the substrate and is therefore inactive. binding of inhibitor (fig. 5b, green triangle) to the monomer results in monomer to dimer switch leading to the formation of a dimer that contains inhibitor bound in one of the active sites. once the dimer is formed, the substrate binds in the second active site and catalysis takes place. under high inhibitor concentrations, however, the inhibitor molecule directly competes with substrate for the free dimer active site, and inhibition of the enzymatic activity is observed as a result. we would also expect to observe induced dimerization and activation in the presence of the substrate. indeed, the monomer-dimer kinetic studies performed in fig. 2 were performed at a fixed concentration of substrate at 2 m. in this experiment, the k d value for the mers-cov 3cl pro dimer was determined to be 7.8 m, which is lower than the k d value determined in the absence of substrate using auc, thereby supporting substrateinduced dimerization. given the high k m value of 3cl pro for the peptide substrate (51-54), even higher substrate concentrations would be required to observe substrate activation in a plot of catalytic activity versus substrate concentration. however, we are limited to use our fret-based substrate only at low concentrations due to a significant inner filter effect at higher concentrations of substrate. therefore, a compound that both mimics substrate and has higher binding affinity can act as a useful surrogate for the substrate, allowing the observation of ligand-induced dimerization and activation even at low substrate concentrations. x-ray structure of mers-cov 3cl pro in complex with compound 6-to gain atomic level detail and molecular insight into the mechanism for substrate-induced dimerization of mers-cov 3cl pro , we attempted to crystallize and determine the x-ray structures of the unliganded mers-cov 3cl pro monomer and the mers-cov 3cl pro covalently modified with compound 6. unfortunately, we were unable to crystallize the unliganded mers-cov 3cl pro monomer after multiple attempts, but we were able to crystallize and determine the x-ray structure of mers-cov 3cl pro in complex with compound 6 to a resolution of 1.6 å. the statistics for x-ray data collection, processing, and refinement are summarized in table 4 . the mers-cov 3cl pro and 6 complex crystallized as a biologically relevant, symmetrical dimer in space group c2 with one monomer in the asymmetric unit. electron density for the entire protein was clearly visible and strong electron density (f o ϫ f c ͼ4) was present for compound 6 within the active site (fig. 6a) . mers-cov 3cl pro has a smaller s 2 pocket than sars-cov 3cl pro -the active site of mers-cov 3cl pro bound with compound 6 is shown in fig. 6, a and b. compound 6 is covalently bound to the active site cysteine (cys-148) via a 1.8 å bond between the ␥-sulfur and the electrophilic ␤-carbon of the michael acceptor. the pј 1 -ethyl ester carbonyl, which mimics the carbonyl of the scissile bond in a substrate, forms a hydro-shaded box) . b, kinetic model describing the equilibrium between different species of mers-cov 3cl pro that are formed in the absence (blue box) and presence (green box) of a ligand is shown. based on the auc-calculated k d value of ϳ 52 m, mers-cov 3cl pro primarily exists as a monomer in solution in the absence of a ligand. upon ligand binding (inhibitor i in our case) to the monomer, the monomer-dimer equilibrium shifts toward dimer formation. next, under lower inhibitor concentrations (cyan-shaded box), substrate (s) binds in the second active site and catalysis takes place. however, under higher inhibitor concentrations (yellow-shaded box), inhibitor directly competes with the substrate for the second active site, and inhibition of the enzymatic activity is observed. gen bond with the backbone nh of gly-146 that forms part of the oxyanion hole (fig. 6b) . within the s 1 subsite, the p 1 -lactam carbonyl, which is a surrogate for the amide of p 1 -glutamine of substrates, participates in a hydrogen bonding interaction with the imidazole ring of his-166, and the p 1 -lactam nh forms a hydrogen bond with the carboxylate oxygen of glu-169. the p 2 -backbone amide nh forms a hydrogen bond with the side chain carbonyl of gln-192 (fig. 6b ). the p 2 -leucine side chain atoms of the inhibitor make hydrophobic contacts with the side chains of met-168 and leu-49 that line the s 2 subsite pocket. moreover, compared with the equivalent residue thr-25 in sars-cov 3cl pro , met-25 in the s 2 pocket of mers-cov 3cl pro is expected to reduce the size of the hydrophobic pocket, which is supported by our observed sar described above. the smaller size of the s 2 pocket in mers-cov 3cl pro is also consistent with the preference for a smaller leucine residue at the p 2 position of cleavage sites instead of a bulkier phenylalanine or methionine residue. indeed, analysis of the preference for leucine or phenylalanine at the p 2 position for the 11 3cl pro cleavage sites within the polyprotein of mers-cov shows that none of the 11 cleavage sites contain a phenylalanine residue at this position (fig. 6c) . leucine is the predominantly favored residue at this position followed by methionine. analysis of the cleavage sites from sars-cov, hku4-cov, and hku5-cov shows that none of the 11 cleavage sites from group 2c members (mers-cov, hku4-cov, and hku5-cov) contain a phenylalanine residue at the p 2 position; however, the sars-cov nsp52nsp6 cleavage site contains a phenylalanine residue at this position. other interactions are also observed to play a significant role in stabilizing the mers-cov 3cl pro -compound 6 complex. the p 3 -carbonyl and p 3 -nh participate in hydrogen bonding interactions with the backbone nh and carbonyl of glu-169. the p 4 -serine side chain is within hydrogen bonding distance of the side chain carboxamide of gln-195 and the backbone carbonyl of lys-191. x-ray structure of mers-cov 3cl pro in complex with a noncovalent inhibitor-we were also able to obtain diffraction quality crystals of mers-cov 3cl pro in complex with compound 11, which has an almost identical chemical structure as that of compound 10 (fig. 6d) . we previously showed that compounds similar to 10 and 11 act as potent noncovalent inhibitors of 3cl pro from sars-cov (33) . the x-ray structure of compound 11 bound to mers-cov 3cl pro was determined to a resolution of 2.1 å and the x-ray data collection, processing, and refinement statistics are summarized in table 4 . the mers-cov 3cl pro and 11 complex crystallized in space group p2 1 with two biologically relevant dimers in the asymmetric unit. the overall root mean square deviation between the c-␣ atoms of the four chains was less than 1 å, with the highest c-␣ root mean square deviation of 0.719 å between chains c and d. strong electron density (f o ϫ f c ͼ4) was present for compound 11 within all the four active sites of the two dimers (fig. 6d) . the binding orientation for compound 11 in the active site of mers-cov 3cl pro is similar to the binding orientation of related compounds in the active site of sars-cov 3cl pro (pdb code 4mds). the benzotriazole group binds in the s 1 subsite; phenyl propionamidyl occupies the sј 1 -s 2 subsite, and the thiophene group binds in the s 2 subsite. compound 11 also forms two direct and one water-mediated hydrogen bond interactions with amino acids in the mers-cov 3cl pro active site (fig. 6e) . the n3 of the benzotriazole ring forms a hydrogen bond with the side chain ⑀-nitrogen of conserved his-166, and the central acetamide oxygen forms a hydrogen bond with the backbone nh of conserved glu-169. the nh of the phenyl propionamidyl group interacts with backbone carbonyl oxygen of the catalytic his-41 residue through a water-mediated hydrogen bond, and the imidazole ring of his-41 engages with the phenyl ring of phenyl propionamidyl group through t-shaped stacking. the phenyl ring also form hydrophobic contacts with leu-49. interactions at the 3cl pro dimer interface-analysis of the mers-cov 3cl pro and 6 and mers-cov 3cl pro and 11 crystal structures reveals key differences between the dimer interface of mers-cov and sars-cov 3cl pro (pdb code 2alv) figure 6. x-ray crystal structure of mers-cov 3cl pro in complex with inhibitors. a, solvent-accessible surface (gray-shaded surface) of mers-cov 3cl pro and compound 6 complex. compound 6 is displayed in ball and stick model with atoms colored as follows: carbons (orange), nitrogens (blue), and oxygens (red). electron density associated with compound 6 is shown as an f o ϫ f c electron density difference map contoured to 3 (green mesh). substrate binding pockets s 4 -sј 1 are labeled, where asterisk indicates the electrophilic carbon of compound 6 that forms a c-s covalent bond with the active site cysteine cys-148. b, mers-cov 3cl pro and compound 6 complex with the mers-cov 3cl pro backbone represented as a ribbon model and relevant amino acids that interact with compound 6 represented as ball and sticks. mers-cov 3cl pro carbon atoms are colored blue, and compound 6 carbon atoms are colored orange. nitrogen atoms are colored blue, and oxygen atoms are colored red. catalytic residues cys-148 and his-41 are also shown. hydrogen bonds are depicted as red dashed lines. c, sequence logos showing amino acid conservation for the 11 polyprotein cleavage sites of different 3cl pro enzymes (mers-cov, hku5-cov, hku4-cov, and sars-cov), generated using the weblogo server (63) . residues p 2 -pј 1 are shown. height of each letter corresponds to the amino acid conservation at that position. d, solvent-accessible surface (gray-shaded surface) of mers-cov 3cl pro and compound 11 complex. compound 11 is displayed in ball and stick model. electron density associated with compound 11 is shown as a 2f o ϫ f c electron density difference map contoured to 1.5 (green mesh). functional groups of compound 11 with their corresponding binding pockets are highlighted in yellow, green, and blue ellipses. chemical structure of compound 11 is shown in the inset. e, interactions between mers-cov 3cl pro and compound 11 are illustrated. catalytic residues cys-148 and his-41 are also shown. hydrogen bonds are depicted as red dashed lines. ( fig. 7) (30) . two arginine residues, arg-4 and arg-298 (fig. 7 , a-c), form some of the key interactions at the dimer interface of sars-cov 3cl pro , and mutation of either of these amino acids results in a drastic loss of dimerization in sars-cov 3cl pro (36, 38) . interestingly, these two arginine residues (arg-4 and arg-298) are substituted in mers-cov 3cl pro by two hydrophobic residues (val-4 and met-298) that are unable to participate in the formation of hydrogen bonds or salt bridges. therefore, we initially thought that the loss of these key interactions might simply explain the ͼ100,000-fold weaker dimerization observed for mers-cov 3cl pro compared with sars-cov 3cl pro . surprisingly, however, structural analysis of the dimer interface from the available x-ray structure of hku4-cov 3cl pro (pdb code 2ynb; fig. 7, b and c) , and primary sequence alignment of 3cl pro from mers-cov, hku5-cov, hku4-cov and sars-cov (fig. 8) revealed that val-4 and met-298 are conserved between all the ␤-cov 2c members studied here. substantial differences between the ability of mers-cov 3cl pro and hku4/hku5-cov 3cl pro to dimerize, despite their high sequence identity, led us to the hypothesis that nonconserved residues between mers-cov and other ␤-cov 2c members that are remote from the dimer interface may play a significant role in dimer formation. analysis of nonconserved residues of mers-cov 3cl pro -analysis of our current crystal structures does not reveal a clear mechanism for the monomer to dimer switch of mers-cov 3cl pro upon ligand binding. therefore, we attempted to identify the nonconserved residues in mers-cov 3cl pro that might affect enzymatic activity due to their proximity to key residues involved in substrate binding and/or dimer formation. based on a sequence alignment, mers-cov 3cl pro contains ϳ24 nonconserved amino acids (pink arrows in fig. 8 ). upon analyzing the position of these amino acids in the crystal structure, we observed that a remarkable number of these amino acids are present in the loop regions. fig. 9a illustrates the nonconserved residues present in the loop regions as gray (monomer a) and pink (monomer b) spheres. interestingly, we also observed that there are hot spots in the protein structure where most of these amino acids are clustered. these hot spots include the n-terminal region, the active site region, the interdomain loop (loop between the catalytic fold and domain iii), and the domain iii. in mers-cov 3cl pro , nonconserved amino acid his-8, which forms van der waals contacts with lys-155 of the same monomer and thr-128 of the other monomer, is present at the end of the n-terminal finger (fig. 9 , b and c), whereas amino acids asp-12 and ala-15 are part of the n-terminal helix (fig. 9b) . additionally, amino acids thr-128, lys-155, and ser-158 are present within 6 å of the n-terminal region (fig. 9b ). substitution to these amino acids in mers-cov 3cl pro might have changed the protein dynamics in a way that only ligand binding populates the monomer conformation, which is more amenable to dimer formation. we also observe that some of the nonconserved residues in mers-cov 3cl pro are located in proximity to the substratebinding site and might contribute toward ligand-induced dynamic changes favorable for dimer formation. for example, nonconserved amino acid met-61 forms hydrophobic interactions with met-43, which in turn is in close proximity to the catalytic residue his-41 (fig. 9d) . residue ala-171 is present on a loop, and this loop, along with conserved residues his-166 and his-175, forms the s 1 subsite for binding the p 1 amino acid of the substrate (fig. 9e ). in addition to its influence on substrate binding, ala-171 may also contribute toward dimer formation upon substrate binding due to its close proximity with glu-169. this glutamate residue in sars-cov 3cl pro (glu-166) has been established as a key residue linking the substrate-binding site to the dimer interface (56) . val-132 forms hydrophobic interaction with other nonconserved residue ala-114 within domain ii (fig. 9f) . additionally, val-132 is present within van der waals contact distance of glul-290 from extrahelical domain iii (fig. 9f) . it is noteworthy that glu-290 forms a salt bridge with arg-4 across the dimer interface in sars-cov 3cl pro . however, this interaction is not formed in mers-cov 3cl pro due to the substitution of arg-4 with val-4. tyr-137 forms hydrophobic contacts with the conserved residue tyr-185 (fig. 9g) . besides amino acid val-132 that connects domains ii and iii, residue tyr-185, along with two other nonconserved residues, thr-183 and met-189, is present on the inter-domain loop that connects the catalytic fold (domains i and ii) with the extra-helical domain iii (fig. 9g) . flexibility within these residues might affect the orientation of domain iii required for dimer formation. model for regulation of the enzymatic activity of mers-cov 3cl pro during polyprotein processing-enzymatic activity of coronavirus 3cl pro is required for the processing of viral polyproteins at 11 distinct cleavage sites, allowing the release of nonstructural proteins that subsequently form a replication complex for virus genome replication. because of its indispensable role in the virus life cycle, regulation of the enzymatic activity of 3cl pro is instrumental for efficient replication of coronaviruses. based on our experimental results, we propose a model to explain the mechanism for regulating the enzymatic activity of mers-cov 3cl pro in the context of polyprotein processing during virus infection (fig. 10) . a number of in vitro studies performed on sars-cov 3cl pro have established the mechanism for 3cl pro auto-release from the polyprotein (34, 39, 40) . based upon these studies and our data on mers-cov 3cl pro , we propose the polyprotein processing model in fig. 10 . the steps proposed for auto-release of mers-cov 3cl pro from the polyprotein (steps 1-4, fig. 10 ) have been adapted from chen et al. (39) , where it is suggested that the n-terminal auto-processing does not require the formation of a mature 3cl pro dimer for sars-cov. based on the differences between the properties of sars-cov 3cl pro and mers-cov 3cl pro , as highlighted in our studies, we added two additional steps (steps 5 and 6, fig. 10 ) that mers-cov 3cl pro may need to utilize for efficient polyprotein processing. in fig. 10, step 1 , two immature mers-cov 3cl pro monomers in the polyprotein approach each other and form an immature dimer via interactions between domain iii, which allows each of the monomers to insert their n termini into the active site of the other monomer. in step 2, the n termini are cleaved, and the dimer with uncleaved c termini adopts a conformation similar the n and c termini are labeled, and the yellow cylinder labeled s represents a ligand that can be a peptide inhibitor, peptide substrate, or 3cl pro cleavage sites in the polyprotein. various steps required for the auto-release of 3cl pro from the polyprotein and subsequent processing of the polyprotein cleavage sites are described in the text. suggested by our auc and kinetic studies, the shaded region (steps 5 and 6) highlights the additional steps mers-cov 3cl pro would undertake during polyprotein processing and have been described in the kinetic model depicted in fig. 5b. to the mature dimer. our observation of auto-cleavage of the n-terminal his 6 tag from mers-cov 3cl pro during expression in bacterial cells supports steps 1 and 2, where formation of an immature dimer capable of auto-processing the n terminus occurs. in step 3, two dimers with uncleaved c termini approach each other, followed by insertion of the c terminus from one dimer into one of the active sites of the other dimer. in step 4, the c termini are cleaved and mature dimer is released from the polyprotein. for sars-cov, the 3cl pro dimer formed in step 4 continues to process cleavage sites in the polyprotein, effectively skipping steps 5 and 6 (red arrow in fig. 10 ) because the dimer is tightly associated. however, the high k d value of mers-cov 3cl pro dimer suggests that the active and mature dimer may dissociate into inactive, mature monomers in the absence of any ligand (step 5). in order for polyprotein processing to proceed, another step (step 6) must occur. in step 6, a substrate s, e.g. one of the 11 polyprotein cleavage sites, would induce dimer formation and hence activate catalysis and cleavage at the substrate recognition sites. our auc results and the kinetic activation studies performed in the absence and presence of inhibitors support steps 5 and 6 where the inactive but mature monomers require binding of a ligand to undergo ligand-induced dimerization and formation of an active, mature dimer that can then process the polyprotein cleavage sites. nonconserved amino acids of mers-cov 3cl pro may regulate the dimer formation-long range interactions have been reported to modulate dimerization and activity of 3cl pro enzymes. barrila et al. (57) demonstrated that mutation of a conserved amino acid ser-147, which is distant from the dimer interface, results in a total loss of dimerization and enzymatic activity of sars-cov 3cl pro . although ser-147 does not form direct interactions at the dimer interface, disruption of the dimer upon mutation stems from the fact that ser-147 makes several interactions with other residues involved in forming a hydrogen bonding network within sars-cov 3cl pro . site-directed mutagenesis studies on domain iii of sars-cov 3cl pro , where n214a and s284a/t285a/i286a mutants were characterized, revealed that despite being present on an entirely different domain, these residues affect catalysis through a network of residues undergoing correlated motions across the entire protease (58, 59) . utilizing 3cl pro temperature-sensitive mutants of mhv, stobart et al. (60) have also demonstrated that second-site mutation physically distant from the temperature-sensitive mutation suppresses the temperature-sensitive phenotype through long range interactions, thereby regulating 3cl pro enzymatic activity during polyprotein processing and virus replication. our studies also suggest that long range interactions among the nonconserved residues can significantly alter the properties of mers-cov 3cl pro . a detailed analysis of nonconserved residues of mers-cov 3cl pro among ␤-cov 2c members identified hot spots, including the n-terminal finger and helix, the active site region, the inter-domain loop, and the domain iii, where these residues are clustered. several studies done on sars-cov 3cl pro have demonstrated that amino acids from the n-terminal finger, the n-terminal helix, and domain iii significantly contribute toward dimer formation. in addition to the direct interactions at the dimer interface, correct orientation between the catalytic fold and domain iii is also crucial for dimer formation. wu et al. (61) showed that the mostdramaticdifferencebetweenthecrystalstructuresofmonomer and the ligand-bound dimer of the r298a mutant of sars-cov 3cl pro was a 33°rotation of domain iii (38) . this rotation results in a steric clash between domain iii from two monomers and would essentially block dimer formation. however, upon addition of a ligand, domain iii of the r298a mutant adopts the correct orientation and results in the formation of a dimer structure. similar to the sars-cov 3cl pro r298a mutant, ligand binding into the active site of the mers-cov 3cl pro monomer possibly stabilizes the inter-domain loop conformation that maintains domain iii in the correct orientation for dimer formation. most of the nonconserved residues within domain iii are present on the surface and also are distant from the dimer interface. these residues may be involved in providing the flexibility required for conformational changes during the monomer to dimer switch. we have identified several amino acids in mers-cov 3cl pro that may contribute to the dimer formation upon ligand binding. however, single amino acid mutagenesis alone is unlikely to reveal significant differences in the dimerization properties. as demonstrated by myers et al. (62) for ornithine decarboxylase, the response of single amino acid to ligand binding may be limited to only local conformational changes and may not have significant contribution toward dimer stability. however, local conformational changes in a network of residues may propagate larger effects that stabilize dimer formation upon ligand binding. analysis of the nonconserved residues of mers-cov 3cl pro discussed here sets forth a framework to perform systematic single or multiple mutagenesis studies to gain insights into the mechanism for ligand-induced dimerization of the enzyme. development of 3cl pro inhibitors with broad spectrum specificity-insights into the mechanistic and structural similarities as well as differences between 3cl pro enzymes from different coronavirus subgroups are instrumental for the development of 3cl pro inhibitors with broad spectrum specificity. to evaluate the broad spectrum specificity of our peptidomimetic compounds, we determined their inhibitory activity against 3cl pro from mers-cov, sars-cov, hku5-cov, and hku4-cov. our inhibitory data and k i values clearly show that compounds 6 -9 inhibit all the 3cl pro enzymes tested here. the x-ray structure of mers-cov 3cl pro in complex with compound 6 revealed that out of eight direct hydrogen bonds formed between compound 6 and mers-cov 3cl pro , four of these hydrogen bonds involve interactions with conserved structural elements of the peptide backbone of the enzyme. furthermore, the amino acids that form hydrogen bonds with compound 6 through side chain interactions are conserved in all the coronavirus 3cl pro enzymes evaluated here, as well as 3cl pro enzymes from other ␤-coronaviruses like mhv, oc43, and hku1. these results suggest that canonical structural features exist among the 3cl pro enzymes that can be exploited for structure-based design of broad spectrum inhibitors. for the noncovalent inhibitor compound 11, the x-ray structure reveals two direct hydrogen bonding interactions between the compound and mers-cov 3cl pro . one of the hydrogen bonds forms with the side chain ⑀-nitrogen of conserved his-166, and the second involves the backbone nh of conserved glu-169. we speculate these interactions remain conserved in other 3cl pro enzymes as well, because his-166 and glu-169 amino acids are conserved in all 3cl pro enzymes. in fact, the crystal structure of sars-cov 3cl pro in complex with an inhibitor similar to compound 11 (pdb code 4mds) reveals that the interactions of the inhibitor with the amino acids his-166 and glu-169 are conserved. the identification of 3cl pro -inhibitor interactions utilizing conserved elements of the protein structure, including the peptide backbone and conserved side chains of active site residues, suggests that the development of broad-spectrum inhibitors of coronavirus 3cl pro is feasible. our studies here demonstrate the unique properties of mers-cov 3cl pro among ␤-cov 2c members, evident from the requirement for a ligand to induce dimerization. although the peptidomimetic compounds containing a michael acceptor group (for example, compounds 6 -9) induce dimer formation of mers-cov 3cl pro , the irreversible nature of their reaction with the active site cysteine ensures complete inhibition of the enzyme at stoichiometric ratios in a time-dependent manner. on the contrary, noncovalent peptidomimetic compounds (for example, compounds 10 and 11) inhibit the enzymatic activity of mers-cov 3cl pro only at high compound concentrations. based on these observations, compounds that irreversibly modify the 3cl pro active site may serve as better candidates for the development of inhibitors for mers-cov 3cl pro . potential complexity in the development of mers-cov 3cl pro inhibitors as antiviral agents-induced dimerization of mers-cov 3cl pro , as seen in the presence of peptidomimetic inhibitors, has significant implications in the development of antiviral agents targeting mers-cov 3cl pro . as a consequence of enzyme activation, the development of an effective antiviral agent may necessitate the development of a compound that can inhibit the mers-cov 3cl pro monomer and stabilize it without inducing dimerization and/or inhibit the active sites of the dimer at low doses, ensuring inactivation of both the active sites within the dimer. on the contrary, it is also possible that the presence of an inhibitor could enhance the activity of mers-cov 3cl pro to an extent that results in a complete loss of the temporal and spatial regulation of the enzymatic activity, thereby disrupting viral genome replication. ramifications of ligand-induced dimerization and activation of mers-cov 3cl pro , as seen in the presence of lower concentrations of inhibitor, will need to be further explored in virus-infected cells. bovine coronavirus as the causative agent of winter dysentery: serological evidence disease in a dairy herd associated with the introduction and spread of bovine virus diarrhoea virus coronaviruses post-sars: update on replication and pathogenesis growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease community-wide outbreak of infection with a 229e-like coronavirus in tecumseh, michigan a new virus isolated from the human respiratory tract characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia identification of a new human coronavirus human coronaviruses hcov-nl63 and hcov-hku1 in hospitalized children with acute respiratory infections in beijing, china isolation of a novel coronavirus from a man with pneumonia in saudi arabia clusters of coronavirus cases put scientists on alert genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? human betacoronavirus 2c emc/2012-related viruses in bats middle east respiratory syndrome coronavirus in bats middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart hospital outbreak of middle east respiratory syndrome coronavirus interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 the molecular biology of coronaviruses virus-encoded proteinases and proteolytic processing in the nidovirales coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor drug design targeting the main protease, the achilles' heel of coronaviruses design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors ) design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived sars-cov 3clpro inhibitors discovery, synthesis, and structure-based optimization of a series of n-(tert-butyl)-2-(n-arylamido)-2-(pyridin-3-yl) acetamides (ml188) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (sars-cov) 3cl protease discovery of n-(benzo[1,2,3]triazol-1-yl)-n-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (sars-cov) 3clpro inhibitors: identification of ml300 and noncovalent nanomolar inhibitors with an induced-fit binding mechanism of the maturation process of sars-cov 3cl protease quaternary structure, substrate selectivity and inhibitor design for sars 3c-like proteinase residues on the dimer interface of sars coronavirus 3c-like protease: dimer stability characterization and enzyme catalytic activity analysis evaluating the 3c-like protease activity of sars-coronavirus: recommendations for standardized assays for drug discovery mechanism for controlling the dimer-monomer switch and coupling dimerization to catalysis of the severe acute respiratory syndrome coronavirus 3c-like protease liberation of sars-cov main protease from the viral polyprotein: n-terminal autocleavage does not depend on the mature dimerization mode maturation mechanism of severe acute respiratory syndrome (sars) coronavirus 3c-like proteinase x-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases a mouse model for betacoronavirus subgroup 2c using a bat coronavirus strain hku5 variant structurebased design, synthesis, and biological evaluation of peptidomimetic sars-cov 3clpro inhibitors is dimerization required for the catalytic activity of bacterial biotin carboxylase? processing diffraction data with mosflm processing of x-ray diffraction data collected in oscillation mode phenix: a comprehensive python-based system for macromolecular structure solution features and development of coot molprobity: all-atom structure validation for macromolecular crystallography the pymol molecular graphics system biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors high-throughput screening identifies inhibitors of the sars coronavirus main proteinase a new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods characterizing proteinprotein interactions by sedimentation velocity analytical ultracentrifugation mutation of glu-166 blocks the substrate-induced dimerization of sars coronavirus main protease long-range cooperative interactions modulate dimerization in sars 3clpro dynamically-driven inactivation of the catalytic machinery of the sars 3c-like protease by the n214a mutation on the extra domain dynamically-driven enhancement of the catalytic machinery of the sars 3c-like protease by the s284-t285-i286/a mutations on the extra domain temperaturesensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3clpro) define residues involved in long-distance communication and regulation of protease activity mechanism for controlling the monomer-dimer conversion of sars coronavirus main protease long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function we-blogo: a sequence logo generator multiple sequence alignment with hierarchical clustering deciphering key features in protein structures with the new endscript server key: cord-298974-69xjc5yq authors: adegboye, oyelola a.; elfaki, faiz title: network analysis of mers coronavirus within households, communities, and hospitals to identify most centralized and super-spreading in the arabian peninsula, 2012 to 2016 date: 2018-05-07 journal: can j infect dis med microbiol doi: 10.1155/2018/6725284 sha: doc_id: 298974 cord_uid: 69xjc5yq contact history is crucial during an infectious disease outbreak and vital when seeking to understand and predict the spread of infectious diseases in human populations. the transmission connectivity networks of people infected with highly contagious middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia were assessed to identify super-spreading events among the infected patients between 2012 and 2016. of the 1379 mers cases recorded during the study period, 321 (23.3%) cases were linked to hospital infection, out of which 203 (14.7%) cases occurred among healthcare workers. there were 1113 isolated cases while the number of recorded contacts per mers patient is between 1 (n=210) and 17 (n=1), with a mean of 0.27 (sd = 0.76). five super-important nodes were identified based on their high number of connected contacts worthy of prioritization (at least degree of 5). the number of secondary cases in each sse varies (range, 5–17). the eigenvector centrality was significantly (p < 0.05) associated with place of exposure, with hospitals having on average significantly higher eigenvector centrality than other places of exposure. results suggested that being a healthcare worker has a higher eigenvector centrality score on average than being nonhealthcare workers. pathogenic droplets are easily transmitted within a confined area of hospitals; therefore, control measures should be put in place to curtail the number of hospital visitors and movements of nonessential staff within the healthcare facility with mers cases. middle east respiratory syndrome coronavirus (mers-cov) is a contagious respiratory pathogen that is contracted via close contact with infected individuals. interactions among individuals can aid super-spreading of infectious diseases in humans or animals, and it is usually highest among individuals in close proximity with one another. mers-cov was first reported in a 60-year-old man in bisha area of the kingdom of saudi arabia in 2012 [1] . it has now spread across 27 countries in 4 continents. e most index case-patients have either resided in or have travelled to areas neighbouring the arabian peninsula [2] . from the intermittent transmission that had occurred in animal-to-human [1, 3] , many human-to-human cases have also been documented within families and healthcare facilities [3] [4] [5] [6] [7] . during an infectious disease outbreak such as mers-cov (or mers for short), the transmission usually forms networks of infected individuals (cluster of outbreaks) probably because of the way the virus crosses from one infected individual to another susceptible individual. in other words, the source of infection may be the direct or indirect connection [8] . in network analysis, the aim is to identify the most crucial infected patients (also called nodes), who are important in the super-spreading and to use the location of the node in the network to predict which patients (nodes) are likely to be infected [9] . ese important patients are influential in that they infect disproportionately large numbers of secondary contacts [10, 11] . super-spreading events (sses) are as a result of individuals (agents of sse) harbouring the virus who infect disproportionately more secondary contacts, as compared to most others [7, 10, 11] . erefore, an sse consists of a large cluster of infection in which some individuals infect many more other individuals, thereby acting as agents for an sse. in this study, super-spreading was defined as having at least five contacts. sses have been documented in other infectious disease outbreaks such as sars in beijing 2003 [12] and more recently ebola in west africa, 2014-2015 [13, 14] , and mer-cov in south korea, 2015 [10] . e knowledge of people's connectivity network is very crucial in the spread of infectious diseases transmitted via pathogenic droplets such as respiratory infection, mers, and ebola. as in the case of mers, occasional larger cluster sizes should not be unexpected, such as the outbreak in south korea [7] . although there have been variations in the size of human-human transmission of mers, its high variability and heterogeneity in the transmission potential have been underscored [15, 16] . over the years, researchers have been exploring how the knowledge of network structures could influence public health measures. for example, network analysis based on connectivity centrality was used to identify high-risk people for targeted vaccination in an effort to contain the spread of infectious disease [17] . network analysis was used to investigate whether the density of the network contacts of persons infected by mycobacterium tuberculosis was more likely to be tested positive for tuberculosis (tb) compared to the occurrence of tb clusters detected through network connections with clusters detected by molecular genotyping [18] . similarly, the effect of protein-protein interactions within the host-pathogen interactome was explored via network analysis on pathogen fitness during infection [19] . e focus of this study was to map and measure relationships and flows between people infected with mers and to investigate the structure of mers transmission with the help of network and graphs. patient's interactions and links were obtained through contact tracing within 14 days prior to the onset of the disease. e structure of network connectivity will assist in identifying the most influential contacts, while network centrality metrics were used to investigate the contribution and characteristics of the agents of super-spreading to the infection and spread of mers. and lastly, the role played by patient's attributes (node property) was investigated as an epidemic amplifier or attenuator via hypotheses testing. is study is based on case-by-case list of clinical-confirmed mers cases provided by dr. rambaut [20] . e focus is on the 1379 mers cases reported in the kingdom of saudi arabia between june 2012 and september 2016. e variables considered in this study were age, gender, patient type (whether the patient is a healthcare worker (hcw) or nonhealthcare worker), health outcome (dead or alive) as at the last day of follow-up, patient comorbidity status, types of exposure to known risk factors (animal contact and camel contact indirectly or directly or through consumption of camel products), and place of infection (classified as hospital, community, and household/ family). e data set was cross validated with information from who disease outbreak news and saudi arabia moh mers command and control website. e kingdom of saudi arabia (ksa) is the main part of the peninsula bounded by the red sea on the west, gulf of aden on the south, gulf of oman on the south east, and arabian gulf on the east. e ksa measures about 2,150,000 square kilometres and shares its border with several countries, such as jordan and iraq in the north, qatar, bahrain, kuwait and united arab emirates in the east, and oman and yemen in the south. e country is situated on latitude 15.66-32.15 and longitude 34.5-55.67 ( figure 1 ). e ksa is divided into 13 regions (manatiq, administrative level 1) and 118 governorates (muhafazat, administrative level 2). e world bank as of 2014 estimated its population to be 30.89 million [21] . variables. mers patients were clinically confirmed via realtime rna-positive strand virus using reverse transcription polymerase chain reaction (rt-pcr) with a positive pcr on at least two specific genomic targets, upstream e protein (upe) and orf1a, or a single positive target (upe) with sequencing of a second target rdrpseq assay or n gene (nseq assay) [6, 22] . patients' contact investigation was conducted by hospital officials by tracing patient's history of exposure to other known risk factors such as contact with other laboratory-confirmed mers cases, animal/camel contact, or visiting other places known to be linked to mers cases 14 days prior to the onset of symptoms. an animal contact patient implies a patient with historical contact with animals, while camel contact patients were those who work in a camel market or have history of contact with camels, or consumed camel products in 14 days prior to the onset of symptoms. a healthcare worker is anyone who works in a healthcare facility (all personnel, such as doctors, nurses, laboratory staff, securities, and receptionists). a patient is said to have comorbidity if he or she has coexisting chronic diseases or medical conditions or has been admitted to the hospital due to unrelated medical conditions. place of exposure is classified as (a) hospital infection if the infection occurred in a hospital, for example, a healthcare worker/hospital visitor/outpatient contracting the disease in a healthcare facilities, (b) family infection if the infection was through a family member or within the household, or (c) community infection if the infection is contracted outside the hospital or household, such as in schools, workplace, hajj tents, etc. descriptive analysis was conducted on some sociodemographic variables presented as mean and standard deviation for age and frequencies and percentages for other categorical variables. prevalence of mers disease across communities, household, and hospitals was tested via the chi-square statistic for categorical variables while the t-test was used for continuous variables (table 1) . e units of network analysis in this study are the nodes representing individuals infected with mers within families, hospitals, or communities which are connected via edges. e outbreak network visualization and network analyses were conducted in r package "igraph" [23] and ucinet 6.0 version 1.00 [24] . we used centrality metrics to measure the structural importance of patients (nodes) in a network. e node "degree centrality" was used to reveal the most active nodes in the network and how well a node is connected with its neighbours-a node degree is the number of edge incidents on a node. e "betweenness centrality" was used to measure how many pairs of nodes a node can be connected to through a shortest path, while the "closeness centrality" was used to measure how contagious an infected patient (a node) is to others [9, 17, 24, 25] . similarly, "2-reach centrality" was used to explore the proportion of nodes that can reach a given node in 2 steps or less while "eigenvector centrality" was used to measure the importance of a node depending on the importance of its neighbours. finally, the effects of patient's attributes on their position in a network (measured by network centrality metrics) were investigated, and the relationship between two (or more) network centrality metrics was explored. we used permutation tests in ucinet 6.0 version 1.00 [24] to test specifically the following aspects: (1) whether more central patients are hcws or not, (2) whether more central figure 3 ). mers fatality increases with age, 11% of the infection occurred in older males aged 55-59 years and more fatal in older men (above 50 years). similarly, 85% of fatality in females occurred in women above 50 years. a high proportion (about 79%) of patients had some kind of prior medical condition (comorbidity). e number of patients with comorbidity varied slightly across the regions. ere was higher fatal outcome in patients with comorbidity than patients without any comorbidity. about 47% of those with some kind of medical condition died of the disease compared to only 17% of patients without medical condition. e results showed that about half of cases (629 (45.6%)) were linked to at least one place of exposure where 321 (23.3%) of them were linked to hospital outbreaks (table 1) . ere were 750 (54.4%) cases, whose contact history of infection was unknown. of the 321 cases linked to hospital outbreaks, more than one-third (n � 119) occurred among healthcare workers, indicating that about 58.7% of all cases involving healthcare workers happened in the hospital (data not shown in table 1 ). figure 2 : illustration of (a) degree-, (b) betweenness-, (c) closeness-, and (d) eigenvector-centrality metrics for sample mers infection network. each of the patient number represents a node connected by links called edges. e square nodes represent males while the circle nodes represent females. grey represents healthcare workers and dark colours are nonhealthcare workers while white is unknown. e node sizes indicate the centrality values in table 2 . e contact structure of mers cases in the ksa between june 2012 and september 2016 is displayed in figure 4 . isolated nodes (n � 1113 (80.7%)) with degree of zero are not shown in the figure. ere were 1113 isolated cases while the number of recorded contacts per mers patient is between 1 (n � 210) and 17 (n � 1), with a mean of 0.27 (sd � 0.76). ere were a total of 266 connections-110 primary cases and 156 secondary cases. e majority of the first line secondary cases did not produce any further secondary cases of their own. e largest cluster has a wheeland-spoke configuration in which patient number 1664 is in the centre and linked to 17 other secondary cases (6.4% of the total cases in the network) (figure 4 ). e results showed that most of the outbreaks occurred in the hospital (indicated by dark line in figure 4 ) and that most of the infected patients were nonhealthcare workers with comorbidity (indicated by green nodes in figure 4 ). table 2 presents the measures of network centrality estimates for the 10 most important nodes (subsequently called patients) selected by each network metric. a total of 31 nodes worthy of prioritization were selected based on these network metrics (table 2 ). it was revealed that 9 out of 31 (29%) of the cases are healthcare workers and 8 (25.8%) cases had some kind of comorbidity. about half of the prioritized nodes (n � 15 (48.4%)) occurred in hospital settings while 7 (22.6%) cases were fatal. based on degree centrality metrics, the top five most important cases were identified based on at least five secondary cases, indicating their high number of connected contacts worthy of prioritization (table 2 ). e number of secondary cases in each sse varies slightly (range, 5-17). closeness and 2-reach network metrics ranked the same patients (1664, 1672, 1673, 1674, 1681, 1682, 1683, 1684 , 1685, and 1686) among the top 10 cases worthy of prioritization while the eigenvector metrics list was also very similar. e betweenness scores, which indicate the number of other nodes infected by the given node, indicated that patient 1522, 1521, 897, 895, and 910 had the top 5 highest betweenness score of a range of 6 to 9. among the important cases according to degree centrality, four resulted in fatality. patient 1664 was favoured (based on degree, closeness, betweenness, and eigenvector network centrality metrics) as the most important in the transmission network by having the highest number of secondary cases. however, the cases selected using betweenness centrality were slightly different from other metrics with patient 1522 topping the list. e degree and betweenness centrality estimates are depicted in figures 5(a) and 5(b), respectively. e larger node area shows the level of worthiness of prioritization. centrality. when network centrality metrics were used to investigate the association between patient's attributes and patient's position in a network, only eigenvector centrality was significantly associated with two patient's attributes. e eigenvector centrality was significantly (p < 0.05) associated with place of exposure, with hospital infection having on average significantly higher eigenvector centrality than other places of exposure. similarly, being a healthcare worker was significantly associated (p < 0.05) with eigenvector centrality. to answer the questions about the relationships among the network centrality metrics, the results suggest that patient's degree centrality explains 90.2% of the variation in patient's closeness centrality, while 14% and 9.3% of the variation in closeness centrality were explained by eigenvector and betweenness centrality, respectively. furthermore, we investigated whether super-spreading patients with high network centrality metrics (e.g., degree, betweenness, closeness, eigenvector, and 2-reach) have similar attributes. significant differences were observed in age, gender, healthcare worker, and fatal cases across the regions; however, no difference was observed in the proportion of patients with comorbidities. in this study, several network centrality metrics (degree, betweenness, closeness, eigenvector, and 2-reach) were used to quantify the connectivity among mers cases and to identify which patient requires prioritization for intervention. usually the patient with the highest degree has the most ties to other patients in the network [9] . saudi arabia is said to be facing continuous risk of mers outbreaks [26] . e findings emphasize the importance of patient's level characteristics in understanding their level of infectiousness. results show that healthcare facilities and healthcare workers are the most crucial factors in driving national epidemics of mers. although healthcare workers are at higher risk of mers infection due to their proximity to infected patients, previous studies have shown that cases among healthcare workers are less serious [27] with few fatalities [28, 29] . hospital infections display higher interconnectivity and, on average, are linked to patients that are more connected to highly connected patients than nonhospital infections. recent studies have reported the outbreaks of mers in hospitals [5, [30] [31] [32] [33] . e overcrowding in hospitals due to easy access to medical care caused mers to move quickly throughout korea [5] . virtually, all cases of mers in korea occurred in a hospital-to-hospital type of transmission [5] . similarly, healthcare workers are more connected to important nodes themselves than nonhealthcare workers. in terms of the number of connected contacts (via degree centrality), nodes 1664, 1025, 124, 133, and 897 were the top 5 most active mers cases, but they do not reflect the spread of mers. e most influential node is patient 1664 based on degree centrality. most of the secondary cases connected to patient 1664 were either attended to by the same healthcare worker(s) or are themselves healthcare workers that attended to patient 1664. patients who are strongly tied in a network are more likely to be similar to each other than different. patient 1664 was a 47-year-old female admitted to the hospital with unrelated symptoms [36] , and she was associated with 14 healthcare worker cases and 3 household cases because many healthcare staff treated her at the initial stage during her hospitalization in a vascular surgery ward and initially in an open ward, stressing the importance of limiting access to other patients and quarantines. regarding infectivity, when determining the strength of a patient in a network through not only its connectivity but also the interconnectivity of its secondary cases, the betweenness metric provides better estimate. e low betweenness score of patient 1664 indicates that while patient 1664 connects many other patients, it is not a pathway to further infection. nodes 1522, 1521, 897, 895, and 910 were identified as critical in the spread of mers to other cases using the betweenness metric. ese patients acted as a bridge connecting other smaller secondary cases and thus the spread of mers infection by further connecting with other secondary cases who also connect other important cases. it was found that the individuals who connect 2 or more separate contacts have an increased likelihood to connect to multiple contacts [37] . patient 1522 is a 26-yearold female with no comorbidities was linked to three secondary cases and five other indirect offspring. e major limitation in this study lies in the data sources and contact tracing accuracy. e analysis is based on retrospective data rather than prospective data collected from multiple sources which are publicly available. e accuracy of some of the information provided by the patient may not be verifiable especially during the early outbreaks; however, the reporting has been improved upon over the years with coordination between saudi ministry of health and regional who office. similarly, while it is acknowledged that most reported clusters occurred in the hospital as a result of contact tracing, this is not surprising because of adequate monitoring and data collection in the hospital that revealed large secondary cases in the healthcare facilities. lastly, as in the case of publicly available data, these study data are characterized by missing data. while the problems associated with inference-drawn, publicly available epidemiological data are acknowledged, the results are meaningful and suggest that structure and characteristics of contact network can indeed have significant effect on the rate of transmission of mers disease. e present study highlighted the importance of contact network in the spread of infectious disease. ese results provide interesting findings. ey show that real-time network analysis can provide insight into the structure of transmission of infectivity to identify important players for isolation and selective treatment. moreover, the results present rational estimate of the size of outbreak and the underlying structural characteristics of the group [37] . patients with high degree of centrality played a powerful role as epidemic attenuators. patients with high degree of centrality played a powerful role as epidemic attenuators as they are linked to many secondary cases that did not produce any further secondary cases of their own. while patients with high betweenness are epidemic amplifiers, they are a pathway to infect other patients. is study has shown that most important nodes are those within the hospital, and healthcare workers are more prone to the infection. pathogenic droplets are easily transmitted within the confined areas of hospitals, and control efforts should be put in place at different layers of hospitals. reducing contact formation especially within the hospital by restricting hospital visitation for mers patient families and reducing the number of healthcare workers with access to mers patients will certainly have significant effect on the spread of mers disease. saudi arabia is said to be facing continuous risk of mers outbreaks [26] . if control measures are not put in place to curtail the number of hospital visitors and movements of nonessential staff within healthcare facility, with their connectivity to people within the community, the disease will speed up further national/international outbreaks. future research should focus on epidemiological analysis exploring the role of a healthcare facility in the spread mers disease. e authors declare no conflicts of interest. mers coronavirus: diagnostics, epidemiology and transmission infectious diseases epidemic threats and mass gatherings: refocusing global attention on the continuing spread of the middle east respiratory syndrome coronavirus (mers-cov) transmission characteristics of mers and sars in the healthcare setting: a comparative study mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study 2015 mers outbreak in korea: hospital-to-hospital transmission epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study e role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission spatial covariate adjusted survival rates for middle east respiratory syndrome (mers) coronavirus in the arabian peninsula transmission network analysis to complement routine tuberculosis contact investigations mers, sars, and ebola: the role of super-spreaders in infectious disease super-spreaders in infectious diseases superspreading sars events spatial and temporal dynamics of superspreading events in the 2014-2015 west africa ebola epidemic ebola superspreading assessing the risk of observing multiple generations of middle east respiratory syndrome (mers) cases given an imported case nuanced risk assessment for emerging infectious diseases infectious disease containment based on a wireless sensor system transmission network analysis in tuberculosis contact investigations centrality in the host-pathogen interactome is associated with pathogen fitness during infection mers-cov spatial, temporal and epidemiological information infection prevention/control and management guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection e igraph software package for complex network research ucinet for windows: software for social network analysis, analytic technologies network centrality and super-spreaders in infectious disease epidemiology a pandemic risk assessment of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia risks of death and severe disease in patients with middle east respiratory syndrome coronavirus spatial modelling of contribution of individual level risk factors for mortality from middle east respiratory syndrome coronavirus in the arabian peninsula building predictive models for mers-cov infections using data mining techniques description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabi hospitalassociated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description e critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study it feels like i'm the dirtiest person in the world.": exploring the experiences of healthcare providers who survived mers-cov in saudi arabia an assessment of the level of concern among hospital-based health-care workers regarding mers outbreaks in saudi arabia knowledge, attitude and practices of healthcare providers towards mers-cov infection at makkah hospitals, ksa disease outbreak news: middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia using social network and ethnographic tools to evaluate syphilis transmission is study was supported by research grant qust-cas-spr-2017-25 from qatar university. e authors would also like to thank majeed adegboye for his help with the mers data management. high connectivity among healthcare workers and their proximity to mers cases is not surprising because of the nature of their job. in general, healthcare workers across saudi arabia have negative attitude toward mers infection [34, 35] . although infectious disease epidemiological plans were put in place in some hospitals, outbreaks of mers still occurred due to the failure to adhere to the infection control measures [33] . a high proportion of healthcare workers felt at risk of contracting the disease but obliged to care for mers patients [33, 34] . similarly, a high percentage of healthcare workers do not feel safe at work using standard precautions [34] . table 2 . larger sized nodes implies (a) degree centrality and (b) betweenness centrality. e circle nodes are females while square nodes are males. e node colours represent contact history: pink is the index patient, green is hospital linked, blue is community or family linked, red denotes the primary contact, and white nodes represent unknown contact history. key: cord-290744-m0vpizuh authors: kindler, e.; thiel, v.; weber, f. title: interaction of sars and mers coronaviruses with the antiviral interferon response date: 2016-09-09 journal: adv virus res doi: 10.1016/bs.aivir.2016.08.006 sha: doc_id: 290744 cord_uid: m0vpizuh severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) are the most severe coronavirus (cov)-associated diseases in humans. the causative agents, sars-cov and mers-cov, are of zoonotic origin but may be transmitted to humans, causing severe and often fatal respiratory disease in their new host. the two coronaviruses are thought to encode an unusually large number of factors that allow them to thrive and replicate in the presence of efficient host defense mechanisms, especially the antiviral interferon system. here, we review the recent progress in our understanding of the strategies that highly pathogenic coronaviruses employ to escape, dampen, or block the antiviral interferon response in human cells. coronaviruses have made a remarkable career. originally recognized as viral pathogens of veterinary importance but little medical (i.e., human) relevance, the appearance of sars-cov causing a worldwide epidemic with a large number of fatalities has changed everything. in 2003, the virus emerged in chinese animal markets to circle the world in just a few weeks, teaching us important new lessons on perceived "differences" between animal and human pathogens. just in case someone did not get the message, mers-cov repeated the coronavirus wake-up call 10 years later, providing yet another example for how easily animal viruses may be transmitted and adapt to new hosts including humans. often, the tricks and strategies that viruses evolved to propagate in specific animal hosts may only need some fine-tuning (if at all) to enter the wide world of human crowds, air travel, and camel races. here, we will summarize the insights gathered so far on an important aspect of virulence and host adaptation, the interactions of sars-cov and mers-cov with antiviral interferon (ifn) responses of human cells. the coronavirus genome is composed of a linear, single-stranded, monopartite rna with a cap structure at its 5 0 end and a polya tail at the 3 0 end . the 5 0 -terminal two-thirds of the cov genome contain the open reading frames (orf) 1a and 1b that together constitute the viral replicase gene. translation is initiated at the start codon of orf1a and may continue to orf1b via a ribosomal frameshift mechanism, ultimately giving rise to two overlapping replicase polyproteins pp1a and pp1ab perlman and netland, 2009; snijder et al., 2003; thiel et al., 2003) . virus-encoded proteinases, namely two papain-like cysteine proteases (pl1 pro and pl2 pro ), residing in nonstructural protein (nsp) 3 and a 3c-like cysteine protease (3cl pro ) associated with nsp5, proteolytically process the polyproteins into nsps 1-16 (anand et al., 2003; schiller et al., 1998; thiel et al., 2003; ziebuhr et al., 2007) . a multitude of functions and enzymatic activities associated with specific nsps have been identified over the past years (for reviews, see masters and perlman, 2013; ziebuhr, 2005) . moreover, orf1b harbors several rna-processing enzymes, including a 3 0 -5 0 exonuclease and a guanosine n7-methyltransferase (associated with the n-and c-terminal domains, respectively, of nsp14), an endoribonuclease (nsp15) and a 2 0 -o-methyltransferase (nsp16) (chen et al., 2009; decroly et al., 2008; ivanov et al., 2004; kindler and thiel, 2014; minskaia et al., 2006; perlman and netland, 2009; snijder et al., 2003; thiel et al., 2003; zust et al., 2011) . the 3 0 orfs are translated from a set of subgenomic (sg) rnas and yield on one hand four canonical structural proteins like the spike protein (s), the envelope (e), the membrane (m), and the nucleoprotein (n). moreover, sgrnas express accessory genes, which vary in function and number between different cov strains and are interspersed between the structural genes perlman and netland, 2009; snijder et al., 2003; thiel et al., 2003) . specifically, the genome of sars-cov expresses eight different accessory genes (3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b), while mers-cov encodes five accessory genes (3, 4a, 4b, 5 , and 8b). the schematic overview of the genome organization of sars-cov and mers-cov is depicted in fig. 1 . the cov life cycle starts with the attachment of the viral spike protein to particular cellular receptors, subsequently leading to fusion between the viral envelope and the plasma membrane or the endosome membrane of the host. cov uses a range of receptors, with sars-cov employing angiotensinconverting enzyme 2 (ace2) and mers-cov employing dipeptyl peptidase 4 (dpp4) (li et al., 2003; raj et al., 2013) . following membrane fusion, the viral rna genome is delivered into the host cytoplasm, where translation of the two 5 0 -terminal orfs 1a and 1b is accomplished by the cellular translation machinery. most of the newly synthesized nsps assemble with the n protein into a replicase-transcriptase complex (rtc) responsible for viral genome replication and transcription. at the site of replicative organelles , the rtc initiates minus-strand synthesis using the full-length genome as template, thereby either copying the entire template to generate full-length minus strands or to move discontinuously along the template to produce a nested set of sgrnas with negative polarity. the minus strands of genomic and sgrnas are subsequently used as templates to synthesize positive sense strands (mrnas), specifically the genomic rna (genome replication) and sg mrnas (transcription) (sawicki et al., 2007) . the n protein then encapsidates the newly synthetized rna genome and thereby forms a helical nucleocapsid. virion assembly is triggered by the action of the m protein, which assists in incorporating the nucleocapsid, the envelope and the spike into virus particles. budding takes place between the endoplasmic reticulum and the golgi and new viruses are released by exocytosis (mcbride et al., 2014; neuman et al., 2011; ruch and machamer, 2012; ujike and taguchi, 2015) . the antiviral ifn (ifn-alpha/beta) system confers an important part of the innate immune defense in chordates (tenoever, 2016) . ifns are cytokines that are produced and secreted by cells encountering viruses or parts thereof (fig. 2) . humans are able to express one ifn-beta, 13 subtypes of ifnalphas, and one each of ifn-kappa and ifn-omega (schneider et al., 2014) . all nucleated cells are able to respond to them as they express the ifn receptor (composed of the two subunits ifnar-1 and ifnar-2) on their surface (bekisz et al., 2004) . the docking of ifn-alpha/beta onto its cognate receptor activates the so-called jak-stat pathway. thereby, the janus kinases jak1 and tyk2 are waiting to be activated on the cytoplasmic side of ifnar2 and 1, respectively. the activated kinases then phosphorylate the signal transducers and transcription factors stat1 and stat2, which form a complex with irf9 (isgf3) that enters the nucleus to transactivate promoters of an antiviral gene expression program. genes that are specifically upregulated by ifns are collectively called isgs (ifn-stimulated genes). the alpha/beta-ifns are classified as type i ifns, since they had been discovered first (isaacs and lindenmann, 1957) . the type ii ifns use a different receptor and consist of only one member, ifn-gamma. ifn-gamma also confers some antiviral activity but is regarded more of an immunoregulator (produced by specialized immune cells) than a general antiviral mediator (schneider et al., 2014) . it signals through a jak/stat pathway that partially overlaps with one of the type i ifns. ifn-gamma will not be further discussed here as it is not to the core of the antiviral ifn response to coronaviruses. recently, the ifn family was extended by the newly discovered type iii ifns, consisting of ifn-lambda 1-4 (schneider et al., 2014) . type iii ifns resemble type i ifns in that they also trigger stat1/2 phosphorylation via jak1 and tyk2. they employ however a different receptor which is only expressed by epithelial cells (sommereyns et al., 2008) . thus, type i and type iii ifns trigger largely overlapping sets of isgs, but while the former constitute a major, general antiviral cytokine system, the latter are mainly restricted to mucosal sites (galani et al., 2015) . the molecular events leading to the upregulation of type i ifns are well established. as indicated earlier, molecular structures that are specific for virus infections (often called pamps, for pathogen-associated molecular patterns) are sensed by pathogen recognition receptors (prrs) of the host, that in turn are triggering the upregulation of ifn genes. a prototypical pamp relevant for coronaviruses is double-stranded rna (dsrna), a by-product of genome replication and transcription (weber et al., 2006; zielecki et al., 2013) . dsrna can be sensed by toll-like receptor 3 (tlr3) in the endosome, and in the cytoplasm by the rna helicases rig-i (retinoic acid-inducible gene i) and mda5 (melanoma differentiation antigen 5), as well as by the kinase pkr (protein kinase, rnaactivated) (rasmussen et al., 2009; yim and williams, 2014; yoneyama et al., 2016) . rig-i is thereby specific for long dsrna molecules and short dsrnas bearing a tri-or di-phosphorylated 5 0 end, whereas mda5 senses long dsrnas, preferentially with a higher-order structure (binder et al., 2011; goubau et al., 2014; pichlmair et al., 2009; schlee, 2013) . pkr is activated by dsrna as well as by short stem-loop rnas bearing a 5 0 triphosphate end (dabo and meurs, 2012; nallagatla et al., 2011) . also specific single-stranded rnas (ssrnas) can act as pamps, either if they are in the wrong location or if they display particular features. tlr7 senses gu-rich ssrna in the endosome (heil et al., 2004) . rig-i can be triggered by polyu/uc rich or 3 0 monophosphorylated ssrnas stretches, and mda5 was found to bind ssrna stretches of negative-sense rna viruses and hypomethylated 5 0 capped mrnas (luthra et al., 2011; malathi et al., 2007 malathi et al., , 2010 rasmussen et al., 2009; runge et al., 2014; saito et al., 2008; zust et al., 2011) . depending on the particular prr, various-partially cross-talking-signaling pathways lead to the transactivation of promoters for antiviral genes (o'neill et al., 2013) . the endosomal prr tlr3 engages the intracellular adapters trif (tir domain-containing adapter protein inducing ifnbeta) and traf3 (tnf receptor-associated factor 3) to activate the kinases tbk1 (tank-binding kinase 1) and ikkepsilon (inhibitor of nf-kappab kinase epsilon). the kinases tbk1 and ikkepsilon then phosphorylate irf3 (ifn regulatory factor 3), a transcription factor that activates genes for ifns and other immunoregulatory cytokines. signaling by tlr7, by contrast, requires the adaptor proteins myd88 (myeloid differentiation primary-response protein 88) and traf3 to channel to the kinase ikkalpha. this kinase then phosphorylates irf7, a transcription factor that covers a gene spectrum similar to irf3. tlr7/myd88 also recruits the adaptor protein traf6 that eventually activates the transcription factor nf-kappab via the kinases ikkalpha and ikkbeta. nf-kappab drives transcription of genes for proinflammatory cytokines but also enhances ifn gene expression. in the cytoplasm, rna sensing by the two prrs rig-i and mda5 (collectively termed rig-i-like receptors, rlrs) converges on the adaptor protein mavs (mitochondrial antiviral signaling protein) that uses various trafs (traf 2, 3, 5, 6) to trigger tbk1/ikkepsilon and ikkalpha/ ikkbeta. these kinases then activate irf3 and nf-kappab, respectively (belgnaoui et al., 2011; liu et al., 2013) . besides the rlrs, pkr contributes to ifn induction in the cytoplasm. pkr is a master regulator of mrna translation (see later), but several lines of evidence indicate a role in activation of nf-kappab and irf3 via traf2/6, ikkalpha/beta, antiviral stress granule formation, and ifn-alpha/beta mrna stability (gil et al., 2004; onomoto et al., 2012; pfaller et al., 2011; pham et al., 2016; schulz et al., 2010; zamanian-daryoush et al., 2000) . thus, several types of prrs are constantly surveying the extracellular and intracellular space to detect virus infections in a timely and sensitive manner. importantly, tlrs are preferentially expressed by immune cells, especially myeloid dendritic cells (mdcs) and plasmacytoid cells (pdcs), whereas rlrs and pkr are thought to be active in all nucleated cells. detection of viral rna in mdcs is mainly mediated by tlr3 (and some tlr7), and in pdcs by tlr7 (and tlr8 in human pdcs) (schreibelt et al., 2010) . pamp sensing by prrs eventually culminates in activation of irf3, irf7, and nf-kappab, as described earlier, the transcription factors driving the expression of genes for ifn-beta, ifn-alpha, and various proinflammatory and immunomodulatory cytokines (belgnaoui et al., 2011) . signaling by both type i and type iii ifns triggers the formation of isgf3 (see section 3.1), the heterotrimeric transcription factor complex consisting of phosphorylated stat1 and stat2, and irf9 (schneider et al., 2014) . isgf3 binds to the isres (for ifn-stimulated response element), specific promoter sequences of the so-called isgs. of note, there are actually several types of "isres" that are responding to different types of triggers and transcription factors. first, there are the isres that purely respond to ifn signaling and isgf3, as it would be expected from the name. a prominent example is given by the promoter of the human antiviral protein mxa (holzinger et al., 2007) . second, there are the-somewhat mislabeled-isres that do not respond to ifn at all, but only to the irf3-, irf7-, and nf-kappab-related signal transduction that occurs much earlier, directly after virus infection has triggered a prr. the ifn-beta promoter belongs to this class of isres (freaney et al., 2013; schmid et al., 2010) . third, there are mixed-type isres that can be activated by both virus infection and ifns. an example is the promoter of the gene for the antiviral protein ifit1 (also known as isg56) (fensterl and sen, 2015) . the different isre classes can be distinguished as irf-specific isres (responding only to prr signaling), isgf3-specific isres (responding only to type i or type iii ifns), and universal isres (responding to both infection and ifns) (schmid et al., 2010) . many isgs are controlled by additional promoter elements ensuring basal levels of expression already in the absence of ifn. moreover, low levels of ifn itself are constitutively secreted by many tissues (tonic ifn), ensuring physiological homeostasis and priming of cells for a rapid response against pathogens (gough et al., 2012) . it is estimated that, depending on the ifn subtype, dose, and cell type, ifns regulate hundreds, if not thousands of genes (rusinova et al., 2013) . many of the isgs (i.e., those genes that are upregulated by ifns) are known to have antiviral, immunomodulatory, or antiproliferative function (samuel, 2001; stark and darnell, 2012) . the broad antiviral activity of ifns occurs on several levels, namely virus entry, viral polymerase function, host cell translation, rna availability, rna stability, particle budding, apoptosis, or general boosting of innate and adaptive immune responses. high-dose ifn treatment (type i and type iii) has clear effects against sars-cov and mers-cov in cell culture cinatl et al., 2003; falzarano et al., 2013; kindler et al., 2013; spiegel et al., 2004; stroher et al., 2004; zielecki et al., 2013) , in animal experiments (channappanavar et al., 2016; frieman et al., 2010; haagmans et al., 2004; mahlakoiv et al., 2012; mordstein et al., 2008) , and possibly also in patients (loutfy et al., 2003; omrani et al., 2014; strayer et al., 2014) . remarkably, mers-cov was found to be substantially more ifn sensitive than sars-cov in cell culture . the cellular basis for the relatively low (sars-cov) and high (mers-cov) ifn sensitivity is currently unknown. several prominent (i.e., potent) isg products were studied in the context of human pathogenic coronaviruses, but only some of them were found to have an effect. the ifn-induced transmembrane (ifitms) proteins 1, 2, and 3 restrict the entry of many enveloped viruses including sars-cov (huang et al., 2011b) as well as reoviruses . they act by altering the site of membrane fusion, but the exact mechanism remains to be elucidated . strikingly, while ifitms are inhibitory for the highly pathogenic sars-cov, they appear to boost infection with the related, low pathogenic coronavirus hcov-oc43 (zhao et al., 2014) . in particular, ifitm2 or ifitm3 acts as entry factor for hcov-oc43 by facilitating-rather than impeding-membrane fusion. human mxa (for myxovirus resistance protein a) is a well-known antiviral host factor with activity against a wide range of (mostly) rna viruses (haller et al., 2015) . it blocks early replication steps of influenza viruses but was found have no effect on sars-cov (spiegel et al., 2004) . the kinase pkr is an isg product acting as a signaling prr on one hand (see earlier), but its main function in antiviral defense is the inhibition of protein synthesis. after binding viral dsrna, pkr undergoes autophosphorylation to activate itself, and subsequently phosphorylates eif-2alpha that is thereby converted from a translation initiation factor to a translation inhibitor (yim and williams, 2014) . pkr has a broad antiviral spectrum. nonetheless, pkr has no bearing on the replication of sars-cov, although it is involved in virally induced apoptosis (krahling et al., 2009) . also the 2 0 -5 0 oligoadenylate synthetase (oas) family members are triggered by viral dsrna (chakrabarti et al., 2011) . in the dsrnabound state they synthesize short chains of 2 0 -5 0 oligoadenylates that activate the latent rnase l. rnase l then cleaves virus and host ssrnas, predominantly at single-stranded ua and uu dinucleotides (wreschner et al., 1981) . interestingly, the small 3 0 -monophosphorylated cleavage products of rnase l are recognized by the prrs rig-i and mda5, thus amplifying the ifn response in an infection-dependent manner (malathi et al., 2007) . polymorphisms of the oas-1 gene might affect susceptibility to sars-cov (hamano et al., 2005) , but to our knowledge, there is no direct data on antiviral effects of the oas/rnase l system on human coronaviruses. for the mouse coronavirus mhv-a59, however, it was shown that mutants deficient in the ns2 gene are highly sensitive against rnase l (zhao et al., 2012 ) (see also later). as mentioned, there are several hundreds of isgs, of which about 40 were characterized as being antiviral (schneider et al., 2014) . it is in a way remarkable that relatively little is known about isgs that impede human pathogenic coronaviruses. most likely, active and passive evasion mechanisms such as the ones described later are responsible for the relative insensitivity of at least sars-cov against ifn and potent antiviral isgs. although our review will focus on the human pathogenic coronaviruses sars-cov and mers-cov, we will draw additional conclusions from well investigated other coronaviruses whenever adequate. viral evasion strategies against the ifn response can act on several levels, namely the induction of ifn, ifn signaling, or antiviral action of individual isg products (gack, 2014; kindler and thiel, 2014; vijay and perlman, 2016; weber and weber, 2014; wong et al., 2016; zinzula and tramontano, 2013) . the viruses can thereby actively sequester or destroy key regulators, or otherwise interfere with the ifn system. moreover, several aspects of the viral replication cycle can be regarded as a passive ifn evasion. the strategies described later are also summarized in three tables. both sars-cov and mers-cov induce very little-if any-ifn in most cell types cheung et al., 2005; kindler et al., 2013; lau et al., 2013; menachery et al., 2014a; spiegel et al., 2005; zhou et al., 2014; ziegler et al., 2005; zielecki et al., 2013) . in fact, it was recently shown in a mouse model of sars that the delay in ifn induction is responsible for the activation of proinflammatory monocyte-macrophages and cytokines in the lung, resulting in vascular leakage and impaired adaptive immune responses (channappanavar et al., 2016) . thus, the high levels of dsrna that are produced during replication (weber et al., 2006; zielecki et al., 2013) do not result in an adequate ifn induction. one of the reasons (besides the active measures described later) is certainly the storage of coronaviral dsrna inside double-membrane vesicles van hemert et al., 2008; versteeg et al., 2007) . moreover, the n protein sequesters ifninducing rna pamps lu et al., 2011) . however, the fact that infection with coronaviruses activates the cytosolic dsrna-sensing host factors pkr and oas (birdwell et al., 2016; krahling et al., 2009; zhao et al., 2012) , as well as the existence of numerous mechanisms dedicated to suppress dsrna-dependent ifn induction (see later) strongly suggest that dsrna stashing alone is not sufficient and that some dsrna or other pamps are exposed to prrs, thus necessitating the presence of additional, active mechanisms. while most cell types remain ifn-silent after infection, a notable exception are pdcs, which express high levels of ifn-alpha/beta in response to infection with both sars-cov and mers-cov (cervanteschannappanavar et al., 2016; scheuplein et al., 2015) . for the mouse coronavirus mhv-a59 it was shown that ifn induction in pdcs occurs through tlr7 (cervantes, suggesting the same to be true for sars-cov and mers-cov. indeed, gu-rich ssrnas from the sars-cov genome were shown to activate an excessive innate immune response via tlr7 . moreover, the membrane (m) protein and the envelope (e) protein of sars-cov are able to activate a tlr-like pathway and nf-kappab signaling, respectively (dediego et al., 2014; wang and liu, 2016) . the mouse coronavirus mhv-a59 also naturally induces ifn in brain macrophages/microglia, with mda5 being the responsible prr (birdwell et al., 2016; roth-cross et al., 2008) . also in oligodendrocytes ifn induction by mhv occurs through both mda5 and rig-i (li et al., 2010) . interestingly, a general (i.e., not restricted to particular cell types) mda5-dependent ifn induction can be obtained by ablating the ribose 2 0 -o-methylation activity of the nsp16. as it was shown for mhv-a59, sars-cov, and the mildly human pathogenic coronavirus hcov-229e, nsp16-mediated 2 0 -o-methylation of viral mrna cap structures prevents recognition by mda5 (menachery et al., 2014b; zust et al., 2011) . besides these "hiding" or "disguising" strategies, active mechanisms targeting specific host factors are in place (table 1 ). sars-cov was shown to inhibit irf3 by preventing its hyperphosphorylation, dimerization, and interaction with the cofactor cbp (spiegel et al., 2005) . curiously, irf3 initially enters the nucleus of infected cells, but later returns to the cytoplasm. sars-cov also inhibits the nuclear import of the related transcription factor irf7 (kuri et al., 2009) . in this context, the papain-like protease (pl pro ) domain of nsp3 (the largest coronaviral protein) of sars-cov and the mildly pathogenic hcov-nl63 both interact with irf3 and block its activation (devaraj et al., 2007; frieman et al., 2009) . moreover, pl pro was shown to drive the deubiquitination (or inhibit ubiquitination) of rig-i, tbk1, and irf3 (clementz et al., 2010; devaraj et al., 2007; frieman et al., 2009; sun et al., 2012) . irf3 activation is also prevented by the m protein of sars-cov through inhibiting complex formation between traf3 and tbk1 (siu et al., 2009) . since m was also found to activate a tlr-like signaling pathway (wang and liu, 2016) , a final picture of m protein function in the context of ifn induction/inhibition remains to be provided. ifn induction is also disturbed by the sars-cov nsp1, nsp7, nsp15, orf3b, orf6, and orf9b proteins, respectively (frieman et al., 2009; kopecky-bromberg et al., 2007; shi et al., 2014; zust et al., 2007) . the anti-ifn function of nsp1 is based on its ability to mediate host mrna degradation, while sparing viral mrnas at the same time, and to block host mrna translation (huang et al., 2011a; narayanan et al., 2008; tanaka et al., 2012) . nsp1 also has a function in evasion from ifn signaling (see later), providing a possible reason why nsp1 mutants are particularly ifn sensitive (wathelet et al., 2007; zust et al., 2007) . while the mechanisms of other sars-cov ifn induction antagonists like nsp7, nsp15, orf3b, and orf6 proteins remain to be characterized, for the orf9b protein it was shown that it drives degradation of mavs, traf3, and traf6 by interacting with the host factors pcbp2 and the e3 ubiquitin ligase aip4 (shi et al., 2014) . also for mers-cov, the reason for the low levels of ifn produced by infected cells kindler et al., 2013; lau et al., 2013; menachery et al., 2014a; zhou et al., 2014; zielecki et al., 2013) was further investigated. the orf4a protein inhibits ifn induction by interaction with dsrna and the rlr cofactor pact (niemeyer et al., 2013; siu et al., 2014) . like the orf4a, the orf4b, 5, and m proteins of mers-cov were shown to prevent irf3 translocation (yang et al., 2013) . the orf4b protein, in particular, inhibits ifn induction by binding to tbk1 and ikkepsilon (matthews et al., 2014; yang et al., 2015) . in agreement with the data on sars-cov, the pl pro of mers-cov has deubiquitinating activity and inhibits ifn induction (bailey-elkin et al., 2014; mielech et al., 2014) , and the nsp1 mediates host mrna degradation (lokugamage et al., 2015) . in contrast to sars-cov, however, infection with mers-cov additionally activates repressive histone modifications that downregulate isg expression (menachery et al., 2014a) . several proteins of sars-cov and mers-cov were found to interfere with the signal transduction chain that leads from ifn docking onto its receptor to the upregulation of isgs by isgf3, the stat1/stat2/ irf9 complex ( table 2 ). the orf3a protein was shown to decrease levels of ifnar, most probably by ubiquitination and proteolytic degradation (minakshi et al., 2009 ). the orf6 protein was the first factor described for sars-cov that affects ifn signaling in infected cells, disrupting nuclear import of stat1 kopecky-bromberg et al., 2007) . the orf6 protein binds to the nuclear import factor karyopherin alpha 2 and tethers it (together with karyopherin beta 1) to intracellular membranes . there, they become unavailable for their normal cellular function, the import of, e.g., stat1. the phosphorylation of stat1 is impeded by the multifunctional nsp1 protein of sars-cov, which otherwise drives degradation of host mrnas and inhibits translation (see earlier) (wathelet et al., 2007) . for mers-cov, the orf4a, 4b, and m proteins inhibit isre activation after stimulation with ifn (yang et al., 2013) . the mechanisms are currently unknown. the orf4a protein, which also acts as an inhibitor of ifn induction (see earlier), had the strongest activity. lastly, the repressive modifications that are imposed by mers-cov onto the cellular histones are also a strategy to dampen isg expression (menachery et al., 2014a) . despite having some sensitivity toward ifn, especially mers-cov (see section 4), viral strategies to increase ifn resistance are also in place ( table 3 ). the sequestration of viral dsrna in dmvs van hemert et al., 2008; versteeg et al., 2007) not only reduces cytoplasmic exposure to prrs and hence ifn induction but also limits activation of antiviral dsrna-responsive isg products like pkr. however, pkr is eventually activated by sars-cov infection, but has no effect on viral replication (krahling et al., 2009) . interestingly, other coronaviruses kuri et al. (2011) cope differently with pkr. the avian infectious bronchitis virus (ibv) expresses a weak inhibitor or pkr (nsp2) and additionally upregulates the phosphatase subunit gadd34 to reduce phosphorylation of the pkr downstream target eif-2alpha (wang et al., 2009) . by contrast, the porcine reproductive and respiratory syndrome virus (prrsv; a member of the arteriviridae that are related to the coronaviridae and other nidoviruses) does not inhibit but rather requires pkr for optimal replication and gene expression . thus, the interactions and interdependencies of coronaviruses with pkr are complex and far from being understood. with respect to the antiviral oas/rnase l system that is also activated by dsrna, the mouse coronavirus mhv-a59 was shown to expresses an ns2 protein that antagonizes by degrading the product of the oas enzyme, 2 0 -5 0 oligoadenylate that would activate rnase l (zhao et al., 2012) . sars-cov and mers-cov do not possess an ns2 homolog (silverman and weiss, 2014) , but the mers-cov ns4b was recently demonstrated to cleave 2 0 -5 0 oligoadenylate (thornbrough et al., 2016) . although ns4b-mutated mers-cov was not attenuated in cell culture, it provoked increased rnase l activity in infected cells (thornbrough et al., 2016) . a critical factor for ifn resistance of sars-cov (and of the low pathogenic hcov-229e) is the adp-ribose-1 00 -monophosphatase (adrp) domain that is contained within the nsp3 protein (kuri et al., 2011) . virus mutants lacking a functional adrp domain (also called macrodomain) display an increased ifn sensitivity. adrp-like macrodomains are encoded by other coronaviruses and several other positive-strand rna viruses (gorbalenya et al., 1991) . also for mhv-a59, a role of the adrp domain in pathogenesis was shown (eriksson et al., 2008; , but this seems not be related to ifn sensitivity. the last 10+ years have seen tremendous progress toward the identification of ifn antagonists of human coronaviruses (de diego et al., 2014; gralinski and baric, 2015; kindler and thiel, 2014; perlman and netland, 2009; thiel and weber, 2008; totura and baric, 2012; vijay and perlman, 2016; wong et al., 2016) . for sars-cov, the catalogue of ifn antagonists may be nearly complete by now and that of mers-cov may follow soon. nonetheless, we are still far from comprehensively understanding the manifold interactions of human pathogenic coronaviruses with the ifn system. many of the factors described here were identified by overexpression studies, and still lack the final biological assessment through generation and characterization of adequate virus mutants. it would also be interesting to see at which infections stage, in which subcellular compartment, and with which comparative intensity the ifn antagonists act, and whether and how they interact with each other. it is however safe to state that coronaviruses, which have the largest rna genome known to date, do not rely on single virulence factors but employ several layers of anti-ifn strategies. otherwise they would not be able to exist, thrive, and even expand to new hosts in the presence of powerful antiviral ifn responses. coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs ifitm-family proteins: the cell's first line of antiviral defense crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression human interferons alpha, beta and omega orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (mavs) adapter molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) activation of rnase l by murine coronavirus in myeloid cells is dependent on basal oas gene expression and independent of virus-induced interferon control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon new insights into the role of rnase l in innate immunity tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis treatment of sars with human interferons deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases dsrna-dependent protein kinase pkr and its role in stress, signaling and hcv infection coronavirus virulence genes with main focus on sars-cov envelope gene coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'o)-methyltransferase activity inhibition of nf-kappab-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus mouse hepatitis virus liver pathology is dependent on adp-ribose-1 00 -phosphatase, a viral function conserved in the alpha-like supergroup inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin coronaviruses: an overview of their replication and pathogenesis the nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis interferon-induced ifit proteins: their role in viral pathogenesis extensive cooperation of immune master regulators irf3 and nfkappab in rna pol ii recruitment and pause release in human innate antiviral transcription severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism mechanisms of rig-i-like receptor activation and manipulation by viral pathogens crossroads between innate and adaptive immunity v traf family proteins link pkr with nf-kappa b activation putative papain-related thiol proteases of positive-strand rna viruses-identification of rubivirus and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubivirus, alpha-and coronaviruses antiviral immunity via rig-i-mediated recognition of rna bearing 5 0 -diphosphates constitutive type i interferon modulates homeostatic balance through tonic signaling molecular pathology of emerging coronavirus infections pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques mx gtpases: dynamin-like antiviral machines of innate immunity polymorphisms of interferon-inducible genes oas-1 and mxa associated with sars in the vietnamese population species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 induction of mxa gene expression by influenza a virus requires type i or type iii interferon signaling sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus virus interference. i. the interferon major genetic marker of nidoviruses encodes a replicative endoribonuclease to sense or not to sense viral rna-essentials of coronavirus innate immune evasion efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase r but is resistant to its antiviral activity interferon priming enables cells to partially overturn the sars coronavirusinduced block in innate immune activation the adp-ribose-1 00 -monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus 229e mediate resistance to antiviral interferon responses delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 extraordinary gu-rich single-strand rna identified from sars coronavinis contributes an excessive innate immune response mavs recruits multiple ubiquitin e3 ligases to activate antiviral signaling cascades middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome-a preliminary study sars-cov nucleocapsid protein antagonizes ifnbeta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism activation of ifn-beta expression by a viral mrna through rnase l and mda5 combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread small self-rna generated by rnase l amplifies antiviral innate immunity rnase l releases a small rna from hcv rna that refolds into a potent pamp coronaviridae the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling the coronavirus nucleocapsid is a multifunctional protein pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2 0 -o-methyltransferase activity mers-cov papain-like protease has deisgylating and deubiquitinating activities the sars coronavirus 3a protein causes endoplasmic reticulum stress and induces ligandindependent downregulation of the type 1 interferon receptor discovery of an rna virus 3 0 !5 0 exoribonuclease that is critically involved in coronavirus rna synthesis interferon-lambda contributes to innate immunity of mice against influenza a virus but not against hepatotropic viruses regulation of innate immunity through rna structure and the protein kinase pkr severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells a structural analysis of m protein in coronavirus assembly and morphology middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study the history of toll-like receptorsredefining innate immunity critical role of an antiviral stress granule containing rig-i and pkr in viral detection and innate immunity coronaviruses post-sars: update on replication and pathogenesis protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response pkr transduces mda5-dependent signals for type i ifn induction activation of mda5 requires higher-order rna structures generated during virus infection dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc innate recognition of intracellular pathogens: detection and activation of the first line of defense murine coronavirus mouse hepatitis virus is recognized by mda5 and induces type i interferon in brain macrophages/microglia the coronavirus e protein: assembly and beyond in vivo ligands of mda5 and rig-i in measles virus-infected cells interferome v2.0: an updated database of annotated interferon-regulated genes innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna antiviral actions of interferons a contemporary view of coronavirus transcription high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus processing of the coronavirus mhv-jhm polymerase polyprotein: identification of precursors and proteolytic products spanning 400 kilodaltons of orf1a master sensors of pathogenic rna-rig-i like receptors transcription factor redundancy ensures induction of the antiviral state interferon-stimulated genes: a complex web of host defenses toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome viral phosphodiesterases that antagonize doublestranded rna signaling to rnase l by degrading 2-5a severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage ifn-lambda (ifn-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo the antiviral effect of interferon-beta against sars-coronavirus is not mediated by mxa protein inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 the jak-stat pathway at twenty sensitivity of sars/mers cov to interferons and other drugs based on achievable serum concentrations in humans severe acute respiratory syndrome related coronavirus is inhibited by interferon-alpha coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mrna the evolution of antiviral defense systems interferon and cytokine responses to sars-coronavirus infection mechanisms and enzymes involved in sars coronavirus genome expression middle east respiratory syndrome coronavirus ns4b protein inhibits host rnase l activation sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon incorporation of spike and membrane glycoproteins into coronavirus virions sars-coronavirus replication/transcription complexes are membraneprotected and need a host factor for activity in vitro group 2 coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition middle east respiratory syndrome and severe acute respiratory syndrome the membrane protein of severe acute respiratory syndrome coronavirus functions as a novel cytosolic pathogen-associated molecular pattern to promote beta interferon induction via a toll-like-receptor-related traf3-independent mechanism inhibition of protein kinase r activation and upregulation of gadd34 expression play a synergistic role in facilitating coronavirus replication by maintaining de novo protein synthesis in virusinfected cells protein kinase r (pkr) plays a proviral role in porcine reproductive and respiratory syndrome virus (prrsv) replication by modulating viral gene transcription severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain segmented negative-strand rna viruses and rig-i: divide (your genome) and rule doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses a molecular arms race between host innate antiviral response and emerging human coronaviruses interferon actionsequence specificity of the ppp(a2 0 p)na-dependent ribonuclease the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets protein kinase r and the inflammasome regulation of antiviral innate immune signaling by stress-induced rna granules nf-kappab activation by double-stranded-rna-activated protein kinase (pkr) is mediated through nf-kappab-inducing kinase and ikappab kinase antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology interferon induction of ifitm proteins promotes infection by human coronavirus oc43 active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis the coronavirus replicase human coronavirus 229e papain-like proteases have overlapping specificities but distinct functions in viral replication severe acute respiratory syndrome coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines ribose 2 0 -o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 acknowledgments f.w. is supported by the sfb 1021 and grant we 2616/7-1 (spp 1596) of the deutsche forschungsgemeinschaft. e.k. and v.t. were supported by the swiss national science foundation (snf grant 149784).disclosures: no conflicts of interest declared. key: cord-227268-8k9zaqsy authors: wick, w. david title: stopping the superspreader epidemic: the lessons from sars (with, perhaps, applications to mers) date: 2013-08-29 journal: nan doi: nan sha: doc_id: 227268 cord_uid: 8k9zaqsy i discuss the so-called superspreader epidemic, for which sars is the canonical examples (and, perhaps, mers will be another). i use simulation by an agent-based model as well as the mathematics of multi-type branching-processes to illustrate how the ss epidemic differs from the more familiar uniform epidemic (e.g., caused by influenza). the conclusions may surprise the reader: (a) the ss epidemic must be described by at least two numbers, such as the mean reproductive number (of"secondary"cases caused by a"primary case"), r0, and the variance of same, call it v0; (b) even if r0>1, if v0>>r0 the probability that an infection-chain caused by one primary case goes extinct without intervention may be close to one (e.g., 0.97); (c) the ss epidemic may have a long"kindling period"in which sporadic cases appear (transmitted from some unknown host) and generate a cluster of cases, but the chains peter out, perhaps generating a false sense of security that a pandemic will not occur; (d) interventions such as isolation (or contact-tracing and secondary case isolation) may prove efficacious even without driving r0 below one; (e) the efficacy of such interventions diminishes, but slowly, with increasing v0 at fixed r0. from these considerations, i argue that the ss epidemic has dynamics sufficiently distinct from the uniform case that efficacious public-health interventions can be designed even in the absence of a vaccine or other form of treatment. abstract i discuss the so-called superspreader (ss) epidemic, for which sars is the canonical example (and, perhaps, mers will be another). i use simulation by an agent-based model as well as the mathematics of multi-type branchingprocesses to illustrate how the ss epidemic differs from the more-familiar uniform epidemic (e.g., caused by influenza). the conclusions may surprise the reader: (a) the ss epidemic must be described by at least two numbers, such as the mean reproductive number (of "secondary" infections caused by a "primary" case), r 0 , and the variance of same, call it v 0 ; (b) even with r 0 > 1, if v 0 r 0 the probability that the infection-chain caused by one primary case goes extinct without intervention may be close to one (e.g., 0.97); (c) the ss epidemic may have a long "kindling period" in which sporadic cases appear (transmitted from some unknown host) and generate a cluster of cases, but the chains peter out, perhaps generating a false sense of security that a pandemic will not occur; (d) interventions such as isolating primary cases (or contact-tracing and secondary-case isolation) can be efficacious even without driving r 0 below one; (e) the efficacy of such interventions diminishes, but slowly, with increasing v 0 at fixed r 0 . from these considerations, i argue that the ss epidemic has dynamics sufficiently distinct from the uniform case that efficacious public-health interventions can be designed even in the absence of a vaccine or other form of treatment. this is a mathematical-modeling paper about certain types of epidemics and corresponding public-health interventions, other than by vaccines or drugs. my desire is to communicate the main points to a wider audience then just my fellow math-modelers: in particular, infectious-disease doctors and public-health officials, who are charged with responding to, and, if possible, halting, a future epidemic of a deadly disease. so i have chosen to relegate the mathematical and computational details to appendices (which are meant to be accessible to anyone with some background in math modeling or computer science, with no prerequisites.) i also eschew the usual introduction/methods/results/discussion format and adopt the form of an essay, because i believe the flow of the argument will be easier for the general reader to follow. to begin, recall sars, the acronym for "severe acute respiratory syndrome," an atypical form of pneumonia caused by a coronavirus that generated a gobal pandemic in 2003. the large-scale spread of sars began on february 21 of that year, when a professor of medicine in guangdong province, china, who had been treating patients with pneumonia, traveled to hong kong and checked into the metropole hotel downtown. for reasons still unclear, the professor passed the virus on to about a dozen persons staying at the hotel. 1 then the professor, feeling unwell, checked himself into the teaching hospital and told the staff that he had a dangerous infectious disease and should be isolated, which the staff accomplished. (the professor died of the disease in hospital.) when another patient from the hotel cluster arrived at the hospital, the staff made a medical mistake (administering an expectorant or performing aspiration), and subsequently every doctor, nurse, medical student, and orderly who entered the room-50 or more-developed sars. this was the first "superspreader" (ss) event of the epidemic. more such events followed: 15+ infected on a plane to beijing, 37 at a hospital in vietnam, and so forth. but the godzilla of all known ss events occurred at an apartment complex in hong kong called the amoy gardens, where a sick person visited for one night and generated 300+ secondary cases. 2 virologists eventually traced the source of sars to bats, with an intermediate host, the civet cat, which was sold in live-animal markets in guangdong province. the final toll (november 2002 to july 11, 2003, the date of the last known case) was: 29 countries, including canada and the united states, affected; 8098 cases; and 774 deaths. (thus the case mortality for sars was around 10 percent; compare to the two percent estimated for the 1918 flu.) the pandemic is thought to have been brought under control by introduction of better hospital procedures for rapidly isolating cases; most clusters were "nosocomial" (occurring in, and specific to, a medical setting) with few secondary clusters, beyond those mentioned above, occurring in the community. as i write, a new disease caused by a different coronovirus, called the middle east respiratory syndrome (mers) virus, has appeared and generated cases in saudi arabia, jordan, qatar, and other countries of the arabian penninsula (at this time, all cases outside the region have occurred in persons who had recently visited the middle east). a steady drumbeat of sporadic cases (91 since september 2012, as of 25 july 2013), some generating small ss events (mostly nosocomial), have been reported to the world health agency (and can be followed on the listservice of the international society for infectious diseases called promed-mail). the current estimate of mers case mortality is a frightening 50 percent (but "case mortality" has a denominator problem: the "cases" are hospitalized patients, and we do not as yet know how many milder cases are occurring in the community). sars, mers, and (in one theory) hiv 3 all represent a different kind of epidemic than usually treated in textbooks (particularly those that emphasize modeling). i will refer to a "superspreader epidemic," sse for shorthand. much of the conventional wisdom about epidemics does not apply to the sse case. next, i informally describe models for an sse, leaving details about software implementation and mathematical issues to appendices. the approbathrooms had floor drains that were supposed to be isolated by u-traps, but these only function if filled with water; but no one had told the residents to regularly dump a gallon of water on their bathroom floors. 3 studies of transmission of hiv between an infected and an uninfected partner in a long-term relationship found very low transmission rates; e.g., one transmission in 1,000 unprotected sex acts. this gave rise to the theory that hiv is an ss epidemic; the candidates for the superpreaders are: (a) persons in the primary retroviral-infection period that lasts a few weeks, who have a thousand times the level of virus in blood and semen found in chronically-infected patients; and (b) cases like "patient zero," the canadian airline attendant with an impressive rolodex of sexual partners in many cities, described in 'randy shilts's 1987 book, and the band played on. priate kind of model is called a "stochastic multi-type branching-process." the adjective "stochastic" refers to random events, as in a dice game; in computer terms, when simulating the model the program makes calls on the random number generator, abbreviated rng (supplied with your operating system), when making updates. the "branching-process" was introduced by mathematicians in the 1920s to describe, among other things, epidemics. (other applications include demographic population growth and nuclear chain-reactions.) in a branching-process, some entity produces a (random) number of "offspring" at some rate, not depending on the number of other entities existing, for some (also possibly random) reproductive period; the offspring, which may be of various types, can generate descendants in a similar manner. (the particular sort of branching-processes i use may differ from the kind described in math texts, however; see the appendices for the details.) mathematical biologists also introduced a deterministic model of epidemics dubbed "sir," for susceptible-infected-recovered, appropriate to describe a measles or influenza epidemic. here i am interested in interventions that bring the disease to a halt before it establishes an epidemic, so the susceptible population can be regarded as fixed. (by the time a moderate fraction of the population of a major city or country had sars or mers, the pandemic would long since have been declared by who and, given the modern phenomenon of "jet spread," airport closures and international panic would soon follow.) in addition, as the reader will understand from the results described below, a deterministic model of an sse is totally inappropriate. 4 this is true for much of biology, despite the widespread use of deterministic equations, called by the ancient acronym "odes" whose significance nobody recalls, by mathematical biologists; see my book, [5] . now we come to the famous "r 0 ," also called the basic, or mean, reproduc-4 full disclosure: in march of 2003, the author and two colleagues sent a paper to science proposing a stochastic branching-process model of the sars epidemic, concluding that the epidemic could be controlled by rapid isolation of infected patients if r 0 was not too large. science rejected the paper, with the sole reviewer's comment being: "the model looks right, the conclusion looks right, but it doesn't penetrate!" in fact, the outcome of the epidemic was as we predicted. later that spring, science published papers from two modeling groups; one used odes, [3] , which is absurd, and another, [4] , a model like the authors', with similar conclusions. the latter group had access to case incidence data from hong kong, which our group lacked (we derived our model from published accounts, mostly in newspapers and on who and cdc websites). later, the present author fit a stochastic model to the hong kong data, see [5] , chapter 6, in order to derive disease transmission parameters and demonstrate a new fitting technique. tive number of the virus, which was historically the original "tipping-point." it is defined as the average number of secondary cases caused (infected directly by) a primary case, in the absence of any treatment or intervention. as we will see, that number of secondary infections should be thought of as a random variable, so it has a variance as well as a mean; to uphold tradition, i denote it by v 0 . now we must carefully distinguish two epidemic scenarios, which i will call the "uniform" (or poisson) and the superspreader (ss) epidemics. in the former, the number of secondary cases of a primary case (assumed for this discussion to have a fixed infectious period) is a poisson random variable, which is the name given in probability theory for the number of events in a random but constant-rate accrual process, like the number of hits inside the ring by a darts-player of little skill. the poisson random variable is characterized by v 0 = r 0 and a (super-exponential) fall off beyond the mean; e.g., if r 0 = 2, the probability of the primary case producing 10 secondary infections is infinitesimal. models in which such accruals have larger variances are said to possess "extra-poisson variation" (epv). what could be the meaning of this epv? the more-infectious case-i will also refer to this person as a superspreader-might have a special biological ability to spread the infection (perhaps through vomiting, diarrhea, or just a deep cough), or might be situated in some place that facilitates transmission (e.g., in a crowded icu, but not in isolation; on a plane; or visiting the amoy gardens). the simplest example, that i use throughout this paper for illustrations, is an epidemic model with two types of infected persons, each making a poissondistributed number of secondary cases. the person of type one has an average number of secondary cases of r l (the subscript meaning lower-value), and the type-two person, r h (high-level); these occur with probabilities p l and p h , so the population average is: for instance, suppose r l = 0.7 and r h = 30.0, with p l = 0.9556 and p h = 0.04437; then r 0 = 2.0, while (easy computation) v 0 = 38.4. mathematicians proved in the 1920s that a branching-process with r 0 > 1 can grow, while one with r 0 ≤ 1 eventually dies out. but it is often overlooked that a branching-process with r 0 > 1 can nevertheless go extinct. the outcome of a branching-process with r 0 > 1 (mathematicians use the phrase, "supercritical") is dichotomous: either heading for infinity or destined to die out, with certain probabilities. for instance, the process with the r's above plus assumptions about incubation periods, infectious periods, etc., resulting in various models; (see the appendices) will disappear eventually with probability p e in the range 0.88-0.91. for the uniform case, r l = r h = 2.0, p e is about 0.2. as a rule, p e increases with increasing v 0 for fixed r 0 . this is easy to understand: the most probable "index" (epidemiology jargon for initial) case is a low-infectivity patient, and, although one or more secondary cases might follow while the patient remains infectious (even if r l < 1), the infection-chain is likely to die out unless preserved by the appearance of a superspreader. see table 1 for some illustrative examples. the simulation technique i used is described in appendix one, and the parameters are listed there in table a . i also included results in the table from an exactly-solvable model, meaning explicit formulas exist for extinction probabilities (see appendix two), called the markov case. i included it to satisfy mathematicians and to check the software, by comparing probabilities from repeated simulations to exact answers; but it has a feature that renders it dubious for use in biology. the markov case is the model in which all waiting times-times to end of the incubation period; to end of the infectious period; and to generate the next secondary case-have exponential distributions (see appendix two for the explanation of why the famous "markov property" requires exponential distributions for all waiting times). but only for the last mentioned is this a realistic choice: the exponential law was derived from physics, where it represents radioactive decay; but people are not atoms. for applications i assumed, pending more data, normal distributions for the incubation and infectious periods (conditional on non-negativity, of course). for these models, simulation must be used to compute probabilities (for reasons spelled out in appendix two). so the last column in the table represents the biologically more-realistic case. the reason that the entries in this column are the smallest in the rows is that the markov case has more epv; see appendix two. next consider mers, for which (as i write) another sporadic case apparently shows up on average every three days. (a "case" means laboratoryconfirmed in hospital, which may represent the very sick and potential superspreaders; there are probably more cases appearing in the community.) let "sp-int" stand for the average interval between appearances of sporadic infections. sp-int is hard to estimate, because it is difficult to distinguish sporadic from secondary cases; e.g., if in a family a father and two children become infected and the father becomes ill first, should the children be counted as secondary cases, or were they exposed to the same external source of virus and so additional sporadic cases? thus sp-int might be considerably larger than three days. the law for first-time-to-a-bernoulli-event gives: e.g., if p e = 0.98 and sp-int is 3 days, the mean initiation time is 150 days (but it could be much longer). because of the dichotomy and the tendency of most infection-chains to go extinct, the sse with large epv seeded by sporadic cases can be very surprising. for instance, figure 1 shows an epidemic with r's as in the example above; for several months the epidemic seems indolent, then around 70 days, it takes off. figure 2 shows another epidemic with parameters from the last row (most extreme case) of the table; nothing exciting happens for a year, which might cause premature optimism among public-health officials; then comes disaster. (rerunning with the same parameters but different "seeds" for the rng, the timing of the pandemic in the figures is highly variable.) note that no spontaneous modifications, genetic or otherwise, are assumed in the virus for these simulations; disease parameters are fixed. the patterns are entirely due to epv and chance. now to interventions. neither vaccines nor therapeutic drugs currently exist for human coronaviruses; considering that the sars virus has disappeared while mers remains contained, and the enormous cost of developing, testing, and licensing a new vaccine or drug, this unfortunate situation is likely to persist. therefore, let us explore other interventions that could prevent an sse pandemic by these viruses. the interventions are restricted to isolation of cases, preventing some secondary infections. the intervention might be primary: rapidly isolating an infected patient in hospital in a negativepressure room and requiring all attendant personel to wear gowns and p99 masks, hopefully decreasing the patient's infectious period; or secondary: tracing contacts of the primary case and getting as many as possible to go into isolation at home, thus diminishing secondary infections. the secondary intervention has one advantage over the primary: it can be instigated even if laboratory tests are delayed beyond the lifetime, or infectious period, of the patient, which may be a consideration early in the epidemic. both interventions have the same effect: decreasing the effective "r" of the patient. an interesting special case, which i will refer to as the "ss intervention" (ssi), limits the intervention to high-infectivity cases. possible scenarios where the ssi is effective might include: ss patients are sicker and usually hospitalized, while low-infectivity cases remain in the community; and where ss patients can be detected by some physiological or virological measurement. in most discussions of interventions in infectious-disease epidemiology, it is assumed that an efficacious intervention is one that drives r 0 below one. i argue that, for the sse, the proper quantity to look at is not r 0 but the extinction probability of the infection-chain, which i have denoted by p e . thus the goal of the intervention must be to increase p e ; indeed, i define intervention-efficacy (ie) by: where p e,i stands for the extinction probability in the presence of the intervention. thus ie = 1.0 if the intervention drives p e,i all the way to one, and zero if p e,i = p e . of course, if the intervention should push r 0 below one, then ie will be one, but ie could be, e.g., 0.75 even with an r 0 still greater than one after the intervention is active (as we will see below). ie depends on both r 0 and v 0 , as we will see. if the reader is dubious about this redefinition of efficacy, consider (a) the disease introduced into 1000 cities. if, e.g., p e = 0.7 while p e,i = 0.9 (efficacy, 67 percent; these numbers are not implausible, see table 1 ), the effect of the intervention is to spare 200 cities on average from a local epidemic. more convincing, perhaps, is to think about (b): the time before the epidemic takes off in a given region, for the sporadically-appearing disease. in the scenario in table 1 fourth row, the epidemic takes off in a few months, but with the intervention increasing p e to the value in the last row, it takes roughly a half-year-perhaps enough time for epidemiologists or veterinarians to find and eliminate the source of the virus. i therefore explored efficacy for such interventions through simulating an sse epidemic with ssi's of various efficiencies (at lowering r h ). see figures 3 and 4 . efficacy is partially mediated by decreasing the effective (withintervention) r 0 , which in any case remains above one in the figures. note that, for a high-v 0 epidemic, even an intervention with efficiency in the 60-70 percent range can be quite valuable. i next asked whether it is easier or harder to stop an epidemic with a large v 0 (equivalently here, large r h ) than an epidemic with a smaller value, at fixed intervention. the question is hard to answer through introspection or analysis. on the one hand, a larger r h should mean that the ssi intervention is more relevant; on the other hand, the explosive nature of case growth suggests the intervention would have to be larger. one thought suggests that efficacy should be an increasing function of r h , the other that it should be decreasing. see figures 5 and 6 : in fact, efficacy in the models does diminish with increasing r h at fixed intervention efficiency, but falls off remarkably slowly. as of august 2013, mers has not generated a pandemic after 10 months of sporadic cases. the obvious explanation is that r 0 is less than one-but that could be an illusion; recall figure 2! in july 2013, a group of epidemiologists published in lancet, [1] an estimate, based on 55 cases (there were more as of july but the authors culled some due to ambiguities), of r 0 as about 0.7, which i used for the lower figure (r l ) in the simulations. however, they made an "occam's razor" simplifying assumption which may have biased their analysis. despite being critical of the authors' methodology, i agree with their conclusion that r 0 of mers is currently less than one. (the best fit in my model-class to the cluster-data reported in [1] had r 0 = 0.813; r l = 0.402; r h = 17.9; normal periods; and sp-int = 3 days. i.e., an sse but not a pandemic. see appendix three.) whatever the truth may be, in two ) saudi arabia has a population of 26 million, so this represents less than an eight percent increase; however, these pilgrims will be congregating in a few sites where there may be increased opportunity for viral transmission from infected cases, possibly locally raising r 0 and, more likely, v 0 . also at present the source of the sporadic cases is under investigation, with reports of a related virus in a bat (not found in the middle east, however), and antibodies to coronaviruses in camels (but not in saudi arabia, and without recovery of a virus; because of cross-reactivity the latter report may be noise). the situation is too uncertain to make definite predictions about a mers epidemic at this time. here is a summary of my conclusions about the ss epidemic and interventions, based on the modeling exercise. first, it is better not to be complacent about an often-fatal disease occurring at low frequency and generating small clusters of secondary cases. despite appearances, it may represent the "kindling" phase of a future pandemic-which may develop even in the absence of genetic modification in the pathogen increasing transmission rates to or among humans (as occurred a decade ago in sars; but an event, given the current state of genetics, which nobody can predict). second, the sse has very different dynamics from more uniform epidemics caused by, e.g., influenza or measles; remarkably, this permits the design of interventions which are not available in the latter cases. for instance, because the superspreader is rare and possibly detectable, a specifically targeted intervention may at least delay the onset of a pandemic long enough to allow epidemiologists to locate and eliminate the source of sporadic infections. even detection after the end of a superspreader's infectious period can be efficacious (through contact-tracing). finally, for my fellow math-modelers: the story related here is an instance of the important role played by heterogeneity, and stochasticity, in biology. (for other examples, see my books [5] and [6] .) a desire for simplicity in modeling is not an excuse for overlooking these possibilities. the model is of "agent-based" type, which means that the characteristics of each case are stored separately in computer memory. 5 the descriptors of each case are: infectious status (in incubation period, so not infectious; or infectious); infectious type; and some integers assigned at creation for identification purposes (including the index of the primary, if a secondary case; and an indentifier for cluster). in addition, for each case some floating-point numbers are stored: when a case is created in the incubation phase, a predicted (clock) time for entering the infectious phase; once in the latter, the time of creation of a secondary case (updated when that event occurs); and the time of death or end of infectious period. because of the agent-based modeling, these times can be generated with any desired distributions. for the time-of-next-secondary-case, an exponential random variable is appropriate (consistent with the assumption that the infections caused in a fixed period have a poisson distribution). but an exponential random variable is a poor choice for the other waiting times (time-to-infectivity and to death or end-ofinfectious period), because the exponential law has no memory. conventional mathematical models assume that all waiting times are exponentials, because the markov property then holds, permitting mathematicians to write down and solve equations for quantities like extinction probabilities; see the math appendix for discussion. however, the convenience of mathematicians should not be a determining element in science. i assumed that the waiting times other than for secondary infections had normal distributions (conditional on being non-negative). the simulation starts by filling in the characteristics and times for the index case (and, for the sporadic scenario, a conjectured time for the appearance of the next sporadic case). then the smallest waiting time is found; the corresponding changes are made, including creation of a new case and its characteristics and waiting times if the event is a sporadic-or secondaryinfection. the clock time is moved forward to this event time. at subsequent iterations the program runs through all the stored waiting times looking for the next one; then performs those changes, etc. in other words, the process is simulated in the most straightforward manner, with no mathematical approximations whatsoever except for the belief that the rng provides truly random digits (about which john von neumann made his famous joke: "anyone who believes a computer can generate a random variable is living in sin"), which is unlikely to cause trouble here. when computing extinction probabilities, the process was run until either extinction occurred, 500 incident (incubation or infectious) cases existed on a single day (after which i assumed extinction was unlikely), or the clock time reached four years. 10,000 samples were used in simulations. table 1 reports the comparisons of the solvable (markov) case with simulations of same; rerunning some cases with 100,000 repetitions, even the third decimal came out right. table a reports the parameters used. when more data about, e.g., mers, becomes available, it will be interesting to fit the model in order to estimate parameters; a convenient method was introduced in my book [5] , chapter 5. the program was written in the c programming language, runs on anything, and is available from the author by request. (but if the reader is interested in modeling the sse, it is always better to write your own program then rely on somebody else's.) i am an ex-mathematical physicist. so why the resort to programming the laptop? my motivation is simple: i intend to use the model to make predictions about mers and other ss epidemics, once transmission parameters become available. in my philosophy, the proper goal of any scientist is to make predictions for future experiments or observations, because that is ultimately the only way to be sure that you are not modeling moonshine. so marc kac's fabled advice for mathematical physicists ("mutilate, mutilate until you can solve the equations") cannot be accepted. i may need to employ all sorts of peculiar distributions taken from empirical data. however, it is useful to solve even an unrealistic scenario because it provides a formula to compare to the output of the software for that case, helpful to allay the suspicion that the results in this paper are due to bugs or numerical issues. the markov case is the model in which all waiting times-times to end of the incubation period; to end of the infectious period; and to generate the next secondary case-have exponential distributions. as pointed out in the text, only for the last mentioned is this a realistic choice. but it is required by the markov property ("the future and the past are independent, given the present") because only the exponential is memoryless: provided τ is an exponential random variable: p [ τ > t ] = exp(−λ t). with the exponential for incubation and infectious periods, the number of secondary cases of a primary case is actually geometric rather than poisson, with a larger variance (so this case has epv even even without multiple types). it is, of course, the markov property that makes it easy to derive equations for probabilities associated to stochastic processes. here is a simple (non-rigorous?) derivation of the extinction probabilities for the markov version of the ss epidemic without sporadic cases. let ex stand for the event: extinction of the infection-chain; p k for probabilities generated by the process begun by a type-k case; and r k for the corresponding extinction probability, so: r k = p [ extinction probability given one initial case, type k ] then by conditioning on the first secondary case, if there is one: where γ k is the rate of generating secondary cases by a primary case of type k and ρ is the reciprocal of the time-to-end of the infectious period. thus we obtain a quadratic system, easily reduced to a single quadratic for the two-level case of the text (below). however, the reader may doubt whether this is rigorous. there is another (long-winded but fully rigorous) approach for the markov case, which is to note that it is identical to a markov birth-and-death compartmental jump process. here we have to distinguish incubation and infectious stages for each type; call the respective compartments (populations) x k and y k . the rates of jumps for this process are: next given numbers 0 < r k < 1 and 0 < s k < 1 introduce the momentgenerating function (mgf): where e denotes expectation. differentiating using kolmogorov's forward equation (just a consequence of jumps occurring at given rates): now make the substitutions: r k ∂/∂r k −→ x k and s k ∂/∂s k −→ y k to obtain the pde: solve this system by the method of characteristics; that is, define functions of time r k (t) and s k (t) to satisfy the ode system: now note that φ is a function of r = {r k }, s = {s k } and t and from the above φ(r(t), s(t); 0) = φ(r(0), s(0); t). also from its definition, if we take as initial conditions x k = 1 and y k = 0 so setting the right-hand sides of the odes, (13), equal to zero to find the fixed point gives the required extinction probabilities. the resulting equations are the same as (9). for the two-level case, these equations are, using r k = γ k /ρ: which after a little algebra yields the quadratic: this is solved for ψ (taking the negative sign in the quadratic formula and rationalizing the numerator) by: note that if the extinction probabilities r 1 and r 2 are less than one, ψ is negative and vice versa; hence that is the case if and only if r 0 > 1. given ψ, r 1 and r 2 can then be found from (16); p e = r 1 , r 2 , or p 1 r 1 + p 2 r 2 , depending on how you choose the initial condition. (the third is the realistic case.) we used these results to reproduce tables and figures from the text, comparing to output obtained by implementing exponentials for all waiting times and repeating the simulations. why can't we use the informal argument for the general case (without requiring everything to be exponential)? because there were several hidden assumptions in deriving (9). for one, in (6) we used the exponential integral: which in a non-exponential case would have to be done numerically. worse, in (8) there was a concealed use of the markov property; i erased conditioning as follows: p [ ex | first secondary case is of type j, at time τ ] = p [ ex | first secondary case is of type j ] , reasoning that the time of creation of the first secondary infection was not informative about the time left in the infectious period, which invokes the memorylessness of the exponential law, or just the markov property. in a non-markov case, that time would be informative, so one would have to evaluate that conditional probability as stated somehow, and (9) becomes a system of integral equations which would have to be solved on the computer by discretizing the time, solving matrix equations, then letting the discretization interval shrink, controlling for numerical error, etc. relative to simulating the process on a fast computer, it doesn't seem worth the effort. appendix 3: the lancet transmission paper, [1] the lancet authors, breban et al., based their analysis on certain caseclusters (epidemiologically-linked cases), which they summarized in a table (the scenarios concern how the authors interpreted the clusters in terms of sporadic vs. secondary cases): table b . distribution of cluster sizes size scenario 1 scenario 2 1 17 11 2 4 2 3 3 3 4 1 1 5 0 2 24 1 1 converting from numbers of clusters to frequencies: in their mathematical appendix, the lancet authors begin by citing a paper in plos by others [2] who showed how to use branching-processes and bayesian methodology to estimate r 0 from cluster data, allowing for some extra variance in reproductive number, namely by assuming a certain distribution (called the "negative binomial") that interpolates between poisson and geometric. in my terminology, those two cases represent the uniform epidemic with either fixed or exponential infectious period-so much smaller epv than allowed in my two-level model (which in fact has three free parameters, see last paragraph). the lancet authors apply bayesian analysis to the plos authors' distribution but reject it as unable to resolve both parameters (r 0 and a dispersion parameter affecting v 0 ) because the latter "could be very large, even 1000." it is here that they abused poor occam 6 and retreated to an analysis using poisson and estimating a single parameter, r 0 . of course, with this assumption the big cluster in the last row must include tertiary, etc., cases, because a cluster of 24 secondary cases of one primary case, with an r 0 of 0.7, is virtually impossible. here are simulated cluster frequencies under some models described in the text (10,000 simulations, each run to 91 cases, per model; a "cluster" consists of the tree generated by a sporadic primary case): not surprisingly, the uniform (poisson) model with r 0 = 0.7 has difficulty fitting the big cluster of 24 cases in the last line of tables b/c; indeed, it appears as if models b-d can be rejected, say by a fisherian p-value test. this suggests that what happened in the lancet authors' bayesian fitting procedure is that their single-type, poisson likelihood settled for fitting the small clusters and ignored the the big one. which could render their logic circular (r 0 is less than one because, in a model with no higher-infectious types and ignoring one data point . . . r 0 comes out less than one). however, does that mean the authors' conclusion is wrong? to answer that question, i utilized the nknn method (advertised in my book [5] ) to search for best-fitting models in my model-class. this venerable method selects among various theories, given samples from each, that best explains a given data-set; it is commonly used in pattern-recognition. (it reduces to maximum likelihood in the limit of infinitely-large samples from each theory; i.e., if you have an infinitely-fast computer to perform the simulations.) here the samples were 1,000 simulated epidemics, and corresponding clusterfrequencies, from models chosen at random with 0.2 < r l < 1, 1 < r h < 30, and r l < r 0 < min(r h , 2.0), with 1,000 repetitions. the data-set was column one of table c . the search turned up excellent fits for the frequency data (smallest sum-of-squares, 0.052): on the basis of this result, i must agree that r 0 < 1 for mers at present. note however that the good fit of table e to table c is attained because an ss model reproduces the big cluster in the last row of the latter. the lancet authors' choice to limit their model-class to a poisson distribution for secondary cases ruled out this possibility-a superspreader, multiple-levels, epidemic model, but with r 0 less than one-a priori. on the other hand, "fitting" a three-parameter model to data consisting of seven frequencies cannot be recommended either. now a bit of preaching about modeling traps. when you have a model with free parameters and a data-set, there are two ways to delude yourself. methods like bayesian analysis, possible if you have an explicit likelihood, or my nknn method, which works for anything provided you can simulate fast enough, will generate a "best-fitting model." if some parameter (like the plos authors' dispersion parameter) fluctuates so much that it can't be pinned down, you can choose to omit it-i.e., mutilate the model in order to be able to "estimate the parameters." or you can keep the troublesome parameter and let the software choose a "best model." either way, the model selected may be false; but you won't discover that until more data arrives. in my book, [5] , i labeled these two pitfalls the "simple-is-best trap" and the "etch-a-sketch trap" and argued that the modeler is required to steer between these two hazards, like odysseus sailing between skylla and charybdis. fall in the first, and your parameter estimates will be wrong or biologically unintelligible; fall in the other, and your model can "explain" anything (which karl popper decried as not science). ultimately, the best approach is to make a prediction-even when it fails, you will have learned something. (unfortunately, on the basis of the data thus far available, making a prediction about mers is impossible.) interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk inference of r 0 and transmission heterogeneity from the size distribution of stuttering chains transmission dynamics and control of severe acute respiratory syndrome. science transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions fitting non-linear, stochastic models to data in war in the body: the evolutionary arms race between hiv and the human immune system and the implications for vaccines key: cord-279255-v861kk0i authors: dhama, kuldeep; khan, sharun; tiwari, ruchi; sircar, shubhankar; bhat, sudipta; malik, yashpal singh; singh, karam pal; chaicumpa, wanpen; bonilla-aldana, d. katterine; rodriguez-morales, alfonso j. title: coronavirus disease 2019–covid-19 date: 2020-06-24 journal: clin microbiol rev doi: 10.1128/cmr.00028-20 sha: doc_id: 279255 cord_uid: v861kk0i in recent decades, several new diseases have emerged in different geographical areas, with pathogens including ebola virus, zika virus, nipah virus, and coronaviruses (covs). recently, a new type of viral infection emerged in wuhan city, china, and initial genomic sequencing data of this virus do not match with previously sequenced covs, suggesting a novel cov strain (2019-ncov), which has now been termed severe acute respiratory syndrome cov-2 (sars-cov-2). although coronavirus disease 2019 (covid-19) is suspected to originate from an animal host (zoonotic origin) followed by human-to-human transmission, the possibility of other routes should not be ruled out. compared to diseases caused by previously known human covs, covid-19 shows less severe pathogenesis but higher transmission competence, as is evident from the continuously increasing number of confirmed cases globally. compared to other emerging viruses, such as ebola virus, avian h7n9, sars-cov, and middle east respiratory syndrome coronavirus (mers-cov), sars-cov-2 has shown relatively low pathogenicity and moderate transmissibility. codon usage studies suggest that this novel virus has been transferred from an animal source, such as bats. early diagnosis by real-time pcr and next-generation sequencing has facilitated the identification of the pathogen at an early stage. since no antiviral drug or vaccine exists to treat or prevent sars-cov-2, potential therapeutic strategies that are currently being evaluated predominantly stem from previous experience with treating sars-cov, mers-cov, and other emerging viral diseases. in this review, we address epidemiological, diagnostic, clinical, and therapeutic aspects, including perspectives of vaccines and preventive measures that have already been globally recommended to counter this pandemic virus. o ver the past 2 decades, coronaviruses (covs) have been associated with significant disease outbreaks in east asia and the middle east. the severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) began to emerge in 2002 and 2012, respectively. recently, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causing coronavirus disease 2019 (covid19) , emerged in late 2019, and it has posed a global health threat, causing an ongoing pandemic in many countries and territories (1) . health workers worldwide are currently making efforts to control further disease outbreaks caused by the novel cov (originally named 2019-ncov), which was first identified in wuhan city, hubei province, china, on 12 december 2019. on 11 february 2020, the world health organization (who) announced the official designation for the current cov-associated disease to be covid-19, caused by sars-cov-2. the primary cluster of patients was found to be connected with the huanan south china seafood market in wuhan (2) . covs belong to the family coronaviridae (subfamily coronavirinae), the members of which infect a broad range of hosts, producing symptoms and diseases ranging from the common cold to severe and ultimately fatal illnesses, such as sars, mers, and, presently, covid-19. sars-cov-2 is considered one of the seven members of the cov family that infect humans (3) , and it belongs to the same lineage of covs that causes sars; however, this novel virus is genetically distinct. until 2020, six covs were known to infect humans, including human cov 229e (hcov-229e), hcov-nl63, hcov-oc43, hcov-hku1, sars-cov, and mers-cov. although sars-cov and mers-cov have resulted in outbreaks with high mortality, others remain associated with mild upper-respiratory-tract illnesses (4) . newly evolved covs pose a high threat to global public health. the current emergence of covid-19 is the third cov outbreak in humans over the past 2 decades (5) . it is no coincidence that fan et al. predicted potential sars-or mers-like cov outbreaks in china following pathogen transmission from bats (6) . covid-19 emerged in china and spread rapidly throughout the country and, subsequently, to other countries. due to the severity of this outbreak and the potential of spreading on an international scale, the who declared a global health emergency on 31 january 2020; subsequently, on 11 march 2020, they declared it a pandemic situation. at present, we are not in a position to effectively treat covid-19, since neither approved vaccines nor specific antiviral drugs for treating human cov infections are available (7) (8) (9) . most nations are currently making efforts to prevent the further spreading of this potentially deadly virus by implementing preventive and control strategies. in domestic animals, infections with covs are associated with a broad spectrum of furthermore, it acts as a critical factor for tissue tropism and the determination of host range (45) . notably, s protein is one of the vital immunodominant proteins of covs capable of inducing host immune responses (45) . the ectodomains in all covs s proteins have similar domain organizations, divided into two subunits, s1 and s2 (43) . the first one, s1, helps in host receptor binding, while the second one, s2, accounts for fusion. the former (s1) is further divided into two subdomains, namely, the n-terminal domain (ntd) and c-terminal domain (ctd). both of these subdomains act as receptorbinding domains, interacting efficiently with various host receptors (45) . the s1 ctd contains the receptor-binding motif (rbm). in each coronavirus spike protein, the trimeric s1 locates itself on top of the trimeric s2 stalk (45) . recently, structural analyses of the s proteins of covid-19 have revealed 27 amino acid substitutions within a 1,273-amino-acid stretch (16) . six substitutions are located in the rbd (amino acids 357 to 528), while four substitutions are in the rbm at the ctd of the s1 domain (16) . of note, no amino acid change is seen in the rbm, which binds directly to the angiotensinconverting enzyme-2 (ace2) receptor in sars-cov (16, 46) . at present, the main emphasis is knowing how many differences would be required to change the host tropism. sequence comparison revealed 17 nonsynonymous changes between the early sequence of sars-cov-2 and the later isolates of sars-cov. the changes were found scattered over the genome of the virus, with nine substitutions in orf1ab, orf8 (4 substitutions), the spike gene (3 substitutions) , and orf7a (single substitution) (4) . notably, the same nonsynonymous changes were found in a familial cluster, indicating that the viral evolution happened during person-to-person transmission (4, 47) . such adaptive evolution events are frequent and constitute a constantly ongoing process once the virus spreads among new hosts (47) . even though no functional changes occur in the virus associated with this adaptive evolution, close monitoring of the viral mutations that occur during subsequent human-to-human transmission is warranted. the m protein is the most abundant viral protein present in the virion particle, giving a definite shape to the viral envelope (48) . it binds to the nucleocapsid and acts as a central organizer of coronavirus assembly (49) . coronavirus m proteins are highly diverse in amino acid contents but maintain overall structural similarity within different genera (50) . the m protein has three transmembrane domains, flanked by a short amino terminus outside the virion and a long carboxy terminus inside the virion (50) . overall, the viral scaffold is maintained by m-m interaction. of note, the m protein of sars-cov-2 does not have an amino acid substitution compared to that of sars-cov (16) . the coronavirus e protein is the most enigmatic and smallest of the major structural proteins (51) . it plays a multifunctional role in the pathogenesis, assembly, and release of the virus (52) . it is a small integral membrane polypeptide that acts as a viroporin (ion channel) (53) . the inactivation or absence of this protein is related to the altered virulence of coronaviruses due to changes in morphology and tropism (54) . the e protein consists of three domains, namely, a short hydrophilic amino terminal, a large hydrophobic transmembrane domain, and an efficient c-terminal domain (51) . the sars-cov-2 e protein reveals a similar amino acid constitution without any substitution (16) . the n protein of coronavirus is multipurpose. among several functions, it plays a role in complex formation with the viral genome, facilitates m protein interaction needed during virion assembly, and enhances the transcription efficiency of the virus (55, 56) . it contains three highly conserved and distinct domains, namely, an ntd, an rna-binding domain or a linker region (lkr), and a ctd (57) . the ntd binds with the 3= end of the viral genome, perhaps via electrostatic interactions, and is highly diverged both in length and sequence (58) . the charged lkr is serine and arginine rich and is also known as the sr (serine and arginine) domain (59) . the lkr is capable of direct interaction with in vitro rna interaction and is responsible for cell signaling (60, 61) . it also modulates the antiviral response of the host by working as an antagonist for interferon (ifn) and rna interference (62) . compared to that of sars-cov, the n protein of sars-cov-2 possess five amino acid mutations, where two are in the intrinsically dispersed region (idr; positions 25 and 26) , one each in the ntd (position 103), lkr (position 217), and ctd (position 334) (16) . besides the important structural proteins, the sars-cov-2 genome contains 15 nsps, nsp1 to nsp10 and nsp12 to nsp16, and 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14) (16) . all these proteins play a specific role in viral replication (27) . unlike the accessory proteins of sars-cov, sars-cov-2 does not contain 8a protein and has a longer 8b and shorter 3b protein (16) . the nsp7, nsp13, envelope, matrix, and p6 and 8b accessory proteins have not been detected with any amino acid substitutions compared to the sequences of other coronaviruses (16) . the virus structure of sars-cov-2 is depicted in fig. 2 . sequence percent similarity analysis. we assessed the nucleotide percent similarity using the megalign software program, where the similarity between the novel sars-cov-2 isolates was in the range of 99.4% to 100%. among the other serbecovirus cov sequences, the novel sars-cov-2 sequences revealed the highest similarity to bat-sl-cov, with nucleotide percent identity ranges between 88.12 and 89.65%. meanwhile, earlier reported sars-covs showed 70.6 to 74.9% similarity to sars-cov-2 at the nucleotide level. further, the nucleotide percent similarity was 55.4%, 45.5% to 47.9%, 46 .2% to 46.6%, and 45.0% to 46.3% to the other four subgenera, namely, hibecovirus, nobecovirus, merbecovirus, and embecovirus, respectively. the percent similarity index of current outbreak isolates indicates a close relationship between sars-cov-2 isolates and bat-sl-cov, indicating a common origin. however, particular pieces of evidence based on further complete genomic analysis of current isolates are necessary to draw any conclusions, although it was ascertained that the current novel sars-cov-2 isolates belong to the subgenus sarbecovirus in the diverse range of betacoronaviruses. their possible ancestor was hypothesized to be from bat cov strains, wherein bats might have played a crucial role in harboring this class of viruses. splitstree phylogeny analysis. in the unrooted phylogenetic tree of different betacoronaviruses based on the s protein, virus sequences from different subgenera grouped into separate clusters. sars-cov-2 sequences from wuhan and other countries exhibited a close relationship and appeared in a single cluster (fig. 1 ). the covs from the subgenus sarbecovirus appeared jointly in splitstree and divided into three subclusters, namely, sars-cov-2, bat-sars-like-cov (bat-sl-cov), and sars-cov (fig. 1) . in the case of other subgenera, like merbecovirus, all of the sequences grouped clinical microbiology reviews than italy. a john hopkins university web platform has provided daily updates on the basic epidemiology of the covid-19 outbreak (https://gisanddata.maps.arcgis.com/ apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6) (238) . covid-19 has also been confirmed on a cruise ship, named diamond princess, quarantined in japanese waters (port of yokohama), as well as on other cruise ships around the world (239) (fig. 3) . the significant events of the sars-cov-2/covid-19 virus outbreak occurring since 8 december 2019 are presented as a timeline in fig. 5 . at the beginning, china experienced the majority of the burden associated with covid-19 in the form of disease morbidity and mortality (65), but over time the covid-19 menace moved to europe, particularly italy and spain, and now the united states has the highest number of confirmed cases and deaths. the covid-19 outbreak has also been associated with severe economic impacts globally due to the sudden interruption of global trade and supply chains that forced multinational companies to make decisions that led to significant economic losses (66) . the recent increase in the number of confirmed critically ill patients with covid-19 has already surpassed the intensive care supplies, limiting intensive care services to only a small portion of critically ill patients (67) . this might also have contributed to the increased case fatality rate observed in the covid-19 outbreak. the novel coronavirus was identified within 1 month (28 days) of the outbreak. this is impressively fast compared to the time taken to identify sars-cov reported in foshan, guangdong province, china (125 days) (68) . immediately after the confirmation of viral etiology, the chinese virologists rapidly released the genomic sequence of sars-cov-2, which played a crucial role in controlling the spread of this newly emerged novel coronavirus to other parts of the world (69) . the possible origin of sars-cov-2 and the first mode of disease transmission are not yet identified (70) . analysis of the initial cluster of infections suggests that the infected individuals had a common exposure point, a seafood market in wuhan, hubei province, china (fig. 6 ). the restaurants of this market are well-known for providing different types of wild animals for human consumption (71) . the huanan south china seafood market also sells live animals, such as poultry, bats, snakes, and marmots (72) . this might be the point where zoonotic (animal-to-human) transmission occurred (71) . although sars-cov-2 is alleged to have originated from an animal host (zoonotic origin) with further humanto-human transmission (fig. 6 ), the likelihood of foodborne transmission should be ruled out with further investigations, since it is a latent possibility (1). additionally, other clinical microbiology reviews potential and expected routes would be associated with transmission, as in other respiratory viruses, by direct contact, such as shaking contaminated hands, or by direct contact with contaminated surfaces (fig. 6) . still, whether blood transfusion and organ transplantation (276) , as well as transplacental and perinatal routes, are possible routes for sars-cov-2 transmission needs to be determined (fig. 6 ). from experience with several outbreaks associated with known emerging viruses, higher pathogenicity of a virus is often associated with lower transmissibility. compared to emerging viruses like ebola virus, avian h7n9, sars-cov, and mers-cov, sars-cov-2 has relatively lower pathogenicity and moderate transmissibility (15) . the risk of death among individuals infected with covid-19 was calculated using the infection fatality risk (ifr). the ifr was found to be in the range of 0.3% to 0.6%, which is comparable to that of a previous asian influenza pandemic (1957 to 1958) (73, 277) . notably, the reanalysis of the covid-19 pandemic curve from the initial cluster of cases pointed to considerable human-to-human transmission. it is opined that the exposure history of sars-cov-2 at the wuhan seafood market originated from humanto-human transmission rather than animal-to-human transmission (74) ; however, in light of the zoonotic spillover in covid-19, is too early to fully endorse this idea (1). following the initial infection, human-to-human transmission has been observed with a preliminary reproduction number (r 0 ) estimate of 1.4 to 2.5 (70, 75) , and recently it is estimated to be 2.24 to 3.58 (76) . in another study, the average reproductive number of covid-19 was found to be 3.28, which is significantly higher than the initial who estimate of 1.4 to 2.5 (77) . it is too early to obtain the exact r 0 value, since there is a possibility of bias due to insufficient data. the higher r 0 value is indicative of the more significant potential of sars-cov-2 transmission in a susceptible population. this is not the first time where the culinary practices of china have been blamed for the origin of novel coronavirus infection in humans. previously, the animals present in the liveanimal market were identified to be the intermediate hosts of the sars outbreak in china (78) . several wildlife species were found to harbor potentially evolving coronavirus strains that can overcome the species barrier (79) . one of the main principles of chinese food culture is that live-slaughtered animals are considered more nutritious (5) . after 4 months of struggle that lasted from december 2019 to march 2020, the covid-19 situation now seems under control in china. the wet animal markets have reopened, and people have started buying bats, dogs, cats, birds, scorpions, badgers, rabbits, pangolins (scaly anteaters), minks, soup from palm civet, ostriches, hamsters, snapping turtles, ducks, fish, siamese crocodiles, and other animal meats without any fear of covid-19. the chinese government is encouraging people to feel they can return to normalcy. however, this could be a risk, as it has been mentioned in advisories that people should avoid contact with live-dead animals as much as possible, as sars-cov-2 has shown zoonotic spillover. additionally, we cannot rule out the possibility of new mutations in the same virus being closely related to contact with both animals and humans at the market (284) . in january 2020, china imposed a temporary ban on the sale of live-dead animals in wet markets. however, now hundreds of such wet markets have been reopened without optimizing standard food safety and sanitation practices (286) . with china being the most populated country in the world and due to its domestic and international food exportation policies, the whole world is now facing the menace of covid-19, including china itself. wet markets of live-dead animals do not maintain strict food hygienic practices. fresh blood splashes are present everywhere, on the floor and tabletops, and such food customs could encourage many pathogens to adapt, mutate, and jump the species barrier. as a result, the whole world is suffering from novel sars-cov-2, with more than 4,170,424 cases and 287,399 deaths across the globe. there is an urgent need for a rational international campaign against the unhealthy food practices of china to encourage the sellers to increase hygienic food practices or close the crude live-dead animal wet markets. there is a need to modify food policies at national and international levels to avoid further life threats and clinical microbiology reviews economic consequences from any emerging or reemerging pandemic due to close animal-human interaction (285) . even though individuals of all ages and sexes are susceptible to covid-19, older people with an underlying chronic disease are more likely to become severely infected (80) . recently, individuals with asymptomatic infection were also found to act as a source of infection to susceptible individuals (81) . both the asymptomatic and symptomatic patients secrete similar viral loads, which indicates that the transmission capacity of asymptomatic or minimally symptomatic patients is very high. thus, sars-cov-2 transmission can happen early in the course of infection (82) . atypical clinical manifestations have also been reported in covid-19 in which the only reporting symptom was fatigue. such patients may lack respiratory signs, such as fever, cough, and sputum (83) . hence, the clinicians must be on the look-out for the possible occurrence of atypical clinical manifestations to avoid the possibility of missed diagnosis. the early transmission ability of sars-cov-2 was found to be similar to or slightly higher than that of sars-cov, reflecting that it could be controlled despite moderate to high transmissibility (84) . increasing reports of sars-cov-2 in sewage and wastewater warrants the need for further investigation due to the possibility of fecal-oral transmission. sars-cov-2 present in environmental compartments such as soil and water will finally end up in the wastewater and sewage sludge of treatment plants (328) . therefore, we have to reevaluate the current wastewater and sewage sludge treatment procedures and introduce advanced techniques that are specific and effective against sars-cov-2. since there is active shedding of sars-cov-2 in the stool, the prevalence of infections in a large population can be studied using wastewater-based epidemiology. recently, reverse transcription-quantitative pcr (rt-qpcr) was used to enumerate the copies of sars-cov-2 rna concentrated from wastewater collected from a wastewater treatment plant (327) . the calculated viral rna copy numbers determine the number of infected individuals. the increasing reports of virus shedding via the fecal route warrants the introduction of negative fecal viral nucleic acid test results as one of the additional discharge criteria in laboratory-confirmed cases of covid-19 (326) . the covid-19 pandemic does not have any novel factors, other than the genetically unique pathogen and a further possible reservoir. the cause and the likely future outcome are just repetitions of our previous interactions with fatal coronaviruses. the only difference is the time of occurrence and the genetic distinctness of the pathogen involved. mutations on the rbd of covs facilitated their capability of infecting newer hosts, thereby expanding their reach to all corners of the world (85) . this is a potential threat to the health of both animals and humans. advanced studies using bayesian phylogeographic reconstruction identified the most probable origin of sars-cov-2 as the bat sars-like coronavirus, circulating in the rhinolophus bat family (86) . phylogenetic analysis of 10 whole-genome sequences of sars-cov-2 showed that they are related to two covs of bat origin, namely, bat-sl-covzc45 and bat-sl-covzxc21, which were reported during 2018 in china (17) . it was reported that sars-cov-2 had been confirmed to use ace2 as an entry receptor while exhibiting an rbd similar to that of sars-cov (17, 87, 254, 255) . several countries have provided recommendations to their people traveling to china (88, 89) . compared to the previous coronavirus outbreaks caused by sars-cov and mers-cov, the efficiency of sars-cov-2 human-to-human transmission was thought to be less. this assumption was based on the finding that health workers were affected less than they were in previous outbreaks of fatal coronaviruses (2) . superspreading events are considered the main culprit for the extensive transmission of sars and mers (90, 91) . almost half of the mers-cov cases reported in saudi arabia are of secondary origin that occurred through contact with infected asymptomatic or symptomatic individuals through human-tohuman transmission (92) . the occurrence of superspreading events in the covid-19 outbreak cannot be ruled out until its possibility is evaluated. like sars and mers, covid-19 can also infect the lower respiratory tract, with milder symptoms (27) . the basic reproduction number of covid-19 has been found to be in the range of 2.8 to 3.3 based on real-time reports and 3.2 to 3.9 based on predicted infected cases (84) . coronavirus infection in humans is commonly associated with mild to severe respiratory diseases, with high fever, severe inflammation, cough, and internal organ dysfunction that can even lead to death (92) . most of the identified coronaviruses cause the common cold in humans. however, this changed when sars-cov was identified, paving the way for severe forms of the disease in humans (22) . our previous experience with the outbreaks of other coronaviruses, like sars and mers, suggests that the mode of transmission in covid-19 as mainly human-to-human transmission via direct contact, droplets, and fomites (25) . recent studies have demonstrated that the virus could remain viable for hours in aerosols and up to days on surfaces; thus, aerosol and fomite contamination could play potent roles in the transmission of sars-cov-2 (257) . the immune response against coronavirus is vital to control and get rid of the infection. however, maladjusted immune responses may contribute to the immunopathology of the disease, resulting in impairment of pulmonary gas exchange. understanding the interaction between covs and host innate immune systems could enlighten our understanding of the lung inflammation associated with this infection (24) . sars is a viral respiratory disease caused by a formerly unrecognized animal cov that originated from the wet markets in southern china after adapting to the human host, thereby enabling transmission between humans (90) . the sars outbreak reported in 2002 to 2003 had 8,098 confirmed cases with 774 total deaths (9.6%) (93) . the outbreak severely affected the asia pacific region, especially mainland china (94) . even though the case fatality rate (cfr) of sars-cov-2 (covid-19) is lower than that of sars-cov, there exists a severe concern linked to this outbreak due to its epidemiological similarity to influenza viruses (95, 279) . this can fail the public health system, resulting in a pandemic (96) . mers is another respiratory disease that was first reported in saudi arabia during the year 2012. the disease was found to have a cfr of around 35% (97) . the analysis of available data sets suggests that the incubation period of sars-cov-2, sars-cov, and mers-cov is in almost the same range. the longest predicted incubation time of sars-cov-2 is 14 days. hence, suspected individuals are isolated for 14 days to avoid the risk of further spread (98) . even though a high similarity has been reported between the genome sequence of the new coronavirus (sars-cov-2) and sars-like covs, the comparative analysis recognized a furin-like cleavage site in the sars-cov-2 s protein that is missing from other sars-like covs (99) . the furin-like cleavage site is expected to play a role in the life cycle of the virus and disease pathogenicity and might even act as a therapeutic target for furin inhibitors. the highly contagious nature of sars-cov-2 compared to that of its predecessors might be the result of a stabilizing mutation that occurred in the endosome-associated-protein-like domain of nsp2 protein. similarly, the destabilizing mutation near the phosphatase domain of nsp3 proteins in sars-cov-2 could indicate a potential mechanism that differentiates it from other covs (100) . even though the cfr reported for covid-19 is meager compared to those of the previous sars and mers outbreaks, it has caused more deaths than sars and mers combined (101) . possibly related to the viral pathogenesis is the recent finding of an 832-nucleotide (nt) deletion in orf8, which appears to reduce the replicative fitness of the virus and leads to attenuated phenotypes of sars-cov-2 (256) . coronavirus is the most prominent example of a virus that has crossed the species barrier twice from wild animals to humans during sars and mers outbreaks (79, 102) . the possibility of crossing the species barrier for the third time has also been suspected in the case of sars-cov-2 (covid19) . bats are recognized as a possible natural reservoir host of both sars-cov and mers-cov infection. in contrast, the possible intermediary host is the palm civet for sars-cov and the dromedary camel for mers-cov infection (102) . bats are considered the ancestral hosts for both sars and mers (103) . bats are also considered the reservoir host of human coronaviruses like clinical microbiology reviews hcov-229e and hcov-nl63 (104) . in the case of covid-19, there are two possibilities for primary transmission: it can be transmitted either through intermediate hosts, similar to that of sars and mers, or directly from bats (103) . the emergence paradigm put forward in the sars outbreak suggests that sars-cov originated from bats (reservoir host) and later jumped to civets (intermediate host) and incorporated changes within the receptor-binding domain (rbd) to improve binding to civet ace2. this civetadapted virus, during their subsequent exposure to humans at live markets, promoted further adaptations that resulted in the epidemic strain (104) . transmission can also occur directly from the reservoir host to humans without rbd adaptations. the bat coronavirus that is currently in circulation maintains specific "poised" spike proteins that facilitate human infection without the requirement of any mutations or adaptations (105) . altogether, different species of bats carry a massive number of coronaviruses around the world (106) . the high plasticity in receptor usage, along with the feasibility of adaptive mutation and recombination, may result in frequent interspecies transmission of coronavirus from bats to animals and humans (106) . the pathogenesis of most bat coronaviruses is unknown, as most of these viruses are not isolated and studied (4) . hedgehog coronavirus hku31, a betacoronavirus, has been identified from amur hedgehogs in china. studies show that hedgehogs are the reservoir of betacoronavirus, and there is evidence of recombination (107) . the current scientific evidence available on mers infection suggests that the significant reservoir host, as well as the animal source of mers infection in humans, is the dromedary camels (97) . the infected dromedary camels may not show any visible signs of infection, making it challenging to identify animals actively excreting mers-cov that has the potential to infect humans. however, they may shed mers-cov through milk, urine, feces, and nasal and eye discharge and can also be found in the raw organs (108) . in a study conducted to evaluate the susceptibility of animal species to mers-cov infection, llamas and pigs were found to be susceptible, indicating the possibility of mers-cov circulation in animal species other than dromedary camels (109) . following the outbreak of sars in china, sars-cov-like viruses were isolated from himalayan palm civets (paguma larvata) and raccoon dogs (nyctereutes procyonoides) found in a live-animal market in guangdong, china. the animal isolates obtained from the live-animal market retained a 29-nucleotide sequence that was not present in most of the human isolates (78) . these findings were critical in identifying the possibility of interspecies transmission in sars-cov. the higher diversity and prevalence of bat coronaviruses in this region compared to those in previous reports indicate a host/ pathogen coevolution. sars-like coronaviruses also have been found circulating in the chinese horseshoe bat (rhinolophus sinicus) populations. the in vitro and in vivo studies carried out on the isolated virus confirmed that there is a potential risk for the reemergence of sars-cov infection from the viruses that are currently circulating in the bat population (105) . the disease caused by sars-cov-2 is also named severe specific contagious pneumonia (sscp), wuhan pneumonia, and, recently, covid-19 (110) . compared to sars-cov, sars-cov-2 has less severe pathogenesis but has superior transmission capability, as evidenced by the rapidly increasing number of covid-19 cases (111) . the incubation period of sars-cov-2 in familial clusters was found to be 3 to 6 days (112) . the mean incubation period of covid-19 was found to be 6.4 days, ranging from 2.1 to 11.1 days (113) . among an early affected group of 425 patients, 59 years was the median age, of which more males were affected (114) . similar to sars and mers, the severity of this ncov is high in age groups above 50 years (2, 115) . symptoms of covid-19 include fever, cough, myalgia or fatigue, and, less commonly, headache, hemoptysis, and diarrhea (116, 282) . compared to the sars-cov-2-infected patients in wuhan during the initial stages of the outbreak, only mild symptoms were noticed in those patients that are infected by human-to-human transmission (14) . the initial trends suggested that the mortality associated with covid-19 was less than that of previous outbreaks of sars (101) . the updates obtained from countries like china, japan, thailand, and south korea indicated that the covid-19 patients had relatively mild manifestations compared to those with sars and mers (4). regardless of the coronavirus type, immune cells, like mast cells, that are present in the submucosa of the respiratory tract and nasal cavity are considered the primary barrier against this virus (92) . advanced in-depth analysis of the genome has identified 380 amino acid substitutions between the amino acid sequences of sars-cov-2 and the sars/sarslike coronaviruses. these differences in the amino acid sequences might have contributed to the difference in the pathogenic divergence of sars-cov-2 (16) . further research is required to evaluate the possible differences in tropism, pathogenesis, and transmission of this novel agent associated with this change in the amino acid sequence. with the current outbreak of covid-19, there is an expectancy of a significant increase in the number of published studies about this emerging coronavirus, as occurred with sars and mers (117) . sars-cov-2 invades the lung parenchyma, resulting in severe interstitial inflammation of the lungs. this is evident on computed tomography (ct) images as ground-glass opacity in the lungs. this lesion initially involves a single lobe but later expands to multiple lung lobes (118) . the histological assessment of lung biopsy samples obtained from covid-19-infected patients revealed diffuse alveolar damage, cellular fibromyxoid exudates, hyaline membrane formation, and desquamation of pneumocytes, indicative of acute respiratory distress syndrome (119) . it was also found that the sars-cov-2infected patients often have lymphocytopenia with or without leukocyte abnormalities. the degree of lymphocytopenia gives an idea about disease prognosis, as it is found to be positively correlated with disease severity (118) . pregnant women are considered to have a higher risk of getting infected by covid-19. the coronaviruses can cause adverse outcomes for the fetus, such as intrauterine growth restriction, spontaneous abortion, preterm delivery, and perinatal death. nevertheless, the possibility of intrauterine maternal-fetal transmission (vertical transmission) of covs is low and was not seen during either the sars-or mers-cov outbreak (120) . however, there has been concern regarding the impact of sars-cov-2/covid-19 on pregnancy. researchers have mentioned the probability of in utero transmission of novel sars-cov-2 from covid-19-infected mothers to their neonates in china based upon the rise in igm and igg antibody levels and cytokine values in the blood obtained from newborn infants immediately postbirth; however, rt-pcr failed to confirm the presence of sars-cov-2 genetic material in the infants (283) . recent studies show that at least in some cases, preterm delivery and its consequences are associated with the virus. nonetheless, some cases have raised doubts for the likelihood of vertical transmission (240) (241) (242) (243) . covid-19 infection was associated with pneumonia, and some developed acute respiratory distress syndrome (ards). the blood biochemistry indexes, such as albumin, lactate dehydrogenase, c-reactive protein, lymphocytes (percent), and neutrophils (percent) give an idea about the disease severity in covid-19 infection (121) . during covid-19, patients may present leukocytosis, leukopenia with lymphopenia (244), hypoalbuminemia, and an increase of lactate dehydrogenase, aspartate transaminase, alanine aminotransferase, bilirubin, and, especially, d-dimer (244) . middle-aged and elderly patients with primary chronic diseases, especially high blood pressure and diabetes, were found to be more susceptible to respiratory failure and, therefore, had poorer prognoses. providing respiratory support at early stages improved the disease prognosis and facilitated recovery (18) . the ards in covid-19 is due to the occurrence of cytokine storms that results in exaggerated immune response, immune regulatory network imbalance, and, finally, multiple-organ failure (122) . in addition to the exaggerated inflammatory response seen in patients with covid-19 pneumonia, the bile duct epithelial cell-derived hepatocytes upregulate ace2 expression in liver tissue by compensatory proliferation that might result in hepatic tissue injury (123) . coronavirus can cause disease in several species of domestic and wild animals, as well as humans (23) . the different animal species that are infected with cov include horses, camels, cattle, swine, dogs, cats, rodents, birds, ferrets, minks, bats, rabbits, snakes, and various other wild animals (20, 30, 79, 93, 124, 125, 287) . coronavirus infection is linked to different kinds of clinical manifestations, varying from enteritis in cows and pigs, upper respiratory disease in chickens, and fatal respiratory infections in humans (30) . among the cov genera, alphacoronavirus and betacoronavirus infect mammals, while gammacoronavirus and deltacoronavirus mainly infect birds, fishes, and, sometimes, mammals (27, 29, 106) . several novel coronaviruses that come under the genus deltacoronavirus have been discovered in the past from birds, like wigeon coronavirus hku20, bulbul coronavirus hku11, munia coronavirus hku13, white-eye coronavirus hku16, night-heron coronavirus hku19, and common moorhen coronavirus hku21, as well as from pigs (porcine coronavirus hku15) (6, 29) . transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), and porcine hemagglutinating encephalomyelitis virus (phev) are some of the coronaviruses of swine. among them, tgev and pedv are responsible for causing severe gastroenteritis in young piglets with noteworthy morbidity and mortality. infection with phev also causes enteric infection but can cause encephalitis due to its ability to infect the nervous system (30) . bovine coronaviruses (bocovs) are known to infect several domestic and wild ruminants (126) . bocov inflicts neonatal calf diarrhea in adult cattle, leading to bloody diarrhea (winter dysentery) and respiratory disease complex (shipping fever) in cattle of all age groups (126) . bocov-like viruses have been noted in humans, suggesting its zoonotic potential as well (127) . feline enteric and feline infectious peritonitis (fip) viruses are the two major feline covs (128) , where feline covs can affect the gastrointestinal tract, abdominal cavity (peritonitis), respiratory tract, and central nervous system (128) . canines are also affected by covs that fall under different genera, namely, canine enteric coronavirus in alphacoronavirus and canine respiratory coronavirus in betacoronavirus, affecting the enteric and respiratory tract, respectively (129, 130) . ibv, under gammacoronavirus, causes diseases of respiratory, urinary, and reproductive systems, with substantial economic losses in chickens (131, 132) . in small laboratory animals, mouse hepatitis virus, rat sialodacryoadenitis coronavirus, and guinea pig and rabbit coronaviruses are the major covs associated with disease manifestations like enteritis, hepatitis, and respiratory infections (10, 133) . swine acute diarrhea syndrome coronavirus (sads-cov) was first identified in suckling piglets having severe enteritis and belongs to the genus alphacoronavirus (106) . the outbreak was associated with considerable scale mortality of piglets (24,693 deaths) across four farms in china (134) . the virus isolated from the piglets was almost identical to and had 95% genomic similarity with horseshoe bat (rhinolophus species) coronavirus hku2, suggesting a bat origin of the pig virus (106, 134, 135) . it is also imperative to note that the sads-cov outbreak started in guangdong province, near the location of the sars pandemic origin (134) . before this outbreak, pigs were not known to be infected with bat-origin coronaviruses. this indicates that the bat-origin coronavirus jumped to pig by breaking the species barrier. the next step of this jump might not end well, since pigs are considered the mixing vessel for influenza a viruses due to their ability to be infected by both human and avian influenza a viruses (136) . similarly, they may act as the mixing vessel for coronaviruses, since they are in frequent contact with both humans and multiple wildlife species. additionally, pigs are also found to be susceptible to infection with human sars-cov and mers-cov, making this scenario a nightmare (109, 137) . it is only a matter of time before another zoonotic coronavirus results in an epidemic by jumping the so-called species barrier (287) . the host spectrum of coronavirus increased when a novel coronavirus, namely, sw1, was recognized in the liver tissue of a captive beluga whale (delphinapterus leucas) (138) . in recent decades, several novel coronaviruses were identified from different animal species. bats can harbor these viruses without manifesting any clinical disease but are persistently infected (30) . they are the only mammals with the capacity for self-powered flight, which enables them to migrate long distances, unlike land mammals. bats are distributed worldwide and also account for about a fifth of all mammalian species (6) . this makes them the ideal reservoir host for many viral agents and also the source of novel coronaviruses that have yet to be identified. it has become a necessity to study the diversity of coronavirus in the bat population to prevent future outbreaks that could jeopardize livestock and public health. the repeated outbreaks caused by bat-origin coronaviruses calls for the development of efficient molecular surveillance strategies for studying betacoronavirus among animals (12) , especially in the rhinolophus bat family (86) . chinese bats have high commercial value, since they are used in traditional chinese medicine (tcm). therefore, the handling of bats for trading purposes poses a considerable risk of transmitting zoonotic cov epidemics (139) . due to the possible role played by farm and wild animals in sars-cov-2 infection, the who, in their novel coronavirus (covid-19) situation report, recommended the avoidance of unprotected contact with both farm and wild animals (25) . the live-animal markets, like the one in guangdong, china, provides a setting for animal coronaviruses to amplify and to be transmitted to new hosts, like humans (78) . such markets can be considered a critical place for the origin of novel zoonotic diseases and have enormous public health significance in the event of an outbreak. bats are the reservoirs for several viruses; hence, the role of bats in the present outbreak cannot be ruled out (140) . in a qualitative study conducted for evaluating the zoonotic risk factors among rural communities of southern china, the frequent human-animal interactions along with the low levels of environmental biosecurity were identified as significant risks for the emergence of zoonotic disease in local communities (141, 142) . the comprehensive sequence analysis of the sars-cov-2 rna genome identified that the cov from wuhan is a recombinant virus of the bat coronavirus and another coronavirus of unknown origin. the recombination was found to have happened within the viral spike glycoprotein, which recognizes the cell surface receptor. further analysis of the genome based on codon usage identified the snake as the most probable animal reservoir of sars-cov-2 (143) . contrary to these findings, another genome analysis proposed that the genome of sars-cov-2 is 96% identical to bat coronavirus, reflecting its origin from bats (63) . the involvement of bat-derived materials in causing the current outbreak cannot be ruled out. high risk is involved in the production of bat-derived materials for tcm practices involving the handling of wild bats. the use of bats for tcm practices will remain a severe risk for the occurrence of zoonotic coronavirus epidemics in the future (139) . furthermore, the pangolins are an endangered species of animals that harbor a wide variety of viruses, including coronaviruses (144) . the coronavirus isolated from malayan pangolins (manis javanica) showed a very high amino acid identity with covid-19 at e (100%), m (98.2%), n (96.7%), and s genes (90.4%). the rbd of s protein in cov isolated from pangolin was almost identical (one amino acid difference) to that of sars-cov-2. a comparison of the genomes suggests recombination between pangolin-cov-like viruses with the bat-cov-ratg13-like virus. all this suggests the potential of pangolins to act as the intermediate host of sars-cov-2 (145) . human-wildlife interactions, which are increasing in the context of climate change (142) , are further considered high risk and responsible for the emergence of sars-cov. covid-19 is also suspected of having a similar mode of origin. hence, to prevent the occurrence of another zoonotic spillover (1), exhaustive coordinated efforts are needed to identify the high-risk pathogens harbored by wild animal populations, conducting surveillance among the people who are susceptible to zoonotic spillover events (12) , and to improve the biosecurity measures associated with the wildlife trade (146) . the serological surveillance studies conducted in people living in proximity to bat caves had earlier identified the serological confirmation of sars-related covs in humans. people clinical microbiology reviews living at the wildlife-human interface, mainly in rural china, are regularly exposed to sars-related covs (147) . these findings will not have any significance until a significant outbreak occurs due to a virus-like sars-cov-2. there is a steady increase in the reports of covid-19 in companion and wild animals around the world. further studies are required to evaluate the potential of animals (especially companion animals) to serve as an efficient reservoir host that can further alter the dynamics of human-to-human transmission (330) . to date, two pet dogs (hong kong) and four pet cats (one each from belgium and hong kong, two from the united states) have tested positive for sars-cov-2 (335) . the world organization for animal health (oie) has confirmed the diagnosis of covid-19 in both dogs and cats due to human-to-animal transmission (331) . the similarity observed in the gene sequence of sars-cov-2 from an infected pet owner and his dog further confirms the occurrence of human-to-animal transmission (333) . even though asymptomatic, feline species should be considered a potential transmission route from animals to humans (326) . however, currently, there are no reports of sars-cov-2 transmission from felines to human beings. based on the current evidence, we can conclude that cats are susceptible to sars-cov-2 and can get infected by human beings. however, evidence of cat-to-human transmission is lacking and requires further studies (332) . rather than waiting for firmer evidence on animal-to-human transmission, necessary preventive measures are advised, as well as following social distancing practices among companion animals of different households (331) . one of the leading veterinary diagnostic companies, idexx, has conducted large-scale testing for covid-19 in specimens collected from dogs and cats. however, none of the tests turned out to be positive (334) . in a study conducted to investigate the potential of different animal species to act as the intermediate host of sars-cov-2, it was found that both ferrets and cats can be infected via experimental inoculation of the virus. in addition, infected cats efficiently transmitted the disease to naive cats (329) . sars-cov-2 infection and subsequent transmission in ferrets were found to recapitulate the clinical aspects of covid-19 in humans. the infected ferrets also shed virus via multiple routes, such as saliva, nasal washes, feces, and urine, postinfection, making them an ideal animal model for studying disease transmission (337) . experimental inoculation was also done in other animal species and found that the dogs have low susceptibility, while the chickens, ducks, and pigs are not at all susceptible to sars-cov-2 (329) . similarly, the national veterinary services laboratories of the usda have reported covid-19 in tigers and lions that exhibited respiratory signs like dry cough and wheezing. the zoo animals are suspected to have been infected by an asymptomatic zookeeper (335) . the total number of covid-19-positive cases in human beings is increasing at a high rate, thereby creating ideal conditions for viral spillover to other species, such as pigs. the evidence obtained from sars-cov suggests that pigs can get infected with sars-cov-2 (336). however, experimental inoculation with sars-cov-2 failed to infect pigs (329) . further studies are required to identify the possible animal reservoirs of sars-cov-2 and the seasonal variation in the circulation of these viruses in the animal population. research collaboration between human and animal health sectors is becoming a necessity to evaluate and identify the possible risk factors of transmission between animals and humans. such cooperation will help to devise efficient strategies for the management of emerging zoonotic diseases (12) . rna tests can confirm the diagnosis of sars-cov-2 (covid-19) cases with real-time rt-pcr or next-generation sequencing (148, 149, 245, 246) . at present, nucleic acid detection techniques, like rt-pcr, are considered an effective method for confirming the diagnosis in clinical cases of covid-19 (148) . several companies across the world are currently focusing on developing and marketing sars-cov-2-specific nucleic acid detection kits. multiple laboratories are also developing their own in-house rt-pcr. one of them is the sars-cov-2 nucleic acid detection kit produced by shuoshi biotechnology (double fluorescence pcr method) (150) . up to 30 march 2020, the u.s. food and drug administration (fda) had granted 22 in vitro diagnostics emergency use authorizations (euas), including for the rt-pcr diagnostic panel for the universal detection of sars-like betacoronaviruses and specific detection of sars-cov-2, developed by the u.s. cdc (table 1) (258, 259) . recently, 95 full-length genomic sequences of saras-cov-2 strains available in the national center for biotechnology information and gisaid databases were subjected to multiple-sequence alignment and phylogenetic analyses for studying variations in the viral genome (260) . all the viral strains revealed high homology of 99.99% (99.91% to 100%) at the nucleotide level and 99.99% (99.79% to 100%) at the amino acid level. overall variation was found to be low in orf regions, with 13 variation sites recognized in 1a, 1b, s, 3a, m, 8, and n regions. mutation rates of 30.53% (29/95) and 29.47% (28/95) were observed at nt 28144 (orf8) and nt 8782 (orf1a) positions, respectively. owing to such selective mutations, a few specific regions of sars-cov-2 should not be considered for designing primers and probes. the sars-cov-2 reference sequence could pave the way to study molecular biology and pathobiology, along with developing diagnostics and appropriate prevention and control strategies for countering sars-cov-2 (260) . nucleic acids of sars-cov-2 can be detected from samples (64) such as bronchoalveolar lavage fluid, sputum, nasal swabs, fiber bronchoscope brush biopsy specimen, pharyngeal swabs, feces, blood, and urine, with different levels of diagnostic performance (table 2) (80, 245, 246) . the viral loads of sars-cov-2 were measured using n-gene-specific quantitative rt-pcr in throat swab and sputum samples collected from covid-19-infected individuals. the results indicated that the viral load peaked at around 5 to 6 days following the onset of symptoms, and it ranged from 10 4 to 10 7 copies/ml during this time (151) . in another study, the viral load was found to be higher in the nasal swabs than the throat swabs obtained from covid-19 symptomatic patients (82) . although initially it was thought that viral load would be associated with poor outcomes, some case reports have shown asymptomatic individuals with high viral loads (247) . recently, the viral load in nasal and throat swabs of 17 symptomatic patients was determined, and higher viral loads were recorded soon after the onset of symptoms, particularly in the nose compared to the throat. the pattern of viral nucleic the results of the studies related to sars-cov-2 viral loads reflect active replication of this virus in the upper respiratory tract and prolonged viral shedding after symptoms disappear, including via stool. thus, the current case definition needs to be updated along with a reassessment of the strategies to be adopted for restraining the sars-cov-2 outbreak spread (248) . in some cases, the viral load studies of sars-cov-2 have also been useful to recommend precautionary measures when handling specific samples, e.g., feces. in a recent survey from 17 confirmed cases of sars-cov-2 infection with available data (representing days 0 to 13 after onset), stool samples from nine cases (53%; days 0 to 11 after onset) were positive on rt-pcr analysis. although the viral loads were lower than those of respiratory samples (range, 550 copies per ml to 1.21 ϫ 10 5 copies per ml), this has essential biosafety implications (151) . the samples from 18 sars-cov-2-positive patients in singapore who had traveled from wuhan to singapore showed the presence of viral rna in stool and whole blood but not in urine by real-time rt-pcr (288) . further, novel sars-cov-2 infections have been detected in a variety of clinical specimens, like bronchoalveolar lavage fluid, sputum, nasal swabs, fibrobronchoscope brush biopsy specimens, pharyngeal swabs, feces, and blood (246) . the presence of sars-cov-2 in fecal samples has posed grave public health concerns. in addition to the direct transmission mainly occurring via droplets of sneezing and coughing, other routes, such as fecal excretion and environmental and fomite contamination, are contributing to sars-cov-2 transmission and spread (249) (250) (251) (252) . fecal excretion has also been documented for sars-cov and mers-cov, along with the potential to stay viable in situations aiding fecal-oral transmission. thus, sars-cov-2 has every possibility to be transmitted through this mode. fecal-oral transmission of sars-cov-2, particularly in regions having low standards of hygiene and poor sanitation, may have grave consequences with regard to the high spread of this virus. ethanol and disinfectants containing chlorine or bleach are effective against coronaviruses (249) (250) (251) (252) . appropriate precautions need to be followed strictly while handling the stools of patients infected with sars-cov-2. biowaste materials and sewage from hospitals must be adequately disinfected, treated, and disposed of properly. the significance of frequent and good hand hygiene and sanitation practices needs to be given due emphasis (249) (250) (251) (252) . future explorative research needs to be conducted with regard to the fecal-oral transmission of sars-cov-2, along with focusing on environmental investigations to find out if this virus could stay viable in situations and atmospheres facilitating such potent routes of transmission. the correlation of fecal concentrations of viral rna with disease severity needs to be determined, along with assessing the gastrointestinal symptoms and the possibility of fecal sars-cov-2 rna detection during the covid-19 incubation period or convalescence phases of the disease (249) (250) (251) (252) . the lower respiratory tract sampling techniques, like bronchoalveolar lavage fluid aspirate, are considered the ideal clinical materials, rather than the throat swab, due to their higher positive rate on the nucleic acid test (148) . the diagnosis of covid-19 can be made by using upper-respiratory-tract specimens collected using nasopharyngeal and oropharyngeal swabs. however, these techniques are associated with unnecessary risks to health care workers due to close contact with patients (152) . similarly, a single patient with a high viral load was reported to contaminate an entire endoscopy room by shedding the virus, which may remain viable for at least 3 days and is considered a great risk for uninfected patients and health care workers (289) . recently, it was found that the anal swabs gave more positive results than oral swabs in the later stages of infection (153) . hence, clinicians have to be cautious while discharging any covid-19infected patient based on negative oral swab test results due to the possibility of fecal-oral transmission. even though the viral loads in stool samples were found to be less than those of respiratory samples, strict precautionary measures have to be followed while handling stool samples of covid-19 suspected or infected patients (151) . children infected with sars-cov-2 experience only a mild form of illness and recover immediately after treatment. it was recently found that stool samples of sars-cov-2-infected children that gave negative throat swab results were positive within ten days of negative results. this could result in the fecal-oral transmission of sars-cov-2 infections, especially in children (290) . hence, to prevent the fecal-oral transmission of sars-cov-2, infected covid-19 patients should only be considered negative when they test negative for sars-cov-2 in the stool sample. a suspected case of covid-19 infection is said to be confirmed if the respiratory tract aspirate or blood samples test positive for sars-cov-2 nucleic acid using rt-pcr or by the identification of sars-cov-2 genetic sequence in respiratory tract aspirate or blood samples (80) . the patient will be confirmed as cured when two subsequent oral swab results are negative (153) . recently, the live virus was detected in the selfcollected saliva of patients infected with covid-19. these findings were confirmative of using saliva as a noninvasive specimen for the diagnosis of covid-19 infection in suspected individuals (152) . it has also been observed that the initial screening of covid-19 patients infected with rt-pcr may give negative results even if they have chest ct findings that are suggestive of infection. hence, for the accurate diagnosis of covid-19, a combination of repeated swab tests using rt-pcr and ct scanning is required to prevent the possibility of false-negative results during disease screening (154) . rt-pcr is the most widely used test for diagnosing covid-19. however, it has some significant limitations from the clinical perspective, since it will not give any clarity regarding disease progression. droplet digital pcr (ddpcr) can be used for the quantification of viral load in the samples obtained from lower respiratory tracts. hence, based on the viral load, we can quickly evaluate the progression of infection (291) . in addition to all of the above findings, sequencing and phylogenetics are critical in the correct identification and confirmation of the causative viral agent and useful to establish relationships with previous isolates and sequences, as well as to know, especially during an epidemic, the nucleotide and amino acid mutations and the molecular divergence. the rapid development and implementation of diagnostic tests against emerging novel diseases like covid-19 pose significant challenges due to the lack of resources and logistical limitations associated with an outbreak (155) . sars-cov-2 infection can also be confirmed by isolation and culturing. the human airway epithelial cell culture was found to be useful in isolating sars-cov-2 (3). the efficient control of an outbreak depends on the rapid diagnosis of the disease. recently, in response to the covid-19 outbreak, 1-step quantitative realtime reverse transcription-pcr assays were developed that detect the orf1b and n regions of the sars-cov-2 genome (156) . that assay was found to achieve the rapid detection of sars-cov-2. nucleic acid-based assays offer high accuracy in the diagnosis of sars-cov-2, but the current rate of spread limits its use due to the lack of diagnostic assay kits. this will further result in the extensive transmission of covid-19, since only a portion of suspected cases can be diagnosed. in such situations, conventional serological assays, like enzyme-linked immunosorbent assay (elisa), that are specific to covid-19 igm and igg antibodies can be used as a high-throughput alternative (149) . at present, there is no diagnostic kit available for detecting the sars-cov-2 antibody (150) . the specific antibody profiles of covid-19 patients were analyzed, and it was found that the igm level lasted more than 1 month, indicating a prolonged stage of virus replication in sars-cov-2-infected patients. the igg levels were found to increase only in the later stages of the disease. these findings indicate that the specific antibody profiles of sars-cov-2 and sars-cov were similar (325) . these findings can be utilized for the development of specific diagnostic tests against covid-19 and can be used for rapid screening. even though diagnostic test kits are already available that can detect the genetic sequences of sars-cov-2 (95), their availability is a concern, as the number of covid-19 cases is skyrocketing (155, 157) . a major problem associated with this diagnostic kit is that it works only when the test subject has an active infection, limiting its use to the earlier stages of infection. several laboratories around the world are currently developing antibody-based diagnostic tests against sars-cov-2 (157). chest ct is an ideal diagnostic tool for identifying viral pneumonia. the sensitivity of chest ct is far superior to that of x-ray screening. the chest ct findings associated with covid-19-infected patients include characteristic patchy infiltration that later progresses to ground-glass opacities (158) . early manifestations of covid-19 pneumonia might not be evident in x-ray chest radiography. in such situations, a chest ct examination can be performed, as it is considered highly specific for covid-19 pneumonia (118) . those patients having covid-19 pneumonia will exhibit the typical ground-glass opacity in their chest ct images (154) . the patients infected with covid-19 had elevated plasma angiotensin 2 levels. the level of angiotensin 2 was found to be linearly associated with viral load and lung injury, indicating its potential as a diagnostic biomarker (121) . the chest ct imaging abnormalities associated with covid-19 pneumonia have also been observed even in asymptomatic patients. these abnormalities progress from the initial focal unilateral to diffuse bilateral ground-glass opacities and will further progress to or coexist with lung consolidation changes within 1 to 3 weeks (159). the role played by radiologists in the current scenario is very important. radiologists can help in the early diagnosis of lung abnormalities associated with covid-19 pneumonia. they can also help in the evaluation of disease severity, identifying its progression to acute respiratory distress syndrome and the presence of secondary bacterial infections (160) . even though chest ct is considered an essential diagnostic tool for covid-19, the extensive use of ct for screening purposes in the suspected individuals might be associated with a disproportionate risk-benefit ratio due to increased radiation exposure as well as increased risk of cross-infection. hence, the use of ct for early diagnosis of sars-cov-2 infection in high-risk groups should be done with great caution (292) . more recently, other advanced diagnostics have been designed and developed for the detection of sars-cov-2 (345, 347, (350) (351) (352) . a reverse transcriptional loopmediated isothermal amplification (rt-lamp), namely, ilaco, has been developed for rapid and colorimetric detection of this virus (354) . rt-lamp serves as a simple, rapid, and sensitive diagnostic method that does not require sophisticated equipment or skilled personnel (349) . an interactive web-based dashboard for tracking sars-cov-2 in a real-time mode has been designed (238) . a smartphone-integrated home-based point-of-care testing (poct) tool, a paper-based poct combined with lamp, is a useful point-of-care diagnostic (353) . an abbott id now covid-19 molecular poct-based test, using isothermal nucleic acid amplification technology, has been designed as a pointof-care test for very rapid detection of sars-cov-2 in just 5 min (344) . a crispr-based sherlock (specific high-sensitivity enzymatic reporter unlocking) diagnostic for rapid detection of sars-cov-2 without the requirement of specialized instrumentation has been reported to be very useful in the clinical diagnosis of covid-19 (360) . a crispr-cas12-based lateral flow assay also has been developed for rapid detection of sars-cov-2 (346) . artificial intelligence, by means of a three-dimensional deep-learning model, has been developed for sensitive and specific diagnosis of covid-19 via ct images (332) . tracking and mapping of the rising incidence rates, disease outbreaks, community spread, clustered transmission events, hot spots, and superspreader potential of sars-cov-2/covid warrant full exploitation of real-time disease mapping by employing geographical information systems (gis), such as the gis software kosmo 3.1, web-based real-time tools and dashboards, apps, and advances in information technology (356-359). researchers have also developed a few prediction tools/models, such as the prediction model risk of bias assessment tool (probast) and critical appraisal and data extraction for systematic reviews of prediction modeling studies (charms), which could aid in assessing the possibility of getting infection and estimating the prognosis in patients; however, such models may suffer from bias issues and, hence, cannot be considered completely trustworthy, which necessitates the development of new and reliable predictors (360) . recently emerged viruses, such as zika, ebola, and nipah viruses, and their grave threats to humans have begun a race in exploring the designing and developing of advanced vaccines, prophylactics, therapeutics, and drug regimens to counter emerging viruses (161) (162) (163) 280) . several attempts are being made to design and develop vaccines for cov infection, mostly by targeting the spike glycoprotein. nevertheless, owing to extensive diversity in antigenic variants, cross-protection rendered by the vaccines is significantly limited, even within the strains of a phylogenetic subcluster (104) . due to the lack of effective antiviral therapy and vaccines in the present scenario, we need to depend solely on implementing effective infection control measures to lessen the risk of possible nosocomial transmission (68) . recently, the receptor for sars-cov-2 was established as the human angiotensin-converting enzyme 2 (hace2), and the virus was found to enter the host cell mainly through endocytosis. it was also found that the major components that have a critical role in viral entry include pikfyve, tpc2, and cathepsin l. these findings are critical, since the components described above might act as candidates for vaccines or therapeutic drugs against sars-cov-2 (293) . the majority of the treatment options and strategies that are being evaluated for sars-cov-2 (covid-19) have been taken from our previous experiences in treating sars-cov, mers-cov, and other emerging viral diseases. several therapeutic and preventive strategies, including vaccines, immunotherapeutics, and antiviral drugs, have been exploited against the previous cov outbreaks (sars-cov and mers-cov) (8, 104, (164) (165) (166) (167) . these valuable options have already been evaluated for their potency, efficacy, and safety, along with several other types of current research that will fuel our search for ideal therapeutic agents against covid-19 (7, 9, 19, 21, 36) . the primary cause of the unavailability of approved and commercial vaccines, drugs, and therapeutics to counter the earlier sars-cov and mers-cov seems to owe to the lesser attention of the biomedicine and pharmaceutical companies, as these two covs did not cause much havoc, global threat, and panic like those posed by the sars-cov-2 pandemic (19) . moreover, for such outbreak situations, the requirement for vaccines and therapeutics/drugs exists only for a limited period, until the outbreak is controlled. the proportion of the human population infected with sars-cov and mers-cov was also much lower across the globe, failing to attract drug and vaccine manufacturers and clinical microbiology reviews producers. therefore, by the time an effective drug or vaccine is designed against such disease outbreaks, the virus would have been controlled by adopting appropriate and strict prevention and control measures, and patients for clinical trials will not be available. the newly developed drugs cannot be marketed due to the lack of end users. the s protein plays a significant role in the induction of protective immunity against sars-cov by mediating t-cell responses and neutralizing antibody production (168) . in the past few decades, we have seen several attempts to develop a vaccine against human coronaviruses by using s protein as the target (168, 169) . however, the developed vaccines have minimal application, even among closely related strains of the virus, due to a lack of cross-protection. that is mainly because of the extensive diversity existing among the different antigenic variants of the virus (104) . the contributions of the structural proteins, like spike (s), matrix (m), small envelope (e), and nucleocapsid (n) proteins, of sars-cov to induce protective immunity has been evaluated by expressing them in a recombinant parainfluenza virus type 3 vector (bhpiv3). of note, the result was conclusive that the expression of m, e, or n proteins without the presence of s protein would not confer any noticeable protection, with the absence of detectable serum sars-cov-neutralizing antibodies (170) . antigenic determinant sites present over s and n structural proteins of sars-cov-2 can be explored as suitable vaccine candidates (294) . in the asian population, s, e, m, and n proteins of sars-cov-2 are being targeted for developing subunit vaccines against covid-19 (295) . the identification of the immunodominant region among the subunits and domains of s protein is critical for developing an effective vaccine against the coronavirus. the c-terminal domain of the s1 subunit is considered the immunodominant region of the porcine deltacoronavirus s protein (171) . similarly, further investigations are needed to determine the immunodominant regions of sars-cov-2 for facilitating vaccine development. however, our previous attempts to develop a universal vaccine that is effective for both sars-cov and mers-cov based on t-cell epitope similarity pointed out the possibility of cross-reactivity among coronaviruses (172) . that can be made possible by selected potential vaccine targets that are common to both viruses. sars-cov-2 has been reported to be closely related to sars-cov (173, 174) . hence, knowledge and understanding of s protein-based vaccine development in sars-cov will help to identify potential s protein vaccine candidates in sars-cov-2. therefore, vaccine strategies based on the whole s protein, s protein subunits, or specific potential epitopes of s protein appear to be the most promising vaccine candidates against coronaviruses. the rbd of the s1 subunit of s protein has a superior capacity to induce neutralizing antibodies. this property of the rbd can be utilized for designing potential sars-cov vaccines either by using rbd-containing recombinant proteins or recombinant vectors that encode rbd (175) . hence, the superior genetic similarity existing between sars-cov-2 and sars-cov can be utilized to repurpose vaccines that have proven in vitro efficacy against sars-cov to be utilized for sars-cov-2. the possibility of cross-protection in covid-19 was evaluated by comparing the s protein sequences of sars-cov-2 with that of sars-cov. the comparative analysis confirmed that the variable residues were found concentrated on the s1 subunit of s protein, an important vaccine target of the virus (150) . hence, the possibility of sars-cov-specific neutralizing antibodies providing cross-protection to covid-19 might be lower. further genetic analysis is required between sars-cov-2 and different strains of sars-cov and sarslike (sl) covs to evaluate the possibility of repurposed vaccines against covid-19. this strategy will be helpful in the scenario of an outbreak, since much time can be saved, because preliminary evaluation, including in vitro studies, already would be completed for such vaccine candidates. multiepitope subunit vaccines can be considered a promising preventive strategy against the ongoing covid-19 pandemic. in silico and advanced immunoinformatic tools can be used to develop multiepitope subunit vaccines. the vaccines that are engineered by this technique can be further evaluated using docking studies and, if found effective, then can be further evaluated in animal models (365) . identifying epitopes that have the potential to become a vaccine candidate is critical to developing an effective vaccine against covid-19. the immunoinformatics approach has been used for recognizing essential epitopes of cytotoxic t lymphocytes and b cells from the surface glycoprotein of sars-cov-2. recently, a few epitopes have been recognized from the sars-cov-2 surface glycoprotein. the selected epitopes explored targeting molecular dynamic simulations, evaluating their interaction with corresponding major histocompatibility complex class i molecules. they potentially induce immune responses (176) . the recombinant vaccine can be designed by using rabies virus (rv) as a viral vector. rv can be made to express mers-cov s1 protein on its surface so that an immune response is induced against mers-cov. the rv vector-based vaccines against mers-cov can induce faster antibody response as well as higher degrees of cellular immunity than the gram-positive enhancer matrix (gem) particle vector-based vaccine. however, the latter can induce a very high antibody response at lower doses (167) . hence, the degree of humoral and cellular immune responses produced by such vaccines depends upon the vector used. dual vaccines have been getting more popular recently. among them, the rabies virus-based vectored vaccine platform is used to develop vaccines against emerging infectious diseases. the dual vaccine developed from inactivated rabies virus particles that express the mers-cov s1 domain of s protein was found to induce immune responses for both mers-cov and rabies virus. the vaccinated mice were found to be completely protected from challenge with mers-cov (169) . the intranasal administration of the recombinant adenovirus-based vaccine in balb/c mice was found to induce long-lasting neutralizing immunity against mers spike pseudotyped virus, characterized by the induction of systemic igg, secretory iga, and lung-resident memory t-cell responses (177) . immunoinformatics methods have been employed for the genomewide screening of potential vaccine targets among the different immunogens of mers-cov (178) . the n protein and the potential b-cell epitopes of mers-cov e protein have been suggested as immunoprotective targets inducing both t-cell and neutralizing antibody responses (178, 179) . the collaborative effort of the researchers of rocky mountain laboratories and oxford university is designing a chimpanzee adenovirus-vectored vaccine to counter covid-19 (180) . the coalition for epidemic preparedness innovations (cepi) has initiated three programs to design sars-cov-2 vaccines (181) . cepi has a collaborative project with inovio for designing a mers-cov dna vaccine that could potentiate effective immunity. cepi and the university of queensland are designing a molecular clamp vaccine platform for mers-cov and other pathogens, which could assist in the easier identification of antigens by the immune system (181) . cepi has also funded moderna to develop a vaccine for covid-19 in partnership with the vaccine research center (vrc) of the national institute of allergy and infectious diseases (niaid), part of the national institutes of health (nih) (182) . by employing mrna vaccine platform technology, a vaccine candidate expressing sars-cov-2 spike protein is likely to go through clinical testing in the coming months (180 (298) . the process of vaccine development usually takes approximately ten years, in the case of inactivated or live attenuated vaccines, since it involves the generation of long-term efficacy data. however, this was brought down to 5 years during the ebola emergency for viral vector vaccines. in the urgency associated with the covid-19 outbreaks, we expect a vaccine by the end of this year (343) . the development of an effective vaccine against covid-19 with high speed and precision is the combined result of advancements in computational biology, gene synthesis, protein engineering, and the invention of advanced manufacturing platforms (342) . the recurring nature of the coronavirus outbreaks calls for the development of a pan-coronavirus vaccine that can produce cross-reactive antibodies. however, the success of such a vaccine relies greatly on its ability to provide protection not only against present versions of the virus but also the ones that are likely to emerge in the future. this can be achieved by identifying antibodies that can recognize relatively conserved epitopes that are maintained as such even after the occurrence of considerable variations (362) . even though several vaccine clinical trials are being conducted around the world, pregnant women have been completely excluded from these studies. pregnant women are highly vulnerable to emerging diseases such as covid-19 due to alterations in the immune system and other physiological systems that are associated with pregnancy. therefore, in the event of successful vaccine development, pregnant women will not get access to the vaccines (361) . hence, it is recommended that pregnant women be included in the ongoing vaccine trials, since successful vaccination in pregnancy will protect the mother, fetus, and newborn. the heterologous immune effects induced by bacillus calmette guérin (bcg) vaccination is a promising strategy for controlling the covid-19 pandemic and requires further investigations. bcg is a widely used vaccine against tuberculosis in high-risk regions. it is derived from a live attenuated strain of mycobacterium bovis. at present, three new clinical trials have been registered to evaluate the protective role of bcg vaccination against sars-cov-2 (363) . recently, a cohort study was conducted to evaluate the impact of childhood bcg vaccination in covid-19 pcr positivity rates. however, childhood bcg vaccination was found to be associated with a rate of covid-19-positive test results similar to that of the nonvaccinated group (364) . further studies are required to analyze whether bcg vaccination in childhood can induce protective effects against covid-19 in adulthood. population genetic studies conducted on 103 genomes identified that the sars-cov-2 virus has evolved into two major types, l and s. among the two types, l type is expected to be the most prevalent (ϳ70%), followed by the s type (ϳ30%) (366) . this finding has a significant impact on our race to develop an ideal vaccine, since the vaccine candidate has to target both strains to be considered effective. at present, the genetic differences between the l and s types are very small and may not affect the immune response. however, we can expect further genetic variations in the coming days that could lead to the emergence of new strains (367) . there is no currently licensed specific antiviral treatment for mers-and sars-cov infections, and the main focus in clinical settings remains on lessening clinical signs and providing supportive care (183) (184) (185) (186) . effective drugs to manage covid-19 patients include remdesivir, lopinavir/ritonavir alone or in a blend with interferon beta, convalescent plasma, and monoclonal antibodies (mabs); however, efficacy and safety issues of these drugs require additional clinical trials (187, 281) . a controlled trial of ritonavirboosted lopinavir and interferon alpha 2b treatment was performed on covid-19 hospitalized patients (chictr2000029308) (188) . in addition, the use of hydroxychloroquine and tocilizumab for their potential role in modulating inflammatory responses in the lungs and antiviral effect has been proposed and discussed in many research articles. still, no fool-proof clinical trials have been published (194, 196, 197, (261) (262) (263) (264) (265) (266) (267) (268) (269) (270) (271) (272) . recently, a clinical trial conducted on adult patients suffering from severe covid-19 revealed no benefit of lopinavir-ritonavir treatment over standard care (273) . the efforts to control sars-cov-2 infection utilize defined strategies as followed against mers and sars, along with adopting and strengthening a few precautionary measures owing to the unknown nature of this novel virus (36, 189) . presently, the main course of treatment for severely affected sars-cov-2 patients admitted to hospitals includes mechanical ventilation, intensive care unit (icu) admittance, and symptomatic and supportive therapies. additionally, rna synthesis inhibitors (lamivudine and tenofovir disoproxil fumarate), remdesivir, neuraminidase inhibitors, peptide (ek1), antiinflammatory drugs, abidol, and chinese traditional medicine (lianhuaqingwen and shufengjiedu capsules) could aid in covid-19 treatment. however, further clinical trials are being carried out concerning their safety and efficacy (7) . it might require months to a year(s) to design and develop effective drugs, therapeutics, and vaccines against covid-19, with adequate evaluation and approval from regulatory bodies and moving to the bulk production of many millions of doses at commercial levels to meet the timely demand of mass populations across the globe (9) . continuous efforts are also warranted to identify and assess viable drugs and immunotherapeutic regimens that revealed proven potency in combating other viral agents similar to sars-cov-2. covid-19 patients showing severe signs are treated symptomatically along with oxygen therapy. in such cases where the patients progress toward respiratory failure and become refractory to oxygen therapy, mechanical ventilation is necessitated. the covid-19-induced septic shock can be managed by providing adequate hemodynamic support (299) . several classes of drugs are currently being evaluated for their potential therapeutic action against sars-cov-2. therapeutic agents that have anti-sars-cov-2 activity can be broadly classified into three categories: drugs that block virus entry into the host cell, drugs that block viral replication as well as its survival within the host cell, and drugs that attenuate the exaggerated host immune response (300) . an inflammatory cytokine storm is commonly seen in critically ill covid-19 patients. hence, they may benefit from the use of timely anti-inflammation treatment. anti-inflammatory therapy using drugs like glucocorticoids, cytokine inhibitors, jak inhibitors, and chloroquine/hydroxychloroquine should be done only after analyzing the risk/benefit ratio in covid-19 patients (301). there have not been any studies concerning the application of nonsteroidal anti-inflammatory drugs (nsaid) to covid-19-infected patients. however, reasonable pieces of evidence are available that link nsaid uses with the occurrence of respiratory and cardiovascular adverse effects. hence, as a cautionary approach, it is better to recommend the use of nsaids as the first-line option for managing covid-19 symptoms (302) . the use of corticosteroids in covid-19 patients is still a matter of controversy and requires further systematic clinical studies. the guidelines that were put forward to manage critically ill adults suggest the use of systemic corticosteroids in mechanically ventilated adults with ards (303) . the generalized use of corticosteroids is not indicated in covid-19, since there are some concerns associated with the use of corticosteroids in viral pneumonia. stem cell therapy using mesenchymal stem cells (mscs) is another hopeful strategy that can be used in clinical cases of covid-19 owing to its potential immunomodulatory capacity. it may have a beneficial role in attenuating the cytokine storm that is observed in severe cases of sars-cov-2 infection, thereby reducing mortality. among the different types of mscs, expanded umbilical cord mscs can be considered a potential therapeutic agent that requires further validation for managing critically ill covid-19 patients (304) . repurposed broad-spectrum antiviral drugs having proven uses against other viral pathogens can be employed for sars-cov-2-infected patients. these possess benefits of easy accessibility and recognized pharmacokinetic and pharmacodynamic activities, stability, doses, and side effects (9) . repurposed drugs have been studied for treating cov infections, like lopinavir/ritonavir, and interferon-1␤ revealed in vitro anti-mers-cov action. the in vivo experiment carried out in the nonhuman primate model of clinical microbiology reviews common marmosets treated with lopinavir/ritonavir and interferon beta showed superior protective results in treated animals than in the untreated ones (190) . a combination of these drugs is being evaluated to treat mers in humans (miracle trial) (191) . these two protease inhibitors (lopinavir and ritonavir), in combination with ribavirin, gave encouraging clinical outcomes in sars patients, suggesting their therapeutic values (165) . however, in the current scenario, due to the lack of specific therapeutic agents against sars-cov-2, hospitalized patients confirmed for the disease are given supportive care, like oxygen and fluid therapy, along with antibiotic therapy for managing secondary bacterial infections (192) . patients with novel coronavirus or covid-19 pneumonia who are mechanically ventilated often require sedatives, analgesics, and even muscle relaxation drugs to prevent ventilator-related lung injury associated with human-machine incoordination (122) . the result obtained from a clinical study of four patients infected with covid-19 claimed that combination therapy using lopinavir/ritonavir, arbidol, and shufeng jiedu capsules (traditional chinese medicine) was found to be effective in managing covid-19 pneumonia (193) . it is difficult to evaluate the therapeutic potential of a drug or a combination of drugs for managing a disease based on such a limited sample size. before choosing the ideal therapeutic agent for the management of covid-19, randomized clinical control studies should be performed with a sufficient study population. several classes of routinely used antiviral drugs, like oseltamivir (neuraminidase inhibitor), acyclovir, ganciclovir, and ribavirin, do not have any effect on covid-19 and, hence, are not recommended (187) . oseltamivir, a neuraminidase inhibitor, has been explored in chinese hospitals for treating suspected covid-19 cases, although proven efficacy against sars-cov-2 is still lacking for this drug (7) . the in vitro antiviral potential of fad-approved drugs, viz., ribavirin, penciclovir, nitazoxanide, nafamostat, and chloroquine, tested in comparison to remdesivir and favipiravir (broad-spectrum antiviral drugs) revealed remdesivir and chloroquine to be highly effective against sars-cov-2 infection in vitro (194) . ribavirin, penciclovir, and favipiravir might not possess noteworthy in vivo antiviral actions for sars-cov-2, since higher concentrations of these nucleoside analogs are needed in vitro to lessen the viral infection. both remdesivir and chloroquine are being used in humans to treat other diseases, and such safer drugs can be explored for assessing their effectiveness in covid-19 patients. several therapeutic agents, such as lopinavir/ritonavir, chloroquine, and hydroxychloroquine, have been proposed for the clinical management of covid-19 (299) . a molecular docking study, conducted in the rna-dependent rna polymerase (rdrp) of sars-cov-2 using different commercially available antipolymerase drugs, identified that drugs such as ribavirin, remdesivir, galidesivir, tenofovir, and sofosbuvir bind rdrp tightly, indicating their vast potential to be used against covid-19 (305). a broadspectrum antiviral drug that was developed in the united states, tilorone dihydrochloride (tilorone), was previously found to possess potent antiviral activity against mers, marburg, ebola, and chikungunya viruses (306) . even though it had broad-spectrum activity, it was neglected for an extended period. tilorone is another antiviral drug that might have activity against sars-cov-2. remdesivir, a novel nucleotide analog prodrug, was developed for treating ebola virus disease (evd), and it was also found to inhibit the replication of sars-cov and mers-cov in primary human airway epithelial cell culture systems (195) . recently, in vitro study has proven that remdesivir has better antiviral activity than lopinavir and ritonavir. further, in vivo studies conducted in mice also identified that treatment with remdesivir improved pulmonary function and reduced viral loads and lung pathology both in prophylactic and therapeutic regimens compared to lopinavir/ritonavir-ifn-␥ treatment in mers-cov infection (8) . remdesivir also inhibits a diverse range of coronaviruses, including circulating human cov, zoonotic bat cov, and prepandemic zoonotic cov (195) . remdesivir is also considered the only therapeutic drug that significantly reduces pulmonary pathology (8) . all these findings indicate that remde-sivir has to be further evaluated for its efficacy in the treatment of covid-19 infection in humans. the broad-spectrum activity exhibited by remdesivir will help control the spread of disease in the event of a new coronavirus outbreak. chloroquine is an antimalarial drug known to possess antiviral activity due to its ability to block virus-cell fusion by raising the endosomal ph necessary for fusion. it also interferes with virus-receptor binding by interfering with the terminal glycosylation of sars-cov cellular receptors, such as ace2 (196) . in a recent multicenter clinical trial that was conducted in china, chloroquine phosphate was found to exhibit both efficacy and safety in the therapeutic management of sars-cov-2-associated pneumonia (197) . this drug is already included in the treatment guidelines issued by the national health commission of the people's republic of china. the preliminary clinical trials using hydroxychloroquine, another aminoquinoline drug, gave promising results. the covid-19 patients received 600 mg of hydroxychloroquine daily along with azithromycin as a single-arm protocol. this protocol was found to be associated with a noteworthy reduction in viral load. finally, it resulted in a complete cure (271) ; however, the study comprised a small population and, hence, the possibility of misinterpretation could arise. however, in another case study, the authors raised concerns over the efficacy of hydroxychloroquine-azithromycin in the treatment of covid-19 patients, since no observable effect was seen when they were used. in some cases, the treatment was discontinued due to the prolongation of the qt interval (307) . hence, further randomized clinical trials are required before concluding this matter. recently, another fda-approved drug, ivermectin, was reported to inhibit the in vitro replication of sars-cov-2. the findings from this study indicate that a single treatment of this drug was able to induce an ϳ5,000-fold reduction in the viral rna at 48 h in cell culture. (308) . one of the main disadvantages that limit the clinical utility of ivermectin is its potential to cause cytotoxicity. however, altering the vehicles used in the formulations, the pharmacokinetic properties can be modified, thereby having significant control over the systemic concentration of ivermectin (338) . based on the pharmacokinetic simulation, it was also found that ivermectin may have limited therapeutic utility in managing covid-19, since the inhibitory concentration that has to be achieved for effective anti-sars-cov-2 activity is far higher than the maximum plasma concentration achieved by administering the approved dose (340) . however, ivermectin, being a host-directed agent, exhibits antiviral activity by targeting a critical cellular process of the mammalian cell. therefore, the administration of ivermectin, even at lower doses, will reduce the viral load at a minor level. this slight decrease will provide a great advantage to the immune system for mounting a large-scale antiviral response against sars-cov-2 (341). further, a combination of ivermectin and hydroxychloroquine might have a synergistic effect, since ivermectin reduces viral replication, while hydroxychloroquine inhibits the entry of the virus in the host cell (339) . further, in vivo studies and randomized clinical control trials are required to understand the mechanism as well as the clinical utility of this promising drug. nafamostat is a potent inhibitor of mers-cov that acts by preventing membrane fusion. nevertheless, it does not have any sort of inhibitory action against sars-cov-2 infection (194) . recently, several newly synthesized halogenated triazole compounds were evaluated, using fluorescence resonance energy transfer (fret)-based helicase assays, for their ability to inhibit helicase activity. among the evaluated compounds, 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-iodophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol and 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-chlorophenyl) hydrazinyl]-4h-1,2,4-triazole-3-thiol were found to be the most potent. these compounds were used for in silico studies, and molecular docking was accomplished into the active binding site of mers-cov helicase nsp13 (21) . further studies are required for evaluating the therapeutic potential of these newly identified compounds in the management of covid-19 infection. monoclonal antibodies (mabs) may be helpful in the intervention of disease in clinical microbiology reviews cov-exposed individuals. patients recovering from sars showed robust neutralizing antibodies against this cov infection (164) . a set of mabs aimed at the mers-cov s protein-specific domains, comprising six specific epitope groups interacting with receptor-binding, membrane fusion, and sialic acid-binding sites, make up crucial entry tasks of s protein (198, 199) . passive immunization employing weaker and strongly neutralizing antibodies provided considerable protection in mice against a mers-cov lethal challenge. such antibodies may play a crucial role in enhancing protective humoral responses against the emerging covs by aiming appropriate epitopes and functions of the s protein. the cross-neutralization ability of sars-cov rbd-specific neutralizing mabs considerably relies on the resemblance between their rbds; therefore, sars-cov rbd-specific antibodies could cross-neutralized sl covs, i.e., bat-sl-cov strain wiv1 (rbd with eight amino acid differences from sars-cov) but not bat-sl-cov strain shc014 (24 amino acid differences) (200) . appropriate rbd-specific mabs can be recognized by a relative analysis of rbd of sars-cov-2 to that of sars-cov, and cross-neutralizing sars-cov rbd-specific mabs could be explored for their effectiveness against covid-19 and further need to be assessed clinically. the u.s. biotechnology company regeneron is attempting to recognize potent and specific mabs to combat covid-19. an ideal therapeutic option suggested for sars-cov-2 (covid-19) is the combination therapy comprised of mabs and the drug remdesivir (covid-19) (201) . the sars-cov-specific human mab cr3022 is found to bind with sars-cov-2 rbd, indicating its potential as a therapeutic agent in the management of covid-19. it can be used alone or in combination with other effective neutralizing antibodies for the treatment and prevention of covid-19 (202) . furthermore, sars-cov-specific neutralizing antibodies, like m396 and cr3014, failed to bind the s protein of sars-cov-2, indicating that a particular level of similarity is mandatory between the rbds of sars-cov and sars-cov-2 for the cross-reactivity to occur. further assessment is necessary before confirming the effectiveness of such combination therapy. in addition, to prevent further community and nosocomial spread of covid-19, the postprocedure risk management program should not be neglected (309) . development of broad-spectrum inhibitors against the human coronaviral pathogens will help to facilitate clinical trials on the effectiveness of such inhibitors against endemic and emerging coronaviruses (203) . a promising animal study revealed the protective effect of passive immunotherapy with immune serum from mers-immune camels on mice infected with mers-cov (204) . passive immunotherapy using convalescent plasma is another strategy that can be used for treating covid-19-infected, critically ill patients (205) . the exploration of fully human antibodies (human single-chain antibodies; huscfvs) or humanized nanobodies (single-domain antibodies; sdab, vh/vhh) could aid in blocking virus replication, as these agents can traverse the virus-infected cell membranes (transbodies) and can interfere with the biological characteristics of the replicating virus proteins. such examples include transbodies to the influenza virus, hepatitis c virus, ebola virus, and dengue virus (206) . producing similar transbodies against intracellular proteins of coronaviruses, such as papain-like proteases (plpro), cysteinelike protease (3clpro), or other nsps, which are essential for replication and transcription of the virus, might formulate a practical move forward for a safer and potent passive immunization approach for virus-exposed persons and rendering therapy to infected patients. in a case study on five grimly sick patients having symptoms of severe pneumonia due to covid-19, convalescent plasma administration was found to be helpful in patients recovering successfully. the convalescent plasma containing a sars-cov-2specific elisa (serum) antibody titer higher than 1:1,000 and neutralizing antibody titer more significant than 40 was collected from the recovered patients and used for plasma transfusion twice in a volume of 200 to 250 ml on the day of collection (310) . at present, treatment for sepsis and ards mainly involves antimicrobial therapy, source control, and supportive care. hence, the use of therapeutic plasma exchange can be considered an option in managing such severe conditions. further randomized trials can be designed to investigate its efficacy (311) . potent therapeutics to combat sars-cov-2 infection include virus binding molecules, molecules or inhibitors targeting particular enzymes implicated in replication and transcription process of the virus, helicase inhibitors, vital viral proteases and proteins, protease inhibitors of host cells, endocytosis inhibitors, short interfering rna (sirna), neutralizing antibodies, mabs against the host receptor, mabs interfering with the s1 rbd, antiviral peptide aimed at s2, and natural drugs/medicines (7, 166, 186) . the s protein acts as the critical target for developing cov antivirals, like inhibitors of s protein and s cleavage, neutralizing antibodies, rbd-ace2 blockers, sirnas, blockers of the fusion core, and proteases (168) . all of these therapeutic approaches have revealed both in vitro and in vivo anti-cov potential. although in vitro research carried out with these therapeutics showed efficacy, most need appropriate support from randomized animal or human trials. therefore, they might be of limited applicability and require trials against sars-cov-2 to gain practical usefulness. the binding of sars-cov-2 with ace2 leads to the exacerbation of pneumonia as a consequence of the imbalance in the reninangiotensin system (ras). the virus-induced pulmonary inflammatory responses may be reduced by the administration of ace inhibitors (acei) and angiotensin type-1 receptor (at1r) (207) . several investigations have suggested the use of small-molecule inhibitors for the potential control of sars-cov infections. drugs of the fda-approved compound library were screened to identify four small-molecule inhibitors of mers-cov (chlorpromazine, chloroquine, loperamide, and lopinavir) that inhibited viral replication. these compounds also hinder sars-cov and human covs (208) . therapeutic strategies involving the use of specific antibodies or compounds that neutralize cytokines and their receptors will help to restrain the host inflammatory responses. such drugs acting specifically in the respiratory tract will help to reduce virus-triggered immune pathologies in covid-19 (209) . the later stages of coronavirus-induced inflammatory cascades are characterized by the release of proinflammatory interleukin-1 (il-1) family members, such as il-1 and il-33. hence, there exists a possibility that the inflammation associated with coronavirus can be inhibited by utilizing anti-inflammatory cytokines that belong to the il-1 family (92) . it has also been suggested that the actin protein is the host factor that is involved in cell entry and pathogenesis of sars-cov-2. hence, those drugs that modulate the biological activity of this protein, like ibuprofen, might have some therapeutic application in managing the disease (174). the plasma angiotensin 2 level was found to be markedly elevated in covid-19 infection and was correlated with viral load and lung injury. hence, drugs that block angiotensin receptors may have potential for treating covid-19 infection (121) . a scientist from germany, named rolf hilgenfeld, has been working on the identification of drugs for the treatment of coronaviral infection since the time of the first sars outbreak (19) . the sars-cov s2 subunit has a significant function in mediating virus fusion that provides entry into the host cell. heptad repeat 1 (hr1) and heptad repeat 2 (hr2) can interact and form a six-helix bundle that brings the viral and cellular membranes in close proximity, facilitating its fusion. the sequence alignment study conducted between covid-19 and sars-cov identified that the s2 subunits are highly conserved in these covs. the hr1 and hr2 domains showed 92.6% and 100% overall identity, respectively (210) . from these findings, we can confirm the significance of covid-19 hr1 and hr2 and their vital role in host cell entry. hence, fusion inhibitors target the hr1 domain of s protein, thereby preventing viral fusion and entry into the host cell. this is another potential therapeutic strategy that can be used in the management of covid-19. other than the specific therapy directed against covid-19, general treatments play a vital role in the enhancement of host immune responses against the viral agent. inadequate nutrition is linked to the weakening of the host immune response, clinical microbiology reviews making the individual more susceptible. the role played by nutrition in disease susceptibility should be measured by evaluating the nutritional status of patients with covid-19 (205) . for evaluating the potential of vaccines and therapeutics against covs, including sars-cov, mers-covs, and the presently emerging sars-cov-2, suitable animal models that can mimic the clinical disease are needed (211, 212) . various animal models were assessed for sars-and mers-covs, such as mice, guinea pigs, golden syrian hamsters, ferrets, rabbits, nonhuman primates like rhesus macaques and marmosets, and cats (185, (213) (214) (215) (216) (217) (218) . the specificity of the virus to hace2 (receptor of sars-cov) was found to be a significant barrier in developing animal models. consequently, a sars-cov transgenic mouse model has been developed by inserting the hace2 gene into the mouse genome (219) . the inability of mers-cov to replicate in the respiratory tracts of animals (mice, hamsters, and ferrets) is another limiting factor. however, with genetic engineering, a 288-330 ϩ/ϩ mers-cov genetically modified mouse model was developed and now is in use for the assessment of novel drugs and vaccines against mers-cov (220). in the past, small animals (mice or hamsters) have been targeted for being closer to a humanized structure, such as mouse dpp4 altered with human dpp4 (hdpp4), hdpp4-transduced mice, and hdpp4-tg mice (transgenic for expressing hdpp4) for mers-cov infection (221) . the crispr-cas9 gene-editing tool has been used for inserting genomic alterations in mice, making them susceptible to mers-cov infection (222) . efforts are under way to recognize suitable animal models for sars-cov2/covid-19, identify the receptor affinity of this virus, study pathology in experimental animal models, and explore virus-specific immune responses and protection studies, which together would increase the pace of efforts being made for developing potent vaccines and drugs to counter this emerging virus. cell lines, such as monkey epithelial cell lines (llc-mk2 and vero-b4), goat lung cells, alpaca kidney cells, dromedary umbilical cord cells, and advanced ex vivo three-dimensional tracheobronchial tissue, have been explored to study human covs (mers-cov) (223, 224) . vero and huh-7 cells (human liver cancer cells) have been used for isolating sars-cov-2 (194) . recently, an experimental study with rhesus monkeys as animal models revealed the absence of any viral loads in nasopharyngeal and anal swabs, and no viral replication was recorded in the primary tissues at a time interval of 5 days post-reinfection in reexposed monkeys (274) . the subsequent virological, radiological, and pathological observations indicated that the monkeys with reexposure had no recurrence of covid-19, like the sars-cov-2-infected monkeys without rechallenge. these findings suggest that primary infection with sars-cov-2 could protect from later exposures to the virus, which could help in defining disease prognosis and crucial inferences for designing and developing potent vaccines against covid-19 (274). in contrast to their response to the 2002 sars outbreak, china has shown immense political openness in reporting the covid-19 outbreak promptly. they have also performed rapid sequencing of covid-19 at multiple levels and shared the findings globally within days of identifying the novel virus (225) . the move made by china opened a new chapter in global health security and diplomacy. even though complete lockdown was declared following the covid-19 outbreak in wuhan, the large-scale movement of people has resulted in a radiating spread of infections in the surrounding provinces as well as to several other countries. large-scale screening programs might help us to control the spread of this virus. however, this is both challenging as well as time-consuming due to the present extent of infection (226) . the current scenario demands effective implementation of vigorous prevention and control strategies owing to the prospect of covid-19 for nosocomial infections (68) . follow-ups of infected patients by telephone on day 7 and day 14 are advised to avoid any further unintentional spread or nosocomial transmission (312) . the availability of public data sets provided by independent analytical teams will act as robust evidence that would guide us in designing interventions against the covid-19 outbreak. newspaper reports and social media can be used to analyze and reconstruct the progression of an outbreak. they can help us to obtain detailed patient-level data in the early stages of an outbreak (227) . immediate travel restrictions imposed by several countries might have contributed significantly to preventing the spread of sars-cov-2 globally (89, 228) . following the outbreak, a temporary ban was imposed on the wildlife trade, keeping in mind the possible role played by wild animal species in the origin of sars-cov-2/covid-19 (147) . making a permanent and bold decision on the trade of wild animal species is necessary to prevent the possibility of virus spread and initiation of an outbreak due to zoonotic spillover (1) . personal protective equipment (ppe), like face masks, will help to prevent the spread of respiratory infections like covid-19. face masks not only protect from infectious aerosols but also prevent the transmission of disease to other susceptible individuals while traveling through public transport systems (313) . another critical practice that can reduce the transmission of respiratory diseases is the maintenance of hand hygiene. however, the efficacy of this practice in reducing the transmission of respiratory viruses like sars-cov-2 is much dependent upon the size of droplets produced. hand hygiene will reduce disease transmission only if the virus is transmitted through the formation of large droplets (314) . hence, it is better not to overemphasize that hand hygiene will prevent the transmission of sars-cov-2, since it may produce a false sense of safety among the general public that further contributes to the spread of covid-19. even though airborne spread has not been reported in sars-cov-2 infection, transmission can occur through droplets and fomites, especially when there is close, unprotected contact between infected and susceptible individuals. hence, hand hygiene is equally as important as the use of appropriate ppe, like face masks, to break the transmission cycle of the virus; both hand hygiene and face masks help to lessen the risk of covid-19 transmission (315) . medical staff are in the group of individuals most at risk of getting covid-19 infection. this is because they are exposed directly to infected patients. hence, proper training must be given to all hospital staff on methods of prevention and protection so that they become competent enough to protect themselves and others from this deadly disease (316) . as a preventive measure, health care workers caring for infected patients should take extreme precautions against both contact and airborne transmission. they should use ppe such as face masks (n95 or ffp3), eye protection (goggles), gowns, and gloves to nullify the risk of infection (299) . the human-to-human transmission reported in sars-cov-2 infection occurs mainly through droplet or direct contact. due to this finding, frontline health care workers should follow stringent infection control and preventive measures, such as the use of ppe, to prevent infection (110) . the mental health of the medical/health workers who are involved in the covid-19 outbreak is of great importance, because the strain on their mental well-being will affect their attention, concentration, and decision-making capacity. hence, for control of the covid-19 outbreak, rapid steps should be taken to protect the mental health of medical workers (229) . since the living mammals sold in the wet market are suspected to be the intermediate host of sars-cov-2, there is a need for strengthening the regulatory mechanism for wild animal trade (13) . the total number of covid-19 confirmed cases is on a continuous rise and the cure rate is relatively low, making disease control very difficult to achieve. the chinese government is making continuous efforts to contain the disease by taking emergency control and prevention measures. they have already built a hospital for patients affected by this virus and are currently building several more for accommodating the continuously increasing infected population (230) . the effective control of sars-cov-2/covid-19 requires high-level interventions like intensive contact tracing, as well as the quarantine of people with suspected infection and the isolation of infected individuals. the implementation of rigorous control and preventive measures together might control the r 0 number and reduce the transmission risk (228) . clinical microbiology reviews considering the zoonotic links associated with sars-cov-2, the one health approach may play a vital role in the prevention and control measures being followed to restrain this pandemic virus (317) (318) (319) . the substantial importation of covid-19 presymptomatic cases from wuhan has resulted in independent, self-sustaining outbreaks across major cities both within the country and across the globe. the majority of chinese cities are now facing localized outbreaks of covid-19 (231) . hence, deploying efficient public health interventions might help to cut the spread of this virus globally. the occurrence of covid-19 infection on several cruise ships gave us a preliminary idea regarding the transmission pattern of the disease. cruise ships act as a closed environment and provide an ideal setting for the occurrence of respiratory disease outbreaks. such a situation poses a significant threat to travelers, since people from different countries are on board, which favors the introduction of the pathogen (320). although nearly 30 cruise ships from different countries have been found harboring covid-19 infection, the major cruise ships that were involved in the covid-19 outbreaks are the diamond princess, grand princess, celebrity apex, and ruby princess. the number of confirmed covid-19 cases around the world is on the rise. the success of preventive measures put forward by every country is mainly dependent upon their ability to anticipate the approaching waves of patients. this will help to properly prepare the health care workers and increase the intensive care unit (icu) capacity (321) . instead of entirely relying on lockdown protocols, countries should focus mainly on alternative intervention strategies, such as large-scale testing, contract tracing, and localized quarantine of suspected cases for limiting the spread of this pandemic virus. such intervention strategies will be useful either at the beginning of the pandemic or after lockdown relaxation (322) . lockdown should be imposed only to slow down disease progression among the population so that the health care system is not overloaded. the reproduction number (r 0 ) of covid-19 infection was earlier estimated to be in the range of 1.4 to 2.5 (70); recently, it was estimated to be 2.24 to 3.58 (76) . compared to its coronavirus predecessors, covid-19 has an r 0 value that is greater than that of mers (r 0 ͻ 1) (108) but less than that of sars (r 0 value of 2 to 5) (93) . still, to prevent further spread of disease at mass gatherings, functions remain canceled in the affected cities, and persons are asked to work from home (232) . hence, it is a relief that the current outbreak of covid-19 infection can be brought under control with the adoption of strategic preventive and control measures along with the early isolation of subsequent cases in the coming days. studies also report that since air traffic between china and african countries increased many times over in the decade after the sars outbreak, african countries need to be vigilant to prevent the spread of novel coronavirus in africa (225) . due to fear of virus spread, wuhan city was completely shut down (233) . the immediate control of the ongoing covid-19 outbreaks appears a mammoth task, especially for developing countries, due to their inability to allocate quarantine stations that could screen infected individuals' movements (234) . such underdeveloped countries should divert their resources and energy to enforcing the primary level of preventive measures, like controlling the entry of individuals from china or countries where the disease has flared up, isolating the infected individuals, and quarantining individuals with suspected infection. most of the sub-saharan african countries have a fragile health system that can be crippled in the event of an outbreak. effective management of covid-19 would be difficult for low-income countries due to their inability to respond rapidly due to the lack of an efficient health care system (65) . controlling the imported cases is critical in preventing the spread of covid-19 to other countries that have not reported the disease until now. the possibility of an imported case of covid-19 leading to sustained human-to-human transmission was estimated to be 0.41. this can be reduced to a value of 0.012 by decreasing the mean time from the onset of symptoms to hospitalization and can only be made possible by using intense disease surveillance systems (235) . the silent importations of infected individuals (before the manifestation of clinical signs) also contributed significantly to the spread of disease across the major cities of the world. even though the travel ban was implemented in wuhan (89) , infected persons who traveled out of the city just before the imposition of the ban might have remained undetected and resulted in local outbreaks (236) . emerging novel diseases like covid-19 are difficult to contain within the country of origin, since globalization has led to a world without borders. hence, international collaboration plays a vital role in preventing the further spread of this virus across the globe (237) . we also predict the possibility of another outbreak, as predicted by fan et al. (6) . indeed, the present outbreak caused by sars-cov-2 (covid-19) was expected. similar to previous outbreaks, the current outbreak also will be contained shortly. however, the real issue is how we are planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or even sooner (fig. 7) . several years after the global sars epidemic, the current sars-cov-2/covid-19 pandemic has served as a reminder of how novel pathogens can rapidly emerge and spread through the human population and eventually cause severe public health crises. further research should be conducted to establish animal models for sars-cov-2 to investigate replication, transmission dynamics, and pathogenesis in humans. this may help develop and evaluate potential therapeutic strategies against zoonotic cov epidemics. present trends suggest the occurrence of future outbreaks of covs due to changes in the climate, and ecological conditions may be associated with humananimal contact. live-animal markets, such as the huanan south china seafood market, represent ideal conditions for interspecies contact of wildlife with domestic birds, pigs, and mammals, which substantially increases the probability of interspecies transmission of cov infections and could result in high risks to humans due to adaptive genetic recombination in these viruses (323) (324) (325) . the covid-19-associated symptoms are fever, cough, expectoration, headache, and myalgia or fatigue. individuals with asymptomatic and atypical clinical manifestations were also identified recently, further adding to the complexity of disease transmission dynamics. atypical clinical manifestations may only express symptoms such as fatigue instead of respiratory signs such as fever, cough, and sputum. in such cases, the clinician must be vigilant for the possible occurrence of asymptomatic and atypical clinical manifestations to avoid the possibility of missed diagnoses. the present outbreak caused by sars-cov-2 was, indeed, expected. similar to clinical microbiology reviews previous outbreaks, the current pandemic also will be contained shortly. however, the real question is, how are we planning to counter the next zoonotic cov epidemic that is likely to occur within the next 5 to 10 years or perhaps sooner? our knowledge of most of the bat covs is scarce, as these viruses have not been isolated and studied, and extensive studies on such viruses are typically only conducted when they are associated with specific disease outbreaks. the next step following the control of the covid-19 outbreak in china should be focused on screening, identification, isolation, and characterization of covs present in wildlife species of china, particularly in bats. both in vitro and in vivo studies (using suitable animal models) should be conducted to evaluate the risk of future epidemics. presently, licensed antiviral drugs or vaccines against sars-cov, mers-cov, and sars-cov-2 are lacking. however, advances in designing antiviral drugs and vaccines against several other emerging diseases will help develop suitable therapeutic agents against covid-19 in a short time. until then, we must rely exclusively on various control and prevention measures to prevent this new disease from becoming a pandemic. history is repeating itself: probable zoonotic spillover as the cause of the 2019 novel coronavirus epidemic return of the coronavirus: 2019-ncov china novel coronavirus investigating and research team. 2020. a novel coronavirus from patients with pneumonia in china evolutionary perspectives on novel coronaviruses identified in pneumonia cases in china a novel coronavirus emerging in china-key questions for impact assessment bat coronaviruses in china drug treatment options for the 2019-new coronavirus (2019-ncov) comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov recent discovery and development of inhibitors targeting coronaviruses epidemiology, genetic recombination, and pathogenesis of coronaviruses understanding bat sars-like coronaviruses for the preparation of future corona virus outbreaks-implications for coronavirus vaccine development coronavirus infections reported by promed origin and evolution of the 2019 novel coronavirus clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses genome composition and divergence of the novel coronavirus (2019-ncov) originating in china genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province this scientist hopes to test coronavirus drugs on animals in locked-down wuhan coronavirus infection in equines: a review 2020. design, synthesis and molecular docking of novel triazole derivatives as potential cov helicase inhibitors una nueva zoonosis viral de preocupación global: covid-19, enfermedad por coronavirus coronavirus pathogenesis coronavirus infections and immune responses who. 2020. coronavirus disease 2019 (covid-19) situation report-114 severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group emerging coronaviruses: genome structure, replication, and pathogenesis coronaviridae: the viruses and their replication discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus coronaviruses: an overview of their replication and pathogenesis genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan coronavirus genome structure and replication viral and cellular mrna translation in coronavirus-infected cells ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex identification of novel subgenomic rnas and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus emerging novel coronavirus (2019-ncov)-current scenario, evolutionary perspective based on genome analysis and recent developments identification of a novel coronavirus causing severe pneumonia in human: a descriptive study bat origin of human coronaviruses discovery of bat coronaviruses through surveillance and probe capture-based next-generation sequencing bats, civets and the emergence of sars middle east respiratory syndrome coronavirus and the one health concept the molecular biology of coronaviruses mechanisms of coronavirus cell entry mediated by the viral spike protein architecture of the sars coronavirus prefusion spike structure, function, and evolution of coronavirus spike proteins isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor bat origin of a new human coronavirus: there and back again a structural analysis of m protein in coronavirus assembly and morphology differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e a conserved domain in the coronavirus membrane protein tail is important for virus assembly coronavirus envelope protein: current knowledge severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis structure and inhibition of the sars coronavirus envelope protein ion channel a severe acute respiratory syndrome corona virus that lacks the e gene is attenuated in vitro and in vivo modular organization of sars coronavirus nucleocapsid protein analysis of preferred codon usage in the coronavirus n genes and their implications for genome evolution and vaccine design the coronavirus nucleocapsid is a multifunctional protein the covid-19 clinical microbiology reviews nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties identification of in vivointeracting domains of the murine coronavirus nucleocapsid protein specific interaction between coronavirus leader rna and nucleocapsid protein subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin the first 2019 novel coronavirus case in nepal the covid-19 epidemic economic impacts of wuhan 2019-ncov on china and the world china critical care clinical trials group (cccctg). 2020. intensive care during the coronavirus epidemic preparedness and proactive infection control measures against the emerging wuhan coronavirus pneumonia in china emerging viruses without borders: the wuhan coronavirus china coronavirus: what do we know so far? the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan, china outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle the rate of underascertainment of novel corona virus (2019-ncov) infection: estimation using japanese passengers data on evacuation flights initial cluster of novel coronavirus (2019-ncov) infections in wuhan, china, is consistent with substantial human-to-human transmission china coronavirus: cases surge as official admits human to human transmission preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak the reproductive number of covid-19 is higher compared to sars coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china identification of alpha and beta coronavirus in wildlife species in france: bats, rodents, rabbits, and hedgehogs. viruses diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement interpretation of guidelines for the diagnosis and treatment of novel coronavirus (2019-ncov) infection by the national health commission (trial version 5) sars-cov-2 viral load in upper respiratory specimens of infected patients preliminary prediction of the basic reproduction number of the wuhan novel coronavirus 2019-ncov bat-origin coronaviruses expand their host range to pigs the global spread of 2019-ncov: a molecular evolutionary analysis receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars the next big threat to global health? 2019 novel coronavirus (2019-ncov): what advice can we give to travellers? interim recommendations january 2020, from the latin-american society for travel medicine (slamvi) going global-travel and the 2019 novel coronavirus severe acute respiratory syndrome middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission mast cells contribute to coronavirus-induced inflammation: new anti-inflammatory strategy consensus document on the epidemiology of severe acute respiratory syndrome (sars) epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong a new emerging zoonotic virus of concern: the 2019 novel coronavirus (covid-19) isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus (2019-ncov) outbreak middle east respiratory syndrome coronavirus (mers-cov) does sars-cov-2 has a longer incubation period than sars and mers? the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade covid-2019: the role of the nsp2 and nsp3 in its pathogenesis coronavirus covid-19 has killed more people than sars and mers combined, despite lower case fatality rate from sars to mers, thrusting coronaviruses into the spotlight another decade, another coronavirus a decade after sars: strategies for controlling emerging coronaviruses a sars-like cluster of circulating bat coronaviruses shows potential for human emergence origin and evolution of pathogenic coronaviruses identification of a novel betacoronavirus (merbecovirus) in amur hedgehogs from china who mers global summary and assessment of risk livestock susceptibility to infection with middle east respiratory syndrome coronavirus overview of the 2019 novel coronavirus (2019-ncov): the pathogen of severe specific contagious pneumonia (sscp) 2019-ncov: new challenges from coronavirus a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china feng z. 2020. early transmission dynamics in epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan sars-cov, mers-cov and now the 2019-novel cov: have we investigated enough about coronaviruses? a bibliometric analysis early detection and disease assessment of patients with novel coronavirus pneumonia pathological findings of covid-19 associated with acute respiratory distress syndrome potential maternal and infant outcomes from (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury the keypoints in treatment of the critical novel coronavirus pneumonia patient exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia fenner's veterinary virology sars-cov related betacoronavirus and diverse alphacoronavirus members found in western old-world genomic characterization and phylogenetic classification of bovine coronaviruses through whole genome sequence analysis biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child feline coronaviruses: pathogenesis of feline infectious peritonitis canine enteric coronaviruses: emerging viral pathogens with distinct recombinant spike proteins canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex emergence of avian infectious bronchitis virus and its variants need better diagnosis, prevention and control strategies: a covid-19 clinical microbiology reviews global perspective pathogenesis and diagnostic approaches of avian infectious bronchitis the biology and pathogenesis of coronaviruses fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome the pig as an intermediate host for influenza a viruses between birds and humans sars-associated coronavirus transmitted from human to pig identification of a novel coronavirus from a beluga whale by using a panviral microarray rapid classification of betacoronaviruses and identification of traditional chinese medicine as potential origin of zoonotic coronaviruses novel coronavirus: from discovery to clinical diagnostics a qualitative study of zoonotic risk factors among rural communities in southern china brazil burning! what is the potential impact of the amazon wildfires on vector-borne and zoonotic emerging diseases? a statement from an international experts meeting homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human viral metagenomics revealed sendai virus and coronavirus infection of malayan pangolins (manisjavanica). viruses 11:979 isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins a strategy to prevent future epidemics similar to the 2019-ncov outbreak serological evidence of bat sars-related coronavirus infection in humans efficient management of novel coronavirus pneumonia by efficient prevention and control in scientific manner evolving status of the 2019 novel coronavirus infection: proposal of conventional serologic assays for disease diagnosis and infection monitoring measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in wuhan viral load of sars-cov-2 in clinical samples consistent detection of 2019 novel coronavirus in saliva molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing emergence of a novel coronavirus disease (covid-19) and the importance of diagnostic testing: why partnership between clinical laboratories, public health agencies, and industry is essential to control the outbreak molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia labs scramble to produce new coronavirus diagnostics therapeutic and triage strategies for 2019 novel coronavirus disease in fever clinics radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study outbreak of novel coronavirus (covid-19): what is the role of radiologists? advances in developing therapies to combat zika virus: current knowledge and future perspectives advances in designing and developing vaccines, drugs, and therapies to counter ebola virus nipah virus: epidemiology, pathology, immunobiology and advances in diagnosis, vaccine designing and control strategies-a comprehensive review a dna vaccine induces sars coronavirus neutralization and protective immunity in mice role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings anti-sars coronavirus agents: a patent review (2008 -present) characterization of the immune response of mers-cov vaccine candidates derived from two different vectors in mice the spike protein of sars-cov-a target for vaccine and therapeutic development one-health: a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity identification of the immunodominant neutralizing regions in the spike glycoprotein of porcine deltacoronavirus t-cell immunity of sars-cov: implications for vaccine development against mers-cov an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies use of the informational spectrum methodology for rapid biological analysis of the novel coronavirus 2019-ncov: prediction of potential receptor, natural reservoir, tropism and therapeutic/vaccine target sars vaccine development immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length spike protein of middle east respiratory syndrome coronavirus epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases analysis of the genome sequence and prediction of b-cell epitopes of the envelope protein of middle east respiratory syndromecoronavirus niaid. 2020. developing therapeutics and vaccines for coronaviruses cepi. 2020. cepi to fund three programmes to develop vaccines against the novel coronavirus, ncov-2019 moderna announces funding award from cepi to accelerate development of messenger rna (mrna) vaccine against novel coronavirus novel inhibitors of severe acute respiratory syndrome coronavirus entry that act by three distinct mechanisms treatment with interferon-␣2b and ribavirin improves outcome in mers-cov-infected rhesus macaques middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread covid-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics potential antiviral therapeutics for 2019 novel coronavirus a novel coronavirus outbreak of global health concern coronavirusesdrug discovery and therapeutic options treatment with lopinavir/ritonavir or interferon-␤1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-␤1b (miracle trial): study protocol for a randomized controlled trial clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses chloroquine is a potent inhibitor of sars coronavirus infection and spread breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the mers-coronavirus spike glycoprotein characterization of novel monoclonal antibodies against merscoronavirus spike protein crossneutralization of sars coronavirus-specific antibodies against bat sars-like coronaviruses new coronavirus threat galvanizes scientists potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection potential interventions for novel coronavirus in china: a systemic review human transbodies that interfere with the functions of ebola virus vp35 protein in genome replication and transcription and innate immune antagonism inhibitors of ras might be a good choice for the therapy of covid-19 pneumonia screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein animal models for sars and mers coronaviruses recent advances in the vaccine development against middle east respiratory syndromecoronavirus virology: sars virus infection of cats and ferrets a live attenuated severe acute respiratory syndrome coronavirus is immunogenic and efficacious in golden syrian hamsters animal models and vaccines for sars-cov infection infection with mers-cov causes lethal pneumonia in the common marmoset vaccines for the prevention against the threat of mers-cov molecular basis of coronavirus virulence and vaccine development mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection genetically engineering a susceptible mouse model for mers-cov-induced acute respiratory distress syndrome prospects for a mers-cov spike vaccine a mouse model for mers coronavirus-induced acute respiratory distress syndrome replicative capacity of mers coronavirus in livestock cell lines entry of human coronavirus nl63 into the cell china's response to a novel coronavirus stands in stark contrast to the 2002 sars outbreak response novel coronavirus is putting the whole world on alert early epidemiological analysis of the coronavirus disease 2019 outbreak based on crowdsourced data: a population-level observational study estimation of the transmission risk of the 2019-ncov and its implication for public health interventions the mental health of medical workers in wuhan, china dealing with the 2019 novel coronavirus the progress of 2019 novel coronavirus (2019-ncov) event in china nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study response to the emerging novel coronavirus outbreak potential for global spread of a novel coronavirus from china novel coronavirus, poor quarantine, and the risk of pandemic novel coronavirus outbreak in wuhan, china, 2020: intense surveillance is vital for preventing sustained transmission in new locations risk for transportation of 2019 novel coronavirus disease from wuhan to other cities in china infections without borders: a new coronavirus in wuhan, china an interactive web-based dashboard to track covid-19 in real time limiting spread of covid-19 from cruise ships-lessons to be learnt from japan clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records a case of 2019 novel coronavirus in a pregnant woman with preterm delivery clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study a pregnant woman with covid-19 in central america latin american network of coronavirus disease 2019-covid-19 research (lancovid-19). 2020. clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests detection of sars-cov-2 in different types of clinical specimens a well infant with coronavirus disease 2019 with high viral load clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster public health might be endangered by possible prolonged discharge of sars-cov-2 in stool screening of faecal microbiota transplant donors during the covid-19 outbreak: suggestions for urgent updates from an international expert panel evidence for gastrointestinal infection of sars-cov-2 enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? viral load kinetics of sars-cov-2 infection in first two patients in korea sars-cov-2 cell entry depends on ace2 and tm-prss2 and is blocked by a clinically proven protease inhibitor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein discovery of a 382-nt deletion during the early evolution of sars-cov-2 aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 laboratory diagnosis of emerging human coronavirus infections-the state of the art covid-19) emergency use authorization (eua) information-in vitro diagnostic euas the establishment of reference sequence for sars-cov-2 and variation analysis chloroquine and hydroxychloroquine as available weapons to fight covid-19 a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? aminoquinolines against coronavirus disease 2019 (covid-19): chloroquine or hydroxychloroquine of chloroquine and covid-19 in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) covid-19: a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression increased incidence of gastrointestinal side effects in patients taking hydroxychloroquine: a brand-related issue? clinical trials registry-chloroquine. covid-19 province and health commission of guangdong province for chloroquine in the treatment of novel coronavirus. 2020. expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia covid-19 clinical microbiology reviews results of an open-label non-randomized clinical trial in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 reinfection could not occur in sars-cov-2 infected rhesus macaques severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection community-acquired respiratory viruses in transplant patients: diversity, impact, unmet clinical needs respiratory infections in the u.s. military: recent experience and control middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease two years after pandemic influenza a/2009/h1n1: what have we learned? emerging respiratory viruses: challenges and vaccine strategies virus receptors: implications for pathogenesis and the design of antiviral agents an update on sars-cov-2/covid-19 with particular reference on its clinical pathology, pathogenesis, immunopathology and mitigation strategies-a review can sars-cov-2 infection be acquired in utero? more definitive evidence is needed wuhan virus: chinese animal markets reopened with almost no precautions china must close down "wet markets will they ever learn? chinese markets are still selling bats and slaughtering rabbits on blood-soaked floors as beijing celebrates "victory sars-cov-2: jumping the species barrier, lessons from sars and mers, its zoonotic spillover, transmission to humans, preventive and control measures and recent developments to counter this pandemic virus novel coronavirus outbreak research team. 2020. epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient detectable sars-cov-2 viral rna in feces of three children during recovery period of covid-19 pneumonia quantitative detection and viral load analysis of sars-cov-2 in infected patients ct screening for early diagnosis of sars-cov-2 infection characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies insights into cross-species evolution of novel human coronavirus 2019-ncov and defining immune determinants for vaccine development vaccine designers take first shots at covid-19 precision vaccinations. 2020. coronavirus vaccines draft landscape of covid-19 candidate vaccines-20 features, evaluation and treatment coronavirus (covid-19) therapeutic opportunities to manage covid-19/sars-cov-2 infection: present and future the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the experience of clinical immunologists from china non-steroidal anti-inflammatory drugs and covid-19 surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) expanded umbilical cord mesenchymal stem cells (uc-mscs) as a therapeutic strategy in managing critically ill covid-19 patients: the case for compassionate use ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study tilorone: a broad-spectrum antiviral invented in the usa and commercialized in russia and beyond no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection the fdaapproved drug ivermectin inhibits the replication of sars-cov-2 in vitro prevention of nosocomial sars-cov-2 transmission in endoscopy: international recommendations and the need for a gold standard treatment of 5 critically ill patients with covid-19 with convalescent plasma a novel treatment approach to the novel coronavirus: an argument for the use of therapeutic plasma exchange for fulminant covid-19 coronavirus (covid-19) outbreak: what the department of endoscopy should know covid-19: face masks and human-tohuman transmission. influenza other respir viruses does hand hygiene reduce sars-cov-2 transmission? reply to "does hand hygiene reduce sars-cov-2 transmission? clinical characteristics of 54 medical staff with covid-19: a retrospective study in a single center in wuhan revisiting the one health approach in the context of covid-19: a look into the ecology of this emerging disease emerging coronavirus disease (covid-19), a pandemic public health emergency with animal linkages: current status update covid-19, an emerging coronavirus infection: current scenario and recent developments-an overview cdc cruise ship response team, california department of public health covid-19 team, solano county covid-19 team. 2020. public health responses to covid-19 outbreaks on cruise ships-worldwide preparing for the covid-19 pandemic: our experience in new york covid-19: extending or relaxing distancing control measures sars-cov-2: an emerging coronavirus that causes a global threat the 2019 novel coronavirus disease (covid-19) pandemic: a review of the current evidence profile of specific antibodies to sars-cov-2: the first report sars-cov-2 and the hidden carriers-sewage, feline, and blood transfusion first confirmed detection of sars-cov-2 in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid-19 in the community what do we know about the sars-cov-2 coronavirus in the environment? susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 a critical needs assessment for research in companion animals and livestock following the pandemic of covid-19 in humans we should err on side of caution with covid-19 advice can cats become infected with covid-19? can companion animals become infected with covid-19? leading veterinary diagnostic company sees no covid-19 cases in pets avma. 2020. sars-cov-2 in animals coronavirus disease 2019 (covid-19) outbreak: could pigs be vectors for human infections? xenotransplantation 27:e12591 infection and rapid transmission of sars-cov-2 in ferrets current therapeutic applications and pharmacokinetic modulations of ivermectin hydroxychloroquine and ivermectin: a synergistic combination for covid-19 chemoprophylaxis and/or treatment? the approved dose of ivermectin alone is not the ideal dose for the treatment of covid-19 ivermectin and covid-19: a report in antiviral research, widespread interest, an fda warning, two letters to the editor and the authors' responses rapid covid-19 vaccine development global efforts on vaccines for covid-19: since, sooner or later, we all will catch the coronavirus abbott-launches-molecular-point-of-care-test-to -detect-novel-coronavirus-in-as-little-as-five-minutes era of molecular diagnosis for pathogen identification of unexplained pneumonia, lessons to be learned crispr-cas12-based detection of sars-cov-2 diagnostic testing for severe acute respiratory syndrome-related coronavirus-2: a narrative review artificial intelligence distinguishes covid-19 from community acquired pneumonia on chest ct development of a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov-2 global threat of sars-cov-2/covid-19 and the need for more and better diagnostic tools diagnosing covid-19: the disease and tools for detection in vitro diagnostic assays for covid-19: recent advances and emerging trends point-of-care rna-based diagnostic device for covid-19 rapid colorimetric detection of covid-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (rt-lamp) diagnostic plat-form: ilaco mapping the incidence of the covid-19 hotspot in iran-implications for travellers covid-19: preparing for superspreader potential among umrah pilgrims to saudi arabia geographical tracking and mapping of coronavirus disease covid-19/severe acute respiratory syndrome coronavirus 2 (sars-cov-2) epidemic and associated events around the world: how 21st century gis technologies are supporting the global fight against outbreaks and epidemics policy decisions and use of information technology to fight 2019 novel coronavirus disease prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal consider pregnancy in covid-19 therapeutic drug and vaccine trials rational vaccine design in the time of covid-19 bacillus calmette guérin (bcg) vaccination use in the fight against covid-19 -what's old is new again? sars-cov-2 rates in bcg-vaccinated and unvaccinated young adults high throughput and comprehensive approach to develop multiepitope vaccine against minacious covid-19 on the origin and continuing evolution of sars-cov-2 covid-19: the race for a vaccine all authors substantially contributed to the conception, design, analysis, and interpretation of data and checking and approving the final version of the manuscript, and we agree to be accountable for its contents.this compilation is a review article written, analyzed, and designed by its authors and required no substantial funding to be developed.all authors declare that there are no existing commercial or financial relationships that could, in any way, lead to a potential conflict of interest. with 25 years of research and teaching experience in the areas of microbiology, immunology, virology, public health, medicine, and biomedicine as an eminent researcher, he has developed several diagnostics, vaccines, immunomodulatory modules, and hypotheses to counter infectious diseases of animals, poultry, and public health concerns. he has to his credit 600 publications, 6 books, and 65 book chapters. dr. dhama has been recognized as an extremely productive researcher in the journal nature. he has been honored with 50 best paper awards and other recognitions. he is an naas (national academy of agricultural science, india) associate and has worked as nodal officer, wto, and member, wildlife health specialist group (iucn). he is actively serving as editor-in-chief, co-eic, editor, and member, editorial board, of nearly 20 scientific journals. his google scholar h-index is 47 and scopus h-index is 31. sharun khan, m.v.sc., is currently working as a researcher in the stem cell laboratory, division of surgery, icar-indian veterinary research institute, izatnagar, india. his area of interest is regenerative medicine with a focus on understanding cell biology and molecular pathways involved in the maintenance and differentiation of stem cells originating from different tissues. he has particular interest and knowledge in the fields of veterinary medicine, pharmacology, infectious diseases of animals, wildlife diseases, diagnosis and therapy of animal diseases, nutrition, and biomedicine. with excellent academic records, he has received awards and recognitions (fellowships and scholarships) and participated in national and international workshops, training programs, and courses. he has a keen interest in learning excellent scientific writing skills and has published 30 papers, including in international journals of repute. he is highly enthusiastic about gaining knowledge of advancements in educational and scientific research areas.ruchi tiwari is currently working as assistant professor in the department of veterinary microbiology, college of veterinary sciences, duvasu, mathura, india. she is currently pursuing her ph.d. (hons) degree from duvasu. with an excellent academic record and 10 years of research and teaching experience, she has expertise in the field of diagnosis, prevention, and control of important livestock/poultry diseases/pathogens having public health significance, along with particular reference to veterinary microbiology, immunology, ethnoveterinary medicine, alternative and complementary therapies, and bacteriophage therapy. dr. tiwari has published 150 research/review articles and 5 book chapters. she has been honored with the young scientist award, best paper awards (10) key: cord-286631-3fmg3scx authors: pormohammad, ali; ghorbani, saied; khatami, alireza; farzi, rana; baradaran, behzad; turner, diana l.; turner, raymond j.; bahr, nathan c.; idrovo, juan‐pablo title: comparison of confirmed covid‐19 with sars and mers cases ‐ clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta‐analysis date: 2020-06-05 journal: rev med virol doi: 10.1002/rmv.2112 sha: doc_id: 286631 cord_uid: 3fmg3scx introduction: within this large‐scale study, we compared clinical symptoms, laboratory findings, radiographic signs, and outcomes of covid‐19, sars, and mers to find unique features. method: we searched all relevant literature published up to february 28, 2020. depending on the heterogeneity test, we used either random or fixed‐effect models to analyze the appropriateness of the pooled results. study has been registered in the prospero database (id 176106). result: overall 114 articles included in this study; 52 251 covid‐19 confirmed patients (20 studies), 10 037 sars (51 studies), and 8139 mers patients (43 studies) were included. the most common symptom was fever; covid‐19 (85.6%, p < .001), sars (96%, p < .001), and mers (74%, p < .001), respectively. analysis showed that 84% of covid‐19 patients, 86% of sars patients, and 74.7% of mers patients had an abnormal chest x‐ray. the mortality rate in covid‐19 (5.6%, p < .001) was lower than sars (13%, p < .001) and mers (35%, p < .001) between all confirmed patients. conclusions: at the time of submission, the mortality rate in covid‐19 confirmed cases is lower than in sars‐ and mers‐infected patients. clinical outcomes and findings would be biased by reporting only confirmed cases, and this should be considered when interpreting the data. during the last two decades, coronaviruses have been recognized as one of the most critical human pathogenic viruses that affect global health and cause concern in the world health system. 1 coronavirus is classified into four genera: alpha, beta, delta, and gamma. major (about 4% mortality). 8 there is no vaccine or targeted treatment currently available for covid-19 infection. treatment is mostly supportive, although multiple experimental antiviral medications are being evaluated. 9, 10 thus, prevention and rapid diagnosis of infected patients are crucial. the trigger for rapid screening and treatment of covid-19 patients is based on clinical symptoms, laboratory, and radiographic findings that are similar to sars and mers infections. in this study, we attempted to distinguish the clinical symptoms, laboratory findings, radiographic signs, and outcomes of confirmed covid-19, sars, and mers patients. all findings are compared to determine unique features among each of them. these data could be helpful in the early diagnosis and prevention of infection as well as providing more reliable epidemiological data on a large-scale for health care policies and future studies. this study was conducted according to the preferred reporting items for systematic reviews and meta-analyses statement (prisma) guidelines, and it has been registered in the prospero database (id 176106). 11 we searched all studies published up to 28 february 2020, from the following databases: embase, scopus, pubmed, web of science, and the cochrane library. search medical subject headings (mesh) terms used were: "covid-19," "coronavirus," "severe acute respiratory syndrome," "sars virus," "severe acute respiratory syndrome coronavirus 2", "coronavirus infections," "middle east respiratory syndrome coronavirus," and all their synonyms like "wuhan coronavirus," "sars-cov-2," and "covid-19," "2019-ncov" and mers. moreover, we searched for unpublished and grey literature with google scholar, centre for disease controls (cdc) and who databases. we also examined references of included articles to find additional relevant studies. there was no language restriction, and all included studies were written in english or chinese languages; the latter was translated by https://translate.google.com/. additional search strategy details are provided in table s1 . duplicate studies were removed using endnote x7 (thomson reuters, new york, ny, usa). records were initially screened by title and abstract by independently four authors (ap, sg, ak, and rf). the full-text of potentially eligible records was retrieved and examined, and any discrepancies were resolved by consensus. studies had to fulfill the following predetermined criteria to be eligible for inclusion in our meta-analysis. all case-control, cross-sectional, cohort studies, case reports, and case series peer-reviewed studies were included if they reported the number of confirmed cases of patients with demographic data, [and] [or] clinical data, [and] [or] laboratory data, [and] [or] risk factor data. studies were excluded if they did not report the number of confirmed cases. letters to the editor, individual case reports, review articles, and news reports were also excluded. duplicate information from the same patient was combined and counted as a single case when the data was reported twice. all covid-19 included publications were published in 2020, and all patients were from china. the following items were extracted from each article: first author, center and study location in china, countries, sample collection time, patient follow-up time, the reference standard for infection confirmation, number of confirmed cases, study type, and all demographic, clinical, laboratory data, and risk factor data. three of our authors (sg, ak, and rf) independently extracted data, and all extracted data were checked randomly by another author (ap); differences were resolved by consensus. quality assessments of studies were performed by two reviewers independently according to the critical appraisal checklist recommended by the joanna briggs institute, 12 and disagreements were resolved by consensus. the checklist is composed of nine questions that reviewers addressed for each study. the "yes" answer to each question received one point. thus, the final scores for each study could range from zero to nine (table s2 ). data cleaning and preparation were done in microsoft excel 2010 (microsoft©, redmond, wa, usa), and further analyses were carried out via comprehensive meta-analysis software version 2.0 (biostat, englewood, nj). determination of heterogeneity among the studies was undertaken using the chi-squared test (cochran's q) to assess the appropriateness of pooling data. depending on the heterogeneity test, we used either random or fixed-effect models for pooled results. in the case of high heterogeneity (i2 > 50%), a random effect model (m-h heterogeneity) was applied, while in low heterogeneity cases (i2 < 50%), a fixed-effect model was used. 13 percentages and means ± sds were calculated to describe the distributions of categorical and continuous variables, respectively. p values reflect study heterogeneity with <.05 being significant. we also used the funnel plot, begg's, and egger's tests based on the symmetry assumption to detect publication bias ( figure s1 ). the process of study selection is displayed in figure 1 quality assessment of included studies was performed based on the critical appraisal checklist, and the final quality scores of the included studies are represented in table s2 . in brief, studies by chen et al, 14 wang et al, 17 huang et al, 18 guan et al, 19 zhang et al, 24 cheng et al, 25 li et al, 28 xu et al, 30 figure s2 ). cough was the second most common symptom presenting in covid-19 63% (95% ci 55.5-70, p < .001), sars 54.2% (95% ci 49-59, p < .001), and mers 61% (95% ci 51-70, p < .001) of patients ( figure s3 ). age is an exception, presented in mean age in years. (95% ci 2.7-13, p < .001), or runny nose 6% (95% ci 1-14, p < .001). more detail information about demographics and clinical characterization of covid-19 (table s3) , sars (table s4) , and mers patients (table s5 ) demonstrated in the supplementary material. the greatest risk for covid-19 patients 69.5% (95% ci 54.5-81, p < .001) up to 28 february 2020, is a history of recent travel to wuhan, contact with people from wuhan, or were wuhan residents, and 24.3% (95% ci 9.6-49, p < .001) had exposure at the seafood market(s (table s9) , sars (table s10) , and mers patients (table s11) is demonstrated in the supplementary material. most covid-19 confirmed patients required hospitalization 85.4% (95% ci 68-94, p < .001) and 20.6% (95% ci 6.7-48, p < .001) were deemed to be in critical condition. the mortality rate of covid-19 confirmed cases was 5.6% (95% ci 2.5-12.5, p < .001), sars 13% (95% 9-17, p < .001), and mers 35% (95% ci 31-39, p < .001) (figure 2 ). the laboratory findings showed that among a subset of patients 4.5% increased the percentage value. the actual mortality rate from covid-19 is almost certainly much lower than that found in this study. as more data emerges from screening asymptomatic or mildly symptomatic individuals in china and around the world, the exact mortality rate will be better understood. among covid-19, sars, and mers patients, leukocytosis was found in 13.3%, 28%, and 30%, respectively, and leukopenia in 26%, 32%, and 41%, respectively. most of the patients with coronavirus had abnormal chest radiological findings. on the other hand, runny nose and rhinorrhea are less common symptoms in coronavirus-infected patients, 127 and that it is typically expressed on pulmonary alveolar epithelial cells. 128 another study reported that following covid-19 infection deregulated cytokine/chemokine response and higher virus titer causes an inflammatory cytokine storm with lung immunopathological injury. 129 inflammation related to the cytokine storm in the lungs may then spread throughout the body via the circulation system. covid-19 patients have been reported to have increased plasma concentration of inflammation-related cytokines, including interleukin (il)-2,6,7,10, tumor necrosis factor-α (tnf-α), and monocyte chemoattractant protein i (mcp-i) especially in moribund patients. 130 several limitations of this study exist. publication bias and study heterogeneity are unavoidable in this type of study. therefore, it should be considered when interpreting the outcomes of the reports and our final data set. furthermore, this study likely overestimates disease severity due to a lack of screening for asymptomatic or mildly symptomatic individuals and subsequent publication bias related to these factors. likely, many infected persons have not been detected, thus falsely elevating the rates of hospitalization, critical condition, and mortality. the lower quality analysis and reporting in some of the included publications is another limitation of the study. to prevent language bias, we included reports in languages other than english. additionally, we searched for a variety of sites and databases to prevent internet platform bias. using egger's regression test, we did not find significant publication bias. journal bias is an issue facing those who carry out a meta-analysis, yet it does not usually affect the general conclusions. 132 however, we cannot reject the occurrence of other biases in this study, such as choice bias, since several journals are not indexed in embase, scopus, pubmed, web of science, and the cochrane library and unpublished data from some regions of the world. fever and cough are the most common symptoms of covid-19-, sars-, and mers-infected patients. the mortality rate in covid-19 confirmed cases was lower than sars-and mers-infected patients. clinical outcomes and findings may be biased by reporting only confirmed cases, and it should be considered when interpreting the data. a novel coronavirus outbreak of global health concern three emerging coronaviruses in two decades: the story of sars, mers, and now covid-19 severe acute respiratory syndrome (sars): epidemiology and clinical features middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster responding to covid-19-a once-in-a-century pandemic? rational use of personal protective equipment for coronavirus disease 2019 (covid-19) virtual screening of an fda approved drugs database on two covid-19 coronavirus proteins potential covid-2019 3c-like protease inhibitors designed using generative deep learning approaches preferred reporting items for systematic reviews and meta-analyses: the prisma statement the development of a critical appraisal tool for use in systematic reviews: addressing questions of prevalence statistical aspects of the analysis of data from retrospective studies epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study early epidemiological analysis of the 2019-ncov outbreak based on a crowdsourced data clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 2019 novel coronavirus infection in china epidemiological and clinical features of the 2019 novel coronavirus outbreak in china analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia. chin j tuberc respir dis [zhonghua jiehe he huxi zazhi chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia clinical characteristics of 140 patients infected by sars-cov-2 in wuhan kidney impairment is associated with in-hospital death of covid-19 patients. medrxiv imaging profile of the covid-19 infection: radiologic findings and literature review characteristics of covid-19 infection in beijing early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan, china clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series emerging 2019 novel coronavirus (2019-ncov) pneumonia ct imaging features of 2019 novel coronavirus (2019-ncov) characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case coronavirus-positive nasopharyngeal aspirate as predictor for severe acute respiratory syndrome mortality severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of 144 patients with sars in the greater toronto area investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto 34% mortality rate from sars in critically ill patients at 28 days in toronto coronavirus as a possible cause of severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) clinical manifestations, laboratory findings, and treatment outcomes of sars patients clinical presentations and outcome of severe acute respiratory syndrome in children severe acute respiratory syndrome: radiographic appearances and pattern of progression in 138 patients severe acute respiratory syndrome: lessons from singapore a cluster of cases of severe acute respiratory syndrome in hong kong cluster of cases of severe acute respiratory syndrome among toronto healthcare workers after implementation of infection control precautions: a case series epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients a major outbreak of severe acute respiratory syndrome in hong kong a prediction rule for clinical diagnosis of severe acute respiratory syndrome severe acute respiratory syndrome: clinical outcome and prognostic correlates haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital: prospective observational study accuracy of clinical diagnosis versus the world health organization case definition in the amoy garden sars cohort description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china national microbiology laboratory, canada; canadian severe acute respiratory syndrome study team.identification of severe acute respiratory syndrome in canada viral loads in clinical specimens and sars manifestations clinical description of a completed outbreak of sars in vietnam in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds clinical picture, diagnosis, treatment and outcome of severe acute respiratory syndrome (sars) in children clinical course and management of sars in health care workers in toronto: a case series detection of sars coronavirus in patients with suspected sars serology of severe acute respiratory syndrome: implications for surveillance and outcome severe acute respiratory syndrome among children prevalence of subclinical infection and transmission of severe acute respiratory syndrome (sars) in a residential care home for the elderly seroprevalence of sars coronavirus among residents near a hospital with a nosocomial outbreak identification and containment of an outbreak of sars in a community hospital an outbreak of severe acute respiratory syndrome among hospital workers in a community hospital in hong kong severe acute respiratory syndrome: radiographic review of 40 probable cases in toronto clinical and laboratory features in the early stage of severe acute respiratory syndrome princess margaret hospital sars study group.outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients viral load distribution in sars outbreak acute renal impairment in coronavirus-associated severe acute respiratory syndrome severe acute respiratory syndrome in taiwan: analysis of epidemiological characteristics in 29 cases cluster of sars among medical students exposed to single patient detection of sars-associated coronavirus in throat wash and saliva in early diagnosis investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study assessing the detection of middle east respiratory syndrome coronavirus igg in suspected and proven cases of middle east respiratory syndrome coronavirus infection surveillance and testing for middle east respiratory syndrome coronavirus seroprevalence of middle east respiratory syndrome coronavirus (mers-cov) in public health workers responding to a mers outbreak in seoul, republic of korea epidemiological status of the middle east respiratory syndrome coronavirus in 2019: an update from current epidemiological status of middle east respiratory syndrome coronavirus in the world from 1.1. 2017 to 17.1. 2018: a cross-sectional study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome in 3 persons, south korea ksa mers-cov investigation team.hospital outbreak of middle east respiratory syndrome coronavirus multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia increase in middle east respiratory syndrome-coronavirus cases in saudi arabia linked to hospital outbreak with continued circulation of recombinant virus clinical characteristics and outcome of icu admitted mers corona virus infected patients outbreak of middle east respiratory syndrome at tertiary care hospital case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region, saudi arabia mers-cov outbreak in jeddah-a link to health care facilities epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital middle east respiratory syndrome coronavirus infections in health care workers the korean society of infectious diseases.clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection mortality risk factors for middle east respiratory syndrome outbreak, south korea association between severity of mers-cov infection and incubation period middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients the predictors of 3-and 30-day mortality in 660 mers-cov patients diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia preliminary epidemiologic assessment of mers-cov outbreak in south korea clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea transmission of merscoronavirus in household contacts association of higher mers-cov virus load with severe disease and death, saudi arabia predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia hospital outbreaks of middle east respiratory syndrome middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data spatial modelling of contribution of individual level risk factors for mortality from middle east respiratory syndrome coronavirus in the arabian peninsula presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon covid-19: who declares pandemic because of "alarming levels" of spread, severity, and inaction comparison of severe and non-severe covid-19 pneumonia: review and meta-analysis. medrxiv single-cell rna expression profiling of ace2, the putative receptor of wuhan immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients angiotensin-converting enzyme 2 in acute respiratory distress syndrome empirical assessment of effect of publication bias on meta-analyses supporting information section at the end of this article. how to cite this article comparison of confirmed covid-19 with sars and mers cases -clinical characteristics, laboratory findings, radiographic signs and outcomes: a systematic review and meta-analysis none. the authors have declared that no conflict of interests. key: cord-262542-vevsgkp6 authors: alharbi, naif khalaf; padron-regalado, eriko; thompson, craig p.; kupke, alexandra; wells, daniel; sloan, megan a.; grehan, keith; temperton, nigel; lambe, teresa; warimwe, george; becker, stephan; hill, adrian v.s.; gilbert, sarah c. title: chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice date: 2017-06-27 journal: vaccine doi: 10.1016/j.vaccine.2017.05.032 sha: doc_id: 262542 cord_uid: vevsgkp6 abstract the middle east respiratory syndrome coronavirus (mers-cov) has infected more than 1900 humans, since 2012. the syndrome ranges from asymptomatic and mild cases to severe pneumonia and death. the virus is believed to be circulating in dromedary camels without notable symptoms since the 1980s. therefore, dromedary camels are considered the only animal source of infection. neither antiviral drugs nor vaccines are approved for veterinary or medical use despite active research on this area. here, we developed four vaccine candidates against mers-cov based on chadox1 and mva viral vectors, two candidates per vector. all vaccines contained the full-length spike gene of mers-cov; chadox1 mers vaccines were produced with or without the leader sequence of the human tissue plasminogen activator gene (tpa) where mva mers vaccines were produced with tpa, but either the mh5 or f11 promoter driving expression of the spike gene. all vaccine candidates were evaluated in a mouse model in prime only or prime-boost regimens. chadox1 mers with tpa induced higher neutralising antibodies than chadox1 mers without tpa. a single dose of chadox1 mers with tpa elicited cellular immune responses as well as neutralising antibodies that were boosted to a significantly higher level by mva mers. the humoral immunogenicity of a single dose of chadox1 mers with tpa was equivalent to two doses of mva mers (also with tpa). mva mers with mh5 or f11 promoter induced similar antibody levels; however, f11 promoter enhanced the cellular immunogenicity of mva mers to significantly higher magnitudes. in conclusion, our study showed that mers-cov vaccine candidates could be optimized by utilising different viral vectors, various genetic designs of the vectors, or different regimens to increase immunogenicity. chadox1 and mva vectored vaccines have been safely evaluated in camels and humans and these mers vaccine candidates should now be tested in camels and in clinical trials. middle east respiratory syndrome (mers) is caused by a novel betacoronavirus (mers-cov) that was isolated in late 2012 in saudi arabia [1] . the syndrome (mers) is described as a viral infection that causes fever, cough, and/or shortness of breath and to a lesser extent gastrointestinal symptoms such as diarrhea [2] . severe disease from mers-cov infection can cause respiratory failure and organ failure, and cases can be fatal, especially in patients with co-morbidities such as diabetes and cardiac complications. however, the infection can be asymptomatic or mild in many cases [3] [4] [5] [6] [7] . mers-cov has spread to 27 countries and infected more than 1900 humans with a mortality rate of 40% [2] . dromedary camels, especially juveniles, contract the infection and shed the virus, without notable symptoms of disease; this is now known to have been occurring since the early 1980s [8] [9] [10] [11] [12] [13] . the mechanism of camel to human transmission is still not clear, but several primary cases have been associated with camel contact, which is considered an important risk factor [14] [15] [16] . therefore, camels http [8] [9] [10] [11] [12] [13] . other livestock animals such as sheep, goats, cows, chicken, and horses have proved seronegative in many studies [17] [18] [19] [20] . further, these animals did not productively contract mers-cov when they were inoculated experimentally [21, 22] . therefore, to date, dromedary camels are the only confirmed animal reservoir. there is currently no approved vaccine against mers-cov for camels or humans despite active vaccine research and development. a number of vaccine candidates have been developed using various platforms and regimens and have been tested in several animal models [23] . viral vectors are potent platform technologies that have been utilised to develop vaccines against malaria, tuberculosis, influenza, hiv, hcv, ebola, and many viral pathogens. these vectors include adenoviruses, poxviruses, yellow fever viruses, and alphaviruses [24, 25] , and they are preferred for their ability to induce cellular immune responses in addition to humoral immunity. here, we report development of mers-cov vaccine candidates that are based on two different viral vectors: chimpanzee adenovirus, oxford university #1 (chadox1) [26] and modified vaccinia virus ankara (mva) [27, 28] . each viral vector was developed by generating two alternative versions, resulting in four vaccine candidates that all encode the same complete mers-cov spike gene (s). the two chadox1 based vaccines were produced with or without the signal peptide of the human tissue plasminogen activator gene (tpa) at the n terminus. previous studies have shown that encoding tpa upstream of recombinant antigens enhanced immunogencity, although results differed depending on the antigens employed. the tpa encoded upstream of influenza a virus nucleoprotein, in a dna vector, enhanced both cellular and humoral immune responses in mice [29, 30] , whereas the same leader sequence resulted in increased humoral sequences but decreased cellular responses to hiv gag [30] . the two mva based vaccines were produced with either the mh5 or f11 poxviral promoter driving antigen expression, both including the tpa sequence at the n terminus of mers-cov spike protein. previously, we reported the ability of the strong early f11 promoter to enhance cellular immunogenicity of vaccine antigen candidates for malaria and influenza, as compared to utilising p7.5 or mh5 early/late promoters which resulted in a lower level of gene expression immediately after virus infection of target cells, but higher levels at a later stage [31] . here, we continue to assess the f11 promoter in enhancing cellular immunogenicity, and to investigate its ability to impact on humoral immune responses. the four vaccine candidates were evaluated in a number of different regimens in mouse models that showed a single dose of chadox1 mers inducing higher cellular and humoral immunogenicity than a single dose of mva mers, or equivalent to two doses of mva mers. chadox1 based vaccines have been tested in different animal models, including camels [32] , and in human clinical trials and proved safe and immunogenic [33] . therefore, based on our data, chadox1 mers can be readily developed for use as a mers vaccine in humans. furthermore, utilising chadox1 mers for camel vaccination can serve the one-health approach whereby blocking mers-cov transmission in camels is expected to prevent human infections. the spike (s) gene of mers-cov camel isolate (genbank accession number: kj650098.1) was synthesised by geneart gene synthesis (thermo fisher scientific). the s transgene was then cloned into four shuttle plasmid vectors following in-fusion cloning (clontech). two plasmids contained the s transgene within the e1 homologous region of chadox1, driven by the human cytomegalovirus major immediate early promoter (ie cmv) that includes intron a. one of the chadox1 shuttle plasmids was designed to include the tpa signal sequence upstream of the transgene sequence while the second plasmid did not contain the tpa. the chadox1 shuttle plasmids contained the s transgene within gateway ò recombination cassettes. to construct mva mers, one of the shuttle plasmids for mva was designed to have the upstream and downstream (flanks) of the f11l orf as homologous sequence arms. inserting the s transgene within these arms enabled the utilisation of the endogenous f11 promoter, which is part of the right homologous arm, while deleting the native f11l orf. this resulted in the shuttle vector for generation of f11-mva mers (f11 shuttle vector). the mh5 promoter sequence was subcloned upstream of the s transgene; and this mh5-s transgene was then subcloned into the f11 shuttle vector. this resulted in the shuttle vector for generation of mh5-mva mers (f11/mh5 shuttle vector). mh5-mva mers contained the mh5 promoter at the f11l locus, however, the endogenous f11 promoter is intact and located upstream of the mh5 promoter. the endogenous f11 promoter could not be replaced with the mh5 since it is part of the essential upstream orf. the chadox1 shuttle plasmid, described above, was used to validate the expression of mers-cov spike protein in vitro. an african green monkey kidney cell line (vero cells) was seeded into 6-well plate to 80% confluence. then the plasmid dna was transfected into vero cells using lipofectamine ò 2000 (thermo fisher scientific) following manufacturer's instruction. twenty-four hours after transfection, cells were fixed, permeabilised, and immunostained using a rabbit polyclonal anti-mers-cov spike antibody, following standard protocols. dapi stain was used to label nuclei. the chadox1 mers vaccines were prepared by gateway ò recombination between the chadox1 destination dna bac vector (described in [26] ) and entry plasmids containing the coding sequence for mers-cov spike gene (chadox1 shuttle vectors explained above), according to standard protocols. chadox1 mers genomes were then derived in hek293a cell lines (invitrogen, cat. r705-07), the resultant viruses were purified by cscl gradient ultracentrifugation as previously described [34] . the titres were determined on hek293a cells using anti-hexon immunostaining assay based on the quicktiter tm adenovirus titer immunoassay kit (cell biolabs inc). for mva mers vaccines chicken embryo fibroblast cells (cefs) were infected with mva parental virus that encodes dsred marker instead of the native f11l orf and transfected with mva shuttle plasmids containing mers-cov spike gene (explained above) to allow recombination with the mva genome and deletion of dsred marker whilst keeping the f11 promoter sequence. recombinant mva expressing mers-cov s protein was purified by plaque-picking and fluorescent selection using the sorting function of cyclone robotic module of a moflo flow cytometer (dako cytomation, denmark) as previously described [31] . f11-mva mers and mh5-mva mers were confirmed to lack the native f11l orf (and the dsred marker), and contain mers-cov s by pcr (identity and purity pcr screening). the sequence of the s transgene amplified from these vaccines was confirmed. the recombinant viruses (vaccines) were amplified in 1500 cm 2 monolayers of cefs cells, partially purified over sucrose cushions and titrated in cefs cells according to standard practice, and purity and identity were again verified by pcr. female balb/c mice (harlan, uk) aged 6-8 weeks were immunised intramuscularly (i.m.) in the upper leg (total volume 50 ml) with a total of 10 8 iu of chadox1 mers with or without tpa or with a total of 10 6 pfu of either f11-mva mers or mh5-mva mers. for induction of short-term anaesthesia, animals were anaesthetised using vaporised isofloh. in prime only regimens, mice were vaccinated with chadox1 with blood samples taken at 14 days post immunisation (d.p.i) or 28 d.p.i. for serum isolation; and spleens were collected at 28 d.p.i. in heterologous prime-boost regimens, mice were vaccinated with chadox1 mers and boosted with mva mers at 28 d.p.i; mice were bled at 28 d.p.i. splenocytes were harvested for analysis by ifn-c elispot or intracellular cytokine staining (ics) and flow cytometry as previously described [35, 36] , using re-stimulation with 2 mg/ml s291 mers-cov s-specific peptide (vydtikyysiiphsi); for vaccine cellular immunogenicity [37] ); or 1 mg/ml e3 and f2(g) mva vectorspecific peptides [38] (for anti-mva immune responses). in the absence of peptide re-stimulation, the frequency of ifn-c + cells, which was typically 0.1% by flow cytometry or less than 50 sfc by elispot, was subtracted from tested re-stimulated samples. 2 lg/ml with capturing antigen (s1 recombinant protein from mybiosource, ca, usa) were used to coat elisa plates, and standard endpoint elisa protocol was followed, as previously described [39] . sera were prepared in a 10-fold serial dilution in pbs/t and then 50 ll were plated in duplicate wells. serum from a naïve balb/c mouse was included as a negative control. goat anti-mouse total igg conjugated to alkaline phosphatase (sigma) and pnpp tablet (20 mg p-nitrophenylphosphate, sigma) substrate were used in the assay. mers pseudotyped viral particles (merspp) were produced and titrated using huh7.5 cell line as described previously [40] . for the merspp neutralisation assay, serum samples were serially diluted in 96-well white plates (nunc). a standard concentration of the merspp were added to the wells and plates were incubated for 1 h at 37°c. after incubation, huh7.5 cells (10,000 cells per well) were added to the plate in duplicates. following 48 h incubation, cells were lysed and luciferase activity was measured. ic90 neutralisation titres were calculated for each mouse serum sample using graphpad prism. induction of virus-neutralising antibodies was confirmed according to previously published protocols [37, 41] . briefly, mouse serum samples were tested for their capacity to neutralise mers-cov (emc isolate) infections in vitro with 100 50% tissue culture infective doses (tcid 50 ) in huh-7 cells. sera of non-immunised mice served as negative control. graphpad prism (graphpad software) was used for statistical analysis and to plot data. all animal procedures were performed in accordance with the terms of the uk animals (scientific procedures) act (aspa) for the project licenses 30/2414 or 30/2889 and were approved by the university of oxford animal care and ethical review committee. all mice were housed for at least 7 days for settlement prior to any procedure in the university animal facility, oxford, uk under specific pathogen free (spf) conditions. the spike gene from a camel isolate (camel/qatar_2_2014 mers-cov isolate, genbank accession number kj650098.1) was cloned into four shuttle vectors that facilitate homologous recombination with the genome of chadox1 or mva. four recombinant viral vectors, two chadox1 and two mva, were derived as described in the materials and methods. chadox1 based vaccine candidates were generated with or without the signal peptide of the human tissue plasminogen activator gene (tpa). the spike transgene expression in chadox1 mers vaccine candidates is under the control of the human cytomegalovirus major immediate early promoter (cmv ie) that includes intron a. in mva mers vaccine candidates, the tpa was also inserted upstream of the spike transgene, which was under the control of either the ectopic mh5 promoter or the endogenous f11 promoter (fig. 1a) . all of our mers-cov vaccine candidates contain the same codonoptimized spike transgene. the expression of the newly synthesized transgene was first tested by transfection of an african green monkey kidney cell line (vero cells) with the adenovirus shuttle vector, and immunofluorescence staining of the transfected cells ( fig. 1b and 1c) . this was performed to confirm the expression of the codon optimized spike transgene in mammalian cells. the level of transgene expression from the four vaccine candidates was not evaluated in vitro. we have previously reported that differences in mva promoter activity detectable in vitro does not correlate with in vivo immunogenicity [31] , and that only in vivo expression correlates with the in vivo immunogenicity. to evaluate humoral immune responses to chadox1 mers with or without tpa, balb/c mice were vaccinated with 1 â 10 8 iu of chadox1 intramuscularly. serum samples from 14 and 28 d.p.i. were collected and evaluated by elisa. both vaccine candidates induced a high level of s1-specific antibodies (mean endpoint titre (log 10 ) = 4.8 with tpa, 4.7 without tpa), unlike the control vaccine, chadox1 encoding enhanced green fluorescent protein (chadox1-egfp, mean endpoint titre (log 10 ) = 1). these antibody levels were similar between the two candidates (with or without tpa) at day 14. however, at 28 d.p.i. chadox1 mers with tpa induced significantly higher s1-specific antibodies than chadox1 mers without tpa (mean endpoint titre (log 10 ) = 5.13 with tpa, 4.6 without tpa, fig. 2a ). serum samples from day 28 were selected for merspp neutralisation assay. serum antibodies induced by chadox1 mers with tpa showed significantly higher neutralisation activity than without tpa (mean titre ic 90 (log 10 ) = 2.8 with tpa, 2.2 without tpa; fig. 2b ). in order to confirm that the psuedotyped virus neutralisation assay was producing biologically relevant results, serum samples from mice immunised with chadox1 mers with tpa were also tested in a neutralisation assay utilising wildtype mers virus. this assay confirmed the neutralisation activity of mouse antibodies (nab) with a median of 360 vnt (virus neutralization test antibody titre; fig. 2c ). we therefore continued to evaluate chadox1 mers with tpa in addition to generating mva mers vaccine candidates with tpa. having established the utility of tpa in chadox1 mers vaccines (referred to as chadox1 mers in the rest of this report) at increasing humoral responses, spleens were collected at 28 d.p.i. from immunised balb/c mice. splenocytes were processed to evaluate cellular immune responses to chadox1 mers in elispot and intracellular cytokine staining (ics). peptide s291, described by others [37] , was used to re-stimulate the cells in both assays and elispot data showed a high level of ifn-c secreting splenocytes (median = 1300 sfu/10 6 splenocytes; fig. 3a ). ics data confirmed the ifn-c secreting cd8 + splenocytes also secreted tnf-a and il-17 (fig. 3b ). to evaluate humoral immune responses to heterologous primeboost vaccination, balb/c mice were immunised with chadox1 were collected and evaluated by elisa and merspp neutralisation assay. at 28 d.p.i. chadox1 mers induced similar levels of s1-specific antibodies and nab as observed previously ( fig. 4a and b) . at 42 d.p.i. s1-specific antibodies were boosted to a higher level (mean endpoint titre (log 10 ) = 5 by chadox1 mers boosted to 5.8 by mh5-mva mers or 5.9 by f11-mva mers); fig. 4a ) with nab also enhanced to a statistically significant level (mean titre ic 90 (log 10 ) = 2.87 by chadox1 mers boosted to 3.3 by mh5-mva mers or 3.5 by f11-mva mers; fig. 4b ). there was no difference in antibody levels induced using either the f11 or mh5 promoter in the mva. at 42 d.p.i. splenocytes were also processed to evaluate cellular immune responses to chadox1 mers mva mers prime-boost vaccination in elispot and ics as shown in fig. 3 . the t cell responses to mers s were boosted by the mva vaccinations; in the ics experiments, f11-mva and mh5-mva boosted the percentage of ifn-c + splenic cd8 + t cells to 7.3 and 5.2% respectively (fig. 4d) whereas the percentage was 2.5% after chadox1 mers prime in fig. 3b . the percentage of tnf-a + splenic cd8 + t cells were also increased by mva boost (comparing fig. 3b and 4d) . utilising the f11 promoter resulted in a trend towards greater cell-mediated immunogenicity ( fig. 4c and d) . splenocytes were also re-stimulated with mva backbone-specific e3 and f(g)2 peptides and evaluated in ics. both mva based vaccines induced similar responses to e3 or to f(g)2 peptides, 2 weeks after mva vaccination (fig. 4e and f) . this similarity confirmed the efficiency of vaccine titration, vaccination, and sample processing because responses to each of those peptides are not expected to be different unless there is variation in the doses administered or sample preparation. overall, mva mers vaccines were able to boost the humoral and cellular immune responses to cha-dox1 mers prime vaccination. there was no difference between the f11 and mh5 promoter in the resulting antibody titres after cha-dox1 prime/mva boost, but there was a trend towards increased cellular immunogenicity when the f11 promoter was used. to evaluate humoral immune responses to a homologous mva mers prime-boost vaccination, two groups of balb/c mice were immunised with f11-mva mers or mh5-mva mers and boosted with the same vaccine after three weeks. serum samples from 21 d. antibodies (mean endpoint titre (log 10 ) = 3.2 and 2.8 respectively; fig. 5a ). at 42 d.p.i s1-specific antibody levels had increased to 4.7 and 4.8 respectively (fig. 5a) . the titres of nab (mers pp assay) were also similar for both vaccines (mean titre ic 90 (log 10 ) = 2.71 (f11-mva mers) and 2.76 respectively; fig. 5b ). utilising different promoters in mva vectors did not result in differences in the induced antibody levels. however, at 42 d.p.i. ifn-c secreting splenocytes induced by f11-mva mers were statistically significantly higher than those of mh5-mva mers ((median = 525 and 249 sfu/10 6 splenocytes, respectively, fig. 5c ). both mva vaccines induced similar vector-specific immune responses as expected ( fig. 5d and e) . vaccines against mers-cov have been developed and tested in a number of animal models (including non-human primates [42] [43] [44] and camels [45] ) as well as in human clinical trials [46] . all vaccine candidates focused on the spike antigen because it contains the receptor-binding domain used for cell entry by the virus, against which neutralising antibodies may be induced, and it is conserved. therefore, the improvement of mers-cov vaccines focuses on platform and vaccination regimens rather than antigen selection and optimisation. here, we focused on using the same antigen (transgene) to develop a vaccine against mers-cov, and to assess different vectors, different versions of each vector, and different vaccination regimens. we generated a number of mers-cov vaccine candidates based on the same codon optimized spike transgene and ensured its expression in vitro before we evaluated the humoral and cellular immunogenicity in a pre-clinical balb/c mouse model. chadox1 based vaccine candidates were produced with or without tpa. the tpa signal peptide was predicted to enhance the humoral immunogenicity of encoded vaccine antigens, based on previous reports [29] . our data supported this hypothesis and showed a significant increase in the s1-specific antibody levels at 28 d.p.i. the level of neutralising antibodies was also increased when tpa was utilised. however, chadox1 mers without tpa was still a potent vaccine candidate, inducing a high level of both s1-specific binding antibodies and mers-cov neutralising antibodies. neutralisation activity of mouse serum antibodies was assayed by using mers-cov pseudotyped viral particles (merspp), an approach used by a number of researchers for other human pathogens such as hiv, influenza, and hcv to overcome the necessity of handling bsl-3 viruses [40] . additionally, we confirmed the ability of serum samples from vaccinated mice to neutralise live mers virus. we therefore selected chadox1 mers with tpa (simply referred to chadox1 mers) for further evaluation. chadox1 mers also induced cellular responses for mers s, with polyfunctional cd8 + t cells detected in the spleen of immunised mice. this supports the potency of the chadox1 viral vector in inducing t cellular immunity, observed previously in animal models [26, 32, 47] as well as in humans [33] . following chadox1 prime/mva boost, mva significantly boosted the neutralising antibody titres to higher levels. no difference in humoral immunity was found when either the f11 or mh5 promoter was used. regarding the promoter effect on mva cellular immunogenicity, we have previously reported that utilising the f11 promoter enhanced malaria and influenza antigens in mva [31] . here, we again report that f11-mva mers induced higher t cell responses than mh5-mva mers in a homologous prime-boost mva mers vaccination. all of our vaccine candidates induced humoral (with nab) and cellular immune (with polyfunctional cd8 + t cell) responses against mers-cov spike antigen. modest effects on immunogenicity of different versions of the vaccines were noted, with the use of the tpa leader sequence in chadox1, and the use of the f11 promoter in mva producing small increases in immunogenicity compared to no leader sequence, or the mh5 promoter. the protective level of either antibodies or cellular immunity required to counter mers-cov infection in humans or in animal models is not yet defined, despite some efforts [48] [49] [50] [51] . the ideal vaccine would provide rapid onset of immunity and complete protective efficacy after a single dose, with a long duration of immunity. complete protective efficacy of one dose of chadox1 expressing the external glycoprotein of rift valley fever virus has been demonstrated in multiple species and it is already known that chadox1 rvf is highly immunogenic in camels [32] . to date, the only vaccine against mers to be tested in camels is an mva vectored vaccine [41] which was protective in hdpp4 transgenic mice immunised with a homologous prime/boost regimen [37] but in camels required two doses given both intranasally and intramuscularly to provide partial protection and reduction of virus shedding [45] . here we find that a single dose of chadox1 mers is as immunogenic as two doses of mva mers, suggesting that this regimen should be tested for protective efficacy in camels. however if this is not completely protective, administration of mva mers as a heterologous boost should be considered next. in our hands one dose of mva resulted in an endpoint titre of 3 logs, two doses of mva produced 4.7 logs, one dose of chadox1 produced 5 logs, and chadox1/mva prime boost produced 5.9 logs. if a single dose of chadox1 mers is not protective and a two dose regimen is required, chadox1/mva would be more likely to provide complete protection than mva/mva. chadox1 mers should now be evaluated for immunogenicity and efficacy in larger animal species, including both camels and humans. scg is a co-founder of, consultant to and shareholder in vaccitech plc which is developing vectored influenza and mers vaccines. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates mers-cov outbreak in jeddah-a link to health care facilities state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus infections in health care workers mers coronavirus neutralizing antibodies in camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity. euro surveillance: bull eur sur les maladies transmissibles = presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan risk factors for primary middle east respiratory syndrome coronavirus illness in humans human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection mers-cov at the animal-human interface: inputs on exposure pathways from an expert-opinion elicitation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study livestock susceptibility to infection with middle east respiratory syndrome coronavirus inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding vaccines against middle east respiratory syndrome coronavirus for humans and camels viruses as vaccine vectors for infectious diseases and cancer yellow fever 17d as a vaccine vector for microbial ctl epitopes: protection in a rodent malaria model a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity clinical applications of attenuated mva poxvirus strain clinical development of modified vaccinia virus ankara vaccines immunization with plasmid dna encoding influenza a virus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immune responses and protection against h5n1 challenge in mice post-translational intracellular trafficking determines the type of immune response elicited by dna vaccines expressing gag antigen of human immunodeficiency virus type 1 (hiv-1) enhancing cellular immunogenicity of mva-vectored vaccines by utilizing the f11l endogenous promoter chimpanzee adenovirus vaccine provides multispecies protection against rift valley fever clinical assessment of a novel recombinant simian adenovirus chadox1 as a vectored vaccine expressing conserved influenza a antigens preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors single-dose protection against plasmodium berghei by a simian adenovirus vector using a human cytomegalovirus promoter containing intron a recombination-mediated genetic engineering of a bacterial artificial chromosome clone of modified vaccinia virus ankara (mva) protective efficacy of recombinant modified vaccinia virus ankara (mva) delivering middle east respiratory syndrome coronavirus spike glycoprotein poxvirus cd8 + t-cell determinants and cross-reactivity in balb/c mice effective induction of high-titer antibodies by viral vector vaccines an optimised method for the production of mers-cov spike expressing viral pseudotypes middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge evaluation of candidate vaccine approaches for mers-cov an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels army scientists begin first mers vaccine clinical trial. dod news, defense media activity immunogenicity and efficacy of a chimpanzee adenovirusvectored rift valley fever vaccine in mice characterization of anti-mers-cov antibodies against various recombinant structural antigens of mers-cov in an imported case in china comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity kinetics of serologic responses to mers coronavirus infection in humans viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection key: cord-293938-40zyv1h8 authors: jonsdottir, hulda r.; dijkman, ronald title: coronaviruses and the human airway: a universal system for virus-host interaction studies date: 2016-02-06 journal: virol j doi: 10.1186/s12985-016-0479-5 sha: doc_id: 293938 cord_uid: 40zyv1h8 human coronaviruses (hcovs) are large rna viruses that infect the human respiratory tract. the emergence of both severe acute respiratory syndrome and middle east respiratory syndrome covs as well as the yearly circulation of four common covs highlights the importance of elucidating the different mechanisms employed by these viruses to evade the host immune response, determine their tropism and identify antiviral compounds. various animal models have been established to investigate hcov infection, including mice and non-human primates. to establish a link between the research conducted in animal models and humans, an organotypic human airway culture system, that recapitulates the human airway epithelium, has been developed. currently, different cell culture systems are available to recapitulate the human airways, including the air-liquid interface (ali) human airway epithelium (hae) model. tracheobronchial hae cultures recapitulate the primary entry point of human respiratory viruses while the alveolar model allows for elucidation of mechanisms involved in viral infection and pathogenesis in the alveoli. these organotypic human airway cultures represent a universal platform to study respiratory virus-host interaction by offering more detailed insights compared to cell lines. additionally, the epidemic potential of this virus family highlights the need for both vaccines and antivirals. no commercial vaccine is available but various effective antivirals have been identified, some with potential for human treatment. these morphological airway cultures are also well suited for the identification of antivirals, evaluation of compound toxicity and viral inhibition. respiratory diseases caused by human coronavirus infection are of both medical and socio-economic importance. currently, they are studied in various model systems, ranging from cell lines to animal models. originally, the importance of hcovs in the burden of human disease was underestimated and as a result, no general therapy exists to treat coronavirus induced disease in humans. furthermore, no commercial vaccine is available leaving the human population vulnerable to emerging coronavirus infections. both the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses have recently crossed the species barrier and entered the human population to cause severe disease. in this review, we summarize the current knowledge on human coronavirus infection emphasizing the usefulness of organotypic human airway cultures as a model system. coronaviruses (covs), a subfamily of the coronaviridae family, are positive strand rna viruses with the largest genome of all known rna viruses (≥27 kb). the genomic rna is capped, polyadenylated and associated with nucleocapsid proteins within an enveloped virion. the envelope is covered by the characteristic surface glycoprotein that gives the virus particles their characteristic crown-like (latin: corona) appearance [1] . all covs share a common genome organization where the replicase gene encompasses the 5′-two thirds of the genome and is comprised of two overlapping open reading frames (orfs), orf1a and orf1b that encode for up to 16 non-structural proteins. the structural gene region, which covers the 3′-third of the genome, encodes the canonical set of structural protein genes in the order 5′ -spike (s) -envelope (e) -membrane (m) and nucleocapsid (n) -3′. the structural gene region also harbors several orfs that are interspersed along the structural protein coding genes. the number and location of these accessory orfs vary between the cov species [2, 3] . in animals, cov infections are mainly associated with respiratory and enteric disease and can have large economical impact on the veterinary industry, e.g. porcine epidemic diarrhea virus (pedv) causes gastrointestinal disease in pigs [4] , infectious bronchitis virus (ibv) causes severe kidney and respiratory disease in chicken [5] and bovine coronavirus (bcov) causes both respiratory disease and diarrhea in cattle [6] . additionally, cov infections can have other disease manifestations, such as central nervous system (cns) involvement, hepatitis and peritonitis [7] [8] [9] [10] . in humans, cov infections are mainly associated with respiratory diseases that are considered to have a large impact on the economy due to reduced productivity of the working population. currently, 6 coronaviruses that cause disease in humans have been discovered. four of those are commonly circulating and two have caused epidemics of severe acute respiratory disease. the first human coronavirus (hcov), b814, was described in 1965. in the following years, over 30 additional strains were characterized. ten of those strains could only be isolated from primary embryonic tracheal organ culture. others were readily isolated from monolayer cultures and were antigenically related to the prototype strain hcov-229e. hcov-oc43, for organ culture 43, was isolated and found to be distinct from the 229e prototype strain [11, 12] . in the subsequent decades, research on hcovs would center on these two distinct viruses. however, in 2002, an unknown respiratory illness, termed severe acute respiratory syndrome (sars), surfaced in asia. research determined it to be caused by a novel coronavirus [13, 14] . at the end of the epidemic, this virus had infected over 8000 people, most in china, and caused 774 deaths [15] . following the discovery of this virus, two additional covs causing human disease were identified. hcov-nl63 was isolated in the netherlands in 2004 from an infant with bronchiolitis [16] and hcov-hku1 in 2005 from a patient with pneumonia in hong kong [17] . in 2012, another respiratory hcov, middle east respiratory (mers)-cov, was isolated from a patient with pneumonia in saudi-arabia [18] . unlike sars-cov, this virus is still intermittently present in the human population and most recently caused a large outbreak in south-korea [19] . to date, there have been over 1600 cases and almost 600 deaths related to mers-cov infection [20] . out of the 6 known human coronaviruses, hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1 are commonly circulating in the human population and usually cause general respiratory illness and cold symptoms in healthy individuals [21] [22] [23] . like influenza, these viruses are capable of causing more severe disease in the immunocompromised and the elderly [24] . they infect the human airway from the luminal side and progeny viruses are released from the same side facilitating spread through coughing and sneezing [25, 26] . these coronaviruses are responsible for approximately 5-10% of all upper and lower respiratory tract infections [27] [28] [29] but the interactions between them and their natural host cells are poorly understood. currently, it is hypothesized that most of the human coronaviruses may have originated from bats [30, 31] . for example, hcov-229e is believed to originate from african hipposiderid bats possibly using camelids as intermediate hosts [32] . in the last 15 years, two coronaviruses have crossed the species barrier and caused severe and fatal disease in humans. sars-cov surfaced in 2002 and mers-cov in 2012 [13, 14, 18] . as opposed to the commonly circulating viruses, which generally only cause mild respiratory symptoms, these viruses presented with higher case fatality ratios, around 10 and 20-50% respectively [33, 34] . currently, there is abundant phylogenetic evidence for the bat origin of sars-cov, based on sequences of sars-like viruses found among bats in the recent years [35] [36] [37] . the initial transmissions of sars-cov from animals to humans were traced back to the live animal wet markets and it was hypothesized that the virus made its way into the human population using the civet cat as an intermediate host. however, successful isolation of sars-like viruses from bats [38] and the fact that a contemporary bat sars-like virus can infect human airway cultures [39] suggest that an intermediate host between humans and bat might not have been needed for the transmission of sars-cov. the evolutionary origin of mers-cov is less clear but it has been speculated to be bats as well. characterization of an african bat virus closely related to mers-cov shows that both the human and camel strains belong to the same viral species and phylogenetic analysis suggests that mers-cov infection in camels predates that in humans, suggesting that camels infect humans and not the other way around. furthermore, the bat virus roots the phylogenetic tree providing further evidence for the bat origin of mers-cov [40] . additionally, human-tohuman transmission, although not robust, seems to happen simultaneously as camel-to-human transmission. therefore, any further adaptation of mers-cov to the human host must be monitored carefully and intermediate hosts identified [41] . many bat coronaviruses have been identified in the recent years further highlighting the zoonotic potential of this family of viruses [30] . given the documented history of coronaviruses overcoming the species barrier and causing severe disease in humans, it is important to investigate the zoonotic potential of close evolutionary relatives of common hcovs in a culture model that recapitulates the aspects of the human airway, e.g. morphology and receptor distribution. it's important to study the mechanisms of pathogenesis and the evolution of zoonotic viruses in detail in order to identify molecular determinants that affect either transmission or pathogenesis. it's also important to elucidate whether coronaviruses currently circulating in animals are a potential danger to the human population. all of the known cellular receptors of hcovs belong to the same protein family, the membrane ectopeptidases. interestingly, the catalytic activity of these peptidases is not required for viral entry but rather the co-expression of other host peptidases activates the hcov spike proteins [42, 43] . it has been established that the human transmembrane serine proteases tmprssii and hat cleave and activate the hcov-229e, sars-and mers-cov spike proteins during viral entry [44, 45] . out of the four commonly circulating coronaviruses, hcov-229e is the only one that infects non-ciliated cells using the human aminopeptidase n (hapn) as its receptor [46] . this peptidase is predominantly expressed on non-ciliated cells in the human bronchus [47] . sars-cov and hcov-nl63 both utilize the angiotensin converting enzyme 2 (ace2) for cellular binding [48, 49] . ace2 is expressed on ciliated bronchial cells along with endothelial cells and both type i and ii alveolar cells [50] . mers-cov was found to use a different receptor than sars-cov, namely the dipeptyl-peptidase 4 (dpp4) [51] . dpp4 is widely expressed in endothelial cells and various epithelial tissues in the human body [52] . in ex vivo human lung organ cultures, different tropism of sars-and mers-covs was observed. mers-cov can actively replicate in both bronchial and alveolar tissue while sars-cov primarily replicates in alveolar tissue [53] . the wide cellular tropism of mers-cov might contribute to its associated disease severity and high mortality rate whereas the alveolar replication of sars-cov would explain why it generally presents with pneumonia. the cellular surface receptors for hcov-oc43 and hcov-hku1 are currently unknown but receptor determinants for these two viruses have been identified as n-acetyl-9-o-acetylneuraminic acid and o-acetylated sialic acid, respectively [54, 55] . all of these viruses can be successfully cultured and investigated in hae cultures [56, 57] . the discovery of hcovs, their receptor usage, cell tropism and receptor binding domain (rbd) is summarized in table 1 . furthermore, established reverse genetic systems for hcov-229e [58] , hcov-oc43 [59] and hcov-nl63 [60] allow for controlled virus mutation and fluorescent transgene insertion to better understand the interaction of these viruses with their pulmonary host cells. traditionally, respiratory viruses are studied in animal models, usually mice and ferrets [48, 61] . however, it is not always possible to correctly recapitulate human infection and disease in animal models. the establishment of transgenic animal models for human disease is attainable when either the virus receptor has been identified, which is not the case for all hcovs, or when viruses can be adapted to a different host. an adapted human virus may not share the same properties as the original human virus. sars-cov was found to replicate naturally in various strains of inbred mice but to enhance clinical signs of disease the hace2 was introduced into these mice. this resulted in murine models with varying degree of human disease similarity. since sars-cov already replicated in mouse cells, adapting it to the murine host was quite successful. this resulted in three mouse adapted strains that caused disease in mice similar to severe sars-cov cases in humans [62] . in an effort to establish a mouse model for hcov-229e infection transgenic hapn mice were created. however, the insertion of the hapn into mouse cells is not enough to establish robust hcov-229e infection in vivo. nevertheless, cells isolated from these transgenic animals could be infected in vitro [63, 64] . the emergence of both sars-and mers-covs emphasized the importance of establishing animal models for human coronaviruses. currently, a few animal models for mers-cov have been established. mice carry their own variant of the viral receptor ddp4 that differs from the human in regions important for mers-cov spike interaction and by replacing this receptor with the human one, mers-cov can infect mouse cells but the method of hdpp4 insertion has an effect on the degree of pathogenesis observed in these mice [65, 66] . various non-human primates (nhps) can be naturally infected with both sars-and mers-covs. however, disease presentation and pathogenesis differs between the different subspecies and nhp models are expensive, although ideal to study human infection due to their genetic similarity [62] . to establish a link between the research conducted in animal models and humans, an organotypic airway culture system resembling the human airway epithelium has been developed. this model is a universal platform to study human respiratory viruses [67] [68] [69] [70] . they have been used successfully for infection studies with all known human coronaviruses [56, 57] . furthermore, the cultures can be inoculated with a low infectious dosage to mimic natural infection in the human airway. whereas, animal models often require both high dosage and artificial inoculation routes. organotypic cell cultures are becoming increasingly common. different cell culture models exist to depict different epithelial tissues [71] . these cultures closely resemble their tissue of origin and contain various different cell types with distinctive roles in the polarized tissue. currently, various organotypic cell culture models exist to represent the different areas of the human airways. the human lungs span a long anatomical distance and carry out different functions depending on anatomical location [72, 73] . the structure of the epithelium also differs the further you descend into the airways. tracheal and bronchial epithelium is columnar and pseudostratified, with every cell in contact with the basement membrane, while the epithelium in the alveoli is comprised of a single cell layer to facilitate air-exchange [74] . tracheobronchial cells are one of the first targets of human respiratory viruses and can be cultured in airliquid interface (ali) where the apical side of the cell layer is exposed to air while the basolateral side is submerged in medium. tracheobronchial cells cultured in that way form a pseudostratified epithelial layer that both morphologically and functionally resembles the human upper conducting airway (fig. 1a) [75, 76] . after differentiation, these cultures contain many different cell types such as basal, ciliated and goblet cells. they also produce protective mucus, much like in vivo epithelium. when compared to primary bronchial cells in submerged two-dimensional culture, the gene expression of primary ali cultures differs significantly. however, the expression pattern of primary human bronchial ali cultures is comparable to that of in vivo epithelium. the human bronchial cell line calu-3 has been used as a culture model for respiratory epithelium but its gene expression in ali cultures is more similar to submerged bronchial cell cultures than differentiated epithelium [77] . disruption of the cell layer [57] . therefore, the primary tracheobronchial ali culture model is especially fitting for human respiratory virus research since it accurately recapitulates the primary entry point for these viruses. by using these cultures, virus replication and host interactions can be studied in natural target cells. further establishing the usefulness of this system hcov-hku1 was propagated for the first time in ciliated cells of bronchial hae cultures in 2010 after culturing it in conventional cell lines had failed [26] . alveolar epithelial ali cultures (fig. 1b) can also be used for virus-host interaction studies and are especially applicable when a viral infection causes pneumonia and alveolar damage [79] . hcov-hku1 has also been propagated in alveolar hae cultures and exhibits a strong tropism for alveolar type ii cells and causes large syncytia formation upon infection [80] . when compared to traditional two dimensional cell cultures, the hae cultures are more cumbersome and their preparation is time consuming but they do have an advantage over traditional monolayer cell cultures when it comes to virus-host interaction studies. different types of ali cultures used for virus research are summarized in table 2 . within the respiratory epithelium the innate immune system has a major protective role as the first line of defense against respiratory pathogens. in particular, the interferon (ifn) system orchestrates hundreds of different cellular effector proteins that (i) protect the epithelial barrier by altering the physiological and cellular environment, (ii) impair virus propagation, spread and transmission, and (iii) shape the host's adaptive immune response. recent publications have demonstrated that the innate immune system is functional in the hae cell culture system and that most pathogen recognition receptors are expressed and up-regulated upon treatment with exogenous stimuli [57, 81] . in general, hcovs do not elicit a strong innate immune response in primary target cells of the human airway early during infection. despite the presence of all major pathogen recognition receptors, no elevated expression of ifn beta, pro-inflammatory cytokines or interferon stimulated genes can be observed up to 12 h post-infection in haes infected with hcov-229e, mers-or sars-covs [57] . this is most likely due to the intrinsic cov properties harbored in the replicative non-structural proteins that actively aid in avoiding recognition by the host innate immune system. for example, the 5′ termini of the viral mrna are capped making them indistinguishable from the host cellular mrnas and no longer detectable by cellular sensors. furthermore, cov replication is associated with the appearance of double membrane vesicles (dmvs) in the host cell cytoplasm, which might serve as a protective shield for viral rna to prevent recognition by cytoplasmic rna sensors [82] [83] [84] [85] . in addition to the non-structural proteins, various cov accessory proteins have been discovered to inhibit interferon signaling at different stages of the host innate immune response. for example, mers-cov accessory protein 4a inhibits innate antiviral signaling by suppressing the activation of mda5 and rigi [86, 87] whereas 4b inhibits the induction of the ifn-beta promoter [88] . while orf 4a and 4b are ifn antagonists in the genome of mers-cov, sars-cov orf3b antagonizes ifn signaling through mavs/rigi [89] . whereas sars-cov orf6 disrupts ifn signaling by blocking the nuclear translocation of stat1 [89, 90] . these discoveries highlight that hcovs employ similar yet different strategies despite that respiratory infections with hcovs can result in severe respiratory disease there are currently no effective prophylactic or therapeutic treatment options available. however, the emergence of novel coronaviruses has emphasized the need to develop effective treatment options. for example, vaccines using the spike proteins of both sars-and mers-covs have proven protective in animal models [91, 92] suggesting that a vaccine against hcovs for human use might be achievable. additionally, various drugs that inhibit hcov infection at different stages of the replication cycle have been reported and some could potentially serve as treatment options for hcov associated severe respiratory disease. for example, patients presenting with severe respiratory disease, caused by sars-or mers-covs, are generally treated with steroids and interferon, sometimes in combination with the antiviral drug ribavirin [93] [94] [95] [96] . this treatment, however, is not especially effective highlighting the need for hcov specific antivirals. many different compounds have been determined to have anti-hcov activity. for example, protease inhibitors which suppress hcov entry [97] [98] [99] , cyclosporin a (csa) treatment blocks the replication of coronaviruses from all subgroups [100] and non-immunosuppressive derivatives of csa represent a possible therapeutic option for both human and animal cov infections. hcov infection can also be inhibited by pre-treating hae cultures with either recombinant ifn alpha or lambda [57] . similar effect has also been shown for recombinant ifn alpha and beta which could inhibit mers-cov in ex vivo lung cultures [53] . as previously described, ifn treatment of active hcov infection is not particularly effective in vivo. therefore, the use of ifn in humans might be limited to prophylactic treatment of exposed persons and/or health care workers treating infected patients. screenings of compound libraries have also resulted in the identification of some hcov specific antivirals. for example, a novel small compound inhibitor (k22) has been identified, and showed to be effective against a broad spectrum of covs and could inhibit both hcov-229e and mers-cov in hae cultures [101] . additionally, hcov-nl63 has been inhibited in hae cultures with polymer-based compounds [102] . to date, most treatment and inhibitor studies have been conducted in hcov susceptible cell lines. however, the hae cultures represent an ideal system to test the application and efficacy of those already identified, and new, antiviral compounds against hcovs in cells that represent the primary site of replication. furthermore, the hae cultures are heterogenous, containing many different cellular sub-populations, and would allow for the evaluation of compound toxicity and effect in a differentiated layer similar to human airway epithelium. compounds already shown to inhibit hcovs in cell lines should be applied to hae cultures as well before any animal or human trials. hcov induced respiratory diseases are of both medical and socio-economic importance. the emergence of sars-and mers-cov and the yearly circulation of the four common hcovs highlight the importance of elucidating the different mechanisms employed by hcovs to evade the host immune system as well as identifying antiviral compounds and human vaccine candidates. the hae culture system is based on primary human cells offering a unique platform to study respiratory viruses in cells representing the primary entry point of these viruses, bronchial epithelial cells, or investigate the interaction of hcovs and the distal airways, in type i and ii alveolar cells. additionally, the inclusion of airway epithelial cultures for other species enables the study of zoonosis and animal-to-human transmission. currently, many aspects of hcov infection and pathogenesis remain to be determined. the hae culture system, both tracheobronchial and alveolar, represents a unique platform to study virus-host interaction in natural target cells at the molecular level. these cultures are becoming more common and more relevant to hcov research. especially, for those viruses for which there is no animal model, as they provide an organotypic substitute for virushost interaction studies. the authors declare no competing interests. authors' contribution hrj wrote the review, designed tables and figures. rd revised the text, tables and figures. both authors read and approved the final manuscript. this work was supported by the 3r foundation, switzerland (project 128-11) and the university of bern initiator grant. received: 17 december 2015 accepted: 27 january 2016 coronaviruses: an overview of their replication and pathogenesis coronavirus genome structure and replication the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses a new coronavirus-like particle associated with diarrhea in swine a nomenclature for avian coronavirus isolates and the question of species status enteric disease in postweaned beef calves associated with bovine coronavirus clade 2 cleavage of a neuroinvasive human respiratory virus spike glycoprotein by proprotein convertases modulates neurovirulence and virus spread within the central nervous system the chemokine receptor cxcr2 and coronavirus-induced neurologic disease acute hepatic failure in ifn-gamma-deficient balb/c mice after murine coronavirus infection detection of feline coronavirus spike gene mutations as a tool to diagnose feline infectious peritonitis a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome summary of probable sars cases with onset of illness from 1 identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus outbreak in the republic of korea effects of a "new" human respiratory virus in volunteers enzyme-linked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus strain 229 e the behaviour of recent isolates of human respiratory coronavirus in vitro and in volunteers: evidence of heterogeneity among 229e-related strains the "common cold" in frail older persons: impact of rhinovirus and coronavirus in a senior daycare center human coronavirus 229e infects polarized airway epithelia from the apical surface culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures human coronavirus-associated influenza-like illness in the community setting in peru high incidence but low burden of coronaviruses and preferential associations between respiratory viruses the pediatric burden of human coronaviruses evaluated for twenty years ecology, evolution and classification of bat coronaviruses in the aftermath of sars evidence supporting a zoonotic origin of human coronavirus strain nl63 evidence for an ancestral association of human coronavirus 229e with bats estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea the emergence of the middle east respiratory syndrome coronavirus (mers-cov). pathogens and disease orf8-related genetic evidence for chinese horseshoe bats as the source of human severe acute respiratory syndrome coronavirus identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in china novel sars-like betacoronaviruses in bats, china isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor a sars-like cluster of circulating bat coronaviruses shows potential for human emergence rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat spread, circulation, and evolution of the middle east respiratory syndrome coronavirus ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence membrane ectopeptidases targeted by human coronaviruses. current opinion in virology tmprss2 activates the human coronavirus 229e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response human aminopeptidase n is a receptor for human coronavirus 229e expression of aminopeptidase n and dipeptidyl peptidase iv in the healthy and asthmatic bronchus severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures human coronavirus hku1 spike protein uses o-acetylated sialic acid as an attachment receptor determinant and employs hemagglutinin-esterase protein as a receptor-destroying enzyme analysis of cellular receptors for human coronavirus oc43 isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus recovery of a neurovirulent human coronavirus oc43 from an infectious cdna clone systematic assembly of a full-length infectious clone of human coronavirus nl63 animal models for the study of influenza pathogenesis and therapy animal models for sars and mers coronaviruses development of a transgenic mouse model susceptible to human coronavirus 229e cells of human aminopeptidase n (cd13) transgenic mice are infected by human coronavirus-229e in vitro, but not in vivo rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium human bocavirus 1 infects commercially available primary human airway epithelium cultures productively infection of human airway epithelium by human and avian strains of influenza a virus human bocavirus can be cultured in differentiated human airway epithelial cells non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology airway epithelial cells: current concepts and challenges alveolar development and disease building and maintaining the epithelium of the lung serial culturing of human bronchial epithelial cells derived from biopsies ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface the air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia bilateral entry and release of middle east respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome-coronavirus human coronavirus hku1 infection of primary human type ii alveolar epithelial cells: cytopathic effects and innate immune response transcriptome analysis reveals a classical interferon signature induced by ifnlambda4 in human primary cells sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum 2'-o methylation of the viral mrna cap evades host restriction by ifit family members ribose 2'-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 middle east respiratory syndrome coronavirus 4a protein is a double-stranded rnabinding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study hostdirected therapies for improving poor treatment outcomes associated with the middle east respiratory syndrome coronavirus infections ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus novel polymeric inhibitors of hcov-nl63 isolation from man of "avian infectious bronchitis virus-like" viruses (coronaviruses) similar to 229e virus, with some epidemiological observations human coronavirus 229e: receptor binding domain and neutralization by soluble receptor at 37 degrees c growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease the sars-cov s glycoprotein: expression and functional characterization identification of residues in the receptor-binding domain (rbd) of the spike protein of human coronavirus nl63 that are critical for the rbd-ace2 receptor interaction the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome we would like to thank prof. dr. volker thiel for his careful review of the manuscript and dr. eveline kindler for providing components for figures. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-282279-zmfcfbo8 authors: lee, sang min; kang, won sub; cho, ah-rang; kim, tae; park, jin kyung title: psychological impact of the 2015 mers outbreak on hospital workers and quarantined hemodialysis patients date: 2018-11-30 journal: comprehensive psychiatry doi: 10.1016/j.comppsych.2018.10.003 sha: doc_id: 282279 cord_uid: zmfcfbo8 abstract objectives this study aimed to assess the immediate stress and psychological impact experienced by quarantined patients undergoing hemodialysis and university hospital workers who treated patients middle east respiratory syndrome (mers) during its outbreak. design the group of subjects consisted of 1800 hospital practitioners and 73 quarantined patients undergoing hemodialysis. the impact of events scale–revised (ies-r) was administered to the practitioners twice, once during the hospital shutdown and again one month after the shutdown. the mini international neuropsychiatric interview and hospital anxiety and depression scale were administered to patients undergoing hemodialysis. results during the initial stages of the mers outbreak, healthcare workers who performed mers-related tasks scored significantly higher on the total ies-r and its subscales. in the second assessment of the high-risk group, the sleep and numbness subscale scores from the ies-r differed depending on the implementation of home quarantine, and the intrusion subscale scores differed depending on the performance of mers-related tasks. conclusion medical staff that performed mers-related tasks showed the highest risk for post-traumatic stress disorder symptoms even after time had elapsed. the risk increased even after home quarantine. prompt and continuous psychiatric intervention is needed in high mortality infectious disease outbreaks. an outbreak of the middle east respiratory syndrome coronavirus (mers-cov) infection occurred in korea from may to december 2015, resulting in 186 cases of infection, 38 deaths (20.4% of total cases), and 16,692 exposed individuals experiencing home quarantine for two weeks [1] . it was defined as a disaster and the management of the outbreak was led by the korean centers for disease control and prevention (kcdc), which was then responsible for the oversight of our hospital. about 3.8% of 186 cases of mers and 2% of 16,692 home quarantine cases were linked to our hospital. 0.1% of all hospital workers were confirmed to be mers positive and 18.9% were home quarantined. kyung hee university hospital at gangdong, located in seoul, shut down its artificial kidney center and implemented cohort quarantine after a patient undergoing hemodialysis for renal failure was confirmed on june 17, 2015 to have mers, the first in our hospital and the 165th patient nationally. after the 167th patient with mers was confirmed on june 19, 2015, it was decided, for the first time in korea, that all 104 patients actively undergoing treatment in the artificial kidney center at x hospital would be hospital quarantined in private rooms and receive inpatient care, and the hospital was immediately closed until july 13, 2015. according to research on other infectious diseases, including severe acute respiratory syndrome (sars) and ebola virus disease, many healthcare workers experience severe emotional stress during the outbreak [2] [3] [4] . it has also been shown that healthcare workers experience burnout, traumatic stress, anxiety, and depressive symptoms even after the outbreak [4] . previous research has found that the prevalence of post-traumatic stress disorder (ptsd) was also increased among survivors of infectious diseases [5] . although the emergence of sars stimulated related research, there remains little research on the psychological impact of people directly exposed to infectious disease outbreaks [6] . there are few known studies till to date about the effect of mers infection on mental health [7] . those studies that are available have analyzed factors related to symptoms of anxiety and anger after home quarantine during the mers outbreak [8] and reported that individuals experienced increased anxiety with a higher degree of knowledge about the disease during the initial stages of the mers outbreak, and a greater trust in unofficial information [9] . the present study aimed to understand the immediate stress and psychological impact experienced by healthcare workers who treated patients mers during the outbreak period, and hospital quarantined patients, especially undergoing hemodialysis, who had been exposed to mers. at the time of the mers outbreak, 1800 hospital workers were on duty at kyung hee university hospital at gangdong. case of mers was identified in the artificial kidney center and a total of 73 patients undergoing hemodialysis were quarantined in the hospital. these hospital workers and patients were eligible for inclusion in this study, which was approved by the ethics review committee of the medical research institute, kyung hee university hospital at gangdong (irb no. 2017-09-029). the impact of events scale-revised (ies-r) korean version was used to assess the psychological distress in hospital workers. the ies-r, developed by weiss and marmar is a 22-item, 5-point scale (0, not at all; 1, a little bit; 2, moderately; 3, quite a bit; 4, extremely) with a cronbach's alpha of 0.93. the ies-r korean version yields a total score (ranging from 0 to 88) and subscale scores which can be calculated for the hyperarousal (consisting of the following items: 4, 10, 14, 18, 19, 21) , avoidance (consisting of the following items: 5, 8, 11, 12, 17, 22) , intrusion (consisting of the following items: 1, 3, 6, 9, 16) , and sleep and numbness (consisting of the following items: 2, 7, 13, 15, 20) subscales of ptsd [10] . the internal consistency of the ies-r korean version was 0.69-0.83 [11] . in the korean version, a total score equal to or n25 was suggested to indicate a diagnosis of ptsd, and a score equal to or n18 was indicative of the presence of ptsd-like symptoms. the hospital anxiety and depression scale (hads) has 14-items and a 4-likert scale (0-3) and was administered to the 73 patients in hospital quarantine undergoing hemodialysis [12] . the cronbach's alphas were 0.89 and 0.86 [13] . patients were then assessed on the mini international neuropsychiatric interview (mini). an abbreviated self-rated korean version of the mini was developed for the screening of common anxiety disorders (panic disorder, general anxiety disorder, and social anxiety disorder) and major depressive disorder (mdd). participants were suggested of having a panic disorder if they scored n4 points, general anxiety disorder (gad) if the score was n3 points, social anxiety disorder if the score was n4 points, and mdd if the score was n5 points in the respective modules. the kappa values were in the expected range of 0.49 for panic disorder, 0.60 for gad, 0.60 for social anxiety disorder, and 0.59 for mdd [14] . the ies-r was administered via email and mobile devices to 1800 practitioners that were on duty during the outbreak period. the survey was administered twice, once during the hospital shutdown and once a month after the shutdown was cleared (fig. 1) . the hads was administered during the admission process to the 73 patients undergoing hemodialysis that were in quarantine at the hospital. the mini was assessed by telephone during the patients' hospitalization period. differences across symptoms in the ies-r scores depending on home quarantine and the performance of mers-related tasks were examined using a t-test or anova. post-hoc comparisons were conducted to examine the differences across job types. the statistical significance level was set at 1% for two-sided tests. the software package spss 11.0 for windows was used for statistical analyses. of the 1800 hospital practitioners who were on duty during the mers outbreak, 359 completed the first survey. the total response rate was 19.9%. the response rates stratified by employment group were as follows: doctor (33.1% of total workers), 5%; technician (10.4%), 29.4%; nurse (31.5%), 34.6%; pharmacist (2.1%), 21.7%; administrative (10.1%), 17%; others (12.8%), 17%. the first survey consisted of 294 female (81.9%) and 65 male (18.1%) respondents, with 182 respondents in their 30s (50.7%), the most well-represented age group. there were 196 nurses (54.6%), who formed the majority of respondents, followed by 55 medical technicians (15.3%), 31 administrators (8.6%), 30 doctors (8.4%), 8 pharmacists (2.2%), and 39 others (10.9%). ninety-two respondents (25.6% of total respondents) experienced home quarantine, and 185 (51.5%) performed mers-related tasks. the mean ies-r score was 26.3 ± 19.09. of all respondents, 230 (64.1%) received a score of 18 or higher, indicating the presence of ptsd-like symptoms, while 183 respondents (51.5%) exceeded the cut-off score of 25 for a diagnosis of ptsd. those with ies-r scores of 25 or higher in the first survey were designated as the high-risk group, and administered a second survey after six weeks, to which 77 responded (response rate 43.8%). the effect size between the first and second survey was 1.18 (cohen's d, t = 10.07, p b .001). the second survey was comprised of 70 female (90.9%) and 7 male (9.1%) respondents, with 44 respondents in their 30s (57.1%), the largest age group. most respondents in the second survey were nurses, who accounted for 53 responses (68.8%), followed by 7 medical technicians, 7 administrators (9.1% each), 3 doctors, 3 pharmacists (3.9% each), and 4 others (5.2%). twenty-three (29.9%) respondents in the second survey experienced home quarantine, and 50 (64.9%) performed mers-related tasks. the ies-r identified 42 respondents (54.5%) as having the presence of ptsd-like symptoms, and 31 (40.3%) as being eligible for a diagnosis of ptsd. in the mean ies-r scores of the first survey respondents, there was a significant difference between those who performed mers-related tasks and those who did not [related vs unrelated, 30.02 ± 19.55 vs 22.33 ± 17.70, t = −3.894, p b .001]. post-hoc comparisons were conducted to examine differences across job types. while the difference between nurses and doctors approached the p value of .048, no statistically significant differences were found (table 1) . we compared ies-r scores and characteristics between respondents who completed the second survey and those who did not: ies-r score ( 14) . no significant differences were found between the two groups. for respondents in the first and second surveys, ies-r scores were compared across symptoms, depending on the implementation of home quarantine and performance of mers-related tasks. the proportion of staff involved in caring for mers patients was 18.89%; 27.1% of them participated in the survey. in the first survey, the healthcare workers who performed mersrelated tasks had significantly higher total ies-r scores (t = 3.894, p b .001) and sub-scores, including hyperarousal (t = 3.535, p b .001), avoidance (t = 3.573, p b .001), intrusion (t = 3.756, p b .001), and sleep and numbness (t = 3.583, p b .001), than those who did not ( table 2 ). in the second survey, the home quarantined healthcare workers showed higher scores in the sleep and numbness sub-score (p = .03) than those who were not quarantined, and the healthcare workers who performed mers-related tasks had higher scores in the intrusion sub-score (p = .03) than those who did not; however, these showed no statistical significance. during the mers outbreak, a total of 73 patients undergoing hemodialysis were quarantined in the hospital, including 41 male (56.2%) and 32 female (43.8%) patients. their mean age was 61.3 ± 13.03 years. during the hospital quarantine, 62 patients (84.9%) were hospitalized for 7 to 14 days. the mini was administered to the patients undergoing hemodialysis; 4 (5.5%) scored 5 or above on the a -mdd item and 8 (11%) scored 3 or above on the b -gad item. in the hads, 8 patients (11%) scored 8 or above on the anxiety item and 11 (15.1%) scored 8 or above on the depression item (table 3) . the main results of the present study indicate that during the initial stages of the mers outbreak, healthcare workers who performed mersrelated tasks scored significantly higher on the total ies-r and all symptom subscales; this suggests that they be the main targets of psychiatric assessment and care. these results are consistent with the findings from research regarding the 2003 sars outbreak demonstrating that 18 to 57% of healthcare workers experienced severe emotional distress during and immediately after the outbreak [2, 3, [15] [16] [17] . during the acute infection stage, performing mers-related tasks could have resulted in serious psychological distress. that is, in the present study, the group that performed mers-related tasks during the acute infection stage scored significantly higher on the ies-r than the group that did not perform such tasks (mean ± standard deviation, 30.02 ± 19.55 vs 22.33 ± 17.70). the higher scores persisted above 25 even after time had elapsed (related vs unrelated, 26.08 ± 22.15 vs 17.0 ± 14.09). thus, this suggests that hospital workers who performed mers-related tasks were at risk of experiencing ptsd-like symptoms even after some time had passed. while the trauma appears to play the most important etiological role in the ptsd symptoms, there are some predisposing vulnerability factors suggested, such as childhood trauma, personality disorder traits, an inadequate support system, comorbid psychiatric illness, and excessive alcohol intake [18] . based on this, we assume that some respondents with persisting high ies-r scores during the second survey might have these vulnerability factors rather than the trauma itself, mers-related task. there were no differences in the ies-r scores in the first survey respondents between those who did and did not experience home quarantine. however, both the home quarantine and the non-quarantine groups had mean ies-r scores of 25 or above, which can be categorized as reaching the threshold for ptsd diagnosis. this may be due to the fact that the first survey was conducted at the acute infection stage of the mers outbreak, and that overall, respondents had high scores with a mean of 26.3. although the mean ies-r scores in the second survey differed between respondents who were and were not home-quarantined (28.3 ± 20.19 vs 20.74 ± 19.74), the difference was not statistically significant. however, among the second survey respondents, the home-quarantined group had a mean ies-r score of 25 or higher, suggesting that the effect of home quarantine is strong enough to result in levels that would categorize them with a ptsd diagnosis, even after the acute infection period. in the assessment of the high-risk group determined from the first survey, the ies-r sleep and numbness subscores tended to be higher in the home-quarantined group (p = .03) during the second survey. this is consistent with the results of a study conducted during the 2014 nigerian ebola viral disease outbreak, which examined the psychological distress of survivors and their close contacts and found that loss of sleep over worry was the second most frequent complaint [19] . thus, taken together, we suggest that even after time has passed following the acute infection period, sleep and numbness symptoms are persistent in healthcare workers and survivors, emphasizing the importance of assessment and management. a number of recent, large, and well-conducted studies have confirmed markedly increased rates of depression among those with chronic kidney disease, with a meta-analysis suggesting the prevalence of interview-defined depression to be approximately 20% [20] . a german explorative study on the frequency of mdd and gad reported that gad was diagnosed in 9 patients (17%), while 12 patients (23%) scored higher than seven points on the hads anxiety subscale [21] . in the present study of hospital quarantined renal patients, the proportion of depression assessed using an abbreviated mini was 5.5%, while when it was assessed using hads was 15.1%. the proportion of patients with gad assessed using the abbreviated mini and hads was 11%. patients undergoing hemodialysis, who received care while being quarantined for a month at the hospital after the mers outbreak, showed a good level of adjustment and had low rates of reporting psychological distress (table 3 ). it appears that this is because the quarantined patients wanted to receive inpatient care and hemodialysis in the first place, and because psychological support was provided by the hospital psychiatrists immediately after patients were quarantined. the limitations of the present study are as follows. first, there are limitations to generalizing the sample, as the response rates by employment group were different, which may be due to a restricted sampling during the outbreak, as the subjects were healthcare workers and patients at a local hospital that treated mers patients. second, as only a survey method was used for the hospital workers, qualitative methods, including interviews, could not be simultaneously conducted. third, the survey was limited to the mers outbreak, and did not consider other potential variables, such as preexisting psychiatric conditions or individual personality issues before the outbreak. another limitation is that the sampling of this study was voluntary and conducted by e-mail and mobile devices. therefore, the possibility of a self-selection sampling bias as a factor accounting for the prevalence of psychiatric morbidity should be considered. despite these limitations, this study examined the medical staff and patients at a hospital directly affected by the mers outbreak, and demonstrated the effect of stress caused by mers on their sociopsychological health. the results of this study suggest that medical staff that performed mers-related tasks were at a higher risk of displaying symptoms of ptsd even after time had passed, and the risk was increased in sleep and numbness-related symptoms in particular with the implementation of home quarantine. previously, when sars occurred in south korea, the awareness regarding mental health issues related to infectious diseases was low and no psychological guidelines existed for large-scale quarantine and isolation situations during the pandemic period [16, 22] . therefore, there should be more awareness regarding this population as targets for psychiatric care, and prompt and continuous psychiatric intervention for outbreaks of life-threatening, epidemicpotential infectious diseases, in which the psychiatrist's role should be emphasized. korea ministry of health and welfare and center for disease control and prevention. updates on mers: for press release severe acute respiratory syndrome (sars) in hong kong in 2003: stress and psychological impact among frontline healthcare workers factors associated with the psychological impact of severe acute respiratory syndrome on nurses and other hospital workers in toronto prevalence of psychiatric disorders among toronto hospital workers one to two years after the sars outbreak posttraumatic stress disorder in convalescent severe acute respiratory syndrome patients: a 4-year follow-up study long-term psychiatric morbidities among sars survivors middle east respiratory syndrome in south korea during 2015: risk-related perceptions and quarantine attitudes mental health status of people isolated due to middle east respiratory syndrome worry experienced during the 2015 middle east respiratory syndrome (mers) pandemic in korea assessing psychological trauma and ptsd: a practitioner's handbook a study on reliability and validity of the korean version of impact of event scale-revised the hospital anxiety and depression scale a study on the standardization of the hospital anxiety and depression scale for koreans: a comparison of normal, depressed and anxious groups a validation study of the abbreviated self-rated korean version of mini (mini patient health survey) psychological impact of the 2003 severe acute respiratory syndrome outbreak on health care workers in a medium size regional general hospital in singapore coping responses of emergency physicians and nurses to the 2003 severe acute respiratory syndrome outbreak psychosocial effects of sars on hospital staff: survey of a large tertiary care institution kaplan & sadock's synopsis of psychiatry: behavioral sciences/clinical psychiatry an evaluation of psychological distress and social support of survivors and contacts of ebola virus disease infection and their relatives in lagos, nigeria: a cross sectional study-2014 depression and chronic kidney disease: a review for clinicians prevalence and correlates of anxiety and depression in patients with end-stage renal disease (esrd) emergency preparedness and response: crisis and emergency risk communication (cerc) this research did not receive a specific grant from any funding agencies in the public, commercial, or not-for-profit sectors. key: cord-289311-0wgafqdz authors: kim, jee-eun; heo, jae-hyeok; kim, hye-ok; song, sook-hee; park, sang-soon; park, tai-hwan; ahn, jin-young; kim, min-ky; choi, jae-phil title: neurological complications during treatment of middle east respiratory syndrome date: 2017-06-30 journal: j clin neurol doi: 10.3988/jcn.2017.13.3.227 sha: doc_id: 289311 cord_uid: 0wgafqdz background and purpose: middle east respiratory syndrome (mers) has a high mortality rate and pandemic potential. however, the neurological manifestations of mers have rarely been reported since it first emerged in 2012. methods: we evaluated four patients with laboratory-confirmed mers coronavirus (cov) infections who showed neurological complications during mers treatment. these 4 patients were from a cohort of 23 patients who were treated at a single designated hospital during the 2015 outbreak in the republic of korea. the clinical presentations, laboratory findings, and prognoses are described. results: four of the 23 admitted mers patients reported neurological symptoms during or after mers-cov treatment. the potential diagnoses in these four cases included bickerstaff's encephalitis overlapping with guillain-barré syndrome, intensive-care-unit-acquired weakness, or other toxic or infectious neuropathies. neurological complications did not appear concomitantly with respiratory symptoms, instead being delayed by 2–3 weeks. conclusions: neuromuscular complications are not rare during mers treatment, and they may have previously been underdiagnosed. understanding the neurological manifestations is important in an infectious disease such as mers, because these symptoms are rarely evaluated thoroughly during treatment, and they may interfere with the prognosis or require treatment modification. since the first case of middle east respiratory syndrome (mers) was reported in saudi arabia in 2012, 1,826 laboratory-confirmed cases have been documented in 27 countries, and 35.5% of these patients have died from this novel virus. 1,2 in may 2015, the republic of korea suffered the largest outbreak of mers outside of the middle east, in which 186 patients were infected and 38 deaths were confirmed. 3 patients with mers-coronavirus (cov) infections typically exhibit fever, myalgia, cough, and dyspnea, which typically proceed to pneumonia. although some infections are asymptomatic, many cases present with severe symptoms that can result in acute respiratory distress syndrome (ards), septic shock, multiorgan failure, and death. 4 mers-cov is in the betacoronavirus genus, whose species are known to be potentially neuroinvasive. 5 however, very little information is currently available on the neurological manifestations of mers and their incidence rates. a retrospective study in saudi arabia found that 25.7% of mers patients developed confusion and 8.6% experienced a seizure. 6 only four cases with central nervous system involvement (acute disseminated encephalomyelitis, stroke, and encephali-jee-eun kim a jae-hyeok heo a hye-ok kim b sook-hee song b sang-soon park a tai-hwan park a jin-young ahn a min-ky kim a jae-phil choi c jcn tis) and one case with critical-illness polyneuropathy have been reported for patients with mers. 7, 8 here we describe four patients with neurological manifestations that developed while they were being treated for mers at a single hospital designated to treat mers during the 2015 outbreak in the republic of korea. we retrospectively reviewed the clinical records and laboratory and radiological findings of all laboratory-confirmed mers-cov-infected patients who were admitted to seoul medical center (korea), which was one of the main designated isolation hospitals for the treatment of patients with severe mers during the korean mers outbreak in may and june 2015. specifically, the medical records of the four patients who experienced neurological complications were evaluated in detail by two experienced neurologists. the presence of mers-cov infection was confirmed by applying real-time reverse-transcription polymerase chain reactions (rt-pcr) to specimens from the lower respiratory tract (collected sputum and endotracheal aspirates) according to the recommendations of the world health organization. 9 clinical stages of sepsis were defined according to the third international consensus definitions for sepsis and septic shock (sepsis-3). 10 we calculated the pneumonia severity index and the simplified acute physiology score ii to identify the severity of pneumonia and the condition of each patient upon admission to the intensive care unit (icu). 11, 12 this study was approved by the institutional review board of seoul medical center (approval no. 2015-069). twenty-three laboratory-confirmed mers-cov-infected patients were admitted during the study period. the virus transmission was associated with health-care facilities in all of the identified patients. the mean follow-up period was 49 days [interquartile range (iqr)=12-290 days]. of the total 23 confirmed cases, 19 patients survived following treatment with a combination of antiviral agents and maximal supportive care. a triple antiviral treatment regimen comprising subcutaneous pegylated interferon alpha-2a (180 µg per week for 2 weeks), high-dose oral ribavirin [2,000 mg loading dose, followed by 1,200 mg every 8 h (q8h) for 4 days and then 600 mg q8h for 4-6 days], and oral lopinavir/ritonavir (400 mg/100 mg q12h for 10 days) was administered to all patients regardless of disease severity, which was in accordance with the interim recommendations generated during the early period of the korean mers epidemic. 13 the demographic characteristics and clinical outcomes of all patients are presented in table 1 . four patients died due to early or late respiratory failure caused by the progression of the disease. four of the 23 included patients (2 men and 2 women) complained of neurological symptoms during or after mers-cov treatment, and all of these patients were referred to a neurologist. their median age was 46 years (iqr=27-46 years). the clinical presentations of these four patients and the therapies applied to them are summarized in tables 2 and 3 . a 55-year-old man who may have been exposed to mers-cov in a hospital or through household contact between 5 and 18 days prior to his presentation at our hospital was admitted because an rt-pcr of his sputum revealed positivity for mers-cov. he had a history of atrial fibrillation, diabetes mellitus, hypertension, chronic kidney disease, and hypothyroidism. at admission he denied any clinical symptoms, no *sepsis is defined as life-threatening organ dysfunction from a dysregulated host reaction to an infection. septic shock represents a subtype of sepsis that is accompanied by severe circulatory, cellular, and metabolic abnormalities that increase mortality. 10 gi: gastrointestinal, hfnc: high-flow nasal cannula oxygen therapy, ifn: type 1 interferon, ivig: intravenous immunoglobulin, lr: lopinavir/ritonavir, mers: middle east respiratory syndrome, psi: pneumonia severity index, rb: ribavirin, saps ii: simplified acute physiology score ii. but chest radiography revealed ill-defined opacities in both of his lower lungs. a triple antiviral regimen was initiated. he began to complain of dyspnea on hospital day (hd) 2, and his respiratory deterioration then progressed very rapidly. antimicrobial therapy was added to his treatment regimen. he was intubated, and a mechanical ventilator was added on hd 10. acute respiratory failure was followed by ards, septic shock, and multiorgan dysfunction syndrome. his respiratory status began to improve on hd 16, and he was taken off the mechanical ventilator on hd 28. the patient remained drowsy and exhibited bilateral ptosis up to 31 h after the administration of the sedative midazolam was stopped on hd 25. a neurological examination revealed complete external ophthalmoplegia and mild limb ataxia. weakness was also suspected in all four limbs [medical research council (mrc) grade 4]. nystagmus, sensory changes, and oropharyngeal or facial palsy were not observed. deep tendon reflexes were decreased in all limbs. the results of brain magnetic resonance imaging (mri) and cerebrospinal fluid (csf) studies were normal, including a negative csf mers-cov rt-pcr assay. an electroencephalogram exhibited diffuse slow-wave activity. nonspecific results were obtained in laboratory studies performed at the onset of neurological symptoms, including in assays to determine the glucose, thiamine, blood gas, ammonia, electrolyte, and creatinine levels. he was diagnosed with bickerstaff's encephalitis (bbe) overlapping with guillain-barré syndrome (gbs). igm/igg anti-gq1b and igm/igg anti-gm1 antibody titers were analyzed on hd 39, and all were negative. the findings of nerve conduction studies performed on hd 46 were normal. his neurological complications began to improve on hd 30, and he had fully recovered by approximately hd 60 (fig. 1) . a 43-year-old woman who had been diagnosed with a laboratory-confirmed mers-cov infection was referred to our hospital. it was suspected that she had had contact with mers-cov-infected patients in another hospital 10 days before the onset of her symptoms. no underlying medical problems were present, but she initially presented with severe myalgia, chills, fever, cough, and headache. after 1 week (hd 2), she developed gastrointestinal symptoms, including nausea, vomiting, and anorexia. on hd 3, she developed acute respiratory failure, and the flow rate of her high-flow nasal cannula oxygen therapy was increased to 60 l/min with an inspiratory fraction of oxygen of 1.0. a chest radiograph displayed bilateral diffuse ground-glass opacities and dense consolidation. a triple antiviral regimen was initiated on hd 1, with other treatments only including antiemetic, antitussive, and nonsteroidal anti-inflammatory drugs. her clinical and radiological findings began to improve on hd 5, and oxygen therapy was stopped on hd 10. on the date of discharge (hd 10), she described a stinging pain in both hands and below the knees. she felt that her legs were weakened, and she had difficulty walking for 1 km independently. a neurological examination revealed symmetric proximal dominant lower leg weakness, with her proximal and distal muscles at mrc grades 4-and 4, respectively. she experienced normal sensations to pinprick, temperature, vibration, and proprioception. her deep tendon reflexes were mildly diminished in both legs. laboratory findings did not indicate the presence of any underlying autoimmune, infectious (except for a previous mers infection), or nutritional diseases. her creatine kinase level and the results of nerve conduction, electromyography, and evoked potential studies were normal. a csf study was not performed. her weakness began to improve 17 days after the original onset of the neurological symptoms, and had largely disappeared jcn after 53 days. however, tingling and pain in the four distal limbs continued until the last follow-up (approximately 7 months after symptom onset). this patient was presumed to have icu-acquired weakness or gbs. a 46-year-old man with hypertension and a history of pulmonary tuberculosis was admitted due to infection with laboratory-confirmed mers-cov. he had previously experienced contact with patients with mers in another hospital. at 4 days after this exposure, he developed a fever, coughing, chest discomfort, dyspnea, and stool loss. a chest radiograph displayed ground-glass opacities in his left lung. a triple antiviral regimen was initiated. no other drugs were administered that could cause peripheral neuropathy. oxygen therapy was provided via a nasal prong and was increased to 5 l/min during admission. his pneumonia began to improve at 13 days after the onset of symptoms (hd 4), and the oxygen therapy was tapered off on hd 10. during this period he began to feel some tingling in the distal parts of his hands and feet. he denied experiencing any sensory symptoms prior to this admission. a neurological examination revealed normal mentation, cranial nerves, and muscle strength. he had bilateral decreases in pinprick and temperature sensations up to the knees and elbows. his reflexes were decreased at both knees but were normal in both upper extremities. his other neurological findings were unremarkable. the results of nerve conduction studies and quantitative sensory tests were normal. he was assumed to have infectious or toxic polyneuropathy, and his sensory symptoms gradually improved over 6 months. a 38-year-old woman developed a cough, sore throat, and fever 3 days after being exposed to patients with mers in another hospital. a chest radiograph exhibited diffuse groundglass opacities in both lungs. a triple antiviral regimen was started. she reported tingling in both hands at approximately 3 weeks after the onset of her respiratory symptoms. she denied experiencing any sensory symptoms prior to her most recent admission. these sensory symptoms lasted for more than 4 months. a neurological examination showed that her cranial nerves and muscle strength were normal. she experienced normal sensations in pinprick, temperature, vibration, and proprioception tests. her reflexes were normal in all four extremities. a nerve conduction study was not performed because the patient was lost to follow-up. she was tentatively diagnosed with acute sensory neuropathy caused by a toxin or infection. we have presented four patients with mers-cov infections who showed specific neurological manifestations during their treatment. these patients probably had bbe overlapping with gbs, icu-acquired weakness, or acute sensory neuropathy that resulted from a toxin or infection. a particularly interesting finding was that almost one in five patients with mers-cov infections displayed neurological symptoms during or after the infection was confirmed. although data were collected at a single institution, this represents a considerable proportion of infected patients, and suggests that such cases could have been underdiagnosed previously. patient 1 showed hypersomnolence, ophthalmoplegia, and weakness in all four limbs after a severe viral infection, and we therefore needed to differentiate between various conditions including gbs variant, wernicke encephalopathy, prolonged neuromuscular blockade, and critical-illness polyneuropathy/myopathy. because this patient had a preceding infection history, relative symmetric motor weakness, and a monophasic course with a clinical nadir within 4 weeks followed by a plateau, we considered gbs variant as a possible diagnosis. 14 bbe, one of the gbs variants, is considered a subtype of gq1b antibody syndrome and is classically diagnosed based on its characteristic clinical features of progressive symmetric external ophthalmoplegia, ataxia, and impaired consciousness. 15 because patient 1 had the classical triad of bbe accompanied by limb weakness, a diagnosis of bbe overlapping with gbs was suggested. 14 we excluded many bbemimicking conditions in this patient based on his clinical history, brain mri, and csf findings. the presence of antiganglioside antibodies and albuminocytologic dissociation in the csf supported the diagnosis of bbe as part of the gbs spectrum. nevertheless, a diagnosis of bbe is largely dependent on its clinical presentation. the absence of laboratory findings does not rule out a bbe diagnosis. 16 wernicke's encephalopathy was an important condition to eliminate in this patient because it also frequently manifests with varying degrees of encephalopathy, ophthalmoplegia, and ataxia. however, we considered it unlikely that wernicke's encephalopathy was present in patient 1 for several reasons: 1) he showed none of the typical changes associated with wernicke's encephalopathy in brain mri, which has particularly high sensitivity (97-100%) in patients without alcohol abuse, 17 2) the ptosis and complete ophthalmoplegia that were observed in this patient are rare in wernicke's encephalopathy, 18 and 3) thiamine deficiency was not confirmed in laboratory tests and the patient did not have dietary deficiencies or alcoholism. critical-illness polyneuropathy/myopathy appears frequently in icu patients but was not thought jcn to be present in patient 1 because ophthalmoparesis and ptosis are very rare in critical-illness polyneuropathy/myopathy. 19 prolonged neuromuscular blockade was another possible consideration in this clinical setting, but a neuromuscular blocking agent was not administered in patient 1. the limb and ocular weakness had lasted for almost 2 months, and this duration would be an unusual response to the prolonged administration of a neuromuscular blocking agent. neurological signs typically completely recover within 1-2 weeks in patients with prolonged neuromuscular blockade. 19 we hypothesized that patient 2 had icu-acquired weakness or gbs. icu-acquired weakness is diagnosed when a critically ill patient has limb weakness or ventilator dependency without heart or lung disease. 20 gbs was another possible diagnosis, since the patient experienced symmetric limb weakness following viral infection with a typical monophasic disease course. 14 these two diseases are differentiated by the presence of albuminocytologic dissociation in the csf, positivity for antiganglioside antibodies, or demyelinating or axonal patterns in electrophysiology. 18, 19 patient 2 was evaluated neurologically after discharge, when her neurological symptoms had substantially improved; a lumbar puncture was therefore not performed, and critical information from laboratory studies was also not available. however, we cautiously supported a diagnosis of icu-acquired weakness in patient 2, because a paraparetic presentation (as observed in this patient) is extremely rare in gbs and because dysautonomia and cranial nerve palsy, which frequently accompany gbs, were not observed in this patient. 18, 21 acute sensory neuropathy was the probable diagnosis in patients 3 and 4, which may have resulted from a toxin and/ or drug or viral infection. these two patients both received pegylated interferon alpha-2a, ribavirin, and lopinavir/ritonavir to treat mers-cov. interferon alpha-2a rarely induces peripheral neuropathy, 22 and several studies have found complications associated with sensory neuropathy, vasculitic neuropathy, bell's palsy, gbs, chronic inflammatory demyelinating polyneuropathy, and autonomic neuropathy. 23, 24 ribavirin is not associated with peripheral neuropathy, 24 while lopinavir/ritonavir is another possible causative drug. however, many people living with human immunodeficiency virus have been treated with this protease inhibitor for many years, and the risk of peripheral neuropathy in patients receiving lopinavir/ritonavir remains unclear. 25 covs are a group of enveloped rna viruses that include the alphacoronavirus and betacoronavirus [including mers-cov and severe acute respiratory syndrome (sars) cov] genera. 26 these viruses have neurotrophic and neuroinvasive characteristics, and cov rna has been detected in the central nervous systems of patients with various neurological diseases. 26, 27 a recent in vitro study evaluated the human tissue tropism of mers-cov in diverse cell lines, and revealed that it has the ability to infect human neuronal cells (nt2). 28 the neuropathological effects of mers-cov infections are suggested to result from immune-mediated processes, either directly by viral invasion or via molecular changes that arise from systemic inflammatory response syndrome. the delayed onset of neurological complications, the absence of the virus in the csf, and the development of gbs-which is one of the prototypical immunological diseases observed in our patients-might support the immunological mechanisms of these phenomena. this topic requires further microbiological and pathological studies. the structure of sars-cov is very similar to that of mers-cov, and these two viruses share many clinical characteristics. 26 the neurological manifestations observed in patients with sars were revealed during the taiwan outbreak. four patients who showed critical-illness polyneuropathy/myopathy have been described, 29 while other groups have also reported central nervous involvement in ischemic stroke in patients with sars. 30 patients with mers-cov infections have a high probability of experiencing neurological complications, similar to other covs. in conclusion, we encountered four neurological manifestations during mers treatment. the most likely diagnoses were gbs (including its variant), bbe, as well as icu-acquired weakness and toxins (including drugs) or virus-related sensory neuropathy. it was particularly interesting that the neurological complications did not appear concomitantly with respiratory symptoms, instead being delayed by 2-3 weeks. understanding that neurological complications are not rare and may be delayed is important because it might be difficult for patients with mers-cov infections to contact a neurologist while they are in isolation and because thorough neurological evaluations of these patients are rarely performed. furthermore, some neurological complications interfere with the prognosis and require appropriate treatment, such as immunoglobulin or plasmapheresis for gbs. middle east respiratory syndrome coronavirus infections-disease outbreak news isolation of a novel coronavirus from a man with pneumonia in saudi arabia summary of mers statistics in the republic of korea state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) neurological complications of middle east respiratory syndrome coronavirus: a report of two cases and review of the literature technical guidance on laboratory: middle east respiratory syndrome coronavirus (mers-cov) the third international consensus definitions for sepsis and septic shock (sepsis-3) a prediction rule to identify low-risk patients with communityacquired pneumonia a new simplified acute physiology score (saps ii) based on a european/north american multicenter study antiviral treatment guidelines for middle east respiratory syndrome guillain-barré and miller fisher syndromes--new diagnostic classification clinical features and prognosis of miller fisher syndrome bickerstaff brainstem encephalitis and fisher syndrome: anti-gq1b antibody syndrome efns guidelines for diagnosis, therapy and prevention of wernicke encephalopathy mimics and chameleons in guillain-barré and miller fisher syndromes clinical approach to the weak patient in the intensive care unit critical illness polyneuropathy and myopathy: a major cause of muscle weakness and paralysis paraparetic guillain-barré syndrome peripheral neurotoxicity of pegylated interferon alpha: a prospective study in patients with hcv chronic inflammatory demyelinating polyneuropathy after treatment with interferon-alpha acute inflammatory demyelinating polyneuropathy associated with pegylated interferon alpha 2a therapy for chronic hepatitis c virus infection human immunodeficiency virus protease inhibitors and risk for peripheral neuropathy middle east respiratory syndrome neuroinvasion by human respiratory coronaviruses differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation neuromuscular disorders in severe acute respiratory syndrome large artery ischaemic stroke in severe acute respiratory syndrome (sars) the authors have no financial conflicts of interest. key: cord-291694-nokowfdi authors: wickramage, kolitha; peiris, sharika; agampodi, suneth b title: “don’t forget the migrants”: exploring preparedness and response strategies to combat the potential spread of mers-cov virus through migrant workers in sri lanka date: 2013-07-29 journal: f1000res doi: 10.12688/f1000research.2-163.v1 sha: doc_id: 291694 cord_uid: nokowfdi from september 2012 to july 2013, 81 laboratory-confirmed cases of infection with middle east respiratory syndrome coronavirus (mers-cov), including 45 deaths (a case fatality ratio of 55%) have been reported from eight countries. human-to-human transmission is now confirmed showing potential for another pandemic of zoonotic disease, with an extremely high mortality rate. effective surveillance strategies are required in countries with a high influx of migrants from the middle east to mitigate the probable importation of mers-cov. we discuss here the risk of mers-cov in major labor sending countries and list the probable strategies for control and prevention of mers-cov using sri lanka as an example. it is conservatively estimated that 10% of sri lanka’s population work as international labor migrants (1.8 to 2 million workers), with 93% residing in the middle east. an average of 720 workers depart each day, with the majority of these workers (71%) departing to the kingdom of saudi arabia (the country with 81.5% of total mers-cov cases). we also describe other inbound migration categories such as tourists and resident visa holders relevant to the context of preparedness and planning. the importance of partnerships between public health authorities at national and regional levels with labor migration networks to establish institutional and/or policy mechanisms are highlighted for ensuring effective preparedness and response planning. strategies that can be taken by public health authorities working in both labor sending and labor receiving counties are also described. the strategies described here may be useful for other labor sending country contexts in asia with a high frequency and volume of migrant workers to and from the gulf region. the global health community is experiencing one of the deadliest coronavirus outbreaks that has been reported in recent times. the first case of middle east respiratory syndrome coronavirus (mers-cov) infection was reported in september 2012 from the kingdom of saudi arabia (ksa) 1 . since then, 81 laboratory-confirmed cases of infection with 45 deaths were reported by eight countries, of which 66 (81.5%) were from the ksa 2 (table 1) . even though france, germany, italy, tunisia and the united kingdom have also reported laboratoryconfirmed cases, these patients had been either transferred to these countries from hospitals in the middle east for specialist care or had returned from the middle east and subsequently became ill. hitherto, there have been no cases reported in asia. coronaviruses have long been known to cause widespread human infections such as the common cold and global pandemics such as severe acute respiratory syndrome (sars) 3 . mers-cov has not been identified previously among humans 4 , thus knowledge about the natural history of the disease is still limited. the clinical syndrome of mers-cov is primarily a respiratory disease including fever, cough and shortness of breath, resembling sars. more than half of cases develop life threatening complications, such as respiratory failure 5,6 , acute respiratory distress syndrome (ards) 6-8 , renal failure [4] [5] [6] 8 , and consumptive coagulopathy 8 . studies of clusters of cases suggest that the spread may occur by both large and small aerosols and possibly via the faecal-oral route 9 . the pathogenesis of mers-cov is not fully understood. it appears to cause respiratory problems by attacking and infecting the cells in the nasopharynx; laboratory studies show that the virus has the ability to cause profound apoptosis of human bronchial epithelial cells 10 . all confirmed cases have had respiratory disease and most have developed pneumonia 11 . complications during the course of illness have included severe pneumonia with respiratory failure requiring mechanical ventilations, ards with multi-organ failure, renal failure requiring dialysis, consumptive coagulopathy and pericarditis 11 . hitherto, 45 out of 81 cases (55%) have died as a result of infection (table 1) . the rapid transmission and high attack rate in hospital settings have raised concerns about the risk of health care associated transmission of this virus 12 . although the transmission of the disease is still not as rapid as seen during the sars epidemic in 2003 13 , human to human transmission of mres-cov has now been established 5 . given the high case fatality rate compared to previous coronavirus pandemics, continued risk assessment, surveillance, and preparedness measures by all countries are required to minimize the impact of a probable global pandemic of mers. the who encourages "all member states to continue their surveillance for severe acute respiratory infections (sari) and to carefully review any unusual pattern" 2 . the annual hajj pilgrimage, attended by 3 million pilgrims from all over the globe, has been identified as a potential threat for major spread 14 . a recent study has shown evidence of rapid acquisition of respiratory viruses among pilgrims during their stay during the hajj in the ksa, most notably rhinovirus 14, 15 . the authors highlight the potential of spreading these infections in the pilgrims' home countries upon their return. memish and colleagues also suggest a 'high degree of clinical vigilance' required for the possibility of mers-cov infection in patients with respiratory infections who have visited the middle east in the preceding 10 days 6 . despite these concerns, the who does not recommend changing travel plans for hajj or umrah because of mers-cov. however, at a recent meeting organized by the who in cairo (june, 2013), public health officials specifically emphasized the importance of preparedness and response at hajj and contexts of mass gatherings 'as a priority action', with member states of who agreeing to develop specific plans for mers-cov 16 . no emphasis at this meeting or in peer-reviewed literature has been made in relation to the large volumes and frequent travel patterns of international labor migrant workers to the middle eastern countries, especially from asia 17 . labor migration from asia to the middle east involves the movement of contractual workers from many 'labor sending' nations such as the philippines, india, sri lanka and indonesia, to 'labor receiving' ones, mainly within the middle eastern region 18 . estimates of total migrant workers by the international labor organization for 2010 were 105.5 million, 30 million of which were from within asia 19 . it is estimated that there is a net annual outflow of two million migrant workers from the 'top five' south asian labor sending countries of sri lanka, india, bangladesh, nepal and pakistan 20 ( table 2) . unregistered 'irregular' migrant workers also contribute to this outflow of contractual workers from asia, although estimates are difficult to assess due to the clandestine nature of their travel. it is important to highlight that remittance from labor migrants contribute significantly to the economic growth of most developing countries in asia. the sri lankan economy is highly dependent on foreign exchange earnings from its migrant workforce, with remittance from workers in middle eastern countries alone contributing 58.9% of all total foreign exchange earned in 2011 21 . although the who has not yet issued a travel health warning for any country, nor recommended conducting on-arrival screenings at ports of entry, the infectious nature of mers-cov means that there is a risk of contracting the disease through infected individuals who have visited the middle east in the preceding 10 to 14 days. health authorities in some countries in the region have already begun making advanced arrangements for the diagnostic test kits developed by the cdc for mers-cov to be made available to national reference laboratories 16 . ensuring guidance for health care professionals regarding case definition, diagnosis and management for mers-cov infection, and establishing an active surveillance system for 'influenza-like' illnesses in hospitals are essential steps for surveillance. elaborating pandemic preparedness and response measures are not the focus of this current paper since these have already been well described and indeed established in sri lanka through previous efforts against sars and h1n1 22 . rather, this article will focus on understanding the importance of the large volumes of migration categories and their dynamics, which may yield more specific and targeted public health and screening interventions for mers-cov. inbound migration refers to the flow of persons traveling into a country 23 . we identify five major inbound migrant flows from the middle east to sri lanka with the potential of introducing mers-cov ( figure 1 ). ksa, qatar, kuwait, uae and jordan are the major destination (labor receiving) countries, encompassing 85% of sri lanka's total international labor migrant force (1.8 to 2 million workers in 2011) 21 . each day, around 720 migrant workers leave sri lanka to the middle east as labor migrants through bandaranaike international airport 24 . over 93% of the 262,960 labor migrants were employed in middle eastern countries in the year 2011 (table 2 ). female participation in foreign employment is 48.3% of the total departures during the same year, and 85% of them worked as domestic housemaid 25 . the recent evidence of virus spreading within family clusters may be a significant factor in determining household transmission 6 . data on patterns of returning migrant workers are not available since there is no registry of returning workers. however, inflow is expected to be greater than outflow considering both the cyclical nature of labor migration (where a worker usually returns to the country for a short period before departing again -a cycle which can last 10 years or more), and the large stock total of formally registered workers from sri lanka. every year, muslims from all over the world converge in ksa to take part in the annual hajj (pilgrimage). ksa hosted 2.5 million pilgrims in 2009 amidst the h1n1 pandemic 27 . in 2013, the hajj is expected to fall between the 13-18 october. a quota system operates to limit the number of people from each country visiting mecca each year based on the number of muslims in each country. the sri lankan quota for 2013 is currently set at 2,800 28 . a residence visa is a permit for non-sri lankan citizens to obtain residence facilities for purposes of long stays, work and study. the numbers of both residency visa holders and tourists visiting sri lanka from the middle east, disaggregated by country of residence, are shown in table 3 . both ksa and the uae remain the primary source countries of migrants within this inbound category. if a highly conservative estimate on the number of labor migrants returning from the middle east is placed at 220,000 persons per year, then based on data from the five major categories of migrant flows presented here, an estimated 280,901 persons will travel from the middle east to sri lanka. this number does not account for the number of returning sri lankan tourists and irregular migrants from the middle eastern region. based on the fact that 71% of the current caseload of sri lankan migrant workers depart for the ksa, it is expected that the majority of inbound migrants will be traveling from the same country. it is important to note that the following recommendations are suggested as a way of enhancing, not substituting, existing frameworks on pandemic disaster preparedness and response. there are currently no established guidelines for mers-cov established at country level, unlike in other settings 29 . a number of prevention and screening strategies for migrant workers are presented here, classified according to the three phases of migration: 'pre-departure' (departing sri lanka), 'at destination' (time spent in the gulf states) and 'upon-arrival' (arrival back in sri lanka). each stage in the migration cycle offers unique opportunities for public health action/intervention based on enabling mechanisms and capacities harnessed in routine migrant worker pathways ( figure 2 ). these may be useful in refining into other country contexts. a. strategies at the 'pre-departure' phase. the majority of labor receiving countries require pre-departure health assessment as a pre-requisite for a work visa. migrant workers to gulf state countries are expected to undertake a mandatory pre-departure medical examination in sri lanka to ensure their 'fitness to travel' and fulfillment of health assessment criteria set by the recipient country. health care workers could provide health information on mers-cov to potential migrant workers during the medical examination. the gulf-approved medical centers association (gamca) has a network of 13 private medical centers in sri lanka, which are accredited to conduct health assessments of sri lankan migrant workers prior to departure to the gamca countries ksa, kuwait, bahrain, qatar, uae and oman. as a preparedness measure, medical staff at these health assessment centers can be trained with up-to date information on mers-cov and be encouraged to disseminate language specific information-exchange communication (iec) materials on signs, symptoms and preventative actions for the migrant worker 30 . b. strategies at the 'destination' phase. sri lankan embassies and diplomatic missions at destination countries could disseminate public health service messages in relation to mers-cov in singhalese/tamil languages via embassy welfare programs, social networks and through ethno-specific radio programs. it is vital that local health authorities and employers provide access for migrant workers to seek primary health care and that they are supported with specialized/referral care within the health system in the gulf states. the importance of health accessibility, irrespective of visa status, for migrant workers to primary and specialized health care facilities in these destination countries also needs to be emphasized through state-to-state and regional advocacy mechanisms. it is recommended that public health authorities and global bodies such as the who and the international organization for migration utilize the support of existing inter-regional and trans-national migrant worker networks such as the members of the 'colombo process' and 'abu-dhabi process' in order to promote effective public health messages and strategies 31 . the sri lanka bureau of foreign employment (slfbe) which provides policy direction and regulation of labor migrants has a dedicated 24-hour administrative desk at sri lanka's bandaranaike international airport, to manage grievances from returning migrant workers. a worker welfare center to house migrant workers in need of support managed by the slfbe is also available near the airport. currently there are no medical personnel attached to the slfbe services for on-arrival phase. it is recommended that the ministry of health make arrangements to establish a coordination mechanism with the slfbe and with airport health authorities, which currently have no linkage to migrant worker programs. a rotating roster of trained health professionals allocated at the health center at the airport could ensure each returning worker completes the rapid symptom checklist (see assessment algorithm in figure 3 ). the algorithm was developed after augmenting the guidance frameworks for mers-cov created by the public health authorities in canada 29 and the cdc 32 . it is important for port health authorities to also build effective partnerships and protocols with immigration control officers at 'on arrival counters'. this will ensure referral of travelers returning from the middle east where cases of mers-cov have been reported to the health screening desk. leaflets advising travelers of symptoms of the influenza-like illness could also be distributed at the immigration counter to arriving passengers. managing risk communication also forms a vital strategy for any form of public health preparedness and response. studies have shown that when responding to a novel infectious disease outbreak, policy and planning decisions can limit the ability to control the outbreak and result in unintended consequences including lack of public confidence 33 . communication of risk to target populations needs to be carefully planned to avert maladaptive behaviors due to fear and defensive avoidance (the motivated resistance to the message, such as denial or minimization of the threat 34 ). individuals may defensively avoid a message by being inattentive to the communication (e.g., looking away from the message), or by suppressing any thoughts about the threat over the long term. mitigating such threats through targeted communication strategies to migrant workers and other categories such as those described above may be useful 35 . the strategies outlined above do not warrant large scale 'national level' awareness campaigns, which may exacerbate anxiety and induce maladaptive rather than positive health seeking behaviors 36 . it has been one year since mers-cov was discovered, yet many questions remain unanswered about its pathogenesis, host reservoirs and transmission dynamics. what is clear from global health authorities is that countries need to plan for preparedness and response planning 29 . we recommend partnerships between public health authorities, at national and regional levels, with the labor migration industry and migrant worker networks in establishing both institutional and policy mechanisms to ensure effective preparedness and response planning in response to a potential mers-cov threat through labor migrants from south asia. has passenger traveled to, or resided in, the arabian peninsula or a neighboring country in the 14 days prior to onset of illness? these points can be presented, however, with much greater clarity and brevity. the authors occasionally use hyperbole and repetition to make misleading assertions. for example, "the extremely high" mortality rate they cite has been falling as more cases are identified and will likely continue along this trend as milder cases are discovered. claims of "rapid transmission" and a "high attack rate" are made without any epidemiologic basis or support from literature. throughout the commentary, the authors also make redundant, disorganized, and extra-contextual statements about mers clinical features, labor sending/receiving nations, the hajj pilgrimage, and strategies for controlling a potential mers epidemic. the only way this manuscript can be salvaged is if it is refashioned as a brief communication of no more than a few hundred words with one figure or table. the manuscript should be narrowly focused on migrant workers between gulf states and southeast asia and the threat they may pose to the spread of the virus. the additional commentary on screening and surveillance adds nothing to the existing who report and essentially boils down to education, awareness, and appropriate referral. the following are specific points of concern: instead of presenting the number of cases and deaths in each country in tabular format, the authors should provide a link to who's running tally as the authors numbers will be (as they are now) inaccurate and out-of-date. remove alarmist statements such as "extremely high mortality rate", "deadliest coronavirus outbreaks" and platitudes such as "the infectious nature of mers-cov means that there is a risk of contracting the disease through infected individuals" . i'm confused by the last sentence of the introduction's first paragraph: "there have been no cases reported in asia." the second paragraph of the introduction repeats the clinical features of mers. this should be corrected. the hajj pilgrimage is discussed briefly in the introduction, dropped, and then brought up again without any context later in the manuscript. the same is true regarding labor migration patterns and surveillance strategies. there is no real coherent flow to any of these topics. the terms created for the different phases of labor migration cycle make no sense. to the best of my understanding, "pre-departure" means departure and "upon-arrival" means return. these terms are superfluous and are more jargon than they are informative. figure 3 is unclear. is this for anybody returning from a gulf state or only those returning with symptoms? the first diamond makes this unclear. i have read this submission. i believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however i have significant reservations, as outlined above. no competing interests were disclosed. competing interests: counsel patient on referral and test procedure make arrangements to contact family if not present undertake medical escort patient to isolation ward at national infectious disease hospital (idh) in colombo (1 hr from airport) governing international labour migration: current. issues, challenges and dilemmas. routledge/ripe studies in global political economy trends and outlook for labour migration in asia annual statistics report on foreign employment, sri lanka bureau of foreign employment: battaramulla 2011. reference source 22 ministry of health, national migration health policy (draft) annual statistics report on foreign employment. sri lanka bureau of foreign employment: battaramulla annual statistics report on foreign employment. sri lanka bureau of foreign employment: battaramulla asian development bank institute and organization for economic coorporation and development, managing migration to support inclusive and sustainable growth tokyo: asian development bank institute 56 2013 a mass gathering experience at the 2009 pilgrimage in makkah, saudi arabia, during the 2009 novel influenza a (h1n1) pandemic provincial infectious diseases advisory committee tools for preparedness: triage, screening and patient management for mers-cov infections in acute care settings reference source 31. international organization for migration. regional consultative process (rcp) on the management of overseas employment and contractual labor for countries of origin in asia interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov). middle east respiratory syndrome pandemic h1n1 in canada and the use of evidence in developing public health policies -a policy analysis hospital outbreak of middle east respiratory syndrome coronavirus clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage respiratory tract infections during the annual hajj: potential risks and mitigation strategies public health officials agree priority actions to detect and control mers-cov. 2013. reference source 17. international organization for migration international labour migration dynamics and inequality in southeast asia maria van kerkhove faculty of medicine, school of public health no competing interests were disclosed. the author(s) declared that no grants were involved in supporting this work.author contributions kw conceived the paper and drafted the first version of the manuscript. sp contributed in the concept and manuscript preparation. sba revised and edited and finalized the manuscript for submission. 11 november 2013 referee report: i only have one major edit that i would recomend for this article; table 1 is not necessary and can be removed. no competing interests were disclosed. key: cord-304054-sn7rswab authors: khan, gulfaraz; sheek-hussein, mohamud title: chapter 8 the middle east respiratory syndrome coronavirus: an emerging virus of global threat date: 2020-12-31 journal: emerging and reemerging viral pathogens doi: 10.1016/b978-0-12-819400-3.00008-9 sha: doc_id: 304054 cord_uid: sn7rswab abstract middle east respiratory syndrome (mers) is a viral respiratory illness caused by a coronavirus (cov), first identified in saudi arabia in 2012. since then, almost 2000 cases have been reported from 27 countries, with saudi arabia being the epicenter. this newly emerging virus is highly pathogenic and has a case mortality rate of 35%. it is similar to the cov causing severe acute respiratory syndrome cov (sars-cov) in that both belong to the genus beta covs that are of zoonotic origin and cause lower respiratory infection. the natural reservoir for mers-cov remains unknown. serological studies indicate that most dromedary camels in the middle east have been infected with this virus, and they maybe the potential intermediate host. however, the mode of transmission from camels to humans is poorly understood. the majority of confirmed human cases have resulted from human-to-human transmission, most probably via respiratory route. patients most at risk of developing severe mers-cov infection appear to be those with underlying conditions such as diabetes, hypertension, obesity, cardiac diseases, chronic respiratory diseases, and cancer. unlike sars-cov, mers-cov is considered an ongoing public health problem, particularly for the middle east region. in this chapter, we outline the prevailing information regarding the emergence and epidemiology of this virus, its mode of transmission and pathogenicity, its clinical features, and the potential strategies for prevention. renal failure (zaki et al., 2012; khan, 2013) . from 2012 to the end of 2017, the world health organization reported that a total of 2123 laboratories confirmed the cases of mers-cov infection and at least 740 deaths in 27 countries (case fatality rate 35%). although sizable outbreaks have been noted in several countries, the latest being in south korea (186 cases and 35 deaths) (arabi et al., 2017) , the vast number of cases ( . 80%) have been reported from saudi arabia (fig. 8.1 ) (who, 2017) . this newly emerging, highly pathogenic respiratory virus is closely related to the virus that caused an outbreak of severe acute respiratory syndrome (sars) in 2002à03. both viruses are beta covs of zoonotic origin and cause similar clinical presentations. although the natural reservoir of mers-cov infection and mode of transmission to humans is not known, one factor appears to be common to all primary cases; they are epidemiologically linked to the middle east region. most secondary cases, on the other hand, have occurred as a result of human-to-human transmission. indeed, several well-documented outbreaks have occurred in healthcare settings, often in elderly men with comorbidities (arabi et al., 2017; chafekar and fielding, 2018) . unlike sars-cov, mers-cov is an ongoing public health threat, particularly for the middle east. the fact that there is no effective antiviral drug or approved vaccine available against mers-cov makes the threat even more worrisome (zumla et al., 2016) . mers-cov is an enveloped, single-strand, and positive-sense rna virus, which belongs to the coronaviridae family. although covs are very common and can infect a variety of different animals, including cats, pigs, and bats, they rarely jump species barrier and infect humans. human covs (hcovs) were first isolated in mid-1960s, and until 2002, only two viruses, namely, hcov-229e and hcov-oc43, were known to infect humans (forni et al., 2017) . currently, six covs have been shown to infect humans. except for mers-cov and sars-cov, all others are associated with mild illnesses resembling common cold. covs are grouped into four genera, α, β, γ, and δ. the β-covs are further subgrouped in four lineages or clades, aàd (forni et al., 2017; milne-price et al., 2014) . although mers-cov and sars-cov belong to the same genus and both cause severe lower respiratory tract infection in humans, phylogenetic and sequencing data suggests that mers-cov is in fact more closely related to several bat covs (btcovs) than to sars-cov ( fig. 8 .2) (forni et al., 2017; milne-price et al., 2014) . these findings suggest that mers-cov probably is originated from a btcov ancestor (omrani et al., 2015; chan et al., 2015a,b) . the fact that covs are rna viruses exhibiting high rates of mutation and recombination, and a propensity to cross species barrier, increases the risk of new variants emerging with higher virulence and transmission sabir et al., 2015) . the replication cycle of mers-cov consists of a number of important steps: attachment and entry into host cell, uncoating and release of viral rna, transcription and translation of viral specific genes, replication of viral genomic rna, and assembly and release of progeny virions from the infected cell. as is typical of most rna viruses, all of these steps take place in the cytoplasm of the host cell (de wit et al., 2016) . the initial attachment of mers-cov to its susceptible host cells is mediated by the viral envelop spike glycoprotein s binding to its cellular receptor, cd26 (also known as dipeptidyl peptidase 4, dpp4) (lu et al., 2013; raj et al., 2013) . a number of different cell types express dpp4 and hence are susceptible to mers-cov infection including pneumocytes, alveolar macrophages, bronchial epithelia, vascular endothelium, as well as a subset of mononuclear cells (meyerholz et al., 2016; yu et al., 2017) . following attachment, the virus enters the susceptible cell by fusion of its envelope with the plasma membrane and/or via receptor-mediated endocytosis (de wit et al., 2016) . once in the cytoplasm of the target cell, the virus particle uncoats and the positive-sense viral rna binds to ribosomes, and the viral rna-dependent rna polymerase is translated. this enzyme in turn transcribes full-length negative-sense rna that forms the template for the production of positive-sense viral genome. the viral polymerase also generates various individual mrnas that are translated into viral proteins. viral structural proteins and viral genomic rna are assembled into new virus particles in the rough endoplasmic reticulum-golgi intermediate compartment and eventually released out of the cell by exocytosis. from the infected host, it appears that the virus is shed in nasal secretions (adney et al., 2014) . interestingly, in bats, a recent study revealed that dpp4 receptor is rarely expressed in epithelial cells of respiratory tract, but highly expressed in epithelial cells of intestinal tract, indicating that fecalàoral is probably the main mode of transmission in bats (widagdo et al., 2017) . of all the documented cases to date, there is no evidence for the transmission of the virus from bats or their droppings directly to humans. we also have limited data on the survival of the virus outside its host. when the virus was added to milk from dromedary camels, goats, or cow and stored at 4 c or 22 c, the virus could be recovered up to 72 and 48 hours, respectively (van doremalen et al., 2013) . pasteurization of the milk, however, completely destroyed mers-cov infectivity (van doremalen et al., 2013) (table 8 .1). the current prevalent view is that mers-cov is a zoonotic virus that entered the human population in the arabian peninsula, via direct or indirect contact with infected dromedary camels. studies indicate that the virus had been circulating in the camel population for decades, and only recently "jumped" the species barrier to infect humans. what are the factors that precipitated the virus to cross the species barrier are unknown. most of the confirmed cases of mers-cov infection in humans have been via person-to-person transmission. the epidemiological elements in the transmission of mers-cov appear to be factors related to the virus, the host, and the environment. cases have occurred as sporadic infections, family clusters, or outbreaks in healthcare settings (kim et al., 2017; oboho et al., 2015) . although the infection is limited and nonsustained, outbreaks in healthcare settings have been particularly extensive and worrisome. the nonspecific initial symptoms, late diagnosis, and inadequate infection control measures have all contributed to the outbreaks in healthcare settings (oboho et al., 2015; hunter et al., 2016; kim et al., 2017) . although mers-cov cases have been detected in many countries around the world, almost all have been directly or indirectly linked to the middle east region (table 8 .2). one of the most notable outbreaks outside the middle east occurred in south korea in may 2015 (kim et al., 2017; lee and wong, 2015) . a single infected man returning from the middle east caused a hospital outbreak in which 185 individuals were infected (kim et al., 2017) . the epidemiological pattern observed in the korean outbreak was similar to that observed in the middle east; more males than females were affected, most of the 38 patients who died had underlying conditions such as respiratory disorders, cancer, hypertension, cardiovascular problems, or diabetes (kim et al., 2017) . it is noteworthy that the death rate was lower in the cases from south korea compared to those reported from saudi arabia (23% vs 47%) (virlogeux et al., 2016) . the reason for this is not clear. although more than 80% of mers-cov cases have occurred in saudi arabia, the virus clearly has the potential of spreading to other countries. thus there is an obvious need to detect, respond, and contain any outbreak of mers-cov cases if we want to prevent the global spread of the virus. unfortunately, this is easier said than done. there are a number of risk factors prevalent in some of the countries of the middle east which support the emergence and reemergence of infectious diseases (buliva et al., 2017) . these risk factors include political instability, famine and war, less developed healthcare infrastructure, weak public health and surveillance systems, increased population growth and mobility, climate change, and urbanization (buliva et al., 2017) . in order to prevent the emergence and spread of infectious diseases such as mers-cov, it is essential to address the underlying causes and risk factors. needless to say, these are major challenges for any country, let alone the eastern mediterranean region. to successfully address these challenges, it will require not only funding, establishment of robust and effective surveillance systems, and national and international corporations but also above all, peace and security in the region. infection with mers-cov, in its initial description, resembled "sars-like" illness (chan et al., 2015a,b) . further analysis of the epidemiological, virological, and clinical aspects of mers-cov and sars-cov revealed important differences between the two viruses. identifying unique aspects of mers-cov helped to explain how the epidemic evolved and the steps that could be taken to prevent its spread (chan et al., 2015a,b) . serological studies have indicated that most dromedary camels in africa and the middle east, but not other animals such as sheep, goats, and cows, were seropositive for mers-cov . moreover, seroprevalence in dromedary camels appears to vary, with high rates reported in animals from egypt, ethiopia, nigeria, and sudan and lower rates in animals from tunisia (ali et al., 2017) . intriguingly, dromedaries from australia, canada, the united states, germany, netherlands, and japan have been reported to be seronegative for mers-cov (omrani et al., 2015) . importantly, population-based seroepidemiologic studies indicated that the seroprevalence of the virus was several folds higher in people who have been exposed to camels compared to those in the general population ( accumulating serologic and molecular evidence indicates that the virus in dromedaries is genetically similar to mers-cov in humans, supporting the notion that dromedary camels could be the potential source of infection to human memish et al., 2014a,b,c; sabir et al., 2015) . indeed, mers-cov antibodies have been isolated in dromedary camels across the arabian peninsula, north africa, and eastern africa dating from as far back as the 1990s (milne-price et al., 2014; omrani et al., 2015) . this finding suggests that mers-cov may have been circulating in dromedaries for over 20 years before it was first recognized as a cause of human infection (aly et al., 2017) . in a recent study, a fatal case of mers-cov infection was reported in an individual who had direct contact with a dromedary camel (azhar et al., 2014) . sequence analysis of the virus isolated from the case and the camel was identical, clearly indicating that mers-cov can indeed be transmitted from camels to human (azhar et al., 2014) . it appears that active infection with release of the virus in nasal secretions, particularly during the incubation period, is important for the transmission of the virus to humans . where and how the camels acquired the infection remains unknown. it has been hypothesized that bats could be the potential source (fig. 8.3 ) (anthony et al., 2017; mohd et al., 2016; omrani et al., 2015) . indeed, mers-cov-like viruses have been identified in certain species of bats (anthony et al., 2017; woo et al., 2006) . the bats are present in most parts of the world and often infected with various zoonotic viruses. thus it is plausible that at some point in the past, camels acquired the infection from bats, leading to a sustained infection in the camel population (fig. 8.3) . mers-cov rna has been identified in the milk, nasal secretion, and feces of dromedary camels (omrani et al., 2015) . since camels and humans are often in close contact, particularly in the arab gulf states, humans would be at increased risk of contracting the virus from actively infected animals (mackay and arden, 2015; reusken et al., 2015) . indeed, mers-cov seropositivity in shepherds and those working in slaughterhouses in saudi arabia has been reported to be an order of magnitude higher than in the general population (arabi et al., 2017) . although possible, no evidence currently exists to support the transmission of mers-cov from bats to humans directly. what is certain is that transmission of the virus can occur from camels to humans, but the process is still not fully understood (al hammadi et al., 2015; memish et al., 2014a,b,c) . one possibility is that some species of covs from camels and humans could recombine leading to the emergence of a new virus that can infect both, camels and human (sabir et al., 2015) . most mers-cov infections in humans occur through human-tohuman contact (arabi et al., 2017; zumla et al., 2016) . available data on epidemiologic observations suggest that human-to-human transmission occurs primarily through close contact with an infected individual. the mode of transmission is presumed to be via respiratory droplets or aerosols, with higher risk in situations where aerosols are generated, and inadequate personal protection or proper room ventilation is not present (kutter et al., 2018) . in the south korean outbreak a total of 185 individuals were infected; 136 of whom were directly infected by just 3 cases, the so-called super spreaders. late diagnosis, lack of infection control measures, poor communication and healthcare management procedures, and failure to quarantine the "super spreaders" were identified as major factors contributing to this large nosocomial outbreak (kim et al., 2017) . cov is a common cause of mild respiratory tract infection manifesting as common cold. it is estimated that approximately one-third of all upper respiratory tract infections in adults are due to covs. sars-cov and mers-cov are the exceptions. both of these viruses have a high propensity to infect the lower respiratory track and lead to severe disease and death (de wit et al., 2016) . the finding that both of these viruses, but in particular, mers-cov, are able to evade the body's immune responses and infect a broad range of cells, explaining the widespread infection and development of severe disease (mackay and arden, 2015) . it is noteworthy that, even in the absence of viral shedding in the upper respiratory tract, most symptomatic patients have abnormal chest radiographs (fig. 8.4) (assiri et al., 2013; de wit et al., 2016) . the incubation period for mers-cov infection is about 5à6 days with most patients showing symptoms within 14 days of exposure (de wit et al., 2016; virlogeux et al., 2016) . the initial clinical symptoms of mers-cov infection can range from asymptomatic to low-grade fever, cough, sore throat, myalgia, and less frequently diarrhea and vomiting. progression to more severe disease is characterized by the symptoms of shortness of breath, severe pneumonia, respiratory distress syndrome, multiorgan failure, and death (arabi et al., 2017; de wit et al., 2016; guery et al., 2013) . the severity of the infection appears to vary depending on the age of the patient and any underlying conditions. adults over the age of 50 years and with comorbidities such as diabetes, hypertension, chronic renal or lung disease, cancer, and heart disease are at increased risk of developing severe disease and death (badawi and ryoo, 2016) . although the vast majority of confirmed cases have been in male adults, children are also susceptible to infection, albeit at lower rate and with milder disease . based on limited data, mers-cov infection in pregnancy can also lead to maternal and perinatal disease and death assiri et al., 2016) . not surprisingly, the severity of infection and the risk of transmission of mers-cov are significantly increased in environments such as hospitals (cho et al., 2016; hastings et al., 2016) . the clinical symptoms of mers-cov infection, especially in early stages of the infection, are typically nonspecific and can resemble a number of acute respiratory tract infections. however, acute febrile respiratory illness in a patient with a recent travel history to the middle east or direct/indirect contract with a confirmed case of mers-cov should be enough suspicion to request laboratory testing for mers-cov. studies indicate that viral replication and shedding is higher in lower compared to upper respiratory tract (de wit et al., 2016; memish et al., 2014a,b,c) . hence, for laboratory diagnosis, lower respiratory tract specimens such as tracheal aspirate, bronchoalveolar lavage, or pleural fluid are preferred over upper respiratory tract specimens such as nasopharyngeal swab (mackay and arden, 2015; memish, et al., 2014a,b,c) . it is essential that appropriate personal protective equipment (ppe) and infection control measures are implemented when dealing with suspected cases. the assay of choice for the laboratory diagnosis of mers-cov is reverse transcriptase real-time polymerase chain reaction (rt-pcr) on respiratory samples. this assay was established soon after the identification of mers-cov back in 2012 (zaki et al., 2012) . rt-pcr is not only very sensitive but also importantly a fairly rapid technique, which is essential for early diagnosis and quarantine implementation. the virus can also be cultured. a number of different cell lines are susceptible for in vitro infection, including vero and llc-mk2 cells (zaki et al., 2012) . however, cell culture approach is very slow and not easily adaptable to every diagnostic laboratory, hence the preference of rt-pcr. for determining past infection or for surveillance studies, the detection of antibodies to mers-cov using serological assay, such as enzyme linked immunosorbent assay (elisa), can be performed (mackay and arden, 2015) . currently, no specific antiviral therapy or vaccine is available for the treatment and prevention of mers-cov infection. supportive care and prevention of complications are the main management options that are available. however, efforts are underway for the development of therapeutic and vaccine candidates. in a marmoset model of mers-cov infection, several compounds, including ribavirin, lopinavir/ritonavir, interferon-β1b, and interferon-α2b, alone or in combinations, have shown varying degree of success (chan et al., 2015a,b; falzarano et al., 2013) . similarly, passive immunotherapy with neutralizing antibodies against mers-cov has also shown some therapeutic value in inhibiting viral replication (luke et al., 2016) . in terms of vaccines, several potential candidates have been developed and are in different stages of clinical testing (du et al., 2016) . the possibility of developing an effective vaccine based on mers-cov spike protein is promising . in the absence of licensed antiviral or vaccine, current strategies of combating mers-cov infection are aimed at reducing the risk of animal-to-human and human-to-human transmissions. strategies recommended include avoidance of drinking unpasteurized camel milk, limiting direct contact with a sick animal, avoidance of close contact, and sharing of utensils with an infected individual, using ppe when in direct contact with an infected person and proper hand hygiene. in addition, early recognition and laboratory confirmation of infected cases, segregation/isolation of infected cases, and contact tracing and strict implementation of infection control measures in healthcare settings are all essential for controlling and preventing of mers-cov infection and spread. replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from 2012 to 2015 asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia evidence for camel-to-human transmission of mers coronavirus prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis emerging and reemerging diseases in the world health organization (who) eastern mediterranean region-progress, challenges, and who initiatives mers-cov: understanding the latest human coronavirus threat middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study sars and mers: recent insights into emerging coronaviruses transmission of mers-coronavirus in household contacts vaccines for the prevention against the threat of mers-cov treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques molecular evolution of human coronavirus genomes the spike-protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2 and is targeted by neutralizing antibodies clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels outbreak of middle east respiratory syndrome at tertiary care hospital transmission of middle east respiratory syndrome coronavirus infections in healthcare settings a novel coronavirus capable of lethal human infections: an emerging picture middle east respiratory syndrome infection control and prevention guideline for healthcare facilities middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications transmission routes of respiratory viruses among humans probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo mers coronavirus: diagnostics, epidemiology and transmission screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome human infection with mers coronavirus after exposure to infected camels, saudi arabia jumping species-a mechanism for coronavirus persistence and survival dipeptidyl peptidase 4 distribution in the human respiratory tract the emergence of the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: references emerging and reemerging viral pathogens a nationwide, cross-sectional, serological study mers-cov outbreak in jeddah-a link to health care facilities middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction evidence of person-to-person transmission within a family cluster of novel coronavirus infections dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study occupational exposure to dromedaries and risk for mers-cov infection co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia confirmed global cases of mers-cov mers-cov global summary and assessment of risk tissue distribution of the mers-coronavirus receptor in bats molecular diversity of coronaviruses in bats comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to sars-coronavirus coronaviruses-drug discovery and therapeutic options key: cord-288389-z0sz1msj authors: fanoy, ewout b; van der sande, marianne ab; kraaij-dirkzwager, marleen; dirksen, kees; jonges, marcel; van der hoek, wim; koopmans, marion pg; van der werf, douwe; sonder, gerard; van der weijden, charlie; van der heuvel, jet; gelinck, luc; bouwhuis, jolande w; van gageldonk-lafeber, arianne b title: travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia, may 2014 date: 2014-10-17 journal: emerg themes epidemiol doi: 10.1186/1742-7622-11-16 sha: doc_id: 288389 cord_uid: z0sz1msj background: in may 2014, middle east respiratory syndrome coronavirus (mers-cov) infection, with closely related viral genomes, was diagnosed in two dutch residents, returning from a pilgrimage to medina and mecca, kingdom of saudi arabia (ksa). these patients travelled with a group of 29 other dutch travellers. we conducted an epidemiological assessment of the travel group to identify likely source(s) of infection and presence of potential risk factors. methods: all travellers, including the two cases, completed a questionnaire focussing on potential human, animal and food exposures to mers-cov. the questionnaire was modified from the who mers-cov questionnaire, taking into account the specific route and activities of the travel group. results: twelve non-cases drank unpasteurized camel milk and had contact with camels. most travellers, including one of the two patients (case 1), visited local markets, where six of them consumed fruits. two travellers, including case 1, were exposed to coughing patients when visiting a hospital in medina. four travellers, including case 1, visited two hospitals in mecca. all travellers had been in contact with case 1 while he was sick, with initially non-respiratory complaints. the cases were found to be older than the other travellers and both had co-morbidities. conclusions: this epidemiological study revealed the complexity of mers-cov outbreak investigations with multiple potential exposures to mers-cov reported such as healthcare visits, camel exposure, and exposure to untreated food products. exposure to mers-cov during a hospital visit is considered a likely source of infection for case 1 but not for case 2. for case 2, the most likely source could not be determined. exposure to mers-cov via direct contact with animals or dairy products seems unlikely for the two dutch cases. furthermore, exposure to a common but still unidentified source cannot be ruled out. more comprehensive research into sources of infection in the arabian peninsula is needed to strengthen and specify the prevention of mers-cov infections. in 2012, the middle east respiratory syndrome coronavirus (mers-cov) was isolated for the first time from the sputum of a 60-year-old man who presented with an acute pneumonia in kingdom of saudi arabia (ksa) [1] . as of june 24 2014, 707 laboratory-confirmed cases of mers-cov infection have been reported to the who including 252 (36%) fatal cases [2] . following the first upsurge of cases in spring 2013, a second wave occurred in 2014, mainly in ksa [3] . most cases so far either resided in or travelled to the arabian peninsula and neighbouring countries or were close contacts of these cases. furthermore, travel related cases have been reported from the united states of america (usa), the united kingdom (uk), germany, france, tunisia, italy, greece, algeria, egypt, the philippines and malaysia [3] [4] [5] [6] [7] . so far, mainly sporadic cases are reported as well as a few hospital outbreaks, which is in line with a low reproduction rate (r0) of mers-cov, estimated to range from 0.4 to 1.5 [8] [9] [10] . the median incubation period is estimated to be 5.2 days (95% ci 1.9-14.7 days) [8] . for most non healthcare associated cases no clear source of infection could be identified, although camels are considered a reservoir as the virus and antibodies against mers-cov have been identified in camels (camelus dromedarius) and in their milk [9, 10] . the exact modes of transmission of the virus to humans remain unclear [11] [12] [13] [14] , leaving the possibility for other yet unidentified sources of infection. to stop or reduce transmission of mers-cov and prevent new human infections, it is important to identify all potential sources of infections as well as risk factors and route(s) of transmission. in may 2014, mers-cov infection was diagnosed in two dutch residents. these cases travelled with a group of 29 other people, returning to the netherlands from pilgrimage to medina and mecca, ksa [15] . we conducted a comprehensive epidemiological assessment of the travel group aiming to identify the likely source(s) of infection and presence of potential risk factors for these two cases. we compiled a questionnaire based on sample questionnaires originating from two who-protocols aimed to asses risk factors and investigate contacts of patients with mers-cov infection [16, 17] . we adapted the questionnaire to the specific route and activities of the dutch travellers. our questionnaire covered human, animal and food exposures to mers-cov during the whole trip to ksa. the questionnaires consisted of 28 open and closed questions, and it took approximately 20 minutes to complete the face-to-face interview with the travellers (additional file 1). nurses of the public health services administered the questionnaires approximately 3 weeks after return to the netherlands. for the two cases the questionnaire was completed with data on the travel route and stay in ksa collected by the national coordination centre for communicable disease control. the patients were a 70-year-old man (case 1) and his 73-year-old sister (case 2), both having underlying cardiovascular co-morbidities and diabetes mellitus. the remaining travel group included 29 persons (41% male) aged between 10 and 70 years (median age: 55 years). seventeen (59%) of the non-cases did not report any comorbidities (table 1 ). upon identification of case 1, throat swabs of all contacts were tested by pcr to assess other mers-cov cases. apart from case 2, who had reported mild respiratory complaints starting on the 5 th of may (t = day 9), all other 29 travellers tested negative and did not develop symptoms. the group made a pilgrimage to medina and mecca, ksa ( figure 1 ). they arrived on the 26th of april 2014 (day 0) in medina, left by private bus to mecca on the 4 th of may (day 8) and returned to amsterdam on the 10 th of may (day 14). case 1 started to feel feverish and had diarrhoea on the 1 st of may (day 5). respiratory complaints started on the 10 th of may (day 14). mers-cov was identified in case 1 on the 13 th of may (day 17) and in case 2 on the 14 th of may (day 18). detailed case reports of the two patients are described by kraaij et al. [15] . during their pilgrimage, the whole group stayed in the same hotels and had breakfasts together. there was no fixed collective travel programme, but they had some joint visits to several mosques. the travellers also spent time alone or in smaller subgroups, visiting different mosques, markets and restaurants. the exposures for both the cases and 29 asymptomatic travellers during their visit to saudi arabia are summarized in table 2 . on the 3 rd of may (day 7), 12 travellers (excluding the two cases) made a trip to the touristic valley wadi-e-jinn, and stopped when they came across a herd of camels laying behind an improvised razor fence. four travellers reported direct contact with the camels. eight travellers mentioned indirect contact, for example via the fences surrounding the animals. eleven travellers consumed unpasteurized camel milk, offered to them by the caretakers of the camels. besides contact with camels, two travellers reported that they spotted many pigeons in the cities and fed them. no other direct contact with animals was mentioned. page 2 of 6 http://www.ete-online.com/content/11/1/16 as mentioned above, eleven travellers, excluding the two cases, consumed unpasteurized camel milk. furthermore, 22 travellers, including case 1, visited one or more local markets in medina. seven of them, excluding case 1, bought and consumed a range of fruits such as prunes and dates. furthermore, 14 travellers bought souvenirs, such as dates, perfume and clothing on these markets. case 1 and his son visited two hospitals in medina on the 29 th of april (day 3) because of eye complaints of the son. as mentioned before, they spent 45 minutes in a crowded waiting room of a general hospital where they were exposed to coughing patients. subsequently, the son was referred to a specialized eye clinic where they spent no time in a waiting room and did not notice any coughing patients. on the 5 th of may (day 9), case 1, accompanied by his son, visited an emergency department of a general hospital in mecca because of acute malaise, weakness, nausea and diarrhoea (without respiratory complaints). these symptoms started on may 1 (day 5). case 1 and his son spend 30-45 minutes in the waiting room, though they had no specific exposure to coughing people. on the 7 th of may (day 11) case 1 returned to another hospital in mecca, accompanied by his son, where he only briefly stayed in the waiting room, received antibiotic treatment and was observed for approximately three hours. four other travellers, including his son, accompanied him. two of them did not notice any coughing persons, while the other two did not answer this question. case 2 did not visit a hospital during the pilgrimage or prior to her diagnosis in the netherlands. two travellers (case 1 and his son) waited 45 minutes in a crowded waiting room of a general hospital in medina (29th of april, day 3) where they were exposed to coughing patients. one other traveller, who did not enter a hospital, reported coughing persons outdoors. the duration and intensity of this contact was not clarified in more detail. case 1 and 2 had been in close contact with each other during the entire trip, as they shared hotel rooms. all 29 travellers had been in contact with both cases during the trip. seventeen travellers had minimal social contact with the two cases, for example only brief eye contact in the hotel lobby or in the bus. twelve travellers reported daily contact, such as visits to the hotel room of case 1 while he was sick, or accompanying him while shopping. the son of case 1 had the most intensive contact sharing a hotel room with both cases. nevertheless, his throat swabs tested negative to mers-cov by pcr during the follow-up after the identification of case 1. we found no indications that the two cases were infected via contact with specific animals or dairy products. infection during a hospital visit could be a source for case 1. sequence analysis of parts of the viral genome collected from both patients had shown that the strains were nearly identical, suggesting that the cases have infected each other, or that they were exposed to a common source. in the latter case, exposure during a hospital visit is unlikely because case 2 did not visit a hospital before onset of disease. the possibility of asymptomatic infections and transmission among the accompanying travellers cannot be ruled out, but seems unlikely in view of the negative pcr results. subsequently, although considered unlikely, we cannot exclude the possibility of asymptomatic travellers being a source of infection for case 1 and/or 2. no other common sources were identified although we cannot exclude the possibility of lack of accurate recall or indirect exposure to animal excreta. it might be possible that one or both cases were infected during non-specific contacts with the local population or the environment. if initial zoonotic introductions of mers-cov from camels have been followed by low level presence among the (asymptomatic) population or in the environment at large, this might result in an ongoing community-based outbreak, which, unlike sars, cannot be controlled by rigorous hospital hygiene alone. both patients were of older age and had comorbidities. in general, older age and comorbidity are considered risk factors for symptomatic mers-cov infections, although this was not the case in all outbreaks [18, 19] . in this study, the distribution of symptoms towards higher age and comorbidity fits this assumption. ongoing serological analysis upon travellers and people exposed to the case in the netherlands may reveal to what extent asymptomatic infection has occurred. the remainder of the travellers did not develop symptomatic mers-cov and did not have positive pcr-test results despite exposure to camels, dairy products and the two cases. this could point to a low transmissibility of the virus, which is also illustrated by the limited number of secondary infections among contacts of import cases described elsewhere, with the reservation that asymptomatic infections among the remaining travellers cannot be excluded fully as final serologic results are not available [5, 6] . currently, there is only limited epidemiological information published considering potential sources and transmission dynamics of mers-cov infection within the arabian peninsula. descriptive studies among a well-defined group of travellers can therefore provide relevant epidemiologic information, even when the number of travellers is too small to draw definite conclusions. more in depth epidemiological investigation of comparable travel groups will add to the body of evidence, although the diversity of activities during a relatively short trip makes accurate recall of potential exposure challenging. alternatively, prospective cohort studies among travellers to affected areas, whereby travellers are asked to keep a daily diary, are likely to be more informative and complete. due to the uncertainty about the source, the detailed questions of the who-questionnaire were a very useful guide to decide on the most relevant exposures to be explored, adapted to the specific route of travel. the exact source of infection remains difficult to identify. case 1 could have been infected during his visit to a page 4 of 6 http://www.ete-online.com/content/11/1/16 hospital and subsequently have infected case 2. however, exposure to a common source for both case 1 and 2 cannot be ruled out. besides, case 1 may have been exposed to a yet unidentified source and subsequently have infected case 2, or vice versa. more research to relevant sources in the arabian peninsula is needed. the suggested role of underlying disease to develop mers-cov infection is in line with the age and comorbidity of the two dutch cases and the absence of symptomatic mers-cov infections among the younger and healthier travellers. additional file 1: the questionnaire. isolation of a novel coronavirus from a man with pneumonia in saudi arabia organization wh: coronavirus infections global alert and repsonse (gar) first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities euro surveillance: bulletin europeen sur les maladies transmissibles = european communicable disease bulletin investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in a case of imported middle east respiratory syndrome coronavirus infection and public health response clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands world health organization: seroepidemiological investigation of contacts of middle east respiratory syndrome coronavirus (mers-cov) patients world health organization: case-control study to assess potential risk factors related to human illness caused by middle east respiratory syndrome coronavirus (mers-cov) al raiy b: clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description submit your next manuscript to biomed central and take full advantage of: â�¢ convenient online submission â�¢ thorough peer review â�¢ no space constraints or color figure charges â�¢ immediate publication on acceptance â�¢ inclusion in pubmed, cas, scopus and google scholar â�¢ research which is freely available for redistribution 11 medical centre haaglanden, the hague, the netherlands. 12 isalaklinieken, zwolle, the netherlands. 13 national institute for public health and the environment, epidemiology and surveillance unit, antonie van leeuwenhoeklaan 9, bilthoven, ma 3721, the netherlands. the authors declare that they have no competing interests. all readers have read and approved the manuscript.authors' contributions ef compiled the questionnaires, analysed the data and drafted the manuscript. ms is in charge of the epidemiology department; she designed and coordinated the study and reviewed the manuscript. mk assessed travel data and reviewed the manuscript. kd coordinated interviews with travellers and reviewed the manuscript. mj was responsible for laboratory testing and reviewed the manuscript. wh is in charge of respiratory epidemiology department and reviewed the manuscript. mpgk is involved in laboratory testing and reviewed the manuscript. dw coordinated interviews with travellers and reviewed the manuscript. gs coordinated interviews with travellers and reviewed the manuscript. cw coordinated interviews with travellers and reviewed the manuscript. jh coordinated interviews with travellers and reviewed the manuscript. lg was involved in clinical care and interviewed a case. jb was involved in clinical care and interviewed a case. ag was leading the study, including the study design and analysis and she reviewed the manuscript. all authors read and approved the final manuscript. key: cord-297652-ut6e1ysz authors: vanden eynde, jean jacques title: covid-19: a brief overview of the discovery clinical trial date: 2020-04-10 journal: pharmaceuticals (basel) doi: 10.3390/ph13040065 sha: doc_id: 297652 cord_uid: ut6e1ysz the outbreak of covid-19 is leading to a tremendous search for curative treatments. the urgency of the situation favors a repurposing of active drugs but not only antivirals. this short communication focuses on four treatments recommended by who and included in the first clinical trial of the european discovery project. covid-19 (coronavirus disease 2019) is a worldwide outbreak of pneumonia and acute respiratory distress syndrome. it is unambiguously linked to the human-human transmission of a novel coronavirus. mutagenomic analyses [1] demonstrated that 79.6% of the genome sequences were identical to those found in the coronavirus responsible for the severe acute respiratory syndrome (sars), which spread in 2002. hence the classification as 2019-ncov (n for novel) and the name sars-cov-2 [2] . combatting sars-cov-2 has emerged as a tremendous challenge, gathering efforts from academia, pharmaceutical companies, hospitals, international organizations, as well as governments and philanthropic associations (e.g., the council on foundations [3] ). in this opinion, we give a brief overview of some current and future clinical trials, as they can be found in the u.s. national library of medicine of the national institutes of health (bethesda, md, usa) [4]. as of march 28, 202 clinical trials could be retrieved with the search term "covid19 more than 100 studies involving other small molecules. a quick reading of these projects [4] allows one to sort out the monoclonal antibodies, antiviral drugs, and other small molecules that are most cited in the trials. the list can be found in table 1 . on march 13, the united nations foundation, the swiss philanthropy foundation, and the world health organization (who) have created the solidarity response fund in order to raise money to support studies on covid-19 [5]. on march 18, who indicated that the first trial supported by the fund would be an adaptative study performed in ten countries, namely argentina, bahrain, canada, france, iran, norway, south africa, spain, switzerland, thailand. india joined the trial on march 27. on march 22, the french institut national de la santé et de la recherche médicale (inserm) announced a european counterpart, named discovery, and aimed at a study of four treatments on 3100 patients in seven countries, namely france, spain, the united kingdom, germany, luxembourg, the netherlands, and belgium [6]. the four treatments to be analyzed are: the combination lopinavir/ritonavir; • the combination lopinavir/ritonavir with the addition of interferon β-1a; • hydroxychloroquine, eventually associated with an antibiotic (azithromycin) in add-on studies. the study is entitled "trial of treatments for covid-19 in hospitalized adults (discovery)" (nct04315948) [4] . it started in three french hospitals: centre hospitalier régional universitaire de lille (lille, france), centre hospitalier universitaire de nantes (nantes, france), and assistance publique hôpitaux de paris -bichat claude bernard (paris, france). the structure and some characteristics of the four small molecules involved in that clinical trial are collected in figure 1 . the formula of chloroquine is added for comparison. lille (lille, france), centre hospitalier universitaire de nantes (nantes, france), and assistance publique hôpitaux de paris -bichat claude bernard (paris, france). the structure and some characteristics of the four small molecules involved in that clinical trial are collected in figure 1 . the formula of chloroquine is added for comparison. it has been announced that the discovery project is an adaptive study. that means that some treatments can be abandoned during the study if results are not encouraging, whereas new treatments could be added. some noticeable characteristics of each of the four actual treatments are given in table 2 . remdesivir is an investigational drug developed by gilead sciences. before the outbreak of sars-cov-2, it has been the subject of only one clinical trial, following the u.s. national library of medicine of the national institutes of health [4]. that study (nct03719586) was entitled "investigational therapeutics for the treatment of people with ebola virus disease" and started on november 21, 2018. three monoclonal antibodies (zmapp for the control group, regn-eb3, mab114) and remdesivir were separately administered to a total of 673 participants. an initial dose of 200 mg of remdesivir was administered on day 1, followed by a daily maintenance dose of 100 mg for 9-13 days. results were disappointing. after 28 days, the highest number of deaths (53.1%) was it has been announced that the discovery project is an adaptive study. that means that some treatments can be abandoned during the study if results are not encouraging, whereas new treatments could be added. some noticeable characteristics of each of the four actual treatments are given in table 2 . remdesivir is an investigational drug developed by gilead sciences. before the outbreak of sars-cov-2, it has been the subject of only one clinical trial, following the u.s. national library of medicine of the national institutes of health [4]. that study (nct03719586) was entitled "investigational therapeutics for the treatment of people with ebola virus disease" and started on november 21, 2018. three monoclonal antibodies (zmapp for the control group, regn-eb3, mab114) and remdesivir were separately administered to a total of 673 participants. an initial dose of 200 mg of remdesivir was administered on day 1, followed by a daily maintenance dose of 100 mg for 9-13 days. results were disappointing. after 28 days, the highest number of deaths (53.1%) was observed in the group treated with remdesivir, a bit more than in the control group (49.7%). more encouraging results were obtained in the two other groups (regn-eb3: 33.5 %; mab114: 35.1%) [7] . in contrast, in the cases of sars-cov and mers-cov, remdesivir exhibited an excellent activity, in vitro, in the submicromolar range with ec 50 values of 0.07 µm for both coronaviruses [8] . results were confirmed in vivo in the case of an infection by mers-cov in mice [9] and rhesus macaque models [10] . however, pharmaceuticals 2020, 13, 65 4 of 8 the conclusion of sheahan [9] regarding sars-cov in a mouse model warned that "these data suggest that reductions in viral load after peak lung titers were achieved were insufficient to improve outcomes after the immunopathological phase of disease had been initiated. thus, in the mouse, if given prior to the peak of sars-cov replication and peak damage to the airway epithelium, gs-5734 (remdesivir) can improve pulmonary function, reduce viral loads and diminish disease." an ec 50 value of 1.76 µm for remdesivir against sars-cov-2 in vero e6 cells has been measured by wang et al. [11] . there is a study involving only one human: the first patient suffering from covid-19 in the united states [12] . he was hospitalized on january 19 in the state of washington. for the first 6 days of hospitalization (days 5-10 of the illness), he received 650 mg of acetaminophen every 4 h, 600 mg of ibuprofen every 6 h, 600 mg of guaifenesin and 6 liters of normal saline. on day 6 of hospitalization, he was treated with vancomycin (1750 mg then 1 g every 8 h) and cefepime (every 8 h). the next day, remdesivir was administered for compassionate use, whereas the antibiotics were discontinued. the authors concluded their article by writing [12] : "on hospital day 8 (illness day 12), the patient's clinical condition improved. supplemental oxygen was discontinued, and his oxygen saturation values improved to 94% to 96% while he was breathing ambient air. the previous bilateral lower-lobe rales were no longer present. his appetite improved, and he was asymptomatic aside from intermittent dry cough and rhinorrhea. as of january 30, 2020, the patient remains hospitalized. he is afebrile, and all symptoms have resolved with the exception of his cough, which is decreasing in severity. . . . although a decision to administer remdesivir for compassionate use was based on the case patient's worsening clinical status, randomized controlled trials are needed to determine the safety and efficacy of remdesivir and any other investigational agents for treatment of patients with 2019-ncov infection.". it is actually too early to calculate the price of an eventual treatment of covid-19 with remdesivir, but some thoughts can be explored. the dosage that will be evaluated in the discovery project (nct04315948 [4,13]) is 200 mg iv initially, then 100 mg od for 2-10 days. the product, as a research derivative, is actually sold at 854 usd for 10 mg by medchemexpress (monmouth junction, nj, usa; cat # hy-104077) [14] and 330 usd for 1 mg by biovision inc (milpitas, ca, usa; cat # b2997-1000) [15] , it is not pessimistic to suggest a retail price higher than 5000 usd. the commercial combination (kelatra®) comprises lopinavir and ritonavir in a weight ratio 4:1; it is available for the treatment of hiv infection. it has been the subject of more than 400 registered clinical trials [4] . in vitro, the combination is more than 100-fold less effective than remdesivir against strains of sars-cov (ec 50 = 17.1 µm [16] ) and mers-cov (ec 50 = 8.0 µm [16] ). other data indicated some success regarding the use of the combination in marmosets (mers) [17] and in infected human patients (sars as well as mers) [18] . however, some authors remain skeptical and are not confident in the success of kelatra®for treating covid-19 [19] [20] [21] [22] . in the discovery project (nct04315948 [4,13]), the suggested dose of the combination is 500 mg (400 mg lopinavir + 100 mg ritonavir) every 12 h for 14 days. on the basis of the lowest retail price found on the website pharmacychercker.com [23] , the cost of the treatment, if the drug combination proves to be efficacious in humans, should be estimated at 61 usd for the generic and 215 usd for the original brand. interferon β-1a is an efficient inhibitor of the multiplication of sars-cov [24, 25] and mers-cov [26] in cell cultures. in a mers-cov mouse model, the combination lopinavir/ritonavir/interferon β-1a, used as prophylactic and curative treatments, revealed no significant decrease in the viral load [9] . in a marmoset model [17] , the combination lopinavir/ritonavir/interferon β-1b (bacterial origin, whereas interferon β-1a is produced by chinese hamster ovary cells [27] ) opened some hope for curing mers-cov-infected animals. the results of an ongoing clinical trial (nct pharmaceuticals 2020, 13, 65 5 of 8 02845843 [4]), entitled "mers-cov infection treated with a combination of lopinavir/ritonavir and interferon beta-1b", are eagerly awaited, but will not be disclosed before 2021. the study suggested a dose of lopinavir/ritonavir (400/100 mg) twice daily for 14 days, and interferon β-1b 0.25 mg subcutaneous every alternate day for 14 days [4]. interferon β-1a is clinically used for the treatment of relapsing multiple sclerosis. it was approved by the fda on 05/17/1996 under the brand name avonex®and is sold by biogen under the orphan drug designation. it is also known as rebif®from serono inc (fda-approved on 03/07/2002). in the discovery project, interferon β-1a will be added to the treatment lopinavir/ritonavir (see section 4.2) and administered subcutaneously at the dose of 44 µg for a total of three doses in 6 days. that will represent an additional cost of almost 2000 usd for rebif®, following [23] . there is a huge confusion in the public, as well as among politicians and even some scientists, about those two molecules. that confusion ultimately led to people accidently overdosing [28] . both molecules can be classified as 4-aminoquinolines, and both have anti-plasmodial activities, but they remain two different molecules with some different metabolites. chloroquine has been widely used since the 1950s for prophylactic and curative treatments of malaria. its extensive use has led to the emergence of chloroquine-resistant strains of plasmodium falciparum, the parasite responsible for the severest form of malaria. such strains are also resistant to the structurally related hydroxychloroquine. regardless, the latter is sometimes preferred because of its lower (retinal) toxicity [29, 30] . chloroquine (aralen®) is indicated for the treatment of malaria and extraintestinal amebiasis. hydroxychloroquine (plaquenil®) is also prescribed for the treatment of malaria. it is also used successfully to treat lupus erythematosus and rheumatoid arthritis [27] . in addition, it should be noted that chloroquine, essentially, and hydroxychloroquine, to a lesser extent, have been clinically tested for their anti-hiv activity [4, 31] . as recently as 2006, the anti-coronavirus properties of hydroxychloroquine and chloroquine were reported by biot et al. [32] . those authors observed that, in vitro, both molecules were active against sars-cov with ec 50 values of 34 and 6.5 µm, for hydroxyquinoline (4) and chloroquine (5), respectively. the difference is less marked in the study of dyall et al. [33] who reported ec 50 values of 7.97 µm (4) and 6.54 µm (5) against sars-cov and 8.28 µm (4) and 6.28 µm (5) against mers-cov. the trend is reversed when dealing with sars-cov-2, as values related by yao et al. [34] were 0.72 µm (4) and 5.47 µm (5). a comparable efficacy of 6.9 µm was measured by wang et al. [11] for chloroquine (5). on february 19, gao et al. [35] recorded, in china, 15 clinical trials involving chloroquine. on march 28, the fda issued an emergency use authorization for use of hydroxychloroquine and chloroquine for patients who do not have access to the drugs via clinical trials [36] . as underlined by a reviewer of this manuscript, this essentially means that lots of anecdotal testimonies as to the effects of these two drugs will be released until properly controlled clinical trials are done. as of 8 april, 58 clinical trials concern hydroxychloroquine, compared to 19 on march 28 [4] . in france, controversial results have been published, in march, on a treatment associating the same dosing regimen of hydroxychloroquine and azithromycin. the treatment was administered to small cohorts (less than 25 people) of patients hospitalized for severe covid-19 infection [37, 38] . due to such confusion, in france and belgium (and probably in other countries), politicians, their advisors, and even some physicians called for caution. they suggested that preliminary reports describing successful outcomes in the use of hydroxychloroquine or chloroquine in hospitals must be carefully considered, either because those two molecules are plagued by side-effects or because of a lack of scientific rigor in the studies. contrastingly, the bill and melinda gates foundation announced that it had launched and financially supported two trials aiming at a study on the use of hydroxychloroquine as a prophylactic agent for covid-19 [39] . in the discovery project, the suggested dosage for hydroxychloroquine is 400 mg for the initial dose, followed by 400 mg 12 h later and then 200 mg bid for up to 4 days. using the same metric [23] to evaluate the cost of the treatment, the price should not exceed 4.1 usd. alternatively, a higher dosage is proposed for chloroquine: 600 mg for the initial dose, followed by 600 mg 12 h later and then 300 mg bid for up to 4 days. considering the commercial availability of chloroquine as a diphosphate salt, the retail price would not exceed 6.6 usd [23] . the first cases of covid-19 emerged in wuhan, china, in late 2019, and to date there is no approved specific drug to cure patients infected by sars-cov-2. politicians and physicians suggest that we should wait for clinical trials' data before considering an eventual treatment. in europe, the discovery project (nct04315948) has a study start date of march 22, 2020 and will enroll 3100 patients. the first results should be available within time frames of 15 and 29 days, but no publication date has been announced. the estimated study completion date has been set in march 2023. as of april 8, there are 388 ongoing studies on covid-19 that are recorded in the u.s. national library of medicine of the national institutes of health [4]: a breath of hope. a novel coronavirus from patients with pneumonia in china the species severe acute respiratory syndrome related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a call to action: philanthropy's commitment during covid-19 foundation and partners launch first-of-its-kind covid-19 solidarity response fund a randomized, controlled trial of ebola virus disease therapeutics broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel coronavirus in the united states sciensano epidemiologie des maladies infectieuses. available online screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease-19 treatment option systematic review of treatment effects covid-19-the search for effective therapy a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 why re-purposing hiv drugs lopinavir/ritonavir to inhibit the sars-cov2 protease probably won't work-but re-purposing ribavirin might since it has a very similar binding site within the rna-polymerase pharmacy checker helping people safely find more affordable medicine the antiviral effect of interferon-beta against sars-coronavirus is not mediated by mxa protein interferon-β 1a and sars coronavirus replication interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays husband and wife poison themselves trying to self-medicate with chloroquine comparison of hydroxychloroquine and chloroquine use and the development of retinal toxicity pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against calaria caused by plasmodium vivax chloroquine and beyond: exploring anti-rheumatic drugs to reduce immune hyperactivation in hiv/aids design and synthesis of hydroxyferroquine derivatives with antimalarial and antiviral activities repurposing of clinically developed drugs for tteatment of middle east respiratory syndrome coronavirus infection in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies request for emergency use authorization hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 infection the bill and melinda gates foundation. three weeks, two drug trials: an update on the therapeutics accelerator with trevor mundel funding: this research received no external funding. the author declares no conflict of interest. key: cord-260334-xo8ruswo authors: new, r.r.c.; moore, b.d.; butcher, w.; mahood, r.; lever, m.s.; smither, s.; o'brien, l.; weller, s.a.; bayliss, m.; gibson, l.c.d.; macleod, c.; bogus, m.; harvey, r.; almond, n.; williamson, e.d. title: antibody-mediated protection against mers-cov in the murine model() date: 2019-07-09 journal: vaccine doi: 10.1016/j.vaccine.2019.05.074 sha: doc_id: 260334 cord_uid: xo8ruswo murine antisera with neutralising activity for the coronavirus causative of middle east respiratory syndrome (mers) were induced by immunisation of balb/c mice with the receptor binding domain (rbd) of the viral spike protein. the murine antisera induced were fully-neutralising in vitro for two separate clinical strains of the mers coronavirus (mers-cov). to test the neutralising capacity of these antisera in vivo, susceptibility to mers-cov was induced in naive recipient balb/c mice by the administration of an adenovirus vector expressing the human dpp4 receptor (ad5-hdpp4) for mers-cov, prior to the passive transfer of the rbd-specific murine antisera to the transduced mice. subsequent challenge of the recipient transduced mice by the intra-nasal route with a clinical isolate of the mers-cov resulted in a significantly reduced viral load in their lungs, compared with transduced mice receiving a negative control antibody. the murine antisera used were derived from mice which had been primed sub-cutaneously with a recombinant fusion of rbd with a human igg fc tag (rbd-fc), adsorbed to calcium phosphate microcrystals and then boosted by the oral route with the same fusion protein in reverse micelles. the data gained indicate that this dual-route vaccination with novel formulations of the rbd-fc, induced systemic and mucosal anti-viral immunity with demonstrated in vitro and in vivo neutralisation capacity for clinical strains of mers-cov. the middle east respiratory disease syndrome (mers) first emerged in 2012 in saudi arabia [1, 2] . since then, there have been an estimated 2311 laboratory-confirmed cases with 811 deaths, reported from a total of 27 countries in the eastern mediterranean region and from 12 countries elsewhere [1] . saudi arabia, however, remains the main focus of infection and a disease outbreak in south korea involving 186 cases was traced back to an index case who had travelled from saudi arabia. whilst the incidence of mers cases in saudi arabia peaked in 2014, there are still a significant number of cases reported from the country and in the period september 2012-may 2018, there were 1844 cases including 716 deaths with a case fatality rate of 38.8% [1] . mers coronavirus (mers-cov) is a member of the betacoronavirus genus [3] and as for other betacoronaviruses, bats may provide a natural reservoir for the virus [3, 4] , but high levels of antibodies to mers-cov in dromedary camels [5] suggest that the dromedary camel is the principal source for animal-to-human transmission of mers-cov [6] . however, evidence of human-to-human transmission comes from the reporting of outbreaks in countries remote from saudi arabia such as the uk, europe, usa, and china where small outbreaks have also occurred [1] . mers-cov is an enveloped, positive-sense, single-stranded rna virus [3] . the virus possesses an envelope-anchored trimeric spike protein which binds to the human receptor dipeptidyl peptidase 4 (dpp4 or cd26) and gains host cell entry by the fusion of viral and host membranes [7] . the spike protein comprises an s1 sub-unit and a membrane fusion s2 sub-unit. in the coronaviruses, the s1 sub-units are further divided into n-terminal and c-terminal subdomains and for mers-cov, it is the c-terminal sub-domain that comprises the receptor-binding domain (rbd) [8] . the rbd also incorporates a receptor-binding motif at its c-terminal and the crystal structures of mers-cov rbd [8] and of the rbd bound to the extracellular domain of human dpp4 have been reported [9] . the rbds of the coronaviruses represent vaccine and therapeutic targets and the rbd of mers-cov as a vaccine antigen has been demonstrated to induce neutralising antibody [10] and to protect mice transduced with a viral vector expressing hdpp4, or nonhuman primates from viral challenge [11] [12] [13] [14] [15] . there are significant ongoing efforts to develop vaccines for mers-cov infection, predominantly involving live attenuated viral vectors such as adenovirus, modified vaccinia ankara or measles [16] to induce anti-viral immunity and some of these vaccines are already in clinical trials. here, we were interested to determine the relative importance of inducing systemic and/or mucosal immunity in vaccination to protect against mers-cov, an infection predominantly of the respiratory tract and lungs. to this end, we have used a dual route immunisation regimen in balb/c mice to induce both systemic and mucosal immunity, to generate rbd-specific murine antisera. initially, we immunised balb/c mice sub-cutaneously (s.c.) with rbd-fc in the mf59 adjuvant to induce rbd-specific igg. subsequently, we have immunised further groups of balb/c mice by s.c. priming and per oral (p.o.) boosting with the rbd-fc, to induce both systemic igg and mucosal iga responses. to do this, we have used novel formulations of rbd-fc coated onto microcrystals formed from histidine or glutamine and also incorporating calcium phosphate for sub-cutaneous priming [17] , whilst the formulation for oral boosting comprised rbd-fc in reverse micelles dispersed in a self-emulsifying oil phase, which has been optimised from previous formulations [18, 19] . the advantages of these formulations are that they are very stable under extremes of temperature [20] . furthermore, on translation to the clinic, only one injected priming dose would be required, followed by a p.o. booster dose; the latter could be self-administered in capsule form. we have compared the relative abilities of the two sources of antiserum to neutralise clinical isolates of mers-cov in vitro. to do this, we have used two clinical strains of mers-cov (erasmus medical center 2012 or emc2012 and london1-2012:), each of which were derived from severely-ill individuals who had contracted the virus in the middle east in 2012. subsequent sequencing of the polymerase gene from these isolates indicated them to be newly-emerged members of the betacoronavirus genus with a close sequence homology and phylogenetic relationship to the bat coronaviruses hku4 and hku-5 [21, 22] . mice are not naturally susceptible to mers-cov infection, but susceptibility can be induced by the administration of an adenovirus vector which induces expression of the human receptor (hdpp4/cd26) for the virus in vivo for a limited time, providing a non-lethal murine model of the disease [23] . we have used this transduced mouse model to test the capacity of the antiserum derived from the dual route immunisation to neutralise mers-cov in vivo, by passive transfer prior to challenge with the emc2012 strain and we have demonstrated a significant reduction in viral load in lung tissue in transduced mice. the rbd was synthesised and expressed according to methods adapted from du et al. [10] . in brief, a single dna fragment containing an in-frame fusion of the coding sequences for the human il2 signal peptide, the rbd and human igg1-fc was synthesised. this was transferred into the plasmid pef-dest51 (invitrogen) so that the target sequence was expressed as a secreted protein with a c-terminal human igg1 fc tag. this construct was transfected by cationic transfection into human embryo kidney (hek) cells in suspension (fshek) or adherent hek cells stably expressing the sv40 large t antigen (293ft), using serum-free media and incubated for 4-7 days. small scale purifications of rbd-fc were performed using protein a chromatography. for this, medium from the transfected cells was treated with ammonium sulphate to precipitate the protein, prior to dialysis and resuspension in buffers for binding to protein a beads. the latter were washed and eluted with buffer containing 1 m urea. protein concentration was determined by uv absorbance spectroscopy and purity was estimated by sds-page with coomassie staining and subsequent optical densitometry using a syngene g:box imaging system. the rbd-fc was incorporated on glutamine calcium phosphate (cap) microcrystals for s.c. immunisation, using methodology adapted from [17] . briefly, aqueous mixtures of rbd-fc with sodium orthophosphate and glutamine were precipitated as cap protein-coated microcrystals (cap-pcmc), by addition to of a 19fold excess of isopropanol containing dissolved calcium chloride. the resultant suspension contained self-assembled microcrystals comprising a glutamine core with the rbd-fc protein embedded in a thin surface layer of cap (now termed rbd-fc-pcmc). the pcmc were isolated by vacuum filtration and dried to a powder. protein content and integrity was determined by elisa and sds-page. for oral dosing, the rbd-fc was incorporated in mineral oil with added excipients using methodology adapted from [18, 19] . the oral formulation comprised rbd-fc with the mucosal adjuvant cholera toxin b sub-unit (ctb), retinoic acid (ra), vitamin d, e and trehalose debehenate (tdb), a synthetic analog of the mycobacterial trehalose dimycolate [25] and imiquimod, a tlr 7/8 agonist [26] . specific pathogen-free female balb/c mice (6-8 weeks of age) were obtained from a commercial breeder and used throughout this study. on receipt, mice were randomised for allocation to cages and given free access to food, water and environmental enrichment. mice were fully acclimatised to the animal housing facility for at least five days prior to any procedure. all animal procedures were performed in accordance with uk legislation as stated in the uk animal (scientific procedures) act 1986. the institutional animal care and use committee approved the relevant project licence. naïve mice were randomised for allocation to a treatment group (typically 5 per group) and immunised in one of two regimens: either with a s.c. priming dose followed by two s.c. doses, given at 10 and 31 days after the prime; alternatively, mice received a s.c. priming dose followed by an oral or s.c. booster dose 21 days after the prime (table 1) . for s.c. immunisation, mice received 2.5 lg of rbd-fc-pcmc in 0.1 ml pbs injection volume, whereas for all per oral (p.o.) dosing, mice received 25 lg of rbd-fc in a total volume of 0.1 ml mineral oil (mo), by oral gavage. where rbd-fc was administered s.c. in the conventional adjuvants mf59 or alhydrogel, mf59 (novartis, us) was used in a 1:1 ratio by volume with rbd-fc in pbs, whilst alhydrogel (brenntag biosector, denmark) was used in a 1:5 ratio with rbd-fc by volume in pbs. at selected intervals after dosing, mice were blood-sampled from the tail vein for assay of specific antibody titre. at the end of the immunisation schedule, individual mice were terminally anaesthetised for collection of blood by cardiac puncture, then culled prior to removal of small and large intestines for collection of faecal pellets for extraction of iga. titres of rbd-fc-specific antibody in serum samples were determined by elisa. in brief, test sera were bound to microtitre plates pre-coated with rbd-fc and antibody binding was detected with an hrpo-labelled secondary antibody to mouse igg, igg1, igg2a or iga (bio-rad). a standard curve for calibration comprising the relevant murine ig isotype (sigma) captured with an anti-fab reagent, was included on each plate. plates were developed by the addition of 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts) substrate (sigma) and optical density (od) was read at 414 nm (multiskan plate reader). for assay of antibody in faecal samples, faecal pellets were extracted in supplemented pbs as described previously [20] . in brief, 10 ml of cold pbs was prepared, supplemented with 1 tablet of complete mini protease inhibitor cocktail (sigma) and 5 ll tween 20 were added. to 0.1 g faecal pellets, 1 ml of supplemented pbs was added and left at room temperature for 5 min. samples were vortexed for approximately 30 s, incubated on ice for a further 20 min. and then centrifuged (15,000g, 5 min.). supernatants were retained and stored at à80°c pending assay. the faecal extracts were assayed for specific igg and iga content, by elisa, as for serum samples. antibody concentrations in all samples were determined from the relevant standard curves using ascent software with fourparameter logistic curve-fitting and reported in ng/ml or lg/ml serum or faecal extract, as appropriate. to determine if the antibody induced by immunisation with to rbd-fc was neutralising for mers-cov in vitro, plaque assays were performed. for this, two strains of mers-cov were used: london1-2012 (genbank accession number kc164505.2) [22] and erasmus medical center (emc2012 genbank accession number jx869059) (21) . the london1-2012 strain was obtained from the national collection of pathogen viruses, phe porton, salisbury, uk and the emc2012 strain was kindly provided by the erasmus university medical center rotterdam, the netherlands. both strains were prepared in serum-free media (gibco) at a multiplicity of infection (moi) of 0.01, equivalent to 10 3 plaque-forming units (pfu). the murine antiserum for testing was prepared at a dilution range from undiluted to 1:10 in pbs. virus was incubated overnight (4°c) with murine antiserum, negative control antibody (nibsc, uk) or media, prior to infection of a confluent monolayer of vero e6 cells (ecacc, salisbury uk) with 200 ll of the mixture. the neutralising ability of the murine antiserum was tested in duplicate or triplicate at each dilution. after incubation (1 h, 37°c), an overlay comprising a 1:1 dilution of carboxymethyl cellulose with serum-free media was added to the cells and incubation continued for a further 4 days (37°c) prior to fixing (7.4% formaldehyde) and staining (0.2% crystal violet) with enumeration of the number of plaques per ml. mice are not naturally susceptible to infection with mers-cov, since they lack the human dpp4 receptor. to induce transient susceptibility in balb/c mice, we used an ad5 construct (oxford genetics) to express the human dpp4 receptor (ad5hdpp4), as previously described (23) . mice were administered the ad5hdpp4 construct (2.5 â 10 8 pfu in 50 ll) by the intra-nasal (i.n.) route under light sedation with inhalational isofluorane and then monitored by serial blood sampling for serum levels of hdpp4/cd26 by elisa (thermoscientific). at peak levels of expression of hdpp4 (days 5-7), mice were lightly sedated as before and challenged by the i.n. route with mers-cov (emc2012 strain) at 10 4 pfu in 50 ll per mouse. mice were weighed prior to challenge on each subsequent day to monitor changes in body weight during infection. to test the in vivo neutralising capacity of murine antiserum raised to the rbd-fc construct, naïve mice (n = 10 per treatment group) were passively immunised by the i.p. route at 24 h. prior to i.n. challenge with the mers co-v (emc2012 strain), as described above. the murine antiserum, pooled from 4 mice who had been primed with rbd-fc pcmc and boosted orally (regimen 2, treatment group 2), was delivered at a dilution of 1:10 in pbs and delivered in a total volume of 100 ll per mouse. a further group of 10 mice received a purified polyclonal human igg at a single dose level (150 lg/mouse in 100 ll i.p.), which had been raised to inactivated mers-cov. control mice received a non-specific human igg at a single dose-level (200 lg/mouse in 100 ll, i.p.). both sets of human igg (specific and non-specific) were raised in a bovine transchromosomal model and purified prior to use. a further group of 10 negative control mice were included, which received pbs in place of either the ad5dpp4 construct or the mers-cov-specific antibody, and were also challenged i.n. with mers-cov (emc2012 strain) at 10 4 pfu/mouse. to determine the protection afforded by the passive immunisation, pairs of mice from each treatment group were culled on days 1-8 after challenge and their lungs were removed and weighed and then rapidly frozen (à80°c) prior to the determination of viral load. pairs of lungs from each of 2 mice per treatment group were individually thawed and homogenised in serum-free media (2 ml). rna was extracted from 140 ll of each homogenate using the qiaamp viral rna kit (qiagen), following the manufacturers' instructions. real-time pcr was conducted on duplicate 5 ll the amount of virus in tested samples was determined in duplicate using the standard curve and reported as pfu/g lung tissue. all data were analysed using graph pad prism software v.6 and expressed as mean ± s.e.m. statistical comparisons were made using one-way anova or unpaired t-test. the rbd-fc protein was expressed in both adherent 293ft and suspension human embryo kidney (hek) cells, but with greater expression in adherent cells (fig. 1) . purification of protein from adherent cells with protein a was very effective, yielding protein which was >99% pure, with molecular weight of approximately 100 kda (fig. 1a) . the use of 1 m urea for elution was optimum, as it was sufficient to solubilise the protein without denaturing it, yielding rbd-fc in optimum yield (0.2 mg/ml) and predominantly in a dimeric form (fig. 1b) . this method of protein purification was therefore selected for forward use. rbd-fc, formulated for either sub-cutaneous (s.c.) or per oral (p.o.) immunisation, was tested for immunogenicity and the formulations optimised in an iterative approach. initially, a s.c. dosing regimen was used in which rbd-fc was formulated in either alhydrogel or mf59 to deliver 2.5 lg of protein on each of three occasions at 0, 10 and 31 days. mice were monitored for 26 days after the final boost and igg titre determined ( fig. 2a) . at day 70, the total igg titres achieved with rbd-fc in alhydrogel or mf59 did not differ significantly. to determine if the presentation of rbd-fc in either alhydrogel or mf59 influenced the ability to develop virus-neutralising antibody, antisera were selected from 2 mice in each immunisation group and tested in a plaque assay for neutralisation of both the emc2012 and london1-2012 strains of mers-cov ( fig. 2b and c) . all four sera gave some neutralisation of viral activity, although at a 1:20 dilution, sera 136 and 169 were most potent, against both viral strains. sera 136 and 169 were derived from the treatment group immunised with mf59adjuvanted rbd-fc, whereas sera 132 and 150 were derived from alhydrogel-adjuvanted rbd-fc ( fig. 2a) . based on this pilot data, we subsequently used mf59 as the conventional adjuvant for rbd-fc, to compare with some novel formulations. having demonstrated to proof-of-principle that the rbd-fc, when delivered in mf59 can induce a high titre of antibody with neutralising activity, we next investigated how to tailor an rbd-fc vaccine optimally to induce both systemic and mucosal immunity, with the aim also of reducing to a 2-dose immunisation regimen and increasing functional antibody. for this, we selected novel formulations in which rbd-fc protein was presented as rbd-fc-pcmc for s.c. priming and incorporated into mineral oil (mo) for p.o. boosting. we compared the serum igg response achieved from this 2-dose dual route immunisation with that induced to rbd-fc delivered in mf59 in a 2-dose s.c. regimen (fig. 3) . at 1 month after the booster dose, at day 49, there was no significant difference in the serum igg titres achieved, so that the 2-dose dual-route immunisation with rbd-fc-pcmc for s.c. priming and incorporated in mo with excipients for p.o. boosting, was just as immunogenic as the 2-dose s.c. immunisation with rbd-fc in mf59 (fig. 3a) . at day 49, the serum response to rbd-fc in the dual-route regimen was predominantly igg1 biased, whereas s.c. dosing with rbd-fc in the presence of mf59 induced both igg1 and igg2a (fig. 3b) . since dual route immunisation effectively induced serum igg to rbd-fc, it was of interest to determine whether it could also effectively induce mucosal immunity. in this study, the rbd-fc-specific iga response was determined in serum and in faecal pellet extracts from individual animals on day 49. in this case, the rbd-specific iga responses of mice immunised in the 2-dose dual route regimen were compared with that of mice immunised by the oral route twice, and with mice immunised by the s.c. route in mf59 twice, on exactly the same days (0,21) (fig. 4) . this comparison showed that s.c. immunisation in mf59 did not induce serum iga. however s.c. priming with rbd-fc-pcmc with p.o. boosting effectively induced rbd-fc-specific iga and was not inferior to oral priming and boosting in this effect in either serum (fig. 4a ) or faecal extracts (fig. 4b ). however mice primed and boosted orally did not develop rbd-specific systemic igg (data not shown). additionally, day 49 sera from mice in all treatment groups were tested for their ability to neutralise either strain of mers-cov in vitro (table 2) . from this it can be seen that sera from 4 out of 5 mice in the dual route regimen were fully neutralising neutralisation of mers-cov in vitro. in in vitro for both strains (emc2012 and london1-2012), when tested at 1:60 dilution ( fig. 5a and b) . in order to test whether the in vitro neutralising activity translated into viral neutralisation in vivo, sera from these 4 mice (highlighted in table 2 ) were pooled in equal aliquots at 1:10 dilution to enable a subsequent passive transfer study. in order to design the passive transfer study, it was necessary to define the duration of expression of cd26 in murine lungs in vivo, following induction with the ad5hdpp4 construct. mice dosed with ad5hdpp4 i.n. at t 0 were culled in pairs and lung homogenates prepared and assayed for cd26 expression. cd26 in lung tissue was expressed in a time-dependent manner, with levels peaking at day 3 and declining to day 17 (fig. 6a) , setting a sufficient window to use the model for the determination of the protection against viral challenge afforded by the passive transfer of mers-specific antibody. to determine the protection afforded by the passive transfer of murine antiserum raised in the dual route immunisation regimen, against infection, susceptibility to mers-cov was induced at t 0 with i.n. administration of ad5hdpp4 to groups of 10 mice. passive transfer by the i.p. route of the pooled serum sample derived from the 4 mice highlighted in table 2 , which had previously been shown to be neutralising in vitro (table 2 ) was conducted 5 days later and mice were challenged after a further 24 h with mers-cov emc2012. additional groups of mice, which had been transduced with ad5hdpp4, were passively immunised with a mers-cov specific human igg and a non-specific human igg. at 1-8 days after challenge, pairs of mice were culled for the determination of viral load in lungs, which was determined to peak at 3 days p.i. (data not shown). at 3 days p.i., the pooled murine antiserum significantly reduced viral titres in lungs, to the same extent as the specific human igg, and contrasting with the negative control human igg, demonstrating significant in vivo neutralising activity (fig. 6b ). fig. 6 . a. expression of cd26 was induced in lung tissue by the administration of ad5hdpp4 (2.5 â 10 8 pfu) to mice by the i.n. route at t 0 . subsequently, mice were culled in pairs on the days shown and their lungs assayed for the expression of cd26. the plot shows the time-course of cd26 expression from 3 to 17 days post-induction. all data points were normalised for background values from control mice. 6b: content of mers-cov (emc2012 strain) in murine lungs (pfu/g tissue) determined by rt-pcr at day 3 post-infection, (equivalent to day 4 after passive transfer with murine antisera to rbd-fc which had previously been shown to neutralise the emc2012 strain in vitro). mice received either a mers-cov-specific human igg (150 lg) or non-specific human igg (200 lg) in 100 ll /mouse i.p.; or murine antisera to rbd-fc, which had been pooled from 4 murine donors and which was delivered at 1:10 dilution (100 ll/mouse i.p.). negative control mice received pbs in place of ad5hdpp4 or antiserum all mice were challenged with mers-cov emc2012 i.n. at 10 4 pfu/mouse. statistical significance was determined at the p < 0.05 level by one way anova and unpaired t-test. no significant differences in body weight were detected between treatment groups challenged with mers-cov, which was attributed to the short time period of the study. mers is a serious endemic respiratory infectious disease for which there is no licensed vaccine, although there are several vaccines in clinical trial currently including adenovirus-vectored delivery of the spike protein and sub-units [27] , dna vaccines and nanoparticle-delivered sub-unit approaches [28] . we were interested in determining the advantage of r a vaccine which could induce mucosal as well as specific systemic immunity to the key target, the rbd protein, in order to achieve optimum protective efficacy. vaccination to induce effective immunity at mucosal surfaces, should prime the immune system to respond rapidly to invading pathogens such as mers-cov. previous studies have used adenovirus delivery of the mers spike protein with intra-muscular (i.m.) or intra-gastric (i.g.) delivery to induce neutralising systemic igg, but not iga; further, whilst i.m. delivery also induced specific t-cell immunity, i.g. delivery did not [29] . others have shown that intra-nasal delivery of a live attenuated adenovirus-vectored subunit vaccines does induce specific mucosal as well as systemic immunity, although translation of this approach to the clinic may raise safety issues [30] . here, we have relied on novel formulations of a sub-unit protein to enable dual route vaccination (parenteral and oral) to induce mucosal as well as systemic immunity. in this study, we have achieved the expression and purification of a recombinant rbd-fc protein in milligram quantities. we have also demonstrated that when formulated as a sub-unit vaccine, the construct induced murine antibody which effectively neutralised two different clinical strains of mers-cov in vitro. additionally, we have shown that these murine antisera, when passively transferred into naïve mice transduced to express the hdpp4 /cd26 receptor, conferred protection against viral challenge in the recipients, with significantly reduced viral loads in the lung tissue of the recipient mice. whilst the use of conventional adjuvants such as mf59 or alhydrogel to formulate the rbd-fc protein resulted in high titres of specific igg in serum, the mf59 formulation did not induce specific iga in serum. in order to promote both systemic and mucosal immunity to rbd-fc, we have formulated this protein for injected priming and p.o. boosting, entailing the optimisation of the cap pcmc and of the reverse micelles in oil emulsion, respectively. this has enabled the achievement of a vaccination regimen comprising only two doses and rapidly inducing rbd-fc-specific systemic and mucosal immunity. whilst the pcmc formulation of rbd-fc was as effective as rbd in mf59 in inducing a primary igg response, we have shown that an oral formulation of rbd-fc in mineral oil with selected immunostimulants was as effective as mf59 when used as a booster immunisation. additionally, we have shown that non-invasive oral priming and boosting is as effective at inducing a specific mucosal response, measured as specific iga in serum and faeces, as is injected priming with rbd-fc in the pcmc formulation together with oral boosting, leading to the exciting concept of a potential orally-dosed sub-unit vaccine for mers. whilst both alhydrogel and the combination of pcmc and oral formulations are th2-polarising, as evidenced by the predominantly igg1 titres raised to rbd-fc, the influence of mf59 on the response to rbd-fc was a mixed th-2/th1 effect, with a significant induction of specific igg1 and igg2a. to counter a viral infection, it would be expected that a th1 response would be most appropriate. however, the fact that neutralising antibody to rbd-fc was raised under either th1 or th2-polarising influences, suggests that either isotype can be protective and primes the immune system sufficiently and would allow for cross-presentation to occur on subsequent exposure to the virus [31] . in this study, we have not examined the induction of a cell-mediated memory response to the rbd-fc protein, although this will play a significant role in protection against the virus. currently, we are presenting the rbd protein in our formulations with an fc tag, derived from human igg1 and useful in purifying the protein. the fc tag may contribute additional adjuvantising activity by engaging antigen-presenting cells in the vaccinee [32] and it may aid mucosal immunity since the fc receptor, an mhc1 transmembrane protein, is also expressed at mucosal surfaces e.g. in the respiratory tract [33] . vaccination of the zoonotic host, the dromedary camel, may also effectively curb outbreaks of mers in endemic regions and limit the risk of viral recombination [34] and significant progress with an orthopox-vectored vaccine for mers has recently been reported [35] . the potential use of a sub-unit vaccine for mers in camel vaccination could be aided by varying the sequence of the rbd protein [36] and substituting the human fc tag with an alternative tag recognised by the camel, to design approaches tailored for animal vaccination, bearing in mind that a single dose vaccine would be ideal in this context. however, future work in our laboratory will also address the value of retaining or removing the fc tag from the rbd protein for clinical or veterinary iterations of the vaccine. in this study we have determined vaccine efficacy by demonstrating in vitro and in vivo neutralising ability of murine antisera raised in the dual route two-dose regimen against two virulent clinical strains of mers-cov which have greater than 99% genome sequence homologyy. in future work, it will be worthwhile to test the efficacy of this approach against other clinical isolates of mers-cov. this is the first report of a dual route dosing regimen applied to a sub-unit vaccine for mers-cov. future development of this approach would require the direct testing of efficacy in the immunised transduced mouse model. as well as giving a direct readout of vaccine efficacy, this will enable the identification of the immune correlates of protection, ready for transitioning this candidate vaccine into more extensive pre-clinical testing and clinical development. the authors declare that all the data supporting the findings of this study are available within the paper. ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov origin and animal reservoir novel betacoronavirus in dromedaries of the middle east middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction spiking the mers-coronavirus receptor crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transduced mice from mers-cov infection evaluation of candidate vaccine approaches for mers-cov introduction of neutralising immunogenncity index to the rational design of mers coronavirus sub-unit vaccines recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge mers-cov spike protein: targets for vaccines and therapeutics toward developing a preventive mers-cov vaccine-report from a workshop organized by the saudi arabia ministry of health and the international vaccine institute protein coated microcrystals formulated with model antigens and modified with calcium phosphate exhibit enhanced phagocytosis and immunogenicity a new oil-based antigen delivery formulation for both oral and parenteral vaccination reverse micelle-encapsulated recombinant baculovirus as an oral vaccine against h5n1 infection in mice dual route vaccination for plague with emergency use applications genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans severe respiratory illness caused by a novel coronavirus rapid generation of a mouse model for middle east respiratory syndrome real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus cutting edge: mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog trehalose-dibehenate the tlr7 agonists imiquimod and gardiquimod improve dc-based immunotherapy for melanoma in mice chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice novel chimeric virus-like particles vaccine displaying mers-cov receptor-binding domain induce specific humoral and cellular immune response in mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus a tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus intracellular recycling and cross-presentation by mhc class i molecules fc-fusion proteins: new developments and future perspectives fc-fusion proteins and fcrn: structural insights for longer-lasting and more effective therapeutics co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants the authors acknowledge with thanks the expert technical assistance of lin eastaugh, louise thompsett and vicky roberts. this work was supported by sbri awards 972228 and 971527 from innovate uk to rrcn and na, respectively and on independent research commissioned from na and funded by the nihr policy research programme, [971527]. the views expressed in the publication are those of the author(s) and not necessarily those of the nhs, the nihr, the department of health, 'arms' length bodies or other government departments. the authors declare no conflict of interests. supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.05.074. key: cord-299621-m4kdkmey authors: kumar, a.; chaterjee, souranshu title: outbreak of middle east respiratory syndrome coronavirus, saudi arabian experience date: 2017-08-31 journal: current medicine research and practice doi: 10.1016/j.cmrp.2017.07.006 sha: doc_id: 299621 cord_uid: m4kdkmey abstract objective mers-cov infection is uncommonly identified among patients visiting healthcare facilities & we were vigilant in screening all patients entering our hospital to prevent cross infection among patients, visitors & healthcare providers and aiming to prevent potential outbreaks with mers-cov infection. in spite of our efforts on practicing failure mode effect analysis for mers-cov infection management we ended up having an outbreak with mers-cov. based on our experience, we actively implemented policies, procedures & practices on early identification & appropriate isolation practices along with supplemental infection prevention & control measures for preventing future outbreaks at our healthcare facility. methods retrospectively we analyzed our failure in preventing the outbreak of mers-cov infection among our hospitalized patients by identifying the outbreak & actively intervening as a team to control the outbreak with the support of hospital higher management, administrators, quality improvement team, infection prevention & control team & the active support of all healthcare workers of the facility. results following the early identification of mers-cov outbreak, we could successfully prevent large scale outbreak both in the hospital & the community. conclusion continuous implementation of infection prevention & control standards along with early clinical diagnosis of mers-cov infections based on the case definition as laid out by the ministry of health will prevent infectious outbreaks at healthcare facilities. middle east respiratory syndrome coronavirus (mers-cov) was first identified from a 60-year-old saudi male patient admitted to a private hospital in jeddah, saudi arabia on june13, 2012, with history of fever, cough, expectoration, and shortness of breath who eventually expired 11 days after admission from progressive respiratory failure. 1 sputum sample had tested negative for common respiratory viruses viz: influenza a and b, parainfluenza virus types1-3, respiratory syncytial virus, and adenovirus. inoculation of processed sputum sample from the patient on viral cell cultures (llc-mk2 and vero) produced specific cytopathic effect in a netherlands virology centre known as erasmus medical center and so the identified virus was initially labelled as human coronavirus erasmus medical center. 1 in september 2012, similar virus was recovered from a patient with severe respiratory symptoms who had a history of travel to saudi arabia and had been transferred from a hospital in qatar to a hospital in london and so named as human coronavirus england 1. 2 this new disease was also traced to an earlier time period. in april 2012, a cluster of cases of pneumonia occurred in health care workers of an intensive care unit in a hospital in zarqa, jordan, of which 2 patients died, both of whom were confirmed to be infected with the novel coronavirus by retrospective analysis of stored sample. 3 understanding of epidemiology of mers-cov increased after a large hospital outbreak in al-hasa in the eastern province of saudi arabia where cluster of mers-cov infections and health careassociated human-to-human transmission of mers-cov were reported. the case fatality rate in the outbreak was 65% and it was found that the disease presentation ranged from mild to fulminant in clinical severity. clinical syndrome was found to be similar to severe adult respiratory syndrome (sars), with initial presentation as nonspecific fever and non-productive cough, later progressing to pneumonia. significant number of patients with mers-cov infection also had gastrointestinal symptoms (vomiting and diarrhoea), a finding similar to that with sars. in the majority of patients in the study, fever was high, but the pulmonary involvement on chest radiography was variable. the survival rate was higher among patients who were identified by active surveillance during the outbreak than among those patients who were identified clinically. the incubation period of mers-cov was estimated to have an incubation period of 14 days, with 5% of cases developing within 1.8 days and 95% within 10.6 days. 6 despite failure mode and effect analysis (fmea) 7 that was conducted to prevent mers-cov outbreak in our healthcare facility which is also located in the same geographical location where increasing number of mers-cov outbreaks are noted. our hospital is a 150 bedded secondary care level facility and an outbreak with mers-cov among 5 patients occurred in october 2015 where the 1st patient was diagnosed on the 2nd of october 2015. all the 5 patients were confirmed by virological diagnosis and all these patients succumbed to their illness at a government referral centre dedicated to care for patients with mers-cov infection. the cause of death was respiratory failure along with renal shutdown. during this outbreak, 2 nursing staff who cared for the above patients in the medical intensive care unit were also infected with mers-cov. of the 2 nursing staff, one was asymptomatic and the other was severely symptomatic who needed ventilator support as she progressed to respiratory failure. both the nursing staff recovered completely with 3 negative virology tests on their respiratory samples. as a result of mers-cov outbreak at our facility, we had to face the following adverse effects: the hospital was kept closed for new admissions for 40 days until the hospital was declared free of new mers-cov cases on the 10th of november 2015. the regional community and our hospital healthcare workers (hcws) developed anxiety to the risk of developing mers-cov infection. following the opening of our facility for new patient admissions, significant reduction in patient visits and admission was noted which costs the hospital management, significant financial loss and loss of reputation. hence, we from the infection prevention and control (ipc) services initiated a collaborative approach with the support of the following departments; hospital quality improvement, nursing services, chief executive officer, medical director and the hospital higher management, to focus on the practice of hcws in managing suspected mers-cov infection. a team was formulated to come up with the analysis, results, and effectiveness of planned solutions to the problem with implementation of the following preventive measures. scheduled monthly meeting of project team members was conducted and was continuously reviewed. review and update of mers-cov policy and procedure along with structural compliance was adhered to. intensive onsite education to all hcws of the hospital and subsequent competency test results were tabulated and reported to the moh preventive medicine department. hcws with low competency test results were re-educated along with hands on training. triage teams were formed and got posted at all hospital entry points to screen all patients, visitors and hcws for symptoms (fever, cough, difficulty in breathing, diarrhoea, vomiting, headache, bodyache) and signs (evidence of pneumonia or acute respiratory distress syndrome, leucopenia, thrombocytopenia) correlating with mers-cov infection. any epidemiological history of contact with a confirmed or suspected mers-cov infected patient, camels or its products in the previous 14 days were to be considered for mers-cov screening. the virus gets suspended in the air for at least 36 h and commonly spreads by air borne route especially when aerosol generating procedures are conducted 6 . hence, individuals identified with the above signs, symptoms and epidemiological link are promptly transferred to air borne infection isolation rooms (aiirs) for further evaluation by the physicians. new respiratory illness room with portable high efficiency particulate arrestors (hepa) was created to isolate suspected mers-cov infected patients within the emergency medical services. the physicians were provided with hands on training on proper collection of nasopharyngeal samples to prevent occurrence of false negative virology results. hcws prior to entering the patient room were strictly supervised for donning the personal protective equipments (ppe) in sequence after adequate hand hygiene. sequences of wearing the ppe are as follows; clean gown, n95 mask, face shield and gloves. removal of the ppe after completion of patient care was also done in a sequence in the reverse order and special emphasis was laid on performing hand hygiene between each ppe removal. the hospital management of information system installed video programs on all infection prevention practices including the case definitions for mers-cov on the hospital portal and all televisions displayed at all patient care locations. social workers and patient educators were trained and encouraged to provide education to all visitors, patients and their families on mers-cov infection and prevention methods. the hospital higher management fully supported the interventional program conducted by the infection prevention and control team. the ipc team recommended installing fumigation machines delivering a combination of hydrogen peroxide with silver ions for disinfection of patient rooms following patient discharge. the air ventilation system within the entire hospital building was cleaned and disinfected along with appropriate change of hepa filters. the housekeeping staff had been provided with adequate training on the process of terminal cleaning and subsequent use of the fumigation system. portable radiological machines such as ultrasound, carm machines have been used for all clinically suspected mers-cov patients. the hospital construction services created additional aiirs which totaled to 22. in addition, portable hepa filter machines (8) were procured to be used in respiratory illness rooms identified at high risk patient care locations viz: haemodialysis unit, outpatient services, special care baby unit, paediatric services, obstetric and internal medicine wards. the ipc team underwent re-training with regular refresher courses on the management of mers-cov infections with the preventive medicine services of moh. the moh team also conducted multiple unannounced visits to the hospital for conducting audits on infection control practices by the healthcare providers. any identified deficiency was promptly corrected with the active support of the hospital medical director. since then, the hcws along with the active support of ipc services are performing continuous surveillance on suspected mers-cov patients, actively isolating them, performing adequate and appropriate respiratory sample collection and prompt transport to the regional virology laboratory. the virology test results are made available within 24 h. until date (july 2017), there are no healthcare associated mers-cov infection among patients, visitors and hcws of our hospital and improved compliance with the ipc policies and procedures were achieved. the emergence of mers-cov nearly 10 years after sars-cov disappearance has shown that pathogenic coronaviruses may continue to spill over from zoonotic sources into the human population. surveillance studies of viruses in animal species, including bats, rodents, and livestock, will help us to understand the potential human pathogens that exist in the environment before they can spill over human population. there are no antiviral medications available to treat mers-cov infections and the patient management is purely symptomatic. there is a need to develop vaccines and therapeutic strategies to prepare for the emerging coronaviruses. the sooner we understand the dynamics of current threats, the more we will be better equipped to save people from infection and disease. isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov) -update. geneva: world health organization ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndromecoronavirus coronavirus: diagnostics epidemiology and transmission none. key: cord-286472-pqtem19t authors: mcfee, r.b. title: middle east respiratory syndrome (mers) coronavirus date: 2020-07-28 journal: dis mon doi: 10.1016/j.disamonth.2020.101053 sha: doc_id: 286472 cord_uid: pqtem19t nan ) (1) was isolated from a patient who died from severe pneumonia and multi-organ failure in saudi arabia (2, 3) . this newly identified respiratory viral illness was caused by a novel coronavirus, which was initially designated as human betacoronavirus (2) (3) (4) (5) , but was eventually named middle east respiratory syndrome coronavirus (mers cov). reminiscent of, and worth considering as a caution for greater vigilance towards emerging pathogens, the suddenness that sars cov emerged as a new cause of severe pulmonary illness, has been replicated in this new aggressive respiratory illness mers cov. unlike sars cov, it has not caused the thousands of cases over a short period of time. but it also differs from sars cov in that it carries a much higher case fatality rate, and causes more severe illness (6, 7) . unlike sars which seems to have gone quiescent, new mers cases have been reported well after the initial outbreak, including december 2019 (7b) . since september 2012 there have been cases reported in 27 countries across 4 continents, with most human cases occurring in saudi arabia (map 1 and 2) (6) . while the greatest spike in cases occurred near 2014, new cases to date continue to be reported. consider by the end of 12/19 the number of mers cases 2492, with 858 deaths, resulting in a case fatality rate of 34.43% (7b, 7c) . earlier data from world health organization (who) as of 12/05/16 there have been 1917 laboratory confirmed cases of mers cov since 09/12, reported from 27 countries (maps 1 and 2), resulting in 677 deaths, yielding a significant case fatality rate (~35%) (6); a fatality rate for a coronavirus that is significantly greater than that associated with sars cov delta (~9%), at least among confirmed cases (6) (7) (8) . these data demonstrate mers continues to cause illness, though thankfully not at pandemic rates in the same manner as covid-19. as with other cov, including sars cov and covid-19, the exact epidemiology, including the magnitude of mild or asymptomatic illness, remains unknown. that said, among those presenting with mers, males over 60 yrs of age seem to be at higher risk of severe disease symptoms and death among those infected. the risk associated with age seems consistent with covid-19 findings as well. abnormalities associated with mers cov include thrombocytopenia, lymphopenia, leukopenia, elevated serum lactate dehydrogenase (ldh), elevated aspartate aminotransferase (ast), elevated alanine aminotransferase (alt), along with abnormal renal function tests (5, 29, 30) . superinfection from viral or bacterial causes has been found in some patients and may complicate the course of the disease. severe cases of mers may require intensive care and mechanical ventilation, with a relatively large number of patients progressing to respiratory or renal failure. a retrospective radiology review of ct findings in laboratory confirmed cases of mers cov noted that cough, fever, and dyspnea were the most common presenting symptoms. three of the seven subjects died (31) . the high case fatality rate associated with mers cov has sparked interest in the role of virus on the immune system. one area of interest is the potential for cytokine storm, which is also found in covid-19 patients, and currently being studied extensively (31b) . some studies have shown mers cov is associated with an attenuated interferon (ifn) response, and does not induce inflammatory cytokines (7, 32, 33 chest xrays should be obtained early in the course of progressive pulmonary illness ( figure 2 ) (31). radiographic findings may include unilateral or bilateral patchy densities or opacities, interstitial infiltrates, consolidation, and pleural effusions. rapid progression to acute respiratory failure, acute respiratory distress syndrome (ards), refractory hypoxemia, and extrapulmonary complications (acute kidney injury requiring renal replacement therapy, hypotension requiring vasopressors, hepatic inflammation, septic shock) has been reported. key to appropriate testing is a thorough history and physical. it is important to consider multisystem function as well as pulmonary status in patients with severe respiratory illness, including suspected mers cov, especially those returning from regions where aggressive pathogens are noted. involving infectious disease and pulmonologist specialists early on, as well as good critical care management are essential. as noted earlier, laboratory findings at admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels (ldh). renal and hepatic function tests, at least for baseline information are worth obtaining. in the ajlan study (31) , all seven patients had lymphopenia. elevated creatinine developed after admission in all patients. ast also were elevated in all patients. of note, co-infection with other respiratory viruses and a few cases of co-infection with communityacquired bacteria at admission has been reported in mers cov patients. not surprisingly, nosocomial bacterial and fungal infections have also been reported in mechanically-ventilated patients. according to clinical experience reported to cdc, mers-cov virus can be detected with higher viral load and longer duration in the lower respiratory tract compared to the upper respiratory tract, and has been detected in feces, serum, and urine. however, very limited data are available on the duration of respiratory and extrapulmonary mers-cov shedding. as mentioned earlier, aggressive testing, isolation, and management are critical in treating highly pathogenic coronaviruses, especially mers, which among patients who present for medical care, has a high case fatality rate, extrapulmonary involvement in some cases, and rapid deterioration. this includes early radiographic studies (7b). according to the aljan radiology study (31) , chest xray findings were most notable for airspace and interstitial opacities with mers cov. radiographic findings such as airspace opacities are nonspecific, whereas the interstitial changes were described as reticular or reticulonodular. total lung opacification and thickening of the bronchovascular markings have been reported as well. previous chest ct findings with mers have been reported as bilateral patchy or extensive opacities (5, 7b) . not unexpected, imaging that had features consistent with acute respiratory distress syndrome were typically identified in sicker patients, and can be found in covid-19 patients as well (table 1 ). in aljan (31) mers cov the most common findings on ct were shows that airspace opacities on ct are common in patients hospitalized with mers-cov infection. airspace opacities are suggestive of an organizing pneumonia pattern. it was also described in h1n1 influenza a viral infections. in most of our patients, ground-glass opacities were more extensive than consolidation. septal thickening and pleural effusions also were demonstrated. importantly, tree-in-bud pattern, cavitation, and lymph node enlargement were not seen in our cohort. chest xrays and ct scanning ( figure 2 ) are recommended depending upon the presentation, and clinical course. in the aljan study (31) the most common ct finding in hospitalized patients with mers -cov infection reveals predominantly subpleural and basilar airspace changes, with more ground glass opacities than consolidation. it is important to note this is a small study. nevertheless this pattern is consistent with organizing pneumonia. patients recently returning from the middle east, presenting with significant respiratory illness, with ct findings of peribronchial region abnormalities, organizing pneumonia, should be considered for mers cov infection, and if possible, queried about international travel and occupational exposures. 27-year-old man with middle east respiratory syndrome patient was a smoker who was healthy otherwise. ct was performed 8 days after admission, and 20 days after onset of symptoms. patient was eventually discharged. lower lung ct image show large right lower lobe and small focal left lower lobe subpleural consolidations the cdc provides case definitions. the reader is requested to review periodically this site for updates as the mers cov situation can change (8, 34) . although emergency testing can be possible for suspected asymptomatic cases, the cdc guidelines for sending samples for laboratory confirmation are predicated upon a combination of clinical and epidemiological factors to identify persons under investigation (pui) table 1 . the other two categories per cdc are: a confirmed case is a person with laboratory confirmation of mers-cov infection. confirmatory laboratory testing requires a positive pcr on at least two specific genomic targets or a single positive target with sequencing on a second. laboratory confirmation of mers cov can be obtained by real time polymerase chain reaction (rt pcr) a probable case is a pui with absent or inconclusive laboratory results for mers-cov infection who is a close contact 3 of a laboratory-confirmed mers-cov case. examples of laboratory results that may be considered inconclusive include a positive test on a single pcr target, a positive test with an assay that has limited performance data available, or a negative test on an inadequate specimen. nb the reader is advised to check for updated testing procedures with the cdc (35) . this is an updated version of the interim guidance document issued by the centers for disease control and prevention (cdc) january 2014 based on input from public health partners, healthcare providers, professional organizations, and others. cdc will continue to update the document as necessary to incorporate new information that increases our understanding of mers-cov. updates: minor changes were made to clarify specimen type and collection procedures. of note -respiratory specimens should be collected as soon as possible after symptoms beginideally within 7 days. however, if more than a week has passed since symptom onset and the patient is still symptomatic, respiratory samples should still be collected, especially lower respiratory specimens since respiratory viruses can still be detected by rrt-pcr. for example -if symptom onset for a pui with respiratory symptoms was less than 14 days ago, a single serum specimen, an np/op specimen and lower respiratory specimen should be collected for cdc mers rrt-pcr testing. 1. if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was 14 or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. 2. if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was 14 or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. for short periods (≤ 72 hours), most specimens should be held at 2-8°c rather than frozen. for delays exceeding 72 hours, freeze specimens at -70°c as soon as possible after collection (with exceptions noted below). label each specimen container with the patient's id number, specimen type and the date the sample was collected. broncheoalveolar lavage, tracheal aspirate, pleural fluid collect 2-3 ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. have the patient rinse the mouth with water and then expectorate deep cough sputum directly into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. nasopharyngeal swab and oropharyngeal swab (np/op swab) use only synthetic fiber swabs with plastic shafts. do not use calcium alginate swabs or swabs with wooden shafts, as they may contain substances that inactivate some viruses and inhibit pcr testing. place swabs immediately into sterile tubes containing 2-3 ml of viral transport media. np/op specimens can be combined, placing both swabs in the same vial. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. nasopharyngeal swab -insert a swab into the nostril parallel to the palate. leave the swab in place for a few seconds to absorb secretions. swab both nasopharyngeal areas. oropharyngeal swab (e.g., throat swab) -swab the posterior pharynx, avoiding the tongue. nasopharyngeal wash/aspirate or nasal aspirate collect 2-3 ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. for serum antibody testing: because we do not want to delay detection of mers infection and since the prevalence of mers in the us is low, serologic testing on a single serum sample collected 14 or more days after symptom onset may be beneficial. this is in contrast to serologic testing for many other respiratory pathogens which require collection and testing of acute and convalescent serum specimens. serologic testing is currently available at cdc upon request and approval. please be aware that the mers-cov serologic test is for research/surveillance purposes and not for diagnostic purposes -it is a tool developed in response to the mers-cov outbreak. contact cdc's emergency operations center (eoc) (770-488-7100) for consultation and approval if serologic testing is being considered . a single serum specimen collected optimally during the first 10-12 days after symptom onset is recommended. note: the kinetics of mers-cov are not well understood. once additional data become available, these recommendations will be updated as needed. the minimum amount of serum required for mers-cov testing (either serologic or rrt-pcr) is 200 µl. if both mers-cov serology and rrt-pcr tests are planned, the minimum amount of serum required is 400 µl (200 µl for each test). serum separator tubes should be stored upright for at least 30 minutes, and then centrifuged at 1000-1300 relative centrifugal force (rcf) for 10 minutes before removing the serum and placing it in a separate sterile tube for shipping (such as a cryovial). refrigerate the serum specimen at 2-8°c and ship on ice-pack; freezing and shipment of serum on dry ice is permissible. children and adults: collect 1 tube (5-10 ml) of whole blood in a serum separator tube. a minimum of 1 ml of whole blood is needed for testing pediatric patients. if possible, collect 1 ml in a serum separator tube. specimens from suspected mers cases must be packaged, shipped, and transported according to the current edition of the international air transport association (iata) dangerous goods regulations. shipments from outside of the united states may require an importation permit that can be obtained from cdc. specimens should be stored and shipped at the temperatures indicated above. if samples are unable to be shipped within 72 hours of collection, they should be stored at -70°c and shipped on dry ice. when shipping frozen specimen from long distances or from international locations, it is best to use a combination of dry ice and frozen gel ice-packs. the gel ice-packs will remain frozen for a day or two after the dry ice has dissipated. all specimens must be pre-packed to prevent breakage and spillage. specimen containers should be sealed with parafilm® and placed in ziplock bags. place enough absorbent material to absorb the entire contents of the secondary container (containing primary container) and separate the primary containers (containing specimen) to prevent breakage. send specimens with cold packs or other refrigerant blocks that are self-contained, not actual wet ice. this prevents leaking and the appearance of a spill. when large numbers of specimens are being shipped, they should be organized in a sequential manner in boxes with separate compartments for each specimen. saturday delivery is planned, special arrangements must be made with the shipping company. there are no commercially available or fda approved therapeutics specifically designated as mers cov antivirals. as of 2020 there remain a variety of candidate therapeutics in development against covid019 specifically, highly pathogenic coronaviruses such as sars and mers, and coronaviruses in general, including those that cause "common cold-like" symptoms, but none have emerged as fully capable of treating these viruses with the same consistency as neuraminidase antivirals have against influenza. and even in that context there are some clinical considerations, and limitations. there continues to be ongoing research into repurposing antivirals that are approved for other clinical indications such as hiv, influenza, and other illnesses, as possible treatments -monotherapy or in combination, against covid-19 and other coronaviruses. brief updates are provided below. covid-19 has reenergized research into greater study of the pathogenicity of coronaviruses, and newly or better described characteristics of highly pathogenic coronaviruses has led to increased insights into additional antiviral and vaccine targets. significant research into a wide array of therapeutic approaches is undergoing at unprecedented levels internationally. as will be seen in the covid-19 therapeutics section of this article, multiple options are being clinically tested that warrant consideration for the clinician faced with treating covid-19 patients. the mainstay of therapy for mers cov remains supportive care, especially respiratory care, while managing circulatory, renal, hepatic and neurological function, as well as protecting against secondary infections. although attempted in both the sars cov and early mers cov outbreaks, immune based interventions -interferon -resulting in equivocal outcomes. non human primate studies using ifn a2b and ribavirin against mers cov demonstrated improved outcomes but it is worth noting treatment was initiated soon after viral infection initiated; this is unlikely going to be the case with human infection, where a delay in presentation to health care, or delayed diagnosis will likely preclude the same rapidity of treatment that occurred in the study (6 -8, 32, 33) . the role for interferon in mers cov remains unclear; clinical trials are needed to better characterize successful strategies. but the limited number of cases make such studies difficult. in addition to various interferon treatment protocols, ribavirin -a potent nucleoside analog -has been utilized against rna viruses with varying degrees of success (7,32,33, 33b, 33c) . unfortunately ribavirin presents a risk for adverse effects including hemolytic anemia. interferon is not without side effect risk either. the early use of corticosteroids in sars cov infected patients resulted in increased viral load, critical care/intensive care unit admission, and death (7, 33c). other approaches to cov include neutralizing antibodies from convalescent plasma or hyperimmune globulin; these may hold some promise in reducing the cfr (33c, [36] [37] [38] . convalescent plasma was shown to decrease mortality in sars cov patients if provided to patients within 14 days of illness (36) . however in order for this to be effective, rapid diagnosis of patients, and survivors is required in order to obtain, and utilize these interventions. towards that end, a network for convalescent plasma is being assembled, allowing safety, effectiveness and logistic feasibility testing. (37) . that said, to date no host derived interventions have been shown to consistently confer significant benefit to severely ill mers cov patients via controlled study. while to date there are no antivirals currently licensed for use against mers cov, some candidate drugs are in development. two categories of candidates showed early promise. an adenine analogue that can be incorporated into viral rna, which can disrupt virus replication, and has shown in vitro activity against mers cov, as well as benefit against ebola in non human primates (nhps). also there is a nucleoside analogue for treatment of filoviruses, cov, and other rna viruses. researchers are working on other molecular approaches to treat cov (7, 39, 40) . ribavirin is a guanine, oral nucleoside analogue, which inhibits viral rna-dependent rna polymerase. in trials exploring the use of ribavirin against mers, it was often in combination with other therapeutics, including interferon. evidence revealed it did not confer significant clinical benefit on outcomes or viral clearance (33c, 41, 42) . it has shown some in vitro activity against sars, where high concentrations/high doses were required to inhibit viral replication (1.2 g to 2.4 g po every 8 hours), and combination therapy via intravenous or enteral administration (33b, 33c, 43) . studies on the use of ribavirin as a treatment for sars were either inconclusive in terms of clinical benefit, or suggested possible harm referable to adverse events, which included hepatic and hematologic deleterious effects (33c, 43). ribaviran as a potent nucleoside analogue has been, and continues to be used with varying degrees of success against rna viruses, but there is the potential for adverse side effects including hemolytic anemia, metabolic derangements. interferon can also elicit adverse effects, although they have demonstrated value against viral infection (44) (45) (46) (47) . another repurposing of antivirals involved lopinavir-ritonavir combination therapy trialed against mers. the medication is an oral protease inhibitor (33b, 33c). early success with treating sars in 2003, using lopinavir/ritonavir and ribavirin was suggested (33b, 48) . mortality and need for intensive respiratory support was noted (33b, 48) . in an animal study involving marmosets, lopinavir-ritonavir or interferon beta -1b reduced viral load and improved lung pathology (33b, 49) . combination antiviral therapy for patients hospitalized with severe influenza was noted in some cases to confer greater results, for those with high viral loads at presentation (33b, 50, 51) . it is worth noting that studies have shown both sars coronavirus and mers coronavirus their viral loads peak at ~7 -10 days after symptoms begin compared to covid-19 coronavirus where viral loads seem to peak at the time of clinical presentation (33b, 52,53) . this adds another level of clinical challenge in terms of managing this new, highly pathogenic coronavirus, and underscores the importance of time sensitivity in the administration of potentially lifesaving medications. another promising approach includes monoclonal antibodies (mabs). mabs have already demonstrated benefit against certain cancers and autoimmune disorder management (7, 38) . to date the only pathogen for which there is a licensed mab is respiratory syncytial virus (rsv). who recommends standard precautions with all patients, especially those displaying early signs of respiratory illness, as the diagnosis -whether common cold, or mers cov or rsv or pertusis or other pathogen related infection. also droplet precautions should be added to standard precautions when providing care to such patients, including those with acute respiratory illness. although eye protection is recommended for probable or confirmed cases of mers cov, and airborne precautions for aerosol generating procedures, this would seem prudent even in cases clearly not mers cov or sars cov. from an infection control perspective, among the known human coronaviruses they are capable of surviving on environmental surfaces for up to 3 hours (11) . the sars and mers experiences, as well as previous hcov, underscore the threat of transmissibility; coronaviruses may be transmitted from person-to-person by droplets, hand contamination, fomites, and small particle aerosols. avoiding camels, especially dromedary camels, farms, as well as not consuming raw milk, urine, or meat are also worth considering. camels infected with mers cov may not appear ill. if contact with animals, especially dromedary camels cannot be avoided, strict hand washing is recommended. personal protection, including face/eye if working with camels or other animals. patients who must travel to the region are encouraged to present to a health care facility especially if fever and respiratory symptoms develop. according to a who update, multiple cases from the united arab emirates, qatar, oman, kuwait, and bahrain have links to dromedary camels. these patients were thought to be infected via contact with infected dromedaries or close primary cases (uae). a good practice for anyone who works with potential contaminants -whether in the us or middle east farm -remove work clothes that may carry materials from farms and animal contact (poster 1) (53). to date few cases have occurred in europe or the us. however with the emergence of medical tourism -cosmetic and advanced surgeries -patients who reside in the middle east may bring with them diseases endemic to the region, including mers cov. the astute clinician will be aware of potential importation of illness. as of 2020 there are sporadic cases of mers-cov, which continues to cause illness since its emergence in 2012, and with a mortality rate estimated at greater than 30%. mers still remains a potential outbreak threat, and is a dangerous disease causing pathogen. as with sars cov and other viral threats of public health concern, there is a great need for both prophylactic measuresvaccines, and more targeted therapies. there is significant research effort underway internationally to develop an effective vaccine. as with the sars cov, there are a variety of approaches. one attempt which shows early animal efficacy is by a vaccine candidate consisting of chimeric virus-like particles (vlp) expressing the receptor binding domain (rbd) of mers-cov. the researchers have fused canine parvovirus (cpv) vp2 structural protein gene with the rbd of mers-cov; it self-assembles into chimeric, spherical vlp (svlp). this svlp retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to cpv virions. immunization with svlp induced rbd-specific humoral and cellular immune responses in mice. svlp-specific antisera from these animals were able to prevent pseudotyped mers-cov entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. of note, interferon (ifn) -ifn-γ, il-4 and il-2 secreting cells induced by the rbd were detected in the splenocytes of vaccinated mice by elispot. mice given svlp or an adjuvanted svlp vaccine elicited t-helper 1 (th1) and t-helper 2 (th2) cell-mediated immunity. although early stage, resarch svlp displaying the rbd of mers-cov may become, or lead to an effective vaccine against mers-cov. human research is needed (55) . of note, several of these approaches will be applied to developing vaccines against covid-19, which will be discussed in the next section (33c). -or -a member of a cluster of patients with severe acute respiratory illness (e.g., fever 1 and pneumonia requiring hospitalization) of unknown etiology in which mers-cov is being evaluated, in consultation with state and local health departments in the us. and close contact 3 with a confirmed mers case while the case was ill. fever may not be present in some patients, such as those who are very young, elderly, immunosuppressed, or taking certain medications. clinical judgement should be used to guide testing of patients in such situations countries considered in the arabian peninsula and neighboring include: bahrain; iraq; iran saudi arabia; syria; the united arab emirates meters), or within the room or care area, of a confirmed mers case for a prolonged period of time (such as caring for, living with, visiting, or sharing a healthcare waiting area or room with, a confirmed mers case) while not wearing recommended personal protective equipment or ppe (e.g., gowns, gloves, nioshcertified disposable n95 respirator, eye protection); or b) having direct contact with infectious secretions of a confirmed mers case (e.g., being coughed on) while not wearing recommended personal protective equipment. see cdc's interim infection prevention and control recommendations for hospitalized patients with mers for detailed information regarding healthcare personnel (hcp) please review cdc interim u.s. guidance for monitoring and movement of persons with potential middle east respiratory syndrome (mers-cov) exposure for more information: call 800-cdc-info (232-4636) or visit www.cdc.gov/travel poster 1 (54) mers references 1. image of middle east respiratory syndrome (mers) coronavirus as isolated by the national institute of allergy and infectious diseases isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus in a patient transferred to the united kingdom from the middle east middle east respiratory syndrome coronavirus (mers -cov) announcement of the cornoavirus study group epidemiological, demographic, and clnical characteristics of 47 cases of midle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study world health organization (who) mers update and global summary treatment strategies for middle east respiratory syndrome coronavirus radiology perspective of coronavirus disease 2019 (covid-19): lessons from severe acute respiratory syndrome and middle east respiratory syndrome ajr middle east respiratory syndrome coronavirus (mers-cov) differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation world health organization middle east respiratory syndrome coronavirus emergency preparedness response survival of human coronaviruses 229e and oc43 in suspension after drying on surfaces: a possible source of hospital-acquired infections middle east respiratory syndrome coronavirus (mers cov): what lessons can we learn? assessment of the middle east respiratory syndrome coronavirus (mers -cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach estimation of mers -coronavirus reproductive number and case fatality rate for the spring 2014 saud arabia outbreak insights from publicly available data hadi mohamed rae. outbreak of middle east respiratory syndrome coronavirus in saudia arabia: a retrospective study middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia serological evidence of mers cov antibodies in dromedary camesl (camelus dromedaries) in laikipia county, keny isolation of mers coronavirus from a dromedary camel risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia a more detailed picture of the epidemiology of middle east respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide cross sectional serological study disocvery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruess as the gene source of alphacoronavirus and betacoronavirus and avian cornnaviruses as the gene source of gammacoronavirus and deltacornavirus evidence of the recombinant origin of a bat severe acute respiratory synddrome osars) like coronavirus and its implications on the direct ancestor of sars coronavirus delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle easst respiratory syndrome coronavirus: implications for pathogenesis and treatment middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd middle east respiratory syndrome coronavirus (mers cov) infection family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middlle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers cov): evidence and speculation middle east respiratory syndrome coronavirus the pathogenesis and treatment of the 'cytokine storm' in covid-19 treatment with interferon-alpha2b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha 2a or ifn beta 1 in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study triple combination of interferon beta-1b, lopinavirritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open label randomized, phase 2 trial pharmacologic treatments for coronavirus disease 2019 (covid-19); a review centers for disease control (cdc) mers clinical laboratory testing recommendations the effectiveness of vonvalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis feasibility, safety, clinical and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol a conformation dependent neutralizing monoclonal antibody specifically targeting receptor binding domain in middle east respiratory syndrome coronavirus spike protein nucloeotide prodrug gs-5734 is a broad spectrum filovirus inhibitor that provides complete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx4430 in a non-human primate model of filovirus infecton clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and metaaanalysis ribavirin and interferon therapy for critically ill patients with mers: a multicenter observational study sars: systematic review of treatment effects modjarrad k treatment strategies for middle east respiratory syndrome coronavirus treatment with interferon-alpha2b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha 2a or ifn beta 1 in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment withlopinavir/ritonavir or interferon b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset antiviral combinations for severe influenza efficacy of clarithromycin-naproxen-oseltamivir combination in the treatment of patients hospitalized for influenza a (h3n2) infection: an open-label randomized, controlled, phase iib/iii trial temporal profiles of viral load inposterior oropharyngeal saliva sample and serum antibody responses during infection by sars cov-2: an observational cohort study medical treatment of viral pneumonmia including sars in immunocompetent adult novel chimeric virus-like particles vaccine displaying mers-cov antiviral res key: cord-305317-08a1oin2 authors: maltezou, helena c.; tsiodras, sotirios title: middle east respiratory syndrome coronavirus: implications for health care facilities date: 2014-12-31 journal: american journal of infection control doi: 10.1016/j.ajic.2014.06.019 sha: doc_id: 305317 cord_uid: 08a1oin2 background middle east respiratory syndrome coronavirus (mers-cov) is a novel coronavirus that causes a severe respiratory disease with high case fatality rate. starting in march 2014, a dramatic increase of cases has occurred in the arabian peninsula, many of which were acquired in health care settings. as of may 9, 2014, 536 laboratory-confirmed cases and 145 deaths have been reported globally. methods review of publicly available data about mers-cov health care–associated transmission. results we identified 11 events of possible or confirmed health care–associated transmission with high morbidity and mortality, mainly among patients with comorbidities. health care workers are also frequently affected; however, they tend to have milder symptoms and better prognosis. gaps in infection control were noted in all events. currently, health care–associated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic in countries in the arabian peninsula. conclusion there is a need to increase infection control capacity in affected areas and areas at increased risk of being affected to prevent transmission in health care settings. vaccines and antiviral agents are urgently needed. overall, our knowledge about the epidemiologic characteristics of mers-cov that impact health care transmission is very limited. as the mers-cov epidemic continues to evolve, issues concerning best infection control measures will arise, and studies to better define their effectiveness in real life are needed. middle east respiratory syndrome coronavirus (mers-cov) is a novel betacoronavirus of the coronaviridae family that causes a severe respiratory disease with a high case fatality rate. [1] [2] [3] [4] the virus was isolated for the first time in september 2012 from a 60-year-old patient with fatal pneumonia in saudi arabia. 5 however, the earliest identified human cases were traced back to march 2012, to a cluster of severe respiratory infections in a hospital in jordan. 6 up until now, all mers-cov infected cases are directly or indirectly linked to the middle east; therefore, the name mers-cov was established. 4 over the first 2 years after the emergence of mers-cov, the world health organization (who) has been notified of 191 laboratory-confirmed cases, of which 82 were fatal. 7 however, starting in mid-to late march 2014, a dramatic increase of cases has been recorded, many which were acquired in health care settings and concerned health care workers (hcws). 8 as of may 9, 2014, 536 laboratory-confirmed cases and 145 deaths have been reported to the who globally. 8 as a result, concerns have been expressed about the possibility of a virus genetic change conferring increased transmissibility, and the novel virus received media attention globally. in the context of uncertainties about its epidemiology, the high case fatality rate, the urgent need for a specific antiviral treatment, and the unavailability of a vaccine, mers-cov has been a major public health concern of global dimensions. given the current local epidemiologic trends of mers-cov 8 and the large numbers of travelers that fly out of the arabian peninsula, 9 it is almost certain that an increasing number of cases will be exported to other countries; these cases, especially when that patient is seriously ill, will require medical attention and hospitalization. herein, we review publicly available data about mers-cov focusing on health careeassociated transmission. aspects relevant to infection control are also discussed. we searched pubmed from september 2012 through june 19, 2014, using the terms middle east respiratory syndrome, mers, and novel coronavirus. the abstracts of articles identified through the first pubmed search were screened, and articles presenting original data on health careeassociated infections and outbreaks were included. the reference lists of these articles were also reviewed as were any relevant review articles. in addition, we searched the web sites of the who, united states centers for disease control and prevention (cdc), and european centre for disease prevention and control (ecdc). in total, we reviewed 252 articles on mers-cov and identified 10 articles presenting original data about 11 possible or confirmed health careeassociated transmission events. details about the health careeassociated transmission ranged widely among these articles. to the best of our efforts, we avoided presenting duplicated data. in addition, we selected 30 original and review articles. all articles were studied by both authors independently. mers-cov infection so far has been described in 10 countries in the middle east (saudi arabia, united arab emirates, qatar, jordan, oman, kuwait, egypt, yemen, lebanon, iran), 6 countries in europe (united kingdom, germany, france, italy, greece, the netherlands), 2 country in africa (tunisia, algeria), 2 countries in asia (malaysia, the philippines), and 1 county in the americas (united states). 10 molecular analyses of mers-cov or similar viruses from bats and camels suggest that these 2 species are the natural reservoirs of the virus. 11, 12 whole genome sequencing showed that human and camel viruses from saudi arabia are indistinguishable. 12 multiple transmission routes are suspected; however, their exact contribution has not been elucidated so far. a phylogenetic study of 21 mers-cov genomes from saudi arabia suggested that both humanto-human transmission and sporadic zoonotic events occur. 13 the stability of the virus for prolonged periods in camel milk suggests the potential of excretion of the virus into camel milk and spread through consuming raw milk. 14 the upsurge of cases since mid-march in the arabian peninsula (mainly in saudi arabia) is possibly attributed to an increase in the number of primary cases and hospital-acquired cases, some as a part of mainly small (1-2 cases), but in a few instances large, outbreaks. 8 a change in the transmissibility pattern of the virus and increased efficacy for sustained transmission could facilitate inhospital transmission; however, epidemiologic and molecular data to this effect do not exist. family clusters of mers-cov have been recorded. 15 the secondary attack rate in families was 1.35% in saudi arabia in 2014. 8 among imported travel-associated cases, very few instances of person-to-person transmission have been verified. [16] [17] [18] recent phylogenetic analysis using human sequences from jeddah suggests that the virus has not changed from previous strains. 8 overall, it seems unlikely that the virus has increased its transmissibility or patterns of transmission. the basic reproductive number has been estimated to be <1 using real-time data until june and august 2013, respectively, 19,20 even though the upper range of estimates exceeded 1 in a scenario where infection control was not implemented. 20 these findings indicate no pandemic potential for mers-cov so far. recently, a committee appointed by the who concluded that the conditions for a public health emergency of international concern have not yet been met. moreover, increased testing rates of less ill or asymptomatic cases may have contributed to the upsurge of detected cases. regarding characteristics of affected patients, most are men (male-to-female ratio: 2:1), with a median age of 49 years (range, 9 months-94 years). 8 the spectrum of mers-cov infections ranges from asymptomatic infection to very severe pneumonia with acute respiratory distress syndrome, septic shock, and multiorgan failure resulting in death. in an analysis of 144 confirmed and 17 possible cases, symptomatic patients typically had fever and cough, chills, sore throat, myalgia, and arthralgia, whereas vomiting and diarrhea were present in at least one third of patients. 21 in the same study, 63.4% of patients developed severe respiratory disease. it appears that severe disease predominantly occurs in patients with comorbidities; 76% of the patients in this report had at least 1. 21 the overall case fatality with the latest who figures is 27%. 8 from the very first events of the mers-cov epidemic, the virus showed its health careeassociated dynamic. 6 apart of sporadic community cases and family clusters, health careeassociated transmission has been reported on several occasions during the last 2 years, indicating human-to-human, although inconsistent, transmission (table 1) . 2, 3, 6, 8, [22] [23] [24] [25] [26] [27] gaps in infection control were the common denominator in the events of health care associatede transmission. 2, 3, 6, 8, 22, 24 during the largest so farepublished outbreak of mers-cov that occurred in al-hasa, saudi arabia, in 2013, 4 health care facilities were affected through transfer of patients but also possibly because of repeated introductions of cases from the community. 3 the outbreak extended for almost 2 months and involved 34 cases, including 2 hcws. most cases were confined in the hemodialysis unit with rapid transmission and high attack rates. 3 this outbreak gave the opportunity to elucidate several epidemiologic parameters of secondary mers-cov infection, such as the incubation period (5.2 days; 95% confidence interval, 1.92-14.7 days), serial interval (7.6 days; 95% confidence interval, 2.5-23.1 days), and heterogeneity in transmission, with many infected patients not transmitting the infection at all and 1 infected patient transmitting the infection to 7 others. 3 moreover, this outbreak raised the possibility of transmission through direct or indirect contact and between rooms in the same ward. 3 a recent study showed that mers-cov remained viable for up to 48 hours under specific environmental conditions, which mimic the hospital environment (20 c with 40% relative humidity), whereas its stability was not reduced during aerosolization. 28 these data show that mers-cov has the potential to spread through contact or fomites caused by prolonged survival. a model-based study found that the virus structural characteristics render it very likely to remain viable in the environment for a long period and support fecal-oral transmission. 29 vomiting and diarrhea are common in patients with mers-cov 1,22,27 and may contribute to transmission. the mers-cov case imported in france shared his bathroom with the secondary hospital-acquired case, which raises the possibility of spread through stools. 22 mers-cov is predominantly shed through respiratory secretions during cough. mers-cov has been detected through polymerase chain reaction for up to 16 days in respiratory specimens and stools and up to 13 days in urine. 22, 30, 31 our knowledge about virus shedding and viral load kinetics throughout the clinical course of ill patients is scarce and therefore can provide limited guidance about the duration of implementation of infection control measures. 31 the possibility of prolonged shedding under an immunocompromised status should also be investigated and considered for infection control purposes. health careeassociated mers-cov infections and outbreaks have been associated with high morbidity, high rates and prolonged use of mechanical ventilation, and fatality rates up to 65%. 2, 3, 23 given the fact that health care services are often used by older people with comorbidities and in association with the severe course in the description of demographics of secondary mers-cov cases, a drop of the median age from 59 to 43 years old compared with primary cases has been reported. 21 this depends on the conditions of each outbreak and may be affected by the preponderance of affected hcws in each instance. for example, in the most recent who report, 8 the hcws who tested positive for mers-cov in the 2014 jeddah outbreaks were more likely to be younger, women, and to exhibit mild or no symptoms compared with primary cases. however, 15% of hcws developed a severe disease, which resulted in admission to an intensive care unit or death. 8 unsuspected cases are the main source for the introduction of mers-cov virus from the community or another health care facility. 3 although such patients may present with compatible symptoms, the diagnosis may not be considered early or symptoms may be mild. 2, 24, 33 in the hospital outbreak that occurred in saudi arabia in 2013, 3 patients exhibited no fever during initial presentation. 3 hcws may acquire mers-cov infection either in the community or through occupational exposure. 2, 3, 32 nurses are mostly affected, which is attributed to their prolonged, repeated, and closer physical contact with patients. hcws may continue working despite being symptomatic. 6 an asymptomatic or mildly symptomatic course has been described in hcws, 2, 8, 24 which raises the possibility of transmission of the infection to their vulnerable patients during an asymptomatic phase or early incubation. patient-to-patient transmission has been noted as well. 3, 22 currently, health careeassociated outbreaks are playing a pivotal role in the evolution of the mers-cov epidemic. 8 in the recent mission report by the who authorities evaluating data on 128 laboratory-confirmed cases in 14 hospitals in jeddah, saudi arabia, with onset of symptoms between february 17 and april 26, 2014, one-third of the cases were considered to be primary cases (some of the investigations are still ongoing), whereas >60% of the cases (including 39 hcws) were classified as hospital acquired. 8 in the rest of saudi arabia, 26 out of 127 (20.5%) recent cases were identified in hcws. 8 overall, 65 of the 290 (22.4%) cases reported from saudi arabia from march 27 to may 9, 2014, were hcws. 23 in mecca, another large outbreak in a hospital was described with 28 laboratory-confirmed cases, including 27 hcws. 8 both outbreaks were larger than the originally described outbreak in saudi arabia. 3 in the united arab emirates, hcws accounted for more than twothirds of 37 cases reported during the same period. 8 although the who points to infection control gaps for the recent propagation of mers-cov within health care facilities in saudi arabia and the united arab emirates, we do not know if this concerns the use of personal protective equipment, hand hygiene, procedures, environmental cleaning, or triage. given that no vaccines or specific antiviral prophylaxis against mers-cov are available, 35,36 the prevention and control of transmission of mers-cov within health care facilities relies solely on early detection, isolation, and strict implementation of infection control measures. rapid and accurate diagnosis is crucial to trigger contact tracing in the hospital and the community and should be ordered as soon as possible in the context of a relevant epidemiologic profile but also in the event of a health careeassociated cluster of severe respiratory illness cases. patients with confirmed or suspected mers-cov infection should be cared under contact and droplet precautions until testing results. in accordance with who guidelines, a high protection mask (eg, n95 respirator) along with eye goggles, gowns, and gloves should be used during aerosol-generating procedures; the latter should be performed in an adequately ventilated room (minimum of 6-12 air changes per hour) (airborne infection isolation room). 37 for consistency with the recommendations during the 2009 h1n1 pandemic, the united states cdc recommends the use of n95 respirators in all contacts with a laboratory-confirmed or suspected mers-cov infected case. 38 the rationale for this recommendation relies on the gaps of knowledge about the potential for airborne transmission of the novel coronavirus. however, n95 respirators are less tolerated by hcws and are more expensive. 39 the united states cdc also recommends that patients with confirmed or suspected mers-cov infection are placed in an airborne infection isolation room. 38 hcws with mers-cov infection should be strictly excluded from patient care, even with mild symptoms. the role of asymptomatic hcws is under question. overall, there is a need to increase infection control capacity in affected areas and areas at increased risk of being affected to prevent transmission in health care settings. our knowledge about the epidemiologic characteristics of mers-cov that impact health care transmission is very limited. to interrupt in-hospital transmission, routes of efficient exposure and virus shedding should be well studied. the contribution of primary cases to the so-called hospital-acquired cases in the recent upsurge of detected cases in the arabian peninsula is still unclear, and further epidemiologic data and analyses are necessary. in 1 analysis, 60 of 95 (63.2%) cases with evidence of secondary transmission acquired the infection in the hospital environment; nevertheless, 49 of them had additionally reported exposure to animals, therefore not eliminating an alternative source of infection. 21 the stability of the proportion of asymptomatic versus symptomatic cases is an argument against increasing testing as a possible explanation for either primary or secondary cases. 10 on the other hand, a reverse scenario could be that additional cases are missed because cases at the early incubation period or with low viral loads may be missed with molecular testing. a transmission event under similar circumstances has been described in the community for the first imported mers-cov case in the united states that tested negative by molecular assays but subsequently tested positive by serology. 25, 40 research for the future active surveillance and testing are of outmost importance to provide answers about the epidemiology of mers-cov and evolution of the current epidemic. case-control, serologic studies in exposed hcws are needed to better define the effectiveness of infection control measures. transmission of the virus via asymptomatic shedding in feces or other routes (eg, fomites, environment) is another topic for investigation. studies of viral kinetics in affected patients with molecular analyses of samples from various body sites will provide answers for infection control as well. a vaccine against mers-cov should be developed along with specific antiviral agents. there is no doubt that mers-cov remains a serious threat and has exhibited a significant public health impact in the affected countries. currently, health careeassociated transmission plays a pivotal role in the evolution of the mers-cov epidemic in countries in the arabian peninsula. a significant cost has been encountered in terms of personnel and time required for contact tracing and means of implementing infection control and prevention measures in health care settings. so far, there is no evidence of sustained humanto-human transmission. however, significant concerns exist in terms of the increased number of health careeassociated cases, gaps in knowledge regarding transmission routes, and limited infection control capacity in affected countries. as the mers-cov epidemic continues to evolve, vaccine and specific antiviral agents against mers-cov are urgently needed. studies about the effectiveness of infection control measures will provide answers and eventually promote safety in health care facilities both for patients and hcws. epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirus (mers-cov) e update middle east respiratory syndrome coronavirus (mers-cov) summary and literature updateeas of 9 potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study stability of middle east respiratory syndrome coronavirus in milk family cluster of middle east respiratory syndrome coronavirus infections investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov). ninth update interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case middle east respiratory syndrome coronavirus infections in health care workers first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description a case of imported middle east respiratory syndrome coronavirus infection and public health response stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions prediction of intrinsic disorder in mers-cov/ hcov-emc supports a high oral-fecal transmission severe respiratory illness caused by a novel coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study contact investigation of a case of human novel coronavirus infection treated in a german hospital current advancements and potential strategies in the development of mers-cov vaccines broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) preventing the spread of influenza a h1n1 2009 to health-care workers illinois resident who had contact with indiana mers patient tests positive for mers coronavirus key: cord-294854-rvrgcugn authors: hu, biying; huang, shaoying; yin, lianghong title: the cytokine storm and covid‐19 date: 2020-06-27 journal: j med virol doi: 10.1002/jmv.26232 sha: doc_id: 294854 cord_uid: rvrgcugn coronavirus disease 2019 (covid‐19), which began in wuhan, china in december 2019 has caused a large global pandemic and poses a serious threat to public health. more than four million cases of covid‐19, which is caused by the severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), have been confirmed as of may 11, 2020. sars‐cov‐2 is a highly pathogenic and transmissible coronavirus that primarily spreads through respiratory droplets and close contact. a growing body of clinical data suggests that a cytokine storm is associated with covid‐19 severity and is also a crucial cause of death from covid‐19. in the absence of antivirals and vaccines for covid‐19, there is an urgent need to understand the cytokine storm in covid‐19. here, we have reviewed the current understanding of the features of sars‐cov‐2 and the pathological features, pathophysiological mechanisms, and treatments of the cytokine storm induced by covid‐19. additionally, we suggest that the identification and treatment of the cytokine storm are important components for rescuing patients with severe covid‐19. this article is protected by copyright. all rights reserved. an outbreak of coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has rapidly spread throughout the world 1 . the initial symptoms of covid-19 mainly include fever, cough, myalgia, fatigue, or dyspnea. in the later stages of the disease, dyspnea may occur and gradually develop into acute respiratory distress syndrome (ards) or multiple organ failure (mof) 2 . it has been reported that a cytokine storm is accepted article associated with the deterioration of many infectious diseases, including severe acute respiratory syndrome (sars) 3 and middle east respiratory syndrome (mers) 4 . the cytokine storm caused by has been suggested to be associated with covid-19 severity 2, 5 . however, there is currently a limited understanding of the cytokine storm in severe covid-19. therefore, here, we have discussed the current findings and treatment strategies for the cytokine storm in severe covid-19. sars-cov-2 is the newest coronavirus known to infect humans. sars-cov, mers-cov, and sars-cov-2 cause severe pneumonia, while other human coronaviruses, including 229e, oc43, hku1, and nl63, cause only the common cold 6 . sars-cov-2 belongs to the genus betacoronavirus, which also includes sars-cov and mers-cov, both of which have caused sars and mers, respectively 7 . sars-cov-2, sars-cov, and mers-cov have similarities and differences. genetic sequence analysis has revealed that sars-cov-2 shares 79% sequence identity with sars-cov and 50% identity with mers-cov 8 . the genomes of sars-cov-2 and the bat coronavirus ratg13 are 96.2% homologous 9 . as of may 11, 2020, covid-19 has resulted in 278,892 deaths and 4,006,257 cases, with an approximate case fatality rate of 7.0% 10 . sars-cov and mers-cov had a case fatality rate of 9.6% (774/8,096) and 34.4% (858/2,494), respectively 11 . the reproduction number (r0) of covid-19 is thought to be this article is protected by copyright. all rights reserved. accepted article between 2 and 2.5 12 , which is slightly higher than of sars (1.7.1.9) and mers (<1) 13 . covid-19 appears to be more infectious than sars and mers, but may be less severe. the origins of sars-cov, mers-cov, and sars-cov-2 are considered to be zoonotic. both sars-cov and mers-cov originated in bats and spread directly to humans from marked palm civets and dromedary camels, respectively 14 . however, the origin of sars-cov-2 remains unclear. sars-cov-2 has been reported to be transmitted between humans through direct contact, aerosol droplets, the fecal-oral route, and intermediate viruses from symptomatic and asymptomatic patients 15 . sars-cov and mers-cov are also thought to spread from infected to non-infected individuals through direct or indirect contact 15 19 proposed that sars-cov-2 infection triggers an excessive immune response known as a cytokine storm in cases of severe covid-19. a cytokine storm is a potentially fatal immune disease characterized by the high-level activation of immune cells and excessive production of massive inflammatory cytokines and chemical mediators 20 . it is considered to be the main cause of disease severity and death in accepted article covid-19 patients 5 , and is related to high levels of circulating cytokines, severe lymphopenia, thrombosis, and massive mononuclear cell infiltration in multiple organs 21 . it has been found that sars-cov-2 genomes from different parts of the world have evolved in different clusters 22, 23 . forster et al. 23 reported there to be at least three central variants of sars-cov-2 globally, named a, b, and c. the a type is the most similar to the bat coronavirus and is mainly found in the united states and australia. the b type is more common in east asia and has evolved through several mutations. the c type is primarily found in europe. different viral isolates exhibit significant differences in pathogenicity and viral load 24 . notably, given the diverse clinical symptoms of patients, it will be challenging to establish a genotype-phenotype relationship. with new sequences being uploaded to the giasid (global initiative on sharing all influenza data) every day, new results may be produced as more data become available. the emergence of variants may add to the challenges of vaccine development. pathological alterations in covid-19 patients include pulmonary edema, diffuse alveolar injury with the formation of hyaline membranes, the presence of reactive type ii pneumocyte hyperplasia, proteinaceous aggregates, fibrinous accepted article exudates, monocytes and macrophages within alveolar spaces, and inflammatory infiltration of interstitial mononuclear cells [25] [26] [27] [28] . electron microscopy has revealed the presence of sars-cov-2 virus particles in bronchial and alveolar type ii epithelial cells, but not in other tissues 26, 27 . therefore, although a pcr test may be negative from blood or throat swabs, sars-cov-2 viral inclusions may be detected in the lungs. immunohistochemical staining indicated that cd68 + macrophages, cd20 + b cells, and cd8 + t cells infiltrated the alveolar cavity and alveoli 26 . the levels of cd8 ＋ t cells may be slightly higher than that of cd4 ＋ t cells within the alveolar septa 29 cellular entry of sars-cov-2 depends on the binding of s proteins covering the surface of the virion to the cellular ace2 receptor 9, 33 and on s protein this article is protected by copyright. all rights reserved. priming by tmprss2, a host membrane serine protease 33 table 1 , and cytokine secretion patterns based on covid-19 severity are shown in table 2 . prevention and mitigation of the cytokine storm may be the crux to saving patients with severe covid-19. currently, many therapies are being evaluated in clinical trials due to a lack of high-quality evidence. corticosteroids inhibit the host inflammatory response and suppress the immune response and pathogen clearance 46 overall, although evidence indicates a potential role for the use of corticosteroids this article is protected by copyright. all rights reserved. in patients with severe covid-19, caution should be exercised given the possibilities of viral rebound and adverse events. given their in vitro antiviral effects and anti-inflammatory properties, cq and its analog hcq are considered to be potential therapies for covid-19. considering the severe side effects of cq, hcq may be a better therapeutic option. cq and hcq are able to reduce cd154 expression in t cells 52 however, the risks associated with tcz (e.g., severe infections, thrombocytopenia, neutropenia, liver damage) should also be noted 64 . it is unclear whether there are different effects between il-6 antagonists (siltuximab) and il-6r antagonists (tcz). siltuximab binds to sil-6 and inhibits only cis and trans signaling. tcz binds to both mil-6r and sil-6r and inhibits both cis and trans signaling and trans presentation 40, 65 . of note, il-6 inhibitors are not able to bind to il-6 produced by viruses such as hiv and human herpesvirus-8 65 . currently, the application of tcz for covid-19 treatment is under study. the three drugs mentioned above (corticosteroids, hcq, tcz) are immunosuppressants. owing to overall damage to the immune system caused by autoimmune diseases and the iatrogenic effects of immunosuppressants, the risk of infection in patients with autoimmune diseases will be increased compared to the general population. currently, rheumatology societies 66-69 recommend the use of immunosuppressive drugs (except glucocorticoids) to be suspended in covid-19 patients. mscs have a wide range of immune regulatory functions and can inhibit the abnormal activation of t lymphocytes and macrophages and the secretion of pro-inflammatory cytokines 70 . msc therapy was found to significantly reduce the mortality of patients with h7n9-induced ards and had no harmful side effects 71 . a clinical trial of msc therapy revealed that mscs were able to rapidly and significantly improve the clinical symptoms of covid-19 without any observed adverse effects 72 . although side effects of msc treatment are rarely reported, the safety and effectiveness of this treatment require further investigation. anakinra, an il-1 receptor antagonist that blocks the activity of pro-inflammatory cytokines il-1α and il-1β, has been reported to improve respiratory function and increase the survival rate of covid-19 patients 73 . il-1 receptor antagonists increase the risk of bacterial infections, but this is extremely rare for anakinra 65 . janus kinase (jak) inhibitors can inhibit inflammatory cytokines and reduce the ability of viruses to infect cells 74 table 3 . the cytokine storm leads to deleterious clinical manifestations or even acute mortality in critically ill patients with covd-19. impaired acquired immune this article is protected by copyright. all rights reserved. responses and uncontrolled inflammatory innate responses may be associated with the mechanism of the cytokine storm in covid-19. early control of the cytokine storm through therapies such as immunomodulators and cytokine antagonists is essential to improve the survival rate of covid-19 patients. although many research articles are published each month, majority of the existing literature about covid-19 comes from descriptive works. additionally, high-quality evidence will be necessary to understand and treat the cytokine storm of covid-19. potential for global spread of a novel coronavirus from china clinical features of patients infected with 2019 novel coronavirus in wuhan an interferon-gamma-related cytokine storm in sars patients active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis covid-19: consider cytokine storm syndromes and immunosuppression chapter eight -hosts and sources of endemic human coronaviruses coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin world health organization w. coronavirus disease (covid-19)situation report -112 genetic recombination, and pathogenesis of coronaviruses report of the who-china joint mission on coronavirus disease 2019 (covid-19) 2020 covid-19, sars and mers: are they closely related? origin and evolution of pathogenic coronaviruses the covid-19 pandemic: a comprehensive review of coronaviruses -drug discovery and therapeutic options pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology clinical characteristics of coronavirus disease 2019 in china covid-19 with different severity: a multi-center study of clinical features mapping the innate signaling cascade essential for cytokine storm during influenza virus infection pathological inflammation in patients with covid-19: a key role for monocytes and macrophages analysis of the mutation dynamics of sars-cov-2 reveals the spread history and emergence of rbd mutant with lower ace2 binding affinity phylogenetic network analysis of sars-cov-2 genomes patient-derived mutations impact pathogenicity of sars-cov-2 pathological findings of covid-19 associated with acute respiratory distress syndrome pathological evidence for residual sars-cov-2 in pulmonary tissues of a ready-for-discharge patient sars-cov-2 and viral sepsis: observations and hypotheses histopathologic changes and sars-cov-2 immunostaining in the lung of a patient with covid-19 immune response to sars-cov-2 and mechanisms of immunopathological changes in covid-19 the clinical pathology of severe acute respiratory syndrome (sars): a report from immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor cellular and molecular pathways of covid-19 and potential points of therapeutic intervention aberrant pathogenic gm-csf+ t cells and inflammatory cd14+cd16+ monocytes in severe pulmonary syndrome patients of a new coronavirus neutrophil extracellular traps in covid-19. jci insight covid-19: a new virus, but a familiar receptor and cytokine release syndrome understanding angiotensin ii type 1 receptor signaling in vascular pathophysiology. hypertension (dallas, tex: 1979) pleiotropy and specificity: insights from the interleukin 6 family of cytokines cytokine release syndrome in severe covid-19 plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile clinical and immunological features of severe and moderate coronavirus disease 2019 longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients a dynamic immune response shapes covid-19 cell host & microbe covid-19 infection and rheumatoid arthritis: faraway, so close! autoimmun rev treatment of severe acute respiratory syndrome with glucosteroids: the guangzhou experience on the use of corticosteroids for 2019-ncov pneumonia clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis. the journal of infection hydroxychloroquine inhibits cd154 expression in cd4 t lymphocytes of systemic lupus erythematosus through nfat, but not stat5, signaling effects of chloroquine on viral infections: an old drug against today's diseases? in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial hydroxychloroquine versus covid-19: a rapid systematic review and meta-analysis chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of accepted article coronavirus (sars-cov-2) infection: preliminary safety results of a randomized, double-blinded, phase iib clinical trial (clorocovid-19 study) safety of hydroxychloroquine, alone and in combination with azithromycin, in light of rapid wide-spread use for covid-19: a multinational, network cohort and self-controlled case series study the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality chinese clinical trail registry. a multicenter, randomized controlled trial for the efficacy and safety of tocilizumab in the treatment of new coronavirus pneumonia effective treatment of severe covid-19 patients with tocilizumab improved survival outcome in sars-cov-2 (covid-19) acute respiratory distress syndrome patients with tocilizumab administration rational use of tocilizumab in the treatment of novel coronavirus pneumonia the society for immunotherapy of cancer perspective on regulation of interleukin-6 signaling in covid-19-related systemic inflammatory response bsr guidance for patients during covid-19 outbreak acr guidance for patients during covid-19 outbreak 2020 australian rheumatology association guidance for patients during covid-19 outbreak 2020 the immunomodulatory function of mesenchymal stem cells: mode of action and pathways clinical study of mesenchymal stem cell treating acute respiratory distress syndrome induced by epidemic influenza a (h7n9) infection, a hint for covid-19 treatment transplantation of ace2(-) mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome,and hyperinflammation: a retrospective cohort study ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study baricitinib therapy in covid-19: a pilot study on safety and clinical impact. the journal of infection baricitinib as potential treatment for 2019-ncov acute respiratory disease the cytokine storm of severe influenza and development of immunomodulatory therapy epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study immunomodulatory therapy for the management of severe covid-19. beyond the anti-viral therapy: a comprehensive review the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection we thank all the doctors and nurses who have bravely fought the virus during the covid-19 pandemic. the authors declare no conflicts of interest. 1 key: cord-272932-devmy5yx authors: wang, wen ling; wang, hui juan; deng, yao; song, tie; lan, jia ming; wu, gui zhen; ke, chang wen; tan, wen jie title: serological study of an imported case of middle east respiratory syndrome and his close contacts in china, 2015 date: 2016-03-31 journal: biomedical and environmental sciences doi: 10.3967/bes2016.027 sha: doc_id: 272932 cord_uid: devmy5yx the first imported middle east respiratory syndrome (mers) case in china was identified in may 2015. we determined the kinetics of antibody (igg and igm) and neutralizing antibodies against mers-coronavirus (mers-cov) in this case before discharge. moreover, no seroconversion was found among 53 close contacts by anti-mers igg antibody enzyme-linked immunosorbent assay (elisa) of paired serum samples. these findings suggest that neither community nor nosocomial transmission of mers-cov occurred in china. middle east respiratory syndrome (mers), caused by the mers-coronavirus (mers-cov), is an emerging respiratory disease of global public health concern with a high mortality rate [1] . on may 29, 2015, a business traveler from the republic of korea was identified as the first imported case of mers-cov infection in china [2] [3] . the world health organization (who) received notifications of confirmed cases of mers-cov infection from the republic of korea mid may 2015 [4] [5] . the who was notified later that one close contact of the index mers case in the republic of korea travelled to guangdong province, china, by way of hong kong. the first mers-cov case in china imported from the republic of korea was confirmed by the national health and family commission of the people's republic of china on may 29, 2015 [2] [3] . the patient was admitted to huizhou municipal central hospital, huizhou, guangdong province on may 28, 2015 and was discharged after recovery on june 26, 2015. a set of venous blood samples was collected after his admission. to confirm the specificity of the inactivated mers-cov virion-based elisa and the background of normal sera, anti mers-cov igm and igg in 10 sera from the newborns and 40 sera from normal healthy adults were detected by elisa. to evaluate both the serological response of this mers patient before discharge and the risk of transmission, a set of serum samples from the patient and paired serum samples collected at least 14 d apart from the close contacts of the mers patient during his trip and hospital admission in china, were tested for mers-cov using an inactivated mers-cov-based elisa. meanwhile to confirm the specificity and sensitivity of inactivated mers-cov-based elisa, a commercially available (euroimmune ag, lübeck, germany) mers-cov s1 elisa was performed as reference. the assay included a calibrator for defining the upper limit of the reference range of non-infected persons (cut-off) recommended by euroimmun. values above the indicated cut-off are considered as positive, those below as negative. mers-spike pseudoparticle neutralization test (ppnt) based on env-defective, a reporter gene of luciferase-expressing hiv-1 genome (pnl4-3r-e-luc) were performed as previoiusly described [6] , neutralizing antibody titers were defined as the highest serum dilutions that resulted at 50% reduction in relative luciferase units. to strengthen infection control measures and identify possible person-to-person transmission, close contacts of the mers patient were also sampled. a total of 78 close contacts of the mers case during his journey were identified and monitored in isolation for 14 d after their last contact with the mers case, they included hotel staff, company employees, restaurant waiters, bus passengers and plane passengers. of these close contacts, including those who had face-to-face contact with the patient as well as more distant contacts, none presented symptoms compatible with mers, and all throat swab samples from the close contacts were negative for mers-cov by real-time rt-pcr [3] . only 53 paired serum samples were finally available for the close contacts. these paired serum samples from close contacts of the mers-cov patient were collected 16 days apart (june 3 and 19, 2015). the blood samples were processed within 24 h of collection, and sera were stored at -80 °c. all serum samples from the close contacts tested negative for mers-cov by real time rt-pcr. we used an inactivated mers-cov particle-based elisa to analyze serum samples from the imported mers-cov patient and his close contacts for the presence of igg against mers-cov. all experiments involving live mers-cov were conducted according to the standard operating procedure of the biosafety level 3 facilities at the chinese center for disease control and prevention. normal controls comprised serum samples from 40 healthy blood donors; these were used to determine background values and calculate the cut-off level. anti mers-cov igm and igg in the 10 sera from newborns and 40 normal control sera were detected at dilution 1:80 by elisa. the average od450 values for the newborns and normal adult controls (x) were calculated to be 0.13 (range 0.05-0.25) and 0.15 (range 0.09-0.27) for igm, with no difference existing between the two groups (t-test, p>0.05); and 0.16 (range 0.01-0.28) and 0.20 (range 0.12-0.38) for serum igg, with no difference existing between the two groups (t-test, p>0.05) (figure 1 ), and the cut-off value was determined to be 2.1-fold x, i.e., 0.32 for igm and 0.42 for igg. anti-mers-cov titers were determined in serial serum samples from the mers patient. his serum igm and igg level on the day following admission (may 29, 2015) was markedly lower than the cut-off value, exceeded the cut-off value 11 d after admission and plateaued 15 days after admission (figure 2a) . the serum igm and igg titer increased to 0.62 and 0.77 immediately before discharge (june 24, 2015) (figure 2a) . the igg titer of the patient shortly after admission were both 1:40, compared with 1:320 (8-fold higher) and 1:640 (16-fold higher) before discharge (data not shown), which demonstrated mers-cov seroconversion. mers s1-based elisa results showed that the od450 value for the calibrator recommended by euroimmun was 0.43 which was defined as the cut-off value, consequently, anti-mers s1igg in the mers patient exceeded the cut-off value 8 days after admission ( figure 2b) , consistent with the results reported by guan et al. [7] . although seroconversion of anti-mers-cov s1 igg appeared two days earlier than that of inactivated mers-cov, similar antibody kinetics based on mers s1 and inactivated mers-cov appeared and there was high correlation coeffieient of 0.9331 between them ( figure 2c) . a lentivirus-based mers-cov pseudovirus neutralization test was performed to confirm the presence of mers-cov-specific antibodies in serum samples from the patient. the neutralizing antibody titer was tested as 1:141 6 days after admission and peaked 15 d after admission ( figure 2d ). the results of the neutralization tests correlated well with the elisa results. all 53 paired serum samples from the close contacts were below the cut-off value, negative for mers-cov by elisa (figure 3 ). the average od450 values of the serum samples collected on june 3 and 19, 2015 were 0.18 and 0.19, respectively. no seroconversion was detected in the 53 close contacts. lentivirus-based mers-cov pseudovirus was pre-incubated with serially diluted sera from the imported mers-cov patient at 37 °c for 1 h before addition to cells. after 24 h of incubation, fresh medium was added to the culture, followed by incubation for a further 48 h. cells were washed with pbs and lysed using the lysis reagent included in the luciferase kit (promega). aliquots of cell lysates were transferred to 96-well costar flat-bottom luminometer plates (corning costar), followed by addition of luciferase substrate (promega). rfi values were determined immediately using a gaomax luminometer (promega). a 50% rfi reduction was used to calculate mers-cov-specific neutralizing antibody titers in serum samples. fifty-three paired serum samples from 53 close contacts that may have been exposed to the imported mers-cov patient were collected at an interval of at least 14 d. all of the sera were diluted 1:80 and subjected to inactivated mers-cov-based elisa. hrp-labeled goat anti-human igg was used as the secondary antibody, with tmb as the substrate, and the absorbance was read at 450 nm. od450 values higher than the cut-off value were considered to be indicative of mers-cov positive. the korean outbreak showed that mers-cov can result in an epidemic despite its lack of apparent mutations [2] , and thus it represents a global threat to public health. many laboratory tests-such as immunofluorescence assays, elisa, microneutralization assays and real-time rt-pcr assays-have been used to confirm mers-cov infection in mers patients and their close contacts [8] . however, data regarding the dynamics of anti-mers-cov igg and mers-cov neutralizing antibodies are lacking. in this study, we developed methods to detect antibodies to mers-cov using an inactivated mers-cov particle-based indirect enzyme-linked immunosorbent assay (elisa) and lentivirus-based mers-cov pseudovirus assay. the serum anti-mers-cov igg level of the mers patient immediately improved 16-fold before discharge, demonstrating seroconversion. his serum igm and igg level reached a plateau at 15 d after admission. we further determined the kinetics of mers-cov neutralizing antibody; the titer was highest (approximately 1:10,000) at 15 d after admission, in agreement with the igg curve, and then declined gradually to approximately 1:4500 before discharge. south korea experienced a mers outbreak representing the largest cluster of mers cases outside the middle east. as of october 31, 2015, 186 cases (including the mers case exported to china) of mers-cov infection have been associated with the south korea outbreak, 36 of whom died [4] [5] . there have been several reports of household or nosocomial transmission of mers-cov to healthcare workers in hospitals with mers-cov patients and/or delayed implementation of effective infection control practices [5, 8] . when the imported mers-cov patient in china was admitted on june 28, he was suffering from high fever, and real-time rt-pcr indicated a high viral load in serum and throat swab samples. seroconversion often occurs 10-14 d after exposure, so we performed serological detection on paired serum samples to minimize the possibility of missing asymptomatic infection. however, none of the 53 close contacts were positive for mers-cov, which suggest that none community transmission of mers-cov occurred in china. the lack of transmission of mers-cov from the patient to the close contacts in this study may have been due to several factors: 1) the short duration of exposure, and more importantly, 2) implementation by china of strict infection prevention and control practices immediately after being notified of importation of the mers-cov patient. these findings are similar to the results of an investigation of healthcare workers following importation of the first mers case into the usa [9] . in conclusion, we first determined the kinetics of antibody (igg and igm) and neutralizing antibodies against mers-cov for the first imported mers case in china. the data from the close contacts investigation indicated that the ability is limited for human-to-human transmission of mers-cov. however, every year approximately 10,000 residents of china travel to and return from mecca, saudi arabia for the ramadan period, so travel-associated importation and transmission of mers-cov remains a possibility. to prevent mers-cov infection, early screening and routine supervision are required, especially for confirmed and suspected mers cases and close contacts thereof [10] [11] . isolation of a novel coronavirus from a man with pneumonia in saudi arabia origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis imported case of mers-cov infection identified in china middle east respiratory syndrome -advancing the public health and research agenda on mers -lessons from the south korea outbreak tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptorbinding domain as an antigen characteristics of traveler with middle east respiratory syndrome, china hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description lack of transmission among close contacts of patient with case of middle east respiratory syndrome imported into the united states mers prevention and control plan china upgrades surveillance and control measures of middle east respiratory syndrome (mers) we thank the staffs in huizhou central hospital and guangdong provincial center for disease control and prevention for providing the information and samples from this imported case and close contacts. none key: cord-286683-mettlmhz authors: ortiz-prado, esteban; simbaña-rivera, katherine; gómez-barreno, lenin; rubio-neira, mario; guaman, linda p.; kyriakidis, nikolaos c; muslin, claire; jaramillo, ana maría gómez; barba-ostria, carlos; cevallos-robalino, doménica; sanches-sanmiguel, hugo; unigarro, luis; zalakeviciute, rasa; gadian, naomi; lópez-cortés, andrés title: clinical, molecular and epidemiological characterization of the sars-cov2 virus and the coronavirus disease 2019 (covid-19), a comprehensive literature review date: 2020-05-30 journal: diagn microbiol infect dis doi: 10.1016/j.diagmicrobio.2020.115094 sha: doc_id: 286683 cord_uid: mettlmhz abstract coronaviruses are an extensive family of viruses that can cause disease in both animals and humans. the current classification of coronaviruses recognizes 39 species in 27 subgenera that belong to the family coronaviridae. from those, at least seven coronaviruses are known to cause respiratory infections in humans. four of these viruses can cause common cold-like symptoms. those that infect animals can evolve and become infectious to humans. three recent examples of these viral jumps include sars cov, mers-cov and sars cov-2 virus. they are responsible for causing severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and the most recently discovered coronavirus disease during 2019 (covid-19). covid-19, a respiratory disease caused by the sars-cov-2 virus, was declared a pandemic by the world health organization (who) on 11 march 2020. the rapid spread of the disease has taken the scientific and medical community by surprise. latest figures from 20th may 2020 show more than 5 million people had been infected with the virus, causing more than 330,000 deaths in over 210 countries worldwide. the large amount of information received daily relating to covid-19 is so abundant and dynamic that medical staff, health authorities, academics and the media are not able to keep up with this new pandemic. in order to offer a clear insight of the extensive literature available, we have conducted a comprehensive literature review of the sars cov-2 virus and the coronavirus diseases 2019 (covid-19). the viral membrane contains the spike (s) glycoprotein that forms the peplomers on the virion surface, giving the virus its 'corona'or crown-like morphology in the electron microscope. the membrane (m) glycoprotein and the envelope (e) protein provide the ring structure. within the virion interior lies a helical nucleocapsid comprised of the nucleocapsid (n) protein complexed with a single positive-strand rna genome of about 30 kb in length [16] . the first genome of sars-cov-2 named wuhan-hu-1 (ncbi reference sequence nc_045512) was isolated and sequenced in china in january 2020 [12, 16] . the sars-cov-2 genome has similarities to other viruses: approximately 96% similarity to the bat coronavirus batcov rath13; an estimated 80% similarity with sars-cov [16] , and an estimated 50% identity with mers-cov [17, 18] . sars-cov-2 has a positive-sense single-stranded rna genome. it is approximately 30,000 bases in length and comprises of a 5′ terminal cap structure and a 3′ poly a tail. according to wu et al. [19] , this novel coronavirus (ivdc-hb-01/2019 strain) has 14 open reading frames (orfs) encoding 29 proteins. the 5' terminus of the genome contains the orf1ab and orf1a genes. orf1ab is the largest gene and encodes the pp1ab protein that contains 15 non-structural proteins named nsps (nsp1-nsp10 and nsp12-nsp16). orf1a encodes the pp1a protein and also has 10 nsps (nsp1-nsp10) [19] . the 3' terminus of the genome contains four structural proteins: spike (s) glycoprotein; envelope (e) protein; membrane (m) glycoprotein and nucleocapsid (n) phosphoprotein. it also contains 8 accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14) [20] (figure 3b ). the global scientific community from 58 countries have united to study this novel coronavirus by sequencing and submitting 12,059 sars-cov-2 genomes to the global initiative on sharing all influenza data (gisaid) (https://www.gisaid.org/) between december 2019 and april 2020 [21, 22] . sars-cov-2 has accumulated mutations in its rna genome as the outbreak progresses. as an intracellular obligate microorganism, the coronavirus exploits the host cell machinery for its own replication and spread. since virus-host interactions form the basis of diseases, knowledge about their interplay is of great importance, particularly when identifying key targets for antivirals. j o u r n a l p r e -p r o o f ace2 is a type i membrane protein that participates in the maturation of angiotensin, a peptide hormone that controls vasoconstriction and blood pressure [30] . in the respiratory tract, ace2 is widely expressed on the epithelial cells of alveoli, trachea, bronchi, bronchial serous glands [31] , and alveolar monocytes and macrophages [32] . xu et al. reported the [33] rna-seq profiling data of 13 organs with para-carcinoma normal tissues from the cancer genome atlas (tcga; https://www.cancer.gov/tcga) and 14 organs with normal tissue from fantom5 cage (https://fantom.gsc.riken.jp/). these were used to validate the expression of the human cell receptor ace2 in the virus and may indicate the potential infection routes of sars-cov-2 [34] . interestingly, the ace2 receptor is expressed more in oral cavity than lung. this potentially could indicate that susceptibility and infectivity of sars-cov-2 is greater from oral mucosa surfaces [33] . following the binding of the rbd in the s1 subunit to the receptor ace2, sars-cov-2 s protein is cleaved by the cell surface-associated transmembrane protease serine 2 tmprss2, which activates s2 domain for membrane fusion between the viral and cell membrane [35] . a functional polybasic (furin) cleavage site was found at the s1-s2 boundary through the insertion of 12 nucleotides [13, 29, 36] . the s673, t678 and s686 residues of o-linked glycans flank the cleavage site and are unique in sars-cov-2 [29] . in addition to the s glycoprotein -ace2 receptor complex, wang following the release and uncoating of viral rna to the cytoplasm, coronavirus replication starts with the translation of orf1a and orf1b into polyproteins pp1a and pp1ab via a frameshifting mechanism (figure 4 ) [38] . subsequently, polyproteins pp1a and pp1ab are processed by internal viral proteases, including the main protease m pro , a potential drug target whose crystal structure was recently determined for sars-cov-2 [15] . polyprotein cleavage yields 15 mature replicase proteins, which assemble into a replicationtranscription complex that engages in negative-strand rna synthesis. both full-length and multiple subgenomic negative-strand rnas are produced. the former serves as template for new full-length genomic rnas and the latter template the synthesis of the subgenomic mrnas required to express the structural and accessory protein genes residing in the 3′proximal quarter of the genome [37] . coronavirus rna replication occurs on a virusinduced reticulovesicular network of modified endoplasmic reticulum (er) membranes [39] . the assembly of virions is quickly ensued with the accumulation of new genomic rna and structural components. the n protein complexes with genome rna, forming helical structures. then, the transmembrane m protein, localized to the intracellular membranes of the er -golgi intermediate compartment (ergic) , interacts with the other viral structural proteins (s, e and n proteins) to allow the budding of virions [40, 41] . following assembly and budding, virions are transported in vesicles and eventually released by exocytosis. normal immune responses against the majority of viruses involve a rapid containment phase mediated by innate immunity components and -if necessary-a delayed, yet more sophisticated adaptive immunity phase that should be able to eradicate the pathogen andhopefully-generate long-lasting immunological memory. the former includes antiviral type i interferons (ifns), macrophage and neutrophil activation that leads to proinflammatory cytokine production and nk cells on the other hand, anti-viral adaptive immune responses involve a virus-tailored coordinated attack by antigen specific cd8+ cytotoxic t cells (ctls), the th1 subset of cd4+ t helper cells that orchestrates the j o u r n a l p r e -p r o o f immune response against viruses and other intracellular pathogens, specific antibody producing plasma cells, and finally the production of memory t and b cell subsets. immune responses following sars-cov-2 infection can be a double-edged sword. a rapid and robust type i ifn orchestrated response can lead to virus clearance and -given that antiviral lymphocytes are activated and expanded-immune memory. conversely, a late activation of innate immunity, possibly owing to is usually associated with severe pathology that can lead to pneumonia, ards, septic shock, multi-organ failure and, eventually, death. in this line, a delayed type i ifn response and inefficient sars-cov-2 clearance by alveolar macrophages can promote excessive viral replication that can lead to severe pathology accompanied by increased viral shedding and, thus, viral transmissibility. accordingly, in patients whose immune system is weakened or otherwise dysregulated, such as older men with comorbidities, severe covid-19 is clearly more likely to occur [42] [43] [44] . a recent study has demonstrated that the average duration of sars-cov-2 viral shedding was 20 days after covid-19 onset, raising a debate as to the optimal time of patient isolation [45] . however, in terms of transmission viral shedding seems to be more relevant in the early phases of the infection as it can precede covid-19 symptoms by 2-3 days whilst up to 50% of infections are associated with viral shedding by asymptomatic cases [46] [47] [48] therefore, individuals that mount efficient containment-phase immune responses accompanied by decreased inflammatory responses will not experience infection-or immune response-mediated overt manifestations, but may be important silent spreaders of sars-cov-2. type i ifns are mainly produced by plasmacytoid dendritic cells (pdcs) and have a plethora of antiviral effects such as blocking cell entry and trafficking of viral particles, inducing rnase and dnase expression to degrade virus genetic material, enhancing presentation of viral antigens by mhc-i, inhibiting protein synthesis, inducing apoptosis of j o u r n a l p r e -p r o o f infected cells and activating anti-viral subsets such as macrophages and cytotoxic nk cells and t lymphocytes [49] . pathogen recognition receptors like cytosolic rig-i and mda-5 [50, 51] or endosomal toll like receptors (tlrs) 7 and 8 that recognize viral rna [52] are responsible for the activation of signaling cascades that activate the transcription factors nf-kb, interferon regulatory factor (irf) 3 and irf7 that translocate to the nucleus and induce proinflammatory cytokines and type i interferon (ifn) production. in turn, type i ifns activate the downstream jak-stat signal pathway resulting in expression of ifnstimulated genes (isgs) [53, 54] . our experience from sars-cov and mers-cov infection has shown that delayed type i ifn production and excessive recruitment and activation of infiltrating proinflammatory cells (neutrophils and monocytes-macrophages) are possible mediators of lung dysfunction and bad prognostic factors for the outcome of the infection. delayed type i ifn production allows for highly efficient viral replication that, in turn, results in recruitment of hyperinflammatory neutrophils and monocytes. therefore, the pathogen recognition receptors (prrs) of these proinflammatory cells recognize high numbers of their ligands and subsequently secrete excessive amounts of proinflammatory cytokines that lead to septic shock, lung pathology, pneumonia or acute respiratory distress syndrome. it has been shown that in severe cases both sars-cov and mers-cov fruitfully employ an immune evasion mechanism whereby early type i ifn responses to viral infection are dampened [54] . this can be achieved by blocking signaling both upstream, as well as downstream of type i ifn expression. sars-cov can inhibit irf3 nuclear translocation, whereas mers-cov can impede histone modification [56] . additionally, both viruses can inhibit ifn signaling by decreasing stat1 phosphorylation [57] . due to the many sequence similarities of sars-cov-2 with sars-cov and mers-cov it would be enticing to speculate that similar mechanisms are also present, however further studies are needed to shed light to this hypothesis. hyperactivated neutrophils and monocytes-macrophages are the usual source of the cytokine storm. in this aspect, absolute neutrophil counts and neutrophil to lymphocyte j o u r n a l p r e -p r o o f ratio (nlr) were strongly associated with disease severity in a large cohort of covid-19 patients and were proposed as markers of adverse disease prognosis [58] . interestingly, the increased amounts of proinflammatory cytokines in serum associated with pulmonary inflammation and extensive lung damage described both in sars [59] and mers diseases [60] were also reported in the early study of 41 patients with covid-19 in wuhan [41] . evidence shows that the leading cause of covid-19 mortality is respiratory failure caused by acute respiratory distress syndrome (ards). there is an association with a cytokine storm mediated by high-levels of proinflammatory cytokines including il-2, il-7, il-10, g-csf, ip-10, mcp-1, mip-1a and tnf-α. ards was associated with increased fatality and subsequent studies confirmed il-6 and c-reactive protein are significantly upregulated in patients that died compared to convalescent patients [56] moreover, a recent study of 452 patients in wuhan identified that severe cases showed significantly higher cytokines and chemokines such as tumor necrosis factor-α (tnf-α), il-2, il-6, il-8 and il-10 expressed [58] . in accordance with these findings, therapeutic strategies are being tested. a phase 3 randomized controlled trial of il-1 blockade (anakinra) in sepsis has shown significant survival benefit in patients with hyperinflammation, without apparent increased adverse events [61] . currently, a multicenter, randomized controlled trial of tocilizumab (il-6 receptor blockade, licensed for cytokine release syndrome), is being trialed in patients with covid-19 pneumonia presenting with high levels of il-6 in china (chictr2000029765) [62] . moreover, several clinical trials are exploring if the well-established antiviral [63] and anti-inflammatory effects of hydroxychloroquine will be effective in treating patients with covid-19 as has previously been suggested for sars-cov infection [64] . this has also been demonstrated in vitro for sars-cov-2 [65] . in contrast, janus kinase (jak) inhibition has been proposed as a potential treatment in order to reduce both inflammation and cellular viral entry in covid-19 [66] . thus, it comes as no surprise that in a recent correspondence, lancet authors have identified the following potential therapeutic options for cytokine storm syndrome including ards the use of corticosteroids, selective cytokine blockade (eg, anakinra or tocilizumab) and jak inhibition [67] . j o u r n a l p r e -p r o o f virus presentation to the different t cell subsets stands on the crossroads between innate and adaptive immune responses. studies on sars-cov and mers-cov [72] presentation have identified several susceptibility and protection conferring hla alleles. the dearth of similar data regarding sars-cov-2 antigen presentation to t cells and possible virus evasion mechanisms of this process suggests it is a virgin investigation field to be explored. apart from the sustained inflammation and cytokine storm, lymphopenia has been implicated as a major risk factor for ards and mortality in the context of covid-19 [73] . similar findings were described for sars-cov infected patients who had considerable decreases of cd4+ t and cd8+ t cells [69] . however, in convalescent patients specific tcell memory responses to sars-cov were still found six years post infection [74] . showed no reactivity with viral antigens. however, the small number of sera used (n=4) implies that further investigation is needed to corroborate these results [78] . nonetheless, since we are currently in early stages of sars-cov-2 pandemic more studies need to be carried out to shed light on antibody persistence (both igm and igg) and protective effects. recently, macaques re-challenged with sars-cov-2 after a primary infection did not show signs of re-infection, suggesting that protective immunity and memory responses were fruitfully mounted. this finding can also impact vaccine production strategies [79] . importantly, covid-19 convalescent sera was shown to hold promise as a passive immune therapy alternative to facilitate disease containment [80] . to the best of our knowledge, at j o u r n a l p r e -p r o o f least one pharmaceutical company, takeda, is preparing to purify antibody preparations from covid-19 convalescent sera against sars-cov-2 [81] . a recently published case report of a patient with mild-to-moderate covid-19 revealed the presence of an increased activated cd4+ t cells and cd8+ t cells, antibody-secreting cells (ascs), follicular helper t cells (tfh cells), and anti-sars-cov-2 igm and igg antibodies, suggesting that both cellular and humoral responses are important in containing the virus and inhibiting severe pathology [82] . antibody dependent enhancement (ade) is a mechanism whereby non-protective antibodies produced during an infection with an agent either mediate increased uptake of this agent into target cells or cross-recognize a different pathogen and facilitate its entrance to target cells [83] . evidence emerging over the past two decades suggests that antibodies against different coronavirus can cross-react to some extent and mediate ade [84] . ade in the context of sars-cov was thought to be mediated by antibodies produced against 229e-cov [85] and was contemplated as contributing to high mortality rates in china [86] . the described mechanism suggests that low affinity or low title anti-spike protein antibodies rather than neutralizing the virus result in fc receptor mediated infection of immune cells, further aggravating the dysregulation of anti-sars-cov immune responses [87] . indeed, in vitro as well as in vivo experimental models have shown that ade hinders the ability to manage inflammation in the lung and elsewhere. this may lead to ards and other hyperinflammation-induced clinical manifestations also observed in several of the documented cases of severe covid-19 [88, 89] . while the molecular and immunological host response to sars-cov-2 infection has not yet been fully elucidated to confirm ade is occurring, anti-sars-cov-2 antibodies have been shown to partially cross-react with sars-cov, suggesting enhancement is a possibility. with this in mind, ade in populations previously exposed to other coronavirus can partially explain the geographic discrepancies observed in covid-19 pathogenesis and severity. finally, ade can have several implications in vaccine development as low-affinity or low-titer antibody producing vaccines can increment susceptibility rather than confer protection against the pathogen as has previously been described for a dengue vaccine [90] [91] [92] . j o u r n a l p r e -p r o o f detection methods based on nucleic amplification are often used in the case of sars-cov, mers-cov and other viruses, because have high sensitivity and specificity, particularly in the acute phase of infection [93] . case identification and surveillance of covid-19 spread although rt-qpcr assay is considered the gold-standard method to detect viruses such as sars-cov and mers-cov [94, 95] , currently available rt-qpcr assays targeting sars-cov-2 have important considerations. firstly, due to the genome similarity of j o u r n a l p r e -p r o o f sars-cov-2 to sars-cov (82% of nucleotide identity [96] ), some of the primer-probe sets described by different groups and listed in the who coronavirus disease (covid19) technical guidance [97] , have cross-reaction with sars-cov and other bat-associated sars-related viruses, therefore, it is important to run confirmatory tests. loop-mediated isothermal amplification (lamp) is a one-step isothermal amplification reaction that couples amplification of a target sequence with four to six primers, to ensure high sensitivity and specificity, under isothermal conditions (63-65°c), using a polymerase with high strand displacement activity [129] . in the case of an rna sample, lamp, is preceded by the reverse transcription of the sample rna. rt-lamp has been used before for the detection of various pathogens [130] . including sars-cov-2 and other respiratory viruses [131, 132] . recently, it received emergency use authorization (eua) from the u.s. j o u r n a l p r e -p r o o f serological tests also, called immunoassays, are rapid and simple alternatives for screening of individuals that have been exposed to sars-cov-2 based on the qualitative or quantitative detection of sars-cov-2 antigens and/or anti-sars-cov-2 antibodies. there are several types of serological tests available, including elisa (enzyme-linked immunosorbent assay), iift (indirect immunofluorescence test), lateral flow immunoassays and neutralization tests. immunoassays assays are very useful because they allow to study the immune response(s) to sars-cov-2 in a qualitative and quantitative manner. in addition, help to determine the precise rate of the infection [78, 133] , and to determine more precisely the fatality rate of the infection [78] . several sars-cov-2 targeted serological tests are commercially available or in development [134] . a recently developed kit, reported a sensitivity of 88.66% and specificity of 90.63% [135] using sars-cov-2 igg-igm combined antibody rapid (within 15 minutes) test [135] . despite their simple and fast readout and their potential for being used outside laboratory environments (bedside, small clinics, airports, train stations, etc.), serological tests have a critical disadvantage; given the fact that antibodies specifically targeting the virus would normally appear after 6 days or longer [136] after the illness onset [137] , tests based on this principle have a lag period of approximately 4 to 7 days post-infection. during this lag period, infected and non-infected individuals will both result in a negative output. in addition, it is important to highlight that because serological tests depend on the ability to produce antibodies, intrinsic immunological differences and/or responses between individuals, can significantly affect the outcome of these tests. recently, some commercially available immunoassays received ce mark for professional use [138, 139] , and therefore are registered as in vitro diagnostic devices. currently, there are a plethora of antibody tests for covid-19 with variable performance (sensitivity varying from 45 to 100%, specificity from 96 to 100%, reviewed in (foundation for innovative new diagnostics) [134] . different manufacturers of serological assays declare that their assays have no cross reactivity to other human coronaviruses and other respiratory viruses. however, despite the data provided by manufacturers, recent studies highlighted that several of the commercially available tests have sensitivity and/or specificity issues that should be considered for using and analyzing results of many of these tests [140] [141] [142] [143] [144] . j o u r n a l p r e -p r o o f as mentioned before, immunoassays -particularly tests detecting anti-sars-cov-2 igm and/or igg-indicate that the person has been exposed to the virus. in the case of other viral infections, having antibodies targeting a pathogen has often been considered an indication of having immunity against that pathogen [145] . based on this idea, some governments have suggested using serological tests, to determine who has developed immunity against sars-cov-2 and provide positive individuals a "risk-free certificate" or "immunity passports", which would enable them to travel or to return to work, assuming that they are protected against re-infection [146]. however, based on the limited knowledge of how immunity to this virus works [147] , there is not enough evidence to declare a person immune, or to confirm that people who have anti-sars-cov-2 antibodies are protected from a re-infection. even though covid-19 can be diagnosed using qpcr as the gold standard, inadequate access to reagents and equipment has slowed disease detection even in developed countries such as the us. several low cost and rapid tests using different approaches have been described. unlocking) technique for the detection of covid-19 and the detectr (developed by mammoth biosciences) prototype rapid detection diagnosis kit using crispr to detect the sars-cov-2 in human samples have been described [148] . the use of rna aptamers, have recently emerged as a powerful background-free technology for live-cell rna imaging due to their fluorogenic properties upon ligand binding, a technology that could be of use to diagnose sars-cov-2 infection [149] . finally the use of next generation sequence (explify®) might be used to detect and identify bacterial, viral, fungal, and parasitic pathogens by their unique genome sequences [107] . in covid-19 symptomatic infection, the clinical presentation can range from mild to ventilation assistance [151] . the spectrum of symptoms of covid19 infection are characteristic of a mild disease in most of the cases, however, it is important to point that the progression could lead to a severe respiratory distress. asymptomatic infection (while incubation occurs) was described both in the first cases in wuhan and in other cohorts. a group of isolated patients were screened for sars-cov-2, where 17% (629 cases) were positive for the test, and half of these cases had no symptoms. on the other hand, there are reports of cases without overt symptoms in which there were ground glass images in the chest tomography in up to 50% of patients [152] . of the asymptomatic cases studied in wuhan city, the 2.5% of people exposed developed specific symptoms in 2.2 days, and the remaining 97.5% were symptomatic in the following 11.5 days (ci, 8.2 to 15.6 days). the median estimated incubation period was 5.1 days (95% ci, 4.5 to 5.8 days) [153] . some patients with initially mild symptoms had symptom progression over the course of one week [154] . the descriptive studies available so far have concluded that the majority of cases are mild infections (more than 80% of cases); with up to 15% of patients being sever j o u r n a l p r e -p r o o f in most cohorts, and less than 5% have been considered as critical cases with high vital risk [155] . in a study describing 138 patients with covid-19 pneumonia in wuhan, the most common clinical characteristics at the onset of the disease were described. this is consistent with other international cohorts (table 3 ) [150] . it is important to note that fever is not always present and up to 20% of patients could had a low grade temperature between 37.5 to 38 degrees celsius or normal temperature [156] . if these patients required hospitalization, 89% developed a fever during the course of the illness. rarer accompanying symptoms included headache without warning signs, odynophagia and rhinorrhea. gastrointestinal symptoms such as nausea and watery diarrhea were relatively rare [151] . dyspnea develops after a median of 5 to 8 days from the onset of symptoms. it is important to notice that, if dyspnea is an important clinical finding, not all the patients with this j o u r n a l p r e -p r o o f symptom will develop severe respiratory distress or require oxygen supplementation [150] . according to world health organization (who) guidelines, covid-19 infection can present as pneumonia without signs of severity, and could be managed in the outpatient setting. this is applicable to those patients who do not need supplemental oxygen [157] . as previously mentioned, the most serious manifestation of covid 19 infection is pneumonia, characterized by cough, dyspnea, and infiltrates on chest images; the latter is indistinguishable from other viral lung infections. acute respiratory distress syndrome (ards) is a major complication of covid pneumonia in patients with severe disease. this develops in 20% after a median of eight days. mechanical ventilation is implemented in 12.3% of cases [158] . in different case reports, the need for supplemental oxygen via the nasal cannula was required in approximately 50% of hospitalized patients. 30% required non-invasive mechanical ventilation, and less than 3% required invasive mechanical ventilation with or without extracorporeal membrane oxygenation (ecmo) [159] . it is important to mention that the proportion of severe cases is highly dependent on the study population and may be related to the epidemiological behavior of the infection in each country. additionally, the number of people tested will influence the denominator. in italy, the average age of people infected with covid-19 is between 60 and 65 years, and 16% of those hospitalized require admission to the intensive care unit (icu) [160]. the who recommendations had established that severe covid-19 disease could be defined by the following parameters in table 4 [157] . [162] . among the established risk factors for the development of ards is age greater than 65 years, diabetes mellitus and hypertension, in at least 40% of patients [151] . it should be clarified that, although advanced age is identified as a risk factor for a severe infection, those of any age may suffer from severe illness from covid-19. the descriptions made so far of the patients from china have determined that almost 90% of the patients were between the ages of 30 and 79 years (cohort of 44,500 cases) [155] . in other population settings, such as in the united states, more than 60% of confirmed the clinical characteristics of symptomatic cases and their severity has been described. in addition to the symptoms reported by the patients, the findings on physical examination may be absent during mild coivd-19 infection. those with moderate to severe covid-19 infection have various signs during pulmonary auscultation, however the most common findings include: wet rales; global decrease in respiratory sounds and increased thrill [164] . early recognition is essential to classify cases as potential cases and initiate one of the most important measures to contain the pandemic, isolation. 2. anyone who has resided or been traveling in areas where widespread community transmission has been reported. 3. any patient who has had potential exposure through attending events or has spent time in specific settings where cases of covid-19 have been reported. the scenarios described respond to the context of a high suspicion of covid-19 infection. the world health authorities (cdc, who) continually update these contexts. there have been multiple case definitions and clarifications regarding when to perform a covid-19 test: • they have pointed out the importance of fever, cough and dyspnea as sentinel symptoms, since these should form part of the clinical judgment that guides doctors. this allows to expand the group of suspicious patients. • in cases of severe respiratory distress of undetermined etiology and that do not meet the previously indicated criteria, a screening for covid-19 would be indicated. • in areas of limited resources, the suggestion is to prioritize cases that require hospital care, and in this way guide the epidemiological fence to order isolation and protect the most vulnerable people (chronically ill and over 65 years of age), as well as test those with the greatest possibility of exposure (travelers and health personnel). currently, there is no laboratory data profile that is framed in covid 19 infection. from a cohort of 43 patients confirmed with covid 19, these findings were classified as mild, moderate and severe disease [165] . high levels of d-dimer and more severe lymphopenia have been associated with mortality due to a prothrombotic state that determines multi-organ failure. in general, leukopenia and / or leukocytosis can be found in the interpretation of blood biometry, however, the most widely described finding is lymphopenia [166] . it should be considered that in the context of viral pneumonia biomarkers such as procalcitonin and pcr are not useful, as often these biomarkers are in the normal range for patients with covid-19. among other findings, descriptive studies have reported considerable elevations of lactate dehydrogenase and ferritin as well as alteration in aminotransferases; although elevation ranges for these parameters have not been established [167] . about the imaging findings, covid 19 viral pneumonia has similar features on imaging to other viral infections. although computed tomography (ct) is the test of choice, it is not useful for a definitive diagnosis due to the wide variety of images that can be found in patients with covid 19 infection. this statement is derived from a large cohort of more than 1000 wuhan patients, where rt-pcr confirmation of covid 19 and chest ct images of these patients were correspondingly analyzed. ct images were determined to have a sensitivity of 98%; however, the specificity was only 25% [168] . in general, the majority of descriptive studies concur that the finding of ground glass opacifications is most common. it is typically basal and bilateral, and rarely associated with underlying consolidation. a multicenter chinese study that retrospectively reviewed the ct scans of 101 patients found that 87% had typical ground-glass images and up to 53% had this finding along with consolidations. these findings were more frequent in the most j o u r n a l p r e -p r o o f severe and older age groups of patients [169] . pleural effusion (4%), and lymphadenopathy (2.7%) [170] . the emergence and outbreak of sars-cov-2, the causative agent of covid-19, has rapidly become a global concern that highlights the need for fast, sensitive, and specific tools to monitor the spread of this infectious agent. diagnostic protocols to detect sars-cov-2 using real-time quantitative polymerase chain reaction (rt-qpcr) were listed on the world health organization (who) website as guidance, however, various institutions and governments have chosen to establish their own protocols that might not be publicly available or listed by who. there are important challenges associated with close surveillance of the current sars-cov-2 outbreak. firstly, the rapid increase of cases has overwhelmed diagnostic testing capacity in many countries, highlighting the need for a high-throughput, scalable pipelines for sample processing [171, 172] . secondly, given that sars-cov-2 is closely related to other coronaviruses [96] , some of the currently available nucleic acid detection assays can result in false positives [173] . thirdly, critical concern for molecular detection is the low sensitivity reported for rt-qpcr assays [168] and serological tests [135] , particularly in the early stages of infection. additionally, most of the available rt-qpcr assays require sample processing and equipment only available in diagnostic and/or research laboratories. it is important to mention that coinfection is a possibility, as some reports from italy and in spain, less than 1% of cases in a cohort correspond to plwh, which have had a satisfactory evolution and less than half required an intensive care unit [177] . in the us, of the 5,700 hospitalized patients in the new york area, only 47 patients had hiv, while in san francisco, data was published on 1,233 people who had diagnosed with sars-cov-2 infection, of which less than 3% had hiv and none of them developed severe covid-19 [178] . despite the existence of in vitro studies on the efficacy of the use of lopinavir / ritonavir, it is currently known that its effect in cases of moderate and severe covid-19 is null, and therefore at the moment no recommendation can be given nor how treatment, much less as prophylaxis [179] . this clarification is important given that there is a belief that plwhs could be protected if they take antiretroviral therapy. therefore, current recommendations for plwhs are to maintain antiretroviral therapy with the goal of controlling hiv as well as following the same standards of care as the general population to avoid acquiring a sars-cov-2 infection [180] . regarding sars-ncov infection in pregnant women, there is currently limited evidence about the effect of the virus on the mother or fetus. however, due to the physiological changes typical of pregnancy, especially on the immune system (immunosuppression) and the cardiopulmonary system, pregnant women are thought to be more susceptible to developing severe symptoms when they acquire the viral respiratory disease. in 2009, when influenza a h1n1 infection occurred, only 1% of the infected population were pregnant, yet they accounted for 5% of infection-related deaths [181] . pregnancy (25%) [182] . in another study of 11 pregnant patients infected with mers-cov, 9 presented adverse results (91%), 6 neonates were admitted to the neonatal intensive care unit (55%) and three of them died (27%) [183] . however, it is important to note the small sample size which could increase the risk of bias and low power of the study. with information obtained so far from the wuhan sars-cov-2 outbreak, the infection appears to be less severe for pregnant women, compared to previous sars-cov and mers-cov outbreaks [181] . however, it is important to take into account that the data from covid-19 infection should be monitored with a doppler ultrasound every two weeks, due to the risk of developing intrauterine growth restriction [187, 188] . the time of termination of the pregnancy, as well as the method, also depend on several factors, including gestational age, maternal condition in relation to sars-cov-2 infection, presence of maternal comorbidities, and fetal condition. decisions must be made collaboratively during multidisciplinary team discussions, with individualized management plans established for each patient [189] . a diagnosis of covid-19 alone is not an indication for the termination of pregnancy, rather it should be made in combination with consideration of morbidity and mortality of both the fetus and mother. after delivery, the use of corticosteroids is recommended for antenatal fetal lung maturation, with betamethasone or dexamethasone [190] ; taking special care in critically nursing patients, as this may worsen their condition, and may delay delivery, which is necessary for the management of these patients [187, 191] . the symptoms children present with are similar to adults, as is the incubation period ranging from 1 to 14 days (mean of 5.2). a cough is the most frequent presenting symptom (65%) followed by fever (60%). there is a higher occurrence of gastrointestinal symptoms including diarrhea (15%), nausea, vomiting (10 %) and abdominal pain. these gastrointestinal symptoms are usually more variable in children than adults and are sometimes the only clinical manifestation in associations with fevers. [192, 193] . the clinical progression and disease severity in pediatric patients is markedly different from that of adults. over 90% of affected children are asymptomatic or have mild to moderate disease [192] . the majority of serious cases in children are related to those with significant comorbidities such as heart disease, immunosuppression, etc. to date of this review, only a few cases of children without underlying comorbidities have died as a result of covid 19 have been reported. this difference of severity of illness between adults and j o u r n a l p r e -p r o o f children has not been clarified, however, several theories have been postulated. these include that children express more ace2 receptors in their lungs which confer some protection to severe injuries such as those caused by rsv and which would decrease dramatically with age [194, 195] . immunological factors may also influence outcomes, as in childhood we are most exposed to frequent challenges including recent seasonal viruses such as rsv in the winter months. most likely, it is multifactorial and depends on factors from both the host and the virus itself [195] . abnormal radiological (ct) findings are found in asymptomatic children and consist of bilateral lung lesions (50%). elevated crp (creactive protein), procalcitonin pct (80%), and liver enzymes are present in most affected children, unlike adults in whom pct is not a reliable marker. virus elimination via the stool even after the negativity in the nasopharyngeal mucosa and the disappearance of symptoms makes them a potential source of contagion through the fecal-oral route [196] . patients with cancer are generally more susceptible to infections than healthy people, because they have a state of systemic immunosuppression that is exacerbated during chemotherapy or radiotherapy [197] . in china, according to national surveillance data, coronavirus infection occurs in 1.3% of patients with malignant tumors. this is a much higher proportion than the general incidence of 0.3% [198] . p<0.0001) even after adjusting for age [197] . further research, completed in a tertiary hospital in wuhuan -china similarly found that 25% of patients with cancer and sars-cov-2 infection died, most of them over 60 years of age [199] . due to these findings, it has been proposed by many international entities that during the pandemic, for prevention, an individualized plan based on the patient's specific conditions is required, with the aim to minimize the number of visits to health institutions.  for early-stage patients with need of post-surgical adjuvant chemotherapy, especially those whose clinical, pathologic, and molecular biologic staging suggest a better prognosis, the start time of adjuvant chemotherapy may be delayed up to 90 days after surgery without affecting the overall effect of treatment [200] .  for patients with advanced cancer, the main approach should be to minimize hospitalization in covid-19 positive installations. replacing the existing intravenous treatment regimen with oral chemotherapy during this special period may be considered, to ensure that treatment is not interrupted for a long time during the pandemic [201] . however, if there is a suspicion of covid-19 infection in this population group, the same updated diagnostic guidelines and the corresponding management should be followed depending on their severity of illness. moreover, an individualized follow-up plan should be outlined due to higher likelihood of complications in this group of population [202] . it should be noted that patients attending out-patient appointments for cancer have higher levels of anxiety, depression and other mental health problems than the general population. studies have shown that approximately 50% of malignant tumor survivors have a moderate to severe fear of tumor recurrence [203] . for this reason, psychologist surveillance of outpatients in quarantine or during hospitalization should be considered. reported complications derived from covid-19 describe a severe disease that requires management in an intensive care unit (icu) in approximately 5% of proven infections. j o u r n a l p r e -p r o o f appear to be most susceptible to the life-threatening complications. the risk of patient-topatient transmission in the icu is currently unknown, therefore adherence to infection control precautions is paramount [204, 205] . progressive deterioration of respiratory function is undoubtedly the most common and lifethreatening complication of the infection. the prevalence of hypoxic respiratory failure in covid-19 patients is 19%, and it can progress to acute respiratory distress syndrome (ards), with the need of mechanical ventilation support at 10.5 days on average. one study found that between 10 and 32% of hospitalized patients require admission to the icu due to respiratory deterioration [205] . as respiratory complications are the most common cause of severe deterioration, early identification of them will undoubtedly help in timely support. support provided should be adapted to take into account risk factors such as advanced age, neutrophilia and organic dysfunction for the development of ards. the diagnostic support of pulmonary tomography is undoubtedly a valid tool; images in patients with different clinical types of covid-19 have characteristic manifestations, but it can become an operational problem due to the difficulty in performing imaging on critically ill patients. on the contrary, lung ultrasound at the bed-side could provide an alternative to radiographs and tomography during the diagnosis of covid-19 [206, 207] . since more than 70% of hospitalized patients will require supplemental oxygen, it is recommended that oxygen should be started when pulse oximetry values fall below 90%. an upper-limit of 96% saturation should be established, since higher values have been shown to be harmful [208, 209] . hemodynamic deterioration has a variability of presentation, this depends on the study population and the definition [223] , the presence of shock in the intensive care unit may be present between 25 to 35% [204, 224] . cardiomyopathy related to viral infection is one of j o u r n a l p r e -p r o o f the main causes of hemodynamic detriment, occurring in up to 23% of patients with covid-19 [43] . hemodynamic failure is one of the main causes of death in these patients, with percentages of up to 40%, inconclusive risk factors are associated to date such as diabetes, hypertension, lymphopenia, and elevation of d-dimer [225] . acute kidney injury (aki) is present in up to 12% of critically ill patients, podocytes and proximal tubule cells are potential host cells for sars-cov-2, caused by the virus induced cytopathic effect. the diagnosis is based on markers of early kidney injury and urinary output [210] . initial management of shock is based on fluid resuscitation, based on the application of dynamic parameters to predict response to fluids, such as variation in stroke volume (svv), variation in pulse pressure volume (ppv) and change in stroke volume with passive leg elevation or fluid challenge above static parameters [225] . variables such as skin temperature, capillary refill time and/or serum lactate measurement are currently valid tools to assess shock. the volume of liquids used in resuscitation should be restricted and administered in relation to dynamic assessment. a liberal water resuscitation strategy is not recommended, rather a balance of crystalloids over colloids as resuscitation liquids should be encouraged and avoiding the use of hydroxyethyl starches, albumin, dextrans or gelatins [226, 227] . indirect evidence suggests that the target mean arterial pressure (tam) for patients with septic shock is 65 mmhg using vasoactive support [228] . the recommendation of norepinephrine use as the first agent is maintained. if norepinephrine is unavailable, vasopressin or epinephrine could be used, avoiding the use of dopamine as the initial vasopressor due to the potential development of arrhythmias [229, 230] . in patients with covid-19 and shock with evidence of cardiac dysfunction and persistent hypoperfusion despite fluid resuscitation and norepinephrine use, dobutamine as inotropic is recommended. given the development of refractory septic shock, the suggestion of the use of hydrocortisone in continuous infusion is maintained, as indirect evidence, this in favor of reducing the length of stay in the icu and the resolution time of the shock [229] . according to the investigative mission of the who in china, the case-fatality rate ranged other reports from china have coincided with this clinical risk profile, for example, a study that included 41 confirmed cases, 12 patients who had ards had as main underlying diseases: diabetes and high blood pressure. of these cases, 6 patients died [156] . according to who, the recovery time is estimated to be two weeks for patients with mild infections and three to six weeks for those with serious illnesses. on the other hand, cdc established that people who had symptoms in the mild to moderate spectrum and maintained home isolation have a resolution of 3 days after the fever decrease, and there was a substantial improvement in respiratory symptoms, even without use of medications. isolation may be limited to 7 days from resolution of symptoms, however, it must be adapted to the population circumstances of the epidemic [158] . the evolution of the epidemiological curve in covid-19 outbreak makes consider containment strategies in china primarily, and other countries based on nonpharmaceutical interventions (npis). according to the who, the most effective measure is hands washing [231] . combination as public health measures reduced contact rates in the population and therefore reduce virus transmission (table 6 ) [232] . table 6 non -pharmacological measures. increasing the level of hand cleanliness to 60% in places with a high concentration of people, like all airports in the world would have a reduction of 69% in the impact of a potential disease spreading [233] . the epidemiological evolution of the covid-19 pandemic through phases has required the application of specific measures according to the time or phase in which the virus is found in each country. the evolution in the non-pharmacological measures has been as variable as the pharmacological ones, in such a way, since january to march, it was ensured that the use of face masks was limited only to people who had contact with epidemiological foci, not to healthy people [234] . this concept was also reinforced by cdc, in order to optimize the use of masks for health workforce. definitely the course of the pandemic was changing rapidly, which also demanded the change from containment measures to mitigation. the recommendations in the current context remain regarding the use of a facial mask in the community, but its optimization is important for health professionals. the use of the mask is not a substitute for handwashing and social distancing measures, as these ones together allow avoiding viral particles in aerosols or drops, as a low cost and accessible measure for general population [235, 236] . there is still non-specific information for the recommendation of masks, in general, having in several studies claims that surgical or cotton cloth masks do not prevent the spread of the sars-cov-2 virus [237] . the evidence about the transmission of the virus in the asymptomatic period also changed the containment measures, suggesting the community use of masks. it is from this that the recommendations for the rational use of masks arise since in some j o u r n a l p r e -p r o o f countries the massive use of n95 masks was reported, masks indicated for the use of medical personnel [238] . regarding to this non pharmaceutical recommendations, the studies suggest to priories the resources on vulnerable population, in endemic areas, older people, adult with comorbidities and health workforce. studies are still needed on the duration of the protective effect of the masks and above all the possibility of their reuse for resource optimization. meanwhile the most important recommendation continues to be its use in addition to hand hygiene and social distancing [238] . therapeutic j o u r n a l p r e -p r o o f this effect is reinforced by azithromycin. there were the best results in terms of viral load reduction, even though is mentioned some limitations in the study like small sample size, a short long-term outcome follow-up, and dropout of six patients from the study [246] . concerning to mortality rates, a study was conducted in new york with 1376 patients (0.63-1.85). thus, it concludes that the use of hcq is not associated with either a decreased or increased clinical impairment, intubation or death [247] results reinforced by other study in 1438 hospitalized patients with covid-19 diagnosis in new york city, whom received treatment with hydroxychloroquine, azithromycin, or both drugs was not associated with significantly reduction in mortality [248] . relating to safety of this drug, in a study carried out in 200 patients in which the theoretical complications of the use of hcq and its combination with macrolides (azithromycin) were assessed by serial electrocardiograms, the following results were obtained. in 5% of patients (10) received chloroquine, 95% (191) received hcq, and 59% (119) [252] . supportive therapies in immune regulation, together with the use of antivirals, are important to take into account, especially in those patients in a serious and critical state, in which they could improve the clinical response and perhaps avoid residual lung injuries. the convalescent plasma is extracted from recovered individuals from an infection, being an antibody transfer medium to provide passive immunity (neutralizing antibodies and globulin). the goal is to provide a rapid immune response until the patient can develop their own active immune response in the hope that there will be clinical improvement [253] . improvement in most individuals, as well as viral suppression 7 days after treatment [255] . in the same country, at the shenzhen hospital, 5 cases of patients with severe covid-19 were reported who met criteria for acute respiratory distress syndrome (ards), who were administered convalescent plasma (titration greater than 1: 1000 and neutralizing antibodies greater than 40). it was found that clinical recovery occurred approximately 12 days after the transfusion (4 patients) and 3 of the 5 patients were discharged 55 days after admission. it is important to mention that this group of patients also received antivirals, methylprednisolone and all the necessary support measures in intensive care [256] . other drugs like ivermectin, nitazoxanide, and others have been studied in the context of covid-19 treatment, but the results are inconsistent. all of the clinical trials evidence, supporting or against the use of mentioned drugs are detailed in (supplementary table 2 ). this review summarized some drug repurposing agents previously known to has efficacy against other virus like sars-cov, mers-cov, influenza. actually, exist some new drugs with high potential action on targets for covid-19 therapeutics. it is important to notice that there is no specific treatment for the coronavirus approach. in context of the scientific evidence exposed and the particular clinical features of each patient, the reader will be able to make the best clinical and therapeutic decisions. when it comes to vaccine design and manufacturing, the main objectives are to ensure its safety, its efficacy in activating specific adaptive immune responses and the production ofideally-long term memory. thus, eliciting protective immune responses including neutralization antibodies and/or ctl generation is of paramount importance. huge challenges need to be tackled in order to minimize the long and cumbersome process of vaccine generation. among them, candidate antigen targets need to be identified, immunization routes and delivery systems investigated, animal models set, adjuvants optimized, scalability and production facility considered, target population selected, and vaccine safety and long-term efficiency evaluated. currently there are no approved vaccines against any human coronavirus, suggesting that their generation is quite novel. several candidate vaccines against sars-cov had shown promise reaching phase i or phase ii clinical trials [258, 259] , but the rapid containment of sars-cov expansion rendered them redundant, did not allow for a test population for phase iii trials and, therefore, put their further assessment to a halt. ctl memory could last up to 11 years after infection [260] . these data suggest that vaccine strategies employing viral structural proteins that can elicit effective, long-term memory t cell responses could yield fruitful results. on the other hand, the s1 spike protein region containing the ace receptor binding domain (rdb) is the obvious option when neutralizing antibody responses are considered [261] [262] [263] . indeed, a candidate sars vaccine antigen consisting of the rbd of sars-cov spike protein was created and found it could elicit robust neutralizing antibody responses and long-term protection in vaccinated animals [264] . the fact that covid-19 convalescent sera shows potential as a therapeutic approach [80] aligns with the theory that efficient b cell responses are mounted and lead to production of protective antibodies. two different groups, using an immunoinformatic approach mapped several ctl and b cell epitopes on different proteins of the virus [265, 266] . moreover, various ctl epitopes were found to be binding mhc class i peptide-binding grooves via multiple contacts, illustrating their probable capacity to elicit immune responses [265, 266] . consequently, these identified b and t cell epitopes could be potential targets for therapeutic vaccines. however, important safety considerations should be taken into account before releasing a new vaccine in the market. previous studies on macaque models have shown that a vaccineinduced anti-spike protein antibody at the acute stage of sars-cov infection can provoke severe acute lung injury [267] . similar observations of sars-cov vaccine-induced pulmonary injury have also been described in multiple several murine and monkey animal models [268] . an additional factor that needs to be checked in phase ii and iii trials is that the vaccine does not cause ade of the pathogen, as has previously been described. such concerns have risen in the context of a dengue vaccine [269] . the pharmaceutical companies that are currently on a race to produce a vaccine for covid-19 along with the vaccine developing strategies they are using are summarized in table 8 and figure 7 . as can be easily deduced from table 8 hospitalization and admission to already heavily charged icus due to these pathologies that could prove critical for weaker health systems that would struggle to carry the burden of combined outbreaks. moreover, vaccinating health care workers is crucial for reducing the risk of absence due to disease, thereby strengthening the healthcare workforce and minimizing the risk to infect covid-19 hospitalized patients with additional pneumoniacausing pathogens. lastly, covid-19 patients vaccinated for influenza and streptococcus pneumoniae allow their immune system to focus on one pathogen and, therefore, give it a better fighting chance against sars-cov-2 infection [276] . high risk groups prioritized for vaccination for these two pathogens include pregnant women, persons with immunocompromised immune systems (either due to congenital or acquired immunodeficiencies), children, adults ≥ 65 years and health care professionals. j o u r n a l p r e -p r o o f heat or chemical treatment inactivation. f) attenuated live pathogen vaccine strategies consist in administering a live pathogen that due to cell culture passaging has lost its virulence. they usually elicit robust and long-term memory immune responses without the need to administer an adjuvant. g) in dna vaccines the dna codifying a highly immunogenic antigen is administered and captured by professional antigen presenting cells (apcs) leading to antigen production and presentation by these cells. h) moderna's vaccine candidate already in phase i clinical trials uses an mrna vaccine approach whereby the genetic information codifying for the s protein of sars-cov-2 is delivered in lnps to enhance absorption by apcs. once uptaken by apcs the mrna induces the expression of s antigen that is subsequently mounted on and presented by mhc molecules to elicit adaptive immune response. numerous studies confirm that climate has an impact on virus (i.e., influenza, coronavirus, etc.) spread through manipulating the conditions of i) its diffusion, ii) the virus survival outside the host, and iii) the immunity of host population [277] . meteorological conditions, such as temperature, humidity, wind speed and direction, atmospheric pressure, solar radiation (including ultraviolet (uv) spectrum) and precipitation amount and intensity depend on the latitude and the elevation of the location, thus creating distinct climatic zones in the planet. while in some regions, such as temperate climate zones, human influenza peaks have clear seasonal cycles, in others it is not as predictable [276] [277] [278] [279] [280] . an array of studies, investigating the relationship between climatic factors and the activity of influenza all over the world, concluded that at the high latitudes of the world the peaks of influenza correlate with cold and dry weather conditions (i.e., winter season), while around the equatorial zone, it is more common during the months of high humidity and precipitation [282] [283] [284] [285] [286] [287] [288] . essentially, it depends on explicit threshold conditions based on monthly averages of specific humidity and temperature. when specific humidity drops below 11-12 g/kg and temperature drops below 18-21°c, the peak of influenza is stimulated during the cold-dry season, however, for tropical and subtropical (always humid and warm) regions, it is likely to prevail during the high precipitation (≥150 mm) months the possible origins of the new coronavirus sars-cov genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding when darkness becomes a ray of light in the dark times: understanding the covid-19 via the comparative analysis of the dark proteomes of sars-cov-2, human sars and bat sars-like coronaviruses novel coronavirus (sars-cov-2) epidemic: a veterinary perspective the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak an interactive web-based dashboard to track covid-19 in real time interim analysis of pandemic coronavirus disease 2019 (covid-19) and the sars-cov-2 virus in latin america and the caribbean: morbidity, mortality and molecular testing trends in the region epidemiological, socio-demographic and clinical features of the early phase of the covid-19 epidemic in ecuador. medrxiv severe acute respiratory syndrome a novel coronavirus from patients with pneumonia in china structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis for the recognition of the sars-cov-2 by full-length human ace2 crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. the lancet return of the coronavirus: 2019-ncov. viruses genome composition and divergence of the novel coronavirus (2019-ncov) originating in china analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods disease and diplomacy: gisaid's innovative contribution to global health global initiative on sharing all influenza data -from vision to reality pre-fusion structure of a human coronavirus spike protein cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a new coronavirus associated with human respiratory disease in china receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars the proximal origin of sars-cov-2. virological a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa a promoter-level mammalian expression atlas sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor article sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade host factors in coronavirus replication human coronaviruses: a review of virus-host interactions sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus a structural analysis of m protein in coronavirus assembly and morphology epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical features of the 2019 novel coronavirus outbreak in china. medrxiv clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study temporal dynamics in viral shedding and transmissibility of covid-19 presumed asymptomatic carrier transmission of covid-19 potential presymptomatic transmission of sars-cov-2 interferon-stimulated genes and their antiviral effector functions rig-i in rna virus recognition rna recognition and signal transduction by rig-i-like receptors recognition of viral single-stranded rna by toll-like receptors mechanisms of innate immune evasion in re-emerging rna viruses viral innate immune evasion and the pathogenesis of emerging rna virus infections immunopathogenesis of coronavirus infections: implications for sars pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology middle east respiratory syndrome interaction of sars and mers coronaviruses with the antiviral interferon response sars and mers: recent insights into emerging coronaviruses dysregulation of immune response in patients with covid-19 in wuhan plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile interleukin-1 receptor blockade is associated with reduced mortality in sepsis patients with features of the macrophage activation syndrome: re-analysis of a prior phase iii trial a multicenter, randomized controlled trial for the efficacy and safety of tocilizumab in the treatment of new coronavirus pneumonia (covid-19) effects of chloroquine on viral infections: an old drug against today's diseases chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) baricitinib as potential treatment for 2019-ncov acute respiratory disease covid-19: consider cytokine storm syndromes and immunosuppression. the lancet novel immunodominant peptide presentation strategy: a featured hla-a* 2402-restricted cytotoxic t-lymphocyte epitope stabilized by intrachain hydrogen bonds from severe acute respiratory syndrome coronavirus nucleocapsid protein association of human leukocyte antigen class ii alleles with severe acute respiratory syndrome in the vietnamese population epidemiological and genetic correlates of severe acute respiratory syndrome coronavirus infection in the hospital with the highest nosocomial infection rate in taiwan in 2003 human-leukocyte antigen class i cw 1502 and class ii dr 0301 genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection association of human leukocyte antigen class ii alleles with severe middle east respiratory syndrome-coronavirus infection a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome profile of specific antibodies to the sars-associated coronavirus antibody responses to sars-cov-2 in patients of novel coronavirus disease a serological assay to detect sars-cov-2 seroconversion in humans. medrxiv reinfection could not occur in sars-cov-2 infected rhesus macaques the convalescent sera option for containing covid-19 drugmaker takeda is working on coronavirus drug breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 a tale of two viruses: does heterologous flavivirus immunity enhance zika disease? evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus neutralizing antibody response and sars severity anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcγr pathway severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice licensed dengue vaccine: public health conundrum and scientific challenge dengue vaccine efficacy: not a zero sum game critique of world health organization recommendation of a dengue vaccine an updated roadmap for mers-cov research and product development: focus on diagnostics identification of a novel coronavirus in patients with severe acute respiratory syndrome assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan covid-19) technical guidance: laboratory testing for 2019 novel coronavirus (2019-ncov) in suspected human cases specific primers and probes for detection 2019 novel coronavirus. novel primers and probes for detection of novel coronavirus in coronavirus detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr detection of 2019 novel coronavirus (2019-ncov) in suspected human cases by rt-pcr protocol: real-time rt-pcr assays for the detection of sars-cov-2 detection of second case of 2019-ncov infection in japan rt-pcr protocol for the detection of 2019-ncov centers for disease control and prevention. real-time rt-pcr panel for detection2019-novel coronavirus. 2020 coronavirus in california | covid-19 testing. carbon health xpert® xpress sars-cov-2 has received fda emergency use authorization. cepheid. 2020 cdc. coronavirus disease 2019 (covid-19): information for laboratories: 2019-ncov coronavirus update | hologic molecular test receives fda emergency use authorization. hologic. 2020 genmark receives fda emergency use authorization for its eplex® sars-cov-2 test 2019-ncov cdc-qualified probe and primer kits for sars-cov-2 new york sars-cov-2 real-time reverse transcriptase (rt)-pcr diagnostic panel coronavirus disease (covid-19) | labcorp covid-19 : genesig new coronavirus (2019-ncov) nucleic acid detection kit fda oks cellex's antibody-based test for covid-19 flexible testing options for sars-cov-2 coronavirus mesa biotech covid-19 test receives fda emergency use authorization | genomeweb neumodx secures fda emergency use authorization for coronavirus test qiagen launches qiastat-dx test kit for detection of sars-cov-2 coronavirus in europe following ce marking loopmediated isothermal amplification of dna development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria development of reverse transcription loop-mediated isothermal amplification (rt-lamp) assays targeting sars-cov-2. biorxiv recent advances and perspectives of nucleic acid detection for coronavirus potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review sars-cov-2 diagnostic pipeline development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis pm 5:15. labs scramble to spot hidden coronavirus infections mers-cov diagnosis: an update covid-19 coronavirus rapid test cassette bioeasy novel coronavirus (2019-ncov) test kits antibody testing for covid-19: a report from the national covid scientific advisory panel immunological assays for sars-cov-2: an analysis of available commercial tests to measure antigen and antibodies antibody tests in detecting sars-cov-2 infection: a meta-analysis evaluation of nine commercial sars-cov-2 immunoassays test performance evaluation of sars-cov-2 serological assays immunity and immunopathology to viruses: what decides the outcome? the trinity of covid-19: immunity, inflammation and intervention rapid detection of 2019 novel coronavirus sars-cov-2 using a crispr-based detectr lateral flow assay live cell imaging of single rna molecules with fluorogenic mango ii arrays hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series cr ip t ce pt e d us cr ip t ac ce pt us the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan who-2019-ncov-clinical-2020.4-eng clinical characteristics of coronavirus disease 2019 in china risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease /fulltext?utm_campaign=tlcoronavirus20&utm_source=twitter&utm_medium=social. the lancet clinical management of severe acute respiratory infection ( sari) when covid-19 disease is suspected: interim guidance management of critically ill adults with covid-19 if clinically indicated:" is it? a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) diagnostic utility of clinical laboratory data determinations for patients with the severe covid-19 severe acute respiratory syndrome-related coronavirus -the species and its viruses, a statement of the coronavirus study group clinical characteristics of coronavirus disease 2019 in china correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases relation between chest ct findings and clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter study performance of radiologists in differentiating covid-19 from viral pneumonia on chest ct as deaths mount, china tries to speed up coronavirus testing. the new york times fielddeployable viral diagnostics using crispr-cas13 clinical diagnosis of 8274 samples with 2019-novel coronavirus in wuhan. medrxiv detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr differential diagnosis of illness in patients under investigation for the novel coronavirus (sars-cov-2) hiv-1 did not contribute to the 2019-ncov genome covid-19 in patients with hiv: clinical case series presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 enfermedad por coronavirus (covid-19) and hiv: asuntos y acciones claves novel coronavirus infection and pregnancy pregnancy and perinatal outcomes of women with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical manifestations and outcome of sars-cov-2 infection during pregnancy coronavirus in pregnancy and delivery: rapid review coronavirus disease 2019 (covid-19) and pregnancy: what obstetricians need to know guidelines for pregnant women with suspected sars-cov-2 infection analysis of the pregnancy outcomes in pregnant women with covid-19 in hubei province novel corona virus disease (covid-19) in pregnancy: what clinical recommendations to follow? isuog interim guidance on 2019 novel coronavirus infection during pregnancy and puerperium: information for healthcare professionals pediatric patients with 2019 coronavirus disease in china characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding angiotensin-converting enzyme 2 inhibits lung injury induced by respiratory syncytial virus clinical and ct features in pediatric patients with covid-19 infection: different points from adults are children less susceptible to covid-19? cancer patients in sars-cov-2 infection: a nationwide analysis in china surgical treatment strategy for digestive system malignancies during the outbreak of novel coronavirus pneumonia sars-cov-2 transmission in patients with cancer at a tertiary care hospital in wuhan, china delayed initiation of adjuvant chemotherapy among patients with breast cancer health management of breast cancer patients outside the hospital during the outbreak of 2019 novel coronavirus disease managing oncology services during a major coronavirus outbreak: lessons from the saudi arabia experience stress, depression, the immune system, and cancer clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study care for critically ill patients with covid-19 the role of ultrasound lung artifacts in the diagnosis of respiratory diseases is there a role for lung ultrasound during the covid-19 pandemic? mortality and morbidity in acutely ill adults treated with liberal versus conservative oxygen therapy (iota): a systematic review and meta-analysis the search for optimal oxygen saturation targets in critically ill patients: observational data from large icu databases practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019-ncov) patients the impact of high-flow nasal cannula (hfnc) on coughing distance: implications on its use during the novel coronavirus disease outbreak exhaled air dispersion during high-flow nasal cannula therapy versus cpap via different masks staff safety during emergency airway management for covid-19 in hong kong noninvasive ventilation in critically ill patients with the middle east respiratory syndrome. influenza other respir viruses noninvasive ventilation for patients with hypoxemic acute respiratory failure ventilation with lower tidal volumes versus traditional tidal volumes in adults for acute lung injury and acute respiratory distress syndrome. cochrane database syst rev treatment for severe acute respiratory distress syndrome from covid-19 beyond low tidal volume ventilation: treatment adjuncts for severe respiratory failure in acute respiratory distress syndrome optimal plateau pressure for patients with acute respiratory distress syndrome: a protocol for a systematic review and metaanalysis with meta-regression prone position for acute respiratory distress syndrome. a systematic review and meta-analysis formal guidelines: management of acute respiratory distress syndrome low-dose and short-term application of corticosteroid treatment in patients with severe covid-19 pneumonia: single-center experience from wuhan characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intensive care med conservative fluid management or deresuscitation for patients with sepsis or acute respiratory distress syndrome following the resuscitation phase of critical illness: a systematic review and meta-analysis colloids versus crystalloids for fluid resuscitation in critically ill people effect of reduced exposure to vasopressors on 90-day mortality in older critically ill patients with vasodilatory hypotension: a randomized clinical trial scandinavian ssai clinical practice guideline on choice of inotropic agent for patients with acute circulatory failure vasopressors for hypotensive shock water, sanitation, hygiene and waste management for covid-19 impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand hand-hygiene mitigation strategies against global disease spreading through the air transportation network rational use of face masks in the covid-19 pandemic advice on the use of masks in the community, during home care and in healthcare settings in the context of the novel coronavirus (covid-19) outbreak wearing face masks in the community during the covid-19 pandemic: altruism and solidarity. the lancet. 2020;0 effectiveness of surgical and cotton masks in blocking sars-cov-2: a controlled comparison in 4 patients rational use of face masks in the covid-19 pandemic research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial. the lancet systematic review of the efficacy and safety of antiretroviral drugs against sars, mers or covid-19: initial assessment a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 an exploratory randomized controlled study on the efficacy and safety of lopinavir/ritonavir or arbidol treating adult patients hospitalized with mild/moderate covid-19 (elacoi) efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial a pilot study of hydroxychloroquine in treatment of patients with moderate covid-19 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial observational study of hydroxychloroquine in hospitalized patients with covid-19 association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in new york state the effect of chloroquine, hydroxychloroquine and azithromycin on the corrected qt interval in patients with sars-cov-2 infection clinical trials on drug repositioning for covid-19 treatment the antiviral effect of interferonbeta against sars-coronavirus is not mediated by mxa protein what's new | coronavirus disease covid-19. covid-19 treatment guidelines effectiveness of convalescent plasma therapy in severe covid-19 patients the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory metaanalysis effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma effect of convalescent plasma therapy on viral shedding and survival in covid-19 patients from sars to mers, thrusting coronaviruses into the spotlight recent advances in the vaccine development against middle east respiratory syndrome-coronavirus memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection immunogenicity of candidate mers-cov dna vaccines based on the spike the spike protein of sars-cov -a target for vaccine and therapeutic development receptor-binding domain of sars-cov spike protein induces long-term protective immunity in an animal model yeast-expressed recombinant protein of the receptor-binding domain in sars-cov spike protein with deglycosylated forms as a sars vaccine candidate immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge ade and dengue vaccination safety and immunogenicity of an anti-zika virus dna vaccine -preliminary report mrna vaccines -a new era in vaccinology mrna as a transformative technology for vaccine development to control infectious diseases mrna vaccine delivery using lipid nanoparticles first fda-approved vaccine for the prevention of ebola virus disease, marking a critical milestone in public health preparedness and response could enhanced influenza and pneumococcal vaccination programs help limit the potential damage from sars-cov-2 to fragile health systems of southern hemisphere countries this winter? global influenza seasonality: reconciling patterns across temperate and tropical regions epidemiology of seasonal influenza in the middle east and north africa regions, 2010-2016: circulating influenza a and b viruses and spatial timing of epidemics. influenza other respir viruses influenza seasonality: timing and formulation of vaccines roles of humidity and temperature in shaping influenza seasonality seasonality of influenza and its association with meteorological parameters in two cities of pakistan: a time series analysis environmental predictors of seasonal influenza epidemics across temperate and tropical climates influenza epidemics in the united states global patterns in seasonal activity of influenza a/h3n2, a/h1n1, and b from 1997 to 2005: viral coexistence and latitudinal gradients seasonality of influenza in the tropics: a distinct pattern in northeastern brazil influenza surveillance in pune, india, 1978-90 epidemiological and virological influenza survey in dakar, senegal: 1996-1998 quantifying the role of weather on seasonal influenza influenza seasonality: underlying causes and modeling theories stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation an explanation for the seasonality of acute upper respiratory tract viral infections epidemic influenza and vitamin d 25-dihydroxyvitamin d3 is a potent suppressor of interferon γ-mediated macrophage activation seasonal variation in host susceptibility and cycles of certain infectious diseases influence of extreme weather and meteorological anomalies on outbreaks of influenza a (h1n1) key: cord-293127-c27qh5y7 authors: monteleone, pedro aa; nakano, mayra; lazar, victor; gomes, alecsandra p; de martin, hamilton; bonetti, tatiana cs title: a review of initial data on pregnancy during the covid-19 outbreak: implications for assisted reproductive treatments date: 2020 journal: jbra assist reprod doi: 10.5935/1518-0557.20200030 sha: doc_id: 293127 cord_uid: c27qh5y7 the current outbreak of the novel 2019 coronavirus disease (covid-19) started in china in december 2019 and has since spread to several other countries. on march 25, 2020, a total of 375,498 cases had been confirmed globally with 2,201 cases in brazil, showing the urgency of reacting to this international public health emergency. while in most cases, mild symptoms are observed, in some cases the infection leads to serious pulmonary disease. as a result, the possible consequences of the covid-19 outbreak for pregnant women and its potential effects on the management of assisted reproductive treatments, demand attention. in this review, we summarize the latest research progress related to covid-19 epidemiology and the reported data of pregnant women, and discuss the current evidence of covid-19 infections during pregnancy and its potential consequences for assisted reproductive treatments. reported data suggest that symptoms in pregnant women are similar to those in other people, and that there is no evidence for higher maternal or fetal risks. however, considering the initial data and lack of comprehensive knowledge on the pathogenesis of sars-cov-2 during pregnancy, human reproduction societies have recommended postponing the embryo transfers and do not initiate new treatment cycles. new evidence must be considered carefully in order to adjust these recommendations accordingly at any time and to guide assisted reproductive treatments. the current outbreak of the novel 2019 coronavirus disease (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in china in december 2019 and subsequently spread to many other countries. on january 30 2020, the emergency committee of the world health organization (who) declared a global health emergency, and less than one month later, almost 80,000 cases were confirmed in china and more than 7,000 cases outside of china. numerous countries have reported increasing numbers of confirmed cases and deaths per day, and despite all efforts, spreading of covid-19 currently continues; therefore, on march 11 2020, the who declared covid-19 a pandemic (who, 2020a) . in numerous countries, current disease dynamics resemble those observed in china following the emergence of covid-19. on march 5 2020, an issue of euro surveillance journal reported the first confirmed covid-19 case in europe (spiteri et al., 2020) , according to the who case definition (who, 2020b) . on march 15 2020, covid-19 cases have been detected in all 30 countries of the european union/european economic area and in the united kingdom with a total of 39,768 cases and 1,727 deaths, of which 17,775 cases and 1,441 deaths occurred in italy alone (european centre for disease prevention and control, 2020) . the brazilian health ministry confirmed the first covid-19 case on february 26 2020 in são paulo. the patient was a 65-year-old man who had recently returned from northern italy, where a significant outbreak had occurred. this was also the first confirmed case in south america, a continent with a population of over 640 million people who have previously experienced significant outbreaks of infections such as zika, dengue, and measles. the number of covid-19 cases has been increasing constantly since the first reported case, and while initial cases were associated with travelers arriving from countries with ongoing covid-19 epidemics, since march 13 2020, the stage of 'community transmission' was announced to be reached in são paulo and rio de janeiro. at the time this manuscript was drafted (march 25 2020), 2,201 cases have been confirmed in brazil, and the pandemic continues to increase worldwide, with currently 375,498 confirmed cases and 16,362 deaths in 195 countries (who, 2020c) . sars-cov-2 shows rapid community transmission, and nosocomial infections are common (novel coronavirus pneumonia emergency response epidemiology, 2020). evidence suggests that when the number of initial cases reaches 40, the likelihood of losing control is high, and the number of infections should double within a week, on average, which highlights the urgency of early detection and rapid response measures (hellewell et al., 2020) . china enacted a program of strict social isolation to control further spreading of covid-19, with measures including isolation of cases and limited contact, lock-down of cities, mass quarantine, social distancing mandates, school closures, and intense case identification and contact tracing executed by health care professionals (chen et al., 2020a) . these steps helped control local outbreaks, and chinese authorities reported absence of community transmission cases in china as of march 19 2020. following this strategy, numerous countries also adopted social isolation regulations to contain the pandemic (who, 2020b). however, it is important to emphasize that transmissibility of sars-cov-2 is high and infection growth rates are exponential, ranging from 2.2 to 3.6 . the rapid increase in suspected and confirmed cases of covid-19 suggests that virus transmission may occur by droplet infection and a fecal-oral route. moreover, transmission from people with mild or no symptoms or before symptom onset may reduce the effect of such isolation strategies (khan et al., 2020; niud & xu, 2020; rothe et al., 2020) . coronaviruses are a large family of viruses known to cause symptoms ranging from a common cold to more severe diseases, such as the severe acute respiratory syn a review of initial data on pregnancy during the covid-19 outbreak: implications for assisted reproductive treatments pedro aa monteleone 1,2 , mayra nakano 1,2 , victor lazar 1 , alecsandra p gomes 1 , (drosten et al., 2003; ksiazek et al., 2003) , and mers coronavirus (mers-cov) was the pathogen responsible for severe respiratory disease outbreaks in the middle east in 2012 (zaki et al., 2012) . sars-cov-2 is the seventh identified member of the family of coronaviruses which infect humans, and the main symptoms including fever, cough and fatigue are similar to those following sars-cov and mers-cov infection (liu et al., 2020a) . coronaviruses are large, enveloped, positive-sense single-stranded rna viruses that infect humans and a wide range of animals. sars-cov-2 belongs to the genus beta-coronavirus, and it may originate from a virus of bats, as the genome sequence of sars-cov-2 is to approximately 90% identical with that of a bat coronavirus. in contrast, sars-cov-2 sequences show only about 80% sequence identity with sars-cov and about 50% with mers-cov zhou et al., 2020) . sars-cov-2 has four key structural proteins: the nucleocapsid protein (n), spike protein (s), small membrane protein (sm), and membrane glycoprotein (m). the angiotensin-converting enzyme 2 (ace2), which is expressed on type-i and type-ii alveolar epithelial cells, is the main sars-cov-2 receptor, and infection causes respiratory symptoms and eventually the acute respiratory syndrome. this receptor is also expressed in the gut, albeit at a low abundance, and infection may lead to diarrhea and vomiting, despite it is less frequent. the s protein is required for the virus to fuse with the host cell through the receptor-binding domain. this protein includes two subunits, s1 and s2, and while s1 determines cellular tropism, s2 mediates virus-cell membrane fusion. after membrane fusion, viral rna is released into the cytoplasm, and viral replication is initiated. newly formed viral particle buds then fuse with the plasma membrane through virion-containing vesicles to release the virus sun et al., 2020) . it is noteworthy that sars-cov also uses ace2 as a receptor for cell entry; however, receptor binding ability of sars-cov-2 is 10-to 20-fold higher than that of sars-cov, and the number of infections with sars-cov-2 has exceeded that of sars infections during the outbreak in china in 2002/2003, indicating higher transmission rates. moreover, men typically have higher ace2 levels than women, and asians show higher levels of ace2 expression in alveolar cells than caucasian and african american people, which would suggest asian males to be most susceptible to infection . sars-cov-2 is predominantly transmitted from person to person through droplets and close contact . after contact with a virus-shedding patient, the mean incubation period is about 5 days, ranging from 1 to 14 days . the spectrum of clinical presentations of sars-cov-2 infections has been reported to range from asymptomatic infections to severe respiratory failure. however, most cases experience a similar course of disease as sars and mers patients, with the most common symptoms including fever and cough which frequently leads to lower respiratory tract disease with poor clinical outcomes in elderly patients and in those with preexisting health conditions. confirmation of infection requires nucleic acid testing of respiratory tract samples (e.g., pharyngeal swabs), whereas clinical diagnoses can be made based on symptoms, exposure to infection, and chest imaging (wu & mcgoogan, 2020) . the study describing approximately 45.000 cases of covid-19 in china showed that in most cases (86%), the course of disease was mild (i.e., no or mild pneumonia), and it was severe in 14% (i.e., dyspnea, respiratory frequency ≥ 30/min, blood oxygen saturation ≤ 93%, partial pres-sure of arterial oxygen to fraction of inspired oxygen ratio <300, and/or lung infiltrates >50% within 24-48 hours) and critical in 5% (i.e., respiratory failure, septic shock, and/or multiple organ dysfunction or failure). the overall case fatality rate was 2.3%, but it was 8.0% in patients aged 70-79 years and 14.8% in patients aged 80 years and older; no deaths occurred in patients aged 9 years or younger and among mild and severe cases. however, case fatality rates were higher in patients with preexisting comorbid conditions, varying from 5.6% to 10.5%, depending on comorbidity (wu & mcgoogan, 2020) . sars and mers infections, which also showed widespread transmission, produced fatality rates of 9.6% and 35%, respectively . despite considerably higher case-fatality rates in sars and mers patients, covid-19 seems to be more transmissible and has led to a higher number of deaths due to the large number of cases. in addition, the true number of covid-19 cases can be presumed to be higher than the number of reported cases owing to inherent difficulties in identifying mild and asymptomatic cases in addition to insufficient covid-19 testing capacities in all affected countries (wu & mcgoogan, 2020) . immunosuppression and other physiological changes during pregnancy cause high susceptibility to respiratory pathogens and severe pneumonia in pregnant women (jamieson et al., 2006) which may require hospitalization in intensive care units and ventilatory support (goodnight & soper, 2005) . hormone levels and immune competence show considerable variation throughout pregnancy. early pregnancy seems to be more risk-prone due to adaptive changes in response to fetal antigens, but conditions typically stabilize with gradual adjustment of the mother's immune and endocrine systems, with highest stability in the late stages of pregnancy. early pregnancy is a crucial period of fetal organ development, and the immune system is particularly sensitive at this stage, which likely affects the course of infections (wong et al., 2004) . experience with previous respiratory virus epidemics may offer some insights regarding covid-19 susceptibility and complication rates during pregnancy. swine-origin influenza a (h1n1) virus is an influenza virus type a, which also causes respiratory disease that can develop into an acute respiratory syndrome. during the h1n1 epidemic in 2009, pregnant women were found to be at higher risk of complications as they were four times more likely to be hospitalized than the rest of the population (jamieson et al., 2009) . regarding other coronaviruses, the sars epidemic in 2002/2003 produced 8,442 cases and 916 deaths, and studies have shown that clinical outcomes during this epidemic were worse in pregnant women than in non-pregnant women. in addition, increasing rates of premature births and abortions have been associated with sars-cov infections (schwartz and graham, 2020) . approximately 50% of pregnant women suffering from sars required intensive care, and approximately 33% needed mechanical ventilation. the death rate of pregnant women suffering from sars reached 25% (wong et al., 2004) . from the mers epidemic which produced 2,500 confirmed cases and caused 858 deaths, we can affirm that mers progresses much more quickly to respiratory failure and results in higher mortality rates than sars. however, there was no evidence of vertical transmission of mers or sars. based on this evidence, there is no doubt that sars-cov and mers-cov infections, as even the h1n1, are associated with higher rates of complications in pregnant women (schwartz and graham, 2020) . despite that covid-19 epidemic is ongoing and the data are limited, recent reports indicate that clinical char covid-19 and assisted reproductive treatments -monteleone et al. jbra assist. reprod. | v.24 | nº2 | apr-may-jun/ 2020 acteristics reported in pregnant women with confirmed sars-cov-2 infections are similar to those of non-pregnant women with covid-19 pneumonia, and no evidence of vertical transmission of sars-cov-2 in late pregnancy has been produced so far. nine studies reported covid-19 in pregnant women, with a total of 69 patients; however, some of these patients may have been included in more than one study (table 1 ). the reported cases included five patients in the second trimester of pregnancy, and all other patients were in the third trimester. most women showed mild or moderate symptoms, and three of them required intensive care. preterm birth occurred in 8 out of 61 women who gave birth (chen et al., 2020b; chen et al., 2020c; fan et al., 2020; liu et al., 2020b; liu et al., 2020c; wang et al., 2020a; wen et al., 2020; zhu et al., 2020) . a joint investigation carried out by the who and china evaluated 147 pregnant women in china (64 confirmed and 82 suspected covid-19 cases and an asymptomatic patient), 8% showed severe symptoms, and 1% showed a critical course of disease. it was concluded that pregnant women with covid-19 were not at a higher risk of developing severe symptoms (who, 2020d). most likely, there are many other pregnant women with no or mild symptoms who were not included in these statistics. one case of a neonate infected with sars-cov-2 was confirmed 36 hours after birth; however, it is unclear whether this was due to vertical transmission from mother to child (wang et al., 2020b) . regardless of the small number of reported cases, the combined data suggest that susceptibility to infection and frequencies of severe courses of disease due to sars-cov-2 infection in pregnant women are similar to those in other young adults, and no case of vertical transmission has been reported. moreover, according to the who definition of preterm delivery as birth occurring before 37 weeks of gestation and an estimated rate of preterm births of 10% (who, 2018), preterm birth rates in pregnant women affected by covid-19 seems to follow the general rate. regarding premature births, it should be considered that many pregnant patients who are hospitalized near term presenting symptoms of covid-19, the anticipation of delivery by elective cesarean section can be a medical decision that is influenced by the patient and epidemic pressure and is not necessarily a result of current sars-cov-2 infection. no studies on severe covid-19 and obstetric complications during the first trimester of gestation are available so far; therefore, we lack information on potential effects of infection on pregnancy during the initial stages. regarding other coronaviruses, sars and mers epidemics showed no correlation with frequencies of malformations. moreover, data from the current epidemic should be considered for managing covid-19 infections during pregnancy, as the clinical course of this disease and the response to treatments seem to differ from those of previous outbreaks of other types of coronaviruses (chen et al., 2020d; liang & acharya, 2020) . further research is needed in order to understand pathogenesis and epidemiology of sars-cov-2 during pregnancy, including aspects such as the time of maternal infection, gestational age, effects of comorbidity factors, and frequencies of adverse outcomes; however, preliminary observations of pregnant women infected with sars-cov-2 suggest an optimistic outlook regarding the clinical course. it is important to consider that the covid-19 pandemic elicited psychological stress and anxiety in the general population, including pregnant women. several concerns regarding potential infection during pregnancy have been raised, including (i) presence of family members given quarantine constraints, (ii) potential sars-cov-2 exposure during visits to physicians, (iii) potential requirement of early termination of pregnancy through elective cesarean section; (iv) constant use of sodium hypochlorite and alcohol as disinfectants which may exert toxic effects, and (v) potential postpartum complications, e.g., during breastfeeding or neonatal care (rashidi fakari & simbar, 2020) . in view of the current covid-19 pandemic and uncertainties regarding effects of sars-cov-2 on mothers and fetuses during pregnancy, human reproduction societies published suggestions for managing patients who currently are or will be undergoing infertility treatments through assisted reproductive technologies (art). the international federation for fertility societies (iffs) recommended on march 12 2020 that patients who are considering pregnancy or who are currently undergoing fertility therapies should consult with their personal physician for planning further steps (iffs, 2020). the same day, the american society for reproductive medicine (asrm) published a bulletin suggesting that patients who are highly likely to suffer from covid-19 (i.e., patients who were tested sars-cov-2 positive or who have been exposed to confirmed covid-19 cases within 14 days of onset of their symptoms) should consider freezing oocytes or embryos and avoid embryo transfer until they are symptom-free; however, this recommendation was emphasized to not necessarily apply to suspected covid-19 cases as symptoms of covid-19 closely resemble those of other more common forms of respiratory disease (asrm, 2020a). on march 17 2020, the asrm published a new document named "patient management and clinical recommendations during the coronavirus (covid-19) pandemic" in which the key recommendations were: 1. suspend initiation of new treatment cycles, including ovulation induction, intrauterine inseminations (iuis), in vitro fertilization (ivf) including retrievals and frozen embryo transfers, as well as non-urgent gamete cryopreservation. 2. strongly consider cancellation of all embryo transfers whether fresh or frozen. 3. continue to care for patients who are currently "in-cycle" or who require urgent stimulation and cryopreservation. 4. suspend elective surgeries and non-urgent diagnostic procedures. 5. minimize in-person interactions and increase utilization of telehealth (asrm, 2020b). the european society of human reproduction and embryology (eshre) issued a statement on march 14 2020 detailing that so far, only few cases of covid-19 during pregnancy have been reported, thus the respective data must be interpreted with caution as no information is available regarding potential effects of covid-19 infection during the initial stages of pregnancy; furthermore, medical treatment administered to severe covid-19 cases may include drugs that are contraindicated during pregnancy (eshre, 2020). the same publication advised that all patients considering or planning treatments, independently of confirmation or suspicion of covid-19 infections, should avoid becoming pregnant at this time and consider deferring pregnancy by freezing oocytes or embryos for embryo transfer at a later point (eshre, 2020) . the brazilian society for human reproduction (sbrh), the brazilian society for assisted reproduction (sbra), and the latin american network of assisted reproduction (redlara) also published statements concerning patients undergoing assisted reproductive treatments. the sbrh stressed that there is no cause for panic in pregnant women and urged that the asrm recommendations for women undergoing or planning infertility treatments be followed. however, treatment plans should be individually discussed with physicians as postponing of treatments may, in some cases, reduce chances of success (sbrh, 2020). the sbra and the redlara published a joint note on march 17 2020, firstly suggesting that infertility treatments should continue as planned to avoid reducing prospects of success in infertile women but that the advice of international societies to postpone embryo transfers should be followed (sbra & redlara, 2020) . then, an update launched on march 20 2020 recommended that ongoing cycles should be finalized and new procedures should not be initiated. embryo transfer must be assessed individually with strict controls on the patients and teams involved and the exceptions lies on oncological and others situations in which the postponement may cause loss to the patient, since the decision need to be shared (sbra & redlara, 2020) . it is important to cite the brazilian health ministry published a technical note on covid-19 and pregnancy on march 25 2020. based on available evidences until this moment reporting no difference of clinical course, as well as rates of complications and evolution to severe diseases of pregnant compared to young adults; it was recommended that the covid-19 diagnostic in pregnant women follow the protocol for the general adult population, as the prenatal care for all asymptomatic pregnant women. however, it is highlighted the importance the prevention of agglomerations, best hygiene practices and home screening and isolation of suspected cases of flu syndrome. also, despite the literature shows the unlikely vertical transmission of sars-cov-2, they suggests it is prudent to perform morphological ultrasonography in the second trimester in mothers with sars-cov-2 infection, when available, since the data in infected women in the first trimester of pregnancy are not available (brasil -ministério da saúde, 2020). taken together, we currently face a pandemic with a novel virus, and considerable uncertainty remains regarding sar-cov-2 infections and consequences of infection during pregnancy. hence, most of human reproduction societies are restrictive suggesting to postpone embryo transfer independently of confirmation or suspicion of covid-19 and suspend all cycle's initiations, with rare exceptions. however, whilst every effort must be made to reduce services over coming weeks or maybe months, it is necessary to think forwards towards a resumption of services and a number of questions need to be answered around the management of art: should we cancel all art cycles at this moment? should we keep the treatments and postpone all embryo transfers? should we keep only embryo transfers for couple whose success of treatment can be impaired or those who desire transfer the embryos? based on most recent epidemiologic data on covid-19 and pregnancy, there is no evidence to suggest increased risk for mothers or fetuses. it appears that the course of disease after infection with sars-cov-2 in pregnant women does not differ from that in other young adults. moreover, recent evidence suggests no association of vertical transmission and malformations, and the management of pregnant patients should be individualized based on obstetrical indications and maternal/fetal health status. it is important to consider that the current covid-19 pandemic causes psychological stress and anxiety in pregnant women, which may exert adverse effects. furthermore, it is important to emphasize the recommendations regarding social isolation and quarantine as issued by health authorities in order to avoid further spreading of sars-cov-2. therefore, deciding between initiating/resuming or postponing assisted reproductive treatments depends more strongly on social isolation than on covid-19 and its potential effects during pregnancy, bearing in mind potential emotional effects on patients. however, considering the lack of knowledge regarding sars-cov-2 pathogenesis during pregnancy, the current pandemic requires caution and human reproduction societies generally recommended postponing embryo transfers of current cycles and do not initiate any new cycles, with rare exceptions. nevertheless, we must be alert to new evidence, which can change these recommendations at any time, in order to adjust the management of assisted reproductive treatments. asrm -american society for reproductive medicine. covid-19: suggestions on managing patients who are undergoing infertility therapy or desiring pregnancy. 2020a. available at statements/patient-management-and-clinical-recommendations-during-the-coronavirus-covid-19-pandemic brasil -ministério da saúde. secretaria de atenção primária à saúde a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster early containment strategies and core measures for prevention and control of novel coronavirus pneumonia in china pregnant women with new coronavirus infection: a clinical characteristics and placental pathological analysis of three cases expert consensus for managing pregnant women and neonates born to mothers with suspected or confirmed novel coronavirus (covid-19) infection identification of a novel coronavirus in patients with severe acute respiratory syndrome perinatal transmission of covid-19 associated sars-cov-2: should we worry? the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status feasibility of controlling covid-19 outbreaks by isolation of cases and contacts the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan, china iffs -international federation for fertility societies. up-dates and resources related to the coronavirus pandemic and covid-19. 2020. available at novel influenza a (h1n1) pregnancy working group. h1n1 2009 influenza virus infection during pregnancy in the usa emerging infections and pregnancy the emergence of a novel coronavirus (sars-cov-2), their biology and therapeutic options sars working group. a novel coronavirus associated with severe acute respiratory syndrome early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia novel corona virus disease (covid-19) in pregnancy: what clinical recommendations to follow? overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov pregnancy and perinatal outcomes of women with coronavirus disease (covid-19) pneumonia: a preliminary analysis clinical manifestations and outcome of sars-cov-2 infection during pregnancy genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding deciphering the power of isolation in controlling covid-19 outbreaks novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china coronavirus pandemic and worries during pregnancy; a letter to editor. arch acad emerg med transmission of 2019-ncov infection from an asymptomatic contact in germany redlara -red latinoamericana de reproducion assistida covid-19: acompanhamento de pacientes submetidas às terapias de reprodução assistida ou que desejam engravidar potential maternal and infant outcomes from (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections first cases of coronavirus disease 2019 (covid-19) in the who european region understanding of covid-19 based on current evidence a case of 2019 novel coronavirus in a pregnant woman with preterm delivery a case report of neonatal covid-19 infection in china a patient with sars-cov-2 infection during pregnancy in qingdao who -world health organization. preterm birth coronavirus disease 2019 (covid-19) situation report -41. 2020a. available at technical-guidance/surveillance-and-case-definitions who -world health organization. novel coronavirus (covid-19) situation. 2020c pregnancy and perinatal outcomes of women with severe acute respiratory syndrome characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention. jama. 2020. epub ahead of print isolation of a novel coronavirus from a man with pneumonia in saudi arabia preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia the authors gratefully acknowledge the contributions of the team of monteleone centro de reprodução humana, são paulo, brazil, for technical and emotional support with patients and procedures during the covid-19 pandemic. the authors declare no conflict of interest. key: cord-301633-t8s4s0wo authors: gralinski, lisa e.; menachery, vineet d. title: return of the coronavirus: 2019-ncov date: 2020-01-24 journal: viruses doi: 10.3390/v12020135 sha: doc_id: 301633 cord_uid: t8s4s0wo the emergence of a novel coronavirus (2019-ncov) has awakened the echoes of sars-cov from nearly two decades ago. yet, with technological advances and important lessons gained from previous outbreaks, perhaps the world is better equipped to deal with the most recent emergent group 2b coronavirus. the third zoonotic human coronavirus (cov) of the century emerged in december 2019, with a cluster of patients with connections to huanan south china seafood market in wuhan, hubei province, china. similar to severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) infections, patients exhibited symptoms of viral pneumonia including fever, difficulty breathing, and bilateral lung infiltration in the most severe cases [1] . news reports of patients with an unknown pneumonia were first identified on 31st december with the wuhan municipal health commission saying they were monitoring the situation closely ( figure 1 ). on 1st january 2020, the seafood market was closed and decontaminated while countries with travel links to wuhan went on high alert for potential travelers with unexplained respiratory disease. after extensive speculation about a causative agent, the chinese center for disease control and prevention (cdc) confirmed a report by the wall street journal and announced identification of a novel cov on 9th january [2] . the novel cov (2019-ncov) was isolated from a single patient and subsequently verified in 16 additional patients [3] . while not yet confirmed to induce the viral pneumonia, 2019-ncov was quickly predicted as the likely causative agent. the first sequence of 2019-ncov was posted online one day after its confirmation on behalf of dr. yong-zhen zhang and scientists at fudan university, shanghai [4] . subsequently, five additional 2019-ncov sequences were deposited on the gsaid database on 11th january from institutes across china (chinese cdc, wuhan institute of virology and chinese academy of medical sciences & peking union medical college) and allowed researchers around the world to begin analyzing the new cov [5] . by 17th january, there were 62 confirmed cases in china and importantly, three exported cases of infected travelers who were diagnosed in thailand (2) and japan (1) [6] . the sequences of these exported cases and several additional 2019-ncov isolated in china have also been deposited on the gsaid database [5] . diagnostic tests have subsequently been developed and some are being used on suspect cases identified in other locations including vietnam, singapore, and hong kong [7] . to date there have been twenty-six fatalities associated with 2019-ncov infection, many of these cases had significant co-morbidities and were older in age (>50). a range of disease has been observed highlighted by fever, dry cough, shortness of breath, and leukopenia; patients have included mild cases needing supportive care to severe cases requiring extracorporeal membrane oxygenation; however, compared to sars-cov (10% mortality) and mers-cov (35% mortality), the 2019-ncov appears to be less virulent at this point with the exception of the elderly and those with underlying health conditions. initial monitoring of case close contacts had not revealed any further 2019-ncov cases. however, modeling analysis based on official case numbers and international spread suggested that there may be cases going undetected [8] . on 19th january, these fears were seemingly confirmed as an additional 136 cases were added from further surveys raising the total in wuhan to 198 infected patients [9] . among the 198 total cases in wuhan, 170 remained in hospitals, 126 mostly with mild symptoms, 35 in serious condition, and 9 in critical condition. the expanded numbers and extended range of onset dates (12 december 2019-18 january 2020) suggested likely human to human transmission or ongoing transmission from a market or other primary sources. on 20th january, the outbreak was further expanded to other parts of china (beijing, shanghai, & shenzhen) as well as another exported cases to south korea. as of january 24, the total case number has expanded to at least 870 total cases with 26 deaths across 25 provinces in china and 19 exported cases in 10 countries [10] . public health authorities have quarantined travel from wuhan to limit the spread of the virus and reports indicate other chinese cities have also been isolated [11] . with the heavy travel season for lunar new year underway in asia, major concerns exist for the 2019-ncov outbreak to continue and spread. the source of the 2019-ncov is still unknown, although the initial cases have been associated with the huanan south china seafood market. while many of the early patients worked in or visited the market, none of the exported cases had contact with the market, suggesting either human to human transmission or a more widespread animal source [6] . in addition to seafood, it is reported on social media that snakes, birds and other small mammals including marmots and bats were sold at the huanan south china seafood market. the who reported that environmental samples taken from the marketplace have come back positive for the novel coronavirus, but no specific animal association has been identified [6] . an initial report suggested that snakes might be the possible source based on codon usage [12] , but the assertion has been disputed by others [13] . researchers are currently working to identify the source of 2019-ncov including possible intermediate animal vectors. a zoonotic reservoir harkens back to the emergence of both sars-and mers-cov. sars-cov, the first highly pathogenic human cov, emerged in 2002 with transmission from animals to humans occurring in wet markets. surveillance efforts found sars-cov viral rna in both palm civets and raccoon dogs sold in these wet markets [14] ; however, sars-cov was not found in the wild, suggesting that those species served as intermediary reservoir as the virus adapted to more efficiently infect humans. further surveillance efforts identified highly related covs in bat species [15] . more recent work has demonstrated that several bat covs are capable of infecting human cells without a need for intermediate adaptation [16, 17] . additionally, human serology data shows recognition of bat cov proteins and indicates that low-level zoonotic transmission of sars-like bat coronaviruses occurs outside of recognized outbreaks [18] . mers-cov is also a zoonotic virus with possible origins in bats [19, 20] , although camels are endemically infected and camel contact is frequently reported during primary mers-cov cases [21] . for sars-cov, strict quarantine and the culling of live markets in se asia played a major role in ending the outbreak. with the cultural importance of camels, a similar approach for mers-cov was not an option and periodic outbreaks continue in the middle east. these lessons from sars and mers highlight the importance of rapidly finding the source for 2019-ncov in order to stem the ongoing outbreak. with limited patient data, it is difficult to make robust declarations about populations that may be most susceptible to 2019-ncov. however, disease severity following sars-and mers-cov corresponded strongly to underlying host conditions including age, biological sex, and overall health [22] . early patient reports from 2019-ncov find similar trends. severe illness with 2019-ncov has been associated with elderly patients (>60 years old), including twenty-six lethal cases. these findings correspond to increased severity and death in people over the age of 50 following both sars and mers-cov infection [23, 24] . similarly, the underlying health of the patient likely plays a critical role in overall susceptibility. for the 2019-ncov, limited comorbidity data is available; however, the twenty-six patients that have succumbed to the novel cov had significant health conditions including hypertension, diabetes, heart and/or kidney function issues that may have made them more susceptible. for the mers-cov outbreak, smoking, hypertension, diabetes, cardiovascular disease, and/or other chronic illnesses have been present in the majority of deaths and correspond to findings in animal models [25] . the results indicate vigilance is necessary for these vulnerable patients following 2019-ncov infection. the rapid sequencing of the nearly 30,000 nucleotide 2019-ncov genome by dr. zhang's group at fudan university and several other groups in china illustrate the dedication and increased capacity of the scientific infrastructure in china [4, 5] . for sars-cov, the causative agent was unknown for months and subsequently took over four weeks until a full genome was released [26] . similarly, mers-cov was only identified after several months of testing and a full-length genome available about a month later [27] . in contrast, time from the first date of patient onset (12 december 2019) to the report of several 2019-ncov full-length genomes took less than one month. combined with the immense pressure of an ongoing outbreak with an unknown agent, the effort of these scientists should be considered nothing less than remarkable. building from the sequence, the nucleotide alignment quickly distinguished the novel virus as a group 2b cov, distinct from the sars-cov strains [4, 5] . examining the whole genome, 2019-ncov maintains~80% nucleotide identity to the original sars epidemic viruses. its closest whole genome relatives are two bat sars-like covs (zc45 and zxc21) that shared~89% sequence identity with 2019-ncov; these cov sequences were deposited in early 2018 from zhejiang province in r. sinicus bats in china. comparing across the deposited 2019-ncov strains finds > 99.5% conservation; the lack of diversity suggests a common lineage and source with emergence not likely having occurred that long ago [28, 29] . a recent report has subsequently identified a bat cov sequence, ratg3, with 92% sequence identity with the novel virus which argues for bat origins for the 2019-ncov [30] . we next shifted analysis to the nucleocapsid (n) protein, the most abundant protein produced in covs. generally, the n protein is well conserved across cov families including group 2b [31] . the n protein for 2019-ncov is no exception with~90% amino acid identity to the sars-cov n protein. while less conserved than other group 2b covs like hku3-cov and shc014-cov, 2019-ncov antibodies against the n protein would likely recognize and bind the sars-cov n protein as well. n antibodies do not provide immunity to 2019-ncov infection, but the cross reactivity with sars-cov n protein would allow a serum based assay to determine exposure to the novel cov in asymptomatic cases. while previous studies have found serum reactivity to group 2b virus n proteins in chinese populations [18] , exposure to 2019-ncov should increase the dilution factor substantially if exposure/infection had occurred. importantly, this information may provide insights about susceptibly and potential routes of spread through asymptomatic carriers. examining further, we next compared the spike proteins, the critical glycoprotein responsible for virus binding and entry. overall, the 2019-ncov spike protein has roughly 75% amino acid identity with sars-cov, which is less conserved than other group 2b covs including hku3-cov [31] . however, narrowing analysis to the spike receptor binding domain (rbd) of sars-cov (amino acids 318-518), the 2019-ncov rbd is 73% conserved relative to the epidemic rbd. this conservation level places the 2019-ncov rbd between hku3-4 (62.7% conservation), a bat virus that cannot use human ace2, and rshc014 (80.8%), the most divergent bat cov spike known to use human ace2 for entry [16, 32] . importantly, the key binding residues for sars-cov have been identified [33] ; among these fourteen residues predicted to interact directly with human ace2, the receptor for sars-cov, eight amino acids are conserved in 2019-ncov. notably, several of these residues are also conserved relative to wiv1-and wiv16-cov, two bat strains closely related to sars-cov and known to use human ace2 [17, 34] . initial structural modeling suggest that the 2019-ncov may be able to use human ace2 as a receptor, although its affinity m be reduced relative to the epidemic sars-cov strains [35] . a subsequent report demonstrated that the receptor binding domain of 2019-ncov was capable of binding ace2 in the context of the sars-cov spike protein [36] . in addition, another rapid report links demonstrates 2019-ncov uses ace2 receptors from human, bat, civets, and swine [30] . together, the modeling, pseudotyping, and infection data provide strong evidence for human ace2 being the receptor for 2019-ncov. traditional identification of a microbe as the causative agent of disease requires fulfillment of koch's postulates, modified by rivers for viral diseases [37] . at the present time, the 2019-ncov has been isolated from patients, detected by specific assays in patients, and cultured in host cells (one available sequence is identified as a passage isolate), starting to fulfill these criteria. given the recentness of the 2019-ncov outbreak, at this point there is no animal model available to fulfill the remaining criteria: 1) testing the capability of 2019-ncov to cause respiratory disease in a related species, 2) re-isolating the virus from the experimentally infected animal and 3) detection of a specific immune response. these efforts will surely be an area of intense research in the coming months both in china and in cov research laboratories around the world. notably, generating small animal models of coronavirus disease can be difficult. while sars-cov readily infected laboratory mice, it does not cause significant disease unless the virus is passaged to adapt to the mouse host [38] . infection of primates produces a more mild disease than that observed in humans, although fever and pulmonary inflammation were noted [39, 40] . mers-cov is incapable of infecting rodent cells without engineering changes in critical residues of the receptor protein, dpp4 [41, 42] . however, mers-cov does infect non-human primates [43] . as such, mers mouse models of disease required a great deal of time to develop and are limited in the types of manipulations that can be performed [41] . at this point, the infectious capability of the 2019-ncov for different species and different cell types is unknown. early reports suggest that the virus can utilize human, bat, swine, and civet ace2 [30] ; notably, the group found mouse ace2 was not permissive for 2019-ncov infection dissemination of virus stocks and/or de novo generation of the virus through reverse genetics systems will enable this research allowing for animal testing and subsequent completion of koch's postulates for the new virus. while the huanan seafood market in wuhan has been associated with the majority of cases, many of the recent cases do not have a direct connection [9] . this fact suggests a secondary source of infection, either human to human transmission or possibly infected animals in another market in wuhan. both possibilities represent major concerns and indicate the outbreak has the potential to expand rapidly. for human to human transmission, there was limited data in the initial set of cases; one family cluster is of three men who all work in the market. similarly, a husband and wife are among the patients, with the wife claiming no contact with the market. in these cases, direct human to human infection may have been possible; alternatively, a contaminated fomite from the market may also be responsible as surfaces all around the market were found to test positive 2019-ncov. however, the major increase in the number of cases, the lack of direct connection to the wuhan market for many cases, and the infection of health care works all suggest human to human spread is likely [9, 44] . importantly, until the source of the virus is found, it will be difficult to distinguish zoonotic versus human to human spread. in the early part of the outbreak, the absence of infection in health care workers argued for inefficient human to human spread and distinguished 2019-ncov from both sars-cov and mers-cov. in the two prior cov epidemics, health care settings served as a major transmission point fueling both outbreaks. based on who data, 1 in 10 mers-cov cases have been found to be health care workers; these patients generally have reduced disease and death likely due to younger age and absence of existing health conditions. the recent reports of numerous infected health care workers in wuhan indicate human to human infection can occur with 2019-ncov and may be the product of a super spreading patient [44] . however, while large swaths of healthcare workers are not getting sick as seen with sars and mers-cov, it may be too early to rule out their potential exposure to the novel cov as their disease may be asymptomatic. while not described during the sars-cov outbreak, asymptomatic cases ranged from 12.5% to 25% in some mers-cov studies [45] . a similar phenomenon may be occurring with 2019-ncov and would make stopping the outbreak even more difficult to contain. another parameter to consider is the possibility of super spreading in the context of 2019-ncov. super spreading is the amplified transmission of a virus by individuals in a population and has been suggested by at least one news report [44] . both sars-and mers-cov outbreaks had documented evidence of super spreading patients [46] . in general, both epidemic covs maintain a low r 0 , the rate spread from an individual infected patient. however, roughly 10% of sars-and mers-cov patients have been associated with super spreading and an r 0 > 10. these cases seeded a significant portion of the epidemic around the world. notably, neither mutations in the viruses nor severity of disease were found to be associated with super spreading, implying that host factors contribute to the phenotype [47] . for 2019-ncov, contact tracing to date suggest limited human to human spread and a low r 0 . however, the recent increase in cases, both in and outside wuhan could signal the existence of super-spreading individuals fueling the outbreak. alternatively, super spreading could occur from the zoonotic source which has been seen in other disease outbreaks [10] . in any event, the possibility of super spreading may continue to play a role in this ongoing 2019-ncov outbreak. news of the 2019-ncov came to widespread attention through the internet. over the years, websites like flutrackers.com, promed (promedmail.org), and others have permitted the collection of disease information from around the world and facilitated dissemination to interested parties. in 2012, mers-cov first drew attention as a "novel coronavirus" entioned on promed mail and subsequently through conversation on twitter between science journalists, virologists, and public health experts. eight years later, a more connected network quickly dissected statements from the wuhan municipal health commission and speculated about possible causes. early during an outbreak, it can be difficult to distinguish between rumors with elements of truth versus baseless fear mongering. this fact can be exacerbated by language barriers and off the record sources. however, in this case, speculation of a novel coronavirus was fed by carefully worded statements that specifically excluding some virus families (influenza, adenovirus), but only excluded sars-cov and mers-cov for coronaviruses. coupled with memories of the sars outbreak, many worried that the truth may be held back. when the agent was finally confirmed as a cov, the world acted with both worry and relief: the outbreak would not be hidden. while far from perfect, the government response to 2019-ncov provides a stark contrast to the sars outbreak at the beginning of the century. the rapid release of 2019-ncov sequences permitted the research community to quickly become engaged, providing analysis and developing diagnostic tests. both the chinese cdc and the wuhan municipal health commission have posted regular updates of confirmed case numbers and patient statuses enabling public health authorities to monitor the situation in real time. researchers from around the world have connected on social media to compare updated sequence information and highlight key unknowns about the outbreak. while not always provided in a timely manner, the ability to share news updates and data in real time with researchers and public health officials around the world signals a major change in the response to outbreaks. this connectivity has facilitated awareness as well as new collaborations and a rapid response by the global research community. while there are many unknowns with 2019-ncov, the world is engaged and prepared to battle the newest emergent virus strain. perhaps this means the lessons from the sars outbreak have truly been learned. wuhan municipal health commision. wuhan municipal health and health commission's briefing on the current pneumonia epidemic situation in our city world health organization. who statement regarding cluster of pneumonia cases in wuhan gsaid database. 2020 coronavirus. available online world health organization. novel coronavirus-japan (ex-china) world health organization. laboratory testing for 2019 novel coronavirus (2019-ncov) in suspected human cases estimating the potential total number of novel coronavirus (2019-ncov) cases in wuhan city wuhan municipal commission of health and health on pneumonia of new coronavirus infection super-spreaders in infectious diseases wuhan virus: china locks down huanggang, shuts down railway station in ezhou after wuhan lockdown homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human ncov's relationship to bat coronaviruses and recombination signals no snakes molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence serological evidence of bat sars-related coronavirus infection in humans further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome: emergence of a pathogenic human coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study risk factors for fatal middle east respiratory syndrome coronavirus infections in saudi arabia: analysis of the who line list severe acute respiratory syndrome preliminary phylogenetic analysis of 11 ncov2019 genomes genomic epidemiology of novel coronavirus (ncov) using data generated by fudan university, china cdc, chinese academy of medical sciences, chinese academy of sciences and the thai national institute of health shared via gisaid discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin jumping species-a mechanism for coronavirus persistence and survival synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice the s proteins of human coronavirus nl63 and severe acute respiratory syndrome coronavirus bind overlapping regions of ace2 bat severe acute respiratory syndrome-like coronavirus wiv1 encodes an extra accessory protein, orfx, involved in modulation of the host immune response evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission functional assessment of cell entry and receptor usage for lineage b î²-coronaviruses, including 2019-ncov viruses and koch's postulates a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys newly discovered coronavirus as the primary cause of severe acute respiratory syndrome host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques 15 medical staff in wuhan confirmed new coronavirus pneumonia, and another suspected asymptomatic middle east respiratory syndrome coronavirus (mers-cov) infection: extent and implications for infection control: a systematic review the role of super-spreaders in infectious disease analysis of intrapatient heterogeneity uncovers the microevolution of middle east respiratory syndrome coronavirus key: cord-295633-vkjcheaz authors: hao, xin‐yan; lv, qi; li, feng‐di; xu, yan‐feng; gao, hong title: the characteristics of hdpp4 transgenic mice subjected to aerosol mers coronavirus infection via an animal nose‐only exposure device date: 2019-10-30 journal: animal model exp med doi: 10.1002/ame2.12088 sha: doc_id: 295633 cord_uid: vkjcheaz background: middle east respiratory syndrome coronavirus (mers‐cov), which is not fully understood in regard to certain transmission routes and pathogenesis and lacks specific therapeutics and vaccines, poses a global threat to public health. methods: to simulate the clinical aerosol transmission route, hdpp4 transgenic mice were infected with mers‐cov by an animal nose‐only exposure device and compared with instillation‐inoculated mice. the challenged mice were observed for 14 consecutive days and necropsied on days 3, 5, 7, and 9 to analyze viral load, histopathology, viral antigen distribution, and cytokines in tissues. results: mers‐cov aerosol‐infected mice with an incubation period of 5‐7 days showed weight loss on days 7‐11, obvious lung lesions on day 7, high viral loads in the lungs on days 3‐9 and in the brain on days 7‐9, and 60% survival. mers‐cov instillation‐inoculated mice exhibited clinical signs on day 1, obvious lung lesions on days 3‐5, continuous weight loss, 0% survival by day 5, and high viral loads in the lungs and brain on days 3‐5. viral antigen and high levels of proinflammatory cytokines and chemokines were detected in the aerosol and instillation groups. disease, lung lesion, and viral replication progressions were slower in the mers‐cov aerosol‐infected mice than in the mers‐cov instillation‐inoculated mice. conclusion: hdpp4 transgenic mice were successfully infected with mers‐cov aerosols via an animal nose‐only exposure device, and aerosol‐ and instillation‐infected mice simulated the clinical symptoms of moderate diffuse interstitial pneumonia. however, the transgenic mice exposed to aerosol mers‐cov developed disease and lung pathology progressions that more closely resembled those observed in humans. middle east respiratory syndrome coronavirus (mers-cov), which was first identified in saudi arabia in 2012 and causes acute respiratory illness, multiorgan failure, shock and even death, is an important highly pathogenic coronavirus that is similar to severe acute respiratory syndrome coronavirus (sars-cov) and produces severe infections with a high mortality rate. [1] [2] [3] at the end of may 2019, there were a total of 2428 laboratory-confirmed cases of mers with 838 associated deaths (case-fatality rate: 34.5%, which is higher than the fatality rate of sars) worldwide according to world health organization (who) statistics. 4 mers cases have been reported in 27 countries, including countries in the middle east, africa, europe, asia, and north america as well as australia, and case numbers continue to increase, posing a global threat to public health. in china, the first patient infected with mers-cov from south korea was diagnosed in may 2015, 5 and it will be extremely important to prevent, control, and treat mers-cov infections during any future outbreaks. hence, effective small animal models are needed to investigate viral pathogenesis and evaluate mers-cov therapeutics and vaccines. nonhuman primate animal models of mers-cov in both rhesus macaques and common marmosets were established in previous reports, 6, 7 however, these models are limited by restricted availability, high costs, expert husbandry requirements, and ethical concerns. 8, 9 traditional small animals such as mice, hamsters, and ferrets cannot be infected with mers-cov owing to absence of the necessary dipeptidyl peptidase 4 (dpp4) receptor that interacts with the receptor binding domain of the mers-cov spike protein (s protein) [10] [11] [12] mers-cov fails to replicate in mice, which are readily available, have a defined genetic background and low cost and are frequently used in infectious disease research, due to variations in the dpp4 receptor. previous studies showed that transgenic mice expressing the human dpp4 (hdpp4) receptor could be infected intranasally with mers-cov and developed acute pneumonia. [13] [14] [15] therefore, hdpp4 transgenic mice were selected for exposure to mers-cov-containing aerosols using an animal nose-only exposure device. there are two modes of mers-cov infection, animal-to-human and human-to-human transmission. 16 some reports have found that airborne transmission via the coughing and sneezing of infected dromedary camels or contact with respiratory secretions and consumption of unsterilized milk from infected camels can significantly increase the risk of mers-cov infection in humans. 17, 18 kim et al 19 discovered extensive viable mers-cov contamination in the air and surrounding environment in mers isolation wards. according to the who, it has been suggested that human-to-human transmission, to a very limited extent, is caused by inhalation of droplets or airborne virus and close contact with patients. 20 the above studies have demonstrated that mers-cov has a risk of aerosol transmission. in addition, aerosol inhalation is the main clinical route of infection for viral respiratory illnesses. there are different clinical presentations in animal models established by different infection routes. comparative studies using approaches with different perspectives will contribute to a deeper understanding of mers. in this work, to simulate the aerosol transmission route for comparison with the instillation route, hdpp4 transgenic mice were exposed to mers-cov aerosols by an animal nose-only exposure device. after infection, we analyzed the mouse characteristics of weight loss, survival, viral replication, tissue pathology, viral antigen distribution, and cytokine and chemokine profiles, which provide additional data to investigate the pathogenesis of mers-cov-induced disease and evaluate relevant therapeutics and vaccines. specific pathogen-free transgenic c57bl/6 mice expressing hdpp4 all animals were fed under absl-3 conditions for 3 days before the start of the study. on two consecutive days prior to infection, each mouse was trained in an animal nose-only aerosol device. infected mice were kept in the absl-3 laboratory throughout the study and observed daily to ensure that they had enough water and food. mers-cov (human betacoronavirus 2cemc/2012, complete genome genbank: jx869059.2) was provided by the ilas, cams. the virus was propagated and expanded in vero e6 cells (american type culture collection, usa) cultured and passaged at 37°c and 5% co 2 by routine methods. purified and concentrated progeny viruses were titrated using vero e6 cell-based infectivity assays, and viral titers are expressed in units of 50% tissue culture infectious dose per 100 microliters (tcid 50 /100 μl). mers-cov stocks at a concentration of 10 6.8 tcid 50 /100 μl were stored at −80°c. an animal nose-only aerosol exposure device (in-tox products) was located in an absl-3 laboratory and comprised a nose-only exposure chamber and nebulizer inside a class ⅱ biological safety cabinet (bsc ⅱ), a control box, mouse restraint tubes, a clean compressed air tank and a vacuum pump ( figure 1 ). the exposure device, which exposed only the mouse nose, generated mers-cov aerosol particles of 1.27 ± 0.61 μm to infect transgenic mice expressing hdpp4 and simulated the natural route of infection. 21 as shown in table 1 , transgenic mice were randomly assigned to an aerosol group, an instillation group, an aerosol control group, and an instillation control group, and the body weight of each mouse was measured on the day of infection (day 0). each group contained 17 mice; five mice in each group were used to analyze clinical symptoms, weight loss and survival, and three mice in each group were randomly chosen for necropsy on days 3, 5, 7, and 9 postinfection. mers-cov virus suspensions (10 6.5 tcid 50 ) and serum-free dulbecco's modified eagle medium (dmem) were separately added to the nebulizer reservoir to infect exposed mice in the aerosol and control aerosol groups, respectively, for 30 minutes. according to the instructions of the exposure device and mouse respiratory rate (25 ml/min per mouse), the nebulizer flow rate was set to 0.24 l/ min, the diluter flow rate was set to 6.8 l/min, and the nebulizer pressure was set to 20 psi. mice were anesthetized with 1.2% tribromoethanol (0.2 ml/10 g of body weight, intramuscular (im)) for intranasal inoculation with 10 6.5 tcid 50 of mers-cov in the instillation group and serum-free dmem in the instillation control group. infected mice were observed for 14 consecutive days to analyze the clinical symptoms of disease, weight change, and survival. the mice were euthanized with 1.2% tribromoethanol (0.2 ml/10 g of body weight, im) when they reached 25% weight loss. aerosol control dmem aerosol to analyze clinical signs, weight loss, and survival 5 a instillation control dmem suspension to analyze clinical signs, weight loss, and survival on days 3, 5, 7, and 9 postinfection, three animals randomly selected from each group underwent necropsy to obtain tissue specimens for assessing viral distribution, associated histopathology, and cytokine levels using quantitative reverse transcription-pcr (qrt-pcr), hematoxylin and eosin (h&e) staining, immunohistochemistry (ihc), and enzyme-linked immunosorbent assay (elisa). total viral rna was extracted from tissues (lungs, brain, kidneys, spleen, liver, heart, and intestine) homogenized using the rneasy control. 23 a standard curve was generated for pcr using 10-10 7 copies of a qualified standard plasmid to calculate copy numbers for each reaction. formalin-fixed lung, brain, and kidney samples were embedded in paraffin wax and sectioned at an approximately 5-μm thickness. deparaffinized and hydrated tissue sections were routinely stained with h&e to examine histopathological changes. immunohistochemical staining was performed to assess the expression of a viral antigen using a rabbit two-step detection kit (zhongshan golden bridge biotechnology co., ltd) with a rabbit polyclonal anti-mers-cov nucleoprotein (np) antibody (sino biological inc). visualization was then performed by dab staining and hematoxylin counterstaining. supernatants of tissue homogenates from infected mice (50 µl) were added to the bottom of an antibody-coated plate. the levels of interleukin (il)-1β, il-6, il-8, il-10, tumor necrosis factor (tnf)-α, interferon (ifn)-γ, and ifn-β were assayed using elisa kits (kete biotechnology co., ltd). chemokine and cytokine concentrations were recorded as pg/ml of homogenate or ng/l of homogenate. data were analyzed using spss 21 or graphpad prism 5.0 software. the experimental results are presented as the mean plus standard deviation (sd). one-way anova was used to assess differences in body weight, viral load, and cytokine levels among different groups. student's t test was performed for two-group comparisons. p < .05 was considered statistically significant. the infected mice in both the aerosol and instillation groups displayed significant clinical symptoms, such as huddling, hunching, ruffled fur, weight loss, and death. there were significant differences in weight change (p < .001) and survival (p < .0001) between the mers-cov infection groups and the control groups. the incubation period, however, was 5-7 days after aerosol infection and 1 day after instillation inoculation. after mers-cov aerosol exposure, hdpp4 transgenic mice showed profound clinical signs on days 5-7, rapid weight loss on days 7-9 and 60% survival by day 11 (acute death or euthanasia at 25% weight loss). the intranasally infected transgenic mice displayed rapid weight loss on days 1-5 and 0% survival by day 5 (acute death or euthanasia at 25% weight loss). there were significant differences in disease progression (p < .01) after challenge between the aerosol group and the instillation group. transgenic hdpp4 mice infected with mers-cov aerosols exhibited milder disease and slower disease progression than did those inoculated intranasally (figure 2a,b) . no obvious abnormalities, including weight loss or signs of clinical illness, were detected in the aerosol control and instillation control groups. there were no significant differences in weight change or survival rates between mice inoculated with dmem in the above two control groups (p > .05; figure 2c ). based on qrt-pcr analyses of tissue rna contents, we identified high viral loads in the lungs and brain in mice and a small amount of viral rna in other tissues after mers-cov infection via the aerosol or instillation route ( figure 3a ,b). however, there were significant differences in the tissue viral loads of infected mice between the two groups (p < .0001). after mers-cov aerosol infection, high viral loads were detected in the lungs at 3-9 days and in the brain at 7-9 days. viral loads were high in the lungs and brain of intranasally infected mice at days 3 and 5. there were significant differences (p < .0001) in the viral loads in the lungs and brain between the two groups at days 3 and 5. the viral loads in the lungs and brains of the mice in the aerosol group were significantly lower than those of the mice in the instillation group. high levels of viral rna accumulated more slowly in the tissues of the mers-cov aerosol-exposed mice than in those of the mice infected intranasally ( figure 3 ). as shown in figure the data are presented as the mean change ± sd for each group (n = 5). mice in the instillation group died acutely or were euthanized when they reached 25% weight loss; these mice had a 0% survival rate by day 5, which produced no results for weight loss on days 7 and 9. a key indicating the color coding for the groups is provided in the figure. *p < .05, **p < .01, ***p < .001, and ****p < .0001 mice in the instillation group died acutely or were euthanized when they researched 25% weight loss; these mice had a 0% survival rate by day 5, so no tissue lesion results were available on days 7 and 9 mice infected with mers-cov via the aerosol inhalation or intranasal instillation route, but no obvious lesions were found in other tissues. there were no abnormalities in the tissues of the normal control group. it was clear that the appearance of the lungs exhibited obvious congestion and dark brown regions on days 7-9 in the aerosol group. the mers-cov-intranasal mice showed gross lung lesions on day 3 and more severe lung lesions on day 5. gross lung lesions developed more slowly and were milder in the aerosol group than in the instillation group ( figure 4a ). microscopically, challenged mice developed moderate acute interstitial pneumonia and brain pathology, but no pathological changes were detected in other tissues in the mice. in the aerosol group, the lungs of the exposed mice showed alveolar septal widening, inflammatory cell infiltration, and vessel dilatation and congestion at 3-9 days, gradual development of severe pathological changes and inflammatory cell infiltration in perivascular regions at 5-9 days, focal hemorrhages at 7-9 days, and an expanded pathology range at day 9 ( figure 4b ). dilatation and congestion of the cerebral vessels were not clearly observed until day 7, and few areas of neuron deformation necrosis were found in the cerebral cortex, hippocampus, and thalamus before day 9 ( figure 3c ). on days 3 and 5 after intranasal infection, we found moderate acute interstitial pneumonia and brain lesions ( figure 4b ,c). tissue lesions, however, were milder in the aerosol group than in the instillation group. furthermore, there were significant differences in the progression of lung and brain lesions in the two infected groups. tissue lesion progression was slower in the aerosol-infected mice than in the instillation-infected mice ( table 2 ). the expression of a mers-cov antigen was primarily evaluated using ihc assays and was found in endothelial cells and alveolar pneumocytes in the lungs and in cerebral cortical neurons, dendrites, axons, microglia and the hippocampus in the brains of aerosol-and instillation-challenged mice but not in control mice ( figure 5a,b) . prominent mers-cov expression was also observed in renal tubular epithelial cells ( figure 5c ). however, there were significant differences in the timing of virus expression in the tissues of the mice postinfection. after mers-cov infection, ihc assays with a rabbit polyclonal anti-mers-cov np antibody found that viral antigens predominantly appeared in tracheal endothelial cells at day 3 postinfection in the lungs of the aerosol-infected mice and in both tracheal endothelial cells and pneumocytes in the lungs of the aerosol-infected mice at 5-9 days; these changes were observed in the lungs of the instillation-infected mice at 3 and 5 days, respectively. in addition, the mers-cov antigen was discovered in the brain and kidneys in the aerosol group at 5-7 days and in the instillation group at 3 and 5 days. based on these results, we concluded that the distribution of the mers-cov antigen in the lungs, brain and kidneys after infection was slower in the aerosol group than in the instillation group. there were significant differences in the level of related proinflammatory cytokine and chemokine profiles, including il-1β, il-6, il-8, il-10, tnf-α, and ifn-γ, between infectious groups (the aerosol group and instillation group) and the control group (p < .05). significantly elevated levels of il-1β, il-6, il-8, il-10, tnf-α, and ifn-γ were discovered in the lungs and brains of mice in the aerosol group with increased cxcl-1 at 3-9 days (p < .05) postchallenge and in those of mice the instillation group at 3 and 5 days postchallenge ( figure 6 ). in the aerosol group, the exposed mice showed peak il-10 and concentration in the lungs and il-10 and cxcl-1 concentrations in the brain at 5-9 days, and peak tnf-α and ifn-γ levels in the lungs and brains with peak il-6 level at a -, no apparent changes; +, mild alveolar septum widening; ++, moderate alveolar septum widening; and +++, severe alveolar septum widening. b -, no apparent changes; +, infiltration of a few interstitial inflammatory cells; and ++, some interstitial inflammatory cell infiltration. c -, no apparent changes; and +, a small amount of exudate in alveoli. d -, no apparent changes; +, mild dilatation and congestion of vessels; and ++, moderate dilatation and congestion of vessels. e -, no apparent changes; and +, mild hemorrhage. f nd, not done. mice in the instillation group died acutely or were euthanized when they reached 25% weight loss, which occurred by day 5. f i g u r e 5 immunohistochemical staining of mouse tissue samples after infection. a, immunohistochemical staining of the lungs of infected mice. b, immunohistochemical staining of the brains of infected mice. c, immunohistochemical staining of the kidneys of infected mice. mice in the instillation group died acutely or were euthanized when they researched 25% weight loss; these mice had a 0% survival rate by day 5, so no tissue lesion results were available on days 7 and 9 7-9 days. after intranasal infection, however, the levels of il-1β, il-6, il-10, tnf-α, and ifn-γ in the lungs and il-6, il-8, il-10, and ifn-γ in the brains peaked at 3-5 days. the secretion of some cytokines and chemokines in the aerosol group was slower than that in the intranasal group (p < .05). the mice in the instillation group died acutely or were euthanized when they researched 25% weight loss; these mice had a 0% survival rate by day 5, so no results were available on days 7 and 9. the results represent the mean ± sd for each group (n = 3). *p < .05, **p < .01, ***p < .001, and ****p < .0001 distribution in tissues between the aerosol-and instillation-challenged mice (table 3) . after mers-cov infection, the disease progression in the mice in the aerosol group was slower than that in the mice in the instillation group. sanders et al showed that virus droplets were deposited and concentrated in the lungs through the respiratory tract of mice inoculated intranasally, resulting in fast disease onset. 29, 30 correspondingly, after instillation infection with mers-cov, we found that mice with a short airway and high concentration of virus deposited in the lungs displayed weight loss at day 1 and lung lesions at day 3, consistent with intranasal mouse models established by adam, agrawal and li et al [31] [32] [33] ; these mice also exhibited 0% survival by day 5. previous studies reported that aerosol particles ≤5 μm penetrated the respiratory tract to reach the alveoli and were diffusely distributed in the lungs. 30, 34 in addition, virus aerosols entered the blood circulation through the alveoli, and other viruses slowly replicated in the lungs after mice inhaled mers-cov-containing aerosols (particle size: 1.27 ± 0.61 μm). compared with instillation-inoculated mice with virus deposition in the lungs, aerosol-exposed mice displayed slower disease progression with an incubation period of 5-7 days, lung lesions on day 7, continuous weight loss on days 7-11, milder clinical signs, and 60% survival on day 11. we found that the progressions of virus replication and lung lesions in challenged mice were slower in the aerosol group than in the instillation group. based on high viral loads in the lungs and brain of challenged mice, which was consistent with previous reports, 35 and acute renal failure in mers patients, we carried out h&e staining to assess histopathological changes and immunohistochemical staining with a specific antibody to further characterize mers-cov expression in the lungs, brain, and kidneys. a relatively high viral load in the lower respiratory tract is associated with severe illness in viral respiratory diseases. 36, 37 at 3-5 days postinfection, mice in the intranasal group, which had high viral loads in the lungs and brain at 3-5 days, exhibited acute interstitial pneumonia and pathological brain changes. in the aerosol group, mice developed acute interstitial pneumonia at 3-9 days and pathological brain changes at 7-9 days, which were caused by high levels of virus rna in the lungs at 3-9 days and in the brain at 7-9 days, respectively. higher virus rna levels in the instillation group might contribute to the more severe high level on days 3 and 5 high level on days 7 to 9 high level on days 3 and 5 histopathology lungs moderate acute interstitial pneumonia on days 3 to 9 moderate acute interstitial pneumonia on days 3 to 5 brain relatively mild brain lesion on days 7 and 9 brain lesions on days 3 and 5 lungs in bronchial endothelial cells on day 3 in both tracheal endothelial cells and alveolar pneumocytes in the lungs on days 5 to 9 in both tracheal endothelial cells and alveolar pneumocytes in the lungs on days 3 and 5 in cerebral cortical neurons, dendrites, axons, glial cells, and the hippocampus on days 5 to 9 in cerebral cortical neurons, dendrites, axons, glial cells, and the hippocampus on days 3 and 5 in renal tubular epithelial cells on days 5 to 9 in renal tubular epithelial cells on days 5 to 9 cytokines and chemokines b high levels on days 3 to 9, including cxcl-1 high levels on days 3 to 5 middle east respiratory syndrome patients exhibit a median incubation period of 5-7 days, with a range of 2-14 days. 41 we discovered that instillation-inoculated mice exhibited clinical signs within 1 day but that the incubation period of aerosol-exposed mice was 5-7 days, which more closely resembled the period observed in humans. mers-cov binds to hdpp4 receptors that are primarily expressed in the lower respiratory tract and alveoli, resulting in a wide range of disease symptoms in patients, from no symptoms to mild respiratory illness or severe acute pneumonia, which rapidly progresses to acute lung damage, multiorgan failure and even death. 42, 43 clinically, chest radiography and chest computed tomography (ct) show no lung lesions in patients in the early stages of illness, but pneumonia is identified during the course of the disease and includes patchy densities, extensive diffuse and focal alveolar space opacities, interstitial infiltrates, and consolidation. 44 pulmonary pathological changes in aerosol-infected mice were similar to those noted in patients with respiratory tract infection. additionally, immunohistochemical staining revealed that a mers-cov antigen was expressed in alveolar pneumocytes and endothelial cells, the brain, and the kidneys in challenged transgenic mice. studies of a fatal case of mers-cov infection evidenced that the expression of a mers-cov antigen was predominantly localized in pneumocytes and endothelial cells, resulting in cell necrosis and pneumocyte damage; however, no viral antigens were detected in other tissues in the fatal case. 47 as demonstrated in previous studies, we also discovered high viral loads, pathological changes and the expression of a mers-cov antigen in the brain of challenged mice; and no brain lesions, but multiorgan damage, were observed in mers patients. 35, 48, 49 zhou et al demonstrated that human dendritic cells and macrophages were permissive to mers-cov replication, indicating that the multiorgan injury induced by mers-cov may be associated with the distribution of the hdpp4 receptor in many cell types that are spread throughout multiple organs. 38 some studies have indicated that mers-cov has cell and tissue tropisms, especially tropisms for pneumocytes and neurons, and synapses may be one of the structures by which viruses diffuse through the brain after mers-cov infection. 31, 35 the mechanisms underlying the brain lesions and death induced by mers-cov infection in hdpp4 transgenic mice remain complex and complicated and need to be further investigated. hdpp4 transgenic mice were successfully infected with mers-cov aerosols by an animal nose-only exposure device, and aerosol-and instillation-infected mice all simulated the clinical symptoms of moderate diffuse interstitial pneumonia. compared to instillation-infected mice, aerosol-infected mice more closely resembled infected humans in terms of the progression of disease and pathology in the lungs, which provided additional data for studying pathogenesis and evaluating the efficacy of preventive and therapeutic agents for mers-cov. the current work was supported by the national science and technology major projects of infectious disease (grant number 2018zx10734401-011). none. hg was the principal investigator, designed and supervised the study, and wrote the grant application. xyh performed the main experiments. xyh and ql performed the cell experiments. xyh and fdl conducted the animal experiments. yfx completed the pathology experiments. xyh and hg conceived the experiments, analyzed the data and wrote the paper. all authors read and approved the final manuscript. xin-yan hao https://orcid.org/0000-0001-7664-9568 middle east respiratory syndrome: emergence of a pathogenic human coronavirus nurses' experiences of care for patients with middle east respiratory syndrome-coronavirus in south korea middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease middle east respiratory syndrome coronavirus (mers-cov). mers monthly summary discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus recent aspects on the pathogenesis mechanism, animal models and novel therapeutic interventions for middle east respiratory syndrome coronavirus infections searching for animal models and potential target species for emerging pathogens: experience gained from middle east respiratory syndrome (mers) coronavirus. one health host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc a rapid scoping review of middle east respiratory syndrome coronavirus in animal hosts clinical and biological character in mouse models for middle east respiratory syndrome generated by transduction with different doses of dpp4 molecule elevated human dipeptidyl peptidase 4 expression reduces the susceptibility of hdpp4 transgenic mice to middle east respiratory syndrome coronavirus infection and disease acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with middle east respiratory syndrome coronavirus emergencies preparedness, response. frequently asked questions on middle east respiratory syndrome coronavirus (mers-cov) modeling the spread of middle east respiratory syndrome coronavirus in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards infection prevention and control during health care for probable or confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection. world health organization inhalation exposure systems: design, methods and operation influenza a virus challenge models in cynomolgus macaques using the authentic inhaled aerosol and intra-nasal routes of infection laboratory testing for middle east respiratory syndrome coronavirus. interim guidance replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques a spike-modified middle east respiratory syndrome coronavirus (mers-cov) infectious clone elicits mild respiratory disease in infected rhesus macaques a comparative review of animal models of middle east respiratory syndrome coronavirus infection what have we learned about middle east respiratory syndrome coronavirus emergence in humans? a systematic literature review. vector borne zoonotic dis intranasal influenza infection of mice and methods to evaluate progression and outcome comparison of traditional intranasal and aerosol inhalation inoculation of mice with influenza a viruses a mouse model for mers coronavirus-induced acute respiratory distress syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease mouseadapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice distribution of aerosols in mouse lobes by fluorescent imaging multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus an acute immune response to middle east respiratory syndrome coronavirus replication contributes to viral pathogenicity association of higher mers-cov virus load with severe disease and death, saudi arabia active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis sars and other coronaviruses as causes of pneumonia delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment association between severity of mers-cov infection and incubation period clinical implication of radiographic scores in acute middle east respiratory syndrome coronavirus pneumonia: report from a single tertiary-referral center of south korea mortality rate of icu patients with the middle east respiratory syndrome -coronavirus infection at king fahad hospital middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov key: cord-308340-p2iqzyv4 authors: devitt, elizabeth title: lack of small animal model hinders mers coronavirus research date: 2013-08-06 journal: nat med doi: 10.1038/nm0813-952 sha: doc_id: 308340 cord_uid: p2iqzyv4 nan when reports of a new coronavirus trickled out of saudi arabia last year, scientists leveraged a decade of experience studying sars (severe acquired respiratory syndrome) to quickly find ways to stop infections from the deadly pathogen. but research efforts are stalled because of one key difference between the two coronaviruses: unlike sars, which readily infects a menagerie of animals, the virus responsible for so-called 'middle east respiratory syndrome' , or mers, doesn't seem to cause disease in small lab animals. fortunately for mers researchers laboring to develop an animal model before the viral outbreak gets out of hand, time seems to be on their side. the mers coronavirus doesn't seem to be spreading between people fast enough to warrant fears of a pandemic, according to an analysis published 5 july in the lancet 1 . still, with more than half of the 80-plus people known to be afflicted with mers dying at the hands of the virus, public health officials remain on high alert. last month, the world health organization (who) established new clinical guidelines for reporting cases of mers and appointed a committee of 15 experts to review what scientists know about the virus and establish a plan in case a pandemic begins. the committee convened its first two meetings on 9 and 17 july and plans to meet again in september, or sooner if needed. "epidemiology is crucial now," says bart haagmans, a virologist at the erasmus medical center in rotterdam, the netherlands, who is not part of the who panel. "if you can pinpoint where the virus is coming from, you can really get a grip on the problem." as the epidemiological data trickle in, some scientists are searching for the virusand clues about its character-in nonhuman animals. by sequencing the virus's genome, haagmans and his colleagues showed last year that the mers pathogen is closely related to coronaviruses carried by two bat species 2 . bats are natural reservoirs of many deadly viruses, including sars-like coronaviruses. however, researchers don't yet have direct evidence for a bat source of transmission for mers. in april, an international team of virus hunters traveled to saudi arabia, where more than 80% of the human infections to date have occurred, to take blood samples from bats, as well as camels, sheep and goats. their plan is to look for the virus or antibodies that would indicate previous exposure to the virus in these species. at press time, an analysis of those sampleswhich is being conducted in ian lipkin's laboratory at the center for infection and immunity in the mailman school of public health at columbia university in new york-was still pending. in the meantime, other researchers are working to establish laboratory models with which to study the how the virus takes its toll on the body and to test the efficacy of potential therapeutics. earlier this year, a team led by haagmans identified dipeptidyl peptidase 4 (dpp4; also known as cd26) as the surface receptor employed by the mers coronavirus to gain entry into host cells, including human ones 3 . using crystallization techniques, george gao and his colleagues at the chinese academy of sciences in beijing on 7 july described the molecular basis of the binding between the virus and dpp4 (ref. 4) . and also last month, a group led by shibo jiang published a study demonstrating the immunogenic potency of a 286-amino acid stretch on the mers coronavirus's binding domain 5 -a first step toward an eventual mers vaccine. "our previous work with sars helped us find a critical target quickly," says jiang, head of the laboratory of viral immunology at the new york blood center' . "the next step is to optimize the immune response." but evaluating such a vaccine will require an animal model. and even though ddp4 has been identified in the lung cells of many rodents, including syrian hamsters, researchers at the laboratory of virology of the us national institute of allergy and infectious diseases (niaid) in hamilton, montana, could not get the mers coronavirus to replicate in this common infection model 6 . the virus successfully causes illness in rhesus macaques, though. the same niaid team, led by heinz feldmann, showed in april that pneumonia-like symptoms develop in macaques infected with mers within 24 hours of infection, resulting in a respiratory disease similar to, but less severe than, that found in people with mers 7 . the macaque system is not as practical and widely applicable as a smallanimal model, and not that many research groups are working with it. but the niaid team, for one, is now moving ahead with drug tests in the monkeys. specifically, the researchers are evaluating whether two antiviral compounds-alpha-interferon and ribavirin, both of which are used to treat hepatitis and other infections-can clear mers coronavirus infections. both drugs inhibit viral replication in monkey cell lines with synergistic effects when administered together, feldmann and his colleagues have shown 8 . whether or not drugs like those are ultimately needed in response to a pandemic, niaid director anthony fauci stresses the importance of being prepared. "we can't take the attitude that we escaped a bullet with sars," he told nature medicine. "the mers outbreak has rekindled the importance of sticking with our study of coronaviruses." elizabeth devitt science source breathe uneasy: the mers virus key: cord-285039-9piio754 authors: zhou, haixia; zhang, shuyuan; wang, xinquan title: crystallization and structural determination of the receptor-binding domain of mers-cov spike glycoprotein date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_4 sha: doc_id: 285039 cord_uid: 9piio754 three-dimensional structures of the receptor-binding domain (rbd) of mers-cov spike glycoprotein bound to cellular receptor and monoclonal antibodies (mabs) have been determined by x-ray crystallography, providing structural information about receptor recognition and neutralizing mechanisms of mabs at the atomic level. in this chapter, we describe the purification, crystallization, and structure determination of the mers-cov rbd. the first three-dimensional structure of the mers-cov spike glycoprotein receptor-binding domain (rbd), providing the molecular basis of viral attachment to host cells, was determined in the complex with it cellular receptor dipeptidyl peptidase 4 (dpp4, also called cd26) by x-ray crystallography [1] . because of the significance in receptor recognition and specific pathogenesis, rbd became a hot spot in the study of mers-cov. a number of structures of rbd bound by monoclonal antibodies (mabs) have also been determined and deposited in the protein data bank (pdb, http://www.rcsb.org/pdb/) [2] [3] [4] [5] [6] [7] [8] . our group determined the rbd structures in complex with dpp4 and the mabs mers-27, mers-4 and mers-gd27, respectively [2] [3] [4] 9] . all the three-dimensional structures of mers-cov rbd have been determined by x-ray crystallography, which is a powerful method for determining molecular structures at atomic resolution. briefly, the ordered and repeated atoms in a single protein crystal can diffract the incident x-ray beam into many specific directions. the angles and intensities of these diffracted x-rays can be collected and measured in an x-ray diffraction experiment. after obtaining the phases of these diffracted x-rays by heavy-atom derivative, anomalous scattering or molecular replacement methods, a protein crystallographer then calculates the density of electrons with the protein crystal and builds a structural model based on the density map. for details on the principles and methodology of protein crystallography, please refer to the range of other excellent textbooks. in this chapter, an overview of the standard method of protein crystallography is briefly introduced, focusing on crystallization and structural determination of mers-cov rbd using the molecular replacement method. prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a resistivity of 18 mω cm at 25 c) and analytical grade reagents. when dealing with waste, we strictly follow all waste disposal regulations. 1. pfastbac vector containing the mers-cov rbd gene. 3. lb liquid medium: 10 g tryptone, 5 g yeast extract, 10 g nacl, and 1 l ultrapure water; sterilize by high-pressure steam. 4. liquid lb selection medium: lb liquid medium, 50 μg/ml kanamycin, 7 μg/ml gentamicin, and 10 μg/ml tetracycline. 5. bacmid selection lb agar plate:10 g tryptone, 5 g yeast extract, 10 g agar powder, 10 g nacl, and 1 l ultrapure water. sterilization at high-pressure steam. 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml x-gal, 40 μg/ ml iptg mix and pout into sterile plates (see note 1). mer-cov rbd can be expressed using the bac-to-bac baculovirus expression system (fig. 1 ), collected and captured using nta sepharose (ge healthcare) and then further purified by gel filtration chromatography using a superdex 200 high performance column (ge healthcare). crystallization trials are set up using the hanging-drop or sitting-drop vapor diffusion method in conjunction with the sparse-matrix crystal screening kits. the structure of mers-cov rbd in complex with mers-4scfv was determined using the molecular replacement method. when the total volume remaining is reduced to 50 ml, add 100 ml hbs buffer to collect all the liquid in the system in a beaker. then dispense into high-speed centrifuge tubes, centrifuge at 3000 â g for 1 h at 4 c. 4. the supernatant after centrifugation is loaded onto the nickel-nta beads equilibrated with 30 ml of hbs buffer. mers-cov rbd with a his tag could be captured by nickel-nta beads. repeat loading the sample once more. 5. add wash buffer to the beads to remove the nonspecifically bound proteins until the flow-through is not able to discolor the coomassie brilliant blue g250 solution. 6. after adding elution buffer, the target protein will dissociate from the beads; collect it in a 10 kda millipore concentrating tube. similarly, detect protein with coomassie brilliant blue g250. the concentrating tube containing the protein sample is centrifuged at 3000 â g to concentrate the sample to less than 7. mers-covrbd is further purified by gel filtration chromatography. the sample is loaded onto the superdex 200 column pre-equilibrated with hbs buffer. fractions containing rbd are collected and the protein's purity is confirmed by sds-page (fig. 2) . 8. dilute the protein to 1 mg/ml, and digest with endoglycosidase f1 and f3 (f1/f3: rbd at the ratio of 1:100) at 18 c overnight. the digested protein is concentrated and purified by gel filtration chromatography same as above (optional). 9. after preparing the mers-cov rbd protein and mers-4scfv protein (see note 11) detect the absorption of the protein sample at 280 nm (a280). according to the molecular weight and extinction coefficient, the molar concentration can be calculated. the two proteins were mixed at molar ratio of 1:1, incubated on ice for 1 h, and purified using a superdex 200 column. collect the fractions containing the complex and confirm the protein purity by sds-page. sartorius centrifugal concentrator to concentrate to 10-15 mg/ml. after mixing and aspirating, centrifuge at 10,000 â g for 10 min at 4 c. 3. use ttp labtech's mosquito crystallization setup for automated crystallography. absorb 3 μl protein on the 8-well 5 μl micro-reservoir strip (fig. 3a) . then the needles aspirate the protein from the strip onto the swissci plate with 200 nl of protein per well (fig. 3b) , using the sitting-drop vapor diffusion method by mixing 200 nl reservoir and 200 nl reservoir (fig. 3c,d) . 4. seal the plate with tape and gently place it in an 18 c room. 5. check the sample drops under a microscope at 20-100â magnifications after 3 and 7 days (and if necessary, after 1 and 2 weeks, and 1, 3, and 6 months). 6 . a week later, we found crystal growth in the peg/ion, pegrx and jcsg+ kits. specific conditions were as follows (fig. 4a,b) 2. the diffraction images should be collected on the bl17u beamline (fig. 4c) . rotate the mounted crystal and the x-ray diffraction patterns should be recorded at 1 per image, and collected for 360 . 3. the diffraction images in a dataset should be processed with hkl2000 [10] including auto-index, refinement, integration, and scaling steps. after data processing, the crystal unit cell parameters, crystal space group, miller indexes of reflections, intensities, and error estimates of reflections should be determined and stored in a * .sca file, which provides the dataset applicable to structure determination. 4. using ccp4 suite solve the structure as follows: export the * . sca file to a * .mtz file using the program scalpack2mtz. use the * .mtz file to calculate the solvent via matthews_-coef. run with phaser mr (see note 12) with the mers-cov rbd structure (pdb id: 4l72) and the structures of the variable domain of the heavy and light chains available in the pdb with the highest sequence identities as search models (see note 12) [11] . when the phases are determined, the electron density map can be calculated, from which the molecular model can be constructed. 5. subsequent model building and refinement were performed using coot and phenix, respectively (see note 13) [12, 13] . 6. many validation programs are used to check the structure, until the investigator is satisfied, and then the structure can be deposited in the pdb. 1. weigh 10 g tryptone, 5 g yeast extract, 10 g agar powder, and 10 g nacl, and add ultrapure water to 1 l. after sterilization by high-pressure steam, wait until the temperature of the medium drops to about 60 c, add the required antibiotics, inducers etc. (50 μg/ml kanamycin,7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml x-gal, 40 μg/ml iptg). mix evenly and then pour into sterile plates. when the culture medium has cooled and solidified, store the bacmid selection lb agar plate at 4 c. 6. endoglycosidase f1 and f3 are expressed and purified from e. coli by our laboratory. the endoglycosidase was added into the reaction system according to the mass ratio of 1:100. 7. the coding sequence of the mers-cov rbd (emc strain, spike residues 367-588) was ligated into the pfastbac-dual vector (invitrogen) with a n-terminal gp67 signal peptide to enable the protein secreting outside the cell and a c-terminal his-tag to facilitate further purification processes. 8. allow the pellet to dissolve for at least 10 min at room temperature. to avoid shearing the dna, pipet only 1-2 times to resuspend. store the bacmid at 4 c and use it as soon as possible, usually within 1 week. aliquot the bacmid dna into separate tubes and store at à20 c (not in a frost-free fridge). avoid multiple freeze/thaw cycles as this decreases the transfection efficiency. 9. characteristics of infected cells: a 25-50% increase in cell diameter can be seen and the size of cell nuclei increases at the early stage. cells release from the plate and appear lysed, showing signs of clearing in the monolayer. 10 . p0 virus can be stored for years, adding 2% (v/v) fbs at 4 c, protected from light. 11. the expression of mers-4scfv protein was conducted in 293f cells transiently transfected with plasmid dna. after 72 h, the supernatant was collected and concentrated. the purified mers-4scfv protein was obtained by ni-nta affinity chromatography and superdex 200 size-exclusion chromatography. the purification method is the same as that of rbd protein. 12. if the crystal structure of the same protein or a similar protein has been solved, the molecular replacement method can be applied. after obtaining the solutions of the rotation and translation functions, initial phases can be calculated from the reference model, after which the electron density can be calculated. 13. the accuracy of the constructed model is confirmed by the crystallographic r-factor and r-free, which indicate the discrepancy between the calculated and observed amplitudes. the stereo-chemical parameters of the model can also be checked using programs such as molprobity, procheck, or rampage. molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 structural definition of a unique neutralization epitope on the receptorbinding domain of mers-cov spike glycoprotein ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient evaluation of candidate vaccine approaches for mers-cov junctional and allelespecific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software coot: modelbuilding tools for molecular graphics phenix: building new software for automated crystallographic structure determination key: cord-294831-pem059zk authors: zhang, ling-pu; wang, meixian; wang, yanping; zhu, jun; zhang, nannan title: focus on a 2019-novel coronavirus (sars-cov-2) date: 2020-06-11 journal: future microbiology doi: 10.2217/fmb-2020-0063 sha: doc_id: 294831 cord_uid: pem059zk a new coronavirus, severe acute respiratory syndrome coronavirus 2, was first discovered in wuhan, china, in december 2019. as of april 7, 2020, the new coronavirus has spread quickly to 184 countries and aroused the attention of the entire world. no targeted drugs have yet been available for intervention and treatment of this virus. the sharing of academic information is crucial to risk assessment and control activities in outbreak countries. in this review, we summarize the epidemiological, genetic and clinical characteristics of the virus as well as laboratory testing and treatments to understand the nature of the virus. we hope this review will be helpful to prevent viral infections in outbreak countries and regions. clinical symptoms fever (98%), sough (77%), dyspnea (63.5%), myalgia (11.5%), malaise (35%) and so on fever (ͼ99%), cough (62%-100%),chills or rigor (15%-73%), diarrhea 20%, dyspnea (40%) fever (77%), cough (90%), dyspnea (68%), sputum production (40%), odynophagia (39%), digestive system /signs (20%), hemoptysis (4.3%), myalgia (43%) and headache (20%) [53, 69, 85] radiology critically ill patients with bilateral multiple lobular and subsegmental areas of consolidation; mild patients with bilateral ground-glass opacity and subsegmental areas of consolidation almost 100% patients with abnormal ct unilateral/bilateral ground-glass opacities or focal unilateral/bilateral consolidation. chest radiography or ct abnormal rate was ͼ94% unilateral/bilateral patchy densities or infiltrates, bilateral hilar infiltration, segmented/lobar opacities, ground-glass opacities and possible small pleural effusions. chest radiography or ct abnormal rate was between 90% to 100% was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by the international committee on taxonomy of viruses on 11 february 2020 [1] . the disease caused by sars-cov-2 was named covid-19 by the world health organization (who). on 30 january 2020, the who declared the outbreak of covid-19 to be a global health emergency and further labeled it a pandemic on 11 march 2020. the virus can be transmitted not only from animals to humans but also from humans to humans [23] . a lancet report demonstrated that the virus could have recently acquired the ability to transmit between humans [6] . a report of five patients in a family cluster who traveled to wuhan and were infected with sars-cov-2 was the first report directly illustrating that the virus is capable of person-to-person transmission in hospital and family settings [23] . sars-cov-2 can spread via direct contact and respiratory droplets. respiratory particles are spread while breathing, speaking, coughing or sneezing [24] . in addition, aerosol and fomite transfer may promote transmission of the virus according to a study in the new england journal of medicine [25] . aerosolized virus may be generated by respiratory and surgical procedures. the study showed that the half-life of the virus is about 1.1-1.2 h, and it remained viable for 3 h in aerosols. meanwhile, the virus on plastic, stainless steel, copper and cardboard remained stable for 4-72 h [25] . these results indicate that aerosol or fomites may be able to spread sars-cov-2 [25] . a fluid-resistant (type-r) surgical face mask is used to protect against droplets. fecal-oral transmission may also play an important role in sars-cov-2 spread [26] . xiao and colleagues showed that 53.42% of 73 hospitalized covid-19 patients had sars-cov-2 rna in stool specimens, and the duration time of positive stool results ranged from 1 to 12 days [27] . in addition, viral nucleic acid in 64.29% patients remained positive in the feces after sars-cov-2 rna in pharyngeal swabs turned negative. furthermore, positive sars-cov-2 rna in stool specimens was not associated with gastrointestinal symptoms [28] . together, these findings indicate the possibility of sars-cov-2 via the fecal-oral transmission. the basic reproduction number (r0) of this virus reflects the dynamics of transmission during this coronavirus outbreak. the who has estimated that sars-cov-2 has a reproduction number of 1.4-2.5. however, a recent study shows the average r0 to be 3.28 (median: 2.79; interquartile range [iqr]: 1.16), an r0 considerably higher than the who estimate at 1.95 [29] . in an earlier phase of the outbreak, li et al. reported that the mean incubation period of the virus is 5.2 days (95% ci: 4.1-7.0), the epidemic doubled in size every 7.4 days, and the r0 was estimated to be 2.2 (95% ci: 1.4-3.9) [30] . zhao and collaborators estimated an r0 ranged from 2.24 (95% ci: 2.49-2.63) to 3.58 (95% ci: 2.89-4.39) [31] . in another study, r0 was computed to oscillate between 3.30 (95% ci: 2.73-3.96) and 5.47 (95% ci: 4.16-7.10) [32] . in addition, the study reported a transmission rate within wuhan of 1.94 days (95% ci: 1.25-6.71), an infectious period of 1.61 days (95% ci: 0.35-3.23) and an r0 value of 3.11 (95% ci: 2.39-4.31) [32] . based on the nowcasting and forecasting approach, wu et al. showed an estimated reproduction number of 2.68 (95% ci: 2.47-2.86), and an epidemic doubling time of 6.4 days [33] . recently, tang et al. calculated the r0 as 6.47 (95% ci: 5.71-7.23), using mathematical seir-type epidemiological model [34] . nevertheless, sars-cov-2 has demonstrated a higher transmission rate than that of sars-cov and mers-cov. variation in viral transmissibility should be considered, and estimates of the reproduction number may change in the future. sars-cov-2 is similar to sars-cov. both single-stranded rna viruses share 82% nucleotide identity, and sars-cov-2 shares 89% identity with sars-like covzxc21 [35] . the genome of sars-cov-2 has 29,891 nucleotides encoding 9,860 amino acids. genetically, sars-cov-2 is similar to sars-cov (about 79%) and mers-cov (about 50%) [36] . the virus contains a replicase, spike (s) protein, envelope (e) protein, membrane (m) protein and nucleocapsid [35] (figure 1a & b) . however, sars-cov-2 lacks the hemagglutinin-esterase gene, which is found in lineage a β-covs. sars-cov-2 has 12 open reading frames (orfs) encoded by nine subgenomic mrnas that carry nine transcription regulatory sequences, two terminal untranslated regions (utr) and a conserved leader sequence [37] . the large replicase polyprotein pp1a contains ten nonstructural proteins (nsp1-nsp10), and pp1ab contains 15 nonstructural proteins (nsp1-nsp10, nsp12-nsp16), which are all encoded by orf1a and orf1ab [38] (figure 1a ). with the exception of nsp3 and nsp5, which are cysteine proteases, most nonstructural proteins play an important role in the transcription and replication of sars-cov-2 [39] . pp1ab has different lengths in covid-19, sars-cov and mers-cov of 29,844 bp (7096 aa), 29,751 bp (7073 aa) and 30,119 bp (7078 aa), respectively ( figure 1a) . furthermore, there is no obvious difference between sars-cov-2and sars-cov nonstructural proteins and orfs. the spike glycoprotein plays an important role in binding to receptors on host cells and, therefore, is involved in host tropism [40] . sars-cov-2, sars-cov and mers-cov have s proteins containing 1,273, 1,255 and 1,270 aa, respectively ( figure 1a ) [36] . the s protein mediates entrance into human respiratory epithelial cells by interacting with the cell-surface receptor angiotensin-converting enzyme 2 (ace2) [35, 41] . it is comprised of s1 and s2 subunits ( figure 1c ), and the s1 subunit shares approximately 70% identity with that of human sars-cov and bat sars-like covs (sl-covzxc21 and zc45). the s1 subunit has an n-terminal domain and a receptorbinding domain (rbd) that are both responsible for the binding of virions to host cells [16] . both sars-cov-2 and sars-cov bind to ace2 through the c-terminal domains (ctd) of their s1 subunits, and mers-cov utilizes the ctd to bind proteinaceous dipeptidyl peptidase 4 (dpp4) [42] . the rbd of sars-cov-2 has 73% identity to that of sars-cov [43] . the transmissibility of the sars-cov-2 virus is greater than that of sars-cov, which may be because the rbd of sars-cov-2 is slightly different from that of sars-cov. the s2 subunit shares 99% identity with two bat sars-like covs and human sars-covs [38] . the s2 subunit contains a fusion peptide (fp) and heptad repeats (hrs) 1 and 2 ( figure 1c ). after the s1 rbd binds to the ace receptor on the host cell, the fp of s2 is inserted into the host cell membrane, and then hr1 and hr2 form a six-helix bundle (6-hb), which helps the virus fuse with host cell membranes [16, 44] . sars-cov-2 envelope (e) protein, matrix protein, accessory proteins p6 and p8, nonstructural protein 7 (nsp7), and nsp13 are homologous with those of sars virus [39] . therefore, the sars-cov-2 virus has a high level of identity with sars-cov. this suggests that an anti-sars-cov antibody, which could cross-react with the sars-cov-2 s protein, may be useful to treat patients with the virus. sars-cov-2 infection has caused clusters of severe respiratory illness similar to that of sars-cov. the virus causes symptoms such as fever, cough, shortness of breath, leukopenia and pneumonia in both lungs [6] . the symptoms are observed approximately 5.2 days after the sars-cov-2 infection [5] . in a study published in the lancet, 41 of 41 patients who were identified as positive for sars-cov-2 infection presented with pneumonia and abnormal chest computed tomography (ct) [6] . covid-19 symptoms included fever (98%), cough (76%) and myalgia or fatigue (44%). less common symptoms such as sputum production (28%), headache (8%), hemoptysis (5%) and diarrhea (3%), were also observed [6] . another clinical study containing 138 patients showed that common symptoms were fever, fatigue, dry cough, lymphopenia, prolonged prothrombin time and an increased lactate dehydrogenase level (table 1 & figure 2 ) [5] . common complications included shock, acute respiratory distress syndrome, acute renal injury, acute liver failure, arrhythmia, rnaaemia and acute cardiac injury [45] . in addition, it is now understood that sars-cov-2 can infect children as well as adults [46] . one study showed that during the early infection period, most patients had normal white blood cell counts; however, 56.8% of patients had leukopenia in cases of serious infection [46] . patients with dyspnea were more frequently admitted to the intensive care unit (icu) [46] . most chest computed tomography (ct) showed bilateral patchy shadows or ground-glass opacity (ggo) in the lungs [5, 6] . in another study, 86% of patients showed ggo by chest ct, and 29% of patients had consolidation [47] . following the appearance of ggo, 75% of patient lung cts showed reticular or interlobular septal thickening [48] . however, no direct cavitation, pleural effusion, lymphadenopathy or nodules were observed in the lungs of covid-19 patients [49] (table 1 ). in view of the large amounts of cytokines produced during sars-cov infection, infection with sars-cov-2 similarly induces the production of proinflammatory cytokines such as, interleukin 1 beta (il-1β), interferon gamma (ifn-γ), ip10 and monocyte chemoattractant protein 1 (mcp). moreover, the levels of granulocyte colony stimulating factor (gcsf), ip10, mcp1, mip1α and tumor necrosis factor-alpha (tnf-α) were found to be higher in intensive care unit (icu) than non-icu patients [50] . however, secretion of immunosuppressive cytokines (e.g.,interleukin 4 [il-4] and interleukin 10 [il-10] by t-helper type 2 [th2] cells was also increased during sars-cov-2 infection [6] ) ( table 1) . the mortality rate of sars-cov-2-infected patients varies in different studies. a study of 41 patients showed a mortality rate of 15% [6] , and the fatality of 138 patients in a clinical study was 4.3% [5] . however, chen showed that the mortality was 3%, a number closer to the official national statistics of china (2.01%) [51] . these differences in mortality rates are possibly due to the difference in sample size. the reported mortality rate of sars-cov was 10%, and that of mers-cov was about 36% [11] . the currently estimated mortality rate of sars-cov-2 is therefore lower than that of sars-cov and mers-cov [51, 52] (table 1 ). in summary, sars-cov-2 spreads more rapidly but has a relatively lower fatality rate as compared with two other related coronaviruses. the number of total leukocytes, lymphocytes and monocytes has been detected from hospitalized patients with covid-19 [53] . approximately 25% of cases formed leucopenia [6] . moreover, lymphopenia was observed in 63-70.3% of patients [5, 6] . cd4 + or cd8 + t-cell numbers decreased as the disease severity increased [6, 54] . patients with severe cases had more prominent abnormalities than those with non-severe cases. patients infected with covid-19 had some unique clinical features including rhinorrhoea, sneezing, sore throat. most patients had haemoptysis dyspnea, fever, headache, fatigue, sputum production, pneumonia and ground-glass opacities. however, only a low percentage of patients developed intestinal symptoms such as diarrhea and vomiting. leucopenia, lymphopenia, pro-inflammation cytokines increasing, acute respiratory distress syndrome and acute organs damages (such as cardiac, liver or kidney) were common features in some intensive care unit patients. virus from throat swabs, blood, urine, stool or respiratory tracts have been assessed by fluorescent reverse transcription-polymerase chain reaction (rt-pcr) methods [55] . primers and probes targeting rdrp/helicase (hel), e, s, n and replicase orf 1a/b genes were designed and tested for sars-cov-2 [55] . rt-pcr showed that primers of the e and rdrp genes demonstrated better sensitivity than that of the n gene, and the limit of detection (lod) of rdrp/hel and n gene was lower than that of s gene and rdrp-p2 [18] . chan et al. showed that the covid-19-rdrp/hel assay (rdrp/hel probe, fam-ttaagatgtggtgcttgcatacgtagac-labkfq) was significantly more sensitive than the rdrp-p2 assay for the detection of sars-cov-2 rna, and detection targeted orf1a/b, orf1b-nsp14, rdrp, s, e or n genes was with less specificity for sars-cov-2 [18] . moreover, a previous study showed that the specific probe rdrp sarsr-p2 (fam-caggtggaacctcatcaggagatgc-bbq) detected only the sars-cov-2 rna transcript but not the sars-cov rna [55] . however, a novel rt-pcr assay showed that targeting rdrp2 was a non-specific assay for sars-cov-2, because this detected other betacoronaviruses such as sars-cov [18] . the covid-19-rdrp/hel assay had the lowest lod in vitro and higher sensitivity and specificity, which helped to reduce the false-negative rate and improve the laboratory diagnosis of covid-19 [18] . sars-cov-2 viral load is detectable from throat-and lung-derived samples; however, blood and urine have not yet yielded virus [56] . patients with covid-19 produced the highest viral load near symptom presentation, which may be the reason for the fast spread of the virus [57] . an observational cohort showed that viral load in saliva was highest following symptom onset in the first week, then gradually declined with time [56] . endotracheal aspirate viral load was available from day 8 after symptom onset and did not significantly decline thereafter [57] . following symptom onset, virus load in 33% patients could be detected for 20 days or longer in that study. viral load in respiratory tract specimens was about sixfold higher than that in the nonrespiratory tract specimens [57] . in addition, elder patients reportedly had a greater virus load than that of younger patients. studies showed that higher initial viral load was related to the severity of covid-19 symptoms [57] . although the positive test ratio was only 47.4% in previous study, it was improved by novel assay methods [58] . the sample quality, collection time, detection kits and technical abilities of clinical doctors may affect the accuracy of detection. therefore, precise diagnosis of covid-19 should be combined with ct scans and nucleic acid testing. bronchoalveolar lavage fluid or throat swabs from patients have been sequenced, and viral genomes were searched via blast with the sars-cov-2 sequence [38, 39] . genome sequencing has also been used to identify patients with suspected infection. in addition to hematological detection and nucleic acid testing, serological diagnosis is important for patients who present late with a very low viral load, below the detection limit of rt-pcr assays [59] . igm and igg titers were relatively increased on day 5 and rapidly raised 10 days after symptom onset in most patients [59, 60] . the igm-positive rate increased from 50 to 94%, whereas the igg-positive rate increased from 81 to 100% [60] . in addition, the igm-and igg-positive rates were not significantly different before and after patients were found to be virally negative [61] . therefore, virus-specific igm and igg serological testing may be used to confirm current or previous infection with sars-cov-2. an ideal animal model for covid-19 would reflect the clinical signs, viral replication and pathology displayed in humans. non-human primate models (rhesus, cynomolgus macaques, african green monkeys, common marmoset, squirrel monkeys and mustached tamarins) have all been evaluated as models of sars-cov infection [62] . all non-human primates had pneumonia, cough and respiratory distress after virus infection [15] . mice (balb/c, c57bl6 and 129s strains) supported sars-cov replication and showed clinical signs of sars [15] . rag1 −/− mice, cd1 −/− mice, stat1 −/− mice and beige mice have been used to determine the role of immune effectors of the virus [63] . ac70 and ac63 transgene-positive mice also showed clinical manifestation after sars-cov infection, demonstrating their usefulness to study the pathogenesis and evaluation of vaccines and other therapeutics [63] . hamsters also have been used to study immuno-prophylaxis and drug research as they harbored high viral titers and pulmonary histopathology upon virus infection [63] . ferrets are another appropriate animal model to study this respiratory virus because the clinical symptoms, viral titers and histologic changes were similar to those of patients with virus infection [63] . ace2, the receptor of sars-cov, was also identified as the functional receptor for sars-cov-2, therefore, mice, hamsters and ferrets may be animal models for studying the sars-cov-2 [63] . an article reported in science shows that sars-cov-2 can replicate in the upper respiratory tract of ferrets, indicating that ferrets represent an ideal animal model for evaluating antiviral drugs or vaccine candidates against covid-19 [64] . in addition, the domestic cat has shown multifocal pulmonary consolidation with infection of sars-cov, and sars-cov-2 can replicate efficiently in cats and transmit between cats via the airborne route [64] . by contrast, dogs, pigs, chickens and ducks are poorly susceptible to sars-cov-2 [64] . no specific therapeutic medicine has been approved for the treatment of sars-cov-2 infection. all patients are given empirical antibiotic or antiviral drugs. moxifloxacin or levofloxacin are empirically used to treat early coinfections with bacteria [65] . linezolid is effective against streptococcus pneumoniae and staphylococcus aureus and is combined with nemonoxacin in cases of severe infection [65] . a combination of lopinavir and ritonavir has been used to treat covid-19 because the lopinavir/ritonavir (lpv/r) combination has been confirmed to be effective against sars-cov and mers-cov [6, 66, 67] . another antiviral drug, remdesivir (rdv), was predicted to be efficacious against covid-19 by target-based virtual ligand screening [68] . rdv is a novel nucleotide analog prodrug that is in development and has demonstrated effective pan-cov therapy. the first case of sars-cov-2 infection in the usa was successfully treated with rda. moreover, a randomized, double-blind, parallel-controlled phase iii clinical trial was conducted to recruit sars-cov-2-infected patients [4, 69] . favipiravir is a nucleoside analog that can lead to lethal viral mutagenesis, chain termination or the inhibition of nucleotide biosynthesis [70] . favipiravir in combination with oseltamivir, which was given to patients infected with sars-cov-2, has been used to treat severe influenza [45] . oseltamivir, a neuraminidase inhibitor, is recommended as an antiviral treatment for influenza and has been widely used to inhibit covid-19 in china [45, 70] . other neuraminidase inhibitors, zanamivir and peramivir, are also effective treatments for mers-cov [71] . at this time, these are all empirical therapies for covid-19. however, whether oseltamivir or zanamivir are effective treatments for covid-19 also needs further study. arbidol (arb) was licensed for the treatment of influenza and other respiratory viral infections in russia and china [72, 73] . blaising and coauthors consider arb to be a broad spectrum antiviral drug [72] . in addition, arb has been reported to inhibit sars-cov in vitro. therefore, a clinical trial of arb-treated covid-19-positive patients has been registered [65] . glucocorticoids have been commonly used in patients with sars-cov or mers-cov infection [74, 75] . glucocorticoids prolonged the survival time of sars cases [75] . patients with covid-19 were given glucocorticoids in some hospitals in china. however, the mortality rate did not decrease with corticosteroid treatment in patients infected with sars-cov-2, and viral clearance was not delayed [6, 74] . therefore, it is still controversial whether corticosteroids should be used to treat sars-cov-2 infections [69] . chloroquine/hydroxychloroquine was used as an antimalarial, broad spectrum antiviral drug, and has been broadly used in autoimmune diseases including lupus and rheumatoid arthritis [76] . a recent study indicates that chloroquine and the antiviral drug rdv-inhibited sars-cov-2 in vitro [77] . clinical symptoms of patients treated with chloroquine were obviously relieved: for example, more rapid decline in fever and improvement of lung ct [77] . chloroquine was suggested to treat covid-19 in the sars-cov-2 treatment guidelines by the chinese medical advisory. chloroquine has been highly effective in reducing sars-cov-2 viral replication by increasing endosomal ph and interfering with the glycosylation of cellular receptors [76] . moreover, chloroquine was probably the first molecule to be used to treat covid-19. another antiviral drug, nelfinavir, an hiv protease inhibitor, was predicted to be a potential inhibitor of covid-19 [26] . in addition, cytokine immunotherapy with ifn-α, a broad spectrum antiviral drug, has been used to inhibit hbv. ifn-α was used to treat patients infected with sars-cov-2 according to established guidelines [70] . ifn-α combined with lpv/r was shown to be beneficial for treatment of covid-19 [78] . tocilizumab is a humanized anti-il-6-receptor (il-6r) monoclonal antibody that inhibits il-6 signaling and is used as a treatment in rheumatoid arthritis [79] . il-6 is one of the most important cytokines involved in covid-19-induced cytokine storms. tocilizumab (tcz) has been used to treat covid-19 in china and italy. tcz was recommended for covid-19 patients to prevent or treat cytokine storms and could reduce the mortality of covid-19 [80] . other drug types such as rna synthesis inhibitors (tdf and 3tc) and an fp (ek1), have also effectively inhibited sars-cov-2 in vitro. in addition, traditional chinese medicines (lian hua qing wen capsules, shu feng jie du capsules) have also been used to treat covid-19 in the latest version of the diagnosis and treatment of pneumonia induced by covid-19 [81] . in addition, human seroalbumin and γ-immunoglobulin were given to some patients with severe infections [82] . in conclusion, there are specific vaccines or antiviral drugs for covid-19. all of the drugs described above have shown some usefulness in treating sars-cov-2 infections, and their efficacy merits further study. covid-19 is a serious human infectious disease of global concern. as of 7 april 2020, sars-cov-2 has infected a total of 134,784 patients globally at least ( figure 3d & g) . the covid-19 outbreak poses a serious challenge to china and the whole world; it has profoundly affected public health. although the overall mortality rate of sars-cov-2 appears to be lower than that of sars-cov and mers-cov (table 1) , the transmissibility of covid-19 is more rapid. moreover, the fatality rate of elderly patients with reduced immunity or chronic diseases is as high as 15% [6] . in addition, asymptomatic carriers may be a potential source of infection, sustaining a local 10 epidemic and global spread [83] . therefore, covid-19 may cause disruptions to the global public health system for an extended period of time. the outbreak of covid-19 has shown that there are still shortcomings in the prevention of public health diseases such as the lack of awareness of the frontline doctors and the early vigilance and attention from the government. meanwhile, the awareness of the general public to health concerns also must be improved. individuals should develop good living habits: for example, keeping away from wild animals, not consuming wild animals, maintaining hand hygiene -among others. in addition, the development of targeted antiviral drugs should be anticipated and accelerated in the future. certainly, vigorous measures to prevent contagion have been taken in china and other countries. to prevent the spread of infection, the chinese government imposed a full lockdown and canceled public events such as the new year festival; contact with wild animals was also restricted, and travel was reduced with screening at airports, railway stations and subway stations [84] . moreover, two emergency hospitals in wuhan were constructed, and army medical units and medical staff from other areas were deployed to help prevent infections in wuhan [84] . the who and various governments advised people to reduce public activity, maintain social distance, wear masks, wash their hands frequently, and practice respiratory hygiene. as of 7 april 2020, the number of fully recovered patients was 277,420 worldwide ( figure 3e & h) , and the number of recovered patients was 77,468 in china ( figure 3b ). therefore, we are confident that the outbreak of covid-19 will be effectively curbed. most countries have isolated any suspected cases as rapidly as possible to contain infection and prevent local outbreaks. the ability to rapidly test patients suspected of having a sars-cov-2 infection is the cornerstone of case isolation. the experience gained from sars-cov and mers-cov by the health community over the last 20 years could also help in dealing with sars-cov-2 infections throughout the world. to date, no sars-cov-2-specific antiviral drugs or vaccines have been described for covid-19. therefore, a safe and stable vaccine for covid-19 is urgently needed, and it is expected to be ready within 18 months [81] . vaccines specific for sars-cov-2 will immunize people worldwide in the future. it is hoped that drugs specifically targeted for covid-19 also will be widely developed. meanwhile, the origin, intermediate host, structure and pathogenesis of sars-cov-2 will be the focus of future research. moreover, prediction of whether the similar coronaviruses can infect humans will be import for all the world. we hope that vaccines specific for the similar coronaviruses will be developed in advance to prevent epidemics in the world. • coronavirus disease 2019 (covid-19) was first reported in china and currently poses a serious challenge worldwide. the pneumonia was caused by a novel coronavirus named sars-cov-2. • more than 1,347,804 cases of covid-19 and 74,596 deaths have been reported as of april 7, 2020 according to data from johns hopkins resource center. • sars-cov-2 belongs to betacoronavirus, a large genus of viruses prevalent in nature. sars-cov-2 has 82% nucleotide identity with human sars-cov and 96% nucleotide identity with bat sars coronavirus (sarsr-cov-ratg13). • the fatality rate of covid-19 has been approximately 3.4% and the r0 has ranged from 1.4 to 6.4. compared with previous coronavirus outbreaks, has been reported to have a lower mortality rate and more rapid transmissibility and caused severe acute respiratory syndrome similarly to sars. • the main symptoms are fever, cough, shortness of breath, leukopenia and pneumonia. • diagnostic laboratory testing of covid-19 includes hematology testing, nucleic acid testing, viral genome sequencing and serology testing. to date, no specific antiviral drugs or vaccines for covid-19 have been developed. therefore, empirical antiviral drugs (lopinavir/ritonavir, favipiravir, oseltamivir, zanamivir and peramivir, arbidol), antibiotic drugs (moxifloxacin, levofloxacin, linezolid), chloroquine/hydroxychloroquine, glucocorticoids, monoclonal anti-inflammatory antibody (tocilizumab), have been used to treat sars-cov-2 infection. traditional chinese medicines are also used for therapy during infection with sars-cov-2. • this review is presented in the hope of helping the public effectively recognize and combat covid-19 and to provide a reference for future studies or outbreaks. the authors gratefully acknowledge all doctors who participate in the fight against covid-19 on the frontline. financial & competing interests disclosure no writing assistance was utilized in the production of this manuscript. 10.2217/fmb-2020-0063 future microbiol. (epub ahead of print) future science group 52279 ireland:5364 norway:5865 netherlands:18926 germany:28700 belgium:3986 turkey:1326 iran:24236 china:77468 us 5373 france:8911 belgium:1632 germany:1810 ltaly:16523 china 2019 novel coronavirus-important information for clinicians estimating the unreported number of novel coronavirus (2019-ncov) cases in china in the first half of january 2020: a data-driven modelling analysis of the early outbreak lessons learned from the 2019-ncov epidemic on prevention of future infectious diseases coronavirus 2019-ncov: a brief perspective from the front line clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan • provides the first evidence for the diagnosis and treatment of covid-19 patients. the common symptoms among 41 patients were fever, cough, myalgia, fatigue and pneumonia clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study • shows the epidemiological and clinical features of pediatric patients with covid-19. the route of transmission in children was by close contact with family members (89%) or a history of exposure to the epidemic area or both exposures return of the coronavirus: 2019-ncov bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond clinico-epidemiological characteristics of acute respiratory infections caused by coronavirus oc43, nl63 and 229e epidemiology, genetic recombination, and pathogenesis of coronaviruses human coronavirus circulation in the united states 2014-2017 recently discovered human coronaviruses epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method development of animal models against emerging coronaviruses: from sars to mers coronavirus an emerging coronavirus causing pneumonia outbreak in wuhan, china: calling for developing therapeutic and prophylactic strategies 2019 novel coronavirus: where we are and what we know improved molecular diagnosis of covid-19 by the novel, highly sensitive and specific covid-19-rdrp/hel real-time reverse transcription-pcr assay validated in vitro and with clinical specimens spike (s) and nucleocapsid (n) genes of sars-cov-2 with that of the reported rdrp-p2 assay. the results showed that the covid-19-rdrp/hel assay had the lowest limit of detection in vitro and did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens. the positive rate of rdrp/hel assay is higher than that of rdrp-p2 assay. the study may provide a sensitive rdrp structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a pneumonia outbreak associated with a new coronavirus of probable bat origin cross-species transmission of the newly identified coronavirus 2019-ncov the continuous evolution and dissemination of 2019 novel human coronavirus a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster this research reported that covid-19 could transmit from person to person in hospital and family settings, which provided the evidence that covid-19 could spread from person to person. it is very important for ministries of health and individuals in other countries outside of china personal protective equipment during the covid-19 pandemic -a narrative review aerosol and surface stability of sars-cov-2 ascompared with sars-cov-1 this research showed that aerosol and fomite transmission of sars-cov-2 is plausible, as the virus can remain viable and infectious in aerosols for hours and on surfaces for up to days. these findings echo those found with sars-cov, in which these forms of transmission were associated with nosocomial spread and super-spreading events the epidemic of 2019-novel-coronavirus (2019-ncov) pneumonia and insights for emerging infectious diseases in the future evidence for gastrointestinal infection of sars-cov-2 the presence of sars-cov-2 rna in feces of covid-19 patients the reproductive number of covid-19 is higher compared to sars coronavirus early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study estimation of the transmission risk of the 2019-ncov and its implication for public health interventions evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission genotype and phenotype of covid-19: their roles in pathogenesis genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and re0eptor binding this research showed that sequences of sars-cov-2 obtained from nine patients had 88% identity to two bat-derived sars-like coronaviruses, bat-sl-covzc45 and bat-sl-covzxc21, collected in 2018 in zhoushan, eastern china, but were more distant from sars-cov (about 79%) and mers-cov (about 50%) genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan genome composition and divergence of the novel coronavirus (2019-ncov) originating in china the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade structure, function, and evolution of coronavirus spike proteins coronavirus membrane fusion mechanism offers a potential target for antiviral development potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody the spike protein of sars-cov -a target for vaccine and therapeutic development epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan, china • shows the characteristic chest ct imaging features of covid-19 patients. out of 21 covid-19 patients, 71% had involvement of more than two lobes at chest ct, 57% had ground-glass opacities, 33% had opacities with a rounded morphology, 33% had a peripheral distribution of disease, 29% had consolidation with ground-glass opacities and 19% had crazy-paving pattern emerging 2019 novel coronavirus (2019-ncov) pneumonia chest ct findings in 2019 novel coronavirus (2019-ncov) infections from wuhan, china: key points for the radiologist the novel chinese coronavirus (2019-ncov) infections: challenges for fighting the storm pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses assessing the pandemic potential of mers-cov severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr virological assessment of hospitalized patients with covid-2019 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests the laboratory diagnosis of covid-19 infection: current issues and challenges molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 the battle against sars and mers coronaviruses: reservoirs and animal models animal models for sars and mers coronaviruses susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 investigates the susceptibility of ferrets and animals in close contact with humans to sars-cov-2 and found that sars-cov-2 replicates poorly in dogs, pigs, chickens and ducks, but ferrets and cats are permissive to infection therapeutic and triage strategies for 2019 novel coronavirus disease in fever clinics a systematic review of therapeutic agents for the treatment of the middle east respiratory syndrome coronavirus (mers-cov) sars: systematic review of treatment effects the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in china clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study drug treatment options for the 2019-new coronavirus (2019-ncov) clinical management of respiratory syndrome in patients hospitalized for suspected middle east respiratory syndrome coronavirus infection in the paris area from arbidol as a broad-spectrum antiviral: an update antiviral activity of arbidol hydrochloride against herpes simplex virus i in vitro and in vivo clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury what caused lymphopenia in sars and how reliable is the lymphokine status in glucocorticoid-treated patients? new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? chloroquine for the 2019 novel coronavirus sars-cov-2 the correlation between viral clearance and biochemical outcomes of 94 covid-19 infected discharged patients the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality tocilizumab treatment in covid-19: a single center experience review of the 2019 novel coronavirus (sars-cov-2) based on current evidence a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) a novel coronavirus (covid-19) outbreak: a call for action novel coronavirus pneumonia emergency in zhuhai: impact and challenges focus on middle east respiratory syndrome coronavirus (mers-cov) key: cord-301016-9t7v7ipt authors: forni, diego; filippi, giulia; cagliani, rachele; de gioia, luca; pozzoli, uberto; al-daghri, nasser; clerici, mario; sironi, manuela title: the heptad repeat region is a major selection target in mers-cov and related coronaviruses date: 2015-09-25 journal: sci rep doi: 10.1038/srep14480 sha: doc_id: 301016 cord_uid: 9t7v7ipt middle east respiratory syndrome coronavirus (mers-cov) originated in bats and spread to humans via zoonotic transmission from camels. we analyzed the evolution of the spike (s) gene in betacoronaviruses (betacovs) isolated from different mammals, in bat coronavirus populations, as well as in mers-cov strains from the current outbreak. results indicated several positively selected sites located in the region comprising the two heptad repeats (hr1 and hr2) and their linker. two sites (r652 and v1060) were positively selected in the betacovs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. during the most recent evolution of mers-cov, adaptive mutations in the hr1 (q/r/h1020) arose in camels or in a previous host and spread to humans. we determined that different residues at position 1020 establish distinct interand intra-helical interactions and affect the stability of the six-helix bundle formed by the hrs. a similar effect on stability was observed for a nearby mutation (t1015n) that increases mers-cov infection efficiency in vitro. data herein indicate that the heptad repeat region was a major target of adaptive evolution in mers-cov-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function. camel to human transmission rather than vice versa 10 . therefore, the most likely scenario envisages that a bat-derived mers-cov spread to humans via the zoonotic transmission from dromedary camels. coronaviruses use their spike (s) protein to bind a host receptor and to promote membrane fusion. the spike protein assembles as a trimer on the viral surface and belongs to the class i fusion protein family 11 . class i fusion proteins are found in many other virus genera including retroviruses, orthomyxoviruses, paramyxoviruses, and filoviruses and share similar domain organization, as well as common functional properties 12 . host proteases cleave the cov spike protein into two functionally distinct domains: the n-terminal region (usually referred to as the s1 subunit) contains the receptor binding domain (rbd), whereas the c-terminal portion (s2 subunit) includes the fusion peptide, two heptad repeats (hr1 and hr2), and the transmembrane (tm) domain 12 (see fig. 1a ). following receptor binding, membrane fusion is mediated by a major conformational rearrangement that exposes the fusion peptide and results in the formation of a six-helix bundle (6hb) 13, 14 . the core of the 6hb is a triple-stranded coiled coil formed by the hr1s of the three spike subunits forming the trimer; the hr2 elements pack within the grooves of the coiled coil in an antiparallel direction 13, 14 . because of its central role in membrane fusion, a number of antiviral peptides that interfere with the 6hb formation have been developed as potential therapeutic compounds against hiv 15 , ebola virus 16 , sars-cov 17, 18 , and mers-cov 14 . although the rbd of spike proteins is generally considered the major determinant of host range, several reports have suggested that variation in the c-terminal portion of spike proteins, particularly in the hr1 and hr2, determine host range expansion 12 . moreover, recent works indicated that mers-cov and ty-batcov hku4 bind dpp4 both of human and of bat origin [7] [8] [9] . in particular, although mers-cov binds human dpp4 with higher affinity than ty-batcov hku4, which shows a preference for the bat receptor, the rbds of the two viruses engage human dpp4 via a similar binding mode 9 . these observations suggest that mers-cov and related viruses have the potential to shift host range with little adaptation of the rbd. motivated by the notion that evolutionary analyses can provide information on the molecular events that underlie host shifts and, more generally, host-pathogen interactions 19 , we investigated the evolutionary history of s proteins in mers-cov and related betacovs. specifically, we aimed to determine whether natural selection drove the evolution of specific regions and sites that may contribute to variation in host range or replication efficiency. thus, using different strategies, we analyzed mers-cov strains isolated from human and camels, as well as mers-cov-related viruses from other mammals. data indicate the hr1 to hr2 region as a major target of adaptive evolution in these viruses. positive selection shaped the evolution of clade c betacov spike protein. we first investigated whether positive selection drove the evolution of mers-cov-related coronavirus spike proteins. previous phylogenetic analyses of s genes of viruses isolated from humans/camels (mers-cov), bats, and hedgehogs (supplementary table. s1) indicated that the s1 and s2 regions display different tree topologies (fig. 1b) , possibly as a result of recombination 6 . because recombination can inflate estimates of positive selection 20 , we separately analyzed the two regions. we pruned the s1 and s2 multiple sequence alignments (msas) from unreliably aligned codons and we screened them for evidence of additional recombination events using gard (genetic algorithm recombination detection) 21 , which detected no breakpoint. the saturation of substitution rates represents a major problem in the detection of positive selection among distantly related sequences. computation of the nonsynonymous (dn) and synonymous (ds) substitution rates over whole phylogenies allows breaking of long branches, resulting in improved rate estimation. thus, evidence of episodic positive selection was searched for by using the codeml branch-site test 22 , which is relatively insensitive to the saturation of substitution rates 23 . in the s1 and s2 regions, 3 and 4 branches yielded statistically significant evidence of positive selection under different codon frequency models (fig. 1b , table 1 and supplementary table s3 ). positively selected sites along these branches were detected using the beb (bayes empirical bayes) procedure and validated using the mixed effects model of evolution (meme) 24 . one positively selected site was found in the s1 region, 7 sites in the s2 subunit (fig. 1a , table 1 ). the r652 selected site (in s1) almost corresponds to two mutations that independently arose in the sars-cov spike gene as a result of in vitro adaptation of zoonotic strains to primate cells 25 (fig. 1a) . in s2, most selected sites are located in the hr1, hr2, and in the intervening linker. remarkably, position 1060 is the almost exact counterpart of aminoacid changes that expand the host range or cell-type tropism of infectious bronchiolitis virus (ibv, l857f) and murine hepatitis virus (mhv, e1035d) ( fig. 1a) 26, 27 . no positively selected site was found to be located in the rbd (fig. 1a) . nevertheless, the pruning of unreliably aligned codons operated on the msa left a minority of rbd sites available for analysis. we thus repeated the branch-site test on a subset of more closely related sequences (fig. 1b) . this procedure decreased divergence and pruning in the rbd and allowed analysis of most codons; even with this procedure, no positively selected site was detected (not shown). indicated that positive selection targeted the spike protein and particularly its s1 region 28 . nevertheless, recombination was not accounted for in that analysis. we thus analyzed the sequence alignments of the hong kong isolates for the presence of recombination breakpoints using gard. the algorithm detected 9 recombination breakpoints for the pi-batcov hku5 alignment (fig. 1a) and none for ty-batcov hku4. the ty-batcov hku4 spike gene was therefore analyzed using the codeml site models, which test the hypothesis that a subset of codons evolve with dn/ds > 1. no evidence of positive selection was found, even using the relatively non-conservative model m7/model m8 comparison (supplementary table s4 ). as for the pi-batcov hku5 spike gene, rampant recombination prevents application of a similar approach. we thus resorted to the simultaneous estimation of selection and recombination using omegamap 29 . this analysis confirmed high recombination along the whole gene ( supplementary fig. s1 ) and detected a single positively selected site in the s1 region (fig. 1c ). the same site (codon 198, position relative to the mers-cov spike sequence) was also detected by meme, which was run by incorporating the alternative phylogenies detected by gard. most of the previously reported selected sites 28 were not detected using other methods that account for recombination (supplementary table s5 ). we thus consider that robust inference of positive selection in pi-batcov hku5 spike genes can only be made for position 198, which lies outside the rbd (fig. 1a ). positive selection in the mers-cov heptad repeat 1. we next wished to determine whether positive selection occurred at the spike gene of mers-cov viruses circulating in the recent outbreak. a previous study suggested that positive selection drove the evolution of two codons in the mers-cov spike gene (positions 509 and 1020) 30 . nevertheless, in that study only one method was used to infer selection and sequences isolated from camels were not included. we thus retrieved 54 fully or almost fully spike sequences of mers-cov isolated from camels or humans (supplementary table s1 ). alignments for the s1 and s2 regions were separately analyzed and screened for the presence of recombination. no breakpoint was detected and the codeml site models were applied. for the s2 region, two models of gene evolution that allow a class of codons to evolve with dn/ds > 1 (nssite models m2a and m8) showed better fit to the data than the null models (nssite models m1a and m7), strongly supporting the action of positive selection ( table 2, supplementary table s6 ). no evidence of selection was detected for the s1 portion ( table 2, supplementary table s6 ). in s2, both beb and meme detected one selected site: position 1020 in the hr1 (fig. 1a) . interestingly, three different residues are observed at this site in both camel-and human-derived viruses (fig. 1a) . mers-cov is thought to have spread from camels to humans; the presence of the three alternative residues in viruses isolated from camels suggests that adaptive evolution at this site occurred prior to the infection of humans. interestingly, the 1020 variant is in proximity to a mutation (t1015n) (figs 1a and 2a) that arose during tissue-culture adaptation of mers-cov (strain emc2012) and increases replication efficiency 31 . through its side chain, the q1020 residue forms hydrogen bonds with d1024 and interacts with m1266 in hr2 ( fig. 2a,b) . replacement of the glutamine residue with histidine or arginine (observed in the camel-and human-derived viruses) results in loss of side chain interactions with m1266 and variably affects hydrogen bonds with d1024 (fig. 2b) . to gain further insight into the effect of adaptive evolution at position 1020, we performed a stability computational analysis after in silico mutagenesis. q1020 was replaced with all other possible aminoacids: even if changes of different magnitude in δ g were obtained using three different methods 32-34 , trends were very consistent (fig. 2c ). in particular, replacement with histidine or arginine residues resulted in mild destabilization (fig. 2c) . as a comparison, the same analysis was performed for position 1015. replacement of the threonine residue with asparagine, which was previously associated with increased replication efficiency in vitro 31 , resulted in a similar level of destabilization as observed for q1020h/r (fig. 2c ). we analyzed the evolution of the s protein in betacovs, in bat ty-batcov hku4 and pi-batcov hku5 viral populations, as well as in mers-cov isolates from the current outbreak. different strategies were applied, as appropriate depending on divergence and recombination. results indicated that several adaptive changes are located in the s2 region, with fewer in the s1 domain and none of these within the rbd. it should be noted, though, that in all analyses we applied quite conservative approaches and we intersected two different methods to declare a site as positively selected. whereas this approach was meant to limit the false positive rate it may have yielded some false negative results. in particular, the branch-site test we used to analyze the s1 and s2 regions of betacovs is robust to saturation issues and has a minimal false positive rate, but lacks power 23 . moreover, due to the high divergence and the consequent need of alignment pruning, analysis of the rbd was performed on a shallower phylogeny compared to the other regions. this procedure is expected to reduce power, but is nonetheless necessary. in fact, alignment errors, together with unrecognized recombination, inflate estimates of positive selection and represent major sources of false positive results in evolutionary analyses 20, 35 . consistently, when we accounted for recombination in pi-batcov hku5 sequences most previously described selection signals disappeared, including those in the rbd 28 . thus, whereas we cannot exclude that adaptive variants in the rbd of betacovs were missed by our approach, we conclude that the more recent evolution of mers-cov, ty-batcov hku4, and pi-batcov hku5 was not driven by positive selection in this domain. conversely, as previously shown for sars-cov 25 , our data support a role for the s1 region separating the rbd and fusion peptide as a determinant of betacov host range expansion. analysis of both betacovs and mers-cov strains revealed evidence of positive selection in the s2 region. most positively selected sites were found to be located either in the heptad repeats or in the intervening linker. among these sites, position 1060 is particularly interesting, as it almost corresponds to substitutions that modify the host range and/or cell tropism in mhv and ibv 26, 27 . both viruses belong to the coronavirus genus. the beaudette strain of ibv has been adapted to embryonated chicken eggs; following passages in culture, the strain was further adapted to infect vero cells (from african green monkey) and primary chicken kidney cells 26 . the l857f mutation was shown to represent a major determinant of the fusogenic activity in these cell types 26 table 2 . likelihood ratio test statistics for models of variable selective pressure among sites in mers-cov isolates. 1 m1a is a nearly neutral model that assumes one dn/ds (ω ) class between 0 and 1, and one class with ω = 1; m2a (positive selection model) is the same as m1a plus an extra class of ω > 1. 2 m7 is a null model that assumes that 0 < ω < 1 is beta distributed among sites; m8 (positive selection model) is the same as m7 but also includes an extra category of sites with ω > 1. 3 2δ lnl: twice the difference of the natural logs of the maximum likelihood of the models being compared. 4 positions are relative to the mers-cov sequence (emc/2012). mutation was recovered after passages in mouse liver and was shown to contribute significantly to the hepatotropism and hepatic virulence of a previously attenuated strain (mhv-a59) 27 . additional variants in the hr1 and fusion peptide of mhv strains cooperate with changes in the s1 region, resulting in a broadening of receptor usage (to heparan sulfate) and, consequently, an extension of the host range 36 . finally, in the mhv-a51 strain the ability to bind human caecam receptors is strongly influenced by mutant residues located in the fusion peptide, hr1 and hr1/hr2 linker 37 . similar observations have been reported for viruses that do not belong to the coronavirus genus, but that use a class i fusion protein. for instance, one single mutation in the hr1 of a simian-human immunodeficiency virus (shiv) strain (kb9) increases by two-to three-fold infection efficiency in cells expressing the marmoset cellular receptors 38 , whereas a nearby hr1 change in siv contributes to macrophage tropism 39 . overall, these findings pinpoint the relevance of changes in the hr1 and hr2 as modifiers of host range and cell-type tropism. the molecular mechanisms underlying the altered phenotype of hr1 and hr2 mutants remain to be determined in all these instances, although changes in conformational structure have been suggested as a possible explanation. unfortunately, no coronavirus s protein fusion intermediate or pre-fusion state has been solved to date, hampering investigation of molecular interactions. we therefore analyzed the effect of variation at the 1020 position of mers-cov on the stability of the 6hb in the post-fusion conformation 14 . the presence of a q1020 had previously been suggested to confer higher stability to the mers-cov 6hb compared to sars-cov 14 . indeed, replacement of this residue results in loss of intraand inter-helical interactions. in line with these observations, three different methods used for stability analysis were concordant in showing that the alternative arginine and histidine residues at position 1020 result in a moderate and similar level of destabilization. although the observed δ δ g is relatively small, it was calculated on the single monomer, and is expected to be multiplied in the trimer. the observation whereby mildly destabilizing variants are favored by selection may seem counterintuitive. nonetheless, we show that a similar level of destabilization is observed for a mutation in mers-cov hr1 (t1015n) that increases infection efficiency, at least in vitro 31 . mutagenesis of hr1 in retroviral type i fusion proteins has indicated that, whereas strong destabilization of the 6hb (as measured by circular dichroism) almost inevitably results in reduced infectivity, a minor stability decrease is not necessarily associated with defects in cell fusion and infection efficiency [40] [41] [42] . for instance, different aminoacid replacements at the same hr1 position in hiv-1 gp41 result in decreased stability, but unaffected or even increased infectivity 40 . in the case of eiav (equine infectious anemia virus), destabilized hr1 mutants were found to display different infection phenotypes depending on temperature 42 . this observation may be extremely interesting in the context of mers-cov, as both bats and dromedary camels display adaptive heterothermy (i.e. sensible daily or season variation in body temperature). coronavirus spike proteins are highly exposed on the virus surface and represent major targets for antibody response 43 , raising the possibility that adaptive evolution of the s protein is driven by the host immune system. this hypothesis is difficult to address due to the paucity of information concerning the specific mers-cov epitopes recognized by human antibodies. recently, different studies identified human antibodies againts mers-cov from non-immune human antibody libraries: all of them were directed against the rbd, suggesting that this region represents a major target for the host immune system 43 . nevertheless, data on the humoral immune response to mers-cov in infected subjects are presently lacking, whereas such information is richer for sars-cov. indeed, analysis of antibodies derived from a patient who recovered from sars indicated that some of them recognize epitopes in the hr2 region 44 . their neutralizing effect was ascribed to interference of the interaction between hr2 and hr1. whether antibodies against the s2 region also arise in human subjects (or other mammalian hosts) infected with mers-cov and related betacovs remains to be determined; if this were the case, some of the selected sites we identified may be under selective pressure to evade recognition. finally, it is worth noting that hrs have been studied in different viruses because synthetic peptides interfering with 6hb formation are promising antiviral molecules [15] [16] [17] [18] . this is also the case for mers-cov, and hr2-like peptides were recently shown to be effective in vitro 14 . these peptides were tested against a mers-cov strain carrying q1020 and all include the interacting m1266 residue 14 . these antivirals may display decreased activity depending on the mers-cov strain and its aminoacid status at the selected 1020 position. sequences and alignments. virus sequences were retrieved from the ncbi database and a list of accession numbers is provided as supplementary table s1 . sequences of ty-batcov hku4 and pi-batcov hku5 isolated in hong kong were derived from a previous work 28 . errors in the inferred multiple sequence alignment (msa), which may be common when highly divergent sequences are analyzed, can inflate estimates of positive selection. we therefore used prank 45 for building the msa and guidance 46 for filtering unreliably aligned codons (i.e. we masked codons with a score < 0.90), as suggested 35 . detection of recombination and positive selection. to detect positive selection at the s gene of clade c betacovs we applied the branch-site test from the paml suite 22 . the test compares a model (ma) that allows positive selection on one or more lineages (foreground lineages) with a model (ma1) that does not allow such positive selection. twice the difference of likelihood for the two models (δ lnl) is then compared to a χ 2 distribution with one degree of freedom 22 . specifically, the internal branches of previously reconstructed 6 bayesian phylogenies of the s1 and s2 regions were set as the foreground lineages in independent tests. a false discovery rate (fdr) correction was applied to account for multiple hypothesis testing (i.e. we corrected for the number of tested branches), as suggested 47 . positively selected sites were identified through the beb analysis (with a p value cutoff of 0.90), which calculates the posterior probability that each site belongs to the site class of positive selection on the foreground branch(es). sites were validated using meme (with the default cutoff of 0.1), which allows the distribution of dn/ds (also referred to as ω ) to vary from site to site and from branch to branch at a site, therefore allowing the detection of episodic positive selection 24 . the site models implemented in paml were applied -independently-for the analysis of hku4 and mers-cov sequences, which display very limited divergence and do not suffer from saturation problems. to detect selection, site models that allow (m2a, m8) or disallow (m1a, m7) a class of sites to evolve with ω > 1 were fitted to the data 48 . trees were generated by maximum-likelihood using the phyml program 49 with a gtr model of nucleotide substitution and γ distributed rates. positively selected sites were identified using the beb analysis (from model m8) 50 . again, sites were validated using meme. to assure consistency, all models were run using the f3 × 4 and the f61 codon frequency models. msas were screened for the presence of recombination using gard. recombination breakpoints were considered significant if the hk (kishino-hasegawa) p value was < 0.01. simultaneous inference of selection and recombination for analysis of positive selection was performed using omegamap 29 , a program for detecting natural selection and recombination based on a model of population genetics and molecular evolution. the model uses a population genetic approximation to the coalescent with recombination. this latter is estimated from patterns of linkage disequilibrium assuming that recombination events occur only between codons and not within them. omegamap applies reversible-jump markov chain monte carlo (mcmc) to perform bayesian inferences of both ω and the recombination parameter ρ , allowing both parameters to vary along the sequence. an average block length of 10 and 30 codons was used to estimate ω and ρ , respectively. to determine the influence of the choice of the priors on the posteriors, analyses were repeated with alternative sets of priors (supplementary table s2 ). three independent omegamap runs, each with 500,000 iterations and a 50,000 burn-in iteration, were compared to assess convergence and merged to obtain the posterior probability estimate. the rel (random effects likelihood) analysis models variation in both dn and ds across sites according to a predefined distribution with different rate classes; positively selected sites are identified through an empirical bayes method 51 . the default criterion of a bayes factor > 50 was used to identify positively selected sites. for gard, meme, and rel the nucleotide substitution models were chosen using a genetic algorithm implemented in the datamonkey suite 52 . all analyses were performed through the datamonkey server 53 (http://www.datamonkey.org). in silico analysis of hr1 variants. the crystal structure of mers-cov hr1 and hr2 region was obtained from pdb (pdb id: 4mod). histidine or arginine residues were introduced at positions 1020 and suitable rotamers were sampled through the rapid torsion scan utility in maestro (maestro. 9.1; schrodinger). intraprotein interactions were calculated with pic (protein interaction calculator) 54 . because programs that calculate stability changes achieve only moderate accuracy 55 , we used three different methods to assure reliability. these approaches are based on different principles. specifically, popmusic uses statistical potentials and takes into account amino acid volume variation upon mutation 33 ; foldx uses an empirical force field and evaluates the energetic effect of point mutations and the interactions contributing to the stability of proteins 32 . finally, i-mutant 2.0 is based on a neural network approach to evaluate the free energy change after a single point mutation with incorporation of information on the three-dimensional structure of the protein 34 . in foldx and i-mutant the δ δ g values are calculated as follows: δδg = δg mutant − δg wild-type . in foldx and i-mutant δ δ g values > 0 kcal/mol indicate mutations that decrease protein stability, whereas in popmusic δ δ g values > 0 kcal/mol are mark of mutations increasing protein stability. therefore, popmusic δ δ g values were multiplied by − 1 to obtain homogeneous results. in the analysis carried out with foldx 3d, the three-dimensional structure of the protein was repaired using the < repairpdb> command. mutations were introduced using the < buildmodel> command with < numberofruns> set to 5 and < vdwdesign> set to 0. temperature (298k), ionic strength (0.05 m) and ph (7) were set to default values and the force-field was used to predict the water molecules on the protein surface. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs prevalence and genetic diversity of coronaviruses in bats from rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution a predicted receptor-binding and critical neutralizing domain in s protein of the novel human coronavirus hcov-emc recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor hiv-1 inhibition by a peptide functional importance of the coiled-coil of the ebola virus glycoprotein interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides the evolution and genetics of virus host shifts effect of recombination on the accuracy of the likelihood method for detecting positive selection at amino acid sites automated phylogenetic detection of recombination using a genetic algorithm evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level the branch-site test of positive selection is surprisingly robust but lacks power under synonymous substitution saturation and variation in gc detecting individual sites subject to episodic diversifying selection synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells murine coronavirus evolution in vivo: functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus estimating diversifying selection and functional constraint in the presence of recombination spread, circulation, and evolution of the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus the foldx web server: an online force field popmusic 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality 0: predicting stability changes upon mutation from the protein sequence or structure improving the performance of positive selection inference by filtering unreliable alignment regions cooperative involvement of the s1 and s2 subunits of the murine coronavirus spike protein in receptor binding and extended host range amino acid substitutions in the s2 subunit of mouse hepatitis virus variant v51 encode determinants of host range expansion adaptation of the human immunodeficiency virus type 1 envelope glycoproteins to new world monkey receptors mechanisms for adaptation of simian immunodeficiency virus to replication in alveolar macrophages detailed mechanistic insights into hiv-1 sensitivity to three generations of fusion inhibitors the fusion activity of hiv-1 gp41 depends on interhelical interactions structural and biochemical insights into the v/i505t mutation found in the eiav gp45 vaccine strain development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge an algorithm for progressive multiple alignment of sequences with insertions guidance: a web server for assessing alignment confidence scores multiple hypothesis testing to detect lineages under positive selection that affects only a few sites paml 4: phylogenetic analysis by maximum likelihood estimating maximum likelihood phylogenies with phyml accuracy and power of bayes prediction of amino acid sites under positive selection not so different after all: a comparison of methods for detecting amino acid sites under selection codontest: modeling amino acid substitution preferences in coding sequences datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology pic: protein interactions calculator performance of protein stability predictors host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein vice rectorate for graduate studies and scientific research in king saud university (ksu) key: cord-305175-1wg0wodr authors: dolzhikova, i. v.; grousova, d. m.; zubkova, o. v.; tukhvatulin, a. i.; kovyrshina, a. v.; lubenets, n. l.; ozharovskaia, t. a.; popova, o.; esmagambetov, i. b.; shcheblyakov, d. v.; evgrafova, i. m.; nedorubov, a. a.; gordeichuk, i. v.; gulyaev, s. a.; botikov, a. g.; panina, l. v.; mishin, d. v.; loginova, s. y.; borisevich, s. v.; deryabin, p. g.; naroditsky, b. s.; logunov, d. y.; gintsburg, a. l. title: preclinical studies of immunogenity, protectivity, and safety of the combined vector vaccine for prevention of the middle east respiratory syndrome date: 2020 journal: acta naturae doi: 10.32607/actanaturae.11042 sha: doc_id: 305175 cord_uid: 1wg0wodr the middle east respiratory syndrome (mers) is an acute inflammatory disease of the respiratory system caused by the mers-cov coronavirus. the mortality rate for mers is about 34.5%. due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of mers beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of mers and reduce its death toll. we have developed a combined vector vaccine for the prevention of mers based on recombinant human adenovirus serotypes 26 and 5. studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. studies of the cellular immune response in mice after vaccination showed the emergence of a specific cd4(+) and cd8(+) t cell response. a study of the vaccine protectivity conducted in a model of transgenic mice carrying the human dpp4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the mers-cov virus (mers-cov emc/2012, 100ld(50) per mouse). studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing. the middle east respiratory syndrome (mers) is an acute inflammatory disease of the respiratory system that was first diagnosed in june 2012 in saudi arabia [1, 2] . the disease is caused by the mers-cov coronavirus, a member of the genus betacoronavirus of the family coronaviridae. one-humped camels are the natural reservoir of the virus; human infection occurs through contact with camels and consumption of unpasteurized camel milk; an aerosol transmission of infection is also possible [3, 4] . according to the who, a total of 2,458 laboratory-confirmed cases of mers had been registered by september 12, 2019, 848 of which resulted in a fatal outcome (a 34.5% mortality rate) [5] . most mers cases were registered in saudi arabia [6] . however, the disease was also detected in 27 other countries (the united arab emirates, south korea, yemen, etc.); cases of imported infection were reported in europe, north africa, and north america [5] . because of the lack of effective preventive and therapeutic drugs for mers, the high mortality rate of the disease, and the widespread character of the infection reservoir, who experts classify mers-cov as a virus with the potential to cause a pandemic. there have been no cases of mers in russia. however, due to the high mortality of mers and the continuing threat that it could spread outside the endemic areas [5] , development of a vaccine is an urgency. vaccination can limit the spread of mers and reduce its mortality [7] . to date, several candidate vaccine preparations based on a protective antigen, mers-cov s glycoprotein and its derivatives (s1 subunit, receptor-binding domain), are known: vector vaccines (based on recombinant adenoviruses and vaccinia virus), a dna vaccine based on plasmid dna, as well as vaccines based on recombinant proteins and virus-like particles [8] [9] [10] [11] [12] [13] [14] [15] . since the formation of a humoral and cellular immune response is important to protect against mers-cov, the use of recombinant viral vectors for antigen delivery seems promising for the development of anti-mers vaccines. these vectors provide long-term expression of the antigen in the cells of the immunized organism, which results in a protective immune response as early as after the first or second immunization. repeated vaccination is effective in inducing the most pronounced and lasting immune response, while heterologous vaccination involving the use of different viral vectors for primary and secondary immunization is the most optimal regimen. this regimen was successfully implemented in the development of a vaccine against the disease caused by the ebola virus; the vaccine has been registered in the russian federation for medical use and already undergone post-registration clinical trials in the african republic of guinea [16] . we have developed a combined vector vaccine for the prevention of mers based on recombinant human adenovirus serotypes 26 and 5 expressing mers-cov glycoprotein (mers-cov emc/2012 isolate). here, we present the results of a study of the post-vaccination humoral and cellular immune responses in mice and primates, as well as the results of preclinical studies of the safety of the developed vaccine against mers. both components are lyophilisates for the preparation of solutions for intramuscular administration. the drug was obtained in compliance with the conditions of biotechnological production at the medgamal branch of the gamaleya national research center for epidemiology and microbiology of the ministry of health of the russian federation. all experiments on animals were carried out in strict accordance with the recommendations of the national standard of the russian federation (gost r 53434-2009, "principles of good laboratory practice"). sixweek-old female c57bl/6 mice (18-20 g) were purchased from the pushchino breeding facility (russia). transgenic f1 hybrid mice were obtained by crossing transgenic homozygous +/+ males carrying the human dpp4 receptor gene (hdpp4) (medical university of texas, usa) and non-transgenic c57bl/6 females (pushchino, russia). expression of the transgene in f1 hybrid mice was confirmed by immunoblotting. all mice had free access to water and food and were housed in an isocage animal housing system (tecniplast, italy). common marmosets (callithrix jacchus) were born and kept in a specialized animal facility at the chumakov federal scientific center for research and development of immune-and-biological products ras (moscow, russia). the animals were kept at the laboratory for modeling immunobiological processes with the experimental clinic of callitrichidae (chumakov federal scientific center for research and development of immune-and-biological products ras) in accordance with the requirements for housing laboratory primates. all experimental procedures with marmosets were carried out by a specialist who had received certification from the federation of european laboratory animal science associations (felasa) and completed a course on working with primates ("laboratory animal science for researchers: non-human primates," karolinska institute, stockholm, sweden). all animals were identified by a radio chip implanted subcutaneously and having a unique 15-digit code (globalvet, moscow). the mice were immunized intramuscularly using the widest possible dose range, 5×10 11 to 10 5 v.p. per mouse. immunization was carried out twice successively with component 1 and then component 2 with a 21-day interval. mouse serum samples were collected at the following time points: 14 and 28 days, three and six months after immunization. the marmosets were immunized intramuscularly at a dose of 10 11 v.p. per animal. immunization was conducted twice successively with component 1 and then component 2 with a 21-day interval. plasma samples were collected at the following time points: before immunization, seven and 24 days, as well as three and six months, after immunization. the titer of glycoprotein-specific antibodies in serum/ plasma was determined by enzyme immunoassay. the following recombinant proteins were used: s glycoprotein (40069-v08b; sino biological, china) and rbd (40071-v08b1; sino biological). a pbs solution in 0.1% tween-20 (pbs-t) containing 5% non-fat dry milk (a0830; applichem, spain) was used for blocking. serum/plasma was titrated in two steps in a pbs-t solution containing 3% non-fat dry milk. anti-mouse igg horseradish peroxidase-linked secondary antibodies (nxa931; ge healthcare, usa) were used to detect mouse igg. serum of a rabbit immunized with marmoset igg and anti-rabbit igg horseradish peroxidase-linked secondary antibodies (na934v; ge healthcare, usa) were used to detect marmoset igg. a tetramethylbenzidine solution (niiopik, russia) was used as a chromogenic agent. the reaction was stopped by adding 1 m h 2 so 4 ; optical density was measured at 450 nm (od 450 ) using a multiskan fc microplate reader (thermo fisher scientific, usa). the igg titer was defined as the maximum serum dilution at which the od 450 value of the serum sample from the immunized animal exceeded that of the control serum/plasma (serum/plasma of the control animal or animal before immunization) more than twofold. the titer of virus-neutralizing antibodies (vnas) in the plasma of immunized animals was determined in a neutralization reaction (nr) by suppressing the cytopathic effect caused by the mers-cov virus (mers-cov emc/2012) in the monolayer of vero b cells. the neutralization reaction was carried out in the "constant viral dose/serum dilution" mode. monkey plasma was incubated at 56°c for 30 min to remove non-specific inhibitors. all serum samples were diluted in a dmem medium supplemented with 2% inactivated fetal bovine serum, starting from the 1 : 10 ratio, then with two-fold dilution to 1 : 5,120. dilutions of the mers-cov virus suspension were prepared in a dmem medium supplemented with 2% inactivated fetal bovine serum. the concentration of the mers-cov virus in the prepared dilution was 1,000 tcid 50 /ml. a mixture of equal volumes of plasma and the virus suspension was incubated at 37°c for 60 min. vero b cells were plated in 96-well plates at 4 × 10 4 cells per well at a volume of 100 µl and then supplemented with 100 µl of the mixture of plasma and the virus suspension. the cytopathic effect was assessed after four days. the vna titer of the studied plasma was defined as its highest dilution at which the cytopathic effect was suppressed in two out of three wells (compared to the control serum samples). the mice were euthanized on day eight after immunization. the spleens were collected and homogenized through a 100-µm sieve in sterile pbs. splenocytes were isolated by ficoll (1.09 g/ml; paneco, russia) density gradient centrifugation (800 g, 30 min). for t cell proliferation assay, the splenocytes were stained with carboxyfluorescein using the carboxyfluorescein succinimidyl ester (cfse) tracer kit (invitrogen, usa) as previously described [17] . cells were seeded in 96-well plates at 2 × 10 5 cells per well in a rpmi 1640 medium and re-stimulated with the recombinant mers-cov s protein (40069-v08b; sino biological) at 1 µg per well. after 72 h, the media were collected for a ifn-gamma analysis and the cells were harvested, washed with pbs, stained with antibodies specific to cd3, cd4, and cd8: allophycocyanin (apc)-labelled anti-cd3, apc-cy7-labelled anti-cd8, and phycoerythrin-labelled anti-cd4 (bd biosciences, usa), and then fixed in 1% paraformaldehyde. proliferating cd4 + and cd8 + t lymphocytes were evaluated in the cell mixture using a bd facs aria iii flow cytometer (bd biosciences). the resulting percentage of proliferating cells (x) was determined using the following formula: x = %st -%, where %st is the percentage of proliferating cells after splenocyte re-stimulation with recombinant mers-cov s glycoprotein, and % is the percentage of proliferating cells in the absence of splenocyte re-stimulation (intact cells). the concentration of ifn-gamma in the medium was measured by elisa using a commercial kit (mouse ifn-γ elisa kit; invitrogen) according to the manufacturer's protocol. the increase in the concentration of ifn-gamma was determined using the following formula: x = cst/cint, where x is the fold increase in the concentration of ifn-gamma, cst is the concentration of ifn-gamma in the medium of stimulated cells (pg/ ml), and cint is the concentration of ifn-gamma in the medium of unstimulated (intact) cells (pg/ml). the protective efficacy of the vaccine was studied in a model of lethal infection in transgenic mice carrying the human dpp4 receptor gene and obtained by crossing homozygous transgenic hdpp4 +/+ males and non-transgenic c57bl/6 females. the animals were immunized intramuscularly twice successively with component 1 and then component 2 with a 21-day interval. seven days after the injection of component 2, the mice were infected intranasally with the mers-cov virus (mers-cov emc/2012) at a dose of 100 ld 50 per animal, and then the survival rate was analyzed for a period of 30 days. preclinical studies of the safety of the combined vector vaccine against mers were conducted in collaboration with the autonomous non-commercial organization "institute of biomedical research and technology" and fsaei he i.m. sechenov first moscow state medical university, in compliance with the guidelines for preclinical trials of medicinal products [18] and guidelines for experimental (preclinical) study of new pharmacological substances [19] . the safety study included the analysis of the toxicity of a single and repeated administration, as well as sn assessment of the reproductive and ontogenetic toxicity, immunogenicity, and allergenicity. a total of 670 mice, 725 rats, 24 rabbits, 120 guinea pigs, and six common marmosets were used in the preclinical safety study. tolerability of the vaccine in primates was analyzed daily by assessing the physical condition of the animals and based on the presence of general symptoms of in-toxication, which included an assessment of behavior, appearance, and physiological functions. vaccine tolerance in marmosets was studied in the laboratory by monitoring the body weight, rectal temperature, and blood biochemical parameters: total bilirubin, direct bilirubin, aspartate aminotransferase (ast), alanine aminotransferase (alt), creatinine, total protein, and alkaline phosphatase (alp). the studies were carried out on fully automatic analyzers ca-180 and b-200 (furuno, japan) using diasys reagent kits (germany). statistical analysis of the data was performed using the graphpad 7.0 software. either the student's t-test for independent samples or the mann-whitney u-test was used for the analysis of the data of unpaired samples depending on the data distribution normality [20] . either the student's t-test for paired samples or wilcoxon's test was used for the analysis of the data of related samples depending on the data distribution normality [20] . distribution normality was determined using the generalized d'agostino-pearson test [21] . in order to select an effective dose, mice were immunized intramuscularly with the vaccine at doses of 10 5 -10 10 , 5 × 10 10 v.p. per mouse; serum samples were collected, and the titers of glycoprotein-specific antibodies were analyzed two and four weeks after immunization. next, the intensity of the post-vaccination humoral immune response was assessed based on the titer of glycoprotein-specific igg (fig. 1) . analysis of the obtained results demonstrates a dose-dependent increase in the serum titer of glycoprotein-specific igg. the minimum dose of the combined vector vaccine required to induce a robust humoral immune response was 10 6 v.p. per mouse for all vaccinated animals. analysis of the duration of post-vaccination humoral immunity showed that glycoprotein-specific antibodies were detected at a high titer in the mouse serum six months after immunization (the geometric mean titer was 1 : 182,456, fig. 2) . next, we studied the level of humoral immunity in primates vaccinated with the developed vaccine. in order to determine the level of humoral immunity in common marmosets (c. jacchus), they were immunized with the combined vaccine according to the regimen intended for clinical use, i.e. successively with component 1 (at a dose of 10 11 v.p. per animal) and then com-ponent 2 (at a dose of 10 11 v.p. per animal) with a 21-day interval. further, plasma samples were collected from animals for the analysis of the titer of glycoproteinspecific igg seven and 24 days, as well as three and six months, after the boosting of immunization (fig. 3a) . immunization of primates was shown to induce robust humoral immunity, which persists for at least six months. for instance, the titers of glycoprotein-specific igg in primates six months after immunization did not differ from the titers after three months, which is an indication of the induction of long-term immunity. analysis of the titer of neutralizing antibodies to the mers-cov virus in the plasma of immunized monkeys showed that vnas were detected in the animals as early as seven days after booster immunization, while virus-neutralizing antibody titers are shown on the ordinate axis; time after immunization is represented on the abscissa axis. individual titers for each studied animal and the geometric mean titer (n = 3) are indicated the maximum vna titer was reached three and six months after immunization (fig. 3b) . no vnas were detected in the plasma of the control animals and the animals before immunization. thus, our analysis of the level of post-vaccination immunity showed that immunization of mice and primates induces a robust humoral immune response, which persists for at least six months after immunization. in order to assess the level of post-vaccination cellular immunity, the mice were immunized with the candidate vaccine against mers once at a dose of 10 7 v.p. per mouse. spleens were collected from the animals 18 days after immunization; splenocytes were isolated, and the number of proliferating cd4 + and cd8 + t lymphocytes was determined in the splenocyte culture in vitro after cell re-stimulation with the recombinant mers-cov s protein (fig. 4) . the obtained data demonstrate that introduction of the combined vector vaccine induces the formation of s-specific cd4 + and cd8 + t cells. activation of cellular immunity was also analyzed by measuring the expression of ifn-gamma. the results of the study of an increase in the ifn-gamma concentration in an in vitro culture of mouse splenocytes after repeated stimulation of the cells with the recombinant mers-cov s protein are presented in fig. 5 . administration of the vaccine increased the concentration of ifn-gamma in the medium upon stimulation of the splenocytes of immunized mice with the mers-cov s glycoprotein. the concentration of ifn-gamma in the medium increased by an average of 22 times. summarizing the data of our analysis of the antigenspecific lymphoproliferative activity of cd4 + and cd8 + t cells and the level of ifn-gamma expression by restimulated splenocytes, we can conclude that immunization of animals with the combined vaccine against mers results in the formation of glycoprotein-specific cellular immunity. the study was carried out in a model of lethal infection caused by mers-cov in transgenic mice carrying the human dpp4 receptor gene. mice were immunized successively with component 1 and then component 2 with a 21-day interval. one week after administration of component 2 of the vaccine, animals were infected intranasally with the mers-cov virus (mers-cov emc/2012) at a dose of 100 ld 50 per animal, and the survival rate was assessed during 30 days. immunization of the animals with the combined vector vaccine was shown to protect 100% of animals from lethal infection caused by the mers-cov virus. all control (unvaccinated) animals died (fig. 6) . irritation effect, immunotoxicity, allergenic properties, and reproductive toxicity) were evaluated in rodents (mice, rats, and guinea pigs) and large animals (rabbits). the combined vector vaccine against mers did not cause any toxic effects, did not have an allergenic or immunotoxic effect, did not affect the generative function, did not have a local irritation effect, and can be recommended for clinical studies. vaccine tolerability was also studied in primates. no abnormalities in the analyzed parameters of physical condition (behavioral reactions, appearance, and physiological functions) were found in the animals immunized with the combined vector vaccine against mers and the control animals during the observation period. rectal temperature, changes in body weight, and biochemical parameters were within the normal range for the species in all animals during the experiment (fig. 7) . summarizing the obtained data, we can conclude that the combined vector vaccine against mers has shown good tolerability in the common marmoset model. currently, there are no specific prophylactic and therapeutic agents against the middle east respiratory syndrome in the world. intensive studies on the development of vaccines for this disease are currently underway in the united states, germany, korea, china, great britain, and other countries. among the prophy-lactic drugs with the highest efficiency demonstrated in preclinical studies, the following candidate vaccines can be mentioned: vaccines based on adenoviral vectors (ad5, ad41, chadox1) [22, 23] , modified vaccinia virus ankara (mva) [24] encoding mers-cov protective antigen s, as well as preparations of recombinant mers-cov protective antigen s [25, 26] . two drugs are currently undergoing clinical trials: two vaccines based on recombinant viral vectors mers001 (based on chimpanzee adenovirus, phase 1) [27] and mva-mers-s (based on vaccinia virus, phase 2) [28] . clinical studies of the first phase of a vaccine based on plasmid dna (gls-5300), as well as a vaccine based on a vaccinia virus (mva-mers-s), have been completed [29, 30] . all vaccines that have reached clinical trials are based on mers-cov s glycoprotein. this glycoprotein performs one of the most important roles in the viral life cycle: it enables virus internalization via interaction with the dpp4 receptor on the cell surface. neutralization of this interaction limits penetration of the virus into the cell, thus decreasing its replication. since the formation of not only a humoral, but also cellular immune response is important for protection against mers, the development of vaccines based on recombinant viral vectors seems promising. such vectors effectively deliver antigen-encoding genetic material to the cells, which results in the cellular expression of the antigen and induction of a robust cellular and humoral immunity. an important property of recombinant viral vectors is that they induce protective immunity as early as after the first or second immunization, which is extremely important when developing a vaccine for the prevention of dangerous and extremely dangerous infections and is intended for use during an epidemic or in the case of an infection that spreads beyond non-endemic areas. we have conducted a study of the immunogenicity of various forms of mers-cov s glycoprotein: fulllength glycoprotein (s), full-length glycoprotein with the transmembrane domain of the g protein (s-g) of the vesicular stomatitis virus, a secreted glycoprotein receptor-binding domain (rbd), a secreted glycoprotein rbd fused to the fc fragment of human igg1 (rbd-fc), and the membrane form of the glycoprotein rbd (rbd-g) [31] . the obtained data demonstrated that the membrane form of the rbd is the most effective in inducing a robust cellular immune response, while full-length glycoprotein is most efficient in inducing a robust cellular immunity. when choosing an immunization regimen, one should take into account the fact that repeated heterologous vaccination, which involves the use of two different recombinant viral vectors for primary and secondary immunization, is advisable for inducing long-term immunity. for this studies of the immunogenicity of the combined vector vaccine revealed the induction of long-term humoral immunity in mice, while the mean titer of glycoprotein-specific antibodies equaled 1 : 121,775 two weeks after vaccination at a dose of 10 7 v.p. per mouse. a similar antibody titer was observed by alharbi et al. in mice 28 days after immunization with a vaccine against mers based on chimpanzee adenovirus chadox1 mers [12] ; however, the authors used a dose of 10 8 v.p. per mouse for immunization. in another study by munster et al. [13] , immunization of transgenic mice carrying the human dpp4 receptor gene with a chadox1 mers vaccine at a dose 10 8 v.p. per mouse was shown to protect 100% of the animals from a lethal infection with mers-cov. hashem et al. developed a rad5-based drug carrying the mers-cov s1 sequence and demonstrated that repeated immunization of mice with the drug at a dose of 10 9 v.p. per mouse induced a humoral immune response [32] . the titer of glycoprotein-specific igg was 1 : 70,000 three weeks after the second immunization (one and a half months from the beginning of immunization); the drug also provided 100% protection to animals from mers-cov infection [32] . glycoprotein-specific antibodies were found at a titer range of 1 : 25,600 to 1 : 102,400 in the plasma of the animals as early as a week after the boosting of immunization. it is important to note that muthumani et al. [33] detected glycoprotein-specific antibodies at a titer of 1 : 20,000 for a period of six weeks in primates after long-term thrice immunization with a dna vaccine; the authors also showed that immunization of primates with the dna vaccine protects them from a mers-cov infection. the study of post-vaccination humoral immunity in mice and primates demonstrated that an intense humoral immune response persists in the animals for at least six months after vaccination. analysis of the cellular component of the immunity in the mice showed that administration of the developed vaccine induces a robust cellular response. it is important to note that not only a cd4 + but also cd8 + t cell response is observed, which can play an important role in protection against mers-cov [34, 35] having completed studies of the immunogenicity of the combined vector vaccine in a model of lethal infection in transgenic mice carrying the human dpp4 receptor gene, we studied the protective effect of the vaccine. the vaccine was shown to provide 100% protection to animals from lethal infections of mers-cov. our series of preclinical studies of vaccine safety revealed no contraindications for the clinical testing of the developed vaccine. in this work, we have studied the immunogenicity and safety of a combined vector vaccine for the prevention of the middle east respiratory syndrome. the following conclusions were obtained: vaccination of animals with the vaccine induces a robust humoral immune response to the mers-cov s glycoprotein persisting for at least six months. vaccination of animals induces a robust cellular immune response to the mers-cov s glycoprotein. vaccination of animals induces a protective immune response, which protects 100% of animals from a lethal infection of mers-cov. preclinical studies of the vaccine safety did not reveal any contraindications to clinical testing. middle east respiratory syndrome coronavirus (mers-cov) annual review of diseases prioritized under the research and development blueprint) international multicenter study of the immunogenicity of medicinal product gamevac-combi. clinicaltrials.gov identifier: nct03072030 guidelines for preclinical trials of medicinal products. // moscow: grif i k guidelines for experimental (preclinical) study of new pharmacological substances // moscow: medicine // ekologiya cheloveka with the english safety and immunogenicity of a candidate mers-cov vaccine (mers001) phase ib study to assess the safety and immunogenicity of mva-mers-s_df-1. clinicaltrials.gov identifier: nct04119440 url phase i, open label dose ranging safety study of gls-5300 in healthy volunteers clinicaltrials.gov identifier: nct02670187 url proc. natl. acad. sci. usa. 2014. v. 111. № 13 key: cord-024569-d9opzb6m authors: seo, mihye title: amplifying panic and facilitating prevention: multifaceted effects of traditional and social media use during the 2015 mers crisis in south korea date: 2019-07-26 journal: journal mass commun q doi: 10.1177/1077699019857693 sha: doc_id: 24569 cord_uid: d9opzb6m in the context of the 2015 middle east respiratory syndrome (mers) outbreak in south korea, this study examines the multifaceted effects of media use considering the current complex media environment. analysis of a two-wave online panel survey found that traditional media use had a positive influence on mers knowledge while social media use did not. however, knowledge did not facilitate preventive behaviors. in contrast, negative emotional responses due to media use stimulated desirable behaviors. furthermore, social media use directly influenced behavioral responses but traditional media use did not show the same effects. different functions of traditional and social media during an epidemic are discussed. changed that understanding considerably (petersen, hui, & zumla, 2015) . this unforeseen crisis induced not only morbidity and mortality but also fear and panic in korea. in fact, the panic epidemic caused more widespread damage than the disease itself by slowing the economy and interfering with people's daily routines. in times of crisis, the importance of the media is heightened. government and responsible organizations consider media to be an essential part of crisis management (reynolds & seeger, 2005) , and the public relies on the media to make sense of confusing or chaotic situations (tai & sun, 2007; zhang, kong, & chang, 2015) . given the importance of media in times of crisis, scholarly attention has been largely paid to the following questions: (a) how government and other organizations work (or should work) with media to prepare for and respond to crises and (b) how the media reports (or should report) on crises. relatively less attention, however, has been paid in existing research to examining informational, emotional, and behavioral consequences of individuals' media use in times of crisis. as the importance of social media has risen in general, its importance in the context of crises has also increased. evidence shows that many people turn to social media to seek crisis-related information, such as safety instructions and news updates (veil, buehner, & palenchar, 2011) , which stands to promote proper behavioral responses to facilitate effective crisis management. however, both researchers and practitioners caution that media-social media in particular-may create misperceptions and amplify public fears by fostering public panic and proliferating unverified information (kasperson, 1992) . in comparison to traditional media, social media use is particularly susceptible to the aforementioned concerns due to enhanced speed of information transmission and distinctive features of open access platforms (zeng, starbird, & spiro, 2016) . in the context of the 2015 mers outbreak in korea, this study provides an empirical examination of the multifaceted effects of media use in times of crisis in the complex and dynamic media environment of today. using two waves of online panel data collected at two different time points during the mers crisis, i investigate how individuals' traditional and social media use during the crisis produced various consequences, including increased mers knowledge, negative emotions such as fear and anxiety, and direct and indirect facilitation of mers preventive behaviors. i also scrutinize the differences in these effects caused by traditional and social media use. the term crisis is defined as "some breakdown in a system that creates shared stress" (coombs, 2014, p. 2) , which includes a very broad range of situations. an infectious disease outbreak is a typical example of crisis in the public health context. prior research has focused on how governments or other responsible organizations can achieve positive relationships with the public in managing a particular crisis. based on the organization-public relationship (opr) approach, scholars have theorized and investigated various (pre)conditions, attributes, and communication strategies of organizations to bring about positive relational outcomes with the public, such as satisfaction, commitment, trust, and mutual understanding (s.-u. yang, 2018) . with respect to the mers crisis in korea, s.-u. yang (2018) showed that the government's lack of dialogic competency negatively affected government-public relationships. those findings indicate that the korean government's lack of mutuality and openness weakened the credibility of its risk information, which produced negative relational consequences such as distrust and dissatisfaction and the intent to dissolve the relationship. cooperation with the media on the part of government and responsible organizations is a major portion of effective crisis management processes (coombs, 2014) . from a crisis management perspective, prior research has mainly focused on how to understand and work with the media to accomplish various goals (reynolds & seeger, 2005) . for instance, researchers have identified the kinds of communication strategies that work best to reduce public-relations damage and generate compliance with desired behaviors in hazardous situations (glik, 2007) . for instance, seeger, reynolds, and sellnow (2009) emphasized the importance of coordinating specific communication tasks for each crisis phase in the context of hurricane katrina and the h5n1 outbreak. based on the existing literature and case studies, scholars have also attempted to provide guidelines for best practices in crisis communication (veil et al., 2011) , which could also be used as evaluative criteria in crises (plattala & vos, 2012) . another line of research focuses on how media channels cover crises by analyzing the content of crisis reporting and discussing its implications. as manifested by terms such as disaster marathon (liebes, 1998) , the unexpected and impending nature of crises triggers media hype, which produces a prolific amount of reporting. much research has investigated the characteristics of crisis coverage (shih, wijaya, & brossard, 2008) . shih and colleagues (2008) , for instance, found that the coverage of epidemics showed common patterns across discrete diseases, such as a high eventbase and emphasis on newly identified cases and government actions. with respect to the mers crisis in korea, jin and chung (2018) performed semantic network analysis of korean and foreign media coverage of the crisis. they examined the most frequently used words (e.g., patient, hospital, infection, government, and case) and concluded that korean media focused heavily on the number of cases and the government's responses, consistent with shih and colleagues' findings (see kwon, 2016, for similar findings) . based on content analyses of crisis reporting, past research has also identified persistent problems in crisis reporting, such as excessiveness (rezza, marino, farchi, & taranto, 2004) , inaccuracy (auter, douai, makady, & west, 2016) , and sensationalism (moeller, 1999) . korean media's mers reporting was not exempt from sensational and excessive coverage of the contagious nature of the disease and patient counts (kim, 2016; kwon, 2016) . as population mobility and trade in goods and services have increased, newly emerging infectious diseases have become global public health concerns. some emerging infectious diseases have derived from a known infection, such as influenza, and have spread into new populations. the mers outbreak in korea can be understood as one such example. an outbreak of infectious disease causes not only human casualties but also massive economic harm. different from chronic health risks, infectious pandemics trigger spontaneous and intense media attention (posid, bruce, guarnizo, taylor, & garza, 2005) , which could create cascading effects in various public responses. however, relatively little empirical research has considered the various consequences of media use by individuals during a public health crisis. one of the most desirable public responses to a public health crisis is engaging in preventive behaviors (mitroff, 2004) . public adoption of precautionary behaviors is critical to preventing large outbreaks of infectious disease, particularly in densely populated countries such as korea. people need to behave in ways that prevent the spread of infectious disease and its consequences, and the media plays an important role in facilitating those behaviors (gammage & klentrou, 2011; zhang et al., 2015) . learning from media is one potential pathway to engagement in preventive behaviors (sayavong, chompikul, wongsawass, & rattanapan, 2015) . besides cognitive responses, another way to galvanize preventive behaviors could be through emotional responses, which could alarm people enough to take proper actions with respect to a given risk. research on risk and health communication has offered various theoretical models and empirical evidence for each approach (boer & seydel, 1996; griffin, dunwoody, & neuwirth, 1999 ). yet, there has been little research testing and comparing the two potential paths to preventive behaviors in the context of a pandemic crisis. first, media use could increase knowledge about a crisis, which could stimulate the public to enact preventive behaviors. the heavy emphasis on knowledge is largely drawn from the traditional knowledge deficit model of communication (rutsaert et al., 2014) , which claims that a lack of understanding is the major obstacle to reasonable public responses to a risk or crisis. therefore, it accentuates the scientific knowledge transfer from experts to the layperson and media have been regarded as a major conduit of knowledge transfer (hilgartner, 1990) . thus, when a crisis happens, government and responsible organizations attempt to work with media to disseminate crisis-related information, and the general public turns mainly to the media to acquire the information to deal with the atmosphere of uncertainty. despite that widespread expectation, relatively little empirical attention has been given to whether public crisis knowledge is indeed increased by media use or whether understanding of a crisis indeed facilitates preventive behaviors in times of crisis. media is known to be more suitable for diffusion of knowledge than other channels, such as interpersonal communication (price & oshagan, 1995) . prior research shows that media use increases health knowledge in the general public, which in turn encourages desirable health behaviors (gammage & klentrou, 2011; sayavong et al., 2015) . little empirical evidence, however, has been collected in the context of urgent public health crises such as epidemics. on the contrary, disaster studies have extensively examined individual and group responses to impending threats (e.g., natural disasters or terrorist attacks). according to that body of research, in the face of an impending threat, people become more sensitive to cues about social environments and engage in searches for information as a basis for protective behaviors (lindell & perry, 2004) . however, despite the known contribution of media channels to these disaster research models, media variables have received insufficient attention in explaining the behavioral responses of individuals to crises. second, media use could stimulate proper behavioral responses via mobilizing information (mi). in the health communication literature, mi is designed to encourage a specific health behavior (friedman & hoffman-goetz, 2003) . applied to a crisis context, mi offers specific "how to" and "where to" information, such as checklists for preparedness supplies, evacuation information, phone numbers or websites for further information, or specific instructions for precautionary behaviors, meant to encourage people to take specific actions (tanner, friedman, & barr, 2009) . facing a crisis, the public needs to learn about both the nature of the crisis and how to mitigate its effects and defend themselves (guion, scammon, & borders, 2007) . a handful of prior studies in the communication discipline have documented the direct and indirect effects of media use on preventive behaviors via knowledge in a crisis situation. ho (2012) , for example, found that attention to newspaper and television news increased public knowledge about the h1n1 pandemic. zhang et al. (2015) also found that media use potentially influenced h1n1 preventive behaviors through fear and perceived knowledge. the results of 36 national surveys in the united kingdom indicate that exposure to media coverage or advertising about swine flu increased the adoption of recommended preventive behaviors (g. rubin, potts, & michie, 2010) . lin and lagoe (2013) also showed that tv and newspaper use increased h1n1 risk perception and vaccination intent in media users. based on those discussions and findings, it is expected that media use will facilitate public understanding of an emerging infectious disease and encourage appropriate precautionary behaviors. media use in large-scale emergencies, however, still requires empirical scrutiny because of the many unexpected twists that characterize fluid crisis situations. human beings facing a crisis often experience a range of negative emotions. the intense uncertainty inherent to a crisis situation galvanizes fear, worry, and panic (sandman & lanard, 2003) . in the outbreak of an unfamiliar contagious disease, both the unknown cause and fatal outcome and the interruptions of daily routines and stigma could strengthen negative emotional responses (lee, kim, & kang, 2016) . prior research indicates that individuals often feel more threatened during crises than is warranted by the actual risk level (coombs & slovic, 1979) . according to social amplification theory, the risk people feel when facing a crisis could be amplified or weakened by exchanging various forms of information via the news media or informal networks (renn, burns, kasperson et al., 1992) . once a perceived risk is officially acknowledged, the distortion and exaggeration of information tend follow (song, song, seo, jin, & kim, 2017) , feeding a range of negative emotional responses. prior research has found that the media tends to overemphasize risk and sensationalize crises. for instance, the media overstresses the horrific symptoms of contagious diseases regardless of facts about the prevalence of those symptoms in a time of outbreak (moeller, 1999; ungar, 2008) . the media also tends to focus more on the spread of a disease and the body counts rather than scientific causes (d. rubin & hendy, 1977) . these types of sensationalism can produce disproportionate public fear and panic responses to infectious diseases. scholars such as muzzatti (2005) have gone a step further and demonstrated that the media can actually manufacture threats to public health. some prior works have examined the effects of media use on emotional reactions toward a crisis. hoffner, fujioka, ibrahim, and ye (2002) , for instance, found that people who learned about the september 11 terrorist attacks through mass media were more likely to report negative emotions than those who heard the news interpersonally. they attributed that difference to the nature of the live pictures and content in mass media crisis reporting. people's negative emotional responses as influenced by media use could lead to inappropriate behaviors, such as avoidance of precautionary behaviors or unnecessary or excessive behavioral reactions (liu, hammitt, wang, & tsou, 2005) . however, other convincing literature has claimed that emotions can trigger behavioral responses that are benign and adaptive (baumeister, vohs, dewall, & zhang, 2007) . negative emotional experiences can stimulate people to seek pertinent information (e.g., the risk information seeking and processing (risp) model, griffin et al., 1999) and encourage them to take preventive actions (e.g., protection motivation theory [pmt], boer & seydel, 1996) . unlike the cognition-based approach, which emphasizes the role of knowledge, these theoretical models focus on how negative emotions work as motivational drivers to guide people to protect themselves. empirical work has supported those claims by showing that negative emotion can drive positive behavioral responses, including adopting recommended health behaviors (ruiter, abraham, & kok, 2001) and engaging in information seeking (z. j. yang & kahlor, 2013) . as discussed, korean media coverage of the mers crisis did not deviate wildly from the patterns reported in the literature (kim, 2016; kwon, 2016) . the terms most frequently and centrally mentioned by the major news outlets mainly related to the contagious nature of the disease and patient counting (kwon, 2016) . sensational reporting and delivering government press releases without critical validation were also characteristic of the mers coverage (kim, 2016) . in addition, poor government handling of that crisis reduced the credibility of the information it provided (s.-u. yang, 2018) . against that backdrop, it is worth investigating the potential association between media use related to mers and the negative emotional responses of media users to determine the extent to which they influenced the behavioral responses of those users. along with traditional media channels such as television and newspapers, the importance of social media increases during large-scale events. these trends are particularly salient in the context of crises, which are traditionally marked by high levels of information seeking by the general population. evidence shows that people turn to social media during times of crisis to find information about safety instructions, news updates, and damage reports. increasingly, the public expects even official agents to respond to public requests via social media in times of emergency, concurrent with traditional crisis management (veil et al., 2011) . these heightened expectations are due in large part to perceptions of the benefits of social media for crisis communication. it is believed that social media can accelerate information dissemination in crisis situations by linking end users directly to critical information sources in real-time (hughes & palen, 2012) . during the 2009 h1n1 virus outbreak, people exchanged information and experiences through social media (chew & eysenbach, 2010) . social media, such as twitter, has been used to quickly share initial information and updates during various types of crises, as well as to encourage specific actions, such as volunteering and precautionary behaviors (potts, 2014) . the presence of social media as an information source becomes salient when traditional media provides limited information. for instance, tai and sun (2007) showed that, during the severe acute respiratory syndrome (sars) epidemic in 2003, chinese people turned to the internet and message services to find information unavailable from the traditional media, which operate under close censorship by the chinese government. likewise, at the beginning of the mers crisis, the korean government withheld the list of hospitals affected by mers, and the mainstream media adhered to the information embargo requested by the government. limited access to needed information fueled fear, so people turned to social media instead of traditional media outlets (kim, 2016) . social networking service (sns) users are likely to be exposed to messages that affect their perceptions and their use of preventive behaviors related to health risks and the crisis event. vos and buckner (2016) found that sns messages in the 2013 outbreak of the h7n9 virus consisted mainly of sense-making messages to educate users about the nature of the risk and efficacy messages to encourage appropriate responses. in a similar line, yoo, choi, and park (2016) found that receiving information about mers through sns directly encouraged mers preventive behavioral intentions. social media is filled not only with information but also emotional expressions (chew & eysenbach, 2010) . negative emotions are more likely than positive emotions to be floating around social media during an infectious outbreak, which could increase risk perception (choi, yoo, noh, & park, 2017) . for instance, song et al. (2017) found that negative emotions (e.g., anxiety or fear) prevailed in online boards and social media during the mers crisis. often, information intertwined with emotional markers travels around social media, and emotionally charged messages are more likely than purely informational posts to be shared and diffused (pfeffer, zorbach, & carley, 2014; stieglitz & dang-xuan, 2013) . s. choi, lee, pack, chang, and yoon (2016) mined internet media reports about mers in 2016 and examined their effects on public emotion expressed online, which they captured using a sentiment analysis. they mined all mers-related news articles from 153 media companies, including the comments about each one. in a time-series analysis, they found a flow from internet media to public fear. according to song et al. (2017) , negative emotion accounted for 80% of all posts throughout online networks, especially twitter, during the mers crisis. therefore, i have drawn the following four hypotheses. i expected that both traditional and social media use in times of crisis could directly and indirectly facilitate preventive behaviors (via mers knowledge) and negative emotional responses to the mers situation. emerging social media channels have reshaped the crisis information context, which potentially complicates the relationships among media use and informational, emotional, and behavioral responses in the general public. although social media has been considered a powerful tool for disseminating information, both researchers and practitioners have cautioned against the propensity of social media to proliferate inaccurate data, unverified rumors, and even malicious misinformation (zeng et al., 2016) . because information disseminated through social media is often unverified, identifying accurate data and valid sources can be challenging and could both undermine relevant knowledge and exacerbate the consequences of emotional and behavioral responses. together with the characteristics of open access platforms, this concern is heightened by the dynamic nature of crisis communication and information overload in times of crisis (zeng et al., 2016) . social media networks are mostly composed of acquaintances and thus share common characteristics with interpersonal channels. prior works have shown that interpersonal channels tend to show stronger effects on behavioral change than traditional media, mainly through normative pressure (price & oshagan, 1995) . in addition, social media generally deal with a rapid exchange of information, which could be more appropriate for short and clear mi than for scientific knowledge about a crisis. for instance, won, bae, and yoo (2015) conducted an issue word analysis of sns messages during the mers outbreak and reported the six most frequently mentioned words on sns at that time. two of the six words were mask and hand sanitizer, which are clearly related to preventive behaviors. the other four words were hospital, infection, coughing, and checkup, which are also at least indirectly related to preventive behaviors. however, a big data analysis examining online news sites found that posts about symptoms, government reaction, disease treatment, business impacts, and rumors ranked higher than prevention-related information (song et al., 2017) . therefore, the effects of social media use on crisis knowledge and preventive behaviors might differ from those of traditional media. in terms of emotional responses, social media and traditional media might also show some differences. social media is known for its ability to spread information in a speedy and viral manner, but that information tends to be integrated with emotions. furthermore, emotionally charged messages are more likely to be shared than neutral messages (pfeffer et al., 2014; stieglitz & dang-xuan, 2013) . recent works on the mers crisis have shown that about 80% of the buzzwords on social media during the mers crisis were negatively charged emotional words such as fear and anxiety (s. choi et al., 2017; song et al., 2017) . those works suggest that social media might be more likely than traditional media to magnify negative emotional responses. on the contrary, some prior works have found that people use social media for social support in crisis situations (y. choi & lin, 2009) . that phenomenon could lessen the negative emotional responses in social media users. therefore, it is worth investigating how the cognitive, emotional, and behavioral responses to social media use differ from those to traditional media use, which raises the following research question: are there any differences between traditional media use and social media use in terms of cognitive, emotional, and behavioral responses during the mers crisis? the proposed research model is based on the preceding discussion (see figure 1) . specifically, i expect that both traditional and social media use related to mers was associated with mers knowledge, and that it directly and indirectly encouraged mers preventive behaviors in media users. i also expect that traditional and social media use about the korean mers crisis stimulated negative emotional responses, which in turn influenced both precautionary and panic behaviors in media users. finally, i examine whether social media use showed informational, emotional, and behavioral consequences similar to those of traditional media use. the data for this study were obtained from a two-wave online panel survey. wave 1 survey was conducted in the first few days of june 2015, when hype about the mers situation was escalating, and wave 2 survey was conducted about 1 month later. the respondents were adult members of an online panel recruited and maintained by a survey company in seoul, korea. about 1 million online users compose the company's panel, and participants were randomly drawn from that list to meet the specified constraints (e.g., areas of residence based on census composition). the selected panel members received a soliciting e-mail from the company and had full liberty to decline participation. the first page of the online survey included an explanation of the study purpose and researcher contact information. study participants gave informed consent by clicking the start survey button. they were free to stop taking the survey at any time, and they received small monetary incentives (e.g., cash-equivalent points) from the company. a total of 1,187 members of the online panel participated in wave 1 survey. about 1 month later, the same respondents were recontacted by the survey company to participate in wave 2 survey. the number of participants who completed both waves 1 and 2 online surveys was 969, which is the final subject sample used for the analyses herein. the final sample was 50.7% male, with a mean age of 43.88 years (sd = 12.67). with regard to level of education, 50.5% of the sample had some level of college education or a bachelor's degree, 17.1% had a 2-year college degree, and 20.9% of the sample had some level of high school education or were graduates of high school. the median monthly household income was between 3,500,000 and 4,000,000 won. of the total sample, 64.4% were married, with an average of 1.7 children (sd = .84). traditional news use. traditional news use of mers-related content was measured based on media use frequency questions. specifically, on a 7-point scale (1 = never to 7 = very often), respondents were asked to measure how much they relied on tv news or tv news websites and newspapers and newspaper websites in the context of the korean mers crisis (r = .37, m = 4.20, sd = 1.25). social media use. social media use of mers-related content was measured using three 7-point scales (1 = never to 7 = very often) in wave 1 survey. the specific use behaviors were (a) seeking mers-related news or information using social media, (b) receiving mers-related news or information from others via social media, and (c) talking about mers with others via social media (î± = .79, m = 3.72, sd = 1.48). negative emotional reactions to the mers situation. negative emotional reactions to the mers situation were measured in both waves 1 and 2 surveys. on a 7-point scale (1 = not at all to 7 = feel strongly), respondents were asked to report how strongly they felt fear and anxiety about the mers situation (wave 1, r = .85, m = 5.42, sd = 1.47; wave 2, r = .85, m = 5.08, sd = 1.53). mers knowledge. mers knowledge was measured using six quiz-type questions in both waves 1 and 2 surveys. knowledge items covered mers symptoms, lethality, maximum latent period, and institutions where a formal diagnosis of mers could be made during the outbreak. for each question, correct answers were coded as 1, and incorrect answers were coded as 0. subsequently, the answers to those six questions were combined and used as the mers knowledge measure (wave 1, with scores ranging from 0 to 6, m = 3.09, sd = 1.17; wave 2, with scores ranging from 0 to 6, m = 3.75, sd = 1.26). wave 2 survey using five items. on a 7-point scale (1 = never to 7 = very often), respondents were asked how often they engaged in behaviors to prevent mers: wearing a mask when they were out, washing hands frequently, avoiding contact with people with mers symptoms, avoiding hospitals, and avoiding daily activities such as grocery shopping to avoid crowds (î± = .77, m = 4.18, sd = 1.43). before running the path model to test hypotheses, a confirmatory factor analysis (cfa) was conducted to test validity of the measurement model. two knowledge measures were excluded from cfa because measures were composed of all dichotomized items. model modification was not utilized: ï� 2 (67) = 301.95, p = .00; normed fit index (nfi) = .95; incremental fit index (ifi) = .96; comparative fit index (cfi) = .96; root mean square error approximation (rmsea) = .06 with 90% confidence interval (ci) = [.05, .07]. factor loadings ranged from .51 to .98 and two factor loadings were rather low of .56 (one of mers preventive behaviors) and .51 (newspaper use item). however all ave (average variance extracted) were higher than .50 and factor loadings were still within acceptable range (fornell & larcker, 1981) . therefore, all items remained. path analysis was conducted to test the hypotheses with latent variables and two observed knowledge variables. the tested model included two control variables from wave 1 data. specifically, mers knowledge and negative emotions about the mers situation measured in wave 1 controlled each of the wave 2 variables accordingly. the research model showed a reasonably good fit according to commonly used criteria (hu & bentler, 1999) : ï� 2 (90) = 359.76, p = .00; nfi = .96; ifi = .96; cfi = .96; rmsea = .056 with 90% ci = [.050, .062]. figure 2 presents the overall results with path coefficients of the hypothesized model. h1 predicted that the more individuals use traditional news media (h1a) and social media (h1b), the more mers knowledge they would acquire. as expected, mers knowledge increased with increasing use of mers-related traditional news media (î² = .13, p < .01), which supported h1a. on the contrary, mers-related social media use was not significantly associated with mers knowledge (î² = â��.04, ns), which did not support h1b. therefore, h1 was only partially supported. h2 predicted that two types of media use would be positively associated with negative emotional responses to the mers situation. results showed that the more traditional media one used, the more anxious and worried about mers one became (î² = .09, p < .05), which supported h2a. however, mers-related sns use and negative emotions was not significantly associated (î² = .05, ns), which failed to support h2b. therefore, h2 was also partially supported. the next set of hypotheses predicted that positive effects of mers knowledge (h3a) and negative emotional responses (h3b) on mers preventive behaviors. as predicted, negative emotional response was positively related to engagement in mers preventive behaviors (î² = .54, p < .001). on the contrary, mers knowledge was not significantly associated with mers preventive behaviors (î² = .05, ns). therefore, h3 was also partially supported. the final set of hypotheses expected that both traditional news media use (h4a) and social media use (h4b) would have direct positive effects on mers preventive behaviors. results showed that only social media use showed note. dotted arrows denote paths that are not statistically significant or only marginally significant. the numbers presented in figure 2 are the standardized path coefficients. all exogenous variables, including mers knowledge (w1) and negative emotions (w1), were correlated with one another. no error term was correlated. w1 = wave 1; w2 = wave 2; mers = middle east respiratory syndrome. *p < .05. **p < .01. ***p < .001. direct positive effects on mers preventive behaviors (î² = .23, p < .001) not traditional media use (î² = .05, ns). finally, rq1 asked whether traditional media use and social media use show any differences in terms of cognitive, emotional, and behavioral responses. results showed that individuals during the mers crisis showed different responses depending on two types of media use. with respect to cognitive responses, as reported above, only traditional media use was positively associated with mers knowledge. likewise, in terms of emotional responses, traditional news media use showed a positive association with negative emotional reactions. with respect to behavioral responses, only social media use showed significant positive direct effects on mers preventive behaviors. wald tests comparing the path coefficients confirmed that the difference between effects of social media and traditional media on behavioral responses (wald test = â��6.51, p < .001) was statistically meaningful. wald test result also validated that the difference in cognitive response between social and traditional media use was also statistically significant (wald test = â��2.29, p < .01). despite a consensus that the media play an important role in times of crisis, the informational, emotional, and behavioral effects of traditional and social media use by individuals have been understudied in previous research. using two sets of data collected at two different time points during the 2015 mers crisis in korea, i investigated how traditional and social media use influenced mers knowledge, fear and anxiety about the mers situation, and adoption of preventive behaviors. to reflect the complexity of today's media environment in times of crisis, i focused on potential differences between the effects of traditional and social media use. first, this study found that traditional media use and social media use have different effects. as expected, traditional media use galvanized public understanding of mers. the more people read newspapers and watched tv news about mers, the more knowledge they acquired about mers symptoms, lethality, and maximum latent period. social media use neither increased nor decreased mers knowledge. the role of social media in times of crisis has received growing attention, at least in part because of its potential for viral information transmission. however, i did not find significant cognitive effects from social media use. compared with traditional media, which mainly report information verified by expert sources (lowrey, 2004) , social media can not only convey knowledge but also disseminate false or unverified information during a crisis. the null effect of social media use on mers knowledge might thus result from the conflicting content of social media. this speculation, of course, should be verified with a robust empirical examination. second, i found that negative emotions played a prominent role in facilitating preventive behaviors in the mers crisis. experiencing fear and anxiety is a natural reaction when people are faced with a crisis. a lack of familiarity with newly emerging infectious diseases such as mers tends to deepen fear and anxiety (sandman & lanard, 2003) , and media use could amplify those emotional experiences (kasperson, 1992) . finding of this study also indicate that traditional media use increased negative emotional responses. the more media respondents consumed, the more fear and anxiety they felt about the mers situation. more importantly, i found that the negative emotional responses people experienced when facing a crisis caused them to protect themselves from mers instead of pushing them into illogical or destructive behaviors. the positive role of negative emotions in promoting adaptive behaviors is consistent with what theoretical frameworks such as pmt and risp suggest. however, most prior studies based on pmt have been developed and tested within the context of public health campaigns. compared with carefully designed campaign messages intended to scare people to act properly with respect to a given risk, media messages could more likely be crude and mixed with various intentions (e.g., sensational reports to secure audience attention). indeed, there is a lack of empirical research examining what kinds of crisis-specific responses could be elicited from the media individuals choose to use in times of crisis. in addition, risp research provides a useful theoretical framework to predict that negative emotions could motivate people to seek information about a risk. however, this framework did not pay attention to media use, which can lead to behavioral consequences such as preventive behaviors beyond information seeking. this study addressed these gaps in the literature by directly testing the roles of negative emotions and media as potential sources of those emotions during a pandemic, which has been rarely examined in previous studies. this finding has clear implications for governmental communication strategies and media reporting. this study's results suggest that it may not be effective crisis management to brush off negative emotions among the public as illogical overreactions. instead, admitting fears and anxiety people may feel and designing messages channeling those emotions into desirable health behaviors needs to be considered even in times of crisis. sympathizing with people's feelings can be an effective means to boost the evaluation of communicators' dialogic competency which was found to be positively related to information credibility and trust in the government during the mers endemic (s.-u. yang, 2018) , which in turn may promote government and media health message effectiveness. this seemingly positive role of negative emotions, however, does not advocate for sensationalizing crisis situations. instead, both government and media may consider applying knowledge about the potentially positive function of negative emotion to public communication during a crisis. for instance, risk and health communication research has developed guidelines on how to write a good fear appeal message in the public health campaign context. government and news media may consult with these guidelines to compose a message to help people to cope with an epidemic risk. third, the findings of this study indicate that the role of knowledge for preventive behaviors in the 2015 mers crisis was rather limited. only traditional media use significantly increased the public's knowledge about mers but the increased understanding did not facilitate precautionary behaviors. disseminating knowledge has been a top priority in times of crisis under the assumption that understanding could lead the public to protective behavioral responses. the results of this study imply that filling the deficit of knowledge may not be the most efficient way to promote behavioral responses. the findings here suggest that an emotional pathway may work more efficiently than cognitive drives in promoting the adoption of preventive behaviors during a public health crisis. however, it is also important to point out that the findings related to knowledge should be interpreted with caution in the context of the 2015 mers crisis. s.-u. yang (2018) noted that koreans in general tended to perceive the government's mutuality and openness in communication somewhat unfavorably, which often negatively affected government-public relationships. as s.-u. yang (2018) showed, the korean government's lack of mutuality and openness seems to have deteriorated the credibility of its risk information, which may have worsened the public's trust and satisfaction with the government. this unique situation might have lowered not only the credibility of government information but also the credibility of information from news media that relied heavily on the government for information. accordingly, the low credibility of public information on mers can explain the limited role of knowledge in promoting desirable behavioral responses. prior work examining a different epidemic crisis found that one material factor influencing preventive action was respondent opinion about authorities (quah & hin-peng, 2004) . my finding thus suggests that simple acquisition of disseminated scientific knowledge might not necessarily produce desirable behavioral responses from the public. it also implies that contextual factors, such as government dialogic competency, should be considered to fully grasp the role of knowledge in times of crisis. fourth, the findings of this study show that, unlike traditional media use, social media use produced strong direct behavioral responses during the mers crisis. traditional media use did not show direct effects on preventive behaviors. this distinction between social media use and traditional media use could be explained in the following two ways. first, compared with traditional media, which is known to be powerful in knowledge diffusion, the interpersonal channel has been considered effective on behavioral responses via the normative route (price & oshagan, 1995) . the role of normative pressure on behavioral adaptation has been well documented in the health communication literature (e.g., planned behavioral intention model, ajzen, 1991) . given that social media networks are mainly composed of acquaintances, it is possible that social media might have formed specific behavioral norms related to the mers crisis (e.g., avoidance of hospitals and crowded places). the other explanation is related to potential differences in the content of traditional media and social media. social media might have delivered more mi than traditional media. indeed, a prior work analyzing social media content during the mers crisis reported that preventive behavior-related terms (e.g., masks and hand sanitizers) were the most frequently mentioned words (won et al., 2015) . furthermore, the most needed information at the beginning of the mers crisis was the list of mers-affected hospitals, which the korean government had withheld. given the government's unwillingness to share the list of hospitals, the public resorted to social media to actively seek, exchange, and share the hospital-related information. that piece of information could single-handedly trigger one preventive behavior (i.e., avoiding specific hospitals to protect themselves). indeed, hospital was one of the terms most frequently used on social media during the mers crisis (won et al., 2015) . however, a big data analysis of traditional media reported that words such as infection, confirmed cases, and death were centrally located in traditional media content (kwon, 2016) . in short, compared with traditional media, social media might play an essential role in virally diffusing mi (specific behavioral information, including where not to go and what to do). given the importance of dialogic competency for effective crisis communication (s.-u. yang, 2018) , some technological features of social media may have enhanced communication mutuality and openness which the korean government and media lacked. communication through social media may allow users to share empathy and social support. at the same time, social media-mediated communication can be perceived as more accessible and open than media-mediated communication. strong mutuality and openness perceived through communication with network members on social media could have increased the credibility of mi which could have enhanced behavioral responses at least during the mers crisis in korea. another notable finding regarding social media is that social media did not generate public anxiety and fear, at least during the mers crisis in korea. this is quite an interesting finding because the korean government strongly criticized media, especially social media, for releasing unverified rumors and fears. the lack of association between social media use and negative emotional responses could be a result of canceling out effects. in other words, social media might induce fear and anxiety, but social media might effectively cancel out the negative emotions by providing wanted social supports or mi, which should be under solid empirical scrutiny in the future study. in the interest of future research, it is important to discuss some of the limitations of this study. the data are not based on a representative sample, which means the study findings must be interpreted with caution. for instance, online panels tended to include the younger and the more educated compared with the general population, which could influence the interpretation of the role of social media and traditional media use during the mers crisis in korea. the measurement of key variables also has limitations. for example, traditional media use was assessed only for newspaper and television. omitting radio and general internet use should be recognized as another major limitation of the study. also, using quiz-type questions to tap mers knowledge without close validation and planning might have contributed to the low correlation between the two mers knowledge measures, which also raises a concern about a test-retest error involved in the panel data. these measurement issues could produce errors, which would be carried into the hypothesized path model this study proposed. in addition, wave 1 data did not include mers preventive behavior measures, which made it impossible to control all the endogenous variables in wave 1 data. therefore, it is hard to argue that an ideal panel data analysis was conducted for testing the proposed hypotheses. with these limitations in mind, this study contributed to the scholarship by testing the predictions drawn from existing theories that have rarely been examined in the context of a real epidemic crisis. major scholarly focus in the communication field has been on how government and responsible organizations manage or should manage crises. shifting away from that focus, this study investigated the associations between individual media use and consequent responses which have been relatively understudied. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. mihye seo https://orcid.org/0000-0002-5734-3181 the theory of planned behavior. organizational behavior and human decision process circulating health rumors in the "arab world": a 12-month content analysis of news stories and reader commentary about middle east respiratory syndrome from two middle eastern news outlets how emotion shapes behavior: feedback, anticipation, and reflection, rather than direct causation predicting health behaviour: research and practice with social cognition models pandemics in the age of twitter: content analysis of tweets during the 2009 h1n1 outbreak mining internet media for monitoring changes of public emotions about infectious diseases consumer responses to mattel product recalls posted on online bulletin boards: exploring two types of emotion the impact of social media on risk perceptions during the mers outbreak in south korea ongoing crisis communication: planning, managing, and responding newspaper coverage of causes of death evaluating structural equation models with unobservable variables and measurement error cancer coverage in north american publications targeting seniors predicting osteoporosis prevention behaviors: health beliefs and knowledge risk communication for public health emergencies proposed model of the relationship of risk information seeking and processing to the development of preventive behaviors weathering the storm: a social marketing perspective on disaster preparedness and response with lessons from hurricane katrina the dominant view of popularization: conceptual problems, political uses the knowledge gap hypothesis in singapore: the roles of socioeconomic status, mass media, and interpersonal discussion on public knowledge of the h1n1 flu pandemic emotion and coping with terror cutoff criteria for fit indexes in covariance structure analysis: conventional criteria versus new alternatives the evolving role of the public information officer: an examination of social media in emergency management semantic network analysis of domestic and overseas media coverage regarding korea mers the social amplification of risk: progress in developing an integrative framework in social theories of risk an essay on korean media's coverage of middle east respiratory syndrome coronavirus a study of semantic network analysis of newspaper articles on mers situation: comparing conservative and progressive news media the emotional distress and fear of contagion related to middle east respiratory syndrome (mers) on general public in korea television's disaster marathons effects of news media and interpersonal interactions on h1n1 risk perception and vaccination intent the protective action decision model: theoretical modifications and additional evidence valuation of the risk of sars in taiwan media dependency during a large-scale social disruption: the case of crisis leadership: planning for the unthinkable compassion fatigue: how the media sell disease, famine, war and death bits of falling sky and global pandemics: moral panic and severe acute respiratory syndrome (sars). illness middle east respiratory syndrome-advancing the public health and research agenda on mers-lessons from the south korea outbreak understanding online firestorms: negative word-of-mouth dynamics in social media networks quality indicators for crisis communication to support emergency management by public authorities sars: mobilizing and maintaining a public health emergency response social media in disaster response: how experience architects can build for participation social-psychological perspectives on public opinion crisis management and management during sars outbreak the social amplification of risk: theoretical foundations and empirical application crisis and emergency risk communication as an integrative model sars epidemic in the press swine influenza and the news media the impact of communications about swine flu (influenza a h1n1v) on public responses to the outbreak: results from 36 national telephone surveys in the uk scary warnings and rational precautions: a review of the psychology of fear appeals social media as a useful tool in food risk and benefit communication? a strategic orientation approach fear is spreading faster than sars and so it should knowledge, attitudes and preventive behaviors related to dengue vector breeding control measures among adults in communities of vientiane, capital of the lao pdr crisis and emergency risk communication in health contexts: applying the cdc model to pandemic influenza media coverage of public health epidemics: linking framing and issue attention cycle toward an integrated theory of print news coverage of epidemics social big data analysis of information spread and perceived infection risk during the 2015 middle east respiratory syndrome outbreak in south korea emotions and information diffusion in social media-sentiment of microblogs and sharing behavior media dependencies in a changing media environment: the case of the 2003 sars epidemic in china disaster communication on the internet: a focus on mobilizing information global bird flu communication: hot crisis and media assurance a work-in-process literature review: incorporating social media in risk and crisis communication social media messages in an emerging health crisis: tweeting bird flu issue words analysis according to the mers outbreak on sns middle east respiratory syndrome coronavirus (mers-cov)-republic of korea effects of government dialogic competency: the mers outbreak and implications for public health crises and political legitimacy what, me worry? the role of affect in information seeking and avoidance the effects of sns communication: how expressing and receiving information predict mers-preventive behavioral intentions in south korea media use and health behavior in h1n1 flu crisis: the mediating role of perceived knowledge and fear #unconfirmed: classifying rumor stance in crisis-related social media messages mihye seo (phd, the ohio state university) is an associate professor in the department of media and communication at sungkyunkwan university, seoul, south korea. her research focuses on media use and its influence on individuals' daily life and on community-building efforts. key: cord-288589-bt9429bh authors: habibzadeh, farrokh title: hadj ritual and risk of a pandemic date: 2013-12-31 journal: am j infect control doi: 10.1016/j.ajic.2013.08.011 sha: doc_id: 288589 cord_uid: bt9429bh nan at mass gatherings such as the hadj, one of the greatest islamic rituals, pilgrims are exposed to various infectious agents. 1 the pilgrims bring home not only souvenirs but also various infectious agents, as is evident from an increase in the incidence of infectious diseasesdmainly flu-like syndromesdin returning pilgrims every year. from early october 2013, saudi arabia will be hosting millions of muslims from different parts of the world who travel to saudi arabia to participate in the hadj. however, since november 2012, saudi arabia has also hosted a new fatal viral infection: the middle east respiratory syndrome coronavirus (mers-cov). coronaviruses are enveloped, single-stranded, positive-sense rna viruses. the first known occurrence of mers-cov in human was reported in a patient with severe acute respiratory infection in april 2012, in jordan. 2 later, person-to-person transmission was reported, 3 which is very important because it might result in a switch from aborted outbreaks to a pandemic, which was probably the case for the severe acute respiratory syndrome (sars) coronavirus outbreak in 2003-2004. as of august 13, 2013, 94 laboratory-confirmed cases of human infection with mers-cov had been reported to the world health organization, 46 (49%) of which have been fatal. the age of the affected patients ranged from 14 months to 94 years (median, 50 years). the incubation period of the infection is estimated at 1 week, based on the available data. 4 mers-cov has a high mortality rate of almost 50%, and thus the possibility of a pandemic is worrying. soon after pilgrims return home, they hold ceremonies and invite relatives and friends to celebrate their return from hadj, which provide further opportunities for the spread of the virus. there are many reasons to convince us that mers-cov represents a high risk: a deadly virus that can be transmitted from person to person, a mass gathering of millions of people from different parts of the world at the epicenter of the infection, an incubation period that provides enough time for pilgrims to return home and disseminate the virus, ceremonies that place relatives and friends in close contact with infected individuals when they return, and signs and symptoms that can easily be mistaken for common postpilgrimage flu-like syndromes. 5 this year, health care workers should be more alert to identify the probable cases of mers-cov infection as early as possible. the total risk may not be very high, but, considering the devastating outcome for the middle east in particular, and world at large, it would be wise to take into account even minute risks and take all possible safety precautions into action. emergence of medicine for mass gatherings: lessons from the hajj isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus (mers-cov), update middle east respiratory syndrome coronavirus: the unseen menace key: cord-255628-bm4nogig authors: su, shuo; wong, gary; liu, yingxia; gao, george f; li, shoujun; bi, yuhai title: mers in south korea and china: a potential outbreak threat? date: 2015-06-11 journal: lancet doi: 10.1016/s0140-6736(15)60859-5 sha: doc_id: 255628 cord_uid: bm4nogig nan first reported in september, 2012, human infections with middle east respiratory syndrome coronavirus (mers-cov) can result in severe respiratory disease, characterised by life-threatening pneumonia and renal failure. 1 countries with primary infections of mers-cov are located in the middle east, but cases have been occasionally exported in other countries (fi gure). human-to-human infections of mers-cov are rare 2 and confi rmed cases are usually traced back to contact with camels, an intermediate host species for mers-cov. 3 as of may 24, 2015, worldwide, a total of 1134 cases and 427 deaths (case fatality rate 37·7%) have been reported, according to who. 4 there is no approved vaccine or treatment. on may 11, 2015, a 68-year-old male in south korea developed symptoms and sought medical care at a clinic between may 12-15, before admittance into hospital on may 15. 4 the patient had been travelling between april 18-may 3 through bahrain, the united arab emirates, saudi arabia, and qatar. he was asymptomatic upon return to south korea on may 4, but tested positive for mers-cov on may 20, along with two additional cases: his 64-year-old wife, and a 76-year-old male who was a fellow patient. 4 concerns of further mers-cov spread were confirmed when a 71-year-old male fellow patient, the daughter of the 76-year-old case, and two medical staff developed symptoms and were diagnosed with mers-cov infection (appendix). as of may 29, 2015, south korea has 12 laboratoryconfirmed cases of mers-cov, and more than 120 additional contacts under surveillance. 5 on may 28, a 44-year-old male traveller from south korea to huizhou, china was admitted into hospital. mers-cov infection was confi rmed on may 29, marking the fi rst laboratoryconfirmed case in china (appendix), and the patient was immediately put in isolation. this patient was the son of the 76-year-old south korean patient. he had visited his father in the hospital on may 16, developed symptoms on may 21, 6 and travelled to hong kong by plane on may 26 before arriving by road into mainland china via shenzhen. 6 in response, the chinese health authorities promptly placed 38 high-risk contacts under surveillance, but it is not known whether additional contacts exist and further mers-cov infections in china remains a possibility. this series of events highlighted issues with the current surveillance system put in place to prevent the importation of infectious diseases. the diagnosis for mers-cov infection was made on may 20 for the 76-year-old patient. his 44-year-old son should have been monitored as a close contact of the laboratory-confi rmed case, with provisional quarantine and testing upon development of symptoms and isolation upon a positive diagnosis. such a high-risk case should not be travelling until after the incubation period, which is between 2-15 days for mers-cov. 2 non-compliance by the patient regarding travel advice likely contributed to this scenario. 5 these events serve as a timely reminder that natural geographical barriers against pathogens can now be easily overcome through trade and travel, and marks the fi rst mers-cov import case that did not come directly from the middle east. these developments are worrisome given favipiravir-a prophylactic treatment for ebola contacts? since the ebola outbreak began in march, 2014, 25 178 cases of ebola have been reported. 1 to control spread of ebola in west african communities, vaccination campaigns have been proposed. however, the efficacy of candidate ebola vaccines for primary prevention has not been proven. 2 furthermore, in communities in which ebola transmission might be ongoing, an important question is: how will such a vaccination be perceived if a vaccinated person develops ebola? such a scenario is possible in people who contract ebola virus before vaccination. if a person is infected with ebola virus before vaccination, the vaccine might have a post-exposure prophylactic effect. however, how effective this prophylaxis might be is unknown. 2 moreover, if someone is infected more than 48 h before vaccination, the post-exposure prophylactic eff ect is likely to be insuffi cient, leading to possible development of ebola after vaccination. this scenario is likely to result in serious issues relating to community trust and acceptance of an ebola vaccine. 3 how to exclude ebola among people presenting with post-vaccination fever is also an issue. 2 we make a case for the study of favipiravir (toyama chemical, japan), administered as directly observed therapy for contacts of patients with ebola. favipiravir has increased benefit in patients with low ebola viraemia compared with patients with high viraemia. 4 as such, this drug could have a post-exposure prophylactic effect among recently infected contacts and a pre-exposure prophylactic eff ect among contacts exposed to, but not yet infected by, ebola virus. additionally, fever has not been reported as a side-effect of favipiravir (clinicaltrials.gov, nct02329054). furthermore, oral administration of prophylactic favipiravir gives people the choice to interrupt treatment if wanted. additional effects of prophylactic favipiravir might include increased openness of communities to use alert systems and to support contact tracing services (ie, contacts might be receptive to daily follow-up visits). finally, to reduce incidence of malaria, prophylactic artesunateamodiaquine could be administered to the contacts of patients with ebola. one disadvantage of proposed favipiravir prophylaxis might be the need to exclude pregnant women. to mitigate this problem, pregnancy tests could be included as a routine part of the favipiravir prophylaxis package. finally, prophylactic favipiravir could be field tested by measurement of incidence of ebola among contacts of patients with ebola before and after favipiravir is introduced. we declare no competing interests. that hong kong airport is a major international transport hub, and thus any potential infections can travel worldwide in a short time. after dealing with several pandemic threats over the past 15 years, notably severe acute respiratory syndrome coronavirus (sars-cov) in 2003, h1n1 influenza in 2009, and ebola virus in 2014-15, authorities now have ample experience in outbreak response compared with past years. in addition to the need for increased vigilance from health authorities, compliance by the public is crucial for the eff ective implementation of outbreak responses. everyone is responsible for upholding the principles of public health, and must play their part to minimise the chances of disease transmission across borders. middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus (mers-cov)-republic of korea international society for infectious diseases. mers-cov (50): south korea middle east respiratory syndrome coronavirus (mers-cov)-china we declare no competing interests. key: cord-297954-87w2itin authors: memish, ziad a.; al-tawfiq, jaffar a.; alhakeem, rafat f.; assiri, abdullah; alharby, khalid d.; almahallawi, maher s.; alkhallawi, mohammed title: middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases date: 2015-07-15 journal: travel med infect dis doi: 10.1016/j.tmaid.2015.06.012 sha: doc_id: 297954 cord_uid: 87w2itin since the initial description of the middle east respiratory syndrome (mers) in september 2012, a total of 1038 cases of mers-cov including 460 deaths have been reported from saudi arabia. from august 24, 2013 to september 3, 2013, a total of 397 patients and contacts were tested for mers-cov. of those tested, there were 18 (4.5%) mers-cov cases reported in al-madinah al-munawwarah with one large cluster. in this report, we describe the outcome, epidemiology and clinical characteristics of this cluster of which 4 cases involved healthcare workers. fourteen cases appeared to be linked to one cluster involving healthcare workers (hcws), family and patient contacts. of the 18 cases, five (including 2 hcws) were community acquired, two were household contacts, and 11 were healthcare associated (including 4 hcws). all except 4 cases were symptomatic and the case fatality rate was 39% (7 of 18). the outbreak resulted in human to human transmission of an estimated 6 cases. contact screening showed positive test in 1 of 56 (1.8%) household contacts, and 3 of 250 (1.2%) hcws. summary since the initial description of the middle east respiratory syndrome (mers) in september 2012, a total of 1038 cases of mers-cov including 460 deaths have been reported from saudi arabia. from august 24, 2013 to september 3, 2013, a total of 397 patients and contacts were tested for mers-cov. of those tested, there were 18 (4.5%) mers-cov cases reported in al-madinah al-munawwarah with one large cluster. in this report, we describe the outcome, epidemiology and clinical characteristics of this cluster of which 4 cases involved healthcare workers. fourteen cases appeared to be linked to one cluster involving healthcare workers (hcws), family and patient contacts. of the 18 cases, five (including 2 hcws) were community acquired, two were household contacts, and 11 were healthcare associated (including 4 hcws). all except 4 cases were symptomatic and the case fatality rate was 39% (7 of 18). the outbreak resulted in human to human transmission of an estimated 6 cases. contact screening showed positive test in 1 of 56 (1.8%) household contacts, and 3 of 250 (1.2%) hcws. ª 2015 elsevier ltd. all rights reserved. since middle east respiratory syndrome (mers) was described in september 2012, a total of 1038 cases of mers-cov including 460 deaths have been reported from saudi arabia [1] . the current case fatality rate is lower than the initial rate of 65% [2] . mers-cov is known to cause three patterns of transmissions [2e8]: sporadic cases, community-transmission [9] and healthcare associated transmissions such as the case in the zarqa, jordan [10, 11] , al-hasa, saudi arabia [12] and jeddah, saudi arabia [13, 14] . the exact source of the infection for most patients remains unknown. in this report, we describe the outcome, epidemiology and clinical characteristics of this cluster of mers-cov in al-madinah al-munawwarah of which 4 cases involved healthcare workers. all samples were tested in jeddah regional lab. we included all mers-cov cases reported from al-madinah al-munawwarah between august 24, 2013 and september 3, 2013. a confirmed case of mers cov is defined as an isolation of mers cov from a nasopharyngeal or a respiratory sample by real time reverse transcriptase pcr, as described previously [12, 15] . clinical information included demographic data, clinical symptoms and signs, co-morbidities, and contact with animals. during the study period, a total of 397 patients and contacts were tested for mers-cov. there were 18 (4.5%) mers-cov positive cases reported in al-madinah al-munawwarah with one large cluster. of those cases, 15 (83%) were male and 3 (17%) were females. twelve of the cases were saudis (67%) and 6 were non-saudis 33%. there were two possible clusters and two cases were sporadic in nature. the largest cluster included 15 cases and was thought to be initiated by a 55 year-old male resident. he was in the same hospital ward of a 74 year-old saudi male who was thought to acquire the infection in the healthcare setting. transmission then occurred in an additional 12 cases as illustrated in fig. 1 . another case was from qatar, the son of a patient sharing a room with the second case although the father tested negative for mers-cov. the second cluster was from the city of hanakia located 100 km from madina, and involved a 56 year-old male healthcare worker (hcw), who then infected another 39 year-old hcw. there was one sporadic case, a 50 year-old hcw, who had no contacts with other cases. the majority of the cases (61.1%) were healthcare associated infections and primary cases constituted 27.8% and intra-familial transmission was only 11.1%. the case fatality rate was 39% (7 of 18 cases). of the 14 symptomatic cases, 11 (78.5%) had at least two of the following chronic diseases: diabetes mellitus, hypertension, end stage renal disease, cardiac disease, sickle cell anemia, obesity, or smoking. only one patient had contact with animals, he was a healthcare worker and was asymptomatic. all of the 14 symptomatic cases had fever (100%), 64% had shortness of breath, 50% had cough, 35% had nausea, 35% headache, and 21% had sore throat. a total of 250 hcws were screened and 4 (1.6%) were positive. in addition, 56 family contacts were screened and 1 (1.8%) was positive. the current report illustrates the pattern of transmission of mers-cov. our data harmonizes with the previously described pattern of transmission of mers-cov [2e8]. the majority of the patients (61.1%) were healthcare associated infections and primary cases constituted 27.8% and intrafamilial transmission was only 11.1%. the recent jeddah outbreak in 2014 was documented to be secondary to intrahospital and inter-hospital transmissions [13, 14] . fig. 2 shows major mers-cov outbreaks in kingdom of saudi arabia. the rate of community infections seem to be low with expansion of the infection in the healthcare setting [5] . animal contact, especially with camels is uncommon among primary cases, and in our series only one patient had camel contact [3, 7] . the case fatality of these cases was 39%, compared to the overall case fatality in ksa of 44% [1] of the 14 symptomatic cases, 78.5% had at least two of underlying chronic diseases. the presence of comorbidities predisposes to increased risk of mers-cov and was shown to also correlate with case fatality rates [16] . screening of contacts yielded less than 2% positivity among hcws and family contacts. in a large screening of contacts, mers-cov was detected in 1.12% of hcws contacts and in 3.6% of family contacts [15] . however, the majority of the cases were acquired within healthcare facilities similar to the al-hasa and jeddah outbreak [12e14]. an interesting observation in this report is the link of one of the mers cases from qatar to this healthcare associated cluster. travel associated mers cases were reported from: turkey, austria, united kingdom, germany, france, greece, the netherlands, tunisia, algeria, malaysia, philippines, china, and the united states of america [17] . the recent occurrence of an outbreak in the republic of korea was started with a returning traveler [17e19]. the patient traveled to bahrain (18e29 april), the united arab emirates (29e30 april), bahrain (30 aprile1 may), saudi arabia (1e2 may), bahrain (2 may) and qatar (2e3 may) [17, 19] . the outbreak spanned 72 healthcare facilities which have treated patients and six healthcare facilities have documented nosocomial transmission [19] . as of june 24, 2015, this outbreak had caused 179 cases including 27 deaths [19] . the outbreak highlights the importance of infection control and early recognition and isolation of suspected cases [5] . the kingdom of saudi arabia also hosts one of the largest mass gathering in the world hosting millions of pilgrims during the annual hajj where pilgrims visit the holly cities of makkah and al-madinah [7] . the occurrence of mers-cov transmission during the annual hajj and subsequent development of a global epidemic is of a great concern. respiratory samples were obtained from all mers suspected cases during 2013 hajj season and all samples tested negative for mers-cov [20] . a cohort of 129 french hajj pilgrims were systematically sampled in 2013 with screened for mers-cov using nasal swabs prior to returning to france [21] . although, the majority (90.7) had respiratory symptoms, none was tested positive for mers-cov [21] . in 2012 and 2013 hajj season, a total of 5 million pilgrims from 184 countries visited makkah and al-madinah and no cases of mers-cov were detected during or after the hajj [22] . screening of 5235 adult pilgrims from 22 countries in 2013 showed no positive mers cases using nasopharyngeal swabs [23] . although only rare cases have been associated with the umrah pilgrimage so far, there is a need for continuing surveillance among travelers, pilgrims and hcw attending pilgrims [24] . middle east respiratory syndrome coronavirus: epidemiology and disease control measures coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers an update on middle east respiratory syndrome: 2 years later middle east respiratory syndrome coronavirus infection control: the missing piece? middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution travel implications of emerging coronaviruses: sars and mers-cov middle east respiratory syndrome coronavirus (mers-cov) in healthcare setting family cluster of middle east respiratory syndrome coronavirus infections epidemiological findings from a retrospective investigation hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia mers-cov outbreak in jeddahea link to health care facilities screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov)-17th update middle east respiratory syndrome coronavirus (mers-cov): summary and risk assessment of current situation in the republic of korea and china etiology of severe community-acquired pneumonia during hajjdpart of the mers-cov surveillance program lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms the hajj pilgrimage and surveillance for middle east respiratory syndrome coronavirus in pilgrims from african countries prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj imported cases of middle east respiratory syndrome: an update we are grateful to the staff of the ministry of health in al-madinah area and the staff of the regional laboratory, kingdom of saudi arabia. middle east respiratory syndrome coronavirus none. key: cord-305871-w1quh4fx authors: hindawi, salwa i.; hashem, anwar m.; damanhouri, ghazi a.; el‐kafrawy, sherif a.; tolah, ahmed m.; hassan, ahmed m.; azhar , esam i. title: inactivation of middle east respiratory syndrome‐coronavirus in human plasma using amotosalen and ultraviolet a light date: 2017-12-14 journal: transfusion doi: 10.1111/trf.14422 sha: doc_id: 305871 cord_uid: w1quh4fx background: middle east respiratory syndrome‐coronavirus (mers‐cov) is a novel zoonotic pathogen. although the potential for mers‐cov transmission through blood transfusion is not clear, mers‐cov was recognized as a pathogen of concern for the safety of the blood supply especially after its detection in whole blood, serum, and plasma of infected individuals. here we investigated the efficacy of amotosalen and ultraviolet a light (uva) to inactivate mers‐cov in fresh‐frozen plasma (ffp). study design and methods: pooled ffp units were spiked with a recent clinical mers‐cov isolate. infectious and genomic viral titers were determined in plasma before and after inactivation with amotosalen/uva treatment by plaque assay and reverse transcription–quantitative polymerase chain reaction, respectively. in addition, residual replicating or live virus after inactivation was examined by passaging in the permissive vero e6 cells. results: the mean mers‐cov infectious titer in pretreatment samples was 4.67 ± 0.25 log plaque‐forming units (pfu)/ml, which was reduced to undetectable levels after inactivation with amotosalen/uva demonstrating a mean log reduction of more than 4.67 ± 0.25 pfu/ml. furthermore, inoculation of inactivated plasma on vero e6 cells did not result in any cytopathic effect (cpe) even after 7 days of incubation and three consecutive passages, nor the detection of mers rna compared to pretreatment samples which showed complete cpe within 2 to 3 days postinoculation and log viral rna titer ranging from 9.48 to 10.22 copies/ml in all three passages. conclusion: our data show that amotosalen/uva treatment is a potent and effective way to inactivate mers‐cov infectious particles in ffp to undetectable levels and to minimize the risk of any possible transfusion‐related mers‐cov transmission. the mean mers-cov infectious titer in pretreatment samples was 4.67 6 0.25 log plaqueforming units (pfu)/ml, which was reduced to undetectable levels after inactivation with amotosalen/ uva demonstrating a mean log reduction of more than 4.67 6 0.25 pfu/ml. furthermore, inoculation of inactivated plasma on vero e6 cells did not result in any cytopathic effect (cpe) even after 7 days of incubation and three consecutive passages, nor the detection of mers rna compared to pretreatment samples which showed complete cpe within 2 to 3 days postinoculation and log viral rna titer ranging from 9.48 to 10.22 copies/ ml in all three passages. conclusion: our data show that amotosalen/uva treatment is a potent and effective way to inactivate mers-cov infectious particles in ffp to undetectable levels and to minimize the risk of any possible transfusion-related mers-cov transmission. t ransfusion of blood components saves millions of lives by controlling bleeding due to accidents, surgeries, or other disease complications. however, transmission of pathogens is one of the biggest risks of transfusion of labile blood components. therefore, a key mission of blood transfusion services is to provide safe blood and blood products. screening of blood products has reduced the spread of known blood-borne pathogens such as hepatitis b and c viruses (hbv and hcv), human immunodeficiency virus (hiv), and human t-lymphotropic virus (htlv). 1,2 however, other known or unknown pathogens pose a threat to the blood supply with regional differences that is difficult to address. the number of pathogens screened in blood banks is limited by the number of blood screening assays that are commercially available. therefore, pathogen inactivation offers an appealing alternative to blood screening (serology or nucleic acid testing [nat] ) because of its proactive nature and the broad spectrum of protection it offers without a priori characterization of unknown pathogens. indeed, pathogen inactivation technologies have been developed to provide safe blood products while reducing the need for the implementation of additional screening assays. 3 the middle east respiratory syndrome-coronavirus (mers-cov) is a zoonotic pathogen that is endemic in saudi arabia and other countries in the arabian peninsula since 2012. 4 as of february 24, 2017, mers-cov has been responsible for 1905 confirmed infections including 677 deaths (approx. 35.5% mortality rate) in 27 countries. 5 most cases (1552 confirmed infections) were reported in saudi arabia with a mortality rate of approximately 41.9%. 6 mers-cov causes respiratory illness in humans varying from asymptomatic or mild to severe pneumonia. 4, 7 the elderly and patients suffering from comorbidities are the most at risk for death outcome after development of severe clinical symptoms such as severe pneumonia and extrapulmonary manifestations. 8 the major route of transmission for mesr-cov is via droplets, fomites, and person-to-person contact through the respiratory system as well as via direct or indirect contact with zoonotic sources. so far, most mers cases are linked to residence in or travel to saudi arabia. 9 several hospital and household outbreaks have been reported in saudi arabia as well as south korea due to nosocomial transmission and close contact with patients. 9,10 however, the number of asymptomatic cases is estimated to be much higher than the reported cases, 11 which together with mild cases may increase the spread of the virus and pose a significant risk for blood safety. indeed, presymptomatic and asymptomatic donors may be allowed to donate blood while their donation has the potential to carry the virus, although there is no report on mers-cov detection in blood from presymptomatic and asymptomatic patients. while most of the reported mers-cov cases were due to human-to-human transmissions especially under inadequate infection control measures, dromedary camels are believed to be the reservoir host and an important source of human infection. 9, [12] [13] [14] [15] [16] [17] specifically, primary or index cases that had no contact history with infected individuals are more likely to have had direct or indirect contact with dromedaries. interestingly, current regulations of blood donation do not consider recent history of contact with dromedaries as a reason for deferral of blood donation. mers-cov has been detected in a variety of samples from infected patients including respiratory samples, serum, plasma, urine, and stool. [18] [19] [20] [21] [22] while detection of mers-cov rna is most frequent and persistent in respiratory secretions with high viral loads, detection of viral rna in plasma or serum was documented in up to onethird of the patients and showed association with disease severity. [20] [21] [22] this is reminiscent of the severe acute respiratory syndrome-coronavirus (sars-cov) outbreak which started in china in 2003 and led to global human-tohuman spread with more than 8000 confirmed cases and approximately 10% death rate. during the sars outbreak, viral rna was detected in serum from several patients but no transfusion-associated transmission was reported. 23, 24 while sars-cov disappeared quickly, mers-cov continues to be endemic in the arabian peninsula for more than 4 years now. similar to sars-cov, there is no proven evidence so far of transfusion-transmitted mers-cov infections, 25 but the presence of viral rna in plasma and serum of acute patients raises this concern especially in endemic areas like saudi arabia. in fact, it is not known whether presence of viral rna in blood products could lead to transmission of mers-cov or not. therefore, it is important to find a method to mitigate the risk associated with mers-cov rna presence in blood components to proactively prevent any possible transmission of mers-cov through contaminated blood products in endemic regions and elsewhere. the intercept blood system (ibs) inactivates pathogens by forming crosslinks or monoadducts on nucleic acids using amotosalen, a photoactive psoralen, after illumination with low-energy ultraviolet a (uva) light. during this highly specific targeted process, an adduct is formed at high frequency in the nucleic acids, thus inhibiting transcription and replication. 26 the ibs is a food and drug administration-approved pathogen reduction system that has been shown to inactivate a broad spectrum of viruses, bacteria, and parasites in plasma as well as other blood products [27] [28] [29] [30] without affecting the plasma efficacy and patient safety as demonstrated in clinical evaluation 31 and by hemovigilance data from multiple countries. 32, 33 until now there has been no data available on the inactivation of mers-cov with ibs. however, the inactivation of sars-cov with ibs, which is related to mers-cov, was successfully shown, 27 raising the expectation of a sufficient inactivation efficacy. therefore, the aim of this study was to evaluate the efficacy of amotosalen/uva treatment to inactivate mers-cov in human apheresis plasma concentrates. african green monkey kidney-derived vero e6 cells (atcc #1568) were grown and maintained as previously described in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs). 34 the human mers-cov isolate used in this study was mers-cov/hu/taif/sa/2015, which was characterized previously. 34 all experiments involving live virus were conducted in a biosafety level 3 facility at the special infectious agents unit at king fahd medical research center, king abdulaziz university, jeddah, kingdom of saudi arabia, following the recommended safety precautions and measures. mers-cov amplification and culture was done as previously described. 35 virus was inoculated on 90% to 95% confluent vero e6 cells in a t175 tissue culture flask at multiplicity of infection of 1 and incubated in a humidified 5% co 2 incubator at 378c for 1 hour with gentle shaking every 15 to 20 minutes. subsequently, the inoculum was replaced by 25 ml of viral inoculation medium (dmem with 2% fbs, 1% penicillin/streptomycin, and 10 mmol/l hepes [ph 7.2]) and the cells were incubated in a humidified 5% co 2 incubator at 378c for 72 hours or until 80% to 90% of cells showed a cytopathic effect (cpe). supernatant was collected and centrifuged to remove cellular debris for 5 minutes at 500 3 g at room temperature. virus was then aliquoted and stored at -808c, and the titer was determined by plaque assay. whole blood units (450 ml 6 10%) were collected and prepared at king abdulaziz university hospital, transfusion services, jeddah, kingdom of saudi arabia, from voluntary donors. briefly, blood units were centrifuged at 544 3 g for 10 minutes to separate the platelet (plt)-rich plasma. the plt rich plasma was then centrifuged at 2305 3 g at 208c for 10 minutes. the plasma was then separated into the plasma bag and kept at not more than -188c. all blood units were screened routinely for hcv antibody or antigen, hbsag, hbc antibody, hiv (1/2) antibody, htlv (1/2) antibody, and syphilis as well as hcv, hbv, and hiv by nat. two units of fresh-frozen plasma (210 ml each) were pooled for each experiment. the count of red blood cells was less than 4 3 10 6 /ml in the pooled plasma. in total, four pools (n 5 4) were used in this study. pooled plasma units were then inoculated with approximately 4 ml mers-cov stock (1:100 dilution). plasma units were inactivated with the intercept processing set for plasma and the intercept illuminator according to the manufacturer's instructions. residual amotosalen and the free photoproducts were removed with an intercept compound adsorption device according to the manufacturer's instructions. samples collected for testing were as follows ( fig. 1) : a positive control sample from the virus stock, a negative control sample from the plasma pool before inoculation, a 2-to 3-ml pretreatment sample from the inoculated plasma pool, and an inactivated sample from the treated pooled plasma. all samples were stored at -808c until testing by plaque and reverse transcriptionquantitative polymerase chain reaction (rt-qpcr) assays. for the detection of mers-cov replication, all samples were diluted at 1:10 dilution in dmem with 10% fbs, inoculated on 90% confluent vero e6 cells in six-well plates, and incubated for 3 to 7 days at 378c. supernatants were collected, diluted 1:10 in dmem with 10% fbs, and blindly passaged for two more times. each passage was observed for cpe and all collected supernatants were used for viral rna quantification by rt-qpcr. vero e6 cells were prepared at 1 3 10 5 /ml in dmem growth medium and 2 ml were seeded in each well in sixwell plates and incubated overnight at 378c. samples was then serially diluted in inoculation dmem starting from 10 21 , and 200 ll of each dilution were applied to the confluent vero e6 cell monolayers in each well and incubated for 1 hour at 378c with gentle shaking every 15 to 20 minutes. after 1 hour, inoculum was removed and replaced with overlay medium (dmem with 0.8% agarose) and incubated for 3 to 4 days at 378c. after incubation, the overlay was removed and cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and stained with crystal violet stain. plaques were examined and counted to calculate titer as plaque-forming units (pfu)/ml as previously described. 35 in some experiments, 15 to 30 ml of ibs-treated plasma was tested in plaque assay to detect any residual infectious virus and to increase the dynamic range of the assay in which 200 ll of neat plasma were inoculated in each well. for all samples (positive, negative, pretreatment, and inactivated samples) and cell supernatants, rna was extracted as previously described 12 using viral rna mini kit (qiagen) according to the manufacturer's instructions. real-time rt-qpcr was performed using primers and probe targeting mers-cov n gene as previously described. 36 specifically, the following forward primer 5 0 -caaaaccttccctaagaaggaaaag-3 0 , reverse primer 5 0 -gctcctttggaggttcagacat-3 0 , and probe 5 0 -fam-acaaaaggcaccaaaagaagaatcaacagacc-bhq1-3 0 were used. real-time rt-qpcr was performed in 96well plates on a fast real-time pcr system (model 7500, applied biosystems) using an rt-pcr kit (quantitect probe, qiagen) according to the manufacturer's instructions in a total reaction volume of 25 ll. rna transcript corresponding to the same target region was generated using a cdna synthesis kit (superscript rt iii, invitrogen) from a plasmid containing the mers-cov n gene according to the manufacturer's instructions and used to generate a standard curve to estimate the viral rna copy number in each sample as previously described. 37, 38 each run included a positive viral template control and no-template negative control. each sample was tested in duplicate and the mean is reported as log copies/ml. samples were reported as negative if the cycle threshold values were higher than 36 cycles. the study was approved by the biomedical ethics committee unit of king abdulaziz university hospital (approval 257-16). to evaluate the potential of the ibs in inactivating mers-cov in pooled plasma, mers-cov virus was spiked into pooled plasma units and treated with amotosalen and uva light. the viral titer in pretreatment samples ranged from 4.51 to 5.04 log pfu/ml with mean titer of 4.67 6 0.25 log pfu/ml ( table 1 ). the treatment resulted in no detection of viable viruses in inactivated samples by plaque assay with a mean reduction of 4.67 6 0.25 log pfu/ml in viral infectivity titer from four independent experiments (experiments a-d) and fig. 2 shows representative results from the plaque assay. therefore, larger volumes from inactivated samples were tested to increase the sensitivity and the dynamic range of the assay as shown in table 1 . nonetheless, no viable virus was detected post pathogen inactivation treatment even when 15 to 30 ml of plasma was tested. the viral genomic titer was not reduced post treatment as expected (table 2) . together, these data show that amotosalen/uva light pathogen inactivation technology inactivated all mers-cov infectious particles in the spiked plasma units. next, we analyzed the presence of any potential low level of replicating mers-cov in inactivated plasma units taking into consideration that genomic viral titer did not change before and after treatment ( table 2) . to this end, vero e6 cells were inoculated with plasma samples of pathogen-inactivated units and incubated for 3 days, followed by two additional passages with 3 days of incubation. no cpe was observed from the negative control or inactivated samples even on day 7 postinoculation or after passaging for three times compared to the positive or pretreatment samples, which completely destroyed all cells by day 3 (data not shown). to further confirm these results, the viral genomic rna titer was measured by rt-qpcr from supernatant collected on day 3 from all samples in the three consecutive passages. as shown in table 3 , viral genomic titer in positive control and pretreatment samples ranged from 9.03 to 10.51 and 9.48 to 10.22 log rna copies/ml, respectively, in the three passages indicating presence of replicating viruses in these samples. on the other hand, no viral rna was detected in the supernatant collected from any of the three passages from the negative control or inactivated samples, confirming the complete inactivation of the infectious mers-cov in plasma. currently, there are more than 70 known pathogens that have been recognized by the aabb as pathogens of concern for blood transfusion; 25 however, blood donations are only screened for a small number of pathogens on a regular basis. although such specific pathogen screening and testing of blood products have significantly reduced transfusion-related infections, transmission of wellrecognized pathogens such as hiv, hbv, and hcv is still being reported in many parts of the world. 39, 40 furthermore, emerging and/or reemerging pathogens which are usually of zoonotic nature pose a high risk for blood safety. 41 the unpredictable nature of such outbreaks and the erratic dynamics of their spread represent a challenging hurdle to preparedness plans and implementation of safety measures. a recent example is the emergence of zika virus, a mosquito-borne flavivirus that is transmissible through blood transfusion. 42 outbreaks of coronaviruses with high mortality in humans have been described for sars-cov in china and southeast asia 23,24 and mers-cov in the middle east and south korea. 9 the detection of viral rna in the blood of patients infected with sars-cov and mers-cov suggests a potential risk for their transmission through transfusion. [21] [22] [23] [24] during a mers-cov outbreak in south korea, levels of viral load ranged from 3.3 to 4.2 log genomic copies/ml in whole blood and from 2.7 to 4.0 log genomic copies/ml in serum, 21 while another study reported up to 6 logs of rna copies/ml in serum from infected individuals. 20 that corresponds to an approximate infectious titer of less than 1.2 log in whole blood and less than 3 logs in serum according to our data which shows 3-log difference between genomic and infectious titers (tables 1 and 2 ). using ibs, we were able to show a mean reduction of 4.67 6 0.25 log in mers-cov infectious titers (ranging from 4.51 to 5.04) in plasma (table 1 ) which is approximately 1.5 logs higher than the minimum reduction titer recommended by the european committee of blood transfusion, 43 suggesting an inactivation capacity of mers-cov in plasma with a high safety margin. in contrast to the infectious titer, the genomic titer was only affected slightly by the pathogen inactivation treatment ( table 2 ). the amotosalen/uva process effectively crosslinks nucleic acids, but does not break the strands. therefore, to ensure that there are no remaining amounts of infectious virus particles, the collected supernatants were passaged three times after inactivation for 3 days. the absence of cpe even after 7 days of incubation in cultures inoculated with the posttreatment samples suggests that there is no infectious mers-cov remaining in these samples. no viral genomes were detected in the supernatant of cell cultures inoculated with posttreatment samples even after multiple passages mostly due to nonreplicating viral genome and degradation of viral rna during the incubation period. this is in contrast to the pretreatment and the positive control samples that showed a high level of viral rna after incubation in all three passages. this demonstrates the loss of infectious virus through passaging and the successful inactivation of infectious particles by the ibs treatment. so far, there have been no reports of any transmission of mers-cov or sars-cov via blood transfusion despite the detection of their viral rna in serum from infected individuals. 20 nonetheless, the aabb still lists both of these viruses as pathogens of concern for blood safety. 25 mers-cov rna detection persisted in patient serum up to 14 days after diagnosis, 20 and there is evidence that mers-cov could infect and replicate in macrophages, dendritic cells, and t cells from the peripheral blood. 36, 44, 45 therefore, it is not really clear whether presence of mers-cov rna in blood, serum, or plasma could have any consequences on the transmission risks of mers-cov during transfusion. apart from that, a recent large blood virome study found that blood from 42% of the study population (8240 healthy blood donors) contained genetic material from 19 46 many of these viruses are of great importance in transfusion medicine although their detection in blood does not necessarily imply infectivity since only genetic material was detected. furthermore, 75 other nonhuman viral species have also been detected in blood from many individuals, which were attributed to contamination from commercial reagents or the environment. 46 nonetheless, identification of other viruses such as the sewage-associated gemycircularvirus was suggested to be due to contamination during phlebotomy or plasma pooling processing. therefore, targeted testing of blood products might not be the best economic or logistic option to ensure blood safety. here, we were able to show efficient inactivation of mers-cov in therapeutic human plasma units of 4.67 logs, far above the expected viral load of mers-cov in human blood and serum described until now. a recent study investigated the inactivation of mers-cov in plasma using riboflavin and uv light. 47 the study showed a reduction of at least 4.07 and at least 4.42 log in viral titers for pooled and individual donor plasma, respectively. however, passaging experiments after pathogen reduction were not conducted, so complete inactivation of all infectious particles cannot be taken for granted. several previous studies have also proved the efficiency of ibs in inactivating a large number of pathogens (viruses, bacteria, and parasites). the study described here adds mers-cov to the list of pathogens that can be inactivated by ibs. taken together with previously published work, our data show that ibs could represent an economically and logistically efficient protective solution to reduce the risk associated with the circulation of mers-cov in the arabian peninsula and the potential transmission of not only mers-cov but also other known or unknown pathogens for which there is no commercially available screening assays. comparison of elisa and rapid screening tests for the diagnosis of hiv, hepatitis b and hepatitis c among healthy blood donors in a tertiary care hospital in mumbai seroprevalence of hiv, hbv, hcv and syphilis infections among blood donors at gondar university teaching hospital, northwest ethiopia: declining trends over a period of five years the expansion of vector-borne diseases and the implications for blood transfusion safety: the case of west nile virus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus world health organization riyadh: saudi ministry of health, command & control center (ccc) middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients middle east respiratory syndrome middle east respiratory syndrome coronavirus: a comprehensive review estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia evidence for camelto-human transmission of mers coronavirus mers coronavirus neutralizing antibodies in camels saudi arabia (1993) and australia (2014) and characterisation of assay specificity middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia risk factors for primary middle east respiratory syndrome coronavirus hindawi et al. illness in humans, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications ifn-a2a or ifn-b1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome (sars)-paradigm of an emerging viral infection current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections pathogen inactivation of platelet and plasma blood components for transfusion using the intercept blood system tm amotosalen photochemical inactivation of severe acute respiratory syndrome coronavirus in human platelet concentrates inactivation of dengue virus in plasma with amotosalen and ultraviolet a illumination inactivation of zika virus in plasma with amotosalen and ultraviolet a illumination pathogen inactivation technologies for cellular blood components: an update a randomized, controlled phase iii trial of therapeutic plasma exchange with fresh-frozen plasma (ffp) prepared with amotosalen and ultraviolet a light compared to untreated ffp in thrombotic thrombocytopenic purpura an active hemovigilance program characterizing the safety profile of 7483 transfusions with plasma components prepared with amotosalen and uva photochemical treatment independent evaluation of tolerance of therapeutic plasma inactivated by amotosalen-hcl-uva (intercept tm ) over a 5-year period of extensive delivery immunogenicity of candidate mers-cov dna vaccines based on the spike protein growth and quantification of mers-cov infection active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections hiv transmission through transfusion-missouri and colorado blood transfusion safety in africa: a literature review of infectious disease and organizational challenges emerging pathogens: the epidemiology and evolution of species jumps evidence for transmission of zika virus by platelet transfusion european directorate for the quality of medicines and healthcare. guide to the preparation, use, and quality assurance of blood components productive replication of middle east respiratory syndrome coronavirus in monocytederived dendritic cells modulates innate immune response middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways the blood dna virome in 8,000 humans inactivation of middle east respiratory syndrome coronavirus (mers-cov) in plasma products using a riboflavin-based and ultraviolet light-based photochemical treatment we thank edwin raj from the blood transfusion services, king abdulaziz university hospital, king abdulaziz university, jeddah, saudi arabia, for the technical help with plasma collection and screening. the authors have disclosed no conflicts of interest. key: cord-286703-ipoj13va authors: de wilde, adriaan h.; falzarano, darryl; zevenhoven-dobbe, jessika c.; beugeling, corrine; fett, craig; martellaro, cynthia; posthuma, clara c.; feldmann, heinz; perlman, stanley; snijder, eric j. title: alisporivir inhibits mersand sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model date: 2017-01-15 journal: virus res doi: 10.1016/j.virusres.2016.11.011 sha: doc_id: 286703 cord_uid: ipoj13va currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like sarsand mers-coronavirus. we here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin a-analog, inhibit the replication of four different coronaviruses, including mersand sars-coronavirus. ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of sars-cov infection in a mouse model. nevertheless, our data provide a basis to further explore the potential of cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication. the outbreak of the severe acute respiratory syndromecoronavirus (sars-cov) in 2003 and the continuing circulation of the middle east respiratory syndrome-coronavirus (mers-cov; since 2012) have highlighted the potentially lethal consequences of zoonotic coronavirus infections in humans. approximately 8000 people were infected during the sars-cov epidemic (∼10% mortality; http://www.who.int/csr/sars/en/), while the global mers tally is now over 1800 laboratory-confirmed cases, with a mortality of about 35% (http://www.who.int/csr/disease/coronavirus infections/en/). in mid-2015, an air travel-related outbreak in south korea (186 confirmed cases, 36 deaths, and about 17,000 potentially exposed individuals quarantined) again illustrated the socioeconomic impact of this kind of emerging pathogen, also outside regions in which the virus is endemic (http://www.who.int/ csr/don/07-july-2015-mers-korea/en/). the lack of effective methods to prevent or treat coronavirus infections in humans remains a serious public health concern, especially given increasing evidence that sars-like coronaviruses continue to circulate in bats and may have the potential to readily cross the species barrier and emerge as human pathogens (ge et al., 2013; menachery et al., 2015) . data from cell culture infection models (chan et al., 2013a (chan et al., , 2013b de wilde et al., 2013b; falzarano et al., 2013a; kindler et al., 2013; zielecki et al., 2013) and experiments in rhesus macaques (falzarano et al., 2013b) and marmosets (chan et al., 2015) suggested that interferons (ifns) are potent inhibitors of mers-cov replication. the outcome of the clinical use of interferons was variable, with some reports questioning the long-term survival benefits (al-tawfiq et al., 2014; omrani et al., 2014; shalhoub et al., 2015) , whereas others suggested that further investigation is warranted (khalid et al., 2016) , possibly in combination with the use of lopinavir/ritonavir (kim et al., 2015) . unfortunately, some of the negative outcomes are confounded by the late-stage treatment of critically-ill patients. since the development and registration of novel therapeutic compounds is generally time consuming, the repurposing of approved drugs offers one of the few shortcuts to establishing anticoronavirus therapy. several fda-approved compounds have been reported to inhibit mers-cov and sars-cov replication in cell culture (de wilde et al., 2014; dyall et al., 2014; hart et al., 2013) , but their efficacy in animal models remains to be determined. previously, the fda-approved drug cyclosporin a (csa) was shown to inhibit the replication of a variety of viruses (reviewed in nagy et al., 2011) , including coronaviruses (carbajo-lozoya et al., 2014; de wilde et al., 2013b de wilde et al., , 2011 kim and lee, 2014; pfefferle et al., 2011; http tanaka et al., 2012) . csa targets members of the cyclophilin (cyp) family, which are peptidyl-proline isomerases (ppiases) that act as chaperones in protein folding and function (davis et al., 2010) . since the immuno suppressive properties of csa (schreiber and crabtree, 1992) are an undesirable side-effect in the context of antiviral therapy, numerous non-immunosuppressive cyp inhibitors have been developed. among these, alisporivir (alv) showed increased sustained viral response during treatment of chronic hepatitis c virus (hcv) infection in phase iii clinical trials, when treatment with ribavirin and pegylated interferon was combined with alv (buti et al., 2015; flisiak et al., 2012; pawlotsky et al., 2015; zeuzem et al., 2015) . in this study, we have first investigated whether alv inhibits mers-and sars-cov replication in cell culture. we employed previously described cell-culture based screening assays relying on the rapid cytopathic effect (cpe) observed in coronavirus-infected cells (de wilde et al., 2013b (de wilde et al., , 2014 . first, the viability of mers-cov-infected vero cells (moi 0.005) treated with increasing alv concentrations was assessed at 3 days post infection (p.i.). we found that virus-induced cpe was prevented by alv in a dosedependent manner (ec 50 3.6 m; table 1 . we next established that alv treatment also reduced the yield of infectious mers-cov progeny. vero and huh7 cells were infected with mers-cov (moi 0.01) and treated with increasing but noncytotoxic alv concentrations (for cell viability data, see fig. 1 ; grey lines) from 1 h post infection (p.i.) onward. progeny virus from mers-cov-infected vero cells was harvested at 48 h p.i. and titrated by plaque assay, which revealed a ∼2 log reduction when using 6.3 m alv ( fig. 2a ). this effect was more pronounced in huh7 cells, as a comparable decrease in progeny titer was already observed when using 3.1 m alv (fig. 2b ). similar results were obtained using fully differentiated primary human airway epithelial (hae) cells, of which the non-ciliated cells were shown to be the primary target for mers-cov infection (raj et al., 2013) . first, air-liquid interface cultures derived from two donors were cultured for 14 days on semi-permeable transwell membranes (van wetering et al., 2007) . next, these hae cell cultures were pretreated for 16 h with 25 m alv or 0.13% etoh (vehicle control) and infected with mers-cov isolate emc/2012 (moi of 2; titer determined on vero cells). after a 48-h alv treatment, mers-cov progeny titers were reduced by 1 or 3 log, depending on the donor (fig. 2c-d) . in a second laboratory, the ability of alv to inhibit mers-cov replication was assessed independently in vero, llc-mk2, and huh7 cells. cells were inoculated with mers-cov (emc/2012) at an moi of 0.001 and medium containing between 0.625 and 20 m of alv was added at 1 h p.i. supernatant was collected on day 3 and levels of infectious virus progeny assessed by tcid 50 assay. in parallel, toxicity was assessed using a cell viability assay. on day 3, a 4-to 5-log decrease in infectious progeny was observed at a dose of 10 m alv and ec 50 values of 3.9, 2.8, and 4.0 m were calculated for vero, llc-mk2, and huh7 cells, respectively ( fig. s1 and table 1) . sars-cov infection (moi 0.01) in vero cells was sensitive to 3.1 m alv (fig. 2e ), resulting in a near-complete block when using 6.3 m alv. a ∼1.5 log reduction in virus progeny could be achieved in veroe6 cells using the same dose (fig. 2f) , suggesting that alv treatment in veroe6 cells was twice less effective compared to vero cells. for both sars-cov and mers-cov, differences in alv sensitivity in various cell lines were observed. one explanation for such difference would be that alv uptake differs per cell line. interestingly, we also observed remarkable difference in e.g. sars-cov-induced cell death between veroe6 and vero cells, ranging from severe cpe in the former to minimal cpe in the latter after the same incubation period (unpublished observations). this suggests that these related cell lines can respond quite differently to virus infection. to investigate the potential of alv as a broad-spectrum coronavirus inhibitor, we assessed the effect of alv treatment on the replication of two additional coronaviruses, murine hepatitis virus (mhv; strain a59) and human coronavirus 229e (hcov-229e). following infection with an moi of 0.01, media were harvested at 16 h p.i. for mhv on 17cl1 cells or 48 h p.i. for hcov-229e on huh7 cells ( fig. 2g and h) . when using an alv dose of 6.3 m, mhv progeny titers were decreased by 1 log, while a 25-m dose resulted in a ∼2log reduction without showing any signs of toxicity in uninfected cells (cell viability values >85% of untreated 17cl1 control cells were observed for all alv concentrations tested; data not shown). hcov-229e production was reduced to a similar extent (∼1.5 and ∼2-log reduction at 6.3 and 12.5 m alv, respectively). as ribavirin has previously been reported to inhibit mers-cov replication (falzarano et al., 2013a) and alv and ribavirin have been used together during clinical trials for hepatitis c treatment (pawlotsky et al., 2015) , this combination was tested in llc-mk2 cells. combining ribavirin and alv treatment primarily had an additive effect on antiviral activity, with the exception of the combination of 25 g/ml ribavirin and 5 m alv, which had a synergy value of 1.51 (fig. 3a) as calculated using macsynergy ii (prichard and shipman, 1990) . increasing concentrations of ribavirin gradually lowered the cell viability measured without affecting cellular morphology when assessed microscopically (fig. 3b) . the above findings suggested that a combination approach might have a chance of success in animal models. therefore, we assessed whether similar inhibitory effects could be observed in a mouse model. we employed the mouse-adapted ma15 strain of sars-cov (roberts et al., 2007) and animals were treated daily with alv (60 mg/kg) and/or ribavirin (50 mg/kg). unfortunately, treatment with alv alone did not enhance survival (data not shown) and combined therapy with alv and ribavirin did not prevent weight loss (fig. 4a ) or enhance survival (fig. 4b) . since the alv concentration required for sars-cov inhibition in cell culture was >100-fold higher than that required for inhibition of hcv replication (coelmont et al., 2010; paeshuyse et al., 2006; puyang et al., 2010) , the negative outcome of this animal experiment may not be too surprising. nevertheless, the use of cyp inhibitors remains a promising and innovative antiviral strategy. this class of drugs can potentially inhibit against broad range of pathogenic viruses, including hepatitis b virus (phillips et al., 2015 ; ec 50 of 4.1 m), hcv (coelmont et al., 2010; paeshuyse et al., 2006; puyang et al., 2010; ec 50 values between 0.02 and 0.23 m), and human immunodeficiency virus 1 (ptak et al., 2008; ec 50 values in the low-nanomolar range). we previously reported that also the replication of two arteriviruses, which are distantly related to coronaviruses, can be blocked by the alv-related nonimmunosuppressive csa analog debio-064 (de wilde et al., 2013a). mock-infected cells that did not receive alv or solvent were used as a reference for cell viability (their relative viability was set at 100 %). cells were incubated for 3 days with the exception for mers-cov emc/2012 in huh7 cells (2 days) and cell viability was monitored using the celltiter 96 ® aqueous non-radioactive cell proliferation assay (promega). in addition, the (potential) toxicity of alv treatment was monitored in parallel in mock-infected cell cultures. graphs show the results (average and sd) of a representative experiment that was performed in quadruplicate. all virus-cell combinations were tested at least twice. lines represent relative cell viability in the absence of infection (alv toxicity control); bars represent relative cell viability after infection and alv treatment. i., virus titers in the culture medium were determined by plaque assays as described before (van den worm et al., 2012) . (c,d) hae cells from two different donors were cultured on semi-permeable 12-well transwell membranes for 14 weeks. about 16 h prior to infection, 25 m alv, 0.13% etoh, or medium was added to the basal side of the cell layer. subsequently, cells were apically infected with mers-cov (moi of 2; titer determined on vero cells). after 48 h, virus release from the apical side of the cell layer was determined by harvesting the mucous fluid and subsequent plaque assay. (e, f) sars-cov-infected (e) vero or (f) veroe6 cells (moi 0.01) were treated with various concentrations of alv from 1 h p.i. onwards, and virus titers in the culture medium at 32 h p.i. were determined by plaque assay. (g) 17cl1 cells infected with mhv-a59 (moi 0.01) or (h) huh7 cells infected with hcov-229e (moi 0.01) were treated with alv from 1 h p.i. onwards, and infectious progeny titers were determined at 16 h p.i. and 48 h p.i., respectively. the graphs show the results of one representative experiment (mean ± sd, n = 3). for all experiments, control infections include cells that remained untreated ("-") or are treated with an amount of etoh equalling that present at the highest alv concentration used. two-sided student's t test (graphpad prism 7 software) was used to determine the significance of inhibition of virus replication between etoh-treated and alv-treated samples ( * p < 0.05; ** p < 0.005; *** p < 0.001; n.s. not significant). our study reveals that alv is a broad-spectrum coronavirus inhibitor in cell culture, as it inhibits the replication of both alphaand betacoronaviruses (figs. 1 and 2 and carbajo-lozoya et al., 2014) . further research is needed to elucidate the exact mechanism of action underlying alv's interference with coronavirus replication, as well as the involvement of cyps in the coronavirus replication cycle. for hcv, alv has been shown to disrupt functional interactions between cyclophilin a (cypa) and viral proteins and/or rna (coelmont et al., 2010; garcia-rivera et al., 2012; nag et al., 2012) . interestingly, although alv has a ∼4 times higher affinity for cypa compared to csa (unpublished data), the ec 50 values for both inhibitors are similar, leaving the possibility that alv and other cyp inhibitors target cov replication independently of cypa. in a previous study, a 4-log reduction in hcov-nl63 virus progeny was reported upon treatment of infected caco-2 cells with 10 m alv (ec 50 0.8 m; carbajo-lozoya et al., 2014). the observed variations in ec 50 values suggest that different coronaviruses may not be equally sensitive to the drug, although they may also reflect differences in e.g. experimental design, cyp expression levels, and/or alv uptake or turnover in different cell lines. the lack of alv activity in the sars-cov animal model suggests that the drug itself may not be suited for the treatment of coronavirus infections. nevertheless, cyp inhibitors remain interesting leads for the development of host-directed anti-coronavirus therapy, as well as interesting tools to study the role of cyps in coronavirus replication in more detail. data are from two independent laboratories. ec50 and cc50 values were calculated as described previously (de wilde et al., 2014; falzarano et al., 2013a) . the selectivity index (si), the relative efficacy of a compound in specifically inhibiting virus replication, was calculated as cc50/ec50. statistical analyses were performed using the results of at least two independent experiments. a ec50 and cc50 values are means (± se) from a representative experiment (n = 4) that was repeated at least twice. b si: selectivity index, calculated as cc50/ec50. c data is presented in fig. 1 . d virus yield is determined by tcid50 assay (data is presented in fig. s1 ; falzarano et al., 2013a) . e experiments performed to independently confirm the antiviral effect of alv in a second laboratory. to 100 g/ml ribavirin from 1 h p.i. onwards. virus titers in the culture medium at 3 days p.i. were determined by tcid50 as previously described (falzarano et al., 2013a) . (b) in parallel, control cells were treated with the same compound concentrations to determine cytotoxicity with a celltiter 96 aqueous one solution cell proliferation assay. ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study alisporivir with peginterferon/ribavirin in patients with chronic hepatitis c genotype 1 infection who failed to respond to or relapsed after prior interferon-based therapy: fundamental, a phase ii trial human coronavirus nl63 replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine a-derivatives including alisporivir broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus tropism and innate immune responses of the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset deb025 (alisporivir) inhibits hepatitis c virus replication by preventing a cyclophilin a induced cis-trans isomerisation in domain ii of ns5a structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques update on alisporivir in treatment of viral hepatitis c multiple mutations in hepatitis c virus ns5a domain ii are required to confer a significant level of resistance to alisporivir isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards porcine epidemic diarrhea virus induces caspase-independent apoptosis through activation of mitochondrial apoptosis-inducing factor combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome: a case report efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential a sars-like cluster of circulating bat coronaviruses shows potential for human emergence suppression of viral rna binding and the assembly of infectious hepatitis c virus particles in vitro by cyclophilin inhibitors emerging picture of host chaperone and cyclophilin roles in rna virus replication ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study the non-immunosuppressive cyclosporin debio-025 is a potent inhibitor of hepatitis c virus replication in vitro alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis c virus genotype 2 or 3 infection the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors alisporivir inhibition of hepatocyte cyclophilins reduces hbv replication and hepatitis b surface antigen production a three-dimensional model to analyze drug-drug interactions inhibition of human immunodeficiency virus type 1 replication in human cells by debio-025, a novel cyclophilin binding agent mechanism of resistance of hepatitis c virus replicons to structurally distinct cyclophilin inhibitors dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice the mechanism of action of cyclosporin a and fk506 ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study isolation of a novel coronavirus from a man with pneumonia in saudi arabia randomised clinical trial: alisporivir combined with peginterferon and ribavirin in treatment-naive patients with chronic hcv genotype 1 infection (essential ii) human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus cyclosporin a inhibits the replication of diverse coronaviruses cyclophilin inhibitors block arterivirus replication by interfering with viral rna synthesis mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture epithelial differentiation is a determinant in the production of eotaxin-2 and −3 by bronchial epithelial cells in response to il-4 and il-13 we thank dr. frauke fischer, dr. nikolai naoumov (novartis, switzerland) and dr. grégoire vuagniaux (debiopharm, switzerland) for helpful discussions and providing alisporivir. we supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2016.11. 011. key: cord-294800-akr4f5p8 authors: kabir, md. tanvir; uddin, md. sahab; hossain, md. farhad; abdulhakim, jawaher a.; alam, md. asraful; ashraf, ghulam md; bungau, simona g.; bin-jumah, may n.; abdel-daim, mohamed m.; aleya, lotfi title: ncovid-19 pandemic: from molecular pathogenesis to potential investigational therapeutics date: 2020-07-10 journal: front cell dev biol doi: 10.3389/fcell.2020.00616 sha: doc_id: 294800 cord_uid: akr4f5p8 in december 2019, a severe acute respiratory syndrome coronavirus 2 (sars-cov-2)-related epidemic was first observed in wuhan, china. in 2020, owing to the highly infectious and deadly nature of the virus, this widespread novel coronavirus disease 2019 (ncovid-19) became a worldwide pandemic. studies have revealed that various environmental factors including temperature, humidity, and air pollution may also affect the transmission pattern of covid-19. unfortunately, still, there is no specific drug that has been validated in large-scale studies to treat patients with confirmed ncovid-19. however, remdesivir, an inhibitor of rna-dependent rna polymerase (rdrp), has appeared as an auspicious antiviral drug. currently, a large-scale study on remdesivir (i.e., 200 mg on first day, then 100 mg once/day) is ongoing to evaluate its clinical efficacy to treat ncovid-19. good antiviral activity against sars-cov-2 was not observed with the use of lopinavir/ritonavir (lpv/r). nonetheless, the combination of umifenovir and lpv/r was found to have better antiviral activity. furthermore, a combination of hydroxychloroquine (i.e., 200 mg 3 times/day) and azithromycin (i.e., 500 mg on first day, then 250 mg/day from day 2–5) also exhibited good activity. currently, there are also ongoing studies to evaluate the efficacy of teicoplanin and monoclonal and polyclonal antibodies against sars-cov-2. thus, in this article, we have analyzed the genetic diversity and molecular pathogenesis of ncovid-19. we also present possible therapeutic options for ncovid-19 patients. coronaviruses (covs) belong to the large family of positivesense, enveloped, highly diverse, and single-stranded rna viruses (fehr and perlman, 2015) . indeed, covs have been found to infect both humans and animals, therefore causing various respiratory, gastrointestinal, neuronal, and hepatic diseases (weiss and leibowitz, 2011; chan et al., 2013; zumla et al., 2016) . former epidemics of covs include severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov, these outbreaks caused severe health problems in humans . a group of individuals was admitted to hospitals in late december of 2019 with a primary diagnosis of pneumonia due to an unknown cause (bogoch et al., 2020; lu et al., 2020) . it was assumed by the earlier reports that the onset of a potential cov epidemic provided the estimation of a reproduction number for the 2019 novel coronavirus (ncovid-19, named by world health organization (who) on feb 11, 2020) which was thought to be considerably >1 (ranges from 2.24-3.58) . this severe acute respiratory syndrome coronavirus 2 (sars-cov-2) can be transmitted largely via droplets and due to the close contact. it has been found that elderly people and individuals with chronic diseases or comorbidities are particularly high-risk populations (li et al., 2020a) . there are various symptoms of ncovid-19 including cough (68%), fever (88%), diarrhea (3.7%), and vomiting (5%) (mungroo et al., 2020) . the mode of transmission of sarscov-2 is supposed to take place from human to human through respiratory secretions released by the infected people when sneezing and coughing (mungroo et al., 2020) . ncovid-19 patients can be asymptomatic, which is making the control of the transmission more difficult (gao et al., 2020; li et al., 2020a) . since february of 2020, strict infection control approaches were executed by the centers for disease control (cdc) in order to limit the spread of sars-cov-2. in a recent study, mentioned that ncovid-19 patients had the highest viral load (measured in saliva samples) near presentation. they also summarized that as viral load is quite high during the time of hospital admissions, use of potent antiviral agents at an early stage might prove abbreviations: ace2, angiotensin converting enzyme 2; ap, antigen presentation; apcs, antigen presentation cells; apn, aminopeptidase n, arbs, angiotensin ii receptor blockers; ards, acute respiratory distress syndrome; cdc, centers for disease control; ncovid-19, novel coronavirus disease 2019; covs, coronaviruses; dpp4, dipeptidyl peptidase 4; dsrna, double-strand rna; ec 50 , half maximal effective concentration; ed, emergency department; elisa, enzymelinked immunosorbent assay; eua, emergency use authorization; fda, food and drug administration; ggo, ground-glass opacity; hcv, hepatitis c virus; hiv, human immunodeficiency virus;, mhc, major histocompatibility complex; or hla, human leukocyte antigen; icu, intensive care unit; il-6, interleukin 6; lpv/r, lopinavir/ritonavir; mabs, monoclonal antibodies; mers, middle east respiratory syndrome; n7-mtase, n7-methyltransferase; nsaids, nonsteroidal anti-inflammatory drugs; prrs, pattern recognition receptors; pui, patient under investigation; rdrp, rna-dependent rna polymerase; rsv, respiratory syncytial virus; s protein, spike protein; sam, s-adenosyl-methionine; sars, severe acute respiratory syndrome; sars-cov-2, severe acute respiratory syndrome coronavirus 2; tmprss2, transmembrane serine protease 2; who, world health organization. beneficial in managing the severity of ncovid-19 infection . previously, sars was found to be partially linked with environmental factors (lin et al., 2006) . in a study, it was revealed that air pollution was linked with mortality in sars patients in china (cui et al., 2003) . in this regard, it was mentioned that lung functions can be compromised owing to long-or shortterm exposure to certain environmental pollutants (cui et al., 2003) . air temperature is another factor that is also needed to be considered. it has been revealed by lin et al. (2006) that the occurrence of sars was much higher (18 times) at lower air temperatures as compared to higher temperatures. researchers also showed that respiratory disorders are more likely to take place in colder environments since virulence of agents are likely to deteriorate at higher air temperatures because they might not endure the alterations in the environment (d'amato et al., 2018) . in addition to this, they also summarized that sars-cov's transmissibility is comparable with the transmissibility of influenza virus. moreover, the occurrence of influenza markedly elevates with high relative humidity and low temperatures (park et al., 2020) , which is further suggesting that viral transmission can be significantly affected by environmental factors. there are no therapeutic agents that have been approved to treat ncovid-19. various medicines including immunomodulatory or antiviral drugs such as remdesivir, favipiravir, ribavirin, chloroquine, hydroxychloroquine, azithromycin, nitazoxanide, teicoplanin etc. have been advised as potential investigational drugs, many of which are now being studied in animals and humans who, 2020c) . on march 28, 2020, the food and drug administration (fda) gave an emergency use authorization (eua) for emergency use of oral administrations of chloroquine phosphate and hydroxychloroquine sulfate to treat sars-cov-2 infection (fda, 2020) . along with oxygen and mechanical ventilation, a guideline has also been published by belgium which involved recommendations from four other european countries, including switzerland, netherlands, france, and italy that recommended the use of remdesivir, lopinavir/ritonavir, tocilizumab, and chloroquine or hydroxychloroquine (sciensano, 2020) . in addition, japan and china approved the use of favipiravir (an antiviral agent) to treat influenza, which is now under investigation to treat ncovid-19 (fujifilm, 2020) . in this article, we have critically appraised the genetic diversity, molecular pathogenesis, symptoms, diagnosis, and prevention of ncovid-19. furthermore, we also specially reviewed the mechanisms, efficacy, and use of various drugs that might be beneficial in combating ncovid-19 infection. in nature, nucleotide substitution is considered as a vital step for viral evolution (lauring and andino, 2010) . the rapid spreading of sars-cov-2 raised a suspicion that mutations are driving its evolution. in a recent study, from gisaid, phan (2020a) collected 86 complete or near-complete sars-cov-2 genomes to estimate its genetic variation. in addition to this, these strains of sars-cov-2 were identified in patients with confirmed ncovid-19 from usa (11), china (50), japan (5), australia (5), england (2), singapore (3), france (4), germany (1), belgium (1), south korea (1), vietnam (1), and taiwan (2). clustalx2 was used to align the pair-wise nucleotide sequence (saitou and nei, 1987) . as a reference genome, the sequence of the strain "china/whu01/2020/epi_isl_406716" was used. interestingly, similar to other beta coronaviruses, the genome of sars-cov-2 contains a long orf1ab polyprotein at the 5 ′ end, followed by 4 main structural proteins, such as nucleocapsid protein, matrix protein, small envelope protein, and spike surface glycoprotein (phan, 2020b) . in addition to this, it was also observed that there were 3 deletions in the genomes of sars-cov-2 from australia (victoria), usa (wisconsin), and japan (aichi). in contrast, 1 deletion (10 nucleotides) was found in the 3 ′ end of the genome, while 2 deletions (2 nucleotides and 24 nucleotides) were found in the orf1ab polyprotein. furthermore, it was also observed from the nucleotide sequence alignment that there were 93 missense mutations in the entire genomes of novel coronavirus (table 1) . except for the envelope protein, 42 mutations were detected in all of the main structural and non-structural proteins. whereas, 4 missense mutations were observed in the nucleocapsid protein, 1 in the matrix protein, 29 in the orf1ab polyprotein, and 8 in the spike surface glycoprotein. interestingly 3 mutations (i.e., phe 367 , tyr 364 , and asp 354 ) were found in the spike surface glycoprotein receptor-binding domain. indeed, spike surface glycoprotein contributes significantly in binding to receptors on the host cell and eventually regulates host tropism (fung and liu, 2019) . furthermore, this spike glycoprotein is the main target of neutralizing antibodies . conformational changes of spike glycoprotein can be induced by the mutations, which can lead to altered antigenicity. up until now, no study has identified the amino acids that are involved in conformational alterations of spike glycoprotein. therefore, further studies are required to identify these important amino acids. various wild and domestic animals such as bats, cats, cattle, and camels might play a role as hosts for coronaviruses (adhikari et al., 2020) . in general, animal coronaviruses do not spread among human beings (cdc, 2020a) . nevertheless, exceptions have been noticed in case of mers and sars, where these diseases were found to be transmitted owing to the contact with respiratory droplets from sneezing or coughing of ncovid-19 patients. initial ncovid-19 patients were detected in china, where there was an association with the seafood market of wuhan, which is indicating that these initial infections took place because of the animal-to-person transmission. later on, ncovid-19 was also detected in healthcare professionals and also in other individuals where there was no history of contact with that affected area of wuhan, which is further suggesting the human-to-human transmission (gralinski and menachery, 2020; huang et al., 2020; li et al., 2020b; liu et al., 2020; who, 2020d) . as per the recent guidelines from health authorities of china (adhikari et al., 2020; who, 2020e) , there are 3 major routes of ncovid-19 transmission including droplets transmission, aerosol transmission, and contact transmission. transmissions via droplets were found to take place when respiratory droplets of infected individuals are inhaled or ingested by people who are in close contact. whereas, contact transmission might take place when a person touches a virus-contaminated-object or surface and then that person touches his/her nose, mouth, or eyes. on the other hand, aerosol transmission might take place when respiratory droplets mix into the air, thus forms aerosols and might result in infection when a high dose of aerosols are inhaled into the lungs in a comparatively closed environment (adhikari et al., 2020; who, 2020e) . in a study, it was revealed that the digestive system is also a possible route for sars-cov-2 transmission. symptoms like diarrhea and abdominal discomfort have been observed in individuals with confirmed ncovid-19, this observation led to studies which revealed that ace2 (to which sars-cov-2 binds) is highly expressed in enterocytes of colon and ileum . the effect of temperature on the health of humans can be varied depending on the countries or even areas (hajat and kosatky, 2010) . in line with this aforesaid finding, it was also reported that temperature can affect the transmission of respiratory syndromes-causing viruses including sars-cov-2 (ma et al., 2020) and influenza virus (park et al., 2020) . studies have also revealed that novel coronavirus and influenza virus can survive only in some specific environmental conditions and their transmissions also depend on temperatures (chan et al., 2011; jaakkola et al., 2014) , which is also applicable for sars-cov-2 transmission (wang et al., 2020c) . it was observed in case of influenza virus that it can transmit more readily at lower temperatures (lowen and steel, 2014) , since host immunity is likely to remain weakened in cold weather, this can further increase the vulnerability toward infection (kudo et al., 2019) . as the transmission process of coronaviruses is comparable with the influenza virus transmission (lin et al., 2006) , thus it can be expected that these processes are also applicable for the sars-cov-2 transmission (wang et al., 2020c) . several other environmental factors can affect the link between mortality and temperature including air pollution (cai et al., 2007) , humidity (jaakkola et al., 2014; kudo et al., 2019) , latitude (bao et al., 2016) . in this regard, socio-demographic factors including income, age, and gender (bao et al., 2016) have also been reported to play roles. in a study, chan et al. (2011) revealed that individuals who live at lower latitudes showed a strong adaptive capacity toward heat, and a relatively weak adaptive capacity was observed toward cold. these researchers also observed that the viability of sars-cov was much lower at higher relative humidity and higher temperatures (for example, relative humidity: over 95%, and temperature: 38 • c). in a different study, it was revealed that humidity and temperature are linked with an increased risk of ncovid-19 (wang et al., 2020c) . interestingly, coronaviruses can persist on inanimate surfaces including plastic, glass, or metal for up to 9 days (kampf et al., 2020) . ncovid-19 patients exhibit various clinical symptoms including cough, fever, fatigue, radiographic evidence of pneumonia, dyspnea, decreased or normal leukocyte counts, and myalgia . these aforesaid symptoms are also similar to mers-cov and sars-cov infections (peiris et al., 2004) . even though ncovid-19 pathogenesis is not well-understood, however the similar mechanisms used previously by mers-cov and sars-cov can provide a lot of information regarding sars-cov-2 pathogenesis (figure 1 ). spike protein (s protein) of coronavirus determines the viral entry into the host cells (de wit et al., 2016) . interestingly, the envelope spike glycoprotein binds to its cellular receptor, angiotensin converting enzyme 2 (ace2) for sars-cov (li et al., 2003b) and sars-cov-2 (figure 1) , dipeptidyl peptidase 4 for mers-cov (raj et al., 2013) , and cd209l for sars-cov [26] . although it was initially identified that sars-cov enters into cells by direct fusion of plasma membrane and virus (simmons et al., 2004) . however, belouzard et al. (2009) revealed that a vital proteolytic cleavage process takes place at sars-cov s protein at position (s2 ′ ) that facilitated the membrane fusion and infectivity of the virus. for membrane fusion, mers-cov also has evolved an aberrant 2 steps furin activation (mille and whittaker, 2014) . other than membrane fusion, entry of sars-cov was also found to be mediated by the clathrin-independent and -dependent endocytosis (wang et al., 2008; kuba et al., 2010) . following the entry of virus into the cells, rna genome of sars-cov is released into the cytoplasm and is translated into 2 polyproteins and structural proteins, subsequently the viral genome starts to replicate (perlman and netland, 2009 ). the newly generated envelope glycoproteins are then inserted into the membrane of the endoplasmic reticulum or golgi apparatus, and the it has been found that s protein is cleaved into s1 and s2 by a cell-derived protease, where s1 binds with ace2 receptor, and s2 is activated by the host serine protease tmprss2 and results in a fusion with the cell membrane. following the entry into the host cell, sars-cov-2 takeovers the host cell machinery to transcribe, replicate, and translate its rna genome and structural proteins before being reassembled, encapsulated, and exocytosed from the host cell. following exocytosis, sars-cov-2 is presented to host antigen presenting cells (apcs), which eventually leads to the generation of various cytokines including, tnf-α, cxcl-10, il-1, and il-6 (invivogen, 2020 when the sars-cov-2 enters into the cells, its antigen will be presented to the antigen presentation cells (apcs) (figure 1) , this process is crucial for the anti-viral immunity of the human body (kumar et al., 2020) . peptides of antigens are presented via major histocompatibility complex (mhc; or human leukocyte antigen (hla) in humans) and then identified by virusspecific cytotoxic t lymphocytes. therefore, the understanding of antigen presentation (ap) of the virus will provide a better understanding of the pathogenesis of ncovid-19. however, not much information is available regarding this, thus we can obtain information from previous studies on mers-cov and sars-cov. ap of sars-cov-2 mostly relies on mhc i molecules (liu et al., 2010) , nonetheless mhc ii also plays roles in its presentation. former studies revealed that many hla polymorphisms associate with the susceptibility of sars-cov, for instance hla-cw * 0801 (chen et al., 2006b) , hla-b * 0703, hla-dr b1 * 1202, and hla-b * 4601 (keicho et al., 2009) , while hla-a * 0201, hla-dr0301, and hla-cw1502 alleles are associated with the protection from sars infection (wang et al., 2011) . in case of mers-cov, it was observed that mhc ii molecules (for example hla-dqb1 * 02:0 and hla-drb1 * 11:01) were linked with the susceptibility to mers-cov infection (hajeer et al., 2016) . other than mannose-binding lectin gene polymorphisms linked with ap are associated with the risk of sars-cov infection (tu et al., 2015) . indeed, the aforementioned findings will give us an important idea regarding the mechanism, prevention, and treatment of ncovid-19. ap subsequently induces the human body's humoral and cellular immune responses, which are then facilitated via virus-specific b and t cells. like other common acute viral infections, antibodies including igg and igm are produced against sars-cov virus. it is estimated that at the end of week 12, sars-specific igm antibodies disappear. whereas, sars-specific igg antibody can stay for a longer period, which is suggesting that igg mainly has a protective function (li et al., 2003a) . furthermore, it was also found that sars-specific igg antibodies mainly are nspecific and s-specific antibodies (de wit et al., 2016) . most of the studies have focused on cellular immune responses, as compared to the humoral immune responses in case of coronavirus. recent findings have revealed that the levels of cd8 + and cd4 + t cells in the peripheral blood of ncovid-19 individuals were significantly decreased, as confirmed by increased percentages of cd38 (cd8 39.4%) and hla-dr (cd4 3.47%) double-positive fractions . likewise, acute phase response in individuals with ncovid-19 is linked with a marked decrease of cd8 + t and cd4 + t cells. interestingly, it was found that although there is no presence of antigen, cd8 + , and cd4 + memory t cells can last for 4 years in individuals who have recovered from sars-cov and can perform ifnγ generation, delayed-type hypersensitivity response and t cell proliferation (fan et al., 2009) . after 6 years of infection with sars-cov, specific t-cell memory responses to the sars-cov s peptide library can still be identified in 14 of 23 recovered sars individuals (tang et al., 2011) . in mouse models, specific cd8 + t cells also exhibited similar activity in the clearance of mers-cov (zhao et al., 2014) . indeed, these results might be useful in the rational designing of an effective vaccine against sars-cov-2. acute respiratory distress syndrome (ards) is considered as the major cause of ncovid-19-related death. in the early stages of the epidemic, 6 out of the 41 admitted patients with confirmed ncovid-19 died owing to ards . this ards is found to be the main immunopathological characteristic of sars-cov, mers-cov, and sars-cov-2 infections . cytokine storm is the major characteristic of ards. this storm is a fatal uncontrolled systemic inflammatory response that takes place because of the high secretions of chemokines (i.e., c-x-c motif chemokine 10 like sars-cov, mers patients showed increased levels of cxcl-10, cxcl8, ccl5, ifn-α, and interleukin 6 (il-6) in serum as compared to individuals with the mild to moderate disease (min et al., 2016) . in the human body, a powerful cytokine storm will induce an aggressive attack by the immune system, which will lead to multiple organ failure and ards, and will ultimately result in death in severe novel coronavirus infection, as like mers-cov and sars-cov infection . various strategies are used by viruses including sars-cov and mers-cov to evade immune responses for their better survival in host cells. the pattern recognition receptors (prrs) can identify the evolutionarily conserved microbial structures called pathogen-associated molecular patterns. nonetheless, mers-cov and sars-cov can stimulate the generation of doublemembrane vesicles lacking prrs and subsequently can replicate in these vesicles, thus evading the host detection of their doublestrand rna (dsrna) (snijder et al., 2006) . ifn-i (ifn-β and ifn-α) plays a protective function on mers-cov and sars-cov infection, however the ifn-i mechanism is suppressed in infected mouse models (channappanavar et al., 2016 (channappanavar et al., , 2019 . interestingly, by directly interacting with the dsrna, mers-cov's accessory protein 4a might block the stimulation of ifn at the level of melanoma differentiation-associated protein 5 activation (niemeyer et al., 2013) . ifn β promoter activation and transportation of ifn regulatory factor 3 to the nucleus can be inhibited by the orf5, orf4b, orf4a, and membrane proteins of mers-cov (yang et al., 2013) . sars-cov-2 can also affect the ap. in this regard, for instance, gene expression associated with ap is downregulated following mers-cov infection . therefore, it is vital to terminate the immune evasion of coronavirus to develop specific and effective therapies. following an incubation period of around 5.2 days, the symptoms of sars-cov-2 infection appear . it takes around 6 to 41 days from the first appearance of the symptoms to death, along with a median of 14 days (wang et al., 2020d) . however, the aforesaid durations depend on various factors including the patient's age and status of the immune system. this duration was found to be shorter for individuals older than 70-years old as compared to the individuals who are under the age of 70 (wang et al., 2020d) . at the onset of the disease, the most commonly observed symptoms are cough, fatigue, and fever (figure 2 ). in addition to this, various other symptoms including headache, lymphopenia, dyspnea, sputum production, diarrhea, and hemoptysis (graham carlos et al., 2020; huang et al., 2020; ren et al., 2020; wang et al., 2020d) . pneumonia has also been identified by computed tomography scan in ncovid-19 patients, unfortunately, various aberrant clinical features including ground-glass opacity (ggo), acute cardiac injury, and ards led to death . occasionally, in subpleural areas of both lungs, the multiple peripheral ggos were detected (lei et al., 2020) and these triggered both localized and systemic immune responses, which collectively raised the level of inflammation. unfortunately, treatment with interferon inhalation did not result in any clinical benefit, rather it aggravated the condition via facilitating pulmonary opacities (lei et al., 2020) . indeed, some of the symptoms of ncovid-19 are similar to the earlier betacoronavirus including dyspnea, dry cough, fever, and bilateral ggos . nonetheless, there are some unique clinical manifestations of ncovid-19 such as sore throat, sneezing, rhinorrhea (lee et al., 2003; assiri et al., 2013) . as revealed by chest radiographs following admission, in some cases it was observed that an infiltrate in lung's upper lobe is linked with growing dyspnea with hypoxemia (phan et al., 2020) . although ncovid-19 exhibited digestive disorders like diarrhea, only a small proportion of sars-cov or mers-cov showed similar gastrointestinal symptoms. thus, testing urine and fecal samples are important to eliminate a possible alternative mode of transmission (lee et al., 2003; assiri et al., 2013) . henceforth, developing methods to detect different routes of transmission for example urine and fecal samples are immediately required to develop ways to suppress and/or minimize the transmission and also to discover therapies to treat ncovid-19. recently, it has been observed that ncovid-19 might predispose to both arterial and venous thromboembolic disease because of immobilization, hypoxia, inflammation, and diffuse intravascular coagulation guan et al., 2020; klok et al., 2020; wang et al., 2020a; zhou et al., 2020) . furthermore, it was also revealed that respiratory failure in the disease is not only driven by the ards, rather microvascular thrombotic activities might also contribute in this regard (grillet et al., 2020) . therefore, klok et al. (2020) have strongly suggested to administer pharmacological agents in a prophylactic manner to all the intensive care unit (icu) ncovid-19 patients. for any given emergency department (ed) visiting patients with the symptoms of fever and respiratory diseases, healthcare workers must need to get a travel history in detail from that patient. if a patient shows flu-like symptoms and has a travel history to a country or area with confirmed ncovid-19 cases or if the patient came into close contact with a confirmed ncovid-19 patient in the last 14 days then the patient ought to be considered as a patient under investigation (pui) (npr, 2020) . it needs to be noted that here close contact means any individual who was within six feet of an individual with confirmed ncovid-19 for an extended period. furthermore, any individual who came into direct contact with the secretions of any ncovid-19 patient will also be considered as a close contact. individuals who have traveled from high-risk countries or areas with confirmed ncovid-19 cases and members of a family who are suffering from ncovid-19 and not staying at home care or not maintaining isolation precautions are regarded as highrisk exposures. while medium risk exposures involve individuals who have traveled from low-risk countries or areas and family members are stringently maintaining appropriate home care and adhering with proper isolation precautions (who, 2020e). in contrast, low-risk exposures involve those individuals who were in the same indoor environment (for example in a waiting hall) for a longer period with ncovid-19 patients but did not come into close contact. molecular assays of respiratory specimens are performed for diagnosis purposes usually at the regional referral laboratories designated by who (kaiser health news, 2020). for regional testing, the cdc started distributing ncovid-19 test kits on february 7 (who, 2020a). ncovid-19 test is getting more widely available day by day. for hospitals or institutions where ncovid-19 test is not available, the only option is the testing by cdc. ncovid-19 should be tested on an urgent basis for the pui cases. an individual should be removed from pui status only if that individual is fully evaluated clinically and has consulted with proper healthcare professionals. the mode of sars-cov-2 transmission is still complex. guidelines for ncovid-19 prevention is mainly based on the previously developed guidelines for sars and mers and also on the intervening guidelines provided by cdc and who (cdc, 2020a,b; who, 2020a) . before or upon arrival in ed, a pui ought to be identified by the hospitals to protect the healthcare professionals and other patients. prevention measures should involve maintaining hand and respiratory hygiene and also screening questions including travel history. following a pui identification, both local health department and hospital infection control ought to be immediately notified to avert further spread among healthcare professionals and other patients. a surgical mask must need to be given to any pui and need to be isolated in a private room or if possible in a negative pressure room (who, 2020a). as like sars and mers, ncovid-19 is also found to spread through the airborne route. therefore, surgical face masks might be beneficial to prevent sneeze and cough-related larger fluid droplets, however they are less likely to prevent small airborne contaminants (yee et al., 2020) . in this regard, respirators containing air filters and adequate seal should be more beneficial (tran et al., 2012; smith et al., 2016) . in healthcare settings, right use of respirators and personal protective equipment and proper hand hygiene are likely to prevent transmission (cowling et al., 2009; radonovich et al., 2019; yee et al., 2020) . if a patient requires hospital admission and there is no private or separate room for that patient, then that patient needs to be taken to an adequate facility containing institution. isolated rooms and care provide would need to be customized in a way that reduces the exposure of healthcare providers to the patient. indeed, along with an eye shield, all the healthcare providers must take measures to prevent contact with droplets and to maintain airborne precautions. since the risk of transmission is much higher during the aerosol-generating procedures (such as intubation), in these cases the importance of ppe is enormous (raboud et al., 2010; cdc, 2020a) . still, it remains not known, regarding how long ncovid-19 can stay airborne following a patient leaves the room. respiratory protection is essential to enter into the vacated room. since still there is no specific drug to treat ncovid-19, therefore the best approach will be taking preventative measures at a personal level including avoiding public transport, unnecessary travel, contact with ncovid-19 suspected individuals, and so on. indeed, the significance of maintaining frequent and proper hand hygiene is paramount. like other coronaviruses, sars-cov-2 has a lipid envelope, thus proper hand-washing with soap can break apart that lipid envelope and therefore can make it difficult or even impossible for the virus to infect humans. so far, this proper hand-washing is considered as the most effective preventative measure. in addition, duration of hand-washing with soap is also equally important. cdc has recommended that effective hand-washing should last at least for 20 s. in a study, borchgrevink et al. (2013) showed that out of 3,749 individuals in a college town environment, only 5% of those individuals properly followed the hand-washing rules (i.e., washing, rubbing, and rinsing). this finding indicates that there is a poor understanding of the significance of proper handwashing among the general people. therefore, awareness among people should be increased about the importance of frequent and proper hand-washing. in order to form a physical barrier, the who has recommended the use of a face mask by those individuals who are showing respiratory symptoms (who, 2020b). however, healthy people are not required to use face masks. a typical surgical mask only provides one-way protection and can avert the spreading of droplets during coughing and sneezing to the surrounding areas. healthcare professionals who are treating or in contact with a suspected or confirmed ncovid-19 patient must need to wear a specialized respirator (for example n95 or its equivalent) to effectively prevent the droplets entry and thus can reduce the chance of acquiring the infection (bae et al., 2020; who, 2020b) . strict precautionary measures must need to be taken by the individuals during handling affected individual's body secretions including sputum, urine, or stools (yeo et al., 2020) . remdesivir out of all the investigational drugs, remdesivir (figure 3) has appeared as the most effective and promising antiviral drug (li and de clercq, 2020) . this antiviral drug targets rnadependent rna polymerase (rdrp) of the virus while escaping proofreading via viral exoribonuclease, (agostini et al., 2018) which can ultimately lead to early termination of viral rna transcription as given in figure 4 . interestingly, remdesivir is a phosphoramidate prodrug and has a wide range of activities against numerous virus families, such as pneumoviridae, paramyxoviridae, filoviridae, and orthocoronavirinae (for example pathogenic mers-cov and sars-cov) (sheahan et al., 2017; martinez, 2020) . in a covid-19 mouse model, when remdesivir was administered prophylactically and as early therapeutic intervention, it significantly decreased the pulmonary viral load, which ultimately reduced the progression of the disease and significantly improved respiration (sheahan et al., 2017) . in tissue culture models, brown et al. revealed that remdesivir showed half-maximal effective concentration (ec 50 ) of 0.074 mm and 0.069 mm for mers-cov and sars-cov, successively . furthermore, remdesivir (within the submicromolar ec50s) also effectively inhibited zoonotic cov and human covs (hcov-229e and hcov-oc43) ko et al., 2020) . similar results were also observed when remdesivir was administered therapeutically (12 h post-inoculation) and prophylactically (24 h before prior inoculation) in mers animal (rhesus macaque) model (de wit et al., 2020) . even 2 amino acid substitutions (v553l and f476l) in the non-structural protein 12 polymerase were found to show lower-level of resistance toward remdesivir (agostini et al., 2018) . in humans, pharmacokinetic data of remdesivir is not available. however, it has been revealed in rhesus monkeys that intravenous remdesivir administration at the dose of 10 mg/kg increased the intracellular concentration (over 10 mm) of active triphosphate form in peripheral blood mononuclear cells for a minimum of 24 h, which is indicating its clinical significance in ncovid-19 treatment. furthermore, human safety data of remdesivir are available online (mulangu et al., 2019) . in usa, the first patient with confirmed ncovid-19 was effectively treated with remdesivir for the advancement of pneumonia on 7th day of hospital admission in january, 2020 (holshue et al., 2020) . moreover, to assess its efficacy and safety to treat individuals with confirmed ncovid-19, phase iii clinical trials (clinicaltrials.gov, 2020e) have been started in march 2020. in that study, individuals received 200 mg of remdesivir on first day, subsequently received 100 mg/day. although remdesivir showed promising in vitro and clinical activity against coronavirus (sheahan et al., 2017; holshue et al., 2020) , recently it has been reported that there are some uncertainties because of its multiple adverse effects including hepatotoxicity, rectal hemorrhage (jean et al., 2020b) . in japan, favipiravir (figure 3) was primarily developed and approved as an anti-influenza drug (shiraki and daikoku, 2020; wang et al., 2020b) . this antiviral drug has a wide range of activities against various rna viruses including rhinovirus, respiratory syncytial virus (rsv), and influenza. former studies revealed that favipiravir was successfully used to treat infections associated with rabies, lassa virus, and ebola virus (shiraki and daikoku, 2020) . furthermore, favipiravir was also found to be effective to treat severe fever with thrombocytopenia syndrome (shiraki and daikoku, 2020) . nevertheless, favipiravir was found to be ineffective against dna viruses. favipiravir is a potent antiviral drug that selectively suppresses the rdrp of rna viruses (figure 4) favipiravir is likely to produce resistant viruses, as compared to oseltamivir (shiraki and daikoku, 2020) . indeed, this feature of favipiravir can be beneficial in the treatment of ncovid-19. to treat influenza, favipiravir's recommended oral dose is 1,600 mg two times on first day, subsequently 600 mg twice/day from day 2 to 5, and 600 mg once/day on the sixth day. in recent times, initial findings of clinical trials have revealed that favipiravir exhibited significant activity in treating chinese ncovid-19 patients ( table 2 ) (xinhua news agency). in china, favipiravir has been approved to treat ncovid-19 in march 2020. furthermore, in china, randomized controlled trials involving ncovid-19 patients are also assessing the efficacy of favipiravir plus baloxavir marboxil (an antiviral drug) and favipiravir plus ifn-α (arab-zozani et al., 2020). ribavirin (figure 3) is a rdrp inhibitor (figure 4 ) used to treat various viral infections, for example, infections caused by rsv and hepatitis c virus (hcv) (ogawa and morisada, 2002) . it was revealed by in vitro studies that when ribavirin was administered at a concentration of 50 mg/ml, it showed effective antiviral activity against sars-cov (chan et al., 2015) . unfortunately, this antiviral drug was found to decrease the level of hemoglobin, therefore it can be detrimental for individuals with respiratory distress (martinez, 2020) . umifenovir umifenovir (figure 3) is a potent antiviral agent that has a wide-range of activities against various viruses including hcv, influenza a and b viruses (boriskin et al., 2008) . umifenovir's mechanism slightly varies with different viruses. it has been revealed that umifenovir suppresses the fusion of the virus with the host cell membrane (figure 4) , thus the subsequent viral entry into the host cell is inhibited (boriskin et al., 2008) . in a clinical trial, it has recently been observed that lopinavir/ritonavir (lpv/r, figure 3 ) protease inhibitors that are mainly used in human immunodeficiency virus (hiv) treatment did not significantly improve the ncovid-19 symptoms . furthermore, in a different study, effect of umifenovir plus lpv/r was compared with the sole treatment with lpv/r to treat ncovid-19 (deng et al., 2020) . the findings of that study revealed that better effects were observed with the treatment of umifenovir plus lpv/r in comparison with the sole lpv/r treatment (deng et al., 2020) . however, more studies are required to evaluate the incidence of resistance and efficacy. as coronavirus becomes activated on the membrane of the host cell, thus combination of lpv/r and umifenovir are likely to inhibit/prevent the viral entry into the host cell (figure 4) . besides, there is also a need regarding a better chloroquine chloroquine (figure 3) is mainly used as an antimalarial drug. furthermore, chloroquine is also used to treat various autoimmune disorders including rheumatoid arthritis and lupus erythematosus. in an animal model, it has recently been observed that chloroquine can also play a role as a potent antiviral drug against various viruses including influenza h5n1 (yan et al., 2013) . interestingly, chloroquine can prevent the viral fusion with the cell membrane of host cell by increasing endosomal ph (figure 4) . glycosylation of sars-cov's cellular receptors can also be interfered by chloroquine (vincent et al., 2005; wang et al., 2020b) . even though findings from in vitro studies regarding chloroquine is auspicious (ec 90 = 6.90 mm, used ncovid-19-infected vero e6 cells), however use of chloroquine to treat ncovid-19 infection is a completely off-label use. furthermore, this drug is not strongly indicated due to some of its safety reasons including qt prolongation with ventricular dysrhythmia and adverse reactions on the renal, hepatic, and hematologic systems (cortegiani et al., 2020) . hydroxychloroquine (a chloroquine derivative, figure 3 ) is also mainly used as antimalarial and anti-inflammatory drugs (sinha and balayla, 2020) . it has been proposed that hydroxychloroquine controls cytokine storm (figure 4) , which takes place in critically ill late phase ncovid-19 patients (yao et al., 2020) . as compared to chloroquine, hydroxychloroquine is more potent and their ec 50 values are 5.47 and 0.72, successively. in addition to this, hydroxychloroquine is less likely to interact with other drugs as compared to chloroquine. moreover, in comparison with chloroquine phosphate, pharmacokinetic data confirmed that hydroxychloroquine is much more effective (5 days before) at inhibiting sars-cov-2 in vitro (yao et al., 2020) . it has been declared on march 26, 2020 by taiwan cdc that hydroxychloroquine has a significant role in the treatment of ncovid-19 patients. however, treatment with hydroxychloroquine is contraindicated for the patients who are pregnant or breastfeeding, allergic to hydroxychloroquine, glucose-6-phosphatase deficient, and for individuals with prolonged qt interval in electrocardiograms and retinopathy (gautret et al., 2020) . azithromycin previously, azithromycin (figure 3) showed excellent in vitro activity against ebola virus (madrid et al., 2016) . it was found that azithromycin was administered to individuals with viral infection, it prevented severe infections of respiratory tract in pre-school children (bacharier et al., 2015) . in a recent study, when azithromycin was administered (i.e., 500 mg on first day, then 250 mg per day from day 2-5), it remarkably reinforced the hydroxychloroquine's efficacy (when 200 mg was administered 3 times/day for 10 days) to treat 20 severely ill ncovid-19 patients (figure 4) . the mean serum concentration of hydroxychloroquine was 0.46 ± 0.20 mg/ml. it is assumed that this excellent virus eliminating activity was achieved owing to the use of the aforesaid combination therapy (gautret et al., 2020) . therefore, use of azithromycin along with hydroxychloroquine can be an effective future alternative to remdesivir in ncovid-19 treatment. however, in this regard, a possible complication related to prolonged qt interval should be taken into consideration. teicoplanin is a glycopeptide antibiotic and it has been revealed by zhou et al. (zhou et al., 2016 ) that teicoplanin exerted inhibitory activity (ic 50 as low as 330 nm) against replication-and transcription-competent virus-like particles. studies confirmed that teicoplanin can suppress the entry of mers and sars envelope pseudotyped viruses zhou et al., 2016) . in terms of its mechanism, teicoplanin can selectively suppress the effects of cathepsins b and l in host cell. these proteases are involved with cleaving the viral glycoprotein permitting exposure of the receptor-binding domain of its core genome and then release into the cytoplasm of host cells (zhou et al., 2016; baron et al., 2020) . therefore, teicoplanin blocked the entry of ebola virus in the late endosomal pathway. also, the derivatives of teicoplanin including telavancin, dalbavancin, and oritavancin, were also found to block the entry of sars, mers, and ebola viruses (zhou et al., 2016) . collectively, these findings suggest that teicoplanin and its derivatives might play a vital role in inhibiting the viruses that are dependent on cathepsin l ( table 2) . ivermectin ivermectin (figure 3) is an antiparasitic agent and it has broadspectrum of activity (caly et al., 2020) , recent in vitro studies have revealed that this drug also has an antiviral effect against dengue and hiv viruses . it has been found that the preformed impα/β1 heterodimer is accountable for the transport of viral protein into the nucleus and ivermectin can dissociate this heterodimer. since this transport of viral protein into the nucleus is important for the replication cycle and suppression of the host's antiviral response, thus targeting this viral protein transport might prove as a significant target in the development of therapeutic agents against rna viruses (caly et al., 2012; yang et al., 2020a) . following 48 h of ncovid-19 infection, a recent in vivo study has been demonstrated that ivermectin can decrease the level of viral rna (figure 4 ) up to 5,000-times (caly et al., 2020) . since ivermectin has an established safety profile as an antiparasitic agent, thus now it is needed to establish a safe and effective dose of this drug in clinical trials to treat ncovid-19 infection. nitazoxanide (figure 3) is an effective antiparasitic and antiviral drug (rossignol, 2016) . this drug has a broad-spectrum in vitro antiviral activity against a range of viruses including rsv, rotavirus, parainfluenza, influenza, and coronavirus (rossignol, 2016) . in vero-e6 cells, nitazoxanide exerted a potent in vitro antiviral activity against sars cov-2 (ec 50 = 2.12 µm, at 48 h) . furthermore, this strong antiviral effect is in line with the observed ec 50 values for nitazoxanide (ec 50 = 0.92 µm) and tizoxanide (an active metabolite of nitazoxanide) (ec 50 = 0.83 µm) against mers-cov in llc-mk2 cells (rossignol, 2016) . in terms of its mechanism of action, it is believed that nitazoxanide has potent antiviral effect because of its ability to interfere with the host-regulated pathways associated with viral replication instead of the virus-specific pathways (rossignol, 2016) . therefore, studies were carried out to evaluate the ability of this drug to treat influenza and other related acute respiratory infections. in the phase iib/iii of a clinical trial, positive effects of nitazoxanide were observed in the management of influenza symptoms, where 600 mg of nitazoxanide was orally administered twice a day (haffizulla et al., 2014) . unfortunately, in phase ii clinical trial it was observed that nitazoxanide neither alleviated the symptoms nor decrease the length of stay in hospitals of individuals infected with respiratory viruses (gamiño-arroyo et al., 2019). however, in vitro data regarding the activity of nitazoxanide against coronavirus is promising. therefore, further studies are required to estimate its potential in ncovid-19 treatment. baricitinib most commonly used in rheumatoid arthritis treatment. this drug is a reversible and selective inhibitor of janus kinase 1 (jak1) and jak2. it has been found that these latter mentioned enzymes transduce intracellular signals for growth factors and cytokines associated with immune response, inflammation, and haematopoiesis. moreover, this jak inhibitor blocks the activities of ap2-associated with protein kinase 1, which ultimately prevents viral binding with the alveolar epithelium (mayence and vanden eynde, 2019) . it has also been indicated that baricitinib might be used as an additional therapy for the covid-19 treatment (richardson et al., 2020) . in order to determine the safety and efficacy of sarilumab, hydroxychloroquine, lopinavir/ritonavir, and baricitinib to treat 1,000 hospitalized covid-19 patients, a non-randomized phase ii clinical study has recently been started (scavone et al., 2020) . other selective jak inhibitors including ruxolitinib, fedratinib, and sunitinib might also be effective against covid-19 in decreasing endocytosis of virus, inflammation, and levels of cytokines including il-6 and ifn-γ (bekerman et al., 2017; clinicaltrials.gov, 2020f; favalli et al., 2020; scavone et al., 2020; stebbing et al., 2020) . previously, convalescent plasma therapy was used as a terminal therapy to increase the survival rate of individuals with a range of viral infections including sars, severe infection caused by ebola virus, pandemic 2009 influenza a h1n1, h5n1 avian influenza shen et al., 2020) . convalescent plasma therapy can be effective because viremia can be suppressed due to the presence of plasma immunoglobulin antibodies in recovering patients. in a study, shen et al. (2020) evaluated the effect of convalescent plasma therapy in 5 severely ill ncovid-19 patients with ards. in that study, convalescent plasma was transfused in those patients with a novel coronavirus-specific antibody (neutralization titer >40 and binding titer > 1:1000). the used convalescent plasma of that study was obtained from five ncovid-19-recovered individuals. the obtained convalescent plasma was then administered to the 5 patients (in between 10 and 22 days following admission) along with methylprednisolone and antiviral drugs. after convalescent plasma transfusion, clinical conditions of the patients were found to be improved, including decreased viral loads (patients became ncovid-19 negative within 12 days), elevated level of sars-cov-2-specific enzyme-linked immunosorbent assay (elisa), neutralizing antibody titers, normalized body temperature (within 3 days in four/five patients), improved ards (four patients at 12 days following transfusion), successful weaning from mechanical ventilation (three participating individuals within 2 weeks of therapy), increased partial pressure of oxygen/fraction of inspired oxygen, and reduced score in sequential organ failure assessment. out of the 5 participants, 2 of them were in stable condition (at 37 days following transfusions), while 3 of them were discharged from the hospital (following 51, 53, and 55 days of staying in the hospital) (shen et al., 2020) . finally, the researchers summarized that although there were a small number of participants in this study, they suggested the therapy with convalescent plasma can be effective in the ncovid-19 treatment (shen et al., 2020) . as a prophylactic measure and therapy, monoclonal and polyclonal antibodies (targeting hemagglutinin binding) have been recommended to treat various viral infections including influenza (beigel et al., 2019) . the effectiveness of these antibodies against mers-cov largely encouraged the recent efforts to develop monoclonal and polyclonal antibodies against coronaviruses (sheahan et al., 2017) . for instance, in a phase i trial, sab-301 (a human polyclonal antibody) which was produced in transchromosomic cattle was found to be safe and better tolerated in healthy participants (beigel et al., 2019) . in a study, cockrell et al. (2016) revealed in mouse models that human monoclonal antibodies (mabs)-based immunotherapy only mediated protection in the early stage of mers (martinez, 2020) . many in vitro analyses showed that s protein of sars-cov is crucial to mediate the viral entry into the host cells. in addition to this, the cleavage and subsequent activation of the s protein of sars-cov via a host cell's protease is vital for the entry of the virus (glowacka et al., 2011) . in cell cultures, it has been noticed that transmembrane serine protease 2 (tmprss2) is a vital protease of host cells that causes activation of s protein of sars-cov, therefore it was studied as an important target for antiviral drugs (sheahan et al., 2017) . previously, camostat mesylate (an inhibitor of serine protease) showed inhibitory activity against tmprss2 (kawase et al., 2012) . furthermore, k11777 (a cysteine protease inhibitor) exhibited significant inhibitory activity (at submicromolar range) against replication of mers-cov and sars-cov (zhou et al., 2015) . sarilumab is a human monoclonal antibody and 3 clinical trials are ongoing to assess the safety and efficacy of this antibody (alone or along with other standard therapies) in nearly 1,500 covid-19 patients (clinicaltrials.gov, 2020a,d,f; scavone et al., 2020) . eculizumab (a monoclonal antibody) is approved to treat neuromyelitis spectrum disorders, refractory generalized myasthenia gravis, and atypical hemolytic uraemic syndrome. this monoclonal antibody inhibits the terminal portion of the inflammatory response-associated complement cascade. although the function of the complement cascade in ncovid-19 pathogenesis is not clear, numerous studies revealed that its suppression may effectively function as a therapeutic technique (ip et al., 2005; yuan et al., 2005; gralinski et al., 2018) . due to these findings, eculizumab will be tested in the solid-c19 clinical trial to treat individuals with severe ards and ncovid-19 (clinicaltrials.gov, 2020b). currently, emapalumab (a monoclonal antibody) is being studied in an openlabel, randomized, phase ii/iii study to evaluate the safety and efficacy of this antibody in decreasing respiratory distress and hyper-inflammation in ncovid-19 patients (clinicaltrials.gov, 2020c) . in china, stem cells are currently being studied as a treatment for ncovid-19. tocilizumab (a mab) is an immunosuppressive agent and is used to treat rheumatoid arthritis (kaneko, 2013) . this agent was designed to suppress the il-6 binding with its receptors to alleviate cytokine storm syndrome. tocilizumab is now being studied as a potential ncovid-19 treatment (jean et al., 2020b; slater, 2020) . in ncovid-19 high-risk populations, traditional chinese medicines were also regarded as a preventative measure, based on the traditional uses and anecdotal evidence of prevention of h1n1 pdm09 and sars. nonetheless, there is a lacking of clinical data regarding the effectiveness of these herbal medicines as an ncovid-19 treatment (cunningham et al., 2020; luo et al., 2020) . in china, several traditional medicines were widely used during the ncovid-19 epidemic and 6 of these herbal medicines include lianqiao (fructus forsythia), jinyinhua (lonicerae japonicae flos), gancao (glycyrrhizae radix et rhizoma), baizhu (atractylodis macrocephalae rhizoma, rhizome of atractylodes macrocephala koidz), fangfeng (saposhnikoviae radix, dried root from the perennial herb saposhnikovia divaricate), and huangqi (astragali radix, dried root of astragalus membranaceus bge. var. mongholicus). indeed, stringent clinical studies are required with a large number of participants to demonstrate the preventive role of these traditional chinese medicines (cunningham et al., 2020; luo et al., 2020) . the occurrence of co-infection can widely vary among the patients with confirmed ncovid-19. various reports suggest that several co-pathogens including viruses (such as rhinovirus, influenza, and hiv) and bacteria (for example candida species, mycoplasma pneumonia) can co-exist in these patients. among them, influenza a virus was most commonly found to coexist (jean et al., 2020b) . furthermore, ncovid-19 patients with pneumonia were found to be commonly treated by the coadministration of anti-influenza drugs and antibiotics (jean et al., 2020b) . therefore, careful selection of potential broadspectrum antibiotic(s) is required for the long-stay (over 6 days) hospitalized patients (chou et al., 2019; jean et al., 2020a) . mixed clinical findings were observed with the use of corticosteroids to treat sars-cov infections. although various reports suggested that there was no significant contribution of corticosteroids in clinical outcomes (stockman et al., 2006) . in contrast, it was suggested by a report that decreased mortality rate was observed due to the use of corticosteroids in critically ill patients (chen et al., 2006a; wu et al., 2020a) . unfortunately, several reports suggested worse outcomes including longer time for viral clearance, or elevated composite endpoint of icu admission or even death, owing to the use of corticosteroids (auyeung et al., 2005) . in a cohort (n = 309), a longer time in viral clearance was observed in the corticosteroids-receiving mers-cov patients (arabi et al., 2018) . nevertheless, in the same study, it was observed that there was an insignificant decrease in 90-day mortality in corticosteroids-receiving patients. recent reports suggested that there was a decreased rate of mortality in ncovid-19 patients with ards due to the use of corticosteroids (wu et al., 2020a) . these findings suggest that use of corticosteroids resulted in inconsistent outcomes. however, corticosteroids might be beneficial for patients with cytokine-linked lung injury and those who might rapidly develop progressive pneumonia . indeed, healthcare professionals need to carefully assess the risk and benefit ratio of corticosteroid use for each patient. this necessity to assess risk and benefit of corticosteroid use in individual patients and its careful dose consideration has been demonstrated in diagnosis and treatment guidelines from china's national health commission. as per that guideline, glucocorticoid (equivalent to methylprednisolone 1-2 mg/kg per day for three-five days or less) may be considered based on chest imaging and respiratory distress. large-dose of glucocorticoids can suppress the immune system, this can result in delayed sars-cov-2 clearance (mccreary and pogue, 2020) . recently, chinese thoracic society recommended a lower dose of methylprednisolone (≤ 0.5-1 mg/kg per day) for a maximum of 7 days in selected patients, prior to treatment these selected patients should be carefully assessed for potential risks and benefits . more clinical studies are immediately required to elucidate the function of corticosteroids in ncovid-19. in a study, yang et al. (2020b) mentioned that diabetes and cerebrovascular diseases were the commonly observed comorbidities in the non-survivors of ncovid-19 in icus. furthermore, guan et al. (2020) also observed similar results in their study and these ncovid-19 patients received angiotensin ii receptor blockers (arbs) or ace inhibitors. indeed, sars-cov-2 and sars-cov can bind with the ace2 receptors on the epithelial cells of lung, kidney, and intestine (fang et al., 2020) . therefore, when ards is not present, arb or ace inhibitors can be administered to ncovid-19 patients. increased activity of ace2 was found to be linked with decreased severity of ards among individuals with rsv-caused lower respiratory tract infection (wösten-van asperen et al., 2013) . interestingly, fedson (2016) revealed in their study that statins mainly target host response to infection, instead of the virus itself. these researchers also indicated that combination therapy with statins and arb may induce the reversal of homeostatic processes, which will allow the self-recovery of individuals (fedson et al., 2020) . there is an argument regarding the usage of non-steroidal anti-inflammatory drugs (nsaids) like ibuprofen since it can increase the ace2 receptors (day, 2020) . if the severely ill ncovid-19 individuals suffer from fever, acetaminophen can be a good option to control body temperature as compared to other nsaids (therapeutics initiative, 2020). tang et al. (2020) confirmed that anticoagulant therapy by heparin (an anticoagulant) specially with low molecular weight heparin improved the prognosis in severely ill patients with ncovid-19. furthermore, 28-day mortality of heparin receivers was found to be lower as compared to the non-users among individuals with sepsis-stimulated coagulopathy scores 4 or ddimer > 6-times the upper limit of normal . in human body, vitamin a plays various important functions including protecting mucosal and epithelium integrity, mediating growth and development, and proper maintenance of vision (huang et al., 2018) . vitamin a is also essential for enhancing immune response and maintaining regulatory action in both humoral and cellular immune responses (huang et al., 2018) . in case of infants, supplementation with vitamin a was found to ameliorate antibody response following several vaccines including anti-rabies (siddiqui et al., 2001) and measles vaccination (huang et al., 2018) . moreover, an improved immune response to influenza virus vaccination has also been reported in children (2-8 years) who had a deficiency of vitamin a and d at baseline, following supplementation with vitamin a and d (patel et al., 2019) . vitamin d has a significant contribution in modifying both adaptive and innate immune responses (aranow, 2011) . it has been revealed by epidemiological studies that there is a link between deficiency of vitamin d and elevated susceptibility to acute viral respiratory infections (monlezun et al., 2015) . it has also been suggested that vitamin d significantly modulates the innate immune responses against various viral respiratory infections including rsv, parainfluenza 1 and 2, and influenza a and b (zdrenghea et al., 2017) . indeed, studies have revealed that there is a strong relationship between vitamin d deficiency and elevated risk of both lower and upper respiratory tract infections (jolliffe et al., 2013) . nonetheless, conflicting and heterogeneity in dosage regimens and baseline vitamin d conditions in study populations were observed in randomized controlled trials (rcts) (jolliffe et al., 2013) . in a study, aglipay et al. (2017) observed no significant difference between the action of high-dose (2000 iu per day) vs. standard-dose (400 iu per day) vitamin d supplementation on viral upper respiratory tract infections (aglipay et al., 2017) . nevertheless, only one-third of the study subjects received vitamin d at doses below 30 ng/ml. vitamin d increased the plasma level of tgfβ without ameliorating antibody generation in a rct on the effect of vitamin d administration on influenza vaccine response in deficient elderly person (goncalves-mendes et al., 2019) . in addition to this, it was also indicated in the latter mentioned rct that vitamin d administration perhaps directed the polarization of lymphocyte toward a tolerogenic immune response (goncalves-mendes et al., 2019) . in a different rct, monthly administration of high-dose of vitamin d (100,000 iu/month) decreased the occurrence of acute respiratory infections in older long-term care residents as compared to a standard dose group (12,000 iu/month) (ginde et al., 2017) . therefore, it is quite clear that the effect of vitamin d on antiviral immunity against respiratory infections is dependent on an individual's vitamin d status. moreover, it has been confirmed that vitamin d supplementation is also useful in case of other viral infections, for instance, vitamin d addition to conventional peg-α-2b/ribavirin therapy for treatment-naive individuals with chronic hcv genotype 1 infection considerably ameliorated the viral response (abu-mouch et al., 2011) , and similar action was also seen in individuals with hcv genotype 2-3 (nimer and mouch, 2012) . vitamin e possesses strong antioxidant property and it can modify host immune responses [14] . the deficiency of this vitamin can lead to impairment of both cellular and humoral immune responses (moriguchi and muraga, 2000) . some studies revealed that administration of vitamin e may exert harmful activities in case of infectious disease. vitamin e increased the risk of pneumonia among 50-69 years old adult smokers (hemilä and kaprio, 2008) . similarly, vitamin e supplementation (200 iu/day) did not significantly reduce the respiratory tract infections in elderly nursing facility residents (meydani et al., 2004) . nevertheless, in a small pilot rct, positive activities of vitamin e were seen in the treatment of chronic hepatitis b, where vitamin e administration markedly normalized the liver enzymes and hbv-dna negativization (andreone et al., 2001) . similarly, in a rct, vitamin e supplementation increased anti-hbe seroconversion and virological response in the pediatric population (fiorino et al., 2017) . vitamin c plays a significant role as an enzymatic cofactor in numerous physiological reactions including immune potentiation, collagen synthesis, and hormone generation . in mouse models, it was revealed that vitamin c plays important role in the antiviral immune responses against the influenza a virus (h3n2) via the elevated generation of ifn-α/β, particularly at the early stages of infection . nonetheless, no significant benefit has been observed in using mega-dose of vitamin c as a prophylactic measure to lower the incidence of common cold caused by viral infections (hemilä and chalker, 2013) . zinc (an essential trace element) contributes significantly in the growth, development, and maintenance of immune responses (prasad, 2013; read et al., 2019) . the deficiency of zinc is linked with an enhanced susceptibility toward infectious diseases, for example, viral infections. an individual's zinc status is a vital factor that can affect the immune response against viral infections. indeed, zinc-deficient individuals are at greater risk of developing infections including hcv or hiv (read et al., 2019) . acevedo-murillo et al. (2019) reported that there was a noticeable clinical improvement in the 103 children (1 month−5 years) with pneumonia in the zinc-receiving group as compared to placebo (acevedo-murillo et al., 2019) . the researchers also confirmed that there was a rise in the cytokine response in th1 pattern (inf-γ and il-2) only in the zinc-receiving group, along with th2 cytokines (il-10 and il-4) being increased or remained elevated in both groups. following stem cell transplantation, oral administration of a high dose of zinc (150 mg/day) increased thymic activity and output of new cd4 + naive t cells, which eventually helped in the prevention of torque teno virus reactivation (iovino et al., 2018) . nonetheless, provinciali et al. (1998) summarized that prolonged administration of zinc (400 mg/day) or zinc plus arginine (4 d/day) in the elderly (age 64-100 years) people restored zinc concentrations in plasma, which was ineffective in stimulating or improving the antibody response or number of cd3, cd4, or cd8 lymphocytes following influenza vaccination. selenium (a trace element) also exerts a range of important functions including antioxidant effects, various pleiotropic activities, and anti-inflammatory effects (rayman, 2012) . selenium deficiency is found to be linked with cognitive impairment, poor immune response, and elevated risk of mortality, whereas an increased level of selenium or treatment with selenium has exhibited antiviral actions (rayman, 2012) . broome et al. (2004) assessed whether an increased selenium administration (50-100 µg/day) ameliorated immune response in adults with a borderline concentration of selenium (broome et al., 2004) . treatment with selenium elevated the plasma selenium levels, and also increased the activities of cytosolic glutathione peroxidase and lymphocyte phospholipid. furthermore, selenium also increased the cellular immune responses (elevated level of ifn-γ and other cytokines), along with an increased level of t-helper cells and earlier peak tcell proliferation. nonetheless, it was observed that humoral immune responses were not affected (broome et al., 2004) . moreover, selenium treatment in participants also induced rapid poliovirus clearance. copper (another essential trace element) has a significant contribution in the differentiation and development of immune cells (li et al., 2019) . it has also been confirmed that copper exerted in vitro antiviral effects. intracellular copper was found to regulate the life cycle of influenza virus (rupp et al., 2017) , while thujaplicin-copper chelates inhibited the replication of human influenza viruses (miyamoto et al., 1998) . in a study, turnlund et al. (2004) determined the effects of chronic administration of copper on immune response, oxidative stress, and indices of copper status. these researchers observed that when copper was administered at a dose of 7.8 mg/day, copper significantly increased the level of superoxide dismutase, benzylamine oxidase, and plasma ceruloplasmin activity as compared to 1.6 mg/day dose, which further suggesting an enhancement in antioxidant status. nonetheless, increased copper administration (7.8 mg/day) markedly decreased the proportion of antibody titer, serum il-2r, and circulating neutrophils against the beijing strain of influenza (turnlund et al., 2004) . magnesium (an essential mineral) has a significant contribution in regulating immune response via significantly affecting the t helper-b cell adherence, macrophage response to lymphokines, immunoglobulin m (igm) lymphocyte binding, adherence with immune cells, antibody-dependent cytolysis, and immunoglobulin synthesis (liang et al., 2012) . it has also been reported in in vivo and in-vitro studies that magnesium may have a contribution in the immune function against viral infections (chaigne-delalande et al., 2013) . still now there is no specific antiviral drug to treat ncovid-19, but some of the investigational drugs were found to be useful. various drugs are being analyzed in vitro studies or clinical trials. although ribavirin is a potent antiviral drug, its clinical effects are not clear and its side effects ought to be carefully considered. on the other hand, chloroquine has been studied in 15 interventional studies. furthermore, in the chinese clinical trial registry, the derivatives of chloroquine were prospectively registered; and more studies are required to evaluate their antiviral effects and to estimate the recommended dose in ncovid-19 patients . along with antiviral drugs, glucocorticoids ought to be utilized carefully and in a timely manner in ncovid-19 patients. in addition to this, extracorporeal support need to be considered under strict contraindications and indications, otherwise, there will be numerous additional complications and also a waste of resources . in order to manage the current ncovid-19 outbreak, extensive measures are needed to be taken to lower the personto-person transmission of the virus. in addition to this, special efforts and attention are required to reduce or protect the susceptible populations such as elderly people, health care providers, and children. more studies are also essential to understand the mechanisms related to ncovid-19 pathogenesis. this better understanding will help the development of specific and effective therapies against sars-cov-2. since the respiratory tract is mainly affected by sars-cov-2, thus special consideration is required to deliver the drug into the respiratory tract. more studies in animals and clinical trials on drug repositioning can also be considered to identify potential drugs to treat ncovid-19. still there is no available specific drug or vaccine to treat ncovid-19, thus effective preventative measures are recommended. specific drugs are urgently required to inhibit the entry of the virus and subsequent replication to overcome this outbreak. currently, as mentioned in this article, multiple investigational drugs and clinical trials are ongoing. the discovery of new drugs will ultimately enable us to better control this outbreak. furthermore, in silico studies can also be considered to faster the drug development process. finally, sharing findings or data will be effective to fight against ncovid-19 globally. mk and mu conceived the original idea and designed the outlines of the study and prepared the figures for the manuscript. mk wrote the initial draft of the manuscript. mu revised and improved the draft. mh, ja, ma, ga, sb, mb-j, ma-d, and la participated in the literature review of the manuscript. all authors have read and approved the final manuscript. vitamin d supplementation improves sustained virologic response in chronic hepatitis c (genotype 1)-naïve patients zinc supplementation promotes a th1 response and improves clinical symptoms in fewer hours in children with pneumonia younger than 5 years old epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (covid-19) during the early outbreak period: a scoping review effect of high-dose vs standard-dose wintertime vitamin d supplementation on viral upper respiratory tract infections in young healthy children coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease vitamin e as treatment for chronic hepatitis b: results of a randomized controlled pilot trial corticosteroid therapy for critically ill patients with middle east respiratory syndrome favipiravir for treating novel coronavirus (covid-19) patients: protocol for a systematic review and meta-analysis of controlled trials. medrxiv vitamin d and the immune system epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses a randomized clinical trial effectiveness of surgical and cotton masks in blocking sars-cov-2: a controlled comparison in 4 patients the influence of temperature on mortality and its lag effect: a study in four chinese cities with different latitudes teicoplanin: an alternative drug for the treatment of covid-19? advances in respiratory virus therapeutics -a meeting report from the 6th isirv antiviral group conference anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites pneumonia of unknown aetiology in wuhan, china: potential for international spread via commercial air travel hand washing practices in a college town environment arbidol: a broadspectrum antiviral compound that blocks viral fusion an increase in selenium intake improves immune function and poliovirus handling in adults with marginal selenium status broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro nuclear trafficking of proteins from rna viruses: potential target for antivirals? human immunopathogenesis of severe acute respiratory syndrome (sars) a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 cases in the u.s. available online at infection control guidance for healthcare professionals about coronavirus (covid-19) mg2+ regulates cytotoxic functions of nk and cd8 t cells in chronic ebv infection through nkg2d treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns the effects of temperature and relative humidity on the viability of the sars coronavirus dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-covinfected mice ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology convalescent plasma as a potential therapy for covid-19 treatment of severe acute respiratory syndrome with glucosteroids: the guangzhou experience clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study epidemiological and genetic correlates of severe acute respiratory syndrome coronavirus infection in the hospital with the highest nosocomial infection rate in taiwan in 2003 recommendations and guidelines for the treatment of pneumonia in taiwan cohort multiple randomized controlled trials openlabel of immune modulatory drugs and other treatments in covid-19 patients -sarilumab trial -corimuno-19 -sari eculizumab (soliris) in covid-19 infected patients efficacy and safety of emapalumab and anakinra in reducing hyperinflammation and respiratory distress in patients with covid-19 infection evaluation of the efficacy and safety of sarilumab in hospitalized patients with covid-19 study to evaluate the safety and antiviral activity of remdesivir (gs-5734tm) in participants with moderate coronavirus disease (covid-19) compared to standard of care treatment treatment of moderate to severe coronavirus disease (covid-19) in hospitalized patients a mouse model for mers coronavirus-induced acute respiratory distress syndrome a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial air pollution and case fatality of sars in the people's republic of china: an ecologic study treatment of covid-19: old tricks for new challenges the impact of cold on the respiratory tract and its consequences to respiratory health covid-19: ibuprofen should not be used for managing symptoms, say doctors and scientists prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection sars and mers: recent insights into emerging coronaviruses arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study characterization of sars-cov-specific memory t cells from recovered individuals 4 years after infection are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? covid-19 infection and rheumatoid arthritis: faraway, so close! request for emergency use authorization for use of chloroquine phosphate or hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of, 019 coronavirus disease treating the host response to emerging virus diseases: lessons learned from sepsis, pneumonia, influenza and ebola hiding in plain sight: an approach to treating patients with severe covid-19 infection coronaviruses: an overview of their replication and pathogenesis vitamin e for the treatment of children with hepatitis b e antigen-positive chronic hepatitis: a systematic review and meta-analysis global fujifilm accelerates production of its influenza antiviral drug "avigan r tablet" for covid-19 human coronavirus: hostpathogen interaction efficacy and safety of nitazoxanide in addition to standard of care for the treatment of severe acute respiratory illness a systematic review of asymptomatic infections with covid-19 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial high-dose monthly vitamin d for prevention of acute respiratory infection in older long-term care residents: a randomized clinical trial evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response impact of vitamin d supplementation on influenza vaccine response and immune functions in deficient elderly persons: a randomized placebo-controlled trial novel wuhan (2019-ncov) coronavirus return of the coronavirus: 2019-ncov complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis acute pulmonary embolism associated with covid-19 pneumonia detected by pulmonary ct angiography clinical characteristics of coronavirus disease 2019 in china effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial heat-related mortality: a review and exploration of heterogeneity association of human leukocyte antigen class ii alleles with severe middle east respiratory syndrome-coronavirus infection vitamin c for preventing and treating the common cold vitamin e supplementation and pneumonia risk in males who initiated smoking at an early age: effect modification by body weight and dietary vitamin c first case of 2019 novel coronavirus in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan role of vitamin a in the immune system spotlight on covid-19: infection high-dose zinc oral supplementation after stem cell transplantation causes an increase of trecs and cd4+ naïve lymphocytes and prevents ttv reactivation mannose-binding lectin in severe acute respiratory syndrome coronavirus infection decline in temperature and humidity increases the occurrence of influenza in cold climate epidemiology, treatment, and prevention of nosocomial bacterial pneumonia treatment options for covid-19: the reality and challenges vitamin d in the prevention of acute respiratory infection: systematic review of clinical studies cdc coronavirus testing decision likely to haunt nation for months to come persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents tocilizumab in rheumatoid arthritis: efficacy, safety and its place in therapy simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry association of human leukocyte antigen class ii alleles with severe acute respiratory syndrome in the vietnamese population vitamin c is an essential factor on the anti-viral immune responses through the production of interferon-α/β at the initial stage of influenza a virus (h3n2) infection incidence of thrombotic complications in critically ill icu patients with covid-19 arguments in favour of remdesivir for treating sars-cov-2 infections trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters low ambient humidity impairs barrier function and innate resistance against influenza infection host immune response and immunobiology of human sars-cov-2 infection quasispecies theory and the behavior of rna viruses a major outbreak of severe acute respiratory syndrome in hong kong ct imaging of the 2019 novel coronavirus (2019-ncov) pneumonia. radiology the role of copper homeostasis at the host-pathogen axis: from bacteria to fungi profile of specific antibodies to the sars-associated coronavirus therapeutic options for the 2019 novel coronavirus (2019-ncov) scientific research progress of covid-19/ sars-cov-2 in the first five months early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus magnesium affects the cytokine secretion of cd4+ t lymphocytes in acute asthma environmental factors on the sars epidemic: air temperature, passage of time and multiplicative effect of hospital infection novel immunodominant peptide presentation strategy: a featured hla-a * 2402-restricted cytotoxic t-lymphocyte epitope stabilized by intrachain hydrogen bonds from severe acute respiratory syndrome coronavirus nucleocapsid protein time-varying transmission dynamics of novel coronavirus pneumonia in china roles of humidity and temperature in shaping influenza seasonality outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle can chinese medicine be used for prevention of corona virus disease 2019 (covid-19)? a review of historical classics, research evidence and current prevention programs effects of temperature variation and humidity on the death of covid-19 in wuhan evaluation of ebola virus inhibitors for drug repurposing compounds with therapeutic potential against novel respiratory 2019 coronavirus baricitinib: a 2018 novel fdaapproved small molecule inhibiting janus kinases coronavirus disease 2019 treatment: a review of early and emerging options mers-cov and h5n1 influenza virus antagonize antigen presentation by altering the epigenetic landscape vitamin e and respiratory tract infections in elderly nursing home residents: a randomized controlled trial host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity thujaplicin-copper chelates inhibit replication of human influenza viruses vitamin d status and acute respiratory infection: cross sectional results from the united states national health and nutrition examination survey vitamin e and immunity a randomized, controlled trial of ebola virus disease therapeutics novel coronavirus: current understanding of clinical features, diagnosis, pathogenesis, and treatment options middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist vitamin d improves viral response in hepatitis c genotype 2-3 naïve patients coronavirus has now spread to all regions of mainland china novel mode of action of ribavirin (rebetol r ), a drug for the treatment of chronic hepatitis c: inducting the mutation of rna viruses effects of temperature, humidity, and diurnal temperature range on influenza incidence in a temperate region baseline serum vitamin a and d levels determine benefit of oral vitamin a&d supplements to humoral immune responses following pediatric influenza vaccination severe acute respiratory syndrome coronaviruses post-sars: update on replication and pathogenesis importation and human-to-human transmission of a novel coronavirus in vietnam genetic diversity and evolution of sars-cov-2 novel coronavirus: from discovery to clinical diagnostics discovery of human zinc deficiency: its impact on human health and disease effect of zinc or zinc plus arginine supplementation on antibody titre and lymphocyte subsets after influenza vaccination in elderly subjects: a randomized controlled trial outcome reporting from protocols of clinical trials of coronavirus disease 2019 (covid-19): a review. medrxiv risk factors for sars transmission from patients requiring intubation: a multicentre investigation in toronto n95 respirators vs medical masks for preventing influenza among health care personnel: a randomized clinical trial dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc coronavirus infections: epidemiological, clinical and immunological features and hypotheses selenium and human health the role of zinc in antiviral immunity identification of a novel coronavirus causing severe pneumonia in human baricitinib as potential treatment for 2019-ncov acute respiratory disease nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus host cell copper transporters ctr1 and atp7a are important for influenza a virus replication the neighbor-joining method: a new method for reconstructing phylogenetic trees current pharmacological treatments for covid-19: what's next? interim clinical guidance for patients suspected of confirmed with covid-19 on the use of corticosteroids for 2019-ncov pneumonia broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses treatment of 5 critically iii patients with covid-19 with convalescent plasma favipiravir, an anti-influenza drug against life-threatening rna virus infections the role of vitamin a in enhancing humoral immunity produced by antirabies vaccine characterization of severe acute respiratory syndromeassociated coronavirus (sars-cov) spike glycoprotein-mediated viral entry hydroxychloroquine and covid-19 fda approves phase iii clinical trial of tocilizumab for covid-19 pneumonia. cancer net effectiveness of n95 respirators versus surgical masks in protecting health care workers from acute respiratory infection: a systematic review and meta-analysis ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex covid-19: combining antiviral and anti-inflammatory treatments sars: systematic review of treatment effects lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy acetaminophen vs. nsaids during covid-19 pandemic temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection long-term high copper intake: effects on indexes of copper status, antioxidant status, and immune function in young men chloroquine is a potent inhibitor of sars coronavirus infection and spread ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of hiv-1 and dengue virus clinical characteristics of 138 hospitalized patients with 2019 novel coronavirusinfected pneumonia in wuhan sars coronavirus entry into host cells through a novel clathrin-and caveolaeindependent endocytic pathway remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro temperature significant change covid-19 transmission in 429 cities human-leukocyte antigen class i cw 1502 and class ii dr 0301 genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan teicoplanin inhibits ebola pseudovirus infection in cell culture therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys coronavirus disease (covid-19) technical guidance: laboratory testing for 2019-ncov in humans advice on the use of masks in the context of covid-19 landscape analysis of therapeutics as 21st novel coronavirus -china report of the who-china joint mission on coronavirus disease 2019 (covid-19) the mercurial nature of neutrophils: still an enigma in ards? imbalance between pulmonary angiotensinconverting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china a new coronavirus associated with human respiratory disease in china favipiravir shows good clinical efficacy in treating covid-19: official pathological findings of covid-19 associated with acute respiratory distress syndrome anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model the broad spectrum antiviral ivermectin targets the host nuclear transport importin α/β1 heterodimer clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) novel coronavirus 2019 (covid-19): emergence and implications for emergency care enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in wuhan influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome vitamin d modulation of innate immune responses to respiratory viral infections controversial treatments: an updated understanding of the coronavirus disease 2019 the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes rapid generation of a mouse model for middle east respiratory syndrome preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) protease inhibitors targeting coronavirus and filovirus entry coronaviruses-drug discovery and therapeutic options the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 kabir, uddin, hossain, abdulhakim, alam, ashraf, bungau, bin-jumah, abdel-daim and aleya. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-278839-uu2wlpmp authors: alberca, ricardo wesley; pereira, nátalli zanete; oliveira, luanda mara da silva; gozzi-silva, sarah cristina; sato, maria notomi title: pregnancy, viral infection, and covid-19 date: 2020-07-07 journal: front immunol doi: 10.3389/fimmu.2020.01672 sha: doc_id: 278839 cord_uid: uu2wlpmp pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov-2) can induce the coronavirus diseases-2019 (covid-19) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid-19-positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid-19 during pregnancy, to establish a correlation and possible implications of covid-19 during pregnancy and neonatal's health. pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov-2) can induce the coronavirus diseases-2019 (covid19) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid-19-positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid-19 during pregnancy, to establish a correlation and possible implications of covid-19 during pregnancy and neonatal's health. keywords: covid-19, sars-cov-2, pregnancy, neonatal, immunology pregnancy pregnancy comprises a unique immunological condition, to protect the fetus from maternal rejection, allowing adequate fetal development and protection against microorganisms (1, 2) . the maternal immune system is challenged by paternal alloantigens expressed both by the fetus and the placenta. however, through a complex range of cells and molecules, the mother does not develop a classic response to this allograft (3) . during pregnancy, fetal microquimerism occurs, where fetal cells, such as nucleated erythrocytes, trophoblastic cells, and leukocytes (3), cross the placental barrier and expose the mother to fetal alloantigens. these cells can remain in the bloodstream and maternal tissues many years after delivery (4, 5) . in comparison to the post-partum period, pregnancy increases monocytes, granulocytes, pdcs, mdcs in the blood, peaking during 2 trimesters. simultaneously, during pregnancy occurs a reduction in cd3, cd4, and cd8 t cells in comparison with post-partum. b cells are decreased during the third trimesters. nk cells cd56 dim are reduced in the second and third trimester of pregnancy in comparison with the first trimester and post-partum period. during the second and the third trimesters, nk and cd4 t cells present a reduction in the production of ifn-γ, tnf, il-6 cells, compared with post-partum (6) but the variability and contradictory reports are noted (7) . maternal monocytes do not show differences in absolute numbers, however, they show some phenotypic changes including an increase in the expression of adhesion molecules (cd11a, b; cd54), and the high-affinity igg receptor, fcγr-i (cd64) (8) . the absolute number of nk cells in maternal blood increases in the first trimester of pregnancy (9) . like lymphocytes, b cells are decreased during pregnancy and remain lower until 1 month after delivery. in vitro, b cells of pregnant women were less responsive, with suppression of lymphopoiesis and exclusion of autoreactive b cells (10). despite this, vaccine response during pregnancy remains effective (11, 12) . from the 13th week of gestation, maternal peripheral blood monocytes also undergo phenotypic and functional changes. there is an increase in the ability to produce cytokines il-1β and il-12 and a reduction in the potential for tnf-α secretion (13) . the placenta is a transient chimeric organ that develops from the uterine wall and can express different receptors and dynamically delivered microvesicles through pregnancy (14) . this organ mediates hormonal, nutritional, and oxygen support to the fetus while modulating maternal's immune response (15) . the placental maternal face is formed from decidual cells, with the presence of wide range of immune cells, including uterine natural killer (unk), dendritic cells (dcs), and regulatory t cells (tregs). the fetal face consists of the placental villus, which contains fetal blood vessels surrounded by fibroblasts and placental villous macrophages of fetal origin, hofbauer cells (16, 17) . treg cells are crucial for proper gestational development and are numerically elevated during pregnancy, in peripheral, deciduous and umbilical cord blood (18) . paternal hla-c is a crucial molecule that can elicit allogeneic immune responses by maternal cell and aid in the development of maternal-fetal tolerance (19) , also t reg may regulate cd4 + and cd8 + t lymphocyte activation through the expression of il-10 and tgfβ (20) . another striking feature of the maternal-fetal interface is the accumulation of nk cells, which comprise up to 70% of deciduous leukocytes in early pregnancy (21) . these cells are important for the regulation of cytokines production, especially il-10, and act in the production of angiogenic factors, chemokines, controlling the invasion of trophoblasts and availability of adequate maternal blood at the implantation site (17, 21, 22) . during pregnancy, hormonal variations can modulate immune responses, generating a reduction in the number of dcs and monocytes, and a decrease in the activation of macrophages, t, and b cells (23) . to better establish the tolerogenic milieu, estrogen induces efficiently foxp3 t regs cells (24) (25) (26) . changes in hormonal levels and immune system function generated by pregnancy may increase women's vulnerability to infections. pregnant women show higher mortality rates and complications associated with viral infections compared to the general population (27, 28) . for example, varicella disease in children is mild, but primary infections during pregnancy can progress to varicella pneumonia and death (29) . in 2009, during the h1n1 flu pandemic, an increased ratio of female to male cases was verified, in which pregnant women developed more complications, as severe acute respiratory syndrome, and higher mortality compared to the general population (30, 31) . similarly, in 1918 the pandemic spanish flu, among 1,350 reported cases of influenza in pregnant women, 27% died as a result of the infection (32) . in 1957, with the h5n1 pandemic, 50% of influenza deaths in women of reproductive age in minnesota occurred in pregnant women (33) . although influenza viruses are restricted to maternal lungs, inflammatory cytokines can lead to fetal complications mainly preterm birth and fetus miscarriage (34, 35) . in the ebola epidemic in 1995, 46% of infected women (out of a total of 177) were pregnant (36) . some evidence suggests that during pregnancy there is a greater risk of developing serious illnesses, spontaneous abortion, hemorrhage, and death when infected with the ebola virus (37) . additionally, infection by the lassa virus in pregnant women shows high levels of placental replication, and the risk of maternal-fetal mortality increases with the duration of pregnancy (38, 39) . viruses can gain access to the decidua and placenta by ascending from the lower reproductive tract or via hematogenous transmission, viral tropism for the decidua and placenta is then dependent on viral entry receptor expression in these tissues as well as on the maternal immune response to the virus (16) . a range of viral infections in pregnancy are associated with specific placental findings, including lymphoplasmacytic villitis with associated enlargement of villi and intravillous hemosiderin deposition in the setting of maternal cytomegalovirus infection (40) , as well as rare reports of intervillositis in the setting of zika virus (41) and dengue virus (42), among others. although there is little knowledge about placental findings associated with the common coronaviruses, ng et al. reported placental pathology in seven women with sars infection in hong kong (43) . in three placentas delivered in the acute stage of sars, demonstrated increased perivillous or subchorionic fibrin, while in two women who had recovered from third-trimester infection by the time of delivery, there were large zones of avascular villi, with one of the two additionally demonstrating a large villous infarct; both contained increased nucleated red blood cells in the fetal circulation. none of the seven placentas examined had any acute or chronic inflammatory processes (43) . the covid-19 pandemic is still in its early stages, with preliminary case series of infection in pregnant women available. a study of three placentas delivered from pregnant women with sars-cov-2 infection, infected in their third trimester with emergency cesarean section, describe various degrees of fibrin deposition. the fibrin deposition occurred inside and around the villi with local syncytial nodule increases in all three placentas, multiple villous infarcts in one placenta, and a chorangioma in another case. all samples from three placentas were negative for the nucleic acid of sars-cov-2 (44) . another study with 16 placentas from patients with sars-cov-2 were examined and the most significant finding is an increase in the rate of features of maternal vascular malperfusion (mvm), most prominently decidual arteriopathy including atherosis, fibrinoid necrosis, and mural hypertrophy of membrane arterioles (45). maternal hypertensive disorders, including gestational hypertension and preeclampsia, are the major risk factors for mvm (46) , although only 1 of the patients was hypertensive in this study. notwithstanding, sars-cov-2 is a virus that is expected to induce inflammation, it is relevant that neither acute inflammatory pathology (aip) nor chronic inflammatory pathology (cip) were increased in covid-19 patients relative to the controls. however, none of the covid-19 patients in this study were severely ill or undergoing a cytokine storm and it may be possible that cip could be induced in those cases of severe systemic inflammation (45). there few knowledges about miscarriage in women with covid-19, one case was a pregnant woman with symptomatic coronavirus disease who experienced a second-trimester miscarriage. a stillborn infant was delivered vaginally and swabs from the axillae, mouth, meconium, and fetal blood obtained within minutes of birth tested negative for sars-cov-2 and bacterial infection. the fetal autopsy showed no malformations, and fetal lung, liver, and thymus biopsies were negative for sars-cov-2. furthermore, amniotic fluid and vaginal swabs sampled during labor tested negative for sars-cov-2 and bacterial infection. placental histology demonstrated mixed inflammatory infiltrates composed of neutrophils and monocytes in the subchorial space and unspecific increased intervillous fibrin deposition (47) . during the worldwide sars-cov-1 (severe acute respiratory syndrome coronavirus-1) epidemic in 2003, a notable increase in mortality and morbidity was documented in pregnant patients (48) . agreeing with previous observations that the risk of viral pneumonia is significantly higher among pregnant women compared to the rest of the population (49) . in 2012, infection with the middle east respiratory syndrome (mers-cov) coronavirus in saudi arabia after the isolation of a male patient who died of severe pneumonia (50, 51) . data on the effects of mers-cov on pregnancy are limited, whereas there is a description of stillbirth at 5 months of gestation (52) . between 2012 and 2016, the ministry of health of saudi arabia reported the occurrence of 1,308 cases of mers-cov infection, five of which were pregnant (53) . despite the few descriptions, the immunological changes in pregnancy may alter the susceptibility to mers-cov and the severity of the clinical disease (51) . in a mice model of herpes virus infection, even in the absence of herpes virus placental passage, there was a marked increase in the levels of pro-inflammatory cytokines, including ifn-γ and tnf-α, as well as changes in fetal development (30) . this scenario may result from the placenta's pro-inflammatory response generated by the infection, or it may be due to other physiological changes in the mother or placenta related to the infectious process (54) . placental cells, predominantly trophoblasts, express tlr (toll-like receptors) and this expression varies according to the gestational age and the differentiation stage of these cells. viral infections can disturb the fine immune regulation at the maternal-fetal interface and lead to fetal damage, even without the virus reaching it directly (55) . for example, tlr-3 expressed by trophoblasts in the first trimester of pregnancy (56) , mediates rapid antiviral response (57) , and induces the production of cytokines, type i interferon (ifn) and type iii ifn (58) . tlr7 is also expressed in trophoblasts, which induces the synthesis of anti-viral cytokines and plays a role in preventing intrauterine transmission of hbv (59) . however, these inflammatory responses can be associated with complications in pregnancy, such as pre-eclampsia and/or intrauterine growth deficit (1) . in general, cytokines and ifns are important mediators in a healthy pregnancy, due to their role in the regulation of cell function, proliferation, and gene expression. however, when dysregulated, they have the potential to interrupt fetal and placental development pathways (60). the world health organization (who) estimates that about 2.5 million children died within the first month of life in 2018. every day ∼7,000 newborns die, amounting to 47% of all child mortality under the age of 5 years (61) . the majority of all neonatal's deaths are due to preterm birth, intrapartum-related complications (birth asphyxia or lack of breathing at birth), infections and birth defects. regarding the highest incidence of infection observed in early-life, it is generally attributed to an immature immune system during the transitional post-natal period (62) . innate immune cells are composed of specialized cells, such as granulocytes (e.g., neutrophil), monocytes, macrophages, dcs and innate lymphocytes. around 5 weeks gestation, neutrophils are present in human fetal liver parenchyma (63) , when compared to the adult response, neonatal neutrophils have qualitative and quantitative impairments in the response under stress conditions, including reduced chemotaxis, respiratory burst, and extracellular traps formation (64) . the cytokine profile produced by antigen-presenting cells (apcs) monocyte/macrophage and dcs in newborn differs from those produced by adults. typically, apcs from neonates produce less pro-inflammatory cytokines like il-1β, tnf-α, il-12p70, and type i ifn upon stimulation on tlrs (65) . otherwise, it produces great amounts of th17-promoting cytokines (il-6 and il-23) when compared with adult cells (66) . following, the importance of anti-inflammatory response in early life is highlighted through the great amount of il-10 produced by newborn monocyte/conventional dc (cdc) compared to adults (67) . the pattern of innate cytokine response can be attributed to two mechanisms: (i) high mononuclear cell levels of intracellular cyclic adenosine monophosphate (camp), a secondary messenger that suppresses th1 but enhances th2 and anti-inflammatory cytokine production (68) and (ii) altered dna binding capacity of transcription factors, such as irf3 to the promoter regions of cytokine genes secondary to age-specific chromatin (69) . curiously, neonates' dcs activation with clr agonist dectin or macrophage-inducible c-type lectin (mincle), simultaneously with tlr7/8 potently drives caspase-1 and nf-kb activation and th1-supporting cytokine production (including il-12p70), overcoming the age-specific epigenetic barrier in early life for irf3 function and leading to a th-1 phenotype (70, 71) . on 14 weeks of gestation, mature fetal αβ t lymphocytes can be detected. during the second and third trimesters of gestation, the repertoire of fetal t cell receptors diversifies (72) . generally, neonates have a limited th1 profile response to some vaccines and pathogens, agreeing with a lower capacity of cd4 t cells to produce ifn-γ and of apcs to produce th1-skewing cytokines (73) . although there are some situations where the responsiveness of the th1 profile is efficient, for example, neonates and infants develop adult-like th1 responses to bcg or pertussis vaccines, and a fetus can develop th1 responses in congenital cmv infection (74) (75) (76) . recent studies suggested that the early life immune system could present advantages for the elicitation of broadly neutralizing antibodies (bnabs), a response highly desired for an hiv vaccine. in fact, hiv-infected children develop bnabs responses earlier and more frequently than infected adults (77) . congenital and perinatally acquired viral infections do occur and may lead to major disabilities in infancy and childhood, the main causes can be attributed to pathogens like toxoplasma gondii, rubella virus, cytomegalovirus (cmv), herpes viruses, syphilis, and zika virus (78) . while congenital rubella virus syndrome is no longer seen in countries with compulsory immunization against this virus, an outbreak of zika virus (zikv) recently occurred in brazil resulting in the zikv syndrome, with brain lesions comparable to, but more severe than congenital cmv infection (79) . neonates display an immature immune response, the first exposition to an environmental stimulus can shape the lung's immune response (80) . furthermore, there is a predominant type 2 immune response in the lungs (81), these characteristics make infants susceptible to respiratory viral infections, a common cause of infant's death (82) . rsv is an important cause of lower respiratory tract illness in infants globally and is responsible for one-third of deaths due to lower respiratory tract infections in children <1 year of age (83) . pregnant women are considered at high risk for severe influenza disease, for this reason, influenza vaccination has been recommended for pregnant women and introduced into immunization programs (84) . influenza vaccination is safe and protective on preterm birth (ptb) and low birth weight (lbw) (85) . one of the benefits of maternal immunization has also been shown to extend to neonates through the transfer of maternal antibodies, providing passive immunization against the influenza virus (86). on the severe 2009 pandemic h1n1 influenza illness, some studies suggested an association between severe h1n1 disease, preterm birth, and fetal death; however, these limited data do not permit firm conclusions (35) . sars-cov-1 infected ∼100 pregnant women during the pandemic (87), causing a high lethality and miscarriage rate (88) , but no neonatal infection has been reported (88) . in 2017, cynthia maxwell postulated possible intensive care and procedures to properly manage maternal and neonatal sars-cov-1 infections (89) . vertical transmission of mers has not been documented. in a case report by alserehi et al., a mother was diagnosed with mers, treated and a cesarean section was performed to deliver a healthy preterm baby with 32 weeks of gestation (52) . hon et al. described 14 children with mers, that presented persistent fever and cough, after treatment no fatal case was reported. all children in this report obtained the infection via adult-to-children transmission, and no children-tochildren transmission was reported (90) . iqbal et al. reported a case of spontaneous vaginal delivery in covid-19-positive pregnant, with no signs of neonatal infection up to 7-days post-partum (91) . nevertheless, it is important to highlight contact precautions were made in this report to prevent post-partum transmission. in late 2019, a respiratory infectious disease began to be investigated in wuhan, china (92) . at first, contagion occurred through contact with some infected animals but, soon there were the first reports of human-to-human transmission (93), the virus was identified as belonging to the coronaviridae family and was designated sars-cov-2 (severe acute respiratory syndrome coronavirus-2) (94). like other members from this viral family, mers and sars-cov-1, the new coronavirus causes a respiratory disease, named covid-19 (coronavirus disease−2019) (95) . although very similar, sars-cov-1 and sars-cov-2 impacted the world differently. sars-cov-1 emerged in 2002 and killed almost 800 people in 26 countries (96) and, even without a vaccine, it was taken preventive actions as patient isolation. the new coronavirus has killed more than 480,000 people in just 6 months and has spread to 5 continent (97). sars-cov-2 shares genetic similarities between sars-cov-1 and mers, 79 and 50%, respectively (98) . sars-cov-2 is an enveloped single-stranded rna virus and has a genome of ∼30,000 nucleotides that encode structural and accessory proteins-the largest known viral rna genome (99) . sars-cov-1 and sars-cov-2 enter the host's cells via the ace2 receptor (angiotensin-converting enzyme 2) (100). in the lung, the most affected organ among those infected, the main target is the type 2 alveolar cell (101) . the ace2 receptor is also expressed in cells from kidneys, esophagus, heart (102) . moreover, a small percentage of monocytes and macrophages express the ace2 receptor (94, 99) . thus, there may be another alternative receptor or infectious pathway, such as antibody-dependent enhancement (ade). however, unlike other coronaviruses, limited to respiratory disorders, sars-cov-2 caused multiple organ failure. furthermore, this receptor is more expressed in the elderly, which associated with immunosenescence and other comorbidities common among the elderly may justify the high lethality rate in this age group (103) . the viral load peaks occur during the first week of infection and then gradually decrease over the next few days. in addition, the viral load is correlated with the patient's age. igg and igm antibodies start to increase 10 days after disease and most patients are seroconverted in the first 20 days (104) . moreover, in vitro assays, has shown that the serum from sars-cov-2-infected patients were able to neutralize the virus (101) . thereby, the humoral response can be another antiviral strategy via plasma transfer (105) . in sars-cov-1 and mers, as a viral escape mechanism, the virus can suppress ifn type i response, either by cytosolic sensors of ubiquitination, inhibiting nuclear factors translocation or decreasing stat1 phosphorylation (106) . neutrophils, c-reactive protein and several cytokines (as il-6, tnf, il-10) are increased in covid-19, and this elevation is correlated with disease severity and death (97) . in serious illness, the same protein levels were detected and inflammatory cytokines increase is correlated with t cd4 + and t cd8 + lymphocytes decrease and lower ifnγ production. b-lymphocytes do not appear to be affected by the disease, regardless of severity (92, 103, 107) . these characteristics observed in patients indicate that a covid-19 can be mediated by an intense inflammatory process that follows the disease severity. as with sars-cov-1 and mers, this increase in cytokine levels-known as a cytokine storm-can be involved with the pathogenesis of the disease (92). to defend itself against an aggressive agent (such as infection, trauma, acute inflammation, among others) the body produces an exaggerated response to localize and then eliminate the damage. this response is known as the systemic inflammatory response syndrome (sirs) or, if the source infection sepsis (108) , this process leads to the release of acute-phase proteins and endocrine, hematological and immunological changes, among them, the cytokine storm can lead to tissue damage and even death (109) . cytokine storm is produced, mainly, by highly activated macrophages and can cause lung damage and start viral sepsis (110) . this inflammation leads to other complications, such as acute respiratory distress syndrome (ards) and respiratory and cardiac failure (48, 111) . studies in mice infected with sars-cov-1, also demonstrate the cytokine storm dampening adaptive immunity (112) . other factors may also influence the susceptibility for covid-19 infected persons, and some gene polymorphisms, well-documented for other viral infections (113) . at the moment no vaccine or specific treatments are available for disease control of the sars-cov-2. in pregnancy, pneumonia infections may trigger an increased mortality risk to the mother and fetus (114) , which can also lead to complications as preterm birth and small for gestational age (115) . placental syncytiotrophoblast cells express the ace2 receptor and this receptor is highly expressed in the first months of pregnancy. associated with placental immaturity, the early ace2 expression can make the first trimester the most likely period for sars-cov-2-infection (14) . a serine protease, tmprss2, is also required for viral entry (100, 116) and there is still no consensus about placenta expression. some studies report low, but present, mrna expression in human placentas (117) , others describe that expression is not detectable (118) . the association of tmprss2 and ace2 expression, in the first months of pregnancy, would make this phase more susceptible to sars-cov-2-infection. blood tests in pregnant women revealed regular covid-19 markers, such as lymphopenia, neutrophilia, and elevated c-reactive protein level in pregnant women (119, 120) . some reports also verified an increase in alt, ast, and d-dimer (120) (121) (122) ). an important report verified that 3 mothers developed anemia and dyspnea, which could potentially be a risk factor during c-section labor (123) . chen and collaborators, verified alteration in calcium and albumin levels in the blood of pregnant women with sars-cov-2 infection (124) , which could potentially increase the severity in covid-19 (125) . furthermore, in a recent report involving maternal death in consequence to covid-19, 2 cases reported a low number of platelets, which is associated with an increase in mortality by covid-19 (126, 127) . it is still under investigation the effects of sars-cov-2infection in the maternal-fetal context ( table 1) . some reports describe that symptomatic infected-mothers did not transmit the virus during pregnancy. in a case report of seven cases, showed that three babies were tested to sars-cov-2 and only 1 was positive 36 h post-partum (138) . on the other hand, another report shows increase in inflammatory cytokines and virus-specific igm levels in newborns, from infected-mothers, 2 h after birth (120) , and in another report, newborns presented virus-specific igm and igg, but no sars-cov-2-infection ( table 1 ) (128) . this lead to the possibility of the activation of the maternal immune system by sars-cov-2 may have some implication of the offspring's health and immune system development. although the number of pregnant women with covid-19 studies is limited, there is no conclusive report of vertical transmission ( table 1 ) (129, 139) . a recent case report, was described two cases of rashes and one with facial ulcerations (123) . another important factor, besides the immune activation, the maternal usage of antiviral drugs can also permanently affect the offspring's immune response (140) , as there is no current standard protocol of treatment regarding the usage of antibiotics or antivirals ( table 1 ) (115) . only a fraction of patients infected with sars-cov-2 develops severe respiratory disorders, it is unknown whether the pregnant could be more susceptible to pulmonary diseases. covid-19 can progress to a severe lung inflammation that can progress to life-threatening illness at the severe stage (141) . this inflammatory process is associated with high plasma levels of cytokines, as cytokines storm, including il-2, il-7, il-10, g-csf, ip-10, mcp-1, mip-1a, and tnfα (92) . this might play an important role in pregnancy as il-2 has been implicated to be upregulated in pre-eclampsia (142) and miscarriage (143) and il-7/il-7r signaling pathway in fetal miscarriage (144) , due to the upregulation in the ratio of th17/treg cells (145) . another relevant aspect is the possible implication of polymorphisms in covid-19 diseases, as is well-documented for other viral infections (114) . also, cytokines polymorphisms, such as tnf-α 308g/a (rs1800629) polymorphism is associated with recurrent miscarriage (146) . in fact, tnf-α and tnf-α receptor play an important role in the development of the fetus, being present in the ovary, endometrium, placenta, and fetus, and in the amniotic fluid in different concentration (147) . this increase in tnf-α during pregnancy may implicate in different health outcomes depending on the gestational period (148) , leading to tissue necrosis in the placenta and hypoxia (149) . interestingly, an acute increase of this cytokine during pregnancy in animals may cause abortion (7) . moreover, alteration in the health status of the mother during pregnancy can have long-term effects on the offspring's health (150) . inflammatory processes during pregnancy can also impact women's health, as the increase in tnf-a during pregnancy can also lead to impaired insulin sensitivity (151) and gestational diabetes mellitus (152) . in animal models, inflammation during pregnancy has been shown to alterations in the behavior (153, 154) fetal brain development (155) (156) (157) , metabolic disturbance (158, 159) , and shape offspring's immune response to antigens and infections (160, 161) . the physiological response, as stress and the control of temperature, during the infection may present a long-term effect in pregnant women with covid-19. the increase in stressrelated hormones can also affect the offspring's immune system (162) and fever during pregnancy increase the chances of neural disorders in the children (163) . moreover, an increase in anti-inflammatory cytokine il-10 in covid-19 mothers is probably a regulatory mechanism crucial to regulate the inflammation (164) and pregnancy maintenance (165). even though no vertical transmission for covid-19 has been reported until now, several reports of early-life infections have been described with very low death rates (98, 119) . reports with recommendations to the treatment of pregnant women with covid-19 (166) and for neonates with covid-19 have been published (167, 168) . another possible route for sars-cov-2 is oral transmission by fecal samples (169) , and via breastfeeding from a sars-cov-2 infected mother. regarding breastfeeding, a small study found no evidence of covid-19 in breast milk, of six patients (139) . however, the primary concern is whether an infected mother can transmit the virus through respiratory droplets during breastfeeding. other viruses in the past have also caused concern in pregnant women. the zika virus has been linked to several cases of microcephaly in newborns during an epidemic in 2015 in brazil (170) . the infection had a high point in the first trimester of pregnancy, where there were more favorable conditions for its entry and replication in placental cells. in the case of sars-cov-2, it has not yet possible due to the time of infection occurs in the world, to observe the consequences of infection in the firsttrimester pregnancy. taking into account the early pregnancy, the placental tissue immaturity together with the up-regulation of ace2 expression in placental cells, perhaps the more susceptible period for sars-cov-2 infection is around the first trimester of pregnancy. it is important to highlight that after the 2009 influenza pandemic there have been reports of reduced cytokine response to bacterial infections. this leads to the hypothesis that covid-19 can lead to impairments of the immune response to other pathogens and vaccines in the future. future investigations are needed to identify the possible implications of sars-cov-2/covid-19 in pregnancy, the possible infection of the placenta in the first trimester of pregnancy and implications of the cytokine storm to the neonatal's health. ra and ms: write, conception, and review. np, lo, and sg-s: write and review. all authors contributed to the article and approved the submitted version. ra holds a post-doctorate fellowship from fapesp (19/02679-7) and lo also holds a post-doctorate fellowship from fapesp (19/07976-0). sg-s holds a master degree fellowship from fapesp (19/22448-0). the immune system in pregnancy: a unique complexity immune mechanisms at the maternal-fetal interface: perspectives and challenges self-recognition and the role of fetal microchimerism male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum chimerism occurs in thyroid, lung, skin and lymph nodes of women with sons characterizing the pregnancy immune phenotype: results of the viral immunity and pregnancy (vip) study how immune mechanisms are affected by pregnancy the transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women maternal b lymphocytes specific for paternal histocompatibility antigens are partially deleted during pregnancy influenza immunization in pregnancy-antibody responses in mothers and infants neonatal outcomes after antenatal influenza immunization during the 2009 h1n1 influenza pandemic: impact on preterm birth, birth weight, and small for gestational age birth monocytes are progressively activated in the circulation of pregnant women the expression and localization of the human placental prorenin/renin-angiotensin system throughout pregnancy: roles in trophoblast invasion and angiogenesis? regulation of placental development and its impact on fetal growth-new insights from mouse models risks associated with viral infections during pregnancy hla-g: at the interface of maternal-fetal tolerance membrane progesterone receptors in human regulatory t cells: a reality in pregnancy the dual role of hla-c in tolerance and immunity at the maternal-fetal interface new paradigm in the role of regulatory t cells during pregnancy single-cell reconstruction of the early maternal-fetal interface in humans endocrine factors modulating immune responses in pregnancy enhanced foxp3 expression and treg cell function in pregnant and estrogen-treated mice induction of regulatory t cells by physiological level estrogen cutting edge: estrogen drives expansion of the cd4 + cd25 + regulatory t cell compartment viral infections during pregnancy the association between viral infections, maternal and fetal mortality/morbidity intrauterine infection with varicellazoster virus after maternal varicella preparing for influenza after 2009 h1n1: special considerations for pregnant women and newborns pandemic 2009 influenza a(h1n1) virus illness among pregnant women in the united states influenza occurring in pregnant women: a statistical study of thirteen hundred and fifty cases deaths from asian influenza associated with pregnancy the influence of pregnancy on systemic immunity maternal influenza and birth outcomes: systematic review of comparative studies ebola hemorrhagic fever and pregnancy what obstetrician-gynecologists should know about ebola: a perspective from the centers for disease control and prevention a prospective study of maternal and fetal outcome in acute lassa fever infection during pregnancy lassa fever in pregnancy: report of 2 cases seen at the university college hospital demethylation profile of the tnf-α promoter gene is associated with high expression of this cytokine in dengue virus patients pathology of congenital zika syndrome in brazil: a case series sickle-cell erythrocytes in the placentas of dengue-infected women the placentas of patients with severe acute respiratory syndrome: a pathophysiological evaluation pregnancy with new coronavirus infection: clinical characteristics and placental pathological analysis of three cases placental histopathological lesions in correlation with neonatal outcome in preeclampsia with and without severe features second-trimester miscarriage in a pregnant woman with sars-cov-2 infection a casecontrolled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome pneumonia in pregnancy sars and mers: recent insights into emerging coronaviruses potential maternal and infant outcomes from (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome hospital outbreak of middle east respiratory syndrome coronavirus viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor toll-like receptors and pregnancy: trophoblast as modulators of the immune response a role for tlrs in the regulation of immune cell migration by first trimester trophoblast cells recognition of doublestranded rna and activation of nf-kappab by toll-like receptor 3 type iii interferons produced by human placental trophoblasts confer protection against zika virus infection maternal-derived hepatitis b virus e antigen alters macrophage function in offspring to drive viral persistence after vertical transmission protecting the newborn and young infant from infectious diseases: lessons from immune ontogeny ontogeny of myeloid cells neutrophil production and function in newborn infants innate cellular immune responses in newborns innate immunity of the newborn: basic mechanisms and clinical correlates regulatory activity of autocrine il-10 on dendritic cell functions the adenosine system selectively inhibits tlr-mediated tnfalpha production in the human newborn age-related gene expression differences in monocytes from human neonates, young adults, older adults dectin-1 activation unlocks il12a expression and reveals the th1 potency of neonatal dendritic cells age-specific adjuvant synergy: dual tlr7/8 and mincle activation of human newborn dendritic cells enables th1 polarization timely and spatially regulated maturation of b and t cell repertoire during human fetal development unbalanced neonatal cd4(+) t-cell immunity newborns develop a th1-type immune response to mycobacterium bovis bacillus calmette-guérin vaccination bordetella pertussis infection in 2-monthold infants promotes type 1 t cell responses functional exhaustion limits cd4 + and cd8 + t-cell responses to congenital cytomegalovirus infection immunological mechanisms of inducing hiv immunity in infants treatment of perinatal viral infections to improve neurologic outcomes viral infections and the neonatal brain age-specific incidence of influenza a responds to change in virus subtype dominance pulmonary susceptibility of neonates to respiratory syncytial virus infection: a problem of innate immunity? respiratory viruses and sudden infant death evaluation of maternal mortality under-reporting in the heights of chiapas using the ramos and modified ramos strategies maternal immunisation: what have been the gains? where are the gaps? what does the future hold? the safety of inactivated influenza vaccines in pregnancy for birth outcomes: a systematic review influenza vaccination of pregnant women and protection of their infants consensus document on the epidemiology of severe acute respiratory syndrome (sars) pregnancy and perinatal outcomes of women with severe acute respiratory syndrome 225-management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) clinical presentations and outcome of severe acute respiratory syndrome in children an uncomplicated delivery in a patient with covid-19 in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster a novel coronavirus from patients with pneumonia in china coronaviruses as the cause of respiratory infections world health organization. coronavirus disease (covid-2019) situation reports 158 coronavirus disease (covid-19) and neonate: what neonatologist need to know genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection clinical and immunologic features in severe and moderate coronavirus disease 2019 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study convalescent plasma as a potential therapy for covid-19 interaction of sars and mers coronaviruses with the antiviral interferon response the pathogenesis of spinal cord involvement in dengue virus infection definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis the systemic inflammatory response syndrome the landscape of lung bronchoalveolar immune cells in covid-19 revealed by single-cell rna sequencing mers-cov infection in humans is associated with a proinflammatory th1 and th17 cytokine profile dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice host single nucleotide polymorphisms modulating influenza a virus disease in humans pneumonia in pregnancy pneumonia and pregnancy outcomes: a nationwide population-based study tmprss2 isoform 1 activates respiratory viruses and is expressed in viral target cells expression of transmembrane serine protease tmprss2 in mouse and human tissues prostatelocalized and androgen-regulated expression of the membrane-bound serine protease tmprss2 a case report of neonatal covid-19 infection in china possible vertical transmission of sars-cov-2 from an infected mother to her newborn clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china infants born to mothers with a new coronavirus (covid-19). front pediatr clinical analysis of pregnant women with 2019 novel coronavirus pneumonia mortality from covid-19 increases with unsaturated fat, and may be reduced by early calcium and albumin supplementation association between platelet parameters and mortality in coronavirus disease 2019: retrospective cohort study thrombocytopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a meta-analysis antibodies in infants born to mothers with covid-19 pneumonia lack of vertical transmission of severe acute respiratory syndrome coronavirus 2, china first case of neonatal infection due to covid 19 in spain covid-19 vaginal delivery-a case report analysis of the pregnancy outcomes in pregnant women with covid-19 in hubei province coronavirus disease 2019 (covid-19) during pregnancy: a case series covid-19 in a 26-week preterm neonate covid-19 infection in first trimester of pregnancy marked by a liver cytolysis: a case report evidence of mother-to-newborn infection with covid-19 maternal death due to covid-19 clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records testicular effects following in utero exposure to the antivirals acyclovir and ganciclovir in rats pathological findings of covid-19 associated with acute respiratory distress syndrome interleukin-2 receptor serum concentrations in normal pregnancy and pre-eclampsia the involvement of inflammatory cytokines in the pathogenesis of recurrent miscarriage abnormal il-2 receptor levels in non-pregnant women with a history of recurrent miscarriage il-7/il-7r signaling pathway might play a role in recurrent pregnancy losses by increasing inflammatory th17 cells and decreasing treg cells association of tumor necrosis factoralpha 308g/a polymorphism with recurrent miscarriages in women human tumour necrosis factor: physiological and pathological roles in placenta and endometrium tumor necrosis factor-α and pregnancy complications: a prospective study placental tnf-α signaling in illness-induced complications of pregnancy programming of fetal insulin resistance in pregnancies with maternal obesity by er stress and inflammation tnf-alpha is a predictor of insulin resistance in human pregnancy maternal circulating concentrations of tumor necrosis factor-alpha, leptin, and adiponectin in gestational diabetes mellitus: a systematic review and meta-analysis prenatal immune challenge affects growth, behavior, and brain dopamine in offspring in utero exposure to virus infections and the risk of developing anorexia nervosa maternal infection: window on neuroimmune interactions in fetal brain development and mental illness prenatal exposure to maternal infection alters cytokine expression in the placenta, amniotic fluid, fetal brain prenatal lps-exposurea neurodevelopmental rat model of schizophrenia-differentially affects cognitive functions, myelination and parvalbumin expression in male and female offspring prenatal lipopolysaccharide exposure promotes dyslipidemia in the male offspring rats prenatal viral exposure followed by adult stress produces glucose intolerance in a mouse model prenatal initiation of endotoxin airway exposure prevents subsequent allergeninduced sensitization and airway inflammation in mice thrown off balance: the effect of antenatal inflammation on the developing lung and immune system maternal stress during pregnancy increases neonatal allergy susceptibility: role of glucocorticoids hertz-picciotto i. is maternal influenza or fever during pregnancy associated with autism or developmental delays? results from the charge (childhood autism risks from genetics and environment) study effect of anti-tnf-α on the development of offspring and pregnancy loss during pregnancy in rats sars-cov-2 infection during pregnancy. information and proposal of management care emergency plan for inter-hospital transfer of newborns with sars-cov-2 infection chinese expert consensus on the perinatal and neonatal management for the prevention and control of the 2019 novel coronavirus infection detectable sars-cov-2 viral rna in feces of three children during recovery period of covid-19 pneumonia zika virus in the americas: early epidemiological and genetic findings the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 alberca, pereira, oliveira, gozzi-silva and sato. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-289096-wuegn0jg authors: wang, liang; su, shuo; bi, yuhai; wong, gary; gao, george f. title: bat-origin coronaviruses expand their host range to pigs date: 2018-04-18 journal: trends microbiol doi: 10.1016/j.tim.2018.03.001 sha: doc_id: 289096 cord_uid: wuegn0jg infections with bat-origin coronaviruses have caused severe illness in humans by ‘host jump’. recently, novel bat-origin coronaviruses were found in pigs. the large number of mutations on the receptor-binding domain allowed the viruses to infect the new host, posing a potential threat to both agriculture and public health. liang wang, 1 shuo su, 2 yuhai bi, 1,3 gary wong, 3 and george f. gao 1,3,4, * infections with bat-origin coronaviruses have caused severe illness in humans by 'host jump'. recently, novel bat-origin coronaviruses were found in pigs. the large number of mutations on the receptor-binding domain allowed the viruses to infect the new host, posing a potential threat to both agriculture and public health. the host range expansion of coronaviruses (covs) from wildlife to humans via genetic recombination and/or mutations on the receptor-binding domain in the spike (s) gene is well established and results in several diseases with high fatality rates, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [ [4] . thus, pigs are regarded as mixing vessels for iavs. however, pigs were not known to be susceptible to bat-origin coronaviruses until recently, when two independent groups reported the detection of novel swine enteric alphacoronaviruses (seacovs) distinct from known swine coronaviruses (with one group successfully isolating live virus). the seacovs were found to be phylogenetically close to bat coronavirus hku2 [5, 6] . this suggests that bat-origin coronaviruses may have 'jumped' the species barrier to infect pigs. in february 2017, outbreaks of severe watery diarrhea of suckling piglets were reported in commercial pig farms in guangdong province, china. the disease had high case fatality rates (cfrs, over 35% for <10-day-old suckling piglets), and none of the animals were positive for known pathogens responsible for porcine diarrhea [6] . instead, two genomes of novel seacov were detected in the ill piglets by two independent groups, and preliminary analysis showed that the seacovs possibly originated from bat hku coronaviruses [5, 6] . it is currently unclear whether seacovs had been circulating undetected in pigs, if the viruses had originated from cross-species transmission, or if the seacovs were the result of viral recombination. to understand the molecular origin and evolution of sea-covs, we performed a detailed phylogenetic analysis at the genomic level by using all known alphacoronaviruses and bat-origin coronaviruses which are known to cause severe diseases, such as sars and mers. a total of 224, 312, and 778 complete genomes from mers-cov, sars-cov, and alphacoronaviruses, respectively, were used. phylogeny was reconstructed using conserved regions in genomes by the maximum likelihood method with 300 replicates ( figure 1a) . consistent with previous studies, phylogenetic analysis shows that seacovs were closely related to the rhinolophus bat coronavirus hku2 isolated in southern china ( figure 1a ). seacov was found to share a common ancestor with human coronavirus 229e/nl63, but these viruses are distant from other known swine alphacoronaviruses, indicating their different origins. further analysis on the s gene, which determines virus attachment, host cell entry, and 'host jump' of coronaviruses [7] , showed that domain 0 in the s1 subunit has structural similarity to that of nl63. the rest of the domain on the s1 subunit was similar to that of murine hepatitis coronavirus (betacov) [6] . there were 11 residues of the receptor-binding domain (rbd, also called c-terminal domain, ctd) of seacov directly in contact with its receptor (angiotensin-converting enzyme 2) that were mutated or deleted [7] (figure 1b) . seven mutations (a deletion of four amino acids, and three substitutions) among the 11 sites in seacov were also found in [ 8 1 _ t d $ d i f f ] 229e/nl63, indicating similarities in the receptor-binding mechanism between seacov and [ 8 1 _ t d $ d i f f ] [ 7 9 _ t d $ d i f f ] 229e/nl63. therefore, seacov may be able to infect humans and should be closely monitored. infection of seacov in vero cells (a primate cell line) will provide experimental evidence to support this possibility [6] . another requirement for the host range expansion of viruses is physical contact between different host species. the novel seacovs were both detected in guangdong province, whereas closely related bat coronaviruses were also isolated from guangdong or hong kong ( figure 1a) . in guangdong province, the high density of pig slaughterhouses and the wide distribution of bat species (figure 2a ) promote the possibility of viral cross-species transmission. additionally, bats have a wide geographical distribution in southern china, with extensive species diversity, unique behaviors (characteristic flight patterns, diet, roosting, and mobility) [8] , and constant interactions with both pigs and humans (figure 2a ). pigs are well established as mixing vessels and as intermediate hosts for iavs, and coronaviruses have already been shown to possess potential for recombination in animals [9] . given that pigs are in frequent contact with human and multiple wildlife species, and that pork is one of the most commonly consumed meats in non-muslim countries, it is important to assess whether pigs could be mixing vessels for the emergence of novel coronaviruses with high agricultural impact and risks to public health. it has already been reported that pigs are susceptible to infection with human sars-cov [10] and mers-cov [11] . additionally, the cd26 receptor sequence alignment of pigs and humans shows 94.5% similarity, which is sufficient for potential cross-species transmission [7] . in southern china, the unique climate, the high density of domestic as well as wild pigs, and extensive bat distribution, together with bats carrying large numbers of recombinant novel coronaviruses [12] , could lead to the emergence of more novel coronaviruses in the future. the isolation of seacov from ill piglets expands our knowledge of the host range of bat-origin coronaviruses, and potentially poses a threat to public health. despite considerable progress in characterizing cross-species transmission for coronaviruses, several areas need to be addressed, including: (i) whether other unknown coronaviruses are circulating in pigs; (ii) whether pigs are mixing vessels for coronaviruses; (iii) whether seacov infects humans and causes severe disease; and (iv) whether seacov vaccines should also be developed to control the spread of this virus in pigs. in-depth epidemiological investigation and comprehensive analysis of these novel coronaviruses should be performed to answer these urgent questions. using metagenomics to characterize an expanding virosphere epidemiology, genetic recombination, and pathogenesis of coronaviruses molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 sars and mers: recent insights into emerging coronaviruses the pig as an intermediate host for influenza a viruses between birds and humans a new bat-hku2-like coronavirus in swine discovery of a novel swine enteric alphacoronavirus (seacov) in southern china bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond bats: important reservoir hosts of emerging viruses co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia sars-associated coronavirus transmitted from human to pig livestock susceptibility to infection with middle east respiratory syndrome coronavirus key: cord-309734-m8miwtha authors: vergara‐alert, j.; raj, v. s.; muñoz, m.; abad, f. x.; cordón, i.; haagmans, b. l.; bensaid, a.; segalés, j. title: middle east respiratory syndrome coronavirus experimental transmission using a pig model date: 2017-06-26 journal: transbound emerg dis doi: 10.1111/tbed.12668 sha: doc_id: 309734 cord_uid: m8miwtha dromedary camels are the main reservoir of middle east respiratory syndrome coronavirus (mers‐cov), but other livestock species (i.e., alpacas, llamas, and pigs) are also susceptible to infection with mers‐cov. animal‐to‐animal transmission in alpacas was reported, but evidence for transmission in other species has not been proved. this study explored pig‐to‐pig mers‐cov transmission experimentally. virus was present in nasal swabs of infected animals, and limited amounts of viral rna, but no infectious virus were detected in the direct contact pigs. no virus was detected in the indirect contact group. furthermore, direct and indirect contact pigs did not develop specific antibodies against mers‐cov. therefore, the role of pigs as reservoir is probably negligible, although it deserves further confirmation. middle east respiratory syndrome coronavirus (mers-cov) was first detected in 2012 in saudi arabia, and it causes severe acute respiratory illness with fever, cough and shortness of breath (zaki, van boheemen, bestebroer, osterhaus, & fouchier, 2012) . up to date, it has caused 1952 human infections, including 693 related deaths (world health organization (who), 2017). dromedaries are the natural reservoir of mers-cov (sabir et al., 2016) . however, other animal species such as non-human primates (rhesus macaques and common marmosets), members of the family camelidae (alpacas and llamas), rabbits and pigs have been demonstrated to be susceptible to mers-cov infection (crameri et al., 2016; falzarano et al., 2014; haagmans et al., 2015; vergara-alert, van den brand, et al., 2017; de wit et al., 2013 de wit et al., , 2017 . the finding that pigs can be infected with mers-cov would suggest that other suidae might be susceptible to the virus. indeed, common warthogs (phacochoerus africanus), bushpig (potamochoerus larvatus) and wild boars are commonly found in the greater horn of africa or the middle east, sharing the same habitats and water sources with dromedaries (cumming, 2008; vergara-alert, vidal, bensaid, & segal es, 2017) . a recent study in alpacas demonstrated efficient animal-to-animal transmission (adney, bielefeldt-ohmann, hartwig, & bowen, 2016) but, to our knowledge, evidence for transmission between animals from other species has not been reported. to study whether mers-cov might be transmitted between pigs, an experimental transmission study in this animal model was designed and performed under direct and indirect contact settings. 2.1 | experimental design 3 (bsl-3) animal facilities (irta-cresa, barcelona, spain), and divided into three groups: g1, mers-cov-inoculated pigs (p1-p5); g2, direct contacts (p6-p10); g3, indirect contacts (p11-p15). three extra animals were used as negative controls (g4, p16-p18). animals from g1, g2 and g3 were housed in the same experimental box unit but placed in two different pens. the pens were separated by two fences with a 30 cm distance among them ( figure 1 ). tarpaulin, from the ceiling to the floor, was used to avoid contact between pen 1 and pen 2. tarpaulin was also placed in the front doors of both pens. at the beginning of the experiment, g1 was housed in pen 1, and g2 and g3 in pen 2. g1 was inoculated with 10 7 tcid 50 (50% tissue culture infectious dose) mers-cov (passage 7 human isolate hcov-emc/2012) in 3 ml saline solution via intranasal route (1.5 ml in each nostril). two days later, all tarpaulins were removed and g2 pigs were moved from pen 2 to pen 1 until the end of the study. all animals were monitored daily for clinical signs (sneezing, coughing, nasal discharge and/or dyspnoea), as well as rectal temperatures until day 10 post-inoculation (pi). nasal swabs (ns) were obtained on days 0, 1, 2, 3, 4 pi from all animals and at days 7, and 10 pi from g1. animals from g2 and g3 were also sampled at 5, 6, 9 and 12 days pi, corresponding to days 3, 4, 7 and 10 after direct (g2) and indirect (g3) contact with g1. two independent ns were collected and placed in pbs (for pcr analysis) and dmem containing antimicrobial drugs (for detection of infectious virus); swabbing was performed deep in both sides of the nasal cavity. sera were obtained before challenge and at 7, 15 and 26 days pi, and they were subsequently used to detect the presence of mers-cov-specific antibodies. negative control pigs were sampled (ns and sera) and euthanized before the start of the experiment. daily environmental samples (es) between day 0 and 10 pi were obtained from air sampling and wall surface swabbing (figure 1 ). briefly, swabs pre-moistened with transport medium (copan universal transport medium utm-rt system) were collected from walls in pen 1 and 2 (es1 and es2). air sampling was performed using an air sampler (airport md8 sartorius device) located between pens, which suctioned 50 l/min air volume for 20 min through a gelatin membrane filter (es3). air from the box unit was sampled with 10 9 10 cm dry membrane filters located in the air extraction of the box (es4). es were tested for the presence of viral rna. viral rna from ns and es was extracted with nucleospin â rna virus kit (macherey-nagel, germany) following the manufacturer's f i g u r e 1 schematic representation of the experimental animal box. boxes in the animal facility of the biosafety level 3 at irta-cresa are behind two sets of doors (with a shower in between) following the standards of a negative pressure room. animals were distributed into two pens separated by two fences with a 30 cm distance between them. g1 (p1-p5) was allocated in pen 1 and g2 (p6-p10) and g3 (p11-p15), in pen 2. two days after inoculation of g1 with mers-cov, g2 was cohoused with g1 until the end of the experiment. tarpaulin was used to prevent contact between g1 and the other two groups during the firsts 2 days after inoculation. environmental samples (es) were obtained from different locations, as represented in the scheme [colour figure can be viewed at wileyonlinelibrary.com] vergara-alert et al. instructions. the rna extracts were tested by the upe pcr (raj et al., 2013) , and the techniques were carried on as previously (vergara-alert, van den brand, et al., 2017) . ns were also evaluated for the presence of infectious virus by titration in vero cells, following previous protocol (vergara-alert, van den brand, et al., 2017). serum samples from days 0, 7, 15 and 26 pi were tested to determine the specific s1-antibodies by a mers-cov s1-elisa, and by a specific virus neutralization assay, as previously described (haagmans et al., 2016) . similar to a previous experiment (vergara-alert, van den brand, et al., 2017) , none of the pigs had appreciable rise in rectal temperature upon challenge, nor any clinical signs (data not shown). all mers-cov-experimentally infected animals (p1-p5) shed viral rna at least from 1 to 4 days pi, and three of five pigs had detectable viral rna until 7 days pi (figure 2a ). most importantly, all five animals shed infectious virus during the first 4 days pi (figure 2b ). viral rna was detected in four of five cohoused, direct contact animals (g2) at least one time pi. the mers-cov rna load of g2 pigs, however, was lower than those of g1 (figure 2a) . no viral rna or infectious virus was detected in swabs from g3 (p11-p15) and control pigs (p16-p18).to test whether seroconversion occurred, serum samples were tested with a specific recombinant mers-cov s1-elisa and for neutralizing antibodies against mers-cov. all five mers-cov infected animals (p1-p5) had detectable levels of s1-antibodies 2-and 3-weeks after the infection (figure 2c ). the specificity of the response was confirmed by virus neutralization assay. in p1-p4 (but not in p5), serum neutralizing mers-cov-specific titres (1:40-1:160) were detected at 1-and 2-week pi (figure 2d) . however, at week 3 pi, the virus neutralizing antibodies decreased (1:20-1:40). no mers-cov-specific antibodies were detected in serum of g2, g3 and control pigs. in environmental samples, very low levels of viral rna were detected at different time points, with a peak at day 5 pi (table 1) . other livestock besides dromedaries are susceptible to mers-cov infection (crameri et al., 2016; falzarano et al., 2014; haagmans et al., 2015; munster et al., 2013; vergara-alert, van den brand, et al., 2017; de wit et al., 2013 de wit et al., , 2017 ; thus, they might be potential intermediate we thank all animal caretakers from the irta-cresa biosecurity level 3 laboratories and animal facilities for technical assistance. this work was performed as part of the zoonotic anticipation and preparedness initiative (zapi project) [innovative medicines initiative (imi) grant 115760] with assistance and financial support from imi and the european commission and contributions from efpia partners. the funding from cerca programme/generalitat de catalunya to irta is also acknowledged. infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus phacochoerus africanus. the iucn red list of threatened species infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels pneumonia from human coronavirus in a macaque model dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia livestock susceptibility to infection with middle east respiratory syndrome coronavirus. emerging infectious diseases searching for animal models and potential target species for emerging pathogens: experience gained from middle east respiratory syndrome (mers) coronavirus. one health domestic pig unlikely reservoir for mers-cov middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques middle east respiratory syndrome coronavirus (mers-cov): infection, prevention and control measures are critical isolation of a novel coronavirus from a man with pneumonia in saudi arabia how to cite this article: vergara-alert j, raj t a b l e 1 viral rna from air sampling and wall surface swabbing at different times after mers-cov infection. swabs were collected from the walls in pen 1 and pen 2 (es1 and es2), air sampling was performed with an air device (es3), and circulating air from the box was sampled with filters located in the ceiling air extraction of the room (es4) key: cord-298350-pq1dcz3a authors: ryan, jeffrey r. title: category c diseases and agents date: 2016-03-25 journal: biosecurity and bioterrorism doi: 10.1016/b978-0-12-802029-6.00005-0 sha: doc_id: 298350 cord_uid: pq1dcz3a this chapter covers category c diseases and agents. these emerging diseases present a very unique challenge to public health officials and infectious disease specialists. perhaps they have been with us for millions of years, lurking in a dark corner of the environment, waiting for an opportunity to jump from their natural cycle of transmission to a human host. or they may represent something totally new. regardless of their origin, an emerging disease pathogen must be characterized quickly by molecular biologists and microbiologists. the dynamics of disease transmission must be investigated by teams of epidemiologists. treatment regimens must be formulated by clinicians working on the frontlines of the outbreak. disease prevention strategies and risk communications must be quickly formulated by public health officials. finally, media attention for emerging disease outbreaks forces government officials at all levels to address the problem with planning and preparedness activities aimed at preserving the health of the public. specific examples explored in this chapter include nipah virus, hantavirus, west nile fever virus, and the coronaviruses that cause severe acute respiratory syndrome and middle east respiratory syndrome. category c comprises newly emerging diseases and pathogens. no disease outbreak or epidemic escapes media coverage, whether it is an enteric disease due to contaminated lettuce, foodborne botulism, or the threat of "bird flu." as laurie garrett points out in her thought-provoking book the coming plague (1995) , we seem to be living in a world that is out of balance. our era may very well be characterized by eruptions of new diseases, epidemics of old diseases moving into new areas, diseases that are sparked by advances in technology, and diseases that come to us from insects and animals as civilization invades ecologies where humans have not trodden before (garrett, 1995) . although these encounters have been occurring throughout history, technology now enables us to define the problems with great acuity. our ability to determine the causal relationship among pathogen, host, and disease relies heavily on state-of-the-art technologies brought to us by advances in science and engineering. still, to this day the microscope remains a primary tool in the toolbox. moreover, electron microscopy provides researchers with high-powered visual proof of the existence of the pathogens that cause disease (fig. 5.1) . the category c list changes with the world disease outbreak situation. bioterrorism coordinators, emergency managers, and public health officials should periodically check the centers for disease control (cdc) website for updates. consider how much attention has been brought to the emergence and spread of the deadly h5n1 influenza virus, also known as bird flu. in the last 12 years there have been a little more than 800 confirmed cases of h5n1 bird flu in humans. however, more than 50% of those human cases resulted in death and approximately 400 million birds have died either directly from the illness or indirectly from the control efforts aimed at eliminating the problem. public health officials fear that h5n1 may spark the next pandemic. bird flu due to the h5n1 virus is covered in chapters biological threat to agriculture and recent animal disease outbreaks and lessons learned because of the effect that this outbreak has had on the poultry industry. refer to table 5 .1 for a snapshot of the category c pathogens that have the attention of public health officials in 2015. a would-be terrorist or rogue state might take in 1998 a mysterious outbreak in the peninsular region of malaysia caught public health officials by surprise (centers for disease control and prevention, 1999) . the outbreak took place over an 8-month period. when it was over, more than 1 million pigs had to be destroyed. moreover, there were 257 human cases from the same pathogen, which caused 105 deaths (a 41% case-fatality rate). most human cases were from people employed in the swine industry who had direct contact with live pigs (parashar et al., 2000) . the outbreak had a profound psychological and economic impact on the country. the outbreak was due to nipah virus, a pathogen completely unknown to scientists before this outbreak (see fig. 5 .2). the pathogen was fully characterized by molecular methods as a paramyxovirus in the genus henipavirus (wong et al., 2002) . nipah virus is very similar to another recently emergent pathogen, hendra virus, which causes severe respiratory and encephalitic disease in horses and humans. nipah virus causes severe, rapidly progressive encephalitis in humans. in pigs, nipah virus causes severe respiratory illness with neurological complications. transmission of the virus to humans is associated with close contact with infected pigs. the survivability of nipah virus outside of the host has not been determined. in september 1998 the malaysian ministry of health (moh) began to receive reports from three geographic locations of several human cases of febrile encephalitis with high mortality. moh authorities initially believed the outbreak was due to japanese encephalitis (je) virus, a mosquito-borne ribonucleic acid (rna) virus. however, je vaccination and mosquito control efforts conducted over several months failed to halt the epidemic. numerous features of this outbreak's epidemiology were inconsistent with past je outbreaks. remarkably, researchers noted that human case patients had an association with infected animals from a concurrent and severe outbreak of respiratory disease in pigs and that there was a notable absence of illness in children. subsequently, serological testing coupled with epidemiological findings showed that the malaysians were not dealing with je. tissue culture isolation eventually led them to the actual etiologic agent of the outbreak (chua et al., 1999) . nipah virus gets its name from the village (sungai nipah) where the first cases were reported. in march 1999 a related outbreak occurred in singapore, where abattoir workers were exposed to swine imported from malaysia. the outbreak was quickly identified and stamped out. the natural cycle of transmission of nipah virus involves flying foxes (fruit bats, pteropus) as reservoirs and pigs as amplifying hosts (calisher et al., 2006) . hendra virus also is found in fruit bats. many species of fruit bats are found in malaysia. two species of flying fox, the island flying fox (pteropus hypomelanus) and malayan flying fox (pteropus vampyrus), have been shown to be asymptomatic carriers of the virus. no secondary hosts have been implicated. researchers have not yet determined how nipah virus is transmitted from bats to pigs. they suspect that fruit trees close to pig pens are foraged by the bats and the virus is spread by this close proximity (urine or saliva on partially eaten fruit). most human cases (93%) had direct contact with pigs or secondary contact with body fluids, urine, or feces (calisher et al., 2006) . person-to-person transmission has not been established or related to any cases from the 1998-99 outbreak. in the malaysian outbreak of nipah virus, the contagion was rapidly spread from farm to farm by the movement of infected pigs. malaysia's domestic pig population before the outbreak was 2.4 million. the total annual value was estimated to be approximately $400 million, with a total export value of $100 million (us dollars). as a result of the outbreak, approximately 1.1 million pigs were culled to prevent the spread of the disease. this alone resulted in an estimated loss of $217 million. during the outbreak, pork consumption in malaysia dropped by almost 80% (food and agriculture organization of the united nations, 2002). in 2004 an outbreak of nipah virus occurred in bangladesh. only 34 human cases were identified; however, more than 75% of the case patients died (hsu et al., 2004) . another outbreak of nipah virus occurred in early 2005 in the tangail district of bangladesh, when 13 people became ill and lost consciousness after drinking palm fruit juice. the fruit may have been contaminated with infected fruit bat droppings. blood samples from case patients were sent to the cdc to confirm nipah virus infection; one was confirmed positive (luby et al., 2006) . the incubation period for nipah virus in humans is believed to be 3-14 days. initial symptoms are fever and headache. nipah virus produces widespread effects and induces necrosis of endothelial cells in vasculature and neuronal damages. subsequently, case patients experience dizziness, drowsiness, disorientation, and vomiting. endothelial cells are involved in vascular permeability, and damage to the cells leads to vessel leaking and ultimately to hypovolemic shock. the most severely affected develop encephalitis, seizures, and coma. complications noted during the malaysian outbreak included septicemia, intestinal bleeding, and renal impairment (wong et al., 2002) . laboratory diagnostic methods for nipah virus infections now include serology, histopathology, immunohistochemistry, real-time polymerase chain reaction (pcr), and virus isolation. the virus is classified biosafety level (bsl)-4. there is no cure for nipah; however, a vaccine is in development. current treatment involves supportive care. nipah virus has been listed by the cdc as a category c potential bioterrorist agent. this is described as an emerging pathogen that has a potentially high morbidity and mortality as well as a major health impact. currently, spread of the disease involves close contact with pigs. however, aerosolization may be a possible bioterrorist method of dispersal. in addition, the potential for this virus to infect a wide range of hosts and produce significant mortality in humans makes this emerging virus one of public health concern. because of the need to cull infected pigs, attack with this agent could produce a great economic impact to a nation's pork industry. during the nipah outbreak in malaysia, widespread panic and fear occurred until the outbreak was brought under control. an outbreak of a cluster of serious febrile illness occurred in new mexico in 1993. a task force of scientists, public health experts, and epidemiologists discovered that the outbreak was due to hantavirus. hantavirus is the causative agent of hantavirus pulmonary syndrome (hps) and hemorrhagic fever with renal syndrome (hfrs) in humans (duchin et al., 1994) . from what we know now, this disease agent occurs naturally throughout most of north and south america, it is airborne, and in the absence of prompt medical attention infections are usually fatal. this agent serves as a perfect example of a pathogen that makes a dramatic entry into modern-day society, bringing with it numerous challenges. for its attributes and its sudden arrival, hantaviruses were placed in department of health and human services category c. hantavirus is a three-segmented rna virus in the family bunyaviridae. several rodent species act as the reservoir for these viruses in nature. rodents transmit the disease horizontally within their species and vertically to humans through aerosolized virus particles from their dried feces and urine (leduc et al., 1992) . the hantavirus-caused diseases hfrs and hps are considered to be pan-american zoonoses. more than 25 antigenically different viral species make up this group. table 5 .2 provides a breakdown of hantavirus by type, endemic region, and rodent host. hantaviruses are encapsulated in a lipid envelope; therefore they are easily destroyed by common disinfectants, such as acetone, iodine, ethanol, and chlorine (kraus et al., 2005) . in addition, hantaviruses are deactivated by ultraviolet light, low ph, and temperatures above 37°c (98.6°f). hantaviruses previously recognized as causing hfrs in the old world are dobrava, hantaan, puumala, and seoul. infected rodents remain so for life, yet they are often unaffected by the virus and will transmit it among themselves. it is unknown whether animals other than the natural rodent hosts are epidemiologically important. many other hantaviruses have been isolated and characterized but not linked to human illness. most human infections with hantavirus in north america are associated with rodents of the subfamily sigmodontinae (childs et al., 1994) . as many as three hantaviruses have been found circulating in one location, each with its own rodent reservoir. rodents other than the primary reservoirs may play an important role as a carrier (a common reservoir for hantavirus is shown in fig. 5 .3). disease outbreaks that occurred during the american civil war are now believed to have been due to hantavirus. in addition, there are records of hfrs from both world wars. what is thought to be the first outbreak of hantavirus causing hfrs was recorded in russia in 1913. it is reported that japanese troops in manchuria experienced cases in 1932. in the early 1950s western physicians recorded more than 3200 cases of an acute, debilitating, febrile illness in un forces fighting in the korean war. the illness, known as korean hemorrhagic fever, affected soldiers living out of foxholes along the contested border between north and south korea (ricketts, 1954) . because the mortality rate was high (10-15%), the us army medical department formed the hemorrhagic fever commission to conduct an epidemiological investigation. results of the commission indicated that a field mouse (apodemus agrarius coreae) was harboring the infectious agent. however, it was not until 1977 that the infectious agent was isolated and named hantaan for the river that runs near the 38th parallel, which separates north and south korea . the hemorrhagic fever commission preserved more than 600 serum samples from 245 soldiers. later, in 1990, the serum samples from these 245 soldiers were screened for antibody to the hantaan virus. almost 40 years after the outbreak, investigators detected an antibody to hantaan virus in 94% of the samples. in 1979 a virus similar to hantaan caused hemorrhagic fever in laboratory workers. this virus, named seoul virus after the site of the initial studies, infected norway rats and roof rats shipped to japan and europe. shipping these laboratory animals led to the dissemination of the seoul virus. in 1993 an outbreak of illness resulted in several fatalities on the navajo nation indian reservation in new mexico. as the outbreak spread, cases were distributed around the four corners region of the united states (chapman and khabbaz, 1994) . the region gets its name from the perfectly formed grid square that marks the boundaries among the states of arizona, colorado, new mexico, and utah. these cases, later described as hps, were attributed to a new species of hantavirus. actually, nothing was new about it. the virus had probably been in the region for many years but had not been the cause of so much notable human illness in the past. several young navajo tribal members presented to the indian health service physicians with sudden onset of respiratory failure in may 1993. by june of that year, 12 people had succumbed to the illness. the illness was initially diagnosed as unexplained acute respiratory distress syndrome. patients presented with abrupt fever, severe headache, myalgia, and cough followed by rapidly progressive pulmonary edema (stelzel, 1996) . within 2-10 days, this condition led to respiratory failure, hypotension, and death. new mexico public health officials teamed with cdc investigators to set up surveillance and laboratory testing. the team found that serum from the patients showed crossreactive antibodies to hantaan, seoul, and puumala virus antigens. however, the condition in these patients had developed into a pulmonary form unlike any other clinical presentation of hantavirus infection. the investigation also focused on the wildlife and domestic animals in the area of the outbreak (calisher et al., 1999) . epidemiologists were quick to discover that the virus was being spread through the dried feces and urine of peromyscus mice. unfortunately, the navajo believe that mice are responsible for bringing seeds to the earth, which enables humans to survive. the fact that mice are highly respected in navajo culture made disease control efforts difficult. tribal members had to accept all forms of rodent population management efforts, which include trapping, housecleaning, and reduction of harborage. outbreaks of hantavirus-related diseases are often attributed to weather patterns. drought causes plants to die, which leads to a decrease in rodent populations. conversely, heavy snowfall and spring rains allow plants to flourish and rodent populations to surge. these surges in the rodent population cause rodents to compete for food and protection in the dryer months that follow. the competition puts pressure on some infected rodents, forcing them into peridomestic environments, putting more people at risk for infection. hps is most common in spring and summer months, when rodents are most active. although the overall risk for hps in endemic areas is relatively low, infections are associated with an increased population of rodents in and around the house. activities that put people in contact with rodent droppings, urine, or nesting materials place the individual at higher risk for infection. indoor exposures have been linked to rodents in the home or near dwellings, especially in colder months. cleaning buildings that have been closed up for a period of time, such as cabins, barns, and storage facilities, also increases the risk of exposure. persons living in squalid housing conditions, those employed in agriculture, and those participating in wilderness camping or other outdoor activity in endemic areas are at greater risk for hantavirus infections. hikers and campers may be exposed when they use infested trail shelters or camp near rodent harborage. construction and utility workers can be exposed when they work in crawl spaces under houses that may have a rodent population. research shows that many hps case patients acquired the virus after having been in frequent contact with rodents or their droppings for long periods (centers for disease control and prevention, special pathogens branch, 2007) . worldwide, approximately 150,000 hospitalizations due to hfrs are reported each year. most cases come from china, where hantavirus was first recognized in 1931. approximately 500 cases of korean hemorrhagic fever are reported annually from south korea. many hfrs outbreaks occurring in asia and europe are due to people planting and harvesting crops having contact with field rodents. normally, humans become infected with hantavirus by the inhalation of aerosolized virus particles from rodent excreta. transmission of hantavirus begins with a chronically infected rodent. horizontal transmission occurs between rodents of the same species. infections in rodents are asymptomatic and not deleterious to the rodent, making them ideal reservoirs. rodents shed the virus in their urine, feces, and saliva. humans become infected when they disturb the infected rodent's environment and breathe the infected particles, a process called aerosolization. although less likely, transmission may occur through breaks in the skin. virus particles may contaminate food sources and people may become infected through consumption. very rarely, a bite from an infected rodent may cause disease. person-to-person transmission of hantaviruses have not been reported. several laboratory-acquired cases of hfrs have been reported. hantaviruses are categorized as bsl-4 agents when propagating them in culture or passing them through laboratory animals known to be efficient reservoirs. the incubation period for hantavirus infection is believed to be 14-17 days. initially, hps case patients experience headache, fatigue, fever, increased respiratory and heart rates, and myalgia of the large muscles in the thighs, hips, back, and shoulders. approximately half of all patients experience dizziness, chills, and various gastrointestinal symptoms, such as nausea, vomiting, diarrhea, and abdominal pain. four to ten days after the initial presentation, patients begin to experience coughing, rales, and shortness of breath due to severe hypotension and rapidly progressive pulmonary edema, requiring immediate hospitalization and ventilation. approximately 40% of hps case patients die within the first 48 h because of hypoxia and shock. the cdc maintains a national surveillance program for hps. to assist physicians with the recognition of this illness, a specific case definition for hps was published: a previously healthy person presenting with a febrile illness with a temperature at or above 101°f (38.3°c), unexplained acute respiratory distress syndrome, radiographic evidence of bilateral interstitial infiltrate that develops within 1 week of hospitalization, and respiratory compromise that requires supplemental oxygen. if sudden death occurs before supplemental oxygen and noncardiogenic pulmonary edema is present on autopsy without an identifiable, specific cause of death, then diagnosis can be made (centers for disease control and prevention, 1997) . confirmatory diagnosis of hps requires meeting specific inclusion and exclusion criteria plus laboratory confirmation. the cdc uses enzyme-linked immunoabsorbent assay to detect the presence of hantavirus-specific immunoglobulin (ig) m in acutephase serum or a 4-fold increase in titers of igg from acute-and convalescent-phase sera. immunohistochemistry can be used on formalin-fixed tissues to detect hantavirus antigen when serum is unavailable. virus detection by pcr or isolation from whole blood or serum may also be useful. treatment of hps requires early and aggressive intensive care, focusing on oxygenation of the blood, electrolyte balance, and maintaining blood pressure. there was a grave prognosis for many of the initial victims of the four corners outbreak because healthcare providers did not know what they were dealing with. now, history of exposure leads physicians in endemic areas to a more rapid diagnosis. early aggressive supportive care is needed for a successful resolution of symptoms. without treatment, the prognosis for hps is grave. with supportive care and symptom-targeted therapy, patients can recover from the disease. chronic lung and heart damage may result depending on the aggressiveness of supportive care. wnv is a single-stranded rna virus in the genus flavivirus (family flaviviridae). wnv is a member of the je virus antigenic complex of mosquito-borne flaviviruses. also included in this complex are saint louis encephalitis virus, kunjin virus, and murray valley encephalitis virus. wnv was initially isolated in 1937 from a febrile patient in the west nile district of uganda. since then, wnv has been isolated from mosquitoes, humans, birds, and other vertebrates in africa, eastern europe, western asia, and the middle east (murgue et al., 2002) . from 1975 to 2015 there were several significant outbreaks of west nile fever. studies conducted in egypt in the 1950s showed that the natural history of this disease can dramatically vary. at one extreme are areas where wnv circulates routinely with uncomplicated west nile fever manifesting as a mild and common childhood disease, which is easily confused with other febrile conditions. in this situation, the heightened infection rate improves background immunity and increases with age. hence, west nile fever epidemics and west nile encephalitis are rare. the other extreme exists in industrialized urban areas, where little or no previous wnv activity has occurred. here, aging and immunologically naïve populations are likely to encounter wnv for the first time. this leads to west nile fever epidemics with numerous cases of west nile encephalitis (knudsen et al., 2003) . there have been many west nile outbreaks throughout the world. similar to egypt, israel experienced outbreaks in the 1950s. in 1957 nursing homes in israel reported severe neurologic disease and death associated with west nile fever. an outbreak in romania is believed to be the catalyst for several outbreaks in large industrialized urban areas. wnv was first discovered in the united states in 1999. sixty-two cases and seven deaths (11% case-fatality rate) resulted from it in new york city and the surrounding area. horses, crows, and exotic birds from a zoo were also found to be infected. initially, saint louis encephalitis virus was believed to be the cause of the human infections until wnv was isolated from the human and animal specimens. this discovery marked the first appearance of wnv in the western hemisphere (jia et al., 1999) . naturally, there has been some speculation as to how wnv was introduced into the united states. no one really knows for sure. however, the isolates characterized from the 1999 outbreak were shown to be antigenically similar to a strain that circulated in israel from 1997 to 2000 (ebel et al., 2001) . in 2002 officials at the cdc affirmed that they provided scientists in iraq with wnv isolates in the 1980s and 1990s. this has led some to believe that the introduction was intentional. the more plausible explanation for a mosquito-borne disease such as this is that the introduction was accidental. perhaps infected mosquitoes or a reservoir host gained access to the united states via international transportation or trade. since the first detected case in 1999 the number of cases and deaths in humans increased dramatically almost every year until reaching a peak in 2006. table 5 .3 presents a summary of west nile cases in the united states reported between 1999 and 2014. horses are affected by wnv infections more often than any other domestic animals. in 2003 there were 4554 horses diagnosed with clinically apparent wnv infection. ravens, jays, and crows (corvids; family corvidae) serve as a reservoir for wnv. certain female mosquito species that feed primarily on birds (ornithophilic) enable maintenance of wnv in avian hosts. other female mosquito species that are not so particular in their blood feeding take up the virus from infected birds and pass them along to other animals. reservoir competency and field studies suggest that horses or other mammals do not serve as reservoirs for infection, which makes them incidental hosts (mclean et al., 2001) . west nile fever burned like a slow-moving wildfire across the united states from new york city to the california coastline in approximately 4 years. clinicians, public health officials, mosquito control specialists, and animal health professionals all had to come together to mitigate the impact of this emerging disease in the united states. although much has been done, this serious disease appears to have a foothold on us soil, and cases increased dramatically from 2006 to 2007. when the outbreak was first realized in 1999, some government officials were left to wonder how the pathogen made entry into the united states. could it have been due to an intentional act? worldwide, many different species of mosquitoes have been found to transmit wnv. wnv has been isolated from ticks in eurasia, but their role in natural transmission of the virus remains elusive. in north america, wnv has been detected in more than 40 different species of mosquitoes. mosquitoes in the genus culex are the most important vectors for maintaining wnv in nature, but no one knows which species are most responsible for transmission to humans. as to how wnv persists in the environment is not exactly known. however, studies have shown that several possible mechanisms may work together to provide the opportunities necessary for the virus to survive and thrive. environmental surveillance studies conducted in new york city after 1999 showed that culex mosquitoes were capable of overwintering in the new york city sewer system. laboratory studies have shown that transovarial transmission of wnv is possible with culex vishnui mosquitoes. studies done with birds indicate that contact transmission between birds may occur and that migratory birds may play a role in transporting wnv and its vectors to unaffected regions (centers for disease control and prevention, 2000) . laboratory-acquired infections have occurred with wnv. in 2002 the cdc documented west nile fever in two laboratory workers. the first became infected through a wound sustained from a scalpel while removing the brain from an infected blue jay. the second case was from a needle stick to a worker harvesting wnvinfected mouse brains. in 2002 wnv was found to be present in the blood supply. twenty-three cases of wnv infection were due to infected blood components from 16 wnv-viremic blood donors. this finding prompted blood collection agencies to begin screening blood donations. the following year, 737 donor samples were found to be wnv positive, prompting blood bank officials to discard their donations. despite the screening, two cases of confirmed blood transfusion-associated west nile fever were documented in 2003. nationwide blood screening for wnv has been successful in preventing transfusion-transmitted wnv (stramer et al., 2005) . however, as with all blood donation screening, infections can be transmitted to transfusion recipients on rare occasions despite negative donor test results. although wnv transmission by blood transfusion is rare, the few cases seen since 2002 underscore the importance of clinical recognition, effective wnv blood screening strategies, and investigation coordination (pealer et al., 2003) . in august 2002 four patients receiving organ transplants from one organ donor were diagnosed with wnv infection. one of the transplant recipients subsequently died from the infection. the organ donor had received blood products from 63 blood donors to help combat injuries sustained in an accident. ironically, the last blood transfusion received had been from a wnv-viremic blood donor. although believed to be rare, transplacental transmission of wnv is possible, with one confirmed case taking place in 2002. the incubation period for wnv is approximately 3-14 days. epidemiologists believe that approximately 80% of people infected with wnv are asymptomatic. approximately 20% of those infected develop a mild illness, termed west nile fever. uncomplicated west nile fever typically begins with sudden onset of fever, headache, lymphadenopathy, and myalgia, often accompanied by gastrointestinal symptoms. the acute illness usually lasts 3-6 days, but prolonged fatigue is common. in earlier epidemics in which west nile fever cases predominated, nearly half of all case patients presented with a maculopapular rash. less than 1% of wnv infections result in severe neurological disease. the more severe form of the disease is referred to as west nile encephalitis, west nile meningitis, or west nile meningoencephalitis. encephalitis refers to an inflammation of the brain, meningitis to an inflammation of the membrane around the brain and the spinal cord, and meningoencephalitis to inflammation of the brain and the membrane surrounding it. the symptoms of these severe infections include headache, high fever, neck stiffness, stupor, disorientation, coma, tremors, convulsions, muscle weakness, and paralysis. severe neurological disease due to wnv infection may occur in patients of all age groups. yearround transmission is possible in some areas. sars appeared as an outbreak in china very suddenly in 2003. sars serves as an example of a category c disease because of the many challenges government agencies faced from a newly emerging disease, which seemingly came out of nowhere. what caused it was never seen before coronavirus (fig. 5.4) . the coronavirus that causes sars is highly infectious. some of the patients from this outbreak were referred to as superspreaders because they shed so much virus they infected many other people. the initial outbreak that had the world's attention occurred in may 2003 in hong kong, but an epidemiological investigation showed the true origin led back to china's guangdong province. guangdong is one of the more prosperous areas of china. it can be characterized as an area dotted with industrial complexes surrounded by fertile farmlands, where people work and live in close proximity to their animals. animals are an important part of life in guangdong. in fact, much of south china is known for its live animal markets. in this region, the chinese believe that eating freshly killed wild animals promotes vitality and good health. in the live animal markets, you may purchase cats, dogs, snakes, bats, and civets. once you make your purchase the animal is butchered for the customer to take home. sars is believed to have developed here in the live markets or farm settings of guangdong province. on november 16, 2002, a 45-year-old man in foshan, a guangdong city of 3.4 million, became ill with an unusual respiratory illness (knobler et al., 2004) . no one knows exactly where or how he contracted the illness. he had no travel history, but he had recently prepared chicken, cat, and snake for household meals. an epidemiological investigation showed that many of the earliest sars case patients had possible associations with the use of wild animal food sources. the man, a local leader in the province, was married with four children. within weeks, his wife, a niece, an aunt, and her husband also became ill (xu et al., 2004) . the initial case patient and his four family members are thought to have been the first cluster of a disease that infected 8096 people around the world and killed 774 before ebbing in the summer of 2003 (9.5% case-fatality rate). guangdong was especially hard-hit, accounting for more than 1500 probable cases and 58 deaths. it took months after this first known infection for health authorities throughout the world to identify the disease as something new, learn its characteristics, and determine how to deal with it (goldsmith et al., 2004) . in the early days of sars, little was known by anyone anywhere about this mysterious disease. medical workers had no diagnostic criteria and no clinical test, and the incubation period was unknown. the method of transmission was uncertain, as was the effectiveness of protective equipment and safety requirements. sars spread from foshan into other areas of guangdong. by january 2003 it was seen in guangzhou, the provincial capital, where workers in the health industry began to fall ill. sars was a tragedy. in the space of a few months the deadly virus emerged from the jungles of central china and moved to several other countries by various means of transportation. in canada sars caused severe illness in more than 3300 people. southern ontario was the worst-affected jurisdiction outside of asia, with sars infecting 375 people and killing 44 (sars commission, 2006) . it caused untold suffering to its victims and their families, forced thousands into quarantine, brought the health system in the greater toronto area and other parts of the province to its knees, and seriously affected health systems in other parts of the country. in addition, travel advisories issued by the world health organization and the cdc, advising people to avoid all travel to ontario, caused the local economy there to suffer great losses. nurses lived daily with the fear that they would die or infect their families with a fatal disease. respiratory technicians, doctors, hospital workers, paramedics, and home care workers lived with the same fear. of the almost 375 people who contracted sars in ontario, 72% were infected in a health-care setting. of this group, 45% were health workers (mcdonald et al., 2004) . most of these workers were nurses whose jobs brought them into the closest contact with sick patients. this does not show the full burden of sars on nurses, paramedics, and other health workers. in many cases nurses sick with undetected sars brought illness, and in some cases death, home to their families (sars commission, 2006) . as mysteriously as it appeared the deadly sars virus was contained and put to rest. hundreds of cases were dealt with in several countries connected by international travel routes to china. case-fatality rates were very high. infection control procedures, isolation, and quarantine all were needed to contain the problem. however, the global public health community managed to muster an amazing effort, which now speaks volumes about the importance of public health education, surveillance, and modern technology. is this indicative of how all new emerging disease threats will be handled? • despite the unwillingness of the chinese government to share information from the initial cases in this outbreak, the world health community collaborated in an unprecedented manner. a consortium of laboratories managed to sequence the genome of this newly discovered pathogen in a few days to develop rapid diagnostic and surveillance tools. • international travel played a tremendous role in the spread of sars. this shows us that the connectedness of our cities and populations can spread a deadly pathogen from one side of the world to the other in hours. • health-care facilities played an important role in the epidemiology of sars. patients infected with the sars-coronavirus disease are likely to present to health-care facilities. if unrecognized as sars, then these patients may transmit sars to health-care workers and other patients. health-care workers accounted for a significant percentage of cases in most major sars outbreaks reported. • is coronavirus just a relic? a coincidence? some people with the disease were not presenting with antibodies to coronavirus, whereas there were some people showing no signs of the disease that were positive for antibodies to coronavirus. this finding is still very puzzling to many. because the outbreak was so short-lived we do not know how this disease might affect a large population or what role asymptomatic or subclinical patients play in the dynamics of disease transmission. • aids patients did not seem to be affected by the sars coronavirus. that fact is very puzzling to researchers. mers is a respiratory illness caused by a coronavirus called middle east respiratory syndrome coronavirus (mers-cov). refer to fig. 5 .5 for a colorized electron micrograph of mers-cov. similar to sars, mers patients develop severe respiratory illness. nearly 40% of mers cases are fatal (jalal, 2015) . this emerging disease was first reported from an outbreak of serious respiratory case patients in saudi arabia in september 2012 (hui, 2013) . however, through retrospective investigations, health officials later identified that the first known cases of mers occurred in jordan in april 2012. since the initial outbreak, mers-cov has spread to many other countries in the arabian peninsula and to europe, the united states, and south korea. the primary means of transmission of mers-cov is from person to person, primarily through coughing. mers-cov has spread from ill people to others through close contact, such as caring for or living with an infected person. infected people have spread mers-cov to others in health-care settings, such as hospitals. researchers studying mers have not seen any ongoing spreading of mers-cov in the community (cotton et al., 2013) . most infected people either lived in the arabian peninsula or recently traveled from the arabian peninsula before they became ill. a few people became infected with mers-cov after having close contact with an infected person who had recently traveled from the arabian peninsula. so far all cases of mers have been linked to countries in and near the arabian peninsula. most people confirmed to have mers-cov infection have had severe acute respiratory illness with the symptoms of cough, high fever, and shortness of breath. some mers patients also have gastrointestinal symptoms of diarrhea, nausea, and vomiting. the most serious cases come from the development of severe complications, such as kidney failure and pneumonia. in fact, most of the people who died had an underlying medical condition. patients with mild symptoms recover well (hui, 2013) . it is currently believed that people with preexisting medical conditions (eg, diabetes) are more likely to become infected with mers-cov or have a severe case. individuals with weakened immune systems are also at higher risk for getting mers or having a severe case. the incubation period for mers is thought to be 5 or 6 days, but it can range from 2 to 14 days (zumla et al., 2015) . there is no us food and drug administration approved test for mers. instead, experimental assays are run in cdc or who reference laboratories. molecular tests, such as real-time reverse-transcription polymerase chain reaction (rrt-pcr) assays, are used to diagnose active infection in mers case patients. the who's current case definition for laboratory confirmation of mers-cov infection requires either a positive rrt-pcr result for at least two specific genomic targets or a single positive target with sequencing of a second target. serological testing is used to detect previous infection (antibodies to mers-cov) in people who may have been exposed to the virus. the presence of antibodies to mers-cov indicates that a person had been previously infected with the virus and developed an immune response. there is no specific antiviral treatment recommended for mers. patients need to be identified rapidly and put into medical isolation. current treatment for severe cases is supportive care with emphasis on support of vital organ (liver, kidneys, lungs, etc.) functions. emerging diseases present a very unique challenge to public health officials and infectious disease specialists. perhaps they have been with us for millions of years, lurking in a dark corner of the environment, waiting for an opportunity to jump from their natural cycle of transmission to a human host. or they may represent something totally new. regardless of their origin, an emerging disease pathogen must be characterized quickly by molecular biologists and microbiologists. the dynamics of disease transmission must be investigated by teams of epidemiologists. treatment regimens must be formulated by clinicians working on the front lines of the outbreak. disease prevention strategies and risk communications must be quickly formulated by public health officials. finally, media attention for emerging disease outbreaks forces government officials at all levels to address the problem with planning and preparedness activities aimed at preserving the health of the public. category c agents may be exploited in much the same way that hoax powder incidents followed the 2001 amerithrax event. in addition, terrorist groups and rogue states might take advantage of the emergence of one of these special pathogens to intentionally introduce an emerging disease into an area, thereby causing fear, panic, and social disruption. • emerging disease. any disease, of various causes, that has newly appeared or is rapidly expanding its range in the human species. • hantavirus. one of the four genera of the family bunyaviridae. hantaviruses are spread by rodents and target the kidneys, lungs or pulmonary system, and heart. bats: important reservoir hosts of emerging viruses natural history of sin nombre virus in western colorado case definitions for infectious conditions under public health surveillance outbreak of hendra-like virus-malaysia and singapore update: surveillance for west nile virus in overwintering mosquitoes etiology and epidemiology of the four corners hantavirus outbreak serologic and genetic identification of peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in the southwestern united states fatal encephalitis due to nipah virus among pig-farmers in malaysia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease partial genetic characterization of west nile virus strains regional office for asia and the pacific animal production (rap) and health commission for asia and the pacific. manual on the diagnosis of nipah virus infection in animals. rap publication no. 2002/01. regional office for asia and the pacific animal production the coming plague: newly emerging diseases in a world out of balance. farrar, straus and giroux ultrastructural characterization of sars coronavirus nipah virus encephalitis reemergence, bangladesh. emerging infectious diseases tracking the transmission and evolution of mers-cov the emerging threat of mers genetic analysis of west nile new york 1999 encephalitis virus learning from sars: preparing for the next disease outbreak. workshop summary, forum on microbial threats, board on global health death from the nile crosses the atlantic: the west nile fever story inactivation of hantaan virus-containing samples for subsequent investigations outside biosafety level 3 facilities the hantaviruses, etiologic agents of hemorrhagic fever with renal syndrome: a possible cause of hypertension and chronic renal disease in the united states isolation of the etiologic agent of korean hemorrhagic fever foodborne transmission of nipah virus sars in healthcare facilities, toronto and taiwan. emerging infectious diseases west nile virus transmission and ecology in birds the ecology and epidemiology of west nile virus in africa case-control study of risk factors for human infection with a new zoonotic paramyxovirus, nipah virus, during a 1998-1999 outbreak of severe encephalitis in malaysia transmission of west nile virus through blood transfusion in the united states in 2002 report of the clinical and physiological research at hemorrhagic fever center, korea, fall of 1953 spring of fear. the story of sars hantavirus pulmonary syndrome: epidemiology, prevention, and case presentation of a new viral strain west nile virus among blood donors in the united states nipah virus infection pathology and pathogenesis of an emerging paramyxoviral zoonosis epidemiologic clues to sars origin in china. emerging infectious diseases middle east respiratory syndrome key: cord-264408-vk4lt83x authors: ruiz, sara i.; zumbrun, elizabeth e.; nalca, aysegul title: animal models of human viral diseases date: 2017-06-23 journal: animal models for the study of human disease doi: 10.1016/b978-0-12-809468-6.00033-4 sha: doc_id: 264408 cord_uid: vk4lt83x as the threat of exposure to emerging and reemerging viruses within a naïve population increases, it is vital that the basic mechanisms of pathogenesis and immune response be thoroughly investigated. recent outbreaks of middle east respiratory syndrome corona virus, ebola virus, chikungunya virus, and zika virus illustrate the emerging threats that are encountered. by utilizing animal models in this endeavor, the host response to viruses can be studied in a more complex and integrated context to identify novel drug targets, and assess the efficacy and safety of new products rapidly. this is especially true in the advent and implementation of the fda animal rule. although no one animal model is able to recapitulate all aspects of human disease, understanding the current limitations allows for a more targeted experimental design. important facets to consider prior to an animal study are route of viral exposure, species of animal, biomarkers of disease, and a humane endpoint. this chapter covers the current animal models for medically important human viruses, and demonstrates where the gaps in knowledge exist. well-developed animal models are necessary to understand disease progression, pathogenesis, and immunologic responses to viral infections in humans. furthermore, to test vaccines and medical countermeasures, animal models are essential for preclinical studies. ideally, an animal model of human viral infection should mimic the host-pathogen interactions and the disease progression that is seen in the natural disease course. a good animal model of viral infection should allow assay of many parameters of infection, including clinical signs, growth of virus, clinicopathological parameters, cellular and humoral immune responses, and virus-host interactions. furthermore, viral replication should be accompanied by measurable clinical manifestations and pathology should resemble that of human cases such that a better understanding of the disease process in humans is attained. there is often more than one animal model that closely represents human disease for a given pathogen. small animal models are typically used for first-line screening, and for testing the efficacy of vaccines or therapeutics. in contrast, nonhuman primate (nhp) models are often used for pivotal preclinical studies. this approach is also used for basic pathogenesis studies, with most studies in small animal models when possible, and studies in nhps to fill in the remaining gaps in knowledge. the advantages of using mice to develop animal models are low cost, low genetic variability in inbred strains, and abundant molecular biological and immunological reagents. specific pathogen free (spf), transgenic and knockout mice are also available. a major pitfall of mouse models is that the pathogenesis and protection afforded by vaccines and therapeutics cannot always be extrapolated to humans. additionally, blood volumes for sampling are limited in small animals, and viruses often need to be adapted through serial passage in the species to induce a productive infection. the ferret's airways are anatomically and histologically similar to that of humans, and their size enables collection of larger or more frequent blood samples, making them an ideal model for certain respiratory pathogens. ferrets are outbred, with no standardized breeds or strains, thus greater numbers are required in studies to achieve statistical significance and overcome the resulting variable responses. additionally, spf and transgenic ferrets are not available, and molecular biological reagents are lacking. other caveats making ferret models more difficult to work with are their requirement for more space than mice (rabbit-style cages), and the development of aggressive behavior with repeated procedures. nhps are genetically, the closest species to humans, thus disease progression and host-pathogen responses to viral infections are often the most similar to that of humans. however, ethical concerns pertaining to experimentation on nhps along with the high cost and lack of spf nhps raise barriers for such studies. nhp studies should be carefully designed to ensure the fewest number of animals are used, and the studies should address the most critical questions regarding disease pathogenesis, host-pathogen responses, and protective efficacy of vaccines and therapeutics. well-designed experiments should carefully evaluate the choice of animal, including the strain, sex, and age. furthermore, depending on the pathogen, the route of exposure and the dose should mimic the route of exposure and dose of human disease. the endpoint for these studies is also an important criterion. depending on the desired outcome, the model system should emulate the host responses in humans when infected with the same pathogen. in summary, small animal models are helpful for the initial screening of vaccines and therapeutics, and are often beneficial in obtaining a basic understanding of the disease. nhp models should be used for a more detailed characterization of pathogenesis and for pivotal preclinical testing studies. ultimately, an ideal animal model may not be available. in this case, a combination of different well-characterized animal models should be considered to understand the disease progression and to test medical countermeasures against the disease. in this chapter, we will be reviewing the animal models for representative members of numerous virus families causing human diseases. we will focus on viruses for each family that are of the greatest concern for public health worldwide. norovirus, the genus of which norwalk is the prototypic member, is the most common cause of gastroenteritis in the united states (hall et al., 2013) . there are five distinct genogroups (gi-gv) and numerous strains of norwalk virus, including the particularly significant human pathogens gi.1 norwalk virus, gii.2 snow mountain virus, and gii.1 hawaii virus. in developing countries, norwalk virus, also known as "winter vomiting virus," is responsible for approximately 200,000 deaths annually (patel et al., 2008) . a typical disease course is self-limiting, but there have been incidences of necrotizing enterocolitis and seizures in infants (chen et al., 2009; lutgehetmann et al., 2012; turcios-ruiz et al., 2008) . symptoms of infection include diarrhea, vomiting, nausea, abdominal cramping, dehydration, and fever. incubation is normally 1-3 days, with symptoms persisting for 2-3 days (koopmans and duizer, 2004) . viral shedding can range from 6 to 55 days in healthy individuals (atmar et al., 2014) . however, longer illness duration can be indicative of immunocompromised status, with the elderly and young having a prolonged state of shedding (harris et al., 2008; rockx et al., 2002) . interestingly, individuals vary greatly in susceptibility to norovirus infection depending on their fucosyl transferase 2 (fut2) allele functionality and histoblood group antigen status, with type a and o individuals susceptible and types ab and b resistant (hutson et al., 2005) . transmission occurs predominately through the oralfecal route with contaminated food and water being a major vector (atmar and estes, 2001; becker et al., 2000; koopmans and duizer, 2004) . vomiting results in airborne dissemination of the virus with areas of 7.8 m 2 being contaminated and subsequent transmission from oral deposition of airborne particles or contact with contaminated fomites, which can remain contaminated for up to 42 days (makison booth, 2014; tung-thompson et al., 2015) . each vomiting event in a classroom setting elevates the risk of norovirus illness among elementary students with proximity correlating with attack rates (evans et al., 2002; marks et al., 2003) . viral titers in emesis and fecal suspensions are as high as 1.2 × 10 7 and 1.6 × 10 11 ges (genomic equivalent copies per milliliter), respectively and the 50% infectious dose is 1320 ges (atmar et al., 2014) . therefore, outbreaks can be extremely difficult to contain. therapeutic intervention consists of rehydration therapy and antiemetic medication (bucardo et al., 2008; moe et al., 2001) . no approved vaccine or therapeutic is available, and development has been challenging given that immunity is short-lived after infection, new strains rapidly evolve and the correlates of protection are not completely understood (chen et al., 2013) . however, one promising strategy utilized a virus-like particle (vlp)-based vaccine that protected or reduced infection by almost 50% in human volunteers (aliabadi et al., 2015; atmar et al., 2011) . given the relatively benign disease in adults, experimental challenge has been carried out on human volunteers (ball et al., 1999; tacket et al., 2000) . viral titers are determined by shedding in feces and sera with histopathology changes monitored by biopsies particularly of the duodenum. the ph of emesis samples collected containing virus is consistent with viral replication in the small intestine with reflux to the stomach (kirby et al., 2016) . additionally, norwalk virus has been shown to bind to duodenal tissue (chan et al., 2011) . however, this type of research is technically difficult and expensive, and thus other models have been developed. a major hindrance to basic research into this pathogen is the lack of permissive cell culture systems or animal models for norwalk virus. nhps including marmosets, cotton-top tamarins, and rhesus macaques infected with norwalk virus are monitored for the extent of viral shedding; however, no clinical disease is observed in these models. disease progression and severity is measured exclusively by assay of viral shedding (rockx et al., 2005) . incidentally, more viruses were needed to create an infection when challenging by the oral route than by the intravenous (iv) route (purcell et al., 2002) . chimpanzees were exposed to a clinical isolate of norwalk virus by the iv route (bok et al., 2011) . although none of the animals developed disease symptoms, viral shedding within the feces was observed within 2-5 days postinfection and lasted anywhere from 17 days to 6 weeks. viremia never occurred and no histopathological changes were detected. the amount and duration of viral shedding was in-line with what is observed upon human infection. as such, chimeric chimpanzee-human antinorovirus neutralizing antibodies have been explored as a possible therapeutic strategy (chen et al., 2013) . a recently identified calicivirus of rhesus origin, named tulane virus, has been used as a surrogate model of infection. unlike norwalk virus, tulane virus can be cultured in cells. rhesus macaques exposed to tulane virus intragastrically developed diarrhea and fever 2 days postinfection. viral shedding was detected for 8 days. the immune system produced antibodies that dropped in concentration within 38 days postinfection, mirroring the short-lived immunity documented in humans. the intestine developed moderate blunting of the villi as seen in human disease (sestak et al., 2012) . a murine norovirus has been identified and is closely related to human norwalk virus (karst et al., 2003) . however, clinically the virus presents a different disease. the murine norovirus model does not include observable gastrointestinal clinical signs, possibly in part because rodents lack a vomiting reflex. additionally, mice infected with norovirus develop a persistent infection in contrast to human disease (hsu et al., 2006 (hsu et al., , 2007 khan et al., 2009) . porcine enteric caliciviruses can induce diarrheal disease in young pigs, and an asymptomatic infection in adults (wang et al., 2006 . gnotobiotic pigs can successfully be infected with a passaged clinical norovirus isolate by the oral route. diarrheal disease developed in 74% of the animals and virus was detected in the stool of 44% of the animals. no major histopathological changes or viral persistence was noted (cheetham et al., 2006) . calves are naturally infected with bovine noroviruses (scipioni et al., 2008) . experimental challenge of calves by oral inoculation with a bovine isolate resulted in diarrheal disease 14-16 h postinfection. recovery of virus was achieved after 53.5 and 67 h postinfection (otto et al., 2011) . eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), and venezuelan equine encephalitis virus (veev) present with near synonymous symptoms. the majorities of human cases are asymptomatic, but can present as a flu-like illness progressing to central nervous system (cns) involvement to include seizures and paralysis. mortality rates vary among the virus, with the highest reported for eev at 36%-75% followed by weev and lastly veev at less than 1% (ayers et al., 1994; griffin, 2007; steele and twenhafel, 2010) . there are currently no licensed vaccines or therapies but a recent phase 1 clinical trial of a veev dna vaccine resulted in veev-neutralizing antibody responses in 100% of the subjects (hannaman et al., 2016) . mouse models have been developed for numerous routes of infection including cutaneous, intranasal (in), intracranial (ic), and aerosol. eeev susceptibility in mouse models is correlated with age, with younger mice being more susceptible than adults. importantly, eeev pathogenesis is dependent on route of infection with delayed progression upon subcutaneous (subq) exposure (honnold et al., 2015) . newborn mice display neuronal damage with rapid disease progression, resulting in death (murphy and whitfield, 1970) . similarly, eeev produces fatal encephalitis in older mice when administered via the intracerebral route, while inoculation via the subq route causes a pantropic infection eventually resulting in encephalitis (liu et al., 1970; morgan, 1941) . a general drawback to the usage of the mouse model is the lack of vascular involvement during the disease course (liu et al., 1970) . after subq inoculation with weev, suckling mice started to show signs of disease by 24 h and died within 48 h (aguilar, 1970) . the heart was the only organ in which pathologic changes were observed. conversely, adult mice exhibited signs of lethargy and ruffled fur on day 4-5 postinfection. mice were severely ill by day 8 and appeared hunched and dehydrated. death occurred between days 7 and 14 with brain and mesodermal tissues, such as heart, lungs, liver, and kidney involvement (aguilar, 1970; monath et al., 1978) . intracerebral and in routes of infection resulted in a fatal disease that was highly dependent on dose while intradermal (id) and subq inoculations caused only 50% fatality in mice regardless of the amount of virus (liu et al., 1970) . comparing susceptibility of inbred and outbred strains revealed that cd-1, balb/c, a/j, and c57bl6 mice were all highly susceptible to experimental infection via subq inoculation when challenged prior to 10 weeks old with cns involvement and lethality (blakely et al., 2015) . subq/dermal infection in the mouse model results in encephalitic disease very similar to that seen in horses and humans (macdonald and johnston, 2000) . virus begins to replicate in the draining lymph nodes at 4 h postinoculation. eventually, virus enters the brain primarily via the olfactory system. furthermore, aerosol exposure of mice to veev can result in massive infection of the olfactory neuroepithelium, olfactory nerves, and olfactory bulbs and viral spread to brain, resulting in necrotizing panencephalitis (charles et al., 1995; steele et al., 1998) . aerosol and dermal inoculation routes cause neurological pathology in mice much faster than other routes of exposure. the clinical signs of disease in mice infected by aerosol are ruffled fur, lethargy, and hunching progressing to death (charles et al., 1995; steele and twenhafel, 2010; steele et al., 1998) . in challenge of c3h/hen mice with high dose veev caused high morbidity and mortality (julander et al., 2008b) . viral titers in brain peaked on day 4 postchallenge and remained elevated until animals succumbed on day 9-10 postchallenge. protein cytokine array performed on brains of infected mice showed elevated il-1a, il-1b, il-6, il-12, mcp-1, ifnγ, mip-1a, and rantes levels. this model was used successfully to test antivirals against veev (julander et al., 2008a) . additionally, a veev vaccine inactivated with 1,5-iodonaphthyl azide v3526 protects against both footpad and aerosol challenge with virulent veev in a mouse model (gupta et al., 2016) . guinea pigs and hamsters have also been developed as animal models for eeev studies (paessler et al., 2004; roy et al., 2009) . guinea pigs developed neurological involvement with decreased activity, tremors, circling behavior, and coma. neuronal necrosis was observed in brain lesions in the experimentally challenged animals (roy et al., 2009) . subq inoculation of eeev produced lethal biphasic disease in hamsters with severe lesions of nerve cells. the early visceral phase with viremia was followed by neuroinvasion, encephalitis, and death. in addition, parenchyma necrosis were observed in the liver and lymphoid organs (paessler et al., 2004) . harlan sprague-dawley hamsters develop viremia and progress to respiratory, gastrointestinal, and nervous system involvement when inoculated via subq route. vasculitis and encephalitis were both evident in this model, which mirrors the human disease clinical spectrum (paessler et al., 2004) . weev is highly infectious to guinea pigs and has been utilized for prophylactic screening (sidwell and smee, 2003) . studies demonstrated that although the length of the incubation period and the disease duration varied, weev infection resulted in mortality in hamsters by all routes of inoculation. progressive lack of coordination, shivering, rapid and noisy breathing, corneal opacity, and conjunctival discharge resulting in closing of the eyelids were indicative of disease in all cases (zlotnik et al., 1972) . cns involvement was evident with intracerebral, intraperitoneal (ip), and id inoculations (zlotnik et al., 1972) . ip inoculation of weev is fatal in guinea pigs regardless of amount of virus inoculum, with the animals exhibiting signs of illness on day 3-4, followed by death on day 5-9 (nalca, unpublished results) . id, im, or iv inoculations of eeev in nhps cause disease, but does not reliably result in neurological symptoms (dupuy and reed, 2012) . intracerebral infection of eeev produces nervous system disease and fatality in monkeys (nathanson et al., 1969) . the differences in these models indicate that the initial viremia and the secondary nervous system infection do not overlap in nhps when they are inoculated by the peripheral route (wyckoff, 1939) . in and intralingual inoculations of eeev also cause nervous system symptoms in monkeys, but are less drastic than intracerebral injections (wyckoff, 1939) . the aerosol route of delivery will result in uniformly lethal disease in cynomolgus macaques (reed et al., 2007) . in this model, fever was followed by elevated white blood cells and liver enzymes. neurological signs subsequently developed and nhps became moribund and were euthanized between 5-9 days postexposure. meningoencephalomyelitis was the main pathology observed in the brains of these animals (steele and twenhafel, 2010) . similar clinical signs and pathology were observed when common marmosets were infected with eeev by the in route (adams et al., 2008) . both aerosol and in nhp models had similar disease progression and pathology as seen in human disease. very limited studies have been performed with nhps. reed et al. exposed cynomolgus macaques to low and high doses of aerosolized weev. the animals subsequently developed fever, increased white blood counts, and cns involvement, demonstrating that the cynomolgus macaque model could be useful for testing of vaccines and therapeutics against weev (reed et al., 2005) . veev infection causes a typical biphasic febrile response in nhps. initial fever was observed at 12-72 h after infection and lasted less than 12 h. secondary fever generally began on day 5 and lasted 3-4 days (gleiser et al., 1961) . veev-infected nhps exhibited mild symptoms, such as anorexia, irritability, diarrhea, and tremors. leukopenia was common in animals exhibiting fever (monath et al., 1974) . supporting the leukopenia, microscopic changes in lymphatic tissues, such as early destruction of lymphocytes in lymph nodes and spleen, a mild lymphocytic infiltrate in the hepatic triads, and focal myocardial necrosis with lymphocytic infiltration have been observed in monkeys infected with veev. surprisingly, characteristic lesions of the cns were observed histopathologically in monkeys in spite of the lack of any clinical signs of infection (gleiser et al., 1961) . the primary lesions were lymphocytic perivascular cuffing and glial proliferation and generally observed at day 6 postinfection during the secondary febrile episode. similar to these observations, when cynomolgus macaques were exposed to aerosolized veev, fever, viremia, lymphopenia, and clinical signs of encephalitis were observed but the nhps did not succumb to disease (reed et al., 2004) . a common marmoset model was utilized for comparison studies of south america (sa) and north america (na) strains of eeev (adams et al., 2008) . previous studies indicated that the sa strain is less virulent than na strain for humans. common marmosets were infected in with either the na or sa strain of eeev. na strain-infected animals showed signs of anorexia and neurological involvement and were euthanized 4-5 days after the challenge. although sa strain-infected animals developed viremia, they remained asymptomatic and survived until the end of study. chikungunya virus (chikv) is a member of the genus alphaviruses, specifically the semliki forest complex, and has been responsible for a multitude of epidemics centered within africa and southeast asia (griffin, 2007) . the virus is transmitted by aedes aegypti and aedes albopictus mosquitoes. given the widespread endemicity of aedes mosquitoes, chikv has the potential to spread to previously unaffected areas. this is typified by the emergence of disease reported for the first time in 2005 in the islands of south-west indian ocean, including the french la reunion island, and the appearance in central italy in 2007 (charrel et al., 2007; rezza et al., 2007) . the incubation period following a mosquito bite is 2-5 days, leading to a self-limiting acute phase that lasts 3-4 days. symptoms during this period include fever, arthralgia, myalgia, and rash. headache, weakness, nausea, vomiting, and polyarthralgia have all been reported (powers and logue, 2007) . individuals typically develop a stooped posture due to the pain. for approximately 12% of infected individuals, joint pain can last months after resolution of primary disease, and has the possibility to relapse. underlying health conditions including diabetes, alcoholism, or renal disease, increase the risk of developing a severe form of disease that includes hepatitis or encephalopathy. children between the ages of 3 and 18 years old have an increased risk of developing neurological manifestations (arpino et al., 2009) . there is currently no approved vaccine or antiviral. wild-type c57bl/6 adult mice are not permissive to chikv infection by id inoculation. however, it was demonstrated that neonatal mice were susceptible and severity was dependent upon age at infection. six-dayold mice developed paralysis by day 6, and all succumbed by day 12, whereas 50% of 9-day-old mice were able to recover from infection. by 12 days, mice were no longer permissive to disease. symptomatic mice developed loss of balance, hind limb dragging, and skin lesions. neonatal mice were also used as a model for neurological complications (couderc et al., 2008; ziegler et al., 2008) . an adult mouse model has been developed by injection of the ventral side of the footpad of c57bl/6j mice. viremia lasted 4-5 days accompanied with foot swelling and noted inflammation of the musculoskeletal tissue morrison et al., 2011) . adult ifnα/βr knockout mice also developed mild disease with symptoms including muscle weakness and lethargy, symptoms that mirrored human infection. all adult mice died within 3 days. this model was useful in identifying the viral cellular tropism for fibroblasts (couderc et al., 2008) . icr and cd-1 mice can also be utilized as a disease model. neonatal mice inoculated subq with a passaged clinical isolate of chikv developed lethargy, loss of balance, and difficulty walking. mortality was low, 17 and 8% for newborn cd-1 and icr mice, respectively. the remaining mice fully recovered within 6 weeks after infection (ziegler et al., 2008) . a drawback of both the ifnα/βr and cd-1 mice is that the disease is not a result of immunopathogenesis as occurs in human cases, given that the mice are immunocompromised (teo et al., 2012) . a chronic infection model was developed using recombinant activating gene 1 (rag1 −/− ) knockout mice. in this study, mice inoculated via the footpad lost weight in comparison to the control group. both footpad and subq injected mice developed viremia 5-6 days postinfection, which was detectable up to 28 days postinfection. inflammation was evident in the brain, liver, and lung of the subq inoculated animals at 28-56 days postinfection. despite minimal footpad swelling on day 2 postinfection, on day 14 there was severe muscle damage noted at necropsy, which resolved by day 28 (seymour et al., 2015) . golden hamsters serve as another option for small animal modeling. although hamsters do not appear to develop overt clinical symptoms following subq inoculation, viremia developed in the majority of animals within 1 day postinfection with clearance following from day 3 to 4. histologically, inflammation was noted at the skeletal muscle, fascia, and tendon sheaths of numerous limbs. this study was limited in the number of animals utilized, and more work is needed to further develop the hamster model (bosco-lauth et al., 2015) . nhp models of disease include adult, aged, and pregnant rhesus macaques in addition to cynomolgus macaques (broeckel et al., 2015) . differing routes of infection (subq, iv, and im) have been successfully administered, although there is not a clear understanding of the role that route of transmission plays in subsequent pathogenesis and clinical symptoms. typically, viremia is observed 4-5 days postinfection with a correlation between infectious titer and time to viremia observed in cynomolgus but not rhesus (labadie et al., 2010; messaoudi et al., 2013) . fever began at 1-2 days postinfection and persisted for 2-7 days and 3-7 days in cynomolgus and rhesus, respectively and coincided with rash (chen et al., 2010; labadie et al., 2010; messaoudi et al., 2013) . overall blood chemistries changed in conjunction with initiation of viremia, and returned to baseline 10-15 days postexposure (chen et al., 2010) . cns involvement has been difficult to reproduce in nhp models, although it was reported that high inoculum in cynomolgus did result in meningoencephalitis (labadie et al., 2010) . the nhp models have been utilized to conduct efficacy testing on novel vaccines and therapeutics (broeckel et al., 2015) . dengue virus (denv) is transmitted via the mosquito vectors a. aegypti and a. albopictus (moore and mitchell, 1997) . given the endemicity of the vectors, it is estimated that half of the world's population is at risk for exposure to denv. this results in approximately 50 million cases of dengue each year, with the burden of disease in the tropical and subtropical regions of latin america, south asia, and southeast asia (gubler, 2002) . it is estimated that there are 20,000 deaths each year due to dengue hemorrhagic fever (dhf) (guzman and kouri, 2002) . there are four distinct serotypes of denv, numbered 1-4, which are capable of causing a wide clinical spectrum that ranges from asymptomatic to severe with the development of dhf (world health organization, 1997) . incubation can range from 3 to 14 days, with the average being 4-7 days. the virus targets dendritic cells and macrophages following a mosquito bite (balsitis et al., 2009) . typical infection results in classic dengue fever (df), which is self-limiting and has flu-like symptoms in conjunction with retroorbital pain, headache, skin rash, and bone and muscle pain. dhf can follow, with vascular leak syndrome and low platelet count, resulting in hemorrhage. in the most extreme cases, dengue shock syndrome (dss) develops, characterized by hypotension, shock, and circulatory failure (world health organization, 1997) . thrombocytopenia is a hallmark clinical sign of infection, and aids in differential diagnosis (gregory et al., 2010) . severe disease has a higher propensity to occur upon secondary infection with a different denv serotype (thein et al., 1997) . this is hypothesized to occur due to antibody dependent enhancement (ade). there is no approved vaccine or drug, and hospitalized patients receive supportive care including fluid replacement. in order to further progress toward an effective drug or vaccine, small human cohort studies have taken place. however, to provide statistically relevant results, testing must progress in an animal model. in developing an animal model, it is important to note that mosquitoes typically deposit 10 4 -10 6 pfu, and is considered the optimal range during experimental challenge . denv does not naturally replicate effectively in rodent cells, creating the need for mouse-adapted strains, engineered mouse lines, and a variety of inoculation routes to overcome the initial barrier. several laboratory mouse strains including a/j, balb/c, and c57bl/6 are permissive to dengue infection. however, the resulting disease has little resemblance to human clinical signs, and death results from paralysis (huang et al., 2000; paes et al., 2005; shresta et al., 2004) . a higher dose of an adapted denv strain induced dhf symptoms in both balb/c and c57bl/6 souza et al., 2009) . this model can also yield asymptomatic infections. a mouse-adapted strain of denv 2 introduced into ag129 mice developed vascular leak syndrome similar to the severe disease seen in humans (shresta et al., 2006) . passive transfer of monoclonal dengue antibodies within mice leads to ade. during the course of infection, viremia was increased and animals died due to vascular leak syndrome (balsitis et al., 2010) . another mouse-adapted strain injected into balb/c caused liver damage, hemorrhagic manifestations, and vascular permeability (souza et al., 2009) . ic injection of suckling mice with denv leads to death by paralysis and encephalitis, which is rare in human infection (lee et al., 2015; parida et al., 2002; zhao et al., 2014a) . immunocompromised mice have also been used to gain an understanding of the pathogenesis of denv. the most well-defined model is ag129 which is deficient in ifnα/β and γ receptors and can recapitulate dhf/dss if a mouse-adapted strain is utilized yauch et al., 2009) . scid mice engrafted with human tumor cells develop paralysis upon infection, and thus are not useful for pathogenesis studies (blaney et al., 2002; lin et al., 1998) . df symptoms developed after infection in nod/scid/il2rγko mice engrafted with cd34 + human progenitor cells (mota and rico-hesse, 2011) . rag-hu mice developed fever, but no other symptoms upon infection with a passaged clinical isolate and labadapted strain of denv 2 (kuruvilla et al., 2007) . a passaged clinical isolate of denv 3 was used to create a model in immunocompetent adult mice. ip injection in c57bl/6j and balb/c caused lethality by day 6-7 postinfection in a dose dependent manner. the first indication of infection was weight loss beginning on day 4 followed by thrombocytopenia. a drop in systolic blood pressure along with noted increases in the liver enzymes, ast and alt, were also observed. viremia was established by day 5. this model mimicked the characteristic symptoms observed in human dhf/dss cases (costa et al., 2012) . vascular leakage was also observed when c57bl/6 were inoculated with denv 2 (st john et al., 2013) . a murine model was developed that utilized infected mosquitoes as the route of transmission to hu-nsg mice. female mosquitoes were intrathoracically inoculated with a clinical isolate of denv 2. infected mosquitoes then fed upon the mouse footpad to allow for transmission of the virus via the natural route. the amount of virus detected within the mouse was directly proportional to the number of mosquitoes it was exposed to, with 4-5 being optimal. detectable viral rna was in line with historical human infection data. severe thrombocytopenia developed on day 14. this model is notable in that disease was enhanced with mosquito delivery of the virus in comparison to injection of the virus (cox et al., 2012) . nhp models have used a subq inoculation in an attempt to induce disease. although the animals are permissive to viral replication, it is to a lower degree than that observed in human infection (marchette et al., 1973) . the immunosuppressive drug, cyclophosphamide enhances infection in rhesus macaques by allowing the virus to invade monocytes (marchette et al., 1980) . throughout these preliminary studies, no clinical disease was detected. in order to circumvent this, a higher dose of denv was used in an iv challenge of rhesus macaques. hemorrhagic manifestations appeared by day 3 and resulted in petechiae, hematomas, and coagulopathy; however, no other symptoms developed (onlamoon et al., 2010) . a robust antibody response was observed in multiple studies (marchette et al., 1973; onlamoon et al., 2010) . marmosets also mirror human dengue infection, developing fever, leukopenia, and thrombocytopenia following subq inoculation (omatsu et al., 2011 (omatsu et al., , 2012 . nhps are able to produce antibodies similar to those observed during the course of human infection, making them advantageous in studying ade. sequential infection led to a cross-reactive antibody response which has been demonstrated in both humans and mice (midgley et al., 2011) . this phenotype can also be seen upon passive transfer of a monoclonal antibody to dengue and subsequent infection with the virus. rhesus macaques exposed in this manner developed viremia that was 3-to 100-fold higher than previously reported, however, no clinical signs were apparent (goncalvez et al., 2007) . the lack of inducible dhf or dss symptoms hinders further examination of pathogenesis within this model. west nile virus (wnv) was first isolated from the blood of a woman in the west nile district of uganda in 1937 uganda in (smithburn et al., 1940 . after the initial isolation of wnv, the virus was subsequently isolated from patients, birds, and mosquitoes in egypt in the early 1950s (melnick et al., 1951; taylor et al., 1953) and was shown to cause encephalitis in humans and horses. wnv is recognized as the most widespread of the flaviviruses, with a geographical distribution that includes africa, the middle east, western asia, europe, and australia (hayes, 1989) . the virus first reached the western hemisphere in the summer of 1999, during an outbreak involving humans, horses, and birds in the new york city metropolitan area (centers for disease control and prevention, 1999; lanciotti et al., 1999) . since 1999, the range of areas affected by wnv quickly extended. older people and children are most susceptible to wnv disease. wnv generally causes asymptomatic disease or a mild undifferentiated fever (west nile fever), which can last from 3 to 6 days (monath and tsai, 2002) . the mortality rate following neuroinvasive disease ranges from 4% to 11% (asnis et al., 2000; hayes, 1989; hubalek and halouzka, 1999; komar, 2000) . the most severe complications are commonly seen in the elderly, with reported case fatality rates from 4% to 11%. hepatitis, myocarditis, and pancreatitis are unusual, severe, nonneurologic manifestations of wnv infection. inoculation of wnv into nhps intracerebrally resulted in the development of either encephalitis, febrile disease, or an asymptomatic infection, depending on the virus strain and dose. viral persistence is observed in these animals regardless of the outcome of infection (i.e., asymptomatic, fever, encephalitis) (pogodina et al., 1983) . thus, viral persistence is regarded as a typical result of nhp infection with various wnv strains. after both intracerebral and subq inoculation, the virus localizes predominantly in the brain and may also be found in the kidneys, spleen, and lymph nodes. wnv does not result in clinical disease in nhps although the animals show a low level of viremia (lieberman et al., 2009; pletnev et al., 2003) . this is mirrored in new zealand white rabbits in that they only develop fever and low levels of viremia following inoculation via footpad (suen et al., 2015) . id inoculation of both marmosets and rhesus macaques did not yield any clinical signs of disease including fever. viremia was detected in both nhp species, but marmosets developed a higher titer for a greater duration than rhesus (verstrepen et al., 2014) . wnv has also been extensively studied in small animals. all classical laboratory mouse strains are susceptible to lethal infections by the intracerebral and ip routes, resulting in encephalitis and 100% mortality. id route pathogenesis studies indicated that langerhans dendritic cells are the initial viral replication sites in the skin (brown et al., 2007; johnston et al., 1996) . the infected langerhans cells then migrate to lymph nodes and the virus enters the blood through lymphatic and thoracic ducts and disseminates to peripheral tissues for secondary viral replication. virus eventually travels to the cns and causes pathology that is similar to human cases (byrne et al., 2001; cunha et al., 2000; diamond et al., 2003; fratkin et al., 2004) . the swiss mouse strain was inoculated ip in order to screen a variety of viral lineages to assess differences in pathogenesis (bingham et al., 2014) . tesh et al. developed a model for wn encephalitis using the golden hamster, mesocricetus auratus. hamsters appeared asymptomatic during the first 5 days, became lethargic at approximately day 6, and developed neurologic symptoms between days 7 and 10 . many of the severely affected animals died 7-14 days after infection. viremia was detected in the hamsters within 24 h after infection and persisted for 5-6 days. although there were no substantial changes in internal organs, progressive pathologic differences were seen in the brain and spinal cord of infected animals. furthermore, similar to the previously mentioned monkey experiments by pogodina et al. (1983) , persistent wnv infection was found in the brains of hamsters. zika virus recently came to the forefront of public health concerns with the outbreak in brazil at the end of 2015. the clinical disease spectrum is highly variable with reports of a flu-like illness accompanied by rash, guillan-barre syndrome, and microcephaly in newborns (ramos da silva and gao, 2016) . to date, a correlation between gestational age at which exposure to the virus occurs and severity of microcephaly is not fully understood (brasil et al., 2016) . however, a recent study of pregnant women in columbia found that infection with zika virus during the third trimester was not associated with any obvious structural abnormalities of the fetus (pacheco et al., 2016) . transmission of the virus occurs via the bite from an infected a. aegypti or a. albopictus (ramos da silva and gao, 2016) . other reported routes of exposure include sexual transmission and blood transfusion (cunha et al., 2016; d'ortenzio et al., 2016; hills et al., 2016; mccarthy, 2016) . the emergence of this virus with no approved vaccine or therapy, and few diagnostic options demonstrates the utility of well-characterized animal model development. it was first demonstrated in 1956 that experimentally infected mosquitoes could be used to transmit the virus to mice and nhps (boorman and porterfield, 1956) . a129 mice were susceptible to nonadapted zika virus infection following subq inoculation of the limbs. mice began to lose weight 3 days postinfection and met euthanasia criteria by day 6. microscopic lesions within the brain were noted upon necropsy. in conjunction, viral rna was detected in the blood, brain, ovary, spleen, and liver of the infected mice. wild-type 129sv/ev mice were also challenged with no observable clinical disease. however, viral rna was detected at day 3 postinfection in the blood, ovary and spleen, and then remained at detectable levels in the ovaries and spleen on day 7 (dowall et al., 2016) . footpad inoculation of the virus leads to a fatal disease in ag129 mice by day 7 postinoculation with significant histopathological changes in the brain noted at necropsy (aliota et al., 2016) . ag129 mice were also observed to develop neurologic disease by day 6 postexposure (rossi et al., 2016) . immunocompetent mice are resistant to infection via the subq route (rossi et al., 2016) . recently, a mouse model was identified to verify vertical transmission of the virus. pregnant c57 mice were injected either ip or in utero into the lateral ventricle of the fetal brain. ip inoculation induced transient viremia in the pregnant mice on day 1. viral rna was detected in five out of nine placentas on day 3 postinfection. the virus was able to infect the radial glia cells in the fetal brain and leads to a reduction in the cortical neural progenitors . viral exposure via cerebroventricular space/ lateral ventricle of the fetal brain exhibited small brain size at day 5 postexposure in addition to cortical thinning (cugola et al., 2016; li et al., 2016a) . ifnar1 −/− pregnant mice exposed to the virus had nonviable fetuses. in the same study, wild-type mice were given an anti-ifnar antibody prior to and during infection resulting in detectable virus in the fetal head with mild intrauterine growth restriction (miner et al., 2016) . all of these murine studies will further study of the pathogenesis of vertical transmission and the resulting neurological disorders in conjunction with screening novel countermeasures. nhp studies are currently ongoing for animal model development. numerous viruses from the coronavirus (cov) family exist that infect a wide range of animals. six species have been identified that can infect humans. two of these are alpha coronavirues: hcov-229e and hcov-nl63. four are beta coronavirueses: hcov-oc43, hcov-hku1, hcov-sars, and mers-cov. hcov-229e and hcov-oc43 were first detected in the 1960s from the nasal passages of humans with the "common cold" (gaunt et al., 2010) . hcov-nl63, which was first isolated in 2004, causes upper and lower respiratory infections of varying intensity and has been continuously circulating among humans (van der hoek et al., 2006) . hcov-hku1, first isolated in 2002, has been identified more sporadically but also causes respiratory infections (lau et al., 2006) . a significant portion of common cold infections in humans are caused by coronaviruses. in 2002 and 2012, two human coronaviruses, sars-cov and mers-cov, emerged that caused a great deal of alarm since these infections have resulted in nearly 10 and 40% fatality, respectively (assiri et al., 2013; peiris et al., 2004) . the etiologic agent of severe acute respiratory syndrome (sars), sars-cov, emerged in 2002 as it spread throughout 32 countries in a period of 6 months, with 8437 confirmed infections and 813 deaths (roberts and subbarao, 2006; world health organization, 2003) . no additional cases of community acquired sars-cov infection have been reported since 2004. the natural reservoir of sars-cov is the horseshoe bat and the palm civet is an intermediate host (lau et al., 2005) . the main mechanism of transmission of sars-cov is through droplet spread, but it is also viable in dry form on surfaces for up to 6 days and can be detected in stool, suggesting other modes of transmission are also possible (pearson et al., 2003; rabenau et al., 2005; rota et al., 2003) . sars-cov infection has a 10% case fatality with the majority of cases in people over the age of 15 (peiris et al., 2003; wang et al., 2004) . after an incubation period of 2-10 days, clinical signs of sars include general malaise, fever, chills, diarrhea, dyspnea, and cough (drosten et al., 2003) . in some sars cases, pneumonia may develop and progress to acute respiratory distress syndrome (ards). fever usually dissipates within 2 weeks and coincides with the induction of high levels of neutralizing antibodies (tan et al., 2004) . in humans, sars-cov replication destroys respiratory epithelium, and a great deal of the pathogenesis is due to the subsequent immune responses (chen and subbarao, 2007; perlman and dandekar, 2005) . infiltrates persisting within the lung and diffuse alveolar damage (dad) are common sequelae of sars-cov infection (perlman and dandekar, 2005) . virus can be isolated from secretions of the upper airways during early, but not later stages of infection as well as from other tissues (cheng et al., 2004) . sars-cov can replicate in many species, including: dogs, cats, pigs, mice, rats, ferrets, foxes, and monkeys (roper and rehm, 2009) . no model captures all aspects of human clinical disease (pyrexia and respiratory signs), mortality (∼10%), viral replication, and pathology (roberts et al., 2008) . in general, the sars-cov disease course in the model species is much milder and of shorter duration than in humans. viral replication in the various animal models may occur without clinical illness and/or histopathologic changes. the best-characterized models utilize mice, hamsters, ferrets, and nhps. mouse models of sars-cov typically are inoculated by the in route under light anesthesia (roberts et al., 2005) . young, 6-to 8-week-old balb/c mice exposed to sars-cov have viral replication detected in the lungs and nasal turbinate, with a peak on day 2 and clearance by day 5 postexposure (mcauliffe et al., 2004) . there is also viral replication within the small intestines of young balb/c mice. however, young mice have no clinical signs, aside from reduced weight gain, and have little to no inflammation within the lungs (pneumonitis) . in sars-cov infection of c57bl/6 (b6), also yields reduced weight gain and viral k. viral disease replication in the lungs, with a peak on day 3 and clearance by day 9 (glass et al., 2004) . in contrast, balb/c mice 13-14 months of age show weight loss, hunched posture, dehydration, and ruffled fur on day 3-6 postexposure (bisht et al., 2004) . interstitial pneumonitis, alveolar damage, and death also occur in old mice, resembling the age-dependent virulence observed in humans. 129s mice and b6 mice show outcomes to sars-cov infection similar to those observed for balb/c mice but have lower titers and less prolonged disease. while the aged mouse model is more frequently used then young mice, it is more difficult to obtain large numbers of mice older than 1 year (table 33 .1). a number of immunocompromised knockout mouse models of in sars-cov infection have also been developed. 129svev mice infected with sars-cov by the in route develop bronchiolitis, with peribronchiolar inflammatory infiltrates and interstitial inflammation in adjacent alveolar septae . viral replication and disease in these mice resolves by day 14 postexposure. beige, cd1 −/− , and rag1 −/− mice infected with sars-cov have similar outcomes to infected balb/c mice with regard to viral replication, timing of viral clearance, and a lack of clinical signs (glass et al., 2004) . stat1 ko mice infected in with sars-cov have severe disease, with weight loss, pneumonitis, interstitial pneumonia, and some deaths . the stat1 ko mouse model is therefore useful for studies of pathogenicity, pathology, and evaluation of vaccines. angiotensin converting enzyme 2 (ace2) and cd209l were identified as cellular receptors for sars-cov, with affinity for the spike (s) protein of the virus (jeffers et al., 2004) . the variations in the ace2 sequence across animal species could partially explain the differences in infection severity (li et al., 2016b; sutton and subbarao, 2015) . since mice in particular have a greater number of sequence differences in ace2, transgenic mice were created that express human ace2 (mccray et al., 2007; netland et al., 2008; yang et al., 2007) . unlike other murine models of sars-cov, mice expressing hace2 had up to 100% mortality, with severity correlating to the level of hace2 expression (tseng et al., 2007) . with high levels of hace2 expression, mice developed a severe lung and brain infection. however, cns k. viral disease infection is only rarely observed in humans infected with sars-cov. syrian golden hamsters (strain lvg) are also susceptible to in exposure of sars-cov. after the administration of 10 3 tcid 50, along with a period of transient viremia, sars-cov replicates in nasal turbinates and lungs, resulting in pneumonitis (roberts et al., 2005) . there are no obvious signs of disease, but exercise wheels can be used to monitor decrease in nighttime activity. limited mortality has been observed, but it was not dose dependent and could have more to do with genetic differences between animals because the strain is not inbred (roberts et al., 2008) . damage is not observed in the liver or spleen despite detection of virus within these tissues. several studies have shown that intratracheal (it) inoculation of sars-cov in anesthetized ferrets (mustela furo) results in lethargy, fever, sneezing, and nasal discharge (skowronski et al., 2005) . clinical disease has been observed in several studies excluding one, perhaps due to characteristics of the inoculating virus (kobinger et al., 2007) . sars-cov is detected in pharyngeal swabs, trachea, tracheobronchial lymph nodes, and high titers within the lungs. mortality has been observed around day 4 postexposure as well as mild alveolar damage in 5%-10% of the lungs, occasionally accompanied by severe pathology within the lungs (martina et al., 2003; ter meulen et al., 2004) . with fever, overt respiratory signs, lung damage, and some mortality, the ferret intratracheal model of sars-cov infection is perhaps most similar to human sars, albeit with a shorter time course. sars-cov infection of nhps by intransal or it routes generally results in a very mild infection that resolves quickly. sars-cov infection of old world monkeys, such as rhesus macaques, cynomolgus macaques (cynos), and african green monkeys (agms) have been studied with variable results, possibly due to the outbred nature of the groups studied or previous exposure to related pathogens. clinical illness and viral loads have not been consistent; however, replication within the lungs and dad are features of the infections for each of the primate species. some cynos have no illness but others have rash, lethargy, and respiratory signs and pathology martina et al., 2003; mcauliffe et al., 2004; rowe et al., 2004) . rhesus have little to no disease and only have mild findings upon histopathological analysis (rowe et al., 2004) . agms infected with sars-cov have no overt clinical signs but dad and pneumonitis has been documented (mcauliffe et al., 2004) . viral replication has been detected for up to 10 days in the lungs of agms; however, the infection resolves, and does not progress to fatal ards. farmed chinese masked palm civets, sold in open markets in china, were involved in the sars-cov outbreak. it and in inoculation of civets with sars-cov results in lethargy, decreased aggressiveness, fever, diarrhea, and conjunctivitis . leucopenia, pneumonitis, and alveolar septal enlargement, with lesions similar to those observed in ferrets and nhps, have also been observed in laboratory-infected civets. squirrel monkeys, mustached tamarinds, and common marmosets have not been susceptible to sars-cov infection (greenough et al., 2005; roberts et al., 2008) . vaccines have been developed for related animal covs in chickens, cattle, dogs, cats, and swine, and have included live-attenuated, killed, dna and viral-vectored vaccine strategies (cavanagh, 2003) . an important issue to highlight from work on these vaccines is that cov vaccines, such as those developed for cats, may induce a more severe disease (perlman and dandekar, 2005; weiss and scott, 1981) . as such, immune mice had th2type immunopathology upon sars-cov challenge (tseng et al., 2012) . severe hepatitis in vaccinated ferrets with antibody enhancement in liver has been reported (weingartl et al., 2004) . additionally, rechallenge of agms showed limited viral replication but significant lung inflammation, including alveolitis and interstitial pneumonia, which persisted for long periods of time after viral clearance (clay et al., 2012) . mouse and nhp models with increased virulence may be developed by adapting the virus by repeated passage within the species of interest. mouse-adapted sars with uniform lethality was developed from 15 serial passages in the lungs of young balb/c mice (mccray et al., 2007; roberts et al., 2007; rockx et al., 2007) . middle east respiratory syndrome (mers-cov) emerged in saudi arabia and is associated with fever, severe lower respiratory tract infection, and oftentimes renal failure (al-tawfiq et al., 2016; omrani et al., 2015) . mers patients can also occasionally manifest with neurological symptoms. mers-cov infection has a high fatality rate. infections in humans can also be asymptomatic. as of october 2015, there were 1589 confirmed cases and 567 deaths (li et al., 2016b) . bats serve as the likely natural reservoir since virus with 100% nucleotide identity to the index case was isolated from egyptian tomb bats (memish et al., 2013) . spread to humans likely comes from infected dromedary camels (adney et al., 2014; azhar et al., 2014) . the host range for mers-cov is dependent on the binding of the viral s protein to the host receptor, which is human dipeptidyl peptidase four (hdpp4), also known as cd26 (raj et al., 2013) . the expression and distribution of dpp4 in the human respiratory tract has recently been well characterized (meyerholz et al., 2016) . interestingly, dpp4 expression is preferentially localized to alveolar regions, perhaps explaining why mers predominantly manifests as an infection of the lower respiratory tract. humans with preexisting pulmonary disease have increased dpp4 expression in alveolar epithelia. small animals typically used for viral disease research, such as mice, hamsters, guinea pigs, and ferrets are naturally nonpermissive to mers-cov infection due to a low binding efficiency of the viral s protein to the host dpp4 (sutton and subbarao, 2015). in contrast the rhesus macaque and common marmoset have complete homology to human dpp4, allowing productive mers-cov infection to occur falzarano et al., 2014; munster et al., 2013; yao et al., 2014) . new zealand white rabbits can be infected with mers-cov, and virus was isolated from the upper respiratory tract, but there were no clinical symptoms or significant histopathological changes (haagmans et al., 2015) . due to the lack of strong binding affinity of the mers-cov s protein to the murine dpp4 receptor, wildtype mice are not susceptible to mers-cov infection. as such, several approaches have been used to create susceptible murine animal models of mers-cov infection by inducing the expression of hdpp4. one approach utilized an adenovirus vector expressing hdpp4 to transduce mice (zhao et al., 2014b) . these mice developed pneumonia but survived mers-cov infection. in mers-cov infection of mice with global expression of hdpp4 resulted in id 50 and ld 50 values of <1 and 10 tcid 50 , respectively (tao et al., 2016) . thus, mers-cov infection of these transgenic mice can be either sublethal or uniformly lethal depending on the dose. inflammatory infiltrates were found in the lungs and brain stems of mice with some focal infiltrates in the liver as well. another strategy uses transgenic mice expressing hdpp4 under either a surfactant protein c or cytokeratin 10 promoter (li et al., 2016b) . in mers-cov infection in these mice resulted in a uniformly lethal disease characterized by alveolar edema and microvascular thrombosis and mononuclear clear cell infiltration in the lungs. the brain stem was also impacted by the infection. dpp4 expression with an ubiquitously expressing promoter from cytomegalovirus also had a uniformly lethal infection with predominant lung and brain involvement, but numerous other tissues were also impacted and contained virus (agrawal et al., 2015) . common marmosets infected with 5.2 × 10 6 tcid 50 (emc-2012) mers-cov by the combined in, oral, ocular, and it routes capitulate the severe disease in human infections (falzarano et al., 2014) . the animals manifested moderate to severe clinical disease, with interstitial infiltration of both lower lung lobes. two of nine animals became moribund between days 4 and 6. viral rna was detected in nasal and throat swabs, various organs, and in the blood of some animals, indicating a systemic infection. histologically, animals showed evidence of acute bronchial interstitial pneumonia as well as other pathological defects. infection of rhesus macaques with mers-cov results in a mild clinical disease characterized by a transient lung infection with pneumonia. rhesus macaques were inoculated with at least 10 7 tcid 50 (emc-2012) mers-cov either by the it route or a combined in, it, oral, and ocular inoculation . the result was a mild respiratory illness including nasal swelling and a short fever with all animals surviving. viral rna was recovered from nasal swab samples and replicating virus was found in lung tissue . mild pathological lesions were found only in the lungs. radiographic imaging of the lungs revealed interstitial infiltrates, which are signs of pneumonia (yao et al., 2014) . interestingly, mer-cov infection is more severe in marmosets compared to rhesus macaques (falzarano et al., 2014) . this is despite the finding that both species have complete homology with humans within the dpp4 domain that interacts with the viral s protein. other host factors influencing disease severity have not yet been identified. transgenic mouse models expressing hdpp4 are ideal for initial development and screening of mers-cov countermeasures, and marmosets can be used for final selection and characterization. filoviridae consists of three genera, ebolavirus and marburgvirus, and a newly discovered group, cuevavirus (kuhn, 2008) . it is thought that various species of bats are the natural host reservoir for these viruses that have lethality rates from 40% to 82% in humans. there is evidence that the egyptian rousette bat (rousettus aegyptiacus) is the natural reservoir for marburgviruses but may not be for ebolaviruses (jones et al., 2015) . marburg virus (marv) first emerged in 1967 in germany when laboratory workers contracted the virus from agms (chlorocebus aerthiops) that were shipped from uganda. ebolaviruses sudan and zaire (sudv and ebov) caused nearly simultaneous outbreaks in 1976 in what is now the democratic republic of congo (drc). the most recent outbreak of ebov in west africa was by far the largest with over 28,000 suspected, probable and confirmed cases and over 11,000 deaths. bundibugyo virus (bdbv) first emerged in 2007 in bundibugyo, uganda with 56 confirmed cases . two other ebolaviruses are known: taï forest (tafv) (previously named cote d'ivoire) (ciebov) and reston (restv), which have not caused major outbreaks or lethal disease in humans. filovirus disease in humans is a characterized by aberrant innate immunity and a number of clinical symptoms: fever, nausea, vomiting, k. viral disease arthralgia/myalgia, headaches, sore throat, diarrhea, abdominal pain, and anorexia as well as numerous others (mehedi et al., 2011; wauquier et al., 2010) . approximately 10% of patients develop petechia and a greater percentage, depending on the specific strain, may develop bleeding from various sites (gums, puncture sites, stools, etc.) (table 33 .2). natural transmission in an epidemic is through direct contact or needle sticks in hospital settings. however, much of the research interest in filoviruses primarily stems from biodefense needs, particularly from aerosol biothreats. as such, im, ip, and aerosol models have been developed in mice, hamsters, guinea pigs, and nhps for the study of pathogenesis, correlates of immunity, and for testing countermeasures . since filoviruses have such high lethality rates in humans, scientists have looked for models that are uniformly lethal to stringently test efficacy of candidate vaccines and therapeutics. one issue to take note of in animal model development of filovirus infection is the impact of particle to plaque-forming unit (pfu) ratios on lethality, wherein it is possible that increasing the dose could actually decrease infectivity due to an immunogenic effect produced by inactive virions in the stock. additionally, the plaque assay used to measure live virions in a stock may greatly underestimate the true quantity of infectious virions in a preparation (alfson et al., 2015; smither et al., 2013a) . immunocompetent mice have not been successfully infected with wild-type filoviruses due to the control of the infection by the murine type 1 interferon response (bray, 2001) . however, wild-type inbred mice are susceptible to filovirus that has been mouse adapted (ma) by serial passage in mice (bray et al., 1999) . marv angola was particularly resistant to adaptation, but after 24 serial passages in scid mice, infection caused severe disease in balb/c and c57bl/6 mice when administered in or ip (qiu et al., 2014) . these mice had pathology with some similarities to infection in humans including lymphopenia, thrombocytopenia, liver damage, and viremia. balb/c mice, which are the strain of choice for ip inoculation of ma-ebov, are not susceptible by the aerosol route (bray et al., 1999; zumbrun et al., 2012a) . for aerosol infection of immunocompetent mice, a panel of bxd (balb/c x dba) recombinant inbred strains were screened and one strain, bxd34, was particularly susceptible to airborne ma-ebov, with 100% lethality to low or high doses (approximately 100 or 1000 pfu) ( zumbrun et al., 2012a) . these mice developed weight loss of greater than 15% and succumbed to infection between days 7 and 8 postexposure. the aerosol infection model utilizes a whole-body exposure chamber to expose mice aged 6-8 weeks to ma-ebov aerosols with a mass median aerodynamic diameter (mmad) of approximately 1.6 µm and a geometric standard deviation (gsd) of approximately 2.0 for 10 min. another approach uses immunodeficient mouse strains, such as scid, stat1 ko, ifn receptor ko, or perforin ko with a wild-type ebov inoculum by ip or aerosol routes (bray, 2001; lever et al., 2012; zumbrun et al., 2012a) . mice are typically monitored for clinical disease "scores" based on activity and appearance, weight loss, and moribund condition (survival). coagulopathy, a hallmark of filovirus infection in humans, has been observed, with bleeding in a subset of animals and failure of blood samples to coagulate late in infection (bray et al., 1999) . liver, kidney, spleen, and lung tissue taken from moribund mice have pathology characteristic of filovirus disease in nhps (zumbrun et al., 2012a) . while most mouse studies have used ma-ebov or ebov, an ip mouse-adapted marv model is also available (warfield et al., 2007 (warfield et al., , 2009 ). ma-marv and ma-ebov models are particularly useful for screening novel antiviral compounds (panchal et al., 2012) . recently, a model was created using immunodeficient nsg [nonobese diabetic (nod)/scid/il-2 receptor chain knockout] mice with transplanted human hematopoietic stem cells from umbilical cord blood. these mice were susceptible to lethal wt (nonadapted) ebov by ip and in exposure (ludtke et al., 2015) . the transplanted mice had all of the cellular components of a fully functional adaptive human immune system and upon ebov (brannan et al., 2015; lever et al., 2012) . interestingly, inoculation of infa/br −/− mice with tafv and restv does not result in clinical signs. yet another strategy uses knockout mice lacking possible receptors for filovirus entry, such as niemann-pick c1 and c2 (npc1 and npc2). npc2 (−/−) mice were fully susceptible to infection with ebov but npc1 (−/−) mice were completely resistant (herbert et al., 2015) . hamsters are frequently used to study cardiovascular disease, coagulation disorders, and thus serve as the basis for numerous viral hemorrhagic fever models (gowen and holbrook, 2008; herbert et al., 2015) . an ip ma-ebov infection model has been developed in syrian hamsters gowen and holbrook, 2008; herbert et al., 2015; tsuda et al., 2011) . this model, which has been used to test a vesicular stomatitis virus vectored vaccine approach, utilizes male 5-to 6-week-old syrian hamsters which are infected with 100 ld 50 of ma-ebov. virus is present in tissues and blood collected on day 4 and all animals succumbed to the disease by day 6. infected hamsters had severe coagulopathy and uncontrolled host immune responses, similar to what is observed in primates. (ebihara et al., 2010) guinea pig models of filovirus infection have been developed for ip and aerosol routes using guinea pigadapted ebov (gp-ebov) and marv (gp-marv) (choi et al., 2012; connolly et al., 1999; twenhafel et al., 2015; zumbrun et al., 2012c) . guinea pig models of filovirus infection are quite useful in that they develop fever, which can be monitored at frequent intervals by telemetry. additionally, the animals are large enough for regular blood sampling in which measurable coagulation defects are observed as the infection progresses. a comparison of ip infection of outbred guinea pigs with guinea pig-adapted marv angola and marv ravn revealed similar pathogenesis (cross et al., 2015) . infection with either strain resulted in features of the disease that are similar to what is seen in human and nhp infection, such as viremia, fever, coagulopathy, lymphopenia, elevated liver enzymes (alt and ast), thrombocytopenia, and splenic, gastrointestinal and hepatic lesions. gp-marv-ravn had a delayed disease progression relative to gp-marv-ang. hartley guinea pigs exposed to aerosolized gp-ebov develop lethal interstitial pneumonia. this is in contrast to subq infection of guinea pigs, aerosol ebov challenge of nhps, and natural human infection (twenhafel et al., 2015) . both subq and aerosol exposure of guinea pigs to gp-ebov resulted in only mild lesions in the liver and spleen. by aerosol exposure, gp-ebov is uniformly lethal at both high and low doses (100 or 1000 pfu target doses) but lethality drops with low (less than 1000 pfu) presented doses of airborne gp-marv and more protracted disease is seen in some animals (our unpublished observations) (zumbrun et al., 2012c) . weight loss of between 15% and 25% is a common finding in guinea pigs exposed to gp-ebov or gp-marv. fever, which becomes apparent by day 5, occurs more rapidly in gp-ebov exposed guinea pigs than with gp-marv exposure. lymphocytes and neutrophils increase during the earlier part of the disease, and platelet levels steadily drop as the disease progresses. increases in coagulation time can be seen as early as day 6 postexposure. blood chemistries (i.e., alt, ast, alkp, and bun) indicating problems with liver and kidney function are also altered late in the disease course. transmission of ebov has been documented from swine to nhps via the respiratory tract (kobinger et al., 2011) . as such, guinea pigs have been used to establish transmission models (wong et al., 2015a,b) . nonexposed guinea pigs were placed in the cages with infected guinea pigs 1 day postexposure to gp-ebov. guinea pigs challenged intanasally were more likely to transmit virus to naive cagemates than those that were exposed by the ip route. nhp models of filovirus infection are the preferred models for more advanced disease characterization and testing of countermeasures because they most closely mimic the disease and immune correlates seen in humans (dye et al., 2012) . old world primates have been primarily used for development of ip, im, and aerosol models of filovirus infection ( twenhafel et al., 2013) . uniformly lethal filovirus models have been developed for most of the virus strains in cynomolgus macaques, rhesus macaques, and to a lesser degree, agms (alves et al., 2010; carrion et al., 2011; davis et al., 1997; hensley et al., 2011a; reed et al., 2007; zumbrun et al., 2012b) . low-passage human isolates that have not been passaged in animals have been sought for development of nhp models to satisfy the food and drug administration (fda) animal rule. ebov-makona, the strain responsible for the recent large outbreak in west africa, was compared to the "prototype" 1976 ebov strain (marzi et al., 2015) . the disease in cynos was similar for both viruses, but disease progression was delayed for ebov-makona. this delay as well as the lower fatality rate in the 2014 epidemic compared to the 1976 outbreak suggest that ebov-makona is less virulent. the large number of cases in the 2014-15 ebov outbreak brought to light previously underappreciated eye pathology and ocular viral persistence in survivors. while survivors of nhp filovirus infection are infrequent, necrotizing scleritis, conjunctivitis, and other ocular pathology has been observed in ebov-infected animals (alves et al., 2016) . prominent features of the filovirus infections in nhps are onset of fever by day 5 postexposure, viremia, lymphopenia, tachycardia, azotemia, alteration in liver function enzymes (alt, ast, and alkp), decrease in platelets, and increased coagulation times. petechial rash is a common sign of filovirus disease and may be more frequently observed in cynomolgus macaques than in other nhp species (zumbrun et al., 2012b) . immunological parameters have been evaluated and t, b, and natural killer cells are greatly diminished as the infection progresses (fernando et al., 2015) . a cytokine storm occurs with rises in ifnγ, tnf, il-6, and ccl2 (fernando et al., 2015) . however, there is also evidence from transcriptional profiling of circulating immune cells that the early immune response is skewed toward a th2 response (connor et al., 2015) . strikingly, animals surviving challenge may have a delay in the production of inflammatory cytokines and chemokines (martins et al., 2015) . clinical disease parameters may have a slightly delayed onset in aerosol models. dyspnea late in infection is a prominent feature of disease after aerosol exposure (zumbrun et al., 2012b) . aerosol filovirus infection of nhps results in early infection of respiratory lymphoid tissues, dendritic cells, alveolar macrophages, blood monocytes, and fibroblastic reticular cells followed by spread to regional lymph nodes then multiple organs (ewers et al., 2016; twenhafel et al., 2013) . a number of pronounced pathology findings include multifocal hepatic necrosis and fibrin accumulation, particularly within the liver and the spleen. for aerosolized marv infection of rhesus, the most significant pathology included destruction of the tracheobronchial and mediastinal lymph nodes (ewers et al., 2016) . lymphocytolysis and lymphoid depletion are also observed (alves et al., 2010) . multilead, surgically implanted telemetry devices are useful in continuous collection of temperature, blood pressure, heart rate, and activity levels. as such, blood pressure drops as animals become moribund and heart rate variability (standard deviation of the heart rate) is altered late in infection (zumbrun et al., 2012b) . the most recently developed telemetry devices can also aid in plethysmography to measure respiratory minute volume for accurate delivery of presented doses for aerosol exposure. standardized filovirus-infected nhp euthanasia criteria have also been developed to enhance reproducibility for studies that evaluate therapeutic and vaccine countermeasures (warren et al., 2014) . filovirus infection of common marmosets (callithrix jacchus) is also a viable model to study the disease course. respiratory infection of marmosets with marv results in a lethal infection with fever, hemorrhaging, transient rash, disseminated viral infection, increases in liver function enzymes, coagulopathy, hepatitis, and histological lesions particularly in the kidney and liver (smither et al., 2013b) . marmosets are similarly susceptible to infection with ebovkikwit (smither et al., 2015) . thus, ebov or marv infection of marmosets produces features of the disease that are very similar to that of other nhps and humans. hendra and nipah virus are unusual within the paramyxoviridae family given that they can infect a large range of mammalian hosts. both viruses are grouped under the genus henipavirus. the natural reservoirs of the viruses are the fruit bats from the genus pteropus. hendra and nipah have the ability to cause severe disease in humans with the potential for a high case fatality rate (rockx et al., 2012) . outbreaks due to nipah virus have been recognized in malaysia, singapore, bangladesh, and india, while hendra virus outbreaks have yet to be reported outside of australia (luby et al., 2009a,b) . hendra was the first member of the genus identified and was initially associated with an acute respiratory disease in horses. all human cases have been linked to transmission through close contact with an infected horse. there have been no confirmed cases of direct transmission from bat to human. nipah has the distinction of transmission among, although the exact route is unknown (homaira et al., 2010) . the virus is susceptible to ph, temperature, and desiccation, and thus close contact is hypothesized as needed for successful transmission (fogarty et al., 2008) . both viruses have a tropism for the neurological and respiratory tracts. the incubation period for hendra virus is 7-17 days and is marked by a flu-like illness. symptoms at this initial stage include myalgia, headache, lethargy, sore throat, and vomiting (hanna et al., 2006) . disease progression can continue to pneumonitis or encephalitic manifestations, with the person succumbing to multiorgan failure (playford et al., 2010) . nipah virus has an incubation period of 4 days to 2 weeks (goh et al., 2000) . much like hendra, the first signs of disease are nondescript. severe neurological symptoms subsequently develop including encephalitis and seizures that can progress to coma within 24-48 h (lo and rota, 2008). survivors of infection typically make a full recovery; however, 22% suffer permanent sequelae, including persistent convulsions (tan and chua, 2008) . at this time, there is no approved vaccine or antiviral, and treatment is purely supportive. animal models are being used to not only test novel vaccines and therapeutics, but also deduce the early events of disease because documentation of human cases is at terminal stages. the best small animal model is the syrian golden hamster due to their high susceptibility to both henipaviruses. clinical signs upon infection recapitulate the disease course in humans including acute encephalitis and respiratory distress. challenged animals died within 4-17 days postinfection. the progression of disease and timeline is highly dependent on dose and route of infection. in inoculation leads to imbalance, limb paralysis, lethargy, and breathing difficulties whereas ip resulted in tremors and paralysis within 24 h before death. virus was detected in lung, brain, spleen, kidney, heart, spinal cords, and urine, with the brain having the highest titer. this model is used for vaccination and passive protection studies (guillaume et al., 2009; rockx et al., 2011; wong et al., 2003) . the guinea pig model has not been widely used due to the lack of a respiratory disease upon challenge (torres-velez et al., 2008; williamson et al., 2001) . inoculation with hendra virus via the subq route leads to a generalized vascular disease with 20% mortality. clinical signs were apparent 7-16 days postinfection with death occurring within 2 days of cns involvement. higher inoculum has been associated with development of encephalitis and cns lesions. id and in injection does not lead to disease, although the animals are able to seroconvert upon challenge. the inoculum source does not affect clinical progression. nipah virus challenge only causes disease upon ip injection and results in weight loss and transient fever for 5-7 days. virus was shed through urine and was present in the brain, spleen, lymph nodes, ovary, uterus, and urinary bladder (hooper et al., 1997) . ferrets infected with hendra or nipah virus display the same clinical disease as seen in the hamster model and human cases (bossart et al., 2009; pallister et al., 2011) . upon inoculation by the oronasal route, ferrets develop severe pulmonary and neurological disease within 6-9 days including fever, coughing, and dyspnea. lesions do develop in the ferret's brains, but to a lesser degree than seen in humans. cats have also been utilized as an animal model for henipaviruses. disease symptoms are not dependent upon the route of infection. the incubation period is 4-8 days and leads to respiratory and neurological symptoms (mungall et al., 2007; johnston et al., 2015; westbury et al., 1996) . this model has proven useful for vaccine efficacy studies. squirrel and agms are representative of the nhp models. for squirrel monkeys, nipah virus is introduced by either the in or iv route and subsequently leads to clinical signs similar to humans, although in challenge results in milder disease. upon challenge, only 50% of animals develop disease manifestations including anorexia, dyspnea, and acute respiratory syndrome. neurological involvement is characterized by uncoordinated motor skills, loss of consciousness, and coma. viral rna can be detected in lung, brain, liver, kidney, spleen, and lymph nodes but is only found upon iv challenge (marianneau et al., 2010) . agms are very consistent model of both viruses. it inoculation of the viruses results in 100% mortality, and death within 8.5 and 9-12 days postinfection for hendra and nipah viruses, respectively. the animals develop severe respiratory and neurological disease with generalized vasculitis rockx et al., 2010) . the reservoir of the viruses, gray-headed fruit bats, has been experimentally challenged. due to their status as the host organism for henipaviruses, the bats do not develop clinical disease. however, hendra virus can be detected in kidneys, heart, spleen, and fetal tissue, and nipah virus can be located in urine . pigs develop a respiratory disease upon infection with both nipah and hendra viruses (berhane et al., 2008; li et al., 2010; middleton et al., 2002) . oral inoculation does not produce a clinical disease, but subq injection represents a successful route of infection. live virus can be isolated from the oropharynx as early as 4 days postinfection. nipah virus can also be transmitted between pigs. nipah virus was able to induce neurological symptoms in 20% of the pigs, even though virus was present in all neurological tissues regardless of symptoms (weingartl et al., 2005) . within the pig model, it appeared that nipah virus had a greater tropism for the respiratory tract, while hendra for the neurological system. horses are also able to develop a severe respiratory tract infection accompanied with fever and general weakness upon exposure to nipah and hendra viruses. oronasal inoculation led to systemic disease with viral rna detected in nasal swabs within 2 days (marsh et al., 2011; williamson et al., 1998) . animals died within 4 days postexposure and have interstitial pneumonia with necrosis of alveoli (murray et al., 1995a,b) . virus could be detected in all major systems. mice, rats, rabbits, chickens, and dogs have been tested but are nonpermissive to infection (westbury et al., 1995; wong et al., 2003) . suckling balb/c mice succumb to infection if the virus is inoculated intracranially (mungall et al., 2006) . in exposure with nipah does not induce a clinical disease; however, there is evidence of a subclinical infection in the lungs following euthanasia of the mice (dups et al., 2014) . in addition, a human lung xenograph model in nsg mice demonstrated that the human lung is highly susceptible to nipah viral replication and damage (valbuena et al., 2014) . embryonated chicken eggs have been inoculated with nipah virus leading to a universally fatal disease within 4-5 days postinfection (tanimura et al., 2006) . annually, respiratory syncytial virus (rsv) is responsible for the lower respiratory tract infections of 33 million children under the age of 5, which in turn results in 3 million hospitalizations and approximately 200,000 deaths (nair et al., 2010) . within the united states, hospital costs alone amount to over 600 million dollars per year (paramore et al., 2004) . outbreaks are common in the winter (yusuf et al., 2007) . the virus is transmitted by large respiratory droplets that replicate initially within the nasopharynx and spreads to the lower respiratory tract. incubation for the virus is 2-8 days. rsv is highly virulent leading to very few asymptomatic infections (collins and graham, 2008) . disease manifestations are highly dependent upon the age of the individual. rsv infections in neonates produce nonspecific symptoms including overall failure to thrive, apnea, and feeding difficulties. infants present with a mild upper respiratory tract disease that could develop into bronchiolitis and bronchopneumonia. contracting rsv at this age results in an increased chance of developing childhood asthma (wu et al., 2008) . young children develop recurrent wheezing while adults have exacerbation of previously existing respiratory conditions (falsey et al., 2005) . common clinical symptoms are runny nose, sneezing, and coughing accompanied by fever. mortality rates from rsv in hospitalized children are 1%-3% with the greatest burden of disease seen in 3-4 month olds (ruuskanen and ogra, 1993). hematopoietic stem cell transplant patients, solid organ transplant patients, and copd patients are particularly vulnerable to rsv infection and have mortality rates between 7.3% and 13.3% upon infection (anderson et al., 2016) . although there are almost 60 rsv vaccine candidates which are in preclinical and clinical phases, there is no licensed vaccine available and ribavirin usage is not recommended for routine treatment (american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis, 2006; higgins et al., 2016; kim and chang, 2016) . animal models of rsv were developed in the hopes of formulating an effective and safe vaccine unlike the formalin-inactivated rsv (fi-rsv) vaccine. this vaccine induced severe respiratory illness in infants whom received the vaccine and were subsequently infected with live virus (kim et al., 1969) . mice can be used to model rsv infection, although a very high in inoculation is needed to achieve clinical symptoms (jafri et al., 2004; stark et al., 2002) . strain choice is crucial to reproducing a physiological relevant response (stokes et al., 2011). age does not affect primary disease manifestations (graham et al., 1988) . however, it does play a role in later sequelae showing increased airway hyperreactivity . primary rsv infection produces increased breathing with airway obstruction (jafri et al., 2004; van schaik et al., 1998) . virus was detected as early as day 3 and reached maximum titer at day 6 postinfection. clinical illness is defined in the mouse by weight loss and ruffled fur as opposed to runny nose, sneezing, and coughing as seen in humans. a humanized mouse model was recently developed by in inoculation. the challenged mice experienced weight loss and demonstrated a humoral and cellular immune response to the infection (sharma et al., 2016) . cotton rats are useful given that rsv is able to replicate to high titers within the lungs and can be detected in both the upper and lower airways after in inoculation (boukhvalova et al., 2009; niewiesk and prince, 2002) . viral replication is 50-to 1000-fold greater in the cotton rat model than mouse model (wyde et al., 1993) . the cotton rats develop mild to moderate bronchiolitis or pneumonia (grieves et al., 2015; prince et al., 1999) . although age does not appear to factor into clinical outcome, it has been reported that older cotton rats tend to take longer to achieve viral clearance. viral loads peak by the 5th day, dropping to below the levels of detection by day 8. the histopathology of the lungs appears similar to that of humans after infection (piazza et al., 1993) . this model has limited use in modeling the human immune response to infection as challenge with the virus induces a th2 response in cotton rats, whereas humans tend to have a response skewed toward th1 (culley et al., 2002; dakhama et al., 2005; ripple et al., 2010) . fi-rsv disease was recapitulated upon challenge with live virus after being vaccinated twice with fi-rsv. chinchillas have been challenged experimentally with rsv via in inoculation. the virus was permissive within the nasopharynx and eustachian tube. the animals displayed an acute respiratory tract infection. this model is therefore useful in studying mucosal immunity during infection (gitiban et al., 2005) . ferrets infected by it were found to have detectable rsv in throat swabs up to day 7 postinfection, and positive qpcr up to day 10. immunocompromised ferrets were observed to have higher viral loads accompanied with detectable viral replication in the upper respiratory tract (stittelaar et al., 2016) . chimpanzees are permissive to replication and clinical symptoms of rsv including rhinorrhea, sneezing, and coughing. adult squirrel monkeys, newborn rhesus macaques, and infant cebus monkeys were also challenged but did not exhibit any disease symptoms or high levels of viral replication (belshe et al., 1977) . bonnet monkeys were developed an inflammatory response by day 7 with viral rna detected in both bronchial and alveolar cells (simoes et al., 1999) . the chimpanzee model has been proven useful for vaccine studies (hancock et al., 2000; teng et al., 2000) . sheep have also been challenged experimentally since they develop respiratory disease when exposed to ovine rsv (meyerholz et al., 2004) . lambs are also susceptible to human respiratory syncytial infection (olivier et al., 2009; sow et al., 2011) . when inoculated intratracheally, the lambs developed an upper respiratory tract infection with cough after 6 days. some lambs went on to develop lower respiratory disease including bronchiolitis. the pneumonia resolved itself within 14 days. rsv replication peaked at 6 days, and rapidly declined. studying respiratory disease in sheep is beneficial given the shared structural features with humans (plopper et al., 1983; scheerlinck et al., 2008) . the influenza viruses consist of three types: influenza a, b, and c, based on antigenic differences. influenza a is further classified by subtypes; 16 ha and 9 na subtypes are known. seasonal influenza is the most common infection and usually causes a self-limited febrile illness with upper respiratory symptoms and malaise that resolves within 10 days (taubenberger and morens, 2008) . the rate of infection is estimated at 10% in the general population and can result in billions of dollars of loss annually from medical costs and reduced work-force productivity. approximately 40,000 people in the united states die each year from seasonal influenza (dushoff et al., 2006) . thus, vaccines and therapeutics play a critical role in controlling infection, and development using animal models is ongoing (braun et al., 2007b) . influenza virus replicates in the upper and lower airways, peaking at approximately 48-h postexposure. infection can be more severe in infants and children under the age of 22, people over the age of 65, or immunocompromised individuals where viral pneumonitis or pneumonia can develop or bacterial superinfection resulting in pneumonia or sepsis (barnard, 2009; glezen, 1982) . pneumonia from secondary bacterial infection, such as streptococcus pneumonia, streptococcus pyogenes, and neisseria meningitides, and more rarely, staphylococcus aureus, is more common than viral pneumonia from the influenza virus itself, accounting for ∼27% of all influenza associated fatalities (alonso et al., 2003; ison and lee, 2010; speshock et al., 2007) . death, often due to ards can occur as early as 2 days after onset of symptoms. lung histopathology in severe cases may include dad, alveolar edema and damage, hemorrhage, fibrosis, and inflammation (taubenberger and morens, 2008) . the h5n1 avian strain of influenza, has lethality rates of around ∼50% (of known cases), likely because the virus preferentially binds to the cells of the lower respiratory tract, and thus the potential for global spread is a major concern (matrosovich et al., 2004; wang et al., 2016) . h7n9 is another avian influenza a strain that infected more than 130 people and was implicated in 37 deaths. approximately 75% of infected people had a known exposure to birds. there is no evidence of sustained spread between humans but these viruses are of great concern for their pandemic potential (zhang et al., 2013) . the most frequently used animal models of influenza infection include mice, ferrets, and nhps. a very thorough guide to working with mouse, guinea pig, ferret, and cynomolgus models was published by kroeze et al. (2012) . swine are not frequently utilized but are also a potentially useful model for influenza research since they share many similarities to human anatomy, genetics, susceptibility, and pathogenesis (rajao and vincent, 2015). lethality rates can vary with virus strain used (with or without adaptation), dose, route of inoculation, age, and genetic background of the animal. the various animal models can capture differing diseases caused by influenza: benign, severe, super infection, and sepsis, severe with ards, and neurologic manifestations (barnard, 2009) . also, models can utilize seasonal or avian strains and have been developed to study transmission, important for understanding the potential for more lethal strains, such as h5n1 for spreading among humans. mouse models of influenza infection are very predictive for antiviral activity and tissue tropism in humans, and are useful in testing and evaluating vaccines (gilbert and mcleay, 2008; hagenaars et al., 2008; ortigoza et al., 2012) . inoculation is by the in route, utilizing approximately 60 µl of inoculum in each nare of anesthetized mice. exposure may also be to small particle aerosols containing influenza with a mmad of <5 µl. most inbred strains are susceptible, with particularly frequent use of balb/c followed by c57bl/6j mice. males and females have equivalent disease but influenza is generally more infectious in younger 2-to 4-week-old (8-10 g) mice. mice are of somewhat limited use in characterizing the immune response to influenza. most inbred laboratory mice lack the mxa gene which is an important part of human innate immune response to influenza infection. the mouse homolog to mxa, mx1 is defective in most inbred mouse strains (staeheli and haller, 1987) . mice with the knocked-in mx1 gene have a 1000-fold higher ld-50 for an influenza a strain (pr8) than wildtype background c57bl/6 mice (grimm et al., 2007) . weight loss or reduced weight gain, decreased activity, huddling, ruffled fur, and increased respiration are the most common clinical signs in influenza infected mice. for more virulent strains, mice may require euthanasia as early as 48 h postexposure, but most mortality occurs from 5 to 12 days postexposure accompanied by decreases in rectal temperature (sidwell and smee, 2000). pulse oximeter readings and measurement of blood gases for oxygen saturation are also used to determine the impact of influenza infection on respiratory function (sidwell et al., 1992) . virus can be isolated from bronchial lavage (bal) fluids throughout the infection and from tissues after euthanasia. for influenza strains with mild to moderate pathogenicity, disease is nonlethal and virus replication is detected within the lungs, but usually not other organs. increases in serum alpha-1-acidglycoprotein and lung weight also frequently occur. however, mice infected with influenza do not develop fever, dyspnea, nasal exudates, sneezing, or coughing. mice can be experimentally infected with influenza a or b, but the virus generally requires adaptation to produce clinical signs. mice express the receptors for influenza attachment in the respiratory tract; however, the distribution varies and sa 2,3 predominates over sa 2,6 which is why h1, h2, and h3 subtypes usually need to be adapted to mice and h5n1, h2, h6, and h7 viruses do not require adaptation (o'donnell and subbarao, 2011). to adapt, mice are infected intratracheally or intranasally by virus isolated from the lungs, and reinfected into mice and then the process is repeated a number of times. once adapted, influenza strains can produce severe disease, systemic spread, and neurotropism. h5n1 and the 1918 pandemic influenza virus can cause lethal infection in mice without adaptation (gao et al., 1999; taubenberger, 2006) . h5n1 infection of mice results in viremia and viral replication in multiple organ systems, severe lung pathology, fulminant diffuse interstitial pneumonia, pulmonary edema, high levels of proinflammatory cytokines, and marked lymphopenia ( dybing et al., 2000; gubareva et al., 1998; lu et al., 1999) . as in humans, the virulence of h5n1 is attributable to damage caused by an overactive host immune response. additionally, mice infected with the 1918 h1n1 influenza virus produce severe lung pathology and oxygen saturation levels that decrease with increasing pneumonia (barnard et al., 2007) . reassortment influenza viruses of the 2009 h1n1 virus and a low-pathogenicity avian h7n3 virus can also induce disease in mice without adaptation . in superinfection models, a sublethal dose of influenza is given to mice followed 7 days later by in inoculation of a sublethal dose of a bacterial strain, such as s. pneumoniae or s. pyogenes (chaussee et al., 2011) . morbidity, characterized by inflammation in the lungs, but not bacteremia, begins a couple of days after superinfection and may continue for up to 2 weeks. at least one transmission model has also been developed in mice. with h2n2 influenza, transmission rates of up to 60% among cagemates can be achieved after infection by the aerosol route and cocaging after 24 h (schulman, 1968). rats (f344 and sd) inoculated with rat-adapted h3n2 developed inflammatory infiltrates and cytokines in bronchoalveolar lavage fluids, but had no lethality and few histopathological changes (daniels et al., 2003) . additionally, an influenza transmission model has been developed in guinea pigs as an alternative to ferrets (lowen et al., 2006) . cotton rats (sigmodon hispidus) have been used to test vaccines and therapeutics in a limited number of studies (eichelberger et al., 2004) . cotton rats have an advantage over mice in that the immune system is similar to humans (including the presence of the mx gene) and influenza viruses do not have to be adapted (eichelberger et al., 2006; ottolini et al., 2005) . nasal and pulmonary tissues of cotton rats were infected with unregulated cytokines and lung viral load peaking at 24 h postexposure. virus was cleared from the lung by day 3 and from the nares by day 66, but animals had bronchial and alveolar damage, and pneumonia for up to 3 weeks. there is also a s. aureus superinfection model in cotton rats (braun et al., 2007a) . coinfection resulted in bacteremia, high bacterial load in lungs, peribronchiolitis, pneumonitis, alveolitis, hypothermia, and higher mortality. domestic ferrets (mustela putorius furo) are frequently the animal species of choice for influenza animal studies because the susceptibility, clinical signs, peak virus shedding, kinetics of transmission, local expression of cytokine mrnas, and pathology resemble that of humans (lambkin et al., 2004; maines et al., 2012; mclaren and butchko, 1978) . like humans, ferrets exclusively express neu5ac, which acts as a receptor for influenza a virus, a feature likely contributing to the susceptibility of ferrets to human-adapted influenza a virus strains (ng et al., 2014) . the glycomic characterization of ferret respiratory tract tissues demonstrated some similarities and some differences to humans in terms of the potential glycan binding sites for the influenza virus (jia et al., 2014) . ferrets also have airway morphology, respiratory cell types, and a distribution of influenza receptors (sa 2,6 and sa 2,3) within the airways similar to that of humans (van riel et al., 2007) . influenza was first isolated from ferrets infected in with throat washes from humans harboring the infection and ferret models have since been used to test efficacy of vaccines and therapeutic treatments (huber et al., 2008; lambkin et al., 2004; maines et al., 2012) . when performing influenza studies in ferrets, animals should be serologically negative for circulating influenza viruses. infected animals should be placed in a separate room from uninfected animals. if animals must be placed in the same room, uninfected ferrets should be handled before infected ferrets. anesthetized ferrets are experimentally exposed to influenza by in inoculation of 0.25-0.5 ml containing approximately 10 4 -10 6 egg id 50 dropwise to each nostril. however, a larger inoculum volume of 1.0 ml has also been explored as being more appropriate, yielding more severe and consistent respiratory tract pathology, likely because the larger inoculum is more widely distributed in the lower respiratory tract (moore et al., 2014) . video tracking to assign values to activity levels in ferrets can aid ferret studies, eliminating the need for collection of subjective and arbitrary clinical scores (oh et al., 2015) . viral replication in the upper respiratory tract is typically measured by nasal washes, but virus can also be measured in bronchoalveolar lavage fluid using a noninvasive technique (lee et al., 2014) . influenza types a and b naturally infect ferrets, resulting in an acute illness, which usually lasts 3-5 days for mild to moderately virulent strains (maher and destefano, 2004) . ferrets are more susceptible to influenza a than influenza b strains and are also susceptible to avian influenza h5n1 strains without adaptation (zitzow et al., 2002) . however, the localized immune responses within the respiratory tract of ferrets infected with influenza a and b have been characterized and are similar (carolan et al., 2015) . virulence and degree of pneumonitis caused by different influenza subtypes and strains vary from mild to severe and generally mirrors that seen in humans (stark et al., 2013) . nonadapted h1n1, h2n2, and h3n2 have mild to moderate virulence in ferrets. the sequencing of the ferret genome has allowed for the characterization of the ferret host response using rnaseq analysis . distinct signatures were obtained depending on the particular influenza strain to inoculate the ferrets. also helpful is the sequencing and characterization of the influenza ferret infectome during different stages of the infection in naïve or immune ferrets (leon et al., 2013) . since influenza infection is particularly devastating to the elderly population, an aged ferret model of h1n1 influenza infection was developed (paquette et al., 2014) . features associated with increased clinical disease are weakened hemagglutinin antibody generation and attenuated th1 responses. pregnant and breastfeeding women and infants are also susceptible to more severe illness from influenza virus. to study this dynamic, a breastfeeding mother-infant ferret influenza infection model was created (paquette et al., 2015) . notably, the mammary gland itself harbored virus and transcript analysis showed downregulation of milk production genes. in support of the development of therapies, the ferret influenza model for pharmacokinetic/pharmacodynamics studies of antiviral drugs as also been developed (reddy et al., 2015) . critical to this model is ensuring pronounced clinical signs and robust viral replication upon influenza infection. strains of low virulence have predominant replication in the nasal turbinates of ferrets. clinical signs and other disease indicators in ferrets are similar to that of humans with mild respiratory disease, sneezing, nasal secretions containing virus, fever, weight loss, high viral titers, and inflammatory infiltrate in the airways, bronchitis, and pneumonia (svitek et al., 2008) . replication in both the upper and lower airways is associated with more severe disease and greater mortality. additionally, increased expression of proinflammatory mediators and reduced expression of antiinflammatory mediators in the lower respiratory tract of ferrets correlates with severe disease and lethal outcome. h5n1-infected ferrets develop severe lethargy, greater interferon response, transient lymphopenia, and replication in respiratory tract, brain, and other organs (peng et al., 2012; zitzow et al., 2002) . immunocompromised humans have influenza illness of greater duration and complications. immunocompromised ferrets infected with influenza similarly had prolonged virus shedding (van der vries et al., 2013) . interestingly, antiviral resistance emerged in both humans and ferrets with immunocompromised status infected with influenza. alveolar macrophage depleted of ferrets infected with 2009 pandemic h1n1 influenza also had a more severe disease with greater viral replication in the lungs and greater induction of inflammatory chemokines (kim et al., 2013) . a superinfection model resembling that of mice has been developed by in instillation of influenza in 6-to 8-week-old ferrets followed by in inoculation of s. pneumonia 5 days later (peltola et al., 2006) . this typically resulted in otitis media, sinusitis, and pneumonia. transmission models in ferrets have recently met with worldwide media attention and controversy with regard to the study of h5n1 (enserink, 2013; fouchier et al., 2012; herfst et al., 2012; oh et al., 2014) . in general, some subtypes, such as the 2009 h1n1, can transmit easily through aerosol and respiratory droplets (munster et al., 2009) . of concern, h7n9 isolated from humans was more pathogenic and readily transmissible between ferrets by larger respiratory droplets and smaller particle aerosols (kreijtz et al., 2013; richard et al., 2013; zhang et al., 2013) . h5n1 became transmissible by adopting just four mutations, spreading between ferrets in separate cages (imai et al., 2012) . transmission occurs more readily at the height of pyrexia, but for the 2009 h1n1 in particular, can occur before fever is detected (roberts et al., 2012) . ferret-to-ferret transmission of a mouseadapted influenza b virus has also been demonstrated (kim et al., 2015) . since ferrets can be expensive and cumbersome, influenza infection has been characterized and a transmission model developed in the guinea pig; however, this is a newer model with infrequent utilization thus far (lowen et al., 2014) . old and new world primates are susceptible to influenza infection and have an advantage over ferret and mouse models which are deficient for h5n1 vaccine studies because there is a lack of correlation with hemagglutination inhibition (murphy et al., 1980) . of old world primates, cynomolgus macaque (macaca fascicularis) is most frequently utilized for studies of vaccines and antiviral drug therapies (stittelaar et al., 2008) . h5n1 and h1n1 1918 infection of cynos is very similar to humans (rimmelzwaan et al., 2001) . cynos develop fever and ards upon in inoculation of h5n1 with necrotizing bronchial interstitial pneumonia . nhps are challenged by multiple routes k. viral disease (ocular, nasal, and tracheal) simultaneously 1 × 10 6 pfu per site. virus antigen is primarily localized to the tonsils and pulmonary tissues. infection of cynos with h5n1 results in fever, lethargy, nasal discharge, anorexia, weight loss, nasal and tracheal washes, pathologic and histopathologic changes, and alveolar and bronchial inflammation. the 1918 h1n1 caused a very high mortality rate due to an aberrant immune response and ards and had more than 50% lethality (humans only had a 1%-3% lethality) (kobasa et al., 2007) . ards and mortality also occur with the more pathogenic strains, but nhps show reduced susceptibility to less virulent strains, such as h3n2 (o'donnell and subbarao, 2011) . influenza-infected rhesus macaques represent a mild disease model for vaccine and therapeutic efficacy studies (baas et al., 2006) . host microarray and qrt-pcr proved useful for analysis of infected lung tissues. other nhp models include influenza infection of pigtailed macaques as a mild disease model and infection of new world primates, such as squirrel and cebus monkeys (baskin et al., 2009) . domestic pig models have been developed for vaccine studies for swine flu. pigs are susceptible in nature as natural or intermediate hosts but are not readily susceptible to h5n1 (isoda et al., 2006; lipatov et al., 2008) . while pigs infected with influenza may have fever, anorexia, and respiratory signs, such as dyspnea and cough, mortality is rare (van der laan et al., 2008) . size and space requirements make this animal difficult to work with, although the development of minipig models may provide an easier to use alternative. cat and dog influenza models have primarily been utilized to study their susceptibility to h5n1 with the thought that these animals could act as sentinels or could serve to transmit the virus to humans (giese et al., 2008; rimmelzwaan et al., 2006) . these models are not generally used to better understand the disease in humans or for testing vaccines or antivirals. rift valley fever virus (rvfv) causes epizootics and human epidemics in africa. rvfv mainly infects livestock, such as sheep, cattle, goats, etc. after 2-4 days incubation period, animals show signs of fever, hepatitis, and abortion, which is a hallmark diagnostic sign known among farmers (balkhy and memish, 2003) . mosquito vectors, unpasteurized milk, aerosols of infected animal's body fluids, or direct contact with infected animals are the important routes of transmission to humans (abu-elyazeed et al., 1996; mundel and gear, 1951) . after 2-to 6-day-incubation period, rvfv causes a wide range of signs and symptoms in humans ranging from asymptomatic to severe disease with hepatitis, vision loss, encephalitis, and hemorrhagic fever (ikegami and makino, 2011; laughlin et al., 1979; peters and linthicum, 1994) . depending on the severity of the disease when the symptoms start, 10%-20% of the hospitalized patients might die in 3-6 days or 12-17 days after the disease onset (ikegami and makino, 2011) . hepatic failure, renal failure or dic, and encephalitis are demonstrated within patients during postmortem examination. live domestic animals especially sheep and goats were used to develop animal models of rvfv (weingartl et al., 2014) . this study indicated that goats were more resistant to the disease compared to sheep. the viremia in goats was lower and had a shorter duration with only some animals developing fever. the susceptibility is influenced by route of infection, breed of animals, the rvfv strain, and growth conditions as well as the passage history. therefore, it might be difficult to establish an animal model with domestic ruminants. mice are one of the most susceptible animal species to rvfv infection. several mouse models including balb/c, ifnar −/− , mbt/pas, 129 and c57bl/6 were exposed to rvfv via parental or aerosol routes of infection (ross et al., 2012) . subq or ip routes of infection cause acute hepatitis and lethal encephalitis at a late stage of the disease in mice (mims, 1956; smith et al., 2010) . mice start to show signs of decreased activity and ruffled fur by day 2-3 postexposure. immediately following these signs, they become lethargic and generally die 3-6 days postexposure. ocular disease or the hemorrhagic form of the disease has not been observed in mouse models so far (ikegami and makino, 2011) . increased viremia and tissue tropism were reported in mice with (smith et al., 2010) increased liver enzymes and lymphopenia observed in sick mice. aerosolized rvfv causes faster and more severe neuropathy in mice compared to the parental route (dodd et al., 2014; reed et al., 2014) . the liver is a target organ following aerosol exposure and liver failure results in fatality. rats and gerbils are also susceptible to rvfv infection. the rat's susceptibility is dependent on the rat strain utilized for the challenge model and route of exposure. there is also noted age dependence in the susceptibility of rats. while wistar-furth and brown norway strains, and young rats are highly susceptible to rvfv infection, fisher 344, buffalo and lewis strains, and old rats demonstrated resistance to infection via subq route of infection (findlay and howard, 1952; peters and slone, 1982) . similar pathologic changes, such as liver damage and encephalopathy were observed in both rats and mice. the recent study by bales et al. (2012) showed that aerosolized rvfv caused similar disease outcome in wistar-furth and aci rats while lewis rats developed fatal encephalitis which was much more severe than the subq route of infection. there was no liver involvement in the gerbil model and animals died from severe encephalitis. the mortality rate was dependent on the strain used and the dose given to gerbils (anderson et al., 1988) . similar to the rat model, the susceptibility of gerbils was also dependent on age. natural history studies with syrian hamsters indicated that the liver was the target organ with highly elevated alt levels and viral titers (scharton et al., 2015) . lethargy, ruffled fur, and hunched posture were observed in hamsters by day 2 post-subq inoculation and the disease was uniformly lethal by day 2-3 postexposure. this model has been successfully used to test antivirals against rvfv (scharton et al., 2015) . studies thus far showed that rvfv does not cause uniform lethality in a nhp model. ip, in, iv, and aerosol routes have been utilized to develop nhp model. rhesus macaques, cynomolgus macaques, african monkeys, and south american monkeys were some of the nhp species used for this effort . monkeys showed a variety of signs ranging from febrile disease to hemorrhagic disease and mortality. temporal viremia, increased coagulation parameters (pt, aptt), and decreased platelets were some other signs observed in nhps. animals that succumbed to disease showed very similar pathogenesis to humans, such as pathological changes in the liver and hemorrhagic disease. there was no ocular involvement in this model. smith et al. compared iv, in and subq routes of infection in common marmosets and rhesus macaques (peng et al., 2012) . marmosets were more susceptible to rvfv infection than rhesus macaques with marked viremia, acute hepatitis, and late onset of encephalitis. increased liver enzymes were observed in both species. necropsy results showed enlarged livers in the marmosets exposed by iv or subq routes. although there were no gross lesions in the brains of marmosets, histopathology showed encephalitis in the brains of in challenged marmosets. a recent study by hartman et al. (2014) demonstrated that aerosolized rvfv only caused mild fever in cynomolgus macaques and rhesus macaques, while agms and marmosets had encephalitis and succumbed to disease between days 9 and 11 postexposure. in contrast to other lethal models, the brain was the target organ in agms and marmosets. although no change was observed in ast levels, alp levels were increased in marmosets. little or no change was observed in hepatic enzyme levels in agms. lack of information regarding human disease concerning the aerosol route of exposure makes it difficult to evaluate these animal models. crimean-congo hemorrhagic fever virus (cchfv) generally circulates in nature unnoticed in an enzootic tick-vertebrate-tick cycle and similar to other zoonotic agents, appears to produce little or no disease in its natural hosts, but causes severe disease in humans. cchfv transmits to humans by ixodid ticks, direct contact with sick animals/humans, or body fluids of animals/humans (ergonul and whitehouse, 2007) . incubation, prehemorrhagic, hemorrhagic, and convalescence are the four phases of the disease seen in humans. the incubation period lasts 1-9 days. during the prehemorragic phase, patients show signs of nonspecific flu-like disease for approximately a week. the hemorrhagic period results in circulatory shock and dic in some patients (mardani and keshtkar-jahromi, 2007; swanepoel et al., 1989) . over the years, several attempts have been made to establish an animal model for cchf in adult mice, guinea pigs, hamsters, rats, rabbits, sheep, nhps, etc. (fagbami et al., 1975; nalca and whitehouse, 2007; shepherd et al., 1989; smirnova, 1979) . until recently, the only animal that manifests disease is the newborn mouse. infant mice ip infected with cchfv resulted in fatality around day 8 postinfection (tignor and hanham, 1993) . pathogenesis studies showed that virus replication was first detected in the liver, with subsequent spread to the blood (serum). virus was detected very late during the disease course in other tissues including the heart (day 6) and the brain (day 7). the recent studies utilizing knockout adult mice were successful to develop a lethal small animal model for cchfv infection (bente et al., 2010; bereczky et al., 2010) . bente et al. infected stat1 knockout mice by the ip route. in this model, after the signs of fever, leukopenia, thrombocytopenia, viremia, elevated liver enzymes and proinflammatory cytokines, mice were moribund and succumbed to disease in 3-5 days postexposure. the second model was developed by using interferon alpha/beta (ifnα/β) receptor knockout mice (ifnar −/− ) (bereczky et al., 2010) . similar observations were made in this model as in the stat1 knockout mouse model. animals were moribund and died 2-4 days after exposure with high viremia levels in liver and spleen. characterization studies with ifnar −/− mice challenged with different routes (ip, in, im, and subq) showed that cchfv causes acute disease with high viral loads, pathology in liver and lymphoid tissues, increased proinflammatory response, severe thrombocytopenia, coagulopathy, and death, all of which are characteristics of human disease . proinflammatory cytokines and chemokines, such as g-csf, ifnγ, cxc-cl10, ccl2 increased dramatically day 3 postchallenge and gm-csf, il-1a, il-1b, il-2, il-6, il-12p70, il-13, il-17, cxcl1, ccl3, ccl5, and tnf-α concentrations were extremely elevated at the time of death/euthanasia. this model is also utilized to test therapeutics, such as ribavirin, arbidol, and t-705 (favipiravir) successfully (oestereich et al., 2014) . experimental vaccines developed for cchf were evaluated in this model provided protection compare to unvaccinated mice (buttigieg et al., 2014; canakoglu et al., 2015, p. 725) . thus, the ifnar −/− mouse model would be a good choice to test medical countermeasures against cchfv, although they have an impaired ifn and immune response phenotype. other laboratory animals, including nhps, show little or no sign of infection or disease when infected with cchfv (nalca and whitehouse, 2007) . butenko et al. utilized agms (cercopithecus aethiops) for experimental cchfv infections. except one monkey with a fever on day 4 postinfection, the animals did not show signs of disease. antibodies to the virus were detected in three out of five monkeys, including the one with fever. fagbami et al. (1975) infected two patas monkeys (erythrocebus patas) and one guinea baboon (papio papio) with cchfv. whereas all three animals had low-level viremia between days 1 and 5 after inoculation, only the baboon serum had neutralizing antibody activity on day 137 postinfection. similar results were obtained when horses and donkeys have been used for experimental cchfv infections. donkeys develop a low-level viremia (rabinovich et al., 1972) and horses developed little or no viremia, but high levels of virus-neutralizing antibodies, which remained stable for at least 3 months. these studies suggest that horses may be useful in the laboratory to obtain serum for diagnostic and possible therapeutic purposes (blagoveshchenskaya et al., 1975 (blagoveshchenskaya et al., ). shepherd et al. (1989 infected 11 species of small african wild mammals and laboratory rabbits, guinea pigs, and syrian hamsters with cchfv. whereas scrub hares (lepus saxatilis), cape ground squirrels (xerus inauris), red veld rats (aethomys chrysophilus), white-tailed rats (mystromys pumilio), and guinea pigs had viremia; south african hedgehogs (atelerix frontalis), highveld gerbils (gerbilliscus brantsii), namaqua gerbils (desmodillus auricularis), two species of multimammate mouse (mastomys natalensis and mastomys coucha), and syrian hamsters were negative for virus. all species regardless of viremia levels developed antibody responses against cchfv. iv and intracranially infected animals showed onset of viremia earlier than those infected by the subq or ip routes. the genus hantavirus is unique among the family bunyaviridae in that it is not transmitted by an arthropod vector, but rather rodents (schmaljohn and nichol, 2007) . rodents of the family muridae are the primary reservoir for hantaviruses. infected host animals develop a persistent infection that is typically asymptomatic. transmission is achieved by inhalation of infected rodent saliva, feces, and urine (xu et al., 1985) . human infections can normally be traced to a rural setting with activities, such as farming, land development, hunting, and camping as possible sites of transmission. rodent control is the primary route of prevention (lednicky, 2003) . the viruses have a tropism for endothelial cells within the microvasculature of the lungs (zaki et al., 1995) . there are two distinct clinical diseases that infection can yield: hemorrhagic fever with renal syndrome (hfrs) due to infection with old world hantaviruses or hantavirus pulmonary syndrome (hps) caused by new world hantaviruses (nichol, 2001) . hfrs is mainly seen outside of the americas and is associated with the hantaviruses dobrava-belgrade (also known as dobrava), hantaan, puumala, and seoul (lednicky, 2003) . incubation lasts 2-3 weeks and presents as flu-like in the initial stages that can further develop into hemorrhagic manifestations and ultimately renal failure. thrombocytopenia subsequently develops which can further progress to shock in approximately 15% patients. overall mortality rate is 7%. infection with dobrava and hantaan viruses are typically linked to development of severe disease. hps was first diagnosed in 1993 within southwestern united states when healthy young adults became suddenly ill, progressing to severe respiratory distress and shock. the etiological agent responsible for this outbreak was identified as sin nombre virus (snv) (centers for disease control and prevention, 1993) . this virus is still the leading cause within north america of hps. hps due to other hantaviruses has been reported in argentina, bolivia, brazil, canada, chile, french guiana, panama, paraguay, and uruguay (padula et al., 2000; stephen et al., 1994) . the first report of hps in maine was recently documented (centers for disease control and prevention, 1993). andes virus (andv) was first identified in outbreaks in chile and argentina. this hantavirus is distinct in that it can be transmitted between humans (wells et al., 1997) . the fulminant disease is more lethal than that observed of hfrs with a mortality rate of 40%. there are four phases of disease including prodromal, pulmonary, cardiac depression, and hematologic manifestation (peters and khan, 2002) . incubation typically occurs 14-17 days following exposure (young et al., 2000) . unlike hfrs, renal failure is not a major contributing factor to the disease. there is a short prodromal phase that gives way to cardiopulmonary involvement accompanied by cough and gastrointestinal symptoms. it is at this point that individuals are typically admitted to the hospital. pulmonary function is hindered and continues to suffer within 48 h after cardiopulmonary involvement. interstitial edema and air-space disease normally follow. in fatal cases, cardiogenic shock has been noted (hallin et al., 1996) . syrian golden hamsters are the most widely utilized small animal model for hantavirus infection. hamsters inoculated im with a passaged andes viral strain died within 11 days postinfection. clinical signs did not appear until 24 h prior to death at which point the hamsters were moribund and in respiratory distress. mortality was dose dependent, with high inoculums leading to a shorter incubation before death. during the same study, hamsters were inoculated with a passaged snv isolate. no hamsters developed any symptoms during the course of observation. however, an antibody response to the virus that was not dose dependent was determined via elisa. hamsters infected with andv have significant histopathological changes to their lung, liver, and spleen. all had an interstitial pneumonia with intraalveolar edema. infectious virus could be recovered from these organs. viremia began on day 8 and lasted up to 12 days postinfection. infection of hamsters with andv yielded a similar clinical disease progression as is seen in human hps including rapid progression to death, fluid in the pleural cavity, and significant histopathological changes to the lungs and spleen. a major deviation in the hamster model is the detection of infectious virus within the liver . normally, snv does not cause a disease in hamsters (wahl-jensen et al., 2007) . but a recent study showed that immunosuppression with dexamethasone and cyclophosphamide in combination causes lethal disease with snv in hamsters (brocato et al., 2014) . the disease was very similar to the disease caused by andv in hamsters. lethal disease can be induced in newborn mice, but does not recapitulate the clinical symptoms observed in human disease (kim and mckee, 1985) . the disease outcome is very much dependent on the age of the mice. younger mice are much more susceptible to virus than the adult mice. adult mice exposed to hanta virus leads to a fatal disease dependent upon viral strain and route of infection. the disease progression is marked by neurological or pulmonary manifestations that do not mirror human disease (seto et al., 2012; wichmann et al., 2002) . knockout mice lacking ifnα/β are highly susceptible to hanta virus infection (muller et al., 1994) . in a study of panel of laboratory strains of mice, c57bl/6 mice were most susceptible to a passaged hanta viral strain injected ip. animals progressed to neurological manifestation including paralyses and convulsions, and succumbed to infection within 24-36 h postinfection. clinical disease was markedly different from that observed in human cases (wichmann et al., 2002) . in a recent study, 2-weekold icr mice was exposed to htnv strain aa57 via the subq route (seto et al., 2012) . mice started to show signs of disease by day 11 postinoculation. piloerection, trembling, hunching, loss of body weight, labored breathing, and severe respiratory disease were observed in mice. studies to develop nhp models were not successful until recently. nhps have been challenged with new world hantaviruses; however, no clinical signs were reported (hooper et al., 2006; mcelroy et al., 2002) . cynomolgus monkeys challenged with a clinical isolate of puumala virus developed a mild disease (klingstrom et al., 2002; sironen et al., 2008) . challenge with andv to cynomolgus macaques by both iv and aerosol exposure led to no signs of disease. all animals did display a drop in total lymphocytes within 5 days postinfection. four of six aerosol exposed monkeys and 8 of 11 iv injected monkeys developed viremia. infectious virus could not be isolated from any of the animals. in a recent study, rhesus macaques were inoculated by the intramuscular route with snv passaged only in deer mice (safronetz et al., 2014) . characteristics of hps disease including rapid onset of respiratory distress, severe pulmonary edema, thrombocytopenia, and leukocytosis were observed in this promising model. viremia was observed 4-10 days prior to respiratory signs of the disease that were observed on days 14-16 postinoculation. with all aspects, this animal model would be very useful to test medical countermeasures against hanta virus. the family arenaviridae is composed of two serogroups: old world arenaviruses including lassa fever virus and lymphocytic choriomeningitis virus and the new world viruses of pichinde virus and junin virus. all of these viruses share common clinical manifestations (mccormick and fisher-hoch, 2002) . lassa fever virus is endemic in parts of west africa and outbreaks are typically seen in the dry season between january and april (curtis, 2006) . this virus is responsible for 100,000-500,000 infections per year, leading to approximately 5000 deaths (khan et al., 2008) . outbreaks have been reported in guinea, sierra leone, liberia, nigeria, and central african republic. however, cases have sprung up in germany, netherlands, united kingdom, and the united states due to transmission to travelers on commercial airlines (amorosa et al., 2010) . transmission of this virus typically occurs via rodents, in particular the multimammate rat, mastomys species complex (curtis, 2006) . humans become infected by inhaling the aerosolized virus or eating contaminated food. there has also been noted human-to-human transmission by direct contact with infected secretions or needle-stick injuries. the majority of infections are asymptomatic; however, severe disease occurs in 20% of individuals. the incubation period is from 5 to 21 days and initial onset is characterized by flu-like illness. this is followed by diarrheal disease that can progress to hemorrhagic symptoms including encephalopathy, encephalitis, and meningitis. a third of patients develop deafness in the early phase of disease that is permanent for a third of those affected. the overall fatality is about 1%; however, of those admitted to the hospital it is between 15% and 25%. there is no approved vaccine and besides supportive measures, ribavirin is effective only if started within 7 days (mccormick et al., 1986a,b) . the primary animal model used to study lassa fever is the rhesus macaque (jahrling et al., 1980) . aerosolized infection of lymphocytic choriomeningitis virus has been a useful model for lassa fever. both rhesus and cynomolgus monkeys exposed to the virus developed disease, but rhesus mirrored more closely the disease course and histopathology observed in human infection (danes et al., 1963) . iv or intragastric inoculation of the virus led to severe dehydration, erythematous skin, submucosal edema, necrotic foci in the buccal cavity, and respiratory distress. the liver was severely affected by the virus as depicted by measuring the liver enzymes ast and alt (lukashevich et al., 2003) . disease was dose dependent with iv, intramuscular, and subq inoculation requiring the least amount of virus to induce disease. aerosol infections and eating contaminated food could also be utilized, and mimic a more natural route of infection (peters et al., 1987) . within this model, the nhp becomes viremic after 4-6 days. clinical manifestations were present by day 7 and death typically occurred within 10-14 days (lukashevich et al., 2004; rodas et al., 2004) . intramuscular injection of lassa virus into cynomolgus monkeys also produced a neurological disease due to lesions within the cns (hensley et al., 2011b) . this pathogenicity is seen in select cases of human lassa fever (cummins et al., 1992; gunther et al., 2001) . a marmoset model has recently been defined utilizing a subq injection of lassa fever virus. virus was initially detected by day 8 and viremia achieved by day 14. liver enzymes were elevated and an enlarged liver was noted upon autopsy. there was a gradual reduction in platelets and interstitial pneumonitis diagnosed in a minority of animals. the physiological signs were the same as seen in fatal human cases (carrion et al., 2007) . mice develop a fatal neurological disorder upon intracerebral inoculation with lassa, although the outcome of infection is dependent on the mhc background, age of the animal, and inoculation route (salvato et al., 2005) . stat1 knockout mice inoculated ip with both lethal and nonlethal lassa virus strains develop hearing loss accompanied by damage to the inner ear hair cells and auditory nerve (yun et al., 2015) . guinea pig inbred strain 13 was highly susceptible to lassa virus infection. the outbred hartley strain was less susceptible, and thus strain 13 has been the preferred model given its assured lethality. the clinical manifestations mirror those seen in humans and rhesus (jahrling et al., 1982) . infection with pichinde virus passaged in guinea pigs has also been used. disease signs include fever, weight loss, vascular collapse, and eventual death (lucia et al., 1990; qian et al., 1994) . the guinea pig is an excellent model given that it not only results in similar disease pattern, viral distribution, histopathology, and immune response to humans (connolly et al., 1993; katz and starr, 1990) . infection of hamsters with a cotton rat isolate of pirital virus is similar to what is characterized in humans, and the nhp and guinea pig models. the virus was injected ip resulting in lethargy and anorexia within 6-7 days. virus was first detected at 3 days, and reached maximum titers within 5 days. neurological symptoms began to appear at the same time, and all animals died by day 9. pneumonitis, pulmonary hemorrhage, and edema were also present (sbrana et al., 2006) . these results were recapitulated with a nonadapted pichinde virus (buchmeier and rawls, 1977; gowen et al., 2005; smee et al., 1993) . the lentiviruses are a subfamily of retroviridae, which includes human immunodeficiency virus (hiv), a virus that infects 0.6% of the world's population. a greater proportion of infections and deaths occur in subsaharan africa. worldwide, there are approximately 1.8 million deaths per year with over 260,000 being children. transmission of hiv occurs by exposure to infectious body fluids. there are two species, hiv-1 and hiv-2, with hiv-2 having lower infectivity and virulence (confined mostly to west africa). the vast majority of cases worldwide are hiv-1 (de cock et al., 2011) . hiv targets t-helper cells (cd4+), macrophages, dendritic cells (fields et al., 2007) . acute infection occurs 2-4 weeks after exposure, with flu-like symptoms and viremia followed by chronic infection. symptoms in the acute phase may include fever, body aches, nausea, vomiting, headache, lymphadenopathy, pharyngitis, rash, and sores in the mouth or esophagus. cd8+ t-cells are activated which kill hiv-infected cells, and are responsible for antibody production and seroconversion. acquired immune deficiency syndrome (aids) develops when cd4+ t-cells decline to less than 200 cells/µl; thus cell-mediated immunity becomes impaired and the person is more susceptible to opportunistic infections as well as certain cancers. hiv has a narrow host range likely because the virus is unable to antagonize and evade effector molecules of the interferon response (thippeshappa et al., 2012) . humanized mice, created by engrafting human cells and tissues into scid mice, have been critical for the development of mouse models for the study of hiv infection. a number of different humanized mouse models allow for the study of hiv infection in the context of an intact and functional human innate and adaptive immune responses (berges and rowan, 2011) . the scidhu hiv infection model has proven useful, particularly in screening antivirals and therapeutics (denton et al., 2008; melkus et al., 2006) . a number of different humanized mouse models have been developed for the study of hiv, including rag1 −/− γc −/− , rag2 −/− γc −/− , nod/scidγc −/− (hnog), nod/scidγc −/− (hnsg), nod/scid blt, and nod/scidγc −/− (hnsg) blt (karpel et al., 2015; li et al., 2015; shimizu et al., 2015) . cd34+ human stem cells derived from umbilical cord blood or fetal liver are used for humanization (baenziger et al., 2006; watanabe et al., 2007) . hiv-1 infection by ip injection can be successful with as little as 5% peripheral blood engraftment (berges et al., 2006) . vaginal and rectal transmission models have been developed in blt scid hu mice in which mice harbor human bone marrow, liver, and thymus tissue. hiv-1 viremia occurs within approximately 7 days postinoculation . in many of these models, spleen, lymph nodes, and thymus tissues are highly positive for virus, similar to humans (brainard et al., 2009) . importantly, depletion of human t-cells can be observed in blood and lymphoid tissues of hivinfected humanized mice and at least some mechanisms of pathogenesis that occur in hiv-infected humans, also occur in the hiv-infected humanized mouse models (baenziger et al., 2006; neff et al., 2011) . the advantage of these models is that these mice are susceptible to hiv infection and thus the impact of drugs on the intended viral targets can be tested. one caveat is that while mice have a "common mucosal immune system," humans do not, due to differences in the distribution of addressins (holmgren and czerkinsky, 2005) . thus, murine mucosal immune responses to hiv do not reflect those of humans. another strategy uses a human cd4-and human ccr5-expressing transgenic luciferase reporter mouse to study hiv-1 pseudovirus entry (gruell et al., 2013) . hiv-1 transgenic (tg) rats are also used to study hiv related pathology, immunopathogenesis, and neuropathology (lentz et al., 2014; reid et al., 2001) . the clinical signs include skin lesions, wasting, respiratory difficulty, and neurological signs. brain volume decreases have been documented and the hiv-1 tg rat is thus used as a model of neuropathology in particular. there are a number of important nhp models for human hiv infection (hessell and haigwood, 2015) . an adaptation of hiv-1 was obtained by four passages in pigtailed macaques transiently depleted of cd8(+) cells during acute infection (hatziioannou et al., 2014) . the resulting disease has several similarities to aids in humans, such as depletion of cd4(+) t-cells (kimata, 2014) . simian immunodeficiency virus (siv) infection of macaques has been widely used as a platform for modeling hiv infection of humans (demberg and robert-guroff, 2015; walker et al., 2015) . importantly, nhps have similar, pharmacokinetics, metabolism, mucosal tcell homing receptors, and vascular addressins to those of humans. thus, while the correlates of protection against hiv are still not completely known, immune responses to hiv infection and vaccination are likely comparable. these models mimic infection through use of contaminated needles (iv), sexual transmission (vaginal or rectal), and maternal transmission in utero or through breast milk (keele et al., 2009; miller et al., 2005; stone et al., 2009) . there are also macaque models to study the emergence and clinical implications of hiv drug resistance (van rompay et al., 2002) . these models most routinely utilize rhesus macaques (macaca mulatta), cynomolgus macaques (m. fasicularis), and pigtailed macaques (macaca nemestrina). all ages are used, depending on the needs of the study. for instance, use of newborn macaques may be more practical for evaluating the effect of prolonged drug therapy on disease progression; however, adult nhps are more frequently employed. female pigtailed macaques have been used to investigate the effect of the menstrual cycle on hiv susceptibility (vishwanathan et al., 2015) . studies are performed in bsl-2 animal laboratories and nhps must be simian type-d retrovirus free and siv seronegative. siv infection of pigtailed macaques is a useful model for hiv peripheral nervous system pathology, wherein an axotomy is performed and regeneration of axons is studied (ebenezer et al., 2012) . exposure in model systems is typically through a single high-dose challenge. iv infection of rhesus macaques with 100 tcid 50 of the highly pathogenic siv/ deltab670 induces aids in most macaques within 5-17 months (mean of 11 months) (fuller et al., 2012) . peak viremia occurs around week 4. aids in such models is often defined as cd4+ t-cells that have dropped to less than 50% of the baseline values. alternatively, repeated low dose challenges are often utilized, depending on the requirements of the model (henning et al., 2014; moldt et al., 2012; reynolds et al., 2012) . since nhps infected with hiv do not develop an infection with a clinical disease course similar to humans, siv or siv/hiv-1 laboratory-engineered chimeric viruses (shivs) are used as surrogates. nhps infected with pathogenic siv may develop clinical disease which progresses to aids, and are thus useful pathogenesis models. a disadvantage is that siv is not identical to hiv-1 and is more closely related to hiv-2. however, the polymerase region of siv is 60% homologous to that of hiv-1 and it is susceptible to many reverse transcriptase (rt) and protease inhibitors. siv is generally not susceptible to nonnucleoside inhibitors, thus hiv-1 rt is usually put into siv for such studies (uberla et al., 1995) . sivmac239 is similar to hiv in the polymerase region and is therefore susceptible to nucleoside, rt, or integrase inhibition (witvrouw et al., 2004) . nhps infected with sivmac239 have an asymptomatic period and disease progression resembling aids in humans, characterized by weight loss/wasting, cd4+ t-cell depletion. additionally, sivmac239 utilizes the cxcr5 chemokine receptor as a coreceptor, similar to hiv, which is important for drugs that target entry (veazey et al., 2003) . nhps infected with shiv strains, may not develop aids, but these models are useful in testing vaccine efficacy . for example, rt-shivs and env-shivs are useful for testing and evaluation of drugs that may target the envelope or rt, respectively (uberla et al., 1995) . one disadvantage of the highly virulent env-shiv (shiv-89.6 p), is that it uses the cxcr4 coreceptor. of note, env-shivs that do use the cxcr5 coreceptor are less virulent; viremia develops then resolves without further disease progression (humbert et al., 2008) . simian-tropic (st) hiv-1 contains the vif gene from siv. infection of pigtailed macaques with this virus results in viremia, which can be detected for 3 months, followed by clearance (haigwood, 2009) . a number of routes are utilized for siv or shiv infection of nhps, with iv inoculation the most common route. mucosal routes include vaginal, rectal, and intracolonic. mucosal routes require a higher one-time dose than the iv route for infection. for the vaginal route, female macaques are treated with depo-provera (estrogen) 1 month before infection to synchronize the menstrual cycle, thin the epithelial lining of the vagina, and increase susceptibility to infection by atraumatic vaginal instillation (burton et al., 2011) . upon vaginal instillation of 500 tcid 50 of shiv-162p3, peak viremia was seen around 12 days postexposure with greater than 10 7 copies/ml and dropping thereafter to a constant level of 10 4 rna copies/ml at 60 days and beyond. in another example, in an investigation of the effect of vaccine plus vaginal microbicide on preventing infection, rhesus macaques were vaginally infected with a high dose of sivmac251 (barouch et al., 2012) . an example of an intrarectal model utilized juvenile (2-year-old) pigtailed macaques, challenged intrarectally with 10 4 tcid 50s of siv mne027 to study the pathogenesis related to the virulence factor, vpx (belshan et al., 2012) . here, viremia peaked at approximately 10 days with more than 10 8 copies/ml. viral rna was expressed in the cells of the mesenteric lymph nodes. the male genital tract is seen as a viral sanctuary with persistent high levels of hiv shedding even with antiretroviral therapy. to better understand the effect of haart therapy on virus and t-cells in the male genital tract, adult (3-to 4-year-old) male cynomolgus macaques were intravenously inoculated with 50 aid50s of sivmac251 and the male genital tract tissues were tested after euthanasia by pcr, ihc, and in situ hybridization (moreau et al., 2012) . pediatric models have been developed in infant rhesus macaques through the infection of siv, allowing for the study of the impact of developmental and immunological differences on the disease course (abel, 2009) . importantly, mother-to-infant transmission models have also been developed (jayaraman et al., 2004) . pregnant female pigtailed macaques were infected during the second trimester with 100 mid 50 shiv-sf162p3 by the iv route. four of nine infants were infected, one in utero and three either intrapartum or immediately postpartum through nursing. this model is useful for the study of factors involved in transmission as well as the underlying immunology. nhps infected with siv or shiv are routinely evaluated for weight loss, activity level, stool consistency, appetite, virus levels in blood, and t-cell populations. cytokine and chemokine levels, antibody responses, and cytotoxic t-lymphocyte responses may also be evaluated. the ultimate goal of an hiv vaccine is sterilizing immunity (preventing infection). however, a more realistic result may be to reduce severity of infection and permanently prevent progression. strategies have included live attenuated, nonreplicating, and subunit vaccines. these have variable efficacy in nhps due to the genetics of the host (mhc and trim alleles), differences between challenge strains, and challenge routes (letvin et al., 2011) . nhp models have led to the development of antiviral treatments that are effective at reducing viral load and indeed transmission of hiv among humans. one preferred variation on the models for testing the long-term clinical consequences of antiviral treatment is to use newborn macaques and treat from birth onward, in some cases more than a decade (van rompay et al., 2008) . unfortunately, however, successes in nhp studies do not always translate to success in humans, as seen with the recent step study which used an adenovirus-based vaccine approach (buchbinder et al., 2008) . vaccinated humans were not protected and may have even been more susceptible to hiv, viremia was not reduced, and the infections were not attenuated as hoped. with regard to challenge route, iv exposure is more difficult to protect than mucosal exposure and is used as a "worst case scenario." however, efficacy at one mucosal route is usually comparable to other mucosal routes. human and animal papillomaviruses cause benign epithelial proliferations (warts) and malignant tumors of the various tissues that they infect (bosch and de sanjose, 2002) . there are over 100 human papillomaviruses, with different strains causing warts on the skin, oropharynx, nasopharynx, larynx, and anogenital tissues. approximately one third of papillomaviruses are transmitted sexually. of these, virulent subtypes, such as hpv-16, hpv-18, hpv-31, hpv-33, and hpv-45 place individuals at high risk for cervical and other cancers. up to 35% of head and neck cancers are caused by hpv-16, particularly oropharyngeal cancers. major challenges in the study of these viruses are that papillomaviruses generally do not infect any other species outside of the natural hosts and can cause a very large spectrum of severity. thus, no wild-type animal models have been identified that are susceptible to hpv. however, a number of useful surrogate models exist which use animal papillomaviruses in their natural host or a very closely related species (borzacchiello et al., 2009; brandsma, 1994; campo, 2002) . these models have facilitated the recent development of useful and highly effective prophylactic hpv vaccines (rabenau et al., 2005) . wild-type inbred mice cannot be used to study disease caused by papillomaviruses unless they are engrafted with relevant tissue, orthotopically transplanted or transgenic, but they are often used to look at immunogenicity of vaccines (jagu et al., 2011; oosterhuis et al., 2011) . transgenic mice used for hpv animal modeling typically express the viral oncogenes e5, e6, e7, or the entire early region of hpv-16 from the keratin 14 promoter which is only active in the basal cells of the mouse epithelium (chow, 2015) . cancers in these models develop upon extended estrogen exposure (maufort et al., 2010; ocadiz-delgado et al., 2009; stelzer et al., 2010; thomas et al., 2011) . transgenic mice with constitutively active wnt/b-catenin signaling in cervical epithelial cells expressing the hpb oncoprotein e7 develop invasive cervical squamous carcinomas (bulut and uren, 2015) . the tumors occur within 6 months approximately 94% of the time. another model uses c57bl/6 mice expressing the hpv16-e7 transgene which are then treated topically with 7,12-dimethylbenz(a)anthracene (dmba) (de azambuja et al., 2014) . these mice developed benign and malignant cutaneous lesions. cervical cancers can also be induced in human cervical cancer xenografts transplanted onto the flanks of athymic mice and serially transplanted thereafter ( hiroshima et al., 2015; siolas and hannon, 2013) . a wild-type immunocompetent rodent model uses m. coucha, which is naturally infected with mastomys natalensis papillomavirus (mnpv) (vinzon et al., 2014) . mnpv induces papillomas, keratoacanthomas, and squamous cell carcinomas and provides a means to study vaccination in an immunocompetent small animal model. wild cottontail rabbits (sylvilagus floridanus) are the natural host for cottontail rabbit papillomavirus (crpv), but this virus also infects domestic rabbits (oryctolagus cuniculus), which is a very closely related species ( breitburd et al., 1997) . in this model, papillomas can range from cutaneous squamous cell carcinomas on one end of spectrum, and spontaneous regression on the other. lesions resulting from crpv in domestic rabbits do not typically contain infectious virus. canine oral papillomavirus (copv) causes florid warty lesions in mucosa of the oral cavity within 4-8 weeks postexposure in experimental settings (johnston et al., 2005) . the mucosatrophic nature of these viruses and the resulting oropharyngeal papillomas that are morphologically similar to human vaginal papillomas caused by hpv-6 and hpv-11 make this a useful model (nicholls et al., 1999) . these lesions typically spontaneously regress 4-8 weeks after appearing; this model is therefore useful in understanding the interplay between the host immune defense and viral pathogenesis. male and female beagles, aged 10 weeks to 2 years, with no history of copv, are typically used for these studies. infection is achieved by application of a 10 µl droplet of virus extract to multiple 0.5 cm 2 scarified areas within the mucosa of the upper lip of anesthetized beagles (nicholls et al., 2001) . some investigators have raised concerns that dogs are not a suitable model for high-risk hpv-induced oral cancer (staff, 2015) . bovine papillomavirus (bpv) has a wider host range than most papillomaviruses, infecting the fibroblasts cells of numerous ungulates (campo, 2002) . bpv-4 infection of cattle feeding on bracken fern, which is carcinogenic, can result in lesions of the oral and esophageal mucosa that lack detectable viral dna. bpv infections in cattle can result in a range of diseases, such as skin warts, cancer of the upper gastrointestinal tract and urinary bladder, and papillomatosis of the penis, teats, and udder. finally, rhesus papillomavirus (rhpv), a sexually transmitted papillomaviruses in rhesus macaques and cynomolgus macaques is very similar to hpv-16 and is associated with the development of cervical cancer ( ostrow et al., 1990; wood et al., 2007) . monkeypox virus (mpxv) causes disease in both animals and humans. human monkeypox, which is clinically almost identical to ordinary smallpox, occurs mostly in the rainforest of central and western africa. the virus is maintained in nature in rodent reservoirs including k. viral disease squirrels (charatan, 2003; khodakevich et al., 1986) . mpxv was discovered during the pox-like disease outbreak among laboratory monkeys (mostly cynomolgus and rhesus macaques) in denmark in 1958. no human cases were observed during this outbreak. the first human case was not recognized as a distinct disease until 1970 in zaire (the present drc) with continued occurrence of a smallpox-like illness despite eradication efforts of smallpox in this area. during the global eradication campaign, extensive vaccination in central africa decreased the incidence of human monkeypox, but the absence of immunity in the generation born since that time and increased dependence on bush meat have resulted in renewed emergence of the disease. in the summer of 2003, a well-known outbreak in the midwest was the first occurrence of monkeypox disease in the united states and western hemisphere. among 72 reported cases, 37 human cases were laboratory confirmed during an outbreak (nalca et al., 2005; sejvar et al., 2004) . it was determined that native prairie dogs (cynomys sp.) housed with rodents imported from ghana in west africa were the primary source of outbreak. the virus is mainly transmitted to humans while handling infected animals or by direct contact with the infected animal's body fluids, or lesions. person-to-person spread occurs by large respiratory droplets or direct contact (jeézek and fenner, 1988) . most of the clinical features of human monkeypox are very similar to those of ordinary smallpox (breman and arita, 1980) . after a 7-to 21-dayincubation period, the disease begins with fever, malaise, headache, sore throat, and cough. the main sign of the disease that distinguishes monkeypox from smallpox is swollen lymph nodes (lymphadenitis), which is observed in most of the patients before the development of rash (di giulio and eckburg, 2004; jeézek and fenner, 1988) . a typical maculopapular rash follows the prodromal period, generally lasting 1-3 days. the average size of the skin lesions are 0.5-1 cm and the progress of lesions follows the order: macules, papules, vesicles, pustules, umblication then scab, and desquamation and lasts typically 2-4 weeks. the fatality rate is 10% among the unvaccinated population and death generally occurs during the 2nd week of the disease (jeézek and fenner, 1988; nalca et al., 2005) . mpxv is highly pathogenic for a variety of laboratory animals and many animal models have been developed by using different species and different routes of exposure (table 33 .3). due to unavailability of variola virus (smallpox) to develop animal models and similar disease manifestations in humans that are similar, mpxv is one of the pox viruses that are utilized very heavily to develop a number of small animal models via different routes of exposure. wild-derived inbred mouse, stat1-deficient c57bl/6 mouse, icr mouse, prairie dogs, african dormice, ground squirrels, and gambian pouched rats are highly susceptible to mpxv by different exposure routes (americo et al., 2010; falendysz et al., 2015; hutson et al., 2009; osorio et al., 2009; sbrana et al., 2007; schultz et al., 2009; sergeev et al., 2016; stabenow et al., 2010; tesh et al., 2004; xiao et al., 2005) . cast/eij mice, one of the 38 inbred mouse strains tested for susceptibility to mpxv, showed weight loss and dose dependent mortality after in exposure to mpxv. studies with ip route of challenge indicated a 50fold higher susceptibility to mpxv when compared to in route (americo et al., 2010) . scid-balb/c mice were also susceptible to the ip challenge route and the disease resulted in mortality on day 9 postinfection (osorio et al., 2009) . similarly, c57bl/6 stat1 −/− mice were infected in with mpxv and the infection resulted in weight loss and mortality 10 days postexposure. recently sergeev et al. (2016) showed that in challenge of icr mice with mpxv resulted in purulent conjunctivitis, blepharitis, and ruffled fur in these mice although there was no death. the mouse models mentioned here are very promising for screening therapeutics against poxviruses but testing in additional models will be required for advanced development. high doses of the mpxv by ip or in routes caused 100% mortality in 6 days postexposure and 8 days postexposure, respectively, in ground squirrels (tesh et al., 2004) . the disease progressed very quickly and most of the animals were lethargic and moribund by day 5 postexposure without any pox lesions or respiratory changes. a comparison study of usa mpxv and central african strain of mpxv strains in ground squirrels by the subq route resulted in systemic disease and mortality in 6-11 days postexposure. the disease resembles hemorrhagic smallpox with nosebleeds, impaired coagulation parameters, and hemorrhage in the lungs of the animals. another study by sergeev et al. (2017) showed that in challenge with mpxv caused fever, lymphadenitis, and skin rash in ground squirrels 7-9 days postexposure. mortality was observed in 40% of the animals 13-22 days postexposure (sergeev et al., 2017) . since mpxv was transmitted by infected prairie dogs in the us outbreak, this animal model has been more thoroughly studied and utilized to test therapeutics and vaccines compared to other small animal models ( hutson et al., 2009; keckler et al., 2011; smith et al., 2011; xiao et al., 2005) . studies using in, ip, and id routes of exposure showed that mpxv was highly infectious to prairie dogs, ip infection with the west african mpxv strain caused a more severe disease and 100% mortality than challenge by the in route. anorexia and lethargy were common signs of the disease for both exposure routes. in contrast to ip route, the in route of exposure caused severe pulmonary edema and necrosis of lungs in prairie dogs, while splenic necrosis and hepatic lesions were observed in ip-infected animals (xiao et al., 2005) . hutson et al. (2009) african and congo basin strains and showed that both strains and routes caused smallpox-like disease with longer incubation periods and most importantly generalized pox lesions. therefore, this model has the utility for testing therapeutics and vaccines against pox viruses. furthermore, mpxv challenged prairie dogs were used to perform in vivo bioluminescent imaging (bli) studies (falendysz et al., 2015) . bli studies showed real time spread of virus in prairie dogs as well as potential routes for shedding and transmission. the african dormouse is susceptible to mpxv by a footpad injection or in routes (schultz et al., 2009) . mice had decreased activity, hunched posture, dehydration, conjunctivitis, and weight loss. viral doses of 200 and 2000 pfu provided 100% mortality with a mean time to death of 8 days. upper gastrointestinal hemorrhage, hepatomegaly, lymphadenopathy, and lung hemorrhage were observed during necropsy. with the hemorrhage in several organs, this model resembles hemorrhagic smallpox. in a recent study, comparison of the disease pathogenesis was performed by using live bioluminescence imaging in the cast/eij mouse and african dormouse challenged with low dose of mpxv (earl et al., 2015) . following in challenge, mpxv dissemination occurred through the blood or lymphatic system in dormice compared to dissemination that was through the nasal cavity and lungs in cast/eij mice. the disease course was much faster in cast/eij mice (earl et al., 2015) . considering the limited availability of prairie dogs, ground squirrels and african dormice, lack of reagents specific for these species, and not having commercial sources of these species, these small animal models are as attractive for further characterization and vaccine, and countermeasure testing studies. nhps were exposed to mpxv by several different routes to develop animal model for mpxv (edghill-smith et al., 2005; johnson et al., 2011; nalca et al., 2010; stittelaar et al., 2006; zaucha et al., 2001) . during our studies using an aerosol route of exposure, we observed that macaques had mild anorexia, depression, fever, and lymphadenopathy on day 6 postexposure (nalca et al., 2010) . complete blood count and clinical chemistries showed abnormalities similar to human monkeypox cases with leukocytosis and thrombocytopenia (huhn et al., 2005) . whole blood and throat swabs had viral loads peak around day 10, and in survivors, gradually decrease until day 28 postexposure. since doses of 4 × 10 4 pfu, 1 × 10 5 pfu, or 1 × 10 6 pfu resulted in lethality for 70% of the animals, whereas a dose of 4 × 10 5 pfu resulted in 85% lethality, survival was not dose dependent. the main pitfall of this model was the lack of pox lesions. with the high dose, animals succumbed to disease before developing pox lesions. with the low challenge dose, pox lesions were observed but they were few in comparison to the iv model. a recent study also evaluated the cytokine levels in aerosol challenged animals. (tree et al., 2015) . tree et al. (2015) showed that ifnγ, il-1rα, and il-6 increased dramatically on day 8 postexposure the day that death was most likely to occur, and viral dna was detected in most of the tissues. these results support the idea of a cytokine storm causing mortality in monkeypox disease. mpxv causes dose dependent disease in nhps when given by the iv route (johnson et al., 2011) . studies showed that a 1 × 10 7 pfu iv challenge results in systemic disease with fever, lymphadenopathy, macula-papular rash, and mortality. an it infection model skips the upper respiratory system and deposits virus into the trachea, delivering the virus directly to the airways without regard to particle size and the physiological deposition that occurs during the process of inhalation. fibrinonecrotic bronchopneumonia was described in animals that received 10 7 pfu of mpxv intratracheally (stittelaar et al., 2006) . although a similar challenge dose of it mpxv infection resulted in a similar viremia in nhps to the aerosol route of infection, the timing of the first peak was delayed by 5 days in intratracheally exposed macaques compared to aerosol infection, and the amount of virus detected by qpcr was approximately 100-fold lower. this suggests that local replication is more prominent after aerosol delivery compared to the it route. an intrabronchial route of exposure resulted in pneumonia in nhps (johnson et al., 2011) . delayed onset of clinical signs and viremia were observed during the disease progression. in this model, similar to aerosol and it infection models, the number of pox lesions was much less than in the iv infection model. a major downside of the iv, it, and intrabronchial models is that the initial infection of respiratory tissue, incubation, and prodromal phases are circumvented with the direct inoculation of virus to the blood stream or to the lung. this is an important limitation when the utility of these models is to test possible vaccines and treatments in which the efficacy may depend on protecting the respiratory mucosa and targeting the subsequent early stages of the infection, which are not represented in these challenge models. although the aerosol model is the natural route of transmission for human varv infections and a secondary route for human mpxv infections, the lack of pox lesions is the main drawback of this model. therefore, when this model is used to test medical countermeasures, the endpoints and the biomarkers to initiate treatment should be chosen carefully. hepatitis b virus (hbv) is one of the most common infections worldwide with over 400 million people chronically infected and 316,000 cases per year of liver cancer due to infection (lee, 1997) . the virus can naturally infect both humans and chimpanzees (guha et al., 2004) . hbv is transmitted parenterally or postnatally from infected mothers. it can also be transmitted by sexual contact, iv drug use, blood transfusion, and acupuncture (lai et al., 2003) . the age at which one is infected dictates the risk of developing chronic disease (hyams, 1995) . acute infection during adulthood is self-limiting and results in flu-like symptoms that can progress to hepatocellular involvement as observed with the development of jaundice. the clinical symptoms of hbv infection last for a few weeks before resolving (ganem and prince, 2004) . after this acute phase, lifetime immunity is achieved (wright and lau, 1993) . of those infected, less than 5% will develop the chronic form of the disease. chronicity is the most serious outcome of the disease as it can result in cirrhosis or liver cancer. hepatocellular carcinoma is 100 times more likely to develop in a chronically infected individual than a noncarrier (beasley, 1988) . the viral determinant for cellular transformation has yet to be determined, although studies involving the woodchuck hepatitis virus suggest that x protein may be responsible (spandau and lee, 1988). many individuals are asymptomatic until complications emerge related to chronic hbv carriage. chimpanzees have a unique strain that circulates within the population (hu et al., 2000; . it was found that 3%-6% of all wild-caught animals from africa are positive for hbv antigen ( lander et al., 1972) . natural and experimental challenge with the virus follows the same course as human disease; however, this is only an acute model of disease (prince, 1972) . to date, chimpanzees are the only reliable method to ensure that plasma vaccines are free from infectious particles (prince and brotman, 2001 ). this animal model has been used to study new therapeutics and vaccines. chimpanzees are especially ideal for these studies given that their immune response to infection directly mirrors humans (nayersina et al., 1993) . recent regulations by the national institute of health (nih) and restrictions to use great apes as animal models forced researches to find alternate models for hbv infection. other nhps that have been evaluated are gibbons, orangutans, and rhesus monkeys. although these animals can be infected with hbv, none develops hepatic lesions or liver damage as noted by monitoring of liver enzymes (pillot, 1990 ). mice are not permissible to infection, and thus numerous transgenic and humanized lines that express hbv proteins have been created to facilitate their usage as an animal model. these include both immunocompetent and immunosuppressed hosts. the caveat to all of these mouse lines is that they reproduce only the acute form of disease (guha et al., 2004) . recently, the entire genome of hbv was transferred to an immunocompetent mouse line via adenovirus. this provides a model for persistent infection (huang et al., 2012) . another model that has been developed is hydrodynamic injection of hbv genomes in the liver of mice (liu et al., 1999; yang et al., 2002) . although this model is very stressful to mice and has liver toxicity, it is successfully used to evaluate antivirals against hbv (mccaffrey et al., 2003) . liver chimeric mouse models are an additional set of surrogate models for hbv infection (dandri and lutgehetmann, 2014) . in these models human hepatocytes are integrated into the murine liver parenchyma (allweiss and dandri, 2016) . this model might be used to test antivirals as well as to study the molecular biology of hbv infection. hbv can also be studied using surrogate viruses, naturally occurring mammalian hepadna viruses (mason et al., 1982) . the woodchuck hepatitis virus induces hepatocellular carcinoma (summers et al., 1978) . within a population, 65%-75% of all neonatal woodchucks are susceptible to chronic infection (cote et al., 2000) . a major difference between the two hepatitis isolates is the rate at which they induce cancer; almost all chronic carriers developed hepatocellular carcinoma within 3 years of the initial infection in woodchucks, whereas human carcinogenesis takes much longer (gerin et al., 1989) . the acute infection strongly resembles what occurs during the course of human disease. there is a self-limiting acute phase resulting in a transient viremia that has the potential of chronic carriage (tennant, 2001) . challenge with virus in neonates leads to a chronic infection while adults only develop the acute phase of disease (buendia, 1992) . a closely related species to the woodchuck is the marmota himalayan. this animal is also susceptible to the woodchuck hepadna virus upon iv injection. the marmot himalayan develops an acute hepatitis with a productive infection (lucifora et al., 2010) . hepatitis d virus (hdv) is dependent upon hbv to undergo replication and successful infection in its human host (gerin, 2001) . there are two modes of infection possible between the viruses: coinfection where a person is simultaneously infected or superinfection in which a chronic carrier of hbv is subsequently infected with hdv (purcell et al., 1987) . coinfection leads to a similar disease as seen with hbv alone; however, superinfection can result in chronic hdv infection and severe liver damage (guilhot et al., 1994) . both coinfection and superinfection can be demonstrated within the chimpanzee and woodchuck by inoculation of human hepatitis d (ponzetto et al., 1991) . a recently published report demonstrated the use of a humanized chimeric upa mouse to study interactions between the two viruses and drug testing (lutgehetmann et al., 2012) new models ranging from nhps to small animals and representing the disease characteristics in humans are necessary to study viral and host factors that drive disease pathogenesis and evaluate medical countermeasures. the ideal animal model for human viral disease should closely recapitulate the spectrum of clinical symptoms and pathogenesis observed during the course of human infection. whenever feasible, the model should use the same virus and strain that infects humans. it is also preferable that the virus is a low passage clinical isolate thus animal passage or adaptation should be avoided if model species can be identified that are susceptible. ideally, the experimental route of infection would mirror that occurs in natural disease. in order to understand the interplay and contribution of the immune system during infection, an immunocompetent animal should be used. the aforementioned characteristics cannot always be satisfied; however, and often virus must be adapted, knockout mice must be used, and/or the disease is not perfectly mimicked in the animal model. well-characterized animal models are critical for licensure to satisfy fda "animal rule." this rule applies to situations in which vaccine and therapeutic efficacy cannot safely or ethically be tested in humans; thus licensure will come only after preclinical tests are performed in animal models. many fields in virology are moving toward standardized models that can be used across institutions to test vaccines and therapeutics. a current example of such an effort is within the filovirus community, where animal models, euthanasia criteria, assays, and virus strains are in the process of being standardized. the hope is that these efforts will enable results of efficacy tests on medical countermeasures compared across institutions. this chapter has summarized the best models available for each of the viruses described. the rhesus macaque pediatric siv infection model-a valuable tool in understanding infant hiv-1 pathogenesis and for designing pediatric hiv-1 prevention strategies prevalence of anti-rift-valley-fever igm antibody in abattoir workers in the nile delta during the 1993 outbreak in egypt common marmosets (callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels. emerg generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pathological changes in brain and other target organs of infant and weanling mice after infection with nonneuroadapted western equine encephalitis virus particle-to-pfu ratio of ebola virus influences disease course and survival in cynomolgus macaques progress toward norovirus vaccines: considerations for further development and implementation in potential target populations characterization of lethal zika virus infection in ag129 mice experimental in vitro and in vivo models for the study of human hepatitis b virus infection a model of meningococcal bacteremia after respiratory superinfection in influenza a virus-infected mice middle east respiratory syndrome coronavirus: current situation and travel-associated concerns aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques necrotizing scleritis, conjunctivitis, and other pathologic findings in the left eye and brain of an ebola virus-infected rhesus macaque (macaca mulatta) with apparent recovery and a delayed time of death american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models the gerbil, meriones unguiculatus, a model for rift valley fever viral encephalitis morbidity and mortality among patients with respiratory syncytial virus infection: a 2-year retrospective review chikungunya and the nervous system: what we do and do not know the west nile virus outbreak of 1999 in new york: the flushing hospital experience hospital outbreak of middle east respiratory syndrome coronavirus diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses norovirus vaccine against experimental human norwalk virus illness determination of the 50% human infectious dose for norwalk virus an epizootic attributable to western equine encephalitis virus infection in emus in texas evidence for camel-to-human transmission of mers coronavirus integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics disseminated and sustained hiv infection in cd34+ cord blood cell-transplanted rag2 −/− gamma c −/− mice choice of inbred rat strain impacts lethality and disease course after respiratory infection with rift valley fever virus rift valley fever: an uninvited zoonosis in the arabian peninsula recombinant norwalk virus-like particles given orally to volunteers: phase i study tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining lethal antibody enhancement of dengue disease in mice is prevented by fc modification animal models for the study of influenza pathogenesis and therapy effect of oral gavage treatment with znal42 and other metallo-ion formulations on influenza a h5n1 and h1n1 virus infections in mice macaque studies of vaccine and microbicide combinations for preventing hiv-1 sexual transmission early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus hepatitis b virus. the major etiology of hepatocellular carcinoma transmission of norwalk virus during football game vpx is critical for sivmne infection of pigtail macaques experimental respiratory syncytial virus infection of four species of primates pathogenesis and immune response of crimean-congo hemorrhagic fever virus in a stat-1 knockout mouse model crimean-congo hemorrhagic fever virus infection is lethal for adult type i interferon receptor-knockout mice the utility of the new generation of humanized mice to study hiv-1 infection: transmission, prevention, pathogenesis, and treatment hiv-1 infection and cd4 t cell depletion in the humanized rag2 −/− gamma c −/− (rag-hu) mouse model bacterial infections in pigs experimentally infected with nipah virus evaluation of a mouse model for the west nile virus group for the purpose of determining viral pathotypes severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice study of susceptibility to crimean hemorrhagic fever (chf) virus in european and long-eared hedgehogs. tezisy konf manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells chimpanzees as an animal model for human norovirus infection and vaccine development a simple technique for infection of mosquitoes with viruses; transmission of zika virus human papillomavirus research: do we still need animal models? human papillomavirus in cervical cancer development of a hamster model for chikungunya virus infection and pathogenesis a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection the cotton rat model of respiratory viral infections correlates of immunity to filovirus infection filovirus vaccines induction of robust cellular and humoral virusspecific adaptive immune responses in human immunodeficiency virus-infected humanized blt mice animal models of human-papillomavirus-associated oncogenesis interferon alpha/beta receptor-deficient mice as a model for ebola virus disease zika virus outbreak in rio de janeiro, brazil: clinical characterization, epidemiological and virological aspects co-infection of the cotton rat (sigmodon hispidus) with staphylococcus aureus and influenza a virus results in synergistic disease effectiveness of influenza vaccination the role of the type i interferon response in the resistance of mice to filovirus infection a mouse model for evaluation of prophylaxis and therapy of ebola hemorrhagic fever the rabbit viral skin papillomas and carcinomas: a model for the immunogenetics of hpv-associated carcinogenesis the confirmation and maintenance of smallpox eradication a lethal disease model for hantavirus pulmonary syndrome in immunosuppressed syrian hamsters infected with sin nombre virus nonhuman primate models of chikungunya virus infection and disease tissue tropism and neuroinvasion of west nile virus do not differ for two mouse strains with different survival rates pediatric norovirus diarrhea in nicaragua efficacy assessment of a cell-mediated immunity hiv-1 vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial variation between strains of hamsters in the lethality of pichinde virus infections hepatitis b viruses and hepatocellular carcinoma generation of k14-e7/n87betacat double transgenic mice as a model of cervical cancer limited or no protection by weakly or nonneutralizing antibodies against vaginal shiv challenge of macaques compared with a strongly neutralizing antibody a novel vaccine against crimean-congo haemorrhagic fever protects 100% of animals against lethal challenge in a mouse model interleukin-1beta but not tumor necrosis factor is involved in west nile virusinduced langerhans cell migration from the skin in c57bl/6 mice animal models of papillomavirus pathogenesis immunization of knock-out alpha/beta interferon receptor mice against high lethal dose of crimean-congo hemorrhagic fever virus with a cell culture based vaccine characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses lassa virus infection in experimentally infected marmosets: liver pathology and immunophenotypic alterations in target tissues a small nonhuman primate model for filovirus-induced disease severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus outbreak of acute illness-southwestern united states outbreak of west nile-like viral encephalitis-new york, 1999. mmwr morb. mortal in vitro whole-virus binding of a norovirus genogroup ii genotype 4 strain to cells of the lamina propria and brunner's glands in the human duodenum animal models for studying dengue pathogenesis and therapy us doctors investigate more than 50 possible cases of monkeypox mechanism of neuroinvasion of venezuelan equine encephalitis virus in the mouse chikungunya outbreaks-the globalization of vectorborne diseases inactivated and live, attenuated influenza vaccines protect mice against influenza: streptococcus pyogenes super-infections pathogenesis of a genogroup ii human norovirus in gnotobiotic pigs the immunobiology of sars* induction of tetravalent protective immunity against four dengue serotypes by the tandem domain iii of the envelope protein norovirus infection as a cause of diarrhea-associated benign infantile seizures comparative pathogenesis of epidemic and enzootic chikungunya viruses in a pregnant rhesus macaque model development of norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of norwalk virus gastroenteritis viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome a single sublingual dose of an adenovirus-based vaccine protects against lethal ebola challenge in mice and guinea pigs model systems to study the life cycle of human papillomaviruses and hpv-associated cancers primary severe acute respiratory syndrome coronavirus i nfection limits replication but not lung inflammation upon homologous rechallenge viral and host factors in human respiratory syncytial virus pathogenesis pathogenesis of pichinde virus infection in strain 13 guinea pigs: an immunocytochemical, virologic, and clinical chemistry study pathogenesis of experimental ebola virus infection in guinea pigs transcriptional profiling of the immune response to marburg virus infection the use of a neonatal mouse model to study respiratory syncytial virus infections a model of denv-3 infection that recapitulates severe disease and highlights the importance of ifn-gamma in host resistance to infection effects of age and viral determinants on chronicity as an outcome of experimental woodchuck hepatitis virus infection a mouse model for chikungunya: young age and inefficient type-i interferon signaling are risk factors for severe disease mosquito bite delivery of dengue virus enhances immunogenicity and pathogenesis in humanized mice comparison of the pathogenesis of the angola and ravn strains of marburg virus in the outbred guinea pig model the brazilian zika virus strain causes birth defects in experimental models age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood lassa fever encephalopathy: clinical and laboratory findings profound and prolonged lymphocytopenia with west nile encephalitis first complete genome sequence of zika virus (flaviviridae, flavivirus) from an autochthonous transmission in brazil viral haemorrhagic fevers caused by lassa, ebola, and marburg viruses the enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and il-13 production mouse models of hepatitis b and delta virus infection [experimental inhalation infection of monkeys of the macacus cynomolgus and macacus rhesus species with the virus kinetic profile of influenza virus infection in three rat strains pathology of experimental ebola virus infection in african green monkeys. involvement of fibroblastic reticular cells validation of an hpv16-mediated carcinogenesis mouse model middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques selection of unadapted, pathogenic shivs encoding newly transmitted hiv-1 envelope proteins b-cells and the use of non-human primates for evaluation of hiv vaccine candidates antiretroviral pre-exposure prophylaxis prevents vaginal transmission of hiv-1 in humanized blt mice innate and adaptive immune responses determine protection against disseminated infection by west nile encephalitis virus rift valley fever virus encephalitis is associated with an ineffective systemic immune response and activated t cell infiltration into the cns in an immunocompetent mouse model evidence of sexual transmission of zika virus a susceptible mouse model for zika virus infection identification of a novel coronavirus in patients with severe acute respiratory syndrome subclinical infection without encephalitis in mice following intranasal exposure to nipah virus-malaysia and nipah virus-bangladesh nonhuman primate models of encephalitic alphavirus infection: historical review and future perspectives mortality due to influenza in the united states-an annualized regression approach using multiple-cause mortality data distinct pathogenesis of hong kong-origin h5n1 viruses in mice compared to that of other highly pathogenic h5 avian influenza viruses postexposure antibody prophylaxis protects nonhuman primates from filovirus disease comparative live bioluminescence imaging of monkeypox virus dissemination in a wild-derived inbred mouse (mus musculus castaneus) and outbred african dormouse (graphiurus kelleni) siv-induced impairment of neurovascular repair: a potential role for vegf smallpox vaccine does not protect macaques with aids from a lethal monkeypox virus challenge influenza-induced tachypnea is prevented in immune cotton rats, but cannot be treated with an anti-inflammatory steroid or a neuraminidase inhibitor distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats an outbreak of viral gastroenteritis following environmental contamination at a concert hall natural history of aerosol exposure with marburg virus in rhesus macaques experimental congo virus (ib-an7620) infection in primates further assessment of monkeypox virus infection in gambian pouched rats (cricetomys gambianus) using in vivo bioluminescent imaging respiratory syncytial virus infection in elderly and high-risk adults infection with mers-cov causes lethal pneumonia in the common marmoset immune response to marburg virus angola infection in nonhuman primates fields' virology the susceptibility of rats to rift valley fever in relation to age henipavirus susceptibility to environmental variables pause on avian flu transmission research aetiology: koch's postulates fulfilled for sars virus spinal cord neuropathology in human west nile virus infection therapeutic dna vaccine induces broad t cell responses in the gut and sustained protection from viral rebound and aids in siv-infected rhesus macaques hepatitis b virus infection-natural history and clinical consequences biological heterogeneity, including systemic replication in mice, of h5n1 influenza a virus isolates from humans in hong kong chikungunya virus arthritis in adult wild-type mice epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method development of an acute and highly pathogenic nonhuman primate model of nipah virus infection animal models of hepatitis delta virus infection and disease hepadnavirusinduced liver cancer in woodchucks experimental infection and natural contact exposure of dogs with avian influenza virus (h5n1). emerg megaribavirin aerosol for the treatment of influenza a virus infections in mice discovery of novel human and animal cells infected by the severe acute respiratory syndrome coronavirus by replication-specific multiplex reverse transcription-pcr chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice studies on the virus of venezuelan equine encephalomyelitis. i. modification by cortisone of the response of the central nervous system of macaca mulatta serious morbidity and mortality associated with influenza epidemics a novel respiratory model of infection with monkeypox virus in cynomolgus macaques clinical features of nipah virus encephalitis among pig farmers in malaysia monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention animal models of highly pathogenic rna viral infections: hemorrhagic fever viruses interferon alfacon-1 protects hamsters from lethal pichinde virus infection primary respiratory syncytial virus infection in mice pneumonitis and multiorgan system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area-puerto rico acute and chronic airway disease after human respiratory syncytial virus infection in cotton rats (sigmodon hispidus) alphaviruses replication fitness determines high virulence of influenza a virus in mice carrying functional mx1 resistance gene antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal hiv-1 infection in a transgenic mouse model characterization of influenza a/hongkong/156/97 (h5n1) virus in a mouse model and protective effect of zanamivir on h5n1 infection in mice epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century cell culture and animal models of viral hepatitis. part i: hepatitis b expression of the hepatitis delta virus large and small antigens in transgenic mice acute hendra virus infection: analysis of the pathogenesis and passive antibody protection in the hamster model lassa fever encephalopathy: lassa virus in cerebrospinal fluid but not in serum 1, 5-iodonaphthyl azide-inactivated v3526 protects against aerosol challenge with virulent venezuelan equine encephalitis virus dengue: an update pegylated interferonalpha protects type 1 pneumocytes against sars coronavirus infection in macaques asymptomatic middle east respiratory syndrome coronavirus infection in rabbits head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model update on animal models for hiv research norovirus disease in the united states. emerg cardiopulmonary manifestations of hantavirus pulmonary syndrome serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus hendra virus infection in a veterinarian a phase 1 clinical trial of a dna vaccine for venezuelan equine encephalitis delivered by intramuscular or intradermal electroporation deaths from norovirus among the elderly, england and wales. emerg aerosolized rift valley fever virus causes fatal encephalitis in african green monkeys and common marmosets hiv-1-induced aids in monkeys west nile fever short communication: viremic control is independent of repeated low-dose shivsf162p3 exposures pathogenesis of marburg hemorrhagic fever in cynomolgus macaques pathogenesis of lassa fever in cynomolgus macaques niemann-pick c1 is essential for ebolavirus replication and pathogenesis in vivo airborne transmission of influenza a/ h5n1 virus between ferrets animal models in hiv-1 protection and therapy advances in rsv vaccine research and development-a global agenda transmission of zika virus through sexual contact with travelers to areas of ongoing transmissioncontinental united states establishment of a patient-derived orthotopic xenograft (pdox) model of her-2-positive cervical cancer expressing the clinical metastatic pattern resolution of primary severe acute respiratory syndromeassociated coronavirus infection requires stat1 mucosal immunity and vaccines nipah virus outbreak with person-to-person transmission in a district of bangladesh eastern equine encephalitis virus in mice i: clinical course and outcome are dependent on route of exposure the lesions of experimental equine morbillivirus disease in cats and guinea pigs a lethal disease model for hantavirus pulmonary syndrome hantaan/ andes virus dna vaccine elicits a broadly cross-reactive neutralizing antibody response in nonhuman primates persistent infection with and serologic cross-reactivity of three novel murine noroviruses molecular characterization of three novel murine noroviruses identification of hepatitis b virus indigenous to chimpanzees manifestation of thrombocytopenia in dengue-2-virusinfected mice transfer of hbv genomes using low doses of adenovirus vectors leads to persistent infection in immune competent mice west nile fever-a reemerging mosquito-borne viral disease in europe. emerg live, attenuated influenza virus (laiv) vehicles are strong inducers of immunity toward influenza b virus clinical characteristics of human monkeypox, and risk factors for severe disease shiv-1157i and passaged progeny viruses encoding r5 hiv-1 clade c env cause aids in rhesus monkeys norwalk virus infection associates with secretor status genotyped from sera a prairie dog animal model of systemic orthopoxvirus disease using west african and congo basin strains of monkeypox virus risks of chronicity following acute hepatitis b virus infection: a review the pathogenesis of rift valley fever experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets pathogenicity of a highly pathogenic avian influenza virus, a/chicken/yamaguchi/7/04 (h5n1) in different species of birds and mammals respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice a multimeric l2 vaccine for prevention of animal papillomavirus infections lassa virus infection of rhesus monkeys: pathogenesis and treatment with ribavirin pathogenesis of lassa virus infection in guinea pigs perinatal transmission of shiv-sf162p3 in macaca nemestrina human monkeypox and other poxvirus infections of man cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus glycomic characterization of respiratory tract tissues of ferrets: implications for its use in influenza virus infection studies comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route phenotypic changes in langerhans' cells after infection with arboviruses: a role in the immune response to epidermally acquired viral infection? protection of beagle dogs from mucosal challenge with canine oral papillomavirus by immunization with recombinant adenoviruses expressing codon-optimized early genes detailed analysis of the african green monkey model of nipah virus disease experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera treatment of venezuelan equine encephalitis virus infection with (-)-carbodine c3h/hen mouse model for the evaluation of antiviral agents for the treatment of venezuelan equine encephalitis virus infection blt humanized mice as a small animal model of hiv infection stat1-dependent innate immunity to a norwalk-like virus pichinde virus infection in strain 13 guinea pigs reduces intestinal protein reflection coefficient with compensation establishment of the black-tailed prairie dog (cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high-and low-dose monkeypox virus challenges low-dose rectal inoculation of rhesus macaques by sivsme660 or sivmac251 recapitulates human mucosal infection by hiv-1 new opportunities for field research on the pathogenesis and treatment of lassa fever gastrointestinal norovirus infection associated with exacerbation of inflammatory bowel disease isolation of monkeypox virus from wild squirrel infected in nature in hot pursuit of the first vaccine against respiratory syncytial virus pathogenesis of hantaan virus infection in suckling mice: clinical, virologic, and serologic observations respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine the severe pathogenicity of alveolar macrophage-depleted ferrets infected with 2009 pandemic h1n1 influenza virus mouse adaptation of influenza b virus increases replication in the upper respiratory tract and results in droplet transmissibility in ferrets stepping toward a macaque model of hiv-1 induced vomiting as a symptom and transmission risk in norovirus illness: evidence from human challenge studies wild-type puumala hantavirus infection induces cytokines, c-reactive protein, creatinine, and nitric oxide in cynomolgus macaques aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques replication, pathogenicity, shedding, and transmission of zaire ebolavirus in pigs west nile viral encephalitis foodborne viruses: an emerging problem low pathogenic avian influenza a(h7n9) virus causes high mortality in ferrets upon intratracheal challenge: a model to study intervention strategies filoviruses: a compendium of 40 years of epidemiological, clinical, and laboratory studies pathology of human influenza a (h5n1) virus infection in cynomolgus macaques (macaca fascicularis) dengue virus infection and immune response in humanized rag2(-/-)gamma(c) (-/-) (rag-hu) mice chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages viral hepatitis b strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine origin of the west nile virus responsible for an outbreak of encephalitis in the northeastern united states antibody to hepatitis-associated antigen. frequency and pattern of response as detected by radioimmunoprecipitation severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus hku1 and other coronavirus infections in hong kong epidemic rift valley fever in egypt: observations of the spectrum of human illness hantaviruses. a short review hepatitis b virus infection quantitative measurement of influenza virus replication using consecutive bronchoalveolar lavage in the lower respiratory tract of a ferret model characterization of the activity of 2'-c-methylcytidine against dengue virus replication diffusion tensor and volumetric magnetic resonance measures as biomarkers of brain damage in a small animal model of hiv sequencing, annotation, and characterization of the influenza ferret infectome lethality and pathogenesis of airborne infection with filoviruses in a129 alpha/beta −/− interferon receptor-deficient mice experimental inoculation study indicates swine as a potential host for hendra virus early initiation of antiretroviral therapy can functionally control productive hiv-1 infection in humanized-blt mice zika virus disrupts neural progenitor development and leads to microcephaly in mice middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys study of dengue virus infection in scid mice engrafted with human k562 cells a comparative study of the pathogenesis of western equine and eastern equine encephalomyelitis viral infections in mice by intracerebral and subcutaneous inoculations hydrodynamics-based transfection in animals by systemic administration of plasmid dna the emergence of nipah virus, a highly pathogenic paramyxovirus the guinea pig as a transmission model for human influenza viruses transmission in the guinea pig model a mouse model for the evaluation of pathogenesis and immunity to influenza a (h5n1) viruses isolated from humans transmission of human infection with nipah virus recurrent zoonotic transmission of nipah virus into humans the effect of an arenavirus infection on liver morphology and function hepatitis b virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model ebola virus disease in mice with transplanted human hematopoietic stem cells arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation humanized chimeric upa mouse model for the study of hepatitis b and d virus interactions and preclinical drug evaluation role of dendritic cell targeting in venezuelan equine encephalitis virus pathogenesis detection of hepatitis b virus infection in wild-born chimpanzees (pan troglodytes verus): phylogenetic relationships with human and other primate genotypes proportion of deaths and clinical features in bundibugyo ebola virus infection the ferret: an animal model to study influenza virus local innate immune responses and influenza virus transmission and virulence in ferrets vomiting larry: a simulated vomiting system for assessing environmental contamination from projectile vomiting related to norovirus infection studies on the pathogenesis of dengue infection in monkeys. 3. sequential distribution of virus in primary and heterologous infections studies on dengue 2 virus infection in cyclophosphamide-treated rhesus monkeys crimean-congo hemorrhagic fever experimental infection of squirrel monkeys with nipah virus. emerg a school outbreak of norwalk-like virus: evidence for airborne transmission virology: sars virus infection of cats and ferrets characterization of clinical and immunological parameters during ebola virus infection of rhesus macaques delayed disease progression in cynomolgus macaques infected with ebola virus makona strain. emerg asymmetric replication of duck hepatitis b virus dna in liver cells: free minusstrand dna human and avian influenza viruses target different cell types in cultures of human airway epithelium a role for hpv16 e5 in cervical carcinogenesis replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys inhibition of hepatitis b virus in mice by rna interference zika virus was transmitted by sexual contact in texas, health officials report lassa fever. effective therapy with ribavirin lassa virus hepatitis: a study of fatal lassa fever in humans lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus andes virus infection of cynomolgus macaques regional t-and b-cell responses in influenza-infected ferrets clinical aspects of marburg hemorrhagic fever humanized mice mount specific adaptive and innate immune responses to ebv and tsst-1 isolation from human sera in egypt of a virus apparently identical to west nile virus middle east respiratory syndrome coronavirus in bats, saudi arabia. emerg chikungunya virus infection results in higher and persistent viral replication in aged rhesus macaques due to defects in anti-viral immunity reduced clearance of respiratory syncytial virus infection in a preterm lamb model dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome experimental nipah virus infection in pigs and cats experimental nipah virus infection in pteropid bats (pteropus poliocephalus) an in-depth analysis of original antigenic sin in dengue virus infection propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus rift valley fever virus in mice. i. general features of the infection zika virus infection during pregnancy in mice causes placental damage and fetal demise outbreaks of acute gastroenteritis associated with norwalk-like viruses in campus settings a nonfucosylated variant of the anti-hiv-1 monoclonal antibody b12 has enhanced fcgammariiiamediated antiviral activity in vitro but does not improve protection against mucosal shiv challenge in macaques flaviviruses experimental studies of rhesus monkeys infected with epizootic and enzootic subtypes of venezuelan equine encephalitis virus necrotizing myocarditis in mice infected with western equine encephalitis virus: clinical, electrocardiographic, and histopathologic correlations aedes albopictus in the united states: ten-year presence and public health implications. emerg severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume impact of short-term haart initiated during the chronic stage or shortly post-exposure on siv infection of male genital organs influence of age on susceptibility and on immune response of mice to eastern equine encephalomyelitis virus a mouse model of chikungunya virusinduced musculoskeletal inflammatory disease: evidence of arthritis, tenosynovitis, myositis, and persistence dengue virus tropism in humanized mice recapitulates human dengue fever functional role of type i and type ii interferons in antiviral defense the occurrence of human cases in johannesburg. s feline model of acute nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine vertical transmission and fetal replication of nipah virus in an experimentally infected cat pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets pneumonia from human coronavirus in a macaque model eastern equine encephalitis virus infection: electron microscopic studies of mouse central nervous system evaluation of three strains of influenza a virus in humans and in owl, cebus, and squirrel monkeys a novel morbillivirus pneumonia of horses and its transmission to humans. emerg a morbillivirus that caused fatal disease in horses and humans global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis cchf infection among animals reemergence of monkeypox: prevalence, diagnostics, and countermeasures experimental infection of cynomolgus macaques (macaca fascicularis) with aerosolized monkeypox virus eastern equine encephalitis. distribution of central nervous system lesions in man and rhesus monkey hla a2 restricted cytotoxic t lymphocyte responses to multiple hepatitis b surface antigen epitopes during hepatitis b virus infection an aptamer-sirna chimera suppresses hiv-1 viral loads and protects from helper cd4(+) t cell decline in humanized mice severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 ferrets exclusively synthesize neu5ac and express naturally humanized influenza a virus receptors field's virology naturally occurring, nonregressing canine oral papillomavirus infection: host immunity, virus characterization, and experimental infection regression of canine oral papillomas is associated with infiltration of cd4+ and cd8+ lymphocytes diversifying animal models: the use of hispid cotton rats (sigmodon hispidus) in infectious diseases induction of focal epithelial hyperplasia in tongue of young bk6-e6/e7 hpv16 transgenic mice the contribution of animal models to the understanding of the host range and virulence of influenza a viruses evaluation of antiviral efficacy of ribavirin, arbidol, and t-705 (favipiravir) in a mouse model for crimean-congo hemorrhagic fever evaluation of oseltamivir prophylaxis regimens for reducing influenza virus infection, transmission and disease severity in a ferret model of household contact a novel video tracking method to evaluate the effect of influenza infection and antiviral treatment on ferret activity human respiratory syncytial virus a2 strain replicates and induces innate immune responses by respiratory epithelia of neonatal lambs common marmoset (callithrix jacchus) as a primate model of dengue virus infection: development of high levels of viraemia and demonstration of protective immunity changes in hematological and serum biochemical parameters in common marmosets (callithrix jacchus) after inoculation with dengue virus middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction dengue virus-induced hemorrhage in a nonhuman primate model preclinical development of highly effective and safe dna vaccines directed against hpv 16 e6 and e7 a novel small molecule inhibitor of influenza a viruses that targets polymerase function and indirectly induces interferon comparison of monkeypox viruses pathogenesis in mice by in vivo imaging a rhesus monkey model for sexual transmission of a papillomavirus isolated from a squamous cell carcinoma infection of calves with bovine norovirus giii. 1 strain jena virus: an experimental model to study the pathogenesis of norovirus infection the cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis zika virus disease in colombia-preliminary report genetic diversity, distribution, and serological features of hantavirus infection in five countries in south america liver injury and viremia in mice infected with dengue-2 virus the hamster as an animal model for eastern equine encephalitis-and its use in studies of virus entrance into the brain a recombinant hendra virus g glycoprotein-based subunit vaccine protects ferrets from lethal hendra virus challenge identification of an antioxidant small-molecule with broad-spectrum antiviral activity impaired heterologous immunity in aged ferrets during sequential influenza a h1n1 infection influenza transmission in the mother-infant dyad leads to severe disease, mammary gland infection, and pathogenesis by regulating host responses economic impact of respiratory syncytial virus-related illness in the us: an analysis of national databases inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type-2 replication systematic literature review of role of noroviruses in sporadic gastroenteritis. emerg sars: what have we learned? the severe acute respiratory syndrome severe acute respiratory syndrome bacterial sinusitis and otitis media following influenza virus infection in ferrets neuropathology of h5n1 virus infection in ferrets the draft genome sequence of the ferret (mustela putorius furo) facilitates study of human respiratory disease immunopathogenesis of coronavirus infections: implications for sars hantavirus pulmonary syndrome: the new american hemorrhagic fever rift valley fever inbred rat strains mimic the disparate human response to rift valley fever virus infection experimental studies of arenaviral hemorrhagic fevers experimental rift valley fever in rhesus macaques bovine respiratory syncytial virus protects cotton rats against human respiratory syncytial virus infection human hendra virus encephalitis associated with equine outbreak molecularly engineered live-attenuated chimeric west nile/dengue virus vaccines protect rhesus monkeys from west nile virus structure as revealed by airway dissection. a comparison of mammalian lungs study on west nile virus persistence in monkeys experimental hepatitis delta virus infection in the animal model changing patterns of chikungunya virus: re-emergence of a zoonotic arbovirus grune and stratton. hepatitis and blood transfusion perspectives on hepatitis b studies with chimpanzees pulmonary lesions in primary respiratory syncytial virus infection, reinfection, and vaccine-enhanced disease in the cotton rat (sigmodon hispidus) experimental hepatitis delta virus infection in the chimpanzee relative infectivity of hepatitis a virus by the oral and intravenous routes in 2 species of nonhuman primates cardiovascular and pulmonary responses to pichinde virus infection in strain 13 guinea pigs establishment and characterization of a lethal mouse model for the angola strain of marburg virus stability and inactivation of sars coronavirus possibility of extracting hyperimmune gammaglobulin against chf from doneky blood sera dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc swine as a model for influenza a virus infection and immunity zika virus: an update on epidemiology, pathology, molecular biology, and animal model oseltamivir population pharmacokinetics in the ferret: model application for pharmacokinetic/pharmacodynamic study design aerosol exposure to western equine encephalitis virus causes fever and encephalitis in cynomolgus macaques severe encephalitis in cynomolgus macaques exposed to aerosolized eastern equine encephalitis virus differences in aerosolization of rift valley fever virus resulting from choice of inhalation exposure chamber: implications for animal challenge studies an hiv-1 transgenic rat that develops hiv-related pathology and immunologic dysfunction a trivalent recombinant ad5 gag/pol/nef vaccine fails to protect rhesus macaques from infection or control virus replication after a limiting-dose heterologous siv challenge infection with chikungunya virus in italy: an outbreak in a temperate region a single dose of an iscom influenza vaccine induces long-lasting protective immunity against homologous challenge infection but fails to protect cynomolgus macaques against distant drift variants of influenza a (h3n2) viruses influenza a virus (h5n1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts immunomodulation with il-4r alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease animal models for sars aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice transmission of a 2009 h1n1 pandemic influenza virus occurs before fever is detected, in the ferret model experimental norovirus infections in non-human primates synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice a novel model of lethal hendra virus infection in african green monkeys and the effectiveness of ribavirin treatment clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection recent progress in henipavirus research: molecular biology, genetic diversity, animal models mucosal arenavirus infection of primates can protect them from lethal hemorrhagic fever sars vaccines: where are we? animal models of rift valley fever virus infection characterization of a novel coronavirus associated with severe acute respiratory syndrome macaque model for severe acute respiratory syndrome pathogenesis of aerosolized eastern equine encephalitis virus infection in guinea pigs pathophysiology of hantavirus pulmonary syndrome in rhesus macaques virulence and pathophysiology of the congo basin and west african strains of monkeypox virus in non-human primates arenaviridae. virus taxonomy, viiith report of the international committee on taxonomy of viruses clinical laboratory, virologic, and pathologic changes in hamsters experimentally infected with pirital virus (arenaviridae): a rodent model of lassa fever comparative pathology of north american and central african strains of monkeypox virus in a ground squirrel model of the disease biomedical applications of sheep models: from asthma to vaccines bunyaviruses the use of an animal model to study transmission of influenza virus infection experimental infection of an african dormouse (graphiurus kelleni) with monkeypox virus animal noroviruses human monkeypox infection: a family cluster in the midwestern united states the possibility of using the icr mouse as an animal model to assess antimonkeypox drug efficacy using the ground squirrel (marmota bobak) as an animal model to assess monkeypox drug efficacy experimental inoculation of juvenile rhesus macaques with primate enteric caliciviruses a rodent model of chikungunya virus infection in rag1 −/− mice, with features of persistence, for vaccine safety evaluation respiratory syncytial virus (rsv) pulmonary infection in humanized mice induces human anti-rsv immune responses and pathology viremia and antibody response of small african and laboratory animals to crimean-congo hemorrhagic fever virus infection early activation of natural killer and b cells in response to primary dengue virus infection in a/j mice murine model for dengue virus-induced lethal disease with increased vascular permeability in vitro and in vivo assay systems for study of influenza virus inhibitors viruses of the bunya-and togaviridae families: potential as bioterrorism agents and means of control potential role of immunomodulators for treatment of phlebovirus infections of animals patient-derived tumor xenografts: transforming clinical samples into mouse models treatment of lethal pichinde virus infections in weanling lvg/lak hamsters with ribavirin, ribamidine, selenazofurin, and ampligen a comparative study of the crimean hemorrhagic fever-congo group of viruses the pathogenesis of rift valley fever virus in the mouse model effective antiviral treatment of systemic orthopoxvirus disease: st-246 treatment of prairie dogs infected with monkeypox virus a neurotropic virus isolated from the blood of a native uganda comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity experimental respiratory marburg virus haemorrhagic fever infection in the common marmoset experimental respiratory infection of marmosets (callithrix jacchus) with ebola virus kikwit essential role of platelet-activating factor receptor in the pathogenesis of dengue virus infection respiratory syncytial virus is associated with an inflammatory response in lungs and architectural remodeling of lung-draining lymph nodes of newborn lambs trans-activation of viral enhancers by the hepatitis b virus x protein filamentous influenza a virus infection predisposes mice to fatal septicemia following superinfection with streptococcus pneumoniae serotype 3 contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage. elife 2, e00481. stabenow correction: a retrospective investigation on canine papillomavirus 1 (cpv1) in oral oncogenesis reveals dogs are not a suitable animal model for high-risk hpv-induced oral cancer clinical profiles associated with influenza disease in the ferret model comparative neurovirulence and tissue tropism of wild-type and attenuated strains of venezuelan equine encephalitis virus administered by aerosol in c3h/hen and balb/c mice a mouse model for human anal cancer first reported cases of hantavirus pulmonary syndrome in canada antiviral treatment is more effective than smallpox vaccination upon lethal monkeypox virus infection evaluation of intravenous zanamivir against experimental influenza a (h5n1) virus infection in cynomolgus macaques ferrets as a novel animal model for studying human respiratory syncytial virus infections in immunocompetent and immunocompromised hosts differential pathogenesis of respiratory syncytial virus clinical isolates in balb/c mice limited dissemination of pathogenic siv after vaginal challenge of rhesus monkeys immunized with a live, attenuated lentivirus experimental west nile virus infection in rabbits: an alternative model for studying induction of disease and virus control a virus similar to human hepatitis b virus associated with hepatitis and hepatoma in woodchucks development of animal models against emerging coronaviruses: from sars to mers coronavirus severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction the clinical pathology of crimean-congo hemorrhagic fever human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes nipah virus encephalitis profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers distribution of viral antigens and development of lesions in chicken embryos inoculated with nipah virus characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the origin and virulence of the 1918 "spanish" influenza virus the pathology of influenza virus infections isolation of west nile virus from culex mosquitoes recombinant respiratory syncytial virus that does not express the ns1 or m2-2 protein is highly attenuated and immunogenic in chimpanzees animal models of hepadnavirus-associated hepatocellular carcinoma mouse models for chikungunya virus: deciphering immune mechanisms responsible for disease and pathology human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets experimental infection of ground squirrels (spermophilus tridecemlineatus) with monkeypox virus. emerg persistent west nile virus infection in the golden hamster: studies on its mechanism and possible implications for other flavivirus infections risk factors in dengue shock syndrome breaking barriers to an aids model with macaque-tropic hiv-1 derivatives dominant role of hpv16 e7 in anal carcinogenesis ribavirin efficacy in an in vivo model of crimean-congo hemorrhagic fever virus (cchf) infection histopathologic and immunohistochemical characterization of nipah virus infection in the guinea pig sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus protective efficacy of a bivalent recombinant vesicular stomatitis virus vaccine in the syrian hamster model of lethal ebola virus infection persistence of human norovirus rt-qpcr signals in simulated gastric fluid outbreak of necrotizing enterocolitis caused by norovirus in a neonatal intensive care unit pathology of experimental aerosol zaire ebolavirus infection in rhesus macaques experimental aerosolized guinea pig-adapted zaire ebolavirus (variant: mayinga) causes lethal pneumonia in guinea pigs animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors a human lung xenograft mouse model of nipah virus infection human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals virulence and reduced fitness of simian immunodeficiency virus with the m184v mutation in reverse transcriptase chronic administration of tenofovir to rhesus macaques from infancy through adulthood and pregnancy: summary of pharmacokinetics and biological and virological effects use of a small molecule ccr5 inhibitor in macaques to treat simian immunodeficiency virus infection or prevent simian-human immunodeficiency virus infection experimental infection of rhesus macaques and common marmosets with a european strain of west nile virus protective vaccination against papillomavirus-induced skin tumors under immunocompetent and immunosuppressive conditions: a preclinical study using a natural outbred animal model cataloguing of potential hiv susceptibility factors during the menstrual cycle of pig-tailed macaques by using a systems biology approach temporal analysis of andes virus and sin nombre virus infections of syrian hamsters anti-alpha4 integrin antibody blocks monocyte/macrophage traffic to the heart and decreases cardiac pathology in a siv infection model of aids clinical manifestations, laboratory findings, and treatment outcomes of sars patients. emerg prevalence of noroviruses and sapoviruses in swine of various ages determined by reverse transcription-pcr and microwell hybridization assays porcine enteric caliciviruses: genetic and antigenic relatedness to human caliciviruses, diagnosis and epidemiology avian influenza viruses, inflammation, and cd8(+) development of a model for marburgvirus based on severe-combined immunodeficiency mice development and characterization of a mouse model for marburg hemorrhagic fever euthanasia assessment in ebola virus infected nonhuman primates hematopoietic stem cell-engrafted nod/scid/ il2rgamma null mice develop human lymphoid systems and induce long-lasting hiv-1 infection with specific humoral immune responses the magnitude of dengue virus ns1 protein secretion is strain dependent and does not correlate with severe pathologies in the mouse infection model human fatal zaire ebolavirus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis immunization with modified vaccinia virus ankarabased recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets invasion of the central nervous system in a porcine host by nipah virus development of a rift valley fever virus viremia challenge model in sheep and goats antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever an unusual hantavirus outbreak in southern argentina: person-to-person transmission? hantavirus pulmonary syndrome study group for patagonia. emerg equine morbillivirus pneumonia: susceptibility of laboratory animals to the virus susceptibility of cats to equine morbillivirus hantaan virus infection causes an acute neurological disease that is fatal in adult laboratory mice a north american h7n3 influenza virus supports reassortment with 2009 pandemic h1n1 and induces disease in mice without prior adaptation transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats a guinea-pig model of hendra virus encephalitis susceptibility of hiv-2, siv and shiv to various anti-hiv-1 compounds: implications for treatment and postexposure prophylaxis a golden hamster model for human acute nipah virus infection ebola virus transmission in guinea pigs intranasal immunization with an adenovirus vaccine protects guinea pigs from ebola virus transmission by infected animals characterization and experimental transmission of an oncogenic papillomavirus in female macaques dengue haemorrhagic fever: diagnosis, treatment, prevention and control, second ed. world health organization, geneva. world health organization clinical aspects of hepatitis b virus infection civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates evidence of a causal role of winter virus infection during infancy in early childhood asthma vertical transmission of zika virus targeting the radial glial cells affects cortex development of offspring mice the antiviral activity of sp-303, a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type 3 viruses in cotton rats experimental infection of prairie dogs with monkeypox virus. emerg epidemiological studies of hemorrhagic fever with renal syndrome: analysis of risk factors and mode of transmission hydrodynamic injection of viral dna: a mouse model of acute hepatitis b virus infection mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection an animal model of mers produced by infection of rhesus macaques with mers coronavirus a protective role for dengue virus-specific cd8+ t cells the incubation period of hantavirus pulmonary syndrome animal model of sensorineural hearing loss associated with lassa virus infection the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus. epidemiol hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease the pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (macaca fascicularis) hiv-1 infection and pathogenesis in a novel humanized mouse model h7n9 influenza viruses are transmissible in ferrets by respiratory droplet induction of neutralizing antibodies against four serotypes of dengue viruses by mixbiediii, a tetravalent dengue vaccine rapid generation of a mouse model for middle east respiratory syndrome an animal model for studying the pathogenesis of chikungunya virus infection pathogenesis of avian influenza a (h5n1) viruses in ferrets lethal crimean-congo hemorrhagic fever virus infection in interferon alpha/beta receptor knockout mice is associated with high viral loads, proinflammatory responses, and coagulopathy the pathogenesis of western equine encephalitis virus (w.e.e.) in adult hamsters with special reference to the long and short term effects on the c.n.s. of the attenuated clone 15 variant development of a murine model for aerosolized filovirus infection using a panel of bxd recombinant inbred mice a characterization of aerosolized sudan ebolavirus infection in african green monkeys, cynomolgus macaques, and rhesus macaques characterization of disease and pathogenesis following airborne exposure of guinea pigs to filoviruses manuscripts in preparation opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the us army. key: cord-018239-n7axd9bq authors: rusoke-dierich, olaf title: travel medicine date: 2018-03-13 journal: diving medicine doi: 10.1007/978-3-319-73836-9_32 sha: doc_id: 18239 cord_uid: n7axd9bq before travelling to other countries, thorough travel advice should be provided. not only information about diseases of specific countries but also general advice for travelling should be given on this consultation. before travelling to other countries, thorough travel advice should be provided. not only information about diseases of specific countries but also general advice for travelling should be given on this consultation. the following topics should be included in the travel advice consultation: 5 vaccinations (general and country specific) 5 country-specific diseases 5 malaria prophylaxis 5 mosquito prophylaxis (wearing bright long-sleeved clothes, avoiding perfume, staying in air-conditioned rooms, using a mosquito net, using insect repellents, staying inside at dawn and dusk) 5 food consumption and drinking overseas (no consumption of ice cubes, uncooked meals, salads and food, which is exposed to flies, limited alcohol consumption) 5 uv protection (using sun cream, avoiding sun exposure between 11.00 and 15.00 o' clock, remaining in shaded areas, wearing a hat and covering skin) 5 fitness assessment for travelling, flying and diving 5 challenges of different climates and their effects on the personal health (dehydration, hyperthermia) 5 medications 5 thrombosis counselling 5 counselling on symptoms on return, which require review (fever, skin changes, abnormal bleeding, lymphadenopathy, diarrhoea) 5 sexual transmitted diseases 5 contraception 5 rabies the following items should be asked to enable to give the appropriate advice: 5 risk assessment of the travel in a particular country (transport, area of stay/ rural or resort, reason for travelling, appropriate conduct overseas, pre-existing diseases and medications) 5 vaccination status 5 accomodation and stopovers 5 duration of the stay the vast majority of up-to-date travel information and information about tropical disease are available on who (world health organization) or cdc (centres for disease control and prevention) websites. information on these websites are frequently updated. before giving appropriate advice based on these online resources, it should be checked, which medications are available in the particular countries. hence, recommendations need to be adjusted individually. usually, a medication record is required at the customs. however, it might be sufficient, if the original medication box has the patients and prescribing doctors details (. table 32 .1). malaria is a tropical disease transmitted by the female anopheles mosquito. the distribution of malaria is primarily in the tropics and subtropics of africa, central and south america, asia, papua new guinea and the western pacific islands. as popular diving spots are located in these areas, malaria prophylaxis and advice should be given. the who (world health organization) estimates the worldwide number of people affected by malaria with about 198 million and 1,200,000 deaths (2013). the plasmodium parasites need temperatures above 20 °c in order to complete the entire growth cycle. therefore, malaria occurs in some places only seasonal. additionally, there are differences in anopheles species regarding the affinity to the host and their local distribution. some genetic factors are protected against malaria. for example, sickle cell anaemia gives a certain protection against p. falciparum and duffy negative blood group against p. vivax. it appears that after recurrent malaria infections, the body adapts to the disease. this means that an infection is possible, but the symptoms of malaria seem to be reduced. children and pregnant women have an increased risk of being affected by malaria. additionally, children have a high mortality rate. during pregnancy the resistance against malaria is reduced. it also poses an increased risk for the unborn child (low birth weight). anopheles is active especially at sunrise and sunset. different kinds of mosquitoes are rather active during the day and can transmit other diseases such as dengue. especially p. falciparum and p. vivax have resistances against antimalaria drugs. there are different plasmodium pathogens: 5 p. falciparum: worldwide tropical and subtropical distribution, mainly in africa; pathogen of severe malaria causes 1 million deaths per year; rapid growth in the blood with haemolysis and emboli due to cytoadherence of affected erythrocytes; 7-30 days of incubation, irregular fever spikes. 5 p. vivax: mainly in asia, latin america and some countries in africa; the disease can be activated after months or years. incubation period of 12-18 days; fever spikes every 2 days. 5 p. ovale: mainly west africa and the western pacific islands. similar to the p. vivax, it can also infect people with duffy-negative blood group; incubation period of 12-18 days; fever spikes every 2 days. 5 p. malariae: worldwide distribution; typical 3-day cycle, untreated can lead to lifelong chronic malaria; incubation period 16-50 days; fever spikes every 3 days. 5 p. knowlesi: southeast asia, mainly infected animals. after the anopheles mosquito aspirates with gametocytes infected blood, the gametocytes develop to gamete in the mosquito's intestines. in the blood of the mosquito, the microgametes (male) penetrate the macrogametes (female), forming zygotes. then cells are changed to an elongated, motile ookinete. this evolves into an oocyst. after the oocyst bursts, sporozoites are released and get in the saliva of the mosquito. the entire cycle inside the mosquito takes 8-16 days. if sporozoites enter the human bloodstream through the saliva of the . symptoms of malaria appear after the incubation period. the incubation period varies depending on the pathogen. it can be between a few weeks and also takes up to several months or even a year (p. vivax or occasionally p. ovale). malaria can be divided in three different forms: 5 malaria tertiana: pathogen: p. vivax and p. ovale; fever every second day with one day without fever, spontaneous remission after max. 5 years 5 malaria quartana: pathogen: p. malaria; fever every third day with 2 days without fever, no spontaneous remission 5 malaria tropica: pathogen: p. falciparum, irregular fevers due to the lack of synchronisation of the parasite reproduction, severe form of malaria (malaria maligna) with high fatality, recurrence up to 2 years the fever has a specific pattern. in the first hour, strong rigors and increasing fever typically develop. the fever can reach 40 °c and more for duration of about 4 h. it is often associated with flushing, vomiting and nausea. the fever stage is followed by an approximately 3-h stage of severe sweating with decreasing fever. severe forms of malaria can be fatal in within few days. causes of death are cerebral malaria, respiratory failure with adrs and kidney failure. the main reason of these complications is the cyto-adherence ("bonding") of the erythrocytes. it results in a failure of the microcirculation followed by ischaemia of vital organs. . the treatment depends on the severity and the pathogen. in complicated malaria, admission to the intensive care should be considered, if more than one of the following criteria exists: 5 inability of the oral intake of medication 5 parasite load of erythrocytes >2% 5 severe symptoms of malaria (see table above) the treatment options of complicated malaria are: 5 artesunate (allowed only in some countries): 2.4 mg/kg/bw iv; first dose on admission, repeated after 12 and 24 h, minimum duration of therapy 24 h and then once a day, till oral therapy is tolerated. or 5 combination of quinine + doxycycline or clindamycin. 5 quinine: ȥ first dose: 20 mg/kg/bw iv over 4 h or 7 mg/kg/bw iv over 30 min with subsequent administration of 10 mg/kg/bw iv over 4 h. ȥ maintenance therapy: 10 mg/kg/bw iv over 4 h three times a day, beginning 4 h after the completion of the first dose. ȥ exemption: if the patient received three or more doses of quinine in the last 48 h or had an mefloquine prophylaxis in the last 24 h or received a mefloquine treatment in the last 3 days. + 5 doxycycline: 100 mg iv twice daily for 7 days (iv or oral) or 5 clindamycin: ȥ initial dose: 10 mg/kg/bw ȥ maintenance dose: 5 mg/bw every 8 h for 7 days (iv or oral) 5 after clinical improvement medication can be changed to a complete cycle of the oral therapy of an uncomplicated malaria (riamet ® or quinine with doxycycline or clindamycin). uncomplicated malaria can be handled on the normal ward. outpatient therapy with close supervision can be considered under the following conditions: 5 parasite load of erythrocytes <1%. 5 age > 12 months. 5 no co-morbidity. 5 pregnancy is excluded. 5 ability of oral medication intake. 5 p. falciparum is excluded. 5 clinically stable under medical therapy for the last 24 h. a daily blood smear is necessary during treatment to follow the process of the disease. the patient can be discharged from the hospital and continue treatment at home; if oral therapy is tolerated, a clinical improvement is achieved and the parasite count decreases. a week and a month after discharge, blood smears should be repeated. primaquine as eradication therapy is approved in some countries. it is the only drug that can be used to eliminate hypnozoites, which are the dormant forms of the malaria parasites that occur with p. ovale and p. vivax. because primaquine causes haemolysis in g-6-pd deficiency, g-6-pd status prior therapy needs to be established. if an eradication with primaquine is required in patients with g-6-pd deficiency, a dose up to 45 mg weekly for 8 weeks, with monitoring for haemolysis, could be considered. in children methaemoglobinaemia can be provoked by giving primaquine. a single dose of primaquine 45 mg for p. falciparum, p malaria and p. knowlesi can be given to sterilise the gametocytes. if malaria caused by p. vivax or p. ovale or co-infection with these parasites is suspected, a 14-day treatment with 15 mg of primaquine twice a day is recommended. before commencing holidays overseas, medical advice should be given in order to assess the malaria risk of the particular country. in nearly all tropical areas, there is a risk of getting infected with malaria. in some tourist areas, this risk might be small, but infection is still possible. in particular day trips to more remote areas pose a risk. some areas have malaria outbreaks and therefore should be avoided. in general, mosquito bites should be avoided to minimise the risk of any mosquitoborne infections. mosquitoes transmitting malaria are mainly active at night, sunrise and sunset. however, mosquito bites are also possible throughout the day. long-sleeved shirts, long pants and closed shoes cover the skin and provide protection against insect bites. insect repellent for the skin and clothes offer additional protection. higher concentrations offer better and longer protection. the protection period of a normal insect repellent lasts usually only 1-2 h. slow release products can prolong the effect. mosquitoes avoid air-conditioned rooms. so staying in air conditioned rooms itself provides certain protection. spraying insecticides in rooms and surroundings can be helpful to repel and minimise the quantities of mosquitoes. the bed should be covered with a mosquito net (. fig. 32 .2). chemoprophylaxis is important, because the main cause of malaria deaths is still inadequate chemoprophylaxis. there are different drugs for chemoprophylaxis available. they are subject to the travel location and the parasite's resistances to certain drugs. in addition, they differ in side effects, dosage and cost. except malarone ® , all other drugs for the chemoprophylaxis against malaria have to be taken 4 weeks after leaving the country as they aren't sufficiently effective against the primary liver stages of malaria. mefloquine (lariam ® ) is the only malaria prophylaxis without absolute contraindication in pregnancy. diving (decrease in vigilance); 2-3 weeks (at least 1 week) before entering the malaria-endemic country and 4 weeks after return; lariam ® is a category b medication and is the only medication against malaria without absolute contraindication in pregnancy. the use in the first trimester should only be considered, if the expected benefits justify the potential risk to the foetus. however, recent studies suggest that even in the first trimester this medication is safe to take. the dengue virus is an arbovirus. it has four different serotypes (denv 1-4). dengue has a worldwide distribution in the tropics and subtropics, especially in asia and south america. approximately 50-100 million cases and about 100,000 with serious complications per year occur. there is a 10% mortality, which can be reduced to 1% with timely diagnosis and appropriate treatment. it has an increased risk for children under 15 years and persons with previous dengue infections. the dengue virus is transmitted by the aedes aegypti mosquitoes. these mosquitoes mainly bite at day and in twilight (. fig. 32 .3). z symptoms the incubation period is 2-10 days. there is a wide range in severity of dengue symptoms. the majority of infections cause minor symptoms. but dengue infections can be also quite severe (. table 32 .3). in particular recurrent infections with dengue are associated with complications and severity of the disease. it is important for the treating doctor to remember that after the initial fever, the critical phase follows. therefore, the patient must be monitored closely during this time. the disease goes through three stages: 5 fever phase (day 1-3): sudden high fever 40 °c occasional associated with bradycardia; myalgia mainly in the spine, arms and legs ("breakbone fever"), headache; retrobulbar pain; rigors; metallic/bitter taste; vomiting; and dehydration. . 5 critical phase (day 4-5): normal temperature with possible mild fever later on, leucopenia, exanthema, petechiae and lymphadenopathy. severe dengue: abdominal pain, spontaneous bleeding, volume shift in to the peritoneal space ("plasma leak"), pleural effusion, hepatomegaly (≥2 cm), rapid increase in haematocrit and decreasing thrombocytes, shock (dengue haemorrhagic shock = dhs or dengue shock syndrome = dss), increased bleeding (dengue haemorrhagic fever = dhf) and organ failure (particularly liver). 5 remission (after 6 days lasting sometimes for weeks): risk of hyperhydration is given when extravascular fluid is reabsorbed without reducing the intravenous fluid administration. in particular in long remissions, fatigue and depression may be present. normally there are no long-term damages after a dengue infection, and the vascular changes recover completely. z treatment there is no medication available to treat dengue directly. the diagnosis of dengue can be demonstrated by pcr in the initial phase and using igm and igg a few days later. due to severe complications, the haematocrit, coagulation parameters, leukocytes and platelets have to be tested daily. thrombocytes <100,000 cells/mm 3 can rise the suspicion of dhf. if pleural effusion is suspected, a cxr should be obtained. by tightening a blood pressure cuff petechiae can be provoked (medium pressure of the systolic and diastolic pressure for 5 min). this can be used as a diagnostic tool. an increase of the haematocrit of >20%, pleural effusion, ascites or hypoproteinaemia could be a sign for extravascular fluid loss. the extravascular fluid loss is typically found in the initial phase. hence, fluid replacement therapy is crucial in this phase. as the extravascular fluid loss can come to an end quite quickly, a complication of the fluid replacement therapy is hyperhydration. decrease of haematocrit of >20% after fluid administration can represent a fluid excess and hyperhydration. hence, careful monitoring of the fluid balance and weight are necessary. the therapy is adjusted according to its severity. if necessary, dic, blood loss or shock require specific treatment. like dengue, chikungunya is a mosquitoborne disease. the species transmitting the chikungunya virus (chikv) are aedes aegypti in the tropics and subtropics and aedes albopictus in colder regions (. fig. 32 .4). these mosquitoes bite day and night, but mainly in the early morning hours and late afternoon. the incubation period is between 2 and 12 days. the symptoms are similar to that of dengue. patients suffer from sudden fever with headache, skin rash, fatigue, strong limbs and muscle pain. affected joints often are swollen. the symptoms generally last for few days but can persist for weeks and years. the disease has no long-term effects. for diagnosis rt-pcr and virological methods can be used in the initial phase. later, it can be diagnosed by igm and igg. igm peaks after 3-5 weeks and can be detected up to 2 months. the treatment requires analgesia only. . yellow fever is a disease transmitted mainly by the aedes aegypti mosquito but also by other mosquitoes or ticks. the pathogen is a rnacontaining flavivirus. it has approximately 200,000 infections with approximately 30,000 deaths annually. 90% of cases occur in africa and the remaining 10% in south america. the risk of getting infected with yellow fever is with 1:200-2000 in africa and higher than 1:20000 in south america (. fig. 32 .5). the transmission occurs in rainforest areas (jungle or sylvatic cycle), where mosquitoes transfer the virus from monkeys to humans, in endemic areas of the savannah (savannah or intermediate cycle) either transferred from monkeys or human to humans via mosquitoes or in urban areas from human to human via mosquitoes. the incubation period is 3-6 days. the disease has two phases. the acute phase comes with fever, headache, myalgia, headand backache, loss of appetite, nausea, vomiting and diarrhoea. the second phase occurs only in approx. 15% of infected humans within the next 24 h. jaundice, abdominal pain and vomiting are rapidly developing, followed by diffuse bleeding (epistaxis and gi bleeding) and multi-organ failure (mainly kidneys). if symptoms of the more severe second phase develop, 50% of the patients die within the next 10-15 days. patients who survive usually recover without significant organ damage. the diagnosis can be made via a blood or tissue biopsy of the liver. there is no cure for yellow fever and only supportive measures can be taken. however, a very effective life-vaccination (stamaril ® ) is available. only authorised doctors are authorised to prescribe and give the vaccine. severe side effects of these vaccinations are severe allergic reaction (1:55000), vaccine-associated neurotropic disease/post-vaccinal encephalopathy (1:125000) and vaccine-associated viscerotropic disease/ multi-organ failure (1:400000). for travelling into countries where yellow fever is endemic, vaccination is mandatory. the side effects seem to be age-related and occur increasingly with progressive age or in young children. the vaccination is contraindicated in children 32.2 · other mosquito-borne diseases below 9 month and during pregnancy. analysis of yellow fever vaccines adverse events demonstrated an increased frequency of serious adverse events in persons age 60 years and older. the risk of viscerotropic side effects in <65 years is 1:400000, in a population of 65-75 years of age 1:40000 and in >75 years of age 1:4000. a failure to be vaccinated or being documented can lead to a refusal of entry into other countries or to a certain time in quarantine when leaving the area where yellow fever occurs. if there is a clinical indication against receiving yellow fever vaccine (e.g. children <9 month or poor immune status), a written medical exemption can be granted, to enable to travel to these countries without vaccination. absolute contraindications for a yellow fever vaccination are: 5 allergy against the vaccine or egg protein 5 age < 6 months 5 immunodeficiency 5 neoplasia 5 transplantations 5 immunosuppressive therapies relative contraindications for a yellow fever vaccination are: 5 age 6-9 months 5 age > 60 5 asymptomatic hiv infections and cd4+ t lymphocytes 200-499/mm 3 (15-24% of the total in children <6 years of age) 5 pregnancy 5 lactation aedes aegypti spreads also the zika virus. however, it is also sexually, intrauterine and perinatal transmitted. currently the main distribution is countries in south and north america as well as the caribbean islands, singapore and some countries in south pacific islands. symptoms of zika infection may be fever, rash, arthralgia, myalgia, headache and conjunctivitis. but in most cases, an infection is asymptomatic (~80%). these symptoms are lasting for several days to a week. the incubation period is 3-14 days but is likely to be a few days to a week. the diagnosis can be made via pcr or serology. blood pcr can be detected only in the first week of the disease. urine pcr can detect the virus up to 2 weeks. there is no specific treatment available. deaths are unlikely. there is a potential risk during pregnancy, as microcephaly or other birth defects (~20%) may develop. the zina virus cane be also transferred via semen and can affect unborn life. ross river virus (rrv) is transmitted by the bites of culex annulirostris, aedes vigilax, aedes normanensis and aedes notoscriptus in australia, papua new guinea, parts of indonesia and the western pacific islands. the main transmission time is in the humid summer month from december till march. the main symptoms are fever, rash, headache, myalgia, arthralgia and fatigue. the initial symptoms with fever last usually for 1-2 weeks. myalgia and arthralgia usually last longer. symptoms of fatigue and depression can be late complications. the incubation time is between 3 days and 3 weeks. the diagnosis is made with igm. there is only symptomatic treatment available. barmah forest virus (bfv) is transmitted by the same species as the rrv. it mainly can be found in australia. many people don't develop any symptoms. the incubation time is 3-11 days. if symptoms appear, they are similar to the one of rrv. the initial symptoms last for 1-2 weeks, and the arthralgia and myalgia may last for 6 months. the diagnosis is made with igm. there is only symptomatic treatment available. sindbis virus (sinv) is related to the chikungunya virus. it is mainly transmitted via the culex and culiseta mosquitoes. it can be found in europe, africa, asia and oceania. the symptoms and the duration of the symptoms are quite similar to rrv and bfv. the diagnosis is made with igm. there is only symptomatic treatment available. the o'nyong-nyong virus (onnv) is related to the chikungunya virus but is restricted to africa. it has similar symptoms as the chikungunya virus but has additionally mainly cervical lymphadenopathy, and the affected joins rarely show signs of an effusion. most of the gastrointestinal tract infections are caused by poor hygienic conditions of the travel destination. occasionally ingested seawater can cause intestinal infections too. the main transmission routes are either food-borne or by contact. however, the most common cause for gastrointestinal infections is eating contaminated food. old, warmed up food, salads, unpeeled fruits, poorly cooked food, contaminated water (ice and already opened bottles with refilled water) and ice cream often have substantial quantities of pathogens and pose a risk. hence, the best protection against gi infections is avoiding contaminated food or drinks. usually gastrointestinal infections last for a few days and are self-limiting. if diarrhoea contains blood or mucus in combination of high fever for more than 2 days, more thorough assessment is required. blood and mucus without fever are most likely related to a parasitic disease. if fever is present, it's most likely a bacterial or viral disease. but also climate change by itself or dehydration may be caused by autonomic dysregulation gastrointestinal symptoms such as nausea, weakness, vomiting and diarrhoea. with dehydration the dci risk increases. rehydration and supply of certain electrolytes such as sodium, chloride and potassium are the most important treatments for gastroenteritis. fatigue is a common associated symptom. tannins of black tea boiled for more than 10 min might be beneficial for diarrhoea. the consumption of bananas is recommended because of the high content of potassium. but the best options are rehydration preparations in form of drinks, powders or icy poles. loperamide may slow down the peristaltic and give some relief from diarrhoea. probiotics may support recovery. a low fibre diet is rec-ommended in the active phase of diarrhoea. administration of antibiotics is rarely necessary and indicated. it only is used for serious illnesses or symptoms. 5 reservoir: poultry or meals prepared with egg 5 incubation: 8-48 h 5 symptoms: fever, vomiting nausea, diarrhoea, occasionally blood and mucous in the stool 5 duration: 4-7 days 5 treatment: symptomatic; azithromycin 1 g od for 5 days or ciprofloxacin 500 mg bd for 7 days or ceftriaxone 2 g od 5 reservoir: water and food 5 incubation: 1-2 weeks 5 symptoms: headache, myalgia, bradycardia, roseola in the abdominal area, continuous fever 39-40 °c, porridge -like diarrhoea, intestinal bleeding and decrease of the fever after 4 weeks 5 treatment: symptomatic; azithromycin 1 g od for 5 days or ciprofloxacin 500 mg bd for 7 days or ceftriaxone 2 g od; vaccination available 5 reservoir: human, flies, food and faeces 5 incubation: 2-5 days 5 symptoms: fever, diarrhoea, sometimes with blood and mucus in the stool and severe abdominal pain 5 treatment: symptomatic; ciprofloxacin 500 bd for 5 days, norfloxacin 400 mg bd for 5 days or bactrim 160/800 mg bd for 5 days 5 reservoir: food and water 5 incubation: 0-2 days 5 symptoms: mild to severe diarrhoea with fever and blood and mucous in the stool, most common cause for diarrhoea overseas 5 treatment: symptomatic; norfloxacin 400 mg od and ciprofloxacin 500 mg od 5 reservoir: food (particular strawberries) and water 5 incubation: 1-4 weeks 5 symptoms: diarrhoea like raspberry jelly, no fever! blood and mucous in the stool, risk for developing a liver abscess 5 treatment: symptomatic, asymptomatic carrier, paromomycin 500 mg tds for 7 days; invasive, tinidazole 2 g od for 3 days or metronidazole 400 mg tds for 7 to 10 days 32.3.6 cholera (vibrio cholerae) 5 reservoir: contaminated food and water 5 incubation: 0-5 days 5 symptoms: often mild gi symptoms, 5-10% develop severe symptom with nausea vomiting, rice water-like diarrhoea and severe dehydration, mortality risk of 25-50% 5 treatment: rehydration, electrolyte substitution; vaccination available; azithromycin 1 g single dose, ciprofloxacin 1 g single dose 5 reservoir: food (in particular sea food) and water 5 incubation: 15-50 days 5 symptoms: initial phase (2-7 days)flulike symptoms, gastrointestinal, hepatomegaly; hepatic manifestation (4-8 weeks), no jaundice (approx. 70%), jaundice (30%) with dark urine, jaundice, pruritus; hepatitis a has no chronic form, rarely fatal (fatality is age dependent) 5 treatment: symptomatic, bed rest, avoidance of liver toxic substances (alcohol, medication); vaccination available japanese encephalitis is caused by a flavivirus, which is transmitted by mosquitoes (culex particularly c. tritaeniorhynchus). the hosts are usually pigs and water birds. in humans there are usually not sufficiently high concentrations of virus to serve as a host. the distribution is the asia, especially in rural areas. epidemics occur every 2-15 years (. fig. 32.6 ). the transmission can occur throughout the year but frequently peaks in the rainy season. there are about 68,000 cases per year. only about 1% of the patients are symptomatic. however, if symptoms develop, the mortality rate is 20-30%. approx. 30-50% of patients who survive have long-term neurological or psychiatric complications. mild courses of japanese encephalitis may be accompanied by mild fever and headache. severe cases show high fever, neck stiffness, photophobia, headache, disorientation, coma, convulsions, spastic paralysis or death. consequential damages may be behavioural disorders, convulsions, paralysis and speech disorders. the diagnosis can be established with blood tests and lumbar puncture. there is currently no treatment option. the vaccination is usually well tolerated and available for prophylaxis. there are various tropical diseases, which are present in poorer countries causing more or less severe symptoms. these diseases are termed "neglected tropical diseases" (ntd). the more common ntds are summarised in this chapter. there are three main conditions caused by these pathogens. the african trypanosomiasis (sleeping sickness) is transmitted by the tsetse fly. the distribution is only in some countries of the sub-saharan africa. seventy percent occur in the democratic republic of congo. tsetse flies are mainly found in rural areas. there are two forms causing sleeping sickness, t. brucei rhodesiense and t. brucei gambiense. t. brucei gambiense has an incubation period of months to years and t. brucei rhodesiense weeks to months. the initial phase is the haemolytic-lymphatic phase, in which pathogens replicate in tissues, blood and lymphatic tissues. symptoms are intermittent fever, headache, myalgia and pruritus. additionally, a painless, indurated chancre on the skin 5-15 days after the bite and lymphadenopathy (axillary and inguinal) can be associated. in the second phase, the cns affected causes continuous headache, behavioural disorders (mood swings and depression), delirium, sensitivity disorders, coordination problems and disruptions of the sleeping cycle (daytime somnolence). the diagnosis is mainly made clinically. only for the t. b. rhodesiense, a blood test (centrifuged or wet preparation) to data . detect the parasite is available. examination of buffy coat increases sensitivity. a biopsy of the lymph node to detect the pathogens can be diagnostic for t. brunei gambiense or be used for a culture and pcr. the card agglutination test for trypanosomiasis (catt) is a field test suitable for mass population screening in endemic areas for t. b. gambienses but has a low specificity and is hence only used for identifying suspected cases. all diagnosed patients need to have their cerebrospinal fluid examined for staging, which influences treatment options (. table 32 .4). the treatment is dependent on the pathogen and the staging. if untreated, infections of both forms lead to coma and death. leishmaniasis has three forms: visceral, cutaneous and mucosal (kala-azar). there are about 30 different pathogens, from which approx. 20 are held responsible for these diseases. the disease is transmitted by mosquitoes or sandflies (phlebotomus and lutzomyia). the cutaneous form is the most common one, which causes skin ulcerations. typically this form appears weeks to months after the initial mosquito bite. initially papules are formed, which later ulcerate. they can be painful or painless. the visceral form affects organs, especially the liver, spleen and bone marrow. therefore, this form can be quite dangerous. the changes occur within months and years. hepatosplenomegaly and pancytopenia develop. the mucus form is rare. ulcerative changes of the mucous membranes (e.g. nose, mouth and throat) are typical for this. endemic areas for leishmaniasis are east africa, some arabic countries, india, bangladesh, brazil and some other south american countries. historically, the diagnosis was made by taking a biopsy (skin, bone marrow or other tissues) for culture. now pcr or serological testing with high sensitivity replaced biopsies for making diagnosis. as the visceral disease is fatal without treatment, it needs to be treated in any case. all other forms require normally no treatment. following medication is available: 5 pentavalent antimonial (sb v ) compounds (20 mg per day iv or im for 28 days) 5 liposomal amphotericin b (3 mg od iv on day 1-5, 14 and 21) 5 miltefosine (in adults > 45 kg 50 mg 3 times daily for 28 days) 5 azoles (fluconazole 200 mg od for 6 weeks, itraconazole 200 mg bd for 28 days, ketoconazole 600 mg od for at least 28 days) 5 paromomycin (uncommonly used) 5 pentamidine isethionate (uncommonly used) the chagas' disease is transmitted via an insect bite ("kissing bug") or by contaminated food. it occurs in central and south america. it has . an acute and chronic phase. in the acute phase within 1-2 weeks after the infection, localised swelling of the area of the insect bite (skin or mucous membranes), lymphadenopathy, bilateral orbital oedema, meningoencephalitis and myocarditis can occur. 20-30% of all infections become chronic, causing arrhythmias with risk of "sudden death", cardiomyopathy and enlargement of the oesophagus (megaoesophagus) or of the colon (megacolon) even after years or decades. the cardiomyopathy consists of fibrosing myocarditis, causing arrhythmia (rbbb, left anterior fascicular block, st changes, premature ventricular beats and bradycardia) and ventricular failure. the diagnosis in the acute phase is made by a blood smear (thick and thin) to visualise the parasite. a serological test is also available. treatment is recommended in the acute phase and in patient up to the age of 50 and no advanced cardiomyopathy with chronic chagas' disease (. table 32 .5). in age groups above 50, benefits and risk need to be outweighed. worm infections are a major problem in underdeveloped countries. they occur mainly in rural areas. these conditions may cause insignificant symptoms but also lead to serious consequences or even cause death. because some dive sites are located far away from tourist centres, these infections should be discussed before travelling. this kind of roundworm is found in the tropical and subtropical regions of africa and southeast asia. the transfer follows on oral intake of eggs by contaminated food. the larvae are entering the bloodstream after hatching in the intestine. they reach the lungs via the blood and penetrate the lung tissue, and the larvae can be coughed up. if the sputum is swallowed again, the larvae reach the intestine, mature there within the next 2-3 months and lay eggs, which are then excreted via the faeces. the adult worms live about 1-2 years. infection is usually asymptomatic. however, abdominal pain, flulike symptoms, allergic skin manifestations, malnutrition, productive cough and a stridor can occur. the diagnosis can be made by examining the faeces (eggs, worms) or sputum (larvae). hookworms are found in tropical and subtropical regions of africa and latin america. the transmission is percutaneously or orally by ingestion of contaminated soil. in contaminated soil the larva is able to survive for about 3-4 weeks. larvae can penetrate the skin and enter the blood and reach the alveoli in the . lungs. from there they ascend in the airways, are swallowed again and finally get into the intestines. there larvae mature to adult worms. the worms attach themselves to the wall of the intestine and feed on blood. the eggs are excreted in the faeces and reach again the soil. the eggs can survive up to 2 years. common symptoms are pruritus and rash at the entry site, abdominal pain, diarrhoea, weight loss, anaemia and extreme fatigue. the diagnosis can be made of the faeces. z filariasis filariasis has a worldwide distribution in tropical and subtropical regions. it is caused by wuchereria bancrofti and brugia malayi. it is transmitted by mosquitoes. the infective filariform grow inside mosquitoes and enter via its saliva during the bite. they migrate to the lymphatic vessels and lymph nodes where they develop into adults. they can live there for about 6 years. the female worms produce microfila, which are circulating in the blood. absorbed by mosquitoes they develop within 1-2 weeks to the infective filariform. initially there are no symptoms. later lymph oedema in extremities or genitals is a common symptom. in men hydrocele can develop. the skin typically swells and hardens ("elephantiasis"). the diagnosis is made via the blood. detection in the blood smear has to be performed at night, as larvae only circulate in the blood at night. there is also a serological detection of anti-filaria igg4 available for diagnosis. the treatment with dec is the drug of choice. concurrent disease of loa loa or onchocerciasis is a contraindication for dec, because of the serious side effects (encephalopathy and deaths). ivermectin is used as a prophylaxis, but not as a therapy. z schistosomiasis (bilharziose) schistosomiasis can be found in tropical and subtropical regions worldwide. in addition to malaria, it is the most common parasitic disease. the parasite schistosoma is housed in freshwater snails. by being exposed to freshwater in these regions, infections can occur. the eggs are excreted in urine or faeces of the host. they hatch under optimal conditions and release miracidia. these miracidia infect freshwater snails and develop into sporocysts. these develop into cercariae and get released into the water, where they can penetrate the skin of the host. there, they shed their tail and become schistosomulae and migrate to the liver. in the liver they mature into adults. the paired adult worms migrate to the bowel and bladder, where they lay the eggs. a rash ("swimmers itch") may develop at the entry site on the skin. suprapubic pain and haematuria, abdominal pain, myalgia, fever, swelling of the lymph nodes, liver and spleen enlargement and eosinophilia can be additional symptoms. the risk of bladder cancer is increased with schistosomiasis. the diagnosis can be made in the stool and urine. the maximum excretion of eggs in the urine is between 12 and 3 pm. z trichuriasis (whipworm) whipworms have a worldwide distribution in the humid tropics. the eggs are orally absorbed via soil or unwashed vegetables or fruits. the whipworm grows in the large intestine. the eggs are excreted via the faeces. in the soil the eggs pass through various stages before getting absorbed again. the symptoms are abdominal pain, chronic diarrhoea, nausea, vomiting, inflammation of the intestine, anaemia and eosinophilia. the diagnosis is made with a stool sample. the treatment on the infection is dependent on the parasite (. table 32 .6). leptospirae are long, motile spirochetes. they have a worldwide distribution, but infections occur more commonly in tropical and subtropical regions. they spread through infected urine, which enters water or soil. leptospires can survive for several weeks and months. infections can be caused by contact with either direct contact with the urine or other body fluids except saliva as well as with contaminated soil and water. the bacteria enter the body through the skin or mucous membranes. a broken skin increases the risk of infection. increased risk is after heavy rainfall or flooding. the incubation period is usually 5-14 days, but can range from 2-30 days. symptoms vary greatly. usually sudden onset of headaches, fever, chills, myalgia, nausea and vomiting, diarrhoea, rash and jaundice are common signs of the first phase for 3-8 days. if the patient doesn't recover the second phase (weil's disease) develops, with renal failure, ards, hepatomegaly, jaundice, haemorrhage and meningitis. this has a fatality rate of 1-5%. untreated symptoms can persist for several months. treatment is either doxycyclin 100 mg bd or benzylpenicillin 1.2 g qid or ceftriaxone 1 g od for 7 days. infections caused by rickettsia, orienta, ehrlichia, neorickettsia, neoehrlichia and anaplasma are summarised as rickettsial infections. rickettsias are divided into the typhus group and the spotted fever group. orienta make up the typhus group. the reservoir is found in mainly animals, like rodents, but some species are found in fish. the vector is commonly ticks. in scrub typhus the vectors are larval mites. others have fleas and lice as a vector. infection occurs either by bites of the vectors or by direct contact, inoculation or inhalation of contaminated fluids or faeces. the clinical presentation varies. mild symptoms are headache, myalgia, abdominal pain, cough and rash. some rickettsial infections, . q-fever is a zoonosis caused by the protozoa coxiella burnetii. the bacterium is quite resilient due to its sporelike life cycle and remains virulent for months even up to more than a year. the primary reservoir is cattle, goats, sheep and other wildlife like kangaroos, rats and cats. rarely is it transmitted by tick bites or by ingestion of unpasteurised milk or dairy products. the incubation time is usually 2-3 weeks but can range from 2 days to 6 weeks. the initial acute q-fever comes with sudden onset of high fever up to 40 °c, headache (retrobulbar), myalgia, chills, non-productive cough and sweats. the symptoms settle within 5-14 days. 50% of all infections are however asymptomatic. often thrombocytopenia and abnormal lfts are found. complications are ards, endocarditis and meningoencephalitis. the diagnosis is based on detecting phase ii and phase i antibodies (igg) 4 weeks apart. the initial test (phase ii) should be taken at the end of the first week of illness. igm and igg rise almost at the same time. a fourfold rise is diagnostic. an initial negative titre doesn't rule out q-fever. seroconversion occurs usually between days 7 and 15 but is almost always present by 21 days. pcr testing can be used in the first 2 weeks but before antibiotic administration. however, a negative pcr result doesn't rule out q-fever. chronic q-fever develops in 0.2-4%. it can result in endocarditis, aneurysms, osteomyelitis, hepatitis, neurogic (mononeuritis, optic neuritis), pulmonary (interstitial fibrosis, pseudotu-mor) and renal (glomerulonephritis) disease. chronic q-fever usually develops shortly after the infection. however, chronic endocarditis may not come apparent until 2-4 years or even longer. chronic fatigue syndrome is described in approx. 10%. typically in chronic q-fever, the initial igg titre is increasing (>1:800). the treatment for acute q-fever is doxycyclin 100 mg bd for 14 days or for at least 3 days after fever subsides and until clinical improvement. as serological confirmation takes time, treatment should not be delayed. early treatment is effective at preventing severe complications. for chronic q-fever, 18 months of doxycyclin 100 mg bd and hydroxychloroquine 200 mg tds is recommended as standard treatment. rabies has an almost worldwide distribution. more than 95% of deaths occur in africa and asia. about 40% are children under 15 years of age. dogs are the main vectors. in asia, there is also a risk of transmission through monkeys. in addition to other diseases, like the lyssavirus, bats or flying foxes can transfer rabies. it is transmitted by bites or scratch wounds but also by inoculation of saliva onto mucous membranes or eye of an infected animal. thorough cleaning of the wound and vaccination within hours can prevent the disease. the incubation period is usually 1-3 months but can be less than 1 week and more than a year. initial symptoms include paraesthesia in the wound area. the disease can pass in two forms. the hyperactive form (70%) shows up with hyperactivity, manic behaviour, paranoia, hallucinations, delirium, hydrophobicity and occasionally aerophobia (triggered by the extremely painful spasms in the larynx area). the paralytic form (30%) is characterised by a slow but steady increasing paralysis. the paralysis begins in the area of the infection. the diagnostics can be established on the animal that has inflicted the wound. the tissue samples of the animal are taken from the brain (brainstem and cerebel-lum). the diagnosis in humans is difficult and unreliable. investigation of blood (antibodies), saliva (pcr), spinal fluid (antibodies) and skin biopsies (rabies antigen) are available. the vaccine and the immune globulin can be given during pregnancy. typical side effects of the vaccine are headache, myalgia, malaise, fatigue and nausea. treatment after potential infection (postexposure prophylaxis pep) includes: 5 irrigation of the wound for a minimum of 15 min and washing of the wound with water, soap, iodine or other disinfecting substances 5 rabies vaccine 5 rabies immunoglobulin into the wound area within 7 days after the first vaccination following data should be recorded when a rabies vaccine is given overseas: 5 address, email and telephone of the practice or hospital 5 date of vaccinations 5 batch number, name of the vaccine and manufacturer 5 how many vaccinations are given 5 application: subcutaneous or intramuscular injection who recommends the following approach with potential rabies after animal contact: vaccination against rabies is recommended for: 5 travellers, who for more than 1 month in areas, in which rabies is present 5 professions that deal with bats or fruit bats 5 professions, in which might get with rabies in contact (e.g., veterinary surgeon or nurse) 5 laboratory workers who handle objects with rabies or lyssavirus 5 after animal contact category 2 + 3 pre-exposure prophylaxis (prep) includes three vaccinations on day 0, 7 and 21-28. the dose is 0.1 ml intramuscularly or subcutaneously. the vaccination lasts for 10 years. follow-up vaccinations (post-exposure prophylaxis = pep) include four vaccinations on day 0, 3, 7 and 14. the dose is 1.0 ml intramuscularly. immunocompromised patients should receive five vaccinations with an additional vaccination on the 28th day. with previous vaccinations, two vaccinations are recommended on day 0 and 3 after exposure. it is not recommended to change the brand or the manufacturer during the course of vaccinations. however, it is possible, if that particular vaccine is not available. immunoglobulin should be administered with the first vaccination. the dose is 20 iu/kgbw. the immunoglobulin preferably should be given in proximity of the wound. the immunoglobulin can be diluted, if the wounds is large, to enable to cover the entire wound area. the immunoglobulin is not recommended, if the first vaccination was given more than 7 days ago, if prep or pep was completed or if an adequate serologic detection of vnab titres (≥0.5 iu/ml) is present. to avoid infection, no animals should be fed. bringing your own food or carrying items like handbags, water bottles, etc. should be avoided, if you stay in the range of monkeys. distance should be maintained to stray cats and dogs. the middle east respiratory syndrome (mers) is caused by a corona virus. corona viruses can cause mild flulike symptoms but also severe symptoms like the severe acute respiratory syndrome (sars). the mers-cov occurs 32.7 · mers mainly on the arabian peninsula (iran, jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates and yemen). but through international travel, it can spread worldwide. recently it resulted in some cases in korea. mers has 37% mortality. the disease is transmitted through droplets or direct contact. the mers-cov also has a wide range of symptoms, from mild common cold symptoms and infections of the upper respiratory tract to a rapidly progressive pneumonitis, respiratory failure, septic shock and multi-organ failure. it seems the mers-cov has a low virulence, since the transmission occurs usually only through close contact by human to human, such as the care of a person suffering from mers. camels seem to be the original reservoir. mild forms with fever and mild respiratory symptoms, mers should be considered, if close contact with infected people existed prior to these symptoms. mers can be asymptomatic but also lead to respiratory failure and death. typical symptoms include fever, cough and shortness of breath. pneumonia or pneumonitis is often associated with mers. sometimes gastrointestinal symptoms such as diarrhoea and vomiting can occur. it has a high mortality of 36%. the treatment depends on the severity of the disease. caution in contact with camels in affected countries should be taken. eating insufficient heated camel meat and milk should be avoided. a suspicion of mers should be considered in individuals with the following risk profile: 5 fever and pneumonia/pneumonitis and stay in endemic areas or contact with a symptomatic person from an endemic area within 14 days before onset of symptoms 5 fever and pneumonia/pneumonitis and hospitalisation in endemic areas or contact with camels and camel products in an endemic area within 14 days before onset of symptoms 5 fever and pneumonia/pneumonitis and contact with a mers diseased person within 14 days before onset of symptoms 5 cluster of patient (especially medical personnel) with severe respiratory symptoms with unclear aetiology tuberculosis is caused by an acid-resistant mycobacterium. m. tuberculosis is responsible for tuberculosis in more than 95%. it has global distribution but occurs more frequently in countries with low hygienic standards. tuberculosis spreads around the globe through international travel and immigration. it also shows a rising rate of resistances to conventional therapies. the time between the initial infection and tuberculin conversion takes approx. the diagnosis can be made with the tuberculin skin test (tst/mendel mantoux). 3 days after the strictly intradermal injection of the substance, the induration at the injection site is measured. an induration of >5 mm may be suggestive of tuberculosis. it is considered a positive test if either the patient has a radiological proof, had close contact with someone with tuberculosis, and has symptoms of tuberculosis, is hiv positive or suffers from immunodeficiency. an induration >10 mm is considered as positive, when the patient who travelled to a country with high tb prevalence is an iv. drug user, homeless and a resident of nursing home or prison and has diabetes mellitus, silicosis, m. hodgkin's or end-stage renal failure. an induration >15 mm is considered as evidence of tuberculosis without any risk factors or symptoms. the tst can be negative in the first 8 weeks after an infection as well as in patients suffering from miliary tuberculosis, m. hodgkin, sarcoidosis, viral infections, and lowered immunity, receiving an immunosuppressive therapy or at high age. a false-positive test can occur after multiple tsts, after vaccination against tuberculosis and infection of other mycobacteria. the interferon-γ test (quantiferon ® tb gold) offers an alternative testing method. this test has the same sensitivity as the tst but a higher specificity. moreover, this test is a confirmation test and isn't affected by previous bcg-immunisations. it consists of three parts, the control (to determine the baseline-interferon-γ), mitogen control (determining the ability of an immune response) and antigen detection (detection of prior infections). a cxr may demonstrate caverns or hilar lymph nodes, but is not a diagnostic tool to exclude tuberculosis. the treatment duration of uncomplicated tuberculosis is 6 months, of complicated tuberculosis 9-12 months (. table 32 .7). it's a combination treatment of different drugs. medications for the tuberculosis treatment are: 5 isoniazid: 5 mg/kgbw, max. 300 mg /d; side effects: elevated serum transaminases, polyneuropathy, prophylaxis to avoid side effects of pyridoxine 40-80 mg/d a vaccination bcg vaccine is not recommended due to its side effects and the lack of efficacy. all vaccinations should be given 28 days before travelling. minimum time for a sufficient protection is 2 weeks (. divers alert network (dan) is a non-profit organisation for divers. they provide medical information and articles, diving insurance, life insurance and travel insurance. they also offer courses, support and research. dan has an international hotline for support and coordination of diving accidents but also for general medical advice overseas. european underwater and baromedical society (eubs) is a european organisation for diving and hyperbaric medicine. they provide guidelines for hyperbaric treatment and training of medical professionals for the hyperbaric medicine. the german organisation for diving and hyperbaric medicine is the "gesellschaft für tauch-und überdruckmedizin" (getüm). . . single dose certificate is valid for 10 years, a new vaccination may be required after 10 years to renew the certificate they provide guidelines for hyperbaric treatment and training of medical professionals for the hyperbaric medicine. brazil; office: tel: +1-919-684-2948, emergency-hotline: +1-919-684-9111. japan: japan marine recreation association, kowa-ota-machi bldg office: tel: +81-45-228-3066, f ax southern africa: private bag x197, halfway house, midrand 1685 eubs: webmaster@eubs.org 5 gtuem: c/o bg-unfallklinik, professor-kuentscher-str. 8, d-82418 murnau spums: 630 st kilda road uhms: 631 us highway 1, suite dan: 7 www. diversalertnetwork. org 5 dan europe: 7 www. daneurope. org 5 emedicine yellow fever chikungunya virus middle east respiratory syndrome (mers) parasites -african trypamosiasis (also known as sleeping sickness) parasites -american trypanosomiasis (also known as chagas disease) parasites -trichuriasis (also known as whipworm) air embolism of the brain in rabbits pretreated with mechlorethamine an examination of the critical released gas concept in decompression sickness accessed 12 middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus (mers-cov) key: cord-303786-snch80z7 authors: kim, hyun-chung; yoo, so-young; lee, bun-hee; lee, so hee; shin, hyoung-shik title: psychiatric findings in suspected and confirmed middle east respiratory syndrome patients quarantined in hospital: a retrospective chart analysis date: 2018-03-30 journal: psychiatry investig doi: 10.30773/pi.2017.10.25.1 sha: doc_id: 303786 cord_uid: snch80z7 objective: little is known about the psychiatric complications or risk factors for depression in suspected or confirmed middle east respiratory syndrome (mers) patients quarantined in hospital. methods: a retrospective chart review was performed of all the patients admitted to the acute mers inpatient unit at the nmc during the 2015 outbreak. results: 30 (75%) were confirmed to be mers-cov positive among 40 admitted cases. among the 24 mers survivors, 17 (70.8%) exhibited psychiatric symptoms and 10 (41.7%) received a psychiatric diagnosis and medication during their hospital stay. suspected mers patients did not exhibit psychiatric symptoms or receive a psychiatric diagnosis. 27 suspected or confirmed mers patients (age 41.15±18.64, male 37.0%) completed psychological assessments. a multiple linear regression analysis revealed that the korean national health and nutrition examination survey-short form and the impact of event scale-revised scores were significantly positively correlated with patient health questionnaire-9 scores. conclusion: our findings indicate that the acute treatment of mers-cov infections in quarantine had a significant impact on the patients’ mental health. furthermore, assessment of the risk factors for depression may identify vulnerable patients who require psychiatric care and attention during hospital quarantine. the study was approved by the institutional review board for human subjects at the nmc in seoul, korea (h-1508-057-004). we reviewed the psychiatric charts for all consecutive admissions for suspected or confirmed mers to the acute mers inpatient unit at the nmc, from may to july 2015 to obtain information concerning demographic characteristics, previous medical conditions, previous or present psychiatric disorders or psychiatric symptoms, and the results of psychological assessments. furthermore, descriptions of the experiences of patients with suspected and confirmed mers were collected through retrospective chart review. we as psychiatrists thought it important to make good rapport with the patient from the beginning, as soon as they entered the mers ward for the first time. to achieve this goal each psychiatrist was assigned to a patient from admission and each psychiatrist, wearing the proper protective equipment, introduced ourselves to the assigned patient face-to-face and informed the patient of daily telephone interviews and symptom screening. this helped to build acquaintance with the patient and give personal familiarity when receiving their first telephone call. daily telephone calls were offered as psychiatric rounding to check any sleep or mood disturbances. further face-to-face interview was done if requested by the patient or when acute psychiatric symptoms emerged. these two modes of mental health services were offered to all of the mers inpatients from day 1 in order to give support and to detect and treat possible psychiatric symptoms. there were two protocols. one protocol was used for the daily psychiatric telephone rounding and one was a package of checklists for the patients. the checklist package was given to the patient after medical examinations and history taking was done. a psychologist called all of the patients to ask the questions on the checklist. this allowed collecting and assessing the checklist without direct contact with the patient or the contaminated checklist package. the patients completed the patient health questionnaire-9 (phq-9), the impact of event scale-revised (ies-r), the korean national health and nutrition examination survey (knhanes)-short form, and the peritraumatic dissociation-posttraumatic negative beliefs-posttraumatic social support (ptd-ptnb-ptss) scale upon admission to the quarantine ward. a psychologist collected data on the second hospital day using a smartphone. patients with poor physical or mental status were excluded from the psychological assessment using the structured instruments. the phq-9 is used to monitor the severity of depression. the questionnaire consists of nine items based on the diagnostic and statistical manual of mental disorders, fourth edition (dsm-iv) criteria for depression, each scored from 0 to 3 providing a severity score ranging from 0 to 27. accordingly, depression severity was defined as: no depression (1-4), mild depression (5-9), moderate depression (10-14), moderately severe depression (15-19), and severe depression (20-27). the korean version of the phq-9 has been shown to be a reliable and valid tool for the screening and assessment of depressed patients (cronbach's alpha coefficient=0.81). 3 the ies-r was designed to assess symptoms associated with post-traumatic stress disorder (ptsd) following a traumatic life event (mers infection in our study). symptoms are measured on 22 self-reported items for a total subjective stress assessment that can be further divided into intrusion, avoidance, and hyperarousal subscales. individual items describe difficulties associated with stressful events, and participants were asked to indicate the extent to which each item distressed or bothered them over the last 7 days using a rating scale of 0-4. the korean ies-r has shown good reliability (cronbach alpha coefficient=0.93), test-retest reliability (cronbach alpha coefficient=0.91), and validity for the assessment of ptsd symptom severity. 4 the knhanes-short form is a shortened version of the knhanes developed by the korea centers for disease control and prevention to assess current stress perception. most measures of distress are derived from pre-existing structured tools that capture the amount of stress or change associated with stressful events that have occurred over the past month. the knhanes has been shown to be reliable and valid, suggesting that it may be a useful instrument for assessing current feelings of stress such as burn-out, depression, and anger. 5 the knhanes-short form is composed of nine items and responses are rated using a five-point likert scale. the ptd-ptnb-ptss is a shortened version of the posttrauma risk checklist (prc), which is a validated korean instrument used to identify the risk factors for ptsd. 6 the prc consists of 57 items that measure ptd, ptnb, and ptss. the ptd-ptnb-ptss scale comprises 18 items selected from the prc (3 ptd, 10 ptnb, and 5 ptss items) that have been converted from yes/no answers to a five-point likert scale. patients were divided into two groups based on phq-9 scores: those experiencing mild-to-severe depression and those without depression. an independent-sample t-test was used to test for differences in continuous variables between quarantined patients with mild-to-severe depression and those without depression. pearson's correlation analysis was used to examine the correlations among psychological measures. a multiple linear regression analysis was performed to identify the risk factors for depression in patients with suspected or confirmed mers. the null hypothesis was rejected at p<0.05. all statistical tests were conducted using the statistical package for the social science (spss) for windows (ver. 18.0; spss inc., chicago, il, usa), and the threshold for twosided significance tests was p<0.05. a total of 40 confirmed and suspected mers cases were admitted to a quarantine ward at the nmc. of those, 30 (75%) were confirmed to be mers-cov positive. among the con-firmed mers patients, 24 survived and 6 died. of the 34 surviving confirmed or suspected mers patients, 12 (35.3%) were male and 22 (64.7%) were female and had a mean age of 46.15±20.41 (range, 21-86) years. in total, 20 (58.8%) of the surviving confirmed and suspected mers patients reported previous medical conditions and three (8.8%) reported previous psychiatric disorders. among the confirmed mers patients, 17 (70.8%) exhibited psychiatric symptoms and 10 (41.7%) received a psychiatric diagnosis and were prescribed medication during their hospital stay (figure 1 ). suspected mers patients did not exhibit psychiatric symptoms or receive a psychiatric diagnosis. of the 17 patients with psychiatric symptoms, 14 complained of experiencing: insomnia (n=7), depressive mood (n=5), and tension (n=9), and three were disoriented (n=2), had impaired memory (n=2), auditory hallucinations (n=2), and aggressive outbursts (n=2). dsm-iv-text revision criteria were used to diagnose three patients with adjustment disorders, two with depressive disorders, and two with acute stress disorders after they recovered from delirium. two patients were diagnosed with anxiety disorders, one of whom had comorbid mild neurocognitive disorder. the cognitive function of a patient previously diagnosed with dementia deteriorated further after the mers infection and isolation. moreover, a patient who lost her father to mers experienced bereavement during isolation. most of the patients had no knowledge of mers. some asked whether they would carry the virus and be infectious for life. some patients feared impending death, and two patients previously diagnosed with panic disorder experienced panic attacks although the panic disorder had been in remission before contracting mers. several patients reported distress from not knowing the effects of mers and feelings of isolation, as well as concerns about their family and workplace colleagues. several patients reported feelings of guilt and fear of spreading mers. one patient felt that he was being punished for something as an explanation for why he had been infected. one patient had been infected by her father, whom she had nursed in the hospital. her father had died of mers-related complications. she received this news while in isolation herself. she was unable to attend his funeral, as all deceased patients were cremated shortly after their deaths and formal funerals were impossible. she felt depressed and sad. an elderly patient did not understand why she was restricted to a room after she had recovered consciousness. she cried every day, begged the nurses to let her go home, and occasionally expressed anger and made suicidal threats. a blind patient who had lost contact with his healthcare aide was so mistrustful of the medical staff that he initially refused admission. psychological data were not obtained from 7 of the 34 survivors due to intubation, delirium, cognitive dysfunction, or noncompliance. thus, 27 (79.4%) survivors completed the psychological assessment and were included in the analysis. of those, 18 (66.7%) were confirmed and 9 (33.3%) were suspected mers cases, 10 (37.0%) patients were male and 17 (63.0%) were female, and the mean age was 41.15±18.64 (range, 21-83) years. in total, 15 (55.6%) patients reported previous medical conditions and 2 (7.4%) reported previous psychiatric disorders (table 1) . according to the phq-9 criteria, 11 (40.7%) participants were depressed (phq-9 ≥5) and 16 (59.3%) were not depressed. patients with mild-to-severe depression had significantly higher mean scores on the knhanes-short form (t= 4.169, p< 0.001), ies-r (t=2.262, p=0.017), and ptd (t= 2.922, p=0.007) than those without depression. however, the mean ptn and ptss scores were not significantly different between groups ( table 2 ). the phq-9 scores were significantly positively correlated with the knhanes-short form (r=0.814, p<0.001), ies-r (r=0.679, p<0.001), ptd (r=0.627, p<0.001), and ptnb (r= 0.454, p=0.017) scores. conversely, the phq-9 scores were not significantly correlated with the ptss scores (table 3) . we performed a multiple linear regression analysis to assess the impact of the psychological factors measured by the ies-r, ptd, ptnb, and knhanes-short form on depression severity in suspected and confirmed mers patients. the ies-r, ptd, ptnb, and knhanes-short form scores yielded an adjusted r 2 value of 0.693 (f=12.747, p<0.001) . the analysis revealed that only the knhanes-short form and ies-r scores were significantly positively correlated with phq-9 scores (table 4 ). we found that about 70% of the confirmed mers patients exhibited psychiatric symptoms and about 40% received psychiatric diagnoses and were prescribed medication during hospital quarantine, whereas none of the suspected mers patients exhibited psychiatric symptoms. furthermore, we found that the level of daily distress over the past month and posttraumatic stress symptoms may be risk factors for depression .100 *patients with depression (phq-9 ≥5) (n=11) included 8 subjects (29.6%) with mild depression (phq-9, 5-9), 2 (7.4%) with moderate depression (phq-9, 10-14), and 1 (3.7%) with severe depression (phq-9, 20-27). phq-9: patient health questionnaire-9, ies-r: the impact of event scale-revised, ptd-ptnb-ptss: peri-traumatic dissociation-post-traumatic negative beliefs-post-traumatic social support, knhanes: national health and nutrition examination survey a previous study found that seven of ten sars patients with psychiatric complications were deemed to have mild psychiatric problems such as anger, anxiety, suicidal ideas, and depressive reaction, whereas the remaining three patients had more severe psychiatric disorders with hallucinatory and manic features. similarly, we found that 14 of 24 mers patients had mild psychiatric symptoms such as insomnia, depressed mood, and tension and that three had severe psychiatric disorders with hallucinations and psychotic features. the recent outbreaks of sars in asia and canada provide the most recent data on emerging infectious diseases (eids). psychiatric disorders and chronic fatigue were prevalent among sars survivors 3 years after contracting the disease and were associated with various functional impairments. 7 however, to our knowledge, our study is the first to use selfreported measures to assess risk factors for the development of psychiatric symptoms during the acute treatment period. about 35% of sars survivors reported moderate-to-severe or severe anxiety and/or depressive symptoms 1 month after recovery. 8 moreover, sars survivors had elevated levels of psychological distress 1 year after the outbreak, and 64% scored above the general health questionnaire cut-off for psychiatric morbidity. 9 the current prevalence for any psychiatric disorder 30 months post-sars is 33.3%. 10 a biopsychosocial model may explain the development of psychiatric symptoms in patients with mers. relevant biological factors may be weight loss of 10 kg to 20 kg, 11 antiviral agent side effects, 12 pre-existing disabilities (e.g., blindness or hearing impairment), or a history of psychiatric disorders. relevant psychological factors include tension, fear, anger, mistrust, and depressed mood due to mers and subsequent isolation during quarantine. 2 additionally, we found that the level of daily distress over the past month and post-traumatic stress symptoms may be risk factors for depression during acute hospital quarantine. social factors that may have led to unhappiness during treatment include financial losses attributable to isolation and separation from young children or parents at home. given the nature and characteristics of mers, we established a patient-centered consultation program to monitor the safety issues that arose during the period of total isolation and offered mental health services to all mers inpatients, regardless of whether they were diagnosed with psychiatric disorders. our guiding principle was centered on the need for early detection of psychiatric symptoms including those related to difficulty in accessing mers wards. our study has potential limitations. we used a retrospective chart review design performed in a single center with a small study population. furthermore, our study was not designed to compare depressed and non-depressed patients. additionally, the small sample size may have affected the prevalence rate of psychiatric symptoms and treatment. despite these limitations, our findings indicate that the acute treatment of mers-cov infections in quarantine had a significant impact on the patients' mental health. furthermore, assessment of the risk factors for depression may identify vulnerable patients who require psychiatric care and attention during hospital quarantine. our findings suggest the need to increase mental health services during the acute treatment phase of eids and to conduct follow-up studies in survivors. mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study psychiatric complications in patients with severe acute respiratory syndrome (sars) during the acute treatment phase: a series of 10 cases the phq-9: a new depression diagnostic and severity measure disaster psychiatry committee in korean academy of anxiety disorders. reliability and validity of the korean version of the impact of event scale-revised development and posttrauma risk checklist mental health impact of severe acute respiratory syndrome: a prospective study psychological distress and negative appraisals in survivors of severe acute respiratory syndromes (sars) stress and psychological distress among sars survivors 1 year after the outbreak long-term psychiatric morbidities among sars survivors the effect of nutritional management on the mood of malnourished patients current status and challenges of antiretroviral research and therapy key: cord-308061-hz7fsn2g authors: drosten, christian title: is mers another sars? date: 2013-09-30 journal: the lancet infectious diseases doi: 10.1016/s1473-3099(13)70159-2 sha: doc_id: 308061 cord_uid: hz7fsn2g nan almost all individuals with mers-cov infection had fever as the main symptom on admission. however, occurrence of fever is not surprising in (mostly) self-reporting patients; in studies of the clinical features of sars, with a few exceptions, equivalent selection biases were noted. 4,5 furthermore, similar to sars, only a few people with mers had upper-respiratorytract symptoms such as sore throat and rhinorrhoea, providing a means to discriminate mers from the common cold in adults. a striking diff erence to sars is the high rate of underlying comorbidity in patients with mers. a virus not yet fully adapted to human infection might be more likely to cause illness in people weakened by pre-existing disease. however, caution is necessary when interpreting comorbidity data, because we should compare rates in aff ected patients with those in the exposed population. in a study of more than 6000 adults attending an outpatient department in riyadh, 30% had diabetes overall, including 63% of those older than 50 years. 6 in assiri and colleagues' report, 32 (68%) of 47 patients (most of whom were older than 50 years) had diabetes, a prevalence that does not seem high in view of the background rate. furthermore, about half the patients included were from an outbreak centred around a haemodialysis unit. 7 the rates of chronic kidney disease (49%) and hypertension (34%) noted would, therefore, be expected in this overall context. since communitybased studies are unavailable for comparison, we have no reason to regard mers as a disease restricted to people with underlying disorders. an unfortunate fi nding from assiri and colleagues' study is the rapid progression to respiratory failure and intubation in individuals with mers, occurring about 1 week after onset of symptoms, up to 5 days earlier than in sars. 5 this fi nding accords with the high rate of haemoptysis seen in patients with mers, suggesting severe lung injury. data of a preliminary infection study in lung explants indeed indicate that mers-cov reaches higher replication levels and shows broader cell tropism in the lower human respiratory tract than does sars-cov. 8 even capillary endothelial cells of the lung became infected. post-mortem analyses and further experimental studies are needed to understand why mers-cov can induce lung failure so rapidly and with such severity. use of recommended real-time rt-pcr assays 9,10 in assiri and colleagues' study means we can compare virological features of mers-cov infection between cohorts. this situation is much diff erent from the sars experience, during which the diversity of home-brewed rt-pcr formulations made crossstudy comparisons very diffi cult. a key question is whether we can use upper-respiratory-tract samples for rt-pcr-based diagnostics? on the basis of our experience, 9, 10 we would judge eight of the 37 upperrespiratory-tract samples obtained in assiri and colleagues' study diffi cult to interpret, namely those with a cycle threshold (ct) value greater than 37 (ct is a technical surrogate for viral load; higher numbers indicate lower viral loads). this rate is better than that noted in patients with sars: only a third of laboratoryconfi rmed cases yielded virus in upper-respiratory-tract samples. 5 the values reported by assiri and colleagues also show that viral loads in the upper respiratory tract are higher than in patients with mers who were treated overseas and, thus, who were investigated later in the disease course. 2, 11, 12 of note, in seven of 37 upper-respiratory-tract swabs (ct <24), we estimate that viral loads could exceed fi ve million viral genome copies per sample, implying effi cient viral shedding from the upper respiratory tract in only some patients, as assumed in mathematical projections of mers epidemics. 13 to ascertain relevant data for mers epidemiology, we need to develop serological assays using samples from well defi ned groups of patients, such as described here. population-based antibody testing could establish the extent of mers-cov infection, instead of only seeing the tip of the iceberg represented by cases admitted, such as those summarised in this important paper. institute of virology, university of bonn medical centre, bonn, germany drosten@virology-bonn.de i declare that i have no confl icts of interest. in the lancet infectious diseases, catherine ison and colleagues 1 describe recent dissemination (2007-11) of the pena mosaic gene in neisseria gonorrhoeae in england and wales, particularly among men who have sex with men. their fi ndings indicate a creeping resistance to cefi ximethe former fi rst-line treatment for gonorrhoea-and much slower development of ceftriaxone resistance, both with breakpoints of 0·25 mg/l. this decreased susceptibility has been reported previously in the usa, 2 most typically in men who have sex with men and with similar relation to the mosaic pena resistance gene. isolated cases of resistance to these drugs have also been recorded in france and spain. 3 in japan, although cefi xime was not used as standard treatment until the mid 2000s, a sporadic case of resistance was noted in a man in 2001. 4 however, in general, the prevalence of ceftriaxone isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study the severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study prevalence of diabetes mellitus in a saudi community hospital outbreak of middle east respiratory syndrome coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection and the mers-cov study group. clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk key: cord-294349-ps3qlho2 authors: al-sharif, eman; strianese, diego; almadhi, nada h.; d’aponte, antonella; dell’omo, roberto; di benedetto, rita; costagliola, ciro title: ocular tropism of coronavirus (covs): a comparison of the interaction between the animal-to-human transmitted coronaviruses (sars-cov-1, sars-cov-2, mers-cov, cov-229e, nl63, oc43, hku1) and the eye date: 2020-09-03 journal: int ophthalmol doi: 10.1007/s10792-020-01575-2 sha: doc_id: 294349 cord_uid: ps3qlho2 purpose: several studies have reported conflicting results on ocular manifestations and transmission of coronavirus disease 2019 (covid-19) whose causative virus, sars-cov-2, belongs to the coronavirus family, the seventh recognized as a human pathogen and the third causing a severe clinical syndrome. covid-19 primarily affects the lungs, similar to the other human coronaviruses. comparing the relation between the animal-to-human transmitted coronaviruses (sars-cov-1, sars-cov-2, mers-cov, cov-229e, nl63, oc43, hku1) and the eye may contribute to determining their actual eye-tissue tropism and risk of ocular transmission. methods: literature review was conducted via pubmed.gov, google scholar and medrixv using the following keywords: covid-19, sars-cov-2, sars-cov-1, mers-cov, cov-229e, nl63, oc43, hku1, conjunctivitis, tear swab, ocular expression, ocular symptoms and human angiotensin converting enzyme-2 expression. studies with lack in methodology were excluded. results: sixteen observational studies were selected. the range for detection of viral rna in tears was 0–8% for sars-cov-1 and 0–5.3% for sars-cov-2, while no reports were found for other coronaviruses. ocular manifestations have been reported for nl63 and sars-cov-2. ocular symptoms in the form of conjunctivitis/conjunctival congestion predominantly were detected in 65 (3.17%) out of 2048 reported patients with covid-19 (range of 0.8–32%). eye symptoms were not reported for the other coronaviruses. conclusions: data aggregation for coronaviruses shows a relatively low eye-tissue tropism. conjunctival congestion is an uncommon manifestation of covid-19 similar to all human coronaviruses’ infections. in a low percentage of patients, the virus can be excreted in ocular fluids at different stages of the infection, regardless of positive sars-cov-2 throat swab. albeit high viral loads in ocular tissue seem to have relatively low prevalence, the eye should be regarded as a potential source of infection dissemination for covid-19. the eye may serve as a site of virus replication and as a gateway for virus transferring to extraocular sites, for instance the respiratory system. this is achieved primarily by the nasolacrimal system, which provides an anatomical bridge between ocular and respiratory tissues. this anatomical conduct may cause drainage of immunizing agents to nasal tissue following topical ocular administration as well as the spread of intranasally administered solutions to the conjunctival mucosal surface. linkage of the ocular mucosal immune system, which includes conjunctiva, cornea, lacrimal glands and lacrimal drainage system, with nasal cavity-associated lymphoid tissue in the nasolacrimal ducts, further supports the immunological interdependence between ocular and respiratory tract tissues. ocular tropism of respiratory viruses has been extensively studied and reported [1] . the coronaviruses (covs) have become the major pathogens of emerging respiratory disease outbreaks in the last 20 years. they are a large family of singlestranded rna viruses (? ssrna) that can be isolated from different animal species. among coronaviruses which have circulated in the human population, two were identified in the 1960s (hcov-229e and hcov-oc43), and two others (hcov-hku1 and hcov-nl63) were identified recently [1] . sars-associated coronavirus was first identified in 2003, whereas (mers-cov) was first identified in saudi arabia in 2012 [2, 3] . for reasons yet to be explained, these viruses can cross species' barriers and can cause, in humans, illness ranging from the common cold to the more severe diseases such as the middle east respiratory syndrome (mers) and the severe acute respiratory syndrome (sars). the potential for these viruses to cause a pandemic worldwide seems to be a serious public health risk. coronavirus disease 2019, known as covid-19, is an emerging infection which is caused by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) that was first reported in wuhan city, china, late in december 2019 [4] . since then, the coronavirus outbreak has been spreading rapidly and extensively across continents and the number of infected cases and ensuing deaths is continuously rising worldwide. this led the world health organization (who) to proclaim it a global pandemic earlier in march 2020. since the very beginning of this pandemic, warnings regarding an impending epidemic were raised up by a young ophthalmologist, dr. li wenliang, who passed away because of infection by covid-19 on february 7, 2020 [5] . thereafter, several other ophthalmologists became ill in different parts of the world. this raised a lot of concerns among both ophthalmologists and patients. thus, several studies have been published in the last weeks to better address the relation between sars-cov-2 and the eye. given the relatively shortterm experience with this pandemic, data available are still too scarce to draw robust conclusions. nevertheless, we thought it would be useful to conduct a qualitative systematic review of the literature focusing on the relation between the known epidemic zoonotic coronaviruses and the eye, examining the spectrum of ocular manifestations and routes of transmission, in the attempt to aid the understanding of the actual coronavirus-eye interaction. all types of published observational studies (case reports, case series and cross-sectional studies) published in the period between january 1, 2002, and april 30, 2020, and reporting ocular manifestations/ ocular transmission of coronaviruses-related infection were reviewed. studies judged as having relevant flaws in methodology were excluded. only studies published in the english language were considered. this was accomplished via pubmed, google scholar and medrixv e-databases, using and matching the following keywords: covid-19, sarscov2, sars, sarscov1, mers, mers-cov, cov-229e (alpha coronavirus), nl63 (alpha coronavirus), oc43 (beta coronavirus), hku1 (beta coronavirus), eye symptoms, conjunctivitis, tear swab, ocular expression, ocular symptoms, ophthalmology and human angiotensin-converting enzyme 2 (ace2) expression. a team of three reviewers (es, ds and ad) searched the specified databases and reviewed all the eligible articles independently. all included articles were agreed upon by all researchers. criteria for selection were as follows: homogeneity in the directions and degrees of results and retrospective observational studies with good reference standards. the following information was derived from each of the included studies: study design, the total number of included patients, number of patients with ocular manifestations, number of patients undergoing pcr testing from conjunctival secretions or tears and number of patients undergoing laboratory confirmative tests of their coronavirus infection. one researcher completed data abstraction (es) using a standardized abstraction form to collect the study results. we then summarized nonnumerically the key findings. sixty-one papers were reviewed; of these, 16 studies reporting the ocular findings and conjunctival/tear swab pcr results of patients affected by coronaviruses were considered for analysis ( fig. 1 ). among these, four studies (three case series and one longitudinal cross-sectional study) reported the relationship between the eye and sars-cov-1 (table 1) . clinical ocular manifestations were absent in all sars-cov-1 patients, and viral rna was detected in the conjunctival secretions and tears in three cases out of 120 (2.5%) with a range of 0-8% [6] [7] [8] [9] . conversely, 12 observational studies (three case reports, three case series and six cross-sectional studies) reported the relationship between the eye and the emerging sars-cov-2 (table 2) [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] . overall, ocular symptoms, in the form of conjunctivitis/conjunctival congestion predominantly, were detected in 65 out of 2048 examined patients (3.17%) with a range of 0.8-32%. in almost all cases, the diagnosis of covid-19 was confirmed by nasopharyngeal swabs or other methods. a total of 414 patients underwent pcr testing of samples obtained from ocular fluids but the viral rna was detected in 11 patients collectively, accounting for a rate of 2.7% (range of 0-5.3%). liang and wu [10] reported the presence of viral rna in the conjunctival secretions of a patient diagnosed with severe covid-19 using real-time polymerase chain reaction (rt-pcr) assay. of note, this patient demonstrated no conjunctivitis clinically. the authors further hypothesized the proportionate association between the viral load and the severity of disease. in comparison, wu and associates reported positive conjunctival reverse-transcriptase polymerase chain reaction results (rt-pcr) only in two out of 12 patients with ocular symptoms and positive sars-cov-2 nasopharyngeal swabs [11] . similarly, a small study testing the conjunctival secretions and tears (collected twice over 2-3 days) of 30 confirmed covid-19 patients demonstrated the presence of viral rna (in both samples) in one patient only who also showed clinical signs of conjunctivitis [12] . in an attempt to study the possible ocular transmission of covid-19, zhou et al. retrospectively tested by pcr the conjunctival swabs of 67 patients with suspected (n = 4) or confirmed (n = 63) covid-19 and found that three confirmed cases with no ocular symptoms tested positive (one confirmed and two probable positive pcr assays). though, the only patient presenting with symptoms of mild conjunctivitis, an anesthesiologist who acquired the infection from a confirmed covid-19 patient during intubation, had negative conjunctival swab results [13] . a recent report from the national institute for infectious diseases ''lazzaro spallanzani'' describes a 65-year-old woman, who traveled from wuhan, china, to italy in january 2020 and was admitted with respiratory symptoms after one week from her arrival. she presented with nonproductive cough, sore throat, coryza and bilateral conjunctivitis. infection with sars-cov-2 was confirmed by performing rt-pcr assay on sputum samples. on day 3 after hospital admission, owing to the persistence of conjunctivitis, an ocular swab was collected and viral rna was detected. subsequent ocular samples collected with almost daily frequency were positive up to day 21, with declining virus concentrations. conjunctivitis greatly improved at day 15 and apparently resolved at day 20. however, sars-cov-2 rna was detected again at day 27 in conjunctival swabs after it was undetectable in nasal swabs [20] . on the other hand, yu et al. [14] compared viral shedding in tears with the nasopharyngeal swabs' results of 17 patients during the course of covid-19 infection. no evidence was found of sars-cov-2 shedding in tears through the course of the disease even when nasopharyngeal swabs' samples continued to show positive results. only one patient showed ocular symptoms during the disease course, but no evidence of sars-cov-2 could be found in this patient's tear samples. in terms of ocular manifestations, a study published by wu and colleagues on ocular findings in covid-19 patients showed that approximately one-third of patients (12/38, 31.6%) had eye manifestations such as conjunctival redness, chemosis, tearing and increased secretions. interestingly, the majority of these patients were considerably ill (four were studies included in quanɵtaɵve synthesis (meta-analysis) (n = not applicable) considered as moderate, two severe and six critical based on pc-ncp guidelines). among these patients, eye symptoms were the first sign of covid-19 only in one patient (2.5%). almost all patients with eye findings (11 out of 12) had positive nasopharyngeal swabs, and two of these tested positive for covid-19 by pcr of conjunctival swabs. this study also suggested that patients developing eye manifestations were more likely to have abnormal blood tests (white blood cell count, c-reactive protein, lactate dehydrogenase and prolactin) compared to patients without ocular symptoms. in spite of the limited sample size (n = 38 patients), this study is among the first to report a direct clinical relationship between the eye and covid-19 [11] . based on this study, it seems that the presence of ocular findings might be predictive of a more severe clinical course. the incidence of conjunctival congestion was estimated to be less than 1% based on the report of who-china joint mission report on covid-19 that studied 55,924 laboratory-confirmed cases [22] . another study that comprehensively reviewed the clinical presentation of 1099 confirmed cases of covid-19 showed that conjunctivitis is an uncommon presentation occurring in only 0.9% of the patients [15] . a recent study conducted by chen and associates on 534 patients with covid-19 (64% positive by nasopharyngeal swab pcr) showed that 25 (4.68%) patients had conjunctival congestion that lasted from 2 to 12 days (average duration: 4.9 ± 2.6 days). in fact, conjunctival congestion was the initial presenting manifestation in three of these patients. other common reported manifestations were as follows: dry eye (112, 20.97%), blurred vision (68, 12.73%), foreign body sensation (63, 11.80%), tearing (55, 10.3%), itching (53, 9.9%), conjunctival secretions (52, 9.7%), ophthalmoplegia (22, 4 .1%) and photophobia (15, 2.8%). some of these ocular manifestations, such as dry eyes, blurred vision and foreign body sensation, were reported for the first time in this study. notably, 60.11% (321) of these patients spent a considerable amount of their day, more than four hours, on short-distance reading and this could explain the high incidence of these dryness-related symptoms reported in this study. bilateral lung infiltrates on ctscan were detected in 68% of the patients with conjunctival congestion, and this supports the assumption of possible correlation between disease severity and occurrence of ocular manifestations [18] . an nl63-related conjunctivitis was reported in two cases. the first case was a 7-month-old child with bronchitis and conjunctivitis [23] . the second case was associated with kawasaki disease. however, it is unclear if conjunctivitis in hcov-nl63-infected patients was due to the coronavirus itself or rather represented a manifestation of disease caused by an unrelated pathogen or disease [24] . concerning the other coronaviruses, i.e., mers-cov, 229e (alpha coronavirus), oc43 (beta coronavirus), hku1 (beta coronavirus), we did not find any studies in the literature regarding ocular manifestations or conjunctival/tear swab pcr results. covs are positive-stranded rna viruses with a crown-like appearance under an electron microscope (coronam is the latin term for crown) due to the presence of spike glycoproteins on their envelope. the subfamily orthocoronavirinae of the coronaviridae family (order nidovirales) classifies into four genera of covs: alphacoronavirus (alphacov), betacoronavirus (betacov), deltacoronavirus (deltacov) and gammacoronavirus (gammacov) [25] . genomic characterization has shown that probably bats and rodents are the gene sources of alphacovs and betacovs. on the contrary, avian species seem to represent the gene sources of deltacovs and gam-macovs [26, 27] . to date, seven human covs (hcovs) have been identified as capable of causing respiratory, enteric, hepatic and neurological diseases in different animal species, including camels, cattle, cats and bats. large studies report that 2% of the population are healthy carriers of a cov and that these viruses are responsible for about 5-10% of acute respiratory infections [26, 27] . common human covs listed by the centers for disease control and prevention (cdc) are 229e (alphacov); nl63 (alphacov); oc43 (betacov); and hku1 (betacov) [23, [28] [29] [30] . they can cause common colds and self-limiting upper respiratory infections in immunocompetent individuals; however, in immunocompromised subjects, elderly and infants, lower severe respiratory tract infections are also reported. conversely, the sars-cov-1 (betacovs, b lineage), sars-cov-2 (be-tacovs, b lineage) and mers-cov (betacovs, c lineage) are the causative agents of large epidemic/pandemic outbreaks with variable clinical severity featuring respiratory and extra-respiratory manifestations [26, 27] . interestingly, these latter viruses (sars-cov-1 and mers-cov) have probably originated from bats and then moved into other mammalian hosts, the himalayan palm civet for sars-cov-1 and the dromedary camel for mers-cov, before jumping to humans [31, 32] . the dynamics of sars-cov-2 are currently unknown, but there is speculation that it also has an animal origin. some evidence, based on the results of viral genome sequencing, suggests that covid-19 is transmitted to humans from bats which are likely to be the natural hosts of sars-cov-2; however, the potential amplifying mammalian host, intermediate between bats and humans, is not known. since the mutation in the original strain could have directly triggered virulence towards humans, it is not certain that this intermediary exists [33] . at the cellular level, both sars-cov-1 and sars-cov-2 employ the human angiotensin-converting enzyme 2 (ace2) as a surface receptor to gain access into the human cells and then subsequent viral replication ensue [26, 34] . these viruses attach to cells via their surface spike-like s-glycoprotein consisting of two subunits, s1 and s2, that are responsible for binding to the host cells' receptors and fusion with its membrane, respectively [33] . cellular expression of ace2 contributes to the viral cell-line tropism. alveolar, bronchial and tracheal epithelial cells express ace2; therefore, these viruses tend to disturb the respiratory system [27, 33] . in the eye, ace2 is found mainly in the posterior segment (retina) and aqueous humor [35, 36] . sun et al. had shown previously the weak binding capability of sars-cov-1 to ace2 expressed on the in vitro cultured conjunctival fibroblasts and epithelial cells as well as corneal epithelial cells [37] . these cellular receptors are also expressed in other organs such as the kidney, intestine, liver, intestinal tract, t lymphocytes and others. this might explain the wide range of manifestations accompanying these infections. once the virus gains access into the host cells, it starts replicating to produce new virus particles that infect more cells. this infection triggers the innate immune system resulting in the release of inflammatory mediators and cytokines [33] . mers-cov utilizes a different receptor, dipeptidyl peptidase 4 (dpp4), also known as cd26, which is found predominantly in lung alveolar cells in addition to epithelial cells of various organs such as upper respiratory tract, kidney, small intestine and liver [38] . there are no reports of expression of cd26 at the eye level. reflecting on the ocular tropism of the well-known zoonotic coronaviruses might help us in understanding the behavior of the current sars-cov-2. among the zoonotic coronaviruses that were studied intensively is the mouse hepatitis virus (mhv), strain jhm. this murine cov is not only neurovirulent causing demyelinating disease, but it can also affect both anterior and posterior eye segments. this had led robbins et al. in 1990 to inoculate this virus intravitreally in mice eyes to study its effect on retinal degeneration, and this animal model was later recognized as the experimental coronavirus retinopathy (ecor). the authors observed mild anterior uveitis followed shortly by infection of almost all retinal layers with progressive retinopathy lasting 6-7 weeks post-infection [39] . even when the virus was introduced by other routes into the eye, it still demonstrated retinotropism (neural retina and retinal pigment epithelium) [40] . the viral antigen was detected in the ganglion cells 10 days after inoculation, and degenerative changes continued up to 14 weeks after inoculation. these two studies demonstrate the biphasic nature of the disease which starts with an acute inflammatory phase and then a prolonged degenerative phase associated with retinal autoantibodies production [41] . elevated levels of tnf-a and tnf receptors were observed in animal models, and this further supports the autoimmune reactivity during the second phase [42] . recent data have shown that the ocular manifestations of sars-cov-2 in human beings are mild, which implicates low ocular tropism compared to the ecor model. nevertheless, the ocular immune responses observed in the ecor model might carry some important therapeutic implications for sars-cov-2. based on animal models, neri et al. speculated that this autoimmune response starts within 10-14 days of infection and this might represent a golden period to suppress the immune system by steroids or monoclonal agents preventing the subsequent cytokines storm and permanent multi-organ damage [43] . despite that, the ace-2 receptor, the main receptor for virus cellular entry uncovered so far, is present in a higher concentration in the posterior segment of the eye; yet, the vast majority of reported cases had anterior ocular manifestations, mostly conjunctivitis. this could be explained by the different binding ability of the virus to this receptor depending on the tissue type which expresses the receptor. another explanation might be the low retinal tropism for this virus as sars-cov-2 can potentially access the posterior eye segment due to the resulting viremia, especially in immunocompromised patients; however, no evidence of retinal inflammation has been reported so far. indeed, retinal inflammation in severely ill patients in the icu might be also overlooked or poorly detected being due to their life-threatening condition. performing retinal examinations on these patients would have been valuable to see if any changes were present. one more explanation could be the inability of the virus to induce a vigorous immune response even when it gains access to the posterior eye. eventually, in months or years to come, we might expect some patients to develop an immune late response possibly characterized by destruction and fibrosis of neural cells, which might ultimately include the retinal layers, as already observed for some postinfective neurological conditions, such as canine distemper, the human subacute sclerosing panencephalitis and the encephalitis lethargica of von economo-whose relation with the 1918/1919 pandemic influenza is still under debate [44] eye as a route of transmission the world is currently facing an outbreak crisis caused by sars-cov-2 which started more than 3 months ago. this infection primarily affects the respiratory system; however, the ocular involvement and ocular transmissibility of this infection are gaining more importance as this pandemic continues. recognizing the routes of transmission of sars-cov-2 is of utmost importance to help mitigate its spread. the most important human-to-human route of transmission is through direct contact with viruscontaminated respiratory secretions (droplets and aerosols) [26] . additionally, contact with virus-contaminated fomites, objects and environmental surfaces may result in transmission [45] . whether or not the eye is a possible route of spread is still debatable. nevertheless, evidence of possible ocular transmission is growing in the literature as the pandemic resumes universally. in this systematic review, the rate of sars-cov-2 viral shedding in tears and conjunctival secretions tested by pcr was 2.7%. these results should be interpreted with caution as the time when samples were obtained from patients varied between the studies. hypothetically, samples obtained late during the disease course might not yield positive results due to decreasing virus concentrations and seroconversion. additionally, the viral load and severity of infection might affect the results. albeit the study includes a single patient, the report of the spallanzani institute with the sars-cov-2 traced in the tears from the onset until day 27, after it has been tested negative for 5 days, has two relevant caveats: firstly, ocular involvement of sars-cov-2 may occur early in the covid-19 course and secondly, the virus may continue to replicate in the conjunctival tissue even after the complete clinical recovery of the patient [20] . however, in other studies, patients with symptoms of upper respiratory tract infection did not demonstrate any viral shedding in tears, suggesting that the hypothesis of the lacrimal duct acting as a viral conduit may not be true and that transmission through tears regardless of the phase of infection is likely low [10-14, 16, 20] . indeed, studies evaluating the ocular secretions of sars-cov-1 patients during the outbreak had also conflicting results. loon et al. from singapore reported the detection of viral rna in tear samples for three out of 33 patients using pcr early during the course of infection (within 9 days) [6, 46] . in contrast, chan et al. reported the absence of sars-cov-1 rna in tears and conjunctival scrapings obtained from 20 infected patients. the authors attributed this to decreased pcr sensitivity resulting in false-negative rates, and this can be lessened by repeated testing. another proposed explanation is the presence of viral rna in the tears for a short period only [7] . similarly, leong et al. reported no viral rna in conjunctival swabs obtained from 64 sars patients during the convalescent phase [8] . there are no reports on the presence of mers-cov rna in tear samples or on ocular surface. this is consistent with the absence of the dpp4 in the upper respiratory tract and presumably on ocular surface too. since the conjunctiva is an exposed mucous membrane, theoretically, it is a possible portal of entry for the virus. multiple studies emphasized the possibility of conjunctival contamination by respiratory droplets expelled by infected individuals. this may result in spreading the virus from the ocular surface mucous membrane through the tear nasolacrimal drainage system and down into the lower respiratory tract [17, 47] . what further supports this notion is the transmission of covid-19 to one of the national expert panel members inspecting wuhan during the outbreak in whom conjunctivitis preceded the onset of pneumonia. he first experienced unilateral conjunctivitis, then respiratory symptoms and fever followed. this occurred in spite of adherence to precautionary measures except for eye protection, and this signifies the possibility of ocular transmission and importance of wearing protective goggles or face shields [17] . moreover, a recent study evaluating the replication of sars-cov-2 and sars-cov-1 in ex vivo human conjunctival cells showed that both viruses demonstrated positive replication with sars-cov-2 revealing more extensive infectivity than sars-cov-1 [48] . this again further emphasizes the eye being a potential entry site for the virus. despite the controversy of the currently available evidence, eye fluids should be regarded as a potential source of viral transmission until proven otherwise. in ophthalmology, physicians and other healthcare providers are in close contact with patients during ocular examination and drops instillation. this serves as a possible source of infection transmission from patients to physicians due to very close contact with ocular surface and secretions, particularly during slitlamp examination, direct and indirect ophthalmoscopy, contact intraocular pressure acquisition. in addition, physicians may also transmit the infection to other patients via direct contact and contaminated instruments if they do not adhere to infection control measures. interestingly, an observational study showed that participants touched common surfaces and their mouth/nasal mucosa between 3.3-3.6 touches per hour [49] . covid-19 has a wide clinical spectrum ranging from mild flu-like symptoms to severe pneumonia and acute respiratory distress syndrome. the case fatality rate seems to be 2.3% with death occurring mainly in the elderly and those with preexisting morbidities [50] . however, the actual casualty rate is not yet determined as having been reported with a wide geographical variation. concerning sars-cov and mers-cov, the mortality rates are up to 10% and 35%, respectively [51, 52] . the common presenting manifestations of covid-19 include fever, dry cough, shortness of breath and lung ground-like opacities on chest ct-scans [53] [54] [55] . the disease was further categorized by the chinese cdc into mild, severe and critical disease based on the clinical presentation. the majority of patients (81%) had mild disease (mild pneumonia to no pneumonia), whereas 14% suffered of severe disease manifesting with dyspnea, increased respiratory rate (c 30/min), decreased blood oxygen saturation (spo2 b 93%) and/or lung infiltrates ([ 50% within 1-2 days). a minority of patients (5%) were critically ill where they developed respiratory failure, septic shock and/or multiple organ failure [50] . the systemic presentation of sars-cov-1 and mers-cov is similar to covid-19 except for the upper respiratory tract symptoms and gastrointestinal symptoms (ex: diarrhea) that are much less frequently seen with covid19 [45] . eye symptoms have not been described previously with both sars-cov-1 and mers-cov [6] [7] [8] [9] [56] [57] [58] [59] . there were no ocular manifestations detected in 45 patients with acute sars who were examined at baseline and then at twoand three-month follow-up [9] . no ophthalmological signs have been reported for the other covs, 229e (alpha coronavirus), oc43 (beta coronavirus), hku1 (beta coronavirus), as well. the number of infected healthcare workers is on the rise on a daily basis worldwide, with currently over 9000 healthcare workers infected by sars-cov-2 internationally with the highest rates being in china, italy and recently the usa [60] . as healthcare workers are at the frontline in combating the covid-19 outbreak, it is crucial to ensure their safety, not only to maintain continuous care, but also to prevent further transmission. recommendations on precautionary measures to promote occupational safety and health were recently published by several international organizations such as the cdc and the who [61] [62] [63] [64] . the standard universal precautions instructed to all healthcare facilities regardless of patients' infection statuses (suspected or confirmed) are as follows: limiting healthcare facilities crowding, safe hand hygiene practices, physical and social distancing, respiratory etiquette such as covering the mouth when coughing and the use of personal protective equipment (ppe) when dealing with suspected or infected patients [61] . as the world continues to investigate this emerging covid-19 infection, lessons can be learned from our previous experience with sars-cov-1 and mers-cov-1 in the context of infection control and prevention as all diseases are highly contagious with presumably similar routes of transmission. in a previous case-control study conducted in china among healthcare providers who were taking care of sars patients, all the 69 providers adhering to the four infection control measures (gown, gloves, mask and hand hygiene practices) did not contract the infection, while all the 13 infected staff omitted at least one of the four control measures [65] . inadequate implementation of infection control measures was deemed to be an imperative risk factor for mers-cov infection among healthcare workers [66] . covid-19 was classified as group b infectious disease by the recent guidelines of the china national health commission [67, 68] . however, they suggested that all healthcare workers should take protective measures ''make no mistake approach'' similar to group a infectious diseases (for example: plague and cholera) due to the current lack of evidence on this novel virus. these data necessitate an aggressive preparedness to hopefully mitigate the spread of sars-cov-2 among the healthcare community. many covid-19 patients are asymptomatic on initial presentation, and one would likely present to ophthalmology clinics without respiratory symptoms or fever [17, [69] [70] [71] [72] . actually, reports published recently on the death of dr. li wenliang claim that he mostly acquired the infection while treating his infected glaucoma patient who did not demonstrate any covid-19 symptoms [17, 59] . thus, all patients whether presenting to ophthalmic or nonophthalmic clinics should be questioned about any recent history of travel, exposure to infected or suspected individuals within the past 14 days. ophthalmologists must be very vigilant when approaching a patient with a red eye because covid-19 patients may present initially with conjunctivitis prior to the onset of respiratory symptoms [11] . although wu and his colleagues found only 5% positive conjunctival specimens in patients with sars-cov-2, it was still suggested to consider taking conjunctival swabs for rt-pcr when available to address whether sars-cov-2 is found in any patient who presents with conjunctivitis with no travel history or catarrhal symptoms. this could actually aid in early detection of subclinical cases and subsequent containment of the disease [73] . ophthalmologists were alleged to be among the highest risk subspecialties susceptible to contract the infection due to the face-to-face proximity during slitlamp biomicroscopy examination, direct and indirect ophthalmoscopy which highly expose them to aerosolized particles [74] . therefore, a disinfection protocol was advised in clinic settings to clean potentially contaminated surfaces like slit lamps, lenses and goldman tonometer tips using 70% alcohol-based wipes that are effective against coronaviruses, unlike adenoviruses that are more resistant to these alcoholbased disinfectants [75] . also, large breath shields mounted on slit lamps are being currently used in many ophthalmic institutes as rationally they might be beneficial in minimizing droplet exposure. nevertheless, these barriers can also get contaminated if not properly sterilized between patients, and in the meantime, there is no scientific evidence supporting the definite use of these shields [76] . it was also advised by many organizations to carry out a focused sufficient examination on those with respiratory symptoms and fever but yet demand an urgent ophthalmic assessment while using protective equipment, goggles and mask to both the patient and examiner. additionally, patients should be kindly instructed to refrain from speaking during examination to decrease the risk of exposure [61, 62] . wan et al. also suggested temporarily replacing the use of direct ophthalmoscope with binocular indirect ophthalmoscope as the former has shorter working distance [73] . the american academy of ophthalmology has listed numerous precautionary measures that should be implemented in ophthalmic institutes, specifically in clinics, operating theater and emergency room [76] . they also advised using the following interim guidance for triaging patients seeking ophthalmic assessment based on the clinical situation: • postpone any nonurgent ophthalmic issues until this outbreak is under control • follow the standard precautions, including hand hygiene and eye/nose/mouth protection, when dealing with patients with urgent ophthalmic conditions but no symptoms suggestive of covid-19 • employ extra precautions for patients with urgent ophthalmic conditions and respiratory symptoms only such as goggles, face shield, gloves, surgical mask or n-95 mask if an aerosol-generating procedure will be performed, masking the patient, immediate attendance to the patient and disinfection of the room after the patient departs • treat patients with suspected or confirmed covid-19 and concurrent urgent ophthalmic conditions in a hospital setting with firm compliance with the cdc and/or hospital precautionary guidelines for care of these patients along with curfew and travel restrictions mandated globally, nonurgent and elective ophthalmology clinic appointments and operations are being postponed, especially for elderly and patients with comorbidities who are at increased risk of severe disease [63, 74, 76] . the cdc also recommended delaying all elective ambulatory provider visits, rescheduling elective and nonurgent admissions and procedures. these recommendations are also consistent with the recommendations of the american academy of ophthalmology, the royal college of ophthalmologists, american college of surgeons and many other organizations. if the patient were to have a clinic appointment, it is advisable to contact them before their appointments ahead of time to ask about tocc (travel, occupation, cluster and contact), fever and respiratory symptoms. if a patient had any of the above-mentioned, the appointment should be postponed for at least 14 days if possible. a well-equipped special clinic with a separate waiting area is mandated if appointment is urgent [63] . other measures advised by many medical societies include the following: limiting the number of patients' companions, noncontact temperature measurement before entering the health institute, spacing out patients' appointments to minimize clustering in the waiting hall and social distancing; these are all measures taken and advised by many health societies [62, 63, 73, 74] . telemedicine is also gaining popularity among the different medical specialties to minimize the risk of exposure for both patients and healthcare workers. the european society of ophthalmic plastic and reconstructive surgery has also recommended deferring any nonurgent procedure that requires general anesthesia as this carries increased risk of microaerosol generation. also, they recommended that all patients scheduled to undergo high-speed procedures (for example, orbital decompression with bone drilling) should undergo prior testing for sars-cov-2 whenever possible. nasal procedures like nasopharyngeal swabbing and endoscopic dacryocystorhinostomy should also be avoided or performed under high protection measures because of direct exposure to respiratory tract droplets [77, 78] . when an urgent ophthalmic operation needs to be done, local anesthesia is preferable to avoid general anesthesia as much as possible, as endotracheal intubation might irritate the mucosa which may generate respiratory tract particles [77] . if general anesthesia is essential, it is advised to have a thorough multidisciplinary assessment by an anesthesiologist, internist and ophthalmologist. in order to further minimize exposure among healthcare workers in ophthalmic institutes, some facilities are advocating assigning healthcare providers into smaller groups with different working hours' shifts and schedules. this certainly facilitates proper tracing and isolation among these smaller clusters in case asymptomatic infected individuals were detected [64, 74] . when exposure to a suspected covid-19 patient does occur, healthcare workers are instructed to report immediately to the infection control personnel and isolate themselves at home away from other family members while monitoring themselves for any symptoms. a pcr test is usually performed as well [62] . overall, conjunctivitis/conjunctival congestion is an uncommon manifestation of covid-19 as well as for other coronaviruses' infections that showed a low eyetissue tropism. although ocular transmission remains enigmatic, the most up-to-date evidence proved that the virus might be excreted in tears and conjunctival secretions and also replicate in conjunctival tissue up to 27 days after the onset of the disease; hence, the eye should be regarded as a potential source of infection dissemination. until further evidence becomes available on ocular tissue tropism and permissiveness to the coronaviruses, ophthalmologists and ophthalmic-care providers must heighten their precautionary measures given that ophthalmic examinations and procedures necessitate close contact with patients. ocular tropism of respiratory viruses the severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a pneumonia outbreak associated with a new coronavirus of probable bat origin duncan powers sl (2020) ophthalmologists are more than eye doctors-in memoriam li wenliang the severe acute respiratory syndrome coronavirus in tears tears and conjunctival scrapings for coronavirus in patients with sars virus-specific rna and antibody from convalescent-phase sars patients discharged from hospital ocular screening in severe acute respiratory syndrome there may be virus in conjunctival secretion of patients with covid-19 characteristics of ocular findings of patients with coronavirus disease 2019 (covid-19 evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva. preprint, ophthalmology. epub ahead of print 12 assessing viral shedding and infectivity of tears in coronavirus disease clinical characteristics of coronavirus disease 2019 in china the infection evidence of sars-cov-2 in ocular surface: a single-center crosssectional study. preprint, public and global health. epub ahead of print 26 2019-ncov transmission through the ocular surface must not be ignored ocular manifestations and clinical characteristics of 534 cases of covid-19 in china: a cross-sectional study. preprint, ophthalmology. epub ahead of print 16 ocular findings and proportion with conjunctival sars-cov-2 in covid-19 patients sars-cov-2 isolation from ocular secretions of a patient with covid-19 in italy with prolonged viral rna detection keratoconjunctivitis as the initial medical presentation of the novel coronavirus disease 2019 (covid-19) who. who announces covid-19 outbreak a pandemic identification of a new human coronavirus association between a novel human coronavirus and kawasaki disease 2020) features, evaluation and treatment coronavirus (covid-19 human coronavirus: host-pathogen interaction mers, sars and other coronaviruses as causes of pneumonia: mers sars and coronaviruses cdc. human coronavirus types characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia human coronaviruses: what do they cause close relative of human middle east respiratory syndrome coronavirus in bat south africa wildlife and emerging zoonotic diseases: the biology, circumstances and consequences of cross-species transmission the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus ocular renin-angiotensin system with special reference in the anterior part of the eye angiotensin ii and its receptor subtypes in the human retina mechanism of the action between the sars-cov s240 protein and the ace2 receptor in eyes dipeptidyl peptidase 4 distribution in the human respiratory tract retinopathy following intravitreal injection of mice with mhv strain jhm ocular tropisms of murine coronavirus (strain jhm) after inoculation by various routes retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinopathy retinal degeneration in experimental coronavirus retinopathy (ecor) is associated with increased tnf-a, soluble tnfr2 and altered tnf-a signaling covid-19 and the eye immunity: lesson learned from the past and possible new therapeutic insights influenza caused epidemic encephalitis (encephalitis lethargica): the circumstantial evidence and a challenge to the nonbelievers role of the eye in transmitting human coronavirus: what we know and what we do not know. preprint, medicine and pharmacology. epub ahead of print 17 sars: a timely reminder the possibility of covid-19 transmission from eye to nose tropism, replication competence, and innate immune responses of the coronavirus sars-cov-2 in human respiratory tract and conjunctiva: an analysis in ex-vivo and in-vitro cultures facing ubiquitous viruses: when hand washing is not enough characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention in the absence of sars-cov transmission worldwide: guidance for surveillance, clinical and laboratory evaluation, and reporting clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan china clinical features of patients infected with 2019 novel coronavirus in wuhan middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings middle east respiratory syndrome coronavirus in pediatrics: a report of seven cases from saudi arabia middle east respiratory syndrome (mers): a new zoonotic viral pneumonia novel coronavirus disease 2019 (covid-19): the importance of recognising possible early ocular manifestation and using protective eyewear covid-19: protecting health-care workers who. coronavirus disease (covid-19) outbreak: rights, roles and responsibilities of health workers, including key considerations for occupational safety and health stepping up infection control measures in ophthalmology during the novel coronavirus outbreak: an experience from hong kong the royal college of ophthalmologists protecting patients, protecting staff. the royal college of ophthalmologists effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu announcement of national health commission of the people's republic of china protecting healthcare workers from subclinical coronavirus infection a confirmed asymptomatic carrier of 2019 novel coronavirus who (2020) report of the who-china joint mission on coronavirus disease 2019 covid-19: identifying and isolating asymptomatic people helped eliminate virus in italian village presumed asymptomatic carrier transmission of covid-19 effects of the first month of lockdown for covid-19 in italy: a preliminary analysis on the eyecare system from six centers oculoplastic management of patients in the covid-19 era: experience from an italian tertiary referral center efficacy of various disinfectants against sars coronavirus important coronavirus updates for ophthalmologists aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review recommendations for oculoplastic surgeons during the covid-19 pandemic funding none. conflicts of interest all authors of this study have no conflict of interest to disclose. key: cord-305745-9lngdjow authors: solnier, julia; fladerer, johannes-paul title: flavonoids: a complementary approach to conventional therapy of covid-19? date: 2020-09-18 journal: phytochem rev doi: 10.1007/s11101-020-09720-6 sha: doc_id: 305745 cord_uid: 9lngdjow covid-19, the highly contagious novel disease caused by sars-cov-2, has become a major international concern as it has spread quickly all over the globe. however, scientific knowledge and therapeutic treatment options for this new coronavirus remain limited. although previous outbreaks of human coronaviruses (covs) such as sars and mers stimulated research, there are, to date, no antiviral therapeutics available that specifically target these kinds of viruses. natural compounds with a great diversity of chemical structures may provide an alternative approach for the discovery of new antivirals. in fact, numerous flavonoids were found to have antiviral effects against sars-and mers-cov by mainly inhibiting the enzymes 3-chymotrypsin-like protease (3clpro) and papain-like protease (plpro). in this review, we specifically focused on the search for flavonoids, polyphenolic compounds, which are proven to be effective against human covs. we therefore summarized and analyzed the latest progress in research to identify flavonoids for antiviral therapy and proposed strategies for future work on medicinal plants against coronaviruses such as sars-cov-2. we discovered quercetin, herbacetin, and isobavachalcone as the most promising flavonoids with anti-cov potential. historically, viral diseases have always emerged and posed major issues to public health. several viral outbreaks-such as the severe acute respiratory syndrome coronavirus (sars-cov) in 2002 -2003 , the h1n1 influenza virus in 2009 , and the middle east respiratory syndrome coronavirus (mers-cov) in 2012-have caused serious global health concerns in recent years (cascella et al. 2020) . over the past 50 years, there has been a noticeable increase in the emergence of different novel coronaviruses responsible for a wide range of human and veterinary diseases (fehr and perlman 2015) . most recently, a new viral epidemic with numerous cases of unexplained low respiratory tract infections occured in wuhan, hubei province, china, as it was first reported to the world health organization (who) on 31 december 2019 (world health organization 2020b). the novel virus strain was identified as the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) triggering coronavirus disease 2019 (he et al. 2020a ). on 11 march 2020, the who declared covid-19 a pandemic (world health organization 2020a). coronaviruses (covs) are highly diverse, enveloped, positive-sense, single-stranded rna viruses (? ssrna) (he et al. 2020a) , which constitute the biggest group of viruses within the nidovirales order, containing the largest genomes for rna viruses (fehr and perlman 2015) . in total, about 30 covs have so far been recognized to be able to infect different species, including humans, mammals, fowl, and other animals ). among them, seven human covs, belonging to the alpha-and beta-covs groups , have been identified as being capable of infecting humans, including 229e, nl63, oc43 hku1, mers-cov, sars-cov and the novel sars-cov-2 (centers for disease control and prevention 2020; fehr and perlman 2015; zhu et al. 2020 ). the name 'coronavirus' is inspired by its most defining feature: the club-shaped spikes projecting from the surface of the virion. the spikes sticking out of the envelope's surface give the virus the appearance of a crown (fehr and perlman 2015) . the nucleocapsids of covs, enclosing the genomic rna, are helically symmetrical. this is in fact unusual for positive-sense rna viruses, and far more common for negative-sense rna viruses (fehr and perlman 2015) . the two overlapping open-reading-frames (orf1a and orf1b) of sars, translated into the viral enzymes 3c-like protease (3clpro) and papainlike protease (plpro), which are vital for virus multiplication, constitute approximately two-thirds of the genome (adedeji et al. 2012 ). the other onethird of the genome encodes structural proteins of the virus, such as the spike (s), envelope (e), membrane (m) and nucleocapsid (n) proteins (adedeji et al. 2012) . the interaction between the s-protein and the receptor is the primary determinant for a coronavirus to infect a host species (lim et al. 2016) . to date, it is known that sars-cov attaches to its receptor angiotensin-converting enzyme 2 (ace2), while mers-cov was found to bind to dipeptidyl-peptidase 4 (dpp4) in order to penetrate human cells (fehr and perlman 2015) . so far, it has been observed that the new coronavirus sars-cov-2 behaves much like sars by using the same entry mechanism to human cells (rabi et al. 2020 ) and sharing a 79.5% genome sequence identity to sars-cov zhou et al. 2020) . several studies have demonstrated that novel sars-cov-2 likely binds to the human ace2 receptor, but with a higher affinity than the original sars virus strain (gurwitz 2020; letko et al. 2020; rabi et al. 2020; wrapp et al. 2020; xu et al. 2020) . genetic data demonstrated that sars-cov-2 possesses overlapping open-reading-frames (orf1a and orf1b) similar to those of sars-and mers-cov ( fig. 1) , translated into the viral enzymes 3clpro and plpro. sars-and sars-cov-2 share a 3clpro sequence similarity of 96%, and a plpro sequence identity of 83% (mckee et al. 2020) . therefore, 3clpro and plpro present two key targets for the development of anti-sars-cov-2 therapeutics as both are crucial for viral replication; and they share significant homology with proteases of several other related coronaviruses (goetz et al. 2007 ). even though the sars and mers outbreaks stimulated research on human covs, there are, to date, no antiviral therapeutics available that specifically target these viruses (rabaan et al. 2020) . several potential vaccines, including recombinant attenuated viruses, live virus vectors, or individual viral proteins expressed from dna plasmids, have been developed for sars-cov; however, none of them are yet approved for clinical use (fehr and perlman 2015) . there are several reports which propose potential drugs, although their clinical efficacy has not yet been confirmed for sars-cov-2 infection and covid-19 disease. these drugs include: chloroquine, lopinavir/ ritonavir, remdesivir, umifenovir, nucleoside analogs, neuraminidase inhibitors, dna synthesis inhibitors (e.g. tenofovir disoproxil, and lamivudine), ace2based peptides, novel vinylsulfone protease inhibitors, teicoplanin, 3-chymotrypsin-like protease (3clpro)and papain-like protease (plpro) inhibitors mckee et al. 2020) . to date, the application of remdesivir appears to be the most promising strategy for covid-19 . in preclinical studies, it has been shown that remdesivir (gs5734)-an inhibitor of rna polymerase with in-vitro activity which was used against various rna viruses, including ebola-could be effective for both prophylaxis and therapy against human cov infections (gordon et al. 2020) . alpha-interferon and lopinavir/ritonavir have also been suggested for the treatment of covs (cascella et al. 2020 ). since therapy options for coronaviruses, such as for covid-19, comprise only preventive and supportive measures, natural products may have a fundamental role in supportive and prophylaxis treatments, and present an alternative approach for cov-management. flavonoids form the largest group of polyphenolic compounds in higher plants (nileeka balasuriya and vasantha rupasinghe 2011) , with more than 9000 structures identified . they represent an important class of plant secondary metabolites, widely distributed throughout the plant kingdom . flavonoids are categorised into several subgroups, which include chalcones, flavanes, flavanols, flavanones, flavanonols, flavones, flavonols, isoflavones or catechins, and procyanidins, all of them consisting of a common flavan (2-phenylchroman) basic structure (fig. 2) . these polyphenolic substrates perform a series of protective functions in the human body. many of them are bioactive compounds capable of interfering with nucleic acid or proteins, meaning that they have diverse pharmacological properties (panche et al. 2016) . it has been reported that flavones and catechins appear to be the most powerful antioxidants, preventing the effects of reactive oxygen species in the body (nijveldt et al. 2001; panche et al. 2016) . there are numerous studies highlighting the broad range of biological activities of flavonoids, including antioxidant (d'amelia et al. 2018 ), anti-cancer (lejeune et al. 2015 , antimicrobial (abreu et al. 2017; solnier et al. 2020) , antiviral (wang et al. 1998) , and antiinflammatory (catarino et al. 2016 ) activities (panche et al. 2016) . more importantly, various flavonoids have been found to inhibit different targets of coronaviruses sars and mers , such as blocking the enzymatic activites of viral proteases like 3-chymotrypsin-like protease (3clpro), papain-like protease (plpro) and helicase or interfering with spike (s) proteins. a few flavonoids were shown to suppress the activity of angiotensin-converting enzyme (ace) (guerrero et al. 2012 ; nileeka balasuriya and vasantha rupasinghe 2011), which not only plays an important role in cardiovascular diseases such as in hypertension, but may also represent a key determinant in viral infections, and pneumonia (actis-goretta et al. 2006) . another important issue presents the anti-inflammatory potential of flavonoids in viral diseases, such as activating and stimulating the host immune response to viral infections (dong et al. 2014 ), but also suppressing overwhelming inflammatory reactions, which are often associated with a higher mortality rate of sars-cov-2 infections (mckee et al. 2020) . for instance, some flavonoids have been reported to interfere with the activation of nlrp3 inflammasome (lim et al. 2018 ) which upregulates the production of inflammatory cytokines, and thus can cause respiratory distress syndrome that frequently occurs within sars coronavirus diseases (chen et al. 2019) , and sars-cov-2 infections (shah 2020) . however, most studies on flavonoids-showing their numerous health-beneficial properties-are conducted in vitro based on the fact that these polyphenolic compounds often deal with low bioavailability, little stability and poor distribution when tested in vivo using animal and/or human cell models (nileeka balasuriya and vasantha rupasinghe 2011) . there are different strategies reported to enhance these functions, such as the introduction of structural modifications (srinivas 2009) , absorption enhancers or nanotechnology (ajazuddin and saraf 2010; zhao et al. 2019) . the aim of this review is to evaluate the information on flavonoids as possible leads for developing therapeutics to treat sars-cov-2. therefore, we searched pubmed for studies reporting on the effects of flavonoids on human coronaviruses. most of the studies targeted the enzymatic activities of the viral proteases 3cl and pl in vitro using fret (fluorescence resonance energy transfer)-based methods. 3cl and pl proteases represent valuable targets for the development of anti-coronaviral drugs, as these proteins are essential for the viral transcription and replication complex of all coronaviruses (anand et al. 2003; chen et al. 2005b; park et al. 2016) , translated from two open reading frames orf1a and orf1b which are found in sars-, sars-2-, and mers-cov (chuck et al. 2011; lin et al. 2004 ). according to the literature, we were able to identify 47 flavonoids which might present potential agents to treat sars-cov-2 (table 1) . detailed analysis of their structureactivity relationships, has ultimately led to the identification of the three most promising compounds with broad-spectrum antiviral activity. the various studies have focused primarily on the interference of flavonoids with some viral proteases such as 3cl and pl of sars-and mers-cov by using common enzymatic, fluorogenic (fret)-based methods and molecular docking studies, as summarized in table 2 . 3clpro and plpro are both key targets as they process several viral polyproteins which are involved in the replication and transcription of the genomic rna within host cells (mckee et al. 2020) . further, they share significant homology with viral proteases of several other coronaviruses-especially with those of sars-, sars-2-, and mers-cov . besides 3cl protease, papain-like protease has been the focus of numerous studies on the development of chemotherapeutic drugs against cov-induced diseases. plpro is not only responsible for processing viral polyproteins, but is also involved in deubiquitination (cleaving ubiquitin chains) and deisgylation, which represent relevant factors in the host immune response to viruses (cho et al. 2013) . by removing ubiquitin and isg15 proteins from host cell proteins, the innate host immune response is likely to be compromised (ratia et al. 2014; xian et al. 2020 ). plpro of both sars-and mers-cov has been shown to possess deubiquitinating activities (park (park et al. 2016 ) and may, therefore, have an important part in the virus life cycle. hence, antiviral therapeutics targeting plpro could also prevent the antagonist activities of plpro on host innate immune response (ratia et al. 2014 ). in fret-based assays, the proteolytic activity is detected through cleavage of a fluorogenic peptide and measuring the increase in fluorescence intensity by continuously monitoring the reaction . fret is a non-destructive method, widely exploited to study protein interactions (margineanu et al. 2016) , and is also applied to detect signals in living cells ). while the cell-free fret assays can provide easy and fast access to proteins including some difficult targets (sierecki et al. 2013) , the cell-based fret methods enable in situ analysis of a variety of biological targets and proteinprotein interactions in a more natural environment (silverman et al. 1998 ). the studies of park et al. (2017) , lin et al. (2005) and others could prove that flavonoids tested in cell-based assays generated higher activity against 3cl and pl, than excerting unspecific activity against other enzymes. furthermore, the unspecific aggregation of proteins often caused by flavonoids could be reduced significantly by the addition of triton x without affecting anti-coronaviral activity in these studies. in a number of studies molecular docking technology was used for screening purposes. despite the valuable use of this method to rapidly discover novel inhibitors of sars-cov-2, there are some disadvantages which include low accuracy and high rate of false-positive results ). however, compounds which are identified through such virtual screening methods can be further examined in highthroughput enzymatic in-vitro assays, followed by more in-depth in vivo investigations, such as studies on efficacy and toxicity. in general, the combination of docking studies and bioactivity assays can improve screening accuracy by providing supportive data. chalcones isolated from angelica keiskei were shown to inhibit both sars-cov proteases plpro and 3clpro in enzymatic, fret-based (table 2) and molecular docking studies (park et al. 2016 ). inhibition was achieved through a competitive manner against 3clpro, and a non-competitive mode against plpro (park et al. 2016) . xanthoangelol e, an alkylated chalcone substituted with a -ooh group (table 1) , exerted the most relevant inhibition against 3clpro (ic 50 = 11.4 and 7.1 lm) and plpro (ic 50-= 1.2 lm) using fluorogenic methods (park et al. 2016 ) ( table 2 ). analyses of the relationships of the alkylated chalcones (tables 1 and 2) against 3clpro showed that xanthoangelol e substituted with 2-perhydroxyl-3-methyl-3-butenyl (pmb) group was more effective than xanthoangelol d with a 2-hydroxy-3methyl-3-butenyl group (ic 50 = 26.6 and 9.3 lm). similarly, the hydroxyl group of the a-ring caused apparently higher activity than when substituted with a methyl group. comparing xanthoangelol d and 4-hydroxyderricin, it seemed that the substitution of the a-ring with a 2-hydroxy-3-methyl-3-butenyl group (xanthoangelol d, table 1 ) generated higher inhibition than when substituted with the dimethylallyl group (4hydroxyderricin: ic 50 = 81.4 and 50.8 lm, table 2 ) (park et al. 2016 ). based on the results it can be assumed that a perhydroxyl group on the substituted hemiterpene might be crucial for enzyme binding and may affect conformational stabilization of the polyhydroxylated chain through intramolecular hydrogen bonding (park et al. 2016) . in general, the bioactivity of highly potent compounds against sars plpro apparently depended on modifications in their molecular structure, such as prenylation in the a and b ring and methylation. for instance, the prenyl moiety of the 3 0 -prenylated chalcones isobavachalcone and xanthoangelol e led to considerable inhibition against sars plpro (ic 50-= 7.3 and 1.2 lm), highlighting the importance of the hydrophobic substituent. further, it is generally assumed that prenylation of flavonoids may enhance their bioactivity and bioavailability (grienke et al. 2016; mukai 2018) , presumably by altering lipophilicity and thereby affinity for membrane targets (shen et al. 2012) . thus, chemical modifications of flavonoids, such as the introduction of hydrophobic substituents, may also demonstrate a reasonable strategy to enhance their antiviral activity against sars-cov-2. some flavonoids isolated from broussonetia papyrifera have been proposed as useful lead-compounds for the development of anti-cov agents against sars and mers ) and therefore, might be also considered to treat sars-cov-2. among them, broussochalcone a, broussochalcone b, and 4-hydroxyisolonchocarpin derived from b. papyrifera showed proteolytic activity against both proteases 3cl and pl of sars-and mers-cov (table 2) through non-competitive inhibition. as table 2 demonstrates, the compounds showed generally higher inhibitory potential against sars-cov plpro than when tested against the other viral proteases using fluorogenic methods, which is likely related to genomic variations in the single amino acid sequences. broussochalcone b was found to be the most effective substrate for inhibiting 3clpro of mers-cov (ic 50 = 27.9 lm) in the study of park et al. (2017) . in addition to inhibitory effects on cysteine proteases, the compounds isolated from b. papyrifera caused inhibition against alpha-glucosidase . inhibition of glycosidases, especially of alpha-glucosidases, affects maturation, transport, secretion, and the functioning of glycoproteins which can enhance cell-cell and/or cell-virus recognition processes (ryu et al. 2010b ). therefore, it plays a relevant role in the treatment of viral infections, but also in other diseases such as diabetes mellitus type 2, and cancer (ryu et al. 2010b ). in accordance with a previous study (ryu et al. 2010b) , the alpha-glucosidase inhibitory activity of the substrates from b. papyrifera depends on the number and positions of prenyl groups present in the molecule . kim et al. (2014) could demonstrate that isobavachalcone and 4 0 -o-methylbavachalcone extracted from the seeds of cullen corylifolia have great inhibitory potential against sars-cov plpro. both compounds inhibited plpro in a dose-dependent manner, with ic 50 values of 7.3 and 10.1 lm (table 2 ) (kim et al. 2014) . isobavachalcone (2 0 ,4,4 0 -trihydroxy-3 0 -(3-methyl-2-butenyl) chalcone) also showed inhibitory activity against mers-cov 3clpro (ic 50-= 35.85 lm, table 2 ), when tested within the screening of a flavonoid library using a fluorogenic (fret-based) method (jo et al. 2019) . based on the results summarized in table 2 , the chalcones isobavachalcone, 4 0 -o-methylbavachalcone, broussochalcone a and b, including xanthoangelols, can be considered as prominent inhibitory compounds of sars-cov plpro (ic 50 b 12.5 lm, table 2 regarding the broad-spectrum activity of chalcones, isobavachalcone presents the major compound in this group -with a good scaffold to bind with proteases of both sars-and mers-cov. hence, further in-vitro investigations with isobavachalcone on viral proteins of sars-cov-2, as well as some pharmacological studies in-vivo regarding toxicity and bioavailability of the compound, might be a valuable approach in the field of research for covid-19. there are a few plant sources of isobavachalcone other than those mentioned in table 2 , as it is abundantly found in species belonging to plant families fabaceae (e.g., anthyllis hermanniae, glycyrrhiza glabra, glycyrrhiza uralensis, sophora prostrata) and moraceae (e.g., dorstenia poinsettifolia, dorstenia turbinata, maclura tinctoria, treculia acuminata) (kuete and sandjo 2012). the flavanes 3 0 -(3-methylbut-2-enyl)-3 0 ',4,7-trihydroxyflavane, broussoflavan a, kazinol a and kazinol b isolated from broussonetia papyrifera interfered with viral proteases 3cl and pl of both sars-and mers-cov (table 2) through non-competitive inhibition ). among all flavanes tested, 3 0 -(3-methylbut-2-enyl)-3 0 ,4,7-trihydroxyflavane, a c5alkyl group (prenyl)-substituted flavan (table 1) , exhibited the most potent inhibition against sarsand mers-cov 3clpro (ic 50 = 30.2 lm and 34.7 lm). considering the structure-activity relationships of flavonoids from b. papyrifera (tables 1 and 2) , it was observed that molecules having a c4-oh group and saturated chromenone with a dihydroxy group at ring c have stronger inhibitory potential than those with a closed prenyl group such as kazinol b (ic 50 = 233.3 lm, table 2) . given the results, the flavane 3 0 -(3-methylbut-2enyl)-3 0 ,4,7-trihydroxyflavane may serve as good template for continuing in-vitro experiments on viral proteases of sars-cov-2 due to similar efficacy against proteases of both sars-and mers-cov, and the homology of proteases from the sars-cov-2 strain (fig. 1) . epigallocatechin gallate (egcg) and gallocatechin gallate (gcg), two flavanol derivatives, demonstrated inhibitory activity against sars-cov 3clpro, when tested in proteolytic (fret-based) assays and molecular docking studies of nguyen et al. (2012) . the catechin gcg (ic 50 = 47 lm) inhibited 3clpro activity (table 2 ) by interacting with amino acid residues in the active site pocket of 3clpro (nguyen et al. 2012 ). analyses of structure-activity relationships of these compounds (tables 1 and 2) suggest that the galloyl moiety at position 3-oh of egcg and gcg (table 1) , which is absent in the other catechins, seemed to be relevant for causing 3clpro inhibition. gcg (2s, 3r type), which is a c-2 epimeric isomer of egcg (2r, 3r type), generated higher 3clpro inhibition than that of egcg (ic 50 = 73 lm). in a following docking study, it was shown that the galloyl group of gcg, was essential for hydrogen bond interactions with the amino residues leu141, gly143, ser144, and his163 of the 3clpro active pocket site (nguyen et al. 2012) . for further studies with these compounds on proteases of sars-cov-2 it should be considered that different conformation and structural modifications in the molecules likely increase antiviral activity. in a study investigating the chinese medicinal plant isatis tinctoria root-which was used for the prevention of sars-cov during the sars-outbreaks, and is known for its antiviral properties against influenza, hepatitis a and japanese encephalitis infections (lin et al. 2005; wu et al. 1997 )-hesperetin was discovered as highly potent inhibitor of sars-cov 3clpro (ic 50 = 8.3 lm) when tested in a cell-based cleavage assay (lin et al. 2005) . in comparison with other reported inhibitors of sars-cov 3clpro, such as cinanserin and cinanserin hydrochloride (ic 50-= 31 lm and 34 lm, respectively) (chen et al. 2005a) , hesperetin may present a promising lead in the development of sars-cov-2 3cl protease inhibitors. however, further screenings on proteolytic activity of hesperetin against plpro might be also important. several citrus fruits are the major plant sources of flavanones, including hesperetin-the aglycone of hesperidin. bavachinin, a flavanone extracted from the seeds of cullen corylifolia, was found to have inhibitory potential against sars-cov plpro (ic 50 = 38.4 lm, table 2 ) (kim et al. 2014) . amelopsin, also known as dihydromyricetin, had only weak inhibitory activity against sars-cov 3clpro (ic 50 = 364 lm, table 2 ) when tested in a proteolytic (fret-based) assay and molecular docking studies (nguyen et al. 2012 ). in the same study, it was observed that the additional oh group at 5 0position in the b ring as well as the absence of 2,3double bonds in the c-ring of amelopsin (table 1) considerably reduced 3clpro inhibitory activity when compared to other compounds tested, such as quercetin (ic 50 = 73 lm, table 2 ) (nguyen et al. 2012 ). amentoflavone, a biflavone isolated from torreya nucifera, demonstrated a prominent inhibitor of sars-cov 3clpro (ic 50 = 8.3 lm) in a fretbased assay (ryu et al. 2010a) . in that study, amentoflavone proved to be far more potent than the parent compound apigenin (ic 50 = 280.8 lm), and the other flavone luteolin (ic 50 = 20 lm), as well as the flavonol quercetin (ic 50 = 23.8 lm). by comparing their structures (table 1) , those compounds with a c-3 0 -substituted hydroxyl group, such as luteolin and quercetin, excerted stronger inhibition than apigenin. furthermore, biflavone derivatives of amentoflavone with methylation of 7-, 4 0 -, and 4 000 -hydroxyl groups, decreased inhibitory activity (ryu et al. 2010a) . based on the prominent in vitro inhibition activity of amentoflavone against 3clpro of sars-cov, the compound may serve as a good starting point for further investigations on 3clpro of sars-cov-2due to 79.5% genome sequence identity zhou et al. 2020) , including more than 95% similarity of 3clpro amino acid sequences between sars-cov and sars-cov-2 (mckee et al. 2020). however, the biflavonoid should be also tested against other proteases of sars-and mers-cov in order to determine a broad spectrum antiviral efficacy against sars-cov-2. there are numerous plant sources from which amentoflavone can be isolated, these include for instance gingko biloba, hypericum perforatum, lobelia chinensis, polygala sibirica, ranunculus ternatus, and several plants from selaginella species, such as selagenella tamariscina, selaginella nipponica, and selaginella pulvinata (yu et al. 2017) . two other flavones, pectolinarin and rhoifolin, and the flavonol herbacetin were discovered as inhibitors of sars-cov 3cl enzyme when tested in proteolytic assays (ic 50 b 37.78 lm, table 2 ) and induced-fit docking experiments (jo et al. 2020) . in docking studies, jo et al. showed that the additional 8-hydroxyl group of herbacetin seems to be crucial for its high binding affinity to the polar s1 site and the hydrophobic s2 site of sars-cov 3clpro (jo et al. 2020) . furthermore, carbohydrate groups of rhoifolin and pectolinarin were responsible for the high affinity to sars-cov 3clpro (jo et al. 2020 ). in another screening using a fret-based method, herbacetin (3,4 0 ,5,7,8-pentahydroxyflavone, table 1 ) also caused inhibition against 3clpro of mers-cov at an ic 50 value of 40.59 lm (table 2) (jo et al. 2019) . since herbacetin shows similar inhibitory effects against both 3cl proteases of sars-and mers-cov (ic 50 = 33.17 and 40.59 lm), it may also demonstrate proteolytic activity when tested against sars-cov-2 3cl protease. several 3clpro inhibitors that have been suggested as candidates for sars-cov-2 3clpro, were found to be effective against 3clpro of both sars-and mers-cov (he et al. 2020b ). herbacetin can be found, for instance, in ephedrae herba (hyuga et al. 2013) , linum usitatissimum (qiu et al. 1999) and rhodiola rosea (pã©ter zomborszki et al. 2019) . papyriflavonol a, a double prenylated flavone derivative isolated from broussonetia papyrifera, presents one of the most significant inhibitors of sars plpro at an ic 50 value of 3.7 lm (table 2) , causing higher activity than other flavonols such as kaempferol (ic 50 = 16.3 lm), quercetin (ic 50-= 8.6 lm), and quercetin-beta-galactoside (ic 50-= 51.9 lm). similarly, geranylated flavonols from the fruits of paulownia tomentosa were found to be the main group of active constituents that successfully interferes with sars-cov plpro (cho et al. 2013 ). among twelve plpro-inhibitory flavonoids isolated, five new geranylated flavonols named tomentin a-e bearing an unusual 3,4-dihydro-2h-pyran motif with a cyclized geranyl chain (table 1) caused high inhibition (ic 50 values 5-12.5 lm) within a fret-based assay (table 2) . prenylated and geranylated flavonoids like papyriflavonol a, and tomentins (ic 50 b 7.3 lm, table 2 ) can be proposed as interesting leads for sars-cov-2 plpro inhibitors due to 83% homology of plpro sequences between sars-cov and sarscov-2 (mckee et al. 2020) . it can be assumed that the hydrophobic substituents of prenylated flavonoids e.g., papyriflavonol a, show higher affinity to sars-cov plpro than to other proteases, which might be due to certain structural differences in the protein sequences. further, the polarity of the compounds such as an increased number of hydroxyl groups, including the addition of sugar residues in the flavone backbone (table 1) , seems to affect inhibitory potential, as demonstrated by kaempferol (ic 50 = 16.3 lm), quercetin (ic 50-= 8.6 lm), and quercetin-b-galactoside (ic 50 = 51.9 lm). comparing quercetin with quercetin b-glycosides-the sugar moieties such as that of quercetin-3-b-galactoside, and quercetin 3-b-d-glucoside did not enhance inhibitory activity against sars-and mers-cov proteases. in molecular docking studies, quercetin-3-b-galactoside created hydrophobic interactions with residues asn142, glu166, leu141 and met165 of sars 3clpro (chen et al. 2006) . in particular, the residue q189 represented a principle factor in the binding between quercetin-3-b-galactoside and sars 3clpro (chen et al. 2006) . analyses of structure-activity relationships of quercetin-3-b-galactoside derivatives suggested that hydroxyl groups on the quercetin moiety are crucial for inhibitory activity, which enable compound-target interaction through hydrogen bonding (chen et al. 2006) . quercetin and quercetin 3-b-glucoside (3,3 0 ,4 0 ,5,7pentahydroxyflavone 3-b-glucoside) exerted inhibition when tested against mers 3clpro with respective ic 50 values of 34.8 and 37.03 lm (table 2) (jo et al. 2019) . in a following docking study, jo et al. showed that a rhamnose substitution instead of glucose (in quercetin 3-b-glucoside), results in a stronger binding with mers-cov 3clpro (jo et al. 2019) . in general, quercetin causes a broad range of activities against proteases of sars-and mers-cov (table 2) . based on that, and the number of sources cited, such as the ic 50 values reported in different studies, quercetin is among the most promising agents to treat sars-cov-2. quercetin represents one of the most abundant dietary flavonoids that naturally occurs in a great variety of plant products (medicinal herbs, food, beverages, etc.) (anand david et al. 2016; pandey and rizvi 2009) . a few of these rich sources from which quercetin can be isolated include: allium cepa, allium fistulosum, asparagus officinalis, camellia sinensis, capparis spinosa, coriandrum sativum, ginkgo biloba, hypericum perforatum, moringa oleifeira, punica granatum (howell and d'souza 2013) , sambucus canadensis (anand david et al. 2016; li et al. 2016) . the isoflavones, daidzein (ic 50 = 351 lm) and puerarin (ic 50 = 381 lm) lacking the b-ring in their 3-phenyl-4h-1-benzopyran-4-one backbone (table 1) , showed only weak inhibition of sars-cov 3clpro, as demonstrated in table 2 . neobavaisoflavonestructurally related to daidzein-prenylated at c3 0 , and corylifol a extracted from the seeds of cullen corylifolia, were found to have good inhibitory potential against sars-cov plpro (ic 50 = 18.3 and 32.3 lm) (kim et al. 2014) . as mentioned before, flavonoids with lipophilic substituents like prenyl or geranyl side chains in the a or b ring (table 1) appear to be very good scaffolds to bind with pl proteases, and may therefore be considered as templates for the development of sars-cov-2 plpro inhibitors. isoflavonoids are predominantly found in legumes; prenylated derivatives can be mainly isolated from the plant familiy fabaceae (å  mejkal 2014). flavones and flavonols a screening of 64 natural compounds against sars-cov helicase led to the identification of scutellarein and myricetin-two strong inhibitors of sars nsp13 (table 2) using an atp hydrolysis assay yu et al. 2012) . the non-structural proteins (nsps), which are processed by the viral proteases pl (residing in nsp3) and 3cl (residing in nsp5), comprise the rna-dependent rna polymerase and the ntpase/helicase-two essential determinants for virus replication (adedeji et al. 2012) . the sars helicase nsp13 protein has been shown to have doublestrand (ds) rna and (ds) dna unwinding activity and can translocate along the nucleic acids by hydrolyzing atp . both compounds, the flavone scutellarein and the flavonol myricetin, successfully impaired atpase activity of nsp13 with respective ic 50 values of 0.86 lm and 2.71 lm (table 2 ). this effect was mediated through inhibition of atpase activity, but did not affect dsdna-unwinding activity of sars helicase, as it was demonstrated in a fluorometric, fret-based, dsdna unwinding assay of yu et al. (2012) . myricetin naturally occurs in high concentrations in different fruits (e.g., cranberry) (singh et al. 2009 ), and vegetables such as spinach, cauliflower etc. (sultana and anwar 2008) . scutellarein can be found in members of the genus scutellaria such as scutellaria baicalensis-an important traditional chinese plant used against diverse infections including inflammatory or respiratory diseases (zhao et al. 2016) . another study evaluating 24 derivatives of 7-oaryl-methylquercetin for their antiviral activity against sars-cov, three derivatives with 3 0 '-cl, 3 0 '-cn, and 4 0 '-cl aromatic substituents showed selective inhibition of sars-cov ntpase/helicase activity by performing a fret-based dsdna unwinding assay (park et al. 2012) . the introduction of an arylmethyl substituent, such as 3-clphch2, 3-cnphch2 and 4-clphch2, at the position 7-oh of quercetin (table 1) , led to stronger inhibition against sars-cov helicase (ic 50 values of 5.2, 2.7, 4.1 lm, respectively) than that of quercetin (ic 50 = 8.1 lm) (park et al. 2012 ). the spike (s) protein of covs is responsible for viral entry into target cells through binding to the angiotensin-converting enzyme 2 (ace2) receptor on host cell membranes , and thus presents another important target in the anti-sars-cov-2 drug development. besides the inhibitory potential of luteolin against sars 3cl protease (ic 50 = 20 lm, table 2 ), the flavone was found to successfully impede the entry process of sars-cov into host cells by interfering with the s protein of sars-cov (ec 50 value = 10.6 lm) in a two-step screening method combining frontal affinity chromatography-mass spectrometry (fac/ms) and pseudotyped virus-infection assay (yi et al. 2004 ). likewise, quercetin was found to block host cell entry (ec 50 = 83.4 lm) when tested in hiv-luciferase/ sars pseudotyped virus assay (yi et al. 2004 ). the use of pseudotyped viruses is reported to be a powerful, rapid, and highly sensitive method for studying the entry process of viruses to host cells (yi et al. 2004) . since sars-cov and sars-cov-2 share high sequence similarity of the s proteins, these compounds may be also expected to impede the entry of sars-cov-2 into host cells. furthermore, it is proven that the s protein of sars-cov-2 binds with 10-20-fold higher affinity to ace2 than that of sars-cov (mckee et al. 2020). thus, inhibition of ace2 via a competing substance, seems to be a reasonable approach for preventing sars-cov-2 infections. quercetin had potent inhibitory effects on ace in vitro, and in vivo when tested in rat models (actis-goretta et al. 2006; al shukor et al. 2013; guerrero et al. 2012; nileeka balasuriya and vasantha rupasinghe 2011) . however, ace inhibitors-which are standard therapeutics for the treatment of high blood pressure (actisgoretta et al. 2006) , and may have a vital role in viral infections and pneumonia (henry et al. 2018)-have not yet been shown to inhibit ace2. baicalin, a flavone glycoside and major constituent of scutellaria baicalensis, was found to have antiviral in vitro activity against sars-cov (12.5 to 25 lg/ml after 48 h; and 25 to 50 lg/ml after 72 h) when screened against 10 clinical isolates of sars-cov with foetal rhesus kidney-4 cells tested in a plaque reduction assay . for comparison, lopinavir-a protease inhibitor used against hiv infections, and which is currently in clinical trials for anti-sars-cov-2 infections-showed antiviral efficacy against sars-cov infected fetal rhesus kidney-4-cells at a concentration of 4 lg/ml, using the same assay (chu et al. 2004) . baicalin is also a known inhibitor of hiv-1 reverse transcriptase (li et al. 2000) , and was shown to possess inhibitory potential against influenza in vitro and in vivo by acting as immune modulator promoting ifn-c production in mice and human cd4 ? and cd8 ? t cells (chu et al. 2015; ding et al. 2014) . in general, some hiv protease inhibitors e.g., lopinavir, ritonavir or nelfinavir could interfere with sars-cov replication in-vitro, and had binding affinity to sars-cov 3clpro ). based on available evidence, it is controversial whether antiretroviral drugs can be suggested for the treatment of coronavirus diseases such as sars-cov-2. however, flavonoids which were found to have anti-hiv activity may be still worth investigating for their anti-sars-cov-2 potential. for example, baicalein, another major component from scutellaria baicalensis, has been described for its anti-hiv properties such as its binding to hiv-1 integrase (hu et al. 2010; zhao et al. 2016) , and thus might be proposed as interesting lead compound for sars-cov-2 drug discovery. in addition to the inhibitory potency of kaempferol against sars-cov proteases (table 2) , the flavonol was able to block the 3a ion channel of sars-cov, a protein encoded by the open-reading-frame (orf) 3a, which is involved in the virus release mechanism (schwarz et al. 2011). schwarz et al. demonstrated that glycosides of kaempferol are stronger inhibitors, highlighting the importance of sugar residues (schwarz et al. 2014) . the kaempferol glycoside juglanin having an arabinose residue, was shown to be the most prominent substrate (ic 50 value = 2.3 lm) (schwarz et al. 2014) . procyanidins such as procyanidin a2, and procyanidin b1 isolated from cinnamomi cortex had inhibitory potential against wild-type sars-cov using a plaque reduction assay (ic 50 = 29.9 and 41.3 lm, table 2 ). however, it was reported that the interplay of all procyanidins in the extracts from cinnamomi cortex appeared to be crucial for causing strong activity against wild-type sars-cov (zhuang et al. 2009 ). plaque reduction assays do not indicate any specific inhibition mechanism of the compounds, but provide a fast and high throughput pre-screening method. analyses of 17 studies based on flavonoids as anti-cov agents revealed 47 compounds (table 2) as possible agents against sars-cov-2. among them, 16 compounds were investigated in at least two independent studies. 4 of these compounds, quercetin, herbacetin, isobavachalcone, and kaempferol, show activity against at least two viral targets. according to the high ic 50 values of kaempferol (116.3 lm against sars-cov 3clpro, and 206.6 lm against mers-cov plpro), we do not consider this compound a promising candidate to treat sars-cov-2. therefore, the flavonols, herbacetin and quercetin, along with isobavachalcone ( fig. 2) were identified to be the most attractive antiviral leads against sars-cov-2.-due to their ic 50 values and broad-spectrum activity against proteases of sars-and mers-cov described in different studies (table 2) , as well as their availability from different plant sources. given the similarity of the genomic sequences encoding the 3clpro and plpro catalytic sites of sars-cov-2, sars-cov, and mers-cov (fig. 1) , 3clpro and plpro inhibitors of sars-and mers-cov are expected to be valuable leads for treating sars-cov-2 infections and covid-19. for instance, remdesivir-one of the most important antiviral drugs which has been suggested for therapeutic use against sars-cov-2-showed broad-efficacy against both sars-and mers-cov (sheahan et al. 2017 (sheahan et al. , 2020 . based on the high cc 50 values of the active flavonoids, such as that of herbacetin cc 50 = 293.7 lm ) and quercetin cc 50 = 385.5 lm (chiow et al. 2016) , they can be used in higher concentrations in therapeutic treatments without causing any substantial cytotoxic effects. prenylated flavonoids like isobavachalcone, are reported be relatively non-toxic to non-cancer cells (å  mejkal 2014) , and can be found in significant amounts in the plant families moraceae, and fabaceae (kuete and sandjo 2012) . as quercetin, herbacetin and isobavachalcone are readily available in high amounts from several plant sources, this might be also beneficial for future investigations on sars-cov-2, and the development of anti-cov therapeutics. although the daily intake of plant food may appear to be a readily accessible source of active flavonoids, there is no scientific evidence that a high consumption of flavonoid-rich food and/or supplements, like that of quercetin, provides significant protection against viral diseases in humans e.g., against sars-cov-2. due to low bioavailability, fast metabolism and elimination of flavonoids, the biological functions of these dietary polyphenols in-vivo are likely to be compromised. on the other hand, there are some external, non-host related, factors limiting the bioavailability of dietary flavonoids which include: environmental factors (i.e., storage, sun exposure), food processing factors (i.e., cooking), the interaction with other polyphenols present in plant-based food (i.e., antagonistic actions), the chemical structure (i.e., polymer or in glycosylated structure) and the concentration of the dietary compounds (d'archivio et al. 2010) . besides strong inhibitory effects of flavonols and chalcones on sars-and mers-cov, these flavonoids also have some anti-inflammatory (choy et al. 2019; ur rashid et al. 2019 ) and immune-modulating properties (hosseinzade et al. 2019) , which can be highly beneficial in the host immune response to viral infections. as an example, quercetin which is welldocumented for its wide spectrum of antiviral functions (zakaryan et al. 2017) , had antiviral effects against rhinovirus (rv) infections in-vitro and in-vivo using rv-infected mice, by reducing the expression of pro-inflammatory cytokines and chemokines (ganesan et al. 2012 ). hence, due to different important functions they can be valuable for either preventing and treating viral infections, as well as for alleviating symptoms (e.g., fever, cough, etc.) that may occur during the course of viral infections like that of sars-cov-2. in general, quercetin can be considered a highly potent candidate for treating sars-cov-2, as it has affinity to a variety of anti-cov drug targets. the flavonol interfered with the viral replication of sars-cov by blocking the enzymatic activities of 3clpro, plpro (table 2 ) and helicase (ic 50 = 8.1 lm) ), but also impaired sars plpro cleavage activity of ubiquitin and interferon-stimulated gene (isg) 15 (ic 50 = 20.7 and 34.4 lm, respectively) , which can be important for both the viral replication and the host immune response to viruses (cho et al. 2013) . moreover, quercetin interacted with sars-cov at host cell entry level by binding to the s glycoprotein (ec 50 = 83.4 lm) that docks to the host receptor ace2 ). further, quercetin demonstrated proteolytic activity against mers-cov 3clpro (ic 50 = 34.8 lm, table 2 ). for that purpose, there are a few issues that need to be addressed. since polyphenolic compounds are known for their aggregating tendency, doubts have been raised about the reliability of in vitro bioassay data of flavonoids, as false-positive results may occur through non-specific binding of phenolic compounds to proteins (pohjala and tammela 2012) . the formation of aggregates can cause a non-specific inhibition by sequestering enzyme molecules, absorbing or adsorbing them within their structure and thus causing denaturation (coan et al. 2009; pohjala and tammela 2012) . quercetin, which is one of the best studied flavonoid, showing virucidal activity against various enveloped viruses (e.g., influenza (wu et al. 2015) , parainfluenza type 3, herpes simplex (chen et al. 2006) ), was among the flavonoids that form large aggregates and thus may operate as a promiscuous inhibitor affecting various unrelated targets (pohjala and tammela 2012) . however, it has been reported that the addition of triton x-100, a solubilizing agent, to proteolytic assays, can help to reduce aggregate formation and complexation, such as that of flavonoids (jo et al. 2020; pohjala and tammela 2012) . besides the limitations of the bioassays, there is another aspect-the in-vitro molecular assays and cell-based assays do not provide any information about the bioavailability of the compounds. however, this aspect is not within the scope of the review, but for developing flavonoids further, this aspect should be considered. therefore, it is important to address the bioavailability of flavonoids as one of the critical limiting factors in their therapeutic use. different strategies to enhance the stability, solubility and systemic distribution of flavonoids have been reported, which can be employed. these include nanotechnology, co-crystallization, absorption enhancers and structural transformations (e.g. prodrugs, glycosylation) (ajazuddin and saraf 2010; zhao et al. 2019) . the use of the flavonoids quercetin, herbacetin, and isobavachalcone in adjuvant therapy with other proposed antiviral drugs may present another interesting approach to combat sars-cov-2 infections. combination therapy often leads to better outcomes in antiviral treatments. for instance, it was found that synergistic effects between quercetin and the antiviral agent aciclovir resulted in enhanced antiviral activity against pseudorabies herpesvirus infection in-vitro (ahmad et al. 2015) . similarly, a-glucoside inhibitors applied together with ribavirin, a standard antiviral drug, could improve antiviral efficacy against dengue infection in vitro and in vivo (chang et al. 2011 ). furthermore, it should be also evaluated whether the pure flavonoid itself like quercetin, herbacetin, and isobavachalcone, or adminstered in combination with other flavonoids and/or natural compounds can achieve optimal health benefits. the increasing development of drug-resistance in antiviral treatment might be another critical matter for the therapeutic use of flavonoids. literature search in pubmed, has led to the identification of numerous flavonoids exerting antiviral in vitro activity against sars and/or mers coronavirus, primarily by inhibiting the enzymatic activity of 3cl and pl. flavonols and chalcones were found to be the main groups of flavonoids containing the highest number of effective compounds against 3cl and pl proteases. in particular, herbacetin, quercetin and isobavachalcone (fig. 3) were identified as promising antiviral leads against sars-and mers-cov based on their broad-spectrum activity against the viral proteases 3cl and pl of both covs, the number of relevant literature data, and the availability of the compounds from different plant sources. considering the fact that quercetin could interfere with sars-cov at different levels, such as at the viral entry and replication process, as well as with 3cl protease of mers-cov, we specifically propose this compound to be a highly valuable anti-sars-cov-2 candidate. quercetin may prevent host cell entry of sars-cov through binding to the s protein and inhibiting ace. further, the flavonol can impair the viral replication of sars-and mers-cov by blocking the enzymatic activites of 3cl and pl including sars-helicase, and can inhibit the deubiquitination and deisgylation process of sars plpro, which might be also an issue in the host immune response. other potent substrates able to interact with the enzymatic activity of sars-and mers-cov proteases, were found to be prenylated flavonoids (e.g., isobavachalcone). however, despite some promising inhibitory activities of flavonoids against sars-and mers-cov in vitro, none of these compounds have been tested in vivo using animal and/or human cell models. therefore, more detailed investigations on pharmacological mechanisms, long-term toxicology, bioavailability, as well as some additional studies on possible herb-drug interactions are required. in fact, natural compounds like flavonoids, continue to be a wealthy source for the discovery of novel antiviral agents. it was reported that natural substrates from traditional chinese medicine (tcm) seemed to have a positive impact on the recovery process of patients suffering from sars-cov diseases, by mitigating possible side effects of conventional therapeutics . since flavonoids are well-documented for their broad spectrum of health-beneficial properties, especially anti-inflammatory and antioxidative effects, they could be of great value for strengthening the host immune response to viral diseases, and alleviating infection-related symptoms, such as down-regulating overwhelming inflammatory responses (e.g. cytokine production). for instance, besides the antiviral effects of quercetin, the flavonol was found to prevent tissue damage by scavenging free radicals, and could reduce the release of inflammatory cytokines such as interleukine il-8 (kinker 2014) . due to high similarities of sars-cov, sars-cov-2, and mers-cov-sharing several homologous viral proteases, the proposed flavonols herbacetin and quercetin, as well as isobavachalcone may demonstrate attractive antiviral substrates against sars-cov-2 and/or other coronaviruses. however, further research and more detailed pharmacological investigations in-vivo, particularly on the bioavailability of these compounds, appear to be a promising approach for the discovery of novel herbal substrates used as adjunctive therapeutics in the treatment of coronavirus diseases, such as covid-19. furthermore, greater attention should be paid to combinatory effects of flavonoids, especially when used together with other standard antiviral drugs. looking to nature for a new concept in antimicrobial treatments: isoflavonoids from cytisus striatus as antibiotic adjuvants against mrsa inhibition of angiotensin converting enzyme activity by flavanol-rich foods mechanism of nucleic acid unwinding by sars-cov helicase therapeutic potential of flavonoids and their mechanism of action against microbial and viral infections-a review applications of novel drug delivery system for herbal formulations angiotensinconverting enzyme inhibitory effects by plant phenolic compounds: a study of structure activity relationships coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs overviews of biological importance of quercetin: a bioactive flavonoid the sarscoronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds di napoli r (2020) statpearls: features, evaluation and treatment coronavirus the antiinflammatory potential of flavonoids human coronavirus types: common human coronaviruses combination of a-glucosidase inhibitor and ribavirin for the treatment of dengue virus infection in vitro and in vivo in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds cinanserin is an inhibitor of the 3c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro inhibition of sars-cov 3c-like protease activity by theaflavin-3,3'-digallate (tf3) binding interaction of quercetin-3-beta-galactoside and its synthetic derivatives with sars-cov 3cl(pro): structure-activity relationship studies reveal salient pharmacophore features severe acute respiratory syndrome coronavirus viroporin 3a activates the nlrp3 inflammasome evaluation of antiviral activities of houttuynia cordata thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection geranylated flavonoids displaying sars-cov papain-like protease inhibition from the fruits of paulownia tomentosa flavonoids as natural anti-inflammatory agents targeting nuclear factor-kappa b (nfjb) signaling in cardiovascular diseases: a mini review role of baicalin in anti-influenza virus a as a potent inducer of ifn-gamma role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings chuck promiscuous aggregate-based inhibitors promote enzyme unfolding the antioxidant properties of plant flavonoids: their exploitation by molecular plant breeding antiviral activity of baicalin against influenza a (h1n1/h3n2) virus in cell culture and in mice and its inhibition of neuraminidase a dual character of flavonoids in influenza a virus replication and spread through modulating cell-autonomous immunity by mapk signaling pathways coronaviruses: an overview of their replication and pathogenesis quercetin inhibits rhinovirus replication in vitro and in vivo substrate specificity profiling and identification of a new class of inhibitor for the major protease of the sars coronavirus the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus discovery of prenylated flavonoids with dual activity against influenza virus and streptococcus pneumoniae inhibition of angiotensinconverting enzyme activity by flavonoids: structure-activity relationship studies angiotensin receptor blockers as tentative sars-cov-2 therapeutics coronavirus disease 2019 (covid-19): what we know? potential of coronavirus 3c-like protease inhibitors for the development of new anti-sars-cov-2 drugs: insights from structures of protease and inhibitors immunomodulatory effects of flavonoids: possible induction of t cd4? regulatory cells through suppression of mtor pathway signaling activity the pomegranate: effects on bacteria and viruses that influence human health. evid based complement alternat med computational investigation of the anti-hiv activity of chinese medicinal formula three-huang powder current and emerging approaches for studying inter-organelle membrane contact sites herbacetin, a constituent of ephedrae herba, suppresses the hgf-induced motility of human breast cancer mda-mb-231 cells by inhibiting c-met and akt phosphorylation neuraminidase inhibitory activities of flavonols isolated from rhodiola rosea roots and their in vitro anti-influenza viral activities characteristics of flavonoids as potent mers-cov 3c-like protease inhibitors inhibition of sars-cov 3cl protease by flavonoids development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia quercetin: a promising treatment for the common cold isobavachalcone: an overview asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths investigation of the pharmacophore space of severe acute respiratory syndrome coronavirus (sars-cov) ntpase/ helicase by dihydroxychromone derivatives cooperative translocation enhances the unwinding of duplex dna by sars coronavirus helicase nsp13 mechanism of action of two flavone isomers targeting cancer cells with varying cell differentiation status functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses coronavirus infections and immune responses flavonoid baicalin inhibits hiv-1 infection at the level of viral entry flavonoids interfere with nlrp3 inflammasome activation human coronaviruses: a review of virus-host interactions characterization of trans-and ciscleavage activity of the sars coronavirus 3clpro protease: basis for the in vitro screening of anti-sars drugs anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plant-derived phenolic compounds disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes screening for protein-protein interactions using fã¶rster resonance energy transfer (fret) and fluorescence lifetime imaging microscopy (flim) candidate drugs against sars-cov-2 and covid-19 prenylation enhances the biological activity of dietary flavonoids by altering their bioavailability flavonoidmediated inhibition of sars coronavirus 3c-like protease expressed in pichia pastoris flavonoids: a review of probable mechanisms of action and potential applications plant flavonoids as angiotensin converting enzyme inhibitors in regulation of hypertension flavonoids: an overview plant polyphenols as dietary antioxidants in human health and disease chalcones isolated from angelica keiskei inhibit cysteine proteases of sars-cov evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors synthesis and antiviral evaluation of 7-o-arylmethylquercetin derivatives against sars-associated coronavirus (scv) and hepatitis c virus (hcv) rhodiosin and herbacetin in rhodiola rosea preparations: additional markers for quality control? aggregating behavior of phenolic compounds-a source of false bioassay results isolation and characterization of flaxseed (linum usitatissimum) constituents sars-cov-2/covid-19 and advances in developing potential therapeutics and vaccines to counter this emerging pandemic virus-a review sars-cov-2 and coronavirus disease 2019: what we know so far structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease biflavonoids from torreya nucifera displaying sars-cov 3cl(pro) inhibition polyphenols from broussonetia papyrifera displaying potent alpha-glucosidase inhibition emodin inhibits current through sars-associated coronavirus 3a protein kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus novel coronavirus-induced nlrp3 inflammasome activation: a potential drug target in the treatment of broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers a cell-free approach to accelerate the study of protein-protein interactions in vitro new assay technologies for high-throughput screening isolation of specific cranberry flavonoids for biological activity assessment. isolation of specific cranberry flavonoids for biological activity assessment å  mejkal k flavonoids as novel efflux pump inhibitors and antimicrobials against both environmental and pathogenic intracellular mycobacterial species structurally modified 'dietary flavonoids': are these viable drug candidates for chemoprevention? flavonols (kaempeferol, quercetin, myricetin) contents of selected fruits, vegetables and medicinal plants promising anti-inflammatory effects of chalcones via inhibition of cyclooxygenase, prostaglandin e2, inducible no synthase and nuclear factor jb activities bioactive flavonoids in medicinal plants: structure, activity and biological fate study on structure activity relationship of natural flavonoids against thrombin by molecular docking virtual screening combined with activity evaluation in vitro recent advances in the discovery and development of flavonoids and their analogues as antitumor and anti-hiv agents world health organization (2020a) coronavirus disease (covid-19) pandemic world health organization (2020b) novel coronavirus (2019-ncov): situation report-1 21 cryo-em structure of the 2019-ncov spike in the prefusion conformation new alkaloids from isatis indigotica quercetin as an antiviral agent inhibits influenza a virus (iav) entry analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods bioactive natural compounds against human coronaviruses: a review and perspective evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase a review on the phytochemistry, pharmacology, and pharmacokinetics of amentoflavone flavonoids: promising natural compounds against viral infections old drugs as lead compounds for a new disease? binding analysis of sars coronavirus main proteinase with hiv, psychotic and parasite drugs scutellaria baicalensis, the golden herb from the garden of chinese medicinal plants improvement strategies for the oral bioavailability of poorly water-soluble flavonoids: an overview a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements open access funding provided by university of graz is greatly acknowledged. author contributions julia solnier and johannes-paul fladerer contributed equally to this work.funding open access funding provided by university of graz. conflict of interest the authors declare that they have no conflict of interest.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. key: cord-303917-2tu707ng authors: zhang, lei; liu, yunhui title: potential interventions for novel coronavirus in china: a systematic review date: 2020-03-03 journal: j med virol doi: 10.1002/jmv.25707 sha: doc_id: 303917 cord_uid: 2tu707ng an outbreak of a novel coronavirus (covid‐19 or 2019‐cov) infection has posed significant threats to international health and the economy. in the absence of treatment for this virus, there is an urgent need to find alternative methods to control the spread of disease. here, we have conducted an online search for all treatment options related to coronavirus infections as well as some rna‐virus infection and we have found that general treatments, coronavirus‐specific treatments, and antiviral treatments should be useful in fighting covid‐19. we suggest that the nutritional status of each infected patient should be evaluated before the administration of general treatments and the current children's rna‐virus vaccines including influenza vaccine should be immunized for uninfected people and health care workers. in addition, convalescent plasma should be given to covid‐19 patients if it is available. in conclusion, we suggest that all the potential interventions be implemented to control the emerging covid‐19 if the infection is uncontrollable. coronaviruses (covs) belong to the subfamily orthocoronavirinae in the family of coronaviridae in the order nidovirales, and this subfamily including α-coronavirus, β-coronavirus, γ-coronavirus, and deltacoronavirus. 1 coronaviruses primarily cause enzootic infections in birds and mammals and, in the last decades, have shown to be capable of infecting humans as well. 2 the outbreak of severe acute respiratory syndrome (sars) in 2002 and middle east respiratory syndrome (mers) in 2012 has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans. 2 belong to the β-coronavirus family. 3 recently, a novel flu-like coronavirus related to the mers and sars coronaviruses was found at the end of 2019 in china 4,5 and the evidence of human-to-human transmission was confirmed among close contacts. 6 the genome of covid-19 is a single-stranded positive-sense rna. 7 the sequence analysis showed that the covid-19 possessed a typical genome structure of coronavirus and belonged to the cluster of β-coronaviruses including sars-cov and mers-cov. 7 covid-19 was more than 82% identical to those of sars-cov. 8 vitamin a is the first fat-soluble vitamin to be recognized and β-carotene is its plant-derived precursor (table 1 ). there are three active forms of vitamin a in the body, retinol, retinal, and retinoic acid. vitamin a is also called "anti-infective" vitamin and many of the body's defenses against infection depend on an adequate supply. researchers have believed that an impaired immune response is due to the deficiency of a particular nutritional element. 10 vitamin a deficiency is strongly involved in measles and diarrhea 11 and measles can become severe in vitamin a-deficient children. in addition, semba et al 12 had reported that vitamin a supplementation reduced morbidity and mortality in different infectious diseases, such as measles, diarrheal disease, measles-related pneumonia, human immunodeficiency virus (hiv) infection, and malaria. vitamin a supplementation also offers some protection against the complications of other life-threatening infections, including malaria, lung diseases, and hiv. 13 jee et al 14 had reported that low vitamin a diets might compromise the effectiveness of inactivated bovine coronavirus vaccines and render calves more susceptible to infectious disease. the effect of infection with infectious bronchitis virus (ibv), a kind of coronaviruses, was more pronounced in chickens fed a diet marginally deficient in vitamin a than in those fed a diet adequate in vitamin a. 15 the mechanism by which vitamin a and retinoids inhibit measles replication is upregulating elements of the innate immune response in uninfected bystander cells, making them refractory to productive infection during subsequent rounds of viral replication. 16 therefore, vitamin a could be a promising option for the treatment of this novel coronavirus and the prevention of lung infection. b vitamins are water-soluble vitamins and work as part of coenzymes. each b vitamin has its special functions. for example, vitamin b2 (riboflavin) plays a role in the energy metabolism of all cells. vitamin b2 deficiency had been suspected to occur among us elderly. 17 keil et al 18 had reported that vitamin b2 and uv light effectively reduced the titer of mers-cov in human plasma products. vitamin b3, also called nicotinamide, could enhance the killing of staphylococcus aureus through a myeloid-specific transcription factor and vitamin b3 was efficacious in both prophylactic and therapeutic settings. 19 moreover, vitamin b3 treatment significantly inhibited neutrophil infiltration into the lungs with a strong anti-inflammatory effect during ventilatorinduced lung injury. however, it also paradoxically led to the development of significant hypoxemia. 20 vitamin b6 is also needed in protein metabolism and it participates in over 100 reactions in body tissues. in addition, it also plays important role in body immune function as well. as shortage of b vitamins may weaken host immune response, they should be supplemented to the virus-infected patients to enhance their immune system. therefore, b vitamins could be chosen as a basic option for the treatment of covid-19. vitamin c is another water-soluble vitamin and it is also called ascorbic acid, which means "no-scurvy acid." vitamin c is best known for its role in the synthesis of collagen in connective tissues and acts as an antioxidant. vitamin c also supports immune functions and protects against infection caused by a coronavirus. 21 infection. vitamin c may also function as a weak antihistamine agent to provide relief from flu-like symptoms such as sneezing, a running or stuffy nose, and swollen sinuses. 23 three human controlled trials had reported that there was significantly lower incidence of pneumonia in vitamin c-supplemented groups, suggesting that vitamin c might prevent the susceptibility to lower respiratory tract infections under certain conditions. 24 the covid-19 had been reported to cause lower respiratory tract infection, so vitamin c could be one of the effective choices for the treatment of covid-19. vitamin d is not only a nutrient but also a hormone, which can be synthesized in our body with the help of sunlight. in addition to its role in maintaining bone integrity, it also stimulates the maturation of and omega-6 pufas predominantly promote anti-inflammatory and pro-inflammatory effects. they are precursors of resolvins/protectins and prostaglandins/leukotrienes, respectively. 31 begin et al 32 had studied plasma lipids levels in patients with aids and had found that a selective and specific lack of the long-chain pufas of omega-3 series, which are found in high concentrations in fish oils. in addition, protectin d1, the omega-3 pufa-derived lipid mediator, could markedly attenuate influenza virus replication via rna export machinery. in addition, treatment of protectin d1 with peramivir could completely rescue mice from flu mortality. 33 leu et al 34 had found that several pufas also had anti-hepatitis c virus (hcv) activities. therefore, omega-3 including protectin d1, which served as a novel antiviral drug, could be considered for one of the potential interventions of this novel virus, covid-19. selenium is an essential trace element for mammalian redox biology. 35 the nutritional status of the host plays a very important role in the defense against infectious diseases. 36 nutritional deficiency impacts not only the immune response but also the viral pathogen itself. 10 dietary selenium deficiency that causes oxidative stress in the host can alter a viral genome so that a normally benign or mildly pathogenic virus can become highly virulent in the deficient host under oxidative stress. 10 deficiency in selenium also induces not only impairment of host immune system, but also rapid mutation of benign variants of rna viruses to virulence. 37 beck et al 38 had reported that selenium deficiency could not only increase the pathology of an influenza virus infection but also drive changes in genome of coxsackievirus, permitting an avirulent virus to acquire virulence due to genetic mutation. 39 it is because that selenium could assist a group of enzymes that, in concert with vitamin e, work to prevent the formation of free radicals and prevent oxidative damage to cells and tissues. 37 it was reported that synergistic effect of selenium with ginseng stem-leaf saponins could induce immune response to a live bivalent infectious bronchitis coronavirus vaccine in chickens. 40 therefore, selenium supplementation could be an effective choice for the treatment of this novel virus of covid-19. zinc is a dietary trace mineral and is important for the maintenance and development of immune cells of both the innate and adaptive immune system. 41 zinc deficiency results in dysfunction of both humoral and cell-mediated immunity and increases susceptibility to infectious diseases. 42 zinc supplement given to zinc-deficient children could reduce measles-related morbidity and mortality caused by lower respiratory tract infections. 43 increasing the concentration of intracellular zinc with zinc-ionophores like pyrithione can efficiently impair the replication of a variety of rna viruses. 44 in addition, the combination of zinc and pyrithione at low concentrations inhibits the replication of sars coronavirus (sars-cov). 44 56 in addition, interferons have also been found to be potent inhibitors of mers-cov replication. 57 moreover, the combination of interferon-α-2a with ribavirin was administered to patients with severe mers-cov infection and the survival of these patients was improved. 57 these findings suggest that these approved ifn's could be also used for the treatment of this novel coronavirus. intravenous gammaglobulin (ivig) was first developed in the late 1970s 58 and is probably the safest immunomodulating drug available for long-term use in all ages. however, it does have adverse reactions. during the sars outbreak in 2003, ivig was used extensively in singapore. however, one-third of critically ill patients developed venous thromboembolism including pulmonary embolism despite the use of lowmolecular weight heparin prophylactic. 59 it was due to the ivig-induced increase of viscosity in hypercoagulable states of sars patients. 60 thymosin α-1 (ta1) is a thymic peptide hormone and it has a peculiar ability to restore the homeostasis of the immune system. 61 it is was first isolated from thymic tissue in the mid-sixties and it had gained much attention for its immunostimulatory activity. 62 it was chemically synthesized and used in diseases where the immune system was hindered or impaired. 63 besides its role in thymocyte development, thymosin α-1 could also increase resistance to glucocorticoid-induced death of the thymocyte. 64 thymosin α-1 could also be used as immune enhancer to sars patients and it was effective in controlling the spread of the disease. 65, 66 methylprednisolone was often used during the current treatment of covid-19 and the side effect of corticoid-induced death of thymocytes should be considered. so, it is wise to use thymosin α1 before the administration of methylprednisolone. thymopentin (tp5, munox), a synthetic pentapeptide corresponding to the active site of thymopoietin, had been shown to restore antibody production in old mice. 67 additionally, it could enhance the antibody response in humans when it was applied subcutaneously three times a week at doses of 50 mg. 68 moreover, thymopentin could also be used as an adjuvant treatment for non-responders or hyporesponders to hepatitis b vaccination. 69 levamisole, a synthetic low-molecular-weight compound, is the first member of a new class of drugs that can increase the functions of cellular immunity in normal, healthy laboratory animals. 70 medicine could also be considered as a choice to enhance host immunity against the infection of covid-19. in summary, the general treatment for viral infection including nutritional interventions and all kinds of immunoenhancers has been used to enhance host immunity against rna viral infections. therefore, they may also be used to fight covid-19 infection by correcting the lymphopenia of patients. chymotrypsin-like (3c-like) and papain-like protease (plp) are coronavirus encoded proteins (table 2) . they have an essential function for coronaviral replication and also have additional function for inhibition of host innate immune responses. targeting 3c-like protease (3 clpro) and papain-like protease (plpro) are more attractive for the treatment of coronavirus. 77 cinanserin cinanserin, an old drug, is well-known for serotonin receptor antagonist. it could inhibit the 3 chymotrypsin-like (3c-like) protease and was a promising inhibitor of replication of sars-cov. 78 the 3clpro was also been found to be encoded in covid19. 7 therefore, cinanserin may be a better choice for the treatment of covid-19 infection. papain-like protease (plp) of human coronavirus is a novel viralencoded deubiquitinase and is an ifn antagonist for inhibition of host innate antiviral immune response. diarylheptanoids is a natural product and is extracted from the stem bark of alnus japonica. it had been found to be able to inhibit papainlike protease of sars-cov. 77 therefore, cinanserin together with flavonoids and other natural compounds could be chosen as alternative choices to fight covid-19 infection through targeting coronaviral proteases. angiotensin-converting enzyme-2 (ace2) is a type i integral membrane protein which functions as a carboxypeptidase and is the first human homolog of ace. 84 ace2 efficiently hydrolyzes the potent vasoconstrictor angiotensin ii to angiotensin (1-7) and it has been implicated in hypertension, cardiac function, heart function, and diabetes. 84 in addition, ace2 is also a functional receptor of sars-cov and it mediates virus entry into the cell through binding with spike (s) protein. 85 chloroquine is a 9-aminoquinoline known since 1934. apart from its well-known antimalarial effects, the drug also has many interesting biochemical properties including antiviral effect. in addition, it had been used against viral infection. 90 moreover, chloroquine was also found to be a potent inhibitor of sars coronavirus infection through interfering with ace2, one of cell surface binding sites for s protein of sars-cov. 91 emodin is an anthraquinone compound derived from genus rheum and polygonum and it is also a virucidal agent. 92 emodin could significantly block the interaction between the s protein of sars-cov and ace2. therefore, emodin might abolish sars-cov infection by competing for the binding site of s protein with ace2. 93 promazine, anti-psychotic drug, shares a similar structure with emodin. it has been found to exhibit a significant effect in inhibiting the replication of sars-cov. 94 as compared to emodin, promazine exhibited potent inhibition of the binding of s protein to ace2. these findings suggested that emodin and promazine might be able to inhibit sars-cov infectivity through blocking the interaction of s protein and ace2. 93 therefore, the monoclonal antibody (scfv80r), chloroquine, emodin, and promazine could be used as alternative choices for the treatment of covid-19. nicotianamine is an important metal ligand in plants 95 and it is found a novel angiotensin-converting enzyme-2 inhibitor in soybean. 96 so, it is another potential option to be used to reduce the infection of covid-19. ribavirin, a broad-spectrum antiviral agent, is routinely used to treat hepatitis c (table 3 ). during the outbreak of sars, ribavirin was used extensively for most cases with or without concomitant use of steroids in hong kong. 97 however, there was considerable skepticism from overseas and local experts on the efficacy of ribavirin. 98 because there was a report mentioned that ribavirin had no significant activity against sars-cov in vitro 52 and the use of ribavirin was found to be associated with significant toxicity, including hemolysis (in 76%) and decrease in hemoglobin (in 49%). 99 remdesivir (rdv), a nucleoside analog gs-5734, had been reported to inhibit human and zoonotic coronavirus in vitro and to restrain severe acute respiratory syndrome coronavirus (sars-cov) in vivo. 104 recently, the antiviral activity of rdv and ifn-β was found to be superior to that of lpv/rtv-ifn-β against mers-cov in vitro and in vivo. 101 in addition, rdv could improve pulmonary function and reduce lung viral loads and severe lung pathology in mice, which was impossible for lpv/rtv-ifn-β. 101 recently, a first covid-19 nelfinavir is a selective inhibitor of hiv protease, which is responsible for posttranslational processing of hiv propeptides. 106 yamamoto et al 107 had found that nelfinavir could strongly inhibit the replication of sars-cov. therefore, nelfinavir could also be an option for the treatment of covid-19. arbidol (arb) is a russian-made small indole-derivative molecule and is licensed in russia and china for prophylaxis and treatment of influenza and other respiratory viral infections. 108 arbidol had been found to be able to block viral fusion against influenza a and b viruses as well as hepatitis c virus. 109 arbidol could also inhibit hepatitis c virus by blocking hepatitis c virus entry and replication in vitro. 110 in addition, arbidol and its derivatives, arbidol mesylate, had been reported to have antiviral activity against the pathogen of sars in the cell cultures and arbidol mesylate was nearly 5 times as effective as arbidol in reducing the reproduction of sars virus in the cultured cells. 111 nitric oxide (no) is a gas with diverse biological activities and is produced from arginine by no synthases. no is able to interact with superoxide, forming peroxynitrite, which, in turn, can mediate bactericidal or cytotoxic reactions. 112 in addition, no had played an important role in regulating airway function and in treating inflammatory airway diseases. 113 rossaint et al 114 reported that the beneficial effects of no inhalation could be observed in most patients with severe acute respiratory distress syndrome. no was also found to inhibit the synthesis of viral protein and rna. 115 (table 3 ). 117 ala, as an antioxidant, has played a pivotal role in scavenging free radicals to protect against oxidative damage in several diseases. 118 in addition, ala also had its capability to enhance intracellular glutathione (gsh) levels 118 and to normalize the oxidative stress induced by dexamethasone in chicken. 119 wu et al 120 also reported that the oxidative stress in host cells was an important factor in the infectivity of human coronavirus 229e and the glucose-6-phosphate dehydrogenase (g6pd) deficiency was another factor that enhanced human coronavirus 229e infection. the addition of α-lipoic acid to g6pd-knockdown cells could attenuate the increased susceptibility to human coronavirus 229e infection. 120 interestingly, baur et al 121 also found that α-lipoic acid was effective to inhibit the replication of hiv-1. in summary, we speculate that ala could be also used as an optional therapy for this new virus. females, generally, mount more robust immune responses to viral challenges than males, which can result in more efficient virus clearance. 122 epidemiological studies showed that males experiencing a higher rate of incidence and case fatality compared with females after sars-cov infection. 123, 124 during the mers outbreak, the disease occurrence rate in men was almost twice as much as in women and the case fatality rate was the same as the occurrence rate among men and women. 125 in addition, channappanavar et al had reported that male mice were more susceptible to sars-cov infection compared with age-matched female mice. however, the mortality was increased in female mice when the ovariectomy was done or the estrogen receptor antagonist was given. 126 wei et al 127 also found that serum levels of prolactin, follicle-stimulating hormone, and luteinizing hormone of zhang and liu | 485 sars patients were significantly higher than those of control groups, while estradiol (e2), pregnancy hormone, and thyroid-stimulating hormone were considerably lower than those of normal controls. interestingly, estrogenic compounds had been found to reduce influenza a virus replication in primary human nasal epithelial cells derived from female, but not male, donors. 128 in addition, resveratrol, a phytoestrogen from grape seeds and red wine, had been reported to be a potent anti-mers agent in vitro. 129 therefore, 17β-estradiol or phytoestrogen could also be an alternative option to be considered for the treatment of covid-19. mucroporin-m1 is a scorpion venom-derived peptide and has broadspectrum virucidal activity against many viruses including measles, influenza h5n1 viruses, and sars-cov. 130 therefore, this peptide could also be used for the treatment of covid-19 infection as well as the new drug design to target covid-19. in this review, we summarize all the potential interventions for regarding short-term protection and prevention of viral infection, passive immunotherapy should not be neglected. 135 monoclonal antibody therapy is one of the best forms of passive immunotherapy. a human igg1 mab, cr3014, had been generated and it had been found to be reactive with whole inactivated sars coronavirus. in addition, cr3014 could be used as prophylaxis for sars coronavirus infection in ferrets. 136 however, cr3014 was found to be able to block the interaction in parent sars-cov strain, but not in escape variants. this led to the ineffectiveness of cr3014 to prevent infection in humans. cr3022 was another monoclonal antibody and it had been found to neutralize cr3014 escape viruses. 136 the combination of cr3014 and cr3022 had also been reported to have the potential to control immune escape. 135 however, the clinical trial of cr3022 with cr3014 had never been tried due to the high cost of manufacturing. convalescent plasma can also be called passive immunotherapy. it is usually chosen when there are no specific vaccines or drugs available for emerging infection-related diseases. 137 arabi et al had tested the feasibility of convalescent plasma therapy as well as its safety and clinical efficacy in critically ill mers patients. they found that convalescent plasma had an immunotherapeutic potential for the treatment of mers-cov infection. 138 in addition, convalescent plasma from recovered sars patients had also been reported to be useful clinically for treating other sars patients. 139, 140 importantly, the use of convalescent plasma or serum was also suggested by the world health organization under blood regulators network when vaccines and antiviral drugs were unavailable for an emerging virus. in summary, these findings suggest that the current children's rna-virus-related vaccines are the best alternative methods to be used to vaccinate the uninfected people and health care workers. convalescent plasma should be routinely used for the treatment of covid-19 infected critically sick patients if it is available. the avian ibv vaccine is also another choice for clinical trials if its safety has been approved in monkeys. therefore, we suggest that all the potential interventions be implemented to control the emerging covid-19 if the infection is uncontrollable. the authors declare that there are no conflict of interests. yunhui liu http://orcid.org/0000-0002-9920-9933 bats and coronaviruses coronavirus envelope protein: current knowledge middle east respiratory syndrome new sars-like virus in china triggers alarm a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia coronaviruses: genome structure, replication, and pathogenesis recent advances in the detection of respiratory virus infection in humans genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan selenium, selenoproteins and viral infection importance of vitamin a deficiency in pathology and immunology of viral infections vitamin a and immunity to viral, bacterial and protozoan infections vitamin a supplements ameliorate the adverse effect of hiv-1, malaria, and diarrheal infections on child growth effects of dietary vitamin a content on antibody responses of feedlot calves inoculated intramuscularly with an inactivated bovine coronavirus vaccine epithelia-damaging virus infections affect vitamin a status in chickens retinoids inhibit measles virus through a type i ifn-dependent bystander effect riboflavin (vitamin b-2) and health inactivation of middle east respiratory syndrome coronavirus (mers-cov) in plasma products using a riboflavin-based and ultraviolet light-based photochemical treatment c/ebpepsilon mediates nicotinamide-enhanced clearance of staphylococcus aureus in mice nicotinamide exacerbates hypoxemia in ventilator-induced lung injury independent of neutrophil infiltration the effect of ascorbic acid on infection chick-embryo ciliated tracheal organ cultures by coronavirus nutrients and their role in host resistance to infection vitamin c intake and susceptibility to pneumonia vitamin d insufficiency among free-living healthy young adults sunlight and vitamin d for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease acute phase response elicited by experimental bovine diarrhea virus (bvdv) infection is associated with decreased vitamin d and e status of vitamin-replete preruminant calves vitamin e metabolic effects and genetic variants: a challenge for precision nutrition in obesity and associated disturbances vitamin e deficiency intensifies the myocardial injury of coxsackievirus b3 infection of mice increased virulence of coxsackievirus b3 in mice due to vitamin e or selenium deficiency macrophage-derived extracellular vesicles induce long-lasting immunity against hepatitis c virus which is blunted by polyunsaturated fatty acids plasma fatty acid levels in patients with acquired immune deficiency syndrome and in controls the lipid mediator protectin d1 inhibits influenza virus replication and improves severe influenza anti-hcv activities of selective polyunsaturated fatty acids selenium and human health micronutrients and host resistance to viral infection review: micronutrient selenium deficiency influences evolution of some viral infectious diseases selenium deficiency increases the pathology of an influenza virus infection rapid genomic evolution of a non-virulent coxsackievirus b3 in selenium-deficient mice results in selection of identical virulent isolates combined adjuvant effect of ginseng stem-leaf saponins and selenium on immune responses to a live bivalent vaccine of newcastle disease virus and infectious bronchitis virus in chickens zinc and immunity: an essential interrelation zinc deficiency zinc supplementation for the treatment of measles in children zn(2+) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture crossing the iron gate: why and how transferrin receptors mediate viral entry childhood iron deficiency anemia leads to recurrent respiratory tract infections and gastroenteritis chicken interferon type i inhibits infectious bronchitis virus replication and associated respiratory illness prevention of experimental coronavirus colds with intranasal alpha-2b interferon ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds interferon priming enables cells to partially overturn the sars coronavirus-induced block in innate immune activation inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis c: a randomised trial pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques proposal for vaccination against sars coronavirus using avian infectious bronchitis virus strain h from the netherlands interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review uses of intravenous gammaglobulin in immune hematologic disease acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome strokes, thromboembolic events, and ivig: rare incidents blemish an excellent safety record thymosin alpha 1 and hiv-1: recent advances and future perspectives a reappraisal of thymosin alpha1 in cancer therapy serum thymosin alpha 1 levels in normal and pathological conditions thymosin alpha 1 antagonizes dexamethasone and cd3-induced apoptosis of cd4+ cd8+ thymocytes through the activation of camp and protein kinase c dependent second messenger pathways clinical investigation of outbreak of nosocomial severe acute respiratory syndrome anti-sars coronavirus agents: a patent review (2008-present) modulation of immune response in aged humans through different administration modes of thymopentin phase variation in the modulation of the human immune response thymopentin as adjuvant in non-responders or hyporesponders to hepatitis b vaccination the general immunopharmacology of levamisole lymphocyte subsets in measles. depressed helper/inducer subpopulation reversed by in vitro treatment with levamisole and ascorbic acid systemic cyclosporine and corneal transplantation nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a factor in virus replication and potential target for anti-viral therapy the sars-coronavirus-host interactome: identification of cyclophilins as target for pancoronavirus inhibitors glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus diarylheptanoids from alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus cinanserin is an inhibitor of the 3c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro flavonoids: an overview flavonoids from pterogyne nitens inhibit hepatitis c virus entry inhibition of sars-cov 3cl protease by flavonoids characteristics of flavonoids as potent mers-cov 3c-like protease inhibitors biflavonoids from torreya nucifera displaying sars-cov 3cl(pro) inhibition angiotensin-converting enzyme-2: a molecular and cellular perspective angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus the secret life of ace2 as a receptor for the sars virus characterization of severe acute respiratory syndromeassociated coronavirus (sars-cov) spike glycoprotein-mediated viral entry severe acute respiratory syndrome coronavirus entry into host cells: opportunities for therapeutic intervention potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association effects of chloroquine on viral infections: an old drug against today's diseases? chloroquine is a potent inhibitor of sars coronavirus infection and spread membrane-related effects underlying the biological activity of the anthraquinones emodin and barbaloin emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction old drugs as lead compounds for a new disease? binding analysis of sars coronavirus main proteinase with hiv, psychotic and parasite drugs the transition metal chelator nicotianamine is synthesized by filamentous fungi nicotianamine is a novel angiotensin-converting enzyme 2 inhibitor in soybean managing sars amidst uncertainty critics slam treatment for sars as ineffective and perhaps dangerous clinical features and short-term outcomes of 144 patients with sars in the greater toronto area comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio first case of 2019 novel coronavirus in the united states a review of its therapeutic efficacy in hiv infection hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus arbidol as a broad-spectrum antiviral: an update arbidol: a broadspectrum antiviral compound that blocks viral fusion the synthetic antiviral drug arbidol inhibits globally prevalent pathogenic viruses nitric oxide nitric oxide and airway disease efficacy of inhaled nitric oxide in patients with severe an overview on severe acute respiratory syndrome (sars) nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus efficacy of thioctic acid in the therapy of peripheral diabetic neuropathy biochemical and clinical relevance of alpha lipoic acid: antioxidant and anti-inflammatory activity, molecular pathways and therapeutic potential effects of dietary vitamin c, vitamin e, and alpha-lipoic acid supplementation on the antioxidant defense system and immune-related gene expression in broilers exposed to oxidative stress by dexamethasone glucose-6-phosphate dehydrogenase deficiency enhances human coronavirus 229e infection alphalipoic acid is an effective inhibitor of human immuno-deficiency virus (hiv-1) replication sexual dimorphism in innate immune responses to infectious organisms do men have a higher case fatality rate of severe acute respiratory syndrome than women do? sars in singapore--predictors of disease severity the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection endocrine cells of the adenohypophysis in severe acute respiratory syndrome (sars) estrogenic compounds reduce influenza a virus replication in primary human nasal epithelial cells derived from female, but not male, donors effective inhibition of mers-cov infection by resveratrol virucidal activity of a scorpion venom peptide variant mucroporin-m1 against measles, sars-cov and influenza h5n1 viruses severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus protection from sars coronavirus conferred by live measles vaccine expressing the spike glycoprotein live-attenuated bivalent measles virus-derived vaccines targeting middle east respiratory syndrome coronavirus induce robust and multifunctional t cell responses against both viruses in an appropriate mouse model measles-derived vaccines to prevent emerging viral diseases human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets convalescent plasma: new evidence for an old therapeutic tool? feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol use of convalescent plasma therapy in sars patients in hong kong retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients key: cord-296237-i9cti2ok authors: díez, josé-maría; romero, carolina; vergara-alert, júlia; belló-perez, melissa; rodon, jordi; honrubia, josé manuel; segalés, joaquim; sola, isabel; enjuanes, luis; gajardo, rodrigo title: cross-neutralization activity against sars-cov-2 is present in currently available intravenous immunoglobulins date: 2020-06-19 journal: biorxiv doi: 10.1101/2020.06.19.160879 sha: doc_id: 296237 cord_uid: i9cti2ok background there is a crucial need for effective therapies that are immediately available to counteract covid-19 disease. recently, elisa binding cross-reactivity against components of human epidemic coronaviruses with currently available intravenous immunoglobulins (ivig) gamunex-c and flebogamma dif (5% and 10%) have been reported. in this study, the same products were tested for neutralization activity against sars-cov-2, sars-cov and mers-cov and their potential as an antiviral therapy. methods the neutralization capacity of six selected lots of ivig was assessed against sars-cov-2 (two different isolates), sars-cov and mers-cov in cell cultures. infectivity neutralization was measured by determining the percent reduction in plaque-forming units (pfu) and by cytopathic effects for two ivig lots in one of the sars-cov-2 isolates. neutralization was quantified using the plaque reduction neutralization test 50 (prnt50) in the pfu assay and the half maximal inhibitory concentration (ic50) in the cytopathic/cytotoxic method (calculated as the minus log10 dilution which reduced the viral titer by 50%). results all ivig preparations showed neutralization of both sars-cov-2 isolates, ranging from 79 to 89.5% with prnt50 titers from 4.5 to >5 for the pfu method and ranging from 47.0%-64.7% with an ic50 ~1 for the cytopathic method. all ivig lots produced neutralization of sars-cov ranging from 39.5 to 55.1 % and prnt50 values ranging from 2.0 to 3.3. no ivig preparation showed significant neutralizing activity against mers-cov. conclusion in cell culture neutralization assays, the tested ivig products contain antibodies with significant cross-neutralization capacity against sars-cov-2 and sars-cov. however, no neutralization capacity was demonstrated against mers-cov. these preparations are currently available and may be immediately useful for covid-19 management. the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which causes the respiratory disease covid-19 was declared a pandemic by the who in march 2020. most infected patients (80%) have mild symptoms. however, about 20% of covid-19 patients can progress to severe pneumonia and to acute respiratory distress syndrome (ards) which is associated with multiorgan failure and death 1 . the current critical situation demands an effective and reliable therapy that is immediately available to control the progression of the disease. convalescent plasma or plasma-derived immunoglobulin (ig) (either polyvalent ig prepared from healthy donors or hyperimmune ig prepared from donors with high antibody titers against a specific antigen) have been historically used as a readily available therapeutic option in outbreaks of emerging or re-emerging infections 2 . to date, seven human coronaviruses (hcov) have been identified. four of them are endemic and globally distributed (hcov-229e, hcov-nl63, hcov-oc43 and hcov-hku1) 3 . these viruses typically cause mild symptoms and are associated with about 15% of common colds 4 [5] [6] [7] . more recently (december 2019), the novel coronavirus sars-cov-2 emerged in china and because of its extraordinary human-tohuman transmissibility is currently causing an unprecedented pandemic 8 . although several therapeutic approaches against sars-cov-2 are under investigation, therapeutic agents of proven efficacy are still lacking. interestingly, coronaviruses share some morphological and functional properties that may be associated with cross-reactive immune responses. this cross-reactivity may have important therapeutic implications 9 . sars-cov, sars-cov-2, and mers-cov are classified within the family coronaviridae, genus betacoronavirus, subgenera sarbecovirus (sars-cov, sars-cov-2) and merbecovirus (mers-cov). the spike protein (s), which is exposed on the virion surface, is the main determinant of the coronavirus entry into the host cell and is also the major target of neutralizing antibodies 10 . spikes are formed by trimers of protein s, which is in turn formed by subunit (s1) that mediates the binding to the cell receptor and a membraneanchored subunit (s2) that mediates the fusion of the virus with cell membranes 11 . potent neutralizing antibodies often target the receptor interaction site on s1. however, the s1 subunit shows a higher variability than s2. antibodies targeting s1 are often virus-specific making s2 a better target for cross-neutralizing antibodies 12, 13 . the amino-acid sequence identity among the s proteins of human betacoronaviruses causing mild (hcov-oc43 and hcov-hku1) and severe (sars-cov, sars-cov-2, and mers-cov) respiratory infections varies between 22% and 33% 10 . however, the s proteins of sars-cov and sars-cov-2 share 77% amino-acid identity 14 and more than 90% rna sequence homology 15 . cross-reactivity in antigenic responses has been described among human coronaviruses of the same genus, particularly betacoronaviruses. crossreactivity between sars-cov, mers-cov and other endemic human coronaviruses has been reported in some neutralization assays 16-18 . recently, cross-reactivity in elisa binding assays against antigens of sars-cov, sars-cov-2, and mers-cov has been reported with currently available intravenous immunoglobulins (ivig) such as gamunex-c and flebogamma dif 19 . in this study, the neutralization capacity of the ivig products gamunex-c and flebogamma dif against these epidemic human coronaviruses -sars-cov, sars-cov-2, and mers-cov-was evaluated. ivig products used in this study were flebogamma ® dif 5% and 10% (instituto grifols s.a., barcelona, spain) and gamunex ® -c 10% (grifols therapeutics inc., raleigh nc, usa), two highly purified (≥ 98%-99%igg), unmodified human immunoglobulins. each product is manufactured from plasma collected from thousands of donors in the us and/or several european countries. igg concentrations in flebogamma dif products were 50 mg/ml and 100 mg/ml (5% and 10%) and in gamunex-c, the concentration was 100 mg/ml (10%). to ensure a virus-free product, both ivig manufacturing processes contain dedicated steps with high pathogen clearance capacity, such as solvent/detergent treatment, heat treatment, caprylate treatment and planova™ nanofiltration down to 20 nm pore size. the plasma used to manufacture the ivig lots tested was collected from march 2018 to october 2019. six different lots of flebogamma dif and gamunex-c were tested at several dilutions for cross-reactivity against sars-cov, sars-cov-2, and mers-cov by: i) elisa techniques; and ii) well-stablished neutralization assays in cell cultures. lots were identified as f1 and f2 for flebogamma 5% dif, f3 and f4 for flebogamma 10% dif and g1 and g2 for gamunex-c. each experiment was performed in duplicate. handling of viruses and cell cultures was carried out at the recombinant sars-cov was generated from urbani strain using a previously described reverse genetic technique 20 . two different sars-cov-2 isolates were tested: a) sars-cov-2 mad6 isolated from a covid-19 patient in spain; and b) sars-cov-2 (accession id epi_isl_418268 at gisaid repository: http://gisaid.org) isolated from a covid-19 patient in spain. both stock viruses (a and b) were prepared by collecting the supernatant from vero e6 cells, as previously described 21 . recombinant mers-cov was generated using a previously described reverse genetic system 22 from the reference sequence of mers-cov isolated from the index patient emc/2012 (genebank jx869059) 23 . huh7 is a well differentiated human hepatocyte-derived carcinoma cell line, kindly provided by dr. luis carrasco vero e6 is a cell line isolated from kidney epithelial cells extracted from an african green monkey. vero e6 is composed of epithelial-like cells susceptible to infection by sars-cov and sars-cov-2 25 . at cnb-csic, vero e6 cell lines were kindly provided by dr. eric snjider (university of leiden medical center, the netherlands). both huh7 and vero e6 cell lines were cultured in dulbecco-modified eagle medium (dmem) supplemented with 25 mm hepes buffer, 2 mm l-glutamine (sigma-aldrich, st. louis, mi, usa), 1% nonessential amino-acids (sigma-aldrich), 10% fetal bovine serum (fbs; biowhittaker, inc., walkersville, md, usa). in the postinfection semisolid medium, the percentage of fbs was reduced to 2%, and deae-dextran was added to a final concentration of 0.08 mg/ml. at irta-cresa, vero e6 cells were obtained from the atcc (atcc crl-1586) and cultured in dmem (lonza, basel, switzerland) supplemented with 5% (fbs (euroclone, pero, italy), 100 u/ml penicillin, 100 µg/ml streptomycin, and 2 mm glutamine 8 (all thermofisher scientific, waltham, ma, usa). in the post-infection medium, the percentage of fbs was reduced to 2%. qualitative determination of igg class antibodies crossreactivity against antigens of the tested coronaviruses was performed using elisa techniques. ivig samples were serially diluted using the buffer solutions provided in each igg elisa kit. the following kits were used for the qualitative determination of igg class antibodies in the experimental ivig lots: sars coronavirus igg elisa kit (creative diagnostics, shirley, ny, usa), against virus lysate; human anti-sars-cov-2 virus spike 1 [s1] igg elisa kit (alpha diagnostic intl. inc., san antonio, tx, usa), against s1 subunit spike protein; rv-402100-1, human anti-mers-np igg elisa kit (alpha diagnostic intl. inc.), against n protein; rv-402400-1, human anti-mers-rbd igg elisa kit (alpha diagnostic intl. inc.), against receptor-binding domain (rbd) of s1 subunit spike protein (s1/rbd); rv-402300-1, human anti-mers-s2 igg elisa kit (alpha diagnostic intl. inc.), against s2 subunit spike protein; rv-405200 (formerly rv-404100-1). in all cases the determinations were carried out following the manufacturer's instructions. reactivity was rated as negative if no reaction was observed with neat ivig or positive if the lowest ivig dilution demonstrated reactivity. aliquots of 50 µl of each ivig dilution-virus complex were added in duplicate to confluent monolayers of vero e6 cells (for sars-cov and sars-cov-2) or huh7 (for mers-cov), seeded in 12-well plates and incubated for 1 h (37°c; 5% co2). after this adsorption time, the igg-virus complex inoculum was removed, and a semi-solid overlay was added (dmem 2% fbs + 0.6% agarose). cells were incubated for 72 h at 37ºc. the semi-solid medium was removed, and the cells were fixed with 10% neutral buffered formaldehyde (sigma-aldrich) for 1 hour at room temperature and stained with 0.2% aqueous gentian violet for 10 min, followed by plaque counting. the sensitivity threshold of the technique was 20 pfu per ml. the neutralization potency of the ivig products was expressed in two ways: 1) percent reduction in pfu calculated from the pfu count after neutralization by ivig relative to initial pfu count inoculated onto the cells; and 2) plaque reduction neutralization test (prnt50) value, calculated as the -log10 of the reciprocal of the highest ivig dilution to reduce the number of plaques by 50% compared to the number of plaques without ivig. a fixed concentration of a sars-cov-2 stock (10 1.8 tcid 50/ml, a concentration that achieves 50% cytopathic effect) was mixed with decreasing concentrations of the ivig samples (range 1:10 to 1:5120), each mixture was incubated for 1 h at 37º c and added to vero e6 cells. to assess potential plasma-induced cytotoxicity, vero e6 cells were also cultured with the same decreasing concentrations of plasma in the absence of sars-cov-2. uninfected cells and untreated virus infected cells were used as negative and positive infection controls, respectively. plasma from a covid-19 positive patient with a high half-maximal inhibitory concentration (ic50) was included as an active positive control (expressed as the -log10 of the reciprocal of the dilution). all the cultures were incubated at 37ºc and 5% co2 for 3 days. cytopathic or cytotoxic effects of the virus or plasma samples were measured at 3 days post infection, using the cell titer-glo luminescent cell viability assay (promega, madison, wi, usa). luminescence was measured in a fluoroskan ascent fl luminometer (thermofisher scientific). neutralization curves are shown as nonlinear regressions. ic50 values were determined from the fitted curves as the plasma dilutions that produced 50% neutralization. details of the technique are available elsewhere 21 . ivig products showed consistent reactivity to antigens of sars-cov (culture lysate) at 10-100 mg/ml igg, sars-cov-2 (s1 subunit protein) at 100 µg/ml igg, and mers-cov (n protein, s1 subunit/rhd protein and s2 subunit protein) at 50 µg/ml igg (table 1) . all the assayed ivig preparations had neutralizing activity against sars-cov ranging from 39% to 61% (figure 1 ). all 10% igg ivig preparations (f3, f4, g1, and g2) showed prnt50 neutralization titers between 2.0 and 3.3, corresponding to 50-61% pfu reduction ( figures 1b, 1c) . the highest pfu reductions, 59.3% and 61.9% (prnt50 neutralization titers of 3.2 and 3.3), were observed with lots f4 and g1, respectively, at 1 and 0.1 mg/ml igg (dilution factors 2 and 3). the f1 and f2 lots, (5% igg) showed a lower neutralization capacity with pfu reductions of 39.5% and 43.3%, respectively ( figure 1a ). for sars-cov-2 mad6 isolate, all ivig lots, except f1 (inconclusive results) showed a significant neutralizing activity and reached prnt50 titers ranging from 4.5 to >5 (figure 2 ). pfu reductions ranging from 78.2% to 82.5% were observed with lots f2, f3 and f4 at a dilution factor of 1. even at the highest dilution factor (5 = 0.5 and 1 µg/ml), the pfu reduction ranged from 38.5% to 50.9% corresponding to prnt50 titers of 4.5-5.0 (figures 2a, 2b) . for lots g1 and g2, the pfu reduction was even higher, ranging from 88.5% -89.5% at a dilution factor of 1 to 61.7% -62.5% at a dilution factor of 5 with prnt50 titers greater than 5 ( figure 2c ). for the sars-cov-2 epi_isl_418268 isolate, f4 and g1 lots neutralized 58.4% and 64.7%, respectively, tcid50 counts at a dilution factor of 1 ( figure 3 ). as shown in figure 3 , one replicate of f4 product failed to demonstrate neutralization. no ivig lot showed any significant pfu reduction (i.e., >10%) on mers-cov even at the lowest dilution factor (10 mg/ml igg). the results presented here demonstrate, for the first time, significant cross-neutralization activity against sars-cov and especially sars-cov-2 in therapeutic ivig concentrates (flebogamma dif and gamunex-c). this neutralizing activity correlates with the cross-reactivity to different coronavirus antigens observed in elisa binding assays with ivig, as shown in a previous study 19 . the plasma used to manufacture the tested ivig lots was collected prior the detection of sars-cov-2 in europe and the usa. therefore, these results should be ascribed to cross-reactivity against sars-cov-2 by antibodies against endemic human coronaviruses in the human population at large. similar results have been reported for sars-cov and mers-cov. [16] [17] [18] these neutralization studies showed that ivig products contain antibodies with cross-neutralizing capacity against sars-cov (40-60%) and sars-cov-2 (80%-90%), but not against mers-cov (<10%). these results suggest that the cross-neutralizing antibodies target antigenic regions more conserved in sars-cov and sars-cov-2 than in mers-cov. no significant differences in the neutralizing capacity were observed among ivig lots regardless the country of origin for the plasma. this reinforces the broad applicability of these results. two different neutralization techniques were used for sars-cov-2 and both techniques showed not only the ivig neutralization capacity, but also the reliability of the results. in addition, results obtained with two different sars-cov-2 isolates confirm that the neutralization capacity is not dependent on the isolate. this was not unexpected since no significant sequence differences have been observed among sars-cov-2 isolates currently circulating throughout the world. the percentage of sars-cov-2 cross-neutralization was higher in the pfu reduction technique than in the cytopathic effect/cytotoxic technique with very low or negative values in some few cases (inconclusive for lot f1 by the pfu study, and cytopathic effect in one replicate of lot f4). this suggests that the technique used and/or slight variations in methodology may significantly influence the nature or magnitude of the results. therefore, further evaluation this cross-neutralizing activity should be conducted. cross-neutralization is gaining attention as a protective mechanism against viral infection in the context of the covid-19 health emergency. the results of this study are in agreement with recent studies that describe crossneutralization of sars-cov-2 by monoclonal antibodies from memory b cells of an individual who was infected with sars-cov 26 . furthermore, sars-cov-2-reactive cd4+ t cells have been detected in around half of unexposed individuals, suggesting that there is cross-reactive t cell recognition between circulating common cold coronaviruses and sars-cov-2 27 . however, the levels of crossneutralizing antibodies against sars-cov-2 in the sera of sars-cov patients can be highly variable 28 . ivig products are prepared using plasma from thousands of different donors, hence containing a broad representation of the state of immunity in the population at that time. this is consistent with the low rate of variability found among the different lots of ivig products tested. nevertheless, greater variability is expected among individuals with respect to infection by a given endemic human coronavirus. therefore, it has been hypothesized that the diversity of symptoms observed in sars-cov-2-infected individuals and even the potential for getting infected may depend on pre-existing cross-immunity due to previous exposure to other endemic human coronaviruses. in this regard, a detailed study of the state of immunity in the general population distinguishing those affected and not affected by the sars-cov-2 may be warranted. the higher cross-neutralizing capacity of the tested ivig preparations against sars-cov and sars-cov-2 than mers-cov may be explained by higher sequence identity of the s proteins of circulating mild human coronaviruses (hcov-oc43 and hcov-hku1) and sars-cov and sars-cov-2 compared to mers-cov (32%-33% vs. 23%-25) 14, 15 . additionally, differences in specific domains of the s protein between sars-cov and sars-cov-2 might explain higher cross-reactivity of the tested ivig against sars-cov-2 compared to sars-cov (80%-90% vs. 40%-60%). the absence of cross-neutralization against mers-cov despite the cross-reactivity observed in elisa assays suggest that these antibodies are not neutralizing. this does not necessarily indicate that the antibodies are not functional by another mechanism. for example, these non-neutralizing antibodies could be labelling the virion for identification by immune cells and subsequent destruction 29 . despite the limitations of the in vitro nature of this study, the clinical implications of the findings are encouraging. although ivig are considered a therapeutic option for hyperinflammation in patients with severe covid-19 30 , the results of this study may support the use of high dose ivig as a therapy for covid-19. positive results have already been reported for ivig in case studies 31, 32 . ivig is being tested in an ongoing clinical trial 33 . further studies looking at the functionality of these antibodies could improve our understanding the human coronavirus acquired immunity. this could pave the way for ivig (and other igg products such as intramuscular or subcutaneous preparations) as a potential therapeutic/prophylactic approach to fight future epidemics by emerging human coronaviruses. in conclusion, under the experimental conditions of this study, ivig (flebogamma dif and gamunex-c) contained antibodies with significant neutralization capacity against sars-cov and sars-cov-2, but not against mers-cov. additional research is warranted to advance ivig towards clinical use for covid-19. strategies for the prevention and management of coronavirus disease 2019 use of human immunoglobulins as an anti-infective treatment: the experience so far and their possible re-emerging role genetic recombination, and pathogenesis of coronaviruses update on human rhinovirus and coronavirus infections sars: prognosis, outcome and sequelae sars-cov-2/covid-19: viral genomics, epidemiology, vaccines, and therapeutic interventions middle east respiratory syndrome european centre for disease prevention and control. covid-19. situation update worldwide global patterns in coronavirus diversity. virus evolution identification of the receptor-binding domain of the spike glycoprotein of human betacoronavirus hku1 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-cov-2, sars-cov, and mers-cov: a comparative overview phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop profiling early humoral response to diagnose novel coronavirus disease (covid-19) an outbreak of human coronavirus oc43 infection and serological cross-reactivity with sars coronavirus cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests antigenic crossreactivity between severe acute respiratory syndromeassociated coronavirus and human coronaviruses 229e and oc43 currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus 2 antigens construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis search for sars-cov-2 inhibitors in currently approved drugs to tackle covid-19 pandemia engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans comparative host gene transcription by microarray analysis early after infection of the huh7 cell line by sars coronavirus and human coronavirus 229e the life cycle of sars coronavirus in vero e6 cells crossneutralization of sars-cov-2 by a human monoclonal sars-cov antibody targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals lack of crossneutralization by sars patient sera towards sars-cov-2 protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts covid-19: consider cytokine storm syndromes and immunosuppression high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid-19 the efficacy of intravenous immunoglobulin therapy for severe 2019-ncov infected pneumonia jordi bozzo phd, cmpp and michael k. james phd (grifols) are acknowledged for medical writing and editorial support in the preparation of this manuscript. contribution from antonio páez md (grifols) who provided his expert opinion is acknowledged. the authors acknowledge the expert technical assistance from daniel casals, eduard sala, judith luque, laura gómez, and gonzalo mercado (grifols, viral and cell culture laboratory). neutralization experiments were funded by grifols, the manufacturer of flebogamma ® dif and gamunex ® -c. jva, jr, mbp, jmh, is, and le declare having no other conflict of interest. jmd, cr and rg are full-time employees of grifols. key: cord-304057-d2r92nji authors: harrath, rafik; abu duhier, faisel m. title: sero‐prevalence of middle east respiratory syndrome coronavirus (mers‐cov) specific antibodies in dromedary camels in tabuk, saudi arabia date: 2018-04-26 journal: j med virol doi: 10.1002/jmv.25186 sha: doc_id: 304057 cord_uid: d2r92nji the middle east respiratory syndrome coronavirus (mers‐cov) is a novel coronavirus which was responsible of the first case of human acute respiratory syndrome in the kingdom of saudi arabia (ksa), 2012. dromedary camels are considered as potential reservoirs for the virus and seem to be the only animal host which may transmit the infection to human. further studies are required to better understand the animal sources of zoonotic transmission route and the risks of this infection. a primary sero‐prevalence study of mers‐cov preexisting neutralizing antibodies in dromedary camel serum was conducted in tabuk, western north region of ksa, in order to assess the seopositivity of these animals and to explain their possible role in the transmission of the infection to human. one hundred seventy one (171) serum samples were collected from healthy dromedary camels with different ages and genders in tabuk city and tested for specific serum igg by elisa using the receptor‐binding s1 subunits of spike proteins of mers‐cov. 144 (84,21%) of the total camel sera shown the presence of protein‐specific antibodies against mers‐cov. these results may provide evidence that mers‐cov has previously infected dromedary camels in tabuk and may support the possible role of camels in the human infection. blood sera were separated, diluted at 1:100 and analyzed for mers-cov specific antibodies using the anti-mers-cov elisa camel (igg) kit manufactured by euroimmun ag (lübeck, germany). this test is based on the recombinant mers-cov spike protein subunit-1 and has successfully been used by other authors evaluating mers-cov in camels. 16 optical density (od) was measured at 450 nm using a mindray mr-96 elisa reader. statistical analysis was performed on spss v. 22.0 software (spss inc., chicago, il). data were expressed as percentage for continuous variables, which were normally distributed, or as percentages of total for categorical variables. pearson χ 2 test was used to assess intergroup significance. this research was ethically approved by the research ethic committee at the university of tabuk. one hundred seventy one serum samples were collected from three areas in tabuk a primary study was conducted in tabuk instructions. this test has been successfully used and evaluated by many authors for mers-cov detection in camels. 11, 16, 17 results have shown that a high number (85%) of dromedary camels from the different farms of tabuk riyadh and screened by elisa test showed that 74% of the animals were found to have antibodies to mers-cov. 7 in the same study, 264 archived serum samples collected from dromedary camels from 1992 to 2010 in riyadh and kharj were also analyzed by elisa and showed a high seroprevalence (92%) of mers-cov neutralizing antibodies. 7 our data agree also with previous studies reporting wide antibody prevalence in camels in many countries, including egypt and oman. 12, 19 in another study conducted in oman, all serum samples from 50 dromedary camels were positive for mers-cov specific antibodies. 12 similar results were also reached from a larger study conducted in united arab emirates (uae), where 500 dromedary camels' sera screened in 2013 revealed 96% seropositivity. 18 in africa, an outbreak investigating serum samples for mers-cov assessment in camels has also shown similar results in nigeria (94%), tunisia (48.5%), and ethiopia (96.3%). 20 likewise, in a study conducted on 189 archived camel sera samples collected in 1997 from egypt and between 1983 and 1984 from sudan and somalia, 81% were found to have neutralizing antibodies to mers-cov. 16 similar results were also obtained in a study from kenya. 17 while camels in the middle east and africa were highly exposed to mers-cov infection, camels from europe and australia which are geographically isolated were not exposed to the virus. 12 however, serum samples collected from 105 dromedary camels living in the archipelago of canary islands, between 2012 and 2013 showed that 14% have antibodies against mers-cov. 12 in another study carried out in the same islands, 170 dromedary camel sera were analyzed and only 4.1% were seropositive for mers-cov. data showed that all the seropositive dromedary camels for mers-cov were all imported from africa. in addition, 307 dromedary camels' sera collected from different regions between 2013 and 2014 in the islands were all tested negative for specific mers-cov antibodies. 21 all these findings indicate that active mers-cov infection did not occur in the canary islands. 22 the same author explained the increase of the seropositivity in adult camels by the age range of the animals which were exposed for long time to the virus infection. 24 for young camels, specific mers-cov antibodies could be acquired from their mothers as it was reported by kamber 25 who suggests that maternal igg antibodies in camels are acquired through the colostrum intake during the first 24 h post-parturition and not via the trans-placental route. after 24 h, antibody levels in the dam's milk decrease rapidly, and igg levels in serum cease to rise. 25 we also suggest that maternal antibodies might not have been sufficient to mediate protective immunity for them. animals would then be exposed to new mers-cov infection and specific antibodies would consequently increase in 1-2 years after, which can explain the higher seroprevalence in juveniles then in adults. the predominance of viral antibodies in young camels could also be explained by animal exposure to the virus few times postparturition and not by mother immunity, which will stimulate antibody secretion in the blood. in the two last possibilities, young camels could contract viral infection after 2012, period of epidemic mers circulation in ksa, and production of specific antibodies would consequently increase in some months after birth. respiratory specimens, as nasal swabs, and rectal swabs will be required for analysis to know whether anti-mers antibodies are issued from recent viral infection. in relation to sex, our results have shown a high seropositivity in both males (82,8%) and females (85.9%) without a significant difference between the two genders. this information was not reported in previous studies and indicates that the sex cannot be a limiting factor in camel's infection by the virus. in conclusion, our findings in dromedary camel's sera, even if limited to serology, constitute a strong argument suggesting that mers-cov has previously infected dromedary camels in tabuk region. we speculate that dromedary camels may support the role of these animals as potential reservoirs for human infection but we cannot confirm this relationship from the current data alone. further studies including more animals and respiratory samples are needed for a larger evaluation of camel population in this region. in addition, a whole genome sequencing of camel mers-cov is required to confirm its incrimination in human infection. the work was financially supported by the deanship of scientific research (dsr) at the university of tabuk. http://orcid.org/0000-0002-9071-2970 middle east respiratory syndrome coronavirus (mers-cov) update severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study disappearance of antibodies to sars-associated coronavirus after recovery assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections specific serology for emerging human coronaviruses by protein microarray antibodies against mers coronavirus in dromedary camels antibodies against mers coronavirus in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt geographic distribution of mers coronavirus among dromedary camels absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands time course of mers-cov infection and immunity in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia studies on the supply of immunoglobulin g to newborn camel calves (camelus dromedarius) how to cite this article: harrath r, abu duhier fm. seroprevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in key: cord-287953-prn8cnvo authors: shin, nina; kwag, taewoo; park, sangwook; kim, yon hui title: effects of operational decisions on the diffusion of epidemic disease: a system dynamics modeling of the mers-cov outbreak in south korea date: 2017-05-21 journal: journal of theoretical biology doi: 10.1016/j.jtbi.2017.03.020 sha: doc_id: 287953 cord_uid: prn8cnvo abstract we evaluated the nosocomial outbreak of middle east respiratory syndrome (mers) coronavirus (cov) in the republic of korea, 2015, from a healthcare operations management perspective. establishment of healthcare policy in south korea provides patients’ freedom to select and visit multiple hospitals. current policy enforces hospitals preference for multi-patient rooms to single-patient rooms, to lower financial burden. existing healthcare systems tragically contributed to 186 mers outbreak cases, starting from single “index patient” into three generations of secondary infections. by developing a macro-level health system dynamics model, we provide empirical knowledge to examining the case from both operational and financial perspectives. in our simulation, under base infectivity scenario, high emergency room occupancy circumstance contributed to an estimated average of 101 (917%) more infected patients, compared to when in low occupancy circumstance. economic patient room design showed an estimated 702% increase in the number of infected patients, despite the overall 98% savings in total expected costs compared to optimal room design. this study provides first time, system dynamics model, performance measurements from an operational perspective. importantly, the intent of this study was to provide evidence to motivate public, private, and government healthcare administrators’ recognition of current shortcomings, to optimize performance as a whole system, rather than mere individual aspects. in 2015, korea was faced with an unexpected yet one of the largest outbreaks, worldwide, of middle eastern respiratory system (mers). a total of 186 individuals were infected, starting with a single "index" patient (a.k.a., "patient zero"), ultimately causing 38 fatalities within a two-month time period ( choi et al., 2015 ) . regardless of numerous scientific journals estimation of a mers reproduction rate < 1, implying sub-epidemic potential ( breban et al., 2013; chowell et al., 2015 ) , the mers outbreak in korea has placed historical significant impact and global recognition of the vulnerability of the present-day korean healthcare system. while korea has enjoyed "top-tier" overall healthcare status, based on its placement in the organization for economic cooperation and development (oecd) rankings ( park and jang, 2012 ) , the recent mers outbreak revealed shortcomings of korean hospital and government management systems, leading to poor mers patient care in outbreaks, as well as other ongoing patient care. serendipitously, last year's outbreak of mers is now being discussed as the sole responsibility of the hospital, instead of the healthcare system as a whole. the single index patient in the 2015 korea mers outbreak, who returned from a trip to the middle east, visited hospital a , at which he developed a cough and fever. hospital a referred the index patient to hospital b . then hospital b referred inpatients, who made close contacts with the index patient, to different hospitals. through referring process from hospital to hospital, facilities render vulnerability to a complex contagious disease, creating a mers into a severe hospital-acquired infection (hai). for example, assiri et al., (2013) 's study identified the 21 of the 23 confirmed cases of mers-cov infection were acquired through personto-person transmission in patient care units and three different hospital facilities in saudi arabia, 2013. similarly in korea, within 10 days between the date of symptom onset to the identification of the index patient, 28 first generation mers cases were identified ( korea centers for disease control and prevention, 2015 ) . overview of the mers spreading process across 15 hospitals (excluding unidentified cases), and 186 confirmed cases, is summarized in fig. 1 . despite the high regard for korea's level of medical practice, a defect in one medical specialty infectious disease was the primary catalyst for the rapid spread of mers -ultimately causing social, economic and psychological impact nationwide ( kim, 2015b ) . however, a number of hypotheses were generated to explain the spread, including: excessive patients' freedom in seeking medical care at only large hospitals, inadequate quarantine, questionable government transparency, such as belated reports of infected hospital names, and the cultural social norm of visiting patients as standard etiquette ( choe, 2015a ( choe, , 2015b ; korea centers for disease control and prevention, 2015 ) . the significance of hospital operation flaws became apparent in the mers breakout when hospital b infected over 80 people, through ineffective actions and lack of protocol within emergency room ( korea centers for disease control and prevention, 2015 ) . consequently, due to healthcare system shortcomings, there is a strong need for operational review of the mers outbreak, and we undertook this study to contribute to "preventive" epidemiological effort s in f orest alling infectious disease spread, in parallel to "cure" efforts from epidemiologists and/or hospital physicians and staff. our study aimed to address the following research objectives: (i) systematic examination and measurement of the flaws of existing healthcare operation that hypothetically affected the mers outbreak, and (ii) development and provision of operational guidelines that can alleviate future outbreaks as continuous improvement efforts. the study is organized as follows. the next section presents the background of mers as an hai, followed by a methodology section that describes both the qualitative and quantitative approaches we undertook, followed by the results and discussion. finally, we conclude with a summary to show the needs for current shortcomings in korean healthcare system and to optimize performance as a whole system, rather than mere individual aspects. hospital-acquired infections (hais) or healthcare-acquired infections (hcais) issues have been quantified as a 1 in 10 hospitalized patients acquiring infection after admission ( graves, 2004 ) . hand hygiene has been acknowledged as the most common source of infection transmission from one patient to another ( allegranzi and pittet, 2009 ) and as the foundation of healthcare associated infection preventions ( kretzer and larson, 1998; pittet et al., 2006 ) . however, korea currently practices controversial healthcare settings for patients with infectious diseases, e.g., excess installation of multi-patient beds per rooms and encouragement of family visitor caregiving for infected patients. as a part of the korean healthcare system's efforts to provide citizens with complete medical care, the system is also under pres-sure to lower medical costs. the korean government has been trying to optimize patient room design by increasing numbers of "multi-patient rooms" rather than "single-patient rooms", in order to minimize overall healthcare cost for its citizens, as announced at the 2015 health insurance policy review committee conference in seoul. consequently, the existing system neglects operational perspectives and raises concerns by allowing 50% of hospital rooms to have more than four beds per room ( ki, 2015 ) . under another global view, the canadian standards association argued that expansion of single patient rooms is no longer an option but a necessary decision ( stall, 2012 ) , highlighting the relationship between patient room design and increased hand-touch dimensions within specific, patient-proximal areas. stall (2012) reported risks of infections (such as clostridium difficile , methicillin-resistant staphylococcus aureus , and vancomycin-reistant enterococcus ) increased by 10-11% with new exposure to hospital roommate. therefore, noted that with an increasing deadly threat of nosocomial infections, rooms with four or more beds expose patients to potential infections and, thus, the installation of single patient rooms is vital. thus, infection control activities have been emphasized, such as the isolation of high-risk patients or patient with epidemiologically significant pathogens ( huang and platt, 2003; marshall et al., 2004 ) . furthermore, bootsma et al. (2006) developed rapid diagnostic testing (rdt) for prediction of mrsa (methicillin-resistant staphylococcus aureus) to a below 1%, using monte carlo simulation model. while prevention of hais has been previously studied and applied elsewhere, its need for application in korean policy remains unaddressed. family visiting and caregiving is a culturally active norm in the korean healthcare system. however, the active family care has increased the risks of disease transmission, through frequent contacts with patients and hand-touch areas. hand-touch sites are pathways to transmit pathogens from patient to patient, healthy visitors, caregivers, and/or hospital staff ( oelberg et al., 20 0 0; rheinbaben et al., 20 0 0 ) . even though ward cleaners are given specific cleaning specifications of general areas and bathrooms, these do not cover all hand-touch sites that are specifically near patients, such as bed rails and nurse call buttons ( dancer, 2004; dancer et al., 2008; white et al., 2008 ) . consequently, frequent hand-sites provide imminent risks for patients and visitors, especially the sites that are closely situated beside patients. bhalla et al. (2004) performed a two-week experiment using organisms such as vancomycin-resistant enterococcus and methicillin-resistant staphylococcus aureus to examine the frequency of hand acquisition of pathogens, after contact with surfaces near patients, and found that "hand imprint cultures were positive for one or more of the pathogens after contacting surfaces in 53% of occupied patient rooms, and 24% of rooms that had been cleaned after patient discharge." their research concluded that the environment could play a significant role in the contamination of medical staff. korea, where families are obligated to provide partial patient caregiving as a social norm ( ki, 2015 ) , is in dire need for social education in the negative influence of frequent visits, and excessive care of patients, leading to hais. besides the hand hygiene and excessive family contact frequencies as the main causes of hais, inadequate systems for quarantine, public health emergencies, and patient referral have been identified as critical causes of the mers outbreak in korea ( kim, 2015a ) . under global standards, infection control needs to be applied throughout the hospital management structure, to ultimately change behavior and culture to prevent further spread of, and better treatment of, infected patients ( brannigan et al., 2009 ) . the importance of an infection control standard of protocols is emphasized in friesema et al. (2009) study, describing that the establishment of a speedy protocol is equally effective as the enhancement of hygienic measures. similarly, koopmans (2009) concluded that ineffective control of outbreaks raises the overall financial burden on both the patient and the healthcare facilities and administration (e.g., added costs for cleaning, disinfection, closure of wards including intensive care units, and postponement of surgery). in another example, lee and ki (2015) reported a high-quality operational management as a requirement for an effective 4-step epidemiologic investigation, whose objective was to verify and quantitatively measure the size of an outbreak through the following steps: (1) identification of suspected (or confirmed) cases; (2) case definition of initial case (or index patient) and estimation of the number of total cases; (3) decision of outbreak public announcement, based on the expected number of cases; and (4) determination of the appropriate response(s). additional examples of applicable methodologies for preventing and controlling infections have been developed for real-time patient-tracking technologies, which can ultimately aid epidemiology from local to national settings ( pearson, 2009 ), in addition to using routine sampling techniques from the food industry for initiating environmental screens as a preventive measure for outbreaks ( griffith, 2006 ) . within the simulation domain, cooke et al. (2010) identified causes for poor patient treatment delays, using system dynamics modeling, and discovered a need for robust and long-term solutions (rather than local solutions), for understanding the underlying dynamics of the system. while system dynamics model does not aim to reflect the consequences of short-term variability, or specific answers to policy changes at the tactical level, its essence lies in providing effective aids for a variety of research objectives in the healthcare industry ( wolstenholme, 1993 ) . healthcare system modeling studies are generally partitioned into two categories: (1) a macro-level, which mainly uses system dynamics model for high-level modeling; and (2) a micro-level, which incorporates discrete-event simulation and queuing analysis, for in-depth modeling of specific healthcare functions and patient pathways ( rohleder et al., 2013 ). using a macro-level system dynamics modeling approach, our study intends to investigate the effect of operational decisions, such as patient-room design, occupancy control at emergency room and patient-visitor management, on the patient-care performance, such as number of infected patients (secondary infections) and financial burden on patients. we identified comprehensive constructs of stocks and flows, in accordance with sterman's (20 0 0) suggestions for high-level model building, based on a semi-structured interview process. then, we referenced previous transmission dynamics studies of severe acute respiratory syndrome (sars), which reflects similar pertinent variables such as infectivity rate, the likelihood of an outbreak, and the impact of control measures (e.g., anderson et al., 2004; lipsitch et al., 2003; riley et al., 2003 ) . lastly, based on wolstenholme's (1993) thinking system approach, we took effort s to sequence customized epidemiology investigation, specifically mers-cov case management, to systematically identify the status of each patient (i.e., susceptible or infected). we adopted a qualitative analysis, derived from bhakoo & choi (2013) 's semi-structured interview protocol, to build our sys(2) discussions of operational variables of patient care process , followed by collaborative design of a system model, and modification to questionnaires; (3) additional discussion until no new key operational aspects are discovered; and lastly (4) confirmation of the system model that represents dynamic hypotheses relevant to the case study. the final interview questionnaire is shown in appendix a . we next compiled a comprehensive list of quotes from official investigators of the korean mers outbreak, korea healthcare system experts, and reports based on hospital staff and patients who were involved in the outbreak ( bae, 2015; choi et al., 2015; kang, 2015 ) . table 1 illustrates representative quotes relevant to the study analysis. finalized key operational factors that affected 2015 korea mers outbreak in emergency room have been summarized in fig. 2 . we combined the abovementioned qualitative findings with quantitative findings from the case of mers outbreak in korea, in order to finalize the model in preparation for quantitative analysis. our accumulated data of infection cases was based on a publicly available site, jointly operated by the ministry of health and welfare and korean centers for disease control and prevention, and peer-reviewed articles ( table 2 ) . table 3 summarizes the classifications of 186 mers confirmed cases by the infection generation, infected patient type, infected location, and possession of preexisting illnesses. with a single index patient (generation zero), a total of 3 generations of secondary infections occurred: 1st generation of 28 patients infected by the index patient, 2nd generation of 125 patients infected by the 1st generation, 3rd generation of 32 patients infected by of 2nd generation. not only was the infection transmitted between patients (60.8%) and visitors (17.2%), mers also infected medical staff members, including doctors, nurses, and even ambulance drivers (21.5%). moreover, various outbreak locations showed a clear indication of nosocomial infection with a significant spread rate in patient rooms (14.5%), emergency rooms (44.1%), and patient wards (37.1%). table 4 summarizes the characteristics of the various infected generations of 183 confirmed cases (excluding two unidentified cases and the index patient) by infection location and patient type. the center of infection spread was general wards for the 1st and 3rd generation (with 20 cases and 16 cases, respectively), and emergency room for 2nd generation (with 73 cases). the majority of infected personnel were inpatients (61%), followed by medical staffs (22%), and visitors (17%). system dynamics modeling was chosen to visually demonstrate the stock and flow perspective of the susceptible and potential infected patients. using vensim ple 6.3g modeling software, we illustrated a stock and flow system, with patients representing the flow, to aid in designing appropriate disease control action plans for varying environmental hospital settings. the model illustrated in fig. 3 depicts a high-level overview dynamics model of causal relationships between operational decisions (patient room designs, occupancy control at emergency room, patient-visitor management) and patient care performance (number of infected patients and average cost per patient). the aim of this model was to applicably aid decision processing, addressing dire needs for evaluating overall causal relationships and system design. we chose exogenous and endogenous variables in the simplest form, rather than trying to accurately mimic the mers outbreak. thus, the sole purpose of the study was to simulate different results dependent on different operational decisions, and to close the gaps among epidemiologists, policy makers, and healthcare operation experts in understanding the mers outbreak. while an actual outbreak prediction model, representing the actual event, would have been ideal, such an endeavor exceeds the scope of this study. we followed cooke et al.'s (2010) processing scheme, prior to finalizing the model. first, we performed the discussion among the healthcare operation management experts. after the discussion, the model was modified with information representing the opinions of various groups. second, we consulted systems engineering consultants to finalize the logic of the healthcare model. lastly, we then developed two models, mainly focused on overcrowding variables and patient room design, to evaluate changes in the infection spread rate. the listed exogenous variables for the system were set up such as to represent general hospital settings in korea ( table 5 ) . input variables were estimated based on the representative cases of hospital a and hospital b. hospital a , the representative setting of the general ward (gw) environment, is where the index patient stayed on both the 7th and 8th floors. a total of 929 (263 patients, 389 caregivers, and 277 medical staff members) subjects were exposed to mers transmission . as we discovered during the interview process, each floor supports an average of 40 beds, and the occupancy rate of gw ( α) was about 85%. hospital b , the representative setting of the emergency room (er) environment, was where one of the super spreaders (who transmitted the infection to more than four patients) patient 14 stayed, supporting an average of 60 beds in the er. the occupancy rate of er ( α) was 111% which placed within top 10 overcrowded national university hospitals in korea (avg. = 105%, low = 79%, high = 182%). .01 low ; .05 base ; .10 high additional details for involved hospitals and mers infectivity rate are as follows: • the number of visitors ( β) was conservatively estimated, with a minimum of three visitors per patient in the gw, and three visitors per patient in the er. the cultural norm allows for family members' participation in caregiving to the patient, regardless of the availability of nurses and other professional caregivers. • the contact frequency ( contact ) was estimated based on the provided number of close contacts by the korean centers for disease control and prevention (2015). based on contact investigations of patient 176, average of 7 direct contacts with family members and visitors per day have been estimated. • the infectivity rate ( λ) was estimated based on drosten et al. (2014) finding of a 5% secondary transmission rate among household contacts of patients with mers-cov. while kim et al.'s (2015) study estimated 3.9% attack rate (or infection rate) at the residence of the index patient, they also found range of 1.4%-14.3% attack rate without significant pattern in its environmental settings or infection spread patients' characteristics. thus, while considering 5% as our base infectivity rate, we also examined effects of different infectivity rates further in this study. the comprehensive parametric settings of the initial model are demonstrated in table 6 . collecting and developing more detailed input variables that lead to accurate prediction of the infection spread should be the aim of future studies, but goes beyond our goal. fig. 4 shows the first secondary infected population model in this study. in brief, first, total population is the summation of patients' arrival dependent to occupancy rate at the interested environmental setting, and number of visitors dependent to number of patient arrival. total population is established as a susceptible population contributing to the estimation of probability of contact with infected patient, arising of a single infected patient. second, cumulative estimation of the first secondary infected patients, as of day 1, is calculated based on the function of the infectivity of mers and the contacts between infected and susceptible population. lastly, model is ran and observed for the number of interested days or until no susceptible population is available. positive sign ( + ) indicate whether the origin event/population positively contributes to the next event/population. the parametric settings of (i) number of patients and visitors are estimated based on patient's hospital information, and (ii) number of contacts made per day, infectivity rate of mers and occupancy rate are presented in table 7 . we performed "white box" analysis, also known as structurebased validation, for verifying the model ( brailsford et al., 2004; lane et al., 20 0 0 ), using information from official investigators of the mers outbreak, to ensure the accuracy of the model's representation in fig. 4 . the objectives of this test were to gain qualitative insight into the system, and to include the appropriate perceptions of the actual processes in "real world" settings. the comparison of fig. 4 simulation result with the actual data of patient 1, within general ward (gw), and actual data of patient 14 within emergency room (er) are shown in table 8 . simulation results of infected patients are shown as 25 and 77, respectively to the finalized system dynamics models of a single hospital ( figs. 5 and 7 ) illustrate feedback loops dependent on operational decision variables (patient-room design, er occupancy control, patientvisitor management), which also drive the system behavior represented by the total number of infected patients (secondary infection). while the abovementioned general transmission dynamics model illustrates a causal loop diagram of first secondary infection model, this section constructs and analyzes models that incorporate operational decision variables and closely observe secondary infection outcomes. the right-hand side of the model represents increasing population infected with mers, while the left-hand side of the model represents a decreasingly susceptible population. the nature of the loop does not account for the cure rate, but solely focuses on the sensitivity of the change in the exogenous variable. we performed two main scenario-based simulations and evaluated (1) the effect of er overcrowding on secondary infection outcomes; and (2) the effect of patient room design on total patientcare performance. to demonstrate the effect of er occupancy rate on the patient care performance, we evaluated varying potential numbers of infected patients by the er occupancy rate ( α low = .79, α high = 1.82), the number of visitors per patient ( β low = 1, β base = 3, β high = 5), and the infectivity rate ( λ low = .01, λ base = .05, λ high = .07) as shown in fig. 5 . briefly, the first secondary infected population model, added the emergency occupancy rate and number of contacts made in the er within the model). we specifically focused on day 10 in accordance with the super spreaders' average exposed table 9 rate of secondary infection transmission in high (vs. low) er occupancy circumstance. low infectivity x 1.9 x 1.9 x 1.9 base infectivity x 9.2 x 10.4 x 10.9 high infectivity x 8.9 x 13.1 x 15.3 duration of 10 days ( korea centers for disease control and prevention, 2015 ). table 8 summarizes the estimated number of infected patients on day 10. under all infectivity scenarios, the numbers of infected patients appeared insensitive to the low er occupancy-(no. infected patients avg. = 1.7 ± 0 low ; 11.1 ± .2 base ; 24.0 ± 1.3 high ) compared to the high er occupancy-circumstance (no. infected patients avg. = 3.3 ± 0 low ; 112.6 ± 12.3 base ; 201.1 ± 92.3 high ). consequently, these results highlight the importance of er overcrowding management as a preventive measure for outbreaks. table 9 demonstrates how the number of visitors, per patient, increased the speed of secondary infections in high (compared to low) er occupancy circumstance. the change in the secondary infection speed between low-and high occupancy circumstances becomes apparent by comparing low infectivity (x1.9 to x1.9) and high infectivity (x8.9 to x15.3) scenarios. specifically, under base infectivity scenario, allowing 5 visitors per patient (vs. 1 visitor) contributed to an estimated 23.4 (27%) more infected patients. fig. 6 (a) and (b) visually confirm a sharp increase in the numbers of infected patients under the high occupancy ( fig. 6 (b) ) vs. low occupancy-circumstances ( fig. 6 (a) ). moreover, the increased number of visitors, per patient, affected overall secondary infection outcome more severely, in both high-(min. = 201.3, max. = 383.3) and low-occupancy circumstances (min. = 22.6, max. = 25.0). lastly, fig. 6 (c) illustrates the comparison of the two extreme worst and best case scenarios under base infectivity scenario. the worst case scenario represents high er occupancy, with high numbers of visitors per patient, and vice-versa for the best case scenario. consequently, the visible gap in secondary infection outcome starts widening on day 5, reaching its maximum of estimated 122.8 infected patients, on day 10. to capture the effect of the patient room design on the total estimated number of infected patients on day 10 in gw setting, we assigned fractions of susceptible contacts made at each room (e.g., contact = 1 for single-room, 4 for 4-patients room, and 6 for 6-patients room) as shown in fig. 7 . briefly, the first secondary infected population model, added the fractions of single patients, four patients and to six patients. details of patient room design, costs, and estimated number of infected patients are shown in table 10 under the parametric settings of the number of beds ( beds = 40), the occupancy rate ( α = .85), the number of visitors per patient ( β = 3), and the infectivity rate ( λ low = .01, λ base = .05, λ high = .07). number of patients' arrival, patient capacity, and beds are assumed equal across varying design types (a through f), while number of rooms will differ (i.e., design a may offer 6 single-patient rooms when design f offer a single room with 6 beds). we estimated costs of private rooms and multipatient rooms, based on the data of representative three hospitals in seoul, as publicly provided on the national health insurance review and assessment service website ( health insurance review and assessment service, 2016 ). the patient charge of private rooms was significantly higher than multi-patient rooms, as private rooms are not covered by national healthcare insurance. thereby, the average costs of single-, 4-, and 6-patient rooms were estimated to be 40 0,0 0 0krw (approx. 333 usd), 20,0 0 0 krw (approx. 16 usd), and 10,0 0 0 krw (approx. 8 usd), respectively. while the average expected cost per patient could be as low as 8 usd, based on a 100% 6-patient room economic design, a maximum of 29.0 infected patients from one-hospital model were estimated in high infectivity situations. by merely increasing the fraction of total beds of single-patient rooms to 25% and reducing 6-patient rooms to 50%, the newly changed patient room design effectively lowered the number of infected patients by more than 50%, for an additional $83 usd per patient in both base and low infectivity scenarios. while the decrease in numbers of infected patients seemed invariable in both the low and base infectivity cases ( fig. 8 ) , the decrease in proportion of infected patients is significant ( table 11 ) . by considering design type c in placement of type f, the simulation result shows 25% and 69% decrease in total number of infected patients in low and base infectivity scenarios, respectively. additionally, in high infectivity scenario, both the curvature and rapid increase in percentages clearly indicate significant effect of design type on secondary infection outcome. overall, the estimated number of infected patients sharply decreased as the total cost of the patient rooms increased by multiples of, and total costs of, patient rooms. our system dynamics model was built to incorporate and minimize the gap among perspectives of epidemiologists, policy makers, and healthcare operation experts in korea. it is essential to examine the means to lower viral infectivity rates and the need for operational examination became inevitable, as shown in our study. the comparison of economic and optimal patient room design scenarios showed that under the low infectivity scenario, patient room designs did not show significant number of infected patients, over a 10-day of period. however, under the all infectivity case scenarios, the financial savings of economic design were offset by the increased proportion of infected patients, thus worsening patient care outcome. while it is impractical to advocate exclusive singlepatient room installations, alternative designs to current economic designs are necessary. secondly, excessive er occupancy rates, at a 182% maximum, based on a recent report on overcrowding in korea hospitals ( oh, 2016 ) , has become an imminent problem in korea. while the secondary infection outcomes under low er occupancy circumstance showed a minimal effect, high er occupancy circumstance displayed maximum of 92.3 infected patient differences. the restriction of occupancy rates appeared significantly dependent on number of visitors, per patient, under base and high infectivity scenarios lastly, frequent visits from families and friends highly affected the infection spread-rate. korean cultural tradition encourages social etiquette for families to act as caregivers when their immediate family member is hospitalized. consequently, more than 20 percent of the confirmed infected cases in the korean 2015 mers outbreak were family members of inpatients, and it became clear that family caregivers could have transmitted pathogens to other inpatients within the same hospital environments. in support of the korean healthcare organization's statement that the cultural tendency of frequent visits to patients needs to be reassessed ( choe, 2015b ) , our simulation result demonstrated that a decrease in number of visitors from 5 to 1 resulted in a dramatic decrease in the potential number of infected patients. moreover, comparing the best-and worst-case scenarios illustrated a gap of approximately 122.8 patients, by day 10. while optimal patient care-centric hospital designs may place financial burdens on individual patients, successfully decreasing the number of total potentially infected patients, from the entire system's perspective. while operational decisions may not have directly mitigated the mers infectivity rate, it could significantly improve patient-care quality by alleviating rate of spread within medical centers ( vincent et al., 2008 ) . it is important for healthcare management and healthcare participants to consider optimizing overall system's benefit over individual's healthcare benefit. a lack of systematic guidelines in handling infectious disease outbreaks, across different hospital tiers, represents vulnerability in korean healthcare operations. no specific guidelines were provided for the transfer of patients from one facility to another, causing misplacement of critical information. besides environmental factors, intrinsic factors, such as lack of knowledge of infectivity, proper guidelines, prior to patient admission or transfer, could also effectively minimize the rate of infection transmission. we further showed that by pairing thorough interviews with healthcare professionals, in addition to the use of simulation tools (e.g., stock and flow charts and causal loop diagrams), integrative efforts will contribute to the development of a comprehensive qualitative model of prevention. kim et al. (2015a) reported comprehensive infection control measures and recommendations for healthcare facilities highlighting procedures for hand hygiene and use of personal protective equipment (hand hygiene), testing procedure of radiological examination and diagnostic collection, packaging, transportation (laboratory management), following with isolation procedure for outpatient, emergency room patient, and visitors (patient management) for the recommendation. study includes surgery procedure and postoperative removal procedure (surgery on suspected or infected patient) in guideline to improve emergency response to outbreaks and to prevent of mers cov. our study adds the values in the operational factors as an importance of patient room design (layout management) and family caregiving and visit frequencies (occupancy control management), consequently adding additional values to previously reported guidelines. the objective of this endeavor was to understand the effect of operational decisions on the infection spread process, which could potentially halt or reduce deadly outbreaks. built upon discussions with representatives of mers outbreak researchers and korean healthcare system experts, we designed an applicable initial model that incorporates fundamental perspectives of healthcare operations. the healthcare industry, made up of different tiers of organizations with different objectives, can truly benefit from the application of system dynamics models, which effectively consider different participants and multitudes of interactions. in our view, this approach can effectively close the gap between understanding and preferred solutions by different disciplinary experts, for overall healthcare quality management. future studies on the investigation of optimal healthcare operations management could benefit by integrating other key determinants of hospital-acquired infections provided by kim et al. (2015a) (i.e., contact control via personal protective equipment, disinfection, and environmental cleaning). second, the model used in this research currently assumes the hospital is able to provide equal number of beds whether they are economic design (all 6-patients room) or optimal design (all single patient room) through the unrestricted ward capacity. following assumption could impact the economic assumption, thus, future studies should consider the inclusion of financial perspective for the healthcare operational cost optimization. lastly, future building of system dynamics model in healthcare should consider the addition of "black box" testing as part of the model validation process ( brailsford et al., 2004 ) . while we did consider black box testing, which quantitatively measures and compares the simulation result with data from the actual event, we found that such analysis was beyond the purview of our current study. however, researchers, whose main objectives are to closely model the actual outbreak, could benefit from adopting such validation processes. role of hand hygiene in healthcare-associated infection prevention epidemiology, transmission dynamics and control of sars: the 20 02-20 03 epidemic hospital outbreak of middle east respiratory syndrome coronavirus the duty to prevent" during an epidemic situation like 2015 korean mers outbreak the iron cage exposed : institutional pressures and heterogeneity across the healthcare supply chain acquisition of nosocomial pathogens on hands after contact with environmental surfaces near hospitalized patients controlling methicillinresistant staphylococcus aureus: quantifying the effects of interventions and rapid diagnostic testing emergency and ondemand health care: modelling a large complex system where does infection control fit into a hospital management structure interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk press releases experts fault south korean response to mers outbreak mers virus's path: one man, many south korean hospitals [www document epidemiological investigation on the 119th confirmed mers-cov case with the indefinite mode of transmission in pyeongtaek outbreak transmission characteristics of mers and sars in the healthcare setting: a comparative study a dynamic model of the systemic causes for patient treatment delays in emergency departments how do we assess hospital cleaning? a proposal for microbiological standards for surface hygiene in hospitals monitoring environmental cleanliness on two surgical wards transmission of mers-coronavirus in household contacts norovirus outbreaks in nursing homes: the evaluation of infection control measures economics and preventing hospital-acquired infection haccp and the management of healthcare associated infections: are there lessons to be learnt from other industries uninsured medical examination fee risk of methicillin-resistant staphylococcus aureus infection after previous infection or colonization an interview of mers epidemic investigator mers outbreak in korea : hospital-to-hospital transmission structural factors of the middle east respiratory syndrome coronavirus outbreak as a public health crisis in korea and future response strategies lessons learned from new emerging infectious disease, middle east respiratory syndrome coronavirus outbreak in korea middle east respiratory syndrome infection control and prevention guideline for healthcare facilities epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek noroviruses in healthcare settings: a challenging problem middle east respiratory syndrome coronavirus outbreak in the republic of korea behavioral interventions to improve infection control practices looking in the wrong place for healthcare improvements: a system dynamics study of an accident and emergency department strengthening epidemiologic investigation of infectious diseases in korea: lessons from the middle east respiratory syndrome outbreak transmission dynamics and control of severe acute respiratory syndrome control of endemic mrsa -what is the evidence? a personal view 20 0 0. detection of pathogen transmission in neonatal nurseries using dna markers as surrogate indicators severity of er overcrowding at university hospitals (korean) the diagnosis of healthcare policy problems in korea historical and changing epidemiology of healthcare-associated infections evidence-based model for hand transmission during patient care and the role of improved practices transmission of viruses via contact in a household setting: experiments using bacteriophage ??x174 as a model virus transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions coordinating health services: an operations management perspective private rooms: a choice between infection and profit 20 0 0. business dynamics: systems thinking and modeling for a complex world is health care getting safer are hygiene standards useful in assessing infection risk? a case study in community care using systems thinking the authors thank the institute of management research at seoul national university for supporting this research. note that the interpretation and conclusions contained herein do not represent those of ministry of health and the korean centers for disease control. all of the data used within the study were from public sources. supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jtbi.2017.03.020 . the authors declare no conflicts of interest regarding the contents of this manuscript. all authors contributed to the discussions and design of the framework. nina shin and yon hui kim designed and developed the research. taewoo kwag conducted the interview and contributed the analysis. nina shin and sangwook park conceived, developed, and analyzed the system dynamics model. nina shin and yon hui kim wrote the manuscript. sangwook park and yon hui kim contributed materials and analysis tools. all authors read and approved the final manuscript. key: cord-293505-1t3hg4wi authors: bernard-stoecklin, sibylle; nikolay, birgit; assiri, abdullah; bin saeed, abdul aziz; ben embarek, peter karim; el bushra, hassan; ki, moran; malik, mamunur rahman; fontanet, arnaud; cauchemez, simon; van kerkhove, maria d. title: comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from 2015 to 2017 date: 2019-05-14 journal: sci rep doi: 10.1038/s41598-019-43586-9 sha: doc_id: 293505 cord_uid: 1t3hg4wi since its emergence in 2012, 2,260 cases and 803 deaths due to middle east respiratory syndrome coronavirus (mers-cov) have been reported to the world health organization. most cases were due to transmission in healthcare settings, sometimes causing large outbreaks. we analyzed epidemiologic and clinical data of laboratory-confirmed mers-cov cases from eleven healthcare-associated outbreaks in the kingdom of saudi arabia and the republic of korea between 2015–2017. we quantified key epidemiological differences between outbreaks. twenty-five percent (n = 105/422) of mers cases who acquired infection in a hospital setting were healthcare personnel. in multivariate analyses, age ≥65 (or 4.8, 95%ci: 2.6–8.7) and the presence of underlying comorbidities (or: 2.7, 95% ci: 1.3–5.7) were associated with increased mortality whereas working as healthcare personnel was protective (or 0.07, 95% ci: 0.01–0.34). at the start of these outbreaks, the reproduction number ranged from 1.0 to 5.7; it dropped below 1 within 2 to 6 weeks. this study provides a comprehensive characterization of mers hca-outbreaks. our results highlight heterogeneities in the epidemiological profile of healthcare-associated outbreaks. the limitations of our study stress the urgent need for standardized data collection for high-threat respiratory pathogens, such as mers-cov. such large healthcare-associated (hca) outbreaks have mainly been limited to the kingdom of saudi arabia (ksa) and the united arabian emirates (uae) until the spring 2015, when a single imported case of mers returning from the middle east initiated a cluster of 186 cases in the republic of korea (rok) across at least 17 hospitals and much of the country 18 . super spreading events in healthcare settings has been described for several previous mers outbreaks, including an outbreak in al-hasa governorate in 2013 and during the outbreak in rok, where approximately 80% of the transmission events were epidemiologically linked to five mers cases 14, 18, 23 . superspreading events in health care facilities have been observed in similar high threat respiratory disease pathogens, such as severe acute respiratory syndrome (sars) in canada, china, singapore [24] [25] [26] . while more than half of the laboratory confirmed mers-cov infections reported globally to date are associated with human-to-human transmission in healthcare settings 27 , there has been little human-to-human transmission reported in household settings 28 . outbreak investigations and scientific studies conducted during or after mers hospital outbreaks have identified that aerosol-generating medical procedures with improper or inadequate personal protective equipment place medical personnel and patients sharing wards with mers patients and family visitors at higher risk for mers-cov infection 29, 30 , with exposure to infectious droplets being the likely source of contamination. although close unprotected contact with a mers patient is generally considered necessary for human-to-human transmission 31 , several studies have revealed that mers-cov particles can persist on surfaces as long as several days, raising the possibility of a role of fomites in transmission 32, 33 . fomite transmission is further supported by observed viral spreading between rooms that were clearly separated 15, 18 and outbreaks that occurred in hemodialysis units 14, 15 . factors leading to healthcare-associated outbreaks include overcrowding in emergency departments, slow triage and isolation of suspected patients and inadequate compliance to infection prevention and control procedures 17, 23, 34 . however, few studies have described or compared the characteristics of hca-outbreaks as a whole in terms of their size, epidemiologic factors 34, 35 , or the role of interventions to stop transmission 23, 36 . here, we provide the largest comprehensive study of eleven healthcare-associated outbreaks that occurred between 2015 and june 2017. we carried out a comparative analysis of these outbreaks in terms of epidemiological profiles, demographic characteristics and clinical outcome. study design. we analyzed epidemiological datasets of laboratory-confirmed mers patients and focused our study on eleven healthcare-associated outbreaks that were reported in ksa and rok since 2015, when policies and procedures for case identification and comprehensive contact identification and follow up became systematic and were implemented by affected countries. the data used documented mers-cov infections reported to who under the international health regulations (2005). we only included clusters of cases/outbreaks that were linked to healthcare facilities. supplemental rok case-based data were provided as a detailed line list of the korean mers cases included in a published study 17 . we defined laboratory-confirmed mers-cov infection as following who guidelines 4, 37 . we defined a hca-outbreak as the occurrence of 5 or more laboratory-confirmed mers-cov infections with reported epidemiologic links between cases and during which the human-to-human transmission events were documented within a single healthcare facility, with no more than 14 days apart between cases symptom onset. the mers outbreak in the republic of korea in 2015 is treated as a single outbreak. individual-level variables included information on age, sex, nationality, occupation (healthcare personnel (hcp) yes/no), dates of symptom onset, date of notification to who, presence of any pre-existing co-morbid conditions, and clinical outcome. in case of missing or conflicting information and when information from the country was not available, we considered the data as missing. statistical analysis. descriptive analysis was performed by hca-outbreak (outbreak-level analysis) using aggregated data, and for all cases (individual-level analysis). all analyses were conducted using stata, version 14 (college station, tx: statacorp lp), microsoft excel (version 15.35 2017, jones, chicago usa) and r. outbreak-level analysis. we calculated the duration, size and case fatality ratio for each outbreak. the duration of an outbreak was calculated as the number of days between the date of symptom onset of the first reported case to the date of symptom onset of the last reported case. we obtained weekly smoothed estimates of the case reproduction number based on the approach developed by wallinga and teunis 38,39 using the r 0 package. we assumed that the serial interval of mers-cov had a gamma distribution with a mean of 6.8 days and a standard deviation of 4.1 days, as described elsewhere 40 . individual-level analysis. we summarized case characteristics as frequencies and proportions for categorical variables, as median and interquartile ranges (iqr) for continuous variables. chi-square tests were used to compare subgroups of cases when appropriate. a p value of less than 0.05 was used to indicate statistical significance. univariate analysis identified variables significantly associated with fatal outcome, which were included in a multivariable model. model selection was performed using a multilevel mixed-effects logistic regression with backwards selection taking into account clustering of individuals by outbreak. for the variable "age", the cut-off was fixed at 65, based on the results of the univariate analysis. variables with p-values < 0.05 were retained in the final model. all data used in these secondary analyses were de-identified data obtained from who or datasets from peer-reviewed literature. as such, these data were deemed exempt from institutional review board assessment. (2019) 9:7385 | https://doi.org/10.1038/s41598-019-43586-9 www.nature.com/scientificreports www.nature.com/scientificreports/ general characteristics of hca-outbreaks. since 1 january 2015 to 1 october 2018, 2,260 laboratory-confirmed mers-cov infections have been reported to who. figure 1a illustrates the global epidemic curve since mers was first identified in humans. each peak is associated with a health care associated outbreak (colored lines, fig. 1a ). from 2015, affected countries implemented systematic contact tracing and follow up (including laboratory testing), investigation and data collection of mers suspect cases 41, 42 . in our analysis, a total of 423 laboratory-confirmed mers cases from eleven distinct hca-outbreaks during 2015-2017 were included ( table 1 ). the eleven hca-outbreaks varied in terms of duration, size and epidemiological profile ( table 1, fig. 1b) . the median number of total reported cases per outbreak was 10 (interquartile range, [iqr] 6-27), ranging from 5 to 186 cases. the median duration was 20 days (iqr 16-23), ranging from 10 to 57 days. three outbreaks began with sporadic cases during the first two to five weeks of the outbreak, while the other eight displayed a rapid increase to the peak. the median time between onset of symptoms of the first reported case and the peak of incidence was 3 weeks (iqr 2-3.75), ranging from 2 to 6 weeks. the case fatality ratio (cfr) in outbreaks was 28% (116 reported deaths among 423 cases), compared with the global overall cfr of 35.5% (800 reported deaths among 2,254 cases reported as of 1 october 3 (table 1) . during hca outbreaks, cfr ranged from 0 to 75% (p < 0.01) and cfr was significantly lower among hcp mers-cov infections compared to non-hcp mers-cov infections (2% vs. 36% p < 0.01). demographic and clinical characteristics. the demographic and clinical characteristics of the cases from hca outbreaks included in our analyses are summarized in table 1 . the median age was 54 (iqr, 36-65), and significantly varied by outbreak (p < 0.001). five outbreaks had a median age <40 and the other 6 outbreaks had a median age ≥50. the majority of cases were male (57%, n = 243/423), and the sex ratio among cases differed significantly between outbreaks (p < 0.001). the overall proportion of hcp was 25% (n = 105/422). this proportion varied significantly by outbreak (p < 0.001), from 13% to 89% (table 1) . median age was significantly lower among hcp than non-hcp cases (35 iqr 29-46 vs 58 iqr 45-70, p < 0.001) and the proportion of females was higher among hcp than non-hcp (70% vs 33%, n = 422, p < 0.001). more than half (57%, n = 214/377) of cases had at least one underlying co-morbid condition (table 1) , and this was significantly lower among females compared to males (46% vs 64%, respectively, n = 377, p < 0.001) and among hcp compared to non-hcp (13% vs 70%, n = 376, p < 0.001). sixteen percent (n = 67/419) of cases were asymptomatic at time of reporting (table 1 ). this proportion varied significantly between outbreaks ranging from 0% to 87% (n = 419, p < 0.001, fig. 1c ). median age of asymptomatic cases was 34 (iqr, [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] , the majority of whom were females (70%, n = 47/67) and had no underlying co-morbid conditions (78%, n = 29/37). the proportion of hcp among asymptomatic infections was high (70%, n = 47/67), and the cfr was null. the median duration between symptom onset and case notification to who was 5 days (iqr 3-8). risk factors associated with fatal outcome. in univariate analysis, fatal outcome was significantly associated with age (p < 0.001), presence of underlying comorbidities (p < 0.001), non-hcp status (p < 0.001), and male sex (p < 0.001, estimation of time-varying reproduction number. at the start of each hca outbreaks, the case reproduction number r (t) ranged from 1.0 (95% ci 0.7-1.3) to 5.7 (95% ci 3.0-9.0) ( table 1 and fig. 2 ). estimates of r (t) dropped below 1 within 2 to 6 weeks from the first reported case in the outbreak (n = 11 outbreaks, median 3 weeks, iqr 2-4). year of outbreak period of time* www.nature.com/scientificreports www.nature.com/scientificreports/ we provide here a comparative characterization of mers hca-outbreaks and report substantial heterogeneity between hca-outbreaks illustrating the complexity of the factors contributing to the emergence of a cluster of cases associated with nosocomial transmission. the duration and epidemic profiles of outbreaks varied; some started with an apparent sharp increase in incidence while others began more slowly with isolated cases emerging intermittently for a few weeks before a cluster of cases appeared in a healthcare facility. some outbreaks had a sharp decline in cases, while others experienced a long tail lasting several weeks after the peak. the median estimates of the reproduction number r(t) in the early stages of outbreaks included in our analyses reached as high as 5.7 in the republic of korea, as has been found by others 43 , likely facilitated by multiple superspreading events at two hospitals 43 . what is perhaps most informative from a public health perspective is the length of time it took to bring the outbreaks under control. all of outbreaks reached r t values below 1 within 2 to 6 weeks after the first cases were identified, highlighting that the time frame in which hospital and ministry officials can implement control measures to stop nosocomial outbreaks. factors explaining differences in hca outbreak size and duration might include variations in the speed in which cases were suspected and timing of interventions implemented in healthcare settings, including contact identification, management and isolation of patients, improved infection prevention and control measures and in some cases, the requirement to close departments [14] [15] [16] [17] [18] 29 . in this study, we were not able to evaluate the impact of interventions in these outbreaks. prevention of large hca outbreaks since 2014 (fig. 1a) , may be, in part, explained by improvements in contact tracing policies implemented in 2015. in 2015, contact tracing became more systematic with the identification and follow up of high (close, unprotected contact) and low risk contacts (protected hcw). in affected countries, national ministries of health and hospital staff comprehensively list all contacts of known mers patients, including healthcare workers at all facilities/departments the patient visited, patients who shared wards/ rooms with mers patients, family and visitors and occupational contacts. follow up of contacts includes the testing of all high-risk contacts, regardless of the development of symptoms. recommendations stated that positive contacts are placed in quarantine (home or hospital isolation for asymptomatic or symptomatic secondary cases, respectively) until they test negative 41, [44] [45] [46] . additionally, affected countries enhanced infection prevention and control procedures education, and training, and implemented visual triage systems 41 to reduce delays in testing, isolation and care of suspected mers-cov patients. this has again been recently illustrated by the lack of secondary cases following the identification of a confirmed case of mers in korea in september 2018 47 was due to the rapid and comprehensive isolation, treatment and management of contacts of the patient. the variation in outbreak size and duration is also affected by superspreading events early in some outbreaks, during which a limited number of cases generated a disproportionately large proportion of the secondary cases under specific conditions in hospitals, occurring in some outbreaks [48] [49] [50] . two super spreading events have been documented in ksa and in the republic of korea. in the republic of korea, the practice of "doctor shopping", extended stays in overcrowded emergency departments, cultural practices of large numbers of family members visiting sick relatives, and environmental contamination amplified transmission from some patients to others 14, 17, 18, 51 . during the outbreak in ksa in 2015 at the ministry of the national guard hospital, a high number of secondary cases were among hcp very quickly after the hospitalization and a surgical procedure of the index case 16 . these events triggered comprehensive review ipc in hospitals, emergency department layout, movements of patients, triage of respiratory visits, duration of emergency department stay, training of hospital staff and disinfection of healthcare facilities. our study confirmed that age and presence of comorbidities are linked to increased risk of death, similar to previously published results 52, 53 whereas being hcp was protective. the protective effect of hcp could be explained by the fact that hcp are more likely to be younger (<60 years old) and have fewer underlying medical conditions than hospitalized patients, but also that they are likely to be identified earlier or seek medical care soon following contact with a confirmed patient. the proportion of asymptomatic secondary cases identified during outbreaks has increased since 2014. there is no evidence to suggest that this represents changes in virus pathogenicity, epidemiology or transmission www.nature.com/scientificreports www.nature.com/scientificreports/ patterns of mers in recent years. however, the increase in the number of reported asymptomatic cases is hypothesized to be due to earlier detection efforts from more aggressive contact identification and testing during hca-outbreaks since 2015 as testing policies adopted and implemented by ksa and other countries have changed following the large outbreaks in jeddah/riyadh in 2014 3, 41, 54 . in 2017, 40-80% of the laboratory confirmed hcp secondary cases experienced no symptoms and were detected as part of a policy to test all contacts irrespective of symptoms (table 1) . we believe that the identification of hcp asymptomatic cases, and their subsequent isolation, has had a strong impact on prevent further human to human transmission in health care settings. this is visually demonstrated in fig. 1c by the outbreak labelled sau16_2, which included 26 (of 30 www.nature.com/scientificreports www.nature.com/scientificreports/ reported cases) asymptomatic cases. while this is a large number of secondary cases, we argue that the early identification, isolation and recovery of these asymptomatic/mildly symptomatic cases effectively stopped human to human transmission. our study has several limitations due to variability in the completeness and quality of case-based data provided to who since 2012 and also due to the lack of detailed information on the timing specific interventions were implemented in relation to each outbreak. without detailed information on the timing of interventions in each health care facility it was not possible in our analyses to determine which intervention or combination of interventions had the greatest impact on stopping the mers outbreaks. moreover, prior to 2015, contacts without symptoms were not tested for mers-cov infection, thus the rate of identification of secondary cases was drastically different prior to 2015, which complicates the comparison of data collected before and after 2015. the improvements in data reporting on cases (e.g., more systematic reporting of underlying conditions, reported exposures, contacts between patients) from 2015 allowed us to perform better analyses with less missing values. we continue to encourage the policy of identifying, following and testing of all high risk contacts of mers patients in hca-outbreaks 3, 41, 55 . the natural history of asymptomatic infection and role of asymptomatic or mildly symptomatic hcp in transmission of the virus between patients, requires detailed scientific studies to better understand their potential role in transmission 15 . the sharing of outbreak experiences between affected hospitals within and between countries and a detailed evaluation of the impact of non-therapeutic interventions is critical to our understanding and for the prevention of nosocomial outbreaks of respiratory pathogens. health care professionals and hospitals currently have tools to limit the extent and impact of such events, which include early identification and isolation of suspect patients and strict adherence to standard infection prevention and control measures. these are the hallmark of effective mers-cov control. a combination of interventions including the efficient triage of patients with respiratory symptoms at hospital entry; limiting wait times and overcrowding in waiting areas; isolation of suspected and confirmed cases; appropriate use of droplet personal protection equipment by hcp; basic hand hygiene; increased protective aerosol precautions for hcp during aerosol-generating medical procedures; efficient surface and environmental decontamination of areas with mers patients, and extensive contact tracing, can prevent human to human transmission in health care settings. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) who|middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia outbreak of middle east respiratory syndrome-coronavirus causes high fatality after cardiac operations an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region, saudi arabia increase in middle east respiratory syndrome-coronavirus cases in saudi arabia linked to hospital outbreak with continued circulation of recombinant virus identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus outbreak in the republic of korea case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from mers: progress on the global response, remaining challenges and the way forward outbreak of middle east respiratory syndrome at tertiary care hospital mers-cov outbreak in jeddah-a link to health care facilities a dynamic compartmental model for the middle east respiratory syndrome outbreak in the republic of korea: a retrospective analysis on control interventions and superspreading events sars outbreak in the greater toronto area: the emergency department experience superspreading sars events understanding the super-spreading events of sars in singapore middle east respiratory syndrome coronavirus (mers-cov): current situation 3 years after the virus was first identified transmission of mers-coronavirus in household contacts middle east respiratory syndrome infection control and prevention guideline for healthcare facilities evidence for zoonotic origins of middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions interpreting results from environmental contamination studies of middle east respiratory syndrome coronavirus preventing healthcare-associated transmission of the middle east respiratory syndrome (mers): our achilles heel comparative epidemiology of human infections with middle east respiratory syndrome and severe acute respiratory syndrome coronaviruses among healthcare personnel outcome of strict implementation of infection prevention control measures during an outbreak of middle east respiratory syndrome middle east respiratory syndrome coronavirus. case definition for reporting to who different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures the r0 package: a toolbox to estimate reproduction numbers for epidemic outbreaks unraveling the drivers of mers-cov transmission ministry of health, kingdom of saudi arabia. infection prevention and control guidelines for the middle east respiratory syndrome coronavirus (mers-cov). moh.gov.sa available at surveillance for human infection with middle east respiratory syndrome coronavirus (mers-cov) high reproduction number of middle east respiratory syndrome coronavirus in nosocomial outbreaks: mathematical modelling in saudi arabia and south korea management of asymptomatic persons who are rtpcr positive for middle east respiratory syndrome coronavirus (mers-cov) home care for patients with middle east respiratory syndrome coronavirus (mers-cov) infection presenting with mild symptoms and management of contacts ?menuids=home002-mnu0576-mnu0586&fid=8652&q_type=&q_value=&cid=140796&pagenum= middle east respiratory syndrome coronavirus (mers-cov) infection -republic of korea assessing the risk of observing multiple generations of middle east respiratory syndrome (mers) cases given an imported case the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea transmission characteristics of mers and sars in the healthcare setting: a comparative study risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea middle east respiratory syndrome risks of death and severe disease in patients with middle east respiratory syndrome coronavirus asymptomatic infection of middle east respiratory syndrome coronavirus using serologic survey in korea world health organization. disease outbreak news the authors would like to thank the many individuals involved in the collection of case-based data and in the care competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-304030-6ve5plea authors: aboagye, james odame; yew, chow wenn; ng, oi-wing; monteil, vanessa m.; mirazimi, ali; tan, yee-joo title: overexpression of the nucleocapsid protein of middle east respiratory syndrome coronavirus up-regulates cxcl10 date: 2018-10-17 journal: biosci rep doi: 10.1042/bsr20181059 sha: doc_id: 304030 cord_uid: 6ve5plea middle east respiratory syndrome coronavirus (mers-cov) causes respiratory diseases in humans and has a high mortality rate. during infection, mers-cov regulates several host cellular processes including antiviral response genes. in order to determine if the nucleocapsid protein of mers-cov (mers-n) plays a role in viral–host interactions, a murine monoclonal antibody was generated so as to allow detection of the protein in infected cells as well as in overexpression system. then, mers-n was stably overexpressed in a549 cells, and a pcr array containing 84 genes was used to screen for genes transcriptionally regulated by it. several up-regulated antiviral genes, namely tnf, il6, il8, and cxcl10, were selected for independent validation in transiently transfected 293ft cells. out of these, the overexpression of mers-n was found to up-regulate cxcl10 at both transcriptional and translational levels. interestingly, cxcl10 has been reported to be up-regulated in mers-cov infected airway epithelial cells and lung fibroblast cells, as well as monocyte-derived macrophages and dendritic cells. high secretions and persistent increase of cxcl10 in mers-cov patients have been also associated with severity of disease. to our knowledge, this is the first report showing that the mers-n protein is one of the contributing factors for cxcl10 up-regulation during infection. in addition, our results showed that a fragment consisting of residues 196–413 in mers-n is sufficient to up-regulate cxcl10, while the n-terminal domain and serine-arginine (sr)-rich motif of mers-n do not play a role in this up-regulation. middle east respiratory syndrome coronavirus (mers-cov) is a nidovirus and etiologic agent for respiratory disease. mers-cov emerged in saudi arabia in 2012 and is still causing respiratory infections with 2220 and 790 (35%) reported cases and deaths respectively [1] . manifestations of mers disease are similar to severe acute respiratory syndrome (sars) with patients usually developing acute pneumonia that progresses to respiratory failure and acute respiratory disease syndrome [2] . patients also exhibit extrapulmonary manifestations include renal failure, hepatic dysfunction, and diarrhoea with some severe cases of deranged coagulation profile and hematological changes [3] [4] [5] . mers-cov is an enveloped, single-stranded, positive-sense rna virus, approximately 30 kb in length with two-thirds of its genome encoding 15-16 non-structural proteins (nsp) [6, 7] . the remaining one-third encodes for structural proteins interspersed with accessory proteins. the structural proteins include the spike (s), matrix (m), envelope (e), and nucleocapsid (n) proteins [6, 7] . for coronavirus, n primarily encapsidates the viral genome but also plays important roles in viral replication, virus particle assembly and release [8, 9] . additionally, the n protein of sars-cov has been reported to regulate host functions such as immune interference, apoptosis, proliferation, and cell cycle [10, 11] . the n protein of mers-cov (to be referred to as mers-n herein) is a 413 amino acids protein and has been reported to share some characteristics with n of other coronaviruses. for example, crystallography and small angle x-ray scattering experiment have shown that the n-terminal region of mers-n exists as a monomer and has structural features that are similar to other coronavirus [12] . like mouse hepatitis virus, porcine epidemic disease virus and sars-cov, mers-n has been reported to be adp-ribosylated [13] and acts as a viral suppressor of rna silencing in mammalian cells [14] . however, the latter noted that mers-n showed lower activity than the n proteins of other coronavirus tested. similar to mouse hepatitis virus and sars-cov, mers-n has been found to be essential for the packaging of viral rna into virus-like particles [15] . during infection, the host defenses are activated to produce antivirals and proinflammatory cytokines and chemokines to help eliminate the infection. interferon treatment has been reported to effectively subdue mers-cov replication [16] ; however, like any successful viral infection, mers-cov has developed mechanisms to evade or dampen the activity of the host immune responses. studies have demonstrated that mers-cov infection shows a delayed but marked induction of proinflammatory cytokines/chemokines [17] . in the present study, we aim to determine if the mers-n protein is involved in regulating host antiviral response to infection. we showed that the mers-n protein could contribute to the regulation of several host antiviral response genes including cxcl10. importantly, up-regulation of cxcl10 has been reported in mers patients [18, 19] as well as mers-cov infected cells [3, 17, 20] . to our knowledge, this is the first time that mers-n has been shown to be one of the contributing factors for cxcl10 up-regulation during infection and this suggests that mers-n may contribute to viral pathogenesis. 293ft cells (invitrogen) were cultured in dulbecco's modified eagle's medium (dmem, hyclone), while a549 (american type culture collection) was maintained in minimal essential medium (hyclone). all media were supplemented with 10% fbs (hyclone) and 1% penicillin-streptomycin (sigma-aldrich). the mers-n gene corresponding to the emc-2012 strain was chemically synthesized (genscript), while n genes of other coronaviruses were purchased (origene). all were subcloned into either pxj40 or pxj40-flag vector and used for transfection with xtreme gene r (sigma-aldrich). the c-terminal fragment (residues 196-413) of mers-n was fused to the glutathione s-transferase (gst) protein by cloning into the pgex-6p1 vector (ge healthcare). the protein was expressed in escherichia coli and purified using gsh-sepharose beads (ge healthcare). the gst-fusion protein was then used to immunize mice and generate hybridoma as previously described [21] . all mice were handled according to national advisory committee for laboratory animal research (naclar) guidelines. mouse monoclonal antibody (mab) 7h6 was purified from the culture supernatant of a selected hybridoma by using a hitrap protein g column (ge healthcare). synthetic 15-mer peptides with ten amino acids overlapping sequences were generated (gl biochem). binding of the mab to the peptides was screened by elisa. briefly, the plates were coated with peptides overnight, washed, and blocked for 30 min with 1% bovine albumin serum in 1× pbs. the mab was then added and incubated at room temperature for 2 h. this was followed by washing, addition of secondary antibody goat anti-mouse hrp (bio-rad), and incubation at room temperature for 1 h. plates were washed again, incubated with tetramethylbenzidine substrate (pierce), and subsequently, the reaction was stopped with 2 m sulphuric acid. the absorbance was read at 450 nm. mers-n was cloned into the plenti6.3 vector (thermo fisher scientific). 293ft cells were seeded at 3 × 10 6 cells into 10-cm dishes and incubated at 37 • c in 5% co 2 overnight. a plasmid mixture containing 2 μg each of phdm-tatib, phdm-hgpm2, phdm-vsvg, and pprb-cmv plasmids; and 8 μg of plenti6.3-lacz or plenti6.3-mers-n plasmid was prepared. the plasmid mixture was added to 500 μl of opti-mem (gibco) and 16 μg of xtreme gene r . the mix was incubated at room temperature for 15 min, added to the seeded cells, incubated overnight, and replaced medium. the medium was harvested after 48 h and centrifuged at 4000 rpm at 4 • c for 10 min. the supernatant containing the lentiviral vector was collected, filtered, aliquoted, and frozen at −80 • c. a549 cells were seeded at 300,000 cells in six-well plates and left overnight. cells were infected with lentiviral vector for 24 h and followed by changing of culture medium. after another 48 h, the medium was changed to medium containing 6 μg/ml blasticidin (selective medium). cells were cultured in selective medium and expanded into t-75 flask at day 7. after day 10, cells were maintained in medium supplemented with 4 μg/ml blasticidin. cells were harvested at day 2 and 10 for rna extraction and immunofluorescence assay (ifa). total rna was isolated from a549 cells stably expressing mers-n (test sample) or lacz (control sample) proteins using rneasy mini kit (qiagen) with genomic dna (gdna) removal with rnase-free dnase set (qiagen). rnas were quantitated and used only when the absorbance ratio of od 260 nm /od 280 nm was at least two. total rna of 3 μg was reverse transcribed into complementary dna using the rt 2 first strand kit (sa, biosciences), mixed with the qpcr mastermix containing sybr green, and used on human antiviral response rt 2 profiler pcr arrays according to the manufacturer's protocol (sa, biosciences). the abi steponeplus tm real-time pcr system was used to run the qpcr cycling program. the samples were repeated in two independent experiments. then, c t values were exported and analyzed by rt 2 profiler pcr array data analysis software version 4. a549 transduced cells were grown on coverslips. approximately 24 h later, the medium was aspirated, and the cells were rinsed twice with pbs, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% triton x-100 for 10 min. coverslips were then blocked in 1% bovine albumin serum for 30 min, and cells were incubated with mab 7h6 for 1.5 h. after washing, cells were incubated with alexa fluor 488-conjugated goat anti-mouse igg secondary antibodies (invitrogen) for 1 h. after washing, cells were stained with dapi before mounting. images were captured with an olympus fluoview fv1000 laser-scanning confocal microscope. vero cells (atcc) were seeded at 40,000 cells per well on a nunc tm lab-tek tm chamber slide (thermofisher scientific) in dmem (gibco) supplemented with 10% fbs (gibco). after 24 h, cells were infected with mers-cov (erasmus mc isolate) at a multiplicity of infection of 1 in dmem for 1 h or mock infected. after 1 h, the cells were washed three times with pbs and followed by addition of dmem with 10% fbs. after 48 h post-infection, the medium was removed. the cells were washed three times with pbs, fixed, and stained as described above. all virus work was performed in a biosafety level 3 (bsl-3) laboratory. cell lysates were separated by sds/page, and proteins were transferred on to nitrocellulose membranes. the membranes were blocked with tbst (20 mm tris [ph 7.5], 150 mm nacl, and 0.1% tween 20) containing 5% skim milk for 1 h. the blot was then incubated with primary antibody at 4 • c overnight and washed thrice with tbst. this was followed by incubating with secondary antibody for 1 h, washed again with tbst, and finally developed through enhanced chemiluminescence. the primary antibodies used in the study included mab 7h6 (as described above), anti-gapdh polyclonal (santa cruz biotechnology), and anti-flag polyclonal (sigma) antibodies. secondary antibodies used were hrp-conjugated goat anti-mouse and goat anti-rabbit igg (bio-rad). the sandwich elisa for cxcl10 (bd biosciences) was used according to the manufacturer's protocol. the unpaired two-tailed t-test was used to evaluate the significant differences of datasets obtained from at least three independent experiments. p<0.05 was considered statistically significant. with the aim to generate an antibody that can be used to detect the expression of mers-n protein in infected cells or transfected cells, a fragment corresponding to residues 196-413 in n was successfully expressed and purified from e. coli (data not shown). the purified protein was used to immunize mice and subsequently, mab 7h6 (isotype of igg2b) was produced. as shown in figure 1a , mab 7h6 bound to full-length mers-n and the c-terminal fragment of mers-n. as it would be expected, mab 7h6 did not bind to the n-terminal fragment consisting of residues 1-195 in mers-n. the human coronaviruses (hcovs), including sars, 229e, hku1, nl63 and oc43, cause respiratory diseases. therefore, there is a need to determine the cross-reactivity of mab 7h6 to the n proteins of other hcovs. as shown in figure 1b , mab 7h6 did not recognize n proteins of the other hcovs indicating its specificity to the mers-n protein. next, mab 7h6 was used on mers-cov infected cells. as shown in figure 1c , specific staining was observed in cells infected with mers-cov indicating that mab 7h6 could detect mers-n expressed during infection. mers-n was observed to localize throughout the cytoplasm as well as in punctate cytoplasmic organelles. no staining was observed in mock-infected cells. in order to determine the binding site for mab 7h6, 15-mer peptides with ten overlapping amino acids from the c-terminal fragment of mers-n were synthesized. based on elisa, mab 7h6 bound significantly to peptides containing residues 381-395, 386-400, and 391-405 ( figure 1d) , and alignment of these peptides indicates that residues 391-395 (sitqr) are essential for the interaction with mab 7h6 ( figure 1e ). western blot analysis showed that mab 7h6 did not bind to a mutant mers-n 391-395aa, confirming that the sitqr motif is essential for binding ( figure 1f ). to determine if the mers-n protein can modulate antiviral response genes, it was overexpressed in a549 cells using a lentiviral system. with the availability of mab 7h6, n was expressed without any tag. the a549 transduced cells expressed relatively high mers-n protein on both day 2 and 10 post-selection in an antibiotics medium as shown in figure 2a . this suggests that mers-n protein can be expressed and maintained for a period of time without cytotoxicity. at day 2 and 10 post-selection, rna transcripts were obtained from transduced cells, and the expression of 84 human antiviral response genes were analyzed by rt-qpcr. fold regulations of antiviral response genes by mers-n protein was compared with negative control cells, which were expressing lacz, by using the 2 − c t method. as shown in supplementary tables s1 and s2, several genes were found to be differentially regulated in the mers-n expressing cells when compared with negative control cells. a fold regulation cutoff of four was used to select for genes that were up-or down-regulated at both day 2 and 10. nine genes, namely ccl5, mx1, il6, cxcl10, isg15, oas2, ifih1, tnf and il8, were found to be up-regulated in both days as represented in the venn diagram ( figure 2b ). all of these genes with exception of ifih1 maintained or had increases in fold regulation between day 2 and 10 suggesting that mers-n persistently regulates these cytokines/chemokines (supplementary tables s1 and s2). a transient expression system was then used to express mers-n so as to rule out host gene expression changes that may be caused by prolonged antibiotics selection. a highly transfectable cell 293ft was used to express flag-tagged or untagged mers-n. as shown in figure 3a , expression of flag-tagged mers-n was detected at 24-72 h post-transfection, and the level of expression increased with time. out of the nine host genes identified using stably transduced a549 cells, proinflammatory cytokine/chemokines tnf, il6, il8, and cxcl10 were reported to be up-regulated in mers-cov infected cells [3, 17, 20] . hence, their levels in cells expressing flag-mers-n were determined using rt-qpcr and normalized to vector-transfected cells. as shown in figure 3b , all four genes were significantly up-regulated in cells expressing flag-mers-n. similar results were obtained when untagged mers-n was expressed in 293ft (supplementary figure s1) , suggesting that the flag tag at the n-terminal of mers-n does not affect its function and can be used when analyzing mutants of mers-n. next, the secretions of these cytokines/chemokines into the culture supernatant were measured. secretions of cxcl10 were observed in mers-n expressing cells at 72 h post-transfection when compared with vector-transfected cells ( figure 3c ). although cxcl10 secretion from mers-n expressing cells was relatively low, a consistent increase of approximately 2-fold was observed in different experiments when normalized with the vector-transfected cells ( figure 3d ). transient transfection of a549 cells was also performed and high expression of mers-n was achieved ( figure 4a) . similarly to the observation in 293ft cells, the overexpression of mers-n in a549 cells resulted in a based on alignment with the n proteins of other covs, mers-n is proposed to be organized into two main folded domains, which are likely to be independent of each other, separated by a linker region that contains a sr-rich motif [12] . the n-terminal domain consists of residues 37-164, while the c-terminal domain consists of residues 239-362. in addition, residues 1-36 and 363-413 are classified as intrinsically disordered regions. here, two fragments of mers-n, namely n1-195 and n196-413, were constructed and found to be expressed at similar levels as full-length n when transfected into 293ft cells ( figure 5a ). as shown in figure 5b , the n196-413 fragment was able to up-regulate cxcl10 transcriptionally, while the n1-195 did not have a significant effect. consistently, both full-length n and n196-413, but not n1-195, increased the secretion of cxcl10 when compared with vector-transfected cells ( figure 5c ). as n1-195 contains n-terminal domain and sr-rich motif, it appears that these domains are not involved in the up-regulation of cxcl10. n196-413 contains part of the linker region, c-terminal domain and the intrinsically disordered region at the c-terminal tail and further studies are required to determine which of these domains are required for up-regulation cxcl10 expression. mers-cov has broad tissue tropism including lower airway, intestinal tract, liver, kidney, neuronal, monocyte, and t-lymphocyte cells [5, 22] . upon infection by mers-cov, cytokine/chemokine regulations have been reported in airway epithelial cells and lung fibroblast cells [17] as well as monocyte-derived macrophages and dendritic cells [3, 20, 23 ]. in the present study, we showed that the overexpression of mers-n protein in a549 (lung) and 293ft (kidney) up-regulated tnf, il6, il8, and cxcl10 on the transcriptional levels, suggesting that mers-n protein may be able to regulate multiple antiviral genes in different tissues. on the protein level, the overexpression of mers-n protein in 293ft also increased the secretion of cxcl10, albeit at low levels, but not tnf, il6, and il8. it is not clear that why the secretion of cxcl10 was low despite a robust up-regulation on mrna levels by approximately 10-fold in mers-n expressing cells, but one possible reason could be the low efficiency of cytokine/chemokine secretion from 293ft cells. similarly, the overexpression of mers-n protein in a549 cells also led to transcriptional and translational up-regulation of cxcl10. as mers-cov has been reported to infect immune cells, further studies could be performed to study the effects of mers-n on the expression of antiviral genes in different types of immune cells. cxcl10 is a cytokine belonging to the cxc chemokine family and binds to the cxcr3 receptor to induce chemotaxis, apoptosis, cell growth, and angiostasis. expression levels of cxcl10 have been associated with inflammatory diseases including infectious diseases, immune dysfunction, and tumor development [19, 24] . high expressions of cxcl10 have been reported in individuals infected with pathogens including viruses, bacteria, fungi, and parasites [24] . cxcl10 regulation has been reported in mers patients [18, 19] as well as mers-cov infected cells [3, 17, 20] . our results suggest that the mers-n protein is one of the contributing factors for cxcl10 up-regulation during infection. in addition, a fragment of mers-n consisting of residues 196-413 is sufficient to up-regulate cxcl10. future works will focus on identifying the exact motif in mers-n that is contributing to the up-regulation of cxcl10, and delineating the relative contribution of mers-n and different viral proteins to this up-regulation which may have implication for viral pathogenesis. who. mers-cov fact sheet pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis pathogenesis of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease the coronavirus nucleocapsid protein is dynamically associated with the replication-transcription complexes structural basis for the identification of the n-terminal domain of coronavirus nucleocapsid protein as an antiviral target the coronavirus nucleocapsid is a multifunctional protein nucleocapsid proteins: roles beyond viral rna packaging structural characterization of the n-terminal part of the mers-cov nucleocapsid by x-ray diffraction and small-angle x-ray scattering the coronavirus nucleocapsid protein is adp-ribosylated the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells nucleocapsid protein-dependent assembly of the rna packaging signal of middle east respiratory syndrome coronavirus a review of treatment modalities for middle east respiratory syndrome delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response. virology 454-455 an antibody against a novel and conserved epitope in the hemagglutinin 1 subunit neutralizes numerous h5n1 influenza viruses differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation this is an open access the authors declare that there are no competing interests associated with the manuscript. gst, glutathione s-transferase; cxcl10, c-x-c motif chemokine 10; il6, interleukin-6; il8, interleukin-8; hcov, human coronavirus; ifa, immunofluorescence assay; mab, monoclonal antibody; mers, middle east respiratory syndrome; mers-cov, mers-coronavirus; n, nucleocapsid; rt-qpcr, reverse transcription-quantitative pcr; sars, severe acute respiratory syndrome; sr, serine-arginine; tnf, tumor necrosis factor. key: cord-303289-qoukiqr7 authors: hemida, m. g.; chu, d. k. w.; perera, r. a. p. m.; ko, r. l. w.; so, r. t. y.; ng, b. c. y.; chan, s. m. s.; chu, s.; alnaeem, a. a.; alhammadi, m. a.; webby, r. j.; poon, l. l. m.; balasuriya, u. b. r.; peiris, m. title: coronavirus infections in horses in saudi arabia and oman date: 2017-03-13 journal: transbound emerg dis doi: 10.1111/tbed.12630 sha: doc_id: 303289 cord_uid: qoukiqr7 equine coronaviruses (ecov) are the only coronavirus known to infect horses. so far, data on ecov infection in horses remain limited to the usa, france and japan and its geographic distribution is not well understood. we carried out rt‐pcr on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in saudi arabia and oman. we document evidence of infection with ecov and hku23 coronavirus by rt‐pcr. there was no conclusive evidence of middle east respiratory syndrome coronavirus infection in horses. serological data suggest that lineage a betacoronavirus infections are commonly infecting horses in saudi arabia and oman but antibody cross‐reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. infect or cause disease in horses (balasuriya, 2013) . it belongs to the genus betacoronavirus lineage a, as does human coronavirus oc43, bovine coronavirus and porcine hemagglutinating encephalomyelitis virus. equine coronavirus was first isolated from faeces of a diarrhoeic foal in 1999 (ecov-nc99) in north carolina, usa (guy, breslin, breuhaus, vivrette, & smith, 2000) , and was initially believed to only affect foals. since 2010, there have been several reports of ecov-associated respiratory and enteric infections in adult horses in japan, europe and the united states, but its global distribution is still poorly defined (kooijman, mapes, & pusterla, 2016; miszczak et al., 2014; oue, morita, kondo, & nemoto, 2013; pusterla, holzenkaempfer, mapes, & kass, 2015) . middle east respiratory syndrome coronavirus (mers-cov) is a betacoronavirus within lineage c first identified in humans in 2012 and continues to pose a threat to global health (who, 2016 may 27) . the evidence points to dromedary camels as a natural host and a major source for human mers-cov infections (alexandersen, kobinger, soule, & wernery, 2014; chu et al., 2015; gutierrez, tejedor-junco, gonzalez, lattwein, & renneker, 2015; memish et al., 2014; perera et al., 2013; reusken et al., 2014) . there is no convincing evidence of mers-cov infections in other domestic livestock species so far (adney et al., 2016; alexandersen et al., 2014; hemida et al., 2013; meyer et al., 2015; perera et al., 2013) . sequence similarity comparisons of dipeptidyl peptidase-4 (dpp4 [cd26]), the functional receptor for mers-cov, have revealed that equine dpp4 is phylogentically closely related to human dpp4 and the binding affinity of mers-cov spike s1 domain for equine dpp4 is similar to that of human and camel dpp4, raising the possibility that mers-cov infections may occur among horses (barlan et al., 2014) . serological studies of horses originating from spain and the united arab emirates gave negative results for mers-cov (alexandersen et al., 2014; meyer et al., 2015) , but it is still relevant to investigate for evidence of mers-cov in horses in areas endemic for mers-cov. we have tested for coronavirus infections in apparently healthy horses in saudi arabia and oman by testing nasal and rectal swabs by rt-pcr assays for mers-coronavirus and using a pan-coronavirus rt-pcr with potential to detect all coronaviruses, hitherto known and unknown. following the detection of ecov and hku23 coronavirus (hku23) by the pan-coronavirus rt-pcr, we tested the swab specimens with specific rt-pcr assays for ecov and hku23 and tested the sera with serological assays to detect ecov, bovine cov (bcov) (closely related to hku23), as well as mers-cov. (guy et al., 2000; zhang et al., 2007) and the bovine coronavirus (atcc brcv-ok-0514-2). viral rna was extracted from nasal and rectal swabs using the qiaamp viral rna minikit (qiagen, hilden, germany) following the manufacturer's instructions. rna extracts were tested for evidence of conserved coronavirus nucleic acid genetic sequences using previously reported rt-pcr assays (chu et al., 2014) , rtqpcr assay for mers-cov upe gene (corman et al., 2012) , rtqpcr assay for ecov (miszczak et al., 2014) , and a rtqpcr assay for hku23 reported below. rna was reverse transcribed in a 20 ll reaction mixture containing 19 first-strand buffer, 5 mm dtt, 0.5 mm deoxynucleotide triphosphates (dntp), 2.5 ng/ll random hexamers and 200 units of superscript iii (life technologies). the pan-coronavirus nested pcr targeted the rna-dependent rna polymerase (rdrp) gene of coronaviruses developed by us as previously described (chu et al., 2014) . using the cdna synthesized as described above, first round pcr was carried out using forward primer 5 0 -ggktgggaytaycckaartg-3 0 (position 15,287 of ecov strain nc99; genbank accession number ef446615) and reverse priof ecov strain nc99) and 5 ll of cdna as template for the reaction. pcr products with expected size of 658 bp were purified for dna sequencing using the forward and reverse primers. we developed a two-step real-time quantitative rt-pcr (rt-qpcr) assay for the detecting hku23 targeting n gene of the virus. virus rna was reverse transcribed as detailed above. real-time pcr the details of all rt-pcr and real-time pcr assays used in this study are summarized in table s1 . the primers used for sequencing were those used to generate the pcr products. dna amplicons were purified and sequenced in both forward and reverse directions with the pcr primers using bigdye all sera were tested using previously validated mers-cov spike pseudoparticle neutralization test (ppnt) (hemida et al., 2014; perera et al., 2013) , and the positive sera were confirmed using microneutralization (mn) test and plaque reduction neutralization tests (prnt) using live mers-cov strain emc in a biosafety level 3 containment laboratory (park et al., 2015) . selected sera were tested for antibodies to ecov and bcovs . the north american ecov-t a b l e 1 geographic location of specimens collected and animal management practices bcov is ubiquitous in cattle worldwide and is genetically and antigenically related with hku23, a coronavirus endemic in dromedaries (woo et al., 2014 (woo et al., , 2016 . antibody to bcov was tested using mn assay as described previously (park et al., 2015; perera et al., 2013) . serology for bcov and ecov was carried out with culture medium without foetal calf serum. known positive and negative control sera were included in all serology assays. (woo et al., 2014) . (table 4 ). in the confirmatory prnt which is considered the "gold standard" serological test for mers-cov, horse serum 9-2 reduced plaque numbers by 90% (prnt 90 ) up to a titre of 40. this serum was negative in ecov and bcov mn assays (table 4 ). this serum was from a racing horse stable in al-qassem and may represent a rare example of transmission of mers-cov to horses or cross-reaction with another yet undocumented coronavirus infecting horses. this horse was not known to frequently come into contact with camels. three of the other sera (serum id nos 5.35; 5.41; 2.12) positive in the mers-cov ppnt assay had detectable ecov mn antibody and one of these also had detectable bcov mn antibody (table 4 ). thus, these may well represent evidence of cross-reactive responses. t a b l e 5 cross-neutralization titres (denoted as reciprocal titres) for middle east respiratory coronavirus (mers-cov), bovine coronavirus (bcov) and equine coronavirus (ecov) in hyperimmune or naturally infected sera known to be positive for different coronaviruses nr460pig antiserum to porcine respiratory coronavirus 1,200 a <20 <20 <20 <20 nr2518 -guina pig antisera feline infectious peritonitis virus 2,000 a <20 <20 <20 <20 nr458pig antisera for porcine transmissible gastroenteritis virus 1,400 a <20 <20 <20 <20 ecov-negative sera 2 (horse, hong kong) not relevant <20 <20 <20 <20 bcov-positive camel serum #740293 e not relevant <20 nd 640 80 bcov-positive camel serum #467468 e not relevant <20 nd 80 <20 c99-10 -bcov antisera from guinea pig 20,480 b <20 <20 320 <20 bcov antisera from germ free bovine calf 580 bneutralisation titre <20 <20 1,280 80 nr456 -bcov antisera from gnotobiotic calf 10,000 a <20 <20 80 <20 nrc772 -rabbit antisera sars s protein high titre 640 <20 <20 <20 <20 mouse hepatitis virus(jhm strain) hyperimmunized mouse dam 1 1,778 cneutralisation titre <20 <20 <20 <20 mouse hepatitis virus(jhm strain) hyperimmunized mouse dam 2 363 cneutralisation titre <20 <20 <20 <20 mouse hepatitis virus(a59 strain)-infected mice 1,000 cneutralization titre <20 <20 <20 <20 nrc774antisera for sars coronavirus sero titre <10 <20 <20 <20 <20 nrc769 -rabbit antisera to sars s protein sero titre <10 <20 <20 <20 <20 gamma coronavirus nr2515-guina pig antiserum to infectious bronchitis virus 50,000 a <20 <20 <20 <20 7-3 <20 <20 7-4 <20 20 7-1 <20 <20 7-2 <20 <20 al-qassem 9 (racing) 9-a3 20 <20 9-a4 <20 40 9-a5 <20 <20 9-b1 <20 <20 9-c1 20 20 9-c2 80 640 9-e1 <20 <20 9-e2 <20 <20 qateef 10 (farming) 10-13 <20 80 10-14 <20 20 10-15 <20 80 10-16 20 160 11 (farming) 11-8 80 40 11-9 <20 320 11-11 <20 <20 11-12 <20 <20 11-13 <20 80 (continues) the extent of the serological cross-reactivity of mers-cov, ecov and bcov was systematically investigated. we did not have an isolate of hku23 virus available for serological testing and bcov was used as a virus that is genetically closely related to hku23 with pairwise amino acid similarity of 94.1% in the spike protein gene, the determinant of antigenic cross-reaction in neutralization tests (woo et al., 2014) . horse sera obtained from the usa reported to be strongly reactive by elisa (optical density ranging from 1.458 to 1.939) for ecov showed high titre (>1,280) for ecov in mn assay as expected, but also had high antibody titres (ranging from 160 to 1,280) in bcov mn tests (table 5) . as these horse sera were from horses in the "field," they may well have been exposed to multiple coronaviruses. thus, these results may reflect cross-reactivity or coinfection of us horses with ecov-and bcov-like viruses. two of these sera showed low titres (20) in the mers-cov ppnt assay but were negative in the more stringent mers-cov mn assay. as mers-cov is not circulating in the usa, these results suggest crossreactivity between mers-cov and ecov or related covs may occur, albeit at low titre. similarly, a bcov immune serum from an experimentally infected gnotobiotic calf showed detectable, but 16-fold reduced antibody titre with ecov but no cross-reaction with mers-cov. spike proteins of equine coronavirus and bovine coronavirus have an overall amino acid similarity of 80.8% and 73.1% similarity of the s1 region which contains the receptor-binding domain and neutralization epitopes. this probably explains the large difference in neutralization titres observed in table 5 . a dromedary serum from kazakhstan, a region known to be free of mers-cov activity (miguel et al., 2016) , had detectable antibody to bcov (probably reflecting hku23 or bcov infection) and also to ecov. we cannot conclude whether this reflects cross-reactivity or infection with bcov, hku23 and/or ecov. none of the beta coronavirus immune sera to sars-cov or mouse hepatitis virus, nor immune sera to alpha or gamma coronaviruses gave cross-reactions in bcov or ecov mn assays (table 5) . as had been previously reported, none of these dromedary sera cross-reacted with mers-cov (hemida et al., 2014) . fifty-four horse sera from saudi arabia and oman were selected to represent different collection locations, excluding those five sera previously reported to be mers-cov ppnt positive (table 6 ). these sera were seronegative for mers-cov (as expected); 40 (74%) of them had detectable mn antibody to ecov (titres ranging from <20 to 640; median 40), and 18 (33.3%) had mn antibodies to bcov (titres ranging from <20 to 160; median <20). the reciprocal geometric mean antibody titres (assigning sera with titres <20 the value of 10 for computational purposes) for ecov and bcov were 44 and 25.5, respectively. there were twenty-four sera with undetectable antibody to bcov with detectable ecov antibody titres ranging from 20 to 640. the titres to ecov were higher than or equal to titres to bcov, with two exceptions where the titre to bcov (20) was marginally higher than that with ecov (table 6 ). these results are compatible with the rt-pcr detection of ecov in rectal swabs in horses and probably suggest that ecov or a closely related virus is commonly infecting horses in many regions of the arabian peninsula. two of the five horses in whom ecov rna was detected by rtqpcr had low ecov mn antibody titres (20) while the other three had titres ranging from 160 to 640 (table 6 ). this may reflect virus shedding persists into convalescence or that re-infection of previously seropositive horses can occur. the rtqpcr detection of two hku23-like viruses indicates that hku23 which is common in dromedaries in the region is also infecting horses. in the absence of an hku23 isolate for use in neutralization tests and given the potential serological cross-reactivity between these three viruses, it is difficult to conclude how commonly equines are infected with hku23. the antibody titres to bcov, which is genetically closely related to hku23 in the spike protein gene, may suggest that bcov-or hku23-like virus infection of horses does occur in saudi arabia. mers-cov did not result in virus replication or seroconversion (adney et al., 2016) . in conclusion, the serological data suggest that lineage a betacoronaviruses are commonly infecting horses in saudi arabia and oman but antibody cross-reactions between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. rt-pcr detection of ecov and hku23 in equine swabs confirms the circulation of these two viruses in horses in saudi arabia. cocirculation of related viruses may provide potential for recombination, a potential means of generating genetic diversity and facilitating host jumps in coronaviruses. this is the first report of hku23 being detected in horses and the first detection of ecov in asia outside of japan. inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding middle east respiratory syndrome coronavirus antibody reactors among camels in dubai coronaviridae receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands characterization of a coronavirus isolated from a diarrheic foal seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses human infection with mers coronavirus after exposure to infected camels serologic assessment of possibility for mers-cov infection in equids absence of middle east respiratory syndrome coronavirus in camelids first detection of equine coronavirus (ecov) in europe epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in 2012 kinetics of serologic responses to mers coronavirus infection in humans seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt prevalence of equine coronavirus in nasal secretions from horses with fever and upper respiratory tract infection geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus (mers-cov) isolation and characterization of dromedary camel coronavirus uae-hku23 from dromedaries of the middle east: minimal serological cross-reactivity between mers coronavirus and dromedary camel coronavirus uae-hku23 novel betacoronavirus in dromedaries of the middle east supporting information additional supporting information may be found online in the supporting information tab for this article. how to cite this article key: cord-287761-73qgx58i authors: aly, mahmoud; elrobh, mohamed; alzayer, maha; aljuhani, sameera; balkhy, hanan title: occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update date: 2017-10-13 journal: plos one doi: 10.1371/journal.pone.0183850 sha: doc_id: 287761 cord_uid: 73qgx58i the emergence of the middle east respiratory syndrome coronavirus (mers-cov) infections has become a global issue of dire concerns. mers-cov infections have been identified in many countries all over the world whereas high level occurrences have been documented in the middle east and korea. mers-cov is mainly spreading across the geographical region of the middle east, especially in the arabian peninsula, while some imported sporadic cases were reported from the europe, north america, africa, and lately asia. the prevalence of mers-cov infections across the gulf corporation council (gcc) countries still remains unclear. therefore, the objective of the current study was to report the prevalence of mers-cov in the gcc countries and to also elucidate on its demographics in the arabian peninsula. to date, the world health organization (who) has reported 1,797 laboratory-confirmed cases of mers-cov infection since june 2012, involving 687 deaths in 27 different countries worldwide. within a time span of 4 years from june 2012 to july 2016, we collect samples form mers-cov infected individuals from national guard hospital, riyadh, and ministry of health saudi arabia and other gcc countries. our data comprise a total of 1550 cases (67.1% male and 32.9% female). the age-specific prevalence and distribution of mers-cov was as follow: <20 yrs (36 cases: 3.28%), 20–39 yrs (331 cases: 30.15%), 40–59 yrs (314 cases: 28.60%), and the highest-risk elderly group aged ≥60 yrs (417 cases: 37.98%). the case distribution among gcc countries was as follows: saudi arabia (1441 cases: 93%), kuwait (4 cases: 0.3%), bahrain (1 case: 0.1%), oman (8 cases: 0.5%), qatar (16 cases: 1.0%), and united arab emirates (80 cases: 5.2%). thus, mers-cov was found to be more prevalent in saudi arabia especially in riyadh, where 756 cases (52.4%) were the worst hit area of the country identified, followed by the western region makkah where 298 cases (20.6%) were recorded. this prevalence update indicates that the arabian peninsula, particularly saudi arabia, is the hardest hit region regarding the emerging mers-cov infections worldwide. gcc countries including saudi arabia now have the infrastructure in place that allows physicians and scientific community to identify and immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. immediately respond to the potential risks posed by new outbreaks of mers-cov infections in the region. given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of mers-cov in our region. the emergence of mers-cov dates back to july 2012 when an elderly patient of age 60 years died from an acute pneumonia in saudi arabia, and a new coronavirus strain was isolated from his lung tissue [1] . another case of acute respiratory disease was diagnosed in a 49-year old male in london who was from qatar and a new strain of coronavirus was isolated from this patient as well [2] . shortly after, the entire genome of the new coronavirus was sequenced and deposited in the genebank database under the number jx869059, kc164505.2. the phylogenetic analysis of the new virus genome revealed that homology of the nucleotide sequence between two cases was 99.5% and the isolates were closely related to bat coronavirus (bat-cov) which belongs to group 2c of β-coronavirus [3] . according to the recommendations by the international committee on taxonomy of viruses (ictv), the new coronavirus was named as 'middle east respiratory syndrome coronavirus' (mers-cov) [4] . although, initially reported from the middle east, mers-cov exported cases have also been observed worldwide. with regard to viral origin and transmission, the first case of mers-cov infection did not relate it to any particular contact with animals before the disease onset; however, other studies did link it to dromedary camels [5] [6] [7] [8] . beta-coronaviruses are strongly associated with bats serving as reservoir host, particularly, the african neoromicia bats were speculated to be the natural reservoir of mers-cov [9] [10] [11] [12] . notably, serological evidence suggests that mers-cov has been in the circulation for at least 2-3 decades in dromedary camels [13] . given that, the ancestral origin of mers-cov links it to african bats whereas, dromedary camels have been functioning as an intermediate host for this virus for a significantly long period of time [14] [15] [16] . health facilities, hospitals and households with mers patients are considered to be the epidemic centers of mers-cov outbreaks. mers-cov is mainly spreading across the geographical region of the middle east while only sporadic cases are reported in the europe, north america, africa, and lately asia. this may be because of the widespread population of dromedary camels in the middle east; however, the scientific proof of evidence that camel farms are a potential source of mers-cov infections still remains to be established. besides, the typical seasonality pattern is not seen in case of mers-cov infections and only one report links it to camel breeding season. the modes of mers-cov transmission by droplet, contact, or airborne are not yet confirmed as well and thus its transmission among animals and from animals to human and human to human remains unclear. there is also no documentation available regarding mers-cov transmission during airplane flights. therefore, standard infection prevention and control procedures are followed including droplet and airborne precautions. the viral incubation period is from 2 days to 2 weeks. the viral cytopathic effects clearly show prominent syncytium formation in humans as well as non-human primates. mers-cov targets directly the lower respiratory tract (pneumocytes) in dromedary camels and continues to replicate preferentially in the airway cells of the upper respiratory tract. the clinical manifestations of mers-cov infections represent a wide spectrum ranging from asymptomatic cases to the ones with severe respiratory indexes. according to the who, mers-cov infection is an acute respiratory infection involving pyrexia of 38˚c or more, cough with radiologic pulmonary presentation and also the history of the patients originating from or travel to the arabian peninsula and its neighboring countries within 10 days of symptoms. mers-cov cases have been identified as both community-and hospital-acquired, mainly among the aged population and in patients with multiple comorbidities such as acute pneumonia, upper respiratory tract infections, influenza-like illness, or asymptomatic infection(s) in children and immunocompromised hosts. moreover, the common extra-pulmonary symptoms include diarrhea and acute renal failure. the early clinical diagnostic changes include the impaired liver and renal functions, lymphopenia, leukopenia and thrombocytopenia whereas leukocytosis, and neutrophilia are linked to progressive infections. the gold standard for diagnosis is detection of viral rna by rt-pcr in compliance with the who guidelines for positive case criteria. the virus is found to be present in different diagnostic specimens such as the lower respiratory tract, sputum, endotracheal aspirate, bronchoalveolar lavage; upper respiratory tract, nasal or nasopharyngeal swabs, urine, feces, and blood. nevertheless, positive biopsy and autopsy tissue specimens still remain to be reported. direct or indirect contact seems to explain a part of the transmission kinetics observed between dromedary camels and humans. in the general population, transmission is rather inefficient (r0<0.7) and it was reported that mers-cov mortality rate is 35% [17] . however, once the virus is introduced into hospital setting with large numbers of susceptible patients at risk, the virus appears to be transmitted very efficiently among such vulnerable host populations. regarding viral evolution and natural reservoir, dromedary camels are the natural reservoir for mers-cov and given the multitude of different clades found in both dromedary camels and human outbreaks, it appears that virus evolution takes place in the reservoir host rather than in humans. the study objective was to report the prevalence of mers-cov infections in the gcc countries and to also investigate its demographics in the arabian peninsula. the data for the last 4 years were collected form the king abdulaziz medical city, riyadh, ksa. further data were collected from who s1 table and also from ministry of health portals of the gcc countries as follows: bahrain http://www.moh.gov.bh/, kuwait www.moh.gov.kw, oman www.moh.gov.om, qatar www.moph.gov.qa, united arab emirates http://www.moh. gov.ae/, and saudi arabia http://www.moh.gov.sa/. we also consulted the gcc countries' reports and websites for the incidence of mers-cov infections between june 2012 and july 2016. furthermore, we searched pubmed database for articles form gcc countries reporting mers-cov infections. epidemiological data including age, sex, symptoms, date of onset, and date of sampling were collected and entered into excel worksheets. descriptive analysis, frequencies and percentages were calculated using spss vr. 20 statistical software. between june 2012 and july 2016, a total of 1797 confirmed mers-cov cases were reported worldwide with a mortality rate of 38.2% (n = 687). the regional distributions of mers-cov were as follows: middle east had the highest number cases (88.4%), followed by asia (10.7%), europe (0.8%) and usa with only 2 cases officially reported (0.1%) the data are summarized in table 1 and fig 1a. one hundred fifty five patients out of the total 1797 confirmed cases (8.6%), reported their exposure to animals, of which, 130 out of 155 cases (83.9%) were exposed to camels, while 25 out of 155 (16.1%) stated exposure to other animals including sheep, cows and poultry ( table 2 ). despite the fact that 674 out of 1797 mers-cov cases (37.5%) were health careassociated infections and 284 out of 1797 cases (15.8%) involved contact with an infected family member, 147 cases (8.2%) were still reported with no exposure to any of the above. the exposure data were found missing for the remaining 537 (29.9%) patients. the distributions of mers-cov infections among 6 gulf countries are illustrated in table 3 and fig 1b. the majority of mers-cov infections (93%) reported between the time period from june 2012 to july 2016, were from saudi arabia. while, the remaining 5 gcc countries contributed only 7% of the cases with the distributions as follows: united arab emirates (5.0%), qatar (1.0%), oman (0.5%), kuwait (0.2%) and bahrain (0.06%). gender analysis shown in table 3 reveals that 60% of the patients were male and 31% were female. moreover, saudi arabia. next, we sought out the detailed demographic distributions of reported cases among 14 governorates in saudi arabia. as shown in table 4 and fig 1c, most of the cases (52%) were reported in al riyadh region, making it the worst hit area in the country followed by makkah (20%), ash sharqiyah (11%), al madinah (4%) and najran (3%). the remaining 9 regions together contributed to 5% of the cases. to date, cases are still logged from ksa and newly-diagnosed positive cases are on the rise. bahrain. to date, there was only one case reported from bahrain in manama region. herein, a 61-year-old saudi male was admitted on the 29 th of march, 2016 to a health care facility in bahrain for an unrelated medical condition. this person was later on tested as positive for mers-cov ( table 1) . state of kuwait. according to kuwait ministry of health, a total of 4 cases were confirmed as mers-cov infections. the first case was reported form the capital (kuwait city) on and an 85-year-old female from abu dhabi were traced to another confirmed mers-cov patient with no history of exposure to camels or other risk factors. finally, a 37-year-old expat male from abu dhabi developed symptoms on the 9 th of june, 2016 and was later tested positive for mers-cov. finally, we searched for the pattern of mers-cov infections over the months in order to identify seasonality relationship. as shown in fig 2, the average of the reported cases during this 4 years period shows that 60-80 cases are reported in the period between february and may, while about 90-100 are reported in august and september. in addition, a low point of infection occurs in the period of october-january and another one in june. the highest number of cases were reported overall during the summer time. over the past 4 years or so, increasing numbers of mers-cov infections have been reported from the middle eastern region [1] . herein, we present a prevalence update on the current status of mers-cov infections in the gcc countries. the data collected over a time period from june 2012 and july 2016 show that the highest number of cases (1441) were reported from saudi arabia (93%) among a total of 1797 cases reported worldwide. overall, a total of 1550 cases were reported only from the gcc region. the saudi arabian capital city of riyadh with 756/1441 (52.4%) cases remained the hardest hit area for mers-cov outbreaks. the incidence of mers-cov infections was found to be highest among the elderly population aged 60 yrs or above. moreover, the gender analysis showed that there were twice more number of males infected (871/1317) than females (446/1317). there is no evidence that mers-cov has gender predisposition [18] . the observed gender-related rates could be simply due to the higher probability of male exposure to camel population than females in the region [18, 19] . furthermore, over one third (37.5%) of mers-cov patients received intensive care among all hospitalized cases [20] . one could argue that the hot climate shared by arabian peninsula and sub-saharan african region could contribute to the spread of mers-cov infections across these geographical regions. although lesser in number, there are still numerous mers-cov infections recorded in saudi arabia during the winter time as compared with summer. the seasonality pattern analysis identifies a 2-phase annual cycle wherein the outbreaks occur during the winter and summer months. altogether, the summer time represents the peak season for mers-cov infections and transmission. the evolutionarily related bat virus might have undergone modifications and adaptation in order to be able to successfully infect and multiply in the camel as an intermediate host before transmission to the human host [21] . although, camel is a well-known animal that is widely colonized in the gulf region, it is also reared and maintained in other parts of the world. we speculate that there might be certain conditions or factors involved with regard to camel herding and shepherding exclusively in saudi arabia that would have facilitated and contributed to the survival of pathogen and fast spread of mers-cov infections from camels to humans across all over the country. there are some reports showed that human consumption of unpasteurized camel milk and or other camel products maybe a reason for the zoonotic transmission of mers-cov in the region [22] [23] [24] . on the other hand, there is strong evidence that mers-cov has been circulating in the dromedary camel population for more than 2 decades [25] . yet, the reason that first human infected case was identified in 2012 remains unclear. we found that mortality rates were higher among the elderly group for both genders which was also concordant with a previous study [26] . the possible explanation for the enhanced mortality in aged patients could be the presence of senescence-associated immune vulnerability in these individuals and suboptimal immune reactivity following a systemic challenge by exposure to mers-cov natural infection. other gulf countries show a few sporadic cases which may be due to the missing data that still have to be set straight or it could possibly be due to small size of camel populations is wide spread across vast geographical region. there may be still other factors involved that remain hidden at present but contribute significantly to the survival, transmission and pathogenesis of this relatively newly identified pathogen in this region of the world. notably, it appears as if coronaviruses are able to cause serious viral infections when transferred from their reservoir (wild bat) host to the human host as observed previously for ebola virus as well which was transmitted from wild bats to humans in africa. actually, many of mers-cov cases are initiated in rural areas and following hospitalization, further cases were reported. the majority of the mers-cov outbreak cases took place at health facilities; index cases are very crucial and they raise the question of the route of transmission of this zoonotic virus. this warrants caution that strict healthcare protocols and guidelines need to be followed and practiced by health care personnel in order to prevent the new outbreaks of mers-cov. in conclusion, mers-cov infections were reported to occur in saudi arabia during the whole year whereas the incidence of human outbreaks peaked in winter and summer months. the disease incidence was also highest among the elderly population aged 60 yrs and above. besides, the fact that majority of these cases were due to human to human interaction i.e. especially among the hospitalized icu patients and not due to camel to human transmission, the local health sectors need to be made aware to mandate implementation of effective control strategies and stringent compliance with better standards of health and hygiene nationwide. despite all whistle blowing efforts aimed at raising awareness of the magnitude of the problem at home, further efforts are still needed for proper treatment and care of mers-cov-infected patients in this country. last but not least, the availability detailed reports of each and every case of mers-cov infection globally, and the gcc region particularly will provide valuable information to the scientific community that may be used to track, contain, and eradicate this disease more effectively. supporting information s1 table. isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate infection control and mers-cov in health-care workers mers coronovirus has probably been present in bats for many years, research shows link to mers virus underscores bats' puzzling threat genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia mers coronavirus neutralizing antibodies in camels animal models of middle east respiratory syndrome coronavirus infection mers-cov an emerging viral zoonotic disease: three years after and counting. recent pat antiinfect drug discov middle east respiratory syndrome prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis middle east respiratory syndrome coronavirus: review of the current situation in the world mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir human infection with mers coronavirus after exposure to infected camels, saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels mers coronaviruses in dromedary camels a lesson learned from middle east respiratory syndrome (mers) in saudi arabia the authors thank the kaimrc members and infectious disease research unit for their help and support. this study was approved by the irb and supported by funds from king abdullah international medical research center kaimrc (grant #rc15/128). conceptualization: mahmoud aly. key: cord-278939-z6kiee09 authors: mani, janice s.; johnson, joel b.; steel, jason c.; broszczak, daniel a.; neilsen, paul m.; walsh, kerry b.; naiker, mani title: natural product-derived phytochemicals as potential agents against coronaviruses: a review date: 2020-04-30 journal: virus res doi: 10.1016/j.virusres.2020.197989 sha: doc_id: 278939 cord_uid: z6kiee09 coronaviruses are responsible for a growing economic, social and mortality burden, as the causative agent of diseases such as severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), avian infectious bronchitis virus (ibv) and covid-19. however, there is a lack of effective antiviral agents for many coronavirus strains. naturally existing compounds provide a wealth of chemical diversity, including antiviral activity, and thus may have utility as therapeutic agents against coronaviral infections. the pubmed database was searched for papers including the keywords coronavirus, sars or mers, as well as traditional medicine, herbal, remedy or plants, with 55 primary research articles identified. the overwhelming majority of publications focussed on polar compounds. compounds that show promise for the inhibition of coronavirus in humans include scutellarein, silvestrol, tryptanthrin, saikosaponin b(2), quercetin, myricetin, caffeic acid, psoralidin, isobavachalcone, and lectins such as griffithsin. other compounds such as lycorine may be suitable if a therapeutic level of antiviral activity can be achieved without exceeding toxic plasma concentrations. it was noted that the most promising small molecules identified as coronavirus inhibitors contained a conjugated fused ring structure with the majority being classified as being polyphenols. the dramatic change of events with the recent unprecedented coronavirus pandemic declared by the world health organisation (who) has prompted an exponential increase of scientific interest in coronaviruses globally. as of april 22 nd 2020, the pandemic has resulted in 2,553,112infections, with 177,286 deaths worldwide, which continues to drastically increase as we write (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). coronaviruses (covs) belong to the family coronaviridae, subfamily coronavirinae and are large (genome size 26-32 kb; wu et al., 2020a) , enveloped, positive-sense single-stranded ribonucleic acid (rna) viruses that can infect both animals and humans ( figure 1 ). based on their genotypic and serological characteristics, the viruses are subdivided into four genera: alpha-, beta-, gamma-, and deltacoronavirus (chu et al., 2020; lu et al., 2015) . at present, all identified covs that are capable of infecting humans belong to the first two genera. these include the alphacoronaviruses (αcovs) hcov-nl63 (human cov-nl63) and hcov-229e and the betacoronaviruses (βcovs) hcov-oc43 (human cov-oc43), hku1 (human cov), sars-cov (severe acute respiratory syndrome cov), and mers-cov (middle eastern respiratory syndrome cov) (lu et al., 2015) . in the past two decades there have been three epidemics caused by the betacovs, namely sars in 2002-03, mers in 2012 and covid-19, first identified in 2019 . sars-cov emerged in 2002-03 in southern china, causing a global threat and infecting more than 8,000 people, with approximately 800 fatalities recorded, largely in china and the surrounding regions (lu et al., 2015; paraskevis et al., 2020) . mers-cov emerged in the middle east, spreading to several countries to infect close to 2,300 individuals, resulting in 845 deaths as of july 2019 (world health organization, 2019) . the present cov pandemic resulting from sars-cov-2, which causes covid-19 (coronavirus disease), was identified in wuhan city, in the hubei province of southern mainland china on the 31 st december 2019 (sohrabi et al., 2020) . the genome of sars-cov-2 is approximately 70% identical to that of sars-cov , hence leading to its current name. the major druggable targets of sars-cov-2 include 3-chymotrypsin-like protease (3clpro), papain like protease (plpro), rna-dependent rna polymerase, and spike (s) proteins (wu et al., 2020b) . the s proteins interact directly with human angiotensin-converting enzyme (ace) 2, allowing the virus to enter the cells. at present, no preventive vaccines or established antiviral therapies are available for coronaviruses (sohrabi et al., 2020) . however, several synthetic compounds have shown promise, including hydroxychloroquine and choloroquine phosphate (cortegiani et al., 2020; gao et al., 2020) , which act through several mechanisms, including alkalisation of the host cell phagolysosomes. newer antiviral medications such as lopinavir (yao et al., 2020) , remdesivir (holshue et al., 2020; wang et al., 2020) , and arbidol (khamitov et al., 2008) also show promise. other suggested treatment options include lopinavir/ritonavir, nucleoside analogues, neuraminidase inhibitors, and peptide ek1 (lu, 2020) . a detailed list of current and planned clinical trials investigating various drugs for the treatment of sars-cov-2 was provided by pang et al. (2020) , with updated results available from clinicaltrials.gov (2020). in addition, traditional herbal medicines and purified natural products may guide the development of novel antiviral drugs. in other words, more efficient drugs can often be designed based on the structure of natural compounds that exhibit the desired activity. classic examples of this drug discovery pathway include emetine, an isoquinoline alkaloid isolated from cephaelis ipecacuanha and used as an amoebicidal drug; quinine, derived from the bark of cinchona trees; and numerous other drugs modified from natural compounds, such aspirin, morphine and paclitaxel, an antineoplastic drug used for the treatment of cancer (ganjhu et al., 2015) . indeed, half of all drugs approved between 1981 and 2014 were derived from or mimicked a natural compound (newman and cragg, 2016) . furthermore, in the current outbreak of covid-19, many patients appear to be turning to complementary or traditional medicinal therapies, albeit using them almost exclusively in conjunction with western medicine. for example, one study suggested that almost 92 % of 135 hospitalised patients in northeast chonqing (china) received traditional chinese medicine in addition to western medicine . however, based on the many studies conducted on this topic, it is hard to separate the potential effects of, and interaction between, traditional chinese herbal medicine and western medicine. recent reviews have suggested that traditional chinese medicine could be used for the prevention or treatment (yang et al., 2020a ) of covid-19; while still acknowledging that many studies involving clinical trials are poorly designed or controlled, and the choice of treatments is largely empirically based. as previous work has highlighted the potential of traditional chinese medicines as a source of potential novel drugs (ling, 2020) , we have not included details on such studies investigating the antiviral activity of remedies comprising portions of numerous plant species in this review. rather, our aim is to collate data on the broad spectrum of natural phytochemicals from individual plant species that may have therapeutic potential. naturally occurring antiviral agents acting against general coronaviruses were briefly reviewed by lin et al. (2014) six years ago, while more recent reviews by pang et al. (2020) and lu (2020) on therapies for covid-19 made only brief mention of natural therapeutics and did not explore the active compounds or their mechanism of action. in light of the current covid-19 pandemic, this review aims to gather and consolidate information on extracts and compound(s) derived from natural products which show potential antiviral bioactivity for the inhibition of coronaviruses. it is hoped that the information presented may guide the naturally-derived drug discovery process in finding a treatment for sars-cov-2. the pubmed database (www.ncbi.nlm.nih.gov/pubmed/) was used to locate articles including the following combination of terms: (coronavirus, sars or mers) and (traditional medicine, herbal, remedy or plants). papers primarily focussed on the antiviral activity of prepared chinese traditional medicines, which typically comprise multiple plant species, were considered out of scope of this review. all articles up to and including 25 march 2020 were considered, yielding a total of 659 results. two of the authors independently screened the results and identified relevant articles, yielding a total of 35 primary articles on human coronaviruses and 22 on animal coronaviruses were found to be pertinent and thus included in this review (total = 58 papers). of these, two papers (6%) were on sars. the majority of studies on human coronaviruses (69%; n=24) included sars-cov, with only 3 (9%) including mers-cov and 8 (23%) other human coronaviruses. it should be noted that one study included both sars-cov and mers-cov (o'keefe et al., 2010) while another included both mers-cov and hcov-229e (müller et al., 2018) , hence these percentages do not add to 100%. table 1 summarises the studies reporting the inhibition of various human coronavirus strains using compounds derived from plant sources. the table is arranged by viral strain in order to better compare the bioactivity of compounds from different studies upon the same viral genotypes. where identified, the key compounds responsible for the antiviral activity and their identified mechanisms of action are presented. it should be noted the term ec50 (effective concentration) applies to cell-based assays, while ic50 (inhibitory concentration) applies to enzyme-or biochemical-based assays. few studies report on sars-cov-2, as expected given the short time since its emergence. however, a number of studies report on use of computer modelling for screening purposes (liu and zhou, 2005; lung et al., 2020; toney et al., 2004; wang et al., 2007; zhang et al., 2020) . typically, these models determine the free binding of energy between a ligand and a receptor (forli et al., 2016) , with a lower free binding energy indicating a stronger bond between the ligand and receptor. although obtaining consistent results via different modelling approaches can be challenging (aldeghi et al., 2016) , computer modelling nevertheless allows for comparison of the relative binding affinity of bank of molecules toward the receptor in question. in addition to reducing the high costs and length of time associated with physically screening large banks of compounds or plant extracts for bioactivity (chen et al., 2017) , the speed and versatility of this method may be particularly valuable for rapidly finding a potent inhibitor of sars-cov-2. compounds that are highlighted through this method can then be forwarded on cell-based assays to assess their in vitro effectiveness and toxicity, before continuing to animal and clinical trials. lung et al. (2020) virtually screened 83 compounds found in chinese traditional medicines for activity against the rna-dependent rna polymerase of sars-cov-2, identifying theaflavin, an antioxidant polyphenol, as a potential inhibitor. similarly, zhang et al. (2020) virtually screened 115 compounds found in chinese traditional medicines, highlighting 13 for further studies. several of these were naturally occurring polyphenolic compounds such as quercetin and kaempferol, which have already received considerable interest for the treatment of other disease types (cassidy et al., 2019; khan et al., 2019; tome-carneiro and visioli, 2016) . given the relatively large amount of research that has been performed searching for inhibitors of sars-cov, antiviral agents that successfully inhibit this viral strain may provide a good starting point for identifying compounds that are active against sars-cov-2. several authors have utilised virtual computer docking models to screen for potential compounds that could bind to and inhibit key proteins present in sars-cov (liu and zhou, 2005; toney et al., 2004; wang et al., 2007) , highlighting the potential antiviral activity of compounds such as sabadinine and aurantiamide acetate. compounds may be screened against a number of binding sites in order to test for potential inhibition of coronaviruses; the main sites that are typically used are the chymotrypsinlike protease (3clpro), papain like protease (plpro), spike proteins and rna-dependent rna polymerase. several large in vitro screening studies searching for inhibitory activity of naturally occurring compounds against sars-cov have been performed, mainly on chinese medicinal herbs (li et al., 2005; wang et al., 2003) . while the results highlight the potential of selected plant extracts against sars-cov, they also demonstrate that such work can be akin to searching for a 'needle in a haystack'. for example, li et al. (2005) screened over 200 ethanol/chloroform extracts of chinese medicinal herbs and found only four (lycoris radiata, artemisia annua, pyrrosia lingua and lindera aggregata) with moderate to high antiviral activity using a cpe assay (ec50 values ranging from 2.4 ± 0.2 to 88.2 ± 7.7 µg/ml). of these, a single compound (lycorine) from one plant species (l. radiata) was earmarked as a potential drug candidate against sars-cov. the antiviral efficacy of lycorine was quite high (ec50 of 15.7 ± 1.2 nm), with a selectivity index greater than 900. although the authors did not make mention of this fact, lycorine can cause toxic effects at low dosage levels (around 1 mg/kg in dogs) (kretzing et al., 2011) , hence great caution would be required for further development of this compound as a drug candidate. yu et al. (2012) screened the activity of a library of 64 naturally occurring compounds against sars-cov helicase, which plays a key role in the viral genome replication, transcription, and translation. the polyphenolics myricetin and scutellarein ( figure 2 ) were identified as the most promising candidates (ic50 values of 2.71 ± 0.19 and 0.86 ± 0.48 µm, respectively). although the antiviral activity of the compounds was not assessed in cell-based assays, the authors did report that neither compound was toxic to normal (non-tumorigenic) breast epithelial cells. myricetin is found in reasonably high concentrations in fruits such as cranberry (häkkinen et al., 1999) as well as in several vegetables such as calamus scipionum and garlic (miean and mohamed, 2001) . scutellarein was isolated from scutellaria baicalensis (chinese skullcap), which has been traditionally used in the treatment of inflammation and respiratory infections, amongst other uses (zhao et al., 2016) . both compounds were found to inhibit sars-cov helicase (nsp13) through the inhibition of atpase activity, but did not directly inhibit helicase activity. this appeared to be the only publication identifying naturally occurring compounds as inhibitors of sars-cov helicase. the need to isolate and synthesise more active agents as part of the development pipeline was highlighted in a study by runfeng et al. (2020) . the authors reported that the chinese herbal medicine lianhuaqingwen (comprised of a mixture of plant species) showed antiviral activity against sars-cov-2; however, the ec50 was quite high (~411 µg/ml). for comparison, the commercial drug remdesivir showed an ec50 of 0.651 µm (approx. 0.39 µg/ml) using the same assay (runfeng et al., 2020) . this also underscores the potential lack of potency in some traditional medicines promoted for the treatment of coronavirus symptoms. one family of compounds that demonstrate antiviral activity across a number of studies is the polyphenols. for example, quercetin showed an ic50 of 8.6 ± 3.2 µm against sars-cov plpro (park et al., 2017) . no cell-based assay of antiviral activity was performed. quercetin ( figure 2 ) is a flavonoid found in many foods, but in particularly high levels in certain berries and herbs (justesen and knuthsen, 2001; kaack and austed, 1998) . as previously mentioned (section 3.2.2), the structurally similar polyphenolics myricetin and scutellarein ( figure 2 ) display reasonable levels of inhibitory activity against sars-cov helicase (yu et al., 2012) . bioassay-guided fractionation of the ethanolic extract obtained from psoralea corylifolia seeds has also identified polyphenolics as the bioactive compounds responsible for the activity of this plant species against sars-cov plpro (kim et al., 2014) . furthermore, six phenolic phytochemicals were isolated from the ethanolic extracts -identified as bavachinin, neobavaisoflavone, isobavachalcone, 4'-o-methylbavachalcone, psoralidin and corylifol a -with their antiviral activity varying widely (ic50 values between 4.2 -38.4 µm). again, no cell-based antiviral assays were performed. isobavachalcone and psoralidin (figure 2 ) showed the greatest antiviral activity, with both found to be mixed, reversible inhibitors of plpro through a type i mechanism (i.e. preferentially bind to the free enzyme, rather than the enzyme substrate complex) (kim et al., 2014) . plant lectins, which are proteins that can bind specifically and reversibly to carbohydrate groups (mitchell et al., 2017) , are another group of naturally occurring compounds that may inhibit sars-cov. lectins have shown promise as antiviral agents against viruses such as influenza and herpes simplex virus (hwang et al., 2020) , as well as ebola (covés-datson et al., 2019; michelow et al., 2011) . remarkably, elevating the plasma levels of recombinant human mannose-binding lectin in mice allowed them to survive otherwise fatal ebola infections (michelow et al., 2011) . keyaerts et al. (2007) screened the activity of a broad range of plant lectins (33 in total) against sars-cov using a cytopathicity effect (cpe) assay, finding ec50 values as low as 0.45 ± 0.08 µg/ml for lycoris radiata agglutinin. although the exact mechanism of action was not determined, activity at the stage of viral attachment or the end of the infectious viral cycle were deemed to be the most likely targets. in clinical trials, other lectins have demonstrated reasonable to good tolerability (petersen et al., 2006) , hence with further testing, they may prove to be one of the more promising classes of naturally derived compound(s) for the treatment of sars-cov-2 and other coronavirus infections. whilst many natural derived compounds show considerable promise for the inhibition of sars-cov and other human coronaviruses, few approach the level of efficacy and/or selectivity required for commercial drugs. for example, the calpain inhibitor mdl28170 has been shown to have an ic50 value of just 2.5 nm (~1 ng/ml) in its activity against the sars-cov enzyme cathepsin-l (simmons et al., 2005) . cathepsin-l is a cysteine protease which is an important lysosomal endopeptidase enzyme involved in the initiation of protein degradation (sudhan and siemann, 2015) and mediates entry and infection of sars-cov in its host cells (huang et al., 2005) . chemical modification of naturally-derived compounds may be required to increase the potency of their antiviral activity to levels suitable for therapeutic application. for the drug discovery process, beginning with the understanding of the structural conformity (i.e. structure including potential isomers) of the naturally derived compound(s) can reduce timelines and greatly increase the chance of finding effective viral inhibitors. working on this principle, hoever et al. (2005) found that modification of glycyrrhizin, naturally found in liquorice and previously used for the treatment of sars-cov (haiying et al., 2003) , could increase its viral inhibition activity. for example, adding 2-acetamido-β-d-glucopyranosylamine to the glycoside chain of glycyrrhizin increased its antiviral activity in a cpe assay by 10-fold (decrease in ec50 from 365 ± 12 µm to 40 ± 13 µm), through increased attraction to the s proteins. amides and conjugates with amino acid residues and free cooh increased activity of glycyrrhizin by up to 70-fold (ec50 for cpe assay ranging from 5 ± 3 µm to 139 ± 20 µm), albeit with significantly reduced selectivity. cho et al. (2013) demonstrated that tomentins a-e (all naturally occurring compounds) showed viral inhibition activity against sars-cov greater as compared to their non-geranylated precursor compounds. similarly, quercetin-7-rhamnoside ( figure 2 ) demonstrates over 100 times higher antiviral activity against an animal coronavirus strain compared to its parent compound, quercetin (choi et al., 2009) . while the examples of modifications presented here vary, it should not be considered that substitutions are added in a random fashion, but rather performed in the context of targeting a specific receptor or biochemical pathway. for example, the addition of glycosides and specific amino acid residues to glycyrrhizin was performed with the intent of increasing its affinity for the highly glycosylated spike proteins of sars-cov. with this in mind, future studies that identify an effective natural inhibitor of coronavirus should also consider the design and testing of potential modifications or synthetic derivatives that could increase its desired activity. a difference in the efficacy of natural therapeutics, mirroring that observed for commercially/synthetically available drugs, has been observed between coronavirus species/strains. for example, park et al. (2017) found that the compound 3'-(3-methylbut-2-enyl)-3',4,7trihydroxyflavane, isolated from broussonetia papyrifera, showed good inhibition of plpro from sars-cov (ic50 of 3.7 ± 1.6 µm), but not against plpro from mers-cov (ic50 of 112.5 ± 7.3 µm). in contrast, the polyphenolics kazinol f and broussochalcone a isolated from the same species showed better efficacy against mers-cov plpro. similarly, o'keefe et al. (2010) found that the efficacy of the compound griffithsin against sars-cov was highest for the urbani and tor-ii strains (ec50 of 0.61 µg/ml using a cpe inhibition assay) and lowest for the frank strain (ec50 of 1.19 µg/ml). griffithsin, isolated from the red alga griffithsia sp., is a lectin possessing three large identical carbohydratebinding domains orientated as an equatorial triangle, which enable multivalent binding (o'keefe et al., 2010) . although the source of these observed differences in the inhibition activity of griffithsin against different sars-cov strains was not explored by o' keefe et al., (2010) , it may be attributable to genomic differences in the amino acid sequences of the spike proteins between sars-cov strains, leading to varying multivalent interactions with the carbohydrates-binding domains and thus differing affinity for binding to the spike proteins. nevertheless, these studies highlight the importance of conducting in vitro tests of potential naturally-derived therapeutics on sars-cov-2 prior to animal or clinical trials. only a handful of studies have investigated the potential of natural products as therapeutic agents against mers-cov. silvestrol (figure 2) , a phytochemical from aglaia sp., was found to be a more potent inhibitor of mers-cov replication (ec50 of 1.3 nm) (müller et al., 2018) . however, no cell-based antiviral assays were performed. silvestrol is a specific inhibitor of rna helicase eif4a, thus inhibiting viral replication and leading to the inhibition of expression of cov proteins and preventing the formation of replication/transcription complexes (müller et al., 2018) . griffithsin (figure 2) , a 12.7 kda lectin found in the griffithsia genus (red algae), is one of the most promising inhibitors of mers-cov. it comprises three carbohydrate-binding domains which allow it to bind specifically to glycans on cov protein spikes and inhibit viral attachment to host cells, with high potency found in in vitro trials against mers-cov (ec50 of ~0.125 µm) (o'keefe et al., 2010) and several hcov strains (ec50 of 0.0032-0.33 µm) (millet et al., 2016) . griffithsin also appears to have a low systematic toxicity, with its specificity index against hcov cells (compared to human colorectal adenocarcinoma or fibroblast cell lines) has been estimated at between 30 -3100 (o' keefe et al., 2010) , hence showing the potential to be considered as one of the prime candidates for animal and clinical trials against sars-cov-2. a recent study highlighted the potential anti-hcov-nl63 activity of the methanolic extract of strobilanthes cusia leaf and its major phytochemicals (tsai et al., 2020) . the s. cusia extract effectively reduced virus yield (ec50 = 0.64 µg/ml) in cells infected with hcov-nl63 in a dose-dependent manner. of the six key compounds isolated, purified and identified via nmr spectroscopy, two exhibited the most potent antiviral activity against hcov, namely tryptanthrin ( figure 2) , a natural alkaloid containing the basic indoloquinazoline moiety, and indigodole b (5ar-ethyltryptanthrin), an indole alkaloid derivative. the ec50 values against virus yield from infected cells were 1.52 µm and 2.60 µm for tryptanthrin and indigodole b, respectively. the increased antiviral activity of tryptanthrin in comparison to indigodole b may result from the double bond at c5a in the former, as compared to the additional ethyl moiety in the latter. this highlights that for compounds based on a tryptanthrin structural conformity, addition of a double bond in the quinazoline ring could significantly increase their antiviral activity. tryptanthrin has previously been found to have a broad spectrum of biological activities including anticancer, anti-inflammatory, antiprotozoal, antiallergic, antioxidant and antimicrobial action (kaur et al., 2017) . through manipulation of varying modes of time-ofaddition/removal assay, it was found that tryptanthrin prevented the early and late stages of hcov-nl63 replication, particularly by blocking the viral rna genome synthesis and papain-like protease 2 activity, and inhibiting the post-entry stage of hcov replication (tsai et al., 2020) . intriguingly, both tryptanthrin (ec50 = 0.06 µm) and indigodole b (ec50 = 2.09 µm) exhibited strong viricidal activity directly against hcov-nl63. as the hcov-nl63 spike protein (s protein) targets the ace2 receptor, showing a highly conserved sequence and structural similarity to sars-cov and sars-cov-2 (letko et al., 2020) , tryptanthrin has the potential to be explored as a possible bioactive agent against sars-cov-2 and other human coronaviruses. another recent study on hcov-oc43 focussed on the antiviral activities of the bis-benzylisoquinoline alkaloids tetrandrine, fangchinoline, and cepharanthine (cpe assay ec50 values of 0.33 ± 0.03, 1.01 ± 0.07 and 0.83 ± 0.07 µm, respectively) (kim et al., 2019) . these compounds were the primary bioactive phytochemicals identified in stephania tetrandra and related species. all compounds were found to inhibit virus-induced cell death, through suppressing viral replication, expression of viral s and n proteins (nucleocapsid protein), and the virus-induced host response. however, the effective inhibitory concentrations were not reported for the inhibition of viral protein expression or host response. in addition, tetrandrine activated the p38mapk pathway in mrc-5 cells, improving the aforementioned host response. weng et al. (2019) tested the ethanolic extracts of sambucus formosana (elderberry) stems against the human coronavirus strain hcov-nl63, finding quite high efficacy (ec50 of 1.17 ± 0.75 µg/ml) for viral yield reduction. with further investigation of the phenolic composition of the extracts and antiviral assays performed on the main individual phenolic acids present, caffeic acid ( figure 2 ) was identified as the most potent compound present (ec50 of 3.54 ± 0.77 µm; or ~0.64 ± 0.14 µg/ml). although no specific binding sites were identified, caffeic acid was found to inhibit the attachment of hcov to host cells, indicating potential binding to s proteins. caffeic acid has also been found to inhibit other viruses such as hepatitis b (wang et al., 2009) . notably, extracts of sambucus nigra (black elderberry), another species in the same genus as s. formosana, have been commercialised for the treatment of cold and flu symptoms. therefore, it is likely that extracts from s. formosana would prove similarly nontoxic and suitable for human use, although clinical trials would be required to confirm this. nevertheless, the bioavailability/delivery mechanism of such extracts would need to be considered in order to reach therapeutic plasma concentrations for viral inhibition (wittemer et al., 2005) . cheng et al. (2006) examined the anticoronaviral activity of saikosaponins (a, b2, c and d) and their mode of action against hcov-229e in vitro. saikosaponins represent a group of pentacyclic triterpenoid derivatives, usually present as glycosides, that have been isolated from medicinal plants such as bupleurum spp., heteromorpha spp. and scrophularia scorodonia, previously found to possess efficacy against several viruses, including human immunodeficiency virus (hiv) (chiang et al., 2003; ushio and abe, 1992) . all saikosaponins tested showed good to moderate anti-coronavirus activity, with saikosaponin b2 (figure 2) showing the greatest potency (ec50 of 1.7 ± 0.1 µm). subsequent timeof-addition studies indicated that saikosaponin b2 inhibited viral attachment and penetration. interestingly, the same compound has been found to inhibit hepatitis c entry (lin et al., 2015) as well as multidrug resistance-associated drug transporters present on the cell surface (zhao et al., 2019) , indicating that it has potential to display a broad spectrum of bioactivity. this may be advantageous in developing nations where hepatitis c prevalence is high (pawlotsky, 2014) . finally, two compounds that have shown efficacy against mers-cov (section 3.3) also show promise against hcov. müller et al. (2018) found that silvestrol (figure 2 ) inhibited the translation of hcov-229e proteins, with an ec50 of 3 nm. a follow-up study confirmed that silvestrol can inhibit hcov-229e in an ex vivo bronchial epithelial cell system, with its mechanism of action being the specific inhibition of rna helicase eif4a (müller et al., 2020) . another compound active against mers-cov, griffithsin, also has shown great efficacy against several hcov strains (ec50 of 0.0032-0.33 µm) (millet et al., 2016) . this again underscores the prospect of exploring griffithsin for antiviral activity against sar-cov-2. animal coronavirus strains are responsible for severe morbidity events across a wide range of domestic animals and livestock, incurring major economic demise worldwide (jackwood et al., 2010; lelesius et al., 2019; mccutcheon et al., 1995) . the genomic diversity, coupled with the ability of coronaviruses to rapidly adapt and mutate, presents unique challenges in the development of novel antiviral agents, hence exploring alternative methods of controlling these viruses could potentially be effective across many or all serotypes. as natural phytochemicals have been shown to have activity across a wide range of viral pathogens (chen et al., 2014) , they form the basis of the studies reviewed here. the majority of plant-derived compounds considered in this review are direct-targeting antivirals, acting through direct inhibition of some part of the virus, such as proteases or spike proteins. for example, silvestrol prevents viral replication occurring through the specific inhibition of rna helicase eif4a (müller et al., 2018) , while griffithsin binds directly to the s protein, preventing viral entry to the host cell (o'keefe et al., 2010) . however, host-targeting antivirals form another important group of antiviral compounds. for example, extracts of cinnamomi sp. inhibit the clathrindependent endocytosis pathway, thus preventing viral entry to the host cells (zhuang et al., 2009) . other host-targeting antiviral compounds may stimulate the immune response (lau et al., 2008) . table 2 presents a summary of studies reporting anti-viral activity of plant-derived isolates against a range of animal coronavirus strains. where available, the key compounds responsible for the antiviral activity and their mechanism of action are provided. the antiviral activity of extracts from plant species against the avian ibv viral strains have been extensively studied (chen et al., 2014; jackwood et al., 2010; lelesius et al., 2019; li et al., 2011; nguyen et al., 2015; yang et al., 2010; yang et al., 2011; yin et al., 2011) . considering all studies on avian ibv that have established the mechanism of viral inhibition, the main mechanisms of action appear to be through viral envelope disruption or interference with the spike protein (table 2) . a notable exception are the terpenoid compounds 1,8-cineole, (-)-α-pinene and (-)-β-pinene, which bind to the ibv nucleocapsid protein (n-protein) (yang et al., 2010; yang et al., 2011) , inhibiting its interaction with the viral genomic rna and breaking the ibv replication cycle. these observations were reinforced by conformational models of the terpenoids binding to the active site at the n terminus of the n protein, which indicated that these compounds have the potential to bind strongly to five amino acid residues at this location (tyra92, proa134, phea137, aspa138 and tyra140) (yang et al., 2010; yang et al., 2011) . as these residues are highly conserved between ibv strains (yang et al., 2010) , these terpenoids would be expected to show high efficacy against most or all ibv strains, making them a sensible target for further research. nevertheless, the envelope protein (e protein) of avian ibv remains the key target for most researchers. the coronavirus e protein is integral to many stages of the viral life cycle, including assembly, budding, envelope formation, and pathogenesis (schoeman and fielding, 2019) . interestingly, chen et al. (2014) reported the inactivation of two distinct enveloped viruses (avian ibv and influenza) following treatment with sambucus nigra extract, suggesting that s. nigra extract may have the potential to display a broad spectrum of antiviral effects against many other enveloped viruses. although the authors raised the possibility of synergistic action of inhibitory compounds within the extract, no fractionation, identification or characterisation of the components was performed. however, flavonoids or lectins were suggested as the likely aetiological agents of the antiviral activity. feline coronavirus is another coronavirus in the alphacoronavirus group, causing a fatal disease in cats with no effective antiviral treatments available. in one study, galanthus nivalis agglutinin, another lectin, was identified as a potent inhibitor of feline coronavirus (fcov) (hsieh et al., 2010) . with an ec50 of just 0.0088 nm and a high selectivity index (>218), this carbohydrate-binding protein outperformed all synthetic antiviral agents tested for comparison purposes. for example, the protease inhibitor nelfinavir displayed an ec50 of 8.19 µm and a selectivity index of just 1.4. as with other agglutinins, galanthus nivalis agglutinin binds to the spike and membrane proteins of fcov, preventing their attachment to the host cells. mcdonagh et al. (2014) conducted a screening study against 19 compounds, focusing on those demonstrated to have previous antiviral effects against coronaviruses or other rna viruses. several naturally occurring compounds were included in this study, such as quercetin, curcumin, rutin, glycyrrhizic acid, hesperidin, hesperitin, baicalin and artemisinin. however, none of these compounds reached ec50 at the concentrations tested. glycyrrhizic acid was the most promising, causing a 26.7% reduction in cpe at a concentration of 25 µm. as only a single concentration was tested for each compound (ranging from 10-50 µm, depending on the compound), further investigation of these compounds against fcov is required. theerawatanasirikul et al. (2020) adopted a computer-aided approach to their screening study against feline infectious peritonitis virus (fipv), a mutated form of the parental enteric form of fcov. firstly, compounds from a chemical library were virtually screened for potential binding to the protease 3cl. the 15 most promising compounds were then evaluated in vitro using a protease inhibitor assay against fipv 3cl pro . of the naturally occurring compounds tested, the lowest ic50 values were shown by 7-methylluteolin (28.5 ± 4.2 µm) and stictic acid (29.4 ± 4.6 µm). quercetin 7-rhamnoside ( figure 2 ) also showed moderate inhibition (ic50 of 77.2 ± 13.8 µm); however 7-benzyl luteolin and steviol showed no inhibition (ic50 > 500 µm). subsequent testing of the active compounds using a cytopathic effect (cpe) assay indicated that only stictic acid protected cells from viral-induced death (ec50 for previral entry treatment of 16.24 ± 1.33 µm; selectivity index of 23). porcine endemic diarrhoea virus is another serious coronavirus from the alphacoronavirus group. several studies have investigated the activity of phytochemicals against murine coronaviruses (strain mhv-a59), the most extensive of which were performed by kim et al. (2008) and kim et al. (2010) . nevertheless, none of the studies of this viral strain have conclusively managed to determine the specific compounds responsible for viral inhibition, but rather suggested possible compounds or classes of compounds based on their abundance in the extracts tested. the low inhibitory concentrations (<1 µg/ml) of extracts obtained from sophorae sp., acanthopanacis sp. and torilis sp. appear to show significant potential. in particular, the high viral specificity (si = 696) of sophorae sp. root extracts suggests that it could be considered as a prime candidate for future studies on the screening and isolation of compounds from the aforementioned species. the authors suggested that the antiviral activity of these three species are likely to be occurring through inhibition of rnadependent rna polymerase or other protease activity. six studies considered natural agents active against porcine endemic diarrhea virus (pedv), with the majority of these identifying the compounds with for their antiviral activity. in particular, the anti-pedv activity of griffithsin and quercetin ( figure 2 ) and its derivatives deserve particular discussion, given that these compounds have also been found to have antiviral activity against human covs (millet et al., 2016; o'keefe et al., 2010; wen et al., 2011) . notably, quercetin was also one of the compounds shortlisted by zhang et al. (2020) in their virtual screening of inhibitors of sars-cov-2 proteases. against pedv, quercetin-7-rhamnoside, a disaccharide glucoside, provided 50 % inhibition of viral activity at a concentration of just 0.03 µm, approximately 187 times lower than quercetin alone (choi et al., 2009) . significantly, the specificity of quercetin-7-rhamnoside was exceptionally high (si = 7143), indicating its potential for application in future animal or clinical trials. this study again highlights the importance of considering all possible structural conformities of a compound, including glycosides, in order to identify the most bioactive chemical species. although the mechanism of action was not determined for quercetin or quercetin-7-rhamnoside against pedv, previous computer modelling work has indicated that quercetin binds to and inhibits the sars-cov proteases plpro and 3clpro . as previously determined, griffithsin binds to the spike protein of cov, preventing attachment to host cells (millet et al., 2016; o'keefe et al., 2010) . in general, most authors recommend testing multiple coronavirus strains when searching for antiviral activity in naturally occurring compounds. similarly important is determining their specific mechanism of action. as highlighted by several studies, a selection of compounds that are active against animal coronaviruses are also active against human coronavirus strains (e.g. griffithsin, quercetin). this underscores the potential of utilising compounds with identified activity against animal coronaviruses to guide the discovery of drugs against human coronaviruses, in addition to identifying drugs for the treatment of economically significant animal coronaviruses such as pedv and avian ibv. across both human and animal cov strains, a clear trend toward the use of chemically polar compounds is evident (figure 2) . of the 30 studies that specified the solvent extraction protocols used, ethanol or an ethanol/water combination was the most commonly used (50 % of all studies), followed by methanol or a methanol/water combination (27 %). a further 17 % of relevant literature used a water-based extraction protocol, with only three studies using relatively non-polar solvents (diaminopropane, chloroform and acetone). this is consistent with a wide body of research indicating that relatively polar extracted fractions generally contain a greater level of bioactive and antimicrobial compounds compared to their nonpolar counterparts (han et al., 2007; tian et al., 2009; wigmore et al., 2016) . this may also be indicative of increased bioactivity of highly polar glycosylated compounds, similar to the vast difference in antiviral activity observed between quercetin and quercetin-7rhamnoside (choi et al., 2009 ). in the case of compounds being administered orally, more polar compounds are subject to compartmentalisation within the body, reducing their rate of elimination. however, the potential chemical changes that could occur as a result of highly acidic stomach conditions and the level of absorption in the intestine would need to be considered individually for each compound. naturally occurring phytochemicals provide a valuable and powerful resource of chemical compounds displaying antiviral properties. further chemical modification of these structures, guided by computerbased docking simulations, may also increase their potency and/or selectivity. some of the key compounds that show promise for the treatment of coronavirus in humans include scutellarein, silvestrol, tryptanthrin, saikosaponin b2, lectins such as griffithsin, lycorine and polyphenolicsincluding quercetin, myricetin, caffeic acid, psoralidin and isobavachalcone. needless to mention, these compounds may be toxic at certain levels, and hence in vitro and in vivo testing is required to determine safe and therapeutic levels for each compound before clinical trials in humans can be performed. initial studies could focus on compounds which have previously been approved for drug use, or are generally regarded as safe by the fda or other national organisations, as is the case for some polyphenolic compounds. it is hoped that researchers will be guided by this information presented here in the process of developing safe, effective anti-coronavirus therapeutic agents from naturally derived compounds. the authors declare that no conflict of interest exists. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. accurate calculation of the absolute free energy of binding for drug molecules oxidative stress in alzheimer's disease: a review on emergent natural polyphenolic therapeutics sambucus nigra extracts inhibit infectious bronchitis virus at an early point during replication toona sinensis roem tender leaf extract inhibits sars coronavirus replication data resources for the computer-guided discovery of bioactive natural products antiviral effects of saikosaponins on human coronavirus 229e in vitro cytotoxicity and anti-hepatitis b virus activities of saikosaponins from bupleurum species geranylated flavonoids displaying sars-cov papain-like protease inhibition from the fruits of paulownia tomentosa antiviral activity of quercetin 7-rhamnoside against porcine epidemic diarrhea virus molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia biopolymeric nano/microspheres for selective and reversible adsorption of coronaviruses covid-19 -list results a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 inhibition of ebola virus by a molecularly engineered banana lectin computational proteinligand docking and virtual drug screening with the autodock suite herbal plants and plant preparations as remedial approach for viral diseases breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies severe acute respiratory syndrome-related coronavirus -the species and its viruses, a statement of the coronavirus study group the curative effects of glycyrrhizin on patients with sars. annual meeting of the society of infectious and parasitic diseases content of the flavonols quercetin, myricetin, and kaempferol in 25 edible berries bioactivity-guided fractionation for anti-inflammatory and analgesic properties and constituents of xanthium strumarium l emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction antiviral activity of glycyrrhizic acid derivatives against sars-coronavirus first case of 2019 novel coronavirus in the united states synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan characterization of a novel mannose-binding lectin with antiviral activities from red alga, grateloupia chiangii avian coronavirus infectious bronchitis virus susceptibility to botanical oleoresins and essential oils in vitro and in vivo composition of flavonoids in fresh herbs and calculation of flavonoid intake by use of herbs in traditional danish dishes interaction of vitamin c and flavonoids in elderberry (sambucus nigra l.) during juice processing jubanines f-j, cyclopeptide alkaloids from the roots of ziziphus jujuba recent synthetic and medicinal perspectives of tryptanthrin plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures polyphenols in the treatment of autoimmune diseases natural bis-benzylisoquinoline alkaloids-tetrandrine, fangchinoline, and cepharanthine, inhibit human coronavirus oc43 infection of mrc-5 human lung cells phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia medicinal herbal extracts of sophorae radix, acanthopanacis cortex, sanguisorbae radix and torilis fructus inhibit coronavirus replication in vitro in vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, cimicifuga rhizoma, meliae cortex, coptidis rhizoma, and phellodendron cortex dosedependent emetic effects of the amaryllidaceous alkaloid lycorine in beagle dogs inhibition of severe acute respiratory syndrome coronavirus replication in a lethal sars-cov balb/c mouse model by stinging nettle lectin, urtica dioica agglutinin immunomodulatory and anti-sars activities of houttuynia cordata porcine epidemic diarrhea virus infection: inhibition by polysaccharide from ginkgo biloba exocarp and mode of its action in vitro antiviral activity of fifteen plant extracts against avian infectious bronchitis virus functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses forsythoside a inhibits the avian infectious bronchitis virus in cell culture in vitro antiviral activity of griffithsin against porcine epidemic diarrhea virus identification of natural compounds with antiviral activities against sarsassociated coronavirus anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plant-derived phenolic compounds saikosaponin b2 is a naturally occurring terpenoid that efficiently inhibits hepatitis c virus entry antiviral natural products and herbal medicines traditional chinese medicine is a resource for drug discovery against 2019 novel coronavirus (sars-cov-2) sars-cov protease inhibitors design using virtual screening method from natural products libraries phytochemical analysis and in vitro antiviral activities of the essential oils of seven lebanon species bat-to-human: spike features determining 'host jump'of coronaviruses sars-cov, mers-cov, and beyond drug treatment options for the 2019-new coronavirus (2019-ncov) the potential chemical structure of anti-sars-cov-2 rna-dependent rna polymerase can chinese medicine be used for prevention of corona virus disease 2019 (covid-19)? a review of historical classics, research evidence and current prevention programs anti-sars coronavirus 3c-like protease effects of rheum palmatum l. extracts antiviral screening of british columbian medicinal plants identification and characterisation of small molecule inhibitors of feline coronavirus replication investigation of the influence of eps(r) 7630, a herbal drug preparation from pelargonium sidoides, on replication of a broad panel of respiratory viruses high-dose mannose-binding lectin therapy for ebola virus infection flavonoid (myricetin, quercetin, kaempferol, luteolin, and apigenin) content of edible tropical plants middle east respiratory syndrome coronavirus infection is inhibited by griffithsin antiviral lectins: selective inhibitors of viral entry broad-spectrum antiviral activity of the eif4a inhibitor silvestrol against corona-and picornaviruses comparison of broad-spectrum antiviral activities of the synthetic rocaglate cr-31-b (−) and the eif4a-inhibitor silvestrol natural products as sources of new drugs from 1981 to 2014 antiviral cystine knot alpha-amylase inhibitors from alstonia scholaris broadspectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event tanshinones as selective and slow-binding inhibitors for sars-cov cysteine proteases evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors new hepatitis c virus (hcv) drugs and the hope for a cure: concepts in anti-hcv drug development phase i safety, tolerability, and pharmacokinetic study of recombinant human mannanbinding lectin lianhuaqingwen exerts anti-viral and anti-inflammatory activity against novel coronavirus (sars-cov-2) biflavonoids from torreya nucifera displaying sars-cov 3clpro inhibition sars-cov 3clpro inhibitory effects of quinone-methide triterpenes from tripterygium regelii coronavirus envelope protein: current knowledge bioactive pyranoxanthones from the roots of calophyllum blancoi inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) quercetin 7-rhamnoside reduces porcine epidemic diarrhea virus replication via independent pathway of viral induced reactive oxygen species cathepsin l targeting in cancer treatment influenza virus variation in susceptibility to inactivation by pomegranate polyphenols is determined by envelope glycoproteins in silico and in vitro analysis of small molecules and natural compounds targeting the 3cl protease of feline infectious peritonitis virus antioxidant and antimicrobial activities of consecutive extracts from galla chinensis:the polarity affects the bioactivities polyphenol-based nutraceuticals for the prevention and treatment of cardiovascular disease: review of human evidence sabadinine: a potential non-peptide antisevere acute-respiratory-syndrome agent identified using structure-aided design autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading anthemis hyalina (ah) and citrus sinensis (cs) extracts on the replication of coronavirus and the expression of trp genes family inactivation of measles virus and herpes simplex virus by saikosaponin d clinical features and treatment of covid-19 patients in northeast chongqing anti-hepatitis b virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro virtual screening for finding natural inhibitor against cathepsin-l for sars therapy screening of traditional chinese remedies for sars treatment traditional chinese medicine herbal extracts of cibotium barometz, gentiana scabra, dioscorea batatas, cassia tora, and taxillus chinensis inhibit sars-cov replication antiviral activity of sambucus formosana nakai ethanol extract and related phenolic acid constituents against human coronavirus nl63 antimicrobial activity of extracts from native plants of temperate australia bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of artichoke leaf extracts in humans who mers global summary and assessment of risk genome composition and divergence of the novel coronavirus (2019-ncov) originating in china analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods bioactive coumarins from boenninghausenia sessilicarpa traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in china anti-infectious bronchitis virus (ibv) activity of 1,8-cineole: effect on nucleocapsid (n) protein comparative anti-infectious bronchitis virus (ibv) activity of (-)-pinene: effect on nucleocapsid (n) protein a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease-19 treatment option small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells in vitro and in vivo effects of houttuynia cordata on infectious bronchitis virus antioxidative and antiviral properties of flowering cherry fruits (prunus serrulata l. var. spontanea) identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus scutellaria baicalensis, the golden herb from the garden of chinese medicinal plants saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection (hoever et al., 2005) page 27 of 34 clpro = chymotrypsin-like protease; cpe assay = cytopathogenic effect assay; n/a = not applicable to this study; nd = no data; plpro = papain-like protease. page 31 of 34 (li et al., 2019) key: cord-295375-nakxfhxk authors: yu, yang; shi, qianling; zheng, peng; gao, lei; li, haiyuan; tao, pengxian; gu, baohong; wang, dengfeng; chen, hao title: assessment of the quality of systematic reviews on covid‐19: a comparative study of previous coronavirus outbreaks date: 2020-04-28 journal: j med virol doi: 10.1002/jmv.25901 sha: doc_id: 295375 cord_uid: nakxfhxk several systematic reviews (srs) have been conducted on the covid‐19 outbreak, which together with the srs on previous coronavirus outbreaks, form important sources of evidence for clinical decision and policy making. here, we investigated the methodological quality of srs on covid‐19, severe acute respiratory syndrome (sars), and middle east respiratory syndrome (mers). online searches were performed to obtain srs on covid‐19, sars, and mers. the methodological quality of the included srs was assessed using the amstar‐2 tool. descriptive statistics were used to present the data. in total, of 49 srs that were finally included in our study, 17, 16, and 16 srs were specifically on covid‐19, mers, and sars, respectively. the growth rate of srs on covid‐19 was the highest (4.54/month) presently. of the included srs, 6, 12, and 31 srs were of moderate, low, and critically low quality, respectively. srs on sars showed the optimum quality among the srs on the three diseases. subgroup analyses showed that the sr topic (p < .001), the involvement of a methodologist (p < .001), and funding support (p = .046) were significantly associated with the methodological quality of the sr. according to the adherence scores, adherence to amstar‐2 items sequentially decreased in srs on sars, mers, and covid‐19. the methodological quality of most srs on coronavirus outbreaks is unsatisfactory, and those on covid‐19 have higher risks of poor quality, despite the rapid actions taken to conduct srs. the quality of srs should be improved in the future. readers must exercise caution in accepting and using the results of these srs. address a specific research question. practitioners mainly rely on srs for evidence to provide evidence-based recommendations. before the covid-19 pandemic, outbreaks of severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) were observed in 2003 and 2012, respectively; substantial numbers of srs have been conducted on these outbreaks. the causative agents of these three outbreaks belong to the same genus of coronavirus (cov), and sars-cov-2 shares a 79.5% sequence identity with sars-cov. thus, the available srs on sars and mers are helpful in guiding covid-19 management. however, the quality of these srs was unclear. in addition, besides these existing srs, we also found that a large number of newly-conducted srs with or without meta-analyses have been published rapidly, shortly after the covid-19 outbreak, and the number of such srs keeps rising. as we know, at the preliminary stage of any public emergency, primary studies are usually lacking, and most studies are observational. thus, it is not easy to conduct an sr. these recent srs on covid-19 are more likely to have the potential methodological flaws. therefore, we hypothesized that there may be a difference in methodological quality between these srs on covid-19 and previous srs on sars and mers. up to now, no studies appraising the methodological quality of the srs on covid-19 and previous mers and sars outbreaks are available. in this comparative study, we investigated the present status of conducting srs on covid-19, mers, and sars, appraised the methodological quality of these srs using the a measurement tool to assess systematic reviews (amstar 2), and performed a preliminary examination of the potential risk factors associated with the quality of srs, with the aim of providing suggestions from the aspects of methodological quality for conducting and using srs during the covid-19 pandemic. we searched for eligible srs (and/or meta-analyses) in both english we included all srs which discussed one of the following diseases: covid-19, sars, and mers. srs were identified based on the following criteria: (a) label of "systematic review" in the title, abstract, or full text; and (b) a literature search was performed. there were no restrictions on the sr topic. srs with or without meta-analysis were both eligible. only srs published in academic journals were included. studies were excluded based on the following criteria: (a) letters, editorials, and expert opinions; case reports and literature reviews; and other narrative reviews; (b) abstract only or data unavailable; or (c) studies in non-english or non-chinese languages. when a duplicate or updated publications were identified, only the most recent one was included. two reviewers independently screened the titles and abstracts of all articles identified in the initial search and then checked the full texts of potentially eligible studies. any disagreement was resolved by discussion or consultation with a third reviewer. the same reviewers used a predesigned table to collect the following information on the included srs: first author, publication year, the growth rate of srs, region or country in which the study was performed, study topic the methodological quality of the included srs was independently assessed by two well-trained reviewers using the amstar-2 tool. 5, 6 any disagreement between the two reviewers was resolved by discussion, and in case of persistent disagreement, a resolution was arrived at by consulting with a third person. the amstar-2 is a critical appraisal tool for srs of randomized control trials and/or observational studies. 6 the amstar-2 contains 16 items, of which seven are critical domains (table 1) . a questionspecific point scale ("yes, no;" "yes, partial yes, no;" or "yes, no, no meta-analysis") was used to score each item. based on the degree of weaknesses detected in critical and noncritical items, the amstar-2 classifies the overall confidence in the results of the srs into four levels: high, moderate, low, and critically low. we graded each included sr on the amstar-2 official website and generated the overall confidence using their online calculator. in addition, to compare adherence to items among srs, a total score was calculated for each sr according to the response to each item, that is, "yes," "partial yes," "no meta-analysis," and "no" were scored as "+1," "+0.5," "0," and "−1," respectively. descriptive statistics were used to present the characteristics and methodological quality of the included srs. categorical variables were expressed as frequencies and percentages. the response to each amstar-2 item was recorded and tabulated for all included srs. overall adherence to the 16 items were analysed with a percent stacked bar chart. the adherence scores in each disease group were presented as mean ± standard deviation (sd). subgroup analysis was performed to compare the differences in quality among subgroups with different study characteristics. statistical significance was tested using the kruskal-wallis rank test and two-sample mann-whitney u test. p < .05 was considered to be statistically significant, and all p values were two-sided. excel 2019 and spss v25 were used for all data management and analyses. were supported by at least one funding source ( were of critically low quality. not a single sr was rated as having high quality. the quality of all srs on covid-19 was rated as low (29%, n = 5) or critically low (71%, n = 12), similar to that of srs on mers (low, 25%, n = 4; critically low, 75%, n = 12) but inferior to that of srs on sars (moderate, 38%, n = 6; low, 19%, n = 3; critically low, 44%, n = 7). the potential factors affecting the quality of srs were investigated in all samples. subgroup analyses of different variables showed that the topic of the sr, involvement of a methodologist, and funding support were significantly associated with the methodological quality of the srs ( details of the assessment of each item are described in the table s1 . srs provide the highest level of evidence in evidence-based medicine, and they are an important source of information for clinical practitioners and policy makers. however, due to the relatively low requirements and costs of conducting srs, the number of srs published in various fields is quite high. the increase in quantity has also brought concerns about the quality of srs. 56 in this study, we found that a large number of srs were conducted de novo after the for unknown emerging diseases, the scarcity of large samples and high-quality original reports are major challenges in scientific research. in particular, in the early stages of disease outbreaks, evidence is mostly acquired from observational studies, 58 srs specific to covid-19, sars, and mers are being heavily relied upon during the current covid-19 pandemic. the methodological f i g u r e 2 adherence to each item in amstar-2. amstar, a measurement tool to assess systematic reviews; ma, meta-analysis quality of most srs is unsatisfactory, and those on covid-19 have higher risks of poor quality, despite the rapid actions taken to conduct srs. teams that may want to conduct a sr should focus on the study design and focus on improving the quality of the sr. sr findings should be used more cautiously, and it is not advisable that users accept the results of a single sr without critical appraisal. covid-19) situation cochrane handbook for systematic reviews of interventions scientific journal rankings2018 a guide to the core journals of china development of amstar: a measurement tool to assess the methodological quality of systematic reviews amstar 2: a critical appraisal tool for systematic reviews that include randomised or non-randomised studies of healthcare interventions, or both asymptomatic middle east respiratory syndrome coronavirus (mers-cov) infection: extent and implications for infection control: a systematic review update on therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis prevalence of diabetes in the 2009 influenza a (h1n1) and the middle east respiratory syndrome coronavirus: a systematic review and meta-analysis a systematic, thematic review of social and occupational factors associated with psychological outcomes in healthcare employees during an infectious disease outbreak effect of integrated traditional chinese medicine and western medicine on the treatment of severe acute respiratory syndrome: a meta-analysis what have we learned about middle east respiratory syndrome coronavirus emergence in humans? a systematic literature review. vector borne zoonotic dis a rapid scoping review of middle east respiratory syndrome coronavirus in animal hosts analyzing on the clinical symptoms and t cell subsets of patients with severe acute respiratory syndrome meta analysis on t cell subsets of patients with severe acute respiratory syndrome meta-analysis of comparative studies of integrative traditional chinese medicine with western medicine and western medicine alone for sars clinical symptoms and peripheral white blood cell counts of patients with severe acute respiratory syndrome: meta analysis meta-analysis on risk factors of sars patients epidemic models of contact tracing: systematic review of transmission studies of severe acute respiratory syndrome and middle east respiratory syndrome seroprevalence of igg antibodies to sars-coronavirus in asymptomatic or subclinical population groups a meta-analysis to evaluate the effectiveness of real-time pcr for diagnosing novel coronavirus infections chinese herbal medicine for severe acute respiratory syndrome: a systematic review and metaanalysis chinese herbs combined with western medicine for severe acute respiratory syndrome (sars) middle east respiratory syndrome coronavirus: current knowledge and future considerations clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis a systematic review of therapeutic agents for the treatment of the middle east respiratory syndrome coronavirus (mers-cov) therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta-analysis femoral head necrosis after severe acute respiratory syndrome: etiology and treatment mers transmission and risk factors: a systematic review global status of middle east respiratory syndrome coronavirus in dromedary camels: a systematic review sars: systematic review of treatment effects a systematic review of assessing the effect of integrated traditional chinese medicine with western medicine for severe acute respiratory syndrome effect of integrated traditional chinese and western medicine on sars: a review of clinical evidence systematic assessment on the effect of integrated chinese traditional medicine with western medicine in the treatment of severe acute respiratory syndrome (sars) steroid therapy and the risk of osteonecrosis in sars patients: a dose-response meta-analysis transmission of middle east respiratory syndrome coronavirus infections among healthcare personnel in the middle east: a systematic review evidence-based rapid review on possibility of treatment of 2019-ncov with subcutaneous α-interferon a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 the possibility of using lopinave/ litonawe (lpv/r) as treatment for novel coronavirus (2019-ncov) pneumonia: a quick systematic review based on earlier coronavirus clinical studies prevalence and impact of cardiovascular metabolic diseases on covid-19 in china covid-19 patients' clinical characteristics, discharge rate and fatality rate of meta-analysis sanchis-gomar f. cardiac troponin i in patients with coronavirus disease 2019 (covid-19): evidence from a metaanalysis procalcitonin in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis thrombocytopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a metaanalysis potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis coronavirus disease 2019 (covid-19): a systematic review of imaging findings in 919 patients clinical characteristics of hospitalized patients with sars-cov-2 infection: a single arm metaanalysis prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease-19 treatment option potential interventions for novel coronavirus in china: a systematic review traditional chinese medicine syndromes of the novel coronavious pneumonica: a systemic review and meta-analysis the possibility of ribavirinas in the treatment of the coronavirus disease 2019: a systematic review the influence of the team in conducting a systematic review analysis of the time and workers needed to conduct systematic reviews of medical interventions using data from the prospero registry european centre for disease prevention and control. evidence-based methodologies for public health-how to assess the best available evidence when time is limited and there is lack of sound evidence. stockholm: european centre for disease prevention systematic reviews: rationale for systematic reviews the methodological quality of robotic surgical meta-analyses needed to be improved: a cross-sectional study an international registry of systematic-review protocols assessment of the quality of systematic reviews on covid-19: a comparative study of previous coronavirus outbreaks the authors declare that there are no conflict of interests. yy and hc conceived the idea for this study. yy designed the study. http://orcid.org/0000-0002-2409-3681 key: cord-299519-hfgmmuy6 authors: alenazi, thamer h.; arabi, yaseen m. title: severe middle east respiratory syndrome (mers) pneumonia date: 2019-10-26 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.11488-6 sha: doc_id: 299519 cord_uid: hfgmmuy6 middle east respiratory syndrome (mers) is a viral respiratory infection, which ranges from asymptomatic infection to severe pneumonia and multiorgan failure, caused by a novel coronavirus named middle east respiratory syndrome coronavirus (mers-cov). majority of cases have been reported from saudi arabia. mers cases occur as sporadic cases or as clusters or hospital outbreaks. dromedary camels are thought to be a host for mers-cov. direct contact with dromedary camels within 14 days prior to infection was identified as an independent risk factor for mers. diagnosis of mers is based on a positive real-time reverse transcriptase polymerase chain reaction (rrt-pcr), obtained from a respiratory specimen. the mainstay of management of mers-cov infection is supportive care. there is no specific antiviral therapy for mers-cov infection at present, although several modalities of treatment options have been examined or are under investigation. middle east respiratory syndrome (mers) is a viral respiratory infection, which ranges from asymptomatic infection to severe pneumonia, caused by a novel coronavirus named middle east respiratory syndrome coronavirus (mers-cov). coronaviruses are a family of viral pathogens that could cause animal and human disease. mers-cov is closely related to the severe acute respiratory syndrome coronavirus (sars-cov) but from a different lineage. the objective of this chapter is to describe the epidemiology, virology, clinical manifestations, management and prevention of mers. between september 2012, until the end of may 2019, the world health organization (who) has been notified of 2374 laboratoryconfirmed case of mers-cov infection from 27 countries with 823 associated deaths resulting in a case fatality rate of 35%. saudi arabia has been the major reporting country with a total number of 2008 cases and 749 deaths (a case fatality rate of 37.3%) ( table 1) (world health organization, 2019, mar 31) . the virus was first isolated in june 2012 from respiratory specimens of a saudi patient from jeddah, saudi arabia, who presented with severe pneumonia that progressed to acute respiratory distress syndrome (ards), renal failure, multi-organ failure and eventually lead to death (zaki et al., 2012) . in the same week, a patient with a recent history of travel to the united kingdom (uk) in august 2015, with 16 cases across three hospitals, and seven deaths (43.7% case fatality rate) (payne et al., 2018) . smaller outbreaks continued to occur in 2016-18 although the number of patients and the magnitude of the outbreaks were less compared to earlier years, presumably due to better infection control practices and earlier identification of cases. transmission of mers-cov in humans occurs through animal-to-human transmission or, human-to-human transmission in the community. additionally, nosocomial transmission of mers-cov occurs frequently. all transmission described up-to-date, occurred in residents in or travelers to the arabian peninsula, or are traced to contact with patients with a history of recent travel to the arabian peninsula. animals seem to play an important role in the transmission of the mers-cov. earlier studies have suggested that bats might be the potential reservoir of mers-cov. this hypothesis that was based on the close proximity of mers-cov-phylogenetically-to tylonycteris bat coronavirus hku4 (ty-batcov hku4) and pipistrellus bat coronavirus hku5 (pi-batcov hku5) (woo et al., 2012) . a study from saudi arabia, a phylogenetically mers-cov identical short gene segment, was detected in a fecal sample of one of the 29 captured bats near the home of a laboratory-confirmed mers-cov patient . however, live mers-cov has never been recovered from bats. further studies are needed to further establish the role of bats in transmission to humans including larger surveillance studies with full viral genome sequencing. epidemiologically, it seems unlikely that bats are the direct source of human cases, since none of the community-acquired laboratory-confirmed mers cases had clear bat exposure. dromedary camels are thought to be a host for mers-cov. direct contact with dromedary camels within 14 days prior to infection was identified as an independent risk factor for mers (gossner et al., 2016) . camel-human transmission was also suggested in a 44-year-old, previously healthy man from jeddah, saudi arabia who was admitted to the intensive care unit (icu) with severe mers pneumonia, and died 15 days after admission. the patient had owned a herd of 9 camels and used to visit them daily until 3 days prior to his admission. four out of the nine camels were sick with nasal discharge, 1 week prior to the patient's onset of symptoms. the patient had significant contact with camels' excretions. respiratory specimens from the patient and one of his camels showed identical mers-cov full genome sequencing. moreover, serum antibodies for mers-cov were positive in both the patient and the camel, with the camel seropositivity preceded the patient's seropositivity suggesting that direction of transmission was from the camel to the patient. a large cross-sectional study from saudi arabia identified mers-cov infected patients who had a history of camel contact. the investigators obtained nasal swabs and serum samples from 584 dromedary camels and found that 12.6% of the studied camels were mers-cov polymerase chain reaction (pcr) positive, and 70.9% of them were mers-cov antibodies positive. furthermore, 10 of the full genome sequences of the camel mers-cov were identical to their contacted patients (kasem et al., 2018) . this data suggests an important role for camels in the transmission of mers-cov. however, in a cohort of 1125 patients with laboratory-confirmed mers, camel contact was reported only in 235 patients (20.9%), denied by 276 patients (24.5%), and not reported in the other 614 patients (54.6%) (conzade et al., 2018) . hospital-based outbreaks and community-based clusters described above suggest strongly that human-human transmission does occur. the transmission was more commonly observed in healthcare-based outbreaks, compared to community clusters. the number of close contacts who got infected by patients with confirmed mers-cov appears to be low, although, it was evident that some patients were spreading the infection to a disproportionally large number of individuals (super spreaders) (hui, 2016) . this phenomenon was clearly described in more than one outbreak. the first outbreak which identified the super spreader phenomena was the korean outbreak, in which a single imported index case resulted in a total of 186 cases. it was thought that 83% of transmission in the korean outbreak was linked epidemiologically to five super spreaders (korea centers for disease and prevention, 2015) the same phenomenon was also described in a large outbreak in riyadh, saudi arabia, where 6 out of the 130 cases, contributed to 58.7% of the transmission (alenazi et al., 2017) . however, it remains unclear if an asymptomatic individual who carries mers-cov can transmit the virus to others. the first family cluster was reported from riyadh, saudi arabia, where three laboratory-confirmed cases and one probable case were diagnosed, and two out of the four patients died . in a study that investigated 26 index cases of mers and their 280 household contacts, the secondary transmission rate was found to be 4% ([95% ci, 2 to 7] . as described above, transmission was more commonly seen in hospital-based outbreaks compared to family community transmission, particularly in emergency department (ed). this was clearly illustrated in the korean outbreak, where a single imported case had led to a total of 186 cases, 185 of which were nosocomial transmission (kim et al., 2017) the main identified reasons for hospitals-based transmission were over-crowdedness of ed, late recognition of suspected mers cases and inadequate infection control measures and proper isolation of suspected cases (stone et al., 2016) . environmental surfaces in hospitals is a potential source of transmission. in one study, a viable mers-cov was detected in 15 out of 68 surface swabs collected from patient's rooms, restrooms and common corridors (kim et al., 2016) . mers-cov is the sixth coronavirus that affects humans. it lies within the lineage c of the genus betacoronavirus (cov) in the family coronaviridae under the order nidovirales. it has close phylogenetic proximity to two bat coronaviruses, tylonycteris bat cov hku4 (ty-batcov-hku4) and pipistrellus bat cov hku5 (pi-batcov-hku5). like the other coronaviruses, it is an enveloped single-stranded rna virus which replicates in the host-cell cytoplasm. the size of its rna genome is approximately 30 kb. it has structural proteins, called the e, m, and n proteins, and membrane protein called the spike (s) protein, which plays an important role in the virus attachment and entry into the host cells. due to the large increase in the number of diagnosed cases in april 2014, there was a concern that mers-cov could have undergone mutation that led to increased virulence or transmissibility of the virus; however, this assumption was proven unlikely (drosten et al., 2015) . the pathogenesis and histopathology of mers-cov is poorly understood and understudied. post-mortem autopsies were rarely performed on mers patients due to cultural reasons in the arabian peninsula. most of the knowledge we have about the histopathology of mers-cov comes from in vitro, ex vivo, animal experiments and limited post-mortem reports. in a 33-year-old male, who died of mers-cov infection, post-mortem analysis of histopathology finding of pulmonary and extrapulmonary tissue were examined under transmission electron microscopy which showed necrotizing pneumonia, pulmonary diffuse alveolar damage, acute kidney injury, portal and lobular hepatitis and myositis with muscle atrophic changes. the brain and heart were histologically unremarkable. ultra-structurally, viral particles were localized in the pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells and macrophages infiltrating the skeletal muscles (alsaad et al., 2018) . in the beginning of the outbreak, the who had proposed a case definition for mers-cov infection for epidemiological purposes, that was last updated on july 27, 2018. the united states (us) center of disease control and prevention (cdc) and the saudi ministry of health (moh), each had developed a case definition for suspected, confirmed and probable mers-cov infection ( table 2) . in one of the earlier outbreaks in saudi arabia, the median incubation period for mers-cov infection was 5.2 days (95% ci 1.9-14.7 days) (assiri et al., 2013b) . similarly, in the south korean outbreak, in 2015, the median incubation period was 6.3 days (95th percentile 12.1 days) (korea centers for disease and prevention, 2015) . therefore, mers should be suspected in patients presenting with respiratory infection, and residence in or travel to the arabian peninsula within the last 14 days prior to onset of symptoms. most of reported mers patients have been in the adult age group. only 31 pediatric cases were reported, most of which were detected on contact tracing screening (42% were asymptomatic), and among symptomatic cases, presence of comorbidities like congenital disease were commonly present (al-tawfiq et al., 2016) . the mean age in one of study was 56.3 years . in another study, that described the epidemiological, clinical characteristics and demographics of 47 mers-cov infected patients, 82.9% of laboratory-confirmed cases were more than 40 years of age with a median age of 56 years. the male: female ratio was 3.3:1. eighty nine percent of patients required icu admission, and the median time to death was 14 days (ranging from 5 to 36 days) (assiri et al., 2013a) . one study from saudi arabia, have compared critically ill mers-cov patients with critically ill non-mers-cov patients, and had found that mers-cov patients tend to be younger, more likely to require mechanical ventilation and had higher mortality . there were eight reported mers-cov infection during pregnancy, from jordan, united arab emirates and saudi arabia, three of them ended with maternal death . in the beginning of the epidemic, the typical presentation of reported mers was severe pneumonia, with acute respiratory distress syndrome (ards) with or without acute kidney injury, but as the surveillance and testing had increased, milder or even asymptomatic cases have been described. in a cohort of 47 patients, with mers-cov infection, the clinical presentation were fever (93%), cough (83%), shortness of breath (72%), myalgia (32%), diarrhea (26%), sore throat (21%), vomiting (21%), abdominal pain (17%) and hemoptysis (17%) (assiri et al., 2013a) . a study that compared 330 critically ill mers-cov infected patients with 220 critically ill patients with non-mers severe acute respiratory infection (sari) found that mers patients were younger than non-mers sari patients (median [q1, q3] 58 [44, 69] vs 70 [52, 78] and were more likely to be males (68.2% vs 58.1%) and to be healthcare workers (9.7% vs 0.0%). chronic comorbidities were prevalent (any comorbidity, 80.3% in mers sari, 91.4% in non-mers sari). after onset of symptoms, mers-cov patients presented to er with a median of 5 days and admitted to icu after 7 days, which was 2 days longer compared to non-mers-cov sari patients. mechanical ventilation was required for 85.2% of patients with mers-cov patients. at the time of icu admission, patients with mers-cov were more likely to be hypoxemic, compared with non-mers sari patients (ratio of arterial oxygen partial pressure to fractional inspired oxygen-pao2/fio2: 106.3 [66.2, 160] vs 176 [104, 252] ) . respiratory distress syndrome) and direct epidemiologic link with a laboratory-confirmed mers-cov case and testing for mers-cov is unavailable, negative on a single inadequate specimen or inconclusive. a febrile acute respiratory illness with clinical, radiological, or histopathological evidence of pulmonary parenchymal disease (e.g. pneumonia or acute respiratory distress syndrome) that cannot be explained fully by any other etiology and the person resides or traveled in the middle east, or in countries where mers-cov is known to be circulating in dromedary camels or where human infections have recently occurred and testing for mers-cov is inconclusive. an acute febrile respiratory illness of any severity and direct epidemiologic link (2) many cases of mers present with gastrointestinal manifestations with or without respiratory symptoms. among the critically ill patients, the most described extra-pulmonary manifestations were acute kidney injury and shock (arabi et al., 2014) . very few patients were reported to have neurological symptoms, in addition to the pneumonia (arabi et al., 2015) . primary infections were more likely to be severe, as opposed to secondary cases. secondary mers infection tends to cause a milder or asymptomatic disease, however severe disease has been described. secondary cases are more likely to be younger with no comorbidities. asymptomatic infections have bee also described in patients with dromedary camel's contacts who were identified during surveillance (al hammadi et al., 2015) . mortality rates were reported to be higher in older age group, male gender and patients with comorbidities (assiri et al., 2013a) . numerous cases of mers occurred among healthcare-workers; leading in some of them to severe illness resulting in admission to icu. in a study that examined 32 critically ill healthcare-workers with mers, 43.75% were nurses and 25% were physicians. 34.4% were having comorbidities, mainly chronic kidney disease (15.6%). fever at presentation, was found in 30/32 (93.8%), cough in 25/32 (78.1%), and gastrointestinal symptoms in11/32 (34.4%). eight out of the 32 (25%) healthcare workers died (shalhoub et al., 2018) . among all hospitalized patients with severe mers pneumonia, the most commonly observed laboratory abnormalities were lymphopenia (34%), thrombocytopenia (36%) and raised lactate dehydrogenase (ldh) (49%). other abnormalities like leukopenia (14%), lymphocytosis (11%), raised aspartate aminotransferase (ast) (15%), raised alanine aminotransferase (alt) (11%), and raised lactate dehydrogenase (49%) were also observed (assiri et al., 2013a) . in a cohort of 330 critically ill mers-cov patients, leukopenia was observed in 20.2%, thrombocytopenia in 58.7%, raised alt in 56.3%, and raised ast in 86.8% . most of the reported symptomatic cases with severe mers pneumonia had abnormal chest-x-ray. abnormalities ranged from mild to extensive changes. peripheral ground-glass opacities were the most frequently found abnormality on cxr, in 55 studied case (das et al., 2015) other findings include, unilateral or bilateral airspace opacities, increased broncho-vascular markings, patchy infiltrates, interstitial changes, nodular opacities, reticular opacities, reticulo-nodular shadowing, pleural effusions, and ards pattern. among inpatients who had chest computed tomography scan (ct scan), the most frequent findings were peripheral and bibasilar opacities bilaterally. in patient presenting with severe pneumonia, mers should be suspected based on the presence epidemiologic links (residence or travel from the arabian peninsula especially if there is history of contact with camels, contact with a person infected with mers or working or being treated in a hospital where mers patients are managed). such links should lead to application of appropriate infection control measures (see below) and to initiate diagnostic work up for mers. diagnosis of mers is based on a positive real-time reverse transcriptase polymerase chain reaction (rrt-pcr), obtained from a respiratory specimen. nasopharyngeal or oropharyngeal swab of the upper respiratory tract are often used in patients who are unable to produce lower respiratory samples. however, lower respiratory samples (sputum, endotracheal aspirate, or bronchoalveolar lavage) are preferred as they generally have better yield. in patients with suspected mers, it is recommended to send more than one specimen since a negative test does not exclude the diagnosis. in a cohort of critically ill patients with mers pneumonia, the diagnosis of mers was based on samples from the nasopharynx in 167 of 311 (54%) and from the lower respiratory tract (sputum, endotracheal aspirates, or broncho-alveolar lavage) in 144 of 311 (46%). the diagnosis was established from the first sample in 76% of patients, from the second sample in 16% of patients and from 3 to 5 repeat samples in 8% of the patients. initial negative samples collected before positive ones were predominantly from the upper respiratory tract (81.5%) . several serological assays have been used including enzyme-linked immune sorbent assay (elisa) and immunofluorescence assay (ifa), which are typically used for screening, and neutralization techniques which are used for confirmation. a three different, indirect elisa have been developed and validated based on mers-cov nucleocapsid protein (n), spike (s) ectodomain (amino acids 1-1297) and s1 subunit (amino acids 1-725) (hashem et al., 2019) . a single positive serological test, in the absence of positive pcr is considered a probable case, in the setting of suspected mers-cov. however, a four-fold increase in mers-cov antibody titer by neutralization tests is considered a confirmed case. viral pathogens were identified in 5% of critically ill patients with mers pneumonia which included other coronaviruses, respiratory syncytial virus, and influenza a virus. bacterial co-infections are described in 18% of critically ill patients with mers pneumonia, with acinetobacter species, pseudomonas species, klebsiella pneumoniae and staphylococcus aureus being the most frequent isolates . there is no specific antiviral therapy for mers-cov infection up to date, although several modalities of treatment options have been tried or are under investigation. the mainstay of management of mers-cov infection is supportive care. patients with suspected severe mers pneumonia-cov infection might have other respiratory pathogens as a cause of their symptoms. therefore, the who recommends starting appropriate empirical antimicrobial therapy as soon as possible, to cover community acquired or nosocomial associated pathogens, based on the presentation from the community or the hospital and based on local epidemiology and guidelines, until the microbiological diagnosis is confirmed. supportive therapy is the mainstay of management of severe mers pneumonia, which includes mechanical ventilation, vasopressor support, and renal replacement therapy if needed. oxygen rescue therapy like extracorporeal membrane oxygenation (ecmo) has been used in patients with refractory hypoxemia. in one case-control study of patients with mers, the rescue use of ecmo compared to a matched control with no-ecmo was associated with reduced in-hospital-mortality (65% compared 100%) (alshahrani et al., 2018) . another retrospective study, found that critically ill healthcare workers who died because of mers were more likely to have received ecmo than not, probably because the severity of pneumonia that led to use of rescue therapy, rather than use of ecmo itself (shalhoub et al., 2018) . corticosteroids have been used frequently in mers patients. a study that accounted for time-varying confounding demonstrated that corticosteroid use was not associated with difference on mortality although it was associated with prolongation of viral rna shedding (arabi et al., 2018b) . data on other human coronaviruses, and in vitro activity of specific therapies were used to identify potential new therapy for mers-cov. examples of those include: combination of ribavirin and interferon, lopinavir-ritonavir, mycophenolate mofetil, convalescent -plasma, and, monoclonal and polyclonal antibodies ( table 3) . the efficacy of ribavirin/interferon combination was suggested to be promising in vitro and animal experiments and cell culture. in a study where two cell viral cultures lines grew mers-cov, high concentrations of ribavirin or interferon alpha 2 b were needed to inhibit viral replication, when each of the drugs was used alone, however, comparable inhibition was observed when combing them at a lower concentration (falzarano et al., 2013a) . similar findings were observed in rhesus macaques model of mers-cov infection. among animals who received combination of ribavirin and interferon alpha 2 b 8 hours after inoculation did not develop respiratory symptoms and had no or very minimal chest x-ray findings of infiltrate compared to the control group. also, the treated group had a moderately lower viral genome copies and fewer severe lung histopathological changes (falzarano et al., 2013b) . data on humans are based on retrospective studies. in retrospective cohort of 20 patients with severe mers-cov pneumonia, ribavirin and interferon combination therapy started at median day three after diagnosis, showed improved 14-day survival, compared to 24 patients who received only supportive therapy, however 28-day survival was not different between the 2 groups (omrani et al., 2014) . other retrospective studies showed no difference in mortality between patients treated with ribavirin interferon combination, and patients who received supportive therapy shalhoub et al., 2015) . the largest cohort study which adjusted for time-varying confounders showed that ribavirin with interferons (alpha 1a and 1b and beta 1a) was not associated with difference in mortality or viral shedding. none of the patients received interferon beta 1b (arabi et al., 2019) . lopinavir-ritonavir efficacy was studied in-vitro in animals with severe mers-cov infection, in which it showed favorable outcome (chan et al., 2015) . there is an ongoing randomized placebo controlled trial evaluating oral lopinavir-ritonavir in combination with subcutaneous interferon beta-1b in hospitalized patients with mers (nct02845843) (arabi et al., 2018a) . the use of passive immune therapy with convalescent plasma was suggested as a potential therapeutic option. a study that examined the feasibility of convalescent plasma therapy for mers was limited by the small pool of donors with sufficient titers of mers-cov antibodies which may be related to the short-lasting immune response . several monoclonal antibody preparations have been developed. humanized bovine transchromosomal polyclonal antibodies against mers-cov have been developed and undergone testing in a phase i trial (beigel et al., 2018) . a phase ii trial in humans is being planned. mycophenolate mofetil efficacy against mers-cov was suggested in vitro studies. however, it was associated with harm in a marmoset model (chan et al., 2015) . remdesivir (gs-5734) which is the monophosphoramidate prodrug of the c-adenosine nucleoside analog gs-441524, has recently been reported to inhibit sars-cov, mers-cov and bat-cov, in vitro. it has also been found to be therapeutic and prophylactic in sars-cov infected mouse modules (agostini et al., 2018) . most of the reported hospital-based outbreaks were attributed to lack of adherence to proper infection control practice, delayed suspected cases identification, and to overcrowded emergency room and inappropriate triage. addressing issues related to infection control practice and proper triaging of patients with suspected mers-cov, had resulted in a decline in the number and the magnitude of hospital outbreaks . the who and us cdc have published guidance for mers prevention in healthcare institutes. as per the who recommendations, patients who have probable or confirmed mers should be under contact and droplet precautions with eye protection. the patient should be under airborne precaution, when performing an aerosol generating procedure (agp) like tracheal intubation or bronchoscopy. the us cdc, on the other hand, recommends contact and airborne precautions for all suspected or confirmed mers-cov patients. viral shedding from respiratory secretions has been found to be at least 3 weeks from onset of symptoms. therefore isolation precaution should not be discontinued until a negative pcr is obtained. patients with suspected or confirmed mers-cov, who does not require admission, can be isolated at home. it is recommended to avoid contact with camels, both direct or indirect contact like consuming raw camel's milk or meat. this is particularly for high risk individuals, such as patients with heart failure, chronic lung disease and immunosuppression. people who have to be in contact with camels should observe infection control precautions, including washing hand before and after contact, and use of appropriate personal protective equipment's (ppe) when dealing of a suspected or confirmed infected camels. it is important to note that the infected camels may not be symptomatic or might only have mild symptoms. the saudi authorities had made certain measures to reduce camel-human transmission, like banning camels in the holy areas, and moving the camels markets outside the cities. the who did not place any travel restriction to any country that have reported mers-cov cases. saudi arabia, where most of the laboratory-confirmed cases have been reported, annually hosts millions of muslims to perform hajj and omrah (pilgrimage), with no documented related cases of mers to date. there were 2 dutch patients who developed mers after returning from 2014 hajj, but the two cases were thought to be acquired from a camel market and raw milk consumption rather than human-human transmission during hajj. table 3 summary of treatment options for mers-cov. ribavirin/interferon combination ribavirin/interferon showed efficacy in and in vitro and in a rhesus macaques model. data in humans are based on retrospective studies. the largest cohort that accounted for time-varying confounders did not demonstrate efficacy. there may be differences in efficacy among different interferons, as interferon beta-1b has the lowest inhibitory concentrations in vitro falzarano et al. (2013a) and falzarano et al. (2013b) lopinavir-ritonavir lopinavir-ritonavir showed efficacy in in vitro and in a marmoset model. it is being tested in combination with interferon beta-1b in a randomized controlled trial (mers-cov infection treated with a combination of lopinavir /ritonavir and interferon beta-1b (miracle), nct02845843) chan et al. (2015) convalescent plasma the feasibility of the option is limited due to the paucity of donors arabi et al. (2015) monoclonal antibodies several monoclonal antibodies exist with promising efficacy in in vitro and in animal studies polycolonal antibodies demonstrated promising efficacy in in vitro and in animal studies. phase i trial has been completed. plans for phase ii trial are undergoing beigel et al. (2018) mycophenolate mofetil efficacy has been suggested in vitro but harm in a marmoset model chan et al. (2015) remdesivir this new drug has promising efficacy in in-vitro and in animal studies. phase i trial has been completed. phase ii trial is ongoing in ebola survivors agostini et al. (2018) there is no licensed human vaccine for mers-cov till now, however, many experimental candidate vaccines are under development. another approach is to vaccinate camels, as the source of infection for many human cases, and good progress has been made in this area (alharbi, 2017) . as of end of december 2018, the global case fatality rate for mers-cov infection was reported as 35.3% (806/2279). it is thought this number overestimates the case fatality rate of the disease, because milder and asymptomatic cases are likely to be underrepresented in the reported cases. this was suggested in a study that estimated the number of undetected human symptomatic cases to be 62% (cauchemez et al., 2014) . in a cohort of 47 mers-cov infected patients, case fatality rate was higher with increasing age (assiri et al., 2013a) . in another study that studied 939 mers-cov infected patients, independent risk factors for mortality were, age more 80 years, underlying cardiac comorbidity or cancer, and healthcare acquisition of the virus (alsahafi and cheng, 2016) . in a south korean cohort of 159 patients, risk factors for death were older age and underlying comorbidities (majumder et al., 2015) . coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department vaccines against middle east respiratory syndrome coronavirus for humans and camels histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-beta1b (miracle trial): study protocol for a randomized controlled trial corticosteroid therapy for critically ill patients with middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group transmission of mers-coronavirus in household contacts an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection development and validation of different indirect elisas for mers-cov serological testing super-spreading events of mers-cov infection clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia mers outbreak in korea: hospital-to-hospital transmission extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications middle east respiratory syndrome coronavirus outbreak in the republic of korea mortality risk factors for middle east respiratory syndrome outbreak, south korea middle east respiratory syndrome coronavirus in bats, saudi arabia family cluster of middle east respiratory syndrome coronavirus infections mers-cov outbreak in jeddah-a link to health care facilities ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study multihospital outbreak of a middle east respiratory syndrome coronavirus deletion variant ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study critically ill healthcare workers with the middle east respiratory syndrome (mers): a multicenter study unique case of papillary fibroelastoma originating from the right interatrial septum middle east respiratory syndrome (mers patient with new strain of coronavirus is treated in intensive care at london hospital genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 middle east respiratory syndrome coronavirus (mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia key: cord-307405-qk1ruj5q authors: hall, aron j.; tokars, jerome i.; badreddine, samar a.; saad, ziad bin; furukawa, elaine; al masri, malak; haynes, lia m.; gerber, susan i.; kuhar, david t.; miao, congrong; trivedi, suvang u.; pallansch, mark a.; hajjeh, rana; memish, ziad a. title: health care worker contact with mers patient, saudi arabia date: 2014-12-17 journal: emerg infect dis doi: 10.3201/eid2012.141211 sha: doc_id: 307405 cord_uid: qk1ruj5q to investigate potential transmission of middle east respiratory syndrome coronavirus (mers-cov) to health care workers in a hospital, we serologically tested hospital contacts of the index case-patient in saudi arabia, 4 months after his death. none of the 48 contacts showed evidence of mers-cov infection. to investigate potential transmission of middle east respiratory syndrome coronavirus (mers-cov) to health care workers in a hospital, we serologically tested hospital contacts of the index case-patient in saudi arabia, 4 months after his death. none of the 48 contacts showed evidence of mers-cov infection. cov) was initially isolated in september 2012 from a 60-year-old man from bisha, saudi arabia (1). in june 2012, this index case-patient was hospitalized for severe respiratory disease in jeddah at dr. soliman fakeeh hospital and subsequently died (1) . as of november 3, 2014, a total of 897 laboratory-confirmed cases of mers-cov infection, including 325 deaths, had been reported to the world health organization; >85% of reported mers-cov cases and deaths have occurred in saudi arabia (2) . the clinical syndrome among hospitalized mers-cov patients includes severe acute respiratory illness, sometimes associated with hypoxemic respiratory failure and extrapulmonary organ dysfunction (3); however, milder illness and asymptomatic infections have been identified through contact investigations (4-6). transmission of mers-cov to health care workers (hcws) has been reported (5,6), although no sustained community transmission has been identified. a zoonotic origin of mers-cov has been hypothesized; camels potentially play a role in transmission (7), although the specific types of exposure associated with primary cases remain unknown. there remains a dearth of information on how mers-cov is spread and on transmission risks to hcw or other close contacts. our objectives were to evaluate the degree and nature of hcw contact with the mers-cov index case-patient and to serologically assess hcws for mers-cov infection. the field investigation was performed in october 2012; we awaited development and validation of mers-cov serologic assays before completing the study. the index case-patient was hospitalized on june 13, 2012, with a 7-day history of fever, cough, sputum expectoration, and shortness of breath. precautions to prevent airborne transmission were taken by placing the patient in a private room with negative pressure for the first 2 days of hospitalization. after an infectious disease consultation on day 2, airborne-transmission precautions were replaced with droplet-transmission precautions; after a multidrug-resistant organism was isolated on day 4, contact-transmission precautions were implemented (8) . the case-patient remained in a private room, under standard and contact-transmission precautions, throughout hospitalization until he died on day 11. using dates and units in which the case-patient received care, hospital staff initially identified hcws who had had contact with the case-patient (came within 2 meters of the case-patient or his bedding, equipment, or body fluids). a comparison group of approximately equal numbers of hcws (preferentially with similar job responsibilities) was selected from hcws who had had no known contact with the case-patient. hospital infection control staff administered a brief, standardized questionnaire to both groups of hcws. information was collected on hcw demographics, job duties, and symptoms of respiratory disease during june 15-july 4, 2012, which corresponds to the period when the case-patient was hospitalized and an incubation period of 2-10 days, based on mers-cov natural history information available at the time of investigation. specific information about circumstances of case-patient contact and potential for mers-cov exposure was collected from hcws who had had contact with the case-patient. in october 2013 (4 months after the case-patient's death), a blood specimen (<20 ml) was collected from each hcw and transported first to the ministry of health western regional laboratory in saudi arabia and then to the us centers for disease control and prevention for mers-cov testing. upon receipt, specimens were gamma-irradiated on dry ice and stored at -70°c. all specimens were tested by hku5.2n nucleocapsid enzyme immunoassay (eia) (9); incubations and substrate development were conducted at 37°c. mers-cov antibody positivity was defined as positive hku5.2n screening eia results and confirmed by mers-cov immunofluorescence or microneutralization assay (9,10); specimens with negative eia results were considered antibody negative. of 56 hcws identified as having had contact with the mers-cov case-patient, 5 were unavailable for interview and 3 refused serum collection, leaving 48 for the final analysis. among hcws who had had case-patient contact, median age was 30.5 (range 22-57) years; 29 (60%) were female; 24 (50%) were nurses; 14 (29%) were physicians; 7 (15%) were respiratory technicians; and 1 each was a housekeeping, radiology, or infection control staff member. six (13%) hcws reported having a chronic medical condition (e.g., asthma, diabetes, hypertension), and 6 (13%) reported smoking tobacco. according to body mass index (bmi) calculations from self-reported height and weight, nearly half of hcws were overweight (bmi 25.0-29.9, n = 11 [23%]) or obese (bmi ≥30.0, n = 12 [25%]). most of the 48 hcws had reportedly come within 1 meter of the case-patient (89%); touched the case-patient (85%); or touched the case-patient's bedding, equipment, or body fluids (62%) (table) . during a single shift, most (60%) hcws reported <1 hour of case-patient contact, but 23% reported >5 hours of contact. hcws reported having been present during airway suction (50%), nebulizer treatment (30%), sputum induction (23%), bronchoscopy (9%), and intubation (6%). infection control precautions reportedly used by hcws during contact with the case-patient included hand hygiene (100%) and/or wearing of gloves (94%), surgical mask (87%), and gown (40%); however, among those reporting use of these precautions, some admitted to <100% compliance and none used eye protection. respiratory disease symptoms during june 15-july 4 were reported by 10 (21%) hcws who had had case-patient contact. among these, symptoms included cough (40%), sore throat (30%), myalgia (30%), rhinorrhea (20%), self-reported fever (10%), diarrhea (10%), and sneezing (10%); 2 (20%) of these 10 hcws sought medical care and received a diagnosis of pharyngitis. for comparison, respiratory symptoms were reported during the same period by 16 (33%) hcws who had not had case-patient contact. results of eia testing of serum collected during october 14-17, 2012, from all 48 hcws who had and all 48 who had not had case-patient contact were negative for mers-cov. immunofluorescence assay of 13 randomly selected serum specimens also gave negative mers-cov results, supporting interpretation of eia-negative specimens as antibody negative. this investigation provides indicators of transmission potential during the initial emergence of mers-cov. the lack of mers-cov transmission among hcws in this study is similar to results of some published contact investigations (11, 12) but different from others that reported transmission to hcw contacts (5, 6) . recovery of cultivable virus from the sputum of the case-patient reported here (1) and the severity of illness suggest some potential for virus transmission. we did not assess use of recommended personal protective equipment during each episode of casepatient contact; however, the reported lack of use during every episode of patient contact suggests that some hcws might have been exposed to mers-cov. nonetheless, the infection control precautions that were used might have contributed to the demonstrated lack of transmission. serologic assays to determine past infections are useful for assessing the risk for mers-cov transmission (13) . the assays used in this study have previously detected mers-cov-specific antibodies <2 weeks and >13 months after illness onset with >97% specificity (9, 14) . although no severely ill hcws were identified during this investigation, real-time reverse transcription pcr detection of mers-cov in respiratory tract specimens from asymptomatic or mildly ill hcws during other contact investigations has been described (5, 15) . the timing of this investigation 4 months after hospitalization of the case-patient precluded use of real-time reverse transcription pcr and potentially introduced recall bias during hcw interviews. despite numerous case-patient contact episodes among hcws, we found no serologic evidence suggesting health care-associated transmission of mers-cov from the index case-patient. rapid identification of potentially infected patients and implementation of infection control precautions can help protect hcws. us centers for disease control and prevention recommendations for management of hospitalized patients with known or suspected mers-cov infection include implementing standard, contact-and airborne transmission precautions (15) . isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-update. disease outbreak news epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study evidence of person-to-person transmission within a family cluster of novel coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infections in health care workers human infection with mers coronavirus after exposure to infected camels, saudi arabia guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description stillbirth during infection with middle east respiratory syndrome coronavirus the united kingdom public health response to an imported laboratory first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission consortium for the standardization of influenza seroepidemiology. novel coronavirus (mers-cov) clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus (mers-cov) into the united states first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities we are extremely grateful for the collaboration of the participating hcw in this investigation. additionally, we thank idris sulemann for assistance with data entry and management and jennifer l. harcourt, hayat caidi, and azaibi tamin for performing the serologic evaluations. key: cord-265769-96p07nyz authors: perlman, stanley; zumla, alimuddin title: mers-cov in africa—an enigma with relevance to covid-19 date: 2020-10-06 journal: lancet infect dis doi: 10.1016/s1473-3099(20)30578-8 sha: doc_id: 265769 cord_uid: 96p07nyz nan three novel zoonotic coronaviruses that have jumped the species barrier and caused lethal disease in humans have focused global public health attention in the past two decades: severe acute respiratory syndrome coronavirus (sars-cov) in 2003, middle east respi ratory syndrome coronavirus (mers-cov) in 2012, 1 and sars-cov-2 in late december 2019 2 -the cause of the ongoing covid-19 pandemic. 2 all these coronaviruses are featured in the who blueprint list of priority pathogens for research and development, because they threaten global health security, they have epidemic potential, and there are no effective treatments or vaccines. 3 in africa, only sars-cov-2 has caused significant outbreaks and disruption to health services. while african governments implement national mitigation and containment strategies 4 to control the spread of covid-19, mers-cov continues to circulate in the middle east causing intermittent outbreaks with up to 34% mortality. 1 mers-cov is highly prevalent in dromedary camels on the arabian peninsula and, they are the main sources of primary human mers-cov infections. 5 however, there have not been any reported clinical cases from africa of confirmed human mers, despite 70% of the world's dromedaries residing in africa (with high mers-cov infection rates) and frequent occupational and domestic contact between dromedaries and humans. 6 little serological evidence of human infection exists. 7, 8 this anomalous disparity between human mers-cov infections in the arabian peninsula and africa has not been easily explained and there has been a longstanding need for further studies of mers-cov at the animal-human interface. in the lancet infectious diseases, chris ka pub mok and colleagues 7 present data that indicate that human mers-cov infection is an occupational hazard in dromedary abattoir workers in nigeria. they focus our attention on the potential for mers-cov transmission and human infection and consequent threat to people living in africa and to public health services. their observational cohort study of people working at a slaughterhouse in kano, nigeria, identified mers-cov-specific cd4+ or cd8+ t cells in pbmcs obtained from 61 workers with close contact to dromedaries, but not in 20 other workers from the same slaughterhouse with no dromedary contact. these results agree with others showing that mers-cov virus-specific t-cell responses are more sensitive than serological tests for detecting past infection. 8 thus, zoonotic mers-cov infections of dromedary-exposed individuals are probably taking place in nigeria, and, by extrapolation, the incidence of human mers infections in all regions of africa with dromedaries has probably been underestimated. further studies are required to determine the extent of human mers-cov infections across africa. genetic and phenotypic differences in west african viruses might also be relevant to zoonotic outbreak potential. the great diversity of mers-cov lineages, and the large number of mers-cov infected dromedaries in africa, 5 might, with time, lead to mers-cov adapting and transmitting more efficiently, with epidemic potential. the findings of ka pub mok and colleagues 7 also have implications for ongoing efforts on covid-19 diagnostics development, testing, and tracing in africa and for vaccine development in general. 4 mild cases of mers and covid-19 induce antibody responses that are transient or wane rapidly. 8 specific diagnostic tests for mers-cov and sars-cov-2 infected individuals based on relevant antigenspecific cd4+ or cd8+ t-cell responses could allow for more sensitive detection of past or present infection than serological tests and identify additional human mers and covid-19 cases. t-cell responses appear to be more stable and useful for detection of previous infection or vaccine response in settings where virusspecific antibody titres are absent. it would also be interesting to know if dromedary-exposed workers with mers-cov-specific t-cell responses are protected from developing severe mers on rechallenge, and by extrapolation, whether t-cell responses are protective against severe covid-19 disease, even if virus-specific antibody is not detectable. to confirm these results, these studies should be repeated at other sites in africa and with larger numbers of samples. furthermore, no mers-specific t-cell responses were detected in abattoir workers with no dromedary exposure. by contrast, low numbers of sars-cov-2-specific t cells have been detected in uninfected individuals. 9 these apparent differences in pre-existing immunity to the two coronaviruses are unexpected and need to be flickr -jean & nathalie reconciled. the results also suggest that covid-19 and mers vaccines should be formulated to induce t-cell responses to maximise the likelihood of long-term protection. covid-19 and mers provide unique opportunities for african and middle eastern countries with relevant stakeholders to align and synergise ongoing covid-19 and mers surveillance, diagnostics, vaccine development, and evaluation, with basic science and translational research activities. 1,10 african and middle eastern countries must invest more in surveillance and urgent priority research to fill major knowledge gaps in the epidemiology, transmission, pathogenesis, and evolution of mers-cov and sars-cov-2, especially because they are co-circulating. the need for an effective one human-environmental-animal health multidisciplinary consortium across africa, middle east, and asia to tackle the ever-present threat of lethal coronaviruses and other emerging infections remains a global priority. 10 middle east respiratory syndrome another decade, another coronavirus prioritizing diseases for research and development in emergency contexts. world health organization from easing lockdowns to scaling-up community-based covid-19 screening, testing, and contact tracing in africa-shared approaches, innovations, and challenges to minimize morbidity and mortality enzootic patterns of middle east respiratory syndrome coronavirus in imported african and local arabian dromedary camels: a prospective genomic study mers-cov in camels but not camel handlers t-cell responses to mers coronavirus infection in people with occupational exposure to dromedary camels in nigeria: an observational cohort study recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals confronting the persisting threat of the middle east respiratory syndrome to global health security we have a special interest in coronaviruses and infectious diseases with epidemic potential. we declare no competing interests. key: cord-301547-d4wt9dqp authors: seng, j. j. b.; yeam, c. t.; huang, w. c.; tan, n. c.; low, l. l. title: pandemic related health literacy a systematic review of literature in covid-19, sars and mers pandemics date: 2020-05-11 journal: nan doi: 10.1101/2020.05.07.20094227 sha: doc_id: 301547 cord_uid: d4wt9dqp background: health literacy plays an essential role in ones ability to acquire and understand critical medical information in the covid-19 infodemic and other pandemics. purpose: to summarize the assessment, levels and determinants of pandemic related health literacy and its associated clinical outcomes. data sources: medline, embase, psychinfo, cinahl, arxiv, biorxiv, medrxiv, and social science research network. the start date was unrestricted and current as of 22 april 2020. study selection studies which evaluated health literacy related to novel coronavirus disease 2019 (covid-19), severe acute respiratory syndrome (sars) or middle east respiratory syndrome (mers) data extraction data on the characteristics of study designs, instruments, participants and level of health literacy were collected. items used in instruments were grouped under the themes of knowledge, attitudes and practices. determinants of health literacy were grouped into five domains (socio-demographic, medical, psychological/psychiatric, health systems related and others). data synthesis: of 2,065 articles screened, 70 articles were included. 21, 17 and 32 studies evaluated health literacy related to covid-19, sars and mers, respectively. the rates of low pandemic health literacy ranged from 4.3 to 57.9% among medical-related populations and 4.0% to 82.5% among non-medical populations. knowledge about symptoms and transmission of infection; worry about infection and, practices related to mask usage and hand hygiene was most frequently evaluated. socio-demographic determinants of health literacy were most studied, where higher education level, older age and female gender were associated with better health literacy. no studies evaluated outcomes associated with health literacy. limitations non-english articles were excluded. conclusion: the level of pandemic related health literacy is sub-optimal. healthcare administrators need to be aware of health literacy determinants when formulating policies in pandemics. with the rapid progression of the novel coronavirus disease 2019 (covid-19) into a pandemic infecting over 2.5 million patients worldwide, the need to gather and synthesize health-related information to make timely behaviour changes among people has become quintessential. (1, 2) this comes in the wake of an "infodemic" with evolving scientific knowledge about infections being generated daily, which has led to reversals in infection prevention recommendations made within a short span of time. (2) (3) (4) for example, the use of cloth masks during the early stages of the covid-19 pandemic was discouraged by the world health organisation due to uncertainty about its efficacy. (3) however, its potential use in slowing the spread of covid-19 has led to subsequent recommendations by the us centers for disease control and prevention for it to be worn by healthy individuals. (4) the ease of access to information via social and online media platforms has also become a double-edged sword in this pandemic where there has been substantial propagation of misinformation. (5) faced with the continuous influx of information related to this pandemic, an individual's level of health literacy exerts a vital role in one's ability to acquire, discern and understand accurate medical information. health literacy is broadly defined as the "level of capacity one has to obtain, process and understand basic health information and services needed to make appropriate health decisions." europe where 47% of the population were shown to have limited health literacy. (8) in the setting of non-communicable diseases, the association between health literacy with increased healthcare costs, morbidity and mortality is well-established. (9) the equal importance of health literacy in communicable diseases was highlighted in the recent covid-19 crisis and previous coronavirus pandemics such as the severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). (1, 10) in contrast to general health literacy required for the prevention or management of chronic diseases, these pandemics require an individual's readiness and adaptive ability in developing their pandemic related and critical health literacy quickly. this is critical as the rapid and successful implementation of infectious diseases control measures requires the collective compliance of all individuals. (11, 12) network (ssrn) were extracted for evaluation. keywords employed in the search strategy included terms related to health literacy as well as the viruses and syndromes implicated in the three coronavirus pandemics which were namely covid-19, mers and sars. terms related to health literacy were adapted from reviews which evaluated health literacy in other patient populations. (18, 19) the full search strategy was detailed in supplementary file 1. the start date of the search was unrestricted and current as of 22 april 2020. full-text articles, both peer-reviewed and non-peer reviewed in the english language were retrieved from the eight databases. studies which evaluated health literacy related to covid-19, sars or mers among adult participants aged ≥ 18 years old from the general population, healthcare sectors and infected patients were included. for the study designs, both interventional and observational studies such as cohort, cross sectional and case control studies were included. case series, case reports, other irrelevant meta-analyses and systematic reviews were excluded. we also excluded studies which evaluated paediatric populations and non-human subjects. two independent reviewers (jjb seng and ct yeam) performed the screening and inclusion of articles. all disagreements encountered during the review process were discussed. in situations where the disagreements could not be resolved, a third independent reviewer (cwh huang) arbitrated to achieve consensus. data extracted included the socio-demographic and clinical characteristics of the study participants such as their age, race/ethnicity, education levels, income levels, study designs, instruments used for assessment of health literacy, the definition of health literacy used in studies, level of health literacy, factors associated with health literacy and clinical outcomes associated with health literacy. for the risk of bias assessment, the quality assessment tool for studies by national health, lung and blood institute was adopted to evaluate the methodological quality of included articles.(20) each study is rated as low, moderate and high risk of bias by the two independent reviewers (jjb seng and ct yeam) based on the responses obtained from the ten items. in situations where insufficient information was available to score an item, the authors of the study were contacted for clarification. if the authors could not be contacted, the item was rated as high risk of bias. all disagreements were resolved via discussion between the two reviewers. only studies which were rated as low and moderate risk of bias were included in this review. descriptive statistics were used to summarize the characteristics of included studies. with regards to the level of health literacy, we reported the average percentage of correct answers or the percentage of participants with low health literacy as defined by cut-offs described in each study, where available. as there are no gold-standard health literacy instruments developed for covid-19, sars or mers(21), significant heterogeneity is expected in the types of tools used for assessment of health literacy across participants. consequently, meta-analysis could not be performed. questions from instruments used across included studies were classified into three main themes, which were 1) knowledge, 2) attitudes and 3) practices, to help guide future development of standardised covid-19 and pandemic health literacy tools. the analyses were segregated by medical and non-medical populations due to the expected differing levels of health literacy in the two populations. for studies where the questionnaires were not available, study authors were contacted for the questionnaire. if there were no replies from the authors, the themes were extracted from the description of the questionnaires in the main text. a framework for core items to be included in pandemic health literacy tools was also proposed based on common themes assessed across studies. for factors associated with better health literacy, they were categorized into five domains which encompassed socio-demographic, medical, psychological/psychiatric, health systems related and others. a narrative review was provided for the factors evaluated among included studies. clinical outcomes associated with poor health literacy among patients infected with covid-19, mers and sars included time from illness onset to seeking medical treatment, hospitalisation and duration of hospitalisation, admission to intensive care units and length of icu stay, need for ventilator support, recovery from infection and re-infection. this study was not funded by any organisation. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 11, 2020. . https://doi.org/10.1101/2020.05.07.20094227 doi: medrxiv preprint figure 1 shows the flowchart for the inclusion of articles. a total of 1,965 published articles and 40 pre-prints were retrieved. after removal of duplicates, exclusion of irrelevant articles and inclusion of articles identified from hand-searching, a total of 70 articles were included in this review. the percentage of concordance during the initial article screening was 90%. details pertaining to the study designs and characteristics of participants among included studies were reported in supplementary file 2. for the risk of bias, 48 (68.5%) and 22 (31.4%) studies were rated as low and moderate risk of bias. no studies were rated as high risk of bias (supplementary file 3). table 1 shows a summary of the characteristics of the included studies. majority of included studies were cross-sectional in design (n=65, 92.9%) and were conducted during the pandemics (n=69, 98.6%). 21 (30%) studies recruited more than 1000 participants. a total of 21 (30.0%) studies examined health literacy related to covid-19 ( table 1) . majority of the studies were conducted in asia (71.4%) and north america (14.3%). most studies were conducted among the general population (n=10, 47.6%). the primary mode of health literacy assessment across studies was via online questionnaires (n=20, 95.2%). aspects of health literacy that were assessed in the instruments included knowledge (n=20, 95.2%), attitudes (n=17, 81%) and practices (n=14, 66.7%), of which only 7 (33.3%) studies performed validation of their questionnaire. most questionnaires (n=8, 38.1%) contained 11-20 items. pertaining to health literacy, the average percentage of correct answers among medical personnel ranged from 67.0 to 94.8%, and low health literacy was reported among 5. table 1) . majority of studies were mostly performed in asia (82.4%), europe (11.8%) and north america (5.9%). the most common groups of study participants included the general population (n=8, 58.8%) and healthcare professionals (n=3, 17.6%). for the assessment of health literacy, these were conducted primarily via . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 11, 2020 among the pandemics, the most number of studies examined health literacy related to mers (n=32, 45 .7%) ( table 1 ). the studies were mostly conducted in asia (n=30, 93.8%) and europe (n=2, 6.3%) where the majority of them were conducted in saudi arabia (n=27, 84.4%). the most common group of participants recruited comprised of healthcare professionals (n=11, 34.4%) and medical students among the three themes, pandemic related knowledge was most studied, followed by practices and attitudes (tables 2-4 ). in the knowledge domain, symptoms (13, 14, 16, , transmission (14, 16, 22, 23, 25-29, 31-34, 37-49, 51, 52, 55-57, 59-78) and incubation period of the virus (16, 23, 26-28, 32, 37, 38, 41, 42, 46-50, 52-57, 59-64, 66-69, 78) ; management and treatment options (14, 16, 22-24, 26, 27, 29, 37, 39-43, 46, 48-51, 55, 56, 58, 60, 62, 64, 66-70, 78, 79) ; and clinical outcomes associated with infection (13, 16, 23-27, 29, 34, 37, 38, 40, 48, 50-52, 55, 56, 59, 61, 63-67, 69, 74-76, 78-81) ; high risk populations for infection (16, 23, 26, 27, 29, 37, 39-41, 48, 49, 55. cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may 11, 2020. . 1 0 59, 63, 65, 67, 68, 72, 78, 79) ; availability of vaccine (16, 29, 37, 39, 40, 42-44, 48, 52, 55, 56, 58, 60-63, 65, 68, 70, 74-76, 79 ); role of hand hygiene (14, 16, 24, 25, 28, 29, 42, 43, 45, 47, 48, 50, 52, 55-58, 60, 64, 66, 69-71, 82, 83) was most studied for medical and non-medical staff. (table 2 ) for medical related populations specifically, knowledge about epidemiology (29, 37, 39, 42, 44, 53, 58, 59, 61-63, 65, 66, 78, 79) and diagnosis of infection (16, 46, 48, 49, 55, 56, 58-63, 67, 76) were also frequently evaluated. for attitudes about pandemics, worry/fear/helplessness about pandemic (13, 14, 22, 28, 31, 33, 49, 51, 52, 60, 61, 63, 65, 68, 72, 76-78, 81, 83, 84) , confidence in governments' ability to manage pandemic (13, 23, 28, 30, 33, 41, 61, 63, 65, 68, 70, 77, 78, 82) and perceived severity of infection as a public health problem (13, 29, 39, 41, 44, 45, 59, 65, 70, 72) was most commonly assessed. (table 3) for practices in pandemics, behaviours related to mask utilization (14, 16, 23, 26, 28, 30, 32, 33, 35, 38, 40, 48, 59, 61, 63, 65, 70, 73, 80, 82, (84) (85) (86) , hand hygiene (14, 16, 28, 32, 33, 35, 38, 40, 41, 46, 59-61, 63, 65, 70, 73, 77, 85, 86) , personal hygiene (16, 30, 33, 35, 38, 41, 48, 60, 61, 63, 65, 67, 70, 73, 80, 82, (84) (85) (86) and information seeking (16, 25, 28-30, 33, 35, 36, 42, 44, 48-51, 53-56, 58, 59, 63, 65, 69, 70, 72, 75, 76, 80, 84, 85, 87) were most commonly studied. (table 4 ) figure 2 shows the proposed framework (pandemic-hl) for items to be included in generic pandemic health literacy tools. across the five domains for health literacy related factors, 34 factors were identified. (table 5 ) among these, socio-demographic-economic and health systems-based domains were the most studied. sociodemographic factors which were commonly associated with better health literacy included higher educational level (23, 26, 27, 33, 34, 36, 37, 39, 40, 42, 43, 45, 72-77, 80-82, 88, 89) , increased age (23, 26, 30, 37, 40, 43, 48, 50, 63, 65, 72, 74, 75, 78, 84, 85) and female gender (13, 16, 23, 26, 33, 34, 38, 40, 41, 44, 45, 60, 62, 72, 74, 78, 80, 81, 84, 85, 89) . for health systems-based factors, increased experience in the healthcare system (48, 56, 63, 76, 77, 89) and attendance in health education programs (28, 33, 58, 60, 71, 78) were associated with better health literacy. for medical and psychiatric/psychological factors, increased general health literacy (13, 30) and increased anxiety about the spread of infection (28, 33, 80, 84) were associated with better health literacy. lastly, other factors 1 1 associated with better health literacy included the use of traditional sources of information such as newspaper or television (28, 33, 75) . among the included studies, no studies evaluated clinical outcomes related to covid-19, sars or mers. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . https://doi.org/10.1101/2020.05.07.20094227 doi: medrxiv preprint for populations at increased risk of poor clinical outcomes of infections such as the elderly, immunocompromised patients, human-immunodeficiency virus or with multiple comorbidities, they form high priority populations where the levels of pandemic health literacy should be assessed. (95, 96) in the context of the current covid-19 pandemic, our review only found 1 study which specifically evaluated the health literacy related to covid-19 among these high-risk populations. (13) it is imperative that future research is undertaken to evaluate the health literacy among these patient populations for targeted interventions to be designed for patients if required. significant heterogeneity in the instruments used for the assessment of pandemic related health literacy was noted in our review. currently, there is no gold-standard instrument which evaluates pandemicrelated health literacy. given the need for validated and standardised tools to be created to facilitate the evaluation of pandemic related health literacy, the pandemic-hl framework was proposed to guide the selection of topics to be addressed in instruments. it was modelled after psychosocial models of health behaviours and encompassed key topics which were frequently evaluated in instruments across the three pandemics. (97) it is hoped that the framework will serve as a foundation for facilitating the development of health literacy tools for future pandemics. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . https://doi.org/10.1101/2020.05.07.20094227 doi: medrxiv preprint this may limit the generalizability of the study results. (98) additionally, the validity of these study results may be affected as these surveys commonly suffer from poor response rates. (98) with regards to the determinants of pandemic related health-literacy, higher education levels, older age, female gender, and being employed were the most studied factors associated with higher pandemic . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . the main strength of this review was that health literacy in previous coronaviruses related pandemics such as sars and mers were evaluated to provide a more comprehensive overview of pandemic related health literacy. however, the findings from this review should also be interpreted with the following limitations. firstly, while we adopted a reasonably comprehensive search strategy, potentially relevant articles may have been missed. finger searching within the references of included articles was performed to minimize this omission of potentially relevant articles. secondly, we were only able to include articles in the english language due to the language limitations of the authors. thirdly, we were not able to perform meta-analyses for the overall level of pandemic related health literacy and their determinants due to the heterogeneity in instruments. with the development of a standardised instrument for the assessment of health literacy related to pandemics, future studies should consider using meta-analyses to compare the level of health literacy across different populations. lastly, the full questionnaires could not be accessed for 27 studies. while themes described in the main text of these articles were carefully extracted, we could not rule out the omission of themes which were not described. future health literacy studies should append their questionnaires to allow meaningful evaluation of the study results. overall, the level of pandemic related health literacy remains sub-optimal among both the medical and non-medical population. this is worrisome given the critical role health literacy serves in reducing the spread of contagion and mitigating the effects of pandemics. there is an urgent need to develop up-todate, validated and standardised questionnaires for the rapid assessment of pandemic-related health literacy. important determinants associated with better levels of health literacy such as older age, female gender, employment status and education level were highlighted in this review. healthcare administrators and policymakers need to be mindful of these determinants when formulating dissemination of critical pandemic related information and interventions to improve the health literacy of the population. more studies are required to evaluate the clinical outcomes associated with pandemic related health literacy. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . jjb seng was the study's principal investigator and was responsible for the conception, initial literature review and design of the study. ct yeam, cw huang, nc tan and ll low were the co-investigators. jjb seng, ct yeam and cw huang were responsible for the screening and inclusion of articles and data extraction. all authors contributed to the data analyses and interpretation of data. jjb seng prepared the initial draft of the manuscript. all authors revised the draft critically for important intellectual content and agreed to the final submission. . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . https://doi.org/10.1101/2020.05.07.20094227 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . . it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. definitions of terms used in precautionary measures e.g. "safe distance", "close contacts", " transient contacts" is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. control measures implemented by government e.g. restriction of travel to china repercussions associated with non-compliance with measures implemented by government or authorise control measures implemented by healthcare institutions it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. belief that infection is a biochemical weapon developed by foreign countries or terrorists feelings of fatigue after outbreak is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. . it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may 11, 2020. covid-19: health literacy is an underestimated problem. the lancet public health how to fight an infodemic. the lancet advice on the use of masks the community, during home care and in health care settings in the context of the novel coronavirus (2019-ncov) outbreak 2020 use of cloth face coverings to help slow the spread of covid-19 coronavirus disease 2019: the harms of exaggerated information and non-evidence-based measures office of disease prevention and health promotion. national action plan to improve health literacy the prevalence of limited health literacy health literacy in europe: comparative results of the european health literacy survey (hls-eu) low health literacy: implications for national health policy determinants of health literacy and health behavior regarding infectious respiratory diseases: a pathway model controlling epidemic spread by social distancing: do it well or not at all. bmc public health critical health literacy and the covid-19 crisis. health promotion international awareness, attitudes, and actions related to covid-19 among adults with chronic conditions at the onset of the u.s. outbreak: a cross-sectional survey study of knowledge, attitude, anxiety & perceived mental healthcare need in indian population during covid-19 pandemic. asian journal of psychiatry the singaporean response to the sars outbreak: knowledge sufficiency versus public trust middle east respiratory syndrome (mers): comparing the knowledge, attitude and practices of different health care workers determinants of health literacy in the general population: results of the catalan health survey evidence on the effectiveness of health literacy interventions in the eu: a systematic review protocol for a systematic review of interventions addressing health literacy to improve asthma self-management. npj primary care respiratory medicine measuring health literacy regarding infectious respiratory diseases: a new skills-based instrument 2019-novel coronavirus survey: knowledge and attitudes of hospital staff of a large italian teaching hospital among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online cross-sectional survey use of rapid online surveys to assess people's perceptions during infectious disease outbreaks: a crosssectional survey on covid-19 a preliminary assessment of novel coronavirus (covid-19) knowledge and perceptions in nigeria knowledge and behaviors toward covid-19 among u.s. residents during the early days of the pandemic knowledge and practices towards covid-19 during its outbreak: a multinational cross-sectional study population anxiety and positive behaviour change during the covid-19 epidemic: cross-sectional surveys in singapore, china and italy evaluation of preparedness of healthcare student volunteers against middle east respiratory syndrome coronavirus (mers-cov) in makkah, saudi arabia: a cross-sectional study an outbreak of the severe acute respiratory syndrome: predictors of health behaviors and effect of community prevention measures in hong kong, china experiencing sars: perspectives of the elderly residents and health care professionals in a knowledge and practice concerning severe acute respiratory syndrome among the institutionalized elderly in hong kong public knowledge, attitude, behaviour and response to the sars outbreak in singapore attitude and practice towards sars sars knowledge, perceptions, and behaviors: a comparison between finns and the dutch during the sars outbreak in 2003 a study on sars awareness and health-seeking behaviourfindings from a sampled population attending national healthcare group polyclinics awareness among a saudi arabian university community of middle east respiratory syndrome coronavirus following an outbreak awareness, attitudes, and practices related to coronavirus pandemic among public in saudi arabia effectiveness of an education health programme about middle east respiratory syndrome coronavirus tested during travel consultations is the saudi public aware of middle east respiratory syndrome? middle east respiratory syndrome risk perception among students at a university in south korea public awareness of coronavirus in al-jouf region, saudi arabia knowledge of middle east respiratory syndrome coronavirus (mers-cov) and its management: a survey among saudi people in taif; kingdom of saudi arabia middle east respiratory syndrome-corona virus: knowledge and attitude of qassim university students, ksa bin saeed a. knowledge, attitude and practice of secondary schools and university students toward middle east respiratory syndrome epidemic in saudi arabia: a cross-sectional study covid-19 and iranian medical students; a survey on their related-knowledge, preventive behaviors and risk perception dentists' awareness, perception, and attitude regarding covid-19 and infection control: cross-sectional study among jordanian dentists knowledge, attitude, practice and perceived barriers among healthcare professionals regarding covid-19: a cross-sectional survey from pakistan a survey-based study on the knowledge, attitude, and the practices pertaining to the 2019 novel corona virus infection amongst undergraduate medical students in india assessment of iranian nurses' knowledge and anxiety toward covid-19 during the current outbreak in iran. archives of clinical infectious diseases knowledge of severe acute respiratory syndrome among community physicians, nurses, and emergency medical responders severe acute respiratory syndrome (sars): knowledge, attitudes, practices and sources of information among physicians answering a sars fever hotline service knowledge and attitude of dental health professionals about middle east respiratory syndrome in saudi arabia knowledge and attitude of healthcare workers about middle east respiratory syndrome in multispecialty hospitals of qassim, saudi arabia knowledge and practices of dentists regarding mers-cov. a cross-sectional survey in saudi arabia knowledge and practices of primary health care physicians regarding updated guidelines of mers-cov infection in abha city attitudes and behaviours of healthcare workers in the kingdom of saudi arabia to mers coronavirus and other emerging infectious diseases. international journal of environmental research and public health middle east respiratory syndrome-related knowledge, preventive behaviours and risk perception among nursing students during outbreak raising awareness of health care providers about merscov infection in public hospitals in mecca, saudi arabia are saudi medical students aware of middle east respiratory syndrome coronavirus during an outbreak? attitude and practices of healthcare providers towards mers-cov infection at makkah hospitals awareness about middle east respiratory syndrome-corona virus (mers-cov) among dental students in riyadh, saudi arabia knowledge, attitude, and practice toward mers-cov among primary health-care workers in makkah al-mukarramah: an intervention study knowledge and attitude toward middle east respiratory syndrome coronavirus among heath colleges' students in najran, saudi arabia. international journal of community medicine and public health attitude and practice on mers-cov among nursing students at hail university exploring knowledge and attitude toward middle east respiratory syndrome-coronavirus (mers-cov) among university health colleges' students, saudi arabia: a cross-sectional study. american journal of infectious diseases assessment of the awareness level of dental students toward middle east respiratory syndrome-coronavirus perceptions of the adult us population regarding the novel coronavirus outbreak improving older adults' knowledge and practice of preventive measures through a telephone health education during the sars epidemic in hong kong: a pilot study knowledge of and attitudes toward severe acute respiratory syndrome among a cohort of dental patients in hong kong following a major local outbreak. community dent health the knowledge level and precautionary measures taken by older adults during the sars outbreak in hong kong mers-cov infection: mind the public knowledge gap adequacy of public health communications on h7n9 and mers in singapore: insights from a community based cross-sectional study assessing knowledge, attitudes and practices of dental practitioners regarding the covid-19 pandemic: a multinational study knowledge, attitude and practice regarding covid-19 among health care workers in henan knowledge and attitudes towards middle east respiratory sydromecoronavirus (mers-cov) among health care workers in south-western saudi arabia knowledge and awareness of middle east respiratory syndrome coronavirus among saudi and non-saudi arabian pilgrims sars risk perception, knowledge, precautions, and information sources, the netherlands. emerging infectious diseases knowledge and apprehension of dental patients about mers-a questionnaire survey an evaluation of information dissemination during the severe acute respiratory syndrome (sars) outbreak among selected rural communities in kuala kangsar sars risk perceptions in healthcare workers behaviour of singaporeans during the sars outbreak: the impact of anxiety and public satisfaction with media information impacts of sars on health-seeking behaviors in general population in hong kong epub 2005/05/27 hajj pilgrims knowledge about middle east respiratory syndrome coronavirus knowledge and attitudes of medical staff in chinese psychiatric hospitals regarding covid-19 knowledge and beliefs towards universal safety precautions to flatten the curve during novel coronavirus disease (ncovid-19) pandemic among general public in india: explorations from a national perspective awareness of droplet and airborne isolation precautions among dental health professionals during the outbreak of corona virus infection in riyadh city, saudi arabia health literacy interventions and outcomes: an updated systematic review health literacy now: developing a web site for communicating clearly with patients health literacy in the ehealth era: a systematic review of the literature. patient education and counseling smog grading-a new readability formula covid-19) -united states coronavirus disease 2019 (covid-19) pandemic and pregnancy world health organisation. a guide to developing knowledge, attitude and practice surveys when to use web-based surveys gender differences in health literacy among korean adults: do women have a higher level of health literacy than men? won s-y, pascall g. a confucian war over childcare? practice and policy in childcare and their implications for understanding the korean gender regime education, social status, and health health literacy and older adults: a systematic review a cross-sectional study of pandemic influenza health literacy and the effect of a public health campaign literacy and health outcomes: a systematic review of the literature health literacy and access to care guarantor's name jjb seng is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. key: cord-287159-bjccnp7u authors: yavarian, jila; shafiei jandaghi, nazanin zahra; naseri, maryam; hemmati, peyman; dadras, mohhamadnasr; gouya, mohammad mehdi; mokhtari azad, talat title: influenza virus but not mers coronavirus circulation in iran, 2013–2016: comparison between pilgrims and general population date: 2017-10-12 journal: travel med infect dis doi: 10.1016/j.tmaid.2017.10.007 sha: doc_id: 287159 cord_uid: bjccnp7u background: the pilgrimage to mecca and karbala bring many muslims to a confined area. respiratory tract infections are the most common diseases transmitted during mass gatherings in hajj, umrah and karbala. the aim of this study was to determine and compare the prevalence of middle east respiratory syndrome coronavirus (mers-cov) and influenza virus infections among iranian general population and pilgrims with severe acute respiratory infections (sari) returning from mecca and karbala during 2013–2016. methods: during 2013–2016, a total of 42351 throat swabs were examined for presence of influenza viruses and mers-cov in iranian general population and pilgrims returning from mecca and karbala with sari by using one step rt-pcr kit. results: none of the patients had mers-cov but influenza viruses were detected in 12.7% with high circulation of influenza a/h1n1 (47.1%). conclusion: this study showed the prevalence of influenza infections among iranian pilgrims and general population and suggests continuing surveillance, infection control and appropriate vaccination especially nowadays that the risk of influenza pandemic threatens the world, meanwhile accurate screening for mers-cov is also recommended. the middle east respiratory syndrome coronavirus (mers-cov) was first identified in a patient from kingdom of saudi arabia (ksa) in june 2012 [1] . according to world health organization (who) report, until 21 september 2017, the number of laboratory-confirmed cases of mers-cov was 2081, with 722 deaths. most of the cases originated from or had a history of travel to middle-east. mecca and karbala are places in the middle-east which are visited by muslims especially during hajj, umrah and arbaeen. ksa hosts about 2.5 million muslim pilgrims from more than 180 countries during the hajj pilgrimage annually. hajj is one of the largest mass gatherings of its kind in the world. umrah is a visit to the holy sites in ksa the same as hajj but it can be occurred at any time during the year. during the hajj, respiratory tract infections are the leading cause of hospitalization in ksa [2, 3] . karbala is a holly place in iraq which muslims visit there during the year especially arbaeen. arbaeen is a shia muslim ritual that occurs forty days after the day of ashura (10th day of the month of muharram). it celebrates the death of hussein ibn ali, the grandson of prophet mohammad, who was killed on the day of ashura. arbaeen is the world largest annual pilgrimage as more than 20 millions of shia muslims gather in the city of karbala in iraq. mass gathering of people in a confined area specially hajj and arbaeen increases the risk of respiratory tract infections which are very common and responsible for most of the hospital admissions. after june 2012 global concern was about the potential for mers-cov spreading by travelers returning from the pilgrimage. for early detection of emerging respiratory viruses, the international health regulations emerging committee established a program for all countries (especially those with returning pilgrims) to strengthen their surveillance to detect and report any new cases. however ksa has been reported the majority of mers-cov cases (> 80%) since 2012, but in the 6.5 million pilgrims in hajj 2012 and 2013 no mers-cov cases were reported [4] . influenza viruses are important human respiratory pathogens with high morbidity and mortality that cause both seasonal and endemic infections. nowadays emergence of h5n1 and h7n7 is the concern for https://doi.org/10.1016/j.tmaid.2017. 10 [5, 6] but there is no published data about the prevalence of respiratory virus infections during arbaeen. among hajj pilgrims, influenza is the most common vaccine preventable virus infection, but its epidemiology is poorly understood in mass gatherings [7] . beside detection of mers-cov, we designed this study to investigate about the importance of influenza vaccination in general population and pilgrims. in iran, the influenza season starts in late november and lasts until late april, peaking in january and february. the national influenza center (nic) in iran, located at virology department, school of public health, tehran university of medical sciences, examines clinical samples from patients with severe acute respiratory infections (sari) for influenza virus surveillance throughout the year in general population and/or pilgrims. after mers detection in 2012, all suspected cases were tested in nic and the first mers case, a 52 year old woman with a history of hypertension, was confirmed in may 2014, iran [8] . with continues surveillance totally six mers cases were identified in iran which the last one was in march 2015. the study's primary aim was screening the iranian pilgrims and general population with sari for detection of mers-cov during 2013-2016. the second aim was to assess the prevalence of influenza virus infections in these patients and the final aim was to comparison of influenza and mers-cov circulation between general population and pilgrims. throat swab specimens according to ministry of health protocol were collected from a total of 42351 patients with saris. of them, 38511 specimens were collected from general population and 3840 specimens were taken from arriving pilgrims at emam khomeini airport in tehran, 2013-2016. throat swabs were collected in viral transport media and immediately transported to nic, school of public health, tehran university of medical sciences. total nucleic acids were purified from a 200 μl sample using high pure viral nucleic acid kit (roche, germany) according to the manufacturer's instructions. each sample was tested independently in a 25 μl reaction for influenza a/b and mers-cov using quantifast probe rt-pcr kit (qiagen, germany). mers-cov was tested with targeting the upstream region of the e gene (upe) for screening and the open reading frame 1b for confirmation [9] . in total 42351 patients with saris were included in this study which 3840 were returning iranian pilgrims from mecca and karbala and 38511 were patients with sari who admitted to local hospitals. iranian pilgrims had symptoms upon arrival or a week later, thereby indicating that the respiratory infections were acquired during the pilgrimage. of 3840 pilgrims, 499 (13%) were positive for influenza viruses. during the years of study in all patients, circulating influenza strains differed but the pattern was similar in both pilgrims and general population. in in 2016 just in non-pilgrim patients three dual infections of influenza a/h1n1 and a/h3n2 viruses were detected in november. during the years of this study from 3840 iranian pilgrims, 46.1% (1773/3840) returned from karbala, 35.2% (1355/3840) came from umrah and 18.7% arrived from hajj. we did not have any pilgrims returning from mecca in 2016 but just 4.8% (185/3840) came from karbala. more information about the prevalence of different influenza strains in hajj, umrah, karbala and general population are shown in table 1 . this paper showed the results of study of mers-cov and influenza virus infections among pilgrims and non-pilgrim patients with sari during 2013-2016. each year more than 5 million muslims travel from all over the world to participate in hajj and umrah. approximately more than one million pilgrims travel from iran to ksa annually. in recent years more than 10 million iranian pilgrims have been gathering during arbaeen in karbala. in this study 46.1% (1773/3840) of pilgrims returned from karbala which 13.6% were influenza positive with a/h1n1 predominance. in a study on 177 iranian pilgrims to karbala who admitted to iraqi hospitals, 3.39% suffered from respiratory infections [10] . in another study from a total of 26574 pilgrims admitted to iranian clinics in iraq, the main cause was acute respiratory infections (48%) [11] . generally performing the pilgrimage in a confined area is associated with an increased occurrence of respiratory infections in the pilgrims. transmission of different infectious diseases during mass gatherings in holly places has a global effect when pilgrims return to their country. in 1989 a meningococcal disease outbreak and its global spread during the hajj lead to this fact that meningococcal vaccine became a mandatory vaccine for all pilgrims [12] . according to the vaccination protocol in iran, all pilgrims had received meningococcal vaccination, but influenza vaccination is not mandatory and we do not have data about its vaccination in this group. however in a review by gautret et al. no remarkable effect of influenza vaccination on the influenza infection of pilgrims was found. apparently this lake of efficiency of influenza vaccine might be the result of mismatch between circulating influenza viruses with vaccine strains [2] . influenza viruses are common respiratory viruses with high mortality and morbidity especially in young children and elderly. in iran influenza viruses are circulating throughout the year with a big peak during cold months. since 2012 besides influenza virus screening nic examines clinical samples for mers-cov detection from suspected patients throughout the year in general population and/or pilgrims. we previously reported that a cluster of mers-cov was detected in kerman/iran in 2014 among nonpilgrims [8] . current study showed that among the population screened, no cases were positive for mers-cov. these results were in accordance with previous studies which have performed among pilgrims of different countries. a cohort of 5235 pilgrims attending the 2013 hajj showed the lack of mers-cov in nasal carriage [13] . in a study on 154 french hajj pilgrims in 2012, in spite of high rate of respiratory infections, mers-cov was not detected [14] . these findings suggest that mers-cov in its current form has poor interhuman transmission and may not have the pandemic potential as seen in influenza a/h1n1 in 2009. however investigation about a highly fatal human coronavirus is necessary as it is a challenge and little is known about its importance, epidemiology and zoonotic total patients 544 141 268 8321 87 366 528 6630 724 205 792 14453 185 9107 influenza positive 3 6 21 392 2 137 34 653 54 49 168 2430 19 1372 a/h1n1 -1 5 116 -73 6 125 15 16 133 1577 9 454 a/h3n2 1 3 10 173 1 8 10 272 38 5 15 432 9 717 b 2 2 6 103 1 56 18 256 1 28 20 421 1 201 transmission. in pilgrims of this study influenza b accounted for 27% (135/499) and influenza a for 71.7% (358/499) of positive influenza results in contrast to findings by balkhy et al., in 2003 , that 90% of pilgrims had influenza b and 10% had influenza a [15] . the results of a uk study with paired serum samples collected before and after the hajj using hemagglutination inhibition test, showed that 38% of uk pilgrims had influenza infection during the hajj 2003 [16] . in another study during hajj 2005, 14% of uk pilgrims with respiratory infections had influenza virus [17] . rashid et al., in 2008 performed a comparative study in symptomatic uk and saudi pilgrims which found infections in 25% and 13% of their pilgrims respectively. rhinoviruses were detected in half of uk pilgrims, followed by influenza virus but in saudi pilgrims 78.5% had influenza virus infection [18] . in 2009, alborzi et al. reported that 9.8% of iranian hajj pilgrims with respiratory infections had influenza [19] . in 2012, 305 iranian pilgrims with respiratory infections returning from hajj were assessed for detection of a/h1n1pdm which just five patients (1.69%) were positive [20] . in a survey on serum samples of 338 iranian pilgrims before and after hajj with elisa, 3.6% were influenza positive [21] . in another iranian study on serum samples of hajj pilgrims in 2004-2005, before departure and two weeks after respiratory infections, there was a 21.5% seroconversion for influenza viruses. while virus culture on their sputum was 13.3% influenza positive [22] . in a study on 275 symptomatic iranian hajj pilgrims, 25 (9.1%) were influenza positive by virus culture whereas 33 (12%) had influenza with rt-pcr test [23] . the findings of this research showed that influenza virus infection was the cause of respiratory infections in 499 of 3840 (13%) of iranian pilgrims. in a similar study in kashmir, north india during 2014-15 among returning hajj and umrah pilgrims with respiratory illness, none of the 300 participants tested positive for mers-cov; however, 33 (11%) tested positive for influenza viruses [24] . in general population, of 38511 sari patients, 4868 (12.6%) were influenza positive during the years of this study with different circulation of the subtypes as seen in other studies: timmermans et al. performed a study on 586 outpatients with influenza-like-illness in western cambodia between may 2010 and december 2012. influenza was found in 168 cases (29%). dominant influenza subtypes were a/h1n1 in 2010, influenza b in 2011 and influenza a/h3n2 in 2012 [25] . in a study by mancinelli et al. a total of 133 respiratory specimens positive for the influenza a and b viruses were subtyped during the 2012-2013 influenza season in italy. influenza b was slightly more prevalent (53.38%) than influenza a (46.62%) and the most common subtype was a/h1n1 (87.1%) while only 12.9% were a/h3n2 [26] . in a ten year (2004-2014) study of influenza surveillance in northern italy, the same as our study influenza a/h3n2 was prominent during 2013-2014 [27] . the results of this study showed similar pattern of virus circulation in pilgrims and non-pilgrims sari patients. as influenza has high morbidity and mortality, its vaccination is recommended for general population especially for high risk groups and pilgrims before going to pilgrimage. finally accurate screening and testing for mers-cov and other respiratory viruses including influenza, is necessary for early diagnosis to prevent virus transmission and to do effective treatment. as a final point lack of demographic and clinical data was the most important limitation of this study. jila yavarian performed the analyses of the data and wrote the paper. nazanin zahra shafiei jandaghi reviewed the paper critically, and comments were included. maryam naseri performed the tests. peyman hemmati, mohammadnasr dadras were responsible for epidemiological investigation and data collection. mohammad mehdi gouya and talat mokhtari azad were responsible for study design. none. isolation of a novel coronavirus from a man with pneumonia in saudi arabia hajj-associated viral respiratory infections: a systematic review pattern of admission to hospitals during muslim pilgrimage (hajj) public health management of mass gatherings: the saudi arabian experience with mers-cov influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination enhanced surveillance of influenza and other respiratory viruses among uk pilgrims to hajj pandemic influenza: mass gatherings and mass infection cluster of middle east respiratory syndrome coronavirus infections in iran detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction the most frequent causes of hospitalization of iranian pilgrims in iraq during a 5-month period in 2012, and their outcome prevalence of diseases in pilgrims referring to iranian clinics in iraq travel epidemiology: the saudi perspective prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination influenza among uk pilgrims to hajj influenza and respiratory syncytial virus infections in british hajj pilgrims viral respiratory infections at the hajj:comparison between uk and saudi pilgrims viral etiology of acute respiratory infections among iranian hajj pilgrims pandemic 2009 influenza a (h1n1) infection among 2009 hajj pilgrims from southern iran: a real-time rt-pcr-based study acute respiratory viral infections among tamattu' hajj pilgrims in iran surveying respiratory infections among iranian hajj pilgrims influenza viral infections among the iranian hajj pilgrims returning to shiraz, fars province. iran influenza other respir viruses influenza not mers cov among returning hajj and umrah pilgrims with respiratory illness human sentinel surveillance of influenza and other respiratory viral pathogens in border areas of western cambodia clinical features of children hospitalized with influenza a and b infections during the 2012-2013 influenza season in italy ten influenza seasons in france: distribution and timing of influenza a and b circulation we thank all staff in national influenza center, virology department, school of public health, tehran university of medical sciences. key: cord-293481-bmfj50fb authors: malin, jakob j.; suárez, isabelle; priesner, vanessa; fätkenheuer, gerd; rybniker, jan title: remdesivir against covid-19 and other viral diseases date: 2020-10-14 journal: clin microbiol rev doi: 10.1128/cmr.00162-20 sha: doc_id: 293481 cord_uid: bmfj50fb patients and physicians worldwide are facing tremendous health care hazards that are caused by the ongoing severe acute respiratory distress syndrome coronavirus 2 (sars-cov-2) pandemic. remdesivir (gs-5734) is the first approved treatment for severe coronavirus disease 2019 (covid-19). it is a novel nucleoside analog with a broad antiviral activity spectrum among rna viruses, including ebolavirus (ebov) and the respiratory pathogens middle east respiratory syndrome coronavirus (mers-cov), sars-cov, and sars-cov-2. first described in 2016, the drug was derived from an antiviral library of small molecules intended to target emerging pathogenic rna viruses. in vivo, remdesivir showed therapeutic and prophylactic effects in animal models of ebov, mers-cov, sars-cov, and sars-cov-2 infection. however, the substance failed in a clinical trial on ebolavirus disease (evd), where it was inferior to investigational monoclonal antibodies in an interim analysis. as there was no placebo control in this study, no conclusions on its efficacy in evd can be made. in contrast, data from a placebo-controlled trial show beneficial effects for patients with covid-19. remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. although this is an important milestone in the fight against covid-19, approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable. tients with covid-19. remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. although this is an important milestone in the fight against covid-19, approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable. i n december 2019, a novel coronavirus (ncov), severe acute respiratory distress syndrome coronavirus 2 (sars-cov-2), emerged in wuhan, central china. like in sars-cov, infections with the closely related sars-cov-2 cause a respiratory disease that can progress to viral pneumonia and acute respiratory distress syndrome (ards) (1) . because of its onset in december 2019, the associated disease was called coronavirus disease 2019 . through 26 june 2020, the ongoing pandemic caused more than 9 million confirmed covid-19 cases and nearly 500,000 deaths globally (2). in the light of an uncontrolled expansion and the steadily increasing covid-19 fatalities in january and february 2020, huge efforts were put into the identification of effective antiviral agents against covid-19. nucleoside/nucleotide analogs are one of the most promising antiviral drug classes in general, and significant drug discoveries emerged from this class that today form the basis for treatments against infections with several herpesviruses, human immunodeficiency virus (hiv), hepatitis b virus (hbv), and hepatitis c virus (hcv). remdesivir or gs-5734 is a prodrug of a nucleoside analog with direct antiviral activity against several single-stranded rna viruses, including sars-cov and middle east respiratory syndrome coronavirus (mers-cov). the first cell-based studies of remdesivir also showed antiviral activity against the novel sars-cov-2 (3, 4) . in the absence of any effective treatment options against covid-19, remdesivir has been applied under compassionate use. recently, preliminary data from a randomized placebo-controlled clinical trial showed that remdesivir reduces the time to recovery in patients with covid-19 (5) , leading to an emergency-use authorization (eua) by the u.s. food and drug administration (fda) only 2 days after the first press release from the national institute of allergy and infectious diseases (niaid) (6) . on 3 july, the european medicines agency (ema) granted a conditional marketing authorization for remdesivir, now being the first approved antiviral treatment against covid-19 (7) (fig. 1 ). here, we provide a comprehensive review of results from preclinical and clinical studies on this important novel antiviral drug to understand its clinical significance. in addition, we briefly describe the discovery and molecular mechanism of viral inhibition. nucleoside and nucleotide analogs as small-molecule-based antivirals have been explored for many years and form the backbone of treatment against viral infections, including hiv, hepatitis b virus, and herpesvirus infections (8) (9) (10) . in 2013, the nucleotide analog sofosbuvir was approved by the fda for the treatment of chronic hepatitis c virus infections. the novel compound that targets the rna-dependent viral polymerase (ns5b) revolutionized hcv treatment, as it is able to cure the formerly lifelong chronic progressive disease when combined with other antivirals (11) . in the past years, nucleoside/nucleotide analogs were increasingly recognized as potential antivirals targeting other positive-stranded rna viruses such as members of the flaviviridae, picornaviridae, caliciviridae, and coronaviridae families, as they share relevant amino acid sequences with hcv (12) , and the rna-dependent polymerases are closely related phylogenetically (13, 65) . this supported the assembly of antiviral compound libraries that could be screened against emerging rna viruses. in the past years, several pharmacological advances in the development of nucleoside analogs were made based on structure-to-activity relationship (sar) studies that improved pharmacokinetics, antiviral activity, and selectivity (14) (15) (16) . a comprehensive overview of the medicinal chemistry and pharmacological evolution of antiviral nucleoside analogs can be found elsewhere (17, 18) . nucleoside analogs require intracellular activation by phosphorylation in order to become their active metabolites. one of the most important milestones was the addition of a monophosphate prodrug to the nucleoside, which significantly improved intracellular delivery and activation (19) (20) (21) . this so-called pro-tide approach, developed by mcguigan et al. (22, 23) , was also used to optimize the precursor of remdesivir named gs-441524. the parent molecule of remdesivir, gs-441524, was derived from a small-molecule library of around 1,000 diverse nucleoside and nucleoside phosphonate analogs that were assembled over many years of antiviral research based on their potential ability to target emerging rna viruses such as sars-cov and mers-cov of the coronaviridae or zika and dengue viruses of the flaviviridae family (20) . following the ebolavirus (ebov) epidemic in west africa from 2013 to 2016, a selection of promising leads from this library underwent intensive testing against different types of ebov in collaboration with the centers for disease control and prevention (cdc) and the u.s. army medical research institute of infectious diseases (usamriid), which included studies in nonhuman primates (nhps) (24) . these efforts finally led to the identification of gs-5734, a monophosphate prodrug version of gs-441524, as the most promising lead against ebov. gs-5734, later renamed remdesivir, had a broad antiviral spectrum, including ebov, marburg virus, respiratory syncytial virus (rsv), hcv, and several paramyxoviruses (20, 21, 24) , in vitro. in addition, it demonstrated activity against mers-cov (24-26) and sars-cov (25, 26) . favorable in vitro results stimulated further evaluation in ebov-infected macaques, where remdesivir suppressed viral replication and improved survival, clinical signs of the disease, and pathophysiological blood markers (24) . after its discovery, remdesivir was administered under compassionate use to patients with ebolavirus disease (evd) but stopped after an interim analysis of the first randomized controlled clinical trial (rct) showed an inferiority of remdesivir to treatments with monoclonal antibodies (mab114 and regn-eb3). the trial evaluated the efficacies of 3, 5, 20, 21, and 24-29.) different investigational therapeutics against evd. following the interim analysis, the remdesivir arm was halted for the remainder of the trial (27) . in december 2019, a novel coronavirus, sars-cov-2, emerged and caused a pandemic that is still ongoing. there were strong arguments for the antiviral effect of remdesivir against coronaviruses emerging from multiple cell-based in vitro models, including primary human airway epithelial (hae) cell cultures (25) , and, for mers-cov, from a mouse model of pulmonary infection (28) . in addition, in a rhesus macaque model of mers-cov infection, remdesivir demonstrated strong prophylactic properties, and administration was associated with clinical benefits for treated subjects (29) . the global hazards caused by the pandemic with the novel sars-cov-2 prompted the identification of potential treatment options. given the solid preclinical data, remdesivir was considered one of the most promising candidates that went into clinical testing against covid-19. remdesivir is a monophosphoramidate nucleoside prodrug that undergoes intracellular metabolic conversion to its active metabolite nucleoside triphosphate (ntp). as described for several other direct-acting antivirals, the active metabolite of remdesivir (remdesivir triphosphate [remdesivir-tp] or gs-443902) subsequently targets the machinery responsible for the replication of the viral rna genome, a highly conserved element of the viral life cycle. nucleoside analogs are synthetic compounds that work by competition with endogenous natural nucleoside pools for incorporation into replicating viral rna. while these compounds mimic their physiological counterparts, the incorporation of the analog molecule disrupts subsequent molecular processes. the drug target and the exact processes that lead to the inhibition of viral replication have been studied extensively in ebolavirus (24, 30) . the suggested drug target, the ebov rna-dependent rna polymerase (rdrp) complex, was only recently biochemically purified, which allowed for in-depth molecular analyses. viral rdrp is the target protein for the active metabolite remdesivir-tp. remdesivir-tp acts as the substrate for rdrp where it competes with atp for incorporation into new strands. inhibition of ebov rdrp most probably results from delayed chain termination, a mechanism that is known from approved antivirals against human immunodeficiency virus type 1 (hiv-1) and hbv (31) (32) (33) (34) . in the case of ebov, the incorporation of remdesivir-tp into replicating rna was observed to cause chain termination predominantly at five positions downstream (i ϩ 5) (30) . importantly, the activity of human rna polymerase is not inhibited in the presence of remdesivir-tp (24) . in sars-cov and mers-cov, remdesivir-tp interferes with the nsp12 polymerase, which is a multisubunit rna synthesis complex of viral nonstructural proteins (nsp's) produced as cleavage products of viral polyproteins. as nsp12 is highly conserved across the coronavirus family, it is most likely that the mechanism of action (moa) of remdesivir does not differ significantly among covs (35, 36) . like in ebov, remdesivir-tp efficiently inhibits the replication of sars-cov and mers-cov by causing delayed chain termination when being incorporated into the replicating rna (26) . a recent biochemical analysis revealed that in sars-cov-2, remdesivir-tp causes the termination of rna synthesis at three positions after the position where it is incorporated (i ϩ 3). this mechanism was nearly identical in rdrps of sars-cov and mers-cov (37) . the premature termination of rna synthesis ultimately abrogates further transcriptional and translational processes needed for the generation of new virions (fig. 2) . the resulting antiviral effects of remdesivir have been studied in different cell-based models. remdesivir has been tested against various viruses in different cell-based systems and target cells (table 1) . when comparing the results of antiviral assays, one has to take into account that different assay methodologies and parameters such as the target cell type and virus input used may have significant impacts on the efficacy outcome (38) . a variety of methods are available to assess the antiviral activity of candidate compounds in cell-based models. simplified, susceptible target cells allowing viral replication are infected and subsequently exposed to serial concentrations of the test compounds. historically, compounds were analyzed for their ability to reduce the amount of virus pfu on cellular layers (39) . although this method has the advantage of addressing the complete viral life cycle, it has been widely replaced by molecular methods that allow automated quantification. the antiviral effects of test compounds can be assessed by monitoring viral replication by either quantification of viral rna using real-time pcr (rtpcr) or measurement of fluorescent reporter gene expression (rge) from genetically modified virus strains. a special type of reporter gene assay is viral replicon (repl) assays using genetically modified virus genomes that undergo transcriptional and translational processes inside the target cell but do not yield infectious progeny. often, genes encoding structural proteins are exchanged by reporter genes that enable the monitoring of viral replication. this approach has been used to study the inhibition of hcv replication. alternatively, viral antigens (ags) can be quantified by fluorescence-or chemiluminescence-based immunostaining. antiviral effects can also be measured indirectly by assessing virus-induced cytopathic effects (cpe) in the presence of test compounds. assays measuring cpe can display not only direct antiviral effects but also beneficial effects of compounds with antivirulence properties. this is an advantage for high-throughput screening due to a gain of signal (40) . in addition, cpe can be used to measure antiviral activity when no robust rge-or rtpcr-based assay is available. it is unclear which type of assay provides the best information that can predict in vivo efficacy. however, the comparability of results from direct methods measuring viral replication and from cpe-based assays is limited by the fact that viral loads and related cpe do not automatically have a linear association. the choice of target cell displays another important factor. activity against filoviruses is classically assessed in vero e6 cells that were derived through the immortalization of african green monkey kidney cells. this cell line is known to highly express the angiotensin-converting enzyme 2 (ace-2) receptor, which is required for viral entry of both sars-cov and sars-cov-2 into the target cell (41) . in addition, vero e6 cells support the replication of sars-covs to high titers, which made them a standard cell model to study related pathogens (42) (43) (44) (45) . besides common target cells, the antiviral activity of remdesivir was evaluated in human cell lines and primary cells that represent more clinically oriented in vitro systems. activity against filoviruses was tested in a human liver cancer cell line (huh-7) and human primary macrophages (hpms). anti-cov activity was evaluated in a human lung epithelial cell line (calu-3), primary hae cells, and immortalized human foreskin microvascular endothelial cells (hmvecs). in contrast to sars-cov experiments that were conducted in hae cells, assays against sars-cov-2 were conducted in vero e6 cells, which might explain the 1-log-lower antiviral efficacy measured for sars-cov-2 (3, 25, 26) . a recent comparison of replication kinetics and cpe of sars-cov and sars-cov-2 in vero e6 cells concluded that there were no significant differences in drug sensitivities to remdesivir, thereby supporting this hypothesis (4). in 2016, warren et al. (24) tested the small-molecule nucleoside analog remdesivir (gs-5734) against ebov. the half-maximum effective concentrations (ec 50 s) for ebov inhibition were between 0.06 and 0.14 m in different cell types, including human macrophages and endothelial cells. it was also shown that remdesivir inhibits the replication of other pathogenic rna viruses such as rsv (ec 50 , 0.019 m) and mers-cov (ec 50 , 0.34 m) while having low cytotoxicity in a wide range of human primary cells and cell lines (24) . the characterization of the antiviral spectrum of remdesivir in vitro was subsequently reevaluated and expanded across multiple virus families, including representatives of the filo-, paramyxo-, pneumo-, arena-, rhabdo-, flavi-, and coronavirus families, including zoonotic and epidemic covs (20, 21, 25, 26, 28) . remdesivir effectively inhibited ebov (ec 50 , 0.003 to 0.1 m) and rsv (ec 50 , 0.021), with results being comparable to those reported by warren et al. (24) . in addition, efficacy against marburg virus (ec 50 , 0.014 to 0.19 m) and several paramyxoviruses (ec 50 , 0.018 to 0.79 m) was demonstrated in cell-based assays (21) . the ec 50 s for mers-cov and (46) demonstrated that the antiviral spectrum of remdesivir also includes porcine covs and endemic human covs (hcovs) that are associated with the common cold (hcov-oc43 and -229e). after the outbreak of sars-cov-2 in january 2020, remdesivir was rapidly tested in a vero e6 cell-based model that made use of direct viral quantification by rtpcr along with the antimalaria and immune-modulating drug chloroquine and known antivirals such as ribavirin and penciclovir. remdesivir was active against sars-cov-2, with an ec 50 of 0.77 m, which was slightly lower than that of chloroquine (ec 50 , 1.13 m) and remarkably lower than those of other tested antivirals (ec 50 , 2.12 to 109.50 m). more recently, a comprehensive comparison of the antiviral effects of remdesivir on the replication kinetics and cytopathology of clinical isolates of sars-cov and sars-cov-2 was made in vero e6 cells. here, both viruses showed similar sensitivities to remdesivir, with ec 50 s of 4.3 and 4.9 m, respectively, when measuring the 50% cytopathologic effect (4) . these values are substantially higher than those reported in previous studies that made use of reporter gene or rtpcr assays, which seems to have a methodological background. however, the higher values in this study may also reflect significantly lower susceptibilities of sars-cov and sars-cov-2 clinical isolates to remdesivir. choy et al. (47) recently determined the ec 50 (23.15 to 25 m) for another sars-cov-2 clinical isolate (hong kong/vm20001061/2020) using three different vero e6 cell-based assays. however, the comparability of these results to those of previous studies is limited by viral load calculations fitted to logarithmic scales (log 10 rna copies per milliliter) in order to estimate the effect of increasing drug concentrations. finally, efficacy data were also reported by the manufacturer. gilead's fact sheet on remdesivir reports an ec 50 of 0.137 m (preliminary data), but no information is given on the specific strain that was tested by the china cdc in collaboration with gilead sciences (48) . a complete overview of (peer-reviewed) published in vitro efficacy data is given in table 1 . nucleoside analogs such as remdesivir are generally expected to have a higher barrier to antiviral resistance than other antivirals such as neuraminidase inhibitors due to their well-conserved and vulnerable drug target (19) . however, a general obstacle in the development of antiviral nucleoside analogs against covs is the presence of a potent exoribonuclease (exon)-mediated proofreading function of the rdrp subdomain nsp12, which causes resistance against ribavirin and 5-fluorouracil (26) . exon is able to identify and remove incorporated nucleoside analogs. an important observation was that virus mutants lacking exon are 4-fold more sensitive to remdesivir (ec 50 , 0.019 m) than the wild type with intact proofreading, implying that remdesivir is prone to exon-mediated proofreading. nevertheless, this effect was shown to be concentration dependent, and even with intact exon proofreading, remdesivir inhibits viral replication still at submicromolar concentrations in direct antiviral assays (26) . the relatively modest effect of exon on remdesivir susceptibility was attributed to the fact that the incorporation of remdesivir into replicating rna occurs efficiently in comparison to natural nucleotides and to the mechanism of delayed chain termination that causes nsp12 inhibition by remdesivir (37, 49, 50) . the additional natural nucleotides that are subsequently incorporated may have protective effects against exon activity (49) . although nsp12 rdrp is highly conserved among covs, the amino acid identity still varies between 67 and 100% when including human and zoonotic covs (25) , and variations in the amino acid sequence could have effects on viral susceptibility to remdesivir. therefore, brown et al. (46) tested the antiviral activity of remdesivir in strains with the most divergent rdrp compared to sars-and mers-cov. they included endemic human covs (229e and oc43) and a porcine cov known to harbor a native residue in the nsp12 subunit that confers antiviral resistance in betacoronaviruses. they found that the activity of remdesivir includes both contemporary human and highly divergent zoonotic covs and that natural variations in wild-type rdrp do not confer remdesivir resistance. another important factor regarding the remdesivir drug target is that mutations leading to changes in neighboring amino acids of the nsp12 subunit will most probably result in a substantial loss of viral fitness. agostini et al. (26) demonstrated that mutations in nsp12 can be induced in wild-type murine hepatitis virus (mhv) by serial passage in the presence of increasing concentrations of gs-441524. these mutations (f476l and v553l) led to decreased remdesivir susceptibility, with a 2.4-fold or 5-fold shift in the ec 50 (0.057 to 0.13 m). the introduction of these substitutions into the sars-cov genome had a similar effect on remdesivir susceptibility, but mutants were unable to compete against the wild-type strain in coinfection passages without selective pressure. furthermore, variants carrying f476l or v553l were attenuated in vivo, as shown by decreased lung titers in a mouse model of sars-cov infection. in summary, remdesivir has a high genetic barrier to resistance development, and known resistant variants suffer from a loss of competitive fitness. this may suggest that these mutations will most likely not be maintained in nature and do not favor an uncontrolled spread of remdesivir-resistant variants. compounds that are able to block exon-mediated proofreading would be of interest for combination therapy as they significantly increase virus susceptibility to remdesivir in vitro. ebolavirus disease. the pro moiety of remdesivir can be degraded by serum esterase, which negatively affects efficacy and the pharmacokinetic profile. because of high serum carboxylesterase activity in most rodents primarily used for animal models, initially, there was no suitable rodent model to study the efficacy of remdesivir in vivo (24) . the first in vivo studies were therefore performed in nhps that have lower or no serum esterase activities and are therefore comparable to humans (51) . in an nhp model of fatal evd, rhesus monkeys were inoculated with ebov by intramuscular injection and treated for 12 days with an intravenous (i.v.) vehicle (n ϭ 3), 3 mg/kg of body weight on day 0 or day 2 (n ϭ 6 per group), 3 mg/kg after a 10-mg/kg loading dose on day 2 or day 3 (n ϭ 6 per group), or 10 mg/kg of gs-5734 once daily starting 3 days after inoculation. of 12 animals treated 3 days after virus exposure (10 mg/kg daily or 3 mg/kg with a 10-mg/kg loading dose), 100% of nhps survived the 28-day in-life phase. in contrast, only 8 out of 18 subjects treated on day 0 or 2 survived the infection (all at 3 mg/kg daily; 6 received a 10-mg/kg loading dose). antiviral effects (reduction in plasma viral rna) and reductions in clinical signs were more pronounced in the 10-mg/kg treatment group. warren et al. concluded that there was substantive postexposure protection with daily dosing of 10 mg/kg remdesivir (24) . for mers-cov, a head-to-head assessment of remdesivir versus combinations of lopinavir, ritonavir, and interferon beta was performed in esterase-deficient mice with a humanized dipeptidylpeptidase 4 (ddp4) receptor. humanization of the ddp4 receptor is required for mers-cov infection in mouse models because the wild-type ddp4 receptor does not enable mers-cov spike protein binding. wildtype mice are therefore not susceptible to mers-cov infections (52) . in this study, the therapeutic and prophylactic properties of remdesivir could be reproduced, and in the overall view of the authors, remdesivir was superior to other tested antivirals and combination treatments (28) . remdesivir was also evaluated in a rhesus macaque model of mers-cov infection. the prophylactic administration of 5 mg/kg 24 h before inoculation with mers-cov prevented all six tested animals from developing active disease. therapeutic administration by 12 h postinoculation reduced clinical signs of the disease, viral replication in the lungs, and pathological lung tissue processes in all six animals of the treatment group. those authors therefore considered remdesivir to be a potential candidate against the novel sars-cov-2 (29). given the limited plasma stability of remdesivir in rodents that is caused by high serum esterase activity, its antiviral activity against covs was evaluated in a mouse model of sars-cov infection using carboxylesterase 1c knockout (ces1c ϫ/ϫ ) mice. here, prophylactic and early therapeutic administration of remdesivir (24 h before or 24 h after inoculation) reduced viral loads in the lungs and improved respiratory functions and clinical signs of the disease when given subcutaneously at doses of 25 mg/kg twice daily (25) . in comparison to studies in nhps or humans, high doses of remdesivir are necessary in mouse models, as tissue levels of the active remdesivir metabolite are approximately 10 times lower than those in nhps, even in esterase-deficient mice. in addition, remdesivir-tp is more rapidly degraded in rodent lung tissues than in nhp lungs and primary human airway cells (25, 28) . recently, the efficacy of remdesivir treatment was finally tested in a rhesus macaque model of sars-cov-2 infection (53) . animals were infected with sars-cov-2 (n ϭ 12) by combined intranasal, oral, ocular, and intratracheal inoculations and subsequently treated with intravenous placebo or remdesivir (10-mg/kg loading dose followed by 5 mg/kg daily) for 6 days starting at 12 h postinfection. remdesivir-treated animals did not show signs of respiratory disease and had lower lung virus titers and less lung tissue damage than the placebo group. according to the manufacturer's information on the pharmacokinetic bridge from rhesus monkeys to humans, these doses approximate serum drug exposures that are equivalent to 200 mg and 100 mg, respectively (48) . it is also important to note that the dynamics of acute sars-cov-2 infection progress more rapidly in animal models than in humans and that optimal treatment time points that are calculated based on expected viral load peaks cannot be directly translated to humans (54) . although the results of this study have to be interpreted with caution until final publication, they support early treatment initiation with remdesivir for sars-cov-2 infections. more than 1,200 adult patients, 76 pediatric patients, and 96 pregnant women with covid-19 were treated with remdesivir through the compassionate-use program according to the manufacturer gilead sciences. liver function test abnormalities were reported in 19 of 163 evaluated cases (55) . once the covid-19 epidemic started in china, at a time when there was no clinical trial in preparation, the first observational data for remdesivir arose from patients treated under compassionate use. in a prospective cohort study funded by gilead sciences, 61 patients with covid-19 were treated with remdesivir for a 10-day course (200 mg on day 1, followed by 100 mg daily). clinical improvement was observed in 36 (68%) of 53 evaluable patients (56) . another study from italy reported on 35 patients treated with remdesivir in a general infectious disease ward. interpretations of data from this study are very limited due to the low sample size, as only 22 patients completed a 10-day treatment course. the most frequent adverse events (aes) were elevations of liver transaminase levels (15/35 patients) and acute kidney injury (8/35 patients) (57) . as there were no control groups, no efficacy statements can be made based on these studies. one approach with a simulated control group is currently under peer review and suggests reductions in mortality with remdesivir (58) . several clinical trials were conducted to evaluate its efficacy against evd and covid-19. ebolavirus disease. two studies on remdesivir were conducted in the context of ebolavirus disease, prevail iv (clinicaltrials.gov identifier nct02818582) and the pamoja tulinde maisha (palm) trial (clinicaltrials.gov identifier nct03719586). prevail iv was a small phase 2 study in 38 men with evidence of ebolavirus persistence in their semen, who were formerly included in the observational evd survivor study prevail iii. however, no reliable safety or efficacy data on remdesivir were derived from this study. the first randomized controlled phase 2/3 clinical trial evaluating the efficacy of remdesivir (palm) started in 2018 in the democratic republic of congo during an outbreak of evd. within 9 months, a total of 681 patients were enrolled in the study assessing the efficacies of four different therapeutic strategies in an open-label parallel 1:1:1:1 design. patients received either remdesivir, the single monoclonal antibody mab114, a coformulated composition of 3 human igg1 monoclonal antibodies called regn-eb3, or the triple monoclonal neutralizing antibody complex zmapp (control group). the primary endpoint was death at day 28 of enrollment. an interim analysis on 9 august 2019 that included data from 499 patients showed that the mortality rates with both zmapp (84/169; 49.7%) and remdesivir (93/175; 53.1%) were higher than those with mab114 (61/174; 35.1%) and regn-eb3 (52/155; 33.5%). therefore, randomization into these groups was subsequently stopped. as this study did not include a placebo control arm, no definite conclusions on the clinical efficacy of remdesivir against evd can be made. however, the observed mortality rate of approximately 50% is comparable to that of the natural course of the disease and thus does not suggest a substantial clinical benefit of remdesivir (59) . covid-19. the first phase 3 randomized, double-blind, placebo-controlled trial evaluating the efficacy of remdesivir against covid-19 (clinicaltrials.gov identifier nct04257656) started in february 2020 in wuhan. hospitalized patients with severe covid-19 were enrolled (defined as having hypoxia and radiological signs of lung involvement) and treated for 10 days with standard doses of remdesivir (200 mg on day 1 and 100 mg on days 2 to 10; n ϭ 158) or a placebo (n ϭ 79). due to the rapidly changing dynamics of the outbreak in china, with a local decrease in new includable cases during march 2020, the trial was stopped preterm with only 237 patients enrolled and could not reach the calculated target enrollment size. in the final analysis of the present data set, treatment with remdesivir was not associated with significant clinical improvement in the treatment arm compared to the placebo (hazard ratio [hr], 1.23 [95% confidence interval {ci}, 0.87 to 1.75]). furthermore, there was no significant difference between groups regarding mortality and time to viral clearance. in a subgroup of patients who were treated early within 10 days of symptom onset, remdesivir was associated with a numerical median reduction of 5 days in the time to clinical improvement, but this finding was not statistically significant (hr, 1.52 [95% ci, 0.95 to 2.43]). those authors therefore proposed to evaluate remdesivir earlier in the course of covid-19 (60) . the adaptive covid-19 treatment trial (actt) started at the end of february 2020 and included a total of 60 study sites globally. a total of 1,063 hospitalized patients with all stages of covid-19 that included signs of lower respiratory tract involvement (hypoxia or radiological evidence) were enrolled until 19 april 2020. patients received either standard doses of remdesivir (n ϭ 541) or a placebo (n ϭ 522) for 10 days in a double-blind design. the primary outcome was time to recovery, defined as discharge from the hospital or continued hospitalization for infection control purposes only (no further medical treatment needed) (5). on 27 april 2020, the data and safety monitoring board (dsmb) concluded that there was a significant effect of remdesivir after reviewing an interim data analysis. based on the available data, the dsmb recommended enabling patients of the placebo arm to benefit from a switch to remdesivir, which required early unblinding for a limited number of patients (61) . the preliminary data have recently been published and show that treatment with remdesivir was associated with a reduction in the time to recovery from a median of 15 to 11 days (recovery rate ratio [rrr], 1.32 [95% ci, 1.12 to 1.55] [p ͻ 0.001]). this effect was independent of symptom duration prior to randomization. but subgroup analyses showed that patients with the need for oxygen therapy (ordinal score of 5) benefit most from the treatment (rrr, 1.47 [95% ci, 1.17 to 1.84] [n ϭ 421]), while no effect could be demonstrated for patients on invasive ventilation and/or extracorporeal membrane oxygenation (ecmo). the mortality rate by 14 days was 7.1% (remdesivir) versus 11.9% (placebo), which was not statistically significant (hr, 0.7 [95% ci, 0.47 to 1.03] [p ϭ 0.06]) (5) . based on the preliminary results of this trial, the fda issued an emergency-use authorization for remdesivir only 2 days after the initial press release from the niaid. while complete results have not yet been published, another clinical trial was conducted, which evaluated the optimal treatment duration with remdesivir. the randomized open-label trial gs-us-540-5773 (previously simple) started in march 2020 and evaluated the efficacy of a 10-day treatment regimen (n ϭ 197) versus a 5-day regimen (n ϭ 200) of remdesivir in severe covid-19. the results were recently published and suggest similar effects of 5-day and 10-day treatments when adjusting for baseline clinical status. clinical improvement, clinical recovery, and mortality by day 14 were assessed. due to the absence of a control group, these results do not permit an overall assessment of the efficacy of remdesivir (62) . in addition to the study of severe covid-19 cases, another open-label trial in patients with moderate covid-19 is ongoing (table 2) . gilead sciences, the manufacturer of remdesivir, conducted four phase 1 clinical trials to evaluate the safety, tolerability, and pharmacokinetics of remdesivir (gs-us-399-1812, -1954, -4231, and -5505) in a total of 138 patients, of whom 131 received remdesivir and 7 received a placebo. overall, the drug is generally well tolerated. adverse events (pooled data) occurred in only a few cases and included phlebitis (8 subjects), constipation (7), headache (6), ecchymosis (5), nausea (5) , and pain in extremities (5) . only a few grade 1 and 2 laboratory abnormalities were detected: transient elevations of alanine aminotransferase (alt)/aspartate aminotransferase (ast) levels from day 5 until day 25 (12 subjects), mild reversible prolongation of the prothrombin time without changes in international normalized ratio (inr) (7 subjects), and mild hyperglycemia (4 subjects). there were no signs of nephrotoxicity in healthy subjects and no patterns of clinically relevant changes in vital signs or electrocardiograms (63) . the available safety data from phase 1/2 studies are provided in a fact sheet for health care providers that was published in the context of the emergency-use authorization issued by the fda and can be downloaded (55) . in the first phase 3 trial of remdesivir that was conducted in the context of evd, one event of hypotension occurred in the remdesivir arm that was judged as not being related to underlying evd by the site investigators. the event occurred during the administration of the loading dose and led to a fatal cardiac arrest. an independent pharmacovigilance committee concluded that the death could not be readily distinguished from underlying fulminant evd (27) . safety data from the compassionate-use study are not conclusive as there was no control group. however, the most frequently reported adverse events in patients treated with remdesivir were increases in hepatic enzyme levels (32 patients; 60%) and diarrhea (5 patients; 9%) (56) . in the chinese phase 3 trial where 155 patients with covd-19 received remdesivir, no deaths occurred that were judged as being possibly related or related to the study drug. the frequencies of adverse events (most frequently constipation, hypoalbuminemia, hypokalemia, and anemia) were virtually identical in the treatment and placebo groups (60) . preliminary data from the actt study do not change this picture. the incidences of most adverse events were not found to be significantly different among the treatment and placebo groups. grade 3 to 4 adverse events in general and some adverse events such as anemia or increased transaminase levels occurred slightly more often in the placebo group (grade 3 to 4 aes in 172 versus 156 with remdesivir). other adverse events occurred slightly more often in the remdesivir group (increased creatinine levels, pyrexia, and hyperglycemia) (5) . in the openlabel trial on patients with severe covid-19, the most common adverse events were nausea (9%), worsening of respiratory failure (8%), elevated ast levels (77%), and constipation (7%) (62) . taken together, at this time, there is no evidence for grade 3 to 4 or even severe adverse events resulting from once-daily doses of remdesivir (75 mg up to 225 mg i.v.) for treatment durations of up to 14 days. the drug seems to be well tolerated. grade 1 and 2 adverse events have been described in healthy volunteers and patients with covid-19 treated with remdesivir and mainly refer to transient elevations of alt or ast levels. there are not sufficient data on the safety of remdesivir in patients younger than nucleoside analogs require active cellular uptake by nucleoside transporters and intracellular activation by cellular and viral kinases to become their active ntp metabolite. this activation process requires three phosphorylation steps, of which the first step is most often inefficient and rate limiting (64) . a common problem of nucleoside analogs is that they yield suboptimal levels of ntp at the site of infection (65) . remdesivir is a monophosphoramidate prodrug of a 1=-cyano-substituted adenosine nucleoside analog that is able to bypass the rate-limiting first phosphorylation step to effectively deliver intracellular ntp (66) . the prodrug component is necessary to mask the negatively charged phosphonate group, which allows faster entry into target cells independently of membrane transporters. phosphonate-containing pronucleotides have the disadvantage that their diacids are deprotonated at a physiological ph (67) . remdesivir has suboptimal oral bioavailability and therefore can be administered only by intravenous infusion in the actual formulation. however, there might be pharmacological approaches to solve this problem. one example of a clinically approved nucleoside phosphonate with an oral formulation is the nucleoside reverse transcriptase inhibitor (nrti) tenofovir (67) , which is one of the drugs most frequently used for therapy of hiv infections. the prodrug remdesivir (gs-5734) has a relatively short systemic half-life (ϳ0.9 h) and is rapidly converted intracellularly into several intermediate metabolites (gs-704277 and gs-4471524) before being converted into the more stable and active tp metabolite (gs-443902) (24, 54) . plasma concentrations of remdesivir that are reached by the administration of therapeutic doses are several times higher than the concentrations required to inhibit sars-cov-2 replication in vitro (48, 54, 68) . a dose of 200 mg remdesivir yields a maximum concentration of drug in serum (c max ) of 9.03 m (area under the concentration-time curve [auc] of 4.85 m), and subsequent dosing of 100 mg daily reaches a c max of 4.33 m (auc of 2.59 m) on day 5 (54) . data on tissue distributions are available from studies with cynomolgus monkeys, where remdesivir and its metabolites were detectable in testes, eyes, and brain 4 h after a 10-mg/kg dose, which is comparable to 200 mg in humans. unfortunately, no data on pulmonary drug delivery were reported in this publication (24) . distribution into lung tissues was recently studied in six rhesus macaques, where the intermediate metabolite gs-441524, which was used as a surrogate for tissue loading, could be detected in all samples 24 h after injection of remdesivir. the intermediate metabolite gs-704277 was not detectable in lung tissue samples (53) . it remains unclear how plasma concentrations of remdesivir and its metabolites correlate with pulmonary drug delivery in humans, and there are speculations of suboptimal exposure in respiratory target cells of sars-cov-2 (69). thus, therapeutic strategies that improve pulmonary drug exposure might be helpful to further improve the clinical efficacy of remdesivir. remdesivir is administered by intravenous infusion over 30 to 120 min. the standard dose for adults and pediatric patients weighing 40 kg and higher is a loading dose of 200 mg followed by once-daily doses of 100 mg. dose adjustments are necessary for pediatric patients weighing less than 40 kg. it is not known if dose adjustments based on kidney or liver function are necessary. administration in patients with a glomerular filtration rate (gfr) below 30 ml/min is not recommended based on the potential accumulation of sulfobutylether-␤-cyclodextrin sodium salt present in both formula-tions of remdesivir (55) . the optimal treatment duration for covid-19 is still unknown. in phase 3 trials, a treatment course of 5 or 10 days was investigated. based on these data, the actual recommendations in the context of emergency authorizations are 5 days for patients who do not require mechanical ventilation, which can be extended up to 10 days if patients do not demonstrate clinical improvement. for patients on mechanical ventilation, the actual recommended treatment duration is 10 days (55) . the in vitro activity of remdesivir against ebov has been demonstrated in various cell-based models and for many different ebov strains (20, 21, 24) . in addition, it showed therapeutic and prophylactic effects in a rhesus monkey model of lethal evd (24) . however, in the palm study, remdesivir was less efficacious than other investigational drugs. as this study did not include a placebo control group, no definite conclusions on the clinical efficacy of remdesivir in evd can be made. the mortality rate among patients treated with remdesivir was approximately 50%, which was similar to that of the control group treated with the triple monoclonal antibody zmapp but significantly higher than those with mab114 (35.1%) and regn-eb3 (33.5%) (27) . in a previous trial on evd, the mortality rates were 22% among zmapp-treated patients and about 37% among patients who received the standard of care only (59) . the reason for these differences in mortality rates is unclear and may result from differences in the virulence of ebov, sample size, patient population, or standard-of-care practices. however, a final interpretation of the clinical effects of remdesivir remains elusive, and based on the results of the palm trial, it is unlikely that remdesivir will be clinically reevaluated for the treatment of evd. remdesivir is active in vitro against various covs, including sars-cov-2 (3, 4, 47, 55) , and its mechanism of action has been studied extensively. animal studies that included nonhuman primate models of mers-cov and, recently, sars-cov-2 support its efficacy, especially when administered early in the course of the disease (29, 70) . finally, a phase 3 trial showed beneficial clinical effects of remdesivir in patients who require supplemental oxygen, while clinical efficacy for critically ill patients who require mechanical ventilation could not be demonstrated (5) . remdesivir reduces the time to recovery by 31%, which is a relatively modest but clearly therapeutic effect (5) . besides these beneficial effects on patients, this may help to reduce the number of inpatient days, with positive effects on hospital costs and capacity issues that have emerged during the covid-19 pandemic in several countries. with regard to mortality, a lower 14-day mortality rate of patients treated with remdesivir was reported from the actt study, which may indicate a beneficial but not statistically significant effect. taking into account that the study was not powered to evaluate mortality, this is still a positive signal that must be further evaluated in large-scale studies. a meta-analysis with pooled data from the two available rcts that is still under peer review concludes a statistically significant reduction in mortality (risk ratio [rr], 0.69 [95% ci, 0.49 to 0.99]) (71) . although the clinical data on remdesivir are not fully published yet, the emergency authorizations and recent approval by the ema are encouraging, as most other investigational drugs have failed until now (72, 73) , leaving remdesivir the only antiviral with clinically proven efficacy against covid-19 to date. complete results of the actt study, including impacts on viral loads and mortality by day 28, as well as results from ongoing trials and meta-analyses will provide more information on the clinical efficacy of remdesivir. after decades of research on direct-acting antiviral drugs, remdesivir is the first nucleoside analog that can be used to treat infections caused by a respiratory virus. in the light of its beneficial clinical effects, its favorable safety profile, and the absence of alternatives to treat covid-19, remdesivir will increasingly be used outside the context of clinical trials or compassionate-use programs. the drug is already available in the united states and japan based on emergency-use authorizations and was recently approved in europe. however, treatment with the antiviral drug remdesivir alone will not be sufficient to reliably save the lives of patients suffering from covid-19 or to solve the hazardous public health issues caused by the ongoing covid-19 pandemic. antiviral therapy in hospitalized patients cannot prevent the virus from being transmitted among communities and cannot reverse pathophysiological processes that have occurred already at the time of diagnosis. in general, prophylactic measures would be much more efficient in reducing covid-19-associated morbidity and mortality as well as economic implications (1) . the prophylactic use of remdesivir might be effective, as it completely protected exposed macaques from mers-cov-induced clinical disease (29) . prophylactic effects are also known from other virostatic-acting drugs like neuraminidase inhibitors that may prevent influenza virus infections and can also be used as postexposition prophylaxis (74) . however, the prophylactic use of remdesivir is generally hampered by its poor oral bioavailability and the absence of an oral formulation. further pharmacological efforts are needed to make the drug accessible to an outpatient population. recently, the manufacturer announced in an open letter that a phase 1 trial with remdesivir inhalation is being planned and already accepted by the fda (75) . interestingly, another sars-cov-2 active nucleoside analog called eidd-1931, which is orally bioavailable, is currently in preclinical evaluation (76) . finally, it should be mentioned that drug pricing will also have significant implications for the possibility of applying remdesivir with a broader scope. the therapeutic efficacy of remdesivir might be improved by the addition of other antivirals or immunomodulatory agents. it has recently been shown that glucocorticoids are able to improve clinical outcomes in cases of severe and critical covid-19 (77) . based on these data, it can be expected that physicians will use both remdesivir and glucocorticoids to treat patients with severe or critical covid-19. however, combination therapy should be used with caution, as drug interactions may occur. in vitro, remdesivir acts as the substrate or inhibitor of several drug-metabolizing enzymes (e.g., cyp3a4), which could influence the exposure levels of other therapeutic agents. in addition, these agents may interfere with the pharmacokinetics of remdesivir. the immunomodulatory drug hydroxychloroquine, for example, seems to reduce the antiviral activity of remdesivir by impairing its intracellular metabolic activation (55) . another approach that may improve clinical outcomes could be combination therapy with direct antiviral drugs that target several processes within the viral life cycle. although this strategy is highly effective in the therapy of chronic infections with hiv and hcv, it is unclear if this is true for acute infections with sars-cov-2. clinical trials evaluating combination therapy are needed to estimate their role in covid-19. manuscript. g.f. and j.r. supervised the manuscript preparation process, were involved in reviewing, and were responsible for manuscript validation. coronavirus disease 2019 -covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro sars-coronavirus-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology remdesivir for the treatment of covid-19 -preliminary report nih clinical trial shows remdesivir accelerates recovery from advanced covid-19 first covid-19 treatment authorised for use in the eu acyclovir: discovery, mechanism of action, and selectivity a 40-year journey in search of selective antiviral chemotherapy antiviral acyclic nucleoside phosphonates structure activity studies sofosbuvir for previously untreated chronic hepatitis c infection common and unique features of viral rnadependent polymerases the phylogeny of rna-dependent rna polymerases of positive-strand rna viruses synthesis and antiviral activity of a series of 1=-substituted 4-aza-7,9-dideazaadenosine c-nucleosides inhibition of hepatitis c virus replication by gs-6620, a potent c-nucleoside monophosphate prodrug discovery of the first c-nucleoside hcv polymerase inhibitor (gs-6620) with demonstrated antiviral response in hcv infected patients the evolution of antiviral nucleoside analogues: a review for chemists and non-chemists. part ii: complex modifications to the nucleoside scaffold the evolution of nucleoside analogue antivirals: a review for chemists and non-chemists. part 1: early structural modifications to the nucleoside scaffold nucleosides for the treatment of respiratory rna virus infections discovery and synthesis of a phosphoramidate prodrug of a pyrrolo[2,1-f][triazin-4-amino] adenine c-nucleoside (gs-5734) for the treatment of ebola and emerging viruses gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses aryl phosphoramidate derivatives of d4t have improved anti-hiv efficacy in tissue culture and may act by the generation of a novel intracellular metabolite application of phosphoramidate protide technology significantly improves antiviral potency of carbocyclic adenosine derivatives therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease palm consortium study team. 2019. a randomized, controlled trial of ebola virus disease therapeutics comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir delayed chain termination protects the anti-hepatitis b virus drug entecavir from excision by hiv-1 reverse transcriptase presteady-state kinetic studies establish entecavir 5=-triphosphate as a substrate for hiv-1 reverse transcriptase in vitro inhibition of hepadnavirus polymerases by the triphosphates of bms-200475 and lobucavir identification of bms-200475 as a potent and selective inhibitor of hepatitis b virus structure of the rna-dependent rna polymerase from covid-19 virus structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors remdesivir is a direct-acting antiviral that inhibits rnadependent rna polymerase from severe acute respiratory syndrome coronavirus 2 with high potency testing therapeutics in cell-based assays: factors that influence the apparent potency of drugs mouse hepatitis virus (mhv-2). plaque assay and propagation in mouse cell line dbt cells cell-based assays to identify inhibitors of viral disease sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor isolation, sequence, infectivity, and replication kinetics of severe acute respiratory syndrome coronavirus 2 enhanced isolation of sars-cov-2 by tmprss2-expressing cells exogenous ace2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication sars-associated coronavirus replication in cell lines broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro fact sheet for health care providers: emergency use authorization (eua) of veklury (remdesivir)-revision 1. us food and drug administration the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus remdesivir and sars-cov-2: structural requirements at both nsp12 rdrp and nsp14 exonuclease active-sites species difference of esterase expression and hydrolase activity in plasma a mouse model for mers coronavirus-induced acute respiratory distress syndrome clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 remdesivir: review of pharmacology, pre-clinical data and emerging clinical experience for covid-19 fact sheet for health care providers: emergency use authorization (eua) of veklury (remdesivir) compassionate use of remdesivir for patients with severe covid-19 icu) and non-icu patients: clinical outcome and differences in post-treatment hospitalisation status efficacy of remdesivir in covid-19 patients with a simulated two-arm controlled study a randomized, controlled trial of zmapp for ebola virus infection remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial the national institute of allergy and infectious diseases decision to stop the adaptive covid-19 trial: on solid ethical and scientific grounds remdesivir for 5 or 10 days in patients with severe covid-19 remdesivir (gs-5734) investigator's brochure version 5.0 advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases inhibition of viral rna polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand rna viruses beyond hepatitis c virus the mechanism of action of ␤-d-2=-deoxy-2=-fluoro-2=-c-methylcytidine involves a second metabolic pathway leading to ␤-d-2=-deoxy-2=-fluoro-2=-c-methyluridine 5=-triphosphate, a potent inhibitor of the hepatitis c virus rna-dependent rna polymerase medicinal chemistry of nucleoside phosphonate prodrugs for antiviral therapy safety, tolerability, and pharmacokinetics of remdesivir, an antiviral for treatment of covid-19, in healthy subjects remdesivir for treatment of covid-19: combination of pulmonary and iv administration may offer aditional [sic] benefit clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 remdesivir use in patients with coronavirus covid-19 disease: a systematic review and meta-analysis a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial neuraminidase inhibitors for preventing and treating influenza in healthy adults and children an open letter from daniel o'day, chairman & ceo an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice dexamethasone in hospitalized patients with covid-19 -preliminary report this research received no specific financial support. j.j.m. and i.s. receive funding from german center for infection research (dzif) stipends ti 07.001_malin_00 and ti 07.001_suarez_00. j.r. receives funding from the dzif thematic translational unit tuberculosis (ttu tb) (grant numbers ttu 02.806 and 02.905) and the german research foundation (dfg ry 159). the funders had no role in data collection, interpretation, or the decision to submit the work for publication.we acknowledge patrick lane, sceyence studios, for graphical enhancement of the presented figures.g.f. has served as an advisor to gilead sciences and has conducted clinical research supported by gilead sciences. j.j.m., v.p., i.s., and j.r. declare no potential conflicts of interest.all authors critically discussed the present study results and their clinical significance. j.j.m. wrote the original draft and was responsible for conceptualization, investigation, and visualization. i.s. and v.p. were involved in reviewing and editing the jakob j. malin (m.d.) obtained an m.sc. in medicine at maastricht university and a doctoral degree in the field of viral infectious diseases at the university of bonn. he works as a medical doctor and clinician-scientist at the university hospital of cologne, completing a clinical residency in internal medicine and infectious diseases. he is involved in coordinating and conducting clinical trials on anti-infectives, including investigational drugs against covid-19. moreover, he contributes to the development of clinical guidelines on covid-19 pharmacotherapy released by the german society of infectious diseases (dgi). dr. malin has a strong translational research focus on antimicrobial drug discovery. since 2012, he has been working on antimicrobial compounds against several pathogens, including opportunistic mycobacteria. he is affiliated with the translational research unit for infectious diseases (tru-id) at the center for molecular medicine cologne (cmmc), where he works as a postdoctoral researcher and is currently expanding his translational working field for sars-cov-2. key: cord-309239-6lso1w0o authors: adney, danielle r.; brown, vienna r.; porter, stephanie m.; bielefeldt-ohmann, helle; hartwig, airn e.; bowen, richard a. title: inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding date: 2016-08-19 journal: viruses doi: 10.3390/v8080230 sha: doc_id: 309239 cord_uid: 6lso1w0o the middle east respiratory syndrome coronavirus (mers-cov) was first recognized in 2012 and can cause severe disease in infected humans. dromedary camels are the reservoir for the virus, although, other than nasal discharge, these animals do not display any overt clinical disease. data from in vitro experiments suggest that other livestock such as sheep, goats, and horses might also contribute to viral transmission, although field data has not identified any seropositive animals. in order to understand if these animals could be infected, we challenged young goats and horses and adult sheep with mers-cov by intranasal inoculation. minimal or no virus shedding was detected in all of the animals. during the four weeks following inoculation, neutralizing antibodies were detected in the young goats, but not in sheep or horses. the middle east respiratory syndrome coronavirus (mers-cov) is an emerging pathogen first described from saudi arabia in 2012 [1] that can cause severe respiratory disease and death in roughly 36% of infected humans [2] . there is considerable field and experimental evidence that dromedary camels serve as an important reservoir host involved in transmission to humans [3] [4] [5] [6] [7] [8] , but whether other livestock such as goats, sheep, and horses play a role in transmission has only been assessed indirectly. the virus is phylogenetically similar to betacoronaviruses previously detected in bats and there has been speculation that this disease originated through a cross-species transmission from bats to camels or humans [9] [10] [11] . serologic testing of sheep, goats, and cattle from jordan [12] and saudi arabia [13] failed to identify animals with neutralizing antibodies to mers-cov. similarly, horses tested in the united arab emirates lacked antibodies to mers-cov [14] . direct contact with dromedaries in saudi arabia was found to be independently associated with mers-cov illness, while contact with goats, sheep, or horses was not associated with human illness [15] . in vitro studies in which replication of mers-cov in cultured cells was assessed have yielded mixed results with respect to species susceptibility. cells from goats, but not sheep or cattle, supported replication of mers-cov [16] , and primary equine kidney cells supported virus replication, albeit at lower levels than observed with vero cells [14] . transfection of the dpp4 receptor from goats, sheep, and horses into non-permissive mouse or hamster cells allowed replication of mers-cov [17, 18] . collectively, these in vitro studies suggest the possibility that some livestock are susceptible to infection, but demonstration of infection in live animals is required to better assess their potential as reservoir hosts. the objective of this study was to determine if goats, sheep, and horses can be infected with mers-cov and assess their potential importance in viral transmission. goats (n = 5) were evaluated for viral shedding, organ burden, and seroconversion and transmission to co-housed goats (n = 2). limited viral shedding was observed without demonstration of viral transmission. due to the lack of transmission, only viral shedding and serology were evaluated in horses (n = 4) and sheep (n = 3). these animals did not become productively infected or seroconvert, indicating that such livestock are unlikely to serve as reservoirs for mers-cov and are unimportant in viral transmission. these studies were approved by the animal care and use committee of colorado state university (approval number 13-4384a) and were conducted in an association for the assessment and accreditation of laboratory animal care, international (aaalac) approved facility. two goats, three sheep, and four horses were purchased in colorado, usa. both of the goats were bred on site and gave birth to either two (doe 1) or three kids (doe 2). all animals were fed a complete pelleted feed supplemented with hay, and were observed at least once daily for nasal discharge, demeanor, food consumption, and clinical status. sheep, goat kids and horses were each inoculated intranasally with 1.4 × 10 6 to 1.9 × 10 6 plaque-forming units (pfu) of a low passage human isolate of mers-cov (strain hcov-emc/2012) propagated in vero e6 cells as described previously [11] . the goat kids were maintained at all times in a room with their mothers, who served as in-contact controls to test for virus transmission. rectal temperature and nasal swabs were taken daily for seven days. one goat kid from each doe was euthanized 5 days post-inoculation (dpi) and the remaining kids and mother goats were euthanized on day 28 post-inoculation. the horses and sheep were monitored for viral shedding and seroconversion, and were euthanized on day 28 post-inoculation, with the exception of horse 4, which was euthanized on day 17 due to an injury. samples of nasal secretions were collected by inserting and rotating a swab into each nare and were immediately placed in viral transport medium and frozen until plaque assay was performed. plaques originating from all animals having low titers of virus were confirmed to be mers-cov by immunofluorescence using a rabbit polyclonal antiserum against hcov-emc-2012 antigen as a primary antibody. nasal turbinates, trachea, larynx, and lung samples were collected from two kids (goat 1c and 2a) on day 5 post-infection and frozen for virus titration or fixed in 10% neutral-buffered formalin for greater than 7 days prior to being embedded in paraffin. tissue sections (hematoxylin/eosin and immunohistochemistry) were prepared and evaluated by a veterinary pathologist as previously described [19] . in order to detect mers-cov antigen immunohistochemical analysis was performed with a previously described rabbit polyclonal antiserum against hcov-emc-2012 antigen [19, 20] . serum was collected immediately prior to inoculation and weekly thereafter until necropsy. neutralizing antibodies in sera were assayed using a plaque reduction neutralization test (prnt) with a 90% neutralization cutoff as described previously [11] . goats were assessed for clinical disease, viral shedding, seroconversion, and viral transmission to their mothers. fevers were not detected in any of the goats, and no nasal discharge was observed. low levels of infectious virus were detected in two of the inoculated goat kids from doe 1 (figure 1 ), but not from either of the adult goats that had intimate contact or the kids from doe 2. serum was collected immediately prior to inoculation and weekly thereafter until necropsy. neutralizing antibodies in sera were assayed using a plaque reduction neutralization test (prnt) with a 90% neutralization cutoff as described previously [11] . goats were assessed for clinical disease, viral shedding, seroconversion, and viral transmission to their mothers. fevers were not detected in any of the goats, and no nasal discharge was observed. low levels of infectious virus were detected in two of the inoculated goat kids from doe 1 (figure 1 ), but not from either of the adult goats that had intimate contact or the kids from doe 2. in order to study acute pathology and determine organ burden, two goats were euthanized on day 5-post infection and nasal turbinates, trachea, and lung were collected. very small but confirmed quantities of virus were isolated from the turbinates of both goats euthanized 5 days post-infection (dpi) (figure 2 ), which may reflect input virus or very low level virus replication. goat kid 1c was histologically unremarkable, however, the turbinates of goat kid 2a had multifocal areas of loss of goblet cells, epithelial necrosis or squamous metaplasia and attenuation and/or erosion of the epithelium, accompanied by mild to moderate neutrophil and monocyte/macrophage infiltration and occasional minimal hemorrhage. small amounts of cellular debris, leukocytes and mucus (exudate) were present in the nasal cavity, mainly associated with the aforementioned affected areas. these tissues were negative for viral antigen by immunohistochemistry (ihc) and the histopathologic lesions were very likely the result of trauma from daily swabbing rather than due to virus replication. in order to study acute pathology and determine organ burden, two goats were euthanized on day 5-post infection and nasal turbinates, trachea, and lung were collected. very small but confirmed quantities of virus were isolated from the turbinates of both goats euthanized 5 days post-infection (dpi) (figure 2) , which may reflect input virus or very low level virus replication. goat kid 1c was histologically unremarkable, however, the turbinates of goat kid 2a had multifocal areas of loss of goblet cells, epithelial necrosis or squamous metaplasia and attenuation and/or erosion of the epithelium, accompanied by mild to moderate neutrophil and monocyte/macrophage infiltration and occasional minimal hemorrhage. small amounts of cellular debris, leukocytes and mucus (exudate) were present in the nasal cavity, mainly associated with the aforementioned affected areas. these tissues were negative for viral antigen by immunohistochemistry (ihc) and the histopathologic lesions were very likely the result of trauma from daily swabbing rather than due to virus replication. the remaining goats were euthanized on day 28 post-infection and the serological status of the experimentally infected kids and their cohoused dams were assessed. each of three kid goats held past day 5 seroconverted, however, neutralizing antibodies were not detected in either of the dams (table 1) . table 1 . neutralizing antibody titers in goats experimentally infected or exposed by contact to mers-cov. mother goats are indicated as 1 or 2, and their corresponded kids are indicated as 1a, 1b, 1c (doe 1), 2a, or 2b (doe 2). titers represent dilutions of serum which neutralized ≥90% of input virus. nd = not done. three sheep were experimentally infected and evaluated for clinical disease, viral shedding, and seroconversion. no nasal discharge or clinical disease was observed and all three sheep maintained consistent food intake and activity levels. a small quantity of virus was detected in nasal swabs from sheep 1 on days 1, 2, 3, 5, and 6 and from sheep 2 on day 6 ( figure 3 ). the remaining goats were euthanized on day 28 post-infection and the serological status of the experimentally infected kids and their cohoused dams were assessed. each of three kid goats held past day 5 seroconverted, however, neutralizing antibodies were not detected in either of the dams (table 1) . table 1 . neutralizing antibody titers in goats experimentally infected or exposed by contact to mers-cov. mother goats are indicated as 1 or 2, and their corresponded kids are indicated as 1a, 1b, 1c (doe 1), 2a, or 2b (doe 2). titers represent dilutions of serum which neutralized ≥90% of input virus. nd = not done. three sheep were experimentally infected and evaluated for clinical disease, viral shedding, and seroconversion. no nasal discharge or clinical disease was observed and all three sheep maintained consistent food intake and activity levels. a small quantity of virus was detected in nasal swabs from sheep 1 on days 1, 2, 3, 5, and 6 and from sheep 2 on day 6 ( figure 3) . unlike the goats, acute pathology in sheep was not evaluated in this study. the sheep were euthanized on day 28 post-infection, and serum samples from each week were assessed for the presence of mers-cov neutralizing antibodies. sheep 2 developed a low titer of neutralizing antibody on day 14 (10), but neutralizing antibodies were not detected in either of the other two sheep at any time-point tested ( table 2 ). unlike the goats, acute pathology in sheep was not evaluated in this study. the sheep were euthanized on day 28 post-infection, and serum samples from each week were assessed for the presence of mers-cov neutralizing antibodies. sheep 2 developed a low titer of neutralizing antibody on day 14 (10), but neutralizing antibodies were not detected in either of the other two sheep at any time-point tested ( table 2) . table 2 . neutralizing antibody titers in sheep experimentally infected with mers-cov. titers were determined by plaque reduction neutralization test (prnt) using a 90% cutoff. <10 despite no detectable rise in rectal temperature or change in appetite and activity, horses 1 and 3 showed mild intermittent nasal discharge prior to inoculation and throughout the 28 days experiment. low levels of virus were detected in nasal swab samples from three of the four inoculated horses. virus was detected on day 3 in horse 2, day 2 in horse 3, and day 1 in horse 4 ( figure 4) . virus was not detected in any of the nasal swab specimens tested from horse 1. virus isolation was performed by plaque assay from nasal swab specimens obtained from sheep experimentally infected with mers-cov. the limit of detection for this assay was 1.5 log 10 pfu/ml, indicated by the dashed line. table 2 . neutralizing antibody titers in sheep experimentally infected with mers-cov. titers were determined by plaque reduction neutralization test (prnt) using a 90% cutoff. despite no detectable rise in rectal temperature or change in appetite and activity, horses 1 and 3 showed mild intermittent nasal discharge prior to inoculation and throughout the 28 days experiment. low levels of virus were detected in nasal swab samples from three of the four inoculated horses. virus was detected on day 3 in horse 2, day 2 in horse 3, and day 1 in horse 4 ( figure 4 ). virus was not detected in any of the nasal swab specimens tested from horse 1. serum was collected weekly until day 28 (with the exception of horse 4, which was euthanized early due to an injury unrelated to the experiment), and evaluated for the presence of mers-cov neutralizing antibodies. unlike the inoculated goat kids, none of the infected horses seroconverted (table 3 ). serum was collected weekly until day 28 (with the exception of horse 4, which was euthanized early due to an injury unrelated to the experiment), and evaluated for the presence of mers-cov neutralizing antibodies. unlike the inoculated goat kids, none of the infected horses seroconverted (table 3) . the objective of this study was to determine if goats, sheep, or horses could be experimentally infected with mers-cov. very limited amounts of infectious virus were detected in nasal swab specimens of some of the experimentally infected animals, but not in uninfected, co-housed goats. it is possible that the infectious virus detected was residual from the input virus, at least on day 1 post-infection. however, a previous study with alpacas re-challenged 80 days after an initial infection was not able to detect infectious virus upon re-challenge indicating that input challenge virus is not detected one day after infection, although the role of secretory antibodies was not addressed in that study [19] . similarly, another study with alpacas re-challenged on day 28 post-infection was unable to detect viral rna upon re-challenge [21] . in comparison to experimentally infected dromedaries, significantly less virus was isolated from the livestock in this study. since the main objective of this study was to determine the role these animals might play in transmission of the virus, we chose to test these samples by plaque assay in order to determine the amount of infectious virus present, rather than by rt-pcr which only reveals the presence of viral rna regardless of infectivity. previous studies of naturally or experimentally infected camels and experimentally infected alpacas showed variable levels of nasal discharge. studies in camels demonstrated that infected camels have nasal discharge while infected alpacas did not have any observable discharge [8, 10, 11, 19] . the goats and sheep in this study did not have any observable discharge; in contrast horses 1 and 3 had discharge throughout the entire study. all animals were examined and healthy prior to the study, but due to the dust associated with the housing of horses we believe this discharge was unrelated to infection and instead an effect of the environment. current evidence suggests that dromedary camels are the primary reservoir of mers-cov. however, elucidating the role that other livestock such as goats, sheep, or horses could play in transmission is important for designing field studies and biosecurity strategies, and in assessing individuals at risk for viral transmission. while in vitro studies suggested that these animals could be naturally or experimental infected, the lack of support from field data coupled with the experimental data presented here suggest that these animals are unlikely to be infected and are not important in viral transmission of mers-cov. the authors declare no conflict of interest. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-to-human transmission of mers coronavirus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia serologic assessment of possibility for mers-cov infection in equids risk factors for primary middle east respiratory syndrome coronavirus illness in humans replicative capacity of mers coronavirus in livestock cell lines host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas infection with mers-cov causes lethal pneumonia in the common marmoset experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus key: cord-294856-eeh2a0t8 authors: lambert, paul-henri; ambrosino, donna m.; andersen, svein r.; baric, ralph s.; black, steven b.; chen, robert t.; dekker, cornelia l.; didierlaurent, arnaud m.; graham, barney s.; martin, samantha d.; molrine, deborah c.; perlman, stanley; picard-fraser, philip a.; pollard, andrew j.; qin, chuan; subbarao, kanta; cramer, jakob p. title: consensus summary report for cepi/bc march 12-13, 2020 meeting: assessment of risk of disease enhancement with covid-19 vaccines date: 2020-05-25 journal: vaccine doi: 10.1016/j.vaccine.2020.05.064 sha: doc_id: 294856 cord_uid: eeh2a0t8 a novel coronavirus (cov), severe acute respiratory syndrome coronavirus 2 (sars-cov-2), emerged in late 2019 in wuhan, china and has since spread as a global pandemic. safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of coronavirus disease 2019 (covid-19) disease and ease the major economic impact. there has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. however, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. a syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. animal models allowed scientists to determine the underlying mechanism for the former in the case of respiratory syncytial virus (rsv) vaccine and have been utilized to design and screen new rsv vaccine candidates. because some middle east respiratory syndrome (mers) and sars-cov-1 vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for sars-cov-2 vaccines. to address this challenge, the coalition for epidemic preparedness innovations (cepi) and the brighton collaboration (bc) safety platform for emergency vaccines (speac) convened a scientific working meeting on march 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. this report summarizes the evidence presented and provides considerations for safety assessment of covid-19 vaccine candidates in accelerated vaccine development. since the identification of a novel coronavirus, sars-cov-2, as the cause of pneumonia in patients from wuhan china, a pandemic has erupted, resulting in enormous health care, social and economic disruption to our global society [1] . as of may 17, 2020 there have been 4,708,415 cases and 314,950 deaths worldwide [2] . in rapid response to the pandemic, academic and industry scientists from around the world have initiated efforts to develop vaccines and therapeutics for disease prevention and patient management. the coalition for epidemic preparedness innovations (cepi), a global partnership between public, private, philanthropic, and civil organizations, is funding work to develop sars-cov-2 vaccines using a variety of technology platforms. several vaccine candidates are already in phase 1 studies with others likely to enter the clinic in the next few months [3] . one of the challenges facing rapid vaccine development for sars-cov-2 is the need to adequately assure the safety of these vaccines. one such safety concern is disease enhancement syndrome that occurred in the 1960s with inactivated rsv and measles vaccines. vaccinemediated disease enhancement is characterized by a vaccine that results in increased disease severity if the subject is later infected by the natural virus. during early trials with inactivated rsv vaccine, the vaccine did not prevent infection, 80% of those infected required hospitalization and two children died [4] . lung pathology in patients showed an unexpected inflammatory response with both neutrophils and eosinophils, evidence of immune complex formation and complement activation in small airways [5] . scientists later learned that the vaccine caused a similar disease enhancement in animals characterized by immunopathology and a t helper cell type 2 (th2) biased response and antibody responses with poor neutralizing activity [6] [7] [8] . since that time, the animal models have been relied upon to predict safety for new 6 rsv vaccines that are developed. of note, the pathogenesis of rsv disease enhancement is distinct from antibody disease enhancement (ade) which occurs for macrophage tropic viruses, demonstrated most notably for dengue in humans and the coronavirus feline infectious peritonitis virus in cats, and is directly caused by non-neutralizing or sub-neutralizing antibodies leading to more efficient viral uptake via fcγ receptor binding [9] . since pathology consistent with the rsv vaccine enhanced disease (and perhaps ade) has been demonstrated for some sars-cov-1 vaccine candidates in animal models, there is also a concern that a similar syndrome could occur in humans immunized with sars-cov-2 candidate vaccines. therefore, cepi and the brighton collaboration safety platform for emergency vaccines (speac) convened a scientific working meeting https://brightoncollaboration.us/brighton-collaboration-cepi-covid-19-web-conference/) on march 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to discuss current knowledge that could form the basis for the assessment of the risk of enhanced disease during sars-cov-2 vaccine development. this consensus report presents considerations for vaccine developers and can serve as a guide for the development and testing of vaccine candidates to avoid these safety concerns. ultimately, the door to clinical trials is controlled by regulators in the context of the risk/benefit for the entire dataset provided by developers and within the local trial context. microbiology and immunology and pediatrics at the university of iowa, both reviewed their work and that of others in animal models developed for sars-cov-1 and mers-cov. the lessons from these models can inform the development priorities for useful sars-cov-2 animal models to address both efficacy and safety. in inbred mouse strains, sars-cov-1 replicates efficiently in the respiratory tract and can cause pneumonitis, but clinical signs and pneumonia were only observed in old balb/c mice [10] . subsequent passage of sars-cov-1 through mouse lungs resulted in the isolation of virus that caused severe disease in both young and old mice [11, 12] . this virus was used in many subsequent studies. ferret models of sars-cov-1 also demonstrate virus replication in respiratory tracts with induction of a neutralizing antibody response but also demonstrated little evidence of clinical disease [13] . hamsters, in contrast to mice and ferrets, demonstrate high levels of viral replication, develop pneumonitis, and can be shown to have clinical signs of disease [14] . following the identification of human ace2 as the receptor for sars-cov-1, transgenic murine models expressing human ace2 receptor (hace2) were developed and shown to develop mild pulmonary disease. of note, these mice also developed lethal viral encephalitis, attributed to viral spread through the olfactory nerve, despite the relative scarcity of hace2 expression in the brain which may have relevance to sars-cov-2 disease [15] . efficacy of several sars-cov-1 vaccines was evaluated in these models with spike (s) protein based vaccines demonstrating neutralizing antibody and protection against pulmonary replication of the challenge virus in mice and hamsters [16] . for dna vaccine studies, it was shown that candidate vaccines encoding the s protein conferred antibody mediated protection from challenge in mice and that vaccines encoding the n protein induced humoral and cellular immunity [17, 18] . for vectored vaccines expressing sars-cov-1 proteins, it was shown that 8 viral proteins were expressed in mice, ferrets, and hamsters. in these studies, neutralizing antibodies were elicited by b/hpiv3, vsv, rabies, mva and adeno viruses expressing s protein, that protected against sars-cov-1 replication in lungs of challenged animals. however, one mva vaccine expressing the s-protein did not protect against infection [16] . in contrast to sars-cov-1, inbred mice were found to be resistant to mers-cov, thus infection was studied by creating models that expressed the mers receptor, human dpp4 (hdpp4). ad5-hdpp4 transduced mice could be infected with mers virus but infection was associated with minimal clinical disease except in immunocompromised mice that developed weight loss after infection. of note, hdpp4-transgenic mice developed lethal viral encephalitis with concurrent inflammatory changes on histopathological examination of the lung, similar to hace2-tg mice with sars-cov-1. subsequently, investigators developed mice "knocked-in" for expression of hdpp4 and after virus passage in these mice, identified mouse-adapted mers strains that caused more severe disease and increased histopathology with more pulmonary edema than those infected with the original mers strain [19] . importantly, mice without functional t cells, such as rag1-/-and tcr alpha-/-, had delayed viral clearance whereas mice that could not produce antibodies, mumt mice, did not show delay in clearance. similar models were developed by crispr/cas9 mutagenesis of two residues in the mouse ace2 molecule, followed by mouse adaptation with serial passage, leading to an ards model of lethal infection [20, 21] . taken together this evidence supports the notion that t cells are important in viral clearance for mers [22] . non-human primate (nhp) models have also been established for both sars-cov-1 and mers-cov. there was evidence of upper respiratory and lower respiratory tract sars-cov-1 replication in african green monkeys to a greater extent than in cynomolgus macaques, and least 9 in rhesus macaques, with little evidence of clinical disease in all three species [23] . of note, consistent with findings in older humans and mice, increased pathology has been documented in aged cynomolgus macaques with sars-cov-1 wild type infection [24] . there is some controversy on the disease severity in the mers models with different groups seeing different levels of pathology. this has not been resolved [25, 26] . both vaccine efficacy and safety have been studied in animal models with many sars-cov-1 candidate vaccines. the group of experts discussed how the vaccine models were utilized to characterize the response of specific vaccines and to examine both disease enhancement and antibody dependent enhancement (ade) signals. there is evidence for disease enhancement in vaccinated animals after challenge with live virus in multiple studies with sars-cov-1 vaccine candidates as summarized in table. we are limiting our comments in this report to data in animal models and not discussing in vitro data except to mention that there is some evidence of ade in human primary monocytes [27, 28] . different animal models exhibit different pulmonary pathology but generally characterized by cellular infiltrates including eosinophils. in this summary, we provide an overview of the consensus opinion on vaccine related outcomes in animal models that were of concern for risk of disease enhancement and could guide assessments of sars-cov-2 vaccine candidates. in murine models, evidence for vaccine related disease enhancement has been demonstrated for inactivated whole vaccine (with and without alum), vectored vaccine expressing n protein (but not seen with vectored vaccine expressing s protein in same report), a replicon particle platform expressing s protein, and a vectored vaccine expressing s proteins. in general, the pathology described included pulmonary infiltrates often with eosinophils observed. th2 dominant responses were documented in some reports by expression of th2 driven cytokines [29] [30] [31] [32] [33] . in a ferret model, hepatitis was demonstrated in animals vaccinated with a recombinant modified vaccinia virus ankara vaccine expressing s protein and then challenged with virus [34] although questions have been raised about this study [35] . [36, 37] . non-human primate models have also produced evidence of enhanced disease after sars-cov-1 vaccine immunization. chinese macaques immunized with a modified vaccinia virus expressing s protein then challenged with sars-cov-1 did not develop clinical disease, but histopathology showed lung injury. this injury was characterized by decreased wound healing, and increased pro-inflammatory macrophages expressing il-6, il-8, and ccl2 [38] . this report also demonstrated that passively administered anti-s antibody was associated with lung pathology after challenge with the live virus although the mechanism may not be through fc receptor and thus not classic "ade". of note, a second report similarly demonstrates the effect with certain anti-s antibody preparations and without fc involvement [39, 40] . the relevance of these reports remains unclear as there are multiple studies with administration of neutralizing monoclonal antibodies to different models that did not induce disease enhancement. other investigators have reported absence of disease enhancement in both hamsters and monkeys immunized with a whole inactivated vaccine although these models differed in a number of ways, most notably by the use of bpl (β-propiolactone) instead of formalin for inactivation of the virus [41, 42] . finally, we note that there has not be an agreed upon positive control applied in these animal studies and thus interpretations are hampered.ba animal models with sars-cov-2 are being rapidly developed by multiple research human ace2 transgenic mice (hace2 tg) aged 4-6 weeks and 6-11 months of age were studied and hace2 expression was observed in lung, heart, kidney and intestinal tissues. following intranasal inoculation with sars-cov-2, weight loss was observed, and viral rna was detected in the lungs as well as in the intestine [43] . gross pathology demonstrated swollen and enlarged lungs with moderate interstitial pneumonia. histological studies documented an accumulation of inflammatory cells including monocytes and lymphocytes in alveolar interstitium, with thickening of alveolar walls. sars-cov-2 s protein was detected by ihc in alveolar macrophages and epithelia [43] . nhp were also infected with sars-cov-2 with 3 rhesus macaques aged 3-4 years inoculated intratracheally and although no fever was observed, weight loss and asthenia were seen on multiple days. viral rna was detected from nasal and throat swabs and to a lesser degree in anal specimens, peaking on days 3 to 7 and lasting until day 11 post infection. one animal was euthanized on day 7 for necropsy and viral rna was detected in multiple organs including cns, skeletal muscle and heart. for the two surviving rhesus macaques, positive neutralization titers were documented by day 11 post infection. there was radiographic evidence of multiple ground glass opacities in the lungs on days 3, 5 and 7 post infection. microscopically the lung lesions represented an acute interstitial pneumonia characterized by mild to moderate thickening of alveolar septum, increased number of macrophages, degeneration of pneumocytes and an inflammatory cell infiltration. presence of viral antigen was confirmed, predominately in alveolar monocytes and macrophages [44] . analysis of blood samples showed a decline in counts of total white blood cells, lymphocytes and monocytes with no observed changes in 13 percentages. a decrease in both cd3+cd4+ and cd3+ cd8+ t-cell counts was observed. importantly, these hematological findings are similar to those seen in sars-cov-2 infected patients. this model could serve as a critical tool for detailed studies of pathogenesis and the evaluation of intervention strategies including vaccines. of note, following the meeting another group has confirmed sars-cov-2 infection in rhesus macaques with viral antigen detected in type i and type ii pneumocytes and diffuse pulmonary alveolar damage noted [45] . experts agreed that these models and others under development should be utilized to evaluate vaccine candidates for any evidence of disease enhancement as specified in later sections. design barney the sars-cov-2 s protein structure was solved shortly after its emergence and shows similar structure and mobility as the sars-cov-1 s [47] . the timing from first knowledge of sars-cov-2 to the beginning of the phase 1 study was a remarkable sixty-five days. the advantages of mrna vaccines include ability to create a highly precise type of protein to elicit the correct antibodies, to elicit t cell responses that are th1 predominant, and the rapidity of manufacturing. of course, disadvantages include the novel nature of both mrna and dna vaccines without any licensed vaccine with either technology to date and lack of experience for mass production. therefore, multiple platforms for sars-cov-2 are under development that mitigate against some of the potential disadvantages of nucleic acid vaccines. although mrna and dna vaccines elicit t cell responses without adjuvants, adjuvants may be important for subunit and whole cell inactivated vaccines to increase their immunogenicity and drive an immune response that could limit the risk of disease enhancement. multiple sars-cov-2 vaccines are in development including vectored vaccines, whole cell inactivated vaccines, and recombinant protein vaccines. the experts discussed how the choice of adjuvants will be important for both efficacy and safety with these platforms. dr. arnaud didierlaurent from the centre of vaccinology at the university of geneva presented background on the effects of different adjuvants on animal and human immune responses. several adjuvants are now being used in commercial vaccines or are in clinical development [48] . oil-in-water emulsions such as mf59 or as03 have been shown to increase the breadth of the antibody repertoire, binding affinity and affinity maturation when compared to unadjuvanted vaccines [49, 50] in human studies with influenza vaccines, h5n1 vaccine adjuvanted with mf59 (squalene-based emulsion) increased the levels of h5-specific antibody for subclasses igg1 and igg3 and the binding to fcγr2 but not to fcγr3 when compared to alum adjuvanted vaccines. this demonstrates that the use of an adjuvant can skew the functionality profile of antigen-specific antibodies, with the potential to activate different innate effectors based on their fcγr expression [51] . use of squalene-based emulsion vaccines for influenza have also been shown to increase cd4+ t cell response frequencies and crossreactivity. even if pre-existing cross-reactive antibodies are present prior to immunization, such adjuvants could activate naïve b cells and promote the adaptability of memory b cells [52] [53] [54] [55] . in addition to antibodies, adjuvants can promote cellular responses. human malaria challenge studies provided early evidence that the choice of adjuvants(combined with the malaria antigen rts,s) was critical in achieving optimal protection and highlighted the importance of cellular response [56] . more recently, studies with hepatitis b surface antigen (hbs) vaccine adjuvanted with as01, as03, as04 or alum showed that vaccines formulated with as01 and as03 induced the highest antibody levels while as01 promoted best hbs-specific cd4 t cell response [57] . these differences were associated with the magnitude of the initial inflammatory 18 response triggered by the different adjuvanted formulations [57, 58] . interestingly, although the level of cd4 t cell response was lower in the alum group compared to the as01 group, both adjuvants led to similar memory subset profiles and cytokine production profiles given the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for subunit vaccines in providing antigen-dose sparing, increased immunogenicity, breadth and duration of response, potentially eliciting cross-protection against new cov strains and minimizing the risk of enhanced disease. following the presentations, attendees participated in discussion of the suggested consensus statements and all attendees were asked to comment on the draft statements available online. these comments were reviewed and discussed again on the second day of the meeting and resulted in the summary consensus statement that follows. • sars-cov-2 has a low affinity for murine ace2 receptor and murine models will require the use of hace2 transgenic mice, preferably with a 'knock-in' approach. preliminary data indicate the possibility of infecting hace2 transgenic mice with demonstration of viral replication and mild lung lesions. mouse adaptation of sars-cov-2, as done with sars-cov-1, will likely be required to obtain a virus that causes more severe disease in mice. models that develop acute lung injury with some lethality and that mimic the human condition will be important for evaluating vaccine safety. • previous studies from sars-cov-1 and mers-cov indicated that some vaccines, especially those using whole inactivated virus, could enhance the disease induced in mice challenged with sars-cov-1 or mers-cov. the lung lesions were highly inflammatory, with a dominance of eosinophil infiltration and occurred in animals despite 20 presence of a neutralizing antibody response and reduced challenge virus replication in the lungs. such studies have not yet been completed for sars-cov2. • in mice, this immunopathology was considered a consequence of a dominant th2 type response to the vaccine antigens. it was not seen after including adjuvants (e.g. cpg) in the vaccine or other vaccine formulations known to drive immune responses towards th1. the timing of challenge after vaccination may be critical. it would be of major interest to explore the outcome following challenge at later timepoints when antibodies are significantly decaying. • one should be aware of the potential confounding effect of cell-culture excipients in the vaccine and challenge strain material. it is known that impurities such as fetal calf serum in the preclinical vaccine preparation may induce eosinophil influx in any mouse model if the challenge strain also contains the same excipients. • in these models, characterization of the immune response to the candidate vaccine (e.g., igg isotypes, th2 markers) may have some predictive value. • other small animal models which can be infected by sars-cov-2 can be considered (e.g. ferret, hamster). development of small animal models of severe disease will also inform studies of vaccine-enhanced disease. • passive transfer in nhps of human antibodies generated during phase 1 trials, followed by viral challenge could be considered to assess the risk of disease enhancement. • challenge of immunized animals with a closely related heterologous cov strains may assess the risk of enhancement during future outbreaks. • in case of disease enhancement, in-depth studies in animal models may give some indications on the mechanism of immunopathology. they can inform human trial designers on the critical immunological risk markers to be monitored in phase 1 trials. • given what will be the unprecedented demand for an effective vaccine, the use of adjuvants may be critical for sub-unit vaccines in providing increased immunogenicity, breadth of response, dose sparing, duration of response, potentially cross-protection against new cov strains, and possibly minimize the risk of enhanced disease. preference should be given to th1-driving adjuvants with an established safety profile in humans. • understanding of the role of cross-reacting antibodies from prior coronavirus infections may have on natural disease caused by sars-cov-2 or influence the risk of enhanced disease following vaccination may inform vaccine design. • data are needed on whether antibody waning could increase the risk of enhanced disease on exposure to virus in the long term. it was the opinion of the experts that animal data to support clinical development could address: • post-vaccination (neutralizing) antibody responses, and t cell analysis to demonstrate a th1 response. • post-vaccination challenge data from nhps with careful evaluation for immunopathology and quantitative virology in the animals. • small animal data may also provide important supporting evidence of safety, and hamster, ferret and mouse models are likely to be available for developers. • where possible, immunopathology experiments with a positive control (e.g., formalin inactivated alum-adjuvanted sars-cov-1 or sars-cov-2 vaccine) and a mockimmunized negative control will provide best guidance. it was felt that it will be important to establish broadly accepted endpoints and scoring systems to allow comparison of various vaccine candidates. who is working on this issue. • for vaccine constructs likely to induce a predominant th2 response, the group felt that animal studies should be considered before entering human phase 1 trials in more than one animal species including nhps where possible. it was noted that the absence of a th2 response does not eliminate the risk of enhanced disease. • since not all studies that have begun or are about to begin will prescreen to determine preimmunization serostatus of participants, although this shall be determined retrospectively, appropriate baseline blood specimens should be obtained and stored. because the virus is spreading rapidly, such specimens will allow assessment of the 25 immune response in both seronegative and seropositive persons as both are likely to be vaccinated. • level of neutralizing antibodies and determination of the relative ratio of binding to neutralizing antibodies will be important to assess the potential risk of enhanced disease. also, detection of initial priming that includes cd8 t cells and/or a cd4 th1 biased response is likely to mitigate the risk of disease enhancement. determination of memory responses will be useful, particularly if sars-cov-2 continues to circulate. • consideration should be given to the use of post-vaccination sera from vaccinees which could be used for antibody transfer studies in animals to look for enhanced disease and for evidence of cross-protection against other coronaviruses. • monitoring for enhanced disease in immunized participants may require longer follow-up than is usual in phase 1 trials but need not delay phase 2 trials. • investigators on the call requested frequent updating with both preclinical and evolving clinical data that are being developed by the different academic and industrial developers to help in decision-making about the various vaccine clinical trials. creation of a central information hub was encouraged for this purpose. • participants on the call expressed the need for standardization of protocols, data collection forms, critical assays (including reagents) and biobanking of samples from initial clinical trials to allow future re-assay once standards are agreed to and enable comparison of results across trials 26 • the group of experts considers that the demonstration of some disease enhancement with any candidate vaccine after viral challenge in animal models should not necessarily represent a no-go signal for deciding whether to progress into early trials in clinical development of a covid-19 vaccine. • continuous monitoring of this risk during clinical trials in an epidemic context will be needed. • each observed effect should be discussed by the developers with their regulators who will ultimately define the actual requirements for clinical studies. the considerations in this document should be interpreted as general scientific remarks based on current knowledge to inform a research agenda that could be beneficial for vaccines in development. these considerations are not of a regulatory nature and cannot in any sense replace the need for proper regulatory consultations on the requirements for vaccines clinical trials. vaccine developers are therefore encouraged to seek individual scientific advice from regulatory authorities. a novel coronavirus from patients with pneumonia in china covid-19 data center developing covid-19 vaccines at pandemic speed respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine a role for immune complexes in enhanced respiratory syncytial virus disease enhanced pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalin-inactivated rsv-immunized balb/c mice is abrogated by depletion of interleukin-4 (il-4) and il-10 pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalin-inactivated rsv-immunized balb/c mice is abrogated by depletion of cd4+ t cells lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease viral-induced enhanced disease illness aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice the sars-cov ferret model in an infection-challenge study severe acute respiratory syndrome coronavirus infection of golden syrian hamsters lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease a dna vaccine induces sars coronavirus neutralization and protective immunity in mice generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice a mouse model for mers coronavirus-induced acute respiratory distress syndrome adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys exacerbated innate host response to sars-cov in aged non-human primates 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012. virology intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcgammar pathway antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets animal models and antibody assays for evaluating candidate sars vaccines: summary of a technical meeting 25-26 immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a highly immunogenic, protective, and safe adenovirus-based vaccine expressing middle east respiratory syndrome coronavirus s1-cd40l fusion protein in a transgenic human dipeptidyl peptidase 4 mouse model anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates correction: immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates immunogenicity and protective efficacy in monkeys of purified inactivated verocell sars vaccine. vaccine immunogenicity and protective efficacy in mice and hamsters of a betapropiolactone inactivated whole virus sars-cov vaccine the pathogenicity of sars-cov-2 in hace2 transgenic mice age-related rhesus macaque models of covid-19 comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen cryo-em structure of the 2019-ncov spike in the prefusion conformation towards an evidence based approach for the development of adjuvanted vaccines mf59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines as03-adjuvanted h5n1 vaccine promotes antibody diversity and affinity maturation, nai titers, cross-clade h5n1 neutralization, but not h1n1 cross-subtype neutralization selective induction of antibody effector functional responses using mf59-adjuvanted vaccination superior antigen-specific cd4+ t-cell response with as03-adjuvantation of a trivalent influenza vaccine in a randomised trial of adults aged 65 and older correlates of adjuvanticity: a review on adjuvants in licensed vaccines investigating the effect of as03 adjuvant on the plasma cell repertoire following ph1n1 influenza vaccination h5n1 influenza vaccine formulated with as03 a induces strong cross-reactive and polyfunctional cd4 t-cell responses protective immunity induced with malaria vaccine, rts,s, is linked to plasmodium falciparum circumsporozoite protein-specific cd4+ and cd8+ t cells producing ifn-gamma impact of adjuvants on cd4(+) t cell and b cell responses to a protein antigen vaccine: results from a phase ii, randomized, multicenter trial different adjuvants induce common innate pathways that are associated with enhanced adaptive responses against a model antigen in humans. front immunol systems analysis of human vaccine adjuvants innate transcriptional effects by adjuvants on the magnitude, quality, and durability of hiv envelope responses in nhps all authors attest they meet the icmje criteria for authorship 28 key: cord-286741-h3oix9zc authors: park, mee sook; kim, jin il; bae, joon-yong; park, man-seong title: animal models for the risk assessment of viral pandemic potential date: 2020-04-22 journal: lab anim res doi: 10.1186/s42826-020-00040-6 sha: doc_id: 286741 cord_uid: h3oix9zc pandemics affect human lives severely and globally. experience predicts that there will be a pandemic for sure although the time is unknown. when a viral epidemic breaks out, assessing its pandemic risk is an important part of the process that characterizes genomic property, viral pathogenicity, transmission in animal model, and so forth. in this review, we intend to figure out how a pandemic may occur by looking into the past influenza pandemic events. we discuss interpretations of the experimental evidences resulted from animal model studies and extend implications of viral pandemic potentials and ingredients to emerging viral epidemics. focusing on the pandemic potential of viral infectious diseases, we suggest what should be assessed to prevent global catastrophes from influenza virus, middle east respiratory syndrome coronavirus, dengue and zika viruses. of the four types of influenza viruses, influenza a virus (iav) and influenza b virus (ibv) cause major respiratory diseases to humans [1, 2] . the iavs can be classified into different subtypes by the antigenicity of surface glycoproteins, hemagglutinin(ha) and na(neuraminidase). so far, 18 and 11 subtypes have been identified from the ha and na proteins, respectively, and the last two subtypes (17 and 18 subtypes in ha and 10 and 11 subtypes in na) were recently discovered from bats [3, 4] . all other subtypes (h1 through h16 and n1 through n9) have been identified in aquatic birds, which are considered as the main reservoirs of iavs [5] . in contrast to the iavs, ibvs are classified into two antigenically distinct lineages, namely victoria and yamagata [1, 5, 6] . while the iavs infect diverse avian and mammalian hosts including humans, the ibvs are circulating mostly among human beings with a few exceptions of spillover cases reported in seals and swine [7] [8] [9] [10] . iav and ibv infections show similar clinical signs of 'influenza-like illness' and outcomes [11] [12] [13] [14] . there have been four major influenza pandemics since 1918 with some glimpses of pandemic-like events in history [15] [16] [17] . the h1n1 influenza pandemic of 1918 (pdm1918) is estimated to have caused up to 50 million human deaths across the globe [18] , symbolizing how devastating one pandemic outbreak can be. it is believed that influenza pandemics can be occurred by antigenic shift, which generally results from the introduction of certain gene segment(s) from nonhuman sources to human infecting iavs through a genetic reassortment process [5, 16] . the efficient human-to-human transmission and lack of immunity against the novel virus in humans can be driving forces to facilitate the dissemination of the virus and then to result in a pandemic. after a pandemic wave, the virus may lose momentum under increasing immune pressures among humans and persist as a seasonal virus. this seasonal virus will retain genetic mutations by circulating season by season, and its viral antigenicity may change, which is so-called antigenic drift, and it is the main reason that the vaccine viruses need updates every year. currently, the h1n1 and h3n2 subtypes of iavs, which are the descendants of 2009 and 1968 influenza pandemics, respectively, and the victoria and yamagata lineages of ibvs are circulating as seasonal viruses in humans. before the h1n1 pandemic in 2009 (pdm2009), an avian h5n1 iav had been remarked as a strong candidate that would cause a next pandemic given accumulating human infection cases with the virus [19, 20] . recently, an avian h7n9 virus has become the focus of attention concerning the increasing number of human infection cases in china [21, 22] . however, it is important to remember that pdm2009 was caused unexpectedly by a swine origin iav [16] , emphasizing the importance of the surveillance of swine iavs [23] . there are also other subtypes of avian ha and na isolated from human influenza cases sporadically [24, 25] . given their pandemic potential, we need to assess these humaninfecting zoonotic iavs in detail by comparing with the viruses that had caused past influenza pandemics. recently, middle east respiratory syndrome coronavirus (mers-cov) is dubbed 'camel-flu' virus [26] . seven years after its first human infection in 2012 [27] [28] [29] , more than 2400 human cases have been reported with approximately 35% case fatality rate [30] . mers-cov has a singlestranded positive-sense rna genome consisting of two partially overlapping large replicase open reading frames (orfs) and at least nine downstream orfs including the orfs encoding the four canonical structural proteins of coronaviruses, the envelope proteins s, e, and m and the n protein [31] . similarity of mers-cov with influenza viruses is not in its genome organization but probably in its respiratory symptoms, zoonotic potential, and the mode of respiratory transmission [32] [33] [34] . in addition to influenza viruses and mers-covs, arthropod-borne viruses, such as dengue and zika, may also pose pandemic threats even though persistent human-to-human transmissions have been rarely reported [35] [36] [37] [38] [39] . in this review, we intend to figure out the recipe and the ingredients of a pandemic by looking into the past pandemic events. iavs have eight segmented genomes of single-stranded, negative-sense rnas, which express similar major proteins, such as polymerase basic 2 (pb2), polymerase basic 1 (pb1), polymerase acidic (pa), ha, nucleoprotein (np), na, matrix 1 (m1) and matrix 2 (m2), nonstructural 1 (ns1), and nonstructural 2 (ns2/nep) [1] . studying the past influenza pandemics helps us to understand the mechanisms of such devastating outcomes. theoretically,144 different iav subtype viruses can be generated by the combinations of 16 ha and 9 na subtypes of avian iavs. however, only the h1n1, h2n2, and h3n2 subtypes have been identified as the causes of human influenza pandemics. of these, the h1n1 subtype caused the 1918 and 2009 pandemics and the 'abortive pandemic'-like swine influenza epidemic in 1976, which hundreds of soldiers at fort dix, new jersey, the united states were infected with [15, 40, 41] (fig. 1) . although diverse iav subtypes have been isolated from swine, h1, h2 and h3, and n1 and n2 subtypes have been mainly established [42] [43] [44] . as mentioned above, the pdm2009 and 1976 h1n1 viruses were swine-origin and readily transmissible among humans [15, 16, 45] . another pandemic h1n1 virus, pdm1918, however, appeared to be closely related with avian strains, which would be ultimately the ancestor of subsequent human and swine h1n1 iavs [46] . since human-infecting avian iavs were shown to be less transmissible among humans [47] [48] [49] , a 'spill-over' infection with avian iavs was not likely to be a fig. 1 timeline of influenza pandemics. each pandemic event is marked by a bold arrowhead on the timeline, which is not to the scale. the open arrowhead designates the 'aborted pandemic'. the animal symbols designate the host origins of the has of the pandemic strains. lines with the diamond heads designate the duration and the end of the circulation. the arrowed lines designate being circulated currently. the dotted lines designate unknown direct source of pdm1918 [50, 51] . as the receptor specificity is considered another prerequisite for avian iavs to infect and transmit to humans, there are some doubts about the avian-origin pdm1918 [52] . the h2 ha of the 1957 h2n2 pandemic and the h3 ha of the 1968 h3n2 pandemic were introduced from the avian reservoir to circulating human iavs [53, 54] , but whether the reassortment events occurred in humans or in other hosts, such as swine, immediately before transmitting to humans remains unanswered [16] . iavs can be largely divided into the human-like and avianlike types given the receptor specificity of their ha proteins. normally, human-like iavs bind to α-2,6 sialic acid (α-2,6 sa) receptor whereas avian-like iavs prefer α-2,3 sa receptor [55, 56] . it has been revealed that an aquatic bird mallard expresses more α-2,3 sa than α-2,6 sa in the respiratory tract but α-2,6 sa is barely expressed in the intestinal tracts [57] . the preference of avian iavs to α-2,3 sa might be related with fecal transmission of the viruses [58, 59] . regarding zoonotic transmission of iavs, swine is considered the intermediate host that can shuffle genetic segments between avian and human iavs to produce a novel strain [54] . because swine expresses both α-2,3 and α-2,6 sas in the upper respiratory tract, avian and human iavs all can infect swine [60] [61] [62] . it has been also challenged by additional studies that demonstrated similar sa distribution between human and swine [63] [64] [65] [66] . however, it has been shown that avian iavs could transmit between swine, and novel strains could be generated from contact swine by the genetic reassortment between avian and swine iavs [67] , which cannot be demonstrated in humans. hence, it appears that swine rather than humans may play a major role for the generation of novel strains at the interface of avian and human iavs [68] [69] [70] , and swine might be considered an adaptation host of iavs, as indicated in the cases of zoonosis [71] [72] [73] [74] [75] [76] and reverse zoonosis of iavs [43, 77] . then, it should be questioned whether avian iavs can be transformed into a pandemic virus by the adaptation only in humans. even though some reports indicated acquired transmissibility of avian iavs through multiple passages in ferrets [78] and transmissibility of avian iavs in swine [67] , it may be limited contact opportunities of the same avian iav to be repeatedly adapted in humans, as demonstrated in herfst et al. [78] . whether a rare adaptive mutant would grow out to dominate in the human host would be another issue. unlike severe symptoms observed in novel avian iav-infected patients, however, swine may be asymptomatic when infected with avian iavs [79, 80] . unless efficient transmissibility of an avian iav in humans was adaptively acquired during a single human infection, there would be only very limited close contact transmission from the patient to the care giver. close contact transmission of avian h7n9 iavs between patients and care givers have been recognized, but the contacted care givers have rarely shown the signs of infection [81] [82] [83] . this may demonstrate why avian iavs have acquired necessary adaptive mutations in swine rather than in humans to be pandemic viruses [69] . from the examples of the past influenza pandemics discussed so far, the recipe of a pandemic may be drawn up. the ingredients of influenza pandemics appear to be (1) non-human animal reservoir(s) that provides novel antigenic sources continuously, (2) adaptation host(s) where accumulated mutations result in host specificity changes or genetic reassortment occurs, (3) proper transmissibility between adaptation host(s) and humans back and forth, (4) efficient human-to-human transmission, and (5) pathogenicity in humans (fig. 2) . the second and third ingredients may work together to generate a virus with the human adapted genes with a new antigenic flavor. we have seen that the influenza pandemics came about with these ingredients and the human activity of socializing and traveling. determining whether a virus has these ingredients might be an important step in assessing its pandemic potential. the sudden reappearance of 1950s seasonal h1n1 strains in 1977 that was dubbed 'russian flu' right after the 1976 swine iav epidemic spurred the awareness of the need for pandemic planning [15, 40] . although the virus had rapidly spread among people under 25 years of age and had been 'drifting' as seasonal strains until the appearance of the pdm2009 [18] , the 1977 h1n1 virus may not be considered a pandemic virus. the virus was only a reappearance of previous human iav most likely by an accidental release and met an immunity gap among young people. the virus was not of a nonhuman reservoir origin, according to the pandemic ingredients summarized above. in case of the h5n1 and h7n9 avian iavs, they may lack efficient transmissibility to and between humans. however, as shown in ferret studies, they should be under close surveillance for their pandemic potential in advance. in addition to these avian iavs, swine iavs should be also monitored because novel strains can be generated by genetic reassortment in swine between avian and human iavs, as presented in the genesis of pdm2009. in contrast to iavs, ibvs do not have animal reservoirs, so they might be considered a virus of less pandemic potential. mers-cov appeared to originate from a bat coronavirus [28, [84] [85] [86] [87] and has become enzootic since a certain time point among dromedary camels [32, [88] [89] [90] , which is readily transmissible to and between humans [34] . unlike other human virus, such as measles [91] , mers-cov might evolve constantly in the dromedary camels [92] , which show a high rate of seroconversion and carry the virus mostly asymptomatically [90] . continuous back and forth transmission between humans and the dromedary camels constitutes very similar situations with iavs. frequent recombination during mers-cov replication in the reservoir host [93] [94] [95] might be used as a tool of adaptation by antigenically novel mers-cov strains or closely related bat coronaviruses, similarly as genetic reassortments of iavs in swine [96] . if there might be a mers-cov pandemic, subsequent mers-cov epidemics by antigenically 'drifted' strains might follow the pattern of seasonal influenza viruses until antigenically shifted (recombined) mers-cov strains hit humans again. mosquitos are a vector and non-human reservoir of dengue and zika viruses. back and forth transmission of these viruses between mosquitos and humans and the 'antibody dependent enhancement' of infection to dengue and zika viruses might potentially support the expansion of susceptible human pools [97] [98] [99] . however, currently there are very limited cases of human-tohuman transmission of these viruses through body fluid contacts [35] [36] [37] [38] [39] , and it is unlikely to result in rapid global spreading of the viruses like iavs, especially since mosquito distribution is geographically limited [100] . hence, possible human-to-human transmission of the dengue and zika viruses is inevitably limited by the requirement of intimate contacts or blood transfusion [101] . even though this ecological limitedness, solid control measures against mosquitos should be implemented to prevent dissemination of these arthropod-borne viral diseases in a global scale. small laboratory animals are surrogate models used in the experiments of human infecting viruses. viral behaviors in a natural host are often different from those in humans. viruses causing serious diseases in humans are often asymptomatic in their natural hosts. this is why natural hosts have a limitation as animal models. in case fig. 2 the ingredients and recipe of influenza virus pandemic. the workings of the five ingredients of iav pandemics are depicted. the ingredients are numbered (1) through (5) . avian iav, swine iav, and human iav are given as aviav, swiav and huiav, respectively. the viruses generated through (1), (2), and (3) may have a diverse range of transmissibility. when a virus with a nonhuman origin ha and an efficient human transmissibility gets transmitted from the adaptation host swine to human (4), a pandemic might ensue (5) of iavs, avian and swine species should be considered the natural reservoir animals, and in case of mers-covs, bats and dromedary camels [32, 87, 90] . viruses causing severe diseases and deaths in humans are the targets of prevention and control. researches using animal models for such viruses are carried out in two main directions: (1) investigation of viral characteristics in hosts, such as replication capacity, cellular tropisms, pathogenicity, and transmission, and (2) development of antivirals and vaccines. no animal models can be a perfect replicate of humans. certain animal models can have advantages in representing viral infection and, at the same time, disadvantages in other aspects. therefore, experimental questions may determine the best animal model, and experiments should be conducted such a way that the results obtained using animal models can be translated to humans. non-reservoir animals used for influenza virus infection experiments include ferrets, mice, guinea pigs, syrian hamsters, and non-human primates [102] [103] [104] [105] [106] [107] [108] [109] . historically, the discovery of the first human influenza virus was made by infecting ferrets with throat washings of influenza patients [110] . ferrets could also be readily infected with swine influenza virus. the different species of guinea pigs, mice, rabbits, hamsters, hedgehogs, and monkeys did not develop flu-like symptoms [103] . however, these asymptomatic animals have become useful for special purposes of influenza virus infection experiments now because virus detection and titration methods have become sophisticated (table 1) . especially, mice and guinea pigs are the most accessible animal models cost-and space-wise. in the case of mice, human infecting influenza viruses do not usually infect mice well, which is overcome by adapting the virus in mice through serial passages [103, 119, 120] . mice express the avian type α-2,3 sa in the lower respiratory tract, similarly as humans, but not the human type α-2,6 sa [121] . this is in line with the tendency of experimentally inoculated avian iavs, regardless of lowpathogenic (lpaiv) or highly pathogenic avian iavs (hpaiv), being mouse lethal with a relatively low 50% mouse lethal dose (mld 50 ) [104, 122] . as far as seasonal isolates of human iavs are concerned, dba/2 mice have been shown to be highly susceptible to diverse strains of un-adapted iavs [123, 124] . ferrets, guinea pigs, and syrian hamsters could be infected with most of iavs without adaptation, but only guinea pigs could be infected with ibv without adaptation and supported the airborne transmission [108, 111] . syrian hamster has been tried to replace mice because iavs could infect it without adaptation and there were airborne transmissions among syrian hamsters [108] . however, since guinea pigs can be infected with un-adapted ibvs as well as iavs and support airborne transmissions, which is readily detectable by the nasal wash titer, any advantage of syrian hamsters over guinea pigs awaits further reports of the use of animal models in influenza virus research. nonhuman primates (nhps) are genetically closest to humans. although nhps are not a readily accessible animal model, nhps are indispensable in the cases of vaccine and antiviral tests, where data relevant to humans in terms of pharmacokinetics and physiology are critical [125] . 'animal efficacy rule' of the united states food and drug administration (fda) requires for the therapeutics to demonstrate efficacy in two animal models manifesting human-like symptoms including at least one non-rodent model [126] . although the needs of testing in nhp models are clearly present [113] [114] [115] [116] [117] , most laboratories cannot afford nhps, and there is other ethical uneasiness about using nhps. how best to do without nhps may be a persisting issue in search of appropriate animal models. in the case of influenza virus research, among the frequently used animal models, ferrets appear to be the only non-rodent model other than nhps [127] . animal models of mers-cov are restricted by the availability of the receptor dpp4 that contains distinct amino acid sequence motif. besides the reservoir host dromedary camels and bats, nhps, rabbits, and other livestock animals, such as goat, cow, sheep, and pig, have been shown to express dpp4 that can bind to mers-cov [85, [128] [129] [130] . however, dpp4 of frequently used small the designation of "o" means that there are many studies using the animal for the purposed study the designation of "?" means that there are no or not many studies using the animal for the purposed study animal models like mouse, hamster, and ferret did not bind to mers-cov [128] . in nhps, mers-cov infection showed similar clinical signs as in humans, ranging from mild to severe, depending on the species of nhps. although dpp4 expressions were similar between rhesus macaques and common marmosets, the disease severity was from mild to moderate and from moderate to severe, respectively [131, 132] . lack of replication of mers-cov in small animal models poses problems of cost and space, especially since experiments using mers-cov must be carried out in an animal biosafety level 3 facility [29, [133] [134] [135] . mouse engineering technology has been deployed in diverse ways to generate mice expressing human type dpp4 (hdpp4) [136] [137] [138] [139] [140] . mers-cov infection in mice having hdpp4 exhibited only moderate signs of respiratory pathology, most likely due to the low level expression of hdpp4 in the mouse lung [135] , but mers-cov could be adapted in these mice to a more pathogenic virus [139, 141] . in addition to nhp and hdpp4-mouse models, rabbits might be a good candidate for mers-cov transmission experiments due to its camel-like receptor distribution in the upper respiratory tract (table 2 ) [142, 150] . however, while dromedary camels and new world camelids could transmit mers-cov upon contact, rabbits could hardly transmit the virus [130, 143] . mers-cov has been shown to use α-2,3 sa as a receptor assistant, which dromedary camels but not rabbits express in the nasal epithelium [130, 151, 152] . humans do not express the primary receptor dpp4 in the upper respiratory tract but transmits mers-cov well [153] . despite the controversies, humans have been reported to express α-2,3 sa in the upper respiratory tract [61, 64] . contribution of the 'pre-attachment' receptor α-2,3 sa or any other 'assistant' receptors to mers-cov transmission might be worth further investigation [151] . as far as the animal efficacy rule of fda is concerned, there appears no other choices but hdpp4-mouse and nhp models in the case of mers-cov studies. human-like symptoms of mers-cov infection have not been reproduced in other animals than hdpp4-mice and nhps. starting from the distinct receptor specificities of the ha proteins between avian and human iavs, host restriction determinants of iavs have been documented [56] . receptor specificity and amino acid signatures at pb2 residue 627 are well established host determinants critical for the interhost transmission of iavs [154] . however, iavs with avian or human receptor specificity can infect swine, as mentioned above. furthermore, the pb2 protein of the triple reassortant swine iav lineage, which comprises a majority of north american swine iavs [155] , retains the avian type e627 (glutamate in the pb2 residue 627) [156] . this was also a part of the molecular signatures of pdm2009 [16] . some human infecting avian iav isolates have shown acquisition of the human type k627 (lysine in the pb2 residue 627) but not acquisition of the human type receptor specificity determinants, and some acquisition of both [80, 157, 158] . iavs were shown to bind cells lacking sialic acid, and replicated efficiently [159] . acquisition of pb2 k627 might be more advantageous than acquisition of human type receptor specificity for avian iavs to replicate in the upper respiratory tract of humans, which is not an optimal temperature for pb2 e627 [157] . it has been also shown that the pb2 e627k mutation can emerge in a human case infected with an avian iav [160] . of note is that, although there have been avian-to-human transmission cases of avian h5n1 and h7n9 iavs, there have been no sustained human-to-human transmission of avian iavs. indeed, it has been reported that an h5n1 hpaiv harboring the human-type pb2 e672k mutation (change from glutamate to lysine in the position 627) and human-type ha q226l and g228s mutations (change from glutamine to leucine and from glycine to serine in positions 226 and 228, respectively, by h3 numbering) by itself could not transmit via airborne droplets between ferrets [78] . this may suggest that airborne transmissibility of avian iavs in humans might not be determined only by the presence or absence of molecular determinants. several studies using reassortant viruses have shown that the competence of reassortant viruses may not be predicted simply by the presence of the specific molecular determinants [161] [162] [163] [164] . an experiment testing a swine 'mixing vessel' hypothesis by co-housing pigs infected with an avian h1n1 strain or with a swine h3n2 strain and naive pigs revealed 40 and 60% transmission efficiency, respectively [67] . in that experiment, the reassortant viruses the designations of "o" and "?" are the same as in table 1 appeared to be well replicated (59/63) in the middle or lower respiratory tract, regardless of the presence of swine pb2 or avian pb2, although all but one reassortants (62/63) contained the swine ha. four out of the 63 reassortants did replicate in the upper respiratory tract and three out of four of those were the swine pb2containing reassortants [67] . what we can learn from this experiment is that the reassortant with an avian ha is not frequently selected in pigs and that the reassortant with swine pb2 is selected for the replication in the upper respiratory tract of pigs. the proportion of iav infection in farmed pigs is relatively low [68] , and the likelihood of co-existing of pigs infected with avian iavs and/or with swine iavs in the same pen may be even lower. however, avian and swine iav reassortants have been established and isolated in pigs [155] , which is the evidence of ongoing 'genetic mixing' of iavs. surveillance of newly emerging iavs may be approached in two ways; isolating and sequencing a virus using nextgeneration sequencing (ngs). as we discussed above, it is not enough to look for the molecular determinants by sequencing. even though full genome sequences are recovered, and their evolutionary relationships are reconstructed, further studies using the viruses should be carried out [165, 166] . the classical method of growing viruses in madin-darby canine kidney (mdck) and human airway epithelial a549 cells might be the first step after a genetic sequence analysis. the mdck cells express both α-2,3 and α-2,6 sas and support replication of influenza viruses ubiquitously due to the lack of the mx protein anti-influenza signaling [167, 168] . therefore, viral growth in the mdck cells is considered to evaluate the inherent growth potential of the viruses under such a condition where no innate and adaptive immune responses of the host are counted in [157] . on the other hand, the a549 cells, expressing more α-2,6 sa than α-2,3 sa like in the human upper respiratory tract, may indicate the growth potential of the viruses in the human upper respiratory tract [169, 170] . any swine isolatesavian, swine endemic, or reassortant originshowing equivalent to or better growth rates in mdck and a549 cells than the swine-origin pdm2009 virus may be further studied for their pathogenicity and transmissibility in animal models, to determine their pandemic potential. viral pathogenicity is related to host cell tropism but may be separate from the susceptibility of hosts to the virus. iavs are pathogenic to humans but not guinea pigs, although both are permissive to the virus. the degree of pathogenicity, the virulence of an iav, is inevitably associated with how well the virus replicates in a tissue or an organ, impairment of which results in a serious disease. in cases of respiratory viruses, those replicating in the lower respiratory tract tend to be more pathogenic than those in the upper respiratory tract [171, 172] . therefore, viral pathogenicity is closely related to inherent replication ability and receptor specificity of the virus. an avian iav with pb2 k627 has been shown to replicate better in both upper and lower respiratory tracts in mice and more pathogenic to the infected mice than with pb2 e627 [157] . however, in case of the pdm2009 isolate a/california/04/09 virus (ca04), the virus lacked previously identified molecular markers of iav virulence or transmissibility [173] , although was more pathogenic to mice than the seasonal h1n1 virus [174] [175] [176] . droplet transmissibility of pdm2009 in ferrets was shown to be slightly lower than the seasonal h1n1 virus whereas their contact transmissibilities were equally efficient [174] . this suggests that antigenic novelty might play a more important role for the pandemic potential of a certain iav than viral transmissibility. ferrets have been known to have human-like glycan distributions in their respiratory tract and may develop respiratory symptom after iav infection [104, 127] . however, although most human iavs including swine iavs are not pathogenic to mice, mice could be a good initial testing model in terms of cost and handling easiness compared with ferrets, especially for an isolate containing avian origin has. since mice have α-2,3 sa in the lower respiratory tract [121] , avian iavs tend to be pathogenic to mice without a prior adaptation [177] . therefore, viral pathogenicity in mice could be an initial pathogenicity indicator of certain iavs. the first pdm2009 isolate ca04 was more pathogenic in balb/c mice than the later pdm2009 isolates [176] . the pdm2009 isolates from fatal cases were also more pathogenic in mice than from the mild cases, and replacing the ha of the mild case isolate with that from the fatal case could make a more pathogenic virus in mice [178] . these experiments show that iav pathogenicity in mice may reflect inherent lung pathogenicity of iavs in humans. however, the results of mouse experiments should be interpreted in comparison with a pathologically well-characterized control. human iavs adapted in mice vs. avian iavs adapted for human-to-human transmission through a serial adaptation process, more pathogenic viral isolates can be recovered. for an iav with the receptor specificity to α-2,6 sa to replicate in mice, where there is hardly any or small amount of α-2,6 sa in the respiratory tract [121, 179, 180] , the virus must have used surrogate receptors such as c-type lectins or else [181] [182] [183] . iavs have been shown to bind to and replicate in sa-free or sialidase treated cells, although to a lower degree than in the untreated cells [159, 183] . abolishing na activity has been shown to be another mechanism of adapting to the host expressing a low level of the specific receptor [184] . indeed, the ha protein of a mouse-adapted pdm2009 has been shown to have acquired higher affinity to α-2,3 sa and lower affinity to α-2,6 sa compared to the wild type [185] . the difference between a human iav adapted in mouse and an avian iav infecting humans may be determined by the usage and availability of appropriate sa or equivalent molecules. to be pathogenic to mice, human iavs with α-2,6 sa specificity must adapt to use non-sa receptors or α-2,3 sa abundant in mice [180] , but avian iavs with α-2,3 sa specificity may have a possibility to replicate in the lower respiratory tract of humans without a prior adaptation. it has been known that humans express α-2,3 and α-2,6 sas in the lower and upper respiratory tracts, respectively [61] . but, it has been also reported that humans express both α-2, 3 and α-2,6 sas at a similar level in the upper respiratory tract [64] . given the availability of animal models that reflect human respiratory diseases, we conceptually suggest human, swine, and ferret respiratory tracts in fig. 3 , based on the study of de graaf et al. [63] . human or swine iavs with α-2, 6 sa specificity would be trapped in the upper respiratory tract of the respective host, where α-2,6 sa is abundant (fig. 3a) . some replicating viruses may overflow down to the lower respiratory tract, where both α-2,3 and α-2,6 sas are expressed. avian iavs with α-2,3 sa specificity would be also trapped in the upper respiratory tract by α-2,3 sa, but replicate poorly due to the unfavorable temperature. under such circumstances, only a high dose of avian iavs allows to escape the trapping in the upper respiratory tract and reach the lower respiratory tract, where the temperature is more favorable for avian iavs. even though avian iavs might overflow from the lower respiratory tract up to the upper respiratory tract, the virus might not replicate there due to the unfavorable temperature, unless there was the pb2 e627k mutation. this may be the reason that avian iavs are not easily transmissible between humans. in terms of viral adaptation, a rare appearance of avian iav mutants with α-2,6 sa specificity may not have special selective growth advantages in the lower respiratory tract of humans due to the overwhelming dominance of α-2,3 sa. only when avian iavs replicating in the upper respiratory tract, although poorly, acquires the pb2 e627k mutation or a reassortment, with or without acquisition of α-2,6 sa specificity at the same time, the variants may grow out well [67] . serial passaging of a wild-type h5n1 hpaiv in ferrets could not make the virus airborne transmissible between ferrets, but only those containing the mutations conferring the human type α-2,6 sa specificity and pb2 e627k could acquire airborne transmissibility after several passages in ferrets [78] . basically, these experiments suggest that, even if a rare mutant retaining the human-type receptor specificity and pb2 determinants might appear during replication of avian iavs, the mutant might not be easily selected to a domination over multiple passages, at least in ferrets. the reason may be that avian iavs have their niche of efficient replication in the lower respiratory tract of human, swine, or ferret (fig. 3) . the issue of avian iav adaptation in humans may not be whether adaptive mutations appears but whether there is a selective force enough for a virus to possess adaptive mutations to grow out to dominance. the ha protein of avian h5n1 iavs has been reported to require both human-type q226l and g228s mutations to bind to both α-2,3 and α-2,6 sas [186] , and that of avian h7n9 iavs only needs the q226l mutation [122, 187] . interestingly, a h7n9 human isolate was contact transmissible in pigs, regardless of pb2 e627 or k627, but only in the acquisition of human-type ha q226l determinant, which indicates the importance of the acquisition of human-type receptor specificity for the avian iav transmission in pigs, potentially also in humans [188] . however, as demonstrated in fig. 3b and c, the acquisition of human-type pb2 k627 would have been more critical due to the presence of saα-2,3 glycans in the upper respiratory tract of pigs. avian iavs with the pb2 e627k mutation should be able to replicate in the upper respiratory tract of humans and may be transformed to be transmissible between humans (fig. 3c) . however, they were poorly transmissible in ferrets (fig. 3d ). there have been no reports that investigated the transmissiblity of avian iavs with the pb2 e627k mutation between humans. all the first three human isolates of avian h7n9 iavs, which contained the human-type pb2 e627k and ha q226l mutations, showed approximately 30% airborne transmissibility in ferrets [48, 80, 122] . these appear to match with the conceptualized transmission in ferrets of fig. 3d . the h7n9 isolates with pb2 k627 and ha l226 would have replicated in the upper respiratory tract of ferrets, but their replication might have been inefficient since the receptor binding of the h7n9 q226l ha was still weaker to α-2,6 sa than to α-2,3 sa [187] . airborne transmission of the h7n9 virus in ferrets lacking α-2,6 sa might be explained by the overflow of the virus replicating in the lower respiratory tract, since ferrets poorly express α-2,3 sa in the upper respiratory tract [189] . this model also indicates similar level of transmission of avian iavs in ferrets without any human-type determinants. therefore, the conceptual model of iav transmission based on the receptor distribution appears to agree only with the ferret experiments, and probably only with h7n9 transmission in ferrets, since avian h5n1 iavs were not transmissible in ferrets [78, 190] . this conceptual model is apparently an oversimplification. there may be more factors involved in the transmission of iavs, such as the functional balance between the ha and na proteins and inherent replicability of iavs [191, 192] . the problem is, due to the discrepancy between what is expected in humans by the conceptual model and the reality, how to translate the results of transmission experiments evaluated in animal models to the natural transmission environments between humans. we may compare the transmissibility of certain viral isolates to that of ca04 in ferrets, but it is difficult to determine what the level of the transmissibility of these viruses indicate in terms of the pandemic potential of the viruses. the reports on the α-2,3 sa expression status in the upper respiratory tract of ferrets, pigs, and humans appear to be inconclusive [63] . determination of iav receptor distributions in humans and animal models appears critical for the interpretation of pathogenicity and transmission experiments assessed in animal models. we have discussed how to approach to the determination of the pandemic potential of iavs. similar principles may apply to mers-covs. previously, mers-cov replication was noted in nhp-derived cell lines, vero, and llc-mk2 cells [28] . it is now known that dpp4 is a functional cellular receptor for mers-covs and that vero cells express dpp4 [193] . vero cells also express α-2,3 sa, which has been shown to assist receptor binding of mers-covs [130, 151, 152, 167] . vero cells are well known for an impairment in the type i interferon pathways [194] . . avian iav with e627 or k627 pb2 might behave similarly in ferrets. an avian iav, without the specific receptor in the upper respiratory tract of ferret, may replicate in the lower respiratory tract but relatively poorly due to relatively low expression of saα-2,3 glycans. the overflowing avian iav, although with a low likelihood, may not be 'cleaned up' due to lack of saα-2,3 glycans in the upper respiratory tract of ferret, and may occasionally get transmitted to nearby ferrets covs in the upper respiratory tract is likely to contribute to their better transmission [143] . therefore, growth characteristics of camel mers-covs in vero cells may give us initial clues about how efficient the transmission of novel strains might be. the pathogenicity of mers-covs is closely associated with dpp4 expression in the lower respiratory tract of humans [153] . like iavs, the pathogenicity of mers-cov in hdpp4 mice may be an initial indicator. while hdpp4 mice produced severe symptoms upon mers-cov infection [140] , mice whose mdpp4 replaced with hdpp4 or modified to contain mers-cov binding hdpp4 motif (m-hdpp4-mice) showed little clinical signs [138, 139] . hdpp4 mice might be good for the evaluation of antiviral candidates, but not as a pathogenicity indicator, due to the ectopic expression of hdpp4. hdpp4 mice might be better to observe increases of growths and clinical signs after mers-cov infection. comparison of viral titers in the lungs and the lung pathology of mers-cov infection with those of the first mers-cov isolate hcov-emc [28] would give clues concerning the replication potential of mers-covs. there are no small animal models for the evaluation of mers-cov transmission yet. even though avian iavs transmit extremely poorly between humans, mers-covs appears highly transmissible between humans. in case of mers-cov outbreaks in korea, 2015, a superspreader individual resulted in 28 infection cases [34] . however, it is difficult to appropriately interpret the transmissibility difference between iavs and mers-covs in ferrets. due to the distribution differences of dpp4 and α-2,3 sa in the lower and upper respiratory tracts in humans and dromedary camels, it might be a major problem in translating the results of transmission experiments using small animal models. hcov-emc exhibited no transmission in rabbits [143] , and a kind of transmission threshold has not been defined, so it would be a problem to determine the pandemic potential of mers-cov isolates. the dpp4 motif of nhps have the same sequence with that of humans [132, 147] . however, no mers-cov transmission has been reported between nhps. hence, some kinds of surrogate measures might be necessary to evaluate the transmissibility of mers-covs. mice are not capable of contact or aerosol transmitting iav but can be infected with aerosolized live-attenuated influenza vaccine virus using a nebulizer [195] . since it has been reported that the lower respiratory tract of hdpp4 mice is very similar to that of humans, replication kinetics of a mers-cov isolate in the lungs of hdpp4 mice would provide a clue concerning transmissibility and virulence of mers-covs. this suggests the feasibility and importance of hdpp4 mice for the evaluation of replication or transmissibility of mers-covs. we have discussed what may be required to be a pandemic virus by analyzing the past influenza pandemics. the uniqueness of the past influenza pandemics is in that the three pools of reservoirs or hosts (avian, swine, and human) keep the persistent potential of generating novel iavs and that no other pathogens are known to bring about pandemics recurrently. moreover, the ingredients of the influenza pandemics and the modes of transmission may apply to other pathogens exhibiting pandemic potential. unlike iavs, mers-covs have not swept global communities and appeared to need a persistent human reservoir. the ultimate goal of mers-cov researches may find a way to prevent a mers-cov pandemic. studying the pandemic viruses, such as pdm1918 and pdm2009, may provide scientific information regarding molecular and viral requirements of potential pandemic viruses. our conceptual interpretation of animal models also underlines the value and importance of preclinical experiments in terms of the translational purposes and insights of the pandemic potential of influenza and other rna viruses. this study was supported by a grant from the national research foundation of korea (nrf) funded by the ministry of science and ict, republic of korea (grant no. nrf-2017r1a2b2003773). the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. availability of data and materials not applicable. influenza a in bovine species: a narrative literature review novel insights into bat influenza a viruses bat-derived influenza-like viruses h17n10 and h18n11 cocirculation of two distinct evolutionary lineages of influenza type b virus since 1983 recurring influenza b virus infections in seals influenza b virus in seals domestic pigs are susceptible to infection with influenza b viruses susceptibility of the domestic pig to influenza b virus comparison of clinical features and outcomes of medically attended influenza a and influenza b in a defined population over four seasons comparative outcomes of adults hospitalized with seasonal influenza a or b virus infection: application of the 7-category ordinal scale comparing clinical characteristics between hospitalized adults with laboratory-confirmed influenza a and b virus infection hospitalization for influenza a versus b influenza pandemics of the 20th century the first influenza pandemic of the new millennium historical thoughts on influenza viral ecosystems, or behold a pale horse, dead dogs, failing fowl, and sick swine emergence and pandemic potential of swine-origin h1n1 influenza virus avian influenza virus h5n1: a review of its history and information regarding its potential to cause the next pandemic pandemic influenza threat and preparedness avian influenza a virus pandemic preparedness and vaccine development. vaccines (basel) the pandemic threat of emerging h5 and h7 avian influenza viruses. viruses phylogenetic analysis of a swine influenza a(h3n2) virus isolated in korea in 2012 recent zoonoses caused by influenza a viruses zoonotic potential of influenza a viruses: a comprehensive overview recent advances in the vaccine development against middle east respiratory syndrome-coronavirus severe respiratory illness caused by a novel coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers: emergence of a novel human coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives high reproduction number of middle east respiratory syndrome coronavirus in nosocomial outbreaks: mathematical modelling in saudi arabia and south korea the clinical and virological features of the first imported case causing mers-cov outbreak in south korea global spread and persistence of dengue prolonged detection of dengue virus rna in the semen of a man returning from thailand to italy threat of dengue to blood safety in dengue-endemic countries effect of acute zika virus infection on sperm and virus clearance in body fluids: a prospective observational study zika virus: an emerging infectious disease with serious perinatal and neurologic complications pandemic influenza planning swine influenza a outbreak diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza a(h1n1) history of swine influenza viruses in asia genetics, evolution, and the zoonotic capacity of european swine influenza viruses sowing the seeds of a pandemic? mammalian pathogenicity and transmissibility of h1 variant influenza viruses from the swine reservoir the origin and virulence of the 1918 "spanish" influenza virus a two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission infectivity, transmission, and pathology of human-isolated h7n9 influenza virus in ferrets and pigs increased acid stability of the hemagglutinin protein enhances h5n1 influenza virus growth in the upper respiratory tract but is insufficient for transmission in ferrets 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds a single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity genesis and pathogenesis of the 1918 pandemic h1n1 influenza a virus the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses evolution and ecology of influenza a viruses receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h3 hemagglutinin based on species of origin host and viral determinants of influenza a virus species specificity distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species shedding light on avian influenza h4n6 infection in mallards: modes of transmission and implications for surveillance isolation of a reassortant h13n2 virus from a mallard fecal sample in south korea molecular basis for the generation in pigs of influenza a viruses with pandemic potential avian flu: influenza virus receptors in the human airway sialic acid tissue distribution and influenza virus tropism. influenza other respi viruses role of receptor binding specificity in influenza a virus transmission and pathogenesis sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses comparative distribution of human and avian type sialic acid influenza receptors in the pig distribution of sialic acid receptors and influenza a virus of avian and swine origin in experimentally infected pigs tissue tropisms opt for transmissible reassortants during avian and swine influenza a virus co-infection in swine the inability to screen exhibition swine for influenza a virus using body temperature experimental infection of pigs with the human 1918 pandemic influenza virus absence of clinical disease and contact transmission of hpai h5nx clade 2.3.4.4 from north america in experimentally infected pigs swine influenza virus infections in humans mammalian pathogenicity and transmissibility of a reassortant eurasian avian-like a(h1n1v) influenza virus associated with human infection in china triplereassortant swine influenza a (h1) in humans in the united states transmission of influenza a viruses between pigs and people influenza a(h3n2) virus in swine at agricultural fairs and transmission to humans outbreak of influenza a(h3n2) variant virus infections among persons attending agricultural fairs housing infected swine -michigan and ohio continual reintroduction of human pandemic h1n1 influenza a viruses into swine in the united states airborne transmission of influenza a/h5n1 virus between ferrets domestic pigs have low susceptibility to h5n1 highly pathogenic avian influenza viruses human infection with a novel avian-origin influenza a (h7n9) virus probable person to person transmission of novel avian influenza a (h7n9) virus in eastern china, 2013: epidemiological investigation limited human-to-human transmission of avian influenza a(h7n9) virus clusters of human infection and human-to-human transmission of avian influenza a(h7n9) virus tissue distribution of the mers-coronavirus receptor in bats replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) cd26/ dpp4 cell-surface expression in bat cells correlates with bat cell susceptibility to middle east respiratory syndrome coronavirus (mers-cov) infection and evolution of persistent infection adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus replication of mers and sars coronaviruses in bat cells offers insights to their ancestral origins middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir biological feasibility of measles eradication mers coronaviruses from camels in africa exhibit region-dependent genetic diversity mers-cov recombination: implications about the reservoir and potential for adaptation evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission the recent ancestry of middle east respiratory syndrome coronavirus in korea has been shaped by recombination reverse zoonosis of influenza to swine: new perspectives on the human-animal interface homotypic dengue virus reinfections in nicaraguan children the possible role of cross-reactive dengue virus antibodies in zika virus pathogenesis zika virusimmune plasmas from symptomatic and asymptomatic individuals enhance zika pathogenesis in adult and pregnant mice an overview of mosquito vectors of zika virus history of arthropod-borne virus infections in french polynesia the contribution of animal models to the understanding of the host range and virulence of influenza a viruses animal models for influenza virus transmission studies: a historical perspective animal models for influenza virus pathogenesis and transmission bronchointerstitial pneumonia in guinea pigs following inoculation with h5n1 high pathogenicity avian influenza virus pathogenesis of 1918 pandemic and h5n1 influenza virus infections in a guinea pig model: antiviral potential of exogenous alpha interferon to reduce virus shedding the guinea pig as a transmission model for human influenza viruses syrian hamster as an animal model for the study of human influenza virus infection animal models in influenza vaccine testing a virus obtained from influenza patients transmission of influenza b viruses in the guinea pig transmission in the guinea pig model the use of nonhuman primates in research on seasonal, pandemic and avian influenza pathogenesis of influenza a (h5n1) virus infection in a primate model experimental production of respiratory tract disease in cebus monkeys after intratracheal or intranasal infection with influenza a/victoria/3/75 or influenza a/new jersey/76 virus african green monkeys recapitulate the clinical experience with replication of live attenuated pandemic influenza virus vaccine candidates evaluation of replication, immunogenicity and protective efficacy of a live attenuated cold-adapted pandemic h1n1 influenza virus vaccine in non-human primates establishment of a cynomolgus macaque model of influenza b virus infection a single amino acid in the polymerase acidic protein determines the pathogenicity of influenza b viruses adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza a viruses influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells pathogenesis and transmission of avian influenza a (h7n9) virus in ferrets and mice dba/2 mouse as an animal model for anti-influenza drug efficacy evaluation the dba.2 mouse is susceptible to disease following infection with a broad, but limited, range of influenza a and b viruses comparison of the pharmacokinetics of several polychlorinated biphenyls in mouse, rat, dog, and monkey by means of a physiological pharmacokinetic model development of animal models against emerging coronaviruses: from sars to mers coronavirus the ferret as a model organism to study influenza a virus infection host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 livestock susceptibility to infection with middle east respiratory syndrome coronavirus host determinants of mers-cov transmission and pathogenesis pneumonia from human coronavirus in a macaque model an acute immune response to middle east respiratory syndrome coronavirus replication contributes to viral pathogenicity permissivity of dipeptidyl peptidase 4 orthologs to middle east respiratory syndrome coronavirus is governed by glycosylation and other complex determinants middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a mouse model for mers coronavirus-induced acute respiratory distress syndrome a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease asymptomatic middle east respiratory syndrome coronavirus infection in rabbits lack of middle east respiratory syndrome coronavirus transmission in rabbits enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus mers coronavirus induces apoptosis in kidney and lung by upregulating smad7 and fgf2 replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein species-specific colocalization of middle east respiratory syndrome coronavirus attachment and entry receptors differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels a single amino acid in the pb2 gene of influenza a virus is a determinant of host range a historical perspective of influenza a(h1n2) virus pb2 residue 271 plays a key role in enhanced polymerase activity of influenza a viruses in mammalian host cells growth of h5n1 influenza a viruses in the upper respiratory tracts of mice characterization of h7n9 influenza a viruses isolated from humans influenza virus infection of desialylated cells emergence of the virulence-associated pb2 e627k substitution in a fatal human case of highly pathogenic avian influenza virus a(h7n7) infection as determined by illumina ultra-deep sequencing reassortment between seasonal h1n1 and pandemic (h1n1) 2009 influenza viruses is restricted by limited compatibility among polymerase subunits reassortment between avian h5n1 and human h3n2 influenza viruses creates hybrid viruses with substantial virulence genetic compatibility and virulence of reassortants derived from contemporary avian h5n1 and human h3n2 influenza a viruses virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (h1n1) 2009 influenza virus and circulating influenza a viruses rescue of influenza a virus from recombinant dna a dna transfection system for generation of influenza a virus from eight plasmids african green monkey kidney (vero) cells provide an alternative host cell system for influenza a and b viruses high yields of influenza a virus in madin-darby canine kidney cells are promoted by an insufficient interferon-induced antiviral state receptor binding specificity of recent human h3n2 influenza viruses differential susceptibility of different cell lines to swine-origin influenza a h1n1, seasonal human influenza a h1n1, and avian influenza a h5n1 viruses pathogenesis of influenza virus infections: the good, the bad and the ugly molecular pathology of emerging coronavirus infections introduction of virulence markers in pb2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses replication and pathogenic potential of influenza a virus subtypes h3, h7, and h15 from free-range ducks in bangladesh in mammals virulence determinants of pandemic a(h1n1)2009 influenza virus in a mouse model sialic acid recognition is a key determinant of influenza a virus tropism in murine trachea epithelial cell cultures effective replication of human influenza viruses in mice lacking a major alpha2,6 sialyltransferase the c-type lectin langerin functions as a receptor for attachment and infectious entry of influenza a virus nlinked glycosylation facilitates sialic acid-independent attachment and entry of influenza a viruses into cells expressing dc-sign or l-sign evolving complexities of influenza virus and its receptors adaptation of influenza a viruses to cells expressing low levels of sialic acid leads to loss of neuraminidase activity adaptation of pandemic h1n1 influenza viruses in mice structure and receptor specificity of the hemagglutinin from an h5n1 influenza virus three mutations switch h7n9 influenza to human-type receptor specificity analysis of recombinant h7n9 wild-type and mutant viruses in pigs shows that the q226l mutation in ha is important for transmission influenza h1n1 a/solomon island/ 3/06 virus receptor binding specificity correlates with virus pathogenicity, antigenicity, and immunogenicity in ferrets experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets functional balance of the hemagglutinin and neuraminidase activities accompanies the emergence of the 2009 h1n1 influenza pandemic effects of ha and na glycosylation pattern changes on the transmission of avian influenza a(h7n9) virus in guinea pigs dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc influenza a virus lacking the ns1 gene replicates in interferon-deficient systems aerosol vaccination induces robust protective immunity to homologous and heterologous influenza infection in mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank sang woo kim, juho do, dahae hong, and eunji choi for technical assistance with the animal experiments.authors' contributions m-sp outlined this review. msp, jik, j-yb, and m-sp wrote this review. the authors read and approved the final manuscript. the authors declare no competing interests.received: 28 january 2020 accepted: 10 march 2020 key: cord-311937-6hadssmh authors: sherbini, nahid; iskandrani, ayman; kharaba, ayman; khalid, ghalilah; abduljawad, mohammed; al-jahdali, hamdan title: middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data date: 2016-06-11 journal: j epidemiol glob health doi: 10.1016/j.jegh.2016.05.002 sha: doc_id: 311937 cord_uid: 6hadssmh background: middle east respiratory syndrome coronavirus (mers-cov), is an emerging virus respiratory infection. it has a high mortality rate and a wide spectrum of clinical features. this study describes the clinical characteristics and outcome of mers infected patients. methods: a retrospective study was conducted of all confirmed mers-cov infections from march 2014 to may 2014 at two tertiary care hospitals in al-madinah region (saudi arabia). we gathered data about demographic, clinical presentation, and factors associated with severity and mortality. results: a total of 29 cases were identified; 20 males (69%) and nine females (31%), age 45 ± 12 years. the death rate was higher for men (52%) than for women (23%). initial presentation was fever in 22 (75%) cases, cough in 20 (69%) cases, and shortness of breath in 20 (69%) cases. associated comorbidities were diabetes mellitus in nine (31%) patients and chronic kidney disease (ckd) in eight (27%) patients. duration of symptoms before hospitalization ranged from 2.9 days to 5 days. elevated liver enzymes were present in 14 (50%) patients and impaired renal profile present in eight (27%) patients. we also describe in this study radiological patterns and factors associated with mortality. conclusion: mers-cov infection transmission continues to occur as clusters in healthcare facilities. the frequency of cases and deaths is higher among men than women and among patients with comorbidities. in saudi arabia, a beta new coronavirus was isolated for the first time at the end of 2012 from a patient who presented with acute community acquired pneumonia [1] . he died 11 days later from progressive severe respiratory failure and acute renal failure (arf) and his sputum sample was negative for respiratory viruses commonly tested. epidemiology of middle east respiratory syndrome coronavirus (mers-cov) was expanded after exploring the large hospital outbreak in al-hasa, saudi arabia [2] . subsequently, the virus was designated as mers-cov [3] . the geographic distribution of the cases has been mainly linked to the arabian peninsula particularly from saudi arabia where most of the cases were reported [4] [5] [6] [7] . however, in some countries in north america, europe, africa, and asia, the disease has been detected in some travelers from endemic countries [3, [7] [8] [9] [10] [11] [12] [13] . the initial occurrence of mers-cov was thought to have particular predominance for male patients and those with comorbid diseases. the male-tofemale ratio was between 2.8:1 and 3.3:1 [2, 6] ; this male predominance might have been related to the nature of the outbreak. initial cases were reported among elderly patients with a median age of 56 years. mers-cov has a very high mortality rate, and complications arising from infection can result in severe respiratory and renal failure [2] . symptoms of mers-cov range from mild upper respiratory symptoms to rapidly progressive severe pneumonia requiring intubation, and multiorgan failure. a significant number of patients may present with nonrespiratory symptoms such as headache, myalgia, and gastrointestinal symptoms of nausea, diarrhea, or vomiting [2, 14] . this study describes the demographic, clinical characteristics, and outcome of mers-cov in al-madinah region, saudi arabia. a retrospective chart review study of all confirmed mers-cov cases recorded by two tertiary hospitals from the madinah region from march 2014 to may 2014. institutional review board approval was obtained for our study from authorities of both hospitals. a case was confirmed as having infection if mers-cov real-time polymerase chain reaction was positive, using the recommended sampling technique (nasopharyngeal swab and tracheal aspirates or bronchoalveolar lavage in intubated patients). extraction of rna was performed with roche magna pure lc (rna viral isolation kit). samples were pretreated with lysis according to the manufacturer's instructions [15] . we obtained data about demographic characteristics, clinical presentation, laboratory results, diagnosis, incubation period, smoking history, comorbidities, and history of contact with camels or mers-cov positive patients in regions within the madinah area. we recorded the duration of the patient's illness, microbiological test results, and reviewed imaging and treatments received. we also recorded the following outcomes: duration of mechanical ventilation, intensive care unit (icu) length of stay, and survival during hospitalization until the patient is discharged from hospital. data were analyzed using ibm spss for windows, version 18.0. the frequency of cases of mers-cov infection and percentage of resulting deaths were calculated. statistical analyses of demographics, clinical, and laboratory descriptive data are tabulated. descriptive statistics such as means and standard deviation mean (±sd) were used to describe the age of the patients, laboratory test results, and duration of illness. frequencies and percentages n (%) were used to describe demographic and mer-cov outcomes. we also did a correlation with the outcome using the t test and fisher's exact test as appropriate with a significant value at p 6 0.05. the total number of cases with confirmed mers-cov infection reported from april 2014 to may 2014 was 29. the majority of patients (60%) were men, and the median age was 45 years. the most common symptoms were fever (75.9%) and cough (69%), shortness of breath (69%), and vomiting and diarrhea (27%). the average duration of symptoms prior to hospitalization was 5 days (range, 1-12 days). demographic and clinical characteristics of patients with confirmed mers-cov are shown in table 1 . mortality rate among patients with confirmed mer-cov was 34%. mortality from mers-cov was significant (p < 0.05) and associated with older age, the presence of gastrointestinal symptoms, longer duration of symptoms prior to hospitalization (8 ± 2.5 days), diabetes mellitus, chronic kidney disease (ckd), smokers, and lower blood pressure, as shown in table 1 . most of the patients were coming from hanakia 11 (38%) and all 11 patients had contact with camels table 2 . close contact with confirmed index mers-cov was documented in five patients, all of whom were healthcare professionals; three staff nurses and two clinicians. most of the patients were mildly hypoxic; oxygen saturation was 88.9 ± 5.4 at presentation with a picture of respiratory acidosis (ph 7.3 ± 0.1 and pco2 57.1 ± 8.2). the basic liver functions show elevated alanine transaminase (98.4 ± 105.4 u/l), aspartate aminotransferase (86.3 ± 93.5 u/l), and creatinine (225.0 ± 115.3 mmol/l). as shown in fig. 1 , which were collected during the patient's initial assessment. all patients had abnormal initial chest radiographs. the predominant finding was bilateral basal consolidations with ground-glass opacities, nodular or/and reticular pattern, and total diffuse multilobar involvement. all patients had appropriate supportive management and received a broad spectrum antibiotic and readjusted based on sputum cultures. among patients who required icu, the mean time of icu stay ranged from 9 days to 55 days (13.7 ± 4.0 days), and mechanical ventilation support was used in nine (31%) patients. mechanical ventilation support and longer stay in icu were significantly associated with death (p < 0.05) (see table 4 ). emerging viral respiratory infections are causing a significant burden on public health and causing significant morbidity and mortality. over the past decades, many viral infection outbreaks have been reported including influenza h7n9 such as h1n1, sars-cov, and the most recent mers-cov infection. the world health organization reported 1368 laboratory-confirmed cases of human infection with mers-cov including at least 487 deaths between 2012 and july 2015 [7] . they reported that 65% of cases were male (n = 1359) and the median age was 50 years (n = 1365) which is similar to our study. similar to a study reported by assiri et al. [2] , our study showed more cases among older patients, but our study showed an association of death with older age. since 2012, 26 countries have been affected, including countries in the middle east, africa, europe, asia, and north america as reported from the world health organization. the majority of cases (75%) have been reported from saudi arabia. in saudi arabia, mortality secondary to mers-cov was 35% [16] and in our study it was 37% (al-madinah). camels have been confirmed as a reservoir for mers-cov, and many hypotheses are behind this zoonotic (camels) transmission [17, 18] . in our study, only five healthcare employees acquired infection from documented contact with an infected patient, but another 24 patients were coming from areas around al-madinah; the largest number of infected patients was from the alhenakia area, where camels are prevalent. the second important mode of transmission is person-toperson transmission (travelers returning from the middle east and close contacts with mers-cov cases) [19] . this type of transmission was confirmed by genome sequencing of mers-cov [19] and isolates from the al-hasa healthcare-associated outbreak [16] . in the uk, mers-cov was transmitted to a family member who visited a patient with confirmed infection and another report from france described patient-to-patient nosocomial transmission of mers-cov. in our study, five (18%) patients had transmission through close contact. approximately 30% of mers-cov patients in our study reported diabetes and ckd, which is similar to those from other observational epidemiological studies in saudi arabia [2, 16] . the high mortalities reported early were probably due to a delay in the diagnosis and presence of comorbidities [20] . however, the large number of mers-cov cases and ckd might have been based on the al-hasa hospital outbreak, which mainly happened in the dialysis center [2] . we may consider the existence of chronic illnesses such as diabetes, hypertension, and ckd increasing the risk of acquiring this infection and categorize them as a high risk group for more complications and worse outcome as was revealed [21] also in our study. we and others have found that the severity of illness associated with mers-cov infection ranges from mild to fulminant [22] . the severity of the respiratory infections caused by mers-cov can progress to hypoxemic respiratory failure which requires the use of mechanical ventilation and death [23] . all of our patients had significant respiratory manifestations requiring admission to the icu but 30% only required mechanical ventilation and died [24] . mers-cov is known to infect cell lines of the intestinal tract [25] , but it is not yet known what proportion of ill patients shed the virus in their stools, which is why some patients presented with gastrointestinal symptoms. identification of the full range of clinical presentations is important so that the mild cases are not missed. mers-cov is detected by reverse transcription polymerase chain reaction. to date, laboratory testing for mers-cov remains not very accurate; the sensitivity and negative predictive values are unknown. development of rapid and accurate diagnostic tests is needed urgently. results of throat swabs were occasionally negative and repeat testing for mers-cov is recommended. it seems difficult to conclude that one negative sample is enough to rule out mers-cov disease when a patient presents with respiratory symptoms and history of exposure. it is also not clear whether nasopharyngeal samples might be superior to throat samples or whether virus is shed more abundantly later in the course of the illness as it is in sars. there is evidence that repeat testing and tests on sputum or bronchoalveolar lavage fluid are of value in improving diagnostic accuracy. microbiological investigations were done routinely to exclude bacterial copathogens with community acquired pneumonia (cap). we had seven patients with methicillin-resistant staphylococcus aureus (mrsa) coinfection, two with streptococcus and one with methicillin-sensitive staphylococcus aureus (mssa) in our study population. assiri et al. [2] stated that none of the 47 samples screened was positive. other investigators found that one patient was coinfected with mssa and influenza b and another with streptococcus pneumonia [23] . there might have been a selection bias because we were only screening critically ill cases, which in turn will lead to detection of more severe cases of mers-cov infection; mild cases may not come to hospital or may not be screened for mers-cov and could lead to false high case-fatality rates. clinical symptoms, laboratory investigations, and imaging findings of mers-cov are similar to those noted in other community-acquired respiratory tract infections. radiological findings in mers-patients tended to range from unilateral focal air-space opacities to multifocal or bilateral lower lobe involvement was seen with a picture of organizing pneumonia which was noted in our patients and other reports [25, 26] . on the basis of findings until now, the clinical features of mers-cov infection have similarities to those seen in patients with sars-cov infection. the initial phase of nonspecific fever, cough, and shortness of breath are the major symptoms in those admitted to hospital; other common symptoms include chills, rigor, headache, myalgia, and malaise which may last for several days before progressing to pneumonia [21, 22] . a significant number of patients had gi symptoms, another important similarity to sars. we found patients with mers-cov who had gi manifestation tend to progress to severe illness and this may be considered one of the poor prognostic factors. the disease may progress rapidly to a critical respiratory failure, requiring mechanical ventilation and lead to death in the icu [5] . in our observations, all of the patients started with symptoms of fever, cough for 5-7 days. our study design has several limitations including that it is a retrospective chart review study with known inherited problems; these include missing data regarding contact with camels and documentation of all comorbidities and availability of follow up data after discharge. despite these limitations, we have been able to highlight some features in the epidemiological, demographic, and clinical characteristics of patients with mers-cov infection in al-madinah regions. the epidemiology and the transmission pattern of mers-cov to date indicate that the majority of cases occur in the healthcare setting. strengthening the infection control measures in the healthcare setting is of great importance. since about 25% of cases are community based, there is a real need to further prevent the animal-to-human transmission of mers-cov. the frequency of cases and deaths is higher among men than women and those around 45 years of age are the most affected patients. the disease had higher mortality in older patients with comorbidities. also, the presence of gastrointestinal manifestations, high liver enzymes, and need for mechanical ventilation or longer stay in icu are all associated with high mortality. there are gaps in our knowledge of the epidemiology, prevalence, clinical characteristics, prognostic factors, and nature of the disease. it is also important to further delineate the transmission routes and the presence of any other animal or intermediate hosts. the influence of geographical distribution and comorbidities on the incidence and outcome of mers-cov patients should be studied further. the authors report no conflicts of interest in this work. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group managing mers-cov in the healthcare setting epidemiological and clinical aspects of coronavirus infection mers-cov middle east respiratory syndrome novel corona mers-cov infection. epidemiology and outcome update middle east respiratory syndrome coronavirus (mers-cov), summary of current situation, literature update and risk assessment. who/mers/ra/151. geneva: who lessons to learn from mers-cov outbreak in south korea epidemiological investigation of mers-cov spread in a single hospital in south korea public health response to two incidents of confirmed mers-cov cases travelling on flights through london heathrow airport in 2014. lessons learnt travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia eds on heightened alert for mers-cov as first cases reach the us middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands middle east respiratory syndrome coronavirus (mers-cov): what lessons can we learn? screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study hospital outbreak of middle east respiratory syndrome coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012 family cluster of middle east respiratory syndrome coronavirus infections prevalence of diabetes mellitus in a saudi community severe acute respiratory syndrome and coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection a major outbreak of severe acute respiratory syndrome in hong kong clinical and laboratory features in the early stage of severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings this study did not receive funding. key: cord-296026-6qtip2ga authors: cho, sung-il title: urgent call for research on middle east respiratory syndrome (mers) in korea date: 2015-07-31 journal: j prev med public health doi: 10.3961/jpmph.15.047 sha: doc_id: 296026 cord_uid: 6qtip2ga nan there have been no additional cases for 22 consecutive days since july 4, leaving a total of 186 laboratory-confirmed cases from the outbreak [1] . there have been 36 deaths (19.4%) among the confirmed cases. a few more deaths might occur from the 12 cases who are still under treatment. starting from the index case, who received confirmatory diagnosis on may 20, the outbreak showed the characteristics of a hospital-associated epidemic. transmission occurred across korea's network of hospitals, as infective patients moved from one hospital to another [2] . hospitals often served as amplification points that generated clusters of new cases. as a result, 82 (44%) of the mers cases were patients being treated in the hospitals, 65 (35%) were family members or visitors of the patients, and the other 39 (21%) were medical personnels including physicians and nurses. public health emergency responses were implemented, including enhanced triage in hospitals, screening of suspected patients, rapid testing, isolation of suggestive cases, information technology-supported contact tracing, and extensive quarantine. however, it took several weeks to catch up with the spread, because of the initial delays in identifying infective cases. overall, 16 693 persons have been cumulatively quarantined because of potential contact with infective cases. significant public fear and economic impact have been documented over the course of the outbreak. what could we have done better? how can we do better next time? our experiences, if painful, are not in vain. we have the journal of preventive medicine and public health (jpmph) will do its best to facilitate efficient sharing of knowledge, experience, and lessons gainied from the mers outbreak in korea. jpmph strongly encourages researchers to submit study results in diverse forms, ranging from case reports, to epidemiologic studies, policy perspectives, economic analyses, and any other types of contributions. jpmph hopes to be a vehicle of scholarly and practical communications and collaborations that will help overcome the challenges of global infectious diseases, including mers-cov. jsp?menuids =&fid=5767&q_type=&q_v alue=&cid=64426&page num =1 mers outbreak in korea: hospital-to-hospital transmission key: cord-290319-decr6wrd authors: kayali, ghazi; peiris, malik title: a more detailed picture of the epidemiology of middle east respiratory syndrome coronavirus date: 2015-05-31 journal: the lancet infectious diseases doi: 10.1016/s1473-3099(15)70128-3 sha: doc_id: 290319 cord_uid: decr6wrd nan the diff erential diagnosis of sepsis or meningitis is broad. third, although the path to commercialisation is underway, it might be challenging. inexpensive, straightforward assays are excellent from public health and global health perspectives, but disruptive new technologies are not always adopted. irrespective of how successful the commercialisation process is, the authors have developed a novel diagnostic test for neisseria meningitidis that is easy to use and provides results within 90 min. in such a timeframe, results can lead to actionable decisionmaking, most likely to halt further diagnostic testing. the niche for lamp in meningococcal disease remains to be established, but the fact that the technique allows diagnostic confi rmation at the treating hospital rather than a reference laboratory is a good start. 3 most cases were in countries in the arabian peninsula, with sporadic travel-related infections occurring elsewhere. although clusters of human-to-human transmission of mers-cov are well documented, transmission originates from zoonotic events, and therefore identifi cation of the animal sources of mers-cov is a priority. genetically, mers-cov is related to bat coronaviruses from china, saudi arabia, europe, and africa. 2,4-6 on the basis of the evidence so far, none of the bats from these countries are likely to be the reservoir for the virus. evidence is accumulating that dromedary camels are a natural host of mers-cov. serological fi ndings suggest that more than 90% of adult dromedaries in the middle east and africa are seropositive for mers-cov. 7 the virus isolated from dromedaries has spike proteins with conserved receptor-binding domains for the human dipeptidyl peptidase-4 receptor, 8, 9 and mers-cov has been detected in camels that were in close contact with people with middle east respiratory syndrome. 10, 11 the role of camels as a source of human infection with middle east respiratory syndrome is controversial because many people who have the disease have had no obvious association with camels. case-control studies that aim to defi ne risk exposures of index patients have not yet been done, and an investigation of mers-cov seroprevalence in people in contact with camels yielded negative results (ie, no association was noted). 8, [12] [13] [14] the study by muller and colleagues is the fi rst population-based, seroepidemiological investigation of mers-cov infection in an area where zoonotic transmission is sustained. the investigators used a cross-sectional design that tested serum samples from about 10 000 people in saudi arabia whose age and sex distribution was similar to that of the general population. by use of a rigorous serological testing algorithm, the investigators identifi ed mers-cov antibodies in 15 (0·15%, 95% ci 0·09-0·24) of 10 009 samples from the general population. men had a signifi cantly higher proportion of infections (11 [0·25%] of 4341) than did women (two [0·05%] of 4378; p=0·025), and more infections were noted in central rural areas than in coastal provinces (14 [0·26%] of 5479 vs one [0·02%] of 4529; p=0·003). the investigators also obtained samples from camel shepherds and slaughterhouse workers, and showed that seroprevalence of mers-cov was 15-23 times higher in camel-exposed individuals than in the general population. the fi ndings from this study suggest that young men in saudi arabia who have contact with camels in cultural or occupational settings are becoming infected with mers-cov, often without being diagnosed, and might proceed to introduce the virus to the general population in which more severe illness triggers testing for the virus and disease recognition. this hypothesis could account for cases of middle east respiratory syndrome without previous animal exposure. when the data from muller and colleagues 1 are put into context with those from studies in camels, a clearer picture of the epidemiology of mers-cov emerges (fi gure). camels seem to be a natural host for mers-cov and transmission within camel herds is well established. 15 however, several questions need to be resolved. the route for camel-to-human transmission is unclear, and could be from one or more of the following: direct contact with infected animals, or consumption of milk, urine, or uncooked meat-all practices that are common in aff ected countries in the middle east. an intermediate host could also transmit mers-cov between camels and human beings. finally, whether dromedaries are the natural reservoir or an amplifi er host is a hypothesis that is open to further investigation. muller and colleagues' study is the fi rst step along the way to addressing these questions. human papillomavirus (hpv) vaccination programmes have been in use in many countries since 2007 following licensing of the bivalent and quadrivalent hpv vaccines. 1 clinical trials have shown that hpv vaccines have more than 90% effi cacy in preventing high-grade cervical lesions caused by human papillomavirus types 16 and 18, 2,3 which are the two hpv types known to cause 70-80% of cervical cancers and large proportions of other anogenital and oropharyngeal cancers. the quadrivalent vaccine has shown similar effi cacy in the prevention of anogenital warts caused by hpv types 6 and 11. furthermore, both vaccines have shown lower-but still substantial-effi cacy against related non-vaccine oncogenic human papillomavirus types. 4,5 therefore, since rollout of these vaccination programmes began, there has been great anticipation to see whether these promising trial results will translate into substantial reductions in hpv-related disease at the population level. 6 in the lancet infectious diseases, mélanie drolet and colleagues present the fi ndings of a timely systematic review and metaanalysis assessing the population-level and herd eff ects of hpv vaccination programmes so far. 7 the key fi ndings of the study suggest that in the preto-post-vaccination period covered by the 19 included studies, hpv type 16 and 18 infections and anogenital warts have decreased by more than 60% in girls (<20 years of age) when high female vaccination coverage (>50%) was reported. the authors also report signifi cant reductions in hpv types 31, 33, and 45 infections in this same group (relative risk [rr] 0·72 [95% ci 0·54-0·96]) and in anogenital warts in boys younger than 20 years of age (0·66 [0·47-0·91]) and in women 20-39 years of age (0·68 [0·51-0·89]), which provides strong evidence of both cross-protection and herd eff ects. smaller, but still signifi cant, reductions in hpv types 16 and 18 infection (rr 0·50 [95% ci 0·34-0·74]) and anogenital warts (0·86 [0·79-0·94]) also occurred in girls and young women in countries with lower vaccination coverage (<50%), but there was no evidence to suggest either cross-protection or herd immunity in these countries. drolet and colleagues aimed to report the populationlevel eff ect of vaccination and, although the results are very encouraging, we need to keep in mind that their study included both clinic-based and population-based studies, of which the clinic-based studies might not be representative of the underlying population. additionally, vaccine coverage for the outcome of hpv prevalence was based on vaccine uptake in the study, which in some cases was substantially larger than that in the underlying population. 8 although the authors did several subanalyses to investigate heterogeneity, we think it would have been helpful to do an additional subanalysis stratifying the data by clinic-based versus population-based study. disappointingly, all the included studies were from high-income countries (nine countries in total) and we are therefore none the wiser regarding the eff ect of vaccination in low-income countries in which the burden of hpv-associated disease is highest. this report shows that the maximum population-level eff ect of vaccination is achieved through delivery at a young age with high vaccination uptake rate. a result we found striking is that the greatest eff ect of vaccination, in both male and female individuals, was recorded for countries such as australia, new zealand, and the uk that have school-based vaccination programmes. this fi nding is corroborated by the results of another recent systematic review that assessed the eff ect of the quadrivalent vaccine on anogenital warts. 9 in this study, mariani and colleagues reported that the greatest reductions in anogenital warts were achieved in countries with high vaccination uptake rates, mostly through school-based vaccination programmes, although some non-school-based programmes also achieved high vaccination uptake rates. one concern with school-based programmes is the presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional serological study isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers-cov): summary of current situation, literature update and risk assessment human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe close relative of human middle east respiratory syndrome coronavirus in bat mers-related betacoronavirus in vespertilio superans bats middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd mers coronaviruses in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012 lack of middle east respiratory syndrome coronavirus transmission from infected camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries key: cord-298535-wmxlu3l1 authors: agnihothram, sudhakar; gopal, robin; yount, boyd l.; donaldson, eric f.; menachery, vineet d.; graham, rachel l.; scobey, trevor d.; gralinski, lisa e.; denison, mark r.; zambon, maria; baric, ralph s. title: evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses date: 2013-11-18 journal: the journal of infectious diseases doi: 10.1093/infdis/jit609 sha: doc_id: 298535 cord_uid: wmxlu3l1 background. middle east respiratory syndrome coronavirus (mers-cov) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between mers-cov and other covs. methods. using synthetic genomics and venezuelan equine encephalitis virus replicons (vrps) expressing spike and nucleocapsid proteins from mers-cov and other human and bat covs, we characterize the antigenic responses (using western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 mers-cov isolates in comparison with those of other human and bat covs. results. serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. cov n proteins within but not across subgroups share cross-reactive epitopes with mers-cov isolates. our findings were validated using a convalescent-phase serum specimen from a patient infected with mers-cov (na 01) and human antiserum against sars-cov, human cov nl63, and human cov oc43. conclusions. vaccine design for emerging covs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct covs. group of phylogenetically diverse enveloped viruses that have the largest plus-strand rna genomes and replicate efficiently in most mammals [1, 2] . human cov (hcov-229e, -oc43, -nl63, and -hku 1) infections typically result in mild-to-severe upper and lower respiratory tract disease [3, 4] . sars-cov emerged in 2002-2003, causing acute respiratory distress syndrome with 10% mortality overall and up to 50% mortality among aged individuals [5] . most recently, middle east respiratory syndrome cov (mers-cov) emerged in the middle east in april 2012, manifesting as severe pneumonia, acute respiratory distress syndrome, and acute renal failure. the virus is still circulating and has caused 136 human infections with 58 deaths (mortality rate, approximately 44%) [6, 7] . phylogenetic analysis groups covs into 4 genera-alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus-and for many mammalian covs, bats are considered reservoirs [6, 8, 9] . sars-cov is closely related to bat cov (btcov) hku 3 [1, [10] [11] [12] , whereas mers-cov is closely related to pipistrellus btcov hku 5 and tylonycteris btcov hku 4 [9] . however, the serologic and antigenic relationship between strains is unclear. given the vast number of genetically distinct covs, well-defined serologic and virologic reagents are needed to rapidly track mers-cov and other cov infections in natural populations and to optimize vaccine and therapeutic designs early in an outbreak setting, especially within and between phylogenetic subgroups. the spike (s) and nucleocapsid (n) proteins are major immunogenic components of covs and are produced in abundant quantities during infection. the s protein is the principle determinant of protective immunity and cross-species transmission in cov [11] . antibodies against s protein protect from homologous and heterologous sars-cov challenge in vivo [13] , whereas n protein-specific immune responses may offer limited protection especially against low-dose challenge [13] . therefore, antibodies against s and n protein have diagnostic and therapeutic potential [14, 15] . in this article, we use alphavirus replicon vaccine vectors to express a panel of recombinant s and n proteins from distantly related alphacoronaviruses and betacoronaviruses, including mers-cov and other subgroup 2c covs. using mouse polyclonal antisera and recombinant proteins, we compare the cross-reactivity and neutralization titers of these antisera between distantly related human and bat covs. our results indicate that the s glycoprotein but not the n protein is the major determinant of the neutralizing antibody response to mers-cov; that the n proteins of covs only cross-react within but not between subgroups; that little if any cross-neutralization or cross-reactivity exists between the s proteins of covs within subgroup 2c or any other subgroup; and that cross-neutralization and cross-reactive patterns were validated with the convalescent-phase serum sample from a patient infected with mers-cov hu/england-n1/2012 and a donor panel of human antisera against 3 different hcovs. our approach provides critical reagents, antisera, and recombinant virus vaccines that allow for rapid diagnosis of and intervention against mers-cov and other zoonotic covs that emerge in the future. mers-cov hu/england-n1/2012 and mers-cov hu/sa-n1/2012 were cultured on vero 81 cells and grown in opti-mem (gibco, carlsbad, ca) with 5% fetal clone serum (hyclone, south logan, ut) and gentamicin/kanamycin (gibco). viral growth assays in vero and calu-3 cells were performed as previously described [16] . genes encoding the indicated s and n proteins were synthesized from bio basic (ontario, canada) and packaged into venezuelan equine encephalitis virus replicon particles (vrps). following vaccination, mouse polyclonal sera were generated from balb/c mice, and neutralization assays involving mers-cov strains and sars-cov were as described previously [17] . for western blots, vrp or virus-infected cell lysates and controls were prepared as described before in detail [8] , and these blots were probed using the indicated mouse polyclonal sera. vero cells inoculated with mers-cov isolates were harvested 12 hours after infection by means of trizol reagent (invitrogen) and were used to perform northern blots [18] . an elisa using indicated virus-infected cell lysates or antigens expressed from vrps was performed as described previously [19] , and the reactivity of mouse or human serum was determined using a chemiluminescent substrate. blocking elisa was performed by sequentially reacting plate-bound mers-cov lysate antigen with convalescent-phase serum obtained from a patient with mers, followed by mouse polyclonal serum raised against vrp-packaged mers-cov n or s proteins. blocking was expressed as the percentage reduction in the reactivity of the mouse serum alone. mers-cov hu/england-n1/2012 (mers eng 1) was isolated from a 49-year-old patient with severe respiratory illness and was transferred to london for treatment [7] . twenty-nine mutations in mers eng 1 at the amino acid level were identified and compared to the published sequence of mers-cov hu/ sa-n1/2012 (mers sa 1; genbank jx869059.2; supplementary figure s2 ). to identify whether these mutations altered virus growth, we analyzed the replication kinetics of the 2 isolates in vero cells and a continuous epithelial cell line, calu-3 ( figure 1a and 1b). although the replication kinetics were slightly different between 2 isolates in vero cells, peak viral titers were equivalent. in contrast, virus growth was markedly distinct in human calu-3 cells and could have represented differences in strain-specific in vitro adaptation phenotypes or resulted from functional differences in the sensitivity to innate immune responses [20] . cov replication and transcription involves production of a nested set of subgenomic messenger rnas (mrnas) [2] , and previous reports predicted 10 open reading frames in mers sa 1 and mers eng 1 [9] . northern blot analysis identified 8 subgenomic mrnas after infection in both viruses ( figure 1c ). the observed nested set of subgenomic mrna expression is consistent with observations for other covs [2, 9] . the open reading frames encoded by each mrna in these isolates are detailed in supplementary table 1 . vrps function as efficient expression and vaccine platforms for a variety of antigens [13, 21] . we generated vrps expressing mers sa 1 s and n proteins and then immunized mice. n protein-specific antiserum recognized a discrete 50-kda band at the predicted molecular weight in lysates from vero cells (supplementary figure 1a) and calu-3 cells ( figure 1d ) infected with vrp-n or with the 2 different mers-cov isolates. for the most part, similar expression patterns were evident between vrps and viruses; however, the n protein of mers eng 1 had a slightly lower molecular weight, which was consistent with amino acid deletions at positions s391 and i392 (supplementary figure 2 ). mouse anti-s serum identified an approximately 180-kda s protein in vrp-s-or mers-cov-infected vero cells (supplementary figure 1a ). the observed molecular weights were consistent with the sizes of the s proteins of other covs [2, 22] . we noted similar results in calu-3 cells ( figure 1d ), and interestingly, an increased amount of a higher-molecular-weight form of s protein (glycosylated dimer) was noted in mers eng 1 in both cell lines. antiserum against mers sa 1 s and n proteins also recognized mers eng 1 in elisas (data not shown), and n and s proteins recognized the mers-cov jordan isolate (supplementary figure 1b) . plaque reduction neutralization tests (prnt 50 ) indicated complete neutralization of both mers-cov isolates (prnt 50 titer, approximately 1:1400 for each; figure 2a ) and the mers-cov jordan isolate (supplementary figure1c) by vrp-s antiserum, whereas no neutralization was observed with n antiserum. similar findings have been reported with sars-cov, as well as with other known human and animal covs [13, 23] . interestingly, serum from aged mice vaccinated with vrp s showed a 6-fold reduction in prnt 50 titers (approximately 1:200), indicating that immunosenescence attenuates vaccine responses to mers-cov antigens, as was noted with sars-cov vaccines [13, 17] . using serum from na 01 patient, elisa demonstrated high reactivity of the patient's serum to n and s antigens of mers sa 1 expressed from vrps ( figure 2b and 2c). titers of antibody against n protein in patient serum peaked 3-5 weeks after onset of illness (which occurred on 3 september 2012) and waned thereafter, but the antibodies were still detected up to 5 months after illness onset. titers of antibody against s protein were consistent from 3 weeks to 5 weeks after illness onset, after which they remained detectable. most importantly, patient serum collected on 16 november 2012 (which contained high titers against s protein) outcompeted the binding of mouse s antiserum to intact virus in a blocking assay ( figure 2d ). these data suggest that different/overlapping epitopes are recognized by human and mouse antisera following virus or vrp-s infection. mers sa 1 and mers eng 1 are closely related to btcov hku 5 and btcov hku 4 ( figure 2e ) [6, 9, 24] . to evaluate antigenic relationships with the subgroup 2c betacoronaviruses, vrps expressing s and n proteins of btcov hku 4.2 and btcov hku 5.5 were inoculated into mice. antisera against both hku 4.2 and 5.5 n proteins recognized the n proteins of both mers-cov isolates, whereas mers sa 1 n antisera also detected the vrp-expressed hku 4.2 and 5.5 n proteins, as revealed by western blot (figure 3a and 3b). we obtained similar results using elisa and immunofluorescence assays (data not shown). in contrast, there was little if any observable cross-reactivity observed between mers sa 1 s antisera with the vrp-expressed s proteins of hku 4.2 and hku 5.5, whereas antisera to hku 5.5 s protein but not hku 4.2 s protein recognized the s proteins of both mers-cov isolates ( figure 3c and 3d). we also measured serologic relationships using elisa, which captures cross-reactivity to conformational epitopes, and confirmed these antigenic relationships ( figure 6c ). consistent with results of serologic tests, antisera against hku 4.2 and hku 5.5 s proteins did not crossneutralize the mers-cov isolates. these data indicate that the n protein but not the s glycoprotein are antigenically conserved within the subgroup 2c betacoronaviruses evaluated in this panel. we then extended our analysis to the highly pathogenic sars-cov and related subgroup 2b betacoronaviruses. polyclonal mouse sera to sars-cov or mers sa 1 n or s proteins exhibited no cross-reactivity to the reciprocal strains ( figure 4a and 4b). we observed very low levels of cross-neutralization of mers sa 1 by mouse antisera to sars-cov, using very high but not low concentrations of serum ( figure 4c ), a finding that is consistent with a recent report [24] . interestingly, elisa results also showed very minimal cross-reactivity of the na 01 patient sera obtained on 23 september 2012 to sars-cov s antigen ( figure 4d ). consistent with this observation, binding of mouse sars-cov s antiserum to sars-cov was not inhibited by na01 patient sera in blocking assays ( figure 2d ), indicating the absence of antibodies to sars-cov in the patient serum. consonant with these findings, no cross-reactivity was observed with antisera against the vrp-expressed n or s glycoproteins of btcov hku 3 and 279 and the mers-cov isolates ( figure 5a-d) . furthermore, no cross-neutralization of mers-cov isolates by hku 3s antiserum was observed, although this serum has previously been shown to neutralize a synthetically resurrected hku 3 variant encoding the sars s glycoprotein receptor binding domain [23] . interestingly, we observed very low levels of cross-neutralization of sars-cov by btcov 279 s antiserum ( figure 4c ). to further elucidate the antigenic relationships between the s glycoproteins of alphacoronaviruses and the mers-cov isolates, we expressed and generated mouse antisera to btcov 1a and btcov hku 2 (group 1b alphacoronaviruses), using the vrp platforms. despite efficient recombinant s glycoprotein expression ( figure 7a and 7b) , none of the recombinant s glycoproteins were recognized by mers sa 1 s antisera. antisera against btcov1a and hku 2 s glycoproteins had little if any cross-reactivity with and did not neutralize mers-cov ( figure 7a -c). we next analyzed the antigenic relationships between vrpderived mouse serum with the following representative hcovs from each subgroup, using elisa ( figure 6c ): mers eng 1, from subgroup 2c; sars-cov, from subgroup 2b; hcov-nl63, from subgroup 1b; and hcov-oc43, from subgroup 2a. mers eng 1 was recognized by antisera targeting the n but not the s glycoprotein of viruses within the subgroup 2c betacoronaviruses. likewise, sars-cov was only recognized by antisera to n but not s glycoproteins of viruses with the subgroup 2b betacoronaviruses. none of the antiserum screened reacted with hcov-nl63 (subgroup 1b) or hcov-oc43 (subgroup 2a). although btcov hku 2 is genetically close to hcov-nl63, we did not observe any cross-reactivity within the s glycoprotein. serum from patients infected with sars-cov, hcov-nl63, or hcov-oc43 was screened against the n proteins from representative subgroup 2c and 2d betacoronaviruses. consistent figure 2 . a, serum from each of 4 young and aged mice immunized with venezuelan equine encephalitis virus replicons (vrps) expressing spike (s) or nucleocapsid (n) proteins were tested in a plaque reduction neutralization test to neutralize middle east respiratory syndrome coronavirus (mers-cov) hu isolates. error bars indicate standard error of the mean. b and c, na01 patient sera collected at indicated dates after hospitalization were analyzed in an enzyme-linked immunosorbent assay, using cell lysates expressing s and n antigens from vrps. d, indicated dilutions of na01 patient sera collected on november 16, 2012 were screened with 1:800 dilutions of mouse antisera to s, n, bat cov (btcov) hku 5.5 n, or sars-cov s in an in vitro competition assay for binding to mers-cov or sars-cov. e, the full-length genome sequences of 51 covs were downloaded from genbank or patric, aligned with clustalx, and phylogenetically compared by maximum likelihood estimation, using 100 bootstraps. the tree shows that covs are divided into 3 distinct phylogenetic groups, defined as î±, î², and î³. this taxonomic nomenclature replaced the former group 1, 2, and 3 designation, respectively. classical subgroup clusters are marked as 2a-2d for the î²-covs and as 1a and 1b for the î±-covs. the tree was generated using maximum likelihood estimation with the phyml package. the scale bar represents nucleotide substitutions. only nodes with bootstrap support of >70% are labeled. accession numbers and definitions of various cov strains will be provided upon request. with our previous findings, human serum to sars-cov recognized btcov hku 3n, btcov 279n, and sars-cov n (subgroup 2b) but did not recognize n proteins from other subgroups ( figure 6b) . similarly, there was no cross-reactivity of the human antisera from hcov-nl63 (subgroup 1b) and hcov-oc43 (subgroup 2a) infections with any of the viral antigens within the panel. serum collected from the patient infected with mers-cov na01 showed cross-reactive binding only to btcov hku 5.5 n (subgroup 2c), and little if any cross-reactivity was noted outside the subgroup ( figure 6a emerging respiratory covs offer a considerable threat to the health of global populations and the economy. platforms for generating well-characterized molecular reagents and recombinant viruses are needed to detect and control the emergence of new strains, especially early in an outbreak, before the development of type-specific serologic reagents and therapeutics. here, we characterized the genome organization, subgenomic mrna expression, and protein expression patterns of 2 isolates of mers-cov. using alphavirus replicon particles and synthetic gene design, we assembled a panel of recombinant proteins from and donor antisera against phylogenetically distant alphacoronaviruses and betacoronaviruses to evaluate the antigenic relationships between strains and to inform vaccine design. mers-cov is a highly pathogenic respiratory cov of humans, causing acute respiratory distress syndrome, with mortality rates approaching 44%. cov primer sets were not successful in diagnosing the etiology of the jordan outbreak in april 2012, demonstrating a critical need for paneled reagent sets of recombinant proteins and sera that allow for serologic evaluations of cases, contact cases, and asymptomatic infections, using western blot or elisa-based techniques [25] . this article is also the first report that describes the serologic characterization of mers-cov and cov reagents. an advantage of the vrp platform is that it can also function as a vaccine vector, affording the rapid production of candidate vaccines against newly emerged strains [23] . using sars-cov and mers-cov as models, we clearly demonstrated that s protein-based recombinant vaccines elicit robust neutralization responses in young and aged rodent models. because vrp-s vectors against sars-cov protected young and aged animals [17] , we have developed a recombinant s vectored vaccine that could likely prove successful in preventing heterologous mers-cov infection in aged individuals, but this remains to be tested. mers eng 1 replicated to lower titers than sa1 in calu-3 cells. as the 2 viruses have different passage histories in vitro, tissue culture adaptive mutations may account for these differences, as reported with many sars-cov isolates [26] . alternatively, 29 amino acid differences have been described, most of which reside in the replicase polyprotein (supplementary figure 2) and may affect replication efficiency. in addition, the s glycoprotein of mers eng 1 differs from that of mers sa 1 by 2 amino acids, l506f and q1020h (supplementary figure 2) , which may account for the increased amount of the higher-molecular-weight form of s protein in mers eng 1 ( figure 1d and supplementary figure 1a) . recent studies indicate that tmprss2 likely plays important roles in viral entry by enhancing fusogenic potential through proteolytic processing of mers-cov s glycoprotein [22] . in addition, we identified a unique mutation, t1015n, in the mers sa 1 isolate but not in the mers eng 1 isolate and showed that this mutation is responsible for increased in vitro fitness and for plaque morphology [20] . it is possible that the presence or absence of 1 or more of the s glycoprotein mutations in mers eng 1 may result in the slower growth phenotype in calu3 cells. alphavirus vrps have considerable potential as recombinant virus vaccine platforms in the absence of preexisting immunity [27] [28] [29] [30] . we demonstrate efficient expression of several cov s and n structural proteins both in vitro and in vivo, resulting in robust serologic responses in vaccinated mice. antiserum to vrp-s glycoprotein but not to vrp-n protein neutralized both isolates of mers-cov. furthermore, we and others have demonstrated that vaccine-induced immunopathology observed after challenge is minimized in vrp-s protein-based vaccines, partly because of the t-helper type 1-biased immune response and high neutralization titers elicited by vrp vectors [13, 31] . importantly, vaccination of aged mice demonstrated that immunosenescence contributes to a reduction in the magnitude of the antibody response to mers-cov s glycoprotein, an important point to be considered in candidate vaccine designs. to date, wild-type and vrp 3526-coated vrp-s vaccines represent one of the few vaccine platforms that functioned well in aged animals, in addition to recombinant subunit-based vaccines and poxvirus-vectored vaccines [21, [32] [33] [34] . in sars-cov pathogenesis, increased age-related susceptibility is linked to increased prostaglandin d2 expression; it remains uncertain whether increased prostaglandin d2 levels have contributed to reduced vaccine performance, as well [35] . because we have not observed mers-cov replication in immunocompetent and immunocompromised mice, these vectors must be tested directly in primates [36] . the safety of the vrp platform has been demonstrated in high-risk human populations and immunosenescent nonhuman primates [27, 28, 37, 38] , and we believe that these vectors will be efficacious in healthcare workers and target populations infected with mers-cov. our results indicate the presence of strongly cross-reactive epitopes in the n protein within a particular subgroup but not between subgroups. under identical conditions, little cross-reactivity or conservation of cross-neutralizing epitopes was observed between s proteins within and across subgroups. similar studies showing strong conservation of cross-reactive epitopes between n proteins, but to a lesser extent between s proteins of the subgroup 2a covs, has been reported [39, 40] . importantly, the pattern of serologic and antigenic relationship observed using the mouse antisera was recapitulated using the human antiserum to 4 different covs. neutralization assays demonstrated little if any conservation of cross-neutralizing epitopes between s glycoproteins of covs within and across subgroups. in particular, the absence of cross-neutralization of mers-cov figure 6 . a, na01 patient serum specimens collected at indicated dates were analyzed in an enzyme-linked immunosorbent assay (elisa), using cell lysates expressing indicated antigens. b, mouse antisera to the indicated antigens were screened in an elisa. c, human antisera to indicated covs were screened in an elisa with cell lysates expressing indicated antigens. isolates by antiserum to hku 4 or hku 5 s glycoprotein and of sars-cov by antiserum to the hku 3 or btcov 279 s glycoprotein suggests very limited conservation or, possibly, the deliberate masking of conserved cross-neutralizing sites within a subgroup. although speculative, these cross-neutralization relationships suggest that at least 3 antigenically distinct covs could emerge from zoonotic viruses circulating within subgroup 1a/b, 2b, and 2c reservoirs and then simultaneously circulate in humans. these findings are evidence that vaccine design for any new emerging cov should either focus on the development of chimeric s glycoproteins containing neutralizing epitopes from multiple strains within or across subgroups or on the development of new paradigms in structure-guided antigen design that improve the presentation of broadly neutralizing epitopes. regions of s glycoprotein are interchangeable between covs within and across subgroups, rendering viable recombinant viruses [23] . inclusion of n protein in such chimeric vaccines may broaden the protective response, although this remains to be tested using lethal challenge viruses. such a vaccine might provide robust protection against several homologous and heterologous viruses within or across genoclusters. after the sars-cov epidemic and in stark contrast to the situation with emerging influenza viruses such as influenza a (h7n9), the research and biomedical communities failed to develop broadly applicable biopreparedness platforms for rapid response against future emerging cov threats. because covs have demonstrated an accelerating pattern of zoonotic emergence since the 1980s [25, 41] , our data indicate that an appropriate diagnostic platform should include a large panel of phylogenetically distinct cov s and n structural proteins, which must be validated using larger panels of antisera against other hcovs in the general population. while molecularbased platforms like polymerase chain reaction and deep sequencing offer clear advantages in early detection of active infections, public health response platforms would be strengthened by the availability of recombinant proteins and subgroup-and type-specific antisera that can track subclinical infections, determine the prevalence of infection in populations, and identify hospital-acquired infections. a recent report identified subclinical cases of mers-cov infection through reverse-transcription polymerase chain reaction, and the screen using the panel of recombinant proteins described here would have provided more-specific information about the presence of other covs in these cases [42] . the vrp platform we describe not only yields high-level expression of key recombinant proteins across the alphacoronaviruses and betacoronaviruses, it also provides the first candidate vaccine vectors with the potential to augment the t-helper type 1-based immune responses to mers-cov infection and to reduce associated immune pathology. the vrp 3526-associated approach is also applicable to improving the public health response to and the control of future outbreaks of other highly pathogenic emerging infectious diseases due to cov in human populations. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission the molecular biology of coronaviruses culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures identification of a new human coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus severe respiratory illness caused by a novel coronavirus evidence supporting a zoonotic origin of human coronavirus strain nl63 genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines sars-cov and emergent coronaviruses: viral determinants of interspecies transmission human coronavirus emc is not the same as severe acute respiratory syndrome coronavirus vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants structural basis for potent crossneutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge tropism and innate immune responses of the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus murine hepatitis virus replicase protein nsp10 is a critical regulator of viral rna synthesis cross-neutralisation of antibodies elicited by an inactivated split-virion influenza a/vietnam/ 1194/2004 (h5n1) vaccine in healthy adults against h5n1 clade 2 strains reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests development of an enzyme-linked immunosorbent assay-based test with a cocktail of nucleocapsid and spike proteins for detection of severe acute respiratory syndrome-associated coronavirus-specific antibody mutational dynamics of the sars coronavirus in cell culture and human populations isolated in 2003 a phase i dose escalation trial of vaccine replicon particles (vrp) expressing prostate-specific membrane antigen (psma) in subjects with prostate cancer phase i safety and immunogenicity evaluations of an alphavirus replicon hiv-1 subtype c gag vaccine in healthy hiv-1-uninfected adults neutralizing antibody responses of humans and mice to vaccination with venezuelan encephalitis (tc-83) virus the distribution and prevalence of group a arbovirus neutralizing antibodies among human populations in southeast asia and the pacific islands immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge recombinant vaccinia virus expressing the pr/8 influenza hemagglutinin gene overcomes the impaired immune response and increased susceptibility of old mice to influenza infection immune responses and protection in different strains of aged mice immunized intranasally with an adjuvant-combined influenza vaccine age-related increases in pgd(2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice pneumonia from human coronavirus in a macaque model venezuelan equine encephalitis virus vaccine candidate (v3526) safety, immunogenicity and efficacy in horses neurovirulence evaluation of venezuelan equine encephalitis (vee) vaccine candidate v3526 in nonhuman primates antigenic and genomic relationships among turkey and bovine enteric coronaviruses antigenic relationships among proteins of bovine coronavirus, human respiratory coronavirus oc43, and mouse hepatitis coronavirus a59 potent and persistent antibody responses against the receptor-binding domain of sars-cov spike protein in recovered patients middle east respiratory syndrome coronavirus infections in health care workers financial support. this work was supported by the national institutes of allergy and infectious diseases, national institute of health (grants ai085524, ai057157, and u19 ai107810) and public health of england (formerly, health protection agency, england).potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-309898-sju15hev authors: hu, yiwen; buehler, markus j. title: comparative analysis of nanomechanical features of coronavirus spike proteins and correlation with lethality and infection rate date: 2020-11-02 journal: matter doi: 10.1016/j.matt.2020.10.032 sha: doc_id: 309898 cord_uid: sju15hev the novel coronavirus disease, covid-19, has spread rapidly around the world. its causative virus, sars-cov-2, enters human cells through the physical interaction between the receptor-binding domain (rbd) of its spike protein and the human cell receptor ace2. here, we provide a novel way in understanding coronavirus spike proteins, connecting their nanomechanical features – specifically its vibrational spectrum and quantitative measures of mobility – with virus lethality and infection rate. the key result of our work is that both, the overall flexibility of upward rbd and the mobility ratio of rbds in different conformations, represent two significant factors that show a positive scaling with virus lethality and an inverse correlation with the infection rate. our analysis shows that epidemiological virus properties can be linked directly to pure nanomechanical, vibrational aspects, offering an alternative way of screening new viruses and mutations, and potentially exploring novel ways to prevent infections from occurring. the novel coronavirus disease, covid-19, has spread rapidly around the world [1] [2] [3] [4] [5] . its causative virus, sars-cov-2, enters human cells through the interaction between the receptor-binding domain (rbd) of its spike protein and the cell receptor ace2 [6] [7] [8] . due to the significant role that coronavirus spike protein plays in receptor recognition, viral fusion and cell entry, it is a promising target for drug and vaccine development. here, we provide a novel way towards better understanding the coronavirus spike proteins, connecting its nanomechanical features -especially their vibrational patterns -with virus lethality and infection rate 6 . in a broader context, the mechanics of proteins has long been a subject of interest, and this study shows how it can be a useful tool to help us understand complex disease etiology by connecting nanoscopic physical features with epidemiological data [9] [10] [11] [12] [13] [14] [15] . to provide a comparative analysis -specifically focused on how nano-level features relate with macroscopic epidemiolocal observables -we focus on different coronavirus types within the same family of pathogens. over the past decades, several types of coronaviruses have emerged. the virus types hcov-nl63 and hcov-hku1 are often reported to cause lower respiratory tract infections, while hcov-oc43 and hcov-229e are usually associated with comparatively mild symptoms similar to the common cold 5, 16, 17 . the ones that threaten public health more seriously are three highly pathogenic human coronaviruses -namely: sars-cov, mers-cov and sars-cov-2. sars-cov was first reported in china in november 2002, then quickly spread globally, resulting in over 8,000 infections with about 800 deaths 18 . mers-cov was first identified in saudi arabia in june 2012, featuring limited transmission with case fatality rate as high as 35% 19, 20 . sars-cov-2 was first reported in china in december 2019 3, 21, 22 . it can easily transmit from human to human, resulting in more than 40 million global cases as of october 2020 23, 24 . the spike protein of the coronavirus plays an essential role in receptor recognition, viral fusion and cell entry 6, 25, 26 . the process represents a complex mechano-chemical process, whereby during the entry into the host cell, the spike protein first binds to a cell receptor through the receptor-binding domain (rbd) and then begins the fusion process. it is believed that the rbds of different coronaviruses recognize different cell receptors 3, 27 . sars-cov, sars-cov-2 and hcov-nl63 recognize angiotensinconverting enzyme 2 (ace2) as their receptor in the human body, while mers-cov recognizes dipeptidyl peptidase 4 (dpp4) as its receptor 1, 7, 8, 28 . in order to successfully bind to the receptor, the spike protein of a specific coronavirus must maintain a receptor-accessible state with at least one rbd in upward conformation. this is because otherwise there would be steric clashes hindering the binding process 4 . in experimental work, this specific type of receptor-accessible state has been captured for mers-cov, sars-cov and sars-cov-2 6, 29 . while the structure of coronavirus spike protein is well studied, much less attention has been focused on the connection between the mechanical features of the virus with virus lethality as well as infection rate. the structural basis of sars-cov viral infectivity has been explored to some extent, pointing out that the trp-rich region of s protein is essential 30 . it has also been observed that cleavage of the spike protein of sars-cov is associated with viral infectivity 31 . however, there has been no lateral comparative study between similar coronaviruses on this type of connection. it remains a question which kind of mechanical and structural properties could possibly relate to the mortality and infection rate of the virus. if successful, this approach may provide an alternative or complementary way to screen viruses or mutations against large-scale epidemiological data, provide additional mechanistic insights into disease etiology, and offer potential targets for therapies or preventive measures. in protein science, normal mode analysis (nma) has long been one of the most comprehensive yet efficient methods to calculate vibrational normal modes and analyze protein flexibility, which provides the rationale for use in this study 32 . another reason behind the broad use of nma is that the lowfrequency modes elucidated by nma could often describe the real-world motions of a protein, and often bear important functional significance 33 . in nma, the atoms are modeled as point masses, which are connected by springs that represents the interatomic force fields. after constructing the hessian matrix based on the second-order partial derivatives of the potential energy function, the normal modes and corresponding frequencies can be directly obtained by diagonalizing the matrix and computing its eigenvalues 34 (further details see experimental procedures). sars-cov-2 enters human cells through the interaction between the receptor-binding domain (rbd) of its spike protein and the cell receptor ace2, as is depicted in the schematic shown in figure 1 . our study hence focuses on the spike protein, which is essential for the infection to take place. the spike protein of coronavirus is composed of an amino (n)-terminal s1 subunit and a carboxyl (c)-terminal s2 subunit. the s1 subunit, which consists of an n-terminal domain (ntd) and three c-terminal domains (ctd), is responsible for recognizing and binding to the host cell receptor. it has been reported that for the betacoronavirus that utilizes ctd1 of its s1 subunit as rbd, there is a prerequisite conformational state for receptor binding where at least one rbd should be upward 4, 6 . in this paper, we refer to this receptoraccessible state as "open state" and receptor-inaccessible state as "closed state". as outlined in figure 1 , we conduct a normal mode analysis (nma) 33 , our open-state analysis is limited to these three highly pathogenic beta-coronaviruses. for closed-state exploration, since there have been no reports about closed conformational state of the mers-cov spike protein, we choose the hcov-nl63 spike protein because it shares the same relevant cell receptor ace2 with sars-cov and sars-cov-2 spike proteins. figure 2 depicts data that shows that the lowest-frequency normal modes of mers-cov, sars-cov and sars-cov-2 spike proteins are all associated with a swing motion of upward receptor-binding domain (rbd) to different extents. this type of global movements corresponds well with the molecular motions directly reported in experiments 4, 6 . considering the required receptor-accessible state for receptor binding, the swing of rbd is of functional significance since it is the likely way by which a spike protein changes from closed state to open state to facilitate the binding of target receptors. from a lateral aspect, this observed shared type of lowest-frequency normal mode movements indicates the structural similarity of mers-cov, sars-cov and sars-cov-2 spike proteins. according to our analysis, while the sequence identity of sars-cov s and sars-cov-2 s is as high as 78.8%, mers-cov s has only about 30% sequence identity with sars-cov s and sars-cov-2 s. this indicates that a small portion of the whole sequence of beta-coronavirus would largely determine the general structure topology and thus the overall global motion (details see supplementary material figure s1 ). figure s1 depicts a sequence alignment of mers-cov s, sars-cov s and sars-cov-2 s, where identical residues are denoted by *. the analysis reported in 36 reveals that the percentage identity in ntd and rbd, which reflect the major parts participating in the lowest normal mode movement, is about 22%. compared with 30% sequence identity of the whole spike protein, this partial percentage identity is lower and confirms the concept that likely only a few sequence pieces ultimately determine the shared global movement of coronavirus spike protein in open state. we further note that the sequence similarity in the s2 subunit could play an important role, as it may contributes to the comparatively higher rigidness of the s2 subunit. while mers-cov, sars-cov and sars-cov-2 spike proteins share the same type of lowestfrequency normal mode movements, their fluctuation profiles differ dramatically, as is illustrated by figure 3 . generally speaking, the s1 subunit of coronavirus spike protein is much more flexible than its s2 subunit. among these beta-coronaviruses, the active rbd of mers-cov spike enjoys super mobility and only sars-cov-2 spike protein and its d614g mutant exhibit a comparatively flexible downward rbd. interestingly, when we focus on fluctuations over the upward rbd, the figure reveals that for all these spike proteins the fluctuations first slowly build up to its maximum and then decrease sharply, which demonstrates that the appearance of large flexibility of upward rbd is based on some common detailed structures of these beta-coronaviruses. notably, panel (d) of figure 3 shows that the d614g mutation decreases the general flexibility of upward rbd and significantly enhances the mobility of a limited region in one downward rbd in the spike protein. it is also shown that the d614g mutation results in a general slight flexibility decrease in the ntds of all three chains, and importantly, no noticeable differences in other areas. using the fluctuation profile data, two significant mechanical factors are identified, which are: (1) the overall flexibility of upward rbd and (2) the mobility ratio of rbds in different conformations. figure 4 provides a correlation diagram for mers-cov, sars-cov and sars-cov-2 spike protein, where the overall flexibility of upward rbd is evaluated by the average fluctuation of open-state rbd and the mobility ratio is quantified as the ratio of maximum fluctuations over upward and downward rbds. the data shows that both factors have positive correlation with case fatality rate and inverse relationship with the virus infectivity. we find that for the flexibility ratio, the smaller it is, the larger the mobility of downward rbd is compared to upward one, which could indicate a larger possibility toward a second standing rbd. this would make it even easier for the spike protein to bind to the host cell receptor and thereby increasing the virus infectivity. on the other hand, if the flexibility of downward rbd is not large enough to generate conformational change, as flexibility ratio decreases, it becomes more difficult for the j o u r n a l p r e -p r o o f receptor to bind with the right rbd since the downward one is quite active. this may provide an explanation for the positive correlation between flexibility ratio and virus lethality that we see in the epidemiological data. one possible reason for the positive relationship between overall flexibility of upward rbd and the mortality rate could be that the flexible upward rbd is more active when binding to the receptor, and may hence benefit the subsequent membrane fusion process. even though there is limited available empirical data at this point, there is actually an intrinsic negative relationship between the mortality rate and infection rate of mers-cov, sars-cov and sars-cov-2, which could help explain why the infectivity is inversely correlated with the general flexibility of upward rbd. while there are many other factors situated between nanomechanical and epidemiological aspects, such as binding affinities 37 and dysregulation of type i interferon responses 38 , the influence of which on different epidemiological characteristics of coronavirus has not been fully explained, and our analysis points out the direct correlation between nanomechanical features and the lethality and infection rate of coronavirus. the goal is to attempt to improve our understanding of the direct relationship between the nanoscale and epidemiological level, not considering internal relationships. as the results show, this perspective provides useful insights into the mechanics of disease relationships ( figure s2 ). figure 5 show different flexibility variations in sars-cov-2, sars-cov and hcov-nl63 spike protein, which share the same human receptor ace2. among them, there exists a sharp increase and large variation in flexibility in rbd of sars-cov-2 s, while sars-cov s appears to have more flexible structural regions. hcov-nl63, the only one in this comparative study that is classified as alpha-coronavirus and cause only mild symptoms, has a different s1 subunit topology, bringing more flexibility to ntd rather than ctd1, where its rbd is situated. thus, our suggestion about the importance of the general flexibility of upward rbd in open-state analysis needs to be expanded. as for closed states, the overall flexibility of rbd in a single protomer shows positive relationship with virus lethality, since the disease severity is regressive in order of sars-cov, sars-cov-2 and hcov-nl63. notably, for these three coronaviruses in closed states, the overall flexibility of rbd of a single protomer is positively associated with their disease severity. panels (e) to (g) in figure 5 provide detailed structures of the rbd of the above virus complexed with their shared receptor human ace2. the ctd1 in s1 subunit of coronavirus often contains a core structure, which is composed of several antiparallel βsheet and short connecting α-helices, and one or more extended loops. theses extended loops, referred to as receptor binding motif (rbm), is located at the edge of the core structure and is usually responsible for realizing the interactions with the receptor if the virus uses ctd1 as its rbd. beta-coronaviruses such as sars-cov and sars-cov-2 have a unique long-extended loop as their rbm, accounting for the most flexible region of their rbd in both open and closed states. as for hcov-nl63, there are three separated short rbms, which are quite restricted and unable to generated large mobility. these insights may also explain its lower-affinity interaction with ace2, at least to some extent. we reported an analysis linking key nanomechanical vibrational features of various coronavirus spike proteins with epidemiological data. as shown in figure 2 , structural similarity and major movement associated with the swing of upward rbd is seen throughout this family of viruses, representing a sort of universal feature of this class of pathogens. the molecular modeling results show that the general motion corresponds with experimental observation and have functional importance. we find that the active rbd of mers-cov enjoys super mobility, whereas only sars-cov-2 and its d614g variant show a comparatively flexible downward rbd. the more recently occurring d614g mutation decreases the general flexibility of upward rbd and largely enhances the mobility of some small region in one downward rbd in the spike protein. as shown in figure 4 , the key result of this study, the general fluctuation profiles of upward rbd and the associated fluctuation ratio have a positive correlation with case fatality rate and inverse relationship with the virus infectivity. our results offer two different explanations for the effects of the flexible downward rbd: (1), the possibility towards a second standing rbd if the mobility is large enough, and (2) that it could indicate difficulty for the receptor to bind with the right rbd if no conformational change happens. we hypothesize that there may be a possible threshold between these two effects, which could be studied in future research. these insights offer several possible applications, including a search for inhibitors that could bind to downward rbd may provide a viable strategy. we find a sharp increase and large variation in flexibility in sars-cov-2 s, whereas more flexible structural regions are present in the closed-state sars-cov s. the long-extended loop is unique for beta-coronaviruses, and also accounts for the most flexible region in open states. hcov-nl63 s has three separated short rbms, which are unable to generate large mobility. this may also explain its lower-affinity interaction with ace2. as for possible applications, we may target the rbm to identify new inhibitors that lock the closed s-protein conformation. this opens a question whether perhaps, we can we utilize the significant flexibility difference for potential inhibitor screening for the development of novel treatment methods or design future experiments for gain-offunction research 39, 40 . future work may address additional influences of temperature dependence (to explore whether seasonal variations of temperature and other environmental factors can be linked to nanoscopic phenomena). other aspects may include a more detailed analysis of intermediate steps in the mechanistic hierarchical progression as schematically outlined in figure s2 , including aspects of the strong age-dependence of covid-19 disease progression. please contact prof. markus buehler via mbuehler@mit.edu. no new materials were generated in this work. all data are available upon request to the lead contact author. to assess the molecular mechanics from an atom-by-atom perspective, we conduct normal mode analyses (nma) of coronavirus spike proteins in receptor-accessible state with one upward rbd as well as receptor-inaccessible state where all three rbds are in downward position. we access the desired threedimensional protein structures from the protein data bank 41 and prepare the atomistic models with visual molecular dynamics (vmd) 2, 42 . before normal mode analysis, 10,000 steps of conjugate gradient energy minimization are performed using nanoscale molecular dynamics (namd) 43 in order to relax the protein structure 44 . no further md simulation is performed since the protein structure from protein data bank is already experimentally equilibrated. a coarse-grained elastic network model (enm) available in the bio3d package in r is employed to analyze the normal modes of coronavirus spike protein structures [45] [46] [47] . this model uses n, ca, c atoms to represent the protein backbone and selects 0 to 2 significant side chains based on their size and distance to ca atoms, proved to have comparable accuracy with all-atom enm. here, the atomic displacements are scaled for temperature 300k. for the sars-cov-2 d614g mutant, we implement the mutation to the open-state spike protein (pdb id: 6vsb) and carry out 10,000 steps of energy minimization and md simulation for 50 ns with namd. we then compute the average residue mean square derivations (rmsd) based on the last 25 ns equilibrium period and pick out 10 frames with the nearest rmsd from the trajectory file so that we could conduct normal mode analysis on them. the fluctuation profile of d614g mutant is calculated as the average of the normal mode fluctuations of these 10 configurations. we notice that some local unfolding occurs at the end of each chain, which induces abnormally high fluctuations in the fluctuation profile. since these local events are far from the rbd, we do not consider them in the analysis and set the fluctuations of the terminal 5-8 residues to zero. for consistency, the same approach is used for the fluctuation difference of sars-cov-2 spike protein and the d614g mutant. the global confirmed case numbers and the case fatality rate presented in figure 4 are updated as of sep 1, 2020. according to the analysis based on gisaid sars-cov-2 sequence database, by aug 31, 2020, 80% of the global sequence database were identified with d614g mutation (71242 sequences counted in total) 48, 49 . based on this estimate we use 80% of the covid-19 global case number as the infection number of sars-cov-2 with g614 and the remaining 20% as the case number of the original sars-cov-2 virus type. since there has been little evidence assessing an association between d614g mutation and disease severity, the same case fatality rate is applied to the original sars-cov-2 spike protein and its d614g variant. the two factors depicted in figure the causative virus of the covid-19, sars-cov-2, enters human cells through the physical interaction between the receptor-binding domain (rbd) of its spike protein and the human cell receptor ace2. while the structure of coronavirus spike protein is well studied, it remains unclear how those mechanical features of the virus affect its epidemiological characteristics. here, we report that both, the overall flexibility of upward rbd and the mobility ratio of rbds in different conformations, represent two significant factors that show a positive scaling with virus lethality and an inverse correlation with the infection rate. our analysis shows that epidemiological virus properties can potentially be linked directly to pure nanomechanical, vibrational aspects, offering an alternative way of screening new viruses and mutations, and perhaps even novel ways to prevent infections from occurring. • this work provides a novel way in understanding coronavirus pathology using mechanics • reports major movement associated with the swing of upward rbd for open-state viruses • links key nanomechanical vibrational features directly to the epidemiological data • provides possibility of screening new viruses or mutations from a mechanical aspect we provide a novel way towards understanding coronavirus spike proteins, connecting their nanomechanical features -specifically its vibrational spectrum and quantitative measures of mobilitywith virus lethality and infection rate. our study shows that the nanomechanics of proteins -captured in their continuous motions -can be a useful tool to help us understand complex disease etiology by connecting nanoscopic physical features with epidemiological data. potential application includes developing mechanical ways of screening new viruses and mutations, and exploring novel treatment methods. crystal structure of nl63 respiratory coronavirus receptor-binding domain complexed with its human receptor sartorius products receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding human coronavirus nl63, a new respiratory virus cryo-em structure of the 2019-ncov spike in the prefusion conformation structural biology: structure of sars coronavirus spike receptor-binding domain complexed with receptor structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor coiled-coil intermediate filament stutter instability and molecular unfolding deformation and failure of protein materials in physiologically extreme conditions and disease triangular core as a universal strategy for stiff nanostructures in biology and biologically inspired materials nanomechanical sonification of the 2019-ncov coronavirus spike protein through a materiomusical approach molecular model of human tropoelastin and implications of associated mutations nanomechanics of functional and pathological amyloid materials sustained release silk fibroin discs: antibody and protein delivery for hiv prevention coronavirus hku1 and other coronavirus infections in hong kong epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method sars and mers: recent insights into emerging coronaviruses isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers in south korea and china: a potential outbreak threat? a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster clinical features of patients infected with 2019 novel coronavirus in wuhan covid-19 map -johns hopkins coronavirus resource center estimates of the severity of coronavirus disease 2019: a model-based analysis pre-fusion structure of a human coronavirus spike protein structural basis for the recognition of the sars-cov-2 by full-length human ace2 structural basis of receptor recognition by sars-cov-2 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains importance of sars-cov spike protein trprich region in viral infectivity cleavage of spike protein of sars coronavirus by protease factor xa is associated with viral infectivity building-block approach for determining low-frequency normal modes of macromolecules global dynamics of proteins: bridging between structure and function analysis of the vibrational and sound spectrum of over 100,000 protein structures and application in sonification dynamut: predicting the impact of mutations on protein conformation, flexibility and stability the embl-ebi search and sequence analysis tools apis in 2019 cell entry mechanisms of sars-cov-2 dysregulation of type i interferon responses in covid-19 might sars-cov-2 have arisen via serial passage through an animal host or cell culture?: a potential explanation for much of the novel coronavirus' distinctive genome ethical and philosophical considerations for gain-of-function policy: the importance of alternate experiments the protein data bank the protein data bank namd: a parallel, object-oriented molecular dynamics program scalable molecular dynamics with namd harmonicity in slow protein dynamics a new approach for determining lowfrequency normal modes in macromolecules building-block approach for determining low-frequency normal modes of macromolecules tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus covid-19 and cardiovascular disease: from basic mechanisms to clinical perspectives we acknowledge support from the mit-ibm ai lab, onr (n000141612333 and n000141912375), afosr (fate muri fa9550-15-1-0514), nih (u01 eb014976), as well as aro (w911nf1920098). the authors declare no competing interests. key: cord-309081-v098m4dc authors: bin saeed, abdulaziz a.; abedi, glen r.; alzahrani, abdullah g.; salameh, iyad; abdirizak, fatima; alhakeem, raafat; algarni, homoud; el nil, osman a.; mohammed, mutaz; assiri, abdullah m.; alabdely, hail m.; watson, john t.; gerber, susan i. title: surveillance and testing for middle east respiratory syndrome coronavirus, saudi arabia, april 2015–february 2016 date: 2017-04-17 journal: emerg infect dis doi: 10.3201/eid2304.161793 sha: doc_id: 309081 cord_uid: v098m4dc saudi arabia has reported >80% of the middle east respiratory syndrome coronavirus (mers-cov) cases worldwide. during april 2015–february 2016, saudi arabia identified and tested 57,363 persons (18.4/10,000 residents) with suspected mers-cov infection; 384 (0.7%) tested positive. robust, extensive, and timely surveillance is critical for limiting virus transmission. m iddle east respiratory syndrome (mers) coronavirus (cov) causes severe respiratory illness in humans, with death occurring in >35% of reported cases (1) . mers has been documented among persons with close contact with known case-patients in healthcare (2) and household (3) settings and among persons with recent contact with dromedaries (4) . proper clinical management of persons with suspected mers-cov infection who seek care in a healthcare setting relies upon adherence to recommended infection-control precautions (5) , which in turn depends upon the early recognition of cases. the international health regulations emergency committee of the world health organization reported that data sharing for this disease, including sharing of surveillance results, "remains limited and has fallen short of expectations" (6) . to determine the extent of mers surveillance in saudi arabia, we reviewed electronic surveillance data collected during april 1, 2015-february 1, 2016, to describe trends in surveillance for mers and to compare demographic and clinical features among persons tested. in saudi arabia, persons who should be tested for mers-cov include suspect case-patients who meet at least 1 of 4 case definition categories (online technical appendix table, https://wwwnc.cdc.gov/eid/article/23/4/16-1793-techapp1. pdf). in brief, the categories are persons with communityacquired pneumonia (category i); healthcare-associated pneumonia (ii); symptoms after exposure to a mers-cov case-patient (iii); or unexplained febrile illness (iv). the case definition was revised in may 2014 (7); additional refinements were made in june 2015 (8) . the 2015 definition included changes to the approach for testing children <14 years of age with nonsevere illness (testing is reserved for children with exposure to camels or camel products or to a confirmed or suspected mers case-patient). in addition to suspected cases, testing is recommended for close contacts of persons with confirmed mers-cov infection, regardless of symptoms, and can also be requested at the discretion of an infectious disease consultant. tests are performed on respiratory specimens at 5 regional laboratories using real-time pcr (9) . since march 7, 2015, official reporting of cases referred for mers-cov testing in saudi arabia has exclusively been documented through the health electronic surveillance network (hesn). when a suspected case-patient is identified for testing, the referring hospital reports demographic and basic clinical data to hesn ( figure 1 ). after specimens are submitted and testing completed, the regional laboratory reports the result to hesn. for positive cases, the referring hospital submits additional clinical information, and the local health affairs directorate (had) initiates an investigation of exposures and contacts. for negative test results, no further action is taken in hesn. surveillance activities occur in each of the 20 local hads and among hajj pilgrims. we analyzed demographic, clinical, and laboratory data for persons reported to hesn during april 1, 2015-february 1, 2016, in aggregate and by had using microsoft excel 2013 (microsoft corp., redmond, wa, usa) and sas version 9.3 (sas institute, inc., cary, nc, usa). a total of 57,363 suspected mers case-patients were identified and tested during the study period; 384 (0.7%) tested positive (table 1 ). among those for whom nationality and sex were known, 70.3% were saudi (compared with 67.3% of the general population) and 54.3% were male. rates of positivity among those with known age differed by age group; highest and lowest rates were among persons 50-65 and <14 years of age, respectively ( table 1) among tested persons for whom the reason for testing was known, 89.0% met the clinical case definition for suspected mers ( table 1 ). the remaining 11.0% were those recommended for testing by an infectious disease consultant and asymptomatic contacts of confirmed case-patients. more than half of those tested (60.2%) met the category i definition (community-acquired pneumonia) for a suspected case-patient; 0.3% tested positive. the highest positivity most tested persons were reported in the course of routine surveillance through a local had. nationwide, 18.4 persons/10,000 inhabitants were tested, and 1.2/100,000 were mers-cov-positive (10) ( table 2 ). rates of testing and positivity varied by had; the highest testing rates were in ahsa had, followed by riyadh had. najran had had the highest percentage of positive persons ( table 2 ). in addition, surveillance during the annual hajj pilgrimage included 888 tested persons during september 2015, representing 4.5 tested persons/10,000 among 1,952,817 pilgrims. none tested positive for mers-cov. among 8,032 children <14 years of age, 10 (0.1%) tested positive, including 5 who were <1 year of age. at least 7 of the 10 children were tested because of exposure to a mers case-patient. the number of tests among children <14 years of age temporarily dropped after the case definition revision in june 2015, which introduced more stringent criteria for testing. surveillance and testing for mers-cov infection is extensive and widespread in saudi arabia. during our study, an average of >5,000 persons per month were identified as being at high risk for infection due to clinical or epidemiologic criteria and were subsequently tested. mers was first recognized in 2012, and as of november 3, 2016, saudi arabia has reported 80.9% of the cases reported worldwide (11); this distinction may be partly due to the country's robust implementation of surveillance practices and the ready availability of testing, which is facilitated by hesn. we found few other published descriptions of surveillance practices for mers-cov (12, 13) . confirmed mers case-patients represented <1% of all tested persons in saudi arabia. most tests were conducted for persons with community-acquired pneumonia, among whom the positivity rate was predictably low. positivity rates were highest among persons tested because of presumed exposure to mers case-patients (i.e., those tested because of healthcare-acquired pneumonia or onset of symptoms following contact with a confirmed case-patient). only 0.1% of children <14 years of age tested positive for mers-cov; this was the lowest rate among all age groups. most mers-cov-positive children <14 years of age were tested because of high-risk exposures, not because they met clinical criteria. although the proportion of positive tests was highest among persons >35 years of age, the number of tests was highest among persons 18-34 years of age, perhaps because of widespread testing of healthcare workers during outbreaks. the largest number of tests was conducted in november, coinciding with the winter respiratory virus season. in comparison, the proportion of positive tests peaked in may and august, coinciding with outbreaks that occurred in ahsa (14) and riyadh (15) , respectively. our analysis had limitations. variations were probably present in the reporting practices of the various data reporters, in the clinical diagnostic practices used across saudi arabia, and among investigation teams. such variations could affect the completeness, accuracy, and timeliness of the data used for this assessment. surveillance and testing for mers-cov throughout saudi arabia is extensive, as documented by hesn; in a single month during this study, >9,000 patients at high risk for mers were investigated. a continued robust approach to the early detection of patients with mers is critical for the prompt implementation of infection-control precautions and the prevention of healthcare-associated transmission of mers-cov. situation update on middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities transmission of mers-coronavirus in household contacts risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection. interim guidance world health organization. who statement on the tenth meeting of the ihr emergency committie regarding mers case definition and management of patients with mers coronavirus in saudi arabia ministry of health kingdom of saudi arabia, scientific advisory board. infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection world health organization. laboratory testing for middle east respiratory syndrome coronavirus (mers-cov). interim guidance kingdom of saudi arabia. statistics book. statistical book for the year 1435 world health organization. confirmed global cases of mers-cov reported to who as of 14 response to emergence of middle east respiratory syndrome coronavirus evaluation of patients under investigation for mers-cov infection an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region, saudi arabia description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia key: cord-298941-xf2ukinp authors: al-abdallat, mohammad mousa; payne, daniel c.; alqasrawi, sultan; rha, brian; tohme, rania a.; abedi, glen r.; nsour, mohannad al; iblan, ibrahim; jarour, najwa; farag, noha h.; haddadin, aktham; al-sanouri, tarek; tamin, azaibi; harcourt, jennifer l.; kuhar, david t.; swerdlow, david l.; erdman, dean d.; pallansch, mark a.; haynes, lia m.; gerber, susan i. title: hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description date: 2014-05-14 journal: clinical infectious diseases doi: 10.1093/cid/ciu359 sha: doc_id: 298941 cord_uid: xf2ukinp background: in april 2012, the jordan ministry of health investigated an outbreak of lower respiratory illnesses at a hospital in jordan; 2 fatal cases were retrospectively confirmed by real-time reverse transcription polymerase chain reaction (rrt-pcr) to be the first detected cases of middle east respiratory syndrome (mers-cov). methods: epidemiologic and clinical characteristics of selected potential cases were assessed through serum blood specimens, medical record reviews, and interviews with surviving outbreak members, household contacts, and healthcare personnel. cases of mers-cov infection were identified using 3 us centers for disease control and prevention serologic tests for detection of anti–mers-cov antibodies. results: specimens and interviews were obtained from 124 subjects. seven previously unconfirmed individuals tested positive for anti–mers-cov antibodies by at least 2 of 3 serologic tests, in addition to 2 fatal cases identified by rrt-pcr. the case-fatality rate among the 9 total cases was 22%. six subjects were healthcare workers at the outbreak hospital, yielding an attack rate of 10% among potentially exposed outbreak hospital personnel. there was no evidence of mers-cov transmission at 2 transfer hospitals having acceptable infection control practices. conclusions: novel serologic tests allowed for the detection of otherwise unrecognized cases of mers-cov infection among contacts in a jordanian hospital-associated respiratory illness outbreak in april 2012, resulting in a total of 9 test-positive cases. serologic results suggest that further spread of this outbreak to transfer hospitals did not occur. most subjects had no major, underlying medical conditions; none were on hemodialysis. our observed case-fatality rate was lower than has been reported from outbreaks elsewhere. in april 2012, the jordan ministry of health (jmoh) investigated a cluster of 13 suspected pneumonia cases among healthcare personnel, of which 2 were fatal, at a hospital in the city of zarqa [1] . despite testing for multiple potential pathogens, the investigation did not identify a known etiology for these infections. following the discovery of middle east respiratory syndrome coronavirus (mers-cov) in september 2012 [2] , specimens from the 2 fatal cases in jordan were retrospectively tested and both yielded positive results for mers-cov by real-time reverse transcription polymerase chain reaction (rrt-pcr), and were reported to the world health organization (who). these were the first confirmed human cases of infection with this emergent virus, which continues to appear as sporadic cases and clusters internationally, and which is now the focus of worldwide public health investigation and response [3, 4] . using newly developed serologic assays to determine mers-cov antibody responses among case contacts in this outbreak, epidemiologists from the jmoh, us centers for disease control and prevention (cdc), and regional partners conducted a retrospective seroepidemiologic investigation to (1) confirm whether surviving outbreak members had presence of antibodies to mers-cov, (2) ascertain whether viral transmission occurred among household contacts or to other healthcare personnel, and (3) describe the clinical features of all detected mers-cov infections in jordan. we interviewed and collected serum specimens from available members of the initial outbreak (who were admitted to the focal outbreak hospital during the period from 15 march to 30 april 2012 with fever and dry cough, and with radiological evidence of pneumonia), their household contacts (who reported usually sleeping under the same roof as a defined outbreak member during february-april 2012), a sample of healthcare personnel from 3 medical institutions that admitted outbreak subjects (nonsystematic enrollment, with preference toward those reporting close contact with outbreak members), and field investigators from the jmoh. hospitalized subjects meeting the initial outbreak case definition were subsequently transferred from the focal outbreak hospital to 2 other hospitals in amman. participating healthcare personnel were employed at one of these hospitals or at jmoh during february-april 2012. epidemiologic data were obtained through medical record reviews and personal interviews during our may 2013 investigation. interviews were conducted in arabic, and documented contact history (with outbreak members, household members, visiting travelers, and animals) and occupational exposures. we conducted medical record reviews and key informant interviews with clinicians who provided medical care to patients with suspected infection and heads of infection control units at each medical institution and at the jmoh. informed consent was obtained prior to serum collection and interviews. as a public health response to a disease outbreak, this investigation did not require institutional review board review. all work with live mers-cov was done in cdc biosafety level 3 (bsl-3) containment facilities in atlanta, georgia. serum samples were inactivated using 2 × 10 6 rads γ-irradiation and stored at −80°c until use. to maximize specificity, we defined mers-cov antibody positivity as subjects having correlated, positive laboratory results from the hku5.2n screening enzyme-linked immunosorbent assay (elisa), as well as confirmed positive results by either the mers-cov immunofluorescence assay (ifa) or the mers-cov microneutralization assay (mnt) (supplementary table 1 ). an initial indeterminate test result was recorded for those subjects having only a single, uncorrelated positive test result. antibody detection by hku5.2 nucleocapsid elisa and mers-cov ifa and mnt genetic sequencing data indicate that mers-cov is a β-coronavirus (subgroup 2c) similar to the bat covs hku4 and hku5. the recombinant bthku5.2 nucleocapsid proteinbased elisa was developed by the cdc to detect the presence of antibodies that cross-react with the hku5.2 n protein in serum samples from possible mers cases. if cross-reactive antibodies were detected in serum samples, then confirmation of mers-specific antibodies was determined by either mers-cov mnt or ifa. pi-batcov hku5.2 nucleocapsid (n) gene in pet-28b (+) plasmid was provided by dr susanna lau, university of hong kong. his-tagged recombinant protein was expressed in escherichia coli and purified by metal affinity chromatography. recombinant hku5.2n protein indirect elisa was developed using a modified version of the severe acute respiratory syndrome (sars) cov n elisa described by haynes et al [5] . sera were considered positive when the optical density (od) values were at or above the 0.43 cutoff value (mean absorbance at 405 nm of sera from us blood donors plus 3 standard deviations). the overall specificity of the assay was determined after screening 545 serum samples from donors in the united states and the middle east and persons with other non-mers respiratory infections (eg, human coronavirus [hcov] oc43, hcov-229e, sars-cov, hcov-nl63, rhinovirus, human metapneumovirus, h1n1). the assay specificity was 96.7% (527/545). serum from hku1 human serum was not available for evaluation; however, hku1 mouse hyperimmune serum did not cross-react with the hku5.2 n protein. at a screening dilution of 1:400, sera with od values at or near the cutoff were titered with serial 4-fold dilutions (1:100-1:6400) and further evaluated using mers-cov (hu/jordan-n3/2012) (genbank kc776174.1) ifa and mnt. indirect immunofluorescence was performed by screening sera at a dilution of 1:50 or 1:100 on paraformaldehyde-fixed, acetone-methanol-permeabilized, mers-cov-infected oruninfected control vero cells. the source of the positive control for this assay was a serum sample from a patient infected with mers-cov hu/england-n1/2012 ( provided by m. zambon, public health england). antihuman immunoglobulin (ig) g, igm, and iga fluorescein isothiocyanate conjugate was used and specific fluorescence was detected under an immunofluorescence microscope. a positive result was scored when fluorescent intensity equaled or was higher than that of the positive control. a weakly positive result was scored when fluorescent intensity was lower than that of the positive control. serum samples were tested for the presence of neutralizing antibodies to mers-cov using a modified mnt method described for sars-cov [6] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell monolayer lysis in at least 1 of the 3 triplicate wells. controls were included for each mnt assay performed, including the input virus back titration and mock-infected cells. all assay results were confirmed in 3 separate assays, and representative data are presented. tests of statistical significance were performed between the mers-cov antibody-positive and -negative subjects, including fisher exact test and χ 2 tests for categorical variables using sas software version 9.3 (sas institute, cary, north carolina). serologic specimens and interviews were obtained from 124 subjects. we obtained serologic specimens and data from 9 of the 11 (82%) surviving members meeting the initial outbreak case definition; the remaining 2 subjects were unable to be interviewed (1 member was lost to follow-up and 1 did not consent) ( figure 1 ). we also enrolled 26 household contacts and 89 subjects who did not meet the initial outbreak case definition who worked in healthcare and allied professions. among the healthcare personnel interviewed, 58% were nurses, 21% were physicians, and the remaining were allied health professionals; approximately half were employed at the focal outbreak hospital. seven of the 124 subjects tested positive for anti-mers-cov antibodies by both hku5.2 elisa and ifa (table 1 and supplementary figure 1) , and all but 1 also had detectable neutralizing antibody titers as determined by mnt. the subject who did not have detectable neutralizing antibodies was testpositive both by hku5.2n elisa and by a confirmative ifa. demographic and epidemiologic comparisons of seropositive and seronegative subjects are provided in supplementary table 2 . sera from the 2 fatal cases (designated outbreak subjects 01 and 12) having positive rrt-pcr tests were also tested by the 3 described serology tests. a serum sample from outbreak subject 01 (taken 16 days after onset of respiratory symptoms) was positive by hku5.2n elisa and ifa and had detectable mers-cov neutralizing antibodies. two serum specimens from outbreak subject 12 (collected 26 and 32 days after onset) were negative for anti-mers-cov antibodies. of the 7 subjects found to be positive for anti-mers-cov antibodies during this investigation, 6 were surviving members of the initial outbreak group and 1 was previously unrecognized. thus, including the 2 fatal cases previously detected and reported, a total of 9 individuals in this outbreak had evidence of mers-cov infections by acute rrt-pcr tests (n = 2) or convalescent antibody tests (n = 7). the case-fatality rate among all test-positive subjects was 22% (2 of 9). we documented that each serologic test-positive subject had unprotected mers-cov exposure(s) to at least 1 rrt-pcr test-positive subject. an additional 8 subjects had single positive test results by either hku5.2n elisa or ifa, but their mers-cov antibody status was considered indeterminate because both tests were not positive (table 1) . we obtained specimens and data from a total of 97 healthcare personnel who worked during february-april, 2012, representing a majority of intensive care (intensive care unit [icu] and coronary care unit [ccu]) personnel at the outbreak hospital as well as other personnel having close contact with initial outbreak investigation members ( figure 1 ). these included 8 surviving outbreak members who were healthcare personnel at the focal outbreak hospital and were not lost to follow-up, 49 other personnel at the focal outbreak hospital, 16 personnel at transfer hospital a, 20 personnel at transfer hospital b, and jmoh's 4 outbreak investigators. of the 57 healthcare personnel at the focal outbreak hospital who survived and the 1 who died, 6 (10%) had cases of mers-cov. our investigation provided no evidence of mers-cov infections or transmission events among personnel at the 2 receiving transfer hospitals, even though some patients were transferred temporally close to their symptom onset dates. interviews with surviving subjects and family members revealed that transmission opportunities among healthcare personnel were not restricted to the workplace. we obtained serologic specimens from members of 11 households, including those from the initial outbreak group and another 26 subjects who had resided in those outbreak member abbreviations: elisa, enzyme-linked immunosorbent assay; ifa, immunofluorescence assay; mers-cov, middle east respiratory syndrome coronavirus; mnt, microneutralization titer. a outbreak member 08 was lost to follow-up, and outbreak member 13 did not consent. outbreak members 01 and 12 were previously laboratory-confirmed positive by real-time reverse transcription polymerase chain reaction (rrt-pcr) and died. serum samples from outbreak members 01 and 12 were collected prior to death and stored. b serum specimens with optical density (od) values ≥0.43 at a 1:400 dilution against hku5.2n elisa were considered to be positive. specimens were further titered against hku5.2n at 1:100, 1:400, 1:1600, and 1:6400 dilutions. the antibody titer was taken to be the highest antibody dilution above the cutoff od that yielded a ratio of the absorbance of the positive serum and negative serum (p/n) > 3. the value is the reciprocal of the dilution. c serum specimens that were positive by hku5.2n elisa were screened at either 1:50 or 1:100 by indirect ifa using mers-cov_jordan-infected vero cells. h outbreak members conformed to the original outbreak definition; however, some were retrospectively determined to be mers-cov test negative. they were part of the original, defined outbreak that our investigation used to trace a priori contacts and exposures, so this descriptive title is retained. i hku5.2 n elisa od values for serum specimens from outbreak members 05, 07, and 10 and from healthcare personnel 308 were near the assay cutoff od value and rescreened by serial dilution. these serum samples were initially weakly positive by ifa and considered initially indeterminate. upon rescreen by ifa, the samples were determined to be negative for the presence of mers-cov antibodies. j although outbreak member 12 was positive for mers-cov by rrt-pcr, his sera were antibody negative. presumably, this subject died before an antibody response was detectable. this case is considered to be confirmed by current who mers-cov diagnostic guidelines. households during the outbreak period. one household was lost to follow-up, and 1 did not consent for participation. from one of these households was the symptomatic wife of an initial outbreak investigation member who tested positive for mers-cov antibodies. twelve household subjects were children <18 years old, all of whom were serologically test negative. a summary of underlying conditions for test-positive subjects, including the 2 fatal cases initially identified by rrt-pcr (outbreak members 01 and 12), is presented in table 2 . of the 9 testpositive subjects, 66% were male, with a median age of 40 years (range, 25-60 years) at illness onset. we found no evidence of underlying immunodeficiency or immunosuppressant medications/therapies among any of these subjects. one subject had an atrial septal defect, 2 had a history of hypertension, 2 were smokers at the time of illness, and 1 reported a pregnancy of 5 months' gestation. although diabetes mellitus has been observed as a potential risk factor for mers-cov [7] , none of the subjects reported here had a prior diagnosis of diabetes mellitus and, based on serum glucose values taken during their hospitalizations, none had indications of undiagnosed diabetes mellitus. the most common presenting symptoms, as documented in medical charts, included fever (89%), cough (89%), dyspnea (56%), chest pain (44%), and malaise (33%). eight subjects presented for hospital care a median of 5 days after symptom onset (range, 1-14 days). of these patients, 7 (88%) had cough, 7 (88%) had documented fever (temperature (≥38.0°c), 6 (75%) had dyspnea, 5 (63%) had chest pain, and 5 (63%) had malaise at some point during their disease course. less common symptoms included chills (38%), wheezing (25%), and diarrhea, vomiting, sore throat, palpitations, and confusion (13% each). seven subjects had abnormal chest radiographic findings reported within 3 days of presentation, and 3 of those 7 had bilateral findings. of the remaining 4 subjects with initial unilateral findings, 3 went on to develop bilateral infiltrates later in their hospitalization, documented either by chest radiography or computed tomography (ct). one subject (outbreak member 12) received an initial diagnosis of pericarditis, and a ct scan with abnormal pulmonary findings was reported 4 days later ( table 3) . seven of the 8 subjects (88%) who presented to medical care were admitted; 1 refused admission. six subjects (75%) required respiratory support with at least supplemental oxygen, and 4 subjects (50%) received intensive care (in ccu or icu), but only the 2 (25%) patients who died required mechanical ventilation, of which 1 patient also required pressor support (dopamine and norepinephrine) for cardiorespiratory failure. complications among hospitalized subjects were also limited to the 2 patients who died, 1 of whom had hyperkalemia with associated ventricular tachycardia, disseminated intravascular coagulation, and eventual cardiac arrest; the other had pericarditis, pericardial and pleural effusions, and supraventricular tachycardia late in the course of illness. although leukopenia (<4.0 × 10 9 /l) was observed in 2 subjects, lymphopenia (<1.5 × 10 9 /l) was observed in 6 of the 7 subjects who had documented complete blood counts with differentials (86%). elevated leukocyte counts (>11 × 10 9 /l) were observed during the course for 2 subjects (25%), both of whom died. these 2 subjects also had laboratory abnormalities consistent with multiorgan system failure late in the course of disease. these included evidence of elevated alanine aminotransferase and aspartate aminotransferase (>40 u/l) and significant coagulopathy with an international normalized ratio of >1.5, as well as thrombocytopenia (<140 × 10 9 /l). in addition, the 2 subjects who died had elevated serum creatinine measurements (≥133 µmol/l) on the day of their deaths. a third case had an isolated elevated creatinine measurement, but had a subsequent normal value the following day. no patient received hemodialysis (table 3) . outbreak member 01 died 17 days after onset of symptoms (on day 11 of hospitalization) and outbreak member 12 died 35 days after onset of symptoms (on day 22 of hospitalization). the remaining 7 subjects survived, and the 5 who were hospitalized were discharged following a median of 8 days (range, 4-16 days). despite having respiratory symptoms, the pregnant household subject did not seek medical care due to concerns regarding receiving chest radiography and medications. this pregnancy resulted in stillbirth during the course of her illness [8] . surviving subjects and the family members of deceased patients reported that contact with animals was rare in this urbanized area, and no contact with camels was identified among subjects having early symptom onsets. furthermore, none of the subjects had traveled to, or had received visitors from, the arabian peninsula shortly prior to symptom onset. at the focal outbreak hospital, there were no physical barriers between ccu and icu beds, spaced approximately 3 meters, with the exception of cloth drapes in the ccu. isolation or negativepressure rooms were not present, and infection control compliance issues were reported during the outbreak. infection control insufficiencies were not noted at the 2 receiving transfer hospitals. we used novel serologic assays to determine antibody responses of subjects from a mers-cov outbreak investigation in jordan, including the earliest cases of this emerging virus yet discovered. in addition to 2 fatal cases confirmed by rrt-pcr and reported to who, we discovered 7 previously unconfirmed and unreported mers-cov infections. detection of these 7 additional antibody-positive subjects, including healthcare personnel from the focal outbreak hospital and a family contact of 1 antibody-positive subject, and the establishment of contacts with mers-cov infected subjects when potentially infectious, suggests that human-to-human transmission of mers-cov occurred. although community exposures were possible, healthcare-associated transmission was a plausible explanation for healthcare personnel infections. mers-cov infections were not detected among healthcare personnel at a transfer hospital having better adherence to infection control measures. compared with published descriptions of saudi arabian and french cases [9] [10] [11] [12] , among the 9 total jordanian cases identified through our collaborative investigation, subjects were younger and had fewer underlying medical conditions, and there was a lower case-fatality rate. although all subjects with mers-cov infection in our investigation had acute respiratory illnesses during the outbreak period, 78% of those who were infected survived. most subjects had no underlying medical conditions and none were on hemodialysis or had indications of diabetes mellitus. one newly detected subject, who was a household contact, did not seek medical care. our data support the probability that, in outbreak settings, infections may remain undetected among subjects who have mild symptoms, lack predisposing conditions, or have barriers to accessing appropriate diagnostic care. therefore, the true mers-cov case-fatality rate may be lower than that based on symptomatic, hospitalized cases alone. the presenting symptoms we observed were largely consistent with those of previously described mers-cov cases [13] [14] [15] [16] and included fever with respiratory symptoms such as cough and dyspnea, and associated infiltrates on chest radiography. on initial presentation, many subjects did not have evidence of bilateral pneumonia. although gastrointestinal symptoms such as vomiting and diarrhea were documented for 2 subjects, we did not observe these as presenting symptoms, as they were in saudi arabian and french cases. once hospitalized, lymphopenia, a prominent laboratory feature among previously described cases, was observed in the majority of our subjects. however, other laboratory abnormalities observed in previous reports, such as thrombocytopenia, were limited mostly to the 2 fatal cases late in the course of illness, consistent with multiorgan system failure. also, unlike previously reported cases, renal failure was not a prominent clinical feature among our subjects, as renal dysfunction was observed only in the 2 fatal cases on the day of death. rapid isolation of patients with suspected mers-cov and rigorous infection control practices at the receiving transfer hospitals may have been important in preventing transmission at these locations. hospitals should have established policies and procedures for the rapid identification of suspected or known mers-cov cases and implementation of appropriate infection prevention measures. the cdc recommends standard, contact, and airborne precautions for the management of hospitalized patients with known or suspected mers-cov infection [17] . one jordanian patient was initially hospitalized with pericarditis, a manifestation similar to 1 mers-cov case occurring in the kingdom of saudi arabia [9] . although this jordanian patient's serologic specimens tested negative for mers-cov antibodies at periods throughout his hospital stay, 1 acute specimen collected several days before death was confirmed positive for the virus by rrt-pcr. these laboratory findings and the patient's exposure in the ccu, where he was situated in the bed directly next to another patient with rrt-pcr-confirmed mers-cov, collectively suggest the likelihood that the patient was nosocomially infected with mers-cov and died before an antibody response was detectable. based on the knowledge of sars-cov antibody responses, igg and neutralizing antibodies to sars-cov peaked 4 months following a patient's recovery from acute infection [18] . antibody levels did decline over time, but detectable sars-cov neutralizing antibodies persisted up to 2 years after onset of sars-cov symptoms [19, 20] . approximately 13 months had passed between our may 2013 investigation and the april 2012 outbreak. although this was sufficient time for infected subjects to produce an antibody response to mers-cov, the role of waning immunity on the antibody response [21] and whether persistence of these antibodies is important for protection from reinfection remain unclear. we implemented a rigorous case definition based on an elisa-positive result plus at least 1 correlating assay result to maximize specificity. infections with sars-cov triggered humoral and cellular immune responses in all studied humans [22] , and high titers of neutralizing antibodies were observed in response to sars-cov infections, but such characteristics of the mers-cov immunologic response remain unknown. as for those indeterminate laboratory findings among subjects with documented mers-cov exposure(s) but having only an elisa-positive result and mild or absent respiratory symptoms, it is possible that the viral exposure to these subjects did not trigger a long-lasting ifa-or mnt-recognizable immune response. because obtaining appropriate lower respiratory specimens from subjects having mild or asymptomatic infections is challenging, the use of serologic assays to identify otherwise undetected cases of mers-cov has been demonstrated to be a useful tool. serological surveys have been conducted in retrospective case investigations around instances of mers-cov importations in europe [23] , as well as for establishing estimates of mers-cov seroprevalence among populations at risk [24] . further validation of serologic assays and assessments of how they complement rrt-pcr testing is needed. our investigation was unable to find evidence of any exposure (either zoonotic contacts, human contacts from the arabian peninsula, or among hospitalized contacts preceding the earliest symptomatic cases) that might explain the origin of the virus. the precise route(s) of mers-cov transmission remains unclear overall, but several mers-cov sequences have been identified in dromedary camel nasal secretions, including one that is indistinguishable from that found in infected humans [25] . in conclusion, the jordan respiratory illness outbreak in april 2012 resulted in a total of 9 test-positive mers-cov subjects. the source of the virus in these earliest known mers-cov cases remains unknown. compared with other reports, the improved survivability we observed is perhaps related to the youth and relative lack of underlying illnesses among the subjects we investigated. infection control practices at both transfer receiving hospitals may have been important in preventing mers-cov transmission in those facilities. since the discovery of the mers-cov, enhanced surveillance for severe acute respiratory illnesses in jordan has been implemented. international severe acute respiratory infection surveillance, collaborative investigations, and vigilance among healthcare providers are necessary components for addressing and preventing the further spread of mers-cov worldwide. supplementary materials are available at clinical infectious diseases online (http://cid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. epidemiological findings from a retrospective investigation isolation of a novel coronavirus from a man with pneumonia in saudi arabia update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide global alert and response (gar): novel coronavirus infection-update (middle east respiratory syndrome coronavirus) recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association middle east respiratory syndrome coronavirus (mers-cov): a case-controlled study of hospitalized patients stillbirth during infection with middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission epidemiological, demographic and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus a patient with severe respiratory failure caused by novel human coronavirus clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection severe respiratory illness caused by a novel coronavirus family cluster of middle east respiratory syndrome coronavirus infections interim infection and prevention control recommendations for hospitalized patients with middle eastern respiratory syndrome coronavirus (mers-cov) disappearance of antibodies to sars-associated coronavirus after recovery two year prospective study of the humoral immune response of patients with severe acute respiratory syndrome longitudinal profile of antibodies against sars-coronavirus in sars patients and their clinical significance neutralizing antibodies in patients with severe acute respiratory syndrome associated coronavirus infection sars immunity and vaccination contact investigation for imported case of middle east respiratory syndrome lack of mers coronavirus neutralizing antibodies in humans middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia author contributions. d. c. p. had full access to all the data in the study and had final responsibility for the decision to submit for publication.disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc.financial support. this work was supported by the us global disease detection operations center outbreak response contingency fund.potential conflicts of interest. all authors: no potential conflicts of interest.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-303941-3lg1bzsi authors: han, hui-ju; wen, hong-ling; zhou, chuan-min; chen, fang-fang; luo, li-mei; liu, jian-wei; yu, xue-jie title: bats as reservoirs of severe emerging infectious diseases date: 2015-07-02 journal: virus research doi: 10.1016/j.virusres.2015.05.006 sha: doc_id: 303941 cord_uid: 3lg1bzsi abstract in recent years severe infectious diseases have been constantly emerging, causing panic in the world. now we know that many of these terrible diseases are caused by viruses originated from bats (table 1), such as ebola virus, marburg, sars coronavirus (sars-cov), mers coronavirus (mers-cov), nipah virus (niv) and hendra virus (hev). these viruses have co-evolved with bats due to bats’ special social, biological and immunological features. although bats are not in close contact with humans, spillover of viruses from bats to intermediate animal hosts, such as horses, pigs, civets, or non-human primates, is thought to be the most likely mode to cause human infection. humans may also become infected with viruses through aerosol by intruding into bat roosting caves or via direct contact with bats, such as catching bats or been bitten by bats. bats have been known to be reservoirs of rabies virus for a long time (pawan, 1959a,b) , and bats have also been considered as natural hosts of many common human and animal viruses, such as measles, mumps, parainfluenza, canine distemper and hepatitis c viruses (drexler et al., 2012; quan et al., 2013) . however, bats have caught our attention very recently, due to their association with several severe emerging infectious diseases. currently, bats have been considered to be natural reservoirs of sars-cov, mers-cov, niv, hev, ebola virus, and marburg viruses. these viruses have attracted global attention in recent years for their severity and/or easy transmission. this article reviews special features of bats in viral transmission and maintenance, bat's role as the reservoirs of some important viruses, the methods for bat-borne viruses' transmission to humans, and ecological drivers of bat-borne emerging infectious diseases. bats belong to the order chiroptera (greek means hand-wing) and can be further classified into two suborders: the yinpterochiroptera (megachiroptera, commonly known as megabats) and the yangchiroptera (microchiroptera, commonly known as microbats) (simmons, 2005) . megabats eat fruit and microbats live on insects, small mammals, fish or blood. megabats have no echolocation and microbats possess echolocation (except for rousettes and its relatives) ( table 1) . bats have many features that enable them to carry a diversity of viruses. with approximately 1240 recognized species worldwide, bats account for about 25% of all mammalian species, making them the second largest order of mammals (jones et al., 2005; turmelle and olival, 2009 ). the huge diversity of bat species can provide a large breeding ground for viruses. in addition, bats are ancient species and can be traced back to 52.5 million years ago (clyde et al., 2001; jepsen, 1966) . the long evolutionary history provides long period for a variety of viruses to co-evolve with bats to make bats their natural reservoirs. in order to facilitate flying, bats developed hollow bones to reduce body mass; as a result, they do not have bone marrow as other mammals to produce b cells. this special immunological characteristics of bats may enable bats to carry a large number of viruses without obvious illness (dobson, 2005) . during winter time, some bats hibernate to conserve energy. reduced body temperature and metabolic rate may suppress immune responses and delay viral clearance from bats (george et al., 2011; sulkin and allen, 1974) . some features of bats may keep viruses circulating among the bat population. bats are social animals, millions of individuals can be found in a single cave. the large number of bats in a colony with physical proximity facilitates viral transmission among bats, maintaining viruses circulating stably among bats. a study showed that several emerging viruses could be amplified in a bat colony during the breeding season (drexler et al., 2011) . microbats developed echolocation for navigation. apart from ultrasonic waves, the vibration of the larynx can also generate aerosols, which may also play an important role in viral transmission among bats (calisher et al., 2006) . some features of bats can contribute to viral dispersal. with a large number of species, bats can be found in all continents except the antarctica and inhabit various ecological niches, including trees, caves, and man-made structures, such as tunnels, deserted houses, even occupied houses in rural areas. the worldwide distribution and various habitats of bats pose the public to a general risk of infection with bat-borne viruses. bats are special as the only mammals that can actually fly. bats fly in their daily quest for food and seasonal migration, some of which can fly up to nearly 2000 km (fleming and eby, 2006) . the ability to fly enables bats to carry viruses to distant areas. the eating behavior of bats can also be linked to viral transmission. fruit bats cannot take a large amount of food, and to meet their demand for energy, instead of swallowing, they just chew to absorb sugars and higher energy components, and spit out the partially digested fruits. discarded fruits contaminated by viruses in bat saliva may be eaten by other animals and infect them (dobson, 2005) . in addition, despite their small size, bats have a relatively long life span, most of the species live for 10-20 years and some can live up to 30 years (brunet-rossinni and austad, 2004) . the longevity of bats also increases the possibility of viral dispersal. hev (formerly known as equine morbillivirus) was first recognized in australia in 1994, when it caused severe respiratory or neurological diseases in horses and humans (murray et al., 1995) . many investigations were done in search for the natural reservoir of this new highly pathogenic virus. initial screening of 2411 horses for hev-specific antibodies showed negative results (ward et al., 1996) , and a later extensive serosurvey of 5264 sera from 46 animal species, including both domesticated and wild animals, showed no evidence of infection. an epidemiological investigation pointed to fruit bats as a candidate natural reservoir, and serosurvey of 224 serum samples of fruit bats showed that 20 contained hevspecific antibodies (young et al., 1996) . three virus isolates were later obtained from urine and lung tissue from two of 460 individual fruit bats, and serological testing and sequencing of 200 nucleotides in the matrix gene indicated that the three isolates were identical to hev (halpin et al., 2000) . isolation of hev, together with serological and epidemiological evidence indicates that fruit bats are natural reservoirs of hev in australia. niv first emerged in malaysia in 1998, when it caused an outbreak of respiratory illness in pigs and encephalitis in humans. niv and hev were classified into a new genus, henipavirus, within the family paramyxoviridae. studies also tend to link niv to bats. serological evaluation in outbreak areas of malaysia showed that domesticated animals, such as dogs, cats and ponies had nivspecific antibodies (chua et al., 2000) , while wild boar, hunting dogs and rodents were all negative (yob et al., 2001 ). an extended survey of 14 species of bats highlighted that two fruit bat species had relatively high seropositivity, 31% and 17% for small flying fox (pteropus hypomelanus) and large flying fox (p. vampyrus), respectively. niv was isolated from p. hypomelanus urine and their partially eaten fruits, which was nearly identical to isolates from pigs and humans (56 nt changes) (abubakar et al., 2004; chua et al., 2002b) . recently, niv was isolated from p. vampyrus, which was about twice the difference between the human and p. hypomelanus isolates (98 nt changes) (rahman et al., 2010) . serological findings and viral isolation indicate that fruit bats are natural reservoirs of niv. sars-cov is the causative agent of sars (severe acute respiratory syndrome), which emerged as a new clinical severe human disease in guangdong province of china in 2002. initially, palm civets sold in live animal markets were demonstrated to carry sars-cov and were considered as the reservoirs of sars-cov (guan et al., 2003) , but later studies pointed to bats as the source of sars-cov. one group of researchers took bat samples from the natural environment, with 408 bats representing nine species. three species of horseshoe bats (genus rhinolophus) showed relatively high seroprevalence of sars-neutralizing antibodies, ranging from 28% to 71%. in addition, bat-cov rna was demonstrated 92% identity with human sars-cov isolates . another group of researchers screened nasopharyngeal and anal swabs of 120 bats and found that bat-cov sequences detected in 23 chinese horseshoe bats (rhinolophus sinicus) manifested 88% identity with the sars-cov, with great differences in spike genes (lau et al., 2005) . these bat sars-covs may be the progenitors of sars-cov, but cannot be closely related, since they were phylogeneticlly disparate from sars-cov and were unable to use the sars-cov cellular receptor molecule, human angiotensin converting enzyme ii (ace2), to enter cells (ren et al., 2008) . later, a study reported the whole-genome sequences of two novel bat-covs detected in chinese horseshoe bats, which were far more closely related to sars-cov than any previously identified ones, especially in the receptor binding domain of the spike protein, and they also isolated live sars-like cov from bat fecal samples. in vitro testing showed that this virus had a broad species tropism and used ace2 from humans, civets and chinese horseshoe bats for cell entry (ge et al., 2013) . thus chinese horseshoe bats are considered to be the natural reservoirs of sars-cov. mers-cov is a novel coronavirus, which was isolated from a man with acute pneumonia and subsequent renal failure in saudi arabia in 2012 . full genome sequence analysis of mers-cov and serological data provided evidence for transmission of mers-cov from camels to humans (azhar et al., 2014) , and mers-cov specific antibodies and rna have also been detected in camels during mers-cov outbreak investigations . however, phylogenetic analysis showed that mers-cov belonged to lineage c of the genus betacoronavirus, along with the bat-cov hku4 and hku5 . hence, bats are supposed to be the natural reservoirs of mers-cov, while camels may just act as intermediate animal hosts that facilitate the spillover from bats to humans. recent studies have revealed a diversity of mers-related covs in bats from saudi arabia , africa (ithete et al., 2013) , europe (annan et al., 2013) , and asia . studies on receptor usage by mers-cov can also give a clue of bat origins. initial study found that the receptor use by mers-cov was different from the one used by sars-cov. the receptor may be a conserved one, since mers-cov can replicate in both bat and human cells . subsequently, dipeptidyl peptidase 4 (dpp4 or cd26) was identified as the cellular receptor for mers-cov . bat coronavirus hku4, which is closely related to mers-cov, can also use dpp4 as receptor to initiate cellular entry, and this discovery indicated that bats might be the natural reservoirs of mers-cov (wang et al., 2014) . ebola virus first came into our knowledge in the democratic republic of congo in 1976 (johnson et al., 1977; report of an international, 1978) . since then, this notorious virus has been decimating gorilla, chimpanzee and human populations in africa with high mortality. during the 2013-2014 epidemic, ebola virus has caused 21,121 human infections and 8304 deaths (as of january 9th, 2015, http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/ index.html). during ebola outbreaks in humans and great apes between 2001 and 2003 in gabon and the republic of congo, to investigate the natural reservoirs, more than 1000 small vertebrates were captured. serum antibodies specific for ebola virus were found in three different bat species with prevalence as high as 25% (leroy et al., 2005) . given the lack of overt disease after being infected, bats are considered as likely natural reservoirs of ebola virus. the viruses discussed above tend to be restricted to certain geographic regions with a particular bat reservoir, such as hev and niv associated with flying foxes in australia and southeast asia and ebola virus associated with egyptian fruit bats in africa. most bat population roost in tropical forests or caves and they do not make frequent contact with people. how bats transmit viruses to humans is a mystery until now. below are the hypotheses of how bat-borne viruses are transmitted to humans. a popular theory for bats to transmit viruses to humans is through intermediate hosts, which are in close contact with humans and can amplify viruses. when fruit bats eat fruit, they contaminate the fruit with viruses in their saliva and urine; discarded fruit fall to the ground and is consumed by intermediate hosts, such as pigs, horses, and nonhuman primates; humans become infected by contact with or consuming products of the intermediate animal hosts. flying foxes are thought to transmit niv/hev in southeast asia and tropical australia. during the 1998 niv outbreak in malaysia, it is thought that pigs were infected by niv by consuming mangoes contaminated by flying foxes. mangoes were a food source for flying foxes, and discarded mangoes contaminated by the saliva and urine of the bats fell into the pigsties and were consumed by pigs, resulting in the cross-species infection of pigs and subsequently humans (chua et al., 2002a) . hev is endemic in flying-foxes in australia. hev spills over from bats to domestic animals, primarily horses, which amplify the virus and subsequently infect humans (murray et al., 1995) . bats contaminate a drip zone around trees where they feed or roost by excreting urine, feces and saliva (with partially eaten fruit). horses may be exposed to hev when consuming contaminated grass, fruit, feed or water; or when browsing or sniffing contaminated surfaces within this drip zone (plowright et al., 2015) . fruit contaminated by bats is also thought to transmit ebola virus to apes in central africa (leroy et al., 2005) . in the case of the 2003 sars outbreak in china, the transmission of sars-cov from bats to humans was made possible by the special taste for wildlife cuisine, including civets, which acted as intermediate hosts for sars-cov. in south china, civets are thought to strengthen health in winter, and the demand was so high that farming of this wildlife species was widespread. trade of civets in live wet markets exposed other susceptible animals. therefore, bat sars-cov was likely introduced into humans through civets and other animals (liu, 2003) . as for mers in middle east, dromedary camels are hypothesized as intermediate animals to transmit mers-covs from bats to humans (memish et al., 2014) . camels are popular in middle east countries both for entertainment and transportation. people may be infected through direct contact with infected camels, which can shed virus in respiratory secretions (azhar et al., 2014) . mers-covs were also detected in camel milk samples from mares infected with the virus (reusken et al., 2014) . whether the viruses is excreted in the milk or is introduced as contaminant during the milking process, it poses a risk for people to become infected by consumption of unpasteurized camel milk. although bats are rarely in contact with people, people may become infected with bat-borne viruses by consuming bat meat. wild animal meat or 'bush meat', including bats, are taken as delicacies in some regions of africa. the capture and selling of wild animals increases the risk of being infected by zoonotic viruses. consumption of infected bats may transmit such bat-borne viruses as ebola virus to humans. in 2007 ebola hemorrhagic fever reemerged in the democratic republic of congo (drc) causing 186 deaths. epidemiological investigation showed that the outbreak was due to consumption of fruit bats, which were migrating towards the southeast and were massively hunted as a major source of protein by local villagers (leroy et al., 2009 ). consumption of well-cooked bat meat may not be a problem, but the handling of bats may increase the risk of infection. in addition, accidental bite by a bat may result in rabies. one example is the case of an elderly man in south africa who died of duvenhage virus (duvv) infection after being scratched by a bat (adjemian et al., 2011) . some bats with a large population roost in caves. bat-borne viruses may contaminate air in caves where bats live and people may become infected by inhaling viruses in the air when they enter caves. research showed that cynomolgus macaques could suffer a lethal viral hemorrhagic fever after exposure to small-particle aerosols containing marv-angola (alves et al., 2010) . there was a report that humans were infected by marburg virus after visiting or working in caves in africa, and transmission by aerosol could not be excluded as a possible mode of infection (timen and koopmans, 2008) . for a spillover to occur, a range of conditions and events must be met. bats must be present and be infected, in most cases shedding viruses. viruses excreted by bats must survive in the environment (if transmitted indirectly), with access to intermediate animals. human or intermediate animals must be exposed to a sufficient dose of virus for an infection to be established, and humans or intermediate animals must be susceptible to the virus (plowright et al., 2015) . however, of all these conditions required for a spillover, it is of utmost importance for bats and humans or animals in close contact with humans to be present in the same habitat, which can be a result of intrusion into each other's living environment. once infected, some factors can accelerate transmission in human society. below we describe ecological factors that increase interaction between bats and humans and factors that accelerate transmission in humans. in human history, the emergence of new pathogens has been accompanied by increased density of humans and other animals. with human population explosion, more space and resources are needed to meet ever-increasing demand for food and housing. factors that contribute to the intrusion of bats into human living environment can be summarized into a 'push' and a 'pull' (brüssow, 2012) . a 'push' refers to the enormous demand for more space and resources brought by the human population explosion, which leads to the destruction of bat habitats and shortage of food. natural environmental changes, such as typhoons and droughts, can also place stresses on bats. a 'pull' involves the living environments built by humans, characterized by urbanization, intensive agriculture and food animal breeding, which attracts bats into human living environments for an abundant of food supply. the 1998 niv outbreak in malaysia was thought to be a combined result of anthropogenic deforestation and drought caused by el nino, which pushed flying fox bats from their natural habitats into farms (chua et al., 2002a) . the rapid development of modern transportation, and the mobility of people, animals and goods lead to the rapid spread of emerging infectious diseases. local transportation is important in sustaining endemics, while international air travel may facilitate cross-continent spread (bobashev et al., 2008) . during the 1998 niv outbreak in malaysia, the transport of infected pigs led to the expansion of outbreak areas in malaysia (lam and chua, 2002) , and the follow-up niv outbreak in singapore among abattoir workers (paton et al., 1999) . due to air transit, the sars outbreak that initially emerged in guangdong, china in november of 2002, quickly spread to 25 countries as far away as canada by the end of march of 2003 (cdc, 2003 . another example is the 2014 ebola hemorrhagic fever outbreak, the largest ebola virus outbreak to date, which quickly spread in west africa and resulted in massively affected areas including guinea, liberia, sierra leone and nigeria. ebola virus also spread to the usa by infected patients who traveled from west africa (von drehle, 2014) . cultural practices can also play an important role in the transmission of emerging infectious diseases. during the 2001 niv outbreak in bangladesh, person-to-person transmission was attributed to the social norms that require family members to maintain close physical contact with the sick one (luby et al., 2009) . in africa, traditional burial practices require the living ones to make contact with the deceased, which also facilitates transmission during ebola virus outbreaks (dowell et al., 1999) . socioeconomic factors may also contribute to transmission, associated with poor health care. one example is the 2014 west africa ebola virus outbreak, first-line health care workers were infected, because of lack of knowledge as well as the unavailability of proper personal protective equipment (kilmarx et al., 2014) . person-to-person transmission during the 2001 bangladesh niv outbreak was also thought to be a result of poverty-induced sharing of eating utensils and food with the infected person (brüssow, 2012) . with ever-increasing interest in bat-borne viruses, and the availability of molecular biotechnology, novel viruses are continually detected or isolated from bats all around the world. in recent years, viral metagenomics may provide a glimpse of bat viral diversity (dacheux et al., 2014; donaldson et al., 2010; ge et al., 2012; he et al., 2013) . so far, viruses detected in bats can be classified into as many as 22 families and many are novel viruses (http://www.mgc. ac.cn/dbatvir/). the pathogenicity of many of these novel viruses for humans remains unknown, and further efforts are needed to determine their potential threats to humans. in addition, previously known bat-borne viruses are also being detected in more bat species, and the geographic distribution of these viruses is also expanding. with human activity increasingly overlapping the habitats of bats, emerging infectious diseases from bat-borne viruses will undoubtedly increase. transmission modes discussed above are just current hypothesis that could theoretically explain some of the spillover events. however, the spillover process is still something of a black box that is scarcely understood and much more research is needed to expand our understanding of the spillover events. there are many questions that remain to be answered about the ecology of bat-borne infections: (1) are bats the natural reservoirs or just transient carriers of these novel viruses? (2) how long can bats harbor these viruses? (3) what are the routes of virus shedding? (4) how long can viruses excreted by bats survive in the environment? (5) and what are the risk factors leading to infection of humans or intermediate animals exposed to bat-borne viruses? with special social, biological and immunological features, bats provide unique niches for many viruses to co-evolve with them. spillover of bat-borne viruses to humans may cause severe diseases and panic. bats have been proposed as the natural reservoirs of viruses causing severe diseases in humans, such as niv and hev in southeast asia and australia, ebola and marburg viruses in africa, sars-cov in asia and mers-cov in middle east. however, the role of bats in transmission of these infectious diseases needs to be further investigated because of lack of direct experimental data on transmission of the viruses from bats to intermediate animal hosts. in addition, bat-borne viruses may have other reservoirs. in a recent study a novel henipa-like virus was detected in rats and was responsible for 3 deaths of mine workers in yunnan province, china, suggesting that bats may not be the only reservoir of these viruses . isolation and molecular identification of nipah virus from pigs outbreak of marburg hemorrhagic fever among miners in kamwenge and ibanda districts, uganda aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques human betacoronavirus 2c emc/2012-related viruses in bats evidence for camel-to-human transmission of mers coronavirus sampling for global epidemic models and the topology of an international airport network on viruses, bats and men a natural history of food-borne viral infections ageing studies on bats: a review bats: important reservoir hosts of emerging viruses cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto, canada nipah virus: a recently emergent deadly paramyxovirus anthropogenic deforestation, el nino and the emergence of nipah virus in malaysia isolation of nipah virus from malaysian island flying-foxes linking the wasatchian/bridgerian boundary to the cenzoic global climate optimum: new magnetostratiographic and isotopic results from south pass, wyoming. paleogeogr a preliminary study of viral metagenomics of french bat species in contact with humans: identification of new mammalian viruses virology. what links bats to emerging infectious diseases? metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat transmission of ebola hemorrhagic fever: a study of risk factors in family members bats host major mammalian paramyxoviruses amplification of emerging viruses in a bat colony ecology of bat migration metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in china isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor host and viral ecology determine bat rabies seasonality and maintenance isolation and characterization of viruses related to the sars coronavirus from animals in southern china isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus virome profiling of bats from myanmar by metagenomic analysis of tissue samples reveals more novel mammalian viruses close relative of human middle east respiratory syndrome coronavirus in bat early eocene bat from wyoming isolation and partial characterisation of a new virus causing acute haemorrhagic fever in zaire bats, clocks, and rocks: diversification patterns in chiroptera ebola virus disease in health care workers -sierra leone nipah virus encephalitis outbreak in malaysia severe acute respiratory syndrome coronaviruslike virus in chinese horseshoe bats human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo fruit bats as reservoirs of ebola virus bats are natural reservoirs of sars-like coronaviruses sars, wildlife, and human health recurrent zoonotic transmission of nipah virus into humans human infection with mers coronavirus after exposure to infected camels middle east respiratory syndrome coronavirus in bats, saudi arabia human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines a morbillivirus that caused fatal disease in horses and humans outbreak of nipah-virus infection among abattoir workers in singapore rabies in the vampire bat of trinidad, with special reference to the clinical course and the latency of infection the transmission of paralytic rabies in trinidad by the vampire bat (desmodus rotundus murinus wagner ecological dynamics of emerging bat virus spillover bats are a major natural reservoir for hepaciviruses and pegiviruses characterization of nipah virus from naturally infected pteropus vampyrus bats dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin ebola haemorrhagic fever in zaire middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study mammal species of the world: a taxonomic and geographic reference, 3 vols virus infections in bats marburg hemorrhagic fever-the netherlands ex uganda correlates of viral richness in bats (order chiroptera) genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans now arriving, the deadly ebola virus lands in america bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 negative findings from serological studies of equine morbillivirus in the queensland horse population novel henipa-like virus, mojiang paramyxovirus, in rats mers-related betacoronavirus in vespertilio superans bats nipah virus infection in bats (order chiroptera) in peninsular malaysia serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors are grateful to dr. david h. walker (department of pathology, university of texas medical branch at galveston, texas 77555-0609) for reviewing out manuscript. this research was supported by grants from shandong province science and technology development program (2014gsf121004) and supported by the shandong university. key: cord-298773-vnmc6nqd authors: pfeiffer, julie k. title: is the debate and “pause” on experiments that alter pathogens with pandemic potential influencing future plans of graduate students and postdoctoral fellows? date: 2015-01-20 journal: mbio doi: 10.1128/mbio.02525-14 sha: doc_id: 298773 cord_uid: vnmc6nqd nan m uch has been said and written about the risks and benefits of certain experiments that alter pathogens with pandemic potential (ppp) (see recent references 1 to 3 and references within). the experiments in question alter transmissibility, virulence, host range, drug susceptibility, immune responses, and/or infectivity or stability. a debate and research "pause" for certain experiments using certain respiratory viruses (influenza, middle east respiratory syndrome [mers] , and severe acute respiratory syndrome [sars] viruses) has electrified the scientific community, with strong "propause" advocates, strong "antipause" advocates, and everything in between. this letter is not about whether the experiments, debate, or pause is good or bad. this letter is about the potential impact of the debate and pause on graduate students and postdoctoral fellows and how their future plans may be affected. policy makers, ethicists, cognitive scientists, epidemiologists, public health experts, biosafety experts, bioterror experts, mathematical modelers, principal investigators, and others have expressed their opinions in multiple forums, including at a recent meeting hosted by the national academy of sciences (15 to 16 december 2014 [http://bit.ly/16o0j5y]). however, graduate students and postdoctoral fellows have not had a voice in this debate. furthermore, virtually nothing is known about how this debate and research pause has influenced trainees and their future plans. the lack of information is a concern, considering that graduate students and postdoctoral fellows are the ones physically performing many of the relevant experiments. furthermore, trainees are the scientists that will populate these fields in the future. to gain initial insight into how the debate and research pause have affected trainees, i created an informal survey 2 days before the national academy of sciences meeting. my goal was simple: begin to gather data on trainee plans to stimulate discussion at the meeting and encourage future study on this topic. the poll is unofficial, relies on self-reporting, and may have a limited respondent pool, and it can be argued that the questions/wording/tone/ advertising need improvement. for full disclosure, i report that this survey was conceptualized and implemented over a 1-h period on a saturday while a beer was consumed. however, the results from this preliminary unofficial poll are worthy of discussion and future study. trainees were asked to complete a surveymonkey poll. as a "reward," i included a link to my "how to find a postdoctoral fellowship" tutorial, which students at ut southwestern have found helpful (http://bit.ly/1gsk7x5). the poll was advertised via twitter (http://bit.ly/1bycimf and http://bit.ly/1qzyyya), the american society for virology facebook page (http://on.fb.me/ 16tkkau), and forwarded e-mails. the twitter announcement reached up to 13,651 people, counting followers of the 16 people who retweeted the advertisement, making it reasonably publicly available. the poll (https://www.surveymonkey.com/s/b3xjp5b) had five questions about the respondent's background and research interests, with one question assessing knowledge level/ awareness about the debate. a paragraph (reproduced below) summarized the debate, and links to four articles (1-4) were given for additional information, followed by two questions assessing the potential impact of the debate and the research pause on future plans. scientists and policy makers are currently debating the pros and cons of performing certain types of experiments using certain pathogens. this debate largely focuses on pathogens with pandemic potential (or ppp), and the most relevant viruses are influenza, mers coronavirus, and sars coronavirus. in october of 2014, the u.s. government implemented a "pause" for certain research projects involving influenza (biosafety level 2 and 3 strains), mers (biosafety level 3), and sars (biosafety level 3). these projects involve a subset of "gain of function" experiments designed to create mouse adapted viral strains, generate drug resistant viruses to understand drug mechanisms of action, understand host immunity by analyzing viruses with resistance to certain host immune pathways, and to study factors that influence transmission by the respiratory route (which was made famous by work from the kawaoka and fouchier labs in 2012). principal investigators of 18 federally funded research projects were told to stop experiments specifically related to the "gain of function" work described above while risks and benefits could be examined. the future of these particular projects is uncertain. work continues on other projects using influenza, mers, and sars. complete poll results from 13 december 2014 to 17 december 2014 are available at the following urls: http://bit.ly/1gus5px (summary of responses) and http://bit.ly/1c2trmg (all individual responses/comments). there was interest in the poll; 156 respondents completed the survey in the first 5 days, with 81 responding over the weekend. of respondents, 72% were ph.d. students and 28% were postdoctoral fellows. a majority, 75% of respondents, said that virology was their field of study, and 48% said that they currently work on a respiratory virus. other viral systems included enteric viruses (20%), "other" mammalian viruses (such as arboviruses, hiv, herpesviruses, and poxvirus [24%]), urogenital viruses (10%), eukaryotic/nonmammalian viruses (3.2%), bacteriophages (1.6%), and plant viruses (1.6%). therefore, respondents study a diverse array of viral systems. importantly, 68% of respondents said that they want to work on a respiratory virus in the future, making this an area of potential growth. career goals among respondents were diverse: academic research faculty (54%), government research (31%), academic research scientist (23%), industry (large company [18%] and small company [16%]), policy or science writing (6.4%), foundation (3.9%), teaching (3.2%), clinical laboratory (1.3%), state public health laboratory (0.6%), consulting or entrepreneurship (0.6%), and technology transfer (0.6%). respondents were knowledgeable about the debate; 61% said that they know a fair amount of information about the debate, 34% said that they have heard of the debate but do not know the details, and 5% said that they had not heard about the debate. after learning more about the debate, 5% said that they were more likely to work on influenza, sars, or mers virus in the future, 28% said that they were less likely to work on influenza, sars, or mers virus in the future, and 51% said that they were equally likely to work on influenza, sars, or mers virus in the future. an additional 16% of respondents said that their opinion was unchanged because they are not planning to work on a virus in the future. finally, for those interested in virology, 12% said that the debate and research pause have changed their future plans/research direction, 51% said that their plans have not changed, and 37% said that the debate and research pause have made them consider other factors in choosing their future plans/research direction. i have a few thoughts about the poll results. first, this was an informal poll developed by a poll-making novice (me) with a limited sample size, quasi-limited advertising, and a self-reporting format. it can be argued that some of the questions/answers/text could have been phrased differently. therefore, there is room for improvement in future surveys that would be developed ideally by trained individuals using something more sophisticated than sur-veymonkey. that said, the data generated by this poll suggest that this is an important topic worthy of follow-up and consideration. i invite others to improve upon this initial effort (see reference 5 for an example). second, trainees are aware of the debate and research pause; 95% had heard about the debate. this was impressive to me, since many of my microbiology faculty colleagues were unaware of the debate until it was mentioned at a faculty meeting this month. perhaps i should not be surprised that trainees are well informed. after all, the millennial generation is the most connected, technologically savvy generation in history. third, the debate and research pause are influencing future plans of virology trainees. twenty-eight percent of respondents (33% of virologists [42 trainees]) said that they are less likely to work on influenza, sars, or mers virus in the future. respiratory viruses are a press-ing global concern, and a potential loss of future investigators is a serious threat. i encourage policy makers to consider trainee impact and potential damage due to lost researchers in the influenza, mers, and sars virus fields. the potential effect of lost future investigators should be factored into current risk-benefit analyses undertaken during the pause. it would be worthwhile for those with expertise in surveys and forecasting to examine this topic with wellaccepted and -established methods to generate data on the potential future impact to the field of virology, particularly for respiratory viruses. for example, factoring in retirement rates and potential loss of future investigators, what will the influenza/ mers/sars virus fields look like in 2030, 2040, or 2050? additionally, trainees are stakeholders in this debate and should have representation in the discussions. finally, i remind trainees and others that this debate and research pause are affecting only a very small subset of experiments with a few respiratory viruses. there are many, many interesting projects and experiments that can and should be done. people on both sides of the debate agree that influenza, sars, and mers viruses are incredibly important human pathogens and should be studied using a variety of approaches. we need talented and devoted scientists studying a diverse array of viruses, including influenza, sars, mers, and other emerging viruses. many thanks go to the 156 trainees that responded so quickly to this survey. good luck with your experiments and best wishes for your exciting careers! gain-of-function experiments: time for a real debate vagueness and costs of the pause on gain-of-function (gof) experiments on pathogens with pandemic potential, including influenza virus moratorium on research intended to create novel potential pandemic pathogens mbio addresses the pause in gain-of-function (gof) experiments involving pathogens with pandemic potential (ppp) a survey of attitudes and actions on dual use research in the life sciences: a collaborative effort of the national research council and the american association for the advancement of science my laboratory studies viral pathogenesis but not respiratory viruses. work in my laboratory has not been impacted by the funding pause or debate. i have funding from the national institutes of health, the american cancer society, and the burroughs wellcome fund. key: cord-269386-bnh65bqg authors: ko, jae-hoon; seok, hyeri; park, ga eun; lee, ji yeon; lee, ji yong; cho, sun young; ha, young eun; kang, ji-man; kim, yae-jean; kang, cheol-in; chung, doo ryeon; song, jae-hoon; peck, kyong ran title: host susceptibility to mers-cov infection, a retrospective cohort study of the 2015 korean mers outbreak date: 2017-12-06 journal: j infect chemother doi: 10.1016/j.jiac.2017.09.008 sha: doc_id: 269386 cord_uid: bnh65bqg to evaluate host susceptibility factors to middle east respiratory syndrome coronavirus (mers-cov) infection, we conducted a retrospective cohort study from the single largest exposure event of the 2015 korean mers outbreak. a total of 175 patients were closely exposed to a super-spreader, 26 of which were infected (14.9%). in a multivariate analysis, history of autologous stem cell transplantation (hr, 31.151; 95% ci, 5.447–178.145; p < 0.001) and tachypnea at ed (hr, 4.392; 95% ci, 1.402–13.761; p = 0.011) were significantly associated with mers-cov infection. close exposure is a critical factor for human-to-human transmission of middle east respiratory syndrome coronavirus (mers-cov), but clinical factors affecting host susceptibility to mers-cov have not been properly identified [1e3] . to identify host susceptibility factors to mers-cov infection, we performed a retrospective cohort study from the largest exposure event during the 2015 korean mers outbreak [3, 4] . a retrospective cohort study was performed to identify risk factors for increased host susceptibility to mers-cov. this exposure event was previously described in detail [3] : while a severelycoughing mers patient stayed at our emergency department (ed) for three days, 675 patients stayed at the ed and had chance to be exposed. after meticulous contact tracing, 175 closely exposed patients were identified and classified into two groups by exposure time and location: group a, patients who stayed in the same examination rooms at the same time with the index case (our ed is composed of zone i~iv for adult patients, and the index case used zone ii~iv); and group b, patients who shared the same radiology room or registration area at the same time period (30 min before and 2 h after) with the index case. exposure sites of group a were subdivided into the zone ii, iii, and iv, as attack rates of each zone were significantly different (23% (13/57), 32% (7/22), and 8% (3/38), respectively; p ¼ 0.047) [3] . included patients were followed for 14 days, which is a previously determined incubation period of mers-cov [1] . the institutional review board of samsung medical center approved this study. for simple comparison of clinical variables, student's t-tests or mann-whitney u tests were used for continuous variables, and chi-square tests or fisher's exact tests for categorical variables. the cox proportional hazard model was used to examine the association of clinical variables with mers-cov infection. variables with statistical significance in univariate analyses were included in the multivariate analysis. the effect of exposure intensity was adjusted by including exposure sites in the multivariate analysis. all p-values were two-tailed, and those <0.05 were considered to be statistically significant. ibm spss statistics version 20.0 for windows (ibm, armonk, ny, usa) was used for all statistical analyses. during the study period, a total of 175 patients were closely exposed to the index case, 26 of whom (14.9%) were infected with mers-cov (3 patients at radiology rooms or registration area, 13 at zone ii, 7 at zone iii, and 3 at zone iv) [3] . baseline characteristics and clinical presentation of the study population at the time of ed visit are presented in table 1 . there was no statistical difference in age, sex, body mass index, and immunosuppressive conditions. patients exposed at zone ii and iii showed higher attack rates as previously described [3] . among underlying diseases, hypertension and cardiovascular diseases were significantly different between infected and non-infected patients (all p < 0.05). significantly higher proportion of mers-infected patients presented with tachypnea (respiratory rate > 24/min) at ed visit, compared to noninfected patients (15.4% (4/26) and 2.7% (4/149), respectively; p ¼ 0.018). the proportion of concomitant infections and laboratory test results were not statistically different between the two groups. to identify possible risk factors for increased host susceptibility to mers-cov, all the obtained clinical variables and exposure sites were evaluated. history of autologous stem cell transplantation (sct), underlying hypertension and cardiovascular disease, tachypnea at ed visit, and exposure sties of zone ii and iii were statistically significantly associated with mers-cov infection in univariate analyses (supplementary table 1 ). in the multivariate analysis, history of autologous sct (hr, 31.151; 95% ci, 5.447e178.145; p < 0.001) and tachypnea at ed visit (hr, 4.392; 95% ci, 1.402e13.761; p ¼ 0.011) were identified as risk factors for increased host susceptibility to mers-cov (table 2 ). significant association of exposure sties of zone ii and iii was relevant with the previous analysis [3] . although many studies have reported poor prognosis factors of mers-cov infection since the discovery of mers-cov in 2012, little has been determined about host susceptibility to the virus [1] . evaluating an exposure cohort for host susceptibility is often not feasible as enough number, medical records, contact tracing, and follow-up of the exposed patients is required. we experienced a unique situation in that discrete population of patients was exposed to the single index case in the defined space of ed, which made it possible to conduct the first cohort study to evaluate host susceptibility to mers-cov. of note, previously reported poor prognostic factors, including old age, male sex, concomitant infection, low albumin concentrations, lymphopenia, diabetes, and multiple comorbidities [3, 5, 6] , were not statistically significant in the multivariate analysis. these factors were evaluated in the present cohort with sufficient numbers, and the analysis indicated that they did not play critical roles in host susceptibility to mers-cov infection. this finding suggests that risk factors associated with poor prognosis and host susceptibility need to be separately considered. meticulous surveillance of mers-cov exposed patients should be conducted regardless of presence of poor prognosis factors. on the other hand, history of autologous sct showed statistical significance in the multivariate analysis. although susceptibility of autologous sct recipients to viral infections after engraftment has not been well evaluated, two of three autologous sct recipients (66.7%) in the present cohort were infected with mers-cov despite relatively low exposure intensity (at radiology room and zone iv). because all the autologous sct recipients in this cohort had underlying lymphoma and had multiple chemotherapy before sct, whether autologous sct per se or heavily-treated aggressive lymphoma would be a risk for increased host susceptibility for mers-cov infection is not conclusive. also, immunocompromising conditions generally associated with viral infections including hematologic malignancies other than lymphoma, allogeneic sct, and corticosteroid use were scarcely included in this cohort [7, 8] . therefore, it would be more relevant to interpret the present data that severely immunocompromised hosts could be more vulnerable to mers-cov infection and need special caution when exposed to the virus. interestingly, tachypnea at ed (hr, 4.392; 95% ci, 1.402e13.761; p ¼ 0.011) was associated with mers-cov infection. although association between respiratory rate and increased susceptibility to a certain respiratory virus has not been evaluated, theoretically patients with tachypnea may inhale more virus-containing droplets or aerosols. a recent study reported isolation of mers-cov from the air in mers outbreak units, suggesting possibility of air-borne transmission [9] . although this association needs to be further evaluated, patients with tachypnea at the time of exposure should to be monitored with caution, especially when the exposure occurs from severely-coughing mers patients at a restricted space. our study has several limitations. although we could not identify entire routes of exposed patients, we adjusted the exposure intensity by specific exposure sites. sizes, structure, crowdedness, exposure time from the index case were different between each site, which resulted in significantly different attack rates by exposure site [3] . in addition, we only evaluated a single exposure cohort, which could be biased by some special situation. in conclusion, in a cohort study from the single largest exposure event, history of autologous sct and tachypnea at ed were identified as host susceptibility factors to mers-cov infection. previously reported poor prognostic factors did not show association with increased host susceptibility to the virus. there are no potential conflicts of interest relevant to this article to report. middle east respiratory syndrome an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study an overview of infectious complications after allogeneic hematopoietic stem cell transplantation clinical presentation and risk factors for cytomegalovirus colitis in immunocompetent adult patients extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards we would like to express our sincerest condolences to the patients and families who suffered from the mers outbreak. we also greatly appreciate the health care personnel and staff members at samsung medical center and all other hospitals who worked together to overcome the mers outbreak. supplementary data related to this article can be found at https://doi.org/10.1016/j.jiac.2017.09.008. key: cord-313517-5ipj2z86 authors: fung, joshua; lau, susanna k. p.; woo, patrick c. y. title: antigen capture enzyme-linked immunosorbent assay for detecting middle east respiratory syndrome coronavirus in humans date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_7 sha: doc_id: 313517 cord_uid: 5ipj2z86 the middle east respiratory syndrome (mers) is the second novel zoonotic disease infecting humans caused by coronavirus (cov) in this century. to date, more than 2200 laboratory-confirmed human cases have been identified in 27 countries, and more than 800 mers-cov associated deaths have been reported since its outbreak in 2012. rapid laboratory diagnosis of mers-cov is the key to successful containment and prevention of the spread of infection. though the gold standard for diagnosing mers-cov infection in humans is still nucleic acid amplification test (naat) of the up-e region, an antigen capture enzyme-linked immunosorbent assay (elisa) could also be of use for early diagnosis in less developed locations. in the present method, a step-by-step guide to perform a mers-cov nucleocapsid protein (np) capture elisa using two np-specific monoclonal antibodies is provided for readers to develop their in-house workflow or diagnostic kit for clinical use and for mass-screening project of animals (e.g., dromedaries and bats) to better understand the spread and evolution of the virus. the middle east respiratory syndrome (mers) is the second novel zoonotic disease infecting humans caused by coronavirus (cov) in this century. to date, more than 2200 laboratory-confirmed human cases have been identified in 27 countries, and more than 800 mers-cov associated deaths have been reported since its outbreak in 2012 [1] . rapid laboratory diagnosis of mers-cov is the key to successful containment and prevention of the spread. nucleic acid amplification test (naat, e.g., real-time reverse transcription quantitative polymerase chain reaction [real-time rt-qpcr]), virus isolation, transmission electron microscopy, immunohistochemistry, and serological methods (e.g., antigen capture enzyme-linked immunosorbent assay [elisa] and immunofluorescence assay [ifa] ) have been developed and used for mers-cov diagnosis [2] [3] [4] [5] [6] [7] . while the "gold standard" for mers-cov diagnosis is naat of the upper region of the envelope gene (up-e) or the nucleocapsid (n) gene as suggested by the world health organization (who), antigen capture elisa assay for mers-cov can also be informative when naat is not available or when the serological assay is used to confirm the findings and aid treatment decision [2, 3] . further to diagnosing possible human infection of mers-cov, this method is also useful for screening the virus in the wildlife or agricultural applications. government agencies and research groups may find serological tests like antigen capture elisa to be more economical than naats for routine screening of mers-cov in farm-held or city-dwelling animals. the antigen capture elisa described in this method offers four significant advantages over traditional naats. firstly, serological screening requires less space in facilities and can be performed in point-of-care locations to minimize sample transporting and reduce turnover time. to avoid crosscontamination from amplicons in naats, the workflow usually requires four separate physical locations: (1) sample preparation (lysis, extraction of nucleic acids, and reverse transcription), (2) naat master mix preparation, (3) template addition, and (4) amplification and analysis. though technologies like real-time rt-qpcr simplify the workflow, such requirements limit the assay to be performed in regional laboratories designed or designated for this application. antigen capture elisa, on the other hand, can be performed on open bench in a single location after virus inactivation, allowing it to be performed in even the most minimally designed facility. secondly, antigen capture elisa can be performed with simple equipment and can be established with limited initial investment. for performing naats at a modern standard, uv cabinets or workstations for master mix preparation and sample addition, thermal cyclers, agarose gel running, and visualization equipment are the least requirement. for more stringent testing and faster turnaround, it calls for a real-time pcr thermal cycler (e.g., roche's lightcycler systems or bio-rad touch detection systems) which requires a fair amount of initial investment and limits the assay from being performed in remote or less developed locations. in contrast, antigen capture elisa and other serological methods can be performed with much simpler equipment. multichannel pipettes, automatic plate washer, and plate reader are the only specialized tools needed for this application and can be purchased with ease if those are not already available. thirdly, much less training is required for technicians to handle serological testing than naats. though naats and elisa are some of the most basic assays performed in a medical laboratory and minimal training is needed for an experienced worker to perform such task, to allow quicker and broader surveillance of mers-cov in human and animal population, it would be beneficial to set up more surveillance facilities in the less developed parts of the world. the time and resources needed to train a novice laboratory worker to perform elisa are much less, as only dilution and pipetting skills are required. fourthly, common nucleic staining chemicals used in naats for amplicon visualization are a possible mutagen and post potential health risk to workers and the surrounding environment; while chemicals and solutions used in elisa are relatively safer. to visualize the amplicons after agarose gel electrophoresis or during the qpcr thermal cycles, dyes like ethidium bromide (etbr), sybr green, or gel red are used; while etbr is a known mutagen, others are a relatively new addition to the market and extensive safety data is not widely available [8] . in comparison, the chemicals and solutions used in elisa are commonly found in clinical and research laboratories and are generally safe when used properly. finally, and most importantly, antigen capture elisa can offer high sensitivity and specificity for mers-cov diagnosis in even early infection and animal samples. we have previously demonstrated that by using two mers-cov nucleocapsid protein (np) specific monoclonal antibodies (mabs) in performing capture elisa, the test can accurately detect mers-cov virus down to 10 tcid 50 /0.1 ml and has a specificity of 100% [3] . as the nasopharyngeal aspirate viral load from patients during acute infection are around 10 6 copies/ml and nasal samples in dromedaries are usually around 10 4 -10 6 copies/ml, this test offers sufficient sensitivity for mer-cov diagnosis and screening [9] [10] [11] . other forms of mers-cov serological diagnostic test have also been developed based on different principles and are designed to fulfill different purposes, one should also review those options and evaluate their needs. to detect seroconversion from previous infection of mers-cov, the who suggests laboratories to perform ifa or elisa together with neutralization assay, the result alone can be used to determine if it is a confirmed case, regardless of the results from naat assay [2] . for rapid on-site diagnosis of mers-cov, we have previously reported the adaptation of the antigen capture elisa in the format of lateral flow immunoassay (lfia). this assay can yield results in under half an hour, requires minimal equipment, training, and can be stored at room temperature, thus allowing it to be performed in the field [12] . this lfia is also able to detect mers-cov-like viruses (e.g., tylonycteris bat cov hku4 and pipistrellus bat cov hku5) and is useful for the research to understand the evolutional history of mers-cov [13, 14] . in the current manuscript, the method for performing np capture elisa using two mers-cov-np-specific monoclonal antibodies (mabs) will be introduced. the general workflow of the assay is summarized in a figure for quick referencing [3, 15] (fig. 1 ). the antigen capture elisa is also known as sandwich elisa and makes use of a "capture" antibody and a "detection" antibody. the capture antibody is coated onto the wells of a microtiter plate before the assay. then following sample processing, the lysate is incubated in the wells of the microtiter plate. if the sample contains peptides from mers-cov (specifically nucleocapsid protein), they will bind with the coated antibody and be "captured" onto the microtiter plate. even minute amount of viral peptide can be retained in the well if the capture antibody has a high affinity to the peptide and was coated at high concentration. unbonded proteins are then washed away before the addition of the second, "detection" antibody. the secondary mab also recognizes the mers-cov np, presumably binds to a distinct epitope, and is conjugated with horseradish peroxidase for detection. the combination of two mabs in an elisa assay offers increased sensitivity for mers-cov np. on the other hand, this "sandwich" approach also allows improved specificity for the mers-cov nucleocapsid protein by combining the specificities of the two mabs, allowing it to differentiate and identify mers-cov spiked sample from other samples from healthy and patients who contracted various respiratory tract infections, as previously demonstrated [3] . in this assay the nucleocapsid protein was selected as the target for generating antibodies to detect mers-cov. according to previous experience when working with sars-cov, we observed that the np is a highly immunogenic and abundantly expressed structural protein, and a more preferable target than the spike (s) protein [16, 17] . working with the hypothesis that the np protein of mers-cov might also be a desirable target when developing an antigen capture elisa for it, we have shown that the assay offers high specificity and sensitivity, as mentioned above. the steps related to the cloning and purification of (his) 6 -tagged recombinant np (rnp) of mers-cov for the generation of anti-mers-cov-rnp mabs will not be described, as there are commercially available antibodies readily available for purchase. the horseradish peroxidase (hrp) system was used for the colorimetric visualization at the final stage of the assay. commercial elisa kits may utilize other detection methods; optimization may be needed. for readers who would like to generate their own hrp conjugated detection antibody, there are also kits available. when preparing solutions and buffers for the assay, investigators should be aware that "old" buffers may be more likely to be contaminated. the accuracy and reproducibility of the assay can be affected, as the peptides from fungus or other microorganisms may compete with the target antigen. prepare fresh solutions periodically (~1 month); autoclave or filter sterilize the buffers if available. if contaminations are a common occurrence, the addition of 0.05% sodium azide (nan 3 ) as a preservative is an option. microtiter plates with antibody 1. dilute the mers-cov-rnp mab 1f6 in blocking buffer. (see note 2). 2. coat the microtiter plates by adding 100 μl of the solution prepared per well. 3. cover the plate with an adhesive plastic cover and incubate at 37 c overnight (see note 3). 4. discard the adhesive plastic cover and remove the solution. 5. wash the plate with 300 μl of washing buffer per well for five times using an automatic microplate washer. 6. dry the plate by patting the plate on a paper towel. 7. allow the plate to air-dry. proceed to the next step or cover the plate with adhesive plastic cover and store at 4 c until use. all processes with potentially infectious mers-cov materials should be handled according to institutional, local, and international regulations, guidelines, and standard operating procedures (sop) to avoid spreading and contamination of the facility. all work with infectious mers-cov was performed inside a biosafety level-2 cabinet with sop in approved biosafety level-3 facilities during development and evaluation of the assay [3, 18, 19 ]. 1. aliquot 50 μl of viral lysis buffer to new 1.5 ml conical screw cap tubes according to the number of samples and controls (see note 4). 2. pipette 100 μl of specimen from the sample collection tube to the 1.5 ml conical screw cap tubes with viral lysis buffer, mix well. allow sufficient time for inactivation. 3. transfer the inactivated sample out of the biosafety cabinet to the general laboratory area, according to established sop. 4. there are many viral lysis buffers available for purchase from bio-reagents vendors, e.g., buffer al from qiagen. readers could request samples and perform their own testing on the conditions required to efficiently inactivate mers-cov. 5 . tmb solutions are normally purchased from bio-reagents vendors at ready-to-use dilations, follow manufacturer's instructions. world health organization: world health organization, 20 avenue appia laboratory testing for middle east respiratory syndrome coronavirus a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt are other fluorescent tags used instead of ethidium bromide safer clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia a highly specific rapid antigen detection assay for on-site diagnosis of mers rapid detection of mers coronavirus-like viruses in bats: pote1n-tial for tracking mers coronavirus transmission and animal origin genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus differential diagnosis of pandemic (h1n1) 2009 infection by detection of haemagglutinin with an enzyme-linked immunoassay sars coronavirus detection methods detection of severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in sars patients by enzyme-linked immunosorbent assay differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests 1. serially dilute the inactivated sample in sample dilution buffer, add 100 μl of the mixture into the wells in duplicates.2. gently shake the plate for 2 min to mix well, and then incubate at 37 c for 30 min while being covered with an adhesive plastic cover.3. discard the adhesive plastic cover and remove the solution.4. wash the plate with 300 μl of washing buffer per well for five times using an automatic microtiter plate washer. 1. dilute the secondary detection antibody (mab 7c4 conjugated with hrp) in enzyme dilution buffer immediately before use.2. add 100 μl of the diluted detection antibody to each well using an 8-channel pipette.3. cover the plate with an adhesive plastic cover and incubate at 37 c for 30 min.4. discard the adhesive plastic cover and remove the solution.5. wash the plate with 300 μl of washing buffer per well for five times using an automatic microtiter plate washer. 1. add 100 μl of 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb) substrate solution to each well (see note 5).2. cover the plate with aluminum foil to protect from light, incubate for 10 min at room temperature.3. add 50 μl of stop solution to each well to stop the reaction.4. read the plate using an automatic plate reader at wavelength 450 nm.5. analyze the data by using the predetermined cutoff value. 1. an automated microtiter plate washer-dispenser would be a good addition to the workflow as the washing steps can be performed in a shorter amount of time and with greater consistency. but multichannel or even single-channel pipettes can be used instead.2. the actual dilution of antibodies depends on the batch and quality of the mabs used. evaluations and characterization to determine the specificity and sensitivity have to be performed to establish the optimal dilution for the highest signal-to-noise ratio.3. prevent the microtiter plates to dry up by placing the plates in a box with some moist tissue paper laying under while storing in an incubator or on a rack in a warm water bath. key: cord-301313-9595vm0k authors: okba, nisreen m.a.; muller, marcel a; li, wentao; wang, chunyan; geurtsvankessel, corine h.; corman, victor m.; lamers, mart m.; sikkema, reina s.; de bruin, erwin; chandler, felicity d.; yazdanpanah, yazdan; le hingrat, quentin; descamps, diane; houhou-fidouh, nadhira; reusken, chantal b. e. m.; bosch, berend-jan; drosten, christian; koopmans, marion p.g.; haagmans, bart l. title: sars-cov-2 specific antibody responses in covid-19 patients date: 2020-03-20 journal: nan doi: 10.1101/2020.03.18.20038059 sha: doc_id: 301313 cord_uid: 9595vm0k a new coronavirus, sars-cov-2, has recently emerged to cause a human pandemic. whereas molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. validated serologic assays are important for contact tracing, identifying the viral reservoir and epidemiological studies. here, we developed serological assays for the detection of sars-cov-2 neutralizing, spikeand nucleocapsid-specific antibodies. using serum samples from patients with pcr-confirmed infections of sars-cov-2, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial elisas. we demonstrate that most pcr-confirmed sars-cov-2 infected individuals seroconverted, as revealed by sensitive and specific in-house elisas. we found that commercial s1 igg or iga elisas were of lower specificity while sensitivity varied between the two, with iga showing higher sensitivity. overall, the validated assays described here can be instrumental for the detection of sars-cov-2-specific antibodies for diagnostic, seroepidemiological and vaccine evaluation studies. in december 2019, a new coronavirus (cov) emerged in china to cause an acute respiratory disease known as coronavirus disease 19 (covid-19) (1) . the virus was identified to be a betacoronavirus related to severe acute respiratory syndrome coronavirus (sars-cov) and thus, was named sars-cov-2 (2) . in less than two decades, this virus is the third known coronavirus to cross the species barrier and cause severe respiratory infections in humans following sars-cov in 2003 and middle east respiratory syndrome in 2012, yet with unprecedented spread compared to the earlier two. due to the rapid rise in number of cases and uncontrolled and vast worldwide spread, the who has declared sars-cov-2 a pandemic. as of march 14th 2020, the virus has infected over 130,000 individuals in 122 countries, 3.7% of which had a fatal outcome (3) . the rapid identification of the etiology and the sharing of the genetic sequence of the virus, followed by international collaborative efforts initiated due to the emergence of sars-cov-2 have led to the rapid availability of real-time pcr diagnostic assays that support the case ascertainment and tracking of the outbreak (4) . the availability of these has helped in patient detection and efforts to contain the virus. however, specific and validated serologic assays are still lacking at the moment and are urgently needed to understand the epidemiology of sars-cov-2. validated serologic assays are crucial for patient contact tracing, identifying the viral reservoir hosts and for epidemiological studies. epidemiological studies are urgently needed to help uncover the burden of disease, in particular, the rate of asymptomatic infections, and to get better estimates on morbidity and mortality. additionally, these epidemiological studies can help reveal the extent of virus spread in households, communities and specific settings; which could help guide control measures serological assays are also needed for evaluation of the results of vaccine trials and development of therapeutic antibodies. among the four coronavirus structural proteins, the spike (s) and the nucleocapsid (n) are the main immunogens (5) . here, we describe development of serological assays for the detection of virus neutralizing antibodies and antibodies to the nucleocapsid (n) protein and various spike (s) domains including the s1 subunit, and receptor binding domain (rbd) of sars-cov-2 in elisa format. using a wellcharacterized cohort of serum samples from pcr-confirmed sars-cov-2 and patients pcr-confirmed to be infected with seasonal coronaviruses and other respiratory pathogens, we validated and tested various antigens in different platforms developed in-house as well as a commercial platform. serum samples were collected from pcr-confirmed mild and severe covid-19 patients ( table 1 ) from france in accordance with the local ethical approvals. samples used for assay validation were from persons pcr-diagnosed infections with of human coronaviruses (hcov-229e, nl63 or oc43), sars, mers, or with a range of other respiratory viruses ( table 1) as published previously (6) . samples from patients with recent cmv, ebv or mycoplasma pneumoniae infection were included as these have a higher likelihood of causing false positive results. we used serum samples from 45 healthy blood donors (cohort a) as negative controls; sanquin blood bank (rotterdam, the netherlands) obtained written informed consent for research use. all samples were stored at -20°c until use. the use of serum samples from the netherlands was approved by the local medical ethical committee (mec approval: 2014-414). serum samples from sars patients (7) were kindly provided by professor malik peiris, hong kong university. all serum samples from covid-19 pcr-confirmed cases ( n = 9) were previously analyzed by recombinant sars-cov-2 spike protein-based immunofluorescence test and plaque reduction neutralization (wölfel et al submitted manuscript, doi:https://doi.org/10.1101/2020.03.05.20030502). sera were tested as part of an extended diagnostic regimen upon informed written consent of patients. all non-sars-cov-2 sera (n=31) stemmed from the serum collection of the national consiliary laboratory for coronavirus detection at charité, berlin, germany and were provided for diagnostic proposes upon informed written consent. the collection contained follow-up sera from pcr-confirmed human cases: n=4 hcov-229e, n=3 hcov-hku1, n=7 hcov-oc43, n=3 mers-cov, n=6 hcov-nl63, n=3 sars-cov and n=6 common cold cov antibody positive sera. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the spike ectodomains of sars-cov-2 (residues 1-1,213; strain wuhan-hu-1; genbank: qhd43416.1), sars-cov (residues 1-1,182; strain cuhk-w1; genbank: aap13567.1) and mers-cov (residues 1-1262; strain emc; genbank: yp_009047204.1) were expressed in hek-293t cells with a c-terminal trimerization motif and strep-tag using the pcaggs expression plasmid. likewise, the sars-cov-2 s1 subunit or its subdomains (s;s1, residues 1-682; s1 a , residues 1-294; rbd, residues 329-538; genbank: qhd43416.1) were expressed in 293t cells as prnt was used as a reference for this study, because neutralization assays are the standard for cov serology. we tested serum samples for their neutralization capacity against sars-cov-2 (german isolate; gisaid id epi_isl 406862; european virus archive global # 026v-03883) by plaque-reduction neutralization test (prnt) as previously described for with some modifications (9) . heat-inactivated samples were 2-fold serially diluted in dmem medium supplemented with nahco3, hepes buffer, penicillin, streptomycin, and 1% fetal bovine serum, starting at a dilution of 1:10 in 50 μl. fifty μl of the virus suspension (400 spot forming units) was added to each well and incubated at 37°c for 1 h. following incubation, the mixtures were added on vero-e6 cells and incubated at 37°c for 1 more hour. the cells were then washed and further incubated in medium for 8 h. after the incubation, the cells were fixed and stained with polyclonal rabbit anti-sars-cov antibody (sino biological). the cells were then fixed and stained using a rabbit anti-sars-cov serum and a secondary peroxidase-labelled goat antirabbit igg (dako). the signal was developed using a precipitate forming tmb substrate (true blue, kpl) and the number of infected cells per well were counted using the immunospot® all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march 20, 2020. . https://doi.org/10.1101/2020.03.18.20038059 doi: medrxiv preprint image analyzer (ctl europe gmbh). the serum neutralization titer is the reciprocal of the highest dilution resulting in an infection reduction of >50% (prnt50). a titer of >20 was considered to be positive. the prnt for the german sera was done as described previously using vero e6 cells after 1 hour at 37°c the supernatants were discarded, the cells were washed once with pbs and supplemented with 1.2% avicel solution in dmem. after 3 days at plates were fixed and inactivated using a 6% formaldehyde/pbs solution and stained with crystal violet. we performed anti-sars-cov-2 igg and iga elisa using beta-versions of two commercial kits (euroimmun medizinische labordiagnostika ag, https://www.euroimmun.com) and performed the assay according to manufacturer's protocol. reagent wells of both assays are coated with recombinant structural protein (s1 domain) of sars-cov-2. the optical density (od) was detected at 450 nm, and a ratio of the reading of each sample to the reading of the calibrator, included in the kit, was calculated for each sample (od ratio). as the beta-version of the kit awaits ce validation, we determined an in-house cut-off value based on the mean background reactivity of all sars-cov-2-negative sera in the study multiplied by 3. in case of iga this was od ratio = 0.9 and for igg od ratio = 0.3. we performed the inhouse elisas by coating 96-well microtiter elisa plates with in-house produced spike antigens (s or s1 of sars-cov-2, sars-cov or mers-cov; or sars-cov-2 s1 a , or rbd proteins) or sars-n (sinobiological) in pbs overnight at 4°c. following blocking, diluted serum (1:100 or 2-fold serially diluted for titers) was added and incubated at 37°c for 1h. antigen-specific antibodies were detected using peroxidase-labeled rabbit antihuman igg (dako, https://www.agilent.com) and tmb as a substrate. the absorbance of each all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march 20, 2020. . https://doi.org/10.1101/2020.03.18.20038059 doi: medrxiv preprint sample was measured at 450 nm. we set cutoffs at 6 standard deviations above the mean value of the negative cohort. serum samples were previously tested for antibodies against the s1 of different coronaviruses as described earlier (6). the correlations between antibody responses detected by different elisas were and those detected by prnt, as the gold standard for cov serology, were analyzed using graphpad prism version 8 (https://www.graphpad.com). we evaluated sars-cov-2 specific antibody responses in severe and mild cases using serum samples collected at different times post-disease onset from three french pcr-confirmed covid-19 patients. we tested sera for sars-cov-2 specific antibodies using different elisas. following infections, all three patients seroconverted between days 13 and 21 post onset of disease (figure 1) , and antibodies were elicited against the sars-cov-2 s and s1 subunit including the n-terminal (s1 a ) domain and the receptor binding domain (rbd). since the n protein of sars-cov-2 is 90% similar to that of sars-cov ( table 2) , we used sars-cov n as an antigen to test for sars-cov-2 n-directed antibodies in an elisa format. we found that, following infection, antibodies were elicited against the n protein and when tested in a prnt assay these sera were able to neutralize sars-cov-2. we observed cross-reactivity with the sars-cov s and s1 proteins, and to a lower extent with mers-cov s protein, but not with the mers-cov s1 protein (figure 1 g-h) . this was evident from analyzing the degree of similarity of the different cov s protein domains to their corresponding sars-cov-2 proteins ( table 2) , where sars-cov showed high similarities in all different s domains. the analysis showed that the spike s2 subunit is more conserved among covs and thus plays a role in the cross-reactivity seen when the whole s was used as an antigen. thus, s1 is a more specific than s as an antigen for sars-cov-2 serological diagnosis. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. we further assessed the specificity of the s1 assay using cohorts a-e (table 1) comprising of sera from healthy blood donors (a), pcr-confirmed acute respiratory non-cov infections (b), acute to convalescent pcr-confirmed alpha-and beta-hcov infections (c), and pcr-confirmed mers-cov (d) and sars-cov (e) infections. none of the sera from specificity cohorts a-d were reactive in our in-house s1 elisa at the set cut-off indicating 100% specificity, whereas sera from sars-cov patients cross-reacted (figure 2a) . additionally, the specificity of s1 as an antigen for sars-cov-2 serology was further supported by the fact that 87-100 % of the cohort a-c sera included in this study were seropositive for the endemic hcovs (hcov-hku1, hcov-oc43, hcov-nl63, and -229e) as determined by the s1 protein microarray (figure 2b) . nonetheless, all were seronegative for sars-cov and mers-cov. using the same cohort, we also validated the specificity of the anti-nucleocapsid and anti-rbd igg elisas for detecting sars-cov-2 specific antibodies. at the set cut-off, except of sars-cov patient sera, none of the control sera tested positive for anti-rbd nor antinucleocapsid antibodies (figure 2 c,d) , whereas we detected seroconversion among the three covid-19 patients. these validated elisas for different antigens can be useful for epidemiological studies as well as for evaluation of vaccine-induced immune responses. next, we validated the sensitivity and specificity of 2 commercial elisa kits for detecting s1-specific igg and iga antibodies using the same cohort (table1, figure 3 ). all three covid-19 patients had reactive antibodies by both the igg (6/10 serum samples) and iga (7/10 serum samples) elisas (figure 3) . while sars-cov patient's sera were reactive as noted earlier, we also detected reactivity of serum samples from the validation cohorts a-d; 10/203 for iga and 7/203 for igg elisas. two hcov-oc43 (a β-cov) patients' sera were reactive in both igg and iga elisa kits. we confirmed the cross-reactivity of the two sera by testing twelve serum samples from both patients collected at different time points, pre-and post-oc43 infection. while all pre-infection sera were negative, all post-infection sera were reactive in both igg and iga based elisas. we have earlier reported cross-reactivity of these sera in a mers-cov s1 igg elisa kit (6) . further validation was also done in a different laboratory (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march 20, 2020. . https://doi.org/10.1101/2020.03.18.20038059 doi: medrxiv preprint nl63, 7x hcov-oc43) as well as mers-cov (n=3) and sars-cov (n=3) infected persons collected 4-56 days post disease onset (figure 4) . all 9 covid-19 patients were previously confirmed to seroconvert at days 6-15 post onset of disease using recombinant immunofluorescence test and prnt. 8/9 seroconverted patients showed reactivity above the implemented cut-off values in the igg and iga elisa. one patient (figure 4 , black line) maintained slightly below the cut-off which might be explained by an overall reduced antibody response of this patient (prnt90=10). overall, the iga-based elisa kit was more sensitive but less specific than the igg based elisa kit. finally, we compared the performance of the different elisas for the detection of antibodies among pcr-confirmed covid-19 patients to that of prnt, as the gold standard for cov serology ( table 3) . prnt50 correlated strongly with the different elisas, with the commercial iga showing the strongest correlation followed by the s and n elisas indicating their capacity to detect sars-cov-2 specific antibodies. however, a larger patient cohort is needed to assess the sensitivity of these platforms. validated sars-cov-2 serological assays are currently lacking, yet urgently needed for contact tracing, epidemiological and vaccine evaluation studies. since the n and the s proteins are the main immunogenic cov proteins, we developed elisa-based assays, which were able to detect antibodies to these two proteins along with the two spike domains, s1 a and rbd. those correlated strongly with virus neutralizing antibodies detected by a prnt50 assay. since the majority of the human population has antibodies against the four endemic human coronaviruses, it was crucial to verify the specificity of these assays to avoid false positive results. additionally, the two zoonotic coronaviruses, sars-cov and mers-cov, are also beta-coronaviruses, raising potential for cross-reactivity. among the spike protein antigens tested, the s1 was more specific than s in detecting sars-cov-2 antibodies, as mers-cov-s cross-reactive antibodies were detected in the serum of one of the covid-19 patients which was not seen when mers-cov s1 was used for testing. this could be explained by the high degree of conservation in the cov s2 subunit relative to the s1 ( table 2) . therefore, consistent with our earlier findings for mers-cov serology (6), s1 is a specific antigen for sars-cov-2 diagnostics. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. when testing the specificity s1 or its rbd for detecting sars-cov-2 antibodies, none of the sera form the validation cohorts (a-e) showed any reactivity; except for sars-cov patients sera. this -not-unexpected -cross-reactivity resulted from the high degree of similarity between the s1 and rbd of the sars-cov and sars-cov-2 ( table 2) . however, sars-cov has not circulated in the human population since 2003, i.e. 17 years ago, and an earlier study reported waning of sars-cov-specific antibodies which made them undetectable in serum samples of 91% (21/23) of samples tested 6 years following infection (11) . it is therefore unlikely that antibodies to this virus are present in the population and thus there could hardly be a chance that false positives result from sars-cov-antibodies reactivity. meanwhile, we made use of the high degree of similarity between the sars-cov and sars-cov-2 proteins for the development of our inhouse n elisa, where we used sars-cov n (90% similar to sars-cov-2) as an antigen. the n-elisa could detect sars-cov-2-specific antibodies with high specificity and sensitivity. using the three different validated elisas, we found that antibody levels were higher following the severe infection compared to the mild ones; similar findings has been reported earlier for mers-cov (12, 13) . however, this needs to be confirmed with a larger cohort of patients with varying degrees of severity, while it still highlights the potential need of a sensitive assay to avoid missing those with milder infections in epidemiological studies. among the 3 inhouse elisas tested, the rbd and n elisas were more sensitive than s1 elisa in detecting antibodies in mildly infected patients and showed stronger correlation with prnt50 titers. therefore, detecting antibodies against two different antigens might be needed to confirm the findings and avoid false negatives in surveillance studies. however, the sensitivities of the assays need to be further validated with a larger cohort. we further validated beta-versions of an iga and an igg s1 commercial elisa in two different labs. while the iga-based elisa showed higher sensitivity than the igg-based elisa, the opposite was true for the specificity where the igg elisa was more specific than the iga elisa. yet, we noted some cross reactivity in both elisas with serum samples from the same two hcov-oc43 patients that cross reacted in a mers-cov s1 igg elisa (6) despite the different antigen coated. this indicates a response to another protein which could be in the blocking or coating matrix, apart from the specific antigen coated, resulting in this consistent false positive result. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. overall, the assays developed and validated in this study could be instrumental for patient contact tracing, serosurveillance studies, as well as vaccine evaluation studies. however, since various studies will be carried out in different labs, it is crucial to calibrate and standardize assays developed by different labs using well defined standard references as a part of diagnostic assay validation. this is not only needed to reduce interassay variability, but to also harmonize the results obtained from different labs using various assays (14) . this is crucial for better comparison and interpertaion of results from different studies as well as evaluation of vaccine trials, allowing for uniform assessment of immunogenicity, efficacy and better understanding of correlates of immune protection (15) . thus, setting up reference panels is a vital element in our preparedness approaches to emerging viruses. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. protein sequences were aligned using clustalw. n, nucleocapsid; s, spike; s1,the n-terminal subunit of the spike protein; s2, the c-terminal subunit of the spike protein; s1a, domain a of the spike s1 subunit; rbd, receptor binding domain; nd, not done. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. p value summary *** **** ns, non-significant p > 0.05; * reflects significance level: **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001 all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march 20, 2020. . https://doi.org/10.1101/2020.03.18.20038059 doi: medrxiv preprint figure 1 : kinetics of antibody responses against sars-cov-2 following infection. we tested one severe (red) and two mild (green and black) sars-cov-2 patients for antibody responses against the (a) spike (s), (b) spike s1 subunit, (c) spike n-terminal (s1 a ) domain, (d) receptor binding domain, (e) nucleocapsid proteins using elisas. (f) virus neutralizing antibodies were tested using the plaque reduction neutralization assay (prnt). reactivities of sera form the three patients at different time points against whole spikes (g) and s1 (h) of sars-cov-2, sars-cov and mers-cov were tested by elisa. sars-cov, severe acute respiratory syndrome coronavirus; mers-cov, middle east respiratory syndrome coronavirus; all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. kinetics of antibody responses in three covid-19 patients (c,d). cross-reactivity of some hcov-oc43 sera in the commercial platforms (e,f). correlation between antibody responses detected by the elisas and the plaque reduction neutralization assay (prnt50; g,h). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . correlation between antibody responses detected by the elisas and the plaque reduction neutralization assay (prnt50; c,d). (e,f) the kits were tested for specificity using18 serum samples from hcov (4x hcov-229e, 3x hcov-hku1, 4x hcov-nl63, 7x hcov-oc43) as well as mers-cov (n=3) and sars-cov (n=3) infected persons. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted march 20, 2020. . https://doi.org/10.1101/2020.03.18.20038059 doi: medrxiv preprint a pneumonia outbreak associated with a new coronavirus of probable bat origin the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. nature microbiology coronavirus disease (covid-2019) situation reports detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr. euro surveillance : bulletin europeen sur les maladies transmissibles = european communicable disease bulletin serological assays for emerging coronaviruses: challenges and pitfalls. virus research sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections. emerging infectious diseases newly discovered coronavirus as the primary cause of severe acute respiratory syndrome dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc blocking transmission of middle east respiratory syndrome coronavirus (mers-cov) in llamas by vaccination with a recombinant spike protein. emerging microbes & infections transmission of merscoronavirus in household contacts. the new england journal of medicine lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study mers-cov antibody responses 1 year after symptom onset, south korea antibody response and disease severity in healthcare worker mers survivors. emerging infectious diseases comparison of serologic assays for middle east respiratory syndrome coronavirus. emerg infect dis international biological reference preparations for epidemic infectious diseases. emerg infect dis this work was supported by the zoonoses anticipation and preparedness initiative (zapi project; imi grant agreement no. 115760), with the assistance and financial support of imi and the european commission, in-kind contributions from efpia partners. key: cord-300950-ag0sql4i authors: lin, john; ouyang, jing; peng, xiao-rong; isnard, stéphane; fombuena, brandon; routy, jean-pierre; chen, yao-kai title: potential therapeutic options for coronavirus disease 2019: using knowledge of past outbreaks to guide future treatment date: 2020-06-05 journal: chin med j (engl) doi: 10.1097/cm9.0000000000000816 sha: doc_id: 300950 cord_uid: ag0sql4i nan in december 2019, initial cases of the novel coronavirus (2019-ncov) infection, termed coronavirus disease 2019 (covid19) , were first reported in wuhan, china. [1] in humans, infections with the human coronavirus 229e, oc43, nl63, and hku1 usually result in mild, selflimiting upper respiratory tract infections. however, other variants have rapid transmission rates and can cause severe respiratory syndrome and death. these variants include severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and the current 2019-ncov. these three coronaviruses (cov) share some similarities in their genomic, clinical, and pathologic features. cov comprise four genera (a-, b-, g-, and d-cov). these three viruses are all b-cov, with mers-cov belonging to lineage c and sars-cov and 2019-ncov belonging to lineage b. animal to human transmission is thought to be at the origin of these three viruses with bats as the possible natural reservoir. mers-cov uses dipeptidyl peptidase 4 as the primary entry receptor, which has been reported to be expressed in the human lower respiratory tract as well as kidney and t-cells but not in the upper respiratory tract. that may explain the relative lower transmission rate and increased mortality rate of mers-cov compared to sars-cov. on the contrary, 2019-ncov was predicted to use the same primary receptors angiotensin-converting enzyme 2 as sars-cov, which is widely expressed in the respiratory tract on epithelial cells and alveolar monocytes and macrophages. moreover, covid-19 mostly resem-bles sars with symptoms from fever, cough, myalgia, fatigue, to severe acute respiratory distress syndrome leading to death. this newly identified virus is spread through close contact, in particular through respiratory droplets from coughs and sneezes. compared with mers-cov and sars-cov, 2019-ncov is believed to have a higher transmission rate and lower mortality rate. a panel of experts from the world health organization (who) estimated that mortality is 2%, in contrast with >10% and >35% for sars and mers, respectively. [2] according to the current data, more than 90,000 people have been reported to be infected and more than 3000 deaths have been reported, much higher than the other two variants. [3] according to the who guidelines, supportive therapies are recommended to treat covid-19 symptoms, as specific treatments for covid-19 do not currently exist. however, direct treatments have been recommended and applied in clinics within china. these schemes include interferon (ifn)a, lopinavir/ritonavir, ribavirin, chloroquine phosphate, arbidol, and traditional chinese medicine. clinical experience showed some benefits from treatments such as chloroquine phosphate and convalescent-phase plasma from recovered patients, but there is no evidence to support the efficacy and safety of these therapies in a large clinical trial. efforts to investigate the history of treatments for similar outbreaks are necessary to extrapolate potential direct anti-viral therapies. since 2019-ncov shares phylogenetic traits with sars-cov and mers-cov despite being genetically distinct, anti-viral treatments that were used to target sars-cov and mers-cov may provide some insight into future 2019-ncov therapies. potential treatments are summarized in table 1 . several case reports including the first report of sars outbreak described the use of the anti-viral drug ribavirin and a corticosteroid in patients with contradictory clinical outcomes. lee et al [4] observed that most patients receiving ribavirin and the corticosteroid prednisolone had their fever and lung opacities resolved within 2 weeks. on the other hand, significant toxicity has been reported due to ribavirin in toronto, canada. the use of ribavirin with ifn was also described in mers-cov patients with no clinical improvement. furthermore, a retrospective cohort study showed that the group receiving ribavirin and ifn therapy had no difference in survival by 28 days compared to the untreated group. [5] thus, for both sars and mers, the use of ribavirin was not beneficial, and may subsequently not be useful for treatment of covid-19. the hiv anti-retroviral drug lopinavir/ritonavir has been used in sars and mers patients with beneficial effects. in several studies, lopinavir/ritonavir was shown to have anti-cov effects in vitro, in mers-infected primate models, and in sars-infected humans. indeed, in vitro, lopinavir/ ritonavir was shown to inhibit the sars-cov protease 3clpro. initial treatment with lopinavir/ritonavir in sars patients was associated with lower intubation rate, lower adverse clinical events and mortality compared to ribavirin and corticosteroid. furthermore, in a single mers patient, a triple-combination therapy of ribavirin, ifn and lopinavir/ ritonavir resolved viremia in 2 days following initiation of treatment. [6] currently, one placebo-controlled, doubleblind randomized controlled trial on the efficacy of lopinavir/ritonavir in mers (no. nct02845843) is ongoing. lopinavir/ritonavir use has also recently been reported for covid-19 treatment. [7] [8] [9] in two reports from china and korea, the use of lopinavir/ritonavir in patients with covid-19 improved recovery and reduced viral load. [7, 8] however, chen et al [9] showed that lopinavir/ ritonavir and the anti-influenza treatment arbidol had no clinically significant improvement in 134 people with mild covid-19. these conflicting results suggest that the use of lopinavir/ritonavir in the treatment of covid-19 may require more research as the outbreak progresses. remdesivir, a recently-described broad-spectrum anti-viral nucleotide analog, has shown potent activity against diverse rna viruses such as ebola virus, and of note, sars-cov and mers-cov. [10, 11] recently, sheahan et al [12] reported that combination remdesivir and ifn-b was superior to lopinavir/ritonavir in reducing lung viral load and restoring pulmonary function in a mers-infected mouse model. in light of the current outbreak, wang et al [13] described the in vitro inhibition of 2019-ncov by remdesivir at lower concentrations compared to ribavirin. holshue et al [14] described the use of intravenous remdesivir in the first covid-19 patient in the united states with no adverse effects and clinical improvement by the next day. these findings suggest that remdesivir may be effective at controlling viral infections at lower concentrations than existing treatments. future anti-viral therapies such as remdesivir may provide some much-needed firepower to combat epidemic coronavirus strains like 2019-ncov. optimistically, two clinical studies on anti-viral covid-19 treatment have recently come into fruition: the use of lopinavir/ritonavir in a phase 4, randomized controlled trial (no. nct04255017); and the use of remdesivir in a phase 3, randomized, double-blind, placebo-controlled study (no. nct04252664). rapid responses such as these may help guide treatment and improve clinical outcome for those affected by 2019-ncov. the natural progression of the host anti-viral response involves the triggering of the type-i ifn-a and -b, which play key roles in viral innate immunity. interestingly, both sars-cov and mers-cov evade or inhibit ifn-a and -b signaling, allowing uncontrolled viral replication and leading to progressive systemic inflammation. the use of exogenous ifn-a and -b in clinical isolates of sars-cov in cell lines showed that both ifns were effective at reducing viral replication, with ifn-b being 5 to 10 times more potent than ifn-a. [15] it was also demonstrated that pre-incubation of ifn-a and -b in cell lines reduced viral replication of clinical sars-cov isolates. further, in a mers-infected rhesus anti-viral anti-cov effects have been observed in sars and mers models. improvement seen in two cases of covid-19. anti-viral potential new therapeutic in covid-19 with beneficial effects seen in sars and mers models. administration in one patient in the us showed clinically significant improvement by the next day. host-targeted type-i ifns, particularly ifnremarkably, hart et al [16] reported that ifn-b alone showed more potent inhibition of mers-cov in vitro, compared to ifn-a-2a and ribavirin. these findings suggest that type-i ifns, in particular ifn-b, may be effective in a timedependent manner in reducing viral replication of sars-and mers-cov, and may be relevant for covid-19 treatment. pneumonia and lung inflammation are common clinical features in severe 2019-ncov, sars-cov, and mers-cov infections. as reported in the covid-19 outbreak, higher levels of pro-inflammatory markers were detected in intensive care unit (icu)-admitted patients compared to non-icu patients. [17] in an effort to combat inflammation and improve clinical outcome, corticosteroid use has been described in sars, mers, and covid-19. notably, huang et al [17] observed that all patients infected with 2019-ncov in their cohort had pneumonia and 22% were receiving corticosteroid treatment. however, the use of corticosteroid in coronavirus infections is controversial and may not benefit patients with coronavirus infections but may instead prolong infection and delay viral clearance. [18] as such, in the context of covid-19, the use of corticosteroids to control inflammation may not be beneficial and other options should be pursued. the incorporation of artificial intelligence into the immunology field has also highlighted potential drugs for covid-19. notably, the existing therapy baricitinib has been pinpointed as a potential host-targeted treatment. [19] baricitinib was predicted to prevent viral endocytosis in lung cells and could be combined with lopinavir/ritonavir or remdesivir. further implementation of new technologies will greatly improve the response to directly targeting new outbreaks such as covid-19. in the future, ongoing efforts to control transmission between humans such as preventing close contact, regular hygienic practices in public places, and widespread education on prevention should be continued. additionally, measures to improve healthcare infrastructure that can withstand the pressure of emerging zoonotic diseases should be implemented to further improve response times and limit transmission. a novel coronavirus from patients with pneumonia in china from sars to mers, thrusting coronaviruses into the spotlight coronavirus 2019-ncov global cases: johns hopkins csse a major outbreak of severe acute respiratory syndrome in hong kong ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen the course of clinical diagnosis and treatment of a case infected with coronavirus disease 2019 case of the index patient who caused tertiary transmission of covid-19 infection in korea: the application of lopinavir/ritonavir for the treatment of covid-19 infected pneumonia monitored by quantitative rt-pcr efficacies of lopinavir/ ritonavir and abidol in the treatment of novel coronavirus pneumonia (in chinese) broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel coronavirus in the united states treatment of sars with human interferons interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays clinical features of patients infected with 2019 novel coronavirus in wuhan, china corticosteroid therapy for critically ill patients with middle east respiratory syndrome baricitinib as potential treatment for 2019-ncov acute respiratory disease potential therapeutic options for coronavirus disease 2019: using knowledge of past outbreaks to guide future treatment none. key: cord-287156-3plpi6i9 authors: lassandro, giuseppe; palladino, valentina; amoruso, anna; palmieri, viviana valeria; russo, giovanna; giordano, paola title: children in coronaviruses’ wonderland: what clinicians need to know date: 2020-07-01 journal: mediterr j hematol infect dis doi: 10.4084/mjhid.2020.042 sha: doc_id: 287156 cord_uid: 3plpi6i9 human coronaviruses (hcovs) commonly cause mild upper-respiratory tract illnesses but can lead to more severe and diffusive diseases. a variety of signs and symptoms may be present, and infections can range in severity from the common cold and sore throat to more serious laryngeal or tracheal infections, bronchitis, and pneumonia. among the seven coronaviruses that affect humans (sars)-cov, the middle east respiratory syndrome (mers)-cov, and the most recent coronavirus disease 2019 (covid-19) represent potential life-threatening diseases worldwide. in adults, they may cause severe pneumonia that evolves in respiratory distress syndrome and multiorgan failure with a high mortality rate. children appear to be less susceptible to develop severe clinical disease and present usually with mild and aspecific symptoms similar to other respiratory infections typical of childhood. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. available data on pediatric coronavirus infections are rare and scattered in the literature. the purpose of this review is to provide to clinicians a complete and updated panel useful to recognize and characterize the broad spectrum of clinical manifestations of coronavirus infections in the pediatric age. structural proteins, including the spike (s), envelope (e), membrane (m), nucleocapsid (n), and sometimes a hemagglutinin-esterase protein (he). the he protein binds to specific receptors and guides membrane fusion; the s protein is responsible for cell entry, the m and e proteins mediate viral assembly process, the inner n protein develops ribonucleoprotein complexes binding to viral rna. [1] [2] [3] [4] [5] to date, seven coronaviruses affect humans: in 1960s hcov-229e and hcov-oc43 were firstly reported; 6, 7 hcov-nl63 and hcov-hku1 were discovered subsequently in 2004 and 2005, respectively. 8, 9 additionally, three hcovs responsible for outbreaks involving high case fatality rates have been detected in humans in the last two decades: the severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the new coronavirus disease 2019 (covid-19) ( table 1) . table 1 . principal features of severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the most recent coronavirus disease 2019 (covid19) . classification beta-cov beta-cov beta-cov in several studies a similar prevalence in the detection of hcovs in patients with respiratory symptoms compared to healthy children has been found. [36] [37] [38] [39] moreover, patients with other underlying medical conditions or immunocompromised appear more susceptible to developing severe infections than healthy patients. [40] [41] [42] [43] additionally, human coronaviruses are responsible for other common childhood diseases such as acute otitis media [44] [45] [46] [47] , asthma exacerbations 48, and conjunctivitis 8 . they have also been involved in nosocomial infections, especially in the neonatal intensive care units (nicu). gagneur et al. in a prospective study determined the incidence of hcovrelated respiratory infections in newborns hospitalized in a nicu. among 64 neonates, seven positive nasal samples for hcovs (11%) were detected. all children were symptomatic. oxygen and ventilatory support were frequently needed. 49 sizun et al. evaluated the clinical role of coronaviruses respiratory infections in premature newborns. all premature infants infected had severe respiratory symptoms, including bradycardia, apnea, and hypoxemia, while chest x-ray revealed diffuse infiltrates. 50 it has also been shown that coronavirus infections are not only responsible for respiratory symptoms but can also affect other organs and systems in children. several studies have also reported that respiratory symptoms caused by coronavirus infection may be associated with central nervous system (cns) involvement. hcovs have an intrinsic capacity to affect neurons and diffuse centrifugally from cns via the transneuronal route. 51, 52 among neurological symptoms, febrile seizures, convulsions, loss of consciousness, encephalomyelitis, and encephalitis have been reported. [53] [54] [55] primarily in 1980, the viral genome was detected post-mortem in the cerebrospinal fluid of two patients with multiple sclerosis (ms). 56 subsequently, the hcovs neuroinvasion capacity was confirmed in a large panel of human brain autopsy samples affected by ms and other neurological diseases. 57 58 in 2017, a prospective study on 192 children with febrile seizures demonstrated that coronaviruses were frequently detected. 59 additionally, hcovs have been implicated as possible causes of many gastrointestinal disorders in children, and gastrointestinal symptoms have been reported in several studies in more than 50% of pediatric patients 28, 60, 61 . firstly, hcovs could be associated with neonatal necrotizing enterocolitis 62 all hcovs can also be detected in stool samples of patients affected by gastroenteritis. 60, 66 moreover, most of the hcovs found were coinfections with well-known gastroenteric viruses, including norovirus and rotavirus. hcovs may also be found occasionally in healthy children's stool samples. 67 although hcovs have always been associated with respiratory symptoms, these findings suggest that other systems may also be involved in children. the absence of serious symptoms may not be coupled with serological negativity. therefore, these viruses should be considered in the differential diagnoses of most of the common diseases of childhood. the 2002-2004 severe acute respiratory syndrome outbreak was a viral respiratory illness caused by sars-cov. the outbreak firstly emerged in the southern chinese province of guangdong in november 2002 and 68 then spread to 29 countries with 8,096 people infected and 774 died. 69 the sars global outbreak was contained in july 2003. since 2004, there have not been any known cases of sars reported anywhere in the world. 70 probably, civet cats or bats could be the initial step of the transmission to humans. humans to humans infection occurs by respiratory droplets or direct contact. healthcare or household contacts are critical routes of transmission. 71, 72 sars-cov infection cases were classified by the world health organization (who) into suspected, probable, and confirmed ( table 2) . 73 the median incubation period ranges between 2-11 days. sars causes atypical pneumonia, which may progress to respiratory failure. symptoms include fever, malaise, myalgia, headache, diarrhea, and rigors. adults are more likely to develop severe illness characterized by dyspnea, lymphopenia, acute respiratory distress syndrome (ards), and a fatal clinical course in 10% of cases. the exact number of children affected by sars worldwide is unknown. however, children appear to be less susceptible to sars with a lower incidence of the disease and no reported mortality. the majority of children had documented exposure to adults with sars, usually a family member. most infected children had previously attended school, but the spread of the infection in the school environment has not been demonstrated, and this could probably be linked to lower infectiousness of the virus among children. 74, 75 children have less severe symptoms than adults, and they rarely need intensive care. however, subclinical and asymptomatic infections appear uncommon. most children reported worldwide were healthy, previously and underlying conditions were infrequently reported. [75] [76] [77] usually, children require hospitalization after 3-4 days the onset of symptoms: fever (90-100%), dry cough (43-80%), sore throat (5-30%), rhinorrhea (33-60%), malaise and myalgia (10-40%), headache (14-40%) are common. dyspnea, tachypnea, and febrile seizures are infrequent. aspecific gastrointestinal symptoms, including abdominal pain, appetite lack, vomiting, and diarrhea, have been reported. physical examination at presentation is negative in the majority of children, and chest auscultation does not reveal significant findings. moreover, sometimes crackles or signs of lung consolidation can be detected. as well as the clinical examination, laboratory findings are not specific in children with sars and can be confused with those of other respiratory infections typical of childhood. commonly lymphopenia, the elevation of transaminases, lactic dehydrogenase, and creatine phosphokinase are detected. other hematological abnormalities such as leukopenia, thrombocytopenia, the elevation of d-dimer levels and mildly prolonged activated partial thromboplastin times are also observed. [78] [79] [80] circulating interleukin (il)-1β levels might be increased, resulting in caspase-1-dependent pathway activation responsible for an exaggerated and persistent inflammatory response and the consequent respiratory failure in severe cases. 81 in children, radiological findings are nonspecific and similar to other viral respiratory abnormalities. high fever (>38 °c) and cough or breathing difficulty and one or more of the following exposures during the 10 days prior to onset of symptoms: close contact with a person who is a suspect or probable case of sars cough or breathing difficulty history of travel, to an area with recent local transmission of sars residing in an area with recent local transmission of sars 1) a suspect case with radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome (rds) on chest x-ray (cxr). 2) a suspect case of sars that is positive for sars coronavirus by one or more assays. see use of laboratory methods for sars diagnosis. 3) a suspect case with autopsy findings consistent with the pathology of rds without an identifiable cause. www.mjhid.org mediterr j hematol infect dis 2020; 12; e2020042 commonly, the chest x-ray shows ground-glass opacity or focal consolidation. linear atelectasis and peribronchial thickening have also been reported. computed tomography (ct) shows more extensive airspace consolidation and ground-glass attenuation than chest x-ray, but it is performed in selective cases in pediatric age. [78] [79] [80] 82 usually, the clinical course is less severe in children compared to adults, and few patients require oxygen supplementation and assisted ventilation but preterm newborns, children younger than one year and older than 12 years of age have more severe symptoms and are likely to develop respiratory distress. [78] [79] [80] in pediatric age, sars infection commonly has a "biphasic" pattern. the first stage of the disease is characterized by virus replication and clinically by the onset of symptoms. the second phase is characterized by pulmonary involvement, which is typically less severe in children than in adults. most children will become afebrile within seven days, and they usually do not progress to respiratory distress, the adult third phase, that is only reported in a minimal number of cases, commonly among teenagers. 83, 84 in pregnant women, sars infection is associated with a high incidence of spontaneous miscarriage, prematurity, and intrauterine growth retardation (iugr). the increased morbidities during pregnancy are likely to be due to the hypoxic state and circulatory insufficiency that worsen placental blood flow and cause miscarriage or iugr. significantly, among pregnant women, mortality is 25%. 85 however, perinatal sars infections have not been documented. in none infants born from pregnant women affected, real-time pcr (rt-pcr) assays and viral cultures conducted on neonatal blood, body secretions and amniotic fluid were positive for sars. in infants, no congenital malformations have been reported. however, in premature newborns, severe gastrointestinal complications such as jejunal perforation and necrotizing enterocolitis have been described 86 . however, it is not known if these neonatal morbidities are related to prematurity or if maternal infection is a factor that increases their incidence. it is unclear why children develop a less serious disease than adults. recurrent viral respiratory infections typical of the pediatric age could be helpful to the immune system in promptly recognizing and defeating new viral pathogens. furthermore, the immaturity of the immune system could be protective because the inflammatory cascade that causes respiratory failure in adults is more difficult to activate. additionally, children generally have fewer comorbidities than adults. children recovered quickly from sars. li et al. assessed the radiological and clinical outcomes of fortyseven children with sars after 6 months from diagnosis. all children were asymptomatic while mild pulmonary abnormalities including ground-glass opacities and air trappings were found at ct in sixteen patients. 87 although clinical and laboratory findings of sars are aspecific in children, certain features can be useful to distinguish sars from other respiratory viral infections. children with sars have a lower incidence of rhinorrhea and productive cough and higher incidence of monocytopenia than children with influenza. 88 additionally, serum lactate dehydrogenase in the presence of a low neutrophil count and low serum creatine phosphokinase could be suggestive of sars infection. 89 sars infections in children appear to be a relatively mild and aspecific disease, and the diagnosis should be accompanied by laboratory assessment. although infants and teenagers are more likely to have a worse clinical course, usually, all pediatric patients recover entirely without significant long-term sequelae. the middle east respiratory syndrome (mers) is a viral respiratory infection caused by the mers-coronavirus (mers-cov). the first identified case occurred in 2012 in saudi arabia. 11, 90 subsequently, a total of 2494 confirmed cases of mers, including 858 associated deaths with a case-fatality rate of 34% were reported globally; the majority of these cases were reported from arabian peninsula, and in the middle east. 91 currently, mers is an extremely rare disease: in the last year mers was signaled only in saudi arabia. 92 mers-cov is a zoonotic virus: dromedary camels are the primary reservoir hosts. humans are infected through contact with infected dromedary camels, animal products, or humans, especially among close contact between family members and health care workers. mers-cov infection cases were classified by the who into suspected, probable, and confirmed ( table 3) . 93 usually, the mean incubation period ranges from 2 to 15 days. clinical severity of the disease varies from asymptomatic to fatal forms, and the impact of asymptomatic spread is unclear. the infection can cause severe pneumonia, which may progress to ards, respiratory failure, and death, particularly in older people, immunocompromised patients, and those with chronic diseases. common symptoms include fever, cough, and shortness of breath. gastrointestinal symptoms (including diarrhea, vomiting, abdominal pain), pericarditis, septic shock and disseminated intravascular coagulation have been reported. [94] [95] [96] [97] children appear to be less susceptible to mers-cov infection, and pediatric cases described in the literature are rare with a low proportion (0.1%-4%) of infected children. 98 age hospitalized with acute respiratory symptoms and/or fever. among these, none of 474 children tested resulted positive for mers-cov. 103 in pediatric age, few cases of mers cov infection have been described. most of the children were asymptomatic and positive during routine screening of mers-cov. al-tawfiq et al. reported a total of 31 pediatric mers-cov cases with a mean age of 10 years. overall, 42% were asymptomatic, while in symptomatic cases, fever and mild respiratory symptoms were common. 104 subsequently, alfaraj et al. reported a total of 7 pediatric mers-cov cases with a mean age of 8 years. in this case series, common symptoms were fever (57%), cough (14%), shortness of breath (14%), and gastrointestinal symptoms (28%). two (28.6%) patients had abnormal chest radiographic findings with bilateral infiltration, one (14.3%) required ventilatory support, and two (28.6%) required supplemental oxygen. 99 four with underlying conditions (cystic fibrosis, nephrotic syndrome, craniopharyngioma, and a right ventricular tumor) had a fatal outcome. these children developed a critical form of mers infection complicated by respiratory and multiorgan failure. frequently, clinical examination revealed bilateral rhonchi and crackles while chest x-ray showed diffuse bilateral infiltrates, ground-glass opacification and pleural effusion. [105] [106] [107] [108] [109] thrombocytopenia, leukopenia, increased creatinine and prolonged prothrombin time were the only laboratory findings reported in literature. 99, 105, 106 mers-cov in children is less frequent than adults and appears to be associated with low mortality unless the patients have underlying comorbidities. few cases of mers-cov have been reported during pregnancy. a pregnant woman, aged 39 years, had a stillbirth at approximately five months of gestation 110 and another woman gave birth to a healthy term baby, but she died after delivery. 107 in conclusion, although mers-cov represents a clinical concern for the adult population with a high fatality rate, it remains a sporadic disease in childhood. clinicians should learn to recognize and suspect mers-cov infection, as the symptoms and signs are nonspecific, based on epidemiological criteria to avoid the spread of the disease in patients at higher risk of worse clinical course. the outbreak of covid-19 infection (coronavirus disease 2019; previously 2019-ncov) began in wuhan, hubei, china, in december 2019, which then spread rapidly to other provinces of china and around the world. 111 on january 30, 2020, the who declared the outbreak of a public health emergency of international concern and, on march 11, 2020, a pandemic. 112 as of june 5, 2020, 188 other countries and regions, with more than 6.669.358 confirmed cases, are declared. among the confirmed cases, 2.904.828 are recovered, and 393.205 died. 113 recent genetic analysis suggests the covid-19 emerged from an animal source. the full genome sequences showed high homology between covid-19, bat coronavirus, and pangolin coronavirus, but further genetic study is required. moreover, according to current evidence, the principal route of transmission of covid-19 is from human to human. 114, 115 covid-19 spread between people through respiratory droplets and contact routes. droplet transmission occurs when there is close contact with a person with respiratory symptoms such as coughing or sneezing, who may spread potentially infectious droplets. transmission may also occur by direct contact with infected persons and indirect contact with infected surfaces or objects. covid-19 can persist on inanimate objects for days but can be efficiently inactivated by common disinfectants. airborne transmission may be possible when a high risk of aerosolization procedures are performed, such as endotracheal intubation and bronchoscopy. the virus is also detected in stool specimens, and consequently, the feco-oral transmission is also hypothesized. [116] [117] [118] [119] the high transmissibility of covid-19 may be explained by its demonstrated presence in the upper respiratory tract of asymptomatic or presymptomatic subjects with viral loads comparable to those detected from symptomatic patients. the real proportion of asymptomatic cases is unclear, ranging from 1% to 78% in different studies. transmission from asymptomatic patients infected with covid-19 most likely contributed to the rapid and extensive spread of pandemic but further studies are needed to more accurately estimate the proportion of genuinely asymptomatic cases and their risk of transmission. [120] [121] [122] [123] [124] [125] [126] covid-19 has been reported among all age groups. the median incubation period of covid-19 infection is 4-5 days with a range up to 24 days. 119, 127 covid-19 infection case is classified by the who into suspected, probable, and confirmed ( table 4) . 128 clinical severity of the infection varies, ranging from asymptomatic forms to critical diseases. common symptoms are fever, dry cough, malaise, lethargy, shortness of breath, sore throat, and myalgia. headache, conjunctivitis, productive cough, and diarrhea are also described. mild forms present as a common cold, and severe cases may worsen in pneumonia that may evolve to ards, shock, and multiple organ dysfunction. more severe clinical pictures are associated with stronger immune response and with the production of proinflammatory cytokines, including il-2, il-7, il-10, and tumor necrosis factor-α (tnf-α). adverse outcomes are common in elderly patients and those with underlying diseases. the need for intensive care admission is in 25-30% of patients. the fatality rate is estimated to range between 2 and 3%. [129] [130] [131] [132] [133] about 2% of covid-19 confirmed cases are children. [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] 135 generally, children appear to be less likely to develop a severe form of covid-19 infection, and commonly they have a mild clinical course with a good prognosis. few children may evolve into lower respiratory infections. probable reasons include having an immune system still immature, healthier respiratory tract, and less underlying conditions than adults. 136 most of them have an infected contact history with family members. moreover, children, especially those with asymptomatic or milder form, may represent significant spreaders. pediatric patients appear to be likely as adults to become infected but are less likely to develop symptoms. however, future studies are needed to understand the role of children in the transmission of the virus. [137] [138] [139] current researches show that the median age of infection in pediatric cases is 6-7 years. in symptomatic cases, symptoms are typical of acute respiratory infections and frequently included fever (59%) and cough (46%), which may be accompanied by nasal congestion, runny nose, conjunctivitis, pharyngitis, wheezing, myalgia, and expectoration. few children have an atypical presentation with gastrointestinal manifestations, including nausea, vomiting, and diarrhea. low oxygen saturation of less than 92%, dyspnea, cyanosis, and poor feeding, are less common than adults. among infants, symptoms such as irritability, reduced response, and poor feeding could be the main signs of infection. family clustering occurred for all infected infants. rarely infants require intensive care or mechanical ventilation or have any severe complications. common symptoms of pediatric age are summarized in figure 1 . the majority of children recovers 1-2 weeks after the onset of the disease. regarding biochemical results, leukopenia and lymphopenia are frequent in children. elevation of transaminases, myoglobin, muscle enzymes, and d-dimers might be seen in severe cases. [140] [141] [142] [143] [144] [145] [146] dong et al. reported that 94% of 2143 pediatric patients affected by covid-19 developed an asymptomatic, mild, or moderate form of infection. a severe disease characterized by dyspnea, central cyanosis, and oxygen saturation of less than 92% was reported in 5% of cases. a critical disease characterized by ards and multiple organs failure was reported in less than 1% of cases. 141 the prevalence of severe and critical disease appears higher in younger children, particularly in children aged <1-year-old and in children with underlying diseases. to date, death was an uncommon event reported in one 10month-old infant with intussusception and multiorgan failure and in one 14-year-old boy. 145, 147 other systemic symptoms appear to be related to the infection, but their link has not yet been demonstrated. since the outbreak of the pandemic, a large number of rashes, urticaria, and vasculitis affecting hands and feet of healthy children and adolescents have been reported as well as itching, burning, difficulty in joint movements and pain. 142 recently, the relationship between covid-19 infection and the development of cardiac diseases in children has been hypothesized. belhadjer et al. have reported a large number of febrile children resulted positive for covid-19 admitted in intensive care units for acute heart failure associated with a multisystem inflammatory state. in most of the children, clinical features appeared similar to those of kawasaki syndrome: lasting fever, cutaneous rash, lymphadenopathy, persistent activation of systemic inflammation and positive response to intravenous immunoglobulin. 148 similar clinical features have subsequently been reported in children with covid-19 positive serology. 149, 150 as in covid-19 infection, kawasaki syndrome is triggered by proinflammatory cascade activated primarily by innate immunity response. however, further studies are needed to establish the real pathogenetic relationship between emerging covid-19 and kawasaki-like syndromes. 151 dufort et al. 152 have recently reported the emergence of a multisystem inflammatory syndrome in children in new york state coincidental with widespread sars-cov-2 transmission, which can better clarify the relationship between kawasaki disease and covid-19. among 191 children admitted to the new york hospitals for multisystem inflammatory syndrome in children (mis-c), 95 patients had a laboratory-confirmed acute or recent severe acute respiratory syndrome coronavirus 2 [sars-cov-2] infection. this hyperinflammatory syndrome manifested with dermatologic, mucocutaneous, and gastrointestinal features associated with cardiac dysfunction. of these 95 patients, a total of 36 patients (37%) received a diagnosis of kawasaki's disease or atypical (or incomplete) kawasaki's disease; 7 of the 9 patients with coronary-artery aneurysms also received a diagnosis of kawasaki's disease. 152 covid-19 infection may also trigger the onset of other immune-mediated diseases such as immune thrombocytopenia, [153] [154] [155] [156] evans syndrome, 157 and autoimmune hemolytic anemia. 158 among radiological findings, ground-glass opacity, mono or bilateral infiltrates, mesh shadows, and tiny nodules are frequently detected. in severe cases, radiological alterations are diffused, presenting as a "white lung." however, radiologic evidence of pneumonia might be absent in 15-20% of children. 139, 140, [159] [160] [161] [162] [163] in selected cases, lung ultrasound might be useful in the managing and follow-up of covid-19 infection. this radiological technique can precociously identify abnormalities including small pleural effusion and subpleural consolidation and appear more available then x-ray and ct. [164] [165] clinical examination appears mostly negative for pulmonary signs, and in rare cases, rales and thoracic retractions have been reported. 161 whether pregnant women and children born to affected mothers are more likely to have a worse outcome is currently unclear. maternal-infant vertical transmission has not been documented. amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples from newborns delivered by infected women were tested for covid-19, and all samples tested negative. 166 data on the maternal and perinatal outcomes of pregnant women infected with covid-19 is limited. most pregnant women with covid-19 present with fever and coughing. severe and critical maternal symptomatology have also been reported, but no women died. the most common adverse pregnancy outcome is preterm birth, occurring in 41% of cases while the rate of perinatal death is 7%, including one case of stillbirth and one neonatal death. there is no data on miscarriage for covid-19 occurring during the first trimester. in more than a third of cases, fetal distress and frequent admission neonatal intensive care units have been reported. 166, 167 rarely, cases of covid-19 positivity in newborns have been reported. common symptoms are fever, cough, lethargy, and vomiting milk. mottled skin and moderate respiratory distress presented with tachycardia, tachypnoea, subcostal retractions, and low oxygen saturation are also described in newborn babies. [169] [170] [171] [172] [173] although it can be severe in some cases, compared with sars-cov and mers-cov, covid-19 causes less severe disease in children. a recent meta-analysis shows that children infected with covid-19 have less fever than that other epidemic hcovs. 174 despite the rapid worldwide spread of covid-19 infection, additional data are needed to define the severity of the disease in children. the severity of the symptoms and the mortality rate will be better assessed in the future. differential diagnosis with common viral respiratory infections of childhood, such as influenza virus, adenovirus, respiratory syncytial virus, and metapneumovirus, should be considered. in the diagnosis of suspected cases, epidemiological and clinical criteria must be assessed. 73, 93, 138 rt-pcr represents the gold standard to confirm the diagnosis of hcovs infections performed on samples of respiratory secretions. [175] [176] [177] [178] [179] [180] [181] the viral load is higher in lower respiratory tract secretion samples than in upper respiratory tract samples. therefore, suspected cases resulted in firstly negative could be re-tested with a second swab, better if with a low respiratory sampling is performed as proved for sars and mers infection. 182, 183 currently, few data have been published about the sensitivity and specificity of rt-pcr nasopharyngeal swabs for covid-19. in vitro analyses suggest that the rt-pcr test is highly specific and sensitive. 184 in vivo, sensitivity is estimated to be higher than 70% but seems to be lower for "mild" cases while specificity is close to 100%. 185, 186 accuracy of rt-pcr swabs in clinical practice differs depending on the site and quality of the sample. taking swabs from children may be more difficult given the intrusive nature of the procedure and further reduce the specificity and sensitivity of the test. rt-pcr of bronchoalveolar lavage fluid appears the most accurate technique of virologic confirmation, but it may not always be easily collected in all patients, especially in pediatric age. although a negative test cannot currently rule out the disease, further studies are needed to define the exact specificity and sensitivity of rt-pcr nasopharyngeal swabs. 187, 188 moreover, rt-pcr appears to be useful in virus detection on stool samples. 116 to date, serology is not considered a diagnostic method. although most patients with covid-19 appear positive for immunoglobulin-g (igg) within 19 days while igm reaches a peak 20-22 days after symptom onset, the serological response is not useful for early individuation of positive patients. 189 additionally, numerous cross-reactions occur between covid-19 and common hcovs 190, and protective immunity against covid-19 is not proved. despite its potential role in supporting rt-pcr in the diagnosis of covid-19, the clinical and immunological meaning of serology is still unclear. 191 the spread of the infection can be prevented if children and family members were educated about proper hygienic practices and infection control measures, including regular hand washing, cover the mouth with napkin or towel when coughing or sneezing, avoid crowded places and contact with sick people. children with hcovs should receive early supportive therapy and continuous monitoring. additional oxygen, caloric, and hydro electrolytic support should be performed if necessary. frequent checks of oxygen saturation and hematological, urinary, and biochemical parameters, including liver, kidney, myocardial enzymes, and coagulation parameters should be analyzed. finally, blood gas analysis and radiological diagnostics of the chest should be done when necessary. this strategy could be useful in the prevention of ards, multiorgan failure, and other nosocomial infections possibly treated, if bacterial, with appropriate antibiotics. in critical cases, mechanical ventilation with endotracheal intubation and other more invasive interventions, such as blood purification and extracorporeal membrane oxygenation (emco), should be adopted. additionally, the use of antiviral drugs in children with severe hcovs infections may help to reduce viral load and the duration of symptoms. however, their safety and real effectiveness have not yet been proven. interferon alfa and beta, corticosteroids, lopinavir/ritonavir, and ribavirin, were used in the treatment of sars-cov and mers-cov in adults and children. 75, 76, 78, 192 however, ribavirin can cause hemolytic anemia and liver dysfunction, as well as corticosteroids, increase the risk of iatrogenic immune immunosuppression. 193 to date, there is no evidence regarding the management and treatment of covid-19 infection in children. in addition to supportive therapy, the use of nebulized interferonalpha-2b and oral lopinavir/ritonavir together with corticosteroids for complications and hydroxychloroquine or intravenous immunoglobulin for severe cases have been suggested. 145, 194, recently, a position paper of the italian society of pediatric infectious disease on the treatment of children with covid-19 infection has been published. 195 in asymptomatic or mild cases, only antipyretic therapy is recommended. in severe or critical cases, the use of hydroxychloroquine ± azithromycin or lopinavir/ritonavir must be considered. immunomodulating therapy with methylprednisolone or tocilizumab or anakinra must be considered in case of the simultaneous presence of ards or progressive deterioration of respiratory function, the elevation of proinflammatory biomarkers and an interval of at least seven days from symptoms onset. supportive therapy should include antipyretic therapy, inhalation therapy with topical steroids and/or bronchodilators and venous thromboembolism prophylaxis therapy. [196] [197] [198] [199] [200] [201] [202] [203] discharge from the hospital is recommended when the patient is without fever for almost three days, respiratory symptoms have improved, and rt-pcr samples are negative. 195 conclusions. most cases of hcovs infection in children have clinically mild symptoms and a relatively short time to resolution. children seem to have a better prognosis compared to adults, and death is a sporadic event. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. learning to recognize pediatric clinical presentations often indefinite or similar to other typical infections of this age, allows clinicians to perform a correct and early diagnosis and prevent the spread of infections in the general population. furthermore, the psychological and social impact of the pandemic outbreak should be considered, especially in the pediatric age. moreover, we think it is necessary to implement innovative clinical tools, such as narrative medicine, to recognize the burden of disease in children and caregivers. [201] [202] [203] [204] references: update on human rhinovirus and coronavirus infections hosts and sources of endemic human coronaviruses mechanisms of coronavirus cell entry mediated by the viral spike protein discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus discovery of a novel coronavirus, china rattus coronavirus hku24, from norway rats supports the murine origin of betacoronavirus 1 and has implications for the ancestor of betacoronavirus lineage a a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronaviruses: a paradigm of new emerging zoonotic diseases incubation periods of acute respiratory viral infections: a systematic review epidemiological and clinical features of human coronavirus infections among different subsets of patients. influenza other respir viruses epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of hcov-oc43 during 2010-2015 in guangzhou human coronavirus infections in israel: epidemiology, clinical symptoms and summer seasonality of hcov-hku1. viruses the role of infections and coinfections with newly identified and emerging respiratory viruses in children species-specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults. influenza other respir viruses viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection comparison of automated microarray detection with real-time pcr assays for detection of respiratory viruses in specimens obtained from children comparison of viral and epidemiological profiles of hospitalized children with severe acute respiratory infection in beijing and shanghai, china two-year prospective study of single infections and coinfections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong, china phylogenetic analysis of human coronavirus nl63 circulating in italy human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays epidemiology of human coronavirus nl63 infection among hospitalized patients with pneumonia in taiwan an outbreak of coronavirus oc43 respiratory infection in epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method human coronaviruses and other respiratory infections in young adults on a university campus: prevalence, symptoms, and shedding. influenza other respir viruses respiratory viruses associated with severe pneumonia in children under 2 years old in a rural community in pakistan croup is associated with the novel coronavirus nl63. version 2 croup is associated with the novel coronavirus nl63. version 2 detection of human coronavirus nl63 in young children with bronchiolitis coronavirus hku1 in an italian pre-term infant with bronchiolitis erdman dd; new vaccine surveillance network. human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls viral respiratory infections in hospitalized and community control children in alaska role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients coronavirus 229e-related pneumonia in immunocompromised patients acute respiratory viral infections in pediatric cancer patients undergoing chemotherapy a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients acute otitis media and respiratory viruses etiology of acute otitis media and characterization of pneumococcal isolates after introduction of 13-valent pneumococcal conjugate vaccine in japanese children genetic characteristics and antibiotic resistance of haemophilus influenzae isolates from pediatric patients with acute otitis media after introduction of 13-valent pneumococcal conjugate vaccine in japan polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion regional, age and respiratorysecretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review coronavirusrelated nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study neonatal nosocomial respiratory infection with coronavirus: a prospective study in a neonatal intensive care unit effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome possible central nervous system infection by sars coronavirus detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients neuroinvasion by human respiratory coronaviruses coronavirus infections in the central nervous system and respiratory tract show distinct features in hospitalized children respiratory and enteric virus detection in children human coronaviruses are uncommon in patients with gastrointestinal illness detection of the new human coronavirus hku1: a report of 6 cases association of coronavirus infection with neonatal necrotizing enterocolitis human coronavirus nl63, france impact of human coronavirus infections in otherwise healthy children who attended an emergency department new vaccine surveillance network. coronavirus infection and hospitalizations for acute respiratory illness in young children the role of human coronaviruses in children hospitalized for acute bronchiolitis, acute gastroenteritis, and febrile seizures: a 2-year prospective study detection of human coronaviruses in children with acute gastroenteritis comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection summary of probable sars cases with onset of illness from coronavirus as a possible cause of severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome [internet]. world health organization (who) epidemiology of severe acute respiratory syndrome (sars): adults and children clinical presentations and outcome of severe acute respiratory syndrome in children other members of the hospital for sick children sars investigation team. children hospitalized with severe acute respiratory syndromerelated illness in toronto risk-stratified seroprevalence of sars coronavirus in children residing in a district with point-source outbreak compared to a low-risk area severe acute respiratory syndrome in children: experience in a regional hospital in hong kong severe acute respiratory syndrome among children severe acute respiratory syndrome-associated coronavirus infection in toronto children: a second look inflammatory cytokine profile in children with severe acute respiratory syndrome severe acute respiratory syndrome (sars): chest radiographic features in children serial analysis of the plasma concentration of sars coronavirus rna in pediatric patients with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study pregnancy and perinatal outcomes of women with severe acute respiratory syndrome infants born to mothers with severe acute respiratory syndrome long-term sequelae of sars in children childhood severe acute respiratory syndrome in taiwan and how to differentiate it from childhood influenza infection a case-control study of sars versus community acquired pneumonia middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) available at: www.emro.who.int/pandemic-epidemic-diseases/mers-cov/merssituation-update middle east respiratory syndrome coronavirus (mers-cov). case definition ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission acute viral respiratory infections among children in mers-endemic riyadh, saudi arabia, 2012-2013 middle east respiratory syndrome coronavirus in pediatrics: a report of seven cases from saudi arabia el hadi mohamed ra. outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study correction: case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from 2012 to 2015 surveillance and testing for middle east respiratory syndrome coronavirus, saudi arabia middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus during pregnancy acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus stillbirth during infection with middle east respiratory syndrome coronavirus china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china second-meeting-of-the-international-health-regulations-(2005)-emergencycommittee-regarding-the-outbreak-of-novel-coronavirus-(2019-ncov) coronavirus covid-19 global cases by the center for systems science and engineering covid-19 infection: origin, transmission, and characteristics of human coronaviruses probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding community transmission of severe acute respiratory syndrome coronavirus 2 modes of transmission of virus causing covid-19: implications for ipc precaution recommendations persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study public health-seattle and king county and cdc covid-19 investigation team. presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths sars-cov-2 viral load in upper respiratory specimens of infected patients characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) the covid-19 task force of the department of infectious diseases and the it service istituto superiore di sanità. integrated surveillance of covid-19 in italy early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia global surveillance for covid-19 caused by human infection with covid-19 virus. case definition zhong ns; china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. 2020:e201585 clinical features of patients infected with 2019 novel coronavirus in wuhan clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series mmwr morb mortal wkly rep are children less susceptible to covid-19? epidemiology and clinical features of coronavirus disease 2019 in children infection in children epidemiology and transmission of covid-19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts sars-cov-2 infection in children: transmission dynamics and clinical characteristics epidemiology of covid-19 among children in china. pediatrics. 2020:e20200702 a new vasculitis at the time of covid-19 a case series of children with 2019 novel coronavirus infection: clinical and epidemiological features diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus novel coronavirus infection in hospitalized infants under 1 year of age in china corona virus disease 2019, a growing threat to children? a 55-day-old female infant infected with covid 19: presenting with pneumonia, liver injury, and heart damage acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov-2 pandemic multisystem inflammatory syndrome in children during the covid-19 pandemic: a case series features of covid-19 post-infectious cytokine release syndrome in children presenting to the emergency department sars-cov-2, which induces covid-19, causes kawasaki-like disease in children: role of proinflammatory and anti-inflammatory cytokines prevention multisystem inflammatory syndrome in children investigation team. multisystem inflammatory syndrome in children in covid-19-associated immune thrombocytopenia immune thrombocytopenia (itp) in a sars-cov-2 positive pediatric patient covid-19 and children with immune thrombocytopenia: emerging issues evans syndrome in a patient with covid-19 covid-19 infection associated with autoimmune hemolytic anemia chest computed tomography in children with covid-19 respiratory infection clinical and ct imaging features of the covid-19 pneumonia: focus on pregnant women and children clinical and ct features in pediatric patients with covid-19 infection: different points from adults ct features of novel coronavirus pneumonia (covid-19) in children radiographic and clinical features of children with 2019 novel coronavirus (covid-19) pneumonia lung ultrasound in the monitoring of covid-19 infection lung ultrasound can influence the clinical treatment of pregnant women with covid-19 clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records maternal and perinatal outcomes with covid-19: a systematic review of 108 pregnancies outcome of coronavirus spectrum infections (sars, mers, covid 1 -19) during pregnancy: a systematic review and meta-analysis perinatal transmission of covid-19 associated sars-cov-2: should we worry? clin infect dis novel coronavirus in a 15-dayold neonate with clinical signs of sepsis, a case report a case report of neonatal covid-19 infection in china clinical characteristics and diagnostic challenges of pediatric covid-19: a systematic review and metaanalysis direct diagnosis of human respiratory coronaviruses 229e and oc43 by the polymerase chain reaction viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome genomic sequencing of the severe acute respiratory syndrome-coronavirus genomic sequencing of a sars coronavirus isolate that predated the metropole hotel case www.mjhid.org evaluation of a real-time reverse transcription-pcr (rt-pcr) assay for detection of middle east respiratory syndrome coronavirus (mers-cov) in clinical samples from an outbreak in south korea in 2015 analytical and clinical validation of six commercial middle east respiratory syndrome coronavirus rna detection kits based on real-time reverse-transcription pcr mers-cov diagnosis: an update respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome viral loads in clinical specimens and sars manifestations detection of 2019 novel coronavirus (2019-ncov) by realtime rt-pcr application and optimization of rt-pcr in diagnosis of sars-cov-2 infection false-negative results of initial rt-pcr assays for covid-19: a systematic review the appropriate use of testing for covid-19 interpreting a covid-19 test result antibody responses to sars-cov-2 in patients with covid-19 antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 sars-cov-2 antibody testing -questions to be asked ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study sars: systematic review of treatment effects. version 2 national health commission of people's republic of china. diagnosis and treatment of pneumonia caused by novel coronavirus china national clinical research center for respiratory diseases chinese medical doctor association committee on respirology pediatrics; china medicine education association committee on pediatrics; chinese research hospital association committee on pediatrics; chinese non-government medical institutions association committee on pediatrics; china association of traditional chinese medicine committee on children's safety medication; global pediatric pulmonology alliance. diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement treatment of children with covid-19: position paper of the italian society of pediatric infectious disease covid-19: ibuprofen should not be used for managing symptoms, say doctors and scientists ema gives advice on the use of nonsteroidal anti-inflammatories for covid-19 recommendations for inhaled asthma controller medications anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy discovering drugs to treat coronavirus disease 2019 (covid-19) delle fave a. perceived well-being and mental health in haemophilia a narrative approach to describe qol in children with chronic itp. front pediatr itp-qol questionnaire for children with immune thrombocytopenia: italian version validation's key: cord-306923-eujbxdqi authors: ahmed, anwar e.; al‐jahdali, hamdan; alaqeel, mody; siddiq, salma s.; alsaab, hanan a.; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; ali, yosra z.; al otaibi, hazza; aloudah, nouf m.; baharoon, salim; al johani, sameera; alghamdi, mohammed g. title: factors associated with recovery delay in a sample of patients diagnosed by mers‐cov rrt‐pcr: a saudi arabian multicenter retrospective study date: 2018-04-25 journal: influenza other respir viruses doi: 10.1111/irv.12560 sha: doc_id: 306923 cord_uid: eujbxdqi background: research evidence exists that poor prognosis is common in middle east respiratory syndrome coronavirus (mers‐cov) patients. objectives: this study estimates recovery delay intervals and identifies associated factors in a sample of saudi arabian patients admitted for suspected mers‐cov and diagnosed by rrt‐pcr assay. methods: a multicenter retrospective study was conducted on 829 patients admitted between september 2012 and june 2016 and diagnosed by rrt‐pcr procedures to have mers‐cov and non‐mers‐cov infection in which 396 achieved recovery. detailed medical charts were reviewed for each patient who achieved recovery. time intervals in days were calculated from presentation to the initial rrt‐pcr diagnosis (diagnosis delay) and from the initial rrt‐pcr diagnosis to recovery (recovery delay). results: the median recovery delay in our sample was 5 days. according to the multivariate negative binomial model, elderly (age ≥ 65), mers‐cov infection, icu admission, and abnormal radiology findings were associated with longer recovery delay (adjusted relative risk (arr): 1.741, 2.138, 2.048, and 1.473, respectively). camel contact and the presence of respiratory symptoms at presentation were associated with a shorter recovery delay (expedited recovery) (arr: 0.267 and 0.537, respectively). diagnosis delay is a positive predictor for recovery delay (r = .421; p = .001). conclusions: the study evidence supports that longer recovery delay was seen in patients of older age, mers‐cov infection, icu admission, and abnormal radiology findings. shorter recovery delay was found in patients who had camel contact and respiratory symptoms at presentation. these findings may help us understand clinical decision making on directing hospital resources toward prompt screening, monitoring, and implementing clinical recovery and treatment strategies. laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) has been documented in more than 2000 cases worldwide, causing 722 related deaths from september 2012 through september 2017. 1 much research evidence is available on factors associated with a poor prognosis in laboratory-confirmed mers-cov [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] and non-mers-cov 9-11 patients. a high mortality rate was systematically recognized in mers-cov patients of old age, 2,12-15 severe illness, 2,12-14 underlying condition, 2,12-14 and respiratory/gastrointestinal symptoms. 2 however, successful management of mers-cov such as clinical recovery and its predictors has not been given sufficient attention despite the virus having been in circulation since 2012. as per the authors' knowledge, two studies have so far addressed clinical improvement on laboratory-confirmed mers-cov patients. 16, 17 the first study, shalhoub et al, 16 was based on a case report in which their observations may not be generalized to a wider mers-cov population. the second study, al-turaiki et al, 17 utilized publicly available data from the saudi ministry of health. the major shortcomings in their study were several potential confounding factors such as underlying medical conditions and a primary or secondary mode of mers-cov transmission that had not been included in the analysis. in addition, the recovery delay was not reported in their study, and thus, factors related to the recovery delay were not examined. as of october 4, 2017, there was no available detailed data on recovery delay of laboratory-confirmed mers-cov and non-mers-cov patients. data on the time intervals between a patient's presentation or admission to a healthcare facility and the first specimen sample have been limited in patients suspected and screened for mers-cov by a real-time reverse-transcription polymerase chain reaction (rrt-pcr) test, as it might correlate with recovery delay intervals. early screening and diagnosis of mers-cov could greatly promote proper control and clinical management of cases, which may reduce the risk of transmission and increase the chance of successful outcomes. [18] [19] [20] this study provides more understanding of how long a period (in days) it may take to recover from mers-cov infection. the authors have studied, retrospectively, a cohort of survivorslaboratory-confirmed mers-cov and non-mers-cov patients-to estimate recovery delay intervals and identify possible associated factors in saudi arabia. the authors assessed whether the time interval between presentation and the initial rrt-pcr diagnosis (diagnosis delay) correlates with the time interval between initial rrt-pcr diagnosis and recovery (recovery delay). we hypothesized that older age, mers-cov infection, icu admission, and abnormal radiology findings might be associated with longer recovery delay. we hypothesized that diagnosis delay might positively correlate with a recovery delay. a multicenter retrospective study reviewed medical records of 829 patients from september 2012 to june 2016 who were admitted to the hospital and had been diagnosed by rrt-pcr assay for suspected mers-cov to have mers-cov and non-mers-cov infection. the study included patients who were admitted through emergency departments (pediatrics and adults) or patients who were admitted through outpatient clinics. screening referrals for mers-cov was made in accordance with the guidelines set by the saudi ministry of health in standard risk assessment algorithms for identifying and managing mers-cov infection. 21 in the study population, the rrt-pcr was used to detect mers-cov in multiple and/or different clinical specimens, including combined nasopharyngeal and throat swabs, sputum, blood, stool, and endotracheal aspirate (eta). the study gathered data from the two largest hospitals in saudi from patient charts, we collected demographic data: age and gender. elderly age was defined by classifying age into two groups using a cutoff of 65 years (≥65 years). the reason behind this classification was to assess the recovery delay for this vulnerable age group, as a previous study reported a high mortality rate in this group. 15 we collected data on route of transmission: camel contact and patient contact. the study authors collected various clinical data: fever (temperature ≥ 38°c); presence of any of the following respiratory symptoms: cough, bloody cough, shortness of breath, or chest pain; presence any of the following gastrointestinal symptoms: diarrhea, vomiting, or nausea; mers-cov infection; intensive care unit (icu) admission; hospital: kamc-riyadh or kfgh-jeddah; abnormal radiology findings; diabetes; renal disease; and hypertension. recovery delay was calculated as the number of days from the initial rrt-pcr diagnosis (±), which was the date found on the pathology report of the first specimen, to the clinical recovery (recovery delay), based on the date of hospital discharge or date of mers-cov or non-mers-cov infection was ruled out. in some cases, the clinical recovery was verified by taking a sample from different types of specimens at varying times. in all patients with initial rrt-pcr result, the medical records were reviewed from the date of presentation/hospital admission until 60 days after the initial rrt-pcr diagnosis. only patients who achieved recovery were analyzed. the study excluded patients with no available clinical recovery records and no discharge records within 60 days after the initial rrt-pcr diagnosis, as well as patients who had died. the final sample included 396 laboratory-confirmed mers-cov and non-mers-cov patients who had recovered and were identified by reviewing patient charts, hospital discharge records, and medical practitioner notes. the analysis was conducted using ibm statistical package for social sciences (spss) (24; spss, chicago, il). patients' characteristics were described by count and percent, and mean (± standard deviation) or median where appropriate. time intervals in days from presentation to initial rrt-pcr diagnosis (diagnosis delay) and from initial rrt-pcr diagnosis to recovery (recovery delay) were analyzed by spearman's correlation coefficient. the poisson and negative binomial models were used to model the frequency of recovery delay in days and identify unadjusted and adjusted factors associated with longer recovery delay. goodnessof-fit measures were used to compare and identify the best model. the model with the smaller deviance, larger log likelihood, smaller akaike information criterion, and smaller bayesian information criterion was considered the better model. in all analyses, a p-value of less than 5% was considered significant. relative risk (rr) and 95% confidence intervals (ci) were used to assess the strength of association between patients' characteristics and longer/shorter recovery delay. a total of 396 patients, suspected and screened for mers-cov by an rrt-pcr test, were analyzed. the average age was 46 years with age ranges between 1 and 95 years. the median recovery delay in our sample was 5 days. of the sample, 57.7% were male and 18.4% were admitted to icu. fever and respiratory symptoms were common presentations, occurring in 66.3% and 84.1% of the patients, respectively. the chest x-ray and/or ct scan were abnormal in almost half of the samples (48.4%). refer to table 1 for other sample parameters. the longer delays in diagnosis were positively correlated with longer recovery delay (r = .421; p = .001). according to univariate negative binomial regression analysis ( a multivariate negative binomial regression analysis (table 2) revealed six independent factors that affect the recovery delay. 22 and necessitate mechanical ventilation, 11 which is a risk factor for hospital mortality. 23 patients admitted to icu admission were at higher risk for longer recovery delay. a previous report showing similar findings, longer icu stay, and high mortality rate was reported in mers-cov patients who were admitted to the icu. 24 such patients would benefit from monitoring their responses to medical support and recognizing potential complications at an early stage. we found that camel contact was associated with shorter recovery delay. studies on recovery delay in patients with camel contact as compared to close contact exposure of a confirmed case or other exposure are lacking; however, camel contact has also been linked to lower 3-and 30-day mortality rates in mers-cov patients. 2 this important association requires more studies to identify whether camel contact is an independent protective factor of shorter recovery delay. in our study, patients with respiratory symptoms are more likely to experience shorter recovery delay than patients without respiratory symptoms. this finding is probably related to the shorter lag time between symptom onset and diagnosis, in which patients with presence of symptoms could be positively affected by an early diagnosis 18 and thus prompt medical support is deemed necessary. the time interval from presentation to initial rrt-pcr diagnosis (diagnosis delay) was positively correlated with the time interval from initial rrt-pcr diagnosis to recovery (recovery delay). early diagnosis is likely to improve clinical outcomes 18 and reduce the economic and physical burden of a disease. 25, 26 early diagnosis requires full utilization of hospital resources. individuals at high risk of mers-cov infection should be promptly screened after arrival at the healthcare facility, monitored for progression, and then having a prompt decision made for whether further rrt-pcr testing is needed. the authors noticed the following limitations. first, the study was based on chart reviews, and findings should be interpreted with caution. second, we did not collect information on the type of antiviral treatment or other supportive treatments given after diagnosis which may have affected clinical outcomes. 27, 28 third, despite this being the first investigation in this population, including a number of potential predictors for recovery delay, additional relevant predictors should be explored, such as the level of camel exposure, for example, hospital-acquired infections. fourth, patients with clinical recovery were identified by reviewing the medical records of the study sample within 60 days after the initial rrt-pcr diagnosis. studies with longer periods of follow-up in a larger population recovering from mers-cov are warranted to assess the long-term successful clinical outcomes. despite the mentioned limitations, data were aggregated directly from medical charts rather than public source databases. this chart review study was based on information from multicenters and a large sample size, and it provides valuable information on factors associated with prolonged or shorter recovery delay of patients suspected and screened for mers-cov by the rrt-pcr test. it is essential to develop interventional programs or guidelines to ensure early diagnosis, as this may reduce recovery delay intervals as well as improve patients' clinical outcomes. this research may enable identification of patients who require receiving appropriate medical support and care according to their illness progression. this also may prevent spread and transmission of the infection as individuals who are still severely ill can be appropriately isolated and managed apart from others who are responding to medical care. the study evidence supports that longer recovery delay was seen in patients with older age, mers-cov infection, icu admission, and abnormal radiology findings in a sample of patients diagnosed by rrt-pcr. recovery delay was significantly shorter in patients who had camel contact and respiratory symptoms at presentation. a prospective study is needed to evaluate the impact of camel exposure on recovery. evidence was found of an increasing recovery delay with a longer diagnosis delay. the findings may help understand clinical decision making as it directs hospital resources toward prompt screening, monitoring, and implementing clinical recovery and treatment strategies. there are no conflict of interests. middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment the predictors of 3-and 30-day mortality in 660 mers-cov patients estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study descriptive epidemiology and characteristics of confirmed cases of middle east respiratory syndrome coronavirus infection in the makkah region of saudi arabia estimation of merscoronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region, saudi arabia cd8 + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study spatial modelling of contribution of individual level risk factors for mortality from middle east respiratory syndrome coronavirus in the arabian peninsula risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia risks of death and severe disease in patients with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus outbreak in the republic of korea successful recovery of mers cov pneumonia in a patient with acquired immunodeficiency syndrome: a case report building predictive models for mers-cov infections using data mining techniques diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia the clinical and virological features of the first imported case causing mers-cov outbreak in south korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications case definition and surveillance guidance -updated early identification of pneumonia patients at increased risk of mers-cov infection in saudi arabia mechanical ventilation in patients in the intensive care unit of a general university hospital in southern brazil: an epidemiological study the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach antibody response and disease severity in healthcare worker mers survivors recovery from the middle east respiratory syndrome is associated with antibody and t cell responses factors associated with recovery delay in a sample of patients diagnosed by mers-cov rrt-pcr: a saudi arabian multicenter retrospective study http://orcid.org/0000-0001-8743-6007 key: cord-312692-jv3425w1 authors: iwata-yoshikawa, naoko; okamura, tadashi; shimizu, yukiko; kotani, osamu; sato, hironori; sekimukai, hanako; fukushi, shuetsu; suzuki, tadaki; sato, yuko; takeda, makoto; tashiro, masato; hasegawa, hideki; nagata, noriyo title: acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with middle east respiratory syndrome coronavirus date: 2019-01-09 journal: journal of virology doi: 10.1128/jvi.01818-18 sha: doc_id: 312692 cord_uid: jv3425w1 middle east respiratory syndrome coronavirus (mers-cov) infection can manifest as a mild illness, acute respiratory distress, organ failure, or death. several animal models have been established to study disease pathogenesis and to develop vaccines and therapeutic agents. here, we developed transgenic (tg) mice on a c57bl/6 background; these mice expressed human cd26/dipeptidyl peptidase 4 (hdpp4), a functional receptor for mers-cov, under the control of an endogenous hdpp4 promoter. we then characterized this mouse model of mers-cov. the expression profile of hdpp4 in these mice was almost equivalent to that in human tissues, including kidney and lung; however, hdpp4 was overexpressed in murine cd3-positive cells within peripheral blood and lymphoid tissues. intranasal inoculation of young and adult tg mice with mers-cov led to infection of the lower respiratory tract and pathological evidence of acute multifocal interstitial pneumonia within 7 days, with only transient loss of body weight. however, the immunopathology in young and adult tg mice was different. on day 5 or 7 postinoculation, lungs of adult tg mice contained higher levels of proinflammatory cytokines and chemokines associated with migration of macrophages. these results suggest that the immunopathology of mers-cov infection in the tg mouse is age dependent. the mouse model described here will increase our understanding of disease pathogenesis and host mediators that protect against mers-cov infection. importance middle east respiratory syndrome coronavirus (mers-cov) infections are endemic in the middle east and a threat to public health worldwide. rodents are not susceptible to the virus because they do not express functional receptors; therefore, we generated a new animal model of mers-cov infection based on transgenic mice expressing human dpp4 (hdpp4). the pattern of hdpp4 expression in this model was similar to that in human tissues (except lymphoid tissue). in addition, mers-cov was limited to the respiratory tract. here, we focused on host factors involved in immunopathology in mers-cov infection and clarified differences in antiviral immune responses between young and adult transgenic mice. this new small-animal model could contribute to more in-depth study of the pathology of mers-cov infection and aid development of suitable treatments. m iddle east respiratory syndrome coronavirus (mers-cov) was originally isolated as a novel coronavirus from a fatal case of acute respiratory distress syndrome and renal failure in 2012 (1). a human receptor for the virus, called human cd26/dipeptidyl peptidase 4 (hdpp4), was identified subsequently (2) . many epidemiological and virological investigations have been undertaken since then; however, information about the pathogenesis of mers-cov is limited. in addition, because mers-cov is endemic in the middle east, the development of effective prophylactic and therapeutic treatment strategies remains a high priority. therefore, appropriate animal models are needed to better understand the pathogenesis of mers-cov and facilitate development of effective vaccines and drugs. some research groups experimentally infected nonhuman primates and small experimental animals with mers-cov (3) (4) (5) (6) (7) . rhesus macaques appear to develop a transient lower respiratory tract infection after a combination of intratracheal, ocular, oral, and intranasal inoculation with mers-cov (3), whereas the common marmoset develops progressive and severe pneumonia, which can be lethal (4) . however, animal models based on nonhuman primates present both ethical and economic problems. thus, establishing a small-animal model of mers-cov infection is desirable. unfortunately, mers-cov does not infect or replicate in small rodents such as syrian hamsters (8) , mice (9) , or rats (10) because they lack a functional mers-cov receptor. zhao et al. described lung infection in a mouse model transduced with an adenovirus expressing hdpp4 (11) ; thus, a transgenic (tg) mouse carrying hdpp4 should be suitable for mers-cov studies (5) . some research groups developed tg mice overexpressing the hdpp4 receptor under the control of cag or cytokeratin 18 promoters (5) (6) (7) . these mice developed severe lung disease, along with infection of the brain. autopsy data are available from only one mers patient; therefore, it is unclear whether mers-cov causes a systemic infection although there is no evidence that mers-cov infects the human brain. other studies describe development of an hdpp4 knock-in mouse (12) (13) (14) . although the tissue distribution and expression levels of hdpp4 in these models are largely equivalent to those of dpp4 in wild-type mice, the phenotype that determines mers-cov susceptibility varies from model to model. the hdpp4 knock-in mouse model described by coleman et al. (12) succumbed to infection with wild-type mers-cov. in contrast, model mice described by cockrell et al. (14) and li et al. (13) are susceptible to infection by serially passaged mers-cov, which induces severe lung pathology and diffuse alveolar damage (dad). these mice would be good models for studying pathogenesis of mers. here, we developed a new tg mouse model expressing hdpp4 under the control of its endogenous promoter to better mimic physiological expression of hdpp4. these tg mice were then backcrossed onto th1prone c57bl/6 mice. after evaluating susceptibility to mers-cov infection, we investigated age-dependent differences in disease pathogenesis because older age is one of the common factors related to mers severity and mortality (15) (16) (17) (18) (19) (20) . both young and adult tg mice infected with mers-cov showed transient weight loss along with moderate pneumonia and mers-cov replication in the lung; however, they did not recapitulate the severe disease and lethal infection seen in humans. young and adult tg mice infected with mers-cov did, however, show different immunopathologies. adult tg mice showed higher levels of proinflammatory cytokine-and chemokinemediated macrophage infiltration of the lungs than young tg mice. taken together, these results suggest that age affects the immunopathology of mers-cov infection in tg mice. the data suggest that other factors are required to recapitulate severe human disease in these tg mice; however, this mouse model will be useful for identifying host mediators that protect against mers-cov infection. this animal model will provide new insight into factors that cause severe mers-cov infection. expression of hdpp4 in tg mouse tissues. to generate tg mice showing tissueor cell-type-specific hdpp4 expression mimicking that in humans, we first looked at research involving tg mice harboring human enterovirus receptors (such as the human poliovirus receptor) and scarb2 receptor-driven endogenous promoters (21, 22) . promoter sequences, which normally include a transcriptional start site, are usually isolated from the upstream regions of endogenous mammalian genes (23) . therefore, we used a bacterial artificial chromosome (bac) clone (rp11-345j9) containing the complete hdpp4 gene and an endogenous promoter to generate tg mice harboring hdpp4 (fig. 1a) . to screen the tg mice generated, we confirmed presence of the transgene by pcr genotyping using two primers sets specific for hdpp4 (table 1, exons 3 and 10) . hdpp4 is a protease expressed on the surface of cells in various organs, including t cells (24, 25) . the enzyme is expressed by approximately 60% of resting t cells isolated from blood (26) . since handling of peripheral blood in a laboratory is relatively simple, we conducted flow cytometry analysis using a fluorescein isothiocyanate (fitc)-conjugated anti-human cd26/hdpp4 monoclonal antibody that does no react with murine dpp4 to detect expression of hdpp4 in mice. cd3-positive lymphocytes from 2/15 tested pups were positive for hdpp4. these mice were then crossed with c57bl/6 mice to establish two independent tg lines (tg1 and tg2), which were maintained as hemizygotes carrying the hdpp4 gene. the tg animals were born at the the open and filled circles denote the centromere (cen) and telomere (tel) of human chromosome 2, respectively. the gray arrows indicate the genes located downstream of the hdpp4 gene (white arrow). the cloned region in the bac construct is denoted by a black line. gcg, glucagon gene; fap, fibroblast activation protein; ifih1, interferon induced with helicase c domain 1. (b) expression of hdpp4 on peripheral blood cd3-positive t lymphocytes from the transgenic (tg) mice. tg1 and tg2, hdpp4 ϩ/ϫ transgenic mouse lines 1 and 2; non-tg, hdpp4 ϫ/ϫ mouse. (c) genomic dna was extracted from tg2 mice, and human dpp4 exons 1 to 26 were subjected to pcr using specific primers. expected mendelian ratio and were outwardly indistinguishable from control littermates. because cd3-positive lymphocytes from peripheral blood of line tg2 showed higher hdpp4 expression than those from line tg1 (fig. 1b) , tg2 was used for further analyses. pcr genotyping using primer sets specific for hdpp4 revealed that the complete hdpp4 gene had integrated into the genome of tg2 mice ( fig. 1c and table 1) . to examine hdpp4 expression in human and tg2 tissues, we first performed western blot analysis with a goat anti-cd26/hdpp4 polyclonal antibody (af1180; r&d systems), which detected hdpp4 but cross-reacted weakly with mouse dpp4. bands of about 110 kda (hdpp4) were detected in all tested human tissues (liver, spleen, kidney, heart, lung, stomach, small intestine, large intestine, pancreas, brain, spinal cord, and skeletal muscle), except brain ( fig. 2a) . all of the tissues from hdpp4 tg mice expressed hdpp4, including liver, spleen, kidney, heart, lung, stomach, small intestine, large intestine, pancreas, brain, spinal cord, and skeletal muscle ( fig. 2a) . these results suggest that the human transgene was expressed in the majority of organs/tissues in tg2 mice. to further determine hdpp4 distribution in tissues, immunohistochemistry (ihc) was performed (fig. 2b ). ihc using a goat anti-cd26/hdpp4 antibody detected hdpp4 antigens in pneumocytes in the lung, in bile capillaries in the liver, in renal tubular epithelium, on the surface of epithelial cells lining the small intestine, in pancreatic islets, in lymphocytes in the lymph nodes, and in several types of endothelial cell and serous membranes (fig. 2b , left column). while hdpp4 expression was undetectable in brain tissue by western blot analysis, ihc revealed that endothelial cells lining blood vessels and leptomeninges of the human brain were positive for hdpp4 although neurons and glia were negative. in tg2 mice, pneumocytes and bronchial epithelial cells in the lungs, bile capillaries in the liver, the renal tubular epithelium, and the surface of epithelial cells in the small intestine were positive for hdpp4 (fig. 2b ). in addition, several types of endothelial cells and serous membranes in all tested tissues, including the central nervous system, from tg2 mice were positive for hdpp4. notably, most lymphocytes in the t cell zones of the spleen and lymph nodes from tg2 mice were positive for hdpp4. staining of tissues from non-tg mice was very weak or absent (except for the small intestine) (fig. 2b) . these data suggest that the pattern of hdpp4 expression in tg2 mice is similar to that in humans (except for pancreas and lymphoid tissues). expression of hdpp4 was higher in lymphocytes from tg2 mice than in those from humans ( fig. 1c and fig. 2b ). therefore, we investigated the immune response profile in tg2 mice. to assess innate immune responses in the lungs of tg2, non-tg, and c57bl/6 mice, all animals received intranasal administration of pbs with or without (b) immunohistochemical analysis of hdpp4 expression in human, tg2, and non-tg mouse tissues stained with an anti-hdpp4 polyclonal antibody. sections were counterstained with hematoxylin. scale bars, 50 m (large images of liver, kidney, small intestine, pancreas, spleen, and lymph node), 20 m (large images of lung and brain), and 25 m (insets). poly(i·c), a synthetic analog of double-stranded rna (fig. 3) . there was no statistically significant difference in cytokine expression levels between tg2, non-tg, and c57bl/6 mice at 24 h after inoculation with poly(i·c) or phosphate-buffered saline (pbs) (fig. 3) . however, when we set expression levels after pbs treatment as 1, we noted that expression of macrophage inflammatory protein 1␣ (mip-1␣), granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin-1␤ (il-1␤), il-12, and il-2 in poly(i·c)treated tg2 mice was 0.8-to 2-fold higher than in the other two strains. these results suggest that hdpp4 expression in mice does not have a marked effect on basal innate immune responses in the three mouse strains; however, tg2 mice show slightly stronger or earlier innate immune responses than c57bl/6 mice and non-tg mice. thus, when we investigated immune responses in this animal model, we made comparisons between mers-cov-infected and noninfected tg mice. susceptibility of hdpp4-tg mice to mers-cov infection. in this experiment, c57bl/6 mice were used instead of non-tg mice because we were unable to prepare a sufficient number of non-tg mice from littermate mice for this experiment. after intranasal inoculation of 10-week-old tg2 and c57bl/6 mice with 10 5 50% tissue culture infectious doses (tcid 50 ) of mers-cov, neither group was lethargic; however, tg2 mice showed mild but transient weight loss from days 6 to 7 postinoculation (p.i.) (tg2 mice, n ϭ 8 [four females and four males]; c57bl/6 mice, n ϭ 10 [five females and five males]) (fig. 4a ). tg2 mice showed seroconversion at 35 days p.i., whereas c57bl/6 mice did not (tg2 mice, n ϭ 5 [three females and two males]; c57bl/6 mice, n ϭ 6 [three females and three males]) (fig. 4b ), suggesting that tg2 mice are susceptible to infection by mers-cov. we next examined viral replication kinetics and sites of viral replication in 10-weekold tg2 and c57bl/6 mice (n ϭ 3-4 [2 females and 1 to 2 males per time point]). tg2 mice and c57bl/6 mice were inoculated intranasally with 10 5 tcid 50 of mers-cov, and tissue specimens (the maxilla [including the nostril], nasal wash fluid, lung, and lung wash fluid) were collected at 6 h p.i. and on days 1, 3, 5, and 7 p.i. the nasal wash fluid from three out of four tg2 mice contained barely detectable levels of infectious virus at days 1 and 5 p.i. (fig. 4c ). supernatants from maxilla tissue homogenates (20%) from tg2 mice contained 10 2.8 tcid 50 /g at day 1 p.i. although the titer fell by 5 days p.i. the viral titers in lung wash fluid and supernatants of lung tissue homogenates (20%) from tg2 mice contained 10 2.3 tcid 50 /ml and 10 4.6 tcid 50 /g, respectively, at 1 day p.i.; these values were significantly higher than those at 6 h p.i. (p ͻ 0.05 and p ͻ 0.01, respectively; one-way analysis of variance [anova]). the virus titers in the lungs were detectable up until 5 days p.i. virus was undetectable in the respiratory tract at 7 days p.i. although ihc revealed that various organs from tg2 mice were positive for the hdpp4 antigen (fig. 2b ), no infectious virus was detected in the liver, spleen, kidney, heart, intestines, and brain up to 7 days p.i. (table 2 ). in contrast, virus was detected in the respiratory tract of c57bl/6 mice at 6 h p.i. only. these observations suggest that mers-cov infects and replicates mainly in the lower respiratory tract of tg2 mice and is eliminated within 7 days of infection. several research groups have developed tg mouse models of mers-cov infection; however, in these models, viral replication and mers-cov rna were detected in the brain (5-7, 27). thus, we next measured the amount of viral rna in the brain of tg2 mice at 6 h and at 1, 3, 5, and 7 days p.i. by real-time reverse transcription-pcr (rt-pcr) using primers specific for mers-cov; however, no mers-cov rna was detected in the brain of tg2 mice. furthermore, another study showed that experimental infection of common marmosets with mers-cov resulted in viremia (4) . quantitative examination of viral rna levels in tg2 mice revealed very low copy numbers of viral rna in the blood at 3 and 5 days p.i., while sera from tg2 mice were negative for the virus ( table 2 ). these results suggest that although intranasal inoculation of tg2 mice with mers-cov causes no neuroinvasion, it may induce viremia. several animal studies have identified mers-cov rna in the lymphoid organs of infected animals (3, 4, 28) , but no infectious virus was detected in the spleen from tg2 mice up to 7 days p.i. (table 2 ). to investigate whether lymphoid organs in tg2 mice are susceptible to mers-cov infection, we harvested splenocytes from tg2 and c57bl/6 mice and estimated infectivity and mers-cov rna levels (fig. 4d) . although the amount of infectious mers-cov in splenocytes was below the detection limit, viral rna was detected in splenocytes from tg2 mice (peaking at 2 days p.i.). thus, lymphoid organs of tg2 mice were as susceptible to mers-cov infection as those reported in other animal studies although lymphoid organs were not a major site of mers-cov replication in tg2 mice after intranasal inoculation. acute inflammatory changes in the lungs of hdpp4-tg mice after mers-cov inoculation. mers-cov infection of the nasal cavity, lungs, brain, spinal cord, liver, spleen, kidney, heart, and gastrointestinal tract of tg2 mice was examined histopathologically on days 1, 3, 5, 7, 14, and 35 p.i (n ϭ 3; one or two females and one or two males per time point). histopathological investigations revealed that tg2 mice showed progressive pulmonary inflammation associated with acute virus infection, from which they recovered (fig. 5 ). on day 1 p.i., there was no obvious infiltration of the lungs in tg2 mice; however, mild cellular degeneration and viral antigen-positive cells were seen in the bronchioles and a few alveolar areas ( fig. 5a to c). double-immunofluorescence staining revealed that mers-cov antigen colocalized with hdpp4-positive bronchioles and alveolar cells on day 1 p.i. (fig. 6 ). on days 3 and 5 p.i., inflammatory reactions, including partial and/or mild perivascular and peribronchiolar infiltration by mononuclear cells and eosinophils in response to viral antigens, were observed in alveolar areas of lung tissue from tg2 mice (fig. 5d to i). from day 3 p.i. onwards, the alveolar area was the main site of inflammatory responses to viral replication (fig. 5f , i, and l). on day 7 p.i., the point at which tg2 mice showed weight loss, there was evidence of severe lung inflammation, including perivascular and alveolar septal thickening, caused by infiltrating mononuclear cells; some alveoli were positive for viral antigens (fig. 5k ). at day 14 p.i., focal cellular infiltration was still evident in the peribronchioles and alveolar septa although viral antigens and inflammatory responses had cleared from the lungs by day 35 p.i. (fig. 5m to p). there was no evidence of diffuse alveolar damage in the lungs up to day 35 p.i. these findings suggest that progressive immunopathology occurred uniformly throughout the lungs but resolved within 14 days after infection. neither histopathology nor ihc detected inflammatory infiltration or viral antigens in the nasal cavity through 35 days p.i. in addition, there was no inflammation or viral antigen expression in the brain through 35 days p.i. i a g i a g i a g i a g i a g i a g i a (fig. 7) . in contrast, c57bl/6 mice showed no histopathological changes or viral antigen in any organ, including the lung. these results indicate that tg2 mice suffer acute pneumonia after infection of the lungs with mers-cov, which is related to expression of hdpp4 in the bronchiolar epithelium and pneumocytes. splenocytes from tg2 mice (fig. 4d) ; however, the spleen and other lymphoid tissues did not harbor viral antigens. furthermore, mers-cov induced t cell apoptosis upon infection in vitro (28) , whereas immunohistochemical staining detected no evidence of apoptosis in lymphoid tissues, including spleen and lymph nodes, of mers-cov-infected tg2 mice. similar to findings in the other mouse models in which mouse dpp4 was replaced with hdpp4 (12), mers-cov replication and pathology were localized in the lungs in tg2 mice. differences in the immunopathology of mers-cov infection between young and adult hdpp4-tg mice. according to an epidemiological study (29) , age (ͼ45 years) is considered to be one of the risk factors for mers-cov infection in humans. therefore, we infected 25-week-old mice with mers-cov. tg2 mice showed significant weight loss from days 7 and 8 p.i. before recovering by day 14 p.i. (tg2 mice, n ϭ 6 [one female and five males]; non-tg mice, n ϭ 7 [three females and four males]) (fig. 8a) . however, 25-week-old tg2 mice showed no obvious clinical signs (such as respiratory illness and mortality). the 25-week-old tg2 mice had seroconverted by 35 days p.i., whereas the c57bl/6 mice had not (fig. 8b) . next, we examined viral replication kinetics and sites of viral replication in 25-week-old tg2 and non-tg mice (n ϭ 4; two females and two males per time point). the nasal wash fluid from one out of four tg2 mice contained barely detectable levels of infectious virus at 1, 3, and 5 days p.i. (fig. 8c) . supernatants from maxilla tissue homogenates (20%) from tg2 mice contained 10 1.7 and 10 2.0 tcid 50 /g at 1 and 3 days p.i., respectively, although the titer was undetectable at 5 days p.i. the viral titers in lung wash fluid from tg2 mice were detectable up until 3 days p.i., while the supernatants from lung tissue homogenates (20%) from tg2 mice showed a high viral load from 1 to 5 days p.i.; infectious virus was detectable up until 7 days p.i. viral loads in the respiratory tract peaked at 3 days p.i. although no virus was detectable in the respiratory tract of 10-week-old tg mice at 7 days p.i., infectious virus was detected in the lungs of 25-week-old tg mice up until 5 days p.i. in addition, infectious virus was detected in the lungs of one of four 25-week-old tg mice (10 2.5 tcid 50 /g) even at 7 days p.i. we also found that viral titers in the nasal wash fluid, maxilla (including nostril), lung wash fluid, and lungs of 25-week-old tg2 mice on day 3 p.i. (10 2.6 tcid 50 /ml, 10 3.3 tcid 50 /ml, 10 2.3 tcid 50 /ml, and 10 4.9 tcid 50 /ml, respectively) were slightly higher than those in 10-week-old tg2 mice (10 1.6 tcid 50 /ml, 10 2.9 tcid 50 / ml, 10 1.8 tcid 50 /ml, and 10 4.5 tcid 50 /ml, respectively) (p ϭ 0.04, student's t test with welch's correction). histopathological analysis revealed that 25-week-old tg2 mice showed delayed and prolonged inflammatory responses in the lung compared with those in 10-week-old tg2 mice (fig. 9) . interestingly, viral antigen-positive cells were seen in the alveolar area on day 1 p.i. and then in the bronchi on day 3 p.i., along with a sparse cellular infiltrate (fig. 9a to f) . cellular infiltration (which included mononuclear cells and polynuclear cells) was observed in the alveoli from 5 days p.i.; this expanded on day 7 p.i. (fig. 9g to l). focal cell infiltration was seen in the alveoli on day 14 p.i., and lymphoid cell aggregates were seen around bronchioles and blood vessels on day 35 p.i. (fig. 9m to r). next, we compared inflammation of the alveoli in 10-week-old tg mice and 25week-old tg mice on day 7 p.i. ionized calcium binding adaptor molecule 1 (iba-1) is expressed specifically by monocytes/macrophages and is upregulated when these cells are activated. the predominant inflammatory infiltrate within the lungs of both 10week-old and 25-week-old tg mice comprised iba-1-positive large cells and cd3positive mononuclear cells (fig. 10) . phagocytic vacuoles were prominent in large iba-1-positive cells from 25-week-old tg mice. (30) . therefore, we measured the levels of 20 cytokines and chemokines in lung samples from both 10-week-old and 25-week-old tg2 mice inoculated with either mers-cov or minimal essential medium (mem). measurements were made at 6 h and at 1, 3, 5, and 7 days p.i. (n ϭ 3 to 4 [2 females and 1 to 2 males per time point] and n ϭ 4 [2 females and 2 males per time point]) ( fig. 11a and b, for 10-week-old and 25-week-old mice, respectively). on day 3 p.i., we observed early expression of gamma interferon (ifn-␥)-induced protein 10 (ip-10) in the lungs of both 10-and 25-week-old tg2 mice, a level which was significantly higher than that in control mice; this increase lasted through day 7. this was followed by a transient increase in expression of interleukin-6 (il-6), il-13, and monocyte chemotactic protein-1 (mcp-1) in lungs from both 10-and 25-week-old tg2 mice at day 5 p.i. high levels of macrophage inflammatory protein 1␣ (mip-1␣) and monokine induced by ifn-␥ (mig) were detected in the lungs of both groups of tg2 mice from days 3 or 5 to 7 p.i., whereas il-12 levels increased at day 7 p.i. ifn-␥ production in the lungs of 10-week-old tg2 mice peaked significantly on day 5 p.i. while high values were seen in both infected and noninfected 25-week-old mice during the observation period. in contrast, expression of ip-10, il-12, and il-1␤ in 25-week-old tg mice was higher than that in 10-week-old tg2 mice. interestingly, il-1␣ and il-17 were detected only in 25-week-old tg2 mice infected with mers-cov. il-1␣, a potent proinflammatory cytokine associated with inflammation and fever, was detected from 3 days p.i. and remained significantly elevated until 7 days p.i.; il-17 (a proinflammatory cytokine that recruits monocytes and neutrophils) was detected from 3 days p.i. and peaked at 5 days p.i. before falling at 7 days p.i. these results indicate that mers-cov infection induces production of acute inflammatory chemokines and cytokines in the lungs. in addition, aging causes more severe immunopathology; this means that young and adult tg mice show different pathologies in the lung after mers-cov infection. furthermore, we measured expression of mrna encoding ifn-␣4 and ifn-␤ (type i ifn with antiviral activity) in the lungs of 10-and 25-week-old mice at 6 h and at 1, 3, 5, and 7 days p.i. by real-time reverse transcription-pcr (rt-pcr) (31). we did not find any evidence of ifn-␣4 or ifn-␤ in the lungs from 10-week-old tg2 mice at early times postinfection; however, we observed a transient increase in ifn-␣4 expression in the lungs of two out of four tg2 mice at 5 days p.i.; this level fell by 7 days p.i. (fig. 11c) . day 5 p.i. was the time point at which the amount of virus in the lungs of tg2 mice began to fall. no ifn-␤ mrna was detectable in lung samples from either group. on the other hand, the lungs of 25-week-old tg2 mice showed a transient increase in ifn-␣4 and ifn-␤ mrna expression at 3 days p.i. (fig. 11c ). in addition, ifn-␣4 and ifn-␤ mrna expression was higher than that in 10-week-old tg2 mice. taken together, these results indicate that both type i and type ii ifn contribute to the immunopathology of the lungs of 25-week-old tg2 mice infected with mers-cov. here, we describe a new hdpp4-tg mouse expressing the human gene under the control of an endogenous human promoter. this mouse model shows a pattern of hdpp4 expression that closely mimics that in human tissues and is similar to that in other recent models (5) (6) (7) . this mouse model also shows susceptibility to infection by mers-cov; this mimics the nonlethal observations in other mouse models (13, 14) . after intranasal inoculation with a human isolate of mers-cov, the tg mice developed acute and mild interstitial pneumonia; however, the infection was nonlethal and so did not mimic severe cases seen in human mers-cov patients. mers-cov infection can cause severe illness, resulting in acute respiratory distress syndrome, although a large number of mers-cov infections follow a mild or asymptomatic course in healthy individuals (32) (33) (34) (35) . thus, this tg mouse model reflects the natural course of a mild mers-cov infection. the majority of severe mers-cov cases occur in middle-aged and older males (36, 37) . therefore, we infected tg2 mice aged 25 weeks with mers-cov. the mice showed prominent proinflammatory responses and prolonged pulmonary inflammation compared with responses in tg2 mice aged 10 weeks. however, none of the infected tg2 mice had a severe outcome, such as respiratory distress, that led to death. epidemiologically, patients with diabetes, kidney failure, or chronic lung disease, all of which might weaken the immune system, tend to have a poor outcome after infection by mers-cov (http://www.who.int/csr/don/23-september-2015-mers-kuwait/en/). thus, it is presumed that a combination of older age and underlying disease might also increase mortality in this animal model. this hdpp4-tg mouse model, which lacks the clinical signs and mortality associated with severe mers-cov infection, is likely to be less advantageous than other lethal mouse models with respect to development of novel vaccines or antiviral agents (12) (13) (14) . when we asked why the tg2 mice showed nonlethal responses to infection, we could not ignore the fact that virus levels in lungs were lower than those reported for other mers mouse models (5, 12, 13, 38, 39) . one reason for this is that the transgene used in this study is a hemizygote, meaning that the copy number or expression level may be lower than that in mice homozygous for hdpp4. in addition, the dpp4 protein is active as a dimer (40) , but the tg2 mice harbor both murine and hdpp4. we presume that the viral yield in the lungs of tg2 mice was low because of heterodimers formed by hdpp4 and murine dpp4. to address this, we constructed structural models of homo-and heterodimers comprising human and mouse dpp4. notably, the estimated interaction energy of the dpp4 heterodimers was greater than that of murine and hdpp4 homodimers (ϫ358.2, ϫ347.6, and ϫ344.6 kcal/mol, respectively). these results suggest that dpp4 heterodimers are as stable as dpp4 homodimers. cockrell et al. reported that mouse dpp4 does not support mers-cov entry (38) . thus, the presence of stable dpp4 heterodimers may be a reason for the lower levels of infection in our mouse model. further study is necessary to clarify this. some research groups generated a mouse-adapted mers-cov for use in severe/ lethal mers-cov infection mouse models (13, 14) . this may be one way to establish severe mers infection in our tg2 mice. while the tg2 mice expressed hdpp4 protein in the liver, spleen, kidney, heart, lung, gastrointestinal tract, pancreas, and brain, viral infection and replication were limited (mainly) to the lower respiratory tract, with little upper respiratory tract involvement, after intranasal inoculation of mers-cov. tg2 mice developed interstitial pneumonia, and mers-cov antigens were detected in the lungs. virus yields in the lung were up to 100-fold higher than those in the upper respiratory tract. most mers patients exhibit a severe lower respiratory tract infection, with little involvement of the upper respiratory tract (36) . this suggests that mers-cov infection in tg2 mice mimics mild infection in humans. in vitro analysis of mers-cov suggests that the virus also infects human t cells and macrophages (28, 41, 42) . we detected mers-cov rna in serum and spleen cells from tg2 mice. these data are similar to those generated from another tg mouse model in which mouse dpp4 was replaced with hdpp4 under the control of the endogenous mouse dpp4 promoter (12) . mers-cov infection of t cells might affect immunopathology or induction of apoptosis in tg mice, but we found no clear evidence of this. although tg2 mice showed systemic viremia, infection of organs (except lung) did not lead to secondary complications. the disease phenotype (including clinical symptoms, viral titer in the lung, and acute pneumonia) appeared to be driven by infiltration by macrophages and lymphocytes; this is similar to the phenotype observed in another tg mouse model harboring hdpp4 under the control of the endogenous mouse dpp4 promoter (12) . the tg2 mice described herein did not show any brain or renal lesions after mers-cov infection. other tg mouse models in which hdpp4 is expressed under a strong ubiquitous promoter show high levels of viral rna and inflammation in the lungs, which are accompanied by brain lesions (5, 7, 11) . a fatal case of human mers-cov infection published by ng et al. showed no sign of mers-cov infection in the brain (43) . to date, no reports suggest that mers-cov shows tropism for brain tissue. the primary target of mers-cov is the lower respiratory tract; however, patients with mers often show signs of acute kidney failure (1, 43). in addition, mers-cov was identified in the urine of mers patients (44, 45) . data from the first autopsy case did find pathological signs in the patient's kidneys although ihc revealed no evidence of mers-cov replication in the kidneys (43) . in addition, acute renal failure in this patient was not caused by mers-cov directly; rather, it was caused by hypotension (43) and/or acute respiratory distress syndrome (46) . histopathological analysis identified cd3-positive t cells and iba-1-positive macrophages in the lungs of tg2 mice on day 7 p.i., which correlated with expression of inflammatory cytokines and inflammatory infiltrates in the lung. tg2 mice aged 10 and 25 weeks showed increased expression of cytokines and chemokines associated with migration of t cells and activation of macrophages, including ip-10, il-6, il-13, mcp-1, ifn-␥, mip-1␣, mig, and il-12, in the lungs at day 5 and/or 7 p.i. this result is the same as that observed in a hdpp4 knock-in mouse model reported by coleman et al. (12) . in this hdpp4 knock-in mouse model, cd8 ϩ t cells and macrophages affected the course of mers-cov-induced disease (12) . in addition, tg2 mice expressed mrna encoding the type i ifn, ifn-␣4, during the early phase of the mers-cov infection. importantly, the pathogenic and immune response data from the knock-in mouse model and our own model are similar. thus, an acute inflammatory reaction (including production of type i and type ii ifns) and infiltration by macrophages might clear the virus from the lung, thereby preventing progression to mers. interestingly, we detected il-1␣ and il-17 in the lungs of 25-week-old tg2 mice, but not those of 10-week-old tg2 mice, after mers-cov infection. both il-1␣ and il-17 are proinflammatory cytokines that attract monocytes and neutrophils; therefore, they may exacerbate immunopathology after infection. these findings support the notion that the severity of mers-cov infection is age dependent. age is one of the most common factors related to severity and mortality of mers infection; however, the underlying pathology is unclear (47) . many studies have examined immune responses of mers patients (30, (48) (49) (50) (51) (52) . indeed, elevated serum levels of il-6, il-12, ip-10, and ifn-␥ are observed in patients during the early period after severe infection (48) (49) (50) (51) . in addition, a prominent proinflammatory th1 and th17 response, including production of ifn-␥, tumor necrosis factor alpha (tnf-␣), il-15, and il-17, is seen in patients during the acute phase of mers-cov infection (52) . in contrast, we found that administration of poly(i·c) to tg2 mice induced a mild increase (or earlier induction) in innate immune responses compared with those in c57bl/6 mice and non-tg mice. this suggests that overexpression of hdpp4 might influence immune responses in tg2 mice. thus, we must exercise caution when assessing the relationship between immunopathology and outcome in patients and animal models of mers-cov; however, proinflammatory responses seem to contribute to immunopathology during the acute phase of mers-cov infection in both patients and mouse models. in summary, we generated an hdpp4-tg mouse model showing mild respiratory infection by mers-cov. while this tg mouse has limitations as a model for human mers (i.e., lower virus titer in the lungs and mild disease), the immunopathology seems to resemble a mild and early stage of infection in humans. even though it has limitations, this tg mouse model will increase our understanding of the mechanisms underlying mers-cov infection. indeed, we recently used this mouse model to confirm a role for transmembrane protease serine type 2 (tmprss2) during mers-cov infection (53) . this animal model may provide new insight into disease pathogenesis and guide development of therapeutic interventions that mitigate mers. ethics statements. experiments using recombinant dna and pathogens were approved by the committee for experiments using recombinant dna and pathogens at the national institute of infectious diseases, tokyo, japan. all animal experiments were approved by the animal care and use committee of the national institute of infectious diseases and the national center for global health and medicine (ncgm) research institute and were conducted in accordance with institutional guidelines for the care and use of animals. all animals were housed in a japan health sciences foundation-certified facility. all human samples used in this study were obtained from us biomax, inc., genetex, inc., alpha diagnostics international, or protein biotechnologies. the protocols were approved by the health insurance portability and accountability act (hipaa) or institutional review board (irb). cells and viruses. mers-cov, hcov-emc 2012 strain, was kindly provided by bart haagmans and ron fochier (erasmus medical center, rotterdam, the netherlands) and was used throughout the study. vero e6 cells, purchased from the american type cell collection (manassas, va), were cultured in eagle's mem containing 5% fetal bovine serum (fbs), 50 iu/ml penicillin g, and 50 g/ml streptomycin (5% fbs-mem). stocks of mers-cov were propagated and titrated on vero e6 cells and cryopreserved at ϫ80°c. viral infectivity titers are expressed as the tcid 50 /milliliter on vero e6 cells and were calculated according to the behrens-kärber method. work with infectious mers-cov was performed under biosafety level 3 conditions. virus isolation and titration. lung wash fluids and liver, kidney, heart, spleen, intestine, and brain tissue samples from tg2, non-tg, and c57bl/6 mice were collected at the time of postmortem examination and stored at ϫ80°c. tissue homogenates (20%, wt/vol) were prepared in 2% fbs-mem, and samples were inoculated onto vero e6 cell cultures, which were then examined for cytopathic effects (cpe) for 5 days. blind passage was performed after freezing and thawing cells from the first-or second-round passages. if mers-cov-specific cpe were not observed in the first-, second-, or third-round cultures, the samples were deemed negative for infectious virus. viral infectivity titers were determined in vero e6 cell cultures using the microtitration assay described above. mers-cov neutralizing assay. blood was obtained from each mouse and allowed to clot. sera were then obtained by centrifugation and inactivated by incubation at 56°c for 30 min. one hundred tcid 50 aliquots of mers-cov were incubated for 1 h in the presence or absence of mouse serum (serially diluted 2-fold) and then added to confluent vero e6 cell cultures in 96-well microtiter plates. the presence of viral cpe was determined on day 5, and the titers of neutralizing antibody were determined as the reciprocal of the highest dilution at which no cpe were observed. the lowest and highest serum dilutions tested were 1:2 and 1:512, respectively. generation of hdpp4-tg mice. to generate tg mice expressing hdpp4, a bac vector carrying the hdpp4 gene (clone rp11-345j9) was purchased from advanced genotechs co., japan. the bac dna was purified using a large-construct kit (qiagen) according to the manufacturer's instructions and suspended in te buffer (10 mm tris-hcl and 0.1 mm edta, ph 7.5) at a concentration of 4 ng/l. tg mice were generated using standard procedures (23). the purified bac clones were microinjected into the pronuclei of fertilized eggs from bdf1ϫc57bl/6ncr mice (slc, inc., hamamatsu, japan) and then transplanted into pseudopregnant icr mice (slc inc.). expression of the transgene was assessed by fluorescence-activated cell sorter (facs) analysis as described below. the tg mice were then backcrossed onto c57bl/6ncr for five generations. after weaning, the mice were tested for tg integration by pcr and facs analysis. briefly, genomic dna isolated from ear punch tissues was subjected to pcr using hdpp4-specific primers ( table 2) , and lymphocytes were isolated from blood taken from the tail vein. lymphocytes were screened for hdpp4 protein expression by flow cytometry analysis. tg mice and their non-tg littermates were used for the mers-cov infection study. flow cytometry analysis. blood was collected from the retro-orbital venous plexus under isoflurane anesthesia using heparinized capillary tubes. the samples were then treated with red blood cell lysis buffer (sigma-aldrich, st. louis, mo) to remove erythroid cells. for immunofluorescence staining, cells kit (thermo fisher scientific). a panel of inflammatory cytokines and chemokines (basic fibroblast growth factor [bfgf], granulocyte-macrophage colony-stimulating factor [gm-csf], ifn-␥, il-1␣, il-1␤, il-2, il-4, il-5, il-6, il-10, il-12p40/p70, il-13, il-17, ip-10, keratinocyte chemoattractant [kc] , mcp-1, mig, mip-1␣, tnf-␣, and vascular endothelial growth factor) was measured according to the manufacturer's protocols. isolation of splenocytes and infection with mers-cov. spleens were removed aseptically from tg2 and c57bl/6 mice (n ϭ 3 each), dissociated in rpmi 1640 medium, and pressed gently through a 40-m-pore-size nylon mesh filter. the cell suspension was centrifuged at 400 ϫ g for 10 min, and red blood cells were lysed with blood cell lysis buffer (final concentrations of 155 mm nh 4 cl, 10 mm khco 3 , and 0.1 mm edta, ph 7.3) at room temperature for 5 min. the cells were washed twice with rpmi 1640 medium and centrifuged at 1,000 ϫ g for 10 min. hdpp4-expressing cd3 ϩ t cells within the splenocyte population were detected by flow cytometry analysis. the percentage of cd3 ϩ t cells was 39.11% ϯ 3.8%, and that of hdpp4-expressing cd3 ϩ t cells was 26.46% ϯ 1.9%. the cells were resuspended in the medium and infected with mers-cov (multiplicity of infection [moi] of 1). viral replication was determined after 1 and 2 days of culture. viral infectivity titers were measured in vero e6 cell cultures using a microtitration assay. to detect the mers-cov genome in splenocytes, rna from splenocytes infected with mers-cov was extracted at 1 and 2 days p.i. and subjected to quantitative real-time rt-pcr (10) . molecular modeling of dpp4 homo-and heterodimers. dpp4 dimer models were constructed using the molecular operating environment (moe) (chemical computing group, inc., montreal, qc, canada) based on the crystal structure of hdpp4 at a resolution of 2.55 å (pdb accession number 2onc). stereochemical quality was assessed using the ramachandran plot and atom clashes applications in moe. interaction energy, which is an indicator of the affinity of the dimer, was calculated using the potential energy application in moe. statistical analysis. data are expressed as the means and standard errors of the means. statistical analyses were performed using graphpad prism, version 5, software (graphpad software, inc., la jolla, ca). intergroup comparisons were performed using one-way and two-way anova or student's t test with welch's correction. a p value of ͻ0.05 was considered statistically significant. 05% nan 3 ), and fc receptors were blocked by incubation for 20 min on ice with an unlabeled anti-mouse cd16/cd32 monoclonal antibody (clone 2.4g2; bay bioscience co., ltd ca) and an allophycocyanin (apc)-labeled anti-human cd3 antibody human skeletal muscle lysates were purchased from protein biotechnologies (ramona, ca) and used under irb-approved protocols. to prepare protein samples from the tg and non-tg mouse organs, tissues were homogenized in 0.5 ml of radioimmunoprecipitation assay (ripa) buffer (50 mm tris/hcl and the protein concentrations were measured using a pierce bicinchoninic acid (bca) protein assay kit after being blocked, the membranes were incubated for 1 h with a goat anti-hdpp4 antibody (0.1 g/ml) (af1180 followed by incubation with a donkey anti-goat horseradish peroxidase (hrp)-conjugated antibody (abcam) and an anti-rabbit hrp-conjugated antibody (abcam). the bands were detected by an immobilon western chemiluminescent hrp substrate (millipore) and an las-3000 apparatus inflammatory cytokine profiles in 20% (wt/vol) lung homogenates were detected using a commercial mouse cytokine 20-plex antibody bead kit (thermo fisher scientific), as described by the manufacturer. inoculation of mice with mers-cov. tg2 and non-tg mice (9 to 10 weeks or 25 weeks old) and tg2-balb mice (12 to 22 weeks old) were anesthetized and inoculated intranasally with 1 ϫ 10 5 tcid 50 (30 l) of mers-cov. body weight was measured daily for 14 days (n ϭ 4 to 8 per group), and animals were sacrificed at 6 h and at 1, 3, 5, 7, 14, and 35 days p.i. to analyze virus replication, hematological parameters, cytokine expression, and disease pathology (n ϭ 3 to 6 per group) for double staining of cd3 (t cells) and iba-1 (macrophages) antigen, we used a rabbit anti-human cd3 antibody tucson, az) and a rabbit anti-human iba-1 antibody diaminobenzidine (dab 0) for 10 min at 121°c. the second staining was performed for iba-1 with vina green. nuclei were counterstained with hematoxylin for 10 s. to detect apoptosis, terminal deoxynucleotidyltransferase-mediated dutp-biotin nick end labeling (tunel) was performed using an in situ cell death detection kit (roche) dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters wild type and innate immune deficient mice are not susceptible to the middle east respiratory syndrome coronavirus no susceptibility of neonatal and adult rats against the middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome cd8 ϩ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice a mouse model for mers coronavirus-induced acute respiratory distress syndrome middle east respiratory syndrome coronavirus outbreak in the republic of korea clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia the predictors of 3-and 30-day mortality in 660 mers-cov patients the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia mortality risk factors for middle east respiratory syndrome outbreak clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea transgenic mice susceptible to poliovirus transgenic mouse model for the study of enterovirus 71 neuropathogenesis overview: engineering transgenic constructs and mice accessory cell signals involved in t-cell activation cd26: a surface protease involved in t-cell activation the t cell triggering molecule tp103 is associated with dipeptidyl aminopeptidase iv activity a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health pathogenesis of middle east respiratory syndrome coronavirus effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine middle east respiratory syndrome coronavirus infections in health care workers state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans estimation of severe middle east respiratory syndrome cases in the middle east a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection one site mutation disrupts dimer formation in human dpp-iv proteins middle east respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates kinetics and pattern of viral excretion in biological specimens of two mers-cov cases clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection updates in the management of acute lung injury: a focus on the overlap between aki and ards mers transmission and risk factors: a systematic review distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? characteristics of traveler with middle east respiratory syndrome comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity immune responses to mers coronavirus during the acute and convalescent phases of human infection mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile tmprss2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection synthetic double-stranded rna poly(i:c) combined with mucosal vaccine protects against influenza virus infection we thank bart haagmans and ron fouchier for providing mers-cov (isolate hcov-emc/2012) and satoshi koike, ken fujii (tokyo metropolitan institute of medical science), and shutoku matsuyama (national institute of infectious diseases) for helpful discussions. we also thank ayako harashima and midori ozaki for technical assistance.this work was supported by the following: a grant-in-aid for research (h25-shinko-wakate-004) from the ministry of health, labor, and welfare, japan; the research program on emerging and reemerging infectious diseases (grants jp17fk0108313, jp18fk0108058, and jp18fk0108072) from the japan agency for medical research and development; grants-in-aid for scientific research from the ministry of education, culture, sports, science and technology, japan (16k09951 and 18h02665), and a grant from the national center for global health and medicine (27a1102). key: cord-313054-w90eitw9 authors: mobaraki, kazhal; ahmadzadeh, jamal title: current epidemiological status of middle east respiratory syndrome coronavirus in the world from 1.1.2017 to 17.1.2018: a cross-sectional study date: 2019-04-27 journal: bmc infect dis doi: 10.1186/s12879-019-3987-2 sha: doc_id: 313054 cord_uid: w90eitw9 background: middle east respiratory syndrome coronavirus (mers-cov) is considered to be responsible for a new viral epidemic and an emergent threat to global health security. this study describes the current epidemiological status of mers-cov in the world. methods: epidemiological analysis was performed on data derived from all mers-cov cases recorded in the disease outbreak news on who website between 1.1.2017 and 17.1.2018. demographic and clinical information as well as potential contacts and probable risk factors for mortality were extracted based on laboratory-confirmed mers-cov cases. results: a total of 229 mers-cov cases, including 70 deaths (30.5%), were recorded in the disease outbreak news on world health organization website over the study period. based on available details in this study, the case fatality rate in both genders was 30.5% (70/229) [32.1% (55/171) for males and 25.8% (15/58) for females]. the disease occurrence was higher among men [171 cases (74.7%)] than women [58 cases (25.3%)]. variables such as comorbidities and exposure to mers-cov cases were significantly associated with mortality in people affected with mers-cov infections, and adjusted odds ratio estimates were 2.2 (95% ci: 1.16, 7.03) and 2.3 (95% ci: 1.35, 8.20), respectively. all age groups had an equal chance of mortality. conclusions: in today’s “global village”, there is probability of mers-cov epidemic at any time and in any place without prior notice. thus, health systems in all countries should implement better triage systems for potentially imported cases of mers-cov to prevent large epidemics. middle east respiratory syndrome coronavirus (mers-cov) infection is considered to cause a new viral epidemic [1] , and was first reported in a patient who died from a severe respiratory illness in a hospital in jeddah, saudi arabia, in june 2012 [2, 3] . from 1.1.2012 to 17.1.2018, world health organization (who) has notified a total of 2143 laboratory-confirmed cases of mers-cov, including at least 750 deaths related to this infection from 27 countries around the world [4] . the origin of mers-cov has been widely discussed. initially, a bat reservoir was posited based on phylogenetic similarity of certain bat coronaviruses with mers-cov. however, there has been no clear bat source of infection or a consistent history of contact with bats in known cases of mers-cov to date [5, 6] . another source such as dromedary was later introduced as a possible reservoir in some studies [7] [8] [9] [10] . some studies have declared that all cases of mers-cov were directly or indirectly linked to residence or travel to 10 countries: saudi arabia, uae, jordan, qatar, kuwait, oman, yemen, egypt, iran, and lebanon [6, 11] . the mers-cov infection has high mortality rates, especially in patients with comorbidities such as diabetes and renal failure, evoking global concern and intensive discussion in the media along with respiratory droplet route of its transmission [12] . laboratory-confirmed mers-cov cases have been reported during hospital-based cluster outbreaks between 1.1.2017 to 17.1.2018, and cases are still detected throughout the year [4] . the occurrence of a large number of mers-cov cases and their associated deaths in the world indicate that this disease must be considered as a severe threat to public health [13] because millions of pilgrims from 184 countries converge in saudi arabia each year to perform hajj and umrah ceremony. upon their return to home, pilgrims hold a ceremony attended by family members and friends. oriental etiquette to share hospitality with others increases the transmission of probable mers-cov cases to others [12, 14] . worldwide awareness of mers-cov is low, the disease has high intensity and lethality with unknown mode of transmission and source of mers-cov infection (i.e. whether zoonotic or human disease) [15] . therefore, it is necessary to design and implement a research to identify some unknown epidemiological aspects and also determine the current epidemiological situation of mers-cov and its mortality risk factors in order to prevent, control and anticipate effective interventions. permission was obtained from who to conduct this analytical-descriptive epidemiological study. using census method, data related to laboratory-confirmed mers-cov cases between 1.1.2017 to 17.1.2018 were extracted from disease outbreak news on mers-cov from who website as follows. demographic information such as age, gender, reporting country, city, health care worker; clinical data and exposure status of mers-cov cases including comorbidities, exposure to camels, camel milk consumption, exposure to mers-cov cases, day/month of symptom onset, day/month of first hospitalization, day/month of laboratory confirmation, final outcome (dead or survived) of mers-cov cases were recorded. all statistical analyses were conducted using spss, version 21 (ibm inc., armonk, ny, usa). quantitative measurement was expressed by medians and qualitative variables were presented as absolute frequency and percentage. logistic regression was used to calculate the odds ratio (or) with a 95% confidence interval in order to assess the probable relationship between risk factors and final outcome (dead/survived) of laboratory-confirmed mers-cov cases. p values of less than 0.05 were regarded as statistically significant. a total of 229 mers-cov cases, including 70 deaths (30.5%), were recorded in the disease outbreak news on who website from 1. the median age of subjects was 53.2 years (range: 10-89 years). to assess the effect of several potential risk factors on death in morbid cases related to mers-cov infection, we used or index in order to better understand the mechanism of this relationship, and we reported both crude and adjusted or. based on this indicator, variables such as comorbidities and exposure to mers-cov cases were significantly associated with mortality in affected people with mers-cov infections ( table 1) . six countries were affected with mers during the period of this study. the majority of cases (approximately 93.9%) with highest mortality (98.6%) as well as 100% of female cases have been reported from saudi arabia ( table 2 ). the epidemic curve of laboratory-confirmed cases of mers between 1.1.2017 and 17.1.2018 is shown in fig. 1 . it can easily be seen that two peaks are evident in this period: the first at the beginning of april 2017 and the second at the beginning of july 2017. our results indicate that the number of mers-cov cases remained constant from the beginning of september 2017 to the end of january 2018. the findings have important implications for infection control practice. especially, we found evidence that was contrary to many studies declaring that the high mortality rates are related to mers infection with increasing age [16] [17] [18] . our results on mers-cov cases in global level showed that all age groups are somewhat at risk of death from this infection. the chance of mortality in mers-cov cases in all age groups is fairly equal. therefore, in the care and treatment of mers-cov cases, our results suggest that this important point is better to be considered on behalf of health care staff. in this study, we observed a higher disease occurrence and death of (table 1) . a possible explanation for a higher disease occurrence and mortality of mers-cov among men is that men are likely to spend more time outdoors and hence have a higher risk of exposure to a source of infection. the evidence linking mers-cov transmission between camels and humans cannot be ignored. several studies have shown that persons with direct and indirect contact with dromedary camels had a significantly higher risk of mers-cov infection. our finding was inconsistent with other studies that did not mention such evidence (table 1) . random error may be one of the reasons for obtaining this result since there were not details of exposure to camels and camel milk consumption for laboratory-confirmed mers-cov cases. our research is consistent with many studies that provided evidence of human-to-human transmission for mers-cov infection [15, 19, 20] . figure 1 shows two peaks during june until september, which coincides with the largest mass gathering of muslims around the world in saudi arabia to perform hajj and umrah ceremony. this finding highlights the effect of congregation in the spread of mers-cov infection. our findings in table 2 and fig. 2 show that most cases are reported from saudi arabia after about 7 years since the start of mers-cov pandemic (june 2012 to january 17, 2018). so, it seems necessary that epidemiologic investigations are conducted by ministry of health in saudi arabia and international partners to better understand the transmission patterns of mers-cov. this study had a number of limitations. assessment of the relationship between mortality related to mers-cov infection and some potential risk factor requires reliable sources of mortality data. we used the data recorded in the disease outbreak news on mers-cov from who website. the quality and accuracy of this data depend primarily on quality of the recorded data reported by national ihr focal point from different countries to who. in this study, the researcher was unable to verify the accuracy of the data, which potentially results in information bias. in addition, information for some of the variables was not available and the number of missing data was high, which might introduce a negligible selection bias in results. another limitation of this research was that possible misclassification of cases may occur due to the respondent's declarations such as exposure to camels, camel milk consumption, and exposure to mers-cov cases, which potentially occurs as a result of measurement bias. despite the above limitations, the current analytical-descriptive epidemiological study may have a number of implications for health care policy by using the global data. it also reminds us that effective national and international preparedness plans should be in place as well as measures to prevent, control and predict such viral outbreaks, improve patient management, and ensure global health security. the results of this analytical-descriptive epidemiological study revealed and confirmed some potential risk factors for mers-cov cases, which were reported as a possible risk factor in previous research studies. in fact, it reminds us that there is probability of mers-cov epidemic at any time and in any place without prior notice in today's "global village". the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea the clinical and virological features of the first imported case causing mers-cov outbreak in south korea middle east respiratory syndrome coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china middle east respiratory syndrome coronavirus: review of the current situation in the world middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae world organization health middle east respiratory syndrome estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia hadj ritual and risk of a pandemic transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome novel corona (mers-cov) infection. epidemiology and outcome update epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission the authors take this opportunity to thank all the who personnel as well as reporting countries with confirmed mers cases for data collection and sending the data to who. we thank rana sidani senior communication officer in who regional office for the eastern mediterranean (cairo, egypt) for their guidance, help and permission to extract the data. this paper is dedicated to arsam ahmadzadeh and anil ahmadzadeh. as a epidemiological analysis on who data, this study did not need financial support. the dataset used and/or analysed during the current study are available from the corresponding author on reasonable request.authors' contributions km and ja designed the study and performed the search and data extraction. ja analyzed the data. km and ja wrote the manuscript. ja edited the draft. both authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-288670-1vlowf2n authors: yang, naidi; shen, han-ming title: targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid-19 date: 2020-03-15 journal: int j biol sci doi: 10.7150/ijbs.45498 sha: doc_id: 288670 cord_uid: 1vlowf2n coronaviruses (covs) are a group of enveloped, single-stranded positive genomic rna viruses and some of them are known to cause severe respiratory diseases in human, including severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and the ongoing coronavirus disease-19 (covid-19). one key element in viral infection is the process of viral entry into the host cells. in the last two decades, there is increasing understanding on the importance of the endocytic pathway and the autophagy process in viral entry and replication. as a result, the endocytic pathway including endosome and lysosome has become important targets for development of therapeutic strategies in combating diseases caused by covs. in this mini-review, we will focus on the importance of the endocytic pathway as well as the autophagy process in viral infection of several pathogenic covs inclusive of sars-cov, mers-cov and the new cov named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and discuss the development of therapeutic agents by targeting these processes. such knowledge will provide important clues for control of the ongoing epidemic of sars-cov-2 infection and treatment of covid-19. coronaviruses (covs) are enveloped viruses with a long single-stranded rna ranging from 26 to 32 kilobases (kb) in size [1] . covs belong to the family coronaviridae in the order nidovirales, and have been organised into 3 groups: α-covs, β-covs, and γ-covs [2] . two of the β-covs including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) caused severe acute respiratory disease outbreaks in china in [2002] [2003] and in the middle east in 2012, respectively [3] . in december 2019, a novel cov outbreak, identified and named as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) started in wuhan, hubei province, china. the sars-cov-2 spread very quickly in china and then to the many other countries, causing coronavirus disease-19 . the clinical futures of covid-19 mainly include fever, cough and pneumonia [4] . up to date, it has already infected more than 90,000 people worldwide and killed more than three thousand patients, mainly in wuhan, china. sars-cov-2 shares a high sequence identity (around 80%) with sars-cov and a 96.2% sequence identity with batcov ratg13, a bat cov [5] . although some initial cases were linked to a local seafood market in wuhan, its origin, intermediate hosts and how it was transmitted to humans are still largely unknown [4] . in this mini-review, we will mainly focus on β-cov, which is inclusive of sars-cov, mers-cov, and the current emerging sars-cov-2 to discuss the implication of the endocytic pathway and autophagy process in the infection of these pathogenic covs and therapeutic potential of targeting these processes. this review will also include the well-studied mouse hepatitis virus (mhv) since it is often used as a safe mode to study cov infection. macroautophagy or autophagy refers to an evolutionarily conserved process in which the intracellular components such as protein aggregates and damaged organelles are engulfed into a double-membrane structure called autophagosome, which eventually fuses with lysosome to form autolysosome for degradation [6, 7] (figure 1) . the whole autophagy process is controlled by a group of proteins encoded by autophagy-related-genes (atgs) in several consecutive stages [8, 9] . first, the induction or initiation stage is controlled by the ulk1/atg1 complex, downstream of the mechanistic target of rapamycin complex 1 (mtorc1). second, the nucleation/expansion/elongation stage is mediated by the atg14-beclin1-hvps34/class iii phosphatidylinositol 3-kinases (pi3k) complex, as well as the two ubiquitin-like conjugation systems (atg5-atg12 and lc3/atg8). the third and last stage of autophagy is the maturation/fusion/degradation in which autophagosome fuses with lysosome to form autolysosome where the luminal contents are degraded (figure 1 ). at present, the biological functions of autophagy have been extensively studied. autophagy plays an important role in various physiological and pathological processes, including cell survival, cell death, aging, immunity and metabolism [10, 11] . more importantly, accumulating evidence has highlighted the importance of autophagy in many human diseases, such as cancer, neurodegenerative diseases, metabolic disorders, as well as immunity and infection [12, 13] . among them, the implication of autophagy in viral infection has also been widely investigated and deeply appreciated. in the course of autophagy, lysosome plays an essential role in the maturation/degradation stage of autophagy, as the contents in the autophagosomes are eventually degraded by lysosomes, via autophagosome-lysosome fusion [14] [15] [16] . lysosome, first discovered by the nobel laureate christian de duve in the 1950s, is the most important digestive organelle present in almost all eukaryotic cells and with an array of important biological functions, including endocytosis, exocytosis, macropinocytosis, plasma membrane repair, defense against pathogens, cell death, signal transduction, and autophagy [14, 17] . lysosome is featured by its acidic internal ph which is generated by the action of a v-atpase, a proton-pumping membrane protein complex [18] . lysosome contains more than 50 acid hydrolases, including proteases, peptidases, phosphatases, nucleases, glycosidases, sulfatases, and lipases designated for all types of macromolecules [19] . on the other hand, lysosome is also a key component of the endocytic pathway, also termed as the endolysosomal network [20] (figure 1) . in addition to autophagy as described above, lysosome receives cargos from other processes including endocytosis through the endocytic pathway. briefly, following endocytosis, internalized cargos first enter the early endosomes (ee) where the cargos are sorted for two destinations: they are either retrieved for recycling to the plasma membrane/the secretory pathway, or are delivered to the late endosome (le) and then fuse with lysosome for degradation [20, 21] . the main biological functions of the endocytic pathway are for retrieval and recycling of internalized cargo proteins, and such functions are known to play critical roles in the pathology of important human diseases, in particularly neurodegenerative diseases and viral infection [22, 23] . in the past one and a half decades, the implication of autophagy in cov infection has attracted substantial attention, figure 1 . involvement of the endocytic pathway and autophagy in the entry and replication of covs in host cells. entry of covs into the host cells is mainly mediated by the endocytic pathway, meanwhile the autophagy has also been implicated in the viral replication in the cells, a process partly related to the formation of dmv in the host cells. as a result, several groups of inhibitors including the lysosomotropic agents such as cq and inhibitors for clathrin-mediated endocytosis such as chlorpromazine have been proposed to have therapeutic efficacy against covs-induced diseases including covid-19. probably due to the sars outbreak in 2002-2003 and the emerging field of autophagy research at the same period. at present, various reports have converged onto two important questions: whether cov induces autophagy and whether the autophagy machinery or atg proteins are involved in the infection and replication of covs. the first report demonstrating the involvement of autophagy in viral replication was based on mhv [24] , in which the authors made several important observations. first, mhv induced the formation of double-membrane vesicles (dmvs), with resemblance to autophagosome, a hallmark of autophagy. second, the viral replication complexes at dmvs co-localized with the autophagy proteins, lc3 and atg12. third and more importantly, mhv replication was impaired in atg5 knockout embryonic stem cells. therefore, the authors concluded that autophagy is implicated in the formation of dmv as well as in the replication of mhv [24] . in a follow-up study, the same group also examined the sars-covs and found similar colocalization of the key viral replication proteins with endogenous lc3, a protein marker for autophagosome [25] , suggesting a similar function of autophagy in the replication of sars-covs. cottam et al used another cov (infectious bronchitis virus, ibv) and found that one of the key viral replicase protein nsp6 is capable of inducing autophagy [26] . notably, this nsp6 also presents in mhv and sars-cov, and thus it would be of interest to further test the effects of nsp6 in these two covs on autophagy. however, several subsequent studies have challenged the notion that autophagy is implicated in cov infection. for instance, in vero cells infected with sars-covs, snijder et al failed to detect colocalization of lc3 or gfp-lc3 with the viral replication-transcription complexes of sars-cov examined using immunofluorescence staining [27] . further studies also demonstrated that either atg5 or atg7, two of the key autophagy proteins in control of autophagosome biogenesis, is not required for viral replication in cells infected by mhv [28, 29] or by sars-covs [30] . in those studies, cells with deletion of either atg5 or atg7 failed to impair the viral replication rate. similarly, virus infection was not inhibited by the knockdown of atg5 [26] . thus, all these observations suggest that the autophagy machinery is not directly implicated in the viral replication process. intriguingly, there is evidence suggesting the possible inhibitory effect of cov on the autophagy process. for instance, a study using sars-cov and mers-cov in hek293t, hela and mcf-7 cells found that overexpression of membrane-associated papainlike protease plp2 (plp2-tm) of sars-cov and mers-cov led to blockage of autophagosomeslysosomes fusion and suppression of the autophagic flux [31] . consistently, a more recent report found that mers-cov blocks the fusion of autophagosomes and lysosomes and induction of autophagy reduces the replication of mers-cov [32] . thus, it appears that there is certain type of interplay between the autophagy machinery and covs, and the exact nature of such interaction remains to be further elucidated. taken together, as summarized in table 1 , it is still controversial whether and how autophagy is implicated in the infection of covs. the discrepancies in the literature is probably due to the different viruses used, different cells tested and even the different techniques used in study of autophagy. more work is thus needed to clarify those important issues. one of the key determining factors in viral infection is the entry of the virus into the host cells. at present, it is widely believed that covs enter the host cells via two routes: (i) the endocytic pathway and (ii) non-endosomal pathway [33] , as partly illustrated in figure 1 . among them, the endocytic pathway is considered to be particularly important and has been extensively studied. as discussed earlier, covs are enveloped and plus-strand rna virus and they contain a set of four proteins that encapsidate the viral genomic rna: the nucleocapsid protein (n), the membrane glycoprotein (m), the envelope protein (e), and the spike glycoprotein (s) [34] . among them, the function of the s protein is mainly involved in the process of viral entry into the host cells via proteolytic cleavage to form two subunits, s1 and s2 [35] . these two subunits have distinct functions: s1 is responsible for receptor-binding, while s2 is mainly for membrane fusion and both are essential for viral entry via the endocytic pathway and infection into the host cells. the first report showing the relevance of endosome-lysosome in covs was from a morphological study in which two covs (ibv and porcine epidemic diarrhea virus (pedv)) were found to accumulate in the lysosomes of cells after infection [36] , indicating that the possible functional implication of lysosome in covs infection. subsequent studies have firmly established that the endocytic pathway is the key mechanisms controlling the entry of covs into the host cells, and thus the endocytic pathway has been widely investigated as the target of anti-viral therapies, as summarized in table 2 . among all these studies, there are several important points to be highlighted. first, different covs including mhv, sars-cov and mers-cov have been consistently demonstrated to engage the endocytic pathway as the main mechanism for viral entry into a variety types of host cells. among them, clathrin-dependent endocytosis and cathepsinmediated s protein cleavage are two critical steps for the viral entry and infection. in fact, this mechanism is also applicable to many other covs such as ibv [37] , which is out of the scope of this review. second, despite the general understanding for the role of the endocytic pathway for viral entry, there are discrepancies of the exact mechanisms among different reports even with the same cov. for instance, wang et al found that sars-covs engage clathrin-and caveolae-independent endocytic pathway as the key mechanism for viral entry [38] , which is inconsistent with an earlier report in which sars-cov entry into hepg2 cells is mostly mediated by the clathrin-dependent pathway [39] . part of the reason for such discrepancies is the different cell types used in their studies, as shown in table 2 . therefore, it is possible that the exact nature of the entry is context-dependent, including the type of the virus and the type of the cells. third, at present, the entry mechanisms and the implication of the endocytic pathway of the new emerging sars-cov-2 have not been reported directly. it is now known that sars-cov-2 utilizes the same receptor of sars-cov, which is angiotensin converting enzyme ii (ace2) for viral entry into the host cells [5, 40] . ace2 transcripts was originally only found in heart, kidney and testis of human [41] . however, it was later found that ace2 protein expresses abundantly in the epithelia of the human lung and small intestine [42] . since sars-cov-2 also binds to the same ace2 receptor as sars-cov [5] and sars-cov-2 is also susceptible to the inhibitory effect of chloroquine (cq), a lysosomotropic agent [43] , it is highly possible that this new cov utilizes the same endocytic pathway for entry into the host cells. understanding this mechanism is important in the search of effective therapeutic agents in the treatment of covid-19 caused by this new cov. finally, in almost all the studies listed in table 2 , different inhibitors of the endocytic pathway have been used in blocking viral entry and infection. among them, three groups of inhibitors are believed to be particularly important and clinically relevant. the first group are the lysosomotropic agents which are capable of accumulating inside and neutralizing the endosome-lysosomal acidic ph and thus blocking the protease activity to inhibit the viral entry. in this group, cq is an ancient anti-malaria drug and clinically available, as shown in figure 1 . the second group are direct endosomal-lysosomal protease inhibitors such as e64d. and the third group are inhibitors for the clathrin-mediated endocytosis such as chlorpromazine, which is also clinically available (figure 1) . the details of such inhibitors are to be discussed in the section below. taken together, establishing the role of endocytic pathway in viral entry is a major breakthrough in the mechanistic understanding of the covs infection, which offers great opportunity in development of novel therapeutic strategies for treatment of diseases such as sars and covid-19. lysosomotropic agents targeting endosomal/ lysosomal ph cq, a well-known anti-malarial drug, is probably the most well-studied lysosomotropic agent that accumulates in the acidic organelles such as endosomes and lysosomes and neutralizes their ph [57] . at present, it has been well studied that cq has a wide-spectrum of anti-viral effects including anti-covs, anti-hiv, and anti-type a and b influenza viruses [58] . the anti-viral effects of cq and its analogs have been reviewed elsewhere [59] . here we would like to focus on the effect of cq on covs, as summarized in table 2 . for instance, cq has been shown to inhibit mers-cov replication in vitro via a screening of an fda-approved compound library [60] . treatment with cq at a clinically relevant concentration, either before or after sars-cov infection into the vero e6 cells, was found to be effective in suppressing viral infection, indicating its application in both prophylactic and therapeutic conditions [47] . similar results were also found in another lysosomotropic agent, ammonium chloride (nh4cl) [47] . it is known that the cleavage of the spike glycoprotein (s protein) by proteases is required for the sars-cov entry to the cells via a ph-dependent manner [45] . mechanistically, it is believed that the neutralization of endo-lysosomal ph by cq inhibits the protease activities and prevents the cleavage of s protein and subsequently impairs the viral entry into the host cell. interestingly, wang et al showed that in cells treated with cq, nh4cl or bafilomycin a1 (an endo/lysosomal v-atpase inhibitor), the viral receptor ace2 was trapped within perinuclear vacuoles [38] , suggesting that these lysosomotropic agents may also affect the function of ace2. since ace2 serves as the viral receptor for both sars-cov and sars-cov-2, such observations further support the notion for the potent anti-viral activity of those lysosomotropic agents. indeed, a very recent study showed that cq inhibits the sars-cov-2 infection at both entry and post-entry stages in vero e6 cells [43] . in addition to its direct effects on covs, there is evidence of the combinational activity of cq with other therapeutic agents for treatment of sars, mers and possibly covid-19. for instance, he et al reported that cq has synergistic effect on glucocorticoid signaling by stabilizing glucocorticoid receptor [61] . since glucocorticoid is one of the recommended therapy for severe sars patients [62] , it is possible that cq may can be used for treatment of covid-19 in combination of glucocorticoids and clinical trials are thus needed to test the efficacy of such combined therapy. at present, cq phosphate has been listed as a new therapeutic in the sixth version of "guidelines for the prevention, diagnosis, and treatment of covid-19" issued by the national health commission of the people's republic of china. and a number of clinical trials with cq have been initiated in china [63] . the current suggested dosage of cq for covid-19 is as high as 500 mg, with treatment not exceeding 10 days. however, the usage of cq phosphate should be evaluated carefully as it may also have side effects such as retinopathy [64] and cautions should be taken for close monitoring of the potential side effect throughout the whole treatment period. the dosage should be reduced or stopped if reduction in haemoglobin concentration, lymphocyte count and platelet count, or the eyesight are observed. in addition, since cq is the substrate of cytochrome p450 (cyp) enzymes which are responsible for the metabolism of multiple drugs, it might interfere with other medications such as digitoxin (a cardiac glycoside) and tamoxifen (used for treatment of breast cancer). more details of the toxicity and precautious of cq can be referred elsewhere [65] . cathepsins are endosomal and lysosomal cysteine proteases that play important roles in protein degradation in various cellular processes including both the endocytic pathway and autophagy [64] . the role of cathepsins in viral infection was first identified by huang et al and they found that one cysteine proteases inhibitor e64d and a specific cathepsin l inhibitor z-fy(t-bu)-dmk are able to block the sars-cov infection [48] . k11777 is another cysteine protease inhibitor that has been reported to block the entry of sars-cov at the sub-nanomolar range [66] . in addition, teicoplanin, a glycopeptide antibiotic and its derivatives inhibit the entry of mers-cov and sars-cov by inhibition of cathepsin l activity [55] . interestingly, a serine protease inhibitor camostat was known to inhibit transmembrane protease serine 2 (tmprss2) and effectively protected the mice against death caused by sars-cov infection [55] . more importantly, a very recent study showed that camostat can also block the entry of sars-cov-2 by inhibiting ace2 and tmprss2 [67] . since camostat is already in clinical use for the treatment of chronic pancreatitis, suggesting its therapeutic potential for treatment of covid-19. as discussed earlier, clathrin-mediated endocytosis is one of the key mechanisms for viral entry into the host cells, including mhv [50, 52, 54] , sars-cov [38] , and mers-cov [53, 54] . therefore, chlorpromazine, an inhibitor for clathrin-dependent endocytosis, have been consistently found to possess significant inhibitory effect on the entry of those covs [39] . in fact, chlorpromazine is a fda-approved drug widely used for treatment of psychotic disorders such as schizophrenia [68] . importantly, it has been well established for its antivirus function for many types of viruses, including sars-cov and mers-covs, as summarized in table 2 . at present, the clinical application of chlorpromazine in treatment of sars and mers has not been reported and it would be of interest to conduct clinical trials for testing the therapeutic effect of chlorpromazine on covid-19. in addition, two cardiotonic steroids ouabain and bufalin, selective inhibitors of the plasma membrane na + /k + -atpase, have been shown to inhibit the mers-cov infection at nanomolar concentrations via affecting the clathrin-mediated endocytosis pathway [54] . since both of them are also fda-approved drugs and clinically available, it would be of interest to test them clinically for treatment of covid-19. the current ongoing epidemic of sars-cov-2 and covid-19 worldwide has emerged as a significant global public health threat. while urgent regulatory measures in control of the rapid spread of this virus are essential, scientists around the world have quickly engaged in this battle by studying the molecular mechanisms and searching for effective therapeutic strategies against this deadly disease. at present, while the exact role of autophagy remains debatable, there is overwhelming evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sars-covs, mers-covs and possibly sars-cov-2. as a result, several inhibitors targeting the endocytic pathway appear to have the therapeutic potential in treatment of covid-19, including a lysosomotropic agent cq and a clathrin-mediated endocytosis inhibitor chlorpromazine [43, 63, 65, 68] . since both are fda-approved and clinically available, clinical trials either as a single therapy or in combination with other anti-viral drugs are much needed. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus coronavirus envelope protein: current knowledge lysosomal proteases are a determinant of coronavirus tropism a novel coronavirus from patients with pneumonia in china a pneumonia outbreak associated with a new coronavirus of probable bat origin a brief history of autophagy from cell biology to physiology and disease biological functions of autophagy genes: a disease perspective autophagy: machinery and regulation the atg conjugation systems in autophagy autophagy in mammalian development and differentiation autophagy in human health and disease autophagy: regulation and role in disease autophagy and human diseases autophagy: a lysosomal degradation pathway with a central role in health and disease at the end of the autophagic road: an emerging understanding of lysosomal functions in autophagy signals from the lysosome: a control centre for cellular clearance and energy metabolism the lysosome as a cellular centre for signalling, metabolism and quality control lysosomal acidification mechanisms proteomics of the lysosome to degrade or not to degrade: mechanisms and significance of endocytic recycling the enigmatic endosome -sorting the ins and outs of endocytic trafficking endocytic membrane trafficking and neurodegenerative disease endosomal vesicles as vehicles for viral genomes coronavirus replication complex formation utilizes components of cellular autophagy identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex coronavirus replication does not require the autophagy gene atg5 coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin1 to negatively regulate antiviral innate immunity skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection coronaviruses -drug discovery and therapeutic options molecular interactions in the assembly of coronaviruses physiological and molecular triggers for sars-cov membrane fusion and entry into host cells significance of lysosomes in the morphogenesis of coronaviruses infectious bronchitis virus entry mainly depends on clathrin mediated endocytosis and requires classical endosomal/lysosomal system sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury a novel angiotensin-converting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro localization of mouse hepatitis virus nonstructural proteins and rna synthesis indicates a role for late endosomes in viral replication characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign chloroquine is a potent inhibitor of sars coronavirus infection and spread sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry role of endocytosis and low ph in murine hepatitis virus strain a59 cell entry amiodarone alters late endosomes and inhibits sars coronavirus infection at a post-endosomal level mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of eps15 coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner atp1a1-mediated src signaling inhibits coronavirus entry into host cells glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism akt inhibition promotes autophagy and sensitizes pten-null tumors to lysosomotropic agents new insights into the antiviral effects of chloroquine targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture identification of a lysosomal pathway that modulates glucocorticoid signaling and the inflammatory response advances in clinical diagnosis and treatment of severe acute respiratory syndrome breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology health commission of guangdong province for chloroquine in the treatment of novel coronavirus p protease inhibitors targeting coronavirus and filovirus entry sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a 2 clinically-proven protease inhibitor cell classics in chemical neuroscience: chlorpromazine the authors have declared that no competing interest exists. key: cord-295846-quhnesbr authors: li, huan; chen, chongxiang; hu, fang; wang, jiaojiao; zhao, qingyu; gale, robert peter; liang, yang title: impact of corticosteroid therapy on outcomes of persons with sars-cov-2, sars-cov, or mers-cov infection: a systematic review and meta-analysis date: 2020-05-05 journal: leukemia doi: 10.1038/s41375-020-0848-3 sha: doc_id: 295846 cord_uid: quhnesbr we performed a meta-analysis to determine safety and efficacy of corticosteroids in sars-cov-2, sars-cov, and mers-cov infections. we searched pubmed, web of science, medline, wanfang chinese database, and zhiwang chinese database using boolean operators and search terms covering sars-cov-2, sars-cov, or mers-cov and corticosteroids to find appropriate studies. review manager 5.3 was used to analyze results of meta-analysis. observational studies were analyzed for quality using the modified newcastle–ottawa scale and randomized clinical trials, using the jadad scale. subjects were divided into those with severe-only and other (severe and not severe) cohorts based on published criteria. efficacy endpoints studied included mortality, hospitalization duration, rates of intensive care unit (icu) admission, use of mechanical ventilation, and a composite endpoint (death, icu admission, or mechanical ventilation). we included 11 reports including 10 cohort studies and 1 randomized clinical trial involving 5249 subjects (2003–2020). two discussed the association of corticosteroids and virus clearing and 10 explored how corticosteroids impacted mortality, hospitalization duration, use of mechanical ventilation, and a composite endpoint. corticosteroid use was associated with delayed virus clearing with a mean difference (md) = 3.78 days (95% confidence interval [ci] = 1.16, 6.41 days; i(2) = 0%). there was no significant reduction in deaths with relative risk ratio (rr) = 1.07 (90% ci = 0.81; 1.42; i(2) = 80%). hospitalization duration was prolonged and use of mechanical ventilation increased. in conclusion, corticosteroid use in subjects with sars-cov-2, sars-cov, and mers-cov infections delayed virus clearing and did not convincingly improve survival, reduce hospitalization duration or icu admission rate and/or use of mechanical ventilation. there were several adverse effects. because of a preponderance of observational studies in the dataset and selection and publication biases our conclusions, especially regarding sars-cov-2, need confirmation in a randomized clinical trial. in the interim we suggest caution using corticosteroids in persons with covid-19. in december 2019, wuhan, hubei province, china, became the epicenter of a pandemic caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) [1] which causes coronavirus infectious disease-2019 . about 15% of cases of covid-19 become severe [2] . about one-half of hospitalized persons with covid-19 treated in the intensive care unit (icu) receive corticosteroids versus 10-20% of hospitalized, non-icu persons [3, 4] . there are several reports of using corticosteroids in the setting of severe coronavirus infections including sars-cov-2, severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) . there are few data on safety and efficacy of corticosteroids in covid-19 and its use in sars and mers infections is controversial [5, 6] . sars-cov-2, sars-cov, and mers-cov shares many genetic features and sars-cov-2 is highly homologous to sars-cov [7] . reports of efficacy of corticosteroid in some persons with sars-cov infections have resulted in widespread use of this therapy to treat covid-19, especially for persons with severe infection hospitalized in the icu. presently, there are few data on safety and efficacy of corticosteroids in this setting [2, [8] [9] [10] . because of the overlapping genetic and clinical feature of sars-cov-2, sars-cov, and mers-cov, we performed a meta-analysis of safety and efficacy of corticosteroid use in these coronavirus infections. our meta-analysis focus on the effects of corticosteroids on virus clearing and mortality in persons infected with sars-cov-2, sars-cov, or mers-cov. we searched pubmed, web of science, medline, chinese database wanfang, and zhiwang using the terms: "coronavirus," or "covid-19," or "2019-ncov," or "sars-cov-2" or "severe acute respiratory syndrome," or "middle east respiratory syndrome," or "sars," or "mers" and "corticosteroid" or "glucocorticoid," "hydrocortisone" or "methylprednisolone" or "dexamethasone" or "steroid" with these boolean operators. searches were done on march 20, 2020. two investigators independently reviewed the identified abstracts and selected articles for full review. discordances were resolved by a third investigator. we identified 8788 publications. we next excluded 2057 duplicate reports. the remaining 6731 publications were scanned by 3 investigators who identified 159 articles relevant publications after considering title and abstract. in total, 11 publications met our selection criteria, 10 cohort studies [2, [8] [9] [10] [11] [12] [13] [14] [15] [16] and 1 randomized clinical trial [6] . we excluded studies from same geographical region to avoid duplication. because most studies were observational the possibility of duplicate reporting was addressed by deleting small studies from the same geographical region. publication flow and reasons for exclusions are displayed in fig. 1 using prizma flow diagram. inclusion criteria included: (1) research articles including observational studies and clinical trials but excluding reviews or case reports on the use of corticosteroids in persons with sars-cov-2, sars-cov, and mers-cov infections; (2) reported outcomes of virus clearance and/or death; and (3) published in english and/or chinese. studies with insufficient data were excluded. for each selected publication the following were extracted: (1) 1st author; (2) publication year; (3) country; (4) sample size; (5) subject-related covariates; (6) dose and duration of corticosteroid treatment; and (7) dose of corticosteroids. these data are displayed in table 1 . primary outcome measures were rate of virus clearing and death. secondary outcomes were duration of hospitalization, use of mechanical ventilation, composite endpoint, and corticosteroid use between severe-only and nonsevere subjects. the study used anonymized, published data requiring no ethics committee approval. the methodological quality of retrospective studies was assessed by the modified newcastle-ottawa scale (nos) [17, 18] which consists of three domains: (1) subject selection; (2) comparability of the study groups; and (3) assessment of outcome(s). a score of 0-9 was allocated to each study except the randomized clinical trial. we considered observational studies with an nos score ≥6 high quality. we included 1 study with an nos score of 5 because of a large sample size (>1000 subjects) but performed sensitivity analyses excluding this study. risk of bias in the randomized clinical trial was assessed according to the jadad scale in the following domains: (1) random sequence generation; (2) allocation concealment; (3) blinding of participants and personnel; and (4) completed withdrawals and dropouts. we pooled data and used risk ratios (rr) with 90 or 95% confidential intervals (ci) for dichotomous outcomes including (1) death; and (2) the composite endpoint (death, icu admission, or mechanical ventilation). we also used rr with 95% ci for dichotomous outcomes including (1) use of mechanical ventilation; and (2) corticosteroid application between severe-only and nonsevere subjects. if a study considered these as separate outcomes, we used death as the endpoint. subgroup analyses divided by severity of disease were performed for these two types of outcomes. definitions of severity of sars used sars guidelines of the ministry of public health of china with a blood arterial oxygenation index < 300 mm hg [15] . severity of covid-19 was based on american thoracic society guidelines [19] . we used mean difference (md) and 95% ci for the continuous outcome including days to sars-cov-2 clearing, duration of hospitalization. funnel plots were used to screen for potential publication bias. statistical analyses used review manager 5.3 (the cochrane collaboration). some studies [2, 11, 14, 15] identified subjects with nonsevere infection which we analyzed separately. the meta-analysis included 10 observational studies and 1 randomized clinical trials involving 5249 subjects. in three studies the publication language was chinese [12] [13] [14] and six, english [2, 6, 8-11, 15, 16] . six studies discussed sars [6, [11] [12] [13] [14] [15] , four, covid-19 [2, [8] [9] [10] and one, mers [16] . studies were published 2003-2020 and were conducted in china (guangzhou [2, 15] , beijing [14] , shanxi [13] , hong kong [6, 11] , wuhan [8] [9] [10] ) and saudi arabia [16] . the jadad scale of the randomized clinical trial was 4 and nos scores of the observational studies was 5-9. one study was considered poor quality evidence because of brief follow-up (table 1) . to test the impact of corticosteroid use on virus clearing we included 2 studies [6, 16] to test impact of corticosteroid use on risk of death, we included 8 studies [8] [9] [10] [11] [13] [14] [15] [16] fig. 3 ). to test impact of corticosteroid use on duration of hospitalization we included 3 studies [12, 13, 16] involving 828 subjects (sars, n = 519; mers, n = 309). the data indicate corticosteroid use increased duration of hospitalization with md = 9.66 days (95% ci, 5.15, 14.18 days); i 2 = 85%; fig. 4 . we were unable to further analyze this endpoint by cohorts because of our study design. to test impact of corticosteroid use on use mechanical ventilation we included 3 studies [11, 14, 16] involving 2887 subjects (covid-19, n = 2578; mers, n = 309). the data indicate corticosteroid use is not associated with use of mechanical ventilation, rr = 1.33 (95% ci, 0.73, 2.42; i 2 = 70%; fig. 5 ). we were unable to determine whether subjects began receiving corticosteroids before or after use of mechanical ventilation because of our study design. to test impact of corticosteroid use on the composite endpoint we included 9 studies [2, [8] [9] [10] [11] [13] [14] [15] [16] supplementary fig. 1 ). to determine if corticosteroid use was more common in subjects with severe versus nonsevere infections we compared frequency of use in these cohorts. we included 4 studies [2, 11, 14, 15] involving 4078 subjects (sars, n = 2979; covid-19, n = 1099). the data indicate subjects with severe-only coronavirus infections were more likely receive corticosteroids with an rr = 1.48 (95% ci, 1.03, 2.13; i 2 = 99%; supplementary fig. 2 ). fig. 2 the effect of corticosteroid on virus clearness. comparison of virus clearness between corticosteroid and comparator. fig. 3 the impact of corticosteroid on the mortality of studied subjects. comparison of mortality between corticosteroid and comparator. h. li et al. we found potential publication bias in mortality studies in subjects receiving or not receiving corticosteroids with some studies falling outside the 95% ci of the funnel plat ( supplementary fig. 3 ). there was publication bias in the studies included in the meta-analysis. sars-cov, mers-cov, and sars-cov-2 induce host immune responses which, when severe, damage the lungs and cause death [3, 20, 21] . corticosteroids are frequently used to treatment persons with these coronavirus infections, especially when severe. whether this strategy is safe and effective is controversial. other approaches include counteracting high interleukin-6 concentrations with drugs such as tocilizumab [22] . the results of our meta-analysis indicate giving corticosteroids in this setting delayed virus clearing of sars-cov and mers-cov. whether this is so for sars-cov-2 is unknown. our analysis of this issue included relatively few subjects so this conclusion should be viewed cautiously. we determined corticosteroid use did not reduce deaths but was associated with increased hospitalization duration. there was no favorable impact on a composite endpoint of death, icu admission, or mechanical ventilation. it was not possible to determine whether corticosteroid use preceded or followed use of mechanical ventilation. it is also important to emphasize associations and correlations do not imply cause-andeffect. because most of the data come from observational databases and because corticosteroids are more likely to be given to persons with severe infections there is selection bias and our conclusions should be viewed cautiously. also, because 6 of the 11 studies are from subjects with sars-cov and mers-cov our conclusions may not apply to sars-cov-2 infection. however, genetic homology of these viruses and similar, albeit not identical, clinical features of these infections make analyses of data from studies of sars-cov and mers-cov infections reasonable proxies. a previous meta-analysis of corticosteroid use in persons with influenza (mostly h1n1) reported increased mortality [23] . acute respiratory distress syndrome (ards) is the typical evolution of infectious pneumonia. it is unclear whether corticosteroids are effective in ards. three cohort studies summarized in a meta-analysis reported corticosteroid use increased mortality in persons with influenzarelated ards [24] . in another study low-but not highdoses corticosteroids reduced mortality in persons with ards [25] suggesting dose may be an important co-variate in data analyses. as indicated, the or for death in subjects with severeonly coronavirus infection receiving corticosteroids is rr = 0.92 (0.64, 1.32; i 2 = 80%). how to interpret this central estimate and 90% ci is controversial. although the ci includes 1 and the i 2 is generally regarded as not significant (using an arbitrary cutoff significance level), the most conservative interpretation is that the results are inconclusive and consistent with as much as a 36% reduction or an almost 1.32-fold increase in risk of death. it is important to consider the absence of a statistically significant or is not definitive evidence of risk or benefit. fig. 4 the association of corticosteroid and hospital stay length. comparison of hospital stay time between corticosteroid and comparator. safety and efficacy of corticosteroids in persons with sars-cov-2 infection covid-19 can only be properly tested in a randomized clinical trial. however, such a trial is unlikely to be done. our meta-analysis may help inform the decision whether to give corticosteroids. corticosteroid use in subjects with sars-cov-2, sars-cov, and mers-cov infections delayed virus clearing and did not convincingly improve survival, reduce hospitalization duration or icu admission rate and/or use of mechanical ventilation. there were several adverse effects. because of a preponderance of observational studies in the dataset and selection and publication biases our conclusions, especially regarding sars-cov-2, need confirmation in randomized clinical trials. in the interim we suggest caution using corticosteroids in persons with covid-19. our results also further consolidate the national institute of health (nih) covid-19 treatment guideline regarding corticosteroid interventions (https:// covid19treatmentguidelines.nih.gov/critical-care/pharma cologic-interventions/). funding sources had input into the design, execution nor analyses of the data nor the decision to publish the results. submission for publication was agreed by all authors all of whom had full access to of the data and take responsibility to submit the typescript for publication. author contributions yl and rpg designed study. hl and cxc searched databases and processed analysis. cxc, hl, yl, and rpg drafted the paper. yl, rpg, cxc, fh, jjw, and qyz revised the final paper. yl and rpg are responsible for the paper. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. a novel coronavirus outbreak of global health concern clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study severe acute respiratory syndrome: report of treatment and outcome after a major outbreak effects of early corticosteroid treatment on plasma sarsassociated coronavirus rna concentrations in adult patients a novel coronavirus from patients with pneumonia in china clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china corticosteroid treatment of severe acute respiratory syndrome in hong kong retrospectivestudy of the effect of glucocorticosteroids on the treatment of severe acute respiratory syndrome evaluating the effects of different treatments on severe acute respiratory syndrome the cox regression analysis on the use of corticosteroids in the treatment of sars treatment of severe acute respiratory syndrome with glucosteroids: the guangzhou experience corticosteroid therapy for critically ill patients with middle east respiratory syndrome effect of arterial revascularisation on survival: a systematic review of studies comparing bilateral and single internal mammary arteries laparoendoscopic single-site nephrectomy compared with conventional laparoscopic nephrectomy: a systematic review and meta-analysis of comparative studies diagnosis and treatment of adults with community-acquired pneumonia. an official clinical practice guideline of the severe acute respiratory syndrome: historical, epidemiologic, and clinical features middle east respiratory syndrome coronavirus (mers-cov) infection effective treatment of severe covid-19 patients with tocilizumab corticosteroids as adjunctive therapy in the treatment of influenza exploring the heterogeneity of effects of corticosteroids on acute respiratory distress syndrome: a systematic review and metaanalysis effect of different doses and time-courses of corticosteroid treatment in patients with acute respiratory distress syndrome: a meta-analysis key: cord-289535-srrfr1es authors: tregoning, j. s.; brown, e. s.; cheeseman, h. m.; flight, k. e.; higham, s. l.; lemm, n.‐m.; pierce, b. f.; stirling, d. c.; wang, z.; pollock, k. m. title: vaccines for covid‐19 date: 2020-10-18 journal: clin exp immunol doi: 10.1111/cei.13517 sha: doc_id: 289535 cord_uid: srrfr1es since the emergence of covid‐19, caused by the sars‐cov‐2 virus at the end of 2019, there has been an explosion of vaccine development. by 24 september 2020, a staggering number of vaccines (more than 200) had started preclinical development, of which 43 had entered clinical trials, including some approaches that have not previously been licensed for human vaccines. vaccines have been widely considered as part of the exit strategy to enable the return to previous patterns of working, schooling and socializing. importantly, to effectively control the covid‐19 pandemic, production needs to be scaled‐up from a small number of preclinical doses to enough filled vials to immunize the world’s population, which requires close engagement with manufacturers and regulators. it will require a global effort to control the virus, necessitating equitable access for all countries to effective vaccines. this review explores the immune responses required to protect against sars‐cov‐2 and the potential for vaccine‐induced immunopathology. we describe the profile of the different platforms and the advantages and disadvantages of each approach. the review also addresses the critical steps between promising preclinical leads and manufacturing at scale. the issues faced during this pandemic and the platforms being developed to address it will be invaluable for future outbreak control. nine months after the outbreak began we are at a point where preclinical and early clinical data are being generated for the vaccines; an overview of this important area will help our understanding of the next phases. in november 2019, a cluster of pneumonia cases was detected in wuhan, china [1] . these were the first cases of covid-19 caused by the novel beta-coronavirus sars-cov-2. the genetic information was made publicly available on 10 january 2020, 54 days after the first declared case. sixty-three days after the sars-cov-2 sequence was published, on 13 march 2020, the first doses of the first human vaccine were being tested. by 24 september 2020, the sars-cov-2 vaccine landscape included 43 candidates being tested in clinical trials and more than 200 candidates. as the results from the phase i trials and earliest phase ii/iii trials emerge, this review will cover the platforms under development, the type of immune response required and the path to a clinical product. coronaviruses are unusually large enveloped rna viruses, with a large positive-sense, single-stranded rna genome. the integrity of this lengthy genome is maintained by a proof-reading replicase. the sars-cov-2 genome encodes 11 open reading frames (orf), many of which have unknown functions (fig. 1 ). orf1a and orf1b both encode polyproteins, which are cleaved into multiple non-structural proteins. orf4 encodes the envelope protein, a viroporin [2] , and orf5 encodes the membrane protein; together, they coordinate viral assembly and release. orf9 encodes the nucleocapsid (n) protein. orf2 encodes the spike (s) surface glycoprotein, the viral entry protein and key antigenic determinant, which binds the angiotensin converting enzyme 2 (ace2) 163 receptor on the host cells. ace2 is commonly found on type ii pneumocyte cells in the airways. sars-cov-2 has a 10-20 times higher affinity for ace2 than the related coronavirus sars-cov-1 [3] , which was responsible for the 2002-04 sars outbreak. sars-cov-2 is able to bind ace2 from a wide range of mammalian species [4] . having bound ace2, spike protein is cleaved by a host cell surface bound proteinase, either furin or tmprss2, enabling entry of the viral capsid. there may be a relationship between the mechanism of viral entry via ace2 and the pathogenesis of disease. human coronaviruses can cause both mild (oc43, hku1, 229e and nl63) and severe (sars-cov-1, sars-cov-2 and mers) disease. for most patients (approximately 80%), sars-cov-2 causes an asymptomatic infection or mild symptoms [5] . the following signs are associated with a virus positive polymerase chain reaction (pcr) test: fatigue, fever, chills, loss of appetite and persistent cough [6] . a striking feature of infection with sars-cov-2 is anosmia, a loss of smell and taste, reported in approximately 64% of cases in one study [7] . whether viral spread to the lower respiratory tract is a precursor for severe disease is unclear; pneumonia with characteristic pulmonary ground glass opacity changes on chest ct scans is common, even in asymptomatic individuals [8] . blood clotting, respiratory compromise, renal damage and cardiovascular collapse are all features of severe disease. the greatest risk factor for severe covid-19 disease is age: the remarkable relationship with age is consistently observed, despite geographic variability in reported case fatality rates [9] . while sars-cov-2 is a new virus and, therefore, the exact correlates of protection are not completely defined, there are precedents from other respiratory infections in general and coronaviruses in particular [10] . there has been discussion that natural immunity to sars-cov-2 declines quickly; whether this is the case is still unclear. it is our speculation that because vaccines aim to evoke an immune response they could be more immunogenic than the virus itself, which might have mechanisms to dampen immune response: whether this speculation is correct or not is yet to be determined. the t cell response is important in the control of other respiratory infections, and therefore likely to be the spike (s) protein is the major antigenic determinant, coat is made of spike (s), membrane (m) and envelope (e) proteins. the rna in encapsulated with the nucleocapsid (n) protein). created with biorender.com important in covid-19 [11] . models of sars-cov-1 indicate that t cells can be protective. cd4 + t cell depletion in mouse models delayed viral clearance and enhanced disease [12] ; similarly, t cell transfer resulted in rapid viral clearance and disease amelioration [13, 14] . sars-cov-1-specific cd8 + t resident memory were protective in a mouse model in the absence of antibody [15] . t cell memory can be long-lived; sars-cov-1 t cells were detected 4 years after infection [15, 16] . for sars-cov-2, t cell responses have been observed to a range of antigens, including s, m, n and other orfs [17] . sars-cov-2-specific t cells have been detected in individuals who had asymptomatic or mild covid-19 [18] and sars-cov-2-specific t cells have been observed in contacts of infected individuals [19] . patients suffering from covid-19 had fewer t cells than healthy controls [20] . t cells, especially cd4 + t cells, can influence the immune response through the production of cytokines, and elevated cytokines have been associated with exacerbated disease [20] . the skewing of the cd4 + t cell response is likely to be important. t helper type 1 (th1) responses are central to the successful control of sars-cov-1 and mers-cov [21] . th17 responses have been speculated to be deleterious [22] , and increased th2 cytokines were seen in severe disease [23] . regulatory t cells are important in the resolution of infection, and were observed to be elevated in covid-19 patients [20] . circulating follicular t helper cells, important in defining recall antibody response to infection, have been observed in a small number of individuals with covid-19 [24] . it is not clear whether the 'cytokine storm' is a cause or effect of disease; understanding this relationship is critical in monitoring vaccine safety. antibody response. the humoral response is pivotal in later stages of infection and helps to inhibit subsequent reinfection. virus-specific antibodies were detectable in 80-100% of sars-cov-1 and mers-cov patients 2 weeks after onset of symptoms [25] [26] [27] [28] [29] [30] [31] , with delayed antibody responses associated with more severe disease. a number of studies have been performed to try to more clearly understand the antibody response to sars-cov-2; a systematic review of studies on antibody to coronaviruses [32] observed that antibody was rarely seen in the first 7 days of infection, but rose in the second and third weeks postinfection. it is unclear whether antibodies correlate with covid-19 severity. antibodies are likely to be an important part of vaccine-induced protection. in sars-cov-1, the antibody response is short-lived [immunoglobulin (ig)m and iga responses last less than 6 months and igg lasts approximately 1 year]; this is possibly the same for sars-cov-2 [33] . human challenge studies using non-covid-19 coronavirus strains suggest that higher antibody levels correlate with protection [32] . these challenge studies have also suggested that reinfection is possible [34] , but the dose in challenge studies may be higher than experienced during natural infection. two recent studies have observed natural reinfections with sars-cov-2, one asymptomatic [35] and one symptomatic [36] , although this is in the context of more than 25 million recorded cases globally, suggesting that it is a rare event. because of the overlap between sars-cov-1 and sars-cov-2 spike proteins, antibodies could be cross-neutralizing [37] . however, the most potent specific, neutralizing monoclonal antibodies against the receptor binding domain (rbd) of sars-cov-1 did not bind to the spike protein of sars-cov-2 [38] . one promising observation is that isolated neutralizing antibodies have minimally mutated vdj genes, which make inducing them possible with fewer rounds of vaccination [39] . most attention has focused upon neutralizing igg antibodies in the serum, but other antibody-mediated mechanisms may be important in disease pathogenesis. fragment crystallizable (fc) and fc receptor (fcr) interactions can regulate the inflammatory response [40] and the sars-cov-2 virus-antibody complex could potentially trigger such fcr-mediated inflammatory responses, causing acute lung injury [41] . the iga response may be important in determining disease severity of covid-19 patients, but remains relatively unexplored so far [42] . one concern with vaccine development for sars-cov-2 is that the immune response can cause disease, often in the act of clearing the infection. understanding vaccineinduced immunopathology is critically important for all emerging infectious diseases. vaccines for emerging infections will, by necessity, require a shorter turn-around from discovery to deployment, and therefore predicting safety early in the process is critical. vaccine-induced immunopathology can either present as an acute response to the vaccine itself or as disease enhancement after viral infection. vaccines can occasionally induce an acute autoimmune disease. this was observed during the 1976 h1n1 swine flu outbreak, where vaccination in the united states led to an increased risk of guillain-barré syndrome (gbs) [43] . the mechanism has not been fully determined, but one suggestion is off-target antibodies against ganglioside gm1. off-target autoimmune effects were also observed during the 2009 h1n1 swine flu pandemic, with narcolepsy observed in a subset of children immunized with a vaccine adjuvanted with as03 [pandemrix; glaxosmithkline (gsk)]. there was a very tight association with hla-dqb1*06:02 [44] . the proposed mechanism is inhibition of the hypocretin signalling pathway. curiously, another swine flu vaccine made by gsk (arepanrix; gsk) using the same adjuvant was not associated with narcolepsy [45] , suggesting that the side effect was not caused by the adjuvant. the level of viral proteins, specifically nucleoprotein, may have been the problem [46] ; anti-nucleoprotein antibodies have been seen to cross-react with hypocretin [47] . these acute events are relatively rare; the rate of gbs was 8 per million individuals vaccinated and narcolepsy at approximately 30 per million individuals vaccinated (all in individuals aged less than 20 years) [48] . the delayed effects of vaccines are difficult to predict; post-licensure monitoring will be critical, especially as the vaccines will potentially have been tested in fewer people during the prelicensure phases than other licensed products. disease enhancement following infection of vaccinated individuals has been seen in other viral diseases; for example, measles, respiratory syncytial virus (rsv) and dengue virus. of children who received formalin-inactivated measles vaccine and were then subsequently exposed to the wild-type measles virus, 15-60% developed a severe form of the disease [49] , causing the vaccine to be withdrawn in 1967. a similar situation was observed with formalin-inactivated rsv vaccination (fi-rsv) in a clinical trial in 1966. the fi-rsv vaccine induced mainly non-protective antibodies, and children who were seronegative to the virus prevaccination had enhanced disease and hospitalization compared to the control groups [50] . vaccine-enhanced disease has also been observed with the live attenuated tetravalent dengue vaccine (dengvaxia; sanofi pasteur inc., swiftwater, pa, usa), specifically in seronegative children [51] . disease enhancement following vaccination can occur by two main mechanisms: priming for a detrimental t cell response and priming for antibodies that can increase the risk of infection or severe disease. the cellular response to vaccination, particularly t cells and eosinophils, and the inflammatory mediators these cells release has been suggested to promote vaccineenhanced disease [52] [53] [54] . whether sars-cov-2 vaccine platforms will have negative outcomes on infection is currently speculative, and draws upon experience with other respiratory viruses. one important factor determining the t cell response is antigen selection. specific epitopes can affect t cell polarization and activation, therefore antigen selection for vaccine applications requires careful consideration [55] . both the s and n proteins of sars-cov-1 have epitopes that are recognized by cd4 + and cd8 + t cells. some vaccines which used the n protein induced an eosinophilic response associated with vaccine-enhanced disease [13] , and post-vaccination challenge of animals immunized with sars-cov-1 n protein induced severe pneumonia [56] . mismatch of epitopes between vaccine and challenge strain can also lead to t cell enhanced disease due to original antigenic sin, as seen in dengue [57] . the vaccine platform may be critical in determining disease outcome on infection. immunopathology in animal models has most commonly been linked to inactivated, alum-adjuvanted vaccines. for example, double inactivation [ultraviolet (uv) and formalin] of sars-cov-1 enhanced the eosinophilic response from the vaccine, eliciting a proinflammatory pulmonary response and failing to provide complete protection [56] . enhanced disease was also observed following immunization with a gamma-irradiated mers-cov vaccine [56] . the mode of inactivation can influence both the quality of antibodies and the polarization of the t cell response to the vaccine. formalin inactivation in particular has been associated with deleterious th2 skewing by the addition of carbonyl groups [58] , and th2 skewing has been seen for a formalin-inactivated vaccine for sars-cov-1 [59] . other methods of inactivation have been explored; for example, beta-propiolactone, uv or gamma radiation, which could prove to be a promising avenue forward for eliciting the correct t cell response [60] . immunopathology is not restricted to inactivated vaccines. it can occur following immunization with a range of vaccine platforms; for example, it has been seen in animal models of rsv vaccination with both viral vectors and dna vaccines [61] . similarly, a range of vaccines against sars-cov-1 induced th2-directed pulmonary immunopathology in mouse models [56] . age at vaccination may also be an important consideration in immunopathology: the fi-rsv vaccine was given to infants. infants have a different immune response to adults, and this may predispose towards a qualitatively different immune memory [62] . the humoral arm of the adaptive immune response can also contribute to disease, called 'antibody-dependent enhancement' (ade). ade has been observed with flaviviruses, coronaviruses and some viruses of the paramyxoviridae family [63] . ade can occur in two ways, either by causing immune complexes or by enhancing infection. antibodies are bispecific molecules; as such, they can form antigen-antibody complexes. these complexes can cause direct damage when complex deposition in the vasculature leads to complement deposition and vessel damage, as seen after the feline coronavirus infection [64] . immune complexes can trigger macrophage activation leading to the release of proinflammatory cytokines. immune complexes have been proposed to have a role in the enhanced disease seen after fi-rsv immunization [65] , and may have a role in sars-cov-2 [41] . antibodies can also increase viral disease by enhancing infection; some viruses utilize antibodies to enter target cells. in the case of dengue virus, pre-existing antibodies for one serotype of the virus can cause enhancement of infection upon subsequent exposure to a new serotype [63] . a number of mechanisms have been proposed: antibody bound to virus could facilitate entry into macrophages through their fcrs [66] and antibody might stabilize viral surface antigen into a mature form [67] . the avidity of the antibody has been suggested as an important factor, with low antibody avidity a risk factor [68] . ade has been reported in sars-cov-1 after viral challenge in mice [69] , ferrets [70] and macaques [71] using a range of different vaccine strategies. in mers-cov, a neutralizing monoclonal antibody targeting the spike protein promoted viral entry via the fc receptor [72] . it is not yet known whether antibodies to sars-cov-2 will enhance disease, but it is something that is being closely monitored [73] . animal models. as coronaviruses have previously been associated with immunopathogenesis, vaccine-enhanced disease is a potential concern for efficient vaccine design for sars-cov-2. the use of models can improve understanding [74] , potentially predicting correlates of protection or disease. the ideal animal model is permissive to infection with the virus and reproduces the pathology and clinical course observed in humans. since the sars-cov-1 outbreak in 2002-04 a range of species, including hamsters, cats, ferrets and non-human primates, have all been used to study pathogenesis of coronaviruses [75, 76] . despite productive infection in a wide range of laboratory species, few displayed overt clinical disease. several inbred mouse strains have been investigated to model sars-cov-1, including balb/c, c57bl/6, rag1 −/− and 129svev mice. although young adult mice infected with varying doses of sars-cov-1 showed evidence of infection, the inbred strains do not accurately reflect the alveolar damage seen in humans [74] . however, aged mice show signs of clinical disease despite, in many cases, the absence of the lung lesions seen in humans [77] , and therefore have been used more extensively than younger mice. transgenic mice expressing human ace2 (hace2) have also been generated; disease severity in transgenic mice largely correlated with the level of hace2 expression, and when challenged with sars-cov-1 they developed severe infection and 100% mortality was reached by day 7 [78] . mers-cov appears to be even more challenging to model, with most species resistant to infection, except for some primate species [79] and camelids [80, 81] . the same models are being used for sars-cov-2. infection of human ace2 transgenic mice with sars-cov-2 led to weight loss and viral rna was detectable in the lungs, as well as lung pathology [82] . symptomatic infection and transmission of sars-cov-2 between animals has been observed in hamsters [83] , and asymptomatic infection and transmission of sars-cov-2 has been observed in ferrets [83] . sars-cov-2 is also infectious in experimental settings using cats, but not dogs, pigs, chickens or ducks [84] . as with sars-cov-1, non-human primates, e.g. rhesus or cynomologous macaques, have been helpful for evaluating immune protection [85] . human challenge. as animal models do not fully recapitulate human disease, alternative strategies may be required. controlled human infection models (chim) are studies in which participants (either vaccinated or not) are intentionally challenged with an infectious organism [86] . chim trials of sars-cov-2 vaccine candidates could be particularly beneficial in vaccine and drug efficacy studies, especially if the community infection rate has declined due to epidemiological interventions [87] . the deliberate exposure of healthy individuals to sars-cov-2 requires a tight ethical and regulatory framework [88] . the major concerns are that we do not have complete understanding of the long-term sequelae of sars-cov-2 infection and there is a lack of rescue therapy to enable the resolution of severe infection, although recent findings suggest that dexamethasone may reduce mortality in severe disease [89] and remdesivir (gilead sciences, foster city, ca, usa) may improve clinical status [90] . the lack of rescue therapy is not unique to a sars-cov-2 chim. rhinovirus and rsv chim do not have a specific anti-viral treatment but are self-resolving, which may also be true for sars-cov-2 in healthy young adults. there are also challenges associated with the manufacture of a challenge virus stock, which requires a high-containment [biosafety level iii (bsliii)] laboratory. at the time of writing, no study had been established, although the world health organization (who) has published guidance [91] and several academic and contract research organizations are investigating the approach [92] . an alternative use of deliberate human infection has been proposed: to infect young, lowrisk individuals to build herd-immunity, and therefore safeguard the unvaccinated, immunocompromised and 167 immunologically naive [93, 94] . however, this strategy is unattractive because the risk factors for severe disease are not fully understood: ethically there are also questions about infecting groups of individuals for the greater benefit, especially if there is a financial incentive. a huge range of vaccine approaches against sars-cov-2 have been proposed (table 1) . these include traditional approaches -inactivated, live attenuated and protein/adjuvant approaches and more novel, as yet, unlicensed approaches -viral vectors and nucleic acids. this has been a rapidly evolving field and some of the vaccines are more advanced than others. we are focusing upon those that are in clinical trials at the time of writing ( table 2) . several factors need to be considered before any vaccine progresses to widespread usage. first and foremost is vaccine safety and efficacy. closely linked is the scope for global scale-up manufacture to produce enough doses to achieve herd immunity. the spike (s) protein. before looking at the platforms being developed, the antigen needs to be considered. based on experience with sars-cov-1, most vaccines target the sars-cov-2 spike protein. within the spike, the receptor binding domain (rbd) responsible for binding to and entering host cells is the primary target of neutralizing antibodies [95] , and some vaccines only include this region. however, a recent study that isolated monoclonal antibodies found that most of them targeted areas outside the rbd [39] . an important consideration is the correct folding of the protein, both during production and when the vaccine is in storage prior to deployment. the coronavirus spike is a type 1 fusion protein and is metastable, undergoing an irreversible conformational change to enable membrane fusion [3, 96, 97] . this may affect the ability of the antigen to induce neutralizing antibodies. a similar effect has been seen with the rsv fusion (f) glycoprotein. antibodies specific to prefusion f (pre-f) have better neutralizing capacity than post-fusion f-specific antibodies [98] [99] [100] [101] : stabilization of the pre-f form can lead to better responses. based on this, prefusion sars-cov-2 spike protein could elicit a more potent immune response and stabilized sars-cov-2 spike proteins have been generated with stabilizing proline mutations in the s2 domain [3, 102, 103] . coronavirus nucleocapsid (n) is also immunogenic: antibodies against the sars-cov-1 n protein are abundant and longer-lived than those against the s protein in recovered patients [104] . interestingly, in model systems of sars-cov-1, immunization with the n protein is associated with vaccine-enhanced disease [105, 106] . it is not known whether the n protein is a potential protective immunogen for sars-cov-2, although vaccine approaches that use whole virus -either inactivated virus or live attenuated approaches -will potentially include n protein. the n protein can be a useful diagnostic for infection during phase iii trials of s protein-based vaccines. while the emphasis has been on the generation of neutralizing antibodies, targeting t cell epitopes may provide additional protection [11] . in other respiratory viruses, for example rsv, t cell only strategies can enhance disease [61] and t cells can be deleterious in dengue [57] , although less evidence of this has been seen with influenza vaccines [107] . it is not yet clear whether sars-cov-2 behaves more like rsv or influenza. drawing on information about sars-cov-1 and mers-cov and using bioinformatics, potential immunogenic epitopes in the sars-cov-2 proteome have been predicted. a total of 781 human leucocyte antigen (hla) class i and 418 hla class ii epitopes common between sars-cov-1 and sars-cov-2 were found [108]. t cell responses against the structural proteins of sars-cov-1 were found to be more immunogenic than non-structural proteins [13] . a wide range of different platforms have been developed, which can be loosely grouped as proteins, inactivated virus, vectored vaccines, live attenuated and nucleic acid (fig. 2) . this is clearly a fast-moving space and the following is based on data accessed in september 2020; an updated website is available at https://vac-lshtm.shiny apps. io/ncov_vacci ne_lands cape/ and the who has a vaccine tracker [109] . as many of the vaccines under development are produced by commercial organizations, peer-reviewed publications concerning their development and efficacy are limited, as such some information has been taken from press releases which may be less robust in their scrutiny. published results from clinical trials are summarized in table 3 . as with other pathogens, recombinantly produced viral surface proteins can safely be used as vaccines for covid-19. although protein vaccines have a good safety profile they can have low levels of immunogenicity, which means that many require adjuvants to improve their efficacy. while bacterial protein vaccines can be made through the purification of whole pathogen preparations, viral subunit vaccines necessitate recombinant genetic 168 engineering. the genes encoding the chosen antigens are cloned or synthesized, expressed and purified using a variety of expression systems, including insect, bacterial, yeast and mammalian cells [110] . bacterial expression systems are often used because they have high levels of expression and are easy to scale-up, with fermenter repurposing relatively easy. however, for viral antigens, where post-translational modification can be important, the use of insect cells or mammalian cells may be preferential [111, 112] . several protein subunit vaccination approaches were under development for sars-cov-1 and mers-cov [113] [114] [115] . a subunit vaccine made up of sars-cov-1 spike protein fragments, expressed in escherichia coli, induced neutralizing pre-clinical (mice) [197] phase i trials [198, 199, 200] curevac pre-clinical (mice) [195] phase i trial [196] people's liberation army/ walvax antibodies in rabbits [116] . neutralizing antibodies were induced in mice after immunization with transgenic plants [117] or mammalian expressed [118] several sars-cov-2 protein vaccines are in development; eight candidates are in clinical trials, but no data are yet available from these trials. two of the earliest to be announced are from clover biopharmaceuticals and the university of queensland. clover biopharmaceuticals has used 'trimer-tag' technology to make a mammalian cellexpressed, spike protein subunit trimer vaccine [120] . this antigen can be recognized by antibodies in the sera of people who have recovered from sars-cov-2 [121] . the vaccine will be given in conjunction with gsk's adjuvant as03 or cytosine-phosphate-guanosine (cpg)/alum during the phase i trial (nct04405908). the university of queensland, funded by the coalition for epidemic preparedness innovations (cepi) has developed a recombinant subunit vaccine using spike protein that has been 'locked' in prefusion conformation using the molecular clamp technique [122] . this is currently being tested with mf59 (nct04495933). sanofi are developing a protein subunit vaccine against sars-cov-2, expressed using a baculovirus platform, funded by the us biomedical advanced research and development authority (barda). this has been reported to be delivered in conjunction with as03 from gsk [122, 123] or potentially one other adjuvant which has not been revealed. phase i clinical trials were initiated on 3 september 2020 (nct04537208), with an aim to make the vaccine available in early 2021 [122] . other protein candidates in clinical trials (table 2 ) are from adimmune (baculovirus-derived, alum adjuvanted), anhui zhifei (rbd only), instituto finlay de vacunas (rbd), kentucky bioprocessing (tobacco-derived protein), medigen (alum/cpg adjuvanted) and vaxxine (advax adjuvanted). differences in cost of manufacturing, location of the manufacturer and impact of the adjuvant will determine which candidates progress beyond clinical trials. virus-like particles (vlps) are a subset of protein vaccines which are artificially produced nanoparticles that resemble viruses. rather than an individual protein, vlps are made up of some or all of the proteins that form the viral capsid [124] . they have some similarities to live attenuated or inactivated vaccines, and can produce strong cellular and humoral immune responses with no risk of reversion, because they contain none of the genetic material of the virus. they are used for a wide range of viruses, including hpv, and a preclinical sars-cov-1 vlp has been tested [125] . vlp nanoparticles are self-assembling protein particles, not necessarily derived from the virus capsid proteins. novavax, funded by cepi and us operation warp speed, have developed a recombinant nanoparticle vaccine (nvx-cov2373) that displays the sars-cov-2 spike protein [122, 126] . this is produced using engineered baculovirus to infect sf9 insect cells [127] . for the clinical trial with nvx-cov2373 novavax are using their own saponin-based matrix-m adjuvant (nct04368988), the data from which have recently been published [128] . the vaccine was immunogenic, but required the addition of adjuvant to achieve 100% seroconversion; two doses were required for neutralising antibody in all individuals. immunized animal models develop spike proteinspecific antibodies that prevent the attachment of the spike protein to host cell ace-2 receptors and also neutralize the wild-type virus [129] . another company (medicago) are using a plant-based system, nicotiana benthamiana, to produce a vlp [130] which is currently in clinical trial in combination with cpg or as03 adjuvant (nct04450004). other groups at the preclinical stage include saiba ag, based in switzerland, who are using a cucumber mosaic virus vlp that is bound to sars-cov-2 rbd, which induced neutralizing antibody in mice [131] . peptide vaccination is based upon the concept that, as induction of t cell responses can be achieved using a fraction of the entire protein [132, 133] , only the minimal immunogenic peptide sequence needs to be included. by selecting conserved epitopes, peptide vaccines can potentially induce broad-spectrum immunity against multiple strains of a given pathogen [134, 135] . peptides are easier to produce than whole protein antigens, as they can be produced synthetically and do not require folding into a tertiary structure. however, peptide vaccines are often weakly immunogenic. this is due to several factors, including the relatively small size of the peptide and differences in mhc processing; they therefore may require carrier proteins or adjuvants [136, 137] . several groups are exploring the use of multi-epitope peptide vaccines against sars-cov-2; following bioinformatic and immune-informatic-based predictions of immunogenic epitopes [138] [139] [140] [141] , the studies are focusing upon t rather than b cell epitopes. ose immunotherapeutics have used a multiepitope peptide approach to induce t cell responses in mice [142] . covaxx and the university of nebraska medical center have recently registered a phase i clinical trial for a multi-epitope peptide vaccine (nct04545749) as has the vector institute (nct04527575) currently in clinical trial. artificial antigen-presenting cells (aapc) are immunotherapeutic agents that can stimulate antigen-specific t cell responses [143] they have been widely explored for cancer vaccines and have also been proposed for infectious disease vaccines. in the case of sars-cov-2, aapcs are transfected with a lentivirus encoding the structural and protease proteins. the cells are then administered via subcutaneous injection [144] . the shenzhen geno-immune medical institute in china are undertaking an ongoing phase i clinical trial with an aapc approach (nct04299724) and a modified dendritic cell platform (nct04276896). aivita biomedical inc. are following a similar platform (nct04386252). due to the need to isolate and purify cells and maintain them at gmp quality, this approach seems impractical for mass vaccination campaigns. isolating and then inactivating a virus, historically with formaldehyde, is one of the oldest methods of viral vaccination. inactivation of viruses has been effective for a range of different viruses. however, there have been major safety concerns relating to sars-cov-1 and mers-cov-inactivated vaccines, reminiscent of fi-rsv, and these concerns are also valid for sars-cov-2. lung pathology of vaccinated animals on virus challenge has been seen for both a gamma-irradiated mers-cov vaccine [56] and a uv irradiation-inactivated sars-cov-1 vaccine [145] . the choice of both the adjuvant and the inactivating agent is important in shaping the immune response. for example, a formaldehyde inactivated mers-cov vaccine adjuvanted with alum and cpg demonstrated enhanced protection without inducing eosinophil-mediated vaccine-related pathology [146] . there are four inactivated vaccine candidates in clinical trials. sinovac biotech are using a platform previously developed for sars-cov-1 [147] . the virus is grown in vero cells and inactivated with beta-propiolactone. the inactivated vaccine was safe and immunogenic in rhesus macaques and offered complete protection against sars-cov-2 challenge, where no virus was detected in the pharynx or lungs [148] . two different versions of this inactivated vaccine have been developed, adjuvanted with either alum or cpg108. this vaccine has completed a phase ii human trial in 600 healthy adults aged 18-59 years (nct04352608), with 90% seroconversion observed after the second dose of vaccine and some neutralizing antibody detected [149] . it is interesting to note that the production method for the virus was changed between phases i and ii trials, and this may have increased immunogenicity. the vaccine has entered phase iii clinical trials in brazil (nct04456595) and indonesia (nct04508075). sinopharm, working with both the beijing institute of biological products and the wuhan institute of biological products, have also developed an inactivated vaccine. this vaccine has now been tested in a phases i/ii clinical trial (chictr2000031809). no serious adverse effects were observed, and more than 95% of individuals seroconverted with detectable neutralizing antibody in the two different trials [150] . the antibody was mainly observed after the second dose. two other organizations, bharat biotech (india) and the institute of medical biology/chinese academy of medical sciences, are running clinical trials of inactivated vaccines, but these are ongoing with no published data as yet. valneva, based in scotland, have just expanded their bsl3 manufacturing capacity and have signed a deal with the uk government for 100 million doses of a formaldehyde-inactivated vaccine adjuvanted with cpg, based on their japanese encephalitis virus vaccine [151] . the use of a live virus to prevent infection is the oldest vaccine approach. the original vaccine, cowpox, used exactly this approach to prevent smallpox. we are grouping two approaches under live viral vaccine platforms: attenuation of the virus or the use of a viral vector to deliver transgenes. live attenuated vaccines closely resemble natural infection. as a result, they are often immunogenic with a single administration without an adjuvant [152] . one consideration is balancing attenuation and replication -over-attenuated vaccines may not replicate enough to be immunogenic, and this balance can vary between different individuals, especially the very young or immunocompromised. historically, serial passage for attenuating mutations has been used; for example, live attenuated influenza vaccine (laiv) is cold-adapted, restricting it to the upper airway. this method requires time and extensive testing: the yellow fever vaccine yf17d was passaged more than 200 times. alternatively, attenuated viruses can be generated by reverse genetics [153] , introducing site-directed mutations into genes associated with virulence. the e protein has been targeted for both sars-cov-1 and mers-cov [154, 155] . however, this method requires the identification of genes that would attenuate viral replication and the mutation(s) inserted to be phenotypically stable [153] . a novel method of codon-pair de-optimization has been developed. the codon de-optimized virus is chemically synthesized to retain 100% amino acid sequence identical to the parent virus, but to contain an increased number of cpg and upa rna dinucleotides to up-regulate host responses. codon-pair de-optimization has been used for attenuating rsv [156] . codagenix and the serum institute of india are developing a live attenuated sars-cov-2 vaccine, using codon de-optimization technology, building on previous experience with rsv and influenza [157] . in vectored vaccines, the antigenic gene of interest is expressed from another micro-organism, either virus or bacteria. adenovirus, vsv and modified vaccinia virus ankara (mva) are some of the common viral vectors used [158] . the vectors can either be replication-deficient, delivering a gene cargo but not growing themselves, or replication-competent, reproducing in the immunization site. the different platforms may alter the reactogenicity and immunogenicity of the vaccine. a recombinant mva expressing the sars-cov-1 s protein delivered via intranasal or intramuscular routes induced protective immunity in mice [159] . an adenovirus vaccine against mers-cov offered complete protection against challenge in mice [152] . as pre-existing immunity against human adenovirus is widespread and can hamper its clinical application as a vector [158] , a chimpanzee adenovirus can be used. a recombinant chimpanzee adenovirus (chadox1) encoding the s protein, known as mers001, was immunogenic in mice and safe in phase i clinical trials in humans [160] . five non-replicating viral vectored vaccines are currently in clinical trials all based around adenoviral vectors. replication-deficient adenoviral vectors lack the e1a and e1b genes; these are the early genes which are essential for reproduction of the virus [161] , and deliver the antigen gene without replicating in the vaccinated individual. building on experience with mers-cov, the university of oxford are developing a chimpanzee adenovirus vaccine vector expressing the wild-type s protein (chadox1 ncov-19, also known as azd1222). the azd1222 vaccine was immunogenic in mice and pigs [162] . in rhesus macaques it reduced viral load and pneumonia after challenge with sars-cov-2 [163, 164] . the azd1222 vaccine entered phase i clinical trial on april 23 2020 in 543 volunteers aged 18-55 years (nct04324606). in this study, there were local and systemic reactions to the vaccine, controlled by paracetamol, but no severe adverse effects. the vaccine was immunogenic, with 91% participants having neutralizing antibody after one dose and 100% after two doses. interferon (ifn)-γ-producing t cells were also detectable [165] . in partnership with astrazeneca, this vaccine received a further $1.2 billion from barda towards its global development, manufacturing and distribution. the vaccine has now progressed into phases ii/iii trials in the united kingdom (nct04444674), brazil (isrctn89951424) and the united states (nct04516746). cansino biologics (china) are developing a human ad5vectored vaccine. in phase i trials (chictr2000030906), the ad5 vectored covid-19 vaccine was tolerable and immunogenic at 28 days post-vaccination [166] . both humoral responses and specific t cell responses were observed in healthy individuals 28 days after vaccination. transient and self-limiting adverse events such as severe fever, fatigue and muscle pain were reported in the high vaccine dose group. similar results were reported after the phase ii trial [167] . the vaccine is now in a phases i/ii trial in canada (nct04398147). [198, 199, 200] cansino biological subset had t cell responses analysed. [128] janssen (part of j and j) are using an experimental, replication incompetent adenovirus vector (advac ® ) in their per.c6 ® cell line technology [168] . this platform has been used for zika, rsv and hiv vaccine candidates. an ebola vaccine (ad26.zebov) using the same platform has been proven safe and immunogenic, and has been used as part of efforts to contain democratic republic of the congo (drc) ebola outbreaks [169] . the vaccine has been seen to be protective against sars-cov-2 challenge in rhesus macaques [170] and is in phase i trials in the united states, belgium (nct04436276) and japan (nct04509947). one other vectored vaccine that has received a great deal of press attention is from the gamaleya research institute, which has been given the tradename sputnik v. this vaccine uses two different adenovirus vectors, ad26 and ad5. to date, two clinical trials have been registered giving individually or as a prime-boost, either as a solution (nct04436471) or lyophilized formulation (nct04437875) in a total of 75 people. the trial recorded mild-moderate systemic effects and mild local effects, including injection site pain; 100% seroconversion rate by binding enzyme-linked immunosorbent assay (elisa) was observed. interestingly, there was also some anti-vector antibody detected after immunization [171] . the registration of this vaccine is presumably subject to larger efficacy trials, with a phase iii trial registered in september 2020 (nct04530396). an alternative to replication deficient vectors is to use a live attenuated vector. merck has recently acquired themis, who have developed an attenuated measles vector vaccine approach using an attenuated strain of measles derived from the original 1954 vaccine strain. themis have previously used this approach to develop a chikungunya vaccine, which was safe and immunogenic [172] . in collaboration with institut pasteur and the university of pittsburgh they are now running clinical trials with these vaccines (nct04497298 and nct04498247). live and vectored vaccines may lend themselves to mucosal delivery which may achieve better local immunity and has been used for other vaccines; for example, intranasal live attenuated influenza vaccine (laiv). however, the enthusiasm for mucosal vaccines based on preclinical data has not always translated into clinical success. symvivo is using oral delivery of a probiotic bacteria, bifidobacterium longum, to deliver the spike transgene (nct04334980). the migal galilee research institute have adopted an existing vaccine against infectious bronchitis virus (ibv), which has been used in a preclinical veterinary trial inducing humoral, cellular and mucosal immunity [173] to be delivered orally, but this is not yet in clinical trials. beijing wantai biological pharmacy and xiamen university have recently registered a phase i clinical trial using an influenza viral vector (chictr2000037782). nucleic acid vaccines have been highlighted for their potential in pandemic situations due to their low cost and potential rapid development, although this potential has yet to be translated into a real-world vaccine [174] . they utilize either plasmid dna or rna, encoding a target antigen. following delivery of the vaccine, the nucleic acid is taken up by the cells and the encoded antigen is expressed. conceptually, one facility can produce any required nucleic acid vaccine and production can be theoretically scaled-up to meet pandemic level demands. the covid-19 pandemic will serve as an important test case for nucleic acid vaccines, with six rna platforms and four dna platforms currently in clinical trial. most dna vaccines are constructed from plasmids that contain prokaryotic sequences that support the plasmids' propagation in e. coli, and a mammalian expression cassette that controls the expression of the target transgene in the vaccinated organism. the expression cassette contains an upstream promoter to drive transgene expression, a kozak sequence, the inserted transgene and a 3′ polyadenylation (polya) tail. following delivery, the dna vaccine is taken up by host cells local at the immunization site or by migrating apcs [175] . to induce an adaptive immune response the dna must enter the cell nucleus. in transiting to the nucleus the dna passes through the cytosol which is inflammatory, being sensed by intracellular pattern recognition receptors, for example sting1 [175] or tbk1 [176] , inducing an innate immune response. the triggering of innate immunity is essential for promoting adaptive immunity to dna vaccines. if apcs are transfected directly with a dna vaccine, they will load vaccine-encoded peptides onto both mhci and mhcii molecules and activate t cells [177] . transfected stromal cells will generate antigen, which will be encountered by apcs and b cells following antigen release from cell exosomes or apoptotic bodies. transit of injected naked dna to the nucleus is highly inefficient, with a large majority of the dna failing to cross the cell membrane or nuclear envelope [178, 179] . to mitigate this loss, dna vaccine programmes employ delivery platforms such as electroporation and bio-injection. preclinical animal studies have demonstrated that dna vaccines encoding the m, n, 3a or s proteins of the sars-cov-1 virus could elicit immune responses [180] [181] [182] . a multivalent dna vaccine encoding s and m protein epitopes could protect from sars-cov-1 cytopathic effects. the s protein is the target of the only sars-cov-1 dna vaccine to progress to phase i clinical trial, delivered by bio-injector, and it was safe and induced neutralizing antibody responses [183] . the leading dna vaccine against mers-cov (ino-4700) was developed by inovio. phase i clinical trials were completed in 2019, with the vaccine showing a good safety profile and inducing humoral immunity and polyfunctional cd8 + t cell responses [184] . the inovio mers ino4700 (gls-5300) vaccine that was due to be taken to phase ii clinical trials (nct03721718) has now been redeployed as ino-4800 (nct04336410) to begin clinical trials for protection against sars-cov-2. in preclinical studies of the ino-4800 vaccine, neutralizing antibody and t cell responses were observed in mice and blocking antibody responses in vaccinated guinea pigs [185] and macaques [186] . the phase i trial (nct04336410) is ongoing, but the data have not yet been published. genexine, in south korea (nct04445389), zydus cadila in india (ctri/2020/07/026352) and osaka university in japan (nct04463472) have initiated phase i trials of dna vaccines. rna vaccines are based on the same premise as dna vaccines of expressing a vaccine antigen transgene in the host cell, but they are one step further along the expression pathway, skipping the transcription step. unlike dna vaccines, expression of rna vaccines begins once they enter the cell cytosol, which can increase the efficiency of expression. as with dna vaccines, the presence of 'foreign' rna is sensed in both the endosome and cytosol [187] , giving rna vaccines a self-adjuvanting effect [188] . however, the early triggering of type i ifn responses can downregulate protein expression [189] . modified nucleosides can be incorporated into the mrna product to create a 'silenced' rna vaccine that avoids detection by tlrs and does not trigger a type i ifn response [190, 191] , but there is a balance between antigen expression from the vaccine construct and triggering enough inflammation to activate the immune response. this balance may be altered by the formulation to deliver the vaccine and can be different between different animal species, making predictions from preclinical studies difficult. there are two primary types of rna vaccine mrna and self-amplifying mrna (sarna). non-replicating mrna vaccines are constructs engineered to encode the gene of interest, and typically have a 5′ cap, utrs flanking the gene of interest and poly a tail. the 5′ cap is essential for mrna to associate with the eukaryotic translation complex. utrs are selected to optimize rna protein expression, avoiding the inclusion of sequences that would hamper translation [192, 193] . mrna vaccine constructs are made using bacteriophagederived rna polymerases and ntps to transcribe linearized dna in vitro. self-amplifying rna vaccines are alphavirus-derived rna replicons modified to encode the antigen of interest in place of rna structural proteins. the viral replicon also contains an open reading frame (orf) that encodes four alphavirus non-structural proteins (nsp1-4) and a subgenomic promoter. the non-structural proteins form an rna-dependent rna polymerase (rdrp). the rdrp complex transcribes more copies of the vaccine in the transfected cell. as a result, sarna vaccines express protein at higher levels and persist for longer than non-replicating rna [194] . as it is a newer technology, rna vaccines were not developed against sars-cov-1. six rna vaccines are in clinical trials for sar-cov-2. moderna fast-tracked their candidate vaccine mrna-1273 and were first to begin clinical trials on 17 march 2020 with the national institute of health's national institute of allergy and infectious diseases (niaid) (nct04283461). this phase i study involved 45 patient volunteers, divided into three group cohorts, as a dose escalation: low (25 µg), middle (100 µg) and high (250 µg) in a prime boost. a preclinical study using the same vaccines was protective in mice against viral challenge [195] . the vaccine was immunogenic, with increasing antibody titres with increasing dose administered; of note, three individuals (of 15, 21%) in the 250-µg group reported severe adverse events, with severity increasing after the second vaccination [196] . the vaccine is now in phase ii (nct04405076) and phase iii (nct04470427) trials, focusing on the 100-µg dose. biontech is collaborating with pfizer to develop four s protein vaccine candidates. they are using a nucleoside modified mrna. phases i/ii clinical trials are running in germany (nct04380701) and the united states (nct04368728), with a multi-site phase iii study planned. the vaccine induced both cellular and humoral responses in mice [197] and induced neutralizing antibody in the clinical study [198] . they have performed a further two clinical studies, observing similar responses in a second study with their initial construct (bnt162b1) which encodes a rbd trimer [199] . in a comparator study they observed similar levels of immunogenicity to a stabilised membrane anchored spike protein (bnt162b2), but with lower levels of reactogenicity [200] . both curevac and the people's liberation army have also developed mrna vaccines that are in clinical trials, but no results have been published as yet. imperial college london and its spin-out social enterprise, vacequity global health, are developing an sarna vaccine encoding the s protein. intramuscular injection with lnp formulation induced high neutralizing antibody titres in mice [103] . tested doses of the preclinical vaccine ranged from 0·01 µg to 10 µg, with a boost of the same dose at week 4 post-vaccination. human trials of the vaccine with 420 participants started in june 2020 (isrctn17072692). arcturus, based in singapore, are also developing an sarna vaccine encoding a prefusion spike, which is in phase i clinical trial (nct04480957). protein vaccines can have low levels of immunogenicity. this can be boosted by adjuvants [201] . adjuvants enhance the immune response through multiple mechanisms, causing a depot effect; up-regulating the production of chemokines and cytokines; enhancing the cellular recruitment to site of injection; increasing antigen uptake and presentation by apcs and increasing inflammasome activation [202] . adjuvants can also tailor the immune response, guiding it towards producing the most effective form of immunity against the specific pathogen being vaccinated against [203] [204] [205] . a range of adjuvants have been proposed for use with sars-cov-2 protein vaccines. these include advax, alum, as03 (gsk), matrix-m (novavax), cpg (dynavax) and mf59 (csl). additional components are also included with nucleic acid vaccines to enhance uptake and immunogenicity. nucleic acids are combined with a range of formulations, including lipid nanoparticles (lnps), liposomes and polyplexes. such formulations are essential for rna vaccines, as 'naked' rna is susceptible to being degraded by extracellular rnases which will prevent efficient cell uptake. lnps have previously been used for other rna therapeutics and moderna, imperial college london, arcturus, curevac and biontech vaccines all utilize this technology. the stability of these formulations can be a concern and may necessitate vaccine storage at a lower temperature which might, in turn, impact access to the vaccine. for dna vaccines, delivery devices are often used to increase uptake. inovio uses an electroporation device (cellectra ® 2000), which delivers an electric current to the site of injection: in a study on acceptability, acute pain (six of 10 on the vas score) was recorded for the first 5 min after immunization, but this receded [206] . a similar effect was observed in a study using a different electroporation device [207] . genexine are also using electroporation in their trial, but they are also comparing with a needle-free biojector. biojector devices have been shown to increase the antibody response to dna vaccines [208, 209] . one of the more experimental approaches proposed to reduce the impact of covid-19 has been the use of other live vaccines as non-specific vaccines [210] . this has been proposed for bacille calmette-guérin (bcg), oral polio vaccine [211] and measles, mumps and rubella (mmr) [212] . the proposed mechanism is described as trained immunity, where exposure to one agent alters the epigenetic profile of innate immune cells, potentially increasing the production of cytokines. in preclinical models, bcg pretreatment has been shown to reduce influenza viral titres [213] . early ecological data (in april 2020) suggested that countries with mandatory bcg vaccination had reduced mortality from covid-19, but this analysis has a number of issues, mainly associated with demographics and the timing of when the virus reached different countries [214] . a more recent study has supported this protective effect [215] . remarkably, a number of randomized clinical trials have been set up to directly test whether bcg can reduce the burden of covid-19. the covid-19 pandemic has led to a surge of different vaccines being rapidly moved to clinical trials. a number of these vaccines have been around for several years as promising preclinical platforms, but not necessarily been attractive enough to generate funding to support human trials. each of the approaches has advantages and disadvantages (table 4 ): which aspects are the most important will only be identified following efficacy studies. live attenuated vaccines have a long track record of safety and efficacy, but they may not be feasible in the current pandemic due to the length of time it takes to generate a candidate and test for attenuation. inactivated vaccines also have a long track record of protective efficacy, and they have the advantage that they are fast to generate; however, they require highcontainment facilities to generate the virus stock. there is also a concern about vaccine-induced immunopathology with an inactivated vaccine, which has been seen for some other respiratory viruses and in preclinical models of sars-cov-1; whether this is the case for inactivated sars-cov-2 vaccines will only be seen after larger and longer phases ii/iii trials. recombinant protein vaccines have been in use since the 1980s; they are a more targeted approach than using a whole virus, which may focus the immune response on a key antigen, but this may lose some breadth of protection. protein candidates were somewhat slower to enter clinical trials but may have a faster route to licensure, being a more known product than newer vaccines. one challenge is to use the correct conformation of the protein, spike is metastable and may be less protective if used in a post-fusion form. one peptide vaccine has registered a clinical trial (nct04527575) from the vector institute in koltsovo, russia; it is adjuvanted with alum. the cellular-based approaches, using aapc, do not seem to be practical for wide-scale rollout. nucleic acid vaccines, both dna and rna, have much potential in terms of speed of response and scale-up: this outbreak will be an important test for whether they can deliver on their promise. dna vaccines have historically been less immunogenic than other platforms, although with alternate delivery devices that may be overcome. rna vaccines have not been widely tested for infectious diseases; this is the first time an sarna vaccine has been trialled. rna may have a slight issue concerning heatstability, necessitating -80˚c storage. viral vectored vaccines are the furthest ahead in clinical trials, with three candidates in later phase clinical trials. they are known to be safe, but may be reactogenic at higher doses. historically, these approaches have had mixed results for efficacy and one concern is pre-existing immunity against the vector, especially when a human viral-derived vector such as ad5 is used. it will be of great interest to see which platforms and candidates are protective in efficacy trials. as of september 2020, the furthest advanced candidates have completed phase i trials (table 3) , although so far not all the organizations involved have published data from completed trials. all the data published so far indicate that the vaccines are safe, but there are more adverse events at higher doses: both the moderna [196] and biontech [198] vaccines had severe adverse effects at the highest doses, leading to a lower dose in later studies; some severe adverse effects were also recorded in the cansino [166, 167] and university of oxford [165] vectored vaccine trials. the vaccines all appear to be immunogenic, although it is hard to compare directly, as different groups will have used subtly different elisa and neutralization assays. a further complication for comparison is when data have been published as press releases rather than peer-reviewed papers. ultimately, vaccine efficacy in a randomized trial is the most important issue, but here again, different primary end-points are being explored. some studies are looking at reduction of disease, while others are looking at reduction of confirmed infections. the covid-19 crisis represents an opportunity for several experimental vaccine platforms to progress to clinical trials. however, there are considerations between a promising preclinical candidate and a global vaccination campaign, including trials, regulation and manufacture. clinical vaccine trials conventionally undergo four broad phases, from early safety in small numbers of volunteers (phase i) to wide-scale post-licensure monitoring (phase iv). usually, each of the phases take months or even years to complete before moving on. in a pandemic setting, there is need to speed up the transition between phases; this has been achieved with co-operation of regulatory agencies and research ethics committees. additionally, phases can be merged, with planning of phases ii/iii trials initiated before the phase i trial has even begun. there is the potential that data obtained from phase i will mean that planned later-phase trials are cancelled due to safety concerns or futility. ultimately, pushing a vaccine through the different stages of a clinical trial does not negate the need for complete safety data sets to be collected, but close co-operation with the researchers and regulators can accelerate a progress that could reduce 10 years to 18 months. one consideration is that due to the accelerated time-scale, postlicensure monitoring will be extremely important. another issue concerns the sample size required for efficacy studies; as cases fall, larger studies will be necessary or an alternate trial design. a ring trial was used in ebola [216] , which allowed efficacy to be assessed in a few individuals. one of the major hurdles to a vaccine relieving the covid-19 pandemic is manufacturing enough doses to achieve global herd immunity. the number of doses needed to achieve global coverage depends upon the regime used, but is potentially as many as 16 billion (assuming a prime boost regime with some contingency). to ensure licensure and prequalification status, good manufacturing process (gmp) standards must be upheld during up-scaled manufacturing and clinical studies. manufacturing a vaccine at a global scale in the timeframe required is a unique challenge. vaccines that are not only safe and effective but also highly scalable, to produce millions or even billions of doses, would be the most desirable tool for curbing the pandemic. logistics are a key consideration, including access to components to manufacture the vaccines -for example, nucleic acid vaccines require nucleotide triphosphates (ntps), which are also in high demand for the diagnostic tests. the vaccines also require plant and materials to fill and finish the final product; one bottleneck is exactly that: glass bottles. in parallel with the accelerated clinical trials, accelerated manufacturing scale-up is required. this telescoped manufacturing process means that investment in the next step is being made before the results of the previous step are known. this has considerable financial risk, especially in terms of setting up the necessary manufacturing plant if it cannot be repurposed, either for other pandemic vaccines or other biologicals. funding is a critical part of the vaccine development process. it remains to be seen whether the total costs of research, development and licensure of any novel vaccine platforms for sars-cov-2 is comparable to traditional vaccines, such as dengvaxia, which costed approximately $1.5 billion until licensure [217] . a range of funding mechanisms have supported vaccine development. one of the major bodies co-ordinating the funding is cepi, which has received funding from multi-national sources, including governments and charities. other vaccine candidate teams are being supported by their governments 'fast-tracking' their candidates through clinical trials and streamlining their manufacturing. for example, operation warp speed in the united states, is supporting six candidates from astrazeneca (azd1222), moderna (rna), pfizer (rna with biontech), merck (vectored vaccine with themis), johnson and johnson (vectored vaccine) and novavax (recombinant protein). a uk vaccine task force was announced on 20 april 2020 [218] to support uk-based candidates and reviewing government regulations to facilitate rapid and safe vaccine trials. several vaccine candidates have never progressed past phase i before, therefore manufacturing gmp material at scale poses new challenges. many of the vaccines are being developed by either academic groups or small-to mediumsized biotech companies, neither of which necessarily have capacity to manufacture at a large scale. one approach is outsourcing to contract manufacturing organizations (cmos), which can have licensing complications. some companies have invested in manufacturing capacity; for example in july 2018, moderna opened a manufacturing facility in norwood (usa) [219] , which produce rna up to the gram scale. however, they still need to work with external companies to complete formulation protocols. new manufacturing facilities are also being constructed elsewhere, including the vaccines manufacture and innovation centre (vmic) in the united kingdom, which has been accelerated and is planned to open in mid-2021, and valneva opened an expanded bsliii facility in scotland in august 2020. larger companies may have more experience and capacity of manufacturing; for example, janssen and astrazeneca are making adenovirus vectors and sanofi is making a protein vaccine. in the initial stages of the outbreak there was relatively little publicized activity from the larger pharmaceutical companies, with most of the attention on smaller biotechs and academic groups. it is not clear why this was the case. one possible reason could have been exemption from liability, although vaccine manufacturers are exempt in the united states under the 2005 public readiness and emergency preparedness, or prep act. integrating national funding programmes with equitable global access to vaccines is vital. there is a concern that wealthy countries will monopolize initial production runs of vaccines, with preorders outstripping manufacturing capacity. alternative models of licensure, social enterprise and spoke and hub manufacture may be necessary. for example, the astrazeneca/oxford vaccine has been licensed to the serum institute of india, the largest global manufacturer of vaccines by doses produced, and imperial college london have established a social enterprise company to enable equitable access. vaccines require regulation to be introduced as a licensed product. there are a range of national and international bodies which cover this process; for example, any product trialled in the united kingdom will be considered for approval by the medicines and healthcare products regulatory agency (mhra). the product is then considered for prequalification by the who for multinational distribution, which has supported the regulation and distribution of vaccines for 33 years [220] . this process can normally take a substantial amount of time; however, during the covid-19 pandemic manufacturers and regulators are striving towards the delivery of a vaccine that is safe and effective within 18 months from february 2020. there is a precedent for accelerated licensure in the context of a pandemic, as seen with the approval of rvsv-zebov [221] as a vaccine for ebola. the who supported the accelerated regulatory approval for rvsv-zebov-gp using an expedited prequalification review following its receiving conditional marketing authorization from the european medicines agency (ema) [222, 223] . the who has issued guidance and recommendations for the regulation of sars-cov-2 vaccines [220] . the issues faced during this pandemic and the vaccine platforms being developed to address it will be invaluable for future outbreak control. while, at this stage, it is not possible to say which platform is best, and what works best for one infection may not be best for all infections or for all populations. one of the biggest considerations of all the platforms is speed into trials versus ability to deploy the vaccine. while some of the high-speed platforms, for example rna, have entered into phase i trials faster than other approaches, their lack of track record means that approaches for global scale-up are potentially slower. older approaches may leapfrog the newer platforms in the scale-up and manufacturing stage. one observation of interest is the speed with which one of the oldest technologies for viral vaccines, inactivation, has been able to move forwards. prior to covid-19, much of the attention for pandemic vaccine preparedness was on newer technologies; however, of the candidates in phase iii in august 2020, two of four are inactivated vaccines. safety and efficacy data from these large studies will be critical. another consideration is that while distributed global manufacture is effective and appropriate for routine scheduled vaccination, local surge manufacturing capacity for new vaccines is important. this capacity may have to be maintained at a loss for large amounts of time, unless alternative commercial contracts can be found for the same facility that can then be replaced at short notice. this reflects a broader consideration that investment in public health, which may appear expensive to begin with, can save a considerable amount in the long term; one study estimated that every £1 spent on public health saves £14 in return [224] . another consideration is for greater standardization of assays and end-points. comparisons of the different trials has been made significantly harder by the use of different methodologies. this is part of the broader global context of a true pandemic. collaborative worldwide action is required to control the virus, which necessitates leadership and a willingness to share by countries with more developed scientific research programmes. ultimately, while lessons will be learned from this pandemic, the next one will be different and sticking rigidly to a plan that controlled this coronavirus will not necessarily work for a different virus, as the german strategist helmuth von moltke 'sort of ' said: 'no plan survives contact with the enemy' . it is still far too early to know what the best approach will be to control covid-19 with vaccines. speculating as to which vaccine platform is 'best' , while academically enjoyable, is not of value here. that so many platforms, both new and old, are moving into efficacy study makes this an extremely exciting time for vaccinology. the outbreak will certainly be a test case for the novel vaccine platforms, particularly nucleic acid vaccines, which have promised much to date but not been licensed for human use. one issue is whether vaccines will play a role in reducing the burden of the pandemic. even at maximum speed, the first efficacy trials will start 9 months after the start of the pandemic and the first licensed doses are unlikely to be ready for 18 months, by which time the virus will have caused a large wave of mortality and a larger wave of global disruption. important questions remain (box 1) regarding what is a successful vaccine, how should it be deployed and who should be prioritized. these will depend in part upon the results of the efficacy studies, although the who has produced some draft guidance [225] . overall, it has been a remarkable chapter in vaccine development, with widespread collaboration and partnership in a race against the virus. a novel coronavirus from patients with pneumonia in china coronavirus envelope protein: current knowledge cryo-em structure of the 2019-ncov spike in the prefusion conformation the sars-cov-2 spike protein has a broad tropism for mammalian ace2 proteins presumed asymptomatic carrier transmission of covid-19 real-time tracking of self-reported symptoms to predict potential covid-19 alterations in smell or taste in mildly symptomatic outpatients with sars-cov-2 infection a new coronavirus associated with human respiratory disease in china opensafely: factors associated with covid-19 death in 17 million patients lessons for covid-19 immunity from other coronavirus infections t cell responses in patients with covid-19 cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4+ t cells are important in control of sars-cov infection how immune t-cell augmentation can help prevent covid-19: a possible nutritional solution using ketogenic lifestyle jri/vol4without altering disease transmission work? how to achieve equitable global distribution? is human challenge part of the pathway to a licensed product? what is the risk of immunopathology? t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection characterization of sars-cov-specific memory t cells from recovered individuals 4 years after infection targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19 intrafamilial exposure to sars-cov-2 induces cellular immune response without seroconversion reactive t cells in covid-19 patients and healthy donors the potential role of th17 immune responses in coronavirus immunopathology and vaccine-induced immune enhancement longitudinal analyses reveal immunological misfiring in severe covid-19 analysis of adaptive immune cell populations and phenotypes in the patients infected by sars-cov-2. medrxiv 2020 two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome persistence of viral rna in stool samples from patients recovering from covid-19 potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus kinetics of serologic responses to mers coronavirus infection in humans converting detachable wheelchair armrests into parallel bars transmission of merscoronavirus in household contacts a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease longitudinal evaluation and decline of antibody responses in sars-cov-2 infection the time course of the immune response to experimental coronavirus infection of man covid-19 re-infection by a phylogenetically distinct sars-coronavirus-2 strain confirmed by whole genome sequencing genomic evidence for a case of reinfection with sars-cov-2 identification of potential crossprotective epitope between a new type of coronavirus (2019-ncov) and severe acute respiratory syndrome virus potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody analysis of a sars-cov-2-infected individual reveals development of potent neutralizing antibodies with limited somatic mutation antibody in statspearls understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools the role of iga in covid-19 guillain-barré syndrome following vaccination in the national influenza immunization program, united states, 1976-1977 narcolepsy and influenza vaccinationthe inappropriate awakening of immunity comparison of pandemrix and arepanrix, two ph1n1 as03-adjuvanted vaccines differentially associated with narcolepsy development antigenic differences between as03 adjuvanted influenza a (h1n1) pandemic vaccines: implications for pandemrix-associated narcolepsy risk antibodies to influenza nucleoprotein cross-react with human hypocretin receptor 2 measles immunization with killed virus vaccine: serum antibody titers and experience with exposure to measles epidemic respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine effect of dengue serostatus on dengue vaccine safety and efficacy protection induced by virus-like particle vaccine containing tandem repeat gene of respiratory syncytial virus g protein alum adjuvant enhances protection against respiratory syncytial virus but exacerbates pulmonary inflammation by modulating multiple innate and adaptive immune cells intranasal nanoemulsion-based inactivated respiratory syncytial virus vaccines protect against viral challenge in cotton rats mechanisms for t cell receptor triggering immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms a potential molecular mechanism for hypersensitivity caused by formalininactivated vaccines formalintreated uv-inactivated sars coronavirus vaccine retains its immunogenicity and promotes th2-type immune responses examination of the effects of virus inactivation methods on the induction of antibodyand cell-mediated immune responses against whole inactivated h9n2 avian influenza virus vaccines in chickens immune responses and disease enhancement during respiratory syncytial virus infection vaccination strategies to enhance immunity in neonates vaccine-induced enhancement of viral infections immunopathogenesis of coronavirus infections: implications for sars a role for immune complexes in enhanced respiratory syncytial virus disease dengue viruses and mononuclear phagocytes. ii. identity of blood and tissue leukocytes supporting in vitro infection new insights into the immunopathology and control of dengue virus infection lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection molecular mechanism for antibodydependent enhancement of coronavirus entry a perspective on potential antibody-dependent enhancement of sars-cov-2 is there an ideal animal model for sars? lovastatin-mediated g1 arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-coa reductase caution urged on sars vaccines aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus development of animal models against emerging coronaviruses: from sars to mers coronavirus blocking transmission of middle east respiratory syndrome coronavirus (mers-cov) in llamas by vaccination with a recombinant spike protein humoral immunogenicity and efficacy of a single dose of chadox1 mers vaccine candidate in dromedary camels the pathogenicity of sars-cov-2 in hace2 transgenic mice sars-cov-2 infection in farmed minks, the netherlands susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 reinfection could not occur in sars-cov-2 infected rhesus macaques ethical guidelines for deliberately infecting volunteers with covid-19 human challenge studies to accelerate coronavirus vaccine licensure sars-cov-2 controlled human infection models: ethics, challenge agent production and regulatory issues effect of dexamethasone in hospitalized patients with covid-19 criner gjet al, for the gsusi. effect of remdesivir vs standard care on clinical status at 11 days in patients with moderate covid-19: a randomized clinical trial world health organization. key criteria for the ethical acceptability of covid-19 human challenge studies accelerating development of sars-cov-2 vaccines -the role for controlled human infection models vaccination-induced herd immunity: successes and challenges dissecting the indirect effects caused by vaccines into the basic elements novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov structural basis for human coronavirus attachment to sialic acid receptors emergence of sars-cov-2 through recombination and strong purifying selection structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody prefusion f-specific antibodies determine the magnitude of rsv neutralizing activity in human sera human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion f protein modified for virion packaging yields protective intranasal vaccine candidates a cysteine zipper stabilizes a pre-fusion f glycoprotein vaccine for respiratory syncytial virus development and characterisation of neutralising monoclonal antibody to the sars-coronavirus self-amplifying rna sars-cov-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice interactions of sars coronavirus nucleocapsid protein with the host cell proteasome subunit p42 t cell-mediated immune response to respiratory coronaviruses prior immunization with severe acute respiratory syndrome (sars)-associated coronavirus (sars-cov) nucleocapsid protein causes severe pneumonia in mice infected with sars-cov influenza vaccines and immunopathology the authors. clinical and experimental immunology published by john wiley & sons ltd on behalf of british society for immunology bioinformatic prediction of potential t cell epitopes for sars-cov-2 world health organization. draft landscape of covid-19 candidate vaccines recent advances in the production of recombinant subunit vaccines in pichia pastoris recombinant vaccines and the development of new vaccine strategies optimizing assembly and production of native bispecific antibodies by codon deoptimization optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design amino acids 1055 to 1192 in the s2 region of severe acute respiratory syndrome coronavirus s protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents severe acute respiratory syndrome (sars) s protein production in plants: development of recombinant vaccine receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines clover initiates development of recombinant subunit-trimer vaccine for wuhan coronavirus (2019-ncov) 12873 644/ vacci nes/clove r-initi ates-devel opmen t-of-recom binan t-subun it-trime r-vacci ne-for-wuhan -coron aviru s-2019-ncov.html clover successfully produced 2019-ncov subunit vaccine candidate and detected cross-reacting antibodies from sera of multiple infected patients 21073 754/vacci nes/clove r-succe ssful ly-produ ced-2019-ncov-subun it-vacci ne-candi date-and-detec ted-cross -react ing-antib odies the pandemic pipeline press release https:// www.gsk.com/en-gb/media/ press -relea ses/sanofi -and-gsk-tojoin-force s-in-unpre ceden ted-vacci ne-colla borat ion-to-fightcovid -19 virus-like particles as an instrument of vaccine production assembly of human severe acute respiratory syndrome coronavirus-like particles novavax awarded funding from cepi for covid-19 novavax recombinant protein nanoparticle vaccine technology phase 1-2 trial of a sars-cov-2 recombinant spike protein nanoparticle vaccine sars-cov-2 spike glycoprotein vaccine candidate nvx-cov2373 elicits immunogenicity in baboons and protection in mice covid-19: medicago's development programs development of a covid-19 vaccine based on the receptor binding domain displayed on virus-like particles synthetic peptide vaccines design and development of synthetic peptide vaccines: past, present and future comparing pooled peptides with intact protein for accessing cross-presentation pathways for protective cd8+ and cd4+ t cells dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and t-cell activation more than one reason to rethink the use of peptides in vaccine design peptide vaccine: progress and challenges preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies development of epitope-based peptide vaccine against novel coronavirus 2019 (sars-cov-2): immunoinformatics approach molecular and structural insights into covid-19 pandemic design of a peptidebased subunit vaccine against novel coronavirus sars-cov-2 tissue-resident memory cd8 t-cell responses elicited by a single injection of a multi-target covid-19 vaccine hla-class ii artificial antigen presenting cells in cd4+ t cell-based immunotherapy covid-19 vaccines: breaking record times to first-in-human trials a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge enhanced protection in mice induced by immunization with inactivated whole viruses compare to spike protein of middle east respiratory syndrome coronavirus immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine development of an inactivated vaccine candidate for sars-cov-2 immunogenicity and safety of a sars-cov-2 inactivated vaccine in healthy adults aged 18-59 years: report of the randomized, double-blind, and placebo-controlled phase 2 clinical trial effect of an inactivated vaccine against sars-cov-2 on safety and immunogenicity outcomes: interim analysis of 2 randomized clinical trials influence of elemental impurities in aluminum hydroxide adjuvant on the stability of inactivated japanese encephalitis vaccine, ixiaro® recent advances in the vaccine development against middle east respiratory syndrome -coronavirus live attenuated vaccines against influenza; an historical review severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates engineering a replicationcompetent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate a codon-pair deoptimized live-attenuated vaccine against respiratory syncytial virus is immunogenic and efficacious in non-human primates codagenix and serum institute of india initiate co-development of a scalable, live-attenuated vaccine against the 2019 novel coronavirus, covid-19. biospace viral vectors for vaccine applications severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice safety and immunogenicity of a candidate middle east respiratory syndrome coronavirus viral-vectored vaccine: a dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial adenovirus vectors for gene therapy, vaccination and cancer gene therapy evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored covid-19 vaccine candidate chadox1 chadox1 ncov-19 vaccine prevents sars-cov-2 pneumonia in rhesus macaques chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial immunogenicity and safety of a recombinant adenovirus type-5-vectored covid-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebo-controlled, phase 2 trial patented technologies to advance disease research nson-johns on-recei ves-posit ive-chmp-opini on-for-janss en%e2%80%99s-inves tigat ional -preve ntive -ebola -vacci ne-regimen single-shot ad26 vaccine protects against sars-cov-2 in rhesus macaques safety and immunogenicity of an rad26 and rad5 vector-based heterologous prime-boost covid-19 vaccine in two formulations: two open, non-randomised phase 1/2 studies from russia immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebocontrolled, active-comparator, first-in-man trial migal's coronavirus vaccine project using plasmids as dna vaccines for infectious diseases predominant role for directly transfected dendritic cells in antigen presentation to cd8+ t cells after gene gun immunization sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity inflammatory signals in dendritic cell activation and the induction of adaptive immunity in vivo kinetics and biodistribution of a hiv-1 dna vaccine after administration in mice high efficiency transformation by direct microinjection of dna into cultured mammalian cells the development of vaccines against sars corona virus in mice and scid-pbl/ hu mice humoral and cellular immune responses induced by 3a dna vaccines against severe acute respiratory syndrome (sars) or sars-like coronavirus in mice induction of specific immune responses by severe acute respiratory syndrome coronavirus spike dna vaccine with or without interleukin-2 immunization using different vaccination routes in mice a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial immunogenicity of a dna vaccine candidate for covid-19 intradermal-delivered dna vaccine provides anamnestic protection in a rhesus macaque sars-cov-2 challenge model rna sensors of the innate immune system and their detection of pathogens adjuvant effects of a sequence-engineered mrna vaccine: translational profiling demonstrates similar human and murine innate response induction of an ifnmediated antiviral response by a self-amplifying rna vaccine: implications for vaccine design suppression of rna recognition by toll-like receptors: the impact of nucleoside modification and the evolutionary origin of rna incorporation of pseudouridine into mrna yields superior nonimmunogenic vector with increased translational capacity and biological stability mammalian micrornas predominantly act to decrease target mrna levels au-rich elements and associated factors: are there unifying principles? self-amplifying rna vaccines give equivalent protection against influenza to mrna vaccines but at much lower doses sars-cov-2 mrna vaccine development enabled by prototype pathogen preparedness an mrna vaccine against sars-cov-2 -preliminary report a single immunization with nucleoside-modified mrna vaccines elicits strong cellular and humoral immune responses against sars-cov-2 in mice phase 1/2 study to describe the safety and immunogenicity of a covid-19 rna vaccine candidate (bnt162b1) in adults 18 to 55 years of age concurrent human antibody and th1 type t-cell responses elicited by a covid-19 vaccine bnt162b2 selected for a pivotal efficacy study adjuvanted influenza vaccines mechanisms of action of adjuvants vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens vaccine adjuvants: putting innate immunity to work evaluation of adjuvants for protein vaccines against tuberculosis in guinea pigs tolerability of intramuscular and intradermal delivery by cellectra(®) adaptive constant current electroporation device in healthy volunteers the safety and immunogenicity of gtu®multihiv dna vaccine delivered by transcutaneous and intramuscular injection with or without electroporation in hiv-1 positive subjects on suppressive art safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials dna vaccine delivered by a needle-free injection device improves potency of priming for antibody and cd8+ t-cell responses after rad5 boost in a randomized clinical trial bcg-induced trained immunity: can it offer protection against covid-19? can existing live vaccines prevent covid-19? could an unrelated live attenuated vaccine serve as a preventive measure to dampen septic inflammation associated with covid-19 infection? nonspecific protection of mice against influenza virus infection by local or systemic immunization with bacille calmette-guerin is global bcg vaccination coverage relevant to the progression of sars-cov-2 pandemic? efficacy and effectiveness of an rvsv-vectored vaccine in preventing ebola virus disease: final results from the guinea ring vaccination, open-label, cluster-randomised trial (ebola ça suffit!) a review of dengvaxia®: development to deployment government launches vaccine taskforce to combat coronavirus 2020 moderna manufacturing: why norwood? world health organization. standardization of vaccines for coronavirus disease (covid-19) the authors. clinical and experimental immunology published by john wiley & sons ltd on behalf of british society for immunology successful ebola vaccine provides 100% protection in trial roadmap for introduction and roll-out of merck rvsv-zebov world health organization. major milestone for whosupported ebola vaccine return on investment of public health interventions: a systematic review legal agreements: barriers and enablers to global equitable covid-19 vaccine access the authors thank dr ed parker, professor beate kampmann and the team at the vaccine centre at the london school of hygiene and tropical medicine for developing and maintaining the covid-19 vaccine tracker tool, which was invaluable for this review. b.f.p. is funded by the key: cord-297853-peqkcix2 authors: khan, raymond m.; al-dorzi, hasan m.; al johani, sameera; balkhy, hanan h.; alenazi, thamer h.; baharoon, salim; arabi, yaseen m. title: middle east respiratory syndrome coronavirus on inanimate surfaces: a risk for health care transmission date: 2016-11-01 journal: american journal of infection control doi: 10.1016/j.ajic.2016.05.006 sha: doc_id: 297853 cord_uid: peqkcix2 the middle east respiratory syndrome coronavirus (mers-cov) has been responsible for multiple health care–associated outbreaks. we investigated whether high-touch surfaces in 3 rooms of laboratory-confirmed mers-cov patients were contaminated with mers-cov rna. we found 2 out of 51 surfaces were contaminated with mers-cov viral genetic material. hence, environmental contamination may be a potential source of health care transmission and outbreaks. meticulous environmental cleaning may be important in preventing transmission within the health care setting. the middle east respiratory syndrome coronavirus (mers-cov) has been responsible for multiple health care-associated outbreaks. we investigated whether high-touch surfaces in 3 rooms of laboratoryconfirmed mers-cov patients were contaminated with mers-cov rna. we found 2 out of 51 surfaces were contaminated with mers-cov viral genetic material. hence, environmental contamination may be a potential source of health care transmission and outbreaks. meticulous environmental cleaning may be important in preventing transmission within the health care setting. © 2016 association for professionals in infection control and epidemiology, inc. published by elsevier inc. all rights reserved. in september 2012, the middle east respiratory syndrome coronavirus (mers-cov) was identified from a patient in saudi arabia. as of march 29, 2016, the world health organization reported 1,698 laboratory-confirmed mers cases in 26 countries, with 609 deaths (36%). 1 in its most recent report, the centers for disease control and prevention has stressed the great importance of personal protective equipment (ppe), source control, and environmental infection control measures to help eliminate the threat of health care-associated outbreaks. 2 most health care-associated mers-cov outbreaks has occurred in saudi arabia. although the precise mechanism of humanto-human transmission has not been elucidated, mers-cov can be recovered from plastic surfaces after 48 hours at 20°c and 40% relative humidity (rh), and the virus is viable for 8 hours at 30°c and 80% rh and for 24 hours at 30°c and 30% rh. 3 further, data from the south korean outbreak (may 2015) demonstrated that several environmental surfaces frequently touched by laboratory-confirmed mers patients and health care workers were contaminated by mers-cov. 4 additionally, viral sheading was detected by viral cultures from respiratory secretions up to 25 days postdisease onset. 4 although mers-cov was isolated from numerous high-touch surfaces in 2 korean hospitals affected by mers outbreak, 4 such data are lacking in the middle east. therefore, the objective of this study was to examine the extent of environmental contamination with mers-cov during an outbreak in a saudi hospital. the study was performed in the intensive care unit (icu) at king abdul-aziz medical city, riyadh, during a mers-cov outbreak from september 1-october 5, 2015. the icu had strict environmental cleaning policies, which included cleaning the rooms at least twice daily using ammonium-based disinfectant and chlorine solution 1:10 or 5,000 ppm, having a checklist, and frequent inspection using fluorescent light or culturing of high-touch areas. three negative-pressure rooms of laboratory-confirmed mers patients (a, b, and c) were selected for this study ( table 1) . the room temperature was 20.0°c-25.0°c, and rh was 30%-40%. the air exchange rate was 12 per hour, and the pressure gradient between the room and its anteroom ranged from 2.5-12.5 pa. sixteen high-touch surfaces were evaluated (table 2) : 14 in the patients' room (bedrails, mechanical ventilator, ventilator tubing, sink, garbage bin, monitor, intravenous poles, intravenous pumps, telephone, door knobs, floor, drapes-blinds, air conditioning vent, and shelf of the surgical boom) and 2 outside (computer and medical chart). environmental samples were collected as described by julian et al. 5 briefly, a sterile swab premoistened with viral transport media was used to swab each surface (at least 10 cm 2 ) horizontally, vertically, and diagonally for 30 seconds. this procedure was repeated using eluents: 1/4 lactated ringer solution and phosphate buffer solution (pbs). virus detection was performed using specific real-time reverse-transcription polymerase chain reaction (pcr) assays for the upstream of the envelope gene and the open reading frame 1a. positive tests were reported as the cycle threshold value for both upstream of the envelope gene (e) and open reading (o) frame 1a. the demographic for the patients are summarized in table 1 . all 3 laboratory-confirmed mers patients were on mechanical ventilators, with an average pao2/fio2 ratio of 196. the mean icu length of stay and time from last positive tracheal aspirate for mers-cov rna to environmental sampling were 9.3 days and 40 hours, respectively. sixteen surfaces were evaluated in each of the 3 icu rooms, with 153 environmental samples processed ( table 2) our study revealed that mers-cov viral rna was isolated from the environmental surfaces of mers patients. currently, much remains uncertain about the transmission mechanism responsible for mers nosocomial outbreaks. it was postulated from the outbreak in al-hasa, saudi arabia, in may-june 2012 that respiratory droplet and airborne transmission during aerosolgenerating procedures were the most likely transmission modes. 6 however, genetic data from a cluster in hafr al-batin, saudi arabia, showed that direct person-to-person contact could not account for all of their cases, 7 therefore raising the likelihood of an alternate transmission mechanism. studies on kinetics and patterns of viral excretion indicate that mers-cov rna was isolated from urine and feces 13 and 16 days, respectively, after initial symptoms. 8 viral shedding from respiratory aspirates may persist up to 33 days after illness onset. 9 prolonged viral shedding 8, 9 and survival on surfaces for 48 hours 3 make it difficult to ignore contaminated environmental surfaces as a potential etiology of hospital outbreaks. the rate of detecting mers-cov in our environmental samples was low (1.3%) compared with recently published data (pcr positive = 20.3%; culture positive = 4.0%), 4 but the current methods for isolating viruses from the environmental surfaces are not optimal. 5 based on reported methodologies, we used a polyester swab, 1/4 lactated ringer solution, 5 pbs 5 and viral transport media 4 because they seem to give the best yield for isolating viruses from fomites. however, we did screening at the tail-end of our outbreak when the patients' viral load might have been low and our infection control practices might have been optimal. additionally, mers patients were managed in our icu since 2013 and were usually cohorted in 1 unit where the staff became very meticulous about ppe use and environmental cleaning. moreover, fairly weak disinfectants, such as povidone iodine, have a rapid virucidal activity (reduction in virus titer by ≥4 log10) against mers-cov, with an exposure time of just 15 seconds. 10 further, leclercq et al demonstrated that at relatively low temperatures of 56°c, only 25 minutes was needed to reduce the initial titer by 4 log10, while at 65°c virucidy dropped significantly to 1 minute. 11 this sensitivity to weak disinfectants could explain why our stringent environmental cleaning policies may have attenuated the recovery of viral genetic material on fomites within the patients' rooms. our finding of mers-cov rna on environmental samples within our icu shows that the viral material may contaminate fomites and can be a theoretical cause of nosocomial infections. however, we did not use viral cultures; therefore, we do not know if the positive pcrs correlate with live viruses or infectivity. despite this, we believe that in addition to proper hand hygiene and correct ppe donning and doffing, meticulous environmental cleaning is of paramount importance to eliminate health care outbreaks. middle east respiratory syndrome coronavirus (mers-cov) cdc's early response to a novel viral disease, middle east respiratory syndrome coronavirus (mers-cov) stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea comparison of surface sampling methods for virus recovery from fomites hospital outbreak of middle east respiratory syndrome coronavirus community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases rapid and effective virucidal activity of povidoneiodine products against middle east respiratory syndrome coronavirus (mers-cov) and modified vaccinia virus ankara (mva) heat inactivation of the middle east respiratory syndrome coronavirus key: cord-293691-ewerquin authors: sauerhering, lucie; kupke, alexandra; meier, lars; dietzel, erik; hoppe, judith; gruber, achim d.; gattenloehner, stefan; witte, biruta; fink, ludger; hofmann, nina; zimmermann, tobias; goesmann, alexander; nist, andrea; stiewe, thorsten; becker, stephan; herold, susanne; peteranderl, christin title: cyclophilin inhibitors restrict middle east respiratory syndrome coronavirus via interferon λ in vitro and in mice date: 2020-07-02 journal: eur respir j doi: 10.1183/13993003.01826-2019 sha: doc_id: 293691 cord_uid: ewerquin rationale: while severe coronavirus infections, including middle east respiratory syndrome coronavirus (mers-cov) cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use. objectives: we elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin a (csa) and alisporivir (alv) restrict mers-cov to validate their suitability as readily-available therapy in mers-cov infection. methods: calu-3 cells and primary human alveolar epithelial cells (haec) were infected with mers-cov and treated with csa or alv or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nfat, or map kinases. novel csa-induced pathways were identified by rna sequencing and manipulated by gene knockdown or neutralising antibodies. viral replication was quantified by qrt-pcr and tcid(50). data were validated in a murine mers-cov infection model. results: csa and alv both reduced mers-cov titers and viral rna replication in calu-3 and haec improving epithelial integrity. while neither calcineurin nor nfat inhibition reduced mers-cov propagation, blockade of c-jun n-terminal kinase diminished infectious viral particle release but not rna accumulation. importantly, csa induced interferon regulatory factor 1 (irf1), a pronounced type-iii-interferon (ifnλ) response and expression of antiviral genes. down-regulation of irf1 or ifnλ increased mers-cov propagation in presence of csa. importantly, oral application of csa reduced mers-cov replication in vivo, correlating with elevated lung ifnλ levels and improved outcome. conclusions: we provide evidence that cyclophilin inhibitors efficiently decrease mers-cov replication in vitro and in vivo via upregulation of inflammatory, antiviral cell responses, in particular ifnλ. csa might therefore represent a promising candidate to treat mers-cov infection. saudi arabia [1] and led to recurring human infections with more than 2,500 laboratory-confirmed cases and high case fatality rates of about 35% [2] . in ex vivo infection of human lung tissue, mers-cov targets bronchial and alveolar epithelial cells (aec) and leads to a detachment and apoptosis of aec [3] . recent reports analyzing autopsy material of deceased mers-cov-infected patients showed mers-cov antigen in aec and epithelial multinucleated syncytial cell conglomerates in vivo [4, 5] . accordingly, severe human infection presents as pneumonia with progression to acute respiratory distress syndrome [4, 5] . to date, no vaccine or specific treatment for mers-cov -or the recently ongoing pandemic caused by the novel severe acute respiratory syndrome cov 2 (sars-cov-2) -is approved and therapy relies on supportive measures only [2, 6] . while in vitro studies and experiments in non-human primates demonstrated benefits of a combination of type-i-interferon and antiviral compounds including ribavirin against mers-cov [7] [8] [9] , results from retrospective patient cohorts applying similar treatment regimens remain controversial [10] [11] [12] . cyclosporin a (csa) has been found to inhibit several human-pathogenic cov in cell lines originating from kidney or liver epithelia [13] [14] [15] [16] . however, the molecular mechanisms by which csa affects cov, including mers-cov, particularly in its primary target cells, the pulmonary epithelium, remain elusive. moreover, preclinical studies addressing the effect of csa or related compounds on mers-cov replication in vivo have been lacking to date. csa is known to block the peptidyl-prolyl cis-trans isomerase (ppi) activity of cyclophilins that is involved in diverse cellular processes (e.g. protein folding, 17). additionally, csa forms together with cyclophilin a (cypa) and calcineurin (cna) a ternary complex which blocks the cna-dependent activation of nfat (nuclear factor of activated t-cells), a process which accounts for the immunosuppressive effect of csa [18] . in addition, csa has also been shown to inhibit the map kinases jnk (c-jun n-terminal kinase) and p38 [19, 20] . here, we aimed to elucidate the distinct signaling pathways by which csa affects mers-cov in clinically relevant models such as primary human aec and a murine mers-cov infection model [21, 22] . we demonstrate that csa blocks mers-cov infectious particle egress, which is dependent on jnk. moreover, we for the first time provide evidence that csa triggered the activation of an antiviral defense state in lung epithelial cells. we show that csa is a potent inducer of interferon regulatory factor 1 (irf1), type-iii-ifn (ifnλ) and multiple interferon-stimulated genes (isgs). additionally, we demonstrate that oral application of csa induced a robust ifnλ response in vivo and, importantly, significantly reduced mers-cov replication and improved disease progression in infected mice. experiments with mers-cov were performed under biosafety level 4 conditions at the institute of virology, philipps-university of marburg, germany. human alveolar epithelial cells (haec) were isolated and cultured as previously described [23] . human lung tissue was obtained from patients who underwent lobectomy after informed written consent (departments of pathology and surgery, university of giessen, approved by the university of giessen ethics committee; az.58/15). calu-3 or haec were infected at a multiplicity of infection (moi) of 0.1 diluted in dmem/f12 without fcs at 37°c for 1 h. cells were washed with dmem/f12 with 10% fcs and supplemented with stimulatory/ inhibitory reagents as indicated. 24 h after infection (pi) cells were processed for quantitative pcr (maxima-sybr/rox qpcr-mastermix, thermo fisher) and supernatant was harvested for virus titration as described previously [24] . all animal experiments were performed in accordance with the german animal protection laws and were authorized by the regional authorities (g73/2017). c57bl/6 mice were purchased from charles river laboratories and housed under pathogenfree conditions. mice were intratracheally inoculated with adenovirus-hdpp4-mcherry (cloned at viraquest inc.) as described [21, 25] . five days post transduction, mice were infected intranasally with 1.5x10 5 tcid 50 /ml mers-cov (emc/2012). 50 mg/kg/day csa diluted in dmso or dmso alone were mixed with a nut/chocolate-creme, and offered to the mice for voluntary uptake. uptake was controlled daily. csa feeding started 2 days before mers-cov challenge. mice were sacrificed 4 or 7 days post mers-cov infection. all data are given as mean ± sem. statistical significance was analyzed by unpaired two-tailed student's t-test or by 1-way anova and post-hoc multicomparison tests as indicated in the respective figures. a p value of less than 0.05 was considered significant. *p < 0.05; **p < 0.01; ***p < 0.005. further experimental details can be found in the online supplement. to address the previously proposed antiviral activity of csa in clinically relevant cells, we infected the human bronchial epithelial cell line calu-3 and primary human alveolar epithelial cells (haec) with mers-cov and analyzed intracellular viral rna and infectious particle release in presence of dmso or csa ( figure 1 ). in both calu-3 and haec, csa treatment led to a >95% decrease of viral rna ( figure 1a ) and a reduction of viral titers in the supernatant by 2.6-2.8 log 10 , respectively ( figure 1b) . interestingly, and in accordance with reports from autopsy material of mers-cov patients [4] , mers-cov-infected calu-3 and primary haec both showed apoptotic cell loss and formation of multinucleated cell foci ( figure 1c ). addition of csa reduced cell foci formation and significantly reduced apoptosis induction ( figure 1c , d). in line, both cftr (cystic fibrosis transmembrane conductance regulator; figure 1e ) and enacβ (epithelial sodium channel beta; supplement fig.e1) protein expression was improved after addition of csa to mers-cov-infected calu-3. moreover, epithelial structural integrity and ability for vectorial water transport were reduced in mers-cov-infected control cells and significantly improved to normal levels in mers-cov-infected, csa-treated cells ( figure 1f , g). csa is known to act via multiple signaling pathways including cyclophilin ppiase activity, the cna-nfat axis as well as map kinase signaling [17] [18] [19] [20] . using specific inhibitors, we aimed to interfere with csa-affected pathways to identify relevant molecular signaling events involved in the csa-mediated reduction of mers-cov infection. inhibition of cna by its specific inhibitor calcineurin inhibitory peptide (cip), as well as inhibition of the downstream transcription factor nfat resulted in minor, statistically non-significant changes in mers-cov viral titers in both calu-3 and haec (figure 2a , b). the non-immunosuppressive derivate of csa, alisporivir (alv), that binds the ppiase but does not induce ternary complex formation of cypa with cna, reduced viral titers to a similar extent as csa, suggesting that the cypa-ppiase activity elicits the restrictive effect on mers-cov replication rather than ternary complex-mediated signaling events. moreover, alv reduced cell foci formation and loss of epithelial integrity to a similar extent as csa (supplement fig. e2 ). applying specific mapk inhibitors against jnk and p38, we revealed that inhibition of the map kinase jnk, but not of p38 reduced mers-cov titers in both calu-3 and haec ( figure 2a our data suggest that, as opposed to its well-known cna/nfatfigure 4c ). these data indicate that csa treatment mounts a distinct interferon-driven antiviral response in lung epithelial cells. to better understand the transcriptional programs leading to ifnλ induction in csa-treated cells, we analyzed the regulation of interferon regulatory factors (irfs). our data reveal significant upregulation of irf1 mrna levels upon csa treatment, but not of irf3, irf7 or irf9 ( figure 5a ). irf1 is known to be a specific activator of ifnl gene expression [26] . accordingly, we identified a significantly increased number of irf1-expressing cells in csa-stimulated calu-3 cells by immunofluorescence ( figure 5b ). in line, irf1 sirna knockdown significantly reduced ifnl mrna levels in csa treated calu-3 cells ( figure 5c ). accordingly, irf1 knockdown inhibited ifnλ release by more than 75 % as compared to control ( figure 5d ). to understand the extent to which the inhibition of mers-cov propagation in csa treated cells was mediated by irf1-mediated production of ifnλ, we performed knockdown of irf1 or neutralized cell-free ifnλ, respectively. our data demonstrated that silencing of irf1 but not treatment by control sirna lead to a significant increase in mers-cov released viral particles in csa-treated cells ( figure 6a , b). moreover, neutralizing antibodies directed against ifnλ1, ifnλ2 and ifnλ3 or against the less induced ifnβ were applied ( figure 6b ). neutralization of ifnβ had no significant impact on mers-cov replication after csa treatment, whereas application of anti-ifnλ1/2/3 treatment significantly increased mers-cov viral titers by 1.05 log 10 level ( figure 6b ). these data indicate that the antiviral effects of csa were at least partially mediated by an irf1-ifnλ signaling axis, and independent of type-i-ifn. as no specific treatment is approved for mers-cov or sars-cov(-2), current treatment strategies are supportive [29, 30] . treatments including recombinant type-i-ifn and antivirals (e.g. lopinavir/ritonavir) have been applied off-label to treat mers-cov and yielded only moderate efficacy with controversial results in retrospective studies, and data from prospective studies or randomized controlled trials are lacking [29, [31] [32] [33] . due to its receptor specificity to the human dpp4, only few animal models to study mers-cov pathogenesis and mers-cov-directed antiviral compounds have been accessible to date. for this study, mers-cov infection in the mouse was facilitated via intratracheal delivery of a human dpp4encoding adenovirus, that might cause low-level inflammation itself and inhomogeneous receptor distribution within the lung, present for a limited time frame. however, even if this model might not fully recapitulate the native cellular distribution or density of the receptor as seen in the human lung, high transduction efficiencies (≥ 95%, data not shown) allow efficient viral spread in the upper and lower respiratory airways with quick progression to severe lung injury [22] and with moderate changes in morbidity [34] . thus, model-specific neurotropism as seen in some of the transgenic hdpp4 mice [35] or the necessity to adapt virus isolates via multiple passages, which might potentially affect its susceptibility to interventional strategies, are circumvented. while prior exposure to adenovirus evokes moderate histological changes including perivascular and bronchiolar lymphocytic infiltration (data not shown), mers-cov infection led to a clearly distinguishable granulocytic, necrotizing interstitial pneumonia with alveolar edema formation as described previously [22] . moreover, we demonstrate that inhibition of cypa via csa or alv, which both potently block the cypa ppiase activity at the used concentrations [42] , results in a pronounced upregulation of type-iii-ifn on both mrna and protein level, which was mediated via irf1 and was accompanied by expression of antiviral isgs. among those, especially ifit1 (interferon-induced protein with tetratricopeptide repeats 1), has been reported to influence the pathogenesis of mers-cov, highlighting the relevance of our findings [43] . of note, type-iii-ifns have recently emerged as key antiviral players in the innate immune response to viral infections at mucosal and epithelial surfaces [44] [45] [46] [47] . they efficiently restrict different respiratory viruses, and act e.g. by limiting spread from the upper to the lower airways [44, [46] [47] [48] . as opposed to type-i-ifn, type-iii-ifn do not trigger detrimental immune responses that contribute to immunopathology in influenza infection [23, 25, 44, 49] . this might prove to be pivotal in the context of csa-dependent stimulation of ifnλ during cov, as severe human cov infections, like mers-cov and-while data are still limitedalso sars-cov-2, are characterized by an immunopathology with a strong cytokine induction [5, 50, 51] . in addition to defining a novel pro-inflammatory, antiviral expression profile induced by csa on lung epithelial cells, this study also demonstrated for the first time gen. virol. 2017. statistical significance was calculated using unpaired two-way student's t-test (a, b, c, d, g) with *p < 0.05. onestep rt-pcr system (invitrogen life technologies) as described previously [2, 3] . intracellular localization of endogenous irf1 protein was analyzed 3 and 4 hour post csa stimulation. dmso-treated cells were used as negative control. stimulated cells were fixed with 4% pfa and permeabilized with methanol/acetone for 10 min. cells were incubated with a rabbit monoclonal-anti-irf1 (1:100; cell signaling) and an alexafluor 594-conjugated secondary antibody (1:400; dianova). cell nuclei were counterstained with dapi. the samples were mounted in fluoprep (biomérieux) and images were recorded with a confocal laser scanning microscope (leica sp5). calu3 cells were seeded in 0.4µm pore size transwell cell culture dishes (corning) and cultured until achieving electrochemical resistances (ter) of ≥800ω /cm 2 as measured by millicell-ers2 device. cells were infected apically with mers-cov at ; as reported previously [4] . for quantification of mers-cov induced apoptosis a caspase 3/7 gloassay® sds-page and western blot was analyzed as described previously [5] . calu-3 cells were infected with mers-cov using a moi of 0.1 and stimulated with csa 1 hour after virus adsorption. 24 h pi cells were scratched off with 500 µl pbs supplemented with protease-inhibitor mix (calbiochem) and centrifuged for 5 min at 5,000 rpm. cell pellets were resuspended in sample buffer [6] containing 4% sds and boiled at 100°c for 10 min. after discharge of the probes out of the bsl4 laboratory another 10 min boiling step was performed before the samples were separated using an 7,5 % sds-gel. after blotting on a nitrocellulose membrane and blocking using pbsdef with 5% milk powder first antibodies (anti-cftr antibody, clone mm13-4 and mouse monoclonal anti-vinculin antibody both sigma-aldrich; enacβ antibody (e-10), sc-48428; santa cruz biotechnology) diluted in pbsdef with 1% milk powder were incubated overnight followed by secondary antibody-incubation for 1 h (goat anti-mouse/hrp and swine anti-rabbit/hrp; both dako). for visualization of the signals image lab software was used. all animal experiments were performed in accordance with the regulations of german animal protection laws and as authorized by the regional authorities for histopathological analyses of formalin-fixed, paraffin-embedded murine lung tissues, sections of 2 µm thickness were cut from four to six evenly distributed planes throughout the entire lungs and mounted on adhesive glass slides. the slides were stained with hematoxylin and eosin and coverslipped. histopathological evaluation was performed using an established four grade scoring scheme [7] including the following parameters: affected area, severity and distribution of interstitial inflammation, infiltration of macrophages, lymphocytes and granulocytes, necrosis, alveolar hemorrhage and edema as well as formation of bronchus-associated lymphoid tissue (balt) and perivascular, lymphocytic cuffing. figure onestep rt-pcr kit as described previously [2, 3] . quantification was carried out using a standard curve based on 10-fold serial dilutions of appropriately cloned rna ranging from 10 2 to 10 5 copies. bar graphs in represent mean ± sem of n = 4 isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study middle east respiratory syndrome treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques inhibition of novel β coronavirus replication by a combination of interferon-α2b coronaviruses -drug discovery and therapeutic options clinical characteristics of coronavirus disease 2019 in china broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses and the miracle trial group. treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial update on therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease discovery of cyclosporine a and its analogs as broad-spectrum anti-influenza drugs with a high in vitro genetic barrier of drug resistance cyclosporin a inhibits hepatitis b and hepatitis d virus entry by cyclophilin-independent interference with the ntcp receptor suppression of hepatitis c virus replication by cyclosporin a is mediated by blockade of inhibition of human immunodeficiency virus and growth of infected t cells by the immunosuppressive drugs cyclosporin a and fk 506 molecular basis of the receptor binding specificity switch of the hemagglutinins from both the 1918 and 2009 pandemic influenza a viruses by a d225g substitution cyclosporin a inhibits rotavirus replication and restores interferon-beta signaling pathway in vitro and in cyclophilin inhibitors: a novel class of promising host-targeting anti-hcv agents middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis. msphere ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment interferon-λ: immune functions at barrier surfaces and beyond. immunity [internet] nih public access ifn-λ prevents influenza virus spread from the upper airways to the lungs and limits virus transmission interferon lambda 4 signals via the ifnλ receptor to regulate antiviral activity against hcv and coronaviruses impact and regulation of lambda interferon response in human metapneumovirus infection interferon-λ mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology activation signals for interleukin-2 production involve activation of mkk6-p38 and mkk7 signaling pathways sensitive to cyclosporin a variability of interferon-λ induction and antiviral activity in nipah virus infected differentiated human bronchial epithelial cells of two human donors a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform. perlman s, editor protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein. perlman s, editor macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection serine-arginine protein kinase 1 regulates ebola virus transcription multivesicular bodies as a platform for formation of the marburg virus envelope spectrum of pathogen-and model-specific histopathologies in mouse models of acute pneumonia work with live mers-cov was performed in the bsl-4 facility of the philipps university, marburg, germany. we thank julia spengler, larissa hamann, stefanie jarmer, dirk becker and marc ringel for excellent technical and experimental assistance. we thank ralf bartenschlager for providing alisporivir. key: cord-305534-936peb1n authors: johnson, kemmian d.; harris, christen; cain, john k.; hummer, cicily; goyal, hemant; perisetti, abhilash title: pulmonary and extra-pulmonary clinical manifestations of covid-19 date: 2020-08-13 journal: front med (lausanne) doi: 10.3389/fmed.2020.00526 sha: doc_id: 305534 cord_uid: 936peb1n the severe acute respiratory syndrome coronavirus−2 (sars-cov-2) has been recently identified as the culprit of the highly infectious, outbreak named coronavirus disease 2019 (covid-19) in china. now declared a public health emergency, this pandemic is present in more than 200 countries with over 14 million cases and 600,000 deaths as of july 18, 2020. primarily transmitted through the respiratory tract, the most common clinical presentations of symptomatic individuals infected with sars-cov-2 include fever, dyspnea, cough, fatigue, and sore throat. in advanced cases, patients may rapidly develop respiratory failure with acute respiratory distress syndrome, and even progress to death. while it is known that covid-19 manifests similarly to the 2003 severe acute respiratory syndrome (sars) and the 2012 middle east respiratory syndrome (mers), primarily affecting the pulmonary system, the impact of the disease extends far beyond the respiratory system and affects other organs of the body. the literature regarding the extrapulmonary manifestations (cardiovascular, renal, hepatic, gastrointestinal, ocular, dermatologic, and neurological) of covid-19 is scant. herein, we provide a comprehensive review of the organ-specific clinical manifestations of covid-19, to increase awareness about the various organs affected by sars-cov-2 and to provide a brief insight into the similarities and differences in the clinical manifestations of covid-19 and the earlier sars and mers. over the last two decades, the coronavirus family has been identified as the source of several highly pathogenic global outbreaks. some of the most notable are the 2003 severe acute respiratory syndrome coronavirus-1 (sars-cov-1), which caused the severe acute respiratory syndrome (sars) outbreak in china, and the 2012 middle east respiratory syndrome coronavirus (mers-cov) which caused the mers outbreak in saudi arabia (1, 2) . the most recent coronavirus outbreak likely developed in a local market ("wet market") in china in december 2019 as a series of acute respiratory disorders [acute hypoxic respiratory failure, pneumonia, acute respiratory distress syndrome (ards) (3, 4) ]. the causative pathogenic agent was found to be an enveloped, non-segmented, positive-sense rna β-coronavirus, now termed severe acute respiratory syndrome coronavirus-2 (sars-cov-2). the disease is referred to as the coronavirus disease 2019 or covid-19 (3) . the most commonly affected organ system by covid-19 is the pulmonary system, with the most frequent clinical manifestations including cough, dyspnea, fever, and sore throat, similar to sars and mers (5, 6) . in the severe disease state, the patient's clinical course is complicated by the development of pneumonia with ards, acute hypoxic respiratory failure, and/or death (7) . while the pulmonary system is most commonly affected, extrapulmonary organs and organ systems (including the cardiac, gastrointestinal, hepatic, renal, ocular, and dermatologic) are also affected by covid-19, which could have significant health consequences. covid-19-related mortality has affected more individuals than its antecedents, sars and mers, combined. the number of identified cases is steadily growing, and the outbreak has rapidly spread to many different areas in china and more than 200 other countries in a short period of time ( figure 1 ). as of july 18, 2020, 14 million cases and 600,000 deaths have been documented globally across over 200 countries and territories (8) . therefore, understanding the clinical manifestations of covid-9 is crucial. in this article, we summarize the clinical manifestations of covid-19 with an organ system-based approach to educate healthcare practitioners about both common and uncommon presentations of covid-19 and to stay vigilant about this new disease (figure 2 ). by far, the pulmonary system is the most common organ system affected by sars-cov-2. several retrospective studies have consistently reported pulmonary manifestations in patients with covid-19, which include cough, shortness of breath, sputum production, respiratory failure, and ards (table 1) (5, 7, (9) (10) (11) (12) (13) (14) (15) (16) (17) . in one large study (n = 1,099) from china, guan et al. reported that 67.8% of patients with covid-19 presented with cough, while 33% had sputum production, and 18.7% experienced shortness of breath (9) . similarly, another study (n = 262) of patients in beijing demonstrated that cough occurred in almost half (45.8%) of patients with covid-19, and dyspnea occurred in nearly 7% of patients (18) . multiple studies conducted in various countries have also demonstrated similar findings, showing that cough is the predominant pulmonary symptom in patients with covid-19 (10, 19, 20) . the main reason for the development of these symptoms is the presence of severe pneumonia in covid-19 patients. however, the pulmonary symptoms can vary in covid-19 patients, possibly due to variation in severity of disease at the time of presentation. in a study (n = 41) by huang et al. on patients with confirmed sars-cov-2 infection, the most common symptoms were fever (98%) followed by cough (76%), with over half (55%) of the patients developing dyspnea (5) . ards is a known severe pulmonary complication of covid-19, where patients experience severe hypoxia refractory to oxygen therapy (9) . further, covid-19 patients with severe pneumonia can deteriorate and develop life-threatening acute respiratory failure and ards, requiring intensive medical care. it has been well-established that the target of entry for sars-cov-2 is the angiotensin-converting enzyme-2 (ace-2) receptors ( figure 3 ) (3, 26, 27) . ace-2 receptors are expressed in type i & ii alveolar cells, and airway epithelial cells (25) . the virus enters these cells using cell-mediated endocytosis and starts a cascade of pro-inflammatory cytokines such as interleukin (il)-1β, il-6, and tumor necrosis factor (tnf) (28) . when sars-cov-2 binds the ace-2 receptor, it reduces the expression of ace-2 (29) . interestingly, sars-cov-1 and sars-cov-2 bind the same ace-2 receptor; however, sars-cov-2 binds this receptor with 10-20 times greater affinity than sars-cov-1 (26) . though respiratory symptoms predominate, gastrointestinal (gi) complications from sars-cov-2 infection have also been described, and may even precede respiratory symptoms (10, 30) . the most frequently reported gi manifestation include nausea, vomiting, diarrhea, and abdominal pain (10, (31) (32) (33) (34) (35) (36) . in a retrospective study (n = 138) of hospitalized covid-19 patients, 10% of patients reported both nausea and diarrhea. abdominal pain and vomiting were recorded in 5 and 3% of patients, respectively (10) . of note, 10% of patients experienced nausea and diarrhea between 1 and 2 days before experiencing respiratory symptoms, suggesting that gi symptoms may atypically present as one of the initial clinical manifestations of covid-19 (10) . similarly, in a large metanalysis of 10 studies with a total of 1995 cases, diarrhea occurred in 4.8% of cases, while nausea and vomiting occurred in 3.9% of cases (10). jin et al. also recorded either diarrhea, nausea, or vomiting in 74 of the 651 infected patients reviewed in their study (32) . interestingly, patients who experienced gi symptoms were more likely than those without gi symptoms to have a more severe disease course, characterized by greater degrees of liver insult (17.57 vs. 8.84%), development of ards (6.76 vs. 2.08%), and icu admission requiring mechanical ventilation (6.76 vs. 2.08%) (32) . further, nearly a quarter (22.97%) of the study population who experienced critical illness reported gi symptoms at initial presentation (32) . the fecal-oral route has been proposed as a potential mechanism of gi infection with sars-cov-2 (37-39) due to the identification of sars-cov-2 rna in the stool specimens of infected patients (40) . xiao et al. studied the rna in feces from 73 patients with covid-19, and 53% of the patients tested positive for sars-cov-2 rna in the stool (41) . additionally, studies have found overexpression of ace-2 in the epithelial cells of the gi tract, suggesting sars-cov-2 replication in the gi tract (42) . a case of positive fecal specimen in a symptomatic covid-19 patient with a negative pharyngeal and sputum specimen has also been published in the literature (43) . the liver is another organ which can be affected by sars-cov-2 (6, (44) (45) (46) . commonly reported hepatic manifestations of covid-19 include elevations in serum levels of alanine transaminase (alt), aspartate transaminase (ast), and bilirubin, while levels of albumin are decreased (6, 44) . in a single-center retrospective study (n = 99) in patients with reverse transcription-polymerase chain reaction (rt-pcr) confirmed covid-19, nearly half (43%) of patients demonstrated abnormal liver chemistries (6) . decreased albumin was noted in 98% of patients, while serum levels of ast, alt, and bilirubin were elevated in 35, 28, and 18% of patients, respectively. similarly, in an analysis of 1,099 patients, increased levels of ast were observed in 18.2% of patients with non-severe disease and 39.4% of patients with severe disease, while increased alt levels were observed in 19.8% of patients with non-severe disease and 28.1% of patients with severe disease (9) . the authors in this study used the 2007 american thoracic society criteria for community-acquired pneumonia to define covid-19 disease severity (47) . with these results, it appears that the degree of liver injury may be associated with covid-19 disease severity. in a recent meta-analysis lippi et al., demonstrated that hepatic factors that were predictive of patients with an unfavorable course of covid-19 requiring icu admission included an increase in levels of alt (1.5-1.8-fold), ast (1.8-fold), total bilirubin (1.2-1.3-fold) and decreased albumin (0.8-fold) (48) . other studies have demonstrated isolated elevations in ast alone. in a study (n = 81) by shi et al., more than 50% of covid-19 patients were observed to have elevated levels of ast with normal alt (44) . similarly, in another study (n = 41), 63% of icu admitted covid-19 patients had elevated ast vs. only 25% of patients who did not require icu care (5) . patients infected with the 2003 sars-cov-1 also experienced liver impairment (49) (50) (51) (52) . similar to the hepatic injuries associated with covid-19, the most frequent gi clinical manifestations of sars included elevations in levels of serum bilirubin, alt and/or ast, and decreased levels of serum albumin (49) (50) (51) (52) . the number of studies to better understand the mechanisms of hepatic injury in patients infected with coronaviruses is limited. one proposed mechanism for hepatic injury with sars-cov-1 includes heightened inflammatory response to the virus infection (53) . this mechanism has been supported by the abnormally high serum levels of cytokines (serum il-1, il-6, il-10) observed in sars patients and deranged liver chemistries (53) . another proposed mechanism is direct hepatic injury by sars-cov-1 via the entry of the ace-2 receptor on hepatic endothelial cells. further, sars-cov-1 viral particles have also been identified in the liver autopsies of deceased sars patients and sars viral genomes have been found by rt-pcr in hepatocytes (54) . contrarily, the proposed mechanism of hepatic injury with mers-cov involves dipeptidyl peptidase-4 (dpp-4) as its entry receptor to establish infection in the hepatocytes (55) . several animal and human studies have demonstrated higher dpp-4 expression in liver, and mers-cov can infect the liver cells via dpp-4 on the cell surface, causing cell damage and mild to moderate liver injury (56) . data regarding the mechanism of hepatic injury by sars-cov-2 is scarce. it has been proposed that sars-cov-2 attaches to ace-2 as its entry receptor, similar to sars-cov-1. a preliminary study by chai et al. showed over expression of ace-2 specifically in cholangiocytes, indicating that the virus may potentially bind to cholangiocytes to cause hepatic dysfunction (57) . further, liver biopsy of a deceased covid-19 patient with deranged liver chemistries showed moderate microvascular steatosis, and mild lobular and portal activity, which was thought to be caused by sars-cov-2 infection. however, more studies are needed to further evaluate the mechanism of injury. it is important to mention that concurrent use of hepatotoxic medications in patients with covid-19 may contribute to liver injury in patients with covid-19 who are receiving treatment, complicating the discovery of the exact etiology of liver injury (58) . the cardiac manifestations of covid-19 include cardiac arrhythmias, myocarditis, pericarditis, acute coronary syndrome (acs), heart failure, cardiogenic shock, and cardiac arrest (figure 4) . though there appears to be no difference in the prevalence of cardiovascular disease (cvd) amongst those with covid-19 compared to the general population, patients with pre-existing cvd are at higher risk of developing severe covid-19 (59, 60) . a meta-analysis of covid-19 patients revealed that the prevalence of hypertension, cardio-cerebrovascular disease, and diabetes mellitus was 17.1, 16.4, and 9.7%, respectively. moreover, the prevalence of hypertension, cardiocerebrovascular disease, and diabetes mellitus was 2-, 3-, and 2-folds in severe/icu cases as compared to non-icu cases, respectively. additionally, the meta-analysis acknowledged and analyzed viral damage to the heart noting that at least 8% of patients with covid-19 suffered acute cardiac injury (59) . the meta-analysis also evaluated the incidence of myocardial injury in severe/icu cases and non-icu cases. acute cardiac injury was assessed using cardiac markers troponin i/t or ck if troponin i/t were not provided (59) . the analysis showed a 13-fold higher incidence of myocardial injury as measured by elevations in troponin i/t or ck in severe/icu cases compared to non-icu covid-19 cases (59) . additionally, in a separate study (n = 187), guo et al. showed that mortality in covid-19 patients during hospitalization was greatly associated with presence of cvd and myocardial injury. the study revealed inpatient mortality of 7.62% for patients without underlying cvd and normal troponin t (tnt) levels, 13.33% for those with underlying cvd and normal tnt levels, 37.50% for those without underlying cvd but elevated tnt levels, and 69.44% for those with underlying cvd and elevated tnts (61) . this data indicate that those with cardiovascular comorbidities are more likely to have a poor outcome with a more severe covid-19 course. similarly, previous sars outbreaks had increased mortality associated with cvd and diabetes in sars (59) . in addition to the myocardial injury evidenced by elevations in troponins, another cardiac manifestation of covid-19 includes arrythmias. in a retrospective study (n = 137) by lui et al., heart palpitations were reported as an initial symptom in 7.3% of patients with covid-19 (19) . similarly, 17% of hospitalized covid-19 patients had unspecified arrhythmias in a separate case series(n = 138) (10) . another study (n = 187) reported ventricular tachycardia/ventricular fibrillation at a rate of 5.9% in hospitalized covid-19 patients in wuhan, china (61) . though there has been no biopsy or cardiac magnetic resonance imaging (cmri) proven fulminant myocarditis or pericarditis, several case series and case reports recognize these as one of the manifestations of covid-19 based on clinical suspicion and objective data (60, 62) . additionally, development of heart failure or cardiogenic shock was observed in several studies. in a retrospective cohort study of 191 hospitalized covid-19 patients at two chinese hospitals, 23% of patients had evidence of heart failure or cardiogenic shock (63) . though the exact mechanism of myocardial injury, development of heart failure, and cardiogenic shock is unknown, there are a number of proposed mechanisms to consider. one of those mechanisms involves direct cardiac myocyte toxicity associated with viral invasion. similar to sars-cov-1, sars-cov-2 has binding affinity for the ace-2 receptor in myocardial cells. zou et al. performed mapping of cells in various organ systems expressing ace-2 with the use of single-cell rna sequencing. cells expressing similar or more ace-2 than lung type ii alveolar cells (at2) were deemed as having the potential for increased vulnerability to sars-cov-2 (64). in their study, >7.5% of myocardial cells displayed ace-2 expression suggesting that the heart may be at high risk for direct cellular toxicity by sars-cov-2 entry and replication. this ability to infiltrate cardiac tissues appears to be similar to mers-cov and sars-cov-1. in an animal model study using transgenic mice, mers-cov rna was detectable in the heart (65) . similarly, in a study from the toronto sars outbreak, rna of sars-cov-1 was found in 35% of cardiac tissues on autopsy (66) . another proposed mechanism of cardiac effects of covid-19 is heightened release of pro-inflammatory cytokines through activation of the innate and adaptive immune system. the increased production of cytokines such as il-6, il-10, and tnfα can lead to multiorgan failure. in the past, il-6 has been associated with cardiomyopathy. additionally, this inflammatory state can promote and contribute to atherosclerotic plaque rupture (63) and acute coronary syndrome. in the setting of critical illness due to covid-19, prolonged exposure to catecholamines and cytokine storm as a response to infection can result in myocardial damage as well as stress-induced cardiomyopathy (59) . due to the respiratory sequelae of covid-19, some patients suffer from hypoxemia which is another proposed mechanism of cardiac injury. as prolonged hypoxemia results in reduced cellular capacity to metabolize aerobically, cells are subsequently switched to anaerobic metabolism. anaerobic metabolism produces a more acidotic state intracellularly due to increased lactic acid production. subsequently, increased free radical production and direct destruction of phospholipid cell membranes occur (59) . hypoxemia can also increase calcium ion influx which may lead to cardiac myocyte apoptosis (59) . demand ischemia associated with critical illness can produce similar mechanisms of cardiac injury. though more research is needed to further assess the pathogenesis associated with covid-19 myocardial injury, data obtained thus far indicates the presence of viral-related heart damage. this damage manifests in a variety of ways including evidence of arrythmias, pericarditis, myocarditis, heart failure, cardiac shock, and cardiac arrest (59, 61) . finally, current data suggests cardiovascular disease, cardiac manifestations, and cardiac injury in the setting of covid-19 are clinically relevant predictors of overall disease severity and mortality (59, 61) . another organ system affected by sars-cov-2 is the renal system with development of acute kidney injury (aki). this can occur in those with chronic kidney disease (ckd) as well as those with no evidence of prior renal impairment. though acknowledged as a rare occurrence in sars, it appears that aki may be more common in covid-19 (67, 68) . in a single center case series (n = 138) assessing clinical characteristics of patients with covid-19, wang et al. noted that ∼4% of these patients with covid-19 had an aki (10). huang et al. determined in a separate study of 41 covid-19 positive patients that ∼7% had evidence of an aki (69) . in a small washington state study consisting of 21 critically ill covid-19 patients, 19 .1% (n = 6) had acute kidney failure according to kdigo guidelines (70) . analysis of 51 critically ill covid-19 patients in a wuhan, china study showed 29% (n = 15) developed an aki (23) . similarly, during the sars outbreak, aki was also observed. in a study performed by chu et al., 6.7% of 537 patients with sars developed an aki in the setting of normal cr on admission (67) . additionally, chu et al. revealed a significantly higher mortality rate (91.7%) associated with those having evidence of renal impairment compared to those with normal renal function in the setting of sars (8.8%). in wang's study of covid-19 patients, aki was observed more in icu than non-icu patients. this might indicate that severity of illness progression associated with covid-19 may be significantly impacted by the presence of renal impairment. several potential pathophysiological explanations have been suggested to explain renal impairment in covid-19. ace-2 receptor have been shown to be highly expressed in the proximal tubules and urothelial cells of the bladder on single cell rna sequencing (63) . the increased susceptibility of the kidney to viral entry associated with ace-2 expression make it a possible target for direct cellular toxicity. moreover, sars-cov-2 has a significantly higher affinity for the ace-2 receptor which could explain the higher incidence of aki in covid-19. another possible mechanism of aki in covid-19 is significantly higher immune response to infection and multiorgan failure. sars-cov-2 induces the release of inflammatory cytokines il-2, il-7, il-10 which are believed to be involved in the pathology of aki (71) . additionally, in critically ill patients, the presence of hypovolemia, rhabdomyolysis, hypoxemia, sepsis, and septic shock associated with this viral illness are likely to contribute significantly to renal impairment. furthermore, the possibility that the etiology of akis seen in covid-19 patients is multifactorial should also be considered. more recently, a wide range of neurological complications have been reported in patients with covid-19 suggesting that sars-cov-2 may affect both the central and peripheral nervous system ( figure 5) . commonly reported central nervous system (cns) manifestations include headache, acute cerebrovascular disease, dizziness, and encephalopathy. in a retrospective study (n = 214) of confirmed covid-19 patients, 36.4% of subjects collectively experienced either dizziness, headache, cerebrovascular disease, and/or reduced consciousness (72) . headache appears to be one of the most common cns symptoms, which has been reported at a rate of 6-13% in patients with covid-19 (5, 6, 23, 73) . dizziness occurred in nearly 9-17% of patients based on recent studies (10, 72) , while reduced levels of consciousness and confusion occurred in 7.5 and 9% of covid-19 patients, respectively (6) . in a recent case series (n = 58) of covid-19 patients who developed ards, several neurologic findings, including encephalopathy, confusion, agitation and corticospinal tract signs were reported in 84% of cases. however, it is unclear whether or not these neurological signs and symptoms were directly related to infection with sars-cov-2, medication withdrawal, or cytokine effects (74) . to a lesser extent, seizure has also been reported in a minority of cases (0.5%) (72) . similarly, rare cases of confirmed viral encephalitis and meningitis have been described in small case reports of patients with sars-cov-2 detected in the cerebrospinal fluid (75, 76) . cerebrovascular disease represents another cluster of cns manifestations of covid-19 that have been cited in the literature. in a retrospective, observational study (n = 221) of patients with covid-19 in wuhan china, acute ischemic stroke (confirmed on head ct) was reported in 5% of subjects. additionally, cerebral venous sinus thrombosis (confirmed with ct venography), and cerebral hemorrhage both occurred at a rate of 0.5% in this cohort (77) . another study demonstrated that ischemic stroke and cerebral hemorrhage (both confirmed on head ct) were collectively noted in 2.8% of 214 patients with covid-19 (72) . even further, one case report described a case of acute necrotizing hemorrhagic encephalopathy associated with covid-19 in a middle aged female who presented with acute encephalopathy (78) . based on recent studies, neurologic manifestations appear to occur more frequently in patients with severe disease courses (79) . mao et al. demonstrated that neurological manifestations (specifically cerebrovascular disease, reduced consciousness and myopathy) occurred more frequently (45.5 vs. 30%) in patients with severe disease compared to those with nonsevere disease. of note, those with more severe disease (as defined by the previously described american thoracic society guidelines) were also noted to have a greater burden of comorbidities. however, it is unclear whether these neurologic manifestations hold any prognostic value in regards to covid-19 mortality (72) . in addition to cns manifestations, peripheral nervous system (pns) findings related to covid-19 have been described in the literature. clinical data has demonstrated that patients with covid-19 may experience changes in smell and/or taste, in addition to polyneuropathy and/or even neuralgia. in a retrospective study of covid-19 patients, hypogeusia, and hyposmia occurred in 5.6 and 5.1% of patients, respectively, while neuralgia or peripheral nerve pain occurred in 2.3% of the study patients. in the same study, changes in smell occurred in 12% of patients prior to any respiratory symptoms (80) , suggesting that hyposmia may uncommonly precede development of any respiratory symptoms. other studies have demonstrated similar findings (72, 81) . another study (n = 60) demonstrated that reduced smell function was prominent (98%) among patients diagnosed with covid-19, but concluded that changes in smell did not hold any prognostic value (82) . in addition to changes in smell, one study showed that up to 88% of patients with covid-19 experienced changes (diminished or complete loss) in taste before and during their disease course (83) . as such, it is reasonable to consider change in smell and/or taste as potential warning signs for covid-19, while neuralgia and peripheral neuropathy may be considered disease manifestations that may along the course of infection. clinicians should conduct a thorough neurological history and physical to identify early signs and symptoms of patients who many warrant covid-19 testing (83) . healthcare professionals can get exposed to ocular secretion of the infected patients and these secretions could become a fomite for viral spread. ocular manifestations such as conjunctivitis, retinitis, anterior uveitis, and optic neuritis have been reported due to infections from the coronaviruses in the past (84) (85) (86) . however, there is paucity of literature regarding the ocular manifestations of covid-19, possibly because these manifestations are under-recognized and under-reported. in a case series of 36 patients with confirmed covid-19, nearly one third (31.6%) of patients developed ocular manifestations such as chemosis, epiphora, and conjunctival congestion. interestingly patients with ocular manifestations experienced a severe disease course. loon et al. published a case series of patients with suspected and probable sars infection who had tear samples collected and analyzed by pcr. using who case definitions of suspected and probable cases, eight patients were classified as probable sars (based on chest imaging suggestive of pneumonia or ards) and 28 were classified as suspects of sars (anyone experiencing fever >100.4 • f, respiratory symptoms and known contact with a confirmed case of sars) (87) . of 36 subjects tested, three with probable sars had positive sars-cov results from their tear samples suggesting that sars-cov-1 can exist in tears and may potentially be a source of spread among healthcare workers and inoculating patients (88) . similarly, another earlier predecessor of the sars-cov-2, the human cov-nl63 virus was isolated from nasopharyngeal aspirate from an infant who had conjunctivitis and bronchiolitis (89) . another retrospective study of 18 children with acute respiratory tract infection due to cov-nl63 showed that three patients also developed conjunctivitis (90) . however, some controversy endures as some authors have proclaimed that ace-2 receptors predominantly exist in the posterior eye, which would not account for the cases of anterior uveitis and conjunctivitis related to sars-cov-1 (91). cutaneous findings have also been reported as a manifestation of covid-19. while there is little data regarding the topic, currently reported manifestations include erythematous rash, vesicular lesions, and urticaria. in a small analysis (n = 88) of patients who tested positive for covid-19, nearly 20% of patients developed skin findings (92) . of the 88 positive patients, eight presented with skin findings at disease onset, while 10 developed skin findings during hospitalization. nearly 16% developed an erythematous rash, while 1.1 and 3.4% developed vesicular lesions and urticaria, respectively. the most commonly affected cutaneous region was the trunk and most lesions resolved within a few days (92) . while preliminary data exists, many more studies are needed to provide additional information regarding the dermatologic manifestations of covid-19. while sars-cov-2 has been shown to affect various organs with a variety of clinical manifestations, some patients with rt-pcr detected sars-cov-2 remain completely asymptomatic. a number of studies report a wide incidence rate of asymptomatic infections, ranging from 1.6 to 56.6% (93) (94) (95) (96) (97) (98) . according to these studies, asymptomatic patients typically experience none of the aforementioned clinical signs and/or symptomology. even further, this subgroup of patients have little to no abnormalities on radiological imaging. while some with asymptomatic infection may develop into symptomatic cases, most progress without clinical deterioration. hu et al. conducted a study (n = 24) in asymptomatic patients (no symptoms at the time of screening) who tested positive for covid-19. of the 24 patients in the study, mortality was not observed in any of the patients, however 20% later developed fever, cough, and/or fatigue during the course of hospitalization (99) . it has been well-established that patients with pre-existing comorbidities generally experience worse health outcomes (higher rates of mortality, icu admission, mechanical ventilation) compared to patients who do not have any underlying health conditions. this predisposition to more severe disease can be attributed to the negative impact that comorbidities have on the individual's immune system and subsequent decreased ability to fight infection (100) . prior to the emergence of covid-19, previous studies have substantiated the notion that patients with sars and patients with mers who also had comorbidities generally experienced poorer health outcomes (101) . these comorbidities commonly included heart disease, diabetes mellitus (dm), chronic obstructive pulmonary disease (copd), cancer, chronic renal disease, hypertension, ischemic heart disease, congestive heart failure, asthma, and cerebrovascular accident (cva). in an analytical study (n = 115) of patients with sars, diabetes, and heart disease were each found to be independent risk factors for mortality. specifically, patients with heart disease and/or diabetes conferred a 12.5-time higher risk of mortality (102) . another retrospective case series (n = 144) showed that sars patients with a diagnosis of diabetes had a 3-fold increased the risk of death, icu admission, or mechanical ventilation, while sars patients with other comorbid conditions like copd, heart disease or cancer had a 2.5 increased risk. in the same study, only one of the 144 subjects, a patient with no known comorbidities (former smoker), experienced mortality (103) . similarly, in patients with mers, pre-existing health conditions were shown to impact the severity of the disease course. in a 2013 saudi arabia study (n = 47) of patients with mers, nearly 64% of the study population with diagnosed comorbidities (diabetes, hypertension, cardiac disease, and chronic renal disease) experienced mortality while only 14% of the study population without comorbidities died (104) . another study (n = 1,743), which investigated the impact of comorbidity on mortality rate in mers patients, found that patients without any comorbidities had a higher 21-day survival rate compared to patients with known comorbidities. further, mers patients with comorbidities had a 4-fold risk for fatal health outcomes compared to those without comorbidities (105) . similar to patients with mers and sars, disparities are seen between health outcomes of covid-19 patients with pre-existing health conditions and those without (12) . in a retrospective analysis (n = 138) of patients with covid-19, nearly half (46.4%) of patients had an underlying health condition. even further, those patients burdened with multiple comorbidities (72.2%) were more likely to require icu admission compared to those with no comorbidities (37.3%) (10) . while it is well-known that having comorbidities establishes an increased risk of disease severity, few studies have previously identified which conditions confer the greater risk of covid-19 disease severity. in a large metanalysis (n = 1,558) of patients with covid-19, hypertension, dm, copd, heart disease, and cerebrovascular disease were all found to be independent risk factors for severe disease (defined by either an icu admission or severity of symptoms), while patients with liver disorders, cancer, or kidney disease experienced no increased risk (106) . in another metanalysis (n = 1,813), jain et al. showed that patients with covid-19 who also had underlying copd, hypertension, and/or cardiovascular disease had a greater risk of requiring icu admission or experiencing severe disease (107) . even further, copd was shown to be the greatest predictive comorbid risk factor for severe disease and icu admission followed by cardiovascular disease and hypertension (107) . covid-19 has become a pandemic and a public health emergency, affecting more individuals than previous coronavirus outbreaks with sars and mers. the clinical manifestations of covid-19 are primarily related to the pulmonary system, and include dyspnea, cough with sputum production, fatigue and in severe cases, ards, respiratory failure, and even death. extrapulmonary clinical manifestations of covid-19 exist and affect multiple other organs and organ systems including cardiovascular, renal, hepatic, gastrointestinal, ocular, dermatologic, and neurological systems. clinicians should be aware of the variable organ and organ systems affected in patients with covid-19 and the potential disease course in patients. a novel coronavirus associated with severe acute respiratory syndrome middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission the covid-19 epidemic early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis clinical characteristics of coronavirus disease 2019 in china clinical characteristics of 138 hospitalized patients with 2019. novel coronavirus-infected pneumonia in wuhan clinical characteristics of 140 patients infected with sars-cov-2 in wuhan prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis imaging and clinical features of patients with 2019 novel coronavirus sars-cov-2: a systematic review and meta-analysis novel coronavirus infection (covid-19) in humans: a scoping review and meta-analysis clinical characteristics of hospitalized patients with sars-cov-2 infection: a single arm meta-analysis clinical characteristics of coronavirus disease 2019. (covid-19) in china: a systematic review and meta-analysis outcome of coronavirus spectrum infections (sars, mers, covid 1−19) during pregnancy: a systematic review and meta-analysis characteristics of covid-19 infection in beijing clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province novel coronavirus 2019-ncov: prevalence, biological and clinical characteristics comparison with sars-cov and mers-cov clinical features of 85 fatal cases of covid-19 from wuhan: a retrospective observational study severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients cryo-em structure of the 2019-ncov spike in the prefusion conformation understanding of covid-19 based on current evidence induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies clinical insights into the gastrointestinal manifestations of covid-19 effect of gastrointestinal symptoms on patients infected with covid-19 epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019. (covid-19) with gastrointestinal symptoms covid-19 and gastrointestinal endoscopies: current insights and emergent strategies covid-19 presenting as acute pancreatitis gastrointestinal bleeding in patients with severe sars-cov-2 taste changes (dysgeusia) in covid-19: a systematic review and metaanalysis enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? putative mechanisms of diarrhea in covid-19 gastrointestinal symptoms and outcomes in hospitalized covid-19 patients covid-19 and the digestive system evidence for gastrointestinal infection of sars-cov-2 receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus covid-19 disease with positive fecal and negative pharyngeal and sputum viral tests radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study increased serum aminotransferase activity and clinical outcomes in coronavirus disease 2019 serum activity of liver enzymes is associated with higher mortality in covid-19: a systematic review and meta-analysis diagnosis and treatment of adults with community-acquired pneumonia. an official clinical practice guideline of the american thoracic society and infectious diseases society of america laboratory abnormalities in patients with covid-2019 infection sequential changes of serum aminotransferase levels in patients with severe acute respiratory syndrome clinical characteristics and mechanism of liver injury in patients with severe acute respiratory syndrome the dynamic change of liver injury in patients with severe acute respiratory syndrome organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc multiorgan damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection. biorxiv liver injury in covid-19: management and challenges prevalence and impact of cardiovascular metabolic diseases on covid-19 in china clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019. (covid-19) coronavirus fulminant myocarditis saved with glucocorticoid and human immunoglobulin clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease sars-coronavirus modulation of myocardial ace2 expression and inflammation in patients with sars acute renal impairment in coronavirus-associated severe acute respiratory syndrome should covid-19 concern nephrologists? why and to what extent? the emerging impasse of angiotensin blockade the controversial condition of cognitive frailty: what it is, what it should be characteristics and outcomes of 21 critically ill patients with covid-19 in washington state neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china potential neurological symptoms of covid-19 neurologic features in severe sars-cov-2 infection nervous system involvement after infection with covid-19 and other coronaviruses a first case of meningitis/encephalitis associated with sars-coronavirus-2 acute cerebrovascular disease following covid-19: a single center, retrospective, observational study covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features the spectrum of neurologic disease in the severe acute respiratory syndrome coronavirus 2 pandemic infection: neurologists move to the frontlines a 55-day-old female infant infected with covid 19: presenting with pneumonia, liver injury, and heart damage anosmia and dysgeusia in the absence of other respiratory diseases: should covid-19 infection be considered? smell dysfunction: a biomarker for covid-19 olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study coronaviruses: a paradigm of new emerging zoonotic diseases morbidity, mortality and coronavirus antigen in previously coronavirus free kittens placed in two catteries with feline infectious peritonitis ocular manifestations of feline infectious peritonitis world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) the severe acute respiratory syndrome coronavirus in tears identification of a new human coronavirus human coronavirus nl63, france the possibility of covid-19 transmission from eye to nose cutaneous manifestations in covid-19: a first perspective epidemiology working group for ncip epidemic response ccfdc, prevention estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) estimating the asymptomatic proportion of coronavirus disease 2019. (covid-19) cases on board the diamond princess cruise ship epidemiologic characteristics of early cases with 2019 novel coronavirus (2019-ncov) disease in korea sars-cov-2 infection in children asymptomatic and presymptomatic sars-cov-2 infections in residents of a long-term care skilled nursing facility -king county clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing the impact of cellular networks on disease comorbidity critically ill patients with severe acute respiratory syndrome short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) clinical features and short-term outcomes of 144 patients with sars in the greater toronto area epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study impact of comorbidity on fatality rate of patients with middle east respiratory syndrome. sci rep does comorbidity increase the risk of patients with covid-19: evidence from meta-analysis predictive symptoms and comorbidities for severe covid-19 and intensive care unit admission: a systematic review and meta-analysis kj, cha, jc, chu, hg, and ap equally contributed to this paper with conception and design of the study, literature review and analysis, drafting and critical revision and editing, and final approval of the final version. all authors contributed to the article and approved the submitted version. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 johnson, harris, cain, hummer, goyal and perisetti. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-299986-wuaxatrb authors: afsar, nasir ali title: the looming pandemic of covid-19: what therapeutic options do we have now? date: 2020-04-21 journal: j chin med assoc doi: 10.1097/jcma.0000000000000310 sha: doc_id: 299986 cord_uid: wuaxatrb nan the world came to know about a new corona virus infection spreading from wuhan, china, in december 2019. over the following 3 months, this respiratory pathogen, named as ncov-2019, sars cov-2, or covid-19, has affected many in the most populous regions of the world and there are growing concerns about it being a pandemic. world health organization (who) has reported infection in all continents in its situation report-42 published on 2 march 2020 and >3000 deaths (https:// www.who.int/docs/default-source/coronaviruse/situationreports/20200302-sitrep-42-covid-19.pdf?sfvrsn=224c1add_2). although it causes a flu-like respiratory illness, the mortality is low among previously healthy, young individuals but high in elderly and critically ill. 1 however, the low percent mortality has already transformed into a big absolute number (>3000 individuals) and counting. each life is precious and there is a global movement to find treatment for this disease. as yet, no definite prevention or cure is available for covid-19. lately, some reports have surfaced about various drugs indicated for other viral illness, being tried for this disease due to existing evidence in two related disorders, sars-cov and mers-cov. to critically evaluate the existing options, an attempt has been made to list the drugs considered potentially useful in corona virus infections including the previous outbreaks of sars and mers and are tabulated ( table 1) to derive lessons from the existing scientific literature. this is highly imperative that although new drug and vaccine development is in progress, existing treatment may be applied to save precious lives. the information in media that corona virus has no effective treatment has already created panic in the masses and resultant lock down in many parts of the world. hence, it could be helpful to mitigate the impression of "no treatment" and resultant fear around the world. www.ejcma.org afsar j chin med assoc zinc supplements may positively influence patient outcome. wen 14 in vitro assay chinese herbs potential for drug development. difficult to assign therapeutic benefit to a single agent. effectively inhibit viral replication; may prove to be beneficial as a herbal product. ifn = interferon; mpa = mycophenolic acid. early epidemiological analysis of the coronavirus disease 2019 outbreak based on crowdsourced data: a population level observational study genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding case of the index patient who caused tertiary transmission of covid-19 infection in korea: the application of lopinavir/ritonavir for the treatment of covid-19 infected pneumonia monitored by quantitative rt-pcr treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide corticosteroid therapy for critically ill patients with middle east respiratory syndrome zinc lozenges and the common cold: a meta-analysis comparing zinc acetate and zinc gluconate, and the role of zinc dosage traditional chinese medicine herbal extracts of cibotium barometz, gentiana scabra, dioscorea batatas, cassia tora, and taxillus chinensis inhibit sars-cov replication key: cord-303915-14yfs4pa authors: almazán, fernando; dediego, marta l.; sola, isabel; zuñiga, sonia; nieto-torres, jose l.; marquez-jurado, silvia; andrés, german; enjuanes, luis title: engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate date: 2013-09-10 journal: mbio doi: 10.1128/mbio.00650-13 sha: doc_id: 303915 cord_uid: 14yfs4pa middle east respiratory syndrome coronavirus (mers-cov) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. the construction of a full-length infectious cdna clone of the mers-cov genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. following transfection with the cdna clone, infectious virus was rescued in both vero a66 and huh-7 cells. recombinant mers-covs (rmers-covs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cdna clones with these genes deleted. the mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for mers-cov replication in cell cultures. in contrast, an engineered mutant virus lacking the structural e protein (rmers-cov-δe) was not successfully rescued, since viral infectivity was lost at early passages. interestingly, the rmers-cov-δe genome replicated after cdna clone was transfected into cells. the infectious virus was rescued and propagated in cells expressing the e protein in trans, indicating that this virus was replication competent and propagation defective. therefore, the rmers-cov-δe mutant virus is potentially a safe and promising vaccine candidate to prevent mers-cov infection. importance since the emergence of mers-cov in the arabian peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. this article describes the development of the first reverse genetics system for mers-cov, based on the construction of an infectious cdna clone inserted into a bacterial artificial chromosome. using this system, a collection of rmers-cov deletion mutants has been generated. interestingly, one of the mutants with the e gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing e protein in trans, whereas it would only survive a single virus infection cycle in vivo. this virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent mers-cov infection. likely have not required hospital assistance. in fact, recent data suggest that mild respiratory illness might also be part of the clinical spectrum of mers-cov infection (3) . in addition to mild or acute respiratory illness, other reported clinical symptoms are abdominal pain and diarrhea, fever, and in some cases, renal failure (9) . many hospitalized cases occurred in persons with chronic underlying medical conditions or immunosuppression (3, 13) . the virus loads are highest in lower respiratory tract samples, although low concentrations of viral rna can also be found in stool, urine, and blood samples (12) . the genome of mers-cov includes more than 30,100 nucleotides and contains at least 10 predicted open reading frames (orf1a, orf1b, s, 3, 4a, 4b, 5, e, m, and n), 9 of which seem to be expressed from a nested set of eight mrnas (14, 15) . interestingly, the partial genome sequences of three independent mers-cov isolates reveal that it evolved following a strict molecular clock model (6) . a functional receptor of mers-cov is dipeptidyl peptidase 4 (dpp-4) from both human and bat (16) . this receptor binds to a 231-residue region in the spike (s) protein of mers-cov (17, 18) , a domain different from the receptor-binding site of other betacoronaviruses (18) . infection of human airways by mers-cov prevents the induction of interferon-regulating factor 3 (irf-3)mediated antiviral alpha/beta interferon (ifn-␣/␤) responses. however, mers-cov was markedly more sensitive to the antiviral state established by ectopic ifn than severe acute respiratory syndrome cov (sars-cov) (14, 19, 20) . soon after mers-cov emergence, a diagnostic assay was designed (21) . similarly, antivirals inhibiting virus replication, such as cyclosporine a, ifn-␣, or ribavirin, have been described (14, 22, 23) . in contrast, reliable vaccines have not yet been developed, although the s protein and the receptor-binding site within this protein induce neutralizing antibodies and, in principle, could serve as a subunit vaccine (17) . covs infect respiratory and enteric mucosal areas, and thus, induction of mucosal immunity is necessary to protect these tissues from infection. live attenuated viruses are expected to elicit mucosal immunity more efficiently than nonreplicating antigens, which elicit reduced secretory immune responses. live attenuated viruses can be generated by the deletion of genes conferring virulence, a procedure that requires the availability of a reverse genetics system for mers-cov. in this article, we describe the construction of an infectious cdna clone of mers-cov using a bacterial artificial chromosome (bac). using this clone, recombinant mers-cov (rmers-cov) deletion mutants were constructed lacking genes nonessential for virus replication. in addition, we deleted the structural envelope (e) protein gene, because in previous work from our laboratory, deletion of the e gene in two other covs led to mutants that were either replication-competent, propagation-defective viruses or attenuated viruses (24) (25) (26) . all deletion mutants efficiently replicated and spread in cell cultures except the one in which the e gene was deleted, which was replication competent but propagation defective. this virus was propagated in cells by providing e protein in trans. therefore, this deletion mutant missing the e gene can serve as the basis for a safe vaccine candidate. an infectious cdna clone was assembled as a bac under the control of the cytomegalovirus (cmv) immediate-early pro-moter, based on the genome sequence of the mers-cov-emc12 strain (genbank accession number jx869059) (15) . to this end, the same approach described for the generation of other cov infectious cdna clones (27) (28) (29) (30) was used. this system allows the efficient intracellular production of viral rna from the cdna clone without the need for in vitro ligation and transcription steps. the bac clone carrying the mers-cov infectious cdna was generated in several steps (fig. 1) . after selection of appropriate restriction sites in the viral genome (fig. 1a) , the intermediate plasmid pbac-mers-5=3= (fig. 1b) was generated as the backbone to assemble the full-length cdna clone. this plasmid contained the first 811 nucleotides of the viral genome fused to the cmv promoter, a multicloning site containing the restriction sites selected in the first step (bamhi, stui, swai, and paci), and the last 4,272 nucleotides of the genome, followed by a 25nucleotide (nt) poly(a) stretch, the hepatitis delta virus (hdv) ribozyme, and the bovine growth hormone (bgh) termination and polyadenylation sequences. finally, the full-length mers-cov infectious cdna clone (pbac-mers fl ) was assembled by sequential cloning of four chemically synthesized overlapping dna fragments (mers-1 to mers-4) into the plasmid pbac-mers-5=3= (fig. 1c) . the full-length clone sequence was identical to that reported for the mers-cov-emc12 strain (15) , with the exception of a silent point mutation (t to c) introduced in the cdna at position 20,761 (fig. 1c ). this mutation, which eliminates an additional swai restriction site at position 20,760, was introduced to facilitate the cloning process and was used as a genetic marker to identify the virus recovered from the cdna clone. the assembled infectious cdna clone was stable during its propagation in escherichia coli dh10b cells for more than 200 generations, as determined by restriction endonuclease analysis (data not shown). rescue of infectious rmers-cov from the cdna clone in vero a66 and huh-7 cells. infectious viruses were recovered from the full-length cdna clone, using susceptible vero a66 and huh-7 cells, with titers of around 10 6 pfu/ml at 72 h posttransfection (h.p.t.). the recovered viruses were cloned by three rounds of plaque purification, and their phenotypic and genotypic properties were determined. viruses rescued from both cell lines (rmers-cov) induced a clear cytopathic effect (cpe), characterized by the induction of cell fusion, which was more apparent in huh-7 cells (fig. 2 ). in addition, the replication of the rmers-cov was confirmed by indirect immunofluorescence microscopy using a nucleocapsid (n) protein-specific antibody, showing a cytoplasmic staining pattern in both cell lines (fig. 2) . to further confirm the identity of the rmers-cov, the fulllength genome sequences of two independent clones rescued in vero a66 and huh-7 cells were analyzed. the recombinant viruses rescued in huh-7 cells presented the same sequence as the cdna clone, including the genetic marker at position 20,761. however, in the case of the viruses rescued in vero a66 cells, several changes in the region of the accessory genes were detected in both clones. one of them presented a deletion of 179 nucleotides (from nucleotide 26,721 to 26,900) that disrupted gene 4b and eliminated the transcription-regulating sequence (trs) and the first 20 amino acids of gene 5. the second clone presented a 1-base insertion at position 27,143 that changed the reading frame and promoted the expression of a truncated gene 5. taking these data into consideration, two new virus clones rescued in vero a66 cells were sequenced, and only one of them presented the wild-type sequence, suggesting that the mers-cov was more stable in huh-7 cells. therefore, huh-7 cells were selected for further work. rescue of infectious rmers-covs lacking accessory genes 3, 4a and 4b, and 5. the availability of the pbac-mers fl infectious clone opened the door to investigate the importance of accessory genes 3, 4a, 4b, and 5 for mers-cov replication. to this end, cdna clones with genes 3 (pbac-mers-⌬3), 4a and 4b (pbac-mers-⌬4ab), or 5 (pbac-mers-⌬5) deleted were constructed from pbac-mers fl (fig. 3a) . the expression of gene 3 was abrogated by deletion of its trs and coding sequence, with the exception of the 3= last 41 nucleotides, containing the 4a trs, in order to preserve the expression of gene 4a. in the case of genes 4a and 4b, the majority of both coding sequences was deleted, except for the last 81 nucleotides of gene 4b, which overlap the gene 5 trs. finally, the expression of gene 5 was avoided by deletion of its trs and complete coding sequence. infectious viruses were recovered in huh-7 cells from plasmids pbac-mers-⌬3, pbac-mers-⌬4ab, and pbac-mers-⌬5 with virus titers similar to that of the parental rmers-cov (around 10 6 pfu/ml). after one passage on fresh cell monolayers, the recombinant viruses were cloned by three plaque isolation steps and their genetic structure was confirmed by sequencing. all the deletion mutant viruses (rmers-cov-⌬3, rmers-cov-⌬4ab, and rmers-cov-⌬5) were identical to the parental virus (rmers-cov) in terms of cpe and plaque morphology (data not shown). the growth kinetics of these viruses were also similar, reaching maximum virus titers at 72 h postinfection (h.p.i.) (fig. 3b ). in the case of rmers-cov-⌬4ab, the viral titer was around 10-fold lower than that obtained from the parental virus (fig. 3b ). these data indicated that the proteins encoded by genes 3, 4a, 4b, and 5 were not essential for mers-cov replication in cell cultures. generation of a rmers-cov mutant lacking the structural e protein gene. based on published data showing that the deletion of cov e protein resulted in either replication-competent, propagation-defective viruses (24) or attenuated viruses (25, 26, 31) , a cdna clone with the e gene deleted (pbac-mers-⌬e) was constructed from pbac-mers fl . the expression of the e gene was abrogated by the deletion of its trs and coding sequence, with the exception of the 3= last 49 nucleotides, in order to preserve the expression of gene m (fig. 4a ). to recover infectious virus, bhk cells were transfected with pbac-mers-⌬e or the full-length cdna clone pbac-mers fl . six h.p.t. the transfected cells were overlayed on vero a66 cell monolayers, and at 72 h.p.t., the supernatants were harvested and serially passaged three times on fresh huh-7 cells. infectious rmers-cov was recovered with titers of around 10 6 pfu/ml, whereas visible plaques were not detected for rmers-cov-⌬e virus throughout these passages (data not shown). since cpe was observed at passages 0 and 1, the cell supernatants from the different passages were titrated in huh-7 cells by limiting dilution. in contrast to the wild-type virus, which was recovered with high titers (around 1 ϫ 10 6 50% tissue culture infection dose [tcid 50 ]/ml), the rmers-cov-⌬e was detected only at passage 0 with apparent low titers (around 2 ϫ 10 3 tcid 50 /ml) (fig. 5a ). this apparent low titer was most probably due to the transfer of detached cells transfected with the pbac-mers-⌬e. these cells were taken with the supernatant used to infect the next cell monolayer and formed syncytia with the nontransfected cells, giving the impression of virus production. in fact, rmers-cov-⌬e virus was lost at subsequent passages in several independent experiments performed in two different cell lines, vero a66 and huh-7 cells (data not shown). the presence of viral proteins was analyzed by immunofluorescence microscopy in huh-7 cells infected with either rmers-cov or rmers-cov-⌬e from passage 0. as expected, e protein was detected in cells infected with rmers-cov but not in those infected with rmers-cov-⌬e (fig. 4b ). viral n protein was detected in the cytoplasm of both rmers-cov-and rmers-cov-⌬e-infected cells (fig. 4b) . interestingly, whereas the n protein was detected all over the cell monolayer in rmers-cov-infected cells, it was only detected in small syncytia in cells infected with rmers-cov-⌬e. altogether, these data suggested that e protein was required for efficient virus propagation. previous reports from our laboratory showed that deletion of the transmissible gastroenteritis coronavirus (tgev) e gene leads to a propagation-defective virus that can only spread from cell to cell by expression of the e protein in trans (24, 32) . to analyze whether rmers-cov-⌬e could also be complemented in cells transiently expressing e protein, the rescue of rmers-cov-⌬e and of rmers-cov as a control was analyzed in huh-7 cells that did not express e protein (e ϫ ) and in cells transiently expressing the e protein (e ϩ ). the transfection efficiencies in e ϩ cells varied between 40 and 50% in each independent experiment. infectious rmers-cov was rescued from both e ϩ and e ϫ cells with virus titers of around 4 ϫ 10 5 tcid 50 /ml and 1 ϫ 10 6 tcid 50 /ml, respectively (fig. 5a ). in contrast, rmers-cov-⌬e was rescued in e ϩ cells with titers of around 1 ϫ 10 3 tcid 50 /ml but not in control e ϫ cells, in which the virus was not detectable from passage 1 (limit of detection, 50 tcid 50 /ml) (fig. 5a) . these data indicated that the e protein was necessary for either viral rna synthesis or virus propagation. to evaluate the role of the e protein in viral rna synthesis, the level of genomic rna (grna) was evaluated by quantitative reverse transcription-pcr (rt-qpcr) at each passage. viral grna was detected for rmers-cov in both e ϫ -and e ϩ -expressing cells, as expected. however, mers-cov-⌬e viral rna was detected at high levels in e ϩ cells at passages 0, 1, 2, and 3, whereas it was only detected at similar levels in e ϫ cells at passage 0, suggesting that mers-cov-⌬e was a replication-competent virus (fig. 5b) . to further confirm these data, viral rna synthesis was analyzed in a single-cycle infection. e ϫ cells were infected with either rmers-cov or rmers-cov-⌬e grown in e ϩ cells. at 5 h.p.i., the levels of grna and subgenomic mrna 8 (sgmrna n) were evaluated by rt-qpcr (fig. 6 ). similar levels of grna and sgmrna n were detected in cells infected with both viruses, indicating that e protein was not required for efficient viral replication and transcription. overall, these data indicated that rmers-cov-⌬e was a replicationcompetent, propagation-defective virus. the emergence of mers-cov represents a public health threat that requires further research to understand the virus biology and provide the basis for the development of control strategies. this paper describes for the first time the construction of a reverse genetics system for mers-cov, using bacs as vectors. the recombinant viruses described in this work were rescued using a combination of synthetic biology and reverse genetics techniques, as previously described for other covs (33). this system constitutes a valuable molecular tool for the rational design and launching of attenuated viruses that may serve as efficient and safe vaccine candidates. in addition, the infectious cdna clone will be useful to study the role of specific viral genes in virus-host interactions in the context of the complete viral cycle. a full-length cdna copy of mers-cov-emc12 was generated from synthetic fragments cloned downstream from the cmv promoter in a bac. the bac-based strategy allows the efficient and reproducible intracellular production of viral rna, since it is first synthesized in the nucleus by the cellular rna polymerase ii (pol ii) and then amplified in the cytoplasm by the viral replicase encoded in the rna itself (27, 28) . the mers-cov infectious cdna was stably maintained in bacteria for more than 200 generations, allowing the easy and direct manipulation of the viral cdna for molecular studies. in addition, this bac-based system allows for the generation of viral replicons that may be used for the screening of drugs affecting viral rna synthesis (27, 34) . the full-length sequence of rmers-cov, recovered from the infectious cdna clone, was completely identical to that published for the original mers-cov-emc12 isolate (15) , except for the silent point mutation introduced as a genetic marker. therefore, both viruses should have the same biological properties. in fact, the growth kinetics and cpe caused by both viruses were similar when the same multiplicity of infection (moi) was used to infect huh-7 cells (14) . interestingly, the rmers-cov sequence seemed more stable in huh-7 cells than in vero a66 cells. however, the data presented in this article are statistically very limited to definitively conclude that virus genome stability depends on the cell type used. based on the preliminary data presented here, it would be interesting to analyze the evolution of the mers-cov sequence in different cell types, including human respiratory epithelial cells. the 3= third of the mers-cov genome contains a set of accessory genes encoding proteins with no similarity to other viral or mammalian known proteins (35) . in general, cov accessory genes are not essential for virus growth in vitro (36) (37) (38) (39) . the reverse genetics system described in this article was used to study the importance of these proteins in cell culture. mers-cov genes 3, 4a, 4b, and 5 were each found to be dispensable for virus replication in tissue cultures. interestingly, some of the rmers-cov viruses recovered from vero a66 cells contained mutations in the accessory gene genome region that would prevent the expression of any of these genes. similar results were previously reported for the original mers-cov-emc12 isolate after passage in vero cells (15) . these data suggested an apparent lack of selection pressure on mers-cov accessory genes during passages in cell culture and reinforced the dispensability of these genes for virus growth in vitro. although not essential in tissue culture, these mers-cov accessory genes could have an important role in virus-host interaction in vivo, leading to attenuated phenotypes. cov accessory genes have been associated with the modulation of viral virulence (40) . among all covs, sars-cov contains the largest number of accessory genes, and it has been proposed that these genes may have important contributions to its high virulence (26, 39) . to date, mouse hepatitis virus (mhv) ns2 and 5a, tgev 7, and sars-cov 3b and 6 proteins have been implicated in the modulation of innate immune responses, using different mechanisms to influence virus virulence (36, (41) (42) (43) (44) . rmers-cov-⌬e was a replication-competent, propagationdeficient virus and was only efficiently disseminated in cells expressing the e protein in trans. in the presence of transiently ex-pressed e protein, rmers-cov-⌬e yielded maximum progeny viral titers of around 10 3 tcid 50 /ml. this modest yield could be improved by the generation of cell lines stably expressing the e protein. in fact, a direct relation between viral titers and the amount of e protein expressed was previously observed for tgev (45) . however, high expression levels of e protein could induce apoptosis, as described for mhv e protein expression (46) . to overcome this potential adverse effect in the case of mers-cov, an inducible system for e protein expression would have to be established. rmers-cov-⌬e did not spread in cells in the absence of e protein, thus constituting a single-cycle replicative virus. however, infected cells produced syncytia, which suggests good expression of viral s protein. in addition, high levels of n protein were observed by immunofluorescence. these data suggested that the high expression levels of viral proteins might serve as potent immunogens to elicit a protective immune response. in the case of sars-cov, it has been shown that nonreplicating sars-cov-like particles bearing the e, s, and membrane (m) proteins induced immune responses that were protective against sars in mice (47, 48) . in addition, sars-cov inactivated viruses induced adaptive immunity that protected against challenge (49) (50) (51) (52) . the potential of rmers-cov-⌬e as a vaccine candidate is reinforced by previous observations indicating that a sars-cov lacking the e gene (sars-cov-⌬e) is attenuated and induces protection in hamsters, transgenic mice, and conventional aged mice (53) (54) (55) . mers-cov infects mucosal areas in the lungs and, probably, the enteric tract. mucosal immunity in a specific tissue, such as in lung infections with mers-cov, is optimally induced by local stimulation. therefore, immunization with live attenuated forms of rmers-cov-⌬e virus grown in a packaging cell line providing the e protein in trans may be a convenient option, particularly in comparison with purified mers-cov antigens, such as the s protein, that could serve as a subunit vaccine. vaccines based on live attenuated viruses may present biosafety problems associated with the possibility of reversion to virulent phenotypes or causing disease in immunocompromised individuals. in this sense, the use of rmers-cov-⌬e would be a safer option, as it does not propagate in the absence of e protein expression, preventing straightforward reversion to virulence. to increase the biosafety of a rmers-cov-⌬e-based vaccine, additional safety guards could be included, such as the previously de-scribed attenuating mutations in distant genomic locations, like those encoding the nsp1 (56, 57) or nsp14 (58) replicase proteins, or by introducing genomic rearrangements (59) . overall, we consider rmers-cov-⌬e a promising vaccine candidate that should be further developed. rmers-cov-⌬e could also be used as the starting point to generate an inactivated vaccine in case of an urgent need to control the disease. in order to guarantee the absence of virulent viruses after an incomplete chemical inactivation due to clump formation, potential noninactivated viruses would be propagation defective and, therefore, attenuated. rmers-cov-⌬e, in which one of the nonessential accessory proteins was deleted, could be considered a marker vaccine, as it will allow the sera of field-infected patients to be distinguished from sera of vaccinated patients, based on the lack of antibodies specific for nonessential viral proteins (60) . the rmers-cov-⌬e could also be considered a viral replicon, as its genome self amplifies in infected cells but infection is not efficiently spread from cell to cell. the construction of a minimal replicon is also possible with reduced effort using the infectious clone, a project that is currently in progress in our laboratory. therefore, the introduction of a reporter gene, such as green fluorescent protein, within this replicon could easily generate a useful tool for mers-cov antiviral drug screening. in this paper, we describe for the first time a reverse genetics system of mers-cov engineered on bacs, which has allowed the generation of the first modified live vaccine candidate to protect against mers-cov. furthermore, this reverse genetics system is a useful tool for the identification of viral genes involved in pathogenesis and the associated signaling pathways. drugs inhibiting these pathways would be potential antivirals. baby hamster kidney cells (bhk-21) were obtained from american type culture collection (atcc ccl-10). human liverderived huh-7 cells were kindly provided by r. bartenschlager (university of heidelberg, germany). african green monkey kidney-derived vero a66 cells were kindly provided by a. carvajal (university of leon, spain). in all cases, cells were grown in dulbecco's modified eagle's medium (dmem) supplemented with 25 mm hepes, 1% nonessential amino acids (sigma), and 10% fetal bovine serum (fbs) (biowhittaker). virus titrations were performed on vero a66 or huh-7 cells following standard procedures and using closed flasks or plates sealed in plastic bags. for plaque assays, infected cells were overlaid with dmem containing 0.6% low-melting agarose and 2% fbs, and at 72 h.p.i., cells were fixed with 10% formaldehyde and stained with crystal violet. for 50% tissue culture infectious dose (tcid 50 ) assays, cpe was recorded at 72 h.p.i. all work with infectious virus was performed in biosafety level 3 facilities by personnel wearing positive-pressure air-purifying respirators (highefficiency particulate air-mate). plasmids and bacteria strains. plasmid pbelobac11 (61), kindly provided by h. shizuya (california institute of technology, pasadena, ca), was used to assemble the mers-cov infectious cdna clone. this plasmid is a low-copy-number plasmid (one to two copies per cell) based on the e. coli f factor (62) that allows the stable maintenance of large dna fragments in bacteria. e. coli dh10b (gibco/brl) cells were transformed by electroporation using a micropulser unit (bio-rad) according to the manufacturer's instructions. bac plasmid and recombinant bacs were isolated and purified using a large-construct kit (qiagen), following the manufacturer's specifications. construction of a full-length cdna clone of mers-cov. based on the data of the full-length sequence of the mers-cov-emc12 strain (genbank accession number jx869059) (15), a mers-cov infectious cdna clone was assembled in bac using a three-step strategy. in the first step, the restriction sites bamhi (genomic position 806), stui (genomic positions 7,620 and 9,072), swai (genomic position 20,898), and paci (genomic position 25,836), present in the viral genome, were selected (fig. 1a) . second, the intermediate plasmid pbac-mers-5=3= was constructed as the backbone for assembly of the full-length cdna clone (fig. 1b) . to generate this plasmid, two dna fragments were generated by chemical synthesis (bio basic, inc.). the first fragment contained the cmv promoter fused to the first 811 nucleotides of the viral genome flanked by sfoi and bamhi sites, and the other one contained a multicloning site with the restriction sites selected in the first step (bamhi, stui, swai, and paci) followed by the last 4,272 nucleotides of the viral genome joined to a 25-nt poly(a), hdv ribozyme, and bgh termination and polyadenylation sequences. the first dna fragment was cloned into pbelobac11 ϫstui (a pbelobac without the stui restriction site) and digested with sfoi and bamhi to generate the plasmid pbac-mers-5=, and then the plasmid pbac-mers-5=3= was generated by cloning the second dna fragment, digested with bamhi and sfii, into pbac-mers-5= digested with the same restriction enzymes. finally, the third step was the assembly of the full-length cdna clone (pbac-mers fl ) by sequential cloning of four overlapping dna fragments (mers-1 to mers-4) into the multicloning site of the intermediate plasmid pbac-mers-5=3= (fig. 1c) . the overlapping dna fragments flanked by the appropriate restriction sites were generated by chemical synthesis (bio basic, inc.). in the case of fragment mers-3, a silent mutation (t to c) was introduced at position 20,761 in order to eliminate the swai restriction site at position 20,760 and to use it as a genetic marker. the genetic integrity of the cloned dnas was verified throughout the assembly process by extensive restriction analysis and sequencing. construction of mers-cov cdna clones lacking accessory genes 3, 4a, 4b, and 5. the deletion of gene 3 was generated by pcr-directed mutagenesis using the plasmid puc-mers-1 (a puc plasmid containing the mers-1 fragment spanning nucleotides 20,898 to 25,836 of the mers-cov genome) as the template and the oligonucleotides mers-s-tth111i-vs (5= tgctatttgacaaagtcactatagctgatc 3=, where the restriction site tth111i is underlined) and mers-s-paci-rs (5= cccttaattaactgagtaaccaacgtcaaaaagattcacact attagtgaacatgaaccttatgcggctcgaggtcgtattcc 3=, where the restriction site paci is underlined). the pcr product, including the deletion (from nucleotides 25,518 to 25,803), was digested with tth111i and paci and cloned into the same sites of puc-mers-1, leading to puc-mers-1-⌬3. to generate pbac-mers-⌬3, the swai-paci digestion product from puc-mers-1-⌬3 was cloned into the same restriction sites of pbac-mers fl (fig. 3a) . the deletions of genes 4a, 4b, and 5 were introduced by pcr-directed mutagenesis, using as a template the plasmid pbac-mers-3= (a bac plasmid containing the mers-3= fragment spanning nucleotides 25,836 to 30,107 of the mers-cov genome). for deletion of genes 4a and 4b, overlapping pcr fragments were amplified using oligonucleotides del4ab-vs (5= gaactctatggattacggttgtctccatacggtc 3=) and del4ab-rs (5= gaccgtatggagacaaccgtaatccataga gtt 3=). the final pcr product was amplified with outer oligonucleotides t7 and sa27201rs (5= caaacagtggaatgtagg 3=), digested with paci and nhei, and cloned into the same restriction sites of pbac-mers-3=, leading to pbac-mers-3=-⌬4ab that contains a deletion spanning nucleotides 25,862 to 26,751 of the mers-cov genome. for gene 5 deletion, a pcr fragment lacking gene 5 (nucleotides 26,835 to 27,513) was amplified using oligonucleotides sa25834vs (5= gttaattaacga actctatggattacg 3=, where the restriction site paci is underlined) and del5-sandi-rs (5= cacgggacccatagtagcgcagagctgct gttaaaatcctggatg 3=, where the restriction site sandi is underlined), digested with paci and sandi, and cloned in the same sites of pbac-mers-3=, leading to pbac-mers-3=-⌬5. to generate plasmids pbac-mers-⌬4ab and pbac-mers-⌬5, the paci-rsrii digestion products from plasmids pbac-mers-3=-⌬4ab and pbac-mers-3=-⌬5 were cloned in the same sites of pbac-mers fl (fig. 3a) . all cloning steps were checked by sequencing of the pcr fragments and cloning junctions. construction of a mers-cov cdna clone lacking the structural e gene. the pbac-mers-⌬e, encoding a mers-cov lacking the e gene, was constructed from the full-length plasmid pbac-mers fl . to this end, the sandi-rsrii dna fragment (2,634 bp) from pbac-mers fl was exchanged with a chemically synthesized (bio basic, inc.) sandi-rsrii dna fragment with a deletion from nucleotides 27,580 to 27,786 that included the trs core sequence and the first 197 nucleotides of the e gene (fig. 4a) . the genetic integrity of the cloned dna was verified by restriction analysis and sequencing. recovery of recombinant viruses from the cdna clones. to recover infectious virus, bhk cells were grown to 95% confluence in a 12.5-cm 2 flask and transfected with 6 g of the infectious cdna clone using 18 g of lipofectamine 2000 (invitrogen) according to the manufacturer's specifications. at 6 h.p.t., cells were trypsinized, plated over a confluent monolayer of either vero a66 or huh-7 cells grown in a 12.5-cm 2 flask, and incubated at 37°c for 72 h. the cell supernatants were harvested and passaged once on fresh cells, and the recovered viruses were cloned by three rounds of plaque purification, following standard procedures. virus genome sequencing. the complete genome sequence of each rescued recombinant mers-cov was determined by sequencing overlapping rt-pcr fragments of 2.5 kb covering the full-length viral genome. reverse transcription and pcrs were performed with specific oligonucleotides using thermoscript reverse transcriptase (invitrogen) and the expand high-fidelity pcr system (roche), respectively, following the manufacturers' recommendations. the genomic 5=-and 3=-terminal sequences were determined using the 5=/3= race (rapid amplification of cdna ends) kit (roche) according to the manufacturer's specifications. sequence assembly and comparison with the consensus sequence of the mers-cov-emc12 strain were performed with the seqman and megalign programs (lasergene, madison, wi). for the generation of huh-7 cells transiently expressing e protein, cells were nucleofected with the plasmid pcdna3-e (expressing the mers-cov e protein under the cmv promoter) by using a 4d nucleofector device (lonza) and the buffer and program recommended by the manufacturer. for the construction of plasmid pcdna3-e, the e gene was amplified by pcr using pbac-mers fl as the template and the specific oligonucleotides e1-ecori-vs (5= gtgctggaattcgccgccatgttacccttt gtccaagaacgaa 3=, restriction site ecori is underlined) and e249-xhoi-rs (5= cgcccagctcgagttaaacccactcgtcaggtgg 3=, restriction site xhoi is underlined) and cloned into the plasmid pcdna3 (invitrogen) digested with ecori and xhoi. analysis of viral rna synthesis by rt-qpcr. total intracellular rna was extracted from transfected or infected cells with the rneasy miniprep kit (qiagen) according to the manufacturer's specifications. in the case of transfected cells, the residual dna was removed from samples by treating 7 g of each rna with 20 u of dnase i (roche) in 100 l for 30 min at 37°c, and dna-free rnas were repurified using the rneasy miniprep kit (qiagen). viral rna synthesis was quantified by rt-qpcr. total cdna was synthesized with random hexamers from 100 ng of total rna using a high-capacity cdna reverse transcription kit (invitrogen). using this cdna, the viral rna synthesis was analyzed using two custom taqman assays specific for mers-cov grna (forward primer 5= gcacatctgt ggttctcctctct 3=, reverse primer 5= aagcccaggccctactat tagc 3=, and mgb probe 5= tgctccaacagttacac 3=) and sgmrna n (forward primer 5= cttcccctcgttctcttgca 3=, reverse primer 5= tcattgttatcggcaaaggaaa 3=, and mgb probe 5= ctttgattttaacgaatctc 3=). data were acquired with an applied biosystems 7500 real-time pcr system and analyzed with abi prism 7500 software, version 2.0.5. the relative quantifications were performed using the cycle threshold (2 ϫ⌬⌬ct ) method (63) . to normalize for differences in rna sampling, the expression of eukaryotic 18s rrna was analyzed using a specific taqman gene expression assay (hs99999901_s1; applied biosystems). generation of polyclonal antisera specific for mers-cov n and e proteins. rabbit polyclonal antisera (pab) specific for mers-cov n and e proteins were purchased from biogenes. in brief, peptides ntgrs-vyvkfqdskppl (corresponding to e protein amino acids 60 to 76) and aaaknkmrhkrtst (n protein amino acids 244 to 257) were synthesized and used to immunize two rabbits with each peptide according to the company's standard protocol. the polyclonal antisera obtained were evaluated by enzyme-linked immunosorbent assay (elisa) using the synthetic peptides, leading to titers ranging from 1:150,000 to 1:200,000 in all cases. indirect immunofluorescence assay. vero a66 and huh-7 cells were grown to 80% confluence on glass coverslips and infected with the recombinant mers-covs. at 48 h.p.i., cells were fixed either with 4% paraformaldehyde in phosphate-buffered saline (pbs) at room temperature for 20 min or with methanol at ϫ20°c for 15 min. for n protein immunodetection, paraformaldehyde-fixed cells were permeabilized with 0.2% saponin in pbs containing 10% fbs for 20 min and incubated with mers-cov n protein pab (dilution 1:200) in pbs containing 10% fbs at room temperature for 90 min. for e protein immunodetection, methanol-fixed cells were incubated with mers-cov e protein pab (dilution 1:500) in pbs containing 10% fbs overnight at 4°c. coverslips were washed 4 times with pbs and incubated at room temperature for 45 min with goat anti-rabbit antibody conjugated to alexa fluor 488 (invitrogen) diluted 1:500 in pbs containing 10% fbs. nuclei were stained using dapi (4=,6=-diamidino-2-phenylindole) (1:200, sigma). to fully inactivate the samples' infectivity, methanol-fixed cells were treated with 4% paraformaldehyde in pbs as described above. finally, coverslips were mounted in prolong gold antifade reagent (invitrogen) and analyzed on a leica sp5 confocal microscope. images were acquired with the same instrument settings and analyzed with leica software. novel coronavirus associated with severe respiratory disease: case definition and public health measures isolation of a novel coronavirus from a man with pneumonia in saudi arabia update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group human betacoronavirus 2c emc/2012-related viruses in bats full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus detection of alpha and betacoronaviruses in multiple iberian bat species pneumonia from human coronavirus in a macaque model ksa mers-cov investigation team. 2013. hospital outbreak of middle east respiratory syndrome coronavirus evidence of person-to-person transmission within a family cluster of novel coronavirus infections first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the receptor binding domain of the new mers coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo pathogenicity of severe acute respiratory coronavirus deletion mutants in hace-2 transgenic mice construction of a sars-cov infectious cdna clone and a replicon to study coronavirus rna synthesis engineering the largest rna virus genome as an infectious bacterial artificial chromosome molecular characterization of feline infectious peritonitis virus strain df-2 and studies of the role of orf3abc in viral cell tropism recovery of a neurovirulent human coronavirus oc43 from an infectious cdna clone the small envelope protein e is not essential for murine coronavirus replication absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice the nucleoprotein is required for efficient coronavirus genome replication comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features coronavirus gene 7 counteracts host defenses and modulates virus virulence the group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host engineering the transmissible gastroenteritis virus genome as an expression vector inducing lactogenic immunity severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice coronaviruses post-sars: update on replication and pathogenesis accessory protein 5a is a major antagonist of the antiviral action of interferon against murine coronavirus severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists cell type-specific type i interferon antagonism influences organ tropism of murine coronavirus alphacoronavirus protein 7 modulates host innate immune response transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer chimeric coronavirus-like particles carrying severe acute respiratory syndrome coronavirus (scov) s protein protect mice against challenge with scov immune responses against severe acute respiratory syndrome coronavirus induced by virus-like particles in mice comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine immunogenicity, safety, and protective efficacy of an inactivated sars-associated coronavirus vaccine in rhesus monkeys a live attenuated sars coronavirus is immunogenic and efficacious in golden syrian hamsters immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in e protein protects against lethal respiratory disease complete protection against severe acute respiratory syndrome coronavirus-mediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking e protein severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease coronaviruses maintain viability despite dramatic rearrangements of the strictly conserved genome organization vaccines to prevent severe acute respiratory syndrome coronavirusinduced disease complete nucleotide sequence of two generations of a bacterial artificial chromosome cloning vector cloning and stable maintenance of 300-kilobase-pair fragments of human dna in escherichia coli using an f-factor-based vector analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method we thank n. m. beach for critical reading of the manuscript. we also thank milagros guerra for skillful technical assistance. key: cord-292041-a65kfw80 authors: orienti, isabella; gentilomi, giovanna angela; farruggia, giovanna title: pulmonary delivery of fenretinide: a possible adjuvant treatment in covid-19 date: 2020-05-27 journal: int j mol sci doi: 10.3390/ijms21113812 sha: doc_id: 292041 cord_uid: a65kfw80 at present, there is no vaccine or effective standard treatment for severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection (or coronavirus disease-19 (covid-19)), which frequently leads to lethal pulmonary inflammatory responses. covid-19 pathology is characterized by extreme inflammation and amplified immune response with activation of a cytokine storm. a subsequent progression to acute lung injury (ali) or acute respiratory distress syndrome (ards) can take place, which is often followed by death. the causes of these strong inflammatory responses in sars-cov-2 infection are still unknown. as uncontrolled pulmonary inflammation is likely the main cause of death in sars-cov-2 infection, anti-inflammatory therapeutic interventions are particularly important. fenretinide n-(4-hydroxyphenyl) retinamide is a bioactive molecule characterized by poly-pharmacological properties and a low toxicity profile. fenretinide is endowed with antitumor, anti-inflammatory, antiviral, and immunomodulating properties other than efficacy in obesity/diabetic pathologies. its anti-inflammatory and antiviral activities, in particular, could likely have utility in multimodal therapies for the treatment of ali/ards in covid-19 patients. moreover, fenretinide administration by pulmonary delivery systems could further increase its therapeutic value by carrying high drug concentrations to the lungs and triggering a rapid onset of activity. this is particularly important in sars-cov-2 infection, where only a narrow time window exists for therapeutic intervention. coronaviruses (covs) are rna viruses. they may infect both humans and animals, leading to lethal and contagious diseases, such as severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). the recent severe acute respiratory syndrome coronavirus-2 (sars-cov-2) (or coronavirus disease-19 ) is characterized by high genetic homology with sars-cov and mers-cov [1] , sharing with them 79.0% and 51.8% nucleotide identity, respectively. several studies have shown that sars-cov predominantly infects airway and alveolar epithelial cells, vascular endothelial cells, and macrophages. sars-cov-2 and sars-cov use the same receptor, angiotensin-converting enzyme 2 (ace2), for infection, indicating that the same cell types are targeted and infected [2, 3] . in sars-cov-2 infection, the early onset of a rapid viral replication may cause extensive apoptosis of epithelial and endothelial cells and vascular leakage. this frequently leads to acute lung injury and lethal inflammatory responses [4] . although antiviral drugs, glucocorticoids, and mechanical ventilation have been used, there is no specific treatment for covid-19 at the moment [5] . therefore, in the absence of a standard therapeutic intervention, the urgent treatment of pulmonary inflammation to prevent acute lung injury is needed. pulmonary formulations of anti-inflammatory drugs could represent a good option in combination with systemic antiviral drugs or glucocorticoids. in pulmonary administration, the drugs are directly carried to the airway and alveolar epithelia in high concentration, providing rapid onset of the therapeutic response with prompt relief of lung occlusion and respiratory distress symptoms. nevertheless, the drugs used by pulmonary administration in covid-19 should be characterized by low toxicity to avoid injury to the airway and alveolar epithelia already damaged by the viral infection. fenretinide-(n-(4-hydroxyphenyl) retinamide)-is a semisynthetic derivative of all-trans-retinoic acid. it is endowed with many pharmacological features, including anti-inflammatory and antiviral activities, prevention of obesity and type-2 diabetes [6] , and the well-known antitumor activity on a wide range of tumors [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . moreover, its low toxicity profile has been proved in many clinical trials, also in long term treatments [12, 17, 18] . therefore, due to its poly-pharmacology, fenretinide administration by pulmonary formulations may be expected to be protective against acute lung injury (ali)/ acute respiratory distress syndrome (ards) caused by sars-cov infection and could represent a useful tool in a multimodal therapy aimed at establishing a rapid anti-inflammatory and antiviral effect. pulmonary hyper inflammation is a common feature in covid-19 patients. when sars-cov is inhaled and infects epithelial cells in the lungs, an immune response is activated by the local dendritic cells that phagocytose the virus and circulate to the regional lymph nodes where they present antigens to t cells. activated t cells migrate to the lungs, where they mount the immune response. helper t cells secrete cytokines that regulate and assist the immune response; cytotoxic cd8 + t cells (ctls) recognize and kill the infected cells. they release pro-inflammatory cytokines, perforins, and granzymes to induce programmed cell death [19] . however, the cytokines produced by ctls can damage uninfected as well as virus-infected cells, and their massive production may cause detrimental effects, leading to acute lung injury. sars-cov-2 infection is characterized by a rapid viral replication that triggers an amplified immune response with the activation of a cytokine storm. the consequent massive inflammation and increase in vascular permeability induce an abnormal accumulation of neutrophils, macrophages, inflammatory monocytes, and lymphocytes in the lung alveoli, further increasing cytokine production [20] . in this condition, the regulation of the immune response is lost, and the cytokine storm is further activated. without therapeutic intervention, the development of ali/ards and permanent alterations in lung functions may result in dire consequences [4] (figure 1) . moreover, the circulation of cytokines to other organs can lead to multi-organ damage. 1 figure 1 . schematic representation of the progression of coronavirus disease-19 . after an incubation period, severe acute respiratory syndrome coronavirus-2 (sars-cov-2) starts a rapid replication in the lung airway and alveolar epithelial cells. this triggers an immune response with cytokine production, excessive inflammation, and further amplification of the immune response that triggers the cytokine storm. acute lung injury (ali)/ acute respiratory distress syndrome (ards) may arise with dire consequences. fenretinide is the most investigated retinoid due to a significant antitumor activity on a wide range of tumor types [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] combined with a favorable toxicological profile [12, 17, 18] . besides its antitumor activity, multimodal interactions with several biological mechanisms endow fenretinide with other pharmacological properties, including anti-inflammatory activity, antiviral activity, and prevention of obesity and type-2 diabetes [6] . the anti-inflammatory activity of fenretinide has been largely proved, and its mechanism of action has been characterized in different pathological conditions. in inflamed tissues, where the macrophages are characterized by a significantly higher ratio of arachidonic acid (aa) vs. docosahexaenoic acid (dha) than in normal tissues, fenretinide induced normalization of the aa/dha ratio, thus inhibiting phosphorylation of erk1/2 and decreasing the expression of inflammatory cytokines [21] . moreover, fenretinide's ability to downregulate the production of aa and increase the levels of dha provided attenuation of inflammation in cystic fibrosis [22] , osteoporosis [23] , and in a model of spinal cord injury [24] . in a mouse model of allergic asthma, fenretinide inhibited lypopolysaccaride (lps)-induced expression of various th1 and th2 cytokines, including ccl5, ccl7, ccl11, cxcl1, cxcl2, cxcl9, cxcl10, interleukin (il)-6, tnf-α, and inos [25] . in lps-exposed mouse brain microvascular endothelial cells (bend.3 cells), fenretinide showed inhibitory effects on the release of pro-inflammatory cytokines (il-1β, mcp1, inos, and tnf-α). fenretinide may inhibit nf-κb signaling by reducing its nuclear translocation via downregulation of ikkβ and iκbα phosphorylation [26] . this hampers the secretion of inflammatory modulators that is closely associated with the activation of nf-κb signaling. in mouse monocyte/macrophage cells infected with aggregatibacter actinomycetemcomitans, fenretinide suppressed jak-stat, pi3k-akt, pkc, and downstream nf-κb signaling pathways, therefore attenuating il-1β, il-6, and pge2 proinflammatory cytokine expression [27] . fenretinide has been demonstrated to induce reactive oxygen species (ros) generation and oxidative stress in tumor cells as a part of its cytotoxic mechanism [28, 29] . in contrast, it has not increased ros in normal cells, such as fibroblasts, peripheral blood mononuclear cells [29] , and normal and activated peripheral lymphocytes [30] . therefore, its anti-inflammatory activity in non-tumor tissues is not counterbalanced by the ros-inducing activity, and no detrimental effect can be expected when fenretinide is applied to non-tumor tissues. the antiviral activity of fenretinide has been proved in different virus types. in a screening study using a library of pharmacologically active compounds (lopac) of bioactive compounds against the respiratory syncytial virus (rsv), fenretinide was identified as a specific blocker [31] . in the dengue virus (denv), fenretinide has shown high activity both in vitro and in vivo [32] . its mechanism of action includes inhibition of denv non-structural protein 5 (ns5) recognition by host nuclear import proteins importin-α/β1; upregulation of transcripts representing the protein kinase r-like endoplasmic reticulum kinase (perk) arm of the unfolded protein response (upr); phosphorylation of eukaryotic translation initiation factor 2α (eif2α) with consequent translation attenuation and induction of an antiviral state [33] . fenretinide inhibited the steady-state accumulation of viral genomic rna and reduced viremia when orally administered in a murine model of denv infection. the molecular target responsible for this antiviral activity was distinct from other known inhibitors of denv and appeared to affect other members of the flaviviridae, including the west nile, modoc, and hepatitis c viruses [34] . fenretinide inhibited the zika virus (zikv) in mammalian cell culture and reduced both viremia and brain viral burden in a murine model. this antiviral activity was correlated with a significant decrease in the abundance of zikv rna. it was found that fenretinide reduced rna synthesis without direct inhibition of the viral polymerase or chemical destabilization of membrane-associated replication complexes, rather, fenretinide acted via an indirect mechanism mediated by a host factor [35] . in both zikv and denv, fenretinide inhibited the non-structural protein 5 (ns5), which contributed to pathogenesis, by antagonism with the production of interferon-i (ifn-i) [36] . as it is well-known, ifn-i promotes an early protective host mechanism against viral infection, by driving an antiviral state in non-immune cells and activating antiviral immune responses. therefore, the reestablishment of ifn-i production by fenretinide can block the early onset of viral infection [37] . the delayed release of ifn-i is well known in sars-cov and mers-cov infections as mechanisms to hinder the body's antiviral response [38] . the viral mechanisms of ifn-i evasion are multifaceted, including sequestering and shielding rna within double-membrane vesicles, modification of viral mrna 5 -cap structures, and specific targeting of antiviral cellular pathways [39, 40] . in sars-cov and mers-cov, inf-i production is protective only at the early stages after infection; at later time points, on the contrary, when exaggerating immune response takes place, ifn-i and inflammatory cytokines become pathogenic [41] . no studies on the ability of fenretinide to block inf-i evasion in coronavirus have been reported so far, but, by analogy with flaviviridae, some fenretinide activity on the mechanisms regulating the inf-i evasion in coronavirus might be expected. fenretinide treatment has been found to inhibit hiv infection by perturbing cell membrane structure and inhibiting crucial early events in the hiv fusion process [42] . fenretinide indeed is known to modify the biosynthesis and metabolism of ceramides, therefore, changing localized membrane domain organization. cell membrane modifications might alter the trafficking of virions through the endocytic pathway, thus leading to non-productive infection [42, 43] . sars-cov-2 predominantly infects airway and alveolar epithelial cells, vascular endothelial cells, and macrophages by linking angiotensin-converting enzyme 2 (ace2) that is abundantly expressed in the human lungs. after linkage to ace2, the viral entry into the host cells is mainly mediated by endocytosis. consequently, inhibition of cell entry may be expected by treatment with fenretinide. it has been observed that the endocytic pathway of sars-cov-2 also involves autophagy with the formation of autophagosomes and autolysosomes [44] . autophagy represents an essential cellular process, decreasing virus infections. beclin1 (becn1) is one of its key regulators. mers-cov replication results in reduced becn 1 levels, with a consequent block of the fusion of autophagosomes and lysosomes [45] . thus, autophagy-inducing, becn1-increasing agents may have antiviral effects [46] . fenretinide has demonstrated autophagy-inducing ability in different cell types. in human mammary carcinoma cell line mcf-7, fenretinide triggered an autophagic cell death pathway mediated by an increase in beclin 1 expression [47] . in human pancreatic cancer cells, fenretinide induced autophagy by ros increase [48] . in gastric carcinoma hcg27, autophagy was stimulated by the accumulation of dihydroceramide induced by fenretinide [49] . another target for antiviral activity is the akt1/mtor pathway. indeed, a kinome analysis of mers-cov-infected cells has detected phosphorylation changes of several regulatory kinases, including akt1 and mtor. these changes have been correlated to autophagy [50, 51] . the pharmacological use of akt1-and mtor-pathway inhibitors has shown promise for antiviral therapy against several viruses, including mers-cov [51] . the inhibitory effect of fenretinide in akt1 and mtor pathways has been reported in tumor cells [52] and in monocytes/macrophages [27, 53] . all these findings clearly show the involvement of fenretinide in multiple pathways, controlling viral replication. therefore, even in the absence of specific studies on sars-cov-2, an indirect antiviral activity of fenretinide may be supposed through the induction of an "antiviral environment", hindering supportive steps of viral replication, both at a cellular level and in the infected tissues. fenretinide has been well-tolerated in many clinical trials aimed at evaluating its antitumor activity in pediatric and adult cancers. oral administrations of fenretinide in corn oil gelatin capsules (4000 mg/m 2 for 28 days) or in a lipid matrix (lym-x-sorb, 61 mg/kg for 7 days) have been well-tolerated at high doses and for protracted periods of time. no significant hematological alterations or dose-limiting side effects have been obtained [9, 54] in these studies. many other clinical trials with fenretinide have confirmed its tolerability [17, 55, 56] . a phase ii study reported thrombocytopenia, anemia, hepatosplenomegaly in patients with myelodysplastic syndromes orally treated with fenretinide at 300 mg/day for 4 weeks and escalated to 400 mg/day for other 8 weeks [57] . thrombocytopenia was also observed in a phase i study in some patients with hematologic malignancies after administration of fenretinide (905 mg/m 2 ) as a continuous intravenous infusion for five consecutive days [58] . in a very recent study in mice, oral fenretinide led to splenomegaly and increased the circulating leukocytes [59] . a trial aimed at evaluating the effect of fenretinide as a chemopreventive agent included 1435 patients that received a 5-year fenretinide oral treatment. endpoints considered for safety assessment indicated that fenretinide was endowed with good tolerability. indeed, the most common adverse events were diminished dark adaptation (19.0%) and dermatologic disorders (18.6%). less common events were gastrointestinal symptoms (13.0%) and disorders of the ocular surface (10.9%). these symptoms occurred during fenretinide treatment and recovered over time [12, 60] . in covid-19 patients, the rapid evolution of the disease requires prompt treatment because only a narrow time window exists for therapeutic intervention. in this regard, pulmonary drug delivery offers the advantage of carrying the bioactive molecule in direct contact with the pathological lung epithelia, thus ensuring a rapid onset of the therapeutic response. high local drug concentrations may be easily obtained by pulmonary administration with a concomitant increase of the pharmacological effect but without the side effects elicited by other administration routes. drugs administered by enteral or parenteral routes, indeed, need to reach the blood circulation to be distributed to tissues and organs and enter the pathological site. this general distribution may provide extensive side effects, particularly when high drug administration doses are required to achieve a therapeutically active concentration of the drug in the pathological site. the increased pharmacological effect provided by high drug local concentrations and the decreased toxicity due to lack of a systemic distribution may improve the overall therapeutic efficiency of the drugs administered by pulmonary route, as demonstrated in several respiratory diseases, such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease [61] . the inhalation devices play a crucial role in the effectiveness of pulmonary drug administration. the most common devices are nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers), metered-dose inhalers, and dry powder inhalers [62] . the selection of the inhalation device depends on the physicochemical characteristics of the drug and its formulation. liquid formulations are administered by nebulizers and metered-dose inhalers, and solid formulations by dry powder inhalers. in each case, the inhalation device provides aerosol particles whose size can control the extent of inhaled drug accumulation and the site of drug deposition within the airways. smaller particles achieve a greater total drug accumulation in the lungs and farther distal airway penetration compared with larger particles. particles smaller than 5 µm in diameter may flow in the airstream beyond the retro-pharynx and reach the trachea. particles of 2-5 µm in diameter are deposited in the upper respiratory tract at the level of the trachea and tracheal bifurcation. particles smaller than 2 µm in diameter deposit in the lower airway and alveolar epithelia [63, 64] . then, the modulation of intrapulmonary deposition through the control of the aerosol particle size can appreciably improve the inhalation drug therapy (figure 2 ). the pulmonary administration of drugs mainly provides a local therapy but may also provide a systemic therapy when the physicochemical characteristics of the drugs can support their absorption through the alveolar epithelium at extents suitable to elicit systemic effects. indeed, the large surface area, extensive vascularization, and single-cell barrier in the alveoli make the lungs an appropriate portal for the systemic absorption of molecules, such as insulin, human growth hormone, etc. [65] . pulmonary delivery of fenretinide could be a valuable tool in covid-19 due to the possibility of obtaining a very high drug concentration in the airway and alveolar epithelia, thus triggering a rapid onset of local anti-inflammatory response. at the same time, the ability of fenretinide to induce an "antiviral environment" could further enhance its therapeutic efficacy (figure 3 ). in order to be effective, pulmonary fenretinide formulations should provide aerosol particle size smaller than 2 µm, for deposit in the lower airway and alveolar epithelia, where the infection process is amplified by the extensive vascularization. moreover, after deposition, they should trigger a rapid drug release to speed up the onset of the therapeutic activity. such formulations require fenretinide solubilization in an aqueous phase, and the adequate solubilization degree to provide high concentrations of the bioavailable drug, in the lungs, after inhalation. unfortunately, the hydrophobic character of fenretinide strongly hinders its aqueous solubilization. moreover, the possibility to use solubilizing agents, such as tensides or water-mixable co-solvents, is severely restricted, by tolerability issues, in the formulations destined to inflamed lungs. highly tolerated, aqueous fenretinide formulations have been obtained by complexation with cyclodextrins [52] or encapsulation in nanomicelles [66] . complexation with 2-hydroxypropyl beta-cyclodextrin has increased fenretinide aqueous solubility from 0.017 mg/ml (pure drug) to 2.41 mg/ml (complex). the aqueous formulation of the complexed drug, administered by the parenteral route, was well-tolerated and increased the drug bioavailability and antitumor activity in mouse models of different tumor types [52] . nanoencapsulation in phosphatidylcholine-glyceryltributyrate nanomicelles has increased fenretinide's aqueous solubilization up to 3.88 mg/ml (nanoencapsulated drug). the intravenous administration of the nanomicelles in mice bearing tumor xenografts showed enhanced drug bioavailability and antitumor activity [66] . moreover, high tolerability was demonstrated by the absence of adverse effects after repeated administrations and for protracted periods of time. therefore, the in vivo tolerability and the ability to provide high fenretinide solubilization levels suggest that complexation with cyclodextrins [52] or encapsulation in nanomicelles [66] can be valuable means for preparation of safe and efficient pulmonary fenretinide formulations. there is presently no vaccine or documented specific, standard treatment against covid-19. most of the potential drugs are being investigated for safety and efficacy in sars-cov-2 infection, but, until now, no drugs are validated to have significant efficacy in the clinical treatment of covid-19 patients in large-scale studies. current treatments are using drugs that were originally designed for other pathologies and have been repurposed for covid-19 trials. these include antiviral and immunomodulating drugs designed to boost the innate immune response or to inhibit the inflammatory processes, causing lung injury. drugs for symptomatic control are also employed. antiviral drugs act on the coronavirus by direct mechanisms, inhibiting key viral enzymes responsible for genome replication or hindering viral entry to human cells. remdesivir is the most promising, among the antiviral drugs. it exhibits broad-spectrum antiviral activity against rna viruses. in previous studies, it showed antiviral activities, both in vitro and in vivo, against different coronaviruses, including sars-cov and mers-cov [67, 68] . in a recent study, remdesivir inhibited sars-cov-2 in vivo [69] . ivermectin is an anti-parasitic agent, which has proved to exert antiviral activities toward both hiv and dengue virus [70] . recently, an in vivo study put in evidence its capability to reduce viral rna up to 5000 fold in sars-cov-2 infection [71] . lopinavir/ritonavir are protease inhibitors. they are used in combination in the treatment of patients with hiv infection. several clinical, preclinical, and in vitro studies performed on sars-cov and mers-cov proved that the lopinavir/ritonavir combination was effective against these viruses [72] [73] [74] . hydroxychloroquine is active against malaria as well as autoimmune diseases (such as rheumatoid arthritis, lupus erythematosus). it has been recently investigated as a potential broad-spectrum antiviral drug for its ability to increase the endosomal ph, which prevents virus-cell fusion [75] . hydroxychloroquine has been shown to specifically inhibit the replication of sars-cov by interfering with the glycosylation of its cellular receptor ace2 [76] . recent in vitro studies revealed its ability to effectively reduce the viral copy number of sars-cov-2 [77] . immunomodulating drugs able to stimulate innate antiviral immune responses are now repurposed for the treatment of covid-19. natural killer (nk) cells represent a highly specialized lymphoid population of the innate immune system with potent activity against virus-infected cells. migration of nk cells and macrophages to the lungs has been demonstrated to play a major role in the clearance of sars-cov [78] . indeed, the innate response itself can control sars-cov infection, without the help of ctls and antibodies, particularly in the early stages of the infection process. type i interferons are secreted by virus-infected cells and represent a host protective mechanism. recombinant interferons, used alone or in combination with other drugs, have provided a broad-spectrum antiviral activity against hcv, respiratory syncytial virus, sars-cov [79] , and mers-cov [73] . recent trials are assessing their safety and efficacy in covid-19. drugs interfering with the il-6 pathway are used to attenuate the inflammatory response in covid-19 [80] . elevated il-6 levels are predictive of poor prognosis in patients with ards [81] . the pathway of il-6 signaling occurs through il-6 receptors, which are expressed predominantly on neutrophils, monocytes, macrophages, and some lymphocytes [82] . tocilizumab and siltuximab are monoclonal antibodies against il-6. tzls-501 is an anti-il-6 receptor antibody, and sarilumab an il-6 receptor antagonist. they all are designed to inhibit the binding of interleukin-6 to its receptors, thus alleviating cytokine release syndrome. thalidomide has been repurposed for the treatment of covid-19 due to its anti-angiogenic, anti-inflammatory, and anti-fibrotic activity. its anti-inflammatory activity is mainly based on the inhibition of tnf-α production. thalidomide has shown promise in the treatment of multiple inflammatory diseases, such as lupus erythematosus and crohn disease [83] . preclinical studies proved that thalidomide was effective in treating mice infected with influenza virus h1n. it decreased the infiltration of inflammatory cells and inhibited the production of inflammatory cytokines [84] . recent studies are evaluating its efficacy in sars-cov-2 [85] . lenalidomide is a derivative of thalidomide. it is currently used in the maintenance therapy of multiple myeloma due to its immunostimulatory effect on nk cell number and function combined with an acceptable toxicity profile. lenalidomide has been proven to limit the amount of pro-inflammatory cytokines, such as tnf-α, il-12, il-1, il-6, and increased il-2 and ifnγ [86, 87] . many studies have demonstrated the lenalidomide's strong anti-angiogenic activity [88] [89] [90] [91] . with respect to thalidomide, lenalidomide is expected to provide a superior activity and decreased side effects in covid-19 treatment. glucocorticoids have been used in sars-cov and mers-cov infections to restrain lung inflammation and immune responses [92] [93] [94] . their use has provided side effects, such as secondary bacterial infections, osteoporosis, and prolonged viral clearance. glucocorticoids, such as methylprednisolone, are now under evaluation in covid-19 for effectiveness and safety [85] . pulmonary administration of fenretinide, in combination with the drugs currently used in covid-19 treatment, could represent a new, effective tool to improve the efficacy of the single drugs and produce a strengthened overall pharmacological response. fenretinide is particularly suitable to be used in combination because its poly-pharmacology may reinforce the activity of several drugs, and its high tolerability may enable large adjustments of the administered dose without toxicity concerns. the combination of fenretinide with the antiviral drugs used in covid-19 is expected to improve the treatment efficacy due to the ability of fenretinide to induce an "antiviral environment" that can behave as an adjuvant to the specific antiviral activity of the drugs. in combination with the immunomodulating drugs, fenretinide may contribute to enhance the innate immune response due to its stimulating effect on nk cells' proliferation and cytotoxicity [95, 96] . moreover, its anti-inflammatory activity, largely demonstrated in several models, may boost the attenuation of the inflammatory response induced by the immunomodulating drugs. in particular, the ability of fenretinide to decrease il-6 [25, 27] should improve the efficacy of the drugs blocking the il-6 pathway, such as tocilizumab, siltuximab, sarilumab, tzls-501. the combinations of thalidomide or lenalidomide with fenretinide are expected to be very effective due to the multiplicity of mechanisms involved in the anti-inflammatory activity of both types of drugs. particularly, the decrease of tnf-α [25, 26] and the antiangiogenic effect [97, 98] provided by fenretinide, added to the same effects provided by thalidomide and lenalidomide, should boost the anti-inflammatory response elicited by the combination. fenretinide and lenalidomide were evaluated, by intravenous administration, in a mouse model of neuroblastoma. high tolerability and antitumor activity were obtained with this combination [66] . in regard to the administration route, the drugs to combine with the pulmonary fenretinide are expected to be administered by enteral or parenteral routes, depending on the form of the formulations available on the market. however, the pulmonary administration route would be the best option because, as already reported for fenretinide, the local drug administration in the pathological site would provide a rapid onset of the therapeutic response. moreover, in addition to local therapy, systemic therapy may also be obtained if the drug absorption through the alveolar epithelium can take place at a suitable extent. the utility of fenretinide anti-inflammatory activity, associated with its high tolerability, has been repeatedly demonstrated in lung-related pathologies, such as cystic fibrosis, allergic asthma, and chronic lung infections. its antiviral activity in the zika virus, dengue virus, respiratory syncytial virus, hepatitis c virus, and hiv has been largely proved and attributed to several mechanisms of action. although there is no direct evidence of fenretinide application in covid-19, the multiple inhibition mechanisms demonstrated in the studied viruses and the involvement of fenretinide in multiple pathways controlling viral replication strongly suggest that fenretinide could provide an indirect antiviral activity towards sars-cov-2, as well as other virus types, by means of the induction of an "antiviral environment" hindering infection. therefore, both the anti-inflammatory activity and the ability to induce an "antiviral-environment" indicate that fenretinide may have supportive adjuvant utility in treating covid-19. fenretinide administration by means of pulmonary formulations may further improve its therapeutic value by providing high drug concentrations in the pathological lung epithelia and a fast onset of the drug activity, which is particularly important in sars-cov-2 infection, where only a narrow time window exists for therapeutic intervention. moreover, the pulmonary administration of fenretinide, in combination with the drugs that are currently used in sars-cov-2 infection, could represent a new, effective tool in covid-19 treatment. the authors declare no conflict of interest. identification of a novel coronavirus causing severe pneumonia in human single-cell rna expression profiling of ace2, the receptor of sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools clinical features of patients infected with 2019 novel coronavirus in the mechanisms of fenretinide-mediated anti-cancer activity and prevention of obesity and type-2 diabetes clinical development of fenretinide as an antineoplastic drug: pharmacology perspectives phase i trial and pharmacokinetics of fenretinide in children with neuroblastoma phase i trial of fenretinide delivered orally in a novel organized lipid complex in patients with relapsed/refractory neuroblastoma: a report from the new approaches to neuroblastoma therapy (nant) consortium. pediatr. blood cancer a phase ii study of fenretinide in patients with hormone refractory prostate cancer: a trial of the cancer therapeutics research group phase ii trial of fenretinide (nsc 374551) in patients with recurrent small cell lung cancer fifteen-year results of a randomized phase iii trial of fenretinide to prevent second breast cancer phase ii study of oral capsular 4-hydroxyphenylretinamide (4-hpr/fenretinide) in pediatric patients with refractory or recurrent neuroblastoma: a report from the children's oncology group high plasma levels of fenretinide (4-hpr) were associated with improved outcome in a phase ii study of recurrent ovarian cancer: a study by the california cancer consortium phase ii study of fenretinide (nsc 374551) in adults with recurrent malignant gliomas: a north american brain tumor consortium study phase ii trial of fenretinide in advanced renal carcinoma. investig. new drugs phase i trial of oral fenretinide in children with high-risk solid tumors: a report from the children's oncology group (ccg 09709) phase i clinical trial of fenretinide (nsc374551) in advanced solid tumors quis custodiet ipsos custodes? regulation of cell-mediated immune responses following viral lung infections new insights into the immune molecular regulation of the pathogenesis of acute respiratory distress syndrome fenretinide corrects the imbalance between omega-6 to omega-3 polyunsaturated fatty acids and inhibits macrophage inflammatory mediators via the erk pathway cystic fibrosis fatty acid imbalance is linked to ceramide deficiency and corrected by fenretinide fenretinide prevents the development of osteoporosis in cftr-ko mice fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice fenretinide prevents inflammation and airway hyperresponsiveness in a mouse model of allergic asthma fenretinide attenuates lipopolysaccharide (lps)-induced blood-brain barrier (bbb) and depressive-like behavior in mice by targeting nrf-2 signaling fenretinide inhibited de novo ceramide synthesis and proinflammatory cytokines induced by aggregatibacter actinomycetemcomitans preferential eradication of acute myelogenous leukemia stem cells by fenretinide reactive oxygen species-mediated synergism of fenretinide and romidepsin in preclinical models of t-cell lymphoid malignancies implication of mitochondria-derived ros and cardiolipin peroxidation in n-(4-hydroxyphenyl)retinamide-induced apoptosis replication-competent influenza virus and respiratory syncytial virus luciferase reporter strains engineered for co-infections identify anti-viral compounds in combination screens a nuclear transport inhibitor that modulates the unfolded protein response and provides in vivo protection against lethal dengue virus infection novel dengue virus inhibitor 4-hpr activates atf4 independent of protein kinase r-like endoplasmic reticulum kinase and elevates levels of eif2α phosphorylation in virus infected cells the bioactive lipid 4-hydroxyphenyl retinamide inhibits flavivirus replication anti-viral activity of n-(4-hydroxyphenyl) retinamide (4-hpr) against zika virus nuclear import inhibitor n -(4-hydroxyphenyl) retinamide targets zika virus (zikv) nonstructural protein 5 to inhibit zikv infection the many faces of the flavivirus ns5 protein in antagonism of type i interferon signaling ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes to sense or not to sense viral rna-essentials of coronavirus innate immune evasion antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology fenretinide inhibits hiv infection by promoting viral endocytosis proc. natl. acad. sci targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid-19 skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection autophagy and viruses: adversaries or allies? fenretinide induces autophagic cell death in caspase-defective breast cancer cells cytotoxic responses to n-(4-hydroxyphenyl)retinamide in human pancreatic cancer cells dihydroceramide delays cell cycle g1/s transition via activation of er stress and induction of autophagy anti-viral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis a pharmacologic target for autophagy regulation a new bioavailable fenretinide formulation with antiproliferative, antimetabolic, and cytotoxic effects on solid tumors the chemopreventive retinoid 4hpr impairs prostate cancer cell migration and invasion by interfering with fak/akt/gsk3beta pathway and beta-catenin stability pharmacokinetics of oral fenretinide in neuroblastoma patients: indications for optimal dose and dosing schedule also with respect to the active metabolite 4-oxo-fenretinide oral fenretinide in biochemically recurrent prostate cancer: a california cancer consortium phase ii trial results of a phase i-ii study of fenretinide and rituximab for patients with indolent b-cell lymphoma and mantle cell lymphoma phase ii trial of fenretinide [n-(4-hydroxyphenyl) retinamide] in myelodysplasia: possible retinoid-induced disease acceleration phase i study of fenretinide delivered intravenously in patients with relapsed or refractory hematologic malignancies: a california cancer consortium trial fenretinide treatment accelerates atherosclerosis development in apoe-deficient mice in spite of beneficial metabolic effects safety of the synthetic retinoid fenretinide: long-term results from a controlled clinical trial for the prevention of contralateral breast cancer pulmonary administration: strengthening the value of therapeutic proximity to study the attitudes, beliefs and perceptions regarding the use of inhalers among patients of obstructive pulmonary diseases and in the general population in punjab systematic review of errors in inhaler use: has patient technique improved over time? chest drug delivery to the lungs: challenges and opportunities de fougerolles, a. nanobodies® † nanobody is a registered trademark of ablynx nv. as inhaled biotherapeutics for lung diseases a novel nanomicellar combination of fenretinide and lenalidomide shows marked antitumor activity in a broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro ivermectin is a specific inhibitor of importin alpha/beta-mediated nuclear import able to inhibit replication of hiv-1 and dengue virus the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro treatment with lopinavir / ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 effects of chloroquine on viral infections: an old drug against today's diseases chloroquine is a potent inhibitor of sars coronavirus infection and spread positive rt-pcr test results in patients recovered from covid-19 cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4+ t cells are important in control of sars-cov infection treatment of sars with human interferons il-6 trans-signaling via the soluble il-6 receptor: importance for the pro-inflammatory activities of il-6 interleukin-6 displays lung anti-inflammatory properties and exerts protective hemodynamic effects in a double-hit murine acute lung injury the il-6/sil-6r complex as a novel target for therapeutic approaches thalidomide-induced teratogenesis: history and mechanisms anti-inflammatory effect of thalidomide on h1n1 influenza virus-induced pulmonary injury in mice a review of sars-cov-2 and the ongoing clinical trials lenalidomide acts as an adjuvant for hcv dna vaccine anti-inflammatory modulation of human myeloid-derived dendritic cell subsets by lenalidomide mechanism of action of lenalidomide in hematological malignancies antiangiogenic in vivo effect of lenalidomide (cc-5013) in myelodysplastic syndrome with del (5q) chromosome abnormality and its relation to the course of disease orally administered lenalidomide (cc-5013) is anti-angiogenic in vivo and inhibits endothelial cell migration and akt phosphorylation in vitro the anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions systematic review of treatment effects corticosteroid therapy for critically ill patients with middle east respiratory syndrome adjunctive glucocorticoid therapy in patients with septic shock effects of n-(4-hydroxyphenyl)-retinamide on the number and cytotoxicity of natural killer cells in vitamin-a-sufficient and -deficient rats retinoids, breast cancer and nk cells anti-angiogenic properties of chemopreventive drugs: fenretinide as a prototype fenretinide as an anti-angiogenic agent in neuroblastoma key: cord-295559-yc8q62z8 authors: qian, zhaohui; dominguez, samuel r.; holmes, kathryn v. title: role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation date: 2013-10-03 journal: plos one doi: 10.1371/journal.pone.0076469 sha: doc_id: 295559 cord_uid: yc8q62z8 little is known about the biology of the emerging human group c betacoronavirus, middle east respiratory syndrome coronavirus (mers-cov). because coronavirus spike glycoproteins (s) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of mers-cov s protein is a high research priority. mers-cov s on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from new world eptesicus fuscus bats. surprisingly, a polyclonal antibody to the s protein of mhv, a group a murine betacoronavirus, cross-reacted in immunoblots with the s2 domain of group c mers-cov spike protein. mers pseudovirions released from 293t cells contained only uncleaved s, and pseudovirus entry was blocked by lysosomotropic reagents nh(4)cl and bafilomycin and inhibitors of cathepsin l. however, when mers pseudovirions with uncleaved s protein were adsorbed at 4°c to vero e6 cells, brief trypsin treatment at neutral ph triggered virus entry at the plasma membrane and syncytia formation. when 293t cells producing mers pseudotypes co-expressed serine proteases tmprss-2 or -4, large syncytia formed at neutral ph, and the pseudovirions produced were non-infectious and deficient in s protein. these experiments show that if s protein on mers pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin l-dependent manner, but if mers-cov s is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral ph and cause massive syncytia formation even in cells that express little or no mers-cov receptor. thus, whether mers-cov enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion. coronaviruses cause respiratory, enteric, renal and/or neurological disease in humans, many other mammals and birds. in 2002-03 a previously unknown coronavirus emerged from a wild animal reservoir to cause the sars pandemic, with about 8,000 human cases and more than 770 deaths [1, 2] . previously, cross-species transmission from coronaviruses of bat and bovine origin had allowed human respiratory coronaviruses oc43, nl63 and 229e to become established in the human population worldwide [3] [4] [5] [6] [7] [8] . in the arabian peninsula in 2012, another novel human cov, now called middle east respiratory syndrome coronavirus (mers-cov), was isolated in vero e6 cells from sputum from a fatal case of severe respiratory disease with kidney failure. since then, mers-cov rna has been detected by rt-pcr in over 70 patients with severe to moderate respiratory disease, 39 of whom have died [9, 10] . genome sequence analysis showed that mers-cov is a novel betacoronavirus in genogroup c, closely related to two prototype group c betacoronaviruses of asian bats, btcov-hku4 from a tylonycteris pachypus bat and btcov-hku5 from a pipistrellus abramus bat [11] , and to partial sequences of a group c betacoronavirus from a pipistrellus pipistrellus bat in the netherlands [12] . recently group c betacoronaviruses were also detected in a nyctinomops laticaudatus bat in mexico [13] , and nycyteris cf. gambiensis bats in ghana [14] . mers-cov, like sars-cov, is probably a zoonotic betacoronavirus that has spilled over into humans, directly or indirectly, from one of the species of bats that harbor group c betacoronaviruses or from other unknown animal reservoirs [13, 15, 16] . the ~200 kda spike glycoprotein (s) of coronaviruses is an important determinant of virus virulence, tissue tropism and host range. trimers of s form the characteristic large spikes on the coronavirus envelope that bind to receptors, mediate membrane fusion, virus entry and syncytia formation, and elicit virus neutralizing antibodies. coronavirus s proteins are class i viral fusion proteins like the hiv envelope (env), influenza hemagglutinin (ha) and paramyxovirus fusion (f) glycoproteins [17] , which typically require protease cleavage between the s1 and s2 domains ( figure 1a ) to permit conformational changes in s2, activated by receptor binding and/or low ph, that mediate membrane fusion leading to virus entry and syncytia formation [3, 17, 18] . in different cell types and tissues, coronavirus s proteins may be cleaved by a variety of host proteases including furin, trypsin, human airway trypsin-like protease (hat), transmembrane protease serine protease-2 (tmprss-2), tmprss-4, or cathepsins [18] [19] [20] [21] [22] . functional analysis of mers-cov s glycoprotein is needed to identify susceptible cell types and host species that affect viral tissue tropism and host range, and to determine how various host proteases promote mers-cov virus entry and syncytia formation. identification of the receptor or receptors is an important first step in understanding the host range and tissue tropism of coronaviruses. four receptor proteins for spike proteins of different coronaviruses are now known: murine carcinoembryonic antigen cell adhesion molecule 1a (mceacam1a) for mouse hepatitis virus (mhv) [23] , a betacoronavirus in group a; aminopeptidase n (apn) for human coronavirus 229e (hcov-229e) and several other alphacoronaviruses [24, 25] ; and angiotensin-converting enzyme 2 (ace2) for both sars-cov, a betacoronavirus in group b and hcov-nl63, an alphacoronavirus [26, 27] . raj and co-workers [28] recently demonstrated that mers-cov uses dipeptidyl peptidase 4 (dpp4) as a receptor. in contrast, s proteins of several group a betacoronaviruses including bovine coronavirus and hcov-oc43 use sialic acid moieties as receptors [29, 58] . we have used lentivirus pseudotypes with mers-cov spike glycoprotein to identify cells susceptible to infection with mers-cov and to study the role of mers s protein in virus entry and syncytia formation. expression of coronavirus s proteins on 293t cell membranes for incorporation into lentivirus pseudovirions can be enhanced by using codon-optimized spike cdna and deleting an er/golgi retention motif and an endosomal recycling motif from the cytoplasmic tail of s [30] [31] [32] . codonoptimized cdna encoding s of mers-cov (derived from genbank: afs88936) [15] , with the 16 c-terminal amino acids replaced by a linker, ggggs, and a flag tag (here called mers-cov sδ16) ( figure 1a ) was expressed on 293t cell figure 1b, lane 2) . no protease cleavage products of the ~200kda s protein were detected in transfected 293t cells or pseudovirions ( figure 1b) . in marked contrast, the mers lentivirus pseudovirions used to identify cells susceptible to entry of mers-cov in the poehlmann laboratory [33] , contained a high proportion of cleaved mers-cov s protein at about 100 kda. this important difference in the mers pseudovirions is likely due to differences between our 293t cells and those used in the poehlmann laboratory. surprisingly, when these mers pseudovirions and cell lysates were blotted with polyclonal goat antibody ao4 to spikes purified from detergent-disrupted virions of mhv-a59, a betacoronavirus in group a, the mers s protein bands were detected ( figure 1b ). immunoblotting of soluble, truncated mers s proteins with c-terminal flag tags showed that the ao4 antibody did not recognize the s1 domain of mers s ( figure 1c ), so the cross-reactivity between these proteins from betacoronavirus groups a and c must lie within the s2 domain. vero e6 and llcmk2 monkey kidney cell lines are susceptible to infection with mers-cov virus and to sars-cov [10, 34] , and also susceptible to sars pseudovirions and to mers pseudovirions with uncleaved s protein ( figure 2a ). cell entry was quantitated by expression of the luciferase reporter gene in pseudovirus-transduced cells. compared to control pseudovirions with no spike protein, mers pseudovirions showed a 100 to 1,000 fold increase in luciferase activity in vero e6 and llcmk2 cells (figure 2a and b), and sars pseudovirions showed a 1,000 increase in luciferase activity in vero e6 cells. because the uncleaved mers-cov s protein mediated virus entry into vero e6 and llcmk2 cells, transduction by mers pseudovirions was used to identify additional cell lines that express functional receptors for mers-cov [10, 34] . mers pseudovirions detected strong mers-cov receptor activity on the calu3 line of human airway epithelial cells (figure 2a and b) , and weaker receptor activity on the a549 line of human alveolar basal epithelial cells ( figure 2a ) as also shown by mers-cov infection [35] . interestingly, the eff embryo cell line from eptesicus fuscus bats was susceptible to mers pseudovirions, increasing luciferase activity by nearly 100-fold compared to the no spike control, but the tb1lu lung cell line from tadarida brasiliensis bats, murine fibroblasts and hela cells were not susceptible to mers pseudovirions ( figure 2c ). expression of human ace2 in 293t cells did not significantly increase susceptibility to mers pseudovirions ( figure 3a ), although as expected hace2 greatly increased susceptibility of 293t cells to sars pseudovirions ( figure 3a ). figure 3b and c show that neither human ceacam1, or four related human ceacam proteins or human apn functions as a receptor for mers-cov spike protein. these experiments confirm the observation that mers-cov does not use the receptor proteins known for other coronaviruses [33] or related human membrane proteins. instead dpp4 is the principal receptor protein for mers-cov [28] . mers pseudovirions induced a small but consistent 5 to 10-fold increase in luciferase activity in 293t human embryo kidney cells compared to the no spike control virus ( figure 2c ), suggesting that our 293t cells expressed either a low level of dpp4, or an alternative but less efficient receptor, such as cd209l or lsectin for sars-cov [36, 37] . to determine whether entry of mers pseudovirions with uncleaved s protein required endocytosis and acidification in endosomes, the effects of ammonium chloride and bafilomycin a, lysosomotropic agents that inhibit the acidification of endosomes, were studied. in vero e6 cells, 20mm nh 4 cl inhibited entry of sars pseudovirions by about 99.9% compared to entry of sars pseudovirions without inhibitor, and nh 4 cl also inhibited entry of mers pseudovirions by about 99.6% ( figure 3a ). bafilomycin a specifically inhibits the vacuolar-type h+-atpase that is required for acidification of lysosomes. figure 3a shows that bafilomycin a inhibited entry of sars pseudovirions into vero e6 cells by 99.8% as previously reported [21, 38] , and also inhibited entry of mers pseudovirions by more than 99.9% compared to mers pseudovirions without inhibitor. in llcmk2 cells, although bafilomycin a inhibited 99.7% of mers-cov s mediated entry, nh 4 cl reduced mers-cov s-mediated entry only 6-fold (data not shown), suggesting that the inhibition of endosomal acidification by nh 4 cl may be cell type dependent. these experiments show that mers pseudovirions with uncleaved s protein can enter monkey kidney cells only by endocytosis. cathepsins are a diverse group of acid-activated cysteine proteases located within endosomes and lysosomes. cathepsin activity is essential for infection by several viruses that enter by the endosomal route, including reovirus [39] , sars-cov [22] , and ebolavirus [40] . e64d, an inhibitor of the cysteine protease activities of cathepsins b, h, and l and calpain, reduced transduction of vero e6 cells by sars pseudovirions by 80% as previously reported ( figure 3b ) [41] . since cell entry mediated by vsv-g glycoprotein does not require protease activation [17] , e64d treatment of vero e6 ( figure 3b ) and llcmk2 cells (data not shown) did not inhibit entry of vsv pseudovirions. however, e64d decreased entry into vero e6 cells of mers pseudovirions with uncleaved s by 96.7% ( figure 3b ) and llcmk2 cells by 99.2% (data not shown). thus, cleavage of mers-cov s protein by one of the cathepsins or calpain was required for triggering s-mediated membrane fusion and virus entry at low ph in endosomes. as previously reported [41] , in vero e6 cells 10 µm of cathepsin l inhibitor iii, a specific and irreversible inhibitor of cathepsin l, significantly inhibited entry mediated by sars s protein, but did not inhibit vsv-g-mediated entry ( figure 3b ). cathepsin l inhibitor iii reduced entry into vero e6 cells of mers pseudovirions with uncleaved s protein by 97% relative to entry without inhibitor ( figure 3b ), and similar results were seen in llcmk2 cells (data not shown). thus, mers-cov s protein on pseudovirions must be cleaved in endosomes by the acidactivated cysteine protease activity of cathepsin l to trigger receptor-dependent entry into vero e6 and llcmk2 cells. trypsin cleavage of mers-cov s on pseudovirions adsorbed to receptors on the cell surface triggers virus entry at the plasma membrane at neutral ph sars-cov can enter susceptible cells at the plasma membrane, instead of by endocytosis, if virions adsorbed at 4°c to ace2 on the cell membrane are treated with trypsin, then warmed to 37°c in the presence of an inhibitor of endosomal acidification [21] . trypsin treatment at either 4°c or 37°c cleaved the s protein of mers pseudovirions and generated a ~65kda subunit in the s2 domain of the protein recognized by antibody to mhv-a59 s protein ( figure s1 ). mers pseudovirions with uncleaved s protein were adsorbed at 4°c to cell surface receptors on vero e6 cells in the presence of 20mm nh 4 cl, and then the cells with bound virions were briefly treated with trypsin at ph 7.4 at room temperature to cleave the ~200 kda s protein and activate its membrane fusing activity. figures 3a and 4a show that nh 4 cl strongly inhibited infection of vero e6 cells by mers pseudovirions with uncleaved s. however, trypsin treatment of the mers pseudovirions bound at neutral ph and 4°c to the vero e6 cell membrane triggered both virus entry at the plasma membrane and formation of small syncytia by 40 hours post inoculation ( figure 4a and b). thus, receptor binding together with protease cleavage and activation of s at neutral ph was sufficient to trigger entry of mers pseudovirions and syncytia formation. in this experiment membrane fusion did not depend upon synthesis of s protein, but syncytia formation was mediated by the cleaved s protein on pseudovirions adsorbed to virus receptor on the cell membrane. although acidic ph is required to activate the cathepsin l activity that allows mers pseudovirions to enter at endosomes, low ph is not required for the conformational changes in trypsin-cleaved mers-cov s protein that mediate entry at the plasma membrane. 293t cells expressing uncleaved mers-cov s protein or control cells stably transfected with the empty pcdna3.1 vector were overlaid on monolayers of vero e6 cells in the presence or absence of tpck trypsin ( figure 4c ). no syncytia formation was induced by 293t cells with empty vector or 293t cells expressing mers-cov sδ16 without trypsin ( figure 4c ), but addition of tpck trypsin to the medium triggered formation of massive syncytia in the vero e6 cells co-cultured for 20 hr with mers-cov s-expressing 293t cells ( figure 4c , arrows). large syncytia were also formed after even a brief 20 minute trypsin pre-treatment at ph 7.4 and 4°c of 293t cells expressing mers-cov s protein, followed by incubation with a 5-fold excess of soybean trypsin inhibitor before layering the cells over confluent monolayers of vero e6 cells and incubating at 37°c for 20 hours ( figure 4c, lower central panel, arrows) . thus, trypsin cleavage at neutral ph of mers-cov s protein on 293t cells triggered syncytia formation when s was bound to receptors on susceptible vero e6 cells. type ii transmembrane serine proteases, including tmprss-2 and tmprss-4, which like trypsin are expressed in the respiratory tract, play important roles in triggering entry of influenza a virus, human metapneumovirus and sars betacoronavirus in group b [19, 20, [42] [43] [44] [45] . we therefore transfected 293t cells with plasmids encoding tmprss-2 or -4, mers-cov sδ16 protein, pspax2 and plenti-gfp-luc and investigated whether s proteins on pseudovirions produced in these cells were cleaved and whether they could infect vero e6 cells in the presence of nh 4 cl. surprisingly, the pseudovirionproducing 293t cells expressing either tmprss-2 or -4, formed large syncytia by 40 hrs after transfection ( figure 5a ), but the mers pseudovirions produced by these cells could not transduce vero e6 cells in the presence or absence of nh 4 cl ( figure 5b ). in contrast, without tmprss-2 or -4, the 293t cells expressing uncleaved mers-cov sδ16 did not form syncytia, and pseudovirions that they produced efficiently infected vero e6 cells, but virus entry was inhibited by nh 4 cl ( figure 5b ). immunoblots with antibody ao4 to mhv s or anti-flag (data not shown) revealed that the mers pseudovirions produced in 293t cells expressing tmprss-2 or -4 contained little or no immunoreactive s protein or fragments of s. although the novel group c betacoronavirus mers-cov is highly virulent in humans and can infect cells from several different species, including humans, monkeys, pigs, and some species of bats [10, 16, 34] , little is known about the biology of this virus. because the spike glycoprotein is essential for coronavirus entry, elucidating the functions of the mers-cov spike can provide valuable insight into the pathogenesis of mers-cov, and suggest potential therapeutic interventions. here we used lentivirus pseudovirions with mers-cov spike protein to study s-mediated cell entry at biosafety level 2. we found that mers pseudovirions, like infectious mers-cov virions [10, 34] , readily infected the vero e6 and llcmk2 lines of monkey kidney cells, several human respiratory epithelial cell lines, and embryo cells from eptesicus fuscus bats. others have also recently demonstrated that human respiratory tract cells, and also primary human bronchus and alveolar cells are susceptible to mers-cov in accord with the severe respiratory disease in mers patients [10, 33, 35, 46] . muller et al. [34] recently reported that mers-cov can infect cells from four genera of old world bats, rousettus, rhinolophus, pipistrellus, and myotis, and one new world genus, carollia. we found that mers pseudovirions could also infect cells from one new world bat, e. fuscus, but not from another, t. brasiliensis. the ability of mers-cov to infect cells from multiple mammalian species directly and without adaptation [47] , including a diverse array of both old world and new world bats, suggests that the receptor for mers-cov, dpp4 [28] , is broadly conserved among many species, an important property of many emerging viruses [47, 48] . e. fuscus figure 5a were inoculated onto vero e6 cells in the presence (striped bars) or absence (white bars) of 20mm nh 4 cl to inhibit acidification of endosomes. doi: 10.1371/journal.pone.0076469.g005 bats, commonly known as big brown bats, are the bats most commonly encountered by humans in north america, and they are a reservoir for an alphacoronavirus [49, 50] . it will be important to learn whether these new world bats are susceptible to mers-cov or related group c betacoronaviruses. the recent detection in n. laticaudatus bats in mexico of a group c betacoronavirus with 96% similarity to mers-cov [13] , coupled with the diverse array of alphacoronaviruses previously discovered in north american bats [49] [50] [51] [52] , justify increased surveillance to identify additional species of new world bats that may also harbor group c betacoronaviruses like mers-cov or other coronaviruses with the potential to cause severe disease in humans. mers-cov is a betacoronavirus in group c, and we were surprised that its s protein was recognized in immunoblots by a polyclonal antibody to the spike protein of mhv-a59, a group a betacoronavirus. the cross-reactive epitope(s) was mapped to the s2 domain, which is more highly conserved than the s1 domain of betacoronaviruses. chan et al. [33, 53] found that mers-cov s protein was recognized in immunofluorescence and in vitro neutralization assays by sera of some convalescent sars patients, and suggested, based on bioinformatics, that epitope(s) in s2 could account for the observed serological cross-reactivity. these observations that the s protein of mers-cov, a group c betacoronavirus, contains crossreacting epitope(s) with s proteins of both some group b (sars-cov) and group a (mhv) betacoronaviruses, indicate that serological studies may not accurately distinguish between different phylogenetic groups of betacoronaviruses. identification and characterization of the cross-reacting epitope(s) is an important research priority to show whether there is a common epitope in s that could be used as an immunogen to vaccinate against all betacoronaviruses. enveloped viruses infect cells by fusion of the viral envelope with host cell membranes, a process mediated by a series of conformational changes in the viral fusion protein that are regulated by receptor binding, protease activation, and/or ph [17] . the classes of viral fusion proteins are determined based on their structures and conformational changes during membrane fusion. most class i viral fusion proteins require proteolytic cleavage upstream of the hydrophobic fusion peptide in the viral spike protein to enable these conformational changes to occur, as well as subsequent steps that trigger membrane fusion including either binding to receptor like hiv gp120, low ph in endosomes like influenza ha, or both like avian sarcoma leukosis virus (aslv) [9, 17, [54] [55] [56] . coronaviruses in different phylogenetic groups differ in the sequence of steps leading to virus entry [57, 58] . the s protein on virions of group a betacoronavirus mhv-a59 requires protease activation--either by furin during virus maturation [18] , by trypsin or other serine proteases in extracellular fluids before either receptor binding, or by cathepsin in endosomes at acidic ph-to trigger the conformational changes that lead to membrane fusion and virus entry [59] . in contrast, virions of group b betacoronavirus sars-cov that contain uncleaved s [22] , first bind to the viral receptor protein, ace2, and are endocytosed, and then s is cleaved within endosomal vesicles by acid-dependent cathepsin l enabling the conformational changes in s that lead to virus entry [21, 22, 60] . here we analyzed the steps needed to trigger conformational changes in mers-cov s and their roles in virus entry and syncytia formation. in our laboratory, mers pseudovirions released by 293t cells contained only uncleaved s protein, and, as for most coronaviruses, cleavage between s1 and s2 was necessary to enable its membrane fusing activity. the mers pseudovirions bound to receptors on susceptible cells and were endocytized, and within the endosomes cleavage of s by the acid-dependent cysteine protease cathepsin l mediated virus entry. gierer et al [33] reached similar conclusions using mers pseudovirions that, unlike ours, contained more cleaved than uncleaved s protein, although both labs had made the pseudovirions in 293t cells. gierer et al. [33] showed that batches of 293t cells differ markedly in expression of the mers-cov receptor and susceptibility to transduction by mers pseudovirions. in our laboratory, the 293t cells showed minimal susceptibility to transduction with mers-cov pseudovirions with uncleaved s protein. in addition to entry by endocytosis, we showed that, like sars-cov [21, 22] , mers pseudovirions could enter susceptible vero e6 cells at the plasma membrane if virions were first bound to cell surface receptors at 4°c at neutral ph in the presence of nh 4 cl to inhibit acidification of endosomes, and also treated briefly at room temperature with trypsin to cleave the viral s protein. upon warming to 37°c at neutral ph, the mers pseudovirions fused with the plasma membrane and transduced the cells. thus, mers s protein does not require acidification to mediate virus entry, and the acidification required for endosomal entry [33] was required to activate the protease activity of cathepsin. although treatment of coronavirus virions or pseudovirions with proteases can activate virus entry, it may also make them lose infectivity if the cleaved s1/s2 heterodimer dissociates before receptor binding. we found that mers pseudovirions released from cells expressing tmprss-2 contained reduced amounts of s protein and had lost the ability to transduce susceptible cells. we postulate that the mers pseudovirions that contain large amounts of cleaved s protein, detected by a c-terminal tag [33, 53] , could not enter cells at the plasma membrane because s1 may have dissociated from the cleaved spikes on virions. development of antibodies specific for the s1 domain of mers s protein are needed to test this hypothesis. coronavirus s proteins expressed on cell membranes can trigger receptor-dependent syncytia formation if the membranebound s protein is cleaved within the infected cells by furin or other proteases. mers-cov infection of calu-3 and caco-2 cell lines induced syncytia formation [35] . our 293t cells were only minimally susceptible to entry of mers-pseudovirions, and did not form syncytia when producing mers pseudovirions with uncleaved s. however, when 293t cells expressing mers-cov sδ16 protein were co-cultured in the presence of trypsin with vero e6 cells that express the mers-cov receptor, enormous syncytia formed. these observations suggest that in tissues such as the lung, where trypsin, tmprss-2 or -4 and hat and other serine proteases are available, mers-cov virus infection might spread directly from cell to cell by s-mediated, receptor-dependent syncytia formation, potentially escaping from virus-neutralizing antibodies, as do other syncytia-forming viruses such as respiratory syncytial virus, parainfluenza viruses, and measles. it will be important to learn whether syncytia are formed in lungs or other tissues of mers-cov patients or animal models of mers-cov. some coronavirus s proteins can also trigger receptorindependent syncytia formation [61, 62] . when s proteins expressed on the plasma membrane are cleaved, the s1 domain can detach from the spike, exposing the hydrophobic fusion peptide of the membrane-anchored s2 domain that can directly induce fusion with any nearby cell membranes or lipid bilayers even if they lack receptors. this "receptor-independent spread (ris)" allows an infected cell to fuse with adjacent noninfected, receptor-negative cells that can, in turn, produce virus and fuse with additional receptor-negative cells. ris activity depends on the stability of s1/s2 interactions, and low stability of s1/s2 heterodimers correlates with rapid spread of infection through tissues that express little receptor protein [63, 64] . we found that transmembrane serine proteases tmprss-2 and -4 could activate the syncytia forming activity of mers-cov sδ16 protein expressed in 293t cells as these proteases do for class 1 fusion proteins of other respiratory viruses including influenza, sars-cov and human metapneumovirus [19, 20, [42] [43] [44] [45] . we were surprised that 293t cells, which express very little mers-cov receptor, were so extensively fused, and we hypothesize that this syncytia formation may be due to ris. due to its high case fatality rate, therapeutic interventions for mers-cov are urgently needed. pooled purified human immunoglobulin containing neutralizing antibody has been used to treat a variety of infectious diseases. not surprisingly, since mers-cov is an emerging pathogen, we found that human immunoglobulin from the usa could not neutralize the infectivity of mers pseudovirions (data not shown). however, sera from patients infected with either sars-cov or mers-cov contain antibodies that can neutralize mers-cov [33, 53] . most neutralizing antibodies would likely target the receptorbinding s1 domain of mers-cov s, which is less conserved than the s2 domain and can mutate, likely generating antibody escape mutants [65] . here we showed that a polyclonal antibody to the s protein of mhv, a group a betacoronavirus, cross reacts with the s2 domain of mers s protein in immunoblots. as we and others have proposed for sars-covs [66, 67] , the more highly conserved s2 domain, and especially its c-terminal heptad repeat (hrc) region, can be important targets for blocking conformational changes in s, inhibiting syncytia formation and virus entry, and also eliciting neutralizing antibodies. if mers-cov virions made in the lung have uncleaved s protein and must therefore enter cells through endocytosis, then inhibitory hrc peptides, or neutralizing antibodies to hrc might not penetrate into the endosomes to prevent virus entry. however, mers-cov virions in the lung likely have cleaved s protein due to lung proteases, so that virus entry at the plasma membrane might be inhibited by hrc-targeted peptides or antibodies. in summary, we demonstrated that, similar to sars-cov, cleavage of mers-cov-s protein by trypsin, tmprss2 or -4 or cathepsin l is required to activate the membrane fusion activity of s, leading to virus entry and syncytia formation, and that the location of the protease determines whether virus enters via endocytosis or by fusion at the plasma membrane. mers-cov s-mediated binding and entry mechanisms and protease triggering of conformational changes required for mers-cov-s virus entry and syncytia formation present potential targets for development of drugs or vaccines against this newly emerging and lethal group c human betacoronavirus. codon-optimized cdna encoding the spike glycoprotein of mers-cov [15] was synthesized with the c-terminal 16 amino acids replaced with a ggggs linker and a flag tag (genscript, piscataway, nj), and for eukaryotic expression was cloned into pcdna3.1(+) (invitrogen) between the bamhi and noti sites. to make constructs for expression of truncated soluble mers s aa1-351, s aa1-384, and s aa1-748 proteins, pcr reactions were performed using the same forward primer aatgaaaagcttcaccatgattcactccgtgttcctc pairing with the following reverse primers, for s aa1-351: tagttttctagaacttccgcctccaccataa ctacagtggagctggct; for s aa1-384: tagttttctagaacttccgcctccac cgtcgcactccacgccttctgcc; or for s aa1-748 tagttttctagaacttc cgcctccacctggggtcagtgtgctgggggt, and cloned into p3xflag-cmv 14 (sigma, st louis, mo) between hindiii and xbai sites for expression. the vsv-g plasmid and lentiviral packaging plasmid, pspax2, were obtained from addgene (cambridge, ma). the lentiviral reporter plasmid, plenti-gfp-luc, which expresses green fluorescent protein (gfp) and luciferase, was kindly provided by fang li, duke university [68] the vero e6 line of african green monkey kidney cells, the 293t line of human embryonic kidney cells transformed with sv40 large t antigen, the calu 3 line of human airway epithelial cells, the a549 line of human alveolar epithelial cells, and the tb1lu lung cell line from t. brasiliensis bats were obtained from atcc (manassas, va). hela cells stably expressing recombinant human ceacam proteins and the control hela cell line containing the empty vector were kindly provided by scott gray-owen, university of toronto [69] . the bat eff cells were prepared by macerating mid-gestation fetuses of eptesicus fuscus bats, briefly trypsinizing the cells, and plating them for expansion. cells were passaged twice, frozen, and kindly provided by richard bowen, colorado state university. isolation of the eff cells was conducted under approval 03-096a from the colorado state university iacuc. murine nih3t3 cells stably expressing recombinant human aminopeptidase n (hapn) or control cells with empty vector were previously described [70] . these cell lines were maintained in dulbecco's mem with 10% fetal bovine serum (fbs) and 2% penicillin, streptomycin, and fugizone (psf) (life technologies inc, grand island, ny). the llc-mk2 line of rhesus monkey kidney cells from atcc ccl-7 was maintained in opti-mem1 (life technologies inc, grand island, ny) with 10% fbs and 2% psf. pseudotyped lentiviruses were produced as described previously [71] with minor modifications. briefly, plasmids that encode viral spike glycoproteins mers-cov s∆16, sars s∆19, or vsv-g were co-transfected into 293t cells with pspax2 and plenti-gfp-luc using polyetherimide (pei) (polyscience inc, warrington, pa). forty to 60 hr later the supernatant media containing pseudovirions were centrifuged at 800g for 5min to remove debris, and passed through a 0.45µm filter. to quantitate entry of pseudovirions into different cell types, 250µl of pseudovirions with 8 µg/ml of polybrene (sigma) was inoculated onto cells in 24-well plates, incubated overnight at 37°c, and cells were fed with fresh medium. at 40hr post inoculation (pi) cells were lysed at room temperature with 120µl of medium with an equal volume of steady-glo (promega, madison, wi). transduction efficiency was monitored by quantitation of luciferase activity. living cells transduced by pseudovirions were detected by gfp expression. for all experiments triplicate samples were analyzed, data are representative of two or more experiments, and the standard error is shown. pseudovirions with mers-cov sδ16, sars sδ19, or vsvg glycoprotein or control pseudovirions with no spike were centrifuged through a 20% sucrose cushion at 30,000 rpm at 4°c for 3 h in a beckman sw41 rotor [71] . spike proteins in the virions were separated on 4-15% sds page, blotted to nitrocellulose and detected with mouse anti-flag antibody m2 (sigma, st louis, mo) for mers-cov sδ16, or polyclonal goat antibody ao4 to purified spikes from detergent-disrupted mhv-59 virions [72] , followed by horseradish peroxidase (hrp)-conjugated antibody to mouse or goat igg, and visualized with chemiluminescent reagent plus (perkinelmer, boston, ma). vero e6 cells or llcmk2 cells were incubated for 1hr at 37°c with medium alone or medium containing either 20mm nh 4 cl or 200nm bafilomycin a to inhibit acidification of endosomes, and then spin-inoculated with pseudovirions and no spike controls in the presence of either 20mm nh 4 cl or 200nm bafilomycin a for 90 min at 1,200g at 4°c. at 40 hr pi cells were lysed and luciferase activity was quantitated as a measure of virus entry. in figure 3 , the luciferase activities of vero e6 cells transduced with vsv-, sars-, and mers-covspseudovirions without endocytosis inhibitor were 1x10 6 , 1.5-2x10 6 , and 5-6x10 5 , respectively. monolayers of vero e6 or llcmk2 cells were incubated with 20mm nh 4 cl in medium containing 10% fbs for 1hr at 37°c, then shifted for 15min to 4°c with 40mm nh 4 cl. pseudovirions were adsorbed to cells at 4°c by spin-inoculation for 90 min at 1,200g. the virus inocula were removed and replaced with prewarmed, serum-free dmem with or without 20mm nh 4 cl. after 15min at 37°c, the media were replaced with serum-free dmem with or without 15 µg/ml of tpck-trypsin at room temperature to activate the membrane fusing activity of the s protein on virions adsorbed to the plasma membrane. trypsin activity was then inhibited by incubation for 15 min on ice with 75µg/ml of soybean trypsin inhibitor (worthington biochemical corporation, lakewood, nj) in dmem with 10% fbs, and cells were incubated overnight at 37°c, fed with fresh medium, and incubated at 37°c. at 40 hr pi, virus entry was quantitated by luciferase activity and cells were photographed to detect viral cytopathic effects. monolayers of vero e6 cells or llcmk2 cells were preincubated for 2 hrs at 37°c with either 30µm of e64d, that inhibits cathepsin b, h, l and calpain, or 10µm of specific cathepsin l inhibitor iii (millipore, billerica, ma). pseudovirions and no spike controls with or without cathepsin inhibitors were spin-inoculated onto the cells at 4°c, then incubated at 37°c for 5 hours with or without the endocytosis inhibitors. cells were then incubated at 37°c without inhibitors, and at 40 hr pi, luciferase activity in lysed cells was determined. 293t cells in 6-well plates were transfected using pei with 3µg of either empty vector or mers-cov s∆16 plasmid. for brief trypsin pre-treatment, cells were lifted with 1mm edta in pbs and washed, then incubated on ice with 20 µg/ml of tpck trypsin or control medium for 20 min. typsin activity was then inhibited by incubation with a five-fold excess of soybean trypsin inhibitor for 15min. the trypsin-pretreated 293t cells and control cells were then layered over monolayers of vero e6 cells and incubated for 20 hr. syncytia formation was detected by phase contrast microscopy or by imaging fixed cells stained with crystal violet. figure s1 . detection of trypsin-cleaved mers-cov s protein on pseudovirions by antibody to mhv s protein. pseudovirions containing either mers-cov s protein or no spike (control) were incubated with 20 µg/ml of trypsin either at 4°c or 37°c for 20 min. after digestion, glycoproteins on pseudovirions were analyzed by immunoblot using ao4 antibody to the s protein of murine betacoronavirus mhv-a59 that cross-reacts with mers-cov s protein. lanes 1 to 3: no spike control; lanes 4 to 6: mers pseudovirions; lanes 1 and 4: no trypsin; lanes 2 and 5: trypsin at 4°c; lane 3 and 5: trypsin at 37°c. the band at ~90kda was seen in pseudovirions without spike, indicating that it is not due to mers-cov s protein. uncleaved mers-cov s protein, 200kda, on pseudovirions is shown in lane 4, and trypsin treatment cleaved all of the s protein, and generated a subunit of ~65kda that was recognized by ao4 antibody (lanes 5 and 6). (eps) identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission evidence supporting a zoonotic origin of human coronavirus strain nl63 severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event middle east respiratory syndrome coronavirus (mers-cov) isolation of a novel coronavirus from a man with pneumonia in saudi arabia comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans coronaviruses in bats from mexico human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans the new age of virus discovery: genomic analysis of a novel human betacoronavirus isolated from a fatal case of pneumonia structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc crystal structure of bovine coronavirus spike protein lectin domain retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein aromatic amino acids in the juxtamembrane domain of severe acute respiratory syndrome coronavirus spike glycoprotein are important for receptor-dependent virus entry and cell-cell fusion intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus characterization of severe acute respiratory syndromeassociated coronavirus (sars-cov) spike glycoprotein-mediated viral entry cathepsin l and cathepsin b mediate reovirus disassembly in murine fibroblast cells endosomal proteolysis of the ebola virus glycoprotein is necessary for infection sars coronavirus, but not human coronavirus nl63, utilizes cathepsin l to infect ace2-expressing cells matriptase, hat, and tmprss2 activate the hemagglutinin of h9n2 influenza a viruses tmprss2 and tmprss4 facilitate trypsin-independent spread of influenza virus in caco-2 cells proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium efficient multiplication of human metapneumovirus in vero cells expressing the transmembrane serine protease tmprss2 tropism and innate immune responses of the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential global trends in emerging infectious diseases detection of group 1 coronaviruses in bats in north america metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat detection and phylogenetic analysis of group 1 coronaviruses in south american bats alphacoronaviruses in new world bats: prevalence, persistence, phylogeny, and potential for interaction with humans crossreactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests membrane fusion activity of the influenza virus hemagglutinin. the low ph-induced conformational change induction of cd4-dependent cell fusion by the htlv-iii/lav envelope glycoprotein retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein mechanisms of coronavirus cell entry mediated by the viral spike protein ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees c either by soluble murine ceacam1 receptors or by ph 8 proteasemediated entry via the endosome of human coronavirus 229e cell receptorindependent infection by a neurotropic murine coronavirus entry of mouse hepatitis virus into cells by endosomal and nonendosomal pathways the spike glycoprotein of murine coronavirus mhv-jhm mediates receptor-independent infection and spread in the central nervous systems of ceacam1a-/-mice enhanced virulence mediated by the murine coronavirus, mouse hepatitis virus strain jhm, is associated with a glycine at residue 310 of the spike glycoprotein escape from human monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: hydrophilicity/hydrophobicity of side-chains at the n-and c-termini of peptides are dramatically affected by the end-groups and location apoptotic caspases regulate induction of ipscs from human fibroblasts cd66 carcinoembryonic antigens mediate interactions between opaexpressing neisseria gonorrhoeae and human polymorphonuclear phagocytes human myeloid plasma membrane glycoprotein cd13 (gp150) is identical to aminopeptidase n complementation of a binding-defective retrovirus by a host cell receptor mutant isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid we thank scott d. gray-owen (university of toronto) for providing hela cells stably expressing human ceacam proteins, linda shapiro (university of connecticut) for mouse cells producing human apn, richard bowen (colorado state university) for the bat cell lines, stefan poehlmann (hannover medical school and german primate center) for pca7-tmprss2 and pcmv-tmprss4 plasmids, and fang li (duke university) for plenti-gfp-luc. conceived and designed the experiments: zq srd kvh. performed the experiments: zq. analyzed the data: zq srd kvh. contributed reagents/materials/analysis tools: zq srd kvh. wrote the manuscript: zq srd kvh. key: cord-300078-svu06v9c authors: haghani, milad; bliemer, michiel c. j. title: covid-19 pandemic and the unprecedented mobilisation of scholarly efforts prompted by a health crisis: scientometric comparisons across sars, mers and 2019-ncov literature date: 2020-06-01 journal: biorxiv doi: 10.1101/2020.05.31.126813 sha: doc_id: 300078 cord_uid: svu06v9c during the current century, each major coronavirus outbreak has triggered a quick and immediate surge of academic publications on this topic. the spike in research publications following the 2019 novel coronavirus (covid-19) outbreak, however, has been like no other. the global crisis caused by the covid-19 pandemic has mobilised scientific efforts in an unprecedented way. in less than five months, more than 12,000 research items have been indexed while the number increasing every day. with the crisis affecting all aspects of life, research on covid-19 seems to have become a focal point of interest across many academic disciplines. here, scientometric aspects of the covid-19 literature are analysed and contrasted with those of the two previous major coronavirus diseases, i.e. severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). the focus is on the co-occurrence of key-terms, bibliographic coupling and citation relations of journals and collaborations between countries. certain recurring patterns across all three literatures were discovered. all three outbreaks have commonly generated three distinct and major cohort of studies: (i) studies linked to the public health response and epidemic control, (ii) studies associated with the chemical constitution of the virus and (iii) studies related to treatment, vaccine and clinical care. while studies affiliated with the category (i) seem to have been the first to emerge, they overall received least numbers of citations compared to those of the two other categories. covid-19 studies seem to have been distributed across a broader variety of journals and subject areas. clear links are observed between the geographical origins of each outbreak or the local geographical severity of each outbreak and the magnitude of research originated from regions. covid-19 studies also display the involvement of authors from a broader variety of countries compared to sars and mrs. on december 31, 2019 an official case of a novel respiratory diseases of the category of coronaviruses, named covid-19, was reported in wuhan, china, marking the beginning of what proved to be one of the direst and most devastating viral outbreaks in the modern history (sohrabi et al. 2020 ). this was immediately followed by an unprecedented and swift response of the academic community to address various dimensions of this health crisis and prompted an avalanche of scholarly publications on this topic (golinelli et al. 2020 , haghani et al. 2020 , kagan et al. 2020 . in less than five months, more than 12,000 publications on this topic have already been indexed by scopus with the number increasing figuratively every day in considerable increments (torres-salinas et al. 2020) . and this figure does not even include many more publications available in various repositories, including cord-19 (colavizza et al. 2020) , in the form of preprints awaiting peer review by their respective journals. such explosion of research on a single topic and the off-the-charts surge in the rate of publications are arguably unprecedented trends in the history of scholarly publications. an article published by science on may 13, 2020, referred to this phenomenon as one that is "among the biggest explosions of scientific literature ever" (brainard 2020) . it highlighted how, in the face of this phenomenon, it has become extremely challenging for scientists to stay abreast of the latest developments. this has made the importance of research synthesis more tangible than ever and has even resulted in the development of several computational research mining tools for this very topic utilising methods such as artificial intelligence (ai). among such efforts is a research synthesis powered by ai algorithms which has harvested datapoints from a large number the cord-19 articles and categorised them (brainard 2020) . though the impact of the covid-19 health crisis has marked it as a rather unique milestone in the history disease outbreaks, the world, prior to this, was not a stranger with coronavirus disease outbreaks (mcintosh 1974 , myint 1994 , cavanagh 2005 , lim et al. 2016 , chen et al. 2020 . prior to 2020, two major outbreaks of this family of viruses had already been reported with at least one of them carrying the official label of a "global pandemic" ). on november 16, 2002, the first case of the severe acute respiratory syndrome (sars) disease was reported in the guangdong province in southern china, which by 2003, swiftly spread from continent to continent, prompting the world health organisation to declare it as a pandemic. in fact, sars is known to be "the first pandemic of the 21st century" (cherry and krogstad 2004) . nearly ten years later, on june 13, 2012, the first case of the middle eastern respiratory syndrome (mers) disease was discovered in jeddah, saudi arabia. these two constituted the two most major coronavirus outbreaks until covid-19 came along. similar to covid-19, though at a much smaller scale, each of these previous outbreaks generated a literature of their own (kostoff and morse 2011) . in the face of the flood of scholarly outputs on covid-19, and along with the conventional review and research synthesis studies (chang et al. 2020 , chen et al. 2020 , cortegiani et al. 2020 , scientometric (colavizza et al. 2020) and bibliometric methods (bonilla-aldana et al. 2020 , hossain 2020 have also gained traction in documenting and analysing the rapid developments of this literature (chahrour et al. 2020 , dehghanbanadaki et al. 2020 , haghani et al. 2020 , kumar 2020 , le bras et al. 2020 . here in this work, the literatures of these three major coronavirus diseases are disentangled and analysed in a comparative way and from scientometric perspectives. the aim is to discover possible similarities and discrepancies across these three segments of the coronavirus literature, and to discover whether there are recurring patterns in terms of magnitude, temporal evolution and the shape of these three literatures that were each developed in response to a disease outbreak. the main focus of the analyses is on keyword co-occurrences, bibliographic coupling and citation relations of sources and collaborations between countries. to compare the scientometric aspects of the studies on sars, mers and covid-19, three separate datasets of publications on these three topics were retrieved from scopus through three separate search strategies. the decision on which general database to use (e.g. web of science (wos) or scopus) was mainly made based on the number of indexed covid-19 studies as the sector of the literature that is currently emerging (compared to the literatures on sars and mers that are better established). at the time of the data retrieval, wos had indexed slightly less than 5,000 research items on covid-19, while the number of items in scopus neared 12,000. given the fact that the scopus database was considerably more up to date in that area, this database was set as the main source of data extraction in this work. therefore, for the sake of consistency, the data for sars and mers were also extracted from scopus. the search strategies were devised in a way to minimise the possible overlap between the datasets on sars, mers and covid-19 and to disentangle the three datasets to the most possible extent. preliminary inspection of the literature on each three topics determined a set of distinct keywords that would return the target literature with reasonable specificity and sensitivity. in each search, key terms associated with the other literatures were combined with the logical operator "and not" in order to avoid the overlap. the lower bound of the time span for each search was set with consideration of the year where the first outbreak of each virus took place. the query string associated with each dataset are as follows: sars: ( title-abs-key ( ( ( "severe acute respiratory syndrome" or "sars" ) and ( coronavirus* ) ) or ( "sars virus" or "sars disease" or "severe acute respiratory syndrome disease" or "severe acute respiratory syndrome virus" or "sars-cov") ) and not titleor "severe acute respiratory syndrome-2" or "mers" or "middle east respiratory syndrome" ) ) ) and pubyear > 2001 mers: ( title-abs-key ( ( ( "middle east respiratory syndrome" or "mers" ) and ( coronavirus ) ) or ( "mers-cov" or "mers virus" or "mers disease" or "middle east respiratory syndrome virus" or "middle east respiratory syndrome disease" ) ) and not title-abs-key ( ( "ncov" or "covid-19" or "covid19" or "sars-cov" or "sars-cov-2" or "sars" or "severe acute respiratory syndrome" ) ) ) and pubyear > 2011 covid-19: title-abskey ( "covid-19" or "covid19" or "coronavirus disease 2019 " or "2019 ) and pubyear > 2018 the search was last time updated on 24 may 2020 where it returned 5,907 items on sars, 1,752 items on mers and 11,859 items on covid-19. figures 1, 2 and 3 show the distribution of the studies on sars, mers and covid-19 , respectively, across subject areas. figure 4 (a) also shows the composition of the covid-19 literature in terms of the document types, demonstrating that only nearly 50% of the studies on this topic have so far been in the form of full-length articles, while letters, notes, reviews, and other document formats constitute a large portion (i.e. nearly half) of the literature on this topic at the time of this investigation. full records of the three datasets on sars, mers and covid-19 were retrieved in csv excel format from scopus, all on the same day. this included the citation information, bibliographic information, abstract and keywords, funding details and the references. the scopus restriction of maximum 2,000 document to export posed challenges for the retrieval of the sars and covid-19 datasets whose size were bigger than 2,000 documents. for the sars dataset, the challenge was circumvented by further limiting the search to specific year(s), in separate bundles, in a way that the size of each bundle was less than 2,000 items, therefore allowing us to export the items of each bundle separately. the extraction of the covid-19 dataset, however, posed a further layer of complication, given that nearly all studies of covid-19 have been published in one year, i.e. 2020. therefore, the year of publication could not be used as a criterion to form a set of mutually exclusive smaller-size exportable bundles for this literature. to decompose the search outcome to bundles of 2,000 documents or less, the following strategy was adopted. the document type was used to initially limit the search to mutually exclusive (non-overlapping) categories. first, the search was limited to "review or short survey or erratum or conference paper or data paper". this formed a set of 1,267 documents which was extracted in one single export (see figure 4 (a) for details of the number of items within each document type category). subsequently, the search was set back to original and was limited to notes (1,067 items) and then to editorial (1,270 items). with each set of these two subsets being smaller than 2,000, they were exported separately. there were 2,564 documents of letter type. this set was further decomposed to two mutually exclusive subsets based on the publication stage criterion (1,539 article in press, and 1,025 final) and was retrieved in two separate exports. for the remaining 5,691 article documents, the following strategy was devised. of the 5,691 items, 2,944 were article in press and 2,747 were final. first, the 2,944 article in press items were considered. the list of those studies was sorted as first author (a-z) and the first 2,000 items were extracted in one export. then the list was sorted as first author (z-a) and the first 944 items were exported. similar strategy was utilised to extract the remaining 2,747 final documents. a supplementary search was also conducted on the general topic of coronaviruses using the string title-abs-key ( "coronavirus*" ) and pubyear > 1985 which returned 24,620 documents on the same day. only the data related to the number of documents by year was extracted for this search. the increase continued, though at a slower rate, to 2004 and was then followed by a gradual decline till 2012. the 2012 mers outbreak triggered another spike in the number of publications on coronaviruses, though not as large as that of the sars. the intensification of attention to this topic this time lasted for about three years till 2015 before another decline began. the spike of coronavirus studies prompted by the covid-19 outbreak, however, seem to have been occurring at a completely different scale which can be deemed unprecedented in the history of coronavirus studies. the number of studies emerged in the first five months of 2020 nears an equivalent of the 70% of the total sizer of coronavirus literature during more than 50 years . in figure 4 (c), the temporal distribution of the sars, mers and covid-19 studies have been shown separately according to the three datasets explained earlier. note that, the quantities associated with sars and mers are represented by the left vertical axis whereas that of the covid-19 is represented by the right vertical axis, with a scale ten times bigger than the scale of the left axis. the history of previous coronavirus research has suggested that the number of studies will likely keep rising for at least a few years before it peaks. but given the unprecedented magnitude of research and the explosive rate of publications since the begging of 2020, it would be interesting to observe whether this pattern would repeat itself and whether the peak would occur at an earlier or later stage compared to those of the previous outbreaks, a question whose answer will only be determined by time. the co-occurrence of keywords associated with the sars, mers and covid-19 literature were analysed using vosviewer (van eck and waltman 2010). each analysis was performed on the separate set of data associated with the literature of interest. the maps of keyword cooccurrences associated with sars, mers and covid-19 literatures are provided in figures 5, 6 and 7 respectively. the minimum number of occurrences for the keywords to be included in the map was set to 5 in all three cases. the unit of analysis has also been set to all keywords (that includes both author and index keywords) and the method of counting was full counting. figures a1 and a2 in the appendix illustrate the map associated with the sars literature overlaid respectively with the average year of publication and average number of citations associated with the studies where these keywords have occurred. figure a3 and a4 present the counterpart outputs for the mers literature analysis. figure a5 is a heatmap of covid-19 keyword co-occurrence and figure a6 overlays the covid-19 keywords map in figure 7 with the colour-coding of the average number of citations. given that almost all studies of covid-19 are 2020 items, the colour-coding related to the average publication year was forgone in regard to this literature. maps of term occurrences based on the analysis of the title and abstract of studies on sars, mers and covid-19 have also been presented in figures 7, 8 and 9 respectively. while the below analysis focuses mainly on the interpretation of the keyword maps, similar patterns are by-and-large observable through analysis of the title and abstract terms of these studies. with respect to each of the three literatures, three distinct clusters of keywords were identifiable. these clusters showed certain patterns of commonality across the three datasets. each map presents a distinct cluster of keywords that seem to be associable to the studies related to public health emergency management and the prevention of epidemic. this would be the red cluster in figure 5 (sars), the green cluster in figure 6 (mers) and the red cluster in figure 7 . here, this is referred to as cluster (i). in this cluster, one can observe terms such as those associated with general public health including "wold health organisation", "public health", "public health service", "global health", as well as those associated with disease outbreaks including "emergency", "health risk" "epidemics", "pandemic", "outbreak", "viral diseases", "virus infection", "communicable disease", "transmission", "travel". terms representing measures of emergency severity also appear in this cluster including "mortality", "fatality", "morbidity", "infection risk". this cluster also includes terms that are linked to the prediction of disease propagation. these are terms such as "mathematical model", "modelling", "simulation", "statistical model" and "prediction" that have commonly occurred in this cluster. the cluster includes terms affiliated with measures of disease control and spread prevention such as "(social/patient) isolation", "quarantine", "hygiene", "handwashing", "prevention", "infection control", "(population) surveillance", "mass screening", "(face) mask", "contact tracing". the cluster also represents keywords that attributable to public policy making and social protection such as "health care planning", "health care policy", "health care quality", "leadership", "disaster planning", "polices". the cluster (i) of keywords also have distinctly and commonly across all three datasets represented keywords that are attributable to the studies on mental health impact of the epidemic. these are keywords such as "mental health (service)", "psychiatry", "psychology", "mental stress", "anxiety", "fear", "mental disease". these studies have often used methods such as "questionnaire(s)" and "survey(s)" that have commonly reflected in this cluster across the three literatures. issues surrounding the safety of medical facilities and medical staff also appear to have been addressed mainly by studies whose keywords are attributable to this cluster. these studies have generated keywords such as "health care personnel", "nurse(s)", "medical staff", "hospital", "health care facility", "personal protective equipment" that are distinctly observable in cluster (i) of keywords across all three datasets. the economic aspects of the epidemics also seem to have been addressed particularly by covid-19 as reflected in cluster (i) of the covid-19 literature. these have been reflected in terms such as "economics", "economic aspect", which have occurred frequently enough in covid-19 studies for them to appear distinctly on the map. the trace of such cohort of studies is, however, not as clearly identifiable based on the sars and mers maps as is it with respect to the covid-19 literature. this could be explained by the greater magnitude of the societal impact of covid-19 outbreak compared to sars and mers. the names of the countries and regions have almost invariably appeared in cluster (i) across all three datasets. in certain cases, the country names that have occurred most are those from which the outbreaks originated or those that suffered most from the impact of the outbreak. for example, "saudi arabia" appears quite distinctly on the cluster (i) of the mers dataset. similarly, the occurrence of the names of south-east asian countries/regions such as "china", "hong kong", "taiwan", "singapore" on the cluster (i) of the sars map, or the term "wuhan" on the cluster (i) of the covid-19 map are quite notable. the occurrence of the name of the countries also could be a reflection of the early studies with respect to each outbreak that have addressed the local impacts/spread of the outbreaks on their own society. on the issue of early studies, the terms "letters", "editorial", and "review" (which have intentionally been kept on the maps) seem to also have distinctly occurred in cluster (i) of each literature which is another indication that this cluster includes early studies that appeared at a time where the amount of data and clinical trials were insufficient for full-length articles. an inspection of the figures a1 and a3 does, in fact, confirm this hypothesis at least in association with the sars and mers literature, that the cluster (i) of keywords represent studies that on average emerged early during the developments of their respective literatures. figures a2, a4 and a6 that have illustrated the colour-coding of the average number of citations on the maps also show that, although cluster (i) is associated with the early studies that generally predated studies of the two other clusters and although it represents the largest variety of topics compared to the two other clusters, it is also associated with studies that, on average, been the recipient of a lesser number of citations when compared to the two other clusters. this pattern also appears to have commonly occurred across all three datasets. a second cluster of keywords associated with each of the three literatures were also discovered that is attributable to the studies on the chemistry and physiology of the virus, or viral pathogenesis, or in other words, the chemical constitution of the virus (knight 1954 ), a part of virology that investigates the biological processes and activities of viruses that take place in infected host cells and result in the replication of a virus. for the sars map in figure 5 , as well as the covid-19 map in figure 7 , this would be the green cluster, whereas for the mers map ( figure 6 ), this cluster is red. according to the maps, the most distinct terms associated with this cohort of virology studies on sars, mers and covid-19 are terms such as "virus protein", "virus entry", "chemistry", "metabolism", "physiology", "pathology", "cell line", "(virus/viral) protein(s)", "molecular model(s)", "virus genome", "virus rna", "virus replication", "mutation", and "enzyme activity". as this sector of studies often use "animal model(s)", terms such as "animal cell", "animal experiment", "controlled study", "mice" and "mouse" have frequently appeared in cluster (ii) associated with each of the three literatures. in reflection of the fact that these cohort of studies ultimately seek "drug design", in addition to generic common terms such as "drug design/potency/structure/synthesis", the names of the specific potential drugs that have been investigated in relation to each disease have appeared in this cluster. this includes terms such as "hydroxychloroquine" or "remdesivir" on the covid-19 map. an inspection of the maps overlaid with the average year of publications for sars and mers in figures a1 and a3 in the appendix suggests that, on average, this cohort of studies are generally the last to emerge in the published domain compared to the two other major clusters, but they receive relatively high citations on average (according to figures a2, a4 and a6). a third and relatively smaller cluster of keywords was commonly identifiable in relation to each three literatures. this cluster has been visualised in blue colour across all three maps of keyword co-occurrence. the studies represented by this cluster of keywords, here referred to as cluster (iii), appear to have been more closely focused on the developments of antibodies and vaccines. the terms "treatment", "treatment outcome", "disease severity", "antiviral therapy", "prognosis", "drug safety", "prospective/retrospective study", "immunology", "immunotherapy", "innate immunity", "immune response", "virus/viral vaccine(s)", "virus/viral antibody" are notable across these studies. terms affiliated with studies related to treatments and clinical care of respiratory disease patients also appear in this cluster. this includes terms such as "artificial ventilation", "intensive care unit", as well as symptom and organ terminologies associated with each disease, terms such as "fever", "headache", "diarrhea", "lung (injury)", "coughing", "liver injury", "kidney". terms affiliated with cohort analysis studies have appeared in this cluster of the maps associated with each literature. this is reflected in terms such as "female", "male", "child", "infant", "young adult", "adult", "age", "middle aged", "pregnant", "pregnancy". this pattern of the cohort analysis keywords appearing in cluster (iii) is particularly common across the mers and covid-19 studies. for sars, these terms have largely appeared in the red cluster, at the border between the red and blue clusters. bibliographic coupling of the studies on sars, mers and covid-19 were analysed at the level of their sources/journals. figure 8, 9 and 10 show the maps of journal bibliographic coupling associated with sars, mers and covid-10 literatures respectively. the node sizes are proportional to the number of documents published by the corresponding sources and the thickness of the links are proportional to the degree of bibliographic couplings between the sources connected by each link. the minimum number of documents associated with each node/journal to appear on the map has been set to 10. no minimum strength was set for links to be visualised on the map. a first-glance comparison shows that while the maps associated with sars and mers are well connected, connections across the covid-19 map are rather sparse. both the sars and mers maps include three major and distinct clusters of bibliographically coupled journals in addition to one minor and smaller cluster. these clusters show relatively strong degrees of inter-connectivity, whereas, this feature is not shared by the covid-19 map. the observation is understandable in light of the fact that the sars and mers literatures are relatively well established and have each been under development over a period of several years, whereas the covid-19 literature is an emerging field, and newly published studies do not seem to be sharing many references as of yet. the comparison also suggests that the covid-19 studies are generally scattered across a broader variety of journals and subject areas, as opposed to the sars and mers publications that seem to have been concentrated across a smaller set of specialty journals. this is also consistent with our observations from figures 1-3 showing that studies of covid-19 are scattered across a broader variety of subject areas compared to the sars and mers literature. though not shown in figure 3 , due to the respective values being smaller than 1%, journals in the following subject areas (that are deemed minor areas in relation to covid-19 literature) have each published a relatively considerable number of studies on this topics (a phenomenon that is not necessarily common with respect to the literature of other coronaviruses): arts and humanities (110 items 1 , where the most active journal has been social anthropology (24 items) covering topics such as "climate change reactions" (bychkova 2020), or "legal voids linked to declared states of emergency" (karaseva 2020)), economics, econometrics and finance (84 items, with economic and political weekly (36 items) being the most active journal of that category, covering topics such as "food supply chains" (reardon et al. 2020) , "economic stimulus packages" (mulchandani 2020) or "reverse migration" (dandekar and ghai 2020)), physics and astronomy (77 items, where chaos solitons and fractals (16 items) has been the most active publication outlet, covering topics such as "mathematical models for forecasting the outbreak" (barmparis and tsironis 2020, bekiros and kouloumpou 2020, boccaletti et al. 2020 , ndaã¯rou et al. 2020 , postnikov 2020 , ribeiro et al. 2020 , zhang et al. 2020 )), energy (67 items, with international journal of advanced science and technology (44 items) being the most active journal in that category, covering topics such as "flexible work arrangement in manufacturing" (sedaju et al. 2020) ), material sciences (57 items, with acs nano (10 items) being the most active outlet in that category, covering topics such as "3-d printed protective equipment" (wesemann et al. 2020) ), decision sciences (23 items, with lancet digital health (8 items) and transportation research interdisciplinary perspectives (4 items) being the most active outlets in that category, covering topics such as "the effect of social distancing on travel behaviour" (de vos 2020) or "the implementation of drive-through and walk-through diagnostic testing" (lee and lee 2020)), earth and planetary sciences (22 items, with indonesian journal of science and technology (8 items) being most active in that domain, covering topics such as "the deployment of drones in sending drugs and patient blood samples" (anggraeni et al. 2020) ). the analyses of journal citations also showed similar patterns of scatter and relatively unclear clusters in relation to the covid-19 literature compared to well-defined clusters of journal citations for sars and mers literatures. consistent with the previous observation with respect to journal bibliographic coupling, the covid-19 literature seems to be also much less cohesive in terms of its journal citation networks, when compared to the sars and mers literatures. as discussed earlier in relation to bibliographic couplings, this could also be partly explained by the fact that covid-19 papers are scattered across a more diverse range of journals and broader variety of subject categories. for the maps of journal citation relations presented in figures 11, 12 and 13 in figures a10-a14 in the appendix, the nodes of the bibliographic coupling maps have been colour-coded by the average year of publications and the average citations per document associated with the journals that each node represent (except for the covid-19 map that has only been overlaid with the average citations). according to these maps, emerging infectious diseases and the lancet have been a major source of publications for early studies on both sars and mers. this pattern for the lancet seems to have extended to covid-19 studies as well, as this journal has published a substantial portion of early studies on this topic. for sars, the strong representation of chinese medical journal and chinese journal of microbiology and immunology among the journals that published early studies are notable, a pattern that could be explained by the geographical origin of the sars outbreak. such pattern is to a less obvious extent observable in regard to the mers literature through representations of outlets such as eastern mediterranean health journal and saudi medical journal on the bibliographic coupling map associated with this literature by colours associated with relatively early publications. in collaborations of authors aggregated at the level of the countries were also analysed with respect to the sars, mers and covid-19 literatures. outputs of the analysis are presented in figures 14, 15 and 16 for sars, mers and covid-19 respectively. in each map, the size of nodes, each corresponding with a country, are proportional to the number of published documents with an author affiliated with the institutes of those countries. the links connecting the nodes indicate co-authorships between authors residing in the countries, while the thickness of the links represent the strength (i.e. frequency) of the co-authorships. the colour assigned to each node represents the average number of citations that documents authored by the countries have received. the minimum number of documents for country names to appear on the maps has been set to 5. comparison across the three maps of co-authorships shows a pattern of author involvement from the regions where each viral outbreak originated. studies authored by researchers affiliated with chinese institutes are well represented in all three cases, but clearly more so with respect to the sars and covid-19 literature, diseases whose first cases were recorded in china. the involvement of chinese authors is relatively less notable in relation to the mers studies whose origin was in the middle east. instead, with respect to the mers literature, it appears that authors affiliated with institutes in saudi arabia have been exceptionally overrepresented in the publications. this is also, to a lesser extent, the case with egypt being notably presented on the mers map of the country co-authorships. , has a very well spread and rather more evenly distributed network of collaborations with countries across the world, when compared with its network of collaboration on sars and mers. while its strongest collaboration has been with the united states, the names of many other countries appear on its network with no particular country standing out distinctly. italy, as a country that was highly affected by the viral outbreak, has been exceptionally well represented on this map with a very strong link of collaboration with the united states, followed by united kingdom at a smaller scale. this pattern of unique over-representation has to a lesser extent extended to iran, spain, france and brazil as other countries also severely affected by the covid-19 outbreak at early stages of the global spread. the sars studies with involvements of the authors affiliated with the institutes in the netherlands have on average received the highest number of citations and this is followed by authors from germany, as two countries whose authors have both published considerable number of documents and received high number of citations at the same time. this pattern was, to some extent, repeated in relation to the mers literature, with studies from the netherlands, germany and united kingdom having received on average highest number of citations. for studies published on covid-19, studies from china have so far stood out in terms of both the magnitude of research activities and the average number of citations. the map of country co-authorships associated with the sars literature. the map of country co-authorships associated with the mers literature. the map of country co-authorships associated with the covid-19 literature. outbreaks of infectious diseases have often shown a pattern of generating a quick surge of publications on their respective topics, such that they often create an entirely new literature over a short amount of time (olijnyk 2015, tian and zheng 2015) . by all measures, however, the influx of research publications that began to emerge following the 2019 novel coronavirus outbreak outsizes those of the previous cases in the history of coronaviruses, and perhaps arguably, in the history of infectious diaereses (tian and zheng 2015) . this has certainly marked a new milestone in the timeline of research on coronaviruses which dates back to 1968 (almeida et al. 1968 ). according to the editor-in-chief of the journal of virology, as quoted in an article of the scientist magazine (jarvis 2020), this surge of research outputs has been to the extent that has inundated established coronavirus researchers and domain experts with peer review requests to an extent that they are unable to cope. parallel to such intensified efforts in the research, peer review and editorial fronts, widespread efforts are underway in synthesising, summarising and visualising these rapid developments, a pattern that has also been observedthough at much smaller scales-in relation to the previous epidemics of viral diseases (kostoff and morse 2011) . in line with these endeavours, this work also aimed at quantifying and analysing scientometric aspects of the the involvement of authors from various countries on the publications linked to these three diseases seem to be distinctly correlated with the regions where the outbreaks originated, with authors from china, for example, being much more strongly represented on sars and covid-19 studies, two diseases whose origin of outbreaks were attributed to this country. middle eastern countries, on the other hand, are exceptionally represented in the mers literature. the questions of where the covid-19 literature is headed, how big it will grow in the next coming years, at what point in time the rate of publications on this topic are going to slow down (if ever) and how widely this literature is going to spread across journals and subject categories are only a few examples of questions that will be determined by time. these may also be influenced down the line by possible highly sought medical discoveries in relation to vaccine and treatment development, or lack thereof. but given the current rate at which scholarly outputs are emerging and given the extent of studies, projects, and trials that have already been conceived on this topic around the world; and also given the seemingly long-lasting and farreaching consequences of this global emergency which have impacted on aspects of life, it will probably not be so soon before we observe a decline in covid-19 research interest. the map of keyword co-occurrence for sars overlaid with the colour-coding of the average year of publication the map of keyword co-occurrence for sars overlaid with the colour-coding of the average citation number the deployment of drones in sending drugs and patient blood samples covid-19 estimating the infection horizon of covid-19 in eight countries with a data-driven approach sbdiem: a new mathematical model of infectious disease dynamics modeling and forecasting of epidemic spreading: the case of covid-19 and beyond sars-cov, mers-cov and now the 2019-novel cov: have we investigated enough about coronaviruses?-a bibliometric analysis scientists are drowning in covid-19 papers. can new tools keep them afloat? science covid-19 and climate change reactions: sts potential of online research coronaviridae: a review of coronaviruses and toroviruses. coronaviruses with special emphasis on first insights concerning sars a bibliometric analysis of covid-19 research activity: a call for increased output coronavirus disease 2019: coronaviruses and blood safety emerging coronaviruses: genome structure, replication, and pathogenesis sars: the first pandemic of the 21 st century a scientometric overview of cord-19 a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 migration and reverse migration in the age of covid-19 the effect of covid-19 and subsequent social distancing on travel behavior bibliometric analysis of global scientific research on coronavirus (covid-19) the impact of early scientific literature in response to covid-19: a scientometric perspective the scientific literature on coronaviruses, covid-19 and its associated safety-related research dimensions: a scientometric analysis and scoping review current status of global research on novel coronavirus disease (covid-19): a bibliometric analysis and knowledge mapping journals, peer reviewers cope with surge in covid-19 publications scientometric trends for coronaviruses and other emerging viral infections the legal void and covid-19 governance the chemical constitution of viruses structure and infrastructure of infectious agent research literature: sars author productivity of covid-19 research output globally: testing lotka's law. available at ssrn 3603889 2020. visualising covid-19 research. arxiv testing on the move: south korea's rapid response to the covid-19 pandemic human coronaviruses: a review of virus-host interactions coronaviruses: a comparative review. current topics in microbiology and immunology/ergebnisse der mikrobiologie und immunitã¤tsforschung covid-19 crisis: economic stimulus packages and environmental sustainability human coronaviruses: a brief review mathematical modeling of covid-19 transmission dynamics with a case study of wuhan an algorithmic historiography of the ebola research specialty: mapping the science behind ebola estimation of covid-19 dynamics "on a back-of-envelope": does the simplest sir model provide quantitative parameters and predictions? covid-19's disruption of india's transformed food supply chains short-term forecasting covid-19 cumulative confirmed cases: perspectives for brazil flexible work arrangement in manufacturing during the covid-19 pandemic: an evidence-based study of indonesian employees world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) emerging infectious disease: trends in the literature on sars and h7n9 influenza open access and altmetrics in the pandemic age: forescast analysis on covid-19 related literature software survey: vosviewer, a computer program for bibliometric mapping review of the 2019 novel coronavirus (sars-cov-2) based on current evidence 2020. 3-d printed protective equipment during covid-19 pandemic predicting turning point, duration and attack rate of covid-19 outbreaks in major western countries key: cord-286298-pn9nwl64 authors: helmy, yosra a.; fawzy, mohamed; elaswad, ahmed; sobieh, ahmed; kenney, scott p.; shehata, awad a. title: the covid-19 pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control date: 2020-04-24 journal: j clin med doi: 10.3390/jcm9041225 sha: doc_id: 286298 cord_uid: pn9nwl64 a pneumonia outbreak with unknown etiology was reported in wuhan, hubei province, china, in december 2019, associated with the huanan seafood wholesale market. the causative agent of the outbreak was identified by the who as the severe acute respiratory syndrome coronavirus-2 (sars-cov-2), producing the disease named coronavirus disease-2019 (covid-19). the virus is closely related (96.3%) to bat coronavirus ratg13, based on phylogenetic analysis. human-to-human transmission has been confirmed even from asymptomatic carriers. the virus has spread to at least 200 countries, and more than 1,700,000 confirmed cases and 111,600 deaths have been recorded, with massive global increases in the number of cases daily. therefore, the who has declared covid-19 a pandemic. the disease is characterized by fever, dry cough, and chest pain with pneumonia in severe cases. in the beginning, the world public health authorities tried to eradicate the disease in china through quarantine but are now transitioning to prevention strategies worldwide to delay its spread. to date, there are no available vaccines or specific therapeutic drugs to treat the virus. there are many knowledge gaps about the newly emerged sars-cov-2, leading to misinformation. therefore, in this review, we provide recent information about the covid-19 pandemic. this review also provides insights for the control of pathogenic infections in humans such as sars-cov-2 infection and future spillovers. coronaviruses are enveloped, single-strand rna viruses that can infect a wide range of hosts including avian, wild, domestic mammalian species, and humans. coronaviruses are well known for their ability to mutate rapidly, alter tissue tropism, cross the species barrier, and adapt to different coronaviruses are enveloped, icosahedral symmetric particles, approximately 80-220 nm in diameter containing a non-segmented, single-strand, positive-sense rna genome of about 26-32 kb in size [34] . coronaviruses (covs) are one of the largest groups of viruses that belong to the order nidovirales, suborder cornidovirineae, and family coronaviridae. coronaviridae is classified into two subfamilies, namely, letovirinae and orthocoronavirinae. letovirinae includes the alphaletovirus genus, while orthocoronaviridae is further classified on the basis of phylogenetic analysis and genome structure into four genera: alphacoronavirus (αcov), betacoronavirus (βcov), gammacoronavirus (γcov), and deltacoronavirus (δcov), which contain 17, 12, 2, and 7 unique species, respectively (ictv 2018). the most recent classification of the coronaviridae is shown in table 1 . corona in latin means crown, and this name was attributed to the virus due to the presence of spike projections from the virus envelope that give it the shape of a crown under the electron microscope; nido means nest and refers to the ability of the viruses of this order to make a nested set of subgenomic mrna [25, 35] . coronaviruses infect a wide range of wild and domestic animals; αand βcovs infect mammals, while γand δcovs primarily infect birds (table 1) . a human coronavirus (hcov) was first isolated in 1960 from hospitalized patients who suffered from common cold symptoms and was named b814 [36] . so far, the seven different hcovs that infect humans are 229e, nl63, which belong to α covs, and hku1, oc43, sars, mers, sars-cov-2, which belong to βcovs. in 2002-2003, a pandemic caused by sars-cov (lineage b βcov) originated in china [37] . in the middle east, mers-cov (lineage c βcov) emerged in 2012 [27] . in 2019, a newly emerged sars-cov-2, closely related to bat sars-related covs, was clustered with lineage b βcov. chan et al. [13] demonstrated that sars-cov-2 represents a distinct lineage in the subgenus sarbecovirus (previously, lineage 2b of βcov) [38] . additionally, other coronaviruses have caused pandemic diseases in domestic and wild mammals and birds, leading to high mortality rates and severe economic losses. these viruses include ibv in chickens [40] , beluga whale coronavirus sw1 (bwcov-sw1) [41] , bat coronaviruses cdphe15 and hku10 (ictv 2018), porcine epidemic diarrhea virus (pedv), tgev, and sudden acute diarrhea syndrome (sads-cov) [42] . since the emergence of sars-cov-2 in wuhan city, china, in december 2019, many laboratories have been working on sequencing the genome of the causative agent. as of 14 april 2020, there are a total of 7655 complete genomes from 67 countries in the global initiative on sharing all influenza data (gisaid) database [43] (table 2) . a reference genome is now available in the ncbi genome database (29,903 nucleotide, reference sequence: nc_045512.3) [44] . to date, there are a total of 875 sequences including one refseq sequence and 768 complete genomes at ncbi. sars-cov-2 is a monopartite, single-stranded, and positive-sense rna virus with a genome size of 29,903 nucleotides, making it the second-largest known rna genome. the virus genome consists of two untranslated regions (utrs) at the 5 and 3 ends and 11 open reading frames (orfs) that encode 27 proteins (table 3) . the first orf (orf1/ab) constitutes about two-thirds of the virus genome, encoding 16 non-structural proteins (nsps), while the remaining third of the genome encodes 4 structural proteins and at least 6 accessory proteins. the structural proteins are spike glycoprotein (s), matrix protein (m), envelope protein (e), and nucleocapsid protein (n), while the accessory proteins are orf3a, orf6, orf7a, orf7b, orf8, and orf10, as shown in figure 1 [2, 13, 38, 45] . the 5′utr and 3′utr of sars cov-2 are comprised of 265 and 229 nucleotides, respectively. orf1ab is 21,290 nucleotides and encodes either replicase proteins pp1a of 4405 amino acids (aa) (nsp1-nsp11) or pp1ab of 7096 aa (nsp1-nsp16), according to ribosomal frameshift. of these proteins, (1) nsp1 suppresses the antiviral host response, (2) nsp3 is a papain-like protease, (3) nsp5 is a 3clpro (3c-like protease domain), (4) nsp7 makes a complex with nsp8 to form a primase, (5) nsp9 is responsible for rna/dna binding activity, (6) nsp12 is an rna-dependent rna polymerase (rdrp), (7) nsp13 is confirmed as a helicase, (8) nsp14 is a 3′-5′ exonuclease (exon), 9) nsp15 is a poly(u)-specific endoribonuclease (xendou). the remaining nsps are involved in transcription and replication of the viral genome [13, 46, 47] . single-stranded rna viruses exhibit a faster biological mutation rate due to the lack of proofreading activity of viral rna polymerases [48] ; however, unlike other mutation-prone rna viruses, with the exception of the arenaviridae family, covs do have limited proofreading capabilities, with the nsp14 protein allowing for the enhanced genome size of cov family members [49] . recombination is another mechanism of evolution in coronaviruses [50] . a high recombination frequency was demonstrated in murine hepatitis virus during mixed infection, where the majority of viruses recovered after three passages were recombinants [51] . recombination was also reported for mers-cov and sars-cov. seven putative recombination regions were detected in orf1ab and s protein between sars-cov and six other coronaviruses by in silico analysis of their genomes [52] . similarly, bioinformatic analysis of mers-cov genomic data revealed 28 recombinant sequences from humans and camels [53] . recombination in sars-cov-2 is not yet clearly understood. initial studies suggested that it may have occurred in the course of sars-cov-2 evolution [9] , while other researchers excluded the possibility of recombination based on a full genome evolutionary analysis investigating putative recombination events [11] . to better understand the evolution of sars-cov-2, we performed a phylogenetic analysis of 45 representative coronaviruses from 18 countries including sars-cov, sars-cov-2, hcov, bat sars . the other third of sars cov-2 includes four genes (in green) that encode four structural proteins (s, m, e, n), and six accessory genes (in blue) that encode six accessory proteins (orf3a, orf6, orf7a, orf7b, orf8, and orf10). the 5 utr and 3 utr of sars cov-2 are comprised of 265 and 229 nucleotides, respectively. orf1ab is 21,290 nucleotides and encodes either replicase proteins pp1a of 4405 amino acids (aa) (nsp1-nsp11) or pp1ab of 7096 aa (nsp1-nsp16), according to ribosomal frameshift. of these proteins, (1) nsp1 suppresses the antiviral host response, (2) nsp3 is a papain-like protease, (3) nsp5 is a 3clpro (3c-like protease domain), (4) nsp7 makes a complex with nsp8 to form a primase, (5) nsp9 is responsible for rna/dna binding activity, (6) nsp12 is an rna-dependent rna polymerase (rdrp), (7) nsp13 is confirmed as a helicase, (8) nsp14 is a 3 -5 exonuclease (exon), 9) nsp15 is a poly(u)-specific endoribonuclease (xendou). the remaining nsps are involved in transcription and replication of the viral genome [13, 46, 47] . single-stranded rna viruses exhibit a faster biological mutation rate due to the lack of proofreading activity of viral rna polymerases [48] ; however, unlike other mutation-prone rna viruses, with the exception of the arenaviridae family, covs do have limited proofreading capabilities, with the nsp14 protein allowing for the enhanced genome size of cov family members [49] . recombination is another mechanism of evolution in coronaviruses [50] . a high recombination frequency was demonstrated in murine hepatitis virus during mixed infection, where the majority of viruses recovered after three passages were recombinants [51] . recombination was also reported for mers-cov and sars-cov. seven putative recombination regions were detected in orf1ab and s protein between sars-cov and six other coronaviruses by in silico analysis of their genomes [52] . similarly, bioinformatic analysis of mers-cov genomic data revealed 28 recombinant sequences from humans and camels [53] . recombination in sars-cov-2 is not yet clearly understood. initial studies suggested that it may have occurred in the course of sars-cov-2 evolution [9] , while other researchers excluded the possibility of recombination based on a full genome evolutionary analysis investigating putative recombination events [11] . to better understand the evolution of sars-cov-2, we performed a phylogenetic analysis of 45 representative coronaviruses from 18 countries including sars-cov, sars-cov-2, hcov, bat sars cov, bat sars-like cov, and mers-cov. the viral genomes were obtained from the gisaid and ncbi databases. multiple sequence alignment was performed using kalign 3 [54] . a phylogenetic tree was constructed based on whole-genome sequences (coding sequences of all genes) in iq-tree, using the maximum likelihood method, ultrafast bootstrap approximation, and modelfinder [55, 56] . the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. the bootstrap values were determined by 1,000 replicates. the tree was visualized in mega x [57] ( figure 2 ). cov, bat sars-like cov, and mers-cov. the viral genomes were obtained from the gisaid and ncbi databases. multiple sequence alignment was performed using kalign 3 [54] . a phylogenetic tree was constructed based on whole-genome sequences (coding sequences of all genes) in iq-tree, using the maximum likelihood method, ultrafast bootstrap approximation, and modelfinder [55, 56] . the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. the bootstrap values were determined by 1,000 replicates. the tree was visualized in mega x [57] ( figure 2 ). in the analysis we performed, all sars-cov-2 samples from the 18 countries clustered together and were close to bat sars or sars-like coronaviruses, with wuhan bat cov ratg13 being the closest virus. in addition, mers-cov and human cov hku1 were very distant from sars-cov-2 ( figure 2 ). within mers-cov samples, the south china mers-nl13892 clustered separately from other mers-covs. the two bat sars-like covs (bat-sl-covzc45 and bat-sl-covzxc21) were the second closest viruses from bats to sars-cov-2 ( figure 2 ). all sars-covs from china, canada, england, and the us were in a single cluster. the tree was constructed in iq-tree using the maximum likelihood method, modelfinder, and ultrafast bootstrap approximation (1000 replicates). the tree is drawn to scale, with branch lengths (numbers below the branches) measured in the number of substitutions per site. branch lengths less than 0.3 are not shown. numbers above the branches represent the percentage of replicate trees in which the associated viruses clustered together in the bootstrap test. the tree is rooted with two human coronavirus species from the genus alphacoronavirus as an outgroup (hcov-229e and hcov-nl63). in the analysis we performed, all sars-cov-2 samples from the 18 countries clustered together and were close to bat sars or sars-like coronaviruses, with wuhan bat cov ratg13 being the closest virus. in addition, mers-cov and human cov hku1 were very distant from sars-cov-2 ( figure 2 ). within mers-cov samples, the south china mers-nl13892 clustered separately from other mers-covs. the two bat sars-like covs (bat-sl-covzc45 and bat-sl-covzxc21) were the second closest viruses from bats to sars-cov-2 ( figure 2 ). all sars-covs from china, canada, england, and the us were in a single cluster. zhou et al. [9] conducted a phylogenetic analysis of sars-cov-2 against previously identified coronaviruses based on their whole-genome sequences, main structural protein genes, and non-structural protein genes. sars-cov-2 clustering was different depending on whether the whole genome or specific genes were used in the analysis. for example, sars-cov-2 clustered with the members of the subgenus sarbecovirus including the sars-cov (79.5% identical) that caused the global pandemic in 2003 and other bat sars-like viruses (96% identical at the whole-genome level), but the topological position within the sarbecoviruses changed when individual genes (orf1ab, s, e, m, and n) were used for clustering [2, 9] . based on the whole-genome sequence alignment, sars-cov-2 shares 89% identity with bat sars-like covzxc21, 82% with sars-cov, and 96.3% with bat cov ratg13 [11, 13] . alignment of the predicted protein sequences of sars-cov-2 to those of sars-cov or sars-like coronaviruses revealed a total of 380 amino acid substitutions between these viruses [2] . these amino acid substitutions were distributed as follows: 348 mutations in nonstructural proteins (orf1ab, 3a, 3b, 7a, 7b, 9b, and orf14), 27 in s protein, and 5 in n protein. no amino acid substitutions were detected in e or m proteins, indicating that e and m proteins are highly conserved among these viruses. it has been reported that sars-cov-2 uses the same cellular receptor, hace2, as sars-cov to gain entry into the cell [9, 58, 59] . the analysis of the receptor-binding domains (rbd) of sars-cov and sars-cov-2 s protein revealed similar binding affinities [60] . wu et al. [2] found a total of 27 amino acid substitutions in the s protein but not in the receptor-binding motif (rbm) that directly interacts with hace2, which may affect host tropism. these 27 substituted residues were distributed as follows: 17 in the s1 subunit [6 in the rbd and 6 in the subdomain (sd)] and 10 in the s2 subunit. wan et al. [58] reported similarity in the spike protein rbd, including rbm, of both sars-cov and sars-cov-2, in addition to the presence of several residues in sars-cov-2 rbm that favor the interaction with human ace2. these results agree with the genomic analysis of sars-cov-2, according to which the s2 subunit of the spike protein shares 99% identity with those of two bat sars-like covs (sl-covzxc21 and zc45) and of human sars-cov [13] . while the sars-cov-2 s2 subunit was conserved, the s1 subunit shares an overall 70% identity with those of bat and human sars-cov. the rbd core domain of s1 is highly conserved, with most of the amino acid differences located in the external subdomain that is responsible for the direct interaction with host receptors [13] . investigators have also reported the presence of a polybasic cleavage site and predicted o-linked glycans that are unique to sars-cov-2 s protein. differences in sars-cov-2 s protein and the high contagious nature of this virus suggest that sars-cov-2 has evolved via natural selection for binding to human ace2 receptor [61] . orf3b also differs in sars-cov-2. orf3b deletion mutations in sars-cov do not affect viral replication in vitro [62] . orf3b may play a role in viral pathogenicity in addition to its inhibitory effects on interferon (ifn) expression and signaling [63, 64] . recently, a novel short putative protein was identified in orf3b of sars-cov-2 [13] ; however, the function of this novel protein is still not known. sars-cov-2 orf8 is closer to those of bat sars cov zxc21 and zc45 and distant from that of human sars-cov [13] . the assessment of genetic diversity among 86 complete or semi-complete genomes of sars-cov-2 viruses revealed three deletions in the genome of isolates from japan, usa, and australia in addition to many other substitution mutations. the deletion mutations were in the orf1ab gene (3-nucleotide and 24-nucleotide deletion) and at the 3 end of the genome (10-nucleotide deletion). of the 93 substitution mutations, 42 changed the amino acid sequence of structural and non-structural proteins [65] . the 3and 24-nucleotide deletions in orf1ab are expected to reduce the protein sequence by 1 and 8 amino acid residues, respectively, without changing the reading frame, but the functional effects have yet to be investigated. the alignment of sars-cov-2 reference s protein gene against all sars-cov-2 sequenced genomes from china, usa, japan, australia, and taiwan revealed 99.97-100% identity, with 100% query coverage (also confirmed by our phylogenetic analysis, figure 2 ), while the identity and coverage for sars-cov s protein gene were 74.5% and 91%, respectively. also, the s protein gene from bat sars and sars-like coronavirus isolates shared 76.5-83% identity with that of sars-cov-2. this agrees with previous conclusions regarding the evolutionary analysis of sars-cov-2 [11, 44] . in the phylogenetic analysis we performed, sars-cov-2 viruses were in the same cluster regardless of the geographic region ( figure 2 ). these results strongly suggest the possibility of a recent common ancestor for all sars-cov-2 or the transmission of the same virus strain across countries. the outbreak of covid-19 originated from wuhan city, hubei province, in china. fifty-five percent of the infected cases before 1 january 2020 were linked to the huanan seafood wholesale market. however, the first human-to-human case of sars-cov-2 infection reported on 1 december 2019 did not have any exposure to this market [66, 67] . in mid-january 2020, sars-cov-2 spread to other provinces of china due to the spring festival travel season. sars-cov-2 was transmitted from china to other countries via international travelers. on 13 january 2020, the first case of sars-cov-2 infection was confirmed outside china in thailand, and on 16 january 2020 the first infected case was confirmed in japan. these cases were also linked to the huanan seafood wholesale market. by 25 january 2020, the number of confirmed cases had risen to 2062, including 2,016 in china, thailand, hong kong, macau, australia, malaysia, singapore, france, japan, south korea, taiwan, the us, vietnam, nepal, and sweden. on 30 january 2020, china reported a sharp rise in the number of infected cases, with the presence of infection in more than 18 countries. therefore, who declared the sars-cov-2 outbreak to be a public health emergency of international concern [68] . as of 16 march 2020, more than 150 countries and territories have been affected, with major outbreaks in central china, south korea, italy, iran, france, and germany [69] . there were 167,511 confirmed cases of sars-cov-2 infections, with 6606 deaths and about 8% estimated mortality rate. more than 73% of these cases have been reported in mainland china [69] . at this time, the number of global cases has shown a drastic increase within a short time, confirmed cases and deaths in china have not increased too much, while confirmed cases and deaths in other countries have drastically increased ( table 4 ). the number of confirmed cases increased from 2798 to 17,391 in one week (between 27 january and 3 february), and the number of infected countries doubled (from 12 to 24). due to the rapid increase of the number of infected cases and infected countries, the who declared sars-cov-2 a pandemic on 11 march 2020 and on 13 march 2020, the who declared europe to be the new center of the pandemic due to the massive increase of confirmed cases there [70] . on 15 ,889, were in italy). one week later (13 april 2020), the number of confirmed cases of sars-cov-2 increased 1.7 times (up to 524,514 confirmed cases), and the number of deaths increased 2.5 times (up to 20,444 deaths) in the usa alone. the number of confirmed cases, deaths, and infected countries are shown in table 4 . the origins of more than 75% of coronavirus infections are considered zoonotic, i.e., animals are the main source of the outbreaks. for example, sars-cov was transmitted from palm civets to humans, and mers-cov from dromedary camels to humans. bats are currently considered a reservoir for all human coronaviruses, as mentioned above [5, 71] . many coronaviruses are circulating in animals but have not yet infected humans. the type of animal that sars-cov-2 originated from is still unclear. at the beginning of the outbreak in wuhan, china, many patients were linked to the huanan seafood wholesale market, suggesting animal-to-person spread. after retrospectively studying case reports, the number of patients that did not have exposure to animal markets has risen, indicating person-to-person spread was also occurring at that time [66] . sars-cov-2 is closely related to bat coronaviruses and sars-cov [8] . a group of researchers reported early in the outbreak that the novel sars-cov-2 has the highest similarity of codon usage bias with snakes [72, 73] ; however, this method to determine initial host origins is dubious. interestingly, researchers also reported one amino acid difference in the receptor-binding domain of the s protein of pangolin-cov compared to that of sars-cov-2, suggesting that pangolins might play a role as an intermediate host (xiao et al., data currently under review). another group of researchers reported that the virus originated from bats based on the genome sequence of sars-cov-2, which is 96% identical to bat coronavirus ratg13. there were speculations that sars-cov-2 is a laboratory-engineered cov and leaked directly from a laboratory in wuhan where a bat cov (ratg13) was recently reported. however, there is no evidence to support this allegation [74] . recently, a group of researchers found that sars-cov-2 replicates poorly in dogs, pigs, chickens, and ducks but efficiently in ferrets and cats [75] . scientists are still trying to find the main source of the disease outbreak and identify the definitive intermediate hosts. both established (sars-cov, mers-cov) and novel (sars-cov-2) coronaviruses were reported to spread from an infected person to a non-infected person through direct or indirect contact. sars-cov-2 infection was reported to be transmitted directly from person to person like most respiratory viruses via close contact with an infected person or through respiratory droplets (aerosol) produced when an infected person coughs or sneezes. these droplets can be inhaled to reach the lung. the virus can be indirectly transmitted via touching a surface or an object that was previously contaminated with the virus and then touching the face, eyes, or mouth [76] and possibly via the fecal-oral route [77, 78] . asymptomatic carriers (during the incubation period of the virus) and patients after recovery from the acute form of the disease are also considered a potential source of virus transmission to healthy persons [10, 12] . interestingly, human coronaviruses are able to survive on steel, metal, wood, aluminum, paper, glass, plastic, ceramic, disposable gowns, and surgical gloves for 2-9 days. high temperature (≥30 • c) can reduce the persistence period, while low temperature (4 • c) increases the persistence time up to 28 days [79] . transmission of the virus vertically from mother to fetus or via breast milk has not been confirmed yet [80] . the transmission cycle of coronavirus among animals and humans is shown in figure 3 . both established (sars-cov, mers-cov) and novel (sars-cov-2) coronaviruses were reported to spread from an infected person to a non-infected person through direct or indirect contact. sars-cov-2 infection was reported to be transmitted directly from person to person like most respiratory viruses via close contact with an infected person or through respiratory droplets (aerosol) produced when an infected person coughs or sneezes. these droplets can be inhaled to reach the lung. the virus can be indirectly transmitted via touching a surface or an object that was previously contaminated with the virus and then touching the face, eyes, or mouth [76] and possibly via the fecal-oral route [77, 78] . asymptomatic carriers (during the incubation period of the virus) and patients after recovery from the acute form of the disease are also considered a potential source of virus transmission to healthy persons [10, 12] . interestingly, human coronaviruses are able to survive on steel, metal, wood, aluminum, paper, glass, plastic, ceramic, disposable gowns, and surgical gloves for 2-9 days. high temperature (≥30 °c) can reduce the persistence period, while low temperature (4 °c) increases the persistence time up to 28 days [79] . transmission of the virus vertically from mother to fetus or via breast milk has not been confirmed yet [80] . the transmission cycle of coronavirus among animals and humans is shown in figure 3 . there are many factors that affect sars-cov-2 transmission and spread. these factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited wuhan, china, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with sars-cov-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to china such as bats. additionally, the risk of infection is higher for the elderly and for patients suffering from pre-existing illnesses such as cardiovascular disease, hypertension, diabetes, and chronic respiratory disease [66] . the reported fatality rate based on age is 14.8% for people ˃80 years of age, 8% for people between 70 and 79 years, 3.6% for people between 60 and 69 years, 1.3% for people between 50 and 59 years, 0.4% for people between 40 and 49 years, there are many factors that affect sars-cov-2 transmission and spread. these factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited wuhan, china, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with sars-cov-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to china such as bats. additionally, the risk of infection is higher for the elderly and for patients suffering from pre-existing illnesses such as cardiovascular disease, hypertension, diabetes, and chronic respiratory disease [66] . the reported fatality rate based on age is 14.8% for people >80 years of age, 8% for people between 70 and 79 years, 3.6% for people between 60 and 69 years, 1.3% for people between 50 and 59 years, 0.4% for people between 40 and 49 years, 0.2% for people between 10 and 39 years; no fatalities have been reported for children under 10 years of age. notably, the fatality rate is higher in males (2.8%) than in females (1.7%) [81, 82] . the estimated incubation period of the novel coronavirus ranges from 2 to 14 days. however, some cases had an incubation period of 21, 24, or 27 days [83] . the complete clinical picture of sars-cov-2 is still unclear. the disease begins with flu-like symptoms that include fever, fatigue, dry cough, sore throat, shortness of breath, headache, chest tightness, chest pain, and muscle pain. some of sars-cov-2 patients have runny nose, nausea, vomiting, and diarrhea [84] . people can be infected without showing symptoms, which allows the virus to spread more effectively from person to person. complications can occur due to covid-19 leading to severe infections, such as pneumonia (infection of the lungs), kidney failure, and death [8] . the mild phase of the disease can last up to 2 weeks, while severe or critical disease lasts approximately 3 to 6 weeks (this analysis was conducted on 55,924 confirmed cases). additionally, the time from the disease onset to the development of severe disease is one week, while the time from the onset of symptoms to death ranges from 2 to 8 weeks [82] . based on the data analysis of 72,314 confirmed cases of sars-cov-2 in wuhan city, china, by 11 february, 80.9% of the cases were mild with flu-like symptoms, and patients recovered at home, 13.8% were severe with pneumonia and shortness of breath, 4.7% were critical with respiratory failure and septic shock resulting in organs failure, and approximately 2% of the cases were fatal [85] . another study was conducted on 99 hospitalized patients, and symptoms were classified as follow: fever (83%), cough (82%), shortness of breath (31%), muscle ache (11%), confusion (9%), headache (8%), sore throat (5%), runny nose (4%), chest pain (2%), diarrhea (2%), and nausea and vomiting (1%) [8] . the rapid diagnosis of sars-cov-2 infection is the cornerstone of disease control. it depends on several criteria including case history, clinical symptoms, serology, molecular diagnosis, and computed tomography (ct) imaging. on 2 march 2020, who published interim guidance for laboratory testing of suspected human cases, with precautions for specimen collection, packing, shipment, and amplification of nucleic acid to detect viral genes (n, e, s, and rdrp) [82] . sars-cov-2 uses the same cell entry receptor, hace2, as sars-cov. therefore, oral swabs, bronchoalveolar lavage fluid (balf), blood, as well as anal swabs are the best samples used for virus diagnosis [86] . a proper diagnosis depends primarily on the factors described below. the strict monitoring of case history in clinically suspicious patients is considered the first step in the early diagnosis of sars-cov-2 infection. clinically suspicious patients are those who suffer from fever and lower respiratory tract infection symptoms (for details, see the clinical characteristics section) and reside within or have traveled to endemic regions or had close contact with a confirmed or suspected case. additionally, sars-cov-2 can be transmitted by symptomatic and asymptomatic patients especially to the high-risk group mentioned above (for details, see the risk assessment section) [13] . the blood profiles of patients suffering from sars-cov-2 infection revealed the following: (1) increased c-reactive protein and erythrocytes, (2) increased myohemoglobin, liver enzymes, and muscle enzymes, with a high level of d-dimer in severe cases, and (3) normal or decreased white blood cell counts and lymphocytes in the early stage of the disease, with advanced lymphocytopenia in severe cases [13] . in icu patients, high levels of plasma granulocyte colony-stimulating factor (gcsf), ip10, il2, il7, il10, tnf-α, and mip1a were reported [38] . electron microscope examination of sars-cov-2 revealed the typical coronavirus morphology. further, sars-cov-2 was successfully isolated from human respiratory epithelial cells or balf samples of infected patients using huh7 cells and vero e6 cells. the isolated strain was confirmed by immunofluorescent antibody techniques using the cross-reactive nucleoprotein (np) antibody. serum neutralization tests (snt) using vero e6 cells were conducted to confirm the neutralization activity in igg-positive viral samples [87] . igm and igg elisa detection kits using bat sarsr-cov rp3 np were developed with no cross-reaction against human coronaviruses except sarsr-cov [80] . using these serological tools, viral antibody titers were increased in sars-cov-2-infected patients [38] . the procedures of elisa for the determination of sars-cov-2 igg were described before [86] . nucleic acid detection is the main, fastest, and most sensitive test for the diagnosis of sars-cov-2 infection. recently, two nested rt-pcr and two real-time rt-pcr assays have been developed with successful detection of the first 25 positive cases of infection in japan [88] . three real-time rt-pcr techniques have been designed based on the e, rdrp, and n genes [89] . also, scientists established molecular detection tools for sars-cov-2 based on the s gene [86] . chest x-ray examination in the early stage of the disease shows interstitial changes and multiple small plaque shadows. chest ct scans play an important role in the diagnosis of acute respiratory disease syndrome (ards) and pneumonia as well as in the early detection of lung parenchymal abnormalities in patients at risk and provide an impression of secondary infection (figure 4 ). electron microscope examination of sars-cov-2 revealed the typical coronavirus morphology. further, sars-cov-2 was successfully isolated from human respiratory epithelial cells or balf samples of infected patients using huh7 cells and vero e6 cells. the isolated strain was confirmed by immunofluorescent antibody techniques using the cross-reactive nucleoprotein (np) antibody. serum neutralization tests (snt) using vero e6 cells were conducted to confirm the neutralization activity in igg-positive viral samples [87] . igm and igg elisa detection kits using bat sarsr-cov rp3 np were developed with no cross-reaction against human coronaviruses except sarsr-cov [80] . using these serological tools, viral antibody titers were increased in sars-cov-2-infected patients [38] . the procedures of elisa for the determination of sars-cov-2 igg were described before [86] . nucleic acid detection is the main, fastest, and most sensitive test for the diagnosis of sars-cov-2 infection. recently, two nested rt-pcr and two real-time rt-pcr assays have been developed with successful detection of the first 25 positive cases of infection in japan [88] . three real-time rt-pcr techniques have been designed based on the e, rdrp, and n genes [89] . also, scientists established molecular detection tools for sars-cov-2 based on the s gene [86] . chest x-ray examination in the early stage of the disease shows interstitial changes and multiple small plaque shadows. chest ct scans play an important role in the diagnosis of acute respiratory disease syndrome (ards) and pneumonia as well as in the early detection of lung parenchymal abnormalities in patients at risk and provide an impression of secondary infection ( figure 4 ). assessing these lungs parenchymal abnormalities conveys disease severity to clinicians. using artificial intelligence models in the future may be useful in mass screening, to allow risk prioritization and help to minimize turnaround time [90] . pan et al. [91] conducted a retrospective study to elaborate the time course of lung changes during recovery from infection. they described assessing these lungs parenchymal abnormalities conveys disease severity to clinicians. using artificial intelligence models in the future may be useful in mass screening, to allow risk prioritization and help to minimize turnaround time [90] . pan et al. [91] conducted a retrospective study to elaborate the time course of lung changes during recovery from infection. they described findings using international standard nomenclatures such as ground-glass opacity (ggo), consolidation, and crazy paving patterns. they established a semi-quantitative scoring system of 5 grades to quantify the degree of involvement based on an area ranging from 0% to >75%. the total score ranged from 0 to 25 (max), and involvement was subpleural, random, or diffuse. they found that in early stages (0-4 days after the onset of symptoms), ggo was the main finding in lower lung lobes; in progressive stages (5-8 days), the progression of lung disease involved three patterns of ground-glass, consolidation, and crazy paving, while in peak stages (9-13 days) , dense consolidation became the prevalent feature; in absorption stages (>14 days), ggo was detected with no crazy paving and resolution of consolidations [91] . more than 75% of sars-cov-2-affected patients suffered from bilateral lung involvement, and 71% have multilobe involvement. ct examinations of 21 patients showed 29% consolidation and 86% ggo in the chest [8, 9, 92, 93] . another study examined 51 cases by ct and reported that 77% showed pure ggo, 75% exhibited ggo with reticular and/or interlobular septal thickness, 59% had ggo with consolidation, while 55% revealed pure consolidation. bilateral lung involvement was reported in 86% of cases; in 80% of the cases the posterior part of the lung was involved, while in 86%, the periphery was involved [94] . in january 2020, the who issued guidance for the clinical management of sars when sars-cov-2 infection was suspected. in this guidance, the start of emergency treatments, immediate implementation of prevention and control strategies, early supportive therapy and prevention of sars-cov-2 complications were described in detail [15] . so far, there are no approved specific antiviral drugs for sars-cov-2 infection. therefore, preventive measures and inactivation of the virus are essential to stop and control the spread of the disease. human coronaviruses can be inactivated using 0.5% hydrogen peroxide, 62-71% ethanol, 0.1% sodium hypochlorite, 0.7-1% formaldehyde, 2% glutaraldehyde, or 0.23% povidone iodine within 1 minute. other disinfectants such as 0.02% chlorhexidine digluconate, 0.55% orthophtalaldehyde, or 0.05-0.2% benzalkonium chloride are less effective [79] . in light of the urgent clinical demand, many drugs are approved to be used for clinical trials against sars-cov-2 infection, such as lopinavir/ritonavir, arbidol, interferon-alpha, favipiravir, chloroquine phosphate, darunavir/cobicistat, oseltamivir, and methylprednisolone. the most used antiviral drugs [95] are summarized in table 5 . generally, coronaviruses are not sensitive to current antiviral drugs, and high concentrations of drugs effective on these viruses cannot be used in vivo. therefore, combinations of different therapies have been used for the treatment of coronavirus infections [96] . some drug combinations that could be successful for the treatment of sars-cov-2 patients are lopinavir and ritonavir [97, 98] , lopinavir/ritonavir plus arbidol [99] , and ribavirin and interferon [100, 101] . the use of anti-inflammatory drugs such as glucocorticoids, il-6 antagonist, janus kinase inhibitors (jak), and choloroquine/hydrocholoroquine in sars-cov-2 patients is a dilemma, especially in patients suffering from an impaired immune system. balancing the risk-benefit ratio is a critical issue. corticosteroids may delay the elimination of the virus and increase the risk of secondary infection. in addition, drugs targeting pro-inflammatory cytokines can only inhibit specific inflammatory factors and thus may not be very effective in curbing the cytokine storm (excessive and uncontrolled release of pro-inflammatory cytokines). moreover, some anti-inflammatory drugs such as jak block inf-α production, which is important in fighting the virus [102] . additionally, fecal transplantation was approved for clinical trials as a therapeutic option for sars-cov-2-related pneumonia based on the promising results obtained from fecal microbiota transplantation in patients suffering from antibiotic-associated diarrhea, active ulcerative colitis, and other viral infections [103] [104] [105] [106] . recently, it was found that intestinal microbiota-derived ifn in lung stroma confers protection against viral diseases such as avian influenza and respiratory syncytial virus [103] . moreover, based on historical records of the effect of antiviral herbs on sars and influenza h1n1, chinese herbal formulas could be an alternative approach for the prevention of sars-cov-2 in a high-risk population [107] , if no scientifically based therapeutics are available. it was found that sambucus formasana nakai exhibited a strong antiviral effect against human coronavirus nl63 [108]. increases ph in host cell lysosomes and negatively influences virus-receptor binding, as well as interferes with the glycosylation of cellular receptors of sars-cov -exhibited a promising antiviral effect against sars-cov-2 in vitro -improved covid-19-pneumonia patients and shortened the course of the disease [114] remdesivir a monophosphoramidate of adenosine prodrug that incorporates into nascent viral rna chains causing pre-mature termination -used against a wide range of rna viruses such as filoviridae, paramyxoviridae, pneumoviridae, and coronaviridae; used successfully in covid-19 treatment in the united states and showed no adverse events [115] darunavir and cobicistat inhibit 3 c-like protease (3clpro). -used for the treatment of mers-cov in experimental animals -used for the treatment of hiv-1 patients [116] to date, there is no vaccine to prevent sars-cov-2 infection, and trials for vaccine development are in the preliminary stages of research. several vaccine candidates such as live attenuated, adenovirus-vectored, recombinant protein, and nucleic acid (dna and mrna) vaccines are in the pipeline [123] . the epidemiology of sars-cov-2 is still unclear, and data availability is limited. therefore, it is imperative to follow preventive measures and safety precautions issued by health authorities to limit exposure to the virus and to reduce further spread. general hygienic measures should be implemented, such as (1) washing hands often with soap and water or an alcohol-based hand sanitizer, (2) cough or sneeze etiquette, recommending covering of the mouth, (3) avoiding touching eyes, nose, and mouth if the hands are not clean, (4) avoiding close contact with sick persons, (5) avoiding sharing dishes, glasses, bedding, and other household items with sick people, (6) cleaning and disinfection of surfaces that are often touched, and (7) staying home from work, school, and public areas when feeling sick. the transmission route of sars-cov-2 is probably not only through cough, respiratory droplets, and/or contaminated surfaces [13, 124] , but also through fecal-oral transmission [78] . therefore, strict hygienic measures should be followed, especially in dense cities or agricultural spaces [125] . since the sars-cov-2 spread is primarily driven by travel, screening of travelers who arrive at airports from pandemic areas for possible sars-cov-2 infection and entry-screening procedures are necessary. also, general hygienic precautions during travel are highly recommended. travelers who suffered from acute respiratory infection should be tested and reported to the respective public health authorities [73] . in addition, people should be motivated to notify and report about travel history and close contacts in case of sars-cov-2 infection. asymptomatic carriers (during the incubation period) and patients after recovery from the acute form are also considered potential sources of the virus [12, 13] . strict hygienic measures should be implemented to avoid virus transmission to healthcare workers and other contacts, i.e., placement of sars-cov-2 suspected or confirmed patients in single-person rooms and wearing personal protection equipment (ppe) such as masks, goggles, and protective gowns. because early diagnosis and detection of asymptomatic carriers of sars-cov-2 are successful factors for the treatment and prevention of transmission, health authorities should designate laboratories to implement tests for a rapid and accurate diagnosis [126] . the control of coronaviruses is based on biosecurity regarding animals as well as on shifts in food habits, including discouraging the consumption of bushmeat and of animal products without appropriate cooking [127] . ban of wet marketplaces where live or dead animals are handled should be implemented. surveillance among people who have contact with wildlife and improvement of biosecurity regarding wildlife trade are urgently needed to prevent the next pandemic outbreak [128] . the epidemiology of sars-cov-2 is still unclear. many unresolved questions related to sars-cov-2 epidemiology and pathogenicity pose great challenges for researchers. these unresolved questions include: what is the origin of sars-cov-2? what is the intermediate host that transmitted the virus from bats? why does the virus cause severe disease and mortality in the elderly or those with co-morbidities, while it is milder in children? are aerosol, saliva, feces, urine, and foodborne the only routes of transmission? what are the other unknown routes of transmission? control of the sars-cov-2 outbreak and future epidemics requires global efforts among medical and veterinary clinicians, diagnosticians, epidemiologists, public health experts, vaccinologists, pharmaceutical industries, economists, and governments to implement a one-health approach [128, 129] . these measures must include: (1) writing policies and supporting funds required for the implementation of one health, prevention, and control measures, (2) hiring well-trained and professional personnel, (3) performing rapid and accurate diagnosis and treatment of infected persons, (4) developing and providing vaccines for virus control in humans, (5) conducting surveillance among wildlife for the identification and characterization of possible reservoirs and surveillance among people who are in contact with wildlife to identify risk factors in human behaviors and living environment, (6) improving hygienic measures, (7) assessing the social and economic impacts of covid-19 on the population, (8) utilizing veterinary experience in the disinfection of premises and gatherings under the supervision of health authorities to decrease outbreaks in humans, (9) providing antiviral drugs for the treatment of the disease in humans, and (10) increasing public health awareness about the virus and its transmission. the sars-cov-2 outbreak started in wuhan city, china, in december 2019. it is now a global pandemic, with 1,773,084 confirmed cases, 111,652 deaths, and 467,074 recoveries (as of 13 april 2020). the virus has the potential for rapid and extensive spread between people and countries. there are a lot of misleading information and knowledge gaps on the newly emerged sars-cov-2. therefore, we reviewed the latest updates about different aspects including epidemiology, source of infection, transmission dynamics, zoonotic potential, virus characteristics, and discovery of novel strategies for disease control to avoid spillover of infection in the future. bats play an important role in the transmission of the infection to humans. coronaviruses are genetically diverse and have a high tendency towards frequent genetic mutations and gene recombination, which increases the risk of interspecies transmission. information about the incubation period can help in establishing an effective quarantine for asymptomatic carriers, thus preventing the virus spread. from our perspectives and based on the currently available information about the virus and its epidemiology, the control of the sars-cov-2 requires an effective and global disease coordination effort including multidisciplinary research efforts (one-health approach) through collaboration between governments, epidemiologists, virologists, health authorities, veterinarians, and physicians. at this stage of the disease outbreak, developing vaccines is crucial to limit the spread of the infection. genome composition and divergence of the novel coronavirus (2019-ncov) originating in china isolation and characterization of viruses related to the sars coronavirus from animals in southern china supplement to: transmission of mers-coronavirus in household contacts origin and evolution of pathogenic coronaviruses summary of probable sars cases with onset of illness from 1 situation update clinical features of patients infected with 2019 novel coronavirus in a pneumonia outbreak associated with a new coronavirus of probable bat origin a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event transmission of 2019-ncov infection from an asymptomatic contact in germany genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan the epidemic of 2019-novel-coronavirus (2019-ncov) pneumonia and insights for emerging infectious diseases in the future clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-ncov) outbreak molecular evolution of human coronavirus genomes cultivation of the virus of infectious bronchitis a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin: i. isolation and biological properties of the virus history and recent advances in coronavirus discovery cultivation of viruses from a high proportion of patients with colds medicine. a new virus isolated from the human respiratory tract growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease fields virology discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers coronaviruses from camels in africa exhibit region-dependent genetic diversity genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus mers-cov recombination: implications about the reservoir and potential for adaptation origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol the molecular biology of coronaviruses cultivation of a novel type of common-cold virus in organ cultures identification of a novel coronavirus in patients with severe acute respiratory syndrome epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study bat coronaviruses in china evolution of infectious bronchitis virus in china over the past two decades molecular characterization of a new species in the genus alphacoronavirus associated with mink epizootic catarrhal gastroenteritis fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin global initiative on sharing all influenza data a new coronavirus associated with human respiratory disease in china genomic variance of the 2019-ncov coronavirus covid-2019: the role of the nsp2 and nsp3 in its pathogenesis processing of the sars-cov pp1a/ab nsp7-10 region adaptive value of high mutation rates of rna viruses: separating causes from consequences structural and molecular basis of mismatch correction and ribavirin excision from coronavirus rna molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination high-frequency rna recombination of murine coronaviruses testing the hypothesis of a recombinant origin of the sars-associated coronavirus evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission kalign 3: multiple sequence alignment of large datasets fast model selection for accurate phylogenetic estimates iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies molecular evolutionary genetics analysis across computing platforms receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors structure, function and antigenicity of the sars-cov-2 spike glycoprotein the proximal origin of sars-cov-2 severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in vero e6 cells severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists genetic diversity and evolution of sars-cov-2 covid-19) situation summary who. coronavirus disease (covid-2019) situation reports-48. available online who. rolling updates on coronavirus disease (covid-19). available online who. coronavirus disease (covid-2019) situation reports. available online: https director-general's opening remarks at the media briefing on covid-19-11 a comprehensive review of common bacterial, parasitic and viral zoonoses at the human-animal interface in egypt cross-species transmission of the newly identified coronavirus 2019-ncov outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2): increased transmission beyond china-fourth update no credible evidence supporting claims of the laboratory engineering of sars-cov-2 susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 you need to know about coronavirus disease 2019 (covid-19) severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and corona virus disease-2019 (covid-19): the epidemic and the challenges first case of 2019 novel coronavirus in the united states persistence of coronaviruses on inanimate surfaces and its inactivation with biocidal agents diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in covid-19) technical guidance: laboratory testing for 2019-ncov in humans presumed asymptomatic carrier transmission of covid-19 the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes another decade, another coronavirus development of genetic diagnostic methods for novel coronavirus 2019 (ncov-2019) in japan detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr outbreak of novel coronavirus (covid-19): what is the role of radiologists? time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia an interim review of the epidemiological characteristics of 2019 novel coronavirus ct imaging features of 2019 novel coronavirus (2019-ncov) emerging 2019 novel coronavirus (2019-ncov) pneumonia potential antiviral therapeutics for 2019 novel coronavirus der steckbrief des virus ist im fluss. deutsches ärzteblatt jg. 117, heft 6 the course of clinical diagnosis and treatment of a case infected with coronavirus disease 2019 case of the index patient who caused tertiary transmission of covid-19 infection in korea: the application of lopinavir/ritonavir for the treatment of covid-19 infected pneumonia monitored by quantitative rt-pcr efficacy and biological safety of lopinavir/ritonavir based anti-retroviral therapy in hiv-1-infected patients: a meta-analysis of randomized controlled trials ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the experience of clinical immunologists from china microbiota-driven tonic interferon signals in lung stromal cells protect from influenza virus infection rescue fecal microbiota transplantation for antibiotic-associated diarrhea in critically ill patients long-term safety and efficacy of fecal microbiota transplant in active ulcerative colitis microbiota and its role on viral evasion: is it with us or against us? front can chinese medicine be used for prevention of corona virus disease 2019 (covid-19)? a review of historical classics, research evidence and current prevention programs antiviral activity of sambucus formosananakai ethanol extract and related phenolic acid constituents against human coronavirus nl63 role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings inhibition of the infectivity and inflammatory response of influenza virus by arbidol hydrochloride in vitro and in vivo (mice and ferret) favipiravir (t-705), a novel viral rna polymerase inhibitor favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase antiviral activity of ribavirin and favipiravir against human astroviruses antiinflammatory and immunoregulatory effects of total glucosides of yupingfeng powder comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov hiv-1 resistance rarely observed in patients using darunavir once-daily regimens across clinical studies prophylaxis of ferrets with nitazoxanide and oseltamivir combinations is more effective at reducing the impact of influenza a virus infection compared to oseltamivir monotherapy effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients treatment of 5 critically ill patients with covid-19 with convalescent plasma could intravenous immunoglobulin collected from recovered coronavirus patients protect against covid-19 and strengthen the immune system of new patients? expanded umbilical cord mesenchymal stem cells (uc-mscs) as a therapeutic strategy in managing critically ill covid-19 patients: the case for compassionate use the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro the sars-cov-2 vaccine pipeline: an overview importation and human-to-human transmission of a novel coronavirus in vietnam how urbanization affects the epidemiology of emerging infectious diseases infection prevention and control during health care when novel coronavirus (ncov) infection is suspected interim guidance a strategy to prevent future pandemics similar to the 2019-ncov outbreak the one health approach is necessary for the control of rift valley fever infections in egypt: a comprehensive review this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we gratefully acknowledge the authors, originating, and submitting laboratories of the sequences from the gisaid's epicov™ database, on which part of the phylogenetic tree construction is based. the authors declare no conflict of interest. key: cord-309621-6jj19xpr authors: yu, pin; xu, yanfeng; deng, wei; bao, linlin; huang, lan; xu, yuhuan; yao, yanfeng; qin, chuan title: comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus date: 2017-02-24 journal: plos one doi: 10.1371/journal.pone.0172093 sha: doc_id: 309621 cord_uid: 6jj19xpr middle east respiratory syndrome (mers), which is caused by a newly discovered coronavirus (cov), has recently emerged. it causes severe viral pneumonia and is associated with a high fatality rate. however, the pathogenesis, comparative pathology and inflammatory cell response of rhesus macaques and common marmosets experimentally infected with mers-cov are unknown. we describe the histopathological, immunohistochemical, and ultrastructural findings from rhesus macaque and common marmoset animal models of mers-cov infection. the main histopathological findings in the lungs of rhesus macaques and common marmosets were varying degrees of pulmonary lesions, including pneumonia, pulmonary oedema, haemorrhage, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. the characteristic inflammatory cells in the lungs of rhesus macaques and common marmosets were eosinophils and neutrophils, respectively. based on these observations, the lungs of rhesus macaques and common marmosets appeared to develop chronic and acute pneumonia, respectively. mers-cov antigens and viral rna were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, and ultrastructural observations showed that viral protein was found in type ii pneumocytes and inflammatory cells in both species. correspondingly, the entry receptor ddp4 was found in type i and ii pneumocytes, bronchial epithelial cells, and alveolar macrophages. the rhesus macaque and common marmoset animal models of mers-cov can be used as a tool to mimic the oncome of mers-cov infections in humans. these models can help to provide a better understanding of the pathogenic process of this virus and to develop effective medications and prophylactic treatments. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the middle east respiratory syndrome coronavirus (mers-cov) was identified in 2012 in a cell culture taken from a patient who died of pneumonia in saudi arabia [1] . more men than women have become infected with this virus, and the median age of those affected is 47 years old (range: 9 months-94 years), with most of the fatalities occurring in patients over 60 years old [2, 3] . the respiratory symptoms of this infection are primarily related to severe lower respiratory tract complications (e.g., dyspnoea and coughing associated with a fever) that may become fatal, while there is generally little involvement of the upper respiratory tract. a large proportion of severely ill patients require mechanical ventilation [4, 5] . complications that have been described in fatal cases include hyperkalaemia with associated ventricular tachycardia, disseminated intravascular coagulation leading to cardiac arrest, pericarditis and multiorgan failure [6] . mers-cov seems to be widely present in dromedary camels in the middle east and in some parts of africa [7, 8] . zoonotic transmission is likely to have originated from this species and is expected to continue indefinitely in these regions. the entry receptor for mers-cov, dipeptidyl peptidase 4 (ddp4), also named cd26, shows a high similarity in both humans and dromedary camels. most mers patients acquire the infection in the middle east, which subsequently leads to limited human-to-human transmission in local groups and healthcare workers and eventually to travel-related cases outside the region, all of which can result in a mild to severe or even fatal respiratory disease [2] . finding a suitable animal model is a major challenge for understanding the pathogenesis of mers-cov infection, evaluating the safety and efficacy of mers-cov vaccine candidates and developing therapeutic interventions. experimental infections with mers-cov in rhesus macaques (macaca mulatta) [9] , common marmosets (callithrix jacchus) [10] , rabbits (oryctolagus cuniculus) [11] and mice (mus musculus) [12, 13] have been reported in studies on the pathological changes that occur as a result of this viral infection. however, little is known about the pathological changes in the lungs of humans infected with mers-cov, which makes it difficult to interpret data from experimental mers-cov animal models. overall, based on the known clinical aspects of mers-cov infection in humans, useful experimental animal models of mers-cov infection should exhibit a life-threatening lower respiratory tract disease. although there have been several studies in animal models on the pathogenic mechanisms of mers-cov infection, little is known about the comparative pathology and inflammatory cell response in rhesus macaques or common marmosets infected with this virus. therefore, it is vital to study comparative pathology on the association of the mers-cov antigen with its receptor, ddp4, or the histopathological changes in nonhuman primate (nhp) models of mers-cov infection. here, we comprehensively describe the histopathological features of the disease and the distribution of the mers-cov antigen and ddp4 in rhesus macaque and common marmoset models. our findings may contribute to a better understanding of the pathogenic process of mers-cov infection and help in evaluating the suitability and efficacy of the animal models used in the development of effective medications and prophylactic treatments for this disease. this research on the mers-cov virus was discussed among the staff members of the department of pathogen biology at the institute of laboratory animal science (ilas) of the chinese academy of medical sciences and peking union medical college (pumc). the experiments and protocols for this nhp models of mers-cov infection were discussed explicitly and extensively among the staff members of the department of pathogen biology. these discussions were followed by consultations with biosafety officers and facility managers at the ilas of pumc, as well as with numerous specialists in the fields of sars-cov and general infectious disease research throughout china. all research procedures were approved by the ilas institutional animal care and use committee and the laboratory safety committee (lsc). the committee recommended that the number of animals be reduced to comply with the 3r (reduction, replacement, refinement) principles. therefore, our experiment was designed to include three rhesus macaques and three common marmosets to test their effectiveness as animal models of mers-cov infection. two rhesus macaques and two common marmosets were infected with the virus and one individual of each species was left uninfected to serve as a control. the animals were planned to be euthaniazed when they were suffering from fatal respiratory symptom, impending death or 20% of body weight loss, which included fatal dyspnea and infectious shock. the approved registration number for this study is ilas-pc-2013-004. all experiments were conducted within an animal biosafety level 3 (absl-3) facility, which was constructed and accredited based on national standard gb19489 at the ilas of pumc, beijing, china. rhesus macaques and common marmosets were housed in accordance with chinese national standards, which are consistent with the standard set forth in the 8th edition of the nrc guide for the care and use of laboratory animals. because of the infectious nature of this study, nhps were housed individually instead of the generally recommended group or social housing. stainless steel cages measuring 0.5-0.75 m 2 and 0.5-0.6 m depending on the weight of the individual animal, consisted of wire flooring and resting boards or perches. rooms have natural lighting and the photoperiod is supplemented during the winter months with an artificial lighting source to provide a 12: 12 light cycle. temperature and humidity in animal holding rooms are maintained in accordance with recommendations in the chinese national standards for animal care. drinking potable water is obtained from the city of beijing and delivered to the animals via automated watering system (aws). the aws is checked daily to ensure proper operation i.e., water pressure, free flowing exits and absence of leakages. pans were cleaned daily and cages were washed every week by hand. all animals have individual cage id cards which contain the following basic information: study no., sex, weight, principal investigator's name and study protocol number. nhps were fed a measured amount of a commercially available nhp diet (beijing hfk bioscience co., ltd) offered twice daily. fresh fruit (apples, bananas and oranges) are supplemented on alternating days. additional environmental enrichment consists of toys, stainless steel mirrors and heavy-duty dog chew toys (nyla bones or similar), which are provided on a rotating basis. toys are left inside the cages when these are transported out of the room for washing and are sanitized at this time. damaged toys are removed from circulation. soft background music, plants, as well as pictures and photos hung on the animal room walls are provided for relaxation. opportunities for limited social interaction with compatible nhps are also provided at every other cage change when cages of compatible animals are placed in close proximity to each other while avoiding direct physical contact between animals. two rhesus macaques, 2-3 years old, were anesthetised with ketamine hydrochloride (30 mg/kg, i.m) prior to the procedures and intratracheally inoculated with 1 ml of hcov-emc (6.5 ×10 7 tcid 50 /1 ml) diluted in dmem. one mock-infected rhesus macaque was intratracheally inoculated with tissue culture media dmem for use as control. the rhesus macaques were observed twice daily, and clinical signs were recorded. the infected and mock-infected rhesus macaques were anesthetized with pentobarbital sodium (60 mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at 3 days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. two common marmosets, 2-3 years old, were anesthetised with ketamine hydrochloride (120 mg/kg, i.p) prior to the procedures and intratracheally inoculated with 1 ml of hco-v-emc (5 ×10 6 tcid 50 /0.5 ml) diluted in dmem. one mock-infected common marmoset was intratracheally inoculated with tissue culture media dmem for use as control. the common marmosets were observed twice daily, and clinical signs were recorded. the infected and mock-infected common marmosets were anesthetized with pentobarbital sodium (40 mg/kg, i.m) prior to the procedures, and while under deep anesthesia, the animals were sacrificed through femoral artery bloodletting at 3 days post-infection. tissue specimens, including samples from lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. none of the infected animals were euthanized or died without euthanasia prior to their sacrifice at 3 days post-infection. the fixed samples were dehydrated and dewaxed according to conventional procedures, and 4-μm sections were prepared with a microtome. some sections were stained with haematoxylin-eosin (he) using routine methods. two independent pathologists observed all slides and were blinded to the experimental design. lungs were fixed in glutaraldehyde and prepared for ultrastructural observations. transmission electron microscopy was performed essentially as previously described [14] . briefly, serial sections were dewaxed and rehydrated in graded ethanol, and a standard avidinbiotin immunoperoxidase technique was performed [15] . table 1 lists the primary antibodies used for ihc. optimal antibody dilutions were determined in experiments on positive control tissues. negative control sections were prepared using the same steps as described above, but the primary antibodies were derived from an irrelevant sera. sections were dewaxed and rehydrated in a graded ethanol series. ish was carried out using the enhanced sensitive ish detection kit i (boster, china) according to the manufacturer's instructions. endogenous peroxidase activity was quenched with 0.5% hydrogen peroxide in methanol at room temperature for 30 minutes. proteinase k digestion was performed at 37˚c for 20 min. then, pre-hybridization was performed at 37˚c for 3 hours. after removing excess pre-hybridization buffer, 2 μg/ml digoxin (dig)-modified oligo-nucleotide antisense probes (table 2) in the hybridization solution were applied to the sections, followed by incubation at 37˚c overnight. after washing the slides in 2× saline-sodium citrate (ssc), 0.5×ssc, and 0.2×ssc buffer, the sections were incubated in a blocking buffer at 37˚c for 30 min. the sections were then incubated with biotinylated mouse anti-dig at 37˚c for 60 min and with streptavidin biotin peroxidase and biotinylated peroxidase for an additional 20 min, with each incubation followed by three washes in phosphate-buffered saline (pbs). the sections were treated with 3, 3-diaminobenzidine for 2 min, counterstained in haematoxylin for 5 min, dehydrated, and mounted with neutral gum. sections for the negative controls were prepared using the same steps described above, but the antisense or sense probes were replaced with pbs at ph 7.4. rhesus macaques were observed twice daily for clinical signs. the rectal temperature of the infected rhesus macaques increased to 40.5˚c at 1-2 days post-infection, and thereafter turned to normal. the infected common marmosets showed manifest symtoms of viral infection, including severe respiratory symtoms, drastical water intake decrease and overt weight loss, and the maximum body weight loss were about 11%. none of the mock infected nhps showed abnormal clinical signs or died during the expriment. pathological findings in the rhesus macaque tissues he stained tissues from rhesus macaques experimentally infected with mers-cov demonstrate that mers-cov induces lesions that are primarily observed in the lungs, with varying degrees of inflammation, interstitial pneumonia (fig 1a) , pulmonary oedema (fig 1b) , haemorrhaging, degeneration and necrosis of pneumocytes and bronchial epithelial cells (fig 1c) , and the infiltration of inflammatory cells. focal interstitial pneumonia and pulmonary oedema were observed in different parts of the pulmonary lobes, as was mild haemorrhaging. the most prominent pathological effect observed in the lungs of rhesus macaques was diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control rhesus macaque (s1a fig) . pathological findings in common marmoset tissues a histopathological analysis detected numerous lesions in the lungs of the infected marmosets. exudative pathological changes were found, exhibiting haemorrhage, widespread pulmonary oedema and a large number of inflammatory cells. fibrinous exudates were observed in the oedematous alveolar cavities (fig 1d) . diffuse and focal neutrophil infiltration was found in the oedematous alveolar cavities (fig 1e) , bronchial lumen, and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells (fig 1f) . no significant pathological changes induced by viral infection were observed in the other organs, and no obvious pathological changes were identified in any tissues examined from the control common marmoset (s1b fig) . to investigate the infiltration of specific inflammatory cells, ihc was carried out to identify cd68+ macrophages, cd15+ neutrophils, cd57+ natural killer cells, cd20+ b lymphocytes, cd138+ plasma cells, and cd3+, cd4+, cd8+ t lymphocytes. in the lungs of both species, the diffuse infiltration of numerous macrophages (fig 2b and 2d ) was observed in the expanded alveolar septum and the oedematous alveolar cavities. however, in the lungs of rhesus macaques, a large number of diffusely and focally infiltrating eosinophils (fig 2a) were found in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. however, in the lungs of common marmosets, numerous neutrophils ( fig 2c) infiltrated into the oedematous alveolar cavities. in both of the nhp models, other types of inflammatory cells were rarely observed. using immunohistochemical techniques and an ish analysis, we confirmed that mers-cov protein and viral rna were distributed in the lungs of rhesus macaques and common marmosets and that they were primarily located in the pneumocytes and inflammatory cells. in the lungs of rhesus macaques, mers-cov antigens were extensively distributed in type i and ii pneumocytes, alveolar macrophages (fig 3a) , eosinophils and bronchial epithelial cells ( fig 3b) . from the microscopic characteristics, the cuboidal type ii pneumocytes are located on the alveolar cavities, and smaller than macrophages. viral rna was also distributed in pneumocytes and inflammatory cells in the lungs of rhesus macaques (fig 3c) . in the lungs of common marmosets, a moderate level of mers-cov-positive antigens were detected in pneumocytes, and antigens were found more extensively in alveolar macrophages (fig 3d) , especially in the inflammatory cells around the bronchus (fig 3e) . viral rna was massively distributed in pneumocytes and inflammatory cells in the lungs of common marmosets (fig 3f) . no mers--cov-positive antigens or viral rna was detected in the lungs of the control nhps (data not shown). pathological lesions and virus distribution in rhesus macaque and common marmoset animal models are summarized and shown in table 3 . to further determine the effects of mers-cov infection and replication in the lungs of common marmosets, ultrastructural observations were performed on lesions in infected lung samples and on mock-infected samples. virus particles were found in type ii pneumocytes ( fig 4a-4c ) and in inflammatory cells (fig 4d-4f) . under the electron microscope, the characteristic of type ii pneumocytes is lamellar bodies (s2 fig). no viral particles were observed in the lungs of mock-infected common marmosets (data not shown). to elucidate the relationship between mers-cov and its entry receptor, ddp4, we determined the expression pattern of ddp4 in the lungs of rhesus macaques and common marmosets using immunohistochemical techniques. we found that in the lungs of rhesus macaques, ddp4 was strongly expressed in type i and ii pneumocytes, bronchial epithelial cells (fig 5a) , and inflammatory cells, primarily alveolar macrophages (fig 5a) . similarly, in the lungs of common marmosets, ddp4 was widely expressed in type i and ii pneumocytes and alveolar macrophages (fig 5b) . however, ddp4 was only weakly expressed in the bronchial epithelial cells, mainly in basal and ciliated cells (fig 5b) . in the present study, we analysed the histopathological features of mers-cov infection in rhesus macaques and common marmosets. moreover, we compared the distribution of mers-cov antigens, viral rna and ddp4 expression in the infected lungs of these species. we found that the lungs of both species exhibited varying degrees of lesions, including pneumonia, pulmonary oedema, haemorrhaging, degeneration and necrosis of the pneumocytes and bronchial epithelial cells, and inflammatory cell infiltration. comparing the different trends in the two nhp models, it can be seen that varying degrees of inflammation, especially interstitial pneumonia, were found in the lungs of rhesus macaques, indicating mild disease and trend of chronic pneumonia; however, in the lungs of common marmosets, exudative pathological changes were found, exhibiting pulmonary oedema, inflammatory cell infiltration and fibrinous exudates, suggesting acute pneumonia. similar to our results, previous study have also reported that rhesus macaques developed mild disease, and common marmoset exhibited potentially lethal disease [16] . however, in our study we found that the prominent inflammatory cells in the two nhp models were different, which may be the causality of process in mers-cov infection. in our study, the diffuse infiltration of numerous macrophages was observed in the expanded alveolar septa and oedematous alveolar cavities of both species. however, the most prominent pathological effect observed in the lungs of rhesus macaques was a diffuse and focal eosinophil infiltration in the thickened alveolar septum and oedematous alveolar cavities, around the bronchus, and among the necrotic bronchial epithelial cells. in contrast, in the lungs of common marmosets, diffuse and focal neutrophil infiltration occurred in the oedematous alveolar cavities, bronchial lumen and mildly thickened alveolar septum, around the bronchus, and among the necrotic bronchial epithelial cells. these differences in inflammatory cell infiltration suggest that inflammatory cells may function in the development of mers-cov infection. additionally, it is worth noting that eosinophils and neutrophils play important roles in rhesus macaques and common marmosets, respectively, in the development of pulmonary lesions and the pathogenesis of mers-cov infection. in the lungs of common marmoset, pulmonary oedema exhibited much more severe than that in the lungs of rhesus macaques, which may be due to the difference of inflammatory cells in the lungs of nhp models. similar to our results, previous studies have also reported that common marmosets infected with mers-cov exhibit acute bronchointerstitial pneumonia centred at doi:10.1371/journal.pone.0172093.g003 table 3 . pathological lesions and virus distribution in rhesus macaque and common marmoset animal models. pathology of mers-cov infection the terminal bronchioles, with an influx of inflammatory cells, a thickening of alveolar septa, oedema, haemorrhaging and fibrosis in lung tissues [10] . previous studies on rhesus macaques have shown that histological lesions induced by mers-cov infection were limited to the lungs, which exhibited interstitial pneumonia with a thickening of alveolar septa caused by oedema and fibrin accumulation, and small to moderate numbers of macrophages and even fewer neutrophils. in addition, alveoli tissue samples contained moderate numbers of pulmonary macrophages and neutrophils [17] . previous studies on common marmosets infected with mers also showed that marmosets developed a progressive severe pneumonia or interstitial lymphohistiocytic pneumonia, exhibiting hypoproteinemia consistent with high protein pulmonary effusions resulting from alveolar oedema and interstitial lymphohistiocytic pneumonia with type ii pneumocyte and bronchial associated lymphoid tissue hyperplasia [18, 19] . however, rhesus macaque model did not develop breathing abnormalities and showed no-tovery mild radiographic evidence of pneumonia [20] . similar to our study, the common marmoset model of mers-cov infection mimics the acute and severe pathological process, yet the rhesus macaque model represents the mild infection of mers-cov. thus, the nhp models meet different needs of mers-cov researches. fatal human cases of mers-cov infection cause upper respiratory tract illness, severe pneumonia and multiorgan failure. these cases also include exudative-phase diffuse alveolar damage with the denuding of bronchiolar epithelia, the prominent formation of hyaline membranes, alveolar fibrin deposits, type 2 pneumocyte hyperplasia, the occurrence of rare multinucleated syncytial cells, changes in alveolar septa related to oedema and increases in lymphocytes, with fewer plasma cells, neutrophils, and macrophages [21] . these findings provide evidence for the pulmonary tropism of mers-cov infection. the pathological features of mers-cov are shared by other similar respiratory illnesses, such as severe acute respiratory syndrome (sars)-cov [22] . previous studies that have evaluated fatal human cases of sars--cov have described diffuse alveolar damage at various stages as the most characteristic feature pathology of mers-cov infection of the disease, with sars-cov antigens primarily found in alveolar epithelial cells. in this study, we examined the histopathological features and the inflammatory cell response that occurs in the lungs of rhesus macaques and common marmosets experimentally infected with mers-cov. our findings indicate that inflammatory cells may play a crucial role in fatal mers-cov infections and that the progression of this disease in our animal models may mimic the infection in humans, making these models useful for further study of the pathogenesis, prevention and treatment of mers-cov infections. in this study, we analysed the distribution of mers-cov protein and viral rna in the lungs of rhesus macaques and common marmosets. we found that pneumocytes and inflammatory cells were positive for mers-cov antigens and viral rna. similarly, in the lungs of both species, mers-cov antigens were identified in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells, viral rna was distributed in pneumocytes and inflammatory cells, and viral protein were found in type ii pneumocytes and inflammatory cells. based on our observations, we therefore propose that mers-cov may proliferate and spread from the lungs through an inflammatory cell migration pathway. it has been reported that in fatal human cases of mers-cov infection, viral antigens were identified in both unremarkable and necrotic bronchial submucosal glands and in pneumocytes and epithelial syncytial cells. however, macrophages showed no localization with mers-cov antigens in these cases [23] . findings of pulmonary consolidation, diffuse alveolar damage, and pleural effusion are consistent with the clinical features with mers-cov infection [2, 4, 24] . in this study, we also detected mers-cov protein and viral rna in type i and ii pneumocytes, alveolar macrophages and bronchial epithelial cells. therefore, our models of mers-cov infection using rhesus macaques and common marmosets may be suitable for use in the development of effective medications and prophylactic treatment and may serve as a tool to improve our understanding of the pathogenic process of mers-cov infection. dpp4 is a single-pass type ii transmembrane glycoprotein with a short n-terminal cytoplasmic tail. the structural residues comprising the receptor-binding domain have been defined via the co-crystallization of the mers-cov spike glycoprotein and dpp4. dpp4 is typically found in type i and ii cells and alveolar macrophages. it has only rarely been detected in the surface epithelia of the nasal cavity and has also been found in a subset of mononuclear leukocytes and serous cells from submucosal glands [25] . in fatal human cases, dpp4 has been observed in scattered pneumocytes and syncytial cells, bronchiolar epithelia and endothelia, and alveolar macrophages [23, 26, 27] . in this study, we found that in the lungs of the nhps infected with mers-cov, ddp4 was expressed in type i and ii pneumocytes, bronchial epithelial cells, and inflammatory cells, primarily alveolar macrophages. in conclusion, we established animal models of mers-cov infection using rhesus macaques and common marmosets, which mimic the oncome of mers-cov infection in humans and provide a tool that may help in better understanding the pathogenic process of this disease. they may also be suitable models for aiding in the development of effective medications and prophylactic treatments for mers-cov infections. fouchier ra isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease rapid generation of a mouse model for middle east respiratory syndrome rapid nipah virus entry into the central nervous system of hamsters via the olfactory route use of avidin-biotin-peroxidase complex (abc) in immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures an acute immune response to middle east respiratory syndrome coronavirus replication contributes to viral pathogenicity middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study key: cord-305773-ikm1famj authors: lan, bowen; lu, puxuan; zeng, yujing; li, xin; ou, xiaxing; li, jingjing; li, hongjun title: clinical imaging research of the first middle east respiratory syndrome in china date: 2015-11-23 journal: radiol infect dis doi: 10.1016/j.jrid.2015.11.004 sha: doc_id: 305773 cord_uid: ikm1famj middle east respiratory syndrome is a viral respiratory illness caused by a novel human beta-coronavirus. based on the first case of middle east respiratory syndrome found in china, a clinical research in combination with radiological findings was studied. fever was the main clinical manifestation of this patient, and the primary imaging findings were basically the same as viral pneumonia. differential imaging diagnosis on the basis of epidemiological and experimental pathogen detection is helpful for clinical diagnosis of mers, even in distinguishing from sars and pneumonia caused by h7n9 avian influenza. middle east respiratory syndrome (mers), also known as camel flu, is a viral respiratory illness caused by a novel human beta-coronavirus (cov) [1e3] . since it was firstly reported by saudi arabia in september 2012, till june 2015, more than 1200 patients have been detected worldwide, with at least 427 cases of patients died [4, 5] . until june 2015, most of the cases of mers-cov infectors occurred in the middle east, and recently, it is reported that south korea has been sufferring from mers. symptoms may range from mild to severe, including fever, cough, diarrhea, and shortness of breath. although the exact route of transmission is still unclear, the respiratory droplet route is currently most likely [6] . although a few cases were reported in other countries, one was found in china in may 2015. a clinical research in combination with radiological findings was studied. a male korean patient, born in 1971, had a close contact with his father who was diagnosed as the middle east respiratory syndrome. he began to appear back pain on may 21, 2015, without fever, cough and sputum. no special treatment was given. he had a fever on may 25, 2015, with body temperature up to 39.7 c, no chills, no cough, sputum, no shortness of breath, no abdominal pain, no diarrhea, and no sore throat. cold medications were ineffective. on june 26 of 2015, he arrived in hong kong from south korea by flight at 12:50, and then arrived in huizhou city from shenzhen. at 2:00 pm, may 28 of 2015, isolation and treatment was given for him in huizhou designated hospital. during his whole journey in china, there were about 75 close contactors, who were all confirmed to be healthy after the isolated ward. body temperature was 39.0 c, blood pressure was 126/ 78 mm hg, heart rate was 98 beats/min, and breathing was 21 times/min. breath sounds were ruder for the two lungs without coarse rales, heart rate was regular, abdomen was soft, and bowel sounded normal. the blood gas analysis, biochemistry, and blood routine examination after hospitalization were shown in table 1 and table 2 . pathogenic examination (sputum virological detection) showed that mers-cov was positive on june 1 of 2015, and the result changed to negative the second day. mobile dr was implemented by employing the chest semirecumbent on may 28 of 2015 after hospitalization, followed by may 30th, june 1st, june 3rd, june 4th, june 6th and june 8th. obviously, pneumonia of the lower lungs was the primary finding ( fig. 1 ). patients was in fever after may 28, 2015 hospitalized, lasting about a week between 38.0 c and 39.5 c (fig. 2) . then with tamiflu and ribavirin antiviral therapy, broadspectrum antibiotic anti-infective therapy, oxygen therapy and improve immune function with gamma globulin, the virus was negative on june 2. low white blood cell count had been gradually increased to normal in two days after the virus was negative (fig. 3 ). according to 'cases of diagnosis and treatment of middle east respiratory syndrome (2015 edition)' by the national health development planning commission, he was discharged on june 26, 2015. it is well-known that incubation period of mers is 2e14 days. clinically, acute respiratory infection is the primary performance of mers, accompanying with high fever (even reach to 39e40 c), and sometimes with chills, shiver, cough, chest pain, headache, muscle and joint aches, fatigue, loss of appetite and so on. on the basis of pneumonia, the mers rapidly developed into respiratory failure, acute respiratory distress syndrome (ards) or acute renal failure. diarrhea and other atypical clinical manifestations might occur to individual cases (such as immunodeficiency cases). in this study, the patient suffered from fever (up to 39.5 c) and back pain firstly. after hospitalization for one week, his body temperature returned to normal (fig. 2) , but still being in cough with a small amount of yellow phlegm and a little bloodshot; and there is no shortness of breath at rest and oxygen therapy. on the sixth day after his hospitalization, mers-cov was negative via the virological detection of sputum, and his body temperature had decreased to be normal, which indicated that the virus has a direct relationship with the fever. laboratory tests found that the leukocytes count in the peripheral blood had decreased obviously since his hospitalization, and the count was about 3.00 â 10 9 /l lasting for nine days, followed by increasing and till the 27th days it recovered to the normal level. this indicated that mers-cov mainly attacked human immune system, resulting in a significant reduce of leukocytes count; after the virus was cleared, the recovery may be a relatively slow process due to the leukocytes count recovered to the normal range for about two weeks after the virus return to be negative. the camel and bat are always thought to be the main infection source, but the animal to human infection process was not so clear till now. generally, human to human infection should be paid more attention, for mers-cov would spread table 1 the blood gas analysis after hospitalization. results items results on the 7th day after hospitalization, the chest radiograph revealed the little patchy increased density shadow in the two lower lungs near the edge of heart. b. on the 10th day after hospitalization, the chest radiograph revealed two obviously increased patchy shadows in the two lower lungs, the degree of lower right lung was more serious. c. on the 13th day, the patchy lesions progressed to be large patchy consolidation shadows. d. on the 32nd day, chest radiograph showed two obvious absorptions in the lower lung lesions, only small pieces of the grid shadow could be observed. by direct contact of patients' secretions, or by aerosol and droplets. as is reported, there is an evidence of limited personto-person transmission of mers [7, 8] . in this study, 75 people who had more or less contact with the patient were without any further infection. this limited transmissibility is consistent with the data available to date [8] . radiological manifestations of mers are lungs consolidation and ground glass, due to the fact that the mers-cov primary lead to viral pneumonia [3] . imaging findings in this case were characteristic by the following three stages. 1) small pieces of high density shadows in the two lower lungs near the heart edge were observed during the early period via chest x-ray examination, suggesting that it firstly progressed to pneumonia (about one week). 2) subsequently, the lesions gradually expanded. further chest x-ray examination showed large pieces of high density shadows in the middle right lower lung, oval pieces of high density shadows in the left lower lung field, and clear boundaries of the two upper lungs. 3) after the active antiretroviral therapy, the virus turned to be negative, the body temperature decreased to normal level (fig. 2) , and leukocyte count also began to rise (fig. 3) . chest x-ray of deferred examination showed the gradually foci absorbed in both lungs, being consistence with clinical changes. differential diagnosis of pneumonia imaging result from mers, sars or h7n9 avian influenza is necessary. due to the fact that both sars and mers belong to the coronavirus family, their nucleotide homologies for one same pcr fragment are 70%e80%, their radiographic manifestations have in common. both showed ground glass shadows and pulmonary opacities in the middle and lower lung lobes, accompanying with a rapid disease progression. via high-resolution ct scans, septal thickening and bronchiectasis could be both observed. however, ground glass opacities and consolidation of sars were relatively mild, with the lesions of a migratory change [9e11]. as for mers and h7n9 avian influenza, their common characteristics were ground glass shadows and pulmonary opacities in both lower lung lobes, except for that disease progression of h7n9 avian influenza infection might be more rapid [10, 11] . summarily, differential imaging diagnosis on the basis of epidemiological and experimental pathogen detection is helpful for clinical diagnosis of mers, even in distinguishing from sars or pneumonia caused by h7n9 avian influenza. epidemiological findings from a retrospective investigation what can we learn from mers outbreak in south korea? isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mersdcov): an nouncement of the coronavirus study group middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings contact investigation of a case of human novel coronavirus infection treated in a german hospital chest x-ray imaging of patients with sars health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections characteristics of imaging manifestations and dynamic changes in patients with severe pneumonia caused by h7n9 avian influenza virus imaging and pathological analysis of severe pneumonia caused by human infections with avian influenza a (h7n9) relationship of peripheral blood leukocyte counts and viral negative key: cord-317403-1wrsuoy7 authors: yang, jeong-sun; park, sunghan; kim, you-jin; kang, hae ji; kim, hak; han, young woo; lee, han saem; kim, dae-won; kim, a-reum; heo, deok rim; kim, joo ae; kim, su jin; nam, jeong-gu; jung, hee-dong; cheong, hyang-min; kim, kisoon; lee, joo-shil; kim, sung soon title: middle east respiratory syndrome in 3 persons, south korea, 2015 date: 2015-11-17 journal: emerg infect dis doi: 10.3201/eid2111.151016 sha: doc_id: 317403 cord_uid: 1wrsuoy7 in may 2015, middle east respiratory syndrome coronavirus infection was laboratory confirmed in south korea. patients were a man who had visited the middle east, his wife, and a man who shared a hospital room with the index patient. rapid laboratory confirmation will facilitate subsequent prevention and control for imported cases. 18. a nasopharyngeal aspiration specimen was collected for mers-cov laboratory testing on may 19. sputum samples from 2 persons who had been in contact with the index patient were also tested for mers-cov. patient 2 was the 63-year-old wife of the index patient; she had had physical contact with him while caring for him during the 3 days of hospitalization. fever (38°c) and slight oliguria developed in patient 2 on may 19. the other contact, patient 3, was a 78-year-old man who had chronic obstructive pulmonary disease, asthma, and cholangiocarcinoma and who had shared a hospital room with the index patient and had been within 2 meters from him for 4 hours on may 16. fever (37.8°c) and respiratory symptoms developed in patient 3 on may 20. in the hospital room, the index patient did not undergo any aerosol-generating procedures, but a severe cough developed. the same health care workers cared for the index patient and patient 3. laboratory diagnostic methods were performed according to world health organization guidelines for molecular detection of mers-cov (7) (8) (9) . to check for contamination derived from the positive control, we designed and synthesized the mers-cov real-time reverse transcription pcr (rrt-pcr)-positive transcripts for an upstream mers-cov envelope protein gene (upe) and the open reading frame 1a (orf1a) gene containing 50 bp of a foreign gene (centipede). initially, nasopharyngeal samples from the index patient were positive for mers-cov by multiplex rrt-pcr. sputum samples from patients 2 and 3 were also positive, supporting a diagnosis of mers-cov infection. multiplex rrt-pcr results for upe and orf1a were positive (table 1) . according to rrt-pcr, the respiratory samples from the 3 patients were negative for 5 other human coronaviruses (sars-cov and human cov-229e, -oc43, -nl63, and -hku1) and 7 viruses that cause acute respiratory infection (influenza virus a and b; human adenovirus; bocavirus; human parainfluenza virus types 1, 2, and 3; respiratory syncytial virus a and b; human rhinovirus; human metapneumovirus). for the index patient, mers-cov rna was detectable in sputum, throat swab, and serum samples but not in a urine sample collected 9 days after symptom onset ( table 1) . the viral load, indicated by cycle threshold values, was high in the lower respiratory tract sample but almost undetectable in the throat swab and serum samples. after sequential sampling repeated every 2-5 days, mers-cov rna was detected in sputum until 44 days after symptom onset, although viral rna was inconsistently detected and patterns of viral load fluctuated ( 2). other than initial fever (>37°c), clinical features differed for all 3 patients. the index patient had respiratory symptoms with cough, dyspnea, and myalgia. patient 2 did not have a relevant medical history and showed mild symptoms. patient 3 had underlying concurrent conditions and died 16 days after confirmation of mers-cov infection ( figure 1 ). virus isolation on vero cells was attempted for each respiratory specimen from the 3 patients. the culture supernatant after inoculation was serially assessed for virus growth by using rrt-pcr and was used for blind passages every 3-7 days after inoculation. after 3 blind passages, cytopathic effect was observed, and we isolated the mers-cov strain from south korea (kor/knih/002_05_2015) from the vero cells after inoculation by using the sputum from patient 2. we constructed a phylogenetic tree by using the general time reversible plus gamma model of the raxml version 8.8.0 software (10) and figtree version 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree) and by using complete genomes of the mers-cov isolate from south korea (genbank accession no. kt029139) and 67 reference mers-covs (figure 2 ). because, to our knowledge, cases of mers-cov infection in south korea have not been reported, we had to establish laboratory testing protocols to overcome vulnerabilities in the absence of appropriate epidemiologic support (i.e., generate positive controls to check for contamination and repeat testing). positive controls containing foreign genes have been generated to check for laboratory contamination. for patients with an unclear exposure history (such as the index patient) and for patients with short exposure durations and unusual clinical symptoms (such as patients 2 and 3), it would be useful if the positive results of rrt-pcr could be confirmed through agarose gel electrophoresis to exclude contamination from the positive control (3, 4) . the index patient had no history of potential exposure to camels, bats, or their excreta; to symptomatic persons; or to health care workers during his trip to the middle east, including saudi arabia. although the source of infection for the index patient is unclear, phylogenetic analysis of the whole viral genome showed that the isolate from south korea was closely related to the mers-cov strains isolated in saudi arabia in 2015. (8, 9) . ksa, kingdom of saudi arabia; uae, united arab emirates. because the index patient initially concealed his travel history to saudi arabia, united arab emirates, and qatar, mers-cov infection was not considered and the patient was not isolated until mers-cov infection was suspected 7 days after symptom onset. meanwhile, other patients and health care workers had multiple opportunities for exposure to the index patient (3) (4) (5) . the 2 contacts reported here had each been exposed to the index patient. patient 3 was probably infected via droplet transmission in the hospital room. the hospital room, originally built for 6 persons, had been divided into 2 rooms and lacked ventilation. furthermore, an air conditioning unit cycled the air in the room with the door and window closed. thus, poor ventilation might have played a major role in droplet transmission. detection of mers-cov rna in the respiratory tract varies up to day 33 (11) (12) (13) . in this study, virus was detected in the respiratory tract, inconsistently, for up to 44 days. development of effective preventive measures for the mers-cov prevention will require systemic and prospective studies associated with viral shedding and use of specimens in addition to those obtained from the respiratory tract to define the kinetics of mers-cov. rapid detection of mers-cov, using multiplex rrt-pcr to detect upe and orf1a genes, would be helpful for countries outside the arabian peninsula. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. emergencies preparedness, response: middle east respiratory syndrome coronavirus (mers-cov) maps and epicurves korean society for healthcare-associated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea preliminary epidemiological assessment of mers-cov outbreak in south korea probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea south korea): the centers world health organization. laboratory testing for middle east respiratory syndrome coronavirus. interim recommendations (revised) detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases address for correspondence: sung soon kim, kcdc, division of respiratory viruses phylogenetic tree comparing complete genome nucleotide sequences of middle east respiratory syndrome coronavirus (mers-cov) isolate from south korea (kor/ knih/002_05_2015) with those of 67 reference mers-covs (genbank database). the tree was constructed by using the general time reversible plus gamma model of raxml version 8.8.0 software (10) and visualized by using figtree version 1 we thank the division of epidemic intelligence service and national medical center for assistance with identifying patients, abstracting medical records, and collecting specimens. the public health image library (phil), centers for disease control and prevention, contains thousands of public healthrelated images, including high-resolution (print quality) photographs, illustrations, and videos. key: cord-315909-vwugf0wp authors: letko, michael; munster, vincent title: studying evolutionary adaptation of mers-cov date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_1 sha: doc_id: 315909 cord_uid: vwugf0wp forced viral adaptation is a powerful technique employed to study the ways viruses may overcome various selective pressures that reduce viral replication. here, we describe methods for in vitro serial passaging of middle east respiratory syndrome coronavirus (mers-cov) to select for mutations which increase replication on semi-permissive cell lines as described in letko et al., cell rep 24, 1730–1737, 2018. rna viruses are ideal model organisms to study evolutionary genetics under selection. this is due to their large population sizes and short generation times, which are characterized by rapid accumulation of mutations relative to other organisms. given the error-prone nature of viral rna-dependent rna polymerases, viral replication leads to the formation of a quasispecies [1] [2] [3] . rather than one virus producing identical progeny during replication, a population of viruses is produced, each differing from one another by nucleotide substitutions or deletions as a result of errors incorporated by the rna polymerase. while the majority of these mutations will have neutral or negative effects on viral fitness, a small subset of these mutations may prove beneficial and enhance the ability for certain variants to replicate despite selective pressures of interest such as the host immune response or an antiviral drug. forced adaptation experiments have been used to determine viral mutations that facilitate escape from drugs [4] [5] [6] , monoclonal antibodies [7, 8] , host restriction factors [9] [10] [11] , and species variation in host receptors [12] [13] [14] and to elucidate various viral mechanisms of infection and replication [15] [16] [17] . within the laboratory setting, the strength of selective pressure can be adjusted by increasing or decreasing the levels of the restrictive factor, thus facilitating the rapid expansion of viral variants within the population of quasispecies that can overcome the applied selective pressure. the ideal environment is "semi-permissive"-allowing only low levels of wild-type virus replication. below is the method employed to adapt mers-cov to a semi-permissive host receptor, desmodus rotundus dpp4. the techniques described below could be applicable to a wide range of experiments to better understand the adaptive capacity of various coronaviruses under specific selective pressures. 2.1 cell culture 1. semi-permissive cells: baby hamster kidney (bhk) cells which have been transduced to stably express desmodus rotundus dpp4 (drdpp4 [12] . briefly, the coding sequence for drdpp4 was cloned into a lentiviral expression cassette also encoding for mcherry-t2a-puromycin-n-acetyltransferase-p2a (system biosciences) and used to generate lentiviral particles [9] (see note 1). bhk cells were infected with lentiviral particles and then grown in dmem containing puromycin at a final concentration of 1 ug/ml. 2. superscript iv reverse transcriptase cdna production kit. 3. iproof high-fidelity pcr kit. 4. agarose gel purification kit. spike receptor-binding domain sequencing primers (see table 1 ). 6. sequence analysis software capable of multiple sequence alignment and viewing chromatograms. 1. plan number of conditions. at least three replicates (well of semi-permissive cells) for each forced adaptation experiment should be performed in parallel. critically, parental cells or a cell line stably expressing an irrelevant protein should be included to control for any nonspecific cell culture mutations. 2. grow semi-permissive bhk cells to confluency in appropriate format. one 75 cm 2 flask should be sufficient to seed at least three 6-well cluster plates. 3. wash, trypsinize, count, and seed bhk cell lines (parental controls and semi-permissive) in cell culture media (10% fbs) at a density of 1.5 â 10 5 cells/ml in a 2 ml volume in each well of 6-well plates (see note 2). 3.2 infect cells 1. twenty-four hours later, replace media on seeded cell lines with 2 ml of fresh passaging culture media (2% fbs). 2. infect cells with mers-cov/emc2012 at a final moi of 0.01 (fig. 1 ). 3. after 72 h postinfection for previous culture, take a 500 μl of supernatant sample from the infected culture and store for downstream viral sequencing. store supernatants at à80 c. 2. generate cdna from extracted rna using superscript iv, following manufacturer's instructions. 3. amplify select regions from viral cdna using iproof highfidelity pcr polymerase kit (bio-rad). below are example pcr conditions for amplifying the mers-cov receptor-binding domain following the primer numbers listed in subheading 2.2.5 of [12] (see table 2 ). 6. check sanger sequencing chromatograms for overlapping peaks, indicative of mutations within a mixed viral population, as further described in [12] 4 notes 1. importantly, this specific lentivector cassette is expressed under the ef1α promoter, which allows for mid-level expression of the transgene as compared to other popular lentiviral transgene promoters such as cmv or caggs. this midlevel expression is ideal for semi-permissive selective pressure created by the transgene, in this case, drdpp4. 2. the plating density of cells may vary from this suggested value, depending on growth kinetics. in general, cells should be plated to achieve approximately 80-90% confluency on the day of infection. 3. cytopathic effects may be gradual to appear. to increase selective pressures on a viral population which is beginning to show signs of adaptation, one can apply a population bottleneck in the subsequent passage by reducing the amount of viral evolution of mers-cov supernatant passaged to the next cell culture. in this case, we recommend reducing the passage volume by approximately tenfold. 4. in our initial study [12] , cytopathic effects were observed by the eighth passage; however, sequencing from earlier passages showed adaptive mutations emerging in the culture by the third passage. depending on the strength of selection, the number of passages required to elicit adaptive mutations will vary. viral quasispecies viral quasispecies evolution quasispecies theory and the behavior of rna viruses in vitro selection of mutations in the human immunodeficiency virus type 1 reverse transcriptase that decrease susceptibility to (à)-beta-d-dioxolane-guanosine and suppress resistance to 3 0 -azido-3-0 -deoxythymidine in vitro selection of mutations in human immunodeficiency virus type 1 reverse transcriptase that confer resistance to capravirine, a novel nonnucleoside reverse transcriptase inhibitor in vitro passaging of a pandemic h1n1/09 virus selects for viruses with neuraminidase mutations conferring high-level resistance to oseltamivir and peramivir, but not to zanamivir impact of v2 mutations on escape from a potent neutralizing anti-v3 monoclonal antibody during in vitro selection of a primary human immunodeficiency virus type 1 isolate two escape mechanisms of influenza avirus to a broadly neutralizing stalkbinding antibody identification of the hiv-1 vif and human apobec3g protein interface hiv-1 vif adaptation to human apobec3h haplotypes the structural interface between hiv-1 vif and human apobec3h adaptive evolution of mers-cov to species variation in dpp4 persistent infection promotes cross-species transmissibility of mouse hepatitis virus amino acid substitutions in the s2 subunit of mouse hepatitis virus variant v51 encode determinants of host range expansion forced virus evolution reveals functional crosstalk between the disulfide bonded region and membrane proximal ectodomain region of hiv-1 gp41 optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution hiv-1 can escape from rna interference by evolving an alternative structure in its rna genome host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 funding was provided by the intramural research program of the niaid. key: cord-297062-dmiplvt2 authors: almekhlafi, ghaleb a.; albarrak, mohammed m.; mandourah, yasser; hassan, sahar; alwan, abid; abudayah, abdullah; altayyar, sultan; mustafa, mohamed; aldaghestani, tareef; alghamedi, adnan; talag, ali; malik, muhammad k.; omrani, ali s.; sakr, yasser title: presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients date: 2016-05-07 journal: crit care doi: 10.1186/s13054-016-1303-8 sha: doc_id: 297062 cord_uid: dmiplvt2 background: middle east respiratory syndrome coronavirus infection is associated with high mortality rates but limited clinical data have been reported. we describe the clinical features and outcomes of patients admitted to an intensive care unit (icu) with middle east respiratory syndrome coronavirus (mers-cov) infection. methods: retrospective analysis of data from all adult (>18 years old) patients admitted to our 20-bed mixed icu with middle east respiratory syndrome coronavirus infection between october 1, 2012 and may 31, 2014. diagnosis was confirmed in all patients using real-time reverse transcription polymerase chain reaction on respiratory samples. results: during the observation period, 31 patients were admitted with mers-cov infection (mean age 59 ± 20 years, 22 [71 %] males). cough and tachypnea were reported in all patients; 22 (77.4 %) patients had bilateral pulmonary infiltrates. invasive mechanical ventilation was applied in 27 (87.1 %) and vasopressor therapy in 25 (80.6 %) patients during the intensive care unit stay. twenty-three (74.2 %) patients died in the icu. nonsurvivors were older, had greater apache ii and sofa scores on admission, and were more likely to have received invasive mechanical ventilation and vasopressor therapy. after adjustment for the severity of illness and the degree of organ dysfunction, the need for vasopressors was an independent risk factor for death in the icu (odds ratio = 18.33, 95 % confidence interval: 1.11–302.1, p = 0.04). conclusions: mers-cov infection requiring admission to the icu is associated with high morbidity and mortality. the need for vasopressor therapy is the main risk factor for death in these patients. electronic supplementary material: the online version of this article (doi:10.1186/s13054-016-1303-8) contains supplementary material, which is available to authorized users. middle east respiratory syndrome coronavirus (mers-cov) is a novel betacoronavirus that was first reported in september 2012 [1] . by january 6, 2016, a total of 1626 laboratory-confirmed cases of infection with mers-cov, including at least 586 related deaths, had been reported to the world health organization [2] . although mers-cov infections have been reported from 26 countries around the world, the majority of cases have originated in saudi arabia, south korea, the united arab emirates, jordan and qatar [3] . interhuman mers-cov transmission occurs in community and healthcare settings [4] [5] [6] [7] [8] . the exact source and mode of transmission of mers-cov to humans remains uncertain. however, mers-cov circulates among dromedary camels in africa and the middle east with occasions of documented camel-human inter-transmission [9] . there have been several reports outlining the clinical features and outcomes of patients with mers-cov infection [7, [10] [11] [12] [13] [14] [15] . however, very few have focused on critically ill patients in intensive care units (icu) [16] [17] [18] . there is therefore a need for more data to understand the various clinical and prognostic aspects of this potentially lethal disease, particularly for the most severe cases that require admission to the icu. we performed a retrospective study to describe the clinical features and outcomes of patients admitted to our icu with laboratory-confirmed mers-cov infection. the study was approved by the institutional review board of prince sultan military medical city (11159 riyadh, saudi arabia), a large tertiary-care referral center in riyadh, saudi arabia. informed consent was waived due to the retrospective, anonymous nature of data collection. we included all patients aged 18 years or more with confirmed mers-cov infection who were admitted to our 20-bed mixed medico-surgical icu between october 1, 2012 and may 31, 2014. all icu patients with a confirmed diagnosis of mers-cov were registered in a special logbook. for the purpose of the current study, all patients' records were reviewed by a senior intensivist (s. hussain, a. alwan, a. abudayah, s. altayyar, m. mustafa, t. aldaghestani, a. alghamedi, a. talag or m. malik). clinical data and laboratory parameters from confirmed cases of mers-cov were transcribed onto specially developed case record forms. these included the initial manifestations of respiratory infection, the clinical picture on admission to the icu, laboratory indices of organ failure, radiographic findings, interventions during the icu stay, treatment modalities, and final outcome. the acute physiology and chronic health evaluation ii (apache ii) score was calculated from the data obtained within 24 hours of admission to the icu [19] . the sequential organ failure assessment (sofa), score, calculated daily by the physician in charge of the patient, was also noted [20] . since the first reported cases of mers-cov in saudi arabia in september 2012, all suspected cases in our institution are strictly isolated and nasopharyngeal swabs are obtained for initial screening. deep respiratory samples (tracheal aspirates or bronchoalveolar lavage fluid) are obtained from all patients admitted to the icu with suspected respiratory infections, in addition to blood samples to perform cultures and polymerase chain reaction for common respiratory viruses and atypical microorganisms. urinary samples were obtained to detect legionella antigens in only two patients (legionella infections are not common in saudi patients). cultures of tracheal aspirates are analyzed quantitatively and bacterial counts of at least 10 5 colony-forming units are considered positive. these investigations are repeated in the icu whenever secondary infections are suspected. clinical specimens aimed at detecting possible mers-cov infection are processed and analyzed at the national reference laboratory of the saudi ministry of health. mers-cov infections are identified using real-time, reverse transcription polymerase chain reaction (rt-pcr). the standard assays target amplifications of the upstream e protein (upe gene) and open reading frame (orf)1a; both need to be positive to confirm infection, otherwise another sample is required to confirm the diagnosis [21] . the sample requires 2 days of processing for the final results to be available. routine laboratory testing in our icu includes complete blood counts, coagulation profile, electrolytes, renal function, liver profile and arterial blood gases. these parameters are measured on admission to the icu and at least once daily thereafter (at 6:00 am) throughout the icu stay. all patients with suspected or confirmed mers-cov infection were isolated in single rooms, either on the hospital floor or in the icu. patients were admitted to the icu according to the guidelines of the society of critical care medicine for icu admission, discharge, and triage [22] . patients were classified into four categories according to their icu admission priority: priority one comprised critically ill patients who were unstable and need intensive treatment and monitoring, with significant likelihood of recovery; priority two were stable patients who required intensive monitoring because of the possibility of decompensation; priority three were unstable patients who had a low likelihood of recovery because of the severity of acute disease or because of comorbidities; priority four were those who had little or no anticipated benefit from icu admission. patients classified as priority one and two and most of those classified as priority three were admitted to our icu or full critical care services were mobilized and provided for in the isolation ward until a bed was available in the icu. priority four patients were not admitted to the icu and remained in the isolation ward. general ward patients with mers-cov infection were transferred to the icu if their condition deteriorated or organ failure developed. the infection control precautions recommended by the saudi ministry of health guidelines were strictly implemented to prevent possible transmission of mers-cov to other patients or to the healthcare staff [23] . supportive treatment was provided according to our standard operating procedures and in accordance with the surviving sepsis campaign guidelines [24, 25] . antiviral therapies, such as oseltamivir, and ribavirin/interferon alfa-2a, were prescribed at the discretion of the attending physician. protective lung ventilation was applied in mechanically ventilated patients. prone positioning was considered in some patients with severe refractory hypoxemia. extracorporeal membrane oxygenation (ecmo) and high-frequency oscillation were also available as a last resort, when considered necessary by the attending physician. statistical analyses were performed using spss statistics 19 for windows (ibm corp., armonk, ny, usa). the kolmogorov-smirnov test was used to verify whether there were significant deviations from the normality assumption of continuous variables. nonparametric tests of comparison were used for variables evaluated as not normally distributed. difference testing between groups was performed using student's t test, mann-whitney test, chi-square test and fisher's exact test, as appropriate. friedman's test was used to assess the time course of organ function. to identify the risk factors for death in the icu, we performed multivariable logistic regression analyses. due to the relatively small number of deaths in our study, we adjusted only for the severity of illness on admission to the icu (apache ii score) and the degree of organ dysfunction as assessed by admission sofa score. potential risk factors for icu mortality were selected among the demographic characteristics, comorbidities, mode of acquisition of mers-cov, initial manifestations, procedures and therapies, and superimposing infections. variables yielding p <0.2 in the univariate analysis, apa-che ii score and sofa score were included in a multivariable logistic regression analysis. these variables were introduced separately into multivariable models including apache ii and sofa scores on admission to the icu. adjusted odds ratios (or) and 95 % confidence of interval (ci) were computed. none of the covariates simultaneously introduced in a multivariable model were collinear. data are presented as mean ± standard deviation (sd), median value (25th-75th interquartile range [iqr]) or number (%), as appropriate. all statistics were two-tailed and a p < 0.05 was considered statistically significant. during the observation period, 70 cases with confirmed mers-cov infections were diagnosed in our institution [11] (fig. 1) ; 21 patients were managed in the hospital ward, 18 patients were admitted to other icus or received critical care service in the ward, and 31 patients were admitted to our icu (12 between october 1, 2012 and december 31, 2013 and 19 between january 1 and may 31, 2014). patients were admitted to our icu because of respiratory failure (pao2/fio2 < 250 mmhg). the mean age of the patients admitted to our icu (n = 31) was 59 (sd 20) years and 22 (71 %) were males. the characteristics of these patients on admission to the icu are shown in table 1 . eighteen (58.1 %) patients had community-acquired mers-cov infection, while for 13 (47.9 %), including two healthcare staff, infection was acquired in the hospital. twenty-seven patients (87.1 %) had at least one comorbidity. the median number of concomitant comorbid conditions was three (iqr: 2-4). initial clinical manifestations had occurred at a median of 2 days (iqr: 2-4) prior to hospital admission. patients had been treated for a median of 5 days (iqr: 2-9) in general hospital wards before their admission to the icu. only four patients (12.9 %) were admitted to the icu on arrival at the hospital. cough and tachypnea were reported in all patients. other common initial symptoms were fever (87.1 %), abdominal pain (29 %), sore throat (25.8 %), and fatigue (25.8 %) (additional file 1). crackles (93.5 %), tachycardia (67.7 %), and rhonchi (32.3 %) were the most commonly identified initial physical signs. bilateral pulmonary infiltrates were present in the chest x-rays of 24 (77.4 %) patients and lobar infiltrates in six (19.4 %). only one patient had a normal chest x-ray at the time of admission to the icu. on admission to the icu, no patients had microbiologically proven co-existing bacterial pneumonia. secondary infections, as evident from positive quantitative cultures of deep tracheal aspirates, occurred in 18 (58.1) patients within a median of 3 days (iqr: 3-8) after admission to the icu. the most commonly isolated microorganisms were acinetobacter baumannii (25.8 %), only four (12.9 %) patients had positive blood cultures; acinetobacter baumannii (n = 2), escherichia coli (n = 1), methicillin-resistant staphylococcus aureus (n = 1), and vancomycin-resistant enterococcus species (n = 1). invasive mechanical ventilation was applied in 27 (87.1 %) patients during the icu stay; 18 (58.1 %) within 24 hours of admission to the icu, and 14 (45.5 %) patients received noninvasive ventilation (table 2) . eleven (35.5 %) patients were treated with high-frequency oscillation and five (16.1 %) with prone positioning. only one patient received ecmo. the ventilatory parameters are presented in additional file 3. vasopressor therapy using norepinephrine was initiated in 25 (80.6 %) patients (table 2) . oseltamivir was administered to 20 (64.5 %) patients for a median of 5 days (iqr: 3-5). combined ribavirin plus interferon alfa-2a therapy was used in 13 (41.9 %) patients ( table 2 ). all patients received at least one antimicrobial agent during the icu stay (additional file 2). antifungal therapy was only used in four of the five patients with positive cultures for candida but the necessity of this therapy is uncertain. the overall icu mortality rate was 74.2 % (n = 23). the median icu and hospital lengths of stay were 9 (iqr: 4-16) and 12 (iqr: 4-16) days, respectively. the major causes of death were hypoxemic respiratory failure (52.2 %) and refractory septic shock (26.1 %). one patient died from sudden cardiac arrest after icu discharge but while still in the hospital. furthermore, one patient died within 1 year after discharge from the icu because of septic shock related to an infected wound. only one patient was lost to follow-up after hospital discharge. the sofa score and glasgow coma scale (gcs) increased markedly over the first 2 weeks in the icu in the whole cohort, while other parameters of organ function remained largely unchanged (additional file 3). compared with those who were discharged alive from the icu, nonsurvivors were older, had higher apache ii and sofa scores on admission to the icu, and were more likely to require invasive mechanical ventilation and vasopressor therapy and to have been ventilated using highfrequency oscillation (tables 1 and 2 , and additional files 1 and 2). nonsurvivors had a persistently low pao2/fio 2 throughout the first 2 weeks in the icu, whereas survivors showed a slight increase over time (fig. 2) . after adjustment for the severity of illness and the degree of organ dysfunction, the need for vasopressors was the only independent risk factor for death in the icu (or 18.33, 95 % confidence interval 1.11-302.1, p 0.04) (additional file 4). the 31 critically ill patients with confirmed mers-cov infection in our cohort frequently had organ failure with an overall mortality rate greater than 74 %. comorbidities were common in this cohort of patients. not surprisingly, mortality in the icu was associated with older age, severe disease and organ failure. the need for vasopressor therapy was an independent risk factor of death in the icu. since the first reported case of mers-cov infection in 2012, several authors have described various cohorts of patients with this serious infection [8, 10-12, 14, 15, 26] . [16, 18] . our facility is a large tertiarycare medical center in riyadh, central saudi arabia. we herein provide a detailed account of the largest single cohort of critically ill mers-cov infected patients reported thus far. in agreement with previous reports from saudi arabia, comorbid conditions were common in our patients with mers-cov infections with a median of three comorbidities per patient [10, 11, 15, 17] . in contrast, only 54.8 % of the 186 individuals involved in the recent mers-cov outbreak in south korea had any preexisting chronic medical conditions [8] . however, only 29.6 % of patients in the korean outbreak were aged 65 years or older and nearly half (46.2 %) were caregivers or healthcare personnel [8] . the differences in the demographic characteristics of our cohorts and the mode of acquisition of mers-cov infection may explain, at least in part, the discrepancy in the patterns of associated comorbidities between the saudi and korean cohorts. the respiratory manifestations of mers-cov infection in our cohort were similar to those observed in previous reports from saudi patients [10, 11, 14, 15, 17] . cough and tachypnea occurred in all patients and 77 % of cases had bilateral pulmonary infiltrates, denoting severe respiratory illness, which required a median of 5 days to reach the peak of clinical deterioration such that icu admission and organ support therapy were required. gastrointestinal manifestations, such as abdominal pain, diarrhea, vomiting, and abdominal tenderness, were relatively common in our cohort. this was also a common finding in the previous literature in patients with mers-cov infection as well as those with severe acute respiratory syndrome (sars) [10, 11, 15, 17, 28, 29] . our data confirm previous studies that reported a high prevalence of nonrespiratory organ failure in critically ill patients with mers-cov [16, 17] . the mechanisms of organ dysfunction and failure in these patients are yet to be determined. cytokine dysregulation has been suggested to be involved in the pathophysiology of mers-cov-related organ failure. direct viral invasion may also occur as the virus was recovered from urine and stool in one patient [30] . in agreement with the results of the previous reports on critically ill patients with mers-cov infection [16] [17] [18] , more than 80 % of our patients received vasopressor support, underscoring the high prevalence of cardiovascular dysfunction in these patients, and suggesting that disturbances in tissue perfusion may also have been involved in the pathophysiology of the organ failure. lower rates of vasopressor support have been reported in patients with sars [10, 11, 15, 17, 28, 29] with, as a result, lower mortality rates than those reported in patients with mers-cov infections. even though overall mortality rate was high in our cohort, it is still comparable with rates reported in previous studies (58.3-64.3 %) [16] [17] [18] . in all studies, almost all patients had significant comorbidities and median apache ii scores of 25 or higher. we observed significantly higher apache ii and sofa scores in icu nonsurvivors compared to those who survived severe mers-cov infection, underscoring the strong association between mortality and the severity of disease. epidemiological analyses have suggested that mers-cov is unlikely to trigger sustained human epidemics at present [31, 32] . nevertheless, nosocomial outbreaks have resulted in considerable morbidity and mortality, in addition to disruption of medical services and substantial economic losses [9, 33, 34] . the most severe infections usually require icu admission, necessitate major resource utilization and result in high fatality rates. identifying possible risk factors for poor prognosis in patients with mers-cov infection is therefore crucial to enable appropriate allocation of healthcare resources and early transfer of high-risk patients to the appropriate medical facilities. our data show that the need for vasopressor therapy was an independent risk factor for death in the icu. indeed, the major causes of death in our study were hypoxemic respiratory failure and refractory septic shock, which confirm the role of respiratory and cardiovascular system failures as determinants of outcome in this population. this was also evident from the persistent hypoxemia observed in the nonsurvivors. to date, published data on the risk factors for poor prognosis specific to critically ill patients with mers-cov infection are lacking. in cohort studies of patients with any degree of severity of mers-cov infection, older age, diabetes, chronic renal failure, chronic respiratory disease, high viral load in lower respiratory tract samples, shorter incubation period and mers-cov viremia have all identified as independent predictors of mortality [11, 14, [35] [36] [37] . secondary respiratory infections occurred commonly in this cohort, predominantly with gram-negative bacteria. although candida species were frequently isolated, these are probably not relevant as respiratory pathogens and the necessity of antifungal therapy is uncertain. interestingly, acinetobacter baumannii, which is an emerging fatal infection in icu patients worldwide, was isolated from deep tracheal aspirates in one in four patients. this may explain, at least in part, the relatively high mortality rates in this cohort. specific therapeutic options for mer-cov infections are limited and their efficacy is not well established [38] . all patients in this report received antiviral treatment with either oseltamivir or combined ribavirin/interferon alfa-2a therapy; two patients received both. although a previous study from the same institution showed that combined ribavirin/interferon alfa-2a therapy was associated with significant improvement in survival at 14 days, this benefit was not maintained at 28 days after the onset of the disease [39] . the retrospective and observational nature of this study does not allow precise assessment of the efficacy of these therapies. in the absence of a vaccine or a specific treatment, prevention of viral transmission through adequate infection control methods is the mainstay in the management of mers-cov outbreaks. appropriate isolation of patients with suspected or proven infections is crucial. in view of the high fatality rates of these patients in the icu, it may be reasonable to closely monitor patients with suspected infections in the general wards for early signs of organ dysfunction to prevent unnecessary delay in the provision of intensive care services and reduce mortality rates in these patients. our study has some limitations. we included patients with confirmed mers-cov infection from one icu of a large medical center. possible variations in the geographic distribution of the disease and in local practice may hinder extrapolation of these data to other cohorts in saudi arabia and other countries. the relatively low number of patients may have biased the statistical comparisons presented in this report and overestimated mortality rates. multivariable adjustment was also limited to the variables included in the models. collaborative efforts are needed to provide an insight into the risk factors for poor prognosis in these patients. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov european centre for disease prevention and control. epidemiological update: middle east respiratory syndrome coronavirus ?id=1278&list=8db7286c-fe2d-476c-9133-18ff4cb1b568&source=http%3a%2f%2fecdc%2eeuropa%2eeu%2fen% 2fpress%2fepidemiological%5fupdates%2fpages%2fepidemiological%5f updates%2easpx. accessed 23 a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus (mers-cov): what lessons can we learn? epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards apache ii: a severity of disease classification system the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. on behalf of the working group on sepsis-related problems of the european society of intensive care medicine assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections guidelines for intensive care unit admission, discharge, and triage. task force of the american college of critical care medicine, society of critical care medicine infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012 multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd middle east respiratory syndrome in the shadow of ebola middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart spread of mers to south korea and china mers coronavirus: diagnostics, epidemiology and transmission mortality risk factors for middle east respiratory syndrome outbreak, south korea association of higher mers-cov virus load with severe disease and death, saudi arabia association between severity of mers-cov infection and incubation period therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) infection: how close are we? curr treat options infect dis ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study we would like to thank dr. hassane nijimi (free university of brussels) for the statistical revision of this study and dr. karen pickett for editorial assistance with the manuscript. the study was supported only by institutional funds. mers-cov infections requiring admission to the icu are associated with high morbidity and mortality rates. the need for vasopressor therapy is the main risk factor for death in these patients. this report describes the clinical features and outcomes of 31critically ill patients with confirmed middle east respiratory syndrome coronavirus (mers-cov) infection. patients with mers-cov infections frequently had organ failure, and mortality rates were greater than 72 %. the need for vasopressor therapy was an independent risk factor for death in the icu. the authors declare that they have no competing interests.authors' contributions gaa, ym, aso, mma, and ys conceived the study. sh, aal, aab, sa, mm, ta, aalg, at, and mkm participated in data collection. ys processed the data and performed the statistical analyses. ys and gaa drafted the manuscript. all authors read, revised, and approved the final manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-307995-8q7efrqk authors: al-tawfiq, jaffar a.; omrani, ali s.; memish, ziad a. title: middle east respiratory syndrome coronavirus: current situation and travel-associated concerns date: 2016-05-04 journal: front med doi: 10.1007/s11684-016-0446-y sha: doc_id: 307995 cord_uid: 8q7efrqk the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (sars-cov) in 2002. more than 1500 mers-cov cases were recorded in 42 months with a case fatality rate (cfr) of 40%. meanwhile, 8000 cases of sars-cov were confirmed in six months with a cfr of 10%. the clinical presentation of mers-cov ranges from mild and non-specific presentation to progressive and severe pneumonia. no predictive signs or symptoms exist to differentiate mers-cov from community-acquired pneumonia in hospitalized patients. an apparent heterogeneity was observed in transmission. most mers-cov cases were secondary to large outbreaks in healthcare settings. these cases were secondary to community-acquired cases, which may also cause family outbreaks. travel-associated mers infection remains low. however, the virus exhibited a clear tendency to cause large outbreaks outside the arabian peninsula as exemplified by the outbreak in the republic of korea. in this review, we summarize the current knowledge about mers-cov and highlight travel-related issues. the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (sars-cov) in 2002 [1, 2] . as of march 17, 2016 , 1715 mers-cov cases were confirmed with a case fatality rate (cfr) of approximately 40%, whereas 8000 sars-cov cases were recorded in six months with a 10% cfr [1] [2] [3] . an upsurge in the number of cases was observed in march-may 2014 because of the outbreak in large healthcare facilities in jeddah, kingdom of saudi arabia (ksa) [4, 5] . more recently, a larger outbreak occurred in riyadh, ksa and in multiple hospitals in south korea [6] [7] [8] [9] . wolfe et al. [10] described the five stages of pathogen evolution that lead to diseases confined to humans. in stage 1, the pathogen is confined to an animal host. humans become infected by animals only in stage 2. stage 3 is limited human-to-human transmission. in stage 4, long outbreaks with numerous cycles of human-to-human transmission occur. in stage 5, the pathogen exclusively infects humans [10] . mers-cov has not yet reached stage 5. the interest and concern of the public and the health community in emerging infectious diseases stem from the ability of the emerging pathogens to cause pandemics with high fatality rates and the associated economic effects on affected countries. for example, sars-cov caused $30-50 billions of economic losses in mainland and hong kong of china, singapore, and canada, and the pandemic influenza h1n1 in 2009 resulted in $45-55 billion losses worldwide [11] . in this review, we focus on the important aspects of the recently emerged mers-cov, its effects on humans, and the transmission patterns of the disease based on available scientific data. year-old male from bisha [12, 13] . the patient was hospitalized with community-acquired pneumonia; the disease rapidly progressed, resulting in acute renal failure, respiratory failure, and death [13] . a summary of the major events in the development of the mers-cov epidemic is shown in fig.1 . since the first case of mers-cov, a detailed investigation was conducted to determine the environmental and animal source of the virus. extensive contact investigation was carried out on the first case that originated from bisha but was eventually hospitalized in a private hospital in jeddah [12, 14] . the contact investigation started in the patient's hometown of bisha in southern ksa. the investigation included his immediate household contacts (3 wives, 10 sons, 11 daughters, 12 grandchildren, and 1 house maid), as well as the community and healthcare facility in bisha, which included 2 shepherds who took care of his five camels and 14 healthcare workers (hcws) (11 nurses and three physicians) [14] . in total, 53 contacts in bisha were screened and all of them tested negative for mers-cov by polymerase chain reaction (pcr) [14] . an extensive investigation of the 48 out of 56 hcw who had significant contact with the same patient during his 10-day stay in the private hospital in jeddah tested negative [15] . during the october 2012 investigation on the source of the virus, a team representing three agencies, namely, saudi ministry of health, center for infection and immunity of columbia university, and ecohealth alliance, interviewed the family of the index case-patient from bisha. the team also collected samples from bats within 12 km from his home, as well as an abandoned date palm orchard, the area within 1 km from his place of employment, a hardware store that fronted his garden, and a date palm orchard. although none of his family members nor employees recalled seeing bats, the investigating team observed the roosting bats and guano in abandoned wells and ruins within 12 km of his home and insectivorous bats at dusk in the garden behind his store. a sample from a taphozous perforatus bat (egyptian tomb bat) captured in bisha showed 100% identity to the human β-cov 2c emc/2012 cloned from the index case-patient [16] . the largest data set on the contact investigation revealed that the percentage of positive cases was 2.5%, 1.12%, 3.03%, and 2.09% among hospital patients, hcw contacts, family and contacts, and overall [17] . with the expansion of testing to identify the full disease spectrum and the inclusion by the world health organization (who) of the positive cases by serology in july 2014 [18] , the cfr gradually decreased from 60% to 40% as more asymptomatic and mildly symptomatic cases were included [19] . a nationwide serosurvey for mers-cov conducted in the ksa between december 2012 and december 2013 suggested that around 45 000 infected individuals were not aware of their infection, which confirmed an extremely high incidence of asymptomatic to mildly symptomatic disease [20] . a good example of a family cluster of mers-cov infections was published in the new england journal of medicine [21] . once the second case in that cluster was identified, an epidemiologic investigation identified the index case who was the father of the second case who was admitted earlier with congestive heart failure and community-acquired pneumonia. subsequently, the third and fourth cases where recognized [21] . in that cluster, 28 persons lived in the same extended household, including nine children ( < 14 years of age) [21] . aside from the four patients, no other family members had major respiratory symptoms and none of the 124 hcws who managed the index case before the mers-cov diagnosis exhibited symptoms [21] . a second family cluster was also described by omrani et al. [22] . three patients lived in one large house in urban riyadh. none of the other family contacts had positive pcr tests. community outbreak was also described [23] . the first patient infected his cousin, and each of the patients infected their parents. genome analysis showed multiple introduction of the virus and determined three distinct genotypes, which confirm very low patient-to-patient transmission [23] [24] [25] [26] . a number of healthcare-associated outbreaks were reported previously [14, 19, 27, 28] . a cluster of acute respiratory illnesses was reported in the intensive care unit (icu) in zarqa, jordan in april 2012 [29] . thirteen hcw cases were detected (intensivists and icu nurses) with two mortalities. an initial investigation revealed no etiology. after the announcement of the first case of mers-cov in september 2012 in ksa, samples from these patients were retested for mers-cov and confirmed to be positive in two patients by pcr and eight contacts by serology [29, 30] . thus, this case demonstrated mers-cov as a healthcare outbreak. the second large outbreak occurred in al-hasa, ksa in april 2013 [26] . a total of 23 confirmed cases and 11 probable cases were recorded. a detailed transmission map was drawn based on the best available epidemiological data linking these patients epidemiologically [26] . subsequent genotyping showed multiple introduction of the virus leading to the outbreak rather than a single introduction [25] . four of the cases did not match the transmission, which indicates that the disease was not likely transmitted between the cases but had a different genome, thus indicating multiple community introductions [25] . the outbreak was controlled with simple infection control measures in three weeks. mers-cov was shown to have minor clades, and the most recent common ancestor of mers-cov was introduced into humans at the end of 2010 [31] . mers-cov is closely related to pipistrellus bat cov hku5 (pi-batcov hku5) in bats from hong kong and these bats diverged from a common ancestor several centuries ago [32] . in 2014, one of the largest healthcare-associated outbreaks of mers-cov infection took place between february 17 and april 26 in jeddah, ksa [4] . a total of 128 mers-cov patients were treated in 14 hospitals [4, 5] . the number of cases in each hospital varied from 2 to 45 cases [4, 5] . in all the cases, 33% were primary cases and 60% (including 39 hcw) were healthcare-associated infections [33] . the rapid increase in the cases was attributed to more sensitive case detection, active case determination, and contact tracing with changes in testing algorithms [4] . a breakdown in infection control measures was observed with no change in the virus [4] . a near full genome sequence of the three viruses from the early phase of the jeddah outbreak showed a highly similar virus with no genome insertions or deletions [4] . the genetic marker influencing transmissibility was 100% identical to known mers-cov genomes [4] . in 2015, the largest outbreak outside the middle east took place in south korea [6] . the index case was a 68year-old male who visited bahrain, ksa, united arab emirates, and qatar [6] . he developed symptoms on may 11, 2015 and visited multiple hospitals in south korea [6] . he caused an outbreak involving five health care facilities and 63 cases [7] , with 34 cases in hospital b, 18 cases in hospital d, 5 cases in hospital e, and three cases in hospital f [7] . as of june 19, 2015 the outbreak in the republic of korea involved 72 health care facilities, and six facilities exhibited nosocomial transmission [8] . the total number of cases as of july 21, 2015 was 186 cases with 36 deaths [34, 35] . based on epidemiological data monitoring over the last three years, the potential seasonality of mers-cov from march to may and from september to november was observed. in april and may 2014, the number of cases increased [4] . one of the reasons for this increase in the number of cases is the parallel surge in mers-cov tests in jeddah [4] . this increase is also facilitated by an intensified intra-hospital and inter-hospital transmission of mers-cov with no change in the virus genetic composition or ability to cause disease [4, 27] . thus, seasonality is difficult to establish because sporadic cases were documented with amplifications mainly occurring during nosocomial outbreaks. available data to date show that mers-cov behaves differently in various conditions and in different population of patients. isolated sporadic cases, small family clusters, as well as large healthcare-acquired infections and clusters, were recorded. most cases present with respiratory symptoms and about one-third had gastrointestinal symptoms (table 1) [26, [36] [37] [38] [39] [40] . early symptoms are mild and non-specific, which last several days prior to progressing to severe pneumonia. no predictive signs or symptoms exist to differentiate mers-cov from community-acquired pneumonia in hospitalized patients [38] . an apparent heterogeneity in transmission was observed. the severity of the disease is usually seen in primary or index cases, immune-compromised individuals, and people with underlying comorbidities. mild or asymptomatic disease usually occurs in secondary cases and was initially thought to infect the young and previously healthy individuals. however, mortalities and severe cases were observed among primary cases and young individuals [10] . however, person-to-person transmission as a definite route of transmission is still unclear. the median incubation period was 5.2 days (95% ci, 1.9 to 14.7), and the serial interval was 7.6 days (95% ci, 2.5 to 23.1) [26] . cfr is directly related to the number of comorbidities, the increasing detection of asymptomatic to mildly symptomatic cases over the last three years [39] . however, mortalities were reported among healthy individuals. the median time to hospitalization was 4 days, icu admission was 5 days, mechanical ventilation was 7 days, and death was 11.5 days [26, 41] . mers-cov pcr was standardized, and it works extremely well with lower respiratory samples in experienced laboratories. confirmatory testing in national or regional reference laboratories with experience and load of samples will avoid reporting false positive cases. on november 5, 2013, a spanish case from hajj was initially tested positive for mers-cov but was eventually sent to an outside reference laboratory for confirmatory testing; all tests were negative [42] . if mers-cov infection is suspected and initial testing is negative, repeat testing is recommended and lower respiratory tract samples would yield higher positivity [43] . in july 2014, a key change in mers-cov case definition is the inclusion of a confirmed case based on serology [44] . serologic mers-cov confirmation requires sero-conversion in two samples taken at least 14 days apart by a screening (elisa, ifa) and a neutralization assay [44] . since the emergence of mers-cov and till october 2015, 1715 cases were reported in different countries [45] . the cases included 1403 cases in the middle east, 15 cases in europe, 191 in asia, and 7cases in other countries [45] . the who international health regulations (ihr) emergency committee convened a mers-cov emergency committee meeting on multiple occasions; after extensive deliberations and reviews of available data, the diseases weakness did not fulfill the ihr requirements to be defined as a public health emergency of international concern (pheic) and mainly sustained human-to-human transmission [46, 47] (fig.2) . using a mathematical model, the risk of mers-cov was estimated to be one to seven cases per hajj and three to ten umrah pilgrims per year [48] . in another model, 6.2 pilgrims were estimated to develop mers-cov symptoms during the hajj, and 4.0 foreign pilgrims will be infected but return home before developing symptoms [49] . travelrelated mers-cov occurred infrequently among pilgrims performing the umrah [50] . however, millions of pilgrims who performed the annual hajj did not exhibit mers-cov symptoms [51] . a cross sectional study of 839 african hajj pilgrims returning to ghana, west africa in 2013 showed that none of the pilgrims was positive for mers-cov [52] . a cohort of french pilgrims exhibited no mers-cov infection [53] . no mers-cov was detected by pcr among 5235 adult pilgrims from 22 countries [54] . although the risk of travel-associated mers-cov remains low, the potential for healthcareassociated infections in relation with an imported mers-cov is a real concern. this event took place in the republic of korea [6] [7] [8] [9] . thus, all hcws should be vigilant to the importation of mers from returning travelers and healthcare organizations should implement a strategy to screen, isolate, and diagnose these patients. serologic testing allowed the detection of eight of the 124 contacts in the jordan cluster [30] . the positive results were confirmed in six of nine outbreak members, one in 26 household contacts, and one in 89 hcws [30] . interest-ingly, one hcw who tested positive did not recall having respiratory symptoms at the time of the outbreak [30] . a few published serology studies did not present any background on mers-cov. in one study, eight out of 356 abattoir workers and blood donors had weak positive tests by ifa, and none of these individuals tested positive by nt in jeddah and makkah in 2012 [55] . in a second study, none of 268 children with respiratory tract disease and blood donors showed neutralizing antibodies in dammam, ksa in 2010-2012 [55] . an initial study of 268 samples that were tested for mers-cov antibodies in the eastern saudi arabia revealed no positive samples [55] . a serologic evaluation of 280 household contacts of 26 index patients showed 12 probable cases of secondary transmission (4%; 95% confidence interval, two to seven) [56] . a large-scale study of more than 10 000 samples demonstrated that the overall seroprevalence in ksa was 0.15% and detected an increase of 17-and 26-fold of antibody detection rate among camel shepherds and abattoir workers compared to that in the general population [20] . the full genome sequences from mers patients with known dates and locations can help answer these questions: how fast does the virus change? when did the virus begin circulating in its current form? is the virus adapting to humans? can the geographical patterns help locate an animal source? what are the transmission patterns? a previous study showed that sequence success is as function of viral load, which is inversely proportional to the threshold cycle value (ct) in real-time pcr assays [25] . ct values below 33 are associated with good sequencing success rates. thus, ct values are sufficient predictors of the success that is independent on sample type or source [24, 25] . mers-cov was found to be stable in the environment. the virus can survive on plastic and steel for up to 48 h at lower temperatures and humidity; once temperature and humidity increase, the virus becomes less viable [57] . the virus is viable at 20°c and 40% humidity for 48 hours, at 30°c and 30% humidity for 24 hours and at 30°c and 80% humidity for 8 hours only [57] . in another study, increasing the temperature from 25°c to 65°c adversely affects viral infectivity [58] . the who advocates contact and droplet precautions with airborne isolation in hospital settings when dealing with an aerosol-generating procedure [56] ; the united states centers for disease control and prevention (cdc) and the european centre for disease prevention and control (ecdc) call for airborne infection isolation precautions [59] [60] [61] . multiple hospital outbreaks of mers-cov infection in ksa were controlled effectively using infection control precautions recommended by the who and the saudi ministry of health [4, 5, 26, 36] . there are no proven therapeutic agents for the treatment of mers-cov patients. existing antiviral agents can be repurposed against mers-cov [62, 63] . interferon and ribavirin were suggested to be possible therapeutic options based on the sars data [63] . these two agents were used to treat five patients with mers-cov infection [64] . the median time to therapy was 19 days, and no improvement was observed [64] . ribavirin and interferon were used in 20 patients with mers-cov at a median of three days [65] . the 14-day survival was 70% (14 of 20 patients) in the treatment group vs. 29% (7 of 24 of patients) in a historical group (p = 0.004) with no survival improvement at 28 days (30% vs. 17%; p = 0.054) [63] . in an observational study, interferon-α2a with ribavirin and interferon-β1a with ribavirin showed similar results in treating mers-cov [40] . the potential repurposed drugs for mers therapy include ribavirin with or without interferon, hiv protease inhibitors (lopinavir and nelfinavir), cyclophilin inhibitors (cyclosporin and alisporivir), chloroquine, mycophenolic acid, and nitazoxanide [66] . the use of interferon-α2b and ribavirin decreased viral replication in the rhesus macaques model within 8 h of mers-cov infection [67] . in vitro, ribavirin inhibits mers-cov; however, the required doses are extremely high to obtain in vivo [68, 69] . in vitro, nelfinavir and lopinavir achieved inhibitory concentrations against mers-cov [70] . in a primate model, the mortality rate at 36 h post-inoculation was 67% in untreated versus 0%-33% in the lopinavir-or ritonavirtreated and interferon-β1b-treated animals [71] ; the combination was used in another case [72] . to understand the mers-cov disease further, resolving the issues related to the specific host and the specific transmission mode and determining the factors that increase transmission within healthcare environments are crucial. the viral kinetics of mers-cov within different body compartments is another aspect that needs further examination. the optimal therapeutic options and strategies to predict the occurrence and severity of the disease require further analysis. jaffar a. al-tawfiq, ali s. omrani, and ziad a. memish declare that they have no conflict of interest. this manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. travel implications of emerging coronaviruses: sars and mers-cov coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers middle east respiratory syndrome coronavirus (mers-cov) -kuwait an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh mers-cov outbreak in jeddah-a link to health care facilities middle east respiratory syndrome coronavirus (mers-cov) -republic of korea south korea coronavirus mers case list-including imported and exported cases middle east respiratory syndrome coronavirus (mers-cov): summary and risk assessment of current situation in the republic of korea and china -as of 19 middle east respiratory syndrome coronavirus (mers-cov): who statement on the tenth meeting of the ihr emergency committee regarding mers origins of major human infectious diseases the economic and social impact of emerging infectious disease. mitigation through detection, research, and response saudi arabia and the emergence of a novel coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia an update on middle east respiratory syndrome: 2 years later health care worker contact with mers patient, saudi arabia middle east respiratory syndrome coronavirus in bats, saudi arabia screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study middle east respiratory syndrome coronavirus case definition for reporting to who: interim case definition 14 middle east respiratory syndrome coronavirus: epidemiology and disease control measures presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study family cluster of middle east respiratory syndrome coronavirus infections a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study ksa mers-cov investigation team.hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infection control: the missing piece? middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution epidemiological findings from a retrospective investigation hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description molecular epidemiology and genetic analysis-origin and evolution genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 9 middle east respiratory syndrome coronavirus (mers-cov). mers-cov in republic of korea at a glance preliminary epidemiological assessment of mers-cov outbreak in south korea clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 20 respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome world health organization. revised case definition for reporting to who-middle east respiratory syndrome coronavirus severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) 21 st update world health organization. ihr emergency committee concerning middle east respiratory syndrome coronavirus world health organization pilgrims and mers-cov: what's the risk? mers-cov scenario modeling working group. estimating potential incidence of mers-cov associated with hajj pilgrims to saudi arabia imported cases of middle east respiratory syndrome: an update the hajj pilgrimage and surveillance for middle east respiratory syndrome coronavirus in pilgrims from african countries high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana lack of mers coronavirus but prevalence of influenza virus in french pilgrims after prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia transmission of mers-coronavirus in household contacts stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions heat inactivation of the middle east respiratory syndrome coronavirus infection prevention and control of epidemic-and pandemic prone acute respiratory infections in health care interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus treating mers-cov during an outbreak therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon α-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study emerging novel and antimicrobial-resistant respiratory tract infections: new drug development and therapeutic options treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus inhibition of novel coronavirus replication by a combination of interferon-α2b and ribavirin broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-α for middle east respiratory syndrome: a case report key: cord-315234-pqn7qhm8 authors: nan title: an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea, 2015 date: 2015-06-30 journal: infect chemother doi: 10.3947/ic.2015.47.2.120 sha: doc_id: 315234 cord_uid: pqn7qhm8 this report includes a summary of a current outbreak of the middle east respiratory syndrome coronavirus infection in the republic of korea as of june 23, 2015. epidemiologic, clinical, and laboratory investigations of this outbreak are ongoing. between may and june 2015, there was an outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection with a considerable number of cases in the republic of korea. this report includes an overview of the epidemiologic investigations and public health responses in several affected hospitals as of june 23, 2015. epidemiologic, clinical, and laboratory investigations of this outbreak are ongoing. the index patient (patient 1) was a 68-year-old korean man. from april 24 to may 4, 2015, he had traveled to the middle east region (bahrain, united arab emirates, and saudi arabia). on may 11, while in asan-si, chungcheongnam-do, he experienced fever and myalgia. he visited a clinic on may 15, then moved to hospital a in pyeongtaek-si, gyeonggi-do, where inpatient care was advised. persistent fever, myalgia, cough, and dyspnea lead to a diagnosis of pneumonia. he decided to move from hospital a to receive better care, and then visited another clinic and emergency room of hospital b in seoul on may 17. on may 18, he was admitted to hospital b. a meticulous interview regarding his travel history by an infectious disease specialist resulted in the diagnosis of mers-cov infection after pcr confirmation by the korean centers for disease control and prevention (kcdc) on may 20. he was transferred to an isolation unit of hospital b. as of june 23, a cluster of 38 persons including 4 healthcare workers with confirmed mers-cov are known to have had direct or indirect contact with the index patient. among those, five patients (pa-http://dx.doi.org/10.3947/ic.2015.47.2.120 • infect chemother 2015;47(2):120-122 www.icjournal.org 121 tients 6, 14, 15, 16, and 17), who were transferred from hospital a to other hospitals brought about subsequent clusters in five different hospitals. patient 14 had pneumonia and stayed in hospital a between may 13 and 25. he may have been exposed to the index patient on the same floor (eighth) between may 13 and 20. he was transferred to another hospital on may 25, but as his pneumonia deteriorated further; he left the hospital and then came to seoul. he decided to visit the emergency room of hospital b on may 27, but he was intubated on may 29, remaining in the emergency room of hospital b before being transferred to an isolation unit. mers-cov infection was confirmed on may 30. by june 23, 81 persons with confirmed mers-cov are known to have had direct or indirect contact with patient 14. other than clusters from patients 1 and 14, there were several clusters in different hospitals. as of june 23, a total of 175 confirmed mers cases have been reported to the kcdc. these reported mers cases include 27 deaths. cases of mers continue to be reported throughout the republic of korea. the korean government launched a joint task force board called the "immediate response task force for mers (irt-fm)," which was composed of government officials and infectious disease experts and representatives of the korean society for infectious diseases (ksid) and korean society for healthcare-associated infection control and prevention (koshic) for all-out efforts against the epidemic. 1) the irt-fm supported mers hospitals with updated and adapted scientific guidelines for patient care, infection control, and laboratory handling for medium-and small-sized hospitals. 2) the board members voluntarily became involved in mers hospital intervention for infection control, contact tracing policy, and decisions to close hospitals. 3) the ksid and koshic proposed several press releases regarding the mers-cov epidemic situation and the mode of transmission issue in the republic of korea. 4) the ksid and koshic representatives aimed to support their members and interactively shared updates on epidemic data to stop further inter-hospital spreads using social network services (sns). as of june 23, many efforts for contact tracing have identified a total of 14,313 persons who had any close contact with confirmed cases, and these people have been quarantined for 14 days. to control the outbreak, much stronger control measures with contact tracing, quarantine, and contact surveillance continue to be applied. the outbreak is now the second largest worldwide and the largest reported outside the middle east region owing to larg-er population density in the far east region, especially with large hospitals [1, 2] . however, there still is no sound evidence of community transmission; the mers-cov infection in the republic of korea is healthcare-associated, accelerated by inter-hospital spread. on the basis of the reported cases, droplet and contact transmission appear to be the major modes of transmission, and airborne transmission is unlikely in the community [3] . because of unexpected expansion of the epidemic even after proper contact tracing, infection-control measures currently applied in most hospitals focused not only on droplet and contact transmission prevention but also on preventing airborne transmission [3] . the low barrier to healthcare access that leads to easy patient access to hospitals and the crowdedness of emergency rooms and wards in large hospitals, especially in highly populated metropolitan areas, has been suggested to be related with the unexpectedly larger outbreak than in saudi arabia. several additional weeks will be required to confirm whether the outbreak is being controlled [4]. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) we thank jun yong choi, tae hyong kim, young hwa choi, hong bin kim, hee jung yoon, ji hyun yoon, jacob lee, joong sik eom, joon young song, sang-oh lee, won sup oh, kyung mi kim, sun young jeong, hee jin cheong, young goo song, jung-hyun choi, jin-hong yoo, and woo joo kim for their efforts in drafting this report. key: cord-287222-wojyisu0 authors: zhou, min; zhang, xinxin; qu, jieming title: coronavirus disease 2019 (covid-19): a clinical update date: 2020-04-02 journal: front med doi: 10.1007/s11684-020-0767-8 sha: doc_id: 287222 cord_uid: wojyisu0 coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has posed a significant threat to global health. it caused a total of 80 868 confirmed cases and 3101 deaths in chinese mainland until march 8, 2020. this novel virus spread mainly through respiratory droplets and close contact. as disease progressed, a series of complications tend to develop, especially in critically ill patients. pathological findings showed representative features of acute respiratory distress syndrome and involvement of multiple organs. apart from supportive care, no specific treatment has been established for covid-19. the efficacy of some promising antivirals, convalescent plasma transfusion, and tocilizumab needs to be investigated by ongoing clinical trials. currently, coronavirus disease 2019 (covid-19) poses a significant threat to global health. world health organization (who) has declared this outbreak as a "public health emergency of international concern" on january 31, 2020. within the first two months of the outbreak, the epidemic spread rapidly around the country and the world. as of march 8, 2020, a total of 80 868 confirmed cases and 3101 deaths had been reported in chinese mainland by national health commission of china, and 90 other countries are affected. covid-19 as an emerging disease, has unique biological characteristics, clinical symptoms, and imaging manifestations, though considerable progress has been made on the clinical management. this article will summarize the epidemiological, etiological, clinical, pathological, and radiological characteristics of covid-19 and review the latest advancements in the treatment. epidemic curves reflect that this epidemic may be a mixed outbreak pattern, with early cases suggestive of a continuous common source, potentially at huanan seafood wholesale market (hswm), and later cases suggestive of a propagated source as the virus began to be transmitted from person to person [1] . a retrospective analysis on the first 425 patients with confirmed covid-19 showed that during the early stages of this outbreak, the basic reproduction number r 0 was estimated to be 2.2 [2] . another modeling study estimated that the r 0 for covid-19 was 2.68 [3] . considering the strict prevention and control measures implemented by the chinese government, a phase-adjusted estimation of epidemic dynamics assumed that the effective reproduction number r 0 was 3.1 at the early phase of the epidemic, and could be gradually decreased [4] . of the first 99 laboratory-confirmed patients, 49 (49%) had been exposed to hswm, which was reported to be the possible initial source of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) [5] . a shenzhen family cluster without exposure history to wuhan markets or wild animals also proved the possibility of person-to-person transmission [6] . another family cluster of patients provided evidence that asymptomatic carriers may also be potential sources of sars-cov-2 infection [7] . evidence has recently been obtained to suggest transmission along a chain of 4 generations [8] . sars-cov-2 spread mainly through respiratory droplets or close contact. while in the later stage of infection, the virus is also detectable in anal swabs, suggesting the possibility of oral-fecal route transmission [9] . significant environmental contamination by patients carrying sars-cov-2 through respiratory droplets and fecal shedding suggests that the environment serves as a potential medium of transmission and supports the requirement for strict adherence to environmental and hand hygiene [10] . currently, there is no clear evidence of infection caused by vertical transmission or aerosol transmission. sars-cov-2 is the causative pathogen of covid-19, identified as the seventh type of coronavirus to infect humans [11] . six other kinds of coronaviruses are known to cause human disease, including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) with high mortality rate [12] . according to the genome characteristics, coronavirus is separated into four genera: α-cov, β-cov, γ-cov, and δ-cov [12] . deep sequencing revealed that this novel coronavirus isolated from lower respiratory tract samples of patient with covid-19 belongs to β-cov [11] . coronavirus has the appearance of crown under electron microscopy. they are enveloped viruses with a singlestrand, positive-sense rna genome, which is the largest known genome for an rna virus [13] . all coronaviruses share the same genome organization and expression pattern, with two large overlapping reading frames (orf1a/b) which encode 16 nonstructural proteins, followed by orfs for four major structural proteins: spike (s), envelope (e), membrane (m), and nucleocapsid (n) [13] . the sars-cov-2 protein also contains eight accessory proteins [14] . spike protein plays an essential role in binding to receptors and is critical for determining host tropism and transmission capacity. it is functionally divided into s1 domain and s2 domain, responsible for receptor binding and cell membrane fusion respectively. the receptor binding domain (rbd) of β-cov is commonly located in the c-terminal domain of s1 [15] . a team analyzed the cryogenic electron microscopy (cryo-em) structure of the sars-cov-2 spike protein and found that it has 10 to 20-fold higher binding affinity to human angiotensin-converting enzyme 2 (ace2) than sars-cov does [16] . phylogenetic analysis of the evolution history showed that sars-cov-2 shared a closer sequence homology toward the genomes of sars-cov than to that of mers-cov [17] . sars-cov-2 is highly similar to a bat coronavirus ratg13, with an overall genome sequence identity of 96.2% [18] , indicating that bat, which was discovered to be the natural reservoir host of various sars-related coronaviruses [19] , may also be the original host of sars-cov-2. the intermediate host in the process of transmission remains uncertain. clinical manifestation covid-19 has an incubation period of 1-14 days, mostly ranging from 3 to 7 days [20] . the most common symptoms in mild to moderate patients are fever, fatigue, and dry cough, followed by other symptoms including headache, nasal congestion, sore throat, myalgia, and arthralgia [5, [21] [22] [23] . a minority of patients had gastrointestinal symptoms, such as nausea, vomiting, and diarrhea, especially in children. in the study of 1099 covid-19 patients, 43.8% cases presented fever at onset of illness and the percentage further increased to 88.7% during following hospitalization [24] . notably, fever may occasionally be absent from elderly persons or immunocompromised ones. a part of patients may progress to shortness of breath, usually in the second week of the illness, and might be accompanied by or progress to hypoxemia [25, 26] . for patients presenting tachypnea, chest indrawing, or inability to feed or drink, severe pneumonia should be considered. in 10% to 20% of severe patients, the respiratory injury will inevitably develop into acute respiratory distress syndrome (ards) during 8-14 days of the illness, defined as partial pressure of oxygen (pao 2 ) to fraction of inspired oxygen (fio 2 ) ratio lower than 300 mmhg, as well as resultant non-cardiogenic pulmonary edema and mechanical ventilation [24, 25, 27] . ards, as the main cause of respiratory failure, is associated with high morbidity and mortality. risk factors for developing into severe to critical cases include advanced age, underlying comorbidities such as hypertension, diabetes, cardiovascular disease, and cerebrovascular disease [23, 25, 26] . as disease progresses, a series of complications tend to occur, especially in critically ill patients admitted to icu, including shock, sepsis, acute cardiac injury, acute kidney injury, and even multi-organ dysfunction [23, 24, 26] . patients may manifest altered mental status, low oxygen saturation, reduced urine output, weak pulse, cold extremities, low blood pressure, and mottled skin. besides, patients with acute cardiac injury would present tachycardia or bradycardia. critically ill ones may also suffer acidosis and increased lactate [23] [24] [25] . current studies reported the peak value of temperature in non-survivors of covid-19 was significantly higher than that in survivals during hospitalization [23, 24] . thus, patients presenting hyperthermia and chill should exclude the possibilities of co-infection with bacteria or other pathogens. attentions should be paid to prevent hospital-acquired pneumonia (hap) in critical cases and ventilator-associated pneumonia (vap) in those receiving mechanical ventilation. coagulopathy and thrombocytopenia are also common complications for covid-19 infection, which increase the risk of hemorrhage and thrombosis. mottled skin, petechial or purpuric rash, appearance of black stool or hematuresis could be found in some cases. patients with the syndrome of persistent hypoxemia, chest pain, pre-syncope or syncope, and hemoptysis should be suspected of having pulmonary thromboembolism (pte) [28] . the manifestation of limb pain, swelling, erythema, and dilated superficial veins should be suspected of deep vein thrombosis (dvt). nearly 20% of patients had abnormal coagulation function, and most of severe and critical patients presented coagulation disorders and had the tendency to develop into disseminated intravascular coagulation (dic) [5, 25, 26] . in the early stage of infection, the involved lung lobe presented obvious alveolar edema, proteinaceous exudates, and reactive pneumocyte hyperplasia, accompanied by mild inflammatory infiltration [29] . on gross examination, the whole lung showed bronzing surface and diffuse congestive appearance, with partly hemorrhagic necrosis, as same as the cut surfaces. on histological examination, the typical manifestations were extensive proteinaceous and serous exudation in the alveolar, hyaline membrane formation, and inflammatory infiltration with multinucleated syncytial cells. type ii alveolar epithelial cells showed extensive hyperplasia, and some presented necrosis and desquamation. viral inclusions could be identified in epithelium and macrophage. besides, alveolar septal vessels manifested congestion with alveolar edema. the infiltration of monocytes and lymphocytes in alveolar cavity and microthrombosis were prominent. some parts showed alveolar exudate organization and pulmonary interstitial fibrosis. with a fraction of desquamation of mucosal epithelium, bronchi were covered by mucus even mucus plug [20, 30, 31] . in addition, other organs also suffered pathological damage to some extent [20] . the atrophic spleen showed significantly reduced lymphocytes, focal hemorrhage and necrosis, and macrophage hyperplasia. with degeneration and necrosis of cardiomyocytes, a small number of monocytes, lymphocytes, and/or neutrophils were infiltrated in the myocardial interstitium. protein exudation was seen in renal glomerulus and within hyaline cast, and renal tubular epithelium degenerated and desquamated. besides, hepatocytes degeneration, necrosis, and inflammatory infiltration also occurred. the brain presented congestion, edema, and degeneration of some neurons. meanwhile, microthrombosis could be found in multiple organs. radiological images play an important role in the diagnosis and providing guidance for treatment. guan et al. found that 86.2% of patients manifested abnormalities in chest ct images, of whom more than 75% had bilateral lung involvement, mainly with peripheral and diffused distribution [24] . patients of different severity presented significant different lesions on chest ct (fig. 1) . mild patients manifested unilateral and focal ground-glass opacity (ggo) which gradually develops to bilateral or multilobular lesions. as the disease progressed further, ggos evolved to consolidation lesions, presenting mixpattern or pure consolidation, with the latter being more common in critically ill patients admitted to icu [5, 25, 32] . consistent with the interstitial involvement in viral pneumonia, zhao et al. suggested that 48.5% of ct images manifested reticular patterns, and 28.7% presented interlobular septal thickening [33] . unlike influenza pneumonia, which usually exhibited unilateral ggo and significant solid nodules, only 6% of covid-19 patients had solid nodules [32, 34, 35] . moreover, other lesions included adjacent pleura thickening, vascular enlargement, bronchial wall thickening, traction bronchiectasis, air bronchogram, pericardial effusion, etc. [32, 33, 36] . follow-up of ct scan could help to monitor disease changes and evaluate therapeutic effects [32] . some dynamic images fluctuate repeatedly, and showed coexistence of absorption of primary lesions and emergence of new ones. during disease deterioration, increased number of or enlarged lesions could be observed in radiological imaging, and part of them even developed into a "white lung" with diffusely involved lung [37] . a majority of covid-19 patients showed normal leucocyte count, and nearly one third had leucopenia [21, 24] . lymphocytopenia, as one of the most typical laboratory abnormalities, was present in 83.2% of patients, with an even higher proportion in severe ones [24, 26] . in addition, previous studies also revealed that increased ddimer level and prolonged prothrombin time were also common features of covid-19, especially for severe patients [24] [25] [26] . meanwhile, sars-cov-2 might damage liver and myocardium to some extent, showing elevated levels of aminotransferase, creatine kinase, and myoglobin with diverse degrees, as well as increased troponin in critical patients [5, 23, 25] . a few patients had renal dysfunction, presenting increased serum creatinine or blood urea nitrogen [5] . as for infection-related serum biomarkers, our studies have reported that most of patients had increased concentration of c-reactive protein, interleukin-6 (il-6), and erythrocyte sedimentation rate [5] . likewise, huang et al. observed similar phenomenon and proposed that icu patients might suffer severe cytokine storms, with a overproduction of il-7, il-10, gcsf, ip10, mcp1, mip1a, and tnf-α, etc. [25] . multi-drug resistant acinetobacter baumannii and klebsiella pneumoniae have been isolated in covid-19 patients [5, 23] . other identified microorganisms included pseudomonas aeruginosa, aspergillus flavus, aspergillus fumigatus, candida albicans, and candida glabrata [5, 23] . laboratory confirmed covid-19 patients had positive results on real-time reverse transcriptase polymerase chain reaction (rt-pcr) of nasal and pharyngeal swab, sputum, blood, faeces, and urine specimens [25] . the collected clinical specimens need to be transported to designated laboratories promptly, and extracted for rna correctly, followed by rt-pcr detection with primers and probes of appropriate sequences [25] . the value of cycle threshold (ct) was the criterion to determine the detection result, with less than 37 being defined as negative, above 40 as positive and a medium load (37) (38) (39) (40) calling for confirmation by retesting [2] . the detection of sars-cov-2 specific igm and igg antibodies can also be used for diagnosis [20] . covid-19 infection could be determined with one of the following criteria: positive specific igm, the transformation of specific igg from negative to positive, a 4-fold increase in igg titer during recovery period compared with the result of acute phase. although antibody detection was simple, rapid, and inexpensive, it is still not widely used due to inherent limitations, for example, false-negativity resulted from the existence of window period, noncomparable sensitivity and specificity with pt-pcr, absence of exclusion criteria making it a diagnosis tool only. these is no specific antiviral treatment which has been proven to be effective for covid-19. combinations of over three antivirals are not suggested. current treatment options are mainly based on previous experience showing clinical benefits in treating influenza, ebola, mers, sars, and other viral infections. it is reported that most of covid-19 patients received antiviral therapy in china [5, 21, 25] , and here we will introduce some commonly used drugs. ribavirin is representative of nucleoside analogs. the combination of ribavirin and recombinant interferon, a broad spectrum antiviral agent, showed augmentation effect in inhibiting mers-cov replication and reduced doses of both ribavirin and interferon [38] . however, most of clinical experiences in mers patients come from limited case reports and observational studies, making the quality of evidence for ribavirin and interferon treatment efficacy very low [39] . it is recommended to administer ribavirin by intravenous infusion in combination with inhaled interferon-α or oral lopinavir/ritonavir in the 5th version guideline on covid-19 diagnosis and treatment issued by chinese national health commission [20] . notably, ribavirin is not suggested by military medical team coming to hubei [40] and interferon-α inhalation is worried to increase the risk of virus-containing aerosol production and airway stimulation. lopinavir/ritonavir is a combination of a protease inhibitor and a booster used for the treatment of human immunodeficiency virus (hiv) infection. currently, randomized controlled trials for the efficacy of a combination of lopinavir/ritonavir with interferon-α in mild to moderate patients (chictr2000029387) and severe to critical patients with covid-19 (chictr2000029308) are in progress. remdesivir, a novel nucleotide analog rna polymerase inhibitor, is considered as the most promising antiviral drug for the treatment of covid-19. it showed broad spectrum antiviral activities, from inhibition of human and zoonotic coronavirus (including sars-cov-2 [41] as well ebola virus) in vitro, to prophylactic and therapeutic effects in animal model of mers-cov and sars-cov infection [42, 43] . the first covid-19 patient identified in the united states was given remdesivir without obvious adverse reactions. two trials on efficacy of remdesivir have been launched in china among mild to moderate patients (nct04252664) and severe to critical patients (nct04257656) infected with sars-cov-2. neuraminidase inhibitors (nais), such as oral oseltamivir and intravenous peramivir, showed substantial clinical improvement in treating influenza patients [44] . oseltamivir was widely used for suspected and confirmed covid-19 patients in china [26] , however, there is no exact evidence that supports its application. a research team from zhejiang university reported that abidol has the potential to inhibit sars-cov-2, which was previously used for influenza. there is a multicenter, randomized, and controlled trial (chictr2000029573) to evaluate the efficacy of abidol and lopinavir/litonavir, either alone or in combination with a new type of interferon, novaferon. according to current who interim guidance on covid-19 management [27] , corticosteroids were not recommended as routine therapy unless indicated for another reason, because possible harms and higher risk of mortality attributed to corticosteroids therapy have been identified by studies on other coronaviruses and influenza. an epidemiological study conducted in wuhan observed a larger percentage of patients receiving corticosteroids in icu groups when compared with non-icu groups (6 (46%) vs. 3 (11%); p = 0.013), while we still cannot determine the effects of corticosteroids due to the limited sample size [25] . according to the latest guidelines issued by national health commission of china (version 7) [20] and the interim guidance of who [27] , when sars-cov-2 infection is suspected, corticosteroids should be recommended to use with caution. new coronavirus infection diagnosis and treatment scheme (trial version) published by military support hubei medical team also put forward that for mild to moderate covid-19 patients, corticosteroids should not be given principally and highdose corticosteroid pulse therapy was not recommended. only patients presenting ongoing deterioration in oxygenation index, or rapid progression of radiological findings, or excessive activation of immune responses, will be considered to use short-term corticosteroid therapy within 10 days of illness onset. seven designated hospitals in zhejiang province gave patients corticosteroids when they showed increased resting respiratory rate ( > 30 breaths/ minute), drop in oxygen saturation ( < 93%) on room air, or multi-lobular progression ( > 50%) on imaging within 48 h [21] . timely and appropriate use of corticosteroids combined with ventilator support should be considered for severe patients to prevent progression to ards [30] . the pharmacologic use of corticosteroids in covid-19 treatment should vary with severity [20, 40] . for severe cases, it is suggested to start at a dose of 40 to 80 mg/day methylprednisolone and slowly taper over 7 to 10 days, and some suggested for a shorter period of 3 to 5 days. for critically ill cases, a starting dose of 80 to 160 mg/day methylprednisolone, following a slow withdrawal within 7 to 10 days is considered. it is widely recognized that many patients, especially critically ill patients were susceptible to secondary infections. patients receiving corticosteroids had increased risks of developing hap due to the immunosuppression effects, and those who received mechanical ventilation were susceptible to vap. the latest guidelines issued by national health commission of china for the diagnosis and treatment of covid-19 infection (version 7) [20] advise against inappropriate and unnecessary use of antimicrobial therapy, especially combination of broadspectrum antibiotics. if the sputum or blood specimens showed a clear evidence of etiology or the pct levels increased, administration of antimicrobial agents should be considered. as shown in a study of 99 patients with covid-19, acinetobacter baumannii, klebsiella pneumoniae, and aspergillus flavus were simultaneously cultured in one patient. meanwhile, one case of fungal infection was attributed to candida glabrata and three cases of fungal infection were caused by candida albicans [5] . when selecting antimicrobial agents for initial empiric treatment, in addition to the local epidemiological data of hap/vap pathogens, imaging features of pulmonary lesions should also be taken into account [45] . as for fungal infections, voriconazole is recommended for the treatment of aspergillus infections, while fluconazole is more suitable for candida spp. infections. when patients are suspected with pneumocystis pneumonia, sulfamethoxazole and caspofungin should be promptly administrated [45] . in clinical practice, nearly 20% of patients with covid-19 are found to have abnormal coagulation function, and almost all severely and critically ill patients presented coagulation disorders [5, 25, 26] . in view of no relevant experience for reference, anticoagulation should be given with great caution in patients with dic though microthrombosis was observed in lung, liver, and other organs by autopsy. when patients exhibit a bleeding tendency or when surgical treatment is needed, platelet transfusion or administration of fresh-frozen plasma is recommended to correct coagulopathies analogs [46] . low molecular weight heparin (lmwh) can be used for drug prevention. as for subjects with clinical manifestations, clinicians need to be alert to the occurrence of pte, initiate the diagnostic procedures, and develop corresponding treatment strategies based on risk stratification. considering the risk of disease transmission and the false positive results caused by the presence of lung lesions, the diagnosis of pte by pulmonary ventilation-perfusion imaging is not recommended. if the critically ill patients cannot take examination due to specific conditions and the infectivity of covid-19, it is recommended to perform anticoagulant therapy for patients without contraindications. if the condition is lifethreatening and bedside echocardiography indicates new onset of right ventricular volume overload or pulmonary hypertension, thrombolytic therapy or other cardiopulmonary support treatments, such as extracorporeal membrane oxygenation (ecmo) can be initiated with the patient's full informed consent. for mild to moderate patients with hypoxemia, nasal catheters and masks and even high-flow nasal cannula oxygen therapy (hfnc) are advised. while for severe and critical patients with respiratory distress, hfnc, noninvasive mechanical ventilation (niv) or invasive mechanical ventilation, and even ecmo should be considered. hfnc can provide accurate oxygen concentration and a certain positive airway pressure to promote alveolar expansion to improve oxygenation and respiratory distress [47] . however, according to expert consensus on the use of hfnc for covid-19, patients with cardiac arrest, weak spontaneous breathing, pao 2 /fio 2 < 100 mmhg, paco 2 > 45 mmhg and ph < 7.25 and upper airway obstruction are contraindicated. for severe patients with respiratory distress or hypoxemia that cannot be alleviated after standard oxygen therapy, niv can also be considered with close surveillance [24, 26] . dangers et al. considered that changes in dyspnea could be used as a variable to predict the failure of noninvasive ventilation [48] . if the patient continuously deteriorates or the respiratory rate cannot be improved after a short time (about 1-2 h), timely tracheal intubation and invasive ventilation are required [49] . notably, patients with hemodynamic instability, multiple organ failure or abnormal mental status should not receive noninvasive ventilation. lung protection ventilation strategies (small tidal volume, limited plateau pressure, and permissive hypercapnia) are suggested to be adopted in invasive mechanical ventilation to reduce ventilator-related lung injury [50] . compared with niv, invasive mechanical ventilation can more effectively improve the pulmonary ventilation function and respiratory mechanics of patients with acute respiratory failure. it can effectively increase the sao 2 level and is more conducive to lower the plasma bnp level [51] . however, invasive mechanical ventilation requires tracheotomy, or oral/nasal tracheal intubation to establish an artificial airway, which is very likely to cause damage to patients, such as mediastinal emphysema, ventilatorrelated lung injury, and other related complications, such as reduced swallowing function, gastresophageal reflux, infections, etc. what's more, invasive mechanical ventilation also increases the risk of secondary infections transmitted by aerosol particles [52] . for critical patients, crrt can support organ function, reduce cytokine storms and maintain internal environment stability [53] . three clinical studies showed that the incidence of aki in patients with covid-19 was 3% to 7%, and 7% to 9.0% were treated with crrt. in icu, the rate of crrt application was 5.6% to 23.0% and reached as high as 66.7% to 100% in patients with aki [5, 26, 54] . crrt is recommended for patients who exhibit aki indications (hyperkalemia, acidosis, pulmonary edema, severe sodium ion disorders) or patients with ckd who have not undergone hemodialysis. during septic shock, crrt can effectively remove inflammatory mediators and significantly improve hemodynamics. when ards appears in combination with multiple organ dysfunction syndrome (mods), early crrt is recommended [55] . crrt combined with the treatment of ecmo may remove cytokines, reduce the activity of macrophages and monocytes, and better preserve lung parenchyma. some studies reported that early convalescent plasma treatment for influenza and sars-cov infection is associated with decreased viral load and reduction in mortality [56] , however, the studies were heterogeneous and of low quality. the who deemed convalescent plasma transfusion as the most promising therapy for mers-cov infection, while the efficacy remained inconclusive, with a lack of adequate clinical trials [56] [57] [58] . since the virological and clinical characteristics among sars, mers, and covid-19 were comparable [59] , convalescent plasma could have immunotherapeutic potential in covid-19 treatment and further investigations are needed to prove its safety and efficacy. one possible explanation for the efficacy of convalescent plasma therapy is that the neutralizing antibodies from convalescent plasma might suppress viremia [60] , so understanding the antibody response during the course of sars-cov-2 infection could provide strong empirical support for the application of convalescent plasma therapy. a study reported that on day 5 after treatment, an increase of viral antibodies can be seen in nearly all patients, igm positive rate increased to 81%, whereas igg positive rate increased to 100%, which was considered as a transition from earlier to later period of infection [9] . preliminary study has showed that patients who have recovered from covid-19 with a high neutralizing antibody titer and could provide a valuable source of the convalescent plasma. plasma transfusion may cause adverse effects, so convalescent plasma therapy is recommended as a last resort to improve the survival rate of severe patients with covid-19. the optimal dose and treatment time point, as well as the therapeutic indications of convalescent blood products in covid-19 remain uncertain, which need to be further investigated in randomized clinical studies. tocilizumab is a humanized igg1k monoclonal antibody which can specifically bind soluble or membrane-type il-6 receptors (sil-6r and mil-6r), and has been widely used in the treatment of autoimmune diseases such as rheumatoid arthritis [61] , adult-onset still's disease [62] , and large vessel vasculitis [63] . for covid-19 infection, clinical studies have shown that serum levels of inflammatory mediators in severe patients are significantly higher than those in common patients [25] . excessive immune responses can trigger cytokine storms and cause damage to multiple target organs. recent guidelines also point that a progressive rise in il-6 may be a clinical warning indicator for the deterioration of covid-19. a domestic research team found that tocilizumab can block the signaling pathways of two key inflammatory factors, il-6 and gm-csf, and reduce the inflammatory response. a multicenter, randomized, controlled clinical study has been coducted to evaluate the efficacy and safety of tocilizumab in the treatment of moderate patients at high risk to develop into severe and critical patients (registration number: chictr2000029765). for patients with elevated il-6 levels, the efficacy of tocilizumab can be expected. in this review, we gave an overview of epidemiological, etiological, clinical, pathological, and imaging characteristics of covid-19 and introduced the latest advancements in the treatment. this novel virus spread mainly through respiratory droplets and close personal contact. a series of complications tend to develop during disease progression, especially in critically ill patients. pathological studies of autopsy showed typical presentations of acute respiratory distress syndrome and involvement of multiple organs. apart from supportive care, no specific treatment has been established for covid-19. the efficacy of some promising antivirals, convalescent plasma transfusion, and tocilizumab needs to be further validated by ongoing clinical trials. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study phase-adjusted estimation of the number of coronavirus disease epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster a family cluster of sars-cov-2 infection involving 11 patients in nanjing, china. lancet infect dis the novel coronavirus originating in wuhan, china: challenges for global health governance molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china epidemiology, genetic recombination, and pathogenesis of coronaviruses molecular evolution of human coronavirus genomes genome composition and divergence of the novel coronavirus (2019-ncov) originating in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding cryo-em structure of the 2019-ncov spike in the prefusion conformation evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission a pneumonia outbreak associated with a new coronavirus of probable bat origin sars and mers: recent insights into emerging coronaviruses guideline for the diagnosis and treatment of covid-19 infections clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical characteristics of 140 patients infected with sars-cov-2 in wuhan, china clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study zhong ns; china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance esc guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the pulmonary pathology of early phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer pathological findings of covid-19 associated with acute respiratory distress syndrome clinical pathology of critical patient with novel coronavirus pneumonia (covid-19) radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study relation between chest ct findings and clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter study radiographic and ct features of viral pneumonia infectious pulmonary nodules in immunocompromised patients: usefulness of computed tomography in predicting their etiology imaging and clinical features of patients with 2019 novel coronavirus sars-cov-2 novel coronavirus pneumonia (covid-19) ct distribution and sign features. chin j tuberc respir dis (zhonghua jie he he hu xi za zhi) inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin a systematic review of therapeutic agents for the treatment of the middle east respiratory syndrome coronavirus (mers-cov) diagnosis and treatment of disease 2019 novel coronavirus infection suitable for military support hubei medical team remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection influenza virus-related critical illness: prevention, diagnosis, treatment chinese guidelines for the diagnosis and treatment of hospital-acquired pneumonia and ventilator-associated pneumonia in adults the japanese clinical practice guidelines for management of sepsis and septic shock feasibility of high-flow nasal cannula oxygen therapy for acute respiratory failure in patients with hematologic malignancies: a retrospective single-center study research network in mechanical ventilation) and the groupe de recherche en réanimation respiratoire en onco-hématologie (grrroh); list of contributors who included study patients: angers university hospital, angers, france. dyspnoea in patients receiving noninvasive ventilation for acute respiratory failure: prevalence, risk factors and prognostic impact: a prospective observational study european society of intensive care medicine, and society of critical care medicine. an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome diagnosis and treatment in acute respiratory distress syndrome-reply effect of invasive and non-invasive positive pressure ventilation on plasma brain natriuretic peptide in patients with chronic obstructive pulmonary disease and severe respiratory failure severe acute respiratory syndrome (sars): lessons learnt in hong kong coronavirus epidemic: preparing for extracorporeal organ support in intensive care kidney impairment is associated with in-hospital death of covid-19 patients cytokine reduction in the setting of an ards-associated inflammatory response with multiple organ failure convalescent plasma study group. the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis sars: systematic review of treatment effects current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review emerging threats from zoonotic coronavirusesfrom sars and mers to 2019-ncov convalescent plasma as a potential therapy for covid-19 tocilizumab discontinuation after attaining remission in patients with rheumatoid arthritis who were treated with tocilizumab alone or in combination with methotrexate: results from a prospective randomised controlled study (the second year of the surprise study) tocilizumab in patients with adultonset still's disease refractory to glucocorticoid treatment: a randomised, double-blind, placebo-controlled phase iii trial trial of tocilizumab in giant-cell arteritis this work was funded in part by a grant from innovative research team of high-level local universities in shanghai. min zhou, xinxin zhang, and jieming qu declare that they have no conflict of interest. this manuscript is a review article that does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. key: cord-299720-f0ny4ur5 authors: kim, seung woo; park, jung wan; jung, hee-dong; yang, jeong-sun; park, yong-shik; lee, changhwan; kim, kyung min; lee, keon-joo; kwon, donghyok; hur, young joo; choi, boyoul; ki, moran title: risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea date: 2017-03-01 journal: clin infect dis doi: 10.1093/cid/ciw768 sha: doc_id: 299720 cord_uid: f0ny4ur5 background. transmission heterogeneity was observed during the 2015 korean outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection. only 22 of 186 cases transmitted the infection, and 5 super-spreading events caused 150 transmissions. we investigated the risk factors for mers-cov transmission. methods. epidemiological reports were used to classify patients as nonspreaders, spreaders, or those associated with a super-spreading event (5 or more transmissions). logistic regression analyses were used to evaluate the factors for mers-cov transmission. results. compared to nonspreaders, spreaders exhibited a longer interval from symptom onset to isolation (7 days vs 3 days) and more frequent pre-isolation pneumonia diagnoses (68.2% vs 17.1%). spreaders also exhibited higher values for pre-isolation contacts (149 vs 17.5), pre-isolation hospitalization (68.2% vs 16.5%), and emergency room (er) visits (50% vs 7.3%). spreaders exhibited lower cycle thresholds for the upe and orf1a genes (22.7 vs 27.2 and 23.7 vs 27.9, respectively). in multivariate analysis, transmission was independently associated with the cycle threshold (odds ratio [or], 0.84; 95% confidence interval [ci], 0.75–0.96) and pre-isolation hospitalization or er visits (or, 6.82; 95% ci, 2.06–22.84). the super-spreading events exhibited higher values for pre-isolation contacts (777 vs 78), pre-isolation er visits (100% vs 35.3%), and doctor shopping (100% vs 47.1%) compared to non-super-spreading events. conclusions. these findings indicate that transmission is determined by host infectivity and the number of contacts, whereas super-spreading events were determined by the number of contacts and hospital visits. these relationships highlight the importance of rapidly enforcing infection control measures to prevent outbreaks. transmission heterogeneity was a significant characteristic of the 2015 south korean outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection [1] . transmission heterogeneity describes a state in which most transmissions are related to a few patients and most patients do not transmit the disease. numerous other infectious diseases exhibit transmission heterogeneity [2] , and this concept is important for understanding epidemics. the course of an epidemic is influenced by the basic reproduction number (r 0 = the average number of cases that 1 case produces in a susceptible population) and transmission heterogeneity [3] . as r 0 represents an average quantity, it is often insufficient to explain individual variation, and as transmission heterogeneity reflects individual variation, it can help predict the likelihood of super-spreading events. even in instances with a low r 0 , a disease with high transmission heterogeneity (eg, severe acute respiratory syndrome [sars]) can cause super-spreading events [2] , such as the super-spreading that occurred during the 2003 sars outbreak [2, 4] . transmission heterogeneity was observed during early mers-cov outbreaks [1] and became prominent during the 2015 south korean outbreak. among the 186 confirmed korean cases of mers-cov infection, >80% of the transmissions were epidemiologically associated with 5 patients [5] , and almost 90% of the cases caused no transmission. furthermore, a recent study revealed that mers has greater transmission heterogeneity compared to sars [6] . therefore, to successfully control mers-cov infection, it is essential to identify high-risk patients and perform targeted infection control [2] . however, these patients are difficult to identify, as an individual's infectiousness is affected by complex interactions between the pathogen, host, and environment. several researchers have attempted to identify risk factors for super-spreading events during the sars outbreak [3, 4, 7] , although there is little information regarding the high-risk group(s) from the mers-cov outbreak. the recent south korean mers-cov outbreak was triggered by a single imported case, and epidemiological tracing was performed for all laboratory-confirmed cases and their close contacts [5, [8] [9] [10] [11] [12] [13] . thus, it is possible to precisely reconstruct the transmission chain and identify patients who transmitted mers-cov infection. therefore, we analyzed the epidemiological characteristics that were associated with mers-cov transmission and super-spreading events. cases of mers-cov infection were confirmed using real-time reverse-transcription polymerase chain reaction (rt-pcr) assays, regardless of their clinical manifestations. the epidemiological reports were analyzed by epidemic intelligence service officers who participated in the mers-cov outbreak investigation. when a case was exposed to multiple confirmed cases, the transmission was attributed to the case with the highest likelihood of transmission, and any conflicts were resolved through the consensus of the epidemic intelligence service officers. spreaders were defined as confirmed cases of mers-cov infection that were epidemiologically suspected of transmitting mers-cov to 1 or more persons. super-spreading events were arbitrarily defined as transmission of mers-cov infection to 5 or more cases. the patient who triggered the outbreak was defined as patient zero. cases that were infected by patient zero were defined as first-generation cases; cases that were infected by first-generation cases were defined as second-generation cases; and cases that were infected by second-generation cases were defined as third-generation cases [14] . isolation was defined as separating symptomatic patients from others to prevent spreading, and quarantine was defined as separating or restricting the movement of healthy individuals who may have been exposed to the infection within the maximum incubation period. the transmission date was defined as the date of contact between the spreader and suspected secondary case during the spreader's infectious period. in cases with an exposure duration of longer than 1 day, the transmission date was defined as the day with the highest likelihood of transmission or as the median day during the exposure period in cases with consistent contact throughout the exposure. the date of sampling was the day on which the first positive respiratory specimen was collected. close contacts were defined using the guidelines on middle east respiratory syndrome [15] , which include persons who stayed in a room or ward with a confirmed case, who directly contacted respiratory secretions from confirmed cases, or who stayed within 2 m from the confirmed cases without wearing appropriate personal protective equipment. pre-isolation pneumonia diagnoses were based on radiographic evidence. doctor shopping was defined as visiting multiple healthcare facilities without an official interhospital transfer after developing mers-cov symptoms [16] . epidemiological reports from the outbreak were evaluated to collect data regarding basic demographic characteristics, medical history, mers-cov exposure, symptoms and their onset date(s), sampling date(s), contact history, and post-exposure infection control. the reports were drafted during the outbreak based on direct interviews with the confirmed cases and follow-up epidemiological investigations that were performed to identify the exposure route and close contacts. hospital information systems were reviewed to identify patients who stayed in the hospital during the exposure period and healthcare providers who contacted the patient(s). persons who contacted confirmed cases outside healthcare facilities were also traced. data from closed circuit television, credit card transactions, and health insurance services were also reviewed [5] . the numbers of close contacts were calculated based on the number of quarantines during the outbreak. all data were collected as part of the public health response and in accordance with the infectious disease control and prevention act [17] . clinical specimens were collected in sterile containers and immediately transferred to qualified facilities. sputum samples were mixed with 0-1× phosphate-buffered saline and vortexed vigorously to reduce their viscosity. viral rna was extracted from the clinical specimens using a qiagen viral rna mini kit (qiagen, hilden, germany). all laboratory diagnoses of mers-cov were confirmed using the world health organization guidelines [18] and results from real-time rt-pcr assays that target upstream of the mers-cov envelope protein gene (upe) and the open reading frame 1a gene (orf1a) [19] . cycle threshold (ct) values for the upe and orf1a genes were obtained during testing. we analyzed the ct value from the first positive mers-cov specimen (or the specimen obtained immediately after a positive screening test). categorical variables were compared using the χ 2 test and fisher exact test, and the mann-whitney test was used for continuous variables. the variables' associations with mers-cov transmission were evaluated using multiple logistic regression analyses, and covariates were selected based on a p value of < .1 in the univariate analyses. a p value of < .05 was considered statistically significant. all analyses were performed using r software (version 3.2.2; r foundation, vienna, austria). we identified 186 cases of confirmed mers-cov infection. patient zero infected 28 first-generation cases. among the 28 cases, 8 were responsible for transmission to 121 second-generation cases. among the 121 cases, 12 infected 30 third-generation cases. one patient with an unclear source of infection (case 119) transmitted the infection to another patient. four patients exhibited unclear sources of transmission (cases 43, 178, 184, and 185). each confirmed case transmitted the infection to 0-83 secondary cases ( figure 1 ). there were 164 nonspreaders and 22 spreaders (1 or more transmission). of the spreaders, 5 cases transmitted the infection to 5 or more cases (super-spreading event). after excluding the 5 cases with unclear infection sources, we identified 180 transmissions generated by 22 spreaders. a total of 150 transmission events (83.3%) were epidemiologically linked to the 5 super-spreading events. twenty-five transmission events (13.9%) occurred within 3 days after symptom onset, 136 transmissions (75.6%) occurred 4-7 days after symptom onset, and 19 transmissions (10.6%) occurred >7 days after symptom onset. a total of 170 transmission events (94.4%) occurred on the day of or after a radiographically confirmed diagnosis of pneumonia. a total of 173 transmissions (96.1%) occurred before appropriate in-hospital isolation. seven transmissions (3.9%) occurred between confirmed cases and healthcare personnel after in-hospital isolation: 4 (cases164, 169, 181, and 183) were doctors or nurses who managed confirmed cases, 1 (case148) participated in cardiopulmonary resuscitation of a confirmed case, 1 (case 162) involved portable radiography for a confirmed case, and 1 (case 179) rode in an ambulance with a confirmed case during a hospital transfer. ; p = .009). the intervals from symptom onset to diagnosis or obtaining a respiratory specimen were also significantly longer among spreaders (to diagnosis: 9 [5.5-10] days vs 5 [3] [4] [5] [6] [7] [8] days; p = .008 and to sampling: 8 [5-9.3 ] days vs 4 [2] [3] [4] [5] [6] days; p < .001). furthermore, the interval from symptom onset to isolation was longer among spreaders (7 [4.5-9] days vs 3 [1] [2] [3] [4] [5] [6] days; p = .002). spreaders exhibited a significantly higher proportion of pre-isolation pneumonia diagnoses (68.2% vs 17.1%; p < .001) and a longer interval from the pneumonia diagnosis to isolation (4 [3] [4] [5] [6] [7] days vs 1 [0-3] days; p = .008). the overall number of contacts was significantly larger among spreaders compared to nonspreaders (149 [22.3-640.5] vs 17.5 [2-92.5]; p = .004). compared to nonspreaders, spreaders exhibited a significantly higher proportion for pre-isolation hospitalization (68.2% vs 16.5%; p < .001), visiting outpatient clinics (59.1% vs 33.5%; p = .019), and visiting emergency rooms (ers; 50% vs 7.3%; p < .001). we used logistic regression analyses to evaluate the risk factors for transmission (table 2 ). in the univariate analyses, transmission was associated with underlying respiratory disease, ct value, interval from symptom onset to diagnosis, number of contacts, and pre-isolation hospitalization or er visits. in the multivariate analyses, transmission was independently associated with a low ct value for upe (odds ratio [or], 0.84; 95% confidence interval [ci], 0.75-0.96) and pre-isolation hospitalization or er visits (or, 6.82; 95% ci, 2.06-22.84). we compared the epidemiological characteristics of the 5 spreaders with 5 or more transmissions and the 17 spreaders with 4 or fewer transmissions (table 3) . both groups exhibited similar host factors and contact durations. however, spreaders with 5 or more transmissions exhibited higher values for we evaluated the epidemiological characteristics of patients who transmitted mers-cov during the recent south korean outbreak. among the 186 confirmed mers-cov cases, only 22 cases transmitted the infection to other individuals. these spreaders had higher host infectivity and wider and prolonged contacts compared to nonspreaders. the risk factors for super-spreading events included a larger number of contacts and a pre-isolation er visit. doctor shopping was marginally associated with a super-spreading event. however, both spreaders with 5 or more transmissions and spreaders with 4 or fewer transmissions exhibited similar levels of host infectivity. it appears that both host infectivity and the number of contacts influenced mers-cov transmission, whereas super-spreading events were mostly associated with a greater likelihood of encountering other people under diverse environmental conditions. during the 2015 outbreak, approximately 75% of the transmissions occurred during days 4-7 after symptom onset, and this may be a period when the risk of transmission is particularly high. furthermore, this high-risk period was temporally associated with other epidemiological factors. first, the period overlapped with the confirmed cases' visits to healthcare facilities, as hospitalization and er visits peaked during days 4-7 after symptom onset. it is well known that mers-cov outbreaks generally occur in the healthcare setting [1, 5, 13, 20] , and the high-risk period may be associated with healthcare-seeking behaviors. second, the high-risk period was several days (1-4 days) after the radiographic diagnoses of pneumonia, which generally occurred on days 3-4 after symptom onset. although the significance of pre-isolation pneumonia has not been discussed previously, a radiographic diagnosis of pneumonia may influence transmission in 2 ways. first, it may directly increase the chance of transmission by actively generating lower respiratory tract secretions and a productive cough. second, it may be an indirect index of disease severity and hospital visiting status. in our study, cases with pre-isolation pneumonia had lower ct values and more frequent pre-isolation hospital visits. the epidemiological significance of the high-risk period could also be observed when we compared the spreaders and nonspreaders. the spreaders were typically isolated after the high-risk period (median, 7 days after symptom onset and 4 days after a diagnosis of pneumonia), whereas nonspreaders were typically isolated before this period (median, 3 days after symptom onset and 1 day after a diagnosis of pneumonia). similar results were observed in a study of the sars outbreak, which revealed that late admission to healthcare facilities (especially >4 days after symptom onset) was associated with super-spreading events [21] . thus, infection prevention measures should target isolation before this critical period (ie, within 4-7 days after symptom onset and within 1 day after the detection of pneumonia). interestingly, the average duration from symptom onset to isolation dropped to <4 days during the first week of june 2015, and reports of new cases have rapidly decreased since that time. among the host factors that were associated with transmission, only the ct value was statistically significant in the multivariate analyses. the ct value is a semiquantitative continuous variable that is inversely proportional to the viral load. ct values are associated with the severity of mers-cov infection [22] , although its relationship with transmission has rarely been studied. in the present study, spreaders had significantly lower ct values compared to nonspreaders, which suggests that ct values might reliably predict transmission. moreover, the cases with very low ct values (ct <23) tended to transmit the infection in uncommon circumstances. in both the present study and previous studies, mers-cov transmission usually occurred in the hospital setting [1, 11, 13, 23] . in contrast, cases with very low ct values transmitted the infection in more diverse settings in the present outbreak (eg, their household, in an ambulance, in an outpatient clinic, or to healthcare personnel after in-hospital isolation). these findings suggest that cases with very low ct values can potentially transmit the infection in unexpected conditions. however, our data regarding the ct values have several limitations. first, various amounts of phosphate-buffered saline were added to dilute the respiratory specimens, and this there was no multicollinearity between the independent variables (all variables: r score of <0.5). abbreviations: ci, confidence interval; or, odds ratio. may have affected the ct values. second, the ct value is influenced by the specimen type and the interval between symptom onset and sample collection [22, 24] , but various different types of specimens were collected at different time points in the present study. however, we only evaluated 5 nonsputum specimens, and there was no linear correlation between the ct values and the interval from onset to sampling. our comparison of the spreaders with 5 or more transmissions and spreaders with 4 or fewer transmissions revealed that the spreaders with 5 or more transmissions had an approximately 10-fold higher number of contacts. furthermore, there were no significant differences in host infectivity. these findings may suggest that the underlying likelihood of transmission has the greatest influence on super-spreading events rather than an intrinsic difference in host infectivity. a similar finding was observed in a previous study of the sars super-spreading event [4] , with those super-spreaders having 11-74 contacts compared to 1-4 contacts for the spreaders with 1-2 transmissions. our study also revealed that a pre-isolation er visit and doctor shopping were associated with super-spreading events. in addition, super-spreading events were associated with the number of healthcare facilities that each patient visited for hospitalization or er treatment but not with the number of hospitals visited for outpatient treatment. in south korea, patients who seek hospitalization without prior arrangements tend to visit the er, and a history of 2 or more er visits strongly suggests that the patient had been doctor shopping during hospitalization. specific environmental conditions have been suggested to increase the likelihood of a super-spreading event [3] , and doctor shopping may increase the likelihood of encountering these conditions. for example, when a confirmed case changes hospital during hospitalization without an official interhospital transfer, multiple environments are exposed to the infected case (an ambulance, an er, and a ward). thus, doctor shopping can greatly increase the likelihood of encountering conditions that are suitable for a super-spreading event. in the present outbreak, 4 of the 5 super spreaders (cases 1, 14, 16, and 76) transmitted the infection at 2 or more hospitals, as they had visited multiple healthcare facilities. therefore, it is very important to have an early suspicion of mers-cov infection and minimize doctor shopping during the early stage of an outbreak. our study has several limitations. first, some of the confirmed cases had multiple potential sources of infection, and we attributed the transmission to the case with the highest epidemiological probability. the source of infection was clear in >95% of the transmissions, and we excluded 3 cases that had contact with multiple cases and an unclear source of transmission. however, as the analyses of the epidemiological data are ongoing, the list of spreaders may change if new epidemiological evidence is uncovered. second, we did not have access to genomic sequencing data, which might have provided information regarding the relatedness of transmitted strains. third, transmission may be affected by other epidemiological factors, including aerosol-generating procedures, differences in environmental conditions, and variations in crowdedness [3, 13, 25] . however, these factors were not included in the present analysis. fourth, serological testing was not performed for every close contact, and additional asymptomatic cases may have been present. however, the seropositive rate was 0.7% in a recent serological study of close contacts [26] . thus, the absence of serological testing likely did not significantly influence our results. we evaluated the epidemiological risk factors for mers-cov transmission during the recent south korean outbreak. superspreading events were not related to intrinsic host characteristics and were attributable to the likelihood of transmission. therefore, strict er triage and minimizing doctor shopping during an outbreak's early stage may help prevent super-spreading events. hospital outbreak of middle east respiratory syndrome coronavirus superspreading and the effect of individual variation on disease emergence why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? superspreading sars events middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission characteristics of mers and sars in the healthcare setting: a comparative study super-spreaders in infectious diseases mers outbreak in korea: hospital-to-hospital transmission epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital epidemiological investigation on the 119th confirmed mers-cov case with the indefinite mode of transmission in pyeongtaek outbreak the first case of the 2015 korean middle east respiratory syndrome outbreak mers epidemiological investigation to detect potential mode of transmission in the 178th mers confirmed case in pyeongtaek mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study ministry of health and welfare. guidelines on middle east respiratory syndrome doctor shopping: a phenomenon of many themes korea ministry of government legislation infectious disease control and prevention act world health organization. laboratory testing for middle east respiratory syndrome coronavirus. interim recommendations complete genome sequence of middle east respiratory syndrome coronavirus kor/knih/002_05_2015, isolated in south korea mers-cov outbreak in jeddah-a link to health care facilities predicting super spreading events during the 2003 severe acute respiratory syndrome epidemics in hong kong and singapore association of higher mers-cov virus load with severe disease and death, saudi arabia an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity acknowledgements. the authors thank sung soon kim and jeong-gu nam (division of respiratory viruses, center of infectious disease, korea centers for disease control and prevention) for laboratory assistance and technical support in the production of the manuscript. in addition, we thank all administrative and laboratory staff of the korea centers for disease control and prevention who participated in the mers-cov outbreak control effort.author contributions. s. w. k. performed the literature search, study design, data collection, analysis, interpretation, and writing. m. k. contributed to the study design, data interpretation, and writing. j. w. p., y. s. p., c. l., k. m. k., k. j. l., and d. k. contributed to the data collection and interpretation. h. d. j. and j. s. y. contributed to the data collection, mers pcr testing, data interpretation, and analysis. y. j. h. and b. y. c. contributed to the study design, data interpretation, and revising the manuscript. s. w. k. and m. k. revised the manuscript. all authors contributed to writing and approved the final manuscript.potential conflicts of interest. authors certify no potential conflicts of interest. the authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-291679-jfxqipt8 authors: yang, seongwoo; cho, sung-il title: middle east respiratory syndrome risk perception among students at a university in south korea, 2015 date: 2017-06-01 journal: am j infect control doi: 10.1016/j.ajic.2017.02.013 sha: doc_id: 291679 cord_uid: jfxqipt8 background: the 2015 middle east respiratory syndrome (mers) outbreak in south korea was a serious threat to public health, and was exacerbated by the inappropriate responses of major institutions and the public. this study examined the sources of confusion during the mers outbreak and identified the factors that can affect people's behavior. methods: an online survey of the risk perception of university students in south korea was performed after the epidemic had peaked. the questionnaire addressed the major social determinants in south korea during the mers epidemic. the analysis included data from 1,470 subjects who provided complete answers. results: the students had 53.5% of the essential knowledge about mers. women showed higher risk perception than men, and trust in the media was positively associated with risk perception (p < .001). additionally, risk perception was positively associated with overreaction by the public (odds ratio, 2.80; 95% confidence interval, 2.17-3.60; p < .001). these findings suggest that media content affected the public's perception of mers risk and that perception of a high level of risk led to overreaction. conclusions: risk perception was associated with most of the social factors examined and overreaction by the public. therefore, providing accurate information and data to the public, establishing trust, and facilitating the development of an attitude will all be important in future crises. the first middle east respiratory syndrome (mers) case was confirmed in south korea on may 20, 2015. the last case was diagnosed on july 4 and summed up to a total of 186 confirmed cases, almost half of which were in seoul. after that, south korea officially declared the end of the mers epidemic on december 23, 2015. during the mers outbreak, 38 died and 16,752 had been quarantined (fig 1) . 1 the case fatality rate of mers in south korea was approximately 20.4%, lower than that on the arabian peninsula (approximately 45%). 2 public apprehension was exacerbated because the government did not disclose timely information about the status of the epidemic or hospitals' names and procedures related to mers infection. therefore, the public were unaware of the appropriate actions to take, but were provided with relevant information by the media. indeed, several citizens created web sites that listed confirmed and suspected mers patients. 3 however, the public also received inaccurate information from the internet and social media; this increased the level of concern over mers and resulted in rumors. the government of south korea stated that any person who disseminated an untrue rumor would be prosecuted, but this failed to reduce the level of panic. although this action was supposed to prevent secondary damage, it was similar to the censorship of the media in china, where the propaganda departments of the chinese communist party directly supervised the media flow when the severe acute respiratory syndrome (sars) outbreak occurred in 2003. 4 the chinese government attempted to maintain political, social, and economic stability by minimizing the sars crisis through the withholding of information; however, a reverse effect occurred. nevertheless, the chinese government assigned responsibility for censorship of the media, including the internet, to local agencies. 5 in the new media age, in which social media (including the internet, short message services, and mobile applications) are centralized, the government restricts freedom of expression in the same way as it has restricted such freedom among traditional media sources, such as newspapers, radio, and television. 6 the control of the acquisition of information is more difficult in this new age because social media are tools not only for the dissemination, sharing, and seeking of health information but also for the expression of feelings and the sharing of personal experiences and opinions. 7, 8 therefore, organizations need to build effective communication tools to respond to emerging infectious disease (eid) outbreaks because the public may express scientific skepticism about scientific topics and participation in decision-making. 9 during epidemics, people usually require guidance on how to behave from a trusted source. the government and public institutions are the ideal sources because people tend to rely on the national administration. for this reason, trust in these institutions plays a main role in the public's acceptance of policies and actions. 10 the world health organization suggested that outbreak communication should incorporate the following 5 key factors: (1) building, maintaining, or restoring trust; (2) announcing early; (3) maintaining transparency; (4) understanding the public; and (5) planning of all aspects of the response to an outbreak. 11 therefore, risk communication enhances the decision-making ability of laypeople, and can be examined by assessing risk perception. 12 trust in not only the government and public agencies but also in the media and other institutions may be associated with risk perception. therefore, the instigation of fear among the public by the media may contribute to social panic, particularly in emergency situations. the term risk perception usually refers to individuals' judgments about and evaluations of hazards to which they might be exposed. 13 therefore, risk perception might be among the social phenomena related to exposure to the risk of disease. in addition, perceived risk influences health behavior both positively and negatively. 14 during the mers outbreak in south korea, negative behaviors were observed, such as oversensitive or inappropriate reactions. some children of health care workers at hospitals treating mers patients were prevented from attending school. meanwhile, self-quarantined subjects occasionally escaped out of their homes until the level of infection subsided. these reactions of citizens reflect distrust in the government and accelerated noncompliance with the directions provided by the korea centers for disease control and prevention. this finding is important because overreaction is an indicator of the level of trust in the government among the public and may provoke another social problem related to moral panic. 15 fear causing the behaviors described previously mentioned is likely related to risk perception and a low level of trust in the government and society. however, because risk perception may be related to a number of unknown determinants, it is important to identify factors that may affect risk perception. sjöberg examined risk perception using several approaches, 16 and reported that 30%-40% of risk perception could be explained by risk sensitivity, attitude, and a specific fear model. in other models, <20% of risk perception was explained. there were also some studies regarding risk perception. previous studies of risk perception related to nuclear explosions and infectious diseases addressed the relationship between perceived risk and various social predictors, such as knowledge, social trust, and attitude. 17, 18 however, previous literature on the determinants of risk perception has been limited. the aim of this study was to determine whether risk perception was associated with personal and social variables, including trust in the media, the health care field, and government. additionally, we sought to identify the associations of risk perception and social variables with compliance with self-quarantine guidelines and overreaction during the mers epidemic. in this study, knowledge, trust, personal characteristics, and other social determinants were considered the main factors affecting risk perception and overreaction. we developed a questionnaire based on previous studies of perception of the risk of sars and ebola conducted outside of south korea. [19] [20] [21] the questionnaire comprised the following 5 components: knowledge of mers (9 items), risk perceptions about mers (7 items), trust in society and governmental health policy (5 items), preventive behavior (11 items), and particular situations relating to mers (7 items). sociodemographic information was also obtained. korean-and english-language versions of the questionnaire were developed to enable inclusion of foreign students in the survey (supplementary appendix s1). the english-language version of the questionnaire was backtranslated into korean for quality control purposes. questions regarding knowledge of mers were used to estimate the level of accuracy of the following information: the selfrecognized level of knowledge, the concept of mers, route of transmission, the concept of close contact, symptoms, the concept of the incubation period, characteristics of mers, self-quarantine, and treatment of mers. evidence for these questions was from the mers response guidelines of the korea centers for disease control and prevention. 22 the cumulative knowledge score of the participants was calculated (range, 0-8; 1 point for each correct response [ie, in agreement with the evidence]). because all questions referred to information provided in governmental guidelines, the survey was regarded as providing an estimate of respondents' essential knowledge. risk perceptions were assessed using revised versions of the ebola risk perception surveys conducted in germany 20 and israel. 21 risk perception was assessed using levels of agreement with the following statements: "i think that i will contract mers if i come into contact with a mers patient (risk perception 1)," "i think that i might contract mers even if i do not come into contact with a mers patient (risk perception 2)," "my health will be severely damaged if i contract mers (risk perception 3)," "i think mers is more severe than other respiratory diseases (risk perception 4)," "even if i fall ill with another disease, i will not go to hospital because of mers (risk perception 5)," "mers will inflict serious damage on my community (risk perception 6)," and "mers may spread in korea again someday (risk perception 7)." the questions were responded to on a 5-point likert scale. in addition, because risk perception is a continuous variable, it was classified into 4 groups according to quantile (very high, high, low, and very low). a revised questionnaire regarding trust in society, media, and health policy was developed from the korean general social survey (kgss). the kgss has been validated in south korea and is conducted periodically nationwide. several questions in the kgss estimate the levels of trust among respondents in society, the media, central and local government, the national assembly, the medical profession, and nongovernmental organizations (ngos). such trust was expected to be associated with trust in health policy because governmental actions tend to reflect governmental policies. finally, questions regarding particular situations related to mers were classified into the following 2 categories: overreaction and compliance with self-quarantine. a large proportion of the population of south korea was unaware of the appropriate actions during the mers epidemic. these categories measure the characteristics of individuals not inclined to abide by the self-quarantine guidelines. therefore, this section assessed the following personal characteristics: degree of optimism about the health policies of south korea, willingness to sacrifice for society, responsiveness to an emergency situation, and attitude toward self-quarantine and overreaction. attitude toward self-quarantine was measured with a question asking whether respondents were willing to prioritize quarantine adherence above all personal needs (responses were categorized as yes and no). the overreaction section comprised the following 2 questions: (1) how would you react if you realized your children were in the same class as those of someone suspected (or confirmed) to have mers (including school and extracurricular activities)?; and (2) how would you react if you realized your children were in the same class as those of medical workers at hospitals with mers patients (including school and extracurricular activities)? the responses to the formerly involved overreactions, for example, "(do not) allow my relatives or children of my family to attend school," and those to the latter were the opposite, such as "i think children of medical workers at hospitals with mers patients should (not) be allowed to attend school." each question was assumed to indicate overreaction to the behavior of oneself and others. a presurvey including 152 participants was performed to assess the reliability and validity of the questionnaire and the direction of the responses. the presurvey was conducted both in the field using a tablet personal computer or laptop and online using an online survey application (surveymonkey, san mateo, ca). data for the main survey were collected by e-mailing students at seoul national university. the cronbach α values for the 2 new measures developed for this study, trust and risk perception, were 0.8307 and 0.6755, respectively, in the main survey. students at seoul national university were selected for the study. we considered this population appropriate for several reasons. first, they are not likely to include people associated with hospitals. second, younger individuals tend to have access to up-to-date information through smartphones or the internet. third, the subjects had similar levels of academic attainment and demographic characteristics. finally, because of the need for a timely investigation, this setting assured the benefit of accessibility and cost-efficiency. for the main survey, e-mails containing the link connected to the online survey setting were sent to all of the 30,727 students at seoul national university, including undergraduates, postgraduates, and foreign students. survey e-mails were sent on 3 occasions at weekly intervals from october 6-23, 2015. of the 30,727 students, 1,487 (4.8%) provided a complete response. after exclusion of those with missing responses, a total of 1,470 subjects were included in the study. this survey and research were approved by the institutional review board of seoul national university (no. 1508/002-003). relationships between levels of risk perception and demographic factors were evaluated by the χ 2 test, and the mean and se values were estimated. to evaluate correlations among independent variables, spearman correlation coefficients were calculated. correlation coefficients among explanatory variables were all <0.5, which was regarded as not having multicollinearity. 23 variables were selected for inclusion by stepwise calculation of the akaike information criterion. moreover, to prevent multicollinearity, a test of variation inflation factors was performed for each analysis. to assess the associations of demographic factors, knowledge, trust in social organizations, intention to sacrifice, and responsiveness to emergency situations with risk perception, multiple linear regression analyses were used. each question regarding risk perception was aggregated to produce a cumulative score indicative of the overall effect of risk perception. prior to analysis, the variables were tested using a q-q plot to verify the normality of their distribution andwith the exception of knowledge-were confirmed to be continuous because the ordinal values differed only slightly from a normal distribution. 24 risk perception was estimated on a scale of 1-7, and its relationship with predictors was assessed. in addition, to identify the association of risk perception with overreaction to the epidemic and compliance with self-quarantine, multiple logistic regression analyses were performed. odds ratios (ors) were calculated for self-quarantine and overreaction. analyses were conducted using sas version 9.4 (sas institute, cary, nc). sociodemographic variables and levels of risk perception are shown in table 1 . more than half of the participants were men (57.01%), 24-26 years of age (30.54%), citizens of south korea (95.51%), and postgraduate students (52.04%). a high frequency of risk perception was detected in women (n = 152; 52.6%), those 24-26 years of age (n = 97; 33.56%), students from south korea (n = 281; 97.23%), and postgraduate students (n = 166; 57.44%). the mean knowledge score was 5.35 ± 1.41 out of 10. therefore, respondents had approximately 53.5% of the essential knowledge about mers. the mean score for trust in the medical profession was higher than those for other social components (3.63 ± 0.95); the mean score for trust in the central government was lowest (2.37 ± 1.03) ( table 2 ). in terms of the media section, the item regarding trust in newspapers was excluded from the analysis because of its lack of validity according to the variable selection process. women had a higher risk perception score than men, not only overall (by 0.17/5 points; β = 0.17; p < .001), but also in all other risk perceptions from 1-7 (table 3) . older age was negatively associated with risk perception 2 (concern over contracting mers through indirect contact) (β = −0.07; p = .004) and positively associated with risk perceptions 4 (considering mers to be more severe than other respiratory diseases) (β = 0.09; p < .001), 6 (concern over damage to the community because of mers) (β = 0.05; p = .026), and 7 (future reemergence of mers) (β = 0.06; p < .001). in addition, only risk perception 6 (damage to the community) was significantly positively associated with korean ethnicity (β = 0.44; p < .001). knowledge influenced only risk perceptions 1 and 7. that is, knowledge was related to concern over contracting mers through direct contact (β = 0.04; p = .015) and reemergence of mers (β = 0.05; p = .004). knowledge was associated with risk perception, which does not support the hypothesis of this work. trust in the media (broadcasting) was positively associated with cumulative risk perception (β = 0.06; p < .001) and risk perceptions 1 (concern over contracting mers through direct contact), 3 (severity of mers), 4 (considering mers to be more severe than other respiratory diseases), and 6 (concern over damage to the community because of mers). however, trust in the medical profession (β = −0.04; p = .027) and the central government of south korea (β = −0.05; p = .014) was negatively associated with overall risk perception. risk perceptions 2 and 4 (relevant to disease traits) were related to trust in the medical profession. in addition, risk perceptions 5, 6, and 7 (relevant to community traits) were related to trust in the central government. in contrast, trust in local government (β = 0.04; p = .046) and ngos (β = 0.06; p < .001) was positively associated with risk perception. trust in local government and ngos was associated with risk perceptions 5 and 6 and risk perceptions 1, 3, 4, and 6, respectively. compared with the results for trust in the central government, those for trust in local government seemed to have similar effect sizes, but with a different direction of estimates. however, trust in ngos was associated with the perception of the risk of transmission and that to personal health. this suggests that ngos are believed to contribute to communities in various ways, including by providing medical services. trust in society was not associated with the overall risk perception but was negatively associated with concern over damage to the community (β = −0.11; p = .002). finally, trust in health policy was negatively associated with risk perception (β = −0.05; p = .014). risk perceptions 2 and 7 were related to trust in the health policy of the government of south korea. optimism about future health policy (β = −0.04; p = .024), willingness to sacrifice (β = −0.06; p = .004), and active responsiveness to an emergency (β = 0.18; p < .001) were associated with risk perception. only risk perception 3 was associated with optimism regarding health policy (β = −0.09; p = .009). willingness to sacrifice was negatively associated with risk perceptions 1, 3, and 5. in addition, responsiveness to an emergency was positively associated with risk perceptions 1, 2, 3, 4, 6, and 7. therefore, respondents who were pessimistic about health policy and unlikely to sacrifice themselves in specific situations and respond actively to an emergency exhibited a higher perception of risk. ors were calculated to estimate the associations among risk perception, knowledge, personal characteristics, and compliance with self-quarantine ( table 4 ). the proportions of men and women who indicated they would comply with the self-quarantine guideline were 82.7% and 87.2%, respectively. women were more likely to comply with self-quarantine (or, 1.50; 95% confidence interval [ci], 1.10-2.06; p = .010), as were older subjects (or, 1.22; 95% ci, 1.01-1.46; p = .037) and those with a greater level of knowledge (or, 1.22; 95% ci, 1.10-1.35; p < .001). additionally, subjects with more trust in the medical profession (or, 1.35; 95% ci, 1.14-1.60; p = .001) and those who were willing to sacrifice (or, 1.50; 95% ci, 1.20-1.88; p < .001) and actively respond to emergencies (or, 1.31; 95% ci, 1.03-1.67; p = .028) were also more likely to comply with a self-quarantine order. the proportions of positive responses to items about overreactions in one's own behavior (self-behavior) and overreactions in response to the behavior of others were 79.9% and 42.9%, respectively. knowledge (or, 0.90; 95% ci, 0.82-0.99; p = .037) and trust in society (or, 0.81; 95% ci, 0.66-0.98; p = .029) were negatively associated with overreaction in self-behavior (table 5 ). however, responsiveness to an emergency (or, 1.32; 95% ci, 1.05-1.66; p = .016) and risk perception (or, 2.80; 95% ci, 2.17-3.60, p < .001) were positively associated with overreaction in self-behavior. in terms of overreaction to the behavior of others (table 6) , korean respondents (or, 0.486; 95% ci, 0.28-0.84; p = .010), those with a greater level of knowledge (or, 0.79; 95% ci, 0.73-0.86, p < .001), and those with a higher level of trust in the medical profession (or, 0.70; 95% ci, 0.61-0.80, p < .001) were unlikely to overreact to the behavior of others. however, respondents with a higher level of trust in local government (or, 1.19; 95% ci, 1.02-1.38; p = .024) and higher risk perception (or, 1.86; 95% ci, 1.52-2.28, p < .001) were more likely to overreact to the behavior of others. therefore, risk perception was associated with a change in behavior during an epidemic. the findings of this study indicate that risk perception is associated with various social factors. risk perception was correlated with sex and the level of trust in social organizations. these findings indicate that risk perception interacts with demographic and personal attitudinal factors at a collective level, as previously suggested. 25 women had a higher risk perception than men. trust in the media (television), local government, and ngos exhibited positive associations with risk perception, whereas trust in central government, the medical profession, and health policy exhibited negative ones. these results suggest that people are aware of the different roles of central and local government. additionally, these results identify the types of determinant that affect whether people overreact when an infectious outbreak occurs. women and those with low levels of trust in central government, the medical field, and health policy were more likely to overreact. on the other hand, people with high levels of trust in the media, local government, and ngos may be hypersensitive when an eid outbreak occurs. furthermore, the higher risk perception was associated with only overreaction, not compliance to self-quarantine. hypersensitivity that leads to overreaction can be explained by perceived media dependency 26 ; however, this may have been related only to television in this research. moreover, differences in the effects of trust in central and local governments on risk perception may be explained by south koreans' perceptions of the roles of these government bodies. central and local governments do not always appear to smoothly coordinate the emergency response, and this may be related to ongoing challenges about decentralization from the central governmental system. 27 another important finding is related to the role of the media during a crisis. in this study, trust in the media positively affected risk perception and overreaction, supporting that the mass media influence perceptions of disasters and risks; however, they cannot change an epidemic event itself. 4 restrictions on the availability of information by the government can exacerbate the impact of a disaster, especially in the new media age, in which person-centered media is emphasized; this was the case in the 2003 sars outbreak in china and the 2015 mers outbreak in south korea. regardless of the political system (ie, even in democratic states), the absence of risk communication may yield unexpected results. therefore, the world health organization outbreak guidelines 11 regarding building public trust and establishing a transparent flow of information need to be followed. respondents in this study were moderately knowledgeable about mers, possessing 53.5% of the essential knowledge. this finding may reflect a higher mean level of education of the respondents compared with the general population, but indicates that the public is not ignorant about environmental and health-related matters. 28 from another point of view, this finding can be said to contradict the deficit model, which holds that the public lacks knowledge. 29 on the emergence of a novel infectious disease, people tend to be receptive to health-related information conveyed by new media, including the internet and mobile phones. for these reasons, the role of the mass media is particularly important because they usually provide widespread coverage of eids and convey information to the public. 30 furthermore, the level of knowledge about mers was not associated with risk perception. this is not in agreement with a previous report of a correlation between knowledge and perception of the risk of nuclear explosions. 31 notwithstanding these considerations, knowledge of mers was directly associated with overreaction and compliance with self-quarantine. this suggests that greater knowledge of unfamiliar diseases reduces the likelihood of undesirable behavior, which can lead to social problems. personal characteristics, including willingness to sacrifice and responsiveness to an emergency, were negatively and positively, respectively, associated with risk perception. in addition, while responsiveness to an emergency was associated directly with overreaction and compliance with self-quarantine, willingness to sacrifice was associated only with compliance with self-quarantine. personal characteristics were associated with responsive behaviors; however, the reasons for these associations could not be determined. one possibility is nationalism. individuals who regard nations as abstract communities with shared emotional bonds governed by rules and/or norms tend to be altruistic. 32 therefore, people in table 5 associations of risk perception, knowledge, and personal characteristics with overreaction in one's own behavior societies governed by established, systematic rules are less likely to exhibit unusual behaviors that may lead to social panic. moreover, inaccurate information is disseminated through social media platforms, such as twitter, facebook, and google. the enormous influence of social media during the 2014-2015 ebola outbreak in west africa has led to exaggerated concerns even among the population in the united states with negligible risks. 33 indeed, the mass media can also incite panic, which is related to fear and antisocial behavior 34 ; however, when they do their job properly, they can inform the public accurately about current conditions. two studies in which a mobile health communication tool was used demonstrated the effectiveness of a social media-based approach in terms of changing vaccination behavior. 35, 36 furthermore, the provision of appropriate and timely public health information by social media and the mobile health tool could accelerate the detection of disease outbreaks and enhance the public's response, 37 thereby minimizing adverse health and economic effects. 38 the findings of our study are depicted in schematic form in figure 2 . in this model, risk perception was related to various factors such as sex, trust, and other personal characteristics. there was a notable aspect of risk perception: it was directly associated with overreaction, but not with compliance to self-quarantine. this suggests that risk perception motivates self-protective behavior, possibly resulting in overreaction. however, it does not automatically result in compliance to quarantine, which is generally perceived as protecting others, rather than oneself. in this sense, risk perception is a double-edged sword: at an optimal level, it helps the public protect themselves from infection and thereby deter the spread of epidemic; however, when excessive and characterized by fear, it can lead to other social problems, such as overreaction and discrimination. sandman reported that risk perception is comprised of hazard and outrage. 39 the mers outbreak caused fear and anxiety among the population, which might have resulted in an overreaction to both the response of the government and the behaviors of themselves and others. 40 our model also points to the importance of trust in medical experts and proper knowledge. these 2 factors demonstrate the potential to maximize the benefit by promoting compliance and reducing overreaction. therefore, risk communication should not just scare people, but should create a context in which there is trust and a flow of knowledge and clear information between the authorities and the public. these conditions should prevent adverse effects, particularly in democratic states. the existing literature emphasizes these essential components of risk management and risk communication. 41, 42 to date, there are a limited number of studies on risk perception of infectious diseases (including mers); therefore, this work makes an important contribution to the field. several factors affecting perceived risk were evaluated (eg, trust in the media or health policy, personal characteristics), which were not assessed in previous studies. although knowledge and trust have been previously reported to be associated with perceived risk, 17, 18 this study further explored possible effects of risk perception and their implications in responding to an epidemic. eids are unfamiliar to the general public and are likely to be overestimated in the risk, 31 possibly leading to social panic. to prevent this, it is important to boost the risk communication with fostering trust and relevant knowledge, which requires transparency. this study had limitations. first, the survey involved students at a university; therefore, the participants might have different levels of concern about the epidemic compared with the general public. however, the participants represented the younger population with a homogeneous educational level with fewer confounding factors. therefore, the findings were still likely meaningful for understanding the characteristics of risk perception in the essence. second, the response rate was rather low (4.8%). most online surveys have much lower response rates than person-to-person interviews. 43 the students who had a greater perception of the risk might have been better motivated to participate in the study. even if the associations found in this study may be weaker in a larger population, it is informative to identify the direction and structure of the interrelationships among the factors. future studies should investigate whether the findings from this study are applicable in more diverse populations. furthermore, developing and testing more specific approaches to building trust and sharing knowledge during an epidemic would be needed. in responding to the public health emergency, collaboration is important among various social institutions, such as central and local governments, media, and medical community. more research is needed to explore the roles and relationships of these agents for effective risk communication. in conclusion, risk perception was found to be associated with social trust and personal attitudes toward emergency situations in this study. risk perception was associated with overreaction, possibly by fear-induced changes in behavior. however, knowledge about the nature of disease mitigated this possibility and enhanced compliance to quarantine guidelines. understanding the determinants of risk perception contributes to effective communication. building trust and sharing knowledge are important to ensure a rapid response to disease outbreaks, and to prevent unnecessary behaviors among members of the public. taking stock of the first 133 mers coronavirus cases globally-is the epidemic changing? middle east respiratory syndrome in the republic of korea: transparency and communication are key media, crisis, and sars: an introduction how censorship in china allows government criticism but silences collective expression convergence, content industries and media governance public anxiety and information seeking following the h1n1 outbreak: blogs, newspaper articles, and wikipedia visits pandemics in the age of twitter: content analysis of tweets during the 2009 h1n1 outbreak risk communication and infectious diseases in an age of digital media trust and social representations of the management of threatened and endangered species outbreak communication: best practices for communicating with the public during an outbreak risk perception and communication cross-cultural risk perception: a survey of empirical studies risk perception and self-protective behavior moral panic and social theory beyond the heuristic factors in risk perception risk perception of aids and reported sexual behaviour among students in secondary schools and colleges in tanzania epidemics and fear ebola risk perception in germany what does the public know about ebola? perceived threat, risk perception, and efficacy beliefs related to sars and other (emerging) infectious diseases: results of an international survey an introduction to data analysis & presentation when can categorical variables be treated as continuous? a comparison of robust continuous and categorical sem estimation methods under suboptimal conditions society and health: sociology for health professionals no alternatives? the relationship between perceived media dependency, use of alternative information sources, and general trust in mass media public trust in government in japan and south korea: does the rise of critical citizensmatter? citizen science: a study of people, expertise and sustainable development the case for a 'deficit model' of science communication sars wars: an examination of the quantity and construction of health information in the news media knowledge and risk perception among nuclearpower-plant employees why do people sacrifice for their nations? ebola and the social media moral panic: from sociological concept to public discourse assessing vaccination sentiments with online social media: implications for infectious disease dynamics and control stage-based mhealth communication interventions for hpv education social media in public health responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management hazard versus outrage in the public perception of risk. effective risk communication mers-cov outbreak in south korea-public policy trumped by fear and politics social trust and the management of risk risk management in post-trust societies the adequacy of response rates to online and paper surveys: what can be done? we thank all members, hye-rin park, jee-eun yi, kang-jin kim, nam-kyu cho, nara lee, and yunisa astiarani who participated in conducting the survey together in community health field training, one of the programs of a master's course at the graduate school of public health. we also thank the supporting from bk21 plus. supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ajic.2017.02.013. key: cord-303272-1w8epdht authors: reusken, chantal bem; haagmans, bart l; müller, marcel a; gutierrez, carlos; godeke, gert-jan; meyer, benjamin; muth, doreen; raj, v stalin; vries, laura smits-de; corman, victor m; drexler, jan-felix; smits, saskia l; el tahir, yasmin e; de sousa, rita; van beek, janko; nowotny, norbert; van maanen, kees; hidalgo-hermoso, ezequiel; bosch, berend-jan; rottier, peter; osterhaus, albert; gortázar-schmidt, christian; drosten, christian; koopmans, marion pg title: middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study date: 2013-08-09 journal: lancet infect dis doi: 10.1016/s1473-3099(13)70164-6 sha: doc_id: 303272 cord_uid: 1w8epdht background: a new betacoronavirus—middle east respiratory syndrome coronavirus (mers-cov)—has been identified in patients with severe acute respiratory infection. although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of mers-cov. anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. we investigated possible animal reservoirs of mers-cov by assessing specific serum antibodies in livestock. methods: we took sera from animals in the middle east (oman) and from elsewhere (spain, netherlands, chile). cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum igg by protein microarray using the receptor-binding s1 subunits of spike proteins of mers-cov, severe acute respiratory syndrome coronavirus, and human coronavirus oc43. results were confirmed by virus neutralisation tests for mers-cov and bovine coronavirus. findings: 50 of 50 (100%) sera from omani camels and 15 of 105 (14%) from spanish camels had protein-specific antibodies against mers-cov spike. sera from european sheep, goats, cattle, and other camelids had no such antibodies. mers-cov neutralising antibody titres varied between 1/320 and 1/2560 for the omani camel sera and between 1/20 and 1/320 for the spanish camel sera. there was no evidence for cross-neutralisation by bovine coronavirus antibodies. interpretation: mers-cov or a related virus has infected camel populations. both titres and seroprevalences in sera from different locations in oman suggest widespread infection. funding: european union, european centre for disease prevention and control, deutsche forschungsgemeinschaft. in 2012, a new betacoronavirus-middle east respiratory syndrome coronavirus (mers-cov)-was identifi ed in patients with severe respiratory disease in the middle east. as of aug 2, 2013, 94 laboratory-confi rmed cases, including 46 deaths, have been reported to who. 1 illness associated with mers-cov infection is characterised primarily by mild-to-severe respiratory complaints, most requiring hospital admission for acute respiratory distress syndrome. comorbidities and immunosuppression seem to predispose for infection and severe disease, [2] [3] [4] [5] [6] and unpublished serological studies suggest that asymptomatic infections occur. 7 all cases reported so far have been linked to jordan, qatar, saudi arabia, and united arab emirates. humanto-human transmission has been reported, particularly in health-care settings, but on the basis of available evidence the basic reproduction number (r 0 ) is thought to be low, suggesting that the virus is not transmitted readily. 6, 8 therefore, the primary reservoir of mers-cov is probably animals. diff erent coronaviruses have various hosts including wildlife, livestock, poultry, pets, and human beings. coronaviruses can adapt to new host species, as shown by the zoonotic origin of several human coronaviruses. 9 human coronavirus oc43 has recent common ancestry with bovine coronaviruses. 10 rhinolophid bats were identifi ed as a likely reservoir for severe acute respiratory syndrome coronavirus (sars-cov), which emerged in people in 2002-03, through intermediate carnivorous hosts. 11 molecular clock analysis 12 showed that bat and civet strains of viruses closely related to sars-cov only diverged a few years before the outbreak. human coronavirus 229e has a common ancestor with coronaviruses found in ghanaian hipposideros spp bats. 13 mers-cov is able to replicate in various bat cell lines 14 and phylogenetic analyses show that it is closely related to betacoronavirus lineage c viruses from pipistrellus spp bats in europe and asia. [15] [16] [17] [18] molecular clock dating of epidemiologically unlinked isolates of human mers-cov estimated their divergence from a common ancestor in mid-2011, 4, 19 with a cluster of isolates from the eastern arabian peninsula diverging in late 2012. 4 this fi nding could suggest that the diversity of mers-cov in people is the result of multiple independent, geographically structured, zoonotic events in the middle east. 4, 19 possible animal reservoirs need to be identifi ed to determine how circulation of mers-cov is maintained and to break the chain of transmission. 20 mers-cov can infect cells of several species, including human beings and bats. 14 the functional receptor is conserved between species, suggesting that receptor use is not an important barrier to cross-species transmission. 21 data for exposure history of patients are scarce, but suggest contact with livestock, including dromedary camels and goats. 2, 4, 5 food and agriculture organization data from 2011 show that cows, goats, sheep, and dromedary camels are the main sources of meat and milk in jordan, saudi arabia, and united arab emirates. 22 serological studies are best suited to screen animal populations, but have not yet been reported for mers-cov in animals, although several methods have been described for testing antibodies of people. 23, 24 for specifi city, who recommends use of a combination of screening assays with recombinant spike protein, and confi rmatory testing by neutralisation assays. here, we describe antibody profi ling of serum samples from major livestock species that might be relevant to the epidemiology of mers-cov in the middle east, using samples collected from herds inside and outside the region. we sampled a cohort of 105 dromedary camels (camelus dromedarius) from two herds on the canary islands. 50 were male, 55 were female, 88 were adults, nine were age 3-4 years, seven were age 2 years, and one was age 3 months. both herds had the same owner, with frequent exchange of animals between the herds. one herd is from a coastal dune habitat with no other livestock, while the other herd is in an inland valley close to a tropical fruit farm, in particular mango and papaya-which could attract fruit bats-and nearby (roughly 500 m) to horse and goat farms with 25 and 300 animals, respectively. the camels were born in the canary islands except for three adults, which were imported from morocco. 25 the camels are used in the tourist industry. 80 sera were taken april-june, 2012, nine in may, 2013, and 16 paired sera were taken in these months in 2012, and 2013, all for routine veterinary purposes. samples were obtained by jugular puncture. 50 female dromedary camels from oman were sampled in march, 2013. the camels were aged 8-12 years and belonged to diff erent owners from separate locations. the camels are retired racing camels now used for breeding, and blood was taken by jugular puncture for routine screening for brucellosis. omani dromedaries sera were collected for veterinary purposes from two llamas (lama glama), six alpacas (vicugna pacos), and two bactrian camels (camelus bactrianus) in the netherlands. sera were collected for veterinary purposes from two bactrian camels, 18 alpacas, fi ve llamas, and two guanaco (lama guanicoe) in buin zoo in chile. sera from cattle (n=40), domestic goats (n=40), and sheep (n=40) were from routine submission to the dutch animal health service. sera from spanish domestic goats (n=40) were provided by the instituto de investigación en recursos cinegéticos (ciudad real, spain) from submissions for tuberculosis control in 2011. all sera were obtained in agreement with local regulations and dutch import regulations with regard to animal disease legislation. positive human control sera for the three antigens used on the microarray were taken as described previously. 24 all samples were stored at -20°c until testing. we tested the sera for the presence of igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc43 s1 antigens in a protein microarray. the receptor-binding domains, which contain the s1 subunit of spike proteins of mers-cov (residues 1-747), sars-cov (residues 1-676), and human coronavirus oc43 (residues 1-760) were expressed, purifi ed, and spotted on glass slides. slides were incubated with serum and species-specifi c conjugates, as previously described. 24 goat sera were incubated with alexa fluor 647-conjugated rabbit anti-goat igg fc fragment (jackson immuno research, west grove, pa, usa); combinations of the mean fl uorescent intensities of reactions of sera with mers-cov and human coronavirus oc43 antigens from 105 spanish dromedary camels (a). plaque reduction neutralisation tests for bovine coronavirus and mers-cov (b): two representative sera are shown (numbers 15 and 5, corresponding to camel id numbers in table 2) in dilutions of 1/40, 1/160, and 1/640 as well as the virus input control. all samples were tested in duplicates (only one well shown) and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90%. igg reactivity of both camel sera to mers-cov antigen and human coronavirus oc43 antigen in a two-step dilution series in the microarray (c). igg reactivity of two two-step serially diluted omani dromedary camel sera with human coronavirus emc antigen and human coronavirus oc43 antigen in the microarray (d). rfu=relative fl uorescence units. mers-cov=middle east respiratory coronavirus. cow sera with alexa fluor 647-conjugated goat antibovine igg (h+l; jackson immuno research); sheep sera with alexa fluor 647-conjugated donkey anti-sheep igg (h+l; millipore, temecula, ca, usa); and camelid sera with dylight 650-conjugated goat anti-llama igg (h+l; agrisera, vännas, sweden). fluorescence signals were quantifi ed as described previously. 24 we tested the sera for igg reactivity at a dilution of 1/20 and set an arbitrary cutoff at an average signal intensity of 20 000 relative fl uorescence units (rfu). this high cutoff was chosen to reduce cross-reactive false positives. 24 we present results as rfu for each set of quadruplicate spots per antigen. negative fl uorescent intensities (caused by background correction) were assigned to 0. analyses were done with graphpad prism (version 6.02). sera were heat-inactivated before virus neutralisation by incubation for 30 min at 56°c. two-fold serial dilutions of sera were prepared using 96-well plates, starting dilution 1/10. mers-cov was diluted in iscove's modifi ed dulbecco's medium (imdm) supplemented with clemizole penicillin (penicillin g), streptomycin, and 1% fetal bovine serum, to a dilution of 2000 tissue culture infective dose 50 per ml. 50 μl virus suspension was added to the plates and the plates were incubated at 37°c for 1 h. the mixtures of virus and serum were then incubated on 96-well plates containing vero cells for 1 h followed by washing with phosphate buff ered saline and incubation with imdm and 1% fetal bovine serum for 3-4 days, after which endpoint titres were measured. all tests were repeated twice independently. we tested neutralisation activity of sera against mers-cov (erasmus mc isolate) and bovine coronavirus (nebraska strain) by plaque reduction neutralisation test (90% plaque reduction) with african green monkey kidney cells (cell line vero b4; dsmz acc 33) or bovine kidney cells (cell line pt; cclv-rie11) in a 24-well plate format. virus (30-60 plaqueforming units) and heat-inactivated sera (diluted from 1/40 to 1/640) were pre-incubated in 200 μl of serumfree optipro medium (life technologies, karlsruhe, germany) at 37°c for 1 h. virus adsorption was done at 37°c for 1 h. supernatants were removed and overlaid with avicel resin (fcm biopolymer, brussels, belgium). 5 assays were stopped after 3 days by fi xation with 8% paraformaldehyde for 30 min. all samples were tested in duplicate and titres were expressed as the serum dilution resulting in a plaque reduction of at least 90%. the sponsors had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. sera were tested for igg antibodies reactive with mers-cov, sars-cov, and human coronavirus oc43 s1 antigens in a protein microarray (fi gure 1). human coronavirus oc43 is serologically closely related to bovine coronavirus, 26 diverging at the end of the 19th century. 10 bovine coronavirus circulates in cows, sheep, goats, and old and new world camelids. [27] [28] [29] [30] because bovine coronavirus s1 was not available, human coronavirus oc43 s1 antigen was used as a proxy. sera from three llamas, four alpacas, one guanaco, and two bactrian camels reacted with human coronavirus oc43 antigen. one cow and one goat serum reacted with human coronavirus oc43 antigen as did sera from 16 of 105 (15%) spanish dromedary camels. all sera from cattle, sheep, and goats tested negative for mers-cov antigen, but sera from 15 spanish camels (14%) did react with mers-cov antigen (fi gure 1). the reactivity was highly specifi c-the same sera did not bind to sars-cov antigen but a positive control specimen did. no correlation existed between the reactivity of sera with mers-cov antigen and human coronavirus oc43 antigen (fi gure 2). all but one serum sample that reacted with mers-cov antigen were from adult animals. one reactive serum was from a 2-year-old animal. to confi rm the presence of mers-cov specifi c igg in the spanish camel sera, we used a mers-cov neutralisation assay to test a subset of 49 camel sera with diff erent degrees of reactivity with mers-cov and human coronavirus oc43 antigen according to microarray. nine spanish camels had mers-cov neutralising antibodies with titres varying between 1/20 and 1/320 (table 1). three of the 12 sera reacted with mers-cov spike antigen but did not neutralise mers-cov, most likely because of recognition of nonneutralising epitopes. all mers-cov neutralising sera had (almost) saturating reactivity with mers-cov antigen on the microarray, whereas reactivity with human coronavirus oc43 antigen varied from negative to 50% of saturating reactivity (table 1). the variable human coronavirus oc43 signals suggest that mers-cov did not generally cross-react with human coronavirus oc43 or bovine coronavirus antigens. all nine camels with mers-cov neutralising antibodies were born and raised on the canary islands; seven were female, two were male. eight camels were adults, one was 2 years old. to show that the reactivity of the camel sera with human coronavirus oc43 antigen according to the microarray was caused by the presence of bovine coronavirus igg and to further exclude mers-cov neutralising activity caused by cross-neutralisation by the bovine coronavirus antibodies, we tested camels that had suffi cient serum left (n=15) in a comparative mers-cov and bovine coronavirus plaque reduction neutralisation test (fi gure 2, table 2). all camel sera neutralised bovine coronavirus, but with varying titres, suggesting a lower cutoff than 20 000 rfu for oc43 in the microarray (fi gure 1). five camels had high neutralising antibody titres against bovine coronavirus (and a mean signal intensity of greater than 50 000 rfu for human coronavirus oc43 antigen on microarray) but were negative for mers-cov neutralisation, suggesting that cross-neutralisation in this direction did not occur and that the mers-cov neutralising activity was not caused by the presence of bovine coronavirus neutralising antibodies. a serum sample from a patient who had mers, neutralised mers-cov with a high titre (1/640) but neutralised bovine coronavirus less effi ciently (titre 1/80). the latter fi nding was most probably caused by previous infection with human coronavirus oc43-this patient had a high titre (1/>5120) in a human coronavirus oc43 recombinant spike immuno fl uorescence assay and a saturating signal with human coronavirus oc43 antigen in the microarray. two human serum samples positive for human coronavirus oc43 did not neutralise mers-cov, one of which neutralised bovine coronavirus at a titre of 1/80 (table 2) . we tested 50 sera from dromedary camels in oman at a dilution of 1/20 by microarray and mers-cov neutralisation test. all the sera showed saturating reactivity with mers-cov antigen on the microarray, no sars-cov antigen reactivity, and human coronavirus oc43 antigen reactivity varying between negative (below the cutoff of 20 000 rfu) and saturating signals (fi gure 1, table 1). serial dilution of two sera with saturating reactivity for both antigens at the initial dilution of 1/20 showed that mers-cov antigen reactivity was still above the cutoff at 1/5120, whereas human coronavirus oc43 antigen reactivity fell below the cutoff at dilutions of 1/80 to 1/320 (fi gure 2d). consistent with the microarray data, all sera had high mers-cov neutralising capacity, with titres varying between 1/320 (seven of 50 samples) and 1/2560 or more (16 or 50 samples). in this study we describe the presence of mers-cov neutralising antibodies in dromedary camels both in a mers-cov linked (oman) and unlinked regions (canary islands). all the sera from dromedary camels from oman and some from spain had specifi c igg reactivity with the mers-cov receptor binding domain s1. we confi rmed our expectation that another betacoronavirus-bovine coronavirus-circulated in these camelids. 29 spanish camels (9%) had specifi c neutralising antibodies against mers-cov that were clearly not caused by cross-neutralisation by bovine coronavirus antibodies. our study is the fi rst in which animals have been tested for the presence of antibodies specifi c to mers-cov (panel). animal screening is necessary to understand the epidemiology of mers-cov. at present, bats are thought to be the ultimate reservoirs for several established human coronaviruses as well as sars-cov. accordingly, phylogenetic analysis has shown that mers-cov is related to betacoronavirus lineage c viruses found in pipistrellus spp bats. 15, 16 however, direct transmission of mers-cov to people from bats seems unlikely. 4, 19 the identifi cation of possible intermediate hosts that are probably in closer contact with people (eg, livestock) is urgently needed. common livestock species in the middle east include dromedary camels but also cattle, sheep, and goats. based on the available data, we cannot rule out circulation of a mers-related coronavirus in these species-sera were not available from epi demi ologically linked regions. the high prevalence of mers-cov neutralising antibodies in dromedary camels from oman suggests circulation of mers-cov or a closely related virus in this population. however, attempts to identify viral sequences in spanish camel sera and faecal samples using pancoronavirus and specifi c betacoronavirus 2c pcr methods 15, 31, 32 were unsuccessful (unpublished data), as was untargeted amplifi cation followed by deep sequencing of faecal samples (unpublished data). these results imply that the camels were not actively shedding the virus at the time of sampling. less than 10% of the animals in the canary islands had mers-cov neutralising sera with titres up to 1/320. this low seroprevalence means either that exposure of the animals to other putative reservoirs is rare 33 or that the virus is absent in this closed-off population of roughly 2000 animals. 25 we cannot rule out that the population might have once had an outbreak but that by the time of sampling, antibody titres had waned and no new introductions of the virus had occurred. the camels have contact with wild rodents, rabbits, pigeons, and doves and possibly also with bats. seven insectivorous bat species, including three pipistrellus spp, are native to the canary islands, while egyptian fruit bats (rousettus aegyptiacus) have been introduced. 34 the 100% seroprevalence with high titres in omani camels from diff erent owners and locations suggests a diff erent situation in the middle east, with widespread circulation of mers-cov or a closely related virus. this diff erence of epidemiology might be because the virus circulating in the middle east is diff erent to that circulating in spain, with increased animal transmissibility and human infections. 35 in addition, the omani camels were once racing camels now held for breeding and might be kept in circumstances that favour virus transmission. for cattle, a relation has been established between the incidence and eff ects of respiratory diseases, management practices, and animal transport. 36, 37 to our knowledge, the camel populations in oman and the canary islands are not connected. camels on the canary islands were originally imported in the 15th century from the horn of africa for labour and transport. nowadays, import of animals from africa is banned because of the risk of foot-and-mouth disease. only three camels in our study were originally imported from morocco, more than 18 years ago. because the closest relatives of mers-cov were identifi ed very recently in neoromicia zuluensis bats from africa, 38 the introduction of mers-cov or related viruses into some african camel populations could have occurred decades ago, giving a possible explanation for mers-cov antibodies in camels from the canary islands. in the middle east, huge numbers of camels are imported from africa to meet the demand for meat. the top fi ve camel breeding countries are all african, and saudi arabia and united arab emirates are in the top fi ve camel meat producing countries. 22 this increased turnover of animals in the middle east we searched pubmed for "novel coronavirus emc" or "mers-cov", we identifi ed 43 reports in english linked to the middle east respiratory syndrome coronavirus (mers-cov) published before july 22, 2013. none of these reports described a serological study for mers-cov-specifi c antibodies in animals. our report describes the fi rst mers-cov serological study of major livestock relevant to the middle east. our study shows that mers-cov or a related virus has infected dromedary camel populations. both titre levels and seroprevalences in sera from diff erent locations in oman suggest widespread infection of camelids with mers-cov or a closely related virus. targeted studies are needed to confi rm our fi ndings and their possible relevance to human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels from diff erent regions for which epidemiological background information is available, as well as virological assessment of samples from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime, we recommend a detailed case history of confi rmed mers-cov cases, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. compared with the canary islands could also aff ect the epidemiology of a virus, through more frequent infl ux of immunologically naive animals. targeted studies are needed to confi rm our fi ndings and their possible relevance in relation to the human cases of mers-cov. comparative seroprevalence testing of historical and more recent samples from camels for which epidemiological background information is available, as well as virological assess ment of specimens from seroconverting animals are needed to identify and characterise this mers-cov-related virus. in the meantime we recommend a detailed case history of people with mers-cov, with review of any animal exposures including animal products, and targeted, prospective serosurveys to establish whether camels or their products are a potential source of human infections. who. global alert and response (gar): novel coronavirus infection recovery from severe novel coronavirus infection family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov)-update interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk coronavirus diversity, phylogeny and interspecies jumping evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc43 severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats ecoepidemiology and complete genome comparison of diff erent strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe genetic characterization of betacoronavirus lineage c viruses in bats revealed marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications on the origin of the novel middle east respiratory syndrome coronavirus circulation of group 2 coronaviruses in a bat species common to urban areas in western europe comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections specifi c serology for emerging human coronaviruses by protein microarray camel trypanosomosis in the canary islands: assessment of seroprevalence and infection rates using the card agglutination test (catt/t. evansi) and parasite detection tests antigenic and biological relationships between human coronavirus oc43 and neonatal calf diarrhoea coronavirus serum antibodies to bovine coronavirus in swedish sheep serosurveillance of viral diseases in korean native goats (capra hircus) enteric coronavirus infection in a juvenile dromedary analysis of the genome sequence of an alpaca coronavirus the diff erential clinical impact of human coronavirus species in children with cystic fi brosis generic detection of coronaviruses and diff erentiation at the prototype strain level by reverse transcription-pcr and nonfl uorescent low-density microarray forecast and control of epidemics in a globalized world la fauna de quiropteros del archipelgo canario coronavirus genomics and bioinformatics analysis risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds associations between the distance traveled from sale barns to commercial feedlots in the united states and overall performance, risk of respiratory disease, and cumulative mortality in feeder cattle during 1997 to 2009 close relative of human middle east respiratory syndrome coronavirus in bat we thank prof mc horzinek for helpful suggestions. rds was funded by the european public health training program (euphem), ecdc, stockholm, sweden. contributions to the study were funded through the european union fp7 projects emperie (contract number 223498; to blh, sls, ao, cd) and antigone (contract number 278976; to cg, cd, mpgk, ao). work in bonn was also funded by deutsche forschungsgemeinschaft (dfg grant dr772/3-1 to cd). key: cord-312178-tojgojjf authors: segars, james; katler, quinton; mcqueen, dana b.; kotlyar, alexander; glenn, tanya; knight, zac; feinberg, eve c.; taylor, hugh s.; toner, james p.; kawwass, jennifer f. title: prior and novel coronaviruses, covid-19, and human reproduction: what is known? date: 2020-04-16 journal: fertil steril doi: 10.1016/j.fertnstert.2020.04.025 sha: doc_id: 312178 cord_uid: tojgojjf structured abstract objective to summarize current understanding of the effects of novel and prior coronaviruses on human reproduction, specifically male and female gametes, and in pregnancy. design review of english publications in pubmed and embase to april 6, 2020. methods manuscripts were screened for reports including coronavirus, reproduction, including pathophysiology and pregnancy. intervention(s) none. main outcome measure(s) reproductive outcomes; effects on gametes; pregnancy outcomes; neonatal complications. results seventy-nine reports formed the basis of the review. coronavirus binding to cells involves the s1 domain of the spike protein to receptors present in reproductive tissues, including angiotensin converting enzyme-2 (ace2), cd26, ezrin, and cyclophilins. sars-cov-1 may cause severe orchitis leading to germ cell destruction in males. reports indicate decreased sperm concentration and motility for 72-90 days following covid-19 infection. gonadotropin-dependent expression of ace2 was found in human ovaries, but it is unclear whether sars-cov-2 adversely affects female gametogenesis. evidence suggests that covid-19 infection has a lower maternal case fatality rate than sars or mers, but anecdotal reports suggest that infected, asymptomatic women may develop respiratory symptoms postpartum. covid-19 infections in pregnancy are associated with preterm delivery. postpartum neonatal transmission from mother to child has been reported. conclusion covid-19 infection may adversely affect some pregnant women and their offspring. additional studies are needed to assess effects of sars-cov-2 infection on male and female fertility. the rapid spread of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has led to a pandemic of coronavirus disease 2019 (covid-19) across the globe. as of april 8, 2020 , there are over 1.4 million cases and 86,000 deaths attributed to the virus worldwide. as a result, nations have implemented suppression and mitigation strategies to control community spread, including mandated social distancing, restrictions to non-urgent medical care, and closure of non-essential businesses. despite these efforts, the spread of sars-cov-2 is ongoing, creating a public health crisis and impacting the population world-wide. coronaviruses are a group of viruses that can cross species barriers and become human pathogens. all seven identified human coronaviruses originated from animal reservoirs including domestic animals, bats, or mice. while most human coronaviruses cause mild illness, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and the novel sars-cov-2 have been associated with severe lower respiratory tract infections, acute respiratory distress syndrome, and death (1). the novel sars-cov-2 virus spreads rapidly, with 2-3 people infected from every index case, a reproduction number (r 0 ) or transmission rate of 2.24 -3.58 (2) . in contrast, the 2009 h1n1 seasonal influenza had an r 0 of 1.46-1. 48 . the incubation period for sars-cov-2 ranges from 2-14 days, and asymptomatic spread occurs prior to onset of symptoms (3, 4) . transmission is thought to be mainly through respiratory droplets and fomites (5) . in one experiment, viable virus was detected in aerosols for up to three hours, with an estimated half-life of 1.1 hours. in addition, virus was detected on surfaces for days after application, with viable sars-cov-2 identified on plastic and stainless steel up to 72 hours (6). sars-cov-2 rna has also been detected in blood and stool and it is not yet known whether the infection can be acquired through exposure to non-respiratory bodily fluids (7) . symptoms of sars-cov-2 infection include fever, cough, fatigue, shortness of breath, sputum production, headache and myalgias. in addition, patients may report gastrointestinal symptoms (8) or anosmia. the severity of infection ranges from asymptomatic carriers, to mild flu-like disease, to critical illness and death. critically-ill patients may experience respiratory failure, shock or multiorgan dysfunction. approximately 80% of infections are mild with flu-like symptoms, 15-20% are severe, requiring hospitalization and supplemental oxygen, and 5% are critical and require mechanical ventilation (9). risk factors for severe illness include age and underlying medical comorbidities such as cardiovascular disease, diabetes, chronic respiratory disease, hypertension and cancer (10) . death may occur in up to 3% of infections. death from sars-cov-2 is more common in individuals over age 60 or with underlying medical issues but can occur in younger persons, perhaps related to the inoculum. associated cardiac arrhythmias may be fatal. while persons of advanced age are most likely to experience severe symptoms, women of reproductive age are also at risk for development of severe disease and death. furthermore, reproductive age women can act as asymptomatic carriers and increase viral transmission. the aim of this review is to summarize what is currently known about the impact of prior coronaviruses and the novel sars-cov-2 infection on reproduction and pregnancy. a systematic search in the literature published in the pubmed and embase databases was conducted in accordance with the guidelines of the preferred reporting items for systematic reviews and meta-analyses (prisma). literature review of available literature in english, both published and peer-reviewed on-line publications from 1996-april 6, 2020. additional sources were identified from citations of retrieved literature. search strategy for the pubmed and embase searches is shown below. there was no limitation on sample size. published reports of experiences with the sars-cov-2 virus have increased greatly in the past months, but no randomized trials to date regarding possible treatments were identified. most reports pertaining to reproduction or pregnancy involved small cohorts, case reports, guidelines, and editorials. similarly, prior publications of other coronaviruses were limited in number. we reviewed all titles for eligibility. all reports including pregnancy or reproductive tissues were included. we excluded editorials and publications of guidelines. we excluded papers that were duplicates or did not contain information related to pregnancy or reproduction, the presence of virus in reproductive tissues, effects on gametes, pregnancy outcomes, or neonatal complications. case reports were included. because of the limited nature of the reports and the absence of randomized trials we did not use the cochrane rob 2.0 tool. similarly, because of the limited scope of the cohort studies, we did not use the newcastle-ottawa scale to rate the studies. full text articles were then reviewed and evaluated for inclusion by authorship teams. two independent reviewers (j.s. and z.k.) any disagreements were resolved through discussion with third reviewer (j.k.). the authors acknowledge the number of publications about the novel sars-cov-2 virus is increasing at an exponential rate and there may be bias toward reporting of positive findings. we used a broad inclusive search strategy so as not to miss a seminal contribution. the the search revealed 663 manuscripts after removal of duplicates. ninety-seven manuscripts related to pregnancy and coronavirus and only 7 were related to embryos or early reproduction. small cohorts, case reports, comments on guidelines, guidelines, editorials were retrieved. after exclusion, 79 manuscripts were included in the review based on relevance and new data. a review of the coronavirus structure and function help to inform us on the pathophysiology of coronavirus infectivity. coronaviruses are large, single-stranded, enveloped rna viruses approximately 32kb (11) . the viral rna genome is housed inside a nucleocapsid, which itself is contained within a viral envelope (12) . this envelope comprises three distinct proteins: a "membrane protein" and "envelope protein," which are both directly responsible for viral assembly, as well as a "spike protein," which mediates viral entry into host cells (12) . when viewed with electron microscopy, these spike proteins are surface-exposed markers that produce a recognizable "crown-like" appearance to the virus. the spike proteins serve a critical step in initiating human infection, as well as determining host tissue specificity and inducing host immune response (13) (14) (15) . the coronavirus spike protein is composed of two unique subunits that facilitate viral-host binding (16) (17) (18) (19) . the s1 domain of the spike protein functions in viral binding and attachment to the host cell membrane. numerous receptors on the human cell membrane that are involved in s1 subunit binding have been identified to date, including angiotensin converting enzyme-2 (ace2), cd26, ezrin, and cyclophilins (20, 21) . the s2 domain of the spike protein is responsible for fusion of the viral and host cell membranes, allowing the sars-cov-2 viral genome to enter the host cell. this process involves a complex interaction between viral and host machinery, which culminates in rapid viral replication within target cells. international research efforts are currently underway aimed at using the s1 domain of the spike protein as a target for therapeutic anti-viral therapy, as well as vaccine development. the involvement of sars-cov-2 infection within human male and female reproductive systems has yet to be fully elucidated. however, results from other coronavirus subtypes, specifically sars-cov, help inform knowledge of tissue-specific viral pathophysiology. current data suggest that the female reproductive system may be spared from viral infection. immunohistochemical and in situ hybridization studies on tissues from a small cohort of deceased patients that were infected with sars-cov failed to identify sars-cov viral rna within the female reproductive tract, including both ovarian and uterine tissue (22) , though ace2 receptors can be found there. importantly, there is evidence to suggest that coronavirus infection may impact the male reproductive tract. the ace2 protein, a main receptor for coronavirus viral entry, is selectively expressed by leydig cells of the adult testes (23) . there are numerous reports of male reproductive injury after sars-cov infection. leading theories postulate that this is an immune-mediated response to infection since direct inoculation of the coronavirus rna within testicular tissue has not been described. in a 2005 study of eight postmortem sars-cov patients, testicular tissue contained focal atrophy despite lacking identifiable sars viral rna (24) . accordingly, there have been reported cases of sars-cov causing severe orchitis, as evidenced by extensive igg precipitation in testicular interstitial tissue causing germ cell destruction and widespread testicular leukocyte infiltration (25) . further studies on the reproductive involvement of coronavirus infections are warranted, particularly within recovered patients. there are limited data regarding the impact of sars-cov-2 on human reproduction as the virus is novel and has only recently infected humans. to date, there have been no reports of the virus in the female reproductive tract, in vaginal secretions, in amniotic fluid or in peritoneal fluid. although there is nothing to suggest that female or male gametes would be impacted directly by infection with sars-cov-2 or other coronaviruses, there is evidence that fever can impact spermatogenesis. therefore, male fertility may be diminished for 72-90 days following covid-19 due to decreased sperm concentration and motility (26, 27) . the sars-cov-2 virus utilizes ace2 receptors to gain entry into the human cells. the male reproductive system expresses ace2 within adult leydig cells in the testis and there are data to suggest that ace2 plays a role in spermatogenesis. the presence of ace2 receptors is much more prominent in the male reproductive system than the female reproductive system, but gonadotropin-dependent expression of ace2 has been reported in human ovaries (28, 29) . at this time, it is unknown if the sars-cov-2 virus utilizes ace2 receptors in the reproductive system and what, if any, impact this would have on oocyte quality, embryo development, or ensuing pregnancy. gametes obtained from patients with other viral illnesses, such as hiv and hepatitis, must be treated with special precautions aimed at reducing exposure of the non-infected partner and cross contamination of reproductive tissue within the laboratory (30) . these precautions are not currently recommended for sars-cov-2, given the lack of evidence for transmission through blood or sexual contact (31) . similarly, there is no current recommendation for screening oocyte or sperm donors for sars-cov-2. these are areas in which further investigation is necessary in order to assure the safety of stored gametes and the safety of patients undergoing assisted reproduction. the case fatality rate for all reported cases of sars in pregnancy (n=17) was 15% (32-37), and was higher among pregnant women. mechanical ventilation was required three times more often in pregnant versus non-pregnant women. a case control study comparing ten pregnant to forty non-pregnant women affected by sars reported a 60% icu admission rate and a 40% case fatality rate (cfr) in the pregnant group compared to only an 18% and 0% rate in the nonpregnant group (32). four of seven pregnant patients with sars miscarried in the first trimester (37) . four of five women infected after 24 weeks gestation were delivered prematurely, largely due to their deteriorating maternal condition from sars, at 26, 28 and 32 weeks gestation (38) . the infants born at 26 and 28 weeks developed rds requiring surfactant, but were normal weight for gestational age. however, among infants born weeks (5 to 7) after initial infection, fetal growth restriction and fibrin deposition on placental interface was seen in two of three pregnancies, suggesting compromise. there was no evidence of vertical transmission. overall, sars was a very serious disease for pregnant women and their fetuses. among pregnant patients with mers (n=12), 63% were admitted to the icu (7/11) and three died (cfr of 23%) (39) (40) (41) (42) (43) . the only woman known to contract mers in the first trimester went on to deliver a healthy infant at term (44) . of those contracting the disease after the first trimester (n=9), one had a spontaneous loss at 20 week gestation (45) , one presented at 34 weeks gestation with pre-eclampsia and had an intrauterine fetal demise (40) , and a third with preexisting respiratory issues developed mers at 24 weeks gestation and then ards. despite ventilation and cesarean delivery, both the neonate and mother subsequently died (40) . three of nine other patients were delivered preterm due to maternal hypoxia. mers also had a high fatality rate and premature delivery rate. through april 5, 2020, there have been 19 case reports or series and 2 case-control reports of intrauterine growth restriction (iugr), 13 were delivered prematurely, 12 were small for gestational age (sga) and 1 was large for gestational age (lga). one stillbirth and one neonatal death were reported. table 2 summarizes the pregnancy-related experience of the three coronavirus epidemics. peerreviewed case reports to date suggest the following: • covid-19 has a lower maternal case fatality rate than either sars or mers. • pregnant patients display similar signs and symptoms of covid-19 as non-pregnant patients. • pregnant patients are not more susceptible to coronavirus infection, nor at higher risk for severe illness. • severe illness may precipitate premature labor or lead to early delivery. • adverse infant outcomes have been reported, but it is unclear whether these outcomes are directly related to covid-19 infection. given the teratogenicity concerns associated with viruses such as zika, there remains the however, a recent case report in jama (50) suggests that vertical transmission may be possible. in this case report, an otherwise healthy infant was born by cesarean section to a 29-year-old with rt-pcr confirmed sars-cov-2 infection. this infant was immediately placed into isolation and a blood sample at two hours of age was noted to show an elevated sars-cov-2 igg. while the igg can be secondary to transplacental transfer, the infant was also positive for sars-cov-2 igm which cannot be explained by maternal-fetal transfer (50) . furthermore, igm antibodies only appear three-to-seven days after infection. all five rt-pcr tests on the infant were negative for sars-cov-2 (50). the antibody profile of this infant is suggestive of exposure to sars-cov-2 in-utero. a follow-up study done on six infants born to covid-19positive mothers showed positive igm antibodies in two of them. yet, all throat swabs and blood samples from the neonates tested negative for the virus. overall, evidence of vertical transmission in the setting of covid-19 infection is currently inconclusive. continued observation is necessary as more data is gathered during the course of this pandemic. as of march 31, 2020, there were no identified vaccines or targeted therapies for the treatment of covid-19. currently, treatment is aimed at supportive methods such as oxygenation/mechanical ventilation and treating complications. as no effective treatment has been identified, a barrage of potential therapies are being trialed both within and outside of research protocols. although this list is ever-changing, the majority of these medications target the ability of the virus to replicate or are designed to suppress or modulate the immune system, thereby limiting inflammatory damage (67, 68) . during pregnancy these potential remedies fall into three different departments: those established to be safe in pregnancy, those with unknown status, and those which have clear or relative contraindications during pregnancy. it is imperative to consider that although some of these interventions are safe in pregnancy, none so far have been identified as definitive treatment and all have potential risks. these concerns need to be considered as an additional risk assumed by pregnant women; some potential therapies may carry risk to the fetus or be unavailable to pregnant women. several investigated medical treatments for covid-19 are known either to be safe or nonteratogenic in pregnancy. these include various immune modulators such as hydroxychloroquine/chloroquine, methylprednisolone/glucocorticoids, and the anti-viral medications lopinavir-ritonavir (69) (70) (71) (72) (73) . it is important to note that although non-teratogenic, the use of glucocorticoids in pregnancy has been associated with diabetes, weight gain, preterm premature rupture of membranes, hypertension, and intrauterine growth restriction. interferon therapy, often used in hepatitis c treatment, has been well established to be safe in pregnancy (74) . the use of convalescent plasma, or plasma derived from individuals who previously contracted covid-19, has been shown to reduce disease sequela in those with severe disease (75) . given that its risk profile should be similar to a blood transfusion, there are no overt contraindications in pregnancy. east respiratory syndrome coronavirus in monkeys (76) . although current clinical studies exclude pregnant women, remdesivir may be administered in critically-ill pregnant patients (nct0428070500) (77) . to date, no information is available concerning the effects of remdesivir in pregnancy. due to the increased release of cytokines and subsequent inflammatory damage during covid-19, tocilizumab, a monoclonal antibody that binds the receptor il-6, has been used in china, with at least one clinical trial that is ongoing (nct04310228) (78, 79) . a major concern for pregnant patients is that igg isotype antibodies cross the placenta at an increasing rate in accordance with gestational age (79) . however, the effect of each medication on fetal development is unknown. furthermore, in the rheumatology literature it is recommended that tocilizumab be avoided during pregnancy given the lack of data on adverse fetal effects (80) . other anti-viral medications which have been utilized that have clear evidence of teratogenicity, either in humans or animal models, include favipiravir and ribavirin (68, 81, 82) . notably, the aforementioned medications are experimental, thus it is imperative to have a risk-benefit discussion with patients given the medication side-effect profiles, potential effects in pregnancy, and unknown benefits. until randomized trials with adequate controls are completed, it is difficult to draw definitive conclusions concerning the efficacy of any of these treatments (71) . thus far, there has been no indication that infants born to covid-19 positive mothers experience any significant morbidity or mortality. in the aforementioned case series of nine covid-19-positive patients who underwent cesarean section, all of their infants had negative covid-19 testing and had 1-min apgar scores of 8-9 and 5-min apgar scores of 9-10. in addition, none of the infants in this series experienced asphyxia or neonatal death (47) . three newborns have been confirmed with covid-19 and their infection was likely secondary to exposure to infected caregivers. all three neonates experienced either fever, cough, or vomiting, then subsequently recovered with no severe sequelae (67) . given the disease course seen in older children, the disease severity can be expected to be worse for neonates with pre-existing cardiac or pulmonary conditions such as congenital heart disease (67). there are a significant number of unknowns concerning covid-19 and pregnancy, especially regarding the infectivity of a pregnant woman. therefore, the majority of data are extrapolated from previous experience with other coronaviruses, sars-cov and mers-cov, as well as influenza pandemics such as the 1918 flu and the asian flu in the late 1950s (35, 83) . however, pregnant women are not more likely than non-pregnant woman to become infected with sars-cov-2, based on data from regions with the first covid-19 exposures and from previous pandemics (84) . nor do pregnant women appear to suffer a more severe disease course or higher mortality rate compared to non-pregnant women of a similar age. it has been established that sars-cov-2 is spread by respiratory droplets, yet the potential for other routes of transmission, particularly during labor, is unknown (84) . limited data are available for covid-19, but it is possible to extrapolate from limited information available on sars-cov-2 and other coronaviruses. in the sars-cov outbreak, small case reports showed no viral rna in placental, umbilical cord blood, amniotic fluid, or breastmilk (35, 37, 84) . it is important to note that fecal matter that was aerosolized via the flushing of toilets was noted to spread disease (35) . one could hypothesize that amniotic fluid could become aerosolized and infect others if viral rna was present through mixing with fecal matter. mers-cov had an extremely high death toll, however only twelve cases were documented in pregnancy, and no information is available on viral rna in pregnancy-specific tissue (35) . to date, there is limited information concerning which fluids in labor could potentially transmit sars-cov-2 directly or by aerosolization. while a single maternal vaginal swab in the only suspected case of vertical transmission was negative, it is too early to conclude whether transmission can occur via vaginal secretions (50) . rectal swabs have not been obtained in pregnant patients; however, a series of ten pediatric patients did confirm sars-cov-2 rna in rectal specimens (85) . thus, information regarding vaginal deliveries, while limited, remains concerning. as previously noted, case reports have shown no viral rna in amniotic fluid, cord blood, or breast milk (47) . until more information is gathered, it is prudent for personnel present during delivery to treat all fluids as potentially contagious and wear appropriate personal protective equipment. on january 7, 2020 thanks to the initial efforts of the late dr. jixian zhang, the sars-cov-2 virus was identified as a cause of severe cases of pneumonia that began on december 8 in wuhan, china. in the first three months of 2020, the novel virus has infected over 1.4 million people and caused more than 87,000 deaths. declared a pandemic by the who on march 11, 2020, the sickness has overwhelmed healthcare resources as it spread. containment of sars-cov-2 has proven exceptionally difficult due to asymptomatic and pre-symptomatic spread of disease, as well as the relatively high person-to-person transmission. as the virus moved from host to host, as is typical of the single-stranded rna viruses, sars-cov-2 has mutated. notably, there is an enormous phenotypic difference in disease severity from asymptomatic infection to death, but the reasons for this striking variability remains obscure. despite the overwhelming magnitude of the disease and its worldwide prevalence, information regarding the effects of the novel coronavirus on human reproduction are currently limited. this lack of evidence should not be considered reassuring, since only three months have elapsed since the novel coronavirus jumped species and infected humans. there is reason to be concerned based on data from other coronaviruses. as is typical of this family of viruses, the sars-cov-2 spike protein binds the ace2 receptor, a protein found in many reproductive tissues, including the testis. of concern, evidence exists that related coronaviruses have caused severe orchitis. while sperm counts can be reduced by high fever alone, the question of other possible long-term effects on male and female gametes is pressing. specifically, whether there might be shedding of virus in some individuals that might affect the safety and storage of gametes. as noted, evidence continues to emerge regarding effects of the novel coronavirus in pregnancy and some initial reports suggest that complications, particularly after delivery, may be increased. additional studies are urgently needed. because of the risk of viral transmission between patients, staff, physicians and providers, and to comply with local restrictions for non-emergency surgeries, many programs of assisted reproduction have suspended procedures throughout the globe during the height of the pandemic. as the assisted reproductive technology (art) programs resume operations it will be important to gather information regarding the status of individuals infected with the novel coronavirus, and to assess gametes and reproductive outcomes for those who had covid-19. these studies will be enabled by the availability of serologic testing and more widespread rna testing for the novel coronavirus. likewise, researchers should collect data regarding outcomes of pregnancies in women who became infected during pregnancy. while a vaccine may prevent disease, until one is available, information is needed on the safety of medical treatments and outcomes in pregnancy. one limitation of this review is that because of the rapidly increasing number of publications, it is possible that new information may be published contradicting the findings. because of concern about possible adverse effects, there may be a risk of bias toward reporting positive outcomes. additionally, since reports have focused on the acuity of treatment, the reproductive effects may be present, but not yet reported. the strength of the report is that our search was comprehensive and conducted, as much as possible, in accordance with prisma guidelines. the immense impact of the covid-19 pandemic in lives lost, healthcare expenses, and economic consequences of countries and individuals is inestimable because the epidemic is still rampant throughout the globe. while data are limited, and incomplete at this time, there is justifiable concern that reproductive consequences of the novel coronavirus may have lasting effects for male reproduction and for some pregnant women and children. zoonotic origins of human coronaviruses preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 professionals: frequently asked questions and answers clinical characteristics of covid-19 patients with digestive symptoms in hubei, china: a descriptive, cross-sectional, multicenter study interim clinical guidance for management of patients with confirmed coronavirus disease (covid-19) role of changes in sars-cov-2 spike protein in the interaction with the human ace2 receptor: an in silico analysis structure, function, and evolution of coronavirus spike proteins human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptorbinding site in spike protein domain a structural insights into coronavirus entry human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains ezrin interacts with the sars coronavirus spike protein and restrains infection at the entry stage cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways the novel angiotensin-converting enzyme (ace) homolog, ace2, is selectively expressed by adult leydig cells of the testis multiple organ infection and the pathogenesis of sars orchitis: a complication of severe acute respiratory syndrome (sars) history of febrile illness and variation in semen quality influence of genital heat stress on semen quality in humans angiotensin-converting enzymes play a dominant role in fertility recommendations for reducing the risk of viral transmission during fertility treatment with the use of autologous gametes: a committee opinion patient management and clinical recommendations during the coronavirus (covid-19) pandemic a case-controlled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome severe acute respiratory syndrome (sars) in neonates and children sars and pregnancy: a case report potential maternal and infant outcomes from (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections infants born to mothers with severe acute respiratory syndrome pregnancy and perinatal outcomes of women with severe acute respiratory syndrome impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia mers-cov infection in a pregnant woman in korea middle east respiratory syndrome coronavirus during pregnancy emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature stillbirth during infection with middle east respiratory syndrome coronavirus covid-19 in pregnancy: early lessons clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical analysis of pregnant women with 2019 novel coronavirus pneumonia infants born to mothers with a new coronavirus (covid-19). in: front pediatr possible vertical transmission of sars-cov-2 from an infected mother to her newborn should we worry? clin infect dis pregnancy and perinatal outcomes of women with coronavirus disease (covid-19) pneumonia: a preliminary analysis maternal and neonatal outcomes of pregnant women with covid-19 pneumonia: a case-control study premature baby in serious condition after coronavirus infection coronavirus disease 2019 (covid-19) during pregnancy: a case serie a case report of neonatal covid-19 infection in china a case of 2019 novel coronavirus in a pregnant woman with preterm delivery clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study a pregnant woman with covid-19 in central america antibodies in infants born to mothers with covid-19 pneumonia neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia effects of coronavirus infections in children middle east respiratory syndrome an analysis of 38 pregnant women with covid-19, their newborn infants, and maternal-fetal transmission of sars-cov-2: maternal coronavirus infections and pregnancy outcomes coronavirus disease (covid-19) and neonate: what neonatologist need to know development of a standard treatment protocol for severe acute respiratory syndrome immune modulating therapies in pregnancy and lactation a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 treating covid-19-off-label drug use, compassionate use, and randomized clinical trials during pandemics recommendations for the use of antiretroviral drugs in pregnant women with hiv infection and interventions to reduce perinatal hiv transmission in the united states effects of chloroquine on viral infections: an old drug against today's diseases? a systematic review of the fetal safety of interferon alpha treatment of 5 critically ill patients with covid-19 with convalescent plasma prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection adaptive covid-19 treatment trial (actt) favipiravir combined with tocilizumab in the treatment of corona virus disease the eular points to consider for use of antirheumatic drugs before pregnancy, and during pregnancy and lactation favipiravir as a potential countermeasure against neglected and emerging rna viruses the ribavirin pregnancy registry: an interim analysis of potential teratogenicity at the mid-point of enrollment pneumonia in pregnancy interim considerations for infection prevention and control of coronavirus disease 2019 (covid-19) in inpatient obstetric healthcare settings covid-19: faecal-oral transmission? the authors thank drs. catherine racowsky and dr. ricardo azziz. key: cord-312691-ynh84b98 authors: mohd, hamzah a.; memish, ziad a.; alfaraj, sarah h.; mcclish, donna; altuwaijri, talal; alanazi, marzouqah s.; aloqiel, saleh a.; alenzi, ahmed m.; bafaqeeh, fahad; mohamed, amal m.; aldosari, kamel; ghazal, sameeh title: predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia date: 2016-09-24 journal: travel med infect dis doi: 10.1016/j.tmaid.2016.09.008 sha: doc_id: 312691 cord_uid: ynh84b98 background: a case control study to better characterize the clinical features, laboratory, and radiological abnormalities associated with mers-cov infection in order to help with early identification of this syndrome from other respiratory infections. methods: eighty patients admitted to a hospital in riyadh, diagnosed with mers-cov infection based on rt-pcr were matched on age, sex, and the presence of a co-morbid condition on a basis of 1:2 to other patients admitted with respiratory symptoms and tested negative for mers-cov on rt-pcr. results: none of the reported mers-cov presenting symptoms was significantly associated with being infected with mers-cov. on the other hand, wbc count was significantly lower in patients with confirmed mers-cov infection (median 5.7 vs 9.3, p: 0.0004). neutrophil count was as well significantly lower in mers-cov patients (median 3.7 vs 6.7, p: 0.0001). both ast, and alt values were significantly higher in mers-cov infected group (ast median 42 vs 36, p: 0.03, and alt median 33 vs 28, p: 0.003). overall our mers-cov mortality rate was (10%) below the national figure of (40%). conclusions: none of the presenting symptoms are specific for mers-cov infection. and out of all the investigations wbc, neutrophil counts, ast and alt values have some predictive utility. mers-cov is a novel betacoronavirus that was discovered in 2012 after it was isolated from the respiratory secretions of a patient in saudi arabia who died with acute respiratory syndrome [1] . so far, all reported cases were diagnosed in the arabian peninsula (mainly saudi arabia) or epidemiologically linked to it [2e7] . up to april 2016 more than 1360 cases have been reported in saudi arabia alone with a mortality rate that exceeded 40% [8] . though the disease is believed to be zoonotic with strong evidence towards a camel reservoir [9e14], major outbreaks are believed to be nosocomial involving transmission within health care facilities [2,15e18] . early after its discovery, mers-cov infection screening was limited to those critically ill with severe acute respiratory illness [17,19e21] . later on the saudi arabian ministry of health issued guidelines for mers-cov screening in which they limited suspect cases to patients with clinical or radiological evidence of pneumonia, patients with respiratory illness along with a history of possible exposures to a mers-cov patient, and patients with unexplained acute febrile illness with hematological laboratory abnormalities (leukopenia or thrombocytopenia) and gastrointestinal or respiratory symptoms [22] . many studies were conducted in saudi arabia to characterize the illness associated with mers-cov [17,19e21] , but they were limited by the small sample size. studies done elsewhere were limited as well by the extremely small sample size [3e7], except in south korea where a large outbreak occurred between may and july of 2015 due to an imported case from the arabian peninsula [2] . travel associated cases have been observed in europe, notably in uk, france, germany, austria and italy with secondary cases in close contacts of index cases without a travel history suggesting person-to-person transmission [22e25] . in this study we tried to overcome the previous limitation by studying a larger cohort of mers-cov patients at our facility and comparing it to a control group in an attempt to look for predictors of mers-cov infection. in this study we followed a cohort of patients tested for mers-cov infection at prince mohammed bin abdulaziz hospital (pmah) emergency department, a governmental hospital in riyadh, saudi arabia, between april 1st 2014 and september 30th 2015. patients who were felt to have influenza like illness (ili) were screened for mers-cov and all patients who required admission and had any respiratory symptom (cough, shortness of breath, sore throat) were screened for mers-cov infection and followed up until discharged. we also included patients who were called for admission after a positive mers-cov test that was done at an earlier visit to our emergency department and individuals who were hospital quarantined due to a significant exposure history to a mers-cov patient. as pmah is the mers-cov referral center for governmental (public) and private hospitals in riyadh, saudi arabia, we excluded patients who were already diagnosed with mers-cov at other facilities and referred to our hospital for isolation and further management. due to the great heterogeneity of this cohort, and in an attempt to look for predictors of mers-cov infection, we matched the patients who tested positive for mers-cov (cases) to a control group from the rest of the study population who were admitted and repeatedly tested negative for mers-cov. we extracted a matched control for the positive cases based on age (within 5 years of age), gender, and presence of any comorbidity. the attempted matching was 1 case to 2 controls. respiratory samples (nasopharyngeal swapping or tracheal aspirates) were obtained from all patients and tested for mers-cov infection using real-time reverse-transcription polymerase chain reaction (rt-pcr). the specimens were submitted to and testing was carried out at the saudi ministry of health mers-cov regional laboratory. the test amplified both the upstream e protein (upe gene) and orf1a for mers-cov. a positive case was determined if both assays were positive. each patient was tested at least twice, each on a different day. reporting suspected and confirmed cases was the original purpose of data collection which was initially limited to date of admission, sex, age, nationality, designated hospital ward, results/dates of mers-cov testing, and patients' outcomes. data concerning presenting symptoms: fever, sore throat, cough, shortness of breath, and gastrointestinal symptoms (nausea, vomiting or diarrhea), as well as the initial laboratory work up: white blood cell count (wbc) 10 9 per liter, hemoglobin (hgb) grams per deciliter, platelets 10 9 per liter, creatinine micromoles per liter, albumin gram per liter, aspartate aminotransferase (ast) units per liter, alanine aminotransferase (alt) units per liter, and initial chest-x-ray (cxr) results were later extracted from the electronic medical records. we summarized the data by descriptive statistics. frequencies and percentages were calculated for categorical variables. continuous variables (mainly laboratory values) tended to have skewed distributions; thus we used medians rather than means. a conditional logistic regression analysis was conducted to identify the variables that are independently associated with mers-cov infection. the magnitude of association, presented as the odds ratio and 95% ci was also determined this way. all reported p values in this article are also based on the logistic regression. the laboratory measures were found to not be linearly related to the logit of mers-cov infection, so the values were broken into 4 approximately equally sized groups (via quartiles, referred to as quarters) and odds ratios estimated for each as compared to the first quartile as reference. note that for analysis of laboratory measures, a few people had to be excluded due to missing values. we used sas version 9.4 (sas institute, cary nc) to perform all of the analysis. the significance level for all of the statistical tests was set at 0.05. from 24,606 patients who presented to our emergency department between april 1st 2014 and september 30th 2015 with respiratory complaints, 3148 were tested for mers-cov. from those who were screened, only 757 patients were admitted. eighty one patients tested positive for mers-cov and the rest, 676 patients, repeatedly tested negative and were used to find matches to mers-cov cases. see fig. 1 . we were able to match all but two of the confirmed mers patients. for one of them we were only able to find one control, and for the other no match was found. this resulted in a total of 239 patients for analysis. table 1 shows the characteristics of both the mers-cov infected group and the control group. the median age for mers-cov patients was 40 years; 60% were males, 66.2% were saudi, and 16.2% were health care workers. 46% had at least one co morbidity; hypertension and diabetes were the most common. 80% of mers-cov patients were symptomatic; fever was the most common symptom among mers-cov infected group 58.7% (73% of the symptomatic patients), followed by cough 52.5% (73% of symptomatic patients). shortness of breath was the third common symptom that was reported by 37.5% of mers-cov infected patients (47% of the symptomatic patients). around 17.5% of mers-cov infected patients reported gi symptoms (22% of the symptomatic patients). sore throat was reported in 13.7% (17% of the symptomatic patients). in mers-cov infected group the median value for wbcs was 5.7 (iqr: 4.1e8.6), neutrophils 3.6 (iqr: 2.2e5.3), hgb 145 (iqr: 128e157), platelets 205 (iqr: 148e270), albumin 37 (iqr: 34e42), ast 42 (iqr: 26e105), alt 33 (iqr: 21e93), and creatinine 68.1 (iqr: 60.8e81.9). all of the above mentioned medians were within the normal ranges. almost 51% of mers-cov patients had chest x-ray findings upon admission. around 19% were sick enough to be admitted directly to the icu, and around 10% of mers-cov infected patients expired. as discussed above, matching was based on age (within 5 years of age), gender, and presence of any comorbidity. 64.8% of the control group were saudi and11.3% were health care workers. hypertension and diabetes were the most common comorbidities. 95% of the control group patients were symptomatic; fever 64.1% and cough 59.1% were the most commonly reported symptoms in the control group. around 35% complained of shortness of breath, and 14.5% had a sore throat. 13.8% of the control group reported gi symptoms. laboratory analysis showed a median wbcs value of 9.3 (iqr: 6.0e14.2), neutrophils 6.7 (iqr: 3.7e11.1), hgb 138 (iqr: 114e153), platelets 238 (iqr 170e338), albumin 36 (iqr: 32e41), ast 36 (iqr: 23e62), alt 28 (iqr: 14.5e48.5), and creatinine 70.1 (iqr: 61.4e86.9). almost 58% of the control group patients had chest x-ray findings upon admission. around 16.3% of the control group patients were sick enough to be admitted directly to the icu on presentation, and 4.4% of the control group patients expired during their hospital stay. there was no statistical difference in the proportion of saudi nationals (66.2% vs 64.8% or, 1.07; p z 0.82) as well as health care workers (16.2% vs11.3% or, 2.20; p z 0.16) between the confirmed and matched groups. though confirmed mers-cov patients were statistically less likely to be symptomatic (80% vs 95% or: 0.21; p: 0.001), no statistically significant differences between the two groups were found in regards to frequency of a specific symptom (fever, cough, shortness of breath, gastrointestinal symptoms or sore throat with p values of 0.43, 0.35, 0.74, 0.42, and 0.89 respectively). this was as well the case in regards to the presence of chest-x-ray findings upon admission (50.7% vs 57.8%, or: 0.72; p: 0.27). no significant statistical difference was observed between the mers-cov confirmed group and the control group in regards to intensive care unit need upon admission (18.7% vs 16.3%, or: 2.29; p: 0.67). though mortality rate seemed to be higher among mers-cov infected group, this was not statistically significant (10% vs 4.4%, or: 2.29; p: 0.11). the median wbcs counts (5.7 vs 9.3), as well as the median neutrophil counts (3.6 vs 6.7) were both lower in the infected group, and that was statistically significant (p values 0.0004 and 0.0001 respectively). the median alanine aminotransferase (alt) value was higher among mers-cov infected group compared to the control group (33 vs 28, p: 0.003). that was as well the case with aspartate aminotransferase (ast) (42 vs 36, p: 0.031). there was no statistical difference in the median values for hgb, platelets, albumin, and creatinine between the two groups, although the relationship for hgb was marginally significant (p values of 0.07, 0.22, 0.84, and 0.58 respectively). table 2 displays comparative analysis of significant laboratory values between confirmed and suspected cases. the odds of being a confirmed case was significantly lower for those with wbc in the 3rd and 4th quarters (wbcs 8.2) as compared to the reference category of patients with wbc less than the 25th percentile (wbcs < 4.8). the odds of being a confirmed case was significantly lower for those with neutrophil values in the 3rd and 4th quarters as compared to the reference category of patients with neutrophil less than the 25th percentile, with odds ratios 0.22 and 0.18 respectively (quartiles 2.93, 5.06, 9,38) for neutrophil. the median alt values were higher for those with confirmed mers-cov infection as compared to those without (median 33 vs 28 respectively). the odds ratios were significantly increased for patients with confirmed mers when alt values were in the 2nd and 4th quarters (alt 17e29, and alt > 60) as compared to patients with alt values in the lowest quarter (alt < 17); or z 3.72 and 5.94 respectively. patients with values between 29 and 60 did not have statistically significantly increased odds of confirmed mers. patients with confirmed mers had higher median values of ast (42 vs 36). patients with ast greater than 76.5 had statistically significant higher odds of having confirmed mers than those in the lowest quarter (values less than 24) with an odds ratio of 3.31, 95% ci: 1.24, 8.86. as observed in previous studies [17,19e21] , we found that having wbcs and neutrophil counts within the normal range is more likely to be associated with mers-cov. by comparing our cohort of mers-cov patients to another cohort of 47 patients diagnosed with mers-cov between september 2012 and june of 2013 described by assiri et al. [21] we can notice that both cohorts were predominated by male sex (60% vs 77%), though ours had a lower male proportion. male predominance, which was observed in almost every surveillance study [17,19e21] could be related to the culture in saudi arabia, where women wear veils that cover both the nose and mouth and may help protect from exposure, along with decreased outdoor activities compared to men. our patients' median age was 40 years compared to a median age in the range of 50e59 in the previous cohort. fever (59% vs 98%), cough (53% vs 83%), and shortness of breath (38% vs 72%) were the main symptoms, though our patients were less likely to be symptomatic. we also noticed that our patients were less likely to have co morbid conditions (46% vs 96%), less likely to have chest-x-ray abnormalities (51% vs. 100%) and had a significantly lower mortality (10% vs 60%). all this implies that, earlier in the outbreak, screening and diagnoses were limited to the very sick population who subsequently had a high mortality. in our patient population we were liberal in screening any potential admission who complained of respiratory symptoms and due to very strict infection control program we included individuals who were quarantined due to a significant exposure history to a mers-cov patient. by doing so we were aiming at preventing a possible mers-cov outbreak related to inadequate infection control measures. this helped uncover many asymptomatic or mildly symptomatic cases. this might also imply that the true burden of the disease in the kingdom is still uncovered and that we might be just seeing the tip of the iceberg. this theory was first brought up after a nationwide, crosssectional, serological study done between december 1st 2012 and december 1st 2013 in which serum samples from just over ten thousand individuals, whom age and sex distribution largely matched the general population [26] . this report has far reaching implications. in this study we found that none of the presenting symptoms helped distinguish those with mers-cov infection from the matched control group presenting with ili symptoms. almost half of mers-cov patients had no cxr abnormalities on presentation. in addition to raising significant questions on the validity of the current moh suspect case definition, this will challenge the practicing physicians in the emergency room in endemic and non-endemic countries on how to deal with patients presenting with ili symptoms [27, 28] . even with access to full viral panel on all ili patients and with evidence of influenza virus as the etiology, mers-cov can't be ruled out. this is based on data from iran where 3/5 mers cases had concomitant influenza infection [29, 30] . our study has a few limitations, the main being the lack of comprehensive testing for viral respiratory panels for patients admitted with ili symptoms (cases and matched controls). recently this has been added to the testing of all patients admitted with ili. a larger, prospective, multicenter study in the endemic areas is needed to better characterize the illness associated with mers-cov infection and specify its predictors. none. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus (mers-cov) the investigation team. first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission on behalf of the mers-cov outbreak investigation team of the netherlands. middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities -may ccc) statistics. kingdom of saudi arabia: ministry of health (moh) evidence for camel-to-human transmission of mers coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a singlecenter experience in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection imported cases of middle east respiratory syndrome: an update travel implications of emerging coronaviruses: sars and mers-cov middle east respiratory syndrome coronavirus: current situation and travelassociated concerns presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study an adult returned traveler from dubai hospitalized with an influenzalike illness (ili): middle east respiratory syndrome (mers) or influenza? infection control implications from a near mers case middle east respiratory syndrome (mers): a zoonotic viral pneumonia serial intervals of respiratory infectious diseases: a systematic review and analysis cluster of middle east respiratory syndrome coronavirus infections in iran none of the authors declared coi. key: cord-306004-amv0los1 authors: widagdo, w.; sooksawasdi na ayudhya, syriam; hundie, gadissa b.; haagmans, bart l. title: host determinants of mers-cov transmission and pathogenesis date: 2019-03-19 journal: viruses doi: 10.3390/v11030280 sha: doc_id: 306004 cord_uid: amv0los1 middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. in dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. differences in the behavior of the virus observed between individuals, as well as between humans and dromedary camels, highlight the role of host factors in mers-cov pathogenesis and transmission. one of these host factors, the mers-cov receptor dipeptidyl peptidase-4 (dpp4), may be a critical determinant because it is variably expressed in mers-cov-susceptible species as well as in humans. this could partially explain interand intraspecies differences in the tropism, pathogenesis, and transmissibility of mers-cov. in this review, we explore the role of dpp4 and other host factors in mers-cov transmission and pathogenesis—such as sialic acids, host proteases, and interferons. further characterization of these host determinants may potentially offer novel insights to develop intervention strategies to tackle ongoing outbreaks. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that was isolated in late 2012 [1] . since then, the virus has caused multiple outbreaks and infected more than 2000 individuals, [2] who then develop a respiratory infection ranging in severity from asymptomatic to fatal [3, 4] . severe-to-fatal mers-cov patients have a higher chance of transmitting this virus since they shed a higher amount of virus progeny in comparison to the asymptomatic-to-mild ones [5] [6] [7] [8] . identifying and quarantining these patients in healthcare facilities where outbreaks have occurred, together with implementing proper infection control, has been effective in reducing transmission and containing these outbreaks [9, 10] . however, new mers-cov cases are still being reported, especially in the arabian peninsula [2, 11] . this is partly due to the continuous zoonotic introduction of this virus to the human population in this region by dromedaries [12] . the dromedary camel is the only animal species that has been reported to transmit this virus to humans [13] [14] [15] [16] . mers-cov infection in these animals merely causes mild upper respiratory tract infection [17, 18] , but seroepidemiological studies showed that this virus has been circulating in dromedary camels for decades, suggesting the efficient transmission of mers-cov in this species [19] [20] [21] [22] . although the clinical manifestations, as well as transmission, are remarkably different in mers-cov-infected humans and dromedary camels, the viruses isolated from these two species are highly similar, if not indistinguishable [12, 16] . this indicates that host factors play a significant role in mers-cov pathogenesis and transmission. however, the identity of these host factors and how they affect the pathogenesis and transmission of mers-cov are generally not well understood. dipeptidyl peptidase-4 (dpp4)-the mers-cov receptor, sialic acids, proteases, and interferons are ; a cartoon representation of mers-cov s1 protein binding to dpp4 (pdb code 4l72) (b). the s protein consists of the s1 and s2 subunits. the α/β hydrolase domain of dpp4 is indicated in red, β-propeller domain in green, while part of the mers-cov s1 protein is shown in blue. other mers-cov-interacting host factors besides dpp4 are less extensively studied and have mostly been investigated in vitro. glycotopes of α2,3-sialic acids coupled with 5-n-acetylated neuraminic acid are recognized by the s1 protein of mers-cov during attachment [24] . in the absence of these glycotopes, mers-cov entry is reduced but not abolished, indicating their function as an attachment factor rather than a receptor [24] . besides α2,3-sialic acids, ceacam5 and grp78 have also been suggested to be attachment factors for mers-cov, but their roles in vivo during mers-cov infection are not clear at this moment [37, 38] . post attachment, mers-cov uses the c-terminal part of its s protein-known as s2 ( figure 1a )-to interact with host proteases, such as furin, tmprss2, and cathepsins [39] [40] [41] [42] . these proteases cleave the s protein and induce conformational changes, allowing fusion between viral and host cellular membranes, resulting in the release of viral rna into the cell cytoplasm [27] . tmprss2 and dpp4 are held in one complex at the cell surface by a scaffolding protein, the tetraspanin cd9, leading to a rapid and efficient entry of mers-cov into the susceptible cells [43] . once fusion with host cell membranes has occurred, mers-cov subsequently replicates its genetic material and produces viral proteins in the cell cytoplasm to generate new virus progeny. during this stage, mers-cov uses its nsp3-4 polyproteins to build its replication organelles as well as its accessory proteins such as the 4a and 4b proteins to inhibit host anti-viral defense mechanisms [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] . however, the capacity of mers-cov accessory proteins to impede several pathways of host immune response in the lungs may be limited. mers-cov inoculation of macaques and genetically modified mice generally results in limited clinical manifestations; thus, adapting this virus through serial passaging or defecting the type i interferon pathway may be needed to enhance viral replication and pathogenesis in these animals [32, [55] [56] [57] [58] . these observations, together with studies showing type i interferon capacity to inhibit mers-cov infection in vitro [25, 59] , highlight the importance of the innate immune response, especially type i interferon, as an inhibiting factor for mers-cov. so far mers-cov has been isolated from dromedary camels and humans [1, 60] . both species are not only susceptible to mers-cov infection, but also capable of transmitting this virus [7, [12] [13] [14] [15] [16] [17] [18] 22 ]. however, current data indicate that virus spread is more efficient in dromedary camels than in humans [5, 7, [19] [20] [21] 61] . this difference in transmissibility could be partially due to the different tropism of mers-cov in these two species. in dromedaries, mers-cov has been shown to replicate in the nasal epithelium upon experimental in vivo infection [17] , while in humans, mers-cov mainly replicates in the lower respiratory tract, particularly in the bronchiolar and alveolar epithelia [23, [62] [63] [64] [65] . higher viral rna levels in the sputum and lavage samples of mers-cov patients compared to nasal and throat swabs are consistent with the tropism of mers-cov in humans [66] [67] [68] . this different mers-cov tropism in dromedary camels and humans is in line with the localization of dpp4 in the respiratory tract tissues of these two species. in humans, dpp4 is absent in the nasal epithelium but present in the lower respiratory tract epithelium, mainly in type ii pneumocytes [69, 70] . in contrast, dpp4 is expressed in the nasal epithelium of dromedary camels [69] . this difference in dpp4 localization between humans and dromedary camels therefore explains mers-cov tropism in these two species and highlights dpp4 as an essential determinant of mers-cov tropism. dpp4 localization has also been investigated in many other mers-cov-susceptible species. in gambian and egyptian fruit bats, dpp4 is expressed in the respiratory tract and intestinal epithelium, suggesting that mers-cov can target both tissues [71] . in line with this finding, mers-cov inoculation via intranasal and intraperitoneal routes in the jamaican fruit bat led to viral rna shedding both in the respiratory tract and the intestinal tract [72] . in contrast to frugivorous bats, dpp4 is limitedly expressed in the respiratory tract epithelium of two insectivorous bats, i.e., common pipistrelle and common serotine bats, but abundant in their intestinal epithelium [71] . accordingly, sequences of mers-like-covs were mainly obtained from rectal swabs and fecal samples of insectivorous bats [73] [74] [75] [76] [77] [78] [79] [80] . these findings not only support insectivorous bats as the origin host of mers-cov [73] [74] [75] [76] [77] [78] [79] [80] , but also indicate the importance of intestinal tropism and fecal-oral transmission of mers-like-cov in these insectivorous bats. besides bats, humans, and dromedary camels, other animal species have also been proposed as potential hosts of mers-cov. remarkably, dpp4 of horses, llamas, alpacas, pigs, bovines, goats, sheep, and rabbits has been demonstrated to recognize the s protein of mers-cov [81, 82] . in most of these species, there is a preferential upper respiratory tract expression of dpp4 observed. rabbits express dpp4 in the upper and lower respiratory tract epithelium, and thus may allow mers-cov to replicate in both compartments [33, 83] . horses, llamas, and pigs mainly express dpp4 in the upper respiratory tract-particularly the nasal epithelium [84] . upon intranasal mers-cov inoculation, llamas, alpacas, and pigs developed upper respiratory tract infection, while horses did not seroconvert and only shed infectious virus in a limited amount [84] [85] [86] [87] [88] . the reason why horses seem to be less permissive to mers-cov remains to be investigated, but a chronic co-infection in the guttural pouch, a common disease among horses, might be one of the explanations. this guttural pouch infection results in excessive mucus production that might hinder mers-cov from attaching and entering the nasal epithelium [84, 89, 90] . sheep, on the other hand, did not seem to express significant levels of dpp4 in their respiratory tract, and thus did not seroconvert nor shed infectious virus upon experimental mers-cov inoculation [84, 88] . comparable to sheep, goats limitedly shed infectious virus upon experimental infection and did not transmit this virus to other naïve goats upon direct contact [88] . the results of experimental mers-cov infection in livestock animals are in line with data from epidemiological studies. mers-cov seropositive llamas and alpacas are present in the field, while horses, goats, and sheep are generally found to be seronegative [22, 86, 87, [91] [92] [93] [94] [95] [96] [97] [98] . given the fact that experimental in vivo infection studies and dpp4 expression analysis in different animal species revealed that dromedary camels are not the only animals in which mers-cov has an upper respiratory tract tropism [17, 18, 83, 84] , it is then relevant to question whether other animals can potentially spread mers-cov as well. new world camelids, i.e., alpacas and llamas, are able to transmit the virus to respective naïve animals upon contact [86] . pigs and rabbits, on the other hand, hardly transmit the virus-neither by contact nor airborne routes [83, 99] . most likely, this is caused by the fact that pigs and rabbits, unlike dromedary camels, shed low levels of infectious virus upon mers-cov inoculation ( figure 2) . this difference indicates that other host factors besides dpp4 could cause interspecies variation in mers-cov infection. indeed, several glycotopes of α2,3-sialic acids that function as attachment factors of mers-cov are present in the nasal epithelium of dromedary camels but absent in that of rabbits and pigs ( figure 3 ) [24, 100] . the lack of these glycotopes in pigs and rabbits might limit the susceptibility and transmission of mers-cov in these animals. although the role of these glycotopes in mers-cov transmission still requires further investigation, it remains plausible that an efficient transmission of this virus might require the presence of both dpp4 and mers-cov-recognized glycotopes of α2,3-sialic acids (figure 3 ). . it has also been reported that the lysosomal proteases from bat cells support coronavirus spike-mediated virus entry more efficiently than their counterparts from human cells [39] . these observations suggest that host proteases from different host species may determine the species and tissue tropism of mers-cov. because mers-cov has been circulating in dromedary camels for decades before emerging in the human population [19] [20] [21] [22] , it is plausible that this virus inhibits the immune response of dromedary camels more efficiently than that of other species, including pigs and rabbits. the difference in immune response among mers-cov-susceptible species is therefore another factor that might yield interspecies variation in permissiveness to mers-cov. characterizing the difference in host proteases and immune responses among mers-cov-susceptible species, as performed for dpp4 and mers-cov-recognized α2,3-sialic acid glycotopes (figure 3) , has not yet been investigated. these data, however, may further explain interspecies variation in mers-cov infection and transmission. mers-cov causes respiratory infection in humans ranging from asymptomatic to severe pneumonia [3, 4] . however, it is currently unclear what causes this intraspecies variation. epidemiology data indicate that individuals with certain risk factors are at higher risk of developing severe mers-cov infection [4, 102] . this implies that some host factors may dictate the outcome of mers-cov infection, thus rendering intraspecies variation. two of the risk factors, i.e., smoking and chronic obstructive pulmonary disease (copd), have been shown to upregulate dpp4 expression in the lungs [70, [102] [103] [104] , suggesting dpp4 as a possible reason for intraspecies variation observed among mers-cov patients. in healthy human lungs, dpp4 is almost exclusively expressed in type ii pneumocytes [69, 70] . type ii pneumocytes are small cuboidal cells that can regenerate alveolar epithelium upon injury, and roughly cover 2% of the alveolar surface area. meanwhile, around 95% of the surface area of the alveolus is occupied by type i pneumocytes that are morphologically flat and responsible for gas exchange [105, 106] . in the lungs of smokers and copd patients, unlike in healthy human lungs, dpp4 is prominently expressed in both type i and ii pneumocytes, indicating upregulated expression on type i pneumocytes [104] . autopsy reports from fatal mers-cov patients showed that both type i and ii pneumocytes expressed dpp4 and became infected by mers-cov, proposing a role of dpp4-expressing type i pneumocytes in mers-cov pathogenesis [64, 107] . damage to type i cells in the lung alveoli during viral infection may lead to diffuse alveolar damage [108] . in line with observations made in human mers cases, common marmosets that express dpp4 in both type i and ii pneumocytes have been reported to produce more infectious virus upon experimental mers-cov infection, compared to rhesus and cynomolgus macaques that merely expressed dpp4 in type ii pneumocytes [58, [109] [110] [111] [112] . accordingly, these common marmosets developed moderate-to-severe infection, while macaques generally developed mild transient pneumonia [32, 58, [109] [110] [111] [112] . similarly, in genetically modified mice that displayed mers-cov tropism for type ii pneumocytes, only mild clinical manifestations were observed upon mers-cov infection [56, 113] . adapting mers-cov through serial passaging or upregulating dpp4 expression throughout the airway epithelium in mice, however, will induce severe clinical disease [55, 56] . these data altogether support the role of dpp4-expressing type i pneumocytes in the pathogenesis of severe mers-cov infection. the differential expression of host factors that limits the infection should also be taken into account. dpp4 in soluble form has been demonstrated to protect against mers-cov infection in vitro and in a mouse model [23, 114] ; however, its presence in the lungs and role in mers-cov pathogenesis remain to be investigated. the host immune response also has the capacity to inhibit mers-cov infection. mers-cov has been shown to replicate to higher levels in immunocompromised rhesus macaques [115] , consistent with the observation that immunocompromised individuals have difficulties clearing mers-cov upon infection [68, 107, 116] . the survivors of mers-cov infection have been shown to develop virus-specific cd4+ and cd8+ t cell responses, implying the role of t cells in virus clearance [117] . however, the depletion of t cells in mice can either lead to failure in mers-cov clearance or improvement in clinical outcome, depending on the type of mouse model used [57, 118] . therefore, the role of adaptive immune response in mers-cov pathogenesis is currently unclear. on the other hand, one of the main components of the host innate immune response, type i interferon, inhibits mers-cov replication in susceptible cells, partly by inhibiting double membrane vesicles (dmv) formation [25, 57, 59, 119, 120] . the absence of type i interferon signaling in mice also resulted in more severe clinical manifestations and histopathological lesions upon mers-cov infection [57] . advance age, which can cause delayed type i interferon response upon viral infection, is a well-known risk factor for fatal mers-cov infection [4, 102, [121] [122] [123] . collectively, these data highlight the role of host innate immune response as a potent inhibitor for mers-cov infection. it is indubitable that severe mers-cov infection is not solely driven by the pathogen. additional underlying conditions increase mers-cov replication and induce severe-to-fatal clinical manifestations [4, 11, 103, 124, 125] . it is plausible that more than one underlying condition is needed to yield a fatal outcome. dpp4 upregulation in type i pneumocytes and insufficient type i interferon response might be crucial determinants for severe mers-cov infection (figure 4 ). further investigation of the host determinants of mers-cov pathogenesis may offer insights for developing novel therapeutic measures. although mers-cov has been reported to undergo some genotypic changes since it emerged in the human population [12, [126] [127] [128] [129] , this has not resulted in distinct phenotypic changes so far [63, 126] . therefore, host factors remain the most significant determinant in explaining inter-and intraspecies variations observed in mers-cov pathogenesis and transmission. dpp4 and mers-cov-recognized α2,3-sialic acids might partially explain these variations, since their localization has been demonstrated to be variable between mers-cov-susceptible species [69, 71, 84, 100] . dpp4 expression in human lungs has also been shown to vary due to certain comorbidities [70, 96, 104] . nevertheless, it is undoubtable that the inter-and intraspecies variation in mers-cov pathogenesis and transmission is a complex phenomenon influenced by more than one host factor. current data suggest proteases and interferons as other critical host factors, but how they instigate inter-and intraspecies variations, as well as their role in mers-cov pathogenesis and transmission, still remain to be further elucidated. characterization of the host determinants of mers-cov pathogenesis and transmission could potentially offer insight into this virus epidemiology and guide novel therapeutic development. it may also help to identify the most vulnerable individuals to protect against mers-cov infection-for example, by using vaccination. author contributions: all authors contributed to the writing of the manuscript and carefully evaluated the manuscript before submission. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers-coronavirus: from discovery to intervention state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study transmission of mers-coronavirus in household contacts risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea south korea finally mers-free republic of korea-disease outbreak news world health organization mers-cov global summary and assessment of risk mers-cov spillover at the camel-human interface middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment tmprss2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection an overview of their replication and pathogenesis the multifunctional or moonlighting protein cd26/dppiv structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 spiking the mers-coronavirus receptor middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response the endonucleolytic rna cleavage function of nsp1 of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum middle east respiratory syndrome coronavirus ns4b protein inhibits host rnase l activation mers-cov accessory orfs play key role for infection and pathogenesis expression and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice a mouse model for mers coronavirus-induced acute respiratory distress syndrome rapid generation of a mouse model for middle east respiratory syndrome an animal model of mers produced by infection of rhesus macaques with mers coronavirus inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin isolation of mers coronavirus from a dromedary camel mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an in-vitro and ex-vivo study immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in united arab emirates emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs severe respiratory illness caused by a novel coronavirus viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) human betacoronavirus 2c emc/2012-related viruses in bats mers-related betacoronavirus in vespertilio superans bats group c betacoronavirus in bat guano fertilizer detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat close relative of human middle east respiratory syndrome coronavirus in bat further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 domestic pig unlikely reservoir for mers-cov infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding genome specialization and decay of the strangles pathogen, streptococcus equi, is driven by persistent infection strangles: a pathogenic legacy of the war horse middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis serologic assessment of possibility for mers-cov infection in equids coronavirus infections in horses in saudi arabia and oman cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels middle east respiratory syndrome coronavirus experimental transmission using a pig model receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea risk factors for primary middle east respiratory syndrome coronavirus illness in humans dpp4, the middle east respiratory syndrome coronavirus receptor, is upregulated in lungs of smokers and chronic obstructive pulmonary disease patients identification of genes differentially expressed in rat alveolar type i cells renewal of alveolar epithelium in the rat following exposure to no 2 histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset infection with mers-cov causes lethal pneumonia in the common marmoset middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 elevated human dipeptidyl peptidase 4 expression reduces the susceptibility of hdpp4 transgenic mice to middle east respiratory syndrome coronavirus infection and disease pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques atypical presentations of mers-cov infection in immunocompromised hosts recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus ifn production ability and healthy ageing: mixed model analysis of a 24 year longitudinal study in japan age-associated failure to adjust type i ifn receptor signaling thresholds after t cell activation dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice middle east respiratory syndrome the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus mers coronaviruses from camels in africa exhibit region-dependent genetic diversity increase in middle east respiratory syndrome-coronavirus cases in saudi arabia linked to hospital outbreak with continued circulation of recombinant virus multihospital outbreak of a middle east respiratory syndrome coronavirus deletion variant deletion variants of middle east respiratory syndrome coronavirus from humans key: cord-304271-vyayyk50 authors: qin, yuan-yuan; zhou, yi-hong; lu, yan-qiu; sun, feng; yang, sen; harypursat, vijay; chen, yao-kai title: effectiveness of glucocorticoid therapy in patients with severe coronavirus disease 2019: protocol of a randomized controlled trial date: 2020-03-05 journal: chin med j (engl) doi: 10.1097/cm9.0000000000000791 sha: doc_id: 304271 cord_uid: vyayyk50 background: at the end of 2019, a novel coronavirus outbreak emerged in wuhan, china, and its causative organism has been subsequently designated the 2019 novel coronavirus (2019-ncov). the effectiveness of adjunctive glucocorticoid therapy in the management of 2019-ncov-infected patients with severe lower respiratory tract infections is not clear, and warrants further investigation. methods: the present study will be conducted as an open-labeled, randomized, controlled trial. we will enrol 48 subjects from chongqing public health medical center. each eligible subject will be assigned to an intervention group (methylprednisolone via intravenous injection at a dose of 1–2 mg/kg/day for 3 days) or a control group (no glucocorticoid use) randomly, at a 1:1 ratio. subjects in both groups will be invited for 28 days of follow-up which will be scheduled at four consecutive visit points. we will use the clinical improvement rate as our primary endpoint. secondary endpoints include the timing of clinical improvement after intervention, duration of mechanical ventilation, duration of hospitalization, overall incidence of adverse events, as well as rate of adverse events at each visit, and mortality at 2 and 4 weeks. discussion: the present coronavirus outbreak is the third serious global coronavirus outbreak in the past two decades. oral and parenteral glucocorticoids have been used in the management of severe respiratory symptoms in coronavirus-infected patients in the past. however, there remains no definitive evidence in the literature for or against the utilization of systemic glucocorticoids in seriously ill patients with coronavirus-related severe respiratory disease, or indeed in other types of severe respiratory disease. in this study, we hope to discover evidence either supporting or opposing the systemic therapeutic administration of glucocorticoids in patients with severe coronavirus disease 2019. trial registration: clinicaltrials.gov, chictr2000029386, http://www.chictr.org.cn/showproj.aspx?proj=48777. coronaviruses are rna viruses, and may infect the respiratory, gastrointestinal, hepatic and central nervous systems of humans, livestock species, avian species, many mammals, and wild animals. [1] in humans, infection by four coronaviruses, namely hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1, usually lead to mild, self-limiting upper respiratory tract infections. [2] however, other coronaviruses, such as those associated with severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), may cause severe respiratory disease, and have caused a total of more than 10,000 laboratory-confirmed cases globally in the past two decades, with a 10% and 34.4% case-fatality rate, respectively. [3, 4] in december 2019, a novel coronavirus 2019 novel coronavirus (2019-ncov), emerged in wuhan, hubei, china, with clinical manifestations in humans resembling that of viral pneumonia. [5] by february 25, 2020 , about 77,780 confirmed cases have been identified in china, with 2666 deaths. worryingly, 2459 confirmed cases of coronavirus disease 2019 (covid19) have now been identified outside of china. [6] symptoms of infection are non-specific, and include fever, cough, and myalgia, [7] with diarrhea, with or without the subsequent development of dyspnea. [8] severe cases with respiratory distress, sepsis, and septic shock have been reported. [9] during the sars epidemic of 2003, therapeutic systemic glucocorticoids were widely administered in patients who were infected with the severe acute respiratory syndrome coronavirus (sars-cov), and who subsequently developed severe respiratory disease. one cohort study of patients during the sars outbreak showed that the use of pulsed high-dose methylprednisolone was associated with clinical improvement in patients with sars. [10] in another study of 20 patients with sars, certain cytokine levels (interleukin-8, monocyte chemoattractant protein-1, and th1 chemokine interferon-g-inducible protein-10) were found to be reduced after 5 to 8 days of glucocorticoid therapy. [11] however, a previous retrospective study indicated that pulsed doses of methylprednisolone was a risk factor associated with increased 30-day mortality (adjusted odds ratio 26.0; 95% confidence interval, 4.4-154.8; p = 0.001). [12] in addition, a further retrospective observational study found that glucocorticoid therapy in patients with mers was associated with delayed middle east respiratory syndrome coronavirus (mers-cov) rna clearance. [13] the clinical, therapeutic, and other effects of systemic glucocorticoid therapy in patients with covid-19 is not currently clear, and the case for and against the use of systemic glucocorticoids in severe cases of covid-19 warrants further investigation. our study proposes to investigate the effectiveness of glucocorticoid therapy in patients with severe covid-19. our major objective is to observe whether there is a clinical necessity, or therapeutic justification, for the use of systemic glucocorticoids in patients with severe covid-19. the study was approved by the ethics committee of the chongqing public health medical center (no. 2020-003-01-ky). we will publish the results of our study in medical journals at the conclusion of the study. no patient names will appear in any published articles, and no access to the data may be obtained, with the exception of the researchers and members of the hospital ethics committee. this study will be conducted as an open-labeled, randomized, controlled trial. we will enroll 48 subjects from the chongqing public health medical center for this study. this protocol has been written and devised in accordance with the standard protocol items: recommendations for interventional trials statement. [14] the enrolment, intervention, and assessment processes are described in figure 1 . after obtaining informed consent, all subjects in each group will be invited for 28 days of follow-up, which will be scheduled at four consecutive visit points [ table 1 ]. blood and urine samples will be collected for laboratory testing, including hematologic analysis, urinalysis, clinical chemistry studies, electrolytes, coagulation testing, myocardial enzymes, c-reactive protein, erythrocyte sedimentation rate, procalcitonin, cluster of differentiation 4 (cd4) cell counts, cd8 cell counts, blood gas analysis, and 2019-ncov real-time reverse transcriptionpolymerase chain reaction (rt-pcr). all items considered and tested for during the follow-up period are listed in table 1 . adverse events (aes) are defined as undesirable outcomes experienced by patients during the study, whether or not related to treatment with glucocorticoids. all aes described by patients or observed by the medical team will be duly documented. all serious aes will be documented in detail, and the investigators will be notified as soon as possible. aes will be recorded in both the clinical history of the patient and the case report form, with appropriate medical terminology. all aes will be expeditiously resolved. short-term glucocorticoid therapy usually causes mild side effects, including hypokalemia, glucose intolerance, hypertension, pancreatitis, cutaneous, hematologic, immunologic, and neuropsychologic effects. [15] the above mild side effects are reversible upon discontinuation of glucocorticoids. side effects associated with long-term glucocorticoid use are not considered relevant for this study, because of the short duration of the glucocorticoids prescribed in this study (3 days the diagnosis of severe covid-19 in subjects will have to meet the following criteria [16] : (1) identification of 2019-ncov via rt-pcr in blood samples, sputum samples, bronchoalveolar lavage fluid or nasopharyngeal swab samples. (2) having at least one of the following conditions: a. respiratory distress (≥30 times/min); b. oxygen saturation 93% at rest; c. arterial partial pressure of oxygen (pao 2 )/fraction of inspiration o 2 (fio 2 ) 300 mmhg (1 mmhg = 0.133 kpa); d. respiratory failure requiring mechanical ventilation; e. septic shock development; f. critical organ failure requiring intensive care unit care. subjects will be included in our study if they satisfy the following criteria: (1) aged 18 years or over; (2) severe covid-19; (3) be willing to give informed consent. subjects will be excluded from the study if they satisfy the following criteria: (1) are allergic or intolerant to any therapeutic drugs used in this study; (2) pregnant or lactating women; (3) presence of severe systemic illness that may affect the effectiveness or safety evaluation for this study. microsoft excel 2013 (microsoft corporation, redmond, wa, usa) will be used to generate random numbers for each subject with consent. once eligibility has been confirmed, the investigators or designers will randomize the subjects into a control group and an intervention group, at a 1:1 ratio. all results will be recorded and entered twice independently. all data will be documented on case report forms and immediately recorded in the excel database. any missing values will be verified to ensure completeness and accuracy of data as much as possible. data that are obviously abnormal or beyond the upper limit of normal (laboratory items exceeding 20% of the normal value) must be explained, and the necessary explanation must be given by the physician. drop-outs and aes will be recorded in a timely manner, and drugs used for trial will be supplied, stored, distributed, and recycled according to relevant regulations. after the data lock record is signed by the principal researcher, sponsor, statistical analyst, and data manager, the data administrator will perform a database lock. all subjects will receive conventional treatment for covid-19 according to the guidelines for diagnosis and treatment of covid-19 (version 6). patients randomized to the intervention group will receive methylprednisolone (intravenous injection, 1-2 mg/kg/day for 3 days), whereas those in the control group will not receive glucocorticoid therapy. discontinuation of the regimen may occur for those participants who have no substantial improvement after a full course of treatment, or for those subjects whose clinical condition deteriorate during the course of treatment. the primary endpoint is the change in sequential organ failure assessment (sofa) at 3 days after randomization. [17] the secondary endpoints are: proportion of mechanical ventilation use at 2 and 4 weeks, mortality at 2 and 4 weeks, and duration of hospitalization. this trial is a randomized controlled trial with two parallel groups, without masking, and with a 1:1 allocation ratio. up to now, no randomized controlled trials have evaluated changes in sofa scores in patients with 2019-ncov infection with or without glucocorticoid therapy. the sample-size calculation is based on the primary hypothesis of detecting a reduction of 40% in sofa scores between the treatment and non-treatment groups after 3 days of glucocorticoid therapy. pass software version 15 (ncss, llc; kaysville, ut, usa) will be used with a power of 80% and a level of 95% confidence. considering a dropout rate of 5%, the sample size is estimated to be 24 cases in each group. a randomized controlled trial in 2003 aimed to compare the plasma sars-cov rna concentrations in patients in an early hydrocortisone-treated group with patients in a placebo group. [18] this study only enrolled a total of 17 subjects because it was conducted at the end of the sars outbreak, and fewer appropriate patients were available to be recruited. taking into account the uncertainty of the duration of the outbreak, the final sample size may be adjusted. unlike wuhan, chongqing currently has a relatively small number of confirmed cases of covid-19, and an even smaller number of severe cases of covid-19. it is hoped that we do eventually recruit a cohort of 48 patients for our study, but we may have to settle for an even smaller cohort, should suitable subjects not be available. the primary outcome analysis will be presented for the intervention group and control group using descriptive statistics. the normality of the data distribution will be evaluated by the kolmogorov-smirnov test. continuous variables will be described as mean values with standard deviation (mean ± standard deviation), and will be analyzed using the t test for independent samples. categorical variables will be analyzed using the chisquared test or fisher's exact test. to verify the magnitude of the difference, the relative risk of success will be estimated in the intervention group and control group, and its confidence interval will be set at 95%. values of p < 0.05 will be considered to be statistically significance. all the analyses will be performed using the spss software version 24 (spss inc., chicago, il, usa). since 2019-ncov was first reported in december 2019, [19] it has attracted global attention owing to its similarity to sars-cov and mers-cov in causing fatal respiratory disease, and its potential for propagating large-scale human infection and economic disruption. systemic glucocorticoid therapy could be considered for critically ill patients with covid-19, but their impact on clinical outcomes is uncertain. [16] available data on systemic glucocorticoid therapy among coronavirus-infected patients mainly comes from studies of patients with sars and mers, but whether glucocorticoid therapy is definitively beneficial in the clinical management of these two coronavirus infections is still being debated. [13, 20, 21] glucocorticoid therapy was used in the treatment of severe sars because early anecdotal experience supported it, and radiologic findings and histologic features of critically ill patients with sars were similar to those of patients with acute respiratory distress syndrome (ards). [22] [23] [24] [25] as early as march 2003, china first summarized its experience in the management of sars, and suggested that high-dose glucocorticoids should be used if patients had fever persisting for more than 3 days, or radiologic findings were suggestive of persistent lung involvement or progressive deterioration. [26] there were three different regimens used by physicians in hong kong, china including methylprednisolone (1-2 mg/kg qid or 2-4 mg/kg tid iv followed by oral prednisolone for varying periods and of disparate dosages, as per clinical evaluation), hydrocortisone (2 mg/kg qid or 4 mg/kg tid iv followed by oral prednisolone for varying periods and doses as per clinical evaluation), and pulsed methylprednisolone (500 mg iv daily for 5 days followed by maintenance oral prednisolone 50 mg bid, reduced to 20-30 mg daily on day 21 and as per clinical evaluation). [27] some suggested glucocorticoid therapeutic doses during the sars outbreak were very high, approximating the treatment doses for acute severe asthma, and some prescribed doses were similar to those prescribed for organ rejection in transplant patients, or for ards. [23] one study compared pulsed glucocorticoid therapy (methylprednisolone ≥500 mg/day) with non-pulsed glucocorticoid (methylprednisolone <500 mg/day) therapy, finding that pulsed glucocorticoid therapy appeared to be a more efficacious, with patients using pulsed glucocorticoid therapy having lower oxygen requirements, better radiographic outcomes, and decreased likelihood of requiring rescue. unfortunately, the stated study was not a randomized controlled trial, and is thus open to the possibility of bias having an impact on outcomes. [23] however, there were also staunch advocates for not using systemic glucocorticoids in these patients. one systematic review of studies on patients with sars-cov, including 29 studies documenting glucocorticoid use, found 25 studies that were inconclusive regarding the role of the adjunctive use of glucocorticoids, and four studies demonstrated that the use of systemic glucocorticoids in patients with sars may cause possible harm. [28] a prospective, randomized double-blinded, placebo-controlled trial compared early hydrocortisone treatment (before day 7 of the illness) with placebo, finding that early hydrocortisone therapy was associated with a higher subsequent plasma viral load. [18] meanwhile, many possible complications of glucocorticoid use, such as profound immunosuppression, with the possible emergence of severe commensal and other viral and bacterial infections, invasive fungal infections, osteonecrosis, and psychosis may occur with prolonged and high-dose glucocorticoid therapy. [29] [30] [31] [32] [33] [34] glucocorticoid therapy was also commonly used for critically ill patients with mers. in one study, hypoxemic patients with mers-cov pneumonia who had moderate levels of positive end-expiratory pressure were initiated on glucocorticoid therapy. [13] in these patients, there was no difference in 90-day mortality, and these patients were associated with delayed mers-cov rna clearance. many patients with severe mers were treated with systemic high-dose glucocorticoids intending to reverse the progression of respiratory distress and to prevent lung chinese medical journal 2020;vol(no) www.cmj.org fibrosis. however, this approach has not been proven to be successful. [35] one study, including a total of 314 patients with symptomatic mers-cov, found that glucocorticoid use was associated with increased mortality in these patients. [36] a retrospective cohort study compared 151 patients in the glucocorticoid group with 158 patients in the non-glucocorticoid group, finding that mortality in the glucocorticoid group was similar to that of the nonglucocorticoid group, but that glucocorticoid therapy was associated with delayed mers-cov rna clearance, after adjustment for baseline and time-varying confounding factors. [13] after infection with 2019-ncov occurs, some patients develop mild symptoms, but a significant fraction of patients progress to severe ards and require intensive care. [7, 37] the use of glucocorticoids in patients with ards is still controversial because of divergent results in the existing literature. [38] high-dose glucocorticoid is one of the most frequently used adjuncts in ards (17.9%) in 50 countries, even though their effectiveness in the management of acute lung injury is not yet clear. [39] one systematic review conducted an analysis of individual patient data from randomized trials, and demonstrated that compared with the placebo group, prolonged glucocorticoid treatment did improve clinical outcomes. [40] another randomized controlled trial showed that glucocorticoid therapy could down-regulate ards-related inflammation, and was associated with significant improvements in lung and extrapulmonary organ dysfunction, as well as reduce mechanical ventilation duration and intensive care unit hospital admission duration. [41] studies by meduri et al [42, 43] found that prolonged methylprednisolone treatment may complement positive modulation of systemic inflammation by inducing recovery from induced glucocorticoid resistance, and co-existing decreased production of endogenous glucocorticoids. [42, 43] animal experiments in recent years have provided evidence for the use of glucocorticoids to reduce inflammation, the attenuation of acute lung injury, and reduction in mortality during the acute phase of severe disease. [44, 45] however, other studies have suggested that glucocorticoid therapy in ards is not necessary, and may even aggravate the clinical picture. the use of hydrocortisone improves lung function in sepsis-associated ards, but no significant survival benefit has been observed in these patients. [46] a higher mortality rate has been observed in patients with ards who took high-dose glucocorticoids within 7 days after hospital admission, compared with those who were treated without high-dose glucocorticoid. [47] existing studies on ards have not assessed the effects of glucocorticoids in identical types of patients, or identical processes of the disease. [48] therefore, the results for these studies cannot be considered to be congruent. adjunctive glucocorticoid therapy in specific diseases causing ards, such as pneumocystis jirovecii pneumonia, has been shown to reduce mortality in adult patients. [49] additionally, the dosage of the glucocorticoid used needs to be factored in. high-dose glucocorticoid therapy may increase ventilator dependence, and may possibly induce a worse outcome. [50] the development of osteonecrosis was discovered in patients receiving high-dose glucocorticoids during the sars epidemic in 2003, and the number of osteonecrotic lesions was directly related to the dosage of the glucocorticoid prescribed. [51, 52] additionally, high-dose glucocorticoid use is one of the causes for neuropsychiatric outcomes in patients with sars. [53] it has been noted that if lung injury has been effectively controlled, appropriate doses of glucocorticoids based on disease severity may be beneficial for better outcomes of sars. [10, 21] in our study, we will investigate whether the use of mid-dose and shortcourse adjunctive glucocorticoid therapy to treat severe covid-19 is advantageous, safe, and effective. this work was supported by the chongqing special research project for prevention and control of novel coronavirus pneumonia (no. cstc2020jscx-fyzx0074). none. emerging coronaviruses: genome structure, replication, and pathogenesis coronaviruses -drug discovery and therapeutic options who. summary of probable sars cases with onset of illness from middle east respiratory syndrome coronavirus (mers-cov) who. novel coronavirus(2019-ncov)situation report-121 clinical features of patients infected with 2019 novel coronavirus in wuhan, china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster a novel coronavirus outbreak of global health concern severe acute respiratory syndrome: report of treatment and outcome after a major outbreak plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome coronavirus-positive nasopharyngeal aspirate as predictor for severe acute respiratory syndrome mortality corticosteroid therapy for critically ill patients with middle east respiratory syndrome spirit 2013 explanation and elaboration: guidance for protocols of clinical trials side effects of corticosteroid therapy national health commission of the people's republic of china. guidelines for the diagnosis and treatment of novel coronavirus (2019-ncov) infection by the national health commission (trial version 6) 2020 the third international consensus definitions for sepsis and septic shock (sepsis-3) effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov clinical features and short-term outcomes of 144 patients with sars in the greater toronto area corticosteroid treatment of severe acute respiratory syndrome in hong kong development of a standard treatment protocol for severe acute respiratory syndrome high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong a cluster of cases of severe acute respiratory syndrome in hong kong our strategies for fighting severe acute respiratory syndrome (sars) sars: pharmacotherapy sars: systematic review of treatment effects management of severe acute respiratory syndrome: the hong kong university experience fatal aspergillosis in a patient with sars who was treated with corticosteroids osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids avascular necrosis of bone in severe acute respiratory syndrome factors associated with psychosis among patients with severe acute respiratory syndrome: a case-control study pharmacologic treatment of sars: current knowledge and recommendations state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans clinical predictors of mortality of middle east respiratory syndrome coronavirus (mers-cov) infection: a cohort study data sharing and outbreaks: best practice exemplified acute respiratory distress syndrome epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries prolonged glucocorticoid treatment is associated with improved ards outcomes: analysis of individual patients' data from four randomized trials and trial-level meta-analysis of the updated literature methylprednisolone infusion in early severe ards: results of a randomized controlled trial prolonged methylprednisolone treatment suppresses systemic inflammation in patients with unresolving acute respiratory distress syndrome: evidence for inadequate endogenous glucocorticoid secretion and inflammation-induced immune cell resistance to glucocorticoids nuclear factor-kappab-and glucocorticoid receptor alphamediated mechanisms in the regulation of systemic and pulmonary inflammation during sepsis and acute respiratory distress syndrome. evidence for inflammation-induced target tissue resistance to glucocorticoids glucocorticoid attenuates acute lung injury through induction of type 2 macrophage effects of different corticosteroid doses and durations on smoke inhalation-induced acute lung injury and pulmonary fibrosis in the rat hydrocortisone treatment in early sepsis-associated acute respiratory distress syndrome: results of a randomized controlled trial the relationship between high-dose corticosteroid treatment and mortality in acute respiratory distress syndrome: a retrospective and observational study using a nationwide administrative database in japan steroids are part of rescue therapy in ards patients with refractory hypoxemia: we are not sure adjunctive corticosteroids for pneumocystis jiroveci pneumonia in patients with hiv infection cd006150 the negative effect of initial high-dose methylprednisolone and tapering regimen for acute respiratory distress syndrome: a retrospective propensity matched cohort study steroid-induced osteonecrosis: the number of lesions is related to the dosage steroid-induced osteonecrosis in severe acute respiratory syndrome: a retrospective analysis of biochemical markers of bone metabolism and corticosteroid therapy the effects of disease severity, use of corticosteroids and social factors on neuropsychiatric complaints in severe acute respiratory syndrome (sars) patients at acute and convalescent phases effectiveness of glucocorticoid therapy in patients with severe coronavirus disease 2019: protocol of a randomized controlled trial key: cord-291367-rtmsrh16 authors: zumla, alimuddin; rustomjee, roxana; ntoumi, francine; mwaba, peter; bates, matthew; maeurer, markus; hui, david s.; petersen, eskild title: middle east respiratory syndrome need for increased vigilance and watchful surveillance for mers-cov in sub-saharan africa date: 2015-07-02 journal: int j infect dis doi: 10.1016/j.ijid.2015.06.020 sha: doc_id: 291367 cord_uid: rtmsrh16 nan the past two decades have witnessed the emergence of several new and old respiratory tract infectious diseases, which threaten global health security due to their epidemic potential. 1,2 these include multi-drug resistant tb, severe acute respiratory syndrome (sars), avian and swine influenza and more recently the middle east respiratory syndrome (mers). mers is a new zoonotic disease of humans caused by a coronavirus (mers-cov) which was first isolated in september, 2012 from a patient who died from a severe respiratory disease in jeddah saudi arabia. 3 since then mers has attracted global media attention because it is associated with a high mortality (44%) in individuals who have co-morbidities such as diabetes, chronic renal, liver or lung illnesses or in those who are immunocompromised. 4, 5 the recent unprecedented outbreak of the mers 6, 7 in south korea which arose consequential to the importation of mers-cov by a south korean traveler to the middle east, alarmed global public health authorities and highlights the potential of mers-cov to spread across the globe and cause local outbreaks. the who director general convened the ninth meeting of the emergency committee (ec) under the international health regulations regarding mers-cov on 16 june 2015 to discuss the korean outbreak. 8 as of 23 rd june the total number of mers cases reported from the republic of south korea now stands at 175 (94 currently receiving treatment, 54 recovered, 27 deaths. 6, 8 of 175 cases, 80 patients and 33 hospital staff had contracted the virus nosocomially, 62 friends, colleagues and relatives had come in contact within healthcare facilities while visiting family members with mers. virological and serological studies from several middle eastern, west and east african countries indicate that bats and dromedary camels are likely reservoirs of mers-cov. [9] [10] [11] [12] however, human mers-cov infections appear to be endemic only to countries in the middle east where sporadic cases continue to occur in the community throughout the year. 13 13 the outbreak in seoul, republic of korea, has been linked to a single individual who had travelled to saudi arabia. the first mers case in thailand was reported last week and the patient had a history of travel from the sultanate of oman. 13 of note is the striking absence of any mers cases (primary or travel related) reported from sub-saharan african (ssa) countries. 14, 15 the reasons mers-cov predominantly affects humans in the middle east and is not endemic in africa where mers-covinfected camels and bats are present requires further study. a likely explanation may be that this may simply reflect the lack of clinical awareness of exposure risk, diagnosis and treatment of respiratory tract infections largely remains clinically based and empiric in most ssa countries coupled with absence of surveillance. 14 every year an estimated 10 million pilgrims from over 182 countries travel to the kingdom of saudi arabia to participate in hajj pilgrimage, the mini-pilgrimage umrah (which is performed at any time of the year), or for the month of ramadaan. 16 of these, an estimated 1 million pilgrims come from sub-saharan african countries. there were no cases of mers reported during the 2012, 2013 and 2014 hajj pilgrimages or the ramadaan period. [17] [18] [19] however, the risk of mers-cov spreading globally remains due to the continuous influx of pilgrims and the persistent low levels of endemic mers-cov transmission to humans in saudi arabia. there is also the possibility that mers-cov may mutate into a form more adaptable for human to human transmission over time. the potential risk of mers-cov infection to pilgrims who visit saudi arabia from different regions of the world was estimated by coker and colleagues 20 based on overall incidence of mers cases in saudi arabia since its first discovery in 2012. their estimates based on the most likely scenario using recent pilgrim numbers for sub-saharan africa are that there will be at most ten returning pilgrims each year with mers-cov infections. national surveillance systems should be on alert for the low but long-lasting risk of mers-cov infected pilgrims returning from the umrah throughout the year, and also for the large numbers of refugees at several conflict zones in the middle east (those migrating from syria to turkey and from the yemen border into saudi arabia and beyond). the recent mers outbreak in the republic of korea was associated with secondary, tertiary, quarternary and quinary cases of mers-cov transmission, though fortunately there has been no sustained community transmission. 6, 8 the republic of korea mers-cov outbreak has many similarities with that of previously reported mers-cov outbreaks which occurred at healthcare facilities in several cities in saudi arabia and from jordan 21-24 which were all associated with breaches and gaps in infection prevention and control protocols. these lapses in korean hospitals enabled mers-cov infected and uninfected patients, staff and visitors to mix freely in busy and crowded accident and emergency departments, within wards and multi-bed hospital rooms, with no isolation or quarantine of suspected cases. public health measures such as enhanced contact tracing and isolation and quarantine put in place by the korean government to control the outbreak eventually led to the decline in the numbers of mers cases and the outbreak is being brought under control. the importance of infection controls measures was also illustrated by the saudi arabian hospital mers outbreaks, where well-trained health care and workforce brought the hospital outbreaks under control quickly. 21, 22 the who ec meeting noted that 8 the who ec 8 referred to the outbreak as a 'wake-up' call and state that in a highly mobile world, all countries should always be prepared for the unanticipated possibility of outbreaks of mers-cov and other serious infectious diseases. the korean mers outbreak is the largest recorded from outside the middle east and the largest imported from a returning traveller to the middle east, raising several important issues for global surveillance and control. it illustrates that mers-cov, three years after its first discovery remains an important global public health risk with many unanswered questions. 25 further international spread should be anticipated and countries with weaker health systems and lack of laboratory facilities to accurately screen for mers-cov need to be vigilant. this will pose major challenges. 25 there are important lessons here for sub-saharan african and other developing countries from where mers-cov cases have not yet been detected. as the recent ebola virus disease epidemic illustrates, african countries may be very vulnerable to a korea-like mers-cov outbreak, which may arise from returning pilgrims or other travellers from saudi arabia 26 or from traders between saudi arabia and the horn of africa. mers-cov is transmitted through mers-cov-infected respiratory secretions for which contact and droplet precautions are recommended. [27] [28] [29] [30] the korean mers outbreak highlights that hospitals provide ideal conditions for amplifying mers-cov transmission arising from close contact between patients, healthcare and ancillary staff, relatives and other visitors, which enables spread of mers-cov. 6, 8 it is critical that every country should maintain a high level of vigilance and perform mers-cov surveillance according to widely available expert recommendations, 27-30 whether or not mers cases have been detected in their countries, it ensuring infection prevention and control protocols are in place at all health-care facilities. those who travel must be educated to follow basic hygiene measures 30 and those develop ill health during their trip to the middle east, or soon after their return should seek medical care and volunteer the history of travel to their healthcare provider. sub-saharan african governments must pay serious attention to strengthening infection control and public health surveillance systems. all healthcare workers and travellers from africa to the middle east should be aware of the threat to global health security posed by mers-cov. considering a diagnosis of mers at first presentation may be difficult due to non-specific symptoms at clinical presentation. however it is important that prevention and control measures are instituted at first consideration of mers as a diagnosis to prevent spread of mers-cov. hospitals and clinics providing care for patients infected with suspected or confirmed mers-cov infection should take appropriate measures to decrease the risk of mers-cov transmission from the infected patient to other patients, doctors, nurses, allied health-care workers, relatives and visitors. health-care workers should be educated and trained in infection prevention and control and should have continuing professional development on these issues. over the past decade, several surveillance systems have been introduced to monitor the emergence of new infectious pathogens. 31 as the ebola virus epidemic in west africa showed, surveillance systems in african countries for infectious diseases with epidemic potential require strengthening. more effective national, regional, and international surveillance systems are required to enable rapid identification of emerging respiratory epidemics, diseases with epidemic potential, their specific microbial cause, origin, mode of acquisition, and transmission dynamics. in light of the republic of korea mers outbreak increased vigilance and surveillance for mers-cov should be carried out by health services in african countries using current clinical and public health guidelines for mers-cov. 28 although resources may not allow for making an accurate diagnosis of mers, a high degree of awareness of the possibility of mers-cov infection in all returning pilgrims will allow early, isolation of patients and putting in place infection control measures, avoiding a repeat of the korea outbreak. sub-saharan african countries need to protect themselves against the possible outbreaks akin to the korean one. mers-cov should be included in list of pathogens by the african network of influenza surveillance and epidemiology (anise) 32 and mers-cov should be made part of the strengthening influenza sentinel surveillance in africa (sisa)' with national, regional and international reporting mechanisms 33 in liaison with other stakeholders involved in global infectious diseases surveillance. new, low cost, rapid, sensitive and specific diagnostic tests that can be used at all points of healthcare are require for all infectious diseases which threaten global health security. 34 the exact mode of transmission and pathogenesis of mers-cov and other novel respiratory tract viruses such as h7n9 influenza a virus require definition so that more effective prevention and management measures can be developed and introduced. 35 a united and coordinated global response is needed to tackle emerging respiratory tract infections and to fill major gaps 25 in the understanding of the epidemiology, transmission dynamics, pathogenesis prevention and control of these infectious diseases. declaration: all authors declare no conflicts of interest. emerging viral respiratory tract infections-environmental risk factors and transmission emerging infectious diseases and pandemic potential: status quo and reducing risk of global spread isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome severe acute respiratory syndrome vs. the middle east respiratory syndrome spread of mers to south korea and china who -ihr emergency committee concerning middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia geographic distribution of mers coronavirus among dromedary camels mers coronavirus neutralizing antibodies in camels middle east respiratory syndrome coronavirus in dromedary camels:an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia the hajj pilgrimage and surveillance for middle east respiratory syndrome coronavirus in pilgrims from african countries high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana hajj: infectious disease surveillance and control travel implications of emerging coronaviruses: sars and mers-cov etiology of severe community-acquired pneumonia during the 2013 hajj-part of the mers-cov surveillance program an update on middle east respiratory syndrome: 2 years later pilgrims and mers-cov: what's the risk? ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia mers-cov outbreak in jeddah-a link to health care facilities jordan mers-cov investigation team. hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndromeadvancing the public health and research agenda on mers-lessons from the south korea outbreak coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers infection prevention and control of epidemic-and pandemicprone acute respiratory infections in health care -who guidelines. geneva, world health organization infection prevention and control during health care for probable or confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection. update 4 th infection control advice -middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov): prevention in travelers surveillance for emerging respiratory viruses idsr as a platform for implementing ihr in african countries establishing a national influenza sentinel surveillance system in a limited resource setting, experience of sierra leone rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects emerging novel and antimicrobial-resistant respiratory tract infections: new drug development and therapeutic options key: cord-312741-0au4nctt authors: lin, panpan; wang, manni; wei, yuquan; kim, taewan; wei, xiawei title: coronavirus in human diseases: mechanisms and advances in clinical treatment date: 2020-10-01 journal: medcomm (beijing) doi: 10.1002/mco2.26 sha: doc_id: 312741 cord_uid: 0au4nctt coronaviruses (covs), a subfamily of coronavirinae, are a panel of single‐stranded rna virus. human coronavirus (hcov) strains (hcov‐229e, hcov‐oc43, hcov‐hku1, hcov‐nl63) usually cause mild upper respiratory diseases and are believed to be harmless. however, other hcovs, associated with severe acute respiratory syndrome, middle east respiratory syndrome, and covid‐19, have been identified as important pathogens due to their potent infectivity and lethality worldwide. moreover, currently, no effective antiviral drugs treatments are available so far. in this review, we summarize the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. f i g u r e 1 genome organization and structure of hcovs human covs generally cause only mild upper respiratory diseases, which is similar to common flu. 11, 12 a novel cov, named sars-cov-2 by the world health organization (who), has emerged again at the end of 2019, causing more infections and deaths worldwide than ever before. 13 the absence of effective antiviral treatments and serious consequences of these three covs have highlighted the urgent need for novel drug development to prevent the spread of covs. herein, this review mainly focuses on the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. covs possess a nonsegmented, positive, single-stranded rna genome of 26-32 kb. 2, 14, 15 all covs have a similar genome arrangement with a 5′-methylated cap structure along with 3′-polyadenylated tail. the replicase gene, occupying about 20 kb, two-thirds of the genome and comprising two open reading frames (orfs), orf1a and orf1b, is located at the 5′ end. 2 it encodes two large polyproteins (pp) 1a and 1ab that can be cleaved by papain-like cysteine protease (plpro) and 3c-like serine protease (3clpro) into nonstructure proteins, involving some proteases, several rna modification enzymes, as well as rna-dependent rna polymerase (rdrp) and helicase (hel) required for virus replication. 16 additionally, an untranslated region (utr) can also be identified at the 5′-end as same as the 3′terminal. structure proteins, encompassing the 3′-terminal one-third of the genome, are arranged in a certain order of hemagglutinin esterase (he) protein that is present in some beta-covs, spike protein (s), small membrane protein (e), membrane protein (m), and nucleocapsid protein (n). in brief, the arrangement of the cov genome can be shown as 5′-utr-replicase gene (orf 1a and orf 1b)-he protein (if have)-s protein-e protein-m protein-n protein-3′ utr-poly (a) 2 ( figure 1 ). cov is named for the club-shaped projections eradiating from the envelope, which forms its corona or crow-like morphology. the shape of the viral particles is roughly spherical with approximately 80-160 nm in diameters. [17] [18] [19] the nucleocapsid protein and the genome rna intertwine to form a helical structure located inside the envelope. for some covs, the spikes on the surface are not only formed by trimers of s protein, but also he proteins. m protein and e protein, two transmembrane proteins, also participate in the composition of the virus (figure 1 ). s protein, a transmembrane protein, mediates the initiation of cov infection by attaching to the specific receptors on the target cells. [20] [21] [22] for a prototypical cov, s protein is usually cleaved into an extracellular receptor binding subunit (s1) and a membrane-anchored subunit (s2), responsible for virus binding and membrane fusion, respectively. 23, 24 two heptad repeats (hr1 and hr2) and enriched alpha-helices are contained in the s2 domain, a feature typical of fusion protein. [25] [26] [27] the receptor-binding domain (rbd) of s protein specifically binds to target receptors, leading to the conformation change of s1/s2 complex that mediates virus entry. 26 furthermore, the rbd also induces potent neutralizing ab response, which turns s protein into an important antigenic determinant capable of protective immunity induction and provides a vital approach for the development of immunotherapies. [28] [29] [30] [31] [32] cov e protein is an integral membrane protein of 76-109 amino acids [33] [34] [35] and is present in small amount in virions. [36] [37] [38] it contains a short hydrophilic n-terminal followed by a single predicted hydrophobic domain and a hydrophilic c-terminal. although its membrane topology remains unclear, cov e protein is commonly referred to as a transmembrane protein with one transmembrane domain. [39] [40] [41] accordingly, the e protein mainly targets er and golgi-complex and participates in part of the life cycle of the virus, including virus assembly, budding, particles release, envelope formation, and viral pathogenesis. 33, 35, [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] in addition, cov e protein may have a crucial role in virus production and maturation, because recombinant covs lacking the e protein exhibit significantly reduced viral titers and toxicity. [51] [52] [53] [54] [55] the m protein is a small transmembrane protein (25) (26) (27) (28) (29) (30) with three transmembrane segments, an n-terminal ectodomain and a c terminal-endodomain, determining the shape of the virion. 56, 57 it is considered as the most abundant structural protein and plays a pivotal role in virion assembly via interacting with other structural proteins. [58] [59] [60] binding of s protein and m protein is essential to the virus assembly and the maintenance of s protein in the er-golgi intermediate compartment (ergic)/golgi complex. [61] [62] [63] virus-like particles (vlps) are assembled when the combination of m and e proteins occurs, which suggests that they are required for the formation of the envelope. 39, [64] [65] [66] additionally, when expressed alone, it can become a homomultimeric complex, the primary driving force of envelope formation. 43, 60, 67 furthermore, as to n protein, a stable nucleocapsid and internal core of virions can be achieved when combined with m protein. 68, 69 the n protein, combined with viral genomic rna to form a helical nucleocapsid inside the viral envelope, is a multifunctional protein. 70 it contains three highly conserved domains: the n-terminal domain (ntd), responsible for rna binding; a c-terminal domain (ctd) that is a hydrophobic and helix-rich terminal, capable of dimerization and oligomerization; and an intrinsically disordered region (rna-binding domain/domain 2) that is a serine/arginine-rich domain (sr-domain) with a significant phosphorylation potential. 15, [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] phosphorylation of the n protein can initiate structural modification leading to an increased rna-binding affinity. 72, 79, 87, 88 the n protein binds to the genomic rna through a beads-on-astring form. likewise, except for the interaction between n protein and nucleic, the ability of complex oligomerization is another pivotal activity required for the formation of the ribonucleoprotein complexes for viral assembly. 89 in addition to the role of the n protein in viral core formation and assembly, 69, [90] [91] [92] it is also involved in other critical processes of viral life cycle such as virus budding and envelope formation, 21,93-95 genomic mrna replication ,and genomic rna synthesis. 96, 97 3 although the pathogenic mechanisms of human covs have not yet been fully understood, the investigation of their unique characteristics of each cov enables to distinguish the various human covs including sars, mers, and sars-cov-2. the crucial early step of the infection of human covs into susceptible host cells is the interaction between viral s protein and cellular target receptors. one of the subunits of s protein, s1, containing rbd, is responsible for specific recognition and binding of the target receptors. the other subunit, s2, is in charge of the membrane fusion. 22, [98] [99] [100] [101] [102] the tissue tropism, as well as the susceptible host species, is mainly determined by the binding of s protein to target receptors. 102 based on lines of known evidence, human covs utilize multiple and different types of cellular receptors rather than use a common receptor. therefore, multiple cellular receptors have been identified as target receptors for the various human covs to date (table 1) . angiotensin-converting enzyme 2 (ace2), the first known human homolog of angiotensin-converting enzyme and the receptor for hcov-nl63, sars-cov, and sars-cov-2, is a vital component of the reninangiotensin system (ras). [103] [104] [105] [106] [107] [108] [109] it is a secreted enzyme with a transmembrane domain, a single metalloprotease active site and a signal peptide, 110 and predominantly expressed in heart, vascular endothelia, epithelia of the small intestine, kidney, and testis; alveolar macrophage and monocytes of the respiratory tract; and epithelial cells of the trachea, bronchi, and alveoli. 103, [110] [111] [112] in contrast to its homolog ace that contributes to the promotion of lung failure pathogenesis, induction of lung edemas, and impairment of lung function, ace2 plays a protective role in severe acute lung injury (ali). imai et al revealed that the deficiency of ace2 in the murine models of acute respiratory distress syndrome (ards) deteriorated the symptom in lung function, which could be recovered by hcov-nl63 hcov-hku1 the recombinant ace2. 113 thus, downregulation of ace2 expression in sars patients could be used as an indicator of severe clinical outcome. 114 besides lung damage caused by sars-cov infection could be attenuated by blocking the renin-angiotensin pathway. 114 overall, ace2 could serve as a novel therapeutic target for severe respiratory diseases. dipeptidyl peptidase iv (dpp4), a type ii transmembrane protein also known as cd26, is identified as a target receptor for mers. 115 it is a prolyl oligopeptidase expressed in various cells including endothelial cells, epithelial cells, and inflammatory cells in the lung and smooth muscle. [116] [117] [118] the multifunctional protein ddp4 is implicated in the activation of t-cell, the activity regulation of chemokines and growth factors, and the regulation of glucose metabolism. [119] [120] [121] [122] not only can it be embedded in the plasma membrane in the form of a homodimer, but it also presents in extracellular fluid like plasma as a soluble form. 118, 123 although ddp4 is expressed in epithelial cells of the upper respiratory tract in camels, it is principally found in alveoli but rarely in the nasal cavity or conducting airway. 115, 124 accordingly, in a murine mers model, though monocyte infiltration, alveolar edema and microvascular thrombosis were observed in the mers-cov-infected lungs, any symptoms were seldom found in the airways. 125 aminopeptidase n (apn), a type of ii metalloprotease also called cd13, is a ubiquitous enzyme expressed in various organs (lung, intestine, and kidney) and cells (epithelial cells, endothelial cells, leukocyte, and fibroblast). 126, 127 it serves as a target receptor for hcov-229e, 128 but not for hcov-oc43. hcov-oc43 shares the same specific target with hcov-hku1, namely 9-o-acetylated sialic acid. 129,130 virus entry is a finely regulated process requiring a series of interactions between the virion and host cell. [131] [132] [133] following the conjunction with the target receptor, cov fuse its envelope with the membrane of the host cell. these processes are forced by the conformational change of s protein, which is triggered by not only the target receptor binding but also ph acidification and proteolytic cleavage led by cell surface or endosomal proteases such as transmembrane protease serine 2 (tmprss2), furin, cathepsin l, elastase, and trypsin. [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] cleavages of s protein are facilitated at two sites: the boundary between the s1 and s2 subunit (s1/s2) and the conserved site upstream of the fusion peptide (s2'). 138, 144 the former one is aimed at releasing rbd from the membrane fusion subunit, and the latter one is important for the exposure of the fusion peptide, hydrophobic in general, which acts as an anchor to target membrane. 138, 145, 146 then the fusion domain adopts two heptad repeats (hr1/hr2) to form a compact coiled-coil conformation called 6-helix bundle or 6hb. 144, 147 through direct interactions with lipid bilayers, the fusion domain disrupts two apposed membrane layers and fuses the viral envelope to host cell membrane. ultimately, the viral genome is successfully released into the cytosol of the target cell. in addition to the viral infection through the plasma membrane, the entry of covs into cells can be accomplished by the endocytic pathway, depending on the virus strains and host cells. 135,144 sars-cov, whose intermediary host is the palm civet, is a highly pathogenic respiratory virus that emerged in 2002, leading to global pandemic that affected more than 8000 people in 29 countries. 148, 149 patients infected with sars-cov initially present "flu-like" syndrome commonly showing high fever, headache, sore throat, myalgia, and dry cough. [150] [151] [152] during the disease progression, ali or ards is developed in a number of patients. 153 pathological manifestations can be described as three phases. the first step is the disturbance of gas exchange in the first week, owing to the extensive edema, shedding of ciliated epithelial cells, and deposition of hyaline-rich substances on the alveolar membrane. in the next step, pulmonary fibrosis occurs that is characterized as the deposition of fibrin at epithelial cells and the alveolar spaces, as well as the infiltration of inflammatory and fibroblasts. finally, fibrosis of lung tissue, collagen deposition, and proliferation of alveolar and interstitial cells represent the final step of disease, about 6-8 weeks. [154] [155] [156] [157] [158] diffuse alveolar damage (dad) accompanied by hyaline membrane formation as well as interstitial thickening is common characteristics of sars-cov inducing pulmonary damage. 159 although many investigators have devoted to inquiring into the pathogenesis of such virus, it has not yet been fully understood until now. the immune response is the earliest alert system of the host cells warning virus attacks. ironically, it also aids viral pathogenesis. pattern recognition receptors (prrs) that are retinoic acid-inducible gene i protein (rig-i, member of rlrs family) and melanoma differentiation-associated protein 5 (mda5) recognize viral pathogen-associated molecular patterns (pamps), such as viral components and replication intermediates, to initiate signaling cascades against virus infection. 160, 161 once the pamps from invaded viruses are detected, rig-i and mda5 interact with the mitochondrial antiviral signaling protein (mavs) that is a mitochondrial membrane-bound f i g u r e 2 escape mechanisms of innate immune response of sars-cov and mers-cov adaptor molecule, followed by the activation of several kinase complexes and multiple subsequent transcription factors (irf3, irf7, and nf-κb). activation of nf-κb induces the production of proinflammatory cytokines and chemokines in the nucleus that is a substantial cause of the ards. 162, 163 phosphorylation of irf3 and irf7 by the kinase complexes results in homo-or heterodimerization of irf3 and irf7. the dimerization initiates the transcription of type i interferons (ifns, ifn-α and ifn-β), activating the signal transducer and activator of transcription proteins (stats) to mediate antiviral response ( figure 2 ). [164] [165] [166] several defensive approaches are used by sars-cov to avoid the prrs defense system and, ultimately, host innate immune response. one approach is to replicate themselves within the double membrane vesicles (dmvs) that are protected from the prrs. 167, 168 the eukaryotic mrnas contain a 5′ cap that usually lacks in the viral mrnas. however, some viruses such as sars-cov are capable of building the rna cap through nsp14 and nsp16/nap10 complex, helping them bypass the recognition by prrs. [169] [170] [171] [172] in addition, a couple of more proteins encoded by the viruses participate in the suppression of the innate immune response by disrupting the ifn response. among the nonstructural proteins, nsp 1 mainly involves in the degradation of host mrnas, inactivation of host translational machinery as well as the inhibition of stat1 phosphorylating. [173] [174] [175] sars-cov plpro interferes phosphorylation of irf3 and disrupts nf-κb signaling probably via interacting with sting. [176] [177] [178] the nsp7 and nsp15 are also potential ifn antagonists though the mechanism is not clear. 177 structure proteins such as the n protein and m protein are likely to suppress the type i ifn path-ways. because it was known that n protein inhibits the ifn transcription, the n protein could have a strong potential influence on the viral rna. 179 m protein blocks the formation of signaling kinase complexes, and suppresses the irf3 and irf7 activities, suggesting the potential role of the n protein in the viral infection as well. 180 such accessory proteins as orf 3b protein are able to inhibit the rig-i activity and irf3 phosphorylation in addition to the transcription of ifn-stimulated genes (isgs) via the isre promoter, while orf6 blocks the nuclear translocation of stat1. 181, 182 in spite of the number of research findings in vitro, they have not been validated in vivo. therefore, it is urgent to examine the findings in vivo for a clear and solid understanding of the infectious process. besides the immune response of the host, ace2 also plays an essential role in the pathogenesis of sars-cov. as a negative regulator on the ras, ace2 has been closely linked to the pathogenesis of pulmonary diseases and considered as a protective factor for ali. 113 consequently, the downregulation of ace2 mediated by sars-cov binding might give an explanation for the progression of severe lung damage occurred on some sars patients. 114 a decade after sars, another novel cov was identified as the pathogen of mers that caused a higher mortality rate (30%-40%) compared with sars (around 10%). 183, 184 sars-cov and mers-cov are both emerged from bats, and are disseminated to human through palm civets and dromedary camels, respectively. [185] [186] [187] [188] mers-cov and sars-cov share some common clinical manifestations ranging from asymptomatic to severe pneumonia in multiple organs, 150, 184, 189 and pathological features including inflammatory cells infiltration and dad. 183 similar to sars-cov, mers-cov is also capable of causing immune dysregulation by attenuating the innate immune response ( figure 2 ). 190, 191 type i interferon is important for the inhibition of mers-cov replication in host cells probably via the suppression of the dmvs formation. 192, 193 capping viral mrna by nsp14 and nsp16/nap10 complex protects mers-cov, as well as sars-cov, from the prrs recognition since the structure of nsp16/nap10 complex in both viruses are analogous. 194 besides, several proteins of mers-cov are involved in immune escape mechanism by involving in the signaling cascades. it was reported that irf3 nuclear translocation and ifn promoter activation are inhibited by m protein, orf4a, orf4b, and orf5, former three of which also restrained the expression from an the isre promoter. 195 inhibition of the phosphorylation of irf3 might be the mechanism for ifr3 translocation inhibiting. 194 moreover, mers-cov orf4a can interact with ifn-inducible double-stranded dna (dsdna)dependent protein kinase activator a (prkra) and subsequently control the function of rig-i and mda5, resulting in the disruption of the ifn response. 196, 197 orf4a and orf4b are thought to participate in the nf-κb signaling by downregulating the gene stimulated by nf-κb and affecting the kinase complexes, respectively. 198 functions of plpro and nsp1 of mers-cov are analogous to the functions of those in sars-cov. 199, 200 like ace2, the entry receptor dpp4 for mers-cov has also a pivotal role in the disease pathogenesis and is considered as a key factor for the intraspecies variation shown in mers infection. 201, 202 it is usually expressed in type ii pneumocytes that cover 2% of the alveolar surface. 115, 124 approximately 95% of the surface area is occupied by the type i pneumocytes that are responsible for gas exchange. 203, 204 but the autopsy reports indicated that both type i and ii pneumocytes in patients died from mers-harbored dpp4 expression and these pneumocytes were infected by the virus. 205, 206 mers-cov infection of type i pneumocytes might lead to the damage of the cells in the alveoli subsequently causing the dad. 207 it suggests that type i pneumocytes expressing dpp4 might be included in the pathogenesis of the disease. this might explain why chronic obstructive pulmonary disease (copd) patients attacked by mers-cov had poor outcomes, since the expression of dpp4 was predominantly upregulated on type i pneumocytes in such patients. 202, 208 besides, owing to high expression of dpp4 shown in the kidney, the renal dysfunction might be caused by either the direct infection or the hypoxic damage. 116 evidence of tubular injury, such as cell debris and tubular dilation, could be observed in the late stage of the infection in mers-cov-infected mice. however, no virus could be detected in such animals in the early stage after infection, meaning such pathologic changes might be related to the hypoxia. 125 the outbreak of covid-19, whose pathogen is sars-cov-2, now poses a serious threat to the global public health. 209, 210 since the emergence of the virus, sars-cov-2 has affected more than 14 million people with more than 597 thousand deaths worldwide as of july 2020. next-generation sequencing of the novel virus has been developed soon after the outbreak, indicating that sars-cov-2 is closely related to the bat-derived sarslike covs. 107, 211 it is now believed that bats are likely to be the natural reservoir, 212, 213 and pangolins are regarded as intermediary host according to the later studies. typical clinical presentations of sars-cov-2-infected patients include fever, dyspnea, dry cough fatigue, myalgia, pneumonia, and ards symptoms, similar to those of sars and mers patients. [214] [215] [216] however, intestinal disorders such as diarrhea are less frequent in covid-19 patients than sars. 214, 215, 217 furthermore, in spite of the variation of amino acid at some residues, homology modeling informed that sars-cov-2 and sars-cov have an analogous rbd structure, and share the same target cell receptor, ace2, to mediate the viral entry. 107, 108, 216, [218] [219] [220] it is speculated that ace2 is involved in the pathogenesis of sars-cov-2. owing to the current sparsity of data on the pathological characters of sars-cov-2, it is poorly understood. a case report from an infected patient died of this disease showed that dad with hyaline membrane formation, infiltration of inflammatory cells, and pulmonary edema could be found in the samples taken from their lungs, which is notably corresponding with the symptoms of sars and mers patients. 221 additionally, lymphopenia is a common manifestation in covid-19 patients and might be an effective indicator to estimate the severity of hospitalized covid-19 patients. 222 lymphopenia is also supposed to be a vital factor related to the pathogenesis that has not been elucidated so far. 213 moreover, the concentration of some cytokines and chemokines detected in the plasma was higher in covid-19 patients compared with healthy individuals. moreover, higher plasma levels of gscf, il-2, il-7, il-10, mcp1, mip1a, ip10, and tnf-α were linked to the more severe disease. 215 all of the data reveal that immunopathology may occupy a crucial place in the development of the disease, and further researches about the pathogenesis of sars-cov-2 are urgently needed in the future. owing to the fact that no effective specific antiviral therapies are currently available for sars, mers, and covid-19, isolation and symptomatic support cares are the major management strategies for suspected and confirmed cases requiring hospital treatment including oxygen inhalation, fluid management, and rational use of antibiotics, to prevent organ failure and secondary bacterial infection and alleviate the symptoms. 189, [223] [224] [225] thus, the identification of effective agents against human covs is urgently needed in the response to the current covid-19 outbreak. all of sars-cov, mers-cov, and sars-cov-2 encode structure proteins (like s protein), nonstructure proteins (eg, plpro, 3clpro, rdrp, and helicase), and accessory proteins that are essential for the viral life cycle and that are considered as important targets for the development of antiviral agents ( figure 3 ). 226, 227 analyses of genomic sequences and protein structure indicated that the catalytic sites of four crucial enzymes and the key drug-binding pockets in viral enzymes were conserved across sars-cov, mers-cov, and sars-cov-2. 227 therefore, the therapeutic experience based on sars and mers is capable to guide the treatment of covid-19. the idea to disturb the normal life cycle of the virus provides significant insights into the clinical treatment strategy. the s protein is important for the discovery of antiviral agents due to its multifunction in virus infection. rbd located on the s1 subunit can bind to the host cell receptors (ace2 for sars-cov and sars-cov-2, dpp4 for mers-cov) initiating the conformational changes in s2 subunit to get viral and cell membranes closer and trigger membrane fusion. 228 consequently, the interaction between rbd and the host cell receptors serves as a key target for the production of neutralizing antibody followed by the vaccine development. 31, 229 monoclonal antibodies (mabs) and fusion inhibitors against s1 and s2 subunit, respectively, are potential antivirus drugs to conquer the viral infection, and the agents targeting the host receptors also play a similar role. [230] [231] [232] [233] [234] likewise, cleavage at the protease site of the s1/s2 complex by host proteases such as tmprss2 and furin is necessary for the membrane fusion and syncytium formation. 143, 235 the endosomal cysteine protease cathepsins promote the entry of covs into the host cell via the endosomal pathway. 236 inhibitors of these host proteases can potently block the cell entry, which has been proved in vitro and require further validation on animals studies. 237 once entering the host cells, covs release the nucleocapsid and genomic rna into the cytoplasm and start the translation of the replicase gene. the large replicase pp1a and pp1ab are cleaved by plpro and 3clpro to produce nonstructural proteins like rdrp and helicase, forming the replication-transcription complex. 238 numerous agents inhibiting these proteins have shown anti-cov effects in vitro. combination of the hydrophobic domains of the replication-transcription complex to the endoplasmic reticulum membrane can form the typical cov replication structures such as dmvs and convoluted membranes, protecting covs from the detection of prrs. 168, 239 viral rna synthesis produces genomic and subgenomic rnas. then the subgenomic rnas are translated to generate the structural and accessory proteins, participating in the assembly of the virion that is released into the extracellular compartment via exocytosis. 8 small interfering rnas (sirnas) disturbing these processes could be used in the treatment of covs infections. although covs are capable of disturbing the ifn response, they are still sensitive to the ifn treatment in vitro, indicating that augmented host innate ifn response can be an effective strategy to control the viral infection. 207, [240] [241] [242] in addition to the enhancement of inf response, several other cell signaling pathways are also regarded as potential anti-cov targets. these include calcineurin-nuclear factor of activated t cells (nfat) pathway and kinase signaling pathways such as erk/mapk and pi3k/akt/mtor pathways, the inhibitors of which have exhibited anti-cov activities as well. [243] [244] [245] since the discovery of new interventions may take months or even years, a more efficient approach is to repurpose existing antiviral agents approved for treating related viral infections. the followings are approved drugs or preclinical compounds that have potential antiviral abilities against sars, mers, and covid-19. virus-targeted therapeutic strategies all of the major proteases of the virus are attractive druggable targets since they are essential for viral transcription and replication ( table 2) . as a key part of replication-transcription complex, rdrp participates in the production of genomic rna and subgenomic rna. nucleoside analogues targeting rdrp is capable of inhibiting viral rna synthesis in a great variety of rna viruses including covs. [246] [247] [248] [249] [250] favipiravir (t-705), a guanine analogue approved in japan for influenza treatment, has been proven to effectively interfere the rna synthesis of rna viruses such as influenza virus, ebola virus, and other hemorrhagic fever viruses at rdrp level. [251] [252] [253] [254] [255] [256] several studies concluded that favipiravir could inhibit avian influenza a (h5n1) virus and ebola virus infection in mice. 256, 257 also, favipiravir has been proved with a notable effect increasing the survival rate of ebolainfected patients from 35.3% to 56.4% in sierra leone. 258 a recent study ended with the statement that favipiravir owns the ability against sars-cov-2 (ec50 = 61.88um, cc50 > 400um, si > 6.46). 259 covid-19 patients were enrolled in randomized trials for the evaluation of the efficacy of favipiravir plus inf-α or baloxavir marboxil (chictr2000029544 and chictr2000029600). another guanosine derivative with broad-spectrum antiviral activity, ribavirin, has been authorized for hcv and respiratory syncytial virus (rsv) treatment. 260 accurate mechanism of ribavirin against virus infection is not clear, but inhibition of mrna capping and viral rna synthesis could be pivotal to its antiviral activity. 261 although high dose of ribavirin has been applied to sars treatment, the anti-mers-cov effects were moderate at such dose in rhesus macaques infected by mers-cov and no obvious survival benefit has been observed in mers patients. 225, [262] [263] [264] [265] [266] [267] recently, an open-label, randomized phase ii clinical trial (nct04276688) has revealed that triple combination of ribavirin, interferon, and lopinavirritonavir in covid-19 treatment was safe and superior to lopinavir-ritonavir therapy alone in remission of symptoms, shortening virus shedding and promoting discharge of mild to moderate covid-19 patients. 268 remdesivir (gs-5734) is a small-molecule monophosphoramidate prodrug of an adenosine analog with the ability to interfere with the rna polymerase and the proofreading exoribonuclease and terminate the nonobligate chain. 269 on day 1 and 100 mg once-daily maintenance for 9 days. however, the first clinical trial (nct04252664) has been suspended so far and the second trial (nct04257656) with 237 covid-19 patients enrolled finally indicated that remdesivir hardly shown any statistically significant clinical benefits. 274 conversely, a research found that 36 of 53 (68%) hospitalized patients suffered from severe covid-19 and treated with compassionate-use remdesivir could achieve clinical improvement. 275 in addition, a phase iii, randomized, double-blind, placebo-controlled trial (nct04280705) conducted by beigel et al uncovered the fact that remdesivir prevailed over placebo in shortening the time to recovery in adults patients. 276 though remdesivir has been approved by the food and drug administration (fda) to treat severe covid-19 patients, further researches are urgently required to determine the efficacy and the indication of remdesivir therapy. a novel synthesized nucleoside analogue, bcx4430 (galidesivir), is designed to inhibit viral rna polymerase activity via terminating nonobligate rna chain. 277 bcx4430 exhibits a promising antiviral capability against a wide array of . 277 it is currently tested in phase i clinical trial (nct03800173) for marburg virus and can be a potential countermeasure against viral diseases that threaten the public health in the world. a recent study also concluded that penciclovir, another rdrp inhibitor that is approved for hsv, showed effects on reducing sars-cov-2 infection by high-dose administration (ec50 = 95.96 µm, cc50 > 400 µm, si > 4.17). 259 although resistance to nucleoside analogs has rarely been reported, it is worth noting that mutation in rdrp is probably responsible for the acquired resistance and should be monitored for the possible resistance. 278 covs plpro enzymes display proteolytic, deubiquitylating, and deisgylating activities. [279] [280] [281] plpro was first regarded as a druggable target for sars-cov, and then several compounds targeting sars-cov plpro were also found to be effective against mers-cov plpro, recently. 282, 283 though numerous plpro inhibitors have been identified, many of them only inhibit enzymatic activities and do not affect on the viral replication. 284, 285 a study from lin et al suggested that an fda-approved alcoholaversive drug, disulfiram could inhibit sars-cov and mers-cov plpro via different mechanisms. and the synergistic inhibition between disulfiram and known plpro inhibitors, like 6-thioguanine and mycophenolic acid, to mers-cov might offer the potential combination treatments against covs in clinical. 286 another essential protease that cleaves the viral polyproteins during viral replication is 3clpro. similar to plpro, a great many of inhibitors have been identified with the ability to target covs 3clpro. among the 3clpro inhibitors, the human immunodeficiency virus (hiv) protease inhibitors are the most comprehensively studied such as lopinavir, ritonavir, asc09f, as well as darunavir and cobicistat. lopinavir and ritonavir, applied in combination to treat hiv infection, have shown improvement in the outcome of sars patients in nonrandomized trials. 287, 288 though lopinavir alone hardly displayed antiviral activity against mers-cov in vitro, the combination of lopinavir and ritonavir ameliorated the outcome in mers-cov-infected nonhuman primates. 289, 290 therefore, the efficacy of the lopinavir-ritonavir combination in mers patients should be reappraised (nct02845843). however, no benefit was observed in lopinavir-ritonavir treatment compared to standard care in a randomized, controlled, open-label clinical trial (chictr2000029308) involving severe covid-19 patients. 291 further trials are still needed to confirm the therapeutic efficacy. in addition, several other clinical trials have been developed to confirm the efficacy of 3clpro inhibitors on covid-19 (nct04252274, nct04251871, nct04255017, chictr2000029539, nct04251871, nct04255017, and nct04261270), as well as darunavir and cobicistat (nct04252274), asc09f combined with oseltamivir (nct04261270). helicase acts on the duplex oligonucleotides and turns them into single strands in an atp-dependent manner in the cov replication cycle. that helicases in different covs are highly homologous making them potentially strong targets for the covs therapeutic options. based on the mechanism actions, the helicase inhibitors can be approximately classified into two groups. one is able to inhibit both the atpase and helicase activities represented by bananins derivatives, 5-hydroxychromone derivatives, and triazole derivatives (compound 16). 292, 293 the other group including ssya 10-001 and adks has the ability to inhibit the helicase activity with no or little effects on the atpase activity. 294 however, the toxicity of helicase inhibitors needs to be examined before being applied to human patients. the transmembrane glycoprotein, s protein, is also a promising target for antiviral agents' development ( table 2 ). one class of antiviral drugs targeting s protein mostly blocks the spike-mediated membrane fusion. a potent mers-cov inhibitor, nafamostat, has demonstrated to be inhibitive against the sars-cov-2 infection (ec50 = 22.50 µm, cc50 > 100 µm, si > 4.44). 259 griffithsin is a red-alga-derived lectin, which specially binds to oligosaccharides located on the surface of viral glycoproteins, for example, s glycoprotein of sars-cov and hiv glycoprotein 120. 295 a wide range of human covs infection could be inhibited by griffithsin, comprising hcov-229e, hcov-nl63, hcov-oc43, and sars-cov. 295, 296 but the value of griffithsin in the treatment or prevention of covid-19 is needed to be evaluated urgently. s2 subunit of s protein contains two heptad repeat (hr1 and hr2). antiviral peptides analogous derived from these regions exhibited inhibition to the spike protein-mediated cell-cell fusion and viral entry in viruses such as sars-cov, mers-cov, as well as hcov-229e. 231, 297, 298 hr2p, a peptide spanning hr2 sequences of mers-cov s protein, was capable of interacting with hr1 region to form a 6-hb complex, resulting in potent inhibition of viral fusion and replication. its analog hr2p-m2 exhibited an obvious improvement in stability, solubility, and antiviral activity after being modified with hydrophilic residues. 228 additionally, hr2p-m2 intranasal administration effectively prevented experimental mice transduced by adenoviral vectors conveying human dpp4 from mers-cov infection with a >1000-fold decrease in viral titers in the lung, and this protection was intensified via the combination of hr2p-m2 and ifn-β. 299 another newly designed fusion inhibitor from mers-cov called mers-five-helix bundle (mers-5hb), which is derived from the 6-hb, also displayed a potent suppression on s protein-mediated syncytial formation. compared with mers-6hb, mers-5hb lacks one hr2, which endows its capability to interact with viral hr2 to interrupt the membrane fusion. 300 besides, mers-5hb could effectively inhibit the entry of pseudotyped mers-cov with 50% inhibitory concentration (ic50) about 1 µm. 300 altogether, the resistance of these drugs can be overcome by combining antiviral peptides aiming at various domains of s2 subunit, which may also attain synergistic effects in vitro. as for sirnas, which displayed antiviral activities in vitro as well as in sars-cov-infected rhesus macaques, are still under preclinical development and demand further studies to seek out the reliable drug delivery methods in a human before the clinical application. [301] [302] [303] m, n, and e proteins and some accessory proteins are not only vital to the virion assembly but also involved in viral pathogenesis in which they function in the interruption of host innate immune response to assist viral infection. for instance, m protein as well as accessory proteins 4a, 4b, and 5 of mers-cov act as ifn antagonist, and n protein of sars-cov serves as an inhibitor of viral rna. researches carried out by he et al and akerstrom et al emphasized that sirnas silencing m, n, e, orf3a, and orf7a/7b play an important role in the inhibition of viral replication of the sars-cov. 304, 305 but the delivery methods still limit their application in human being. nevertheless, various agents related to these proteins are discovered via functional analysis. one example is resveratrol, a natural stilbene derivative demonstrated to reduce inflammation and exert antiviral activity against multiple viruses. [306] [307] [308] [309] [310] [311] in addition, it exhibited significant inhibition of mers-cov infection and prolonged cellular survival after virus challenge in vitro via deregulating the expression of n protein and the apoptosis induced by mers-cov. 312 alternatively, hexamethylene amiloride, a viroporin inhibitor, is capable to suppress the ion channel activity of e protein of covs such as hcov-229e and sars-cov. 313 identified as dna metabolism inhibitor, gemcitabine hydrochloride is a deoxycytidine analog inhibiting both sars-cov and mers-cov with micromolar ec50 and low toxicity, which suggests its potential antiviral capacity for other human covs. 314 lj001 and jl103, two novel lipophilic thiazolidine derivatives, could induce several changes in membrane properties including the decrease in membrane fluidity, contributing to inhibition of membrane fusion, which made them become broad-spectrum enveloped virus entry inhibitors and potential anti-cov agents. 315 host-cell-targeted therapies the host innate immune response is vital to the interruption of viral replication. recombinant interferons have been applicated in treating various viruses as well as many covs (table 3) . though the host interferon response can be inhibited by the covs, they are still proved effective against covs infection such as sars-cov and mers-cov in vitro and several animal models. 242, 264, 289, 299, 316 recombinant interferons are usually combined with other antiviral agents including ribavirin or lopinavir/ritonavir to treat sars-cov and mers-cov infections, 290, 317 and the anti-covs efficacy of interferons is enhanced when added with ribavirin. 318 a combination of interferon-α2b with ribavirin reduced virus replication and improves clinical outcomes in a rhesus macaque model of mers. 264 however, the effectiveness of combination treatments comprising interferons and ribavirin is still controversial in clinical researches. a study of five patients received interferon-α2b and ribavirin showed no survival, but the finding might be not reliable owing to the delayed administration. 266 contrarily, in another study (n = 44), mortality rates of individuals receiving interferon-α2b and ribavirin exhibited a significant reduction in day 14, compared with the individuals received standard supportive care, but no significant difference was observed in day 28. 265 moreover, no significant difference in mortality rates between interferon-α2b and ribavirin treatment group and interferon-β1a and ribavirin ta b l e 3 host-targeted agents for hcovs treating group was observed in a retrospective study. 267 on the other hand, interferon-β1b displayed stronger inhibition to the mers-cov replication in vitro compared with other interferons, and combined use of interferon-β1b with other antiviral compounds should be evaluated in further research. 289, 290 nitazoxanide, originally identified as an antiprotozoal agent, was later considered as a broad-spectrum antiviral agent able to inhibit the replication of numerous dna and rna viruses such as rsv, parainfluenza, rotavirus, hbv, hcv, hiv, yellow fever, as well as covs in vitro. 319 several clinical trials have confirmed its potential value in treating influenza, chronic hbv, and hcv. [319] [320] [321] moreover, recent research indicated that nitazoxanide was capable of inhibiting sars-cov-2 infection at a low micromolar concentration (ec50 = 2.12 µm; cc50 > 35.53 µm; si > 16. 76) , and the in vivo evaluation of this efficacy is demanded. 259 another type i interferon inducer, polyinosinic:polycytidylic acid (poly(i:c)), is an analog of dsdna with a powerful ability to reduce mers-cov load in animal models. 192 and phase ii clinical trials demonstrated that it was well tolerated by malignant gliomas patients when injected intramuscularly. 322, 323 overall, the use of interferons or interferon inducers may be a valuable strategy against covs infection and should be furtherly accessed in animal models. several host factors are considered essential to covs entry, such as the host receptors that bind to covs s protein, and host proteases that facilitated covs entry through the cell surface or endosomal pathway. thus, these factors become attractive targets for anti-cov agents' development (table 3) . antibodies, peptides, and some functional inhibitors targeting the host receptors can effectively interrupt the binding between s protein and the host cells. one example is that the n-(2-aminoethyl)-1-aziridineethanamine (naae), a small molecular inhibitor, is able to target ace2 leading to the block of s protein-mediated membrane fusion, so does the synthetic peptides analogous, p4 and p5. 324, 325 similar agents were also found in mers treatment, one of which, ys110, was confirmed to be well tolerated in patients with advance solid tumors. 326 owing to their specificity to appointed receptors, they were regarded as narrow-spectrum drugs. however, the efficacy of these receptor-targeted agents have never been evaluated in any covs-infected patients and the safeties of these agents also remain unclear. host protease such as cathepsins and tmprss2 play a key role in the cleavage of s protein and the suppression of these proteases with potent inhibitors can obstruct the virus entry through either endosomal pathway or cell surface pathway. k11777 is a cathepsin inhibitor with broad spectrum against enveloped rna virus including sars-cov and mers-cov. 237, 327, 328 however, the camostat mesylate, applied in chronic pancreatitis treating, is a tmprss2 inhibitor that interrupts the tmprss2mediated cell surface entry. 329, 330 the combination of cathepsin inhibitors and tmprss2 inhibitors is crucial to the complete suppression of mers-cov in vitro. inhibition of another host protease, furin, which is vital to furinmediated s cleavage for covs, also can block membrane fusion and the viral entry like mers-cov. 143 notably, inhibition of host proteases may result in some side effects and need further evaluation. some approved antipsychotic agents (chlorpromazine, triflupromazine, and fluphenazine) and cardiotonic steroids (ouabain and bufalin) can inhibit clathrinmediated endocytosis via affecting the assembly of clathrin-coated pits at the plasma membrane and binding sodium/potassium-transporting atpase subunit α1 (atp1a1), respectively. 314, 331, 332 thus, they are considered as clathrin-mediated endocytosis inhibitors. alternatively, endosomal acidification also has a profound impact on endocytosis. increase of endosomal ph shows an inhibitive effect on virus infection, which has been confirmed by chloroquine, a widely used autoimmune disease and antimalarial agents. 333 chloroquine proves to be active against a wide range of rna viruses including hcov-229e, hcov-oc43, sars-cov, and mers-cov. [334] [335] [336] [337] [338] recently, chloroquine is identified to function at both entry and postentry phase of the sars-cov-2 infection with the ec90 value of 6.90 µm in vero e6 cells. 259 additionally, as an immunoregulator, its antiviral activity may be synergistically intensified in vivo. 259 therefore, chloroquine is suggested as a potential option against the emerging sars-cov-2. significantly, higher dose of chloroquine should not be recommended for severe covid-19 patients owing to the its drug safety, particularly while simultaneously accepting azithromycin and oseltamivir treatment, which was presented by a randomized clinical trial (nct04323527). 339 hydroxychloroquine, a chloroquine analog with lower toxicity, was listed as a potential anti-sars-cov-2 agent and recommended to be applied in covid-19 treatment by chinese national guideline and fda. 340 however, evidence of the benefits and harms of hydroxychloroquine therapy is still insufficient and conflicting. some small studies show that hydroxychloroquine was capable of decreasing sars-cov-2 shedding that could be enhanced by the combination of azithromycin and achieving a shorter time to clinical recovery. 341, 342 but almost no clinical benefit was observed in other studies. 340, 343 therefore, therapeutic efficacy of hydroxychloroquine is still needed to be reconfirmed. moreover, there are several factors required to be reconsidered before efficacy evaluation, such as the severity of illness, definition of the endpoints, and effects of the comorbidities. except for the innate immune response, host receptors, and other factors affecting the endocytosis, some signaling pathways have also been suggested as useful approaches for discovering anti-cov reagents (table 3) . cyclophilins are peptidyl-prolyl isomerases with physiological functions showing as modulating the calcineurin/nfat pathway via reacting with covs nsp1, which is important for the immune cell activation. 244 in addition, they are also required for the replication of numerous viruses including hiv, hcv, as well as covs. 344 consequently, inhibition of cyclophilins by cyclosporines, such as cyclosporin a (csa) and its derivatives, has shown to block the replication of a wide range of covs. 244, 245, 345, 346 however, the obvious immune suppressive properties of csa limit its application in antiviral therapy. but alisporivir, a nonimmunosuppressive cyclosporin a-analog, also displayed the inhibition to the covs replication at a lowmicromolar concentration. 347 additionally, the combined use of cyclosporine and interferon or ribavirin in vitro was beneficial to inhibit sars-cov or mers-cov infection, which needed to be furtherly evaluated in animal models. 347, 348 furthermore, some antiviral agents inhibiting the cellular signaling pathways, in particular, the erk pathway and pi3k/akt pathway, also interrupt the replication of covs. 243, 314, 349 however, the efficacy and safety against covs infection of these agents still need to be reconsidered. corticosteroids, which were used in sars and mers treatment, have been linked to several complications such as psychosis, diabetes, and avascular necrosis. 350, 351 they also were pointed out to be associated with viral replication prolongation in mers patients. 351 however, an update on the efficacy of dexamethasone based on a press release publicized recently indicated that severe covid-19 patients given 6 mg dexamethasone once daily shown a lower mortality (about 8-26%) compared to patients with standard care. 352, 353 besides, another agent, methylprednisolone, also exhibited potential capacity in reducing the mortality rate and achieving better clinical outcomes in severe covid-19 patients. 354, 355 thus, it is wise to apply corticosteroids to the right patients at the right time. but physicians also need to monitor the corticosteroid-related complications. clinical trials of corticosteroid treatments are shown in table 3 . potential immunotherapeutic options s protein of sars-cov proves to be highly immunogenic during infection and responsible for eliciting a humoral immune response in the host. 356 antivirus antibodies could be detected in the plasma of convalescent patients' infected sars-cov and mers-cov. 357, 358 convalescent plasma therapy has been applicated in treating patients infected by numerous viruses involving ebola virus, junin virus, machupo virus, and lassa fever. [359] [360] [361] [362] as for sars-cov, higher day-22 discharge rate and lower mortality rate have been observed among sars patients who received convalescent plasma transfusion before day 14 of the illness. 363, 364 this is consistent with another small cohorts research concluding infected patients with severe conditions who failed to respond to current therapies but finally survived after transfused with convalescent plasma, with no obvious side effects. 358 similar results were found in mers patients. 365 additionally, plasma adoptive therapy with anti-mers-cov antibodies could block the virus adhesion and accelerate the viral elimination from mers-cov-infected animal models. 192 but the efficacy and safety of convalescent plasma therapy in covid-19 patients still need to be reevaluated. although convalescent plasma therapy proves to be a potentially effective therapeutic option for emerging covs, several factors still limit its extensive use in clinical, one of which is enough titers of serum neutralizing antibodies. the development of mabs targeting the s protein of covs is regarded as a remedial strategy (table 4 ). potent mabs against s protein of human covs can be attained via transgenic mice immunization, convalescent b cells immortalization, and cloning of small chain variable regions from naïve and convalescent patients. 366 the majority of mabs interact with the rbd of s protein leading to the interruption of rbd-receptor binding and block the viral attachment. a few mabs react with other regions of s protein besides the rbd. 366 although binding to different epitopes, these mabs exhibit capacity to reduce the viral titers. two rbd-specific neutralizing mabs, mers-4 and mers-27, which were derived from single-chain variable regions, revealed suppressive effects against both mers-cov and pseudotyped mers-cov infection at nanomolar concentrations and were recommended as promising candidates for therapeutic interventions to mers. 232 based on similar mechanisms, other human mabs for mers-cov were also capable of competing with dpp4 for rbd binding and neutralizing the virus. 233, 234, [367] [368] [369] [370] when administrated to individuals at risk, some of the mabs were capable of preventing viral replication and contributing to block the transmission of mers-cov among human. 368 thus, such antibodies could be served as prophylactic strategies in clinical and valuable tools to guild the development of effective anti-covs vaccines. 234,368 from sars-cov to sars-cov-2, the emergence of severe human covs have taught us many lessons about the importance of rapid diagnostics and effective vaccines to control the outbreak caused by these viruses. due to the persistence of zoonotic sources in endemic areas, lethal covs remain existing in human society and may lead to the epidemic at any time. thus, a priority is to develop vaccines targeting conserved alleles and providing broad-spectrum protection against varied viral strains. since the emergence of sars-cov and mers-cov, several strategies were applicated in vaccine design, including inactivated virus vaccines, live-attenuated virus vaccines, viral vector vaccines, nanoparticles, recombinant protein subunits vaccines, and dna vaccines (table 4) . 371, 372 and clinical trials have also been developed to test the efficacy of the novel vaccines (table 5) . effective vaccines are pivotal in blocking the virus spread from animals' reservoirs to human hosts. inactivated virus vaccines, preserving the viral structure and antigenicity but eliminating the infectious ability, could elicit neutralizing antibodies in animal models of sars-cov and show protection against viral replication when administrated with or without adjuvants. 372 which may be related to the disseminated infection observed in immunocompromised patients. in addition, live-attenuated virus vaccines can induce an innate and adaptive immune response and the protective value can last for a long time. 371 besides, other strategies for vaccines development are also evaluated in animal models. based on the experience of sars-cov and mers-cov, the development of novel sars-cov-2 vaccine is currently underway and requires more research. however, several concerns should be addressed about the vaccination. the first is the disease deterioration caused by vaccination. although this situation only appears in a small subset of sars vaccine studies, it is still a significant problem that needs to be properly solved. 372 second, the variability of s protein can mediate covs escape from neutralization, suggesting that recombinant protein subunits vaccines based on s protein may demand multivalent approaches. 376 last but not least, how to define ta b l e 5 important clinical trials with vaccines for sars-cov, mers-cov, and sars-cov-2 the emergence and prevalence of highly pathogenic cov severely threaten public health. a task of top priority is to make clear the viral structural and epidemiological characteristics and block the viral dissemination as well as the progression of the disease, at the first case. to date, further understanding of the life cycle and the pathogenesis of emerging human covs makes current therapeutic strategies of antiviral infection more rational. repurposing existing antiviral drugs is an effective short-term strategy to deal with emerging cov such as the ongoing sars-cov-2. various agents with different targets have been evaluated in vitro and in vivo. but not all antiviral agents are capable of achieving better efficacy than in vitro, and in vivo studies are needed to select optimal agents. suitable animal models are particularly significant. however, there are only a few effective animal models available for the studies of covs treatment, which may postpone the clinical evaluation of drugs. besides, due to the diversity of viruses and the capacity of rapidly mutating, some antiviral reagents available for existing covs may become invalid. accordingly, sequencing more natural specimens and combination therapy with two or more synergistic agents have become promising solutions. furthermore, the efficacy of current antiviral therapies needs to be estimated in larger scale, well-organized randomized clinical trial the efficacy and safety of the most practicable strategies for the ongoing covid-19 are urgently needed to be further assessed in clinical trials, involving remdesivir (gs-5734), lopinavir/ritonavir, lopinavir/ritonavir combined with ifn-β, convalescent plasma, and mabs, which prove to be potentially effective options against sars-cov-2. additionally, to control the future cov pandemics, the development of novel broad-spectrum antiviral agents covering numerous covs will become the ultimate goal. the authors declare no conflict of interest. wei https://orcid.org/0000-0002-6513-6422 coronavirus diversity, phylogeny and interspecies jumping coronavirus pathogenesis is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus interspecies transmission and emergence of novel viruses: lessons from bats and birds the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease cultivation of viruses from a high proportion of patients with colds a new virus isolated from the human respiratory tract epidemiology, genetic recombination, and pathogenesis of coronaviruses molecular evolution of human coronavirus genomes korean society of epidemiology -nco vtft. an interim review of the epidemiological characteristics of 2019 novel coronavirus coronavirus envelope protein: a small membrane protein with multiple functions the molecular biology of coronaviruses proteolytic processing of polyproteins 1a and 1ab between non-structural proteins 10 and 11/12 of coronavirus infectious bronchitis virus is dispensable for viral replication in cultured cells supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion coronavirus immunogens synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of viruslike particles pre-fusion structure of a human coronavirus spike protein sars coronavirus: a new challenge for prevention and therapy cellular entry of the sars coronavirus structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors structural characterization of the sarscoronavirus spike s fusion protein core major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein localization of neutralizing epitopes and the receptor-binding site within the aminoterminal 330 amino acids of the murine coronavirus spike protein identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein exceptional flexibility in the sequence requirements for coronavirus small envelope protein function association of the infectious bronchitis virus 3c protein with the virion envelope the 9-kda hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated mouse hepatitis virus gene 5b protein is a new virion envelope protein infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles a comprehensive comparison of transmembrane domains reveals organelle-specific properties subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer assembly of the coronavirus envelope: homotypic interactions between the m proteins sars coronavirus e protein forms cation-selective ion channels differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e bcl-xl inhibits t-cell apoptosis induced by expression of sars coronavirus e protein in the absence of growth factors the transmembrane domain of the infectious bronchitis virus e protein is required for efficient virus release biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells role of the coronavirus e viroporin protein transmembrane domain in virus assembly severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis heterologous gene expression from transmissible gastroenteritis virus replicon particles generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome the small envelope protein e is not essential for murine coronavirus replication a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway characterization of severe acute respiratory syndrome coronavirus membrane protein sequence and topology of a model intracellular membrane protein, e1 glycoprotein, from a coronavirus genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus the molecular biology of coronaviruses a structural analysis of m protein in coronavirus assembly and morphology envelope glycoprotein interactions in coronavirus assembly efficient assembly and release of sars coronavirus-like particles by a heterologous expression system coronaviruses: an overview of their replication and pathogenesis nucleocapsidindependent assembly of coronavirus-like particles by co-expression of viral envelope protein genes coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins characterization of the coronavirus m protein and nucleocapsid interaction in infected cells the membrane m protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein the nucleocapsid protein of the sars coronavirus is capable of selfassociation through a c-terminal 209 amino acid interaction domain the dimer interface of the sars coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain modular organization of sars coronavirus nucleocapsid protein characterisation of the rna binding properties of the coronavirus infectious bronchitis virus nucleocapsid protein amino-terminal region structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization identification of in vivointeracting domains of the murine coronavirus nucleocapsid protein glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication oligomerization of the carboxyl terminal domain of the human coronavirus 229e nucleocapsid protein solution structure of the c-terminal dimerization domain of sars coronavirus nucleocapsid protein solved by the sail-nmr method high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 theoretical aspects of virus capsid assembly analysis of multimerization of the sars coronavirus nucleocapsid protein mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in rna binding by using surface plasmon resonance mapping of the coronavirus membrane protein domains involved in interaction with the spike protein envelope protein palmitoylations are crucial for murine coronavirus assembly the hydrophobic domain of infectious bronchitis virus e protein alters the host secretory pathway and is important for release of infectious virus the coronavirus e protein: assembly and beyond mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes the intracellular sites of early replication and budding of sars-coronavirus cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer glycan shield and epitope masking of a coronavirus spike protein observed by cryoelectron microscopy cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry highly conserved regions within the spike proteins of human coronaviruses 229e and nl63 determine recognition of their respective cellular receptors genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars angiotensin-converting enzyme 2 (ace2) in disease pathogenesis a novel angiotensinconverting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis angiotensin-converting enzyme 2 protects from severe acute lung failure a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome dipeptidylpeptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv physiology and pharmacology of dpp-4 in glucose homeostasis and the treatment of type 2 diabetes dipeptidyl peptidase iv (dpp-4) inhibition alleviates pulmonary arterial remodeling in experimental pulmonary hypertension inhibitors of dipeptidyl peptidase iv (dp iv, cd26) induces secretion of transforming growth factor-beta 1 (tgf-beta 1) in stimulated mouse splenocytes and thymocytes dpp-4 inhibitors and their potential role in the management of type 2 diabetes revisiting an old acquaintance: cd26 and its molecular mechanisms in t cell function cd26/dipeptidylpeptidase iv-chemokine interactions: doubleedged regulation of inflammation and tumor biology cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 the structure and main functions of aminopeptidase n human aminopeptidase n is a receptor for human coronavirus 229e molecular determinants of species specificity in the coronavirus receptor aminopeptidase n (cd13): influence of n-linked glycosylation human coronavirus hku1 spike protein uses o-acetylated sialic acid as an attachment receptor determinant and employs hemagglutininesterase protein as a receptor-destroying enzyme human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a virus entry: open sesame the cell biology of receptor-mediated virus entry dynamics of virus-receptor interactions in virus binding, signaling, and endocytosis characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the s2 domain cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein mechanisms of coronavirus cell entry mediated by the viral spike protein ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence physiological and molecular triggers for sars-cov membrane fusion and entry into host cells structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme from sars to mers, thrusting coronaviruses into the spotlight isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars: clinical presentation, transmission, pathogenesis and treatment options identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome allelic variation in the toll-like receptor adaptor protein ticam2 contributes to sars-coronavirus pathogenesis in mice. g3 (bethesda) a clinicopathological study of three cases of severe acute respiratory syndrome (sars) lung pathology of fatal severe acute respiratory syndrome the spectrum of pathological changes in severe acute respiratory syndrome (sars) pathology and pathogenesis of severe acute respiratory syndrome evolution of pulmonary pathology in severe acute respiratory syndrome sars: prognosis, outcome and sequelae cytosolic surveillance and antiviral immunity sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion biomarkers in acute respiratory distress syndrome the mercurial nature of neutrophils: still an enigma in ards? sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase in vitro reconstitution of sars-coronavirus mrna cap methylation biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-omethylation by nsp16/nsp10 protein complex ribose 2'-omethylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists pathogenesis of middle east respiratory syndrome coronavirus middle east respiratory syndrome (mers) a review of studies on animal reservoirs of the sars coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camelto-human transmission of mers coronavirus middle east respiratory syndrome tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis rapid generation of a mouse model for middle east respiratory syndrome antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus coronavirus nonstructural protein 16: evasion, attenuation, and possible treatments the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist middle east respiratory syndrome coronavirus 4a protein is a double-stranded rnabinding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea renewal of alveolar epithelium in the rat following exposure to no2 identification of genes differentially expressed in rat alveolar type i cells clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques dpp4, the middle east respiratory syndrome coronavirus receptor, is upregulated in lungs of smokers and chronic obstructive pulmonary disease patients first case of 2019 novel coronavirus in the united states a novel coronavirus from patients with pneumonia in china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan genomic characterization and infectivity of a novel sars-like coronavirus in chinese bats a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia 2019-ncov: new challenges from coronavirus pathological findings of covid-19 associated with acute respiratory distress syndrome lymphopenia predicts disease severity of covid-19: a descriptive and predictive study the novel coronavirus: a bird's eye view medical treatment of viral pneumonia including sars in immunocompetent adult clinical management and infection control of sars: lessons learned coronaviruses -drug discovery and therapeutic options learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor crossneutralization of human and palm civet severe acute respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation protease inhibitors targeting coronavirus and filovirus entry genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans targeting membranebound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis the sars-coronavirus-host interactome: identification of cyclophilins as target for pancoronavirus inhibitors mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment inhibitors of the hepatitis c virus polymerase; mode of action and resistance approved antiviral drugs over the past 50 years new nucleoside analogues for the treatment of hemorrhagic fever virus infections design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity therapeutic options for the 2019 novel coronavirus (2019-ncov) t-705 (favipiravir) induces lethal mutagenesis in influenza a h1n1 viruses in vitro in vitro and in vivo activities of t-705 against arenavirus and bunyavirus infections activity of t-705 in a hamster model of yellow fever virus infection in comparison with that of a chemically related compound, t-1106 post-exposure efficacy of oral t-705 (favipiravir) against inhalational ebola virus infection in a mouse model japanese surveillance systems and treatment for influenza successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model efficacy of orally administered t-705 on lethal avian influenza a (h5n1) virus infections in mice clinical and virological characteristics of ebola virus disease patients treated with favipiravir (t-705)-sierra leone remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide depletion of gtp pool is not the predominant mechanism by which ribavirin exerts its antiviral effect on lassa virus development of a standard treatment protocol for severe acute respiratory syndrome the management of coronavirus infections with particular reference to sars treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial compassionate use of remdesivir for patients with severe covid-19 remdesivir for the treatment of covid-19 -preliminary report protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (t-705), a broad-spectrum antiviral the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deisgylating activities mers-cov papain-like protease has deisgylating and deubiquitinating activities thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus identification and design of novel small molecule inhibitors against mers-cov papainlike protease via high-throughput screening and molecular modeling the sarscoronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 2,6-bis-arylmethyloxy-5-hydroxychromones with antiviral activity against both hepatitis c virus (hcv) and sars-associated coronavirus (scv) design, synthesis and molecular docking of novel triazole derivatives as potential cov helicase inhibitors evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein peptide-based membrane fusion inhibitors targeting hcov-229e spike protein hr1 and hr2 domains protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection identification of a novel inhibitor against middle east respiratory syndrome coronavirus using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque inhibition of sars-cov replication by sirna silencing sars-cov spike protein expression in cultured cells by rna interference inhibition of sars-cov replication cycle by small interference rnas silencing specific sars proteins, 7a/7b, 3a/3b and s development of interfering rna agents to inhibit sars-associated coronavirus infection and replication resveratrol suppresses nuclear factor-kappab in herpes simplex virus infected cells resveratrol decreases nitric oxide production by hepatocytes during inflammation polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression studies on trans-resveratrol/carboxymethylated (1,3/1,6)-β-dglucan association for aerosol pharmaceutical applications resveratrol attenuates inflammatory hyperalgesia by inhibiting glial activation in mice spinal cords resveratrol-mediated gamma interferon reduction prevents airway inflammation and airway hyperresponsiveness in respiratory syncytial virus-infected immunocompromised mice effective inhibition of mers-cov infection by resveratrol hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection singlet oxygen effects on lipid membranes: implications for the mechanism of action of broad-spectrum viral fusion inhibitors treatment of sars with human interferons inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines nitazoxanide: a first-in-class broad-spectrum antiviral agent effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial clinical trial: randomized, double-blind, placebo-controlled study of nitazoxanide monotherapy for the treatment of patients with chronic hepatitis c genotype 4 induction of cd8+ tcell responses against novel glioma-associated antigen peptides and clinical activity by vaccinations with {alpha}-type 1 polarized dendritic cells and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent malignant glioma a multiinstitution phase ii study of poly-iclc and radiotherapy with concurrent and adjuvant temozolomide in adults with newly diagnosed glioblastoma structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor identification of critical determinants on ace2 for sars-cov entry and development of a potent entry inhibitor inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody a cysteine protease inhibitor rescues mice from a lethal cryptosporidium parvum infection cure of hookworm infection with a cysteine protease inhibitor efficacy of camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease pancreatic stellate cells: new target in the treatment of chronic pancreatitis screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture atp1a1-mediated src signaling inhibits coronavirus entry into host cells anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine inhibition of human coronavirus 229e infection in human epithelial lung cells (l132) by chloroquine: involvement of p38 mapk and erk a systematic screen of fda-approved drugs for inhibitors of biological threat agents effects of chloroquine on viral infections: an old drug against today's diseases? chloroquine is a potent inhibitor of sars coronavirus infection and spread effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial hydroxychloroquine and azithromycin as a treatment of covid-19: results of an openlabel non-randomized clinical trial clinical efficacy of hydroxychloroquine in patients with covid-19 pneumonia who require oxygen: observational comparative study using routine care data observational study of hydroxychloroquine in hospitalized patients with covid-19 suppression of coronavirus replication by cyclophilin inhibitors human coronavirus nl63 replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine a-derivatives including alisporivir porcine epidemic diarrhea virus induces caspaseindependent apoptosis through activation of mitochondrial apoptosis-inducing factor alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model effect of interferon alpha and cyclosporine treatment separately and in combination on middle east respiratory syndrome coronavirus (mers-cov) replication in a human in-vitro and ex-vivo culture model abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion factors associated with psychosis among patients with severe acute respiratory syndrome: a case-control study corticosteroid therapy for critically ill patients with middle east respiratory syndrome dexamethasone in the management of covid -19 covid-19: demand for dexamethasone surges as recovery trial publishes preprint risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan a retrospective cohort study of methylprednisolone therapy in severe patients with covid-19 pneumonia identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital the use of lassa fever convalescent plasma in nigeria treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee treatment of argentine hemorrhagic fever with convalescent's plasma. 4433 cases bolivian hemorrhagic fever. a report of four cases retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients use of convalescent plasma therapy in sars patients in hong kong middle east respiratory syndrome virus pathogenesis neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus a conformation-dependent neutralizing monoclonal antibody specifically targeting receptorbinding domain in middle east respiratory syndrome coronavirus spike protein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a decade after sars: strategies for controlling emerging coronaviruses sars vaccines: where are we? inactivated sars-cov vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry a subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease escape from human monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element synthesis and evaluation of phenylisoserine derivatives for the sars-cov 3cl protease inhibitor identification, synthesis and evaluation of sars-cov and mers-cov 3c-like protease inhibitors engineering a novel antibodypeptide bispecific fusion protein against mers-cov. antibodies (basel) identification of a peptide derived from the heptad repeat 2 region of the porcine epidemic diarrhea virus (pedv) spike glycoprotein that is capable of suppressing pedv entry and inducing neutralizing antibodies a novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses antiviral activity of k22 against members of the order nidovirales purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice key: cord-307067-cpc1yefj authors: van doremalen, neeltje; haddock, elaine; feldmann, friederike; meade-white, kimberly; bushmaker, trenton; fischer, robert j.; okumura, atsushi; hanley, patrick w.; saturday, greg; edwards, nick j.; clark, madeleine h. a.; lambe, teresa; gilbert, sarah c.; munster, vincent j. title: a single dose of chadox1 mers provides protective immunity in rhesus macaques date: 2020-06-10 journal: sci adv doi: 10.1126/sciadv.aba8399 sha: doc_id: 307067 cord_uid: cpc1yefj developing a vaccine to protect against the lethal effects of the many strains of coronavirus is critical given the current global pandemic. for middle east respiratory syndrome coronavirus (mers-cov), we show that rhesus macaques seroconverted rapidly after a single intramuscular vaccination with chadox1 mers. the vaccine protected against respiratory injury and pneumonia and reduced viral load in lung tissue by several orders of magnitude. mers-cov replication in type i and ii pneumocytes of chadox1 mers–vaccinated animals was absent. a prime-boost regimen of chadox1 mers boosted antibody titers, and viral replication was completely absent from the respiratory tract tissue of these rhesus macaques. we also found that antibodies elicited by chadox1 mers in rhesus macaques neutralized six different mers-cov strains. transgenic human dipeptidyl peptidase 4 mice vaccinated with chadox1 mers were completely protected against disease and lethality for all different mers-cov strains. the data support further clinical development of chadox1 mers. coronaviruses (cov) pose a continuous emerging virus threat, as demonstrated by the emergence of three previously unidentified corona viruses in the past 18 years. severe acute respiratory syndrome coronavirus (sars-cov) was first detected in 2003 and went on to infect >8000 people, resulting in 774 fatalities (1) . in 2012, middle east respiratory syndrome coronavirus (mers-cov) was first detected. mers-cov continues to infect humans from the dromedary camel host and has thus far infected >2500 people, resulting in 866 fatalities (2). an outbreak of pneumonia with an unknown cause in wuhan, china was first reported in december 2019. although information is still limited, we now know that this outbreak is caused by a third emerging cov. currently, >200,000 cases are associated with this outbreak (3) . the clinical spectrum of mers-cov infection in humans varies from asymptomatic to severe respiratory disease and death. patients present with influenza-like symptoms such as a fever and shortness of breath. thereafter, they may develop pneumonia, which can require mechanical ventilation and support in an intensive care unit (2). human-to-human transmission of mers-cov is relatively limited and occurs mainly in nosocomial settings but has been reported in local communities as well (1) . in 2015, a traveler from the middle east to south korea caused an outbreak involving 186 people and 38 fatalities (4). the nosocomial outbreak lasted from may to july, and 16,752 people were isolated with mers-cov-like symptoms. at least three superspreaders were identified in the outbreak, who infected 28, 85, and 23 patients, respectively (5) . the introduction and spread of mers-cov in south korea underscore the potential of this virus to cause epidemics outside of the arabian peninsula. mers-cov has continued to cause disease in humans, and in 2019, 152 cases have been reported in the kingdom of saudi arabia (ksa), of which 33% were fatal (2). the ongoing circulation of mers-cov and subsequent outbreaks in the human population highlight the need for an efficient mers-cov vaccine. mers-cov is mainly prevalent in the arabian peninsula, with the majority of cases occurring in ksa (84%) (6) . however, phylogenetically diverse mers-cov strains have been isolated from africa and the middle east (7) (8) (9) , and antigenic differences have been reported between spike (s) proteins, the main antigen used in mers-cov vaccines, from the middle east and africa (8) . thus, it is important that a mers-cov vaccine is protective against a variety of diverse mers-cov strains. thus far, only four vaccines have been tested in rhesus macaques. these are a dna vaccine (10); a vaccine based on the receptor-binding domain of the mers-cov s adjuvanted with alum (11); a combination of s plasmid dna and s1 protein (12) ; and virus-like particles based on mers-cov s, matrix, and envelope proteins (13) . of the four vaccine studies, only three studies included challenge studies, all of which used mers-cov isolates from 2012. to date, there have been no reports of efficacy of a single-dose mers-cov vaccine in nonhuman primates (nhps) . we recently demonstrated that vaccination of mice with a replication-deficient simian adenovirus vaccine vector (chadox1) encoding full-length mers-cov s protein (chadox1 mers) elicited high-titer mers-cov-neutralizing antibodies and a robust cd8 + t cell response against the s protein (14) . in addition, chadox1 mers vaccination resulted in full protection of human dipeptidyl peptidase 4 (hdpp4) transgenic mice against a lethal challenge with mers-cov (15) . chadox1 mers vaccination of dromedary camels was immunogenic and reduced mers-cov shedding after challenge in a highly stringent natural transmission model with multiday exposure to infectious mers-cov (16) . chad-vectored vaccines against malaria (17) , hiv (18) , influenza (19) , hepatitis c (20), tuberculosis (21) , ebola (22) , and others show an excellent immunogenicity and safety profile in humans. in the current manuscript, we show that a single dose of chadox1 mers vaccine protects rhesus macaque model against a mucosal challenge with hcov-emc/2012. serum obtained from vaccinated rhesus macaques was able to neutralize six diverse mers-cov strains. furthermore, a single dose of chadox1 mers vaccine protects hdpp4 transgenic mice against all evaluated mers-cov strains. three groups of six animals each were vaccinated with chadox1 mers via a prime-boost regimen [−56 and −28 dpi (days post infection)] or prime-only regimen (−28 dpi) or with chadox1 green fluorescent protein (gfp) (−56 and −28 dpi). animals were then challenged with 7 × 10 6 tcid 50 (median tissue culture infectious dose) of hcov-emc/2012 on 0 dpi via combined intratracheal, intranasal, oral, and ocular route (23) . blood samples were taken at −56, −42, −28, −14, and 0 dpi (fig. 1a) . the humoral immune response to vaccination was examined by enzyme-linked immunosorbent assay (elisa) using s protein and by virus neutralization assay. s proteinspecific immunoglobulin g (igg) antibodies were detected as early as 14 days after vaccination in both chadox1 mers-vaccinated groups. all animals in these groups had s protein-specific antibodies at 0 dpi, whereas no antibodies against s protein were found before vaccination or at any time in the chadox1 gfp-vaccinated group. a significant difference in elisa titers was observed between primeboost and prime-only animals at 0 dpi (fig. 1b) . neutralizing antibodies were detected in 11 of 12 chadox1 mers-vaccinated animals at the time of challenge. one animal in the prime-only vaccination group did not have detectable neutralizing antibodies at this time and had the lowest antibody titer as measured by elisa (1600). a significant difference in vn (virus-neutralizing) titers was observed between prime-boost and prime-only animals at 0 dpi (fig. 1c) . a second chadox1 mers vaccination at −28 dpi resulted in a statistically significant increase in s protein-specific elisa titer (geometric mean titer −28 dpi = 1600; 0 dpi = 10,159; p < 0.0001) and neutralizing antibody titer (geometric mean titer −28 dpi = 24; 0 dpi = 148; p < 0.0001) as determined via two-tailed t test, although neutralizing antibodies against the chadox1 vector could be detected at the time of the second vaccination ( fig. s1a ). this suggests that the presence of neutralizing antibodies against chadox1 does not prevent the vaccine vector from boosting the immune response. vaccination with chadox1 mers reduces disease severity animals vaccinated with chadox1 gfp and challenged with mers-cov showed similar clinical signs as previously reported (23), such as a decreased appetite and increased respiratory rate. clinical signs were scored using a standard nhp scoring sheet, focusing on areas such as general appearance, nasal discharge, and food intake. on average, chadox1 mers-vaccinated animals had a lower clinical score than chadox1 gfp-vaccinated animals ( fig. 2a) . all animals underwent exams on 0, 1, 3, 5, and 6 dpi. no significant changes in weight or body temperature were observed for the duration of the study. peripheral capillary oxygen saturation (spo 2 ) measurements supported a decrease in oxygen saturation from the baseline in chadox1 gfp-vaccinated animals, but not in chadox1 mersvaccinated animals (fig. 2b ). ventrodorsal and lateral thoracic radiographs were collected on all exam days. all animals vaccinated with chadox1 gfp showed moderate to severe pulmonary interstitial infiltrations, whereas animals vaccinated with chadox1 mers showed only mild signs of respiratory disease. a severe collapsed lung was observed on 3 dpi in two animals in the prime-boost group, likely caused by the bronchoalveolar lavage (bal) performed on 1 dpi. full details of all observed signs can be found in table s2 ( fig. 3a and table s2). all lung lobes (right cranial, right middle, right caudal, left cranial, left middle, and left caudal) of each individual animal were scored for severity of disease signs for each day radiographs were taken, and average scores were compared. scores obtained from animals vaccinated with chadox1 gfp were significantly higher from animals vaccinated with a prime-boost regimen of chadox1 mers on 3, 5, and 6 dpi (fig. 3b) . upon necropsy on 6 dpi, gross lung lesions were more prevalent in animals vaccinated with chadox1 gfp than in animals vaccinated with chadox1 mers (fig. 3 , c and d). animals vaccinated with chadox1 gfp showed focally extensive areas of consolidation in all lung lobes and lungs generally failed to collapse. mediastinal lymph nodes were often edematous and enlarged. animals that received a vaccination with chadox1 mers, either via a prime-boost or primeonly regimen, had either no lesions or limited small multifocal areas of consolidation and congestion. an increased lung:body weight ratio is an indicator of pulmonary edema. animals in the control group had significantly higher lung:body weight ratios compared to chadox1 mers-vaccinated animals, and there was minimal difference between prime-boost and prime-only chadox1 mers-vaccinated animals (fig. 3f) . . statistical significance between −28 and −14 dpi in the prime-boost group was determined via one-tailed paired student's t test. statistical significance between prime-boost and prime-only groups on 0 dpi was determined via two-tailed unpaired student's t test. **p < 0.01; ***p < 0.001. lung tissue sections were stained with hematoxylin and eosin or with mers-cov-specific antibodies. all slides were evaluated by a board-certified veterinary pathologist blinded to study group allocations. in animals that received a vaccination with chadox1 mers, either prime-boost or prime-only, a minimal to mild bronchointerstitial pneumonia was present, characterized by mild thickening of the alveolar septae by lymphocytes and macrophages. pulmonary vessels were bound by moderate numbers of lymphocytes. in stark contrast, in lung tissue obtained from animals vaccinated with chadox1 gfp, moderate to marked bronchointerstitial pneumonia was present throughout the lung lobes characterized by thickening of the alveolar septae by lymphocytes and macrophages and edema and fibrin. alveoli contained abundant edema and fibrin and moderate to abundant numbers of alveolar macrophages, neutrophils, and necrotic debris. inflammation often surrounded bronchioles and pulmonary vasculature, and type ii pneumocyte hyperplasia was prominent. in addition, the presence of mers-cov antigen by immunohistochemistry was found only in lungs of animals vaccinated with chadox1 gfp within type i and ii pneumocytes and was not found in lung tissue of chadox1 mers-vaccinated animals. severity of bronchointerstitial pneumonia, type ii pneumocyte hyperplasia and hemorrhages, edema, and fibrin deposits was scored. statistically significant differences between animals vaccinated with chadox1 mers or chadox1 gfp were found for all three categories ( fig. 3 , e, f, and h). thus, vaccination with chadox1 mers, either via a prime-boost or prime-only regimen, significantly decreased the severity of pulmonary pathology and protected rhesus macaques against bronchointerstitial pneumonia. bal was performed on all animals on 1, 3, 5, and 6 dpi, and the amount of viral rna, mrna, and infectious virus was determined. in the prime-boost group, infectious virus was only detected at 1 dpi (n = 3) after inoculation, and in the prime-only group at 1 dpi (n = 6) and 3 dpi (n = 3) but not thereafter. in contrast, infectious virus was detected on all days in bal fluid from the control group (fig. 4a ). viral mrna in bal fluid of animals in the prime-boost group was only detected at 1 dpi (n = 5) and 3 dpi (n = 1). in contrast, viral mrna in bal fluid from animals that received a single vaccination with chadox1 mers mrna was detected on all days, but the amount was reduced compared to animals vaccinated with chadox1 gfp (fig. s1b ). viral rna as measured by upe quantitative reverse transcription polymerase chain reaction (qrt-pcr) assay could be detected in all groups up to 6 dpi. however, the number of genome copies per milliliter detected was lower in animals vaccinated with chadox1 mers compared to animals vaccinated with chadox1 gfp (fig. 4a) . a significant association was found between higher elisa titer or vn titer and lower levels of viral rna, mrna, or infectious virus in bal fluid for all days, except 6 dpi for infectious virus (spearman's rank correlation coefficient; table s2). all animals were euthanized on 6 dpi, and tissues were analyzed for the presence of viral rna, mrna, or infectious virus. infectious virus titers were only found in nasal turbinate tissue (n = 1) and lung lobe tissue (n = 4) from control animals. no viral mrna was found in all tissues obtained from animals that received a prime-boost regimen. in tissue from animals that received a prime-only regimen, limited viral mrna could be found in upper and lower lung lobe tissues (n = 4). in contrast, mrna could be found in respiratory tract tissues of all control animals and in conjunctiva (fig. 4b ). viral rna was detected in tissues from all groups but was mainly found in lung lobes and bronchi. viral load was higher for lower respiratory tract tissue obtained from animals vaccinated with chadox1 gfp (n = 6) than from animals receiving a prime-only (n = 6) or a prime-boost regimen of chadox1 mers (n = 2) (fig. 4b ). the presence of 23 cytokines was evaluated in lung tissue. several cytokines were up-regulated in animals vaccinated with chadox1 gfp compared to animals vaccinated with chadox1 mers, including interleukin-2 (il-2), il-6, il-8, il-18, monocyte chemotactic protein-1 (mcp-1), macrophage inflammatory protein-1a (mip-1), and transforming growth factor- (tgf-), although only il-2 and mcp-1 were significantly different in tissue obtained from control animals compared to vaccinated animals. a significant difference was observed in il-1ra levels in lung tissue obtained from the prime-only and prime-boost groups. it is not clear what the clinical significance of this difference is ( fig. s2 ). overall, these results show local increased immune activity in animals vaccinated with chadox1 gfp, but not in animals vaccinated with chadox1 mers, 6 days after challenge with mers-cov. the likely explanation for this is that vaccinated animals have controlled the infection rapidly, whereas in the control animals, more viral replication has taken place followed animal vaccinated with chadox1 gfp shows focally extensive area of increased pulmonary opacity and deviation of the cardiac silhouette, highlighted in the circle located in the middle and caudal lung lobes. (b) thoracic radiographs of each animal were scored per lung lobe, resulting in a maximum score of 18. values were averaged per group per day (d), and mean with sd is shown (see table s2 for more details). (c) gross pathology of lungs shows no pathologic changes in chadox1 mers-vaccinated animals and focally extensive areas of consolidation in left cranial, middle, and caudal lung lobes in control animals (asterisks). (d) gross lung lesions were scored for each lung lobe, ventral and dorsal. values were averaged per group, and mean with sd is shown. (e) lung tissue sections were stained with hematoxylin and eosin. moderate numbers of lymphocyte accumulation around pulmonary arterioles (asterisks) and mild thickening of alveolar septae by lymphocytes and macrophages (arrows) in lung tissue of animals vaccinated with chadox1 mers. marked bronchointerstitial pneumonia with abundant pulmonary edema and fibrin (asterisks), type ii pneumocyte hyperplasia (arrows), and increased numbers of alveolar macrophages (arrowheads) in lung tissue of control animals. magnification, ×200. (f) lung-to-body weight (bw) ratio was determined for all animals at necropsy. mean with sd is shown. (g) lung tissue sections were stained with antibody against mers-cov antigen, which is visible as a red-brown staining. no immunoreactivity was found in chadox1 mers-vaccinated animals, whereas multifocal immunoreactivity of type i and ii pneumocytes could be found in lung tissue of chadox1 gfp-vaccinated animals. (h) lung tissue sections were scored on severity of lesions (0, no lesions; 1, 1 to 10%; 2, 11 to 25%; 3, 26 to 50%; 4, 51 to 75%; and 5, 76 to 100%) and averaged per group. mean with sd is shown. a, bronchointerstitial pneumonia; b, type ii pneumocyte hyperplasia; c, hemorrhages, edema, and fibrin deposits. statistical significance between groups was determined via two-tailed unpaired student's t test. *p < 0.025; **p < 0.01; ***p < 0.001; ****p < 0.0001. photo credit: neeltje van doremalen, niaid/nih. chadox1 mers is based on the s protein from camel/qatar/2/2014, and we currently do not have access to an isolate. we thus already show cross-protection of chadox1 mers against hcov-emc/2012 (99.78% s protein amino acid identity). here, we extend that analysis to five other strains. we selected six different strains of mers-cov ( fig. s2 ). s protein identity for all strains to the vaccine s protein was >99.3%. amino acid identity was lowest for camel/burkina faso/ cirad-hku785/2015 (99.33%) and highest for hu/korea/seoul/ snu1-035/2015 (99.85%). we tested neutralizing capability of serum obtained at 0 dpi. strains were selected on the basis of geographical location (ksa, south korea, and burkina faso), host (dromedary camel or human), and time of isolation (2012 to 2018). all six strains were neutralized by antibodies elicited by chadox1 mers vaccination. although we were not able to detect neutralizing ability of serum obtained from animal 12 against hcov-emc/2012, antibodies in the serum were able to neutralize four of six tested mers-cov strains (table 1) . to investigate whether vaccination with chadox1 mers vaccination provides protection against a variety of different mers-cov strains, we vaccinated balb/c mice transgenic for hdpp4 with chadox1 mers or chadox1 gfp 28 days before challenge with 10 4 tcid 50 of one of six diverse mers-cov strains ( fig. s3 ) via the intranasal route. all mice vaccinated with chadox1 mers survived challenge with mers-cov, independent of the challenge virus used, whereas most control mice were euthanized because of >20% weight loss or poor body condition (fig. 5a) . four animals per group were euthanized at 3 dpi, and infectious mers-cov titers in lung tissue were evaluated. whereas infectious virus could be found in lung tissue of control animals, we were unable to find infectious virus in lung tissue of chadox1 mers-vaccinated animals (fig. 5b) . thus, chadox1 mers protects against a variety of different mers-cov strains in hdpp4 transgenic mice. (24) . mers is associated with a high case-fatality rate (34.5%), and human-tohuman transmission is a major contributor to patient infections (1). currently, no mers-cov vaccine is available. ideally, such a vaccine would only require a single administration and would protect against a wide variety of different mers-cov strains. several studies have evaluated different types of mers-cov vaccines in animal models, but few have taken these vaccines into nhps. in the current study, we show the efficacy of chadox1 mers in rhesus macaques. unlike other nhp vaccine studies (10-13), we investigate vaccine efficacy after a single dose. animals that received a single dose of chadox1 mers showed an induction of a neutralizing antibody response associated with mostly normal clinical parameters, showing no breathing irregularities or reduced lung function by spo 2 values, limited evidence of infiltration by radiograph analysis after challenge, and no signs of gross pathological lesions. vaccination reduced viral rna load in tissues collected at 6 dpi compared to chadox1 gfp-vaccinated animals by several logs. in contrast to the abundant presence of viral mrna and infectious virus in control animals, we found no evidence of infectious virus, and only a limited presence of viral mrna, in the lungs of prime-only vaccinated macaques. viral mrna and infectious virus was completely absent from animals vaccinated with a prime-boost regimen of chadox1 mers. although both vaccine regimens protected rhesus macaques from the clinical symptoms of mers-cov inoculation, we detected lower virus replication in animals that received a prime-boost regimen compared to animals receiving a prime-only regimen, suggesting that the prime-boost regimen is a superior therapeutic approach. it should be noted that in the current model, animals are inoculated with a high dose of virus (7 × 10 6 tcid 50 per animal). this is likely a higher inoculum than most humans the study was not designed to determine correlates of protection, which must be determined separately for each vaccine candidate, but it is of interest that here, one animal was protected despite not having detectable neutralizing antibodies against hcov-emc/2012, and in general, the neutralizing antibody titer was not high. in clinical trials, chadox1-vectored vaccines prime strong t cell responses against the vaccine antigen (17) (18) (19) (20) (21) (22) . in a clinical study of patients that recovered from mers infection, some had strong cd8 + t cell responses without detectable antibodies (25) . further studies addressing correlates of protection for chadox1 mers should assess cd8 + t cell responses. following the successful induction of protective immunity after vaccination as demonstrated here, the duration of immunity and ability to induce the development of memory b and t cells should be assessed. the phenotype of memory cells induced by vaccination may not necessarily mimic that induced by infection with the pathogen. in the first clinical trial of chadox1 mers (clinicaltrials.gov identifier: nct03399578) both humoral-and t cell-mediated responses were assessed at multiple times and persisted up to 1 year after vaccination. further work will be required to assess memory b and t cell phenotypes. we are currently planning studies to look at long-term protection by chadox1 mers vaccination in our hdpp4 mouse model. a variety of different mers-cov strains have been isolated from dromedary camels and humans over the past 8 years of mers-cov emergence (24) . dromedary camels are distributed throughout africa, the middle east, asia, and australia (26) . although mers-cov has not been detected in dromedary camels in australia (27) , strains have been isolated from africa (9) and the middle east (7), and seropositive dromedary camels have been found in asia (28, 29) . phylogenetic analyses show a clustering of mers-cov by geographical location (8, 9) , and analysis of 219 complete mers-cov genomes, which only included one african strain, showed the presence of two clades, with human isolates in both clades (30) . notably, antigenic differences have been reported between s proteins from the middle east and africa (8), potentially affecting the efficacy of a vaccine based in conclusion, we show that a single vaccination with chadox1 mers results in protection against disease progression and virus replication associated with mers-cov challenge in the rhesus macaque, and a prime-boost regimen reduced viral replication further. furthermore, chadox1 mers vaccination protected against a diverse panel of contemporary mers-cov strains in hdpp4 mice. this is the first time that broad protection after a single vaccination has been shown for any mers-cov vaccine. last, chadox1 vaccines can be produced rapidly, have been shown to be safe in human patients, and are protective against mers-cov in rhesus macaques and hdpp4 mice. we conclude that the chadox1 platform is ideal for the development of vaccines against novel emerging coronaviruses, such as hcov-19/sars-cov-2. animal experiment approval was provided by the institutional animal care and use committee at rocky mountain laboratories. all animal experiments were executed in an association for assessment and accreditation of laboratory animal care-approved facility by certified staff, following the guidelines and basic principles in the national institutes of health (nih) guide for the care and use of laboratory animals, the animal welfare act, the u.s. department of agriculture, and the u.s. public health service policy on humane care and use of laboratory animals. rhesus macaques were housed in individual primate cages allowing social interactions, in a climatecontrolled room with a fixed light-dark cycle (12/12 hours). rhesus macaques were monitored a minimum of twice daily throughout the experiment. commercial monkey chow, treats, and fruit were provided twice daily by a trained personnel. water was available ad libitum. environmental enrichment consisted of a variety of human interaction, commercial toys, videos, and music. the institutional biosafety committee (ibc) approved work with infectious mers-cov virus strains under bsl3 conditions. all sample inactivation was performed according to ibc-approved standard operating procedures for the removal of specimens from high containment. the s protein gene from mers-cov strain camel/qatar/2/2014 (genbank accession no. kj650098.1) was codon optimized for humans and synthesized by geneart (thermo fisher scientific). the synthesized s gene was cloned into a transgene expression plasmid comprising a modified human cytomegalovirus immediate early promoter (cmv promoter) with tetracycline operator sites and the polyadenylation signal from bovine growth hormone. the resulting expression cassette was inserted into the e1 locus of a genomic clone of chadox1 using site-specific recombination (31) . the virus was rescued and propagated in t-rex-293 cells (invitrogen). purification was by cscl gradient ultracentrifugation, and the virus was titered, as previously described (32) . doses for vaccination were based on infectious units (ius) (33) . eighteen adult rhesus macaques (17 males and 1 female) were purchased from morgan island and randomly divided into three groups of six animals each. the group 1 was vaccinated with chadox1 mers at −56 and −28 dpi, the group 2 was vaccinated with chadox1 mers at −28 dpi, and the group 3 was vaccinated with chadox1 gfp at −56 and −28 dpi. all vaccinations were done with 3.9 × 10 8 iu per animal per vaccination. blood samples were obtained before vaccination and 14 days thereafter. animals were challenged with mers-cov strain hcov-emc/2012 on 0 dpi with administrations of 4 ml intratracheally, 1 ml intranasally, 1 ml orally, and 1 ml ocularly of 10 7 tcid 50 /ml virus solution. clinical exams were performed on −56, −42, −28, −14, 0, 1, 3, and 5 and 6 dpi; animals were euthanized at 6 dpi. all exams existed of the following: weight and temperature measurements, radiographs, spo 2 measurements using pulse oximetry, and blood sampling. bal was performed on 1, 3, 5, and 6 dpi by insertion of an endotracheal tube and bronchoscope into the trachea, then past the third bifurcation, and subsequent installation of 10 ml of sterile saline. manual suction was applied to retrieve the bal sample. necropsy was performed on 6 dpi. radiographs were evaluated and scored by a board-certified veterinarian who was blinded to the group assignment of the animals according to the following criteria: 0, normal examination; 1, mild interstitial pulmonary infiltrates; 2, moderate interstitial infiltrates, perhaps with partial cardiac border effacement and small areas of pulmonary consolidation (alveolar patterns and air bronchograms); and 3, pulmonary consolidation as the primary lung pathology, seen as a progression from grade 2 lung pathology (34) . veroe6 cells were maintained in dmem supplemented with 10% fetal calf serum, 1 mm l-glutamine, penicillin (50 u/ml), and streptomycin (50 g/ml). virus titrations were performed by endpoint titration in veroe6 cells, which were inoculated with 10-fold serial dilutions of virus. after 1-hour incubation at 37°c and 5% co 2 , tissue homogenate dilutions were removed, and cells were washed twice with phosphate-buffered saline (pbs) and incubated in 100 l of 2% dmem. cytopathic effect was scored at 5 dpi, and the tcid 50 was calculated from four replicates by the spearman-karber method (33, 35) . sera were heat inactivated (30 min, 56°c), and twofold serial dilutions were prepared in 2% dmem. hereafter, 100 tcid 50 of mers-cov was added. after 60-min incubation at 37°c, virus:serum mixture was added to veroe6 cells and incubated at 37°c and 5% co 2 . at 5 dpi, the cytopathic effect was scored. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, which still inhibited virus replication (33) . chimpanzee adenovirus chadox1-specific neutralizing antibody titers were assessed using a secreted placental alkaline phosphatase (seap) quantitation assay. briefly, griptite msr 293 cells (invitrogen, catalog no. r795-07) were cultured as per manufacturer's instructions and were seeded at 3 × 10 4 cells per well in a 96-well plate the day before starting the assay (24 ± 2 hours). cells were infected with the test sera dilutions (fourfold dilution series) at 1:18, 1:72, 1:288, 1:1152, and 1:4608 in phenol red-free 0% fbs dmem (life technologies, catalog no. 31053028) and the chadox1-seap reporter virus in a 1:1 mixture (pre-incubated for 1 hour to allow any neutralization to occur) for 1 hour before replacing with phenol redfree 10% fbs dmem for a further 24 hours (±2 hours). sample dilutions were tested in duplicate lanes. for each sample, seap concentration was assessed in 50 l aliquots of culture supernatant, with cpsd as an indicator substrate (tropix phospha-light chemiluminescent assay kit, life technologies, catalog no. t1017), using a minor variant of the manufacturer's instructions; luminescence intensity was measured using a varioskan flash luminometer (thermo fisher scientific). serum dilution neutralization titers were measured by linear interpolation of adjacent values (to 50% inhibition) to determine the serum dilution required to reduce seap concentration by 50% compared to wells with virus alone. tissues (30 mg) were homogenized in rlt buffer, and rna was extracted using the rneasy kit (qiagen) according to the manufacturer's instructions. rna was extracted from bal fluid using the qiaamp viral rna kit (qiagen) on the qiaxtractor. the upe mers-cov (36) or mrna (37) detection assay was used for the detection of mers-cov viral rna. five microliters of rna was tested with the rotor-gene probe kit (qiagen) according to the instructions of the manufacturer. dilutions of mers-cov virus stock with known genome copies were run in parallel. genome copies were determined using droplet digital pcr (bio-rad) and the corresponding qrt-pcr. a soluble, trimeric recombinant s protein of mers-cov (isolate ca/jeddah/d42/2014) incorporating amino acids 1 to 1273 and a c-terminal trimerization domain was produced in chinese hamster ovary cells (expicho; thermo fisher scientific) and purified by immunoaffinity chromatography. maxisorp plates (nunc) were coated overnight at room temperature with 5 g of s protein per plate in pbs. plates were blocked with 100 l of casein (thermo fisher scientific) for 90 min at room temperature. serum (2× serial dilution in casein starting at 100× dilution) was incubated at room temperature for 2 hours. antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat anti-monkey igg (seracare, 074-11-021) in casein and 3,3′,5,5′-tetramethylbenzidine (tmb) two-component peroxidase substrate (seracare) and read at 450 nm. all wells were washed three times with pbst (pbs-tween 0.05%) in between steps. threshold for positivity was set at 3× optical density (od) value of negative control (serum obtained from nhps before the start of the experiment). samples for analysis of cytokine/chemokine levels were inactivated with -radiation (2 mrad) according to the standard operating procedures. concentrations of granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage csf, interferon-, il-1, il-1 receptor antagonist, il-2, il-4, il-5, il-6, il-8, il-10, il-12/23 (p40), il-13, il-15, il-17, il-18 mcp-1, and mip-1, mip-1, soluble cd40 ligand (scd40l), tgf-, tumor necrosis factor-, and vascular endothelial growth factor were measured on a bio-plex 200 instrument (bio-rad) using the non-human primate cytokine milliplex map 23-plex kit (millipore) according to the manufacturer's instructions. necropsies and tissue sampling were performed according to ibcapproved protocols. lungs were perfused with 10% formalin and processed for histologic review. harvested tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and then embedded in paraffin. tissues were processed using a vip-6 tissue-tek (sakura finetek, usa) tissue processor and embedded in ultraffin paraffin polymer (cancer diagnostics, durham, nc). samples were sectioned at 5 m, and the resulting slides were stained with hematoxylin and eosin. specific anti-cov immunoreactivity was detected using mers-cov nucleocapsid protein rabbit antibody (sino biological inc.) at a 1:4000. the tissues were processed for immunohistochemistry using the discovery ultra automated ihc/ish staining instrument (ventana medical systems) with a discovery red (ventana medical systems) kit. all tissue slides were evaluated by a board-certified veterinary anatomic pathologist blinded to study group allocations. tukey's multiple comparison test or a two-tailed unpaired student's t test was conducted to compare differences between vaccine groups and the control group. a bonferroni correction was used to control the type i error rate for the two comparisons (group 1 versus control and group 2 versus control), and thus, statistical significance was reached at p < 0.025. spearman's rank correlation coefficient test was used to interfere correlation. supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/6/24/eaba8399/dc1 sars and mers: recent insights into emerging coronaviruses environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedaries in ethiopia is antigenically different from the middle east isolate mers coronaviruses from camels in africa exhibit regiondependent genetic diversity a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge evaluation of candidate vaccine approaches for mers-cov mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp4 mouse model humoral immunogenicity and efficacy of a single dose of chadox1 mers vaccine candidate in dromedary camels phase ia clinical evaluation of the plasmodium falciparum blood-stage antigen msp1 in chad63 and mva vaccine vectors safety and tolerability of conserved region vaccines vectored by plasmid dna, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase i trial heterologous two-dose vaccination with simian adenovirus and poxvirus vectors elicits long-lasting cellular immunity to influenza virus a in healthy adults chronic hepatitis c viral infection subverts vaccine-induced t-cell immunity in humans a phase i trial evaluating the safety and immunogenicity of a candidate tuberculosis vaccination regimen, chadox1 85a prime -mva85a boost in healthy uk adults safety and immunogenicity of a heterologous prime-boost ebola virus vaccine regimen in healthy adults in the united kingdom and senegal middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques mers-cov spillover at the camel-human interface recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (mers-cov) after experimental infection absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread middle east respiratory syndrome coronavirus antibodies in dromedary camels serologic evidence for mers-cov infection in dromedary camels molecular evolution of mers coronavirus: dromedaries as a recent intermediate host or long-time animal reservoir? a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors a single-dose chadox1-vectored vaccine provides complete protection against nipah bangladesh and malaysia in syrian golden hamsters thoracic radiography as a refinement methodology for the study of h1n1 influenza in cynomologus macaques (macaca fascicularis) beitrag zur kollektiven behandlung pharmakologischer reihenversuche detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction growth and quantification of mers-cov infection diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing competing interests: s.c.g. is a board member of vaccitech and named as an inventor on a patent covering use of chadox1-vectored vaccines. the other authors declare that they have no competing interests. data and materials availability: all data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. chadox1 mers can be provided by the jenner institute, university of oxford pending scientific review and a completed material transfer agreement a single dose of chadox1 mers provides protective immunity in rhesus macaques key: cord-288859-19jwawrm authors: choi, s.; jung, e.; choi, b.y.; hur, y.j.; ki, m. title: high reproduction number of middle east respiratory syndrome coronavirus in nosocomial outbreaks: mathematical modelling in saudi arabia and south korea date: 2017-09-25 journal: j hosp infect doi: 10.1016/j.jhin.2017.09.017 sha: doc_id: 288859 cord_uid: 19jwawrm background: effective countermeasures against emerging infectious diseases require an understanding of transmission rate and basic reproduction number (r(0)). r(0) for severe acute respiratory syndrome is generally considered to be >1, whereas that for middle east respiratory syndrome (mers) is considered to be <1. however, this does not explain the large-scale outbreaks of mers that occurred in kingdom of saudi arabia (ksa) and south korean hospitals. aim: to estimate r(0) in nosocomial outbreaks of mers. methods: r(0) was estimated using the incidence decay with an exponential adjustment model. the ksa and korean outbreaks were compared using a line listing of mers cases compiled using publicly available sources. serial intervals to estimate r(0) were assumed to be six to eight days. study parameters [r(0) and countermeasures (d)] were estimated by fitting a model to the cumulative incidence epidemic curves using matlab. findings: the estimated r(0) in korea was 3.9 in the best-fit model, with a serial interval of six days. the first outbreak cluster in a hospital in pyeongtaek had an r(0) of 4.04, and the largest outbreak cluster in a hospital in samsung had an r(0) of 5.0. assuming a six-day serial interval, the ksa outbreaks in jeddah and riyadh had r(0) values of 3.9 and 1.9, respectively. conclusion: r(0) for the nosocomial mers outbreaks in ksa and south korea was estimated to be in the range of 2–5, which is significantly higher than the previous estimate of <1. therefore, more comprehensive countermeasures are needed to address these infections. background: effective countermeasures against emerging infectious diseases require an understanding of transmission rate and basic reproduction number (r 0 ). r 0 for severe acute respiratory syndrome is generally considered to be >1, whereas that for middle east respiratory syndrome (mers) is considered to be <1. however, this does not explain the large-scale outbreaks of mers that occurred in kingdom of saudi arabia (ksa) and south korean hospitals. aim: to estimate r 0 in nosocomial outbreaks of mers. methods: r 0 was estimated using the incidence decay with an exponential adjustment model. the ksa and korean outbreaks were compared using a line listing of mers cases compiled using publicly available sources. serial intervals to estimate r 0 were assumed to be six to eight days. study parameters [r 0 and countermeasures (d)] were estimated by fitting a model to the cumulative incidence epidemic curves using matlab. findings: the estimated r 0 in korea was 3.9 in the best-fit model, with a serial interval of six days. the first outbreak cluster in a hospital in pyeongtaek had an r 0 of 4.04, and the largest outbreak cluster in a hospital in samsung had an r 0 of 5.0. assuming a six-day serial interval, the ksa outbreaks in jeddah and riyadh had r 0 values of 3.9 and 1.9, respectively. conclusion: r 0 for the nosocomial mers outbreaks in ksa and south korea was estimated to be in the range of 2e5, which is significantly higher than the previous estimate of <1. therefore, more comprehensive countermeasures are needed to address these infections. ª 2017 the healthcare infection society. published by elsevier ltd. all rights reserved. the emergence of infectious diseases associated with middle east respiratory syndrome (mers), severe acute respiratory syndrome and ebola has created unprecedented public health challenges. these challenges are complicated by the lack of basic epidemiological data, which makes it difficult to predict epidemics. thus, it is important to quantify actual outbreaks as novel infectious diseases emerge. disease severity and rate of transmission can be predicted by mathematical models using the basic reproduction number (r 0 ) [1] . for example, r 0 has been used extensively to assess pathogen transmissibility, outbreak severity and epidemiological control [2e4] . in previous studies, r 0 for mers has ranged from 0.42 to 0.92 [5e8], which suggests that the mers coronavirus (mers-cov) has limited transmissibility. however, these studies typically considered community-acquired mers infections. in this context, nosocomial infections can exhibit different r 0 values as the transmission routes for community-acquired and nosocomial infections often differ [9] . recent studies have examined large nosocomial outbreaks of mers-cov infection in jeddah and riyadh within the kingdom of saudi arabia (ksa). one study reported higher nosocomial r 0 values than those from community-acquired infections when using the incidence decay with exponential adjustment (idea) model, which yielded values of 3.5e6.7 in jeddah and 2.0e2.8 in riyadh [10] . the idea model is simple because it does not consider the population-level immune status, which makes it especially useful for modelling emerging infectious diseases in resourcelimited settings. the mers outbreak in south korea was associated with nosocomial infections. at that time, the korea centre for disease control and prevention (kcdc) assumed that the outbreak had an r 0 <1. thus, the initial countermeasures were not sufficiently aggressive to prevent the spread of mers-cov infection to other hospitals. therefore, the idea model was used to evaluate and compare the mers r 0 values from the outbreaks in both ksa and south korean hospitals. ksa data were obtained using a line listing of mers-cov cases that was maintained by andrew rambaut (updated on 19 th august 2015). the line listing was created using data from the ksa ministry of health and world health organization (who) report [10] . since only 44% of cases in the ksa listing included the onset date, hospitalization dates or reported dates were used instead. the korean data were obtained from the kcdc. among the 186 mers cases, 178 had confirmed onset dates. the eight cases with unknown onset dates were assigned dates based on laboratory confirmation. all cases in ksa and korea were confirmed based on laboratory findings. study parameters [r 0 and countermeasures (d)] were estimated by fitting a model to the cumulative incidence epidemic curves using matlab (mathworks, natick, ma, usa). the data were narrowed down to the nosocomial cases alone. cases with unknown transmissions were considered to be nosocomial if: (a) the patient was in contact with a healthcare worker and/or hospitalized patients; or (b) the patient was a healthcare worker. cases were excluded if they could not be verified as nosocomial (e.g. zoonotic transmission, family contact or community infection). the idea model was used to estimate r 0 as reported previously [11] , together with publicly available data. the idea model is based on the concept that the number of incident cases (i) in an epidemic generation (t) can be counted as: when an outbreak occurs, epidemic control measures can be implemented, which can, in turn, change r 0 . therefore, the relationship between i and r 0 with d is defined as follows: r 0 and d are estimated by fitting i from eq. (2) to the observed cumulative incidence data of mers using the leastsquares data-fitting method. since the idea model is parameterized using epidemic generation time, incidence case counts were aggregated at serial intervals of six, seven and eight days in the present study [10] . two large outbreaks were considered in each country studied: the outbreaks in riyadh and jeddah for ksa; and those in pyeongtaek st. mary's hospital and samsung seoul hospital for south korea. the term 'resnorm' is defined as the norm of the residual, which is the squared 2-norm of the residual; it measures the difference between observed data and the fitted value provided by a model. however, as residuals can be positive or negative, a sum of residuals is not a good measure of overall error in the fit. therefore, a better measure of error is the sum of the squared residuals (e), which is calculated as follows: the functions to be fit were the given input data (xdata), the observed output data, (ydata) and f(x, xdata), where xdata was an epidemic generation, ydata was the observed cumulative incidence data, and f(x, xdata) was eq. (2). since the generation times and the estimated values differ according to serial interval times, resnorm changes accordingly. therefore, to compare resnorm with the serial interval time, relative resnorm was defined as follows: the idea model was fitted to the cumulative south korean mers-cov case data from the onset date of the first case to the onset date of the last case. the outbreak start date was defined as 11 th may 2015 because that was the symptom onset date for patient 0, who was the index case and caused the outbreak in the pyeongtaek hospital. all data used in these analyses were de-identified publicly available data obtained from who, the ksa ministry of health website or kcdc datasets. as such, these data were deemed to be exempt from institutional review board assessment. ksa outbreaks were relatively large, with 180 cases (over the course of 67 days) in jeddah and 142 cases (over the course of 71 days) in riyadh. the korean outbreaks involved 186 cases (over the course of 55 days), including 36 cases (over the course table i ). the idea model was fitted to the daily ksa and korea mers-cov case data according to the onset date. figure 1 displays the cumulative mers-cov case data for the 2014 ksa and the 2015 south korea mers outbreaks. the date of symptom onset for patient 0 was 11 th may 2015; however, he was admitted to the pyeongtaek hospital on 15 th may 2015. therefore, the outbreak was assumed to start on 15 th may 2015 via a simulation of the pyeongtaek hospital outbreak. the outbreak start date for the samsung hospital was determined to be 25 th may 2015, following the same logic ( figure 1 ). figure 2 shows the results of the 2014 ksa outbreak. squares, circles and asterisks represent data aggregation of the number of cases by serial intervals of six, seven and eight days, respectively; the curves represent model fits for best-fit parameters. the estimated r 0 values for jeddah and riyadh were in the range of 3.95e6.68 and 1.92e2.52, respectively, using serial intervals of six to eight days. the estimated r 0 values for the korea mers outbreak were 3.96, 4.91 and 5.95 for serial intervals of six, seven and eight days, respectively (figure 3 ). since most cases were related to nosocomial infections, r 0 for each hospital was also considered. the outbreak in the samsung hospital was larger than that in the pyeongtaek hospital (the first korean outbreak). the pyeongtaek hospital exhibited best-fit r 0 values of 4.04, 4.23 and 4.39 for serial intervals of six, seven and eight days, respectively, while the samsung hospital exhibited greater r 0 values of 5.0, 6.8 and 8.11 for serial intervals of six, seven and eight days, respectively. figure 3 shows that the idea model provided well-fitted curves for the cumulative data regarding south korean mers symptom-onset dates for all cases. although the idea model seemed to be appropriate, the original data never fit the model precisely. therefore, the appropriateness of the model was assessed. , using the incidence decay with exponential adjustment model. red squares, jeddah, six days; red circles, jeddah, seven days; red asterisks, jeddah, eight days; blue squares, riyadh, six days; blue circles, riyadh, seven days; blue asterisks, riyadh, eight days. evaluated using the relative resnorm to find the best-fit parameters. the results indicated that the best-fit r 0 and serial interval values were 4.9 and seven days for all cases, 4.39 and eight days for the pyeongtaek hospital, and 5.0 and six days for the samsung hospital, respectively. d increased with each serial interval because the daily effort of d was aggregated by serial interval. the clusters of mers-cov cases in ksa healthcare facilities occurred from late march to late may 2014, while the korean outbreaks occurred from mid-may to early july 2015. these hospital-based outbreaks exhibited characteristics different from those of community-based outbreaks (higher r 0 values and case fatality rates) [12, 13] . the estimated r 0 is a basic epidemiological variable that is important for selecting appropriate countermeasure efforts. however, an emerging infectious disease often has unknown epidemiology, making it difficult to model mathematically. several methods have been proposed to address this issue, including the idea model. the richards model can also estimate r 0 using the cumulative daily number of cases and the outbreak turning point (or the peak, t i ) [14] . in this context, hsieh used the richards model to estimate r 0 values for the korean outbreak as 7.0e19.3. however, the richards model does not consider any countermeasures implemented during an outbreak; therefore, it can only be used after an outbreak has peaked. the present study used the idea model to estimate r 0 values from the mers outbreaks in ksa and south korea. the idea model exhibited a good fit: the estimated r 0 values for south korea were 3.9e8.0, and the best-fit r 0 was 4.9 for a serial interval of seven days. conversely, r 0 values for riyadh and jeddah were 1.9e2.5 and 3.9e6.9, respectively, using serial intervals of six to eight days. majumder et al. [10] figure 3 . best-fit reproduction number (r 0 ) by serial intervals of middle east respiratory syndrome in south korea, 2015, using the incidence decay with exponential adjustment model. green squares, total, six days; green circles, total, seven days; green asterisks, total, eight days; red squares, pyeongtaek, six days; red circles, pyeongtaek, seven days; red asterisks, pyeongtaek, eight days; blue squares, samsung, six days; blue circles, samsung, seven days; blue asterisks, samsung, eight days. intervals of six to eight days. however, the estimated r 0 values from the present study were much higher than the previously reported values of <1 for mers (the threshold for an epidemic) [15] . regardless, the korean government assumed that the outbreak had an r 0 value <1 based on the previous research. the initial criterion for quarantine, therefore, was limited to cases of 'close contacts', which were defined as people who were within 2 m of a mers patient for !1 h [16] . these quarantines e established using an incorrectly assumed r 0 e resulted in more mers patients and greater hospital-to-hospital transmission [16] . a serial interval is the interval between successive cases of an infectious disease. this time period depends on the temporal relationship between the infectiousness of the disease, the clinical onset of the source case, and the incubation period of the receiving case [17] . as mers becomes infectious with the onset of clinical symptoms, the mers latency period equals the incubation period. therefore, the shortest serial interval could be the same as the incubation period, and the longest serial interval could be the sum of the incubation period and the maximum duration of infectiousness. during the korean mers outbreak, several superspreading events occurred because the mers cases were not isolated immediately upon presentation of clinical symptoms [18] . thus, these cases contacted susceptible individuals for up to one week after the onset of their clinical symptoms. however, most mers cases with laboratory confirmation were isolated immediately after onset of clinical symptoms [19, 20] . in this study, as the incubation period was two to 14 days (median: six days), the serial interval was slightly longer than the incubation period. the idea model with several serial intervals (four to 12 days) was used and found that intervals of six to eight days provided the best fit. for ksa data, even though the reported date was used instead of the onset date, r 0 was not affected because aggregated data by serial intervals was used in the analysis. the idea model is limited by the fact that d cannot be compared with d of another model. in this context, an increasing d in accordance with increasing serial intervals indicates that the countermeasure efforts are increasing. however, the size of d cannot be compared between two or more models of different outbreaks. nevertheless, the strength of the idea model is its simplicity because r 0 can be estimated using the cumulative number of cases according to the serial interval alone. in conclusion, the estimated r 0 values from the ksa outbreaks (riyadh and jeddah) ranged from 1.9 to 6.9, whereas the estimated values from the south korean outbreaks ranged from 3.9 to 8.0. based on these findings, it appears that nosocomial mers-cov outbreaks in ksa and south korea had higher r 0 values than the previously assumed values of <1. although community-acquired infections are caused by contact, nosocomial infections are caused by a combination of contact and aerosol transmission; therefore, r 0 values for nosocomial infections can be higher than those for communityacquired infections. hence, more comprehensive countermeasures are needed to address nosocomial mers infections and prevent spread. mathematical models to characterize early epidemic growth: a review transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions emergency response to a smallpox attack: the case for mass vaccination could widespread use of combination antiretroviral therapy eradicate hiv epidemics? the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk nuanced risk assessment for emerging infectious diseases middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility identifying determinants of heterogeneous transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, 2015: a retrospective epidemiological analysis estimation of mers-coronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data an idea for short term outbreak projection: nearcasting using the basic reproduction number epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital mortality risk factors for middle east respiratory syndrome outbreak, south korea middle east respiratory syndrome coronavirus (mers-cov) nosocomial outbreak in south korea: insights from modeling a dynamic compartmental model for the middle east respiratory syndrome outbreak in the republic of korea: a retrospective analysis on control interventions and superspreading events mers outbreak in korea: hospital-to-hospital transmission the interval between successive cases of an infectious disease risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study all relevant data are available at http://rambaut.github.io/ mers-tools/cases2.html. key: cord-310071-d195rumq authors: lee, jacob; kim, woo joo title: collaborative intervention of middle east respiratory syndrome: rapid response team date: 2016-06-30 journal: infect chemother doi: 10.3947/ic.2016.48.2.71 sha: doc_id: 310071 cord_uid: d195rumq on may 20th 2015, a 68 year old man was the first to be diagnosed with middle east respiratory syndrome-corona virus (mers-cov) in korea. he travelled to bahrain, saudi arabia, and qatar for 16 days. on may 4th 2015, the patient entered korea, with febrile sense and respiratory symptoms that appeared on may 11th. the mers-cov outbreak became worse and several patients had to be admitted throughout various hospitals starting at the beginning of june. this situation led to a nationwide chaos. the rapid response team (rrt) was organized after the korean government's calling for specialists that were composed of 15 infectious disease doctors and 2 infection control professionals on the 8th of june 2015. the main purpose of the rrt were: 1) consultation to the government controlling mers-cov outbreak. 2) visit hospitals that were exposed to mers-cov infected patients, and to provide advice regarding infection control strategy for rehabilitating of the exposed hospitals. since june 8th, the rrt visited more than 10 hospitals and an effective consultation was carried out. most of the hospitals were recovering from the mers outbreak since early july. cooperation between the government and private sector experts was very effective. the efforts of government and private sector experts overcame the initial chaos situation. it could prevent further deterioration of the mers outbreak. on may 20th 2015, a man at the age of 68 was the first to be diagnosed with middle east respiratory syndrome-corona virus (mers-cov) in korea [1] . he travelled to bahrain, saudi arabia, and qatar for 16 days. on may 4th 2015, the patient entered in korea, with a febrile sense and respiratory symptoms which appeared on may 11th. the mers-cov outbreak became with several patients admitted to local hospitals at the peak of june. this situation led to a nationwide chaos [2] . the rapid response team (rrt) was organized after the korean government's calling for specialists composed of 15 infectious disease doctors and 2 infection control professionals on the 8th june 2015. the purpose of rrt were to consult the government controlling mers-cov outbreak, and visit hospitals that were exposed to mers-cov infected patients and to advise them with an infection control strategy and rehabilitation program that would prevent the exposure of the disease in hospitals. the first task was to create guidelines on the diagnosis, treatment and infection control [3] [4] [5] rrt's individualized strategies for hospitals exposed to mers rrt conducted a consultation directly to hospitals where mers patients occurred. since hospitals were different in size and roles, the mers patients were admitted in various units such as emergency rooms, hospital rooms, intensive care unit (icu) and hemodi alysis (hd) unit, the consultation was conducted according to the characteristics of hospitals. rrt strategies for individual hospitals were also prepared from the failure of mers prevention at st. mary's hospital, pyeongtaek-si [2] . after the index case was confirmed at samsung medical center, isolation for close contact between people began. however, the korea centers for disease control and prevention (kcdc) caught less scope for close contacts. and then, as occurring patients with mers who were not classified to close contacts in various hospitals, mers became an outbreak. as considering kcdc's faults, we began to isolate close contacts early about hospitals where there were mers patients. in order to make hospital rooms for close contact people, we took active steps such as closing emergency rooms and, closing the outpatient department. rrt members, epidemic intelligence service officers, kcdc workers and staffs in community health centers attended onsite rrt. through confirming with hospital officials, we conducted a consultation rapidly about closing the hospitals, isolation of patients and medical staffs and infection control in hospital. from at the early stages of the mers outbreak, small-middle sized hospitals had a number of damages. actually, the small-middle sized hospitals were not sufficient for the infection control practitioners. therefore, they needed to be sent to the infection control practitioners early, so various hospital staffs were isolated who had close contact with the patients. shortage of medical personnel occurred, that led to hospitals needing medical teams from other hospitals. as a result, there were several hospitals which needed a medical team dispatched to. since these hospitals were small, they could not afford to make spare beds and single rooms for close contact people. in some patients, they had to transfer to another hospital. on june 2nd, after discovering the 16th patient on the 5th floor, all patients exposed by the 16th patient was isolated in the cohort room on the 5th floor. on june 5th, 5 patients in every cohort room showed a fever and then were confirmed with mers. rrt modified a guideline that all patients in the cohort area were isolated individually in single bed rooms. because isolating the room was not enough, patients on the 6th and 7th floor (not close contact mers patient) were transferred to daejeon army hospital. patients with close contact with mers patients on the 5th floor were separated into single rooms on the 5th, 6th, and 7th floor. acquiring negative pressure quarantine rooms on the 4th floor were set in the waiting area for mers suspicious patients. confirmed patients were transferred to the mers treatment hospital. because exposed medical staffs were quarantined, there were a lack of medical staffs who arranged patients with mers. the government determined to dispatch army doctors, nurse officers through cooperation of ministry of national defense. on june 10th, the 119th patient was confirmed to be infected. this patient was confirmed at the 7th floor ward. the hospital staff still operated outpatient department and emergency room. rrt recommended individual isolation but only conducted cohort isolation were done on the 7th ward because of small numbers of admitting rooms. medical staffs exposed to mers worked in the cohort area. on the june 18th, a nurse working in the cohort ward was confirmed with mers due to the 119th patient. rrt recommended emergency room and out-patient clinic be closed after coordinating with hospital officials in order to prepare isolating rooms. patients in the cohort ward were sent to a single room. patients who didn`t have close contact on the 5th floor were discharged. wards on the 3rd floor modified to single isolated room. nurses in the 7th floor who had close contact with mers patients and showed mers symptoms were transferred (national medical center, chungju medical center, gonju medical center, and daejeon army hospital). also army medical staffs were dispatched due to lack of medical staffs. on june 13th, an information technology (it) employee who worked at the ' a' hospital (the 143th mers patient) was identified on the 12th floor. the hospital was closed and the rrt guided isolate all patients on the 11th and 12th floor. non-contact patients were discharged or transferred to another hospital. single isolation rooms were prepared on the 11th and 12th floor. contact medical staffs were quarantined, and the 180th patient was identified 8 days after they were admitted with the 143rd patient in same room. rrt conducted the intensive epidemic investigation with closed-circuit television (cctv) monitoring and patients, including the medical staff with close contact without sufficient protective equipment and decided prolonged isolation in the case of suspected medical staffs and patients. on the 21st june, the 170th patient transferred from 'e' hospital admitted for 24hours. this hospital was placed in the commercially used building with wedding room, restaurants and mart. the rrt decided to close the entire commercial building and consider to re-open this building after evaluation of risk and the place was disinfected. d hospital was closed. cohort isolation was impossible due to small medical rehabilitation hospital that had not enough rooms. all 116 patients were transferred to public medical centers. people who had close contact transferred to suwon medical center. patients in the same ward transferred to paju city medical center, pocheon city medical center and daejeon army hospital. after carrying out disinfection, one week later this building was re-open. rrt isolate and control infection while cooperating with division of infectious diseases and infection control unit in the big hospitals affected mers. the rrt defined people who had close contact while cooperating with kcdc workers. hospital should close emergency room and outpatient department in order to acquire available isolating rooms. patients who did not come in contact were early discharged or transferred due to acquiring single isolation rooms. people who had close contact moved to a single isolating rooms. after then, confirmed patients with mers were sent to government designated mers treatment hospital. on the june 19th, the 170th patient was confirmed who was transferred to d hospital, had close contact with the 76th patient in the same ward. the hospital closed the emergency room and outpatient department in order to acquire available isolating rooms. two ward were acquired for preparing isolating rooms and patients who had close contact with the mers were sent to single isolation rooms. no more mers patient were diagnosed. on the june 23rd, the 173rd patient was confirmed on 10th floor of f hospital who was exposed from 76th patient in the emergency room of e hospital. patients moved to the icu because of severe pneumonia from 10th floor. that hospital closed emergency room and outpatient department in order to preparing isolation rooms. not contact patients were dischargedor transferred. contact patients moved to single bed isolating rooms. contact medical staffs had to be self-quarantined. no more mers patient were diagnosed. icu affected mers were 3 hospitals. due to the characteristics of icu, icu patients with close contact with mers were not easy to move another ward. so the rrt decided to define cohort area for all icu areas. in order to detecting patients early, we performed mers pcr surveillance every 2 to 3days to all icu patients. the rrt decided that in case of suspicious mers, we send them to the negative pressure quarantine room in icu and when confirming mers, we transfer them to mers treatment hospitals. fortunately, the three hospitals were not occurred additional mers confirmed patient. in hd unit, patients or medical staffs were confirmed to have mers in two hospitals. no hospitals were equipped with hemodialysis room separate for patients who had close contact. based on the level of exposure, adjustments were made either to send hd patients to single bed room or schedules were adjusted for hd. due to the insufficient of portable dialysis equipment. some portable hd equipment were borrowed from another hospital. as medical staffs exposed from mers in the hd room were isolated, medical teams were dispatched through the korean society of nephrology. since june 8th, 2016, the rrt visited more than 10 hospitals and effective consultation was carried out. most of the hospitals had been recovering from the mers outbreak since early july. cooperation between the government and the private sector experts rrt was very effective. the efforts of govern-ment and private sector experts have overcome the initial chaos situation. it could prevent the further deterioration of mers outbreak. the role of experts in the infection control was important because the mers outbreak occurred in the hospitals. the rrt was very effective and was a professional organization. the end of mers was accelerated through pro-active policy advice and on-site consultation to the affected hospitals with the rrt. kcdc should prepare for a new infectious disease epidemic through the expansion of the experts in infectious diseases. http://orcid.org/0000-0002-7041-065x woo joo kim http://orcid.org/0000-0002-4546-3880 better understanding on mers corona virus outbreak in korea korean society of infectious diseases, and korean society for healthcare-associated infection control and prevention. the same middle east respiratory syndrome-coronavirus (mers-cov) yet different outbreak patterns and public health impacts on the far east expert opinion from the rapid response team of the republic of korea rapid response team. antiviral treatment guidelines for middle east respiratory syndrome middle east respiratory syndrome infection control and prevention guideline for healthcare facilities for laboratory medicine mers-cov task force. guidelines for the laboratory diagnosis of middle east respiratory syndrome coronavirus in korea key: cord-313028-0nhgxoim authors: huang, chaolin; wang, yeming; li, xingwang; ren, lili; zhao, jianping; hu, yi; zhang, li; fan, guohui; xu, jiuyang; gu, xiaoying; cheng, zhenshun; yu, ting; xia, jiaan; wei, yuan; wu, wenjuan; xie, xuelei; yin, wen; li, hui; liu, min; xiao, yan; gao, hong; guo, li; xie, jungang; wang, guangfa; jiang, rongmeng; gao, zhancheng; jin, qi; wang, jianwei; cao, bin title: clinical features of patients infected with 2019 novel coronavirus in wuhan, china date: 2020-01-24 journal: lancet doi: 10.1016/s0140-6736(20)30183-5 sha: doc_id: 313028 cord_uid: 0nhgxoim background: a recent cluster of pneumonia cases in wuhan, china, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-ncov). we report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. methods: all patients with suspected 2019-ncov were admitted to a designated hospital in wuhan. we prospectively collected and analysed data on patients with laboratory-confirmed 2019-ncov infection by real-time rt-pcr and next-generation sequencing. data were obtained with standardised data collection forms shared by who and the international severe acute respiratory and emerging infection consortium from electronic medical records. researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. outcomes were also compared between patients who had been admitted to the intensive care unit (icu) and those who had not. findings: by jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-ncov infection. most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). median age was 49·0 years (iqr 41·0–58·0). 27 (66%) of 41 patients had been exposed to huanan seafood market. one family cluster was found. common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [iqr 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. all 41 patients had pneumonia with abnormal findings on chest ct. complications included acute respiratory distress syndrome (12 [29%]), rnaaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an icu and six (15%) died. compared with non-icu patients, icu patients had higher plasma levels of il2, il7, il10, gscf, ip10, mcp1, mip1a, and tnfα. interpretation: the 2019-ncov infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with icu admission and high mortality. major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. funding: ministry of science and technology, chinese academy of medical sciences, national natural science foundation of china, and beijing municipal science and technology commission. coronaviruses are enveloped non-segmented positivesense rna viruses belonging to the family coronaviridae and the order nidovirales and broadly distributed in humans and other mammals. 1 although most human coronavirus infections are mild, the epidemics of the two betacoronaviruses, severe acute respiratory syndrome coronavirus (sars-cov) [2] [3] [4] and middle east respiratory syndrome coronavirus (mers-cov), 5, 6 have caused more than 10 000 cumulative cases in the past two decades, with mortality rates of 10% for sars-cov and 37% for mers-cov. 7, 8 the coronaviruses already identified might only be the tip of the iceberg, with potentially more novel and severe zoonotic events to be revealed. in december, 2019, a series of pneumonia cases of unknown cause emerged in wuhan, hubei, china, with clinical presentations greatly resembling viral pneumonia. 9 deep sequencing analysis from lower respiratory tract samples indicated a novel coronavirus, which was named 2019 novel coronavirus (2019-ncov). thus far, more than 800 confirmed cases, including in health-care workers, have been identified in wuhan, and several exported cases have been confirmed in other provinces in china, and in thailand, japan, south korea, and the usa. 1013 we aim to describe epidemiological, clinical, laboratory, and radiological characteristics, treatment, and outcomes of patients confirmed to have 2019-ncov infection, and to compare the clinical features between intensive care unit (icu) and non-icu patients. we hope our study findings will inform the global community of the emergence of this novel coronavirus and its clinical features. following the pneumonia cases of unknown cause reported in wuhan and considering the shared history of exposure to huanan seafood market across the patients, an epidemiological alert was released by the local health authority on dec 31, 2019, and the market was shut down on jan 1, 2020. meanwhile, 59 suspected cases with fever and dry cough were transferred to a designated hospital starting from dec 31, 2019. an expert team of physicians, epidemiologists, virologists, and government officials was soon formed after the alert. since the cause was unknown at the onset of these emerging infections, the diagnosis of pneumonia of unknown cause in wuhan was based on clinical characteristics, chest imaging, and the ruling out of common bacterial and viral pathogens that cause pneumonia. suspected patients were isolated using airborne precautions in the designated hospital, jin yintan hospital (wuhan, china), and fit-tested n95 masks and airborne precautions for aerosol-generating procedures were taken. this study was approved by the national health commission of china and ethics commission of jin yin-tan hospital (ky-2020-01.01). written informed consent was waived by the ethics commission of the designated hospital for emerging infectious diseases. local centres for disease control and prevention collected respiratory, blood, and faeces specimens, then shipped them to designated authoritative laboratories to detect the pathogen (nhc key laboratory of systems biology of pathogens and christophe mérieux laboratory, beijing, china). a novel coronavirus, which was named 2019-ncov, was isolated then from lower respiratory tract specimen and a diagnostic test for this virus was developed soon after that. 14 of 59 suspected cases, 41 patients were confirmed to be infected with 2019-ncov. the presence of 2019-ncov in respi ratory specimens was detected by nextgeneration se quencing or real-time rt-pcr methods. the primers and probe target to envelope gene of cov were used and the sequences were as follows: forward primer 5′-acttctttttcttgctttcgtggt-3′; reverse primer 5′-gcagcagtacgcacacaatc-3′; and the probe 5′cy5-ctagttacactagccatccttactgc-3′bhq1. conditions for the amplifications were 50°c for 15 min, 95°c for 3 min, followed by 45 cycles of 95°c for 15 s and 60°c for 30 s. initial investigations included a complete blood count, coagulation profile, and serum biochemical test (including renal and liver function, creatine kinase, lactate dehydrogenase, and electrolytes). respiratory specimens, including nasal and pharyngeal swabs, bronchoalveolar lavage fluid, sputum, or bronchial aspirates were tested for common viruses, including influenza, avian influenza, respiratory syncytial virus, adenovirus, parainfluenza virus, sars-cov and mers-cov using real-time rt-pcr assays approved by the china food and drug administration. routine bacterial and fungal examinations were also performed. given the emergence of the 2019-ncov pneumonia cases during the influenza season, antibiotics (orally and intravenously) and osel tamivir (orally 75 mg twice daily) were empirically administered. corticosteroid therapy evidence before this study human coronaviruses, including hcov-229e, oc43, nl63, and hku1, cause mild respiratory diseases. fatal coronavirus infections that have emerged in the past two decades are severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus. we searched pubmed and the china national knowledge infrastructure database for articles published up to jan 11, 2020, using the keywords "novel coronovirus", "2019 novel coronavirus", or "2019-ncov". no published work about the human infection caused by the 2019 novel coronavirus (2019-ncov) could be identified. we report the epidemiological, clinical, laboratory, and radiological characteristics, treatment, and clinical outcomes of 41 laboratory-confirmed cases infected with 2019-ncov. 27 (66%) of 41 patients had a history of direct exposure to the huanan seafood market. the median age of patients was 49·0 years (iqr 41·0-58·0), and 13 (32%) patients had underlying disease. all patients had pneumonia. a third of patients were admitted to intensive care units, and six died. high concentrations of cytokines were recorded in plasma of critically ill patients infected with 2019-ncov. implications of all the available evidence 2019-ncov caused clusters of fatal pneumonia with clinical presentation greatly resembling sars-cov. patients infected with 2019-ncov might develop acute respiratory distress syndrome, have a high likelihood of admission to intensive care, and might die. the cytokine storm could be associated with disease severity. more efforts should be made to know the whole spectrum and pathophysiology of the new disease. (methylprednisolone 40-120 mg per day) was given as a combined regimen if severe community-acquired pneumonia was diagnosed by physicians at the designated hospital. oxygen support (eg, nasal cannula and invasive mechanical ventilation) was administered to patients according to the severity of hypoxaemia. repeated tests for 2019-ncov were done in patients confirmed to have 2019-ncov infection to show viral clearance before hospital discharge or discontinuation of isolation. we reviewed clinical charts, nursing records, laboratory findings, and chest x-rays for all patients with laboratoryconfirmed 2019-ncov infection who were reported by the local health authority. the admission data of these patients was from dec 16, 2019, to jan 2, 2020. epidemiological, clinical, laboratory, and radiological characteristics and treatment and outcomes data were obtained with standardised data collection forms (modified case record form for severe acute respiratory infection clinical characterisation shared by who and the international severe acute respiratory and emerging infection consortium) from electronic medical records. two researchers also independently reviewed the data collection forms to double check the data collected. to ascertain the epidemiological and symptom data, which were not available from electronic medical records, the researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. to characterise the effect of coronavirus on the production of cytokines or chemokines in the acute phase of the illness, plasma cytokines and chemokines (il1b, il1ra, il2, il4, il5, il6, il7, il8 (also known as cxcl8), il9, il10, il12p70, il13, il15, il17a, eotaxin (also known as ccl11), basic fgf2, gcsf (csf3), gmcsf (csf2), ifnγ, ip10 (cxcl10), mcp1 (ccl2), mip1a (ccl3), mip1b (ccl4), pdgfb, rantes (ccl5), tnfα, and vegfa were measured using human cytokine standard 27-plex assays panel and the bio-plex 200 system (bio-rad, hercules, ca, usa) for all patients according to the manufacturer's instructions. the plasma samples from four healthy adults were used as controls for crosscomparison. the median time from being transferred to a designated hospital to the blood sample collection was 4 days (iqr 2-5). each 80 µl plasma sample from the patients and contacts was added into 240 µl of trizol ls (10296028; thermo fisher scientific, carlsbad, ca, usa) in the biosafety level 3 laboratory. total rna was extracted by direct-zol rna miniprep kit (r2050; zymo research, irvine, ca, usa) according to the manufacturer's instructions and 50 µl elution was obtained for each sample. 5 µl rna was used for real-time rt-pcr, which targeted the np gene using agpath-id one-step rt-pcr reagent (am1005; thermo fisher scientific). the final reaction mix concentration of the primers was 500 nm and probe was 200 nm. real-time rt-pcr was per formed using the following conditions: 50°c for 15 min and 95°c for 3 min, 50 cycles of amplification at 95°c for 10 s and 60°c for 45 s. since we did not perform tests for detecting infectious virus in blood, we avoided the term viraemia and used rnaaemia instead. rnaaemia was defined as a positive result for real-time rt-pcr in the plasma sample. acute respiratory distress syndrome (ards) and shock were defined according to the interim guidance of who for novel coronavirus. 9 hypoxaemia was defined as arterial oxygen tension (pao₂) over inspiratory oxygen fraction (fio₂) of less than 300 mm hg. 15 acute kidney injury was identified and classified on the basis of the highest serum creatinine level or urine output criteria according to the kidney disease improving global outcomes classification. 16 secondary infection was diagnosed if the patients had clinical symptoms or signs of nosocomial pneumonia or bacteraemia, and was combined with a positive culture of a new pathogen from a lower respiratory tract specimen (including the sputum, transtracheal aspirates, or bronchoalveolar lavage fluid, or from blood samples taken ≥48 h after admission). 17 cardiac injury followed the definition used in our previous study in h7n9 patients. 18 in brief, cardiac injury was diagnosed if serum levels of cardiac biomarkers (eg, troponin i) were above the 99th percentile upper reference limit, or new abnormalities were shown in electrocardiography and echocardiography. continuous variables were expressed as median (iqr) and compared with the mann-whitney u test; categorical variables were expressed as number (%) and compared by χ² test or fisher's exact test between icu care and no icu care groups. boxplots were drawn to describe plasma cytokine and chemokine concentrations. a two-sided α of less than 0·05 was considered statistically significant. statistical analyses were done using the sas software, version 9.4, unless otherwise indicated. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. by jan 2, 2020, 41 admitted hospital patients were identified as laboratory-confirmed 2019-ncov infection in wuhan. 20 [49%]) of the 2019-ncov-infected patients were aged 25-49 years, and 14 (34%) were aged 50-64 years (figure 1a). the median age of the patients was 49·0 years (iqr 41·0-58·0; table 1). in our cohort of the first 41 patients as of jan 2, no children or adolescents were infected. of the 41 patients, 13 (32%) were admitted to the icu because they required high-flow nasal cannula or higher-level oxygen support measures to cor rect hypoxaemia. most of the infected patients were men ( ). the symptom onset date of the first patient identified was dec 1, 2019. none of his family members developed fever or any respiratory symptoms. no epidemiological link was found between the first patient and later cases. the first fatal case, who had continuous exposure to the market, was admitted to hospital because of a 7-day history of fever, cough, and dyspnoea. 5 days after illness onset, his wife, a 53-year-old woman who had no known history of exposure to the market, also presented with pneumonia and was hospitalised in the isolation ward. the most common symptoms at onset of illness were fever ( most patients had normal serum levels of procalcitonin on admission (procalcitonin <0·1 ng/ml; 27 [69%] patients; table 2). four icu patients developed secondary infections. three of the four patients with secondary infection had procalcitonin greater than 0·5 ng/ml (0·69 ng/ml, 1·46 ng/ml, and 6·48 ng/ml). on admission, abnormalities in chest ct images were detected among all patients. of the 41 patients, 40 (98%) had bilateral involvement (table 2). the typical findings of chest ct images of icu patients on admission were bilateral multiple lobular and subsegmental areas of consolidation ( figure 3a) . the representative chest ct findings of non-icu patients showed bilateral groundglass opacity and subseg mental areas of consolidation (figure 3b). later chest ct images showed bilateral ground-glass opacity, whereas the consolidation had been resolved (figure 3c). initial plasma il1b, il1ra, il7, il8, il9, il10, basic fgf, gcsf, gmcsf, ifnγ, ip10, mcp1, mip1a, mip1b, pdgf, tnfα, and vegf concentrations were higher in both icu patients and non-icu patients than in healthy adults (appendix pp 6-7). plasma levels of il5, il12p70, il15, eotaxin, and rantes were similar between healthy adults and patients infected with 2019-ncov. further comparison between icu and non-icu patients showed that plasma concentrations of il2, il7, il10, gcsf, ip10, mcp1, mip1a, and tnfα were higher in icu patients than non-icu patients. all patients had pneumonia. ; table 3 ). invasive mechanical ventilation was required in four (10%) patients, with two of them (5%) had refractory hypoxaemia and received extracorporeal membrane oxygenation as salvage therapy. all patients were administered with empirical antibiotic treatment, and 38 (93%) patients received antiviral therapy (osel tamivir). additionally, nine (22%) patients were given systematic corticosteroids. a comparison of clinical features between patients who received and did not receive systematic corticosteroids is in the appendix (pp 1-5). as of jan 22, 2020, 28 (68%) of 41 patients have been dis charged and six (15%) patients have died. fitness for discharge was based on abatement of fever for at least 10 days, with improvement of chest radiographic evidence and viral clearance in respiratory samples from upper respiratory tract. we report here a cohort of 41 patients with laboratoryconfirmed 2019-ncov infection. patients had serious, sometimes fatal, pneumonia and were admitted to the designated hospital in wuhan, china, by jan 2, 2020. clinical presentations greatly resemble sars-cov. patients with severe illness developed ards and required icu admission and oxygen therapy. the time between hospital admission and ards was as short as 2 days. at this stage, the mortality rate is high for 2019-ncov, because six (15%) of 41 patients in this cohort died. the number of deaths is rising quickly. as of jan 24, 2020, 835 laboratory-confirmed 2019-ncov infec tions were reported in china, with 25 fatal cases. reports have been released of exported cases in many provinces in china, and in other countries; see online for appendix some health-care workers have also been infected in wuhan. taken together, evidence so far indicates human transmission for 2019-ncov. we are concerned that 2019-ncov could have acquired the ability for efficient human trans mission. 19 airborne precautions, such as a fit-tested n95 respirator, and other personal protective equipment are strongly recommended. to prevent further spread of the disease in health-care settings that are caring for patients infected with 2019-ncov, onset of fever and respiratory symptoms should be closely moni tored among health-care workers. testing of respiratory specimens should be done immediately once a diagnosis is suspected. serum antibodies should be tested among health-care workers before and after their exposure to 2019-ncov for identification of asymp tomatic infections. similarities of clinical features between 2019-ncov and previous betacoronavirus infections have been noted. in this cohort, most patients presented with fever, dry cough, dyspnoea, and bilateral ground-glass opacities on chest ct scans. these features of 2019-ncov infection bear some resemblance to sars-cov and mers-cov infections. 20, 21 however, few patients with 2019-ncov infection had prominent upper respiratory tract signs and symptoms (eg, rhinorrhoea, sneezing, or sore throat), indicating that the target cells might be located in the lower airway. furthermore, 2019-ncov patients rarely developed intestinal signs and symptoms (eg, diarrhoea), whereas about 20-25% of patients with mers-cov or sars-cov infection had diarrhoea. 21 faecal and urine samples should be tested to exclude a potential alternative route of transmission that is unknown at this stage. the pathophysiology of unusually high pathogenicity for sars-cov or mers-cov has not been completely understood. early studies have shown that increased amounts of proinflammatory cytokines in serum (eg, il1b, il6, il12, ifnγ, ip10, and mcp1) were associated with pulmonary inflammation and extensive lung damage in sars patients. 22 mers-cov infection was also reported to induce increased concentrations of proinflammatory cytokines (ifnγ, tnfα, il15, and il17). 23 we noted that patients infected with 2019-ncov also had high amounts of il1b, ifnγ, ip10, and mcp1, probably leading to activated t-helper-1 (th1) cell responses. moreover, patients requiring icu admission had higher concentrations of gcsf, ip10, mcp1, mip1a, and tnfα than did those not requiring icu admission, suggesting that the cytokine storm was associated with disease severity. however, 2019-ncov infection also initiated increased secretion of t-helper-2 (th2) cytokines (eg, il4 and il10) that suppress inflammation, which differs from sars-cov infection. 22 further studies are necessary to characterise the th1 and th2 responses in 2019-ncov infection and to elucidate the pathogenesis. autopsy or biopsy studies would be the key to understand the disease. in view of the high amount of cytokines induced by sars-cov, 22, 24 mers-cov, 25, 26 and 2019-ncov infections, corticosteroids were used frequently for treatment of patients with severe illness, for possible benefit by reducing inflammatory-induced lung injury. however, current evidence in patients with sars and mers suggests that receiving corticosteroids did not have an effect on mortality, but rather delayed viral clearance. [27] [28] [29] therefore, corticosteroids should not be routinely given systemically, according to who interim guidance. 30 among our cohort of 41 laboratory-confirmed patients with 2019-ncov infection, corticosteroids were given to very few non-icu cases, and low-to-moderate dose of corticosteroids were given to less than half of severely ill patients with ards. further evidence is urgently needed to assess whether systematic corticosteroid treatment is beneficial or harmful for patients infected with 2019-ncov. no antiviral treatment for coronavirus infection has been proven to be effective. in a historical control study, 31 the combination of lopinavir and ritonavir among sars-cov patients was associated with substantial clinical benefit (fewer adverse clinical outcomes). arabi and colleagues initiated a placebo-controlled trial of interferon beta-1b, lopinavir, and ritonavir among patients with mers infection in saudi arabia. 32 preclinical evidence showed the potent efficacy of remdesivir (a broad-spectrum antiviral nucleotide prodrug) to treat mers-cov and sars-cov infections. 33, 34 as 2019-ncov is an emerging virus, an effective treatment has not been developed for disease resulting from this virus. since the combination of lopinavir and ritonavir was already available in the designated hospital, a randomised controlled trial has been initiated quickly to assess the efficacy and safety of combined use of lopinavir and ritonavir in patients hospitalised with 2019-ncov infection. our study has some limitations. first, for most of the 41 patients, the diagnosis was confirmed with lower respiratory tract specimens and no paired nasopharyngeal swabs were obtained to investigate the difference in the viral rna detection rate between upper and lower respiratory tract specimens. serological detection was not done to look for 2019-ncov antibody rises in 18 patients with undetectable viral rna. second, with the limited number of cases, it is difficult to assess host risk factors for disease severity and mortality with multivariableadjusted methods. this is a modest-sized case series of patients admitted to hospital; collection of standardised data for a larger cohort would help to further define the clinical presentation, natural history, and risk factors. further studies in outpatient, primary care, or community settings are needed to get a full picture of the spectrum of clinical severity. at the same time, finding of statistical tests and p values should be interpreted with caution, and non-significant p values do not necessarily rule out difference between icu and non-icu patients. third, since the causative pathogen has just been identified, kinetics of viral load and antibody titres were not available. finally, the potential exposure bias in our study might account for why no paediatric or adolescent patients were reported in this cohort. more effort should be made to answer these questions in future studies. both sars-cov and mers-cov were believed to originate in bats, and these infections were transmitted directly to humans from market civets and dromedary camels, respectively. 35 extensive research on sars-cov and mers-cov has driven the discovery of many sars-like and mers-like coronaviruses in bats. in 2013, ge and colleagues 36 reported the whole genome sequence of a sars-like coronavirus in bats with that ability to use human ace2 as a receptor, thus having replication potentials in human cells. 37 2019-ncov still needs to be studied deeply in case it becomes a global health threat. reliable quick pathogen tests and feasible differential diagnosis based on clinical description are crucial for clinicians in their first contact with suspected patients. because of the pandemic potential of 2019-ncov, careful surveillance is essential to monitor its future host adaption, viral evolution, infectivity, transmissibility, and pathogenicity. bc and jw had the idea for and designed the study and had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. ywa, gf, xg, jixu, hl, and bc contributed to writing of the report. bc contributed to critical revision of the report. ywa, gf, xg, jixu, and hl contributed to the statistical analysis. all authors contributed to data acquisition, data analysis, or data interpretation, and reviewed and approved the final version. all authors declare no competing interests. the data that support the findings of this study are available from the corresponding author on reasonable request. participant data without names and identifiers will be made available after approval from the corresponding author and national health commission. after publication of study findings, the data will be available for others to request. the research team will provide an email address for communication once the data are approved to be shared with others. the proposal with detailed description of study objectives and statistical analysis plan will be needed for evaluation of the reasonability to request for our data. the corresponding author and national health commission will make a decision based on these materials. additional materials may also be required during the process. clinical virology a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group isolation of a novel coronavirus from a man with pneumonia in saudi arabia summary of probable sars cases with onset of illness from 1 who. middle east respiratory syndrome coronavirus who. novel coronavirus -japan (ex-china) novel coronavirus -republic of korea (ex-china) first travel-related case of 2019 novel coronavirus detected in united states a novel coronavirus genome identified in a cluster of pneumonia cases -wuhan relationship between the presence of hypoxemia and the inflammatory response measured by c-reactive protein in bacteremic pneumococcal pneumonia kidney disease: improving global outcomes (kdigo) acute kidney injury work group. kdigo clinical practice guideline for acute kidney injury cdc definitions for nosocomial infections association between cardiac injury and mortality in hospitalized patients infected with avian influenza a (h7n9) virus coronaviruses post-sars: update on replication and pathogenesis a major outbreak of severe acute respiratory syndrome in hong kong epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques sars: systematic review of treatment effects corticosteroids as adjunctive therapy in the treatment of influenza corticosteroid therapy for critically ill patients with middle east respiratory syndrome clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov origin and evolution of pathogenic coronaviruses isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor bats as animal reservoirs for the sars coronavirus: hypothesis proved after 10 years of virus hunting we acknowledge all health-care workers involved in the diagnosis and treatment of patients in wuhan; we thank the chinese national health commission for coordinating data collection for patients with 2019-ncov infection; we thank who and the international severe acute respiratory and emerging infections consortium (isaric) for sharing data collection templates publicly on the website; and we thank prof chen wang and prof george f gao for guidance in study design and interpretation of results. key: cord-291916-5yqc3zcx authors: hozhabri, hossein; piceci sparascio, francesca; sohrabi, hamidreza; mousavifar, leila; roy, rené; scribano, daniela; de luca, alessandro; ambrosi, cecilia; sarshar, meysam title: the global emergency of novel coronavirus (sars-cov-2): an update of the current status and forecasting date: 2020-08-05 journal: int j environ res public health doi: 10.3390/ijerph17165648 sha: doc_id: 291916 cord_uid: 5yqc3zcx over the past two decades, there have been two major outbreaks where the crossover of animal betacoronaviruses to humans has resulted in severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). in december 2019, a global public health concern started with the emergence of a new strain of coronavirus (sars-cov-2 or 2019 novel coronavirus, 2019-ncov) which has rapidly spread all over the world from its origin in wuhan, china. sars-cov-2 belongs to the betacoronavirus genus, which includes human sars-cov, mers and two other human coronaviruses (hcovs), hcov-oc43 and hcov-hku1. the fatality rate of sars-cov-2 is lower than the two previous coronavirus epidemics, but it is faster spreading and the large number of infected people with severe viral pneumonia and respiratory illness, showed sars-cov-2 to be highly contagious. based on the current published evidence, herein we summarize the origin, genetics, epidemiology, clinical manifestations, preventions, diagnosis and up to date treatments of sars-cov-2 infections in comparison with those caused by sars-cov and mers-cov. moreover, the possible impact of weather conditions on the transmission of sars-cov-2 is also discussed. therefore, the aim of the present review is to reconsider the two previous pandemics and provide a reference for future studies as well as therapeutic approaches. coronaviruses (covs) are a group of highly enveloped viruses that are diversely found in humans and wildlife. with their high mutation rate and infectivity, covs are important zoonotic pathogens that can infect animals [1, 2] and humans, leading to 5-10% of acute respiratory syndromes [3] . apart from infecting a variety of economically important vertebrates (such as pigs and chickens), six species have been identified to cause disease in humans [4] . they are known to infect respiratory, gastrointestinal, hepatic and neurologic systems with a wide range of clinical features from asymptomatic course to severe disease that require hospitalization in the intensive care unit [4, 5] . the first human coronaviruses (hcovs), hcov-229e and oc43, shown to be significant respiratory pathogens, were identified in the 1960s [6, 7] . however, it is assumed that the first recorded coronavirus-related disease was feline infectious peritonitis (fip) in 1912 [8] . the "corona"-like or crown-like morphology of these viruses leads to choose the name "coronavirus," in 1968 [6] . coronaviruses were not considered as highly pathogenic for humans before the beginning of the 21st century. afterward, two highly pathogenic hcovs, including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), emerging from animal reservoirs, have led to global epidemics of deadly pneumonia in humans with high morbidity and mortality [9, 10] . in december 2019, seven years after mers outbreak, the third pathogenic hcov emerged in wuhan, the capital city of hubei province in china, causing severe pneumonia [11, 12] . considered as agents that are a great public health threat, an epidemiological alert was placed by the world health organization (who) and this new coronavirus was named sars-cov-2 and the related respiratory disease covid-19 (https://www.who.int). compared with sars-cov, sars-cov-2 appears to be more readily transmitted from human-to-human, spreading to multiple continents and the outbreak of sars-cov-2 was declared on january 30, 2020 [13] (https://www.who.int). in this review, we will introduce the current knowledge on the origin and evolution of sars-cov-2, emphasizing its characteristics and its genetic diversity from previous coronaviruses, with a brief comment on its epidemiology and pathogenesis. we also highlight the environmental factors involved in virus transmission. knowledge about this novel coronavirus is rapidly evolving, and efforts must be implemented in order to protect the populations by reducing transmission and controlling the spread of this fatal disease. according to the international committee on taxonomy of viruses, covs are classified under the order of nidovirales, a family of coronaviridae and subfamily of coronavirinae [14] . based on previous serologic and recent genomic evidences, the family of coronaviridae encompasses two subfamilies: subfamily orthocoronavirinae and subfamily torovirinae ( figure 1) [7, 15] . the subfamily of orthocoronavirinae consists of four genera: alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus [7, 16, 17] . covs can be isolated from different animal species, including birds, livestock and mammals such as camels, bats, masked palm civets, mice, dogs and cats [18, 19] . animal covs are known to cause acute diseases in several animals and could be responsible for economic losses in domestic animals or birds [20, 21] . domestic animals may play an important role as intermediate hosts that enable virus transmission from one species to humans [17] . the genera gamma-and deltacoronavirus infect birds, but some of them can also infect mammals [16] . these animal covs include transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), avian infectious bronchitis virus (ibv)-and more recently-swine acute diarrhea syndrome coronavirus (sads-cov). however, animal covs can also infect humans that can spread the infection through human-tohuman transmission [17, 22] . on the other hand, alpha-and betacoronavirus infect only mammals and usually cause respiratory illness in humans; among these, strains 229e, oc43, hku1 and nl63 are the most widespread infecting young children, infants as well as elderly individuals [23] [24] [25] . the high rates of mutation characterizing all rna viruses [23, 26] , the evolving nature of covs and the simplicity of transmission from one species to another are the most relevant features learned from sars-cov and mers-cov previous outbreaks [15, 23, 25] . importantly, most of alpha-and betacoronavirus were found only in bats, and many genetically diverse coronaviruses phylogenetically related to sars-cov and mers-cov have been discovered in diverse bat species worldwide [17] . therefore, hcovs such as sars-and mers-covs seem to have originated in bats by sequential mutations and recombination, including those occurring in the intermediate hosts, civets and raccoon dogs for sars-cov and camels in the case of mers-cov, finally acquiring the ability to infect humans [15, 17] . comparative genome studies published in recent papers strongly support the hypothesis that sars-cov-2 originated in bats and that pangolins (manis javanica) acted as intermediate mammalian hosts [11, 27] (figure 2) . indeed, the genetic sequence of the sars-cov-2 showed more than 79% nucleotide identity with the sequence of sars-cov and 50% with mers-cov [17, 19] . the high degree of homology of the angiotensin-converting enzyme 2 (ace2) receptor in several animal species can be considered as an additional evidence to support that sars-cov-2 originated from bats [28] . based on findings from molecular studies, the ace2 proteins of non-human primates, pigs, cats and ferrets closely resemble the human ace2 receptor. therefore, these species covs can be isolated from different animal species, including birds, livestock and mammals such as camels, bats, masked palm civets, mice, dogs and cats [18, 19] . animal covs are known to cause acute diseases in several animals and could be responsible for economic losses in domestic animals or birds [20, 21] . domestic animals may play an important role as intermediate hosts that enable virus transmission from one species to humans [17] . the genera gammaand deltacoronavirus infect birds, but some of them can also infect mammals [16] . these animal covs include transmissible gastroenteritis virus (tgev), porcine epidemic diarrhea virus (pedv), avian infectious bronchitis virus (ibv)-and more recently-swine acute diarrhea syndrome coronavirus (sads-cov). however, animal covs can also infect humans that can spread the infection through human-to-human transmission [17, 22] . on the other hand, alphaand betacoronavirus infect only mammals and usually cause respiratory illness in humans; among these, strains 229e, oc43, hku1 and nl63 are the most widespread infecting young children, infants as well as elderly individuals [23] [24] [25] . the high rates of mutation characterizing all rna viruses [23, 26] , the evolving nature of covs and the simplicity of transmission from one species to another are the most relevant features learned from sars-cov and mers-cov previous outbreaks [15, 23, 25] . importantly, most of alphaand betacoronavirus were found only in bats, and many genetically diverse coronaviruses phylogenetically related to sars-cov and mers-cov have been discovered in diverse bat species worldwide [17] . therefore, hcovs such as sars-and mers-covs seem to have originated in bats by sequential mutations and recombination, including those occurring in the intermediate hosts, civets and raccoon dogs for sars-cov and camels in the case of mers-cov, finally acquiring the ability to infect humans [15, 17] . comparative genome studies published in recent papers strongly support the hypothesis that sars-cov-2 originated in bats and that pangolins (manis javanica) acted as intermediate mammalian hosts [11, 27] (figure 2) . indeed, the genetic sequence of the sars-cov-2 showed more than 79% nucleotide identity with the sequence of sars-cov and 50% with mers-cov [17, 19] . the high degree of homology of the angiotensin-converting enzyme 2 (ace2) receptor in several animal species can be considered as an additional evidence to support that sars-cov-2 originated from bats [28] . based on findings from molecular studies, the ace2 proteins of non-human primates, pigs, cats and ferrets closely resemble the human ace2 receptor. therefore, these species may be susceptible to sars-cov-2 infection, as has been shown for sars-cov. although a recent study showed that neither pigs nor chickens are susceptible to sars-cov-2 by intranasal or oculo-oronasal infections, more evidences are needed to exclude pigs as intermediate host of sars-cov-2 [29] . [15] . based on the genetic sequence identity and the phylogenetic reports, sars-cov-2 is sufficiently different from sars-cov; thus, who has classified it as a new betacoronavirus that infects humans [30] . the genome of hcovs is a single-stranded positive-sense rna (+ssrna) (~26-32 kb) with 5′cap structure and 3′-poly a tail, which is among the largest known rna genomes [31] [32] [33] . the typical hcovs gene order is 5′-replicase-s-e-m-n-3′, with numerous (6 to 11) open reading frames (orfs) encoding accessory proteins scattered among the structural genes [34, 35] . the first orfs (orf1a and 1b) comprise two-thirds (approximately 67%) of the genome length and encode 16 nonstructural polyproteins (nsps [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] and are directly translated from the genomic rna [17] . there is a −1 ribosomal frameshift between orf1a and orf1b, leading to the production of two large replicase polypeptides (pp): pp1a and pp1ab. these polypeptides are further processed by two virally encoded cysteine proteases, the papain-like protease (plpro) and a 3-chymotrypsin-like protease (3clpro) into 16 nsps [3, 33, 36] . there are at least four structural proteins encoded by the coronaviral genome: a spike glycoprotein (s), an envelope protein (e), a membrane protein (m) and nucleocapsid protein (n) with short untranslated regions at both termini, required to produce a structurally complete viral particle [37] . the typical coronavirus virion structure and proteins are shown in figure 3 . the m protein is in higher quantities in comparison to any other proteins in the virus particle; with its three transmembrane domains, it shapes virions, promotes membrane curvature and binds to the nucleocapsid [38, 39] . the n protein contains two domains, both of which can bind to nsp3 protein to help tether the genome to replication-transcription complex (rtc) and package viral rna into the viral particle during viral assembly [39, 40] . the e protein is involved in virus assembly and virion release from host cells, while the s protein plays a vital role in attachment to host receptors, viral entry and determines host tropism [41, 42] . additionally, some coronaviruses, such as hcov-oc43 and hcov-hku1, have a hemagglutinin-esterase (he) gene between orf1b and s [43] [44] [45] [46] . this based on the genetic sequence identity and the phylogenetic reports, sars-cov-2 is sufficiently different from sars-cov; thus, who has classified it as a new betacoronavirus that infects humans [30] . the genome of hcovs is a single-stranded positive-sense rna (+ssrna) (~26-32 kb) with 5 -cap structure and 3 -poly a tail, which is among the largest known rna genomes [31] [32] [33] . the typical hcovs gene order is 5 -replicase-s-e-m-n-3 , with numerous (6 to 11) open reading frames (orfs) encoding accessory proteins scattered among the structural genes [34, 35] . the first orfs (orf1a and 1b) comprise two-thirds (approximately 67%) of the genome length and encode 16 nonstructural polyproteins (nsps 1-16) and are directly translated from the genomic rna [17] . there is a −1 ribosomal frameshift between orf1a and orf1b, leading to the production of two large replicase polypeptides (pp): pp1a and pp1ab. these polypeptides are further processed by two virally encoded cysteine proteases, the papain-like protease (plpro) and a 3-chymotrypsin-like protease (3clpro) into 16 nsps [3, 33, 36] . there are at least four structural proteins encoded by the coronaviral genome: a spike glycoprotein (s), an envelope protein (e), a membrane protein (m) and nucleocapsid protein (n) with short untranslated regions at both termini, required to produce a structurally complete viral particle [37] . the typical coronavirus virion structure and proteins are shown in figure 3 . the m protein is in higher quantities in comparison to any other proteins in the virus particle; with its three transmembrane domains, it shapes virions, promotes membrane curvature and binds to the nucleocapsid [38, 39] . the n protein contains two domains, both of which can bind to nsp3 protein to help tether the genome to replication-transcription complex (rtc) and package viral rna into the viral particle during viral assembly [39, 40] . the e protein is involved in virus assembly and virion release from host cells, while the s protein plays a vital role in attachment to host receptors, viral entry and determines host tropism [41, 42] . additionally, some coronaviruses, such as hcov-oc43 and hcov-hku1, have a hemagglutinin-esterase (he) gene between orf1b and s [43] [44] [45] [46] . this hemagglutinin, like the influenza homolog enzyme, binds to sialic acid on host cell-surface glycoproteins and possesses acetyl-esterase activity [47] . besides coronavirus-conserved genes, the sars-cov, sars-cov-2 and mers-cov genomes contain several specific accessory genes including orf3a/b, 4a/b, orf5, orf6, orf7a/b, orf8a/b and 9b ( figure 4) [4, 48, 49] . all the structural and accessory proteins are translated from subgenomic rnas (sgrnas) generated during genome transcription/replication of covs [4] . hemagglutinin, like the influenza homolog enzyme, binds to sialic acid on host cell-surface glycoproteins and possesses acetyl-esterase activity [47] . besides coronavirus-conserved genes, the sars-cov, sars-cov-2 and mers-cov genomes contain several specific accessory genes including orf3a/b, 4a/b, orf5, orf6, orf7a/b, orf8a/b and 9b ( figure 4) [4, 48, 49] . all the structural and accessory proteins are translated from subgenomic rnas (sgrnas) generated during genome transcription/replication of covs [4] . attachment, cell entry, translation of viral replicase, genome replication, translation of structural proteins and virion assembly and release are the phases of coronavirus replication cycle [4, 50] . sars-cov, mers-cov and sars-cov-2 bind to different host receptors to gain entry into host cells [4, 51, 52] . viral entry is mediated by the transmembrane s glycoprotein that comprises two functional subunits (s1 and s2 subunits) responsible for receptor recognition and viral-host cell membranes fusion, respectively [53, 54] . s1 receptor-binding domain (rbd) mediates binding to the cognate host cell receptor; however, the s2 domain mediates the fusion events, between viral envelope and host cell membrane [52, 55, 56] . as recently found, sars-cov-2 uses the same ace2 receptor [57] , as sars-cov, whereas mers-cov uses dipeptidyl peptidase 4 (dpp4, also known as cd26) receptor (table 1 ) [58] . the fusion of the s protein to the plasma membrane of host cell generates a double membrane vesicle in the host cell, thereby allowing release of the nucleocapsid into the cytoplasm, followed by genome transcription [53, 54] . upon entry into the cell, virus-specific rna and proteins are synthesized, probably entirely in the cytoplasm. translation starts with the expression of two polyproteins, pp1a and pp1ab, which undergo co-translational proteolytic processing into the proteins that form the replicase complex. this complex is used to transcribe a 3′-coterminal set of nested subgenomic mrnas, as well as genomic rna that have a common 5′ "leader" sequence derived from the 5′ end of the genome. new virions are assembled by budding into intracellular membranes of the pre-golgi compartment and released through the cell secretory mechanisms [4, 42, 48, 50] . attachment, cell entry, translation of viral replicase, genome replication, translation of structural proteins and virion assembly and release are the phases of coronavirus replication cycle [4, 50] . sars-cov, mers-cov and sars-cov-2 bind to different host receptors to gain entry into host cells [4, 51, 52] . viral entry is mediated by the transmembrane s glycoprotein that comprises two functional subunits (s1 and s2 subunits) responsible for receptor recognition and viral-host cell membranes fusion, respectively [53, 54] . s1 receptor-binding domain (rbd) mediates binding to the cognate host cell receptor; however, the s2 domain mediates the fusion events, between viral envelope and host cell membrane [52, 55, 56] . as recently found, sars-cov-2 uses the same ace2 receptor [57] , as sars-cov, whereas mers-cov uses dipeptidyl peptidase 4 (dpp4, also known as cd26) receptor (table 1 ) [58] . the fusion of the s protein to the plasma membrane of host cell generates a double membrane vesicle in the host cell, thereby allowing release of the nucleocapsid into the cytoplasm, followed by genome transcription [53, 54] . upon entry into the cell, virus-specific rna and proteins are synthesized, probably entirely in the cytoplasm. translation starts with the expression of two polyproteins, pp1a and pp1ab, which undergo co-translational proteolytic processing into the proteins that form the replicase complex. this complex is used to transcribe a 3 -coterminal set of nested subgenomic mrnas, as well as genomic rna that have a common 5 "leader" sequence derived from the 5 end of the genome. new virions are assembled by budding into intracellular membranes of the pre-golgi compartment and released through the cell secretory mechanisms [4, 42, 48, 50] . , the 3′-terminus of the sars-cov-2 and sars-cov genomes contain eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14 and 3a, 3b, p6, 7a, 7b, 8a, 8b and 9b, respectively) while mers-cov genome contains only five (3, 4a, 4b, 5 and 8b). the genes encoding accessory proteins are unique in different coronaviruses in terms of number, genomic organization, sequence and functions (data extracted from [35, 49, 57] the coronavirus genomes encode two replicase polypeptides pp1a and pp1ab translated from orf1a and orf1b; four structural genes encoding for four structural proteins including (s) spike, (m) membrane, (e) envelope and (n) nucleocapsid proteins. the single-stranded rna genomes of sars-cov-2 (~29.8 kb), sars-cov (~29.7 kb) and mers-cov (~30.1 kb) harbor two large genes, the orf1a (red) and 1b (blue) genes encoding accessory genes (nsps 1-16, shades of red and blue). encoded nonstructural proteins: 16 nsps (nsp1-nsp16) in sars-cov-2, sars-cov and mers-cov. along with structural proteins (s, e, m and n), the 3 -terminus of the sars-cov-2 and sars-cov genomes contain eight accessory proteins (3a, 3b, p6, 7a, 7b, 8b, 9b and orf14 and 3a, 3b, p6, 7a, 7b, 8a, 8b and 9b, respectively) while mers-cov genome contains only five (3, 4a, 4b, 5 and 8b). the genes encoding accessory proteins are unique in different coronaviruses in terms of number, genomic organization, sequence and functions (data extracted from [35, 49, 57] virologic as well as genetic studies have demonstrated that bats are reservoir hosts of both sars-cov and mers-cov, but also that they can use other species as intermediate hosts before spreading to humans [59, 60] . the detection of two genomes distinct from known swine in ill piglets were reported by two independent groups [61, 62] . the phylogenetic analyses showed that these novel swine enteric alphacoronaviruses (seacovs) were strongly related to the rhinolophus bat coronavirus hku2 isolated in guangdong province, in southern china [61, 62] . this suggests that coronaviruses of bat origin may have 'jumped' the barrier of the species to infect pigs as intermediate hosts. the cd26 receptor sequence alignment between humans and pigs demonstrates a 94.5% overlap, which is sufficient for the possible cross-species transmission [63] . it has also been documented that pigs are susceptible to human sars-cov [64] and mers-cov infections [65] . the large number of mutations within the rbd enabled viruses to infect new hosts, representing a potential threat for both animal and human health. in southern china, the unique climate, the high density of domestic as well as wild pigs, along with the extensive bat distribution and carriage of tremendous quantities of recombinant novel coronaviruses may result in the appearance of more novel coronaviruses in the future [66] . it is generally acknowledged that numerous viruses have existed and were restricted to their natural reservoirs for lengthy times [17] . the consistent spillover of viruses from natural hosts to humans and other species is essentially related to human activities, including urbanization and modern agricultural practices, leading to the constant human exposure to the ever-changing mutant covs from their reservoirs [15, 17] . the close contact between humans and animals and the practice of eating raw meat are both risk factors for causing a new human cov outbreak [15] . hence, covid-19 should be considered as a zoonotic disease that spread from animals to humans. following the first sars-cov-2 outbreak in seafood and wildlife market in wuhan, secondary cases started to be identified after ten days. although these new patients did not have any contact with the market, they had a history of contact with people who attended the market [60] . therefore, similarly to sars-cov and unlikely to mers-cov, human-to-human transmission for sars-cov-2 has been reported and is currently considered as the main type of transmission worldwide [5, 19] . on january 13, 2020, thailand announced the first non-chinese case of infection that spread from the chinese provinces, to the asian continent [60] . this case was a chinese tourist who has traveled to thailand and did not have any epidemiological link to the market [30] . more recently, forster et al., by using phylogenetic analysis based on nucleotide mutations of 160 complete human sars-cov-2 genomes found that three variants of sars-cov-2 (a as the ancestral type, plus b and c) represent the bat outgroup coronaviruses. in particular, the a and c types were found mostly in european-american patients, whereas the b type was common in east asia suggesting that this kind of analysis could help in following the evolution of sars-cov-2 [67] . it was demonstrated that sars-covs have adapted themselves to bind to human ace2 receptor and infect human cells effectively [68] . this form of adaptation required a series of amino acid changes in the rbd within the s protein of sars viruses that circulated in bats [56, 68] . therefore, the human-to-human transmission that was seen in the course of the sars-cov outbreaks is directly attributable to the ability of sars-covs to adapt their s protein to efficiently bind to human ace2, thus infecting ciliated bronchial epithelial cells and type ii pneumocytes [15, 69] . similar to sars-cov, ace2 is also used by sars-cov-2 as the entry receptor in the ace2-expressing cells, suggesting that sars-cov-2 has a life cycle similar to sars-cov [56, 68] . as outlined before, sars-cov s protein regulates the receptor binding and membrane fusion activities determining host tropism and transmission capacity. several evidences highlighted a higher binding affinity of sars-cov-2 rbd to the ace2 receptor. in particular, molecular and in silico analyses demonstrated that sars-cov-2 rbd conformation and amino acid composition enhance the bonding between the s protein and ace2 receptor [51, 70, 71] . a recent biophysical and structural analysis of the sars-cov-2 s protein showed that it binds to ace2 receptor with about 10-to 20-fold higher affinity than the s protein of sars-cov [52, 72] . this high affinity could account for its extreme infectivity among human populations. another feature of the powerful infectivity of sars-cov-2 is that the shedding pattern of viral particles in sars-cov-2 diagnosed patients is similar to that of influenza patients in which viral loads at the time of symptom onset are higher and gradually decrease within days; interestingly, this pattern seems to be different from that reported for sars-cov patients where the highest shedding is reported 10 days after the onset of symptoms [20, 73, 74] . these results indicate that sars-cov-2 can spread more easily than sars-cov in the community even in the absence of symptoms or when only initial mild symptoms are present [75] . the human-to-human transmission of sars-cov-2 mainly occurs by inhalation of respiratory droplets spread by coughing or sneezing from an infected individual, but also by direct contact of contaminated surfaces and then touching the nose, mouth and eyes [24, [76] [77] [78] . the virus was shown to remain stable in favorable atmospheric conditions on different surfaces for days [79] . additionally, transmission in an unventilated environment or closed spaces due to high aerosol concentrations has been suggested [76, 77] . in agreement, the presence of sars-cov-2 in the surfaces of the houses of confirmed patients was reported, further strengthening this mode of contact transmission. moreover, live viruses were also found in the stool of covid-19 patients, as previously found for both sars-cov and mers-cov [77] . given its capacity for survival in feces and the expression of ace2 within intestine, it was demonstrated that sars-cov-2 can infect these tissues and can be released in feces; therefore, water supply contamination and fecal-oral route transmission is also hypothesized [24, 80] . however, at present, there have not been reported cases of fecal-oral transmission of the virus. studies have also indicated that sars-cov-2 transmission via ocular surfaces should not be overlooked, as contaminated droplets and body fluids could easily infect the human conjunctival epithelium [81] . sars-cov-2 is also responsible for cluster transmission, in particular within family clusters [77] . in some cities, 50% to 80% of all reported cases of covid-19 accounted for cluster transmission [82] . based on the current information, there is no evidence for transplacental transmission from infected pregnant women to their fetus, who underwent caesarean section [24, 78] . therefore, whether transmission during vaginal birth can occur remains to be established, neonatal covid-19 disease as postnatal transmission was documented [83] . although, sars-cov-2 may definitely infect infants, it has been reported that neonates, infants and children develop significantly milder forms of the disease than their adult counterparts [24, 84] . coronaviruses are responsible for 5-10% of acute respiratory illness while it has been estimated that 2% of the population is deemed as an asymptomatic carrier of these viruses. the first discovered hcov was ibv that causes respiratory disease in human whereas, hcov-229e and hcov-oc43, which cause the common cold in humans [15, 26, 85] . they were not considered to be highly pathogenic for humans until the outbreak of sars in guangdong state of china in 2002 and 2003. sars-cov infected more than 8000 people worldwide and caused 916 deaths (table 2) , representing a mortality rate by around 10% [86] . ten years later in 2012, mers-cov emerged in saudi arabia and infected over 2494 people with 858 deaths, accounting for a mortality rate approximately of 35% [9, 24, 87] . starting in china in december 2019, there were reports of patients presenting severe viral pneumonia [15, 88, 89] . this public health concern resulted in many unknown pneumonia cases who were admitted to local hospitals [22, 78] (https://www.who.int). primary etiologic investigations performed in those patients showed that they were epidemiologically linked to a huanan wholesale seafood market that also traded live animals and wildlife [17, 24, 90] . by january 7th, 2020 chinese authorities announced that a new type of coronavirus was isolated [60, 91] . the epicenter of infection was probably linked to a zoonotic pathogen being present in the seafood and exotic animal wholesale market [60, 91] . the rapid increasing numbers and rate of fatalities indicated a second mode of transmission, from human-to-human, that allowed viral spreading primarily in other asian countries such as south korea and iran followed by many countries such as italy, spain, germany, france, brazil and usa [24, 60] . it is very intriguing to note that the sars epidemic in southern china in 2002 and the current outbreak of covid-19 had peaked in mid-february due to exposure to live animals sold in markets. furthermore, similar to the sars outbreak, this epidemic has occurred during the spring festival in china, as the most famous traditional countrywide festival in china, gathering nearly three billion people from different areas. these favorable conditions caused the wide transmission of this fatal pneumonia and severe difficulties for disease control and prevention of the epidemic [92] . based on clinical data of diagnosed patients during the sars-cov-2 outbreak, the basic reproduction number (r0) is estimated to range between 2 and 6.47 in various modeling studies [76, 93] . the sars-cov-2 r0 is in line with the one estimated for sars-covs and mers-covs (from 2 to 5) [94, 95] . currently, increasing countries are experiencing clusters of cases and community transmission following sars-cov-2 pandemic. since its emergence, the covid-19 has drawn well-deserved attention from authorities in order to protect their community and stop or slow down transmission of this disease. at the time of this review, according to the daily report of the world health organization, sars-cov-2 has affected over 17,889,134 people with around 228,611 daily new cases and killed more than 686,145 people all over the world, by august 3rd, 2020 (the up to date fatality rate is reported from https://covid19.who.int). we must take into consideration that these data are relative to laboratories and clinically confirmed cases while the actual number including asymptomatic cases, infected undiagnosed and death patients would be much higher than reported cases. the transmission of seasonal respiratory coronaviruses can be affected by several climate parameters such as temperature and humidity [96] . therefore, understanding the relationship between weather and transmission of covid-19 is the key to forecast the intensity and end time of this pandemic. to this regard, emerging evidence suggests that whether climate conditions may influence the transmission of the sars-cov-2 by boosting the spread (much of the data have not been peer-reviewed yet). to date, covid-19 has had a significant expansion in the northern hemisphere (nh) belt, given that it covers cities and populated areas; conversely, in the belt of the southern hemisphere (sh), which covers very low population and landless areas, covid-19 has not been reported yet. based on climate and era-interim reanalysis dataset in nh belt from november 2019 to march 2020, we compared the average rate of humidity and temperature between five cities in european countries with significant community transmission of covid-19 versus five cities of north africa which are expected to be less exposed to covid-19. the information recorded by the meteorological stations has been used, since these are more accurate than satellite data [97] . as shown in table 3 , the average amount of humidity is very close between european and african selected sites. the main reason is the proximity of these cities to the mediterranean sea coastline. in addition, the north wind, which blows from northern europe to european cities (ecs), increases the humidity of these cities. conversely, there are temperature differences between considered ecs and north african cities (table 3) . thus, temperature and humidity should be considered parameters involved in the transmission of covid-19. up to know, few studies have investigated the association of temperature and humidity with covid-19 incidence and death rates. the first meteorological study was done in 100 different chinese cities each having more than 40 cases of covid-19 in a 3-day period during the end of january [98] . this group showed that high temperature and humidity significantly reduces the transmission of covid-19. their results indicate that the increases of 1 • c in temperature and 1% in relative humidity lower r by 0.0225 and 0.0158, respectively [98] . a preprint study on confirmed covid-19 cases collected from 429 cities showed that every 1 • c increase in the minimum temperature of higher-temperature cities reduced the disease incidence and death rates by 0.86 [99] . another preprint study suggested that the average increase of 1 • c in temperature correlates negatively with the predicted number of cases worldwide [100] . these results are in accordance with wu y et al. who showed that among all confirmed covid-19 new cases and new deaths from 166 countries (excluding china), a 1 • c increase in temperature is associated with a 3.08% reduction in daily new cases and a 1.19% reduction in daily new deaths, whereas a 1% increase in relative humidity was associated with a 0.85% reduction in daily new cases and a 0.51% reduction in daily new deaths [101] . a recent study conducted in italy showed a positive correlation of sars-cov-2 spreading and weather conditions including temperatures ranging 4-12 • c and relative humidity of 60-80% [102] . in a geographic and population modeling study conducted in five largest cities in colombia, the transmission of sars-cov-2 seems to be comodulated by temperature and humidity. their observation revealed a strong reduction of transmission in climates with temperatures above 30 • c and relative humidity below 78% which may comodulate the infectivity of sars-cov-2 within respiratory droplets [103] . table 3 . average humidity and temperature in 10 different cities in europe and north africa between november 2019 to march 2020. the first five cities represent significant communities where transmission of covid-19 was reported, whereas the second 5 cities are expected to be less exposed to covid-19 due to different weather conditions. average humidity (%) temperature ( • c) rome 71 65 66 63 63 15 1 11 13 14 paris 78 76 79 74 66 9 8 8 9 9 madrid 70 67 68 63 61 10 10 9 13 12 milan 77 74 69 58 62 11 8 7 11 11 lisbon 75 74 77 76 71 15 14 12 15 15 rabat 72 71 69 72 74 16 17 14 17 16 algiers 64 62 61 61 66 16 17 15 18 16 tunis 61 66 72 65 70 17 16 14 15 15 tripoli 80 83 73 75 71 15 10 8 9 11 cairo 45 52 55 53 46 25 18 16 18 22 overall, these meteorological analyses support that the combination of temperature and humidity could represent a direct influence on the transmission of the covid-19. it can be assumed that the arrival of summer and rainy season in the nh can effectively reduce the transmission of the covid-19. the distribution of covid-19 across different longitudes and latitudes with a range of temperatures and humidity may help to predict the prevalence of this disease in terms of environmental characteristics. this could lead to a better understanding of how the virus spreads around the world ( figure 5 ). it should be noted that apart from the capability of sars-cov-2 to persist on environmental surfaces under favorable atmospheric conditions, the duration of its persistence may be affected by temperature and humidity. however, caution is needed when considering the implications of these findings, which may be subject to confounding. although warmer climates may slow the spread of sars-cov-2, relying on weather changes alone to slow the transmission of covid-19 are unlikely to be enough. however, using this type of dataset and climate analysis modeling is possible to identify areas that are most likely to be at risk of significant covid-19 cases in the future and serve as an alarm signal to various government departments and agencies to adopt the necessary measures to prevent virus spread [104] . moreover, more data are gathering around the world due to the change of the season and all authors agree that the association between temperature/humidity and sars-cov-2 is an appreciable hypothesis, but not a certainty yet. 19 cases in the future and serve as an alarm signal to various government departments and agencies to adopt the necessary measures to prevent virus spread [104] . moreover, more data are gathering around the world due to the change of the season and all authors agree that the association between temperature/humidity and sars-cov-2 is an appreciable hypothesis, but not a certainty yet. the most common symptoms of patients at onset of covid-19 disease are defined as fever, dry cough, fatigue and less often, symptoms of sputum production, headache, sore throat, myalgia; hemoptysis, dyspnea, diarrhea and lymphopenia were also observed [15, 24, 28] (figure 6 ). the spectrum of clinical features of covid-19 has been found ranging from an asymptomatic state to severe respiratory failure and multiorgan dysfunction [24, 76] . symptomatic people are considered to be more contagious, similar to most viral-related respiratory diseases. however, individuals who remain asymptomatic may also transmit the virus and cases infected by an asymptomatic individual in the prodrome period of covid-19 have also been reported [76] . asymptomatic infections can occur because of weakened immune responses and subclinical manifestations or also because the the most common symptoms of patients at onset of covid-19 disease are defined as fever, dry cough, fatigue and less often, symptoms of sputum production, headache, sore throat, myalgia; hemoptysis, dyspnea, diarrhea and lymphopenia were also observed [15, 24, 28] (figure 6 ). the spectrum of clinical features of covid-19 has been found ranging from an asymptomatic state to severe respiratory failure and multiorgan dysfunction [24, 76] . symptomatic people are considered to be more contagious, similar to most viral-related respiratory diseases. however, individuals who remain asymptomatic may also transmit the virus and cases infected by an asymptomatic individual in the prodrome period of covid-19 have also been reported [76] . asymptomatic infections can occur because of weakened immune responses and subclinical manifestations or also because the virus is waiting for opportunities to invade and reproduce. a recent study has shown that a viral load detected in an asymptomatic patient was just like to the one observed in symptomatic patients, indicating the capability of transmission in asymptomatic patients [74] . according to disease presentation, covid-19 can be classified as mild, moderate, severe and critical ( virus is waiting for opportunities to invade and reproduce. a recent study has shown that a viral load detected in an asymptomatic patient was just like to the one observed in symptomatic patients, indicating the capability of transmission in asymptomatic patients [74] . according to disease presentation, covid-19 can be classified as mild, moderate, severe and critical (table 4 ) [57, 76, 105] . in 81% of all confirmed covid-19 cases. dry cough, mild fever, sore throat, nasal congestion, muscle pain, headache and malaise. absence of serious symptoms like dyspnea, also the absence of radiograph features. it may rapidly deteriorate into severe or critical cases, non-pneumonia or mild pneumonia. dry cough, tachypnea and shortness of breath. acute respiratory distress syndrome (ards), severe pneumonia, severe dyspnea, sepsis or septic shock, tachypnea (respiratory frequency) ≥ 30/min, blood oxygen saturation (spo2) ≤ 93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio (pao2/fio2) < 300, and/or lung infiltrates > 50% within 24 to 48 h. fever can be absent or moderate. in 5% of all confirmed covid-19 cases. respiratory failure, septic shock, rnaemia, cardiac injury and/or multiple organ dysfunction or failure. case fatality rate is 49% (higher case fatality rate for patients with preexisting co-morbidities and lower-case fatality rate (0.9%) for patients without co-morbidities). cardiovascular disease (10.5%), diabetes (7.3%), respiratory disease (6.5%), hypertension (6%) and oncological complications (5.6%). in 81% of all confirmed covid-19 cases. dry cough, mild fever, sore throat, nasal congestion, muscle pain, headache and malaise. absence of serious symptoms like dyspnea, also the absence of radiograph features. it may rapidly deteriorate into severe or critical cases, non-pneumonia or mild pneumonia. dry cough, tachypnea and shortness of breath. acute respiratory distress syndrome (ards), severe pneumonia, severe dyspnea, sepsis or septic shock, tachypnea (respiratory frequency) ≥ 30/min, blood oxygen saturation (spo 2 ) ≤ 93%, partial pressure of arterial oxygen to fraction of inspired oxygen ratio (pao 2 /fio 2 ) < 300, and/or lung infiltrates > 50% within 24 to 48 h. fever can be absent or moderate. in 5% of all confirmed covid-19 cases. respiratory failure, septic shock, rnaemia, cardiac injury and/or multiple organ dysfunction or failure. case fatality rate is 49% (higher case fatality rate for patients with preexisting co-morbidities and lower-case fatality rate (0.9%) for patients without co-morbidities). cardiovascular disease (10.5%), diabetes (7.3%), respiratory disease (6.5%), hypertension (6%) and oncological complications (5.6%). and 95% of patients are likely to experience symptoms within 12.5 days from contact [24, [106] [107] [108] . however, in an asymptomatic carrier the incubation period was 19 days, complicating the challenge to contain the outbreak [109] . the median time between onset of symptoms and dyspnea is 5 days, 7 days for hospitalization and 8 days for acute respiratory distress syndrome (ards) (figure 7 ) [24] (https://www.epicentro.iss.it/coronavirus/sars-cov-2). patients at this stage in intensive care unit (icu) with quarantine facilities may require mechanical ventilation. moreover, bacterial infections can cause a secondary pneumonia [108] . in addition, the period from the beginning of covid-19 symptoms to death varied between 6 and 41 days with an average of 14 days [110] . this period depends on immune system status and the patient's age, being shorter in 70-year-old subjects compared with those younger [78, 110] . in people with compromised immune systems and in elderly patients with underlying health problems, sars-cov-2 is able to infect the lower respiratory tract leading to severe pneumonia [111] . in 25-30% of patients presenting acute lung injury, shock, ards and acute kidney injury, icu admission was absolutely required [24] . recovery started within the 2nd or 3rd weeks with the median duration of hospitalization of 10 days. the virus appears to be more fatal in individuals with underlying co-morbidities (50-75% of fatal cases) [24, 111] . available dataset was obtained from italian istituto superiore di sanità (iss) on 34,026 patients dying in-hospitals ( figure 7) . the mean number of diseases was 3.3 (median 3, sd 1.9). overall, 4.0% of the reported cases has no co-morbidities, 14.0% with a single comorbidity, 20.6% with 2 and 61.4% with 3 or more (https://www.epicentro.iss.it/coronavirus/sars-cov-2). sars-cov-2 infections revealed some unique clinical characteristics that include targeting the lower airway which is evident by symptoms of upper respiratory tract including rhinorrhoea, sneezing and sore throat [78] . chest computed tomography (ct) scans revealed pneumonia in most sars-cov-2 infected patients and several cases showed an infiltrate in the upper lung lobe, which is related to increasing dyspnea with hypoxemia [28, 78, 112] . table 4 describes the full picture of covid-19 clinical manifestation. atypical symptoms include rnaemia, acute cardiac injury, ards and grand-glass opacities that lead to death [113] . it should be noted that covid-19 manifestations such as fever, dyspnea, dry cough and bilateral ground-glass opacities in chest ct scans are similar to the previous betacoronavirus-related diseases [113, 114] . although gastrointestinal symptoms such as diarrhea were reported in sars-cov-2 infected patients, the similar gastrointestinal distress occurred in only a small percentage of mers-cov or sars-cov patients ( figure 6 ) [78] . it was shown that severe cases were characterized by an increased inflammation due to both systemic and localized immune response activation [78, 115] . higher leukocyte numbers, significantly high blood concentrations of cytokines and chemokines were noted in these cases [28, 78] . hence, it is now accepted that high levels of proinflammatory cytokines could worsen the prognosis [28, 113] . the symptoms of sars-cov-2 infection appear after an incubation period of 1 to 14 days, similar to those of sars-and mers-cov infections (median approximately 5.2 days in different studies) and 95% of patients are likely to experience symptoms within 12.5 days from contact [24, [106] [107] [108] . however, in an asymptomatic carrier the incubation period was 19 days, complicating the challenge to contain the outbreak [109] . the median time between onset of symptoms and dyspnea is 5 days, 7 days for hospitalization and 8 days for acute respiratory distress syndrome (ards) (figure 7 ) [24] (https://www.epicentro.iss.it/coronavirus/sars-cov-2). patients at this stage in intensive care unit (icu) with quarantine facilities may require mechanical ventilation. moreover, bacterial infections can cause a secondary pneumonia [108] . in addition, the period from the beginning of covid-19 symptoms to death varied between 6 and 41 days with an average of 14 days [110] . this period depends on immune system status and the patient's age, being shorter in 70-year-old subjects compared with those younger [78, 110] . in people with compromised immune systems and in elderly patients with underlying health problems, sars-cov-2 is able to infect the lower respiratory tract leading to severe pneumonia [111] . in 25%-30% of patients presenting acute lung injury, shock, ards and acute kidney injury, icu admission was absolutely required [24] . recovery started within the 2nd or 3rd weeks with the median duration of hospitalization of 10 days. the virus appears to be more fatal in individuals with underlying co-morbidities (50%-75% of fatal cases) [24, 111] . available dataset was obtained from italian istituto superiore di sanità (iss) on 34,026 patients dying inhospitals ( figure 7) . the mean number of diseases was 3.3 (median 3, sd 1.9). overall, 4.0% of the reported cases has no co-morbidities, 14.0% with a single comorbidity, 20.6% with 2 and 61.4% with 3 or more (https://www.epicentro.iss.it/coronavirus/sars-cov-2). figure 7 . median times, in days, from the onset of symptoms to death, to hospitalization, from hospitalization to death with and without intensive care unit (icu)-admittance (report based on available data on july 9th, 2020 collected from istituto superiore di sanità, iss). sars-cov-2 infections revealed some unique clinical characteristics that include targeting the lower airway which is evident by symptoms of upper respiratory tract including rhinorrhoea, sneezing and sore throat [78] . chest computed tomography (ct) scans revealed pneumonia in most sars-cov-2 infected patients and several cases showed an infiltrate in the upper lung lobe, which is related to increasing dyspnea with hypoxemia [28, 78, 112] . table 4 describes the full picture of covid-19 clinical manifestation. atypical symptoms include rnaemia, acute cardiac injury, ards and grand-glass opacities that lead to death [113] . it should be noted that covid-19 manifestations figure 7 . median times, in days, from the onset of symptoms to death, to hospitalization, from hospitalization to death with and without intensive care unit (icu)-admittance (report based on available data on july 9th, 2020 collected from istituto superiore di sanità, iss). covid-19 clinical evaluation is focused mainly on epidemiological data, clinical symptoms and clinical and laboratory tests. although the scenario is continually changing, several approaches were selected as standard laboratory methods for covid-19 diagnosis. lab tests, differently from clinical-based analyses, immediately reveal sars-cov-2 infected patients. this was particularly important for diagnosis due to the difficulties in detecting specific clinical signs and symptoms in covid-19 patients. moreover, atypical manifestations revealed by pulmonary imaging [116] and the huge number of different clinical signs and symptoms forced the development of molecular-based laboratory tests [117, 118] . lastly, the analysis of personal history of each patient played a fundamental role in covid-19 diagnosis and up to now is considered one of the key information for detecting infected patients also in the early phases of infection. therefore, the epidemiological history together with clinical and laboratory tests are all required for the diagnosis of covid-19. a detailed description focused on clinical diagnostic methods was reviewed by taisheng li [119] . herein, we present an updated overview of the principal techniques used for covid-19 diagnosis. high-throughput sequencing and real time quantitative polymerase chain reaction (rt-qpcr) are the best nucleic acid detection techniques for sars-cov-2. however, in clinical diagnosis, the application of high-throughput sequencing technology is limited due to high cost and its equipment dependency [114, 120, 121] . moreover, to speed up the development of standardized analytic kits for diagnostic application, the quantification of viral load was not considered. therefore, the rt-pcr method was chosen as the gold standard for the detection of sars-cov-2 infections from the commonly used samples such as naso-and oropharyngeal swabs [106, 107, 121, 122] . this molecular method relies on the amplification of up to three sars-cov-2 specific targets including the rna-dependent rna polymerase (rdrp), e and n genes [121] . the who has released numerous rt-pcr protocols for the detection of sars-cov-2 rna at https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance (accessed march 15, 2020). three of those protocols are mentioned below. the us centers for disease control and prevention (cdc) developed an rt-pcr that includes three sets of oligonucleotide primers and probes recognizing three regions of the virus n gene (named n1, n2 and n3) and an additional primer/probe set to detect the human rnase p gene (rp) representing an internal control for rna extraction and retro-transcription. moreover, the positive control consisting in retro-transcribed viral rna is also available at cdc. to report the positivity for sars-cov-2 two out of three n regions must be positive. the chinese center for disease control and prevention (china cdc) recommends the use of specific primers and probes targeting the orf1ab and n gene regions for sars-cov-2 detection by rt-pcr [123] . the positivity is confirmed when both targets are detected. available online: http://ivdc.chinacdc.cn/kyjz/202001/t20200121_211337.html (accessed on 21 january 2020). overall, the who summarized all the primer pairs and probes that can be used to detect sars-cov-2 in clinical specimens with the description of rt-pcr settings and the specificity. apart from the possibility to perform the rt-pcr in house using the selected primer pairs and probes, several ready to use kits were developed for rt-pcr performing. one of the most used is the allplex 2019-ncov (seegene, seoul, south korea) which includes three different viral targets and a positive control [124] . another example is represented by the bgi's real-time fluorescent rt-pcr kit for detecting sars-cov-2 that includes one sars-cov-2 specific target and an internal control of the reaction (bgi, cambridge, ma, usa). both companies declared a sensibility of 100-150 viral copies per ml and a high specificity that excludes most respiratory tract viral and bacterial pathogens. the recommended samples for both in-house and ready-to-use rt-pcr kits include upper and lower respiratory specimens such as throat, nasal nasopharyngeal (np) and/or oropharyngeal (op) swabs, lower respiratory tract aspirates, sputum, bronchoalveolar lavage (bal) fluid and nasopharyngeal wash/aspirate or nasal aspirate. it was observed that samples of the lower respiratory tract provide the higher viral loads [74] . on the other hand, it was shown that in the early stage of infection, the positive rate of rt-pcr was reported to be about 60% for throat swab samples [125] . indeed, although being the gold standard, the rt-pcr presents some drawbacks. one of the most important is related to the sensibility because it was extensively reported that in the presence of low viral load this technique fails in detecting viral genome leading to false negative results [126] . due to this problem, clinical governance as well as kit troubleshooting indicate to retest all the samples showing only single positive target along with patient resampling. to this respect, it should be underlined that operator skills or sampling sources can profoundly affect rt-pcr testing results [22] . finally, during this pandemic several microbiologic labs worldwide are experiencing scarce availability of rna extraction as well as ready-to-use rt-pcr kits increasing the timing of diagnosis confirmation through molecular approaches. very recently, it was reported that the allplex 2019-ncov and the realstar sars-cov-2 rt-pcr kits can amplify the target genes bypassing the rna extraction step for a faster diagnosis [127] . although rt-pcr is specific for the diagnosis of covid-19, its false-negative rate cannot be overlooked due to the severe consequences of missed diagnosis. clinicians have demonstrated the usefulness of ct and chest radiography for the diagnosis of covid-19 associated pneumonia [128] . moreover, the ability of radiologists to diagnose covid-19 pneumonia from chest ct evaluations has been reported to be very high [129] . then a combination between rt-pcr and ct imaging represents the best approach for the correct covid-19 diagnosis. in particular, for early detection and assessment of disease severity, the high-resolution ct (hrct) of the chest is considered necessary [130, 131] . one study analyzed the consistency and diagnostic value of rt-pcr test compared with chest ct in 1014 patients with suspected sars-cov-2 infection. findings indicated that the chest ct sensitivity in suspected patients was 75% based on negative rt-pcr results and 97% based on positive rt-pcr results [132] . moreover, salehi et al. confirmed the higher sensibility of pulmonary imaging with respect to rt-pcr for covid-19 diagnosis and showed a positive correlation between specific ct findings with the different stages of the disease and its severity [116] . the collection of numerous ct images has opened the possibility to build a database of pulmonary images from covid-19 patients. interestingly, the recent progress in integrating artificial intelligence (ai) with computer-aided design (cad) software for diagnostic imaging revealed that ai could be used to support disease diagnosis [133, 134] . ito et al. reviewed the literature on the use of ai for lung diagnostic imaging of covid-19 patients. among the 15 selected studies, 11 used ai for ct and 4 used ai for chest radiography. the number of datasets ranged from 106 to 5941, with sensitivities ranging from 67-100% and specificities ranging from 81-100% for prediction of covid-19 pneumonia. this study revealed the usefulness of ai approach to support the diagnosis of covid-19, but also for future emerging diseases [134] . all the collected knowledge on lung lesions revealed some characteristic ct findings of covid-19 pneumonia: the pulmonary ground-glass opacities in a peripheral distribution and the consolidation referring to an increase in pulmonary parenchymal density [135] [136] [137] . however, chest ct manifestations can vary in different patients and stages of infection, highlighting certain shortcomings of this approach. apart from atypical manifestation that cannot be recognized by radiologists, several lung images are common in viral pneumonia leading to misdiagnosis [138] . soon after the beginning of sars-cov-2 spreading, infected patients underwent antibody research for both basic research and clinical applications. one of the first studies reported the seroconversion of 100% of infected patients (n = 285) within 19 days after symptom onset. seroconversion for igm and igg occurred simultaneously or sequentially and both immunoglobulins titers plateaued within 6 days after seroconversion. importantly, the application of serology testing in surveillance in a cluster of 164 close contacts of covid-19 patients identified 4.6% of positive patients showing negative rt-pcr results [139] . hence, several studies underlined the recommended usage of serology to promote the detection of sars-cov-2 infections where np swab specimens were improperly collected, molecular assays were unsatisfactorily carried out and for determining asymptomatic infections [122] . based on these data, several companies developed kits for igm/igg testing showing a high detection rate of infected patients. basically, there are two different testing methods: the rapid igg-igm test and the classical enzyme-linked immunosorbent assay (elisa)-based test. the rapid test consists in a lateral flow qualitative immunoassay on a strip to detect the presence of both anti-sars-cov-2-igm and anti-sars-cov-2-igg in human specimens such as whole blood, serum and plasma. this igg-and igm-combined antibody test kit has a sensitivity of 88.66% and specificity of 90.63%. results are obtained in 15 min leading to its useful application as point-of-care testing and in supporting rt-pcr-based diagnostic [140] . on the other hand, several elisa-based kits are now commercially available, and their sensitivity and specificity were compared showing an overall high specificity, but a variable sensibility [141] . differently from the rapid tests, the elisa-based test should be performed on serum or plasma samples collected from venous sampling. interestingly, the authors showed the neutralizing capacity of sars-cov-2 specific antibodies on caco-2 cells directly incubating the sera from patients with the cell monolayers [141] . this assay is extremely important for the plasma-based therapies that are successfully used to treat seriously ill patients (see below). finally, recently published papers described the seroconversion of covid-19 patients including the evaluation of iga that seems high in the early stages of infection (about 4 days' post symptom development) [142, 143] . another interesting application of antibody detection is represented by the fluorescence immuno-chromatographic assay for the detection of sars-cov-2 nucleocapsid protein in human specimens such as np swab [144] . it shows the fastness of rapid tests (results in 10 min), the possibility to use the same type of sample that is commonly used for rt-pcr-based diagnosis and high sensibility (detection of the nucleoprotein in all positive samples tested). although these methods were suggested for covid-19 diagnosis, the extent of antibodies production by infected patients is greatly variable. moreover, the delay of antibodies production with respect to the onset of symptoms affects the use of this approach for diagnosis. vice versa, it is reported that several governments, included italy, are using serologic test for population screening to assess the proportion of people that have developed an immunological response against sars-cov-2 (http://www.salute.gov.it/portale/nuovocoronavirus). this screening will help also to detect asymptomatic and/or paucisymptomatic subjects. the rapid spread of sars-cov-2 raises an urgent requirement for effective therapeutic strategies against covid-19. although many efforts have been intended to develop vaccines against hcovs infections in recent years, there is no official and effective treatment against sars-cov-2. however, different considerable options have been applied for possible vaccine validity, efficacy and safety along with speeding up other ongoing searches to discover valuable modalities for dealing with the emerging covid-19 [12, [145] [146] [147] [148] . most of the drugs that are being used to cope with covid-19 epidemic are directed towards specific viral molecular targets and biologic processes through which the virus spreads damaging the host. in line, all available experimental therapies for covid-19 management are based on previous experiences in treating sars-cov and mers-cov infections, such as inhibitors of sars-cov-2 fusion/entry/replication, anti-viral agents against main viral proteases, regulators of sars-cov-2 induced host inflammatory response and direct administration of human monoclonal antibodies (mabs) (figure 8 ) [149] . apart from all these possible therapeutic approaches, it has been reported that the chinese medicine products, as lianhuaqingwen and shufeng jiedu capsules may be helpful for sars-cov-2 treatment [12, 150] . indeed, this product is mainly used to treat upper respiratory tract infections such as the flu, swelling and pain in the throat, mumps and strep throat [151, 152] . moreover, four covid-19 cases have been described to gain improvement after taking combined chinese and western medicine [153] . notably, encouraging progress in deciphering sars-cov-2 genome will lead to new potential therapeutic targets. likewise, more prospective, rigorous population studies are urgently required to confirm the therapeutic effect as well as the safety of new potential therapeutic strategies in order to further implement robust preventive and control measures against sars-cov-2 spread. as outlined above, multiple strategies are aimed at developing covs vaccines, most of which are headed for the surface-exposed spike (s protein) glycoprotein as the major virus-host cell membrane interactor. to this aim, vaccines under study are based on full-length s protein, s1-rbd, expression of virus-like particles (vlp), dna or viral vectors [42, [154] [155] [156] [157] [158] . as outlined above, the s1 includes the rbd that interacts with its host cell receptor, ace2, whereas the s2 mediates fusion between the virus and host cell membranes promoting the entry and subsequent replication of the viral rna into the cytoplasm [158] . the ace2 receptor, as a specific biologic target for vaccine development, is under study in a controlled pilot clinical trial to investigate the effect of recombinant human ace2 (rhace2; gsk2586881) in patients with severe covid-19 (nct04287686) ( figure 8i ) [159, 160] . vice versa, both recombinant proteins containing rbd and the recombinant vectors encoding rbd can be used to generate the effective sars-cov vaccines given the capability of this domain to induce neutralizing antibody [156] . indeed, the first available sars-cov-specific human monoclonal antibody with neutralizing activity against sars-cov, named cr3022, was found to bind potently to sars-cov-2 rbd, in agreement with the high homology shared by this domain with sars-cov homolog [161] . however, it must be taken into account that more than 85% of the rbd antibody epitopes in sars-cov-2 show implicit noticeable changes, indicating the necessity to develop more specific monoclonal antibodies for sars-cov-2 [162] . angiotensin receptor blockers (arbs), such as losartan, valsartan, telmisartan, usually assumed for treating high blood pressure, heart and kidney failure in people with diabetes, have been recently proposed as a novel therapeutic approach to block sars-cov-2 rbd binding to ace2-expressing cells binding, similarly to ace inhibitors [163] . additional targetable epitopes that should be considered are the heptad repeat 1 (hr1) and heptad repeat 2 (hr2) in sars-cov-2 s protein. in fact, the hr2-derived peptides (hr2p) and ek1 (a modified oc43-hr2p peptide), exhibit effective fusion inhibitory activity towards sars-cov-2, suggesting a promising strategy in treating sars-cov-2 infection, although further studies are required to strengthen these hypotheses ( figure 8i ) [164, 165] . lately, immuno-informatics have been employed to identify significant cytotoxic t lymphocyte (ctl) and b-cell epitopes in sars-cov-2 s protein, such as the nucleocapsid (n) protein as well as the potential b cell epitopes of the e protein of mers-cov as likely immunoprotective targets [166, 167] . reverse genetic strategies have been successfully used in live-attenuated vaccines to inactivate the exonuclease effects of non-structural protein 14 (nsp14) or to wipe out the envelope protein in sars [154] . a recent study also revealed that the invasion process requires the priming of the s protein which is facilitated by the host cell produced serine protease tmprss211. the clinically demonstrated serine protease tmprss2 inhibitor camostat mesylate, which partially blocks sars-cov-2 entry into host cells, was shown to be a good target to significantly reduce pulmonary infection in covid-19 affected individuals ( figure 8i ) [168] moreover, it has been suggested that coronavirus entry also involves ph and receptor-dependent endocytosis [169, 170] ; thus, targeting endocytosis may be another assessable option for fighting sars-cov-2 ( figure 8i ). in this view, throughout ai technology, a group of approved drugs, such as the janus kinase (jak) inhibitor baricitinib [171] targeting the ap-2-associated protein kinase 1 (aak1) regulating clathrin-mediated endocytosis, has been developed ( figure 8i ) [172] . furthermore, other drugs such as arbidol (chictr2000029621), a haemagglutinin inhibitor and chloroquine phosphate, a traditional antimalarial drug, have been added to the national health commission of the people's republic of china (nhc) guidelines for covid-19 treatment ( figure 8i ) (http://www.nhc.gov.cn). in particular, in vitro studies have demonstrated that chloroquine as well as hydroxychloroquine could impair the endosome-mediated viral entry or later stages of viral replication [173] . combination of hydroxychloroquine and azithromycin has also been suggested as a valid approach since it showed more rapid resolution of infection than hydroxychloroquine alone [174] ; however, the combined use of azithromycin and hydroxychloroquine seems to be associated with at increased risk of arrhythmias. available online: https://www.acc.org/latest-in-cardiology/articles/2020/03/27/14/00/ventricular-arrhythmia-riskdue-to-hydroxychloroquine-azithromycin-treatment-for-covid-19 (accessed on 29 march 2020). to date, several attempts have also been made in targeting viral main enzymes; in fact, many inhibitory drugs targeting the coronavirus main proteinase 3c-like protease (3clpro) have been validated in clinical trials (e.g., lopinavir/ritonavir; chictr2000029387, chictr2000029468, chictr2000029539) ( figure 8ii ) [175] . moreover, four additional molecules including prulifloxacin, tegobuvir, bictegravir and nelfinavir, detected by high-throughput screening, showed reasonable binding conformations with the viral main protease [176] . moreover, a recent study by performing a virtual screening using a three-dimensional model of the sars-cov-2 3c-like protease (3cl), identified 16 biologic candidates that deserve further consideration. among these, the antivirals ledipasvir or velpatasvir proved to be particularly attracting as therapeutics to combat the new coronavirus showing optimal anti-viral activity and minimal side effects, such as fatigue and headache; also, epclusa (velpatasvir/sofosbuvir) and harvoni (ledipasvir/sofosbuvir) are promising antivirals, not only for their effective and synergic inhibitory activities against two viral enzymes, but also for their minimized possibilities to develop resistance [177] . a certain number of clinical trials on antiviral drugs aimed to arrest sars-cov-2 replication are currently in progress, such as remdesivir (nct04252664, nct04257656) favipiravir (chictr2000029600, chictr2000029544) and asc09 (chictr2000029603) ( figure 8iii ). among these, remdesivir was recently approved for medical use in america and european union and seems to be the most promising antiviral for fighting sars-cov-2 [178] (http://www.who.int), as in vitro studies demonstrated that this molecule, a mono-phosphoramidate prodrug of an adenosine, effectively inhibited sars-cov-2 rna synthesis [179] . targeting the sars-cov-2 rna genome could, therefore, be another potential strategy. in fact, a crispr/cas13d technology, which is an rna-guided rna-targeting crispr system, has been employed to specifically chew up sars-cov-2 rna genome. in this system, a cas13d protein and guide rnas-containing spacer sequences are used to specifically complement the virus rna genome ( figure 8iv ). furthermore, rna genome can be packaged into one adeno-associated virus (aav) vector, making the crispr/cas13d system more efficient for virus elimination and resistance prevention, taking into account that aav has serotypes highly specific to the lung, the main organ infected by sars-cov-2 [180] . in addition to antiviral therapy, a new treatment strategy having a significant impact on clinical outcomes is utmost required. immunomodulatory therapy to downregulate the cytokine storm may provide great benefit to the treatment of covid-19. recently, researchers focused on targeting specific molecular markers involved in inflammatory cytokines-receptors interactions, their correlation in health and disease and drugs in use that can activate or block their actions. a higher concentration of cytokines has been found in the plasma from covid-19 patients in icu compared with the ones from non-icu covid-19 patients, suggesting that the cytokine storm could be linked to the severity of the disease [113] . corticosteroids are among the most commonly used drugs for immunomodulatory therapy of infectious diseases. however, the use of corticosteroids in the treatment of covid-19 can cause host immune suppression and delay of viral clearance. a recent study on 201 patients with ards showed that treatment with methylprednisolone decreased the risk of death (hazard ratio 0.38, 95% confidence interval 0.20-0.72). these findings indicate that using corticosteroids does not influence viral clearance time, length of hospital stays or duration of symptoms in patients with mild covid-19 [181] . thus, the use of corticosteroids is considered beneficial in severe cases of covid-19 (especially in patients with ards), but not in mild cases. accordingly, a recent retrospective study showed the potential benefits from low-dose corticosteroids treatment in a subset of critically sars-cov-2 patients [182] ; these data are in contrast with nhc guidelines that highlight that systematic use of corticosteroids is not recommended for these cases, due to their immunosuppressive effects. however, administration of corticosteroids has been indicated for specific reasons such as exacerbation of asthma or chronic obstructive pulmonary disease (copd), septic shock or severe acute respiratory distress syndrome (ards). further studies are required to find out how and when it is appropriate the use of corticosteroids for covid-19, as there are no available data on the benefits of corticosteroid treatment in sars-cov or mers infection [183] . apart from corticosteroids, il-6 pathway inhibitors such as sarilumab, siltuximab and tocilizumab have been proposed as experimental approach considering the increased il-6 levels that have been observed in patients with severe covid-19 [184] . tocilizumab is a recombinant, humanized monoclonal antibody commonly used for treating patients with rheumatoid arthritis, lupus and psoriasis that binds to il-6 receptors blocking fcr activation; in covid-19 patients, tocilizumab could reduce sars-cov-2-induced inflammatory responses [185] . accordingly, several case reports have referred positive outcomes regarding tocilizumab [113, [186] [187] [188] [189] [190] , but clinical impact of tocilizumab on covid-19 patients as an approved clinical approach has not been evaluated yet. in line, to further investigate the efficacy and safety of tocilizumab in patients with covid-19, a controlled clinical trial is now under way (chictr2000029765) ( figure 8v ). overall, the combination of an immunomodulatory agent to reduce the cytokine storm with an antiviral agent may give physicians more time to provide supportive treatment for patients with covid-19. at the time of writing this review, due to the lack of a specific available therapy, plasma from convalescent patients containing specific antibodies has been proposed as a principal treatment [190, 191] , for patients in rapid disease progression, severe or critical conditions ( figure 8vi ). in a recent retrospective study, one dose (200 ml) of convalescent plasma (cp) collected from 10 severe adult cases has been reported to be tolerated; thus, increasing or maintaining high level of neutralizing antibodies broke down the viral load in seven days, improve clinical symptoms and paraclinical criteria within three days and lung lesions were found to be differently absorbed on radiological examination within seven days [192] . therefore, being cp a promising rescue option for severe covid-19, several clinical trials (chictr2000030010, chictr2000030179, and chictr2000030381) are in progress to investigate the efficacy and safeness of cp direct infusion in covid-19 patients [191] . in addition, combined therapy with mabs and remdesivir seems to be an ideal therapeutic option for covid-19 [193] . pharmaceuticals companies are now focused on searching for specific and effective mabs against covid-19. taking into account that technologies capable of making fully human antibodies such as human single-chain antibody variable fragments (hu-scfvs) or humanized-nanobodies (single-domain antibodies, sdab) able to overpass virus-infected cell membranes (trans bodies) and to interact or interpose with biologic processes required for virus replication are already available [194] . a large number of clinical trials regarding cell-based therapies have been started in china during covid-19 outbreak. among these, mesenchymal stromal cells (mscs)-based therapy displayed strong safety profile and possible efficacy in patients with ards, according to covid-19-related clinical studies listed on the who's international clinical trials registry platform (who ictrp) and national institutes of health's clinical trials.gov databases [195] . nevertheless, further investigations are required to better understand if these therapies could be effective in treating respiratory virus-induced complications. mscs have been largely employed in basic research and clinical trials [196] [197] [198] , and their safeness and effectiveness have been extensively documented especially in immune-mediated inflammatory disorders, such as graft-versus-host disease (gvhd) [199] and systemic lupus erythematosus (sle) [200] . mscs immunomodulatory and differentiation abilities [201] as well as their competency to produce several cytokine types or to directly interact with immune cells have been already described [202] . indeed, they are activated by pathogen-associated molecules (pamps) such as single or double-stranded rnas [203, 204] , priming the immune response during infections. two clinical investigations of systemic msc administration in patients with either covid-19 or avian influenza a (h7n9) have been recently published [205, 206] . the first one, a single-center msc transplantation pilot study, was aimed at exploring mscs therapeutic potentiality in patients with covid-19 pneumonia and conducted at the you'an hospital in beijing, china, from 23 jan 2020 to 16 feb 2020 (chictr2000029990). seven patients with covid-19 pneumonia, sars-cov-2 rna positive, with different degrees of severity, including one critically ill requiring icu care were enrolled and monitored for 14 days after msc injection. a significant improvement of pulmonary function and symptoms were observed two days after msc transplantation characterized by an increase of peripheral lymphocytes and of the anti-inflammatory il-10 levels and a decrease of the c-reactive protein and tnf-α amounts [205] . moreover, an increment of the cd14 + cd11c + cd11b mid regulatory dendritic cell (dc) population and a decrease of cytokine-secreting immune cells such as cxcr3 + cd4 + t cells, cxcr3 + cd8 + t cells, and cxcr3 + nk were detected within 3-6 days in the treated patients compared to the placebo control group [205] . mscs play a role in attenuating cytokine storm, most importantly, because these cells do not express ace2 and tmprss2 viral receptors are insusceptible of sars-cov-2 infection. these observations are in agreement with the knowledge that mscs induce the maturation of dendritic cells into a novel jagged-2-dependent regulatory dendritic cell population [207] , shifting the th1/th2 balance towards th2. thus, from these preliminary results, it seems evident that mscs intravenous transplantation could represent a secure and effective treatment in patients with covid-19 pneumonia, especially those critical. indeed, it inhibits the over activation of the immune system and promotes endogenous repair by preventing pulmonary fibrosis and improving both pulmonary microenvironment and lung function [205] . more than 15 potential vaccine candidates for covid-19 are under development around the world, including inactivated, recombinant subunits, nucleic-acid-based, adenoviral vector, and recombinant influenza viral vector vaccines [208] . moreover, taking into consideration the strong homologies existing among the various coronavirus strains, it was thought that vaccines acting on other coronaviruses, such the avian live ibv vaccine (strain h) directed towards the chicken cov ibv, could be a valuable alternative therapeutic strategy [209] . the coalition for epidemic preparedness innovations (cepi) recently announced that three programs aimed to develop covid-19 vaccines, by utilizing established vaccine platforms, have started [210] . in addition, cepi already financed the company moderna, inc. to compare mrna therapeutics and vaccines, allowing the release of the first batch of mrna-1273 in february 2020, which is an mrna vaccine against sars-cov-2 ready for phase i study in the united states. available online: https://investors.modernatx.com/news-releases/news-release-details/moderna-ships-mrnavaccine-against-novel-coronavirus-mrna-1273 (accessed on 24 february 2020). more recently, scientists from the university of pittsburgh have announced a potential vaccine against sars-cov-2, delivered throughout a fingertip-sized patch, capable of producing sars-cov-2 specific igg antibodies, sufficient for virus neutralization in mice. this vaccine, called pittcovacc (acronym of pittsburgh coronavirus vaccine), is a trimeric recombinant sars-cov-2-s1 subunit vaccine delivered intracutaneously by microneedle arrays (mnas) [211] . delivering vaccine components to a defined skin microenvironment improves safety by reducing systemic exposure, allowing to reach high vaccine concentrations with a relatively low dose of antigen [212, 213] . furthermore, the skin delivery strategy promotes strong and long-lasting antigen-specific antibody responses due to both the high immunogenicity [214] [215] [216] [217] [218] and the redundant immunoregulatory circuits of the skin [217, 219, 220] . given the urgent need for covid-19 vaccines, mnas strategy seems to be a promising immunization approach against coronavirus infection including sars, mers and other emerging infectious diseases. on april 24, the oxford chadox1 ncov-19 vaccine was the first in europe to start human trial stage, with 1110 healthy volunteers enrolled for the tests. oxford scientists have already employed chadox1 in the past to dispense vaccines against ebola, chikungunya, rift valley fever and, above all, mers. chadox1, a chimpanzee-derived adenovirus vector, has been employed to deliver the full-length mers spike gene and shown to induce large amounts of neutralizing antibodies against mers in a mouse model [221, 222] . therefore, the modified chadox1 vaccine, carrying the sars-cov-2 spike gene is under human trial stage. on april 30, the university of oxford has announced a collaboration with the uk-based global biopharmaceutical company astrazeneca for further development, large-scale production and potential delivery of the covid-19 vaccine candidate. available online: https://www. ovg.ox.ac.uk/news/landmark-partnership-announced-for-development-of-covid-19-vaccine (accessed on 30 april 2020). since chadox technology is already available and formerly tested in humans for other vaccines, phase iii will consist in administering vaccine to volunteers following them into their regular environments to ensure that these subjects actually become immune to the disease up to three years. if trials succeed, oxford researchers have proposed to complete testing throughout ring vaccination, namely delivering vaccine to members of the first circle of contacts of covid-19 positive people and then to evaluate if the virus spreads to the second circle, as was previously done during the 2018 ebola epidemic in the democratic republic of the congo. to be a promising immunization approach against coronavirus infection including sars, mers and other emerging infectious diseases. on april 24, the oxford chadox1 ncov-19 vaccine was the first in europe to start human trial stage, with 1110 healthy volunteers enrolled for the tests. oxford scientists have already employed chadox1 in the past to dispense vaccines against ebola, chikungunya, rift valley fever and, above all, mers. chadox1, a chimpanzee-derived adenovirus vector, has been employed to deliver the full-length mers spike gene and shown to induce large amounts of neutralizing antibodies against mers in a mouse model [221, 222] . therefore, the modified chadox1 vaccine, carrying the sars-cov-2 spike gene is under human trial stage. on april 30, the university of oxford has announced a collaboration with the uk-based global biopharmaceutical company astrazeneca for further development, large-scale production and potential delivery of the covid-19 vaccine candidate. available online: https://www.ovg.ox.ac.uk/news/landmark-partnership-announced-fordevelopment-of-covid-19-vaccine (accessed on 30 april 2020). since chadox technology is already available and formerly tested in humans for other vaccines, phase iii will consist in administering vaccine to volunteers following them into their regular environments to ensure that these subjects actually become immune to the disease up to three years. if trials succeed, oxford researchers have proposed to complete testing throughout ring vaccination, namely delivering vaccine to members of the first circle of contacts of covid-19 positive people and then to evaluate if the virus spreads to the second circle, as was previously done during the 2018 ebola epidemic in the democratic republic of the congo. figure 8 . schematic representation of sars-cov-2 infection and virus-induced human immune system response. proposed drugs directed both towards specific sars-cov-2 molecular targets and biologic processes are highlighted: inhibitors of sars-cov-2 fusion/entry targeting ace2 receptor, spike protein, tmprss2 or hr1 and hr2 epitopes and clathrin-mediated endocytosis (i); molecules against sars-cov-2 main protease (ii); molecules against viral genome replication (iii); crispr figure 8 . schematic representation of sars-cov-2 infection and virus-induced human immune system response. proposed drugs directed both towards specific sars-cov-2 molecular targets and biologic processes are highlighted: inhibitors of sars-cov-2 fusion/entry targeting ace2 receptor, spike protein, tmprss2 or hr1 and hr2 epitopes and clathrin-mediated endocytosis (i); molecules against sars-cov-2 main protease (ii); molecules against viral genome replication (iii); crispr technologies targeting sars-cov-2 rna genome (iv); modulators of sars-cov-2 induced inflammatory response (v) and human neutralizing antibodies (vi). ace2, angiotensin-converting enzyme 2; tmprss2, type 2 transmembrane serine proteases; rdrp, rna-dependent rna polymerase; hr1, heptad repeat 1; hr2, heptad repeat 2; hr2p, heptad repeat 2-derived peptides; ek1, a modified oc43-hr2p peptide. adapted from [223] . overall, a joint effort headed to apply both already consolidate and innovative approaches, such as ai to facilitate drug discovery, will be required to develop a broad-spectrum antiviral drugs and vaccines towards existing and potential future coronavirus infections to prevent another highly pathogenic virus epidemic. moreover, continuous collaboration in basic and clinical studies will improve the discovery of new antiviral drugs with therapeutic potentials, decrease the time for drug release on the market and make them affordable for all countries. furthermore, vaccine delivery strategies and cell-based therapies benefit from the significant progresses made by recombinant dna technologies combined with emerging biotechnology and bioengineering methodologies. thus, these approaches can speed up the development and set up of new vaccines and clinical therapies to fight against novel pathogens to protect public health all over the world. this study represents a holistic picture of the current investigations in response to the outbreak of covid-19. the current pandemic is obviously an international public health problem and it remains a challenging task to fight the sars-cov-2 of unknown origin and mysterious biologic features. lesson from the previous two pandemics, mers and sars outbreaks, provide valuable insights about how to manage the current pandemic and provide a reference for future studies to combat disease progression. despite sars-cov-2 rapid transmission, the scale up country readiness, speedy response teams and the capacity of all laboratories are reducing the spread of the virus as well as its mortality rate. as the pandemic is still ongoing and expanding, further studies on all aspects of the disease are needed to better understand the infection, beneficial treatments and development of vaccines. nevertheless, this pandemic, together with the previous ones, have taught us in the harshest possible way that the entire scientific community must be vigilant and ready to advice appropriate containment and screening measures to avoid the spread of any future emerging pathogen. we would like to acknowledge majidi nezhad for providing the meteorological data and climate analyses and gaia scoarughi and adeleh salehi for drawing figures. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: 16 nonstructural polyproteins (nsps 1-16); 2019 novel coronavirus (2019-ncov); 3-chymotrypsin-like protease (3clpro); 3c-like protease (3cl); acute respiratory distress syndrome (ards); adeno-associated virus (aav); angiotensin receptor blockers (arbs); angiotensin-converting enzyme 2 (ace2); ap-2-associated protein kinase 1 (aak1); artificial intelligence (ai); avian infectious bronchitis virus (ibv); basic reproduction number (r0); blood oxygen saturation (spo2); bronchoalveolar lavage (bal); centers for disease control and prevention (cdc); chinese center for disease control and prevention (china cdc); chronic obstructive pulmonary disease (copd); coalition for epidemic preparedness innovations (cepi); computed tomography (ct); computer-aided design (cad); convalescent plasma (cp); coronavirus (cov); cytotoxic t lymphocyte (ctl); dendritic cell (dc); dipeptidyl peptidase 4 (dpp4, also known as cd26); envelope glycoprotein (e); enzyme-linked immunosorbent assay (elisa); european center for medium-range weather forecasts (ecmwf); european cities (ecs); feline infectious peritonitis (fip); graft versus-host disease (gvhd); hemagglutinin-esterase glycoprotein (he); heptad repeat 1 (hr1) and heptad repeat 2 (hr2); high-resolution ct (hrct); hr2-derived peptides (hr2p); ek1 (a modified oc43-hr2p peptide); human coronaviruses (hcovs); human covs (hcovs); human single-chain antibody variable fragments (hu-scfvs); humanized-nanobodies (single-domain antibodies, sdab); intensive care unit (icu); istituto superiore di sanità (iss); janus kinase (jak); membrane glycoprotein (m); mesenchymal stromal cells (mscs); microneedle arrays (mnas); middle east respiratory syndrome coronavirus (mers-cov); monoclonal antibodies (mabs); naso-pharyngeal (np); national health commission of the people's republic of china (nhc); nonstructural protein 14 (nsp14); northern hemisphere (nh); nucleocapsid phosphoprotein (n); open reading frames (orfs); oro-pharyngeal (op); papain-like protease (plpro); partial pressure of arterial oxygen to fraction of inspired oxygen ratio (pao2/fio2); pathogen-associated molecules (pamps); pittsburgh coronavirus vaccine (pittcovacc); polypeptides (pps); porcine epidemic diarrhea virus (pedv); real time quantitative polymerase chain reaction (rt-qpcr); receptor-binding domain (rbd); recombinant human ace2 (rhace2); replication-transcription complex (rtc); rna-dependent rna polymerase (rdrp); rnase p gene (rp); severe acute respiratory syndrome coronavirus (sars-cov); single-stranded positive-sense rna (+ssrna); southern hemisphere (sh); spike glycoprotein (s); subgenomic rnas (sgrnas); swine acute diarrhea syndrome coronavirus (sads-cov); swine enteric alphacoronaviruses (seacovs); systemic lupus erythematosus (sle); transmissible gastroenteritis virus (tgev); virus-like particles (vlp); who's international clinical trials registry platform (who ictrp); world health organization (who). the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns coronaviruses-drug discovery and therapeutic options emerging coronaviruses: genome structure, replication, and pathogenesis human coronavirus: host-pathogen interaction covid-19): a review of clinical features, diagnosis, and treatment cultivation of a novel type of common-cold virus in organ cultures coronavirus genomics and bioinformatics analysis evaluation of risks and benefits associated with vaccination against coronavirus infections in cats identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 an overview of their replication and pathogenesis insights into the recent 2019 novel coronavirus (sars-cov-2) in light of past human coronavirus outbreaks discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus origin and evolution of pathogenic coronaviruses genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding coronavirus as a possible cause of severe acute respiratory syndrome sars and other coronaviruses as causes of pneumonia estimating the potential total number of novel coronavirus cases in wuhan city molecular evolution of human coronavirus genomes a review of coronavirus disease-2019 (covid-19) genetic recombination, and pathogenesis of coronaviruses coronaviruses in avian species-review with focus on epidemiology and diagnosis in wild birds probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak coronavirus disease 2019: what we know? sars-cov-2 in fruit bats, ferrets, pigs, and chickens: an experimental transmission study the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan identification of a new human coronavirus identification of new human coronaviruses a structural view of sars-cov-2 rna replication machinery: rna synthesis, proofreading and final capping accessory proteins of sars-cov and other coronaviruses from sars to mers, thrusting coronaviruses into the spotlight the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins the molecular biology of coronaviruses identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles the spike protein of sars-cov-a target for vaccine and therapeutic development the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage structure, function and evolution of the hemagglutinin-esterase proteins of corona-and toroviruses identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza c virus and bovine coronavirus coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol genome composition and divergence of the novel coronavirus (2019-ncov) originating in china covid-19 infection: origin, transmission, and characteristics of human coronaviruses receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus cryo-em structure of the 2019-ncov spike in the prefusion conformation structural and functional basis of sars-cov-2 entry by using human ace2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry identification and characterization of novel neutralizing epitopes in the receptor-binding domain of sars-cov spike protein: revealing the critical antigenic determinants in inactivated sars-cov vaccine characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72,314 cases from the chinese center for disease control and prevention dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc interaction of sars and mers coronaviruses with the antiviral interferon response novel coronavirus (covid-19) outbreak: a review of the current literature a new bat-hku2-like coronavirus in swine discovery of a novel swine enteric alphacoronavirus (seacov) in southern china bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond sars-associated coronavirus transmitted from human to pig a bat-derived putative cross-family recombinant coronavirus with a reovirus gene phylogenetic network analysis of sars-cov-2 genomes receptor and viral determinants of sars-coronavirus adaptation to human ace2 ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia role of changes in sars-cov-2 spike protein in the interaction with the human ace2 receptor: an in silico analysis structural basis of receptor recognition by sars-cov-2 covid-19 infection and circulating ace2 levels: protective role in women and children. front contact tracing: a game of big numbers in the time of covid-19 sars-cov-2 viral load in upper respiratory specimens of infected patients covid-19, sars and mers: are they closely related? evaluation and treatment coronavirus (covid-19) the transmission and diagnosis of 2019 novel coronavirus infection disease (covid-19): a chinese perspective the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? 2019-ncov transmission through the ocular surface must not be ignored special expert group for control of the epidemic of novel coronavirus pneumonia of the chinese preventive medicine association. an update on the epidemiological characteristics of novel coronavirus pneumonia (covid-19) clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records novel coronavirus infection in hospitalized infants under 1 year of age in china human coronaviruses: a review of virus-host interactions animal models for sars and mers coronaviruses crossref] 89. national health commission of the people's republic of china. pneumonia epidemic situation of new coronavirus infection on 23 pneumonia of unknown aetiology in wuhan, china: potential for international spread via commercial air travel review of early and emerging options. open forum infect. dis. 2020 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study novel coronavirus: where we are and what we know modeling the spread of middle east respiratory syndrome coronavirus in saudi arabia transmission dynamics and control of severe acute respiratory syndrome seasonality of respiratory viral infections assessment of china's offshore wind resources based on the integration of multiple satellite data and meteorological data high temperature and high humidity reduce the transmission of covid-19 temperature significant change covid-19 transmission in 429 cities preliminary evidence that higher temperatures are associated with lower incidence of covid-19, for cases reported globally up to 29 effects of temperature and humidity on the daily new cases and new deaths of covid-19 in 166 countries distribution of the sars-cov-2 pandemic and its monthly forecast based on seasonal climate patterns the transmission of sars-cov-2 is likely comodulated by temperature and by relative humidity wind energy potential analysis using sentinel-1 satellite: a review and a case study on mediterranean islands unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in covid-19 (novel coronavirus 2019) -recent trends presumed asymptomatic carrier transmission of covid-19 updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan return of the coronavirus: 2019-ncov. viruses importation and human-to-human transmission of a novel coronavirus in vietnam clinical features of patients infected with 2019 novel coronavirus in coronavirus disease 2019: initial chest ct findings ct imaging of the 2019 novel coronavirus (2019-ncov) pneumonia. radiology coronavirus disease 2019 (covid-19): a systematic review of imaging findings in 919 patients abdominal and testicular pain: an atypical presentation of covid-19 atypical presentation of covid-19 in a frail older person diagnosis and clinical management of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: an operatioal recommendation of peking union medical college hospital (v2.0) a pneumonia outbreak associated with a new coronavirus of probable bat origin molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes molecular diagnosis of a novel coronavirus (2019-ncov) causing an outbreak of pneumonia sars-cov-2 foch hospital study group. the allplex 2019-ncov (seegene) assay: which performances are for sars-cov-2 infection diagnosis? evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of 2019-ncov infections emergence of a novel coronavirus disease (covid-19) and the importance of diagnostic testing: why partnership between clinical laboratories, public health agencies, and industry is essential to control the outbreak sas-cov-2 detection by direct rrt-pcr without rna extraction radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study imaging of coronavirus disease 2019: a chinese expert consensus statement time course of lung changes at chest ct during recovery from coronavirus disease 2019 (covid-19) initial ct findings and temporal changes in patients with the novel coronavirus pneumonia (2019-ncov): a study of 63 patients in wuhan, china correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases artificial intelligence applications for thoracic imaging a review on the use of artificial intelligence for medical imaging of the lungs of patients with coronavirus disease 2019 radiological role in the detection, diagnosis and monitoring for the coronavirus disease 2019 (covid-19) ct imaging changes of corona virus disease 2019 (covid-19): a multi-center study in southwest china ct imaging features of 2019 novel coronavirus (2019-ncov) ct differential diagnosis of covid-19 and non-covid-19 in symptomatic suspects: a practical scoring method antibody responses to sars-cov-2 in patients with covid-19 development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis brief clinical evaluation of six high-throughput sars-cov-2 igg antibody assays profiling early humoral response to diagnose novel coronavirus disease (covid-19) serum iga, igm, and igg responses in covid-19 diagnosis of acute respiratory syndrome coronavirus 2 infection by detection of nucleocapsid protein recent discovery and development of inhibitors targeting coronaviruses this scientist hopes to test coronavirus drugs on animals in locked-down wuhan emerging novel coronavirus (2019-ncov)-current scenario, evolutionary perspective based on genome analysis and recent developments design, synthesis and molecular docking of novel triazole derivatives as potential cov helicase inhibitors updated approaches against sars-cov-2 traditional chinese medicine for covid-19 treatment complementary and alternative medicine is expected to make greater contribution in controlling the prevalence of influenza application of in vitro bionic digestion and biomembrane extraction for metal speciation analysis, bioavailability and risk assessment in lianhua qingwen capsule clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment a decade after sars: strategies for controlling emerging coronaviruses a dna vaccine induces sars coronavirus neutralization and protective immunity in mice cross-species transmission of the newly identified coronavirus 2019-ncov towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the mers-coronavirus spike glycoprotein angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov angiotensin receptor blockers as tentative sars-cov-2 therapeutics fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion epitope-based vaccine target screening against highly pathogenic mers-cov: an in silico approach applied to emerging infectious diseases analysis of the genome sequence and prediction of b-cell epitopes of the envelope protein of middle east respiratory syndrome-coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway baricitinib as potential treatment for 2019-ncov acute respiratory disease ap-2-associated protein kinase 1 and cyclin g-associated kinase regulate hepatitis c virus entry and are potential drug targets effects of chloroquine on viral infections: an old drug against today's diseases? in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov therapeutic drugs targeting 2019-ncov main protease by high-throughput screening prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3cl pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates compounds with therapeutic potential against novel respiratory remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro virus against virus: a potential treatment for 2019-ncov (sars-cov-2) and other rna viruses corticosteroid treatment of patients with coronavirus disease 2019 (covid-19) potential benefits of precise corticosteroids therapy for severe 2019-ncov pneumonia clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury a systematic review of anakinra, tocilizumab, sarilumab and siltuximab for coronavirus-related infections understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression tocilizumab treatment in covid-19: a single center experience tocilizumab, an anti-il-6 receptor antibody, to treat covid-19-related respiratory failure: a case report first case of covid-19 in a patient with multiple myeloma successfully treated with tocilizumab convalescent plasma: new evidence for an old therapeutic tool? convalescent plasma as a potential therapy for covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients remdesivir as a possible therapeutic option for the covid-19 human transbodies that interfere with the functions of ebola virus vp35 protein in genome replication and transcription and innate immune antagonism current status of cell-based therapies for respiratory virus infections: applicability to covid-19 autologous mesenchymal stem cells for the treatment of secondary progressive multiple sclerosis: an open-label phase 2a proof-of-concept study mesenchymal stem (stromal) cells for treatment of ards: a phase 1 clinical trial mesenchymal stromal cell secretome for severe covid-19 infections: premises for the therapeutic use survival after mesenchymal stromal cell therapy in steroid-refractory acute graft-versus-host disease: systematic review and meta-analysis therapeutic applications of mesenchymal stem cells for systemic lupus erythematosus mesenchymal stromal cells: clinical challenges and therapeutic opportunities mesenchymal stromal cells: sensors and switchers of inflammation tumour viruses and innate immunity mesenchymal stem cells: a double-edged sword in regulating immune responses transplantation of ace2-mesenchymal stem cells improves the outcome of patients clinical study of mesenchymal stem cell treating acute respiratory distress syndrome induced by epidemic influenza a (h7n9) infection, a hint for covid-19 treatment mesenchymal stem cells induce mature dendritic cells into a novel jagged-2-dependent regulatory dendritic cell population potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review potential interventions for novel coronavirus in china: a systematic review cepi. cepi to fund three programmes to develop vaccines against the novel coronavirus microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid dissolving undercut microneedle arrays for multicomponent cutaneous vaccination microneedle delivery of autoantigen for immunotherapy in type 1 diabetes the immunological anatomy of the skin professional antigen-presenting cells of the skin cd4 + t cell responses elicited by different subsets of human skin migratory dendritic cells cutaneous immune responses mediated by dendritic cells and mast cells microneedle patches for vaccination in developing countries organization of the skin immune system and compartmentalized immune responses in infectious diseases neuronal regulation of cutaneous immunity a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice pharmacologic treatments for coronavirus disease 2019 (covid-19): a review key: cord-288409-idq780jb authors: alsahafi, abdullah j.; cheng, allen c. title: knowledge, attitudes and behaviours of healthcare workers in the kingdom of saudi arabia to mers coronavirus and other emerging infectious diseases date: 2016-12-06 journal: int j environ res public health doi: 10.3390/ijerph13121214 sha: doc_id: 288409 cord_uid: idq780jb background: the kingdom of saudi arabia has experienced a prolonged outbreak of middle east respiratory syndrome (mers) coronavirus since 2012. healthcare workers (hcws) form a significant risk group for infection. objectives: the aim of this survey was to assess the knowledge, attitudes, infection control practices and educational needs of hcws in the kingdom of saudi arabia to mers coronavirus and other emerging infectious diseases. methods: 1500 of hcws from saudi ministry of health were invited to fill a questionnaire developed to cover the survey objectives from 9 september 2015 to 8 november 2015. the response rate was about 81%. descriptive statistics was used to summarise the responses. results: 1216 hcws were included in this survey. a total of 56.5% were nurses and 22% were physicians. the most common sources of mers-coronavirus (mers-cov) information were the ministry of health (moh) memo (74.3%). only (47.6%) of the physicians, (30.4%) of the nurses and (29.9%) of the other hcws were aware that asymptomatic mers-cov was described. around half of respondents who having been investigated for mers-cov reported that their work performance decreased while they have suspicion of having mers-cov and almost two thirds reported having psychological problems during this period. almost two thirds of the hcws (61.2%) reported anxiety about contracting mers-cov from patients. conclusions: the knowledge about emerging infectious diseases was poor and there is need for further education and training programs particularly in the use of personal protective equipment, isolation and infection control measures. the self-reported infection control practices were sub-optimal and seem to be overestimated. the kingdom of saudi arabia has experienced a prolonged outbreak of middle east respiratory syndrome (mers) coronavirus since 2012 [1, 2] . healthcare workers (hcws) form a significant risk group for infection [3] [4] [5] . most of the cases in health care workers occurred in the early period of the outbreak [6] . the risk of importation of other emerging infectious diseases, particularly with large population movements during the hajj and umrah is also significant. we aimed to explore the knowledge, attitudes and behaviours of healthcare workers in the kingdom, particularly focusing on the recent disease of international significance mers-coronavirus (mers-cov). a survey was performed of healthcare workers in mecca, medina and jeddah in the kingdom of saudi arabia in 2015. the questionnaire was developed by the primary author and pilot tested on a small number of healthcare workers. participants were recruited from 9 september 2015 to 8 november 2015. the survey was administered on paper in either arabic or english according to respondent preference. the responses entered into an electronic database for analysis. the content areas included mers coronavirus knowledge and sources of information; personal experiences with mers-cov; opinions about the location of management of patients with emerging infectious diseases; attitudes of the hcws to infection control practices; the educational needs of the hcws about emerging infectious diseases; and self-reported infection control practices of the hcws. all responses were anonymous. a chi square test was used to compare differences in the proportions of categorical variables. significance was determined at the 0.05 threshold. ethical permission to conduct the survey was obtained from the department of medical research and studies, jeddah, kingdom of saudi arabia (approval number a00298). this department is registered in saudi national committee for biomedical ethics (registration number h-02-j-002). of the 1500 invited to participate in the survey, responses were received from 1216 health care workers (hcw) included in this survey. this included 267 (22%), medical practitioners, 685 (56.5%) nurses, and 264 other healthcare workers, including health inspectors, pharmacists, lab technicians and radiology technicians. of the participants, 472 (68.9%) of the nurses and 207 (77.5%) of the physicians working in primary health care centres. the majority of survey participants were saudi (87.9%), and had diploma qualifications (64.5%) ( table 1) . almost all participants had heard about mers-cov (98.8%) and understood it to be a problem for the community (86.1%). a significant minority (28.9%) of participants had worked at facilities where mers-cov had been diagnosed and many respondents had personally been tested for mers-cov mostly due to contact with cases within or outside the workplace (table 1) . healthcare workers generally had a good understanding of the requirement to test patients admitted to icu and those who were contacts, but a significant minority felt there was no indication for mers-cov investigation for the patient with acute respiratory illness requiring hospitalisation but not icu. the majority of respondents correctly identified the need for infection prevention measures, patient risk factors and the mode of transmission by close contact. unexpectedly, a significant proportion of respondents thought that mers-cov could be spread through mosquito bites. only (47.6%) of the physicians, (30.4%) of the nurses and (29.9%) of the other hcws were aware that asymptomatic mers-cov was described (table 2) . a significant minority of respondents reporting having been investigated for mers-cov. only about two thirds of the hcws (60.4%) received the result of their investigations in the first two days, it also, shows that, there are 351 (28.9%) of hcws in this study work in places where mers-cov cases had been diagnosed in the last 2 years or less. (62%) of them are nurses, (21%) are physicians and (17%) are other hcws. 145 (11.9) from the hcws in this study were care sharing providers to mers-cov infected patients (table 1) . of these respondents, around half reported that their work performance decreased while they have suspicion of having mers-cov, a similar proportion had disturbances in their social lives, and almost two thirds reported having psychological problems during this period. almost two thirds of the hcws (61.2%) reported anxiety about contracting mers-cov from patients patient and more than half (56.8%) reported avoiding contact with others in public areas (table 1 ). a high proportion of all respondent groups felt that their workplaces were not well prepared to care for patients with emerging infectious diseases, although many respondents indicated that they were personally well prepared. a significant minority of respondents reporting having been investigated for mers-cov. only about two thirds of the hcws (60.4%) received the result of their investigations in the first two days. it also, shows that, there are 351 (28.9%) of hcws in this study work in places where mers-cov cases had been diagnosed in the last 2 years or less. (62%) of them are nurses, (21%) are physicians and (17%) are other hcws. 145 (11.9) from the hcws in this study were care sharing providers to mers-cov infected patients (table 1) . of these respondents, around half reported that their work performance decreased while they have suspicion of having mers-cov, a similar proportion had disturbances in their social lives, and almost two thirds reported having psychological problems during this period. almost two thirds of the hcws (61.2%) reported anxiety about contracting mers-cov from patients patient and more than half (56.8%) reported avoiding contact with others in public areas (table 1) . a high proportion of all respondent groups felt that their workplaces were not well prepared to care for patients with emerging infectious diseases, although many respondents indicated that they were personally well prepared. the majority of respondents believed that patients with mers-cov and other emerging infectious diseases should be managed in specialised centres, but a significant proportion also agreed that general hospitals also had a role in managing such patients. a minority indicated that patients with emerging infectious diseases could be managed in primary healthcare clinics (figure 2) . agreed that general hospitals also had a role in managing such patients. a minority indicated that patients with emerging infectious diseases could be managed in primary healthcare clinics (figure 2 ). it was noted that 45% of physicians, 53% of nurses and 61% of other hcws in the study perceive their knowledge about mers-cov, ebola and others emerging infectious diseases to be low, while 40% of them indicated that it was moderate and ≤7% indicated it was high ( figure 3 ). as expected, the majority of the hcws in the study (≥72.3%) indicated that that they are in need for educational courses and training about the mers-cov, ebola and other emerging infectious diseases (figure 3) . it was noted that 45% of physicians, 53% of nurses and 61% of other hcws in the study perceive their knowledge about mers-cov, ebola and others emerging infectious diseases to be low, while 40% of them indicated that it was moderate and ≤7% indicated it was high ( figure 3 ). agreed that general hospitals also had a role in managing such patients. a minority indicated that patients with emerging infectious diseases could be managed in primary healthcare clinics (figure 2 ). it was noted that 45% of physicians, 53% of nurses and 61% of other hcws in the study perceive their knowledge about mers-cov, ebola and others emerging infectious diseases to be low, while 40% of them indicated that it was moderate and ≤7% indicated it was high ( figure 3 ). as expected, the majority of the hcws in the study (≥72.3%) indicated that that they are in need for educational courses and training about the mers-cov, ebola and other emerging infectious diseases (figure 3) . as expected, the majority of the hcws in the study (≥72.3%) indicated that that they are in need for educational courses and training about the mers-cov, ebola and other emerging infectious diseases (figure 3 ). a large majority of participants reported that they were more eager to apply infection control measures since the onset of mers-cov in ksa. unexpectedly, almost two thirds of respondents were unaware of guidelines or protocols for the care of patients with mers-cov infection. only 22.8% reported having received training about dealing with infectious disease outbreaks, 37.1% reported training in infection control policies and procedures, 54.4% reported training in hand hygiene and 45.6% reported training in n95 mask wearing techniques (table 3) . a high proportion of respondents agreed that emergency department overcrowding, poor hand hygiene and mask use contributed to the risk of hcw being infected with mers-cov. similarly, a high proportion agreed that a lack of knowledge about the mode of transmission, a lack of policies and procedures, and insufficient training in infection control procedures also contributed to the risk (table 3) . self-reported compliance with hand hygiene was moderate, with only about two thirds of the hcws (60.3%) of the physicians, (64.8%) of the nurses and (60.6%) of the other hcws practicing regular hand washing after patient contact. less than half of respondents reported full compliance with use of surgical masks when required, and a similar proportion reported compliance with n95 respirators when required (table 3) . compliance with immunisation recommendations was poor, with only 59.5% self-reporting receipt of annual influenza vaccine within the last 12 months, 74.4% reporting receipt of meningococcal vaccine in the last 3-5 years, and 50.4% reporting have received hepatitis b immunisation or testing for immunity during their work career (table 3) . table 3 . hcws attitudes and barriers to infection control practices following mers-cov outbreak. the control of emerging infectious diseases in the hospitals can be limited by case detection and management using transmission-based precautions to all confirmed and probable cases. for mers-cov in health care settings, this requires early recognition, testing and airborne precautions [7] . in this survey we found that despite a high basic level of awareness about mers coronavirus and the importance of infection control, there remained significant misconceptions. we have previously described more than 171 secondary cases in healthcare workers in the 939 cases reported to july 2015 with another 174 cases acquired by other patients while in hospital [8] . another study suggested that, infected health care workers were an important group involved in disease spread [9] . this survey revealed that, about two third of the hcws whose contact to mers-cov cases were investigated for possible infection, which may reflect a high index of suspicion , the anxiety about infection and accessibility to health services. this study also showed significant proportion with personal experience of mers-cov either as hcw at institutions caring for cases or being investigated for possible infection following contact with cases [10] . a survey of healthcare workers in south korea found a poor level of knowledge of the modes of transmission, which was implicated in the rapid spread of the infection in hospitals. worryingly, more than half of respondents in this survey thought that mers-cov could be spread through mosquito bites [10] . the infection control measures are very crucial for respiratory infectious cases in the healthcare institutes [11] . a high proportion of respondents identified hospital overcrowding, poor hand hygiene and mask use, lack of knowledge about the mode of transmission, a lack of policies and procedures, and insufficient training in infection control procedures also contributed to the risk of spread. self-reported adherence with infection control measures was surprisingly poor, particularly in light of previous studies suggesting that self-reported adherence generally overestimates observed behaviour. the results of this survey suggest that there was poor knowledge about emerging infectious diseases, and self-reported infection control practices were sub-optimal. however, there was recognition in respondents of the need for further education and training, particularly in the use of personal protective equipment despite the high level of trust in official sources of information. system level improvements, such as incorporation of emerging infectious diseases into medical schools and continuous medical education programs, the implementation of isolation and infection control measures, and appropriate nursing-to-patient ratios would also improve preparedness [12] . isolation of a novel coronavirus from a man with pneumonia in saudi arabia first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission contact investigation for imported case of middle east respiratory syndrome hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infections in health care workers first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia institutional preparedness for infectious diseases and improving care healthcare workers infected with middle east respiratory syndrome coronavirus and infection control emerging problems in infectious diseases lessons to learn from mers-cov outbreak in south korea healthcare policy and healthcare utilization behavior to improve hospital infection control after the middle east respiratory syndrome outbreak we thank the department of medical research and studies, jeddah, kingdom of saudi arabia for the ethical approval of this study and general directorate departments of health in makkah, medina and jeddah ministry of health, saudi arabia for facilitating the data collection of this survey. we also appreciate the efforts of ibrahim asiri from jeddah directorate departments of health and tariq al maghamsi from general directorate departments of health in medina for helping us in data collection.author contributions: abdullah j. alsahafi designed the study, obtained ethical approval, collected, entered and analyzed the data and wrote the manuscript; allen c. cheng reviewed and supervised all parts of the work. all authors have read, reviewed and approved the final manuscript before submission. the authors declare no conflict of interest. this work is self-funded and there is no competing financial interest of the authors. key: cord-317232-qk72i0gv authors: gierer, stefanie; müller, marcel a.; heurich, adeline; ritz, daniel; springstein, benjamin l.; karsten, christina b.; schendzielorz, alexander; gnirß, kerstin; drosten, christian; pöhlmann, stefan title: inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity date: 2015-03-15 journal: j infect dis doi: 10.1093/infdis/jiu407 sha: doc_id: 317232 cord_uid: qk72i0gv middle east respiratory syndrome coronavirus (mers-cov) infection is associated with a high case-fatality rate, and the potential pandemic spread of the virus is a public health concern. the spike protein of mers-cov (mers-s) facilitates viral entry into host cells, which depends on activation of mers-s by cellular proteases. proteolytic activation of mers-s during viral uptake into target cells has been demonstrated. however, it is unclear whether mers-s is also cleaved during s protein synthesis in infected cells and whether cleavage is required for mers-cov infectivity. here, we show that mers-s is processed by proprotein convertases in mers-s–transfected and mers-cov–infected cells and that several rxxr motifs located at the border between the surface and transmembrane subunit of mers-s are required for efficient proteolysis. however, blockade of proprotein convertases did not impact mers-s–dependent transduction of target cells expressing high amounts of the viral receptor, dpp4, and did not modulate mers-cov infectivity. these results show that mers-s is a substrate for proprotein convertases and demonstrate that processing by these enzymes is dispensable for s protein activation. efforts to inhibit mers-cov infection by targeting host cell proteases should therefore focus on enzymes that process mers-s during viral uptake into target cells. the emergence and subsequent pandemic spread of the severe acute respiratory syndrome (sars) coronavirus in 2002-2003 caused almost 800 deaths and wreaked enormous economic havoc [1, 2] . the virus was transmitted from bats, potentially via intermediate hosts, to humans, demonstrating that the zoonotic transmission of novel coronaviruses from animal reservoirs to humans can pose a significant threat to public health [3, 4] . a similar outbreak scenario unfolded in 2012, when a novel coronavirus, initially named human coronavirus emc and now termed middle east respiratory syndrome coronavirus (mers-cov), was detected in a patient from jordan hospitalized with a severe and ultimately fatal pneumonia [5] . subsequently, the virus spread within the middle east and, through travel activity, occasionally to europe, africa, asia, and north america [6] [7] [8] [9] . the outbreak has, as of 4 july 2014, entailed 827 laboratory-confirmed infections and 287 deaths [10] , and adaptation of the virus to moreefficient human-to-human spread is a public health concern. therefore, it is imperative to identify and explore novel targets for antiviral therapy. the viral surface protein spike (s), a type i transmembrane protein synthesized in the constitutive secretory pathway of infected cells, mediates coronavirus entry into target cells [11, 12] . for this, the mers-cov spike protein (mers-s) binds to its receptor dipeptidyl-peptidase-4 (dpp4, cd26) on the surface of target cells [13] and drives fusion of the viral envelope with a target cell membrane, which allows delivery of viral proteins and rna into the host cell cytoplasm, the site of viral replication. however, mers-s and other coronavirus s proteins are synthesized as inactive precursors in infected cells and only acquire the ability to drive membrane fusion upon processing into the surface unit (s1) and the transmembrane unit (s2) by host cell proteases [14, 15] . the activity of the responsible proteases is essential for viral infectivity, which makes these enzymes potential targets for antiviral intervention. it has been demonstrated that the cysteine protease cathepsin l [14, 16, 17] and the type ii transmembrane serine protease tmprss2 [14, 16, 17] can activate mers-s during viral binding and uptake into target cells. however, activation of viral glycoproteins, including activation of the s protein of certain strains of the coronavirus infectious bronchitis virus (ibv) [18] , may also proceed in the constitutive secretory pathway of infected cells and is often accomplished by furin and other proprotein convertases [19] [20] [21] [22] . whether mers-s is also cleaved during the passage of the secretory pathway and whether these cleavage events contribute to s protein activation is at present unknown. here, we show that proprotein convertases process mers-s in transfected and infected cells, and we demonstrate that the integrity of several rxxr motifs located at the border of the s1 and s2 subunit is required for s protein processing. treatment of mers-cov-infected cells with a proprotein convertase inhibitor (pci) abrogated s protein cleavage but did not alter viral infectivity, indicating that s protein processing in infected cells is dispensable for mers-s activation. expression plasmids encoding mers-s [14] , vesicular stomatitis virus glycoprotein (vsv-g) [23] , zaire ebolavirus glycoprotein (ebov-gp) [23] , lassa virus glycoprotein (lasv-gpc) [23] , dpp4 [13] , and tmprss2 [24] have been described previously. plasmids pgal4-vp16 [25] , pgal5-luc [25] , pnl4-3-luc-r − e[26] , and p96zm651gag-opt [27] , as well as plasmids encoding mers-s [14] and ebov-gp [28] with a c-terminal v5 tag have also been described. a constructs expressing lasv-gpc and mers-s potential cleavage site mutants (pcm) 1 (626-axxa-629), pcm2 (691-axxa-694), pcm3 (748-axxa-751), and pcm4 (884-axxa-887) with a c-terminal v5 tag were generated by polymerase chain reaction (pcr)based mutagenesis. the integrity of all pcr-amplified sequences was confirmed by automated sequence analysis. 293t cells were propagated in dulbecco's modified eagle's medium (dmem; life technologies) supplemented with 10% fetal bovine serum (fbs; pan-biotech), penicillin, and streptomycin. caco-2 cells were cultured in dmem-glutamax medium (invitrogen) supplemented with 10% fbs, penicillin, and streptomycin. vero b4 cells were cultured in dmem supplemented with 5% fbs, 1% l-glutamine, 1% sodium pyruvate, 1% nonessential amino acids (all from life technologies), and antibiotics as stated above. all cell lines were grown in humidified atmosphere at 37°c and 5% co 2. for the detection of mers-s expression in transfected cells, 293t cells underwent calcium phosphate transfection with the respective plasmids encoding s proteins with a c-terminal v5 antigenic tag. for immunoblotting, the lysates were separated by sodium dodecyl sulfate gel electrophoresis and transferred onto nitrocellulose membranes (hartenstein). mers-s expression was detected using a monoclonal antibody directed against the v5 tag (invitrogen) or a polyclonal antibody directed against the s2 subunit of the mers-s protein (sino biological). for detection of mers-s protein in infected cells, caco-2 and vero b4 cells were infected with mers-cov (human betacoronavirus 2c emc/2012) at a multiplicity of infection (moi) of 0.01 and 5, respectively. at 24 hours after infection, the cells were harvested and treated with nupage lds sample buffer (invitrogen), boiled for 20 minutes at 95°c, and analyzed by western blot as described above. as a loading control, the membranes were incubated with anti-β-actin antibody (sigma). to assess the role of proprotein convertase activity in mers-s processing in transfected 293t cells, the cells were transfected with plasmids encoding mers-s, ebov-gp, and lasv-gpc or with empty plasmid. the medium was changed 6 hours after transfection, and pci (merck) was added to the fresh medium at the indicated concentrations. medium was replaced again 32 hours after transfection, and inhibitor was replenished. cells were lysed 48 hours after transfection, and cleavage of viral glycoproteins was detected by western blot, using a monoclonal antibody recognizing the v5 tag. as a loading control, an anti-β-actin antibody was used. to assess whether proprotein convertase activity is required for mers-s-driven cell-cell fusion, a previously described cellcell fusion assay was used [24] , which is based on mixing effectors cells expressing the trans-activator vp16 with target cells expressing luciferase under the control of a vp16-responsive promoter. specifically, 293t target cells that expressed mers-s or no glycoprotein and effector cells that were transfected to express dpp4 and/or tmprss2 were incubated with 1 µm pci. one day after transfection, the cells were mixed to allow cell-cell fusion, and medium was supplemented with pci at 1 µm final concentration. cell-cell fusion was quantified by determination of luciferase activity in cell lysates at 48 hours after cocultivation, using a commercially available kit (pjk). for analysis of the importance of proprotein convertase activity for mers-s-driven virus-cell fusion, a previously established pseudotyping strategy was used [14] : pnl4-3-luc-r − e − [26] vectors bearing mers-s, lasv-gpc, ebov-gp, or vsv-g or no glycoprotein were generated in the presence or absence of 1 µm pci. as target cells, 293t cells transfected with dpp4 encoding plasmid or empty plasmid, were seeded into 96-well plates and pretreated with 0.5 µm pci for 60 minutes at 37°c. the cells were then incubated with pseudotypes for 8 hours, followed by replacement of infection medium with culture medium containing inhibitor. transduction efficiency was measured after 72 hours by determining luciferase activities in cell lysates. to determine the role of proprotein convertase activity in infection with authentic mers-cov, caco-2 cells seeded in 24-well plates were incubated with dimethyl sulfoxide or rising concentrations of pci for 1 hour at 37°c and were then inoculated with mers-cov (moi 0.01 and 0.001 in quadruplicates) in the presence of inhibitor. after incubation for 30 minutes at 4°c, the cells were washed and again incubated in culture medium with the inhibitor. at 24 hours after infection, the cells were washed and harvested, and the pellet was lysed with ripa lysis buffer, supplemented with 4xnupage (invitrogen) and boiled for 20 minutes at 95°c. s protein expression in lysates was detected by western blot, using a polyclonal antibody directed against the s2 subunit of mers-s (sino biological). in parallel, for quantification of viral rna, 50 µl of the cell supernatant was dissolved in rav1 buffer (macherey-nagel) for rna extraction, followed by quantitative reverse-transcription pcr analysis, using the upe assay as previously described [29] . quantification of infectious particles was done by plaque assay, using vero b4 cells as published elsewhere [6] . briefly, 10-fold dilutions of supernatants were tested in duplicates, using cell monolayers of vero b4 cells. after 1 hour of virus adsorption, cells were washed and overlayed with a 1.2% avicel resin. after 3 days, the plates were fixed with 7% paraformaldehyde and stained with crystal violet solution. to investigate the influence of pci on the formation of mers-covinduced cytopathogenic effects, vero b4 cells were infected at an moi of 0.1 and fixed with 7% paraformaldehyde 42 hours after infection. mers-cov antigen detection was performed by incubation with a serum specimen from a patient with mers as described elsewhere [30] . bound antibodies were detected with a cyanine 2-labeled goat-anti human immunoglobulin g secondary antibody (dianova). nuclei were stained by mounting the slides with dapi containing prolong gold antifade mounting medium (life technologies). to investigate mers-s cleavage in virus producing cells, we determined whether the s protein is cleaved in transfected and infected cells. western blot analysis of s protein transfected 293t cells and mers-cov-infected vero b4 cells with an antibody specific to the s2 subunit of mers-s revealed 2 prominent s protein bands with molecular weights of 170 kda and 90 kda (figure 1 ), in keeping with our previous results [14] . the 170k-da band corresponds to uncleaved mers-s, while the presence of the 90-kda band indicates efficient processing of mers-s into an n-terminal s1 subunit (not detected) and a c-terminal s2 subunit (figure 1 ). several rxxr motifs located at the border of the s1 and s2 subunits are required for processing of mers-s inspection of the sequences located at the border of the s1 and s2 subunits of mers-s revealed the presence of 4 rxxr sequences (figure 2a) , which might represent recognition sites for proprotein convertases [22] . to determine the importance of these motifs for mers-s cleavage, we changed the arginine residues in 626-rxxr-629, 691-rxxr-694, 748-rxxr-751, and 884-rxxr-887 to alanine residues, creating the potential cleavage site mutants pcm1, pcm2, pcm3, and pcm4 (figure 2a ). all s protein mutants were expressed with the same efficiency as wild-type mers-s in transfected cells ( figure 2b ). the only exception was pcm2, for which consistently no expression was detected (not shown). importantly, mutation of potential cleavage sites 3 and 4 alone impacted s protein processing: the presence of the 90-kda band was reduced in cells expressing pcm3, and a band with a molecular weight slightly 293t cells were transfected with a plasmid encoding the mers-s protein or with empty plasmid (pcdna). vero b4 cells were either infected with mers-cov at a multiplicity of infection of 5 or mock infected. subsequently, the cells were lysed and analyzed by western blot, using a polyclonal antibody directed against the s2 subunit of mers-s. a β-actin antibody served as a loading control. similar results were obtained in 2 separate experiments. higher than 90 kda was observed upon expression of pcm4 ( figure 2b ). processing of pcm1 was comparable to that seen for mers-s wild type. however, combined mutation of potential cleavage sites 1 and 3 (pcm1 + pcm3) abrogated s protein processing ( figure 2b ), suggesting that also potential cleavage site 1 contributes to s protein cleavage in this experimental setting. similar effects were seen when potential cleavage sites 3 and 4 were simultaneously altered (pcm3 + pcm4) or when all 3 potential cleavage sites were mutated in parallel figure 2 . rxxr motifs located at the border between s1 and s2 are required for efficient processing of the middle east respiratory syndrome coronavirus spike protein (mers-s). a, the domain organization of the mers-s protein is schematically depicted. the mers-s sequence at the border between the s1 and s2 subunits is shown. rxxr motifs, which constitute potential cleavage sites, are highlighted, and the predicted start of the s2 subunit is underlined. the mutations introduced into the potential cleavage sites in mers-s are shown. b, 293t cells were transfected with expression plasmids coding for mers-s wild type and the indicated mers-s mutants equipped with a c-terminal v5 tag. transfection of empty plasmid (pcdna) served as negative control. expression of s proteins in cell lysates was determined by western blot, using a v5 tag-specific monoclonal antibody. expression of β-actin in cell lysates was assessed as a loading control. the results shown are representative for at least 3 independent experiments. abbreviations: ct, cytoplasmic tail; pcm, potential cleavage site mutant; rbd, receptor binding domain; sp, signal peptide; tm, transmembrane domain. (pcm1 + pcm3 + pcm4). collectively, these results indicate that mers-s cleavage is reduced or altered upon mutation of potential cleavage sites 1, 3, and 4. we next sought to identify the proteases responsible for mers-s cleavage. for this, we investigated whether s protein cleavage can be blocked by a cell-permeable tripeptide derivative containing an arg-x-arg motive, which is known to inhibit several proprotein convertases, including furin [31] . treatment with this pci reduced the production of the 90-kda band in cells transfected to express mers-s ( figure 3) . moreover, the presence of pci diminished processing of the zaire ebolavirus glycoprotein (ebov-gp) into gp1 and gp2 (figure 3) , which depends on the activity of the proprotein convertase furin [32] . in contrast, processing of the glycoprotein of lassa virus (lasv-gpc), which is mediated by the proprotein convertase ski-1/s1p [33] , was not suppressed (figure 3 ). this finding is in keeping with the known differences in substrate specificity (and thus inhibitor sensitivity) of ski-i/sip, compared with proprotein convertases with specificity for basic amino acids, like furin [34] . collectively, these results show that the activity of proprotein convertases is essential for mers-s processing in transfected cells. we next assessed whether proprotein convertase activity is required for s protein-driven cell-cell fusion. for this, we used a previously described assay [24] , which is based on directed expression of s protein and receptor/protease in effector and target 293t cells, respectively. expression of mers-s in effector cells allowed inefficient fusion with control-transfected 293t target cells, which are known to express low amounts of endogenous dpp4 [35] , and the efficiency of cell-cell fusion was markedly increased when target cells were transfected with dpp4 and/or tmprss2 encoding plasmid ( figure 4a ). however, the continuous presence of pci in target and effector cell cultures, before, during, and after mixing had no appreciable effect on fusion efficiency ( figure 4a ), suggesting that proprotein convertase activity is dispensable for mers-s-driven cell-cell fusion. to determine whether proprotein convertase activity is required for mers-s-dependent virus-cell fusion, we used a previously reported vector system [14] . notably, the addition of pci to cells producing mers-s-harboring lentiviral particles reduced transduction of 293t target cells by roughly 100-fold ( figure 4b ). in contrast, infectivity of particles bearing lasv-gpc or ebov-gp was not affected (figure 4b ), in keeping with the findings that processing of lasv-gpc is not inhibited by pci (figure 3 ) and that processing of ebov-gp by proprotein convertases is dispensable for gp-driven virus-cell fusion [36, 37] . of note, no inhibitory effect was detected when 293t cells transfected to express dpp4 were chosen as targets (figure 4b) , indicating that s protein processing in virus-producing cells might be dispensable for infectivity when target cells express robust amounts of dpp4. in sum, these results suggest that proprotein convertase activity is largely dispensable for mers-s-driven cell-cell and virus-cell fusion, at least when target cells produce high levels of dpp4. the lack of a prominent inhibitory effect of pci on mers-s-driven cell-cell and virus-cell fusion might be due to high levels of directed mers-s and dpp4 expression in these experimental systems. therefore, we assessed whether pci inhibits mers-cov spread in target cells expressing endogenous dpp4. pci treatment of caco-2 and vero b4 cells infected with mers-cov did not inhibit total mers-s expression but reduced s protein cleavage efficiently and in a concentration-dependent manner ( figure 5a and data not shown), confirming that mers-s is a substrate of proprotein convertases in infected cells. however, pci treatment did not reduce mers-cov propagation, as determined by the amount of viral rna ( figure 5b and data not shown) or infectious units ( figure 5b and data not shown) present in culture supernatants. similarly, treatment of mers-cov-infected cultures had little if any effect on the formation of cytopathic effects, as demonstrated by comparable destruction of the cell monolayer ( figure 5c ). thus, processing of mers-s by proprotein convertases is dispensable for mers-cov infectivity in cell cultures. the processing of the glycoproteins of human immunodeficiency virus (hiv) [19] and highly pathogenic avian influenza viruses [20] by proprotein convertases is essential for viral infectivity. as a consequence, antiviral strategies aiming at the inhibition of these enzymes are being developed [31] . our results add mers-s to the list of proprotein convertase substrates and show that several rxxr motifs within mers-s are required for efficient s protein processing. however, activity of proprotein convertases was dispensable for infectivity of mers-cov, indicating that, in the context of mers-cov infection, proprotein convertases do not constitute targets for antiviral intervention. the observations that tmprss2 activates mers-cov [14, 16, 17] and other coronaviruses [38] [39] [40] and that knock out of tmprss2 has no phenotype in the absence of infection [41] defined tmprss2 as a target for therapeutic intervention. however, tmprss2 might not be the only cellular protease that constitutes a potential therapeutic target in the context of coronavirus infection. thus, cleavage of mers-s during biogenesis in infected cells might be required for subsequent cleavage and activation of the s protein by tmprss2 during viral uptake into target cells. such a scenario would not be unprecedented, given that entry mediated by the glycoproteins of certain bunyaviruses depends on glycoprotein processing during synthesis in the constitutive secretory pathway and, as suggested by a recent report, during viral uptake into target cells [42] . alternatively, it is conceivable that processing of mers-s in infected cells is dispensable for virus-cell but required for cell-cell fusion, which results in formation of syncytia, a feature of mers-cov pathogenesis [5] . we found that mers-s is efficiently although not completely cleaved in s protein-transfected 293t cells and mers-cov-infected vero b4 and caco-2 cells, and inhibition experiments showed that proprotein convertase activity is essential for mers-s cleavage. the finding that mers-s is efficiently cleaved in transfected or mers-cov-infected cells is in keeping with several studies examining mers-s expression for other purposes [16, 43, 44] . one report showed that mers-s is not cleaved in transfected cells [17] ; however, a mers-s variant with a truncated cytoplasmic tail was examined, and it is conceivable that this deletion interfered with s protein exposure to or recognition by proprotein convertases. alternatively, the cells used for s protein expression might have produced only low amounts of proprotein convertases. robust s protein processing by proprotein convertases has also been reported for mouse hepatitis virus strain a59 [45, 46] and ibv [18, 47] , while cleavage of sars-s by these proteases is inefficient [48] [49] [50] . thus, mers-s belongs to the subgroup of coronavirus s proteins that are substrates of proprotein convertases, and mers-s processing in different cellular systems, including the colon-derived cell line caco-2, is robust. most proprotein convertases cleave the following motif: (r/k)-2nx-r↓, with n standing for 0-3 amino acids [34] . the finding that rxxr motifs located at the predicted border between s1 and s2 subunit are important for mers-s processing by proprotein convertases might therefore not be unexpected. nevertheless, it is noteworthy that at least 2 rxxr motifs had to be mutated in parallel to markedly reduce s protein processing. this suggests that the processing enzyme(s) can recognize >1 cleavage site. indeed, the s2 fragment produced upon cleavage of pcm4 showed a slightly increased molecular weight relative to the fragment generated from mers-s wild type, which would be in keeping with use of an upstream cleavage site, most likely potential cleavage site 3. alternatively, the integrity of the rxxr motifs located at the border of the s1 and s2 subunit might be required to present a single cleavage site in a protease sensitive configuration. interference with ebolavirus glycoprotein processing by proprotein convertases is compatible with robust viral spread in vitro and in vivo [36, 37] . in contrast, proprotein convertase activity is required for full activity of certain mhv and ibv s proteins in cell-cell and virus-cell fusion reactions [18, [45] [46] [47] . moreover, processing of the hiv envelope protein by proprotein convertases is essential for viral infectivity, and it has been suggested that processing of sars-s, although being inefficient, is still required for full viral spread and for the establishment of cytopathic effects in infected cultures [48] . the present study provides evidence that efficient blockade of mers-s processing by pci has no appreciable effects on s protein activity in cell-cell fusion assay and does not modulate mers-cov spread in susceptible cells. in contrast, inhibition of mers-s processing markedly reduced mers-s-driven fusion of virions with 293 t cells expressing low amounts of endogenous dpp4. it is therefore conceivable that processing of mers-s by proprotein convertases is required for optimal spread of mers-cov in target cells expressing low levels of dpp4. such a scenario would be in keeping with the observation that inefficient sars-s-driven cell-cell fusion due to limited receptor expression can be rescued by directed expression of s protein-activating proteases and vice versa [50] . in sum, our results identify mers-s as a substrate of proprotein convertases but indicate that the activity of these enzymes is dispensable for mers-cov spread in target cell lines and potentially also in the infected human host. furthermore, our findings suggest that the mode of s protein activation might depend on the level of receptor expression, a scenario that warrants further analyses. a decade after sars: strategies for controlling emerging coronaviruses from sars to mers: 10 years of research on highly pathogenic human coronaviruses ecology, evolution and classification of bat coronaviruses in the aftermath of sars isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission update 16: summary and literature update as of 11 middle east respiratory syndrome coronavirus (mers-cov) -update mechanisms of coronavirus cell entry mediated by the viral spike protein ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells inhibition of furin-mediated cleavage activation of hiv-1 glycoprotein gp160 influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease furin at the cutting edge: from protein traffic to embryogenesis and disease viral envelope glycoprotein processing by proprotein convertases dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response highly conserved regions within the spike proteins of human coronaviruses 229e and nl63 determine recognition of their respective cellular receptors vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes codon usage optimization of hiv type 1 subtype c gag, pol, env, and nef genes: in vitro expression and immune responses in dna-vaccinated mice comparative analysis of ebola virus glycoprotein interactions with human and bat cells detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections highly potent inhibitors of proprotein convertase furin as potential drugs for treatment of infectious diseases processing of the ebola virus glycoprotein by the proprotein convertase furin the lassa virus glycoprotein precursor gp-c is proteolytically processed by subtilase ski-1/s1p the biology and therapeutic targeting of the proprotein convertases expression profiling of dipeptidyl peptidase 8 and 9 in breast and ovarian carcinoma cell lines reverse genetics demonstrates that proteolytic processing of the ebola virus glycoprotein is not essential for replication in cell culture proteolytic processing of the ebola virus glycoprotein is not critical for ebola virus replication in nonhuman primates tmprss2 activates the human coronavirus 229e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry role of proteases in the release of porcine epidemic diarrhea virus from infected cells phenotypic analysis of mice lacking the tmprss2-encoded protease severe fever with thrombocytopenia virus glycoproteins are targeted by neutralizing antibodies and can use dc-sign as a receptor for ph-dependent entry into human and animal cell lines middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion proteolytic cleavage of the e2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion coronavirus ibv: partial amino terminal sequencing of spike polypeptide s2 identifies the sequence arg-arg-phe-arg-arg at the cleavage site of the spike precursor propolypeptide of ibv strains beaudette and m41 implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry different host cell proteases activate the sars-coronavirus spike-protein for cell-cell and virus-cell fusion acknowledgments. we thank stephan kallies for excellent technical assistance.financial support. this work was supported by fp7-emperie (contract 223498 to c. d. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-294656-sx3tpe0y authors: lee, jonggul; chowell, gerardo; jung, eunok title: a dynamic compartmental model for the middle east respiratory syndrome outbreak in the republic of korea: a retrospective analysis on control interventions and superspreading events date: 2016-11-07 journal: journal of theoretical biology doi: 10.1016/j.jtbi.2016.08.009 sha: doc_id: 294656 cord_uid: sx3tpe0y abstract the 2015 middle east respiratory syndrome (mers) outbreak in the republic of korea has provided an opportunity to improve our understanding of the spread of mers linked to healthcare settings. here we designed a dynamic transmission model to analyze the mers outbreak in the republic of korea based on confirmed cases reported during the period may 20–july 4, 2015. our model explicitly incorporates superspreading events and time-dependent transmission and isolation rates. our model was able to provide a good fit to the trajectory of the outbreak and was useful to analyze the role of hypothetical control scenarios. specifically, we assessed the impact of the timing of control measures, especially associated with a reduction of the transmission rate and diagnostic delays on outbreak size and duration. early interventions within 1week after the epidemic onset, for instance, including the initial government announcement to the public about the list of hospitals exposed to mers coronavirus (mers-cov), show a promising means to reduce the size ( > 71 % ) and duration ( > 35 % ) of the mers epidemic. finally, we also present results of an uncertainty analysis focused on the role of superspreading events. middle east respiratory syndrome (mers) is a fatal respiratory disease caused by a coronavirus that emerged in saudi arabia in 2012 (zaki et al., 2012) . the major reservoir of mers virus (mers-cov) responsible for infections in the human population is likely to be associated with dromedary camels (cauchemez et al., 2014; zumla et al., 2015; sabir et al., 2015) . most individuals infected with mers-cov develop a severe respiratory illness accompanied by cough, fever, shortness of breath, and pneumonia. as of 28 july 2016, a total of 1791 laboratory-confirmed cases including 640 deaths in 27 countries have been reported to the world health organization (who) (world health organization, 2015c) . although countries in africa, asia, europe, and north america have experienced sporadic importations of mers from the middle east, these have not generated local outbreaks thus far. the largest mers outbreak outside saudi arabia occurred in the republic of korea as a result of a single importation from the arabian peninsula in may 2015. as of 4 july 2015, a total of 186 cases have been reported, including 38 deaths. although the person-to-person transmission risk of mers is thought to be not self-sustaining (cauchemez et al., 2014 ; the health protection agency (hpa) uk novel coronavirus investigation team, 2013; chowell et al., 2014; breban et al., 2013) , it has shown potential to be explosive in the nosocomial setting (assiri et al., 2013; oboho et al., 2015) . out of 186 confirmed cases in the control and prevention, 2015, 2016; ki, 2015) and 80 cases (43%) were generated by only one infected case at the same hospital (korea centers for disease control and prevention, 2015, 2016) (fig. 1) . the potential for high variability in the number of secondary cases or superspreading events (sses) is a notable characteristic of infectious diseases (lloyd-smith et al., 2005; galvani and may, 2005) . cases that generate a disproportionate number of secondary cases tend to occur during the early stage of an epidemic (transmission dynamics and control of severe acute respiratory syndrome, 2003; . conversely, unlike "superspreaders", the typical individuals tend to infect only a few or no cases at all. in recent works on the mers outbreak nishiura et al., 2015; kucharski and althaus, 2015; blumberg and lloyd-smith, 2013) , this individual variation has been described by transmission heterogeneity. based on the stochastic approach, it is assumed that the number of secondary cases caused by each infected individual is negative binomial distributed with mean 0 and dispersion parameter k (with lower value representing higher heterogeneity, and vice versa). in this framework, sses during the recent mers outbreaks can be explained by the high dispersion nature of the distribution of the number of secondary cases per case. for example, chowell et al. (2015) estimated that the mean 0 for the mers outbreaks was below the epidemic threshold value of 1 while the dispersion parameter k was estimated at 0.06, indicating high heterogeneity in the potential number of secondary cases. simulations indicated that the probability of observing outbreaks larger than the mers outbreak in the republic of korea is only of the order of 1%. however, this requires careful interpretation because sses during outbreaks might be treated as outliers rather than observations stemming from a highly over dispersed distribution. at the same time, infectious diseases with subcritical 0 and overdispersed k are more likely to subside within just a few disease generations. currently, no vaccine or antiviral treatment against mers-cov infection (world health organization, 2015b) is available. although early intervention strategies such as fast diagnosis and quarantine of suspected cases have proved to be the most effective control measures for rapidly mitigating a mers outbreak (the health protection agency (hpa) uk novel coronavirus investigation team, 2013; breban et al., 2013; nishiura et al., 2015; kucharski and althaus, 2015; banik et al., 2015) . the mean duration from symptom onset to diagnosis of mers-cov infection of the outbreak in the republic of korea was estimated in the range of 4-8 days (korea centers for disease control and prevention, 2015; ki, 2015; cowling et al., 2015) . although it decreased once intense contact tracing activities were implemented, a significant delay in diagnosis was observed in the early stage of the outbreak in the republic of korea, which is one of the critical features that facilitated the outbreak. most studies on the mers outbreak in the republic of korea have focused on inferring the probability of a large outbreak size by analyzing the distribution of cluster sizes kucharski and althaus, 2015) . to the best of our knowledge, there is no dynamic compartmental model for the mers outbreak in the republic of korea that incorporates the role of sses and the timedependent parameters associated with the impact of early interventions. in this work, we develop a mathematical model that is consistent with consolidated retrospective investigations of previous mers outbreaks. our calibrated model provides a basis to analyze the hypothetical impact of intervention strategies. furthermore, by analyzing the variation in infectiousness of the superspreaders, we explore the uncertainty associated with the sses. data on daily laboratory-confirmed mers cases for the outbreak in the republic of korea were obtained from the korea center for disease control and prevention (kcdc) (korea centers for disease control and prevention, 2016 the index case of the mers outbreak in the republic of korea was a businessman who took a trip to the middle east and returned on may 4 cowling et al., 2015; world health organization, 2015a) . showing symptoms of respiratory problems on may 11, he visited several hospitals, was admitted to a hospital on may 15 and discharged on may 17, and finally diagnosed with mers on may 20. consequently, the index case generated multiple exposures, infecting 28 people including the two patients, case 14 and case 16, who in turn generated over 50% of the total cases reported in the republic of korea. most cases were related to nosocomial transmission or hospital-to-hospital transmission in 17 mers-affected healthcare facilities (korea centers for disease control and prevention, 2015, 2016; ki, 2015) . of the 186 cases, 82 were inpatients who shared the same room, ward, or emergency room; 65 were their family members or visitors; and 39 were medical professionals or staff. a number of cases had been exposed in badly ventilated and crowded places such as the emergency room with unisolated patients who had respiratory diseases. more than three healthcare workers were infected by confirmed cases when providing treatment without wearing proper personal protective equipment. in our study, we developed a model for the mers outbreak in the nosocomial setting and investigated the role of superspreaders in the transmission dynamics. we defined the superspreaders as those who transmit the virus to more than 20 patients and have underlying respiratory diseases with a severe cough (korea centers for disease control and prevention, 2015; ki, 2015) . case 14 and case 16 were considered to be "superspreaders" in our model (see fig. 1 ). because our model was simulated based on the data according to the day of lab-confirmation, 28 cases exposed by the index case before may 20, the day on which our simulations start, were used as initial conditions. case 14 and case 16 were inpatients staying with the index case in the same ward at pyengtaek st. mary's hospital and were exposed to mers-cov infection from may 15 to 17. developing mers-cov symptoms with fever, case 14 visited the emergency room at samsung medical center on may 27 and stayed there for 3 days before he was confirmed on may 30. during this period, 80 tertiary cases-approximately 43% of the total cases of mers-cov infection in the republic of koreawere infected in the same hospital by case 14. case 16 developed symptoms on may 20, and he generated a total of 24 tertiary infections at dae cheong hospital and konyang university hospital from may 22 to 30. these two large clusters in the nosocomial setting caused the epidemic peak on june 7. we developed a dynamic transmission model for the mers outbreak in the republic of korea based on a seir compartmental modeling framework that incorporates time-dependent parameters and pulses of intensified transmission that captures sses. we assumed a nosocomial infection in our model. the entire population stays at hospitals and consists of five epidemiological compartments: susceptible (s), exposed (e), infectious (i), isolated (j), and removed (r). therefore, people who stay in a hospital and are not exposed to mers are susceptible individuals, s, such as inpatients, outpatients, family members, healthcare workers, or visitors. the susceptible individuals, s, who have effective contacts with the infected individuals, i, j, and the superspreaders, are exposed to mers-cov. following the mean incubation period, k 1/ , the exposed individuals, e, show symptoms and become infectious individuals, i. note that, in this phase, people with illness might not be entirely under isolation before the case confirmation by laboratory means. after showing symptoms for a mean duration of α 1/ days, patients would be classified as laboratory-confirmed cases, j. then, these individuals are immediately transferred to hospitals for mers-cov treatment and are isolated in an intensive care unit which is restricted to only healthcare staff with personal protective equipment (korea centers for disease control and prevention, 2016). the isolated individuals, j, are discharged from the hospital as cured or dead after γ 1/ days on average. the transmission dynamics of mers-cov shown in fig. 2 is then modeled by the following system of nonlinear ordinary differential equations: where n is the total population, and h(t) is a heaviside function. in this model, there are three ways that the pathogen spreads to the host population: by infectious individuals, isolated individuals, and superspreaders. the i class, who has common initial symptoms including fever, cough, and myalgia, would be misdiagnosed as having a common cold in the beginning of an outbreak for emerging infectious diseases such as the mers epidemic in the republic of korea. because the i class is likely to be isolated improperly, in the nosocomial setting, it can transmit the virus to the s class at transmission rate β. although the j class is isolated to a negative-pressure room, a few of its members could transmit the virus, by accident, to other people such as healthcare workers. we assumed that they have less infectiousness with the reduction factor l. hence, the force of infection by i and j is defined by since the superspreaders played a major role for the mers outbreak in the republic of korea, we considered the force of infection by superspreaders separately in our model. the superspreaders have abnormally high transmissibility because of bad circumstances and personal health status, such as poor ventilation in a ward and excessive clinical symptoms including cough (korea centers for disease control and prevention, 2015; chowell et al., 2015; stein, 2011) . this transmission heterogeneity was deterministically incorporated with a heaviside function during a specific time interval, δt i , representing the exposed period of the i-th sse beginning at time t i . the force of infection by the i-th superspreader is then given by where β * i is the individual transmission rate for the i-th superspreader. note that the probability that a susceptible individual may have contact with a certain superspreader among the total population n is considered by control measures by the government and behavioral changes in communities cause the transmission and isolation rates over time to vary. we considered that the transmission rate, β ( ) t , and isolation rate, α ( ) t , are both defined as a step function, allowing the changes at τ days after the onset of the outbreak, as follows: table 1 ). the sse by the index case was imposed on the initial condition for the mers model (1). because the model was fitted to the data according to day of case confirmation, the secondary cases generated by the index case were considered in the initial condition. the first and second confirmed cases were reported on may 20, 2015. at that time, it was revealed by the kcdc's epidemiological investigation that, of 28 secondary cases, 16 were exposed and 10 were infected (korea centers for disease control and prevention, 2015) . therefore, assuming that there was a total of 10,000 people in healthcare facilities, we set the initial values as in this work, most of the parameters were referred from the report of the kcdc's epidemiological investigation of the mers outbreak (korea centers for disease control and prevention, 2015). the mean incubation period, 1/k, was chosen as 6.83 days (korea centers for disease control and prevention, 2015; ki, 2015; cowling et al., 2015; cho and chu, 2015; park et al., 2015) . the mean period from isolation to discharge, 1/γ, was estimated at 13 days, which is the median for all discharged cases. the transmission rate of the isolated individuals, j, was assumed as 10% of that of the infectious individuals, i. therefore, the reduction factor for the transmissibility of j was set at l ¼0.1. the time when the levels of α ( ) t and β ( ) t are changed, τ, was estimated at 18 days after the outbreak onset, based on the case data for the duration from symptom onset to confirmation (korea centers for disease control and prevention, 2016; ki, 2015) . at that time, the government announced the list of hospitals exposed to mers (korea centers for disease control and prevention, 2016; cho and chu, 2015) , so people in the community or at these hospitals paid more attention to the spread of mers. empirical evidence indicates that behavioral changes could make the nosocomial transmissibility decrease significantly (wallinga and teunis, 2004) . additionally, the government allowed the diagnostic testing for mers to be performed at authorized health facilities in order to shorten the duration of diagnosis (cho and chu, 2015) . the median value of duration from symptom onset to laboratory confirmation was significantly shortened after june 7 (from 6 days to 2 days). the time-dependent transmission rate, β ( ) t , was estimated by fitting the model prediction, days (see fig. 1 ). the basic reproductive number gives us the information whether an infectious disease can spread to a susceptible population in a steady state (diekmann et al., 1990) . generally, 0 could be obtained from the generation matrix for a compartmental disease transmission model (van den driessche and watmough, 2002) . in this work we estimate the effective reproductive number, e , which is the time-dependent reproductive number reflecting the impact of control measures (nishiura and chowell, 2009 ). for our model (1), the effective reproductive number without the pulse of infection is given by for all > t 0 (fig. 3) . to investigate the effects of early interventions, we varied τ and fixed the estimated values of associated parameters with β ( ) t and α ( ) t as the baseline values in table 1 . the total number of confirmed cases and the duration of the outbreak were investigated by varying τ from 1 to 18. the outbreak duration was measured during times until the daily number of new confirmed cases was decreasing and sufficiently small. the proportionate reductions of the outbreak duration and size were calculated by using the baseline results at τ = 18. we assumed that the control measures for β ( ) t and α ( ) t were effectively carried out to prevent transmission of the virus. for example, the list of hospitals that were exposed to mers patients was announced to the public on june 7. cho and chu (2015) after that, people changed their behaviors such as avoiding visiting hospitals, wearing an n95 mask, and using hand sanitizers. at that time, the health authorities expanded the screening capacity for rapid diagnosis. these control measures could help detect suspected cases during their infectious period or even before the onset of symptoms. such interventions could prevent the occurrence of sses because the potential superspreaders were immediately isolated after being confirmed with mers infection. we investigated the uncertainty of sses by using the probability distributions for the timing and size of the events. sses tend to occur during the early stage of an outbreak when the presence of disease is not yet identified by public health authorities (lloyd-smith et al., 2005) . if identification of the superspreaders is delayed, a substantially large number of cases proportional to the duration of exposure are likely to be generated. hence, we assumed that potential sses occur during the first few days of the introduction of the disease. this allows for variations in the timing of occurrence of the sse and in the sizes of secondary cases produced by a superspreader while the duration of exposure was predetermined in simulations. to investigate the uncertainty of sses, we assumed the number of secondary cases by the superspreaders as a uniform distribution in the range of [ ] × * 0.5, 1.5 i . the truncated exponential decay distribution was used for the beginning of a sse. the two superspreaders, case 14 and case 16, were exposed by the index case on may 17. adding the maximum value of the incubation period, 14 days, to the illness onset of the superspreaders, we found the feasible periods for the truncated interval of the exponential decay distribution. because the outbreak began on may 20 (i.e., t ¼0), the truncated interval was determined as [ ] 1, 11 . however, different mean values for the exponential distribution were used in order to consider the individual variability of the sse (see supplementary figure s1 ). note that the abbreviations 'sse 1′ and 'sse 2′ denote the sses caused by case 14 and case 16, respectively. the fit of the model to the temporal evolution of the mers outbreak in the republic of korea from may 20 is shown in fig. 4 . the daily and cumulative numbers of laboratory-confirmed cases for mers-cov from our model (solid curves) showed qualitatively good fit to the data (squares) because the effects of superspreaders were applied to the model at appropriate times. our calibrated model indicates that the epidemic reached its peak on june 7, then gradually decreased to zero as in the real data. in hsieh (2015) , the author deduced that may 27-29 was the period of the turning point for disease infection and the serial interval for the mers outbreak was estimated as 12.5 days in korea centers for disease control and prevention (2015) . then the turning point for the confirmed cases might have occurred during june 8-10. this is similar to the peak of incidence (confirmed cases) by the two superspreaders on june 7. the impact of the control measures was investigated by varying the parameter τ, which plays an important role in the time-dependent parameters, α ( ) t and β ( ) t . fig. 5 shows the impact of τ on the outbreak size and duration. in fig. 5(a) , the cumulative numbers of cases as functions of time after onset are shown for the baseline value, τ = 18 (thick black curve), and smaller values, τ ≤ ≤ 1 17 (thin gray curves). the proportionate reductions of outbreak size (cross) and outbreak duration (square) with respect to τ are plotted in fig. 5(b) . for the baseline value, τ = 18, the outbreak size and duration were 188.7 total cases and 64.9 days, respectively. at a glance, smaller values of τ than the baseline value shows a decreasing effect on outbreak size and duration. especially, when τ ≤ 7, the outbreak sizes are less than 60 total cases (>71% reduction) and the outbreak durations are within 50 days ( >35% reduction). this result suggests how important the early interventions were in mitigating the mers outbreak in the republic of korea. for instance, if the government had announced the list of mers-exposed hospitals to the public less than 1 week after the onset, the outbreak size might have been dramatically reduced. the uncertainty of the sses was investigated by drawing the size of sse from a uniform distribution and/or the timing of the events from a truncated exponential distribution. fig. 6 shows the distributions of outbreak duration and size obtained from the simulations, allowing the variation in the size and initial timing for the occurrence of the sse when τ = 18 (top) and τ = 11 (bottom). the different distributions of sse 1 only (black dot), sse 2 only (green cross), and both (blue plus) are shown. the yellow square represents the outbreak duration and size from the model (1) with baseline parameters. the mean values of the outbreak duration and size for the case of sse 1 and sse 2 are approximately 64 days and 193 total cases at τ = 18, respectively, which are similar to those obtained from the baseline parameter. if there exists only one superspreader, then the mean outbreak duration and size are 61 days and 159 total cases for sse 1, and 52 days and 80 total cases for sse 2, respectively (table 2) . if the intervention is put in place a week earlier, τ = 11, then late-occurring sses are avoided and most outbreaks end with significant reductions in its duration and size. if there exists only one superspreader, the mean outbreak duration and size are 56 days and 131 total cases for sse 1, and 46 days and 67 total cases for sse 2, respectively. if there exist both sse 1 and sse 2, the mean outbreak duration and size are 58 days and 158 total size, respectively (table 2) . we found that 74.5% of the simulated outbreaks with both sse 1 and sse 2 had a shorter duration and a smaller size than the baseline (a yellow square in fig. 6) . notably, about 8% of the simulated outbreaks with both sse 1 and sse 2 are of duration <55 days and of size <100 total cases. scatterplots of the outbreak duration (a) and size (b) are shown when τ = 18 days (fig. 7) and τ = 11 days (fig. 8) . the set of control parameters was obtained from the joint distribution for the initial timing of the sse and the size of secondary cases by the sses. the different distributions of sse 1 only (black dot) and sse 2 only (green cross) are shown with those interpolants (gray). the red curve in fig. 8 highlights the considerable change during days when the sum of the initial timing of the sse and its duration of exposure is equal to the intervention starting time, i.e., δ τ + = t t i i . overall, the outbreak duration and size were positively correlated with the number of secondary cases stemming from the sses. although the outbreak duration was positively correlated with the timing of the sses, the outbreak size was negatively correlated with one. when the control measures to contain more infections by the sse were implemented in the early stage of the spread of mers ( τ = 11), the outbreak duration and size were remarkably reduced. we have developed a mathematical model for the 2015 mers outbreak in the republic of korea, incorporating the time-dependent parameters and the pulse of infections to model sses. assuming a nosocomial setting, the pulse of infection with different transmission rates for the sses was incorporated in the deterministic model. laboratory-confirmed data (korea centers for disease control and prevention, 2015, 2016) were used to estimate the transmission rates for the typical infectious individuals and the superspreaders. to the best of knowledge, this is the first dynamic compartmental model incorporating pulses of infections to model the effect of sses. we estimated e regardless of sses was below 1, which is consistent with previous works on the recent mers outbreak (cauchemez et al., 2014; chowell et al., 2014 chowell et al., , 2015 breban et al., 2013; cowling et al., 2015; majumder et al., 2014) . moreover, the estimated e after the control measures were implemented was substantially decreased. this indicates that the mers outbreak in the republic of korea had a low transmissibility in the absence of the sses (cowling et al., 2015) . however, the reasons for the emergence of the biggest outbreak outside the middle east are attributed to the importation of the virus without awareness of the public health , missed contacts (cho and chu, 2015) , substantial exposure to infection (δt 2 ) caused by delayed diagnosis and isolation , and abnormally high contact rate of the superspreaders ( β * i ) in the crowded hospital setting (korea centers for disease control and prevention, 2015) . of 186 confirmed cases with mers-cov infection in the republic of korea, 153 cases (82.3%) were generated by only 5 cases (2.7%). conversely, this transmission heterogeneity suggests that identifying them in their suspected stage of mers-cov infection could stem the subsequent transmission in over 150 cases in the host population (lloyd-smith et al., 2005; galvani and may, 2005; stein, 2011) . we paid attention to the timing of implementation of control measures associated with the reduction in transmission rate β and diagnostic delay α 1/ . quarantine and isolation turned out to be highly effective control measures for reducing the transmission rate. our results show that the intervention strategies in the early stage of the outbreak (τ ≤ 7) could prevent the occurrence of sses and substantially reduce the outbreak duration and size. in other words, the failure of rapid detection and proper isolation of suspected patients early in the outbreak has contributed to sses, which is in line with the experience of the severe acute respiratory syndrome (sars) outbreak in 2003 (galvani and may, 2005; mcdonald et al., 2004) . our results derived from the uncertainty analysis of the sses suggest that the recent mers outbreak in the republic of korea could have been smaller in size and duration. when τ = 11, it is very likely that outbreaks with both sse 1 and sse 2 have a shorter duration and a smaller size (with mean 58 days and 158 cases, respectively) compared to the baseline outcome. although it is certainly difficult to preemptively identify superspreaders, the implementation of timely interventions (e.g. fast diagnosis and quarantine of suspected cases) can significantly mitigate the chance of sses during an outbreak. case 14 and case 16 were not classified as suspected cases but should have been home-quarantined as soon as they were exposed to mers-cov by the index case. furthermore, the diagnostic delay in nosocomial infection made them become superspreaders. after the health authorities implemented the strong control measures such as contact tracing using cctv surveillance and the gps of mobile phones, the confirmed cases who were not identified in the contact tracing gradually decreased since june 12 (cho and chu, 2015) . our results suggest the importance of the early implementation of such interventions in the rapid containment of the sses and, consequently, in the remarkable reduction in outbreak duration and size. to the best of our knowledge, this is the first dynamic compartmental model that explores the nosocomial outbreak of the mers-cov in the republic of korea including the sses. the outbreak pattern in our results shows a good agreement with the time series of confirmed cases. this implies that our model has captured the contributing main factors such as the delayed diagnosis and the announcement to the public of the list of exposed hospitals during the early phase of the outbreak. this result suggests that our modeling framework could be a useful tool for the prediction or prevention of future emerging infectious diseases that have similar characteristics to mers-cov infection in the republic of korea. hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps inference of r0 and transmission heterogeneity from the size distribution of stuttering chains interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility outbreak of middle east respiratory syndrome in korea? synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission transmission characteristics of mers and sars in the healthcare setting: a comparative study preliminary epidemiological assessment of mers-cov outbreak in south korea on the definition and the computation of the basic reproduction ratio r0 in models for infectious diseases in heterogeneous populations epidemiology: dimensions of superspreading epidemiology and control of sars in singapore middle east respiratory syndrome coronavirus (mers-cov) nosocomial outbreak in south korea: insights from modeling mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respriatory syndrome, press release the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission superspreading and the effect of individual variation on disease emergence estimation of mers-coronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data sars in healthcare facilities the effective reproduction number as a prelude to statistical estimation of time-dependent epidemic trends assessing the risk of observing multiple generations of middle east respiratory syndrome (mers) cases given an imported case mers-cov outbreak in jeddah-a link to health care facilities epidemiological investigation of mers-cov spread in a single hospital in south korea co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia super-spreaders in infectious diseases evidence of person-to-person transmission within a family cluster of novel coronavirus infections reproduction numbers and subthreshold endemic equilibria for compartmental models of disease transmission different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures middle east respiratory syndrome coronavirus (mers-cov) republic of korea middle east respiratory syndrome coronavirus (mers-cov) -fact sheets middle east respiratory syndrome coronavirus (mers-cov) isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome funding: the research work of jung was supported by the korea national research foundation (nrf) grant funded by the korea government (mest) (nrf-2015r1a2a1a15054463). jung's work is also resulted from the konkuk university research support program. we declare we have no competing interests. supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jtbi.2016.08.009. key: cord-312670-hi3fjne4 authors: corman, v. m.; lienau, j.; witzenrath, m. title: coronaviren als ursache respiratorischer infektionen date: 2019-08-27 journal: internist (berl) doi: 10.1007/s00108-019-00671-5 sha: doc_id: 312670 cord_uid: hi3fjne4 background: there are six human pathogenic coronaviruses (cov), which mainly cause infections of the respiratory system. in everyday clinical practice, it is helpful to know the relevance and characteristics of these pathogens. objective: to present the epidemiology, clinical picture and differences of human pathogenic cov and to provide information on the diagnostics and treatment of patients suspected of having cov infections. material and methods: selective literature search, presentation of results and discussion of fundamental works and expert recommendations, including publications by the world health organization (who), the european centre for disease prevention and control (ecdc) and the robert koch institute. results: the four endemic human covs (hcov-nl63, hcov-229e, hcov-oc43 and hcov-hku1) mainly cause mild respiratory tract infections. in addition to these four endemic hcov, the two epidemic cov, severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov can cause severe pneumonia. the sars-cov has not been detected in humans in the last 15 years and mers-cov has been circulating mainly on the arabian peninsula since 2012; however, neither a specific treatment nor approved vaccines exist for any of the six human pathogenic covs. conclusion: all six human covs can be diagnosed using rt-pcr on respiratory specimens but this is rarely necessary for the four endemic strains. in current clinical practice sars-cov has no importance as it has not been detected in humans for 15 years; however, a possible mers-cov infection should be taken into account in patients with typical symptoms and travel history to endemic regions. in this case, rapid diagnostic and general hygiene practices are important to prevent further transmission. es gibt sechs humanpathogene coronaviren (cov), die vornehmlich respiratorische infektionen im menschen auslösen. vier davon kommen weltweit vor und sind sehr häufig. im regelfall rufen diese virustypen nur leichtere respiratorische infektionen hervor. schwere verläufe sind selten und treten am ehesten bei immunsupprimierten auf. zwei weitere typen sind selten, aber für schwere virale pneumonien verantwortlich: das mers-und sars-cov (mers "middle east respiratory syndrome"; sars "severe acute respiratory syndrome" [schweres akutes atemwegssyndrom]). während sars-cov seit 2004 nicht mehr im menschen gefunden wurde, werden infektionen mit mers-cov seit 2012 kontinuierlich detektiert. cov sind behüllte ribonukleinsäure(rna)-viren mit einem plusstrang-rna-genom. sie werden in die unterfamilie orthocoronavirinae (ordnung nidovirales, familie coronaviridae) klassifiziert. die unterfamilie orthocoronavirinae umfasst eine große anzahl an verschiedenen genera, subgenera und virusspezies [12] . darunter sind viele varianten, die in der veterinärmedizin sowohl für haustiere (etwa bezüglich der felinen infektiösen peritonitis bei katzen) als auch für nutztiere (schwere diarrhöen bei rindern und schweinen) von bedeutung sind. bisher wurden sechs verschiedene cov in menschen gefunden, folglich ist nur ein kleiner teil der bekannten cov humanpathogen (. tab. 1). bei symptomatischen infektionen lösen cov im regelfall respiratorische erkrankungen aus. vier der bisher bekannten sechs humanpathogenen cov werden weltweit im menschen gefunden und sind je nach patientenkollektiv für 10-15 % der akuten respiratorischen erkrankungen (are) verantwortlich [10, 15, 24] . zusätzlich zu diesen ständig im menschen zirkulierenden varianten wurden in den vergangenen jahren zwei cov im menschen gefunden, nämlich sars-cov und mers-cov, die aus dem tierreservoir auf den menschen übergegangen sind und bei einem deutlich größeren anteil der infizierten schwere virale pneumonien mit tödlichem verlauf auslösen [25, 28] . mit hcov-oc43 und -229e wurden zwei der vier endemischen humanpathogenen cov (hcov) bereits in den 1960er-jahrenbeschrieben. die zweiweiteren endemischen cov (hcov-nl63 und -hku1) wurden erst deutlich später, in den 2000er-jahren entdeckt [5] . alle vier cov sind etablierte humanpathogene erreger, die weltweit vorkommen und klassischerweise are auslösen können. je nach patientenkollektiv werden hinweise auf eine aktive cov-infektion bei bis zu 20 % der patienten mit akuten ambulant erworbenen respiratorischen erkrankungen gefunden, sowohl bei kindern als auch bei erwachsenen [10, 15, 24] . asymptomatische infektionen wurden ebenfalls beschrieben [23] . obwohl die mehrzahl der infektionen mit den vier endemischen cov nur leichte atemwegserkrankungen verursacht, können alle hcov auch schwere hier steht eine anzeige. zusätzlich zu den vier endemischen hcov haben in den letzten zwei jahrzehnten die beiden epidemischen cov sars-cov und mers-cov beim menschen schwere atemwegserkrankungen ausgelöst. mers-cov ist das zweite hochpathogene cov, das im menschen gefunden wird. es wurde bei menschen erstmalig 2012 im verlauf einer fatalen viralen pneumonie in saudi-arabien entdeckt [11, 14, 28, 30] . seitdem wurden etwa 2500 humane mers-cov-infektionen gemeldet, wovon etwa 30 % tödlich verliefen. über 90 % der infektionen traten in ländern der arabischen halbinsel auf, besonders in saudi-arabien. importierte fälle wurden aber auch in europäischen ländern einschließlich deutschland dokumentiert, insgesamt sind bisher mehr als 25 länder betroffen (. abb. 1b; [28] ). in manchen ländern kam es ausgehend von einem importierten fall auch zu weiteren autochthonen infektionen. besonders zu erwähnen ist hierbei der mers-cov-ausbruchinsüdkorea im jahr2015, bei dem ausgehend von einem einzelnen infizierten reiserückkehrer mehr als 160 weitere nosokomiale infektionen resultierten, unter anderem auch von medizinischem personal [11] . neben mensch-zu-mensch-übertragungen im häuslichen umfeld und im krankenhaus stellen dromedare auf der arabischen halbinsel die wichtigste infektionsquelle dar [11, 14] . mers-cov zirkuliert seit mindestens 2012 kontinuierlich in dieser geografischen region, sodass eine infektion mit mers-cov bei passender anamnese weltweit differenzialdiagnostisch relevant ist [26] . interessanterweise stellen dromedare auch außerhalb der arabischen halbinsel ein reservoir für mers-cov dar. so wurde das virus auch in dromedaren in west-und ostafrika sowie in asien nachgewiesen (. abb. 1b und 2; [2, 13, 31] ). primäre zoonotische infektionen wurden aus diesen regionen allerdings noch nicht bekannt. seit 2015 stehen die beiden pandemischen cov (sars-und mers-cov) auf der von der weltgesundheitsorganisation (who) aufgesetzten liste der "priority diseases". diese liste verzeichnet die acht infektionskrankheiten, bei denen zukünftig eine weltweite epidemie für möglich gehalten wird und für die ein dringender forschungs-und entwicklungsbedarf zur verhinderung einer ausbreitung gesehen wird [17] . pneumonie · akute respiratorische erkrankungen · middle-east-respiratorysyndrome-coronavirus · schweres akutes atemwegssyndrom · erkältung background. there are six human pathogenic coronaviruses (cov), which mainly cause infections of the respiratory system. in everyday clinical practice, it is helpful to know the relevance and characteristics of these pathogens. objective. to present the epidemiology, clinical picture and differences of human pathogenic cov and to provide information on the diagnostics and treatment of patients suspected of having cov infections. material and methods. selective literature search, presentation of results and discussion of fundamental works and expert recommendations, including publications by the world health organization (who), the european centre for disease prevention and control (ecdc) and the robert koch institute. results. the four endemic human covs (hcov-nl63, hcov-229e, hcov-oc43 and hcov-hku1) mainly cause mild respiratory tract infections. in addition to these four endemic hcov, the two epidemic cov, severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov can cause severe pneumonia. the sars-cov has not been detected in humans in the last 15 years and mers-cov has been circulating mainly on the arabian peninsula since 2012; however, neither a specific treatment nor approved vaccines exist for any of the six human pathogenic covs. conclusion. all six human covs can be diagnosed using rt-pcr on respiratory specimens but this is rarely necessary for the four endemic strains. in current clinical practice sars-cov has no importance as it has not been detected in humans for 15 years; however, a possible mers-cov infection should be taken into account in patients with typical symptoms and travel history to endemic regions. in this case, rapid diagnostic and general hygiene practices are important to prevent further transmission. die symptome einer sars-cov-infektion sind vergleichbar mit den symptomen einer mers-cov-infektion. da in den letzten 15 jahren keine menschlichen sars-cov-infektionen mehr bekannt wurden, verzichtet diese kurze übersichtsarbeit auf eine detaillierte betrachtung der sars-cov-infektionen. virale koinfektionen der lunge, unter anderem mit influenza a und b, parainfluenza, rhinovirus und adenovirus, sind sowohl bei der infektion mit endemischen als auch bei infektionen mit mers-und sars-cov beschrieben worden. koinfektionen mit bakterien kommen auch gelegentlich vor. bei einigen patienten mit sars/mers-cov-infektion wurde zusätzlich von sekundär nosokomial erworbenen nichtviralen infektionen (pneumokokken, staphylokokken, klebsiellen, acinetobacter, candida) berichtet. inwiefern diese sekundären infektionen auf die intensivmedizinische behandlung und beatmung zurückzuführen sind oder ein spezifisches grundsätzliches risiko einer cov-infektion darstellen, ist noch nicht verstanden. neben dem regelhaften nachweis von cov im respirationstrakt sind alle endemischen cov auch in stuhlproben nachgewiesen worden; sie sind jedoch typischerweise keine kausale ursache einer gastroenteritis [9, 20] . zudem gibt es hinweise, dass infektionen mit hcov-oc43 eine rolle bei neurologischen erkrankungen spielen könnten [18, 29] . die unterscheidung zwischen cov und anderen erregern, die eine are auslösen, ist anhand der klinischen symptome grundsätzlich nicht möglich. jedoch ist eine spezifische labordiagnostik bei verdacht auf eine infektion mit endemi-schen cov bei harmlosem verlauf und patienten ohne besonderes risiko für die entstehung von komplikationen auch nicht indiziert. bei komplizierten verläufen und schweren infektionen des respirationstrakts ist eine testung auf cov mittels echtzeit-reverse-transkriptase-polymerase-kettenreaktion (rt-pcr) möglich und sinnvoll. geeignetes probenmaterial sind abstriche oder sekrete aus dem oberen, je nach klinik auch aus dem unteren respirationstrakt. eine diagnostik über den nachweis von antikörpern ist aufgrund der häufigen (re-)infektionen nur in ausnahmefällen sinnvoll und begründet. eine spezifische untersuchung auf eine erkrankung durch mers-cov muss beim vorliegen bestimmter kriterien durchgeführt werden. mittel der wahl ist auch für mers-cov der direktnachweis der viralen rna mittels rt-pcr [3, 14, 26, 27] . geeignete materialien sind vor allem sekrete aus den unteren atemwegen (bronchoalveoläre lavage, absaugsekrete, sputum), da dort die höchsten viruskonzentrationen vorliegen [4] . ist die gewinnung und testung dieser materialien nicht möglich, können auch abstriche aus dem oberen respirationstrakt (nasen/rachen) untersucht werden. die antikörperdiagnostik basierend auf der testung von zwei konsekutiven serumproben, beispielsweise im abstand von 4 wochen, ist für den ausschluss einer akuten infektion ungeeignet und nur in bestimmten situationen (umgebungsuntersuchung, postexpositionsuntersuchung) indiziert [26] . aufgrund der interindividuellen heterogenität der antikörperantwort bei mers-cov-infektionen ist die quantifizierung spezifischer antikörpertiter zum nachweis oder ausschluss einer mers-cov-in-fektion ungeeignet. in . abb. 3 ist dargestellt, wann und wie in einem mers-cov-verdachtsfall vorgegangen werden sollte. bei der typischerweise unspezifischen klinik von mers-cov-infektionen sollte auch die möglichkeit einer infektion mit anderen pathogenen in betracht gezogen werden [26] . bei patienten, für die laut mers-cov-falldefinition eine diagnostik indiziert war und von denen proben zum ausschluss einer mers-cov-infektion an das konsiliarlabor für cov gesendet wurden, werden regelmäßig andere erreger als mers-cov als ursächlich für die respiratorische erkrankung identifiziert. häufig handelt es sich dabei um (eigene unpublizierte daten) 4 » mers-cov ist beim menschen hauptsächlich in den tiefen atemwegen zu finden mers-cov kann auch von einem menschen auf einen anderen übertragen werden. allerdings wurde eine kontinuierliche übertragung in der bevölkerung bislang nicht beobachtet; auch im normalen häuslichen umgang finden nur wenige übertragungen statt [6] . diese beobachtung steht im gegensatz zu den beobachteten gravierenden krankenhausassoziierten ausbrüchen auf der arabischen halbinsel und in südkorea. der scheinbare widerspruch lässt sich dadurch erklären, dass mers-cov beim menschen im gegensatz zum auftreten bei kamelen hauptsächlich in den tiefen atemwegen zu finden ist [4] . im rahmen der behandlung eines infizierten patienten, beispiels-weise mit bronchoskopie, beatmung und absaugung, kann es zum kontakt mit sekreten aus den tiefen atemwegen kommen, insbesondere wenn infektionspräventionsmaßnahmen unzureichend umgesetzt werden. treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-beta1b (miracle trial): study protocol for a randomized controlled trial mers coronaviruses from camels in africa exhibit regiondependent genetic diversity assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection hosts andsourcesofendemichumancoronaviruses transmission of mers-coronavirus in household contacts european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) -tenth update incidence, significance, and persistence of human coronavirus infection in hematopoietic stem cell transplant recipients human coronavirusesareuncommoninpatientswithgastrointestinal illness human coronavirus infections in israel: epidemiology, clinical symptoms and summer seasonality of hcov-hku1 middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission international committee on taxonomy of viruses (2019) ictv virus taxonomy detection of distinct mers-coronavirus strains in dromedary camels from kenya mers coronavirus: diagnostics, epidemiology and transmission cocirculation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 fatal outcome of human coronavirus nl63 infection despite successful viral elimination by ifn-alpha in a patient with newly diagnosed all the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts human coronavirus oc43 associated with fatal encephalitis ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study commonly circulating human coronaviruses do not have a significant role in the etiology of gastrointestinalinfectionsinhospitalizedchildren empfehlungen des rki für das management von kontaktpersonen laborbestätigter symptomatischer mers-fälle rki (2015) schwere respiratorische erkrankungen in verbindung mit middle east respiratory syndrome coronavirus (mers-cov). falldefi-nition zur fallfindung, meldung und übermittlung asymptomatic summertime shedding of respiratory viruses frequentdetectionofhumancoronaviruses inclinicalspecimensfrompatientswithrespiratory tract infection by use of a novel real-time reversetranscriptase polymerase chain reaction severe acute respiratory syndrome (sars) -disease outbreak news who (2019) clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance detection of coronavirus in the central nervous system of achildwithacutedisseminatedencephalomyelitis isolation of a novel coronavirus from a man with pneumonia in saudi arabia countrywide survey for mers-coronavirus antibodies in dromedaries and humans in pakistan weitere infos zu e.med finden sie auf springermedizin.de unter "abos" key: cord-294918-lm2ixz8n authors: hotez, peter j.; bottazzi, maria elena; tseng, chien-te k.; zhan, bin; lustigman, sara; du, lanying; jiang, shibo title: calling for rapid development of a safe and effective mers vaccine date: 2014-07-31 journal: microbes and infection doi: 10.1016/j.micinf.2014.05.002 sha: doc_id: 294918 cord_uid: lm2ixz8n abstract the geographic spread and rapid increase in the cases of middle east respiratory syndrome (mers) caused by a novel coronavirus (mers-cov) during the past two months have raised concern about its pandemic potential. here we call for the rapid development of an effective and safe mers vaccine to control the spread of mers-cov. very recently, a new case of middle east respiratory syndrome (mers) coronavirus (mers-cov) was reported in a 44-year-old man living in yemen with no relevant travel history e possibly the first autochthonous case outside of saudi arabia. when considered with recent news of the three reported cases of mers in the united states, there is urgency to consider renewing the global commitment to combat mers and accelerate the development of a mers vaccine. to date, however, the interest and enthusiasm of the global public health community in both mers and a mers vaccine could be described as ambivalent. at last year's world health assembly in geneva, dr. margaret chan, the world health organization (who) director general, announced that mers-cov is "a threat to the entire world". however, later that summer, a special who panel muted such sentiments by indicating that mers did not yet constitute a "public health emergency of international concern" [1]. as of may 23, 2014, 635 laboratoryconfirmed cases of mers-cov infection, including 193 deaths, had been reported to who from seven countries in the middle east, two countries in africa, six countries in europe, two countries in asia, and one country in north america [2] . more worrisome, 429 of these cases have been reported since march 27, 2014. a majority of these newly reported cases are secondary cases or cases with unknown exposure (fig. 1) , partly reflecting increased surveillance for this disease in saudi arabia, but also possibly suggesting an increased ratio of human-to-human transmission of mers-cov. yet a group of who experts during a visit to saudi arabia failed to recommend any new public health measures beyond ongoing surveillance for respiratory infections. moreover, no new travel restrictions including restrictions on trade or the annual pilgrimage have been instituted [3] . of course, this situation could change, pending new developments. similar to how the scientific community responded to the outbreak of severe acute respiratory syndrome (sars) cov that emerged from south china more than a decade ago, several promising avenues for developing a mers vaccine have been explored. for now, however, many of us have received mixed messages on the urgency of advancing a vaccine development program for mers-cov. in 1973, harold bloom, yale university english professor and literary critic, published his famous book entitled the anxiety of influence: a therory of poetry. in essence, bloom advances the hypothesis that poets aspire to write something truly original, but they become hindered in their creative processes by the influence, both conscious and unconscious, of previous works [4] . anxiety of influence similarly affects virus researchers today because of a medical and scientific disaster that struck nih and merck investigators almost 50 years ago during the testing of a first-generation formalin-inactivated respiratory syncytial virus (rsv) vaccine [5] . the vaccine failed to protect infants who received this vaccine in 1966e67, and many became sick from antibody-dependent enhanced respiratory signs and symptoms. two young children died [6] . since then, investigators working with experimental respiratory vaccines have proceeded with great caution and even reluctance based on the prospect of causing another such incident. in laboratory animals antibody-dependent immune microbes [7, 8] , although to a lesser degree. such concerns prompted efforts to develop a more restricted receptor binding domain (rbd) of the s protein as a recombinant vaccine [9, 10] , which elicits highly effective cross-neutralizing antibody responses in the vaccinated animals [11] . an equivalent rbd molecule has now been identified from mers-cov spike protein (fig. 2) [12e14], potentially making it feasible to co-develop this molecule as a recombinant mers vaccine, alongside an rbd-based sars vaccine. to date, however, the level of interest from scientific funding agencies to develop mers vaccines has been modest. when considering the time and expense of developing a safe and effective vaccine against uncertain risk factors, global health policymakers have so far hesitated in prioritizing mers-cov vaccines for the purpose of creating a stockpile in the event of a public health emergency. yet waiting for a full-blown mers epidemic, or even pandemic, to occur before even beginning vaccine development could result in the loss of many lives, especially in saudi arabia. therefore, the current situation of mers in the middle east, especially as we move closer to the hajj pilgrimage in the fall, will continue to require vigilance by the who and other international health agencies, which in turn, must include meaningful and frequent communications with vaccinologists in both academia and industry. the receptor-binding domain (rbd) in the s protein s1 subunit contains major neutralizing epitopes, serving as an attractive target for mers vaccine [14] . mers-cov summary updates the anxiety of influence: a theory of poetry respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine in search of a vaccine for respiratory syncytial virus: the saga continues antisevere acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine proteaseindependent fcgammar pathway immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus the spike protein of sars-cov: a target for vaccine and therapeutic development roadmap to developing a recombinant coronavirus s protein receptorbinding domain vaccine for severe acute respiratory syndrome yeast-expressed recombinant protein of the receptor-binding domain in sars-cov spike protein with deglycosylated forms as a sars vaccine candidate identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development a truncated receptorbinding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines current advancements and potential strategies in the development of mers-cov vaccines hotez** maria elena bottazzi sabin vaccine institute and texas children's hospital center for vaccine development the authors have declared that no conflict of interests exist. key: cord-297418-36j840wm authors: carneiro leão, jair; paula de lima gusmão, teresa; machado zarzar, adriana; leão filho, jair carneiro; barkokebas santos de faria, andreza; morais silva, igor henrique; gueiros, luiz alcino monteiro; robinson, narendran andrew; porter, stephen; de albuquerque tavares carvalho, alessandra title: coronaviridae ‐ old friends, new enemy! date: 2020-05-31 journal: oral dis doi: 10.1111/odi.13447 sha: doc_id: 297418 cord_uid: 36j840wm coronaviridae is a family of single‐stranded positive enveloped rna viruses. this article aimed to review the history of these viruses in the last 60 years since their discovery to understand what lessons can be learned from the past. a review of the pubmed database was carried out, describing taxonomy, classification, virology, genetic recombination, host adaptation, and main symptoms related to each type of virus. sars‐cov‐2 is responsible for the ongoing global pandemic, sars‐cov and mers‐cov were responsible for causing severe respiratory illness and regional epidemics in the past while the four other strains of covs (229‐e oc43, nl63, and hku1) circulate worldwide and normally only cause mild upper respiratory tract infections. given the enormous diversity of coronavirus viruses in wildlife and their continuous evolution and adaptation to humans, future outbreaks would undoubtedly occur. restricting or banning all trade in wild animals in wet markets would be a necessary measure to reduce future zoonotic infections. coronaviruses (covs) are zoonotic viruses that belong to the coronaviridae family with a simple positive-sense rna genome (ksiazek et al., 2003) . covs are similar in the organization and expression of their genome, and are maintained simultaneously in nature, which allows for their constant genetic recombination, resulting in new viruses (su et al., 2016) . the viruses, known since 1960, can infect and cause disease in both animals and humans (siddel et al, 1983; ksiazek et al., 2003) . the types known to cause disease in animals include; infectious bronchitis virus (ibv) (lin & chen, 2017) , canine respiratory coronavirus (crcov) (priestnall, brownlie, dubovi & erles, 2006) , mouse hepatitis virus (mhv) (weiss & leibowitz, 2011) , bovine coronavirus (bcv), turkey coronavirus (tcv) and transmissible gastroenteritis virus (tgev) (szczepanski, owczrek & bzowska, 2019) . viruses of this family were first identified in humans from the nasal secretions of patients with the common cold (tyrrel & byone, 1965) . since four covs are known to produce mild infections in individuals with normal immunity similar to the common 'flu, they are 229-e oc43, nl63, and hku1 (esper et al., 2006; su et al., 2016) . however, in recent years, coronaviruses have given rise to significant diseases such as severe acute respiratory syndrome (sars-cov) and middle eastern respiratory syndrome coronavirus (mers-cov) sars-cov infected 8,000 people, from 2002 to 2003 and had a mortality rate of approximately 10% (marra et al., 2003) . ten years later, in 2012, mers-cov, emerged, and infected more than 1,700 people, with a mortality rate of approximately 36% (zaki et al., 2012) . in 2013, it attacked animals again, with a coronavirus epidemic of porcine diarrhea (pedv) in the united states, causing a mortality rate of almost 100% in piglets and wiping out more than 10% of the swine population in less than a year (mole, 2013) . more recently, a new coronavirus (sars-cov-2) has emerged that is responsible for the current covid-19 global pandemic. this virus was first identified in the chinese province of hubei in december 2019 (gudi & twari, 2020) with possible transmission being suspected to have been from animal to human as the first case was detected from a person working in the local fish and wild animals market -with later transmission from human to human via respiratory droplets or direct contact it is important to appreciate the ecology of covs, its propensity for viral recombination with different covs in animal populations, resulting in new covs that are transmissible and this article is protected by copyright. all rights reserved pathogenic to humans. it is possible that the early cases of the infection went unnoticed as the disease may have been mild, there may have been other comorbidities that would fit with a respiratory infection and/or the initial sporadic distribution of affected individuals may have hidden the insidious nature of this infection. this early period of enigmatic transmission may have allowed sars-cov-2 to acquire the facilities to be both easily acquired by humans and to induce disease that aids its own survival. the coronaviridae study group (csg) of the international virus taxonomy committee (ictv), is responsible for the classification and taxonomy of coronaviruses (gorbalenya et al., 2020 the genera deltacoronavirus and gammacoronavirus have do not infect humans, but cause infections in birds (woo et al., 2012) . the covs in humans hcov-nl63, hcov-229e, hcov-oc43, and hku1 are associated with mild respiratory tract infections (gaunt et al., 2010; forni, cagliani, clerici & sironi, 2017) . hcov-229e is associated with infections in immunocompromised individuals (pene et al., 2003) . sars-cov and mers-cov are highly pathogenic covs for humans and have caused two major epidemics (cui, li & shi, 2019) . there is still no consensus on a taxonomic position for sars-cov-2, but it belongs to a species related to acute respiratory viruses, based on genetic characteristics, but it is a virus independent of sars-cov (gorbalenya et al., 2020) figures 1a and 1b summarizes the main characteristics of human coronaviruses. this article is protected by copyright. all rights reserved coronaviruses are viruses enveloped with positive, non-segmented, single-stranded rna genomes approximately 26 to 32 kilobases in size. they have phenotypic and genotypic variety, due to recombinant rna and large genomes, facilitating, unfortunately, the ability to adapt to new hosts (woo et al., 2012; zaki et al., 2012) . the activation of s proteins of sars-cov-2 occurs through binding with angiotensin-converting enzyme 2 (ace-2) of the host cells that can be found greatly expressed in nasal epithelial cells, allowing the entry of the virus into the host cells, releasing mrna for translation of proteins . the initial portion of the covs genome encodes 16 non-structural proteins while the remaining portion encodes 4 essential structural proteins namely small envelope protein (e), spike glycoprotein (s), matrix (m) protein and nucleocapsid (n) protein . the bat is suspected to be a natural host and origin for sars-cov-2. the present virus may have been transmitted to man via intermediate hosts, such as pangolins. , phylogenetic analysis has shown that sars-cov-2 is approximately 89% similar to two batderived sars coronaviruses: bat-sl-covzc45 (genbank mg772933.1) and bat-sl-covzxc2 (genbank mg772934. 1); and is approximately 79% similar to sars-cov and 50% similar to the middle eastern respiratory syndrome (mers-cov) coronavirus . on the other hand, evolutionary analysis based on the orf1a / 1b, s and n genes suggests that sars-cov-2 is more likely to be a new coronavirus that has been introduced independently from animals to humans due to the inherent mutation property of coronaviruses in nature (lam et al., 2020) . considering genetic investigations and the presence of some bats and other live wild animals in the wuhan market, the virus may have originated from bats or bat droppings associated with contaminated materials (cui, li & shi, 2019 (herrewegh et al; 1998) . this same type of viral recombination seems to have been involved in the emergence of sars-cov (lau et al., 2010) , this article is protected by copyright. all rights reserved another phenomenon supposedly associated with the adaptation of cov to different hosts and cells is the variable deletions that cover more than 600 nucleotides in the s1 globular domain of the spike gene, which is associated with a change in the enteric virus (tgev) to respiratory tract virus (prcv). furthermore, the exclusion of 290 nucleotides from the spike gene in hcov-oc43 compared to its bcov ancestor may have allowed it to adapt to the human host (vijgen et al., 2005) . the pathogenesis of bat coronavirus is poorly understood when compared to other mammalian hosts and there is little evidence of the consequences of clinical infections in bats. in humans, hcovs-nl63, -oc43, -hku1, and -229e circulate constantly in the populations of the world. a comparison and main characteristics of these viruses are shown in table 1 (ogimi et al., 2020) . sars-cov, on the other hand, disappeared a few years after the epidemic, while the new mers-cov seems to have remained. all hcov mainly cause respiratory symptoms in humans (hamre and procknow, 1966; peiris et al., 2003; woo et al., 2005) , although mers-cov has also been associated with severe renal complications (zaki et al., 2012) . hcov is detected in feces (liu et al., 2004) , and there are sporadic reports of covs in humans with gastroenteritis, even though they are not consistently related to gastroenteritis in humans (esper et al., 2010) . profiles. this being due to mutations in nsp2 and nsp3 (angeletti et al., 2020) ; this aspect greatly changed the clinical presentation and hence treatment strategies for each viral infection. although these mutations were smaller when compared to h7n9 mutations, they significantly worsened the clinical landscape (zhang, shen, chen & lin, 2020) . in the chinese population, two prevalent types of sars-cov-2 evolution have now been observed; type l and type s. type l strains derive from type s strains and are the more clinically aggressive and contagious (lai et al., 2020) this article is protected by copyright. all rights reserved italy, and finally one from australia. cluster b is derived from a by two mutations: the mutation synonymous with t8782c and the non-synonymous mutation c28144t, changing a leucine to serine. type c cluster, mainly found in the americas and europe, differs from type b by the nonsynonymous g26144t mutation that alters a glycine in a valine. this genotype has been found in samples from france, italy, sweden, and england, also from california and brazil, singapore, hong kong, taiwan, and south korea but not in mainland china. this phylogenetic classification can be used to design treatment and, eventually, vaccines (foster et al., 2020) . severe acute respiratory syndrome (sars) is a human disease associated with severe pneumonia and as noted above is caused by sars-coronavirus (sars-cov) (drosten et al., 2003) . the disease initially occurred in november 2002 in china, and by february 2003, more than 300 cases had reported (zhong et al., 2003) . during the outbreak in 2002-2003, a total of 8,096 cases were reported, including 774 deaths in 27 countries (ksiazek, et al., 2003) . the outbreak was contained, and since 2004 there have been no known cases of sars-cov. sars-cov was initially detected in masked palm civets and a raccoon dog. antibodies against the virus were detected in chinese ferret badgers in a live-animal market in shenzhen, china (guan, 2003) , but it is believed that bats were the reservoir (drexler, corman & drosten, 2014) . most of the cases were the result of direct transmission by respiratory droplets during close personal contact, and adequate respiratory precautions taken proved to be effective (seto et al., 2003) . of note there was a relatively high incidence of infection in health care professionals managing patients with, or suspected to have sars-cov. peiris et al, (2003) reported that 28% of those infected were health professionals, hospital transmission being a characteristic of the outbreak. the average incubation period depended on the route of transmission but was between 2 to 10 days (donnely et al., 2003) . the clinical course of sars-cov begins with a flu-like prodrome consisting of fever, chills, fatigue, and malaise. towards the end of the first week, there is a deterioration of the clinical picture where the lower respiratory tract is involved, and the patient may present with a dry cough, shortness of breath, and possibly hypoxemia (vijayanand, wilkins & woodhead, 2004) . less common symptoms include watery diarrhea, vomiting, and nausea (peiris et al., this article is protected by copyright. all rights reserved 2003). laboratory findings include lymphopenia, thrombocytopenia, elevated lactate dehydrogenize (ldh), raised creatine kinase, and alanine aminotransferase (aat). infected patients have abnormal chest x-rays in 60-100% of cases, with predominant lower zone and peripheral lung involvement (goh et al., 2003) . other common findings include lack of cavitation, lymphadenopathy, pleural effusion, mainly ground-glass opacification and sometimes consolidation, interlobular septal, and intralobular interstitial thickening . only 20-30% of patients with sars require intensive care . patients with advanced age and male sex are associated with poor disease outcomes, and the mortality rate is approximately 10% (leung et al., 2004) . treatment is mainly symptomatic and supportive, no specific therapy (eg.: antivirals) have proven useful. studies show that mers-cov transmission occurs through direct contact with camel dromedaries, such as the consumption of unpasteurized camel milk or undercooked camel meat. the routes of human to human transmission are not yet fully understood, but possible routes of transmission may occur via human respiratory droplets and contact transmission . another characteristic of mers-cov was hospital transmission with 43.5% to 100% of outbreaks in different places associated with health care (chowell et al., 2015) . among the independent risk factors associated with mers infection are diabetes mellitus, heart disease, smoking, and chronic obstructive pulmonary disease (copd) (al-tawfiq et al., 2014) . common symptoms of people infected with mers-cov are fever, cough, shortness of breath, and diarrhea. pneumonia is not always present and some people are asymptomatic, but test positive on laboratory tests (chafekar & fielding, 2018) . it has an incubation period of this article is protected by copyright. all rights reserved approximately 5 days. abnormal such as chest x-rays and laboratory tests are common the latter include lymphopenia, thrombocytopenia, high elevated levels of lactate dehydrogenase, and liver enzymes. there are reports of some cases of disseminated intravascular coagulation and hemolysis (al-tawfiq et al., 2014) . up to 50 to 89% of patients require intensive care and mechanical ventilation due to acute respiratory distress syndrome (ards). the mortality rate of mers is approximately 35%, higher when compared to sars-cov. there is no specific treatment for mers, the mainstay of management include supportive, symptomatic measures, and the prevention and management of secondary complications ( clinical characteristics and pathogenesis of covid-19 are similar to sars and mers (xie & chen, 2020) , with an incubation period of 4 to 7 days, however, this can be up to 14 days. there are reports of high rates of asymptomatic and subclinical infections . a comparison between these 3 viruses are shown in table 2 . in a study of 1099 patients with covid-accepted article needed oxygen therapy, 6.1% needed mechanical ventilation and 1.4% died. the average hospital stay was 12 days . wang et al (2020) showed that as the number of transmission of covid-19 cases increases, lethality decreases, this would explain the low mortality rate when comparing sars and mers. the most common presentations in symptomatic patients are fever, dry cough, and shortness of breath. other features include gastrointestinal symptoms, such as diarrhea, neurological symptoms (stroke, headache, altered mental status, gbs), cardiovascular events (myocarditis, arrhythmias, heart failure), and ocular manifestations (conjunctival hyperemia, chemosis), anosmia and dysgeusia. . in computed tomography of the chest of covid-19 pneumonia, subpleural ground-glass opacities were initially observed, which increased, with changes in the paving pattern. in recovering patients, the lesions appeared absorbed with the presence of opacities and subpleural parenchymal bands. (xie & chen, 2020) . laboratory findings include leukopenia, lymphocytopenia (sometimes very notable), elevated neutrophilic ratio, c-reactive protein and serum proinflammatory cytokines, the last 3 findings mentioned being associated with more severe disease immune response in covid-19 the initial response to cov infection is carried out through the immune system (frieman & baric, 2008) . toll-type receptors, which are receptors for antigen-presenting cells that detect viral rnas, inducing the signaling cascade of the immune system, consequently increasing cytokines (alexopolou, holt, medzhitov & flavell, 2001; wu & chen, 2014) . to prevent recognition of the host's immune system, the virus forms double vesicles on the outside of the cell, it also has non-structural proteins inhibiting functions of the innate immune system, such as nsp1 to block inf . the increase in pro-inflammatory cytokines, including as ifn-α, ifn-γ, il-1β, il-6, il-12, il-18, il-33, tnf-α, tgfβ and ccl2, ccl3, ccl5 chemokines, cxcl8, cxcl9, cxcl10, is the first line of defense against infection wu & chen, 2014) . however, the massive proliferation of immune cells can result in hyper inflammation of the lungs and other viscera causing acute respiratory distress syndrome (ards) and notable myocardial damage. (huang et al., 2020) asymptomatic cases this article is protected by copyright. all rights reserved as noted above, there are reports of asymptomatic covid-19 cases from several groups. hcov-oc43 the human coronavirus oc43 diverged from the bovine coronavirus in 1890 (vijgen et al., 2005) common cold. 2004 (vijgen et al., 2005) hcov-nl63 human coronavirus nl63 diverged from bat coronavirus 822 years ago (huynh et al., 2012) common cold. 2004 (hu et al., 2015) hcov-hku1 the human coronavirus hku1 diverged from the bat coronavirus (woo et al., 2009) common cold. 2005 (esper et al., 2009) the human coronavirus mers diverged from the bat coronavirus before the 1990s and was transmitted to humans by camels (coman et al., 2016) mers disease. 2012 (hu et al., 2016) sars-cov-2 studies have suggested that the sars-cov-2 virus diverged from the version that parasites bats lu, 2020) and transmitted to humans by an intermediate animal. recent studies indicate that the virus has diverged from the version that parasites pangolins (lam et al., 2020) as it has genetic material 99% equal to the virus found in this animal. covid-19 disease 2019 (who,2020) odi_13447_f1b.docx this article is protected by copyright. all rights reserved accepted article scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia accepted article this article is protected by copyright. all rights reserved recognition of doublestranded rna and activation of nf-κb by toll-like receptor 3 middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients. clinical infectious diseases: an official publication of the infectious diseases society of human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe covid-2019: the role of the nsp2 and nsp3 in its pathogenesis mers-cov: understanding the latest human coronavirus threat epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study kidney impairment is associated with in-hospital death of covid-19 patients transmission characteristics of mers and sars in the healthcare setting: a comparative study link of a ubiquitous human coronavirus to dromedary camels identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229e origin and evolution of pathogenic coronaviruses dyspnoea in patients receiving noninvasive ventilation for acute respiratory failure: prevalence, risk factors and prognostic impact sars and mers: recent insights into emerging coronaviruses epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong accepted article this article is protected by copyright. all rights reserved ecology, evolution and classification of bat coronaviruses in the aftermath of sars identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus hku1 infection in the united states medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation molecular evolution of human coronavirus genomes phylogenetic network analysis of sars-cov-2 genomes mild or moderate covid-19 accepted article this article is protected by copyright. all rights reserved epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial severe acute respiratory syndrome (sars): imaging findings during the acute and recovery phases of disease the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 isolation and characterization of viruses related to the sars coronavirus from animals in southern china clinical characteristics of coronavirus disease 2019 in china preparedness and lessons learned from the novel coronavirus disease guidance for health workers -who accepted article this article is protected by copyright. all rights reserved the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status feline coronavirus type ii strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type i and canine coronavirus an outbreak of severe acute respiratory syndrome among hospital workers in a community hospital in hong kong bat origin of human coronaviruses evidence supporting a zoonotic origin of human coronavirus strain nl63 clinical features of patients infected with 2019 novel coronavirus in wuhan the use of povidone iodine nasal spray and mouthwash during the current covid-19 pandemic may reduce cross infection and protect healthcare workers a novel coronavirus associated with severe acute respiratory syndrome accepted article this article is protected by copyright. all rights reserved severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, selflimiting infection that allows recombination events identifying sars-cov-2 related coronaviruses in malayan pangolins the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus structure, function, and evolution of coronavirus spike proteins infectious bronchitis virus variants: molecular analysis and pathogenicity investigation accepted article this article is protected by copyright. all rights reserved gross examination report of a covid-19 death autopsy drug treatment options for the 2019-new coronavirus (2019-ncov) the genoma sequence of the sars-associated coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia deadly pig virus slips through us borders severe acute respiratory syndrome: radiographic and ct findings what's new with the old coronaviruses? coronavirus 229e-related pneumonia in immunocompromised patients coronavirus as a possible cause of severe acute respiratory syndrome accepted article this article is protected by copyright serological prevalence of canine respiratory coronavirus sars-cov-2 and coronavirus disease 2019: what we know so far. pathogens effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars). the lancet epidemiology, genetic recombination, and pathogenesis of coronaviruses canine respiratory coronavirus human coronavirus oc43: receptors and attachment factors cultivation of a novel type of common-cold virus in organ cultures evolutionary insights into the ecology of coronaviruses accepted article this article is protected by copyright. all rights reserved severe acute respiratory syndrome (sars): a review insight into 2019 novel coronavirus -an updated interim review and lessons from sars-cov and mers-cov advance online publication real-time estimation and prediction of mortality caused by covid-19 with patient information based algorithm. the science of the total environment coronavirus pathogenesis who. middle east respiratory syndrome coronavirus (mers-cov). 2019. available ate haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis comparative analysis accepted article this article is protected by copyright. all rights reserved of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus innate immune sensing and signaling of cytosolic nucleic acids epidemiological and clinical features of the 2019 novel coronavirus outbreak in china. medrxiv isolation of a novel coronavirus from a man with pneumonia in saudi arabia origin and evolution of the 2019 novel coronavirus epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china a pneumonia outbreak associated with a new coronavirus of probable bat origin this article is protected by copyright. all rights reserved table 1 -comparison between hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1 (ogimi et al., 2020) . key: cord-302983-3v5bc80z authors: matterne, uwe; egger, nina; tempes, jana; tischer, christina; lander, jonas; dierks, marie-luise; bitzer, eva-maria; apfelbacher, christian title: health literacy in the general population in the context of epidemic or pandemic coronavirus outbreak situations: rapid scoping review date: 2020-10-10 journal: patient educ couns doi: 10.1016/j.pec.2020.10.012 sha: doc_id: 302983 cord_uid: 3v5bc80z objective: the aim of this rapid scoping review, for which only studies from the general population were considered, was to describe the extent of existing research on hl in the context of previous coronavirus outbreaks (sars-cov-1, mers-cov and sars-cov-2). methods: we searched major databases and included publications of quantitative and qualitative studies in english and german on any type of research on the functional, critical and communicative domains of hl conducted in the context of the three outbreaks in the general population. we extracted and tabulated relevant data and narratively reported where and when the study was conducted, the design and method used, and how hl was measured. results: 72 studies were included. three investigated hl or explicitly referred to the concept of hl, 14 were guided by health behaviour theory. we did not find any study designed to develop or psychometrically evaluate pandemic/epidemic hl instruments, or relate pandemic/epidemic or general hl to a pandemic/epidemic outcome, or any controlled intervention study. type of assessment of the domains of hl varied widely. conclusion: theory-driven observational studies and interventions, examining whether pandemic-related hl can be improved are needed. practice implications: the development and validation of instruments that measure pandemic-related hl is desirable. in late 2019 an outbreak of a new viral disease occurred in wuhan, china and later spread to almost all countries of the world [1] . it is caused by a novel beta coronavirus, the severe acute respiratory syndrome -coronavirus -2 (sars-cov-2), which causes coronavirus disease (covid) -19) [2] . the clinical epidemiology of covid-19 is currently being investigated intensely [3] . course of disease may be very mild, asymptomatic to very severe with respiratory and systemic damage and requiring mechanical ventilation [4] . responses of governments to the covid-19 pandemic have been multifaceted including outbreak management (suppression versus mitigation), provision of adequate clinical treatment facilities for severe cases and measures to alleviate the economic and psychosocial impact of the pandemic and the measures taken to manage it [2] . public health measures implemented in many countries across the globe encompass contact restrictions and physical distancing, hygiene rules (i.e. frequent and thorough handwashing or disinfection), mask wearing, eye protection and recommendations about how to sneeze and cough [3, 5] . some of these measures, particularly contact restrictions, have been law enforced in many countries [6] . relaxing regulations and re-organising social life requires people to voluntarily adhere to the named measures in order to avoid exponential growth of sars-cov-2 to reoccur. further, people who contract sars-cov-2 need to know when and how to seek health care and/or be tested. those who suffer from severe covid-19 and survive will have to seek health care to mitigate the potentially long-lasting physical and psychological sequelae such as kidney damage [7] or post-traumatic stress disorder [8] . in all these and other different scenarios, the concept of health literacy (hl) becomes a vital public health concept that is essential to counterpart on the individual level the social restrictions enforced by law. when restrictions are gradually lifted, the role of individual level hl increases in order to prevent the resumption of these restriction, should infection numbers surge again. the currently prevailing integrated hl notion "entails the motivation, knowledge and competencies to access, understand, appraise and apply health information in order to make judgements and take decisions in everyday life concerning healthcare, disease prevention and health promotion to maintain or improve quality of life throughout the course of life" [9] and is promoted by who (world health organisation) europe [10] . while other notions refer, for instance to hl being the result of health education and distinguishes the distinct concepts of functional, critical and communicative hl [11] the aforementioned hl notion was arrived at by a systematic review and an integration of medical and public health views of hl [9] . in other words, what is necessary beyond governmental regulations and policy, is an increase in the levels of covid-19 related health literacy [12, 13] . we not only need to monitor the pandemic's epidemiology during the course of the pandemic including the creation of herd immunity but also hl and health behavioural responses related to the pandemic in the population [13] . hl is considered a major determinant of a person's health [14, 15] , a factor that contributes to health inequalities [14] , and a person's health behaviour, for instance, healthy diet adherence or non-smoking [15] and health care j o u r n a l p r e -p r o o f utilisation [16] . there is evidence that lower hl is consistently associated with mortality [16] or lower self-rated health status [17] . research suggests that adequate hl may not be as prevalent among populations as might be necessary in order to navigate the increasingly complex healthcare landscape [15, 16, 18] . synthesised evidence suggests a relationship between levels of hl and infectious disease prevention in non-pandemic contexts [19] . inadequate hl was found to be associated with reduced adoption of protective behaviours such as vaccination uptake and poor understanding of antibiotics [19] . large research gaps were found in relation to infectious diseases with a high clinical and societal impact, such as tuberculosis and malaria [19] . for instance, it was emphasised that critical hl, which focuses on supporting effective political and social action, was not considered in any of the reviewed studies [19] . the strengths of this relationship may be exponentially higher under pandemic circumstances, but no synthesised information on this topic appears to exist to date. further, the importance of individual hl in pandemic control has been emphasised more urgently [12, 13] . therefore, the aim of this rapid scoping review, for which only studies from the general population were considered, was to describe the extent of existing research on hl in the context of previous coronavirus outbreaks (sars-cov-1, mers-cov and sars-cov-2). the world health organisation (who) declared only sars-cov-1 a pandemic [1] while mers-cov [20] and sars-cov-1 [21] remained epidemics. facets of hl that were of particular interest were: type of assessment of hl (theory-based versus proxy assessment; validated instrument versus ad hoc assessment), interventions aiming to improve hl during outbreak situations, or hl surveillance during outbreak. this scoping review was performed according to the methodological framework as outlined by khalil et al. [22] . their guidelines regarding scoping reviews build on the work of arksey and o'malley's fivestage scoping review framework [23] , complemented with the joanna briggs institute methodology [24] , in order to (1) identify the research questions, (2) identify relevant studies, (3) select studies, (4) chart the data, and (5) collate and summarise the data. a scoping review's objective is to identify the nature and extent of the existing evidence. unlike other types of review, it does not endeavour to systematically evaluate the quality of available research, but rather seeks to identify the contribution of existing literature to an area of interest [25] . our methodology was also guided by the rapid review approach which inevitably uses less rigor as is required by a traditional systematic review due to the need for production within a short time-frame using limited resources [26] . the protocol for this rapid review was registered at osfregistries on 06/04/2020 [27] . two authors (um, ne) ran the search strategy on pubmed (medline®) and psycinfo® on 20 th april 2020. citations were downloaded to citavi (swiss academic software). we included publications in english and german of quantitative and qualitative studies. the same authors evaluated titles and abstracts excluding any irrelevant ones. full texts of the remaining citations were obtained, and two authors (um, ne) reviewed these, excluding any, which did not meet the inclusion criteria. finally, reference lists of remaining papers were hand-searched for additional relevant studies. we then compared results from full text screening; there were only minor discrepancies, which were resolved through discussion with the whole team. data extraction was carried out by five authors (um, ne, jt, ct, jl) in independent pairs of two. consensus was achieved through discussion and arbitration within the team. the search strategy was informed by hl theory (derivation of search terms) and is displayed in appendix 1. inclusion criteria were: we included reports on any type of research on the functional, critical and communicative domains of hl [11] conducted in the context of sars-cov-2, sars-cov-1 and mers-cov in the general population. this was a rational decision as an initial search using hl as the chief search term in conjunction with the aforementioned coronavirus outbreaks resulted in very few hits. we used the following definitions / concepts of functional, communicative and critical hl: functional hl is broadly compatible with the narrow definition of 'health literacy' which can be considered to consist of healthrelated knowledge, risk perceptions, attitudes, motivation, behavioural intentions, personal skills, or self-efficacy [11] . communicative hl means to be able …'to derive meaning from different forms of communication'…, while the ability to critically analyse information is referred to as critical hl [11] . the following data were extracted from the included studies: authors, publication year, country of study, type of epidemic or pandemic outbreak (sars-cov-2, sars-cov-1, mers-cov), participants (including sample size), design, method, and instruments, and measured constructs including how they were measured (only if applicable e.g. not in qualitative studies). findings were synthesized quantitatively and narratively and reporting followed the guidelines as proposed in prisma-scr [28] . a critical appraisal of the quality of the included studies was not within the scope of this review. we do however, comment on major methodological issues regarding the studies. there was no funding source for this study. the search in pubmed (medline®) and psycinfo® yielded 3394 references, two were obtained from colleagues [29, 30] , leading to 2766 references after removal of duplicates. title and abstract screening all studies, while mainly not explicitly investigating hl, measured one or more components of hl (appendix 2). most studies were observational or short longitudinal (58 cross-sectional, eight pre-post) and six qualitative. all sars-cov-2 studies were conducted during, of the mers-cov studies 27 during, one during (first wave), eight after, of the mers-cov studies 24 during, one after and one both during and after the pandemic/epidemic outbreak. 66 studies used questionnaires, two used focus group discussions, four others used qualitative methods (e.g. interviews) for data collection. 49 studies investigated convenience or opportunity samples and 23 representative samples drawn from general populations. sample size ranged from 19 -222.599 participants. within the nine quantitative sars-cov-2 studies, knowledge was measured in seven, attitude in seven, risk perceptions in four, self-efficacy in three, critical hl in five, communicative hl in three, healthinformation seeking behaviour (hisb) in two, and behavioural aspects in four studies. only one study [29] table 1) . wearing a mask was the most frequently assessed behaviour ( table 1) . 31 quantitative studies were conducted in the context of sars-cov-1. 25 measured knowledge, 28 attitude, 20 risk perception, eight se, 11 critical hl, 12 communicative hl, and 18 behaviour. one study [33] reported all six hl aspects, the others one to five aspects. within knowledge, transmission mode was most often measured. although 25 studies reported knowledge assessment, most studies did not comprehensively assess knowledge ( table 1) . handwashing was the most frequently measured behaviour ( table 1) . of the 26 quantitative mers-cov studies 21 measured knowledge, 17 attitude, 18 risk perceptions, 3 se, 4 critical hl, 17 communicative hl, and 10 behaviour. two studies assessed five of the six hl aspects [34, 35] , the remainder one to four. within knowledge, transmission mode was most often assessed. again, most studies did not comprehensively assess knowledge. handwashing was the most frequently assessed behaviour ( table 1) . the reported measured depth within the domains of hl varied widely among the studies (results not shown). for instance, the number of knowledge components ranged from one to at least eight. hisb was measured in two (sars-cov-2), four (sars-cov-1), two (mers-cov) quantitative studies. our search failed to come across any studies designed to develop or psychometrically evaluate pandemic/epidemic hl instruments or relate pandemic/epidemic or general hl to a pandemic/epidemic outcome. the number of items per hl aspect varied widely (data not shown), hardly any study reported on psychometric properties, two studies from three publications [31, 36, 37] were the notable exception (appendix 3) and a clear distinction between knowledge, attitude, or risk perceptions was sometimes absent. for instance, perceived vulnerability was reported as an attitude [38] . six qualitative studies explored domains of hl in the context of sars-cov-2, sars-cov-1, and mers-cov. one focus group study [39] reported low risk perceptions and a lack of seeking relevant health information in relation to sars-cov-2. one interview study [40] and one focus group study [41] explored risk perceptions and preventive behaviour in relation to sars-cov-1, another interview study [42] explored individual experiences during quarantine. one interview study reported low knowledge about sars-cov-1 and its prevention [43] . another interview study in the context of sars-cov-1 concluded that attitudes towards mask wearing had substantially changed in the post-sars-cov-1 period [44] . while individual hl is recognised as an increasingly important construct in public health [45] , it is of note that only three studies emerged from our extensive search, which explicitly referred to the construct of hl in the context of any of the three coronavirus outbreaks. one used the newest vital sign (nvs), a test measuring nutrition label information processing ability [32] , another study [31] administered a short form of the hls-eu-q47, an hl instrument rooted in testable theory [46] and the third [30] study used a version of the hls-eu-q47 adapted to sars-cov-2. however, the latter provided no evidence on the psychometric properties of the adapted instrument. hence, at present there seems to be no tested instrument designed to measure coronavirus pandemic-related hl. there is, however, one hl instrument assessing print and multimedia literacy in respect to respiratory diseases [47] . most of the other included studies were not theory-based. it is important to highlight that these studies did not purport to measure hl, but were included in this review because the search strategy was based on a pragmatic application of suggested hl components within domains [11, 48] . of those that were theory-driven, the majority employed health-behaviour theory as conceptualised by social cognition models. there is substantial overlap between socio-cognitive predictors of health behaviour and hl. for instance, attitude and self-efficacy (defined as behavioural control) are part of the theory of planned behaviour [49] , risk perceptions part of the health belief model [50] or knowledge part of protection motivation theory [51] . theory-based research allows the formulation of testable a priori hypotheses, and if necessary revision of the theory. nonetheless, the measures obtained from those studies lacking an explicit theoretical foundation can be considered proxies of hl because they constitute or at least contribute to one or more hl domains. while there appears to be no evidence linking validly measured (epidemic or pandemic) hl to coronavirus outbreak/pandemic outcomes there is evidence that hl can be linked to other epidemic outbreaks, e.g. the 2014-2016 ebola epidemic outbreak in west africa resulted among other factors from low health literacy [52] . a center for disease control and prevention campaign, with input from partners, helped increase hl [53] . hl has also been shown to be associated with health and health behaviour in general. hence, one would expect that this association would hold in coronavirus outbreak situations. communicative hl included the measurement of access to different sources of information. whether this had anything to do with better decisions about health in relation to any of the three outbreaks, remained unclear. knowledge items were generally devised by the authors, and very few reported to have items checked against guidelines. this and the lack of an objective standard for cut-offs make knowledge assessment arbitrary as it cannot be established whether knowledge items reflect current and correct evidenced knowledge. similarly, while risk perceptions generally pertain to perceptions of vulnerability/susceptibility to and severity of a disease, they were not always measured accordingly or sometimes subsumed under the term attitudes or knowledge. we also observed very little evidence about the psychometric properties of instruments used to measure socio-cognitive variables such as attitude, risk perceptions, and self-efficacy. it is desirable to know whether measures are reliable and valid, and sensitive to change if the aim is to reflect the effects of health literacy interventions by e.g. education (responsiveness). even if knowledge, attitudinal constructs, risk perceptions or self-efficacy were composed in a clear-cut and unequivocal way and psychometrically sound, uncertainty as to whether hl in its broader definition [9, 54] as a composite/compound construct was measured, would still prevail. hl was proposed to be a latent construct [46] thus indicators for its measurement are necessary. there is the need for the development of adequate measurement models. the present review cannot ascertain, whether established instruments such as tofhla (test of functional health literacy) [55] , or the broader dimension based instruments, for instance the hlq (health literacy questionnaire) [56] could be used to predict a pattern of association between hl and epidemic or pandemic outcomes (and antecedents such as favourable behaviours and practices), because no such investigations appear to have been carried out, yet. the study [31] that used a short form of the j o u r n a l p r e -p r o o f hls-eu-q did not investigate the relationship between hl and pandemic outcome/preventive behaviour but coping responses to the outbreak (depression, quality of life). okan et al. [30] reported individuals' subjective perceptions about how well they could access, understand, appraise and apply information in the sars-cov-2 context but did not test the actual level of what these skills pertain to and whether they are related to better/more favourable behaviour/practices. further, it also not possible to state at present whether pandemic outbreaks require a specific hl instrument, that is able to explain variance in relevant behaviour and practices over and above that of general instruments (i.e. latent trait/construct measured by discrete manifest cognitive antecedents of behaviour). in this rapid review, the systematic search was restricted to two major data bases and no grey literature search was conducted. also, as this review was conducted as a scoping review, we did not look at the strengths of any reported associations between hl aspects and behavioural aspects. further, it is beyond the scope of this review to assess the quality of the reviewed studies according to standard guidelines for observational studies. at present hl in the context of coronavirus outbreaks is at an early stage to inform public health/educational strategies aimed at improving the public's hl in order to contain the spread of pandemics. one study [29] appears to be able to shed light on the question of whether hl related aspects change over the course of the pandemic as its survey is conducted in weekly intervals. we recommend future research be guided by theory from hl research [9, 48] in the much needed work on hl in pandemic outbreak situations. consequently, assessment of hl should be based on the ability to access, understand, critically appraise and eventually apply information to make better choices about one's health in pandemic outbreak situations when viewed as a set of meta-cognitive skills or a latent trait [46] . nevertheless, operationalisations at the manifest level, for example, knowledge, or attitudes (which influence critical appraisal) need to be considered, as latent constructs cannot be directly measured. nevertheless, in the interim, public health communication could benefit from what is generally known from hl research. health information should be clear so that all members of the public can access needed health information for routine and critical decisions [57] . beside theory-driven observational studies, we also need interventions, examining whether coronavirus pandemic-related hl can be improved. in addition, research should also attempt to develop hl instruments that measure coronavirus pandemic-related hl and test the reliability, validity and responsiveness to change. the latter is of particular importance, if we want to be able to examine change during the stages of a pandemic. records identified through database searching (n =3394) novel coronavirus(2019-ncov)situation report-121 can we contain the covid-19 outbreak with the same measures as for sars? severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges covid-19 and italy: what next? physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis covid-19 pandemic: the effects of quarantine on cardiovascular risk kidney disease is associated with in-hospital death of patients with covid-19 psychosocial impact of quarantine measures during serious coronavirus outbreaks: a rapid review health literacy and public health: a systematic review and integration of definitions and models health literacy: the solid facts, world health organization health literacy as a public health goal: a challenge for contemporary health education and communication strategies into the 21st century critical health literacy and the covid-19 crisis covid-19: health literacy is an underestimated problem examining health literacy disparities in the united states: a third look at the national assessment of adult literacy (naal) functional health literacy and health-promoting behaviour in a national sample of british adults the costs of limited health literacy: a systematic review the relationship between health, education, and health literacy: results from the dutch adult literacy and life skills survey the prevalence of limited health literacy health literacy and infectious diseases: why does it matter? world health organisation, middle east respiratory syndrome coronavirus (mers-cov) world health organisation, sars (severe acute respiratory syndrome) an evidence-based approach to scoping reviews scoping studies: towards a methodological framework joanna briggs institute reviewer's manual a typology of reviews: an analysis of 14 review types and associated methodologies evidence summaries: the evolution of a rapid review approach health literacy in the context of pandemic and epidemic (outbreak): rapid scoping review germany covid-19 snapshot monitoring (cosmo germany): monitoring knowledge, risk perceptions, preventive behaviours, and public trust in the current coronavirus outbreak in germany gesundheitskompetenz der bevölkerung im umgang mit der coronavirus-pandemie people with suspected covid-19 symptoms were more likely depressed and had lower health-related quality of life: the potential benefit of health literacy awareness, attitudes, and actions related to covid-19 among adults with chronic conditions at the onset of the u.s. outbreak: a cross-sectional survey sources of information and health beliefs related to sars and avian influenza among chinese communities in the united kingdom and the netherlands, compared to the general population in these countries the effects of repetitive information communication through multiple channels on prevention behavior during the 2015 mers outbreak in south korea exploring the determinants of perceived risk of middle east respiratory syndrome (mers) in korea immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china a longitudinal study on the mental health of general population during the covid-19 epidemic in china your health essential for your hajj": muslim pilgrims' knowledge, attitudes and practices regarding middle east respiratory syndrome coronavirus (mers-cov) during hajj season travel health risk perceptions of chinese international students in australia -implications for covid-19 responses of chinese elderly to the threat of severe acute respiratory syndrome (sars) in a canadian community the perceived threat of sars and its impact on precautionary actions and adverse consequences: a qualitative study among chinese communities in the united kingdom and the netherlands the experience of quarantine for individuals affected by sars in toronto experiencing sars: perspectives of the elderly residents and health care professionals in a hong kong nursing home qualitative study on the shifting sociocultural meanings of the facemask in hong kong since the severe acute respiratory syndrome (sars) outbreak: implications for infection control in the post-sars era health literacy: a prescription to end confusion measuring health literacy in populations: illuminating the design and development process of the european health literacy survey questionnaire (hls-eu-q) measuring health literacy regarding infectious respiratory diseases: a new skills-based instrument advancing health literacy: a global challenge for the 21st century the theory of planned behaviour: reactions and reflections the health belief model: explaining health behavior through expectancies handbook of health behavior research 1: personal and social determinants caring for critically ill patients with ebola virus disease lessons of risk communication and health promotion -west africa and united states a tale of two health literacies: public health and clinical approaches to health literacy the test of functional health literacy in adults: a new instrument for measuring patients' literacy skills the grounded psychometric development and initial validation of the health literacy questionnaire (hlq) health literacy and early insights during a pandemic knowledge and perceptions of covid-19 among the general public in the united states and the united kingdom: a cross-sectional online survey use of rapid online surveys to assess people's perceptions during infectious disease outbreaks: a cross-sectional survey on covid-19 is pakistan prepared for the covid-19 epidemic? a questionnaire-based survey study of knowledge, attitude, anxiety & perceived mental healthcare need in indian population during covid-19 pandemic knowledge, attitudes, and practices towards covid-19 among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online cross-sectional survey knowledge, attitude and practice towards sars media effects on students during sars outbreak the public's response to severe acute respiratory syndrome in toronto and the united states sars risk perception, knowledge, precautions, and information sources, the netherlands improving older adults' knowledge and practice of preventive measures through a telephone health education during the sars epidemic in hong kong: a pilot study psychosocial factors predicting sars-preventive behaviors in four major sars-affected regions restaurant diners' self-protective behavior in response to an epidemic crisis stigmatization of newly emerging infectious diseases: aids and sars social factors associated with aids and sars the singaporean response to the sars outbreak: knowledge sufficiency versus public trust an evaluation of information dissemination during the severe acute respiratory syndrome (sars) outbreak among selected rural communities in kuala kangsar estimation of the impact of providing outpatients with information about sars infection control on their intention of outpatient visit monitoring community responses to the sars epidemic in hong kong: from day 10 to day 62 sars-related perceptions in hong kong sars preventive and risk behaviours of hong kong air travellers the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong longitudinal assessment of community psychobehavioral responses during and after the 2003 outbreak of severe acute respiratory syndrome in hong kong a tale of two cities: community psychobehavioral surveillance and related impact on outbreak control in hong kong and singapore during the severe acute respiratory syndrome epidemic community psychobehavioural surveillance and related impact on outbreak control in hong kong and singapore during the sars epidemic war with sars: an empirical study of knowledge of sars transmission and effects of sars on work and the organisations population-based post-crisis psychological distress: an example from the sars outbreak in taiwan crisis prevention and management during sars outbreak a study on sars awareness and healthseeking behaviour -findings from a sampled population attending national healthcare group polyclinics the knowledge level and precautionary measures taken by older adults during the sars outbreak in hong kong severe acute respiratory syndrome epidemic and change of people's health behavior in china an outbreak of the severe acute respiratory syndrome: predictors of health behaviors and effect of community prevention measures in hong kong, china psychosocial factors influencing the practice of preventive behaviors against the severe acute respiratory syndrome among older chinese in hong kong public perceptions of quarantine: community-based telephone survey following an infectious disease outbreak sars knowledge, perceptions, and behaviors: a comparison between finns and the dutch during the sars outbreak in 2003 practice of habitual and volitional health behaviors to prevent severe acute respiratory syndrome among chinese adolescents in hong kong knowledge of and attitudes toward severe acute respiratory syndrome among a cohort of dental patients in hong kong following a major local outbreak perceived threat, risk perception, and efficacy beliefs related to sars and other (emerging) infectious diseases: results of an international survey bin saeed, knowledge, attitude and practice of secondary schools and university students toward middle east respiratory syndrome epidemic in saudi arabia: a cross-sectional study awareness among a saudi arabian university community of middle east respiratory syndrome coronavirus following an outbreak awareness, attitudes, and practices related to coronavirus pandemic among public in saudi arabia to what extent are arab pilgrims to makkah aware of the middle east respiratory syndrome coronavirus and the precautions against it? association between australian hajj pilgrims' awareness of mers-cov, and their compliance with preventive measures and exposure to camels camel exposure and knowledge about mers-cov among australian hajj pilgrims in 2014 public response to mers-cov in the middle east: iphone survey in six countries knowledge and awareness of middle east respiratory syndrome coronavirus among saudi and non-saudi arabian pilgrims knowledge and apprehension of dental patients about mers-a questionnaire survey mers-cov infection: mind the public knowledge gap hajj pilgrims knowledge about middle east respiratory syndrome coronavirus identification of information types and sources by the public for promoting awareness of middle east respiratory syndrome coronavirus in saudi arabia adequacy of public health communications on h7n9 and mers in singapore: insights from a community based crosssectional study knowledge and practices regarding middle east respiratory syndrome coronavirus among camel handlers in a slaughterhouse the effects of social determinants on public health emergency preparedness mediated by health communication: the 2015 mers outbreak in south korea preventive behaviors by the level of perceived infection sensitivity during the korea outbreak of middle east respiratory syndrome in 2015 tuning in and catching on? examining the relationship between pandemic communication and awareness and knowledge of mers in the usa effectiveness of an education health programme about middle east respiratory syndrome coronavirus tested during travel consultations public awareness of coronavirus in al-jouf region knowledge, attitudes and practices concerning middle east respiratory syndrome among umrah and hajj pilgrims in samsun australian hajj pilgrims' knowledge about mers-cov and other respiratory infections middle east respiratory syndrome risk perception among students at a university in south korea associations between hand hygiene education and self-reported hand-washing behaviors among korean adults during mers-cov outbreak the effects of sns communication: how expressing and receiving information predict mers-preventive behavioral intentions in south korea germany covid-19 snapshot monitoring (cosmo germany): monitoring knowledge, risk perceptions, preventive behaviours, and public trust in the current coronavirus outbreak in germany gesundheitskompetenz der bevölkerung im umgang mit der coronavirus-pandemie to what extent are arab pilgrims to makkah aware of the middle east respiratory syndrome coronavirus and the precautions against it? identification of information types and sources by the public for promoting awareness of middle east respiratory syndrome coronavirus in saudi arabia  yes;  no; n.a.: not applicable; hisb: health information seeking behaviour; n.r.: not reported; * includes related concepts (e.g. outcome expectancies, response efficacy); † perceived vulnerability and/or severity; ‡ skills (self-efficacy, skills, preparedness)j o u r n a l p r e -p r o o f 25 [29, 61] 10 [33, [63] [64] [65] [66] 68, 73, 87, 91, 94] 9 [38, 97, 98, 102, 104, 107, 112, 113, 116] transmission mode 4 [29, 36, 37, 60, 62] 18 [63] [64] [65] 67, 68, [72] [73] [74] [75] [76] [78] [79] [80] [81] [82] [84] [85] [86] [87] 18 [34, 38, [95] [96] [97] [98] [101] [102] [103] [104] [106] [107] [108] [111] [112] [113] 115, 116] symptoms 6 [29, 32, [58] [59] [60] [61] [62] 8 [33, 63, 64, 68, 72, 87, 91, 94] 13 [34, 38, [95] [96] [97] [98] [99] [100] [101] [102] 104, 112, 113, 116] [108] [109] [110] hb: health behaviour; hisb: health-information seeking behaviour; note: cited references do not correspond to number of studies but publications j o u r n a l p r e -p r o o f key: cord-286072-kgpvdb42 authors: sa ribero, margarida; jouvenet, nolwenn; dreux, marlène; nisole, sébastien title: interplay between sars-cov-2 and the type i interferon response date: 2020-07-29 journal: plos pathog doi: 10.1371/journal.ppat.1008737 sha: doc_id: 286072 cord_uid: kgpvdb42 the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) is responsible for the current covid-19 pandemic. an unbalanced immune response, characterized by a weak production of type i interferons (ifn-is) and an exacerbated release of proinflammatory cytokines, contributes to the severe forms of the disease. sars-cov-2 is genetically related to sars-cov and middle east respiratory syndrome-related coronavirus (mers-cov), which caused outbreaks in 2003 and 2013, respectively. although ifn treatment gave some encouraging results against sars-cov and mers-cov in animal models, its potential as a therapeutic against covid-19 awaits validation. here, we describe our current knowledge of the complex interplay between sars-cov-2 infection and the ifn system, highlighting some of the gaps that need to be filled for a better understanding of the underlying molecular mechanisms. in addition to the conserved ifn evasion strategies that are likely shared with sars-cov and mers-cov, novel counteraction mechanisms are being discovered in sars-cov-2–infected cells. since the last coronavirus epidemic, we have made considerable progress in understanding the ifn-i response, including its spatiotemporal regulation and the prominent role of plasmacytoid dendritic cells (pdcs), which are the main ifn-i–producing cells. while awaiting the results of the many clinical trials that are evaluating the efficacy of ifn-i alone or in combination with antiviral molecules, we discuss the potential benefits of a well-timed ifn-i treatment and propose strategies to boost pdc-mediated ifn responses during the early stages of viral infection. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a beta-coronavirus that emerged at the end of 2019 in china and rapidly spread around the world, causing a pandemic [1, 2] . sars-cov-2 infection is responsible for covid-19, a disease associated with mild symptoms in the majority of cases but that can progress to an acute respiratory distress syndrome [1, 3] . so far (july 16th, 2020), the virus has infected more than 13 million people and caused more than 500,000 deaths worldwide. sars-cov-2 is genetically related to other betacoronaviruses that have caused epidemics: sars-cov and mers-cov (for middle east respiratory syndrome-related coronavirus), in 2003 phosphorylation, irf3 and/or irf7 dimerize and translocate into the nucleus, where they induce the expression of ifn-i and a subset of isgs referred to as early isgs (reviewed in [13] ). secreted ifn-i then bind to the interferon alpha and beta receptor (ifnar, composed of the ifnar1 and ifnar2 subunits), leading to the activation of the jak tyrosine kinases tyrosine kinase 2 (tyk2) and janus kinase 1 (jak1), which in turn phosphorylate the signal transducer and activator of transcription (stat)1 and stat2 [14, 15] . phosphorylated stats heterodimerize and associate with the dna binding protein irf9 to form a complex known as ifn-stimulated growth factor 3 (isgf3). the isgf3 complex translocates into the nucleus and binds to interferon-stimulated response elements (isres) in isg promoters, thus inducing the expression of hundreds of isg products that establish the antiviral state at the site of viral infection [15] . the antiviral response is intensified by various signaling factors, including sensors and transcriptional regulators, which are themselves isgs induced by isgf3 and/ or directly by the irf3/irf7 transcriptional activators. aside from the ifn-i response, the recognition of double-stranded viral rna elements by the protein kinase receptor (pkr) triggers a translational arrest in infected cells (reviewed in [8, 16, 17] ). this host response is highly connected to the ifn-i response because pkr is also an isg (reviewed in [16, 18] ). ifn-i response requires fine-tuning because its overactivation is deleterious to the host. notably, some isgs are involved in the regulation of cell metabolism, intracellular rna degradation, translation arrest, and cell death, for which changes can be potentially detrimental to the host. ifn-i also potentiates the recruitment and activation of various immune cells. thus, although a robust ifn-i response is required as a first line of defense against viral infections, systemic/uncontrolled or prolonged ifn-i production can lead to inflammatory diseases. for example, an exacerbated ifn-i response contributes to the development of autoimmune diseases [19] . covid-19 is no exception to the rule, and it is therefore critical to understand the regulation of the ifn-i response upon infection. sars-cov-2 is a poor inducer of ifn-i response in vitro and in animal models as compared with other respiratory rna viruses [20, 21] . ifn-i levels in the serum of infected patients are below the detection levels of commonly used assays, yet isg expression is detected [4, 22] , thus suggesting that a limited ifn-i production could be sufficient to induce isgs. alternatively, ifn-i production could be restricted to specific immune cells, such as plasmacytoid dendritic cells (pdcs). despite a more efficient replication in human lung tissues, sars-cov-2 induced even less ifn-i than sars-cov [4] , which is itself a weak inducer in human cells [23] [24] [25] ]. an ineffective ifn-i response seems to be a hallmark of other coronavirus infections, as observed with mers-cov in ex vivo respiratory tissue cultures [26] and with animal coronaviruses such as the porcine epidemic diarrhea virus (pedv) or the mouse hepatitis virus (mhv), which are alpha-and beta-coronaviruses, respectively [27, 28] . indeed, coronaviruses have developed multiple strategies to escape and counteract innate sensing and ifn-i production. sars-cov encodes at least 10 proteins that allow the virus to either escape or counteract the induction and antiviral action of ifn (fig 2 and table 1 ) [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] . initial observations already suggest that the sars-cov-2 anti-ifn arsenal is at least as efficient as that of sars-cov [4, 20, 22] , although detailed mechanistic studies are required to determine whether the ifn antagonists identified in other coronaviruses have equivalently competent counterparts in sars-cov-2. a virus-cell protein interaction map performed with 26 of the 29 sars-cov-2 proteins expressed in human embryonic kidney (hek)293t identified several innate immune signaling proteins as partners of viral proteins cells (fig 1) [5] . sars-cov-2 orf9b, like sars-cov orf9b, interacts with mavs through its association with tom70, thus suggesting a conserved mechanism of ifn-i evasion [5, 40] (fig 1) . furthermore, sars-cov-2 nsp13 and nsp15 were found to interact with tbk1 and the tbk1 activator ring finger protein 41 (rnf41)/nrdp1, respectively [5] (fig 1) . nsp15, which is a highly conserved endoribonuclease encoded by various coronaviruses, including sars-cov [30, 49, 50], antagonizes the induction of ifn-i by cleaving the 5 0 -polyuridines of the negative-sense viral rna, as demonstrated for mhv and pedv in various cellular models [31, 32, 50] (table 1 and fig 2) . if further validated, the interaction between sars-cov-2 nsp15 and tbk1 may reveal that the viral endoribonuclease antagonizes ifn induction via at least 2 mechanisms. sars-cov orf3b was reported to inhibit ifn induction and to act either on irf3 or possibly on mavs because it translocates to mitochondria when overexpressed in vero cells [36, 42] . despite the fact it encodes a shorter protein than sars-cov, sars-cov-2 orf3b was recently found to suppress ifn induction even more efficiently [51] . by screening 15,000 sars-cov-2 sequences, the authors identified a natural variant encoding a longer orf3b and displaying an even greater inhibitory activity [51] . finally, sars-cov-2 nsp1 was also recently found to bind 40s ribosomal subunits (fig 1) , thus inhibiting host mrna translation, including that of ifn-i on this cartoon are schematically represented the signaling pathways triggered by sars-cov rna recognition by the cytoplasmic rna sensors rig-i and mda5, which leads to ifn induction (a) and subsequent ifn signaling in surrounding cells, resulting in the expression of isgs (b). sars-cov proteins that have been reported to interfere with these pathways are indicated. ifn, interferon; ifnar, interferon alpha and beta receptor; iκb, inhibitor of nuclear factor κb; ikkε, iκb kinase-ε; irf, ifn regulatory factor; isg, ifn-stimulated gene; jak, janus kinase; m, membrane; mavs, mitochondrial antiviral signaling protein; mda5, melanoma differentiation-associated gene 5; n, nucleocapsid; nsp, nonstructural protein; orf, open reading frame; p, phosphate; plp, papain-like protease; rig-i, retinoic acid-inducible gene 1; sars-cov, severe acute respiratory syndrome coronavirus; stat, signal transducer and activator of transcription; tank, traf family member associated nf-κb activator; tbk1, tank-binding kinase 1; traf3, tumor necrosis factor receptor-associated factor 3; tyk2, tyrosine kinase 2. https://doi.org/10.1371/journal.ppat.1008737.g002 [52], a feature that was previously demonstrated for other coronavirus-encoded nsp1, including sars-cov [43, 45] (fig 2 and table 1) . another viral strategy to inhibit ifn-i signaling is to enhance the host retrocontrol of this pathway. several isgs are themselves repressors of the ifn-i response, and their regulatory functions operate at the viral and host mrna transcription and translation steps, acting via a wide-range of mechanisms (reviewed in [7, 53] ). for example, the inducible negative regulators such as the suppressor of cytokine signaling (socs1 and socs3) act at various levels of the jak-stat pathway or by targeting irf7 for degradation [54] . in the context of sars-cov, the s protein induces the expression of socs3 expression in b cells [55] . induction of socs1/ 3 expression is also detected in sars-cov-infected cells, albeit to a lower extent as compared with other respiratory viruses [56] . recent genomic screen approaches identified a set of repressors of the ifn-i response depending on the cell type and activation pathway involved [57] [58] [59] . hence, one might anticipate that distinct repressors of the ifn-i response are induced depending on the cell type targeted by sars-cov-2, the level of replication, and the microenvironment. for example, in the context of coronaviruses, an inefficient detection of mhv infection likely results from an inhibition of the basal levels of sensors mrna expression in several cell types [60] . it is conceivable that this inhibition might involve negative regulators such as the ifn-inducible rnf125, which targets signaling components such as rig-i, mda5, and mavs for degradation [61] . inhibition of protein synthesis is a conserved host response to prevent viral infections. the host translation is dynamically regulated by pkr, activated via recognition of viral rna (reviewed by [62] ). activated pkr inhibits the eukaryotic initiation factor 2 (eif2α), a major regulator of the initiation phase of mrna translation, by phosphorylating its α subunit. the pkr-induced translational arrest shuts down the negative feedback on the ifn-i response, which can thus result in a prolonged and/or amplified ifn-i response [63] . because pkr is an isg, the translational arrest is, in turn, potentiated by the ifn-i response (reviewed in [64] ). this highlights a paradoxical situation in which translation arrest prevents viral replication but also set a threshold of viral detection to commensurate the host transcriptional antiviral response to the level of infection [63] . whether the pkr pathway is modulated by sars-cov2 is unknown, yet different coronaviruses regulate pkr-eif2α axis and host translation. for example, the mers-cov protein 4a (p4a) accessory protein impedes pkr activation [65, 66] . future studies are needed to further uncover the relationship between ifn-i response and host translation and their dynamics in the context of sars-cov2 infection. the ifn-i response varies among different cell types and within different microenvironments. studies at the single-cell level suggest that the amplitude and kinetic of the response is also heterogeneous for a given cell type. mathematic modeling revealed that ifn-i response is, at least in part, stochastic because only a fraction of cells are able to produce ifn-i upon activation by agonists of the sensors and are sensitive to the paracrine stimulation by ifn-i [67] [68] [69] [70] [71] . the heterogeneity of the ifn-i response can be imprinted by the state of the cell at the activation time, including its global translation activity, metabolism, expression levels of signaling molecules (sensors, adaptors, and receptors) [67] [68] [69] [70] . additionally, the distinct onsets of the ifn-i induction depend on the rapidity and amplitude of viral replication. this heterogeneous responsiveness at the individual cell level consequently shapes the dynamics of the host antiviral response at the whole population level [69] [70] [71] . this model of the ifn-i response dynamics yielded in the context of various rna viruses provides a framework likely at play for coronavirus infections. a delayed induction of the isg expression via virus-induced modulation of the basal activity of transcriptional activity of stat1 and pkr pathways leads to a peak of coronavirus replication preceding the isg response [72] . additionally, in vivo study of the dynamic of mhv infection showed that a fast and robust ifn-i production by pdcs down-regulate the ifn-i response by other cells [73] . this suggests that the ifn-i response at play in different cell types might drive the control of coronavirus infection and potentially contribute to the progression of the disease. as mentioned above, coronaviruses possess various mechanisms to defeat the ifn-i response within infected cells, and this inhibition ability is associated with clinical severity (reviewed in [74] ). clinical studies showed that coronaviruses evade innate immunity during the first 10 days of infection, which corresponds to a period of widespread inflammation and steadily increasing viral load [75, 76] . elevated virus replication eventually leads to inflammation and hypercytokinemia, referred to as a "cytokine storm" [77] [78] [79] [80] (fig 3) . the delayed ifn-i response indeed promotes the accumulation of pathogenic monocyte-macrophages [77, 81] . this cell infiltrate results in lung immunopathology, vascular leakage, and suboptimal t cell response [77, 81] . immune phenotypic profiling in peripheral blood mononuclear cells (pbmcs) of covid-19 patients similarly revealed that high viremia is associated with an exacerbated ifn-i response, an aggravated cytokine secretion, and inflammation, driving clinical severity [22] . although the ifnar signaling pathway was up-regulated at an earlier disease stage, down-regulation of isgs, together with exacerbated nf-κb activation, promotes a cytokine storm and hyperinflammation, found in critically ill patients [22] . collectively, these findings highlight the negative impact of a delayed ifn-i response on viral control and disease severity. however, the underlining mechanisms that drive the temporal control of the ifn-i response in patients are still elusive. in particular, the host and viral determinants driving the on/off switch of the ifn-i response in infected cells, noninfected cells, and/or stimulated immune cells need to be investigated. such studies will certainly benefit from longitudinal studies of immune profiling in sars-cov2 infected patients at the single-cell level and in combination with the clinical data. by producing 1,000-fold more ifn-i than any other cell types, pdcs are at the heart of the antiviral ifn-i response [82, 83] . they also produce other proinflammatory cytokines, which contribute to modulate the function of several immune cells, such as the mobilization of natural killer (nk) cells or the licensing of virus-specific t cell responses [82] [83] [84] [85] . because pdcs are refractory to most viral replication, their antiviral response cannot be inhibited by viral proteins [82, 86] . this unopposed response likely contributes to the exceptional magnitude of pdc ifn-i production [82, [87] [88] [89] . pdcs are circulating immune cells; nonetheless, their response is mostly localized at the infection site because their activation requires physical contact with infected cells [82, 83, 90] . the contact site between pdcs and infected cells, which we named the interferogenic synapse, is a specialized platform for pamp transfer from infected cell to the toll-like receptor 7 (tlr7) sensor in pdc, leading to an antiviral response [91] . previous studies on sars-and mers-cov demonstrated that the rapid production of ifn-i by pdcs is essential for the control of potentially lethal coronavirus infections in mouse models [77, 92] . pdcs migrate into the lungs at the early phase of infection (i.e., pdc number peaks at day 2), temporally coinciding with the peak of ifnα production [92, 93] . the pdc number was found to be reduced in blood of covid-19 patients as compared with control patients [22] , potentially resulting from a prior response followed by a vanished number of circulating pdcs and/or their mobilization to the infected site. future studies are needed to address how pdc responsiveness evolves in the course of sars-cov-2 infection and how pdcs respond to contact with coronavirus-infected cells. despite the abovementioned viral inhibitory mechanisms of ifn-i response (table 1) , exogenous ifn-i in cell cultures efficiently inhibit sars-cov, sars-cov-2, and mers-cov spread [5, 20, 26, 51, [94] [95] [96] [97] [98] [99] [100] [101] . consistently, ifn-i was shown to have a protective effect against sars-cov and mers-cov, alone or in combination with other antivirals, in various animal models including mice, marmosets, and macaques [97, 102, 103] . ifn-i and iii interfere with virus infection by inducing the expression of several hundred isgs [7] . numerous welldescribed isgs exhibit direct antiviral activities by targeting specific stages of the viral life cycle, including entry into host cells, replication, protein translation, and assembly of new virus particles. as mentioned above, many signaling regulators are themselves isgs, thus leading to amplification of the antiviral ifn-i pathway. as a first step towards identifying isgs able to restrict sars-cov-2 replication, transcriptomic responses to infection have been analyzed in different cellular models, including primary cells, organoids, and clinical samples [20, [104] [105] [106] , as summarized in table 2 . these studies demonstrate that, despite triggering very little to no ifn expression (table 2) , sars-cov-2 replication induces moderate levels of a limited number of isgs. a small subset of infected cells may be refractory to the antagonistic mechanisms of sars-cov-2, producing minute but sufficient amounts of ifns to trigger isg induction in larger population of cells. alternatively, isgs may be up-regulated in noninfected cells, which were analyzed together with infected ones. indeed, interpretation of genome-wide investigations of virus-pathogen interactions are often obscured by analyses of mixed populations of infected and uninfected cells [107] . of note, by contrast to low-multiplicity of infection (moi) infection of a549 cells expressing angiotensin i converting enzyme 2 (ace2), normal human bronchial epithelial (nhbe) cells, and patient samples, high-moi infections of a549-ace2 and calu-3 cells led to the high induction of ifns and isgs, including isgs with broad antiviral activities [20] ( table 2 ). this discrepancy of ifn production/signaling between the levels of viral replication and/or proportion of infected cells might reflect that the counteraction measures employed by sars-cov-2 are less potent at high moi. alternatively, as suggested by blanco-melo and colleagues, high-moi infections in cell culture may generate more pamps, such as defective noninfectious viral particles, than low-moi infections [108] . despite being expressed at moderate levels in vitro and in vivo, several up-regulated isgs identified by these transcriptomic studies ( table 2 ) exhibit well-characterized broad-spectrum antiviral activities and could thus have additive restrictive effects on sars-cov-2 replication. for instance, the 3 members of the interferon-induced protein with tetratricopeptide repeats (ifitm) family, known to inhibit entry of numerous enveloped rna viruses [109] , similarly restrict entry of sars-cov, mers-cov, and the globally circulating human coronaviruses 229e and nl63 in 293t and a549 cell lines [110, 111] . oas1 and mycovirus resistance protein (mx)a could also contribute to the ifn-i-mediated inhibitory effect on sars-cov-2 because a clinical study revealed that single nucleic polymorphisms in the oas1 3 0 -utr and mxa promoter region appear associated with host susceptibility to sars-cov in the chinese han population [112] . moreover, the fact that mers-cov nonstructural protein 4b (ns4b) is a 2 0 -5 0 oligoadenylate synthetase (oas)-rnase l antagonist [113] suggests that the oas pathway contributes to the antiviral effects of ifns on coronavirus replication. isgs positively potentiating ifn signaling, such as ifih1/mda5, tank, irf7, and stat1, were also increased in the bronchoalveolar lavage fluid (balf) of covid-19 patients as compared with healthy controls [105] and could potentially contribute to the amplification of ifn-i response against sars-cov-2 replication. zinc finger antiviral protein (zap), which is encoded by an isg, contributes to the anti-sars-cov-2 effect of ifns in human lung calu-3 cells [114] . zap is known for restricting the replication of numerous viruses such as retroviruses and filoviruses [115] . the protein recruits the cellular mrna degradation machinery to viral rna via 5 0 -c-phosphate-g-3 0 (cpg) dinucleotide recognition [115] . to further determine which individual isg or combination of isgs mainly restricts sars--cov-2 replication in vitro, several previously established approaches could be used, such as, for example, screening for single or combined isg activity using a lentiviral vector-based library, as successfully performed by schoggins and colleagues for other viral infections [116] [117] [118] [119] . indeed, this library of around 380 human isgs was recently screened in human hepatoma cells for antiviral activity against hcov-229e [120] . the screen identified ifn-inducible lymphocyte antigen 6 complex, locus e (ly6e) as a potent inhibitor of the replication of multiple coronaviruses, including sars-cov, sars-cov-2, and mers-cov, by blocking fusion of viral and cellular membranes [120] . mice studies revealed that ly6e directly protects primary b cells and dendritic cells from murine coronavirus infection [120] . pursuing the identification and characterization of ifn effectors with potent anti-sars-cov-2 activities will reveal weakness points in the life cycle of sars-cov-2 and may lead to the design of drugs that activate antiviral isgs or either mimic or amplify their action. recent advances in systematic screening strategies have revealed the existence of a small subset of isgs exhibiting proviral activities [116, 119] . these proviral isgs act either by exhibiting direct proviral activities such as facilitating viral entry [119] or via their abilities to negatively regulate ifn signaling and facilitate the return to cellular homeostasis. the receptor tyrosine kinase axl is a well-characterized example of an isg that is used by enveloped virus for cellular internalization [121] [122] [123] . alternatively, isgs that possess antiviral activities against a viral family can be hijacked by unrelated viruses to favor infection. this is the case for ifitm2 and ifitm3, which potently block entry of a broad range of enveloped viruses [109] while promoting entry step of human coronavirus oc43 (hcov-oc43) in human cells [124] . sars-cov-2 uses ace2 and transmembrane serine protease 2 (tmprss2) to enter cells [125] . viral tropism is thus largely dictated by ace2 and tmprss2 coexpression. analysis of human, nonhuman primate, and mouse single-cell rna-sequencing (scrna-seq) data sets generated from healthy or diseased individuals revealed that expression of ace2 is primarily restricted to type ii pneumocytes in the lung, absorptive enterocytes within the gut, and goblet secretory cells of the nasal mucosa [126] . interestingly, this meta-analysis identified an association between ace2 expression and canonical isgs or components of the ifn-signaling pathway in different tissues. independent analyzes of publicly available data sets concluded that ace2 expression pattern is similar to isgs [127] . in vitro validations were performed by treating primary human upper airway cells with numerous inflammatory cytokines. ifnα2, and to some extent ifny, led to greater and more significant ace2 up-regulation compared with all other tested cytokines [126] . substantial up-regulation of ace2 was also observed in primary skin and primary bronchial cells treated with either ifn-i or ifn-ii. moreover, ace2 expression was also up-regulated upon ex vivo influenza a infection in human lung explants isolated following surgical resection [126] . because the majority of cells robustly up-regulating ace2 were epithelial, this observation potentially explains why previous analyses to define canonical isgs within immune populations did not identify ace2 as an induced gene [116] . finally, stat1, stat3, irf8, and irf1 binding sites were identified within −1,500 to +500 bp of the transcription start site of ace2 [126] . despite need for direct evidence that ifns up-regulate ace2 in target cells in vivo, altogether these studies suggest that ace2 could be an isg that enhances sars-cov-2 internalization in human epithelial cells [125, 126] . elucidating tissue and cell type specificity of isgs, as well as their mechanisms of action, is essential for understanding the potential dual role of ifns during human sars-cov-2 infection. it may also guide the use of ifns in clinical trials. although ifn-i treatment gave some encouraging results against sars-cov and mers-cov in vitro and in animal models, including mice, marmosets, and macaques [97, 102, 103, 128, 129] , additional knowledge to optimize its therapeutic efficiency in humans is required [130] [131] [132] [133] [134] . previous information yielded from these animal studies provided guidance for treating the current pandemic virus. first, it became clear from these former studies that ifnβ is a more potent inhibitor than ifnα as shown both in vitro and in patients [129, 132] . second, the timing of ifn-i treatment seems determinant for infection outcomes. indeed, as shown in mice and in macaques, ifn-i is protective when administered prior to sars-cov or mers-cov infection or early in the course of infection, whereas late administration could be either ineffective or detrimental [77, 135] . in humans as well, ifn-i-based therapies were not beneficial to critically ill patients with multiple comorbidities and who were diagnosed late with mers-cov, thus pointing out that ifn-i has to be administered early after infection [134, 136] . the first clinical trials using ifn-i alone or in combination with other antivirals are currently carried out in covid-19 patients in several countries. for instance, the multicenter, adaptive, randomized, open clinical trial discovery evaluates, among other treatment, the efficacy of ifnβ as a treatment for covid-19 in hospitalized adults in europe. a recent openlabel, randomized, phase 2 trial performed in adults with covid-19 in hong kong showed that the triple combination of ifnβ-1b, lopinavir-ritonavir, and ribavirin was safe and superior to lopinavir-ritonavir alone in alleviating symptoms and shortening the duration of viral shedding and hospital stay in patients with mild to moderate covid-19 [137] . it has to be noted that the patients were treated in the early stages of the disease because the median number of days from symptom onset to start of study treatment was 5 days, further reinforcing the fact that the timing of ifn-i treatment is key [137] . other therapeutic approaches are under investigation to avoid the adverse effects of ifn-i therapy and/or its potential inefficacity when administrated too late postinfection. one strategy is to use aerosol formulations of recombinant ifn to deliver the cytokine directly inside the lung [138, 139] . this approach has several benefits because it is a noninvasive route of administration, and the local concentration reached in the tissue can be higher than through systemic injection and is thus expected to minimize the adverse effects of ifn. nebulized ifnα-2b was used on covid-19 patients in wuhan, alone or in combination with arbidol [140] . the study, performed on 77 adults, showed a significant reduction of the duration of detectable virus in the upper respiratory tract in ifnα-2b-treated patients, with or without arbidol [140] . another study currently ongoing in beijing aims at evaluating the efficacy and safety of recombinant human ifnα spray in preventing sars-cov-2 infection in highly exposed medical staffs (chictr2000030013). type iii ifns (ifnλs or ifn-iii) are gaining an increased interest in antiviral therapies [141] [142] [143] . like ifn-i, they activate the jak-stat signaling pathway. they do so via a receptor that is largely restricted to cells of epithelial origin, including respiratory epithelial cells (reviewed in [144] ). ifn-iiis are induced upon viral infections, and they are growing evidence that they provide important first-line defense against viral infections of the respiratory and gastrointestinal tracts [145] [146] [147] . in mice, ifn-iii was shown to protect epithelial cells of the respiratory and tract from infections with several respiratory viruses, including mers-cov [147] . a study investigating sars-cov-2 infection of intestinal epithelial cells, using both colon-derived cell lines and primary colon organoids, showed that ifn-iii response was more efficient than ifn-i at controlling viral replication [148] . however, ifn-iiis produced by dendritic cells in the lung were recently shown to cause barrier damage and to compromise host tissue tolerance and predispose to lethal bacterial superinfections [149] . therefore, although the antiviral properties are promising, the benefit of ifn-iii to treat covid-19 patients awaits careful evaluation. the first clinical trials using ifn-iii are ongoing, including one launched at the massachusetts general hospital to evaluate the safety and efficacy of pegylated ifnλ on a small number of covid-19 patients (nct04343976). besides the use of recombinant ifn as a therapeutic treatment, one interesting alternative strategy would be to boost the natural innate immune defenses of covd-19 patients at early stages of the disease. because pdcs are seemingly crucial to control coronavirus infections [77, 92] , a possibility would be to either amplify or prolong their activation to make them produce more ifn-i and ifn-iii. a number of negative feedback loops prevent an exacerbated activation of pdcs, which can be deleterious for the organism in the long term. thus, transitorily inhibiting these negative retrocontrols may increase the antiviral activity of pdcs. for instance, the bone marrow stromal cell antigen 2 (bst2) is an isg that activates the immunoglobulin-like transcript 7 (ilt7) inhibitory receptor expressed by pdcs to interrupt the ifn-i response [150] . the blockade of this interaction using either antibodies or inhibitory molecules should thus increase the duration of pdc activation. one could also envisage to take advantage of viral proteins that counteract the antiviral activity of bst2, such as hiv-1 viral protein u (vpu) [151] . other pdc inhibitory molecules include natural monamines such as histamine, dopamine, or serotonin, which bind to the c-x-c motif chemokine receptor 4 (cxcr4) at the surface of pdcs [152] . because the cxcr4 antagonist amd3100 (also known as plerixafor) blocks the binding of monoamines to pdcs, it can prevent the amine-dependent inhibition of pdc activation [152] . amd3100 is already used in clinics as an immunostimulatory molecule able to mobilize hematopoietic stem cells in cancer patients [153] . finally, we recently reported that the peptidyl-prolyl isomerase peptidyl-prolyl cis-trans isomerase nimainteracting 1 (pin1) switches off the ifn-i expression by pdcs by inducing irf7 degradation [154] . a number of pin1 inhibitors have been developed and could be tested for their potential activity on human pdcs [155] and could represent another possible therapeutic strategy to boost pdc-mediated ifn-i production. sars-cov-2 emerged in the human population around 7 months ago, yet it seems well adapted to avoid and inhibit the ifn-i response in its new host. such efficient strategies allow the virus to replicate and disseminate in infected individuals without encountering the initial host defense. this modest ifn response could explain why viremia peaks at early stages of the disease, at the time of symptoms appearance, and not around 7 to 10 days following symptoms, like during sars-cov and mers-cov infections. ifnβ treatment would be expected to improve the antiviral response of patients at the early stage of covid-19 and, if possible, at plos pathogens the site of infection. indeed, ifnβ appeared to be pivotal to improve patient states in a combined therapy regiment of ifnβ, lopinavir-ritonavir, and ribavirin [137] . nonetheless, ifnresistant viral mutants may arise and be able to control ifn even more efficiently than parental viruses. the exacerbated production of proinflammatory cytokines observed at later stage of covid-19 might challenge the efficiency of an ifnβ treatment administrated after appearance of symptoms. there is indeed an increasing appreciation of the detrimental effects of inappropriate, excessive, or mistimed ifn-i responses in viral infections [156] . the underlying mechanisms by which ifn-i promote disease severity likely include immunopathology due to excessive inflammation and direct tissue damage. at the late stages of covid-19, immunomodulatory drugs that diminish inflammation may benefit patients. the therapeutic benefits of such treatments have been demonstrated in the context of influenza infection in clinical trials [157] and in mice models [158] . discovery of host markers associated with disease progression will be instrumental to defining the appropriate treatment and time of administration. luckily, most covid-19 patients develop no or mild symptoms. in these patients, the virus ends up being cleared by the immune system, possibly through a partial protection conferred by cross-reactive cd4+ t cells that have been found in between 40% and 60% of unexposed individuals [159] . it is therefore probable that a viral replication that is under control thanks to an efficient adaptive immunity prevents a systemic viral spread and the subsequent cytokine storm. prior and coinfections along with age, gender, immunological state, and comorbidities also likely play a key role in the ability of the patients to efficiently respond to sars-cov-2 infection. the current studies that aim to better understand the mechanisms that render some patients particularly sensitive to sars-cov-2 infections raise hope for the possibility of treating patients with drugs that either enhance the ifn response at the early stage of the disease or dampen it at later stages. a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a sars-cov-2 protein interaction map reveals targets for drug repurposing stimulation of innate immunity by host and viral rnas interferon-stimulated genes: what do they all do? nuclear rnf2 inhibits interferon function by promoting k33-linked stat1 disassociation from dna murine coronavirus cell type dependent interaction with the type i interferon response negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 pkr: a kinase to remember protein synthesis inhibition and gadd34 control ifn-beta heterogeneous expression in response to dsrna integration of pkr-dependent translation inhibition with innate immunity is required for a coordinated anti-viral response middle east respiratory coronavirus accessory protein 4a inhibits pkr-mediated antiviral stress responses inhibition of stress granule formation by middle east respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication single-cell analysis reveals that stochasticity and paracrine signaling control interferon-alpha production by plasmacytoid dendritic cells differential induction of interferon stimulated genes between type i and type iii interferons is independent of interferon receptor abundance single-cell analysis shows that paracrine signaling by first responder cells shapes the interferon-beta response to viral infection antiviral interferon response at single-cell resolution multi-layered stochasticity and paracrine signal propagation shape the type-i interferon response pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses a systems immunology approach to plasmacytoid dendritic cell function in cytopathic virus infections sars and mers: recent insights into emerging coronaviruses clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice pathogenesis of severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome how the sars coronavirus causes disease: host or organism? ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes plasmacytoid dendritic cells: development, regulation, and function cell-cell sensing of viral infection by plasmacytoid dendritic cells plasmacytoid dendritic cells control t-cell response to chronic viral infection plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral nk and cd8(+) t cell accrual sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells pacsin1 regulates the tlr7/9-mediated type i interferon response in plasmacytoid dendritic cells ap-3, and hermansky-pudlak syndrome proteins are required for toll-like receptor signaling in plasmacytoid dendritic cells bifurcation of toll-like receptor 9 signaling by adaptor protein 3. science plasmacytoid dendritic cells control dengue and chikungunya virus infections via irf7-regulated interferon responses plasmacytoid dendritic cells and infected cells form an interferogenic synapse required for antiviral responses control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4+ t cells are important in control of sars-cov infection treatment of sars with human interferons interferon-beta 1a and sars coronavirus replication severe acute respiratory syndromerelated coronavirus is inhibited by interferon-alpha inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment sars-cov-2 is sensitive to type i interferon pretreatment antiviral activities of type i interferons to sars-cov-2 infection an infectious cdna clone of sars-cov-2 treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov. nat commun transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients heightened innate immune responses in the respiratory tract of covid-19 patients sars-cov-2 productively infects human gut enterocytes deconvolution of pro-and antiviral genomic responses in zika virus-infected and bystander macrophages the defective component of viral populations ifitm-family proteins: the cell's first line of antiviral defense ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus association of sars susceptibility with single nucleic acid polymorphisms of oas1 and mxa genes: a case-control study antagonism of dsrna-induced innate immune pathways by ns4a and ns4b accessory proteins during mers coronavirus infection the zinc finger antiviral protein restricts sars-cov-2. biorxiv 134379 novel host restriction factors implicated in hiv-1 replication a diverse range of gene products are effectors of the type i interferon antiviral response inhibition of hiv-1 particle assembly by 2',3'-cyclic-nucleotide 3'-phosphodiesterase pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity ly6e mediates an evolutionarily conserved enhancement of virus infection by targeting a late entry step ly6e impairs coronavirus fusion and confers immune control of viral disease enveloped viruses disable innate immune responses in dendritic cells by direct activation of tam receptors zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells the tim and tam families of phosphatidylserine receptors mediate dengue virus entry interferon induction of ifitm proteins promotes infection by human coronavirus oc43 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues increasing host cellular receptor-angiotensin-converting enzyme 2 (ace2) expression by coronavirus may facilitate 2019-ncov infection ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines increased sensitivity of sarscoronavirus to a combination of human type i and type ii interferons ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study sars: systematic review of treatment effects interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial fundamentals of aerosol therapy in critical care the expanding role of aerosols in systemic drug delivery, gene therapy and vaccination: an update interferon-α2b treatment for covid-19 covid-19: lambda interferon against viral load and hyperinflammation weak induction of interferon expression by sars-cov-2 supports clinical trials of interferon lambda to treat early covid-19 covid-19 and emerging viral infections: the case for interferon lambda type iii interferons: balancing tissue tolerance and resistance to pathogen invasion interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness interferon-lambdas: front-line guardians of immunity and homeostasis in the respiratory tract lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections critical role of type iii interferon in controlling sars-cov-2 infection, replication and spread in primary human intestinal epithelial cells type iii interferons disrupt the lung epithelial barrier upon viral recognition. science. 2020: eabc3545 regulation of tlr7/9 responses in plasmacytoid dendritic cells by bst2 and ilt7 receptor interaction epstein-barr virusencoded ebna1 modulates the ap-1 transcription factor pathway in nasopharyngeal carcinoma cells and enhances angiogenesis in vitro natural amines inhibit activation of human plasmacytoid dendritic cells through cxcr4 engagement mozobil(r) (plerixafor, amd3100), 10 years after its approval by the us food and drug administration trim8 is required for virusinduced ifn response in human plasmacytoid dendritic cells development of pin1 inhibitors and their potential as therapeutic agents disease-promoting effects of type i interferons in viral, bacterial, and coinfections a critical role for the sphingosine analog aal-r in dampening the cytokine response during influenza virus infection endothelial cells are central orchestrators of cytokine amplification during influenza virus infection targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals we thank nathalie j. arhel for helpful discussion. key: cord-310297-sbxlz04w authors: al awaidy, salah t.; khamis, faryal title: middle east respiratory syndrome coronavirus (mers-cov) in oman: current situation and going forward date: 2019-05-17 journal: oman med j doi: 10.5001/omj.2019.36 sha: doc_id: 310297 cord_uid: sbxlz04w nan m iddle east respiratory syndrome coronavirus (mers-cov) is a zoonotic viral respiratory illness caused by a novel betacoronavirus, which was first reported in saudi arabia in 2012. 1 since, mers-cov cases have been reported across the arabian peninsula, with occasional cases exported among travelers to other continents. 2 dromedary camels are a major reservoir host, in which the virus causes negligible disease. 3 the virus can spread from dromedary camels to humans, through direct or indirect contact, causing significant morbidity and mortality. 4 the clinical spectrum ranges from asymptomatic illness to septic shock and multiorgan failure. 5 as of january 2019, a total of 2298 laboratoryconfirmed human cases of mers-cov from 27 countries have been reported, including 811 associated deaths giving a fatality rate of 35.2%. eighty percent of cases have been reported from countries of the world health organization eastern mediterranean region. 2 human-to-human transmission of mers-cov has been described in several closed clusters of cases, including a small family cluster of mild disease. 5 two major healthcare-associated outbreaks due to superspreading events led to massive numbers of cases and excessive morbidity and mortality in several countries. 2, 6 currently, there has been no evidence of sustained human-to-human transmission. 2, 6 in oman, the first laboratory-confirmed case of mers-cov was reported in june 2013. 7, 8 up to march 2018, a total of 11 laboratory-confirmed cases have been reported sporadically. 5 ten (91%) cases were males with a mean age of 52±17.7 years (range: 20-75 years). nine cases (82%) were primary and two cases (18%) were secondary. patients were predominately from the north al batinah governorate (average rate: 1 per 100 000 population) with a fatality rate of 9%. eight cases (73%) reported domestic camel exposure. uncontrolled diabetes mellitus was the most common comorbidity in eight cases (73%). no secondary cases were reported among healthcare workers. seroprevalence studies revealed mers-cov exposure among all sampled domestic camels across oman. 9, 10 between 27 january and 12 february 2019, a total of 13 additional human cases of laboratoryconfirmed mers-cov using real-time polymerase chain reaction (rt-pcr) were reported in oman. eight (61%) and four (39%) cases were residents of north al batinah and a'south sharqiyah governorates, respectively. 11 the first cluster of five females (four from the same family), reported on 27 and 28 january 2019 were residents of north al batinah. their mean age was 42±10.8 years (range: 30-59 years). three of the five cases were probably secondary cases exposed to the index case who reported a history of direct contact with camels. none of the other cases had contact with camels. however, four cases resided on a farm where camels were kept, and the fifth case had a history of contact with one of the patient's with mers-cov at the hospital. two of the five cases died (40% fatality rate). uncontrolled diabetes mellitus and hypertension were the most common comorbidities reported among three of the cases. between 12 and 18 february 2019, north al batinah and south a'sharqiyah governorates each reported four additional cases (n = 8). these were sporadic cases with no epidemiological link to the previous cluster. seven cases (88%) were omani nationals. the mean age of cases was 55±17.5 years (range: 30-77 years) with an equal number of males and females. cases from both governorates had a history of contact with camels (community). two cases from south a'sharqiyah were admitted to intensive care units. uncontrolled diabetes mellitus, hypertension, and ischemic heart diseases were the most common comorbidities present and were reported among three cases including the deceased. the case fatality rate was 25%. through contact screening, two asymptomatic healthcare workers, one from each governate tested positive for mers-cov. both were involved in the care of mers-cov patient (one was involved in intubation). a total of 60 familial contacts from both clusters and 119 healthcare worker contacts were screened for mers-cov and monitored for 14 days from the final date of exposure. all tested negative except two. between january and march, the season of dromedary camels breeding, races take place in these governorates. it is possible that during these months there is increased mers-cov circulation in the animal reservoir. the index cases were in contact with or resided on a farm where camels were kept. individuals in close contact with dromedary camels are at an increased risk of acquiring the infection compared with the general population. 12 the transmission from camels to humans can be via direct contact with camels through respiratory droplets or saliva, or the use of camel products. 12 in the current outbreaks, we observed non-linked clusters and sporadic cases, in addition to nosocomial transmission in healthcare facilities with no evidence of sustained human-to-human transmission. 13 although most mers-cov infected healthcare workers are asymptomatic, 13 serious infections can occur, and healthcare workers might play a critical role in spreading the virus within their area of practice in healthcare facilities. 14 several factors could have contributed to the current transmission within healthcare facilities, including delays in suspicion and case detection, and providing close patient care without adherence to infection prevention and control (ipc) measures. hospital transmission of mers-cov has been attributed to suboptimal infection control practices such as lack of personal protective equipment use, poor hand hygiene, delay in timely index case isolation, lack of negative pressure rooms, and performing aerosolizing procedures without appropriate personal protective equipment. thus, there is an urgent need to reinforce the execution of basic ipc measures at all times in all healthcare facilities. other pivotal interventions in reducing nosocomial transmission include prompt triaging, staff cohorting, and excluding non-essential staff and visitors. 6, 15 the current outbreak highlights the need for increased awareness among the public, particularly in individuals with comorbidities, who are at higher risk of complications and death. awareness among this group should focus on avoiding close contact with camels or camel products, particularly in camel race festivals and breeding areas. 12 stringent efforts are required to improve ipc to prevent transmission of the virus within healthcare facilities, to reduce mortality rates, and minimize community transmission. institutional monitoring and training of healthcare workers remain the mainstay of disease prevention in healthcare facilities. 15 furthermore, urgent adoption of the 'one health' strategic approach (the collaboration of multiple disciplines and sectors working locally, nationally, and globally to attain optimal health for people, animals, and the environment), including establishing a robust, timely, integrated surveillance system, and strengthening the governorates capabilities for rapid and efficient investigation of the disease is critical in minimizing the risk of disease spread. r efer ences isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers situation update mers situation update march evidence for camel-to-human transmission of mers coronavirus surveillance for human infection with middle east respiratory syndrome coronavirus (mers-cov), who preliminary epidemiological assessment of mers-cov outbreak in south korea overview of preparedness and response for middle east respiratory syndrome coronavirus (mers-cov) in oman the struggle against mers-cov (the novel coronavirus) middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during 2013-2014: a case-control study infection control influence of middle east respiratory syndrome coronavirus: a hospital-based analysis middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control spread of mers to south korea and china key: cord-293525-c7nwygl1 authors: saldanha, i. f.; lawson, b.; goharriz, h.; rodriguez-ramos fernandez, j.; john, s. k.; fooks, a. r.; cunningham, a. a.; johnson, n.; horton, d. l. title: extension of the known distribution of a novel clade c betacoronavirus in a wildlife host date: 2019-04-03 journal: epidemiol infect doi: 10.1017/s0950268819000207 sha: doc_id: 293525 cord_uid: c7nwygl1 disease surveillance in wildlife populations presents a logistical challenge, yet is critical in gaining a deeper understanding of the presence and impact of wildlife pathogens. erinaceus coronavirus (ericov), a clade c betacoronavirus, was first described in western european hedgehogs (erinaceus europaeus) in germany. here, our objective was to determine whether ericov is present, and if it is associated with disease, in great britain (gb). an ericov-specific bryt-green(®) real-time reverse transcription pcr assay was used to test 351 samples of faeces or distal large intestinal tract contents collected from casualty or dead hedgehogs from a wide area across gb. viral rna was detected in 10.8% (38) samples; however, the virus was not detected in any of the 61 samples tested from scotland. the full genome sequence of the british ericov strain was determined using next generation sequencing; it shared 94% identity with a german ericov sequence. multivariate statistical models using hedgehog case history data, faecal specimen descriptions and post-mortem examination findings found no significant associations indicative of disease associated with ericov in hedgehogs. these findings indicate that the western european hedgehog is a reservoir host of ericov in the absence of apparent disease. the betacoronavirus genus includes multiple bat-associated viruses and several human pathogens, including severe acute respiratory syndrome coronavirus (sars-cov) and middle eastern respiratory syndrome coronavirus (mers-cov) [1, 2] . in 2013, a novel clade c betacoronavirus was discovered in the western european hedgehog (erinaceus europaeus) in germany [3] . the virus was subsequently detected in the same species in france [4] . characterisation of these erinaceus coronavirus (ericov) nucleotide sequences revealed high nucleotide identity to mers-cov [3] , the cause of an acute respiratory syndrome in humans with high case fatality rates [5, 6] . the structural spike protein is key to coronavirus virulence and host specificity [7] and the gene that encodes it plays a pivotal role in coronavirus diversity [8] . analysis of the receptorbinding region in the s protein of ericov revealed that it shared only 36.7% amino acid identity with that of mers-cov [3] . however, there have been no further published ericov whole genome sequencing attempts, and many questions remain over the origins and evolution of ericov [2] . whilst there is no known threat to public health from ericov, further investigation of the virus is warranted to gain a fuller understanding of the impact of coronavirus infection on wildlife populations. bats are widely recognised and accepted as the natural reservoir hosts of a variety of coronavirus species, including sars-cov [9, 10] . both bats and camels have been cited as possible reservoirs of mers-cov [6, 11] . it is considered that bats are the likely natural reservoir of mers-cov [2, 5] , and that dromedaries (camelus dromedarius), and potentially other species, act as an intermediate host for human transmission [6, 12, 13] . mers-cov-like virus sequences have only been detected in one other species, e. europaeus [3] . hedgehogs are members of the order eulipotyphla, which is closely related to chiroptera, the order of bats [14] . this close association is what initially led to hedgehogs being investigated as a potential coronavirus reservoir [3] . many animal species seem to have the capacity for coronavirus infection in the absence of apparent disease, including bats [15] , aquatic birds [16] and rabbits when inoculated with mers-cov [17] . however, dromedaries infected with mers-cov develop pyrexia with mild upper respiratory signs, consisting of serous to purulent nasal discharge [18] . lesions in infected dromedaries are comparable to those seen in the common cold in humans, including inflammation and loss of pseudostratified epithelial cells in the upper respiratory tract. coronaviruses that affect the gastrointestinal system, such as porcine transmissible gastroenteritis virus, typically cause similarly mild lesions, including destruction of enterocytes and villus atrophy [19] . although still widespread throughout much of western and northern europe, the western european hedgehog population in great britain (gb) has seen a rapid decline in recent decades, with an estimated population decrease of 95% between the 1950s and 1990s [20, 21] . the reasons for this decline are not fully understood, and it is likely to be multi-factorial (e.g. habitat loss and fragmentation, road traffic death and predation) [21, 22] . the possible role of infectious disease also needs to be investigated, including the consequences of infection with newlydiscovered viral agents. here, we identify the presence of ericov in british hedgehogs, sequence the full genome, explore the spatial distribution of infection and investigate if there is any association of ericov with disease. a total of 351 samples from individual hedgehogs were tested in this study. of these, 225 were voided faecal samples collected from live hedgehogs that had been found injured, sick or abandoned juveniles and were consequently admitted to wildlife centres for treatment and rehabilitation in 2014 and 2015. a total of nine wildlife centres across gb collected and supplied samples, using the methods described by sangster et al. [23] . a fresh faecal sample was collected non-invasively shortly after admission and associated metadata, including clinical signs, signalment, location and history, were collected where available. faecal samples were stored at 4 or −20°c for a maximum of 1 week at rehabilitation centres prior to submission. a subsample of each sample collected by sangster et al. [23] was used in the current study. an additional four voided faecal samples were collected from the wild as part of a greater london-based hedgehog population survey conducted in 2014 to give a total of 229 faecal samples from live animals. the remaining 122 samples were of distal large intestinal tract contents collected from dead hedgehogs during post-mortem examination (pme) from 2011 to 2015 and comprised animals with infectious and non-infectious (e.g. trauma) causes of death. these hedgehogs had either died in wildlife centres within 48 h of admission or had been found dead by members of the public. where possible, pmes were performed on fresh carcases, but frozen carcases that had been archived at −20°c were also examined. systematic examination of external and internal body systems was performed using a standardised protocol, followed by microbiological, parasitological and histological investigations where indicated by macroscopic findings and when permitted by the state of carcase preservation [24] . the contents of the distal large intestinal tract were sampled to most closely reflect freshly voided faeces, as the latter had contained the highest ericov viral titre in a previous study [3] . faecal sample submission forms provided information on: admittance date, sample collection date, the location where the hedgehog was found, sex, live body weight, faecal characteristics (including colour and consistency) and reason for admittance. pme records were available for 121 of 122 hedgehogs from which samples of distal large intestinal contents were tested. these comprised data on the location where the dead hedgehog was found, the date the carcase was found, sex, age class, carcase weight, subjective assessment of body condition (based on muscle mass and body fat deposits), gross pme findings and the results of ancillary diagnostic tests. rna stabilising agent rnalater (ambion, life technologies europe, nl) was added to, and mixed with, a pea-sized amount of each sample (faeces or distal large intestinal tract contents) in a 1.5 ml microcentrifuge tube. each sample was then stored at either −20 or −80°c. faeces were homogenised and pooled (five samples per pool). each 140 µl of total pooled sample was added to 560 µl buffer avl-carrier rna solution and extracted immediately following the spin protocol alongside negative extraction controls. a guanidine and column-based method (qiaamp viral mini kit qiagen, hilden, de) was used for rna extraction, according to the manufacturer's protocol. rna was then eluted in 60 µl of buffer ave and stored at −80°c. a specific oligonucleotide primer set (ericovr and ericovf, see supplementary table s1 ) was designed based on the ericov rna-dependent rna-polymerase (rdrp) gene sequence of the german virus sequence (kc545383) with oligoarchitect™ (sigma-aldrich®, st louis, mo, usa) and used with the bryt-green based gotaq® 1-step real-time reverse transcription pcr (rt-pcr) system kit (promega corporation, madison, wi, usa) to detect ericov rna in an applied biosystems 7500 fast real-time system. a 10 −2 dilution of extracted rna from a positive ericov sample (r618/14, previously confirmed through sequencing) was used as the positive control. nuclease-free water (promega corporation) was used as the negative control. following the amplification stages, melt curve analysis was performed in order to verify the specificity of the amplicons [25] . in addition, a 110 bp ericov ultramer oligonucleotide was used to quantify the limits of detection and assess the sensitivity of the designed assay. this was a sequence within the ericov primer rdrp target region, but with the removal of 10 nucleotides at the centre to differentiate it from wild-type ericov (see supplementary table s1 ). a 14-fold dilution series was performed and a standard curve was generated using graphpad prism 6 (graphpad software, ca, usa). to assess assay specificity, rna of the gammacoronavirus, infectious bronchitis virus (ibv), was also included as a control. to assess the primer dynamic range and potential rna degradation, a dilution series of rna extracted from faeces was tested. a 'spiked' dilution series was set containing 25% (5 µl in 20 µl) faecal rna extraction from an ericov-negative pool and a subset of negative samples (n = 11) was tested using primers for the 18s ribosomal rna subunit. all samples extracted from faeces produced positive results confirming the presence of amplifiable rna (data not shown). real-time rt-pcr assays using the ericov primers were carried out for all pooled samples. single samples from positive pools were then extracted and tested individually. a subset of amplicons from individual samples (n = 10) was also assessed by gel electrophoresis and compared to the positive control. sanger sequencing was conducted on three amplicons with ericovr and ericovf primers using standard protocols. amplicon sequences were 2 i. f. saldanha et al. manually checked then aligned against the german ericov rdrp gene fragment sequence retrieved from genbank (accession number kc545385) to confirm their identity (mega 6.0). whole genome sequencing was performed on rna extracted from one faecal sample collected in 2014 (r618/14) which was identified as ericov-positive by real-time rt-pcr. sequencing was performed using synthesis technology (illumina, san diego, ca, usa). rna was extracted as before and dna was depleted from this sample using the on-column dnase treatment (rneasy® plus mini kit) following the manufacturer's instructions (qiagen); the resultant rna was then eluted in 30 µl molecular grade water [26] . cdna was synthesised from 50 ng rna using a random cdna synthesis system (cdna synthesis kit, merck, de), according to the manufacturers' instructions. the cdna was purified using ampure xp magnetic beads (beckman coulter, high wycombe, uk), 1 ng of which was processed for sequencing using the nextera xt dna sample preparation kit (illumina). a sequencing library was prepared according to the manufacturers' instructions and sequenced using an illumina miseq with 2 × 150 bp paired end reads following standard illumina protocols. for confirmation of the insertions and deletions of the resulting sequence, seven extra sets of primers were designed (available on request). the sequences of these short amplicons were compared with the sequence derived by illumina sequencing. the total reads (12 908 682) were mapped to a reference ericov sequence from germany (genbank accession number kc545383). a modified samtools/vcfutils script was used to generate an intermediate consensus sequence in which any indels relative to the original reference sequence were appropriately called. the intermediate consensus was used as the reference for subsequent iteration of mapping and consensus calling. the total number of assembled viral reads was 1 217 783 (9.43% of the total reads). despite the low proportion of viral sequence detected within the total data set, coverage of the entire genome was obtained (average coverage depth of 4546 reads). gross pme findings were categorised into whether respiratory abnormalities (including, but not limited to, pneumonia, consolidation, haemorrhage, congestion, granuloma and high crenosoma sp. burden) and/or gastrointestinal abnormalities (including, but not limited to, haemorrhage, inflammation, intussusception, rupture, abscess and faeces of abnormal colour or consistency) were detected. gross pme findings were available for both organ systems from 94 of the 121 hedgehog examined post mortem and were included in these analyses. a subset of hedgehogs (n = 9) examined postmortem that were ericov-positive was selected for histological examination. selection criteria focused on carcases likely to provide the optimal state of tissue preservation from those available, prioritising tissues from carcases examined fresh or when frozen, from carcases with the least evidence of autolysis. a range of available tissue samples from each selected carcase was fixed in neutral-buffered 10% formalin and was prepared for histopathological examination and stained with haematoxylin and eosin using standard techniques. particular attention was paid to examination of the respiratory and alimentary tracts, when available, as these are the organ systems frequently targeted by mammalian covs. associations between ericov-positive status, case history signalment and pathological findings were explored. variables were first re-categorised and coded using microsoft excel before being exported to ibm® spss® version 22 for windows (ibm®, chicago, il, usa) for statistical analyses. cross-tabulation and pearson's χ 2 tests were undertaken for the binary dependent variable (ericov presence or absence) against all other categorical independent variables. in addition, where sample numbers were sufficient, binary logistic regression (blr) was performed to include consideration of continuous independent variables, such as age, and to quantify the strength of association for multiple variables simultaneously (year, region). a p-value of <0.05 was considered statistically significant. arcgis pro version 11 software (esri, redlands, ca, usa) was used to map the distribution of hedgehogs for which location data were available. of 351 samples examined, 38 (24 faecal samples and 14 distal large intestinal tract samples) were positive for ericov, giving a positive percentage of 10.8%. the viral copies detected in positive faecal samples ranged from 1.15 × 10 8 to 2.7x10 4 per 4 µl extracted rna. the limit of detection for the bryt® green i based ericov primer rt qpcr assay was calculated as 2.68 × 10 3 copies per 1 µl of extracted rna sample. ibv rna at 10 −2 dilution was not detected by the ericov-specific assay. the complete genome of the hedgehog ericov from sample (r618/14, genbank accession number: mk679660) is 30 173 nucleotides (nt) in length, which is comparable to those sequenced from germany: kc545383 and kc545386 (30 148 and 30 175 nt, respectively). complete genome comparison with open reading frames (orfs) from the german ericov (kc545383) gave identities ranging from 79% (orf3b) to 98% (orf6, encoding the envelope protein). the genes encoding the spike protein of the british and german ericov viruses shared 93% identity (table 1) . the locations for all hedgehogs with available data (n = 344) are displayed in figure 1 , along with their ericov status. hedgehogs sampled for the study covered a wide area across gb, from devon in south west england to northern scotland. hedgehogs positive for ericov were found in southern england, east of england, north east england and wales. we found no evidence of ericov in hedgehogs from scotland, even though 17.4% (n = 61) of sampled hedgehogs were submitted from this country. there was a bias of submissions from the east of england, accounting for 44.4% (n = 123) of the total, with norfolk alone contributing 23.1% (n = 64) of the hedgehog samples examined, which is explained by a single, large participating wildlife centre in this region. although all regions of gb were represented, some appeared to be under-represented; for example, only 1.1% (n = 3) of submissions came from north east england. the majority of hedgehog samples were submitted in 2014 and 2015. the proportion positive in 2015 was higher (28/144, 19%) than in 2014 (10/182, 5%) but there were too few submissions from other years to determine if there was an association with year of sampling. there was a higher number of juvenile hedgehogs included in the study in 2015 (n = 68) than 2014 (n = 24), but the proportion of juveniles positive in 2015 (19.1%) was comparable to the proportion of adults positive (20.3%). as coronaviruses are associated with diarrhoea in other species, we tested for the possibility of an association between ericov status and abnormal faeces. the proportion of samples from hedgehogs with reportedly normal faecal colour or consistency, which tested positive for ericov (14/102), was not significantly different from the proportion of all faecal samples with abnormal consistency and/or abnormal colour (8/115) (p = 0.099). however, a significant association was observed between ericov-positive hedgehogs and those with green faeces (p = 0.004). a significant association was also observed between ericov-positive hedgehogs and those with yellow faeces (p = 0.034). colour variation within a single sample of hedgehog faeces was not uncommon: 42.6% (n = 20) of hedgehogs with green coloured faeces were also reported to have faeces of brown, yellow or black colouration. no other statistically significant association was shown across the other independent variables, including body condition ( table 2 ). a blr model testing all but the non-mutually exclusive variables also showed no statistical significance. the proportion of hedgehogs with respiratory abnormalities reported at pme that tested positive for eri-cov (5/55) was not significantly different from the proportion of those without respiratory abnormalities that tested positive (7/39) (p = 0.205). the proportion of hedgehogs with gastrointestinal tract (git) abnormalities that tested positive (5/55) was not significantly different from those that had no git abnormalities (6/39) (p = 0.35). a statistically significant association was shown between ericov status and geographical region of origin (p = 0.008) ( table 2 ). the highest proportion of ericov-positive hedgehog samples were submitted from the south of england (34/217, 16%); however, blr showed no significant association (p = 0.678) between ericov infection status and wider region when other factors including age and year were included. histopathological examination was conducted on available tissues from nine of the ericov-positive hedgehogs in this study that were examined post-mortem: results are presented with the signalment, macroscopic findings and other ancillary diagnostic tests for interpretation in supplementary table s2 . interpretation was complicated by the state of tissue preservation in the majority of cases, limiting the ability to appraise evidence of either a host inflammatory response or the presence of mild abnormalities (e.g. loss of pseudostratified epithelial cells in the respiratory tract or intestinal villus atrophy, as has been reported with some coronavirus infections in other species [27] ). examination of the respiratory and gastro-intestinal tract identified frequent metazoan parasite infections of variable severity, sometimes in combination with bronchopneumonia. a range of other significant findings was detected, supported by ancillary testing, including trauma and yersiniosis. we detected ericov, a novel clade c betacoronavirus, in wild western european hedgehogs in gb. this virus had previously only been reported from germany and france; in both instances from this same species. our results, therefore, indicate that ericov is widespread in this european wild mammal. full genome sequence of ericov from gb showed 94% identity with a ericov previously detected in germany, and 78% identity with the virus responsible for mers [3] . there was no evidence to indicate that ericov was causing clinical disease in hedgehogs. the lack of association with detection of ericov and abnormal faecal consistency, poor body condition (i.e. thin or emaciated), respiratory and digestive tract abnormalities suggests ericov is unlikely to be a primary pathogen in this species. this concurs with studies reporting natural cov infections in wild animal species, including bats [15] and aquatic birds [16] , which do not manifest as clinical disease. this is also a common feature of a reservoir or maintenance host [28, 29] . coronavirus infections of man and other animals are known to predispose to secondary infections, for example, a study recently isolated feline coronavirus and several other enteropathogens from cats with diarrhoea [30] , thus making any association difficult to detect should it occur with ericov infection in the hedgehog. in the current study, histopathological examination was complicated by concurrent parasitic and bacterial infections in several cases and limited by freeze-thaw artefact and the state of tissue preservation, therefore microscopic lesions caused by viral infection could have been missed or obscured. experimental infection studies and the development of in situ hybridisation to localise ericov in tissues would be worthwhile in the future to further elucidate the clinical significance of ericov infection. despite the lack of association between ericov and faecal abnormalities overall, there was an apparent association between ericov status and green or yellow faeces. green faeces is not an unusual finding in hedgehogs, and can often be associated with crenosoma sp., isospora or salmonella sp. infection [31] . many of the hedgehogs reported to have green faeces also had faeces of another colour, suggesting that faecal colour change is not unusual. the colour change or the reporting thereof may not be consistent and therefore this result should be interpreted with caution. however, the possibility of a causative relationship between ericov infection and abnormal faeces colour cannot be excluded. significantly stronger amplification of cov rna has previously been described in juvenile and lactating female myotis sp. bats [32] , and younger age has been shown to be associated with increased coronavirus shedding across various bat species [33, 34] . although no evidence for such an association for ericov in the hedgehog was found in the current study, juveniles accounted for only 27.6% of the hedgehogs sampled. examination of a larger sample size of juvenile animals would be worthwhile to further explore the possibility of age-structured prevalence. hedgehogs positive for ericov were detected over a wide region of england and wales, but not from scotland even though 17 .4% of all samples tested were submitted from this country (fig. 1) . inferring population prevalence from these data is inappropriate due to the sampling strategy, relying on convenience sampling and the catchment area of rehabilitation centres. there is apparent sampling bias since 47% of all hedgehog samples were from the east of england. demonstrating regional differences in population prevalence and absence of infection would require a much larger, random sample of the population, and information regarding the population structure of hedgehogs in gb. annual variation in the proportion of ericov-positive samples was found. the proportion of hedgehogs sampled in 2015 that tested positive for ericov was higher than that in 2014; a difference that was consistent irrespective of sample type or hedgehog age class (table 2 ). this temporal variability in detection rate is consistent with previous multi-year studies in bats where significant annual variation in coronavirus detection rate was reported [34] . seasonal variation in contact rates, environmental factors and fluctuating levels of cov immunity in the population have all been postulated as causes for such fluctuations [34] . proving viral persistence and establishing critical community size is challenging for wildlife populations [29] . whilst there is now a wealth of studies investigating bat covs, research is lacking for other wildlife covs. there is evidence that the gregarious roosting habits of some species of bat facilitate cov transmission [35] , but the transmission dynamics of ericov in the more-solitary hedgehog [20] are likely to be very different. establishing whether infection is endemic in british hedgehogs will require longitudinal sampling, augmented by age-specific serology [36] . overall, the percentage of ericov-positive hedgehogs detected, at 10.8%, was lower than that reported previously, where 58.9% (148/248) and 50% (37/74) of specimens tested positive for ericov in germany and france, respectively [3, 4] . this difference could be due to assay sensitivity or sample degradation. although the limit of detection of the previously used assays was not given, average copy numbers in the faecal samples were well over the sensitivity threshold of the assay used here. all three studies included hedgehogs in wildlife centres. in the german study, no details were given for the timing of sampling. in the current and french studies, however, faecal samples were taken soon after admission (typically within 48 h) in order to reduce the possibility of nosocomial transmission. dog kennels and shelters are recognised as important environments for maintaining enteric canine covs, with increased length of stay associated with increased probability of cov infection [37] . however, nosocomial transmission seems to be an unlikely cause of the differences in sample percentage of positive samples in this study. another potential factor is that the relatively high percentage of positive samples in the french and german studies could be simply due to a much higher prevalence of ericov in the mainland europe hedgehog population: the recruited wildlife centres (from which the hedgehogs were sampled) were all located in north and north western france, with one located close to the border with germany [4] . more studies are required to determine this. hedgehogs in northern europe hibernate for 2-5 months of the year [20] . ericov was detected in both 2014 and 2015, suggesting that the virus overwinters in the hedgehog population, but the method of viral persistence is not known. in the current study, no association was detected with the month of sampling but to explore this further, longitudinal sampling of individual hedgehogs could be conducted, including from animals before and after hibernation, to determine if there is persistence of infection in individuals. deriving full genome sequence is an important step in characterising this virus to assess relationships with other covs, including pathogens of domestic animals and man. overall identity to the previous coronavirus detected in hedgehogs from mainland europe was high (94%), but lower than the identity between two german ericov isolates (97%) [3] . previous studies have demonstrated that ericov shares a common ancestor with mers-cov, and bat coronaviruses hku5 and hku4, raising the possibility of a lineage that has been positively selected for and which has evolved with the potential for cross-species transmission. host tropism in the coronaviruses is largely determined by the spike protein and its ability to bind to host cell receptors. both the german and the gb ericovs have <40% amino acid similarity with mers-cov at the crucial receptor-binding domain on the spike protein, suggesting that binding to the human receptor (dipeptidyl peptidase 4) is unlikely. virus culture was not attempted in this study, but was previously unsuccessful and this might be due to the cell tropism of the virus [3] . the detection of ericov in british hedgehogs is a notable finding, and the first report outside mainland europe. wildlife disease research in gb has greatly increased in recent years, particularly for popular wildlife species such as the hedgehog, where citizen science offers a cost-effective approach to achieving national disease surveillance [38] . it is important that this continues if we are to gain a deeper understanding of endemic and emerging pathogens of wildlife and their impact on wildlife, livestock and human populations. a p-value of <0.05 was considered statistically significant. discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus ecology, evolution and classification of bat coronaviruses in the aftermath of sars characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs identification of alpha and beta coronavirus in wildlife species in france: bats, rodents, rabbits, and hedgehogs middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir the coronavirus spike protein is a class i virus fusion protein : structural and functional characterization of the fusion core complex recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission bats are natural reservoirs of sars-like coronaviruses bat origin of human coronaviruses the emergence of the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) a close relationship of chiroptera with eulipotyphla (core insectivora) suggested by four mitochondrial genes ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events avian coronavirus in wild aquatic birds asymptomatic middle east respiratory syndrome coronavirus infection in rabbits replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus (tgev) and porcine respiratory coronavirus (prcv) field isolates co-circulating in a swine herd the state of britain's hedgehogs 2011. people's trust for endangered species national predictors of hedgehog erinaceus europaeus distribution and decline in britain detection and molecular characterisation of cryptosporidium parvum in british european hedgehogs (erinaceus europaeus) streptococcus pyogenes infection in a freeliving european hedgehog (erinaceus europaeus) product differentiation by analysis of dna melting curves during the polymerase chain reaction next generation sequencing of viral rna genomes pathogenesis of coronavirus-induced infections. review of pathological and immunological aspects identifying reservoirs of infection: a conceptual and practical challenge assembling evidence for identifying reservoirs of infection enteropathogen co-infection in uk cats with diarrhoea the importance of faecal indices in assessing gastrointestinal parasite infestation and bacterial infection in the hedgehog (erinaceus europaeus) amplification of emerging viruses in a bat colony influence of age and body condition on astrovirus infection of bats in singapore: an evolutionary and epidemiological analysis alphacoronaviruses in new world bats: prevalence, persistence, phylogeny, and potential for interaction with humans diversity of coronavirus in bats from eastern thailand emerging viruses deciphering serology to understand the ecology of infectious diseases in wildlife cross sectional and longitudinal surveys of canine enteric coronavirus infection in kennelled dogs: a molecular marker for biosecurity. infection citizen science and wildlife disease surveillance key: cord-317061-0bx704ao authors: wu, andong; wang, yi; zeng, cong; huang, xingyu; xu, shan; su, ceyang; wang, min; chen, yu; guo, deyin title: prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus date: 2015-10-02 journal: virus res doi: 10.1016/j.virusres.2015.05.018 sha: doc_id: 317061 cord_uid: 0bx704ao coronavirus 3c-like protease (3clpro) is responsible for the cleavage of coronaviral polyprotein 1a/1ab (pp1a/1ab) to produce the mature non-structural proteins (nsps) of nsp4–16. the nsp5 of the newly emerging middle east respiratory syndrome coronavirus (mers-cov) was identified as 3clpro and its canonical cleavage sites (between nsps) were predicted based on sequence alignment, but the cleavability of these cleavage sites remains to be experimentally confirmed and putative non-canonical cleavage sites (inside one nsp) within the pp1a/1ab awaits further analysis. here, we proposed a method for predicting coronaviral 3clpro cleavage sites which balances the prediction accuracy and false positive outcomes. by applying this method to mers-cov, the 11 canonical cleavage sites were readily identified and verified by the biochemical assays. the michaelis constant of the canonical cleavage sites of mers-cov showed that the substrate specificity of mers-cov 3clpro is relatively conserved. interestingly, nine putative non-canonical cleavage sites were predicted and three of them could be cleaved by mers-cov nsp5. these results pave the way for identification and functional characterization of new nsp products of coronaviruses. middle east respiratory syndrome coronavirus (mers-cov) is an enveloped virus carrying a genome of positive-sense rna (+ssrna). it was identified as the pathogen of a new viral respiratory disease outbreak in saudi arabia in june 2012, named as middle east respiratory syndrome (mers). mers-cov emerged ten years after severe acute respiratory syndrome coronavirus (sars-cov) and quickly spread to several countries in middle east and europe (assiri et al., 2013; tashani et al., 2014) . soon after the first report, the mers-cov genome was sequenced and its genomic organization has been elucidated . this new coronavirus is classified in the lineage c of beta coronavirus, and is close to bat coronavirus hku4 and hku5 (de groot et al., 2013; lau et al., 2013) . like other coronaviruses (hussain et al., 2005; zuniga et al., 2004) , mers-cov contains a 3 coterminal, nested set of seven subgenomic rnas (sgrnas), enabling translation of at least nine open reading frames (orfs). the 5 -terminal two thirds of mers-cov genome contains a large open reading frame orf1ab, which encodes polyprotein 1a (pp1a, 4391 amino acids) and polyprotein 1ab (pp1ab, 7078 amino acids), the latter being translated via a −1 ribosomal frameshifting at the end of orf1a. these two polyproteins were predicted to be subsequently processed into 16 non-structural proteins (nsps) by nsp3, a papain-like protease (plpro), and nsp5, a 3c-like protease (3clpro) (kilianski et al., 2013; van boheemen et al., 2012) . protease plays a key role during virus life cycle. it is essential for viral replication by mediating the maturation of viral replicases and thus becomes the target of potential antiviral drugs (thiel et al., 2003; ziebuhr et al., 2000) . investigating the cleavage sites of coronavirus proteases and the processing of polyproteins pp1a/1ab will benefit to identify the viral proteins and their potential function for viral replication. some cleavage sites have been identified and confirmed by previous studies, including three cleavage sites of plpros of human coronavirus 229e (hcov 229e), mouse hepatitis virus (mhv), sars-cov, mers-cov and infectious bronchitis virus (ibv), whose cleavages release the first 3 non-structural proteins (bonilla et al., 1995; kilianski et al., 2013; lim and liu, 1998; ziebuhr et al., 2007) . the canonical cleavage sites of 3clpros, the sites between the recognized nsps, have also been characterized, including all sites of mhv, ibv, sars-cov and a fraction of sites of hcov 229e which release the non-structural proteins from nsp4 to nsp16 (deming et al., 2007; grotzinger et al., 1996; liu et al., 1994 liu et al., , 1997 lu et al., 1995) . for 3clpro of mers-cov, two cleavage sites releasing nsp4 to nsp6 have been identified (kilianski et al., 2013) . however, other cleavage sites remain to be characterized. furthermore, efforts have been taken to predict these cleavages sites by sequence comparison. gorbalenya et al. (1989) made the first systematical prediction on ibv pp1a/1ab according to the substrate specificity of 3c protease of picornaviruses. however, two of their predicted cleavage sites within nsp6 of ibv were proved uncleavable (liu et al., 1997; ng and liu, 2000) . gao et al. (2003) developed a software (zcurve cov) to predict the nsps as well as gene-encoded orfs of coronaviruses more accurately based on previous studies of 3clpros cleavage sites of ibv, mhv and hcov 229e. later on, non-orthogonal decision trees were used to mine the coronavirus protease cleavage data and to improve the sensitivity and accuracy of prediction (yang, 2005) . however, while these methods focus on the prediction of the canonical cleavage sites and target more and more on prediction accuracy to avoid false positives, potential non-canonical cleavage sites might be neglected. for example, a cleavage site between nsp7 and 8 of mhv strain a59 is not predicted by above methods, but proved to be physiologically important since it produces a shorter nsp7 that can support the growth of mhv carrying a mutation on nsp7-8 cleavage site (deming et al., 2007) . therefore, the substrate specificities of coronaviruses 3clpros are complicated. a 3clpro substrate library of four coronaviruses (hcov-nl63, hcov-oc43, sars-cov and ibv) containing 19 amino acids × 8 positions variants was constructed by making single amino acid (aa) substitution at each position from p5 to p3 , and their cleavage efficiencies were measured and analyzed to find out the most preferred residues at each position (chuck et al., 2011) . however, the non-canonical cleavage site with less preferred residues of 3clpro is adopted by coronaviruses (deming et al., 2007) . thus we speculate that other potential 3clpro cleavage sites may still exist in coronaviruses. in order to set up a more moderate and balanced criteria for protease cleavage site identification, we compared six scanning conditions with different stringency to systematically predict the 3clpro cleavage sites on pp1a/1ab of five coronaviruses including mers-cov. as a representative, the cleavability of the predicted cleavage sites of mers-cov 3clpro was analyzed by the recombinant luciferase cleavage assay and the fluorescence resonance energy transfer (fret) assay. the results showed that all 11 canonical cleavage sites of mers-cov pp1a/1ab were cleavable in our experiments and three of nine predicted non-canonical cleavage sites appeared to be cleavable. our study points out a new direction regarding the prediction and identification of cleavage sites of proteases and contributes to understanding the mechanism of coronaviral polyprotein processing. the genome sequences of 28 coronaviruses were downloaded from genebank database and the sequences of the 3clpro cleavage sites were collected from p4 to p2 (tables s1-s4 ). the substrate profiles of each coronavirus group and the whole coronavirinae were summarized (table s5) . the coding sequence of mers-cov nsp5 (nc 019843) was synthesized chemically by genscript and cloned into vectors pet28a and pgex-6p-1, respectively. the catalytic residue mutation c148a was generated by over lapping pcr with mutagenic primers (table s6 ). all the clones and mutations were confirmed by dna sequencing. the expression vectors were transformed into escherichia coli strain bl21 (de3). the cells were grown at 37 • c in lysogeny broth (lb) medium with antibiotics and induced with 0.2 mm isopropylbd-thiogalactopyranoside (iptg) at 16 • c for 12 h. the cells were harvested and resuspended in lysis buffer (50 mm tris-hcl, ph 7.5, 150 mm nacl, 1 mm edta, 0.05% np40, 0.1 mg/ml lysozyme and 1 mm pmsf) at 4 • c. after incubation for 30 min on ice, 10 mm mgcl 2 and 10 g/ml dnase i (sigma) were added to digest the genomic dna. the supernatant of cell lysate was applied to affinity chromatography column after centrifugation. the recombinant protein with his-tag was bound with nickel-nitrilotriacetic acid (ni-nta) resin (genscript) and washed with buffer a (50 mm tris-hcl, ph 7.5, 150 mm nacl), buffer b (50 mm tris-hcl, ph 7.5, 150 mm nacl, 20 mm imidazole) and buffer c (50 mm tris, ph 7.5, 150 mm nacl, 50 mm imidazole). proteins were eluted with buffer d (50 mm tris, ph 7.5, 150 mm nacl, 250 mm imidazole). gst-tagged protein was bound with gst resin (genscript), washed with buffer a and eluted with buffer a supplemented with 10 mm reduced glutathione (gsh). the purified proteins were desalted and concentrated by ultrafiltration using 30 kda amicon ultra 0.5-ml centrifugal filter (millipore). all the cleavage sites (eight residues, ranging from p5 to p3 ) were inserted into glo-sensor 10f linear vector. comparing to the wild type firefly luciferase (550 aa), glo-sensor luciferase has short truncations at both termini with c-and n-part reversed, resulting in the new 234-aa n-and 233-aa c-terminal region respectively. the inserted sequence and the reversed arrangement of the nand c-terminal regions reduce the luciferase activity dramatically. after the recognition sequence was cut off by nsp5, the luciferase recover its activity and luminescence in the presence of luciferase substrate. a back to front recombinant firefly luciferase inserted with different cleavage sites was expressed when the recombinant plasmids were co-incubated with a cell-free protein expression system extracted from wheat germ (promega). after incubation for 2 h at 25 • c, nsp5 was added into the system and the whole system was incubated at 30 • c for 1 h. then, the reaction system was diluted 20 times and mixed thoroughly with equal volume of luciferase substrate. luciferase luminescence was measured by a luminometer (promega) after incubation for 5 min at room temperature. all the 11 conserved putative recognition sites were designed from p12 to p8 , synthesized and modified with a typical shorter wavelength fret pair, n-terminal dabcyl and c-terminal glu-edans by gl biochem (shanghai). the peptides were completely dissolved in dmso and the final concentration of dmso in the reaction system was 1%. 180 m substrate peptide and 16.3 m tagged nsp5 were mixed in the solution of 50 mm tris, ph 7.5, 1 mm edta, 50 m dtt and incubated at 37 • c for 2 h. to calculate kcat/km, different amounts (7.2-180 m) of substrate peptides were co-incubated with 16.3 m nsp5. the reaction system was placed in giernor black plate and the fluorescence was detected by a microplate reader (molecular devices) with ex/em (nm/nm) = 340/490. relative fluorescence unit (rfu) was collected every 30 s for 2 h. the initial slope (slope a = rfu/min) was generated from the linear interval of the rising stage. then, a linear equation was generated using the rfu at plateau (rfu max ) vs. the concentration of substrate. the slope (slope b = rfu/[s]) indicates the rfu change at per unit change of [s] . the initial reaction velocity (v 0 = [s]/min) was calculated through dividing slope a by slope b. the michaelis-menten kinetic constants were generated by lineweaver-burk plot. the coronavirus 3clpros and their cleavage sites are evolutionarily conserved among different genera. to study the genetic diversity and evolution of 3clpro cleavage sites of coronaviruses pp1a/1ab, 308 primary sequences of 3clpro cleavage sites (ranging from p4 to p2 ) of 28 species of coronaviruses were collected and listed in tables s1-s4, including the predicted and verified cleavage sites. 11 canonical cleavage sites of each coronavirus were joined end to end to produce a spliced sequence which was then used to produce a phylogenetic tree (fig. 1a ). in addition, the sequences of all coronavirus 3clpro were used to generate another phylogenetic tree (fig. 1b) . the analyses showed that the phylogenetic distances and taxonomic positions of each virus, in both phylogenetic trees, were mostly consistent with that classified by the international committee on taxonomy of viruses (ictv) (http://www.ictvonline. org/virustaxonomy.asp). these results implied that the cleavage sites of coronaviral 3clpros might co-evolve with 3clpros, and the genetic diversity of both 3clpro and its cleavage sites are relatively conserved between different genera of coronaviruses. however, on the phylogenetic tree generated with 3clpro cleavage sites (fig. 1a) , the members of the genus gammacoronavirus, although clustered closely, is split into alphacoronaviruses and deltacoronaviruses, suggesting that the cleavage sites of gammacoronaviruses may have undergone recombination events during evolution. in order to develop an optimized method for cleavage site prediction that can cover all possible cleavage sites with fewer false positives, we have set three levels of criteria (stringent, moderate and mild) for cleavage site prediction. in the stringent rules, 3clpro cleavage sites only comprise the most preferred residues at each position based on previous description (chuck et al., 2011) . in moderate rules, 3clpro cleavage sites comprise residues which ever appeared in the cleavage sequences of congeneric coronaviruses at each particular position. as for mild rules, the cleavage sites could comprise any residues ever found in the cleavage sequences of all coronaviruses at each particular position. because the substrate preference at p4 and p2 is not strong, we decided to adopt two different lengths of cleavage sequences for prediction, one containing six residues from position p4 to p2 , and the other containing four residues from position p3 to p1 . these two lengths of cleavage sequences, combining with the three different criteria, made up a total of six search conditions for cleavage site predication with decreasing degree of stringency. the canonical cleavage sites of 3clpro for these seven groups of coronaviruses were summarized in tables s1-s4 and used to set conditions iii to vi. possible residues at each particular position of 3clpro cleavage sites were predicted based on all six conditions to make the cleavage site profile of coronaviruses 3clpro (table s5 ). in principle, when condition i was employed, the least number of possible cleavage sites were identified in a scanned sequence, while condition vi predicted the largest number of possible cleavage sites in a scanned sequence. to the applicability, we applied all the six conditions on five representative coronaviruses, including hcov 229e from alphacoronavirus, mhv from betacoronavirus lineage a, sars-cov from beta coronavirus lineage b, mers-cov from betacoronavirus lineage c and ibv from gammacoronavirus. all possible cleavage sites predicted based on each condition were scanned on pp1a/1ab of five representative coronaviruses and the results were summarized in table 1 . as shown in table 1 , increasing numbers of cleavages sites were found for each coronavirus when conditions from i to vi were applied. the results showed that condition i and ii were too strict to cover all 11 canonical cleavages sites; condition v and vi were too loose so as to produce two to three times more than 11 cleavages sites; condition iii could only cover the canonical cleavage sites for sars cov; only condition iv generates an appropriate number of cleavage sites for all five coronavirus. therefore, search condition iv was chosen for further analysis of the cleavage sites of mers-cov. by applying the search condition iv, 9 putative cleavage sites (pss) as well as 11 canonical cleavage sites (css) were predicted (table 2) . although the canonical cleavage sites of mers-cov 3clpro have been predicted by sequence alignment with other coronavirus , our results suggested that the additional cleavage might occur in the process of mers-cov pp1a/1ab processing. to verify the activity of mers-cov 3clpro and cleavability of the predicted cleavage sites, the biochemical assay systems of mers-cov 3clpro were established. as shown in fig. 2a and b, we first expressed and purified mers-cov 3clpro (nsp5) with different tags and mutation: n-terminally gst-tagged nsp5 (gnsp5, 60.4 kda), n-terminally his-tagged (34 extra amino acids with 6× his tag and linker provided by vector pet-28a) nsp5 (hnsp5, 36.9 kda), hnsp5 with catalytic residue mutation c148a (hnsp5m, 36.9 kda) (kilianski et al., 2013) and gst tag-gvlq-nsp5 with c148a mutation and 6× his tag (gnsp5mh, 61.6 kda), in which the sequence motif gvlq represents the last four residues of mers-cov nsp4, mimicking the cleavage site of mers-cov nsp4/nsp5. in the biochemical assays, the gnsp5mh with catalytic residue mutation c148a could not undergo self-cleavage at the cleavage site to release gst in incubation for 16 h (fig. 2c) , indicating that the 3clpro activity of mers-cov nsp5 in gnsp5mh was inactivated by the mutation c148a. thus, gnsp5mh was used as protease substrate in the following biochemical assays. to verify the 3clpro activity of recombinant nsp5s, gnsp5 and hnsp5 were incubated with substrate gnsp5mh for 5 min to 16 h and analyzed by sds-page (fig. 2d) and western blotting, respectively (fig. 2e) . both gnsp5 and hnsp5 showed the proteolysis activity to cleave the substrate gnsp5mh into two parts: gst (26.0 kda) and nsp5mh (34.1 kda), which were confirmed by the correlation of their molecular weight (fig. 2d and e) . however, the 3clpro activity of gnsp5 was obviously weaker than that of hnsp5, which could entirely cleave the substrate gnsp5mh 2 h post treatment ( fig. 2d and e) . these results could be explained by that the larger fusion tag at the n terminus of mers-cov 3clpro significantly reduced the proteolysis activity of 3clpro, which was consistent with the previous observation (xue et al., 2007) . in the biochemical assays, the the tree was generated by the sequence of nsp5 and the method is the same as described above. the number of cleavage sites in pp1ab of 5 representative coronaviruses predicted by using 6 search conditions. condition iii 11 4 11 5 11 0 11 2 11 3 condition iv 11 10 11 14 11 4 11 9 11 5 condition v 11 9 11 17 11 11 11 12 11 11 condition vi 11 15 11 23 11 19 11 19 11 13 a canonical cleavage sites, which are located between recognized nsps. b putative cleavage sites, which are located inside various nsps. c six search conditions are designed: conditions i, iii and v cover six residues from p4 to p2 ; conditions ii, iv and vi cover four residues from p3 to p1 . conditions i and ii are set to comprise the most preferred residues at each position; conditions iii and iv comprise residues appeared in the cleavage sites of congeneric coronaviruses; conditions v and vi comprise residues appeared in the cleavage sequences of any coronaviruses. relatively lower proteolysis activity of 3clpro will benefit to observe the influence of different substrates. therefore, both recombinant gnsp5 and hnsp5 were used as mers-cov 3clpro in the following studies. to rapidly evaluate the proteolysis activity of mers-cov 3clpro toward the predicted cleavage sites of different substrates, a sensitive luciferase-based biosensor assay was adopted. as shown in fig. 3a , the canonical cleavage sites (cs) of mers-cov nsp4/nsp5 (cs4/5) and nsp5/nsp6 (cs5/6), which were experimentally confirmed in a previous study (kilianski et al., 2013) , were inserted into the inverted and circularly permuted luciferase construct pglo-10f, in which the n-terminal and c-terminal halves of luciferase gene are separated. the resulting luciferase in translation system in vitro was inactive and could convert into an active luciferase when cleaved by recombinant viral protease at the engineered cleavage sites (such as cs4/5 and cs5/6). in this system, the luciferase signals were detected when incubated with both gnsp5 and hnsp5, respectively (fig. 3b) . in contrast, the mutated nsp5 (hnsp5m) could not convert the inactive luciferase into active form (fig. 3b ). this result indicated that the luciferase-based biosensor assay could be used to evaluate the proteolysis activity of mers-cov 3clpro. then, the other nine canonical cleavage sites and nine putative cleavage sites composed with 8 aa from mers-cov pp1a/1ab were inserted into the luciferase construct pglo-10f, and the luciferase-based biosensor assays were performed using hnsp5 and hnsp5m, respectively. as shown in fig. 3c , all the 11 canonical cleavage sites of mers-cov 3clpro generated luciferase signal by hnsp5 at least 6.6 times higher than by the inactive hnsp5m, indicating that all these canonical sites could be cleaved by mers-cov 3clpro. these results experimentally verified the existence of the 11 predicted canonical cleavage sites. interestingly, among the nine putative cleavage sites, the luciferase signals of ps1-1, ps3-1 and ps3-3 remarkably increased more than 70 folds when incubated with hnsp5, indicating that the putative cleavage sites, located inside nsp1 and nsp3 of mers-cov respectively, might be cleavable (fig. 3d) . the other 6 predicted putative sites (ps3-2, ps5-1, ps6-1, ps12-1, ps13-1, and ps16-1) showed less than 2.5 folds increase of luciferase signal when they were treated by hnsp5 comparing with those treated by hnsp5m (fig. 3c and d) . due to high sensitivity of the luciferase-based biosensor assay and the fact that the confirmed verification of the recombinant luciferase assays. inactive luciferase was synthesized in the cell-free translation system and the reaction mixture incubated at 25 • c for 2 h. after that, the protein mixture was divided into four parts and incubated with 1.63 m gnsp5, hnsp5, hnsp5m or h2o, respectively. after incubation for 1 h at 30 • c, the reaction product was diluted 20 times and mixed with equal amount of luciferase substrate. after incubation at room temperature for 5 min, the luciferase luminescence was measured. luciferase activation fold was calculated through dividing the signal value of the reaction system treated with active hnsp5 by the one treated with the inactive nsp5 mutant hnsp5m. (c) the luciferase cleavage assay of predicted 11 canonical cleavage sites and (d) 9 putative cleavage sites. the luciferase expression vector inserted with cleavage sites were added to the wheat germ protein translation mix and incubated at 25 • c for 2 h, and the reaction mixture was divided and treated with hnsp5 and hnsp5m, respectively. the dashed line indicates the lowest fold increase of luciferase signal by cleavage of previously confirmed 3clpro cleavage sites. the data presented here are the mean values ± sd derived from three independent experiments. canonical cleavage sites generated at least 6.6 times increase of luciferase signal, the cleavage signal of these six sites may represent the background level, indicating that they are likely uncleavable per se. these results suggest that previously unrecognized 3clpro cleavage sites may exist inside the nsps, which were regarded as non-canonical cleavage sites. the substrate specificity of coronaviruses 3clpro is determined by the residues from p4 to p2 positions of cleavage sites, especially depending on the p1, p2 and p1 positions, which would benefit the prediction of cleavage site and design the broadspectrum inhibitors of coronaviruses 3clpro (chuck et al., 2011; hegyi and ziebuhr, 2002) . previous studies demonstrated that different canonical cleavage sites of some representative coronaviruses are not equally susceptible to proteolysis by recombinant 3clpro (fan et al., 2004; hegyi and ziebuhr, 2002) . to define the susceptibility of the canonical cleavage sites and substrate specificity of mers-cov 3clpro, 20-mer synthetic peptides representing corresponding canonical cleavage sites of mers-cov 3clpro were synthesized and modified with n-terminal dabcyl and c-terminal glu-edans (fig. 4a) . the fluorophore edans and quencher dabcyl are widely used in the biochemical assays based on the fluorescence resonance energy transfer (fret). as shown in fig. 4b , the peptides represented cleavage sites cs4/5 and cs5/6 were tested to optimize the fret assay, and the relative fluorescence unit (rfu) folds of both sites significantly increased when incubated with gnsp5 and hnsp5. although the fret assay system is more costly and less sensitive than the luciferase-based biosensor assay (figs. 3b and 4b), it provides continuous read signals during the process of reaction, which could measure the kinetic characteristic of protease toward different substrates. the initial reaction rate (rfu/min) of all 11 canonical cleavage sites of mers-cov were measured and shown in fig. 4c . the michaelis constants including kcat, km, kcat/km and relative kcat/km were then calculated (table 3) . as shown in table 3 , the substrate specificity of mers-cov 3clpro is relatively conserved with other coronaviruses as previously reported (fan et al., 2004; hegyi and ziebuhr, 2002; ziebuhr and siddell, 1999) . the relative kcat/km values of cs4/5 and cs5/6 indicated that the cleavage sites flanking mers-cov 3clpro are converted significantly faster than other sites. the efficient proteolysis at the sites flanking nsp5 implies that the nsp5 (3clpro) might be released from the polyprotein 1a/1ab at the very early stage of the maturation of viral nsps, which is similar with the hcov, tgev, sars-cov and mhv (fan et al., 2004; hegyi and ziebuhr, 2002) . however, the relative kcat/km value of cs4/5 is lower than that of cs5/6 (table 3) , which is different from that of the coronaviruses (fan et al., 2004; hegyi and ziebuhr, 2002) . this could be explained by that the residue gly (g) at the p4 of cleavage site between nsp4 and nsp5 of mres-cov reduces the protease activity of 3clpro comparing with the residues ser (s), ala (a) and thr (t) of other coronaviruses (tables s1-s4) as previous described (chuck et al., 2011) . whether such disparity plays any role in the replication and pathogenesis of mers-cov is unknown. the processing of viral polyprotein by 3clpro is essential for the replication of coronaviruses. besides the 11 canonical cleavage sites of coronaviruses, some additional cleavage sites inside nsps, so-called non-canonical cleavage sites, have also been identified (deming et al., 2007) . therefore, more non-canonical 3clpro cleavage sites are to be identified in different coronaviruses. in this study, we designed six search conditions for predicting 3clpro cleavage sites, among which, the search condition iv provides a feasible way to reveal the potential cleavage sites of 3clpro within coronaviruses. based on the genetic diversity of different coronavirus genera (fig. 1) , the scanning condition iv adopted the residues of 3clpro cleavage sites, which ever appeared in the cleavage sequences of congeneric coronaviruses at position p3 to p1 . in contrast, conditions i, ii, iii, v and vi were either too restrictive or generated too many false positive outcomes (table 1 ). in the suggested condition iv, 4 residues from position p3 to p1 were applied to the prediction of 3clpro cleavage site. by measuring the relative protease activities of 3clpro from different coronavirus genera against 19 amino acids × 8 positions of substrate variants, it is shown that the substrate specificity of position p5, p2 and p3 are significantly lower than other positions (chuck et al., 2011) . therefore, the consideration of six or more residues is unnecessary, which could lead to leave-out of potential cleavage sites (table 1) . comparing with the previous researches on the prediction and identification of 3clpro cleavage sites, the scanning condition iv showed its advantages. for example, the two nonexistent putative cleavage sites predicted within nsp6 of ibv (gorbalenya et al., 1989; liu et al., 1997; ng and liu, 1998) were avoided in our prediction method (data not shown). notably, the noncanonical cleavage site at the end of mhv nsp7 identified by deming et al. could be predicted using scanning condition iv. by using the search condition iv, 9 putative cleavage sites were predicted in mers-cov pp1ab in addition to the 11 canonical cleavage sites. the luciferase signal of cs10/12 increased 6.6 fold when treated with nsp5 in the recombinant luciferase cleavage assays, which is the lowest among the 11 canonical cleavage sites (fig. 3c) . therefore, the 6.6 fold increase of luciferase signal was used arbitrarily as a threshold for judging positive and negative. among the nine predicted putative cleavage sites, three sites (ps1-1, ps3-1 and ps3-3) showed obviously increasing signals at least 70 times above the background (fig. 3d ) and therefore were regarded as cleavable sites. the increase of signals of other six predicted putative cleavage sites was less than 2.5 times (fig. 3d) . therefore, they were regarded as non-cleavable sites and thus as false positives from the prediction. interestingly, the homologous sequence of ps1-1 and ps3-1 are conserved in lineage c of betacoronavirus including mers-cov, batcov hku4 and batcov hku5 (fig. 5a and b) . however, ps3-3 is mers-cov unique sequence (fig. 5c) . moreover, the cleavability of a cleavage site in biochemical assays is a necessary but not sufficient condition for its physiological existence in the viral infection. a predicted cleavage site may or may not be accessible by a protease. the 3d structure model of mers-cov adpribose-1-monophosphatase (adrp) domain built by comparative protein modeling and papain like protease (plpro) domain (bailey-elkin et al., 2014) showed that both ps3-1 and ps3-3 are located at the surface of adrp and plpro domain, opposite to the enzymatic active centers ( fig. 5d and e) , suggesting that these two sites are like approachable by the proteinase. most recently, the crystal structure of mers-cov 3clpro was determined (needle et al., 2015) . although ps5-1 is also located at the surface of mers-cov 3clpro, the self-cleavage of mers-cov nsp5 was not observed in this study (fig. 2) . therefore, the threshold we proposed in the luciferase-based biosensor system to exclude the false positive prediction results is reasonable (fig. 3d) . however, further studies are needed to identify the predicted cleavage products from the cells infected by mers-cov. currently, such work with live mers-cov is limited in our research facilities due to the biosafety rules, but it can be addressed in collaboration in the future. notably, the outcomes of the two cleavage assay systems were different. the signal fold change of highly sensitive luciferasebased biosensor assay is dependent on the accumulation of active luciferase cleaved by nsp5 during 1 h (section 2), while the outcome of the fret assay is instant relative fluorescence unit (rfu) signal. the rfu/min is the initial speed of the reaction, which reflects but not equals to the efficiency of the cleavage. these differences may be caused by the steric hindrance of the luciferase subunits, the distance between fluorophore and quencher of substrates for fret assay and substrate solubility. therefore, the activity observed in the two different systems cannot be compared directly. based on the characteristic of the two cleavage assay systems, the highly sensitive luciferase-based biosensor assay might be more suitable to high throughput screen the predicted putative cleavage site of protease while the fret assay better for cleavage kinetic analysis. according to the michaelis constants of mers-cov, the substrate specificity of mers-cov 3clpro is relatively conserved with other coronaviruses (fan et al., 2004; hegyi and ziebuhr, 2002) . notably, the pro (p) has been selected as result of evolution at position p2 of cleavage site between nsp10 and nsp12 (cs10/12) of lineage c betacoronavirus, which is not preferred by the 3clpro based on the previous study (chuck et al., 2011) . however, the relative kcat/km value of mers-cov cs10/12 is 0.053, which is 26.5 fold higher than that of sars-cov (fan et al., 2004) . this indicated that the substrate preferences of some cleavage sites could still be varied among different genera of coronaviruses and the proposed scanning condition iv regarding the residues ever appearing in the cleavage sequences of congeneric coronaviruses is reasonable. in summary, we proposed an optimized search condition for predicting cleavage sites of coronavirus 3clpro. we verified the 11 canonical cleavage sites of pp1ab in biochemical assays. we further identified three non-canonical cleavage sites in the nsps of mers-cov. the results provide clues for possible identification of novel cleavage products of coronavirus nsps and will benefit the studies of the mechanisms of coronavirus replication. processing of polyprotein 1a/1ab by 3clpro is essential in coronavirus life cycle. the 3clpro cleavage site prediction methods established by previous studies are focus on the accuracy, while some noncanonical cleavage sites were missed. in this study, we built a moderate prediction method to balance the accuracy and false positive outcomes. using this method, 9 putative cleavage sites, in addition to the 11 canonical sites, were predicted in mers-cov pp1ab and the cleavability of 3 of them was experimentally confirmed. interestingly, all these 3 non-canonical cleavage sites are located upstream to nsp4, which is in contrast with previous understanding that the coronavirus 3cl protease only cleaves from nsp4 to nsp16. this suggests a novel role of 3clpro in coronavirus pp1a/1ab processing. however, the cleavability of these putative cleavage sites needs to be further verified in the viral proteins of mers-cov-infected cells. finally, the catalytic constants of the 11 canonical cleavage sites of mers-cov 3clpro showed its conservation with the cousins in coronaviridae. hospital outbreak of middle east respiratory syndrome coronavirus crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression characterization of the leader papain-like proteinase of mhv-a59: identification of a new in vitro cleavage site profiling of substrate specificities of 3c-like proteases from group 1, 2a, 2b, and 3 coronaviruses middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group processing of open reading frame 1a replicase proteins nsp7 to nsp10 in murine hepatitis virus strain a59 replication biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase prediction of proteinase cleavage sites in polyproteins of coronaviruses and its applications in analyzing sars-cov genomes coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis characterization of a 105-kda polypeptide encoded in gene 1 of the human coronavirus hcv 229e conservation of substrate specificities among coronavirus main proteases identification of novel subgenomic rnas and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus characterisation of a papain-like proteinase domain encoded by orf1a of the coronavirus ibv and determination of the c-terminal cleavage site of an 87 kda protein a 100-kilodalton polypeptide encoded by open reading frame (orf) 1b of the coronavirus infectious bronchitis virus is processed by orf 1a products proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites identification and characterization of a serine-like proteinase of the murine coronavirus mhv-a59 structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity identification of a 24-kda polypeptide processed from the coronavirus infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites further characterization of the coronavirus infectious bronchitis virus 3c-like proteinase and determination of a new cleavage site australian hajj pilgrims' knowledge about mers-cov and other respiratory infections mechanisms and enzymes involved in sars coronavirus genome expression genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans production of authentic sars-cov m(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction mining sars-cov protease cleavage data using non-orthogonal decision trees: a novel method for decisive template selection isolation of a novel coronavirus from a man with pneumonia in saudi arabia human coronavirus 229e papain-like proteases have overlapping specificities but distinct functions in viral replication processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab virus-encoded proteinases and proteolytic processing in the nidovirales sequence motifs involved in the regulation of discontinuous coronavirus subgenomic rna synthesis supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2015.05. 018 key: cord-305134-s7h6bpof authors: mackman, nigel; antoniak, silvio; wolberg, alisa s.; kasthuri, raj; key, nigel s. title: coagulation abnormalities and thrombosis in patients infected with sars-cov-2 and other pandemic viruses date: 2020-07-13 journal: arterioscler thromb vasc biol doi: 10.1161/atvbaha.120.314514 sha: doc_id: 305134 cord_uid: s7h6bpof the world is amid a pandemic caused by severe acute respiratory syndrome-coronavirus 2. severe acute respiratory syndrome-coronavirus causes serious respiratory tract infections that can lead to viral pneumonia, acute respiratory distress syndrome, and death. some patients with coronavirus disease 2019 (covid-19) have an activated coagulation system characterized by elevated plasma levels of d-dimer—a biomarker of fibrin degradation. importantly, high levels of d-dimer on hospital admission are associated with increased risk of mortality. venous thromboembolism is more common than arterial thromboembolism in hospitalized covid-19 patients. pulmonary thrombosis and microvascular thrombosis are observed in autopsy studies, and this may contribute to the severe hypoxia observed in covid-19 patients. it is likely that multiple systems contribute to thrombosis in covid-19 patients, such as activation of coagulation, platelet activation, hypofibrinolysis, endothelial cell dysfunction, inflammation, neutrophil extracellular traps, and complement. targeting these different pathways may reduce thrombosis and improve lung function in covid-19 patients. in the last century, several new viruses have emerged, including different strains of influenza virus a virus (iav), severe acute respiratory syndrome-coronavirus (sars-cov), middle east respiratory syndrome-coronavirus (mers-cov), and most recently, sars-cov-2, that have caused epidemics and pandemics. iav transmission from zoonotic reservoirs into humans has caused the last 4 influenza pandemics: 1918 h1n1 spanish flu, 1957 h2n2 asian flu, 1968 h3n2 hong kong flu, and 2009 h1n1. [1] [2] [3] the sars-cov epidemic occurred between 2002 and 2004 and infected ≈8000 people with at least 774 deaths worldwide. [4] [5] [6] mers-cov appeared in 2012 and infected ≈2500 people with over ≈850 deaths, and cases still occur. [7] [8] [9] in december 2019, sars-cov-2 emerged in china and quickly spread throughout the world. as of june 17, 2020, there have been over 8 influenza viruses and coronaviruses are enveloped viruses with a single-stranded rna genome (either a positive-or negative-sense rna). influenza viruses enter cells via endocytosis that requires binding and proteolytic cleavage of hemagglutinin on epithelial cells. 10, 11 sars-cov, mers-cov, and sars-cov-2 all belong to the coronavirus family. they are called coronaviruses because they have large spike proteins on the capsid surface that create a crown-like shape. entry of coronaviruses into host cells involves binding of the spike proteins with host receptors, followed by proteolytic cleavage of the spike protein to expose the s2 fusion domain with subsequent membrane fusion. 12, 13 mers-cov uses dpp4 (dipeptidyl peptidase 4) as arterioscler thromb vasc biol. 2020;40:2033-2044. doi: 10.1161/atvbaha. 120 .314514 a cellular receptor, whereas sars-cov and sars-cov-2 use ace2 (angiotensin-converting enzyme 2) as entry receptors. [14] [15] [16] [17] [18] importantly, sars-cov-2 has a stronger binding to ace2 compared with sars-cov. 19 ace2 is predominantly expressed in epithelial cells of subsegmental bronchial branches. 20 interestingly, one study found low levels of ace2 in alveolar epithelial cells and endothelial cells in uninfected control lungs but an increased expression of ace2 in both cell types in the lungs of covid-19 patients. 21 in a physiological setting, ace2 cleaves and inactivates angiotensin i and angiotensin ii and, therefore, plays a critical role in regulating the renin-angiotensin system. 22 differences in tissue expression of these receptors and activating proteases may contribute to unique aspects of the pathophysiology of each virus. super pandemic viruses, such as iav h1n1, sars-cov, mers-cov, and sars-cov-2, cause serious respiratory tract infections that can lead to viral pneumonia and acute respiratory distress syndrome (ards). [23] [24] [25] ards is a type of respiratory failure characterized by widespread local and systemic inflammation. 26 both viral infection of cells and the host response to infection damage the epithelial-endothelial cell barrier that separates the alveoli from capillaries. this injury compromises the lung's ability to exchange oxygen and carbon dioxide. 26 lung stillness, fluid-filled alveoli, and a rise in carbon dioxide levels lead to hypoxemia and respiratory distress. 27 the primary treatment for ards is mechanical ventilation and supportive treatment in an intensive care unit. 28 iav patients with classic ards that requires mechanical ventilation have decreased lung compliance with elevated plateau pressures. 29 one study reported that 46 (23%) of 199 patients hospitalized with sars developed ards, and these patients had a mortality rate of 37% at 28 days. 30 similarly, 20% of hospitalized covid-19 patients in new york required mechanical ventilation. 31 surprisingly, some covid-19 patients with ards have well-preserved lung mechanics despite severe hypoxia. 32, 33 this has led to the suggestion that microvascular thrombosis rather than decreased lung compliance contributes to the impaired oxygenation in covid-19 patients. infection with pandemic respiratory viruses can lead to an overproduction of numerous cytokines that is termed the cytokine storm. [34] [35] [36] this hyperinflammatory response contributes to disease severity and death. tnfα (tumor necrosis factor-alpha), il (interleukin)-1β, and il-6 orchestrate the inflammatory response. 34, 37 both iav and sars-cov infection are associated with a cytokine storm. 38 iav(h1n1)pdm09 patients. seven biomarkers, including il-6, were associated with disease progression in outpatients and inpatients, whereas 5 biomarkers, including tnfα and il-8, were associated with disease progression among hospitalized patients. critically ill patients with iav(h1n1)pdm09 also exhibited higher levels of il-6 compared with patients with bacterial pneumonia. 41 a recent study suggested that monocytes and macrophages play a key role in the hyperinflammatory response in covid-19 patients. 42 indeed, severe sars-cov-2 infection is associated with increased circulating levels of various inflammatory mediators, including il-6 and crp (c-reactive protein). [43] [44] [45] [46] [47] we observed increased levels of crp in severe iav h1n1 patients. 48 zhou et al 47 52 another study observed a higher rate of vte (44%) in hospitalized h1n1 patients (n=36) with severe ards compared with 29% in non-h1n1 patients with ards. 24 thrombotic complications have also been observed in sars-cov patients. 53 a study from a single hospital in singapore reported that one-third of sars-cov patients experienced vte despite the use of low-molecularweight heparin at doses to achieve anti-xa levels of 0.5 to 1.0 iu/ml 54 ; however, no additional details of the vte events were provided. arterial ischemic stroke was observed in a small number of sars-cov patients. 54 surprisingly, perhaps, there are no reports of thrombosis in mers-cov patients. recently, several studies have reported vte rates ranging from 0.9% to 6.5% for noncritically ill hospitalized covid-19 patients and 8% to 69% in covid-19 patients in the intensive care unit (table 1) . [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] rates of pe were between 16.7% and 35% in severely ill covid-19 patients, and rates of deep vein thrombosis were between 0% and 46.1% for nonseverely ill covid-19 patients ( table 1) . rates of arterial thrombotic events were between 2.8% and 3.8%. 57, 62 there are several factors that could explain the wide variation in thrombosis rates in the different studies that include differences in clinical practice, such as if venous ultrasound is performed as a screening strategy or if thromboprophylaxis is routinely used, reporting of symptomatic versus asymptomatic vte, and also differences in patient populations. notably, however, several groups have reported that vte may occur despite standard thromboprophylaxis (table 1) , which is like what was observed in sars-cov infection. although initial reports suggested that covid-19 patients had higher rates of thrombosis compared with patients with other types of pneumonia, a recent study found that the rate of vte in covid-19 patients was 2% compared with 3.6% in patients with non-covid-19 community-associated pneumonia. 65 furthermore, it is important to note that a recent study reported a vte rate of 4.8% and a rate of overall bleeding of 4.8% in covid-19 patients. 62 another study of 353 covid-19 patients in boston found that the cumulative incidence of thrombotic events was 10.2% and major or fatal bleeding of 20.8% in hospitalized covid-19 patients (j. zwicker, unpublished data, 2020). at present, the optimal antithrombotic prophylactic strategy for patients with covid-19 is unclear. a new clinical trial (improve [intermediate or prophylactic dose anticoagulation for venous or arterial thromboembolism in severe covid-19]; https://www.clinicaltrials.gov; unique identifier: nct04367831) will hopefully shed light on this question. several studies have reported pathological findings from autopsies of patients infected with pandemic coronaviruses. pulmonary thromboemboli within the main pulmonary artery or segmental pulmonary arteries, thrombi in small vessels, and fibrin within pulmonary vessels were observed in sars-cov patients. [66] [67] [68] a recent series of studies have described the findings of autopsies of covid-19 patients. 33, [69] [70] [71] fibrin-rich thrombi were found in small vessels and capillaries in the lung, as well as foci of hemorrhages. 33, 70 interestingly, cd61+ megakaryocytes were observed within alveolar capillaries. 70 some fibrin and platelets within small vessels were also associated with neutrophils. intra-alveolar fibrin deposition was observed in a subset of severe covid-19 patients consistent with a loss of vascular integrity. 69 one autopsy study found that 7 of 12 (58%) covid-19 patients had a deep vein thrombosis that was not suspected antemortem, and pe was the direct cause of death in 4 of these patients. 72 a recent study performed autopsies on 7 covid-19 patients and compared the findings to 7 h1n1 patients. 21 there was widespread thrombosis and microangiopathy in the lungs of covid-19 patients, and capillary microthrombi were 9× more prevalent than in h1n1, which suggested a different pathological process. 21 the innate immune response is activated in response to invading pathogens to counteract the infection. this is generally accompanied by activation of coagulation that, in part, serves to localize the infection. 73, 74 however, excessive and widespread activation of coagulation can lead to disseminated intravascular coagulation (dic), defined as fulminant activation of coagulation, consumption of coagulation factors, and bleeding. 75, 76 classic dic caused by bacterial sepsis is associated with prolonged activated partial thromboplastin time, prothrombin time (pt), thrombocytopenia, elevated d-dimer, and microangiopathic thrombosis in multiple organs. 75, 76 d-dimer is a product of plasmin-mediated degradation of cross-linked fibrin. elevated plasma d-dimer is associated with a higher risk of disease progression in hospitalized iav(h1n1) pdm09-infected patients. 40 two other studies of patients with probable iav h1n1 infection found that d-dimer predicted disease progression. 77, 78 elevated plasma levels of d-dimer have also been reported in sars-covinfected patients. 79 d-dimer has attracted attention as a prognostic marker in covid-19 patients. 43, 44, 46, 47, 60, [80] [81] [82] as expected, covid-19 patients with vte had higher d-dimer levels than non-vte patients. 55 a series of articles from china reported higher d-dimer levels in severely affected patients compared with those with a nonsevere disease and higher d-dimer levels in nonsurvivors compared with survivors ( table 2) . [43] [44] [45] [46] [47] 80 similarly, studies from france and italy found high d-dimer levels in covid-19 patients in the intensive care unit (table 2) . 60, 81 two studies found that a higher d-dimer level on admission was associated with increased mortality. 47, 64 one study used 2.0 μg/ml as a cutoff for d-dimer and found a mortality rate of 0.37% (1 of 267 covid-19 patients, <2.0 μg/ml) versus 17.9% (12 of 67 covid-19 patients, ≥2.0 μg/ml). 64 in contrast, a study from france observed a less impressive separation of mortality rates based on the same d-dimer cutoff (10.4% [8 of 77 covid-19 patients], <2.0 μg/ml versus 18.3% [17 of 93 covid-19 patients], ≥2.0 μg/ml) and suggested that the chinese study had selection bias. 83 thrombocytopenia was observed in 45% to 55% of sars-cov patients, but overt dic was rarely observed. 53, 79, 84 thrombocytopenia was also found to be evident in a subset of mers-cov patients. [85] [86] [87] [88] similarly, thrombocytopenia and an elevated pt was observed in 2 fatal cases of mers-cov patients, consistent with a diagnosis of dic. 86 many covid-19 patients have mild thrombocytopenia (100−150×10 9 /l) at most and do not exhibit an increase in pt or decrease in at (antithrombin) levels. 33, [43] [44] [45] [46] 60, [80] [81] [82] 89 these results suggested the absence of a consumptive coagulopathy in most patients. however, several studies found that nonsurviving patients had slightly prolonged pt and a further decrease in platelet count. 46, 47, 80 interestingly, patients with severe sars-cov-2 infection also have elevated levels of fibrinogen ranging from 1.3 to 2.0× above the normal range (2-4 g/l; table 3 ). 60,80-82 we observed increased levels of fibrinogen in severe iav h1n1 patients. 48 ranucci et al 81 showed an association between il-6 and fibrinogen levels. in addition, fviii (factor viii) and vwf (von willebrand factor) levels were increased in covid-19 patients by 2-to 2.3-fold and 3-to 4.1-fold above the normal range, respectively. 60, 82 taken together, these results indicate that most covid-19 patients have an activated coagulation system that is associated with increased levels of d-dimer; however, it is unlike classic dic since there is little change in pt and the thrombocytopenia is generally mild. elevated levels of fviii and fibrinogen likely contribute to the prothrombotic state in covid-19 patients. elevated fviii and vwf may reflect activated/infected endothelium, whereas elevated fibrinogen likely reflects enhanced production by hepatocytes as part of the host's acute phase responses driven by il-6. in the later stages of disease, nonsurviving covid-19 patients may develop classic dic with prolongation of the pt, moderate-tosevere thrombocytopenia (platelet count, <50×10 9 /l), and decreased fibrinogen (<1.0 g/l). several mouse models have been developed to study the pathological changes in the lung associated with infection with iav h1n1, sars-cov, and mers-cov. one study reported that over 3500 genes were differentially regulated in the lungs of mice following sars-cov infection. 90 importantly, mice infected with 1918 and 2009 iav h1n1 strains exhibited similar transcriptional signatures, which suggested a common mechanism of lung injury. 90 infection with iav h1n1, sars-cov, and mers-cov is associated with lung hemorrhages. [90] [91] [92] infection of mice with different coronavirus mouse hepatitis virus strains also caused severe pneumonia and lung hemorrhage. 93 however, thrombosis has also been observed in the lungs of mice expressing human dpp4 infected with mers-cov. 94 at this time, we can only speculate about the mechanisms of thrombosis in covid-19 patients based on the available plasma biomarkers and clinical presentation. several recent comments/reviews have described the coagulation abnormalities and thrombosis occurring in covid-19 patients. [95] [96] [97] [98] [99] there is clear evidence for activation of different cell types, such as lung epithelial cells, macrophages, neutrophils, endothelial cells, and platelets, as well as different systems, such as coagulation, inflammation, and complement, in the lungs of covid-19 patients (figure) . we will briefly summarize some of these pathways and refer to reviews that cover some of the pathways in more detail. aberrant tf (tissue factor) expression is associated with most forms of thrombosis. 100 importantly, tf is a key mediator of activation of coagulation in different forms of ards. [101] [102] [103] [104] viral infection of a variety of cell types, including lung epithelial cells, endothelial cells, and monocytes induces tf expression. 74, 91, 105 in addition, tf expression in endothelial cells is induced by activation of tlr3 (toll-like receptor 3)-a pattern-recognition receptor that detects single-stranded rna. 106, 107 interestingly, tlr3 was shown to protect mice from sars-cov infection. 108 cytokines produced during the cytokine storm (tnfα, il-1β, and il-6) induce tf expression in endothelial cells, and il-6 induces tf expression in mononuclear cells. [109] [110] [111] angiotensin ii can also induce tf expression in vascular smooth muscle cells and endothelial cells. 112, 113 herpes simplex virus infection of endothelial cells increases tf expression. 114 similarly, one would expect that sars-cov-2 infection of the endothelium would increase tf expression and microvascular thrombosis. therefore, there are a variety of mechanisms for increasing tf expression in different cell types in the lung during viral infections. we found that plasma levels of extracellular vesicle tf activity were increased in severe influenza virus patients and were associated with mortality. 48 increased tf is also observed after infection of mice with iav h1n1, sars-cov, and mers-cov 90, 91 (t. sheahan, unpublished data). we found that iav h1n1 infection of mice increases tf expression in lung epithelial cells and activates coagulation. 91 furthermore, both a genetic reduction of tf in epithelial cells and administration of anticoagulants to wild-type mice was associated with increased alveolar hemorrhage. 91, 115 this suggests that tf-dependent activation of coagulation is part of the host innate immune response to viral infection that helps protect against intrapulmonary hemorrhage. however, a complication of this response is thrombosis. therefore, it seems likely that tf plays a central role in thrombosis in covid-19 patients. two recent articles have discussed the role of tf in thrombosis in covid-19 patients. 116, 117 contact activation pathway activation of the contact system leads to thrombin generation and upregulation of the kallikrein-kinin system. 118 kallikrein induces the generation of bradykinin, which increases vascular permeability. in addition, bradykinin interacts with the renin-angiotensin system and increases inflammation, fibrinolysis, and complement severe acute respiratory syndrome-coronavirus 2 (sars-cov-2) infects lung epithelial cells and endothelial cells (ecs), which leads to the recruitment of a variety of immune cells, such as macrophages and neutrophils. activated macrophages and ecs contribute to the cytokine storm. ec activation also increases vascular permeability (vp). neutrophils release neutrophil extracellular traps (nets). activated platelets likely contribute to thrombosis and net formation. tf (tissue factor) expression is likely to be increased in activated epithelial cells, macrophages, and ecs and will activate the coagulation system. similarly, fxiia (factor xiia) can increase coagulation. sars-cov-2 infection also activates the fibrinolytic system and may increase pai-1 (plasminogen activator inhibitor 1), which would reduce fibrin degradation. finally, the complement system is activated in covid-19 patients, and cellular damage would increase the activation of the coagulation system. activation. 119 the effect of targeting the contact system has been studied in animal models of bacterial sepsis. an early study showed that administration of an anti-fxii (factor xii) antibody c6b7 prevented hypotension and extended the life of baboons challenged with escherichia coli but did not prevent dic. 120 in a second study, c6b7 reduced complement activation, neutrophil activation, and the fibrinolytic response (reduced tissue plasminogen activator and plasmin-α2-antiplasmin complexes) but increased pai-1 (plasminogen activator inhibitor 1) in septic baboons. 121 more recently, the effect of blocking the contact pathway using an antibody 3g3 that prevents fxiia activation of fxi (factor xi) was evaluated in a lethal staphylococcus aureus baboon model. 122 pretreatment of the baboons with 3g3 reduced the activation of coagulation, fibrin deposition in tissues, inflammation, neutrophil activation, complement activation, and increased survival. 122 an anti-fxii antibody 3f7 also reduced bradykinin generation and edema in mice. 123 acquired ace2 deficiency also leads to more bradykinin via an unknown mechanism, which would increase vascular permeability. a recent review discussed the potential benefits of targeting the contact activation pathway in covid-19 patients. 124 the fibrinolytic system is activated in ards. 104, 125, 126 elevated levels of pai-1 in ards create a hypofibrinolytic state that leads to increased fibrin deposition within the vasculature and within the alveolar space. high plasma pai-1 levels are associated with mortality in ards patients. 127, 128 one study reported that the plasma pai-1 level was higher in 16 sars-cov patients than 19 patients with other infectious pneumonias and healthy controls. 129 pai-1 expression was increased in sars-cov-infected mice, and pai-1 −/− mice exhibited increased lung hemorrhage and increased mortality. 90 this study suggested that pai-1-dependent inhibition of fibrinolysis is protective against intrapulmonary hemorrhage. a recent review described the fibrinolytic abnormalities associated with ards and discussed the use of thrombolytic drugs to treat covid-19. 130 it was proposed that nebulized plasminogen activators could be used to degrade fibrin in the alveoli and improve oxygenation in covid-19 patients. 130 indeed, a recent study reported that intravenous administration of tissue plasminogen activator temporally improved the respiratory status of 3 patients with severe covid-19 respiratory failure. 131 platelets play an essential role in maintaining vascular integrity but also contribute to thrombosis. more recently, platelets have been found to participate in the immune response to viruses. 132 interestingly, iav particles were observed within platelets from patients with acute influenza infection. 133 in addition, iav engulfment by platelets led to tlr7-dependent release of c3 and subsequent activation of neutrophils and neutrophil extracellular trap (net) release. 133 therefore, platelets participate in the host response to iav infection. however, platelet activation during viral infection may also increase the risk of thrombosis. one study in covid-19 patients found an association between thrombocytopenia and risk of in-hospital mortality. 134 a recent review discussed the potential role of platelets in thrombosis in covid-19. 135 under normal conditions, the endothelium maintains vascular integrity, limits binding and activation of immune cells and platelets, and inhibits coagulation by expression of anticoagulant proteins. however, during infection, the endothelium becomes activated, resulting in a loss of barrier function, expression of adhesion proteins that facilitate the recruitment of immune cells, release of vwf that allows binding of platelets, and expression of tf that activates the coagulation system. one study found that soluble icam-1 (intercellular adhesion molecule 1) and soluble vcam-1 (vascular cell adhesion molecule 1) were associated with disease progression among hospitalized iav(h1n1)pdm09 patients. 40 these biomarkers indicate that the endothelium is activated possibly by circulating inflammatory mediators. although some iav strains have been shown to replicate in human lung microvascular endothelial cells, only avian iav h5n1 has been shown to infect lung microvascular endothelial cells in vivo. 136, 137 importantly, one study found that blocking replication of the highly pathogenic iav strain h5n1 in the endothelium reduced systemic viral spread and mortality without affecting viral replication in the lungs of infected mice. 3 a recent study found that human capillary organoids derived from induced pluripotent stems cells could be infected with sars-cov-2, and this infection was blocked with recombinant, soluble human ace2. 138 interestingly, deceased covid-19 patients had increased ace2 expression in endothelial cells in the lungs compared with noninfected controls. 21 two studies found evidence for direct infection of the endothelium by sars-cov-2 and diffuse endothelial inflammation in the lung, heart, kidney, and liver. 21 hematopoietic changes are observed in sars-cov and mers-cov patients. 53 for instance, sars-cov patients often present with neutrophilia, which is associated with poor outcome. 79, 84 other studies have observed neutrophilia in mers-cov-infected patients. [85] [86] [87] covid-19 patients generally have increased numbers of circulating neutrophils, and an elevated neutrophil count has been associated with poor outcome. [43] [44] [45] [46] [47] neutrophils play a key role in clearing viruses in the lung by phagocytosing viral particles and by releasing nets. [142] [143] [144] however, activated neutrophils can also damage host cells. [145] [146] [147] [148] neutrophils also play a key role in immunothrombosis-a term that has been used to describe the activation of coagulation that accompanies host innate immune defense. 149 importantly, nets may contribute to thrombosis and vascular occlusion. 150, 151 there are several biomarkers used to measure the levels of nets in plasma, including mpo (myeloperoxidase)-dna complexes and citrullinated histone h3. 151 however, many of these assays have low specificity for nets. 151 one study in iav h1n1 and h7n9 patients reported elevated levels of mpo-dna complexes at hospital admission that correlated with disease severity. 152 similarly, serum from severe covid-19 patients contained elevated levels of mpo-dna complexes and citrullinated histone h3. 153 these results suggest that nets may contribute to impairment of blood flow in the lungs of covid-19 patients. 151, 154 the complement system plays a key role in the host immune response to viruses by opsonization of viral particles, recruitment of inflammatory cells, and lysis of infected cells. 155 however, complement activation can also damage host cells. sars-cov infection in mice activates the complement system. 156 c3 −/− mice exhibited reduced recruitment of neutrophils and inflammatory monocytes into the lung and less respiratory dysfunction after sars-cov infection compared with controls. 156 similarly, inhibition of the c5a receptor reduced lung injury in hdpp4 mice infected with mers-cov. 157 these results indicate that the complement system contributed to the lung pathology after sars-cov and mers-cov infection in mice. importantly, significant deposits of terminal complement components have been noted in the lung microvasculature of covid-19 patients. 33 complement system inhibition with eculizumab, which binds to c5, might be beneficial for covid-19-a hypothesis that is currently investigated in a clinical trial (https://www.clinicaltrials.gov; unique identifier: nct04288713). 158 a recent review discussed complement as a target in covid-19. 159 further studies are needed to understand the molecular basis of thrombosis in covid-19 patients and how this contributes to morbidity and mortality. measurement of additional circulating biomarkers of different systems, such coagulation, fibrinolysis, and complement, as well as markers of endothelial cell activation, will provide much needed information on the pathology of covid-19. when optimizing antithrombotic treatment for covid-19 patients, it is important to balance the risk of thrombosis and the risk of bleeding, especially as bleeding has been observed in the lungs of covid-19 patients. it will be also interesting to know whether any of the proposed treatments for covid-19 patients, such as blocking the il-6 receptor or inhibiting complement activation, will reduce thrombosis. received may 18, 2020; accepted june 29, 2020. the 1918 influenza pandemic and its legacy. cold spring harb mortality from pandemic a/h1n1 2009 influenza in england: public health surveillance study endothelial cell tropism is a determinant of h5n1 pathogenesis in mammalian species sars working group. a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group isolation of a novel coronavirus from a man with pneumonia in saudi arabia the middle east respiratory syndrome (mers) functional balance between haemagglutinin and neuraminidase in influenza virus infections role of hemagglutinin cleavage for the pathogenicity of influenza virus different residues in the sars-cov spike protein determine cleavage and activation by the host cell protease tmprss2 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov influenza virus entry middle east respiratory syndrome virus pathogenesis angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin-converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20 th anniversary of the discovery of ace2 genome composition and divergence of the novel coronavirus (2019-ncov) originating in china sars-cov-2 receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase clinical characteristics of 3,062 covid-19 patients: a meta-analysis empirical systemic anticoagulation is associated with decreased venous thromboembolism in critically ill influenza a h1n1 acute respiratory distress syndrome patients coinfection with staphylococcus aureus increases risk of severe coagulopathy in critically ill children with influenza a (h1n1) virus infection acute respiratory distress syndrome: advances in diagnosis and treatment mechanisms and clinical consequences of acute lung injury european society of intensive care medicine, and society of critical care medicine. an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome et al; the northwell covid-19 research consortium. presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area covid-19 does not lead to a "typical" acute respiratory distress syndrome complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases pattern recognition receptors and inflammation cytokine release syndrome cytokine storms in infectious diseases immunotherapeutic implications of il-6 blockade for cytokine storm pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology new fronts emerge in the influenza cytokine storm insight flu 002 & 003 study groups. the association between serum biomarkers and disease outcome in influenza a(h1n1)pdm09 virus infection: results of two international observational cohort studies in vivo platelet activation in critically ill patients with primary 2009 influenza a(h1n1) pathological inflammation in patients with covid-19: a key role for monocytes and macrophages clinical features of patients infected with 2019 novel coronavirus in wuhan clinical and immunological features of severe and moderate coronavirus disease 2019 clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study microvesicle tissue factor activity and interleukin-8 levels are associated with mortality in patients with influenza a/h1n1 infection tocilizumab for the treatment of severe coronavirus disease effective treatment of severe covid-19 patients with tocilizumab thromboprophylaxis in the intensive care unit: focus on medical-surgical patients pandemic h1n1 influenza infection and vascular thrombosis coagulation disorders in coronavirus infected patients: covid-19, sars-cov-1, mers-cov and lessons from the past large artery ischaemic stroke in severe acute respiratory syndrome (sars) prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia incidence of asymptomatic deep vein thrombosis in patients with covid-19 pneumonia and elevated d-dimer levels confirmation of the high cumulative incidence of thrombotic complications in critically ill icu patients with covid-19: an updated analysis lille icu haemostasis covid-19 group. pulmonary embolism in covid-19 patients: awareness of an increased prevalence high incidence of venous thromboembolic events in anticoagulated severe covid-19 patients high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study incidence of venous thromboembolism in hospitalized patients with covid-19 covid and coagulation: bleeding and thrombotic manifestations of sars-cov2 infection pulmonary embolism or pulmonary thrombosis in covid-19? is the recommendation to use high-dose heparin for thromboprophylaxis justified? d-dimer levels on admission to predict in-hospital mortality in patients with covid-19 comparison of venous thromboembolism risks between covid-19 pneumonia and community-acquired pneumonia patients analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis lung pathology of fatal severe acute respiratory syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer vander heide rs. pulmonary and cardiac pathology in african american patients with covid-19: an autopsy series from new orleans multiscale 3-dimensional pathology findings of covid-19 diseased lung using high-resolution cleared tissue microscopy autopsy findings and venous thromboembolism in patients with covid-19 the coagulation system in host defense multiple roles of the coagulation protease cascade during virus infection towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation scientific and standardization committee on dic, and the scientific and standardization committee on perioperative and critical care of the international society on thrombosis and haemostasis. diagnosis and management of sepsis-induced coagulopathy and disseminated intravascular coagulation clinical characteristics of 75 pandemic h1n1 influenza patients from turkey; risk factors for fatality serum d-dimer changes and prognostic implication in a major outbreak of severe acute respiratory syndrome in hong kong abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia the procoagulant pattern of patients with covid-19 acute respiratory distress syndrome hypercoagulability of covid-19 patients in intensive care unit. a report of thromboelastography findings and other parameters of hemostasis uncertainties on the prognostic value of d-dimers in covid-19 patients haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis is mers another sars? hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description clinical and laboratory findings of middle east respiratory syndrome coronavirus infection epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury tissue factor deficiency increases alveolar hemorrhage and death in influenza a virus-infected mice comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in a/j mice middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 coagulation abnormalities and thrombosis in patients with covid-19 supported by the esc working group on pulmonary circulation and right ventricular function. covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review covid-19 update: covid-19-associated coagulopathy covid-19 and its implications for thrombosis and anticoagulation global covid-19 thrombosis collaborative group. pharmacological agents targeting thromboinflammation in covid-19: review and implications for future research tissue factor: an essential mediator of hemostasis and trigger of thrombosis coagulation and inflammation in acute lung injury tissue factor in experimental acute lung injury therapeutic modulation of coagulation and fibrinolysis in acute lung injury and the acute respiratory distress syndrome procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. contribution of tissue factor associated with factor vii the alveolar epithelium can initiate the extrinsic coagulation cascade through expression of tissue factor a key role for toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells tlrs and innate immunity toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection effect of human recombinant interleukin-6 and interleukin-8 on monocyte procoagulant activity procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines transcriptional regulation of tissue factor expression in human endothelial cells at(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor angiotensin ii (at(1)) receptor blockade reduces vascular tissue factor in angiotensin ii-induced cardiac vasculopathy infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression anticoagulation increases alveolar hemorrhage in mice infected with influenza a potential role for tissue factor in the pathogenesis of hypercoagulability associated with in covid-19 thrombotic complications of covid-19 may reflect an upregulation of endothelial tissue factor expression that is contingent on activation of endosomal nadph oxidase the intrinsic pathway of coagulation: a target for treating thromboembolic disease? the kallikrein-kinin and the renin-angiotensin systems have a multilayered interaction the contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia. in vivo use of a monoclonal anti-factor xii antibody to block contact activation in baboons inhibition of factor xii in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase inhibition of contactmediated activation of factor xi protects baboons against s aureusinduced organ damage and death kallikrein-kinin blockade in patients with covid-19 to prevent acute respiratory distress syndrome activation of coagulation and fibrinolysis in acute respiratory distress syndrome: a prospective pilot study local abnormalities of coagulation and fibrinolytic pathways that promote alveolar fibrin deposition in the lungs of baboons with diffuse alveolar damage elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury national heart, lung, and blood institute acute respiratory distress syndrome clinical trials network. pathogenetic and prognostic significance of altered coagulation and fibrinolysis in acute lung injury/acute respiratory distress syndrome analysis of thrombotic factors in severe acute respiratory syndrome (sars) patients fibrinolytic abnormalities in acute respiratory distress syndrome (ards) and versatility of thrombolytic drugs to treat covid-19 tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series circulating platelets as mediators of immunity, inflammation, and thrombosis the role of platelets in mediating a response to human influenza infection thrombocytopenia and its association with mortality in patients with covid-19 potential role of platelets in covid-19: implications for thrombosis endothelial activation and dysfunction in the pathogenesis of influenza a virus infection targeted infection of endothelial cells by avian influenza virus a/fpv/rostock/34 (h7n1) in chicken embryos inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 endothelial cell infection and endotheliitis in covid-19 overcoming barriers: the endothelium as a linchpin of coronavirus disease 2019 pathogenesis? covid-19: the vasculature unleashed neutrophils ameliorate lung injury and the development of severe disease during influenza infection a role for neutrophils in viral respiratory disease neutrophils drive alveolar macrophage il-1β release during respiratory viral infection a systems analysis identifies a feedforward inflammatory circuit leading to lethal influenza infection neutrophil extracellular traps in immunity and disease excessive neutrophil levels in the lung underlie the age-associated increase in influenza mortality nlrp12 mediates adverse neutrophil recruitment during influenza virus infection thrombosis as an intravascular effector of innate immunity host dnases prevent vascular occlusion by neutrophil extracellular traps neutrophil extracellular traps: villains and targets in arterial, venous, and cancer-associated thrombosis high level of neutrophil extracellular traps correlates with poor prognosis of severe influenza a infection neutrophil extracellular traps in covid-19 targeting potential drivers of covid-19: neutrophil extracellular traps clinical promise of next-generation complement therapeutics complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis. mbio blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov will complement inhibition be the new target in treating covid-19-related systemic thrombosis? complement as a target in covid-19? we would like to acknowledge colleagues in the division of hematology key: cord-314357-u1m7yr8f authors: elrggal, mahmoud e.; karami, nedaa a.; rafea, bushra; alahmadi, lama; al shehri, anwar; alamoudi, ruba; koshak, hassan; alkahtani, saad; cheema, ejaz title: evaluation of preparedness of healthcare student volunteers against middle east respiratory syndrome coronavirus (mers-cov) in makkah, saudi arabia: a cross-sectional study date: 2018-04-14 journal: z gesundh wiss doi: 10.1007/s10389-018-0917-5 sha: doc_id: 314357 cord_uid: u1m7yr8f aim: to assess the knowledge and attitude of senior medical, dental, nursing and pharmacy students toward middle east respiratory syndrome-corona virus (mers-cov) in saudi arabia. subjects and methods: a cross-sectional survey using a 21-item questionnaire was conducted for a 3-month period from november 2015–january 2016 in makkah, saudi arabia. the questionnaire was designed to evaluate students’ understanding and perception of mers-cov. an anova test was used to determine the association of study discipline and academic year with the student knowledge score on mers. results: a total of 364 students were assessed during the study. the majority (62%) of the participants were in the 20–22-year age group. more than half (53%) were pharmacy students followed by (22%) medical students. more than two thirds (71%) of the participants were aware that mers is caused by the coronavirus. more than half (59%) of the participants believed that mers can be transmitted through direct or indirect contact with infected camels. a statistically significant association was reported between the study discipline and mean knowledge score (p < 0.0001) with medical students achieving an overall better knowledge score compared with students from other study disciplines. conclusion: overall, students had good knowledge about mers epidemiology, transmission and the recommended protective measures. however, students expressed their reluctance to work in healthcare facilities with inadequate mers infection control isolation policies. since its first detection in saudi arabia in 2012, middle east respiratory syndrome-corona virus (mers-cov) has become a major health problem (bermingham et al. 2012; zaki et al. 2012 ). confirmed cases of mers-cov have been reported across the arabian peninsula including jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates and yemen as well as in asia, europe, africa and north america (usa) (zumla et al. 2015) . however, saudi arabia has reported the highest number of cases affecting 891 people with 372 reported deaths (41.8% mortality) (zumla et al. 2015) . these findings have put saudi arabia at the epicenter of deadly outbreaks of mers-cov. the transmission of mers-cov through person-to-person contact has been confirmed as one of the multiple routes of transmission of mers outbreaks in saudi arabia (assiri et al. 2013; memish et al. 2013a, b) . for example, the hospitalbased emergence of mers during spring 2013 in al-ahsa (eastern province of saudi arabia) was the result of humanto-human transmission with the spread largely suspected to occur through large droplets and contact (assiri et al. 2013) . similarly, the majority of cases detected during the 2014 outbreak of mers in jeddah, saudi arabia, were also suspected to be acquired through human-to-human transmission in healthcare facilities (memish et al. 2013a, b; azhar et al. 2014) . transmission of mers from camels to humans was the other likely source implicated in the mers-covoutbreak in saudi arabia (azhar et al. 2014) . mass gatherings including religious festivals and congregations can carry a potentially huge health risk not only to the attendees but also to the local population. a large congregation of people at one particular place and in close proximity provides an ideal opportunity for the importation and exportation of infectious diseases and facilitates the spread of such diseases through human-to-human transmission to not only the attendees but also the local population (abubakar et al. 2012; al-tawfiq et al. 2013; world health organization 2008) . hajj is one such gathering that every year in makkah, saudi arabia, brings together millions of muslims with multiple ethnicities, races and cultures from all over the world (ahmed et al. 2009 ). it is estimated that more than 2 million muslims from over 184 countries perform the hajj pilgrimage every year (alborzi et al. 2008; gatrad and sheikh 2005) . furthermore, pilgrims performing hajj sacrifice four-footed animals including camels to complete one of the hajj rituals. pilgrims are therefore likely to be exposed to camels, which have been reported to be carriers of mers-cov (azhar et al. 2014) . the convergence of millions of pilgrims at one particular place in close proximity coupled with their exposure to animal carriers of mers-cov puts pilgrims at a major risk of contracting mers, as particularly shown during the outbreaks of mers in saudi arabia in 2013 and 2014. fortunately, no cases of mers were detected in pilgrims who performed hajj during that period (zumla et al. 2015) . nevertheless, the threat of mers becoming a major public health epidemic remains. health science students enrolled in various faculties including medicine, pharmacy, dentistry and nursing at a large public university in makkah often volunteer their services during the hajj season to help pilgrims. these students can help to promote public awareness and understanding of mers and the extent of its potential threat in saudi arabia. however, little is known about the knowledge and perception of saudi health science students concerning mers (kharma et al. 2015) , and more work is required to identify any knowledge gaps. this study therefore aims to assess the knowledge and attitude of senior medical, dental, nursing and pharmacy students toward mers in makkah, saudi arabia. a cross-sectional survey was conducted for a 3-month period from november 2015-january 2016 at umm al qura university, makkah, saudi arabia. a 21-item structured questionnaire was developed using the style and format of some of the questions used in two previous studies (kharma et al. 2015; khan et al. 2014 ). the questionnaire was designed to evaluate students' understanding and perception of mers-cov. although arabic is the national language of saudi arabia, the questionnaire was developed in the english language as this is the official medium of instruction at all healthcare colleges across the kingdom. the questionnaire was piloted among a small number (n = 5) of undergraduate students. the presentation and validity of the questionnaire were undertaken by experienced academic and senior pharmacy students. the study questionnaire comprised four sections containing 21 items. section 1 had six items that explored the demographic information of respondents including age, gender, year of study, study discipline, any healthcare provider in the family and any relatives or friends who suffered from mers. section 2 comprised nine items and was designed to evaluate students' in-depth knowledge about mers including causes, sources of transmission, mortality, clinical manifestations, prevention strategies and risk groups for mers. the knowledge was assessed at three possible levels (yes, no, i do not know). a score of 1 was given for each correct answer. no score was given for an incorrect answer. a maximum of score of 9 was obtainable in this section. section 3 comprised one item and aimed to gather students' sources of knowledge about mers. section 4 comprised five questions and aimed to evaluate students' attitudes and beliefs about mers. attitude questions were designed based on a 5-point likert scale format (1 = strongly agree, 2 = agree, 3 = neutral, 4 = disagree and 5 = strongly disagree). positive statements were scored on a 5 to 1 scale with 'strongly agree' responses yielding 5 points and 'strongly disagree' responses 1 point. similarly, negative statements were scored on a 1 to 5 scale with 'strongly disagree' responses having a maximum score of 5. 'neutral' responses were scored 3. the questionnaire was developed and distributed using google forms. undergraduate students studying medicine, dentistry pharmacy and dentistry were approached and recruited through social networking websites (facebook, twitter and whatsapp). students were eligible to participate if they were in year 4, 5 or 6 of their undergraduate program. the password-protected survey links were posted on various official college social media pages. an introductory paragraph outlining the aims and objectives of the study as well as instructions to complete the questionnaire was posted along with the survey. the data were coded, entered and analyzed using spss. descriptive statistics, frequencies and percentages were used to summarize data. an anova test was used to determine the association of study disciplines and academic year with the knowledge score on mers. p < 0.05 was considered statistically significant. a total of 364 students were assessed during the study. the majority (62%) of the participants were in the 20-22-year age group. more than half (53%) were pharmacy students followed by medical students (22%). most (42%) were 4th and 5th academic year students. just over half (52%) of the participants had a healthcare provider in the family (see table 1 ). overall, medical students achieved significantly better knowledge scores (15.7, sd 3.7) than students from other study disciplines (p < 0.0001). more than two thirds (71%) of the participants were aware that mers is caused by the coronavirus. more than half (59%) of the participants believed that mers can be transmitted through direct or indirect contact with infected camels (see table 2 ). regarding preventive strategies for mers, the majority (90%) of the participants believed that wearing a face mask in a crowded place could prevent the transmission of mers. furthermore, 82% stated that maintaining good hand hygiene can also be helpful in preventing mers (see table 3 ). more than half (60%) of the participants reported that they heard about mers through social media, while (54%) cited tvor radio and (41%) cited posters and brochures as their sources of information (see table 4 ). the majority (72%) of the participants strongly agreed that educating people about mers is important to prevent the spread of the disease. furthermore, just over half (53%) of the participants expressed their level of concern about mers by strongly agreeing or agreeing that they will not do their clinical rotation in a hospital without a clear mers infection control isolation policy (see table 5 ). a statistically significant association was reported between study discipline and mean knowledge score (p < 0.0001). the findings of this study suggest that overall healthcare students have good knowledge and understanding concerning mers. the majority of the participants in this study cited social media as their source of information for mers. study participants' increased use of and access to the internet to seek information have also been reported in previous studies conducted in saudi arabia (al-mohrej et al. 2017; hoda 2016; baseer et al. 2016) . the saudi ministry of health often posts educational programs on infection control on its website (baseer et al. 2016) . such educational programs can be a very useful source for providing information to both the public and various healthcare professionals. similarly, seminars, lectures, conferences and research symposiums can also be effective in raising awareness about mers and other emerging infectious diseases (khan et al. 2014 ). most of the participants correctly responded that maintenance of adequate hand hygiene was paramount in the prevention of mers. lack of proper hand hygiene can potentially increase the risk of mers-associated morbidity and mortality (brug et al. 2004 ). the use of personal face masks was another prevention strategy for mers that was largely supported by the study participants. maintenance of good hand hygiene and the use of face masks and protective equipment are some of the crucial prevention strategies endorsed by the centers for disease control and prevention (cdc) to control mers infection (cdc 2016). other prevention strategies highly supported by the study participants included avoidance of crowded places and close contact with people infected with mers. the role of overcrowding of patients in initiating a potential mers outbreak particularly in hospitals with inadequate infection control measures was also highlighted in a previous study (memish et al. 2013a ). more than half of the participants expressed their apprehension by stating that they would not do their clinical rotation in a hospital without a clear mers infection control isolation policy. the concern showed by participants in this study also reflects their awareness about pathogen transmission (butt et al. 2016) . transmission of mers infection from infected patients to healthcare professionals has been confirmed in previous studies (assiri et al. 2013; memish et al. 2013a, b) . the saudi ministry of health's scientific advisory council has developed mers guidelines for the safer management of mers-infected patients (saudi ministry of health 2014). these guidelines have also clearly outlined the isolation procedures and precautions for the control of mers infection. all healthcare facilities in saudi arabia including the makkah region should therefore strictly adhere to these policies to ensure the protection of not only the public but also healthcare workers. the medical students achieved a better mers knowledge score than their counterparts. this difference may be explained by the fact that medical students have more clinical rotations and therefore have direct contact with the patients compared with pharmacy and dentistry students. furthermore, medical students are often engaged in public health campaigns that provide them with opportunities to improve their knowledge and understanding about potentially epidemic infectious diseases such as mers. there is, however, a need to provide specific courses to students from other study disciplines to improve their awareness of various emerging infection trends and their respective infection control policies and procedures. this study has some limitations. although it suggested a possible association between the study discipline and total knowledge score of students concerning mers, this association could be explained by the risk of confounding. no power calculations were undertaken prior to the commencement of this study. however, it could be argued that this study was a descriptive study with no hypothesis testing. in this study, participants were recruited based on their willingness and ability to participate. therefore, the sample size used in this study was based on available resources. overall, students had good knowledge about mers epidemiology, transmission and the recommended protective measures. however, students expressed their reluctance to work in healthcare facilities with inadequate mers infection control isolation policies. the saudi ministry of health should ensure the strict implementation of clear isolation procedures in all healthcare facilities across the kingdom, including in makkah, to better utilize the services of student volunteers during the umrah and hajj season. funding the study was not funded by any organization. ethical approval ethical approval was obtained from the ethics committee of the university. all information collected from this study was kept strictly confidential. all procedures performed in the study were in accordance with the ethical standards of the university research and ethics committee. consent for participation was understood by completion and submission of the survey. global perspectives for prevention of infectious diseases associated with mass gatherings the quest for public health security at hajj: the who guidelines on communicable disease alert and response during mass gatherings are saudi medical students aware of middle east respiratory syndrome coronavirus during an outbreak? respiratory tract infections during the annual hajj: potential risks and mitigation strategies meningococcal carrier rate before and after hajj pilgrimage: effect of single dose ciprofloxacin on carriage hospital outbreak of middle east respiratory syndrome coronavirus evidence for camel-to-human transmission of mers coronavirus awareness of droplet and airborne isolation precautions among dental health professionals during the outbreak of corona virus infection in riyadh city, saudi arabia severe respiratory illness caused by a novel coronavirus sars risk perception, knowledge, precautions, and information sources, the netherlands infection control and prevention practices implemented to reduce transmission risk of middle east respiratory syndromecoronavirus in a tertiary care institution in saudi arabia interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers) hajj: journey of a lifetime identification of information types and sources by the public for promoting awareness of middle east respiratory syndrome coronavirus in saudi arabia knowledge and attitude of healthcare workers about middle east respiratory syndrome in multispecialty hospitals of qassim, saudi arabia assessment of the awareness level of dental students toward middle east respiratory syndrome-coronavirus family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus infections in health care workers infection prevention/control and management guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection communicable disease alert and response for mass gatherings: technical workshop isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome open access this article is distributed under the terms of the creative comm ons attribution 4.0 international license (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord-313737-cob5hf5q authors: otter, j. a. title: the inaugural healthcare infection society middle east summit: ‘no action today. no cure tomorrow.’ date: 2015-11-30 journal: journal of hospital infection doi: 10.1016/j.jhin.2015.06.021 sha: doc_id: 313737 cord_uid: cob5hf5q nan the healthcare infection society (his) decided to run its spring meeting in dubai this year as the inaugural his middle east summit. the conference was well attended, with delegates from all over the world. most of the presentations can be viewed on the his website. 1 the conference opened with professor tawfik khoja outlining the challenges to infection prevention and control in the middle east. professor khoja focused his thoughts on the impressive joint gulf plan for infection prevention (2011e2016). this strategy document aims to raise the standards of infection prevention and control in the region, and is already yielding success. dr tim boswell then followed with a presentation describing the challenges in europe. among the challenges he covered were public reporting and external scrutiny, hand hygiene, antibiotic resistance, the healthcare environment, surveillance and outbreaks, an increasingly elderly population, new threats [such as ebola and middle east respiratory syndrome coronavirus (mers-cov)], meticillinresistant staphylococcus aureus (mrsa), c. difficile, and invasive devices and new complex equipment. to these i would add the increasingly cost-constrained financial environment that we face in europe. how can we invest in infection control when some hospitals can't afford to buy new pens? the next session covered viruses with pandemic potential: mers-cov, influenza and ebola (although only the first two really have pandemic potential!). dr ali omrani gave an extremely current overview of mers-cov, tracking the outbreak in south korea with up-to-the-minute slides. this illustrated how quickly the picture can change with a pandemic virus such as mers-cov. one striking aspect of dr omrani's talk was findings from saudi arabia that 0.2% of the population are likely to have encountered mers-cov, since this is the proportion of a large community sample who were seropositive for mers-cov antibodies. 2 this suggests a sizeable and previously unrecognized burden of asymptomatic exposure e and possibly shedding. furthermore, animal handlers are more likely to have mers-cov seropositivity, reinforcing the link between animals and mers-cov. 2 dr omrani then discussed sharp spikes of mers-cov in jeddah, saudi arabia in 2014, and the recent outbreak in south korea, attributing both to breakdowns in simple infection control. dr nick phin from public health england (phe) then discussed influenza, and carole fry preparedness for ebola. dr phin highlighted a useful cdc toolkit providing advice on respiratory protection for healthcare workers, and also a recent bmj review concluding that facemasks may help to prevent the spread of respiratory viruses in the community. 3, 4 carole fry added some cultural perspective to ebola considerations: there are still some 'ebola deniers' in west africa! in terms of containing ebola, the british approach of using trexler isolators is safe for staff, but pretty miserable for patients. is this an appropriate trade-off? also, our careful use of personal protective equipment (ppe) for ebola has highlighted our careless use of ppe for other organisms! there are some parallels in preparing for all three of these unlikely but potentially very serious threats (mers-cov, ebola, and influenza). one of the most important challenges is dealing with the paranoia that always seems to engulf these preparations! valerie harmon delivered a useful lecture on achieving hand hygiene compliance. self-reported hand hygiene compliance rates are usually reported to be >90%, but who would believe this with such a huge conflict of interest? the problem with using human beings to monitor hand hygiene compliance is that the moment another person is there, compliance improves! so, automation of hand hygiene compliance monitoring seems the best way forward. valerie's colleagues demonstrated a stateof-the-art automated hand hygiene compliance monitoring approach based on google glass. although the technology was rather prototype, the principle is there, and it seems likely that automated hand hygiene monitoring systems will come into play over the next few years. but this will not alter a fundamental problem: self-protection is a large and rather unhelpful driver for hand hygiene compliance. 5 accurate monitoring of hand hygiene compliance using technology is important, but it must be combined with effective education to help staff to overcome themselves to comply. tim boswell's talk on the environment summarized the evidence that contaminated surfaces make an important contribution to the transmission of key pathogens, including: mrsa, c. difficile, vancomycin-resistant enterococci, norovirus, and acinetobacter baumannii. this is demonstrated most convincingly by the 'prior room occupancy' studies, showing that admission to a room previously occupied by a patient with these pathogens is a risk factor for acquisition for the incoming occupant. underpinning this are the poor levels of conventional cleaning and disinfection, illuminated by techniques such as fluorescent marking of surfaces to evaluate the cleaning process. a failure to eliminate key pathogens from hospital surfaces by cleaning and disinfection processes designed to do just this at the time of patient discharge has been demonstrated for multiple pathogens. automated room disinfection systems (principally hydrogen peroxide and ultraviolet systems) offer the potential to reduce or remove reliance on the operator to assure adequate distribution and contact time of a disinfectant e and can help to reduce the transmission of hospital pathogens. professor tibor pal provided an overview of the concerning epidemiology of multidrug-resistant gram-negative rods (mdr-gnr) in the arabian peninsula: lots of overseas healthcare and travel, and huge antibiotic usage is a toxic mix in this regard. professor pal's view is that all carbapenemases are not equal: oxa-48 is weedy and klebsiella pneumoniae carbapenemase (kpc)/new delhi metallo-beta-lactamase (ndm) are scary! e not least due to the emergence of resistance to last-line agents such as colistin. it does seem from the limited available data that the rate of carbapenem-resistant enterobacteriaceae (cre) in the middle east is considerably higher than in europe, and increasing fast! switching to the non-fermenters, professor pal highlighted the remarkable environmental survival properties of acinetobacter, combined with a propensity towards antibiotic resistance. although data are limited, it seems that around 50% of acinetobacter in the arabian peninsula are carbapenem resistant. i was next to speak, and i outlined approaches to mdr-gnr control in europe based on current guidelines. cre is a big deal in europe, especially in the uk, and has prompted unprecedented action on a national level. one key question is: do we go universal or targeted? there has been much discussion recently about abandoning traditional targeted (also known as vertical) approaches in favour of universal (horizontal). interestingly, all guidelines that i reviewed favoured a targeted approach for mdr-gnr, centred around screening and isolation of carriers. we are hamstrung by the lack of high quality studies telling us with any certainty what works to control mdr-gnr. also, how do you go about producing good guidelines? plus, importantly, how do good guidelines translate through a good policy into good practice? as to which interventions we should use for each organism, this depends on organism and setting, although screening, isolation, stewardship, hand hygiene, and cleaning/ disinfection are the pillars of infection control. but what do we do about the more controversial areas: decolonization, screening of staff, cohorting staff and patients, environment screening, and education? dr muhammad halwani then gave an overview of infection control in the middle east, focusing on acinetobacter and pseudomonas. his comprehensive review set out prevalence and resistance rates across the region, highlighting limited surveillance data e but high rates where data are available. dr halwani then reviewed the guidelines available in the region, most importantly the 2007 guidelines for isolation precautions, and the 2015 gcc strategic plan for combatting antimicrobial resistance (which has a most apt tagline: 'no action today. no cure tomorrow.'). professor david leaper gave an entertaining critique of the evidence base for infection prevention. he began by highlighting the drying of the antibiotic pipeline: the 'variations on a theme' are losing efficacy due to resistance. there is strong evidence for some interventions to prevent infection, for example, the timing of antibiotic prophylaxis prior to surgery, but other areas are more controversial. professor leaper cited the example of nasal decolonization for meticillin-susceptible s. aureus, based on this study being less convincing than you may think. 6 but can we expect a randomized controlled trial for everything? even for parachutes when jumping out of aeroplanes? 7 finally, carole fry and martin kiernan gave an engaging overview with uk perspectives on reductions in mrsa and clostridium difficile infection. carole described the political situation in the early 2000s, with daily stories in the papers about the mrsa 'scandal'. this led to huge scrutiny and political drive for improvement, which has been successful contrary to the predictions of many (most?) experts! but what has caused the rate of mrsa to fall so dramatically? with many interventions during the same period, it is impossible to tell. but the high degree of scrutiny almost certainly played a key role. martin gave some useful guidance about performing an effective root cause analysis (to get to the root of the problem!): make sure the right stakeholders are around the table (junior doctors won't do); be a toddler (ask why? why? why?), document and evaluate improvement. on a personal note, the conference was very enjoyable. it had a good mixture of delegates from the middle east and elsewhere, and the talks prompted much interesting discussion! presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study hospital respiratory protection program toolkit facemasks for the prevention of infection in healthcare and community settings self-protection as a driver for hand hygiene among healthcare workers preventing surgicalsite infections in nasal carriers of staphylococcus aureus parachute use to prevent death and major trauma related to gravitational challenge: systematic review of randomised controlled trials author is a consultant to gama. key: cord-301103-idu4j78a authors: sohrab, sayed s.; azhar, esam i. title: genetic diversity of mers-cov spike protein gene in saudi arabia date: 2019-12-09 journal: j infect public health doi: 10.1016/j.jiph.2019.11.007 sha: doc_id: 301103 cord_uid: idu4j78a background: middle east respiratory syndrome coronavirus (mers-cov) was primarily detected in 2012 and still causing disease in human and camel. camel and bats have been identified as a potential source of virus for disease spread to human. although, significant information related to mers-cov disease, spread, infection, epidemiology, clinical features have been published, a little information is available on the sequence diversity of spike protein gene. the spike protein gene plays a significant role in virus attachment to host cells. recently, the information about recombinant mers-cov has been published. so, this work was designed to identify the emergence of any another recombinant virus in jeddah, saudi arabia. methods: in this study samples were collected from both human and camels and the spike protein gene was amplified and sequenced. the nucleotide and amino acid sequences of mers-cov spike protein gene were used to analyze the recombination, genetic diversity and phylogenetic relationship with selected sequences from saudi arabia. results: the nucleotide sequence identity ranged from 65.7% to 99.8% among all the samples collected from human and camels from various locations in the kingdom. the lowest similarity (65.7%) was observed in samples from madinah and dammam. the phylogenetic relationship formed different clusters with multiple isolates from various locations. the sample collected from human in jeddah hospital formed a closed cluster with human samples collected from buraydah, while camel sample formed a closed cluster with hufuf isolates. the phylogenetic tree by using aminoacid sequences formed closed cluster with dammam, makkah and duba isolates. the amino acid sequences variations were observed in 28/35 samples and two unique amino acid sequences variations were observed in all samples analyzed while total 19 nucleotides sequences variations were observed in the spike protein gene. the minor recombination events were identified in eight different sequences at various hotspots in both human and camel samples using recombination detection programme. conclusion: the generated information from this study is very valuable and it will be used to design and develop therapeutic compounds and vaccine to control the mers-cov disease spread in not only in the kingdom but also globally. coronaviruses (covs), belongs to the family coronaviridae. they have single stranded positive-sense rna genome with ∼26-32 kb in size [1, 2] . the genomic organization and expression pattern are similar in all coronaviruses. it is well known that multiple covs are found naturally and their genetic recombination hap-contact with camels, as well as community settings [6] [7] [8] . mers-cov is responsible for causing lower respiratory infections with fever and cough followed by shortness of breath and organ failure in severe cases with comorbidities [9] [10] [11] . earlier it was believed that inter-human transmission is limited but the nosocomial infection was reported in healthcare facilities due to inadequate infection control which resulted in larger outbreaks with mers-cov confirming human to human infection [12] . mers-cov has been isolated and sequenced from camel and its infected owner patient as well as in air sample collected from the same barn that sheltered the infected camel and showed the identical sequences from both camel and human which confirms the direct transmission from camel to human [7, 13] . mers-cov has been detected in upper and lower respiratory secretions at relatively high virus load and in fecal samples [14] . the identification of mers-cov and the neutralizing antibodies as well as a similar coronavirus has already been identified from camels and bats from multiple locations; ghana, europe, south africa, oman, the canary islands, uae, korea, and egypt [12, 14, 15] . recently, a mutant mers-cov has been identified from south korea. the mutation was observed in ribosomal binding domain (rbd) domain of spike protein gene [16, 17] . additionally, the mers-cov neutralizing antibodies were detected recently in the young goats and sheep from jordan and egypt [15, 18] . recently, a large outbreak observed due to the unusual presentation of mers-cov from riyadh, saudi arabia [19] . the covs have high sequence variation which favors the possible recombination, mutations, and emergence of a novel and recombinant virus with variable characters and extended hosts. the emergence of dominant and recombinant mers-cov which caused a human outbreak in 2015 has been reported from camel [3] . the zoonotic introduction was suspected after the mers-cov identification [18] and in a recent study from uae the genetic diversity of mers-cov full-genome from both human and dromedary camel was analyzed, and very closed sequence similarity was observed which confirms the zoonotic introductions [20, 21] . additionally, the zoonotic introduction time and seasons play an important role in the disease spread. based on the analysis of the distribution of human outbreak cluster size, it has also been demonstrated that the time of zoonotic introduction and season plays a significant role in human outbreak driven by mers-cov in arabian peninsula [22] . the mers-cov is known to have genetic diversity. the spike gene plays a significant role in host cell attachment and the entry of the virus in the host cells [23] . the rbd of spike protein gene mediates the virus interaction to the host cell and binds with dipeptidyl peptidase 4 (dpp4, cd26) known as a cellular receptor which favors the viral entry into the cell and is immunodominant and induces neutralizing antibodies [24] [25] [26] . based on the literature, it is well known that mers-cov spike protein gene has significant genetic variability isolated from both human and camel. in south korea, a recent outbreak occurred with a high fatality rate. the spike gene diversity was identified in many samples and showed interspecific variation with mers-cov isolates from south korea [27] . a novel recombinant mers-cov has already been identified from saudi arabia [28] . recently, in another study, total 530 nucleotides deletion was observed in spike gene from serum samples collected from taif, saudi arabia and a novel genetic variant of mers-cov was designated as a quasispecies [29] . multiple substitutions of amino acids were observed in rbd, part of spike gene from a bat sample collected from uganda and the recombination in the s1 subunit of the spike gene was observed and it was expected that this variation likely to play an important role in the emergence of mers-cov causing disease in human [30] . recently, the mers-cov has been genetically and phenotypically characterized from africa [31] and south korea [32] . the nucleotide substitutions/ amino acid variation of spike gene has significantly affected the virus transmission, disease spread to extended hosts and their evolution in different geographical locations. based on the published information, there is a lack of detail information about spike gene sequence variability of mers-cov from saudi arabia infecting human and camel. so, there is an urgent need to identify the genetic variability of spike protein gene so that it can determine and established the link that how the virus is moving from infected camel to human. based on the above information, this study was conducted. the detection of mers-cov in human and camel determining the genetic diversity among spike gene will further help researchers as well as health authority to design and develop an effective disease management and control strategies in the kingdom of saudi arabia. the mers-cov samples were collected from both human and camel and stored at special infectious agents unit (siau), king fahd medical research centre (kfmrc), king abdulaziz university (kau), jeddah, saudi arabia. the samples (blood and nasal swabs) were isolated from the six patients just after one day after admission into the hospital. all nasal swabs were properly collected and maintained by immersing into viral transport medium in a cold container. all the collected samples were stored for further analysis after proper processing at bsl-3 lab in siau, kfmrc, kau, jeddah, saudi arabia. the freshly prepared vero cells (atccccl-81) were used to inoculate by using 100 l nasal swab and maintained in complete dmem following the described protocol [7] . the inoculated cells were further for virus infection and development of cytopathic effect by incubating at 37 • c with 5% co2. after complete cytopathic effect, the supernatant from cell-culture was collected. the complete sequences of mers-cov were retrieved from national center for biotechnology information (ncbi/pubmed) database. the retrieved sequences were aligned by using bioedit 7.2 software (http://www.mbio.ncsu.edu/bioedit/bioedit.html). the mers-cov spike protein gene-specific primers were designed by using the selected sequences to amplify the complete spike protein gene. the viral rna was purified from culture supernatants and nasal swab by qiaamp viral rna mini kit (qiagen). the mers-cov was detected by real-time rt-pcr using upe gene and further confirmed by orf1a primers, as described previously. briefly, a 25 l reaction was set up containing 5 l of rna, 12.5 l of 2 x reaction buffer from the superscript iii one step rt-pcr system with platinum taq polymerase 1 l of reverse transcriptase/taq mixture from the kit, 0.4 l of a 50 mm mgcl 2 solution (invitrogen), 1 g of non-acetylated bovine serum albumin (sigma), 400 nm of primers emc-orf1a-fwd and emc-orf1a-rev, as well as 200 nm of probe emcorf1a-prb (6-carboxyfluorescein (fam)-ttgcaaattggcttgcccccact -6-carboxy-n,n,n,n-tetramethylrhodamine (tamra). thermal cycling was performed at 55 • c for 20 min for the rt, followed by 95 • c for 3 min and then 45 cycles of 95 • c for 15 s, 58 • c for 30 s. [7, 46] . the purified viral rna was used to amplify the mers-cov spike protein gene. the purified viral rna was reverse transcribed, and the spike protein gene was amplified by rt-pcr. the amplified product was visualized on 1% agarose gel. the pcr product was eluted from the gel and purified with a gel purification kit (qiagen). the pcr amplicon was gel eluted and purified and further sequenced by biveriti thermal cycler (applied biosystems) using spike protein gene-specific primers in siau. the sequence alignment was performed using bioedit, version 7.2.5. and the genetic diversity was determined by analyzing the sequence identity matrix with selected mers-cov sequences from saudi arabia. to explore the phylogenetic relationship of generated mers-cov sequences with sequences were analyzed by megax software programme and a phylogenetic tree was constructed [33] . to analyze the pattern of recombination among the spike protein gene sequences, we have used the recombination detection program (rdp4, v.4.70) [34] . the multiple sequences alignment file was imported to rdp software for recombination detection. the major and minor parent and possible recombinant with recombination breakpoints and hot spots with their start and end sequences were also identified by default settings which include geneconv, bootscan, maxchi, chimaera, siscan, and 3seq, to detect putative recombination events in the spike gene sequence of mers-cov. the putative recombination events were identified and considered significant with the cut off p-value (≤0.05) with standard bonferroni corrections. the structure was predicted by using swiss modeling software (https://swissmodel.expasy.org/) utilizing the spike protein gene of both human and camel samples with selected mers-cov. as it has already been published about the recombinant mers-cov, we selected a few sequences of recombinant virus to compare the protein structure with samples collected from human and camel samples. the nasal swabs collected from both human and camels were found positive by rt-pcr. the vero cells were inoculated with samples obtained from human and camels shoed a cytopathic effect after 3 days of inoculation. the culture supernatants were used to detect the virus by rt-pcr for upe, orf1a, and orf1b regions. the spike protein gene was amplified by using spike gene-specific primers and visualized on 1% agarose gel. the full-genome of spike protein gene was sequenced bidirectionally from both human and camel samples at siau, kfmrc, kau, jeddah and tentatively designated as sample1390-hu-jed and sample-31-cam-jed. the generated sequences have been submitted in genbank with their accession numbers mn403101, mn403102 and used for diversity analysis with previously published sequences. the sample1390-hu-jed was used to analyze the similarity with other sequences and the highest nucleotides (98.4%) and amino acid similarity (99.8%) was observed with many isolates of mers-cov and the lowest nucleotides (64.7%) and amino acid the phylogenetic analysis with spike protein gene sequences (nucleotide and amino acid) of selected mers-cov sequences deposited in genbank was performed. the phylogenetic tree based on nucleotide sequences showed various clusters. the sample1390-hu-jed formed closed cluster with an isolate from buraydah (kt806006-2014) and taif (kr912196) while the sample-31-cam-jed formed the closest cluster with camel isolate from hufuf (kfu-hku-ky706247-2014) there mixed clustering of human and camel isolates were observed in a phylogenetic relationship, fig. 1 . the phylogenetic relationship was also analyzed by using amino acid sequences. the phylogenetic tree was constructed, and an almost similar pattern of clustering was observed with selected virus isolates (fig. 2) . the amino acid sequence variations were observed at multiple locations. total of 28 samples sequences showed sequence transversion of amino acids. interestingly, two common variations were also observed in all 35 samples analyzed as compared to human and camel sample collected and used in this study ( table 2 ). an attempt was made to analyze the nucleotide and amino acid sequence variations with selected recombinant mers-cov as compared to sample 31-camel-jeddah and total nineteen nucleotide sequence variations were observed scattered throughout the spike protein gene, and only three amino acid in camel while two amino acid variations were observed in human sample (fig. 3a, b) . our findings were supported by earlier reports and a unique amino acid substitution was observed in rbd which affecting the binding efficiency (kossyvakis et al., 2015) . additionally, in another study, only five mutations were detected in consensus sequences 473 intrahost and single nucleotide variants were identified (borucki et al., 2016) . the spike protein gene sequences were used to analyze the pattern of recombination in selected mers-cov from saudi arabia. putative recombination events were identified using recombination detection programme (rdp4, v.4.70) with the default settings [34] . based on rdp parameters, no significant recombination was observed. but when the recombination pattern was analyzed by using bootscan, 3seq, and parameters, the recombination was observed in eight, seven and one sequences at multiple positions with the average p-value of 6.870 × 10 −02, 8.031 × 10−03 and 4.589 × 10 −02 respectively with all the sequences analyzed (fig. 4) . recently, a genetic diversity analysis study was conducted from uae and 10 recombination events were observed in the camel samples. the most frequent recombination breakpoint was the junctions between orf1b and spike protein gene [20] . our data also supported by above reports that most of the recombination and sequences diversity have been observed in the spike protein gene. the structure of spike protein using amino acid sequences generated in this study from both human and camel samples and was compared to protein structure with recombinant mers-cov reported recently. the predicted structures of both recombinant as well as our generated sequences from both human and camel sample sequences, are presented in fig. 5 . during modeling, no significant variations were observed among the sequences and 99.4% similarity was observed with the available template in the protein database, but 3 amino acid variations were observed in human and 2 variations in camel samples in this study. the mutation in the rbd of spike protein gene affects the interactions with the human receptor, cd26. the similar kind of mutations and changes in amino acid have been reported earlier and the structural changes has been presented [16, 17] . the mers-cov was identified from jeddah, saudi arabia since 2012 and causes respiratory disease to a human. this virus has spread to 27 countries. the genetic diversity has been reported among covs. mers-cov also has been reported to have genetic diversity across the whole genome and especially in spike protein gene. our study has provided the genetic diversity of the spike protein gene isolated from both camel and human samples from jeddah, kingdom of saudi arabia. the highest sequence identity was observed with the previously reported sequences submitted in the genbank. the phylogenetic tree relationships were also formed a closed cluster with earlier known viruses from various locations in the kingdom. the nucleotide and amino acid variations were scattered throughout the full spike protein genome. interestingly, two common amino acid changes were observed in all the 35 samples analyzed. as it has already been reported about the emergence of the recombinant virus from saudi arabia [19, 28] and mutant virus from south korea [16, 17] and other intrahost variants reported [35] . the transversion of three amino acids was observed in our sample collected from human and two amino acid variation was observed in the camel sample as compared to selected recombinant virus sequences from saudi arabia. the comparative structure of the spike protein gene has been predicted and presented in this study to show how the structural changes as compared to recombinant virus appear. the effect of a mutation in the spike protein gene with the viral entry to the host cell by using cd26 binding s.s. sohrab, e.i. azhar / journal of infection and public health xxx (2019) xxx-xxx requires further detail study. these changes may affect the structural changes of rbd and interactions with the cognate human receptor, cd26 as it has been reported earlier [17] . the most important factor for the emergence of a novel virus is during the recombination step in the life cycle of the virus. the recombination happens with the co-circulating covs in multiple hosts which further increase the rate of recombinant virus emergence. the estimated rate of mutations in covs are known as moderate to high as compared to other known ssrna viruses. the rate of average substitution for covs has been reported as ∼10 −4 substitutions per year per site while the average substitution of s gene in 229e was observed to be ∼3 × 10 −4 per site per year [36] and in saars-cov it was estimated to be 0.80-2.38 × 1 −3 per site per year. in the case of mers-cov, the average rate of substitution was found to be 1.12 × 10 −3 per site per year [37] . recently, the co-circulation of multiple hcov species in camel along with mers-cov has been reported from saudi arabia and resulted into an emergence of a recombinant virus which was responsible for an outbreak in 2015 [28, 38] . these results showed that many cov are circulating in the wild animals originated from human and animal and by this, an opportunity is favored for the genetic recombination, evolution and emergence of potential and recombinant virus lethal to human [3] . by considering the variations in the mers-cov spike protein gene reported so far, it is very important to consider the frequent appearance and conservation of rbd alterations in human infections. it is well reported that the interspecies transmission of covs is mainly mediated by mutations in spike protein gene with a high affinity toward human receptors [28, 39, 40] . the rbd mutations increased the unexpected emergence with reduced affinity to human cd26 in south korean mers-cov outbreak. the identification of recombinant virus from saudi arabia [28] as well as unusual presentation and emergence of mers-cov resulted in an outbreak in riyadh [19] raises several critical questions. based on epidemiologic features, the zoonotic transmission of the mers-cov from the animal reservoir has been suggested by an intermediate animal host. the evidence of dromedary camel as a reservoir for mers-cov has been already reported based on the identical sequences obtained from camel and a patient with close contact with camel nasal secretion which directs cross-species transmission without any intermediate host [7, 13] . indeed, it has been reported that the interfacial residual difference in receptors of various mammalian host species is very important for interspecies transmission of covs [40] . the sequence diversity and homology play an important role in various functions of virus and host cells like cellular fusion and attachment [28, 41] . the viruses have ability to cause various types of diseases including neurological disorders. [42, 43] . the human genome sequence diversity due to gene flow from south east asia to other locations also plays an important role in the disease transmission and spread of pathogens from one location to other as well as intrahost transmission. as it has been shown that the ancient east-asian mtdna hg-m and exhibit the highest nucleotide diversity [44, 45] . however, our data showed no significant amino acid sequences variations in the spike protein gene. finally, there is not much information available about the role of rbd mutation linked with reduced affinity to host receptor cd26 and mers-cov transmissibility to infect human. the genetic diversity was significantly observed in the spike protein gene sequences of both nucleotides and amino acids collected from saudi arabia. the information generated from this study provided an insight to the pattern of sequence diversity which plays an important role in the viral disease spread as well as movement of virus from one host to others. this diversity information will play an important role in designing and development of antiviral drugs, vaccines as well as antiviral compounds against mers-cov. based on the data generated in this study, it is concluded that the genetic diversity of the spike protein gene plays an important role in interaction with cd26. the genetic diversity emerges with the high frequency of recombination events in the covs resulted in the emergence of novel recombinant viruses with unpredictable changes in the virulence during human infection. finally, to elucidate the evolutionary pathway, more detail mutation analysis of spike protein gene needs to be done using more sequences from saudi arabia and we should take lessons from the outbreak caused by mers-cov and saars-cov and we should take necessary measures to combat any further outbreak. sss collected and processed samples, designed the study, analyzed the data, and conceived the idea. eia supervised the research and reviewed the manuscript. deanship of scientific research (dsr), king abdulaziz university, jeddah, saudi arabia. coronavirus genome structure and replication coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus epidemiology, genetic recombination, and pathogenesis of coronaviruses isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus in bats, saudi arabia evidence for camel-to-human transmission of mers coronavirus mers-cov outbreak in jeddah -a link to health care facilities epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection transmission of mers-coronavirus in household contacts probable transmission chains of mers-cov and the multiple generations of secondary infections in south korea detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus crosssectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt variations in spike glycoprotein gene of mers-cov spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction unusual presentation of middle east respiratory syndrome coronavirus leading to a large outbreak in riyadh during 2017 diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae mers-cov spillover at the camelhuman interface mechanisms of coronavirus cell entry mediated by the viral spike protein dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 mers-cov spike nanoparticles protect mice from mers-cov infection variations in spike glycoprotein gene of mers-cov epidemiology of a novel recombinant mers-cov in humans in saudi arabia spike gene deletion quasispecies in serum of patient with acute mers-cov infection further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus mers-cov from camels in africa exhibit region-dependent genetic diversity genetic characterization of middle east respiratory syndrome coronavirus mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets rdp4: detection and analysis of recombination patterns in virus genomes middle east respiratory syndrome coronavirus intra-host populations are characterized by numerous high frequency variants mosaic structure of human coronavirus nl63, one thousand years of evolution spread, circulation, and evolution of the middle east respiratory syndrome coronavirus co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia coronavirus diversity, phylogeny and interspecies jumping bat-to-human: spike features determining "host jump" of coronaviruses sars-cov, mers-cov, and beyond low levels of hiv-1 envelope-mediated fusion are associated with long-term survival of an infected ccr5-/-patient zikv leads to microcephaly inhibition of neurogenesis by zika virus infection austro asiatic tribes are original native inhabitants of india paleolithic spread of y-chromosomal lineage of tribes in eastern and northeastern india assays for laboratory confirmation of novel human coronavirus none declared. not required. key: cord-319006-6f2sl0bp authors: plipat, tanarak; buathong, rome; wacharapluesadee, supaporn; siriarayapon, potjaman; pittayawonganon, chakrarat; sangsajja, chariya; kaewpom, thongchai; petcharat, sininat; ponpinit, teerada; jumpasri, jaruphan; joyjinda, yutthana; rodpan, apaporn; ghai, siriporn; jittmittraphap, akanitt; khongwichit, sarawut; smith, duncan r; corman, victor m; drosten, christian; hemachudha, thiravat title: imported case of middle east respiratory syndrome coronavirus (mers-cov) infection from oman to thailand, june 2015 date: 2017-08-17 journal: euro surveill doi: 10.2807/1560-7917.es.2017.22.33.30598 sha: doc_id: 319006 cord_uid: 6f2sl0bp thailand reported the first middle east respiratory syndrome (mers) case on 18 june 2015 (day 4) in an omani patient with heart condition who was diagnosed with pneumonia on hospital admission on 15 june 2015 (day 1). two false negative rt-pcr on upper respiratory tract samples on days 2 and 3 led to a 48-hour diagnosis delay and a decision to transfer the patient out of the negative pressure unit (npu). subsequent examination of sputum later on day 3 confirmed mers coronavirus (mers-cov) infection. the patient was immediately moved back into the npu and then transferred to bamrasnaradura infectious disease institute. over 170 contacts were traced; 48 were quarantined and 122 self-monitored for symptoms. high-risk close contacts exhibiting no symptoms, and whose laboratory testing on the 12th day after exposure was negative, were released on the 14th day. the omani ministry of health (moh) was immediately notified using the international health regulation (ihr) mechanism. outbreak investigation was conducted in oman, and was both published on the world health organization (who) intranet and shared with thailand’s ihr focal point. the key to successful infection control, with no secondary transmission, were the collaborative efforts among hospitals, laboratories and mohs of both countries. from 2012 to 21 july 2017, there have been 2,040 reported laboratory-confirmed cases and 712 deaths from middle east respiratory syndrome coronavirus (mers-cov) infection in 27 countries [1] . a single imported case of middle east respiratory syndrome (mers) in south korea, identified on 20 may 2015, resulted in 150 laboratory-confirmed cases, amplified by infection in hospitals and the transfer of patients within and between hospitals, and caused 15 deaths within 26 days, mainly among patients, visitors and healthcare personnel [2] . this highlighted the need for vigilant surveillance and the importance of swift and thorough contact tracing. the thai ministry of public health (moph) launched mers surveillance and made mers a notifiable disease in 2012, particularly targeting people travelling into thailand from affected countries. it also initiated a nationwide public education campaign [3] . in 2015, mers-cov infection was classified as a dangerous communicable disease in thailand according to the communicable act b.e. 2523 (ad 1980) . being added to this act required all probable and confirmed cases and their close contacts to be quarantined in a designated area for the duration of the maximum incubation period of 14 days [4] . there have been increasing numbers of incoming travellers from the middle east seeking medical care in thailand in the past decade. more than 1.3 million medical tourists travelled to thailand in 2015, of which 14.2% were from united arab emirates (uae) and oman [5] . despite that, only three cases of mers have been confirmed as of july 2017 [6] . this study shows that, in addition to needing collaboration among different organisations during an outbreak, diagnosis cannot rely only on laboratory examination alone, especially when the specimen was not suitable. a negative laboratory result in a patient from an endemic region with mers-like clinical signs still demands cautious infection control measures in an isolation unit. on 15 june 2015 (day 1), a 75-year old omani man travelled to thailand, seeking treatment for his heart condition. upon arriving at the airport, the patient took a taxi to a hotel and checked in before leaving to a private hospital in another taxi. upon presentation at the emergency room at the private hospital, the patient was promptly diagnosed with heart failure and possible pneumonia. as the patient had travelled from the middle east that day, mers was suspected and he was isolated in a negative pressure room (npu). on day 3, the private hospital notified the bureau of epidemiology, under the thai moph, of the omani patient. rt-pcr for mers-cov on upper respiratory tract samples (nasopharyngeal swabs) that were sent on days 2 and 3 resulted in false negatives, leading to a 48-hour delay in diagnosis and a decision to transfer the patient out of the negative pressure unit (npu) on day 3. subsequent examination of a sputum sample later on day 3 confirmed mers-cov infection in the patient. the patient was immediately transferred back into the npu. on day 4, thailand's moph officially reported the first imported mers case and the over 170 individuals, including 48 with high-risk of exposure were traced. thirty-six high-risk close contacts were quarantined in thailand and 40 low-risk contacts were monitored in oman. another 12 high-risk close contacts (airline crew members) were quarantined in the country they were situated when traced. medical records from the private hospital and bidi under the department of disease control, under the thai moph, were reviewed [7] . further, the patient and their family members were asked to elaborate on the clinical presentations and previous medical care in oman by thai investigators. information from the omani moh was obtained via the ihr mechanism during investigation. in accordance with who interim guidelines for laboratory testing for mers-cov [8] , mers-cov rna was tested in sputum (pre-treated with n-acetylcysteine) and via nasopharyngeal swab (when sputum was not available), using qiaamp viral rna mini kit (qiagen, hilden, germany) for extraction. two real-time rt-pcr assays targeting upstream of envelope (upe) and open reading frame 1a (orf-1a) genes [9, 10] , and one rt-pcr assay for generating amplicons for sequencing, targeting the betacoronavirus rna-dependent rna polymerase (rdrp) gene (rdrpseq assay) [10] , were performed simultaneously by who collaborating centre for research and training on viral zoonoses, faculty of medicine, chulalongkorn university (whocc) to increase efficiency and allow reporting of results within 24 hours of receiving the samples. respiratory specimens (sputum, nasopharyngeal and throat swabs) were collected daily from the index case from the time of patient isolation on day 5 through to day 17. the respiratory samples were sent to three centres, the bidi, the thai national institute of health (nih) and whocc, for parallel real-time pcr testing of mers-cov. additional molecular sequencing was performed by whocc. the whole genome amplification of mers-cov was carried out from extracted viral rna from collected sputum of the index case. seventy sets of specific primer pairs were used to amplify the complete genome as previously described [11] , followed by sanger sequencing. for the analysis, all mers-cov genomes with complete coding sequences available in genbank as of 30 december 2016 (n = 233), were compared with the mers-cov genome obtained in this study. sequences showing less than 35 divergent nt positions and two representatives of the five lineages defined by sabir et al. [12] , were selected and used for phylogenetic analysis. a phylogenetic tree was constructed using the maximum likelihood method based on the general time reversible model and 1,000 bootstrap replicates in mega7 [13] . contact tracing was immediately implemented by the thai moph. contacts were divided into two categories; high-risk and low-risk. a high-risk close-contact was defined as any person who was within 1 m of contact with the index case while the patient was symptomatic, regardless of duration of contact. airline passengers seated in the two rows surrounding the index case's seat were also considered high-risk close contacts as per who guidelines [14] . a low-risk contact was any person who had been in contact with the patient while the patient was symptomatic, but from a distance of more than 1 m. people were considered non-contacts if there was no evidence of direct contact with the patient or if they were not likely to be in contact with respiratory droplets, the means of transmission for mers-cov. several methods of contact tracing and active case finding were applied depending on the nature of contact, contact location, degree of symptoms at the time of contact, etc. at the hospital, attending physicians' and nurses' contact status was determined via airline passengers who sat in the two rows around the patient 14 a quarantined. laboratory testing results for mers-cov were negative. airline crew members 12 identified, but left thailand for their return flight on 15 june 2015 (day 1). self-quarantined and self-monitored for symptoms, being off-service for the duration of the quarantine period. none reported to have developed any illness. healthcare workers at the private hospital 17 all were immediately quarantined in the hospital. their laboratory testing were negative for mers-cov. taxi drivers 2 quarantined. laboratory testing for mers-cov were negative. airline passengers 63 self-monitored for symptoms, with social distancing. hotel staff 6 healthcare workers 53 close relatives 11 assigned for two weeks of follow-up from the last date of exposure. one close-contact was assigned to be tested for mers-cov infection, but refused to cooperate. healthcare contacts at the first hospital 20 healthcare contacts at the second hospital 10 mers: middle east respiratory syndrome; mers-cov: middle east respiratory syndrome coronavirus. a only one of whom was a thai national. interview and the hospital surveillance camera. at the hotel, potential contacts were identified by interviewing the personnel on-duty when the patient checked in and by using the hotel's surveillance camera. the investigation team from the department of disease control (ddc) at the thai moph identified the airport-to-hotel taxi driver using the airport taxi booking slip and the hotel-to-hospital taxi driver by looking at the surveillance camera from the traffic control department. the airline provided the investigation team with the passenger manifest and the thai authorities identified passengers' local address using immigration arrival cards. local health authorities in relevant provinces were informed and asked by the investigation team to locate and contact the identified passengers in their jurisdiction. some passengers voluntarily reported to a hospital or health authority in response to the moph's announcement of first imported mers case in thailand. central authorities were responsible for locating all high-risk close-contacts, while local authorities were responsible for low-risk contacts. the time lapse between the affirmed diagnosis and each contact-tracing varied. contacts at the hospital were identified within a day, while other high-risk close contacts, such as passengers on the flight, were identified within 3 days. other low-risk contacts were traced within 7 days. patients who were in the same npu ward as the index case at the private hospital on day 1 were monitored despite being considered non-contacts because: i) the room for each patient was separated, ii) they had no direct contact with the index case and iii) the known mode of transmission of mers-cov is respiratory droplet. further, patients in the icu, which is where the index case was moved after being taken out of the npu 8 hours before diagnosis on day 3, were monitored, despite being non-contacts. in the event they developed a new episode of fever or respiratory symptoms, samples were collected and sent for testing to rule out mers-cov infection. another concern was icu healthcare workers' simultaneous care of several patients. prompt quarantine and monitoring of patients in the icu was to be implemented if any icu healthcare worker developed any symptoms or was diagnosed with mers-cov infection. most high-risk close contacts were quarantined and all were continuously monitored for 14 days. nasopharyngeal and throat swabs, stored in single viral transport media, from 36 high-risk close contacts were collected on two occasions as per the bureau of epidemiology guidelines [15]: first upon identification as being a high-risk close contact and second on day 12 during the quarantine period. specimens were duplicated and sent to any two of three laboratories (bidi, nih and whocc) for parallel real-time pcr testing of mers-cov, using both who and commercial primers for any given sample. in line with the thai communicable disease act, high-risk close contacts were only released after completing 14 days of quarantine and if laboratory testing on the 12th day of quarantine was negative. sera from three high-risk close contacts (the close relatives who travelled with the patient to thailand) were sent to the institute of virology, university of bonn medical centre, bonn, germany to test for anti-mers igg and igm using mers-cov infected vero cells for immunofluorescence assay (anti-mers-cov iift, euroimmun, lübeck, germany). the real-time rt-pcr results on the patient's nasopharyngeal swabs were negative for upe and orf-1a gene targets on days 2 and 3 (table 1) , and the patient was thus transferred to a non-npu in the icu. however, the third sample from sputum that was taken later on day 3 as the patient's condition deteriorated and that underwent three simultaneous rt-pcr assays at the whocc, was positive for upe, orf-1a and rdrp gene targets. when whocc confirmed sputum was positive for mers-cov infection, the patient was immediately transferred back to the npu that night. mers-cov was confirmed via sequencing within 24 hours by whocc. the patient was referred to and isolated at bidi on the morning day 4. the patient's clinical presentation at that time was diffused bilateral pneumonia with pending acute respiratory distress syndrome [7] . he did not report any previous illnesses pertaining to these symptoms. later the same day, sputum was collected from the patient for reconfirmation, which tested positive for upe and orf-1a genes by four different laboratories: the nih, ramathibodi hospital, bidi and whocc. the thai moph proceeded to publicly announce the first confirmed imported mers case in the evening of 18 june 2015 (day 4). the patient was monitored for mers until 1 july 2015 (day 17) and was discharged on 3 july 2015 (day 19). upon laboratory confirmation on day 3, the case was immediately notified to the who. in order to support local handling of the outbreak, an epidemiologist and a risk communication expert were deployed from the who south-east asia regional office and who headquarters, respectively. the epidemiological investigation revealed retrospectively that on 4 june 2015, 11 days before the admission to the private hospital in thailand, the patient was admitted to a regional hospital in oman, with retrosternal and left-sided chest pain, which was radiating to his left arm (figure 1 ). the condition was associated with shortness of breath on exertion and was considered typical cardiac pain. the patient was diagnosed with acute coronary syndrome. three days later, on 7 june 2015, his condition had improved and he was discharged. a close relative of the patient observed a dry cough of mild degree in the patient since 10 june 2015. on 13 june 2015, he was admitted to a second hospital, displaying signs of somnolence, fatigue and elevated blood sugar level; he was diagnosed with diabetes mellitus on 14 june 2015. there was also decreased air entry in the right lung with fine crepitations. chest x-ray showed opacity in middle and lower zones of right lung. both hospitals' medical records confirmed the patient did not exhibit any fever or cough symptoms. the patient was discharged that day with a follow-up appointment at a regional hospital scheduled for 16 june 2015; however, the patient wished to seek medical care in thailand and flew there on 15 june 2016. the virus sequence (tha/cu/2015) obtained from the patient was submitted to genbank (genbank accession number kt225476). tha/cu/2015 showed closest relations (99.91% nt identity) to three human mers-cov strains isolated in saudi arabia in 2015 (genbank accession numbers kt806044, kt806045 and kt806054). figure 2 shows the phylogenetic tree constructed from the mers-cov whole genome obtained from the patient (tha/cu/2015, 29,809 bp), among the closest relatives and representatives for each mers-cov lineage defined by sabir et al. [12] . correspondences with a close relative of the patient and the omani moh revealed that the patient was a fisherman from ghasil village, south sharqiya governorate, but spent june-august in al mintrib village of bidiya wilayat, north sharqiyah governorate. the patient neither had any history of travel outside oman nor contact with anyone with a history of travel outside oman within the 14 days preceding his travel to thailand. further, the patient had no contact with any person with acute respiratory infection or confirmed mers before developing symptoms. the family used to own one camel but had not had contact with that camel for few months. a close relative who lived near the patient, but did not travel to thailand with the patient, owned and cared for three camels. samples collected from these camels tested negative in oman for mers-cov by rt-pcr on day 15. it is also noteworthy that the patient and the patient's close relatives never consumed raw camel milk or camel urine. a total of 211 contacts of the index case were identified after the patient was confirmed to have mers. in thailand, 170 contacts (excluding the 42 healthcare personnel at bidi, investigated separately in the report by wiboonchutikul et al. [7] ) were identified. of these 170, 48 were high-risk close contacts and 122 were low-risk contacts ( table 2 ). all patients treated in the same ward (icu) at the private hospital of first admission of index case before mers diagnosis were identified as non-contact, however they were fully monitored and followed up for 14 days, as per the thai moph's protocol, as a precautionary measure. in oman, the outbreak investigation determined there to be 41 lowrisk contacts and this information was published on the dedicated who system. fortunately, there was no secondary transmission associated with this case. this study demonstrates the challenges faced by physicians and the cross-border threats that exist with increasing international medical tourism. although precautions such as thermoscan are in place at airports in light of the mers-cov outbreak in the middle east and south korea, cases can slip through checkpoints due to atypical presentations. the patient flew on a commercial airline despite his sickness and was not detected by the thermoscan at the immigration checkpoint in bangkok as he was afebrile. he only had a mild, non-productive cough. this case report also provides important lessons regarding clinical case identification. the clinical diagnosis was complicated due to the existence of congestive heart failure, a condition that predisposes to either community-acquired or nosocomial pneumonia of various aetiologies. furthermore, initial chest radiographs did not show clear signs of interstitial pneumonia as expected with mers-cov infection. various contact tracing methods involving the cooperation of several authorities and business institutes, such as the border control, airline, hotel management, traffic control, local authorities and hospitals, were used to track-down all potential contacts in order to prevent an outbreak. phone calls, passenger manifests, surveillance videos and immigration cards were essential tools for the successful contact tracing. upper respiratory tract samples, such as nasopharyngeal and oropharyngeal, are often used to detect upper respiratory tract illnesses during the acute phase. in mers-cov infection, however, higher viral loads have been found in specimens from the lower respiratory tract [16] , with sputum or endotracheal secretion samples yielding better results [17] . this aligns with the who interim guidelines, which encourage using lower respiratory tract samples if available [8] . physicians, surveillance staff and laboratory personnel must be well-informed about the procedures, reliability and limitations of diagnostic tests, and should be able to recognise signs of mismatch between laboratory results and clinical presentations. in this case, the initial diagnostic testing of upper respiratory tract samples on days 2 and 3 caused a delay in diagnosis that could have facilitated onward transmission. fortunately, the patient was isolated in a npu upon initial admission; however, he could have exposed other patients and hospital workers during the 8 hours he spent in regular icu after the initial false negative test results. the decision to conduct repeated laboratory testing, as well as to test lower respiratory tract samples, was driven by clinical assessment and knowledge of the virus excretion pattern reported in earlier cases. the algorithm for diagnosing mers in the current who interim guidelines for laboratory testing indicates that two positive real-time pcr tests are sufficient in diagnosing mers, i.e. orf-1a and upe. however, we found that performing three simultaneous assays and sequencing for upe, orf-1a and rdrp genes in parallel, allowed for swift in-country confirmation of the presence of mers-cov and reporting within 24 hours, facilitating prompt outbreak control measures. there was no secondary transmission, not even to close relatives or the healthcare workers at bidi where the patient was transferred after the diagnosis was confirmed, despite 170 contacts, including 48 high-risk close contacts [7] . despite the successful outcome of infection control measures, the case provides an example of the risk of mers-cov infection importation. aside from providing technical support through the moph emergency operations centre, deployments of experts from the who south-east asian regional office also greatly facilitated the exchange of information on possible modalities of mers-cov infection with the omani moh through the ihr (2005) mechanism [18] . this study therefore emphasises how important hospital and organisation collaboration, as well as cross-border cooperation, is to successful infection control in the event of an outbreak. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome (mers) in the republic of collaboration preparedness plea announcement nonthaburi: royal thai government gazette medical tourism: falling oil prices & middle east tourism. bangkok post middle east respiratory syndrome coronavirus (mers-cov) -thailand. geneva: who; 26 lack of transmission among healthcare workers in contact with a case of middle east respiratory syndrome coronavirus infection in thailand laboratory testing for middle east respiratory syndrome coronavirus (mers-cov) detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets emergencies preparedness, response: technical guidance on contact tracing, middle east respiratory syndrome coronavirus (mers-cov) department of medical services. [guidelines for diagnosing and treating mers patients, as well as protocols to prevent an outbreak in the hospital clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus: update for clinicians international health regulations none declared. tpl, rb, ps and cp were responsible for the case investigation, sample handling and coordination of sample tests. they were also responsible for contact tracing and quarantine. cs was responsible for the clinical case management and hospital quarantine authority. sw, tk, sp, tpo, jj, yj, aj, sk and drs were responsible for the laboratory testing and sequencing of mers-cov. ar and sg analysed the sequence and submitted it to genbank. rb, sw, sg and th wrote the original manuscript. cd, vmc, rb, sw, sg and th critically reviewed the original manuscript. sg, rb, sw and th critically reviewed the revised manuscript. this is an open-access article distributed under the terms of the creative commons attribution (cc by 4.0) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indicate if changes were made. key: cord-287758-da11ypiy authors: mônica vitalino de almeida, sinara; cleberson santos soares, josé; lima dos santos, keriolaine; emanuel ferreira alves, josival; galdino ribeiro, amélia; trindade tenório jacob, íris; juliane da silva ferreira, cindy; celerino dos santos, jéssica; ferreira de oliveira, jamerson; bezerra de carvalho junior, luiz; do carmo alves de lima, maria title: covid-19 therapy: what weapons do we bring into battle? date: 2020-09-10 journal: bioorg med chem doi: 10.1016/j.bmc.2020.115757 sha: doc_id: 287758 cord_uid: da11ypiy urgent treatments, in any modality, to fight sars-cov-2 infections are desired by society in general, by health professionals, by estate-leaders and, mainly, by the scientific community, because one thing is certain amidst the numerous uncertainties regarding covid-19: knowledge is the means to discover or to produce an effective treatment against this global disease. scientists from several areas in the world are still committed to this mission, as shown by the accelerated scientific production in the first half of 2020 with over 25,000 published articles related to the new coronavirus. three great lines of publications related to covid-19 were identified for building this article: the first refers to knowledge production concerning the virus and pathophysiology of covid-19; the second regards efforts to produce vaccines against sars-cov-2 at a speed without precedent in the history of science; the third comprehends the attempts to find a marketed drug that can be used to treat covid-19 by drug repurposing. in this review, the drugs that have been repurposed so far are grouped according to their chemical class. their structures will be presented to provide better understanding of their structural similarities and possible correlations with mechanisms of actions. this can help identifying anti-sars-cov-2 promising therapeutic agents. the world is facing a huge challenge in the coronavirus disease (covid-19) pandemic: how to fight an enemy without weapons in terms of therapy? unfortunately, even before the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) worldwide spread, there were no clinical treatments nor prevention strategies available for any human coronavirus. 1 it is understandable that both society and researchers urge the discovery of new compounds or even of a drug that is commercially available that can be employed by physicians mainly for patients with the extreme presentation of covid-19. there is also urgency in the discovery of medicine with prophylactic action to prevent the entry of the virus in host cells after exposure. vaccine research experts already indicate that rescue from sars-cov-2 will come from a long but effective journey to produce a vaccine. 2 while this is not a reality, the scientific community, including medicinal chemists and doctors who accompany patients, are trying to identify therapeutic alternatives. this is a meritorious attitude: the commitment with the protection of humanity. nevertheless, the rigorous feature of science in the discovery of a new drug cannot be disregarded, even during a pandemic and in the face of the urgent demand for a treatment, to avoid eventual mistakes and spurious hope. the increase in studies related to sars-cov-2 during the first semester in 2020 has allowed the rather speedy identification of promising therapeutic targets for both developing immunotherapies and producing/identifying antiviral drugs. it is noteworthy the increase in outbreaks of sars-cov (2002) and mers-cov (2013), with accelerated production of knowledge on these hcovs, which has been very useful for ongoing investigations on sars-cov-2. one example is the availability of technological devices that allowed the fast sequencing of sars-cov-2 genome and the elucidation of a promising antigen target, the s glycoprotein. nonetheless, the development of a human vaccine can take years, especially because employing emergent technologies requires extensive safety tests and expansion to large scale production in order to assist the world population, as demanded in the case of the covid-19 pandemic. 3 the development of new medicine also demands many years of research that involve stages of reasonable planning, synthesis, structural characterization, formulation of prototypes, preclinical and clinical trials. therefore, the literature highlights, as alternative treatments for covid-19, the repurposing of drugs, which is fast and useful in emergencies such as the one experienced today. the repurposing of drugs means the use of broad-spectrum medicine for a new disease, once its metabolic characteristics, doses, potential efficacy and adverse effects are pre-established due to drug studies extracellular liquid volume and arterial pressure of the human body. it is largely expressed in fifteen human tissues, including ciliated bronchial epithelial cells and type ii pneumocytes form pulmonary alveoli, the main location of lesions caused by sars-cov-2. 36, 37 after ace2 receptor-binding, a conformational alteration occurs in protein s allowing the fusion between the viral envelope and the host cell membrane via endosomal. then, sars-cov-2 releases the rna into the cytoplasm to be translated into viral replicase polyproteins pp1a and pp1ab, which are processed by 3cl pro and pl pro proteases, respectively. the cleavage products are 16 nsps that form the transcription and replication complex. 38 next, the positive rna strand is translated into a template of negative strand that allows the synthesis of new genomics and sub genomics mrnas. these mrnas are translated and transcribed producing structural and accessory proteins. viral proteins and rna genomic are put together in virions at endoplasmic reticulum and golgi complex, finally transported through vesicles and released from the cell host for infecting new cells. 38, 39 the covid-19 symptomatology starts after the virus is installed in host cells. in general, the symptoms include lasting and high fever, dry cough, shortness of breath, muscles aches or tiredness, sputum production, headaches, and a small percentage of individuals presented gastrointestinal symptoms such as diarrhoea and vomit. 40 the incubation period of sars-cov-2, from exposure to first symptoms, lasts 2 to 14 days. the pre-symptomatic stage lasts from 1-3 days (possibly more) before the beginning of symptoms. the post-symptomatic stage lasts at least 7 days after the beginning of symptoms and 3 days after lowering of fever and improvement of respiratory symptoms. 41 there are many unanswered questions such as the duration of potential immunity of both symptomatic and asymptomatic individuals when infected with sars-cov-2. 31 it is noteworthy that efficient strategies to fight the disease should not depend on the symptoms of patients, once asymptomatic or pre-symptomatic subjects can play an important role in the direct and indirect transmission to others, as demonstrated by arons et al. 42 this investigation reports that half the residents in a nursing facility, who tested positive, were asymptomatic when tested and probably contributed to the transmission to other residents. thus, control strategies focused on symptomatic residents were not sufficient to prevent transmission once sars-cov-2 had been introduced in the facility. laboratory exams of infected patients showed alterations in haematology and biochemistry. it was verified the increase of leukocytes and the reduction of lymphocytes; increased d-dimer and erythrocyte sedimentation rate (esr), prolongation in prothrombin times (pt), followed by increase in bilirubin levels, aspartate transaminase (ast), alanine transaminase (alt), creatinine, lactate dehydrogenase (ldh), protein c reactive (pcr), hypoalbuminemia (low albumin), microcytosis and thrombocytopenia. 43 in addition, inflammatory factors that indicate the immune condition of patients, such as interleukins (il) il-2, il-6, il-7, and il-10 and the tumoral necrosis factor-α (tnf-α) become elevated. plasma levels of granulocyte-colony stimulating factor (gcsf), protein induced by interferon gamma, monocyte chemoattractant protein-1 (mcp-1), macrophages inflammatory protein 1α and tnf-α also display significant increase. 44 potential risk factors or comorbidities that can lead to complications of covid-19 include elderly individuals (specially above 65 years of age), cardiovascular issues, cerebrovascular, chronic pulmonary diseases, immunocompromising, renal problems, hepatic disease, hypertension, diabetes and obesity. [44] [45] [46] [47] [48] [49] there is a notorious concern regard the medicine administered to fight these comorbidities because some of them can lead to greater expression of ace2, such as treatments for diabetes 48 or hypertension. 50 this may favour or even aggravate covid-19 infection. these facts justify the urgency of research that contemplate alternative therapeutic targets such as calcium channels blockers for hypertensive individuals as suggested by fang et al. 48 however, there is little clinical evidence on the risk of treating covid-19 patients with therapies that induce greater expression of ace2. further investigation is necessary to explore whether these medicines inhibit or trigger the viral entry into the cells of an infected host. 51 a frequent report in epidemiological data regarding the mortality of covid-19 concerns the sex of individuals, as men are the predominant fatal victims of the disease. therefore, being of the male sex is considered a bad prognostic factor for infection. 52 ,53 a possible explanation lies in the relation between gonadal hormones and the expression of ace2 enzymes or even an alleged vitamin d deficiency, according to vignera et al. 54 the latter suggest monitoring of serum levels of testosterone and vitamin d in infected patients for a better understanding of the different fatality rates between sexes, including the hypothesis that women's hygiene justify a lesser rate of infection. understanding the pathogenic effects of sars-cov-2 for the different organs affected by the disease has also been object of investigation, such as gut-lung crosstalk. 55 data from research conducted thus far indicate that the infection caused by sars-cov-2 is not only capable of causing pneumonia, but it can also damage other organs such as the heart, the liver, the kidneys and organic systems such as the blood and the immune system. 44, 56, 57 patients with the extreme form of the disease frequently manifest lymphopenia, 30, 57 hepatic insufficiency 58 and viral sepsis diagnostic, 51 whose complications can be related to the severity of the cases and the mortality of patients. 56, 57 there are reports that the eventual death of such patients is due to multiple organ insufficiency, acute respiratory distress syndrome (ards), cardiac insufficiency, arrhythmia and renal insufficiency. 56, 59 therefore, great attention is necessary to the disease's potential damage to multiple organs and to therapeutic alternatives to fight covid-19, 60 given that some of these alternatives can have side effects on organs initially unrelated to the respiratory system, but that may be susceptible to a systemic compromise prompted by the virus once the treatment has begun. hence, it is possible to observe the existence of different forms of aggravating the disease. 41 in this regard, wang et al. 60 recommend the creation of a system to categorize patients with the severe form of covid-19. several investigations report that all hcovs, sars-cov, mers-cov and sars-cov-2 induce exaggerated immune responses in the host, which are associated to the severity of pulmonary pathology and might lead to the development of acute respiratory distress syndrome (ards) or death. 57 the incidence of the extreme form of the infection is associated with cytokine storm syndrome, characterized by high plasma concentration of several interleukin, inflammatory cytokine, inflammatory chemokines, among other factors that cause infiltrated inflammatory in the organs. 44, 61, 62 survivors of this excessive response by the immune system can develop long-term fibrosis and pulmonary damages that might culminate in functional injuries to these organs, thus reducing the patient's quality of life. 63 during the development of drugs to fight microorganisms, the adoption of strategies that allow the design of molecules to act against specific biological targets of bacteria, parasites or viruses is preferred. therapies for covs can be divided into several categories, based on specific paths: (1) covs proteins or functional enzymes that are essential for viral replication; (2) structural proteins of the virus that prevent its binding to the respective receptors in human cells or its assembly process; (3) some viral factor that restores the host's inherent immunity and; (4) host-specific enzymes or receptors, that prevent the entry of the virus in the host cells. 5, 64 so far, structural proteins and enzymes that participate actively in the process of viral replication are the most investigated targets for the development of molecules for anti-covs therapies (fig. 1) . investigations by wu et al. 5 through bioinformatics, analysed possible sars-cov-2 therapeutic targets. the proteins coded by this virus were verified and compared to proteins coded by other covs. the results enabled the detection of structural similarities to sars-cov, from which it was possible to conduct homology modelling to build 19 proteins for sars-cov-2. among the targets were spike (s) glycoprotein, nsp (rna-dependent rna polymerase -rdrp), enzyme helicase (3cl pro and pl pro ), tmprss, orf7a factor and ace2 presents in the host cells. 5 these targets have pivotal roles in the development of the virus and have great influence on its pathogenicity, hence, some details are provided next. molecular modelling showed that spike (s) glycoprotein is a transmembrane protein of approximately 180 to 200 kda type i whose n-terminal turns to the exterior of the virus and its cterminal segment turns to the interior of the virus. the typical structure of covs is given by the assemble of a bulbous projection of a corolla as trimers of protein s and it is cleaved into two important subunits from the pathogenic perspective: s1 and s2. sars-cov and sars-cov-2 (s) glycoprotein share about 76% of amino acid identity and enable the entry of the virus in the host cells. therefore, s glycoprotein present in covs has been considered a promising biological target for antiviral mechanisms. 35, 65 the moment when the virus approximates the target cell prompts the recognition by the receptor-binding domain (rbd) in the s glycoprotein of its receptor, which leads to the binding to subunit s1. next, the subunit s2 allows fusion of viral and cellular membranes, which enables entry in the cell and the release of viral rna genome. 35, 66, 67 some investigations suggest that the strong binding affinity between s protein and ace2 is essential for viral entry, hence, ace2 is also relevant for the development of drugs. 5, 68 molecules that bind to the surface of the virus can destabilize the formation of s glycoproteins and interfere both with the trimerization of the protein and with the continuity of the life cycle of covs. 69 several studies have been conducted on s protein to clarify its sars-cov-2 structure and its binding process as well as to evaluate its relevance as target for in-silico and in-vitro assays on molecules for anti-sars-cov-2 therapies. one study conducted by hoffman et al. 35 investigated how the sars-cov-2 s protein facilitates viral entry in the target cells and how this process could be blocked. results showed that ace2 is used as receptor for the entry of sars-cov-2 in host cells and that the spread of this cov in the infected host depends on the activity of tmprss2 (a cellular serine protease responsible for initiating the binding process between s protein and ace2). this process can be blocked with clinically approved tmprss2 inhibitor. prior to this, the relevance of tmprss2 was highlighted in the dissemination of several types of viruses such as influenza a and other covs, which also makes it a relevant target for covid-19 therapeutic intervention. [70] [71] [72] [73] [74] [75] [76] binding between s proteins and ace2 receptors was corroborated through x-ray crystallography conducted by lan et al. 67 to elucidate the interaction between the sars-cov-2 rbd and ace2 at a higher resolution. in spite of different interactions with ace2, the sars-cov-2 rbd /ace2 and sars-cov rbd /ace2 interfaces share a substantial similarity regarding the surface area, the number of interacting residues and the networks of hydrophilic interactions. such similarity strongly points to a convergent evolution of both sars-cov-2 and sars-cov rbd structure which improves the binding affinity for the same receptor, the ace2. the non-conserved rbd regions in s protein, such as subunit s2, could be potential targets for cross-reactive antibodies. considering rbd as a critical region for receptor binding, antibodies that target the conserved epitopes in the rbd are also good candidates for the development of highly potent cross-reactive therapeutic agents against several species of covs, including sars-cov-2. 67 investigations on ligands obtained from drugbank 5.1 used molecular docking to identify target regions in the pockets of the quaternary structure of sars-cov-2 s glycoprotein (from protein data bank-pdb). six pockets present in s glycoprotein deserve further investigation in medicinal chemistry due to suitable features for small molecule binding. among the six pockets, the eight best ligand candidates from drugbank were all binding pocket #1, which contained residues of amino acids proline, leucine, lysine, asparagine, phenylalanine, glycine, threonine, glutamine, alanine, methionine and tyrosine. one of the best ligands was the drug saquinavir, an antiviral from the class of protease inhibitors, used in anti-hiv therapy. 69 nsps are involved in the rna transcription, translation, protein synthesis, processing and modification, viral replication and infection of the host. significant functional proteins, 3cl pro , pl pro , helicases and rdrp are important targets for the development of small-molecule inhibitors, due to their biological function and vital enzyme active site. 77 factors nsp1, nsp3c and orf7a are related to assistance to the immune evasion of sars-cov-2. interaction between nsp 1 and the host ribosomal subunit induce the degradation of mrna, allowing the virus to develop resistance to the host innate immunity. binding between orf7a and bone marrow matrix antigen 2 (bst-2) inhibits activity and blocks bst-2 glycosylation. these results suggest that all three structures are potential targets for antiviral medicine. 5 proteases pl pro and 3cl pro mediate the proteolytic cleavage of polypeptides produced by βcoronavirus sars after genome transcription, thus generating other proteins. the 3cl pro , known as nsp5, cleavages several non-structural proteins of importance for viral replication and the maturation of nsps, which is essential in the life cycle of the virus. therefore, it is an attractive biological target for that has been inhibited in-silico by several antiviral, anti-inflammatory and anti-hypertensive drugs from the database zinc (fda). 5 in addition, docking and molecular dynamic studies conducted by qamar et al. 78 showed that non-toxic natural products formed strong bonds with sars-cov-2 catalytic dyad cis145-his41 of 3cl pro . moreover, the proteinase pl pro is responsible for cleavages of n-terminus in the replicase polyprotein to release nsp1, nsp2 and nsp3, which are essential for correcting virus replication significant to antagonize the host's innate immunity. analysis of the docking model showed that ribavirin formed hydrogen bonds with residues gly164, gln270, tyr274, asp303 as well as hydrophobic interactions between tyr265 and the pl pro residue. these results indicate ribavirin as a powerful pl pro enzyme inhibitor, which means it has promising features for anti-covid-19 therapy given the inhibition of a likely pl pro therapeutic target. 5 helicase (nsp13) has been identified as a promising target for antiviral drug discovery, particularly against sars-cov-2. it is a multifunctional protein necessary for a wide range of biological processes, such as genome replication, recombination and dislocation of proteins related to chromatin and nucleic acid remodelling. for covs, helicase is indispensable for viral replication. in studies on molecular modelling, several antibacterial, antifungal and antiviral drugs were analysed and presented elevated affinity to helicase, suggesting it as a good target for sars-cov-2 therapy. 5 rna-dependent rna polymerase (rdrp -also nominated nsp12) catalyses the viral rna, which performs a key role in the replication/transcription complex of sars-cov-2, possibly aided by nsp7 and nsp8 complex as cofactor. 5, 79 nsp12 has been studied as potential target for several sars-cov and mers-cov inhibitors, due to its importance for viral control. satisfactory results of rdrp inhibition by several ligands were presented in the modelling studies by gao et al. 79 yin et al. 80 those ligands included antiviral analogous to nucleotides, such as remdesivir, which already shows great potential in the treatment of covid-19 infections. in addition, some nonstructural proteins, including nsp3b, nsp3e, nsp7, nsp8, nsp9, nsp10, nsp14, nsp15 and nsp16, also stood out as useful targets due to their significant role in the synthesis and replication of viral rna. 5 3cl pro is key enzyme for covs, also called main protease (m pro ), that plays a pivotal role in mediating viral replication and transcription, making it an attractive target for anti-sars-cov-2 drugs. such claim is reinforced by studies by jin et al. 81 after the virtual screening of n3 inhibitor. results show that n3 (1) is a time-dependent irreversible inhibitor of this enzyme and that a stable covalent bond is formed between n3 and 3cl pro . high-throughput screening (hts) was applied to 10,000 drugs and drug candidates, demonstrating that ebselen (2), px-12 (3) and carmofur (4) are all able to covalently bind to 3cl pro do sars-cov-2, with ic 50 that varied from 0.67 to 21.4 µm (fig. 2) . it is likely that a part of the hits identified by hts are bonded to the catalytic cysteine of 3cl pro through their sulfhydryl groups. in-vitro studies on antiviral activity were performed to corroborate the results. real time quantitative pcr (qrt-pcr) demonstrated that ebselen and n3 had the strongest antiviral effects at a concentration of 10 μm treatment in sars-cov-2 infected vero cells. after plaque-reduction assay, the dose-response curves suggested that both could penetrate cellular membrane to access their targets. this result strongly supports the hypothesis that developing a single antiviral agent targeting 3cl pro or in combination with other therapies could provide an effective first line of defence against all covs related diseases. in relation to sars-cov-2 therapy, some of the aforementioned targets have been explored for both new drug proposition as well as for sars-cov-2 drug repurposing. our focus is on this last type, and for each medicine, the putative mechanism of action and viral target will be described trying to find an understandable rational therapy even for an immediate illness situation like covid-19 pandemic. carmofur (4). as previously mentioned, sars-cov-2 is an enveloped virus, whose nucleocapsid consists of a positive rna genome surrounded by multiple copies of nucleocapsid protein. this virus, after entry in the host cell, replicates fast the viral genome with new virion production. the rna replication into the cell host depends on enzymes and substrates for rna synthesis, such as ribonucleotides (adenine, guanine, cytosine or uracil) that have nitrogenous bases in the purine or pyrimidine classes. compounds can mimic these chemical structures and interfere with the formation or use of one of these essential normal organism metabolites. the interference is generally prompted by enzyme inhibition in the biosynthetic pathway of the metabolite or by incorporation, as a false building block, into vital macromolecules such as proteins and polynucleotides. so, this class of therapeutic agents is called antimetabolites. 82, 83 diverse antimetabolites have been indicated as promising anti-sars-cov-2. they are described next. pyrimidine derivatives are aromatic organic compounds necessary for all life forms. examples of pyrimidine derivatives are nitrogenous bases cytosine (5), uracil (6) and thymine (7) (fig. 3) . they are found in dna and rna and participate in the metabolic process that involves carbohydrate and lipids. 84, 85 these heterocyclic rings share two nitrogen atoms at 1 and 3 positions, but display variations between themselves, such as an amine group at 4-position in the cytosine and a methyl at 5-position in the thymine. from the pharmacologic perspective, nitrogenous bases are investigated as pharmacophores and are found in the structure of many drugs and experimental substances with various activities, 86 such as antitumoral, 87 antibacterial, 88 antiparasitic, 89 and antiviral. 90, 91 regarding antiviral activity, there are several approved drugs that are classified as pyrimidine nucleotide biosynthesis inhibitors (pnbi) because, after phosphorylation, they are incorporated either into the dna or into the rna and inhibit hosts or pathogenic enzymes, such as polymerases. 85 therefore, the likely mechanism of action of some pyrimidine derivative drugs has been considered for repurposing. some pyrimidine derivatives with antiviral activity are often formulated as prodrugs. this format solves issues of high polarity in its final structure prompted by the phosphonic acid, which interferes with pharmacological properties and causes low cellular permeability and low oral bioavailability. 91 one compound appointed as potential anti-sars-cov-2 is the 5-fluorouracil (8) (5-fu) (fig. 3) , a heterocyclic aromatic amine similar to uracil (u) that presents a fluorine-carbon bond at 5-position. this compound is used in the treatment of oesophageal cancer, 83 stomach cancer, 92 breast and colon cancer. 93 the similarity between 5-fu and uracil allows the direct action on nuclei acid as it is incorporated into the genetic material and inhibits replication. 83 tests with 5-fu as monotherapy confirmed its failure against any coronaviruses. the reason proposed to such failure relied on the fact that coronaviruses rna proofreading activities involve a 3' → 5' exoribonuclease in the nsp14, which removes 5-fu during replication and metabolism. hence, the combination between 5-fu and deoxyribonucleoside and deoxyribose was suggested so that, after its insertion in the rna, it escapes rna proofreading and prompt lethality and/or lethal mutagenesis in the virus. despite the proposition of using a widely marketed drug to treat several types of cancer, which means it has well-established efficiency and safety, no other type of test has been made to confirm its efficacy against sars-cov-2. therefore, further experiments are necessary to explore 5-fu potentialities. 94 another antitumoral drug considered for its anti-sars-cov-2 potential is gemcitabine (gct) (9) (fig. 3) , an analogue of deoxycytidine whose pharmacological action is triggered after the intracellular transformation into triphosphate gemcitabine. the latter competes with endogenous nucleoside triphosphates by incorporation into the genetic material, thus inhibiting dna synthesis. 95 initially, gct was developed for antiviral activity, however, initial results caused it to be redirected for anticancer therapy. it became, then, widely used against non-small cell lung cancer, pancreas, bladder and breast cancers as well. [96] [97] [98] in-vitro analyses of gemcitabine hydrochloride inhibited mers-cov and sars-cov, with a ce 50 of 1.2 μm and 4.9 μm, respectively, in addition to low cell toxicity for vero e6 cells. 99, 100 these data are indicative of a possible activity against sars-cov-2, but complementary preclinical investigations are necessary before clinical trials. albeit considered a safe drug under predetermined doses, gct adverse effects are noteworthy and include myelosuppression and disruption of liver functions. in february 2017, the european union (eu) approved baricitinib (10) as second-line oral treatment for mild to severe active rheumatoid arthritis in adults. 9 a differential feature of baricitinib structure is the azetidine ring bearing an ethylsulfonyl, beyond an acetonitrile group at 3-position. the same ring binds to the n atom at 1-position in the pyrazole, which, in its turn, binds to the pyrimidine conjugated to a pyrrole ring. 101 this medicine can modulate human innate and adaptive immune system. based on this property, presumably, one of the important mechanisms of action of baricitinib in the treatment of rheumatoid arthritis is the inhibition of the il-6 / jak1 / jak2 pathway. 102 the promising nature of baricitinib and other small molecule inhibitors against sars-cov-2 was pointed by richardson et al. 9 through in-silico tests using benevolent ai. the authors evaluated 378 compounds to show that sunitinib (11) and erlotinib (12) inhibit ap2-associated protein kinase 1 (aak1) interrupting the virus entry to the cells and the intracellular assembly of new viral particles (fig. 3) . regarding these two antitumor drugs, it is known that sunitinib is an oral oxindole multitargeted kinase inhibitor that inhibits certain tyrosine kinases including vascular endothelial growth factor receptors (vegfr types 1 and 2), platelet-derived growth factor receptors (pdgfr-α and pdgfr-β), stem cell factor receptor (kit), fms-like tyrosine kinase-3 (flt3), glial cell-line derived neurotrophic factor receptor (ret) and the receptor of macrophage-colony stimulating factor (csf1r). 103 concerning erlotinib, it was developed as reversible and highly specific small-molecule tyrosine kinase inhibitor that competitively blocks the binding of adenosine triphosphate to its binding site in the tyrosine kinase domain of epidermal growth factor receptor (egfr), thereby inhibiting autophosphorylation and blocking downstream signalling. 104 however, these oncological drugs have serious adverse effects such as diarrhoea, loss of appetite and skin rashes. in addition, high doses of these medications can aggravate those effects. in relation to baricitinib, its anti-sars-cov-2 potential was explained in three ways: aak1 inhibition like sunitinib and erlotinib; the kinase associated to cyclin g, which is another endocytosis regulator; and the janus kinase, that inhibits the action of cytosines that triggers the inflammatory process. because baricitinib can inhibit aak1 at the therapeutic dose (2 or 4 mg/day), the drug is indicated for clinical trials. it is highlighted that baricitinib is not indicated for patients with neutropenia or lymphopenia, once it lowers rates of neutrocytes and lymphocytes, which can lead the disease to progress and increase anaemia. furthermore, treatment with baricitinib can reactivate varicella-zoster, herpes simplex and epstein-barr viruses. this implicates in a conflict between the potential effect and the adverse effects of baricitinib against covid-19 to prevent aggravating the disease and the mortality of patients. 105 an analogue of adenosine, galidesivir (gsv) (13) is a broad-spectrum antiviral drug that blocks viral rna polymerase by replacing a natural nucleotide with galidesivir triphosphate. this alteration prompts changes in electrostatic interactions and prevents the formation of the rna elongated strand. 106, 107 adenosine and gsv differ in that galidesivir has one carbon at 7-position in the pyrimidine ring and nitrogen in the ribose ring, whereas adenosine has one nitrogen in the former and oxygen in the latter (fig. 2) . 106 it is noteworthy that gsv has not been approved for clinical trial and is an experimental drug in advanced stages of development. 108 gsv was first developed against hepatitis c (hcv) but first clinical trials were conducted to ensure its safety (in healthy individuals) and efficacy against yellow fever. furthermore, gsv displayed in-vitro and in-vivo antiviral activity against filoviridae, alphavirus, bunyavirus, arenavirus, paramyxovirus, flavivirus, orthomyxovirus, picornavirus and sars and mers coronaviruses. 11, 106, 109 recent in-silico studies have shown the existence of a strong bond between gsv and sars-cov-rdrp to demonstrate the capacity of alterations in rna polymerase, which can eradicate the virus. although, preclinical and clinical trials are necessary to either confirm or deny this hypothesis. 7 it is noteworthy that investigations have pointed the inactivity of gsv against sars-cov-2 at concentrations lower than 100 mμ. 110 the existence of antiviral activity against other coronaviruses indicates that more investigations on gsv against sars-cov-2 are required to elucidate its potential activity in advanced testing. next, sofosbuvir (sbv) (14) is an example of successful nucleotide prodrug, approved by the food drug administration (fda) since 2013, against chronic hepatitis c infections. sbv is also combined with other antiviral drugs, such as ledipasvir, velpatasvir and voxilaprevir. 111, 112 the structural similarity between sbv (fig. 2) and uridine allows that drug to act on hcv rdrp, incorporate itself into the viral rna and terminate the synthesis of the nucleotide sequence. 82 structural analysis of sbv revealed that its elevated potential is partly due to the presence of the 5'phosphate, which terminates the primary enzyme transformation monophosphate inhibitor. 113 the antiviral activity has been explored against other viruses through in-vitro and in-silico studies and shown potential for inhibiting the dengue virus, 114 yellow fever, 115 telbivudine (tbv) (15) (fig. 3) is a thymidine nucleoside analogue used with specific activity against the hepatitis b virus (hbv). it starts acting after phosphorylation by cellular kinases, which results in the active metabolite, telbivudine 5'-triphosphate, enabling dna polymerase and inhibiting viral replication. the hydroxyl at 3-position in the sugar β-l-2'-desoxirribose provides specificity to hbv polymerase. 120 suggesting repurposing tbv to fight covid-19 was prompted by virtual screening to find drugs that act on viral m pro . among other results were ribavirin, tbv and two vitamins, cyanocobalamin (b12) and nicotinamide (b3). researchers suggest that these four drugs can be combined and used against covid-19, once they are safe, marketed and approved by the authorities. 8 notwithstanding, the suggestion of repurposing these drugs requires more information, including on drug interaction parameters. in spite of well-tolerated and safe for monotherapy, associating tbv and ribavirin, another antiviral drug, can increase hepatotoxic activities of tbv. 121, 122 it is also important to consider the elevated risk of resistance to tbv, which and pyrimidine derivatives drugs: 5-fluorouracil (8); gemcitabine (9); baricitinib (10); sunitinib (11); erlotinib (12); galidesivir (13); sofosbuvir (14) ; telbivudine (15). purine is a 5 and 6-membered bicyclic ring. similar to pyrimidines, purine derivatives are essential to life. they are basic constituents of nitrogenous bases adenine (a) (16) and guanine (g) (17) (fig. 4) . 84 the safety of rdv for humans infected with ebov was evaluated in the democratic republic of congo. results confirmed its safety but did not point rdv as the best therapeutic option, once its mortality rate reached 53% of treated group. 133 it has been proved that gs-5734 inhibits epidemic and zoonotic hcov. 134 of viral loads and weight loss in murine. 135 therefore, rdv is a potential drug to treat mers-cov infections. regarding covid-19, rdv was used to treat the first us case. the patient was 35 years old, had slight cough, low fever and no evidence of pneumonia at day 4 of the disease. when the clinical symptoms became worse, the patient was given vancomycin and cefepime. as the symptoms worsened, intravenous treatment with rdv was administered at day 7, and vancomycin and cefepime were no longer administered. at day 8, the patient displayed clinical improvement, unfortunately details on the doses and duration of treatment were not provided. 136 after this first case, a clinical trial with a larger number of covid-19 patients was conducted. 137 this study of efficacy involved 53 patients infected with sars-cov-2 who displayed saturation equal or inferior to 94% while they were breathing ambient air or receiving oxygen support. the treatment lasted 10 days, patients were given 200 mg intravenously on day 1, followed by 100 mg daily for the remaining 9 days of treatment. follow up of patients treated for 18 days indicated that, after the first dose of rdv, 68% improved oxygen support whereas 15% of patients got sicker, 47% were discharged and mortality rate was 13%. the most common adverse events (60% of patients) were increased hepatic enzymes, diarrhoea, rash, renal impairment, and hypotension. some limitations were noted in the study, such as the small size of the cohort, the short duration of follow-up and the lack of information on the patients. hence, the efficacy of rdv requires validation by the ongoing randomized, placebo-controlled trials. one advantage of repurposing rdv is the availability of data on safety and pharmacokinetics, which were obtained previously at phase 1 clinical trial. in addition to the promising results shown by rdv, other purine analogues have been investigated for sars-cov2. ganciclovir (gcv) (19) also named, according to its chemical structure, 9-(1,3-dihydroxy-2-propoxymethyl) guanine (fig. 4) , is a guanine analogue, similar to acyclovir, except for the bond between the methyl group and one hydroxyl. gcv inhibits the human herpesvirus and is also indicated in the treatment of cytomegalovirus infections related to acquired immunodeficiency syndrome (aids). 139 gcv is converted into ganciclovir triphosphate by cellular kinase, which inhibits dgtp and disrupts viral dna synthesis due to substitution of various adenosine bases in the dna chain. 140 recently, gcv was used to treat covid-19 patients in china. 141 the drug was administered with other antivirals, such as oseltamivir and kaletra. as a descriptive study, the relation between gcv as key factor in clinical outcome of the 99 patients (31% of which were discharges) was not possible. 141 therefore, gcv efficacy as monotherapy or part of combined therapy is yet necessary for more robust investigations. valganciclovir (20) (fig. 4) is the antiviral prodrug of gcv taken by mouth. it is indicated for the same treatments as gcv (cytomegalovirus in people who have acquired immunodeficiency syndrome, gastrointestinal disorders related to aids). the drug has great bioavailability and is converted by hydrolysis into ganciclovir. using valganciclovir in its oral form enables clinical treatment and makes patients more comfortable. [142] [143] [144] the mechanism of action is the same of gcv. 145 valganciclovir was computationally evaluated for covid-19. 5 the assay with the main proteins coded for sars-cov-2 allowed the determination of 21 possible binding targets, of which 19 were proteins and 2 host targets. valganciclovir, one of the drugs used in the study, was presented as a possible anti-sars-cov-2 therapeutic drug due to its high binding affinity to two wellestablished viral targets. the first target was pl pro , indispensable enzyme in viral replication; the second, rdrp, conserved nsp12 in coronavirus, which is vital for its replication/transcription. therefore, valganciclovir could be a significant antiviral drug to treat sars-cov-2. but there are no clinical reports on valganciclovir used to treat covid-19 in addition to what has been reported about gcv. 141 hence, its efficacy is yet to be confirmed as anti-sars-cov-2 therapeutic. tenofovir (tfv) (19) is another adenine analogue pointed as promising covid-19 therapeutic (fig. 4) , it is also called tenofovir disoproxil fumarate or alafenamide tenofovir (taf). approved by the fda in 2001, tfv is a prodrug used to treat hiv and cases of nucleoside resistance. 146 tfv is an analogue reverse-transcriptase inhibitor (ntrti). inside cells, tfv is phosphorylated and competes with deoxyadenosine 5'-monophosphate (d-amp), thus preventing the formation of dna. once incorporated into a growing dna strand, it causes premature termination of dna transcription and prevents viral replication. 146, 147 modelling and docking studies evaluated the antiviral effects of tfv and verified a strong bond to sars-cov-rdrp, which can disrupt this polymerase and terminate the viral infection. 7 however, in-vitro tests showed that tfv lacks apparent antiviral effect at concentrations inferior to 100 μm for sars-cov-2. 110 in spite of lukewarm in-vitro and in-silico outcomes, an ongoing clinical study on tfv (chictr2000029468), expected to end in june 2020, aims at assessing the effect of the combination tenofovir + emtricitabine (cytidine analogue) related to lpv/r in covid-19 patients. 131 in addition to the efficacy of treatment, clinical trials can validate the prevalence of adverse effects related to the toxicity of tfv in patients. tfv is also a powerful nephrotoxic drug causing damage to proximal tubular cells. in spite of that, interrupting the treatment is sufficient to improve adverse effects, which makes monitoring of patients essential. 145 heterocyclic compounds with different heteroatoms such as nitrogen, sulphur and oxygen can present different pharmacological properties. one such property is to serve as analogue of nitrogenous bases of nucleic acids, such as triazoles, which have a five-membered ring of two carbon atoms and three nitrogen atoms. this aromatic ring can assume two isometric forms, 1,2,3-triazole and 1,2,4-triazole. the former is stable under acid and basic conditions and becomes more reactive when binding to electronegative elements. 148, 149 triazoles are important and stand out for their various biological activities, such as anticancer, 150 antituberculosis, 151 anti-inflammatory, 152 antimicrobial, 153 and antiviral. 154 specifically for the latter action, triazole-based derivatives have shown promising invitro activity against coronavirus, probably by 3cl pro inhibition. 155 ribavirin (22) (fig. 4) is a powerful triazole-based antiviral analogue to guanosine. it presents a wide range of pharmacological activities related to several viruses, for instance: herpes simplex virus, human immunodeficiency (hvi-1), influenza, respiratory syncytial (rsv) and hepatitis c. 149, 156 the drug was initially used in 1980 to treat syncytial virus in children, generally combined with interferon (inf). however, ribavirin treatment presents undesirable adverse effects, like lowering of haemoglobin, which limit clinical use. 157 its action mechanism relies on the inhibition of enzyme inosine monophosphate dehydrogenase, necessary in the synthesis of guanosine triphosphate, which prevents viral dna and, mainly, rna replication. the necessary concentration for in-vitro inhibition of rsv and influenza ranges from 3-10 μg/ml. 158 site similar to other sars-pl pro inhibitors. the formation of hydrogen bonds and π-π stacking were also predicted. these findings suggest that ribavirin as a powerful pl pro inhibitor. nonetheless, investigation on triazole derivatives for anti-sars-cov-2 therapy are still preliminary. on the other hand, favipiravir (fpv) (23) (fig. 4) is a prodrug, approved in 2014 in japan, to treat cases of influenza a and b that displayed resistance to first line drugs. it has provided results that indicate a promising character and is currently undergoing clinical trials against covid-19. its antiviral efficacy has also been investigated in different countries to fight ebola and lassa, for example. the molecular structure of the drug consists of a pyrazine heterocyclic ring with fluorine at 5-position, carboxamide at 3-position and a double bond between oxygen and carbon 2, which renders its analogue to guanine (17). 162, 163 the metabolization of the prodrug into its active form, favipiravir-ribofuranosyl-5'-triphosphate, requires intracellular ribosylation and phosphorylation. 163 fpv therapeutic targets are rdrp enzymes, necessary in viral transcription and replication, and its inhibition blocks synthesis of viral rna for a spectrum of viruses, including human coronavirus. a different investigation compared patients treated with fpv plus interferon inhalation to lpv/rtv. patients under fpv therapy responded better to the progression of the disease with accentuated viral depuration. also, the incidence of nausea and vomit was higher for lpv/rtv. 166 in addition to these clinical trials, the antiviral activity of fpv against sars-cov-2 was also evaluated but no clear antiviral effect was noted for doses lower than 100 μm. 110 an in-vitro study using molecular docking focused on the binding properties of sars-cov-2 protein structures to 61 antiviral agents, including oseltamivir. the study showed that 37 molecules form bonds to sars-cov-2 crystal proteins. however, data did not show oseltamivir as the best structure because lopinavir, asunaprevir and remdesivir interacted with more than two protein structures in the virus. hence, they are likely more promising than oseltamivir. 178 notwithstanding, we suggest further look into oseltamivir against other enzyme targets since different studies achieved positive results regarding its use as anti-sars-cov-2. nelfinavir (27) (fig. 5) is a safe anti-retroviral drug largely used for hiv-1 protease inhibition with strong in-vivo activity. 179 generally, nelfinavir is combined with other anti-retroviral medication as part of a highly active antiretroviral therapy (haart) that reduces significantly the viral load by increasing cell number to 200 mm -3 cd4(+) lymphocytes. the drug is prescribed for children, young individuals, adults and pregnant women. 180, 181 nelfinavir and its active metabolite m8 strongly bind to serum proteins, displaying optimal tissue distribution. a frequent adverse effect is light to moderate diarrhoea, reported for 15 to 20% of patients. 180 the sars-cov outbreak in several countries triggered the search for antiviral drugs active against the disease. among the 24 drugs likely to inhibit sars-cov, nelfinavir stands out in all assays. 181 the mechanism of action suggested for nelfinavir involves preventing sars-cov replication after its entry in the host cell and disrupting virion production. based on results from previous studies as well, nelfinavir was considered a likely therapy for covid-19 after its indication for clinical trials as a promising anti-sars drug. recently, 1,903 drugs were evaluated for their binding affinity to sars-cov-2 m pro . 182 among the compounds, 15 drugs were selected based on the docking score and three-dimensional atazanavir (28) (fig. 5) is an antiretroviral drug protease inhibitor used to treat hiv infections with in-vitro inhibitory concentration of 2,6-5,3 nmol. compared to other protease inhibitors, atazanavir has the advantage of allowing a daily posology regimen with a favourable metabolic profile and low frequency of adverse effects. 183, 184 several hiv-1 resistant to protease inhibitors are still sensitive to in-vitro atazanavir, which is considered safe and well tolerated. 185 the atazanavir acts to inhibit hiv-1 protease, which is indispensable in the processing of polyproteins precursors of viral structures and prevents the formation of infectious and mature viral particles. 183 the good activities reported for this drug as well as the search for safe and fast therapy for captopril (29) (fig. 5) is an angiotensin-converting enzyme inhibitor (acei). it is a zinc metallopeptidases inhibitor that converts angiotensin-i into angiotensin-ii, an essential function that regulates arterial pressure. it is predominantly indicated as vasodilator in patients with cardiac insufficiency. this drug was suggested as potential antibiotic capable of inhibiting zinc succinyls/dipeptidase by blocking its zinc catalytic center. 189, 190 tolerance to captopril has been largely investigated; its single dose by mouth is well-established and confirms the pharmacological activity in the short term (10-30 minutes) at the cellular level. this capacity is related to captopril transport mainly through plasma proteins such as albumins with absorption rate between 70-75%. reported adverse effects are neutropenia, proteinuria, dysgeusia and cough, but less frequent for low doses. 189, 190 some investigations have suggested captopril as possible covid-19 treatment. serafin et al. 100 indicated captopril as potential for inhibiting the bond between human sars-cov-2 and ace-2 and reduce severe pneumonia symptoms. in-silico studies using molecular docking were conducted with fda-approved drugs capable of binding to the main active site in proteinase 3cl pro . 189 two drugs were identified as ligands for the enzyme active site: captopril and disulfiram. the former binds to the active site at the same position of n3 inhibitor (a standard inhibitor that reacts irreversibly in the same site with 3cl pro cys145). it is, thus, suggested that captopril binds to the same site of n3, obstructing the function of cys145-his41 catalytic dyad. captopril probably inhibits the enzyme in two stages. initially, it establishes non-covalent bonds to sites in the enzyme targets, then, a reaction takes place between the critic groups, which results in a more stable inhibitor complex. the hypothesis is that captopril can bind covalently to 3cl pro cys145. although the potential of captopril on the enzyme has been demonstrated, therapeutic use against covid-19 is controversial, once the drug induces overexpression of ace-2 -the main receptor used by sars-cov-2 to entry the cells. therefore, combination with other drugs, such as angiotensin-ii receptor blockers, needs analysis to clarify the effects of captopril in covid-19 treatment. the cyclosporin a (csa) (30) (fig. 5) is isolated from the fungus beauveria nivea and was approved for use by the fda in 1983. this drug has been used for decades to prevent organ rejection and to treat t cell-associated autoimmune diseases such as behcet's disease, psoriatic arthritis, lupus nephritis, rheumatoid arthritis, systemic lupus erythematosus or interstitial lung disease. such drug exerts its immunosuppressive function and anti-inflammatory effects by inhibiting the transcription of genes required for t cell proliferation, notably interleukin-2. [191] [192] [193] due to the severity of covid-19, csa can be potential to prevent hyperinflammation-induced lung injury. 194 in this regard, it is known sars-cov nps1 induces the expression of interleukin-2 via nuclear factor of activated t cell (nf-at) activation, 195 which can trigger the cytokine storm seen in patients with severe covid-19 status. 138 another advantage presented by csa in relation to other antiinflammatory drugs is its already known anti-cov action against all genus, including sars-cov, [195] [196] [197] at low and non-cytotoxic micromolar concentrations verified in cell culture assays. this antiviral property is thought to be mediated by the inhibition of cyclophilin-a-dependent viral assembly as well as inhibition of the nf-at pathway or even by genetic or pharmacological specific inhibition of cyclophilin-d, hindering the viral replication. 195, 198 as already reported, sars-cov and sars-cov-2 are very similar (79.5% sequence identity). 17 teicoplanin (31) (fig. 6) is an antibiotic used against gram-positive bacteria with 5 major compounds at different side chains. it prevents polymerization of peptidoglycans and inhibits the development of the cell-wall, thus prompting cell death. 203 it is a big molecule that has displayed antiviral activity on an early stage of the viral life cycle by inhibiting the low-ph cleavage of the viral spike protein by cathepsin l in the late endosomes thereby preventing release of viral rna and replication. this compound has already shown inhibitory activity against ebola virus, mers-cov and sars-cov. 203 recent investigations have suggested teicoplanin as alternative treatment for covid-19 after an in-vitro assay achieved ic 50 value of 1.66 µm, thus proving its efficacy against sars-cov-2. these results need to be confirmed through randomized clinical trials, which are still to be conducted. 100, 204, 205 using antibiotics to fight viruses, albeit completely ignored, can become useful to treat covid-19. 206, 207 some studies report the possibility of repurposing drugs like terconazole, which displayed good in-vitro results against mers-cov and sars-cov, 100 dasabuvir (32) (fig. 6) is a drug from the naphthalene class and phenyl-naphthalene subclass due to the bond between its naphthalene ring and a phenyl group. dasabuvir is a first line drug used as combined therapy for chronic hepatitis c. 208 dasabuvir is a non-nucleoside inhibitor that binds to nps5b (non-structural protein 5b -rdrp) and induces conformational change that makes rdrp incapable of elongating the viral rna. 209 repurposing of this drug can be useful as sars-cov-2 therapy due to its antiviral activity. 210 dasabuvir was subjected to docking studies against sars briefly, dasabuvir forms π-cation interactions with lys31a (present in the ace2), π-π interactions with phe170b (s protein residue) and hydrogen bonds to ace2 residues glu35a and asp38a and gly176b and ser174b (s protein residue). the authors highlight the importance of repurposing drugs as new therapeutic alternatives not only for the new coronavirus but for the next viral outbreaks. darunavir (33) (fig. 6) is a benzene derivative that has been evaluated for repurposing against covid-19. this drug is an antiviral used in the treatment of hiv-1 infections. it provides a great genetic barrier to resistance and is highly active against resistant strains of hiv-1 that are not susceptible to other protease inhibitors. 211 darunavir is administered orally as pills or suspension and is often used with low doses of ritonavir as part of a combined art protocol. 212 its mechanism of actions works by protease inhibition. darunavir establishes high affinity bonds to hiv-1 protease forming a stable complex, thereby selectively inhibiting polyprotein gag-pol coded by the virus. this prevents the formation of mature viral particles. 211 fda-approved drugs against 3cl pro , rdrp, helicase, exonuclease 3′ a 5′, endornase e 2′-o-ribose methyltransferase. among the best drugs in the assay, darunavir was a surprise because, despite inhibiting viral proteinase, the study showed that it binds to the replication complex components of sars-cov-2 with inhibitory potency kd < 1000 nm. one example is rdrp, whose kd value was 148.74 nm and exonuclease 3′ to 5′ with k d value of 195.73 nm. a docking study was conducted by sang et al. 215 as in-silico evaluation of anti-hiv drugs in their interaction capacity to proteinase 3cl pro . results suggest that all drugs have higher binding affinity to sars-cov-2 3cl pro than to the homolog sars-cov proteinase. among the evaluated drugs, indinavir and darunavir displayed the highest docking scores, therefore, they were subjected to molecular dynamic (md) simulations, free binding energy calculations and molecular mechanics poisson-boltzmann surface area (mm-pbsa) to detail molecular interactions between inhibitors and proteinase. the data suggest darunavir had better binding affinity to sars-cov-2 3cl pro with binding affinity of -10,24 kj / mol. in addition, darunavir bind to sars-cov-2 3cl pro via 19 contact residues and to sars-cov 3cl pro via 17 residues. this difference explains the lower binding energy values between darunavir and sars-cov 3cl pro . it was also noted 5 hydrogen bonds between darunavir and sars-cov-2 3cl pro but none for indinavir and this proteinase. because hydrogen bonds are important in the stability of the inhibitor-enzyme complex, darunavir is probably more promising against covid-19. finally, a different in-silico assay was conducted by pant at al. 216 to assess a large variety of compounds (300) from several data banks and 66 potential compounds from fda-approved drugs. the compounds were tested against sars-cov-2 3cl pro . darunavir was among the 20 best fda-approved drugs, with a score of -7.208. all data collected from in-silico studies still require experimental studies to validate the anti-sars-cov-2 activity of darunavir. a research conducted by dyall et al. 99 performed a robust in-vitro assay and showed that the drug dasatinib (34) (fig. 6) , a kinase signalling inhibitor developed to treat human cancers, inhibited mers-cov and sars-cov, exhibiting ec 50 values 5.4 and 2.1, respectively. this study also revealed that kinase signalling may also be important for replication of this hcovs. nevertheless, the authors reported that dasatinib may be valuable against coronaviruses infections if a dosing regimen that minimizes immunotoxicity while still blocking viral replication can be defined. results indicated this drug as a likely therapeutic alternative against sars-cov-2 infection. an in-silico study carried out by qiao et al. 217 showed that dasatinib, among others, is one of the most promising drugs for the inhibition of sars-cov-2 3cl pro . more preclinical and clinical studies are required to prove whether dasatinib is really promising for covid-19 patient treatment. imatinib (35) (fig. 6) is an oral anticancer agent that inhibits the activity of some tyrosine kinases, most prominently the bcr-abl fusion oncoprotein (whose overactivation can lead to chronic myeloid leukaemia, cml), c-kit (involved in gastrointestinal stromal tumours development), platelet-derived growth factor receptor (pdgfr), and the native abl kinase, which has a ubiquitous expression and plays important roles in several biological processes. 218 in addition to this well-known antitumor effect, imatinib has also shown in-vitro antiviral properties against several virus, such as infectious bronchitis virus (a viral model for studying the role of tyrosine kinase activity during cov infection), by interfering with virus-cell fusion, 219 and other rna viruses including coxsackie virus, 220 hepatitis c virus, 221 ebola, 222 among others, mainly by blocking viral entry or egress from the host cell. besides, this drug showed activity against sars-cov and mers-cov, 223 both phylogenetically related to sars-cov-2. 24 in this regard, it is reported that imatinib has anti-cov activity in two points of the virus life cycle. in the early phases of infection, it inhibits virion fusion with the endosome and subsequent release into the cytoplasm, thus preventing viral entry and viral replication via abl-mediated cytoskeletal rearrangement. in a later phase of the infection, abl2 protein expression, which is inhibited by this drug, enables sars-cov and mers-cov replication, which suggests that abl2 is a new host cell protein required for viral growth. 223 furthermore, evidences suggest that imatinib can modulate the immune response by sundry mechanisms, [224] [225] [226] for several diseases, such as rheumatoid arthritis, 227 asthma, 228 and crohn's disease. 229 this information insinuates that such drug might perform its potentially beneficial immunomodulatory role as a treatment alternative for covid-19 pneumonia. in addition, the use of imatinib as treatment appears to be reasonable from an economic point of view and its high availability in hospitals, 230 since this drug is well tolerated and the risk of severe adverse effects is relatively low, especially in short-term administration. 231 it is also recognized that adverse effects, mostly mild to moderate in intensity, will be easily controlled by dose reduction or discontinuation. 232 in light of this information, tatar and turhan 233 used the docking methodology to better understand the mechanism of inhibition of the sars-cov-2 n protein with 34 antiviral compounds. based on this study results, imatinib was one of the highly binding affinities performed against the aforementioned target, with the lowest micromolar ki values among the compounds evaluated. in line with this study, an in-vitro research carried out by weston et al. 234 found 17 fda approved drugs that inhibited sars-cov-2 at non-cytotoxic concentrations. the authors indicate imatinib as one of the hits, since it exhibited ic 50 value of 3.24 μm. they subsequently determined the mechanism of action, demonstrating this drug inhibits fusion of covs with cellular membranes, precluding their entry. this result indicates imatinib use against sars-cov-2. however, its efficacy and safety need to be better confirmed in further preclinical and clinical trials in order of elect him as candidate drug in the treatment of covid-19. synthesized for the first time by jean francois rossignol in the beginning of the 1970s, the 2acetyloxy-n-(5-nitro-2-thiazolyl) benzamide, sold under the name nitazoxanide (ntz) (36) (fig. 6) , is the result of a structural modification in the antiparasitic niclosamide when a benzene ring is replaced with a nitrothiazole. 235, 236 developed and sold as antiparasitic, ntz is also a first line broad spectrum antiviral with good results against parainfluenza, coronavirus, rotavirus, hepatitis and other respiratory infections. [236] [237] [238] [239] following oral administration, ntz is absorbed in the intestine, where it is rapidly hydrolysed by plasma esterase into the active metabolite tizoxanide. 236 the mechanism of action of ntz varies according to the pathogen. in relation to antiviral activity, ntz blocks the maturation of the viral hemagglutinin at the post-translation stage in treatments against influenza. in treatments against hcv (hepatitis c), it activates protein kinase r (pkr), which leads to phosphorylation of the eukaryotic initiation factor-2α thereby preventing translation. 240 cao et al. 241 conducted an in-vitro evaluation of ntz against recombinant murine coronavirus expressing the firefly luciferase (mhv-2afls). the strand was pivotal to triage the 727 drugs with likely anti-cov activity. the first assay resulted in 84 molecules among which was ntz. the antiviral effect of ntz verified for mouse astrocytoma (dbt) and fibroblasts (17cl-1). 241 the dbt cells infected with mhv-2afls were treated with ntz at 5 µm for 12 hours, after which the viral titer (tcid 50 ) was determined and viral n protein was subjected to western blot. results show the strong inhibitory effect of ntz on the viral titer. 241 in-vitro studies on ntz or its metabolite tizoxanide were also conducted to verify efficacy against different coronaviruses. the replication of canine coronavirus (strain k378) in a72 cells, for instance, was blocked by the tizoxanide with ic 50 of 1 µg/ml. on the other hand, ntz inhibited the viral n protein in bovine coronavirus l9 (βcov-l9) and human enteric coronavirus 4408 (hecov-4408) with approximate values of 0.3 µg/ml. 239, 241 ntz is also responsible for inhibiting pro-inflammatory cytokines, such as tnf-α, il-2, ilnitazoxanide (36) drugs. more recently, molecules with a quinoline group have been widely investigated as treatment for the new coronavirus (sars-cov-2), such as chloroquine (cq) (37) and hydroxychloroquine (hcq) (38) (fig. 7) that belong to the quinoline class and aminoquinoline subclass. both are quickabsorption synthetic drugs approved to treat malaria (plasmodium falciparum) by several regulating agencies in the world. cq and hcq are water soluble; the latter is more soluble due to presence of hydroxyl group. they are currently used to treat autoimmune diseases such as lupus erythematosus, antiphospholipid syndrome, rheumatoid arthritis as they have immunomodulatory and antithrombosis properties. [243] [244] [245] therefore, these drugs could be useful against covid-19 due to the elevated levels of cytokine caused by cov infections in humans. 246 the mechanism of action of cq for anti-malarial treatment is not entirely clear, but the interference with the digestion of haemoglobin by the parasite has been suggested. 245 hcq has a similar mechanism, however, in regard to sars-cov-2, clinical trials showed it to be safer. [246] [247] [248] therefore, hcq, rather than cq, is used against sars-cov-2. recent studies report antiviral activity of cq and hcq as they impair viral entry and release in different in-vitro and in-vivo models. 244, 249 a factor that can also justify viral mechanisms is the aminoquinoline bioaccumulation in the tissues, as defended by patil, singhal & masand. 243 a factor that facilitates viral replication is the acidic ph of endosomes, lysosomes and golgi complex of the host. thus, cq is promising because it increases the ph of intracellular vacuoles, binds to the cellular receptors, changes the glycosylation and because of its selective and reversible immunomodulator effect on human cd4+ t cells. hcq exerts similar mechanism of action: a) increases the ph; b) modulation of activated immune cells; c) reduces the number of proinflammatory cytokine and other mediators to control inflammation. 243 it has also been suggested by roldan et al. 244 a likely involvement of hcq in iron homeostasis during sars-cov-2 infection, which is a similar mechanism to other viral infections in humans. [250] [251] [252] the little difference between the therapeutic and the toxic dose of cq is also known, and poisoning is related to cardiovascular complications that can be fatal. using either cq or hcq, then, requires strict prescription and self-medication is not advised. 249 based on the recovery data, it was concluded that hcq is not effective in patients admitted with covid-19. this highlights the importance of randomized clinical trials and the collection of clear data on the efficacy and safety of medications. ivermectin (39) (fig. 7) is an fda-approved broad-spectrum antiparasitic agent used in the treatment of tropical diseases, such as onchocerciasis, lymphatic filariasis, strongyloidiasis and lice. there is also evidence of its effectiveness in the management of myiasis, trichinosis, malaria, leishmaniasis, trypanosomiasis, chagas disease and schistosomiasis as well as bed bugs, inflammatory skin lesions, epilepsy, neurological diseases, tuberculosis and some cancers. 266 it is known that ivermectin is capable of inhibiting the bond between a virus and the nuclear transport mediated by the superfamily of importin proteins (imp α/β1) 267 based on the fact that sars-cov-2 is a rna virus deeply related to sars-cov, studies on sars-cov proteins have revealed a potential role for imp α/β1 during infection in signal-dependent nucleocytoplasmic shutting of the sars-cov nucleocapsid protein. 273 furthermore, the sars-cov accessory protein orf6 has been shown to antagonize the antiviral activity of the stat1 transcription factor by sequestering imp α/β1 on the rough er/golgi membrane. 274 considering the ivermectin nuclear transport inhibitory activity, such drug is widely believed as a promising therapeutic approach against sars-cov-2. recently, caly et al. 275 a different mechanism of action that consolidates the use of ivermectin against covid-19 is its immunomodulatory property. the inflammatory response (proinflammatory cytokine) is exaggerated in patients with the extreme case of the disease, which is likely explained by the hypoxiainducible factor (hif-1α) that is activated by the virus when no inhibitory medication is administered. this understanding can be explained by the study conducted by kosyna et al. 276 , whose lab tests with and without ivermectin aimed to examine whether the properties of the bond between hif-1α and imp α/β1 and between hif-1 and nuclear localization signals were affected by the hypoxia mechanism on the cellular level. the authors concluded that ivermectin inhibited both imp α/β1 and hif-1α. regarding this hinders its indication and clinical decisions. thus far, data indicate ivermectin is useful at the early stages of the disease, even the epidemiologic profile of covid-19 shows significant differences regarding the age of patients for the affected countries and patients with comorbidity. 279 therefore, large scale randomized clinical trials are necessary to standardize clinical, laboratory and image evaluations, as well as combined drug therapy with vitamins and zinc, for example. 280 financially, ivermectin is inexpensive and its doses and protocols are well established for different purposes. in addition, this drug has little side effects. 281 indole, also named benzo [b] pyrrole, is a planar bicyclic heteroaromatic, whose ten π electrons move across its structure making this chemical group behave as a weak base. 282, the indole ring is the most abundant heterocyclic in nature and is commonly found in biologically active natural products, such as vegetables and seafood. it is also present in the structure of the essential amino acid tryptophan, which interferes with protein synthesis and with the regulation of physiological mechanisms such as precursors for serotonin and vitamin b3. 283 288 and adding the indole ring to spirothiazolidinones conducted to better influenza a/h3n2 inhibition. 289 now, the antiviral activity of indole and its derivatives for covid-19 therapy is supposed. arbidol (40) is also a promising drug to fight covid-19 (fig. 7) . this drug is classified as antiviral and has been used for 25 there is high expression of eca2, identified as human receptor for virus entry. 290 notwithstanding, a different clinic trial concluded that arbidol monotherapy is best for the patient than lpv/rtv. 291 despite the results, both investigations recognize their own limitations due to small cohort size and lack of placebo-control group. according to the authors, these limitations are inherent to pandemic times when placebo-control groups are difficult to conduct due to life-threatening conditions. most studies with arbidol use 200 mg/3*day 164, 290, 291 following the chinese guidelines. previous investigations on the pharmacokinetics of arbidol in healthy chinese patients showed that a single 800 mg oral dose is sufficient for cmax de ~4,1μm. 293 this value was obtained through invitro assays that pointed the drug as effective and promising against sars-cov-2. hence, clinical trials are still necessary to confirm the efficacy of arbidol at elevated doses to treat covid-19. 293 rizatriptan (rzt) (41) (fig. 7) is used to treat migraine and is a selective receptor of serotonin (5-ht) type 1b and 1d, structurally and pharmacologically related to other selective antagonists at these receptors. 294 its structure is based on an indole ring replaced with methyltriazole at 5-position and unsubstituted ethanamine replaced with methyl at 3-position, the substitution sites are the same of melatonin (mlt). after virtual triage through molecular dock at spike-ace2 interface, ligations π-cation, interactions π-π and hydrogen bonds were identified between rzt and the sars-cov-2 protein complex. as one of the outstanding compounds in the analysis, in-vitro tests are still necessary. 187 it is noteworthy that overdosing of the drug can trigger dizziness, fainting, cardiac issues, hypertension, bradycardia and vomiting. despite the safety of the drug in regular doses, there are no reports on either in-vitro or in-vivo tests to support the theoretical data and the antiviral action thus far. one last indole derivative that could be repurposed to treat covid-19 is melatonin (mlt) (42) (fig. 7) . mlt is classified as a hormone and nutraceutical, as it is naturally produced by the pineal gland and released into the bloodstream. it regulates the sleep-wake cycle as well as our mood, learning and memory, fertility, reproduction and the immune system. from a chemical perspective, it is an indole derivative with a methoxy group at 5-position and one ethylacetamide at 3-position. 295 regarding its antiviral potential, mlt acts indirectly through anti-inflammatory, antioxidant and immune modulating activities. 296 an investigation with murine infected with semliki forest virus (sfv) and west nile virus (wnv) showed the efficiency of mlt in reducing mortality rates for these viruses as well as in reducing the levels of pro-inflammatory cytokines. 297 the anti-inflammatory and antioxidant properties of mlt point to its antiviral effects in humans. 298 based on computational data, zhou et al. 299 suggested using combined medication to fight sars-cov-2. one such combination involves mlt plus mercaptopurine in synergic action against the following targets: pl pro , ace2, c-jun signal and anti-inflammatory vias. therefore, experimental studies on modifications of ace2 pathways caused by mlt are useful to understand this drug. 299 on the other hand, it has been suggested that using this neuro-hormone can mitigate the extreme form of the disease, the acute respiratory syndrome that has caused most deaths by sars-cov-2 cases. despite its safety for humans, the lack of data on the relevance of its use for covid-19 patients emetine (43) is an approved anti-protozoal drug used against amebae with reported inhibitory activity for enterovirus infections, 301 zika virus and ebola by interfering with the process of viral replication and entry in host cells. 302 emetine is an isoquinoline alkaloid that presents 4 methoxy groups in its structure (fig. 7) . studies also confirm emetine has in-vitro activity against coronaviruses, including sars-cov and mers-cov. 99 assays to clarify the activity of these drugs and the mechanism involved in the antiviral action of emetine both in isolation and combined to other drugs. another alkaloid candidate to repurpose against covid-19 is homoharringtonine (hht) (44) (fig. 8) . hht is an fda-approved drug in semi-synthetic form known as omacetaxine. this drug displays antitumoral activity in the treatment of myeloid chronic leukaemia. the mechanism of action implicates the ribosomal bond to prevent protein translation. in addition to antitumor activity, there are data in the literature that describe antiviral activity of hht against several types of viruses including covs. 304, 305 a recent in-vitro evaluation of anti-sars-cov-2 activity displayed ec 50 of 2.10 µm. however, the mechanism of action is not yet clear, which demands further investigation on ideal doses of hht to achieve the clinical results expected of a covid-19 therapeutic drug. 110 the first reports on tetraethylthiuram disulfide, disulfiram (dsf) (45) (fig. 8) , date back to 1881. however, only in the 1940s that dsf would become popular when it was discovered that it could form copper chelates which favoured the death of micro-organisms and enabled treatment of intestinal parasites. [306] [307] [308] in 1945, dsf alcohol sensitivity was discovered accidentally and it was soon used in the clinical treatment of alcohol dependence. 309, 310 dsf is used to treat alcohol dependence because it irreversibly inhibits the acetaldehyde dehydrogenase enzyme and modifies cysteine residues in its active site. this change prompts the formation of a disulphide bond between two cysteine residues in the active site. 311 dsf effectiveness is based on its similarly to several proteins yielding a range of biological activities, such as antitumoral, 312, 313 antimicrobial, 310 and anti-sars and mers-cov. 314 adding to the list of drugs to be repurposed against sars-cov-2, recent studies indicate that dsf is able to inhibit other enzymes, such as methyltransferase, urease and kinase, all by reacting with important cysteine residues that suppress the natural cycle of the enzymes, suggesting broad-spectrum characteristics. 312, 314 covs have two viral enzymes, m pro and pl pro , that are cysteine protease involved in the formation of structural and non-structural proteins that constitute the viruses and favour control of host cells. 315 different assays, such as proteolytic and binding synergy assays, were also conducted and described. 314 although outcomes indicate dsf for anti-mers-cov and anti-sars-cov therapy, to the present date (15 th june 2020), no other article was published claiming the availability of the compound as promising anti-sars-cov-2. recently, an announcement was published on the oxford university website on the results of one randomized evaluation of covid-19 therapy. 317 more specifically, the study focused on dexamethasone (46), a corticosteroid with fluorine at 9-position (fig. 8) . the drug is mostly used as anti-inflammatory, which works by inhibiting vasodilation, reducing leukocyte migration to the inflammation site and increasing vascular permeability. 318 immunotherapy is an effective intervention in viral infections. most attempts at immunotherapy were successful in fighting viruses similar to sars-cov-2. the principal methods include vaccine, neutralizing antibodies (nabs) candidates and convalescent plasma. 35, 61, 67, 68, 156, 319, 320 in addition, according to the evidences from viral infections (ebola, influenza, sars and mers), immunotherapeutic interventions can reduce viral load and mortality rate of patients. 321, 322 development of either monoclonal (mabs) or polyclonal (pabs) neutralizing antibodies is a commonly adopted immunotherapeutic alternative due to its specificity, purity, low contamination by blood-transmitted pathogens and relative safety. however, there are limitations to the use of nabs once its development and large-scale production for clinical use are a complex, expensive and slow process. 321 promising scientific investigations have suggested using mabs or pabs as prophylactic and therapeutic measures against influenza 323 and hcovs, such as mers-cov 324 and sars-cov. 325 targets reported as promising for hcovs immunotherapy were cytokine, 326 s1-receptor-binding domain (s1-rbd), s1 n-terminal domain (s1-ntd) and some other region of subunit s2 in order to block the rbds bonds to their respective receptors and to interfere either with s2-mediated membrane fusion or with the entry in the host cells, thus inhibiting infection. 327 these researchers have encouraged the development of nabs with cross reactivity potential and/or cross neutralization effect on sars-cov-2 infections, as shown by tian et al. 328 data suggest that mab cr3022 can be developed as therapeutic candidate, either isolated or combined with neutralizing antibodies to prevent and treat covid-19, given that it could potently form bonds with sars-cov-2 rbd (kd of 6.3 nm). a different study by wang et al. 329 reported the discovery of a human mab (47d11) that promoted cross neutralization of sars-cov and sars-cov-2 in a culture of cells through an independent receptor-binding inhibition mechanism that targets a conserved epitope on the spike hcovs rbd mentioned above. it is also reported the on-going investigation of convalescent plasma or immunoglobulin as last resource to improve the survival rate of patients with several viral infections such as h 5 n 1 avian influenza, 330 334 a possible explanation for the efficacy of convalescent plasma is that immunoglobulin antibodies in the plasma of recovered patients can suppress viremia. 156 shen et al. 335 reported that five patients with extreme symptoms of covid-19 received blood transfusion containing convalescent plasma with specific sars-cov-2 antibodies. after a series of blood transfusions, the improvement in clinical status of patients was observed. viral loads also decreased and became negative within 12 days after the transfusion, and sars-cov-2-specific elisa and nabs titers increased following the transfusion. in spite of the limited sample, the authors concluded that convalescent plasma transfusion benefited patients infected with sars-cov-2. therefore, testing safety and efficacy of transfusing convalescent plasma in patients infected with sars-cov-2 can be of value. 319, 336 among the modalities of immunotherapy, vaccines are expected to be more promising, hence the global engagement in their production. over the last decade, the scientific community and the vaccine industry had to answer urgently to the epidemics of h1n1, ebola, zika and, more recently, sars-cov-2. vaccine development is an expensive and slow process with high risks of failure, which often motivate developers to follow a linear sequence of steps with several breaks for data analysis and fabrication processes. therefore, it is fundamental that vaccines be developed through faithful methods even if it takes longer to move them onto clinical trials or to make a large number of doses available, a challenge during a pandemic. 337 developing efficient vaccines for sars-cov-2 will be essential to reduce the severity of the disease, viral shedding and transmission to control future outbreaks. prior to the covid-19 pandemic, multiple strategies were used to generate vaccines for the first hcovs (sars-cov and mers-cov). 338 several studies related to sars-cov vaccine production targeting the protein s, due to its function in the receptor binding and fusion to the host membrane, were successful in animal tests against that coronavirus. 339-341 these vaccines employed live-attenuated virus vaccines, killed virus, dna vaccines and viral vector vaccines. theoretically, these techniques could be applied to develop sars-cov-2 vaccines given their similarities from both the genomic perspective and the mechanisms employed in the invasion and infection of host cells. 326 gao et al. 342 promoted the pilotscale production of a purified inactivated sars-cov-2 virus vaccine candidate (picovacc), which induced sars-cov-2-specific nabs in mice, rats and non-human primates. in addition, three immunizations using two different doses (3 μg or 6 μg per dose) in macaques provided partial or complete protection against the sars-cov-2 challenge, respectively, without observable antibodydependent enhancement of infection. these data reinforce the use of picovacc in the next steps of clinical trials targeting sars-cov-2 still for the present year. given the magnitude of the covid-19 pandemic, it has become indispensable to work as fast as possible to develop vaccines for global distribution. however, protocols are necessary to safekeep the population's health. hence, before allowing human testing of covid-19 vaccines, regulatory organizations must evaluate their safety against a series of virus strains and more than one animal model. they also must demand preclinical evidences that experimental vaccines prevent infectioneven if it means waiting weeks or months for models to become available. this is time well-spent, once testing vaccines without investing the due amount of time to completely understand the risks can lead to setbacks for current and future pandemics. 343 repurposing potential relies on the mechanism for other viruses, such as hepatitis c, by inducing conformational changes that compromise rdrp activity. it was also possible to identify benzene derivatives used to treat cancer (dasatinib and imatinib) and one antiviral (darunavir), but predominant in-silico and some in-vitro outcomes demand more conclusive studies. representative of benzoic derivatives, ntz displayed significant inhibitory activity against pro-inflammatory cytokines, which can benefit control of ards, in spite of an unclear mechanism against sars-cov-2. quinoline derivatives, hcq and cq, were some of the first drugs investigated. after several in-silico, in-vitro and in-vivo assays, based on results presented by recovery to the present date, unfortunately, these drugs were proven ineffective in hospitalized covid-19 patients. ivermectin represents macrolide derivatives and is suggested as promising due to both immunostimulatory activity and inhibitory activity on nuclear transport when administered at the early stages of the disease. among indole derivatives, arbidol was pointed out as useful for reducing viral binding and releasing of intracellular vacuoles that contain the virus. nonetheless, clinical trials are still necessary after dose adjustment for better outcomes. both indole derivatives (emetine and hht), in spite of promising results, were only submitted to in-silico and in-vitro tests, thus demanding further investigation on their toxicity and mechanism of control. hence, the currently available data on the classes of drugs investigated here revealed that drugs were considered promising mainly after in-silico tests only. in fact, the inefficacy of some of these drugs became evident after in-vitro or in-vitro tests, as cq and hcq, whose clinical trials failed to confirm the so-expected anti-sars-cov-2 activity. it is necessary to highlight the importance of clinical trials with drugs considered promising in theoretical studies, with due calm and openness to question and refute hypotheses, in the absence of scientific evidence to support their use to treat covid-19. similarly, the careful analysis of practices involving patients with the extreme form of the disease is also important to identify new alternatives, such as the results recently published by recovery on dexamethasone. it is also expected that detailed clinical trials are conducted with some of the drugs described in this article as potential anti-sars-cov-2, for instance sofosbuvir, origin and evolution of pathogenic coronaviruses the covid-19 vaccine development landscape sars-cov-2 vaccines: status report recent discovery and development of inhibitors targeting coronaviruses analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods drug repurposing for viral infectious diseases: how far are we? tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study virtual screening and repurposing of fda approved drugs against covid-19 main protease baricitinib as potential treatment for 2019-ncov acute respiratory disease remdesivir and sars-cov-2: structural requirements at both nsp12 rdrp and nsp14 exonuclease active-sites coronaviruses -drug discovery and therapeutic options development of one-step, real-time, quantitative reverse transcriptase pcr assays for absolute quantitation of human coronaviruses oc43 and 229e coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a review of coronavirus disease-2019 (covid-19) emergence of novel coronavirus 2019-ncov: need for rapid vaccine and biologics development a pneumonia outbreak associated with a new coronavirus of probable bat origin world health organization. coronavirus disease (covid-19) pandemic advances in virus research covid-19: a promising cure for the global panic genotype and phenotype of covid-19: their roles in pathogenesis molecular evolution of human coronavirus genomes recent progress and challenges in drug development against covid-19 coronavirus (sars-cov-2) -an update on the status genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding structure analysis of the receptor binding of 2019-ncov mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence visualizing speech-generated oral fluid droplets with laser light scattering pitfalls of judgment during the covid-19 pandemic a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study origin of viruses: primordial replicators recruiting capsids from hosts the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor renin-angiotensin-aldosterone system inhibitors in patients with covid-19 renin-angiotensin-aldosterone system and covid-19 infection covid-19 infection: origin, transmission, and characteristics of human coronaviruses review of the clinical characteristics of coronavirus disease 2019 (covid-19) mild or moderate covid-19 the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreakan update on the status clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of coronavirus disease 2019 in china clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? comorbidities and multi-organ injuries in the treatment of covid-19 the vasoprotective axes of the renin-angiotensin system: physiological relevance and therapeutic implications in cardiovascular, hypertensive and kidney diseases sars-cov-2 and viral sepsis: observations and hypotheses novel coronavirus infection (covid-19) in humans: a scoping review and meta-analysis sex-specific sars-cov-2 mortality: among hormone-modulated ace2 expression, risk of venous thromboembolism and hypovitaminosis d probiotics and covid-19: one size does not fit all clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study pathological findings of covid-19 associated with acute respiratory distress syndrome liver injury in covid-19: management and challenges endothelial cell infection and endotheliitis in covid-19 comorbidities and multi-organ injuries in the treatment of covid-19 coronavirus infections and immune responses longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients reducing mortality from 2019-ncov: host-directed therapies should be an option ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study coronavirus membrane fusion mechanism offers a potential target for antiviral development pharmacological therapeutics targeting rna-dependent rna polymerase, proteinase and spike protein: from mechanistic studies to clinical trials for covid-19 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structural basis of receptor recognition by sars-cov-2 a possible strategy to fight covid-19: interfering with spike glycoprotein trimerization the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry protease inhibitors targeting coronavirus and filovirus entry structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants structure of the rna-dependent rna polymerase from covid-19 virus structural basis for inhibition of the rna-dependent rna polymerase from sars-cov-2 by remdesivir structure of mpro from sars-cov-2 and discovery of its inhibitors efficiency of incorporation and chain termination determines the inhibition potency of 2′-modified nucleotide analogs against hepatitis c virus polymerase protective effect and potential mechanisms of wei-chang-an pill on high-dose 5-fluorouracil-induced intestinal mucositis in mice higher order structures in purine and pyrimidine metabolism human pyrimidine nucleotide biosynthesis as a target for antiviral chemotherapy pyrrolopyrimidines: an update on recent advancements in their medicinal attributes synthesis and evaluation of anti-tumor activity of novel triazolo[1,5-a] pyrimidine on cancer cells by induction of cellular apoptosis and inhibition of epithelial-to-mesenchymal transition process a facile one pot synthesis of novel pyrimidine derivatives of 1,5-benzodiazepines via domino reaction and their antibacterial evaluation high antiparasitic activity of silver complexes of 5,7-dimethyl-1,2,4-triazolo[1,5 a]pyrimidine new carbocyclic n6-substituted adenine and pyrimidine nucleoside analogues with a bicyclo[2.2.1]heptane fragment as sugar moiety; synthesis, antiviral, anticancer activity and x-ray crystallography new prodrugs of two pyrimidine acyclic nucleoside phosphonates: synthesis and antiviral activity 5-fluorouracil upregulates cell surface b7-h1 (pd-l1) expression in gastrointestinal cancers a new animal model of intestinal mucositis induced by the combination of irinotecan and 5-fluorouracil in mice 5-fluorouracil in combination with deoxyribonucleosides and deoxyribose as possible therapeutic options for the coronavirus, covid-19 infection the safety and efficacy of gemcitabine for the treatment of bladder cancer depletion of sirt7 sensitizes human non-small cell lung cancer cells to gemcitabine therapy by inhibiting autophagy gemcitabine for recurrent ovarian cancer -a systematic review and meta-analysis repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection drug repositioning is an alternative for the treatment of coronavirus covid-19 an efficient synthesis of baricitinib janus kinase inhibitor baricitinib modulates human innate and adaptive immune system in vivo antitumor activity of su11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship the egf receptor family as targets for cancer therapy janus kinase inhibitor baricitinib is not an ideal option for management of covid-19 protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 galidesivir limits rift valley fever virus infection and disease in syrian golden hamsters therapeutic options for the 2019 novel coronavirus (2019-ncov) new nucleoside analogues for the treatment of hemorrhagic fever virus infections remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro genotype and subtype profiling of psi-7977 as a nucleotide inhibitor of hepatitis c virus selected nucleos(t)ide-based prescribed drugs and their multi-target activity mechanism of activation of psi-7851 and its diastereoisomer psi-7977 evaluation of sofosbuvir (β-d-2′-deoxy-2′-α-fluoro-2′-β-cmethyluridine) as an inhibitor of dengue virus replication yellow fever virus is susceptible to sofosbuvir both in vitro and in vivo the fda-approved drug sofosbuvir inhibits zika virus infection beyond members of the flaviviridae family, sofosbuvir also inhibits chikungunya virus replication sofosbuvir as repurposed antiviral drug against covid-19: why were we convinced to evaluate the drug in a registered/approved clinical trial antiviral β-l-nucleosides specific for hepatitis b virus infection. in: frontiers in viral hepatitis treatment of chronic hepatitis b: focus on telbivudine safety and efficacy of telbivudine for the treatment of chronic hepatitis b. ther clin risk manag 9-trisubstituted-9h-purine)-8-chalcone derivatives as potent anti-gastric cancer agents: design, synthesis and structural optimization novel phosphodiesterases inhibitors from the group of purine-2,6-dione derivatives as potent modulators of airway smooth muscle cell remodelling novel butanehydrazide derivatives of purine-2,6-dione as dual pde4/7 inhibitors with potential anti-inflammatory activity: design, synthesis and biological evaluation the purines: potent and versatile small molecule inhibitors and modulators of key biological targets synthesis and biological evaluation of novel hiv-1 protease inhibitors with pentacyclic triterpenoids as p2-ligands seley-radtke, design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity remdesivir in covid-19: a critical review of pharmacology, pre-clinical and clinical studies the epidemiology, diagnosis and treatment of covid-19 therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys a randomized, controlled trial of ebola virus disease therapeutics broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov first case of 2019 novel coronavirus in the united states compassionate use of remdesivir for patients with severe covid-19 pharmacologic treatments for coronavirus disease 2019 (covid-19) oral ganciclovir for patients with cytomegalovirus retinitis treated with a ganciclovir implant drugbank 5.0: a major update to the drugbank database epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study incidence, risk factors, and outcome of cytomegalovirus infection and disease in patients receiving prophylaxis with oral valganciclovir or intravenous ganciclovir after umbilical cord blood transplantation pharmacokinetics of ganciclovir after oral valganciclovir versus intravenous ganciclovir in allogeneic stem cell transplant patients with graft-versus-host disease of the gastrointestinal tract the first 75 days of novel coronavirus (sars-cov-2) outbreak: recent advances, prevention, and treatment tenofovir disoproxil fumarate 3-triazole-containing hybrids as leads in medicinal chemistry: a recent overview medicinal attributes of 1,2,3-triazoles: current developments synthesis and anticancer activity of long chain substituted 1,3,4-oxadiazol-2-thione, 1,2,4-triazol-3-thione and 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine derivatives chemical synthesis and biological evaluation of triazole derivatives as inhibitors of inha and antituberculosis agents synthesis, antiinflammatory and analgesic activity of 2-[4-(substituted benzylideneamino)-5-(substituted phenoxymethyl)-4h-1,2,4-triazol-3-yl thio] acetic acid derivatives synthesis and biological evaluation of some novel triazol-3-ones as antimicrobial agents design, synthesis, antiviral and cytostatic activity of ω-(1h-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues synthesis, biological evaluation and molecular modeling of a novel series of fused 1,2,3-triazoles as potential anti-coronavirus agents treatment options for covid-19: the reality and challenges compounds with therapeutic potential against novel respiratory ribavirin in the treatment of sars: a new trick for an old drug? inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro anti-hcv, nucleotide inhibitors, repurposing against covid-19 ebola virus dynamics in mice treated with favipiravir favipiravir (t-705), a broad-spectrum inhibitor of viral rna polymerase favipiravir versus arbidol for covid-19: a randomized clinical trial. medrxiv [epub ahead of print a possible pharmaceutical treatment for covid-19 experimental treatment with favipiravir for covid-19: an open-label control study. engineering an antiviral for covid-19? covid-19 -the search for effective therapy coronavirus disease 2019 (covid-19): current status and future perspectives role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings improves outcome of mers-cov infection in a nonhuman primate model of common marmoset combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome: a case report a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 triple combination of interferon beta-1b, lopinavirritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an openlabel, randomised, phase 2 trial molecular modeling evaluation of the binding abilities of ritonavir and lopinavir to wuhan pneumonia coronavirus proteases oseltamivir treatment of influenza in children therapeutic management of covid-19 patients: a systematic review in silico studies on therapeutic agents for covid-19: drug repurposing approach synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus nelfinavir was predicted to be a potential inhibitor of 2019-ncov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation atazanavir for the treatment of human immunodeficiency virus infection influence of atazanavir on the pharmacodynamics and pharmacokinetics of gliclazide in animal models therapy with atazanavir plus saquinavir in patients failing highly active antiretroviral therapy: a randomized comparative pilot trial predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov-2) through a drug-target interaction deep learning model déjà vu: stimulating open drug discovery for sars-cov-2 state-of-the-art tools unveil potent drug targets amongst clinically approved drugs to inhibit helicase in sars-cov-2 fda-approved thiolreacting drugs that potentially bind into the sars-cov-2 main protease, essential for viral replication cyclosporin a and cardioprotection: from investigative tool to therapeutic agent modulation of innate immunity by cyclosporine a the use of cyclosporine a in rheumatology: a 2016 comprehensive review cyclosporine a: a valid candidate to treat covid-19 patients with acute respiratory failure? the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors cyclosporin a inhibits the replication of diverse coronaviruses human coronavirus nl63 replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine a-derivatives including alisporivir cyclophilins and cyclophilin inhibitors in nidovirus replication identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs cyclosporin a is a potential inhibitor of sars-cov-2. researchgate (preprint) cyclosporine therapy in cytokine storm due to coronavirus disease 2019 (covid-19) clinically significant drug interactions with cyclosporin glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) discovery and development of safe-in-man broadspectrum antiviral agents teicoplanin: an alternative drug for the treatment of covid-19? fighting viruses with antibiotics: an overlooked path current epidemiological and clinical features of covid-19; a global perspective from china pharmacokinetics and safety of co-administered paritaprevir plus ritonavir, ombitasvir, and dasabuvir in hepatic impairment dasabuvir: a non-nucleoside inhibitor of ns5b for the treatment of hepatitis c virus infection in silico studies on therapeutic agents for covid-19: drug repurposing approach darunavir: a review in pediatric hiv-1 infection darunavir: a review of its use in the management of hiv-1 infection discovering drugs to treat coronavirus disease 2019 (covid-19) therapeutic options and critical care strategies in covid-19 patients; where do we stand in this battle? majallahi danishkadahi pizishkii isfahan insight derived from molecular docking and molecular dynamics simulations into the binding interactions between hiv-1 protease inhibitors and sars-cov-2 3clpro peptide-like and small-molecule inhibitors against covid-19 computational view toward the inhibition of sars-cov imatinib: a breakthrough of targeted therapy in cancer coronavirus s protein-induced fusion is blocked prior to hemifusion by abl kinase inhibitors virus-induced abl and fyn kinase signals permit coxsackievirus entry through epithelial tight junctions abl tyrosine kinase regulates hepatitis c virus entry productive replication of ebola virus is regulated by the c-abl1 tyrosine kinase abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion sars coronavirus spike protein-induced innate immune response occurs via activation of the nf-κb pathway in human monocyte macrophages in vitro imatinib attenuates inflammation and vascular leak in a clinically relevant two-hit model of acute lung injury imatinib stimulates prostaglandin e2 and attenuates cytokine release via ep4 receptor activation imatinib mesylate induces clinical remission in rheumatoid arthritis kit inhibition by imatinib in patients with severe refractory asthma long-standing remission of crohnʼs disease under imatinib therapy in a patient with crohnʼs disease imatinib might constitute a treatment option for lung involvement in covid-19 european leukemianet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia principal long-term adverse effects of imatinib in patients with chronic myeloid leukemia in chronic phase investigation of n terminal domain of sars cov 2 nucleocapsid protein with antiviral compounds based on broad anti-coronaviral activity of fda approved drugs against sars-cov-2 in vitro and sars-cov in vivo nitazoxanide: a first-in-class broad-spectrum antiviral agent nitazoxanide: a new thiazolide antiparasitic agent nitazoxanide/azithromycin combination for covid-19: a suggested new protocol for early management therapeutic potential of nitazoxanide against newcastle disease virus: a possible modulation of host cytokines nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus the anti-hepatitis c agent nitazoxanide induces phosphorylation of eukaryotic initiation factor 2α via protein kinase activated by double-stranded rna activation a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs nitazoxanide suppresses il-6 production in lpsstimulated mouse macrophages and tg-injected mice a systematic review on use of aminoquinolines for the therapeutic management of covid-19: efficacy, safety and clinical trials the possible mechanisms of action of 4-aminoquinolines (chloroquine/hydroxychloroquine) against sars-cov-2 infection (covid-19): a role for iron homeostasis? chloroquine: modes of action of an undervalued drug cardiovascular risks of hydroxychloroquine in treatment and prophylaxis of covid-19 patients: a scientific statement from the indian heart rhythm society chloroquine and hydroxychloroquine as available weapons to fight covid-19 hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro of chloroquine and covid-19 viral infection and iron metabolism hepcidin and the iron-infection axis iron and infection comparison of the antiviral activity in vitro of some non-steroidal antiinflammatory drugs antihistaminics, local anesthetics, and other amines as antiviral agents effect of chloroquine on the growth of animal viruses in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 chloroquine and hydroxychloroquine in the treatment of covid-19 with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov-2 infection hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial hydroxychloroquine in the management of critically ill patients with covid-19: the need for an evidence base association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in new york state observational study of hydroxychloroquine in hospitalized patients with covid-19 this national clinical trial aims to identify treatments that may be beneficial for people hospitalised with suspected or confirmed covid-19 2020 ivermectin: enigmatic multifaceted 'wonder' drug continues to surprise and exceed expectations ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of hiv-1 and dengue virus ivermectin is a potent inhibitor of flavivirus replication specifically targeting ns3 helicase activity: new prospects for an old drug repurposing ivermectin to inhibit the activity of sars cov2 helicase: possible implications for covid 19 therapeutics influenza a viruses escape from mxa restriction at the expense of efficient nuclear vrnp import nuclear localization of dengue virus (denv) 1-4 non-structural protein 5; protection against all 4 denv serotypes by the inhibitor ivermectin the broad spectrum antiviral ivermectin targets the host nuclear transport importin α/β1 heterodimer nucleocytoplasmic transport of nucleocapsid proteins of enveloped rna viruses severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro the importin α/β-specific inhibitor ivermectin affects hif-dependent hypoxia response pathways antiviral treatment of covid-19 hydroxychloroquine and ivermectin: a synergistic combination for covid-19 chemoprophylaxis and treatment? therapeutic potential of ivermectin for covid enhancing immunity in viral infections, with special emphasis on covid-19: a review safety of high-dose ivermectin: a systematic review and meta-analysis five-membered heterocycles: indole and related systems. modern heterocyclic chemistry a review on recent developments of indole-containing antiviral agents discovery of novel multi-target indole-based derivatives as potent and selective inhibitors of chikungunya virus replication medicinal applications of (benz)imidazole-and indole-based macrocycles medicinal chemistry of indole derivatives: current to future therapeutic prospectives synthesis and antiviral activity of some novel indole-2-carboxylate derivatives superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study arbidol monotherapy is superior to lopinavir/ritonavir in treating covid-19 the anti-influenza virus drug, arbidol is an efficient inhibitor of sars-cov-2 in vitro pharmacokinetics of single and multiple oral doses of arbidol in healthy chinese volunteers rizatriptan vs sumatriptan in the acute treatment of migraine ação da melatonina no tecido cartilaginoso covid-19: melatonin as a potential adjuvant treatment protective effects of melatonin in mice infected with encephalitis viruses melatonin: its possible role in the management of viral infections-a brief review network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 lungs as target of covid-19 infection: protective common molecular mechanisms of vitamin d and melatonin as a new potential synergistic treatment emetine protects mice from enterovirus infection by inhibiting viral translation emetine inhibits zika and ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo novel antiviral activities of obatoclax, emetine, niclosamide, brequinar, and homoharringtonine tracing the journey of disulfiram: from an unintended discovery to a treatment option for alcoholism supervised disulfiram as adjunct to psychotherapy in alcoholism treatment the clinical use of disulfiram (antabuse®): a review disulfiram inhibits the in vitro growth of methicillin-resistant staphylococcus aureus disulfiram, an alcohol dependence therapy, can inhibit the in vitro growth of francisella tularensis structural basis for inactivation of giardia lamblia carbamate kinase by disulfiram disulfiram/copper markedly induced myeloma cell apoptosis through activation of jnk and intrinsic and extrinsic apoptosis pathways disulfiram is a direct and potent inhibitor of human o6-methylguanine-dna methyltransferase (mgmt) in brain tumor cells and mouse brain and markedly increases the alkylating dna damage disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes coronaviruses -drug discovery and therapeutic options inhibition of urease by disulfiram, an fda-approved thiol reagent used in humans the microcirculation and inflammation: site of action for glucocorticoids the convalescent sera option for containing covid-19 the possible of immunotherapy for covid-19: a systematic review perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (covid-19) potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review advances in respiratory virus therapeutics -a meeting report from the 6th isirv antiviral group conference a mouse model for mers coronavirus-induced acute respiratory distress syndrome neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential effective treatment of severe covid-19 patients with tocilizumab neutralizing antibodies against sars-cov-2 and other human coronaviruses potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody a human monoclonal antibody blocking sars-cov-2 infection treatment with convalescent plasma for influenza a (h5n1) infection convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection evaluation of convalescent plasma for ebola virus disease in guinea the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol treatment of 5 critically ill patients with covid-19 with convalescent plasma convalescent plasma as a potential therapy for covid-19 developing covid-19 vaccines at pandemic speed a decade after sars: strategies for controlling emerging coronaviruses severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus sars coronavirus: a new challenge for prevention and therapy coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus development of an inactivated vaccine candidate for sars-cov-2 don't rush to deploy covid-19 vaccines and drugs without sufficient safety guarantees the authors would like to thank facepe (fundação de amparo à ciência e tecnologia none. all authors researched data for the article, contributed substantially to discussion of the content, wrote the article and reviewed and edited the manuscript before submission. key: cord-291650-1qy6y7f0 authors: butt, taimur s.; koutlakis-barron, irene; aljumaah, suliman; althawadi, sahar; almofada, saleh title: infection control and prevention practices implemented to reduce transmission risk of middle east respiratory syndrome-coronavirus in a tertiary care institution in saudi arabia date: 2016-05-01 journal: am j infect control doi: 10.1016/j.ajic.2016.01.004 sha: doc_id: 291650 cord_uid: 1qy6y7f0 background: transmission of middle east respiratory syndrome-coronavirus (mers-cov) among health care workers (hcws) and patients has been documented with mortality rate approximating 36%. we propose advanced infection control measures (a-ic) used in conjunction with basic infection control measures (b-ic) help reduce pathogen transmission. b-ic include standard and transmission-based precautions. a-ic are initiatives implemented within our center to enhance effectiveness of b-ic. objective: study effectiveness of combining b-ic and a-ic to prevent transmission of mers-cov to hcws. methods: a retrospective observational study was undertaken. a-ic measures include administrative support with daily rounds; infection control risk assessment; timely screening, isolation, and specimen analysis; collaboration; epidemic planning; stockpiling; implementation of contingency plans; full personal protective equipment use for advanced airway management; use of a real-time electronic isolation flagging system; infection prevention and control team on-call protocols; pretransfer mers-cov testing; and education. results: a total of 874 real-time polymerase chain reaction mers-cov tests were performed during the period beginning july 1, 2013, and ending january 31, 2015. six hundred ninety-four non-hcws were tested, of these 16 tested positive for mers-cov and their infection was community acquired. sixty-nine percent of the confirmed mers-cov-positive cases were men, with an average age of 56 years (range, 19-84 years). of the total tested for mers-cov, 180 individuals were hcws with zero positivity. conclusions: adhering to a combination of b-ic and a-ic reduces the risk of mers-cov transmission to hcws. transmission of middle east respiratory syndrome-coronavirus (mers-cov) among health care workers (hcws) and patients in hospitals within saudi arabia has been documented. 1 of the hcws who have acquired this infection, more than 63% acquired the infection within saudi arabia, where the majority of mers-cov cases have been reported. 2 as of june 20, 2015, 1,338 cases have occurred worldwide, and of these 77.5% (n = 1,038) were identified within saudi arabia. 3, 4 the main contributing factor for health care-associated transmission and causality of outbreaks among saudi hospitals is due to emergency department (ed) overcrowding and poor ventilation. 5 in turn, this can be a reflection of institutional overcrowding, inpatient bed occupancy reaching or exceeding full capacity, and/or a lack of compliance to and understanding of the importance of implementing infection control and prevention (icp) measures. this belief is supported by internationally recognized agencies, including the world health organization (who) and the us centers for disease control and prevention. the organizations support the practice of adhering to icp measures. basic infection control measures (b-ic), defined as standard and transmissionbased precautions, play a major role in preventing and controlling pathogen spread, including adherence to hand hygiene, environment and equipment cleanliness, use of personal protective equipment (ppe) such as high-efficiency particulate respirators (eg, n-95 or r-95), and adhering to respiratory/cough etiquette. 6 we propose that the spread of mers-cov among hcws is preventable when b-ic are used in combination with institution-specific advanced infection control measures (a-ic). in this article, a-ic implemented in a tertiary care hospital during a time of epidemic is described. during the initial and intermediate period after discovering this novel virus during june 2012 it was noted that symptoms and complexity of patients presenting with infection were variable. 7 this resulted in a case definition that required modification over time to allow for broader screening of patients with flu-like illness. the initial case definition mainly encouraged screening of patients with severe acute respiratory illness requiring intensive care unit (icu) admission. 3 in the authors' experience, in dealing with mers-cov cases in the early stages of disease recognition there were poorly established systems in place for identification of suspected/confirmed cases. we believe that this was due to an unclear incubation period, poor rapid implementation of isolation precautions and use of ppe, and the unavailability of diagnostic testing among regional health care facilities. these health care facilities relied on an inconsistent availability of appropriate packaging and transportation of specimens to approved ministry of health (moh) reference laboratories for testing of samples. consequently, there were delays in communication and appropriate isolation of positive cases resulting in spread of infection to hcws. 1 b-ic are well-recognized preventative measures considered the minimum requirement for infection prevention and control. 6, 8 a-ic are institution-specific measures that enhance b-ic to further reduce the risk of transmission. b-ic are based on key components of standard precautions and recommended transmission-based precautions for prevention and control of transmissible diseases. this includes appropriate hand hygiene practices; proper cleaning of the environment and equipment; prompt initiation of transmission-based precautions for suspected/confirmed cases until noninfectious; segregation of confirmed/suspected cases in waiting areas; use of single hospital patient rooms; and proper availability, quality, type, and use of ppe 6,9 (table 1) . a-ic is a group of institution-specific measures that go above and beyond b-ic and enhance their effectiveness. it is vital that administrative support is sought to approve and fund the necessary initiatives. the following outlines the a-ic measures adopted at our institution (table 1). 1. interdepartmental collaborative meetings: as part of the established institutional epidemic plan, using both interdepartmental collaboration and institutional expertise, daily morning meetings are held. these meetings are undertaken at a time of high incidence within the community, other regional institutions, or when an alert has been issued by the moh. key members include hospital administration, senior staff from the infectious diseases department, icp, ed, icu, nursing, microbiology, case management, and others as deemed necessary. their objective is to assess, monitor, and recommend risk mitigation strategies. current regional and international information regarding mers-cov disease activity, institutional preparedness, and its capabilities are discussed. during these meetings initiatives are formulated, implemented if applicable, and evaluated for success and/or modification. measures to reduce risks referred to in this article as a-ic were discussed in these meetings and implemented institution-wide. wide infection control risk assessment to address areas of concern was undertaken. areas of deficiencies addressed at the organization level include insufficient number of staff in highrisk areas, fit testing for high-efficiency particulate respirators (especially for ed, icu, and direct patient care providers), overcrowding in the ed, ventilation systems in the ed, extended turnaround time of mers-cov test results, and awareness of the importance of early identification and isolation of suspected cases. with each automated telephone text message reminder of upcoming clinic or admission came an additional reminder to inform staff of any flu-like illness upon arrival. this is in conjunction with a prescreening process established in the ed and clinic receptions. implemented as an extension of the existing institutional epidemic plan ( e. patient transportation: isolation transportation pods were purchased to be used for transportation of any patient suspected or confirmed positive for an airborne infectious disease, including mers-cov. during epidemic plan activation a disease-specific education campaign is implemented. posters, pamphlets, and the intrahospital television system are used for communication (fig 2) . 14. staff education and communication: staff preparation training and specific education to care for patients with/suspected mers-cov was undertaken. 15. transparency: while maintaining confidentiality, transparency is important to enhance dissemination of accurate information to all staff. this can create an atmosphere of trust, loyalty, and support; therefore, hcws are more likely to adhere to recommendations and reduce their absenteeism. method icis was used to retrieve patient and hcw data. for the purpose of this observational study, data were retrieved retrospectively and collected for a continuous period of 19 months. those tested for mers-cov included patients with suspected/confirmed cases who met the case definition and/or hcws who were tested during an outbreak investigation for protected/unprotected exposure. duplicate tests were removed if testing was performed during the same episode of illness or exposure. all patient and hcw confidentiality and rights were maintained. microsoft office (redmond, wa) and visio (microsoft, redmond, wa) were used to plot results and research advisory committee approval (rac# 2151-194) was obtained to use the data for this publication. a total of 874 cases were tested for mers-cov from the period beginning july 1, 2013, and ending january 31, 2015. of these, 16 (1.8%) were mers-cov positive. they were all non-hcws (fig 3) . during this time period the ed served more than 80,000 patients and institutional inpatient bed capacity was >700 with an occupancy rate consistently >90%. institution employee numbers exceeded 11,000. of the 874 mers-cov samples tested, 180 (21%) were from hcws. hcw testing was due to meeting the case criteria or to protected/ unprotected exposure. exposure occurred in 3 of the confirmed cases as a result of a delay in the initiation of isolation precautions due to lack of early recognition of symptoms or incubation period. testing of the non-hcws (n = 694) was due to case criteria being met during admission or upon presentation to the institution through the ed or clinics, or as part of a screening requirement at the time of admission; that is, direct admission from another hospital per the epidemic plan. the majority of patients with positive cases were men (69%) with a mean age of 56 years (range, 19-84 years). all case patients except 1 had comorbidities and/or immunocompromised status. ten of the 16 patients with confirmed cases died (62%). mortality was directly or indirectly attributed to complications that developed as a result of mers-cov infection. all case patients had respiratory symptoms on initial presentation except for 1 patient who was admitted for elective surgery and developed respiratory symptoms with fever postoperatively on day 7. this was considered community acquired because the incubation period overlapped with the preadmission period. no cases were considered health careassociated infections (table 3) . potential hcw exposure days are calculated from the date of mers-cov symptom onset while in hospital until either the date of negative screening result(s) or death while positive. during the period of study the potential hcw exposure days totaled 338. the mean duration of potential infectivity per case was 21 days (range, 4-57 days). due to the nature of their work, hcws remain at high risk of acquiring communicable diseases from occupational exposure. experience and data collected during the severe acute respiratory syndrome epidemic in hong kong suggests an infection rate of 22% among hcws. 11 during april-august 2014 a number of hospital outbreaks were reported within saudi arabia increasing the risk of hcw exposure. this risk to hcws is supported by ongoing findings reported by the who. the mers-cov situation update report-15 june 2015 12 states that 10%-29% of the total mers-cov cases identified in saudi arabia were in hcws. front-line hcws caring for undifferentiated and severely ill patients who do not adhere to icp measures are at the highest risk. 10 upon the discovery of the novel coronavirus and in anticipation of a local and regional threat, a taskforce was formed to evaluate our institution's readiness to deal with the developing situation and to initiate proactive measures to protect staff, patients, and visitors. this taskforce included representatives from experts from the infectious disease department (adult and pediatric), icp, ed, laboratory, nursing, research, supply chain, and other relevant stakeholders. this article reflects the work undertaken by the taskforce members who recommended and implemented measures to mitigate the risk of viral spread. although institutions aim to protect and safeguard their hcws against communicable diseases, success is not always attainable without a culture of vigilance. the approval by executive management to implement the recommended icp measures was primarily due to recognition of a true potential threat, and in support of recommendations by the taskforce. this prompt action and support is reflective of the mission, vision, and core values of our institution, which supports a culture of safety. due to urgency, time for a prescheduled mock drill to test proposed interventions and make the necessary adjustments based on the results of the simulation was not possible at time of initial implementation. changes or improvements to icp measures occurred in real time with immediate effect. ongoing monitoring for effectiveness occurred through observational hcw compliance monitoring, noting frequency of unprotected exposure events, and screening during outbreak investigations. those working in our institution-especially within high-risk areas (eg, ed and icu) observed an increased level of anxiety reflected by an increased use of masks while not performing direct patient care. in particular, in the use of high-particulate respirators. in our opinion this anxiety was as a result of rumors and misconceptions of mers-cov transmission and number of actual cases within our institution, documented human-to-human and animal-to-human transmission, a high mortality rate (36%) that includes hcws, limited treatment options, and the unavailability of vaccines. 4 communication and transparency helped to reduce levels of anxiety and rumor. our reported mers-cov mortality rate of 62% is higher than that reported nationally and most likely due to the complexity and comorbidities of the patient population served in our major city tertiary care institution. there were 16 confirmed mers-cov cases over a 2-year period with a collective potential infectivity duration of 338 days. none of the confirmed cases were health care-associated infections or hcws from this institution. as reflected in table 3 , cases identified in 2013 from date of admission to date of first mers-cov sample took longer than what was observed in subsequent years. this reflects the successful work of the taskforce. b-ic were already in place but required reinforcement. standard and transmission-based precautions, and hand hygiene are icp key performance indicators for continual compliance monitoring and reporting. 13, 14 a-ic were introduced and reinforced to support the b-ic measures. many of the a-ic measures implemented during this period have now become standard-of-care practices within the institution. without administrative and leadership support, targeted resource allocation, and high hcw compliance rates, icp measures would not be effective. this includes timely information dissemination, investment by the institution on education, ongoing awareness and staff development, adequate and high-quality supplies, and meticulous screening processes. clear and open communication channels are vital between all parties and are a prerequisite for success. close contact with the moh was maintained both in reporting of cases and receiving directives for case identification and management. in addition, updates posted by the who and centers for disease control and prevention were reviewed on a daily basis to remain current and monitor for changes to guidelines, and in the understanding of the disease processes. adjustments were made ac-cordingly to institutional case definitions, management guidelines, and icp measures. no outside consultation was undertaken to develop and refine the a-ic measures. this was a single-center study; therefore, results may not be generalized to other institutions. the actual level of compliance and benefits of implemented icp measures were not systematically measured because a randomized control study would be unethical. seroconversion rates for mers-cov were not undertaken among hcws because this test is currently unavailable within our institution. our institution successfully reduced the transmission risk of mers-cov among hcws and patients. we believe that our success is multifactorial, including a proactive and visionary response by leadership, collaborative efforts by all departments, and staff adherence to both b-ic and a-ic measures. we suggest that other institutions under similar circumstances conduct an internal review as soon as possible and implement appropriate measures accordingly. to our knowledge, a similar experience of preventing the spread of mers-cov through the implementation of both b-ic and a-ic has not been published. ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov)-tenth update infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection coronamap realtime tracking of mers corona virus on world map. as of infection control and mers-cov in health-care workers guideline for isolation precautions: preventing transmission of infectious agents in healthcare setting. the healthcare infection control practices advisory committee state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection interim guidance 6 interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome (mers-cov) pandemic (h1n1) 2009 risk for frontline health care workers clinical management and infection control of sars: lessons learned world health organization. mers-cov situation world health organization. clean care is safer care commentary: protecting health workers from airborne mers-cov-learning from sars, centers for infectious disease research and policy the authors thank elenette prado, department of emergency medicine, for providing secretarial assistance. key: cord-301730-flv5lnv8 authors: pandey, anamika; khan, mohd kamran; hamurcu, mehmet; gezgin, sait title: natural plant products: a less focused aspect for the covid-19 viral outbreak date: 2020-10-15 journal: front plant sci doi: 10.3389/fpls.2020.568890 sha: doc_id: 301730 cord_uid: flv5lnv8 the sudden emergence of covid-19 caused by a novel coronavirus (ncov) led the entire world to search for relevant solutions to fight the pandemic. although continuous trials are being conducted to develop precise vaccines and therapeutic antibodies, a potential remedy is yet to be developed. plants have largely contributed to the treatment of several human diseases and different phytoconstituents have been previously described to impede the replication of numerous viruses. despite the previous positive reports of plant-based medications, no successful clinical trials of phyto-anti-covid drugs could be conducted to date. in this article, we discuss varying perspectives on why phyto-anti-viral drug clinical trials were not successful in the case of covid-19. the issue has been discussed in light of the usage of plant-based therapeutics in previous coronavirus outbreaks. through this article, we aim to identify the disadvantages in this research area and suggest some measures to ensure that phytoconstituents can efficiently contribute to future random viral outbreaks. it is emphasized that if used strategically phyto-inhibitors with pre-established clinical data for other diseases can save the time required for long clinical trials. the scientific community should competently tap into phytoconstituents and take their research up to the final stage of clinical trials so that potential phyto-anti-covid drugs can be developed. nobody knew that the coronavirus disease of 2019 was going to be a greater disaster when compared to the former coronavirus outbreaks, severe acute respiratory syndrome (sars), and middle east respiratory syndrome (mers). the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), that caused covid-19 is alarming to the scientific world as it has resulted in the unexpected viral pandemic. however, was covid-19 an unexpected viral pandemic? the answer is no. the previous two corona outbreaks, sars-cov, and mers-cov, were first reported in guangdong, china in november 2002 (zhong et al., 2003) and saudi arabia in april 2012, respectively (de wit et al., 2016; cui et al., 2019) . after the identification of sars-cov (ksiazek et al., 2003) , several strategies were adopted to eradicate the disease, however, infection control was proven to be more effective than medical intervention leading to the termination of the sars pandemic. although the end of the sars pandemic was announced in july 2003, different types of sars re-occurred in different years either by zoonotic or human-to-human transmission in several countries including canada, vietnam, the middle east, hong kong, south korea, and jordan zaki et al., 2012; hijawi et al., 2013) . later, evidence proved that intermediate hosts may not be required for straight human infection as chinese horseshoe bats are the main reservoirs of sars-cov (ge et al., 2013) . besides, it was suggested that there was a higher risk of recurrence of sars-cov from circulating viruses in bat populations (menachery et al., 2015) . the emergence of sars-cov-2 is just a confirmation of the previous predictions. after previous outbreaks, several studies determined the poor efficacy of therapeutics and both monoclonal antibodies and vaccines against cov infection (menachery et al., 2015) . in this scenario, new measures should have been created to fight the problem. the fact that more than 619,150 deaths around the world have been reported, as of july 23 2020, has proven that we, as a scientific world, were less prepared for such an abrupt viral outbreak (who, 2020, siuation report, 185) . though plants and their extracts have been recognized as effective anti-viral agents for several decades (chantrill et al., 1952) , plant-based medications have been largely ignored. despite the vast research conducted in several directions after the previous cov pandemics, plant-based therapeutics could not achieve a satisfactory level in clinical trials against the disease. medicinal plant extracts have been reported to impede the replication of several viruses including human immunodeficiency virus (hiv), hepatitis b virus (hbv), poxvirus, severe acute respiratory syndrome (sars) virus, and herpes simplex virus type 2 (hsv-2) (vermani and garg, 2002; kotwal et al., 2005; huang et al., 2006) . despite that, there are no reports of plant-based medicines that have been successful in preventing the spread of covid-19 or curing covid-19. thus, further study is required to affirm why plant-based medications could not work in the case of covid-19. in this article, we discuss different aspects of the utilization of plant-based therapeutics in controlling viral outbreaks like covid-19. future research for such viral pandemics should be designed in light of the success stories of previous plant-based medications. the disadvantages in this field of research have been deliberated so that lessons can be learned and the scientific community can prepare for future outbreaks. medicinal plants can be used as anti-viral agents either as firstgeneration drugs where plant crudes are used in their natural forms or as second-generation drugs where the active metabolites of plants which are responsible for the anti-viral activity are employed (jassim and naji, 2003) . however, to be successful, plant-based herbal medicines have to address the issue of genetic variability of viruses, competent replication of dna and rna viruses within the host cells, and their capability to survive in the host cells (wagner and hewlett, 1999; jassim and naji, 2003) . in contrast to synthetic drugs, some of the plantbased metabolites hinder the replication of viruses without disturbing the host metabolism; which consequently have restricted side effects as drugs (hussain et al., 2017) . the interest in anti-viral plant research development started with the suppression of the amplification of the influenza a virus by 12 plant extracts (chantrill et al., 1952) . afterward, continuous efforts have been made to screen different plant sources via in silico, in vitro, and in vivo assays for anti-viral activity towards several viruses such as parainfluenza virus type 3, respiratory syncytial virus, poliovirus type 1, herpes simplex virus (hsv), enteric coronavirus, and rotavirus (rv). (ahmad et al., 1996; rajbhandari et al., 2001; jassim and naji, 2003) . plant crudes contain several metabolites and it is extremely crucial to identify which component makes it a potential candidate for an effective anti-viral drug. different anti-viral compounds of plants including peptides, lignans, terpenoids, polysaccharides, flavonoids, polyacetylenes, and alkaloids are effective against different targets of viruses such as dna, rna genomes, membranes, the replication process, and ribosomal activity (jassim and naji, 2003; ireland et al., 2008; sencanski et al., 2015; vilas boas et al., 2019) . as an example, the strong in vitro activity of extracts of macaranga barteri against e7 and e19 echoviruses suggests that it can be an effective therapeutic agent for enteroviral infections such as encephalitis (ogbole et al., 2018) . however, among all the components, three stilbenoids (especially vedelianin) isolated from m. barteri extracts are largely responsible for the anti-viral activity against echoviruses (segun et al., 2019) . similarly, other stilbenes such as resveratrol and trans-arachidin isolated from grapes and the hairy root culture of peanut are capable of reducing the replication rate of the african swine fever virus (asfv) and rvs, respectively (abba et al., 2015; ball et al., 2015) . coronaviruses, which have the largest genomes among the rna viruses, consist of a long positive-sense rna that behaves like mrna encoding the synthesis of two replicase polyproteins (pp), i.e., pp1a and pp1ab. these polyproteins are processed by a main protease, i.e., chymotrypsin-like (3clpro) protease and papain like protease (plp). while the main protease cleaves at 11 sites, plp cleaves at two or more than two sites on the polyproteins. due to the essential role of these proteases in proteolytic processing during viral replication, these proteases are considered as the main targets for the development of therapeutic drugs (deng et al., 2014) . covs are grouped into four categories: alpha, beta, gamma, and delta types; among which only alpha and beta types are known to infect humans. coronaviruses are difficult to handle because of the higher mutation rates of their nucleotides as compared to other singlestranded rna viruses. before the present sars-cov-2, six different types of human coronaviruses (hcov) had been discovered including hcov-hku1, hcov-oc43, hcov-nl63, hcov-229e, mers-cov, and sars-cov (friedman et al., 2018) . the enveloped hcov viruses fit in to coronaviridae family and are known to develop respiratory diseases (geller et al., 2012) . sars-cov and mers-cov were reported as the most deadly viral corona outbreaks causing an epidemic in different countries with a fatality rate of 9% (during 2002 (during and 2003 (during ) and 35.4% (up to december 2016 , respectively (de wit et al., 2016) . the first outbreak of sars-cov in china led to a sprint of screening of chinese medicinal herbs against the disease and some of them came out as potential anti-viral agents. during the sars outbreak in the absence of effective therapies, ribavirin, a licensed drug for the respiratory syncytial virus (rsv) was commonly suggested as a treatment (van vonderen et al., 2003) . however, it was later found to cause the death of sars patients by inducing anemia and hemolysis (booth et al., 2003; chiou et al., 2005) . glycyrrhizin (triterpene glycoside glycyrrhizic acid), a phytoconstituent extracted from liquorice roots (glycyrrhiza radix) proved to be a more efficient anti-viral agent for sars-cov when compared to ribavirin (cinatl et al., 2003) . glycyrrhizin was tried against the isolates of sars-cov replicated in vero cells (kidney epithelial cells isolated from african green monkeys) and was proven to be the most compelling interceptor of replication of sars-cov when compared to other anti-viral agents such as mycophenolic acid, pyrazofurin, 6-azauridine, and ribavirin (cinatl et al., 2003) . besides it also hinders the entry of the virus which is a main step in the replication cycle. the concentration of glycyrrhizin required to impede the cytopathic effect of a virus in a vero cell culture to 50% of the control value, i.e., ec50, was 300 mg/l. ec50 stands for the effective concentration of a drug that gives a half-maximal response. although the complete effect of glycyrrhizin activity towards sars-cov is not clear, it increases the production of nitrous oxide by the overexpression of nitrous oxide synthase that inhibits the viral replication (jeong and kim, 2002; cinatl et al., 2003) . a concentration of 1000 mg/l of glycyrrhizin was found to be effective in lowering the expression of sars-cov antigens (extracted from patient's serum) in a vero cell culture. further analysis of glycyrrhizin derivatives against sars-cov showed that the addition of different compounds to functional groups may lead to a 10-70 fold increase in the anti-sars activity; however, they may also increase the cytotoxic effects (hoever et al., 2005) . although cytotoxic effects need to be discussed, such studies open the platform for the modification of glycyrrhizin for the production of novel anti-sars-cov drugs with enhanced activity. several plants and their extracts have been reported to have a remedial approach against sars-cov and mers-cov by modulating the immune response. based on a virus-induced cytopathic effect (cpe) assay and an mts [(5-(3carboxymethoxyphenyl)-2-(4,5-dimethyl-thiazoly)-3-(4sulfophenyl) tetrazolium] cell proliferation assay, extracts of linder aggregata, pyrrosia lingua, artemisia annua, and lycoris radiata with ec50 values ranging from 2.4 ± 0.2 to 88.2 ± 7.7 mg/l showed much better anti-sars-cov activity in a vero cell culture when compared to glycyrrhizin (li et al., 2005) . however, among these four extracts, lycoris radiata with an ec50 value of 2.4 ± 0.2 mg/l was found to be the most effective candidate for anti-viral medicine against sars-cov. lycorine, one of the phytoconstituents fractionated from the extract of lycoris radiata is mainly responsible for its anti-sars-cov activity showing a lower ec50 value than its original extract ( ± 0.0012 µm). lycorine is also recognized for its potential inhibition activity against herpes simplex virus (type i) and the poliomyelitis virus. thus, it is crucial to explore the broad antiviral feature of lycorine in detail using real-time pcr to assess its capacity to inhibit viral rna replication and its interface with viral antigens. aescin, an extensively utilized drug in europe, which is extracted from horse chestnut trees and reserpine which is extracted from the rauwolfia species have shown ec50 values of 3.4 µm and 6.0 µm against sars-cov in a vero cell culture, respectively (wu et al., 2004) . in addition, radix ginseng, eucalyptus, and lonicera japonica extracts have shown antiviral activity towards sars-cov at 100 µm. later, ginsenoside-rb1 isolated from the traditional chinese herb, radix ginseng, was reported to lessen acute lung injury in rats by inhibiting the inflammatory signaling pathway (yuan et al., 2014) . many research experiments have been conducted to determine the inhibition capacity of phytoconstituents against the main protease (3clpro) and papain-like protease (plpro) of sars and mers coronaviruses. strangely, even the constituents in tea can be potentially effective against a deadly virus like sars-cov. it is one of the positive points that can be counted on while considering the negative effects of tannic acid in the tea. the 3-isotheaflavin-3-gallate (tf2b), theaflavin-3,3'-digallate (tf3), and tannic acid compounds that are abundant in black tea extracts are potent inhibitors of the main protease of sars-cov, 3clpro at ic50 < 10 µm as determined by an hplc proteolytic assay . ic50 stands for the concentration of an inhibitor where the response is reduced by half. quercetin, a plant flavonoid, and its derivative quercetin-3b-galactoside show potent inhibition of viral replication in sars-cov 3clpro where sugar moiety is crucial for inhibitory action (yi et al., 2004; lin et al., 2005; chen et al., 2006 ). an extract of houttuynia cordata, a traditional chinese medicine, at 200µg/ml had been reported to have an inhibitory effect on the rna-dependent rna polymerase (rdrp) and 3c-like protease (3clpro) of sars-cov which was non-toxic to mice at an oral dosage of 16 g/kg (lau et al., 2008) . scutellarein extracted from scutettaria baicalensis can be a potential sars-cov inhibitor as it hinders the atpase activity of the helicase protein of sars-cov in vitro (yu et al., 2012) . few compounds such as dihydrotanshinone isolated from the root of salvia miltiorrhiza showed anti-viral activity against both sars and mers cov by the inhibition of proteases and hindering of the viral entry, respectively (park et al., 2012; kim et al., 2018) . a phlorotannin, dieckol, extracted from the edible brown algae ecklonia cava showed potential inhibitory effects on the 3clpro of sars-cov at ic50 = 2.7 µm. it is more repressive on the cell-based 3clpro cis-cleavage when compared to the other natural cov protease inhibitors such as quinone-methide triterpene extracted from tripterygium regelii (ryu et al., 2010; park et al., 2013) . not only are these extracts potent against 3clpro but also several natural compounds have been reported to have a greater inhibitory action against plpro. a polyphenol compound, papyriflavonol a, derived from broussonetia papyrifera which hinders the sars-cov plpro with an ic50 value of 3.7 µm can be utilized for the development of anti-cov agents (park et al., 2017) . a cinnamic amide with an infrequent carbinolamide motif derived from the methanol extract of t. terrestris fruit showed effective inhibitory action towards sars-cov plpro with ic50 = 15.8 µm (song et al., 2014) . resveratrol (trans-3, 5, 4′-trihydroxystilbene), a natural stilbene derivative, can be extracted from different plants including cranberry (vaccinium macrocarpon), grape (vitis vinifera), and huzhang (polygonum cuspidatum) is efficient in inhibiting mers-cov replication in vitro by reducing cell death and alleviating the expression of nucleocapsid protein that is required for viral replication (lin et al., 2017) . the inhibition of the nf-kb pathway by resveratrol in signal transduction shows its potential capacity to be an effective broad-spectrum anti-viral agent and thus, its functioning against cov should be investigated in vivo. the anti-viral activity of several medicinal herbal extracts such as sophora subprostrata radix, phellodendron cortex, coptidis rhizoma, meliae cortex, and cimicifuga rhizome was identified against mouse hepatitis virus (mhv) which is widely considered as a prototype of coronavirus. the ec50 values of these compounds is in the range of 2.0 to 27.5 µg/ml suggesting that these can be potent candidates for developing anti-viral therapeutics (kim et al., 2008) . the accidental outbreaks of sars and mers coronaviruses pointed towards the chances of the emergence of novel covs in the future. until 2020, there was a dearth of approved drugs for sars-cov and mers-cov and it emphasized the significance of broad-spectrum viral inhibitors. the extent of conservation in crucial active domains of different human coronaviruses such as rna helicase and 3clpro can be utilized as a target when developing potential broad-spectrum anti-cov drugs. silvestrol, extracted from aglaia sp., inhibits the cap-dependent mrna translation of hcov-229e and mers-cov in human embryonic lung fibroblast (mrc-5) cells with ec50 values of less than 0.003 µm and 0.0013 µm, respectively (muller et al., 2018) . several phytocompounds including mycophenolate mofetil, emetine, and lycorine were identified as the potential broad-spectrum inhibitors that hindered the in vitro replication of four covs; mers-cov, mhv-a59, hcov-nl63, and hcov-oc43-wt with ec50 values less than 5 µm (shen et al., 2019) . among these, the inhibition capacity of lycorine against hcov-oc43 by reducing the viral lethality in the central nervous system of mice was reported in vivo via bioluminescence imaging (shen et al., 2019) . the effectiveness of most of these phytocompounds as broad-spectrum inhibitors has been confirmed in in vitro infection models and was tested in a particular cell line that may be influenced by specific host cell types. the identification of broad-spectrum inhibitors and determining their inhibition capacity in an in vivo system may lead to the production of potential drugs. the emergence of the covid-19 outbreak and its spread around the world led to an urgent search for a solution against the disease. while expected methods such as the combined medication of systematic corticosteroids and anti-viral treatment along with interferons are being tried for the speedy recovery of patients, plant-based treatment regimes are also potentially being explored in the race. the release of the gene sequence of sars-cov-2, the crystallization of its main protease, and its availability in the protein data bank (pdb) showed that the main proteins of sars-cov-2 shares great similarities with those of sars-cov and mers-cov zhou et al., 2020) . it should be considered that despite the reported mutations in the novel coronavirus as compared to sars-cov and mers-cov, the effectiveness of potential plants and their phytoconstituents that were operative against sars-cov and mers-cov could have been employed for sars-cov-2. sars-cov and sars-cov-2 belong to beta coronaviruses with high homology in the genomic sequence at the nucleotide level; however, there are six regions of differences in their genome sequence. these regions can be supportive to developing new drugs for sars-cov-2 (xu et al., 2020) . in addition, proteins of sars-cov-2 share 95% -100% homology with sars-cov with only two non-homologous proteins, orf8 and orf10. the amino acid sequence of orf8 is different in both the viruses (chan et al., 2020) . a blastp comparison of sars-cov and sars-cov-2 showed a more than 95% similarity in helicase, nsp7,8,9,10, 3clike proteinase, 3'-to-5' exonuclease, and rna-dependent rna polymerase (xu et al., 2020) . the antibodies active towards the n protein of sars-cov may have increased the probability of binding to the n protein of sars-cov-2 due to approximately 90% identity in the n protein amino acids of both viruses (gralinski and menachery, 2020) . these similarities and dissimilarities between the two viruses should be considered while utilizing the phyto-inhibitors active against sars-cov for the novel coronavirus (ncov), sars-cov-2. however, not taking potential plants or their phytoconstituents to an effective therapeutic anti-viral drug stage during previous corona outbreaks is one of the major disadvantages of the present scenario. the availability of potent plant-based drugs against sars-cov and mers-cov could have opened new treatment pathways for sudden outbreaks such as covid-19. given this disadvantage, most of the plant-based investigations directed towards covid-19 either focus on bioinformatic tools such as in silico processing, molecular docking, or concentrate on molecular farming such as the production of recombinant proteins involving vaccines and antibodies (islam et al., 2020; rosales-mendoza et al., 2020) . several molecules of known herbal medicines, when docked with the proteins of sars-cov-2, have been reported to inhibit 3clpro, plpro, spike proteins, and viral replication by binding in different domains (islam et al., 2020) . this binding hinders the substrate from going to the enzyme's active sites, prevents dimer formation, or averts viral entry (park et al., 2013; zhang d. h. et al., 2020) . the traditional herbs which contain these potential anti-viral compounds and are regularly used in handling viral respiratory infections might be employed to provide immediate support in the treatment of covid-19. however, clinical manifestations are required for the routine implementation of these phytoconstituents as drugs. additionally, it should also be kept in mind that molecular docking is a crucial process in the identification of potential antiviral compounds and is based on the available genome information of the novel coronavirus. in the case of mutation in the existing sars-cov-2, the suggested compounds may not be effective and new investigations will be required (figure 1) . ul qamar et al. (2020) created a 3d homology model of the 3clpro sequence of sars-cov-2 and highlighted its conserved nature comparable with the main protease sequence of sars-cov which shared a 99.02% sequence similarity. however, 12 point-mutations have been reported in sars-cov-2 which dislocate crucial hydrogen bonds, change the receptor binding site of its main protease, and thus, sars-cov-2 may behave differently towards some phyto-inhibitors that were effective towards sars-cov and need to be tested. a detailed molecular docking-based screening of more than 32,000 phytoconstituents resulted in nine potential compounds (including myricitrin, licoleafol, and amaranthin) that may hinder the activity of the sars-cov-2 3clpro (ul qamar et al., 2020) . another molecular docking-based study using lopinavir and nelfinavir as standards revealed that epicatechingallate, catechin, curcumin, oleuropein, apigenin-7-glucoside, figure 1 | this picture shows the current status of phytoconstituents in the development process of anti-covid-19 drugs. due to the identified mutations in sars-cov-2 as compared to sars-cov, the detection of potent anti-viral compounds is necessary. molecular docking can largely contribute to this process. the phytocompounds (a) that are identified as potential inhibitors of sars-cov-2 should be immediately forwarded to pre-clinical and clinical trials (aanouz et al., 2020; khaerunnisa et al., 2020; ul qamar et al., 2020) . the phytocompounds (b) that were effective against sars-cov and are potent for sars-cov-2 based on in vitro experiments saved the time of target compound selection and extraction assays (nemunaitis et al., 2013; choy et al., 2020) . as their efficacy and safety has already been proven in previous the sars-cov outbreak, these can be directly taken to advanced stages in covid-19 clinical trials. the phytocompounds (c) that are approved for clinical trials or are currently in the process of clinical trials (d) should be taken forward to phase iv as soon as possible so that the wide effect of the developed drugs can be observed before the covid-19 pandemic ends (borba et al., 2020; gautret et al., 2020; qiu et al., 2020) . this is extremely critical for future random viral corona outbreaks. naringenin, demethoxycurcumin, luteolin-7-glucoside, quercetin, and kaempferol compounds extracted from medicinal plants may act as potential inhibitors of the main protease of covid-19 (khaerunnisa et al., 2020) . three phytocompounds, b-eudesmol, digitoxigenin, and ccrocin isolated from lauris nobilis l, nerium oleander, and crocus sativus l, respectively, have been proposed as potential inhibitors of the spike protein of sars-cov-2 based on the molecular docking study (aanouz et al., 2020) (figure 1) . another important aspect while dealing with phyto-inhibitors against covs is that the results obtained from in vitro experiments may be different from the clinical efficacy in in vivo experiments. this may be due to the fact that the oral intake of these phyto-drugs may not reach the expected blood serum concentration as observed in in vitro experiments. emetine, an alkaloid extracted from the root of the plant psychotria ipecacuanha (ipecac root), that was determined to be a broad-spectrum inhibitor regulating different covs in vitro was found to inhibit sars-cov-2 replication at 0.5 mm. however, as a therapeutic its plasma concentration stretches up to 0.156 mm that is much lower than its toxic plasma concentration of 1.04 mm and its ec50 value towards sars-cov-2 (regenthal et al., 1999; choy et al., 2020) . homoharringtonine, isolated from cephalotoxus fortunei, is a broad spectrum anti-viral drug effective against murine hepatitis and porcine epidemic diarrhea coronaviruses and inhibits sars-cov-2 at ec50 = 2.10 mm. however, a semi-synthetic type of homoharringtonine, omacetaxine, is reported to have a therapeutic plasma concentration of 0.066 mm after 11 days of treatment that was much lower than its ec50 value against sars-cov-2 in vitro (nemunaitis et al., 2013; choy et al., 2020) . these results of emetine and homoharringtonine confirmed that plantbased therapeutics which are potentially effective against sars-cov-2 should be immediately considered for dose optimization. isolated from liquorice roots, diammonium glycyrrhizinate combined with vitamin c tablets had been prescribed for common covid-19 symptoms and had been approved for randomized clinical trials qiu et al., 2020) . these results also give us hope that broad-spectrum phyto-antivirals can be effective for sudden viral outbreaks in the future. different researchers are investigating diverse plant forms based on ethnopharmacological data to find effective anti-cov drugs with novel action mechanisms especially targeting viral replication. in this crucial situation, it is required to discuss why phyto-inhibitors could not reach an effective drug level in previous corona outbreaks so that proper strategies could be developed for future viral epidemics. the development of drugs is a costly and long-term process. as the screening of plant-based anti-virals is very similar to the testing procedure of synthetic drugs, a better correlation of their in vitro and in vivo (ivivc) results may hasten their approval process (babar, 2013; bose et al., 2020) (figure 1) . the clinical trials of plants-based anti-virals pass through five phases including the preclinical phase (unrestricted dose on animals or in vitro experiments), phase i, ii, iii, and iv (figure 1) . phase i includes the testing of the drug and its doses on healthy people, while phase ii and phase iii are comprised of screening in patients to check the efficacy, side-effects, and safety issues associated with the drug. the number of participants increases in each phase with approximately 300-3000 patients in phase iii. phase iv is one of the most crucial steps including post-marketing surveillance to observe the safety and long-term effects of the drug when used in public. several potential plant-based anti-virals against different viral diseases have entered into the market of licensed products as they are effective against particular cellular responses without an added destruction of the cell. echinacea purpurea has reached phase iv of nonrandomized clinical trials in spain to illustrate the interaction between the anti-retroviral drug, darunavir, and echinacea purpurea in hiv-1 infected patients (mólto et al., 2010; kurapati et al., 2016) . triptolide woldifiion in china made it up to phase iii in randomized clinical trials and its impact on the hiv-1 reservoir was estimated (li, 2014) . (+)-calanolide a extracted from calophyllum lanigerum hinders hiv-1 reverse transcriptase and was one of the few initial anti-hiv agents that went into a clinical trial. it successfully passed the phase i clinical trial which was performed on healthy people (creagh et al., 2001) ; however, it was not further evaluated for efficacy and safety (usach et al., 2013) . phyllanthus urinaria and phyllanthus niruri were found to block endogenous dna polymerase enzyme necessary for hepatitis b virus (hbv) replication and made it up to clinical trial (jassim and naji, 2003) . however, a randomized controlled trial on 47 patients suffering from chronic hbv showed that 12 months of the intervention of 250 mg capsule of phyllanthus niruri twice in a day did not reduce the virus load and could not clear the hepatitis b antigens. accordingly, phyllanthus niruri was not recommended as a standard drug for chronic hepatitis b patients as its efficiency was found to be wide-ranging according to the variations in the treated populations (baiguera et al., 2018) . similarly, in the case of covid-19, two forms of cinchona bark-based antimalarials, chloroquine diphosphate and hydroxychloroquine have also been the center of interest due to the reported inhibitory effects of these compounds on sars-cov. positive reports of recovery in sars-cov-2 affected patients after hydroxychloroquine treatment are available where a daily dose of 600mg of hydroxychloroquine along with azithromycin significantly reduced virus load after six days of inclusion (gautret et al., 2020) . however, the main drawback of this study was the small sample size and limited time for a long-term follow-up of the patients. another study on chloroquine diphosphate reported a lethality rate of 15% and 39% in lowdosage (450 mg twice a day on the 1st day and once a day for 4 days) and high-dosage (600 mg twice a day for 10 days) groups in critical sars-cov-2 patients after 13 days of treatment (borba et al., 2020) . thus, a higher dosage of chloroquine diphosphate along with azithromycin in critically ill patients especially suffering from cardio disorders has been reported to be unsafe. thus, evaluation of chloroquine as a drug through randomized clinical trials is required. the chances of the effective use of similar phytocompounds in the covid-19 outbreak would have been higher if experiments focusing on these phyto-inhibitors had been performed for and after sars-cov. one of the possible reasons for the failure of such natural products could be the differences in the prepared drug due to the ecological and seasonal variations in the plant growth, genotypic variation, timing of harvest, variations in the storage, and manufacturing conditions (islam et al., 2020) . changes in these factors may influence the production of the main component and contaminants (shimanovskii, 2020) . studies such as that of borba et al. (2020) and gautret et al. (2020) reported a daily requirement of a minimum 600 mg of chloroquine compounds per sars-cov-2 patient. though plants produce enough of these natural products for their own use, it is not sufficient to fulfill the commercial manufacturing needs of pharmaceutical companies. thus, sustainable and reproducible large-scale production of these natural products is another challenge in their successful utilization for the treatment of sars-cov-2. plant cell and tissue culture approaches can be effective for the extraction and multiplication of many of these natural constituents (hussain et al., 2012; shimanovskii, 2020) . extraction of phytocompounds from the tissue culture is quick and efficient when compared to the isolation from whole plants. moreover, plant tissue culture techniques facilitate the production of phytocompounds in completely controlled conditions following the regulations of good manufacturing practices (gmp). phytocompounds extracted from tissue cultures can be free of microbes and other compounds found in soil-grown plants and are protected from climatic changes (hussain et al., 2012) . other than plant tissue culture methods, vertical farming units (vfus) can be promising for the controlled and monitored exponential production of the target crop that prevents the cross-pollination of the target crop with genetically compatible species (ma et al., 2005; buyel et al., 2017; buyel, 2018) . not only for growing and harvesting the plants, gmps must be followed for the extraction and purification of the pure and homogenous phytocompound from the harvested biomass. this downstream processing of phytocompounds may account for approximately 80% of the total production cost depending on the removal of the contaminants and purity of the extracted compound (ma et al., 2005; fischer et al., 2012) . moreover, the chances of success of natural anti-viral products may largely increase if they are prepared in line with ethnopharmacological guidelines. the proper application of in silico and in vitro methods followed by in vivo experiments may smooth the way for clinical trials of phytocompounds. more than a hundred phytocompounds have been found to inhibit different types of coronaviruses either by inhibiting the interaction of the sars-cov (s) protein and the ace2 receptor or by inhibiting the viral replication, cell division, 3cl protease, papain-like protease (pl pro) or by hindering the viral entry (islam et al., 2020) . it should be noted that for sars-cov that emerged in 2003, it was not until 2017 when in vitro studies confirming the action mechanism of natural products were conducted schwarz et al., 2014; park et al., 2017) ; however, the natural products with the same mechanism against sars-cov and even more effective mechanisms were already identified in the initial years of the sars-cov epidemic. if more efforts had been given to those phyto-resources from the start of the sars-cov epidemic, they could have reached successful clinical trials. moreover, the inability of sars-cov phyto-inhibitors-based research to reach an extensive level of in vivo studies reduced the chances of phyto-anti-cov drugs being developed and passing successfully through clinical trials. the emergency sars-cov-2 outbreak led to the utilization of several phytocompounds for the treatment of patients. the direct benefit of most of these compounds is that their effects on the human body and their safety are already established through clinical trials for other diseases. however, utmost care is required while prescribing the dose of these phytocompounds as their uncontrolled use can have long term side-effects on the patient's body. though numerous phytoconstituents were found to be effective against sars and mers covs, they fell behind in the drug development process as most of them could not reach the clinical trial stage. one of the possible reasons for incomplete clinical trials could be the intermittent nature of the sars and mers epidemics. the absence of patients for the advanced phase trials (phase ii, iii, iv) of phytoconstituents may have contributed to their failure as licensed drugs. thus, one of the potential suggestions for covid-19 recovery could be to identify the potential phytoconstituents based on in vitro results and with minimum side effects in phase i trials and take them up to advanced phase trials (phase ii, iii, iv) as soon as possible (figure 1 ). so that, in case the covid-19 pandemic ends abruptly by chance like sars-cov, then we can have at least a few efficient phyto-anti-covid drugs that would have completed randomized clinical trials. such drugs may not only be effective on re-emergence of sars-cov-2 in the coming years but can also be potent against similar viral respiratory outbreaks. moreover, creating an effective phyto-anti-covid drug during this pandemic may provide an idea on the duration and the strategy required for the development of potent plant-based therapeutics in case of such random viral outbreaks (figure 1) . we as plant biologists need to be more concerned and vigilant about the status of the plants, their extracts, and phytoconstituents in developing phyto-anti-viral drugs and controlling pandemics like covid-19. it is as crucial as the production of important cereals in the world. ap and mkk conceived, wrote, and edited the manuscript. mh and sg made intellectual contributions to the manuscript. all authors contributed to the article and approved the submitted version. moroccan medicinal plants as inhibitors of covid-19: computational investigations antiviral activity of resveratrol against human and animal viruses antiviral properties of extract of opuntia streptacantha phyllanthus niruri versus placebo for chronic hepatitis b virus infection: a randomized controlled trial investigation of stilbenoids as potential therapeutic agents for rotavirus gastroenteritis clinical features and short-term outcomes of 144 patients with sars in the greater toronto area effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection application of phytochemicals in pharmaceuticals very-large-scale production of antibodies in plants: the biologization of manufacturing plants as sources of natural and recombinant anti-cancer agents genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan the action of plant extracts on a bacteriophage of pseudomonas pyocyanea and on influenza a virus inhibition of sars-cov 3c-like protease activity by theaflavin-3,3'-digallate (tf3). evid. based binding interaction of quercetin-3-b-galactoside and its synthetic derivatives with sars-cov 3clpro: structure-activity relationship studies reveal salient pharmacophore features a novel combination of vitamin c, curcumin and glycyrrhizic acid potentially regulates immune and inflammatory response associated with coronavirus infections: a perspective from system biology analysis adverse effects of ribavirin and outcome in severe acute respiratory syndrome: experience in two medical centers remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro safety and pharmacokinetics of single doses of (+)-calanolide a, a novel, naturally occurring nonnucleoside reverse transcriptase inhibitor origin and evolution of pathogenic coronaviruses sars and mers: recent insights into emerging coronaviruses coronaviruses resistant to a 3c-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance gmp issues for recombinant plant-derived pharmaceutical proteins human coronavirus infections in israel: epidemiology, clinical symptoms and summer seasonality of hcov-hku1 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies return of the coronavirus: 2019-ncov epidemiological findings from a retrospective investigation antiviral activity of glycyrrhizic acid derivatives against sars-coronavirus inhibition of hepatitis b virus production by boehmeria nivea root extract in hepg2 2.2. 15 cells current approaches toward production of secondary plant metabolites medicinal plants: a repository of antiviral metabolites cyclotides as natural anti-hiv agents natural products and their derivatives against coronavirus: a review of the non-clinical and pre-clinical data novel antiviral agents: a medicinal plant perspective induction of inducible nitric oxide synthase expression by 18b-glycyrrhetinic acid in macrophages potential inhibitor of covid-19 main protease (m pro ) from several medicinal plant compounds by molecular docking study. preprints in vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, cimicifuga rhizoma, meliae cortex, coptidis rhizoma, and phellodendron cortex phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia safe, high-throughput screening of natural compounds of mers-cov entry inhibitors using a pseudovirus expressing mers-cov spike protein anti-hiv, anti-poxvirus, and anti-sars activity of a nontoxic, acidic plant extract from the trifollium species secomet-v/anti-vac suggests that it contains a novel broad-spectrum antiviral a novel coronavirus associated with severe acute respiratory syndrome natural products as anti-hiv agents and role in hiv-associated neurocognitive disorders (hand): a brief overview immunomodulatory and anti-sars activities of houttuynia cordata clinicaltrials.gov, study on the impact of triptolide woldifiion on hiv-1 reservoir in acute hiv-1 infection. available at: clinicaltrials.gov identification of natural compounds with antiviral activities against sars-associated coronavirus anti-sars coronavirus 3c-like protease effects of isatis indigotica root and plantderived phenolic compounds effective inhibition of mers-cov infection by resveratrol plant-derived pharmaceuticals -the road forward a sars-like cluster of circulating bat coronaviruses shows potential for human emergence herb-drug interaction between echinacea purpurea and darunavir-ritonavir in hiv-infected patients broad-spectrum antiviral activity of the eif4a inhibitor silvestrol against corona-and picornaviruses pharmacokinetic study of omacetaxine mepesuccinate administered subcutaneously to patients with advanced solid and hematologic tumors in vitro antiviral activity of twenty-seven medicinal plant extracts from southwest nigeria against three serotypes of echoviruses tanshinones as selective and slow-binding inhibitors for sars-cov cysteine proteases dieckol, a sars-cov 3cl(pro) inhibitor, isolated from the edible brown algae ecklonia cava evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors outcome reporting from protocols of clinical trials of coronavirus disease 2019 (covid-19): a review screening of nepalese medicinal plants for antiviral activity drug levels: therapeutic and toxic serum/plasma concentrations of common drugs what does plant-based vaccine technology offer to the fight against covid-19? vaccines 8 biflavonoids from torreya nucifera displaying sars-cov 3clpro inhibition kaempferol derivatives as antiviral drugs against the 3a channel protein of coronavirus in vitro anti-enteroviral activity of stilbenoids isolated from the leaves of macaranga barteri natural products as promising therapeutics for treatment of influenza disease highthroughput screening and identification of potent broad-spectrum inhibitors of coronaviruses the role of phytoengineering in the preparation and production of herbal medicines papain-like protease (plpro) inhibitory effects of cinnamic amides from tribulus terrestris fruits structural basis of sars-cov-2 3cl(pro) and anti-covid-19 drug discovery from medicinal plants non-nucleoside reverse transcriptase inhibitors: a review on pharmacokinetics, pharmacodynamics, safety and tolerability ribavirin in the treatment of severe acute respiratory syndrome (sars) herbal medicines for sexually transmitted diseases and aids antiviral peptides as promising therapeutic drugs basic virology (p. 656) sars-cov infection in a restaurant from palm civet coronavirus disease 2019 (covid-19): situation report, 185. world health organization phytomedicines for corona virus outbreaks frontiers in plant science | www small molecules targeting severe acute respiratory syndrome human coronavirus systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 attenuating effect of ginsenoside rb1 on lps-induced lung injury in rats isolation of a novel coronavirus from a man with pneumonia in saudi arabia in silico screening of chinese herbal medicines with the potential to directly inhibit 2019 novel coronavirus crystal structure of sars-cov-2 main protease provides a basis for design of improved a-ketoamide inhibitors epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china a pneumonia outbreak associated with a new coronavirus of probable bat origin key: cord-309089-ex9nh1yi authors: coperchini, francesca; chiovato, luca; croce, laura; magri, flavia; rotondi, mario title: the cytokine storm in covid-19: an overview of the involvement of the chemokine/chemokine-receptor system date: 2020-05-11 journal: cytokine growth factor rev doi: 10.1016/j.cytogfr.2020.05.003 sha: doc_id: 309089 cord_uid: ex9nh1yi in 2019-2020 a new coronavirus named sars-cov-2 was identified as the causative agent of a several acute respiratory infection named covid-19, which is causing a worldwide pandemic. there are still many unresolved questions regarding the pathogenesis of this disease and especially the reasons underlying the extremely different clinical course, ranging from asymptomatic forms to severe manifestations, including the acute respiratory distress syndrome (ards). sars-cov-2 showed phylogenetic similarities to both sars-cov and mers-cov viruses, and some of the clinical features are shared between covid-19 and previously identified beta-coronavirus infections. available evidence indicate that the so called “cytokine storm” an uncontrolled over-production of soluble markers of inflammation which, in turn, sustain an aberrant systemic inflammatory response, is a major responsible for the occurrence of ards. chemokines are low molecular weight proteins with powerful chemoattractant activity which play a role in the immune cell recruitment during inflammation. this review will be aimed at providing an overview of the current knowledge on the involvement of the chemokine/chemokine-receptor system in the cytokine storm related to sars-cov-2 infection. basic and clinical evidences obtained from previous sars and mers epidemics and available data from covid-19 will be taken into account. in early december 2019, several pneumonia cases of unknown origin were observed in wuhan (china). the pathogen was identified as a novel enveloped rna β coronavirus that was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [4] . the virus showed phylogenetic similarities to both sars-cov and mers-cov viruses. in view of its similarities to bat coronaviruses, it was postulated that bats could have been the primary hosts of sars-cov-2. this hypothesis suggested that the infection originated via transmission from wild animals illegally sold in the huanan seafood wholesale market. on january, 30 th , 2020, the world health organization (who) declared coronavirus disease 2019 (covid19) a public health emergency of international concern, and on march 11 th , who director general referred to covid-19 as a pandemic. as of may 5 th , 2020, the number of confirmed cases of covid-19 has exceeded 3 million worldwide, with more than 250,000 covid-19-related deaths. the epidemic has put public health systems under severe strain both in western countries and in the developing world. sars-cov-2 displays a more efficient transmission pattern when compared with sars-cov and mers-cov [5] , retaining a high transmission rate also in the asymptomatic incubation period [6] . the clinical spectrum of covid-19 syndrome varies remarkably, going from asymptomatic forms to acute bilateral pneumonias requiring hospitalization. common presenting symptoms include fever, fatigue and dry cough, while laboratory tests often show lymphopenia and elevated lactate dehydrogenase levels. chest computed tomographic scans show a typical pattern of bilateral patchy shadows or ground glass opacity. a significant percentage of cases requires admission to intensive-care-units (icu) due to acute respiratory distress syndrome that requires mechanical ventilation support. a subgroup of patients with severe covid-19 can experience the so-called "cytokine storm syndrome", characterized by a fulminant and fatal hyper-cytokinemia associated with multi-organ failure. the term "cytokine storm" has become increasingly used not only by authors of scientific articles but also by popular media. it is likely that the widespread use of this term is related with its rather immediate meaning, which actually recalls the role of the immune system in producing an uncontrolled and generalized inflammatory response [7] . it seems not casual that the term cytokine storm was first employed in describing j o u r n a l p r e -p r o o f the events modulating the onset of the graft-versus-host disease [8] , a condition characterized by an impressively powerful activation of the immune system. cytokine storms characterize a wide spectrum of infectious and non-infectious diseases, and since 2005, it was associated to the avian h5n1 influenza virus infection [9] . apart from the immediate significance of the term cytokine storm, the biological and clinical consequences of this immune system hyperactivity are by far less known, making it worthwhile to be briefly overviewed. there are several similarities in the clinical features between covid-19 and previously identified beta-coronavirus infections. shared clinical findings include that most patients present with fever, dry cough, dyspnea, and bilateral ground-glass opacities on chest ct scans [10] . however, the physiopathology of the mechanisms through which sars-cov or mers-cov sustain high pathogenicity are yet to be completely unveiled. since the first reports on covid-19 disease, it appeared clear that acute respiratory distress syndrome (ards) accounted for a significant number of deaths among infected patients and that ards should be regarded as the hallmark immune-mediated clinical consequence in sars-cov-2, similarly to what described for sars-cov and mers-cov infections [11] . acute respiratory distress syndrome (ards) is a devastating event, with an estimated mortality of approximately 40%, defined as the presence of bilateral lung infiltrates and severe hypoxemia. ards can occur in a variety of clinical situations, including pneumonia, sepsis, pancreatitis, blood transfusion. ards pathogenesis involves inflammatory injury to the alveolo-capillary membrane, which results in increased lung permeability and the exudation of protein-rich pulmonary edema fluid into the airspaces, leading in the end to respiratory insufficiency [12] . as shown by previous data in the literature, increased circulating levels of pro-inflammatory cytokines (eg, interferon γ, interleukin (il-) 1b, il-6, il-12) and chemokines (cxcl10, and ccl2) are associated with pulmonary inflammation and extensive lung involvement in sars patients, similarly to what happens in mers-cov infection [13] . as far as covid 19 infection is concerned, huang et al recently reported that infected patients also show high levels of pro-inflammatory cytokines and chemokines [4] . the demonstration of increased levels of il-1b, ifnγ, cxcl10, and ccl2 strongly pointed toward an activation of t-helper-1 (th1) cell function. more importantly, the so called "cytokine storm" emerged as a main factor driving a more j o u r n a l p r e -p r o o f severe clinical course. this concept originated from the observation that covid-19 patients requiring icu admission displayed higher concentrations of cxcl10, ccl2 and tnfα as compared to those in which the infection was less severe and did not require an icu admission. to further complicate the issue, it should be highlighted that, in patients with sars-cov-2 infection, at difference from sars-cov infection, there is also an increased secretion th2-immune-oriented cytokines such as il-4 and il-10, whose main effect is to suppress inflammation [14] . taken together, these data clearly indicate that, in sars-cov infection, ards is the ultimate result of a cytokine storm. in this scenario, the release by immune effector cells of large amounts of proinflammatory cytokines (ifnα, ifnγ, il-1β, il-6, il-12, il-18, il-33, tnfα, tgfβ) and chemokines (cxcl10, cxcl8, cxcl9, ccl2, ccl3, ccl5) precipitates and sustains the aberrant systemic inflammatory response [4, 13, 15, 16] . the cytokine storm is readily followed by the immune system "attacking" the body, which in turn will cause ards and multiple organ failure, the final result being death, at least in the most severe cases of sars-cov-2 infection [11] . the cytokine storm, and the consequent ards, results from the effects of a combination of many immune-active molecules. interferons, interleukins, chemokines, colony-stimulating factors and tnf-alpha represent the main components involved in the development of the cytokine storm and will be briefly overviewed. -interferons, a family of cytokines with a central role in virus-directed innate immunity binds specific receptors and result in the expression of genes encoding protein with anti-viral or immunomodulatory properties. this sequence of events supported the therapeutic use of ifns in some viral diseases such as chronic hepatitis, but also in non-viral conditions including leukemia and lymphoma, melanoma and multiple sclerosis [17, 18] . -tumor necrosis factor α (tnfα) is a pyrogen cytokine released from immune cells in the acute phase of inflammation and infection. it is a central cytokine in viral diseases and is associated with a number of chronic inflammatory and autoimmune diseases [19] . j o u r n a l p r e -p r o o f 7 -colony-stimulating factors (csf). these proteins are associated with inflammatory conditions and are components of an amplification cascade which ultimately increases cytokine production by macrophages at sites of inflammation, this effect perpetuates the inflammatory reaction [20] . -interleukins are a family of cytokines involved in immune cells differentiation and activation. they mediate the traffic of immune cells to the site of the infection, induce the increase of the acute phase signaling, activate epithelial cells and mediate the production of secondary cytokines [21] . among them, interleukin-6 (il-6) deserves a more extensive discussion in view of its involvement in the coronavirus-induced cytokine storm. il-6 is crucially involved in acute inflammation due to its role in regulating the acute phase response [21] . it is produced by almost all stromal cells and b lymphocytes, t lymphocytes, macrophages, monocytes, dendritic cells, mast cells and other non-lymphocytic cells, such as fibroblasts, endothelial cells, keratinocytes, glomerular mesangial cells and tumor cells [22] . the production of this cytokine is increased by il-1β and tumor necrosis factor (tnf-α) [23] . il-6 may also be responsible for the activation of t helper 17 (th17) cells in the dendritic cell-t cell interaction [24] . in covid-19 affected patients, a high th17 cells activation could result from a virus-driven increased production of il-6 by the immune system. il-6 plays a key role in the pathogenesis of the cytokine storm owing to its pleiotropic properties. several studies showed that the serum levels of il-6 are increased in covid-19 patients and that its circulating levels are positively related to disease severity [14, 25, 26] . for this reason, high serum il-6 levels were suggested as predictors for disease severity [27, 28] . indeed, in animal models of sars-cov infection, the inhibition of the transcription factor of il-6 and, in turn of its production, was associated with reduced mortality [29] . during the present covid-19 pandemic, the use of tocilizumab as a therapeutic agent was proposed. tocilizumab is a humanized anti-il-6 receptor igg1 monoclonal antibody used for the treatment of rheumatoid arthritis and other chronic inflammatory diseases [14] . by blocking the il-6-receptor interaction, tocilizumab inhibits the il-6-mediated signal transduction. although clinical data on the use of tocilizumab in covid-19 patients derive from small series, some authors recommend its use in critically ill covid-19 patients with significantly elevated il-6 levels. [14] -chemokines are a large family of cytokines characterized by a powerful chemotactic effect. chemokines j o u r n a l p r e -p r o o f act as chemo-attractants in the migration of the immune system cells, but they are also involved in several other processes including the development and function of innate and adaptive immune system, embryogenesis, and cancer metastasis [30, 31] . they are promptly secreted by a variety of cells in response to viral or microbial infections [32] . chemokines act as powerful chemoattractants which recruit inflammatory cells to migrate from the intravascular space across the endothelium and epithelium into the inflammation site, according to a chemokine gradient [33] . the role of one specific chemokine, cxcl10 (previously referred to as interferon-γ inducible protein of 10 kda, or ip-10), has been highlighted in ards in both experimental models and in patients. indeed, in a mouse model of il-2 -induced ards, an up-regulation of the mouse cxcl10 analogue mob-1 mrna was observed at initiation of lung injury [34] . several studies also showed that the intratracheal injection of mob-1 in mice induced pulmonary migration of leukocytes in the alveolar space, with massive recruitment of neutrophils, especially monocytes. this event was rapidly followed by microvascular injury and pulmonary edema typical of ards [35, 36] . cxcl10 signaling appears to be a critical factor for the onset of ards, as shown in mice models of ards induced by either acid aspiration or by viral infection (with influenza h5n1 virus). briefly, ichikawa et al, demonstrated that wild-type mice developing ards had increased levels of cxcl10 mainly due to an increased secretion by infiltrating neutrophils, which induced an autocrine loop mechanism on the chemotaxis of inflamed neutrophils, leading to fulminant pulmonary inflammation. on the contrary, cxcl10 and/or its receptor cxcr3 knock-out mice showed decreased lung injury severity and increased survival in response to both viral and non-viral lung injury [37] . moreover, cxcl10 expression in the lung was significantly up-regulated after induction of ards with lipopolysaccharide (lps) in a mouse model of lung injury, and the neutralization of cxcl10 with anti-cxcl10 antibody lead to amelioration of lung injury [38] . cxcl8 (also referred at as il-8) is another chemokine considered as a potential prognostic bio-marker for ards clinical course [39] . indeed, cxcl8 levels were found to be elevated both in plasma [40] [41] [42] [43] and in the broncho-alveolar lavage fluid [44] [45] [46] of patients with ards. a direct role of cxcl8 in the progression of ards was proven in rabbit with acid-induced ards lead to a 10-fold increase in cxcl8 levels in the j o u r n a l p r e -p r o o f alveolar fluids. of note, pre-treatment with an anti-cxcl8 antibody prevented the development of the typical acute lung injury [47] . although chemokines are crucially involved in the regulation and maintenance of immune responses, their role in the onset of the coronavirus-induced cytokine storm is still poorly investigated. chemokines are a family of low molecular weight proteins expressed, both constitutively and in an inducible manner, by several types of cells. chemokines play an important role in the inflammatory response by attracting leukocytes to sites of infection. these small proteins also contribute to the homeostatic circulation of leukocytes through tissues [31] . at present, 50 chemokines and 20 chemokine receptors have been recognized and classified. chemokines are named according to the most recent nomenclature, which classifies them according to their chemical structure, the c, cc cxc and cx3c families [48] . the binding of chemokines to their receptors is responsible for their chemoattractant ability. the chemokine receptors belong to the seven-transmembrane-spanning, g-protein-coupled receptors, which are expressed primarily on leukocytes but also on other cells, e.g., endothelial cells [49] . the many functions of chemokines include the control of cell proliferation and differentiation, the regulation of angiogenesis and immune and inflammatory responses, tumor growth and metastasis [50] [51] [52] . most recently, several studies investigated the involvement of chemokines in coronavirus-related infective disease. it emerged that specific chemokines could play a crucial role in the development of covid-19-related symptoms, thus confirming what previously known for other types of coronaviruses, such as sars and mers. [13, 53] . these findings could be somehow expected in view of the well-known role of chemokines in viral infections. before addressing the specific relationship between chemokines and coronavirus infections, it is mandatory to briefly overview the general role of chemokines in viral infections and how viruses contrast the actions of chemokines. viruses are infectious agents of small size and simple composition that can multiply only in living cells of animals, plants, or bacteria. all viruses contain a nucleic acid, either -dna (deoxyribonucleic acid) or -rna (ribonucleic acid), and several proteins. viruses should not even be considered organisms since they are not free-living (i.e., they require a host cell), thus viruses need to elude the host immune defense to infect its cells in order to reproduce and survive. [54] . the chemokine/chemokine receptor-related immune defenses are the main obstacles to be by-passed by viruses. some chemokines play a direct anti-viral effect by inducing an array of phenomena that lead cells to determine an "anti-viral "state. these phenomena include activation of apoptosis or direct killing of infected cells by activated immune cells. chemokines also recruit immune cells to the site of infection, which will fight against the intruder [55] . viral infections are associated with enhanced expression of several chemokines, in particular the interferons-inducible ones. interferons, which can be produced by any mammalian cell, are involved in the rapid and efficient host innate response against viruses. a powerful ifn response triggered by the first contact with a virus can slow down viral multiplication and "buy time" for the organism to establish a more efficient adaptive immune response [56] . ifns can stimulate surrounding cells to express potent antiviral proteins including enzymes, transcription factors, cell surface glycoproteins, cytokines and chemokines [57, 58] . moreover, they can inhibit cell proliferation, regulate apoptosis and modulate the immune response [59] . among interferons-induced molecules, the chemokine cxcl10 is currently regarded as a main player in the organism anti-viral response [60] , and particularly in respiratory tract infections. several studies demonstrated that cxcl10 levels, as evaluated in serum, bronchial-alveolar washing fluid or nasal secretions, consistently correlate with the severity and duration of acute respiratory tract infection due to viral infections. [60] [61] [62] also the chemokine cxcl8, is involved in inflammation and immune cell trafficking in the context of viral infections. cxcl8 plays a major role in the initial control of respiratory tract infection due to its chemotactic activity for neutrophils and monocytes [63] . cxcl8 levels in the nasal washing fluid correlate with symptoms severity during acute respiratory tract infections [64] . although in the majority of cases a strong chemokine action can efficiently contrast viral infections, some viruses acquire the capacity of escaping j o u r n a l p r e -p r o o f this surveillance system. furthermore, viruses can use the chemokine system network for their own favor by several strategies: -some viruses "mimic" the components of the chemokine system by producing molecules that are very similar to chemokines and can interact with their receptor. these molecules generate an incongruous signal leading to a disorganized immune response to viruses [65] . -inhibition of the interferon-induced anti-viral response. several viruses do impair the intracellular receptors devoted to pathogen recognition, such as toll like receptors and intracellular rna sensors. [66] taken together, the above data indicate that viruses can interfere with the chemokine/chemokine-receptors system using their own properties to modify intracellular signaling with the final result to further disseminate the infection. the strict relationship between conaviruses infection and chemokines has been thoroughly investigated during both the sars-cov and the mers-cov epidemics, while some initial data are available regarding sars-cov-2 and its related syndrome, covid-19. since the first reports of sars, it seemed clear that the severe clinical manifestations of the disease could not be ascribed only to the viral activity per se, but that an immune-mediated mechanism rather than a direct virus-induced damage would drive the clinical progression [67] . indeed from the physio-pathology point of view, the most interesting observation was the demonstration that viral titers seemed to paradoxically diminish during the most severe phase of the disease both in humans and in several animal models [1] . data in vivo studies showed that several circulating chemokines (cxcl8, ccl2 and cxcl10) and inflammatory cytokines (il-1, il-6 and il-12) were elevated in patients with sars-cov [68, 69] . cxcl10 was also considered an excellent prognostic marker for sars disease progression [70, 71] . in particular, jiang et al showed that cxcl 10 serum levels were significantly increased during the early stage of sars, and remained elevated until resolution. moreover, persistently elevated cxcl10 serum levels during follow-up were predictive of a worse outcome of the infection [71] these findings prompted further in vitro studies aimed at investigating the relationship between sars and the chemokine system. spiegel et al, demonstrated that, in addition to its direct effect on epithelial lung cells, sars-cov could also enter macrophages and dendritic cells [72] . this appeared crucial as viral entrance in these cells lead to an abortive infection (e.g. the virus enters the host-cell but cannot successfully complete replication). yet the virus elicited the secretion of pro-inflammatory chemokines by dendritic and macrophages cells [73] . this finding was confirmed in vivo because the serum levels of a wide spectrum of cytokines and chemokines produced by dendritic cells and macrophages were elevated in sars-cov infected patients [74] . furthermore, the infection with sars-cov of human primary myeloid-derived dendritic cells was followed by an impaired defensive ifnβ response, which was paralleled by a moderate up-regulation of pro inflammatory cytokines (such as tnf-α and il-6) and a much more significant up-regulation of inflammatory chemokines (such as cxcl10, ccl3, ccl5, and ccl2) [75] . the authors suggested that the lack of response to antiviral interferons in the presence of chemokine up-regulation could represent a further mechanism of immune evasion by sars-cov [73] . in line with this hypothesis, the direct exposure of lung epithelial cells [76] or peripheral blood mononuclear cells (pbmcs) [75] coming from sars infected patients to viral proteins (such as s-protein and n-protein) induced a prompt release of several chemokines, including cxcl8 and cxcl10. in vitro gene-expression studies also reported that pbmc from normal healthy donors inoculated with sars-cov showed an early enhancement in the expression of several chemokines belonging both to the cc family (ccl4, ccl20, ccl22, ccl25, ccl27, and their receptors j o u r n a l p r e -p r o o f ccr4, ccr7) and of the cxcl family (cxcl8 and il-17) [77] . additional data came from animal models of sars-cov infection. in mice infected with sars-cov, the clinical features of the syndrome showed an age-dependent increase in severity (similarly to what observed in humans), which was related to an increased level of pro-inflammatory cytokines and chemokines, paralleled by a reduction in t-cell responses [78] . another study showed that in mice infected with sars-cov, robust virus replication accompanied by delayed type i interferon secretion caused a rapidly fatal pneumonia. this delayed type i-interferon signaling promoted the accumulation of pathogenic inflammatory monocyte-macrophages, with resulting increase in cytokine (il-6) and chemokine (ccl2) lung levels, vascular leakage, and impaired virus-specific t cell responses. [79] . these data suggest that coronaviruses, and in particular sars-cov, have a peculiar ability to counteract the antiviral ifn response, pointing toward the fact that the severity of disease might be due to immune dysregulation, rather than to the level of viremia. this dysregulation would be characterized by an insufficient type i interferon response (too little and too late), paralleled by an aberrant pro-inflammatory chemokine secretion by alveolar macrophages, dendritic cells and pneumocytes [66, 80] . in vitro data suggest that this class of viruses, and in particular sars-cov, uses several strategies to avoid type i ifn response, both passive and active [56, 59] . -passive mechanisms include the induction of double membrane vesicles (dmv) at perinuclear sites within the cytoplasm where rna synthesis takes place. this strategy may help to hide and protect rna replication intermediates from being sensed by intracellular rna-sensors, thus avoiding the activation of the ifn cascade [81, 82] . -active mechanisms include a direct action of viral proteins on transcription factors and intracellular signaling molecules that regulate the ifn cascade. in particular, the sars-cov protein orf6 is able to inhibit the action of interferon regulatory transcription factor-3 (irf-3), a transcription factor of the ifn genes [83, 84] . the fact that sars-cov infection would upregulate the transcription of cxcl10, while significantly down-regulating ifns signaling, could seem paradoxical. however, transcriptional enhancement of cxcl10 could be due to a direct effect on the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) [85] triggered by sars-cov [86] , even if other authors did not confirm this early observation [87] . similarly, the up-regulation of the cxcl8 gene expression, could be due to a direct effect of the virus at the cellular level. indeed, intestinal and lung cells lines infected by sars-cov, promptly increase their secretion of cxcl8 [88] . this observation would fit with the notion that the expression of cxcl8 is dependent on the transcription factor activator protein 1 (ap-1), which was shown to be strongly upregulated by sars-cov [86, 89] . during the mers-cov epidemics, several studies were aimed at understanding the pathogenic mechanisms underlying the severe and often fatal pneumonia were performed. available data suggest that mers-cov infection shares some immunological aspects of sars-cov infection, in terms of involvement of the chemokine system. first, an increase in the serum levels of cxcl10 when compared to controls was observed also in mers-cov patients. more importantly, a persistent cxcl10 increase was associated with disease severity [90] . in this regard, the case of two paradigmatic patients diagnosed with mers-cov is worth noting. one had a fatal outcome and experienced an impaired ifn response together with a relevant increase in serum cxcl10 levels. the other one, with a favorable outcome, displayed an up-regulation of ifns and irf3 and a less pronounced increase of serum cxcl10 levels [91] . in vitro studies also support the relevance of chemokines in mers-cov patients. a 2013 study evaluated the expression of several chemokines and cytokines in cell lysates of polarized airway epithelial cells infected with mers-cov or sars-cov. the results showed that cxcl8 was up-regulated to a greater extent by mers-cov infection as compared with sars-cov. at difference, ccl2 and cxcl10 were more strongly up-regulated by sars-cov than mers-cov [92] . in addition, in dendritic cells infected with mers-cov, a significant down-regulation of ifn response paralleled by a striking elevation of cxcl10 was observed [93] . a strong induction of cxcl10 secretion was observed also in monocyte-derived macrophages infected with mers-cov [94, 95] . interesting data come also from an experimental mouse model of mers-cov infection, in which a significant increase in cxcl8 expression was observed in lung and brain tissue after infection with mers-cov [96] . briefly, the inoculation of the two viruses in ex vivo human lung tissue explants showed that sars-cov-2 was more capable, as compared with sars-cov, in both infecting and replicating in human lung. furthermore, sars-cov-2 infection was less able to trigger the expression of any ifns, suggesting that sars-cov and sars-cov-2 might differ in their ability to modulate the production of pro-inflammatory cytokines and chemokines. as an example, it could be worth highlighting that sars-cov infection upregulated 11 out of the 13 pro-inflammatory factors evaluated, while sars-cov-2 upregulated only five of them, (namely, cxcl10, il6, ccl2, cxcl1, cxcl5). interestingly, cxcl8 transcription was upregulated only by sars-cov, but not sars-cov-2 infection, while the opposite was observed for cxcl10 [97] . the potential involvement of the chemokine system during sars-cov-2 infection was already evident from the first covid-19 series described by chinese physicians in early january 2020. it was reported that several pro-inflammatory cytochines and chemokines, including cxcl10, cxcl8, ccl2, tnfα and ifnγ j o u r n a l p r e -p r o o f were higher in the plasma of covid-19 patients as compared to healthy controls. more importantly, among infected patients, cxcl10, ccl2 and tnfα circulating concentrations (but not those of ifnγ) were found to be significantly higher in patients requiring admission to intensive care units as compared to patients experiencing a less severe clinical course [4] . j o u r n a l p r e -p r o o f sars-cov-2, and its related syndrome covid-19, have been known to the scientific community since less than 5 months. clearly much is yet to be understood, and the challenge for the next future will be to increase our understanding the physiopathology of this novel infectious disease. hopefully, the advances in our comprehension of the mechanisms sustaining the clinical course and patients-related factors driving the final outcome will be helpful in developing effective preventive strategies and/or therapeutical options. based on current knowledge, the "cytokine storm" appears as one of the most dangerous and potentially lifethreatening event related to covid-19 sustaining its major clinical consequences. the immune mediated events related to the response to sars-cov-2 infection, and the role of the chemokine/chemokine receptor system, will be further and more extensively characterized with the final goal to identify targeted therapeutic strategies. although lessons from the previous sars and mers epidemics can be drawn, there is still much to do in order to conclude whether sars-cov-2 virus behaves in the same way of its predecessors or if it is characterized by peculiar specificities. clearly, the hide-and-seek challenge between the virus and our immune defenses will also help us understanding the extremely variable spectrum of clinical manifestations of covid-19, which appears to range between asymptomatic cases to possibly lethal bilateral pneumonia with multi-organ failure. coronaviruses: an overview of their replication and pathogenesis the severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features of patients infected with 2019 novel coronavirus in the reproductive number of covid-19 is higher compared to sars coronavirus the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application into the eye of the cytokine storm cytokine storm of graft-versus-host disease: a critical effector role for interleukin-1 human infection by avian influenza a h5n1 pathological findings of covid-19 associated with acute respiratory distress syndrome role of chemokines in the pathogenesis of acute lung injury pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality human immunopathogenesis of severe acute respiratory syndrome (sars) the mercurial nature of neutrophils: still an enigma in ards? clinical uses of interferons interferons at age 50: past, current and future impact on biomedicine an endotoxin-induced serum factor that causes necrosis of tumors colony-stimulating factors in inflammation and autoimmunity evolutionary divergence and functions of the human interleukin (il) gene family interleukin-6 and its receptor: from bench to bedside il-6 as a keystone cytokine in health and disease il-6: regulator of treg/th17 balance the role of cytokines including interleukin-6 covid-19 induced pneumonia and macrophage activation syndrome-like disease hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a meta-analysis interleukin-6 as a potential biomarker of covid-19 progression inhibition of nf-κb-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival role of chemokines in thyroid cancermicroenvironment: is cxcl8 the main player? role of chemokines in endocrine autoimmune diseases chemokine receptors: interaction with hiv-1 and viral-encoded chemokines transepithelial migration of neutrophils: mechanisms and implications for acute lung injury mob-1 expression in il-2-induced ards: regulation by tnf-alpha the novel chemokine mob-1: involvement in adult respiratory distress syndrome mob-1 and tnf-alpha interact to induce microvascular lung injury neutralization can ameliorate lipopolysaccharide-induced acute respiratory distress syndrome in rats biomarkers for the acute respiratory distress syndrome: how to make the diagnosis more precise acute lung injury in patients with traumatic injuries: utility of a panel of biomarkers for diagnosis and pathogenesis national heart, and lung institute acute respiratory distress syndrome network, use of risk reclassification with multiple biomarkers improves mortality prediction in acute lung injury plasma cytokines il-6, il-8, and il-10 are associated with the development of acute respiratory distress syndrome in patients with severe traumatic brain injury pathogenetic and predictive value of biomarkers in patients with ali and lower severity of illness: results from two clinical trials inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups elevated levels of nap-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality acid aspiration-induced lung injury in rabbits is mediated by interleukin-8-dependent mechanisms chemokine/chemokine receptor nomenclature international union of basic and clinical pharmacology chemokine and chemotactic signals in dendritic cell migration chemokines and t lymphocytes: more than an attraction role of chemokine receptors in thyroid cancer and immunotherapy coronavirus infections and immune responses virus classification -where do you draw the line? expression and function of chemokines during viral infections: from molecular mechanisms to in vivo function the interferon response circuit: induction and suppression by pathogenic viruses the interferon in tlr signaling: more than just antiviral links between innate and adaptive immunity via type i interferon inverse interference: how viruses fight the interferon system serum ifn-γ-induced protein 10 (ip-10) as a biomarker for severity of acute respiratory infection in healthy adults host response cytokine signatures in viral and nonviral acute exacerbations of chronic obstructive pulmonary disease serum ip-10 as a biomarker of human rhinovirus infection at exacerbation of copd the pneumococcal polysaccharide capsule and pneumolysin differentially affect cxcl8 and il-6 release from cells of the upper and lower respiratory tract association of interleukin-8 and neutrophils with nasal symptom severity during acute respiratory infection viral mimicry of cytokines, chemokines and their receptors interferon and cytokine responses to sars-coronavirus infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome what does the peripheral blood tell you in sars? an interferon-gamma-related cytokine storm in sars patients characterization of cytokine/chemokine profiles of severe acute respiratory syndrome interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells pathogenesis of severe acute respiratory syndrome expression profile of immune response genes in patients with severe acute respiratory syndrome induction of il-8 release in lung cells via activator protein-1 by recombinant baculovirus displaying severe acute respiratory syndrome-coronavirus spike proteins: identification of two functional regions a human in vitro model system for investigating genome-wide host responses to sars coronavirus infection age-related increases in pgd(2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice coronavirus replication complex formation utilizes components of cellular autophagy ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists one nucleotide in a kappab site can determine cofactor specificity for nf-kappab dimers infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus sars coronavirus and innate immunity inhibition of cytokine gene expression and induction of chemokine genes in nonlymphatic cells infected with sars coronavirus activation of ap-1 signal transduction pathway by sars coronavirus nucleocapsid protein clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection distinct immune response in two mers-covinfected patients: can we go from bench to bedside? perfluoroalkyl acids: a review of monitoring and toxicological findings productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis severe acute respiratory syndrome and the innate immune responses: modulation of effector cell function without productive infection the characteristics of hdpp4 transgenic mice subjected to aerosol mers coronavirus infection via an animal nose-only exposure device comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 clinical and immunological features of severe and moderate coronavirus disease transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients the laboratory tests and host immunity of covid-19 patients with different severity of illness at present she is attending the phd couse in experimental medicine at the university of pavia and she is researcher at the unit of endocrinology of ics maugeri spa -i.r.c.c.s, pavia. her research fields are: clinics of thyroid disease, role of chemokines in autoimmune endocrine disorders and thyroid cancer at present her position is: associate professor in endocrinology at university of pavia. she works at the unit of internal medicine and endocrinology, ics maugeri spa -i.r.c.c.s, pavia. her main research fields are: physiopathology and clinics of thyroid diseases key: cord-287886-41isp0wj authors: al-tawfiq, jaffar a; kattan, rana f; memish, ziad a title: middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia date: 2016-11-08 journal: world j clin pediatr doi: 10.5409/wjcp.v5.i4.391 sha: doc_id: 287886 cord_uid: 41isp0wj aim: to summarize the reported middle east respiratory syndrome-coronavirus (mers-cov) cases, the associated clinical presentations and the outcomes. methods: we searched the saudi ministry of health website, the world health organization website, and the flutracker website. we also searched medline and pubmed for the keywords: middle east respiratory syndrome-coronavirus, mers-cov in combination with pediatric, children, childhood, infancy and pregnancy from the initial discovery of the virus in 2012 to 2016. the retrieved articles were also read to further find other articles. relevant data were placed into an excel sheet and analyzed accordingly. descriptive analytic statistics were used in the final analysis as deemed necessary. results: from june 2012 to april 19, 2016, there were a total of 31 pediatric mers-cov cases. of these cases 13 (42%) were asymptomatic and the male to female ratio was 1.7:1. the mean age of patients was 9.8 ± 5.4 years. twenty-five (80.6%) of the cases were reported from the kingdom of saudi arabia. the most common source of infection was household contact (10 of 15 with reported source) and 5 patients acquired infection within a health care facility. using real time reverse transcriptase polymerase chain reaction of pediatric patients revealed that 9 out of 552 (1.6%) was positive in the kingdom of saudi arabia. conclusion: utilizing serology for mers-cov infection in jordan and saudi arabia did not reveal any positive patients. thus, the number of the pediatric mers-cov is low; the exact reason for the low prevalence of the disease in children is not known. middle east respiratory syndrome-coronavirus (mers-cov) was first isolated in 2012 from a patient in the kingdom of saudi arabia (ksa) [1] . as more cases were reported, the case fatality rate changed to 40% from 60% [2] [3] [4] [5] . in addition, initially there was a predominance of males; later this ratio decreased [2, 6] . mers-cov is characterized by three different patterns of disease: sporadic cases, intra-familial transmission [7] [8] [9] and health care associated infection [2, 3, [10] [11] [12] [13] [14] [15] [16] . despite the increased number overtime and the multiple health care associated outbreaks [17] , the number of pediatric cases remained low during the study period [18] . the initial description of 47 cases included only a 14-year-old child [4] . the first pediatric case was a 2-year-old child reported from jeddah, ksa on june 28, 2013 [19] . later an additional three asymptomatic children were reported [4] . the largest report of childhood mers-cov cases included eleven, of which two patients were symptomatic and nine were asymptomatic [18] . the exact reason for this low prevalence of the disease in children is not known. in this study, we summarize the reported mers-cov cases and the associated clinical presentation and the outcome. we searched the saudi ministry of health website [20] , the world health organization website [21] , the flutracker website [22] , the medical literature and the retrieved published studies for any childhood mers-cov infections. we searched medline and pubmed for the keywords middle east respiratory syndrome-coronavirus, mers-cov, in combination with pediatric, children, childhood, infancy and pregnancy from the initial discovery of the virus in june 2012 until april 19, 2016. the retrieved articles were also read to find other relevant articles. relevant data were placed into an excel sheet and analyzed accordingly. descriptive analytic statistics were used in the final analysis as deemed necessary, including mean and standard deviation when applicable and frequency. the statistical review of the study was performed by a biomedical statistician. statistical review is performed before the submission of the manuscript. from june 2012, to april 19, 2016 , there were a total of 31 pediatric mers-cov cases as shown in table 1 . of all the cases, thirteen (13) or 42% were asymptomatic, and there were 17 males, 10 females and 4 unreported (a male to female ratio of 1.7:1). the mean age of patients was 9.8 + 5.4 (0.75-17) years. twenty-five cases (80.6%) were reported from ksa; the other patients were in jordan, united arab emirates and the republic of korea (table 1 ). the most common source of the infection was household contact (10 of 15 with reported source), and 5 patients acquired the infection within a health care facility. about one half of the cases were reported in 2014, and 29% were reported in 2013 and 22.6% in 2015 ( table 2) . screening of pediatric patients for mers-cov infection using real time reverse transcriptase polymerase chain reaction showed that only 9 out of 552 (1.6%) were positive in ksa [23] . however, serologic testing of pediatric patients admitted with lower respiratory tract infection in jordan and saudi arabia revealed no positive tests [24, 25] (table 3) . the effect of mers-cov infection on the fetus was described in eight cases [26] [27] [28] [29] as summarized in table 4 . the mean age of the mothers was 32.25 + 3.4 years, and the mean gestational age was 28.4 + 6.3 wk. death of the fetus was observed in 3 (37.5%) of the 8 fetuses. despite the total number of mers cases increasing, especially in ksa, the number of pediatric cases remained low during the study period. initially, the testing in ksa was directed towards hospitalized patients with severe pneumonia. in 2015, the saudi ministry of health added a specific case definition for mers-cov infection in children [30] . the definition includes those ≤ 14 years, meets the adult case definition and has either a history of exposure to a confirmed or suspected mers-cov in the proceeding 14 d or a history of contact with camels or camel products in the proceeding 14 d [30] . the case definition also includes children with unexplained severe pneumonia [30] . the 2015 change in the case definition does not account for the low rate of childhood mers-cov infection as 33% of the cases were reported in 2014 before the case definition was changed. one of the reasons for an increased number of cases in 2014 during the jeddah outbreak was increased testing of asymptomatic and mildly symptomatic patients [11] . the pattern of mers-cov pediatric cases was similar to the 2003 sars outbreak. children were less affected than adults and children less than 2 years of age had milder disease [31] . in the largest screening of contacts, the rate of mers-cov positive children (1.6%, 9/616) compared to 2.2% (99/4440) in adults (p = 0.23) [23] . thus, in this study utilizing mers-cov pcr the positivity rate did not differ in children and adults. in adults with mers-cov infections, three patterns of transmissions were observed: sporadic (primary) cases presumed to be due to animal exposure (mainly camels), household contacts or health care associated infections [32] . in ksa, the majority (45%) of cases were health care-associated infections, 38% were primary cases, and 13% were household contacts [32] . in contrast, in the majority of pediatric cases that reported source of acquisition (66.7% of the 15 with reported source), the disease was acquired through household contact. this pattern indicates a low exposure of children to animals and a higher rate of health care associated infections in adult wards. the male to female ratio (2.8:1 and 3.3:1) was initially high [3, 4] . this apparent male predominance could be explained by the nature of hospital outbreaks [2] . eventually the male to female ratio was reduced to 1.3:1 to 1.8:1 [5, 6] . consistent with these studies, the male to female ratio in children with mers-cov was 1.7:1 and may indicate similar exposure of children to index cases in the household settings and differential host factors. possible explanations for the lower number of pediatric cases compared to adults include differential testing of adult patients and milder diseases in children; although, serologic testing of pediatric patients in ksa and jordan did not reveal any positive cases [24, 25] . in the largest sero-epidemiologic survey in ksa, the study did not include children and thus it is difficult to establish the rate of sero-positivity in children [31] . the mers-cov infection rate in children remains low and possible explanations include: a milder disease in children, asymptomatic infection, or the presence of yet to be identified factors. the development of a shorter duration of mers in children is another possible explanation. if this is the case, it may limit the development of a positive serology. in one study, delayed antibody responses as measured with the neutralization test was associated with severe diseases [33] . the longevity of antibodies in mers-cov cases might be limited as was the case with sars [33, 34] . the only study of serology among children was done among hospitalized pediatric cases who presented with lower respiratory tract infections [25] . there is no systematic screening of exposed children using serologic testing; this limited the interpretation of available serologic studies. little data also exist regarding the effect and the likelihood of mers-cov in pregnancy. eight cases were reported [26, 27, 29] . the outcome was favorable in the majority of cases. the exact prevalence of mers-cov antibodies and exposure of pregnant women to mers-cov is not known. in conclusion, the number of mers-cov infections in pediatric patients remains low. possible explanations include low exposure, presence of asymptomatic, mildly symptomatic patients or the presence of yet to be identified factors. the middle east respiratory syndrome-coronavirus (mers-cov) was first isolated in 2012 from a patient in the kingdom of saudi arabia (ksa). despite the increased number of mers-cov cases overtime, the number of pediatric cases remained low. the exact reason for this low prevalence of the disease in children is not known. the aim of this study is to summarize the reported mers-cov cases and the associated clinical presentation and the outcome. the first pediatric case was a two-year-old child reported from jeddah, ksa on june 28, 2013. later an additional three asymptomatic children were reported. the largest report of childhood mers-cov cases included eleven, including nine asymptomatic cases. the number of mers-cov infections in pediatric patients remains low. possible explanations include low exposure, presence of asymptomatic, mildly symptomatic patients or the presence of yet to be identified factors. the immune system predisposing to severe disease and to fatal outcome remains unknown. an exploration of the virus-host interaction may add to the understanding of the low prevalence in this age group. despite the low number of pediatric mers-cov cases, it is important to continue to monitor the development of this disease in this age group and to understand the risk factors. mers-cov is a new emerging virus that was first isolated in 2012. this complication of all known pediatric cases is a useful contribution to the medical literature, and knowing it is possible but rare is important. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: epidemiology and disease control measures hospital outbreak of middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study taking stock of the first 133 mers coronavirus cases globally--is the epidemic changing? state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans family cluster of middle east respiratory syndrome coronavirus infections a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study mers-cov outbreak in jeddah--a link to health care facilities an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia an update on middle east respiratory syndrome: 2 years later middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution epidemiological findings from a retrospective investigation the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea microevolution of outbreak-associated middle east respiratory syndrome coronavirus managing mers-cov in the healthcare setting middle east respiratory syndrome coronavirus disease in children mers-cov summary and literature middle east respiratory syndrome coronavirus (mers-cov) case list of moh/who novel coronavirus mers ncov announced cases screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia al abdallat mm. stillbirth during infection with middle east respiratory syndrome coronavirus impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus during pregnancy middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection severe acute respiratory syndrome coronavirus pathogenesis, disease and vaccines: an update drivers of mers-cov transmission: what do we know? kinetics of serologic responses to mers coronavirus infection in humans disappearance of antibodies to sars-associated coronavirus after recovery middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus (mers-cov) update. disease outbreak news key: cord-320709-2pnqpljt authors: munster, vincent j.; adney, danielle r.; van doremalen, neeltje; brown, vienna r.; miazgowicz, kerri l.; milne-price, shauna; bushmaker, trenton; rosenke, rebecca; scott, dana; hawkinson, ann; de wit, emmie; schountz, tony; bowen, richard a. title: replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) date: 2016-02-22 journal: sci rep doi: 10.1038/srep21878 sha: doc_id: 320709 cord_uid: 2pnqpljt the emergence of middle east respiratory syndrome coronavirus (mers-cov) highlights the zoonotic potential of betacoronaviruses. investigations into the origin of mers-cov have focused on two potential reservoirs: bats and camels. here, we investigated the role of bats as a potential reservoir for mers-cov. in vitro, the mers-cov spike glycoprotein interacted with jamaican fruit bat (artibeus jamaicensis) dipeptidyl peptidase 4 (dpp4) receptor and mers-cov replicated efficiently in jamaican fruit bat cells, suggesting there is no restriction at the receptor or cellular level for mers-cov. to shed light on the intrinsic host-virus relationship, we inoculated 10 jamaican fruit bats with mers-cov. although all bats showed evidence of infection, none of the bats showed clinical signs of disease. virus shedding was detected in the respiratory and intestinal tract for up to 9 days. mers-cov replicated transiently in the respiratory and, to a lesser extent, the intestinal tracts and internal organs; with limited histopathological changes observed only in the lungs. analysis of the innate gene expression in the lungs showed a moderate, transient induction of expression. our results indicate that mers-cov maintains the ability to replicate in bats without clinical signs of disease, supporting the general hypothesis of bats as ancestral reservoirs for mers-cov. scientific reports | 6:21878 | doi: 10 .1038/srep21878 arab emirates and egypt 23, [28] [29] [30] . mers-cov sequences and virus isolates obtained from dromedary camels in qatar and the kingdom of saudi arabia showed high sequence identity with those obtained from epidemiologically linked human cases 28, 31 . together, these data suggest that rather than direct zoonotic transmission from a bat reservoir, dromedary camels are involved as the primary reservoir host for mers-cov. phylogenetic and epidemiological data suggest that rather than a single introduction in the human population, mers-cov appears to continue to be transmitted by multiple independent spillover events from dromedary camels 32, 33 . after the emergence of severe acute respiratory syndrome coronavirus (sars-cov) in 2003, the targeted focus on reservoir studies in bats has resulted in a vast increase of our knowledge on the genetic diversity of bat coronaviruses. despite the increase in genetic data on coronavirus diversity in their natural reservoirs, only very limited data are available on the impact of these viruses on the reservoir host and controlled infection experiments with coronaviruses in their reservoir hosts have not been performed. to understand the drivers of mers-cov emergence, a more comprehensive understanding of the interaction between the virus and its natural and intermediate reservoir hosts is needed. here we present data on the first experimental infection of bats with mers-cov to model the infection kinetics in a coronavirus host species, the jamaican fruit bat (artibeus jamaicensis). artibeus jamaicensis dpp4 receptor and cell susceptibility. the mers-cov receptor dpp4 is the main host restriction factor 34 ; therefore, we first studied the interaction between mers-cov and jamaican fruit bat dpp4. the paj-dpp4 plasmid expressing the dpp4 coding sequence of jamaican fruit bat under control of a cmv promoter was transfected into bhk cells, which are not permissive to mers-cov 34 . the expression of aj-dpp4 in transfected cells was confirmed by flow cytometry, showing the presence of bat dpp4 on the surface of transfected bhk cells by determining the increase over untransfected cells ( figure s1 ). transient expression of bat dpp4 in bhk cells supported mers-cov replication, whereas transient expression of hamster dpp4 in bhk cells did not (fig. 1a) . subsequently, the replication kinetics of mers-cov were compared in llc-mk2 cells (macaca mulatta) and jamaican fruit bat primary kidney cells. mers-cov replicated efficiently to high titers in both cell lines (fig. 1b) , indicating that there is no restriction at the receptor or cellular level for mers-cov replication in jamaican fruit bat cells. clinical signs in bats inoculated with mers-cov. ten adult jamaican fruit bats were inoculated via the intranasal and intraperitoneal routes with 10 5 tcid 50 of mers-cov strain emc/2012; 2 mock inoculated jamaican fruit bats, housed in a separate cage, were used as controls, the mock inoculated animals were inoculated with tissue culture medium via the same routes and volumes. the bats were observed at least once daily for signs of disease. bodyweight and temperature were measured throughout the experiment for a maximum of 28 days post inoculation (dpi) for bat 9 and 10 (mers-cov inoculated) and bat 11 and 12 (mock inoculated controls). none of the bats showed signs of disease, weight loss or increased body temperature throughout the experiment ( figure s2 ). to examine mers-cov shedding in inoculated bats, oral and rectal swabs were collected for the duration of the experiment. mers-cov shedding was first detected on 1 dpi, as indicated by the presence of viral rna in throat and rectal swabs and continued for a maximum duration of 9 days. all animals, except bat 10, shed mers-cov from the respiratory tract ( fig. 2a) ; all bats except 4 and 10, shed mers-cov from the intestinal tract (fig. 2b ). viral loads in swabs collected from the respiratory tract were higher than viral loads in swabs from the intestinal tract. tissues collected at the sequential necropsy dates of 2, 4, 7, 14 and 28 dpi were analyzed for the presence of viral rna, infectious virus, and evaluated by histopathology and immunohistochemistry. mers-cov viral rna was detected in various tissues of all inoculated bats, except bat 8 on 14 dpi and bat 9 on 28 dpi. the highest viral loads were detected in the lower respiratory tract (fig. 3) . mers-cov viral rna was detected at 2 dpi in trachea, lung, liver, spleen, bladder and nasal turbinates; at 4 dpi in lung, spleen, duodenum, colon, bladder, turbinates and brain; at 7 dpi in lung, liver, turbinates and brain; at 14 dpi in heart, lung, liver, spleen and duodenum. no mers-cov viral rna was detected at 28 dpi (fig. 3) . in additon, mers-cov viral rna was detected in blood on 2 and 4 dpi, in bats 1-3, indicative of viremia ( figure s3 ). mers-cov mrna was detected in tissues of bats 1 to 7, confirming mers-cov replication on the transcriptional level (table s2) . infectious mers-cov was isolated from the lungs of bats 1 (2 dpi) and 6 (7 dpi), the bladder and nasal turbinates of bat 7 (14 dpi), and the duodenum of bat 10 (28 dpi), indicating active virus replication, mainly in the respiratory tract. only two of ten bats (bat 3 and bat 5) exhibited histopathology associated with mers-cov infection, which was mild. all mers-cov associated lesions were detected in the respiratory tract of the infected bats (fig. 4 , table s1 ). bat 3 and bat 4 (4 dpi) displayed a mild acute rhinitis, but mers-cov replication was not detected by immunohistochemistry (table s1 and s2). bat 3 and 5 displayed a multifocal interstitial pneumonia that was characterized by minimal alveolar interstitial thickening by small numbers of macrophages and neutrophils (fig. 4 , table s1 ). the adjacent alveolar spaces contained small numbers of alveolar macrophages. mers-cov antigen and rna was detected by immunothroughout the lungs of bat 1 (2 dpi), but no associated pulmonary pathology was detected (fig. 4 , table s2 ). cytokeratin and anti-mers-cov co-staining demonstrated mers-cov antigen in type i pneumocytes of the lungs of bat 1 (fig. 5 ). scientific reports | 6:21878 | doi: 10.1038/srep21878 innate immune response to mers-cov. mers-cov was most consistently detected in the lower respiratory tract of the bats. the mx1, isg56 and rantes gene expression in the lungs of jamaican fruit bats was analyzed as an indicator of the induction of an innate immune response to mers-cov infection. a 6-fold increase in expression of mx1was observed in the lungs of the infected jamaican fruit bats at 2 dpi. a statistically significant upregulation of mx1 gene expression was detected when comparing the lungs of bats collected on 2 dpi and 28 dpi (two-tailed unpaired t-tests, p < 0.034). the maximum isg56 expression of 7.4-fold occurred at 2 dpi. statistically significant differences were observed between the 2 dpi and 7 dpi, 14 dpi and 28 dpi animals (two-tailed unpaired t-tests, p < 0.035, p < 0.0178 and p < 0.0192 respectively. in addition significant differences were observed between the 7 dpi and the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0009 and p < 0.0085). the rantes expression at its peak at 2 dpi was increased 22.5 fold. statistically significant differences were observed between the 2 versus the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0147 and p < 0.0136), the 4 versus the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0092 and p < 0.0075), and the 7 dpi versus the 14 and 28 dpi animals (two-tailed unpaired t-tests, p < 0.0390 and p < 0.0366) (fig. 6 ). antibody response to mers-cov. sera were collected prior to inoculation and at the scheduled necropsy dates. each of the bats was seronegative for mers-cov prior to inoculation. only bat 7 developed a mers-cov specific antibody response, both by elisa and virus neutralization assay. the sera obtained from bat 7 had a neutralizing titer of 320 at 14 days post inoculation. the high sequence similarity of mers-cov to coronavirus sequences detected in bats suggests that mers-cov or its immediate ancestor originated in bats 35 . direct contact between bats and humans is uncommon, and a domestic or peridomestic intermediate species often plays a role in the emergence of zoonotic viruses from natural reservoirs to humans [36] [37] [38] [39] . similar to the emergence of sars-cov in 2002 from the masked palm civet (paguma larvata) as an intermediate host 40 , the dromedary camel appears to have initially served as the intermediate host for mers-cov 41 . several aspects of the emergence of mers-cov are currently still unknown, including the role of the natural reservoir and the relationship between the natural and intermediate reservoirs. with their ability to support efficient replication of mers-cov, the availability of an annotated transcriptome 45 , and the relative easy housing and husbandry practices of jamaican fruit bats suggest that this bat species can become an important model system to investigate the relationship between coronaviruses and their bat hosts 46 . although the jamaican fruit bat is not the direct ancestral reservoir for mers-cov, as it is a new world bat species, generalized responses towards viruses of bat-origin rather than a direct host-pathogen relationships can be modelled. the ability of mers-cov to use dpp4 of multiple species as a receptor, including dpp4 of human, dromedary camel, and bat origin 34, 47 , suggests that no prior adaptation was needed on the dpp4 receptor level for cross-species and zoonotic transmission to occur. with batcov-hku4, a closely related coronavirus, it was shown that replication in human cells required two mutations in the spike protein 48 . these amino acid residues, which are conserved in mers-cov, results in the activation of the batcov-hku4 spike protein by human cellular proteases. this suggests that batcov-hku4 needs these residues for replication in humans. interestingly, our data show that mers-cov replicates efficiently in jamaican fruit bat cells, suggesting that the mers-cov spike can efficiently be processed by jamaican fruit bat cellular proteases and that there is no host restriction on the post-translational modification level of the mers-cov spike in dromedary camels, humans and bats. bat coronaviruses have been primarily detected in fecal samples in field studies suggesting that these viruses have a intestinal tract tropism 9,19,43 . mers-cov was able to replicate to higher titers in the respiratory tract in comparison with the intestinal tract of the jamaican fruit bats. the tissue tropism of mers-cov in jamaican fruit bats is comparable to the respiratory tract tropism observed in dromedary camels and humans 49, 50 . this might suggest that mers-cov, upon cross-species transmission from bats into dromedary camels evolved from a gastrointestinal tract virus into a respiratory tract virus, similar to influenza a viruses 51 . the ability for mers-cov to antagonize the innate immune response appears to correlate with its pathogenic potential in humans. mers-cov and related batcov-hku4 can inhibit innate immune signaling in a variety of human cell lines in vitro via the orf4b-encoded accessory proteins 52 lungs of jamaican fruit bat 5 were stained with α -cytokeratin as an epithelial marker (purple) and with a polyclonal α -coronavirus antibody (brown-red) to demonstrate that viral antigen was located along the basement membrane of alveolar pneumocytes of bat 1 at 2 dpi (indicated by black arrows). original magnification: 40× . with 2% fetal calf serum (hyclone, logan), 1 mm l-glutamine (lonza), 50 u/ml penicillin and 50 μ g/ml streptomycin (gibco). vero e6, llc-mk2, bhk and jamaican fruit bat primary kidney cells were maintained in dulbecco's modified eagle's media (dmem) supplemented with 10% fetal calf serum, 50 u/ml penicillin and 50 μ g/ml of streptomycin. sequencing and cloning of the jamaican fruit bat dpp4 sequence. total rna was extracted from primary kidney cells using the rneasy mini kit (qiagen) and cdna was synthesized using random hexamer primers and superscript iii reverse transcriptase (applied biosystems). dpp4 was then amplified using iproof high-fidelity dna polymerase (biorad) and primers dpp4unvf1 and dpp4unvr12 (primer sequences are available upon request). the obtained dpp4 gene sequence was synthesized in expression plasmid pcdna3.1(+ ) cate with mers-cov with a multiplicity of infection (moi) of 0.01 (cell lines) or 1 (transfected cell lines) 50% tissue culture infectious dose (tcid 50 ) per cell. one hour after inoculation, cells were washed once with dmem and culture medium replaced. supernatants were sampled at 0, 24, 48 and 72 h after inoculation. mers-cov was titrated by end-point titration in quadruplicate in vero e6 cells cultured in dmem supplemented with 2% fetal calf serum, 1 mm l-glutamine (lonza), 50 u/ml penicillin and 50 μ g/ml streptomycin. cells were inoculated with ten-fold serial dilutions of virus, and scored for cytopathic effect 5 days later. the tcid 50 was calculated by the method of spearman-karber. animal experiments. twelve captive-bred jamaican fruit bats were used for this work 46, 57 . ten bats were inoculated with 10 5 tcid 50 emc/2012 via a combination of intranasal (25 μ l each nostril) and intraperitoneal (100 μ l) routes. two mock inoculated bats were included as controls for histopathology and gene expression analyses. mock inoculated bats were inoculated with standard tissue culture media via the same routes and volumes. bats were injected with an iptt-300 temperature transponder (bmds) to monitor body temperature daily. animals were weighed daily and observed for signs of disease. oropharyngeal and rectal swabs were obtained on 1, 2, 3, 4, 5, 6, 7, 9 and 11 dpi and analyzed for the presence of viral rna. on 2, 4, 7, 14 and 28 days post inoculation (dpi), two bats were euthanized and trachea, heart, lung, liver, spleen, kidney, duodenum, colon, bladder, nasal turbinates and brain were collected for virological and histopathological analysis. histopathology. histopathology was performed on bat tissues. after fixation for at least 7 days in 10% neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (h & e) staining. immunohistochemistry was performed using a mers-cov emc/2012 polyclonal rabbit antibody at a 1:1000 dilution and in situ hybridization was performed using probes directed against the mers-cov emc/2012 n gene as described previously 62 . rna extraction. rna was extracted from swab samples using the qiaamp viral rna kit (qiagen). rna was eluted in 60 μ l. tissues (30 mg) were homogenized in rlt buffer and rna was extracted using the rneasy kit (qiagen). rna was eluted in 50 μ l. quantitative pcr. for detection of viral rna in samples, 5 μ l rna was used in a one-step real-time rt-pcr upe assay 63 using the rotor-gene tm probe kit (qiagen) according to the manufacturer instructions. in each run, standard dilutions of a titered mers-cov stock were run in parallel, to calculate tcid 50 equivalents in the samples. for the detection of viral mrna, 5 μ l rna was used in a one-step real-time rt-pcr using the mers-cov m mrna assay in the rotor-gene tm probe kit 64 . artibeus jamaicensis orthomyxovirus resistance gene 1 (mx1) gene expression was determined by qrt-pcr using mx1, isg56 and rantes specific primers (derived from transcriptome sequencing 45 ). the fold-change of each gene was calculated by normalizing the change in ct (cycle threshold) value of mx1 (δ ct) to the ct values for hypoxanthine phosphoribosyltransferase (hprt) as an internal reference gene for each sample and comparing this to the ct values of mock inoculated bats 11 and 12 (2 ∧ (−δ δ ct). mx1 specific primers: 5′ -ccagacctgaccctgataga-3′ , 5′ -tggatgtacttcctgaatgagttg-3′ and 5′ -fam-atctagtgtccgatgtcagctggc-iabkfq-3′ . isg56 specific primers: 5′ -gctgtctatcgtctgaatggg-3′ , 5′ -ttcttgtccgatgtcctgaag-3′ and 5′ -hex-cgatgaggc/zen/attttgtctgcaaaccc-iabkfq-3′ . rantes specific primers: 5′ -agttgtcctaatcacccgaaag-3′ , 5′ -cagagtgttgatgtagtcccg-3′ and 5′ -fam-tgtgccga c/zen/ccggagaagaaat-iabkfq-3′ . hprt specific primers: 5′ -agatggtgaaggtcgcaag-3′ , 5′ -cctgaagtattcattatagtcaaggg-3′ and 5′ -fam-actttgttggatttgaaattccag acaagtttg-bhq1. virus isolation. tissue samples were homogenized in a tissuelyzer ii (qiagen) after addition of 1ml dmem. homogenates were centrifuged to pellet cellular debris and subsequently inoculated onto veroe6 and llc-mk2 cells. after 1hr adsorption, cells were washed once with dmem and media was replaced. elisa. antibody responses were measured in an enzyme-linked immunosorbent assay (elisa) using hcov-emc/2012 as described previously 65 . briefly, emc/2012 containing cell culture supernatant was used to coat immuno 96 microwell maxisorp plates (nunc) at 4 °c overnight and diluted serum samples were added. bound antibodies were detected using a secondary protein a/g conjugated with horseradish peroxidase (hrp; pierce). sera were considered positive when absorbance was higher than three standard deviations above the mean of negative control sera. sera obtained from rabbits immunized with emc/2012 were used as a positive control. virus neutralization assay. two-fold serial dilutions of heat-inactivated sera were prepared in a 96 microwell tissue culture plate and 100 tcid 50 of mers-cov was added and incubated for 1 hour at 37 °c. after incubation the virus-sera mixture was transferred to a 96 microwell tissue culture plate with a 95% confluent scientific reports | 6:21878 | doi: 10.1038/srep21878 monolayer of veroe6 cells. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, that still inhibited emc/2012 virus replication. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who. middle east respiratory syndrome coronavirus (mers-cov) full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans a coronavirus detected in the vampire bat desmodus rotundus highly diversified coronaviruses in neotropical bats close relative of human middle east respiratory syndrome coronavirus in bat novel bat coronaviruses, brazil and mexico detection of a virus related to betacoronaviruses in italian greater horseshoe bats circulation of group 2 coronaviruses in a bat species common to urban areas in western europe human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe coronaviruses in bats from mexico identification of a novel coronavirus in bats molecular diversity of coronaviruses in bats comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features mers-related betacoronavirus in vespertilio superans bats bat coronaviruses and experimental infection of bats, the philippines isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation mers coronavirus in dromedary camel herd, saudi arabia antibodies against mers coronavirus in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study mers coronavirus neutralizing antibodies in camels evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate isolation of mers coronavirus from a dromedary camel transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans the natural history of hendra and nipah viruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china hendra virus: an emerging paramyxovirus in australia discrepancies in data reporting for rabies a decade after sars: strategies for controlling emerging coronaviruses the emergence of the middle east respiratory syndrome coronavirus walker's bats of the world detection and phylogenetic analysis of group 1 coronaviruses in south american bats neotropical bats from costa rica harbour diverse coronaviruses transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) immunology of bats and their viruses: challenges and opportunities dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc two mutations were critical for bat-to-human transmission of mers coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome practical considerations for high-throughput influenza a virus surveillance studies of wild birds by use of molecular diagnostic tests the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling comparison of serological assays in human middle east respiratory syndrome (mers)-coronavirus infection presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study experimental nipah virus infection in pteropid bats (pteropus poliocephalus) pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission tacaribe virus causes fatal infection of an ostensible reservoir host, the jamaican fruit bat experimental inoculation of plants and animals with ebola virus seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection bats and lyssaviruses experimental rabies virus infection in artibeus jamaicensis bats with cvs-24 variants middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction. euro surveillance : bulletin européen sur les maladies transmissibles growth and quantification of mers-cov infection the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the authors would like to thank drs. bart key: cord-307853-m1q1sjr4 authors: majumder, satyabrata; chaudhuri, dwaipayan; datta, joyeeta; giri, kalyan title: exploring the intrinsic dynamics of sars-cov-2, sars-cov and mers-cov spike glycoprotein through normal mode analysis using anisotropic network model date: 2020-10-16 journal: j mol graph model doi: 10.1016/j.jmgm.2020.107778 sha: doc_id: 307853 cord_uid: m1q1sjr4 covid-19 caused by sars-cov-2 have become a global pandemic with serious rate of fatalities. sars-cov and mers-cov have also caused serious outbreak previously but the intensity was much lower than the ongoing sars-cov-2. the main infectivity factor of all the three viruses is the spike glycoprotein. in this study we have examined the intrinsic dynamics of the prefusion, lying state of trimeric s protein of these viruses through normal mode analysis using anisotropic network model. the dynamic modes of the s proteins of the aforementioned viruses were compared by root mean square inner product (rmsip), spectral overlap and cosine correlation matrix. s proteins show homogenous correlated or anticorrelated motions among their domains but direction of c(α) atom among the spike proteins show less similarity. sars-cov-2 spike shows high vertically upward motion of the receptor binding motif implying its propensity for binding with the receptor even in the lying state. mers-cov spike shows unique dynamical motion compared to the other two s protein indicated by low rmsip, spectral overlap and cosine correlation value. this study will guide in developing common potential inhibitor molecules against closed state of spike protein of these viruses to prevent conformational switching from lying to standing state. coronaviruses are zoonotic pathogens which belong to the largest group of nidovirales order [1] and has been shown to infect many avian and mammalian species [2] . severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov) and novel coronavirus (sars-cov-2) are in betacoronavirus family but with different lineages. sars-cov-2, sars-cov belongs to lineage b whereas mers-cov belongs to lineage c but a common factor is that they all cause acute lung injury (ali) and acute respiratory distress syndrome (ards) which leads to pulmonary failure and result in fatality. covid-19 caused by novel coronavirus (sars-cov-2), has now emerged as global pandemic and high rate of fatalities have been reported globally [3, 4, 5] . the major surface protein of all these 3 coronaviruses is called spike glycoprotein (s) which is responsible for host attachment of the virus and entry into cell by membrane fusion. the protein protrudes out of the surface and is found in a trimeric configuration [6] . the protein has 2 subunits called s1 and s2. s1 subunit has the receptor binding domains (rbd) which help to stabilize the prefusion state. the s2 subunit remains anchored to the membrane and contains the fusion machinery and further cleaved to form s2' in certain coronaviruses thus activating the membrane fusion cascade [7, 8] . the receptors recognized by the viruses are also different in case of mers-cov it is 5-n-acetyl-9-o-acetyl-sialosides found on glycoproteins and glyco-lipids at the host cell surface while for sars-cov and sars-cov-2 it is hace2 receptor [9, 10] . spike glycoprotein undergoes conformational switch in order to bind with the receptor. in lying or closed state the receptor binding domain is buried deeply into the trimer core and so it is not accessible to the receptor making the conformational switch essential. in standing or open state one of the rbd protrudes out of the trimer core and becomes accessible to the receptor [11] . a detailed study to explore the intrinsic dynamical motion of the prefusion lying state spike protein trimer is needed for proper understanding of its function from conformation perspective. in recent years characterization of whole protein collective and domain-specific motions have been accomplished using normal mode calculations [12, 13] . coarse-grained normal mode analysis (cg-nma) has now become a valuable tool for studying the biologically relevant conformational motions of large protein or protein complex. although coarse-grained nma (cg-nma) does not capture the detailed local dynamics that are obtained from all atom molecular dynamics simulation, cg-nma has been shown to be equally effective in j o u r n a l p r e -p r o o f demonstrating the global functional motions of proteins [14, 15] . nma with anisotropic network model (anm), a variant of elastic network model (enm), has been proved to be successful in describing the collective dynamics of a wide range of biomolecular systems [16] [17] [18] . notable application of nma using anm especially in membrane proteins has been thoroughly mentioned in the study and review done by bahar et al [19, 20] . in this study we have explored the intrinsic dynamics of the closed or lying state spike (s) glycoprotein of sars-cov-2, sars-cov and mers-cov through nma studies using anm. we found noticeable variations in the dynamics of the proteins in different low frequency modes in terms of both magnitude and direction of displacement vectors. we found that receptor binding motif (rbm) of a single chain of sars-cov-2 spike show high fluctuation in the lying state indicating its high probability to bind with the receptor implying the high infectivity of sars-cov-2 than other two viruses. mers-cov s shows distinct dynamics than sars-cov s and sars-cov-2 s indicates its low sequence similarity and distant phylogenetic relationship with other. detailed domain-wise motion, atomic fluctuation data and quantitative comparison between s protein structures of the mentioned viruses will aid in identifying common potential drug binding hotspots in the receptor binding domain of the protein which will guide in designing common inhibitor or modified version of these molecules through structure-based approach. j o u r n a l p r e -p r o o f all the x-ray crystal structures for sars-cov-2, sars-cov and mers-cov s proteins in the lying state were taken from protein data bank [21] . the corresponding pdb ids are 6vxx, 5x58 and 6q05 respectively [11, 22, 23] . charm-gui was used to prepare the protein (i.e adding missing atoms and assigning correct protonation states) [24] . as a large part of this study is involved in the comparative analysis purposes, we have discarded part of the s2 region of s protein of all viral strains so that each protein model has equal number of cα atoms (1087 in all three chains). s2 region of the protein does not take active part in receptor interaction. detailed mathematical description about normal mode analysis and how it is applicable to protein molecule is given in many studies [20, [25] [26] [27] . nma is a technique to study vibrational motion of harmonic oscillating system near the equilibrium state. the motions are of small amplitude in a potential well and cannot cross energy barriers. at a minimum state , the potential energy v can be obtained from the approximation of taylor series expansion. for the generalized coordinates : where specifies the deviation from equilibrium ( = + ). the kinetic energy t also approximated from taylor expansion. the resulting lagrangian is l = t-v, which results in linear differential equations of motion: assuming oscillatory solution, = cos ( + ) and substituting it in equation 2 results in eigenvalue problem: a is the matrix of amplitudes , and v is the matrix of the second derivative of the potential energy and is referred to as hessian. $ is a diagonal matrix of eigenvalues. the pattern of motions is fully specified by the vibrational normal modes, i.e. the eigenvectors (% ) and their associated eigenvalues ($ ). the vector describes the directionality and magnitude of motion of each particle relative to other particles. here . and . are instantaneous and equilibrium distances between the nodes i and j, ( is here we consider (3n -6) non zero modes, $ corresponds to eigenvalues and the corresponding eigenvectors are 7 . the inverse h -1 is also organized in n x n submatrices of size 3 x 3, each. the ij th submatrix h ij -1 defines the covariance between the fluctuations of residues i and j. the cross-correlation (cc) between the equilibrium fluctuations of residues i and j, 8δ9 • δ9 ;is expressed in terms of the trace (tr) of these submatrices as: we have performed the nma and all the analysis functions including root mean squared inner product (rmsip) [28] , spectral overlap [29] and cosine correlation or overlap [30] using prody python package [31] . rmsip computes quantitative comparison value between the sets of normal modes and expressed as: structural alignments of the models were done using pymol. we have computed the eigenvalues of first hundred non-zero normal modes of the sars-cov-2, sars-cov, and mers-cov spike (s) proteins in the lying state ( fig. 1 ). the energy associated with a given normal mode is directly proportional to its eigenvalue [28] . we have discarded the we have assessed the extent to which the trimer conformation influences the dynamics of each s protein chain (fig. 3) . overall the pattern of the cross correlation matrix (ccm) looks similar for three models and have similar domain wise correlated movement but the magnitude of correlation coefficients (cc) are different to some extent. according to pdb and uniprot protein feature view, the position of s1 ntd, s1 ctd and rbd for the protein models are as follows: sars-cov: 14-290 is s1 ntd (n-terminal domain), 321-513 is s1 ctd (c terminal domain), 306-527 is rbd (binding motif is 424-494); sars-cov-2: 13-303 is s1 ntd, 334-527 is s1 ctd, 319-541 is rbd (binding motif is 437-508); mers-cov: 18-350 is s1 ntd, 381-588 is j o u r n a l p r e -p r o o f spike proteins are shown. color coding is as follows: orange: s1 ntd, violet: s1 ctd, cyan: s2 region and blue: rbd. multiple sequence alignment of the proteins is given in supplementary. emphasizing the motions among the domains, it was found that sars-cov and sars-cov-2 s proteins have homogenous correlated displacements. s1 ctd and ntd of each chain have weak negative correlation with each other (-0.1 to -0.4). s1 ctd of one chain has weak negative correlation with ctd of other chains and same scenario is seen with the ntd. s1 ctd and ntd in each chain are negatively correlated with each other implying the rbm tries to move further from ntd in order to be accessible by the receptor. one of the interesting facts is that s1 ntd of one chain shows strong positive correlation with ctd of at least one of the two other chains which is true for both sars-cov and sars-cov-2 s protein but variation is seen in the ccm of mers-cov s protein (fig. 3c ). s1 ntd of each chain is negatively correlated with ntd of other chains with magnitude range -0. around the interfaces of domains [32] . in order to comprehensibly describe the motion of the trimer chains of the first four low frequency (low eigenvalue) modes (λ7, λ8, λ9, λ10), first we have computed three unique symmetry axis of the spike trimer using vmd symmetry tool. among the three symmetry axis, one pointed outward through the trimer core, which we have named the z axis (default 2 in vmd nomenclature) and the other two axis were named as x and y axis (default vmd nomenclature 1 and 0 respectively). we have used default pdb chain ids of the three spike protein structures to address the chain and trimer motion individually. as previously described we will confined ourselves to discuss the motion of s1 domain of the three chains as it harbors the functional receptor binding domain. in all the structures, the β-strand region (bsr) of s1 dominates overall motions of the trimer in first four low frequency non-zero modes (supplementary 4, 5 and 6). we did not notice any concerted motion of the three chains we have computed the squared atomic fluctuations of the s protein models using first 10 nonzero normal modes (fig. 6) . the amplitude of the fluctuations shows variation within the models. in in order to compare the normal modes between the structures, we have calculated the rmsip, normal mode analysis of equilibrium structures falls in the category of principal component analysis-based methods like singular value decomposition of molecular dynamics or monte carlo trajectories or essential dynamics analysis of the covariance matrices retrieved from md runs [20] . these studies have proven to be useful in studying the equilibrium dynamics of proteins which are spanned by low frequency end of the mode spectrum [33] . anisotropic , which is proportional to energy, will lead to energetically unfavorable motions [20] . spectra of eigenvalues shows that sars-cov-2 s has more collective motion than other two s protein implying sars-cov-2 s protein has higher stability [32] and has lower energetically expensive motion than the latter. this stability of the s trimer of sars-cov-2 in terms of eigenvalue frequency may give a part of the explanation about why sars-cov-2 is more infective than other two but do not provide detailed description. it is shown that in sars-cov s, which is the lying state, the rbd is buried in the s protein trimer therefore interaction with ace receptor is hindered. in order to bind with receptor, s protein must switch to standing state where rbd of any one of the chains is exposed and binds with receptor [8] (fig. 8) . from the homogenous ccm plot of whole trimer (fig. 2) , we hypothesize that the molecular mechanism of this conformational switching at atomic scale may be similar in nature in the three viruses but the variation seen in the ccm of rbm and rbd are responsible for the difference in infectivity. it can be said from the ccm that rbd of all three chains have equal probability to be switched into standing state. from the first four low frequency non-zero modes (λ 7 , λ 8, λ 9, λ 10 ) localized variation in the direction of c α atom motion in the models are worth noticeable (fig. 5 ). we have also described the motions of the c α respect to the symmetry axis of the trimer (supplementary receptor. this leads to more tight binding of sars-cov-2 s protein rbd to the receptor than sars-cov and it explains partially, why sars-cov-2 is 10 to 15 times more infective than sars-cov [31] . cryo-em studies have shown that, in sars-cov spike, the rbd is mostly in the standing state [8, 11] however , in sars-cov-2 spike, the rbd is mostly in the lying state the present comprehensive study demonstrated the intrinsic dynamics of the s proteins from sars-cov-2, sars-cov and mers-cov and their inter-relationship through normal mode analysis using anm. high fluctuation of the rbm of sars-cov-2 spike indicates its propensity to bind with ace2 even in the lying state inferring its higher infectivity than sars-cov and mers-cov. the distant relationship of mers-cov with the sars-cov and sars-cov-2 in phylogenetic tree at sequence level resembles that at the dynamics level due to its unique dynamical motion. sites of very low atomic fluctuation at the s1 site which harbours the rbd could serve as the potential drug binding site. our study could serve as the basis for j o u r n a l p r e -p r o o f designing common potential drugs against the spike of the three mentioned viruses which may inhibit the conformational changes from lying to standing state. covid-19 infection: origin, transmission, and characteristics of human coronaviruses jumping species-a mechanism for coronavirus persistence and survival who | summary of probable sars cases with onset of illness from 1 middle east respiratory syndrome coronavirus (mers-cov) immunogenicity of a dna vaccine candidate for covid-19 structural insights into coronavirus entry pre-fusion structure of a human coronavirus spike protein cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains analysis of domain motions in large proteins analysis of domain motions by approximate normal mode calculations global dynamics of proteins: bridging between structure and function probing the global and local dynamics of aminoacyl-trna synthetases using all-atom and coarse-grained simulations insights into equilibrium dynamics of proteins from comparison of nmr and x-ray data with computational predictions coarse-grained normal mode analysis in structural biology normal mode analysis: theory and applications to biological and chemic anisotropic network model: systematic evaluation and a new web interface normal mode analysis of biomolecular structures: functional mechanisms of membrane proteins protein data bank | nucleic acids research | oxford academic structures of mers-cov spike glycoprotein in complex with receptors structure, function, and antigenicity of the sars-cov-2 spike glycoprotein charmm-gui: a web-based graphical user interface for charmm normal mode analysis as a method to derive protein dynamics information from the protein data bank comparing the intrinsic dynamics of multiple protein structures using elastic network models comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis on the convergence of the conformational coordinates basis set obtained by the essential dynamics analysis of proteins' molecular dynamics simulations convergence of sampling in protein simulations cosine similarity -an overview | sciencedirect topics prody: protein dynamics inferred from theory and experiments -pubmed enhanced receptor binding of sars-cov-2 through networks of hydrogen-bonding and hydrophobic interactions investigating protein dynamics in collective coordinate space anisotropic network model: systematic evaluation and a new web interface -pubmed phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop this study was supported by frpdf grant of presidency university. protocol designed, conceptualized and analyzed by s.m., manuscript preparation done by d.c. and j.d. project was done under the supervision of k.g. the manuscript was reviewed and approved by all authors. the authors declare no conflict of interest. key: cord-303670-fma8wq4z authors: liu, ren-qiang; ge, jin-ying; wang, jin-ling; shao, yu; zhang, hui-lei; wang, jin-liang; wen, zhi-yuan; bu, zhi-gao title: newcastle disease virus-based mers-cov candidate vaccine elicits high-level and lasting neutralizing antibodies in bactrian camels date: 2017-10-31 journal: journal of integrative agriculture doi: 10.1016/s2095-3119(17)61660-5 sha: doc_id: 303670 cord_uid: fma8wq4z abstract middle east respiratory syndrome coronavirus (mers-cov), a member of the coronaviridae family, is the causative pathogen for mers that is characterized by high fever, pneumonia, acute respiratory distress syndrome (ards), as well as extrapulmonary manifestations. currently, there are no approved treatment regimens or vaccines for mers. here, we generated recombinant nonvirulent newcastle disease virus (ndv) lasota strain expressing mers-cov s protein (designated as rla-mers-s), and evaluated its immunogenicity in mice and bactrian camels. the results revealed that rla-mers-s showed similar growth properties to those of lasota in embryonated chicken eggs, while animal immunization studies showed that rla-mers-s induced mers-cov neutralizing antibodies in mice and camels. our findings suggest that recombinant rla-mers-s may be a potential mers-cov veterinary vaccine candidate for camels and other animals affected by mers. middle east respiratory syndrome coronavirus (mers-cov), a member of the c lineage in the genus beta coronavirus, causes high fever, pneumonia, acute respiratory distress syndrome (ards), as well as extrapulmonary manifestations including gastrointestinal symptoms, lymphopenia, mers cases, there is an urgent need to develop vaccines or specific drugs targeted at epidemic mers-cov (modjarrad et al. 2016; zumla et al. 2016) . newcastle disease virus (ndv) belongs to the genus avulavirus in the paramyxoviridae family. ndv is classified as lentogenic (nonvirulent), mesogenic (moderately virulent), or velogenic (highly virulent) according to their pathogenicity in poultry. lentogenic strains, such as the lasota, have also been applied as a vaccine vector targeting human and other animal diseases (ge et al. 2007 (ge et al. , 2010 di napoli et al. 2010a, b) . ndv is innately advantageous as a potential vaccine vector for the following reasons: firstly, ndv is antigenically distinct from the mammalian paramyxoviruses, it does not typically cause an productive infection in mammals. secondly, the pre-existing immunity against mammalian paramyxoviruses does not interfere with the replication capacity of ndv. in addition, the safety profile of ndv has been confirmed in many non-human primates as well as humans (bukreyev et al. 2006; bukreyev and collins 2008; khattar et al. 2010; kortekaas et al. 2010) . as a membrane-anchored structural protein of mers-cov, spike (s) protein mediates viral receptor binding and entry (belouzard et al. 2012; millet and whittaker 2014) . s protein is the primary target for anti-coronavirus vaccine design (zhao et al. 2014) , and studies have demonstrated that s protein is immunogenic and can induce neutralizing antibodies which plays crucial role in anti-cov infection (hofmann et al. 2004; enjuanes et al. 2008; du et al. 2009; pascal et al. 2015) . currently, several mers-cov candidate vaccines, such as dna vaccines (muthumani et al. 2015) , virus like particles (vlps) ) as well as recombinant viral vectored vaccines. of note, the recombinant viral vectored mers vaccines, such as modified vaccinia ankara (song et al. 2013) or adenovirus (kim et al. 2014) demostrated good immunogenicity and provided protection for mice, nonhuman primates (nhps) and camels against mers-cov challenge. herein, we generated a recombinant ndv lasota virus expressing mers-cov s protein and evaluated its immunogenicity in mice and camels. bhk-21 and vero-e6 cells were grown in dulbecco's minimum essential medium (dmem) containing 10% fetal bovine serum (fbs). the ndv vector virus rla was rescued from the genomic cdna of the ndv lasota vaccine strain as previously described (ge et al. 2007) . recombinant ndv was grown and titrated in 9-day-old specific-pathogen-free (spf) embryonated chicken eggs by allantoic cavity inoculation. a recombinant vesicular stomatitis virus (vsv) vectored virus expressing mers-cov s protein and enhanced green fluorescence protein (egfp), designated as vsvδg-egfp-mers, was used to determine the induction of neutralizing antibodies by mers-cov. the recombinant vsv-vectored virus (vsvδg-egfp-mers) was generated by replacing the g gene of the recombinant vsv expressing egfp with the mers-cov s gene as described previously (li et al. 2006; liu et al. 2015) . vsvδg-egfp-mers was grown and titrated in vero e6 cells. modified vaccinia ankara expressing the t7 rna polymerase (kindly provided by dr. bernard moss, the national institutes of health, bethesda, md, usa) was grown and titrated in primary chicken embryo fibroblasts (wyatt et al. 1995) . all viruses were stored at −70°c before use. to construct the full-length recombinant ndv genomic cdna, mers-cov (genbank accession no. kf186567.1) s gene was amplified by pcr from synthesized cdna (invitrogen, shanghai, china) by using the following primers: 5´-gactgtttaaacttagaaaaaatacgggtagaagt gccaccatgatacactcagtgtttctactg-3´, and 5´-gactgtttaaactcattagtgaacatgaaccttatg-cggctcgag-3´, in which the ndv gene end and gene start sequences (underlined), the optimal kozak sequence (italic) and the pmei restriction sites (bold) were introduced. s gene was introduced into ndv genomic cdna through a unique pmei site in the p-m intergenic region. the resultant plasmid was designated as prla-mers-s and used for virus rescue following established protocol as described previously (ge et al. 2007 ). the rescued virus was designated as rla-mers-s. the presence of s gene in the ndv genome was confirmed by sequencing of the entire viral genome. s protein expression in rla-mers-s infected cells was confirmed by indirect immunofluorescence and western blot assay. the pathogenicity of rla-mers-s in poultry was determined by mean death time (mdt), intracerebral pathogenicity index (icpi), and intravenous pathogenicity index (ivpi) in embryonated spf chicken eggs or in spf chickens according to the oie manual (oie 2004) . to assess the pathogenicity of the recombinant viruses in mice, 2 groups of 10 six-week-old female balb/c mice (vital river, beijing, china) were intramuscularly (i.m.) injected with 1×10 8 eid 50 (50% embryo infectious dose) rla-mers-s or rla in 0.1 ml diluted allantoic fluid and intranasally (i.n.) inoculated with 3×10 7 eid 50 rla-mers-s or rla in 0.03 ml diluted allantoic fluid. the third group of 10 mice was i.m. injected with 0.1 ml and i.n. inoculated with 0.03 ml pbs as mock infection control. mice were monitored daily for signs of illness, weight loss, or death. for mouse immunization, 10 six-week-old female balb/c mice (vital river, beijing, china) were i.m. vaccinated with 1×10 8 eid 50 rla-mers-s in 0.1 ml diluted allantoic fluid. three weeks after the first dose, mice were boosted with the same vaccine at the same dose and via the same route. for serological assay, 2 weeks after the first dose (prime) and the second dose (boost), mouse blood samples were collected from the retro-orbital sinuses under isoflurane inhalation anesthesia. camel immunization was carried out in the experimental animal center of the college of veterinary medicine of inner mongolia agricultural university, china. three groups (n=5 per group) of adult bactrian camels (provided by college of veterinary medicine of inner mongolia agricultural university) were immunized. group 1 animals were i.m. immunized with 2×10 9 eid 50 rla-mers-s in 2 ml diluted allantoic fluid. group 2 animals were immunized i.m. with 2×10 9 eid 50 rla in 2 ml diluted allantoic fluid, and group 3 animals were i.m. injected with 2 ml pbs. three weeks after the first dose, the camels were boosted with the same vaccine at the same dose and via the same route. serum was collected before vaccination and at week 0, 3, 5, 9 and 14 after priming. indirect immunofluorescence assay (ifa) was used to detect s protein expression in rla-mers-s infected cells. bhk-21 cells were plated on cover slips in 35-mm-diameter dishes and infected with rla or rla-mers-s at a multiplicity of infection (moi) of 0.1. at 24 h post-infection, cells were fixed in 3% paraformaldehyde in phosphate-buffered saline (pbs) for 15 min at room temperature. cells were blocked in pbs containing 1% (w/v) bovine serum albumin (bsa) at room temperature for 1 h. cells were then incubated with mouse anti-s serum or chicken anti-ndv serum for 1 h at room temperature. cells were then washed 3 times with pbs containing 0.05% tween 20 (pbst) and stained with a fitc-conjugated goat anti-mouse antibody (sigma, st. louis, mo, usa) or an tritc-conjugated rabbit anti-chicken antibody (sigma, st. louis, mo, usa) for 30 min. cell nuclei was stained with dapi after final wash. stained cells were analyzed with a leica tcs sp5 confocal laser microscope (leica microsystems, wetzlar, germany). bhk-21 cells were infected with rla and rla-mers-s at a moi of 2. cells were collected and lysed at 36 h post-infection, cell lysates were subjected to 10% sds-page under denaturing conditions and transferred to polyvinylidene difluoride (pvdf) membrane for western blot assay using chicken anti-ndv serum or mouse anti-s serum as primary antibody and horseradish peroxidase (hrp)-conjugated rabbit-anti-chicken igg or goat-anti-mouse igg as secondary antibodies, respectively (sigma, st. louis, mo, usa). the bands were visualized with ecl plus western blotting detection reagents (ge healthcare life sciences, pittsburgh, pa, usa) on kodak x-ray films. mers-cov s protein-specific igg was measured by elisa as described previously (kong et al. 2012) . briefly, vero-e6 cells were seeded onto two wells of a six-wells plate, and infected with vsvδg-egfp-mers at a moi=0.01. at 48 h post-infection, the cell pellet was collected and lysed by repeat pipetting, and the supernatant was coated in the elisa plate at 4°c overnight. the plates were then washed and blocked with 2% bsa (w/v) at room temperature for 1 h. serially diluted serum was added to the elisa plate, and incubated at room temperature for 1 h. plates were washed three times with pbst, then a 1:3 000 dilution of hrp-labeled goat anti-mouse igg (southern biotech, birmingham, al) was added and incubated for another 1 h at room temperature. the plates were washed 5 times with pbst. for visualization, 50 µl of 3,3´,5,5´-tetramethylbenzidine (tmb) liquid substrate (sigma, st. louis, mo, usa) was added to each well for 5 min at room temperature; 50 µl of 2 mol l -1 sulfuric acid was added to stop the reaction. od values were determined with a model 680 microplate reader (bio-rad, usa) at 450 nm. a standard curve was generated by coating with serially diluted purified unlabeled mouse igg (southern biotech, birmingham, al) at known concentrations. a linear equation was built based on the standard igg concentration and their od values, thus the concentration of mers-specific igg could be determined according to the linear equation. for camel-specific igg, the protocol of elisa was the same as the elisa for mouse serum; the difference was 1:3 000 diluted hrp conjugated rabbit anti-camel igg antibody (neoscientific, ma, usa) was used as secondary antibody. due to the lack of purified camel igg to generate standard curves, all camel sera elisa results were expressed as relative which were used at 1:300 fixed dilutions. to assess the mers-cov specific neutralizing antibodies, 25 µl of 2-fold serially diluted serum (heat inactivated at 56°c for 30 min before use) was mixed with 25 µl of dmem containing 5×10 2 tcid 50 vsvδg-egfp-mers and incubated at 37°c for 1 h. then the mixture was added to bhk-21 cells in quadruplicate wells of a 96-well plate. the egfp-expressing cells were counted at 48 h post-infection under a fluorescence microscope. neutralization titers were determined as the reciprocal of the highest dilution of serum that showed at least a 50% reduction in the number of fluorescent cells as compared with the negative control. a recombinant ndv lasota virus expressing the egfp (designated as rla-egfp) was used to determine the neutralizing antibodies against vector. briefly, 25 µl of 2-fold serially diluted serum was mixed with 25 µl of medium containing 2×10 3 eid 50 of rla-egfp at 37°c for 1 h. after the incubation, 50 µl of the mixture was added to the bhk-21 cell monolayer in quadruplicate wells of a 96-wells plate. the gfp-expressing cells were counted at 16 h post-infection under a fluorescence microscope. neutralization titers were expressed as the reciprocal of the highest dilution of serum that showed at least a 50% reduction in the number of fluorescent cells as compared with the negative control. two-way anova with bonferroni's multiple comparison tests was used for statistical analysis. all p-values were two-tailed and considered statistically significant when the associated probability was less than 0.05. recombinant genomic cdna of rla-mers-s was constructed by inserting the s gene of mers-cov between the p and m gene of ndv ( fig. 1-a) . the resultant recombinant virus, rla-mers-s, was successfully rescued from the full-length genomic cdna clone as described above. s protein expression was confirmed by western blot analysis with mouse anti-s serum ( fig. 1-b) . indirect immunofluorescence further confirmed the s protein expression in infected bhk-21 cells. as shown in fig. 1 -c, non-infected bhk-21 cells (mock) were not stained by either mouse anti-s serum or chicken anti-ndv serum, infected cells were not stained by mouse anti-s serum, but were stained by chicken anti-ndv serum. by contrast, rla-mers-s infected cells were stained by both the mouse anti-s serum and the chicken anti-ndv serum. the growth properties of rla and rla-mers-s in eggs were examined next. as shown in fig. 2 , rla-mers-s reached peak titers of 9.7 logeid 50 ml -1 at 72 h post-inoculation. the stability of the s gene within rla-mers-s was assessed by serial passaging of the virus in spf chicken eggs over repeated passages. after 10 passages, the expression of s gene was assessed by rt-pcr and immunofluorescence, and results confirmed that s gene was stably maintained and expressed (data not shown). to investigate whether expression of s protein altered the pathogenicity of ndv vector, we assessed pathogenicity of rla-mers-s in poultry and mice. according to the oie manual, mdt, icpi, and ivpi tests were used to assess the pathogenicity of ndv strains in poultry . ndv strains can be classified into three groups based on their mdts: velogenic (<60 h), mesogenic (60-90 h), and lentogenic (>90 h). our results showed the mdt of rla and rla-mers-s were both greater than 120 h, indicating that these 2 viruses were lentogenic (fig. 3-a) . all rla and rla-mers-s inoculated chickens remained healthy during the observation period. the icpi values for rla and rla-mers-s were 0.4 and zero, respectively; while the ivpi values for rla and rla-mers-s were both zero. these data indicated that rla-mers-s and rla were of low pathogenicity to spf chickens and embryonated chicken eggs, suggesting that the insertion of the s gene in ndv did not increase the virulence of the ndv vector. next, to investigate the safety of the recombinant viruses in mammals, we inoculated mice i.m. with 1×10 8 eid 50 of rla or rla-mers-s in 0.1 ml diluted allantoic fluid, and at the same time inoculated the mice i.n. with 3×10 7 eid 50 of rla or rla-mers-s in 0.03 ml diluted allantoic fluid. mice were observed daily for two weeks for signs of weight change or other indicators of illness. the rla and rla-mers-s infected mice showed similar changes in body weight to mock-infected mice and no signs of disease were observed in any animals ( fig. 3-b) . mice mers-specific igg1, igg2a and total igg levels were determined by elisa (fig. 4-a, i) . notably, the total igg antibody levels were significantly boosted after the second dose (p<0.01). furthermore, rla-mers-s could induce both the igg1 (th2) (fig. 4-a, ii) and igg2a (th1) antibody responses (fig. 4-a, iii) after the second dose with a slightly th2-biased in immunized mouse. the elisa result was in accordance with the result of ndv and mers vna assays. significant ndv vna was detected in all mice (fig. 4-b) , but mers-cov vna was detected only in the blood of mice inoculated with rla-mers-s, not rla ( fig. 4-a) after the second dose (p<0.01). these results demonstrated that rla-mers-s was highly immunogenic in mice. the levels and duration of the recombinant virus-induced neutralizing antibody responses are shown in fig. 5 . camel igg against s protein was measured by elisa after the primary and secondary immunization (fig. 5-a) . similar to elisa antibodies, the neutralizing antibodies were boosted, and gradually decreased after the second dose. for rla-mers-s immunized camels, mers-cov neutralizing antibodies were detected after the second dose (5 weeks) and lasted for at least 9 weeks (fig. 5-a) . there was no significant difference between 9 and 14 weeks in the level of neutralizing antibodies in the rla-mers-s groups (fig. 5-b , ii). ndv neutralizing antibodies were detected after the primary immunization and were boosted after the second dose ( fig. 5-b ization antibody titers was apparent between the rla and rla-mers-s groups during the 14-week observation period. mers-cov is a deadly emerging infectious pathogen that poses a serious public health threat. development of mers-cov vaccines is important both to protect susceptible animals and to reduce animal-to-human transmission (haagmans et al. 2016; sabir et al. 2016) . in this study, a recombinant ndv, rla-mers-s, expressing mers-cov s protein was constructed. rla-mers-s maintained high growth titers in embryonated eggs and low pathogenicity in poultry and mice. the recombinant viruses induced significant mers-cov-specific neutralizing antibodies in mice and long-lasting mers-cov specific neutralizing antibodies in camels. our findings demonstrated the potential of rla-mers-s as a candidate veterinary vaccine against mers-cov infection. camels and other mers-cov-reservoir animals that are in close contact with humans may facilitate human infection and lead to sustained outbreak during pandemic outbreaks (perera et al. 2013; chu et al. 2014) . the mers-cov reservoir-harboring capacity by camels draws our attention, as to date there are an estimated 300 000 bactrian camels in china. consequently, given the highly threatening zoonotic potential of mers-cov, we previously carried out a serological and virological surveillance study in the camel herds of west inner mongolia autonomous region of china . although our study did not identify any mers-cov rna and antibodies in the camel herds, constant vigilance is still required. furthermore, those results underscore the necessity in developing a reserve vaccine against mers-cov. the role of neutralizing antibody against s protein of mers-cov in controlling viral infections has been well documented. haagmans et al. (2016) showed that orthopoxvirus-vectored mers-cov vaccines (mva-s) were effective in eliciting antibodies against mers-cov and could significantly reduce the amount of excreted infectious virus and viral rna transcripts upon mers-cov challenge. in addition, kim et al. (2014) reported that recombinant adenoviral vectors encoding mers-cov s protein (ad5.mers-s or ad5.mers-s1) could induce neutralizing antibodies in mice. the success of these viral-vectored mers-cov vaccines demonstrated that protection against mers-cov infection in camels could be achieved by live-attenuated vectored-mers-cov vaccines. while both mva-or adenovirus-vectored mers vaccines are safe and effective, their production process is complicated and costly, thus may not be suitable for use in developing countries. in contrast, ndv-vectored vaccines have several advantages: their safety and efficacy profiles have been confirmed in many studies, are easy to culture and can be grown to high titers in chicken eggs, do not require complicated cell culture equipment, and are thermostable when lyophilized. these 2007) through intranasal immunization. in present study, due to the very limited camel numbers and the difficulties to practicing intranasal instillation in camels, we did not use intranasal immunization. while considering mers-cov's pathogenicity respiratory system, it is necessary to further explore and compare the immunogenicity of rla-mers-cov given by intranasal and intramuscular route. these studies were scheduled in our institute's large animal facillity in future. in the present study, we determined that immunization with rla-mers-s induced a slightly higher level of igg1 than igg2a antibodies against s protein of mers-cov. the result is in agreement with the study by zhang et al. (2016) in which the authors demonstrated that igg1 played a more important role in mers-cov protection in dpp4 transgenic mice. furthermore, this observation suggests that both th1 (igg2a) and th2 (igg1) antibody responses may be critical in inhibiting mers-cov infection. however, we cannot exclude the possibility that th2 (igg1) antibodies may play a dominant role in neutralizing mers-cov infection. our results demonstrated that rla-mers-s induced comparable mers neutralizing antibody levels to mva-s, which may indicate that rla-mers-s is protective. due to the unavailability of mers-cov, it was impossible for us to perform neutralization assays using live mers-cov. instead, we used a vsv-based chimeric virus, vsvδg-gfp-mers, to mimic mers-cov in a serum neutralization test. the recombinant virus used the s protein, instead of the g protein, as its sole membrane anchored protein to attach and enter cells. meanwhile, since the virus was engineered to express an egfp gene as an additional transcription unit, the positively infected cells emitted green fluorescence. our results showed that vsvδg-gfp-mers was replication-competent and could stably express s protein and egfp protein. these features render it a reliable surrogate for mers-cov in evaluating the neutralizing antibody capacity in the unavailability of live mers-cov. due to the unavailability of a large animal high-containment facility, it was not possible to conduct a camel challenge study in the current setting, but may be considered in the future. nevertheless, our results thus demonstrated the potential of rla-mers-s to serve as a candidate mers-cov veterinary vaccine. the present study demonstrated that newcastle disease virus-vectored mers-cov vaccine (rla-mers-s) elicits high-level and lasting neutralizing antibodies in mice and camels. the rla-mers-s could be a potential mers-cov veterinary vaccine candidate for camels and other animals affected by mers-cov. newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous h5n1 avian influenza viruses generation and evaluation of a newcastle disease virus-based h9 avian influenza live vaccine an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels s protein of severe acute respiratory syndromeassociated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients immunization of cattle with recombinant newcastle disease virus expressing bovine herpesvirus-1 (bhv-1) glycoprotein d induces mucosal and serum antibody responses and provides partial protection against bhv-1. vaccine immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice newcastle disease virus-vectored nipah encephalitis vaccines induce b and t cell responses in mice and long-lasting neutralizing antibodies in pigs rift valley fever virus immunity provided by a paramyxovirus vaccine vector the ns1 gene contributes to the virulence of h5n1 avian influenza viruses absence of middle east respiratory syndrome coronavirus in bactrian camels in the west inner mongolia autonomous region of china: surveillance study results from protection against respiratory syncytial virus by a recombinant newcastle disease virus vector human infection with mers coronavirus after exposure to infected camels middle east respiratory syndrome coronavirus in bats, saudi arabia host cell entry of middle east respiratory syndrome coronavirus after two-step, furinmediated activation of the spike protein a roadmap for mers-cov research and product development: report from a world health organization consultation a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques mers, sars, and ebola: the role of super-spreaders in infectious disease replication-deficient vaccinia virus encoding bacteriophage t7 rna polymerase for transient gene expression in mammalian cells mers coronavirus induces apoptosis in kidney and lung by upregulating smad7 and fgf2 identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome coronaviruses -drug discovery and therapeutic options key: cord-317647-vcktnsv8 authors: wang, yinhua; li, wen; jiang, zhiming; xi, xiuming; zhu, yibing title: assessment of the efficacy and safety of ribavirin in treatment of coronavirus-related pneumonia (sars, mers and covid-19): a protocol for systematic review and meta-analysis date: 2020-09-18 journal: medicine (baltimore) doi: 10.1097/md.0000000000022379 sha: doc_id: 317647 cord_uid: vcktnsv8 background: the new coronavirus-related pneumonia is causing a global pandemic without specific antiviral drug. ribavirin has activity against extensive rna and dna viruses. we plan to systematically review the use of ribavirin in patients with coronavirus-related pneumonia and meta-analyze the data with updated studies. methods: embase, pubmed, cochrane library, and china national knowledge infrastructure will be searched from 2002 to june 2021 without language restriction to identify randomized controlled trials. subjects consist of patients with coronavirus-related pneumonia. ribavirin of any dose or route will be compared with the control group of other medication, placebo, or no medication. the primary outcome is the hospital mortality. the secondary outcome includes the hospital length of stay, ventilator-free days in 28 days, median time from start of study treatment to negative nasopharyngeal swab, and adverse events. the mantel-haenszel method will be used for analysis of dichotomous data and the risk ratios will be reported with 95% confidence interval; the inverse-variance method will be used for continuous data and the mean differences will be reported. sensitivity and subgroup analyses will be further performed. the funnel plots or egger test will be used for detection of publication bias. the grade methodology will be used for summarizing the quality of evidence. the trial sequential analysis will be conducted to test whether the current meta-analysis is conclusive. results: the efficacy and safety of ribavirin for treatment of coronavirus-related pneumonia will be systematically reviewed and summarized. the forthcoming results of the ongoing studies focusing on ribavirin in patients with the 2019 noel coronavirus disease will also be included. conclusion: the relevant studies will be summarized and advanced evidence will be provided. prospero registration number: crd42020178900 the new coronavirus-related pneumonia, covid-19, is causing a severe public health emergency worldwide. [1] however, no proved vaccines or antivirus treatment has been available so far. [2, 3] ribavirin is a non-interferon-inducing antiviral agent, having activity against extensive rna and dna viruses. [4] during the outbreak of severe acute respiratory syndrome (sars) in 2003 and middle east respiratory syndrome (mers) in 2012, ribavirin was used for antivirus treatment in consideration of its broad-spectrum antiviral activity. [5] [6] [7] the studies on ribavirin during sars and mers suggested a potential efficacy of using ribavirin; however, the quality of evidence remains low. [7] [8] [9] [10] [11] with the previous experience and rationales, ribavirin has also been put into clinical practice in covid-19. the national health commission of china recommended ribavirin intravenous infusion (500 mg per time, 2-3 times per day for <10 days, in combination with interferon or lopinavir/ritonavir) in the latest covid-19 diagnosis and treatment plan (trial version 7). [12] through a search on the trail registration website of clinivaltrials. gov and chinese clinical trail registry (chictr), we found several ongoing randomized controlled trials (rcts). therefore, we aim to systematically review the use of ribavirin on coronavirus-related pneumonia (sars, merss, and covid-19) and meta-analyze the data with the results of the updated rcts to provide advanced evidence. we aim to assess the safety and efficacy of ribavirin in treatment of coronavirus-related pneumonia (sars, mers and covid-19). this meta-analysis has been registered on the prospero (registration number: crd42020178900) on the prisma-p guideline. [13] four electronic databases (pubmed, cochrane library, embase, and china national knowledge infrastructure) will be searched to identify rcts published from 2002 to june 2021 without language restriction. the potentially relevant references will also be searched. a search strategy using a combination of "coronavirus or corona virus or coronavirus-related or sars or severe acute respiratory syndrome or sars-cov mers or middle east respiratory syndrome or mers-cov or 2019ncov or covid-19 or sars-cov-2 or novel coronavirus or ncp" and "ribavirin or virazole" in all fields has been developed. we also plan to re-run the search before the final analysis. the primary outcome is the hospital mortality. the secondary outcome includes the hospital length of stay, ventilator-free days in 28 days, median time from start of study treatment to negative nasopharyngeal swab, and adverse events. the studies only available as the abstract form will be excluded. 3.5. data collection and analysis 3.5.1. study screening. two reviewers (wy and lw) will independently screen the title/abstract of the records after removal of duplicates. the full text will be obtained for further assessment. the potentially relevant studies in the reference lists will also be searched. the flow diagram of selection process will be summarized for report. two reviewers (wy and lw) will independently extract the data of first author, year of publication, study design, demographics of subjects, interventions, and outcomes of included studies to fill in a predesigned form. any discrepancies will be resolved by a consulting group (xx and zy). two reviewers (wy and lw) will independently evaluate the quality of included studies using the cochrane collaboration's tool. [14] the quality of evidence will be assessed using the grade methodology. [15, 16] any discrepancies will be solved by xx and zy. 3.5.4. statistical analyses and data synthesis. review manager 5.3 will be applied for data analyses. for dichotomous data, the mantel-haenszel method will be used for the synthesis of risk ratios. for continuous data, the inverse-variance method will be used for the synthesis of mean differences. a 2-sided p value of <.05 will be considered statistically significant. 3.5.5. assessment of heterogeneity. a x 2 test and the statistic i 2 will be used for assessment of heterogeneities. [17] the i 2 <40% suggests insignificant heterogeneity: 30% to 60% medium; 50% to 90% substantial; 76% to 100% high. [17] clinical and methodological heterogeneities will be assessed by the 2 reviewers (wy and lw) and discussed with the consulting group (xx and zy) when needed. if there is no significant statistical, methodological, or clinical heterogeneity, a fixed-effect model will be used. otherwise, a random-effect model will be chosen. [17] 3.5.6. subgroup and sensitivity analyses. subgroup analyses of pneumonia caused by different viruses, different control groups, and different dosage or route of administration will be performed. the sensitivity analysis will be conducted by excluding each single study to test the robustness of the results. 3.5.7. assessment of publication bias. funnel plots will be used to assess publication bias when there are >10 studies included. otherwise, the egger test will be used. [18, 19] 3.5.8. trial sequential analysis. the trial sequential analysis (tsa) methodology will be used to assess whether the metaanalysis has a sufficient sample size to draw the current conclusion. [20] the copenhagen tsa software will be used for analyses. the in vitro studies showed an antiviral effect on sars coronavirus (sars-cov) and mers coronavirus (mers-cov). [5, 21, 22] the pooled clinical data on ribavirin for treatment of sars or mers suggested a numerical higher survival rate without statistical significance. [8] [9] [10] for safety concerns, there are studies suggesting that ribavirin might be associated with increased incidence of anemia and liver dysfunction. [11, [23] [24] [25] [26] however, the evidence on sars and mers remains mostly based on cohort or case-control studies. [8] [9] [10] [11] 27] for covid-19, only in vitro data on the activity of ribavirin on sars-cov-2 are available so far. [28] till now the possible benefit and/or harm of ribavirin for treatment of coronavirus-related pneumonia are still inconclusive. recently, the epidemic of covid-19 remains serious in various regions and many clinical trials are designed to provide further evidence. some ongoing rcts focusing on ribavirin for covid-19 can be identified on the registration websites. the forthcoming results of these rcts will bring us a more comprehensive understanding on ribavirin and its indications. our systematic review and meta-analysis will include these updated results and re-assess the efficacy and safety of ribavirin in patients with coronavirus-related pneumonia. china novel coronavirus investigating and research team a novel coronavirus from patients with pneumonia in china therapeutic options for the 2019 novel coronavirus (2019-ncov) efficacy and safety of antiviral treatment for covid-19 from evidence in studies of sarscov-2 and other acute viral infections: a systematic review and meta-analysis ribavirin: a clinical overview ribavirin and interferonbeta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy systematic review of treatment effects effectiveness of ribavirin and corticosteroids for severe acute respiratory syndrome investigational use of ribavirin in the treatment of severe acute respiratory syndrome treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia adverse events associated with high-dose ribavirin: evidence from the toronto outbreak of severe acute respiratory syndrome national health commission of the people's republic of china . covid-19 diagnosis and treatment plan preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement the cochrane collaboration's tool for assessing risk of bias in randomised trials grade guidelines: 1. introduction-grade evidence profiles and summary of findings tables grade guidelines: 3. rating the quality of evidence cochrane handbook for systematic reviews of interventions version 5.1.0. the cochrane collaboration available at: www.cochrane-handbook.org. accessed operating characteristics of a rank correlation test for publication bias bias in meta-analysis detected by a simple, graphical test trial sequential analysis may establish when firm evidence is reached in cumulative meta-analysis in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds interferon-b and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada clinical features and short-term outcomes of 144 patients with sars in the greater toronto area severe acute respiratory syndrome: report of treatment and outcome after a major outbreak temporal patterns of hepatic dysfunction and disease severity in patients with sars (4) ifn-a2a or ifn-b1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study infectious diseases society of america guidelines on the treatment and management of patients with covid-19 medicine (2020) 99:38 www.md-journal key: cord-317435-4yuw7jo3 authors: zhou, yadi; hou, yuan; shen, jiayu; huang, yin; martin, william; cheng, feixiong title: network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 date: 2020-03-16 journal: cell discov doi: 10.1038/s41421-020-0153-3 sha: doc_id: 317435 cord_uid: 4yuw7jo3 human coronaviruses (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov) and 2019 novel coronavirus (2019-ncov, also known as sars-cov-2), lead global epidemics with high morbidity and mortality. however, there are currently no effective drugs targeting 2019-ncov/sars-cov-2. drug repurposing, representing as an effective drug discovery strategy from existing drugs, could shorten the time and reduce the cost compared to de novo drug discovery. in this study, we present an integrative, antiviral drug repurposing methodology implementing a systems pharmacology-based network medicine platform, quantifying the interplay between the hcov–host interactome and drug targets in the human protein–protein interaction network. phylogenetic analyses of 15 hcov whole genomes reveal that 2019-ncov/sars-cov-2 shares the highest nucleotide sequence identity with sars-cov (79.7%). specifically, the envelope and nucleocapsid proteins of 2019-ncov/sars-cov-2 are two evolutionarily conserved regions, having the sequence identities of 96% and 89.6%, respectively, compared to sars-cov. using network proximity analyses of drug targets and hcov–host interactions in the human interactome, we prioritize 16 potential anti-hcov repurposable drugs (e.g., melatonin, mercaptopurine, and sirolimus) that are further validated by enrichment analyses of drug-gene signatures and hcov-induced transcriptomics data in human cell lines. we further identify three potential drug combinations (e.g., sirolimus plus dactinomycin, mercaptopurine plus melatonin, and toremifene plus emodin) captured by the “complementary exposure” pattern: the targets of the drugs both hit the hcov–host subnetwork, but target separate neighborhoods in the human interactome network. in summary, this study offers powerful network-based methodologies for rapid identification of candidate repurposable drugs and potential drug combinations targeting 2019-ncov/sars-cov-2. coronaviruses (covs) typically affect the respiratory tract of mammals, including humans, and lead to mild to severe respiratory tract infections 1 . in the past two decades, two highly pathogenic human covs (hcovs), including severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov), emerging from animal reservoirs, have led to global epidemics with high morbidity and mortality 2 . for example, 8098 individuals were infected and 774 died in the sars-cov pandemic, which cost the global economy with an estimated $30 to $100 billion 3, 4 . according to the world health organization (who), as of november 2019, mers-cov has had a total of 2494 diagnosed cases causing 858 deaths, the majority in saudi arabia 2 . in december 2019, the third pathogenic hcov, named 2019 novel coronavirus (2019-ncov/sars-cov-2), as the cause of coronavirus disease 2019 (abbreviated as covid19) 5 , was found in wuhan, china. as of 24 february 2020, there have been over 79,000 cases with over 2600 deaths for the 2019-ncov/sars-cov-2 outbreak worldwide; furthermore, human-to-human transmission has occurred among close contacts 6 . however, there are currently no effective medications against 2019-ncov/sars-cov-2. several national and international research groups are working on the development of vaccines to prevent and treat the 2019-ncov/sars-cov-2, but effective vaccines are not available yet. there is an urgent need for the development of effective prevention and treatment strategies for 2019-ncov/sars-cov-2 outbreak. although investment in biomedical and pharmaceutical research and development has increased significantly over the past two decades, the annual number of new treatments approved by the u.s. food and drug administration (fda) has remained relatively constant and limited 7 . a recent study estimated that pharmaceutical companies spent $2.6 billion in 2015, up from $802 million in 2003, in the development of an fda-approved new chemical entity drug 8 . drug repurposing, represented as an effective drug discovery strategy from existing drugs, could significantly shorten the time and reduce the cost compared to de novo drug discovery and randomized clinical trials [9] [10] [11] . however, experimental approaches for drug repurposing is costly and time-consuming 12 . computational approaches offer novel testable hypotheses for systematic drug repositioning [9] [10] [11] 13, 14 . however, traditional structure-based methods are limited when threedimensional (3d) structures of proteins are unavailable, which, unfortunately, is the case for the majority of human and viral targets. in addition, targeting single virus proteins often has high risk of drug resistance by the rapid evolution of virus genomes 1 . viruses (including hcov) require host cellular factors for successful replication during infection 1 . systematic identification of virus-host protein-protein interactions (ppis) offers an effective way toward elucidating the mechanisms of viral infection 15, 16 . subsequently, targeting cellular antiviral targets, such as virus-host interactome, may offer a novel strategy for the development of effective treatments for viral infections 1 , including sars-cov 17 , mers-cov 17 , ebola virus 18 , and zika virus 14, [19] [20] [21] . we recently presented an integrated antiviral drug discovery pipeline that incorporated gene-trap insertional mutagenesis, known functional drug-gene network, and bioinformatics analyses 14 . this methodology allows to identify several candidate repurposable drugs for ebola virus 11, 14 . our work over the last decade has demonstrated how network strategies can, for example, be used to identify effective repurposable drugs 13, [22] [23] [24] [25] [26] [27] and drug combinations 28 for multiple human diseases. for example, network-based drug-disease proximity sheds light on the relationship between drugs (e.g., drug targets) and disease modules (molecular determinants in disease pathobiology modules within the ppis), and can serve as a useful tool for efficient screening of potentially new indications for approved drugs, as well as drug combinations, as demonstrated in our recent studies 13, 23, 27, 28 . in this study, we present an integrative antiviral drug repurposing methodology, which combines a systems pharmacology-based network medicine platform that quantifies the interplay between the virus-host interactome and drug targets in the human ppi network. the basis for these experiments rests on the notions that (i) the proteins that functionally associate with viral infection (including hcov) are localized in the corresponding subnetwork within the comprehensive human ppi network and (ii) proteins that serve as drug targets for a specific disease may also be suitable drug targets for potential antiviral infection owing to common ppis and functional pathways elucidated by the human interactome ( fig. 1) . we follow this analysis with bioinformatics validation of drug-induced gene signatures and hcovinduced transcriptomics in human cell lines to inspect the postulated mechanism-of-action in a specific hcov for which we propose repurposing (fig. 1 ). to date, seven pathogenic hcovs (fig. 2a, b) have been found: 1, 29 (i) 2019-ncov/sars-cov-2, sars-cov, mers-cov, hcov-oc43, and hcov-hku1 are β genera, and (ii) hcov-nl63 and hcov-229e are α genera. we performed the phylogenetic analyses using the wholegenome sequence data from 15 hcovs to inspect the evolutionary relationship of 2019-ncov/sars-cov-2 with other hcovs. we found that the whole genomes of 2019-ncov/sars-cov-2 had~99.99% nucleotide sequence identity across three diagnosed patients (supplementary table s1 ). the 2019-ncov/sars-cov-2 shares the highest nucleotide sequence identity (79.7%) with sars-cov among the six other known pathogenic hcovs, revealing conserved evolutionary relationship between 2019-ncov/sars-cov-2 and sars-cov (fig. 2a) . hcovs have five major protein regions for virus structure assembly and viral replications 29 , including replicase complex (orf1ab), spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins (fig. 2b) . the orf1ab gene encodes the non-structural proteins (nsp) of viral rna synthesis complex through proteolytic processing 30 . the nsp12 is a viral rna-dependent rna polymerase, together with co-factors nsp7 and nsp8 possessing high polymerase activity. from the protein 3d structure view of sars-cov nsp12, it contains a larger n-terminal extension (which binds to nsp7 and nsp8) and polymerase domain (fig. 2c) . the spike is a transmembrane glycoprotein that plays a pivotal role in mediating viral infection through binding the host receptor 31, 32 . figure 2d shows the 3d structure of the spike protein bound with the host receptor angiotensin converting enznyme2 (ace2) in sars-cov (pdb id: 6ack). a recent study showed that 2019-ncov/sars-cov-2 is able to utilize ace2 as an entry receptor in ace2-expressing cells 33 , suggesting potential drug targets for therapeutic development. furthermore, cryo-em structure of the spike and biophysical assays reveal that the 2019-ncov/sars-cov-2 spike binds ace2 with higher affinity than sars-cov 34 . in addition, the nucleocapsid is also an important subunit for packaging the viral genome through protein oligomerization 35 , and the single nucleocapsid structure is shown in fig. 2e . protein sequence alignment analyses indicated that the 2019-ncov/sars-cov-2 was most evolutionarily conserved with sars-cov (supplementary table s2 ). specifically, the envelope and nucleocapsid proteins of 2019-ncov/sars-cov-2 are two evolutionarily conserved regions, with sequence identities of 96% and 89.6%, respectively, compared to sars-cov (supplementary table s2 ). however, the spike protein exhibited the lowest sequence conservation (sequence identity of 77%) between 2019-ncov/sars-cov-2 and sars-cov. meanwhile, the spike protein of 2019-ncov/sars-cov-2 only has 31.9% sequence identity compared to mers-cov. fig. 1 overall workflow of this study. our network-based methodology combines a systems pharmacology-based network medicine platform that quantifies the interplay between the virus-host interactome and drug targets in the human ppi network. a human coronavirus (hcov)-associated host proteins were collected from literatures and pooled to generate a pan-hcov protein subnetwork. b network proximity between drug targets and hcov-associated proteins was calculated to screen for candidate repurposable drugs for hcovs under the human protein interactome model. c, d gene set enrichment analysis was utilized to validate the network-based prediction. e top candidates were further prioritized for drug combinations using network-based method captured by the "complementary exposure" pattern: the targets of the drugs both hit the hcov-host subnetwork, but target separate neighborhoods in the human interactome network. f overall hypothesis of the network-based methodology: (i) the proteins that functionally associate with hcovs are localized in the corresponding subnetwork within the comprehensive human interactome network; and (ii) proteins that serve as drug targets for a specific disease may also be suitable drug targets for potential antiviral infection owing to common protein-protein interactions elucidated by the human interactome. to depict the hcov-host interactome network, we assembled the cov-associated host proteins from four known hcovs (sars-cov, mers-cov, hcov-229e, and hcov-nl63), one mouse mhv, and one avian ibv (n protein) (supplementary table s3 ). in total, we obtained 119 host proteins associated with covs with various experimental evidence. specifically, these host proteins are either the direct targets of hcov proteins or are involved in crucial pathways of hcov infection. the hcov-host interactome network is shown in fig. 3a . we identified several hub proteins including jun, xpo1, npm1, and hnrnpa1, with the highest number of connections within the 119 proteins. kegg pathway enrichment analysis revealed multiple significant biological pathways (adjusted p value < 0.05), including measles, rna transport, nf-kappa b signaling, epstein-barr virus infection, and influenza (fig. 3b ). gene ontology (go) biological process enrichment analysis further confirmed multiple viral infection-related processes (adjusted p value < 0.001), including viral life cycle, modulation by virus of host morphology or physiology, viral process, positive regulation of viral life cycle, transport of virus, and virion attachment to host cell (fig. 3c ). we then mapped the known drug-target network (see materials and methods) into the hcov-host interactome to search for druggable, cellular targets. we found that 47 human proteins (39%, blue nodes in fig. 3a) can be targeted by at least one approved drug or experimental drug under clinical trials. for example, gsk3b, dpp4, smad3, parp1, and ikbkb are the most targetable proteins. the high druggability of hcov-host interactome motivates us to develop a drug repurposing strategy by specifically targeting cellular proteins associated with hcovs for potential treatment of 2019-ncov/sars-cov-2. the basis for the proposed network-based drug repurposing methodologies rests on the notions that the proteins that associate with and functionally govern viral infection are localized in the corresponding subnetwork ( fig. 1a) within the comprehensive human interactome network. for a drug with multiple targets to be effective against an hcov, its target proteins should be within or in the immediate vicinity of the corresponding subnetwork in the human protein-protein interactome ( fig. 1 ), as we demonstrated in multiple diseases 13, 22, 23, 28 using this network-based strategy. we used a state-of-theart network proximity measure to quantify the relationship between hcov-specific subnetwork (fig. 3a) and drug targets in the human interactome. we constructed a drug-target network by assembling target information for more than 2000 fda-approved or experimental drugs (see materials and methods). to improve the quality and completeness of the human protein interactome network, we integrated ppis with five types of experimental data: (1) binary ppis from 3d protein structures; (2) binary ppis from unbiased high-throughput yeast-two-hybrid assays; (3) experimentally identified kinase-substrate interactions; (4) signaling networks derived from experimental data; and (5) literature-derived ppis with various experimental evidence (see materials and methods). we used a z-score (z) measure and permutation test to reduce the study bias in network proximity analyses (including hub nodes in the human interactome network by literature-derived ppi data bias) as described in our recent studies 13, 28 . in total, we computationally identified 135 drugs that were associated (z < −1.5 and p < 0.05, permutation test) with the hcov-host interactome (fig. 4a , supplementary tables s4 and 5 ). to validate bias of the pooled cellular proteins from six covs, we further calculated the network proximities of all the drugs for four covs with a large number of know host proteins, including sars-cov, mers-cov, ibv, and mhv, separately. we found that the z-scores showed consistency among the pooled 119 hcov-associated proteins and other four individual covs (fig. 4b) . the pearson correlation coefficients of the proximities of all the drugs for the pooled hcov are 0.926 vs. sars-cov (p < 0.001, t distribution), 0.503 vs. mers-cov (p < 0.001), 0.694 vs. ibv (p < 0.001), and 0.829 vs. mhv (p < 0.001). these network proximity analyses offer putative repurposable candidates for potential prevention and treatment of hcovs. to further validate the 135 repurposable drugs against hcovs, we first performed gene set enrichment analysis (gsea) using transcriptome data of mers-cov and sars-cov infected host cells (see methods). these transcriptome data were used as gene signatures for hcovs. additionally, we downloaded the gene expression data of drug-treated human cell lines from the connectivity map (cmap) database 36 to obtain drug-gene signatures. we calculated a gsea score (see methods) for each drug and used this score as an indication of bioinformatics validation of the 135 drugs. specifically, an enrichment score (es) was calculated for each hcov data set, and es > 0 and p < 0.05 (permutation test) was used as cut-off for a significant association of gene signatures between a drug and a specific hcov data set. the gsea score, ranging from 0 to 3, is the number of data sets that met these criteria for a specific drug. mesalazine (an fig. 4 a discovered drug-hcov network. a a subnetwork highlighting network-predicted drug-hcov associations connecting 135 drugs and hcovs. from the 2938 drugs evaluated, 135 ones achieved significant proximities between drug targets and the hcov-associated proteins in the human interactome network. drugs are colored by their first-level of the anatomical therapeutic chemical (atc) classification system code. b a heatmap highlighting network proximity values for sars-cov, mers-cov, ibv, and mhv, respectively. color key denotes network proximity (z-score) between drug targets and the hcov-associated proteins in the human interactome network. p value was computed by permutation test. approved drug for inflammatory bowel disease), sirolimus (an approved immunosuppressive drug), and equilin (an approved agonist of the estrogen receptor for menopausal symptoms) achieved the highest gsea scores of 3, followed by paroxetine and melatonin with gsea scores of 2. we next selected 16 high-confidence repurposable drugs ( fig. 5a and table 1 ) against hcovs using subject matter expertise based on a combination of factors: (i) strength of the network-predicted associations (a smaller network proximity score in supplementary table s4 ); (ii) validation by gsea analyses; (iii) literature-reported antiviral evidence, and (iv) fewer clinically reported side effects. specifically, we showcased several selected repurposable drugs with literature-reported antiviral evidence as below. an overexpression of estrogen receptor has been shown to play a crucial role in inhibiting viral replication 37 . selective estrogen receptor modulators (serms) have been reported to play a broader role in inhibiting viral replication through the non-classical pathways associated with estrogen receptor 37 . serms interfere at the post viral entry step and affect the triggering of fusion, as the serms' antiviral activity still can be observed in the absence of detectable estrogen receptor expression 18 . toremifene (z = -3.23, fig. 5a ), the first generation of nonsteroidal serm, exhibits potential effects in blocking various viral infections, including mers-cov, sars-cov, and ebola virus in established cell lines 17, 38 . compared to the classical esr1-related antiviral pathway, toremifene prevents fusion between the viral and endosomal membrane by interacting with and destabilizing the virus membrane glycoprotein, and eventually inhibiting viral replication 39 . as shown in fig. 5b , toremifene potentially affects several key host proteins associated with hcov, such as rpl19, hnrnpa1, npm1, eif3i, eif3f, and eif3e 40, 41 . equilin (z = -2.52 and gsea score = 3), an estrogenic steroid produced by horses, also has been proven to have moderate activity in inhibiting the entry of zaire ebola virus glycoprotein and human immunodeficiency virus (zebov-gp/hiv) 18 . altogether, network-predicted serms (such as toremifene and equilin) offer candidate repurposable drugs for 2019-ncov/sars-cov-2. angiotensin receptor blockers (arbs) have been reported to associate with viral infection, including hcovs [42] [43] [44] . irbesartan (z = -5.98), a typical arb, was approved by the fda for treatment of hypertension and diabetic nephropathy. here, network proximity analysis shows a significant association between irbesartan's targets and hcov-associated host proteins in the human interactome. as shown in fig. 5c , irbesartan targets slc10a1, encoding the sodium/bile acid cotransporter (ntcp) protein that has been identified as a functional pres1-specific receptor for the hepatitis b virus (hbv) and the hepatitis delta virus (hdv). irbesartan can inhibit ntcp, thus inhibiting viral entry 45, 46 . slc10a1 interacts with c11orf74, a potential transcriptional repressor that interacts with nsp-10 of sars-cov 47 . there are several other arbs (such as eletriptan, frovatriptan, and zolmitriptan) in which their targets are potentially associated with hcov-associated host proteins in the human interactome. previous studies have confirmed the mammalian target of rapamycin complex 1 (mtorc1) as the key factor in regulating various viruses' replications, including andes orthohantavirus and coronavirus 48, 49 . sirolimus (z = -2.35 and gsea score = 3), an inhibitor of mammalian target of rapamycin (mtor), was reported to effectively block viral protein expression and virion release effectively 50 . indeed, the latest study revealed the clinical application: sirolimus reduced mers-cov infection by over 60% 51 . moreover, sirolimus usage in managing patients with severe h1n1 pneumonia and acute respiratory failure can improve those patients' prognosis significantly 50 . mercaptopurine (z = -2.44 and gsea score = 1), an antineoplastic agent with immunosuppressant property, has been used to treat cancer since the 1950s and expanded its application to several autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and crohn's disease 52 . (see figure on previous page) fig. 5 a discovered drug-protein-hcov network for 16 candidate repurposable drugs. a network-predicted evidence and gene set enrichment analysis (gsea) scores for 16 potential repurposable drugs for hcovs. the overall connectivity of the top drug candidates to the hcovassociated proteins was examined. most of these drugs indirectly target hcov-associated proteins via the human protein-protein interaction networks. all the drug-target-hcov-associated protein connections were examined, and those proteins with at least five connections are shown. the box heights for the proteins indicate the number of connections. gsea scores for eight drugs were not available (na) due to the lack of transcriptome profiles for the drugs. b-e inferred mechanism-of-action networks for four selected drugs: b toremifene (first-generation nonsteroidalselective estrogen receptor modulator), c irbesartan (an angiotensin receptor blocker), d mercaptopurine (an antimetabolite antineoplastic agent with immunosuppressant properties), and e melatonin (a biogenic amine for treating circadian rhythm sleep disorders). 53, 54 . mechanistically, mercaptopurine potentially target several host proteins in hcovs, such as jun, pabpc1, npm1, and ncl 40, 55 (fig. 5d) . inflammatory pathways play essential roles in viral infections 56, 57 . as a biogenic amine, melatonin (n-acetyl-5-methoxytryptamine) (z = -1.72 and gsea score = 2) plays a key role in various biological processes, and offers a potential strategy in the management of viral infections 58, 59 . viral infections are often associated with immune-inflammatory injury, in which the level of oxidative stress increases significantly and leaves negative effects on the function of multiple organs 60 . the antioxidant effect of melatonin makes it a putative candidate drug to relieve patients' clinical symptoms in antiviral treatment, even though melatonin cannot eradicate or even curb the viral replication or transcription 61, 62 . in addition, the application of melatonin may prolong patients' survival time, which may provide a chance for patients' immune systems to recover and eventually eradicate the virus. as shown in fig. 5e , melatonin indirectly targets several hcov cellular targets, including ace2, bcl2l1, jun, and ikbkb. eplerenone (z = -1.59), an aldosterone receptor antagonist, is reported to have a similar anti-inflammatory effect as melatonin. by inhibiting mast-cell-derived proteinases and suppressing fibrosis, eplerenone can improve survival of mice infected with encephalomyocarditis virus 63 . in summary, our network proximity analyses offer multiple candidate repurposable drugs that target diverse cellular pathways for potential prevention and treatment of 2019-ncov/sars-cov-2. however, further preclinical experiments 64 and clinical trials are required to verify the clinical benefits of these network-predicted candidates before clinical use. drug combinations, offering increased therapeutic efficacy and reduced toxicity, play an important role in treating various viral infections 65 . however, our ability to identify and validate effective combinations is limited by a combinatorial explosion, driven by both the large number of drug pairs and dosage combinations. in our recent study, we proposed a novel network-based methodology to identify clinically efficacious drug combinations 28 . relying on approved drug combinations for hypertension and cancer, we found that a drug combination was therapeutically effective only if it was captured by the "complementary exposure" pattern: the targets of the drugs both hit the disease module, but target separate neighborhoods (fig. 6a) . here we sought to identify drug combinations that may provide a synergistic effect in potentially treating 2019-ncov/sars-cov-2 with welldefined mechanism-of-action by network analysis. for the 16 potential repurposable drugs (fig. 5a, table 1 ), we showcased three network-predicted candidate drug combinations for 2019-ncov/sars-cov-2. all predicted possible combinations can be found in supplementary table s6 . sirolimus, an inhibitor of mtor with both antifungal and antineoplastic properties, has demonstrated to improve outcomes in patients with severe h1n1 pneumonia and acute respiratory failure 50 . the mtor signaling plays an essential role for mers-cov infection 66 . dactinomycin, also known actinomycin d, is an approved rna synthesis inhibitor for treatment of various cancer types. an early study showed that dactinomycin (1 μg/ml) inhibited the growth of feline enteric cov 67 . as shown in fig. 6b , our network analysis shows that sirolimus and dactinomycin synergistically target hcov-associated host protein subnetwork by "complementary exposure" pattern, offering potential combination regimens for treatment of hcov. specifically, sirolimus and dactinomycin may inhibit both mtor signaling and rna synthesis pathway (including dna topoisomerase 2-alpha (top2a) and dna topoisomerase 2-beta (top2b)) in hcov-infected cells (fig. 6b) . toremifene is among the approved first-generation nonsteroidal serms for the treatment of metastatic breast cancer 68 . serms (including toremifene) inhibited ebola virus infection 18 by interacting with and destabilizing the ebola virus glycoprotein 39 . in vitro assays have demonstrated that toremifene inhibited growth of mers-cov 17,69 and sara-cov 38 (table 1) . emodin, an anthraquinone derivative extracted from the roots of rheum tanguticum, has been reported to have various anti-virus effects. specifically, emdoin inhibited sars-cov-associated 3a protein 70 , and blocked an interaction between the sars-cov spike protein and ace2 (ref. 71 ). altogether, network analyses and published experimental data suggested that combining toremifene and emdoin offered a potential therapeutic approach for 2019-ncov/ sars-cov-2 (fig. 6c) . as shown in fig. 5a , targets of both mercaptopurine and melatonin showed strong network proximity with hcovassociated host proteins in the human interactome network. recent in vitro and in vivo studies identified mercaptopurine as a selective inhibitor of both sars-cov and mers-cov by targeting papain-like protease 53, 54 . melatonin was reported in potential antiviral infection via its anti-inflammatory and antioxidant effects [58] [59] [60] [61] [62] . melatonin indirectly regulates ace2 expression, a key entry receptor involved in viral infection of hcovs, including 2019-ncov/sars-cov-2 (ref. 33 ). specifically, melatonin was reported to inhibit calmodulin and calmodulin interacts with ace2 by inhibiting shedding of its ectodomain, a key infectious process of sars-cov 72, 73 . jun, also known as c-jun, is a key host protein involving in hcov infectious bronchitis virus 74 . as shown in fig. 6d , mercaptopurine and melatonin may synergistically block c-jun signaling by targeting multiple cellular targets. in summary, combination of mercaptopurine and melatonin may offer a potential combination therapy for 2019-ncov/sars-cov-2 by synergistically targeting papainlike protease, ace2, c-jun signaling, and antiinflammatory pathways (fig. 6d) . however, further experimental observations on ace2 pathways by melatonin in 2019-ncov/sars-cov-2 are highly warranted. in this study, we presented a network-based methodology for systematic identification of putative repurposable drugs and drug combinations for potential treatment of 2019-ncov/sars-cov-2. integration of drug-target networks, hcov-host interactions, hcovinduced transcriptome in human cell lines, and human protein-protein interactome network are essential for such identification. based on comprehensive evaluation, we prioritized 16 candidate repurposable drugs (fig. 5 ) and 3 potential drug combinations (fig. 6) for targeting 2019-ncov/sars-cov-2. however, although the majority of predictions have been validated by various literature data (table 1) , all network-predicted repurposable drugs and drug combinations must be validated in various 2019-ncov/sars-cov-2 experimental assays 64 and randomized clinical trials before being used in patients. we acknowledge several limitations in the current study. although 2019-ncov/sars-cov-2 shared high nucleotide sequence identity with other hcovs (fig. 2) , our predictions are not 2019-ncov/sars-cov-2 specific by lack of the known host proteins on 2019-ncov/sars-cov-2. we used a low binding affinity value of 10 μm as a threshold to define a physical drug-target interaction. however, a stronger binding affinity threshold (e.g., 1 μm) may be a more suitable cut-off in drug discovery, although it will generate a smaller drug-target network. although sizeable efforts were made for assembling large scale, experimentally reported drug-target networks from publicly available databases, the network data may be incomplete and some drug-target interactions may be functional associations, instead of physical bindings. for example, silvestrol, a natural product from the flavagline, was found to have antiviral activity against ebola 75 and coronaviruses 76 . after adding its target, an rna helicase enzyme eif4a 76 , silvestrol was predicted to be significantly associated with hcovs (z = -1.24, p = 0.041) by network proximity analysis. to increase coverage of drug-target networks, we may use computational approaches to systematically predict the drug-target interactions further 25, 26 . in addition, the collected virus-host interactions are far from completeness and the quality can be influenced by multiple factors, including different experimental assays and human cell line models. we may computationally predict a new virus-host interactome for 2019-ncov/sars-cov-2 using sequence-based and structure-based approaches 77 . drug targets representing nodes within cellular networks are often intrinsically coupled with both therapeutic and adverse profiles 78 , as drugs can inhibit or activate protein functions (including antagonists vs. agonists). the current systems pharmacology model cannot separate therapeutic (antiviral) effects from those predictions due to lack of detailed pharmacological effects of drug targets and unknown functional consequences of virus-host interactions. comprehensive identification of the virus-host interactome for 2019-ncov/sars-cov-2, with specific biological effects using functional genomics assays 79, 80 , will significantly improve the accuracy of the proposed network-based methodologies further. owing to a lack of the complete drug-target information (such as the molecular "promiscuity" of drugs), the dose-response and dose-toxicity effects for both (see figure on previous page) fig. 6 network-based rational design of drug combinations for 2019-ncov/sars-cov-2. a the possible exposure mode of the hcovassociated protein module to the pairwise drug combinations. an effective drug combination will be captured by the "complementary exposure" pattern: the targets of the drugs both hit the hcov-host subnetwork, but target separate neighborhoods in the human interactome network. z ca and z cb denote the network proximity (z-score) between targets (drugs a and b) and a specific hcov. s ab denotes separation score (see materials and methods) of targets between drug a and drug b. b-d inferred mechanism-of-action networks for three selected pairwise drug combinations: b sirolimus (a potent immunosuppressant with both antifungal and antineoplastic properties) plus dactinomycin (an rna synthesis inhibitor for treatment of various tumors), c toremifene (first-generation nonsteroidal-selective estrogen receptor modulator) plus emodin (an experimental drug for the treatment of polycystic kidney), and d melatonin (a biogenic amine for treating circadian rhythm sleep disorders) plus mercaptopurine (an antimetabolite antineoplastic agent with immunosuppressant properties). repurposable drugs and drug combinations cannot be identified in the current network models. for example, mesalazine, an approved drug for inflammatory bowel disease, is a top network-predicted repurposable drug associated with hcovs (fig. 5a ). yet, several clinical studies showed the potential pulmonary toxicities (including pneumonia) associated with mesalazine usage 81, 82 . integration of lung-specific gene expression 23 of 2019-ncov/sars-cov-2 host proteins and physiologically based pharmacokinetic modeling 83 may reduce side effects of repurposable drugs or drug combinations. preclinical studies are warranted to evaluate in vivo efficiency and side effects before clinical trials. furthermore, we only limited to predict pairwise drug combinations based on our previous network-based framework 28 . however, we expect that our methodology remain to be a useful network-based tool for prediction of combining multiple drugs toward exploring network relationships of multiple drugs' targets with the hcov-host subnetwork in the human interactome. finally, we aimed to systematically identify repurposable drugs by specifically targeting ncov host proteins only. thus, our current network models cannot predict repurposable drugs from the existing anti-virus drugs that target virus proteins only. thus, combination of the existing anti-virus drugs (such as remdesivir 64 ) with the network-predicted repurposable drugs (fig. 5 ) or drug combinations (fig. 6 ) may improve coverage of current network-based methodologies by utilizing multi-layer network framework 16 . in conclusion, this study offers a powerful, integrative network-based systems pharmacology methodology for rapid identification of repurposable drugs and drug combinations for the potential treatment of 2019-ncov/ sars-cov-2. our approach can minimize the translational gap between preclinical testing results and clinical outcomes, which is a significant problem in the rapid development of efficient treatment strategies for the emerging 2019-ncov/sars-cov-2 outbreak. from a translational perspective, if broadly applied, the network tools developed here could help develop effective treatment strategies for other emerging viral infections and other human complex diseases as well. in total, we collected dna sequences and protein sequences for 15 hcovs, including three most recent 2019-ncov/sars-cov-2 genomes, from the ncbi genbank database (28 january 2020, supplementary table s1 ). whole-genome alignment and protein sequence identity calculation were performed by multiple sequence alignment in embl-ebi database (https:// www.ebi.ac.uk/) with default parameters. the neighbor joining (nj) tree was computed from the pairwise phylogenetic distance matrix using mega x 84 with 1000 bootstrap replicates. the protein alignment and phylogenetic tree of hcovs were constructed by mega x 84 . we collected hcov-host protein interactions from various literatures based on our sizeable efforts. the hcov-associated host proteins of several hcovs, including sars-cov, mers-cov, ibv, mhv, hcov-229e, and hcov-nl63 were pooled. these proteins were either the direct targets of hcov proteins or were involved in critical pathways of hcov infection identified by multiple experimental sources, including highthroughput yeast-two-hybrid (y2h) systems, viral protein pull-down assay, in vitro co-immunoprecipitation and rna knock down experiment. in total, the virus-host interaction network included 6 hcovs with 119 host proteins (supplementary table s3 ). next, we performed kyoto encyclopedia of genes and genomes (kegg) and gene ontology (go) enrichment analyses to evaluate the biological relevance and functional pathways of the hcov-associated proteins. all functional analyses were performed using enrichr 85 . here, we collected drug-target interaction information from the drugbank database (v4.3) 86 , therapeutic target database (ttd) 87 , pharmgkb database, chembl (v20) 88 , bindingdb 89 , and iuphar/bps guide to pharmacology 90 . the chemical structure of each drug with smiles format was extracted from drug-bank 86 . here, drug-target interactions meeting the following three criteria were used: (i) binding affinities, including k i , k d , ic 50 , or ec 50 each ≤10 μm; (ii) the target was marked as "reviewed" in the uniprot database 91 ; and (iii) the human target was represented by a unique uni-prot accession number. the details for building the experimentally validated drug-target network are provided in our recent studies 13, 23, 28 . to build a comprehensive list of human ppis, we assembled data from a total of 18 bioinformatics and systems biology databases with five types of experimental evidence: (i) binary ppis tested by high-throughput yeasttwo-hybrid (y2h) systems; (ii) binary, physical ppis from protein 3d structures; (iii) kinase-substrate interactions by literature-derived low-throughput or high-throughput experiments; (iv) signaling network by literature-derived low-throughput experiments; and (v) literature-curated ppis identified by affinity purification followed by mass spectrometry (ap-ms), y2h, or by literature-derived low-throughput experiments. all inferred data, including evolutionary analysis, gene expression data, and metabolic associations, were excluded. the genes were mapped to their entrez id based on the ncbi database 92 as well as their official gene symbols based on genecards (https:// www.genecards.org/). in total, the resulting human protein-protein interactome used in this study includes 351,444 unique ppis (edges or links) connecting 17,706 proteins (nodes), representing a 50% increase in the number of the ppis we have used previously. detailed descriptions for building the human protein-protein interactome are provided in our previous studies 13, 23, 28, 93 . we posit that the human ppis provide an unbiased, rational roadmap for repurposing drugs for potential treatment of hcovs in which they were not originally approved. given c, the set of host genes associated with a specific hcov, and t, the set of drug targets, we computed the network proximity of c with the target set t of each drug using the "closest" method: where d(c, t) is the shortest distance between gene c and t in the human protein interactome. the network proximity was converted to z-score based on permutation tests: where d r and σ r were the mean and standard deviation of the permutation test repeated 1000 times, each time with two randomly selected gene lists with similar degree distributions to those of c and t. the corresponding p value was calculated based on the permutation test results. z-score < −1.5 and p < 0.05 were considered significantly proximal drug-hcov associations. all networks were visualized using gephi 0.9.2 (https://gephi.org/). for this network-based approach for drug combinations to be effective, we need to establish if the topological relationship between two drug-target modules reflects biological and pharmacological relationships, while also quantifying their network-based relationship between drug targets and hcov-associated host proteins (drug-drug-hcov combinations). to identify potential drug combinations, we combined the top lists of drugs. then, "separation" measure s ab was calculated for each pair of drugs a and b using the following method: where d á h i was calculated based on the "closest" method. our key methodology is that a drug combination is therapeutically effective only if it follows a specific relationship to the disease module, as captured by complementary exposure patterns in targets' modules of both drugs without overlapping toxic mechanisms 28 . we performed the gene set enrichment analysis as an additional prioritization method. we first collected three differential gene expression data sets of hosts infected by hcovs from the ncbi gene expression omnibus (geo). among them, two transcriptome data sets were sars-cov-infected samples from patient's peripheral blood 94 (gse1739) and calu-3 cells 95 (gse33267), respectively. one transcriptome data set was mers-cov-infected calu-3 cells 96 (gse122876). adjusted p value less than 0.01 was defined as differentially expressed genes. these data sets were used as hcov-host signatures to evaluate the treatment effects of drugs. differential gene expression in cells treated with various drugs were retrieved from the connectivity map (cmap) database 36 , and were used as gene profiles for the drugs. for each drug that was in both the cmap data set and our drug-target network, we calculated an enrichment score (es) for each hcov signature data set based on previously described methods 97 where j = 1, 2, …, s were the genes of hcov signature data set sorted in ascending order by their rank in the gene profiles of the drug being evaluated. the rank of gene j is denoted by v(j), where 1 ≤ v(j) ≤ r, with r being the number of genes (12,849) from the drug profile. then, es up/down was set to a up/down if a up/down > b up/down , and was set to −b up/down if b up/down > a up/down . permutation tests repeated 100 times using randomly generated gene lists with the same number of up-and down-regulated genes as the hcov signature data set were performed to measure the significance of the es scores. drugs were considered to have potential treatment effect if es > 0 and p < 0.05, and the number of such hcov signature data sets were used as the final gsea score that ranges from 0 to 3. coronaviruses-drug discovery and therapeutic options coronavirus infections-more than just the common cold sars and mers: recent insights into emerging coronaviruses host factors in coronavirus replication epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia putting the patient back together-social medicine, network medicine, and the limits of reductionism the $2.6 billion pill-methodologic and policy considerations silico oncology drug repositioning and polypharmacology individualized network-based drug repositioning infrastructure for precision oncology in the panomics era drug repurposing: new treatments for zika virus infection? a comprehensive map of molecular drug targets network-based approach to prediction and population-based validation of in silico drug repurposing systems biology-based investigation of cellular antiviral drug targets identified by gene-trap insertional mutagenesis understanding human-virus protein-protein interactions using a human protein complex-based analysis framework. msystems computational network biology: data, models, and applications repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection fda-approved selective estrogen receptor modulators inhibit ebola virus infection repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis c virus infection a screen of fda-approved drugs for inhibitors of zika virus infection identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen prediction of drug-target interactions and drug repositioning via network-based inference a genome-wide positioning systems network algorithm for in silico drug repurposing deepdr: a network-based deep learning approach to in silico drug repositioning target identification among known drugs by deep learning from heterogeneous networks network-based prediction of drug-target interactions using an arbitrary-order proximity embedded deep forest network-based translation of gwas findings to pathobiology and drug repurposing for alzheimer's disease network-based prediction of drug combinations molecular evolution of human coronavirus genomes structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors structure of sars coronavirus spike receptor-binding domain complexed with receptor genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-em structure of the 2019-ncov spike in the prefusion conformation transient oligomerization of the sars-cov n protein-implication for virus ribonucleoprotein packaging the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease a structure-informed atlas of human-virus interactions screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture toremifene interacts with and destabilizes the ebola virus glycoprotein the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling the central role of angiotensin i-converting enzyme in vertebrate pathophysiology effect of the angiotensin ii receptor blocker olmesartan on the development of murine acute myocarditis caused by coxsackievirus b3 the impact of statin and angiotensin-converting enzyme inhibitor/angiotensin receptor blocker therapy on cognitive function in adults with human immunodeficiency virus infection irbesartan, an fda approved drug for hypertension and diabetic nephropathy, is a potent inhibitor for hepatitis b virus entry by disturbing na(+)-dependent taurocholate cotransporting polypeptide activity the fda-approved drug irbesartan inhibits hbv-infection in hepg2 cells stably expressing sodium taurocholate co-transporting polypeptide identification of a novel transcriptional repressor (hepis) that interacts with nsp-10 of sars coronavirus host mtorc1 signaling regulates andes virus replication host cell mtorc1 is required for hcv rna replication adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe h1n1 pneumonia and acute respiratory failure middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell bird flu"), inflammation and anti-inflammatory/ analgesic drugs the development of anti-inflammatory drugs for infectious diseases melatonin: its possible role in the management of viral infections-a brief review melatonin in bacterial and viral infections with focus on sepsis: a review ebola virus disease: potential use of melatonin as a treatment one molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species? on the free radical scavenging activities of melatonin's metabolites, afmk and amk anti-inflammatory effects of eplerenone on viral myocarditis remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro systematic identification of synergistic drug pairs targeting hiv antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis differential in vitro inhibition of feline enteric coronavirus and feline infectious peritonitis virus by actinomycin d toremifene is an effective and safe alternative to tamoxifen in adjuvant endocrine therapy for breast cancer: results of four randomized trials mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells emodin inhibits current through sars-associated coronavirus 3a protein emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction calmodulin interacts with angiotensin-converting enzyme-2 (ace2) and inhibits shedding of its ectodomain modulation of intracellular calcium and calmodulin by melatonin in mcf-7 human breast cancer cells activation of the c-jun nh2-terminal kinase pathway by coronavirus infectious bronchitis virus promotes apoptosis independently of c-jun the natural compound silvestrol is a potent inhibitor of ebola virus replication broad-spectrum antiviral activity of the eif4a inhibitor silvestrol against corona-and picornaviruses review of computational methods for virus-host protein interaction prediction: a case study on novel ebola-human interactions pleiotropic effects of statins: new therapeutic targets in drug design integrative functional genomics of hepatitis c virus infection identifies host dependencies in complete viral replication cycle crispr-cas9 genetic analysis of virushost interactions acute eosinophilic pneumonia related to a mesalazine suppository mesalamine induced eosinophilic pneumonia translational high-dimensional drug interaction discovery and validation using health record databases and pharmacokinetics models mega x: molecular evolutionary genetics analysis across computing platforms enrichr: a comprehensive gene set enrichment analysis web server 2016 update drugbank 4.0: shedding new light on drug metabolism therapeutic target database update 2016: enriched resource for bench to clinical drug target and targeted pathway information chembl: a large-scale bioactivity database for drug discovery bindingdb: a webaccessible database of experimentally determined protein-ligand binding affinities the iuphar/bps guide to pharmacology: an expertdriven knowledgebase of drug targets and their ligands uniprot: the universal protein knowledgebase database resources of the national center for biotechnology information conformational dynamics and allosteric regulation landscapes of germline pten mutations associated with autism compared to those associated with cancer expression profile of immune response genes in patients with severe acute respiratory syndrome cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus srebp-dependent lipidomic reprogramming as a broadspectrum antiviral target discovery and preclinical validation of drug indications using compendia of public gene expression data this work was supported by the national heart, lung, and blood institute of the national institutes of health (nih) under award number k99 hl138272 and r00 hl138272 to f.c. the content of this publication does not necessarily reflect the views of the cleveland clinic. key: cord-320909-p93gxjm2 authors: natoli, s.; oliveira, v.; calabresi, p.; maia, l. f.; pisani, a. title: does sars‐cov‐2 invade the brain? translational lessons from animal models date: 2020-05-22 journal: eur j neurol doi: 10.1111/ene.14277 sha: doc_id: 320909 cord_uid: p93gxjm2 the current coronavirus disease (covid‐19) outbreak, caused by the novel severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2), has raised the possibility of potential neurotropic properties of this virus. indeed, neurological sequelae of sars‐cov‐2 infection have already been reported and highlight the relevance of considering the neurological impact of coronavirus (cov) from a translational perspective. animal models of sars and middle east respiratory syndrome, caused by structurally similar covs during the 2002 and 2012 epidemics, have provided valuable data on nervous system involvement by covs and the potential for central nervous system spread of sars‐cov‐2. one key finding that may unify these pathogens is that all require angiotensin‐converting enzyme 2 as a cell entry receptor. the cov spike glycoprotein, by which sars‐cov‐2 binds to cell membranes, binds angiotensin‐converting enzyme 2 with a higher affinity compared with sars‐cov. the expression of this receptor in neurons and endothelial cells hints that sars‐cov‐2 may have higher neuroinvasive potential compared with previous covs. however, it remains to be determined how such invasiveness might contribute to respiratory failure or cause direct neurological damage. both direct and indirect mechanisms may be of relevance. clinical heterogeneity potentially driven by differential host immune‐mediated responses will require extensive investigation. development of disease models to anticipate emerging neurological complications and to explore mechanisms of direct or immune‐mediated pathogenicity in the short and medium term is therefore of great importance. in this brief review, we describe the current knowledge from models of previous cov infections and discuss their potential relevance to covid‐19. highly pathogenic coronavirus (cov) infections are well-established sources of previous epidemics in humans, i.e. severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov). the novel cov named sars-cov-2, which shares a highly homological sequence with sars-cov, is responsible for the current covid-19 outbreak with more than 2 million patients diagnosed and over 146 000 deaths, which exceeds by far the total of sars and mers in 2002 and 2012, respectively [1] [2] [3] . despite the short duration of the current pandemic outbreak, several neurological and neuroradiological phenotypes have been reported [4, 5] , requiring urgent investigation into the mechanisms and etiology underlying the interplay between sars-cov-2 and the central nervous system (cns). a translational neuroscience approach is mandatory to explore the possible cns involvement in cov infections, accelerate scientific knowledge transfer to the clinical frontline and test new disease-oriented treatments. indeed, both clinical features of the previous cov epidemics (sars and mers) and lessons from animal models used in the study of sars and mers constitute valuable tools to understand the viral pathogenesis in the host and to characterize mechanisms of viral access and dissemination in the cns. meanwhile, several laboratories are rushing to study sars-cov-2 in a number of different animals, including primates, mice, rats, hamsters and ferrets [6] . here, we will provide a neurological perspective by analysing the main features of these models and point out relevant similarities and specificities in comparison to sars-cov-2. a comprehensive systematic search of medline, scopus, web of science and https://www.who.int/emerge ncies/diseases/novel-coronavirus-2019/situationreports/ was performed. in 2002, the outbreak of sars in guangdong province, china led to the discovery of sars-cov, a highly pathogenic cov, as the causative pathogen of the epidemic [7] . although the virus is primarily a respiratory pathogen, there are reports of neurological manifestations, such as epileptic seizures and encephalitis, that may suggest a cns involvement of the infection [8, 9] (table 1) . complementing these reports, post-mortem neuropathological studies have detected the sars-cov n protein and rna polymerase gene fragment in neurons of infected patients and pathological changes such as brain tissue edema and vasculitis of cerebral veins [10] . compelling evidence demonstrates that sars-cov attaches to the cell membrane by binding to human angiotensin-converting enzyme 2 (hace2), now also known to be the sars-cov-2 functional receptor [11] . human tissue studies have shown an abundant presence of these receptors not only in the epithelia of the lung and small intestine, but also in arterial and venous endothelial cells and arterial smooth muscle cells in all organs studied, including the brain [12] . based on a transgenic mouse expressing hace2, it was possible to show that angiotensin-converting enzyme 2 (ace2) is also expressed at neuronal level, namely in the cytoplasm of cell bodies [13] . distinctive properties in the structure of mouse ace2 (as compared with hace2 proteins) significantly reduce the virus tropism for mouse tissues. hence, in order to overcome this species-related difference, a transgenic model has been generated in which a vector carrying a hace2-coding sequence was introduced in wild-type mice under control of the human cytokeratin 18 (k18) promoter [14] . notably, when k18-hace2 transgenic mice were infected with sars-cov, the infection would start in the respiratory epithelium and rapidly spread to the alveoli. more importantly, neuroinvasive routes were later explored using the same model, by monitoring the kinetic profile of viral antigen [15] . strikingly, the authors showed that the viral spread started in the olfactory bulb and progressively invaded subcortical and cortical regions. such a trans-neuronal hypothesis could not apply for other infected regions, such as those brainstem nuclei that are not directly connected to the olfactory bulb. the authors raise the possibility that, once the virus is established in the brain, it might spread along specific neurotransmitter pathways or via non-neuronal routes (blood or virchow-robin spaces) [15] . overall, their results showed that, in this model, sars-cov primarily entered the brain via the olfactory nerve. alternatively, other authors theorize that cov can primarily use a hematogenous route to penetrate the cns using dendritic or white blood cells as reservoirs [16] . this presumption is based on pathological studies that have shown that monocytes and macrophages can be infected by sars-cov [17] and on cell-line studies that revealed that dendritic cells (regulators of immune responses) can be infected and impaired by this virus [18] (table 1) . multiple animal models have been explored in the context of sars, including non-human primates, hamsters, ferrets and mice ( table 2) . a comprehensive descriptive review of all suitable models is beyond the scope of this review and we refer the reader to a number of excellent reviews [19] [20] [21] . it can be inferred that there is no single ideal animal model for sars, although the evidence collected so far has significantly contributed to advancing the field. in particular, it is well established that models range from those in which only virus replication is observed (young balb/c, b6 mice) to those in which replication is accompanied by minimal signs and histopathology (such as non-human primates, ferrets and hamsters) [19] . curiously, old immunodeficient balb/c mice exhibit a clinical syndrome, supporting age as a risk factor for more severe clinical phenotypes [19] . transgenic k18-hace2 mice infected with sars-cov develop a severe pulmonary phenotype, starting in the respiratory epithelium with rapid alveolar dysfunction [14] . in this model there is a massive infiltration of macrophages and lymphocytes in the lungs, promoting a release of pro-inflammatory cytokines not only at pulmonary level, but also in the brain. in a relatively short time frame (within 5 days), k18-hace2 mice develop a severe phenotype, that includes a lethargic-like state, suggesting cns involvement. in follow-up studies in the same mouse strain, k18-hace2 [15] , the authors demonstrated an extensive involvement of the transgenic mouse brain. sars-cov produced a widespread infection involving vital brainstem nuclei, such as dorsal motor nucleus of the vagus, nucleus tractus solitarii and area postrema. this model also raised questions as to the cause of neuronal destruction and death in these animals [15] . as there was no pathological evidence of inflammation, the authors considered the possibility of apoptosis as the cause of neuronal death, although this was not confirmed [terminal deoxynucleotidyl transferase (tdt) dutp nick-end labeling (tunel)-positive cells were not detected]. it was proposed that a dysregulated cytokine response could be the cause of death in these animals. at day 4 postinfection, infected k18-hace2 mice had an upregulation of the proinflammatory cytokines interleukin (il)-1, tumor necrosis factor alpha and il-6. the authors also propose a possible direct involvement of the dorsal vagal complex, a vital region of the brain that plays an important role in orchestrating cardiorespiratory function. in fact, animals intracranially inoculated with low-dose virus exhibited limited viral spreading but succumbed rapidly [15] . overall, these data show the relevance of the transgenic approach in converting the mouse response to infection from mild to severe leading to cns human neurons are infectible [53] and ace2 neuronal expression has been identified in human cns [54] capable of infecting human neuronal cells in in-vitro cell lines [55] . ddp4 has a low expression in the brain [56] -neuropathology sars genome sequences detected in the brain in autopsies; also, edema and scattered red degeneration of neurons [17] samples not available for investigation [22, 23] . currently, mers-cov is still a relevant threat for populations in the middle east, with a high lethality (close to 35%) [2] . patients exhibit predominantly pulmonary clinical involvement in contrast to fewer patients presenting neurological manifestations such as coma, ataxia, focal motor deficits and peripheral nerve symptoms [24, 25] . unfortunately, there are no published data regarding human neuropathological findings (table 1) . the mers-cov ex-vivo models supported the clinical tropism for the pulmonary tract by showing that the virus can replicate in human lung cultures (in bronchial, bronchiolar and alveolar epithelial cells) [26] . this cell line susceptibility study also revealed that, although presenting a lower viral expression and no cytopathic effects, mers can infect human neuronal lines. dipeptidyl peptidase-4 (dpp4), also known as cd26, was identified as a functional receptor for mers-cov. dpp4 is generally expressed in human bronchiolar epithelial cells and bronchial lung tissue [27] . it can also be found in the intravascular portion of vascular endothelial cells and in the cerebrospinal fluid [28] . after identification of dpp4 as a functional receptor, which is expressed in the airway epithelia of rodents, it was expected that rodents would have been vulnerable to infection. this turned out to be wrong, as the human binding domain differs from that of rodents [29] . this limitation was overcome by developing mice expressing human dpp4 that exhibited high susceptibility to infection and displayed the features of human disease [30] , including a lethargic state, and showing high mortality and extrapulmonary involvement ( table 2 ). the authors detected a severe lung infection, but brain invasion was not seen until day 4 of infection, suggesting substantially different kinetics of mers-cov infection in the lung and brain [30] . a different animal model using human dpp4 transgenic mice studied the differences in viral replication in animals infected by a clinical aerosol transmission simulator compared with intranasal instillation-inoculated mice [31] . they found that the disease onset, lung lesion and viral replication progression were slower in the mers-cov aerosol-infected mice than in the mers-cov instillation-inoculated mice. furthermore, after aerosol infection, they detected high viral loads after 3-9 days in the lungs versus 7-9 days in the brain. again, although both lungs and brain are infected, the timing is different, with a later infection of the brain [31] . such different kinetics could suggest a hematogenous route of infection. indeed, neuroinvasive routes were not explored in either of these models. in addition, similarly to sars-cov, mers-cov has been shown to replicate in human dendritic cells and macrophages, which would support the hematogenous hypothesis [32] . a number of models have been developed and discussed in detailed review articles [33, 34] (table 2 ). in a non-human primate model of mers, de wit and colleagues inoculated rhesus macaques with mers-cov, which primarily affected the epithelium of the lower respiratory tract, giving rise to a mild-to-moderate interstitial pneumonia [35] . this model was able to replicate virus shedding and replication in tissues, as well as gene expression and cytokine and chemokine profiles. however, despite the mild clinical syndrome, no neurological signs and symptoms were reported. thus, the self-limiting nature of mers-cov infection, as transient patterns at various levels of the model, suggests that this model does not fully resemble the lethal infection observed in humans [35] . it is of note that, when macaques were immunocompromised by immunosuppressive agents, the mers-cov replicated to significantly higher titers and disseminated in other organs (cns not examined). surprisingly, histopathological alterations were reduced in the immunosuppressed animals [36] . together, these data suggest a prominent role of the host response in the manifestation of the disease. the macaque model allowed the testing of a number of potential drugs as novel therapeutics. remdesivir, an antiviral agent used also for covid-19, was able to prevent/treat the histological and radiological signatures of the disease [37] . in studies using transgenic mice expressing human dpp4, it was possible to induce features of human disease in the animals [30] . from the studied cells, pneumocytes, brain microglia, astrocytes and neuronal cells all presented high titers of virus. with regard to pathology, whereas infected mice presented an extensive pulmonary inflammatory infiltrate, the only findings in the brain were a mild perivascular cuffing [30] . however, in a different study using human dpp4 transgenic mice, a few days after the appearance of pulmonary lesions, pathological changes were documented in the brain, with dilatation and congestion of the cerebral vessels and few areas of cellular necrosis in the cerebral cortex, hippocampus and thalamus [31] . as in sars-cov-2, mers-cov infection was also shown to induce a profound acute inflammatory response within the lungs and brain of hcd26 tg mice, with upregulation of multiple genes related to the inflammatory response [30] . clinical and neuropathological features in human patients covid-19 is the most recent and dramatic pandemic, caused by sars-cov-2. registered lethality varies between european countries ranging from 1.5% in germany to over 10% in italy [1] . as in sars and mers, pulmonary clinical involvement is most prominent. however, more recently, neurological phenotypes involving central and peripheral nervous system have emerged and are being increasingly recognized, i.e. anosmia, ageusia, necro-hemorrhagic encephalitis and guillain-barr e syndrome [4] [5] 38] . so far there are no published human neuropathological findings (table 1) . the sars-cov-2 ultrastructure was recently characterized by high-resolution cryo-electron microscopy [39] . remarkably cov spike glycoprotein, by which the virus binds the cell membrane, binds ace2 with a higher affinity compared with sars-cov. in addition, most of the available antibodies to sars-cov targeting acebinding domain were unable to bind the sars-cov-2 spike protein, indicating that binding sites differ between sars-cov and sars-cov-2. such a finding indicates the urgent need for generating specific antibodies for sars-cov-2 binding domain, but might also explain the distinct pathogenic properties of sars-cov-2 [40] . in addition to ace2 receptor, sars-cov-2 uses the serine protease type ii transmembrane serine protease (tmprss2) for spike protein priming [41, 42] . very recently, in a preliminary report, brann et al. took advantage of bulk mouse whole olfactory mucosa (wom) rna sequence data derived from macaque, marmoset and human and found in both mouse and human datasets that olfactory sensory neurons do not express two key genes involved in cov-2 entry, i.e. ace2 and tmprss2 [43] . in contrast, olfactory epithelial support cells and stem cells express both of these genes, as do cells in the nasal respiratory epithelium. taken together, these findings suggest possible mechanisms through which cov-2 infection could lead to anosmia or other forms of olfactory dysfunction. moreover, these findings may question the olfactory bulb as an entry route for covs into the cns [43] . to our knowledge, no study has evaluated, so far, any type of pathway targeted at the cns or peripheral structures. clinical and pathological lessons from animal models several laboratories worldwide are accelerating attempts to develop a suitable animal model for covid-19. experimental infection with sars-cov-2 in these models provides basic information to address a number of fundamental questions regarding its pathogenicity, the interaction with the different hosts and, hopefully, establishing the criteria for prevention and care. in line with observations in sars models, non-human primates and wild-type mice infected with sars-cov-2 exhibit a relatively mild clinical disease, in spite of the evidence that quantitative reverse transcription polymerase chain reaction (rt-qpcr) revealed a massive infection of the respiratory tract [44, 45] (table 2) . rhesus macaques infected with bronchoalveolar lavage fluid obtained from an affected patient developed a histopathologically confirmed interstitial pneumonia, associated with a widespread presence of sars-cov-2 in the respiratory tract. clinical signs were mild and no viral rna was detectable by means of rt-qpcr in the blood of the primates during the whole course of infection (14 days) [44] . these findings demonstrate the causal relationship between sars-cov-2 and interstitial pneumonia, reminiscent of covid-19. moreover, and consistent with observations in sars models, bao et al. [45] used the hace2 transgenic mice and infected them with sars-cov-2 inducing interstitial pneumonia, with typical histopathological elements and, accordingly, viral antigens were found in airway epithelia. these lines of experimental evidence are relevant as they demonstrate the causal relationship between sars-cov-2 and pulmonary involvement, but, unlike in sars models, nervous system involvement was not documented in these experiments. however, it is unclear if brain tissue was systematically assessed and the most susceptible brain regions explored for direct or indirect viral presence. overall, the pathogenicity of sars-cov-2 is lower as compared with sars-cov in mice. indeed, as discussed above, hace2 transgenic mice infected with sars-cov exhibited widespread organ damage, whereas sars-cov-2, at least in this model, was confined to lungs, indicating a differential pathogenicity [45] . these studies reveal important commonalities between sars-cov-2 and sars-cov infection and identify a potential target for antiviral intervention. in fact, very recently, a tmprss2 inhibitor approved for clinical use (camostat mesylate) was tested and blocked sars-cov-2 entry into lung cells [42] . finally, the same authors were able to show that the sera from convalescent patients with sars cross-neutralized sars-2-spike-driven entry [42] . if the same effect occurred in pre-clinical models, then we would be closer to both a preventive and a diseaseoriented treatment. • major routes of cns infection: (i) spread via olfactory bulb and/or (ii) synapse-connected route to the medullary cardiorespiratory center from the mechanoreceptors and chemoreceptors in the lung and lower respiratory airways; and/or (iii) hematogenic via brain endothelial ace2 receptors. • major pathogenic pathways for cns involvement: (i) direct viral pathogenicity; and/or (ii) immunemediated pathogenicity targeting brain tissue; and/ or (iii) inflammatory involvement of brain blood vessels; and/or (iv) intravascular coagulation secondary to the systemic inflammatory response as a mayor cause of thrombosis, hemorrhage and stroke. • host individual susceptibility factors that underlie the variable severity of the disease in human patients. however, a relevant issue is also represented by gender. the clinical observation of a specific involvement of males might suggest a specific protective estrogenic effect. unravelling these points could clarify if cns contributes to respiratory failure in patients with covid-19 [15, 46] and may provide a rationale to preventive and therapeutic strategies for major neurological events such as stroke, encephalitis or other reported complications. established ex-vivo and animal models of cov infection may help to dissect pathogenicity, infective routes and nervous system targets of covs. if we manage to mimic the pathological hallmarks of covid-19 we may have the tools to test treatment efficacy and evaluate the efficacy of vaccines and therapeutics. among limitations, the following emerge as of primary importance. • neurological subtle clinical phenotypes are not easily reproducible in animal models. • neurological severe phenotypes are dependent on using specific transgenic approaches to enhance virulence, which limit direct translation to humans. • severity of the clinical features does not always parallel either the viral replication level or the histopathological findings, hinting at indirect disease mechanisms (such as inflammation and prothrombotic states) that have not been attained in the present models. • innate animal characteristics seem to influence viral infection kinetics leading to faster virus clearance. the ongoing outbreak of sars-cov-2 confirms that human covs are primarily respiratory pathogens and koch postulates have already been fulfilled in this regard [45] . previous reports suggest that sars-cov and mers-cov can occasionally cause clinically relevant cns infections. in fact, animal models suggest invasiveness of these viruses through the cns, either via the olfactory bulb or through blood dissemination of infected and activated monocytes passing through a permeable blood-brain barrier as a consequence of the systemic inflammatory response. with regard to the pathogenesis of immunemediated cns pathology, data derive broadly from mice infected with murine hepatitis virus strains, a beta-cov genetically related to human cov-oc43 [47] . briefly, three mechanisms of immune-mediated cns lesions can be recognized. (i) an excessive host response to the infection can occur resulting in a systemic inflammatory response syndrome that causes a multiple organ dysfunction (including cns). the main pathogenic mechanism in this case includes tissue 'dysoxia' due to intravascular coagulation and dysfunction of the microcirculation homeostasis. (ii) direct viral infection of immune cells, including macrophages, microglia and astrocytes in the cns, may activate glial cells that locally produce pro-inflammatory cytokines, including il-6, tumor necrosis factor alpha, il-1b and il-12 [48] . moreover, activated immune cells may contribute to tissue damage by producing toxic agents, recruiting and activating further immune cells and inducing apoptosis. immune-mediated events, either through t-cells or by means of other cytokine and chemokine pathways, may also eventually lead to demyelination. (iii) an autoimmune reaction is generated by an adaptive immune response directed against host epitopes or proteins either misrecognized by pathogen-directed antibodies or expressed by damaged tissues (and previously cryptic to the adaptive immune system) [49, 50] . in order to speed up clinically useful discoveries, it would be desirable to follow some indications such as: (i) to build a systematic, consecutive, prospective registry including epidemiological data in patients with covid-19 with attention to neurological manifestation to fully understand if sars-cov-2 infections can cause cns involvement and to what extent; (ii) to measure sars-cov-2 rna in the cerebrospinal fluid of symptomatic vs. asymptomatic patients; and (iii) to perform autoptic investigations of patients with covid-19 in order to find and characterize virus distribution across tissues (cerebral blood vessels, endothelia, glia and neurons) and neuropathological consequences such as antibody-based neuroinflammatory responses in gray and white matter, vasculitis, neuroglial death or apoptosis and ischaemic or hemorrhagic events. taking into account the fact that other covs are prone to infecting neurons in animal models as well as in humans [16, 50] we must keep an open mind regarding medium-to long-term sequelae and consequences of the acute infection. therefore, despite immune-mediated control of acute infection being attained, host-mediated immune regulatory mechanisms may fail to clear the virus potentially leading to 'chronic infections' and hence impact chronic neurological diseases, such as parkinson's disease and multiple sclerosis [51] as well as acute disseminated encephalomyelitis [52] . this calls for long-term patient follow-up in the clinics and also exploring the effect of sars-cov-2 in mouse models of neurodegenerative disorders to anticipate the occurrence of chronic sars-cov-2 cns infection. world health organization -coronavirus disease (covid-2019) situation reports world health organization -mers-cov. disease outbreak news summary of probable sars cases with onset of illness from 1 covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features. radiology 2020 neurological manifestations of hospitalized patients with coronavirus disease labs rush to study coronavirus in transgenic animals -some are in short supply a novel coronavirus associated with severe acute respiratory syndrome detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome possible central nervous system infection by sars coronavirus organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis virus transmission pathways angiotensin-converting enzyme 2: a functional receptor for sars coronavirus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis differential expression of neuronal ace2 in transgenic mice with overexpression of the brain renin-angiotensin system lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis multiple organ infection and the pathogenesis of sars interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells is there an ideal animal model for sars? animal models for sars and mers coronaviruses the battle against sars and mers coronaviruses: reservoirs and animal models mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) neurological complications during treatment of middle east respiratory syndrome tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc tackling dipeptidyl peptidase iv in neurological disorders structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease the characteristics of hdpp4 transgenic mice subjected to aerosol mers coronavirus infection via an animal nose-only exposure device active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis a comparative review of animal models of middle east respiratory syndrome coronavirus infection livestock susceptibility to infection with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection guillain-barr e syndrome associated with sars-cov-2 infection: causality or coincidence? cryo-em structure of the 2019-ncov spike in the prefusion conformation potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor non-neural expression of sars-cov-2 entry genes in the olfactory system suggests mechanisms underlying covid-19-associated anosmia infection with novel coronavirus (sars-cov-2) causes pneumonia in the rhesus macaques the pathogenicity of sars-cov-2 in hace2 transgenic mice the neuroinvasive potential of sars-cov2 may be at least partially responsible for the respiratory failure of covid-19 patients neurologic alterations due to respiratory virus infections coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia immunopathogenesis of coronavirus infections: implications for sars coronavirus infection of the central nervous system: host-virus standoff neuroinvasion by human respiratory coronaviruses detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis susceptibility of human and rat neural cell lines to infection by sars-coronavirus quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation unravelling the immunological roles of dipeptidyl peptidase 4 (dpp4) activity and/or structure homologue (dash) proteins we are grateful to drs magdalena mroczek and andrea mancini for sharing the literature on covid-19. this research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. the authors declare no financial or other conflicts of interest that relate to the research covered in this article. data sharing is not applicable to this article as no new data were created or analyzed in this study. key: cord-304227-rbr2un1u authors: nan title: updated information on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection and guidance for the public, clinicians, and public health authorities, 2012–2013 date: 2013-09-27 journal: mmwr morb mortal wkly rep doi: nan sha: doc_id: 304227 cord_uid: rbr2un1u the middle east respiratory syndrome coronavirus (mers-cov) was first reported to cause human infection in september 2012. in july 2013, the world health organization (who) international health regulations emergency committee determined that mers-cov did not meet criteria for a "public health emergency of international concern," but was nevertheless of "serious and great concern". this report summarizes epidemiologic information and provides updates to cdc guidance about patient evaluation, case definitions, travel, and infection control as of september 20, 2013. the middle east respiratory syndrome coronavirus (mers-cov) was first reported to cause human infection in september 2012 (1) . in july 2013, the world health organization (who) international health regulations emergency committee determined that mers-cov did not meet criteria for a "public health emergency of international concern," but was nevertheless of "serious and great concern" (2). this report summarizes epidemiologic information and provides updates to cdc guidance about patient evaluation, case definitions, travel, and infection control as of september 20, 2013. as of september 20, 2013, a total of 130 cases from eight countries have been reported to who; 58 (45%) of these cases have been fatal ( figure 1 ). all cases have been directly or indirectly linked through travel to or residence in four countries: saudi arabia, qatar, jordan, and the united arab emirates (uae) (figure 2 ). the median age of persons with confirmed mers-cov infection is 50 years (range: 2-94 years). the male-to-female ratio is 1.6 to 1.0. twenty-three (18%) of the cases occurred in persons who were identified as health-care workers. although most reported cases involved severe respiratory illness requiring hospitalization, at least 27 (21%) involved mild or no symptoms. despite evidence of person-to-person transmission, the number of contacts infected by persons with confirmed infections appears to be limited. no cases have been reported in the united states, although 82 persons from 29 states have been tested for mers-cov infection. potential animal reservoirs and mechanism(s) of transmission of mers-cov to humans remain unclear. a zoonotic origin for mers-cov was initially suggested by high genetic similarity to bat coronaviruses (3), and some recent reports have described serologic data from camels and the identification of related viruses in bats (4) (5) (6) . however, more epidemiologic data linking cases to infected animals are needed to determine if a particular species is a host, a source of human infection, or both. to date, the largest, most complete clinical case series published included 47 patients; most had fever (98%), cough (83%), and shortness of breath (72%). many also had gastrointestinal symptoms (26% had diarrhea, and 21% had vomiting). all but two patients (96%) had one or more chronic medical conditions, including diabetes (68%), hypertension (34%), heart disease (28%), and kidney disease (49%). thirty-four (72%) had more than one chronic condition (7) . nearly half the patients in this series were part of a health-care-associated outbreak in al-ahsa, saudi arabia (i.e., a population that would be expected to have high rates of underlying conditions) (8) . also, the prevalence of diabetes in persons aged ≥50 years in saudi arabia has been reported to be nearly 63% (9) . it remains unclear whether persons with specific conditions are disproportionately infected with mers-cov or have more severe disease. evaluating patients. cdc has changed its guidance to indicate that testing for mers-cov and other respiratory pathogens* can be conducted simultaneously and that positive results for another respiratory pathogen should not necessarily preclude testing for mers-cov. health-care providers in the united states should continue to evaluate patients for mers-cov infection if they develop fever and pneumonia or acute respiratory distress syndrome (ards) within 14 days after traveling from countries in or near the arabian peninsula. † providers also should evaluate patients for mers-cov infection if they have ards or fever and pneumonia, and have had close contact § with a recent traveler from this area who has fever and acute respiratory illness. cdc continues to recommend that clusters ¶ of patients with severe acute respiratory illness (e.g., fever and pneumonia requiring hospitalization) be evaluated for common respiratory pathogens and reported to local and state public health departments. if the illnesses remain unexplained, particularly if the cluster includes health-care providers, testing for mers-cov should be considered, in consultation with state and local health departments. in this situation, testing should * examples of respiratory pathogens causing community-acquired pneumonia include influenza a and b, respiratory syncytial virus, streptococcus pneumoniae, and legionella pneumophila. † countries considered in or near the arabian peninsula include bahrain, iraq, iran, israel, jordan, kuwait, lebanon, oman, palestinian territories, qatar, saudi arabia, syria, uae, and yemen. § close contact is defined as 1) any person who provided care for the patient, including a health-care worker or family member, or had similarly close physical contact; or 2) any person who stayed at the same place (e.g., lived with or visited) as the patient while the patient was ill. ¶ in accordance with who guidance for mers-cov, a cluster is defined as "two or more persons with onset of symptoms within the same 14-day period who are associated with a specific setting, such as a classroom, workplace, household, extended family, hospital, other residential institution, military barracks, or recreational camp." information available at http://www.who.int/csr/disease/ coronavirus_infections/interimrevisedsurveillancerecommendations_ ncovinfection_27jun13.pdf. be considered even for patients without travel-related exposure. additional information about cdc's interim guidance regarding who should be evaluated for mers-cov infection is available at http://www.cdc.gov/coronavirus/mers/interimguidance.html. case definitions. although cdc has not changed the case definition of a confirmed case, confirmatory laboratory testing now requires a positive polymerase chain reaction of at least two, instead of one, specific genomic targets or a single positive target with sequencing of a second. cdc's definition of a probable case has been changed so that identification of another etiology does not exclude a person with an illness meeting this definition from being classified as having a probable case. additional information about cdc's case definitions is available at http://www.cdc.gov/coronavirus/ mers/case-def.html. travel guidance. the peak travel season to saudi arabia is july through november, coinciding with the religious pilgrimages of hajj and umrah. cdc encourages pilgrims to consider recommendations from the saudi arabia ministry of health regarding persons who should postpone their pilgrimages this year, including persons aged ≥65 years, children, pregnant women, and persons with chronic diseases, weakened immune systems, or cancer (http://www.moh.gov.sa/en/coronanew/news/ pages/news-2013-7-14-001.aspx). who advises that persons with preexisting medical conditions consult a health-care provider before deciding whether to make a pilgrimage (http://www.who.int/ith/updates/20130725/ en). cdc continues to recommend that u.s. travelers to countries in or near the arabian peninsula protect themselves from respiratory diseases, including mers-cov, by washing their hands often and avoiding contact with persons who are ill. if travelers to the region have onset of fever with cough or shortness of breath during their trip or within 14 days of returning to the united states, they should seek medical care. they should tell their health-care provider about their recent travel. more detailed travel recommendations related to mers-cov are available at http://wwwnc.cdc.gov/travel/notices/watch/ coronavirus-arabian-peninsula. infection control. with multiple healthcare-associated clusters identified (8,10), infection control remains a primary means of preventing and controlling mers-cov transmission. cdc has recently made checklists available that highlight key actions that health-care providers and facilities can take to prepare for mers-cov patients (http://www.cdc.gov/coronavirus/mers/preparedness/index. html). cdc's infection control guidance has not changed. standard, contact, and airborne precautions are recommended for management of hospitalized patients with known or suspected mers-cov infection. cdc has determined that federal isolation and quarantine are authorized for mers-cov under executive order 13295 (http://www.cdc.gov/quarantine/aboutlawsregulationsquarantineisolation.html).** at this time, cdc is not restricting the movement of travelers with respiratory illness (that is not confirmed or probable mers-cov infection) arriving from the arabian peninsula. however, persons with illness meeting cdc's definition of a confirmed or probable case of mers-cov infection should remain in isolation until they are no longer considered to be contagious according to current guidance. those who do not adhere to isolation requirements, or who intend to travel, may be subject to additional public health measures. cdc does not recommend quarantine of asymptomatic persons who were exposed to confirmed or probable cases. cdc generally recommends that persons with febrile respiratory illness delay travel until their symptoms resolve. cdc has issued new guidance for care and management of mers-cov patients in the home and guidance for close contacts of these patients (http://www.cdc.gov/coronavirus/ mers/hcp/home-care.html). persons who are confirmed, or being evaluated for mers-cov infection, and do not require hospitalization for medical reasons should be isolated in their homes as long as the home is deemed suitable for isolation. cdc currently recommends mers-cov patients should be isolated at home until public health authorities or a health-care provider determine that they are no longer contagious. persons who might have been exposed † † to mers-cov should be monitored for fever and respiratory symptoms for 14 days after the most recent exposure. asymptomatic exposed persons do not need to limit their activities outside the home. if persons exposed to mers-cov have onset of symptoms, they should contact a health-care provider as soon as possible and follow the precautions for limiting possible exposure of other persons to mers-cov. more detailed mers-cov-related interim guidance about patient evaluation, case definitions, travel, and infection control is available at http://www.cdc.gov/coronavirus/mers/index. html. this guidance might change as cdc learns more about the epidemiology of mers-cov. cdc will continue to post the most current information and guidance on its mers-cov website. state and local health departments with questions should contact the cdc emergency operations center at 770-488-7100. what is already known on this topic? the middle east respiratory syndrome coronavirus (mers-cov) was first reported to cause human infection in september 2012 and is associated with high death rates. all cases have been linked through travel to or residence in saudi arabia, qatar, jordan, and united arab emirates. no cases have been reported in the united states. what is added by this report? this report summarizes epidemiologic information about mers-cov, provides updates to cdc guidance about patient evaluation, case definitions, travel, and infection control as of september 20, 2013, and describes new guidance for home care and management of patients with mers-cov infection. what are the implications for public health practice? cases of mers-cov infection continue to be reported by countries in and near the arabian peninsula. this updated cdc guidance will help health-care providers and state and local health departments prepare for and respond to a possible case in the united states. isolation of a novel coronavirus from a man with pneumonia in saudi arabia persons who might have been exposed to mers-cov include persons who care for or have close contact with someone who has mers-cov infection, persons who recently traveled to countries in or near the arabian peninsula, and persons who were identified as a result of a public health investigation of mers-cov infection cases world health organization. who statement on the second meeting of the ihr emergency committee concerning mers-cov close relative of human middle east respiratory syndrome coronavirus in bat middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in bats, saudi arabia seroepidemiology for mers coronavirus using micronuetralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus prevalence of diabetes mellitus in a saudi community first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission key: cord-294907-i836d6im authors: alabdali, abdullah; almakhalas, kharsan; alhusain, faisal; albaiz, saad; almutairi, khalid; aljerian, nawfal title: the middle east respiratory syndrome coronavirus (mers-cov) outbreak at king abdul-aziz medical city-riyadh from emergency medical services perspective date: 2020-05-20 journal: prehospital and disaster medicine doi: 10.1017/s1049023x20000709 sha: doc_id: 294907 cord_uid: i836d6im middle east respiratory syndrome coronavirus (mers-cov) is a form of an infectious respiratory disease, discovered in november 2012 in saudi arabia. according to the world health organization (who; geneva, switzerland) reports, a total of 2,519 laboratory-confirmed cases and 866 mers-cov-related deaths were recorded as of march 5, 2016.(1) the majority of reported cases originated from saudi arabia (2,121 cases). also, mers-cov is believed to be of zoonotic origin and has been linked to camels in the arabian area.(1,2) in this report, the authors discuss the lessons learned from the mers-cov outbreak at king abdul-aziz medical city-riyadh (kamc-r) from august through september 2015 from the emergency medical services (ems) perspective. the discussion includes the changes in policies and paramedic’s practice, the training and education in infection control procedures, and the process of transportation of these cases. the authors hope to share their experience in this unique situation and highlight the preparedness and response efforts that took place by the division of ems during the outbreak. on december 2019, the first case of a novel coronavirus disease (covid-19) was reported from wuhan, china. after that, a world-wide outbreak has occurred. a global anxiety of medical personnel safety endures. a similar outbreak happened before in saudi arabia that started when saudi arabia had announced in september 2012 that a patient with acute pneumonia was found to have a novel beta-coronavirus (cov); 1,2 later, the patient had died from what is known today as middle east respiratory syndrome coronavirus (mers-cov). 3, 4 since then, there have been clusters of local outbreaks that caused local emergency departments (eds) to be closed, followed by a national emergency in 2014-2015. the mers-cov causes lower respiratory tract disease, such as pneumonia, which may be severe, especially in people with underlying comorbiditiescardiopulmonary disease, or weakened immune systems. patients affected by this disease may present with the following symptoms: fever, cough, shortness of breath, gastrointestinal symptoms including diarrhea and nausea/vomiting, pneumonia, acute respiratory distress syndrome, and kidney failure. 5 the recurrence of outbreaks and saudi ministry of health (riyadh, saudi arabia) policy of designating local centers to manage mers-cov patients have increased the demand on inter-facility transfers (ifts). patients with positive mers-cov in local hospitals are urgently transported by referring facility ambulance services to an identified "mers-cov center" and currently "covid-19 center" to prevent outbreaks in local hospitals. the high mortality rate in mers-cov and high health-care-associated infections, including health care personnel acquired infections, required local hospitals to ensure that ambulance services personnel understand the risk of transporting such patients. this article reported the overall experience of a saudi hospitalbased emergency medical services (ems) system with mers-cov: logistics of transfer, transfer policies, methods of monitoring personnel with cov exposure, and training provided to ems personnel. king abdul-aziz medical city-riyadh (kamc-r), saudi arabia is a 1,501-bed tertiary-care hospital that includes a 150-bed ed that registers 250,000 visits per year; it is the largest trauma center in the region. the ems department is a division of the department of emergency medicine; it is a hospital-based ems service that provides 24 hour/seven days a week care, 365 days a year, under an on-duty board-certified emergency medicine consultant covering all kamc-r facilities. the division has more than 100 personnel (table 1) . minimum manpower shift requirements include one advance life support (als) unit operated with at least one paramedic beside an ambulance driver/basic emergency medical technician, and two basic life support (bls) units operated with at least one basic emergency medical technician and an ambulance driver. in 2015, the services provided emergency and nonemergency coverage with total of 9700calls and with manpower of 106 employees working in the division, which has three subdivisions including operation, logistics, and administration. in 2019, the division transferred 17,402 patients. the als crew transferred 3,442 patients, of those 3,009 were emergency 911 patients. in addition, als crew transported 433 critical care ifts. all the als units are equipped with portable ventilator and infusion pumps. paramedics minimum skill competency is granted through the paramedic license requirements. all paramedics involved in ift of critical patients have received advanced training in operating ventilators and syringe pumps; alsothey are advanced cardiac life support, bls, prehospital trauma life support, and pediatric als providers. early in 2013, kamc-r launched the infection disease epidemic plan (idep), in response to the mers-cov outbreak, which consists of three phases that activate based on the number of confirmed cases reported to/within the hospital. 6 howeveron august 5, 2015, multiple cases were reported within the hospital, including patients and health care workers; phase ii of the idep was immediately activated alerting the outbreak within kamc-r. 7 following thatthe hospital medical director initiated major actions such as isolation rooms for suspected or confirmed case, implementing a screening process for all health care workers and new patients, education and training on identifying symptoms of the disease, and means of protection. patients were categorized based on their presentation or confirmed diagnosis into three categories: low-risk patientshigh-risk patients, and confirmed positive patients. the visual triage checklist for acute respiratory illness was utilized as a triage tool (appendix 1; available online only). at that stage, ems's role began to increase for the aspects of transportation of some cases to different hospitals that are specialized for receiving mers-cov cases. also, responding to emergencies that encounter symptoms of the mers-cov. by august 18, 2015, the number of confirmed cases increased, phase iii of the idep plan was activated, and the hospital completely shut down except for critical services; mutual aid agreements with other health care facilities were agreed upon to receive patients from kamc-r. moreover, a mobile field hospital was deployed outside of the ed to triage patients away to other health care facilities and to identify critically ill patients who required admission to the ed. patients were transported via ems to different destinations based on an algorithm used by dispatchers under medical direction (appendix 2; available online only). in addition to the non-mers-cov patients transported by ems from the mobile field hospital and the ed, the ems happened to transfer a total of 184 confirmed cases to the mers-cov center, the dedicated ministry of health quarantine, specialized in receiving and dealing with infectious diseases. following the activation of phase iii of the idep plan, ems focused on changing ems high-risk practices, implementing immediate education and trainingand changed the process of transportation for these specific cases. with the outbreak of mers-cov, there was great pressure on transporting patients to a previously identified mers-cov center where medical expertise and advance critical interventions such as extracorporeal membrane oxygenation (ecmo) were available. the ems providers were anxious since little was known about this novel cov. the ems department moved quickly to implement certain practices with coordination with the infection control department. mandatory steps implemented from the initial phase of being in contact with a suspected patient throughout the transportation process and upon return of the ambulance crew to the ems station. therefore, the first steps were prevention; this included early identification of any symptoms that might encounter patients or health care workers, screening the ems providers at the beginning of each shift and after each transport through temperature checks, followed by tacking employees sick leave records. the second step was an assessment of the provider's level of knowledge in this specific infectious disease through surveyed questions. along with that, in step three, certain medical protocols were updated and introduced, stopping selective procedures such as using nebulizer treatment on patient (metered-dose inhalers to be used if needed), drawing blood or intubation for patient, and using filter devices for ventilator equipment. furthermore, all ems providers were mandated to use personal protective equipment (ppe; ie, gowns, n95 masks, eye goggles, head covers, and disposable gloves) when attending to all cases. in addition, no food or drinking in the ambulances, even if there were no patients being transported. all ppes and single-use materials were to be disposed of immediately in biohazard bags and bins after each transport. following the recommendations from the centers for disease control and prevention (cdc; atlanta, georgia usa), the department generated a checklist to be used by all ems providers prior to transporting a high-risk patient. 8 any ems provider who had contact with a mers-cov case was monitored closely. they had to log in their temperatures every six hours and were instructed to report any flu-like symptoms. ultimatelythe emergency medical dispatcher (emd) ensured complete information such as patient historycurrent status, safety of patient's location, and the need for special resources. all ems providers were cautioned that every patient could be a potential suspected mers-cov case (appendix 3; available online only). with the mers-cov outbreak, ems along with infection control acted immediately in the training and education aspects. different training sessions were carried out using a variety of teaching methods such as frequent small-group classroom teaching, online videos, simulation, and demonstration sessions in how to use ppe, proper techniques of donning and doffing of ppe, and effective hand washing, hand hygiene, and hands-on practical sessions for mask fitting. also, mandatory n95 mask-fitting sessions applied for all staff to ascertain the best-sized mask for each person along with training sessions on the use of powered air purifying respirator (papr) devices. additionallyems acquired two patient isolation and transportation devices (iso-pods; tradeways, ltd; annapolis, maryland usa) for complete patient isolation during transport. numerous training sessions on this device were conducting including operating of the device, cleaning, and disinfection using high-fidelity simulation. the education process and practical sessions were supervised by either the infection control officers or the ems educators to ensure all personnel had the necessary skills and knowledge. also, the operation leaders were monitoring the staff practice to ensure compliance rates among all staff. there were frequent updates about the mers-cov situation and visits from the infection control department to ensure proper progression throughout the outbreak. also, mers-cov was highlighted during the continuing education sessions for the ems providerscovering the clinical presentations, epidemiological changes of the virus, new definitions or procedures, and risk factors were discussed. the ems played an important role in educating the public about the virus with a focus on recognizing symptoms and proper techniques to prevent transmission of the virus; for example, proper handwashing and avoiding crowded areas. also, they stressed the importance of making an honest declaration when a patient had contact with camels or had a recent history of travel. with the declaration of the mers-cov outbreak in the kamc-r and decision to shut down the hospital and transportation of patients to other hospitals, the ems undertook this role and implemented new transportation schemes. first, ems designated four ambulances (two ambulances for suspected mers-cov non-critical cases and two ambulances for confirmed mers-cov critical/non-critical cases) with one back up ambulance. these ambulances were striped out from supplies and hard equipment only to leave essential equipment. based on the information and request provided to the emdthe designated ambulance was deployed. for the confirmed cases6mil plastic covers were placed in the ambulances covering the entire ambulance cabin for proper isolation. also, for restriction of number of people in the ambulance, relatives were not permitted to accompany the patient in the ambulance, except for certain cases such as female or pediatric patient. in these cases, relatives were asked to sit in the front cab of the ambulance following the safety procedures. any non-critical patient suspected or confirmed to have mers-cov were asked to wear a surgical mask. during transport of any patient, the ambulance door between the driver cab and main cabin was always closed and the air condition was kept off, even though the ambulances have separate ac circuits. all ambulance windows were kept open to help with ventilation and circulating the air at the end of the call. in addition, at the end of every call, the providers were required to implement the cleaning and disinfecting procedure for their ambulance and equipment. the procedure included using the hydrogen peroxide aerosol fogger and leaving all equipment inside the ambulance and to ensure the locking of the ambulance for 60 minutes. after that, the provider would use antiseptic wipes for equipment and mop the floor of the ambulance with antiseptic solution. then the vehicle would be aired, with all its windows and doors opened, for 30 minutes. all providers were mandated to follow the proper hand hygiene, hand washing, and use of ppes with each steps of ambulance cleaning and disinfection. the outbreak of respiratory illness is terrifying. the ems personnel needed an assurance that ems division is supporting their well-being. after meeting with infection control and the medical director who was a board-certified emergency medicine physicianthe following procedures were applied: daily body temperature alabdali, almakhalas, alhusain, et al (oral) monitoring to all ems personnel at the beginning of each shift and hotline operated by infection control to address personnel inquiries. if a person was exposed, a clear pathway was implemented: the person was to report to employee health (during working hours) or to emergency flu clinic (out of working hours) immediately after the exposure, the person is granted an official sick leave, a nasopharyngeal swab would be acquired, a person was not allowed to return to work without the approval of the medical director, and pre-approval to access employees medical records was obtained (the only person allowed to access the record was the medical director). the impact of a community outbreak of respiratory novel virus can be disastrous. the ems agencies are regularly training their employees to function under extraordinary circumstances; however, once unique disasters occur, such as viral outbreak, additional effort is required to ensure that ems will be functioning with maximum capacity during a disaster. the mers-cov is a serious life-threatening virus that has a high mortality (34.4%). 1 it does not seem to be as infectious as covid-19; however, it can be more lethal, the mode of transmission of mers-cov is human to human, and current data show that covid-19 shares the same mode of transmission. 9 consequently, it is logical to conclude that measures taken during transporting mers-cov patients can be applicable to suspected covid-19 patients. there were serious operational and administration issues with the first confirmed mers-cov patient. the hospital administration recognized that the longer the patient stays in the ed the more likely an outbreak will occur. an iso-pod was utilized during the transfer of the first confirmed case. during the attempt to persuade the paramedic to take the patient, it was determined that the staff lacked the minimum infection control techniques needed for such transfer. it turned out that they were not familiar with the iso-pod and it was their first time to operate such device. the extensive training course to all ems personnel was conducted in a week's time, and after that, paramedics dealt with a total of 184 confirmed positive mers-cov patients without significant issues. paramedics are health workers, and with proper training, they can manage such transfers. the ems continuing education and disaster drills might not cover the whole spectrum of emerging novel respiratory illness outbreaks. unique disasters may prevent establishing an internationally recognized recommendation on prehospital continuing education. in this experience, it was clear that paramedics needed training, and once provided, they were confident and competent in transporting critically ill positive mers-cov patients. after reviewing the literatureit was recognized that this program must be designed based on the special needs and with clear justification. 10 the result of that program is ems personnel that are able to manage mers-cov. laterduring the covid-19 outbreak, they were confident and prepared to transport patients. it is reasonable to discuss training needs with ems staff before implementing such programs. occupational safety is a multidisciplinary effort that includes employees and communities. 11 the division recognized that an outbreak of mers-cov to the team members would have disastrous consequences, including the possibility of closing the ems division and failure to respond to any emergency calls. the concern was clearly delivered and discussed with all stakeholders, including hospital administrators and paramedics. the decision was to ensure that enforcement of policies was implemented; also, the compliance was monitored on a daily basis. in addition, paramedics were informed that all measures were taken to protect them, and their compliance was the key to success or fail on this matter. the employees were assured that medical care will be provided immediately in case of exposure. every mers-cov positive patient was pre-planned by transporting team, medical director, sending unit, and receiving unit. the high-risk calls were audited on a daily basis by division quality officer and disciplinary actions were taken. paramedics were required to record their body temperature at the beginning of every shift and strict compliance was monitored by division operation chief. the pre-determined goal in response to mers-cov outbreak was to have zero infection acquired to paramedics. there were three paramedics removed from service due to possible exposures and a nasopharyngeal swab was taken before they were allowed back to service. it cannot be concluded that the rate of zero acquired infection was related to only these policies; however, the assurance of all stakeholders, including providers, that measures were taken and audited was an assistance to achieve success. it is the recommendation that ems agencies train, enforce, and monitor their compliance to achieve the pre-determined goals. this report has some limitations. the paper describes only one service (ems) from the whole experience against mers-cov. in addition, this report and experience was limited to one population. further, this report lacks the patients' outcomes. the outbreak was a true test to the system; it outlined the weakness in ems, as well as the need for proper preparedness and planning. the utilization of the available evidence in the field of ems and previous experiences had an impact on the outcome of the outbreak. this epidemic experience showed the importance of educationcommunication, employee support, and planning. on october 18, 2015, the outbreak was contained and the idep was officially deactivated; due to the dedication of the staff, there was no ems personnel affected by mers-cov during the outbreak. to view supplementary material for this article, please visit https:// doi.org/10.1017/s1049023x20000709 world health organization (who) clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection kingdom of saudi arabia, ministry of national guard taming the beast: hospital management of a nosocomial middle east respiratory syndrome outbreak mers interim guidance for healthcare professionals the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? learning in practice. the need for needs assessment in continuing medical education global occupational health: current challenges and the need for urgent action key: cord-309518-seonrtn3 authors: alraddadi, basem m.; qushmaq, ismael; al‐hameed, fahad m.; mandourah, yasser; almekhlafi, ghaleb a.; jose, jesna; al‐omari, awad; kharaba, ayman; almotairi, abdullah; al khatib, kasim; shalhoub, sarah; abdulmomen, ahmed; mady, ahmed; solaiman, othman; al‐aithan, abdulsalam m.; al‐raddadi, rajaa; ragab, ahmed; balkhy, hanan h.; al harthy, abdulrahman; sadat, musharaf; tlayjeh, haytham; merson, laura; hayden, frederick g.; fowler, robert a.; arabi, yaseen m. title: noninvasive ventilation in critically ill patients with the middle east respiratory syndrome date: 2019-03-18 journal: influenza other respir viruses doi: 10.1111/irv.12635 sha: doc_id: 309518 cord_uid: seonrtn3 background: noninvasive ventilation (niv) has been used in patients with the middle east respiratory syndrome (mers) with acute hypoxemic respiratory failure, but the effectiveness of this approach has not been studied. methods: patients with mers from 14 saudi arabian centers were included in this analysis. patients who were initially managed with niv were compared to patients who were managed only with invasive mechanical ventilation (invasive mv). results: of 302 mers critically ill patients, niv was used initially in 105 (35%) patients, whereas 197 (65%) patients were only managed with invasive mv. patients who were managed with niv initially had lower baseline sofa score and less extensive infiltrates on chest radiograph compared with patients managed with invasive mv. the vast majority (92.4%) of patients who were managed initially with niv required intubation and invasive mechanical ventilation, and were more likely to require inhaled nitric oxide compared to those who were managed initially with invasive mv. icu and hospital length of stay were similar between niv patients and invasive mv patients. the use of niv was not independently associated with 90‐day mortality (propensity score‐adjusted odds ratio 0.61, 95% ci [0.23, 1.60] p = 0.27). conclusions: in patients with mers and acute hypoxemic respiratory failure, niv failure was very high. the use of niv was not associated with improved outcomes. middle east respiratory syndrome (mers) has emerged as a cause of severe respiratory illness in humans. 1, 2 as of march 1, 2019, 2279 cases of mers have been reported including 806 deaths. 3 the disease presentation ranges from asymptomatic infection to severe respiratory illness, multiorgan failure, and death. [4] [5] [6] acute hypoxemic respiratory failure (ahrf) develops in up to 70% of hospitalized patients with mers and is associated with high mortality. 7, 8 to date, there is no specific antiviral therapy for mers of proven effectiveness; supportive therapy remains the cornerstone of management. noninvasive ventilation has been increasingly used in the management of ahrf with variable success. [9] [10] [11] [12] while niv may initially avoid the need for intubation and invasive mechanical ventilation (mv) , several studies have reported high failure rates and the need for invasive ventilation among patients with severe acute respiratory distress syndrome (ards) and an association with increased mortality. 12 in a recent analysis from the lung safe study on unselected patients with ards, niv was associated with higher intensive care unit (icu) mortality in patients with the ratio of partial pressure of oxygen to the fraction of inspired oxygen (pao 2 /fio 2 ) lower than 150 mm hg. 12 the role of niv in ahrf secondary to viral respiratory infections is unclear. although some uncontrolled studies suggested that niv was effective and safe in management of patients with severe acute respiratory syndrome (sars), [13] [14] [15] others have highlighted concern of increased transmission risk to healthcare workers when patients with sars are treated with niv. 16 use of niv in ahrf caused by pandemic h1n12009 virus (pdmh1n1) infection has been reported from several countries, [17] [18] [19] with reported niv failure reaching up to 85%. 17 all studies were limited by their retrospective nature and, often, small sample size. noninvasive ventilation has been used in patients with mers, 5, 8 but its value in preventing intubation and impact on clinical outcomes has not been studied. the objective of this study was to assess the success of niv in mers patients with ahrf in avoiding intubation and its association with mortality and icu and hospital length of stay. our secondary objective was to identify factors associated with niv failure in mers patients. we conducted this analysis on a multicenter retrospective cohort of critically ill mers patients from 14 in this study, we included all patients with ahrf who required mechanical ventilation support in the icu, whether invasively or noninvasively. all patients who were managed initially with niv were compared to those who were managed with invasive mv without niv. for this analysis, we extracted baseline data including demographics, comorbidities, duration from onset of symptoms to emergency room admission, icu admission, and intubation. arterial blood gases, continuous variables were described as medians and interquartile ranges (q1, q3) or means and standard deviations and were tested we performed a secondary comparison of patients who had failed niv to those who had been successfully treated with niv. all statistical tests were two-sided with significance set at α < 0.05. analyses were conducted using sas version 9.2 (sas institute, cary, nc). in 105 patients who were managed initially with niv, niv was used for a median duration of 1 (1, 3) day and 97 patients (92.4%) eventually required intubation and invasive mv ( [11, 35] , p = 0.6). there was no significant difference in the duration of invasive mv and total duration of niv and invasive mv between the two groups, although invasive mv-free days and total niv and invasive mv-free days were significantly longer among niv patients compared to invasive mv patients (table 2, figure s2 ). overall, only 8/105 (7.6%) of the niv patients avoided subsequent intubation (table s1 ). these patients were significantly younger than tables s1 and s2). crude 90-day mortality was significantly higher in patients who failed niv compared with patients successfully treated only with niv (table s3 and figure s3 ). we have shown that among patients with mers-related ahrf, niv was commonly used, but nearly always resulted in subsequent transition to invasive ventilation. our results suggest that while the initial niv use in mers patients was not associated with reduction of mortality or length of icu or hospital stay, these patients had greater requirement for subsequent inhaled nitric oxide. a minority of patients were successfully managed with niv-those who were young and had less severe disease. these findings have important implications for early management of patients infected with mers, specifically, that there is little advantage to initial niv treatment for most patients with mers-related ahrf and that niv may be associated with greater subsequent need for oxygenation rescue therapy such as inhaled nitric oxide. noninvasive ventilation has been proven to be useful as a means to avoid intubation and improve clinical outcomes in certain conditions, generally, with the possibility for rather rapid reversal of respiratory failure-for example, pulmonary edema due to congestive heart failure, and respiratory failure due to copd exacerbations. 22, 23 for conditions that typically worsen or do not improve in the range of many hours (eg, most causes of pneumonia), there appears to be little advantage in using niv as a means to avoid intubation. 9, 24, 25 in choosing to use niv for as initial treatment for patients with hypoxemic respiratory failure, there is a practical risk of patients worsening on niv and requiring intubation at a time when they already have more advanced organ failure. few studies have assessed the effectiveness of niv in patients with ahrf secondary to ards and acute lung injury. the overall effectiveness of niv in reducing intubation rate or improving clinical outcome in these patients remains controversial. [26] [27] [28] post hoc analysis of the lung safe study found that niv was used in 15% of patients with ards and was associated with higher icu mortality in subset of patients with severe ards. 12 a randomized controlled trial of patients niv is generally not recommended for patients with hypoxia secondary to respiratory infections due to lack of efficacy and the potential for pathogen transmission. 16, 31, 32 it is also considered one of the aerosol-generating procedures that may increase risk of transmission to healthcare workers. 32 broad dispersion of exhaled air during niv via a face mask has previously been shown using a simulated patient encounter. 33 in conclusion, we report the results of niv use in mers patients from a large cohort of critically ill patients. we observed that there is little advantage to initial niv treatment for most patients with mers-related ahrf and that niv may be associated with greater subsequent need for oxygen rescue therapy. we would like to thank the international severe acute respiratory and emerging infection consortium (isaric) for their support in the database. the authors have no conflict of interest to disclose. bma: conception and design, data acquisition, analytical plan, interpretation of data for the work, drafting of the manuscript, critical revi middle east respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection mers-cov outbreak in jeddah-a link to health care facilities description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia a multiple-center survey on the use in clinical practice of noninvasive ventilation as a first-line intervention for acute respiratory distress syndrome use of noninvasive ventilation in patients with acute respiratory failure changing use of noninvasive ventilation in critically ill patients: trends over 15 years in francophone countries noninvasive ventilation of patients with acute respiratory distress syndrome. insights from the lung safe study effectiveness of noninvasive positive pressure ventilation in the treatment of acute respiratory failure in severe acute respiratory syndrome non-invasive versus invasive mechanical ventilation for respiratory failure in severe acute respiratory syndrome noninvasive positive pressure ventilation treatment for acute respiratory failure in sars transmission of severe acute respiratory syndrome during intubation and mechanical ventilation critically ill patients with 2009 influenza a(h1n1) infection in canada intensive care adult patients with severe respiratory failure caused by influenza a (h1n1)v in spain clinical findings and demographic factors associated with icu admission in utah due to novel 2009 influenza a(h1n1) infection critically ill patients with the middle east respiratory syndrome (mers): a multicenter retrospective cohort study infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection noninvasive ventilation for acute exacerbations of chronic obstructive pulmonary disease non-invasive positive pressure ventilation (cpap or bilevel nppv) for cardiogenic pulmonary oedema predictors of failure of noninvasive positive pressure ventilation in patients with acute hypoxemic respiratory failure: a multi-center study non-invasive ventilation in community-acquired pneumonia and severe acute respiratory failure noninvasive ventilation for patients with acute lung injury or acute respiratory distress syndrome noninvasive ventilation for acute lung injury a meta-analysis of randomized controlled trials role of noninvasive ventilation in acute lung injury/acute respiratory distress syndrome: a proportion meta-analysis high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure effect of noninvasive ventilation delivered by helmet vs face mask on the rate of endotracheal intubation in patients with acute respiratory distress syndrome: a randomized clinical trial clinical management of pandemic 2009 influenza a(h1n1) infection aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review exhaled air dispersion during noninvasive ventilation via helmets and a total facemask why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article the saudi critical care trials group noninvasive ventilation in critically ill patients with the middle east respiratory syndrome key: cord-326768-uo6482ah authors: hashem, anwar m.; al‐subhi, tagreed l.; badroon, nassrin a.; hassan, ahmed m.; bajrai, leena hussein m.; banassir, talib m.; alquthami, khalid m.; azhar, esam i. title: mers‐cov, influenza and other respiratory viruses among symptomatic pilgrims during 2014 hajj season date: 2019-02-20 journal: j med virol doi: 10.1002/jmv.25424 sha: doc_id: 326768 cord_uid: uo6482ah more than two million muslims visit makkah, saudi arabia, annually to perform the religious rituals of hajj where the risk of spreading respiratory infections is very common. the aim here was to screen symptomatic pilgrims for middle east respiratory syndrome coronavirus (mers‐cov) and other viral etiologies. thus, 132 nasopharyngeal samples were collected from pilgrims presenting with acute respiratory symptoms at the healthcare facilities in the holy sites during the 5 days of the 2014 hajj season. samples were tested using real‐time reverse transcription polymerase chain reactions and microarray. demographic data including age, sex, and country of origin were obtained for all participants. while we did not detect mers‐cov in any of the samples, several other viruses were detected in 50.8% of the cases. among the detected viruses, 64.2% of the cases were due to a single‐virus infection and 35.8% were due to the coinfections with up to four viruses. the most common respiratory virus was influenza a, followed by non‐mers human coronaviruses, rhinoviruses, and influenza b. together, we found that it was not mers‐cov but other respiratory viruses that caused acute respiratory symptoms among pilgrims. the observed high prevalence of influenza viruses underscores the need for more effective surveillance during the hajj and adoption of stringent vaccination requirements from all pilgrims. at the jamaraat pillars in mina. finally, they finish their hajj ritual by going back to the holy mosque in makkah to perform "tawaf". overcrowding of individuals in such confined settings leads to inevitable prolonged close contact and increases the risk of spreading and acquiring respiratory pathogens among pilgrims, which raises global and public health concerns due to the high potential of international spread of such pathogens. [1] [2] [3] [4] another important driver of spreading and acquiring respiratory pathogens during hajj is the great diversity of inbound viruses from around the world that can potentially spread among the immunologically naive hosts. in fact, acute respiratory infections are very common during hajj and represent the leading cause of the most hospitalizations. [3] [4] [5] [6] [7] it has been suggested that more than one-third of pilgrims will suffer from respiratory symptoms during hajj mostly due to the respiratory viruses. 2, [7] [8] [9] [10] [11] [12] most commonly isolated viruses from symptomatic patients during hajj were human rhinoviruses (hrvs), influenza virus and non-mers human coronaviruses (hcovs). 2, [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] the emergence of the novel middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia, its endemicity, and high mortality rates (35%-40%) clearly represent another major public health concern, especially during hajj. since 2012, mers-cov caused more than 2250 confirmed cases in 27 countries in the arabian peninsula, africa, asia, europe, and north america as of december 2018. 21, 22 furthermore, multiple hospitals and household outbreaks have been reported mostly in the saudi arabia. 23 rna extraction was performed using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to manufacturerʼs instructions. extracted rna from all samples was tested for mers-cov using realtime reverse transcription polymerase chain reactions targeting upstream region of the e-gene as described previously. 33 positive and negative (no template) controls were included in all runs. remaining np samples were used for complementary dna (cdna) synthesis and microarray testing as described previously. 34 plus analyzer (autogenomics inc., carlsbad, ca) according to manu-facturerʼs instructions and as previously described. 34 samples were considered positive when the ratio between the virus and background signals was above the calculated threshold. the data were analyzed using the statistical package for the social science software (spss v20.0; spss inc, chicago, il). the χ 2 and fisher exact tests were used to compare the proportions and a twotailed probability value of p < 0.05 was considered statistically significant. table 1 ). as shown in table 1 , although a majority of positive patients were older than 60 years, this difference was not significant. no statistical significance was found for comparisons of infection rates among males and females or the different age groups. one hundred and twenty-nine of the patients in this study were from 37 countries mostly from asia and africa followed by europe and north america. nationalities of three individuals were unknown. as (table 3) . coinfections with more than two viruses were not uncommon. in fact, four patients had triple concurrent infections, and one patient had a quadruple concurrent infection ( table 3 ). the remaining nine coinfections were due to the unique combination of respiratory viruses (table 3) . not surprisingly, flu a, flu b, and hcov oc43 were the most frequently detected viruses in the most age groups ( coinfections with multiple viruses in symptomatic pilgrims were very common in our study and represented more than 35% of the positive cases and more than 18% of the total number of patients. this rate is markedly higher than the previously reported. [10] [11] [12] [13] 17, 18, 38 furthermore, while most of these reports have identified hrvs as the most coinfecting viruses during hajj, our data showed hcov oc43 as the most prevalent coinfecting virus followed by flu a and hrvs. [10] [11] [12] [13] 17, 18, 38 these marked differences most probably were due to technical and methodological differences. however, it is clear that enhanced surveillance using detection assays with high sensitivity and coverage such as microarray and multiplex pcr could enhance our understanding of pathogens involved in respiratory infections during mass gatherings and ultimately lead to better infection control. 12, 19 or hrv c, which could represent a significant number of potential rhinovirus infections among symptomatic pilgrim. in conclusion, we investigated the etiology of acute respiratory infections in symptomatic pilgrims attending 2014 hajj. while mers-cov was not detected in any of the patients, a variety of other respiratory viruses has been found in more than half of the patients with many coinfections with multiple viruses. our observation as well as the previous reports from hajj indicate that enhanced and active surveillance during hajj seasons is critical to recognize the wide variety of pathogens that might be involved in hajj epidemics and to implement proper infection control measures. importantly, there is an evident risk of influenza infection among pilgrims underscoring the need for targeted, active and continuous surveillance for influenza viruses not only to monitor viral circulation but also to characterize circulating viruses to better understand vaccine effectiveness and to recognize the need to improve current influenza vaccination strategies during hajj. hajj: infectious disease surveillance and control hajj-associated viral respiratory infections: a systematic review health risks at the hajj respiratory tract infection during hajj causes of hospitalization of pilgrims in the hajj season of the islamic year infections in travellers returning to turkey from the arabian peninsula: a retrospective cross-sectional multicenter study respiratory tract infections during the annual hajj: potential risks and mitigation strategies the prevalence of acute respiratory symptoms and role of protective measures among malaysian hajj pilgrims influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage respiratory viruses and bacteria among pilgrims during the influenza and respiratory syncytial virus infections in british hajj pilgrims 2 patterns of reported respiratory symptoms and detected respiratory viruses during hajj days. a, bar graph showing the number of pilgrims presented with respiratory symptoms and tested for respiratory viruses with numbers of those tested positive or negative for any virus during daily. b, bar graph showing the number of viruses detected from positive cases during the 5 days of hajj. day 1, makkah then mina in the afternoon; day 2, arafat in the morning then muzdalifah at night; day 3, muzdalifah then mina in the morning detection of respiratory viruses among pilgrims in saudi arabia during the time of a declared influenza a(h1n1) pandemic influenza viral infections among the iranian hajj pilgrims returning to shiraz, fars province, iran. influenza other respir viruses mers-cov but positive influenza viruses in returning hajj pilgrims, china acute respiratory infections among returning hajj pilgrims-jordan active screening and surveillance in the united kingdom for middle east respiratory syndrome coronavirus in returning travellers and pilgrims from the middle east: a prospective descriptive study for the period 2013-2015 etiology of severe community-acquired pneumonia during the hajj-part of the mers-cov surveillance program viral respiratory infections among hajj pilgrims in 2013 isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics, and public health implications lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms lack of mers coronavirus but prevalence of influenza virus in french pilgrims after prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during 2012-2015. influenza other respir viruses influenza not mers cov among returning hajj and umrah pilgrims with respiratory illness influenza a and b viruses but not mers-cov in hajj pilgrims middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands evidence for camel-to-human transmission of mers coronavirus patterns of human respiratory viruses and lack of mers-coronavirus in patients with acute upper respiratory tract infections in southwestern province of saudi arabia influenza virus but not mers coronavirus circulation in iran, 2013-2016: comparison between pilgrims and general population an opportunistic pathogen afforded ample opportunities: middle east respiratory syndrome coronavirus acute respiratory viral infections among tamattu'-hajj pilgrims in iran viral etiology of acute respiratory infections among iranian hajj pilgrims meningococcal, influenza virus, and hepatitis b virus vaccination coverage level among health care workers in hajj pandemic (h1n1) 2009 and hajj pilgrims who received predeparture vaccination pandemic 2009 influenza a (h1n1) infection among 2009 hajj pilgrims from southern iran: a realtime rt-pcr-based study. influenza other respir viruses mismatching between circulating strains and vaccine strains of influenza: effect on hajj pilgrims from both hemispheres respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome mers-cov, influenza and other respiratory viruses among symptomatic pilgrims during 2014 hajj season the authors declared that there is no conflict of interests. http://orcid.org/0000-0002-8471-7011 key: cord-314867-qg3hl5ft authors: yoon, ji hye; lee, jihye; lee, jun young; shin, young sup; kim, dong eon; min, jung sun; park, chul min; song, jong hwan; kim, seungtaek; kwon, sunoh; jang, min seong; kim, hyoung rae title: study on the 2‐phenylchroman‐4‐one derivatives and their anti‐mers‐cov activities date: 2019-07-28 journal: bull korean chem soc doi: 10.1002/bkcs.11832 sha: doc_id: 314867 cord_uid: qg3hl5ft study on the 2‐phenylchroman‐4‐one derivatives and their anti‐mers‐covactivities. [image: see text] middle east respiratory syndrome coronavirus (mers-cov) has been one of the most fearful diseases by its mortality and possibility of global outbreak. until 23 january 2019, 2279 laboratory-confirmed cases of mers-cov, including 806 associated deaths (case-fatality rate: 35.5%) were reported globally. 1 in korea on may 2015, 186 confirmed cases including 38 associated deaths (20.4%) were reported by an outbreak caused by only one infected person from saudi arabia. 2 at present, no effective vaccine or therapeutics are available for the prevention or treatment of mers-cov infection. [3] [4] [5] however, various basic and clinical research are on-going. 6 we screened a variety of natural products against mers-cov as an attempt of developing anti-mers-cov drugs, and many flavonoids showed anti-mers activities. among them, 2-phenylchroman-4-one derivatives e.g., bavachin and bavachnin separated from dry seed of psoralea corylifolia l., korean medicinal herb exhibited comparatively good activities. bavachin derivatives have been reported as their diverse biological activities, including anti-cancer, 7 ppar agonist, 8 anti-inflammatory, 9 anti-alzheimer, 10 immunomodulatory, 11 anti-osteoporosis, 12 and anti-viral (against carp virus 13 and influenza a 14 ) activities. the synthesis of racemic mixtures of bavachin derivatives were reported 15,16 and these compounds have been proven to be ppar agonists 16 and anticancer reagents. 17 however, exchanges of phenol group to aniline group have not been tried. bavachin and bavachinin showed good anti-mers-cov activities of 2.9 and 7.9 μm respectively by phenotypic cellular screening with vero cell. as the small structural difference between two compounds (-oh vs. -ome) could change the anti-viral activity, we tried to synthesize the bavachin derivatives for structure-activity-relationship study (sar study). first, we synthesized racemic bavachinin (1a) according to the procedure reported by du et al. 15 the double bond in the side chain of 1a was reduced by hydrogenation with pd/c to prepare 2a, and oxidation of chromane ring with i 2 was performed to produce 3a (scheme 1). 6a was synthesized by the modified procedure of that of 1a. 15,16 4 was reacted with n-boc-protected 4-aminobenzaldehyde by aldol condensation to give 5, which was cyclized by kf to produce 6a (scheme 3). the further derivatizations of 1a, 2a, 3a, and 6a were performed by the substitutions of phenolic oh (1a, 2a, 3a) and nh 2 (6a) groups. for example, racemic bavachnin, 1a was reacted with iodomethane, 2-bromopropane, and benzyl bromide in the presence of potassium carbonate to afford 1b, 1c, and 1d, respectively. the derivatizations of phenol group of 2a and 3a were performed by the similar procedures as shown in scheme 2 to prepare 2b-2d and 3b. 6b and 6c were prepared by acetylation and propargylation of the amino group of 6a as shown in scheme 3. total 12 compounds of bavachinin derivatives in four core structures were evaluated to figure out their anti-mers-cov activity and cell-cytotoxicity by cellular phenotypic screening method as shown in table 1 . the activity of synthesized racemic bavachin (1a) was around half of the natural bavachin but o-methylated 1a, racemic bavachnin (1b) showed similar activity of natural bavachinin (entry 1 and 2 in table 1 ). the further alkylations of phenolic oh of 1a with isopropyl and benzyl group decreased the anti-mers activities (entry 3 and 4 in table 1 ). 2a was prepared by hydrogenations of external double bonds of 1a, which showed the best anti-mers activities regardless of their o-substituents. interestingly, o-isopropyl note and o-benzyl derivatives (2c and 2d, respectively) showed similar activity with 2a (non-substituted) better than 2b (omethylated), but the cytotoxicity for vero cell also increased. 3a showed good activity similar to that of 2a, however its cytotoxicity was much less than that of 2a. considering activity and cytotoxicity, 3a was thought to be the best compounds for anti-mers drug. the pharmacokinetic study of 1a was performed on rats by iv and po. 1a could not be detected in plasma after 30 min in both iv and po study by its rapid clearance, but its liver microsomal phase i stability (% of remaining after 30 min) in rat was 45%. we decided to change the phenolic oh group to amino group (6a) based on the assumption that phenolic oh group was concerned with its instability in plasma. though bioavailability (4%) was not satisfactory and liver microsomal phase i stability was not improved (36%), the results from pharmacokinetic study of 6a afforded acceptable curves in both iv and po study as shown in figure 1 . the anti-mers activity of 6a was a little decreased compared to 1a, however, the anti-mers activities of its derivatives were conserved. 1a also exhibited anti-viral activity against sars-cov (10.2 μm) in vero cell. as a conclusion, a series of 2-phenylchroman-4-one derivatives were synthesized for the chemical modifications of bavachin, and they exhibited anti-mers activities in vero cell. 2a showed the best activity, but 3a was thought to be the best compounds for anti-mers drug in consideration of cytotoxicity. the modification of phenol group (1a) to amino group (6a) could much improve pharmacokinetic properties. we expect the study on bavachin derivatives can contribute to the development of anti-mers drug. scheme 2. o-alkylations of 1a. reagents and conditions: (a) iodomethane, k 2 co 3 , dmf, rt., overnight, 68% (1b); 2-bromopropane, k 2 co 3 , dmf, 60 c, overnight, 41% (1c); benzyl bromide, k 2 co 3 , dmf, rt., overnight, 58% (1d). for immunofluorescence staining, the fixed cells (above) were permeabilized with 0.25% triton x-100 (sigma-aldrich, st. louis, mo, usa) for 20 min. then the cells were incubated with rabbit anti-mers-cov spike antibody for 1 h at 37 c. after three washes with pbs, the cells were incubated with alexa 488-conjugated goat anti-rabbit igg (h + l) secondary antibody and hoechst 33342 (life technologies, waltham, ma, usa) for 1 h at 37 c. images were acquired by perkin elmer operetta (20×; waltham, ma, usa). the acquired images were analyzed with in-house-developed image-mining 3.0 (im 3.0) plug-in software as previously described (pmid: 24205414). in the analyzed image, the total number of cells and the number of infected cells were determined by counting hoechst-stained nuclei and spike protein-expressing cells, respectively. the infection ratio of each well was normalized using the average infection ratio of mock control as 0% and the average infection ratio of negative control (0.5% dmso) as 100%, in each assay plate. cell ratio was determined according to the number of cells of each well versus the average number of cells of mock control, in each assay plate. all work with mers-cov was performed in an enhanced biosafety level-3 (bsl-3) facility at institut pasteur korea. antimicrob key: cord-305422-t8azymo7 authors: yi, ye; lagniton, philip n.p.; ye, sen; li, enqin; xu, ren-he title: covid-19: what has been learned and to be learned about the novel coronavirus disease date: 2020-03-15 journal: int j biol sci doi: 10.7150/ijbs.45134 sha: doc_id: 305422 cord_uid: t8azymo7 the outbreak of coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome (sars) coronavirus 2 (sars-cov-2), has thus far killed over 3,000 people and infected over 80,000 in china and elsewhere in the world, resulting in catastrophe for humans. similar to its homologous virus, sars-cov, which caused sars in thousands of people in 2003, sars-cov-2 might also be transmitted from the bats and causes similar symptoms through a similar mechanism. however, covid-19 has lower severity and mortality than sars but is much more transmissive and affects more elderly individuals than youth and more men than women. in response to the rapidly increasing number of publications on the emerging disease, this article attempts to provide a timely and comprehensive review of the swiftly developing research subject. we will cover the basics about the epidemiology, etiology, virology, diagnosis, treatment, prognosis, and prevention of the disease. although many questions still require answers, we hope that this review helps in the understanding and eradication of the threatening disease. the spring festival on january 25, 2020 has become an unprecedented and unforgettable memory to all chinese who were urged to stay indoors for all the holiday and for many weeks after due to the outbreak of a novel viral disease. the virus is highly homologous to the coronavirus (cov) that caused an outbreak of severe acute respiratory syndrome (sars) in 2003; thus, it was named sars-cov-2 by the world health organization (who) on february 11, 2020, and the associated disease was named cov disease-19 (covid-19) [1] . the epidemic started in wuhan, china, and quickly spread throughout the entire country and to near 50 others all over the world. as of march 2, 2020, the virus has resulted in over 80,000 confirmed cases of covid-19, with more than 40,000 patients discharged and over 3,000 patients who died. who warns that covid-19 is "public enemy number 1" and potentially more powerful than terrorism [2] . according to pubmed (https://www.ncbi.nlm. nih.gov/pubmed/), in less than two months, over 200 papers have been published on covid-19 including its virology, epidemiology, etiology, diagnosis, and treatment since the first report on january 7, 2020 that determined the sequence of the virus isolated from multiple patients [3] . this review attempts to summarize the research progress in the new and swiftly developing subject area. whenever possible, we will try to compare covid-19 with sars and another cov-caused disease, middle east respiratory syndrome (mers, an outbreak in 2012). we will also discuss what we have learned so far regarding the prevention and prognosis of the disease as well as some remaining yet urgent questions. covs have been traditionally considered nonlethal pathogens to humans, mainly causing approximately 15% of common colds [4] . however, in this century, we have encountered highly pathogenic human covs twice, i.e., sars-cov and mers-cov, which caused an outbreak originally in china in 2003 ivyspring international publisher and saudi arabia in 2012, respectively, and soon spread to many other countries with horrible morbidity and mortality [5] . therefore, the current covid-19 is the third cov outbreak in the recorded history of humans. as shown in fig. 1 , clusters of pneumonia that had unknown origins were first reported from wuhan on december 31, 2019 to the china national health commission [6] . seven days later the sequence of the cov was released [7] . on january 15, 2020 the first fatal case from wuhan was reported [6] . meanwhile, the epidemic rapidly spread to the neighboring cities, provinces, and countries. on january 20, the infection of health-care providers was reported, suggesting that human-to-human transmission was possible [8] . on january 23, the city of wuhan was locked down with all its public transportation stopped. on january 24 the first clinical study on the disease reported that, out of 41 patients with confirmed cases, only 21 had direct contact with the wuhan seafood market that was considered the starting site of the infection from an unknown animal source [6] . on january 30, who declared the outbreak a global health emergency. by the time of this report, the disease has already spread throughout china and near 50 other countries all over the world (fig. 2) . as the situation is rapidly evolving, the final scope and severity of the outbreak remain to be determined. on february 11, 2020, a multi-center study on 8,866 patients including 4,021 confirmed covid-19 patients presented a more updated illustration of the epidemic as follows (https://mp.weixin.qq.com/s/ ulbi-hx_rhpxa1qha2bhda). • sars-cov-2 infected people of all ages, but mainly at the age of 30-65. almost half (47.7%) of the infected individuals were over 50 years old, very few were under 20, and only 14 infected individuals were under the age of 10. • sars-cov-2 infected more men (0.31/100,000) than women (0.27/100,000). • covid-19 expanded in clusters mainly in and around hubei. • covid-19 took an average of 5 (2-9) days from onset to diagnosis. the average incubation period was 4.8 (3.0-7.2) days. the average time from onset to death was 9.5 (4.8-13) days. • the basic reproductive number (r0) was 3.77 (95% ci: 3.51-4.05), and the adjusted r0 was 2.23-4.82. • the number of infected people increased exponentially before 23 jan. 2020, matching the time of massive transportation before the spring festival in china. • the mortality of patients with confirmed cases was 1.44% (95% ci: 1.10-1.86%), and the adjusted mortality of all the patients was 3.06% (95% ci: 2.02-4.59%). • three major risk factors for covid-19 were sex (male), age (≥60), and severe pneumonia. covs are a subfamily of large and enveloped viruses containing a single strand of sense rna. they can be divided into four genera, i.e., alpha, beta, gamma, and delta, of which alpha-and beta-covs are known to infect humans [11] . the envelope spike (s) glycoprotein binds to its cellular receptors angiotensin-converting enzyme 2 (ace2) and dipeptidyl peptidase 4 (dpp4) for sars-cov and mers-cov, respectively, and then membrane fusion occurs [12] . the viral rna genome is released into the cytoplasm; after replication of the viral genome, genomic rna accompanied by envelope glycoproteins and nucleocapsid proteins forms virion-containing vesicles, which then fuse with the plasma membrane to release the virus [13] . the first genomic sequence of sars-cov-2 was reported on january 10, 2020 [3]. sars-cov-2 was found to be a new type of beta-cov with more than 99.98% genetic identity among 10 sequenced samples collected from the original site of the outbreak, the huanan seafood market in wuhan. sars-cov-2 is genetically more similar to sars-cov than to mers-cov [14] [15] [16] . through transmission electron microscopy, sars-cov-2 particles were found in ultrathin sections of human airway epithelium [17] . human ace2 was found to be a receptor for sars-cov-2 as well as sars-cov [16, 18, 19] . however, the s protein of sars-cov-2 binds to human ace2 more weakly than that of sars-cov, which is coincident with the fact that sars-cov-2 causes less severe infection in patients than sars-cov [14] . sars-cov-2 can also form a novel short protein encoded by orf3b and a secreted protein encoded by orf8. the orf3b of sars-cov-2 may play a role in the viral pathogenicity and inhibit the expression of ifnβ; however, orf8 does not contain any known functional domain or motif [20] . on february 18, 2020, zhou, et al., reported the cryo-em structure of the full-length human ace2 at 2.9 å resolution in complex with the amino acid transporter b 0 at1 [21] . they found that the complex, which had open and closed conformations, was assembled as a dimer and the ace2-b 0 at1 complex can bind two s proteins, which provides evidence for cov recognition and infection. b 0 at1 may become a therapeutic target for drug screening to suppress sars-cov-2 infection. it has been known that both sars-cov and mers-cov originated from bats and were transmitted to humans via civet cats and camels, respectively. through a phylogenetic comparison of sars-cov-2 with other covs, bats were considered the native host of sars-cov-2 as the new virus is 96% identical to two sars-like covs from bats called bat-sl-covzx45 and bat-sl-covzx21 [14] [15] [16] 19] . however, what intermediate host helped the virus cross the species barrier to infect humans remains unknown, and the transmission route is yet to be elucidated. ji, et al., proposed snakes as a carrier of the virus from bats to humans which involved homologous recombination within the s protein [22] . according to a study, researchers in guangzhou, china, suggested that pangolins -long-snouted, anteating mammals often used in traditional chinese medicine -are the potential intermediate host of sars-cov-2 based on 99% genetic homology in a cov discovered in pangolins and sars-cov-2 [23] . however, 1% difference spread all over two genomes is still a big difference; thus, conclusive results for concrete evidence are awaited (fig. 3) . the physicochemical properties of sars-cov-2 are largely not yet known. sars-cov and mers-cov can survive in vitro for 48 hours in a dry environment and up to 5 days under 20 °c and 40%-50% humidity [24] [25] [26] . sars-cov-2 may possess similar properties. it has been reported that sars-cov-2 is sensitive to ultraviolet rays and heat at 56 °c for 30 minutes; ether, 75% ethanol, chlorine-containing disinfectant, peracetic acid, chloroform, and other fatty solvents, but not chlorhexidine, can effectively inactivate the virus [27] . the entire human population generally lacks immunity to sars-cov-2 and hence is susceptible to the novel virus. currently, no detailed study has been reported regarding the immunological response to sars-cov-2. thus, we can only refer to previous studies on other covs, especially sars-cov and mers-cov (fig. 4) . in general, after a virus invades the host, it is first recognized by the host innate immune system through pattern recognition receptors (prrs) including c-type lectin-like receptors, toll-like receptor (tlr), nod-like receptor (nlr), and rig-ilike receptor (rlr) [28] . through different pathways, the virus induces the expression of inflammatory factors, maturation of dendritic cells, and synthesis of type i interferons (ifns) which limit the spreading of the virus and accelerate macrophage phagocytosis of viral antigens [28] . however, the n protein of sars-cov can help the virus escape from the immune responses [29] . soon, the adaptive immune response joins the fight against the virus. t lymphocytes including cd4 + and cd8 + t cells play an important role in the defense. cd4 + t cells stimulate b cells to produce virus-specific antibodies, and cd8 + t cells directly kill virus-infected cells. t helper cells produce proinflammatory cytokines to help the defending cells. however, cov can inhibit t cell functions by inducing apoptosis of t cells. the humoral immunity including complements such as c3a and c5a and antibodies is also essential in combating the viral infection [30, 31] . for example, antibodies isolated from a recovered patient neutralized mers-cov [32] . on the other hand, an overreaction of the immune system generates a large number of free radicals locally that can cause severe damages to the lungs and other organs, and, in the worst scenario, multi-organ failure and even death [33] . the sars-cov-2 infection, featured by clustering onset, is more likely to affect elderly people with comorbidities and pregnant women [8] . it is common that for people who are exposed to a large number of viruses or whose immune functions are compromised, they have higher chance to be infected than others. the estimated mean incubation period of sars-cov-2 is 1-14 days, mostly 3-7 days based on a study of the first 425 cases in wuhan [36] . however, a study on 1,099 cases demonstrates that the incubation period was 3 days on average and ranged from 0 to 24 days [8] . a more recent study, as described above, demonstrates that the incubation period was 4.8 (3.0-7.2) days based on the demography of 8,866 cases [37] . it is very important for health authorities to adjust the effective quarantine time based on the most accurate incubation period, thus preventing infected but symptomless people from transmitting the virus to others [38] . as a common practice, individuals exposed to, or infected by, the virus are usually required to be quarantined for 14 days. should the quarantine time be extended to 24 days? fever is often the major and initial symptom of covid-19, which can be accompanied by no symptom or other symptoms such as dry cough, shortness of breath, muscle ache, dizziness, headache, sore throat, rhinorrhea, chest pain, diarrhea, nausea, and vomiting. some patients experienced dyspnea and/or hypoxemia one week after the onset of the disease [8] . in severe cases, patients quickly progressed to develop acute respiratory syndrome, septic shock, metabolic acidosis, and coagulopathy. patients with fever and/or respiratory symptoms and acute fever, even without pulmonary imaging abnormalities, should be screened for the virus for early diagnosis [39] [40] [41] . a demographic study in late december of 2019 showed that the percentages of the symptoms were 98% for fever, 76% for dry cough, 55% for dyspnea, and 3% for diarrhea; 8% of the patients required ventilation support [42] . similar findings were reported in two recent studies of a family cluster and a cluster caused by transmission from an asymptomatic individual [43, 44] . comparably, a demographic study in 2012 showed that mers-cov patients also had fever (98%), dry cough (47%), and dyspnea (55%) as their main symptoms. however, 80% of them required ventilation support, much more than covid-19 patients and consistent with the higher lethality of mers than of covid-19. diarrhea (26%) and sore throat (21%) were also observed with mers patients. in sars patients, it has been demonstrated that fever (99%-100%), dry cough (29%-75%), dyspnea (40%-42%), diarrhea (20-25%), and sore throat (13-25%) were the major symptoms and ventilation support was required for approximately 14%-20% of the patients [45] . by february 14, the mortality of covid-19 was 2% when the confirmed cases reached 66,576 globally. comparably, the mortality of sars by november 2002 was 10% of 8,096 confirmed cases [46] . for mers, based on a demographic study in june 2012, the mortality was 37% of 2,494 confirmed cases [47] . an earlier study reported that the r0 of sars-cov-2 was as high as 6.47 with a 95% confidence interval (ci) of 5.71-7.23 [48] , whereas the r0 of sars-cov only ranged from 2 to 4 [49] . a comparison of sars-cov-2 with mers-cov and sara-cov regarding their symptoms, mortality, and r0 is presented in table 1 . the above figures suggest that sars-cov-2 has a higher ability to spread than mers-cov and sars-cov, but it is less lethal than the latter two [6] . thus, it is much more challenging to control the epidemic of sars-cov-2 than those of mers-cov and sars-cov. clustered onset often happens in the same family or from the same gathering or vehicle such as a cruise ship. patients often have a history of travel or residence in wuhan or other affected areas or contact with infected individuals or patients in the recent two weeks before the onset [50] . however, it has been reported that people can carry the virus without symptoms longer than two weeks and cured patients discharged from hospitals can carry the virus again [51] , which sends out an alarm to increase the time for quarantine. patients have normal or reduced number of peripheral white blood cells (especially lymphocytes) at the early stage. for example, lymphopenia with white blood cell count < 4×10 9 /l including lymphocyte count < 1×10 9 /l, and elevated aspartate aminotransferase levels and viremia were found in 1,099 covid-19 patients [8] . the levels of liver and muscle enzymes and myoglobin were increased in the blood of some patients, and c-reactive protein and erythrocyte sedimentation were increased in the blood of most patients [52] . in patients with severe cases, the level of d-dimer, a fibrin degradation product present in the blood, was elevated, and lymphocyte count was progressively reduced [34] . abnormalities in chest radiography are found in most covid-19 patients and featured by bilateral patchy shadows or ground glass opacity in the lungs. patients often develop an atypical pneumonia, acute lung injury, and acute respiratory distress syndrome (ards) [34] . when ards happens, uncontrolled inflammation, fluid accumulation, and progressive fibrosis severely compromise the gas exchange. dysfunction of type-i and type-ii pneumocytes decreases the surfactant level and increases surface tension, thus reducing the ability of the lungs to expand and heightening the risk of lung collapse [53, 54] . therefore, the worst chest radiographic findings often parallel the most severe extent of the disease [55] . on february 18, 2020, the first pathological analysis of covid-19 demonstrated the desquamation of pneumocytes, hyaline membrane formation, and interstitial lymphocyte infiltration, and multinucleated syncytial cells in the lungs of a patient who died of the disease, consistent with the pathology of viral infection and ards [56] and similar to that of sars and mers patients [57, 58] . the detection of sars-cov-2 rna via reversetranscriptase polymerase chain reaction (rt-pcr) was used as the major criteria for the diagnosis of covid-19. however, due to the high false-negative rate, which may accelerate the epidemic, clinical manifestations started to be used for diagnosis (which no longer solely relied on rt-pcr) in china on february 13, 2020. a similar situation also occurred with the diagnosis of sars [59] . therefore, a combination of disease history, clinical manifestations, laboratory tests, and radiological findings is essential and imperative for making an effective diagnosis. on february 14, 2020, the feng zhang group described a protocol of using the crispr-based sherlock technique to detect sars-cov-2, which detects synthetic sars-cov-2 rna fragments at 20 × 10 -18 mol/l to 200 × 10 -18 mol/l (10-100 copies per microliter of input) using a dipstick in less than an hour without requiring elaborate instrumentation [60] . hopefully, the new technique can dramatically enhance the sensitivity and convenience if verified in clinical samples. due to the lack of experience with the novel cov, physicians can mainly provide supportive care to covid-19 patients, while attempting a variety of therapies that have been used or proposed before for the treatment of other covs such as sars-cov and mers-cov and other viral diseases ( table 2) . these therapies include current and potential treatments with antiviral drugs, immunosuppressants, steroids, plasma from recovered patients, chinese medicine, and psychological support. even plasma from recovered patients was proposed to be used for treatment [61] . pharmaceutical companies are racing to develop antibodies and vaccines against the virus [62] . sars-cov-2 mainly attacks the lungs in the beginning and probably also attacks, to a lesser degree, other organs that express ace2, such as the gastrointestinal system and the kidneys. nevertheless, respiratory dysfunction and failure are the major threat to the patients and the major cause of death. thus, respiratory support is critical to relieve the symptoms and save lives and includes general oxygen therapy, high-flow oxygen, noninvasive ventilation, and invasive mechanical ventilation depending on the severity of the disease. patients with severe respiratory symptoms have to be supported by extracorporeal membrane oxygenation (ecmo), a modified cardiopulmonary bypass technique used for the treatment of life-threatening cardiac or respiratory failure. in addition, the maintenance of electrolyte balance, the prevention and treatment of secondary infection and septic shock, and the protection of the functions of the vital organs are also essential for sars-cov-2 patients [7] . it has been known that a cytokine storm results from an overreaction of the immune system in sars and mers patients [33] . cytokine storm is a form of systemic inflammatory response featured by the release of a series of cytokines including tnfα, il-1β, il-2, il-6, ifnα, ifnβ, ifnγ, and mcp-1. these cytokines induce immune cells to release a vast number of free radicals which are the major cause of ards and multiple organ failure [67] . immunosuppression is essential in the treatment of cytokine storms, especially in severe patients. corticosteroids and tocilizumab, an anti-il6 monoclonal antibody, have been used to treat cytokine storm [68] . other immunosuppression treatments for cytokine storm include the modulation of t cell-directed immune response; the blockade of ifn-γ, il-1, and tnf; jak inhibition [69] ; blinatumomab [70] ; suppressor of cytokine signaling 4 [71] ; and hdac inhibitors [72] . steroids, as immunosuppressants, were widely used in the treatment of sars to reduce the severity of inflammatory damage [65] . however, steroids at high dosages were not beneficial to severe lung injury in sars and covid-19 patients [59, 73] . instead, they may cause severe side effects, especially avascular osteonecrosis, dramatically affecting the prognosis [74] . nevertheless, short courses of corticosteroids at low-to-moderate doses have been recommended to be used prudently for critically ill covid-19 patients [75] . at the time of writing, no effective antiviral therapy has been confirmed. however, intravenous administration with remdesivir, a nucleotide analog, has been found to be efficacious in an american patient with covid-19 [64] . remdesivir is a novel antiviral drug developed by gilead initially for the treatment of diseases caused by ebola and marlburg viruses [76] . later, remdesivir also demonstrated possible inhibition of other single stranded rna viruses including mers and sars viruses [77, 78] . based on these, gilead has provided the compound to china to conduct a pair of trials on sars-cov-2infected individuals [79] , and the results are highly anticipated. in addition, baricitinb, interferon-α, lopinavir/ ritonavir, and ribavirin have been suggested as potential therapies for patients with acute respiratory symptoms [80, 81] . diarrhea, nausea, vomiting, liver damage, and other adverse reactions can occur following combined therapy with lopinavir/ritonavir [80] . the interaction of these treatments with other drugs used in the patients should be monitored carefully. the collection of the blood from patients who recovered from a contagious disease to treat other patients suffering from the same disease or to protect healthy individuals from catching the disease has a long history [82] . indeed, recovered patients often have a relatively high level of antibodies against the pathogen in their blood. antibodies are an immunoglobulin (ig) produced by b lymphocytes to fight pathogens and other foreign objects and they recognize unique molecules in the pathogens and neutralize them directly [83] . based on this, plasma was collected from the blood of a group of patients who recovered from covid-19 and was injected into 10 seriously ill patients. their symptoms improved within 24 hours, accompanied by reduced inflammation and viral loads and improved oxygen saturation in the blood. however, verification and clarification are necessary to propose the method for large-scale use before specific therapies are not yet developed. in addition, given the therapeutic effects, some disadvantages associated with the plasma should be considered carefully. for example, antibodies can overstimulate the immune response and cause cytokine release syndrome, which is potentially a life-threatening toxicity [84] . the concentration of antibodies in the blood is usually low, and the demand for the plasma is large to treat critically ill patients. it is difficult to develop and produce specific antibodies rapidly enough to fight against a global epidemic [62] . thus, it is more critical and practical to isolate b cells from recovered patients and identify the genetic codes encoding effective antibodies or screen for effective antibodies against essential proteins of the virus. this way, we can readily scale up the production of the antibodies. tcm has been used to treat a variety of diseases in china for thousands of years. however, its effects largely rely on a combination of multiple components in a formula that varies depending on the diagnosis of a disease based on the theories of tcm. most of the effective components remain unknown or are vague as it is difficult to extract and verify such components or their optimal combinations. currently, due to the lack of effective and specific therapy for covid-19, tcm has become one of the major alternative treatments for patients with light to moderate symptoms or for those who have recovered from severe stages [85] . for example, shu feng jie du capsules and lian hua qing wen capsules were found to be effective for covid-19 treatment [86] . top cure rates in the treatment of covid-19 patients were observed in several provinces in china that used tcm in 87% of their patients, including gansu (63.7%), ningxia (50%), and hunan (50%), whereas hubei province, which used tcm in only approximately 30% of its covid-19 patients, had the lowest cure rate (13%) [87] . however, this is quite a rough comparison as many other impact factors such as the number and severity of the patients should be included in the evaluation. on february 18, 2020, boli zhang and coworkers published a study to compare western medicine (wm) treatment alone with combined treatment of wm and tcm [88] . they found that the times needed for body temperature recovery, symptom disappearance, and hospitalization were remarkably shorter in the wm+tcm group than in the wm only group. most impressively, the rate for symptomatic worsening (from light to severe) was remarkably lower for the wm+tcm group than for the wm only group (7.4% versus 46.2%) and the mortality was lower in the wm+tcm group than wm only group (8.8% versus 39%). nevertheless, the efficacy and safety of tcm still await more well-controlled trials at larger scales and in more centers. it would also be intriguing to characterize the mechanism of actions and clarify the effective components of tcm treatments or their combinations if possible. patients with suspected or confirmed covid-19 mostly experience great fear of the highly contagious and even fatal disease, and quarantined people also experience boredom, loneliness, and anger. furthermore, symptoms of the infection such as fever, hypoxia, and cough as well as adverse effects of the treatments such as insomnia caused by corticosteroids can lead to more anxiety and mental distress. in the early phase of the sars outbreak, a range of psychiatric morbidities including persistent depression, anxiety, panic attacks, psychomotor excitement, psychotic symptoms, delirium, and even suicidality were reported [89, 90] . mandatory contact tracing and quarantine, as a part of the public health responses to the covid-19 outbreak, can make people more anxious and guilty about the effects of the contagion, quarantine, and stigma on their families and friends [66] . thus, mental health care should be provided to covid-19 patients, suspected individuals, and people in contact with them as well as the general public who are in need. the psychological support should include the establishment of multidisciplinary mental health teams, clear communications with regular and accurate updates about the sars-cov-2 outbreak and treatment plans and the use of professional electronic devices and applications to avoid close contact with each other [66] . effective vaccines are essential for interrupting the chain of transmission from animal reservoirs and infected humans to susceptible hosts and are often complementary to antiviral treatment in the control of epidemics caused by emerging viruses. efforts have been made to develop s protein-based vaccines to generate long-term and potent neutralizing antibodies and/or protective immunity against sars-cov [81, 91] . live-attenuated vaccines have been evaluated in animal models for sars [92] . however, the in vivo efficacy of these vaccine candidates in elderly individuals and lethal-challenge models and their protection against zoonotic virus infection have yet to be determined before a clinical study is initiated. this is probably because sars died down 17 years ago and no new case has been reported since. in contrast, sporadic cases and clusters of mers continue to occur in the middle east and spread to other regions owing to the persistence of zoonotic sources in endemic areas. vaccination strategies have been developed for mers by using inactivated virus, dna plasmids, viral vectors, nanoparticles, virus-like particles and recombinant protein subunits and some have been evaluated in animal models [93] . the development of a safe and effective vaccine against sars-cov-2 for non-immune individuals is an urgent and critical task for controlling the ongoing epidemic. however, it is challenging to overcome the difficulty because of the long period of time (averaged 18 months) needed for vaccine development and the dynamic variations of covs. as a novel disease, covid-19 has just started to manifest its full clinical course throughout thousands of patients. in most cases, patients can recover gradually without sequelae. however, similar to sars and mers, covid-19 is also associated with high morbidity and mortality in patients with severe cases. therefore, building a prognosis model for the disease is essential for health-care agencies to prioritize their services, especially in resourceconstrained areas. based on clinical studies reported thus far, the following factors may affect or be associated with the prognosis of covid-19 patients (table 3) : • age: age was the most important factor for the prognosis of sars [99] , which is also true for covid-19. covid-19 mainly happened at the age of 30-65 with 47.7% of those patients being over 50 in a study of 8,866 cases as described above [37] . patients who required intensive care were more likely to have underlying comorbidities and complications and were significantly older than those who did not (at the median age of 66 versus 51) [34] , suggesting age as a prognostic factor for the outcome of covid-19 patients. • sex: sars-cov-2 has infected more men than women (0.31/100,000 versus 0.27/100,000), as described above [37] . • comorbidities and complications: patients with covid-19 who require intensive care are more likely to suffer from acute cardiac injury and arrhythmia [34] . cardiac events were also the main reason for death in sars patients [55, 65, 99] . it has been reported that sars-cov-2 can also bind to ace2-positive cholangiocytes, which might lead to liver dysfunctions in covid-19 patients [100] . it is worth noting that age and underlying disease are strongly correlated and might interfere with each other [55] . • abnormal laboratory findings: the c-reactive protein (crp) level in blood reflects the severity of inflammation or tissue injury and has been proposed to be a potential prognostic factor for disease, response to therapy, and ultimate recovery [101] . the correlation of crp level to the severity and prognosis of covid-19 has also been proposed [101] . in addition, elevated lactate dehydrogenase (ldh), aspartate aminotransferase (ast), alanine aminotransferase (alt), and creatine kinase (ck) may also help predict the outcome. these enzymes are expressed extensively in multiple organs, especially in the heart and liver, and are released during tissue damage [102, 103] . thus, they are traditional markers for heart or liver dysfunctions. • major clinical symptoms: chest radiography and temporal progression of clinical symptoms should be considered together with the other issues for the prediction of outcomes and complications of covid-19. • use of steroids: as described above, steroids are immunosuppressant commonly used as an adjunctive therapy for infectious diseases to reduce the severity of inflammatory damage [104] . since a high dosage of corticosteroids was widely used in severe sars patients, many survivors suffered from avascular osteonecrosis with life-long disability and poor life quality [105] . thus, if needed, steroids should be used at low dosage and for a short time in covid-19 patients. • mental stress: as described above, during the covid-19 outbreak many patients have suffered from extraordinary stress as they often endured long periods of quarantine and extreme uncertainty and witnessed the death of close family members and fellow patients. it is imperative to provide psychological counseling and long-term support to help these patients recover from the stress and return to normal life [66] . according to demographic studies so far, covid-19 seems to have different epidemiological features from sars. in addition to replicating in the lower respiratory tract, sars-cov-2 can efficiently replicate in the upper respiratory tract and causes mild or no symptoms in the early phase of infection, similar to other covs that cause common colds [106] . therefore, infected patients at the early phase or incubation period can produce a large amount of virus during daily activities, causing great difficulty for the control of the epidemic. however, the transmission of sars-cov was considered to occur when the patients are severely ill, while most transmission did not happen at the early phase [107] . thus, the current outbreak of covid-19 is much more severe and difficult to control than the outbreak of sars. great efforts are currently underway in china including the lockdown of wuhan and surrounding cities and continuous quarantine of almost the entire population in hopes of interrupting the transmission of sars-cov-2. although these actions have been dramatically damaging the economy and other sectors of the country, the number of new patients is declining, indicating the slowdown of the epidemic. the most optimistic estimate is that the outbreak will end by march and the downswing phase will last for 3-4 months [108] . however, some other experts are not that optimistic. paul hunter, et al., estimated that covid-19, which seems substantially more infectious than sars, will not end in 2020 [109] . ira longini, et al., established a model to predict the outcome of the epidemic and suggested that sars-cov-2 could infect two-thirds of the global population [110] . a canadian group reported that sars-cov-2 was detected in both mid-turbinate and throat swabs of patients who recovered and left the hospital 2 weeks earlier [111] , which indicates that the newly identified virus could become a cyclical episode similar to influenza. however, promising signs have occurred in china based on the declining number of new cases, indicating the current strategies might have been working. ebola was originally predicted to cause up to a million cases with half a million deaths. however, via strict quarantine and isolation, the disease has eventually been put under control [112, 113] . it is possible, similar to sars-cov, that sars-cov-2 might become weaker in infectivity and eventually die down or become a less pathogenic virus co-existent with humans. a comparison of the epidemic of covid-19 with that of sars and mers is provided below (fig. 5 ). sars-cov-2 is highly transmittable through coughing or sneezing, and possibly also through direct contact with materials contaminated by the virus [12] . the virus was also found in feces, which raises a new possibility of feces-to-mouth transmission [114] . a recent study on 138 cases reported that 41% of the cases were possibly caused by nosocomial infections, including 17 patients with other prior diseases and 40 health-care providers [115] . thus, great precaution should be used to protect humans, especially health-care providers, social workers, family members, colleagues, and even bystanders in contact with patients or infected people. the first line of defense that could be used to lower the risk of infection is through wearing face masks; both the use of surgical masks and n95 respirator masks (series # 1860s) helps control the spread of viruses [116] . surgical face masks prevent liquid droplets from a potentially infected individual from traveling through the air or sticking onto surfaces of materials, where they could be passed on to others [117] . however, only n95 (series # 1860s) masks can protect against the inhalation of virions as small as 10 to 80 nm, with only 5% of the virions being able to penetrate completely; sars-cov-2 is similar to sars-cov in size and both are approximately 85 nm [117] . since particles can penetrate even five surgical masks stacked together, health-care providers in direct contact with patients must wear n95 (series # 1860s) masks but not surgical masks [118] . in addition to masks, health-care providers should wear fitted isolation gowns in order to further reduce contact with viruses. viruses can also infect an individual through the eyes. on january 22, 2020, a doctor was infected with sars-cov-2 although he wore an n95 mask; the virus might have entered his body through his inflammatory eyes [119] . thus, health-care providers should also wear transparent face shields or goggles while working with patients. for the general public in affected or potentially affected areas, it is highly suggested that everybody wash their hands with disinfectant soaps more often than usual, try to stay indoors for self-quarantine and limit contact with potentially infected individuals. three feet is considered an appropriate distance for people to stay away from a patient [120] . these actions are effective methods to lower the risk of infection as well as prevent the spread of the virus. although sars-cov-2 came as a new virus to the human world, its high homology to sars-cov as reported on 7 january 2020 [3] should have caused high alert to china based on her deep memory of the sars outbreak in 2003. however, not until 19 january 2020 did the director of the center of disease control of wuhan comfort the citizens by saying that the novel virus has low contagiousness and limited reproductivity from human to human and that it is not a problem to prevent and contain the disease. this message remarkably relaxed the alarm of the public, especially when the entire country was preparing for the spring festival, and the critical time was missed to contain the disease at its minimal scale in wuhan. the disease control agencies in china may take this hard lesson and make critical improvements in the future. for example, these agencies should be (1) more careful when making public announcements as every word counts to citizens and can change their attitude and decisions; (2) more sensitive and reactive to unusual information from clinics rather than waiting for formal reports from doctors or officials; (3) more restrictive to contain a potential epidemic at its early stage rather than attempting to comfort the public; and (4) more often to issue targeted and effective drills to increase the public's awareness about epidemic diseases and to test and improve the response system of the society periodically. the outbreak of covid-19 caused by the novel virus sars-cov-2 started in the end of december 2019. in less than two months, it has spread all over china and near 50 other countries globally at the time of this writing. since the virus is very similar to sars-cov and the symptoms are also similar between covid-19 and sars, the outbreak of covid-19 has created a sense of sars recurring. however, there are some remarkable differences between covid-19 and sars, which are essential for containing the epidemic and treating the patients. • covid-19 affects more elderly individuals than youth and more men than women, and the severity and death rate are also higher in elderly individual than in youth. • sars has higher mortality than covid-19 (10.91% versus 1.44%). • covid-19 patients transmit the virus even when they are symptomless whereas sars patients do so usually when they are severely ill, which causes much greater difficulty to contain the spread of covid-19 than sars. this partially explains why sars-cov-2 spread much faster and broader than sars-cov. • the regular rna assay for sars-cov-2 can be negative in some covid-19 patients. on the other hand, cured patients can be positive for the virus again. these findings dramatically increase the risk of virus spreading. given such rapid progress in research on covid-19, several critical issues remain to be solved, as follows: • where did sars-cov-2 come from? although 96% genetic homolog was found between sars-cov-2 and two bat sars-like covs, we still cannot conclude that sars-cov-2 is from bats. • what animal was the intermediate species to transmit the virus from the original host, say bats, to humans? without knowing answers to #1 and 2, we cannot efficiently cut the transmission, and the outbreak can relapse at any time. • although molecular modeling and biochemical assays have demonstrated that sars-cov-2 binds to ace2, how exactly does the virus enter the airway cells and cause subsequent pathological changes? does the virus also bind ace2-expressing cells in other organs [121] ? without clear answers to these questions, we cannot achieve fast and accurate diagnosis and effective treatment. • how long will the epidemic last? how is the virus genetically evolving during transmission among humans? will it become a pandemic worldwide, die down like sars or relapse periodically like the flu? it is essential but may take some time to search for answers to the above and many other questions. however, with whatever expense it may demand, we have no other choice but to stop the epidemic as soon as possible and bring our life back to normal. the covid-19 epidemic a: who warns coronavirus, now dubbed covid-19, is 'public enemy number 1' and potentially more powerful than terrorism sars and mers: recent insights into emerging coronaviruses coronavirus infections-more than just the common cold a novel coronavirus outbreak of global health concern cdc: 2019 novel coronavirus clinical characteristics of coronavirus disease 2019 in china reliefweb: johns hopki university. coronavirus covid-19 global cases by johns hopkins csse an interactive web-based dashboard to track covid-19 in real time host factors in coronavirus replication dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc insights into rna synthesis, capping, and proofreading mechanisms of sars-coronavirus genomic and protein structure modelling analysis depicts the origin and infectivity of 2019-ncov evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. the lancet a novel coronavirus from patients with pneumonia in china functional assessment of cell entry and receptor usage for lineage b β-coronaviruses, including 2019-ncov discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan structure of dimeric full-length human ace2 in complex with b0at1 homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies stability and inactivation of sars coronavirus nhc: diagnosis and treatment of pneumonia caused by 2019 new coronavirus (trial version5 modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the a(2b) receptor sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism molecules great and small: the complement system an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan coronavirus infections and immune responses early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia epidemiological and clinical features of the 2019 novel coronavirus outbreak in china epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study severe acute respiratory syndrome-related coronavirus: the species and its virusesa statement of the coronavirus study group identification of a novel coronavirus causing severe pneumonia in human: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan transmission of 2019-ncov infection from an asymptomatic contact in germany importation and human-to-human transmission of a novel coronavirus in vietnam imaging changes in patients with 2019-ncov summary of probable sars cases with onset of illness from 1 nvember mers situation update estimation of the transmission risk of the 2019-ncov and its implication for public health interventions consensus document on the epidemiology of sars organizaition wh. global surveillance for human infection with novel coronavirus epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study surfactant alteration and replacement in acute respiratory distress syndrome molecular pathology of emerging coronavirus infections clinical manifestations, laboratory findings, and treatment outcomes of sars patients pathological findings of covid-19 associated with acute respiratory distress syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates clinical findings, treatment and prognosis in patients with severe acute respiratory syndrome (sars) a protocol for detection of covid-19 using crispr diagnostics bloomberg: china seeks plasma from recovered patients as virus treatment wired: niiler e. darpa cranks up antibody research to stall coronavirus differential role for t cells in the development of fibrotic lesions associated with reovirus 1/l-induced bronchiolitis obliterans organizing pneumonia versus acute respiratory distress syndrome first case of 2019 novel coronavirus in the united states clinical features and short-term outcomes of 144 patients with sars in the greater toronto area timely mental health care for the 2019 novel coronavirus outbreak is urgently needed into the eye of the cytokine storm current concepts in the diagnosis and management of cytokine release syndrome review: cytokine storm syndrome: looking toward the precision medicine era cytokine release syndrome suppressor of cytokine signaling 4 (socs4) protects against severe cytokine storm and enhances viral clearance during influenza infection hdac inhibitor reduces cytokine storm and facilitates induction of chimerism that reverses lupus in anti-cd3 conditioning regimen clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury osteonecrosis of hip and knee in patients with severe acute respiratory syndrome treated with steroids on the use of corticosteroids for 2019-ncov pneumonia. the lancet therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses the new york times: denise grady. china begins teating an antiviral drug in coronavirus patients ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus coronaviruses -drug discovery and therapeutic options primary immunodeficiency disorders: a historic and scientific perspective immunoglobulins and virus antibody titers: of past needs, current requirements, and future options current concepts in the diagnosis and management of cytokine release syndrome drug treatment options for the 2019-new coronavirus (2019-ncov) covind-19 epidemic situation in real time clinical study on 34 cases of covid-19 treated with combined traditional chinese and western medicine (in chinese) a revisit on older adults suicides and severe acute respiratory syndrome (sars) epidemic in hong kong prevalence of psychiatric morbidity and psychological adaptation of the nurses in a structured sars caring unit during outbreak: a prospective and periodic assessment study in taiwan the spike protein of sars-cov--a target for vaccine and therapeutic development sars vaccines: where are we? antibodies and vaccines against middle east respiratory syndrome coronavirus clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study patient characteristics infected with middle east respiratory syndrome coronavirus infection in a tertiary hospital a comparative study of clinical presentation and risk factors for adverse outcome in patients hospitalised with acute respiratory disease due to mers coronavirus or other causes therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)--possible lessons from a systematic review of sars-cov therapy clinical and laboratory findings of middle east respiratory syndrome coronavirus infection clinical prognostic rules for severe acute respiratory syndrome in low-and high-resource settings specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection applications of acute phase reactants in infectious diseases aspartate aminotransferase: bridging carbohydrate and energy metabolism in plasmodium falciparum lactate dehydrogenase in sickle cell disease induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? avascular necrosis of bone in severe acute respiratory syndrome a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster the severe acute respiratory syndrome reality and future about covid-19 how long will covid-19 outbreak last bloomberg: lauerman j. coronavirus could infect two-thirds of globe, research shows first imported case of 2019 novel coronavirus in canada, presenting as mild pneumonia ebola, the day after the world after ebola: an overview of ebola complications, vaccine development, lessons learned, financial losses, and disease preparedness sciencealert: latest study suggests the new coronavirus is also spreading via feces clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan a quantitative assessment of the efficacy of surgical and n95 masks to filter influenza virus in patients with acute influenza infection do n95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks? protecting healthcare staff from severe acute respiratory syndrome: filtration capacity of multiple surgical masks 2019-ncov transmission through the ocular surface must not be ignored cdc: guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings ace2 expression in kidney and testis may cause kidney and testis damage after 2019-ncov infection y.y. and r.x. conceived and designed the manuscript. y.y., p.l., s.y., e.l. and r.x. wrote the manuscript. r.x gave the final approval of the manuscript. r.x. is a founder of imstem biotechnology, inc., a stem cell company. the other authors declare no competing financial interests. key: cord-317389-trvleobp authors: hoy, carlton f.o.; kushiro, keiichiro; yamaoka, yutaro; ryo, akihide; takai, madoka title: rapid multiplex microfiber-based immunoassay for anti-mers-cov antibody detection date: 2019-10-14 journal: sens biosensing res doi: 10.1016/j.sbsr.2019.100304 sha: doc_id: 317389 cord_uid: trvleobp on-site multiplex biosensors for innate immunity antibodies are ideal tools for monitoring health status of individuals against various diseases. this study introduces a novel antibody immunoassay testing platform incorporating microfiber-based arrays of antigens to capture specific antibodies. the fabrication and setup of the device revolved around electrospun polystyrene (esps) microfibers that act as three-dimensional membrane filters, capable of rapid and multifold analyte capture. in particular, the esps microfibers were patterned through localized oxygen plasma to create hydrophilic zones that facilitate fluid flows and immobilizations of antigens. the bulk of this robust antibody immunoassay platform could be installed into a compact syringe-driven cassette device, which could perform multiplex antibody immunoassay for antibodies specifically against middle east respiratory syndrome coronavirus (mers-cov) with rapid preparation amounting to a total of 5 min, as well as high sensitivity and specificity for the mers-cov down to 200 μg/ml. for the clinical use of immunoassays, these platforms are expected to be high-throughput, rapid and highly sensitive, as these are basic requirements for the necessary performance. thus, assays combining these characteristics are proposed and often realized by the use of microfluidics and nanotechnology [1] [2] [3] [4] [5] [6] [7] [8] . furthermore, many types of multiplex immunoassay platforms have been reported, including microelectromechanical systems (mems) [9, 10] , paper-based [11] [12] [13] [14] , bead-based [15] [16] [17] and array-based platforms [18] [19] [20] [21] . in recent years, one of the most prominent application of such immunoassay platforms from the perspective of healthcare has been virus detection [22, 23] . since 2012, mers-cov has become a prevalent issue affecting multiple countries [24] [25] [26] . this coronavirus has affected individuals with a high mortality rate of about 30% which was reported by the world health organization (who) [27] . furthermore, mers-cov being a relatively recent outbreak, there has been no effective vaccine which is clinically approved to treat an individual [28] . therefore, a means for antibody diagnostics in a simple, robust and rapid manner is necessary to test vaccine or drug efficacies upon patients while also preventing the spread of this infectious disease. in the world of vaccination, blood antibody titres often in the form of enzyme-linked immunosorbent assays (elisa) are common methods to determine whether a patient currently has the necessary antibodies at adequate concentrations. elisa is a highly sensitive method that can achieve limits of detection (lod) below 1 μg/ml for mers-cov, but the test can take days to produce diagnostic results due to the requirements for specialists and large equipment [29] [30] [31] [32] [33] [34] [35] [36] . thus, a more rapid and effective testing methodology that does not require large testing apparatus is desirable to test for vaccine or drug efficacy. in particular, it is important to emphasize the need for low-cost, simple and robust point-of-care (poc) device manufacturing in order to improve global healthcare. here, a rapid immunoassay methodology for antiviral antibody testing that can be conducted within minutes will be introduced. this work illustrates the construction of a novel multiplex antibody immunoassay test which leverages the esps microfiber mats as the capture membrane and pressure-driven convection for ultra-rapid testing. in addition to the previously established fluorescently-linked immunosorbent assay (flisa) microfiber platform [28, 37] , the microfiber mats were pre-patterned with o 2 plasma to create multiple hydrophilic zones with different antigens, and thus creating a multiplex detection system. as a proof of concept, two different antigens designed to individually capture their corresponding antibodies were tested: human https://doi.org/10.1016/j.sbsr.2019.100304 received 20 august 2019; received in revised form 2 october 2019; accepted 3 october 2019 serum albumin (hsa) and mers-cov. hsa is a representative protein that exists in human plasma samples, while mers is a viral agent currently lacking a means of vaccination [28, 38] . the simplistic design, ease of fabrication and setup, the rapid speeds at which diagnostics can be performed for antibody detection with this system can be an integral step forward in creating a low cost commercial device for protecting the general public health in case of mers outbreaks. for the polymeric electrospinning processing, pelletized ps (ps japan) dissolved in a solution containing thf (sigma-aldrich) and dmf (sigma-aldrich). hsa full rapid flisa operations included goat anti-human albumin (bethyl laboratories, a80-129a), human reference serum (bethyl laboratories, rs10-110) and fitc-conjugated goat anti-human albumin (bethyl laboratories, a80-129f), noted as 'anti-hsa', 'hsa' and 'fitc anti-hsa' in this study, respectively. for antibody testing both the hsa reagents mentioned and the following mers reagents: his-mers-np antigen protein (yokohama city university) and anti-mers-np (mab #20, yokohama city university) were utilized and noted in this study as the 'mers' and 'fitc anti-mers' reagents, respectively. anti-mers-np was labeled by the fluorescein labeling kit -nh 2 (dojindo inc.). phosphate buffered saline (pbs, ph 7.4, gibco) with tween 20 (pbs-t, ph 7.4, 0.1 w/v% tween 20) and skim milk (yukijirushi inc.) were utilized for different purposes like diluents, washes and blocking agents. additionally, fitcconjugated bovine serum albumin (bsa-fitc, sigma-aldrich, a9771-1g) and bsa (sigma aldrich, a3608-50g) were utilized in combination with esps fiber mats for fluorescence microscopy. pre-pelletized ps at 10 vol%/vol% was dissolved into a solution of 1:1 thf/dmf. the solutions were left to stir mildly at room temperature for 24 h to allow for the ps to dissolve entirely. the ps solutions were then loaded through a syringe to the electrospinning device. the environmental and processing conditions were set as noted in supplementary material table s1 . after electrospinning, a 'wet-press' technique reported by wu et al. was applied to the microfibers [39] . this technique layered esps fiber mats by stacking 8 layers of fibers atop one another, soaking them in ethanol (etoh) then sandwiching the stack between glass sheets and pressed by a 2 kg weight for 24 h before use. an o 2 plasma cleaner (pdc-001, expanded plasma cleaner, harrick plasma) paired with a gas flowmeter (pdf-fmg, plasmaflo gas flow mixer, harrick plasma) were utilized to treat the esps fiber mats with o 2 plasma. a steel mask with 9 holes (spot size = 2.5 mm) was custommade to create patterns of o 2 plasma treatment on the esps fiber mats. esps 10 wt% fiber mats that were wet-pressed at 8-layers were utilized for spot treatment experimentation. 8-layered 10 wt% esps was used rather than 4-layer samples since the 4-layer samples showed a spreading of added reagents into non-hydrophilic spot regions over time. 8-layer samples showed minimal spread or leak of reagents beyond the scope of the o 2 plasma treated hydrophilic spots. 8-layer esps fiber mats after spot treatment were tested for both protein adsorption and blocking preference by fluorescence microscopy. for protein adsorption esps fiber mats were submerged in a fitc-bsa solution (10 wt% fitc-bsa to bsa in 20 ml pbs) for 1 h. samples were then dip-washed in pbs-t 20 times. blocking effectiveness was tested by comparing bsa, blockingone and skim milk as blocking agents, where three separate esps fiber mats were soaked in the respective blocking solutions: bsa (2 w/v), blockingone (20 v/v), and skim milk (2 w/v) for 1 h before being dip washed in pbs-t 20 times. then the samples were soaked in the same fitc-bsa solution as the previous experiment for 1 h and dip-washed again in pbs-t 20 times. all fluorescence images were measured under an inverted microscope (ckx53, olympus) paired with a fluorescence light source and filter (u-hglgps, olympus). images were taken by the cellsens (olympus) image processing software and measured by imagej (national institutes of health) under the roi manager tool. a 37-mm monitor (advantec) was adapted as the cassette device by replacing the internal membranes with the 8-layered 10 wt% esps samples together with an appropriately cut and shaped filter paper (40 mm, no.5b, kiriyama rohto) layered inside the 37-mm monitor housing environment. the setup had the lower layer as the filter paper with the upper layer as the o 2 plasm-treated esps fiber mat. through the inlet, bulk reagents (pbs wash, antigen, and secondary antibody) were inserted and flushed out the opposite end outlet through syringe pressure. in all immunoassays, the 8-layered 10 wt% esps membranes, which were treated with o 2 plasma treatment for 3 min at 100w, were utilized. rapid immunoassay testing using the cassette platform were performed in following steps: (1) antigen immobilization, (2) blocking, and (3) antibody capture. first, hsa and mers concentrations were optimized independently on the treated esps membranes. hsa concentration in pbs was varied in the range from 0 to 1000μg/ml, using 6 μl per spot. blocking by 2 ml of skim milk at 2% w/v was then applied through the syringe directly into the cassette, where the solution was held for 1 min then pulled through the outlet. finally, 1 ml of fitcconjugated anti-hsa antibody solution at 10 μg/ml was held in the cassette for 1 min and also flushed through. similarly, mers was tested first by immobilizing his-mers-np at concentrations ranging from 0 to 500 μg/ml. after the skim milk blocking procedure, 1 ml of fitcconjugated anti-mers np #20 solution at 11 μg/ml was held in the cassette for 1 min and flushed through. for both hsa and mers testing, washing was conducted with pbs-t with 1 ml volumes flushed through three times through the cassette both after the blocking step and fitcconjugated antibody step. for the multiplex immunoassay, it was conducted identically to the above immunoassay protocol, but with different antigens patterned separately on a single esps membrane: 3 spots were treated with mers (200 μg/ml; 6 μl), 3 spots treated with hsa (200 μg/ml; 6 μl) and 3 spots with no antigen immobilized. again, the blocking was performed with 2 ml of skim milk at 2% w/v was held for 1 min in the cassette and then flushed through. finally, three different test solutions of antibodies were prepared and tested through the multiplex esps membrane: 1 ml of fitc-conjugated anti-hsa solution at 1:100 dilution, 1 ml of fitcconjugated anti-mers np #20 solution at 1:50 dilution, and a 1 ml mixture of fitc-conjugated anti-hsa (1:100 dilution) and fitc conjugated-anti-mers np #20 (1:50 dilution), where the concentrations for each antibody were identical in all cases. fig. 1 and supplementary material table s2 illustrate the overall procedure as well as the final concentrations used for the test protocols. it is critical that as much antigens are immobilized to the surface to enhance the signal of the immunoassay system for the antiviral antibodies. therefore, the amount of protein immobilization was optimized through the microfiber surface hydrophobicity. the 8-layered 10 wt% esps fiber mat samples were treated by o 2 plasma spot treatment for different time lengths, then tested for protein adsorption to the surface. to determine the optimum condition for the largest antibody immobilization to the surface, bsa was used as the model protein. first, the contact angles were measured to confirm the effect of o 2 plasma treatments (fig. 2) . overall, it was confirmed that the hydrophilic areas treated by o 2 plasma showed a decreasing trend in contact angle with increasing treatment time. on the other hand, the contact angles of hydrophobic zones were not significantly altered among the samples. next, the actual amount of bsa adsorption on plasma-treated samples were analyzed to estimate the antigen immobilization capacity. the results for varied o 2 plasma treatment conditions are shown in fig. 3 , where the samples were treated for 1, 3 and 5 min at 100w. it can be seen that the most amount of bsa was adsorbed to the 3-mintreated samples. the lack of sufficient o 2 plasma time at 1 min likely did not allow the oxygen to fully penetrate the fiber thus not yielding adequately-hydrophilic fiber surfaces for the bsa solution to penetrate the fiber and come in contact with the fiber surfaces. on the other hand, the excessive 5-min-treated samples over treated the samples to become super hydrophilic, preventing any hydrophobic bonding between the surface and protein samples. thus, for subsequent experiments, 3 min was chosen as the optimal time for o 2 plasma treatment for protein immobilization to the fiber surfaces. in addition, blocking agents were optimized for the esps samples and thus a similar protein adsorption test was conducted with three types of blocking agents: bsa, blockingone and skim milk (fig. 4) . although all of them displayed efficient blocking of fluorescentlylabeled bsa adsorption, it was demonstrated that skim milk performed the best. therefore, in subsequent experiments, skim milk was used as the blocking agent. to provide a proof of concept study for clinical application as to whether the fabricated esps platform together with the design rapid immunoassay protocol within a device-based environment could be applied to a rapid mers immunoassay, antibodies against hsa (control) and mers-cov (target) were tested using this platform. as mentioned in the introduction, by attaining a rapid detection of desired analytes, in this case antibodies to examine the immune system, a progressive step forward can be made towards preventative diagnostics. first, the optimization of the protocol for antigen immobilization to the esps platform was performed. here, both hsa and mers antigens were tested separately. optimization of hsa adsorption indicated a rapidly increasing trend up until about 200 μg/ml to where increasing amounts of hsa immobilized to the surface has a plateaued effect of detection intensity (fig. 5) . meanwhile, the mers adsorption test demonstrated that detection was limited at concentrations less than 100 μg/ml, thus concentrations above this value were used for subsequent studies. lastly, multiplex rapid immunoassay using 200 μg/ml of hsa and mers antigens was performed (fig. 6) . three separate esps platforms, each with 9 o 2 -plasma treated spots, were all processed under the same conditions with the only difference being the antibody solution having: (1) only fitc-conjugated anti-hsa antibodies, (2) only fitc-conjugated anti-mers antibodies, or (3) a mix of both fitc conjugate anti-hsa and fitc-conjugated anti-mers antibodies. it was successfully observed that, upon the esps platforms tested with fitc-conjugated anti-mers antibody and anti-hsa antibody solutions, each of mers and hsa antibodies could be selectively detected. additionally, when a mixed solution of fitc-conjugated anti-mers and anti-hsa antibodies was added, both antibodies could be simultaneously detected with the multiplex setup. it was also noted that the signals for both antibodies slightly decreased in the mixture test, in comparison to the individual tests, possibly due to the crowding. this multiplex microfiber-based immunoassay platform hinges on several key factors including the patterned o 2 plasma treatment and efficient blocking that warrant some discussions. first, the rationale behind how proteins such as the immobilized antibodies/antigens preferentially adsorb to just the hydrophilic spots is of great importance. generally, the protein adsorption on flat surface is affected by the hydrophobicity and surface potential [40] . in the case of epsp fiber mats, the nature of hydrophobicity arises from cassie-baxter regime consisting of two kinds of hydrophobic materials, which are the untreated ps and air [41] . the o 2 plasma treatment modifies the surface properties of ps rendering it hydrophilic. for flat ps plates, the surface wettability of water changed from 77.7°± 3.0°-25.2°± 3.9°after treatment with o 2 plasma for 5 min. interestingly, in stark contrast to the flat plates, the o 2 -plasma-treated ps microfibers resulted in greater levels of hydrophilicity (4.5°± 4.1°) after the same plasma treatment, as illustrated in fig. 2 . this hydrophilic nature of the esps microfiber mat after o 2 plasma treatment can be explained by the wenzel regime, describing the reduced apparent water contact angles for rough surfaces [41] . indeed, the patterned o 2 plasma treatment on the esps microfiber mats resulted in separate regions with these two different regimes, where only the hydrophilic surfaces promoted solution flow through the plasma-treated spots. in other words, o 2 plasma treatment of ps was suitable for preventing air pockets within microfibers, and thus the protein solution could contact the fiber surface through wetting. on the contrary, the unexposed, hydrophobic surfaces of esps did not allow wetting, so the protein solution could not contact the fiber surfaces in these regions. thus, although the total amount of protein adsorption is regulated by the true contact angle of materials, the apparent contact angle arising of the porous structure of esps microfiber is also a crucial parameter in dictating protein adsorption. next, a brief discussion of skim milk as the blocking agent is of importance. often times, immunoassays are conducted with blocking agents commonly those of proteins which can adhere well to the surface without preventing any other adsorption of proteins to the surface. among the common choices, skim milk, bsa or bsa-based products, such as blockingone exist. as noted in fig. 5 , skim milk had the best results for blocking in the case of the o 2 -plasma-treated samples. the mix of variety of proteins that exist within skim milk could beneficially be penetrating the fiber and thus blocking the surface most effectively [42] [43] [44] . it has been found that casein, within milk, can be an effective blocking agent due to its small protein size. together with other proteins within skim milk a closely packed surface of proteins can effectively prevent non-specific adsorption to the surface. in contrast, bsa has relatively large molecular weight components which can make random close packing of blocking to the surface difficult in comparison to that of skim milk. another interesting point is that skim milk is considered amphipathic having both hydrophilic and hydrophobic parts. as o 2 treatment is introduced to the surface, the hydrophilic nature may have allowed milk to be effectively bound to the surface. lastly, the comparison of the multiplex microfiber platform used in this study to other conventional assays and devices is important. a comparable work to this study was conducted by sato et al. [45] , where a combinatorial assay was conducted utilizing a microfluidic chip together with integrated polystyrene beads to measure iga antibody concentration. similar to how microfibers were utilized in this study to increase the surface to volume ratios, microbeads were packed into a microfluidic device with a filling factor of 60% (bead diameter of 46 μm). although they were able to achieve a high level of sensitivity below 10 μg/ml, their straightforward step-by-step immunoassay procedure of antigen immobilization and then capture of colloid gold labeled-iga antibodies took roughly 1 h, compared to several minutes in this study. thus, sensitivity and speed is often a fine balance for these immunoassays, and priorities should be chosen based on the applications of these assays. the multiplex microfiber platform in this study is intended for antibody detection for poc monitoring of antibody levels. it has recently been reported that the antibody titers of patients infected by mers-cov range from 1: 80 to 1: 800, depending on the time post infection, with the neutralizing antibody titer being more than 1: 800 [46] [47] [48] . considering that elisa is often used for determining antibody titers and its lod is around 1 μg/ml [36] , it is suggested that the required antibody concentration for neutralizing mers-cov would be above 800 μg/ml. thus, the lod of 200 μg/ml for the anti-mers-cov antibody by the multiplex microfiber platform, although not yet as low as other established platforms, would be sufficient to detect the necessary antibody production in mers-infected patients. the multiplex esps fiber system with a housing suited for poc was introduced as a means for rapid mers antibody detection. the capability of a rapid flisa was exhibited by utilizing 8-layered esps fiber mats treated with o 2 plasma. the patterned o 2 plasma treatment method with developed mask showed optimal protein adsorptive results at 3 min at 100w. this in turn created a hydrophilic surface capable of antibody immobilization and, further, rapid flisa testing. in terms of the detection surface, effective detection could be achieved upon this platform within 1 min of operation time. furthermore, the actual setup and preparation of the device could be completed in 5 min, which includes capture molecule immobilization, blocking and washing steps. the results demonstrated the capability of the device for multiplex analysis by detecting antibodies for both hsa and mers-cov concurrently. this microfiber-based multiplex immunoassay platform serves as a stepping stone for the next-generation poc device that would enable preventative diagnostics by accurately and rapidly determining the existence of adequate antibodies and aid general healthcare through appropriate vaccination schemes. rapid automated high sensitivity enzyme immunoassay of c-reactive protein highly sensitive immunoassay of lung cancer marker carcinoembryonic antigen using surface-enhanced raman scattering of hallow gold nanospheres significance of antibody orientation unraveled: well-oriented antibodies recorded high binding affinity bead-based microfluidic immunoassays: the next generation highly sensitive and rapid biosensing on a three-dimensional polymer platform rapid and sensitive detection of protein biomarker using a portable fluorescence biosensor based on quantum dots and a lateral flow test strip rapid lateral-flow immunoassay for the quantum dot-based detection of puerarin microfluidic immunoassays micro total analysis systems. 2. analytical standard operations and applications micro total analysis systems. 1. introduction, theory, and technology diagnostics for the developing world: microfluidic paper-based analytical devices paper-based inkjet-printed microfluidic analytical devices inkjet-printed microfluidic multianalyte chemical sensing paper a perspective on paper-based microfluidics: current status and future trends determination of carcinoembryonic antigen in human sera by integrated bead-bed immunoassay in a microchip for cancer diagnosis a homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology a fully automated immunoassay from whole blood on a disc multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants rapid integrated biosensor for multiplexed immunoassays based on actuated magnetic nanoparticles comparison of multiplex immunoassay platforms development of a bead-based multiplex immunoassay for simultaneous quantitative detection of igg serum antibodies against measles, mumps, rubella, and varicellazoster virus a nanostructured microfluidic immunoassay platform for highly sensitive infectious pathogen detection detection of hepatitis b virus antigen from human blood: sers immunoassay in a microfluidic system development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen isolation of a novel coronavirus from a man with pneumonia in saudi arabia a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus infection with middle east respiratory syndrome coronavirus calling for rapid development of a safe and effective mers vaccine prevalence of antibodies against rubella virus in the netherlands 9 years after changing from selective to mass vaccination antibodies against mumps in the netherlands as assessed by indirect elisa and virus neutralization assay comparison of a neutralization enzyme-immunoassay and an enzyme-linked-immunosorbent-assay for evaluation of immune status of children vaccinated for mumps measles, rubella, mumps, and varicella seroprevalence among health care workers in turkey: is prevaccination screening cost-effective? antibody-response in lyme-disease -evaluation of diagnostic-tests immunization, isolation of immunoglobulins, estimation of antibody titer characterization of novel monoclonal antibodies against the mers-coronavirus spike protein and their application in species-independent antibody detection by competitive elisa an updated roadmap for mers-cov research and product development: focus on diagnostics fabrication and assessment of an electrospun polymeric microfiber-based platform under bulk flow conditions with rapid and efficient antigen capture a rapid, simple measurement of human albumin in whole blood using a fluorescence immunoassay (i) immunoassay on free-standing electrospun membranes understanding how charge and hydrophobicity influence globular protein adsorption to alkanethiol and material surfaces the rigorous derivation of young, cassie-baxter and wenzel equations and the analysis of the contact angle hysteresis phenomenon comparison of blocking agents for an elisa for lps non-specific adsorption of fish immunoglobulin m (igm) to blocking reagents on elisa plate wells phospholipids in milk fat: composition, biological and technological significance, and analytical strategies integration of an immunosorbent assay system: analysis of secretory human immunoglobulin a on polystyrene beads in a microchip viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov): infection, immunological response, and vaccine development we would like to thank dr. satoko matsunaga at the yokohama city university for her kind donations of mers reagents. this work was supported by the grant-in-aid for exploratory research [16k13623] from the ministry of education, culture, sports, science and technology of japan. supplementary data to this article can be found online at https:// doi.org/10.1016/j.sbsr.2019.100304. key: cord-326718-jboiufoq authors: deming, meagan e.; chen, wilbur h. title: covid-19 and lessons to be learned from prior coronavirus outbreaks date: 2020-07-17 journal: ann am thorac soc doi: 10.1513/annalsats.202002-149ps sha: doc_id: 326718 cord_uid: jboiufoq nan a novel coronavirus (cov) was quickly recognized as the cause of a cluster of severe pneumonia cases in china around december 2019. now known as coronavirus disease , the epidemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus rapidly surged to pandemic proportions, with sweeping global public health and economic consequences. in this review, we aim to discuss the emergence of this novel cov in the context of the virus characteristics and pathogenesis, transmission, clinical syndrome, and potential therapeutics or vaccines. covs are large rna viruses that are endemic among bats globally. these bat viruses are known to readily recombine and present an ever-present potential to jump host species, allowing for emergence into novel hosts (1) . four seasonal human covs (hcovs) circulate yearly as mild "common cold" viruses causing upper respiratory symptoms: oc43, hku1, nl63, and 229e. in addition, three novel covs have emerged as zoonotic human infections in the past 17 years; sars-cov, middle east respiratory syndrome cov (mers-cov), and the 2019 novel cov (sars-cov-2) (2) have each been associated with lower respiratory symptoms, progressing in a subset of individuals to acute respiratory distress syndrome (ards) and death. the full genome sequence of sars-cov-2 shares some striking similarities to sars-cov (2). sars-cov-2 is a member of the betacoronavirus 2b clade that includes the original sars-cov (sharing 79.5% sequence homology), as well as a more distant seasonal hcov, oc43 (3). sars-cov-2 also uses the same human host receptor as sars-cov for viral entry, angiotensin converting enzyme 2 ( figure 1 ) (3) . although many questions about the increased pathogenicity of emergent zoonotic covs remain unanswered, the receptors used for host cell entry play a pivotal role. the spike glycoprotein of the virus is responsible for receptor binding and entry, and is the main determinant of host range. both sars-cov and sars-cov-2 use angiotensin converting enzyme 2, whereas mers-cov uses dpp4 (dipeptidyl peptidase 4). interestingly nl63, an hcov that also uses angiotensin converting enzyme 2 as the host receptor, but typically causes mild upper respiratory disease, was the cause of a cluster of severe pediatric pneumonias in china in 2018, during which half of the patients were identified with viruses containing a specific substitution in the spike glycoprotein that enhanced binding to and entry via angiotensin converting enzyme 2 (4). the same substitution does not have a role in the current covid-19 outbreak, as sars-cov-2 has a structurally dissimilar spike glycoprotein and recognizes a different epitope of angiotensin converting enzyme 2 ( figure 1 ). nonetheless, the acquisition of "minor" changes in the spike glycoprotein may contribute to the increased virulence of zoonotic covs. the sars-cov-2 spike binds angiotensin converting enzyme 2 with 10-to 20-fold-higher affinity than sars-cov spike, which may affect transmission or pathogenesis (5). the severe respiratory compromise of sars and covid-19 are likely mediated by mechanisms, including a combination of direct cytopathic effects, immune-mediated pathology, and downregulation of angiotensin converting enzyme 2 within the lung (6) . severe pulmonary damage in sars was associated with increased inflammatory cytokines, recruitment of macrophages and neutrophils to the lungs, and higher viral titers (7) . autopsy data showed histologic evidence of acute lung injury with denuding of the ciliated epithelia, diffuse alveolar damage, and hyaline membrane formation indicative of ards (7) . a pathology report from a single patient with covid-19 shows similar histology (8) . angiotensin converting enzyme 2 is normally expressed on type ii pneumocytes and the apical surface of ciliated airway epithelial cells, serving as an entryway for direct cytopathology (9) . functionally, angiotensin converting enzyme 2 acts as a negative regulator of angiotensin ii in the reninangiotensin system, potentially providing a protective role in ards by promoting antiinflammatory and antifibrotic effects (9) . in animal models, downregulation of angiotensin converting enzyme 2 increased lung pathology (pulmonary edema and acute lung failure), which was restored by supplemental recombinant angiotensin converting enzyme 2 (9). sars-cov infection prompted shedding of the angiotensin converting enzyme 2 ectodomain, removing the catalytic function of angiotensin converting enzyme 2 and possibly potentiating the development of ards (6) . this shedding can be induced by the sars-cov spike glycoprotein alone, and is more rapid than the shedding elicited by the spike glycoprotein of nl63 (seasonal hcov) (6) . it can be hypothesized that the spike glycoprotein of sars-cov-2, with its perspective structural similarity and higher affinity binding to angiotensin converting enzyme 2, provokes a similar mechanism of lung pathology leading to ards with severe covid-19. the overall case fatality rates for sars and mers was 10% and 35%, respectively (10) . although crude case fatality is hovering around 4% for covid-19, this estimation is exaggerated by limitations in testing and underestimated by the lag in deaths, with the adjusted case fatality rate estimated to be between 0.25 and 3% (11, 12) . the three emergent cov infections share the trend of high mortality rates among older adults. the mortality rate was .50% for individuals over 65 years with sars, and a mortality rate of 86.2% was published for individuals over 80 years of age with mers (10). an analysis of 72,314 covid-19 cases by the china centers for disease control showed a strong association between older age and mortality (13) . although individuals under 50 years of age showed a case fatality rate less than 0.5%, mortality increased with each subsequent decade, to 1.3% in those aged 50-59 years up to 14.8% in individuals aged 80-89 years (13) . furthermore, severe outcomes have been observed for both covid-19 and mers in individuals with comorbidities, such as chronic kidney disease or diabetes (13) . in contrast, cases in children appear to be rare and more mild, with asymptomatic cases and no deaths reported for children under 10 years of age (13) (14) (15) . unique among the severe cov outbreaks, sars-cov-2 appears to be efficiently transmitted person to person, including from individuals with minimal symptoms. viral transmissibility is not as simple as the basic reproduction number, or r 0 , but it provides a clue to understand transmission potential. the early r 0 for sars-cov-2 was estimated at 2.2, indicating that, on average, one individual would transmit the virus to 2.2 additional people (16) . the r 0 for sars-cov (2003) was estimated as 3, but severe symptoms typically preceded transmission, thus facilitating epidemiological measures to control the pandemic (17) . in comparison, mers infections have continued in saudi arabia over the past 8 years, without efficient human-to-human transmission (an r 0 below 1), but with ongoing spillover events from camels sustaining the outbreak. epidemiological and social dynamics can further alter the transmission dynamics of an emergent virus. the incubation period of sars-cov-2 is estimated to be approximately 5 days (range, 1.3-11.3 d), and respiratory shedding in mild cases may be as long as 14 days, leading to the current 14-day quarantine recommendation (16) . the transmission of sars-cov-2 has been slowed by either broad-reaching limitation of personal movement and gatherings, as in china, or by aggressive contact tracing and isolation of suspect cases, as in south korea. both strategies result in a lower r 0 and significant decline in covid-19 cases. importantly, early recognition of suspect cases is essential to limit transmission, particularly in hospital environments. hospital employees comprised 29% of the individuals included in one of the early clinical case series, and 3.8% of those identified by records review, emphasizing the importance of early recognition and appropriate personal protective equipment (ppe) to protect healthcare workers (13, 18) . the clinical syndrome of covid-19 can range between asymptomatic or mild illness (e.g., fever with or without cough) to severe respiratory distress, multiorgan failure, and death. currently, 80% of cases are mild, 15% develop lower respiratory tract disease (i.e., worsening pneumonia), and 3-5% require intensive care. for those who progress to severe disease, the clinical course has an insidious onset, with minimal symptomatology progressing to worsening respiratory distress around week 2 of illness (19) . two case series have been published from hospitals in wuhan detailing the clinical course of 99 patients at the jinyintan hospital from january 1 through january 20, and 138 cases at the zhongnan hospital from january 1 through january 28, 2020 (18, 19) . the vast majority of hospitalized patients presented with fever (83-99%) and a cough (59-82%), with 30% in each series having dyspnea on admission. in addition, a subset presented with only diarrhea and nausea as initial symptoms, potentially delaying recognition of infection (18) . in these series, 17-20% of admitted patients had ards, 11-13% required noninvasive ventilation, 4-12% required mechanical ventilation, and 3% were placed on extracorporeal membrane oxygenation (18, 19) . radiologic findings, as described in the above case series and another series of 51 patients with covid-19, demonstrated that the vast majority (>90%) of these hospitalized patients had abnormalities on chest x-ray or computed tomography, usually bilateral (18) (19) (20) . computed tomography findings showed ground-glass opacities, with or without septal thickening, or consolidation, located predominantly in the peripheral or posterior lungs (20) . later in the disease course (after 4 days as inpatient), imaging is more likely to show consolidation (20) . samples from bronchoalveolar lavage fluid appear to have higher viral loads than oropharyngeal washes (3). with higher viral loads detected in deeper lung samples, intubation and bronchoscopy are suspected to be high-risk procedures for providers of patients with covid-19, and therefore should be minimized as able and performed in an airborne isolation room under airborne precautions when necessary. prevention of hospital-acquired infections will require aggressive screening, early recognition and diagnosis, and strict adherence to precautions, particularly for potentially aerosolizing procedures, such as intubation. the demands of airborne isolation precautions for any large number of patients can easily overwhelm medical systems with finite numbers of trained personnel, airborne isolation rooms, ppe, and dedicated equipment. there are no approved drugs or vaccines for hcovs. multiple vaccine candidates using different platforms are in preclinical development, and two have advanced to phase 1 clinical trials. although this speed is unprecedented, progression through the necessary steps of development, safety testing, efficacy analyses, and manufacturing may take over a year until publicly available (21) . in the interim, rapid evaluation of potential therapeutics may provide an earlier intervention to mitigate disease. antivirals targeting the rna-dependent rna polymerase (such as remdesivir) showed in vitro activity, as did the immune modulator, chloroquine (22) . the protease inhibitors, perspective lopinavir and ritonavir, have been used, but they lack a clear antiviral mechanism for cov proteases, and were ineffective in a controlled clinical trial (22) (23) (24) . clinical trials for remdesivir and hydroxychloroquine have begun, and additional therapeutics are in development (22) . host-targeted therapeutics are also under consideration, including inhibitors of host proteases required for viral entry, or anti-il-6 therapeutics that are hypothesized to blunt the cytokine storm in severe cases (25) . based on evidence from sars and mers, current recommendations are to avoid the use of corticosteroids for patients with covid-19 (26) . corticosteroid use for patients with sars-cov was associated with higher plasma rna levels at weeks 2-3 into illness (reflecting likely prolonged viremia) and increased 30-day mortality (adjusted odds ratio, 26; 95% confidence interval, 4.4-154.8) (25) . convalescent sera, including the neutralizing antibodies isolated from recovered cases, is a promising, but not yet scalable, option (27) . sars-cov-2 is the most recent emergent cov, and having already demonstrated a greater facility for transmission than sars-cov or mers-cov, it threatens to be a devastating pandemic. current recommendations to reduce transmission include: social distancing; hand hygiene; cough etiquette; and aggressive recognition and isolation and quarantine of cases and contacts. for the health care environment, early and judicious ppe use to prevent respiratory droplet and short-distance aerosol transmission, and appropriate environmental control of rooms housing patients, are critical. although the majority of infections have been mild, hospitalized patients have high rates of complications, including the need for aggressive supportive care, including mechanical ventilation, continuous renal replacement therapy, and extracorporeal membrane oxygenation. these complications place a heavy burden on hospital systems that may be ill prepared for large numbers of patients who will require airborne isolation and prolonged durations of stay. there are no approved therapeutics, although there are some promising antivirals under study. although the first severe cov epidemic was halted by nonpharmacologic interventions alone, the covid-19 outbreak has become a pandemic due to the efficient transmissibility of the virus. however, several countries have demonstrated that aggressive nonpharmacologic intervention and control measures can slow the spread, blunting the impact on the healthcare systems and allowing the time needed for the testing of potential therapeutics and vaccines. beyond this pandemic, we must continue working toward sustained preparedness against future emergent infectious diseases. n a sars-like cluster of circulating bat coronaviruses shows potential for human emergence coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin discovery of a subgenotype of human coronavirus nl63 associated with severe lower respiratory tract infection in china cryo-em structure of the 2019-ncov spike in the prefusion conformation differential downregulation of ace2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl63 overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov pathological findings of covid-19 associated with acute respiratory distress syndrome renin-angiotensin-system, a potential pharmacological candidate, in acute respiratory distress syndrome during mechanical ventilation the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia coronavirus disease 2019 (covid-19) situation report 69 case-fatality risk estimates for covid-19 calculated by using a lag time for fatality novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia transmission dynamics and control of severe acute respiratory syndrome clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study emerging 2019 novel coronavirus (2019-ncov) pneumonia developing covid-19 vaccines at pandemic speed therapeutic options for the 2019 novel coronavirus (2019-ncov) comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a trial of lopinavirritonavir in adults hospitalized with severe covid-19 coronaviruses: drug discovery and therapeutic options covid-19 treatment guidelines panel. coronavirus disease 2019 (covid-19) treatment guidelines treatment of 5 critically ill patients with covid-19 with convalescent plasma author disclosures are available with the text of this article at www.atsjournals.org. key: cord-307811-6e3j0pn7 authors: hao, wei; ma, bo; li, ziheng; wang, xiaoyu; gao, xiaopan; li, yaohao; qin, bo; shang, shiying; cui, sheng; tan, zhongping title: binding of the sars-cov-2 spike protein to glycans date: 2020-07-02 journal: biorxiv doi: 10.1101/2020.05.17.100537 sha: doc_id: 307811 cord_uid: 6e3j0pn7 the pandemic of sars-cov-2 has caused a high number of deaths in the world. to combat it, it is necessary to develop a better understanding of how the virus infects host cells. infection normally starts with the attachment of the virus to cell-surface glycans like heparan sulfate (hs) and sialic acid-containing oligosaccharides. in this study, we examined and compared the binding of the subunits and spike (s) proteins of sars-cov-2 and sars-cov, mers-cov to these glycans. our results revealed that the s proteins and subunits can bind to hs in a sulfation-dependent manner, the length of hs is not a critical factor for the binding, and no binding with sialic acid residues was detected. overall, this work suggests that hs binding may be a general mechanism for the attachment of these coronaviruses to host cells, and supports the potential importance of hs in infection and in the development of antiviral agents against these viruses. the 2019 novel coronavirus (cov) is the seventh human coronavirus. 1 it is a deadly virus that is affecting the whole world in an unprecedented way. the global impact of the coronavirus disease 2019 (covid19) pandemic is far beyond that of two other major coronavirus outbreaks in the past 20 years, the severe acute respiratory disease (sars) in 2003 2,3 and the middle east respiratory disease (mers) in 2012. 4 given that all three highly pathogenic covs were originated from bats and a large number of closely related covs are present in bats, future outbreak of this type of zoonotic virus remains possible. in order to avoid facing a similar pandemic in the future, it is necessary to develop a better understanding of these covs, especially with regard to effective ways that can help control the current covid-19 pandemic and prevent the second wave of outbreaks. studies showed that the genome of the sars-cov-2 has about 80% nucleotide identity with that of sars-cov. 5 the major differences are found in the regions encoding the structural proteins (envelope e, membrane m, nucleocapsid n, and spike s) and accessory proteins (orf3a/3b, 6, 7a/7b, 8 and 10), not the nonstructural proteins (nsp1 to nsp16). based on this genetic similarity, the 2019 novel cov was named by the international committee on taxonomy of viruses (ictv) as the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). 6 sars-cov-2 is more genetically distant from mers-cov and shares only about 57% genome homology with mers-cov. 5, 7 these similarities and differences are reflected in the viral binding receptors to cell surface receptors. sars-cov-2 and sars-cov use the same receptor angiotensin-converting enzyme 2 (ace2) for entry into target cells, [8] [9] [10] whereas mers-cov uses dipeptidyl peptidase 4 (dpp4, also known as cd26) as the primary receptor. 11 however, there are many important findings that cannot be explained by their genetic relations. for example, it was found that sars-cov-2 has a relatively lower mortality rate (2%) than sars-cov (15%) and mers-cov (35%), but is more transmissible among humans. [12] [13] [14] these findings suggest that more investigations other than genetic analysis need to be carried out to improve the understanding of sars-cov-2 infection. in order to efficiently infect host cells, sars-cov-2 must bind with cell surface molecules in the lungs and other organs to mediate viral attachment and entry into host cells. previous studies of many other viruses suggested that sars-cov-2 s protein may use other molecules on host cell surface as attachment factors to facilitate binding to the high-affinity receptor ace2. 15, 16 examples of such molecules include glycosaminoglycans (gags) and sialic acid-containing oligosaccharides. [17] [18] [19] [20] gags are primarily localized at the outer surface of cells. such a location makes them particularly suitable for acting as attachment factors to recruit viruses to cell surfaces. 21, 22 hs is one of the most prevalent types of gags in mammals. it is a linear and sulfated polysaccharide that is abundantly expressed on the surface of almost all cell types and in the extracellular matrix. 23, 24 the hs chains are mostly covalently linked as side chains to core proteins to form hs proteoglycans (hspgs) (fig. 1 ). a possible mechanism for sars-cov-2 entry and infection. at the early stage of the infection process, sars-cov-2 may first interact with the hspgs on the surface of susceptible cells using the s protein protruding from the virus particle. this initial attachment may promote the subsequent binding of the virus to the high-affinity entry receptor ace2. the serine protease tmprss2 on host cell surface and other host cell proteases may assist in viral entry by cleaving the s protein at the s1/s2 and/or at the s2' sites. hs is synthesized in the golgi apparatus by many different enzymes. during and after its assembly, hs undergoes extensive series of modifications including sulfation, acetylation and epimerization, which leads to glycan structures with high heterogeneity in length, sulfation, and glucuronate/iduronate ratio. 25 considerable variation in the sulfation pattern and degree of hs was noted in different species, organs, tissues, and even at different ages and disease stages. 23, 24, 26 the sequence and sulfation pattern of hs has been shown to be able to regulate the binding of many viruses to host cells during infection. 27, 28 a similar trend was also observed for sialylation patterns of cell surface oligosaccharides. 17, 29, 30 these findings implicate that a possible relationship may exist between the distribution of different types of hs/sialylated glycans and the viral tropism. [31] [32] [33] a better understanding of their relationship can potentially contribute to the design of new antiviral strategies. although currently the data on the viral tropism of sars-cov-2 are limited, results from recent studies suggested that its tropism may not be correlated with the ace2 expression. 34, 35 some other factors, such as proteases and glycans, may be the determinant of cellular susceptibility to the infection with this virus. 36 a recent study suggested that hs may bind to the receptor binding domain (rbd, the c-terminal region of the s1 subunit, fig. 2 ) of the sars-cov-2 spike protein and change its conformation. 37 the intriguing possibility that variation in hs and sialic acid characteristics could impact the tropism of viruses prompts us to investigate the binding of sars-cov-2 toward a series of hs and sialic acid containing oligosaccharides. 38 in this study, we systematically examined and compared the binding of the sars-cov-2 s protein subunits, full-length molecule and its trimer to different hs using microarray experiments (fig. 2) . our results suggested that all the tested protein molecules were capable of binding hs oligomers and had similar binding preferences, with higher affinity toward hs forms with higher degrees of sulfation. the binding was also shown to be positively related to the level of 6-o-sulfation. the hs chain length seemed to be not critical. a long heparin molecule (a highly sulfated hs) and a synthetic heparin pentasaccharide (fondaparinux sodium) were demonstrated to have similar binding affinity to these protein molecules. moreover, our study suggested that the sars-cov-2 s protein might not be able to bind sialic acid residues, or the binding might be too weak to be detected by the microarray technology, and the s proteins of sars-cov and mers-cov have similar hs and sialic acid binding properties as those of the sars-cov-2 s protein. overall, our study laid a foundation for future studies to explore whether the binding specificity to hs can serve as an important contributor to the viral tropism of sars-cov-2 and to explore the possibility of exploiting hs for therapeutic strategies. binding of the subunits and s proteins of sars-cov-2, sars-cov, and mers-cov to a hs microarray. an early study has suggested that the rbd of sars-cov-2 s protein might bind heparin. 9 in order to determine if there is any preference of the sars-cov-2 s protein for particular hs structures, we first investigated the binding of the rbd (here termed as sars-cov-2-rbd) to a hs microarray containing 24 synthetic heparan sulfate oligosaccharides. these oligosaccharides have systematic differences in their length, monosaccharide composition, and sulfation pattern (fig. 3) . the microarray experiment was performed using a previously established standard protocol. 29, 39, 40 briefly, the proteins were labeled with cy3 fluorescent dye and incubated with the microarray at different concentrations. after washing away the unbound hs molecules, a highly sensitive fluorescence method was used to detect the binding of the sars-cov-2-rbd to hs. under the experimental conditions, the binding can be detected at concentrations higher than 0.5 g/ml. increasing the concentration of the protein was not found to produce noticeable changes in binding (supporting information, fig. s1 ). quantification of fluorescence revealed that the sars-cov-2-rbd is able to bind to almost half of the molecules on the microarray, and not surprisingly, the binding is strongly affected by the sulfation level, which is a trend that has been previously noted for many hs-binding proteins. [41] [42] [43] as shown in figure 4a , the hs oligosaccharides with higher sulfation degree, hs020-hs024 (the number of sulfate groups per monosaccharide unit >1.00), exhibit higher fluorescence intensity. the highest fluorescence intensity is observed for hs023 (1.35 sulfate groups per monosaccharide), which is followed by those of hs021 and hs024 (1.25 sulfate groups per monosaccharide) and those of hs020 and hs022 (1.15 sulfate groups per monosaccharide). binding of sars-cov-2-rbd to hs seems to not be affected by the monosaccharide composition (compare hs020 with hs022, and hs021 with hs024). the oligosaccharides with relatively lower sulfation levels (hs001-hs019, the number of sulfate groups per monosaccharide unit <1.00) have lower or almost no fluorescence signals. an analysis of the effect of the variation in sulfation revealed that the position of sulfation is another factor that strongly influence the binding. as shown in the figure 4 , removal of the 6-o-sulfate group from the glucosamine units significantly reduced the binding (compare hs017 with hs020, hs018 with hs021, and hs019 with hs022), suggesting that the 6-o-sulfate in hs plays a crucial role in determining its interaction with sars-cov-2-rbd. the importance of the 6-o-sulfate for binding is further supported by comparing the binding of hs012, hs013, hs014, hs015, and hs016, which shows that the one-by-one addition of sulfate to the 6-oposition of the glucosamine residues gradually increased the binding of hs with sars-cov-2-rbd. the microarray study also indicates that the binding is not positively related to the length of the hs chains. short hs oligosaccharides could have comparable or even better binding properties (compare hs001-hs006, hs007-hs0012, and hs020-hs024). the binding results of the sars-cov-2-s1 follow a similar trend as those of the sars-cov-2-rbd, with only minor differences for a few hs molecules like hs001, hs007, hs016, hs021 and hs024 (fig. 4b) . this is consistent with the assumption that rbd is the major determinant for viral s protein binding to hs. examination of the sequence of the sars-cov-2 s protein revealed the presence of a potential cleavage site for furin proteases (rrars) at the s1/s2 boundary. 9 this is similar to the s protein of mers-cov, which also contain a furin consensus sequence rxxr (fig. 2) . furin cleavage can occur in the secretory pathway of infected cells and breaks the covalent linkage between the s1 and s2 subunits. 44 the s2 subunit functions to fuse the virus to the host cell. it may interact with hs on the cell surface to facilitate the fusion process. therefore, to determine the role of hs in sars-cov-2 infection, it is also important to investigate the binding of the s2 subunit to hs. very interestingly, our results indicate that the sars-cov-2-s2 can also bind to hs, with the binding preferences similar to those of the sars-cov-2-rbd and the sars-cov-2-s1 (fig. 4c) . this suggests that hs may also play an important role during viral membrane fusion after the s1 subunit is removed from the s protein. we also further investigated the binding full-length s protein and its trimer to hs using the same microarray. our data showed that, although the binding to hs020-hs024 still remains the highest, the full-length s protein has increased binding to hs with slightly less sulfation, particularly the molecules in groups hs007-hs012 and hs013-hs016 (fig. 4d, 4e) . additionally, we found that the binding preferences of the rbds, s1 subunits and full-length s proteins of sars-cov and mers-cov are similar to those of the subunits and s protein of sars-cov-2, although small differences are observed (fig. 4f-k) . because the mers-cov s protein also contains a furin cleavage site, we studied the binding of its s2 subunit to the hs microarray. once again, the results are similar to those of the s2 subunit of sars-cov-2. overall, these data support the involvement of hs in the binding with the subunits and s proteins of sars-cov-2, sars-cov and mers-cov and support the importance of hs for the infection of these coronaviruses. to determine the relative binding strength of the subunits and s proteins of sars-cov-2, sars-cov, and mers-cov, we measured and compared the dissociation constants (kd) of the protein molecules studied in the microarray experiment using a real-time spr-based binding assay. a commercially available porcine heparin from sigma-aldrich was used as the first binding partner for the measurement. it is a mixture of highly sulfated hs, with most chains in the molecular weight range of 17 to 19 kilodalton. this long heparin molecule is more similar to the hs molecules on the host cell surface than those on the microarray. in the spr assay, protein molecules were covalently linked to the surface of the cm5 (carboxymethylated dextran) sensor chips by amine coupling. the heparin in various concentrations was then flowed over the immobilized proteins. the changes in refractive index by molecular interactions at the sensor surface were monitored and the dissociation constants were obtained by fitting the results using the software available in the spr instrument (fig. 5) . the comparison of the dissociation constants revealed that all the tested protein molecules can bind to the heparin, but their binding affinities are relatively low (kds are at the micromolar level). this agrees well with previous observations that hs is a weak binder to viral s proteins. 45 the results also showed that rbd has the lowest binding affinity among the tested protein molecules of sars-cov-2 (fig, 5a) . the s protein trimer and s1 subunit have relatively lower binding affinity in comparison with the fulllength s protein and s2 subunit, respectively (fig, 5b-e) . very similar trends were also observed for differences in the binding affinities of the tested protein molecules of sars-cov and mers-cov (fig, 5f-l) . in addition to the long heparin molecule, we also measured the binding of the protein molecules to fondaparinux (arixtra®), which is an ultralow-molecular-weightheparin (ulmwh) containing five monosaccharide units (fig. 6 ). it has a well-defined chemical structure and is currently the only ulmwh that has been clinically approved as an anticoagulant. fondaparinux is very similar to hs023 in size, monosaccharide composition, and sulfation position and degree. the results showed that the kd values of fondaparinux binding to the tested protein molecules are in the same range as those for the long heparin. this agrees well with the observation in the microarray study, suggesting that the length of the hs chains is not a determining factor for binding. despite the similarity, there is a subtle but noticeable difference, which is that the binding affinities of the s1 subunit and rbd to fondaparinux are quite similar. can interact with sialic acid-containing glycans present on the cell surface. 17, 20 such an interaction is normally mediated by the n-terminal domain of the s1 subunit. 46 in order to find out if sars-cov-2 can bind to sialic acid residues, we carried out microarray analyses of its s protein and subunits. the first microarray used contains 100 different n-glycans that may be found on the surface of cells. 49 of them are terminated with α2,3-and α2,6-linked sialic acid, also known as n-acetylneuraminic acid (neu5ac), 8 with α2,3-and α2,6-linked n-glycolylneuraminic acid (neu5gc), and the rest with other glycan residues (supporting information, table s1 ). the experiment was performed in the similar way as described for the hs microarray study. the microarray results showed that both the sars-cov-2-rbd and sars-cov-2-s1 gave no binding signal, suggesting that they may not be able to interact with sialylated n-glycans or the binding signal is too low to be detected. in order to confirm this finding, we also investigated the binding of the full-length s proteins of sars-cov-2, sars-cov and mers-cov to more sialylated glycans, including sialylated n-and o-linked glycans and glycolipid glycans (supporting information, tables s2-4), but again no specific binding was detected. for a virus like sars-cov-2 to establish infection, it must first attach itself to the surface of target cells in different organs and tissues. the s protein plays an essential role in this attachment process. recently, the structure of sars-cov-2 s protein in the prefusion conformation was determined by the cryo-em technique. 10 it shows that the overall structure of the sars-cov-2 s protein is very similar to that of the closely related sars-cov s protein, which is organized as a homotrimer. each monomer can be divided into an n-terminal receptor-binding s1 subunit and a c-terminal fusionmediating s2 subunit. the s1 subunits are located at the apex of the spike, making them more accessible for binding to the proteinaceous receptor, ace2. although similar, there are some notable differences between the sars-cov-2 and sars-cov s proteins. 8, 10 for example, the key amino acid residues involved in the binding of the sars-cov-2 s protein to ace2 are largely different from those of sars-cov. 9, 47, 48 these differences may be related to the observed higher binding affinity of the sars-cov-2 s protein to ace2. another important difference between the sars-cov-2 and sars-cov s proteins is that the former protein contains a multibasic protease recognition motif (rrars) at the junction of s1 and s2. 9 a multibasic cleavage site (rsvrs) was also identified in the mers-cov s protein (fig. 2) . 49 the sars-cov s protein only has a monobasic amino acid (sslrs) at the same site. the multibasic site can be processed by furin or related proprotein convertases, which are widely expressed in different tissues, before the virus is released from the host cell. by contrast, the monobasic site can be cleaved by tmprss2 or other cell-surface proteases (whose expression is confined to certain tissues) only after the virus is released from the host cell. it was reported that the cleavage at the junction of s1 and s2 activates the s protein for virus-cell fusion. thus, the presence of the multibasic cleavage site may partially account for the enhanced infectivity and tropism of sars-cov-2 relative to sars-cov for human cells. 10, 50 however, what is vague is how the virus attaches to the host cells after losing the s1 subunit, which is responsible for the binding to the proteinaceous receptor. in addition to binding protein-based receptors, many viruses can interact with cell surface glycans, including gags and sialic acid-containing oligosaccharides. depending on the virus, the glycan molecules can act as attachment factors, coreceptors or primary receptors. 51 viruses typically bind gags through non-specific charge-based interactions. as one of the most abundant gags, hs appears to be the preferred binding partner for many viruses. 33, [52] [53] [54] [55] sialic acids are normally terminal monosaccharide residues linked to glycans decorating cell surface glycoproteins, glycolipids, or other glycoconjugates. 20, 56 in general, the interactions of viruses with hs or sialic acids are responsible for the first contact with host cells. such contact may serve to concentrate viruses on the surface of target cells, facilitate their binding to more specific high-affinity protein receptors and/or promote their entry into host cells. 57, 58 it has been demonstrated that virus binding and infection can be reduced by enzymatic removal hs or sialic acid from cell surface, or by treating virus with soluble hs or multivalent sialic acid conjugates. 30, [59] [60] [61] therefore, in order to better understand and treat covid-19, it is necessary to carry out research to investigate the possible interactions between sars-cov-2 and hs and sialic acid-containing glycans in the forms of separate subunits and full-length proteins, and to assess if such interactions could represent a target for therapeutic intervention. similar to studies that have been successfully conducted for many other viruses, we used the microarray and spr technology to study the binding of the rbds, s1/s2 subunits and full-length s proteins of sars-cov-2, sars-cov and mers-cov, and a trimer of the sars-cov-2 s protein to hs and sialic acid. 59 the microarray results showed that all the tested protein molecules can bind to about half of the oligosaccharides on the hs microarray. in contrast, only background levels of fluorescence were detected on various sialylated glycan microarrays. this observation suggests all the tested protein molecules are able to bind to hs 45,62 and may not bind to and/or have very low binding affinity to sialylated glycans. this well agrees with previous studies showing that sars-cov can bind to hs 62 and the binding of the mers-cov s protein to sialic acid-containing glycans can only be detected after the s1 subunit was attached to a nanoparticle to enhance its avidity via multivalent interactions. 17 our results also suggested that the binding of the tested proteins to hs is related to hs sulfation position and degree. it seems that more 6-o-sulfate groups and higher sulfation degree normally lead to better binding. because hspgs exhibit different sulfation patterns in different tissues, such a binding specificity may contribute to the tropism of sars-cov-2 for human cells. 63 the length of hs appears not to be a critical factor for the binding. short hs chains could have comparable binding specificity and signals. the comparison of the spr kd values obtained for the long heparin molecule and fondaparinux further support this finding. it implies that it may be possible to reduce the attachment of sars-cov-2 to the surface of host cells by low-molecularweight-heparin (lmwh). this is in agreement with a recent study showing that lmwh treatment may be associated with better prognosis in some severe covid-19 patients. 64 while these initial findings are encouraging, further research is required to determine if the binding to hs could affect the tropism and pathogenesis of sars-cov-2 and to determine if hs could be used for the inhibition of the infection of this virus. our spr data of the tested protein molecules also showed that the s2 subunits could have similar or better binding affinity for hs as compared to those of the s1 subunits. this finding suggests that the cleaved s proteins of sars-cov-2 and mers-cov may depend on hs for interaction with the host cells during viral membrane fusion. in parallel with our study, the linhardt and boons research teams also conducted studies to investigate the binding of s proteins to hs. 45, 65 the absolute values of the dissociation constants determined in our experiments are largely different from those presented by these two teams. this can be seen from the binding of the full-length s protein of sars-cov-2, which was studied by all three teams (supporting information, table s5 ). our kd is one order of magnitude higher than that reported by the boons team, which in turn is three orders of magnitude higher than that reported by the linhardt team. the differences in results may be due to the method of analysis and/or the experimental materials used in the studies. in our study, the tested proteins were immobilized on the surface of cm5 sensor chips, while in their experiments, biotinylated heparin molecules were immobilized on the chips. at the same time, because the s glycoproteins and the heparin molecules were obtained from different sources, their composition may be different from each other. in conclusion, through our study, we provided experimental evidence for whether or not the s protein of sars-cov-2 can bind to two types of cell-surface glycans, hs and sialic acid-containing glycans, which are commonly utilized by human viruses for attachment to target cells. our data revealed that the sars-cov-2 s protein can weakly bind to hs in a sulfation-dependent manner. no binding with sialic acid residues was detected using the microarray assay. the results suggest that hs may act as an attachment factor to concentrate the virus at the cell surface and affect its tropism. through comparison, we found that the s proteins of sars-cov and mers-cov have similar binding properties to hs as that of the sars-cov-2 s protein, indicating that hs binding may be a conserved feature for these three types of coronaviruses. our data also revealed that the s2 subunits could bind equally well as the s1 subunits to hs. this binding may be an important element for viral attachment to the host cell surface after the removal of the n-terminal receptor-binding domains by protease cleavage. overall, our findings support the potential importance of hs in sars-cov-2 infection and in the development of antiviral agents. expression and purification of sars-cov-2-rbd. dna containing the coding sequence for an n-terminal hemo signal peptide, the receptor binding domain (rbd, residues 319-541) of sars-cov-2 s protein and a c-terminal polyhistidine tag was amplified and inserted into a pfasebac1 vector for expression in high-5 insect cells using the bac-to-bac expression system (invitrogen). the resulting recombinant protein, termed sars-cov-2-rbd-his, was secreted into cell culture medium, and subsequently purified on a nickel-nitrilotriacetic acid (ni-nta) affinity column, followed by a superdex 200 gel filtration column (ge healthcare). the final buffer for the protein contains 10 mm hepes (ph=7.0) and 100 mm nacl. the purified sars-cov-2-rbd-his was concentrated to 3.5 mg/ml and flash frozen in liquid n2 and stored at -80 degrees celsius. binding of recombinant proteins to glycan microarrays. the tested protein molecules were first labeled with cy3 fluorescent dye (10 mg/ml in dmso). after dialysis, they were incubated at different concentrations with microarrays for 1 hour in the dark at room temperature. after incubation, the microarray slides were gently washed using washing buffer (20 mm tris-cl containing 0.1% tween 20, ph 7.4) to remove unbound proteins. finally, the slides were scanned with a microarray scanner luxscan-10k/aat an excitation wavelength of 532 nm and evaluated by the microarray image analyzer software. the spr measurements were performed using biacore t200 and s200 (ge healthcare). first, the carboxymethyl dextran matrix on cm5 sensor chip (ge healthcare) was activated by injection of a 1:1 mixture of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (edc) and n-hydroxysuccinimide (nhs). recombinant proteins in 10 mm acetate buffer (ph 4.5, ge healthcare) was then injected over the chip surface at a flow rate of 30 μl/min to couple the amino groups of the recombinant proteins to the carboxymethyl dextran matrix. after the coupling reaction, the remaining activated ester groups were deactivated by ethanolamine. the binding study was carried out at 25°c in pbs-p buffer (ge healthcare). the heparin molecule or fondaparinux at different concentrations were flowed over the immobilized recombinant proteins at a flow rate of 30 μl/min with a contact time of 60 s and a dissociation time of 100 s. the surface was regenerated by injection of 10 mm glycine-hcl (ph 2.5) at a flow rate of 30 μl/min for 30 s. data was collected and analyzed by bia evaluation software (ge healthcare). all authors have given approval to the final version of the manuscript. a novel coronavirus from patients with pneumonia in china identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 the genetic sequence, origin, and diagnosis of sars-cov-2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in the prefusion conformation dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc differences and similarities between severe acute respiratory syndrome (sars)-coronavirus (cov) and sars-cov-2. would a rose by another name smell as sweet? sars case fatality ratio, incubation period middle east respiratory syndrome coronavirus (mers-cov) ebola virus entry requires the cholesterol transporter niemann-pick c1 cell surface α2,3-linked sialic acid facilitates zika virus internalization identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein interaction between respiratory syncytial virus and glycosaminoglycans, including heparan sulfate interaction of zika virus envelope protein with glycosaminoglycans structural basis for human coronavirus attachment to sialic acid receptors heparan sulfate proteoglycans are required for cellular binding of the hepatitis e virus orf2 capsid protein and for viral infection herpes simplex virus types 1 and 2 differ in their interaction with heparan sulfate distribution of heparin and other sulfated glycosaminoglycans in vertebrates tissue specific distribution of sulfated mucopolysaccharides in mammals bio-specific sequences and domains in heparan sulphate and the regulation of cell growth and adhesion heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates? braz n-and 6-o-sulfated heparan sulfates mediate internalization of coxsackievirus b3 variant pd into cho-k1 cells the role of heparan sulfate proteoglycans as an attachment factor for rabies virus entry and infection human parainfluenza viruses hpiv1 and hpiv3 bind oligosaccharides with alpha2-3-linked sialic acids that are distinct from those bound by h5 avian influenza virus hemagglutinin inhibition of viral adhesion and infection by sialic-acidconjugated dendritic polymers herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry heparan sulfate proteoglycans initiate dengue virus infection of hepatocytes heparan sulfate is an important mediator of ebola virus infection in polarized epithelial cells the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status sars-cov-2 and sars-cov differ in their cell tropism and drug sensitivity profiles sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the 2019 coronavirus (sars-cov-2) surface protein (spike) s1 receptor binding domain undergoes conformational change upon heparin binding binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity lectin microarray profiling and relative quantification of glycome associated with proteins of neonatal wt and rd1 mice retinae the sweet spot: defining virus-sialic acid interactions n-terminal syndecan-2 domain selectively enhances 6-o heparan sulfate chains sulfation and promotes vegfa(165)-dependent neovascularization heparan sulfate and heparin interactions with proteins heparan sulfate microarray reveals that heparan sulfate-protein binding exhibits different ligand requirements the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade sars-cov-2 spike protein binds heparan sulfate in a length-and sequence-dependent manner receptor recognition mechanisms of coronaviruses: a decade of structural studies structure of sars coronavirus spike receptor-binding domain complexed with receptor isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor functional analysis of potential cleavage sites in the mers-coronavirus spike protein synthetic analogues of the snail toxin 6-bromo-2-mercaptotryptamine dimer (brmt) reveal that lipid bilayer perturbation does not underlie its modulation of voltage-gated potassium channels glycan-protein interactions in viral pathogenesis human metapneumovirus (hmpv) binding and infection are mediated by interactions between the hmpv fusion protein and heparan sulfate adaptation of tick-borne encephalitis virus to bhk-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo role of heparan sulfate in entry and exit of ross river virus glycoprotein-pseudotyped retroviral vectors role of heparan sulfate in the zika virus entry, replication, and cell death glycosaminoglycans and infection glycosaminoglycans in infectious disease sulfated glycosaminoglycans as viral decoy receptors for human adenovirus type 37 respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus sialic acid is a cellular receptor for coxsackievirus a24 variant, an emerging virus with pandemic potential inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans organ-specific sulfation patterns of heparan sulfate generated by extracellular sulfatases sulf1 and sulf2 in mice anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy glycosaminoglycan binding motif at s1/s2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (sars-cov-2) host cell entry we would like to thank the national natural science the authors declare no competing interests. key: cord-325574-4zf9qtlh authors: farag, elmoubasher; sikkema, reina s.; vinks, tinka; islam, md mazharul; nour, mohamed; al-romaihi, hamad; al thani, mohammed; atta, muzzamil; alhajri, farhoud h.; al-marri, salih; alhajri, mohd; reusken, chantal; koopmans, marion title: drivers of mers-cov emergence in qatar date: 2018-12-31 journal: viruses doi: 10.3390/v11010022 sha: doc_id: 325574 cord_uid: 4zf9qtlh mers-cov (middle east respiratory syndrome corona virus) antibodies were detected in camels since 1983, but the first human case was only detected in 2012. this study sought to identify and quantify possible drivers for the mers-cov emergence and spillover to humans. a list of potential human, animal and environmental drivers for disease emergence were identified from literature. trends in possible drivers were analyzed from national and international databases, and through structured interviews with experts in qatar. the discovery and exploitation of oil and gas led to a 5-fold increase in qatar gdp coupled with a 7-fold population growth in the past 30 years. the lifestyle gradually transformed from bedouin life to urban sedentary life, along with a sharp increase in obesity and other comorbidities. owing to substantial governmental support, camel husbandry and competitions flourished, exacerbating the already rapidly occurring desertification that forced banning of free grazing in 2005. consequently, camels were housed in compact barns alongside their workers. the transition in husbandry leading to high density camel farming along with increased exposure to humans, combined with the increase of camel movement for the racing and breeding industry, have led to a convergence of factors driving spillover of mers-cov from camels to humans. emerging infectious diseases are a cause for increasing global concern, because of their impact on global health and economics [1] . the ebola outbreak in west africa during 2014-2015 showed that pathogens which previously caused small and easy to control outbreaks had the potential to infect thousands of people under the right circumstances [2] . this is also a concern for the middle east respiratory syndrome coronavirus (mers-cov), which until now has been the cause of sporadic cases and hospital outbreaks [3] . to date, there have been 2220 confirmed laboratory cases worldwide, with 790 deaths [4] . all mers index cases are linked to the arabian peninsula. dromedary camels have been identified as a reservoir of mers-cov with occasional zoonotic transmission to humans [5, 6] . human-to-human transmission is also common, with around 30% of the mers cases reported to who being health care associated [7, 8] . however, the source of infection of many index cases remains unclear [9, 10] . studies have shown that mers-cov, or related viruses have been circulating among camels at least since 1983 [11] . since that period, massive changes have occurred in people's lives and in animal husbandry across the arabian peninsula. understanding these changes may help to reconstruct the events that led to the emergence of mers-cov as a human disease. past research identified several drivers of emerging zoonoses, such as urbanisation, population growth and demography, and environmental and agricultural changes [12] [13] [14] . the drivers which could have potentially influenced the mers-cov emergence in humans have only sporadically been investigated [15, 16] . by reviewing changes involving humans and camels over the past 30 years in qatar, this study sought to identify the key drivers of the emergence and spread of mers-cov. potential drivers for disease emergence were identified from literature and from discussions with national and international experts in mers-cov. the final list had the following categories: economic development; human demography and behavior; international travel, commerce, sports and leisure; political environment; agriculture and food industry change, including camel demography, husbandry and movement; changes in climate and land use. data from 1980 onwards were collected from national and international databases. if multiple data sources were available, data from both sources were collected. all data were entered in an excel datasheet and reviewed and discussed with the project team (supplementary 1). qualitative information and remaining data gaps were addressed by interviews with a group of 15 experts and stakeholders from qatar. criteria to select experts included 5 years or more experience in a camel-related business (farming, trading and racing) or professional services related to camels and being familiar with cultural aspects of the qatari community. using a structured interview guide (supplementary 2) and a moderator, a series of 4 interviews were conducted in arabic, each lasting approximately for 3 hours. the main themes that were covered during the interviews included: (changes in) people's living conditions; customs and purposes of camel ownership; cultural habits related to camels; educational level and personal behaviors of camel owners and workers; camel movement; demographic distribution of camels in qatar; camel farming practices: feeding, grazing, and slaughter. a detailed transcript was shared with the experts for authentication. a literature search was done to complement findings from the quantitative and qualitative study, using pubmed, google scholar and the local sources of information including the ministry of public health (moph), ministry of municipality and environment (mme), ministry of development and planning statistics (mdps), and qatar statistical authority (qsa). the funder had no role in study design, data analysis, data interpretation, or writing of the review. historically, qatari inhabitants were mostly bedouins along with a few settled people [17, 18] . the bedouins owned limited numbers of camels, sheep, and goats [19] . camels were used as a source of food (milk and meat) and means for transportation. in 1939, oil and natural gas resources were discovered. however, large-scale exploitation started in the 1950s [20] . from the 1950s onwards, qatar's economy has been steadily growing. however, the year 2000 marked a significant turning point as qatar's gdp almost increased by more than 5-fold during the period 2000-2006 ( figure 1a ) [20, 21] . qatar is currently considered to be one of the wealthiest countries in the world [20] . the thriving economy was paralleled by major demographic and life style changes. in the late 1950s, around 16,000 people lived in qatar [22] . in response to demands for a larger workforce after the exploitation of oil and gas began, foreign laborers started to migrate to qatar from countries in the region, like palestine, oman, iran, and the kingdom of saudi arabia (ksa). later, immigrants from pakistan, india, nepal, sri lanka, bangladesh, the philippines, and indonesia joined the older migrant populations, increasing the number of inhabitants to 369,079 by 1986 and recently to 2,617,634 ( figure 2a ) [23] . in 2016, non-qatari males made up 78% of the residents of working age (15-64) and non-qatari made up more than 90% of the total number of qatar inhabitants older than 15 years of age ( figure 2b ,c) [24] . most recent estimations of the origins of the non-qatari population are that 25% is indian, 11% bangladeshi, 14% nepali, 10% filipinos, 9% egyptian, 5% pakistani, and 2% iranian [25] . the total number of males in qatar increased from 67.2% of the total population in 1986 to 75.5% in 2016 ( figure 2b ). in 2004, almost 50% of residents were between 15 and 39 years old, and this has risen to more than 60% in 2015 ( figure 2a ). detailed accounts on age distribution were not available before 2004 [26] . most people in qatar live in urban areas. the percentage of residents living in cities increased from 85.3% in 1960, to 90.4% in 1986, and 99.3% in 2016 [20] . doha, the capital and the biggest city of qatar, hosts the greatest number of people. however, there has also been a large increase in number of people living in the al-rayan area, where most of the camel farms are located. the number of tourists visiting qatar also increased, especially since 2000. most tourists came from other gcc countries, but the number of visitors from europe and america were also increasing ( figure 2d ) [20, 27] . according to experts, the economic development and population increase coincided with major changes in life style. the bedouin nomadic lifestyle gradually decreased as most of the qatari tribes shifted to an urban, settled lifestyle; cars and planes rapidly replaced camels as transportation means. this transformation to a more sedentary lifestyle is reflected in the profile of comorbidities. more than 70% of adults are overweight and almost half of them obese [28] . male obesity increased from 17% in 1986 to 34% in 2014, which is extremely high compared to the current 11% prevalence in men worldwide [29] . in 1998, 7% of residents above 15 years were hypertensive, rising to 14% in 2006, and 33% in 2012 [30, 31] . prevalence of high blood sugar among adults in 2015 was 14%, compared to a worldwide prevalence of 9% [28] . the qatar stepwise report reported in 2012 that 15% of adults were daily smokers. yet, qatar has a low death rate: 1.49/1000, compared to the worldwide death rate of 7.72/1000, and its healthcare system has developed rapidly over the past twenty years [28, 31, 32] . the increase in the number of dromedary camels reflects the increasing popularity of camels as sports animals ( figure 1b ). with the changing life style and increasing wealth, the purchase and breeding of (expensive) racing camels came within reach of an increasingly large segment of the qatari national population. according to experts, although camel racing has traditionally been part of the bedouin culture, the organized racing business went through major changes over the past decades. this was partly due to financial and regulatory support from the qatari government. this support increased the social and economic value of camels in qatar, further stimulating their popularity. the al-shehaniya camel-racing track, one of the biggest tracks in the gulf, was opened in 1990 [33] . the camel farms that are located near the al-shehaniya camel racing area are mostly used for racing camels. there are about 1500 racing camel holdings at the al-shehaniya camel racing area. some of the camels in qatar are used to compete in camel beauty contests that are organized around the arabian peninsula. according to the fao, in 1960 there were about 6,000 camels in qatar. this rose to over 43,000 in 1992, 50,000 in 2000, and more than 90,000 in 2016 ( figure 1b ). more than 83% of the animals are currently kept for racing [34] . across the gulf region, qatar has the highest camel density, with 6.77 units/km 2 , compared to 4.74 units/km 2 in united arab emirates (uae) and 0.11 units/km 2 in the ksa [35] . in 2005, the total number of camel farms was 1300 and by 2014 it had increased to 9594 [34] . as a result of the loss of traditional methods of rangeland management, the vegetation coverage decreased from 10% to only 1% of total land cover. overgrazing of the green areas due to the increased population of camels and other livestock accelerated the desertification of qatar [36] . therefore, the government decided to assign natural protected areas in 2004 [37, 38] , and started to sanction the free grazing of livestock since 2005 [39] . by 2011, open grazing was completely banned [40] . according to the experts' opinions, this led to changes in farming practices, as herds were then moved outside of qatar to areas where free grazing remained possible. moreover, in qatar, camels are now raised in closed systems and within 1 of 9 designated farming areas (camel complexes) in the residential districts. camel workers also live on the premises of the camel complexes. typically, a camel complex has a reception room (majlis) for social activities of the camel owners. the al-rayyan municipality, where the al-shehaniya camel racing area is also located, currently holds about 83% of the total camel population and 61% of camel holdingss (figure 3 ) [34, 35] . according to the experts, this newly adopted closed farming system led to the increase of disease incidence, especially of parasitic diseases. however, we did not find any disease statistics to substantiate these findings. the increasing focus on camel race competitions caused big changes in camel farming practices. previously, the calves were weaned when the next calf was born. currently, weaning occurs at around 7 months of age. after being weaned, young camels are directly taken for acclimatization (during the period mid-july through mid-august) from the general livestock farms (located across the region) to the racing farms, mostly located within the al-shehaniya area. this involves drastic changes in feeding systems, intense training for races, and mock races alongside camels from other farms and older training camels. the off-season for camel racing is during summer (mid-april to august) ( figure 4 ). during this time, most of the owners travel abroad, the frequency of visits to the farms substantially decreases, and workers are permitted to take annual vacations. from september onward, training intensifies, in preparation of the racing season, which lasts from mid-september through mid-april. during that time, 14,000 registered camels from different origins, ages, gender, nationalities, and breeds compete together at the al-shehaniya camel-racing track. during the racing season, up to 24 rounds take place, approximately five days per week. an unprecedented, increasingly intensified mobility of camels inside and outside qatar has been seen over the recent decades. the domestic and cross border mobility does not only involve camels, but also people who look after the camels to provide care along the journey. import and export of camels have especially increased since the year 2000 ( figure 1c ). the imported camels mainly come from the uae and ksa ( figure 1d) . the dynamics and travel patterns of qatari camels are complex (figure 4 ). camels are transported to and from different locations, for a variety of purposes, and with a noticeable seasonal pattern. mobility gets more intensive during the racing and trading season (september to april). experts believe that the ban of open grazing in qatar played a key role in the intensity and frequency of camel movements. they mention that there has been a remarkable increase after 2011 in numbers of camel workers and owners who cross the borders to and from ksa along with their animals, although this recently stopped with the ksa-qatar political situation. the ban of open grazing stimulated camel owners to establish farms in ksa and uae where open grazing is still permitted. therefore, camels are moved through gulf countries, particularly during the winter season. camel races and beauty contests that are routinely organized in nearly all gulf countries are another factor that boost the national and international movement of camels. compared to other types of camels, racing camels dominate in terms of numbers and frequency of mobility both across borders and domestically, particularly between september and april. as per the records of the camel racing committee, in the 2016 racing competitions, 14,000 camels from qatar and camels from the other gcc countries contested [35] . however, owing to the lack of standardized identification system, it was difficult to determine the exact figures and the extent of these movements. camels are also being mobilized for reproduction purposes (figure 4 ). mating season (also known as camels' honeymoon) starts in the middle of august and continues through february of the next year, with the high season in the september-october period. female camels are usually taken from their own location to other farms where selected males are kept particularly for reproduction purposes. about 14,000 female camels are annually being moved for mating. they spend around 1 week at a breeding farm with male camels before they are taken back to their original farms. programmed mating is exclusively being practiced for race and show camels. the mating season is another seasonal activity that entails intensive movements of camels, camel owners, workers, car drivers and veterinarians. the doha wholesale market constitutes the primary hub for camel trading. in parallel with the increased number of camel races, al-shehaniya city also grew as a market and has become a hub for trade of racing and beauty show camels in qatar. the wholesale market in doha hosts camels and other types of livestock from countries all over the gulf region. the camels typically stay at the market until they are sold. camel workers live at the market premises. camels that are being sold (calves in particular) serve a variety of purposes. they are sold to be slaughtered at the doha wholesale market abattoir, for breeding purposes, to be trained as racing camel, or to be prepared for camel show competitions. in recent years, the doha wholesale market has been surrounded by rapidly growing residential areas. animals in the market are now in close proximity to the residents. as of 2005, slaughter practices were banned inside residential premises, and can only be performed in official slaughterhouses and exclusively by licensed persons. camel meat and milk are no longer part of the daily diet of most qatar inhabitants. nonetheless, camel meat is a fundamental ingredient of qatari social events and family celebrations. production of camel meat and milk has remained stable in the past 30 years. camel milk is generally kept for personal use, particularly for the perceived therapeutic merits of raw camel milk, as well as camel urine. experts state that there is an unshakable belief that the regular consumption of camel milk helps to prevent and control diabetes. it is also widely believed in the qatari community that camel urine and milk can heal skin lesions and other diseases. camel urine is also regularly used to whiten the skin and face and lighten the hair. the majority of camel owners offer camel milk and urine for free, as a practice of generosity. the role of camels in the transmission of mers-cov is well documented [5] . despite the fact that mers antibodies have already been detected in camels since 1983 [11] and human contact with animals is not new, human mers cases were only detected in 2012 [41] . based on institutional and literature data and in-depth interviews with key professionals, this study sought to examine the changes involving the human, animal, and environmental drivers that may have contributed to the spread and virus spillover to humans. our reconstruction of events over the past decades, based on available literature, statistics, and expert opinions, lead to the conclusion that the discovery of oil and natural gas resources has been the starting point of a chain of events that ultimately led to conditions favoring the emergence of mers-cov ( figure 5 ). this discovery led to massive economic growth. owning a camel represents the wealth and status of its owner in arabic culture. governmental sponsorship of camel ownership and camel racing further stimulated the camel industry, especially the camel-racing sector. this in turn lead to an accelerating increase of the camel population, a change in camel farming, and a concomitant increase in the number of camel workers [34] . the human population of qatar has increased by 7-fold over the last decades [23] . this is unlike other high-income countries, that have a yearly overall population growth of only 0.6% [20] . population growth and high population density have been shown previously to be important risk factors for disease emergence [42] . moreover, consistent with the disease profile of wealthy countries where sedentary lifestyle prevails, the prevalence of chronic diseases increased in qatar in accordance with the increasing gpd, ultimately rendering the qatar population not only vulnerable to virus transmission, but also to its deadly complications [43] . the intimate nature and number of interactions between camels and humans has also increased significantly in the past 30 years, increasing the risk of any zoonotic spillover. at camel complexes, workers intimately reside, sleep, and eat with their camels. camel owners, on the other hand, pay regular visits to their barns and stay there for considerable hours every day (even longer during weekends, holidays, and winter season) in the majlis built at the corner of their barns. owners, who are often of advanced age with multiple comorbidities, enjoy drinking fresh camel milk and entertaining guests. those who suffer certain diseases tend to visit the camel barns to use camel urine or drink fresh camel milk for its perceived curative properties. among the variety of changes that involved camel husbandry in qatar, the shift from open grazing to close housing systems seems to be most significant. opportunities for camel-to-camel and camel-to-human spread have greatly increased since then. it is possible that housing camels in barns, with poor biosecurity and hygienic standards, turned these barns into 'melting pots' for the virus that ultimately acquired the ability to cross the human-animal barrier. the increase of cross-border movement of camels increased chances and frequency of (international) virus spread. camels are transported freely across borders for a variety of purposes through multiple routes and means of transportation. when camels and the humans that accompany them, arrive at the site of a race or beauty event in qatar, they are housed with the local camels. owners are welcomed in the majlis at the camel complexes. the mixing of camel and human of different origins further increase chances of virus transmission. viruses 2018, 10, x; doi: for peer review www.mdpi.com/journal/viruses species variation in dpp4 [45] . as such, the increasing human-animal interface that is described in this paper may have facilitated the adaptation of the spike protein to human ddp4. however, much remains unknown, also in view of the findings that mers-cov from east africa were not phenotypically different from the viruses from the middle east, while human mers patients have not been reported from the african continent [46] . finally, the changes in animal husbandry practices, earlier weaning, frequent grouping and transportation of animals, and the introduction of an entirely new feeding system, may induce stress in the camels. these changes and movements often involve young weaned animals, at the same time as maternal antibodies are waning, which are linked to the shedding of the virus [42, 47] . most of the limitations of this study were related to the availability of data. firstly, statistics on animals, import and export, animal workers, and land use were only found since 2000 onwards, limiting the chance although much effort was made to study mers-cov viral sequences and mers-cov transmission between dromedary camels and humans, it is still unknown which genetic mechanisms have caused the viral spillover of dromedary camels to humans. however, the most important determinant of host specificity seems to be the spike s1 protein, that recognizes and binds to host-cell receptor dpp4 [44] . recently it has been shown that the mers-cov spike can rapidly adapt to species variation in dpp4 [45] . as such, the increasing human-animal interface that is described in this paper may have facilitated the adaptation of the spike protein to human ddp4. however, much remains unknown, also in view of the findings that mers-cov from east africa were not phenotypically different from the viruses from the middle east, while human mers patients have not been reported from the african continent [46] . finally, the changes in animal husbandry practices, earlier weaning, frequent grouping and transportation of animals, and the introduction of an entirely new feeding system, may induce stress in the camels. these changes and movements often involve young weaned animals, at the same time as maternal antibodies are waning, which are linked to the shedding of the virus [42, 47] . most of the limitations of this study were related to the availability of data. firstly, statistics on animals, import and export, animal workers, and land use were only found since 2000 onwards, limiting the chance to study the trends and changes prior to that year. secondly, even the available national data on the animals, humans, and environment were found to be sometimes inconsistent, limiting the possibility global trends in emerging infectious diseases the ebola outbreak, 2013-2016: old lessions for new epidemics middle east respiratory syndrome middle east respiratory syndrome coronavirus cross host transmission in the emergency of mers coronavirus evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus transmission coronavirus transmission in extended family mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia the clinical and virological features of the first imported case causing mers-cov outbreak in south korea urbanization and disease emergence: dynamics at the wildlife-livestock-human interface zoonosis emergence linked to agricultural intensification and environmental change reasons for the increase in emerging and re-emerging viral infectious diseases. microbes infect dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) unraveling the drivers of mers-cov transmission divers are a pearl's best friend: pearl diving in the gulf 1840s-1930s. qatar digital library national identity formation in modern qatar: new perspective oil and politics in the gulf qatar country indicators ministry of development planning and statistics. quarterly bulletin for population and social statistics-third quarter population of qatar by nationality-2017 report ministry of development planning and statistics. population and social statistics annual tourism performance report chronic disease risk factor surveillance world health organization. global health observatory (gho) data: overweight and obesity noncommunicable diseases and their risk factors; stepwise approach to surveillance (steps) death rate, crude death the reports of the camel racing organizing committee; ministry of culture and sport department of animal resources animal population; world animal health information database (wahis interface)-version 1 ministry of development planning and statics supreme council for environment and natural reserves opportunities for the sustainable use of the camel in qatar isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond adaptive evolution of mers-cov to species variation in dpp4 replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels mers coronaviruses from camels in africa exhibit region-dependent genetic diversity key: cord-289003-vov6o1jx authors: burdet, c.; guégan, j.-f.; duval, x.; le tyrant, m.; bergeron, h.; manuguerra, j.-c.; raude, j.; leport, c.; zylberman, p. title: need for integrative thinking to fight against emerging infectious diseases. proceedings of the 5th seminar on emerging infectious diseases, march 22, 2016 – current trends and proposals date: 2018-02-28 journal: revue d'épidémiologie et de santé publique doi: 10.1016/j.respe.2017.08.001 sha: doc_id: 289003 cord_uid: vov6o1jx abstract we present here the proceedings of the 5th seminar on emerging infectious diseases, held in paris on march 22nd, 2016, with seven priority proposals that can be outlined as follows: encourage research on the prediction, screening and early detection of new risks of infection; develop research and surveillance concerning transmission of pathogens between animals and humans, with their reinforcement in particular in intertropical areas (“hot-spots”) via public support; pursue aid development and support in these areas of prevention and training for local health personnel, and foster risk awareness in the population; ensure adapted patient care in order to promote adherence to treatment and to epidemic propagation reduction measures; develop greater awareness and better education among politicians and healthcare providers, in order to ensure more adapted response to new types of crises; modify the logic of governance, drawing from all available modes of communication and incorporating new information-sharing tools; develop economic research on the fight against emerging infectious diseases, taking into account specific driving factors in order to create a balance between preventive and curative approaches. the objectives of the 5th edition of the val de grace school's seminar were to take a global, integrative approach to the emergence of new infectious disease agents, putting these in perspective relative to other types of risk, as well as studying crisis situations and the breaches brought on by the occurrence of emerging infectious diseases (eids). two key conferences respectively opened and closed this annual seminar; the first was given by dr. peter daszak (president of the ecohealth alliance and program director of usaid-ept-predict), the second by dr. patrick lagadec (former research professor at the é cole polytechnique, palaiseau). planned for an anticipated up to 200 participants, this seminar is designed for decision-makers, experts, medical doctors and scientists interested in human and animal health, social sciences, environmental sciences, prospective analysis, biosecurity and defense. this day of presentations and debates is presented under the auspices of the french social affairs and health ministries, as well as that of the environment, energy and marine affairs. peter daszak remarks today can be summed up in two primary messages, both personal and that of scientists from the ecohealth alliance. first, we must increase our research capabilities in order to understand the direct and less-direct causes of emerging infections if we hope to fight them once they become responsible for epidemics or pandemics. secondly, this presentation was illustrated with a few projects concerning the economics of eids which reveal the extraordinary costs they incur for national economies. there is at least one obvious reason to speak of this, which is that politicians and public decisionmakers are in fact quite sensitized when the economic damage engendered by the latest epidemics and pandemics are explained to them. if one takes, for example, the sars-cov pandemic, it led to a 1 to 2% decrease in gross domestic product in several southeast asian countries, for an estimated overall cost of 30 to 50 billion us dollars, relative to the total number of approximately 800 people worldwide who were affected. the appearance of new eids appears both more frequent and also vaster in terms of number of people affected these last years, many of these diseases appearing in developing or low-income countries. taking the case of the ebola virus (ebov) epidemic which broke out in west africa in 2014, it was much more widespread than any other epidemic to ever occur in central africa (400 people affected during an epidemic). it is very difficult in the united states -where peter daszak is working -to get public decision-makers interested, as is true with the population which feels very distant from such problems. ecohealth alliance has tried to draw the media and the public's attention to it, in vain! they only received one single email from the management of the boston airport, informing us that our work on the risk of ebola virus propagation via transcontinental air transport was unfounded and that the boston airport could not experience this type of threat. about a month later, when an american citizen was repatriated for treatment, the public began to panic; the media, particularly the tv channel cnn, exaggerated in broadcasting the issue, and the government took several decisions, notably regarding assistance and monitoring of international airports. these measures were decided upon no scientific basis in a moment of panic, and it seems that with every new worrisome eid, it is the same story! the measures taken are often disproportionate to the seriousness of the phenomenon. human demography has exploded in recent years, and populations today are concentrated in megalopolises. this tendency is even more marked in tropical areas in the south where there is also an important biodiversity of animal species. therefore, there exist today more opportunities for a virus or bacteria to pass from an animal to a human, then to propagate among the human population. international air transport makes possible the spread of these new infectious risks on a vast scale. what are governments doing faced with this type of threat when the demands on the part of the population are ever-increasing? vaccines are also considered by the former as a weapon of total destruction, and by the public as a widely-available miracle tool. neither is truly aware that it takes an average of 10 years to produce a vaccine. also, is it the right strategy in that it banks more on cure than on anticipation? in the u.s., president obama opted for a development aid policy through usaid favoring training and improvement of human and technical capacities to prevent future eids in the most needy countries. the american congress, however, did not follow this path, instead directing funds allocated to usaid towards research on a vaccine for and on epidemic management of the ebola virus crisis. recently, in collaboration with economists, ecohealth alliance modeled the risk of a new emergence in a situation identical to that of ebola in 2014-15 in west africa, estimating the economic damage caused, and attempted to deduce the economic cost of a health policy based on epidemic management as opposed to one favoring prevention and training. according to their estimates, a budget of 5 billion us dollars seems to suffice to prevent a new epidemic in west africa in opting for the second solution. their simulations obviously include many underlying hypotheses. all the same, this analysis indicates that if we were to quickly make available 1 billion of the 5 billion dollars for the purchase of equipment, the setting up of working laboratories on site, field hospitals, sending doctors and nurses to the area, training of our partners in affected countries -as military medical services know how to do -such a strategy would represent a real capital investment in managing epidemic propagation. unfortunately, this was not the option chosen by u.s. current governments, which we can only regret. now let us discuss the ecology of ebola virus transmission. the bat species pteropus are possibly the host reservoirs of ebola virus. as for the marburg fever virus, we are now certain that these bats are in fact the reservoir. the ebola virus is clearly present in a few giant bat species in africa. it can also circulate among primate species or indirectly through other infected mammals. we also know which species of bats are affected or not. even if the reservoir question is still under debate, we know a great deal about the rainforest circulation of this virus and the circumstances of its spreading in the human population with high-risk groups such as hunters. based on this, is it possible to prevent the virus' transmission to humans? although it poses a complex question, it is nonetheless possible to offer some avenues of reflection. william karesh, vicepresident of ecohealth alliance and instigator of the onehealth concept, carried out a research project in the democratic republic of congo, which consisted in educating villagers, and particularly hunters, regarding the danger of recovering primate corpses from the forest. this program was a real success, as the villagers changed their behavior and the eating of monkey meat. this represents a low-cost prevention approach, which in the end functions well if properly implemented. the only real solution when faced with this type of health threat is to work in conjunction with populations, and to treat questions of poverty, equality, and use and transformation of land together. these are arduous and complex tasks which must be carried out over the long-term. this type of message is extremely difficult to make heard among politicians and the public, and obviously more complex that announcing or clamoring for a vaccine. we are faced with primordial questions which are more or less difficult to answer. are we witnessing an increased frequency of eids appearance? are there more cases today? can we predict patterns, or rules, of emergence? are there geographic areas in which these emerging infections are more frequently or widely observed today? do we possess the scientific ability to predict exactly where the next infectious epidemic will start? evidently, if we were so able we might allocate the financial resources and technical and scientific support to suspected areas. at the moment, we do just the opposite, or we do nothing, and we are disconcerted at each new emergence. in order to convince governing bodies and public decision-makers, we must demonstrate to them that predictive approaches are less costly than curative ones. using approaches inherited from ecology and biogeography ecohealth alliance has adapted the same principles to understanding the ecology and spatial distribution of eid principles observed over the last decades. their work shows that of the nearly 400 new eids which have appeared, the so-called zoonotic ones originating in wild animals have been in net increase in the last 40 years; 3 out of 5 new infections which appear each year originate in wild fauna. what's more, the risk of infectious transmission is highly and doubly correlated with human density, as are these diseases of animal origin to the areas' biodiversity. they called these areas ''hot-spots'' for disease risks, mostly situated in intertropical regions. they are advocating for these specific zones to be those on which they concentrate our research efforts as well as international public aid for development. in fact, it is not only biological diversity in animals which must be taken into account, but also the evolution of natural ecosystems and their disappearance over recent years. deforestation and land use changes by populations are driving forces in the appearance in new eids. therefore, international politicians must better link economic strategies to those of habitat and biodiversity coordination if we wish to avoid new pandemics. in further use of the formalism of economic models, they have shown that a stable political strategy appears once joint simulations were conducted on economic damage due to a pandemic health crisis and preventive decision-making. even though the financial cost of preventive strategies may initially appear great, an optimal solution based on prevention shows itself over the mid-and long-term to be finally less costly than cure and control-focused strategies. we have need on an international level for the equivalent of the intergovernmental panel on climate change (ipcc) which, in the same way as does the ipcc on climate change scenarios and their impact, would concentrate on eids and their sociopolitical, economic and environmental consequences. the american development aid agency usaid did not originally give priority to eids, but the different public health crises brought on by the h5n1 bird flu virus in recent years have led usaid to modify their strategic orientation, notably with regard to the economic weight they bring to bear on regional economies. the fact that these epidemics appear first in countries situated in strongly species-rich intertropical areas which are developing or low-income, requires a reconsideration of our western policies on development aid in order to better include these notions. for ten years, usaid funded a program on new emerging infection threats, for a total of 1.3 billion u.s. dollars. at ecohealth alliance we have collaborated with this initiative through participation in the predict research project with funding of 45 to 50 million dollars. the goal of the predict program was to identify microorganisms potentially pathogenic for human populations, and which are hosted in animal reservoirs. we took particular interest in three animal groups, not only because it was impossible to work on all the animal groups present, but also because the three groups we choseprimates, rodents and bats -were recognized as being major reservoirs of agents which are pathogenic for humans. these three groups alone make up 75% of the world's mammal species. within the predict program we applied our knowledge of the high-risk emergence zones to draw samples from a large cohort of these three animal groups. over the first five years of the program we sampled 56,000 animals, trained 2500 scientists and medical and administrative personnel, and discovered more than 1000 new viruses belonging to families of viruses known not to be infectious to humans. this in itself represents a major finding! obviously, the discovery of a microorganism does not in and of itself indicates that a new eid might appear. their work in mexico on bat species demonstrated the existence of a dozen viruses which are very like the mers-cov responsible for the respiratory syndrome in the middle east. they therefore believe that the mers-cov is not hosted by dromedaries but rather by bats, as they found it in our work in mexico. some of these new coronaviruses may potentially be pathological for humans. it is clearly impossible to identify all viruses present on the planet. it would also be necessary to do the same for bacteria, parasitic fungi, and protozoa. as they are innumerable, it is preferable to make strategic choices regarding microorganism research and that on the highest-risk animal groups. based on our knowledge of currently known viruses in bangladesh bats, they extrapolated this data using species curve rarefaction, and capture-recapture techniques well known to ecologists, to estimate the total number of viruses to be expected in the totality of known mammal species in the world. they reached a value of 320,000 new viruses which remain to be discovered. were we to carry out biodiscovery research on these viruses, one could catalogue them, classify them in relation to already-known viruses, in particular those known to be pathogenic to humans, and through comparative genome studies assess their pathogenic potential. we are currently in the second phase of the us predict program, with an important accent on our manner of working. in fact we are currently concentrating more on factors involved in emergence and seek particularly to understand three such factors: habitat change and use, intensive agriculture, and the commerce of biodiversity. us development aid policy also focuses on three strategic areas: north africa, west africa, and continued research activity in saudi arabia. these geographic choices are shaped by recent events around the mers-cov and ebola viruses. especially in the case of mers-cov, dromedaries are certainly involved in the transmission cycle of the virus, but we believe that its true reservoir is the bat. thus using the many data at our disposal, we were able to show that the infectious risk of mers-cov for humans is not great in saudi arabia, but it is in other areas of contact between bat, dromedary and human populations, particularly in the horn of africa, especially in somalia. somalia is currently a politically disjointed country, where public health surveillance and care are greatly lacking or simply non-existent. for example, it is currently impossible to state how many mers-cov cases there are in somalia, or how many might exist. our scientific objectives are therefore to better understand the behaviors at the junction of wild or domestic fauna and human populations. for example, understanding human behaviors and practices at the interface of tropical forests and villages could help us to interpret how zoonotic transmission happens. through acting upon these behaviors and habits, we could lower this type of emerging risk. currently ecohealth alliance has research sites in uganda, malaysia and brazil; they are investigating the role played by habitat changes such as deforestation, human interaction with biodiversity through behavior and use. in manaus in brazil for example, the maximum risk of new infection is not in the heart of the city where the major markets are located, but in the peri-urban areas where agriculture and ranching are developing. these zones which ecologists call ''ecotones'', or transition zones, are located near strongly species-rich regions, with high concentrations of livestock which can come into contact with wild fauna, and which are furthermore inhabited by ranchers and farmers. these regions which are now located everywhere in the intertropical world for the purpose of feeding urban populations, are generally those where new infections appear, and where future pandemics will also most likely appear. at the heart of this research is the priority of understanding behaviors, habits and practices of populations with the goal of changing these factors. this is a long-term endeavor which requires developing approaches in the field for communicating with local populations, and for developing community participation in our own research, which we also consider an excellent means of education. o. borraz recalled the fact that we live in a society of risk. it is not that the dangers surrounding us are more numerous or more fearsome than before, but simply that the notion of risk plays a central role in public policies, in public and private organizational management, and in the controversies around new technologies. genetically modified organisms, mobile phones, nuclear waste, urban sanitation sludge; the activities now considered health or environmental risks are countless. this categorization puts public authorities in a position of having to ensure the safety of populations, even as the state itself sometimes represents a risk factor. it is therefore essential to understand how an activity becomes a risk, and how it is then managed by public authorities as well as by companies, associations and local conglomerates. risk from its identification to its management, from its highlighting to its instrumentalization becomes a tool linked to the emergence and expansion of a welfare state. it is used by politicians to justify on the one hand its lack of involvement in risk management, on the other the seizing of power over sectors from which they had previously disengaged. this political power seizure plays on politicians' selective dissemination within society of risk identification and the knowledge required for its management. two different processes exist by which risk represents an organizing principle for political power, a means to assist and contribute to the definition of the state's limits: ''putting at risk'' and ''regulation through risk''. ''putting at risk'' refers to all processes by which an event is described as or constitutes a real or predicted danger, and which thus is categorized as a risk [1] . there are many events, objects, and situations which have historically been categorized as ''putting at risk'' and which can be studied by sociologists (illness, divorce, food crises, unemployment, nuclear risk, chemical substances, technological risks. . .). this putting at risk can be a product of the state or of interest groups, under the influence of lobbying. this has been part of the organization of modern societies since the 20th century, and in particular following world war ii with the expansion of the welfare state which took advantage of different factors putting the population at risk in order to enlarge its sphere of activity and thus its power in the name of a protective mission. this welfare state built itself around risk management in creating and organizing agencies, action plans, drills, and monitoring mechanisms for their application through nationwide inspection bodies. the promotion of ''risk instruments'' and a ''risk-based regulation'' approach makes it possible to deal with risk-creating events. for the last two decades, in a context of decreasing means and in reaction to public-health crises which have led, according to some, to an overprotective state, a new tendency has emerged of governments using ''risk instruments'' to better allocate means and to decrease the state's hold. despite their differences, these two approaches are to the same end, which is to define -or perpetually re-define -the limits of the state. ''risk instruments'' contribute in each ''putting at risk'' situation to determining the state's involvement, the reason for its having competencies and resources, and therefore also the state's limits as opposed to that which concerns the private sector and individuals. the outcome of this defining as ''putting at risk'' and of the application of these risk instruments may be used by the state either to invest, or to disinvest; in the latter case for example entrusting risk management which formerly would have been the realm of institutions to local or private agents or to individuals. in the same way, risk instruments (or risk-based regulation or using risks to rationalize public intervention) may lead to state disinvestment or, on the contrary, be used by the state to seize back the reins in certain areas. paradoxically risk instruments serve in this instance to re-centralize or to ''remote-control'' areas to which the state had granted greater autonomy (universities or hospitals, for example). thus the way in which western societies manage risk, and the way in which these risks -their identification, how politicians and individuals perceive them, and their management -transform the links between the state (the political powers-that-be) and civil society are important to understand for those faced with risk management, especially in public-health risk management. what consequences these risk technologies have from one country to another thus greatly depends on institutions, state structure, professional organizations and their interrelationships, the balance of power and the forces involved, and the legal structurewhich may be more or less open to interpretation according to how it is drafted (more or less restrictive laws). risk management is, after all, at the core of the state and has always been central to its transformations. presenter: alfredo pena-vega (ehess, cnrs) climate change can be interpreted, according to a. pena-vega, as a complex system such as edgar morin defined it in his ''introduction to complex thought'' [2] . the stakes involved in it are multidimensional, and suppose at once the ideas of transformation, uncertainty and unexpectedness, unpredictability and crisis. . . it henceforth seems necessary to arm ourselves with instruments enabling us to understand this reality. the common denominator of such instruments is rationality. because climate change involves stakes which are climatological, economic, social and health-related, as well as questions of governance, ethics, biodiversity etc., cross-disciplinarity appears to be one form of rationality which allows us to avoid the dangers of ''fear-mongering''. a study was conducted among 12,000 high school students from 20 different countries, of which the objective was to understand how younger generations see climate change. the results are quite varied. if a minority of responders question the existence of climate change -with an argument largely based on a social construct communicated by the media -90% of responders claimed to be concerned and aware of the negative consequences of climate change. the vast majority consider it to be a threat to the survival of humanity, in particular due to the multiplication of natural disasters, dwindling biodiversity, the spread of new infectious diseases, notably those with vector-borne transmission, and the inequalities it fosters between humans. we are currently in the ''age of the unthinkable''. today's world constantly exposes us to new crisis situations which we must learn to confront. these situations are all the more difficult to manage in that they most often occur ''out of framework'', or within a framework in which it is difficult to define the outlines delimiting an increasingly ''volatile'' environment. whereas our benchmarks in terms of crisis management are structured according to typologies (natural/biological/social disaster), the boundaries of current crises are unclear, and their typologies intertwined. unexpected and unpredictable, they emerge within a context where uncertainty reigns and where the response is organized according to the logic of competitive leadership. information no longer follows a downward flow from the state to the public, but circulates in a collaborative manner via widespread connectivity, social networks, which compete with institutional media which are sometimes outdated. it is therefore difficult for decision-makers to circumscribe the areas of operation, to isolate causes, and to distinguish the components which permit specific, technical and successive interventions. good judgment thus becomes key. public health crises, and especially those concerning eids, have pointed up the difficulties in predicting their appearance and development. comparisons and connections between these and other types of crises (natural, technological, industrial. . .) and how they are interpreted can thus prove useful in public health crisis management. the unattainable goal of predicting such crises makes it necessary to prepare to meet with unexpected events during the ongoing management of the crisis; it is no longer a question of predicting the unpredictable, but preparing to deal with it. in the context of our intricate, complex society, coordination and communication are of course necessary, but it has also become imperative to have a thorough knowledge of the steering process. in ''out of framework'' situations, strategic thinking capability takes precedence over the quality of available technical expertise. this can take the form of an ''express thinktank'', a group made up of diverse members capable of and trained to work together in situations of uncertainty. whereas in a ''classic'' situation the command functions is a pyramid, in these ''out of framework'' situations, collaborative and flowing cooperation is required, without leadership's impeding the efficaciousness of the response. in the case of hurricane sandy in the u.s., in 2003, several work teams were set up in order to handle the unprecedented nature of the situation: ''real-time innovation'', ''immediate flaw detection'', ''emergency support functions''. these parallel work teams made it possible to optimize management of the disaster by limiting disastrous consequences. it seems essential to develop such networkbased crisis management in france. p. zylberman recalled to mind the definition of an eid as an unexpected infectious -or presumably infectious -phenome-non, affecting humans, animals or both. according to the definition used in the high council of public health's 2011 report [3] , this can entail an infectious clinical entity which has just appeared (''true'' emergence), one previously identified (known emergence) or a known infectious disease whose incidence has increased or whose characteristics have changed (re-emergence). the hiv epidemic in the 20th century or the sars-cov epidemic in the early 21st century are examples of true emergence. the emergence of hepatitis c corresponds to a known clinical entity whose etiological agent was identified at the end of the 1980s. the measles and west nile virus outbreaks on the american continent, in the 19th and end of the 20th centuries respectively, are examples of reemergence. nathan d. wolfe has defined five stages of transformation of an animal pathogen into a specifically human pathogen, resulting in a ''true'' emergence, with the possibility of an evolutionary interruption at each stage. stage 1 corresponds to a situation in which a known virus in animals has never yet been detected in humans in natural conditions. in stage 2, the virus known in animals is capable of infecting humans in natural conditions, but without the capability of person-to-person transmission. in stage 3, some cycles of secondary person-toperson transmission are possible. in stage 4, the virus circulates among humans through several secondary person-to-person transmission of varying duration. stage 5 is reached when the virus becomes exclusively human, and also contagious. on a population-wide scale an epidemic goes through four phases: introduction, propagation, amplification, and regression of the infectious phenomenon. the propagation phase is that during which there are the most widespread and frequent sites of infection. it corresponds (particularly for viruses) to the adaptation of the pathogen to its new host with person-toperson transmission taking effect little by little. it is often at this stage that the epidemic phenomenon is detected, sometimes with a considerable delay relative to the introduction of the pathogen. propagation can take place not only by contiguity but also across vast distances. humans thus play a role through their activities, and are what s. morse refers to as ''microbial traffic engineers'' [4] . what are the major factors in the emergence of new infectious pathogens in humans? a great deal of progress has been made in improving the rapidity in identifying new viruses, as j.-c. manuguerra points out. the time delay between the individualization of a new infectious nosological entity and the identification of the causal agent thus continues to shorten, from 14 years for hepatitis c in the 1970s to 2 years for hiv in 1983, and to 6 weeks for sars in 2003; even less for mers-cov. even so, the discovery or the knowledge of the pathogen's existence does not provide all the answers about the risks posed for or by a host species. various behaviors of pathogens can nonetheless be observed, in particular the pathogen's adaptation may require passing through several intermediary host species before adapting to its final host. moreover, the epidemic potential of a discovered virus is difficult to determine. among the very numerous known arboviruses, most are of anecdotal importance for human pathology, and little has been undertaken upon their discovery in preparation in case the pathogen were to become epidemic. this is true in the case of the zika virus, first isolated over 50 years ago, and currently responsible for a major epidemic in latin america and in the caribbean. the question of the time lapse between the beginning of an epidemic and identification of the pathogen is progressively slipping toward that between the beginning of the phenomenon and its detection by the health system. this period appears to be critical for eradicating the development of an epidemic. is it possible to narrow the time gap between the beginning of an epidemic and its detection by the healthcare services? early identification of an epidemic-level incident mobilizes all health services professionals, both in regards to human and to animal health, according to m. van kerkhove. human and animal health and the state of ecosystems are inextricably linked, and it is believed that nearly 60% of eids, including those re-emergent, are of zoonotic origin. it is through this exploration of the connections between animals and humans that the means of transmission and propagation of mers-cov within the human species could be revealed. this virus is an example of a potentially epidemic emergence. schematically, during the mers-cov epidemic, a limited number of person-to-person transmissions, and sporadic cases between dromedaries and humans, were observed, with a certain degree of increase seen in healthcare facilities, sometimes considerable, as was the case recently in korea. during the epidemic 75% of the mers-cov cases were reported in saudi arabia, and of 1600 cases reported to the who task force, 60% were considered to be primary cases, that is, contracted from an animal host source, and 40% to be secondary cases, or acquired through another human case. every primary case represents an opportunity to understand how the infection was contracted. it gradually seemed necessary to launch a veterinary investigation as soon as a case was diagnosed. this led to the development of animal surveillance, which made it possible to reveal the seropositivity of certain dromedaries, as well as active excretion of the virus in their environment, which made possible its transmission to humans. this improvement in detection of emerging pathogens and early epidemic detection calls for the improvement as well of the veterinary surveillance network, the so-called onehealth approach. in an area with limited resources, where all animals cannot be tested, it is imperative to concentrate on those areas with high concentrations of animals, such as slaughterhouses, and on areas in which humans come into close contact with animals and thus represent a high risk of transmission (see conference by p. daszak). the data collected in these areas of frequent contact between humans and animals can be utilized to issue recommendations for at-risk populations in order to reduce transmission. once the virus has acquired person-toperson transmission capability, its control is far more complex due to the rapidity of propagation following its introduction into the population. it therefore becomes difficult for the healthcare system to improve detection of the epidemic. establishing health policies to optimize the case reporting system is thus critical. how to provide care to patients in the case of new eids? in an epidemic situation, the treatment of affected patients usually is secondary to the need to control the disease, as a. mcgeer stated. nevertheless, an adapted patient care procedure can change the evolution of an epidemic, to varying degrees according to the pre-existing healthcare infrastructure in the given country. in north america, where the services for the monitoring and managing of infectious diseases and public health are separated from the healthcare system, the organization of care for infected patients is usually left to the physicians. the healthcare system is in fact organized around individual patient care, and not oriented toward an approach of global individual and collective care providing. in the case of eids, care of affected patients becomes a political issue in a country graced with a public health system. health is seen as a human right, and governments are judged not only by their ability to prevent and manage epidemics, but also according to their management of care provided to ill patients. the role of the public healthcare system is therefore to advise physicians and to develop recommendations for the detection of cases and their homogeneous treatment. these guiding principles make the physicians the kingpins between healthcare structures and the treatment of patients, and the public healthcare system. better patient care provision thus improves epidemic response. in fact, treating ill subjects also allows the risk of person-to-person transmission and propagation to be reduced. this treatment role attributed to physicians can be variously interpreted in countries in which the healthcare and public health system are underdeveloped or lacking. in fact, the arrival of healthcare personnel and the setting up of precautionary measures required to contain the epidemic may be experienced as an intrusion. the lack of comprehension and communication between medical staff and the affected community, as well as the potential lack of comprehension of the measures set in place can prompt affected individuals to hide, due to the uncertainty of their fate. this was the case for example in west africa during the ebola virus epidemic, during which affected patients were sometimes hospitalized far from their villages without care being taken to inform their families of their clinical progress and outcome. this led to sometimes-violent rejection of the healthcare personnel, which interfered with the measures meant to control the epidemic. another element to take into consideration in the care of affected patients is protection of the healthcare personnel, which has become a recurring problem. most of the microbial forms with epidemic potential propagate within the community. for a long time hospitals were not troubled over the risk of nosocomial transmission of eid agents. care providers were not aware at the time of the risk of contagion that they themselves ran when providing care to patients. this awareness happened during the recent sars, mers-cov and ebola epidemics, which shared certain physio-pathological characteristics different from those involved in previous epidemics. in fact the peak of virus excretion for measles, chicken pox and influenza generally occur before or upon the appearance of symptoms, and the risk of transmission is virtually nil when the patient arrives at hospital. sars, mers-cov and ebola virus have a different viral excretion rhythm: symptoms appear with a weak viral load, and their intensity increases with the level of viral excretion, which reaches its maximum just when the patient requires the most care. the hospital thus becomes a center for the propagation of the pathogen. the hospital system is therefore in danger of breakdown, as it is both the pole for patient care and the new center of the infection's transmission. these new pathogens therefore require a re-thinking of hospital design, in order to optimize both control of epidemics and patient care. what has been, or is, the extent of nosocomial infection's role in the transmission of sars-cov and mers-cov? the new pathogens require us to re-evaluate modes of prevention for nosocomial transmission, confirmed b. guéry. the 2003 sars-cov epidemic serves as a good example of what was learned about the intra-hospital transmission of these new pathogens. person-to-person transmission of sars occurs through droplets, physical contacts and airborne pathways. the sars-cov transmission rate to healthcare providers exclusive of invasive procedures has been estimated at 21%. the main risk factor identified was the lack of protection of the provider's airway through wearing a mask, with an odds ratio estimated at 13 . wearing scrubs and handwashing were also associated with lower transmission risk. sars is a disease which appeared in 2003 in guangdong province in china, then in hong kong, where numerous primary and secondary cases occurred. in total, according to the case index, 716 secondary and tertiary cases occurred, of which 52.3% among healthcare providers. beyond standard hygiene measures, studies conducted on affected healthcare providers revealed that certain categories of personnel, such as technicians and nurse's aids, show an infection rate twice that of the nursing staff, and 6 times greater than the medical personnel. these studies made it possible to identify infection risk factors generally not taken into account in the fight against nosocomial transmission of pathogens: a significantly, greatly increased risk (odds ratio 7.3) was noted in care providers having used precautionary measures against sars transmission for less than 2 hours, as was the case with those not having understood the protective measures (odds ratio 3.1). the factors identified as influencing transmission are patient viral load and patient index distance. the ideal conditions for transmission to occur are those of an infected patient excreting large quantities of virus, presenting with a certain number of comorbidities capable of masking the initial profile, and the existence of multiple close contacts with high-risk procedures such as oro-tracheal intubation, performing a fibroscopy, or the administration of treatments through nebulizers. the idea of a ''super-excreter'' patient was also identified during recent respiratory virus epidemics. this concept could play a crucial role. usually it concerns cases of very serious infection occurring in patients with several co-morbidities. in beijing in 2003, the sars case index was also associated with a large number of secondary cases (76 cases, of which 12 among healthcare personnel). as with mers-cov, despite a relatively low basal reproduction rate (r0), a large number of care providers were infected during the epidemic. this is what happened, for example, in abu dhabi, where 65 cases of mers-cov were diagnosed, of which 42% among healthcare providers. each case of provider infection was followed up through an epidemiological study, and each time an obstacle to the isolation of the patient and to the application of hygiene practices was noted. over 80% of the mers-cov cases identified in korea were thus traceable to 5 ''super-excreter'' patients. this notion remains questionable, as it is reductionist and could lead to the identification only of patients in this category, to the neglect of transmission risks associated with other patients. it is probably more fair to speak of ''hyperexcretion events'' which implies that each patient is at maximum risk, and should be treated using precautionary measures. in conclusion, the intra-hospital control and transmission of eids can only occur in connection with the development of precautionary standards, which should be ongoing over time, and should be applied by all healthcare providers. it is imperative to ensure that caregivers are adequately and regularly trained, and that they constantly keep in mind the importance of isolation of all infected patients. to achieve this, it is probably necessary to resort to specialized units, in reference hospitals, in conjunction with clear decisions at the national level. an integrated approach to health in face of the globalization of risk has been developing over the last few years. the ''onehealth/ecohealth'' concept, or global health, takes into account the fact that human health, animal health and environmental health are inextricably linked, especially in regards to eids, exposure to which is fostered by the multiplication of transcontinental travel, many instances of human-animal contact, and intensive farming and ranching. many recent examples have made it possible to establish the key role played by animal biodiversity in the introduction and transmission of pathogens within human populations. whether it be the role of bats in the 2014 ebola virus epidemic, or dromedaries in the 2012 mers-cov epidemic, the crucial role of animals and of human-animal contact -being wild or domestical animals -in triggering an epidemic has recently been emphasized. primates, rodents and bats are the three mammal groups most likely to be at the origin of future pandemics due to the high proportion of viruses which they share with humans. the inclusion of fields which appear quite unrelated (infectious diseases, animal health and ecological and environmental sciences) should thus be pursued and improved. it has been possible to establish models which allow the prediction of emergence tendencies in infectious diseases, and certain geographical areas at high risk for emergence have been identified: central africa and west africa, southeast asia, central america. these areas correspond to those at high risk of propagation due to underdeveloped or deficient public health systems, and to the absence of epidemiological surveillance. tools necessary for effective prevention of future epidemics are now available. these are all the more critical in that recent increasing tendencies raise fears of a multiplication of the number of emergent epidemics in future. beyond the fight against pathogen propagation, its introduction into the human population is in fact a key step against which ''battle plans'' can be drawn up. in order to perfect pandemic response, it is necessary to improve the coordination and interconnection between individual and institutional participants, such as healthcare providers and public health systems. a global response approach (oneresponse) should be reflected upon and developed. on an institutional level, a first step in bringing together the fields of environment and animal health occurred in 2010 in france with the creation of a national french agency for food, environmental and occupational health & safety (anses), originating in the french agency for food safety (afssa, which also includes the national agency for veterinary drugs) and the french agency for environmental and occupational health & safety (afsset). another case of bringing together the areas of surveillance, prevention and human health intervention occurred in 2016 with the creation of the french public health agency, with the merger of the health surveillance institute (invs), the national institute for prevention and health education (inpes), and the organization for preparedness and response to health emergencies (eprus). the relations between these two new institutions should be developed in order to provide a better-coordinated response to future health crises. the response to an eid should take into account not only factors linked to eids, but also to a constellation of political, economic and socio-cultural constraints. the decision to put in place such a battle is in fact a political decision which involves, beyond the scientific aspects, the intervention's impact upon the popularity of the acting political powers-that-be. if governments are judged according to their ability to prevent epidemic crises, they are equally judged on their ability to avoid expenditures deemed excessive given the existing risk. these political considerations can run counter to the scientific rationale behind the response. this is how, in the case of the 2014 ebola virus epidemic, the united states became involved: through the declaration by the liberian president on august 6, 2014, on the threat to national security posed by ebola, and the danger of the spread of the epidemic to the u.s. soil. in parallel with vaccine research and development, the actions of the u.s., the who and the united nations have focused on treatment of infected patients and the epidemiological securing of burials. in addition, despite a sometimes limited human impact, the economic impact of epidemics involving indirect costs (consequence for certain sectors of activity) has shown a marked increase. however, what characterizes modern epidemics is the duration of the economic ''shock'', in that it is temporary, as opposed to previous epidemics during which the shock tended to be drawn out in particular due to the persistence of infectious sources. there are many examples of this: among others, the ''spanish influenza'' of 1918, the effects of which (company closures, loss of revenue) faded out in 1921, or, more recently, the sars-cov epidemic, when the recession, that had been triggered by alerts against travel to southeast asian destinations communicated in march, ceased once these alerts were lifted two or three months later. it is of note that during the sars-cov epidemic only certain sectors (especially tourism) were affected in asia and in ontario, canada, and not the entire global economy. carrying out preventive measures such as staff training makes it possible to limit the number of crises at a lower cost. the costeffectiveness of such an approach has already been shown. it is henceforth necessary to raise awareness in decision-makers of the importance of prevention relative to risk. beyond these constraints, the decision to intervene is complicated by the heterogeneous nature of potential epidemics of the different pathogens. for this reason, and given the absence of technology permitting the prediction of the epidemic potential of a pathogen, prevention which targets the agent is impossible. prevention must therefore adopt other means, such as training locals in order to improve hygiene conditions. the improvement of transversal knowledge on infectious agents' transmission carries particular importance for the goal of preventing emergence. once an emerging agent is introduced into the human population, the pathogen propagation phase within the human population is the key phase in the development of the epidemic, and the one during which it is still possible to act in order to prevent the amplification of the pathogen in the population. the beginning of the propagation phase can be difficult to identify, and improvement in diagnostic techniques as well as the development of rapid diagnostic tests, and even on the field, makes it possible to accelerate detection of an epidemic signal. moreover, given the key role of certain animal species in the development of new epidemics, the development of animal health surveillance would allow us to further shorten the time lapse between the introduction of the pathogen and its propagation; however, this poses significant problems of wild animal monitoring particularly in southern hemisphere countries. better knowledge of states' operational modes in dealing with risk makes it possible to better understand their interventions and to better adapt the scientific response. the notion of risk has, little by little, become political, and the state uses risk to govern. risk management is therefore at the very heart of the state. recent public health crises have led to risk's taking on a new form, that of the unknown, by the fact of the unpredictability of its appearance and evolution. an interaction between scientific and political approaches to risk is absolutely necessary, in order to better evaluate at-risk situations according to modern methods such as structured decisionmaking, and to better handle risk-management tools. the changing nature of risks calls for the emergence of a new form of governance, which puts the individual back into the center of the state's action, as well as the confronting of arguments with expert committees. civil society's participation in risk management should be developed, and observation should once again play its part in crisis response. younger generations might also take greater part in responding to current crises. seven priority proposals can be outlined as follows: encourage research on the prediction, screening and early detection of new risks of infection; develop research and surveillance concerning transmission of pathogens between animals and humans, with their reinforcement in particular in intertropical areas (''hot-spots'') thanks to public support; pursue aid development and support in these areas of prevention and training for local health personnel, and to foster risk awareness in the population; ensure adapted patient care in order to promote adherence to treatment and to epidemic propagation reduction measures; develop greater sensitization and training among politicians and healthcare providers, in order to better prepare them to respond to new types of crises; modify the logic of governance, drawing from all available modes of communication and incorporating new information-sharing tools; develop economic research on the fight against eids, taking into account specific determining factors in order to create a balance between preventive and treatment approaches. the authors declare that they have no competing interest. introduction à la pensée complexe les maladies infectieuses émergentes : état de la situation et perspectives. available online: www global microbial traffic and the interchange of disease we are grateful to the speakers who generously brought their contributions to this seminar: peter daszak, olivier borraz, we thank corinne jadand for helpful organization and management support. key: cord-304943-thg4fqi2 authors: noor, aziz ullah; maqbool, farhana; bhatti, zulfiqar a.; khan, asmat ullah title: epidemiology of covid-19 pandemic: recovery and mortality ratio around the globe date: 2020-05-17 journal: pak j med sci doi: 10.12669/pjms.36.covid19-s4.2660 sha: doc_id: 304943 cord_uid: thg4fqi2 coronavirus disease 2019 (covid-19) is the third type of coronavirus disease after severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) that appears in human population from the past two decades. it is highly contagious and rapidly spread in the human population and compelled global public health institutions on high alert. due to genetic similarity of this novel coronavirus 2019 with bat virus its emergence from bat to humans is possible. the virus survive in the droplets of coughing and sneezing and spread around the large areas through infected person resulting in its rapid spread among people. clinical symptoms of covid-19 include fever, dry cough, dyspnea, loose stool, nausea and vomiting. the present review discuss the origin of covid-19, its rapid spread, mortality rate and recoveries ratio around the world. since its origin from wuhan, the covid-19 spread very rapidly all across the countries, on april 17, 2020 this disease has affected 210 countries of the globe. the data obtained showed over 2.4 million confirmed cases of covid-19. higher mortality rate was found in algeria and belgium as 15% and 13.95%, respectively. lower mortality rate was found in qatar 0.17% and singapore 0.2%. recovery versus deceased ratio showed that recovery was 68, 59 and 35 times higher than the death in singapore, qatar and thailand respectively. it is concluded that 2019-novel corona virus is a zoonotic pathogen similar to mers and sars. therefore, a barrier should be maintained between and across the human, household and wild animals to avoid such pandemics. coronaviruses (covs) are extremely important, widely distributed pathogens found in both humans and mammals. these are enveloped, single-stranded rna viruses belonging to family coronaviridae, circulating in birds and mammals and affecting human, domestic and wild animals. 1 two coronaviruses related human diseases emerged previously in 2003 and 2012, have been named as sars and mers respectively. these two strains of corona viruses collectively affected 10,000 people with a fatality rate of 10% for sars-cov and 37% for mers-cov. 2, 3 during the last 50 years, the emergence and re-emergence of deadly infectious diseases have increased. sars-cov-1 disease was originated in guangzhou city of china and the start of 2020 was again a challenging year for this country because of extremely contagion 2019-novel coronavirus (2019-ncov) disease outbreak. 4 this virus is also known as sars-cov-2 because of its same place of origin and genetic similarity with slight mutation in sars-cov-1 strain. in december, 2019 a series of new cases of pneumonia were reported in wuhan, china, whose clinical presentation were resembled to viral pneumonia. 5 deep sequencing helped to diagnose the new virus, named as 2019 novel coronavirus (2019-ncov) by world health organization (who). the origin of this virus has been reported in wuhan, china. few recent reports suggested its transmission from animal to human and within humans, which signifies its zoonotic potential. 6 so far, over 2.4 million cases have been reported worldwide with a total fatality reached to over two lac till april 26, 2020. the virus has been reported from many other countries mainly due to the traveling of infected/suspected people from china to these countries. chinese health ministry took immediate action to investigate and control the disease, including quarantine measures, continuous observation of contacts, clinical and epidemiological data collection from infected people and development of diagnostic tools and efficient treatment protocols. beside these measures chinese government had recommended to immigrant not to travel back to home country and should stay in 14 days quarantine before leaving to stop its further spread but many countries have not paid attention on this advice as a result coronavirus spread globally. this review focuses on origin of 2019-ncov, incidence of covid-19 in china, its clinical manifestations, mortality and recovery around the globe. the source of origin of coronavirus is still a mystery, however, early investigations have reported its possible origin from the wuhan seafood wholesale market. most of the early patient history associated with their movement to that seafood market. whereas, there were numerous other patients who have not gone to that market in those days. that association indicated its human to human transmission in spreading the outbreak. few environmental samples taken from the market have been reported positive but no specific animal was identified as its origin. 7 an initial investigation based on codon claimed snake as an origin. 8 it has also been proposed, that 2019-ncov naturally propagates in bats. 9 previous study revealed that wet markets of southern china including wuhan and guangzhou cities have the greater risk of spreading novel corona viruses, because of wild animal trading and the absence of biosecurity measures. 10 the other possibility is that bats and their excrements are commonly used in traditional chinese medicine, which may also be a source of infection. 11 it is also possible that the virus had infected other mammal that was traded at the market and served as the source of the infection to people. two already highly pathogenic coronaviruses were reported to be originated from the animals. the transmission of the first highly pathogenic virus, sars-cov occurred from animal to human in wet markets. the source of sars-cov was bat which transferred this virus to human via civet cat as an intermediate host. 12 the bats were also reported to be the possible origin of mers-cov, which is also a zoonotic virus. 13 however, mers-cov was reported in the patients, having frequent contact with the camels in the middle east. dromedary camel was considered as traditional household animal, to avoid contact with camel is not possible due to which they suffered from the periodic outbreak of mers-cov. 14 in this context, the identification of source animals, responsible for the transmission of covid-19 virus is extremely essential in order to control and prevent any future outbreak. the possible transmission cycle of covid-19 is presented in fig.1 . covid-19 is a great public health and safety concern all across the globe and the chinese government, international agencies and who is aware of the consequences of the outbreak. the first case of covid-19 outside china was reported in thailand. 15 due to rise in infected and suspected cases of covid-19 in china by the end of the third week of january 2020, the chinese government had completely lockdown the wuhan city, hubei province. they partially lockdown other cities at higher risk to stop its further spreading. since the public health emergency declared by who, much more attention at national and international levels has been focused on covid-19. the concept of one health which means that 'the health of people is linked with the animal and connected environment' has gained great importance in this current scenario. 16, 17 the super-spreaders (infected person who travels and infect others) are mostly responsible for large outbreaks. for example, traveling of one infected person with sars-cov from hong kong to toronto infected 128 people in a local health care facility where he visited for treatment. the same situation was with mers-cov, where a single patient from saudi arabia traveled to south korea infected 186 patients with mers-cov. 18 the epidemiological characteristics observed in the case of covid-19 varied to a great extent as compared to the sars-cov. the 2019-ncov replicates efficiently in the upper respiratory tract, but the onset of symptoms appears to be slow. although the infected individual carries a huge number of virus still he/she can perform his/her normal activities, which leads to the spread of this infection to other individuals. whereas, in the sars-cov patients, the transmission of infection is not reported during the prodromal period and transmission occurs when the patient becomes severely ill. 7 various factors affect and determine the spread of disease in an outbreak including transmissibility and severity. morbidity and mortality will rely on the combination of transmissibility and severity and a rapid person to person transmission occurred in this outbreak. the epidemiologists estimated the r o (the basic reproduction number to measure the transmissibility) value would be 2.2 for covid-19. 19 the severity and the case-fatality rate could be underestimated because many infected people have not yet recovered and may deceased. after the onset of the covid-19 outbreak, the potential risk of vertical transmission was a question mark. control and prevention of this novel emerging infection among pregnant women have become a primary concern regarding vertical transmissibility. the latest study published in the lancet gives some insight into the clinical features during pregnancy and potential of vertical transmission covid-19 infection in pregnant women. 20 although the research manipulates only a small number of nine pregnant women with confirmed covid-2019, no evidence for intrauterine infection was reported in late pregnancy in these cases. 21 a sars-cov-2 genomic study suggested that this virus has 88% genetic similarity with two sars-like coronaviruses isolated from a bat, sbat-sl-covzc45 and bat-sl-covzxc21. while in case of sars-cov-1 and mers-cov bat's genome similarities were 79% and 50% respectively. homology studies have shown that sars-cov-2 has a similar receptor binding structure to that of sars-cov-1. so, the ability of vertical transmission of corona-19 could be as low as that of sars-cov-1. 22 although 2019-ncov has adverse effects on newborn including shortening of breathing, premature birth and liver disorder but its vertical transmission has not proved. 23 the clinical findings of covid-19 infected pregnant women were same as non-pregnant women infected with covid-19 infection. 24 in accordance with sars-cov and mers cov, this new virus was observed to affect more males than female individuals. the reduced susceptibility of women can be explained by the fact that the sex hormone and x chromosome play an important role in innate and adaptive immunity. moreover, almost half of the population infected by covid-19 has underlying problems mainly diabetes, cerebrovascular and cardiovascular problems similar to mers-cov. the older people with the weak or compromised immune system are more susceptible to this disease. 24 among the clinical signs; fever, dry cough, dyspnea, and fatigue were common in all cases. upper respiratory tract infection i.e. rhinorrhea, sneezing, or sore throat were also uncommon in covid-19. the laboratory reports of covid-19 patients indicates lymphopenia (decrease in white blood cells) which suggested the destruction of lymphocyte and other immune cells by coronavirus leading to the weakening of cellular immune system. 25 some patients suffer from acute respiratory distress and septic shock which lead to the failure of multiple organs. [24] [25] [26] these cases are of crucial importance to be treated at earlier stages. ct scan indicated ground-glass opacification and occasional consolidation in the patients. 27 most of the deaths have been reported in the patients who have the characteristic of warning signs described by the multi logistic binary search tree analysis (mulbsta) model. these six signs which are included in the mulbsta model are multinodular infiltration, lymphopenia, bacterial co-infection, smoking history, hypertension and age. 28 few or no options are available for treating a viral disease that emerged suddenly, no vaccine has been developed yet for the prevention of covid-19 infection. 26 a combination of antiviral drugs lopinavir/ritonavir, pegylated interferon, and ribavirin has been used in a mers-cov case reported in south korea, that helped in the successful clearance of the virus. 29 another viral drug, remdesivir has been reported to be effective against the viral infection. in-vitro studies indicated that remdesivir has been successful in the termination of viral rna replication, 30, 32 and showed effectiveness against the mers-cov, sars-cov and other bat originated coronaviruses. 31, 33 qamar et al., 2020 screened the database of 32,297 chinese medicinal plants for their antiviral activity. they suggested 9 medicinal plants that might help in the prevention of viral replication. 34 further studies are necessary to figure out the effectiveness of these plants in this infection. another study on virtual screening of a database of more than 3000 food and drug administration (fda) approved drugs was carried out in order to find the possible best available drug. the results suggested that protein inhibitors in human immunodeficiency virus (hiv) drugs might be helpful against the covid-19. 35 recently fda have authorized the use of hydroxychloroquine and chloroquine due to emergency situation without double blind and clinical trial for the treatment of covid-19. 36 since the discovery of the virus, the covid-19 spread very rapidly all across the countries and cases have been reported in 210 countries around the globe (till 10:39 gmt on april 26, 2020) . the data obtained showed over 2.4 million confirmed cases of covid-19. 37 higher mortality rate (15%) was found in algeria, belgium (13.95), italy and united kingdom (13%) and netherland (11.35%). lower mortality rate was found in countries qatar 0.17%, singapore 0.2%, united arab emirate 0.6%, and australia 0.97 . the who keeps on updating and sharing these figures on daily basis and till april 28 th it had iussued ninety seven reports giving countrywise details of number of cases. higher mortality rate is related with the total number of infected cases, as significant positive correlation r=0.9, n=56 was found between confirmed cases and deaths, which showed that disease spread increases the risk of death due to overcrowded hospitals, lower availability of medical facility and other environmental factors. before mitigation measures were taken place covid-19 was already spread in the early stages. 38 countries showed early response suffered less than the countries that did not care in the early stage of this pandemic. yet another reason of this pandemic was as 80% of covid-19 cases are mild or asymptomatic so the symptom base control of this disease is very difficult and less effective. recovery versus deceased ratio was calculated and the data showed that recovery was 68, 59 and 35 times higher than the death in singapore, qatar and thailand respectively. lower value of deceased over recovery ratio was found in united kingdom (0.03), netherland (0.08), ireland (0.16) and norway (0.21). 37 in contrast to covid-19 prevalence, previous study shows that community acquired pneumonia cases were high in male who belonged to lower socio-economic group, illiterate people living in rural areas. 39 patients recoveries are significantly correlated with the number of cases (r = 0.63, n = 56), showed that recoveries are increasing with increase in number of cases. the potential factors involved in the recovery might be strong immune system among the population, good dietry habits and early treatment and bacillus calmette-guérin (bcg) vaccination policy in some country showed less cases than without bcg vaccinated nations. 40 according to reuter the global death due to covid19 disease passed two hundred thousand on april 25 th 2020 and it is expected that the total number of infected cases will cross three million people by the end of april 2020. more than 50% of these deaths have occurred in united states, spain and italy the worst hit countries. till april 25 th united states had reported fifty two thousand four hundred deaths while italy, spain and france have reported between 22,000 and 26,000 fatalities each. of the top twenty most severely affected countires belgium has reported highest number of fatalities per capita with six deaths per ten thousand people compared to 4.9 in spain and 1.6 in the united states. the overall mortality rate of cases reported in united states has been 8% while more than 10% cases reported in spain and italy have resulted in deaths. asia and latin america have each reported more than seven thousand deaths while middle east has reported more than eight thousand eight hundred deaths. 41 the danger of covid-19 can be argued by the fact that this virus has inherited the ability to mutate. it is accepted that most viruses survived in their natural reservoir for a longer time. therefore, the most fruitful method to prevent viral zoonosis is to preserve the barriers between human society and natural reservoir of viruses. global preparedness for any outbreak has been suggested that national health security plan is alarmingly weak around the world and we should prepare ourselves better for the future. coronaviruses in avian species. review with focus on epidemiology and diagnosis in wild birds summary of probable sars cases with onset of illness from 1 middle east respiratory syndrome coronavirus (mers-cov) a novel coronavirus from patients with pneumonia in china 2019 pneumonia of unknown cause -china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster who. novel coronavirus-japan (ex-china) homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human ncov's relationship to bat coronaviruses severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection bats as materia medica: an ethnomedical review and implications for conservation emerging animal coronaviruses: first sars and now mers severe acute respiratory syndrome coronaviruslike virus in chinese horseshoe bats evidence for camel-to-human transmission of mers coronavirus imported cases of 2019-novel coronavirus (2019-ncov) infections in thailand: mathematical modelling of the outbreak the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan, china data sharing and outbreaks: best practice exemplified sars and mers: recent insights into emerging coronaviruses clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan a novel coronavirus outbreak of global health concern clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study t-cell immunity of sars-cov: implications for vaccine development against mers-cov clinical features of patients infected with 2019 novel coronavirus in wuhan imaging profile of the covid-19 infection: radiologic findings and literature review clinical features predicting mortality risk in patients with viral pneumonia: the mulbsta score case report combination therapy with lopinavir/ritonavir, ribavirin and interferon-α for middle east respiratory syndrome therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys initiation, extension, and termination of rna synthesis by a paramyxovirus polymerase broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants virtual screening of an fda approved drugs database on two covid-19 coronavirus proteins. 2020. chem rxiv regulators split on antimalarials for covid-19 covid-19 coronavirus pandemic how will country-based mitigation measures influence the course of the covid-19 epidemic? profiles of community acquired pneumonia cases admitted to a tertiary care hospital correlation between universal bcg vaccination policy and reduced morbidity and mortality for covid-19: an epidemiological study global corona virus death toll hits two hundred thousand. daily dawn april26th 2020 we would like to thanks to dr. mudassar mohiuddin for his editing and final review. none. key: cord-328366-new4d9jg authors: bleibtreu, a.; arias, p.; vallois, d.; debit, a.; lermuzeaux, m.; rioux, c.; cabras, o.; lucet, j. c.; choquet, c.; timsit, j. f.; yazdanpanah, y.; lescure, f. x. title: delayed management of staphyloccocus aureus infective endocarditis in a middle east respiratory syndrome coronavirus possible case hospitalized in 2015 in paris, france date: 2017-06-30 journal: clinical microbiology and infection doi: 10.1016/j.cmi.2016.11.021 sha: doc_id: 328366 cord_uid: new4d9jg nan the risk of emerging infectious diseases such as middle east respiratory syndrome coronavirus (mers-cov) [1] and ebola epidemics is growing not only as the result of changes in demographic, anthropological, ecological and economic conditions but also because of increasing connectedness and speed of movement in the modern world. in response to the risk of hospital transmissions to healthcare workers and other patients, maximum precautions in isolation wards aim to limit transmission. infection due to mers-cov [2] was identified in 2012, and has been responsible for 1733 confirmed cases and 628 deaths to date [3] . in france, since 2012, 1524 patients were classified as possible cases, two were confirmed as mers-cov infection, of which one died [4] . maximum precautions to avoid cross-transmission to healthcare workers may alter the management and care of other lifethreatening infectious diseases. it is of particular importance given the ratio between the number of cases of emerging infectious diseases and the number of 'classical' infections. a man in his sixties with possible mers-cov was admitted to our infectious diseases department at bichat claude bernard hospital in paris in 2016. he lived in the united arab emirates and had returned to france for a holiday 5 days before admission. fever had appeared 3 days earlier. he was initiated on amoxicillin by a general practitioner 2 days before. he was admitted to the emergency department for a persistent fever at 39.5 c with cough, shortness of breath and general weakness. his main medical and surgical history included an aortic prosthesis (bentall surgery) with a mechanical aortic prosthesis, and a pacemaker implanted in 2002. the neutrophil count was 23 230/mm 3 , thrombocytes were low (97 000/mm 3 ) and the c-reactive protein was elevated (386 mg/l). chest x-ray was normal. the first hypothesis raised by the emergency department doctor was an infective endocarditis on the mechanical prosthesis. but the geographic provenance, the presence of fever and cough led to classification as a possible mers-cov, after case review with the 'institut national de veille sanitaire', according to the national recommendation. the patient was placed in the isolation ward 8 hours after admission (h8) through the emergency department. according to french guidelines for laboratory biosafety, initial processing of biological specimens should take place in a class ii or class iii biosafety cabinet. only automatized tests could be collected and analysed. other microbiological tests, such as blood cultures, should not be sent to the laboratory until mers-cov pcr of a superficial or deep airway sample has returned negative [5]. twelve hours after admission (h12), based on the infectious disease clinical assessment, mers-cov diagnosis was no longer considered. isolation procedures were stopped in the emergency department with the agreement of the 'institut national de veille sanitaire' and three blood culture sets were sampled. cloxacillin and gentamicin were initiated targeting an infective endocarditis. eight hours later (h20), blood cultures were positive for staphylococcus aureus with negative meca genexpert. trans-thoracic and transoesophageal echocardiographs revealed a mobile echogenic mass of 10 mm on the cardiac valve, along with a thickened posterior ring. screening for septic embolism showed a cerebral ischaemic and haemorrhagic stroke and fungal aneurysm on the left femoral artery. surgical intervention was performed on day 10 of antibiotic initiation. surgery was followed with diaphragmatic rupture in the pericardia, and retroperitoneal bleeding. after 17 days in the intensive care unit the patient died. this case illustrates the difficulty of managing patients with suspected highly contagious emerging infectious diseases. a final diagnosis of mers-cov requires the exclusion of all other diagnoses [3] . but the diagnosis of mers-cov infection should be considered in a patient returning from epidemic countries with non-specific symptoms. here, the suspicion of mers-cov led to a 12-hour delay in performing blood cultures because of isolation. clinicians did not start antibiotic therapy as quickly as they should. french guidelines for laboratory biosafety in the case of mers-cov suspicion should be discussed again and probably modified. there has been no new mers-cov infection in france since 2012 and these restrictions are more stringent than the who guidelines. suspicion of an emerging infection should not paralyse the clinician to the detriment of the individual in his search for alternative diagnoses. the authors have nothing to disclose. this work was not funded. isolation of a novel coronavirus from a man with pneumonia in saudi arabia case definition and management of patients with mers coronavirus in saudi arabia who. middle east respiratory syndrome coronavirus (mers-cov) available at key: cord-321260-oi37dfsp authors: ahmed, anwar e. title: estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study date: 2017-12-22 journal: epidemiol infect doi: 10.1017/s095026881700293x sha: doc_id: 321260 cord_uid: oi37dfsp although middle east respiratory syndrome coronavirus (mers-cov) has a recorded 5 years of circulation in 27 countries worldwide, there is no international study to assess whether there is variation in mortality by region. neither has there been a comprehensive study detailing how the disease characteristics of mers-cov influence mortality in patients presenting symptoms. this study aimed to assess how region, patient and disease characteristics influence 14and 45-day mortality in mers patients. the author utilised publically available data on mers-cov. the study included 883 mers patients reported between 5 january 2015 and 10 march 2017. data on patient and disease characteristics were collected. the mean age at mers-cov diagnosis was 54.3 years: 69.1% were male, and 86.7% of the cases were reported from saudi arabia. about 40% of mers patients studied were over the age of 60. the study estimated 14and 45-day survival rates after initial onset of symptoms: 83.67% and 65.9%, respectively. saudi arabian mers patients exhibited 4.1 and 5.0 times higher 14-day (adjusted hazard risk (ahr) = 4.1; 95% confidence interval (ci) 1.012–16.921) and 45-day (ahr = 5.0; 95% ci 1.856–13.581) mortality risk compared with mers patients in the republic of korea or other countries. similarly, middle eastern mers patients showed 5.3 and 4.1 times higher 14-day (ahr = 5.3; 95% ci 1.070–25.902) and 45-day (ahr = 4.1; 95% ci 1.288–113.076) mortality risk compared with mers patients in the republic of korea or other countries. the results demonstrated a link between mortality and geography, disease and patient factors such as regions, symptoms, source of infections, underlying medical conditions, modes of transmission, non-healthcare workers and those of older age. educational programmes, access to healthcare and early diagnosis could be implemented as modifiable factors to reduce the higher mortality rates in mers patients. middle east respiratory syndrome coronavirus (mers-cov) is an international public health challenge with considerable differences in methods of reporting death-related within and between countries [1] [2] [3] [4] [5] [6] [7] . the case fatality rate accounted for 30-63% in saudi arabia [1] [2] [3] [4] and 20-63% in the republic of korea [5] [6] [7] . recently, several epidemiological investigations have been published that assessed the factors associated with mortality in mers patients [8] [9] [10] [11] [12] [13] . however, the findings of most of these studies either represent a specific population group such as intensive care unit [8] , a single medical centre [9] or outbreak infection in the republic of korea [10, 11] . one study analysed publicly available data through the saudi ministry of health webpage to identify the factors associated with mortality in saudi arabian patients [12] . one limitation of this study was lack of multivariate risk modelling. rivers et al. [13] used a multivariate poisson regression model to identify factors associated with a high incidence of mortality, but their study excluded all cases (186) from the republic of korea. a recent study by chen et al. compared the risk factors and disease characteristics in south korean and saudi arabian populations, but did not assess the survival rate in these cases [14] . the authors of all previously published studies of mers-cov were not able to assess survival rate in mers cases from various countries that were reported to the world health organization (who). although the mentioned studies provided valuable information in terms of mortality risk in mers patients, a common limitation was noted in these previous studies: no global data were used, such as the associations between different geographical regions and mortality. the findings in these reports were made using data on specific populations or regions. per the author's knowledge, at this time there is no large cross-country comparison study of mers-cov-related mortality, and it is not yet clear whether the mortality rate of mers-cov can be identified by countries and regions. it is important to understand and compare mers-cov patients according to their clinical outcomes between multicountries. such a study may provide a useful multi-country work to reduce the mortality in the mers-cov population worldwide. the current study may provide multivariate risk models to identify patients at high risk of death in order to manage mers-cov patient clinical outcomes. this may improve public health plans by establishing effective programmes to prevent serious outcomes. these programmes can be addressed by the healthcare system or public health policies. the goal of this study was to estimate and compare the variations in the risk-standardised mortality rates by regions and by patients' characteristics among different countries across the world, specifically comparing regions that had the largest number of mers-cov cases. the author hypothesised that people of older age, with underlying medical conditions and from saudi arabia or other middle east countries are at high risk of death related to mers-cov. the study utilised a publicly available mers-cov database of case reports retrieved from the who: http://www.who.int/ csr/don/archive/disease/coronavirus_infections/en/. the who receives situational reports of confirmed mers-cov cases from all countries across the globe. the who provides a routine update through its website on new cases, deaths and current developments. prior to 26 january 2017, all reports were presented in a narrative format, describing case by case the disease characteristics of each patient. the author and research assistant accessed each of these narrative reports, and converted the patient information and disease characteristics into a line listing for analysis. beginning with the reporting period of 2-7 january, who enhanced their reporting practices, including an excel document with patient and disease characteristic data which can be easily analysed. the latest line listing update of mers cases was on april 2017 in a report from qatar. globally, the who report on 4 april 2017 indicated that the overall case fatality rate was 35.6% (690 deaths of 1936 cumulative laboratory-confirmed mers cases) in 27 countries (who, 2017). the author did not include all mers cases reported to the who because the early reports of mers used unstandardised case presentation that lacked important details. this study included mers cases reported to the who between 5 january 2015 and 10 march 2017. another reason for choosing this period to study is that in 2015, the who started reporting cases by the country where the confirmed case has occurred. the author performed quality control checks to detect invalid or missing data. the author excluded some cases from the republic of korea that were reported between 12 june and 21 july 2015, as no patient case report was used. they reported a summary of the updated cases (total cases and deaths) instead of case-by-case details. case #3 reported on 23 march 2016 has been excluded from saudi arabia due to the wrong date of symptom onset stated in his case report. the total cases included in the analysis were 883. the author obtained information on age, gender, date of notification, date of onset of mers symptoms, date of outcome or death, whether a patient or healthcare worker, symptomatic, underlying medical conditions, source of infection and the reported country. in order to assess the impact of older age on mortality among mers patients, patient age was classified into four groups: age <30, 30-59, 60-65 and >65 years. the data retrieved were from 14 countries. the author classified countries into three regions according to the geographical location and the number of cases: (1) saudi arabia 766 (86.7%), (2) middle east, but excluding saudi arabia 44 (5%), and (3) republic of korea or other countries 73 (8.3%). the republic of korea 65 (7.4) and other countries 8 (0.9%) were combined due to the small number of cases in the latter group. saudi arabia was analysed separately from the middle east countries because saudi arabia has a unique situation and recorded the largest number of mers cases. two primary end points were assessed: 14-and 45-day mortality related to mersafter developing symptoms. the study author estimated the survival rates using the time of symptom onset to outcome. mers-cov has an incubation period of 2-14 days, by which time symptoms usually occur. survival rates at the 14-day mark are medically significant as they demonstrate survival at the point at which patients most typically experience statistical analyses were performed with sas version 9.4 software. data from 883 mers-cov patients who reported to the who between 5 january 2015 and 10 march 2017 were retrieved and analysed. descriptive statistics were used to describe the study population (table 1) . unadjusted cox proportional hazards models (cphms) were used to estimate hazard risk (hr) and a 95% confidence interval (95% ci) for 14-and 45-day mortality (tables 2 and 3 ). multivariable cphms were used to estimate adjusted hazard risk (ahr) and 95% ci for 14-and 45-day mortality adjusting for gender, age, region, whether healthcare worker, having comorbidity, being asymptomatic and the source of infection (tables 2 and 3 ). the log-rank tests were used to compare survival curves for demographic and disease characteristics (figs 1-4) . the 14-and 45-day survival rates were estimated using the kaplan-meier estimator. a total of 883 patients with confirmed mers-cov infection were included in the analysis. the characteristics of the population can be found in table 2 illustrates unadjusted and adjusted 14-day hazard risks for all-cause mortality. the unadjusted 14-day analysis shows that age groups 60-65 and >65 years, saudi arabia, the study was conducted to estimate the survival rates in mers patients, specifically 14 and 45 days after experiencing mers-cov symptoms and to determine whether there is a significant epidemiology & infection difference in survival rates by demographic data, disease characteristics and regions using global mers data that were reported to who. the author used the cphms and the kaplan-meier estimator to calculate survival rates. according to the data, the estimated overall survival rate was 63.4% (95% ci 60.15-66.60%). the study estimated 14-and 45-day survival rates were 83.67% (95% ci 81.09-86.07%) and 65.9% (95% ci 62.68-69.04%), respectively. the author calculated region-specific 14-and 45-day survival rates (figs 1 and 3) . for the mers patients studied, the republic of korea or other countries (95.83% and 91.67%) had much higher 14-and 45-day survival rates than the middle east (84.09% and 75.00%) and saudi arabia (82.51% and 62.92%), respectively. the differences in the region-specific survival rates remain significant after accounting for patient and disease factors such as gender, age, comorbidity, symptoms, healthcare worker and source of infection. for instance, the hazard of death on 45-day post-symptoms was 4.1 and 5.0 times higher in the middle east and saudi arabia compared with the republic of korea or other countries, respectively. the disparities in survival rates between regions are probably explained by the delay in reporting mers symptoms, and consequently delay its diagnosis [15] . the median time from date of onset of symptoms to death was lower in the republic of korea or other countries than in the middle east and saudi arabia. more research studies are required to assess whether diagnostic delay [15] and disease characteristics can explain the differences in survival rate by geographical regions. those aged 60 years and older represent a large portion of the mers population (39.5%) and they predominantly contribute a high portion of deaths as well. the findings of this study showed that 14-and 45-day survival rates tend to rise with increasing age (figs 1 and 3) and were similar in women compared with men. the estimate of the 45-day survival rate in patients older than 65 years was 44.86%, in patients aged 60-65 years it was 60.38%, in patients aged 30-50 years it was 74.27% and in young patients aged 29 years or less it was 87.78%. these results support various epidemiological cohorts from saudi arabia and the republic of korea, which stated that older age is a risk factor for death in mers patients [8] [9] [10] [11] [12] [13] [14] . the high death rate among the older age group could be attributable to the greater number of patients with comorbidities in this group. the prevalence of comorbidities increases with age: 28.6% in the young age group <30 years, 62.7% in 30-59 years, 93% in 60-65 years and 97% in older than 65 years. thus, comorbidities may be associated with both age and mortality. several studies [10] [11] [12] [13] , including the current study, suggest that the presence of underlying conditions was associated with an increase in the hazard of death in mers patients. more details on underlying conditions are needed to estimate underlying condition-specific survival rates and would be useful to identify which underlying conditions were associated with the lower survival rates. moreover, prevention and disease management strategies should be assessed as interventions in mers patients with underlying conditions to reduce the mortality rate in this group. in concordance with other studies [9, 13] , being a nonhealthcare worker was associated with lower survival rates (figs 2 and 4) . the study included 116 (13.1%) who were healthcare workers, of which five died. the higher survival rates within the healthcare workers group could be attributed to educating healthcare workers on preparedness, access to healthcare or following proper infection control standards. an explanation for lower survival rates in the non-healthcare workers group is the large gaps in public awareness of the clinical symptoms of mers-cov [16, 17] . public health and health system interventions are needed to reduce the spread of the mers by raising public awareness in identifying the clinical symptoms and by early screening and diagnosis. this could reduce the high rate of mortality in nonhealthcare workers. patients who had acquired the infection from camels, hospitals and unknown sources of infections exhibited 2.5, 3.6 and 3 times higher mortality risk compared with patients who had acquired the infection from a household member, respectively (figs 2 and 4 ). the healthcare system may use this information to properly develop a support care plan to improve patients' outcomes. anwar e. ahmed this study has several limitations. the study used available data on the public source with no details on the underlying conditions. this information could be important to identify which underlying condition is associated with death. patient condition and clinical outcomes may not be final as data are updated routinely. another potential confounding factor was not available, such as access to healthcare, and it may be considered a modifiable factor to account for. finally, an inverse-probability kaplan-meier curve may also be appropriate to model this dataset, as it may provide perspective into the data and a visual way to show adjusted survival rates. despite these limitations, the study author was able to use the estimated 14-and 45-day survival rates, measuring the time from symptom onset to outcome. to date, no study has provided survival estimates and links to regions. the study provided information on region-specific 14and 45-day survival rates, which are found to account for the differences in mortality. the large sample size used was also the main strength of this study. future study could investigate diagnostic delay, which can be defined by the difference between date of symptom onset and the time of diagnosis. this may reduce poor outcomes. the study estimated 14-and 45-day survival rates after initial onset of symptoms: 83.67% and 65.9%, respectively. the results demonstrated a link between mortality and geography, disease and patient factors such as regions, symptoms, source of infections, underlying medical conditions, modes of transmission, non-healthcare workers and older age. educational programmes, access to healthcare and early diagnosis could be implemented as modifiable factors to reduce the higher mortality rates in mers patients. additional files. none. ethical approval and consent to participate. not applicable. consent for publication. the author read and approved the final manuscript. availability of supporting data. the data used for the analysis can be obtained from the study author. declaration of interest. none declared. association of higher mers-cov virus load with severe disease and death, saudi arabia treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia the predictors of 3-and 30-day mortality in 660 mers-cov patients predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a singlecenter experience in saudi arabia real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea mortality risk factors for middle east respiratory syndrome outbreak, south korea risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia risks of death and severe disease in patients with middle east respiratory syndrome coronavirus comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea diagnostic delays in 537 symptomatic cases of mers-cov infection in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov): prevention in travelers acknowledgements. the author acknowledges the who for making mers-cov data publicly available. no funding was provided for this study.authors' information. college of public health and health informatics, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia. key: cord-318448-3bkp1mtj authors: choi, jun yong title: an outbreak of middle east respiratory syndrome coronavirus infection in south korea, 2015 date: 2015-09-01 journal: yonsei med j doi: 10.3349/ymj.2015.56.5.1174 sha: doc_id: 318448 cord_uid: 3bkp1mtj nan between may and july 2015, there was an unexpected outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection in south korea. the outbreak has emerged as the largest one outside the middle east. as of july 20, there have been 186 laboratory-confirmed mers cases, including 36 deaths, 136 recovered individuals discharged from the hospital, and 14 patients who remain in hospitals (fig. 1) . 1, 2 the index patient was a 68-year-old korean man. 2 5 the appearance of mers-cov was unexpected and unfamiliar to most physicians. infection prevention and control measures in hospitals were not optimal. extremely crowded emergency rooms and multi-bed rooms contributed significantly to nosocomial infection in some hospitals. the practice of seeking care at a number of medical facilities may have also been a contributing factor. additionally, the custom of having many friends and family members accompany or visit patients may have contributed to secondary spread of the infection. meanwhile, no evidence of community transmission has emerged. several super-spreading events, which happened within hospitals from patients 1, 14, 16, and 76, contributed to 80% of all subsequent cases. as well, whole genome sequencing of the mers-cov from this outbreak did not identify any major mutations different from global mers-cov. the factors that drove the super-spreading events of the outbreak have not yet been established. medical procedures that can generate aerosols from the lower respiratory tract of an undiagnosed patient with severe pneumonia could contribute as a super-spreading event. in addition, the crowdedness of the hospitals and environmental contamination could other reasons for the special event. strong infection control measures, including robust contact tracing, active surveillance, quarantine and isolation, have been applied to control the outbreak, since the initial recognition of the outbreak by the korean government. as of july 20, a total of 16692 persons have been quarantined for 14 days, and 16671 persons have been discharged therefrom. 1 the infection control measures are anticipated to control this outbreak successfully within several additional weeks. this large and complex outbreak, which arose in crowded hospitals within metropolitan cities, exposed several problems with the korean healthcare system, including emergency preparedness and response systems by the government, as well as infection prevention and control measures in hospitals. to prevent recurrence of a similar situation, we should not only seek to improve and strengthen such systems and measures, but also to develop trained experts and proper facilities. in addition, the outbreak raised several research questions on the epidemiology, virology, pathogenesis, infection control, and treatment of mers-cov infection that await answering. research into the korean outbreak will provide valuable lessons for better global public health. korea ministry of health and welfare and center for disease control and prevention. updates on mers: for press release korean society of infectious diseases; korean society for healthcare-associated infection control and prevention. an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea korea ministry of health and welfare and center for disease control and prevention. identification of imported mers case: for press release middle east respiratory syndrome coronavirus (mers-cov)-republic of korea who recommends continuation of strong disease control measures to bring mers-cov outbreak in republic of korea to an end: for news release the author would like to thank dong-su jang, mfa (medical illustrator, medical research support section, yonsei university college of medicine, seoul, korea), for his help with the illustrations. key: cord-321800-0h28pg3b authors: klingelhöfer, doris; braun, markus; brüggmann, dörthe; groneberg, david a title: coronavirus: an insight into global research until outbreak of covid-19 and its implications for the future date: 2020-09-23 journal: journal of global health doi: 10.7189/jogh.10.020508 sha: doc_id: 321800 cord_uid: 0h28pg3b background: the currently prevailing global threat of covid-19 caused the publication numbers on coronaviruses to explode. the awareness of the scientific and public community is enormous. but what about the sense of all these undertakings and what can be learned about the future for a better understanding? these questions were answered with established bibliometric analyses of the time until the avalanche of publications unfolded. methods: chronological, geographical aspects of publication output on coronavirus were also evaluated under the influence of epidemiological and socio-economic parameters. results: the trend in publication and citation numbers shows the strong influence of the past pandemics sars and mers with an untypical decline afterward. research is becoming increasingly multidisciplinary over time. the usa and china, as the countries with the highest number of publications, are being displaced by other countries in the consideration of socio-economic and epidemiological aspects, which shows the effect of regional interest in corona research. a significant correlation was found between the number of sars cases per country and related publications, while no correlation was found for mers cases and articles. conclusions: the results underline the need for sustainable and forward-looking approaches that should not end with the containment of covid-19. in line with the aphorism: "only those who know the past can understand the present and shape the future", the present study provides background information of research on coronaviruses (cov) as a basis for the scientific situation of the global pandemic covid-19 and a source for a better understanding of research patterns from the time before covid-19. viewpoints research theme 1: were recognized as pathogens causing only mild infections such as common cold and their clinical significance was not recognized. by 2003, sars had infected more than 8000 people in 29 countries and killed 774 people. the pandemic ended abruptly, and no cases occurred later [3] . at an animal market in china, the sars-cov pandemic was found to be transmitted by palm civet cats [4] . more than 87% of all cases worldwide occurred in china. the second cov pandemic mers (middle-east respiratory syndrome) occurred in 2012, but unlike sars, mers did not end suddenly and cases continue to be recorded until now [5] . mers affected more than 2400 people and caused 912 deaths in 27 countries by 2019, according to heath authorities worldwide. more than 77% of the mers cases occurred in saudi arabia, transmitted by dromedary camels [6] . later studies have shown that bats are reservoir hosts for both of these former cov diseases [7, 8] . the current extremely rapid global spread of sars-cov-2 has led to the highly dangerous outbreak of the pandemic covid-19 with daily increasing numbers of new infections and deaths around the world. at this point in time, the peak of the infections has not yet been reached, so the final figures are not predictable -but it seems that they will reach devastating proportions and will certainly change world societies forever. there are no vaccines yet, and treatment options are still limited. only symptomatic treatment or support is possible [9] . due to the enormous spread of infections and the severe course of many cases, the appearance of covid-19 is accelerating research, which is certainly unique in the history of science. almost real-time results provide new insights from all areas of science, from basic to applied research. the financing of cov research is also picking up speed. for the dissemination of this rapidly generated knowledge, international communication is obligatory, and the most common way of disseminating it is the publication of results [10] . can we learn from previous research patterns regarding cov? what influence do they have on future research? how can we use past efforts, their intensification and the influences of research on cov positively to better understand the needs for sustainable and appropriate research? these are compelling questions in light of the currently exploding research output, which, in addition to interesting and meaningful approaches, appears in part excessive, arbitrary and not scientifically sound. although many scientists around the world are giving their best to solve and improve the pandemic, the current development also gives rise to the pressure to be the first to find the solution and not to be overtaken by colleagues. against this background, it is very important to know about previous research. therefore, we have analyzed the global research output on cov in the time before covid-19. in the present study, established bibliometric parameters on chronological and geographical aspects were combined with state-of-the-art visualization techniques based on density equalization principles. additionally, socio-economic, scientific and epidemiological parameters were related to the publication numbers to obtain an even more meaningful picture of the global landscape of cov research. the results may help to find more adequate approaches for future research in the financing, planning, implementation and networking of research based on quality and sustainability. the present study belongs to the established bibliometric platform new quality and quantity indices in science (newqis) [11] , which examines a broad range of biomedical questions with regard to their translational utility [11] [12] [13] . the underlying methodology is constantly evolving and adapted to changing circumstances in terms of global scientific, political and socio-economic characteristics [14] . standardized bibliometric parameters are combined with newly integrated indices and figures on the research topic in order to analyze and discuss the research landscape appropriately. in combination with state-of-the-art visualization, the results are provided convincingly. the default database for retrieving the needed metadata for all newqis studies is the core collection of web of science (wos). not only because of its status as one of the leading online databases for scientific literature, but also because of its listing requirements, which allow only quality work. moreover, the provision of the journal citation report (jcr) enables the analysis of citation-based parameters by specifying all citations received for each publication. viewpoints research theme 1: covid-19 pandemic the search was performed at 18/03/2020. to record the respective articles dealing with cov, the elaboration of an adequate search term is mandatory. the aim is to include as many related entries as possible and to exclude the false entries from the analysis database. therefore, the term must be a combination of all variants or synonyms of the names of the virus or its transmitted diseases. in order to find as many entries as possible, the resulting term was: "*corona virus" or "*coronavirus" or "sars" or "mers" or "covid-19" or "severe acute respiratory syndrome" or "middle east respiratory syndrome". wos offers different modes of search. we applied the title search mode here, because a lot of incorrect entries occur when searching with the topic search mode that also includes abstract and keywords. to further decimate unrelated entries, mostly due to different meanings of the abbreviations sars and mers, a specified topic search was added and combined with the boolean operator "and" searching for the terms: "virus" or "epidem*" or "cov" or "co-v" or "covid-19" or "patient*" or "*coronavirus" or "severe acute respiratory syndrome" or "middle east respiratory syndrome". this strategy ensures content linkage by querying the occurrence of one of these terms either in the abstract or the keywords of the publication. the resulting entries were subsequently filtered by the document type "articles" to include only original papers in this study. the metadata of the articles found in the manner described above were downloaded and recorded in a database that provides a variety of parameters according to the key information encoded by the wos tags. their analyses refer to chronological and geographical parameters, using data on the number of articles in relation to, eg, publication year or country of origin. other advanced parameters include socio-economic [15] [16] [17] and epidemiological data [3, 5] . for the epidemiological evaluation, sub-analyses were performed using the same search term, with the only difference that it was reduced to either sars or mers. furthermore, institutions and research foci were analyzed and the international network was presented. it has to be noticed that the sum of all assigned subject areas of wos must be higher than the number of articles due to multidisciplinary journals assigned to more than one subject area. in this study an article with more than one subject area assignment to assigned to each area and therefore counts several times. citation-based analyses provide information on the recognition of the articles in the scientific community. a threshold was applied to all valuation ratios to reduce distortions of extremely low values, eg, citation rate, and socio-economic ratios. in this way, a concise picture of the global landscape of cov publications and their development could be created. the geographical results were partially visualized with density equalizing map projections (demp) that are distorted maps according to an algorithm developed by gastner & newman [18] . depending on the value of the evaluation parameter, the countries were either enlarged or reduced in size according to the physical principle of density equalization. using the vosviewer application [19] , the results of the keyword analysis to determine research priorities are visualized with a network diagram showing clustered nodes and connections for all terms that occurred at least 150 times. every scientific methodology has some limitations that must be reported on. in this case, the first limitation to be mentioned is due to the characteristics of the data source, as wos does not list all publications in its core collection. wos requires special recognition for listed journals in order to ensure quality, but this led to the fact that some important articles on cov could not be included in the analysis as they are not provided by wos. the title search strategy applied in this study further reduces the database. the advantage, however, is that the included data represent a representative collection that can clearly be used for a valid evaluation. furthermore, the wos is said to give preference to english literature, so that the resulting dominance of english-speaking countries is supported by this fact. another point that should be mentioned is the manual correction method for metadata on institutions and authors. for this, a threshold had to be introduced to make the procedure feasible. as a result, the exact number of institutions and authors publishing on cov could not be given. however, the leading institutions and authors could be determined exactly in this way. viewpoints research theme 1: considering the limitations of citation analyses and their impact on meaningfulness in terms of the quality of publications, the use of several citation parameters is appropriate, which gives more weight to the importance of the analysis results for the resonance of the examined publications in the scientific community. in summary, the applied method has proven to be a valid strategy for the evaluation of bibliometric scientific questions. a total of 6905 articles (n) on cov could be added to the database and form the basis for all analyses except those valid for geographical analyses. analysis of the keywords used (threshold value: 150 occurrences) revealed four thematic clusters of cov articles (figure 1, panel a) . first, the molecular and biological topics form a cluster (red). the second cluster (blue) outlines the articles dealing with the sars epidemic, and the third cluster (yellow) combines the articles dealing with the mers epidemic. the fourth cluster (green) forms an intermediate group that mainly focuses on the spike protein that is characteristic of cov, its pathogenesis, and its connection to the other clusters. in terms of subject areas, the most frequently assigned research fields are virology (n = 2140), infectious diseases (n = 899), veterinary sciences (n = 720), microbiology (n = 622), and immunology (n = 558). looking at the developments over time, it can be seen that the research has become increasingly multidisciplinary ( figure 1 , panel b). especially since the beginning of the 2000s, a change can be observed. veterinary sciences and virology were relatively declining. the relative frequency of articles assigned to infectious diseases increased from this time onwards. other areas of cov research also became more popular. the first article about cov found in this study was published in 1970. from then on, articles were pubit is obvious that the articles from 2003 deal with the new cov and the related disease called sars, which appeared year. working groups from the usa, china, southeast asian countries, germany, france and the netherlands identified and characterized the new virus and its association with sars [20] [21] [22] [23] . canadian scientists worked on the genomic sequence [24] . viewpoints research theme 1: in 2012, the mers coronavirus appeared, leading to this year' s high-ranking publication of dutch and saudi arabian scientists [25] . the 2005 article, which ranks 10 th , stated that bats are natural reservoirs for sars-covs [7] . this was a collaboration between china, australia and the usa. all ten most frequently cited articles were published in renowned journals. the new england journal of medicine and science published four of them each. one each was published in the lancet and nature. the necessary information about the country of origin can be collected through affiliation data. before 1973 this information is not always available and the gaps are too large to be used. exactly n = 50 articles could not be assigned to any country of origin, so that n = 6855 articles are included in the geographical evaluations. from 1970 onwards, the usa was the country with the highest number of publications for the entire evaluation period (n = 2293), followed by china (n = 1707). by a larger margin, germany ranked third (n = 505), followed by canada (n = 488) and the united kingdom (uk) (n = 413) (figure 3 , the leading countries were also represented by the leading publishing institutions ( table 2 ). in terms of citation numbers (c), the landscape looks similar to that of article numbers (figure 4 , when looking at the world map when socio-economic parameters are included in the analysis, the distortion of country sizes shifts again. regarding the ratio of the number of articles and population size in million inhabitants (rpop) of countries with at least 30 articles on cov (threshold) the ranking another socio-economic parameter, the relation of the number of articles on cov to the gross domestic product (gdp) in 1000 billion us-dollars (rgdp) for countries with at least 30 articles (threshold), shows a similar ranking. the rankings of both socio-economic analyses were compromised in table 3 . the inclusion of parameters referring to the research infrastructure of the countries considers two values. first, the gross expenditure for research and development (gerd) in billion ppp$ (purchasing power parity in us-dollars) and the number of researchers in million fte (full time equivalents). again, the number of articles on cov for countries with at least 30 articles (threshold) was set in relation to these parameter (rgerd, rres). table 4 ). the socio-economic parameters gdp, gerd, and number of researchers were significantly correlated with the number of cov articles (p < 0.0001), with correlation coefficients (spearman r) ranging from 0.61 to 0.79. in order to show the publication performance of cov-affected countries in relation to the sars and mers epidemics, the relationship of articles on sars or mers to the respective cases per country was analyzed. according to who [3] 8096 sars cases occurred until the 2004 epidemic came to an abrupt halt. the sub-analysis of the present study resulted in 3039 sars-related articles corresponding to the affected countries. table 5 in total, the who reported 1511 cases of mers worldwide occurring with smaller boosts until today [5] . 1068 articles could be clearly assigned to mers. table 6 viewpoints research theme 1: of the total number of n = 6855 articles for the geographical analyses, n = 1716 articles were prepared in international cooperation. the maximum annual international partnerships for cov research took place in 2004 (n = 135, first publication peak) and in 2016 (n = 126, second publication peak) (figure 1 , panel a). in principle, however, an upward trend can be observed. the usa was at the center of the international network and was involved in the five strongest international collaborations (figure 7) . the most productive bilateral cooperation in cov research was between the usa and china with n = 290 cooperation articles, followed by usa/canada (n = 113), usa -uk (n = 77) and usa -netherlands (n = 75). the non-us partnership with the highest publication volume was between the netherlands and germany (n = 74). especially since the beginning of the covid-19 pandemic, which is causing many serious cases and deaths in many countries, the importance of research on cov has become clear. basic research to date, the identification of novel viruses of the two pandemics sars and mers, has influenced global research efforts. as more is learned about the background, incentives and impact of research, the impact of more focused and improved focus, planning, implementation, collaboration and communication of scientific projects becomes clearer, for all parties involved. this is also shown by the identified research foci, which were analyzed by the keywords used in the articles on cov. however, the proportion of human-related research has increased significantly since the outbreak of sars, and multi-disciplinarity has also become increasingly widespread over time. the currently rampant covid-19 disease and its impact on economic, political and social spheres will provide new impetus for cov research in other scientific fields in the future. the development of publication output on the cov clearly followed a different trend than that of other biomedical topics in general, which usually increase exponentially over the evaluation period [27] and could be shown in other studies on viral diseases [12, 13] . instead, the increase in the number of articles and citations corresponds to the occurrence of the two pandemics sars and mers in the past with peaks at the beginning of each. in 2003, the year in which sars emerged, publication numbers rose at an unprecedented rate in science, with an increase of about 500% within one year. chiu et al. also noted in a 2004 bibliometric study on sars publications that the high publication rate at the beginning of the pandemic resulted in high citation rates due to the immediate recognition in the scientific community [28] . now, 16 years later, the present study confirms this extremely high recognition by the scientific community at the time of the sars outbreak, which even shows a number of citations per publication year, even before the maximum peak in publication numbers is reached. in 2003, the highest citation rate was achieved with cr = 104.83, which means that each article on the cov was cited about 104 times on average. this is certainly an extraordinary result. almost at the same pace, however, the number of publications and citations decreases thereafter, resulting in a value that is below the average biomedical topic. this shows the decline of the scientific interest with the abrupt end of sars. the awareness of the importance of cov research as a preventive measure for future virus infections does not seem to have been present at this time. the progression curves of both publication and citation numbers showed a minimum level at exactly the time when the cited half-life (chl) of publications in biomedical publications is generally reached. basically, the chl is given as 7-8 years that a publication needs to reach half of the expected citations, resulting in a maximum peak of citations up to that point [29] . the outbreak of mers in 2012 caused a second increase, but this time it reached only about one third of the sars-related figures, due to the lower incidence rates compared to the sars pandemics. it also showed a declining trend, but only four years after the outbreak. the longer period during which mers cases occurred also led to a longer lasting interest, but at a significantly lower level. the timeframe of the evaluation was set until march 2020, so that the first months of covid-19 were included in the analysis. the expected exponentially increasing numbers could even be shown in this small time-interval by a further significant increase in the number of publications. the short-term effect of research efforts of earlier cov research and the strengthening of international networking could also be shown with regard to another emerging virus epidemic that occurred in south america in 2015: the zika virus infection. the publication patterns corresponded to those reported here and showed an unsustainable short-term effect of national and international efforts [30] . this similar publication pattern was reinforced by the enormous research incentives of short-term funding and public recognition due to the acute threat. this is also evident in the case of cov research, and the same influences must be considered in future approaches. the most frequently cited publications highlight the outstanding impact of the two pandemics and the years of their outbreaks. most of the ten articles dealing with the identification and characterization of the novel sars virus were published in 2003, which shows the importance of the first articles on sars in the scientific community. one article deals with the isolation of the mers virus, which was published in 2012, and another with the function of bats as natural reservoirs. these articles were from the countries that are considered to be the most publishing countries throughout the assessment period. viewpoints research theme 1: what is also true for the publication output of most life science and biomedical topics is the leading position of the usa in terms of the absolute number of publications. the rapid catch-up trend and the now high numbers of chinese scientists is also not unique for cov research. other bibliometric studies confirmed the ranking of the leading countries in terms of absolute publication figures. bonilla-aldana et al. found the usa publishing 34.9%, china 22.4%, and germany 6.8%. saudi arabia, as the country most affected by mers, contributed only 3.6% to cov publications [31] . in comparison, the percentages revealed in our study were similar: the usa (33.45%), china (24.90%), germany (7.37%), and saudi arabia (4.01%). in 2011, kostoff et al. found in another bibliometric study on sars the declining share of chinese and the increasing trend of us-american studies. they stated a higher percentage of highly cited publication authored by china compared to other research fields [32] . these findings have been confirmed by the present results showing also the percentage decrease of chinese articles. although, a relative downwards trend of us-american articles could also be shown. this is due not only to the decline in absolute figures but also to the efforts of an increasing number of countries worldwide. covid-19 will certainly continue to influence this trend enormously. although the study by chiu et al. showed a low level of international collaboration shortly after the outbreak of the sars pandemic in 2004 [28] , the pace at which international collaboration on cov was established thereafter was remarkable [32] and will certainly continue. as third most publishing country, germany has been involved in cov research from the very beginning in the 1970s, when only the usa and canada was interested in this topic. although germany had its highest share at the beginning of the 1980s, it has been able to maintain its position among the three leading countries to date. the most cited german article by drosten et al. from 2003 ranks second in the overall research on cov and identified a novel coronavirus in a patient with sars [21] . this was a german-french-dutch partnership. the following ranking consists of canada, the uk, taiwan, and the netherlands. all are involved in prominent research on cov and have sufficient scientific resources and infrastructure not only for research on cov. the netherlands -ranked 7 th in absolute numbers -took a leading position when it came to the results of citation-based, socio-economic and science-related parameters. it also participated in three of the most cited articles and thus also in the identification of sars-and mers-cov as a partner country of germany, the usa, and saudi arabia [21, 22, 25] . with a relatively high proportion of articles in the field of molecular biology, the dutch scientists contributed the main research on the structure of the spike glycoprotein, eg, they uncovered the three-dimensional structure [33] . nevertheless, other countries have led the way with respect to the inclusion of socio-economic and science-related. thailand received the highest citation rate, although with n = 38 it was only slightly above the required threshold of 30 articles. thailand' s participation in the most frequently quoted article is therefore responsible for this high value. the article by kziazek et al. [20] , which demonstrates the etiological role of a novel cov in sars, is a joint effort of the sars working group: usa, vietnam, china, thailand, singapore and taiwan. scientists from the center for disease control and prevention (cdc) worked in addition to them from the international emerging infectious disease program in bangkok (thailand) as partners in this study. this program was founded by the ministry of public health (thailand) and the cdc (usa) to establish a prevention and detection system that responds to emerging public health threats. vietnam was also a partner country in this cooperation, but with n = 29 it was just below the threshold and was therefore excluded from the analysis of citation rates. it should nevertheless be mentioned here. scientists from the vietnamese regional division of who were involved in the research. singapore, which was also involved in this successful cooperation via the singapore general hospital, could also be highlighted concerning the analysis of socio-economic and science-related parameters. it received the highest scores in terms of rpop, rgdp, rgerd, and rres. chahour et al. [34] conducted a bibliometric analysis of the first covid-19 publications, which confirmed the leading position of singapore in terms of its demographic characteristics. without applying a threshold, this study ranked mauritius first in terms of economic strength. we could not find an article from mauritius until march 2020, and the chahour study found one publication from mauritius. this shows how important threshold applied in our study is to avoid overestimating countries with such a low publication output. with 238 sars cases, singapore was one of the most affected countries. the resulting scientific interest and the possible in-si-viewpoints research theme 1: covid-19 pandemic tu investigation of the cases caused the publication figures to rise at the beginning of the sars disease and to fall rapidly thereafter. at the time of the covid 19 outbreak, however, the figures from singapore rose strongly again. tan tock seng hospital (ttsh) is at the heart of sars research in singapore, a place where 105 secondary cases mainly affected health workers [35] . the epidemiological impact and the incentive of in-situ research promoted research on both pandemics of the past and showed the clearly centered focus of the publishing countries. the exploding publication numbers in 2003 and the second peak starting after 2012 are clearly caused by the outbreaks of sars and mers. therefore, we defined both the number of sars-and mers-related articles and set them in relation to the number of disease cases of each country. in the case of sars, china had clearly the most cases worldwide and had also written the most articles about it. this was followed by the usa and taiwan. however, the ratio of sars articles from china and taiwan to cases was relatively low. in general, the countries with the most cases did not reach the top ranks, as case numbers varied widely among publishing countries, ranging from more than 7000 in china to only one case in eight countries. therefore, spain and switzerland reached the highest ratios. but with only one identified article, these high rates are not surprising, as both countries were among the 20 most publishing countries. the contribution of literature from both countries to the cov literature was relatively stable and has fluctuated only slightly since the early 2000s at an average level of about 10 articles per year. therefore, the publication efforts of both countries could not be directly linked to the outbreak of sars or mers. with regard to mers, most of the contributions came from the usa, which was also found in a 2016 study [36] . saudi arabia produced the second most articles on mers. as far as the relationship between mers publications and mers cases is concerned, the situation is similar to that of sars. saudi arabia also achieved a relatively low ratio with most mers cases. here, the usa and china are the highest-ranking countries, demonstrating their overall interest in cov research and also focusing on the mers pandemic, despite the relatively low case numbers. egypt also came into the focus of this analysis because of the occurrence of only one case and the associated high ratio. however, egypt' s interest is directly related to the outbreak of mers, as it has seen a significant annual increase in publication numbers from that point on. the occurrence of mers-cov in egyptian dromedary camels and its similarity to the human type influenced the regional research interest [37] . another causal factor is the strong cooperation partnership with saudi arabia and usa in cov research. in addition, egypt ranked 5 th in the inclusion of gerd as a marker of economic strength and was the only country that did not have a high-income status among the top 15 countries. the analysis by chiu et al. from 2004 [28] found no evidence of a correlation between the number of sars cases and the number of publications in the publishing countries [28] , while our results showed a significant correlation. the reason for this discrepancy seems to be the timing of evaluation. in contrast to chiu et al. who consider the beginning of publication activity on sars, the present study shows the results of development 16 years after the appearance of the pandemic. during this period, the affected nations seem to have created an awareness of the importance of sars research and the international interest in cov research that responds to it. however, with regard to mers, our study could not demonstrate a statistical correlation between the number of cases in countries and the number of publications. this could be due to the low number of mers-related cases and the resulting centered interest. taking into account the unusual patterns of previous cov research, the current situation of covid-19-related publication output must be evaluated and discussed accordingly. a short note on the most recent publication figures, which include the output of 2020, already shows 3930 publications on the cov that were found with the same search term (as of 19.05.2020). of these publications, only 1230 are articles (31.3%), which means that almost more than two-thirds of the publication includes other types of documents, especially editorial materials and early access, which are almost invisible in the previous period. as a rule, articles make up the largest share of document types. the most frequently assigned subject area in 2020 was general and internal medicine, which pushed back the fields of virology and infectious diseases as the most frequently assigned areas of previous research, viewpoints research theme 1: thus providing an indication of publication in more interdisciplinary journals. the field of public, environmental, and occupational medicine moved on to third place. these figures imply the enormous interest of the scientific community and the enormous willingness to publish. but it leaves one questioning the priority given to quality and shows the attitude of publishers to value the rapid publication of articles related to the cov. china has the largest share of articles in 2020, followed by the us and italy. this is due to the advance of chinese science and the high number of cases in both china and italy. the results of the present study show the need for sustainable, valid and high-quality research in global cooperation to address not only the current pandemic but also future threats. the increasing divergence of research areas allows for more interdisciplinary approaches, which should be completely free of any blinkered ambitions or dogmatic reservations about the big picture. what concrete effects the current covid-19 pandemic will have on the global landscape of cov research can currently only be estimated. funding is rising exorbitantly, and the first figures for 2020 show an equally exorbitant increase in publications. as a result, the development of vaccines and effective therapeutic methods can be expected in the near future. but the question arises whether this is a short-term effect which, once covid-19 is contained, will lead to an equally sharp decline in publication numbers as was observed with the earlier cov pandemics sars and mers? the need for continued interest and research efforts worldwide is characterized by the characteristics of the past, which lead to a lack of basic knowledge about cov, about advanced therapies and to difficulties in the search for vaccines. the present results show the development and incentives for research in the period before covid-19 and underpin the need for awareness and sustainable international relations for best possible strategies and the benefit of scientific projects. this is also important in view of the fact that the effects of the prevailing climate change will influence the incidence of zoonotic diseases caused by cov through human and animal accumulation. this will certainly lead to the need to combat new groups of viruses in the future. therefore, sustainable and climate-friendly approaches must also be pursued in science, especially if they focus on the newly emerging aspects of cov research in relation to political and economic issues. annual review of diseases prioritized under the research and develpment blueprint detail/30-01-2020-statement-on-the-second-meeting-of-the-international-health-regulations-(2005)-emergencycommittee-regarding-the-outbreak world health organization. summary of prabable sars cases with onset of illness form 1 isolation and characterization of viruses related to the sars coronavirus from animals in southern china middle east respiratory syndrome coronavirus (mers-cov) any researcher with access to the web of science database can obtain the data using the methods described in the paper. readers that do not have access to web of science should contact clarivate analytics to obtain a license draft: dk; writing -review & editing: dk, mb, db, dag; visualization: dk. conflict of interest: the authors completed the icmje unified competing interest form (available upon request from the corresponding author evidence for camel-to-human transmission of mers coronavirus bats are natural reservoirs of sars-like coronaviruses close relative of human middle east respiratory syndrome coronavirus in bat sars and other coronaviruses as causes of pneumonia research collaboration at nordic universities new quality and quantity indices in science (newqis): the study protocol of an international project influenza: a scientometric and density-equalizing analysis respiratory syncytial virus: a systematic scientometric analysis of the global publication output and the gender distribution of publishing authors new quality and quantity indices in science (newqis): results of the first decade-project progress review theworld bank. data, population, total gdp (current us$) diffusion-based method for producing density-equalizing maps software survey: vosviewer, a computer program for bibliometric mapping a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia world bank country and lending groups bibliometric analysis of severe acute respiratory syndrome-related research in the beginning stage journal citation reports: a potential tool for studying journals? 1. description of the jcr journal population based on the number of citations received, number of source items, impact factor, immediacy index and cited half-life the global scientific research response to the public health emergency of zika virus infection mers-cov and now the 2019-novel cov: have we investigated enough about coronaviruses? -a bibliometric analysis structure and infrastructure of infectious agent research literature: sars coronavirus spike protein and tropism changes a bibliometric analysis of covid-19 research activity: a call for increased output the outbreak of sars at tan tock seng hospital -relating epidemiology to control global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis mers coronaviruses in dromedary camels key: cord-324106-unvycvx4 authors: ki, hyun kyun; han, sang kuk; son, jun seong; park, sang o title: risk of transmission via medical employees and importance of routine infection-prevention policy in a nosocomial outbreak of middle east respiratory syndrome (mers): a descriptive analysis from a tertiary care hospital in south korea date: 2019-10-30 journal: bmc pulm med doi: 10.1186/s12890-019-0940-5 sha: doc_id: 324106 cord_uid: unvycvx4 background: in 2015, south korea experienced an outbreak of middle east respiratory syndrome (mers), and our hospital experienced a nosocomial mers infection. we performed a comprehensive analysis to identify the mers transmission route and the ability of our routine infection-prevention policy to control this outbreak. methods: this is a case–cohort study of retrospectively analysed data from medical charts, closed-circuit television, personal interviews and a national database. we analysed data of people at risk of mers transmission including 228 in the emergency department (ed) and 218 in general wards (gw). data of personnel location and movement, personal protection equipment and hand hygiene was recorded. transmission risk was determined as the extent of exposure to the index patient: 1) high risk: staying within 2 m; 2) intermediate risk: staying in the same room at same time; and 3) low risk: only staying in the same department without contact. results: the index patient was an old patient admitted to our hospital. 11 transmissions from the index patient were identified; 4 were infected in our hospital. personnel in the ed exhibited higher rates of compliance with routine infection-prevention methods as observed objectively: 93% wore a surgical mask and 95.6% washed their hands. only 1.8% of personnel were observed to wear a surgical mask in the gw. ed had a higher percentage of high-risk individuals compared with the gw (14.5% vs. 2.8%), but the attack rate was higher in the gw (16.7%; l/6) than in the ed (3%; 1/33). there were no transmissions in the intermediateand low-risk groups in the ed. otherwise 2 patients were infected in the gw among the low-risk group. mers were transmitted to them indirectly by staff who cared for the index patient. conclusions: our study provide compelling evidence that routine infection-prevention policies can greatly reduce nosocomial transmission of mers. conventional isolation is established mainly from contact tracing of patients during a mers outbreak. but it should be extended to all people treated by any medical employee who has contact with mers patients. trial registration: nct02605109, date of registration: 11th november 2015. trial registration: nct02605109, date of registration: 11th november 2015. keywords: middle east respiratory syndrome coronavirus, nosocomial infection, infection control, isolation, hand hygiene background middle east respiratory syndrome (mers) is a respiratory disease caused by a novel single-stranded beta-coronavirus, which was first reported in patients residing in saudi arabia in 2012 [1] . the primary transmission was thought to be from zoonotic exposure in this area [2] . however, some clusters of outbreaks from secondary transmission in the health care setting have been reported, and human-tohuman transmission is considered to play an important role in secondary transmission [3] [4] [5] . until may 2015, outbreaks of mers were mainly restricted to countries in the middle east including saudi arabia, jordan, kuwait, lebanon and qatar [6] . regions outside the middle east, such as europe, usa and some asian countries have not generally experienced noticeable outbreaks of this virus [7] [8] [9] [10] . in 2015, south korea experienced the largest outbreak of mers outside the middle east. a total of 186 patients were serologically confirmed as having mers between may and july 2015 in korea [11] . most of these mers infections were identified as having arisen from human-to-human transmission in the health care setting [12] . given that the emergency department (ed) plays an important role in providing the main care for acutely ill patients, the ed can be an open portal for transmission of pathogens into a hospital system. unrecognized patient who visited in the ed greatly contributed to wide spreading of mers for less time [13] . the first human-to-human transmission of mers in south korea occurred in an old korean business man who travelled to the middle east area in may 2016 ( fig. 1) [12] . he visited a local clinic in pyung-taek because of a high fever and coughing. to provide care for pneumonia, he was admitted to the gw in a local hospital in pyung-taek in gyeonggi province. during the admission, 28 people who had been admitted to this gw in this hospital were infected (1st super-spreading event of the korean outbreak), and some of these people moved to other tertiary hospitals by themselves. among them, a young male patient visited the ed of hospital a in seoul on 27 may (fig. 1) . he complained of severe fever, coughing and sputum and treated during two days. mers was confirmed on 29 may, and a total of 90 transmissions were identified in hospital a (2nd super-spreading event) [14] . an unrecognized patient who was infected in hospital a visited in hospital b and c, and numerous people were exposed directly or indirectly to this patient in the hospital setting. after outbreak, we performed a post-outbreak analysis with detailed observational data from our hospital (hospital c). our report may be helpful for further understanding of mers transmission and the role of routine infectionprevention policies in reducing nosocomial outbreaks of mers in health-care settings. this was a case-cohort study of retrospectively analysed experiences of an in-hospital mers outbreak in hospital c in south korea. the study protocol was reviewed and approved by the institutional review board of hospital c. according to the study protocol, anonymous clinical or public data was used without the need for consent in the interest of public health. we obtained verbal consent from participants to obtain personal information from a closed-loop interview or review of recorded closed-loop video data in the hospital. other public data about mers in korea were obtained from the open data available from an online mers communicating system or the korean health care database. our hospital (hospital c) is an 870-bed university hospital located in the north-east of seoul, south korea. annually, > 50,000 patients visit our ed. the ed is located on the first floor of the main hospital building and has 5 treatment zones: 1 zone containing 4 beds for intensive care, 2 large rooms each containing 10 beds, and one paediatric care room containing 10 beds. for fast radiological evaluation of ed patients, a radiology suite and a computed tomography suite are located adjacent to the ed. the hospital has 7 floors for in-patient care, each of which contains 2 general wards (gws) for admitted patients. each gw comprises 10 rooms with 6 beds each and 7 rooms with 1 or 2 beds each. hospital prevention policy and measures during the preand post-isolation periods before the mers index patient visited our hospital, we had strengthened the triage system for identifying suspected mers patients. we set up the mers triage room outside of the ed. for patients complaining of any symptoms of mers, including fever, cough, sore throat, rhinorrhoea, shortness of breath, nausea, vomiting or diarrhoea, triage staffs routinely checked the histories of exposure to confirmed mers cases. triage staffs also checked for recent travel within the middle east area or visits to hospitals or local clinics where mers cases had been diagnosed during the last two weeks. all suspected mers patients were not permitted to remain in the ed, they were moved directly to the temporary isolation room outside of the ed. given the increasing risk of unrecognized infective patients visiting our hospital, we encouraged all healthcare providers (hcps) to wear surgical mask routinely and gloves during procedures and to use proper hand hygiene in the ed and outpatient department. our hospital also provides a surgical mask routinely to all patients and visitors who stay in the ed, and we encourage frequent hand washing by placing numerous hand sanitizers at all ed stations and beds. after identification of the index patient, we categorized the transmission risk of all possible people (primary interest group) who were suspected of having had contact with the index patient or who had been treated in the same area in the ed or gw during the index patient's stay. all people in the primary interest group were isolated for 14 days according to the national infection control guidelines of the korean ministry of health if they had been in contact with the patient or objects touched by the patient (e.g. bed, blanket or other items) or had stayed within 2 m of the index patient [15] . other people who had not been quarantined were categorized as the active surveillance group and were monitored daily by our hospital or a community health centre. during the 2-3 weeks of close observation, respiratory samples from sputum, naso-or oropharyngeal swabs were acquired for anyone in the isolated group or active surveillance group who displayed fever or any respiratory symptoms. the samples were sent to korean national laboratory centres. mers was confirmed in respiratory samples showing a positive result in a real-time reverse transcriptase polymerase chain reaction assay. if the initial test was negative, an additional assay was conducted using another respiratory sample [15] . post hoc analysis of the transmission tree, personal protection equipment (ppe) and hand hygiene post hoc analysis was performed after the end of the mers outbreak in our hospital. the analysis focused mainly on data for the index patient and all people treated in the ed and gw during the pre-isolation period of the index patient. first, by reviewing the closed-circuit television (cctv) and electronic medical charts, we traced the location and movements of the index patient and drew possible lines and zones of transmission risk. second, we checked the direct transmission trees and contact events. for further analysis, we categorized all persons of interest into three groups according to their location and contact with the index patient as follows [14] . 1) the high-risk group was defined as patients who had been within about 2 m of the index patient. 2) the intermediate-risk group was defined as patients who had stayed in the same room as, but who had had no direct contact with, the index patient. 3) the low-risk group was defined as patients who had been treated on the same floor or department area but who had not shared a room with the index patient. we also collected data about exposure time, duration of contact and suspected route of contact. we also checked ppe data including wearing of masks, gloves, preventive gowns or eye protection and hand hygiene (hand washing) for each person. all data were collected by personal interview and review of hospital cctv, electronic medical records, epidemiological reports from the local community health centre and data reported by the korean centres for disease control and prevention. for statistical analysis, we used ibm spss statistical software package 23.0 (ibm corp., armonk, nj). variables were compared between two groups using an independent two-sample t test or fisher's exact test. two-sided p-values < 0.05 were considered to be significant. the index patient in our hospital was an old patient who was transported to our ed on 6 june for surgery for a fractured femur. this patient had received treatment for malignancy disease in the ed of hospital a between 27 and 28 may. the 2nd super-spreading event of mers occurred in hospital a during the same time ( fig. 1) . after discharge from the ed of hospital a, the index patient was transported to a local primary care facility and was admitted to the long-term care room for supportive care for 10 days (28 may to 5 june). this patient fell during this stay and was diagnosed with a left intertrochanteric fracture on 5 june. the index patient was transferred to the ed of a nearby hospital b for surgery on the inter-trochanteric fracture. the next day (6 may), this patient was transported to our ed at 9:00 am using a personal medical ambulance service at the request of the patient's family (fig. 2) . on arrival at the ed, the patient had a slightly elevated body temperature of 37.5°c, blood pressure of 136/56 mmhg, pulse rate of 76 beats/ min and respiratory rate of ≈16 breaths/min. this patient did not exhibit fever, chilling, dyspnoea, coughing or productive sputum. this patient and caregivers denied any possibility of contact with mers patients. routine examination with chest x-ray in the ed showed suspicious pneumonic consolidation in the left lower lobe. for the elective operation to repair the femur fracture, the patient was admitted to the orthopaedic gw in our hospital at 5:00 pm. the index patient was first admitted to a two-bed hospital room but was then transferred to a single-bed hospital room after five minutes because the caregiver of the other patient staying in this room did not want to share the room with the index patient ( fig. 3) . high fever (39.1°c) was measured in the initial vital sign check at 5:30 pm, and our hospital infection control unit was notified about the patient. the patient was evaluated further and her history taken again. this second evaluation revealed that the index patient had stayed in the ed of hospital a, and this was confirmed by phoning hospital a. the patient was then transferred to a negative-pressure isolation room according to the infection control guideline in our hospital. her respiratory samples were sent to the korean national laboratory centre, which confirmed a positive test result for mers early the next day. after confirmation of mers infection in the index patient, we could not identify those who had never had contact with the index patient or the virus-contaminated environment in the ed because of the frequencies of their movements within and around the ed and crowding. therefore, all 228 people who had stayed in the ed were included as isolation subjects regardless of their time or position when in the ed. a total of 60 people who were suspected of contact with patients (high and intermediate risk group) were isolated at the hospital and 168 (low risk group) were isolated at their home. otherwise, only 8 people (high and intermediate risk group) in the gw who had been in contact or shared a room with the index patient were isolated, because the index patient had stayed in the gw for only 1 h and her movements were limited. other 210 people were not isolated because they had not been in contact with the index patient or virus-contaminated environment. because of concern about indirect transmission by contact through virus shedding, we immediately performed environmental cleaning using bleach and alcohol of all possible areas in which the index patient had stayed or moved. along with the isolations of people and cleaning the environment after identification of the index patient, our infection control team added some new control measures. first, we campaigned for inpatients, hospital employees, and visitors to implement basic infection control including hand washing and wearing surgical masks. second, because some missed cases can transmit mers to other people before isolation is initiated, we performed routine daily checks of body temperature and monitored the onset of new symptoms for all inpatients and hospital employees. table 1 summarizes the data for the 11 mers patients who were infected by the index patient. before the index patient visited our hospital, five (first to fifth patients in table 1 ) were identified as having exhibited mers in the ed of hospital b. two ambulance paramedics (sixth and seventh patients) who had transported the index patient from hospital b to our hospital were also infected with mers (table 1 and fig. 2 ). they had briefly touched the index patient: one had briefly touched her to help her during the ride in the ambulance, and the other had only observed and cared for the patient during the 30 min of transport. neither wore a surgical mask during transportation or when they entered our ed. their recall of hand hygiene was unclear when questioned as part of this study. during the index patient's 8 h stay in the ed, mers transmission within the ed space was not reported. among the 228 isolated people, only 1 radiologist (eighth patient) who performed the x-ray of the index patient while wearing a radiology suit was diagnosed with mers ( fig. 3) . he had briefly touched the index patient (for < 5 min) while taking some simple x-ray. three people at gw were infected with mers during the index patient's 1-h stay in the gw. the first was a visitor (ninth patient) who stood briefly (1-2 min) beside the bed of the index patient. none of the other 7 of the isolated people who had been in the gw at this time were infected. the other 2 mers patients (tenth and eleventh patients) were identified among a group of people who were not initially isolated. one (tenth patient) had been admitted in the same ward area and the other (eleventh patient) had been admitted in another ward on the same floor (fig. 3) . there was no contact or overlap of space between the two patients and the index patient. we checked the movements of these two patients and found no possible indirect transmission via contact with the virus-shedding environment before we cleaned the gw area. both patients were admitted to the orthopaedic department, and they and the index patient had only one thing in common: they were cared for by the same nurse (tenth patient) or same resident and intern (tenth and eleventh patient). further retrospective analysis verified the routine nurse's round for all patients in the same ward station and the routine rounds of the doctors who cared for orthopaedic patients on the same floor on the same evening. before tenth and eleventh patients were isolated, they had walked freely around all areas of the hospital; therefore, numerous instances of contacts or possible viral shedding into the environment were suspected. to protect against further transmissions by infected patients, we closed the hospital over 1 month. during this period, a total of 1019 of people were isolated; (hospital employees were 267) 69 were isolated at the hospital, 950 were isolated at home; 220 (hospital employees were 65) were under active daily surveillance for 3 weeks. however, there was no subsequent mers transmission in these people (fig. 2) . table 2 shows a comparison of variables between the ed and gw groups. the ed group had a higher percentage of high-risk (14.5%) and intermediate-risk (11.8%) people than the gw group (2.8 and 0.9% respectively). people in the ed group had had more frequent direct contact with the index patient. otherwise, in the gw only 8 people (1 patient, 1 visitor and 6 hcps) were categorized into the high-risk group and 96.3% of people were classed as low risk. the overall attack rate was 0.4% in the ed and 1.4% in the gw. for the high-risk group, the attack was higher in the gw (16.7%; l/6) than in the ed (3%; 1/33). there was no transmission in the intermediate-and low-risk groups in the ed. two instances of transmission were identified among the low-risk group in the gw. analysis of ppe usage showed a higher rate of surgical mask wearing in the primary interest group in the ed than in the gw (93.0% vs. 1.8%). in the interviews, near all people recalled that they washed their hands during a stay in the ed or gw stay, but the time and place were unclear. some did not remember exactly and some were dropped from the closed-loop interview process, and we could not obtain correct data for hand washing in the study population. however, by reviewing cctv, we identified obvious hand-rubbing using portable hand sanitizer (hydroalcoholic antiseptic gel for skin and hands) for 218 of the 228 (95.6%) people during their time in the ed stay. wearing gloves was confirmed for all hcps who had been in contact with the index patient during their caring process including changing bedding or bag, blood or urine sampling, physical examination and ecg checking. during ed and gw stays, the index patient had no complaints of respiratory symptoms and there were no aerosol-generating procedures (lung aspiration, suctioning, intubation or bronchoscopy) before the initiation of isolation. after isolation began, two doctors and nine nurses cared for index patient routinely wearing level d equipment (n95 mask, head cover, goggles, surgical glove, disposable coverall and waterproof boots) during all aerosolizing procedures. this outbreak of mers in south korea had entirely nosocomial, human-to-human and hospital-to-hospital transmission patterns. the outbreak was initiated by a failure to identify an imported emerging infectious disease and was then amplified through the unique health care system in south korea [13] . south korea has a nationwide health insurance system, which controls overall medical costs en bloc and provides a flexible medical service-delivery system without barriers between small and large hospitals [11, 13] . all koreans can be treated at a low cost and can easily visit an ed in a tertiary hospital without a referral. to reduce costs, hospital rooms generally have multiple beds per room, as did the ed in our tertiary hospital. this system provides convenient medical service to patients at low cost, but it can be vulnerable to transmission of infectious diseases between hospitals. early in the mers outbreak in south korea, some infected patients visited the ed of a tertiary hospital, but this hospital failed to recognize that the patient had mers because of delayed information for the transmission tree from the national health care ministry and rapid movement of the patient to another hospital before the recognition of mers infection at the previous hospital [16] . mers patients may seek a medical facility early because of serious illness, but they may expose others if the infection is not identified quickly. in the middle east, unrecognized cases are frequently reported and are related to a sudden peak of nosocomial infection [3, 4, 17] . in south korea, lack of awareness about this emerging infection and experience with serious outbreaks of infection may have contributed to the low compliance of hcps for maintaining ppe and hand hygiene in the hospital [11, 13] . therefore, the unrecognized mers patients were easily able to transmit the infection to others during their stay in hospital. in the south korea outbreak, a total of 186 confirmed cases were reported, 153 (83.2%) of which had derived from 5 super-spreading events [16] . in these cases, transmission occurred mainly before these patients were isolated. two of the super-spreading mers middle east respiratory syndrome, cctv the closed-circuit television a all recalled that they washed their hands, but the time and place were unclear events were responsible for many cases of mers transmission in the ed space in hospital a (2nd super-spreading), hospital b and our hospital (index patient in this study). all 3 hospitals were located in seoul with a population of ≈10 million (fig. 1) . the long hospital stays and crowding in these eds contributed strongly to the higher risk of coming into contact with infected patients or exposure to infected droplets. a study of the outbreak in the ed of hospital a initiated from the 2nd super-spreading event revealed a high potential risk of multiple transmissions of mers from an unrecognized patient in a crowded ed [14] . this study showed that tracing of the contact with this patient was important for predicting the attack rate of nosocomial mers infection. because of the close exposure of other patients, there was a high risk of contact or exposure to droplets around this patient, which may have contributed to the high attack rate. in that study, people who stayed in the same zone in the ed (close contact area) had a 20% attack rate (23 of 117). by contrast, the attack rates were only 5% for others who stayed in different zones (3 of 58) despite a time overlap with the mers patient in the ed before that patient was isolated and 1% (4 of 500) in those with no time overlap. this study emphasizes the importance of an isolation and surveillance strategy based on contact tracing. the epidemiological results from our hospital ed contrast with the transmission results from hospital a. in contrast to the high transmission rate from the 2nd super-spreading event in the ed of hospital a, in our hospital, only 1 person, who worked in the radiology suit at ed, was confirmed with mers. although the index patient had a mild fever (37.5°c) and lack of respiratory symptoms, we assumed that the index patient had a sufficient risk of transmission of mers to others when this patient visited our ed. five infected cases were identified at another hospital, and 2 ambulance workers were confirmed as having acquired mers during transportation of the index patient before this patient visited our ed. in our post hoc comparison between the ed and gw, the ed group had a higher overall risk of mers than the gw group. the overlap time with the index patient was much longer in the ed than in the gw (8 h vs. 1 h), and a higher percentage (23.1%) were classified in the highrisk group in the ed group (within 2 m of the index patient). however, the overall occurrence rate was only 0.6% in the ed group and there was no transmission among the 60 people (42.0%) who stayed in the same area of the ed, including the high-risk area within 2 m of the index patient. otherwise gw showed relative higher transmission rate than ed. one of 8 people (16.7%) who stayed in the same room as the index patient in the gw was identified as having mers. surprisingly, two transmissions occurred in the low-risk group (staying on the same floor but no contact), which had been thought unlikely to be infected. these epidemiological features seemed to contradict previous knowledge of mers transmission. our investigation has unique characteristics. the index patient could not walk by herself because of the femur fracture, which allowed us to obtain clear information about patient's movement and contacts during the stay. early acquisition of all available videos and assessment of closed-looped interviews can provide useful information for the investigation of outbreaks in hospitals. using these, we were able to obtain comprehensive information to investigate the direct and indirect transmission risks, including possible virus shedding into the environment. we also obtained objective data by observation of ppe and hand hygiene of hospital staff at the same time. based on these data, we propose three issues for consideration when developing a mers transmission and prevention policy. first, a useful assumption from our analysis is that human-to-human transmission can occur even with a very brief exposure time. generally, a shorter duration of contact time may reduce the risk of transmission because of the lower chance of contact or exposure to droplets [6, 7] . however, more cautious measures should be considered because the duration of exposure to our index patient was very brief. in our case review, all four mers patients who were in contact with or within 2 m of the index patients had a very brief contact time (all < 5 min). the short duration did not translate to a lower risk of mers transmission in this case. this suggests that mers transmission can occur by brief humanto-human contact more easily than has been thought. second, we identified a possible pattern of humanto-human mers transmission via the hands of medical employees who were in contact with an infected patient. in our post-hoc analysis of the tracing process, we missed two case of mers infected form the index patient (tenth and eleventh patient). although these two patients were admitted on the same floor, there was no exposure to the index patient. considering the long incubation period of over 14 days, both patients may have been infected from a contaminated environment on this floor after mers patient was isolated. however, this is seldom a possible route of action because of the following facts. when we identified index patient, we cleaned all environments of hospital. the following day, we began checking daily for presence of the mers virus for several days and confirmed no existence of the mers virus. the transmission routes are not easily identified under current knowledge about contact or droplet transmission or indirect transmission via a contaminated environment. an attending doctor or nurse came into contact with the index patient during the physical examination or patient care and examined other admitted patients, including touching those patients, on the same day. the possible explainable route was transmission through the hands of medical employees who had touched the index patient's body or the virus-shedding environment. unfortunately, we could not perform a serological evaluation of samples from the hands of the hcps and we could not confirm this transmission route. however, we conclude that transmission via medical employees' hands or body was the most likely explanation for mers transmission in these patients. conventionally, isolation of people in response to a mers outbreak is mainly initiated by identifying the epidemiological contact history [18] . however, considering the possibility of indirect transmission via virus shedding into the environment [19, 20] , some researchers have suggested that people who share space and time with a patient should also be included in the quarantine group despite the lack of direct contact. during the early phase of the mers outbreak in south korea, quarantine was mainly confined to people who had come into close contact (within 2 m) with an infected person, but this has limited effects in protecting against mers outbreaks in some hospitals [11, 12, 16] . during an outbreak, most hospitals try to isolate all people who have shared space and time with mers patients based on this belief. however, this strategy does not include the possibility of indirect transmission via medical employees' body or hands, which may mean that some infected people are excluded from the initial isolation process because there was no discernible contact with patients or spatial or time overlap. in contrast to the community setting, more frequent contact may occur between hcps, patients and others in the hospital setting. nosocomial infection may involve the transmission of a pathogen via direct contact between medical staff and other patients. we propose that during a nosocomial mers outbreak the isolation should be extended to people who were treated by any medical employee who contacted mers patients. in our case, we initially missed the tenth and eleventh patients during the first isolation phase because they did not made contact with the index patient and had not shared time and space with the index patient. this had a disastrous outcome. they went around everywhere in the hospital before they were isolated. as a result, numerous people were exposed to the tenth and eleventh mers infected patients or shared space and time with mers patients. although we thought that other people may seldom become infected because of the early isolation of infected patients, we should have performed extensive quarantine procedures for all people in the hospital because of the national fear of mers transmission and the urgency of abating mers transmission. fortunately, among the 1019 people, there were no transmission cases. we believed that following concrete infection control measures may greatly contribute to preventing further transmission of mers. daily monitoring can detect these patients early and minimize the risk of mers transmission. in-hospital campaigns of basic infection control for all people may be another contributing factor that prevented nosocomial infection of mers. however, implementing unnecessarily broad isolations of hospital employees could cause a lack of adequate manpower and increased fatigue among non-isolated medical employees. during the national mers outbreak, most hospitals suffering from nosocomial mers transmission experienced the same, which may have resulted in the crisis of a lack of medical services in some regions. unrecognized mers patient often caused super-spreading events, especially in large volume hospitals, which can threaten not only a hospital system but also the regional medical service system. our study confirmed the importance of a routine basic infection policy for blocking widespread propagation of nosocomial mers infection. our post hoc analysis identified cases involving close contact over the long duration (8 h) of the index patient in the ed. considering the previous report of an unrecognized case in ed [14, 17] , our infection control team predicted widespread nosocomial infection at our ed. however, the outcome turned out to be quite different from what we had anticipated. during the analysis, all researchers agreed that our routine basic infectionprevention policy in the ed may have greatly contributed to the lack of mers transmission in the ed space. being aware of the potential for unrecognized mers patients to enter the ed, we strengthened the initial triage in the ed to select patients at risk of mers and encouraged all involved in the ed, including hcps, patients and visitors, to routinely wear a surgical mask and to wash their hands frequently. although we failed to isolate the index patient at the initial triage, high compliance with routine surgical mask wearing and hand hygiene was observed in nearly all people in the ed. in particular, we placed many hand sanitizers near beds and stations in the ed to allow all hcps and visitors to wash their hands easily at any time. the cctv review showed that > 95% of people in the ed washed their hands using the portable hand sanitizers. the high compliance rate of surgical mask wearing may have contributed to prevention of direct transmission of droplets into the respiratory tract and from hand to mouth or nose [21] . as overseas travel increases, the spread of emerging infections such as mers by returning travellers is a major concern for many countries. although the risk of encountering a patient with an emerging infection in the ed is slight, lack of recognition of emerging infections may cause serious problems for regional and national health care systems. transmission may be extended to include hospital-to-hospital transmission, and a mers outbreak can pose a risk to the national health care system. many studies have reported on the heterogenetic epidemic results of nosocomial mers infection. some hospitals have experienced serious nosocomial outbreaks [3, 4, 14, 17] , but others [7] [8] [9] [10] 22] , mainly outside the middle east, have reported a lack of mers in hospital systems. we postulated that this phenomenon may result from complex interactions between the strong transmission via human-to-human contact and infection-prevention measures in each medical institution. our analysis shows that mers can be easily transmitted by diverse routes in the hospital setting. routine infectionprevention practices, such as wearing a surgical mask and hand hygiene, can reduce the risk of nosocomial infection. routine infection-prevention policies should be established in all medical institutions during a mers outbreak. routine precautions may play an essential role in early protection against transmission. to provide wider protection against infection, specific isolation strategies should be considered in the case of a nosocomial outbreak of mers. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome novel corona mers-cov infection. epidemiology and outcome update hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah--a link to health care facilities hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome lack of transmission among close contacts of patient with case of middle east respiratory syndrome imported into the united states laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response lack of transmission among healthcare workers in contact with a case of middle east respiratory syndrome coronavirus infection in thailand clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome infection control and prevention guideline for healthcare facilities middle east respiratory syndrome coronavirus outbreak in the republic of korea transmission of middle east respiratory syndrome coronavirus infections in healthcare settings control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination viral shedding and environmental cleaning in middle east respiratory syndrome coronavirus infection a cluster-randomised controlled trial to test the efficacy of facemasks in preventing respiratory viral infection among hajj pilgrims health care worker contact with mers patient, saudi arabia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank for the assistance of staffs at konkuk university hospital and the community health centre in republic of korea.authors' contributions sp, hk established the main conception and designed this study. sp, sh and js took part in collecting data. all authors interpreted and analysed the data. jk and sp drafted the manuscript. all authors reviewed and edited the manuscript and approved the manuscript. sp is a guarantor, and takes responsibility for this study as a whole. the authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. the datasets generated and analyzed during the current study are not publicly available due to informations that could potentially identify individuals, but anonymous datasets are available from the corresponding author on reasonable request. the study protocol was reviewed and approved by our institutional review board, approval number kuh1260028. according to the study protocol, anonymous clinical or public data was used without the need for consent in the interest of public health. not applicable. the authors declare that they have no competing interests. key: cord-321651-7e8dwcur authors: jazieh, abdul-rahman; al hadab, abdulrahman; al olayan, ashwaq; alhejazi, ayman; al safi, faisal; al qarni, abullah; farooqui, faisal; al mutairi, nashmia; alenazi, thamer h. title: managing oncology services during a major coronavirus outbreak: lessons from the saudi arabia experience date: 2020-03-27 journal: jco glob oncol doi: 10.1200/go.20.00063 sha: doc_id: 321651 cord_uid: 7e8dwcur outbreaks of infectious etiology, particularly those caused by a novel virus that has no known treatment or vaccine, may result in the interruption of medical care provided to patients with cancer and put them at risk for undertreatment in addition to the risk of being exposed to infection, a life-threatening event among patients with cancer. this article describes the approach used to manage patients with cancer during a large-scale middle east respiratory syndrome–coronavirus hospital outbreak in saudi arabia to ensure continuity of care and minimize harm from treatment interruption or acquiring infection. the approach taken toward managing this high-risk situation (covid-19) could be easily adopted by health care organizations and would be helpful to ensure readiness for the occurrence of future outbreaks of different infectious etiologies like those recent episodes of new coronavirus. the recurrent outbreaks of coronaviruses in different parts of the world pose a major challenge to health care systems and expose patients and staff to serious risk. these outbreaks may lead to an interruption in health care services and the provision of care for patients, posing additional risks to them. these outbreaks pose a greater threat to patients with a chronic disease in terms of morbidity and mortality compared with healthy individuals due to the increased vulnerability of the patient secondary to their disease. 1, 2 patients with cancer are immunocompromised and therefore more vulnerable to infection, which can often end in fatality. the vulnerability of patients with cancer was evident by the high risk of mortality during the previous middle east respiratory syndrome-coronavirus (mers-cov) outbreaks (see the article by jazieh et al 3 in this issue). besides the risk of infection for exposed patients, additional major risks occur because of the interruption of the health care services provided at these facilities when they are subsequently shut down. cancer is a life-threatening disease that should be treated in a timely fashion to avoid a detrimental outcome to patients and minimize their physical and emotional suffering. the provision of cancer care is complex and requires multiple teams of professionals with specialized expertise and access to sophisticated, expensive resources. the complexity of cancer care already presents major challenges to the health care systems in affluent countries; national reports from both the united states and united kingdom identified the crisis situation already faced in the provision of this care. [4] [5] [6] this is the situation internationally in normal daily practice without external complicating factors, such as war or infectious outbreaks. one can imagine the challenges that arise in developing countries while facing such external crises, which present an additional burden to inherently complex care. 7, 8 mers-cov was the cause of multiple outbreaks in saudi arabia, which spread internationally. [9] [10] [11] the outbreak of mers-cov in june 2015 led to the closure of our hospital, as well as other hospitals within the city of riyadh. 12 this crisis posed a serious challenge to our staff and patients. the oncology department, in particular, faced unique challenges because of many factors, including infected oncology patients, a large susceptible patient population, interruption of services provided to our patients, and additional risks to our staff. with recent outbreaks of the new coronavirus in china and other countries, the factors related to oncology patients' care and corresponding outcomes are a major concern for the oncology community; therefore, this article describes the approach used to manage oncology services in response to the mers-cov outbreak and the implications of hospital closure. the real-life experiences and subsequent recommendations may provide some guidance and a supportive framework for oncologists to use in affected areas. in coordination with the organizational leadership, the oncology service leaders developed a plan to manage the crisis with 3 main objectives: to treat affected patients, prevent further infection to patients and staff, and deliver timely, safe cancer care for all patients. the crisis management plan included 5 main components: leadership and communication, patient management, staff management, infection control, and recovery plan (table 1) . this plan presents a comprehensive approach to control the harm that resulted from the outbreak, whether from infection or interruption of treatment. leadership and communication plan. to be effective, leadership must be engaged and visible, and timely communication is imperative for any organization's continuing functionality. these 2 issues are even more relevant during a crisis, making them paramount for crisis management. a leadership committee was formed to manage the oncology services during the crisis; this included the chairman, deputy chairman, section heads, operations administrator, nursing manager, and quality improvement specialist. the main objectives of this committee were to communicate to the oncology department staff the information received from the daily hospital command center meetings, discuss the current status of the outbreak in the whole hospital, and assess the risk for oncology patients. the team discussed the current admitted patients under the oncology service and their management plan, and proposed a contingency plan for their management, if needed. the group met as a whole or as subgroup at least twice a week and discussed any updates related to the outbreak, any changes in patient care, challenges, and the communication plan. the leadership frequently corresponded by using social media tools, such as whatsapp and organizational e-mails. the communication with the remaining departmental staff was performed in person through meetings with the section heads or through departmental e-mails to all staff regarding situational updates and organizational expectations. staff were encouraged to review the mers-cov page within the organization web site and to be aware of the command center correspondence by e-mail disseminated on a daily basis initially to all staff. of note, these communications were bidirectional to ensure better awareness of all aspects of the crisis in a timely fashion (fig 1) . patient management. the patient management plan had 3 main aims: to prevent new infection in the oncology service, to manage currently infected patients, and to provide timely cancer care for the whole patient population. specific plans were developed based on the setting, outpatient services, and inpatient services. a. early in the outbreak, all the appointments were canceled for 4 days to obtain a better understanding of the situation and to develop an appropriate action plan. b. medical records of all scheduled patients were reviewed by the primary oncologist, and patients were divided into 3 categories. • urgent (need to be seen on time): patients who were scheduled for chemotherapy that could not be postponed or were due for disease assessment; we kept the scheduled appointments for these patients. • intermediate: patients who could be rescheduled after a short delay (up to 2-4 weeks); the appointments for these patients were postponed. with recurrent infection outbreaks from different pathogens, patients with cancer are at high risk for harm because of the susceptibility to infections and the interruption of care, in addition to the associated risk of underlying cancer and its treatment. in response to the 2015 coronavirus outbreak in our country, we developed a detailed plan to help manage oncology services to prevent harm to our patients or staff. the plan focused on managing infected patients, preventing any new infections in patients or staff, ensuring continuity of cancer care, and incorporating measures to sustain these interventions far into the postoutbreak period. the described plan may serve as a platform to manage oncology services during similar crises that pose serious risks for patients and staff. • routine follow-up: appointments were rescheduled as far in the future as deemed clinically safe for the patient without any foreseeable negative impact. c. all patients were screened before entering the clinic with a checklist that included clinical and epidemiologic criteria; anyone with a suspected infection was referred to a triage area segregated from the general population. d. the infusion unit working hours were extended from 8:00 am to 8:00 pm to accommodate any delayed patients. medical staff coverage was secured onsite, and the working hours of the satellite pharmacy were extended. e. walk-in patients: patients who showed up to the clinic without an appointment were screened for infection and were either directed to the triage area for suspected infection or seen by physicians covering the clinic, according to the result of the screening checklist. f. oncology physicians managed patients who could be seen in the outpatient setting. patients who required admission were sent to an alternative hospital. patients were transported via hospital ambulance if their condition was unstable; patients in stable condition were transferred by private car. all referred patients were accompanied by a completed transfer form and written medical summary report of their treatment with pertinent data. the inpatient management plan was designed with infection control and nursing. a. every patient's condition was reviewed, and all stable patients were discharged, if possible. the census was reduced from 79 patients on august 19, 2015, to 23 patients on september 10, 2015. b. the patients who were not candidates for discharge remained in the hospital with close monitoring, and if any patient met the definition for suspected infection, she or he was placed on airborne isolation precautions (fig 2) . the organization reached an agreement to use a certain number of beds within an alternative hospital to enable the admission of our patients. a team of physicians and coordinators was assigned to cover the patients we admitted to that hospital. timely information and support were provided to physicians in the other hospitals who were taking care of our oncology patients admitted to their services. this included a detailed treatment plan, updated medical reports, and chemotherapy order protocols. staff management. the aim of the staff management plan was to minimize staff exposure to infection, educate them about the crisis, streamline their work process by clarifying their roles and responsibilities, and minimize their anxiety and stress. minimizing the risk of staff exposure included making sure that all the staff underwent n95 mask fit testing, scheduling all the department's staff to attend the hospitalwide educational training on donning and doffing clothing, applying strict hand hygiene, and implementing infection prevention precautions. all staff (physicians and nurses) were familiarized with the case definition of suspected mers-cov infection. for the clinical staff, we clarified the essential tasks to manage patients, such as reviewing patients' records, prioritizing their care, and providing timely care. all staff were instructed and educated about proper hand hygiene. nonclinical staff were released from duty for a few days to make sure that they were not exposed to the infection, and on their return to work, minimal interaction with clinical staff was advised. all staff with respiratory symptoms were clearly instructed not to report to work; they were sent for further assessment and instructed not to have contact with other health care professionals. infection control management. managing infection control during an outbreak is aimed at stopping the spread of the infection among the patients and staff by ensuring implementation of appropriate policies and procedures. dissemination of the correct knowledge and a thorough understanding of the transmission mechanism and protective measures were paramount to ensure that clinical practice was based on evidence, not myth and rumor. the plan for infection control included: a. timely information was communicated from the leadership committee to the staff and all affected services via e-mail or in face-to-face meetings. b. patient management included screening all patients coming to the outpatient clinic, screening all visitors before entering the oncology wards, and implementation of the mers-cov management plan for inpatient units. c. staff and visitors were screened before entering the oncology wards with a sign-in log. sick staff were kept away from the hospital. all staff were fitted for n95 masks, with training for all staff related to personal protective equipment. all staff were screened by nasal swab and mers-cov polymerase chain reaction testing to identify asymptomatic carriers; this was to ensure exclusion from the workplace and to enforce home isolation if a positive result was obtained. recovery plan. after assurance that the outbreak was under control, we implemented the recovery plan in phases, with the goal of resuming patient care at our facility based on clinical priority. the recovery phases were: • phase 1: immediate phase, 0-4 weeks. resumed the critical services that could not be performed in the alternative hospitals, such as certain specialized surgeries, chemotherapy administration, stem cell transplantation, or radiotherapy application. observed all the precautions for the control of infection. • phase 2: intermediate phase, 5-16 weeks. resumed all services provided before the crisis. • phase 3: long-term phase: strategic transformation of the department to provide better and safer care, integrating all new processes into the workflow to avoid newer outbreaks or in-hospital transmission. the described plan resulted in the prevention of any new infection, manifested by zero cases of in-hospital transmission of mers-cov infection among oncology patients, not just during the months of the crisis, but over the next 4 years (the time of this report). there was a clear transformation of many work processes and departmental functions, especially among those focusing on preventing emergency department overcrowding, which was identified as the primary cause of the outbreak. the described plan was effective in the prevention of further harm to our patients from this deadly virus, a benefit that extended way beyond the outbreak. there are many lessons that can be applied to a similar crisis while taking into account that each health care facility has its own particular circumstances. the application of what we learned and the processes we implemented may vary depending on the health care facility's specifications related to the type of patients served, the facility size and services, the health care system in the locality, and the community at large. collaboration among different health care facilities and providers is critical, including exploring resources available outside the walls of the facility. the key lesson that we learned in managing such outbreaks was the importance of effective communication with all stakeholders and addressing everyone's concerns, while bearing in mind the best interests of our vulnerable patient population. we learned that it is important to have a robust mechanism to prioritize patients to ensure the provision of timely care while preventing further harm by guiding staff to provide care while staying safe. sustaining the improvement in the work process and flow is paramount; this cannot be achieved without support from the organization's top leaders and stakeholders and the commitment and dedication of the staff, especially toward interventions that prevent the causes of the outbreak, such as emergency department overcrowding. middle east respiratory syndrome coronavirus (mers-cov) infection: epidemiology, pathogenesis and clinical characteristics middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea outcome of oncology patients infected with coronavirus confronting a crisis in cancer care delivery delivering high-quality cancer care: charting a new course for a system in crisis committee on improving the quality of cancer care: addressing the challenges of an aging population board on royal college of physicians and royal college of radiology: cancer patients in crisis: responding to urgent needs cancer care at times of crisis and war: the syrian example cancer care in a war zone: radiation oncology in iraq middle east respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): a review description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia the following represents disclosure information provided by authors of this manuscript. all relationships are considered compensated unless otherwise noted. relationships are self-held unless noted. i = immediate family member, inst = my institution. relationships may not relate to the subject matter of this manuscript. for more information about asco's conflict of interest policy, please refer to www.asco.org/rwc or ascopubs. org/go/site/misc/authors.html. open payments is a public database containing information reported by companies about payments made to us-licensed physicians (open payments). key: cord-309010-tmfm5u5h authors: dietert, kristina; gutbier, birgitt; wienhold, sandra m.; reppe, katrin; jiang, xiaohui; yao, ling; chaput, catherine; naujoks, jan; brack, markus; kupke, alexandra; peteranderl, christin; becker, stephan; von lachner, carolin; baal, nelli; slevogt, hortense; hocke, andreas c.; witzenrath, martin; opitz, bastian; herold, susanne; hackstein, holger; sander, leif e.; suttorp, norbert; gruber, achim d. title: spectrum of pathogenand model-specific histopathologies in mouse models of acute pneumonia date: 2017-11-20 journal: plos one doi: 10.1371/journal.pone.0188251 sha: doc_id: 309010 cord_uid: tmfm5u5h pneumonia may be caused by a wide range of pathogens and is considered the most common infectious cause of death in humans. murine acute lung infection models mirror human pathologies in many aspects and contribute to our understanding of the disease and the development of novel treatment strategies. despite progress in other fields of tissue imaging, histopathology remains the most conclusive and practical read out tool for the descriptive and semiquantitative evaluation of mouse pneumonia and therapeutic interventions. here, we systematically describe and compare the distinctive histopathological features of established models of acute pneumonia in mice induced by streptococcus (s.) pneumoniae, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, legionella pneumophila, escherichia coli, middle east respiratory syndrome (mers) coronavirus, influenza a virus (iav) and superinfection of iav-incuced pneumonia with s. pneumoniae. systematic comparisons of the models revealed striking differences in the distribution of lesions, the characteristics of pneumonia induced, principal inflammatory cell types, lesions in adjacent tissues, and the detectability of the pathogens in histological sections. we therefore identified core criteria for each model suitable for practical semiquantitative scoring systems that take into account the pathogenand model-specific patterns of pneumonia. other critical factors that affect experimental pathologies are discussed, including infectious dose, time kinetics, and the genetic background of the mouse strain. the substantial differences between the model-specific pathologies underscore the necessity of pathogenand model-adapted criteria for the comparative quantification of experimental outcomes. these criteria also allow for the standardized validation and comparison of treatment strategies in preclinical models. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 as one of the most frequent infectious diseases, pneumonia causes a tremendous socioeconomic burden in industrialized countries [1] and is the leading infectious cause of death in children worldwide [2] . numerous classes of pathogens can cause acute pneumonia [3] and the risk of pneumonia is greatly enhanced under conditions of impaired pulmonary host defense, including preceding viral infections [4] , mechanical ventilation [5] and sepsis [6] . the leading causative pathogen of community acquired pneumonia (cap) is the gram-positive bacterium streptococcus (s.) pneumoniae [7, 8] which accounts for the majority of bacterial upper and lower respiratory tract infections and is responsible for millions of deaths annually [9, 10] . as another cause of cap influenza a virus (iav) infection leads to rapid progression of lung failure with limited treatment options and frequent fatal outcome [3, 11, 12] . moreover, iav infections are commonly complicated by bacterial superinfection, mostly caused by s. pneumoniae, resulting in severe progressive pneumonia associated with increased mortality [13] . in contrast, the gram-negative and facultatively intracellular bacterium legionella (l.) pneumophila is the causative agent of the severe cap legionnaires' disease, and the second most commonly detected pathogen in pneumonia in patients admitted to intensive care units (icu) in industrialized countries [14, 15] . however, in addition to cap, ventilator-associated pneumonia (vap) is also a major cause of hospital morbidity and mortality in icus [16] and the spectrum of pathogens is shifted in these forms of pneumonia. here staphylococcus (s.) aureus, klebsiella (k.) pneumoniae, acinetobacter (a.) baumannii, and escherichia (e.) coli have been isolated with varying prevalences [17] [18] [19] . more specifically, the gram-negative k. pneumoniae is a significant opportunistic pathogen causing severe life-threatening hospitalacquired respiratory tract infections [20] [21] [22] while s. aureus, a gram-positive bacterium, is one of the most prevalent pathogens of community-and hospital-acquired lower respiratory tract infections in humans and accounts for a significant health and economic burden [23] [24] [25] . a. baumannii and e. coli are ubiquitous gram-negative bacteria which have recently emerged as major causes of community-associated, nosocomial [26, 27] and ventilator-associated pneumonia [19, 28] as well as septicemia induced acute lung injury (ali) [29, 30] . in addition, more recently discovered pulmonary pathogens indicate that novel emerging diseases may add to the list of highly relevant pneumonias that may also be of interest to be studied in animal models. for example, the middle east respiratory syndrome coronavirus (mers-cov) which is transmitted by dromedary camels as vectors [31] has emerged as the cause of severe human respiratory disease worldwide [32, 33] with elderly and immunocompromised individuals particularly in saudi arabia being at highest risk [34] [35] [36] . the various forms of pneumonia have been successfully reproduced in specific murine models of experimentally-induced acute pneumonia [37] [38] [39] . these models have substantially contributed to our understanding of the pathogenesis of community-and hospital-acquired pneumonia as well as emerging lung infections worldwide and are indispensable for the development of novel therapeutic strategies [40] [41] [42] . histopathology has been a powerful, reliable, and reproducible read-out tool for the evaluation of morphological changes in animal lung infection experiments for many decades [43, 44] . qualitative diagnoses are based on a summation of microscopically observable changes in the morphology and cellular composition of the tissue and cell types involved. for a more comparative inclusion of histopathologic information in biomedical research, scoring systems have been widely applied which allow for a first semiquantitative assessment of lesions compared to controls [44, 45] . moreover, all preclinical models used for the development of novel treatment strategies and acceptance by regulatory agencies need to be assessed histopathologically by board certified pathologists as gold standard for qualitative and semiquantitative evaluation of tissue alterations in experimental animals [46] [47] [48] . previous studies have revealed first fundamental differences in histopathologic lesions caused by different pathogens in mouse lungs [38, 41, 42] . however, scoring schemes for acute murine pneumonia existing to date are very superficial, addressing only a few, rather unspecific parameters [45, 49, 50] . more importantly, they hardly allow for a differentiating perspective between distinct pathogens or for group comparisons, e.g., infections of wild type versus genetically modified mice. clearly, there is a strong need for more precise and pathogen-as well as model-specific parameters to allow for an accurate description and semiquantification of the inflammatory phenotype for reliable and reproducible comparisons between experimental groups within each model. therefore, we have recently adapted more specific scoring criteria for s. pneumoniae and s. aureus-induced pneumonia [38, 42] . however, such pathogenspecific scoring criteria have not been employed for other lung pathogens in mice. here, we systematically describe and compare the histopathologies at their peaks of inflammation and injury of nine previously established acute lung infection models induced by s. pneumoniae, s. aureus, k. pneumoniae, a. baumannii, l. pneumophila, e. coli, mers-cov, iav and superinfection with iav and pneumococci. we provide model-specific criteria that can be used for appropriate histological quantitative comparisons, e.g., when different therapeutic interventions are evaluated within these established models. whole mouse lung sections were used to obtain complete overviews, particularly of the distributions of lesions and inflammatory patterns. on the basis of the different and oftentimes quite pathogen-and model-specific changes we identified the most suitable evaluation criteria for each model that will allow for more accurate semiquantitative assessments of the severities and distributions of pneumonic lesions. the lung tissues examined here were derived from experiments primarily conducted for purposes other than this study and most have been published elsewhere [38, 41, 42, [51] [52] [53] , except for the a. baumannii and e. coli experiments that will be published elsewhere. all animal procedures and protocols were approved by institutional ethics committees of charité-university of berlin, justus-liebig university of giessen, philipps university of marburg, university hospital of jena and local governmental authorities (landesamt für gesundheit und soziales (lageso) berlin, regierungspräsidium (rp) gießen and darmstadt, landesamt für verbraucherschutz (tlv) thüringen), respectively. permit numbers were g 0356/10, a-0050/15 (s. pneumoniae), g 0358/11 (s. aureus), g 75/2011, g 110/2014 (k. pneumoniae), a 0299/15 (a. baumannii), g 0175/12 (l. pneumophila), 02-049/12 (e. coli), 114/2012 (mers-cov), g 0152/ 12, and g 0044/11 (iav and superinfection). all animal studies were conducted in strict accordance with the federation of european laboratory animal science associations (felasa) guidelines and recommendations for the care and use of laboratory animals, and all efforts were made to minimize animal discomfort and suffering. all mice, except for mers-cov infected mice, were monitored at 12 hour intervals throughout the experiment to assess appearance, behavior, grooming, respiration, body weight, and rectal temperature. humane endpoints were defined (body temperature <30˚c, body weight loss = 20%, cumbersome breathing, accelerated breathing in combination with staggering, pain or paleness) but not reached by any of the mice at the indicated time points of termination of the experiments. mers-cov infected mice were monitored once daily and appearance, behavior, grooming, respiration and body weight were protocolled. here, a single humane endpoint (loss of body weight of >15%) was defined but not reached by any of the mice employed due to their favourable clinical outcome at the infection dose used. for all experimental infection models, except of k. pneumoniae, female mice (aged 8-12 weeks and weighing 17-22 g) were randomly assigned to groups (n = 2-4) per cage whereas in the k. pneumoniae model female and male mice (aged 23-25 weeks and weighing 22-24 g) were used for model specific reasons [41] . furthermore, for all experimental infection models, specificpathogen-free (spf) mice on c57bl/6 (all except for mers-cov) or balb/c (as previously used for the mers-cov model [52, 54] ) background were used and housed in individually ventilated cages under spf conditions with a room temperature of 22 ± 2˚c and a relative humidity of 55 ± 10%. a 12 hour light/ 12 hour dark cycle was maintained and the animals had unlimited access to standard pellet food and tap water. all experimental details of the infection models compared here were applied following previously published and well established protocols that partly vary in terms of infection doses, routes of infection and time point of examination due to pathogen-or model specific reasons, as given below. for bacterial infections, except for e. coli, mice were anesthetized intraperitoneally with ketamine (80 mg/kg) (ketavet; pfizer, berlin, germany) and xylazine (25 mg/kg) (rompun; bayer, leverkusen, germany). for experimental viral infections, mice were anesthetized using inhalation of isoflurane (forene; abbott, wiesbaden, germany). for lung histology, all mice except of the mers-cov model were humanely euthanized by exsanguination via the caudal vena cava after anesthesia by intraperitoneal injection of premixed ketamine (160 mg/kg) and xylazine (75 mg/kg). mers-cov infected mice were humanely euthanized by cervical dislocation after isoflurane anesthesia. s. pneumoniae (serotype 3 strain, nctc 7978), s. aureus newman (nctc 10833), k. pneumoniae (serotype 2, atcc 43816), a. baumannii (ruh 2037), l. pneumophila (serogroup 1 strain, jr 32) were cultured as described [37, 38, 40, 55, 56] and resuspended in sterile pbs. mice were anesthetized intraperitoneally (i.p.) with ketamine (80 mg/kg) and xylazine (25 mg/kg) and transnasally inoculated with 5 x 10 6 cfu s. pneumoniae (n = 14 mice), 5 x 10 7 cfu s. aureus (n = 4), 5 x 10 8 cfu a. baumannii (n = 8), in 20 μl pbs. mice transnasally infected with l. pneumophila (n = 8) received 1 × 10 6 cfu in 40 μl pbs. mice infected with k. pneumoniae (n = 16) received 3.5 x 10 5 cfu intratracheally in 50 μl nacl (0.9%) via microsprayer1 aerosolizer (model ia-1b, penn-century, inc., wyndmoor, pa) using intubation-mediated intratracheal instillation through intact airways [57] which has previously been optimized for this model [41, [57] [58] [59] . e. coli (atcc 25922) from -80˚c glycerol stock was added to lb broth (carl roth, karlsruhe, germany) and incubated for 12 hours at 200 rpm and 37˚c with 5% co 2 . optical density of 0.03 was adjusted in lb broth followed by incubation until midlogarithmic phase for 1.5 hours at 200 rpm and 37˚c. after centrifugation, the pellet was resuspended in sterile 0.9% nacl at 8 x 10 5 cfu e. coli / 200 μl and administered intraperitoneally (n = 10). for initial transduction of human dpp4 for subsequent infection of balb/c mice with mers-cov (hcov emc) viruses were cultured and prepared as described [52, 54, 60] . mice were transduced transnasally with 20 μl of an adenovirus vector encoding human dpp4 and mcherry with a final titer of 2.5 x 10 8 pfu per inoculum (adv-hdpp4; viraquest inc.), resulting in hdpp4 expression in the epithelial compartment of the lung [60] and transnasally infected with a final titer of 7 x 10 4 tcid 50 of mers-cov in 20 μl dmem (n = 17) under isoflurane anesthesia (forene; abbott, wiesbaden, germany). influenza a/pr/8/34 virus (h1n1; pr8) was grown as described [42] and mice were transnasally infected with 100 pfu pr8 in 50 μl pbs (n = 4) under isoflurane anesthesia. for superinfection experiments, the iav infection procedure was applied as described above with 40 pfu pr8 in 50 μl pbs. 8 days after viral infection, s. pneumoniae was cultured as described [37] and resuspended in sterile pbs. mice were anesthetized intraperitoneally and transnasally inoculated with 1 x 10 3 cfu s. pneumoniae in 20 μl pbs (n = 4). mice were humanely euthanized at model-specific time points as indicated ( table 1 ). between 2 and 6 repetitions of the entire experimental procedures were performed in each model with similar group sizes in each repetition. lungs were carefully removed after ligation of the trachea to prevent alveolar collapse, immersion-fixed in formalin ph 7.0 for 24 to 48 hours (mers-cov for 7 days), embedded in paraffin, cut in 2 μm sections and stained with hematoxylin and eosin (he) after dewaxing in xylene and rehydration in decreasing ethanol concentrations. bacteria were visualized using the giemsa and gram (modified by brown and brenn) stains. for the display of whole lung overviews, he stained slides of entire lung sections were automatically digitized using the aperio cs2 slide scanner (leica biosystems imaging inc., ca, usa) and image files were generated using the image scope software (leica biosystems imaging inc.). three evenly distributed whole-organ horizontal sections throughout the entire lungs were microscopically evaluated to assess the distribution and character of pathologic alterations, generating a modified panel of specific lung inflammation parameters adapted to each pathogen used (table 1 and table 2 ). all examinations were performed by trained veterinary experimental pathologists. for immunohistochemical detection of s. pneumoniae and iav (h1n1), antigen retrieval was performed with microwave heating (600 w) in 10 mm citric acid (750 ml, ph 6.0) for 12 minutes (min). lung sections were then incubated with a purified rabbit antibody polyclonal to s. pneumoniae (1:2,000, kindly provided by s. hammerschmidt) or with a purified goat antibody polyclonal to iav h1n1 (1:4,000, obt155, bio-rad, puchheim, germany) at 4˚c overnight. incubation with an immuno-purified rabbit or goat antibody at the same dilution served as negative controls. subsequently, slides were incubated with a secondary, alkaline phosphataseconjugated goat anti-rabbit (1:500, ap-1000, vector, burlingame, ca) antibody for 30 min at room temperature. the alkaline chromogen triamino-tritolyl-methanechloride (neufuchsin) was used as phosphatase substrate for color development. all slides were counterstained with hematoxylin, dehydrated through graded ethanols, cleared in xylene and coverslipped. transnasal infection of mice with s. pneumoniae, serotype 3 resulted in a broad spectrum of tissue lesions and immune cell infiltrations that are typical of aerogenic bacterial pneumonia. specific for this model, lesions widely expanded down to the periphery of the lung lobes (fig 1a) with inflammation closely surrounding the airways and blood vessels. pneumococcal spread led to an early immune response which was mainly characterized by predominantly intrabronchial ( fig 1b) and intraalveolar ( fig 1c) infiltrations of neutrophils provoking a lobular, suppurative bronchopneumonia with consolidation of affected lung areas. large areas of coagulation and liquefaction necrosis (fig 1d, arrowhead) as indicated by cellular fragmentation, decay, and loss of cellular details, accumulation of cellular and karyorrhectic debris as well as karyorrhexis, karyopyknosis, and karyolysis with consecutive hemorrhage were also present. the perivascular interstitium was widely expanded by edema due to vascular leakage [53] with massive extravasation of neutrophils recruited into perivascular spaces ( fig 1e) . furthermore, suppurative and necrotizing vasculitis accompanied by hyaline thrombi within small-sized blood vessels were occasionally present, indicating early histological evidence of incipient septicemia. increased pulmonary vascular permeability [53] also led to expanded areas of protein-rich alveolar edema which presented as homogenous, lightly pink material within the alveolar spaces in the he stain (fig 1f, asterisk) . a distinctive histopathological feature of pneumococcal pneumonia was the occurrence of massive suppurative to necrotizing pleuritis (fig 1g, arrowhead) and steatitis ( fig 1h) with widespread dispersion of bacteria into the thoracic cavity, likely accounting for the painful and morbid clinical behavior with rapid progression in affected mice. myriads of pneumococci were clearly visible as bluish to purple dots of approximately 1 μm in size in the standard he stain, mostly located on the pleural surface, in the mediastinal adipose tissue or within perivascular spaces in the lungs. + + ++ ++ ++ abscess formation ++ ++ + granuloma formation ++ alveolar edema ++ + ++ + + ++ ++ perivascular edema ++ ++ + + + + perivascular lymphocytic cuffing ++ + ++ ++ ++ + + ++ + vasculitis ++ + + + ++ + fibrinoid degeneration of vascular walls ++ vascular thrombosis +/++ ++ ++ ++ + + + ++ + + pleuritis ++ ++ ++ ++ ++ ++ in contrast, transnasal infection with s. aureus resulted in multifocally extensive but non-expansive bronchopneumonia predominantly located near the lung hilus (fig 2a) , affecting the bronchi, alveoli and interstitium. the main inflammatory cell population consisted of neutrophils, leading to mainly suppurative (fig 2b and 2c ) lesions with a tendency towards abscess formation. in contrast to pneumococci, macrophages were also present albeit at lower numbers than neutrophils. (fig 2c) . similar to klebsiella and streptococci, large areas of necrosis and hemorrhage ( fig 2d) were present. the perivascular areas were predominantly infiltrated by lymphocytes and fewer neutrophils (fig 2e) . compared to the s. pneumoniae model mentioned above [53] , vascular permeability seemed only slightly increased as reported before [38] and perivascular edema (fig 2e) as well as protein-rich alveolar edema (fig 2f, asterisk) were also present albeit to a lesser extent. neither pleuritis nor steatitis were observed consistent with a rather favorable clinical outcome under the conditions used. furthermore, staphylococci were largely undetectable by he stain which was possibly due to the low bacterial spread within the lungs. intratracheal infection of mice with k. pneumoniae resulted in severe widely expansive bronchopneumonia with increased lesion severity in the lung periphery ( fig 3a) . recruited immune cells predominantly consisted of neutrophils, leading to suppurative ( fig 3b) to abscessing ( fig 3c) bronchopneumonia with hemorrhage and necrosis as well as neutrophilic interstitial pneumonia (fig 3d) in less affected areas. increased vascular permeability as reported [40] was associated with massive alveolar (fig 3d, asterisk) and perivascular edema (fig 3e) , admixed with myriads of bacteria easily recognizable as purple dots in the he stain. suppurative to necrotizing vasculitis, pleuritis ( fig 3f, arrowhead) , and steatitis were also present and associated with marked bacterial spread and the rapid lethal clinical outcome. after transnasal infection with a. baumannii, mice developed a widely expansive ( fig 4a) bronchopneumonia with predominantly infiltrating neutrophils causing a suppurative ( fig 4b) to abscessing inflammation with areas of hemorrhage within alveoli and interstitium and large areas of parenchymal necrosis as well as alveolar edema. perivascular spaces had mild to moderate edema and infiltration of lymphocytes and neutrophils ( fig 4c) . vascular thrombosis was a common change (fig 4d, arrowhead) in small-sized blood vessels. similar to staphylococci, acinetobacter was invisible by he stain and neither pleuritis nor steatitis were present. transnasal infection of mice with l. pneumophila resulted in slightly different lesion patterns depending on the time point of examination after infection. at 48 hours after infection, nonexpansive interstitial pneumonia was found in close proximity to the hilus (fig 5a) with comparative histopathology of mouse models of acute pneumonia prominent alveolar wall necrosis (fig 5b) . at the 6 day-time point, specifically the numbers of infiltrating macrophages were clearly increased, leading to accentuated perivascular granuloma formation (fig 5c, arrowhead) . here, marked lymphocytic cuffing of most blood vessels as well as highly activated endothelial cells (fig 5d, arrowhead) were observed. at both time points, neither pleuritis nor steatitis were present. bacteria were invisible in the he stained sections. after intraperitoneal infection, the hematogeneous spread of e. coli to the lungs had resulted in diffuse, interstitial suppurative pneumonia, diffusely affecting the entire lungs, modelling sepsis-induced ali (fig 6a) . the interalveolar interstitium was heavily infiltrated with neutrophils ( fig 6b) with most prominent aggregation around blood vessels (fig 6c) , consistent with bacterial entry via the circulation. numerous hyaline thrombi were present within small-sized blood vessels (fig 6d, arrowhead) , suggestive of disseminated intravascular coagulopathy (dic) due to bacterial septicemia. large, rod-shaped bacteria were easily detectable only outside the lungs, mostly present in the adipose tissue surrounding the esophagus, possibly due to local spread of e. coli via the abdominal cavity. transnasal infection with mers-cov following adenoviral transduction of human dpp4 yielded an expansive, (fig 7a) interstitial pneumonia with severe alveolar epithelial cell necrosis and infiltration of mainly macrophages, lymphocytes, and fewer neutrophils (fig 7b) . only comparative histopathology of mouse models of acute pneumonia moderate peribronchial ( fig 7c) and perivascular (fig 7d) lymphocytic infiltrations were present while most venous blood vessels had marked fibrinoid degeneration and necrosis of comparative histopathology of mouse models of acute pneumonia vascular walls (fig 7d, asterisk) . additional hallmarks of mers-cov infection were large areas of protein-rich alveolar edema (fig 7e, arrowhead) , pronounced hemorrhage within perivascular and alveolar spaces, and interstitium (fig 7f, arrowhead) , and the formation of hyaline thrombi within small-sized blood vessels. after transnasal infection with iav, mouse lungs displayed a diffusely distributed bronchointerstitial pneumonia restricted to single lung lobes only (fig 8a) . alveolar necrosis was prominent and alveolar septae were diffusely distended by infiltrating inflammatory cells (fig 8b) . bronchial epithelial cells were markedly necrotic (fig 8c, arrowhead) and extensively scaled off into the bronchial lumen. alveoli and interstitium were filled with macrophages and lymphocytes as major effector cells ( fig 8d) and prominent perivascular lymphocytic cuffing ( fig 8e) was a characteristic change. furthermore, large areas of alveolar edema (fig 8f, asterisk) and, albeit to a much lesser extent, areas of hemorrhage within alveoli and interstitium were present, suggesting vascular damage and increased permeability. when mice had been infected with iav prior to infection with s. pneumoniae, a combination and exponentiated phenotype of both models was observed 24 hours later. lesions were widely expansive to the lung periphery but restricted to single lung lobes pre-damaged by iav ( fig 9a) . the character of pneumonia included massive infiltration of neutrophils into alveoli ( fig 9b) and bronchi, typical features of severe, suppurative bronchopneumonia. bronchial epithelium was almost entirely necrotic and bronchi were filled up with pus ( fig 9c) . perivascular spaces were edematous and infiltrated by neutrophils and lymphocytes (fig 9d) whereas only mild lymphocytic perivascular cuffing (fig 9e) was present. a severe proteinrich alveolar edema was seen, similar to that seen in the s. pneumoniae mono-infection (fig comparative histopathology of mouse models of acute pneumonia 9f). pneumococci were difficult to visualize by he stain, possibly due to the lower infectious dose used here when compared to the mono-infection. prior to processing for histopathology, small tissue samples from experimentally infected mouse lungs are commonly removed for molecular analyses of gene and/ or protein expression or other readout systems to receive additional information. to obtain representative data from such samples that can be correlated with the histological changes, it is crucial to know about the homogeneity of the distribution of lesions. also, some experimental protocols recommend to use the left and right halves of the lungs, respectively, for different analytical procedures, again anticipating lesion homogeneity and symmetry. however, when we analyzed the distributions and bilateral symmetry of lung lesions for each of the infection models, we found a wide spectrum of distinct distributions and asymmetries (fig 10) . in principle, lesion distributions followed the route of pathogen entry into the lungs. however, the tendencies to spread towards the periphery of the lobes after aerogenous infection varied between different pathogens despite similar aerogenous infection routes. mostly centrally focused lesions induced by s. aureus and l. pneumophila remained close to the hilus with no trend towards peripheral expansion. infection with s. pneumoniae, a. baumannii and mers-cov resulted in lesions closely surrounding major airway segments with centrifugal expansion towards the periphery. in contrast, lesions induced by k. pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus despite aerogenous infection. hematogenously-induced sepsis with e. coli was associated with an entirely diffuse distribution of lesions affecting the whole lung with myriads of inflammatory hot spots, commonly surrounding blood vessels. iav-induced lesions were restricted to individual lung lobes only with a rather homogeneous distribution within affected lobes. superinfection of s. pneumoniae into an iav-pneumonia resulted in a pattern virtually identical to that seen after iav infection alone. except for blood borne e. coli pneumonia which was consistently and evenly distributed throughout the entire lungs, affected areas in all other models tested here were randomly distributed more or less asymmetrically between the right and left halves of the lungs and also between adjacent lobes (fig 10) . for more than 100 years, a wide range of special stains have been used for the histological visualization of pathogens and other relevant structures, based on their more or less specific affinities to certain dyes. here, gram stain modified by brown and brenn was used for the visualization of gram-positive bacteria, including s. pneumoniae (fig 11a, arrowhead) , as easily recognizable, dark blue cocci. in contrast, giemsa stain was conducted predominantly for the detection of gram-negative bacteria such as k. pneumoniae (fig 11b, arrowhead) which then turned into light blue to greenish rods. for more specific pathogen detection on slides, particularly for viruses, immunohistochemistry is the method of choice. here, s. pneumoniae and iav were detected by immunohistochemistry using specific anti-s. pneumoniae or anti-iav antibodies, respectively. s. pneumoniae-positive signals were obtained as myriads of red cocci predominantly in the perivascular interstitium (fig 11c) , within neutrophils in alveoli and interstitium, and on pleural surfaces as well as in mediastinal adipose tissue. in addition, pneumococci were also visualized in the marginal sinuses of tracheal lymph nodes, both in macrophages and extracellularly. iav antigen was localized to the apical surface and cytosol of intact and necrotic bronchial epithelial cells (fig 11d) and within alveolar macrophages. the diversity of lesions and in particular the presence or absence of specific patterns in several of the models used ( table 1 ) strongly suggested that a uniform scoring scheme for the pneumoniae were mostly located in the periphery of the lobes and airways and much weaker adjacent to the hilus. hematogenous infection with e. coli was associated with entirely diffuse distribution of lesions affecting the whole lungs with myriads of inflammatory hot spots, commonly surrounding blood vessels. iav-induced lesions were restricted to individual lung lobes with a rather homogeneous distribution within affected lobes. which lobes were affected followed a rather random and inconsistent pattern. superinfection of s. pneumoniae into an iav-pneumonia resulted in a pattern virtually identical to that seen after iav infection alone. except for e. coli induced pneumonia, virtually all lung lesions were distributed asymmetrically between the left and right lung halves with no tendency of either half to be more often or more strongly affected. https://doi.org/10.1371/journal.pone.0188251.g010 comparative histopathology of mouse models of acute pneumonia semiquantification of mouse pneumonia is inconceivable. instead, scoring systems should take into account the more or less pathogen-specific lesion patterns that can be distilled from the comparative characterizations given above. for this purpose, we carved out the most characteristic lesion patterns that appear suitable for the development of specific scoring schemes for each model (table 2 ). different mouse models of acute pneumonia differ widely, with an obvious strong dependence on pathogen-specific features of virulence and spread, route of infection, infectious dose and other factors. here, we provide a detailed descriptive overview of histopathological features and distributions of lesions within infected lungs and compare them between nine relevant and commonly used infection models at their peaks of injury and inflammation. the models employed here all represent well established protocols that have been optimized and successfully used in previous studies, with model-specific variations in infection doses, routes of pathogen administration and analyzed time points [37-42, 51, 52, 54-56] . our model-specific description parameters (table 2 ) provide a rational for the selection of histopathological quantification criteria, in order to best reflect the model-specific lesion and distribution characteristics, which appear to be most relevant. clearly, the severity of lesions in terms of outcome of quantification systems will depend on several other factors that will have to be addressed separately in each model, such as the infection dose, time point of examination or therapeutic interventions. comparative histopathology of mouse models of acute pneumonia the model-associated characteristics of tissue lesions and immune cell infiltrates are widely consistent with well-established properties of the different pathogens used. for example, the destructive tissue damage with mostly neutrophilic infiltrations as seen in s. pneumoniae, s. aureus, and a. baumannii, are typically seen with extracellular bacteria that express cytotoxic virulence factors, such as pneumolysin and hydrogen peroxide [61] from s. pneumoniae or immunogenic cell wall components such as lipoteichoic acid (lta) from s. aureus [62, 63] . on the other hand, the intracellular pathogen l. pneumophila which primarily infects macrophages [64] resulted in a histiocytic infiltrate at 48 h that developed into granulomatous inflammation at 6 days after infection, typical of a t h 1-response [65, 66] . however, several of the pathogens used were associated with additional distinctive features. for example, histology revealed marked pleuritis and steatitis due to pathogen invasion into adjacent extrapulmonary tissues after infections with s. pneumoniae and k. pneumoniae. this massive bacterial spreading throughout the thoracic cavity was exclusively present in these two models and most likely associated with sepsis, responsible for the rapid clinical progression and unfavourable outcome [41, 67] . however, only k. pneumoniae had the tendency of abscess formation which was not seen in pneumococcal pneumonia. infection with s. aureus and a. baumannii also resulted in similar lesion patterns, except for acinetobacter-induced lesions widely expanding to the lung periphery while staphyloccocus-induced pneumonia was restricted to the lung hilus. a second difference between the two was the presence of prominent vascular thrombosis in a. baumannii-induced pneumonia which was absent from staphylococcus pneumonia. the clinical outcome of mice infected with a. baumannii and s. aureus was more favourable when compared to infection with s. pneumoniae or k. pneumoniae [38] which may be explained by the lack of bacterial spreading throughout the thoracic cavity and adjacent tissues, and possibly sepsis. e. coli infection was included here as a model for sepsis-associated ali [29, 30, 68] and consequently induced wide spread vascular thrombosis and vasculitis, most likely due to its blood borne entry into the lungs and concurrent septicemia with disseminated intravascular coagulopathy and associated vascular lesions. vascular thrombosis with or without vasculitis was also observed in other models, including s. pneumoniae, a. baumannii and mers-cov, however, to a much lesser extent and only within the most strongly affected areas. mers-cov and iav-associated lesions clearly reflected the known cellular tropisms of these viruses with necrosis of alveolar walls or bronchial epithelial cells, respectively, being the most characteristic histopathologic features [69] [70] [71] [72] . typical of virally induced lesions, the inflammatory cell infiltrates in mers-cov and iav infections were dominated by lymphocytes with no or only few neutrophils. nevertheless, the two viral models could be clearly distinguished from each other by additional histological characteristics. only the mers-cov infection led to a marked vascular phenotype with necrosis and degeneration of blood vessels, vasculitis, and consecutive vascular thrombosis as well as pronounced hemorrhages [69, 73] . in contrast, iav-induced pneumonia did not display any of these features, but was dominated by marked perivascular lymphocytic cuffing and alveolar edema [42, 74] . subsequent superinfection with low-dose s. pneumoniae potentiated the severity of the iav-induced lesions and aggravated the course of pneumonia. however, it did not alter the principal histological characteristics of iav-pneumonia. the patterns seen after single infection with s. pneumonia were not repeated in this superinfection model, likely owing to the much lower inoculation dose which is usually rapidly cleared from virus-naive lungs. when the distributions of lesions were compared among the 9 models tested, four distinct patterns could be clearly distinguished. the most common pattern, where lesions were focused around central airways and blood vessels close to the lung hilus with the periphery less or not affected can likely be explained by the aerogenous route of infection and pathogen entry. the opposite pattern characteristic of k. pneumoniae infection where the periphery of the lobes was more strongly affected than their hilus areas despite a similar aerogenous route of infection may be due to the aerosolic intratracheal application [75] of these bacteria which is typical of and necessary in this model [41, [57] [58] [59] . these differences are therefore more likely attributable to the model-specific route of infection rather than pathogen-specific properties. similarly, the very homogenous distribution of e. coli induced pneumonia likely followed the diffuse blood borne entry of the pathogen into the lungs after intraperitoneal infection. again unique among the pathogens tested here, the iav-associated pattern affected entire but only select lung lobes with almost complete sparing of others. this distribution probably followed a random spread of the virus along major airways but why some lobes remained virtually unaffected after transnasal infection remains hard to explain. apart from helping to understand differences in pathogen spread, the uneven and often quite asymmetrical distributions have a tremendous impact in practical terms when acute mouse pneumonia is sampled for molecular studies. when quantitative data on mrna or protein expression levels or other biochemical information are to be compared with one another or with tissue lesions, it is imperative that only identically affected areas are compared. since this is impossible to predict or recognize on the macroscopical level for most models, the practice of sampling different regions of such lungs for different readout systems appears highly problematic. another implication of the distinct lesion characteristics, immune cell reactions and distributions among the different models appears highly relevant for histological scoring systems that aim at first quantitative comparisons [45] . to narrow down the list of parameters appropriate for each pathogen and exclude features that are likely irrelevant for some of the models, we selected 23 single histopathologic criteria for the design of semiquantitative scoring systems suitable for each model. these criteria are partly composed of standard parameters such as the determination of the affected lung area, the distribution of lesions or the type of pneumonia induced. however, numerous other and more model-specific parameters were identified which precisely describe particular aspects and allow for a differentiation between the models, such as the presence of perivascular edema, vascular thrombosis, pleuritis or steatitis. appropriate scoring systems may thus encompass more general parameters when different pathogens are compared to one another or more pathogen-specific parameters in case specific pathogen features are in the focus of an experiment. for example, some of the parameters selected here have proven helpful in the discovery and semiquantification of different phenotypes of mouse pneumonia following genetic engineering of pathogens or mice [38, 51, 76] . however, scoring systems that claim universality for all mouse models of acute pneumonia seem neither generally applicable nor meaningful for all specific experimental goals. even the list of 23 parameters selected here may become inappropriate or insufficient when genetic changes on the pathogen or host side may result in different types of lesions, immune cell responses, time courses or other relevant features. in those cases, the list selected here may have to be adjusted or extended to better meet the specific challenges of each new study. as standard hematoxylin and eosin (he) staining of tissue sections failed to visualize most pathogens, traditional special stains as well as immunohistochemical techniques were employed, depending on specific staining properties of the pathogens and the availability of appropriate antibodies. while s. pneumoniae, k. pneumoniae and e. coli were easily visible in he stained tissue sections in areas with low density of inflammatory cells, e.g., in perivascular spaces, they were very difficult to identify in heavily infiltrated and consolidated lung parenchyma. in contrast, s. aureus, a. baumannii, l. pneumophila and both viruses were entirely invisible by he staining and thus had to be visualized by appropriate histotechnical stains or immunohistochemistry. both approaches will likely also allow for a rough quantification of pathogen numbers in tissues when appropriate image analysis tools are used. in this first comparative study of its kind, we examined previously established models with their optimized routes of infection, time points, and infection doses and volumes specific for each model to reach peaks of lung injury and inflammation. variations of such factors can be expected to result in different lesion severities, composition of the cellular infiltrates, and for some models in different expansions of lesions within the lung. still, the conditions used here are all based on observations that have evolved during extensive previous establishment studies of these models [37-39, 41, 42, 51, 52, 54, 56-58, 60, 77-79] . among the most important reasons, most human pathogens are not pathogenic for mice under non-experimental conditions and the decisive factor for obtaining a useful pneumonia model appears to be the determination of the appropriate infection dose and route of infection. in addition, the exact time points of tissue analysis after infection had to be determined for virtually all models with care to obtain a useful model, including a precise definition of the strain or variant of the pathogen used [51, 53, 80, 81] . another variable to consider is the mouse strain used. except for balb/c mice which were used in the mers-cov infection model here for model-specific reasons [60] , all models were conducted with c57bl/6 mice which is among the most commonly used mouse strain in infection research and therefore allows for comparisons with similar studies. however, variations of the strain or genetic background may have a dramatic impact on the type, severity and outcome of inflammation, particularly in innate immune responses [82] [83] [84] . again, the criteria suggested here for scoring procedures should allow to recognize and quantify such differences related to changes in infection dose and volume, time point of examination, strain and age of mice used, pathogen variant and other variables. histopathology of the lungs may be complex and requires fundamental knowledge in species-specific anatomy, physiology, organ-specific immunology, pathology, and histotechnical procedures. furthermore, various background lesions in mice, including strain specific spontaneous degenerative or inflammatory conditions and the possibility of accidental infections unrelated to the experiment should not be confused with experimental outcome. thus, despite our efforts to specify and simplify the criteria relevant for model-specific assessment and quantification of lesions, it appears crucial that trained histopathology experts be involved in the microscopical examination of mouse lungs [46, 85] . clearly, in addition to descriptive or semiquantitative histology, a number of other parameters may be useful for quantitative comparisons between experimental groups to determine the role of specific cell types, molecules, and therapeutic interventions, depending on the strategy and goal of the study [44] . such parameters could include flow cytometric immune cell identifications and quantifications, elisa or quantitative rt-pcr for the probing of cytokines, chemokines or matrix proteins involved in lung pathology and remodeling, and plaque/colony forming assays for the identification or quantification of pathogens, as previously published for most of the models used here [38, 41, 42, 51-53, 86, 87] . all of these methods, however, lack the spatial resolution that only histological assessments offer. only the combination of these techniques will lead to a better understanding of the disease in the complex context of the entire lung pathology. in conclusion, we have identified a spectrum of pathogen-and model-specific lesion characteristics in mouse models of acute pneumonia. our findings underscore the necessity of model-specific criteria for the accurate histopathological characterization and quantitative assessments of experimental pneumonia. this comparative landscaping of acute mouse pneumonia histology provides a comprehensive framework for future studies on the role of individual pathogen or host factors, complex disease mechanisms, and novel therapeutic strategies that could help to treat pneumonia in human patients. managing community-acquired pneumonia: a european perspective. respiratory medicine fact sheet n˚331 respiratory viral infection predisposing for bacterial disease: a concise review. fems immunology and medical microbiology evidence on measures for the prevention of ventilator-associated pneumonia immunostimulation is a rational therapeutic strategy in sepsis. novartis foundation symposium recent advances in our understanding of streptococcus pneumoniae infection the burden of pneumococcal pneumonia-experience of the german competence network capnetz pneumococcal conjugate vaccine for childhood immunization-who position paper. releve epidemiologique hebdomadaire mortality from invasive pneumococcal pneumonia in the era of antibiotic resistance, 1995-1997 hospitalized patients with 2009 h1n1 influenza in the united states neuraminidase inhibitors for preventing and treating influenza in healthy adults and children. the cochrane database of systematic reviews epidemiology, microbiology, and treatment considerations for bacterial pneumonia complicating influenza legionnaires' disease: update on epidemiology and management options legionella as a cause of severe pneumonia clinical infectious diseases: an official publication of the infectious diseases society of america american journal of respiratory and critical care medicine ventilator-associated pneumonia in a tertiary care hospital in india: incidence and risk factors incidence, bacteriology, and clinical outcome of ventilator-associated pneumonia at tertiary care hospital development of immunization trials against klebsiella pneumoniae the epidemiology of nosocomial infections caused by klebsiella pneumoniae current epidemiology and growing resistance of gram-negative pathogens. the korean journal of internal medicine association between staphylococcus aureus strains carrying gene for panton-valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients archives de pediatrie: organe officiel de la societe francaise de pediatrie european respiratory society: standards for quantitative assessment of lung structure. american journal of respiratory and critical care medicine an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals multiparametric and semiquantitative scoring systems for the evaluation of mouse model histopathology-a systematic review the european college of veterinary pathologists (ecvp): the professional body for european veterinary pathologists a minimum core outcome dataset for the reporting of preclinical chemotherapeutic drug studies: lessons learned from multiple discordant methodologies in the setting of colorectal cancer. critical reviews in oncology/hematology assessment of reproductive toxicity under reach. regulatory toxicology and pharmacology: rtp the virulence variability of different acinetobacter baumannii strains in experimental pneumonia. the journal of infection the role of tlr2 in the host response to pneumococcal pneumonia in absence of the spleen ifns modify the proteome of legionella-containing vacuoles and restrict infection via irg1-derived itaconic acid a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform moxifloxacin is not anti-inflammatory in experimental pneumococcal pneumonia. the journal of antimicrobial chemotherapy protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein flagellin-deficient legionella mutants evade caspase-1-and naip5-mediated macrophage immunity differential roles of cd14 and toll-like receptors 4 and 2 in murine acinetobacter pneumonia. american journal of respiratory and critical care medicine correlation of klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model intubation-mediated intratracheal (imit) instillation: a noninvasive, lung-specific delivery system distinct contributions of neutrophils and ccr2+ monocytes to pulmonary clearance of different klebsiella pneumoniae strains rapid generation of a mouse model for middle east respiratory syndrome streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines. microbiological reviews lipoteichoic acid synthesis and function in gram-positive bacteria. annual review of microbiology role of lipoteichoic acid in infection and inflammation. the lancet infectious diseases interaction between the legionnaires' disease bacterium (legionella pneumophila) and human alveolar macrophages. influence of antibody, lymphokines, and hydrocortisone legionella pneumophila pathogenesis and immunity. seminars in pediatric infectious diseases early recruitment of neutrophils determines subsequent t1/t2 host responses in a murine model of legionella pneumophila pneumonia immunostimulation with macrophage-activating lipopeptide-2 increased survival in murine pneumonia american journal of physiology lung cellular and molecular physiology emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs. american journal of respiratory and critical care medicine differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels avian flu: influenza virus receptors in the human airway influenza virus-induced lung injury: pathogenesis and implications for treatment dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv. critical reviews in clinical laboratory sciences attenuation of immunemediated influenza pneumonia by targeting the inducible co-stimulator (icos) molecule on t cells rnai-mediated suppression of constitutive pulmonary gene expression by small interfering rna in mice moraxella catarrhalis induces an immune response in the murine lung that is independent of human ceacam5 expression and longterm smoke exposure. american journal of physiology lung cellular and molecular physiology multiple myd88-dependent responses contribute to pulmonary clearance of legionella pneumophila chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses surface proteins and exotoxins are required for the pathogenesis of staphylococcus aureus pneumonia cell-specific interleukin-15 and interleukin-15 receptor subunit expression and regulation in pneumococcal pneumoniacomparison to chlamydial lung infection influenza h3n2 infection of the collaborative cross founder strains reveals highly divergent host responses and identifies a unique phenotype in cast/eij mice strain differences in a murine model of air pollutant-induced nonatopic asthma and rhinitis. toxicologic pathology murine strain differences in inflammatory angiogenesis of internal wound in diabetes the ecvp/esvp summer school in veterinary pathology: high-standard, structured training for young veterinary pathologists miniaturized bronchoscopy enables unilateral investigation, application, and sampling in mice il-37 causes excessive inflammation and tissue damage in murine pneumococcal pneumonia the authors thank charlene lamprecht and angela linke for excellent technical assistance and nancy a. erickson for helpful discussions. key: cord-311176-dlwph5za authors: alshahrani, mohammed s.; sindi, anees; alshamsi, fayez; al-omari, awad; el tahan, mohamed; alahmadi, bayan; zein, ahmed; khatani, naif; al-hameed, fahad; alamri, sultan; abdelzaher, mohammed; alghamdi, amenah; alfousan, faisal; tash, adel; tashkandi, wail; alraddadi, rajaa; lewis, kim; badawee, mohammed; arabi, yaseen m.; fan, eddy; alhazzani, waleed title: extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus date: 2018-01-10 journal: ann intensive care doi: 10.1186/s13613-017-0350-x sha: doc_id: 311176 cord_uid: dlwph5za background: middle east respiratory syndrome (mers) is caused by a coronavirus (mers‐cov) and is characterized by hypoxemic respiratory failure. the objective of this study is to compare the outcomes of mers-cov patients before and after the availability of extracorporeal membrane oxygenation (ecmo) as a rescue therapy in severely hypoxemic patients who failed conventional strategies. methods: we collected data retrospectively on mers-cov patients with refractory respiratory failure from april 2014 to december 2015 in 5 intensive care units (icus) in saudi arabia. patients were classified into two groups: ecmo versus conventional therapy. our primary outcome was in-hospital mortality; secondary outcomes included icu and hospital length of stay. results: thirty-five patients were included; 17 received ecmo and 18 received conventional therapy. both groups had similar baseline characteristics. the ecmo group had lower in-hospital mortality (65 vs. 100%, p = 0.02), longer icu stay (median 25 vs. 8 days, respectively, p < 0.01), and similar hospital stay (median 41 vs. 31 days, p = 0.421). in addition, patients in the ecmo group had better pao2/fio2 at days 7 and 14 of admission to the icu (124 vs. 63, and 138 vs. 36, p < 0.05), and less use of norepinephrine at days 1 and 14 (29 vs. 80%; and 36 vs. 93%, p < 0.05). conclusions: ecmo use, as a rescue therapy, was associated with lower mortality in mers patients with refractory hypoxemia. the results of this, largest to date, support the use of ecmo as a rescue therapy in patients with severe mers-cov. middle east respiratory syndrome (mers), which was first described in 2012, is caused by a novel coronavirus (mers-cov). the world health organization (who) as of 5 december 2016 reported 1917 confirmed cases of the mers-cov infection globally with an overall mortality rate of 35% [1] . the majority of cases were reported in saudi arabia, wherein 1567 were confirmed cases, and of which 649 (41%) died [2] . human coronaviruses were first identified in the mid-1960s and usually cause mild upper-respiratory tract illness. in 2012, the first confirmed case of mers-cov was reported from saudi arabia [3] . mers-cov infection is associated with significant mortality related to the virulence of the virus, nature of the disease, and the lack of effective therapy. patients with mers-cov who develop acute respiratory distress syndrome (ards) are at a high risk of dying from refractory hypoxemia, multiorgan failure, and septic shock [4] . current interventions such as lung protective ventilation, prone ventilation, and neuromuscular blocking agents have been shown in randomized trials to improve mortality in patients with ards [5] [6] [7] . however, in some patients, these conventional measures fail to maintain adequate oxygenation; therefore, other rescue therapies are considered, such as different modes of ventilation, inhaled pulmonary vasodilators, and extracorporeal membrane oxygenation (ecmo). anticipated difficulties in patient recruitment, study design, and ethical concerns affect the feasibility of conducting randomized clinical trials that examine the efficacy of ecmo in this population. therefore, observational studies are a reasonable alternative. in this study, we aim to describe the effect of ecmo rescue therapy on patient-important outcomes in patients with severe mers-cov. in response to the large mers-cov outbreak, the saudi ministry of health implemented a national ecmo program in april 2014. the saudi ecmo program provided a rapid transportation chain system (medevac system), isolated intensive care unit (icu) beds, and venovenous (v-v) ecmo machines in selected centers across the country. an ecmo team was created that was available 24 h a day/7 days a week. the team included an intensivist trained in ecmo, a cardiac surgeon, a perfusionist, and ecmo-trained nurses. the intensivist on the ecmo team triaged all calls from other centers based on predefined criteria, wherein patients were predetermined to be candidates to receive ecmo or not. criteria for eligibility to receive ecmo were based on the extracorporeal life support organization (elso) [8] guidelines and are listed below. we retrospectively identified patients who would have been eligible for ecmo but did not receive it because the ecmo program was not available at that time (prior to april 2014). the intervention (ecmo) group was included from five main ecmo centers in three major cities in saudi arabia after the program initiation (april 2014 to december 2015). all participating hospitals were accredited by the joint commission international and had closed icus with 24-h coverage by trained intensivists. we obtained ethics approval from the saudi ministry of health ethics review board and from individual centers' ethics boards. patients were candidates to receive ecmo if they have met the following criteria: the ecmo group included patients who met the above criteria and received ecmo after implementing the ecmo program from april 2014 to december 2015. we included all patients with mers-cov who received ecmo during that period. the control group were patients who met the above criteria but did not receive ecmo in the period prior to the introduction of ecmo program (prior to april 2014). weaning from ecmo was primarily based on clinical improvement demonstrated by adequate oxygenation and gas exchange shown in vital signs, blood gases, and chest x-ray. the decision for readiness of a patient to be weaned from ecmo was left to the judgment of treating clinician and the ecmo team. the weaning process followed the elso criteria as follow: weaning starts by decreasing the flow to 1l/min while keeping the sweep of 100% (to maintain spo2 > 95%). if spo2 remains within target, a trial of clamping the catheters and keeping the patient on the ventilator at appropriate settings was attempted. we designed an electronic pretested data abstraction forms; the forms were pilot tested prior to data collection to ensure accuracy and reproducibility. trained personnel collected the data at each participating center under the supervision of the local principal investigators. research personnel collected data on patients' demographics, comorbidities, acute physiology and chronic health evaluation ii (apache ii) score, laboratory results (hemoglobin concentration, white blood and platelets counts, kidney function, blood gases), ventilator modes and settings, interventions used to treat refractory hypoxemia (prone ventilation, use of neuromuscular blocking drugs, and pulmonary vasodilators), vasoactive support, antimicrobial and antiviral therapy, steroid use, and primary and secondary outcome data. data were tested for normality using the kolmogorov-smirnov test. a repeated-measures analysis of variance was performed. fischer's exact test was used for the categorical data. independent t test was used to compare the continuous variables in the two groups. the mann-whitney u test was performed to compare the nonparametric values of the two groups. data were expressed as median (interquartile range (iqr) [range]), number (proportion), or mean (sd) as appropriate. the volume of cases was not enough to allow a priori power analysis. however, a post hoc power analysis indicated that the current sample size of 35 patients is powered to detect 35% absolute difference in mortality rate, with a type i error of 0.05 and a power of 80%. a value of p < 0.05 was considered statistically significant. eighty patients with confirmed mers-cov infection were admitted to the icus of participating centers from april 2014 to december 2015. thirty-five patients met our eligibility criteria and were included in the analysis, 17 in the ecmo group and 18 in the control group. as shown in table 1 , the baseline characteristics were similar in both groups; the median ages were (46 vs. 50 years), and mean apache ii score (28 vs. 31) were not statistically different. (p = 0.48 and p = 0.12; respectively). adjunctive therapies were used in both groups. ribavirin was used significantly more often in the ecmo group compared to the control group (82 vs. 24%, p = 0.001), interferon was also used more in the ecmo cohort compared to controls (65 vs. 24%, p = 0.016), and the use of steroids was similar in both groups (53 vs. 72%, p = 0.24). at day one of eligibility to ecmo, more patients in the control group required hemodynamic support with norepinephrine compared to ecmo group; however, both groups had similar use of epinephrine and dobutamine, continuous renal replacement therapy (crrt), modes of ventilation, positive end-expiratory pressure (peep), and neuromuscular blocking agents (tables 2 and 3 ). alveolar recruitment maneuver was used in one patient in the ecmo group. none of the patients received prone ventilation. throughout days 1-14, more patients in the control group developed renal impairment and had significantly lower pao2/fio2 ratio (table 3) . other laboratory values were similar between both groups (table 4 ). however, due to the small sample size, it was not feasible to adjust for all confounding factors. in the ecmo group, the v-v mode was used in all patients via the percutaneous cannulation approach for vascular access. femoral-femoral access was used in 65% of patients, while femoral-jugular access was used in 35% of cases. ecmo access was inserted by a cardiac surgeon in 70% of cases and by a cardiac intensivist in the remaining 30%. chest x-ray was used to confirm successful cannulation in 16 patients and transesophageal echocardiography (tee) in one patient. blood flow (l min −1 ), revolutions per minute, and sweep gas among ecmo patients had a mean (sd) of 3.8 (0.77), 3148.7 ecmo-related mechanical complications occurred in 3 (18%) patients; one patient developed pneumothorax that was treated with chest tube insertion, and two patients had major bleeding immediately after the initiation of ecmo. compared to the control group, the ecmo group had significantly lower in-hospital mortality (65 vs. 100%; p = 0.02), longer icu stay (25 vs. 8 days; p = 0.001) ( table 5 and fig. 1 ). less use of norepinephrine at days 1 and 14 (p < 0.05), and better oxygenation (higher pao 2 / fio 2 ratio) throughout days 7-14 (table 2 ). in this retrospective cohort study, we found that ecmo rescue therapy was associated with lower in-hospital mortality, better oxygenation, and fewer organ failures compared to historical control (usual care) in patients with severe mers-cov. however, the length of hospital stay was the same and a possible explanation is that during the crisis phase, patients were mechanically ventilated in the ward when icu beds are full, and it is possible that this could have contributed to similar stay in hospital in both groups. although elso issued guidelines on the use of ecmo in patients with ards, these guidelines do not address specific disease context, and are difficult to generalize to the heterogeneous ards population. therefore, we conducted this observational study to report on the efficacy and safety of ecmo in patients with severe mers-cov infection. there is a single case report in the literature looking at ecmo in mers-cov patients. guery et al. described the use of ecmo in two patients with acute respiratory failure secondary to mers-cov infection in france, where both patients developed severe hypoxia and increasing oxygen requirements, leading to mechanical ventilation and ecmo use. one patient died, and the other survived after approximately 2 months in hospital [10] . ecmo use in respiratory failure has been reported with variable survival rates. the first 2 randomized clinical trials (rcts) failed to prove superiority of ecmo over conventional management [11, 12] . however, the severe adult respiratory failure (cesar) trial showed improved 6-month survival in patients who were referred early to an ecmo center [13] . this was the largest clinical trial to investigate the efficacy of early use ecmo in patients with ards. despite concerns about the trial design and possible differences in steroid use and ventilator strategies, these results contributed to the increasing use of ecmo worldwide. in this study, we observed no significant differences in the use of adjunctive therapies except for ribavirin use in the ecmo group. the benefit of antiviral therapy in mers-cov infection remains unclear. recent korean guidelines published during the 2015 mers-cov outbreak in south korea suggested the use of antiviral therapy in patients with severe mers-cov [14] . in patients with respiratory failure from h1n1 infection who required the use of ecmo, the survival rate varied considerably between studies ranging from 35 to 90% [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . there was a large variation in survival rates, which could be explained by differences in patients' baseline characteristics and severity of illness. in one study, older, obese, diabetic, or immunocompromised patients were found to be at a higher risk of developing severe [28] [29] [30] [31] . in this study, the two groups were comparable at baseline, and there were no significant differences between groups in any of these variables. another large observational study examined the predictors of death in h1n1 patients who underwent v-v ecmo and found that creatinine and bilirubin levels, systemic arterial pressure, hematocrit, and pre-ecmo hospital length of stay were associated with higher mortality [32] . another important factor is the center experience and volume of cases; this could have contributed to the variability in survival rates with ecmo use. a recent study by barbaro et al. [33] demonstrated that centers with > 30 ecmo cases/year had better survival rates than centers with less than 6 cases per year. in saudi arabia, ecmo was not available except in one center until the mers-cov crisis; thereafter, the ecmo program was implemented as a therapeutic option for patients with refractory hypoxemia. ecmo interventions were run in tertiary centers with equipped icus by most experienced intensivists and perfusionists who received training in ecmo prior to the start of the program. although more ecmo patients received ribavirin and interferon therapy, we do not believe that this difference has an impact to our findings. published reports on this therapy are limited, but none showed significant improvement with this combination [34] [35] [36] . the largest study to date published in abstract format [37] showed no reduction in mortality. therefore, we believe that the imbalance of co-interventions between the two groups is unlikely to affect the estimation of treatment effect. in regard to infection control issues, caregivers safety of ecmo patients was organized and maintained by aggressive measures which were applied strictly and monitored closely with all admissions were taken to airborne isolated rooms which impacted the containment of the virus plus applying the universal protective personal measures all the time during the patients encounter. because of these stringent measures, there were no reports by or about any caregiver of any ecmo patient being affected. to our knowledge, this is the largest study to describe outcomes in patients with mers-cov who received ecmo. there are several strengths to our study: the "before and after" design allowed us to compare ecmo cases to a control group with similar demographics and within the same institutions. we also collected data on important variables and confounders, and conducted adjusted analyses to assess the impact on the results. we adhered to the strengthening the reporting of observational studies in epidemiology (strobe) guidelines [38] . despite the strengths of our study, it has several important limitations. first, the retrospective nature of this study renders it at risk of bias. all patients in the control group died, which may be explained by the severity of illness, as these were patients who had ards and were eligible otherwise. we cannot rule out the possibility of selection bias, as we were unable to track all transfer requests due to the outbreak and crisis at the time, leaving us with limited information. in addition, some patients were transferred from non-participating ecmo centers; therefore, baseline pre-ecmo data such as blood gases and ventilator settings could not be obtained. furthermore, due to insufficient documentation during the outbreak and crisis circumstances, we were not able to track the ecmo requests to the referral call center. there were differences in some co-interventions (e.g., antiviral therapy), and the influence of unmeasured confounders cannot be excluded. such concerns can only be addressed in rcts; however, conducting rct is likely to be challenging in the context of epidemics. this study was not designed to compare the cost of 2 interventions; although it is an important outcome that could help the clinicians and stakeholders to make decisions. lastly, the small sample size limited our ability to perform an adequate multivariate analysis. similar to other ecmo studies, it is difficult to determine if the mortality was the result of refractory respiratory failure or other causes like septic shock or other organs failure. in summary, the use of ecmo was associated with lower mortality in patients with severe mers-cov infection and refractory hypoxia. future randomized trials, although challenging to conduct, are highly needed to confirm or dispute these observations. until more data are available, ecmo could be considered as a rescue therapy in selected mers-cov patients with refractory hypoxemia. 10 department of icu national hospital, internal medicine and critical care, riyadh, saudi arabia. 11 critical care medicine department isolation of a novel coronavirus from a man with pneumonia in saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. the acute respiratory distress syndrome network prone positioning in severe acute respiratory distress syndrome neuromuscular blockers in early acute respiratory distress syndrome assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission randomized clinical trial of pressure-controlled inverse ratio ventilation and extracorporeal co2 removal for adult respiratory distress syndrome extracorporeal membrane oxygenation in severe acute respiratory failure. a randomized prospective study efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial antiviral treatment guidelines for middle east respiratory syndrome extracorporeal membrane oxygenation for pandemic h1n1 2009 respiratory failure referral to an extracorporeal membrane oxygenation center and mortality among patients with severe 2009 influenza a(h1n1) extracorporeal membrane oxygenation for 2009 influenza a(h1n1) severe respiratory failure in japan extracorporeal membrane oxygenation for 2009 influenza a(h1n1) acute respiratory distress syndrome ventilatory and ecmo treatment of h1n1-induced severe respiratory failure: results of an italian referral ecmo center extracorporeal membrane oxygenation (ecmo) in patients with h1n1 influenza infection: a systematic review and meta-analysis including 8 studies and 266 patients receiving ecmo extracorporeal membrane oxygenation for pandemic influenza a(h1n1)-induced acute respiratory distress syndrome: a cohort study and propensitymatched analysis long-term quality of life in patients with acute respiratory distress syndrome requiring extracorporeal membrane oxygenation for refractory hypoxaemia extracorporeal membrane oxygenation for 2009 influenza a (h1n1) acute respiratory distress syndrome: single-centre experience with 1-year follow-up extracorporeal membrane oxygenation for critically ill patients with 2009 influenza a (h1n1)-related acute respiratory distress syndrome: preliminary experience from a single center extracorporeal membrane oxygenation for severe influenza a (h1n1) acute respiratory distress syndrome: a prospective observational comparative study extracorporeal lung support in patients with severe respiratory failure secondary to the 2010-2011 winter seasonal outbreak of influenza a (h1n1) in spain the italian ecmo network experience during the 2009 influenza a(h1n1) pandemic: preparation for severe respiratory emergency outbreaks middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus predicting mortality risk in patients undergoing venovenous ecmo for ards due to influenza a (h1n1) pneumonia: the ecmonet score. intensive care med association of hospital-level volume of extracorporeal membrane oxygenation cases and mortality. analysis of the extracorporeal life support organization registry ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome oronavirus:an observational study ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study effect of ribavirin and interferon on the outcome of critically ill patients with mers the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies we wish to acknowledge the saudi ministry of health for implementing a national ecmo program in april 2014 who assisted us throughout the course of this research. we thank our colleagues from all participating icus throughout saudi arabia who provided insight and expertise that greatly assisted the research, and for their comments on an earlier version of the manuscript. the authors declare that they have no competing interests. all data produced and analyzed during this study are included and presented as tables in this manuscript. authors have no objection in granting and assigning the annals of intensive care journal unrestricted right to reproduce, publish, and distribute this manuscript in all forms including electronic form either offline or online media. no patients were involved neither in the design, recruitment, and conduction of this study nor in the development of outcome measures. we plan to disseminate the results of the study in lay language for patient interest groups. we had one institutional review board approval (irb-h-02-j-002) for the ecmo from the saudi arabia ministry of health as all ecmo program during the outbreak was under the umbrella of the ministry. all authors declare that they receive no support from any commercial organization or company. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-318872-0e5zjaz1 authors: park, ji-eun; jung, soyoung; kim, aeran; park, ji-eun title: mers transmission and risk factors: a systematic review date: 2018-05-02 journal: bmc public health doi: 10.1186/s12889-018-5484-8 sha: doc_id: 318872 cord_uid: 0e5zjaz1 background: since middle east respiratory syndrome (mers) infection was first reported in 2012, many studies have analysed its transmissibility and severity. however, the methodology and results of these studies have varied, and there has been no systematic review of mers. this study reviews the characteristics and associated risk factors of mers. method: we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) for englishor korean-language articles using the terms “mers” and “middle east respiratory syndrome”. only human studies with > 20 participants were analysed to exclude studies with low representation. epidemiologic studies with information on transmissibility and severity of mers as well as studies containing mers risk factors were included. result: a total of 59 studies were included. most studies from saudi arabia reported higher mortality (22–69.2%) than those from south korea (20.4%). while the r(0) value in saudi arabia was < 1 in all but one study, in south korea, the r(0) value was 2.5–8.09 in the early stage and decreased to < 1 in the later stage. the incubation period was 4.5–5.2 days in saudi arabia and 6–7.8 days in south korea. duration from onset was 4–10 days to confirmation, 2.9–5.3 days to hospitalization, 11–17 days to death, and 14–20 days to discharge. older age and concomitant disease were the most common factors related to mers infection, severity, and mortality. conclusion: the transmissibility and severity of mers differed by outbreak region and patient characteristics. further studies assessing the risk of mers should consider these factors. middle east respiratory syndrome (mers) was first reported in 2012 in saudi arabia [1] . although most patients are linked to the arabian peninsula geographically, mers has been detected in many other parts of the world [2] . a large mers cluster was also observed in 2015 in south korea [3] . mers causes sporadic infection and intrafamilial and healthcare-associated infection. its symptoms can vary from asymptomatic infection to death. despite the infection's association with high mortality, specified antiviral therapy is lacking, especially for patients with concomitant diseases [2] . many previous studies have assessed the risks of mers, such as factors dictating severity or an infection risk, yet the indices they present vary. for example, the case fatality rate was found to be 25.9% in the middle east area, but 20.4% in south korea [4] . the incubation period was reported to be 6.83-7 days in south korea [4, 5] , but 5.5 in a study using data from multiple areas [6] and 5.2 in saudi arabia [7] . accurate assessment of the risk of mers is essential for predicting and preventing infection. a systematic review of the risk of mers, as covered in previous studies, is potentially helpful for predicting this spread, and its future impact. this study aimed at reviewing the risk of mers, focusing on indices related to infectivity and severity. we searched international (pubmed, sciencedirect, cochrane) and korean databases (dbpia, kiss) using the term "mers" or "middle east respiratory syndrome", encompassing articles published after 2000. the search process was conducted in october 2017. we also manually searched the reference lists of the included studies. human studies were included, while animal studies and reviews were excluded. only articles in english or korean were included. even if a study collected data on humans, such as collecting specimens from religious pilgrims, it was excluded if there were no mers patients in the study sample. additionally, case studies including fewer than 20 mers patients were excluded as they were considered as having insufficient mers patient numbers and representative information. the included studies were classified as epidemiologic studies and those covering risk factors of mers. in the epidemiologic category, indices related to the risk of mers were divided into two categories; related to infectivity and related to severity. the index related to infectivity included the reproduction number (r), attack rate, incubation period, serial interval, and days from onset to confirmation. the index related to severity included the case fatality rate (cfr), days from onset to hospitalization, days from onset to discharge, days from onset to death, and days from hospitalization to death. in the risk factor category, factors related to infection, transmission, severity, and mortality of mers were analysed. even if the included studies investigated factors that were related to mortality, when they did not analyse risk factors of severity or mortality using appropriate statistical methods (e.g., regression analysis, cox proportional hazards model) or only compared prevalence factors, we excluded them from the risk factor category. in all categories, we extracted the study period, number of participants, and geographical region where the data were collected using a data extraction form confirmed after pilot assessment. a total of 3009 studies were searched, and 2717 were reviewed, excluding 292 duplicate studies. after the title and abstract review, a further 1804 and 663 were excluded, respectively. another four studies were included via a manual search, which left a total of 58 studies for analysis ( fig. 1 ). the 38 of total 58 included studies were classified as epidemiologic studies (table 1) . r value, representing the reproduction number, indicates the average number of secondary cases generated by infectious individuals. thirteen studies reported r value of mers. four studies that used data from multiple areas had r < 1.0 [6, [8] [9] [10] . studies using saudi arabia or middle east area data reported r < 1, at 0.45-0.98 [11] [12] [13] [14] , though one reported 1.9-3.9 [15] . studies using south korea data showed higher values, at 2.5-8.09 [16] [17] [18] [19] , in the early stage, and < 1 in the later period [20] or with control intervention [19] . a total of eight studies reported the attack rate. four reported the overall or secondary attack rate, and the other four reported the attack rate of specific participant groups. two studies conducted in saudi arabia showed 0.42% [21] and 4% [22] secondary attack rates. studies in south korea showed secondary attack rates of 3.7% in one study [23] and 14.3-15.8% in another [24] . two studies reported the attack rate among healthcare workers (hcws). one study in south korea reported a mers incidence of 1.5% among hcws [20] , and another study using multiple area data reported a 13.4-13.5% infection rate among hcws [8] . the attack rate among hospital patients was 4% in one study [5] and 22% in the early and 1% in the later period in another [16] . the incubation period is the period between infection and appearance of signs of a disease. a total of 12 studies reported the incubation period of mers. nine used data from south korea and showed a 6-7.8 day incubation period [3] [4] [5] [23] [24] [25] [26] [27] [28] . one study using data from saudi arabia reported a 5.2 day incubation period [7] , and another using data from multiple areas reported a 5.5 day incubation period [6] . sha et al. compared the incubation periods between the middle east area and south korea and reported 4.5-5 and 6 days, respectively [29] . the serial interval of an infectious disease represents the duration between symptom onset of a primary case and of its secondary cases. two studies used south korea data, reporting serial intervals of mers of 12.6 and 14. 6 days, respectively [24, 26] . among five studies reporting days from onset to confirmation, three studies used data from south korea. one study analysing all south korea cases reported 5 days from onset to confirmation [3] . park et al. reported 6.5 days for all cases, 9 for second generation and 4 for third generation [28] . one study from taiwan reported 6 days for hcws and 10 for non-hcws [30] . a study from saudi arabia reported 4 days from onset to confirmation [31] . sha et al. compared the data from middle east and south korea areas and reported 4-8 and 4-5 days, respectively [29] . two studies from saudi arabia reported days from onset to hospitalization. one reported 2.9-5 days [32] , and the other reported 5.3 days [33] . twenty-six studies reported on mers-related mortality. ten reported the mortality rate in south korea as 14.5-47.8% [3, 4, 23-26, 28, 29, 34, 35] ; one of which, including all mers patients in south korea, reported a mortality rate of 20.4% [27] . ten studies analysing data from saudi arabia reported higher mortality rates, of 22-69. 2% [7, 12, 22, [31] [32] [33] [36] [37] [38] [39] , although others reported mortality rates 10% [40] and 19.9% [21] . a taiwanese study reported a mortality rate of 35.6% [30] . studies using data from multiple areas reported mortality rates ranging from 26.6% [29] to 59.4% [9, 41] . three studies reported days from mers onset to discharge. sha et al. reported 14 days in the middle east area and 17 in south korea [29] . one study from saudi arabia reported 17 days [36] , and another in south korea reported 20 [3] . two korean studies reported similar periods of 11-13 days from onset to death: 11-12.5 in park et al. [24] and 13 in ki et al. [3] . although one study from saudi arabia reported longer than 17 days from onset to death [36] , sha et al., comparing data between the middle east and south korea, reported similar periods of 11.5 and 11 days, respectively [29] . one taiwanese study also reported a similar period of 12-13 days [30] . two studies reported a similar length of hospitalization: 15 [33] and 15.2 days [19] . of the 20 studies included in the risk factor category, four were duplicates of studies in the epidemiologic category as they had information regarding the epidemiologic index and risk factors ( table 2) . two studies reported on the risk factors of mers infection. alraddadi et al. [42] analysed the effect of nonhuman contact, including travel history, animal-related exposure, food exposure, health condition, and behaviour and reported direct dromedary exposure, diabetes or heart disease, and smoking as risk factors of mers infection. another study reported older age, outbreak week, and nationality as risk factors [43] . three studies analysed factors associated with spreaders. non-isolated in-hospital days, hospitalization or emergency room visits before isolation, deceased patients, and clinical symptoms, including fever, chest x-ray abnormality in more than three lung zones, and the cycle threshold value, were related to spreaders [34, 44, 45] . four studies reported risk factors of mers severity. the included studies showed that the prnt 50 and cd4 t cell response [46] as well as a high mers virus load [47] were associated with the severity of mers. additionally, male sex; older age; concomitant disease, including hypertension; and symptoms, including fever, thrombocytopenia, lymphopenia, and low albumin concentration, were related to mers severity or secondary disease [47] [48] [49] . fifteen studies reported risk factors of mortality in mers patients. older age [4, 25, 32, [49] [50] [51] [52] [53] [54] [55] and comorbidity [29, [50] [51] [52] 54] , including diabetes [32, 55] , chronic kidney disease [32] , respiratory disease [4, 55] , pneumonia [56] , cardiac disease, and cancer [53] , were the most prevalent in the included studies. male sex was reported as a risk factor in one study [56] . smoking [32, 56] and location of acquisition [51, 53] were also reported. while one study noted that hcw, as a profession, was associated with mortality [53] , non-hcws were reported to be related to mortality in two other studies [50, 51] . additionally, a shorter incubation period [25, 56] , longer duration of symptoms [32] , more days from onset to confirmation [29] , later epidemic period [52] , and longer hospitalized days [29] were reported as mortality risk factors. symptoms at diagnosis, including abnormal renal function [56] , respiratory symptoms [56] , gastrointestinal symptoms [32] , lower blood pressure [32, 55] , and leucocytosis [55, 56] , were also found to be associated with mortality in mers patients. severity of illness, [50, 51] such as need for vasopressors [57] , chest radiographic score [58] , health condition [59] , use of mechanical ventilation [55] , and occurrence of dyspnoea [55] were also found to increase the mortality risk. the characteristics of mers differ between south korea and the middle east area. the r value of mers was reported to be below 1 in the middle east area, except in one study [15] , but was from 2.5-8.1 in south korea [15] [16] [17] [18] [19] . although studies using data from the middle east area reported 0.42-4% secondary attack rates, studies in south korea reported 4-6% secondary attack rates for patients or hospital visitors [5] , and 3.7-15.8% for the overall attack rate [23, 24] . the mers incubation period was reported to be 4.5-5.2 days in the middle east area [7, 29] , but this period was found to be slightly longer in south korea [3] [4] [5] [23] [24] [25] [26] [27] [28] . the severity of mers also differed between the middle east area and south korea. mortality of mers patients was found to be 20.4% in south korea based on a report including all cases [27] , but most studies from saudi arabia reported higher rates, from 22 to 69.2% [7, 22, 33, [37] [38] [39] . days from onset to confirmation were similar, 4-8 days in the middle east area [29, 31] and 4-6.5 days in south korea [3, 28, 29] . days from onset to discharge were slightly longer in south korea, 14-17 days in the middle east area [29, 36] and 17-20 days in south korea [3, 29] (table 3 ). the transmissibility and severity of mers were different by outbreak countries, especially between the middle east area and south korea. the virus, host, and environmental factors may be the causes of the mers outbreakrelated differences between the two regions. from the standpoint of viral factors, there was a mutation of the mers coronavirus (mers-cov) in the south korea outbreak. kim et al. [60] reported a point mutation in the receptor-binding domain of the viral spike protein in mers-cov, and another study showed that mers-cov in south korea had higher genetic variability and mutation rates [61] . individual characteristics can also affect mers transmission. as previous studies showed, there is an association between older age and mers infection [43] , severity [48] , and mortality [4, 50] , and the population structure may be related to transmission and severity. additionally, individuals aware of mers were found to be more likely to practice preventive behaviour [62] , which differed by demographic characteristics [63, 64] . the transmission environment may also contribute to the difference. while many mers cases were contracted through exposure to camels in saudi arabia [42] , the south korea outbreak involved multiple generations of secondary infections caused by intra-hospital and hospital-tohospital transmission [3, 65] . strategies considering various factors are therefore needed to assess the impact of mers and to better control its spread. although several studies have reported the overall r value [9, 10, 14, 19] , others have shown that this value this can be variable based on the generation or a control intervention [11, 16, 19] . especially in the south korea epidemic, the r value was particularly high in the early stage or first generation, at 4.42-5.4, though it later decreased to 0.14-0.39 [16, 19] . further studies should consider and analyse the variation of the r value depending on the period or control intervention. while earlier studies on infectious diseases assumed a homogeneous infection ability of a population, recent studies have shown the existence of so-called super spreaders, individuals with a high potential to infect others in many infectious diseases, including ebola and severe acute respiratory syndrome (sars) [66] . the role of the super spreader is also important in the spread of mers. in south korea, 83.2% of mers patients were associated with five super-spreading events [27] . stein et al. [67] asserted that super spreaders were related with the host, pathogen, and environmental factors, and wong et al. [66] reported that individual behaviours could also contribute to disease spread. there are variations in the mortality and attack rates among studies using south korea data. for example, park et al. [24] reported a 47.8% mers mortality, while reports from the korean ministry of health and welfare showed 20.4% mers mortality. this disparity may, in part, be due to small sample sizes. park et al. [24] included only 23 patients because the study was conducted in an early phase of a mers outbreak. we excluded studies that included cases with < 20 subjects, which were mostly case series, to reduce those types of biases. the present review found that older age and concomitant disease were risk factors of mers infection and mortality. these results are consistent with a recent systematic review that reported older age, male, and an underlying medical condition as predictors of death related to mers [68] ; therefore, these factors should be prioritized in protection and treatment procedures. one limitation of this study was the possibility of subject duplication. especially in south korea, the korean government publishes mers reports that include all patients. the epidemiologic index in other studies might be biased since they included partial korean patients and were analysed in the middle of an outbreak. however, we included those studies because they showed the characteristics of mers in different situations and different stages. we did not conduct a meta-analysis because of the small number of studies for each index, which might be another limitation of this study. although this study reviewed the risk factors of mers and their impact, assessing the effect size of each risk factor is important. more studies investigating the effect of risk factors on mers need to be constantly conducted. most studies on the transmissibility and severity of mers have originated from saudi arabia and south korea. even though the r 0 value in south korea was higher than that in saudi arabia, mortality was higher in saudi arabia. the most common factors behind mers infection and mortality were older age and concomitant disease. future studies should consider the risk of mers based on the outbreak region and patient characteristics. the results of the present study are valuable for informing further studies and health policy in preparation for mers outbreaks. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization; who mers-cov global summary and risk assessment mers outbreak in korea: hospital-to-hospital transmission korea centers for disease control & prevention. middle east respiratory syndrome coronavirus outbreak in the republic of korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study transmission characteristics of mers and sars in the healthcare setting: a comparative study interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department a pandemic risk assessment of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia assessment of the middle east respiratory syndrome coronavirus (mers-cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach high reproduction number of middle east respiratory syndrome coronavirus in nosocomial outbreaks: mathematical modelling in saudi arabia and south korea the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea estimating and modelling the transmissibility of middle east respiratory syndrome coronavirus during the 2015 outbreak in the republic of korea. influenza other respir viruses estimation of basic reproduction number of the middle east respiratory syndrome coronavirus (mers-cov) during the outbreak in south korea modeling the transmission of middle east respirator syndrome corona virus in the republic of korea surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity outcome of strict implementation of infection prevention control measures during an outbreak of middle east respiratory syndrome transmission of mers-coronavirus in household contacts hospital outbreaks of middle east respiratory syndrome outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon association between severity of mers-cov infection and incubation period preliminary epidemiological assessment of mers-cov outbreak in south korea -mnu1154-mnu0005-mnu0010&cid=70039. accessed 10 epidemiological investigation of mers-cov spread in a single hospital in south korea fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea middle east respiratory syndrome and medical students: letter from china diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data clinical characteristics and outcome of icu admitted mers corona virus infected patients risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia descriptive epidemiology and characteristics of confirmed cases of middle east respiratory syndrome coronavirus infection in the makkah region of saudi arabia mers-cov outbreak in jeddah-a link to health care facilities estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia taking stock of the first 133 mers coronavirus cases globally-is the epidemic changing? risk factors for primary middle east respiratory syndrome coronavirus illness in humans outbreak of middle east respiratory syndrome at tertiary care hospital clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea nosocomial amplification of mers-coronavirus in south korea recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses association of higher mers-cov virus load with severe disease and death, saudi arabia predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia spatial modelling of contribution of individual level risk factors for mortality from middle east respiratory syndrome coronavirus in the arabian peninsula the predictors of 3-and 30-day mortality in 660 mers-cov patients impact of comorbidity on fatality rate of patients with middle east respiratory syndrome the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia mortality risk factors for middle east respiratory syndrome outbreak, south korea clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak variations in spike glycoprotein gene of mers-cov association between australian hajj pilgrims' awareness of mers-cov, and their compliance with preventive measures and exposure to camels factors influencing preventive behavior against middle east respiratory syndrome-coronavirus among nursing students in south korea middle east respiratory syndrome-related knowledge, preventive behaviours and risk perception among nursing students during outbreak probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea the role of super-spreaders in infectious disease super-spreaders in infectious diseases clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis authors' contributions jep (corresponding author) designed the study, and conducted the data search and the analysis with jep (1st author). syj and ark participated in the data review. jep (corresponding) drafted the manuscript, and jep (1st), syj, and ark revised it. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. the authors declare that they have no competing interests. key: cord-315437-h6xjudm0 authors: nyon, mun peak; du, lanying; tseng, chien-te kent; seid, christopher a.; pollet, jeroen; naceanceno, kevin s.; agrawal, anurodh; algaissi, abdullah; peng, bi-hung; tai, wanbo; jiang, shibo; bottazzi, maria elena; strych, ulrich; hotez, peter j. title: engineering a stable cho cell line for the expression of a mers-coronavirus vaccine antigen date: 2018-03-27 journal: vaccine doi: 10.1016/j.vaccine.2018.02.065 sha: doc_id: 315437 cord_uid: h6xjudm0 abstract middle east respiratory syndrome coronavirus (mers-cov) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. to address this need, we are developing a subunit recombinant protein vaccine comprising residues 377–588 of the mers-cov spike protein receptor-binding domain (rbd), which, when formulated with the addavax adjuvant, it induces a significant neutralizing antibody response and protection against mers-cov challenge in vaccinated animals. to prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant mers s377-588 protein in chinese hamster ovary (cho) cells. to accomplish this, we transfected an adherent dihydrofolate reductase-deficient cho cell line (adcho) with a plasmid encoding s377-588 fused with the human igg fc fragment (s377-588-fc). we then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. using a gradually increasing methotrexate (mtx) concentration to 5 μm, we increased protein yield by a factor of 40. the adcho-expressed s377-588-fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected hek293t cells. in addition, hcd26/dipeptidyl peptidase-4 (dpp4) transgenic mice vaccinated with addavax-adjuvanted s377-588-fc could produce neutralizing antibodies against mers-cov and survived for at least 21 days after challenge with live mers-cov with no evidence of immunological toxicity or eosinophilic immune enhancement. to prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension cho cell line. with over 712 deaths and 2040 confirmed cases since its original appearance on the arabian peninsula in 2012 [1] , middle east respiratory syndrome (mers) coronavirus (mers-cov) has emerged as an important global pathogen and potential pandemic threat. there remains a critical need for a vaccine targeting mers-cov [2] , and the newly established coalition for epidemic preparedness innovation (cepi) has now designated research and development for the mers-cov vaccine as a global priority [3] . recently, phase i studies of dna-based vaccines against mers-cov showed that 98% of vaccinated volunteers generated antibody against mers cov [4] . however, to date there is no licensed dna vaccine for humans due in part to questions about their long-term safety, and their ability to induce high titers of protective or neutralizing antibodies relative to recombinant protein-based vaccines [5, 6] . a lead candidate for such a protein-based vaccine is the receptor-binding domain (rbd) of the mers-cov spike (s) protein. the mers-cov rbd plays an essential role in host receptor binding, membrane fusion, and cell entry [7] [8] [9] , thus making it an ideal vaccine target. moreover, focusing on the rbd component, rather than the full-length s protein, reduces the likelihood of eosinophilic-or antibody-dependent immune enhancement [10, 11] . expressed and purified as a recombinant fusion protein with the human fc fragment, the addavax(mf59-like)-adjuvanted rbd (residues 377-588) of mers-cov has been shown to elicit high neutralizing antibody titers in both mice and rabbits [9, [12] [13] [14] [15] [16] . these antibodies displayed potent neutralizing activity against almost 20 human and camel mers-cov strains, including those with amino acid mutations in the rbd region of their spike proteins [15, 17, 18] . in a complementary approach, recombinant rbd mutants representing different human and camel virus isolates were all able to elicit broad-spectrum neutralizing antibodies against a wide range of human and camel mers-cov strains [15] . antibodies against the rbd were able to block the binding of the rbd to the mers-cov's cellular receptor, dpp4 [17] , and thus block the viral entry into permissive human cells [18] . most importantly, recent studies found that these neutralizing antibodies were indeed associated with protection, when vaccinated ad5-hdpp4-transduced mice and hdpp4-transgenic mice were found to be immune against lethal mers-cov challenge [14, 19, 20] . although the vaccine antigen has been produced at small, laboratory scale in a transient hek293 cell system [17] , little effort has been put forth to develop and scale up of mers-cov rbd suitable for future vaccine manufacture. therefore, we have now engineered a stable cho cell line suitable for producing this mers-cov protein vaccine antigen. the codon-optimized dna sequence encoding the human igg fc-fused s377-588 and the signaling peptide of interleukin-2 (il2) residing at the n-terminus were synthesized and cloned into pjet2.1 using xbai and noti restriction sites (genescript) (pjet2.1_il2_s377-588-fc). sequences encoding signal peptides derived from igk light chain (igk), human serum albumin (sa), and azurocidin (azu) were incorporated into the mers s377-588-fc by touchdown pcr with ultramer oligomers (table 1, supp. table 1 ). the pcr products were gel purified using a qiaquick pcr purification kit (qiagen) and subsequently cloned into the poptivec-topo vector (invitrogen), followed by escherichia coli top10 transformation. ampicillin-resistant transformants were selected on lb agar plates containing 100 lg/ml of ampicillin and subsequently grown in lb broth. plasmid dna prepared from isolated colonies were sequenced. to construct the expression plasmid with the il2 signal peptide, pjet2.1_il2_s377-588-fc was digested with xbai and noti restriction enzymes, and the gene cassette was gel purified. the expression plasmid poptivec was digested with the same enzymes and gel purified, followed by ligation with t4 dna ligase. adherent, dihydrofolate reductase (dhfr)-deficient cho cells (adcho) (atcc ò crl-9096 tm , cho-dhfr) were cultured in iscove's modified dulbecco's medium (imdm, gibco) supplemented with 2 mm l-glutamine (gibco), 5% fetal bovine serum (fbs, gibco) and 0.1 mm sodium hypoxanthine/0.016 mm thymidine (h/t, gibco). to establish s377-588-fc-expressing adcho cell lines, 1 x 10 6 cells were transfected with 10 lg plasmid dna with different signal peptides using lipofectamine ò 2000 (invitrogen), according to the manufacturer's instructions. all plasmid dnas were linearized with ahdi prior to transfection. stable transfectants were selected by culturing in selective medium (same medium as previously described without h/t supplementation). cells were passaged every 6 days after reaching a maximum cell density of 1.2 â 10 6 viable cells per ml (split ratio 1:15). to investigate the effect of the different signal peptides on protein expression, conditioned medium from each transfection was collected for quantitative analysis. for gene amplification, transfected adcho cells were grown to confluence (6 days) in the selective medium supplemented with mtx. the concentration of mtx was increased gradually during each passage (6 days per passage) of adcho cells (20 nm > 100 nm > 200 nm > 400 nm > 600 nm > 800 nm > 1500 nm > 2000 nm > 3000 nm > 5000 nm, [21] ). the recombinant mers s377-588-fc was loaded onto a hitrap protein-a hp column (ge) at a flow rate of 1 ml/min. the column was washed with 1x pbs (ph 7.5, amesco) for 10 column volumes (cvs) and eluted with 3 cvs of 0-100% elution buffer (100 mm citric buffer ph 4.0), followed by 7 cvs of 100% elution buffer. the elution fractions (0.5 ml) were collected in tubes containing 0.2 ml of 1 m tris-hcl (ph 8.0) to elevate the ph of the eluted protein to ph 7.0. sds-page and western blotting analysis were performed with rabbit-anti-mers-cov rbd (1:2000, [13] ) and rabbitanti-bovine (fab) 2-biotin (1:4000, sigma) antibodies to identify the s377-588-fc protein in the elution fractions. the peak fractions containing the mers s377-588-fc protein were pooled and concentrated to 2 mg/ml with amicon ultra-15 centrifugal filter unit (mwco 10 kda) and buffer-exchanged into 20 mm tris-hcl and 20 mm nacl (ph 7.4). the protein secondary structure was predicted from circular dichroism (cd) spectra. samples for cd experiments were prepared in 20 mm citrate phosphate at a concentration of 0.2 mg/ml. cd spectra were recorded with a jasco j-1500 s spectrophotometer, scanning from 280 nm to 200 nm at 100 nm/min with a bandwidth of 1 nm and response time of 1 s. experiments were performed using one quartz cuvette with a path length of 0.1 cm, keeping a constant temperature of 25°c. the average value was determined after five scans, and the spectrum of the matching 'buffer alone' sample served as the control. the secondary structure was predicted using the cdpro software by comparing with three reference sets (sp43, sdp48 and smp56) and using two data fitting programs (contin and cdsstr). a real-time protein melt experiment was performed using protein thermal shift tm dye (thermo fisher scientific) in an applied biosystems viia 7 realtime pcr system (thermo fisher scientific), yielding a fluorescence table 1 overview of tested signal peptides in the adcho expression system. signal peptide signal peptide sequence ref. profile specific to purified mers s377-588-fc. to evaluate ptm, the purified protein was treated with peptide-n-glycosidase f (pngasef), o-glycosidase and neuraminidase in a nondenatured form and subsequently analyzed by gel electrophoresis. a co-ip assay was carried out to analyze the interactions between adcho-expressed mers s377-588-fc and human dpp4 (hdpp4) receptor in huh-7 cell lysates, using mers s377-588-fc expressed in hek293t cells [13] as a positive control and hek293t-expressing sars-cov rbd-fc as a negative control. the huh-7 cell lysates (5 â 10 7 /ml in 1 ml lysis buffer containing 0.3% n-decyl-d-maltopyranoside-phosphate-buffered saline [22] ) and sars-cov rbd-specific 33g4 mab (anti-sars-rbd, 1 lg/ml, [10] ). flow cytometry analysis was carried out to quantify the binding between mers-cov rbd and hdpp4-expressing huh-7 cells. cells were incubated with s377-588-fc (40 lg/ml), expressed either in adcho cells or hek293t cells, for 30 min at room temperature, followed by addition of fitc-labeled anti-human igg antibody (abcam) for 30 min. cells were then analyzed by flow cytometry. to detect binding between mers-cov rbd and hdpp4 protein, 96-well elisa plates were pre-coated overnight at 4°c with 50 ll of 2 lg/ml purified s377-588-fc protein, expressed either in hek293t cells [13] or in adcho cells, and blocked with 2% fatfree milk at 37°c for 2 h. serial dilutions of hdpp4 protein (histagged, 50 ll/well) were then added to the plates and incubated at 37°c for 1.5 h, followed by four washes with pbs in 0.05% tween-20 (pbst). subsequently, the plates were incubated with mouse anti-his primary antibody (1:2000, sigma) at 37°c for 1.5 h. after four washes with pbst, horseradish peroxidase (hrp)-conjugated anti-mouse igg antibody (1:5000, ge healthcare) was added to the wells and incubated at 37°c for 30 min. finally, plates were washed with pbst, and binding was visualized by adding the colorogenic substrate 3,3 0 ,5,5 0 -tetramethylbenzidine (tmb, sigma). the reaction was stopped after 10 min by adding 1 n h 2 so 4 , and absorbance at 450 nm (a450) was measured on an elisa plate reader (tecan). detection of the binding between mers-cov rbd and rbdspecific neutralizing mabs was performed following a protocol similar to that described above, except that the plates were precoated with 1 lg/ml of purified s377-588-fc proteins, followed by sequential incubation with serially diluted mouse mab (mers-mab1) or human mabs (m336-fab, m337-fab, and m338-fab) [23] [24] [25] and hrp-conjugated anti-mouse igg (1:3000, for mouse mab, ge healthcare) and anti-human-fab (1:5000, for human fab-mabs, sigma) antibodies. the binding between denatured mers-cov rbd and the aforementioned rbd-specific neutralizing mabs or rbd-immunized mouse sera was tested by elisa as described above, except that the plates were pre-coated with s377-588-fc (1 lg/ml) protein treated with dithiothreitol (dtt) (10 mm, sigma) at 37°c for 1 h, followed by incubation with iodoacetamide (50 mm, sigma) at 37°c for 1 h to stop the reaction [25] . after three washes, the elisa was carried out as described above. all in vitro and in vivo studies required the usage of infectious mers-cov (emc/2012 strain) and were conducted within approved biosafety level 3 (bsl-3) and animal bsl-3 laboratories at the galveston national laboratory, strictly following approved notification-of-usage (nou) and animal protocols and the guidelines and regulations of the national institutes of health and aaalac. for a ''proof-of-principal" study to confirm that adchoexpressed mers s377-588-fc is an effective and safe vaccine, two groups of five age-matched cd26/dpp4 transgenic (tg) mice were immunized twice, four weeks apart, via the intramuscular (i.m.) route, with either 10 lg of mers s377-588-fc formulated with addavax (invivogen) or pbs/addavax only (as control). this immunization protocol was selected because it is optimized for mers-cov rbd proteins [16] . the addavax adjuvant was chosen because it promoted the rbd-fc protein to generate the highest neutralizing antibodies among several adjuvants tested in our previous studies [14] . serum specimens were collected at day 28 after the second immunization through the retro-orbital bleeding route to determine the prospective capacity to neutralize infectious mers-cov by using the standard vero e6-based micro-neutralization test. immunized mice were subsequently challenged intranasally (i.n.) with 100x 50% lethal dose (ld 50 ) ($10 3 tcid 50 ) of mers-cov (emc/2012 strain), a gift of heinz feldmann (nih, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, the netherlands), followed by daily monitoring for the onset of clinical manifestations (e.g., weight loss and other clinical manifestations) and mortality. three mice from each group were euthanized at day 3 post-infection (p.i.) to assess lung viral loads by vero e6-based infection assay and quantitative (q) pcr analysis targeting the upe gene of mers-cov for quantifying infectious virus and viral rna, respectively. additionally, de-paraffinized lung tissues were hematoxylin-and-eosin (h&e)-stained for routine histopathologic evaluations, as described. we continued to monitor the remaining two mice in each group for their overall well-being for a total of 3 weeks until terminating the experiment. all methodologies required to assess the immunogenicity (neutralization antibody titers) and efficacy of mers s377-588-fc have been previously reported ( [19, 26] , supplementary methods). suspension cho (cho dg44, gibco, hereinafter termed sus-cho) cells were cultured in cd dg44 medium (gibco) supplemented with 8 mm l-glutamine (gibco) and 0.18% pluronic ò f-68 prior to transfection. transfection was performed by combining 18 lg ahdi-linearized plasmid popti_il2_s377-588-fc and 15 ll of freestyle tm max reagent (invitrogen) in 1.2 ml optipro sfm and incubated at room temperature for 10 min, followed by dropwise addition to 1.5 â 10 7 cells in 30 ml of cd dg44 culture medium (non-selective) according to the manufacturer's instructions. after 48 h, cells were transferred to selective medium (cd opti-cho, invitrogen), supplemented with 8 mm l-glutamine and 0.18% pluronic ò f-68 (gibco), and cultivated until cell viability reached 90%. after selection, stably transfected suscho cells underwent dna amplification by gradually increasing mtx concentration (20-5000 nm) in selective medium. all suspension culture flasks were maintained in a humidified incubator, 37°c/8% co 2 on a shaker, at a constant rotation rate of 135 rpm. the 5 lm mtx-adapted suscho cell pools from serum-free medium were used for single-cell cloning by limited dilution at 0.25-2 cells/well. cloning was performed in 96-well plates (falcon u-bottom untreated), utilizing a cloning medium composed of 80% hybridoma sfm (clonacell) and 20% conditioned media supplemented with 0.5x cho acf supplement (clonacell) at 37°c/5% co 2 for approximately 14 days. conditioned medium from wells with actively growing single colonies was assayed by elisa as follows. a mixture of 10 ll of conditioned medium and 90 ll of coating buffer were added to a 96-well elisa plate (thermo fisher scientific) and incubated at 4°c overnight. after washing the plate with pbst, rabbit anti-human igg was used to detect s377-588-fc protein, followed by a biotinylated goat antirabbit antibody and streptavidin hrp. tetramethylbenzidine (kpl inc., vwr) was added (100 ll) before the reaction was stopped with 100 ll 1 m hcl. elisa plates were read on an epoch microplate spectrophotometer (biotek instruments, inc) at 450 nm. clones which gave high absorbance reading were further propagated in 6-well plates, utilizing a cd opticho medium with 8 mm l-glutamine and 0.18% pluronic ò f-68. conditioned medium from 6-well plates was also screened by elisa for confirmation, and the highest expressing clone was selected and expanded by passaging in shake flasks (37°c, 8% co 2 in air on an orbital shaker platform rotating at 135 rpm). clonal cell lines and heterogeneous suscho cell pools were collected daily (up to 10 days) and counted using the acridine orange (ao) and propidium iodide (pi) nuclear staining dyes (nexcelom bioscience), which enter live cells and dead cells, respectively. conditioned medium from each time point was analyzed by sds-page, and the protein concentration was estimated by densitometry, comparing it to protein standards using the chemidoc tm imaging system (biorad). statistical significance was calculated by student's t test using graphpad prism statistical software. ⁄⁄⁄ indicates p < 0.001. in our previous studies, the human igg fc-fused s377-588 fragment of the mers-cov s protein (genbank afs88936.1) (hereinafter termed mers s377-588-fc) had already been expressed in transiently transfected hek293t cells [17] . however, to establish stably expressing cell clones, we transfected an adherent cho (adcho) with a poptivec construct containing an internal ribosome entry site (ires)-driven dhfr gene for selection and copy number amplification, as well as mers s377-588-fc gene fusion. signal peptides were added to the n-terminal end of the s377-588-fc gene in order to drive secretion into the culture medium (fig. 1a) . addition of gradually increasing methotrexate (mtx) to the culture medium of transfected cells resulted in binding to and inactivation of dihydrofolate reductase (dhfr) activity. transfected adcho cells compensated for this reduced dhfr activity by increasing the dhfr copy number in the genome to overcome inhibition by mtx. since the mers s377-588-fc fusion gene was integrated into the same genetic locus as that of the dhfr gene, the s377-588-fc gene was amplified, as well, leading to increased production of the protein. in the course of developing the adcho cell line expressing the s377-588-fc protein, optimization of a purification protocol using hitrap protein-a hp was performed. on the purification chromatogram, two protein peaks were observed in the elution step (fig. 1b) . denaturing sds-page and western blotting analysis with anti-mers-rbd-specific antibodies confirmed the second peak to be the s377-588-fc fragment (fig. 1c) . further analysis using non-denaturing sds-page and western blotting with rb-anti-bovine (fab)2 antibodies showed that the first peak was mainly contaminating bovine igg (fig. 1d-e) that originated from the fetal bovine serum (fbs) supplemented in the culture medium. we estimated relative productivity of adcho cells by measuring the ratio of the area of first peak and second peak. different signal peptides have been shown to result in different expression levels in cho cell systems [27] . therefore, we transfected adcho cells with linearized mers s377-588-fc expression plasmids with four different signal peptides at the n-terminus. peptides were derived from interleukin 2 (il2), igk light chain (igk), human serum albumin (sa), and azurocidin (azu). conditioned media from confluent monolayers of each transfected cell line were collected, followed by protein purification using protein a to establish yield and estimate relative productivity. in these studies, we found that adcho cells with the signal peptide derived from il2 showed 20-50% more secretion of s377-588-fc than cells with other signal peptides (fig. 2a) . elevated expression levels were achieved by gradually increasing mtx concentration during each cell passage from 20 nm to 5 lm. conditioned medium from transfected adcho cells was collected during the dna amplification process, followed by protein purification using protein a to estimate relative productivity (fig. 2b) . we observed the expected correlation between the expression of s377-588-fc and increased resistance to higher levels of mtx. compared to adcho cells with 20 nm mtx, expression of s377-588-fc was increased 39-fold in the presence of 3-5 lm of mtx (fig. 2c ). the purified protein was analyzed by circular dichroism (cd) spectroscopy. the mers-cov s377-588-fc consists mainly of beta-sheet (39.4%) and loop structures (53%) with limited helices (7.6%) (fig. 3a) . the secondary structure of the protein starts to unfold at 65°c. during thermal melt analysis, mers s377-588-fc had two endothermic transitions: 52.5°c and 67.5°c (fig. 3b) . a comparison between mers s377-588-fc expressed from hek293t cells and adcho cells was carried out using different page analyses. both proteins appeared to be identical following reduced and non-reduced sds-page. on native page and ief gels, adchoexpressed mers s377-588-fc had a lower isoelectric point (pi 6.6) when compared to the protein expressed in hek293t cells (pi 7.2) (fig. 3c) . after removal of n-linked glycans, the molecular size of mers s377-588-fc was slightly smaller than that of the n-linked glycosylated form (fig. 3d) . no change in molecular weight was observed after o-linked deglycosylation and desialylation (reduced sds-page). enzymatic treatment did not affect dimerization of mers s377-588-fc, as determined by nonreducing sds-page. the high pi (>7.4) isomers of s377-588 exhibited a lower shift (between pi 7.4 and pi 6.0) in electrophoretic mobility after n-linked deglycosylation, while no change of band pattern for the protein was seen after treatment with o-glycosidase. after removal of sialic acid through neuraminidase treatment, s377-588-fc isomers shifted to pi higher than 7.4 (fig. 3d) . three assays, including co-immunoprecipitation (co-ip), flow cytometry, and elisa, were performed to detect the binding of mers-cov rbd to its receptor, hdpp4. co-ip demonstrated that similar to the hek293t-expressed mers s377-588-fc protein, the rbd protein expressed in adcho bound strongly to hdpp4expressing huh-7 cells. two clear bands were identified from immunoprecipitated mixture of s377-588-fc and huh-7 cell lysates, and these bands were recognized by both anti-hdpp4 antibody and anti-mers-rbd antibody. in contrast, only one band was identified in huh-7 cells only and s377-588-fc protein only samples, and it was reactive with either anti-hdpp4 antibody or anti-mers-rbd antibody, but not with both antibodies. as expected, sars-cov rbd-fc protein was only recognized by sars-cov rbd-specific mab 33g4 (fig. 4a) . flow cytometry analysis further quantified the binding between mers-cov rbd protein and hdpp4 receptor in huh-7 cells. results showed a similar strong binding for all mers s377-588-fc proteins from adcho and hek293t, but not for sars-cov rbd-fc control (fig. 4b) . the elisa analysis demonstrated a dose-dependent binding between these mers-cov rbd proteins and hdpp4 protein, while no binding was observed between hfc control and hdpp4 (fig. 4c) . the antigenicity of mers-cov rbd proteins was carried out using elisa to test their binding with rbd-specific neutralizing antibodies (mersmab1, m336, m337, and m338), which recognize epitopes at rbd residues f506, d510, r511, w535, d539, y540, r542, or w553, and demonstrate strong activity to block rbd-hdpp4 receptor binding and neutralize mers-cov infection [23] [24] [25] 28] . similar to the s377-588-fc protein expressed in hek293t, the rbd proteins expressed in adcho bound strongly to mouse mab mersmab1 and human mabs m336, m337, and m338 in a dose-dependent manner (fig. 4d) , confirming their antigenicity. while these mabs bound strongly to the non-denatured (no dtt) s377-588-fc proteins expressed in both adcho and hek293t, they had significantly reduced affinity to rbd proteins treated with dtt, a reducing agent cleaving disulfide bonds of rbds and thus disrupting a protein's native conformation (fig. 4e) . these results demonstrate that the neutralizing mabs recognize conformational structures of mers-cov rbd [25] . transgenic mice expressing the human cd26/dpp4 receptor (hdpp4-tg) are a well-characterized animal model with which to evaluate the efficacy of vaccine candidates against mers-cov infection and disease [19, 20] . the immunogenicity of the mers s377-588-fc-based subunit vaccine was verified in hdpp4-tg mice. immune sera obtained from immunized mice were tested by elisa for the binding with denatured and non-denatured s377-588-fc protein or subjected to the vero e6-based micro-neutralization assay to quantify their capacity to neutralize infectious mers-cov. to evaluate protective efficacy against viral infection, viral loads and histopathology of the lungs of three mice in immunized and control groups were measured at day 3 after lethal challenge with 100x ld 50 of mers-cov. the remaining two mice in each group were monitored for morbidity (weight loss) and mortality to determine if this vaccine formulation would sufficiently protect against the disease and lethality caused by mers-cov infection. mers s377-588-fc induced high titers of rbd-specific igg antibodies, which reacted strongly with non-denatured s377-588-fc protein. nevertheless, these rbd-specific antibodies had significantly reduced activity with s377-588-fc treated by dtt (fig. 5) . this suggests that the rbd vaccine-induced antibody response was indeed directed towards one or more conformational epitopes. mice immunized with pbs/addavax uniformly failed to elicit any detectable neutralizing antibodies; however, those immunized with mers s377-588-fc/addavax consistently produced readily detectable titers of neutralizing antibody, ranging from 10-160 and 20-320 of neutralizing titer (nt)-100 (nt 100 ) and nt-50 (nt 50 ), respectively ( table 2) . consistent with the readily detectable neutralization antibodies, mers s377-588-fc/addavaximmunized mice were fully protected against viral infection and disease, as evidenced by the absence of recoverable infectious virus and negligible focal inflammatory responses, if any (supp. fig. 3) , within the infected lungs at 3 days post-infection (dpi). importantly, the remaining two immunized mice did not suffer any significant morbidity (weight loss) and survived until 21 dpi when the experiment was terminated. in contrast, pulmonary infectious viruses, albeit low in titers, were detected from all three unimmunized controls, with an average of 2.7 ± 0.1 tcid 50 /g of mers-cov recovered at 3 dpi, accompanied by mild inflammatory responses. the remaining two control mice suffered profound weight loss prior to succumbing to infection by 8 dpi. taken together, results of this ''proof-of-principle" pilot study indicated not only immunogenicity of mers-cov s377-588-fc, but also its efficacy and safety in the protection of hdpp4-tg mice against lethal challenge with mers-cov. similar to the adcho cell development described earlier, the dna copy number of stably transfected suspension cho dg44 dhfr-cells was amplified by gradual exposure to increased mtx concentrations of up to 5 lm. the resulting heterogeneous suscho cell pools were subsequently cloned by limited dilution to obtain a monoclonal cell population. elisa analysis was performed on the supernatants from individual clones, leading to the identification of clone 1b11 as the highest expressing clone. by comparing the growth curves of clone 1b11 to the heterogeneous (non-clonal) suscho cells, we discovered that clone 1b11 and heterogeneous suscho cells reached their maximum growth on the seventh day with viable cell counts of 8 â 10 6 cells/ml and 7 â 10 6 cells/ml, respectively (fig. 6) . through quantitative analysis using sds-page, we determined that the supernatant of clone 1b11 expressed approximately 97 mg/l of mers s377-588-fc, while the heterogeneous non-clonal cell pools expressed about 65 mg/l ( table 3 , supp. fig. 4 ) on the seventh day. the mers-cov rbd subunit fragment s377-588 has been identified to be a critical neutralizing receptor-binding fragment and an ideal candidate for the development of an effective mers-cov recombinant protein vaccine [13] . our aim here was to optimize expression and purification conditions suitable for pilot scale production of this rbd vaccine candidate. initially, both escherichia coli (bacteria) and pichia pastoris (yeast) expression systems proved well suited for recombinant vaccine production because of low production costs. however, our data showed that e. coli could not produce soluble mers s377-588, and yeast cells could not overexpress mers s377-588. thus, these two systems were not considered suitable to advance the mers vaccine candidate into process development and scale up production (see supplementary information for more detail). in previous reports, mers fc-fused s377-588 had been expressed in transiently transfected hek293t cells. however, transiently transfected cell lines may give low protein yield and potentially lose their production ability over time in continuously expressing recombinant proteins [29, 30] . unlike transient transfection, dna is integrated into cells long-term through stable transfection. despite the fact that the development of stably expressing cell lines is laborious and time-consuming, production with stable cell lines can be scaled up easily and would be suitable for use in manufacturing processes. hence, we generated a stably transfected adcho cell line by transfecting mtxdriven poptivec into cells to produce recombinant mers s377-588-fc protein. fc-fused gp120 protein was first constructed in 1989 as a potential candidate for aids therapy [31] . while there is no fda approved fc-fusion vaccine, vaccine development using fc-fusion proteins is active and ongoing. a number of studies have been initiated on the development of vaccines against ebola, hiv, influenza, as well as tuberculosis [32] [33] [34] [35] . it is noted that adverse side effects with vaccines are likely limited as biotherapeutic fc fusions have been repeatedly shown to be safe and biocompatible in humans. currently, all commercial therapeutics use the fc domain from human igg1, although other options, such as igg3, iga, and igm are also currently being explored [36] . furthermore, the fc domain is known to increase plasma half-life and simplify the purification process [36] . purification of s377-588-fc was performed using a protein a sepharose column, removing most of the impurities from the culture medium. bovine igg originates from fbs in the culture medium in constant amount and was used as a standard to gauge expression levels of the s377-588-fc protein. this estimation method allowed us to select the most suitable signal peptide and to evaluate the effect of the mtx-induced dna amplification process. since the poptivec plasmid has no signal peptide, four different signal peptides were subsequently tested at the n-terminus of the s377-588-fc sequence to drive secretion of the protein, leading to the identification of il2 signal peptide as the most suitable signal sequence. although signal peptides derived from human sa and human azu have been reported to improve production rates in other adcho systems [27] , the yield in our hands was lower than that with the il2 signal peptide. stable and highly productive cell pools were then isolated through a gradual increase in the mtx concentration in the culture [21] . analysis of the relative productivity of adcho cells indicated an increase in s377-588-fc in media proportional to the mtx concentration in the medium, reaching a plateau at 3 lm mtx. the biophysical and biochemical characterization of the mers s377-588-fc protein revealed that is was stable up to a temperature of 52.5°c. after the first major unfolding event at 52.5°c, another unfolding event occurred between 65 and 67.5°c, which could have resulted from destabilization of the ch2-ch2 bond of the fc domain of the recombinant protein [37] . no change in molecular weight was observed after removal of o-linked glycan on mers s377-588-fc, which suggested no o-linked glycosylation in the protein. after neuraminidase treatment, the recombinant protein band pattern showed fewer bands and a pi shift, suggesting that sialic acids might contribute to the charge of the protein. interestingly, although the complexities of the multiple protein bands were greatly reduced after glycosidase and sialidase treatments, the pattern representing charge heterogeneity remained, suggesting the existence of additional ptms, such as mannose-6phosphate, of the recombinant mers s377-588-fc protein. due to the charge differences between adcho-and hek293texpressed s377-588-fc proteins, we evaluated the functionality and antigenicity of the target protein. functionality studies, including co-ip assays, flow cytometry analyses, and elisa binding assay, confirmed that the adcho-expressed mers s377-588-fc protein maintained functionality equal to that expressed in hek293t cells in binding the dpp4 receptor of mers-cov, both of which showed dose-dependent binding with the soluble hdpp4 protein. in addition, mers s377-588-fc expressed in either adcho or hek293t demonstrated similar dose-dependent binding to rbd-specific neutralizing antibodies, an indicator that both s377-588-fc proteins could maintain sufficient antigenicity. we further investigated the protective efficacy of adcho-expressed s377-588-fc protein vaccine in protecting against mers-cov infection in the established transgenic mouse model expressing hdpp4 (hdpp4-tg). by formulating this s377-588-fc protein with adda-vax all vaccinated animals could produce neutralizing antibodies and survive a live viral challenge for 21 days. taken together, we confirmed the absence of functional, antigenic and immunogenic differences between adcho-and hek293t-expressed mers s377-588-fc proteins. moreover, mouse vaccinations with the rbd subunit vaccines did not appear to elicit eosinophilic or antibody-dependent immune enhancement. although we verified adcho-expressed mers s377-588-fc protein as an effective vaccine against mers-cov infection, the use of fbs in the growth medium proved unsuitable for a human vaccine antigen [38] . for both safety and compliance with future regulatory requirements, we therefore developed a stably transfected suspension cho cell line in serum-free medium. the adcho cell development process described here became the foundation for the establishment of the serum-free suspension cho cell line. subsequently, we transfected the poptivec expression plasmid with the il2 signal peptide into suscho cells and carried out the dna amplification as before. from the heterogeneous cell pools adapted to 5 lm mtx, we isolated clone 1b11, which was the highest expressing clone from a two-cycle screening process. in shake flasks, the growth of clone 1b11 was slightly slower than that from the heterogeneous cell pools, but 1b11 expressed 50% more mers s377-588-fc protein than the heterogeneous cell pool. typically, highly productive cell clones have lower growth rates since a significant portion of resources are used for expression of the recombinant protein [39] . additional experiments in the transgenic mice and non-human primate models will be needed to further determine the immunogenicity of mers s377-588-fc protein that produced by suscho. we envision that with a proper production process, the recombinant protein can be scaled up, manufactured, formulated and stockpiled as an efficient countermeasure against future mers-cov outbreaks. table 3 s377-588-fc expressed from heterogeneous non-clonal suscho cell pools and from the monoclonal clone 1b11. the protein concentration (mg protein per liter of culture supernatant) was determined by sds-page gel analysis (supp. fig. 3 ). who. middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus -a continuing risk to global health security vaccine development against prioritized epidemic infectious diseases inovio reports new positive clinical data on vaccine advances in the fight against emerging infectious diseases innate immune signaling by, and genetic adjuvants for dna vaccination the future of human dna vaccines receptor recognition mechanisms of coronaviruses: a decade of structural studies middle east respiratory syndrome: current status and future prospects for vaccine development vaccines for the prevention against the threat of mers-cov yeast-expressed recombinant protein of the receptor-binding domain in sars-cov spike protein with deglycosylated forms as a sars vaccine candidate roadmap to developing a recombinant coronavirus s protein receptor-binding domain vaccine for severe acute respiratory syndrome receptor-binding domain-based subunit vaccines against mers-cov searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus recombinant receptorbinding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines characteristics of early-and lateonset rapid eye movement sleep behavior disorder in china: a case-control study generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease evaluation of stable and highly productive gene amplified cho cell line based on the location of amplified genes a recombinant receptorbinding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection junctional and allelespecific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus optimized signal peptides for the development of high expressing cho cell lines mers-cov spike protein: a key target for antivirals evaluation of transfection methods for transient gene expression in chinese hamster ovary cells gene expression in mammalian cells and its applications designing cd4 immunoadhesins for aids therapy ebola virus glycoprotein fc fusion protein confers protection against lethal challenge in vaccinated mice a neonatal fc receptortargeted mucosal vaccine strategy effectively induces hiv-1 antigen-specific immunity to genital infection adjuvant-free immunization with hemagglutinin-fc fusion proteins as an approach to influenza vaccines apc targeting enhances immunogenicity of a novel multistage fc-fusion tuberculosis vaccine in mice fc-fusion proteins: new developments and future perspectives stabilisation of the fc fragment of human igg1 by engineered intradomain disulfide bonds a plea to reduce or replace fetal bovine serum in cell culture media advances in mammalian cell line development technologies for recombinant protein production identification of a receptorbinding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction this study was supported through the us-malaysian vaccine development program, funded by the university of malaya, and grants from the nih (r01ai098775-03s1 and r21ai128311). we thank drs. dimiter s. dimitrov and tianlei ying for providing m336, m337, and m338 mabs. the authors are involved in the development of a vaccine against mers coronavirus. supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vaccine.2018.02. 065. key: cord-332237-8oykgp0h authors: omrani, ali s; saad, mustafa m; baig, kamran; bahloul, abdelkarim; abdul-matin, mohammed; alaidaroos, amal y; almakhlafi, ghaleb a; albarrak, mohammed m; memish, ziad a; albarrak, ali m title: ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study date: 2014-09-29 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70920-x sha: doc_id: 332237 cord_uid: 8oykgp0h background: middle east respiratory syndrome coronavirus (mers-cov) infection is associated with high mortality and has no approved antiviral therapy. we aimed to compare ribavirin and interferon alfa-2a treatment for patients with severe mers-cov infection with a supportive therapy only. methods: in this retrospective cohort study, we included adults (aged ≥16 years) with laboratory-confirmed mers-cov infection and pneumonia needing ventilation support, diagnosed between oct 23, 2012, and may 1, 2014, at the prince sultan military medical city (riyadh, saudi arabia). all patients received appropriate supportive care and regular clinical and laboratory monitoring, but patients diagnosed after sept 16, 2013, were also given oral ribavirin (dose based on calculated creatinine clearance, for 8–10 days) and subcutaneous pegylated interferon alfa-2a (180 μg per week for 2 weeks). the primary endpoint was 14-day and 28-day survival from the date of mers-cov infection diagnosis. we used χ(2) and fischer's exact test to analyse categorical variables and the t test to analyse continuous variables. findings: we analysed 20 patients who received ribavirin and interferon (treatment group; initiated a median of 3 days [range 0–8] after diagnosis) and 24 who did not (comparator group). baseline clinical and laboratory characteristics were similar between groups, apart from baseline absolute neutrophil count, which was significantly lower in the comparator group (5·88 × 10(9)/l [sd 3·95] vs 9·88 × 10(9)/l [6·63]; p=0·023). 14 (70%) of 20 patients in the treatment group had survived after 14 days, compared with seven (29%) of 24 in the comparator group (p=0·004). after 28 days, six (30%) of 20 and four (17%) of 24, respectively, had survived (p=0·054). adverse effects were similar between groups, apart from reduction in haemoglobin, which was significantly greater in the treatment group than in the comparator group (4·32 g/l [sd 2·47] vs 2·14 g/l [1·90]; p=0·002). interpretation: in patients with severe mers-cov infection, ribavirin and interferon alfa-2a therapy is associated with significantly improved survival at 14 days, but not at 28 days. further assessment in appropriately designed randomised trials is recommended. funding: none. since it was fi rst described in september, 2012, 855 cases of middle east respiratory syndrome coronavirus (mers-cov) infection have been confi rmed, 333 of which were fatal. 1,2 cases occur sporadically, as community clusters or as hospital outbreaks, and range in severity from asymptomatic or mild illness to rapidly progressive and fatal disease. [2] [3] [4] [5] [6] the management of patients with mers-cov infection consists of a combination of supportive measures, antimicrobial therapy for any associated bacterial or viral infections, and strict implementation of appropriate infection control precautions. 7 so far, no antiviral therapy has been approved for the treatment of patients with mers-cov infection. 8 several therapeutic interventions for coronavirus were investigated during the large multinational outbreak of severe acute respiratory syndrome (sars) in 2003. 9, 10 reviews of the available scientifi c literature suggest that a combination of ribavirin and interferon might be of benefi t in patients with severe mers-cov infection. 8, 11, 12 furthermore, this combination was shown to inhibit mers-cov in cell culture and seemed to improve outcomes in an animal study. 13, 14 both agents are associated with substantial potential adverse eff ects and hence their clinical use should be carefully balanced against any potential harm. 8 we aimed to assess outcomes of a treatment programme for patients with severe mers-cov infection that consisted of oral ribavirin and subcutaneous pegylated interferon alfa-2a. we report the results and outcomes in patients given treatment in accordance with this protocol by comparison with a historical group who received supportive therapy only. this single-centre, retrospective cohort study included individuals who were diagnosed with laboratoryconfi rmed mers-cov infection between oct 23, 2012, and may 1, 2014, at the prince sultan military medical city (riyadh, saudi arabia). eligible patients were those aged 16 years or older with severe pneumonia needing invasive or non-invasive ventilation. no exclusion criteria were applied at this stage. mers-cov infection was diagnosed by rt-pcr testing of respiratory tract samples for mers-cov upe, orf 1b, and n genes. 15 all rt-pcr tests for mers-cov were done at the saudi ministry of health regional laboratory in jeddah and riyadh, saudi arabia. pneumonia was defi ned as new, otherwise unexplained, lower respiratory tract symptoms such as cough or shortness of breath with at least one systemic feature such as fever or chills, and new focal chest signs on examination, in addition to new or progressive pulmonary infi ltrates on chest radiograph. 16 from sept 16, 2013, all eligible patients were off ered treatment with oral ribavirin and subcutaneous pegylated interferon alfa-2a after informed written consent had been obtained from the patients themselves or their next of kin. the treatment protocol was approved by pharmacy and therapeutics committee. the study was approved by the research ethics committee at the prince sultan military medical city to allow retrospective access to patients' records and fi les. pegylated interferon alfa-2a (pegasys; roche pharmaceuticals, basel, switzerland) was given by subcutaneous injection at a dose of 180 μg per week for 2 weeks. the dose of oral ribavirin (copegus; roche pharmaceuticals) was adjusted according to calculated creatinine clearance and continued for 8-10 days. 12 patients with a creatinine clearance of greater than 0·833 ml/sec/m 2 received a 2000 mg loading dose, followed by 1200 mg every 8 h for 4 days then 600 mg every 8 h for 4-6 days; those with a creatinine clearance of 0·333-0·833 ml/sec/m² received a 2000 mg loading dose, followed by 600 mg every 8 h for 4 days then 200 mg every 6 h for 4-6 days; and those with a creatinine clearance of <0·333 ml/sec/m² or on dialysis received a 2000 mg loading dose, followed by 200 mg every 6 h for 4 days then 200 mg every 12 h for 4-6 days. patients did not receive ribavirin and interferon alfa-2a therapy if they were diagnosed before sept 16, 2013, or if they declined consent. all patients received appropriate supportive care such as supplementary oxygen, vasopressor therapy, and renal replacement as needed. hydrocortisone 200 mg daily was given to patients with refractory septic shock and continued until vasopressor therapy was no longer needed. 17 in addition to regular clinical monitoring, renal function, liver enzymes, and blood count were assessed at baseline and daily throughout the treatment course. conscious patients were monitored for any clinical signs of depression or acute confusion. patients who received ribavirin and interferon alfa-2a therapy were classifi ed as being in the treatment group and those who did not made up the comparator group. two investigators, aso and kb, both of whom were masked to group allocation and the patients' clinical outcomes, compared baseline characteristics of the two groups. immunosuppressive therapy 1 (5%) 4 ‡ (18%) 0·66 data are number (%) or mean (sd). apache ii=acute physiology and chronic health evaluation ii. sofa=sequential organ failure assessment. *only 19 patients were assessed. †only 13 patients were assessed. ‡only 22 patients were assessed. the primary endpoints for the study were 14-day and 28-day survival from the date of mers-cov infection was diagnosis. we used χ² and fischer's exact tests for categorical variables, whereas we used the student's t test for continuous variables to assess the diff erences in means of the two groups. the log-rank test was used for assessing survival diff erences between the two groups. our cutoff for statistical signifi cance was 0·05. the graphical and statistical tests suggested that the pro portionalhazard assumption was not violated. we did statistical analyses using microsoft excel 2011 and stata statistical software, release 12. no external funding was received for this study. zm had full access to all the data in the study and had fi nal responsibility for the decision to submit for publication. 70 individuals were diagnosed with mers-cov infection between oct 23, 2012, and may 1, 2014. baseline characteristics were generally similar between patients who received ribavirin and interferon alfa-2a therapy and those who did not (table 1) , with the exception that end-stage renal failure was present in three patients who did not receive study treatment and in none who did. after excluding ineligible patients, 44 patients were included in the study: 20 in the treatment group and 24 in the comparator group (fi gure 1). the mean age of all 44 patients was 65·5 years (sd 18·2), and 32 (73%) were men (table 1) mean absolute neutrophil count was signifi cantly lower in the treatment group than in the comparator group (table 1) ; however, no other statistically signifi cant differences in baseline characteristics or support measures were noted between the two groups (tables 1, 2). of all 44 patients, 21 (48%) were still alive 14 days after diagnosis of mers-cov infection, whereas at 28 days only ten (23%) had survived. 14 (70%) of 20 patients in the treatment group were alive 14 days after diagnosis, compared with seven (29%) of 24 in the comparator group (p=0·004). however, six (30%) of 20 patients in the treatment group survived up to 28 days from diagnosis of mers-cov infection, whereas four (17%) of 24 did in the comparator group (p=0·054; fi gure 2). ribavirin and pegylated interferon therapy was well tolerated by the treatment group with no premature discontinuation secondary to adverse eff ects. however, the mean drop in haemoglobin over the treatment course was signifi cantly greater in the treatment group (4·32 g/l [sd 2·47]) than in the comparator group eff ective treatment interventions for patients with severe mers-cov infection are still urgently needed. in critically ill patients with severe mers-cov infection, our study shows that ribavirin and pegylated interferon alfa-2a therapy is associated with a signifi cant 14-day survival benefi t compared with standard treatment. 28-day survival also seemed to improve with ribavirin and pegylated interferon alfa-2a therapy, but the diff erence between groups was not signifi cant (panel). the loss of a signifi cant survival diff erence over time might be partly explained by most patients in our cohort having several comorbidities with high apache ii and sofa scores. mortality is known to be very high in patients with severe mers-cov infection who need critical-care support. 24 therefore, long-term survival benefi t, if present, might be diffi cult to show in smaller studies. treatment with ribavirin and interferon was well tolerated in our study. the only adverse event that was signifi cantly worse in the treatment group was mean decrease in haemoglobin (4.32 g/l in the treatment group compared with 2·14 g/l in the comparator group). anaemia is a well recognised complication of ribavirin therapy and was noted previously in studies investigating the role of ribavirin in the treatment of sars coronavirus infection. 25, 26 of note, receipt of packed red blood cells was not signifi cantly diff erent between the treatment and comparator groups in our study. furthermore, no treatment discontinuations occurred as a result of anaemia. therefore, the risk of ribavirin-associated anaemia-although substantial and in need of careful monitoring-might not hinder the use of ribavirin for patients with severe mers-cov infection, especially if a survival benefi t can be confi rmed. baseline absolute neutrophil count was signifi cantly lower in the treatment group and therefore a signifi cantly lower minimal absolute neutrophil count during the course of the illness is not surprising. several investigators showed that interferon α has useful in-vitro activity against mers-cov. 13, 20, 26 however, when compared with interferon α and interferon γ, interferon β seems to have the most potent inhibitory in-vitro activity against mers-cov. 21 ribavirin has slight anti-mers-cov activity in vitro when used alone or in combination with interferon α. 13, 22 mycophenolic acid is another compound that exhibits signifi cant in-vitro activity against mers-cov. 21 of 290 compounds screened, 60 were active in cell culture against mers-cov. 23 although only the combination of interferon α plus ribavirin has so far undergone in-vivo assess ment against mers-cov, 14 many others are potential candidates for further clinical assessment. one of the limitations of our study is its small size. however, only two previous reports of clinical use of ribavirin and interferon for mers-cov infection have been published. 18, 19 in a retrospective report, fi ve patients with severe mers-cov infection, all of whom had signifi cant comorbidities and needed mechanical ventilation, received a com bination of ribavirin and pegylated interferon alfa-2b a median of 19 days after admission. none of the patients survived and the investigators concluded that late commencement of therapy might not be benefi cial. 18 in another report, 19 a patient with severe mers-cov infection received ribavirin and interferon therapy with good clinical response and no signifi cant adverse eff ects. our study, albeit small, is the largest clinical investigation so far to assess the use of this combination in the treatment of patients with severe mers-cov infection. although baseline characteristics of our treatment and comparator groups seem to be reasonably balanced, substantial diff erences might not be apparent because of the small number of patients in the study. our study is also limited by its retrospective, nonrandomised nature. inevitably, selection and unmeasured confounding bias cannot be completely excluded. undoubtedly, new interventions should ideally be assessed in randomised, controlled clinical trials. however, such an approach is generally accepted to not always be practically feasible in the context of an emerging and relatively uncommon disease such as mers-cov infection. 8 we carefully selected our comparator group, ensuring that the two cohorts were matched as closely as possible in their clinical characteristics and treatment interventions other than the receipt of ribavirin and interferon. we removed three individuals who had outlying baseline serum creatinine from the comparator group to minimise the risk of any spurious conclusions driven by clinical characteristics that might be potentially detrimental to clinical outcome. clinical outcomes for each individual were masked from investigators who selected patients and did matching assessments. the absence of serial viral load measurement in lower respiratory tract samples in our study makes it impossible to show any association between temporal viral load changes and antiviral therapy. such measurements should be included in any future clinical studies exploring the therapeutic benefi t of any antiviral intervention for patients with mers-cov infection. severe mers-cov is associated with poor overall survival. treatment with oral ribavirin and subcutaneous pegylated interferon alfa-2a is associated with signifi cantly improved survival at 14 days, but not at 28 days. the combination is associated with signifi cant falls in haemoglobin, but no other signifi cant adverse eff ects were noted. treatment with ribavirin and pegylated interferon might be considered in patients with severe mers-cov infection, provided that adequate monitoring and assessment can be ensured. further assessment, including in patients with less severe mers-cov infection, in appropriately designed randomised trials, is recommended. this study was initiated and designed by aso, mms, zam, and ama. aso, mms, ab, ma-m, aya, gaa, mma, zam, and ama obtained and collated patient data. kb undertook all statistical analyses for the study. aso and kb prepared all tables and fi gures. aso, mms, zam, and ama wrote the fi rst draft of the manuscript and all authors reviewed and contributed to subsequent drafts and the fi nal report. aso has received consultancy fees from gilead, pfi zer, msd, and viiv; payment for lectures from pfi zer, msd, glaxosmithkline, and sanofi -aventis; and sponsorship to attend international meetings and conferences from msd, pfi zer, biopharma, bristol-myers squibb, and janssen-cilag. ab has received travel funding to attend an international meeting from pfi zer. gaa has received travel funding to attend an international meeting from edwards lifesciences. all other authors declare no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia european centre for disease prevention and control. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov)-11th update epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case hospital outbreak of middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical management of severe acute respiratory infections when novel coronavirus is suspected: what to do and what not to do international severe acute respiratory and emerging infection consortium (isaric) the severe acute respiratory syndrome sars: systematic review of treatment eff ects applying lessons from sars to a newly identifi ed coronavirus therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)--possible lessons from a systematic review of sars-cov therapy inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques assays for laboratory confi rmation of novel human coronavirus (hcov-emc) infections surveillance defi nitions for specifi c types of infections surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon (ifn)-alpha-2b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment interferon-beta and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada severe acute respiratory syndrome: report of treatment and outcome after a major outbreak we thank staff of prince sultan military medical city, riyadh, saudi arabia, for the clinical care given to the patients and for facilitating access to the relevant medical records. we searched pubmed for reports published in english any time before june 24, 2014, with the search term "[(mers-cov or hcov-emc or novel coronavirus) and (therapy or interferon or ribavirin]". we found one animal study, 14 two small case series in human beings, 18, 19 and several in-vitro studies. 13, [20] [21] [22] [23] the data suggested that combination therapy with ribavirin and interferon alfa could have potential benefi ts for patients with severe mers-cov infection. this is, to our knowledge, the largest clinical study done so far assessing the potential benefi t and safety of combination therapy with pegylated interferon alfa-2a plus ribavirin in patients with severe mers-cov infection. because we noted a signifi cant 14-day survival benefi t in patients who received the combination compared with those who received supportive therapy only, but no survival benefi t at 28 days, we recommend further assessment in appropriately designed randomised clinical trials to provide further information about the role of this combination in the treatment of patients with severe mers-cov infection. key: cord-307109-nz8qvuw6 authors: martinez, miguel angel title: compounds with therapeutic potential against novel respiratory 2019 coronavirus date: 2020-04-21 journal: antimicrob agents chemother doi: 10.1128/aac.00399-20 sha: doc_id: 307109 cord_uid: nz8qvuw6 currently, the expansion of the novel human respiratory coronavirus (known as sars-cov-2 [severe acute respiratory syndrome coronavirus 2], covid-2019 [coronavirus disease 2019], or 2019-ncov [2019 novel coronavirus]) has stressed the need for therapeutic alternatives to alleviate and stop this new epidemic. the previous epidemics of infections by high-morbidity human coronaviruses, such as sars-cov in 2003 and the middle east respiratory syndrome coronavirus (mers-cov) in 2012, prompted the characterization of compounds that could be potentially active against the currently emerging novel coronavirus, sars-cov-2. the most promising compound is remdesivir (gs-5734), a nucleotide analog prodrug currently in clinical trials for treating ebola virus infections. remdesivir inhibited the replication of sars-cov and mers-cov in tissue cultures, and it displayed efficacy in nonhuman animal models. in addition, a combination of the human immunodeficiency virus type 1 (hiv-1) protease inhibitors lopinavir/ritonavir and interferon beta (lpv/rtv–ifn-β) was shown to be effective in patients infected with sars-cov. lpv/rtv–ifn-β also improved clinical parameters in marmosets and mice infected with mers-cov. remarkably, the therapeutic efficacy of remdesivir appeared to be superior to that of lpv/rtv–ifn-β against mers-cov in a transgenic humanized mouse model. the relatively high mortality rates associated with these three novel human coronavirus infections, sars-cov, mers-cov, and sars-cov-2, have suggested that proinflammatory responses might play a role in the pathogenesis. it remains unknown whether the generated inflammatory state should be targeted. therapeutics that target the coronavirus alone might not be able to reverse highly pathogenic infections. this minireview aims to provide a summary of therapeutic compounds that have shown potential in fighting sars-cov-2 infections. they mainly produce respiratory tract infections, as observed with sars-cov and middle east respiratory syndrome coronavirus (mers-cov) (7, 8) . sequencing and phylogenetic analyses have shown that the novel sars-cov-2 strain is closely related to a group of human sars-like coronaviruses and bat sars-related coronaviruses (9) (10) (11) . the origin of sars-cov-2 remains unclear; it is unknown how it was first transmitted to humans. the high prevalence of sars-related coronaviruses in bats has suggested that a bat coronavirus might have jumped into a civet or some other mammal, and from there to humans, which started the former 2003 sars (2003-sars) epidemic. initial confirmed cases of sars-cov-2 were associated with huanan seafood and live-animal markets. however, no animal source has been identified to date, and spillover events may continue to occur. although bats might be the source of sars-cov-2, it is critical to identify the intermediate species to stop the current spread and to prevent future human sars-related coronavirus epidemics. a key issue is whether the current sars-cov-2 epidemic is similar to other sars outbreaks or whether it shows different features. the epidemiological and clinical characteristics of sars-cov-2 indicate that this new outbreak is different from the 2003-sars outbreak. sars-cov-2 displays higher transmissibility and lower mortality than 2003-sars (1, 3, 4) . sars-cov-2 has shown efficient intrafamilial spread (4) . the asymptomatic period of sars-cov-2 infections oscillates between 2 and 14 days, and some individuals probably transmit the virus without developing any disease symptoms. it remains to be elucidated whether this virus replicates more readily in the upper airway than sars-cov and mers-cov and whether it is similar to other human coronaviruses (hcovs) that cause colds but not pneumonia. it will be necessary to identify molecular determinants that mediate transmission from animal to human and from human to human. of note, in the novel sars-cov-2 strain, the nucleotide sequence of the external ectodomain in the spike protein receptor-binding domain is different from that of the 2003 sars-cov. when individual bat coronavirus spike genes were introduced into sars-cov infectious clones, the sars-cov/bat-cov spike viruses could bind to the human, bat, or civet angiotensin converting enzyme 2 (ace2) cellular receptor (12) . understanding the interaction between this novel sars-cov-2 spike protein and the host ace2 receptor might reveal how this virus overcame the species barrier between animals and humans. as discussed below, this information might promote the design of effective antivirals. to predict new zoonotic coronavirus jumps across species and to understand the rate of virus spread among people, it is crucial to determine whether sars-cov-2 is mutating to improve its binding to human receptors for infection. as an rna virus, sars-cov-2 has intrinsic genetic variability, which results in a high mutation rate. moreover, coronaviruses have the largest genomes (ϳ30 kb) among rna viruses. however, part of their sequence encodes a proofreading 3= exonuclease that can increase replication fidelity (13) . it has been suggested that any adaptation in the sars-cov-2 sequence that might make it more efficient at transmitting from person to person might also increase its virulence (14) . however, this mechanism could lead to a genetic bottleneck, known as muller's ratchet, which could significantly decrease viral fitness (15) . muller's ratchet predicts that, when mutation rates are high and a significant proportion of mutations are deleterious, a type of irreversible ratchet mechanism gradually reduces the mean fitness of small populations of asexual organisms. because genetic bottlenecks for rna viruses often occur during respiratory droplet transmissions, the sars-cov-2 is expected to become less virulent through human-to-human transmissions (16) . from the public health perspective, we urgently need to develop an effective vaccine and antiviral therapeutics to stop the sars-cov-2 epidemic. moreover, social and economic issues generated by this epidemic also call for rapid interventions. this review focuses on the potential of repurposing preexisting compounds that might provide new opportunities for treating people infected with sars-cov-2. previous work with sars-cov and mers-cov has provided an opportunity to accelerate the identification of meaningful therapies for fighting the novel sars-cov-2 epidemic. neverthe-less, we must be aware that, currently, no compound that targets sars-cov or mers-cov has moved beyond phase 1 trials. the most promising antiviral for fighting sars-cov-2 is remdesivir (gs-5734). remdesivir is an adenosine nucleotide analogue prodrug with broad-spectrum antiviral activity against filoviruses, paramyxoviruses, pneumoviruses, and pathogenic coronaviruses, like sars-cov and mers-cov (17) . pharmacokinetic studies have been completed and clinical trials are ongoing for testing remdesivir efficacy in treating ebola virus (18) . previous studies have indicated that nucleotide analogues generally show low efficacy against coronaviruses, due to the presence of the virus exonuclease proofreading enzyme. nevertheless, remdesivir was found to be effective against sars-cov, mers-cov, and bat cov strains (17) . in tissue cultures, remdesivir displayed half-maximal effective concentrations (ec 50 s) of 0.069 m for sars-cov and 0.074 m for mers-cov. of note, tissue culture studies have shown that remdesivir is also active in the submicromolar ec 50 range against a number of highly divergent coronaviruses, including the endemic human covs oc43 (hcov-oc43) and 229e (hcov-229e). thus, remdesivir has broad-spectrum anti-cov activity (19) . in a mouse model of sars-cov pathogenesis, prophylactic and early therapeutic administration of remdesivir significantly reduced the lung viral load. viral titers were reduced by ͼ2 orders of magnitude on day 4 or 5 postinfection. remdesivir improved the clinical signs of disease and respiratory function compared to untreated control animals (17) . comparable results were obtained with mers-cov in prophylactic studies carried out with a mers-cov mouse transgenic model. in that model, a humanized mers-cov receptor (humanized dipeptidyl peptidase 4 [hdpp4]) was expressed and carboxylesterase 1c (ces1c) was deleted to improve the pharmacokinetics of nucleotide prodrugs (20) . remdesivir specificity for coronavirus was demonstrated by propagating the virus in tissue culture. after 23 passages in the presence of drug, two mutations were identified (f276l and v553l) in the viral rna-dependent rna polymerase gene. these mutations increased the replication capacity of the virus in the presence of remdesivir (21) . however, these amino acid changes decreased the viral fitness and attenuated sars-cov pathogenesis in mice (21) . the efficacy of prophylactic and therapeutic remdesivir treatment was recently tested in a nonhuman primate (rhesus macaque) model of mers-cov infection (22) . when prophylactic remdesivir treatment was initiated 24 h prior to inoculation, mers-cov was prevented from inducing clinical disease and inhibited from replicating in respiratory tissues, which prevented the formation of lung lesions. similar results were obtained when therapeutic remdesivir treatment was initiated at 12 h after virus inoculation (22) . human safety data are available for remdesivir (18) ; thus, human trials can be initiated for testing the efficacy of this compound against novel coronaviruses. therapies that are approved by the food and drug administration (fda) have been evaluated for antiviral activity against sars-cov and mers-cov. for example, lopinavir (lpv), a human immunodeficiency virus 1 (hiv-1) protease inhibitor, was combined with ritonavir (rtv) to increase the lpv half-life. the combination of lpv and rtv (lpv/rtv) was shown to be effective against sars-cov in patients and in tissue culture. the estimated ec 50 in fetal rhesus kidney-4 cells was 4 g/ml (23) . lpv/rtv also reduced weight loss, clinical scores, viral titers, and disease progression in marmosets infected with mers-cov (24) . nevertheless, the antiviral activity of lpv against mers-cov in tissue culture remains controversial. no optimal ec 50 was found in vero cells (25) , but an ec 50 of 8 m was reported in huh7 cells (26) . clinical observations in animals and humans showed that mers-cov infections were mediated by both virus replication and host inflammatory responses. those findings led to explorations of combination therapies that included type i interferon (ifn-i) and ifn-ii. interferon beta (ifn-␤) displayed the best efficacy, with ec 50 s of 1.37 to 17 iu/ml, for reducing mers-cov replication in tissue culture (25, 27) . similarly to lpv/rtv, clinical improvements with ifn-␤ were observed in common marmosets infected with mers-cov (24) . in the kingdom of south arabia, an ongoing randomized control trial (the miracle trial) was initiated to determine whether the combination of lpv/rtv and ifn-␤ could improve clinical outcomes in mers-cov infections (28) . importantly, an-other controlled trial was launched in china to test the efficacy of lpv/rtv and ifn-␣ 2b in hospitalized patients with sars-cov-2 infections (clinicaltrials registration no. chictr2000029308). the prophylactic and therapeutic properties of remdesivir and lpv/rtv-ifn-␤ were previously compared in a humanized transgenic mouse mers-cov infection model (29) . remdesivir improved pulmonary function, reduced lung viral loads, and ameliorated severe lung pathology. in contrast, prophylactic lpv/rtv-ifn-␤ reduced viral loads only slightly and did not impact other disease parameters, and therapeutic lpv/rtv-ifn-␤ improved pulmonary function but did not reduce virus replication or severe lung pathology (29) . overall, these results indicated that remdesivir showed more potential than lpv/rtv-ifn-␤ for treating mers-cov infections. ribavirin, a guanosine analogue, is an antiviral compound used to treat several virus infections, including respiratory syncytial virus, hepatitis c virus, and some viral hemorrhagic fevers. in most cases, ribavirin is combined with ifn. ribavirin was first marketed in 1980 for the treatment of respiratory syncytial virus in children. although promising results were obtained previously with ribavirin and ifn-␣ 2b in a mers-cov rhesus macaque model (30) , data have been conflicting on patients with mers-cov infections that were treated with a combination of ribavirin and ifn (either ifn-␣ 2a or ifn-␤1) (31) . however, ribavirin reduces hemoglobin concentrations, an undesirable side effect in patients with respiratory disorders. this feature reduces its potential as an antiviral against sars-cov-2. work with influenza virus has shown that monoclonal and polyclonal antibodies can be useful prophylactic and therapeutic tools. several antibodies have been shown previously to bind influenza virus hemagglutinin and inhibit virus replication (12) . for example, human immunoglobulin g1 (igg1) monoclonal antibody (mhaa4549a) binds to a highly conserved epitope on the stalk of influenza a virus hemagglutinin. in a phase 2 human influenza a virus challenge study, mhaa4549a significantly reduced the clinical symptoms and viral burden relative to placebo (32) . another example is vis410, a monoclonal antibody engineered to target all known influenza a virus strains. a phase 2a trial showed that vis410 had some clinical benefits (33) . current development efforts in monoclonal and polyclonal antibodies against coronaviruses are mainly targeting mers-cov. in a phase 1 clinical trial, a human polyclonal antibody, sab-301, which is generated in trans-chromosomic cattle, was found to be safe and well tolerated in healthy participants. (34) . however, therapeutic treatment with human monoclonal antibodies did not protect against the severe disease or the loss of lung function induced by mers-cov in animal models (20) . the lack of viral sequence homology among different human coronaviruses suggests that current investigational antibody-based therapeutics will not be effective against novel virus variants. nevertheless, in considering future treatments for novel coronaviruses, immune-based therapies should be not discarded. another potential treatment option could be the use of novel coronavirus sera prepared from the blood of patients in convalescence (convalescent-phase sera). passive immunization is well established for viral infection prophylaxis. polyclonal antibody products have been licensed that target cytomegalovirus, hepatitis b virus, and varicella-zoster virus. a meta-analysis of reports on the 1918 influenza a virus (h1n1) epidemic concluded that early administration of convalescent blood products reduced the absolute risk of pneumonia-related death from 37% to 16% (35) . nevertheless, the appropriate titer of convalescent-phase sera antibody that is required for therapeutic efficacy against sars-cov-2 remains to be determined. moreover, additional studies performed with influenza virus have produced controversial results regarding the clinical benefit of administering high titers of anti-influenza immunoglobulins (36) . finally, it remains unclear whether methods based on the use of a sufficient pool of potential donors are feasible. work carried out with mers-cov showed that sera from patients recovering from infections did not contain sufficient antibody titers for therapeutic use (37) . another interesting therapeutic alternative that was previously explored with influ-enza virus was that of targeting cellular components involved in the host inflammatory response to the infection. for example, the activation of the inflammatory response to an infection can induce a cytokine outburst that results in an acute lung injury. an example of a therapy for this type of infection has been targeting of cellular toll-like receptor 4 (tlr4) with specific antibodies. tlr4 is a transmembrane protein that belongs to the pattern recognition receptor (prr) family. the prototype pathogenassociated molecular pattern (pamp) that tlr4 recognizes is that corresponding to the gram-negative bacterium endotoxin lipopolysaccharide (lps). tlr4 has been implicated in pathology associated with other infections and with tissue damage caused by noninfectious insults. tlr4 activation leads to activation of the nf-b intracellular signaling pathway and to inflammatory cytokine production, which in turn activate the innate immune system. interestingly, tlr4-null mice were highly resistant to infection by the mouse-adapted influenza a virus (38) . thus, protection against influenza virus infections was achieved by targeting tlr4 with small-molecule antagonists, like tak-242, or with anti-tlr4-specific antibodies (39, 40) . indeed, targeting a cellular protein would overcome the drawbacks associated with virus or coronavirus genetic heterogeneity. the high mortality rates observed in some emerging respiratory diseases induced by viruses like mers-cov, sars-cov, and novel influenza a virus strains (h5n1) have given rise to the hypothesis that the proinflammatory response might be involved in the disease pathogenesis. consequently, immunosuppressants (e.g., corticosteroids) might be used as an adjunct for treating severe forms of the disease. however, the therapeutic use of immunosuppressants is not free of controversy. to date, no conclusive results have been found for the effects of immunosuppressants in severe influenza virus infections (12) . furthermore, the use of corticosteroids to treat influenza virus has been associated with an increased risk of superinfection, prolonged viral replication, and an increased risk of death (41) . in contrast, corticosteroid treatment for mers-cov infections was not significantly associated with mortality, although a delay in mers-cov rna clearance was observed (42) . further studies should be performed to clarify the potential clinical benefit of prescribing immunosuppressants for coronavirus infections. to end this minireview, i discuss an interesting potential antiviral strategy. the spike protein of sars-cov mediates viral entry into target cells. intriguingly, cleavage and activation of the sars-cov spike protein by a host cell protease are essential for infectious viral entry (43) . this host protease could be a type ii transmembrane serine protease, tmprss2, which was shown to cleave and activate sars-cov spike protein in cell cultures. therefore, tmprss2 is a potential a target for antiviral interventions. for example, the serine protease inhibitor camostat mesylate inhibits the enzymatic activity of tmprss2 (44) . recently, administration of k11777, a cysteine protease inhibitor, in the subnanomolar range was shown to inhibit sars-cov and mers-cov replication in tissue cultures (45) . moreover, the clinically proven serine protease inhibitor camostat mesylate, which is active against tmprss2, partially blocked sars-cov-2 spike-driven entry into caco-2 and vero-tmprss2 cells (46) . future tissue culture and animal model studies should be conducted to clarify the potential antiviral activity of targeting tmprss2. by the end of february 2020, 2 months after the first cases of sars-cov-2 were reported in china, several hundreds of new infection cases had been registered, mainly in other asian regions and europe. this news has strongly suggested that we are in the thick of a sars-cov-2 pandemic. social alarm and health authorities have called for the development of therapeutic alternatives for fighting the current and, possibly, new coronavirus epidemics. animal models and clinical studies are urgently needed for evaluating the effectiveness and safety of promising antiviral compounds that target the virus and/or the immunopathology involved in the host responses. the identification and characterization of novel compounds and therapeutic alternatives will be required to better control this probable pandemic outbreak. novel coronavirus (2019-ncov) situation reports clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china, 2020. zhonghua liu xing bing xue za zhi (in chinese isolation of a novel coronavirus from a man with pneumonia in saudi arabia identification of a novel coronavirus in patients with severe acute respiratory syndrome genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding china novel coronavirus investigating and research team. 2020. a novel coronavirus from patients with pneumonia in china advances in respiratory virus therapeutics -a meeting report from the 6th isirv antiviral group conference correlation between mutation rate and genome size in riboviruses: mutation rate of bacteriophage q␤ a novel coronavirus outbreak of global health concern fitness of rna virus decreased by muller's ratchet rapid fitness losses in mammalian rna virus clones due to muller's ratchet broadspectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses a randomized, controlled trial of ebola virus disease therapeutics broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase a mouse model for mers coronavirus-induced acute respiratory distress syndrome coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment with lopinavir/ritonavir or interferon-␤1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture interferon-␤ and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays treatment of middle east respiratory syndrome with a combination of lopinavirritonavir and interferon-␤1b (miracle trial): study protocol for a randomized controlled trial comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov treatment with interferon-␣2b and ribavirin improves outcome in mers-cov-infected rhesus macaques effect of ribavirin and interferon on the outcome of critically ill patients with mers phase 2 randomized trial of the safety and efficacy of mhaa4549a, a broadly neutralizing monoclonal antibody, in a human influenza a virus challenge model safety and efficacy of monoclonal antibody vis410 in adults with uncomplicated influenza a infection: results from a randomized, double-blind, phase-2, placebocontrolled study safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-doseescalation study meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a(h1n1) infection feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol the tlr4 antagonist eritoran protects mice from lethal influenza infection tlr4 antagonist fp7 inhibits lps-induced cytokine production and glycolytic reprogramming in dendritic cells, and protects mice from lethal influenza infection a decoy peptide that disrupts tirap recruitment to tlrs is protective in a murine model of influenza corticosteroids as adjunctive therapy in the treatment of influenza. cochrane corticosteroid therapy for critically ill patients with middle east respiratory syndrome evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry protease inhibitors targeting coronavirus and filovirus entry sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor this work was supported by the spanish ministry of science and innovation (saf2016 -75277-r).i declare that i have no conflict of interest. key: cord-322760-tsxniu3j authors: sha, jianping; li, yuan; chen, xiaowen; hu, yan; ren, yajin; geng, xingyi; zhang, zhiruo; liu, shelan title: fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea date: 2016-09-23 journal: arch virol doi: 10.1007/s00705-016-3062-x sha: doc_id: 322760 cord_uid: tsxniu3j middle east respiratory syndrome coronavirus (mers-cov) was first isolated in 2012. the largest known outbreak outside the middle east occurred in south korea in 2015. as of 29 june 2016, 1769 laboratory-confirmed cases (630 deaths; 35.6 % case fatality rate [cfr]) had been reported from 26 countries, particularly in the middle east. however, the cfr for hospital outbreaks was higher than that of family clusters in the middle east and korea. here, we compared the mortality rates for 51 nosocomial outbreaks in the middle east and one outbreak of mers-cov in south korea. our findings showed the cfr in the middle east was much higher than that in south korea (25.9 % [56/216] vs. 13.8 % [24/174], p = 0.003). infected individuals who died were, on average, older than those who survived in both the middle east (64 years [25–98] vs. 46 years [2–85], p = 0.000) and south korea (68 years [49–82] vs. 53.5 years [16–87], p = 0.000). similarly, the co-morbidity rates for the fatal cases were statistically higher than for the nonfatal cases in both the middle east (64.3 % [36/56] vs. 28.1 % [45/160], p = 0.000) and south korea (45.8 % [11/24] vs. 12.0 % [18/150], p = 0.000). the median number of days from onset to confirmation of infection in the fatal cases was longer than that for survivors from the middle east (8 days [1–47] vs. 4 days [0–14], p = 0.009). thus, older age, pre-existing concurrent diseases, and delayed confirmation increase the odds of a fatal outcome in nosocomial mers-cov outbreaks in the middle east and south korea. electronic supplementary material: the online version of this article (doi:10.1007/s00705-016-3062-x) contains supplementary material, which is available to authorized users. the first report of middle east respiratory syndrome (mers) was identified in saudi arabia in june 2012. the middle east respiratory syndrome coronavirus (mers-cov) isolated from this patient was similar to severe acute respiratory syndrome coronavirus (sars-cov), which caused an epidemic in 2002-2003 [49] . both novel viruses are single-stranded rna viruses belonging to the genus betacoronavirus [25, 48] , and the diseases they cause share common clinical characteristics, including fever, cough, diarrhea, and shortness of breath [5] . jianping sha, yuan li, and xiaowen chen equally contributed to this work. as of 29 june 2016, the world health organization (who) had been notified of 1769 laboratory-confirmed cases with mers-cov (globally), including at least 630 deaths; the case fatality rate (cfr) was 35.6 % (630/1769) [46] . a total of 26 countries in the world have been affected, including countries in the middle east (egypt, iran, jordan, kuwait, lebanon, oman, qatar, saudi arabia, united arab emirates, yemen), africa (algeria, tunisia), europe (austria, france, germany, greece, italy, the netherlands, turkey, the united kingdom), asia (china, the republic of korea, malaysia, philippines, thailand) and north america (united states) (http://www.who.int/ emergencies/mers-cov/en/). so far, all cases of mers have been linked through travel to or residence in countries in or near the middle east. generally, the middle east is the primary region where mers-cov originates, circulates and is exported. in contrast, since the first report of sars-cov in china in 2003, a total of 8096 sars cases, including 774 deaths, have been reported to who. these have involved 19 countries, predominantly in south east asia, with only one case identified in kuwait in 2003, and no cases were found in the middle east since then (http:// www.who.int/csr/sars/country/table2004_04_21/en/). the fatality risk for mers-cov is much higher than that for sars-cov, which has a cfr of 9.6 % [9, 24] . furthermore, the cfr for patients with co-morbidities is greater (60 % in mers vs. 46 % in sars) than those without preexisting diseases [49] . generally, the cfr is attributed to both host factors and virus factors (e.g. virus replication and mutation) and local medical expertise [3, 14] . one unique common epidemiological characteristic of these two diseases is that the spread of both mers-cov and sars-cov infection has been largely driven by human-to-human transmission in healthcare settings [25] . failures in infection prevention and control in healthcare settings have occasionally resulted in large numbers of secondary cases in nosocomial outbreaks. the earliest identified nosocomial mers outbreak was traced back to march 2012 from clusters of severe respiratory illness among healthcare personnel (hcp) in jordan [43] . since then, a series of nosocomial mers outbreaks in small numbers have been identified in the middle east (jordan in 2012, saudi arabia in 2014-2015) [1, 6, 10, 18, 36] in this study, we conducted a preliminary mortality risk factor analysis for nosocomial mers-cov outbreaks in south korea and the middle east. the findings from this study might help to reduce the severity and number of deaths from hospital-clustered cases by leading to the adoption of appropriate control measures. in 2015, scientists in the republic of korea and china completed full-genome sequencing of coronaviruses from the mers outbreak in korea. the findings were analysed by a group of virologists convened by who, and preliminary results suggested that the mers cov viruses isolated in the republic of korea were similar to those isolated in the middle east (http://www.who.int/mediacentre/news/ mers/briefing-notes/update-15-06-2015/en/). mers-covs associated with the korean and middle east outbreak belong to lineage 5 of mers-cov, which has been the predominant infectious agent in saudi arabian camels since november 2014 [41] . the mers-cov variants associated with the recent outbreak of human infections in south korea (e.g., chinagd01-v1/2015 and kor/knih/ 002-05/2015) show the highest similarity (99.96-99.98 %, full genome) to a camel virus (camel/riyadh/ry159/2015) sampled in march 2015, followed by the latest strain (kt026453) prevalent in saudi arabia (99.92 % identified) [26] . however, the mers-covs in korea have the ability to cause large outbreaks in environments that are different from that of the middle east (http://www.who.int/emer gencies/mers-cov/en/). the national health and family planning commission of china determined that the collection of data from one human mers-cov infection imported from korea was part of the public health investigation of an outbreak and was exempt from institutional review board assessment. all other mers cases were obtained from publicly available data sources. all data were supplied and analysed without access to personal identifying information. information on all laboratory-confirmed mers cases was obtained from various publicly available sources, including who global alert and response updates, documents officially released by the local health bureau, news releases from middle eastern and south korean authorities, the weekly epidemiological record, promed posts, and literature published from 1 april 2012 to 29 june 2016 (http:// www.who.int/csr/don/archive/disease/coronavirus_infections/ en/). the latest cases that had not been officially announced by who were identified by searching promed posts, which confirmed announcements by individual countries' ministries of health. based on the available data, we initially established a database of a line list of human nosocomial mers outbreaks (supplementary tables s1, s2 and s3). according to the who's 14 july 2015 interim reporting definition (http://www.who.int/csr/disease/coronavirus_ infections/case_definition/en/), a person with mers has a laboratory-confirmed mers-cov infection, irrespective of clinical signs or symptoms. a case may be laboratoryconfirmed by detection of viral nucleic acid or serology. the presence of viral nucleic acid can be confirmed by either a positive reverse transcription polymerase chain reaction (rt-pcr) result on at least two specific genomic targets or a single positive target with sequencing of a second target. a case confirmed by serology requires demonstration of seroconversion in two samples, ideally taken at least 14 days apart, by enzyme-linked immunosorbent assay (elisa), by indirect fluorescent antibody (ifa) screening, or by a neutralization assay [25, 49] . a direct epidemiological link with a confirmed mers-cov patient may include (1) healthcare-associated exposure, including providing direct care for mers-cov patients, working with healthcare workers infected with mers-cov, visiting patients or staying in the same close environment of individuals infected with mers-cov; (2) working together in close proximity or sharing the same classroom environment with individuals infected with mers-cov; or (3) travelling together with individuals infected with mers-cov in any kind of conveyance or living in the same household as individuals infected with mers-cov. in addition, the epidemiological link may have occurred within a 14-day period before or after the onset of illness in the case under consideration [25] . we used a comparative epidemical analysis of the dates of onset of illness and the characteristics of the fatal and surviving cases. all statistical analysis was conducted using the statistical analysis system, version 9.2 (sas institute, cary, nc, usa). quantitative measurements are presented as the median and range of the observed values, and qualitative measurements are presented as relative and absolute frequencies. an analysis of variance (f test) was applied to the measurement data. v 2 tests were used to compare the distribution of the different variables of qualitative measurements between fatalities and survivors. fisher's exact test was used in the analysis of contingency tables when the sample sizes were small (the expected values in any of the cells of a contingency table were below 5; the number of total samples was no more than 40; the data were very unequally distributed among the cells of the table). any p-values given were two-sided and considered statistically significant at 0.05. as of 31 march 2016, we had identified 47 human laboratory-confirmed clusters with mers-cov, involving 179 cases, of which 53 were fatal. all clusters had been reported to who or published by the local authority or in pubmed. these mers-clustered cases were distributed in nine countries: 29 clusters from the kingdom of saudi arabia (ksa), six from the united arab emirates (uae), four from jordan, three from qatar, and one each from france, iran, italy, tunisia, and the united kingdom (uk). the numbers of clusters per year were as follows: three clusters including 18 cases in 2012, 33 clusters including 108 cases in 2014, and 11 clusters including 53 cases in 2014. for the control groups, we chose a total of 504 sporadic cases of mers-cov, composed of 129 fatal and 375 nonfatal cases from the following countries: 350 cases from the ksa, 125 cases from the uae, 10 cases from jordan, 10 from qatar and 9 from tunisia. the numbers of sporadic cases per year were as follows: 110 cases in 2012, 350 cases in 2013 and 44 cases in 2014. fatality risks for nosocomial mers outbreaks 35 the results showed that the percentages of hcp in mers clusters were much higher than those in sporadic cases (32.4 % [58/179] vs. 10.7 % [54/504], p = 0.000) ( table 1 and table s1 ). however, the hcp-specific cfr was much lower than the overall cfr from mers clusters ( , p = 0.000). the median age in fatal cases in hcp was much lower than in fatal cases overall (46.5 years vs. 57 years , p = 0.000) ( table 1) . we stratified the age groups between the fatal and nonfatal cases groups. the results showed a statistical difference in the distribution of the 0-14, 15-29, 30-44, 45-59, and 60? year-olds between the two groups (p = 0.000). males dominated both the fatal and nonfatal groups of the clustered and sporadic cases (p [ 0.05) ( table 1) . a history of exposure to camels prior to onset of disease was not significantly correlated with survival (7. five time periods useful for public health surveillance were evaluated. the median time from onset to confirmation of infection in the fatal groups was much longer than that for survivors in mers clusters (12.5 days [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] vs. 9 days [0-24], p = 0.041) and in sporadic mers cases (12 days vs. 9 days [0-30], p = 0.003). however, there were no statistical differences in the median time from onset to hospital admission, onset to hospital discharge, and subsequent death or the number of hospitalized days between the fatal and nonfatal cases for the two groups (table 1) . by 30 march 2016, we had obtained data on 51 nosocomial outbreaks involved in 216 confirmed cases (all 51 nosocomial outbreaks were from the middle east; the above 47 clusters were not included in these outbreaks), including iran (one cluster), ksa (41 clusters), jordan (three clusters), france (one cluster) and uae (five clusters). we also had one nosocomial outbreak with 174 confirmed cases with mers-cov in south korea (table 2 and table s2 ). the observed average cluster size (174) for mers from south korea was much greater than that for the middle east (4, range 2-28). human nosocomial outbreaks with mers-cov in the middle east occur throughout the year and peak in the spring, especially february to april. mers outbreaks in south korea were reported from march to june 2015, concomitant with peaks in the reporting of mers nosocomial outbreaks in the middle east ( table 2) . the average age in the fatal groups was much higher than that in the survival groups (64 years old vs. table 2) . we found no difference between the fatal and nonfatal cases with respect to exposure to camels and other animals (horses, sheep and goats). in contrast, the level of humanhuman transmission was much higher in the nonfatal cases in the middle east than in the fatal cases (86. table 2) . the middle east group showed a statistical difference between fatal and nonfatal cases for the median days from 165], p = 0.000). however, there were no differences in the percentage of total deaths between the index and secondary cases ( table 3) . the mean age of the deaths was significantly higher than that of the survival cases for the index (64 years vs. 54 years [24-85, p = 0.038) and secondary cases (43 years vs. 37 years , p = 0.030). patients in the age groups c60 and 45-59 years were the most common in the fatal and survival cases, respectively, for the index group, while the 45-59 and 30-44-year age groups were the common groups in the fatal and nonfatal cases, respectively, for the secondary cases. there was no significant difference in gender distribution between the fatal and nonfatal cases in the index and secondary groups ( table 3) . the ratio of co-morbidity was much higher in the fatal groups than in the non-fatal groups from the secondary cases (37.5 % [12/32] vs. 17.1 % [19/111], p = 0.026); however, there was no difference between the fatal and nonfatal groups from the index cases. similarly, a history of exposure prior to onset was common for the fatal and nonfatal groups from the index and secondary cases ( table 3) . there were no differences between fatal and nonfatal cases in the median time from onset to hospitalization, onset to confirmation, onset to discharge or death or hospitalized days (table 3) . however, the median time from onset to hospitalization was shorter in the secondary cases compared to the index cases (3 days the secondary cases was slightly shorter than in the index cases (9 days vs. 14 days , p = 0.033); however, the median time from onset to hospital discharge for secondary survivors was 10 days (6-18), which was significantly shorter than the 14 days (3-31) for index survivors (p = 0.025). acute respiratory tract infections with mers-cov cause considerable morbidity and mortality and pose a threat of repeated outbreaks in healthcare facilities [1, 6, 10, [18] [19] [20] 38] . the resulting transmission among patients, visitors, and hcp has been a defining feature of mers-cov epidemiology since its emergence in 2012 [7] . in this study, we compared the mortality risk factors in two different nosocomial outbreaks, based on 51 nosocomial outbreaks of mers-cov infection in the middle east and one large outbreak identified in south korea. our findings showed the final cfr for the middle east (25.9 %) was significantly higher than that for south korea (13.8 %). both estimated cfrs were significantly lower than that for one hospital outbreak of mers (cfr 65 % [15/23] ) in saudi arabia in 2013 and another nosocomial outbreak (cfr 36.5 % [93/255]) in saudi arabia 2014 [5, 36] . the cfr of this latter outbreak was also much higher than that of one extended family cluster (10.5 % [2/ 19]) in saudi arabia in 2014 [4] . these results demonstrate that the survival rate of clustered patients with mers-cov in korea was higher than in the middle east. there are several possible explanations for the observed differences between the cfrs in south korea and the middle east. first, there may be disparities in national surveillance and available expertise [30] . second, the cfr for the middle east might have been overestimated because a large number of mild and asymptomatic cases are likely to go undetected there [37] . third, it is possible that primary cases accounted for a higher percentage of the cluster patients in the middle east than in south korea [36] . the findings on age were consistent in hospital outbreaks in the middle east and from south korea. our results showed that the median age in fatal cases was much higher than that in nonfatal cases. this is in agreement with a saudi arabian case series report that showed individuals older than 65 years had a greater association with mortality. a multivariate logistic regression model estimated that for every 1-year increase in age, the odds of dying increased by 12 % [29] . in all, this indicates that older age is associated with death in cases of mers-cov infection [12, 17, 44] . in particular, the median age in fatal hcp cases was also much higher than that in nonfatal hcp cases, but lower than the overall average. this is in agreement with the findings of liu et al. [25] . the reasons for the higher fatality rates in older individuals are not understood but have been attributed to cultural practices that result in an increase in the exposure risk that older people are willing to take [37] . in addition, older people may be more likely to smoke and to have underlying diseases and impaired immune functions, which may increase susceptibility and progression of infections and even increase the chance of death [45] . the sex characteristics of mers outbreaks in the middle east are similar to those in south korea. the patients in mers outbreaks in both areas were predominantly male, and the proportion of males in the study populations did not differ [25] . furthermore, there was no difference in the male-specific cfr between the mers clusters of the two groups, a finding that is similar to other reports [1, 2, 10, 18] . our findings suggest that the gender distribution is not linked to a fatal risk factor in mers outbreaks. hcp are at high risk of acquiring emerging mers infections due to occupational exposure and are affected mostly by nosocomial outbreaks [1, 6, 15, 28, 35] [7] . in total, the fatality risk for hcp was significantly lower than the overall fatality risk in the middle east and south korea. these findings can be attributed to three facts: first, the majority of hcp developed asymptomatic or mild symptoms and moderate symptoms [15] ; second, hcp were confirmed as secondary cases under medical investigation, which led to earlier confirmation and good outcomes [32] ; third, epidemiological analysis showed that hcp were much younger and had fewer co-morbidities compared to total mers cases [36] . in contrast with sars, about 75 % of patients with mers had at least one additional illness, and patients who died were more likely to have an underlying condition (86 % of patients who died vs. 42 % of recovered or asymptomatic patients) [47, 49] . similar to the middle east, this study showed that the odds of dying were four times higher for individuals with concurrent health conditions than for those without these conditions in south korea. the odds of fatality were much lower than those estimated by the logistic regression model (seven times) [29] . this is in part due to higher viral loads in the respiratory tract and longer shedding in patients with underlying diseases compared to cases without co-mortalities [33, 49] . human-to-human transmission of mers-cov has been confirmed by epidemiological and genomic studies of cases associated with hospital and household mers outbreaks [13] . spread was assumed to occur largely via large droplets and contact [36] . our study indicated that the percentage of human-to-human transmission in nonfatal cases was slightly higher (92.9 % vs. 64.2 %) than in fatal cases in mers clusters, and two reasons could explain this: first, the survivors in secondary cases were younger and had fewer co-morbidities [11, 19, 20, 29, 38] ; second, most of the secondary cases were under medical investigation, and therefore, the infection could be confirmed early once symptoms were observed, making timely treatment possible [16, 19, 20, 23, 36, 39, 42] . overall, human-to-human transmission seems to have had a positive effect on the outcome of the secondary cases from the mers nosocomial outbreaks in the middle east. rapid diagnosis and providing supportive care may be of marginal consequence in the mers clusters [25, 29] . the progression of illness in fatal and nonfatal infections in nosocomial outbreaks with mers-cov in the middle east does not follow the typical pattern of south korea infections [29] . in the middle east, the median time from onset to confirmation in fatal cases (8 days) was clearly longer than in nonfatal cases (4 days). in south korea, however, there was no difference in the median time between fatal and nonfatal cases. this is consistent with other retrospective studies of mers virus infections [6, 30, 36] . furthermore, the time between suspected symptom onset and laboratory confirmation (6.5 days) in the fatal clusters was also slightly longer than the overall average [38] . in particular, this finding indicated that delayed confirmation is a high-risk factor for human nosocomial outbreaks with mers-cov in the middle east. in conclusion, the overall cfr for nosocomial outbreaks in the middle east was much higher than in south korea. however, the mortality risk factors for mers infections in the middle east were similar to those identified for nosocomial outbreaks in south korea. older age, underlying diseases and delayed confirmation were the major risk factors for fatal outcome in human nosocomial outbreaks. in contrast, person-to-person transmission was associated with a good outcome for secondary cases during nosocomial outbreaks. interestingly, gender, exposure history and median days were not indicators of death with mers nosocomial outbreaks. the severity of nosocomial outbreaks and the risk of fatal infection in hcp were significantly lower than the overall rate in the middle east and south korea. nosocomial outbreaks of mers-cov infection are associated with knowledge deficits, unrecognized disease, insufficient infection control measures, poor compliance, and an overwhelming number of patient cases [21, 22, 34, 40, 45] . therefore, early and rapid detection of suspected cases, especially in older people and hcp, along with appropriate infection control practices, education and timely preparedness, are important strategies to reduce nosocomial transmission and to improve the clinical outcome in health settings in the future [8, 27, 31, 35] . hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome coronavirus transmission in extended family epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia infection control and prevention practices implemented to reduce transmission risk of middle east respiratory syndrome-coronavirus in a tertiary care institution in saudi arabia analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission preliminary epidemiological assessment of mers-cov outbreak in south korea clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia association of higher mers-cov virus load with severe disease and death middle east respiratory syndrome coronavirus (mers-cov) nosocomial outbreak in south korea: insights from modeling fatal outcome of sars in a patient with reactivation of chronic hepatitis b middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia mers outbreak in korea: hospital-to-hospital transmission epidemiologic features of the first mers outbreak in korea: focus nosocomial transmission of sars co-circulation of human metapneumovirus and sarsassociated coronavirus during a major nosocomial sars outbreak in hong kong middle east respiratory syndrome (mers) in asia: lessons gleaned from the south korean outbreak risk factors for sars-related deaths in 2003 comparative epidemiology of human infections with middle east respiratory syndrome and severe acute respiratory syndrome coronaviruses among healthcare personnel complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china infection prevention and control guidelines for patients with middle east respiratory syndrome coronavirus (mers-cov) infection first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission mortality risk factors for middle east respiratory syndrome outbreak, south korea middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome coronavirus infection control: the missing piece? screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome middle east respiratory syndrome corona virus, mers-cov middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications mers-cov outbreak in jeddah-a link to health care facilities a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case epidemiological investigation of mers-cov spread in a single hospital in south korea middle east respiratory syndrome-advancing the public health and research agenda on mers-lessons from the south korea outbreak factors associated with nosocomial sars-cov transmission among healthcare workers in hanoi co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia the mers-cov outbreak: challenges facing the dental profession risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu fatal aspergillosis in a patient with sars who was treated with corticosteroids critical role of nosocomial transmission in the toronto sars outbreak fatality risks for nosocomial mers outbreaks 43 middle east respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mers-related betacoronavirus in vespertilio superans bats middle east respiratory syndrome acknowledgements we thank the reporting countries with the confirmed mers cases. we appreciate their assistance with field investigations, administration and data collection and sending the data to who. conflict of interest none declared. key: cord-337835-78i6j11i authors: alfaraj, sarah h.; al-tawfiq, jaffar a.; alzahrani, nojoom a.; altwaijri, talal a.; memish, ziad a. title: the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus date: 2017-02-09 journal: j infect doi: 10.1016/j.jinf.2017.02.001 sha: doc_id: 337835 cord_uid: 78i6j11i nan ho and colleagues recently drew attention to the consequences of co-infection with influenza and hiv. 1 we present four cases of combined infection with influenza and middle east respiratory syndrome coronavirus (mers-cov) infection. nasopharyngeal swabs or tracheal aspirates were tested for mers-cov using real-time reverse-transcription polymerase chain reaction (rt-pcr). 2,3 samples were tested for influenza a, b and h1n1 by rapid molecular test (gen-exper for detection of flu a, b and 2009 h1n1, cepheid). in the first case, a 39 year-old male, health care worker, an engineer who became ill seven days before admission. he had fever >38 c, cough and sore throat. he had no nausea, vomiting, diarrhoea or shortness of breath (sob). he denied history of travel or contact with positive case or camels. he was febrile with a temperature of 39.5 c. chest x-ray showed non-homogenous opacity at the lower right lung zone. a nasopharyngeal swab was positive for mers-cov with ct value upe gene 34, and orf1a 34 ( table 1 ). the test was negative for influenza but a repeat swab after 48 h was negative for mers-cov and positive for h1n1. the patient received azithromycin, ceftriaxone and oseltamivir. the patient was discharged home after two negative swabs of mers-cov and being asymptomatic for 48 h. in the second case, a 61 year-old female with diabetes mellitus and dyslipidemia was admitted with a three-day history of shortness of breath and productive cough. she had no nausea, vomiting, or diarrhoea. she denied history of travel or contact with positive case or camels. she was afebrile with a temperature of 37 c. chest x-ray showed patchy opacities involving middle and lower zones of both lung fields. a nasopharyngeal swab was positive for mers-cov with ct value upe gene 34, orf1a 35 and negative for influenza. a repeat swab after 48 h was negative for mers-cov but positive for h1n1. she required bipap and she was subsequently intubated and was started on mechanical ventilation. she was extubated after 13 days. the patient received piperacillinetazobactam, and erythromycin. the patient was discharged home after she had 2 negative swabs of mers-cov and being asymptomatic for 48 h. in the third case, a 29 year-old housekeeper female was admitted with two days history of fever and cough. she had no nausea, vomiting, diarrhoea nor shortness of breathing. she had a history of contact with mers-cov positive case. she was afebrile with a temperature of 36.9 c. chest x-ray was normal. a nasopharyngeal swab collected upon presentation was positive for mers-cov with ct value upe gene 32 orf1a 32. the swab was negative for influenza. a repeated swab after 48 h was positive mers-cov and positive for h1n1. the patient received oseltamivir, azithromycin and ceftriaxone. the patient was discharged home after she had 2 negative swab of mers-cov and being asymptomatic for 48 h. in the fourth case, the patient was a 73 year-old female with a history of hypothyroidism, heart failure, lymphoma, and lung fibrosis. she has no history of travel or contact with positive case or camels. four days prior to her presentation, she had productive cough and shortness of breath. she had no fever, diarrhoea, vomiting or nausea. she was afebrile with a temperature of 36.7 c. chest x-ray showed bilateral diffuse infiltrate (fig. 1) . a nasopharyngeal swab was positive for mers-cov with ct value upe gene 37; orf1a 36 and negative for influenza. a repeat swab after 3 days was negative for mers-cov but positive for influenza a. the patient was treated with piperacillinetazobactam for six days and oseltamivir for 5 days. the patient was discharged home after two negative mers-cov and being asymptomatic for 48 h. these patients highlight the co-infection with mers-cov and influenza. the exact reason to have a negative influenza test at the time of positive mers-cov is not completely understood. it is possible that the presence of mers-cov inhibits the pcr reaction for influenza virus. however, an earlier case of mers-cov tested initially positive for influenza a(h1n1)pdm09. 4 on the other hand, the positivity of nasal swabs for influenza is specimen type and technique dependent. 5 thus, initially negative influenza tests could be a false test result. positive results for viral respiratory pathogens should not preclude testing for mers-cov because coinfection can occur. 6 only a small number of mers cases had co-infection with influenza a, parainfluenza, herpes simplex, and streptococcus pneumoniae. 7 in one case, a co-infection with herpes simplex virus type 1 dna13 and rhinovirus rna14 were detected by rt-pcr. 8 the investigation of the first 47 cases showed no co-infection with mers-cov. 2 there is a controversy regarding the risk of increased or decreased severity of co-infections. for example co-infections with respiratory syncytial virus (rsv) and human meta-pneumovirus (hmpv) causes more severe infection than either virus alone with longer hospitalization and oxygen requirement. 9 other studies did not demonstrate these effects. 10 the association and the impact of co-infection with mers-cov and influenza viruses deserve further evaluation and studies. all authors have no conflict of interest to report. completeness of case ascertainment and availability of environmental data in legionnaires' disease enhanced surveillance in england, 2012e2014 to the editor, bai et al. 1 previously highlighted the importance of effective communicable disease surveillance in china for the detection of outbreaks to inform infectious disease prevention and control. to achieve similar aims with relation to the control of legionnaires' disease in england a national enhanced surveillance scheme operates. surveillance data must be both complete and as timely as possible, as highlighted by freeman et al. in this journal. 2 therefore we sought to assess the completeness of case ascertainment in addition to the availability of environmental data reported to the national enhanced legionella surveillance scheme (nelss) for residents in england. the value of environmental data is particularly important for legionnaires' disease in order to inform the attribution of potential environmental sources of cases and clusters of this infection. in england, the national legionella surveillance team (nlst) leads on surveillance and control of legionnaires' disease, while field epidemiology services (fes) teams collect surveillance data and investigate sporadic cases, clusters and outbreaks in conjunction with local health protection teams (hpts). the nlst worked with the fes teams in the north west (nw) and west midlands (wmids) regions of england to audit the reporting of legionella cases with onset dates during each calendar year between 2012 and 2014, inclusive. eligible cases were those cases of legionella spp. infections resident in either of these regions in england, which were laboratory confirmed by urinary antigen testing, culture or serological testing. these cases were identified in the month of february following each calendar year by the nlst and were compared to those known by the fes teams for nw and wmids regions. completeness of case ascertainment was calculated as the proportion of cases recorded by fes teams for cases with onset of symptoms between 2012 and 2014 inclusive which were reported to the nlst. the availability of environmental data reported to the national enhanced surveillance scheme was calculated as the proportion of cases during the same time period with any environmental investigation data reported to the nlst. the results are summarised in table 1 ; in the nw region, a mean of 34 cases were reported per year between 2012 and 2014. in the wmids region, the mean number of cases per year was 46. both regions reported 100% of cases to the nlst. mean availability of environmental data reported was 80.8% in the nw and 42.8% in wmids. however, there was a the impact of hiv on the burden and severity of influenza illness in malawian adults: the bash-flu study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections an adult returned traveler from dubai hospitalized with an influenza-like illness (ili): middle east respiratory syndrome (mers) or influenza? infection control implications from a near mers case saudi ministry of health. case definition and surveillance guidance for mers-cov testing in saudi world health organization. who guidelines for investigation of cases of human infection with middle east respiratory syndrome coronavirus (mers-cov) clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection prospective study of human metapneumovirus infection in children less than 3 years of age the role of infections and coinfections with newly identified and emerging respiratory viruses in children key: cord-328835-r9znjkfo authors: favre, guillaume; pomar, léo; musso, didier; baud, david title: 2019-ncov epidemic: what about pregnancies? date: 2020-02-06 journal: lancet doi: 10.1016/s0140-6736(20)30311-1 sha: doc_id: 328835 cord_uid: r9znjkfo nan members of the coronavirus family responsible for severe acute respiratory syndrome (sars-cov) and middle east respiratory syndrome (mers-cov) are known to be responsible for severe complications during pregnancy. 2, 3 12 pregnant women were infected with sars-cov during the 2002-03 pandemic. 2 four (57%) of seven women in the first trimester had a miscarriage. in the second to third trimester, two (40%) of five women had fetal growth restriction, and four (80%) of five women had preterm birth (one spontaneous; three induced for maternal condition). three (25%) women died during pregnancy. in a review of 11 pregnant women infected with mers-cov, 3 ten (91%) presented with adverse outcomes, six (55%) neonates required admission to the intensive care unit, and three (27%) died. two neonates were delivered prematurely for severe maternal respiratory failure. considering that the 2019-ncov seems to have a similar pathogenic potential as sars-cov and mers-cov, 4 pregnant women are at increased risk of severe infections, there are no specific clinical signs of coronavirus infections preceding severe complications, 5 coronaviruses have the potential to cause severe maternal or perinatal adverse outcomes, or both, 2,3 and the current lack of data on the consequences of a 2019-ncov infection during pregnancy, we recommend systematic screening of any suspected 2019-ncov infection during pregnancy. if 2019-ncov infection during pregnancy is confirmed, extended followup should be recommended for mothers and their fetuses. situation report-14 pregnancy and perinatal outcomes of women with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature china coronavirus: what do we know so far? clinical features of patients infected with 2019 novel coronavirus in wuhan, china we declare no competing interests.guillaume favre, léo pomar, didier musso, *david baud key: cord-291014-cfnoxhtd authors: zheng, jian; perlman, stanley title: immune responses in influenza a virus and human coronavirus infections: an ongoing battle between the virus and host date: 2018-02-28 journal: current opinion in virology doi: 10.1016/j.coviro.2017.11.002 sha: doc_id: 291014 cord_uid: cfnoxhtd respiratory viruses, especially influenza a viruses and coronaviruses such as mers-cov, represent continuing global threats to human health. despite significant advances, much needs to be learned. recent studies in virology and immunology have improved our understanding of the role of the immune system in protection and in the pathogenesis of these infections and of co-evolution of viruses and their hosts. these findings, together with sophisticated molecular structure analyses, omics tools and computer-based models, have helped delineate the interaction between respiratory viruses and the host immune system, which will facilitate the development of novel treatment strategies and vaccines with enhanced efficacy. jian zheng and stanley perlman respiratory viruses, especially influenza a viruses and coronaviruses such as mers-cov, represent continuing global threats to human health. despite significant advances, much needs to be learned. recent studies in virology and immunology have improved our understanding of the role of the immune system in protection and in the pathogenesis of these infections and of co-evolution of viruses and their hosts. these findings, together with sophisticated molecular structure analyses, omics tools and computer-based models, have helped delineate the interaction between respiratory viruses and the host immune system, which will facilitate the development of novel treatment strategies and vaccines with enhanced efficacy. immune response: protective or pathogenic? the host immune system is composed of multiple tissues, cells and molecules and can protect hosts from infectious diseases by recognizing and eliminating pathogens efficiently. in one example, our studies of mice infected with sars-cov showed that the severity of sars correlated with the ability to develop a virus-specific immune response, while inhibitory alveolar macrophages and inefficient activation of dendritic cells (dcs) delayed this process and aggravated disease [1] . in another study, channappanavar et al. further demonstrated that dysregulated type i interferon (ifn) and inflammatory monocytemacrophage responses led to lethal pneumonia in sars-cov-infected mice [2] . in support of these data, inhibition of nuclear factor-kappab (nf-kb)-mediated inflammation in sars-cov-infected mice increased survival [3] . similar to their pathological roles in coronavirus infections, inappropriate or dysfunctional immune responses such as overactivation of nacht, lrr and pyd domains-containing protein 3 (nlrp3), high-mobility group box 1 protein (hmgb-1) and interleukin-1beta (il-1b), have been implicated in host tissue destruction [4-8] and persistent pathological changes in iav-infected hosts [9] . expression of the complex of tumor necrosis factor (tnf) superfamily 10 (tnfsf10), histone deacetylase 4 (hdac4) and hdac5 negatively correlated with the levels of tnfa, nf-kb and cyclooxygenase 2 (cox-2) and increases in their expression was correlated with improved prognosis of iav-infected hosts [10] . in addition to their cell-intrinsic properties, lung macrophage and monocyte heterogeneity in localization in iav infections also contributed to differences in outcomes [11] . of vaccinated iav individuals or mice, in which hemagglutinin (ha), neuraminidase (na) and glycosylation pattern mutations [24] , might hinder an effective antibody and t cell response. the contribution of innate immunity to immune defense is not limited in direct anti-viral effects [34] . innate immune signals such as ifn-i not only interact with other innate immune elements such as monocytes and type-ii ifn to limit iavcaused tissue inflammation [35] , but also directly modulated the adaptive immune response. both ifn-i and toll-like receptor 7 (tlr7) were also found to shape b cell-mediated immune responses against iav [36], while rig-i signaling was critical for efficient polyfunctional t cell responses [37] . moreover, the increased mortality of iav-infected mice in the absence of mitochondrial antiviral signaling (mavs) and tlr7 was found to independent of viral load or myeloid differentiation primary response 88 (myd88)-dependent signaling but dependent on secondary bacterial burden, caspase-1/11, and neutrophil-dependent tissue damage [38 ]. as for innate immune cells, a population of lung-resident innate lymphoid cells (ilcs) in mice and humans that expressed cd90, cd25, cd127 and st2 was found to contribute to airway epithelial integrity and its depletion resulted in diminished lung function and impaired airway remodeling [ broadly neutralizing antibodies generally target conserved functional regions on ha. [21] binding of antibody to an epitope masks the epitope and prevents the stimulation and proliferation of specific b cells. [30] mers-cov recombinant receptor-binding domains of multiple mers-covs induce cross-neutralizing antibodies against divergent human and camel mers-covs. [28] t cell response iav vaccine-generated lung-resident memory cd8 t cells provide heterosubtypic protection to iav infection. [31] potential challenges in translating protective memory cd4 t cell responses in experimental animal models to patients. [19] mers-cov mers-cov efficiently infects human primary t cells and induces apoptosis. [14] sars-cov memory t cell responses targeting the sars coronavirus persist for up to 11 years postinfection. [23] crosstalk between immune components iav cooperativity between cd8+ t cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic iav immunity. [20] antibody specificity plays an important role in the regulation of adcc. cross-talk among antibodies of varying specificities determines the magnitude of fc receptor-mediated effector functions. [17] ige cross-linking impairs monocyte antiviral responses and inhibits iavdriven th1 differentiation. [27] mers-cov recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses. [32] maintenance of immune memory iav levels of neutralizing antibodies against previously encountered iav strains ('original antigenic sin') increase over time. [22] low levels of circulating cd8+ t effector and central memory cells are associated with iav infection severity upon re-challenge. [16] regimen of a ctl-based vaccine/vaccine-component benefits from periodic boosting to prevent clinically evident iav infection. [26] multifunctional cd4+ t-cell responses were maintained only in patients with recurrent infections. [29] immunopathology iav iav-specific cd8+ t cells exacerbate infection following high dose challenge of aged mice. [25] different subsets of cd8+ t cells interact with subsets of innate cells through costimulatory molecules to balance protection and immunopathology. [15] identification of protective and pathogenic t cell epitopes in iav h7n9infected patients. [18] immunotherapy iav, cov high titer anti-iav or cov sera may be useful prophylactically and therapeutically in exposed and infected patients. with the increasing accumulation of knowledge of molecular interactions between host cells and viruses, additional host molecules and normal biological processes [51-65,66 ,67-69] were found to participate in the viral replication cycle (summarized in table 2 ). to clarify the roles of these molecules and processes in virus infection, host genetic determinant screening [70] [71] [72] , immunomics and public health omics [73] , host lipid omics [74 ] and characterization of the epigenetic landscape [75] were used to supplement conventional analyses. moreover, information about interaction between immune and parenchymal cells also facilitated efforts to optimize antiviral response while reducing unwanted side effects [76 ,77 ] . computer modeling of host-pathogen interactions is likely to be used more in the future, as additional parameters are identified. thus, computer modeling helped in prediction of clinical outcomes, demonstrating key roles for the innate immune response and the interval between infections [78] . these novel methodologies are likely to provide additional approaches to identifying targets for novel antiviral therapies. the iav non-structural protein ns1, perhaps the bestcharacterized viral immunoevasive protein, binds doublestranded rna (dsrna) to inhibit host innate immune responses [79] [80] [81] . recently, ns1 was also found to bind cellular dsdna and prevent the loading of transcriptional machinery onto the dna, thus attenuating expression of antiviral genes [82] . meanwhile, the c-terminal domain of ns1 blocked ifn-beta production by targeting tnf receptor-associated factor 3 (traf-3) [83] , while other domains of the protein inhibited interferon regulatory transcription factor 3 (irf3) [84] and rna-dependent protein kinase (pkr) activation [85] . another iav protein, neuraminidase (na), was shown to remove sialic immune responses in respiratory virus infections zheng and perlman 45 table 2 recent findings related to intrinsic molecules and biological processes involved in iav and cov infectionss. ref. cell cycling proteins iav competitive inhibition of iav m1-m2 interaction by cyclin d3 impairs infectious virus packaging, resulting in attenuation apoptosis-related signals iav apoptosis signaling modulates iav propagation, innate host defense, and lung injury. [60] sex hormones-related signals iav progesterone-based contraceptives reduce adaptive immune responses and protection against subsequent iav infections. [59] male mice were more susceptible to sars-cov infection compared with agematched females, while estrogen receptor signaling played a critical role in protecting females from sars-cov-mediated pathogenesis. [53] chd chromatin remodeler iav chd1 is a proviral regulator of iav multiplication. [63] nuclear import and export machinery iav iav have evolved different mechanisms to utilize importin-alpha isoforms, affecting importation on both sides of the nuclear envelope. [65] activation of the interferon induction cascade by iav requires viral rna synthesis and nuclear export. [61] human heat shock protein 40 promotes iav replication by assisting in the nuclear import of viral ribonucleoproteins. [52] preferential usage of importin-alpha7 isoforms by seasonal iav in the human upper respiratory tract makes it a target of selective pressure. [64] vesicular trafficking iav iav infection modulates vesicular trafficking and induces golgi complex disruption. [68] iav enhances its propagation through modulating annexin-a1 dependent endosomal trafficking. [51] iav ribonucleoproteins modulate host recycling by competing with rab11 effectors. [67] sars-cov a predicted beta-hairpin structural motif in the cytoplasmic tail of the sars-cov e protein is sufficient for golgi complex localization of a reporter protein and functions as a golgi complex-targeting signal. [54] mers-cov cd9-facilitated condensation of receptors and proteases allows mers-cov pseudoviruses to enter cells rapidly and efficiently. [56] exosome secretion iav exosome deficiency uncoupled chromatin targeting of the viral polymerase complex and the formation of cellular-viral rna hybrids, which are essential rna intermediates that license transcription of antisense genomic viral rnas autophagy iav autophagy induction regulates iav replication in a time-dependent manner. [58] sars-cov cov nsp6 restricts autophagosome expansion. [55] cellular senescence iav cellular senescence enhances viral replication. [62] coagulation iav beneficial effects of inflammation-coagulation interactions during iav infection [69] acid residues from nkp46, resulting in reduced recognition of ha and enhancing immune evasion of nk cells [86] . in addition, the iav m2 protein was shown to reverse bone marrow stromal antigen 2 (bst-2)-mediated restriction of virus release via proteasomal pathways [87] . to evade the host immune system, iav also inhibits host but not viral mrna nuclear export [88] , without impairing nuclear viral ribonucleoprotein (vrnp) import [89] . in addition, productive viral replication in macrophages resulted in decreased phagocytosis via downregulation of fc receptors cd16 and cd32, potentially playing a role in iav pathogenesis [90] . the crucial role of the ifn response makes it a preferred target for viral evasion. besides ns1, multiple virusencoded molecules including the nucleoprotein [64], the fusion peptide of ha2 (ha2-fp), ha1 and some variants of polymerase subunits pb1-f2, pb1, pb2, pa all counteract the interferon response [91] . interestingly, some host cellular molecules are also utilized by iav to block ifn expression. using rna interference, knockdown of a host factor, the double phd fingers 2 gene (dpf2) [92] , resulted in decreased expression of iav proteins, by releasing ifn-b production from dpf2-mediated suppression [92] . the cov endonuclease, nsp15, efficiently prevented activation of host cell dsrna sensors including melanoma differentiation-associated protein 5 (mda5), 2 0 -5 0 oligoadenylate synthetase (oas) and pkr [93, 94 ] , while coronavirus-encoded proteases countered innate immunity, including the ifn response, through diverse pathways [95] . a recent investigation further showed that sars-cov nucleocapsid inhibited type i interferon production by interfering with tripartite motif protein 25 (trim25)-mediated rig-i ubiquitination [96] . recent studies of cellular metabolic processes [97, 98] and post-transcriptional protein modification [99, 100] identified additional approaches used by viruses to evade host immune responses [101] , and to facilitate optimal replication [102] . for example, iav delayed apoptosis of infected cells by activating a signal transducer and activator of transcription 3 (stat-3)-related pathway, allowing prolonged replication [103] . nicotinamide adenine dinucleotide phosphate (nadph) oxidase 2 (nox2), critical for expression of reactive oxygen species (ros), is often activated in endocytic compartments by rna and dna viruses, exacerbating virus-mediated pathogenicity [104 ] . in addition to important roles for host proteins [105] , the role of lipids [74 ] in respiratory virus infections has also drawn increasing attention. zhao et al. reported that agerelated increases in prostaglandin d2 (pgd 2 ) expression in mouse lungs correlated with a progressive impairment in dc migration to dlns, causing diminished t cell responses upon iav or sars-cov infection [106] . in a subsequent study, vijay et al. demonstrated a critical role for phospholipase a2 group iid (pla 2 g2d) in impaired dc migration to dln and age-related susceptibility to sars-cov infection [107] . pla 2 g2d is upstream of pgd 2 in the prostaglandin synthesis pathway. both molecules may be useful targets for anti-viral therapies. as mentioned above, sars-cov nucleocapsid protein was reported to interfere with rig-i ubiquitination [96] , while decreased deubiquitination mediated by mers-cov nsp3 deubiquitinase also inhibited the host immune response [108] . recently, fehr et al. found that mutation of the macrodomain of nsp3, important for countering adp-ribosylation, resulted in virus attenuation [109] , while another report demonstrated that binding of the methyl donor s-adenosyl-l-methionine (sam) to 2 0 -omethyltransferase nsp16 enhanced mers-cov replication, promoting the recruitment of the allosteric activator nsp10 [110] . on the other hand, iav induced host histone deacetylase 1 dysregulation in lung epithelial cells, inhibiting iav infection [111] while neddylation (conjugation of a ubiquitin-like protein, neural precursor cell expressed developmentally down-regulated 8 (nedd8)) of pb2 protein reduced its stability, suppressing iav replication [112] . nevertheless, the role of epigenetic modification during respiratory virus infection is not well understood; the application of phosphoproteomics to characterization of the human macrophage response to iav infection [113] serves as a model for future studies. other putative targets for modulating the immune response are carbohydrates present on host and viral proteins. recently, integrated omics and computational glycobiology revealed the structural basis for iav glycan microheterogeneity and host interactions [114, 115, 116] . glycosylation of the ha protein not only mediated virus entry into host cells [115] [116] [117] , but also modulated iav replication and transmission [118] , and the immune response against the virus [119] [120] [121] , thus representing a potential target for vaccine and drug development [122, 123 ] . vaccines remain as the most efficient tools for preventing the occurrence and spread of viral respiratory diseases. distinct from conventional adjuvants, novel reagents are being used to shape as well as augment the strength of the induced immune responses. targeting iav ha to the chemokine receptor, xcr1, present on some dendritic cells enhanced protective antibody responses against the virus [124] . in addition, knowledge of the microbiota [125] , and manipulation of apoptosis [126] and mtor (mechanistic target of rapamycin) [127] -related signaling pathways have been used to predict or modulate responsiveness and efficiency of vaccines, respectively. a major goal of iav and cov vaccine development is to develop vaccines able to induce broadly acting antibodies; these efforts require more precise definition of useful conserved protective antibody epitopes [128] . further, monocytederived dendritic cells (modcs) [129] dominated the activation of cd8+ t cells at late times after infection of c57bl/6 mice, triggering a switch in immunodominance from pa to np-specificity. this differential expression of t cell epitopes has implications for dc-based vaccine design. additionally, neutrophil-targeting [42] and th1-targeting strategies [130] might help in establishing tissue-resident memory (trm) and heterosubtypic immunity. inducing effective resident immune memory represents an ideal strategy for protecting the host from respiratory virus infection, especially at very early phases of virus invasion. recent technical advances have facilitated distinguishing tissue-resident cell populations from those in the periphery. in a recent publication, airway memory cd4(+) t cells induced by a single conserved n proteinspecific epitope present in both sars-cov and mers-cov mediated protection against challenge with either pathogen [131 ] . iav-specific resident memory cd8 cells in the upper respiratory tract or bronchoalveolar fluid provided superior protection compared to those in the lung, although some studies questioned whether these were truly resident memory as opposed to memory cells at these sites [132] . in a recent report, slutter et al. delineated the dynamics of iav-induced lung-resident memory t cells that underlies waning heterosubtypic immunity [133] , illustrating the tight collaboration of resident and peripheral t cell memory in respiratory virus control. in the future, use of sophisticated cell sorting methods and mass spectrometric flow cytometry will provide more precise information about resident memory immune cells. the iav-specific antibody response is well known to be a key host factor in protection from subsequent challenge [134, 135] . recent work has identified nasopharyngeal protein biomarkers in immunized mice useful for predicting the severity and outcome of acute respiratory virus infection [136] . other studies have identified an important synergistic role for immune responses in inducible bronchus-associated lymphoid tissue (ibalt) and draining lymph nodes for optimal iav-specific cd4+ t cell responses [137] . in contrast, nasal-associated lymphoid tissues (nalts) have been shown to support the recall but not priming of iav-specific cytotoxic t cells [138 ] . genomics, next-generation sequencing [139] and single cell imaging and analysis [140] facilitated in-depth investigation of the evolution, recombination and spread of infectious pathogens and extend the scope of virology research. in addition to these molecular biology tools, methods such as analyses of dc responses [141] and digital cell quantification (dcq), which combine genome-wide gene expression data with immune cell functional studies, will help identify immune cell subpopulations [142] . especially critical for understanding the ecology of rna viruses, such as iav and cov, will be obtaining respiratory samples from camels (mers-cov) and patients in both cross-sectional and longitudinal studies. these samples will be useful for identifying carriers and understanding virus evolution and transmission dynamics [143, 144] . recent analyses of mers-covinfected camels [145] have also increased our knowledge of virus demography and evolution across diverse populations. although gene sequencing and crystallographic analyses have provided insight into the molecular evolution of iav, the inability to predict future virus evolution remains an obstacle in managing epidemic and pandemic spread. models using canalized evolutionary trajectory induced by selective dynamics [146] , intra-host iav dynamics [147] , sequence based epidemiology [148] , genomic diversification and adaptation during experimental serial passages [149] will help in the development of accurate prediction models. however, successful modeling to prospectively predict the emergence of new virus strains relies on solid experimental data obtained from field investigations. the standardization of protocols and normalization of data are key challenges in developing useful models of virus evolution. this brief review outlines how the host immune response plays both protective and pathogenic roles in respiratory virus infections. to decrease the burden that respiratory viruses place on society, increased understanding of all aspects of the host immune response remains a critical research goal. host genetic determinants of influenza pathogenicity host genetics of severe influenza: from mouse mx1 to human irf7 population diversity and collective interactions during influenza virus replication and evolution influenza a immunomics and public health omics: the dynamic pathway interplay in host response to h1n1 infection integrated omics analysis of pathogenic host responses during pandemic h1n1 influenza virus infection: the crucial role of lipid metabolism crucial role of lipid metabolism in iav infection was illustrated using integrated omics analysis epigenetic landscape during coronavirus infection macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection interaction between macrophage and epithelial cells exacerbated disease severity by inhibiting lung edema clearance alveolar macrophages prevent lethal influenza pneumonia by inhibiting infection of type-1 alveolar epithelial cells aveolar macrophage prevented lethal pneumonia by inhibiting iav infection on epithelial cells innate immunity and the inter-exposure interval determine the dynamics of secondary influenza virus infection and explain observed viral hierarchies non-linear enhancement of mrna delivery efficiencies by influenza a derived ns1 protein engendering host gene inhibition property the rna-and trim25-binding domains of influenza virus ns1 protein are essential for suppression of nlrp3 inflammasome-mediated il-1beta secretion a: induction and evasion of type i interferon responses by influenza viruses influenza virus ns1 protein binds cellular dna to block transcription of antiviral genes the cterminal effector domain of non-structural protein 1 of influenza a virus blocks ifn-beta production by targeting tnf receptor-associated factor 3 the role of n-terminustruncated ns1 proteins of influenza a virus in inhibiting irf3 activation influenza a virus virulence depends on two amino acids in the n-terminal domain of its ns1 protein facilitating inhibition of pkr neuraminidase-mediated, nkp46-dependent immuneevasion mechanism of influenza viruses bst-2 restricts iav release and is countered by the viral m2 protein nuclear imprisonment: viral strategies to arrest host mrna nuclear export influenza a viruses escape from mxa restriction at the expense of efficient nuclear vrnp import influenza overcomes cellular blocks to productively replicate impacting macrophage function to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns1 double phd fingers 2 (dpf2) promotes the immune escape of influenza virus by suppressing interferon-beta production early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication coronavirus nonstructural protein 15 mediates evasion of dsrna sensors and limits apoptosis in macrophages rna-virus proteases counteracting host innate immunity sars coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination autophagosomal protein dynamics and influenza virus infection influenza virus infection induces host pyruvate kinase m which interacts with viral rnadependent rna polymerase structure of human ifit1 with capped rna reveals adaptable mrna binding and mechanisms for sensing n1 and n2 ribose 2 0 -o methylations zbp1/dai ubiquitination and sensing of influenza vrnps activate programmed cell death going against the tide: selective cellular protein synthesis during virally induced host shutoff ha triggers the switch from mek1 sumoylation to phosphorylation of the erk pathway in influenza a virusinfected cells and facilitates its infection highly pathogenic avian influenza h5n1 virus delays apoptotic responses via activation of stat3 endosomal nox2 oxidase exacerbates virus pathogenicity and is a target for antiviral therapy activated nox2 exacerbated virus-mediated pathogenicity via production of reactive oxygen species (ros) proteomic analysis of differential expression of cellular proteins in response to avian h9n2 virus infection of a549 cells age-related increases in pgd(2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice critical role of phospholipase a2 group iid in age-related susceptibility to severe acute respiratory syndrome-cov infection molecular dynamic studies of interferon and innate immunity resistance in mers cov non-structural protein 3 the conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection binding of the methyl donor sam to mers-cov 2 0 -omethyltransferase nsp16 promotes the recruitment of the allosteric activator nsp10 influenza a virus dysregulates host histone deacetylase 1 that inhibits viral infection in lung epithelial cells neddylation of pb2 reduces its stability and blocks the replication of influenza a virus phosphoproteomics to characterize host response during influenza a virus infection of human macrophages integrated omics and computational glycobiology reveal structural basis for influenza a virus glycan microheterogeneity and host interactions glycosylation changes in the globular head of h3n2 influenza hemagglutinin modulate receptor binding without affecting virus virulence permissivity of dpp4 orthologs to mers-coronavirus is governed by glycosylation and other complex determinants influenza virus-glycan interactions progressive glycosylation of the hemagglutinin of avian influenza h5n1 modulates virus replication, virulence and chicken-to-chicken transmission without significant impact on antigenic drift playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection glycosylation characterization of an influenza h5n7 hemagglutinin series with engineered glycosylation patterns: implications for structure-function relationships glycosylation of the ha protein of h5n1 virus increases its virulence in mice by exacerbating the host immune response unmasking stemspecific neutralizing epitopes by abolishing n-linked glycosylation sites of influenza hemagglutinin proteins for vaccine design influenza a surface glycosylation and vaccine design targeting influenza virus hemagglutinin to xcr1+ dendritic cells in the absence of rreceptor-mediated endocytosis enhances protective antibody responses the potential of the microbiota to influence vaccine responses apoptosis and other immune biomarkers predict influenza vaccine responsiveness influenza vaccines: mtor inhibition surprisingly leads to protection evolution in the influenza a h3 stalk -a challenge for broad-spectrum vaccines? monocyte-derived dendritic cells enhance protection against secondary influenza challenge by controlling the switch in cd82 t-cell immunodominance virus-like particle vaccine primes immune responses preventing inactivated-virus vaccine-enhanced disease against respiratory syncytial virus airway memory cd4(+) t cells mediate protective immunity against emerging respiratory coronaviruses conserved epitopes-specific airway memory cd4(+) t cells induced by vaccine protected animals from sars-covs and mers-cov infection resident memory cd8+ t cells in the upper respiratory tract prevent pulmonary influenza virus infection dynamics of influenza-induced lungresident memory t cells underlie waning heterosubtypic immunity heads, stalks and everything else: how can antibodies eradicate influenza as a human disease antibodydependent cellular cytotoxicity and influenza virus inducible bronchus-associated lymphoid tissue (ibalt) synergizes with local lymph nodes during antiviral cd4(+) t cell responses nasal-associated lymphoid tissues (nalts) support the recall but not priming of influenza virusspecific cytotoxic t cells nalts support the recall but not priming of iav-specific cytotoxic t cells new-generation screening assays for the detection of anti-influenza compounds targeting viral and host functions visualization of iav genomes at the single-cell level human dendritic cell response signatures distinguish 1918, pandemic and seasonal h1n1 influenza viruses digital cell quantification identifies global immune cell dynamics during influenza infection natural history of highly pathogenic avian influenza h5n1 middle east respiratory syndrome longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia canalization of the evolutionary trajectory of the human influenza virus within-host influenza dynamics: a small-scale mathematical modeling approach dynamically correlated mutations drive human influenza a evolution quantitative modeling of virus evolutionary dynamics and adaptation in serial passages using empirically inferred fitness landscapes supported in part by grants from the nih (niaid, po1 ai060699, ro1 ai129269). key: cord-329010-n0mz098o authors: mckee, dwight l.; sternberg, ariane; stange, ulrike; laufer, stefan; naujokat, cord title: candidate drugs against sars-cov-2 and covid-19 date: 2020-04-29 journal: pharmacol res doi: 10.1016/j.phrs.2020.104859 sha: doc_id: 329010 cord_uid: n0mz098o outbreak and pandemic of coronavirus sars-cov-2 in 2019/2020 will challenge global health for the future. because a vaccine against the virus will not be available in the near future, we herein try to offer a pharmacological strategy to combat the virus. there exists a number of candidate drugs that may inhibit infection with and replication of sars-cov-2. such drugs comprise inhibitors of tmprss2 serine protease and inhibitors of angiotensin-converting enzyme 2 (ace2). blockade of ace2, the host cell receptor for the s protein of sars-cov-2 and inhibition of tmprss2, which is required for s protein priming may prevent cell entry of sars-cov-2. further, chloroquine and hydroxychloroquine, and off-label antiviral drugs, such as the nucleotide analogue remdesivir, hiv protease inhibitors lopinavir and ritonavir, broad-spectrum antiviral drugs arbidol and favipiravir as well as antiviral phytochemicals available to date may prevent spread of sars-cov-2 and morbidity and mortality of covid-19 pandemic. an exopeptidase expressed on epithelial cells of the respiratory tract, may constitute a pharmacological target to limit cell entry of sars-cov-2. the established antimalarial drugs chloroquine and hydroxychloroquine have been shown to inhibit terminal phosphorylation of ace2 and to elevate the ph in endosomes, respectively. chloroquine and hydroxychloroquine constitute candidate drugs against sars-cov infection and covid-19 disease, and are now investigated for their therapeutic efficacy in international clinical trials with covid-19 patients (i. e. solidarity trial). the glycosylated s protein of sars-cov is highly immunogenic to the host immune system, and murine polyclonal antibodies against sars-co-v s protein potently inhibit sars-cov-2 s-mediated cell entry, indicating that cross-neutralizing antibodies targeting conserved s epitopes can be elicited upon vaccination [9] . similar to the earlier sars and mers beta coronaviruses, sars-cov-2 primarily infects alveolar epithelial cell of the lung, leading to a severe bilateral peripheral pneumonia with ground glass opacity in ct images (covid-19 disease), with a mortality rate of 2 % to 5 % [10] . sars-cov-2 also can contribute to multiple organ failure, affecting heart, liver, kidney, central nervous system and gastrointestinal tract [11] . epidemiology thus far suggests that sars-cov-2 is more contagious than sars-cov or mers-cov [12] . multiple mechanisms now identified in the infective and replication processes of sars-cov-2 offer targets for pharmacological interventions. infection of pneumocytes, macrophages and pulmonary mast cells requires viral s protein. this invasion process which involves attachment of s protein to the ace2 receptor is facilitated by host cell derived serine protease tmprss211 [8] . agents that inhibit tmprss211, such as camostat mesilate, may be useful in blocking viral host cell entry. after host cell entry, the viral single-stranded positive rna, is released for replication of virus rna and translation of virus polyproteins that are finally cleaved into mature effector proteins by virus proteases [13] . the s protein interaction with ace2 on host cell cytoplasmic membrane initiates viral infection. strategies capable of disrupting s protein interaction with ace2 could be of significant therapeutic value, because the binding affinity of sars-cov-2 s protein to ace2 is 10-20-fold higher than for the s protein of sars-cov which may contribute to the higher contagiousness of sars-cov-2 as compared to sars-cov [12] . although sars-cov and sars-cov-2 have only 79% genomic sequence similarity, they share a highly conserved receptor binding domain for their s proteins [1] there is also potential for targeting other highly conserved proteins associated with sars-cov and sars-co-v-2, including rdrp and 3clpro (also termed mpro), which share over 95% similarity between the two viruses, despite only 79% genomic sequence sharing. rdrp is an rna-dependent rna polymerase required for replicating the viral genome within the host cell. 3clpro and plpro are both viral proteases which break down viral polyprotein into functional units within host cells that are finally assembled into new viruses. the 3clpro sequences between the two viruses are 96% similar, the plpro sequence identity is 83%, and their active sites show a high degree of conservation [14] . drugs that have recently been shown to target mers-cov in mice [15] , and to inhibit ebola virus rdrp and sars-cov-2 proteases in humans, such as remdesivir and ritonavir/lopinavir, also constitute candidate drugs against sars-cov-2 and are now investigated for their therapeutic efficacy in covid-19 patients in 2 international clinical trials (solidarity trial and discovery trial). finally, certain phytochemicals and natural products with high antiviral activity should be considered for treatment of sars-cov-2 infection and covid-19 disease. results from previous studies reveal that diverse viruses, including ebola virus, sars-coronavirus (sars-cov), mers-coronavirus (mers-cov) and influenza virus employ host cell proteases for activation of their envelope glycoproteins [16] [17] [18] . cleavage and activation of the spike protein (s protein) of sars-cov that is required for membrane fusion and host cell entry is mediated by transmembrane protease/serine subfamily member 2 (tmprss2), an airway and alveolar cell serine protease [19] [20] [21] . pöhlmann and coworkers recently demonstrated that sars-cov-2 also employs tmprss2 for sars-cov-2 s protein priming and s protein-driven cell entry [8] . using camostat mesilate, a clinically proven and commercial serine protease inhibitor that partially blocks infection by [26, 27] , and chronic pancreatitis [28] [29] [30] [31] . camostat mesilate (ni-03) is manufactured as an oral drug by nichi-iko pharmaceutical co., ltd., and ono pharmaceutical, japan, with a three times daily dose recommendation of 100 mg to 300 mg [30, 31] . in a clinical trial investigating camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease, 95 patients received 200 mg camostat mesilate three times daily for 2 weeks and showed only mild, but no severe adverse effects [28] , indicating that camostat mesilate is a well-tolerated drug. nafamostat mesilate (buipel tm ), (6-amidino-2-naphthyl-4-guanidino benzoate-dimethanesulfonate) (fut-175), (cas number: 81525-10-2), is a clinical proven and synthetic serine protease inhibitor approved in japan for the treatment of acute pancreatitis, disseminated intravascular coagulation and for anticoagulation in extracorporeal circulation [32] [33] [34] . in a screening approach of about 1,100 drugs approved by the fda, nafamostat mesilate has been identified to inhibit mers-cov s protein-j o u r n a l p r e -p r o o f mediated viral membrane fusion with tmprss2-expressing lung calu-3 host cells by inhibiting tmprss2 protease activity [35] . since the s proteins of mers-cov and sars-cov-2 share considerable amino acid sequence homology [1, 9] , nafamostat mesilate may also inhibit cell entry of sars-cov-2. in cell culture experiments with simian vero e6 cells infected with sars-cov-2, nafamostat mesilate was shown to be inhibitive against sars-cov-2 infection at ec50 of 22.50 µm [36] , suggesting that nafamostat mesilate is able to prevent sars-cov-2 infection. in a multicenter, randomized, open-label, phase 2 trial in 19 patients with severe acute pancreatitis, nafamostat mesilate was administered intravenously at a daily dose of 240 mg for 5 days without severe adverse effects [34] . sars-cov and related coronaviruses directly interact via their s proteins with angiotensin-converting enzyme 2 (ace2), a host cell exopeptidase and metallocarboxypeptidase that catalyses the conversion of angiotensin i to the nonapeptide angiotensin and the conversion of angiotensin ii to angiotensin 1-7, to initiate s protein-mediated cell entry [37] [38] [39] . it was demonstrated recently that also sars-cov-2 uses ace2 as a receptor for s protein-driven host cell entry [8, 9] . therefore, ace2 constitute a molecular target to inhibit cell entry of sars-cov-2. unfortunately, ace inhibitors as standard drugs for the treatment of hypertension and chronic heart failure fail to inhibit ace2 [40] , but a number of other drugs and compounds have been shown to inhibit ace2. chloroquine phosphate (resochin tm ) and its derivative hydroxychloroquine (quensyl tm , plaquenil tm , hydroquin tm , dolquine tm , quinoric tm ) have been used for decades for the prophylaxis and treatment of malaria and for the treatment of chronic q fever and various autoimmune diseases [41] , and have recently been demonstrated as potential broad-spectrum antiviral drugs [42, 43] . chloroquine phosphate inhibits terminal phosphorylation of ace2, and hydroxychloroquine elevates the ph in endosomes which are involved in virus cell entry [44, 45] , both mechanisms constitute relevant antiviral mechanisms of chloroquine and hydroxychloroquine. in vivo, hydroxychloroquine is metabolized into chloroquine. chloroquine phosphate has previously been shown to inhibit sars-cov infection and spread in vitro [44, 46] , and results from very recent studies reveal that chloroquine phosphate and, more effectively, hydroxychloroquine also inhibit replication of sars-cov-2 in simian vero cells [46, 47] . by using a physiologically-based pharmacokinetic model for chloroquine phosphate and hydroxychloroquine in human lung fluid, it was demonstrated that the concentrations of hydroxychloroquine recommended for treatment of sars-cov-2 infection comprise an oral loading dose of 400 mg twice daily at day 1, followed by an oral maintenance dose of 200 mg twice daily for 4 j o u r n a l p r e -p r o o f days [47] . these results were deduced from in vitro data obtained from sars-cov-2-infected vero cells treated with hydroxychloroquine [47] . a recent pilot trial conducted in more than 10 hospitals in wuhan, jingzhou, guangzhou, bejing, shanghai, chongqing and ningbo, china, with more than 100 patients with covid-19 disease demonstrated that treatment with chloroquine phosphate is superior to control treatment in inhibiting the exacerbation of pneumonia, improving lung imaging findings, promoting laboratory virus-negative conversion, and shortening the course of covid-19 disease [48] . chloroquine phosphate should be administered as an oral daily dose of 250 mg until clinical convalescence [49] . thus, in view of these results and the urgent clinical demand regarding sars-cov-2/covid-19 pandemia, chloroquine phosphate should be recommended to treat covid-19 associated pneumonia in larger populations [48] . a recent open-label non-randomized clinical trial conducted in march 2020 in france with 20 covid-19 patients treated with daily 600 mg hydroxychloroquine for 6 days demonstrated at day 6 a negative viral load (negative nasopharyngeal pcr) in 57% of the hydroxychloroquine-treated patients, as compared to negative viral load in 12.5% of untreated covid-19 patients (control group, n=16) [50] . in a randomized clinical trial conducted in february 2020 in wuhan, china, sixty two covid-19 patients were randomized to receive either daily 400 mg hydroxychloroquine for 5 days (n=31) or no pharmacological treatment (n=31) [51] . improvement and absorption of pneumonia as analyzed in chest ct at day 6 was observed in 80.6% of the hydroxychloroquine-treated patients vs. 54.8% in the untreated patients [51] . the results from these small studies therefore strongly suggest that hydroxychloroquine has therapeutic efficacy in thus, a considerable number of clinical trials investigating therapeutic efficacy of chloroquine phosphate and hydroxychloroquine in patients with sars-cov-2 infection and covid-19 disease have been initiated in china, great britain, spain and thailand [52] [53] [54] [55] [56] . the triple combination of cepharanthine (an anti-inflammatory alkaloid from stephania cepharantha gx_p2v/2017/guangxi (gx_p2v), whose s protein shares 92.2% amino acid identity with that of sars-cov-2 [60] . further, it was demonstrated that gx_p2v also uses ace2 as the receptor for viral cell entry [60] . two libraries of 2,406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on vero e6 cells by gx_p2v, and only the combination of cepharanthine, selamectin and mefloquine hydrochloride was identified as candidate drug combination against sarsj o u r n a l p r e -p r o o f shortly after the identification of the angiotensin-converting enzyme 2 (ace2), a metallocarboxypeptidase that mediates various cardiovascular and renal functions, peptide inhibitors of the enzyme were developed by selection of constrained peptide libraries displayed on phage [61] . the most potent inhibitor, termed dx600, with the amino acid sequence of acvero-e 6 cells, and inhibits infection of engineered human capillary organoids and kidney organoids by sars-cov-2 isolated from a nasopharyngeal sample of a patient with confirmed covid-19 disease [74] , suggesting that hrsace2 can block host cell entry of sars-cov-2 and early stages of sars-cov-2 infections. remdesivir (gs-5734), (cas number: 1809249-37-3), is a novel small-molecule adenine nucleotide analogue antiviral drug that has shown efficacy against ebola virus in rhesus monkeys. once-daily intravenous administration of 10 mg kg(-1) remdesivir for 12 days resulted in profound suppression of ebola virus replication and protected 100% of ebola virus-infected animals against lethal disease [75] . remdesivir displays antiviral activity against other single stranded rna viruses, including filoviruses, pneumoviruses, paramyxoviruses, and the coronaviruses mers-cov and sars-cov [76] [77] [78] . remdesivir is a prodrug that is metabolized into its active form gs-441524, an adenine nucleotide analogue that interferes with the activity of viral rna polymerase and that promotes evasion of j o u r n a l p r e -p r o o f proofreading by viral exoribonuclease, leading to inhibition of viral rna synthesis [78] . remdesivir acts early in infection, and decreases viral rna levels in a dose-dependent manner that parallels impairment of viral load in vitro [78] . these and related mechanisms of action of remdesivir have been demonstrated in vitro for sars-cov [78] , ebola virus [79] and mers-cov [80] . a recent study demonstrates in cell culture experiments with simian vero e6 cells infected with sars-cov-2 that remdesivir is inhibitive against sars-cov-2 infection at ec90 of 1.76 µm, a concentration achieved in vivo in nonhuman primate models [36] . it was further shown that remdesivir efficiently inhibited sars-cov-2 infection of human liver cancer huh-7 cells, which are sensitive to sars-cov-2 infection [36] . [87, 88] , in the usa [89] , and in france [90, 91] . lopinavir (abt-378) is a highly potent inhibitor of the human immunodeficiency virus (hiv) protease essential for intracellular hiv assembly that was developed in 1998 to circumvent hiv resistance towards the protease inhibitor ritonavir (abt-538), caused by mutation of valine at position 82 (val 82) in the active site of hiv protease in response to ritonavir therapy [92] . because the metabolism of lopinavir is strongly inhibited by ritonavir, concomitant oral administration of lopinavir and ritonavir exceeded the in vitro antiviral ec50 of lopinavir by >50-fold after 8 h in rat, dog, and monkey plasma j o u r n a l p r e -p r o o f [92] . coadministration of 400 mg lopinavir with 50 mg ritonavir enhanced in healthy human volunteers the area under the concentration curve of lopinavir in plasma by 77-fold over that observed after dosing with lopinavir alone, and mean concentrations of lopinavir exceeded the ec50 for >24 h [92] . therefore, the combination of lopinavir and ritonavir (kaletra tm ) has been established as an effective oral drug for the treatment of hiv-infected individuals when used in combination with other antiretroviral agents [93, 94] .an initial study in 2003 demonstrated that lopinavir at 4 µg/ml inhibited the cytopathic effect in a plaque reduction assay with fetal rhesus kidney-4-cells infected with sars-cov (hku-39849 isolate) [95] . in this study, newly diagnosed sars patients infected with sars-cov were treated with the combination of lopinavir (400 mg)/ritonavir (100 mg) orally every 12 hours for 14 days. at day 21, sars patients treated with lopinavir/ritonavir had a milder disease course in terms of diarrhea, recurrence of fever, worsening of chest radiographs and reduction of viral load, compared to a historical control group [95] . in a nonhuman primate model of common marmosets infected with mers-cov, lopinavir/ritonavir-treated animals displayed an improved clinical outcome compared to untreated animals, with improved weight loss, lung imaging and pathological findings, and lower mean viral loads in necropsied lung and extrapulmonary tissues [96] . in response to these findings, an ongoing randomized control trial (miracle trial) was initiated to determine the therapeutic efficacy of lopinavir/ritonavir combined with interferon β-1b in patients infected with mers-cov [97] . in a recent [98] . treatment with lopinavir/ritonavir was not associated with a difference from standard care in the time to clinical improvement, and mortality at 28 days was similar in the lopinavir/ritonavir group and the standard-care group [98] . moreover, treatment with lopinavir/ritonavir treatment did not reduce viral rna loads or duration of viral rna detectability as compared with standard supportive care. sars-cov-2 rna was still detected in 40.7% of the patients in the lopinavir/ritonavir group at the end of the trial at day 28 [98] . however, the numbers of lopinavir/ritonavir recipients who had serious complications (acute kidney injury and secondary infections) or requiring noninvasive or invasive mechanical ventilation for respiratory failure were fewer than in those not receiving lopinavir/ritonavir treatment [98] . these results and observations require additional studies to determine whether treatment with lopinavir/ritonavir given at a certain disease stage can reduce some complications in covid-19 patients [98] . china [99] [100] [101] [102] , hong kong [103] , republic of korea [104] , and in europe (discovery trial), investigating remdesivir, lopinavir/ritonavir, and lopinavir/ritonavir plus interferon β-1a [91] . umifenovir [112] . in view of these promising clinical results, clinical trials with umifenovir alone or in combination with lopinavir/ritonavir, chloroquine phosphate or carrimycin have been recently initiated in china [113] [114] [115] [116] . the human interleukin-6 receptor, tocilizumab [123] or favipiravir in combination with chloroquine phosphate and the viral neuramidase inhibitor oseltamivir [124] have been initiated recently in china. 3clpro (also termed mpro) constitutes the main protease of beta coronaviruses that is essential for processing of polyproteins translated from the viral rna [125] . recently, the x-ray structures of the unligated sars-cov-2 3clpro and its complex with α-ketoamides designed as specific inhibitors of 3clpro were reported [126] . two pyridine-containing α-ketoamides, designated 13a and 13b, displayed favorable pharmacokinetic properties in mice and were detected at sufficient concentrations in lung tissue and broncheo-alveolar lavage fluid within 4 hours to 24 hours after subcutaneous administration, demonstrating lung tropism of the compounds [126] . besides subcutaneous administration, inhalation of nebulized 13b by mice resulted in high and long-lasting (24 hours) concentrations in lung tissue, without any adverse effects [126] , pointing out a role of pyridinecontaining α-ketoamides in covid-19 therapy. in a recent study that employed combined structureassisted drug design, virtual drug screening and high-throughput screening, a mechanism-based inhibitor of mpro, termed n3, was identified by computer-aided drug design [127] . n3, a michael acceptor inhibitor that can inhibit the mpros of sars-cov and mers-cov, was shown to form a covalent bond with and to be an irreversible inhibitor of sars-cov-2 mpro [127] . further, in a highthroughput screening approach for identifying inhibitors of sars-cov-2 mpro, ebselen, an organoselenium compound with anti-inflammatory, anti-oxidant and cytoprotective properties, was identified [127] . in a plaque-reduction assay with simian vero cells infected with sars-cov-2, n3 and ebselen displayed antiviral and cell protection efficacy at ec50 values of 16.77 µm and 4.67 µm, respectively [127] , ultimately demonstrating their antiviral potential against sars-cov-2. natural products can inhibit various steps in viral infection and replication, and many of them have broad-spectrum antiviral effects, the mechanisms of which have not been fully characterized. they also can function as immunomodulators, suppressing inflammatory reaction responsible for the major morbidity and mortality of sars-cov-2 infection. phytochemicals, especially flavonoids, which are widely distributed in food plants and botanicals, have been shown to interfere with nlrp3 inflammasome signaling [128] . the respiratory distress syndrome associated with sars coronaviruses develops in part due to viral activation of the nlrp3 inflammasome within activated macrophages and t helper-1 lymphocytes, which causes increased production of inflammatory cytokines [129] . several flavonoids that interfere with activation of the nlrp3 inflammasome may modulate inflammatory response to sars beta coronaviruses: luteolin [130] , myricetin [131] , apigenin j o u r n a l p r e -p r o o f [132] , quercetin [133] kaempferol [134] , baicalin [135] , and wogonoside [136] . these flavonoids have been shown to be active against a wide variety of viruses, via multiple mechanisms [137, 138] , and are available as nutraceutical supplements at a daily dose ranging from 100 mg to 500 mg. emodin (6methyl-1,3,8-trihydroxyanthraquinone) (cas number: 518-82-1) is an anthraquinone compound found in various chinese herbs and is also produced by many species of fungi, including members of the genera aspergillus, pyrenochaeta, and pestalotiopsis. emodin has been shown to inhibit the interaction of sars-cov s protein with its receptor ace2 in a dose-dependent manner [139] . [140] , suggesting that resveratrol may also be effective against sars-cov-2 infection. the emergence of the novel beta coronavirus sars-cov-2 from wuhan, hubei province, china in december 2019 rapidly led to a pandemic involving more than 2,500,000 infected persons and more proven drugs such as camostat mesilate which prevents virus host cell entry by inhibiting tmprss2 [8] , and chloroquine phosphate which inhibits terminal phosphorylation of ace2, or hydroxychloroquine which is metabolized in vivo to chloroquine [44] . for the treatment of ordinary and severe covid-19 pneumonia, and to lower the mortality rate of covid-19 disease, the antiviral drugs remdesivir, favipiravir, umifenovir, and lopinavir/ritonavir plus interferon β-1a should be administered, in particular after the consideration of (preliminary) results from the recent ongoing clinical trials solidarity and discovery [141, 91] cn and dlm conceived the original idea and wrote large parts of the manuscript. as and us wrote section 3 and prepared table 1 and figure 1 . sl finished the manuscript. none. we have no conflicts of interest. sincerely, genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a novel coronavirus from patients with pneumonia in china genetic evolution analysis of 2019 novel coronavirus and coronavirus from other species global patterns in coronavirus diversity epidemiology, genetic recombination, and pathogenesis of coronaviruses structural insights into coronavirus entry sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease receptor structure, function, and antigenicity of the sars-cov-2 spike glycoprotein, cell (2020) mar 6. pii the outbreak of covid-19: an overview a novel coronavirus from patients with pneumonia in china an updated estimation of the risk of transmission of the novel coronavirus (2019-ncov) the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds effect of a specific serine protease inhibitor on the rat pancreas: systemic administration of camostate and exocrine pancreatic secretion stimulation of pancreatic secretion in man by a protease inhibitor (camostate) efficacy of camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease camostat mesilate, pancrelipase, and rabeprazole combination therapy improves epigastric pain in early chronic pancreatitis and functional dyspepsia with pancreatic enzyme abnormalities a phase 1/2 trial to evaluate the pharmacokinetics, safety, and efficacy of ni-03 in patients with chronic pancreatitis: study protocol for a randomized controlled trial on the assessment of camostat treatment in chronic pancreatitis (tactic) pharmacological studies of fut-175, nafamostat mesilate. v. effects on the pancreatic enzymes and experimental acute pancreatitis in rats plasma collection using nafamostat mesilate and dipyridamole as an anticoagulant continuous regional arterial infusion versus intravenous administration of the protease inhibitor nafamostat mesilate for predicted severe acute pancreatitis: a multicenter, randomized, open-label, phase 2 trial identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro angiotensin-converting enzyme 2 is a functional receptor for the sars coronoavirus isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 aceh/ace2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ace inhibitors, can hydroxychloroquine: from malaria to autoimmunity new insights into the antiviral effects of chloroquine anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model chloroquine is a potent inhibitor of sars coronavirus infection and spread targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies medication for covid-19 -an overview of approaches currently under study hydroxychloroquine and azithromycin as a tratment of covid-19: results of an open-label non-randomized clinical trial efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial, medrxiv (2020) mar 31 doi clinical trial analysis of 2019-ncov therapy registered in china a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 covid-19: an update on the epidemiological, clinical, preventive and therapeutic evidence and guidelines of integrative chinese-western medicine for the management of 2019 novel coronavirus disease cepharanthine: an update of its mode of action, pharmacological properties and medical applications preclinical evaluation of avermectins as novel therapeutic agents for alcohol use disorderspsychopharmacology mefloquine for preventing malaria during travel to endemic areas repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus (2019-ncov) related coronavirus model novel peptide inhibitors of angiotensin-converting enzyme 2 lazartigues, species-specific inhibitor sensitivity of angiotensin-converting enzyme 2 (ace2) and its implication for ace2 activity assays murine recombinant angiotensin-converting enzyme 2: effect on angiotensin ii-dependent hypertension and distinctive angiotensin-converting enzyme 2 inhibitor characteristics on rodent and human angiotensinconverting enzyme 2 identification of critical active-site residues in angiotensin-converting enzyme 2 (ace2) by site-directed mautagenesis identification of critical determinants on ace2 for sars-cov entry and development of a potent entry inhibitor development of potent and selective phosphinic peptide inhibitors of angiotensin-converting enzyme 2 inhibition of angiotensin-converting enzyme 2 exacerbates cardiac hypertrophy and fibrosis in ren-2 hypertensive rats structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor tace antagonists blocking ace2 shedding caused by the spike protein of sars-cov are candidate antiviral compounds protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing role of nicotianamide in the intracellular delivery of metals and plant reproductive development nicotianamide is a novel angiotensin-converting enzyme 2 inhibitor in soybean the "big five" phytochemicals targeting cancer stem cells: curcumin, egcg, sulforaphane, resveratrol and genistein inhibition of sars-cov2 infections in engineered human tissues using clinical-grade soluble human ace2 therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses broadspectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease mechanisms of inhibition of ebola virus rnadependent rna polymerase by remdesivir the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus prophylactic and therapeutic remdesivir (gs-5734) treatment in the 345 rhesus macaque model of mers-cov infection a randomized, controlled trial of ebola virus disease therapeutics compassionate use of remdesivir for patients with severe covid-19 abt-378, a highly potent inhibitor of the human immunodeficiency virus protease safety and antiviral activity at 48 weeks of lopinavir/ritonavir plus nevirapine and 2 nucleoside reversetranscriptase inhibitors in human immunodeficiency virus type 1-infected protease inhibitorexperienced patients kaletra (lopinavir/ritonavir) role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment of middle east respiratory syndrome with a combination of lopinavir/ritonavir and interferon-β1b (miracle trial): statistical analysis plan for a recursive two-stage group sequential randomized controlled trial a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 arbidol as a broad sprctrum antivital: un update characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol arbidol: a broad-spectrum antiviral that inhibits acute and chronic hcv infection arbidol inhibits viral entry by interfering with clathrin-dependent trafficking the synthetic antiviral drug arbidol inhibits globally prevalent pathogenic viruses arbidol and other low-molecular-weight drugs that inhibit lassa and ebola viruses clinical features of 69 cases with coronavirus disease arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study in vitro and in vivo activities of anti-influenza virus compound t-705 favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase the ambiguous base-pairing and high substrate efficiency of t-705 (favipiravir) ribofuranosyl 5'-triphosphate towards influenza a virus polymerase favipiravir as a potential countermeasure against neglected and emerging rna viruses t-705 (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections experimental treatment with favipiravir for ebola virus disease (the jiki trial): a historically controlled, single-arm proof-of-concept trial in guinea coronavirus main protease (3clpro) structure: basis for design of anti-sars drugs crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors structure of mpro from covid-19 virus and discovery of its inhibitors flavonoids interfere with nlrp3 inflammasome activation severe acute respiratory syndrome coronavirus viroporin 3a activates the nlrp3 inflammasome luteolin alleviates nlrp3 inflammasome activation and directs macrophage polarization in lipopolysaccharide-stimulated raw264.7 cells myricetin inhibits nlrp3 inflammasome activation via reduction of ros-dependent ubiquitination of asc and promotion of ros-independent nlrp3 ubiquitination dietary apigenin reduces induction of lox-1 and nlrp3 expression, leukocyte adhesion, and acetylated low-density lipoprotein uptake in human endothelial cells exposed to trimethylamine-n-oxide quercetin and ascorbic acid suppress fructose-induced nlrp3 inflammasome activation by blocking intracellular shuttling of txnip in human macrophage cell lines flavonoids interfere with nlrp3 inflammasome activation baicalin suppresses nlrp3 inflammasome and nuclear factor-kappa b (nf-κb) signaling during haemophilus parasuis infection wogonoside protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting nf-κb and nlrp3 inflammasome activation antiviral efficacy of lavonoids against enterovirus 71 infection in vitro and in newborn mice baicalin, a metabolite of baicalein with antiviral activity against dengue virus emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme 2 interaction effective inhibition of mers-cov infection by resveratrol who launches global megatrial of the four most promising coronavirus treatments use of antiviral drugs to reduce covid-19 transmission virological assessment of hospitalized patients with covid-19 an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice we are grateful to all of the colleagues who have given critical comments on this work.j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f key: cord-318181-xxc7vdnt authors: ahmed, anwar e.; al-jahdali, hamdan; alshukairi, abeer n.; alaqeel, mody; siddiq, salma s.; alsaab, hanan; sakr, ezzeldin a.; alyahya, hamed a.; alandonisi, munzir m.; subedar, alaa t.; aloudah, nouf m.; baharoon, salim; alsalamah, majid a.; al johani, sameera; alghamdi, mohammed g. title: early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia date: 2018-03-14 journal: int j infect dis doi: 10.1016/j.ijid.2018.03.005 sha: doc_id: 318181 cord_uid: xxc7vdnt background: the rapid and accurate identification of individuals who are at high risk of middle east respiratory syndrome coronavirus (mers-cov) infection remains a major challenge for the medical and scientific communities. the aim of this study was to develop and validate a risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. methods: a two-center, retrospective case–control study was performed. a total of 360 patients with confirmed pneumonia who were evaluated for mers-cov infection by real-time reverse transcription polymerase chain reaction (rrt-pcr) between september 1, 2012 and june 1, 2016 at king abdulaziz medical city in riyadh and king fahad general hospital in jeddah, were included. according to the rrt-pcr results, 135 patients were positive for mers-cov and 225 were negative. demographic characteristics, clinical presentations, and radiological and laboratory findings were collected for each subject. results: a risk prediction model to identify pneumonia patients at increased risk of mers-cov was developed. the model included male sex, contact with a sick patient or camel, diabetes, severe illness, low white blood cell (wbc) count, low alanine aminotransferase (alt), and high aspartate aminotransferase (ast). the model performed well in predicting mers-cov infection (area under the receiver operating characteristics curves (auc) 0.8162), on internal validation (auc 0.8037), and on a goodness-of-fit test (p = 0.592). the risk prediction model, which produced an optimal probability cut-off of 0.33, had a sensitivity of 0.716 and specificity of 0.783. conclusions: this study provides a simple, practical, and valid algorithm to identify pneumonia patients at increased risk of mers-cov infection. this risk prediction model could be useful for the early identification of patients at the highest risk of mers-cov infection. further validation of the prediction model on a large prospective cohort of representative patients with pneumonia is necessary. middle east respiratory syndrome coronavirus (mers-cov) was first identified in saudi arabia in 2012. the diagnosis of this infection remains complex (al johani and hajeer, 2016; sung et al., 2016; ahmed, 2017a) and it has a high fatality rate (ahmed, 2017b, c; aleanizy et al., 2017; sherbini et al., 2017; kim et al., 2017) . the early detection and identification of individuals at high risk of a disease is the most effective strategy to improve patient clinical outcomes (ahmed, 2017a) and reduce the costs of testing, both physical and economic (ahmed et al., 2011 (ahmed et al., , 2013 . the real-time reverse transcription polymerase chain reaction (rrt-pcr) has been found to be valid and accurate for the evaluation of respiratory swabs, sputum, and other endotracheal aspirate material (corman et al., 2012a, b) . however, although rrt-pcr is the most accurate and sensitive test available at the authors' centers, absolute identification of mers-cov may require multiple clinical specimens from different sources and take days (corman et al., 2012a, b; anon, 2018a) . the saudi ministry of health (moh) has developed mers-cov visual triage guidelines to identify suspected cases (anon, 2018b) . the current guidelines include fever, respiratory symptoms, gastrointestinal symptoms, chronic diseases, and risk of exposure to mers-cov. in clinical practice, identifying high-risk individuals can be challenging, since most laboratory-confirmed mers-cov patients report common clinical risk indices to patients with other respiratory conditions. for instance, respiratory and gastrointestinal symptoms are common for both mers-cov and non-mers-cov patients (mohd et al., 2016) . thus, further exploration must take place to reduce the risk of mers-cov infection. a risk prediction model is urgently needed to stratify patients with suspected mers-cov. this may decrease the risk of virus transmission to others who are in close contact, for example healthcare workers, patients, and hospital visitors, by allowing the careful management of those who are potentially infected at an early stage of infection. developing a mers-cov prediction model may more efficiently aid physicians in identifying individuals at high risk and selecting the necessary test(s) to improve prevention and control measures. several methodological studies have shown that combining demographic characteristics with clinical, radiological, and laboratory information can improve risk assessment and diagnostic accuracy (ahmed et al., 2011 (ahmed et al., , 2013 sidransky, 2002; etzioni et al., 2003) . these previous studies used a linear combination to develop an algorithm that combines demographic characteristics, symptoms, and clinical, radiological, and laboratory findings to identify the highly suspected mers-cov cases. mers-cov was initially identified in a patient being treated for pneumonia in 2012 (zaki et al., 2012) , and since then, pneumonia and its symptoms have remained common characteristics in mers-cov patients (saad et al., 2014; choi et al., 2016) . the aim of this study was to develop and validate a reliable risk prediction model for the screening of suspected cases of mers-cov infection in patients who have developed pneumonia. a two-center, retrospective case-control study was conducted utilizing data from king abdulaziz medical city in riyadh (kamc-r) and king fahad general hospital in jeddah (kfgh-jed), saudi arabia. the data were obtained between september 1, 2012 and june 1, 2016. kfgh-jed experienced a mers outbreak between march and may 2014 (oboho et al., 2015) , and kamc-r experienced a large mers outbreak between june and august 2015 (al-dorzi et al., 2016) . both study centers applied the saudi moh case definitions (anon, 2018b) to identify suspected individuals in the population studied, and rrt-pcr was used as the gold standard test to diagnose mers-cov in multiple and different clinical specimens when necessary. mers-cov-related symptoms were common at both centers. the project received ethical approval from two independent ethics committees: the saudi moh (irb log number 16-230e) and king abdullah international medical research center (study number rc17/061), riyadh saudi arabia. during the study, the medical records of 829 subjects who were being assessed by rrt-pcr for suspected mers-cov were reviewed. the suspicion of mers-cov infection at both kamc-r and kfgh-jed was determined based on the saudi moh guidelines (anon, 2018b) . two groups were compared: patients who were positive and patients who were negative for mers-cov infection. in an effort to reduce heterogeneity between the study groups, only subjects with a lung infiltrate on chest x-ray and/or computed tomography (ct) scan of the chest were included in the analysis. thus, subjects who had no available results of a chest x-ray or ct scan of the chest were excluded. the initial screening for suspected mers-cov patients includes pneumonia (anon, 2018b) . most of the patients studied were evaluated for pneumonia immediately after presentation. the study excluded subjects who were less than 15 years of age (pediatrics/children), as defined in the saudi moh guidelines (anon, 2018b) , and excluded subjects who had upper respiratory tract infections (respiratory symptoms, positive or negative mers-cov rrt-pcr, and normal chest x-ray and ct scan of the chest). the final sample comprised a cohort of 360 subjects who had a lung infiltrate on chest x-ray and/or a ct scan of the chest, who were classified according to the results of mers-cov rrt-pcr. the case group consisted of 135 pneumonia patients who were positive for mers-cov infection, and the control group consisted of 225 pneumonia patients who were negative for mers-cov infection. cases were defined as patients with mers-cov pneumonia who had positive mers-cov rrt-pcr on nasopharyngeal aspirate and/ or bronchoalveolar lavage in addition to a lung infiltrate on chest xray and/or ct scan of the chest. controls were defined as patients with respiratory symptoms, a lung infiltrate on chest x-ray and/or ct scan of the chest, pneumonia, and negative mers-cov rrt-pcr result of nasopharyngeal aspirate and/or bronchoalveolar lavage. nineteen predictive variables were included: age, sex, fever (temperature !38 c), one composite respiratory symptom (the presence of cough, bloody cough, shortness of breath, or chest pain), one composite gastrointestinal symptoms (the presence of diarrhea, vomiting, or nausea), seven mers-cov potential risk factors (contact with sick patients or camels, severe illness (defined according to the patient's clinical status, 'yes/no', which is based on clinical judgment), diabetes, lung disease, liver disease, renal disease, and heart disease), and seven laboratory measurements (white blood cell (wbc) count (â10 9 /l), platelets (â10 9 /l), creatinine (mmol/l), bilirubin (mmol/l), alanine aminotransferase (alt; u/l), aspartate aminotransferase (ast; u/l), and albumin (g/ l)). the reference ranges for the laboratory measures were as follows: wbc count, 4-11 â10 9 /l; platelets, 150-400 â 10 9 /l; creatinine, 50-98 mmol/l; bilirubin, 3.4-20.5 mmol/l; alt, 5-55 u/l; ast, 5-34 u/l; albumin, 35-55 g/l. no serological tests were available at the centers for these patients. stata statistical software release 15, 2017 (statacorp. llc, college station, tx, usa) was used for the data analysis. the sample characteristics were recorded as the frequency and percentage, or as the mean ae standard deviation (sd). laboratory measurements were summarized as the median and 25th-75th percentiles. a subgroup analysis (chi-square test, independent samples t-test, or mann-whitney u-test) was used to identify unadjusted associations between demographic, clinical, and laboratory measurements according to mers-cov status. the performance of each bivariate predictor was further assessed by unbiased estimate, the area under the curve (auc), and its 95% confidence interval (ci). stepwise binary logistic regression was employed to develop a mers-cov risk prediction model and identify factors that could be used to estimate the probabilities of mers-cov infection. the goodness-of-fit of the final model was tested by hosmer-lemeshow procedure: a large p-value indicates that a model has a good fit. the discrimination ability between high and minimal risk of mers-cov infection of the final model was assessed by the auc and its 95% ci. a receiver operating characteristics (roc) curve was generated for the risk prediction model. two hundred random samples were drawn with replacements from the original study sample (n = 360). the model internal validity was assessed in these 200 samples by the auc and its 95% ci. optimal cut-off values of the probabilities for each model were determined using a generalized youden index (youden, 1950) . at these thresholds, it was possible to achieve the best balance between specificity and sensitivity. a total of 360 pneumonia patients were included in the analysis: 37.5% were confirmed mers-cov-positive and 62.5% were confirmed mers-cov-negative. the mean age at presentation was 55.9 years, with a standard deviation of 20.2 years; age ranged between 16 and 109 years. of the total sample, 6.9% had been in contact with a sick patient or camel, 60% had fever, 84.2% had at least one respiratory symptom, and 18.6% had at least one gastrointestinal symptom. the two groups were similar in the distribution of age (p = 0.220) and sex (p = 0.079). the characteristics of the sample can be found in table 1 . subgroup analyses are presented in tables 1 and 2. the chisquare test or the independent samples t-test revealed that sex (p = 0.079) and age (p = 0.220) were similar in the two groups. the risk of mers-cov infection was similar in patients with and without fever (p = 0.267), respiratory symptoms (p = 0.080), or gastrointestinal symptoms (p = 0.382). severe illness (p = 0.001), contact with a sick patient or camel (p = 0.001), diabetes (p = 0.001), heart disease (p = 0.007), and renal disease (p = 0.025) were significantly associated with an increased risk of mers-cov infection. the independent samples mann-whitney u-test revealed that the wbc count (p = 0.001) and platelet count (p = 0.006) were significantly lower in patients who were positive for mers-cov than in those who were negative for mers-cov infection. in contrast, creatinine (p = 0.001), bilirubin (p = 0.001), ast (p = 0.001), and albumin (p = 0.004) were significantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. alt (p = 0.352) was insignificantly higher in patients who were positive for mers-cov than in those who were negative for mers-cov infection. according to the individual roc curve analysis (table 3) , severe illness, diabetes, wbc count, creatinine, bilirubin, albumin, and ast were the most important predictors of mers-cov infection. a risk prediction model was developed using stepwise binary logistic regression (p 0.05). the model retained seven independent variables that were associated with increased odds of mers-cov (table 4 ). male sex (adjusted odds ratio (aor) 1.883, p = 0.043), contact with a sick patient or camel (aor 21.915, p = 0.001), diabetes (aor 2.703, p = 0.002), severe illness (aor 6.312, p = 0.001), low wbc count (aor 0.897, p = 0.001), high ast (aor 1.007, p = 0.007), and low alt (aor 0.995, p = 0.024) were found to have a significant impact on the prediction of mers-cov. the hosmer-lemeshow test indicated that this model fitted the data well (p = 0.592). this model showed substantial discrimination, with an auc of 0.8162 and a 95% ci of 0.7651-0.8674 ( figure 1 ). the prediction model was validated using the bootstrap procedure. a total of 200 random samples were drawn with replacements from the original sample, and the model showed a substantial ability to assess mers-cov infection in this study population (auc 0.804, 95% ci 0.7838-0.8235). the findings in table 4 were used to create a risk-probability model. the risk prediction for the model can be expressed by the following equation: predicted probability = [1 + exp(1.409-(0.633 â male) à (3.087 â sick patient or camel contact) à (0.995 â diabetes) à (1.842 â severe illness) + (0.109 â wbc count) à (0.007 â ast) + (0.005 â alt))] à1 . a calculator was developed to calculate the potential risk of mers-cov infection in pneumonia patients. we determined the optimal cut-off or threshold values of the probabilities to mark the differences between the high-risk and low-risk groups. when an equal weight was given for sensitivity and specificity (m = 1), the optimal cut-off value (probability !0.33) produced sensitivity and specificity of 0.716 and 0.783, respectively. when more weight was given for sensitivity than specificity (m = 0.50), the optimal cut-off value (probability !0.20) produced sensitivity and specificity of 0.902 and 0.550, respectively. when more weight was given for specificity than sensitivity (m = 1.50), the optimal cut-off value (probability !0.39) produced sensitivity and specificity of 0.647 and 0.831, respectively. based on data from the two largest hospitals in saudi arabia, a risk prediction model was developed for mers-cov infection in pneumonia patients. this model was generated from a retrospective study and should be assessed prospectively for external validation. seven variables were identified as having a great impact on the mers-cov risk assessment prediction. the risk prediction model highlights the strong potential impact of male sex, contact with a sick patient or camel, severe illness, diabetes, low wbc count, high ast, and low alt on mers-cov infection. these few important parameters could be part of routine medical examinations to be performed (for the purpose of identifying highly suspected individuals) in daily clinical practice in order to make a prompt and timely clinical decision. according to the model, high ast was associated with an increased risk of being infected with mers-cov. this finding is similar to that of mohd et al., who noted high ast levels in mers-cov patients (mohd et al., 2016) . unlike their findings, it was noted in the present study that the impact of alt became significantly negative after controlling for several confounders. however, this type of association should be evaluated further in a prospective study in the presence of other unmeasured confounders. although sex was found to have no impact on mers-cov infection in the unadjusted analysis, the multivariate analysis revealed that the risk of mers-cov infection was 88.3% times higher in males than in females. this may be because other factors are playing a role in the development of mers-cov in males, such as camel contact, since males are more likely than females to have contact with camels. in agreement with the recent saudi moh mers-cov visual triage guidelines for the identification of suspected cases (anon, 2018b) , it was found that the odds of being infected with mers-cov were higher in patients with diabetes as compared to those with no diabetes. this also supports the findings of previous studies (badawi and ryoo, 2016; assiri et al., 2013; al-tawfiq et al., 2014; alraddadi et al., 2016) in which researchers systematically recognized that diabetes is a risk factor for mers-cov infection. these findings indicate that more attention should be given to assessing the risk of mers-cov infection in diabetic patients and whether the risk depends on a specific diabetes type or condition in these patients. as asserted in the saudi moh mers-cov visual triage guidelines and many other studies (muhairi et al., 2016; younan et al., 2016; reeves et al., 2015; sabir et al., 2016; azhar et al., 2014a, b) , contact with a sick patient or camel was identified as an independent predictor of mers-cov infection, according to the risk prediction model. it must be noted that the finding in the present study could have been limited by combining camel contact and sick patient contact due to the small sample size of each category. this study shows the importance of incorporating various types of information to improve the performance of the risk prediction. according to the linear combination model, it was found that several of the parameters highlighted in the saudi moh mers-cov visual triage guidelines were not able to distinguish between 'highrisk' and 'low-risk' groups, nor did they help in predicting mers-cov infection. for instance, fever, respiratory symptoms, gastrointestinal symptoms, heart disease, and renal disease were noted to have an insignificant impact on mers-cov infection. however, in agreement with the saudi moh mers-cov guidelines and two other reports (mohd et al., 2016; arabi et al., 2017) , the odds of being infected with mers-cov were associated with a significant risk reduction of 10.3% for each unit increase in wbc count. these results suggest that demographic, clinical, radiological, and laboratory data should be used in routine practice to identify suspected mers-cov cases, as such data could serve as the first line of prevention strategies. it was found that the accuracy of prediction (figure 1 ) was further improved when combining various medical and patient variables as opposed to relying on a single factor (table 3 ). this has been proven theoretically and in application (ahmed et al., 2013 (ahmed et al., , 2011 (ahmed et al., , 2015 etzioni et al., 2003; shen, 2008; pepe and thompson, 2000; su and liu, 1993; thompson, 2003) , where a linear combination has been used to maximize the diagnostic accuracy of a disease of interest. the strength of this study lies in the fact that a simple and applicable predictive model was developed that incorporates demographic, clinical, radiological, and laboratory data, where these were functionally associated and contributed greatly to stratifying and distinguishing between a high and a minimal risk of mers-cov infection. this simple evaluation of suspected mers-cov cases appears promising and could be implemented easily in routine clinical practice. this model could be used as a risk stratification method or a triage tool to help physicians in making an informed decision and planning the next step when deciding whether an rrt-pcr or further investigation is necessary. it was possible to derive a risk probability algorithm (range 0-1), a generalized youden index (choi et al., 2016) was used to determine an optimal cut-off to stratify the risk, and a risk probability of !0.41 was identified as being the optimal cut-off, with a sensitivity of 0.688 and specificity of 0.789. several limitations to the proposed risk prediction model were identified. the study findings were based on a retrospective design; therefore this prediction model should be interpreted with caution. limited information was available on patient variables, clinical variables, and transmission routes. for example, information on primary cases and secondary cases may be supplemented by the results of clinical, radiological, and laboratory data. in this study, 'contact with a sick patient' and 'contact with a sick camel' were combined into one variable due to the small number in each category. severe illness was based on a subjective judgment. an additional potential limitation was that the duration of symptoms was not available for these patients. this study investigated a very specific population (pneumonia) at only two centers, which could compromise the applicability and generalizability of the risk prediction model. moreover, the prediction model may not be generalizable to patients who do not fulfill the moh guidelines. further validation of the prediction model on an external sample and prospective cohort of representative patients with pneumonia is necessary, specifically in relevant settings: emergency, outpatient, inpatient, and community. despite these limitations, the model developed shows promise for the identification of suspected mers-cov cases in clinical practice. this model could be applicable in various healthcare settingsinpatient, outpatient, and emergency departmentsbecause no extensive laboratory testing is required and samples may be available within short turnaround times. this may allow rapid evaluation and improve clinical decision-making. in conclusion, this study provides a simple, practical, and valid algorithm to identify individuals at increased risk of mers-cov infection among patients who have developed pneumonia. this risk model is not only useful for risk stratification and decisionmaking in clinical practice, but it could also be useful in preventing and managing the possible spread of mers-cov. the usefulness of this newly developed tool most be validated in an external prospective study. the project received ethical approval from two independent ethics committees: the saudi ministry of health (irb log number 16-230e) and king abdullah international medical research center (study number rc17/061), riyadh saudi arabia. none. none declared. accuracy and cost comparison in medical testing using sequential testing strategies reducing cost in sequential testing: a limit of indifference approach believe the extreme (be) strategy at the optimal point: what strategy will it become diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study the predictors of 3-and 30-day mortality in 660 mers-cov patients mers-cov diagnosis: an update the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study risk factors for primary middle east respiratory syndrome coronavirus illness in humans laboratory testing for middle east respiratory syndrome coronavirus (mers-cov) case definition and surveillance guidance -updated critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient evidence for camel-to-human transmission of mers coronavirus prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction combining biomarkers to detect disease with application to prostate cancer middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate mers-cov outbreak in jeddah-a link to health care facilities combining diagnostic test results to increase accuracy mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia on the principles of believe the positive and believe the negative for diagnosis using two continuous tests middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data emerging molecular markers of cancer linear combinations of multiple diagnostic markers comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription pcr assessing the diagnostic accuracy of a sequence of tests index for rating diagnostic tests mers and the dromedary camel trade between africa and the middle east isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors acknowledge the saudi ministry of health and king abdullah international medical research center for approving this research project. the authors would like to thank the leaders of king abdulaziz medical city in riyadh and king fahad general hospital in jeddah for their support and understanding. supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ijid.2018.03.005. key: cord-333738-3xtb8gye authors: rabets, a.; bila, g.; grytsko, r.; samborsky, m.; rebets, y.; vari, s.; pagneux, q.; barras, a.; boukherroub, r.; szunerits, s.; bilyy, r. title: development of antibodies to pan-coronavirus spike peptides in convalescent covid-19 patients date: 2020-08-22 journal: nan doi: 10.1101/2020.08.20.20178566 sha: doc_id: 333738 cord_uid: 3xtb8gye coronaviruses are sharing several protein regions notable the spike protein (s) on their enveloped membrane surface, with the s1 subunit recognizing and binding to the cellular receptor, while the s2 subunit mediates viral and cellular membrane fusion. this similarity opens the question whether infection with one coronavirus will confer resistance to other coronaviruses? investigating patient serum samples after sars-cov-2 infection in cross-reactivity studies of immunogenic peptides from middle east respiratory syndrome coronavirus (mers-cov), we were able to detect the production of antibodies also recognizing mers virus antigens. the cross-reactive peptide comes from the heptad repeat 2 (hr2) domain of the mers virus spike protein. indeed, the peptide of the hr2 domain of the mers spike protein, previously proven to induce antibodies against mers-cov is sharing 74% homology with the corresponding sequence of sars-cov-19 virus. sera samples of 47 convalescent sars-cov-2 patients, validated by rt-pcr-negative testes 30 days post-infection, and samples of 40 sera of control patients (not infected with sars-cov-2 previously) were used to establish eventual cross-bind reactivity with the mers peptide antigen. significantly stronger binding (p<0.0001) was observed for igg antibodies in convalescent sars-cov-2 patients compared to the control group. if used as an antigen, the peptide of the hr2 domain of the mers spike protein allows discrimination between post-covid populations from non-infected ones by the presence of antibodies in blood samples. this suggests that polyclonal antibodies established during sars-cov-2 infection has the ability to recognize and probably decrease infectiveness of mers-cov infections as well as other coronaviruses. the high homology of the spike protein domain suggests in addition that the opposite effect can also be true: coronaviral infections producing cross-reactive antibodies affective against sars-cov-19. the collected data prove in addition that despite the core hr2 region being hidden in the native viral conformation, its exposure during cell entry makes it highly immunogenic. since inhibitory peptides to this region were previously described, this opens new possibilities in fighting coronaviral infections. coronaviruses such as the middle east respiratory syndrome coronavirus ( the spike surface glycoprotein (s) plays a key role in mediating virus attachment and fusion and are indeed present in all human infecting coronaviruses. they can be cleaved by host proteases into an n-terminal s1 subunit and a membrane-bound c-terminal s2 region. in order to engage a host receptor, the receptor-binding domain (rbd) of the s1 subunit undergoes conformational movements, which transiently hide or expose the determinants of receptor binding [3, 4] . the heptad repeat 1 (hr1) region in s2 subunits forms a homotrimeric structure, exposing 3 highly conserved hydrophobic grooves on the surface resulting in binding of 3 heptad repeat 2 (hr2) regions and formation of six-helix bundle structure (6-hb). 6-hb is responsible for close approximation of viral and host membranes and their subsequent merging. binding of hr1 and hr2 domains results in the six-helix bundle needed for merging with host cell membrane. thus parts of the hr2 domain specifically binding hr1 can be considered as ideal coronaviral inhibitor strategy preventing cellular entry [5] . further optimization of peptide sequence resulted in pan-coronaviral inhibitors, like ek1 [3] able to inhibit sars-cov-19 pseudovirus infection [6] . the structure of hr2 is poorly resolved during crystallographic assessment due to high level of flexibility [7] . unlike highly mutable receptor-binding domain, the hr1 and hr2 domains are highly conservative between coronaviruses, so form a perfect target for viral neutralization and generation of immunity that latter can be used for viral testing. since hr2 and hr1 domains are merged and surface exposed after s protein cleavage we expected them to be highly immunogenic. this similarity can result in the development of cross-reactive antibodies and protection against other coronaviruses, in case of being infected by another virus species. in this work we would like to establish if sars-cov-2 results in production of antibodies, that are also recognizing mers virus antigens. the complete crystal structure of the hr2 domain of sars-cov-2 remains currently unavailable due to it conformation changes and problems in stabilization [4] (figure 1a) . using seqatoms algorithm [8] the most complete structure of the corresponding region containing defined atomic coordinates was identified to be the one proposed by walls et al [9] , namely a model for hr1 hr2 rearrangements and unfolding accompanying viral entry into host cells for other coronaviruses. this structure clearly demonstrates exposure of the hr2 domains upon cellular binding in trimeric (figure 1b) and monomeric form (figure 1c) . to evaluate the cross reactivity we selected the hr2-specific peptide of the spike protein of mers-cov reported to possessed high immunogenic potential [10] [11] [12] . indeed, ongoing studies showe that it can be also successfully used to raise mers-recognizing antibodies in the presence of neutrophil extracellular traps(net) forming nanoadjuvants [13] . the selected hr2 peptide of the spike protein of mers virus (depicted yellow, figure 1d using pdb deposited crystal structure of 4njl_a, [14] ) shares significant similarities in 3d structure between (the only) known crystal structure of unfolded hr2 domain ( figure 1c ) and with pan-coronaviral inhibitor peptide ek1, using crystal structure 5zvk_a [3] (figure 1e) . protein blast analysis reveales 46% identity and 76% similarity in aminoacid sequence of the mers peptide with the corresponding peptide of the sars-cov-19 spike protein (sequence id qkj68605.1) (figure 1f ). all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . (a) image of spike protein (s1) of sars-cov-2. (b) s1 upon host cell interaction: conformational changes in trimer are occurring, exposing previously hidden hr2 domain regions [4] , (c)monomeric part of (b), exposing domain with structural similarity (yellowidentical, orangesimilar amino acids) towards corresponding mers peptide, depicted on (d) and pan. (f). protein blast all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . analysis of used mers protein is showing similarity towards the sequence in the genome of sars-cov-19. sequence id: qkj68605.1. crystal structure of hr2 domain for sars-cov-19 is currently not available, thus it is represented as rectangle in (a) based on last connected coordinates available in 6vsb structure. having established the structural similarity between the s1 mers peptide with the genome of sars-cov-2, sera of convalescent sars-cov-2 infected patient, who have never suffered from mers-cov infection before, have been collected and tested for the presence of antibodies. the s1 mers-cov specific peptide, nh2-ccttttttsltqinttlldleyemkkleevvkkleesyidlkel-cooh, which we previously successfully used to raise anti-mers antibodies while testing novel net-stimulating adjuvants [13] , was immobilized on elisa plates and incubated with sera samples. as can be seen from figure s1 ). using this s1 mers-cov specific peptide, discrimination of persons that have suffered from sars-cov-2 infection and those who were not in contact with the virus resulted in a predictive value (area under roc curve) equal to 0.823 (figure 2b) , with a specificity and sensitivity of ~60% (95% confidence). sars-cov-2 infections results in the generation of antibodies with significantly strong cross-reactive towards a mers specific peptide with 76% homology. highly conservative region of the exposed domain suggest that the opposite can be truecoronaviral disease can result in some antibodies able to recognize sars-cov-19 epitops circulating in the blood. to determine whether the strong binding with s1 peptide is correlated with higher amount of anti-sars-cov-19 antibodies we used recombinant rbd protein immobilization on elisa plates to incubate with sera samples to evaluate the amount of formed igg type antibodies. as can be seed from figure 2c , the sera samples having shown stronger binding of igg antibodies with anti-hr2 mers spike protein also contained higher igg reactivity towards anti-rbd spike protein of sars-cov-19. the pearsons correlation between the two parameters was 0.5492, p<0.0001. this suggests stronger humoral responses towards one of the virus will be associated with the intensity of the immune response towards other coronaviruses. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . cross-reactivity between coronaviruses has become a critical question, since it brings new promises against covid-19 protection. on the other hand, this cross-reactivity can be negative, since available cross-reactivity towards coronaviruses will make coronaviruses not the best choice for vectors in vaccines, especially taking into account recent data on broad immune cross-reactivity [15] . indeed, it was reported that epitope pools detect cd4+ and cd8+ t cells in 100% and 70% of convalescent covid patients respectively, recognizing s and m proteins, with at least eight sars-cov-2 orfs targeted. t cell reactivity to sars-cov-2 epitopes is also detected in non-exposed individuals [6] . in sars-cov-2 patients s-reactive cd4+ t cells equally target n-terminal and c-terminal parts of the spike protein, whereas in healthy donors s-reactive cd4+ t cells react almost exclusively to the c-terminal part. this part is characterized by a higher homology to spike glycoprotein of human endemic "common cold" coronaviruses, and contains the s2 subunit of s with the cytoplasmic peptide (cp), the fusion peptide (fp), and the transmembrane domain (tm) but not the receptor-binding domain (rbd). s-reactive cd4+ t cells from sars-cov-2 patients are further distinct to those from healthy donors as they co-expressed higher levels of cd38 and hla-dr, indicating their recent in vivo activation [7] . potential preexisting cross-reactive t cell immunity to sars-cov-2 has broad implications, as it could explain aspects of differential sars-cov-2 clinical perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medrxiv preprint outcomes, influence epidemiological models of herd immunity, or affect the performance of sars-cov-2 candidate vaccines. pre-existing memory cd4+ t cells that are cross-reactive with comparable affinity to sars-cov-2 and the common cold coronaviruses hcov-oc43, hcov-229e, hcov-nl63, or hcov-hku1. thus, variegated t cell memory to coronaviruses that cause the common cold may underline at least some of the extensive heterogeneity observed in covid-19 disease [8] . based on these and our data one can assume that the c-terminal part of the spike protein is immunogenic due to exposure upon merging with host cells. its conservative nature provides the background for the development of cross-coronaviral immune responses both cellular, and demonstrated here, by a igg type humoral immune response. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medrxiv preprint goat anti-human igg (h+l)-horseradish peroxidase (hrp) (109-035-003, jackson immunoresearch) was diluted in washing buffer (1:25000), added to the plates and incubated at room temperature for 1 h. after the corresponding washings, the assay was developed with 3,3',5,5'-tetramethybezidine (tmb) containing excess of h2o2 as a substrate (50 µl per well). the reaction was stopped with 50 µl/well of sulfuric acid (1 m). the absorbance was read at 450 nm/600nm using a perkin elmer bioassay reader hst700 (waltham, usa). the protein homology searches were done using blast (ncbi) and pdb databases. in order to include the regions with resolved structures in our searches we had used seqatoms (http://www.bioinformatics.nl/tools/seqatoms/) [8] . protein structures were visualized using pymol (https://pymol.org/). multiple sequence alignments were done using clutalw [16] data analysis. elisa testing was performed in duplicate using 2 technical replicates for each analysis (coefficient of variation [cv] always <3%). the data were normalized between plates using positive controls and corrected for background signal of secondary antibodies, then the mean values were calculated and are shown on the graphs. for comparisons between two groups, the mann-whitney u-test for numerical variables was used. a receiver operating characteristic (roc) curve was generated. the area under the roc (auroc) was calculated to estimate the specificity, sensitivity and usefulness of the binding assays. all analyses were perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medrxiv preprint figure s1 . antibody classes binding peptide of hr2 spike protein of mers virus in convalescent covid-19 sera, from left to right: binding of igm, iga and ige immunoglobulins. all rights reserved. no reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in the copyright holder for this this version posted august 22, 2020. . https://doi.org/10.1101/2020.08.20.20178566 doi: medrxiv preprint middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains seqatoms: a web tool for identifying missing regions in pdb in sequence context tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 aluminum oxide nanowires as safe and effective adjuvants for nextgeneration vaccines structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor key: cord-338564-68z2pxfz authors: kang, min; song, tie; zhong, haojie; hou, jie; wang, jun; li, jiansen; wu, jie; he, jianfeng; lin, jinyan; zhang, yonghhui title: contact tracing for imported case of middle east respiratory syndrome, china, 2015 date: 2016-09-17 journal: emerg infect dis doi: 10.3201/eid2209.152116 sha: doc_id: 338564 cord_uid: 68z2pxfz confirmation of an imported case of infection with middle east respiratory syndrome coronavirus in china triggered intensive contact tracing and mandatory monitoring. using a hotline and surveillance video footage was effective for tracing all 110 identified contacts. contact monitoring detected no secondary transmission of infection in china. because this research was a part of a public health response, our study did not require formal ethical approval from a medical ethics committee. contact tracing was initiated immediately after we detected the case-patient. we communicated with the airline and bus operators to collect passenger information and undertook personal interviews in related communities. a hotline was set up, and the case-patient's travel information was published in the media. we investigated hotline callers and identified suspected contacts. we also reviewed video footage from closed circuit television in hotels and restaurants visited by the case-patient, enabling us to identify contacts and measure duration and distance of exposures. information about bus passengers was limited; consequently, with help from police departments, we analyzed video footage recorded by public surveillance cameras at bus stations and surrounding communities and traced the whereabouts of related passengers. we identified 110 contacts in mainland china: 87 (79%) were from mainland china, 11 (10%) from south korea, 2 (2%) from hong kong, 6 (5%) from taiwan, 3 (3%) from canada, and 1 (1%) from japan. of the 110 contacts, 27 were air travel contacts (passengers onboard the same flight with the case-patient); 24 were land travel contacts (stewards and passengers taking the same buses with the case-patient); and 59 were community contacts (persons who had face-toface contact with the case-patient or who had direct contact with his belongings in hotels, restaurants, and a meeting room) ( table 1) . we found 34 (58%) of the community contacts through personal interviews. the hotline resulted in 16 (59%) air travel contacts and 12 (50%) land travel contacts. reviewing video helped trace 9 (38%) land travel contacts and 25 (42%) community contacts ( table 2) . we located all community, air travel, and land travel contacts within 3 days, 6 days, and 8 days, respectively ( figure) . among 44 contacts whom we classified as close contacts, 6 were air travel contacts who had been seated <3 rows from the case-patient on the flight; 24 were land travel contacts; and 14 were community contacts who had prolonged (>15 minutes) face-to-face (<2 m) contact with the case-patient or direct contact with his belongings. we classified the remaining 66 contacts as common contacts. of the 44 close contacts, 40 were staying in mainland china and were quarantined in designated facilities for 14 days after their last exposure to the case-patient. public health officials checked body temperatures twice daily and monitored symptoms. the remaining 4 close contacts, 2 from taiwan and 2 from south korea, had returned to their countries before they were traced. we notified local health authorities about these 4 contacts. of 66 common contacts, 49 were quarantined in designated facilities, and 17 conducted self-monitoring at home (an alternative for common contacts) for 14 days after their last exposure to the case-patient. public health officials visited them daily. during follow-up, fever developed in 1 contact and 2 others had sore throat. throat swab samples from 106 contacts and serum samples from 53 were obtained on the first and last days of follow-up. an additional set of specimens was collected from the 3 symptomatic contacts immediately after onset east respiratory syndrome, china, 2015 of symptoms. all specimens were tested by real-time reverse transcription pcr, as previously described (4) . no specimens tested positive for mers-cov, and follow-up for contacts ended on june 10, 2015. from the date of his isolation (may 28) until the date of his discharge (june 26), the case-patient received direct medical care and examination from 73 healthcare workers (hcws). these hcws were not considered contacts in our investigation because they all used personal protective equipment, as recommended by the world health organization (5) . the hospital conducted follow-up with all 73 hcws until 14 days after their last interaction with the case-patient. no hcw was symptomatic during followup. throat swab and serum samples were obtained from all hcws on day 10 after the case-patient's admission and on day 14 after his discharge. all specimens tested negative for mers-cov by real-time reverse transcription pcr. follow-up for hcws ended on july 10, 2015. we traced 110 contacts of a patient with an imported case of mers-cov infection in china. follow-up and laboratory testing indicated that no virus transmission occurred among contacts. because of the timely notification from south korea and the world health organization regarding the mers-cov outbreak (4), our hospital was able to prepare in advance for admission of the case-patient. no hcws were infected. our findings indicate that human-tohuman transmission of mers-cov is still limited (6) (7) (8) . to minimize risk of local spreading, china health authorities decided to identify and trace all contacts of the initial case-patient and enforce mandatory monitoring. evidence supports this aggressive policy. first, the mers outbreak in south korea, from where the case-patient traveled, was ongoing, and superspreading was observed there (9,10). second, the patient was symptomatic and potentially infectious during his travel and stay in china. previous clusters have been detected in hospitals and households (11) (12) (13) , and mers-cov transmission might occur in enclosed settings. moreover, because knowledge about mers is still limited, cases could have been easily missed initially. our approach illustrates the feasibility of multiple complementary practices for contact tracing. publicizing information and hotlines can facilitate contact tracing and risk communication and helped us identify >50% of the casepatient's travel contacts. however, we also had to rule out large numbers of false hotline calls that resulted from inaccurate recall and excessive worry. review of video footage *close contacts include 2 from taiwan and 2 from south korea that returned to their countries before they were identified. we notified local health authorities about these 4 contacts. †numbers and percentages for contacts and close contacts exclude 4 contacts who left mainland china before they were identified. is another active solution for identifying contacts, especially for anonymous contacts. in our investigation, we directly located some bus passengers who resided near the station by reviewing footage from surveillance cameras. other bus passengers left the station by private cars, which were captured by cameras. our inquiries into car registration information traced these contacts successfully. reviewing video footage can also measure a contact's exposure objectively and quantitatively. investigators should combine and compare video footage meticulously to gather pieces of information. our contact tracing and monitoring involved challenges. contacts came from different countries and regions, and sites of their exposures varied. also, no identity information for bus passengers was available, and privacy issues were concerns. we spent 8 days tracing these passengers, and some had already left china. furthermore, lack of knowledge about mers made some contacts less willing to comply with mandatory monitoring. nevertheless, we traced and monitored all contacts eventually. we suggest combining multiple approaches and data sources beyond ordinary investigation to trace contacts of persons with imported cases of mers. emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 9, september 2016 world health organization. mers-cov in republic of korea at a glance as of mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov)-china. disease outbreak news imported case of mers-cov infection identified in china infection prevention and control during health care for probable or confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection. interim guidance middle east respiratory syndrome synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea preliminary epidemiological assessment of mers-cov outbreak in south korea jordan mers-cov investigation team. hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus transmission of mers-coronavirus in household contacts we thank all the authorities, the huizhou municipal center for disease control and prevention, the hong kong centre for health protection, and field and laboratory staff for participating in contact tracing and monitoring.dr. kang is a field epidemiologist at the guangdong provincial center for disease prevention and control, guangzhou, china. he is interested in field epidemiology and responses to emerging infectious diseases. key: cord-319501-a2x1hvkk authors: wong, lok-yin roy; lui, pak-yin; jin, dong-yan title: a molecular arms race between host innate antiviral response and emerging human coronaviruses date: 2016-01-15 journal: virol sin doi: 10.1007/s12250-015-3683-3 sha: doc_id: 319501 cord_uid: a2x1hvkk coronaviruses have been closely related with mankind for thousands of years. communityacquired human coronaviruses have long been recognized to cause common cold. however, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses causing severe respiratory diseases. infections by these emerging human coronaviruses are characterized by less robust interferon production. treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. the mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. this review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against sars and mers coronavirus infection are discussed. on the other hand, the counter-measures evolved by sars and mers coronaviruses to circumvent host defense are also dissected. with a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived. [image: see text] coronaviruses (covs) are classified into four genera, namely alpha-, beta-, gamma-and deltacoronavirus, under the family of coronaviridae and the order of nidovirales (woo et al., 2012) . the first three genera were previously known as groups i, ii and iii, respectively (lau et al., 2006; zhong et al., 2012) . covs have been shown to infect many different hosts including bats, birds, dogs, mice and human (woo et al., 2009; de groot et al., 2013) . the infections are commonly zoonotic in nature (chan et al., 2013) . in the past 50 years, several human covs (hcovs) were identified. hcov-229e and hcov-oc43, belonging to alpha-and betacoronavirus respectively, were the first two hcovs identified in the mid-1960s (tyrrell and bynoe, 1965; hamre and procknow, 1966; mclntosh et al., 1967) . healthy individuals infected with either hcov-oc43 or hcov-229e develop illnesses within the range of typical common colds with good prognosis (bradburne et al., 1967) . since the identification of these two hcovs, extensive studies were conducted to understand their pathogenicity. however, almost all studies showed that hcov-oc43 and hcov-229e caused mild illnesses with high titers of neutralizing antibodies (bradburne et al., 1967) . the idea of hcov being a relatively weak respiratory diseasecausing agent was therefore presented to the field. this idea was generally accepted until the outbreak of sars in 2003. sars-cov was the first hcov identified to cause acute respiratory distress syndrome (ar-ds) (cheng et al., 2007; graham et al., 2013) . according to world health organization (who), a total of 8096 cases from 29 countries were reported with a case mortality rate of 9.6%. the sars outbreak changed the landscape of cov studies entirely and marked the new era of combating infectious diseases. tremendous efforts have been put into understanding sars-cov pathogenicity, opening a new page of cov biology. despite advances in infection control and quarantine measures in the past decade, another hcov causing ards was identified in saudi arabia as a novel lineage c betacoronavirus in september 2012 (zaki et al., 2012) . the newly identified hcov was later named mers-cov. up to october 2015, 1611 laboratory-confirmed cases were reported to who with 575 related deaths in 26 countries, including a recent outbreak involving 186 cases and 37 deaths in south korea. mers-cov is closely related phylogenetically to two bat covs, hku4 and hku5, shedding light on the possible zoonotic reservoir of mers-cov (zaki et al., 2012; . together with hcov-hku1 identified in 2005 (woo et al., 2005) and hcov-nl63 discovered in 2004 (fouchier et al., 2004; van der hoek et al., 2004) , six hcovs have been documented up to date. these 6 hcovs present diseases with a range of clinical severity from typical common cold in hcov-oc43, hcov-229e, hcov-hku1 and hcov-nl63 to ards in sars-cov and mers-cov. why these covs show dramatically different pathogenicity in human is an important but unanswered question in the field. one model to explain this difference is based on adaptation and host immunity. according to this model, bats are reservoir of various covs. bat covs constantly emerge in human via intermediate hosts such as civets and dromedaries. exposure of immunologically naïve human populations to these covs commonly causes severe diseases plausibly due to aberrant activation of innate immunity and lack of immune memory. when some covs become better adapted in human by acquiring the ability to transmit from human to human readily, pandemics could arise. meanwhile, as they become fully adapted, the covs might only cause mild diseases in human. existing evidence supports the origin of hcov-oc43, hcov-229e, hcov-hku1 and hcov-nl63 from bats and other animals (woo et al., 2009; huynh et al., 2012; corman et al., 2015) . adaptation and virus-host interaction are also known to be major determinants in cov pathogenesis (pepin et al., 2010; chan et al., 2013) . it will therefore be of great interest to see whether emerging human covs might be particularly capable of evading innate antiviral response while activating pathological inflammation. in other words, we need to determine whether the more severe clinical presentations might be accounted for by the specific interaction between host and emerging human covs, namely sars-cov and mers-cov. in this review, the host innate antiviral response to cov infection is particularly focused. in addition, the viral strategies adopted by sars-cov and mers-cov to subvert innate immunity are also summarized to provide inspiring insights that may explain the discrepancies in virulence (figure 1 ). covs are polycistronic positive-sense single-stranded rna (ssrna) viruses with genomes of about 30 kb in size. the 5′ most two-thirds of cov genome encodes polyprotein 1a (pp1a) and pp1ab replicase polyproteins, which are further cleaved by viral proteases to yield nonstructural proteins (nsps), while the 3′ end of the genome encodes structural and lineage-specific proteins (durai et al., 2015) . the cov life cycle begins with the binding to cellular receptor followed by membrane fusion as well as viral rna and protein synthesis in the cytoplasm. the pp1a and pp1ab polyproteins are co-translationally processed resulting in the formation of the replicase complex. a set of nested subgenomic mrnas and genomic rna, which possess both the same 3′ end and a common 5′ leader sequence derived from the 5′ end of the genome, is then transcribed. normally, only the 5′ end of each mrna is translated. virion assembly is achieved by budding into intracellular membranes and virion release is accomplished through the secretory pathway (cheng et al., 2007; durai et al., 2015) . the coronaviral spike (s) protein is responsible for binding to specific host receptor on cell surface and fusing viral envelope with lipid membrane of host upon infection (bosch et al., 2003; rota et al., 2003; chen et al., 2013) . hcov-nl63 and sars-cov from α-and β-genera respectively recognize angiotensin-converting enzyme 2 (ace2) (li et al., 2003; pyrc et al., 2007; frieman et al., 2008; chen et al., 2013) while mers-cov infects cells through another cell surface enzyme dipetidyl peptidase 4 (dpp4) (chen et al., 2013; raj et al., 2013) . aminopeptidase n (apn) has also been found to be recognized by some α-genus covs like hcov-229e (yeager et al., 1992) . cell surface receptor binding dictates species-specific viral entry as well as tropism. this also confines the direction of cellular antiviral response. we and others have shown the ability of cov s proteins to activate unfolded protein response and endoplasmic reticulum stress fung et al., 2014; siu et al., 2014b) . the activity of s might also be functionally related to coronaviral perturbation of innate antivir-al response including ifn and cytokine production. pattern recognition receptors (prrs) constitute an indispensable part of the host innate immune defense mechan-ism by the detection of foreign, non-self patterns from invading microbes distinct from host. these pathogenassociated molecular patterns (pamps) are usually biomolecules derived from the surface or generated during the life cycle of the microbes. the detection of pamps by host prrs activates innate immune response including the expression of type i ifns and cytokines for clearcycle are exposed to host innate immune sensors, rig-i/mda5 in cytoplasm or toll-like receptors tlr3/7/8 in endosome. activation of these immune sensors initiates a downstream signaling cascade that leads to ifn-β gene expression. rig-i/mda5 conveys signal through a mitochondrial adaptor mavs while tlr signals through trif/myd88. both pathways share the common traf adaptor to activate transcription factors. traf3 serves as an adaptor which activates tank × tbk1/ikkε complex for irf3 phosphorylation and subsequent dimerization, while traf6 is responsible for the activation of ikk complex which phosphorylates the canonical inhibitor of nf-κb (iκb). activated transcription factors are translocated into the nucleus to drive ifn-β expression. (right) ifn-β are secreted into extracellular space and bound to its cognate receptors ifnar to activate downstream jak-stat signaling. receptor-associated tyrosine kinases jak1 and tyk2 are brought to juxtaposition for self-phosphorylation and activation. stats are recruited to and phosphorylated by the tyrosine kinases. phosphorylated stat1/2 with irf9 forms a ternary complex isgf3 which translocates into the nucleus and binds to isre in the promoter region upstream of isg genes. isg genes are expressed consequently to establish an antiviral state in cells. oas is an example of isg which produces 2′, 5′-oligoadenylate (2′, 5′-a) upon detection of dsrna and activates rnase l to cleave viral rna to yield more rlr ligand as a positive-feedback mechanism of ifn production. the cov-encoded proteins shown in red are known to intervene the host innate immune signaling at various action points as evasion mechanisms to sustain viral replication and propagation. the action points at which viral proteins function marked with a question mark (?) represent controversial and inconclusive findings in the field or molecular mechanisms not well studied. mhv: mouse hepatitis virus. arms race between innate immunity and human coronaviruses ance of invading microbes. during cov infection, retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) and toll-like receptors (tlrs) are believed to bear pivotal importance in stimulating host type i ifn induction. it is therefore essential to review the sensing mechanism of the prrs to understand viral evasion mechanisms and provide insights on the development of potential viral antagonists. after viral entry, cov genomes are exposed in the cytoplasm for expression of viral proteins, providing an opportunity for viral rna sensing by host. rlrs are ubiquitously expressed cytoplasmic rna helicases of dexd/h box family responsible for sensing doublestranded rna (dsrna) (yoneyama et al., 2005) . three types of rlrs have been identified up to now, including rig-i, melanoma differentiation-associated gene 5 (mda5) and laboratory of genetics and physiology 2 (lgp2) (loo and gale, 2011) . rig-i and mda5 consist of n-terminal caspase activation and recruitment domain (card) in two tandem copies, a central dexd/h box helicase domain and a c-terminal domain (ctd) (yoneyama et al., 2004 (yoneyama et al., , 2005 . the n-terminal cards are the effector domain of rlrs to mediate downstream transduction, which is held by the ctd when unstimulated (jiang et al., 2011; kowalinski et al., 2011; luo et al., 2011) . however, in the presence of residual amount of cytoplasmic dsrna, rlrs bind to dsrna through the central dexd/h box helicase domain and ctd with atp, causing a conformational change that exposes the n-terminal cards for signal transduction (yoneyama et al., 2004; jiang et al., 2011). lgp2 lacking the n-terminal cards is thought to act as co-factor that augments the function of rig-i and mda5 (satoh et al., 2010; bruns et al., 2014) . exposure of cards leads to oligomerization of rig-i or mda5 to form filamentous structure (berke et al., 2012; peisley et al., 2013; wu et al., 2013) . the card filament recruits and further initiates similar filamentous structure formation of card on mavs, an adaptor protein which further recruits downstream effectors tumor necrosis factor receptor-associated factor 3 (traf3), tank-binding kinase 1 (tbk1) and iκb kinase ε (ikkε) (loo and gale, 2011; wu et al., 2014) . tbk1 and ikkε form a complex of activated protein kinase for phosphorylation and activation of not only mavs adaptor , but also irf3 transcription factor (loo and gale, 2011) . activated irf3 are phosphorylated, dimerized and eventually translocated to the nucleus. on the other hand, traf2/6 is also recruited to mavs for nf-κb activation. specifically, canonical nf-κb inhibitor iκb is phosphorylated and then degraded through proteasomes in a ubiquitinationdependent fashion (loo and gale, 2011) . iκb degrada-tion exposes nuclear localization signal on nf-κb dimer for nuclear translocation. activated irf3 and nf-κb together with other transcription factors including c-jun assemble the enhanceosome that binds to ifn-β promoter for ifn-β expression (ford et al., 2010; loo and gale, 2011) . infection with mouse hepatitis virus induces rig-i expression. in addition, the activation of type i ifn production by this cov in oligodendrocytes requires both rig-i and mda5 . thus, rlrs might play an important role in the sensing of cov infection. several critical questions concerning rlr recognition of covs merit further investigations. first, the role of rlrs in cov sensing should be studied in rlr-null and cov-susceptible cells and animals. when necessary cr-ispr/cas9 technology might be used to disrupt rlr genes in target cells (hsu et al., 2014; yuen et al., 2015) . second, the cov pamps recognized by rlrs should be identified and characterized. particularly, it will be of interest to see whether and how common and highly structured regions in coronaviral genome, such as the aforementioned 5′ leader sequence, might be recognized by rlrs. for example, a polyuridine motif in the 3′ untranslated region of hepatitis c virus genome and the panhandle structure in rna viruses such as influenza a virus have previously been shown to be rig-i agonists (saito et al., 2008; weber et al., 2013; kell et al., 2015; liu et al., 2015b) . in addition, possible involvement of viral proteins such as nucleocapsid (n) in this recognition as in the case of other rna viruses (saito et al., 2008; weber et al., 2013) should also be clarified. finally, comparative analysis of sars-cov, mers-cov and other hcovs for their ability to activate rlrs will shed light on whether rlr activation would be a critical determinant in cov virulence. covs have been observed to infect host cells through more than one pathway. while cov entry by the fusion of viral envelope and host membrane has been described, the endosomal pathway is still considered the classical entry pathway for covs. in this pathway the activation of s protein cleavage by cathepsin l and transmembrane serine protease tmprss2 occurs in the absence of cell surface proteases in certain cell types (shirato et al., 2013; burkard et al., 2014) . in this regard, tlr family may play an essential role in sensing cov infection through the endosomal pathway. tlr family was identified as another prr homologous to drosophila toll receptor (boehme and compton, 2004) , sensing various pamps within the endosome which leads to induction of cytokines and ifns. in human, each of the 11 tlrs is known to specifically recognize a particular pamp and preferentially resides in either plasma or endosomal membrane. the cellular localization of tlrs defines their functions in detecting different pamps. for example, tlrs critically involved in viral nucleic acid sensing, including tlr3 for dsrna, tlr7 and tlr8 for ssrna, and tlr9 for unmethylated cpg island of dsdna viruses, are mainly localized in endosomal membrane while other members having a role in sensing other biomolecules derived from microbial surface components localized to plasma membrane of infected cells (xagorari and chlichlia, 2008; kawai and akira, 2010) . tlr family members being type 1 transmembrane proteins share a similar structure with a single transmembrane domain. tlr specificity is determined by the ectodomain made up of various number of leucine-rich repeats (lrrs) that bind the corresponding pamp directly (boehme and compton, 2004) . signal transduction begins with ligand binding to lrrs in the ectodomain, thus recruiting cytosolic adaptor protein myd88 with cytoplasmic toll/il-1 receptor (tir) domain by homotypic tir-tir domain interaction (xagorari and chlichlia, 2008) . the tlr-myd88 complex then recruits and activates interleukin 1r-associated kinase (irak) by phosphorylation. the activated irak then in turn associates with traf6 and activates a series of downstream effectors leading to the activation of a range of cytokines and ifn-stimulated genes (isgs), while activation of type i ifn expression by tlr3 is independent of myd88 but dependent on trif (boehme and compton, 2004; xagorari and chlichlia, 2008) . tlr pathway is significantly involved in the suppression of cov replication and induction of type i ifn expression. mice deficient of either tlr3 or tlr4 were more prone to sars-cov pathogenesis (mazaleuskaya et al., 2012; totura et al., 2015) . notably, disruption of either myd88 or trif arm of the tlr signaling pathway causes lethal sars-cov disease, indicating the importance of both arms in host innate immunity against sars-cov (totura et al., 2015) . full characterization of the role of tlrs in host innate antiviral response against sars-cov and mers-cov versus other hcovs will not only provide new knowledge about how tlr activation might impact cov pathogenesis, but might also identify new strategies for antiviral and vaccine development. for example, synthetic tlr agonists could potentially serve as antivirals and vaccine adjuvants in the prevention and control of covs. innate antiviral response is the first line of defense against cov infection. type i ifns are important antiviral and immunomodulatory agents. type i ifns function by binding to ifn-α receptor-1 (ifnar-1) and ifnar-2 receptor complex, thus activating janus family tyrosine kinase (jak), leading to the phosphorylation of signal transducer and activator of transcription (stat), a family of transcription factors regulating the expression of isgs. activated stat and irf9 form ifn-stimulated gene factor 3 (isgf3), stimulating expression of isgs by binding to ifn-stimulated response element (isre) in promoters of isgs (levy et al., 2001; samuel, 2001) . viral induction of isgs was abrogated in stat1 -/mice infected with sars-cov. the viral infection could not be cleared resulting in severe disease, extensive lung injury and 100% mortality zornetzer et al., 2010) . this indicates the importance of stat1 in sars-cov pathogenesis. isgs are the workhorses of the innate antiviral response with diverse functions including direct antiviral activities and regulation of adaptive immune system (schneider et al., 2014) . for example, ifn-inducible gene p53 evokes apoptosis in virus-infected cells (takaoka et al., 2003) . ifn-inducible protein kinase pkr, 2′, 5′-oligoadenylate synthetase (oas) and rnase l are important modulators involved in dsrna sensing, viral gene expression and replication. they act sequentially to trigger viral rna degradation and suppression of viral activities (samuel, 2001) . other isgs encoding antiviral effectors such as mx proteins, cholesterol-25-hydrolse, ifitm proteins, trim proteins, viperin, tetherin, cgamp synthase and sting could also be highly relevant to cov infection (schneider et al., 2014; schoggins et al., 2014; ma et al., 2015a; ma et al., 2015b) . inflammatory responses triggered by inflammatory cytokines like tumor necrosis factor α (tnf-α) and ifn-γ are also found to be ifn-dependent (samuel, 2001) . ifns do not only exert antiviral effects through activation of innate immunity but also act as modulators of adaptive immunity. adaptive immune response is activated by increased level of ifns. the levels of major histocompatibility complex (mhc) proteins class i and ii are found up-regulated by ifns. this facilitates efficient antigen presentation and hence cellular immune response to cov infection (samuel, 1991 (samuel, , 2001 ivashkiv and donlin, 2014) . in addition, the roles of non-conventional isgs including micrornas, long non-coding rnas and alternatively spliced isoforms have been increasingly recognized in recent years (schneider et al., 2014) . it will be of importance to determine whether sars-cov and mers-cov might be unique in isg activation as suggested in a recent study, which demonstrated that mers-cov induces repressive histone modifications to down-regulate specific subsets of isgs (menanchery et al., 2014b) . in relation to this, two areas concerning isg activation by covs might require more attention and research efforts. first, unbiased and large-scale screening of antiviral isgs using rna interference or crispr/cas9 technology might be carried out to identify key cellular factors that restrict sars-cov and mers-cov replication and infection. second, small-molecule compounds that activate antiviral isgs could be identified and tested for inhibition of sars-cov and mers-cov replication and infection. for example, establishing the significance of cgas and sting in cov infection might lead to the development of cyclic dinucleotides such as c-di-gmp and cgamp as novel anti-cov agents. covs have been reported to directly or indirectly suppress ifn production and signaling pathways by a subset of viral proteins via various mechanisms. in many cases, infected patients have shown diminished levels of type i ifns. this is especially true for sars and mers patients with severe diseases (faure et al., 2014) . it was also shown that sars-cov and mers-cov were capable of evading type i ifn production and signaling to different extents in cultured cells (kindler et al., 2013) . when the deficiency in type i ifn production in cov-infected cells was remedied by ifn-α treatment, cov replication was inhibited (falzarano et al., 2013) . combination of ifn-α with other antiviral drugs further improves the survival of infected patients (omrani et al., 2014) . this evidence suggests an essential role of type i ifns in the antiviral effect against cov infection. covs have evolved strategies to counter host antiviral response by antagonizing type i ifn production and signaling. cov proteins have been characterized to exhibit innate immunosuppressive effects in cellular models. below we will discuss them in three categories: structural, lineagespecific and non-structural proteins (nsps) (de groot et al., 2013) . nsps of covs are involved in the assembly of the replicase complex for viral rna synthesis (sevajol et al., 2014) . certain nsps have also been reported to possess innate immunosuppressive effect that facilitates viral replication and propagation, although these proteins per se are not required for viral life cycle (narayanan et al., 2008b; lokugamage et al., 2015) . nsps of different covs are more or less evolutionarily conserved suggesting their functional significance, with the exception of nsp1 and nsp2, which are thought to contribute to virulence of certain covs (neuman et al., 2014) . four structural proteins are found in covs, namely s, membrane (m), envelope (e) and n proteins. structural proteins contribute the architecture for virion assembly. accessory proteins are lineage-specific with diverse behaviors in different covs but are not essential for viral replication and propagation (de groot et al., 2013) . cov nsps have shown suppressive effects in various immune pathways including type i ifn production and signaling. sars-cov and mers-cov nsp1 proteins have been shown to selectively induce degradation of host mrna by inducing endonucleolytic cleavage while leaving viral rnas intact (huang et al., 2011; lokugamage et al., 2015) . in addition to the induction of endonucleolytic cleavage of host mrna, general inhibition of host mrna translation is achieved by binding of 40s subunit of ribosome with sars-cov nsp1 (huang et al., 2011) . particularly, sars-cov nsp1 inhibits innate immune response by translational repression of ifn mrna transcripts, hence altering ifn production and signaling (narayanan et al., 2008a; tanaka et al., 2012) . mers-cov nsp1 has also been characterized to specifically induce endonucleolytic cleavage of nuclear transcribed mrna while sparing cytoplasmic host mrna and viral rna (lokugamage et al., 2015) . this suggests a novel mechanism for evading host immune response. cov nsp3 protein has been characterized with a papain-like protease (plpro) domain for enzymatic cleavage of pp1a and pp1ab as well as a plp2 domain with deubiquitinating and deisgylating activity (clementz et al., 2010; mielech et al., 2014) . mers-cov plpro is able to antagonize ifn production induced by rig-i and mda5 as well as nf-κb activation . mers-cov plpro is catalytically more efficient (báez-santos et al., 2014) and its catalytic activity is indispensable for the suppressive effect on rig-i, mda5 and nf-κb (mielesh et al., 2014) . in contrast, sars-cov plpro does not require enzymatic activity for ifn antagonism (clementz et al., 2010) . hcov-nl63 and sars-cov plp2 transmembrane domain can also act as potent ifn antagonists to suppress ifn production induced by rig-in, a dominant active form of rig-i (clementz et al., 2010) . in another view of direct inhibition of ifn induction, nsp3 with deubiquitinating and deisgylating activity may also influence the ubiquitination and isgylation pattern and dynamics thus indirectly hindering innate immune response against cov infections (clementz et al., 2010) . for example, isgylation and ubiquitination of irf3 required for optimal activation is probably altered by plp domain of nsp3. apart from directly manipulating the signaling pathway involved in ifn production, several cov nsps were identified to act on viral rna to minimize ifn stimulation. n7-methylguanosine is the fundamental moiety of eukaryotic mrna cap structure and 2′-o-methylation on this moiety is a representative host signature to avoid prr activation as well as isg action. particularly, viral rna with this modification evades recognition by mda5 or ifit family antiviral factors (züst et al., 2011; daffis et al., 2010) . this is a common immunoevasive mechanism adopted by not only different covs but also other rna viruses. functional screening in yeasts suggested a novel function of sars-cov nsp14 as a guanine-n7-methyltransferase, the activity of which is required for viral replication and transcription (chen et al., 2009) . another nsp of sars-cov, nsp16, also possesses 2′-o-methyltransferase activity (menachery et al., 2014a; menachery et al., 2014c) . structural modeling suggested that sars-cov nsp16 associates with nsp10 in 1:1 ratio to form a complex of mature 2′-o-methyltransferase for viral cap methylation (chen et al., 2011; decroly et al., 2011) . a short peptide derived from nsp10 conserved region has been shown to be a promising nsp16 antagonist which outcompetes native nsp10 to blunt 2′-o-methyltransferase activity and restrict viral replication . plausibly, cov nsps might execute their innate immunosuppressive roles by targeting type i ifn production and signaling. further investigations are required to clarify whether and how far the sensing of cov rna and the induction of innate antiviral response are involved in the inhibitory activity of the nsp antagonists on cov replication. cov structural proteins have been shown to inhibit ifn production and signaling at multiple levels. sars-cov n protein showed inhibitory effects on ifn production induced by sendai virus and dsrna analogue poly(i:c) but no inhibition could be observed when downstream signaling molecules of tlr and rlr pathway were overexpressed. truncation mutant of n protein shows that the c-terminal domain is critical for rna-binding and ifn-antagonizing effect (lu et al., 2011) . this suggests sars-cov n may interfere with rna recognition by host immune sensors such as rig-i and mda5 thus achieving suppressive role in ifn production. other than n protein, sars-cov m protein has been characterized to potently down-regulate ifn production by impeding the formation of traf3·tank· tbk1/ikkε complex through the first transmembrane domain (siu et al., 2009 (siu et al., , 2014a . sars-cov m protein inhibits ifn production possibly through a sequestration model in which components of traf3·tank·tbk1/ikkε complex, an active complex for irf3 phosphorylation, are sequestered to specific locations in the cell (siu et al., 2009) . sars-cov m protein therefore exerts its inhibitory effects by impeding the formation of traf3·tank· tbk1/ikkε complex but not by modulating the catalytic activity of the complex. mers-cov m protein also exhibits ifn-antagonizing effects similar to its counterpart in sars-cov. in a previous study, mers-cov m is shown to impede ifn production by preventing irf3 translocation into the nucleus (yang et al., 2013) . however, the detailed mech-anism of inhibition remains unknown. recently, our group has characterized the mode of inhibition of ifn production by mers-cov m. consistently with previous report, we show that mers-cov m suppresses ifn production by preventing irf3 activation. we showed that mers-cov m interacts with traf3 which impedes the recruitment of tbk1 to traf3 complex. irf3 activation and dimerization have also been hampered as a result. the inhibitory effect is at least in part accounted for by the n-terminal transmembrane domains. despite of the similar behaviors, mers-cov m can only moderately suppress ifn expression when compared to sars-cov m. interestingly, hcov-hku1 m protein does not exert any inhibitory effects on ifn production (siu et al., 2014a) , suggesting that the ifn-antagonizing activity of structural proteins is unique to each cov but not universal. it will be of great interest to see whether this may correlate with the pathogenicity of different hcovs. eight accessory proteins have been identified in sars-cov and five are found in mers-cov (narayanan et al., 2008b) . sars-cov genome encodes orf3a, orf3b, orf6, orf7a, orf7b, orf8a, orf8b and orf9b as accessory proteins (narayanan et al., 2008b) . sars-cov orf3b and orf6 have been found to antagonize type i ifn production and signaling. particularly, sars-cov orf3b and orf6 suppress ifn-β production by perturbing irf3 activation induced by sendai virus infection. sars-cov orf3b and orf6 also suppress ifn-β-induced activation of isre in isg promoters (kopecky-bromberg et al., 2007) , although they are not able to reduce the level of phosphorylation of stat1, a transcription factor that activates isre activity once phosphorylated. however, sars-cov orf6 has been shown to inhibit stat1 translocation for isre activation (kopecky-bromberg et al., 2007) . the findings suggest a mode of inhibition of ifn-β signaling by sars-cov. ifn antagonism of accessory proteins has also been observed in another deadly hcov. mers-cov genome encodes orf3, orf4a, orf4b, orf5 and orf8b (de groot et al., 2013) . among the five accessory proteins, orf4a, orf4b and orf5 show the ability to dampen ifn production (yang et al., 2013) . suppression of ifnβ promoter-driven luciferase activity has been observed in cells transfected with orf4a, orf4b and orf5 plasmids. all these 3 accessory proteins are able to block irf3 translocation to the nucleus to activate ifn promoter (yang et al., 2013) . mers-cov orf4a shows an additional level of inhibition of innate immunity by intervening nf-κb activation. in another study, orf4a has been shown as an antagonist of ifn production by inhib-iting irf3 translocation but has no effect on ifn signaling (niemeyer et al., 2013) . our group demonstrated that mers-cov orf4a interacts with pact, a cellular dsrna-binding protein that optimally activates rig-iand mda5-induced type i ifn production, in an rnadependent manner (siu et al., 2014c) . this suggests that orf4a may compete with rig-i and mda5 for rna, rendering the inactivation of rig-i and mda5. direct interaction of orf4a with pact may also prevent interaction of pact with rig-i and mda5, thus compromising pact-dependent activation of rig-i and mda5 required for optimal induction of ifn production. although we and others have observed the ifn-antagonizing activity of mers-cov orf4b, different activity profiles and mechanisms have been suggested (yang et al., 2013; matthews et al., 2014) . one recent report suggested that orf4b directly interacts with and inhibits tbk1/ikke in the cytoplasm but might also perturb type i ifn production in the nucleus through an unknown mechanism (yang et al., 2015) . mouse hepatitis virus, another betacoronavirus closely related to hcov-oc43 and hcov-hku1, encodes a lineage-specific accessory protein named ns2 with innate immunosuppressive property (zhao et al., 2012) . biochemical assays indicate that ns2 protein has phosphodiesterase activity against 2′, 5′-a, the product of oas . thus, ns2 is a potent inhibitor of an ifn effector molecule and it might represent a new family of viral and cellular proteins with innate immunosuppressive activity gusho et al., 2014) . whether distantly related proteins in hcov-oc43 and hcov-hku1 might have similar activity remains to be determined. more importantly, it will be of interest to see whether sars-cov and mers-cov might encode proteins with similar enzymatic activity. multiple ifn antagonists have been identified and characterized in sars-cov and mers-cov. some differences between these ifn-antagonizing viral proteins and their counterparts in other covs such as the parental bat viruses of mers-cov have also been noticed (siu et al., 2014c) . existing evidence supports several important notions. first, although sars-cov and mers-cov share some features in common, they are distinct and use unique mechanisms for innate immune evasion (perlman and zhao, 2013) . second, both sars-cov and mers-cov are bat-origin covs that are well adapted in bats but newly emerge in human. this provides a golden opportunity for the study of cov-host interaction, cov adaptation as well as the arms race between host innate antiviral immunity and covs. observing how the arms race between the host and sars-cov or mers-cov might evolve when the viruses become adapted to human will be most revealing and could provide important clues as to how a balance of power in this arms race might result in attenuation with increased transmissibility. finally, studies on sars-cov and mers-cov have overturned existing concepts and derived new principles and thoughts to cov biology. particularly, mechanisms by which sars-cov and mers-cov evade innate immunity have attracted increasing attention. however, many key issues remain obscure. particularly, better in vivo evidence should be obtained to clarify whether more potent inhibition of innate ifn production and signaling by sars-cov and mers-cov is a key determinant in virulence and disease severity. covs have drawn a lot of interests in the light of the recent emergence of mers-cov. it remains to be understood whether the emerging deadly covs causing ards might ultimately be established and adapted in human resulting in significant attenuation of virulence. from the identification of the first two hcovs, hcov-229e and hcov-oc43 in the mid-1960s, we learned that hcov was able to cause only common cold. however, the outbreaks of sars and mers that have claimed hundreds of lives revealed the other extreme of cov pathogenicity and raised new questions in cov biology. so far no vaccines have been developed against sars-cov and mers-cov. infection with sars-cov and mers-cov has been accompanied with suppression of innate immune response, most notably with the suppression of type i ifn production and signaling pathways. as the first-line defense in the immune system, suppression of innate immune response by these covs has impeded the host ability to restrict infection, causing significant casualties. although many reports have shed light on the molecular mechanism by which various cov proteins antagonize type i ifn production and signaling, most of the studies were performed with overexpression experiments in cellular models. future emphasis should be put on the characterization of knock-out viruses with which the function of a particular viral gene could be studied in a more physiologically relevant context. infectious clones and replicons for sars-cov and mers-cov have been generated for this reverse genetic approach (yount et al., 2003; almazán et al., 2006 almazán et al., , 2013 almazán et al., , 2014 scobey et al., 2013) . ifn and cytokine profiles of deadly hcovs such as sars-cov and mers-cov can be compared with hcov-229e and hcov-oc43 causing mild diseases. the pivotal significance of type i ifns in innate immune activation and modulation has been discussed in this review. suppression pattern of ifn may provide insights on the high pathogenicity of deadly hcovs. the arms race between host innate antiviral response and emerging human covs might evolve after their introduc-tion and establishment in human populations, with significant impact on virulence, transmissibility and disease severity. emerging human covs remain a potential threat to global public health. new knowledge about the host-cov arms race will provide new ideas, targets and attenuated strains for the design and development of antivirals and vaccines for prevention and control of deadly cov infections. construction of a severe acute respiratory syndrome coronavirus infectious cdna clone and a replicon to study coronavirus rna synthesis engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate coronavirus reverse genetic systems: infectious clones and replicons catalytic function and substrate specificity of the papainlike protease domain of nsp3 from the middle east respiratory syndrome coronavirus mda5 assembles into a polar helical filament on dsrna innate sensing of viruses by tolllike receptors the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex effects of a "new" human respiratory virus in volunteers the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly coronavirus cell entry occurs through the endo-lysosomal pathway in a proteolysis-dependent manner modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein interspecies transmission and emergence of novel viruses: lessons from bats and birds functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2′-o-methylation by nsp16/nsp10 protein complex severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases evidence for an ancestral association of human coronavirus 229e with bats 2′-o methylation of the viral mrna cap evades host restriction by ifit family members crystal structure and functional analysis of the sars-coronavirus rna cap 2′-o-methyltransferase nsp10/nsp16 complex middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? the transcriptional code of human ifn-β gene expression a previously undescribed coronavirus associated with respiratory disease in humans sars coronavirus and innate immunity sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism coronavirus-induced er stress response and its involvement in regulation of coronavirus-host interactions a decade after sars: strategies for controlling emerging coronaviruses murine akap7 has a 2′, 5′-phosphodiesterase domain that can complement an inactive murine coronavirus ns2 gene a new virus isolated from the human respiratory tract development and applications of crispr-cas9 for genome engineering sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage evidence supporting a zoonotic origin of human coronavirus strain nl63 regulation of type i interferon responses structural basis of rna recognition and activation by innate immune receptor rig-i the role of pattern-recognition receptors in innate immunity: update on toll-like receptors pathogen-associated molecular pattern recognition of hepatitis c virus transmitted/founder variants by rig-i is dependent on u-core length efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna coronavirus hku1 and other coronavirus infections in hong kong the virus battles: ifn induction of the antiviral state and mechanisms of viral evasion murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus influenza a virus panhandle structure is directly involved in rig-i activation and interferon induction phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin immune signaling by rig-i-like receptors sars-cov nucleocapsid protein antagonizes ifn-β response by targeting initial step of ifnβ induction pathway, and its c-terminal region is critical for the antagonism structural insights into rna recognition by rig-i positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas positive feedback regulation of type i interferon by the interferonstimulated gene sting the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling protective role of toll-like receptor 3-induced type i interferon in murine coronavirus infection of macrophages recovery in tracheal organ cultures of novel viruses from patients with respiratory disease middle east respiratory syndrome coronavirus in bats, saudi arabia coronavirus nonstructural protein 16: evasion, attenuation, and possible treatments pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2′-o-methyltransferase activity mers-cov papain-like protease has deisgylating and deubiquitinating activities severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells sars coronavirus accessory proteins atlas of coronavirus replicase structure middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner identifying genetic markers of adaptation for surveillance of viral host jumps human coronavirus emc is not the same as severe acute respiratory syndrome coronavirus the novel human coronaviruses nl63 and hku1 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc characterization of a novel coronavirus associated with severe acute respiratory syndrome antiviral actions of interferon interferon-regulated cellular proteins and their surprisingly selective antiviral activities antiviral actions of interferons lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna interferon-stimulated genes: a complex web of host defenses pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus insights into rna synthesis, capping, and proofreading mechanisms of sars-coronavirus middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain comparative analysis of the activation of unfolded protein response by spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus hku1 severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3·tank·tbk1/ikkϵ complex middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response integration of interferon-α/β signalling to p53 responses in tumour suppression and antiviral defence severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mrna toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection cultivation of a novel type of common-cold virus in organ cultures coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis incoming rna virus nucleocapsids containing a 5′-triphosphorylated genome activate rig-i and antiviral signaling characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 molecular imprinting as a signalactivation mechanism of the viral rna sensor rig-i toll-like receptors and viruses: induction of innate antiviral immune responses middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists human aminopeptidase n is a receptor for human coronavirus 229e shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus crispr/cas9-mediated genome editing of epstein-barr virus in human cells isolation of a novel coronavirus from a man with pneumonia in saudi arabia homologous 2′, 5′-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology recent progress in studies of arterivirus-and coronavirus-host interactions transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in stat1 knockout mice during severe acute respiratory syndrome coronavirus infection ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 we thank hinson cheung, kitty fung, edwin kong and sam yuen for reading the manuscript critically. coronavirus research in our laboratory was supported by hong kong health and medical research fund (13121032, 14130822 and hkm-15-m01) and hong kong research grants council (hku1/crf/11g, c7011-15r and t11-707/15-r). the authors declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-332952-d5l60cgc authors: nan title: mers: progress on the global response, remaining challenges and the way forward date: 2018-09-17 journal: antiviral res doi: 10.1016/j.antiviral.2018.09.002 sha: doc_id: 332952 cord_uid: d5l60cgc this article summarizes progress in research on middle east respiratory syndrome (mers) since a fao-oie-who global technical meeting held at who headquarters in geneva on 25–27 september 2017. the meeting reviewed the latest scientific findings and identified and prioritized the global activities necessary to prevent, manage and control the disease. critical needs for research and technical guidance identified during the meeting have been used to update the who r&d mers-cov roadmap for diagnostics, therapeutics and vaccines and a broader public health research agenda. since the 2017 meeting, progress has been made on several key actions in animal populations, at the animal/human interface and in human populations. this report also summarizes the latest scientific studies on mers since 2017, including data from more than 50 research studies examining the presence of mers-cov infection in dromedary camels. since its identification in the kingdom of saudi arabia (ksa) (zaki et al., 2012) and jordan (hijawi et al., 2013) in 2012, middle east respiratory syndrome (mers) has become a global public health threat. typical of an emerging zoonosis, middle east respiratory syndrome coronavirus (mers-cov) has an animal reservoir, i.e. dromedary camels in which the virus causes little to no disease (mohd et al., 2016) . many details about the extent of circulation and the mechanisms of transmission within dromedary camel herds, or factors related to zoonotic transmission and differences in circulating mers-cov strains, remain unknown. the virus has repeatedly spilled over from dromedary camels to humans, principally in countries on the arabian peninsula, causing significant morbidity and mortality (world health organization, 2017a; azhar et al., 2014) . clusters of cases in the community and among family members are rare (world health organization, 2017a; . however, delays in diagnosis in hospitals has sometimes led to secondary cases among health care workers, patients sharing rooms or family members as a result of unprotected direct contact with a patient before isolation. this humanto-human transmission in health care facilities can sometimes be amplified, causing very large outbreaks, as has been seen in the middle east and in the republic of korea, with significant public health and economic impacts (hijawi et al., 2013; assiri et al., 2013; drosten et al., 2015; al hosani et al., 2016; ki, 2015; park et al., 2015) . as of august 2018, more than 2249 human cases from 27 countries have been reported to the world health organization (who) (world health organization, 2017a) . the fao, oie and who tripartite have regularly brought together affected member states, public health and animal officials, and academics to discuss what is known and unknown about the zoonotic origin of mers-cov (world health organization, 2016; fao, 2016 fao, , 2014 ; who regional office for the eastern mediterranean, 2013a). the purposes of these meetings and workshops have been to advocate for more surveillance and research on mers-cov in animals and humans, to share information about how mers-cov is transmitted between animals, from animals to humans and between humans, to describe the diseases it causes, and to develop policies and guidelines for detection, https://doi.org/10.1016/j.antiviral.2018.09.002 received 30 august 2018; accepted 4 september 2018 reporting of animal and human infections, and prevention of human cases and clusters. in the two years since the last international technical consultation on mers-cov in 2016 13 , there have been notable improvements in surveillance and reporting of human cases, multidisciplinary research, cross-sectoral collaboration at country level, public awareness about the disease, and laboratory and surveillance capacity in affected countries. in addition, a number of countries in the arabian peninsula and in africa have engaged in research activities and surveillance of camel populations to shed light on the wider distribution of this virus or investigate transmission patterns and routes for viral shedding. as a follow-up to previous meetings (world health organization, 2016; fao, 2016 fao, , 2014 ; who regional office for the eastern mediterranean, 2014; who regional office for the eastern mediterranean, 2013b, 2013c), fao, oie and who tripartite held a global technical meeting on mers-cov with representatives from ministries of health and ministries of agriculture, subject matter experts, researchers, funders and industrial partners from 25 to 27 september 2017 in geneva, switzerland (see supplementary information) (world health organization et al., 2017) . the objectives were to review the latest scientific evidence on mers-cov, further enhance cross-sectoral collaboration and communication during preparedness and response activities, and identify research priorities given the advancements in our knowledge. with 130 participants, this was the largest mers-cov technical meeting to date and the first meeting attended by representatives from both affected and at risk countries. that is, countries which have reported human infection, countries with evidence of mers-cov in dromedary camels but no reported human cases, and countries at risk for importation (countries without infected camels that have close ties to affected countries through expatriate workers, travel to affected countries for medical procedures and/or frequent international travel). there is strong consensus among all stakeholders that dromedary camels are the main source of transmission to humans. in 2014, oie identified mers-cov as an emerging disease with zoonotic potential in camels and thereby creating expectations of reporting positive camels by countries (oie, 2014a) and recently published a mers-cov case definition (oie, 2017) for the reporting of confirmed and suspected infection in camels. not all countries face the same risks. for example, countries that have the infected reservoir (dromedary camels) differ from those countries in which dromedary camels show no evidence of current or past infection (fig. 1 ). there may also be differences in spillover potential in countries with documented zoonotic transmission, compared to those without, due to several factors including potential differences in husbandry practices, cultural, social, medicinal, occupational exposures, prevalence of underlying chronic medical conditions, or genetic factors in human populations, and mers-cov viral differences (wong et al., 2015) . as such, technical and risk mitigation guidance to protect human health and research priorities differ by region. the findings from the global technical meeting are summarized below: i. surveillance needs: surveillance in animals and humans to limit zoonotic transmission routine human surveillance for mers-cov in ksa (abdulaziz et al., 2017) and throughout the middle east has improved since the identification of the virus in humans in 2012, but there is significant variation in the quality and extent of surveillance between countries. in other parts of the world, surveillance is limited. since it is known that mers-cov is enzootic in areas of africa and asia where dromedary camels are found, heighted awareness and surveillance for zoonotic mers is required. this is currently lacking and remains a knowledge gap. one exception is the notable effort to identify potential mers-cov infection among pilgrims travelling back from the middle east. since 2012, event-based surveillance among pilgrims returning from hajj, umrah and other religious events in ksa has been conducted by ksa and countries sending pilgrims. while many return reporting respiratory symptoms, no mers-cov infections have been identified among returning pilgrims (muraduzzaman et al., 2018; barasheed et al., 2014; atabani et al., 2016; koul et al., 2017; annan et al., 2015; ma et al., 2017; memish et al., 2014a memish et al., , 2014b refaey et al., 2017; al-abdallat et al., 2017; matthew et al., 2015; alqahtani et al., 2016; win et al., 2016; yavarian et al., 2018; kapoor et al., 2014) . among animals, field surveys conducted to date have included several domestic and wildlife species including dromedary camels (camelus dromedarius) and bactrian camels (camelus bactrianus), goats, bats, cattle, sheep, chickens, swine, ducks, buffalo and equids. field studies in dromedary camels have been conducted in a number of countries (table 1) . to date, mers-cov rna or mers-cov-specific antibodies have been identified in dromedary camels a number of countries (table 1 ) except australia (hemida et al., 2014) , kazakhstan (miguel et al., 2016) , and the netherlands (reusken et al., 2013a) . other livestock such as alpacas (vicugna pacos), llamas (llama pacos), young goats, rabbits and pigs have been shown to be susceptible to experimental infection (crameri et al., 2016; adney et al., 2016; vergara-alert et al., 2017) . despite improvements, routine surveillance in dromedary populations is limited. the lack of surveillance information about mers-cov circulation in dromedary camels restricts our understanding of the transmission dynamics and epidemiology in dromedary camel populations. meeting participants agreed that surveillance should be integrated into existing surveillance systems, particularly in at-risk countries, similar to one health approaches developed for avian influenza, and existing human respiratory disease surveillance systems set up for influenza-like illness (ili) or severe acute respiratory infections (sari). currently, a limitation in our ability to mitigate spillover from dromedary camels to humans is a lack of clarity on the mode(s) of transmission between dromedary camels and humans, the extent and epidemiology of mers-cov circulation in dromedary camels in large parts of africa and south asia, and on why zoonotic transmission is limited across africa, large parts of the middle east, and some parts of south asia despite high seroprevalence in dromedary camels (chu et al., 2018) (table 1) . fao has outlined the meeting participants conclusions on priorities for mers-cov surveillance and management of pcr positive dromedary camels, coordinated outbreak investigation of community acquired cases with dromedary exposure, testing of animals at quarantine and entry points, food safety and environmental contamination, risk communication and awareness raising for mers-cov among animal owners and intersectoral collaboration and coordination in an updated doha declaration first published in 2015 (fao, 2015) (ref/hyperlink). in dromedary camels, longitudinal studies to evaluate the natural history, shedding profile and immunity were highlighted as key research priorities. meeting participants agreed that further understanding of differences in viral strains and transmission dynamics, including the role of immunity in acquiring infection and shedding virus, the geographic range of spillover events, and environmental, behavioral or host-related risk factors for zoonotic transmission should be prioritized. countries face significant challenges in the early identification and diagnosis of mers in humans due to the non-specificity of clinical symptoms ( the spectrum of illness ranges from no symptoms (or asymptomatic infection) to severe disease including pneumonia, acute respiratory disease syndrome, organ failure and death, with a case fatality ratio 35.5% among reported cases (world health organization, 2012). the delay in identification and recognition of signs and symptoms compatible with mers and delay in early isolation of patients has reduced the ability to prevent transmission between people in health care settings, notably in emergency departments, cardiac care centers and renal dialysis units (hijawi et al., 2013; assiri et al., 2013; drosten et al., 2015; al hosani et al., 2016; ki, 2015; park et al., 2015; ahmed et al., 2018; amer et al., 2018) . our understanding of human-to-human transmission in health care settings has improved through experimental and observational studies conducted in countries during such outbreaks. for example, studies of respiratory pathogens (yu et al., 2007; tran et al., 2012; thompson et al., 2013) and mers-cov conducted in the middle east (assiri et al., 2013; oboho et al., 2015; hunter et al., 2016; balkhy et al., 2016) and the republic of korea (bin et al., 2016; kim et al., 2016a kim et al., , 2016b nam et al., 2017) illustrate that aerosol-generating procedures and non-invasive ventilation, combined with inappropriate infection prevention and control practices and lack of adherence to standard practices had an important role in facilitating human-to-human transmission in health care settings. the role of environmental contamination has been evaluated in a number of hospitals following the 2015 outbreak in the republic of korea and collaborative, experimental studies are being conducted to evaluate the viability and persistence of mers-cov on surfaces and in the air (bin et al., 2016; kim et al., 2016a; van doremalen et al., 2013) . the role of mild or asymptomatic cases in transmission chains, however, remains unclear (omrani et al., 2013; memish et al., 2014c; al-gethamy et al., 2015; moon and son, 2017; al-abdely et al., 2018) , warranting targeted epidemiological and clinical studies to be conducted among contacts during outbreaks, especially in health care facilities. countries which have reported health care-associated outbreaks have implemented a variety of strategies to improve infection prevention and control and reduce human-to-human transmission in hospitals, including the introduction of a visual triage system prior to entrance to the emergency departments, the restructure of emergency department layouts for better triage of patients with respiratory symptoms, the standardization and training and re-training of infection prevention and control practices at facilities with high hospital staff turnover, and the auditing of health care facilities for adherence to infection prevention and control measures. iii. product research and development needs: clinical management, diagnostics and medical interventions the who r&d blueprint, a global strategy and preparedness plan that allows the rapid activation of r&d of epidemic pathogens, aims to fast-track the development and use of effective point-of-care diagnostic tests, vaccines and medicines that can be used to save lives and avert large scale crises. since 2015, mers-cov has been included in the annual who r&d blueprint list of prioritized pathogens for accelerated research and development on diagnostics, vaccines and therapeutics (world health organization). in addition, mers-cov has a specific roadmap for product research and development, outlined by who in ( the nonspecific, and sometimes unusual, clinical presentation of mers in humans, makes early diagnosis difficult in health care facilities. while several highly specific and sensitive molecular and serologic assays exist for diagnosis in animals and humans corman et al., 2012a corman et al., , 2012b lu et al., 2014; perera et al., 2013a; reusken et al., 2013b; müller et al., 2015; song et al., 2015) , there was a clear call from representatives from affected countries for the development of a rapid diagnostic test to improve identification and isolation of primary human cases in health care facilities. a full landscape analysis of mers-cov diagnostics will be published separately (van kerkhove, personal communication). at the global technical meeting, several therapeutics (including convalescent plasma, lopinavir/ritonavir, ribavirin, interferon and novel therapies including polyclonal antibodies and broad-spectrum antivirals) in development were presented. however, small case numbers make the evaluation of their impact on morbidity and mortality from mers-cov infection difficult (arabi et al., 2017a; arabi et al., 2017b; al-dorzi et al., 2016; sheahan et al., 2017; ko et al., 2018; arabi et al., 2017c) . several pre-clinical and phase i-iii studies are under way or in the design phase (outlined by the who r&d blueprint: http://who-blueprint-mapping-tool.surge.sh/). who is currently evaluating all available evidence on therapeutics to update guidance on clinical management of patients and in the process to develop standardized clinical trial protocols that could be used in affected countries to evaluate promising therapeutic candidates. who has identified target product profiles for mers-cov vaccines which include a dromedary camel vaccine for the reduction of zoonotic transmission, a human vaccine for long term protection of high risk individuals, such as those working with infected dromedary camels or health care workers, and a human vaccine for reactive use in outbreak settings (world health organization, 2017b) . currently, no mers-cov-specific or licensed human vaccines are available (modjarrad et al., 2016; world health organization, 2017c) . several human vaccine candidates for coronaviruses, including mers-cov, are at various stages of development and five general vaccine technology platforms have been developed and target the mers-cov spike protein (modjarrad et al., 2016; okba et al., 2017) . who, the ministry of health in ksa and the international vaccine institute (ivi) have continued to further align efforts to develop coronavirus vaccines (excler et al., 2016) and the coalition for epidemic preparedness and innovation (cepi) has included mers-cov as one of three priority pathogens for financing of a human vaccine. understanding correlates for protection and having a reliable animal model remain essential for evaluating coronavirus vaccine candidates, including mers-cov. there was a clear call from global technical meeting participants to accelerate the development of a dromedary camel vaccine in order to evaluate the potential to reduce spillover transmission to humans. the acceptability, cost-effectiveness and feasibility of a dromedary camel vaccine will also need to be evaluated and compared to other intervention strategies, such as human vaccination of high risk groups (e.g., those with occupational exposure). because mers-cov is endemic in dromedary camel populations in the middle east and elsewhere, multiple intervention strategies, including personal protective measures and the strategic implementation of a camel vaccine, are likely needed to reduce transmission from dromedary camels to humans. (farag et al., 2015) antibodies to mers-cov s1 were found in 100 of 103 (97.1%) dromedary camels tested by micro-array technology (farag et table 2 list of prioritized research and progress on mers-cov research, as discussed at the september 2017 meeting. *based on an enhanced understanding of the virus, the doha declaration (fao, 2015) is undergoing revision with a focus on guiding surveillance techniques, management of dromedary camels shedding the virus, research, regional and inter-sectoral coordination, risk communication, food and environmental safety practices, and biosecurity measures. the update includes explicit guidance on import testing, quarantine procedures, and management of shedding animals. these recommendations and priority actions in the doha declaration will be delivered as a separate document after validation by stakeholders in affected and at risk countries. at least two promising camel vaccine candidates are currently in development and being evaluated in field trials (haagmans et al., 2016; alharbi et al., 2017) . stakeholders agreed that the current funding mechanisms need to include risk-mitigating options that target the animal-human interface to prevent zoonotic transmission. these funding pathways would enable the development of camel vaccine candidates. stakeholders also agreed that prior to camel vaccine implementation, consultation with camel owners and government agencies, feasibility studies including exploring opportunities for commercial manufacturing and incentives for camel vaccination and an assessment of potential trade implications, would all be critical. in june 2018, who and the international vaccine institut (ivi) held a joint workshop update the status of human and animal mers-cov vaccine development, and identify and prioritize activities to accelerate vaccine research and development. the meeting was held in seoul, korea on 26-27 june and included 120 experts and professionals from industry, academia, international organizations and government agencies around the world, including the coalition for epidemic preparedness innovations (cepi), the korean ministry of food and drug safety (kmfds) and the korea centers for disease control and prevention (kcdc). the global technical meeting served as an opportunity to review the available evidence and best practices for control of this epidemic threat six years after the virus was first detected in humans. this covered our understanding of the virus, our ability to detect and respond to cases in animal and human populations, how we communicate our findings and how our work impacts policy decisions to protect animals and prevent human infections. during the global technical meeting, the latest findings from scientific studies and knowledge gained from collaborative research and surveillance were shared across animal, environmental and human sectors. fao, oie and who strongly believe that to effectively address zoonoses, including mers-cov, a one health approach to prevent, detect, contain, eliminate and respond to animal and public health risks from zoonotic high threat respiratory pathogens such as mers-cov and should involve all relevant sectors, the public and animal health and academic research community, industry and affected communities. we acknowledged the progress that has been made, and importantly, discussed the challenges that need to be addressed so that we can minimize the future public health and economic impacts of this epidemic prone virus. our aim was to articulate a clear action plan to address these remaining unknowns and to foster better collaboration between sectors and with subject matter experts willing to support member states. given the marked expansion in research related to mers-cov conducted in the past two years, fao, oie and who agree that global surveillance and research activities must now be focused on achieving the following major public health goals: reducing zoonotic transmission, detecting and identifying suspected cases early, providing safe and effective treatment to reduce human morbidity and mortality, and significantly reducing human-to-human transmission in health care settings. the critical needs for research and technical guidance identified during the global technical meeting have been used to inform the who r&d mers-cov roadmap (modjarrad et al., 2016 ) and a broader research agenda for mers-cov and other high threat coronaviruses. this research agenda serves as a catalyst to focus, align and mobilize partners to address outstanding knowledge gaps in relation to mers-cov across five technical areas: i) virus origin and characteristics, ii) epidemiology and transmission, iii) clinical management and infection prevention and control, iv) product development and implementation (modjarrad et al., 2016) and v) impact of interventions and operational research. the meeting identified a large number of remaining priorities and the organizing committee has summarized the main research needs in table 2 . now is the time to devote more effort to long term planning for mers-cov. we believe more focused efforts in our activities and investments to address scientific and public health research questions, accelerate promising medical interventions and are more strategic on where activities are conducted globally will go further to address remaining public health unknowns. while there have been improvements in the ability to prevent human-to-human transmission once a mers case is identified, these are not sufficient to prevent a large event with substantial public health and economic consequences. surveillance and testing for middle east respiratory syndrome coronavirus, saudi arabia infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia response to emergence of middle east respiratory syndrome coronavirus hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description acute respiratory infections among returning hajj pilgrims-jordan infectious mers-cov isolated from a mildly ill patient, saudi arabia the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker the calm before the storm: clinical observations of middle east respiratory syndrome (mers) patients a cohort-study of patients suspected for mers-cov in a referral hospital in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus antibody reactors among camels in dubai chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice cross-sectional surveillance of middle east r mers-cov) in dromedary camels and other mammals in egypt systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt association between australian hajj pilgrims' awareness of mers-cov, and their compliance with preventive measures and exposure to camels unusual presentation of middle east respiratory syndrome coronavirus leading to a large outbreak in riyadh during 2017 high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study middle east respiratory syndrome corticosteroid therapy for critically ill patients with middle east respiratory syndrome corticosteroid therapy for critically ill patients with the middle east respiratory syndrome middle east respiratory syndrome coronavirus antibodies in dromedary camels hospital outbreak of middle east respiratory syndrome coronavirus active screening and surveillance in the united kingdom for middle east respiratory syndrome coronavirus in returning travellers and pilgrims from the middle east: a prospective descriptive study for the period 2013-2015 evidence for camel-to-human transmission of mers coronavirus description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia viral respiratory infections among hajj pilgrims in 2013 environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through wholegenome analysis and virus cultured from dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels mers-cov infection of alpaca in a region where mers-cov is endemic mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses from camels in africa exhibit region-dependent genetic diversity detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections antibodies against mers coronavirus in dromedary camels experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county transmission of mers-coronavirus in household contacts an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia toward developing a preventive mers-cov vaccine-report from a workshop organized by the saudi arabia ministry of health and the international vaccine institute dromedary camels in northern mali have high seropositivity to mers-cov fao, 2014. fao calls for more surveillance and research on mers. vet. rec. 174, 620. fao, 2015. oie, who. doha declaration. regional workshop on mers-cov and one health fao. understanding mers-cov and the animal-human interface high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels the prevalence of middle east respiratory syndrome coronavirus (mers-cov) antibodies in dromedary camels in israel sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission transmission of middle east respiratory syndrome coronavirus infections in healthcare settings clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia mers outbreak in korea: hospital-to-hospital transmission extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers outbreak units risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience influenza not mers cov among returning hajj and umrah pilgrims with respiratory illness identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus no mers-cov but positive influenza viruses in returning hajj pilgrims, china mers coronavirus in dromedary camel herd, saudi arabia lack of middle east respiratory syndrome coronavirus transmission from infected camels acute respiratory infections in travelers returning from mers-cov-affected areas etiology of severe community-acquired pneumonia during the 2013 hajj-part of the mers-cov surveillance program prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications antibodies against mers coronavirus in dromedary camels absence of middle east respiratory syndrome coronavirus in camelids risk factors for mers coronavirus infection in dromedary camels in burkina faso, ethiopia, and morocco a roadmap for mers-cov research and product development: report from a world health organization consultation middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study mers coronavirus neutralizing antibodies in camels event based surveillance of middle east respiratory syndrome coronavirus (mers-cov) in bangladesh among pilgrims and travelers from the middle east: an update for the period high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels mers-cov outbreak in jeddah -a link to health care facilities group on mers-cov infection in animals infection with coronavirus in camels middle east respiratory syndrome coronavirus (mers-cov) case definition for reporting to oie middle east respiratory syndrome coronavirus vaccines: current status and novel approaches a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae epidemiological investigation of mers-cov spread in a single hospital in south korea seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt isolation of mers coronavirus from a dromedary camel cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during 2012-2015 middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study specific serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia serologic evidence for mers-cov infection in dromedary camels broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans influenza aerosols in uk hospitals during the h1n1 (2009) pandemic -the risk of aerosol generation during medical procedures aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis livestock susceptibility to infection with middle east respiratory syndrome coronavirus deputy director general for animal health, animal health department, public authority for agriculture affairs & fish resources press release: experts meet to discuss novel coronavirus infection in humans press release: countries discuss stepping up preparedness for mers-cov and ebola risk assessment and laboratory investigation of respiratory illness in travellers returning to singapore 2012-2015: experience from the mers-cov surveillance programme mers, sars, and ebola: the role of super-spreaders in infectious disease world health organization. who r&d blueprint list of priority diseases middle east respiratory syndrom coronavirus (mers-cov summary report on the intrenational scientific meeting on middle east respiratory syndrome coronavirus (mers-cov). who regional office for the eastern mediterranean middle east respiratory syndrome coronavirus (mers-cov) who target product profiles for mers-cov vaccines landscape analysis of mers-cov research and project development for diagnostics, theraputics and preventives press release: countries agree next steps to combat global health threat by mers-cov influenza virus but not mers coronavirus circulation in iran, 2013-2016: comparison between pilgrims and general population why did outbreaks of severe acute respiratory syndrome occur in some hospital wards but not in others? isolation of a novel coronavirus from a man with pneumonia in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia we gratefully acknowledge the input from all those who attended the fao-oie-who global technical meeting on mers-cov in september 2017. the authors also thank malik peiris for his review of the final manuscript. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organizations with which they are affiliated. the authors have no competing financial interests. supplementary data to this article can be found online at https:// doi.org/mmcdoino. key: cord-295433-olmein3q authors: banerjee, arinjay; kulcsar, kirsten; misra, vikram; frieman, matthew; mossman, karen title: bats and coronaviruses date: 2019-01-09 journal: viruses doi: 10.3390/v11010041 sha: doc_id: 295433 cord_uid: olmein3q bats are speculated to be reservoirs of several emerging viruses including coronaviruses (covs) that cause serious disease in humans and agricultural animals. these include covs that cause severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), porcine epidemic diarrhea (ped) and severe acute diarrhea syndrome (sads). bats that are naturally infected or experimentally infected do not demonstrate clinical signs of disease. these observations have allowed researchers to speculate that bats are the likely reservoirs or ancestral hosts for several covs. in this review, we follow the cov outbreaks that are speculated to have originated in bats. we review studies that have allowed researchers to identify unique adaptation in bats that may allow them to harbor covs without severe disease. we speculate about future studies that are critical to identify how bats can harbor multiple strains of covs and factors that enable these viruses to “jump” from bats to other mammals. we hope that this review will enable readers to identify gaps in knowledge that currently exist and initiate a dialogue amongst bat researchers to share resources to overcome present limitations. bats are an ancient and diverse group of ecologically important mammals, constituting almost a quarter of all mammalian diversity and inhabiting every continent except antarctica. more than 1300 species of bats belong to the order chiroptera and are further classified into two suborders, yinpterochiroptera and yangochiroptera [1] [2] [3] . the yinpterochiroptera suborder includes the non-echolocating pteropodidae family and the echolocating rhinolophoidea superfamily. yangochiroptera contains the remaining echolocating microbat families. the two suborders diverged over 50 million years ago [4] [5] [6] . in addition to the important role that bats play in preservation of ecological balance, they have also been speculated to harbor a wide variety of viruses. many of the viruses in bats can cause disease in humans and agriculturally important animal species. these viruses include lyssaviruses, filoviruses, henipaviruses and coronaviruses [1, [7] [8] [9] . in this mini-review, we focus on the role of bats as reservoir hosts for important human and animal coronaviruses. we discuss the evidence of coronavirus spillover from bats, how bat ecological niches may contribute to spillover and the need to further explore bat-coronavirus interactions using viruses that have been naturally detected in bats. sars-cov emerged in humans in 2002 and efficient human to human transmission resulted in a global sars epidemic which lasted 8 months [15] . initial studies investigating animal sources of the virus from "wet markets" in the guangdong province of china suggested that himalayan palm civets and raccoon dogs were the most likely hosts responsible for human transmission [22] ; however, the role of bats as the original animal reservoir hosts of sars-cov was speculated as similar viruses were detected in them [27, 28] . years later, during an ecological surveillance of bats in the same region, a sars-like cov that closely matched the human sars-cov was isolated from the chinese horseshoe bat. bat sars-like cov could replicate in hela cells expressing angiotensin-converting enzyme 2 (ace2) receptor from human, civet and bat. the virus replicated in cells derived from human, bat and pig. no civet cells were tested [29] . these data suggest that sars-cov could have spilled over into humans directly from the chinese horseshoe bat while the palm civets in the "wet market" were incidental hosts [30] . however, the exact mechanism by which the zoonotic transmission event to humans occurred is still not clear. retrospective studies have found low levels of seroprevalence of sars-like cov in healthy individuals in hong kong dating back to 2001. interestingly, in 13 of 17 of these seropositive patients, the antibodies responded more strongly against the sars-like-cov isolated from a himalayan palm civet compared to the human sars-cov isolate [31] . these data suggest that low levels of human exposure to zoonotic sars-like covs occurred prior to the sars-cov epidemic that began in 2002, but went unidentified. mers-cov emerged in saudi arabia in 2012 and continues to cause human disease with a case fatality rate of 35% [16] . dromedary camels are a natural reservoir host for mers-cov. in the arabian peninsula and across northern africa, the seroprevalence rate for mers-cov in dromedary camels ranges from 70% to nearly 100% [32] [33] [34] [35] [36] [37] . live mers-cov has been successfully isolated and cultured from camel specimens [38] . approximately 55% of primary mers-cov cases are a result of direct contact with dromedary camels or camel products [39] ; however, the remainder of primary mers-cov cases have no history of contact with camels or infected individuals and thus, where they came into contact with the virus is unknown. a recent study found that 16 out of 30 camel workers surveyed in saudi arabia show evidence of prior mers-cov infection via seroconversion and/or virus-specific cd8+ t cell responses without any history of significant respiratory disease. this study suggests that camel workers with asymptomatic or mild disease may serve as another route of exposure [40] . although camels are thought to be the primary zoonotic reservoir for human transmission, there is strong evidence that bats are the ancestral reservoir host for mers-cov [24, [41] [42] [43] . mers-cov is a group c betacoronavirus and is phylogenetically related to batcovs identified in various bat species that belong to the vespertilionidae family. this includes batcov hku4, batcov hku5, neocov, and pdf-2180 [41, 44, 45] . furthermore, the spike protein from hku4 and mers-cov are highly similar and both use human dipeptidyl-peptidase 4 (dpp4) for virus entry [25, 46, 47] . it is not clear when mers-cov spread from bats to camels, but widespread exposure to the virus in the middle east and north and east africa dates back as early as the 1980s, suggesting that camels have served as a zoonotic reservoir for mers-cov for at least 30 years [33, 34, 37, 40, 48] . in addition to emerging highly pathogenic coronaviruses, human coronaviruses that cause the common cold are also thought to have their origins in bats. hcov-nl63 was first identified in a pediatric patient with bronchiolitis in 2004, but since then it has come to be appreciated that the virus causes approximately 1-9% of the common colds each year and has most likely circulated in humans for centuries with worldwide distribution [49] [50] [51] . a survey of bats in the u.s. found novel alphacoronaviruses, one of which was isolated from the north american tricoloured bat, perimyotis subflavus and was found to be a recent common ancestor of hcov-nl63 with an estimated divergence of~550 years ago [52] . hcov-nl63-like sequences were also identified in bats in africa [53] , further supporting a bat origin for hcov-nl63. although hcov-nl63-like viruses have been identified in bats, these viruses have sequences quite distant from the hcov-nl63 sequences, suggesting a possible intermediate host. hcov-229e also appears to have its origins in bat species. hcov-229e, another cause of the common cold, was first identified in 1967 and has been circulating in the human population for some time [54] . hcov-229e-related viruses have been found in hipposiderid bats during surveillance studies in kenya and ghana [53, 55] . in 2007, a novel alphacoronavirus was identified in an outbreak of respiratory disease in alpacas in the us, which is geographically separated from the bat species that harbor hcov-229e-like viruses in africa [56] . full genome sequencing and phylogenetic analysis of the alpaca cov placed it as an intermediate between the bat hcov-229e-related viruses and hcov-229e from humans [56] . by analyzing more bat, alpaca and human hcov-229e and hcov-229e-related sequences, evidence of genomic changes that occurred between bat and alpaca hcov-229e evolution and subsequently between alpaca and human evolution were identified [57] . interestingly, during tests of dromedary camels for mers-cov, about 6% of the camels studied were positive for hcov-229e [58] . seropositive camels were more prevalent in the arabian peninsula compared to africa and the earliest seropositive sample was from 1997 in a study that looked at samples from 1983 to 2014 [58] . these data all support the notion that hcov-229e has its ancestral origins in bat species while camelids serve as a more recent zoonotic reservoir for human transmission. a recent study has shown that hcov-229e (human strain) is incapable of infecting and replicating in cell lines from multiple bat species [59] . thus it is critical to isolate bat and camel strains of hcov-229e-related viruses to dissect the role of these mammals in the evolution of hcov-229e. porcine epidemic diarrhea (ped) was recognized as an enteric disease in pigs in the united kingdom as early as 1971. pedv was detected in belgium in 1978 [60] . the full-length genomic sequence of the prototype belgian pedv cv777 strain was determined in 2001 [61] . pedv cv777 is more closely related to a scotophilus bat coronavirus (btcov) 512/2005 than to other known alphacoronaviruses, such as transmissible gastroenteritis coronavirus (tgev) and hcov-229e and hcov-nl63, in phylogeny as well as genome organization [21] . this finding suggests that pedv and btcov/512/2005 have a common evolutionary precursor and that cross-species transmission of coronavirus may have occurred between bats and pigs. pedv has since emerged in north america and continues to cause periodic outbreaks that significantly affect producers [18, 62] . multiple pedv vaccine candidates have been shown to provide varying levels of protection in pigs [63, 64] . an effective vaccine may enable control of future pedv outbreaks along with strict biosecurity practices. although pedv propagates in human embryonic kidney cells [65] , no clinical cases of pedv have been reported in humans so far. we (banerjee and misra et al.) have also shown that pedv can infect kidney cells from big brown bats (eptesicus fuscus) [66] . pedv replication in bat cells has not been extensively studied. efforts are focused on designing therapeutics and vaccines to prevent ped in pigs. more recently, a novel hku2-related bat coronavirus, sads-cov has been shown to cause fatal enteric disease in pigs. zhou et al. identified sads-cov as the causative agent for a large-scale outbreak of fatal disease in pigs in china that caused the death of 24,693 piglets across four farms. sads-cov-like viruses with 96-98% similarity to sads-cov were detected in 9.8% of the bats that were sampled in this region [9] . none of the human serum samples that were collected from farm workers were positive for antibodies against sads-cov [9] . thus, sads-cov does not pose a risk for human transmission yet. further studies will be required to confirm the ability of sads-cov to infect and propagate in human cells. understanding how bats maintain a virus within a population is important for predicting spillover transmission events. for many viruses with known or suspected bat reservoirs, spillover transmission events typically occur within a defined time frame and location, which corresponds with higher than normal virus levels in the bat reservoir host. the reason for these "pulses" of virus within the reservoir host population are not clear, but proposed theories and evidence supporting these theories have been reviewed by plowright et al. [67] . in the case of marburg virus (marv), for example, ecological surveillance data shows a clear biannual spike in the prevalence of marv positive bats within the kitaka cave population, which correlates with an increase in the number of juvenile rousettus aegyptiacus bats due to the biannual birthing cycle. this pulse of virus positive bats correlates with an increased incidence of human spillover events [68] . horizontal transmission of marv between r. aegyptiacus bats was confirmed in a controlled experimental setting [69] . furthermore, recent experimental data has shown that bats infected with marv clear infection and maintain long-term immunity. this finding suggests that susceptible naïve juvenile bats are critical for maintaining marv within the population [69] . studies with hendra virus have shown that reproductive and nutritional stress can increase the levels of virus in little red flying foxes (pteropus scapulatus) [70] . the increase in virus replication may enhance the chances of a virus spillover. similar ecological studies need to be undertaken for bats and covs. other stressors, such as secondary infections, may also affect the relationship between bats and their viruses. a recent study by one of our laboratories (misra et al.) suggest that infection of little brown bats (myotis lucifugus) with white-nose syndrome causing fungus (pseudogymnoascus destructans) leads to an increase in replication of a persistently infecting coronavirus in these bats [71] . a recent study by anthony et al. evaluated the global diversity of coronaviruses in almost 20,000 animals and humans. during the course of this study, they found that the diversity of coronaviruses was highly associated with the diversity of bat species and this diversity separated into 3 distinct geographical regions, which mirrored the distribution of different species of bats. the authors report particular associations between bat families and viral sub-clades that suggest co-evolution [72] . a survey of coronaviruses isolated from bats in kenya found a high prevalence of coronaviruses in cardioderma cor, ediolon helvum, epomophorus labiatuc, hipposideros sp., miniopterus minor, otomops martiensseni, rhinolophus hildebrandtii, rhinolophus sp., and triaenops afer. the phylogenetic analysis of these novel covs found a number of cross-species transmission events, although the majority of these events appeared to be transient spillover events [53] . the recombination frequency of coronaviruses, which can be as high as 25% for the entire genome [73] , could lead to bats being an important reservoir for coronavirus recombination and virus evolution, much like birds and pigs are for influenza virus. indeed, there is strong evidence to suggest that a recombination event occurred between hcov-229e-like viruses found in hipposideros bats and hcov-nl63-like viruses found in triaenops afer bats, where the gene encoding for the spike protein is more closely related to the hcov-229e virus [53] . furthermore, the majority of recombination events identified in coronaviruses isolated from bats suggest recombination hotspots around the spike gene [23, 53] . in theory, bats could serve as an important reservoir for coronaviruses and coronaviruses with altered host tropism may very well evolve in bats. although bats are known to harbor a wide variety of coronaviruses, the mechanisms for virus spillover into humans or livestock are widely unknown. there is evidence that there are seasonal fluctuations in virus replication [74, 75] , however, the interconnectedness of virus replication rates and virus spillover have not been explored for bats. typically, coronaviruses found in bats have or require an intermediate host before spilling over into humans, like what is observed with mers-cov and camels. unlike the amount of information available from studies of other bat viruses such as nipah, hendra, ebola, and marburg viruses, we know very little, if anything about how coronaviruses are transmitted directly to humans or if direct human transmission does not occur and spillover via an intermediate host is required. bats are known to harbor a wide range of viruses including many that are highly pathogenic in humans. research to determine the mechanisms by which bats limit disease following virus infection is a relatively new field and can be difficult due to a lack of reagents and the need to develop appropriate in vitro and in vivo systems. even with these limitations, a variety of studies have been performed that evaluate the bat immune response to virus infection at the genomics level, in vitro using cell culture systems, and performing experimental infections in vivo. of note, very few of these studies are focused on coronavirus infections in bats and are rather centered around henipavirus and filovirus infections. future studies evaluating the virus-host interactions of bats and coronaviruses, particularly with bat cov isolates are important in determining why bats serve as important reservoirs for covs and how they control infection to limit severe pathological consequences. multiple studies have elucidated unique adaptations in the antiviral responses of bat cells. the primary bat species being used to study the bat immune response to virus infections in vitro and in vivo are pteropus alecto (black flying fox), rousettus aegyptiacus (egyptian rousette), and artibeus jamaicensis (jamaican fruit bat). papenfuss et al. were the first to sequence the p. alecto transcriptome and identified approximately 500 genes (3.5% of p. alecto transcribed genes) that encode immune-related proteins [76] . a similar number of immune genes were also identified in the transcriptomes of r. aegyptiacus and a. jamaicensis [77, 78] . this included the expression of canonical pattern recognition receptors including toll-like receptors (tlrs) 1-10, retinoic acid-inducible gene i (rig-i), and melanoma differentiation associated protein 5 (mda5) [76, 77] . furthermore, genes for different immune cell subsets, t-cell receptors (tcrs), cytokines and chemokines, and interferon-related genes were detected, while genes encoding for natural killer (nk) cell receptors were largely absent. work has been done to characterize many of these genes in cell lines derived from various bat species including p. alecto [79] [80] [81] . a large amount of interest in bat immune responses has focused specifically on the interferon response. genomic analysis of the interferon loci has shown species-specific evolution in which p. alecto has a contracted type i ifn locus [82] whereas p. vampyrus, m. lucifugus, and r. aegyptiacus have expanded the number of type i ifn genes [83, 84] . it has been observed that there may also be species specific differences in the baseline expression of type i ifns. p. alecto cells constitutively express three different ifnα genes [82] whereas cells generated from r. aegyptiacus do not show constitutive expression of ifnα [84] ; however, baseline expression of interferon alpha/beta receptors, ifnar1 and ifnar2, as well as a variety of interferon-stimulated genes are upregulated in these bat cells compared to human cells [85] . the molecular mechanisms that enable the differential expression pattern of ifns in bats are not known. thus, it is important to acknowledge that different species of bats may have evolved specific strategies to control viruses that they co-evolved with. although it appears that bats have many of the genes that are important for responding to virus infection, how this response compares between human and bat cells is just beginning to be examined. rna sensing and subsequent antiviral responses in bat cells have been studied using viruses known to induce an interferon response, such as sendai virus or newcastle disease virus or by transfecting a synthetic surrogate of viral double stranded rna (poly(i:c)) [80, [86] [87] [88] [89] [90] . these studies show that bat cells respond to rna and induce an antiviral response. many viruses encode proteins that antagonize the host response to infection and dampen the innate antiviral response. it has previously been shown that the v and w proteins of nipah and hendra viruses can inhibit antiviral responses in bat cells, similar to what is observed in human cells [90] . a more recent study showed that marv can inhibit the antiviral response in a r. aegyptiacus bat cell line and that this inhibition is dependent on the viral protein vp35 [85] . coronavirus accessory proteins are dispensable for replication but they play an important role in pathogenesis and virus fitness under the natural environment of a host [13, 91] . multiple studies with pedv, sars-and mers-covs have identified accessory proteins that can effectively inhibit an ifn response in mammalian cells [12] [13] [14] [91] [92] [93] [94] [95] . however, to date, there have been no published studies looking at the role of these accessory proteins in modulating antiviral responses in bat cells. in addition to studying the role of cov proteins in antagonizing the antiviral response in bat cells compared to other mammalian cell lines, it is also important to determine how covs isolated from bats compare to those isolated from humans. coronavirus accessory genes have co-evolved with their natural host for optimum functionality [91] and thus it is important to identify the role of accessory proteins in both their natural and spillover hosts. many of the covs that have been reported in bats, with the exception of few, such as a sars-like cov (bat sl-cov-wiv1) [29] , have been detected by molecular techniques that detect trace amounts of viral nucleic acids. to overcome this limitation, reverse genetics systems using the whole genome sequence from covs isolated from bats could be generated, propagated and evaluated in both bat and human cell lines [96] . this would allow researchers to better understand the role of viral proteins in a species-specific context. the vast majority of studies evaluating the bat host response to virus infection has been performed in cell lines. however, there is a great need to understand what happens during a virus infection in bats in vivo. the ability to study these questions is a daunting task and requires specialized facilities and staff, appropriate species selection especially for covs, and generating the necessary reagents. because of these limitations, only a handful of studies have been performed looking at the in vivo response of bats to virus infection. in fact, there are only two published studies in which experimental infections in bats using covs was performed. the first study was performed in an attempt to rescue a bat cov isolate. watanabe et al. detected covs in 57.1% of insectivorous bats and 55.6% of frugivorous bats; however, they were unable to culture the virus in vitro. to propagate covs detected in a lesser dog-faced fruit bat (cynopterus brachyotis), they administered intestinal samples orally to leschenault rousette bats (rousettus leschenaulti). virus could be detected by quantitative real-time pcr (qpcr) on 2 to 5 days after infection and there was an increase in viral rna while no clinical disease was observed. based on these data, the authors reported that this bat cov replicates in leschenault rousette bats; however, they were not able to isolate live virus [97] . this study emphasizes the importance of bat species selection for studying covs in bats. ideally, we would want to study a bat cov in the same species that it was detected in. the second study aimed to determine if bats could be infected with mers-cov and, if so, what the host response looks like. munster et al. infected ten jamaican fruit bats (artibeus jamaicensis) with mers-cov/emc2012. the authors detected virus shedding in the respiratory and intestinal tracts for 9 days. although the bats showed evidence of virus replication, no overt signs of disease were observed. a moderate and transient induction of the innate immune response was seen, but there were no signs of inflammation. based on their observations, the authors reported that jamaican fruit bats support the replication of mers-cov and thus, bats could be potential ancestral hosts of mers-cov [98] . this study has not been repeated in an insectivorous bat species. although several mers-like viruses have been detected in bats since the study in jamaican fruit bats, none have been successfully isolated [25, 41, 47, 99] . another study focused on looking at species-specific tropism. in this study, the authors focused on the mers-cov receptor dipeptidyl peptidase 4 (dpp4). widagdo et al. mapped the tissue distribution of dpp4 in multiple bat species to identify the differences in tissue tropism of mers-cov. in their study, the authors report that dpp4 in insectivorous bats is primarily detected in the gastro-intestinal (gi) tract and kidneys, whereas frugivorous bats express dpp4 in the respiratory and gi tracts [43] . other studies determined that dpp4 expression in camels is primarily in the upper respiratory tract [100] whereas dpp4 expression in humans is highest in the lower respiratory tract [101] . these data suggest that the tissue tropism in bats may be different than that in other mammalian species and that this may dictate the course of disease and disease severity. the ability of bats to harbor several different coronaviruses may seem like a mystery, but the same is true for rodents. although bats harbor more zoonotic viruses per species, rodents harbor a larger total number of zoonotic viruses [102] . after the sars outbreak, bats have been extensively sampled for coronaviruses and other viruses alike. we may be looking too hard and one may argue that we could find a similar diversity of viruses in other animals if we looked as robustly. metagenomics has enabled us to identify the broad range of viruses in bats and with time, we will expand this to other hosts of zoonotic viruses. for now, we know that bats are major evolutionary reservoirs and ecological drivers of cov diversity [72] . we can leverage this knowledge to design studies that will allow us to identify factors that cause covs to spillover from bats to other hosts. a recent study demonstrated that secondary infection with the white-nose syndrome fungus (pseudogymnoascus destructans) increases cov replication in m. lucifugus [71] . this study opens up a new avenue of investigation in infection dynamics. considering bats harbor multiple viruses, it is necessary to identify the impact these viruses have on each other. how do these viruses modulate the numerous host responses in bats and how does that affect virus replication? several such questions remain and studies are currently delayed due to the inability to isolate bat-covs similar to sars-cov, mers-cov, pedv and sads-cov. ecological and epidemiological studies to identify landscape changes and human practices that could enable a coronavirus to spillover from bats are also necessary [103] . such studies enabled researchers to decipher the transmission cycle of nipah virus in malaysia and bangladesh, which led to public health policies to change farming practices to control the spillover of nipah virus from bats [104] [105] [106] . as the human population expands and societal changes occur, human contact with wildlife will continue to increase. this increases the risk that emerging zoonotic viruses, including covs, pose to human and animal health. surveillance combined with scientific studies to better understand zoonotic covs and spillover will enable us to stay a step ahead of the next epidemic. bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? a molecular phylogeny for bats illuminates biogeography and the fossil record genes, and hearing: implications for the evolution of echolocation in bats primitive early eocene bat from wyoming and the evolution of flight and echolocation the placental mammal ancestor and the post-k-pg radiation of placentals phylogenomic analyses of bat subordinal relationships based on transcriptome data the spread and evolution of rabies virus: conquering new frontiers bats: important reservoir hosts of emerging viruses fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin coronavirus genomics and bioinformatics analysis coronaviruses: important emerging human pathogens accessory proteins of sars-cov and other coronaviruses sars coronavirus accessory proteins the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists severe acute respiratory syndrome middle east respiratory syndrome coronavirus human coronaviruses: a review of virus-host interactions assessment of the economic impacts of porcine epidemic diarrhea virus in the united states assessing the economic burden of avian infectious bronchitis on poultry farms in brazil. revue scientifique et technique de l'oie the database of bat-associated viruses prevalence and genetic diversity of coronaviruses in bats from china isolation and characterization of viruses related to the sars coronavirus from animals in southern china discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia receptor usage of a novel bat lineage c betacoronavirus reveals evolution of mers-related coronavirus spike proteins for human dpp4 binding severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor more evidence emerges that bats may have spread sars sars-related virus predating sars outbreak dromedary camels in northern mali have high seropositivity to mers-cov geographic distribution of mers coronavirus among dromedary camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus antibody reactors among camels in dubai middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study isolation of mers coronavirus from a dromedary camel reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases high prevalence of mers-cov infection in camel workers in saudi arabia further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus middle east respiratory syndrome (mers): bats or dromedary, which of them is responsible? tissue distribution of the mers-coronavirus receptor in bats genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5. emerg. microbes close relative of human middle east respiratory syndrome coronavirus in bat bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 discovery of novel bat coronaviruses in south china that use the same receptor as mers coronavirus identification of a new human coronavirus understanding human coronavirus hcov-nl63 mosaic structure of human coronavirus nl63, one thousand years of evolution evidence supporting a zoonotic origin of human coronavirus strain nl63 surveillance of bat coronaviruses in kenya identifies relatives of human coronaviruses nl63 and 229e and their recombination history recovery in tracheal organ cultures of novel viruses from patients with respiratory disease distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229e evidence for an ancestral association of human coronavirus 229e with bats link of a ubiquitous human coronavirus to dromedary camels replication of mers and sars coronaviruses in bat cells offers insights to their ancestral origins a new coronavirus-like particle associated with diarrhea in swine completion of the porcine epidemic diarrhoea coronavirus (pedv) genome sequence from the field to the lab-an european view on the global spread of pedv evaluation of the efficacy of a commercial inactivated genogroup 2b-based porcine epidemic diarrhea virus (pedv) vaccine and experimental live genogroup 1b exposure against 2b challenge efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs characterization of porcine epidemic diarrhea virus infectivity in human embryonic kidney cells generation and characterization of eptesicus fuscus (big brown bat) kidney cell lines immortalized using the myotis polyomavirus large t-antigen transmission or within-host dynamics driving pulses of zoonotic viruses in reservoir-host populations seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes white-nose syndrome is associated with increased replication of a naturally persisting coronaviruses in bats establishing a genetic recombination map for murine coronavirus strain a59 complementation groups ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events longitudinal surveillance of sars-like coronaviruses in bats by quantitative real-time pcr the immune gene repertoire of an important viral reservoir, the australian black flying fox de novo transcriptome reconstruction and annotation of the egyptian rousette bat transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) molecular characterisation of toll-like receptors in the black flying fox pteropus alecto molecular characterisation of rig-i-like helicases in the black flying fox, pteropus alecto irf7 in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation contraction of the type i ifn locus and unusual constitutive expression of ifn-alpha in bats chiropteran types i and ii interferon genes inferred from genome sequencing traces by a statistical gene-family assembler transcriptomics reveal antiviral gene induction in the egyptian rousette bat is antagonized in vitro by marburg virus infection cloning, expression, and antiviral activity of interferon beta from the chinese microbat lack of inflammatory gene expression in bats: a unique role for a transcription repressor molecular characterization of rig-i, stat-1 and ifn-beta in the horseshoe bat type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum transcriptome profiling of the virus-induced innate immune response in pteropus vampyrus and its attenuation by nipah virus interferon antagonist functions mers-cov accessory orfs play key role for infection and pathogenesis middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase porcine epidemic diarrhea virus inhibits dsrna-induced interferon-beta production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus bat coronaviruses and experimental infection of bats, the philippines replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) rapid detection of mers coronavirus-like viruses in bats: pote1ntial for tracking mers coronavirus transmission and animal origin differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? ecological dynamics of emerging bat virus spillover community intervention to prevent nipah spillover key: cord-323533-otosnjde authors: ekins, sean; madrid, peter b. title: tilorone, a broad-spectrum antiviral for emerging viruses date: 2020-04-21 journal: antimicrob agents chemother doi: 10.1128/aac.00440-20 sha: doc_id: 323533 cord_uid: otosnjde tilorone is a 50-year-old synthetic small-molecule compound with antiviral activity that is proposed to induce interferon after oral administration. this drug is used as a broad-spectrum antiviral in several countries of the russian federation. we have recently described activity in vitro and in vivo against the ebola virus. after a broad screening of additional viruses, we now describe in vitro activity against chikungunya virus (chik) and middle eastern respiratory syndrome coronavirus (mers-cov). i n recent years, we have witnessed major ebola virus outbreaks in africa, the zika outbreak in brazil, and now the novel coronavirus (2019-ncov) in china. these viruses are rarely confined to their original locations and thus create challenges in containment. newer viruses also lack available approved treatments and indicate the need for broader-spectrum antivirals. we now highlight one such molecule, tilorone dihydrochloride (tilorone; amixin or lavomax) which is currently registered for human use in russia, ukraine, kazakhstan, belarus, armenia, georgia, kyrgyzstan, moldova, turkmenistan, and uzbekistan as an antiviral (influenza, acute respiratory viral infection, viral hepatitis, viral encephalitis, myelitis, and others) and immunomodulating medication. it is also included in the list of essential medicines of the russian federation. in vivo efficacy studies dating back to 1970 support possible uses against a broad array of viruses, including influenza a virus, influenza b virus, herpes simplex virus 1, west nile virus, mengovirus, semliki forest virus, vesicular stomatitis virus, and encephalomyocarditis virus (1) (2) (3) . more recently, we recently demonstrated 90 to 100% survival in mice infected with ebola virus and then treated with tilorone (4). these results led us to more broadly profile the antiviral spectrum of activity and focus on chikungunya virus (chik) and middle eastern respiratory syndrome coronavirus (mers-cov). tilorone screening in antiviral assays. tilorone dihydrochloride was purchased from sigma-aldrich (st. louis, mo). tilorone was tested (using the niaid dmid services) against representatives of the herpesviridae, bunyaviridae, togaviridae, arenaviridae, flaviviridae, picornaviridae, and poxviridae; hepatic viruses; respiratory viruses; and other viruses. four-concentration cytopathic effect (cpe) inhibition assays were performed. confluent or near-confluent cell culture monolayers in 96-well disposable microplates were prepared. cells were maintained in minimal essential medium (mem) or dulbecco's minimal essential medium (dmem) supplemented with fetal bovine serum (fbs) as required for each cell line. for antiviral assays, the same medium was used but with fbs reduced to 2% or less and supplemented with 50 g/ml gentamicin. the test compound is prepared at four log 10 final concentrations of 0.1, 1.0, 10, and 100 g/ml or micromolar concentrations. five microwells are used per dilution: three for infected cultures and two for uninfected toxicity cultures. controls for the experiment consist of six microwells that are infected (virus controls) and six that are untreated (cell controls). the virus control and cell control wells are on every microplate. in parallel, a known active drug is tested as a positive-control drug using the same method as is applied for test compounds. the positive control is tested with each test run. the assay was initiated by first removing growth medium from the 96-well plates of cells. then, the test compound was applied in an 0.1-ml volume to wells at 2ϫ concentration. virus, normally at 50% cell culture infectious doses (ccid 50 ) in an 0.1-ml volume, was placed in those wells designated for virus infection (chik, 10 ccid 50 into 4e 4 cells per well ϭ multiplicity of infection [moi] of 0.0003, and mers-cov, 132 ccid 50 into 2e 4 cells per well ϭ moi of 0.007). for chik, compound was added to cells just prior to infection (1to 15-min range, no intended pretreatment), while for mers-cov compound was added to cells 10 to 30 min prior to infection (no intended pretreatment). medium devoid of virus was placed in toxicity control wells and cell control wells. virus control wells were treated similarly with virus. plates are incubated at 37°c with 5% co 2 until maximum cpe is observed microscopically in virus control wells (chik and mers-cov were incubated for 3 days). the plates are then stained with 0.011% neutral red for approximately 2 h at 37°c in a 5% co 2 incubator (5). the neutral red medium was removed by complete aspiration, and the cells were rinsed one time with phosphate-buffered solution (pbs) to remove residual dye. pbs was completely removed, and the incorporated neutral red was eluted with 50% sorensen's citrate buffer-50% ethanol for at least 30 min. the dye content in each well was quantified using a 96-well spectrophotometer at a 540-nm wavelength. the 50% effective (ec 50 , virus-inhibitory) concentrations and 50% cytotoxic (cc 50 , cell-inhibitory) concentrations were then calculated by linear regression analysis. the quotient of cc 50 divided by ec 50 gives the selectivity index (si 50 ) value. ideally, compounds showing si 50 values of ͼ10 are considered active. tilorone has in vitro activity against chik and mers-cov. we identified promising micromolar activities for tilorone against chik and mers-cov with reasonable selectivity indexes ( table 1 ). the in vitro activity against mers-cov also agrees with recent findings of others (6) . these combined observations along with earlier descriptions of many antiviral activities suggest tilorone is a potential broad-spectrum antiviral that may have utility against additional coronaviruses. while this drug is approved in russian federation countries, tilorone has never been evaluated and tested for safety and efficacy under studies that meet current ich and fda guidelines and regulations. recent virus outbreaks such as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) suggest the urgent need for reassessment of this compound as a broadspectrum antiviral as we have yet to fully appreciate the utility of this drug discovered 50 years ago. tilorone hydrochloride: an orally active antiviral agent effects of tilorone hydrochloride on experimental flavivirus infections in mice evaluation of various analogues of tilorone hydrochloride against venezuelan equine encephalitis virus in mice efficacy of tilorone dihydrochloride against ebola virus infection evaluation of cell viability dyes in antiviral assays with rna viruses that exhibit different cytopathogenic properties high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses mindy davis is gratefully acknowledged for assistance with the niaid virus screening capabilities, task order number b22. craig day and colleagues are kindly thanked for providing the experimental data and methodology.we kindly acknowledge nih funding: r21tr001718 from ncats (pi, sean ekins).s.e. is ceo of collaborations pharmaceuticals, inc. key: cord-324978-9qfhsj3n authors: alagaili, abdulaziz n.; briese, thomas; mishra, nischay; kapoor, vishal; sameroff, stephen c.; de wit, emmie; munster, vincent j.; hensley, lisa e.; zalmout, iyad s.; kapoor, amit; epstein, jonathan h.; karesh, william b.; daszak, peter; mohammed, osama b.; lipkin, w. ian title: middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia date: 2014-02-25 journal: mbio doi: 10.1128/mbio.00884-14 sha: doc_id: 324978 cord_uid: 9qfhsj3n the middle east respiratory syndrome (mers) is proposed to be a zoonotic disease; however, the reservoir and mechanism for transmission of the causative agent, the mers coronavirus, are unknown. dromedary camels have been implicated through reports that some victims have been exposed to camels, camels in areas where the disease has emerged have antibodies to the virus, and viral sequences have been recovered from camels in association with outbreaks of the disease among humans. nonetheless, whether camels mediate transmission to humans is unresolved. here we provide evidence from a geographic and temporal survey of camels in the kingdom of saudi arabia that mers coronaviruses have been circulating in camels since at least 1992, are distributed countrywide, and can be phylogenetically classified into clades that correlate with outbreaks of the disease among humans. we found no evidence of infection in domestic sheep or domestic goats. clusters of human infection indicate that human-to-human mers-cov transmission can occur (4, 5) . however, the origin of the infection in most cases remains unknown. analysis of human mers-cov sequences by cotten et al. has revealed the presence of at least three circulating genotypes within the ksa alone (6) . phylogenetic analyses of 13 complete and 8 partial genome sequences enabled estimates of the timing and geographic origins of individual viral clades. the authors proposed that mers-cov emerged in humans in 2011 and noted that sequence divergence between clades is consistent with several sporadic introductions of the virus into the human population, presumably from an animal reservoir. efforts to identify an animal reservoir have focused on bats and camels. bats harbor a wide range of betacoronaviruses (7) ; furthermore, bat cell lines display the mers-cov receptor, dipeptidyl peptidase 4 (8) , and can be experimentally infected. a short sequence fragment consistent with mers-cov was reported in a bat in bisha, ksa, collected in close proximity to the home and workplace of the 2012 index case patient from whom the initial virus isolate was obtained (9) . that same patient owned four pet dromedary camels (dc). serological analysis of those dc revealed the presence of antibodies reactive with mers-cov; however, no mers-cov sequences were found by pcr analysis of nasal or rectal swabs or serum. additional human cases have been associated with exposure to dc, and in some instances, investigators have described both serologic and genetic evidence of mers-cov infection in dc. memish and coworkers reported pcr detection of mers-cov sequences in a dc with respiratory illness owned by an individual with mers-cov who had no history of contact with other infected humans (10). haagmans et al. investigated an outbreak of the disease among humans on a qatari farm and found mers-cov sequences in nasal swabs from 6 of 14 seropos-itive dc. analysis of open reading frame 1a (orf1a) and fragments representing orf1b, spike, and orf4b revealed similarity but not identity to sequences obtained from the mers-covinfected humans at the same farm. the authors provide evidence that mers-cov can infect dc but cautiously conclude that data are insufficient to determine whether the infection spread from dc to humans, from humans to dc, or via another host to both species (11) . several groups have reported serological reactivity with mers-cov or a closely related virus in dc in the middle east (12) (13) (14) (15) (13) . in two regions of the ksa, hemida and colleagues detected antibodies to mers-cov in 90% of 310 dc but not in sheep, goats, cattle, or chickens. the seroprevalence was lower in dc ͻ1 year of age (72% versus 95%), suggesting widespread infection in early life (15) . to determine the prevalence of mers-cov infection in dc throughout the ksa, we undertook a nationwide survey by using both serological and molecular methods. serum, whole blood, and rectal and nasal swabs were freshly collected from dc, sheep, and goats in november and december of 2013 in the southwestern (gizan), western (taif), northwestern (tabuk), eastern (hofuf), and central (unizah, riyadh) regions of the ksa (table 1) . we also collected archived serum samples obtained from dc in 1992 through 2010 (table 2 ). sera were initially tested for the presence of antibodies reactive with mers-cov by using a cell enzyme-linked immunosorbent assay (elisa) based on vero cells infected with mers-cov. subsets of sera positive by elisa were tested in western blot assays that used extracts of vero cells infected with mers-cov and a luciferase immunoprecipitation system (lips) assay based on recombinant mers-cov nucleoprotein. potential serologic cross-reactivity with bovine coronavirus (bo-cov) was addressed by testing for reactivity with bo-cov nucleocapsid protein by lips assay. the presence of viral nucleic acids in rectal and nasal swabs and a subset of serum and whole blood samples was assayed by reverse transcriptionquantitative pcr (rt-qpcr) with primers targeting the upe and orf1a genome regions of mers-cov (16, 17) . one hundred fifty (74%) of 203 dc sampled countrywide in 2013 were found to have antibodies to mers-cov by elisa. the prevalence of seropositivity was higher in adult dc (ͼ2 years of age; 93/98, 95%) than in juvenile dc (յ2 years of age; 57/104, 55%) (p ͻ 0.0001, 2 test). the lowest prevalence of seropositive juveniles was found in the southwestern region, in proximity to the city of gizan (1/21, 5%) (fig. 1a) . a higher prevalence of antibodies to mers-cov in older animals was also seen in samples obtained in years prior to 2012. in 2010, the seroprevalence was 76% (16/21) in juveniles versus 91% (21/23) in adults in the central region (riyadh, kharj). in 2009, the seroprevalence was 72% (40/56) in juveniles versus 92% (24/26) in adults (riyadh, rumah) ( table 2) . seroreactivity was also found in samples collected from dc as early as the 1990s: 1/1 (100%) in 1992, 2/2 (100%) in 1993, 114/123 (93%) in 1994, 6/6 (100%) in 1996, and 6/6 (100%) in 2003. analysis of selected seropositive dc sera by western immunoblot assay indicated two distinct reaction patterns that showed reactivity either to mers-cov spike glycoprotein alone or to spike glycoprotein and nucleocapsid protein. these results were concordant with results of mers-cov nucleocapsid lips assays ( fig. 2 ; see table s1 in the supplemental material). potential serologic cross-reactivity to bo-cov was addressed by analyzing bo-cov nucleocapsid reactivity, which has been shown to be subgroup specific in covs (18) . overall, 17% (35/ 203) of dc were positive for bo-cov, ranging from 3% in the southwest (gizan) to 25% in the east (hofuf) and 20% in adult animals versus 14% in juvenile animals; 2 animals (juveniles in taif) were seropositive for bo-cov exclusively, while the remaining 16% (33/203) were reactive to bo-cov and mers-cov, and 58% (117/203) were reactive to mers-cov alone (see table s1 in the supplemental material). rectal and nasal swabs collected in parallel with serum samples from the same animals were assayed for mers-cov nucleic acids by rt-qpcr. nucleic acids were most frequently detected in nasal swabs; rectal swabs were found positive in only three cases; in two of them, the nasal sample was also positive. the regional distribution of pcr-positive animals is shown in fig. 1b . in contrast to serology, where mers-cov-reactive antibodies were more prevalent in adults than in juveniles (95% versus 55%), mers-cov nucleic acids were found more frequently in juveniles (36/104, 35%) than in adults (15/98, 15%) (p ϭ 0.003, 2 test). the five samples with ͼ10 6 copies were all from juveniles, four of them from seronegative animals. the prevalence of pcr-positive dc ranged from 66% in taif in the west to 0% in gizan in the southwest. pcr analysis of a random selection of serum and whole blood samples collected from nasal or rectal swab pcr-positive, seropositive, and seronegative dc revealed no evidence of viremia (see table s1 in the supplemental material). these included 13 adults and 29 juveniles phlebotomized in 2009, 15 adults and 14 juveniles phlebotomized in 2010, and 8 adults and 13 juveniles phlebotomized in 2013. these animals included the five juveniles with the highest viral genome sequence load in nasal swabs. serum samples collected from goats (n ϭ 36) and sheep (n ϭ 112) in 2013 in the central region (unizah, riyadh) were not immunoreactive with mers-cov but were immunoreactive with bo-cov (25% of goats, n ϭ 36; 54% of sheep, n ϭ 24). nasal swabs from 36 goats and 78 sheep were negative in rt-qpcr assays for mers-cov upe. to test the validity of rt-qpcr results and determine phylogenetic relationships of viral sequences found in dc in the ksa to previously reported sequences, we amplified and sequenced longer regions of the spike, orf1ab, and nucleocapsid genes from rt-qpcr-positive samples (for the sequences of the primers used, see table s2 in the supplemental material). eleven of 13 swab samples with ͼ10 5 copies in upe rt-qpcr yielded products for sequencing. no suitable products were obtained from samples with lighter viral sequence loads (ͻ10 4 copies) (see table s1 in the supplemental material). phylogenetic analysis of a 1,044nucleotide (nt) region of the spike gene and a 2,004-nt region of the orf1ab gene indicated ͻ1% divergence from previously published mers-cov sequences (fig. 3 ) (genbank accession no. kj396756 to kj396771). the nucleocapsid sequence was identical to the previously reported mers-cov sequences. mers-cov is posited to be a zoonosis. however, the evolutionary history of mers-cov and the reservoirs and vectors for human infection remain obscure. early anecdotal reports that some mers-cov victims had exposure to dc led to serologic investigation of dc in spain and oman (14) , jordan (12), egypt (13) , and the ksa (15) that revealed antibodies to mers-cov. definitive evidence that dc can be infected with mers-cov was obtained when viral sequences were detected in nasal swabs from dc sampled in close proximity to outbreaks of the disease among humans in qatar (11) and jeddah, ksa (10) . nonetheless, as noted by nishiura and colleagues, current data do not fulfill the two criteria required to implicate dc as a significant reservoir species in the epidemiology of mers-cov (19) , i.e., (i) that dc are sufficient to maintain mers-cov and (ii) that the presence of dc is essential to the continuous transmission of infection. results presented here do not establish the latter; however, they do provide evidence for the former. our study is the first comprehensive countrywide survey of dc from the ksa, the country with the most recorded mers-cov cases, to use both serological and molecular diagnostic methods. analysis of specimens from western regions of the country, from tabuk in the northwest, taif in the west, and gizan in the southwest, revealed regional differences. although the seroprevalence was high in adults throughout the country at ͼ80%, in juveniles, it ranged from 90% in the east to 5% in the southwest. the sero-prevalence in dc յ2 years of age was lower than that in older animals, confirming the results of hemida et al. (15) . molecular analysis of nasal and rectal swab specimens indicated the highest prevalence of mers-cov sequences in dc in the west and northwest. nasal swabs with heavy sequence loads (ͼ10 5 copies) also clustered in the taif region. a second sample collection in the west (taif) separated from the first by an interval of 2 months confirmed the presence of heavy viral sequence loads in nasal swabs collected from juvenile animals sampled in this area (data not shown). these findings suggest that continuous, longer-term surveillance is necessary to determine the dynamics of virus circulation in dc populations. lower prevalence rates of both mers-cov and bo-cov were evident in samples from the southwest. this may relate in part to the enforcement of restrictions of livestock movement in and out of gizan province implemented after the rift valley fever outbreak in 2000 but also to the generally lower dc population density in this region than in other regions of the ksa. viral nucleic acids were more commonly detected in nasal swabs than in rectal specimens and were more frequent in juvenile than in adult animals. these findings, together with the absence of viremia and the known respiratory tract tropism of several other coronaviruses, suggest that airborne transmission is the most likely mode of mers-cov transmission. although nucleic acid copy numbers were commonly highest in juvenile animals that were seronegative or had low antibody titers, positive findings were also obtained with specimens from highly seropositive and adult animals. our findings in archived dc specimens, although restricted to serology, strongly suggest that mers-cov or a closely related virus has been circulating in dc in the ksa for at least 2 decades. complete genomic sequences of mers-cov found in contemporary dc in the ksa are identical to sequences of viruses recovered from human mers-cov victims (unpublished data). although we speculate that dc are potential reservoirs for human transmission, we cannot prove this relationship from the current data. rigorous epidemiological investigation of the potential for exposure to dc in sporadic cases of mers-cov (those where there is no opportunity for human-to-human transmission) is required to test this model. if dc can be implicated, other questions will arise. did mers-cov truly emerge as a human pathogen in 2012, or were cases of cryptic infection not appreciated because of a lack of suitable diagnostic tests? we may be able to address this conundrum by using archived human materials. if evidence of human mers-cov infections cannot be detected prior to 2012, we must entertain the possibility that mutation facilitated cross-species transmission. however, we see no path to address this possibility absent access to historical dc respiratory tract specimens. the only archived dc specimens we have been able to locate are dc sera; our efforts to recover mers-cov sequences from camel blood have been unsuccessful. what are the roles of bats, if any, as reservoirs of mers-cov? these limitations notwithstanding, the most urgent public health concern, raised in work we and others have reported that focuses on dc infection, is to determine the role of these animals in sporadic human infection. the evidence is clearly sufficient to support targeted investigation of direct or indirect exposure to dc in the disease among humans. sample collection. samples included dc, sheep, and goat sera; whole blood and nasal and rectal swabs freshly collected in 2013; and archived serum samples from 1992, 1993, 1994, 1996, 2004, 2009, and 2010 . two rectal and two nasal swabs were obtained from each animal. one rectal swab and one nasal swab were placed into rnalater (life technologies, carlsbad, ca), and one rectal swab and one nasal swab were placed into viral transport medium (becton dickinson, franklin lakes, nj). all were stored at ϫ80ºc. after 24 h in fixative, plates were rinsed three times with phosphate-buffered saline (pbs) and placed in pbs for storage at 4°c. plates were loaded with infected and noninfected cells in alternating rows to generate a differential reading for each serum tested. positivity was defined as an infected-cell optical density of ͼ0.6 and ͼ3ϫ the noninfected-cell optical density. test sera were diluted 1:3,000 in pbs-0.05% tween 20 -1% bovine serum albumin; secondary antibodies were rabbit anti-goat igg (hϩl)-horseradish peroxidase conjugate (1:3,000; bio-rad, hercules, ca), rabbit anti-sheep igg (hϩl)-horseradish peroxidase conjugate (1:3,000; bio-rad), and anti-llama igg-horseradish peroxidase conjugate (1:10,000; bethyl laboratories, montgomery, tx). western blot. extracts of noninfected vero cells or vero cells infected with mers-cov strain emc were generated at rocky mountain laboratories, loaded onto discontinuous 3 and 7.5% sds gels (bio-rad), and transferred onto nitrocellulose with iblot transfer stacks (invitrogen iblot; life technologies). lanes were loaded with alternating infected and noninfected extract samples, and a pair were cut for incubation with dc sera (1:800 in blocking solution) after blocking of the membrane in pbs-0.05% tween 20 -5% dry milk blocking solution for 1 h. membranes were washed three times with pbs-0.05% tween 20 after a 2-h incubation with serum and then incubated for another 1.5 h with secondary antibody (1:7,000 in blocking solution; anti-llama igg-horseradish peroxidase conjugate; bethyl laboratories). following three more washes, the membranes were developed with westernsure premium chemiluminescent substrate (li-cor, lincoln, ne) and read on a c-digit blot scanner (li-cor). lips assay. the nucleocapsid proteins of bo-cov and mers-cov were pcr amplified with primers introducing appropriate restriction sites for cloning into vector pren-2 fused to the c terminus of the renilla luciferase reporter (20) . sequence-confirmed construct dna was purified from escherichia coli cultures (qiagen, hilden, germany), and transfected into cos-1 cells (african green monkey kidney, atcc crl-1650; 1 g; lipofectamine; invitrogen, life technologies). cells were harvested at 48 h posttransfection in lysis buffer (50 mm tris [ph 7.5], 100 mm nacl, 5 mm mgcl 2 , 1% triton x-100, 50% glycerol, protease inhibitors), and the protein extract was clarified by two rounds of centrifugation at 12,000 ϫ g. the target concentration was determined in relative light units (rlu), and approximately 10 ϫ 10 6 rlu were incubated with test serum (1:100) in a final volume of 100 l buffer a (50 mm tris [ph 7.7], 100 mm nacl, 5 mm mgcl 2 , 1% triton x-100) per well in 96-well plates (catalog no. 249944; thermo/nunc, waltham, ma). after 1 h of incubation at room temperature, protein a/g beads (catalog no. 53135; pierce, junction city, or) were added and the mixtures were transferred to 96-well filter plates (catalog no. msbvn1b50; millipore, billerica, ma) for another hour of incubation at room temperature with shaking. bead-bound anti-gen was washed eight times with buffer a and three times with pbs (tecan hydroflex, maennedorf, switzerland) and then read with coelenterazine substrate (renilla luciferase assay system; promega, madison, wi) on a centro lb960 luminometer (berthold, bad wildbad, germany). nucleic acid extraction and pcr. total nucleic acids were extracted from nasal swabs, serum, and whole blood on a qiacube with cador reagent kits (qiagen) or rneasy reagent kits for extraction of rna from rectal swabs. real-time qpcr used a onestep real-time qpcr buffer (invitrogen, life technologies) and primer/probes upe and orf1a (16, 17) . products for sequencing were generated by rt-pcr. cdna was reverse transcribed with superscript iii and random hexamer primers. pcr was performed with amplitaq gold (life technologies) and primers designed to amplify a 1,044-nt region of the spike gene (heminested pcr), a 913-nt region of the n gene (nested pcr) or a 2,004-nt region of the orf1ab region (heminested pcr). for the sequences of the primers used, see table s2 in the supplemental material. products were purified by agarose gel electrophoresis and with qiaquick gel extraction kits (qiagen) and subsequently sequenced on both strands by the dideoxynucleotide chain termination method (genewiz, south plainfield, nj). supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi:10.1128/mbio.00884-14/-/dcsupplemental. middle east respiratory syndrome coronavirus (mers-cov)-update. world health organization isolation of a novel coronavirus from a man with pneumonia in saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus in bats, saudi arabia mers-cov-eastern mediterranean (85): animal reservoir, camel, suspected, official. promed-mail middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses missing information in animal surveillance of mers-cov antibody-profiling technologies for studying humoral responses to infectious agents we are grateful to his highness, prince bandar bin saud al saud, president, saudi wildlife authority, for his unlimited support and encouragement; to devon welsh and kawthar muhammad for mobile laboratory coordination; to andrew schultz for statistical analysis; to ellie kahn for editorial contributions; to aaloki shah, parisa zolfaghari, mia r. key: cord-320663-xypg6evo authors: market, marisa; angka, leonard; martel, andre b.; bastin, donald; olanubi, oladunni; tennakoon, gayashan; boucher, dominique m.; ng, juliana; ardolino, michele; auer, rebecca c. title: flattening the covid-19 curve with natural killer cell based immunotherapies date: 2020-06-23 journal: front immunol doi: 10.3389/fimmu.2020.01512 sha: doc_id: 320663 cord_uid: xypg6evo natural killer (nk) cells are innate immune responders critical for viral clearance and immunomodulation. despite their vital role in viral infection, the contribution of nk cells in fighting sars-cov-2 has not yet been directly investigated. insights into pathophysiology and therapeutic opportunities can therefore be inferred from studies assessing nk cell phenotype and function during sars, mers, and covid-19. these studies suggest a reduction in circulating nk cell numbers and/or an exhausted phenotype following infection and hint toward the dampening of nk cell responses by coronaviruses. reduced circulating nk cell levels and exhaustion may be directly responsible for the progression and severity of covid-19. conversely, in light of data linking inflammation with coronavirus disease severity, it is necessary to examine nk cell potential in mediating immunopathology. a common feature of coronavirus infections is that significant morbidity and mortality is associated with lung injury and acute respiratory distress syndrome resulting from an exaggerated immune response, of which nk cells are an important component. in this review, we summarize the current understanding of how nk cells respond in both early and late coronavirus infections, and the implication for ongoing covid-19 clinical trials. using this immunological lens, we outline recommendations for therapeutic strategies against covid-19 in clearing the virus while preventing the harm of immunopathological responses. natural killer (nk) cells are a key component of the innate immune system and are critical in the response to many viral infections in humans and animal models (1) (2) (3) . in addition to their beneficial antiviral role, nk cells have also been associated with immunopathology in infections such as respiratory syncytial virus (rsv) (4), influenza a virus (5) (6) (7) (8) , and hepatitis b (9) . additionally, in the context of non-respiratory viral infections by hiv and hcv, nk cells appear to act as a rheostat by eliminating activated cd4+ and cd8+ t cells, thus preventing t cell-mediated autoimmunity (10) . the etiologic agent of the 2019 outbreak of pneumonia in wuhan, china, was identified as belonging to the coronaviridae family and named severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this virus causes the coronavirus disease 2019 (covid19) which was declared a pandemic by the world health organization (who) on march 11th, 2020 (11, 12) . with the paucity of information currently available, there is a lack of consensus on the role played by nk cells in the response to coronavirus (cov) infection. in this review, we will explore evidence for both the protective and pathological role that nk cells may play in cov infection. based on this knowledge we will comment on immune modulating treatment options that are being developed for the current covid-19 crisis. first discovered in the 1960s, covs are part of the coronaviridae family of enveloped positive single-strand rna viruses (13, 14) . the subfamily orthocoronaviridae includes four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (15) . alpha-and betacoronaviruses circulate in mammals, including bats, gammacoronaviruses infect mostly avian species, and deltacoronaviruses infect birds and mammals (15) . low pathogenic human covs (hcovs), such as hcov-299e (16) , infect upper airways and etiological studies suggest they account for 15-30% of common colds (17, 18) . on the other hand, highly pathogenic covs infect the lower respiratory tract and can cause severe pneumonia (19) . these highly pathogenic covs include sars-cov-1, the virus responsible for the 2002-2004 severe acute respiratory syndrome (sars) epidemic, and mers-cov, the virus responsible for the outbreak of middle eastern respiratory syndrome (mers) in 2015 (19) (20) (21) . while highly pathogenic covs have become a relatively recent issue for humans; feline, canine, and bovine covs have long been recognized as significant pathogens with implications in veterinary medicine and agriculture (22, 23) . all covs have a roughly 30 kb genome packed into an enveloped helical capsid ranging from 80 to 120 nm (24) . at minimum, coronaviridae members encode 4 structural and 16 non-structural proteins (14) with the family owing its name to the crown-like appearance produced by their spike (s) proteins (25) . mutations in the s protein have allowed sars-cov1/2 to co-opt ace2 or mers-cov to co-opt dipeptidyl peptidase 4 (dpp4) receptor/cd26 as viral entry receptors, thus facilitating the zoonosis of nonhuman covs (15, (26) (27) (28) . in addition, another mechanism that may have allowed these viruses to adapt to human hosts is through s protein cleavage by host cell proteases to expose the s2 domain fusion peptide, which induces viral and cellular membrane fusion and results in the release of viral genome into the cytoplasm (15) . genetic sequencing revealed sars-cov-2 to be a betacoronavirus that shares 79.0% nucleotide identity with sars-cov-1 and 51.8% identity to mers-cov (29) . the epidemic of sars in 2002-2004 caused by sars-cov-1 illustrated the devastating potential of coronaviruses to cause serious disease in humans (24) . sars ultimately reached 29 countries and 5 continents causing over 8,000 infections and over 900 deaths. the basic reproductive rate (r 0 ) or the number of expected cases arising from one infected individual, ranges from 2 to 4 (20, 30, 31) . with its reservoir in bats, sars-cov-1 is a zoonosis that was transmitted to humans by palm civets (24, 32, 33) . sars-cov-1 infects lung pneumocytes (34) and enterocytes in the digestive tract (35) most often producing flulike symptoms (36, 37) . more severe presentations including pneumonia, pronounced lymphopenia, liver abnormalities, and acute respiratory distress syndrome (ards) were also reported, with most fatalities due to respiratory failure (19, (36) (37) (38) (39) . the subsequent mers-cov outbreak in 2015 also originated in bats, with dromedary camels being the intermediary host (14, 40, 41) . the r 0 for mers-cov is estimated to be under 1 (21) . the extent of mers-cov transmission was more limited than sars-cov-1, but its case fatality rate was greater with 2,494 cases over 27 countries and 858 deaths being reported at the end of 2019 (21) . common presentations for mers-cov include fever, dyspnea, muscle pain, and digestive tract symptoms and disease progression is more likely in those with comorbidities (42) . like sars-cov-1 and mers-cov, sars-cov-2 is thought to have originated in bats through an unknown intermediary host (43) . at the time of writing, the number of global infections is estimated to be over 5,000,000 with over 340,000 deaths (44) and the r 0 is roughly 2.2 (45) . like other diseases caused by infectious covs, most patients present with flu-like symptoms including fever, cough, and lethargy, with the development of pneumonia and ards often proving fatal (46) . furthermore, patients with underlying conditions are at risk for further complications if infected with covid-19, such as those with cardiovascular disease (47) . sars-cov-2 has been posthumously detected in not only the lungs, but the pharynx, heart, liver, brain, and kidneys (48) . transmission of sars-cov-2 is thought to mainly occur through direct contact/inhalation of respiratory droplets and aerosols from infected carriers, but indirect transmission by fomites has also been reported, although less efficient (49, 50) . sars-cov-2 viral entrance is thought to be mediated by binding of the s protein to the ace2 receptor (51, 52) , although this is still under debate (53) . while direct cytopathic effects are thought to play a major role in cov pathology, studies have suggested that a dysregulated immune response resulting in pathological inflammation is also partly responsible (19) . with the current pandemic already surpassing the previous cov outbreaks (54) , rapid deployment of novel approaches to understanding and treating coronavirus infections are needed. innate immunity is essential in disease prevention and viral clearance. among the first responders to viral infections, tissueresident macrophages and dendritic cells (dcs) (55) recognize evolutionarily conserved microbial structures termed pathogenassociated molecular patterns (pamps) via germline-encoded pattern recognition receptors (prrs) (56). in the context of respiratory rna viruses, airway epithelial cells, that also express some prrs (57) , are often infected and have a major role in the first line of defense. tlr3, tlr7, tlr8, mda-5, and rig-i are prr expressed by immune and non-immune cells that are especially relevant in fighting respiratory rna viruses, such as coronaviruses (57) . sensing through prrs results in the transcription of genes involved in the inflammatory response, with type i interferons (ifns) (ifn-α/β) production being a critical part of the antiviral response (58) . type i ifns are produced by many immune and non-immune cells (55, 57, 59) and in addition to eliciting intrinsic antiviral responses (60), they are also essential to prime innate and adaptive lymphocytes, including nk cells (61) . nk cells are cytotoxic lymphocytes that directly target infected, stressed, or transformed cells and play a critical role in bridging the innate and the adaptive immune responses (62) . in humans, mature nk cells comprise 10-15% of total peripheral blood leukocytes and are described phenotypically as cd3 − cd14 − cd19 − cd56 + cd16 +/− (63). nk cells do not undergo clonal selection but instead express several germline-encoded receptors that regulate their activity (62, 64, 65) . upon viral infection, host cells become more susceptible to nk cell killing through: (i) upregulation of self-encoded molecules induced by infection/cellular stress (66, 67) that bind activating nk cell receptors such as natural cytotoxicity receptors (ncrs) (nkp30, nkp44, and nkp46) (68), c-type lectin-like receptors nkg2d and nkp80 (69) , and co-activating receptors such as dnam-1 (70); (ii) downregulation of ligands for inhibitory receptors such as killer immunoglobulin-like receptors (kirs) (71) (72) (73) and the c-type lectin-like receptor cd94-nkg2a (74, 75) which suppress nk cell activation, and; (iii) direct recognition of viral moieties, via engagement of pamps (76) or transmembrane activating receptors such as mouse ly49h (77) or human nkg2c (78) . moreover, nk cells can eliminate virus-infected cells via cd16-mediated antibody-dependent cellmediated cytotoxicity (adcc), which has been shown to be particularly important for herpesvirus clearance (79) . finally, nk cell activity is modulated by cytokines, including, but not limited to, the activating cytokines interleukin (il)-2/12/15/18 (80) and type i ifn, which can be produced by virally infected cells or activated antigen presenting cells (81, 82) . il-2/12/15/18, alone or in combination, promotes nk cell survival, proliferation, cytotoxicity, and cytokine production, including ifn-γ (80) . therefore, nk cells are uniquely equipped to sense and quickly respond to viral infections. nk cells are found in circulation and in peripheral tissues (63) and can be quickly recruited to sites of infection where they facilitate and accelerate viral clearance. in fact, nk cells are not thought to have permanent tissue residency but instead move dynamically between the blood and tissues, such as the lungs (83) . nk recruitment is regulated by chemokine gradients that are sensed via chemokine receptors (84, 85) . activated nk cells induce the apoptosis of target cells through the engagement of death receptors, such as trail and fas (86) or via direct cytotoxicity through ca 2+ -dependent exocytosis of cytolytic granules (perforin and granzymes) (87) . moreover, nk cells secrete cytokines, including ifn-γ, which have key anti-viral properties (88) . in addition to being essential first responders to viral infection, nk cells can elicit a stronger secondary response resembling the memory features of adaptive lymphocytes (89, 90) . nk cell memory has been initially described in mice infected by mcmv, where ly49h + nk cells quickly expand and have stronger responses after a secondary encounter with the virus (91) . interestingly, a similar nk cell subset has been identified in humans, where nk cells expressing nkg2c are expanded and persist in cmv infected patients (92) . both ly49h and nkg2c bind viral determinants, highlighting how nk cell memory is linked with the ability of nk cells to directly recognize viruses (93, 94) . in addition to direct recognition of viral molecules, longlasting changes in nk cells are induced by the cytokine milieu (89, 95) , which can be elicited by viral infection. the relevance of nk cells in fighting viral infections has been highlighted by several studies where nk cells, in mice and humans, were not present or had compromised functions (96) . for example, individuals with nk cell deficiencies (nkd), a subset of primary immunodeficiency diseases, are highly susceptible to viral infection, particularly by herpesvirus and papillomavirus families (96) . the seminal 1989 case of nkd in an adolescent female with severe herpesvirus infections (varicella pneumonia, disseminated cmv, and disseminated hsv) revealed how functional nk cell deficiencies have clinical consequences in terms of viral infections (97) . cancer patients are also at risk of viral infections (98) , which may be explained, at least in part, by an impairment of nk cell responses often observed in humans and in murine tumor models (99) (100) (101) (102) (103) (104) . unsurprisingly, cancer patients are at a significantly increased risk of severe covid-19 (105, 106) . elderly patients are also more susceptible to viral infections (107) . mouse studies highlighted how a decreased number of circulating mature nk cells in aged animals paralleled with increased susceptibility to viral infections (108) . studies in humans suggest that although nk cell numbers can actually increase with aging, nk cell activity declines significantly (109, 110) . przemska-kosicka et al. investigated nk cell function in response to seasonal influenza vaccination in young and old populations and observed quantitative and qualitative changes associated with impaired responses in the nk cell population and this was associated with poor seroconversion in the older population (111) . additionally, obesity, which has been shown to cause systemic nk cell dysfunction (112, 113) , has also been linked to increased covid-19 severity and could be the reason behind the high prevalence of severe covid-19 in younger people (113) . in short, nkd and individuals with reduced nk cell numbers or function are more susceptible to viral infections. unsurprisingly, the cdc has already highlighted a higher risk of infection and severity of covid-19 in older individuals and individuals with comorbidities such as obesity and cancer (114) . however, this point is still controversial as a systematic review showed that primary immunodeficiencies are not linked with increased covid-19 severity (115) , but these data have to be interpreted keeping in mind that a large part of covid-19 pathology is caused by excessive immune activation, which is arguably harder to reach in immunocompromised individuals. given the paradoxical role of the immune response in covid-19 patients, it would be extremely useful to be able to rely on immunological functional biomarkers that could predict the outcome of disease severity. such assays are readily available for determining nk cell activity, e.g., nkvue tm , and there is therefore an opportunity to conduct studies that would link nk cell functions to disease severity. evasion of host immune responses is necessary for the successful propagation of a virus. mechanisms employed by covs to evade the immune response could provide insights into how the immune system, and nk cells in particular, responds to sars-cov-2. covs have been shown to target components of the innate ifn response, employing non-structural proteins (nsps), structural proteins, and accessory proteins to achieve this goal. nsp16 methylates viral rna therefore preventing recognition by mda5 and dampening type i ifn production (116) . nsps also suppress type i ifn responses via the inhibition of the transcription factor stat1 mrna transcription (nsp1) and deubiquitination of transcription factors like interferon regulatory transcription factor (irf)3 (nsp3) (116) . moreover, viral-encoded accessory proteins from sars-cov-1 open reading frame (orf)3b and mers-cov orf4a/4b also block ifn production and signaling (116) . in addition, the mers-cov orf6-encoded protein blocks p-stat1 import, thus blocking ifn signaling (116) . finally, the structural m protein of mers-cov (27) physically sequesters kinase proteins rig-i, tbk1, ikke, and traf3 and the sars-cov-1 n protein inhibits activator protein (ap)-1 signaling, protein kinase r function, and nfκb activation, all of which act to impede ifn responses (117) . in vivo murine studies report young mice rapidly clear sars-cov-1 infection, while old mice do not and that this discrepancy is due to a delay in type i ifn. furthermore, early administration of ifn-β induces a stronger immune response and reduces mortality in old mice (118) . since type i ifns are critical for nk cell activation and effector functions, it is possible that nk cell-mediated clearance of sars-cov-2 is being subverted by these mechanisms. further research into the role of nk cells in cov clearance and potential immune evasion mechanisms are necessary to inform therapeutic development and use. there is currently a paucity of studies into the role of nk cells not only in covid-19 pathophysiology, but also in other coronavirus infections. an in vivo study reported that beige mice on a b6 background cleared sars-cov-1 normally, indicating that functional lymphocytes, including nk cells, may not be required to eliminate sars-cov-1 in murine models (119) . however, in a more recent study characterizing the cellular immune response to sars-cov-1 in 12-14-month old balb/c mice, t cell depletion did not prevent control of sars-cov-1 replication (120), suggesting a role for the innate immune system, and nk cells, in viral clearance. importantly, in this study cd4-depletion resulted in enhanced lung immunopathology and delayed viral clearance, while cd8-depletion did not affect viral replication or clearance, thus highlighting an important role for cd4 + t cells in coronavirus infection. these conflicting results may be due to the inherent limitations of cov murine models. in 4-8 week-old mice, sars-cov-1 is associated only with mild pneumonitis and cytokines are not detectable in the lungs (119, 121, 122) . a sars-cov-1 isolate (ma-15) replicates to a high titer and is associated with viremia and mortality, however the model lacks significant inflammatory cell infiltration into the lungs (123) . thus, mouse models developed for the study of sars fell short in terms of reproducing the clinical and histopathological signs of disease (119, (121) (122) (123) . it is therefore necessary to develop a usable animal model that is capable of reproducing the clinical and histopathological signs on covid-19. israelow et al. recently described a sars-cov-2 murine model based on adeno associated virus (aav)9-mediated expression of human (h)ace2, which replicated the pathologic findings found in covid-19 patients (124) . this model, which overcame the inability of murine (m)ace2 to support sars-cov-2 infection, was used to show the inability of type i ifn to control sars-cov-2 replication (124) . in a similar attempt to overcome the lack of infectability through mace2, dinnon et al. recently described a recombinant virus (sars-cov-2 ma) with a remodeled s protein mace2 interface, which replicated in upper and lower airways in young and aged mice with disease being more severe in aged mice. the authors used this model to screen therapeutics from vaccine challenge studies and assessed pegylated ifn-λ-1 as a promising therapeutic. the authors suggested that this model has greater ease of use, cost, and utility over transgenic hace2 models (125) to evaluate vaccine and therapeutic efficacy in mice (126) . a preliminary analysis of nk cell function and phenotype has been performed by zheng et al. using peripheral blood from covid-19 patients (127). on admission, nk cell levels in the peripheral blood inversely correlated with disease severity. furthermore, covid-19 patients with severe disease had significantly lower numbers of circulating nk cells, as compared to mild disease (p < 0.05) (127) . additionally, circulating nk cells in severe disease displayed increased expression of the inhibitory receptor nkg2a and had an hyporesponsive phenotype with lower levels of ifn-γ, tumor necrosis factor (tnf)-α, il-2, and granzyme b, although degranulation was maintained (127) . finally, as compared to patients with active disease, patients recovering from covid-19 had higher numbers of nk cells and lower nkg2a expression (127) . liao et al. performed singlecell rnaseq on the cells obtained from bronchoalveolar lavage fluid of severe and mild covid-19 patients and found that covid-19 patients had significantly more nk cell infiltrates into the lungs, however patients with severe disease had reduced proportions of nk cells (128) . in addition, klrc1 (nkg2a) and klrd1 (cd94) were highly expressed by nk cells (128) . carvelli et al. analyzed myeloid and lymphoid populations by immunophenotyping from blood and bronchoalveolar lavage fluid (balf) in 10 healthy controls, 10 paucisymptomatic covid-19 patients, 34 pneumonia patients, and 28 patients with ards due to sars-cov-2 and found that absolute numbers of peripheral blood lymphocytes, including nk cells, were significantly reduced in the pneumonia and ards groups compared to healthy controls. furthermore, the proportion of mature nk cells was reduced in patients with ards and nk cells showed increased nkg2a, pd-1, and cd39 (129) . finally, wilk et al. performed single-cell rna-sequencing on 7 covid-19 patients and 6 healthy controls and found that the cd56 bright population was depleted in all covid-19 patients but the cd56 dim population was depleted only in patients with severe covid-19. furthermore, nk cells had increased expression of the exhaustion markers lag3 and havcr2 (130) . nk cell cytopenia seems to be a consistent characteristic among sars-cov-2 infected patients (131). altogether, these data indicate alterations in the nk cell phenotype and functional profile that are consistent with the hypothesis that to establish a productive and lasting infection, sars-cov-2 needs to dampen the nk cell response. nk cell dysfunctions were also observed in patients from the previous cov outbreaks. wang et al. assessed nk cell number and phenotype using peripheral blood from 221 sars patients admitted to hospitals in beijing, china (132) . nk cell proportion and absolute number were significantly reduced in sars patients as compared to healthy donors and patients infected with the bacterium mycoplasma pneumoniae (131). nk cell number correlated inversely with disease severity and patients with anti-sars cov-specific igg or igm antibodies had significantly fewer nk cells (132) . the patients assessed had varied disease duration from 4 to 72 days (mean 31.7 days) and this allowed for patient stratification by disease duration. within the first 10 days of sars-cov-1 infection, nk cell numbers remained high but this period was followed by the development of lymphopenia with levels recovering only around day 40 (132) . dong et al. also observed a reduction of nk cell numbers in sars patients, and these levels were lower in patients with severe, as compared to mild, sars (133) . in addition, mers infection is strongly associated with leuko-and lymphopenia (42, (134) (135) (136) . the mechanisms underlying the reduction of circulating nk cells in patients infected with covs are still unclear. as most studies have focused on peripheral blood nk cells, it is possible that the reduced number of circulating nk cells is due to redistribution of blood nk cells into the infected tissues (137) . while it is hard to assess nk cell migration to infected tissues in covid-19 patients, this hypothesis was corroborated by mouse studies, where nk cells have been shown to migrate to the lungs in cov infected animals (120) . an abundance of inhibitory factors, such as tgf-β, may be partially responsible for the nk cell hyporesponsiveness observed in covid-19 patients. in support of this hypothesis, huang et al. found significantly higher tgf-β levels in sars patients compared to healthy controls and this positively correlated with length of stay (138) . given the importance of tgf-β in suppressing nk cell functions, it is possible that the higher levels of tgf-β (as well as other inhibitory cytokines) in cov patients leads to suppression of nk cell antiviral activity (138) . early studies of covid-19 patients report secondary (super-) infections, including nosocomial pneumonia or bacteremia as a complication of sars-cov-2 infection (138) . since nk cells are critical first responders that play a role in preventing and clearing infections (139) , a poor nk cell count or exhausted phenotype, in addition to negatively influencing covid-19 patient outcomes, could facilitate the development of secondary infections and have a significant negative impact on patient outcomes. one of the main barriers in studying the role of nk cell activation in the early clearance of cov infection in asymptomatic or mildly symptomatic patients is the fact that these individuals are rarely diagnosed in the clinic and therefore an opportunity to collect samples for research does not exist. thus, while there is currently no direct evidence to support a role for nk cells in the clearance of sars-cov-2, evidence showing that viral infection has a negative effect on the nk cell compartment is accumulating. given the importance of nk cell activity in early viral clearance and late immunopathology, having a rapid and reliable test to predict nk cell function, such as nkvue tm (atgen canada/nkmax), whereby whole blood is stimulated by an nk cell-specific activating cytokine mix and activity is measured via ifn-γ production, might allow researchers to predict who will mount an adequate response with asymptomatic or minimally symptomatic viral clearance and who will need icu admission, as has been shown with cancer patients (140) . further research will be required into the innate immune response to cov infection to more fully understand nk cell contributions to viral clearance. in the context of covs, the significant morbidity and mortality associated with severe disease is due to acute lung injury (ali) and the development of ards (19, 141) . pathological analysis of tissues obtained from sars and mers patients showed edematous lungs with areas of consolidation, bronchial epithelial denudation, loss of cilia, squamous metaplasia, pneumocyte hyperplasia, and bronchial submucosal gland necrosis (19, 29) . histological features include diffuse alveolar damage and acute fibrinous and organizing pneumonia (29) . a heightened inflammatory response in the lungs resulting in tissue damage has been hypothesized to explain the development of ali. there are several key factors that may be responsible for the induction of this dangerous inflammation (138) . both sars-cov-1 and mers-cov replicate to high titers early in infection, which could lead to enhanced cytopathic effects and increased production of pro-inflammatory cytokines/chemokines by infected cells. chen et al. developed a pneumonia model where pulmonary replication of sars-cov-1 was associated with histopathological evidence of disease, including bronchiolitis, interstitial pneumonitis, diffuse alveolar damage, and fibrotic scarring (120) . they identified a biphasic cellular immune response in which cytokines (tnf-α and il-6) and chemokines [interferon gamma-induced protein (ip)-10, monocyte chemoattractant protein (mcp)-1, macrophage inflammatory protein (mip)-1a, rantes] were produced early, likely by infected airway epithelial cells, alveolar macrophages, and recruited inflammatory monocyte-macrophages and neutrophils, which have been shown to replace resident alveolar macrophages (19, 142) . sars-cov-1 and mers-cov encode structural and non-structural proteins that antagonize the interferon response, which may initially delay the innate immune response but eventually potentiate inflammatory monocyte-macrophage responses (19) . in covid-19 patients, liao et al. reported increased lung infiltration by macrophages identified via rnaseq analysis of bronchoalveolar lavage fluid. patients with mild cases exhibited infiltration by alveolar macrophages [fatty acid binding protein (fabp)4 + ] while patients with severe ards exhibited infiltration by highly inflammatory [ficolin (fcn1) + ] monocyte-derived macrophages (128) . in the sars-cov-1 pneumonia model, the first wave of cytokines and chemokines induced an accumulation of nk cells, as well as plasmacytoid (p)dcs, macrophages, cd4 + t cells and nkt cells in the lungs. a second wave of inflammatory mediators was detected later on day 7 post-infection [cytokines tnf-α, il-6, ifn-γ, il-2, il-5, and chemokines mcp-1, mip-1a, rantes, monokine induced by gamma interferon (mig), ip-10] and correlated with lung infiltration of t cells and neutrophils (120) . these findings are consistent with studies that have shown increased levels of activating and inhibitory cytokines and chemokines in the blood and lungs of sars patients, as well as histological studies of sars and mersinfected lungs which show extensive cell infiltrates (19, 29, (143) (144) (145) . when huang et al. investigated the cytokine/chemokine profile in the acute phase of sars infection in a cohort of taiwanese patients, they observed an ifn-γ-led cytokine storm (138) . they assessed sera from hospitalized patients prior to the administration of immunomodulators and found significantly increased levels of ifn-γ, il-18, ip-10, mcp-1, mig, and il-8 (138) , which returned to basal levels in convalescent sera. ip-10, mig, mcp-1, and il-18 levels were all significantly increased in death vs. survival groups. interestingly, they found an inverse relationship between ifn-γ levels and lymphocyte numbers and suggested this could either be due to ifn-γ-induced lymphocyte apoptosis or sequestration of chemokine-recruited lymphocytes in the lungs (138) . indeed, this hyper-cytokinemia has been consistently observed in sars-infected patients (146) . however, a recent study found that levels of six pro-inflammatory cytokines (il-1b, il-1ra, il-6, il-8, il-18, and tnf-α) implicated in the cytokine storm in covid-19 patients did not differ significantly from levels in cytokine storms caused by other conditions. they suggest that it is therefore possible that increased levels of proinflammatory cytokines in the context of severe covid-19 may simply reflect an increased viral burden rather than an exuberant immune response and suggest that immunotherapies should therefore be used with caution (147) . altogether these studies show that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce cytokines and chemokines that induce the activation and migration of lymphocytes, including nk cells, to the lungs, where they could be one of the main producers of ifn-γ (148). under normal conditions, human lung nk cells are typically hyporesponsive but dynamically migrate in and out of pulmonary tissues (83) . this supports the hypothesis that during infectious respiratory diseases, an increased recruitment of hyperresponsive nk cells would worsen the festering immunopathology (8) . in fact, through viral-track scanning of unmapped single-cell rnasequencing data, bost et al. showed that patients with severe covid-19 exhibited a hyperinflammatory response with an enriched and highly proliferative nk cell compartment (142) . high levels of ifn-γ leads to epithelial and endothelial cell apoptosis and vascular leakage, suboptimal t cell response, accumulation of alternatively activated macrophages and altered tissue homeostasis, and ards (19) , all of which may contribute to covid-19 disease severity. in summary, the evidence is consistent with the hypothesis that nk cells are involved in the cytokine storm associated with cov infection and that this hyper-cytokinemia contributes significantly to disease severity via inflammation-mediated lung damage (figure 1) . interestingly, this duality of nk cell roles mirrors what is seen in critically ill patients with sepsis. studies suggest that while early nk cell stimulation and ifn-γ production is beneficial to combat infections, excessive and prolonged stimulation of nk cells leads to reduced nk cell numbers and an exhausted phenotype and was associated with increased systemic inflammation in systemic inflammatory response syndrome (sirs)/sepsis and increased mortality (149) (150) (151) (152) . this review of the literature suggests that nk cells may play an important role in both cov clearance and immunopathology. the continued probing of nk cell involvement is essential for a more complete understanding of cov pathophysiology and for the deployment of immunotherapeutics. depending on the patient, the stage of disease, and other still poorly understood factors, it may be necessary to either boost nk cell activity to ensure viral clearance, e.g., at exposure or during early infection, or to finely tune nk cell effector functions in late stage infections to prevent hyper-cytokinemia and inflammatory lung damage. indeed, all covs that infect humans are zoonoses and there is an extensive reservoir of covs that could serve as a source for future pandemics (14, 153) . therefore, a broader understanding of the immune response to coronaviruses and insights into therapeutic implications will be of significant value not only for the current covid-19 pandemic, but also for potential future pandemics. the race to vaccinate and find a cure for covid-19 has resulted in a spectacular effort from researchers and medical practitioners around the world. early attempts at creating targeted therapeutics have mostly relied on historical evidence from related, but not identical, coronaviruses and on the paucity of studies investigating sars-cov-2. these strategies have attempted to combat the virus by targeting various stages of its life cycle starting with neutralizing sars-cov-2 virions using monoclonal antibodies or plasma from convalescent patients (154) . the entry mechanism of covs has been shown to rely on binding the ace2 receptor and using proteases such as tmprss2 for s protein priming (52) . thus, preventing ace2 receptor binding through blocking antibodies or competitive binding with soluble ace2 and tmprss2 protease inhibitors (camostat mesylate) are being tested (155) . upon viral entry, the viral proteolysis or replication cycle can be targeted with protease inhibitors (lopinovir and ritonavir) (156) or rna-dependent rna polymerase inhibitors (remdesivir and ribavirin) (157) . at the time of writing this review, the results of these trials have not been released or are still preliminary and will require further evaluation to assess their clinical efficacy in larger cohort studies. as nk cell activity is critical for viral clearance and may be involved in disease immunopathology, a rapid and reliable predictor of nk cell function may allow for the prediction of clinical progression and the stratification of patients to receive therapeutic intervention. the remainder of this review will discuss the various ways immunotherapies are being deployed to tackle covid-19, with a focus on therapies that use nk cells (table 1) . lastly, while nk cells play an important role in combating viral infections, we also need to be fully cognizant of the potential damage immunotherapies could have in severe cases of covid-19, and how these adverse effects may need to be attenuated ( table 2) . in the absence of a clinically approved vaccine against sars-cov-2, scientists have begun developing therapeutics to halt the spread of covid-19 by alternative strategies. studies have reported that patients infected with sars-cov-2 have lower levels of circulating nk cells and these express a greater level of inhibitory receptors (e.g., nkg2a) while producing less ifn-γ (127, 129, 130) . these findings provide a rationale for pursuing nk cell-based therapies as a tool to fight covid-19. although nk cell-based therapies have mostly been developed for use against cancer, similar concepts and mechanisms could provide guidance in the fight against viruses. therapeutic nk cell products can be thought of as "living drugs" as they generally use either primary nk cells isolated from peripheral blood mononuclear cells (pbmcs) or are generated from stem cell precursors or genetically engineered immortalized human nk cell lines (158) . primary nk cell products are often pre-treated and expanded in vitro with cytokines or via co-culture with target cells before being infused into patients. patients can also receive immune stimulants [e.g. recombinant il-2 (159) or il-15 (160) ] with the goal of improving the in vivo activity and persistence of the nk cell products (161) as is being tested in this covid-19 trial (nct04344548). the first cell-based investigational drug to be approved by the fda for clinical testing in covid-19 patients is an allogeneic, off-the-shelf, cryopreserved nk cell therapy made by celularity (cynk-001), originally developed for cancer immunotherapy (162) . the trial (nct04365101) is split into two phases. phase i will assess the frequency and severity of adverse events in mild, non-icu covid-19 patients (n = 14) following infusion of nk cells derived from placental cd34 + cells. the subsequent phase ii trial will recruit up to 72 patients and include a standard of care comparator at a 1:1 allocation. genetically modified nk cells are also being investigated for efficacy against covid-19. chimeric antigen receptor nk cells (car-nk cells) are engineered to express virtually any receptor(s) of interest and were originally designed to enhance the ability of nk cells to eliminate cancer cells via receptors targeting egfr (163) or cd19 (164) , which are present on many cancer types and b cell hematological malignancies, respectively (164) . although the efficacy of car-nk cells to control viral infections has yet to be rigorously tested in large scale clinical trials, the promising safety profile of car-nk cells in cancer patients, who are often immunocompromised, suggests that car-nk therapy can be well-tolerated in early phase/mild covid-19 patients. notably, car-nk cells are considered "safe" largely because they are less likely to lead to cytokine release syndrome (crs), a severe adverse event of car-t cell therapy (165) . but as these are unchartered waters, it is critical that car-nk cells are used cautiously and not given to late/severe covid-19 patients. a phase i/ii study in early stage covid-19 patients (within 14 days of illness) employing car-nk cell therapy is currently being tested using off-the-shelf nk cells derived from human umbilical cord blood expressing nkg2d and ace2 cars (nct04324996). this complex five-arm study will compare the efficacy of different car-nk constructs: (i) nk cells, (ii) nk cells secreting il-15, (iii) nkg2d car-nk cells, (iv) ace2 car-nk cells, and (v) nkg2d-ace2 car-nk cells. nkg2d car-nk cells have shown promising preclinical results in cancer studies (166) , and although not proven for sars-cov-2, the rationale for expressing nkg2d derives from work showing that nkg2d-ligands (nkg2dl) are upregulated on virally infected cells (167) . similarly, the investigators hypothesize that expressing ace2 on nk cells will facilitate the elimination of sars-cov-2 virions and infected cells by binding the viral spike proteins-but it is unknown whether or not car-nk cells can eliminate virions or if infected cells display sufficient levels of spike protein to be recognized by ace2-nk cells upon viral infection. the investigators also suggest that expressing ace2 on nk cells may also have a secondary benefit as a decoy cell that will be infected by the virus thereby indirectly protecting lung epithelial cells. as described previously, it is unclear whether this strategy will work to stop viral spread to healthy epithelial cells or if it will serve to perpetuate viral spread if the virus can replicate in nk cells. in arms ii-v of this trial, the car-nk cells have been engineered to secrete il-15 based on studies showing improved in vivo persistence of car-nk cells in cancer patients (168) . however, the addition of the proinflammatory cytokine il-15 to this treatment strategy should be monitored closely for life-threatening toxicities, as elevated il-15 has been previously reported to accompany chronic pulmonary inflammatory diseases (169) and mers-cov infection (170) even if no correlation has been reported for sars-cov-2. interestingly, a study compared il-15 levels from lung tissue homogenates following sars-cov infection in aged vs. juvenile monkeys and showed that il-15 concentrations were only elevated in juvenile monkeys 10 days post-infection (171) . this study would suggest that il-15 therapy may be tolerated and effective in older covid-19 patients that may not be able to produce il-15, however this has not been confirmed. lastly, all the car-nk cells in this trial secrete gm-csf neutralizing scfv antibodies, since this cytokine has a known role in crs in cancer patients treated with car-t cells (172) , and has been shown to be correlated with covid-19 disease severity in association with pathogenic cd4 + th1 cells (173) . although nk cell based therapies are versatile, have shown safety and efficacy in cancer patients, and can be utilized in immunocompromised individuals, their potential has yet to be fully realized as an antiviral therapy. furthermore, the logistics of manufacturing nk cell products (cost and time) may pose limitations and barriers to access. for this reason, therapies focused on stimulating a patient's own nk cells offer many advantages over adoptive transfer of nk cells. the importance of the interferon pathway is underscored by the fact that many viruses actively interfere with host interferon responses, for which coronaviruses are a prime example. as described above, covs utilize numerous tactics to avoid elimination by disrupting the host type i ifn response (174) . therefore, since the majority of covs fail to induce any detectable type i ifn response, eliciting a type i ifn response is a very attractive therapeutic strategy (118, 175) . given the robust immunomodulatory nature of type i ifns, uninfected or early symptomatic patients would benefit the most from this therapy to prevent exacerbating immunopathology at later stages of disease. numerous clinical trials have been initiated investigating type i ifns ( table 1) . a large study (nct04320238) of ∼3,000 medical staff allocated participants to two trial arms: (i) low-risk (non-isolated wards or laboratories) or (ii) highrisk (isolated wards in direct contact with covid-19 patients). in addition to the ifn-α-1b nasal drops, high-risk medical staff will also receive the immune-modulating tlr activator, thymosin α1, which indirectly activates nk cells through pdcs (176, 177) . interestingly, reports in sars-cov-1 studies showed that ifn-β therapy had a 50-fold greater anti-viral activity in vero cells than ifn-α treatment (178) . promising results have been published from a phase ii study (nct04276688) (179) , showing that complementing lopinavir-ritonavir and ribavirin with subcutaneous ifn-β-1b in mild-to-moderate covid-19 patients is safe with no serious adverse events reported in the triple combination therapy group, and highly effective, with significant and clinically meaningful reductions in time to complete alleviation of symptoms, hospital length of stay, and time to negative viral load (179) . despite our best efforts in timing type i ifn therapy to mitigate immunopathology, these treatments still increase the risk of excessive activation of proinflammatory signals, which could damage host tissues and perpetuate immunopathology (180, 181) . for this reason, alternative therapeutic avenues to direct type i ifn administration are being explored. type iii ifns can be a valid alternative to type i ifns, because they maintain antiviral functions yet are less toxic and less prone to mediate immunopathology (182) . the type iii ifn, ifn-λ, activates nk cells indirectly (compared to type i ifns which directly act on nk cells), resulting in a less potent and slower immune response (183, 184) . ifn-λ activates nk cells by stimulating macrophages to produce il-12 which in turn induce nk cells to produce ifn-γ (185) . pegylated ifnλ is being tested in covid-19 positive patients with mild symptoms in the absence of respiratory distress (nct04331899). while ifn-λ can lead to the eventual activation of nk cells, its primary utility is in preventing the tissue damaging potential of neutrophils at mucosal surfaces, such as the lungs. however, ifn-λ also has been shown to reduce the rate of tissue repair, which in the context of covid-19 which has a long disease course, could mean greater risk of secondary infections. since exogenous administration of any ifn therapy poses the risk of tipping the balance toward severe covid-19 immunopathology, broggi et al. assessed the levels of ifns in upper and lower respiratory samples from healthy and covid-19 patients. in this preprint, they report that while the upper airway swabs showed similar mrna expression levels of type i and iii ifn compared to healthy controls, the balf samples of severe covid-19 patients had significantly elevated type i and iii ifn levels (186) . therefore, as with all of the therapies discussed in this review, careful consideration about safe and effective timing should guide our design of clinical trials. in addition to ifn cytokine therapy, interleukin cytokine therapy can enhance the effector functions of nk cells (158) . the use of whole, unmodified recombinant cytokines as a monotherapy has resulted in minimal success in humans in cancer immunotherapy. the earliest cytokine therapies to gain fda-approval were ifn-α and recombinant il-2, approved for renal cell carcinoma and metastatic melanoma (187) . although approved, they were limited by their in vivo half-life, marginal anti-tumor activity, and associated toxicities. the next generation of cytokine therapies were created to address these issues by first improving their biological stability through pegylation and fusion to chaperone molecules and secondly improving their specificity by fusing cytokines with antibodies or intratumoral administration. these advances in the field have allowed for the reassessment of the therapeutic potential of specific cytokines (187) . given the importance of il-15 signaling and nk cell function, researchers have developed il-15 "superagonists" which are il-15:il-15r heterodimers that have better in vivo stability and bioactivity compared to monomeric il-15 (168) . although at the time of writing il-15 superagonists are not being studied for their efficacy in covid-19 patients, il-15 superagonists, such as alt-803, are safe in humans (188) and have been used in conjunction with many of the therapies being discussed in this review including: car-nk cell therapy, adoptive nk cell transfers, checkpoint inhibitors, and the bcg vaccine in cancer (189). it should be noted that although the therapeutic potential of cytokine therapy to specifically stimulate nk cells is enticing, exogenous cytokine therapy has a high risk for exacerbating crs if given at the incorrect time. some viruses are known to induce a state of functional hyporesponsiveness in t cells that is essential for the productive establishment of chronic viral infections (190) . a vast body of literature has identified inhibitory checkpoint receptors, including ctla4 and pd-1, as key regulators of this process (191) . interestingly, cancer exploits similar mechanisms to escape the immune response, which provided the rationale for the introduction of antibodies targeting checkpoint receptors for cancer immunotherapy (192) . ctla4 and pd-1/pd-l1 blockade have revolutionized cancer immunotherapy, and their success provides a strong rationale for the use of these drugs in covid-19 patients, where emerging evidence suggests that the immune response is also subverted. a clinical trial (nct04268537) is currently assessing the efficacy of pd-1 blocking antibodies in severe covid-19 patients within 48 h of reported respiratory distress. pd-1 has also been shown to play a role in regulating nk cell responses, in addition to modulating t cell functions (193) (194) (195) (196) (197) , and has been reportedly increased in covid-19 patients (129) . inhibitory receptors on the surface of nk cells regulate nk cell activation and can be targeted by antibody therapy. one of the most promising is certainly the inhibitory receptor nkg2a, which binds to hla-e (74, 198, 199) . nkg2a expression is increased in circulating (127) and balf nk cells from covid-19 patients, in contrast to nkg2c, an activating receptor closely related to nkg2a, which remains unchanged (129) . however, it is unclear whether the observed increase in nkg2a + nk cells is due selective proliferation of nkg2a + cells or if it is the result of nkg2a negative cells migrating out of circulation to infected tissues. circulating nk cells from patients with active hepatitis b disease had higher levels of nkg2a compared to patients without active disease, however antiviral administration was associated with a reduction in nkg2a expression. additionally, blocking nkg2a in vitro with nkg2a monoclonal antibodies led to improved nk cytotoxicity (200) . given the association between nkg2a expression in patients with severe covid-19 (127, 201) , a promising avenue of investigation would be anti-nkg2a therapy, even in light of results showing that nkg2a + nk cells are tuned to present a higher level of responsiveness to stimulation (202) . while nk cells can be stimulated directly by cytokines such as interferons and interleukins, their activity can also be enhanced through a by-stander effect following stimulation of other innate immune cells, such as macrophages and pdcs ( table 1 ). this type of coordinated innate immune response may be more effective at cov viral clearance and mitigation of severe covid-19. trained immunity has been recently described as an epigenetic re-wiring occurring in myeloid cells and progenitors upon stimulation that primes for a stronger response to subsequent stimuli, even of a different nature (90, 203, 204) . whereas, the consensus is that myeloid cells are primarily responsible for trained immunity (205) , it is likely that the resulting alteration in the cytokine milieu also has an effect on nk cells (204, 206) . this is the case for the bcg vaccine, which has been shown to provide non-specific protection against yellow fever viral infection (90, 207, 208) . the bcg vaccine is composed of a live attenuated strain of mycobacterium bovis originally given to young children to protect against tuberculosis (m. tuberculosis) (209) . this vaccine provides an initial boost to innate immunity, but more importantly, results in the secretion of il-1β from monocytes/macrophages, which feeds back to further stimulate the innate response (204) . the use of a heterologous vaccine to provide enhanced protection against non-specific/new pathogens makes this a compelling strategy against covid-19 that warrants thorough investigation in randomized controlled trials (209, 210) . the bcg vaccine is undergoing clinical trials in healthcare workers in the netherlands (nct04328441), australia (nct04327206), egypt (nct04350931), and the usa (nct04348370) to enhance overall innate immunity and provide heterologous protection against sars-cov-2. interestingly, an association was found that linked lower covid-19-attributable mortality rates in countries using bcg in their national immunization schedules (211) . on the contrary, a study that assessed the association of childhood bcg vaccination in adults living in israel did not show a beneficial difference in covid-19 infection rates. the discrepancy between these two reports likely stem from the fact that the latter study only included adults who were previously vaccinated during childhood, supporting the fact that heterologous vaccination may not result in long-term protection (212) . childhood bcg immunization has a limited window of opportunity to protect younger individuals from infection (213) , but it is hypothesized that reducing the number of infected children can have a meaningful impact on curbing the spread of covid-19 to the rest of the population (206, 211) . another heterologous vaccine in the process of clinical trial development for covid-19 studies is imm-101 (cctg id# ic8). created by immodulon therapeutics ltd, imm-101 is composed of heatkilled mycobacterium obuense and may have an improved safety profile over the bcg vaccine (214) . imm-101 has been studied in multiple clinical trials for its non-specific immune stimulating properties as a cancer immunotherapy in pancreatic (215) and melanoma patients (216, 217) . agonists of toll-like receptors (tlrs) have been shown to broadly activate different immune populations and have had both preclinical and clinical success as adjuvants in vaccination and in the treatment of a variety of viral pathogens (218) . for example, cpg oligodeoxynucleotides (cpg odns) are short dna sequences that contain unmethylated cpg dinucleotides which activate tlr9 particularly on dcs and b cells (219). bao et al. showed that their cpg odn construct, bw001, had protective effects against sars-cov-1 in a mechanism that relied on nk cell activation likely through a dc intermediate (220) . amidst the ongoing sars-cov-2 pandemic, two clinical trials (nct04313023, nct04312997) have opened using the tlr2/6/9 agonist, pul-042, in order to prevent infection. ascorbic acid, more commonly known as vitamin c, has been shown to exhibit potent immunomodulatory, antioxidant, and antimicrobial effects (221) . vitamin c has been shown to restore nk cell cytotoxicity in individuals exposed to toxic chemicals through protein kinase c expression, a critical component in lymphocyte metabolism (222) . additional reports have shown that vitamin c also enhances the expression of nkp46, cd69, cd25 and ifn-γ production by nk cells (223) and can increase the expression of irf3 in lung tissues of influenza infected, pneumonia-induced mice (224) . vitamin c also harbors potent antioxidant attributes which can scavenge reactive oxygen species (ros) and prevent lung injury (225, 226) . although ros production is an important component in the host defense response to viruses, they can be harmful to cells and lead to the pathogenesis of viral-induced host injury (227) . the underlying rationale to investigate the therapeutic potential of vitamin c has been based on two key observations: (i) critically ill patients have lower levels of vitamin c (228) (229) (230) and (ii) vitamin c has pleiotropic immunomodulatory, antioxidant, and antiviral effects (221) . it is important to underscore that reports on the clinical outcomes of vitamin c treatment in humans are mixed and context dependent. a thorough metaanalysis on vitamin c supplementation for the common cold has been reported by hemilä and chalker (231) . briefly, they concluded that while the incidence of colds was not reduced, the duration and severity of colds was reduced when assessing studies of regular vitamin c intake (231). interestingly, a separate metaanalysis on vitamin c and cardiac surgery showed a reduction in the length of icu stay and shortened the need for mechanical ventilation (232) . this is an important correlation as clinical trials are currently investigating the efficacy of vitamin c to reduce mortality and hospital burden in covid-19 patients ( table 1) . a phase ii clinical trial (nct04264533) was initiated in wuhan where covid-19 patients will be given a high dose intravenous infusion of vitamin c. lastly, whether oral dosing of vitamin c can achieve therapeutically relevant concentrations, as described in the above studies, is currently unknown, thus caution should be taken as exceeding the recommended dietary allowance of 100-200 mg/day may lead to mild toxicities including abdominal discomfort and diarrhea (231, 233). the main cause of death for covid-19 patients has been pulmonary complications and respiratory failure often as a result of an unregulated cytokine storm (234) . it is unclear whether the hyperinflammation seen in severe cases of covid-19 is the result of the viral replication within pulmonary epithelial cells or an overactive/avalanching immune response. however, studies in sars-cov-1 reported hyperinflammation in later stages of disease progression, despite reduced viral titers, suggesting that the damage was immune-mediated (19) . the most appropriate course of therapy can only be determined by elucidating the pathophysiology of disease progression. scientists and physicians, however, have had to respond quickly to the growing number of severe covid-19 cases and this has resulted in therapy mainly through a combination of anti-inflammatory and anti-viral interventions ( table 2) . as described above, there is a potential for nk cells to contribute to the cytokine storm and therefore the development of ali. a possible explanation for the observed lymphopenia in covid-19 patients is that nk cells and other lymphocytes migrate out of the circulation and into pulmonary tissues to aid in the elimination of infected epithelial cells (235) . this could be the premise for the large, unintended, amount of tissue damage that worsen the respiratory distress (148). for this reason, therapeutics that dampen the immune response have been effective in mitigating immunopathology in severe covid-19 patients. the following review papers have thoroughly discussed many of these immunotherapies already (236) (237) (238) (239) (240) (241) , therefore, this section will focus on immunotherapies and their potential implications on nk cells. the main cytokines responsible for the life threatening respiratory distress seen in reported cases of severe covid-19 are il-2, il-6, il-7, il-10, g-csf, ip-10, mcp-1, mip1a, and tnf-α (234) . many clinical trials have focused on targeting il-6 signaling with anti-il-6r monoclonal antibodies (e.g., tocilizumab, sarilumab, siltuximab) because of the important role il-6 has in propagating crs (242) . tocilizumab, in particular, is being used as the primary therapy in the majority of these trials, likely owing to its fda approved status as a therapeutic for crs in car-t cell therapy (243) . a case report demonstrated the potential for tocilizumab therapy in treating severe covid-19 illness, where a single dose on day 24 of symptoms led to progressive reduction in il-6 levels and resolution of symptoms (244) . a phase iii study (nct04320615) led by hoffman-la roche is recruiting patients to study the safety and efficacy of tocilizumab therapy in a randomized, double-blind, placebocontrolled, multicenter study in over 300 patients with severe covid-19 pneumonia ( table 2) . targeting the il-6 axis in severe covid-19 patients may also serve to improve nk cell functions as cifaldi et al. showed that increased il-6 negatively impacts nk cell function (245) . they also showed that tocilizumab treatment improved nk cell function in vitro (245) . mazzoni et al. recently reported that serum il-6 levels were inversely correlated (p = 0.01) with nk cell function in covid-19 icu patients. additionally, in a small subset of covid-19 icu patients (n = 5), nk cells displayed improved markers of activation (granzyme a and perforin) after tocilizumab treatment (246) . similar therapies have emerged in the fight against covid-19 including an il-1r antagonist (anakinra; nct04330638) (247) and cytosorb (nct04324528) (248) . high dose anakinra therapy has shown promising safety and efficacy in a small retrospective study, as part of the covid-19 biobank study (nct04318366) (247) . cytosorb therapy is used in conjunction with conventional dialysis through a whole blood cartridgebased filtration system designed to remove middle molecular weight molecules (which include inflammatory cytokines <75 kda) through extracorporeal cytokine adsorption (248) . it is reported to be effective at removing ferritin and il-6 in a case study of a 14-year-old with severe crs following car-t cell therapy (249) . jak1/2 inhibitors (jaki) are also undergoing clinical trials in moderate-severe covid-19 patients, such as baricitinib (nct04320277). in addition to their ability to impede the production of il-6, thus curb the excessive inflammation, they may also block clathrin mediated endocytosis-indicating a dual role for jaki (241) . however, jaki can also lead to the transient increase in nk cells as shown in baricitinib treated rheumatoid arthritis patients (250) , which could be detrimental for severe covid-19 patients. corticosteroids have played a key role in the treatment of auto-immune diseases over the past 70 years (251, 252) . whether endogenous or exogenous, corticosteroids decrease the number of circulating monocytes and lymphocytes and decrease synthesis of pro-inflammatory cytokines (il-2, il-6, tnf-α) (251) . their strong anti-inflammatory and immunosuppressive effects make them good candidates for rapidly suppressing inflammation during early auto-immune disease or viral infections. corticosteroids have been shown to inhibit nk cells in ex vivo experiments (253, 254) . while corticosteroids may delay clearance of infections, their major benefit lies in suppressing excessive innate immune responses, thus preventing lung damage and ards commonly present in severe viral infections (255) (256) (257) . in fact, this was the main rationale for the widespread use of corticosteroids during mers and sars infections (255, 256) . specific to covid-19, some groups have advocated for the use of low-dose corticosteroids in a specific subset of critically-ill patients with refractory ards, sepsis, or septic shock ( table 2 ) (257) . there is one known ongoing randomized clinical trial examining the effect of the corticosteroid ciclesonide in adults with mild covid-19 infections (nct04330586). this trial is based on preclinical studies showing in vitro antiviral activity of ciclesonide against sars-cov-2. while there may be a benefit to using corticosteroids in a subset of critically-ill patients with refractory ards or sepsis (257) , their routine use in covid-19 is not recommended outside of clinical trials, based on expert opinion and who recommendations (258) (259) (260) . corticosteroids also cause a multitude of side effects, most notably diabetes mellitus, osteoporosis, and increased risk of infections (251) . controversially, a 2019 systematic review of over 6,500 influenza patients showed that corticosteroids actually led to increased mortality, length of icu stay, and secondary infections (261) . additionally, one retrospective observational study examined the use of corticosteroids in 31 covid-19 patients, and reported no significant association between corticosteroids and viral clearance time, hospital length of stay, or duration of symptoms (262) . these studies highlight the need to be vigilant in our attempts to fight covid-19. healthy, uninfected individuals, who are at a high risk of becoming infected (through situational circumstances such as healthcare workers) would be most fit and suitable to receive investigational prophylactic therapies such as exogenous ifns and heterologous vaccines. (b) individuals who have tested positive for covid-19 that are asymptomatic or have mild to moderate disease progression may benefit from receiving investigational immune stimulating therapies, including nk cell-based therapies. it is critical that investigators must be vigilant to assess the safety profile and potential immunopathologies associated with these immunotherapies. (c) in severe covid-19 patients, the most appropriate therapies to investigate would be those that mitigate immunopathologies, such as anti-inflammatory and immunosuppressive therapies. given the relatively low chance of toxicity and the wide range of beneficial immune effects, natural health products such as vitamin c and vitamin d can be suitable for investigation at all categories of covid-19 patients. non-steroidal anti-inflammatory drugs, or nsaids, are one of the most commonly prescribed drugs for treating fever, pain, and inflammation. nsaids include over-the-counter household names such as ibuprofen, naproxen, and aspirin. given the widespread use of these medications it is appropriate that researchers have investigated the potential benefits and harms of nsaids in patients diagnosed with covid-19. thus far, the evidence for using nsaids in the context of covs are mixed and might not be generalizable to all nsaids as reports tended to focus on specific nsaids. these studies also focused on the potential for nsaids to act as an antiviral, with a potential added benefit of being able to treat inflammatory symptoms. one report showed that the nsaid indomethacin could directly inhibit sars-cov replication in vero cell monolayers in a dosedependent manner (263) . the antiviral properties of naproxen have been described in the context of influenza virus (264, 265) and has prompted the initiation of a clinical trial investigating the efficacy of naproxen as a treatment for critically ill covid-19 infected patients (nct04325633). nsaid therapy should be used with caution as they have been shown to interfere with immune responses and ability to produce antibodies, with ibuprofen having the greatest suppressive effect (266) . furthermore, ibuprofen has been reported to increase the expression of the ace2 receptor (267) which could facilitate sars-cov-2 viral entry. this finding should be considered for any current (nct04334629) and potential covid-19 clinical trial assessing ibuprofen therapy. nsaids also have been shown to have a direct suppressive effect on nk cell ifn-γ and tnf-α production (268) which may be beneficial for late stage covid-19 patients. the relevance of nk cells as antiviral first responders is highlighted in patients with nkd and immunocompromised individuals who show increased susceptibility to viral infections. while there is currently little direct evidence to support a role for nk cells in the clearance of sars-cov-2 there is a paucity of research in this field. however, studies in admitted covid-19 patients with mild and severe disease reported a reduction in circulating nk cell levels and function as compared to healthy individuals. furthermore, reduced nk cell levels and function were inversely correlated with disease severity, suggesting that nk cells may be involved in some capacity. one of the potential mechanisms by which nk cells may become hyporesponsive is via sars-cov-2 interference with type i ifn pathways. in investigating the pathogenesis of other cov infections, namely sars and mers, studies suggest that during acute cov infection, inflammatory monocyte-macrophages and neutrophils accumulate in the lungs and produce chemokines and cytokines that induce nk cell migration and activation. as nk cells are one of the main producers of ifn-γ, they may be involved in the ifn-γ-led cytokine storm that is responsible for the induction of inflammation-mediated ali, ards, and subsequent mortality associated with covid-19. inarguably, more research into the role of nk cells in covid-19 is required. despite the knowledge gaps in covid-19 pathophysiology, there has been a surge of clinical trials as the fda continues to fast-track the approval of investigational therapeutics (269). here we have outlined potential therapeutics with a focus on mediating nk cell activity, including prophylactic treatments that could boost innate immunity in addition to therapeutics that could mitigate the immunopathological consequences of covid-19, thereby relieving the burden on our health care systems (figure 2) . rigorous preclinical testing and thoughtfully designed clinical trials will be necessary for the development of robust therapeutics against sars-cov-2. importantly, we must be aware of the potential dangers immunotherapies may have in potentiating cov immunopathology. the fight against covid-19 is not an easy one. as with any novel disease, we will have to rely on incomplete pictures to guide reasoning for appropriate treatment. we believe that immunotherapeutics targeting the innate immune response, and specifically nk cells, have the potential to flatten the curve and will be important instruments in our armamentarium against this pandemic and the next. natural killer cells as an initial defense against pathogens natural killer cell responses to viral infection control of human viral infections by natural killer cells natural killer cells are involved in acute lung immune injury caused by respiratory syncytial virus infection critical role of natural killer cells in lung immunopathology during influenza infection in mice nk cells exacerbate the pathology of influenza virus infection in mice memory cd4 t cell-derived il-2 synergizes with viral infection to exacerbate lung inflammation influenza a virus infection induces hyperresponsiveness in human lung tissue-resident and peripheral blood nk cells natural killer cells contribute to hepatic injury and help in viral persistence during progression of hepatitis b e-antigen-negative chronic hepatitis b virus infection natural killer cells act as rheostats modulating antiviral t cells a novel coronavirus from patients with pneumonia in china practical considerations for performing regional anesthesia: lessons learned from the covid-19 pandemic coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus epidemiology, genetic recombination, and pathogenesis of coronaviruses insights into the recent 2019 novel coronavirus (sars-cov-2) in light of past human coronavirus outbreaks effects of a "new" human respiratory virus in volunteers a new virus isolated from the human respiratory tract a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology world health organization. consensus document on the epidemiology of severe acute respiratory syndrome (sars) who. middle east respiratory syndrome coronavirus (mers-cov). (2020) available online at bovine coronavirus associated syndromes diagnosis of feline infectious peritonitis: a review of the current literature origin and evolution of pathogenic coronaviruses structure, function, and evolution of coronavirus spike proteins receptor and viral determinants of sars-coronavirus adaptation to human ace2 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov severe acute respiratory syndrome from sars to mers: 10 years of research on highly pathogenic human coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats detection of severe acute respiratory syndrome-associated coronavirus in pneumocytes of the lung severe acute respiratory syndrome associated coronavirus is detected in intestinal tissues of fatal cases a cluster of cases of severe acute respiratory syndrome in hong kong lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore lung pathology of fatal severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome cocirculation of three camel coronavirus species and recombination of mers-covs in saudi arabia mers coronavirus outbreak: implications for emerging viral infections epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding covid-19 map. johns hopkins coronavirus resource center available early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study covid 19 and heart failure: from infection to inflammation and angiotensin ii stimulation. searching for evidence from a new disease multiorgan and renal tropism of sars-cov-2 does covid-19 spread through droplets alone? front public health pathogenesis and transmission of sars-cov-2 in golden hamsters high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the protein expression profile of ace2 in human tissues the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? innate immunity to virus infection innate immune evasion by human respiratory rna viruses type i interferons in infectious disease innate immunity in the lungs type-i interferon responses: from friend to foe in the battle against chronic viral infection nk cells and interferons innate or adaptive immunity? the example of natural killer cells natural killer cells: in health and disease controlling natural killer cell responses: integration of signals for activation and inhibition natural killer cells in immunodefense against infective agents licensed and unlicensed nk cells: differential roles in cancer and viral control nkp44 and natural cytotoxicity receptors as damage-associated molecular pattern recognition receptors nkg2d: a master regulator of immune cell responsiveness dnam-1: would the real natural killer cell please stand up! oncotarget cloning of immunoglobulin-superfamily members associated with hla-c and hla-b recognition by human natural killer cells molecular cloning of nkb1. a natural killer cell receptor for hla-b allotypes molecular clones of the p58 nk cell receptor reveal immunoglobulinrelated molecules with diversity in both the extra-and intracellular domains hla-e binds to natural killer cell receptors cd94/nkg2a, b and c mouse cd94/nkg2a is a natural killer cell receptor for the nonclassical major histocompatibility complex (mhc) class i molecule qa-1(b) tlr-mediated activation of nk cells and their role in bacterial/viral immune responses in mammals peptide-specific recognition of human cytomegalovirus strains controls adaptive natural killer cells ly49h signaling through dap10 is essential for optimal natural killer cell responses to mouse cytomegalovirus infection human nk cells and herpesviruses: mechanisms of recognition, response and adaptation cytokine regulation of natural killer cell effector functions coordinated and distinct roles for ifn-αβ, il-12, and il-15 regulation of nk cell responses to viral infection developmental and functional control of natural killer cells by cytokines. front immunol human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional cd69-cd56dim cells characteristics of nk cell migration early after vaccinia infection the trafficking of natural killer cells keeping nk cells in highly regulated antiviral warfare human nk cell lytic granules and regulation of their exocytosis natural killer cells: development, maturation, and clinical utilization natural killer cell memory in infection, inflammation and cancer about training and memory: nk-cell adaptation to viral infections is there natural killer cell memory and can it be harnessed by vaccination? nk cell memory and immunization strategies against infectious diseases and cancer elusive role of the cd94/nkg2c nk cell receptor in the response to cytomegalovirus: novel experimental observations in a reporter cell system ly49c-dependent control of mcmv infection by nk cells is cis-regulated by mhc class i molecules activating kirs and nkg2c in viral infections: toward nk cell memory? front immunol cytokine-induced memory-like natural killer cells natural killer cell deficiency severe herpesvirus infections in an adolescent without natural killer cells infections in cancer patients with solid tumors: a review the cancer patient supportive care cancer treatment research elevated tgf-beta1 secretion and down-modulation of nkg2d underlies impaired nk cytotoxicity in cancer patients preventing postoperative metastatic disease by inhibiting surgery-induced dysfunction in natural killer cells cytokine therapy reverses nk cell anergy in mhc-deficient tumors natural killer cell ifnγ secretion is profoundly suppressed following colorectal cancer surgery respiratory viral infections in patients with cancer or undergoing hematopoietic cell transplant clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan, china cancer patients in sars-cov-2 infection: a nationwide analysis in china impact of aging on viral infections age-dependent susceptibility to a viral disease due to decreased natural killer cell numbers and trafficking changes in the expression of surface receptors on lymphocyte subsets in the elderly: quantitative flow cytometric analysis the effect of ageing on human lymphocyte subsets: comparison of males and females age-related changes in the natural killer cell response to seasonal influenza vaccination are not influenced by a synbiotic: a randomised controlled trial dysregulation of natural killer cells in obesity obesity could shift severe covid-19 disease to younger ages covid-19) how is immunosuppressive status affecting children and adults in sars-cov-2 infection? a systematic review middle east respiratory syndrome and severe acute respiratory syndrome severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4+ t cells are important in control of sars-cov infection prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice mice susceptible to sars coronavirus a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice mouse model of sars-cov-2 reveals inflammatory role of type i interferon signaling. biorxiv the pathogenicity of sars-cov-2 in hace2 transgenic mice a mouse-adapted sars-cov-2 model for the evaluation of covid-19 medical countermeasures. biorxiv functional exhaustion of antiviral lymphocytes in covid-19 patients single-cell landscape of bronchoalveolar immune cells in patients with covid-19 identification of immune checkpoints in covid-19 a single-cell atlas of the peripheral immune response to severe covid-19. medrxiv the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome dynamics of peripheral blood b lymphocytes and natural killer cells in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome (mers): a new zoonotic viral pneumonia hematologic, hepatic, and renal function changes in hospitalized patients with middle east respiratory syndrome coronavirus the innate immune system: fighting on the front lines or fanning the flames of covid-19? an interferongamma-related cytokine storm in sars patients circulating growth/differentiation factor 15 is associated with human cd56 natural killer cell dysfunction and nosocomial infection in severe systemic inflammation association between natural killer cell activity and colorectal cancer in high-risk subjects undergoing colonoscopy multiple organ infection and the pathogenesis of sars host-viral infection maps reveal signatures of severe covid-19 patients chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4 t cells are important in control of sars-cov infection elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients cytokine profile in plasma of severe covid-19 does not differ from ards and sepsis. medrxiv the role of natural killer cells in sepsis phenotype and functions of natural killer cells in critically-ill septic patients the functional role of natural killer cells early in clinical sepsis the biology of natural killer cells during sepsis molecular evolution of human coronavirus genomes treatment with convalescent plasma for critically ill patients with sars-cov-2 infection angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target race to find covid-19 treatments accelerates tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study nk cells for cancer immunotherapy expansion of highly cytotoxic human natural killer cells for cancer cell therapy first-in-human trial of rhil-15 and haploidentical natural killer cell therapy for advanced acute myeloid leukemia cancer immunotherapy based on natural killer cells: current progress and new opportunities celularity announces fda clearance of ind application for cynk-001 in coronavirus carengineered nk cells targeting wild-type egfr and egfrviii enhance killing of glioblastoma and patient-derived glioblastoma stem cells use of car-transduced natural killer cells in cd19-positive lymphoid tumors car-nk for tumor immunotherapy: clinical transformation and future prospects nk cells expressing a chimeric activating receptor eliminate mdscs and rescue impaired car-t cell activity against solid tumors natural killer cell specificity for viral infections in vivo activation of human nk cells by treatment with an interleukin-15 superagonist potently inhibits acute in vivo hiv-1 infection in humanized mice expression of il-15 in inflammatory pulmonary diseases altered cytokine levels and immune responses in patients with sars-cov-2 infection and related conditions severe acute respiratory syndrome-coronavirus infection in aged nonhuman primates is associated with modulated pulmonary and systemic immune responses gm-csf inhibition reduces cytokine release syndrome and neuroinflammation but enhances car-t cell function in xenografts pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients sars coronavirus and innate immunity type i interferons and natural killer cell regulation in cancer. front immunol thymosin alpha 1 effects, in vitro, on lymphokine-activated killer cells from patients with primary immunodeficiencies: preliminary results immune modulation with thymosin alpha 1 treatment treatment of sars with human interferons triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial type i interferons in viral control and immune regulation type i and iii interferons disrupt lung epithelial repair during recovery from viral infection. biorxiv shared and distinct functions of type i and type iii interferons ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment interferon-λ mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness ifn-λmediated il-12 production in macrophages induces ifn-γ production in human nk cells type iii interferons disrupt the lung epithelial barrier upon viral recognition. biorxiv alt-803, an il-15 superagonist, in combination with nivolumab in patients with metastatic non-small cell lung cancer: a nonrandomised, open-label, phase 1b trial the potential and promise of il-15 in immuno-oncogenic therapies t cell exhaustion during persistent viral infections fundamental mechanisms of immune checkpoint blockade therapy introduction to checkpoint inhibitors and cancer immunotherapy contribution of nk cells to immunotherapy mediated by pd-1/pd-l1 blockade the diverse function of pd-1/pd-l pathway beyond cancer killers 2.0: nk cell therapies at the forefront of cancer control nk cell-based immune checkpoint inhibition anti-nkg2a mab is a checkpoint inhibitor that promotes anti-tumor immunity by unleashing both t and nk cells nkg2a blockade potentiates cd8 t cell immunity induced by cancer vaccines blocking the natural killer cell inhibitory receptor nkg2a increases activity of human natural killer cells and clears hepatitis b virus infection in mice nkg2a and covid-19: another brick in the wall nk cell self tolerance, responsiveness and missing self recognition trained immunity: a program of innate immune memory in health and disease the role of the interleukin-1 family in trained immunity training the trainable cells of the immune system and beyond bcg-induced trained immunity: can it offer protection against covid-19? bcg-induced trained immunity in nk cells: role for nonspecific protection to infection bcg vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity trained immunity: a tool for reducing susceptibility to and the severity of sars-cov-2 infection considering bcg vaccination to reduce the impact of covid-19 differential covid-19-attributable mortality and bcg vaccine use in countries. medrxiv sars-cov-2 rates in bcgvaccinated and unvaccinated young adults bcg vaccination-induced emergency granulopoiesis provides rapid protection from neonatal sepsis have i found the missing ingredient in a coronavirus vaccine? the daily telegraph randomised, open-label, phase ii study of gemcitabine with and without imm-101 for advanced pancreatic cancer five year survival in patients with metastatic melanoma receiving imm-101 enhanced effect of checkpoint inhibitors when given after or together with imm-101: significant responses in four advanced melanoma patients with no additional major toxicity novel drugs targeting toll-like receptors for antiviral therapy cpg dna as a vaccine adjuvant anti-sars-cov immunity induced by a novel cpg oligodeoxynucleotide vitamin c and immune function enhancement of natural killer cell activity and t and b cell function by buffered vitamin c in patients exposed to toxic chemicals: the role of protein kinase red ginseng and vitamin c increase immune cell activity and decrease lung inflammation induced by influenza a virus/h1n1 infection a new mechanism of vitamin c effects on a/fm/1/47(h1n1) virus-induced pneumonia in restraint-stressed mice vitamins c and e: beneficial effects from a mechanistic perspective role of vitamin c in the function of the vascular endothelium ros signaling in the pathogenesis of acute lung injury (ali) and acute respiratory distress syndrome (ards) vitamin c deficiency in elderly hospitalized patients massive and long-lasting decrease in vitamin c plasma levels as a consequence of extracorporeal circulation hemilä h, chalker e. vitamin c for preventing and treating the common cold vitamin c can shorten the length of stay in the icu: a meta-analysis vitamin c in disease prevention and cure: an overview covid-19: consider cytokine storm syndromes and immunosuppression immunology of covid-19: current state of the science pathological inflammation in patients with covid-19: a key role for monocytes and macrophages covid-19: pathogenesis, cytokine storm and therapeutic potential of interferons the trinity of covid-19: immunity, inflammation and intervention the use of antiinflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china adjunct immunotherapies for the management of severely ill covid-19 patients hijaking sars-cov-2? the potential role of jak inhibitors in the management of covid-19 cytokine release syndrome: current perspectives fda approval summary: tocilizumab for treatment of chimeric antigen receptor t cellinduced severe or life-threatening cytokine release syndrome first case of covid-19 in a patient with multiple myeloma successfully treated with tocilizumab inhibition of natural killer cell cytotoxicity by interleukin-6: implications for the pathogenesis of macrophage activation syndrome impaired immune cell cytotoxicity in severe covid-19 is il-6 dependent interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study hemoadsorption by extracorporeal cytokine adsorption therapy (cytosorb) in the management of septic shock: a retrospective observational study multimodal therapeutic approach of cytokine release syndrome developing in a child given chimeric antigen receptor-modified t cell infusion characterization and changes of lymphocyte subsets in baricitinibtreated patients with rheumatoid arthritis: an integrated analysis glucocorticoids-all-rounders tackling the versatile players of the immune system covid-19 infection and rheumatoid arthritis: faraway, so close effects of corticosteroids on natural killer cell activity in systemic lupus erythematosus distinct effects of dexamethasone on human natural killer cell responses dependent on cytokines. front immunol sars: systematic review of treatment effects corticosteroid therapy for critically ill patients with middle east respiratory syndrome potential benefits of precise corticosteroids therapy for severe 2019-ncov pneumonia clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury covid-19 and treatment with nsaids and corticosteroids: should we be limiting their use in the clinical setting? ecancermedicalscience the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and meta-analysis corticosteroid treatment of patients with coronavirus disease 2019 (covid-19) indomethacin has a potent antiviral activity against sars coronavirus structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza a virus naproxen exhibits broad anti-influenza virus activity in mice by impeding viral nucleoprotein nuclear export ibuprofen and other widely used non-steroidal anti-inflammatory drugs inhibit antibody production in human cells are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? office of the commissioner. coronavirus (covid-19) update: fda continues to accelerate development of novel therapies for covid-19. us food and drug administration the authors would like to thank dr. doug gray for his thorough editing, proofreading, and thoughtful suggestions. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 market, angka, martel, bastin, olanubi, tennakoon, boucher, ng, ardolino and auer. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-326864-i1r3bv4p authors: hon, kam lun; leung, karen ka yan; leung, alexander kc; qian, su yun; chan, vivian py; ip, patrick; wong, ian ck title: coronavirus disease 2019 (covid-19): latest developments in potential treatments date: 2020-06-29 journal: drugs context doi: 10.7573/dic.2020-4-15 sha: doc_id: 326864 cord_uid: i1r3bv4p many viral respiratory infections can cause severe acute respiratory symptoms leading to mortality and morbidity. in the spring of 2003, the severe acute respiratory syndrome (sars) outbreak caused by sars-cov spread globally. in the summer of 2012, the middle east respiratory syndrome (mers) outbreak caused by mers-cov occurred in saudi arabia. in the winter of 2019, the coronavirus disease 2019 (covid-19) outbreak caused by a novel coronavirus sars-cov-2 occurred in china which rapidly spread worldwide causing a global pandemic. up until 27 may 2020, there are 5.5 million confirmed cases of covid-19 and 347,587 covid-19 related deaths worldwide, and there has also been an unprecedented increase in socioeconomic and psychosocial issues related to covid-19. this overview aims to review the current developments in preventive treatments and therapies for covid-19. the development of vaccines for sars-cov-2 is ongoing and various clinical trials are currently underway around the world. it is hoped that existing antivirals including remdesivir and lopinavir-ritonavir might have roles in the treatment of covid-19, but results from trials thus far have not been promising. covid-19 causes a mild respiratory disease in the majority of cases, but in some cases, cytokine activation causes sepsis and acute respiratory distress syndrome, leading to morbidity and mortality. immunomodulatory treatments and biologics are also being actively explored as therapeutics for covid-19. on the other hand, the use of steroidal and nonsteroidal anti-inflammatory drugs (nsaids) has been discouraged based on concerns about their adverse effects. over the past two decades, coronaviruses have caused major epidemics and outbreaks worldwide, whilst modern medicine has been playing catch-up all along. viral respiratory infections such as influenza and measles, which can cause severe acute respiratory symptoms, have been responsible for many epidemics. in the spring of 2003, an outbreak of severe acute respiratory infection (sari) spread globally. 1, 2, 3 the world health organization (who) coined the acronym sars (severe acute respiratory syndrome) for this sari and subsequently named the causative coronavirus sars-cov. in the summer of 2012, another sari broke out in saudi arabia, which was found to be caused by a new coronavirus. the who named this respiratory disease middle east respiratory syndrome also known by the acronym mers and called the causative coronavirus mers-cov. in the winter of 2019, another sari outbreak occurred in wuhan, china, which very quickly spread around the world. the culprit was identified as another novel coronavirus, which the who named as sars-cov-2 due to similarities to sars-cov, and the disease was called coronavirus disease 2019 . sars-cov-2 is a newly emergent coronavirus closely related to sars and mers. 4 covid-19 is a respiratory tract infection that causes mild symptoms in the majority of cases, but can also lead to issn: 1740-4398 review -coronavirus disease 2019 : latest developments in potential treatments drugsincontext.com mortality and morbidity. current reports suggest that covid-19 causes milder symptoms in children, including fever and cough, but co-infection has also been observed. 4, 5 however, covid-19 is associated with severe outcomes in the older population, immunocompromised patients, and those with chronic cardiovascular or respiratory conditions. 4 at present, no pharmaceutical products have been shown to be reliable, safe, and effective for treating covid-19. 6 in the midst of the current global pandemic, many research groups around the world are actively developing treatments against this disease to reduce morbidities and mortalities. in the long term, the goal is to develop a vaccine to prevent further infections and future outbreaks. however, an effective vaccine may take years to develop and to manufacture on a global scale. furthermore, it is unknown if newborns and patients already recovered from covid-19 will need to be vaccinated. it is also uncertain if such a vaccine can protect individuals from this novel coronavirus in the future. in this narrative review, we summarise the latest body of evidence and ongoing research on the development of pharmacological therapies for covid-19. the aim is also to review the suggested pathophysiology, prophylactic treatments, and therapeutic modalities for covid-19. articles were retrieved using pubmed clinical queries with the search term 'coronavirus covid-19' regardless of the date of publication. there were 88 articles under clinical study categories (category: therapy; scope: broad) and 11 systematic reviews. the discussion is based on, but not limited to, these search results. severe acute respiratory syndrome is a viral respiratory disease of zoonotic origin caused by sars-cov. the sars outbreak between november 2002 and july 2003 resulted in 8098 cases and 774 deaths in 29 countries around the world, giving a casefatality rate of 9.6%. 7, 8 treatments for sars during the outbreak were mainly supportive, as there were no known effective antiviral agents. the use of broad-spectrum antibiotics to treat secondary bacterial infections was the main treatment regimen. 9 ribavirin, a broad-spectrum purine nucleoside analogue, was empirically used as a broad-spectrum antiviral agent. 10 human immunodeficiency virus (hiv) protease inhibitor lopinavir/ritonavir were also used, as it was found to have weak in vitro antiviral activity on the prototype sars-cov. 10, 11 other therapies included immunomodulators (e.g. corticosteroid, convalescent plasma, and pentaglobulin), interferons, and traditional chinese medicine (tcm). 9, 12 the development of vaccines was underway by the end of the epidemic, but no effective vaccine has since emerged. middle east respiratory syndrome caused by mers-cov may have been transmitted to humans through infected camels. the mers outbreak between september 2012 and january 2020 was reported to have caused 2519 laboratory-confirmed cases and 858 associated deaths globally, giving a case-fatality rate of 34.4%. 13 as of 2019, there is still no effective vaccine or treatment for this disease, although a number of antiviral medications have been investigated. 14 a 2019 systematic review of therapeutic agents against mers-cov showed that there is still no general consensus on the optimal treatment strategy for mers-cov infection. 15 the miracle trial (mers-cov infection treated with a combination of lopinavir/ ritonavir and interferon-β1b) was the first randomised controlled trial to assess the feasibility, efficacy, and safety of a combination of lopinavir/ritonavir and interferon-β1b in hospitalised patients with mers. 16, 17 the trial was started in july 2016 and enrolled 194 participants, although results have yet to be published. 16 sars-cov, mers, and sars-cov-2 are all zoonotic β-coronaviruses that have crossed from animals to humans. 23 the origin of sars-cov is still a mystery and remains a controversial topic. sars-cov is closely related to civet and bat covs, but it is phylogenetically divergent from other coronaviruses associated with human infections, including issn: 1740-4398 review -coronavirus disease 2019 (covid-19): latest developments in potential treatments drugsincontext.com oc43, nl63, 229e, and hku1. 9 the full-length genome sequence of sars-cov-2 shows that it is similar to sars-cov, sharing 79.6% sequence identity. 24 both sars-cov-2 and sars-cov use the same cellular receptor, angiotensin-converting enzyme ii (ace2) receptor, to enter into host cells. 24 the pathophysiology of covid-19 has yet to be confirmed, but it is likely to involve inflammatory processes that can trigger a massive cytokine storm. the cytokine profile of critically ill patients revealed increased levels of interleukin (il)-2, il-7, il-10, granulocyte-colony stimulating factor, interferon-γ inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1-α, and tumour necrosis factor-α. 25 histopathological examination of the lungs of patients with covid-19 revealed immunopathological changes including diffuse alveolar damage, desquamation of pneumocytes, pulmonary oedema, hyaline membrane formation, and interstitial mononuclear inflammatory infiltrates. 26 according to the limited number of reports of biopsy/autopsy results of patients with covid-19, the pathological features resemble those seen in sars and mers virus infections. [26] [27] [28] the similarities between sars-cov and sars-cov-2 suggest that the development of potential prophylactics and therapeutics for covid-19 could be based on research on sars. 3,12,19,29-,32 an important strategy would be to possibly control the viral replication using an effective antiviral agent to minimise the subsequent inflammation and tissue damage due to high viral loads. immunomodulators could play a rescue therapy role, as pathological findings suggest that there is immunopathological damage. 9, 26 although the sars-cov-2 s protein receptor-binding domain has higher affinity than the sars-cov s protein receptor-binding domain, development of vaccines against sars-cov-2 could still be based on research on sars-cov. [33] [34] [35] therapeutics for covid-19 the majority of covid-19 patients, especially children, are either asymptomatic or have mild symptoms, and will likely recover by managing their own symptoms without the need for hospitalisation. 36 38 the who has also launched the 'solidarity' international clinical trial, which is investigating effective treatments and is currently comparing four of the most promising treatment options: remdesivir, lopinavir-ritonavir, lopinavir-ritonavir plus interferon β-1a, and hydroxychloroquine. 39 over 90 countries have joined the 'solidarity' international clinical trial. 39 in the following discussion, we summarise the latest evidence and research progress from the literature, with focus on pharmacological therapies (table 1) . empirical antimicrobials should be started within 1 hour after the detection of sepsis based on clinical diagnosis and local epidemiology. 4 for critically ill covid-19 patients, intensive care treatment can include oxygen therapy, noninvasive mechanical ventilation, invasive mechanical ventilation, and extracorporeal membrane oxygenation. 40 in addition to the standard management for acute respiratory distress syndrome, the literature also suggests lower intubation threshold, early prone positioning, and cautious fluid status management, considering that the incidence of myocardial dysfunction in covid-19 patients is high. 41 further discussion on the intensive care management of covid-19 patients is beyond the scope of this review. remdesivir remdesivir (gs-5734) is a nucleoside analogue prodrug that inhibits viral rna polymerases, and was developed by gilead science in response to the ebola outbreak in 2017. [42] [43] [44] it is considered to be one of the most promising broad-spectrum antivirals for treating covid-19. in vitro antiviral activities of remdesivir have been demonstrated in sars-cov, mers-cov, and sars-cov-2. 45, 46 in early april, a preliminary report describing the clinical outcomes of a cohort of 53 hospitalised covid-19 patients who received remdesivir, 68% of patients showed improvement of their oxygen-support status, but 60% of patients reported adverse events during follow-up. 42 subsequently, a double-blinded randomised controlled trial in 237 adult patients with severe covid-19 showed that remdesivir was not associated with statistically significant clinical benefits whilst adverse events were reported in 66% of patients. 47 separately, the results of an interim analysis of the adaptive covid-19 treatment trial involving 1063 patients are more encouraging, as it indicated that patients who received redmesivir had a 31% faster time to recovery than those who received placebo (median time to recovery was 11 versus 15 days). 48 more recently, a phase 3 simple clinical trial demonstrated that patients receiving a 5-day and 10-day treatment course of remdesivir achieved similar improvements in clinical status. 49 reported in the preliminary studies included elevated hepatic enzymes, diarrhoea, constipation, hypoalbuminaemia, rash, renal impairment, anaemia, thrombocytopenia, and hypotension. 42, 47 potential drug-drug interactions with other medications metabolised through the cytochrome p450 system were also reported. the safety profile of this drug needs to be further evaluated. randomised controlled trials on remdesivir are still ongoing, and two studies are in phase iii of clinical trials to evaluate the safety and efficacy of remdesivir. [50] [51] [52] [53] lopinavir-ritonavir lopinavir-ritonavir is a combination therapy used to treat hiv. ritonavir is an inhibitor of cytochrome p450 and is used to increase the plasma half-life of lopinavir. 54 lopinavir is a protease inhibitor that has been demonstrated to have antiviral effects against sars, mers-cov, and sars-cov-2 in vitro. 54, 55 several trials have been investigating the efficacy of lopinavir-ritonavir compared with other drugs as a treatment for covid-19. 39,56-58 nevertheless, the results so far indicate that lopinavir-ritonavir treatment has little benefit as a standalone therapy against sars-cov-2 infection. in a clinical trial involving 199 patients with laboratory-confirmed sars-cov-2 infection, lopinavir-ritonavir treatment was not associated with any clinical improvements compared with standard care. 54 furthermore, lopinavir-ritonavir treatment caused gastrointestinal adverse events, and nearly 14% of the recipients could not complete the full 14-day course of treatment. 54 other adverse effects included gastrointestinal intolerance, hepatotoxicity, pancreatitis, and qt prolongation. the use of ritonavir can cause severe drug-drug interactions in medications metabolised through the cytochrome system. the tolerance of the side effects might limit the dosage needed, as the concentration necessary to inhibit viral replication is relatively high. 54 other clinical trials have been testing lopinavir-ritonavir combined with different drugs and different combinations such as ritonavir and interferon 1b. 39, 56 early use of lopinavir-ritonavir as an additional treatment to ritonavir-methylprednisolone for sars-cov was associated with a reduction in the overall death rate and intubation rate when compared with a matched cohort. 59 a clinical phase ii trial has been investigating the use of lopinavir-ritonavir combined with interferon β-1b as a treatment for mers since 2016, but the trial is ongoing and results have yet to be published. 60 ribavirin ribavirin is a guanosine analogue that interferes with the replication of rna and dna viruses. 61 in vitro antiviral activities of ribavirin have been demonstrated in sars-cov and mers-cov. 61, 62 from the experiences of sars-cov, the use of ribavirin as a monotherapy was limited as it required high concentration to inhibit viral replication, and ribavirin usage was associated with dose-dependent haemolysis and liver toxicity. 63, 64 in the treatment of sars and mers, ribavirin is used in combination with interferon or lopinavir/ritonavir. 11, 65 the use of ribavirin in combination with interferon or lopinavir/ ritonavir is recommended in the latest chinese national treatment guidelines for covid-19. 66 a recent multicentre randomised phase 2 trial showed triple antiviral therapy of lopinavir/ritonavir, ribavirin, and interferon β-1b was superior to lopinavir-ritonavir alone in alleviating symptoms, shortening the duration of viral shedding and hospital stay in patients with mild-to-moderate covid19. 67 chloroquine and hydroxychloroquine chloroquine and hydroxychloroquine are widely used for the treatment of malaria and certain autoimmune diseases. these drugs have an established safety profile and are readily available at relatively low cost. hydroxychloroquine sulphate has been demonstrated to be less toxic than chloroquine phosphate in animal studies. 68 both these drugs have been reported to have antiviral effects against sars-cov and sars-cov-2 in vitro. their potential mechanisms of action include blocking viral entry into cells by inhibiting glycosylation of host receptors, reducing viral replication, and blocking the export of newly constructed virions. 45, 69 a trial in china involving more than 100 patients with covid-19 showed that chloroquine was better able to inhibit exacerbation of pneumonia and improve lung imaging findings compared to the control treatment. 70 a recent small open-label nonrandomised clinical trial showed patients given daily hydroxychloroquine had significantly reduced viral load measured by nasopharyngeal swab on day 6. 5 however, the results in this study may have been confounded by other factors, as some patients also received azithromycin. 71 the combination of hydroxychloroquine and azithromycin should be used with caution in patients at risk for qt prolongation. the use of chloroquine alone is also a risk factor for qt prolongation. a phase iib clinical trial assessing the safety and efficacy of high-dose chloroquine was terminated early due to prolonged qtc (>500 ms) and high lethality in the high-dose group compared to the lowdose group. 72 a recent analysis of a multinational registry of 96,032 hospitalised covid-19 patients revealed that hydroxychloroquine or chloroquine was associated with decreased in-hospital survival (mortality of 11.1 versus 9.3%) and increased frequency of ventricular arrhythmia. 73 however, concerns have been raised about the veracity of data and analyses in this study and has led to retraction of the article. 74 other adverse effects of chloroquine include retinopathy, rash, nausea, glucose fluctuation and diarrhoea, and use of chloroquine is contraindicated in patients with porphyria. chloroquine or hydroxychloroquine should not be used to treat covid-19 until they have been tested in clinical trials to determine the optimal dosage to balance efficacy and safety. chloroquine or hydroxychloroquine has been suggested as candidate prophylactics for covid-19 in populations at high risk of covid-19 infections, such as italy and new york. 75 other antiviral treatments other antiviral treatments for covid-19 currently undergoing clinical trials include protease inhibitors (nelfinavir, camostat mesilate, ritonavir, danoprevir, darunavir, flavopiridol, relacatib), nucleoside analogues (galidesivir), fusion inhibitors (umifenovir), rna polymerase inhibitors (favipiravir), and neuraminidase inhibitors (oseltamivir). 76 corticosteroids corticosteroid therapies such as intravenous methylprednisolone are currently being tested as a treatment for covid-19. 77 methylprednisolone has already been used in covid-19 patients in combination with antibiotics, oseltamivir, and oxygen therapy. 25 long and colleagues reported that corticosteroid therapy using methylprednisolone, dexamethasone, and hydrocortisone was beneficial in treating issn: 1740-4398 review -coronavirus disease 2019 (covid-19): latest developments in potential treatments drugsincontext.com sars-cov patients, 78 and significantly prolonged survival time in clinical cases. 78 however, other studies reported the use of corticosteroids in early stages of sars infection increased the viral load. 79 furthermore, studies on the use of corticosteroids as an adjuvant therapy against mers-cov infection were unable to prove efficacy because all patients died. 80 based on the recommendation by frontline chinese physicians and local clinical experience during the sars epidemic, a short course of corticosteroids at a low-to-moderate dose is probably justifiable for critically ill patients. 66, 81 corticosteroids are effective in treating the autoinflammatory cytokine activation syndrome. reported side effects such as osteonecrosis are concerning, but data are still conflicting and recent recommendations against the use of corticosteroids are based on circumstantial evidence. routine use of corticosteroids is not recommended as corticosteroids may prolong or worsen the disease, and were found to prolong viral shedding in mers and sars. based on the current body of evidence, corticosteroids may be justified for certain medical indications (i.e. acute respiratory distress syndrome and sepsis) at the discretion of the physician in charge. convalescent plasma might be promising as a postexposure prophylaxis for covid-19 and as a potential treatment after infection, as it was also used as a salvage therapy in the sars and mers epidemics. [82] [83] [84] considering the huge number of people exposed to covid-19, use of convalescent plasma for post-exposure prophylaxis may not be justifiable, but could still be a treatment option. an observational study during the sars epidemic showed a lower mortality rate in those receiving convalescent plasma as a treatment for sars. 82 studies on the clinical effects and outcomes of critically ill covid-19 patients treated with convalescent plasma have so far shown encouraging results, although it should be noted that these studies were based on small case series. 85, 86 furthermore, studies on the use of convalescent plasma in sars patients did not report any significant side effects. 82, 87 randomised trials on convalescent plasma therapy are still needed to eliminate the effects of other treatments and to investigate the safety and efficacy and optimal timing of administration. highly purified preparations of neutralising antibodies against sars-cov-2 would be preferable, as it would be safer and have higher activity, but it might be technically difficult to mass produce. 84 several pharmaceutical companies are starting to develop hyperimmune immunoglobulins against covid-19. 88, 89 monoclonal antibodies the use of tocilizumab, a monoclonal antibody against il-6, has recently been suggested as a treatment for covid-19 patients at risk of cytokine storms. 90 tocilizumab is an il-6 inhibitor that may be effective in treating covid-19. 91 the level of il-6 was found to be correlated with the severity of covid-19 and levels of c-reactive protein, lactate dehydrogenase, d-dimer, and t cells. 90 a retrospective study of 20 covid-19 patients receiving tocilizumab found that 75% of them had decreased oxygen requirement. 92 according to the diagnosis and treatment protocol from the china national health commission and state administration of traditional chinese medicine, tocilizumab can be used in patients with extensive pulmonary lesions and in patients with increased il-6 levels. 66 clinical trials on the efficacy of intravenous tocilizumab as a treatment for covid-19 are ongoing. 93, 94 however, serious adverse effects have been reported including gastrointestinal perforation, anaemia, hepatitis, and infusion reaction. tcm employs phytotherapeutic formulations and cultural concepts that originated more than 5000 years ago. 95 the use of tcm as a coadjuvant therapy in the early stage of the sars infection epidemic was reported to increase oxyhaemoglobin arterial saturation. 95 a cochrane review found that chinese herbs combined with western medicines in sars patients could improve symptoms, quality of life, and absorption of pulmonary infiltration, as well as decrease the corticosteroid usage. 96 specific components such as glycyrrhizin, baicalin, and mol376 were shown to have anti-sars activities in vitro. [97] [98] [99] over 85% of sars-cov-2 patients in china received tcm, which has been reviewed by yang and colleagues. 100 the use of tcm was included in the chinese national treatment guidelines for covid-19 patients and was recommended for different stages and severity of disease (table 2) . 66 however, the effectiveness of tcm in treating covid-19 patients still remains unclear. well-designed, large-scale, randomised, double-blinded, and placebo-controlled studies are necessary to confirm the efficacy of tcm before making any recommendations on their use in the management of patients with covid-19. other complementary therapies such as high doses of vitamins, acupuncture, and exercise have been suggested to promote health. however, there are no reports of specific targeted effects on coronaviruses, and there are no randomised trials of complementary therapies as treatments for covid-19. several case series reported that combination therapy could be effective in treating patients with covid-19 patients. 101 a single-centre case series of 89 hospitalised patients with covid-19 (54 non-icu and 35 icu patients) treated with a combination therapy of moxifloxacin, lopinavir, interferon, and methylprednisolone (given to only icu patients) showed good treatment effects and the mortality rate was less than 1%. 101, 102 in another study of 51 covid-19 patients, 10 (19.6%) patients treated with a combination of traditional chinese medicine, interferon-a-1b, lopinavir, ritonavir, and short-term cold dampness and stagnation lung syndrome raw ephedra 6 g, raw gypsum 15 g, almond 9 g, loquat 15 g, gardenia 15 g, guanzhong 9 g, dilong 15 g, xu changqing 15 g, huoxiang 15 g, peilan 9 g, cangzhu 15 g, yunling 45 g, atractylodes 30 g, jiao sanxian 9 g each, magnolia officinalis 15 g, betel coconut 9 g, yarrow fruit 9 g, ginger 15 g. betel nut 10 g, apple 10 g, magnolia 10 g, zhimu 10 g, scutellaria baicalensis 10 g, bupleurum 10 g, red peony 10 g, forsythia 15 g, artemisia annua 10 g (decocted later), green leaves 10 g, raw licorice 5 g. dampness and stagnation lung syndrome raw ephedra 6 g, bitter almond 15 g, raw gypsum 30 g, raw coix seed 30 g, grass root 10 g, patchouli 15 g, artemisia annua 12 g, polygonum cuspidatum 20 g, verbena 30 g, dried reed root 30 g, gardenia 15 g, orange red 15 g, raw licorice 10 g. cold dampness lung syndrome atractylodes lancea 15 g, chenpi 10 g, magnolia 10 g, aquilegia 10 g, grass fruit 6 g, raw ephedra 6 g, zhihuo 10 g, ginger 10 g, betel nut 10 g. plague poison and lung-closing syndrome raw ephedra 6 g, almond 9 g, raw gypsum 15 g, licorice 3 g, fragrant fragrant 10 g (back), magnolia 10 g, atractylodes 15 g, grass fruit 10 g, pinellia 9 g, poria 15 g, raw rhubarb 5 g (back), mongolian milkvetch root 10 g, gardenia 10 g, red peony 10 g. gypsum (fried first) 30-60 g, zhimu 30 g, raw land 30-60 g, buffalo horn (fried first) 30 g, red sage 30 g, black ginseng 30 g, forsythia 15 g, paeonia 15 g, chinese goldthread rhizome 6 g, peony 12 g, gardenia 15 g, raw licorice 6 g. all the coronaviruses are similar in nature and have defined clinical presentations. the lower prevalence of coronavirus diseases in children might be accounted for by the lower exposure and relatively mild clinical presentation in the paediatric population. many of the mild and asymptomatic cases are often overlooked and therefore go undiagnosed. comparing the immunopathology of sars-cov-2 infection between children and adults could reveal the pathology of the disease and might lead to possible treatment strategies for 'recurrent' novel coronavirus infections. there is currently no approved antiviral treatment for patients suspected of or confirmed with covid-19. research on the sars and mers epidemics and data from in vitro studies show that antiviral therapy may be beneficial. antiviral therapies and adjunctive treatments should, therefore, be considered in covid-19 patients with the unstable clinical condition or clinical deterioration or in patients with comorbidities. as the covid-19 situation develops, we will learn more about the safety and efficacy of specific treatments, and treatment recommendations are likely to change to reflect the latest evidence. at the time of writing, the most urgent issue is to curtail the spread of covid-19. vaccines could be the answer to this crisis, but vaccines are likely many months away and will take time to scale-up and manufacture on a global scale. in the meantime, public health officials should be focusing on nonpharmaceutical interventions. measures such as personal hygiene, wearing masks in the general population, social distancing, surveillance programs for testing suspected cases, lung and spleen qi deficiency syndrome french pinellia 9 g, chenpi 10 g, codonopsis 15 g, sunburn astragalus 30 g, stir-fried atractylodes 10 g, poria 15 g, huoxiang 10 g, amomum villosum 6 g, and licorice 6 g qi and yin deficiency syndrome north and south radix salviae 10 g, ophiopogonis 15 g, american ginseng 6 g, schisandra 6 g, gypsum 15 g, light bamboo leaves 10 g, mulberry leaves 10 g, reed root 15 g, salviae miltiorrhiza 15 g, raw liquorice 6 g. one patient died. 103 in a case report of a 45-year-old woman with covid-19 treated with thalidomide (100 mg orally once a day) and methylprednisolone (40 mg intravenously bid for 3 days then reduced to once a day for 5 days), she had overall improved status, increased oxygen index, decreased symptoms of nausea and vomiting, and lower cytokine levels. 101 however, we cannot draw any definitive conclusions from this single case report as there was no control. although sars-cov-2 is the third re-emergence of a coronavirus in the past two decades and has abruptly put a halt to most social and economic activity around the world, the consequences of which are immeasurable. 122 this pandemic will undoubtedly change our daily habits and social routines and should serve as a huge wake-up call that has shown the vulnerability of the human race. for now, the global battle against covid-19 continues, and together, we will inevitably defeat the coronavirus. hard lessons will be learned that will better prepare us for the next pandemic. early quarantine, vigilant contact tracing, and preventing healthcare-related transmission are key factors to stopping the covid-19 pandemic. 108 although a safe and effective treatment has yet to be found, the sheer number of clinical trials that have started since the beginning of the epidemic is nothing short of impressive. combining advanced technologies in the field of genomics and computer science, potential treatments could be identified through machine learning, complex molecular dynamics, and artificial intelligence. with the joint efforts of research communities around the world, we are hopeful that an effective treatment or vaccine for covid-19 can be developed in the near future. 109 this pandemic should also raise the awareness in governments and pharmaceutical companies contributions: all authors contributed equally to the preparation of this review. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. the authors declare that they have no conflicts of interest. the international committee of medical journal editors (icmje) potential conflicts of interests form for the authors is available for download at: https://www.drugsincontext.com/wp-content/uploads/2020/06/dic.2020-4-15-coi.pdf personal view of sars: confusing definition, confusing diagnoses severe respiratory syndromes: travel history matters just like sars clinical management of severe acute respiratory infection when covid-19 is suspected diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement world health organization. off-label use of medicines for covid-19 world health organization. summary of probable sars cases with onset of illness from 1 the sars epidemic in hong kong clinical management and infection control of sars: lessons learned medical treatment of viral pneumonia including sars in immunocompetent adult role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings management of critically ill patients with severe acute respiratory syndrome (sars) world health organization. mers situation update middle east respiratory syndrome a systematic review of therapeutic agents for the treatment of the middle east respiratory syndrome coronavirus (mers-cov) treatment of middle east respiratory syndrome with a combination of lopinavir/ritonavir and interferon-β1b (miracle trial): statistical analysis plan for a recursive two-stage group sequential randomized controlled trial mers-cov infection treated with a combination of lopinavir /ritonavir and interferon beta-1b (miracle) recent advances in the vaccine development against middle east respiratory syndrome-coronavirus severe acute respiratory symptoms and suspected sars again 2020 mystery deepens over animal source of coronavirus the proximal origin of sars-cov-2 coronavirus covid-19 global cases by johns hopkins csse evaluation and treatment coronavirus (covid-19) a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in wuhan pathological findings of covid-19 associated with acute respiratory distress syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates severe acute respiratory syndrome vs. the middle east respiratory syndrome severe acute respiratory syndrome: "sars" or "not sars typical or atypical pneumonia and severe acute respiratory symptoms in picu characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine cryo-em structure of the 2019-ncov spike in the prefusion conformation evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission q&a: similarities and differences -covid-19 and influenza report of the who-china joint mission on coronavirus disease solidarity" clinical trial for covid-19 treatments a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) intensive care management of coronavirus disease 2019 (covid-19 ): challenges and recommendations compassionate use of remdesivir for patients with severe covid-19 covid-19 treatment: a review of early and emerging options remdesivir as a possible therapeutic option for the covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro remdesivir is a direct-acting antiviral that inhibits rna-dependent rna polymerase from severe acute respiratory syndrome coronavirus 2 with high potency remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial nih clinical trial shows remdesivir accelerates recovery from advanced covid-19 gilead announces results from phase 3 trial of investigational antiviral remdesivir in patients with severe covid-19 study to evaluate the safety and antiviral activity of remdesivir (gs-5734 tm ) in participants with moderate coronavirus disease (covid-19) compared to standard of care treatment study to evaluate the safety and antiviral activity of remdesivir (gs-5734 tm ) in participants with severe coronavirus disease (covid-19 adaptive covid-19 treatment trial multi-centre, adaptive, randomized trial of the safety and efficacy of treatments of covid-19 in hospitalized adults a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro lopinavir/ritonavir, ribavirin and ifn-beta combination for ncov treatment multicenter clinical study on the efficacy and safety of xiyanping injection in the treatment of new coronavirus infection pneumonia (general and severe an investigation into beneficial effects of interferon beta 1a, compared to interferon beta 1b and the base therapeutic regiment in moderate to severe covid-19: a randomized clinical trial (dic) treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial novel coronavirus treatment with ribavirin: groundwork for an evaluation concerning covid-19 sars: systematic review of treatment effects clinical features and short-term outcomes of 144 patients with sars in the greater toronto area pharmacologic treatments for coronavirus disease 2019 (covid-19): a review inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin national health commission & state administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial animal toxicity and pharmacokinetics of hydroxychloroquine sulfate a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (sars-cov-2) infection: preliminary safety results of a randomized, double-blinded, phase iib cl. medrxiv hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid-19: a multinational registry analysis retraction-hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid-19: a multinational registry analysis correspondence chloroquine or hydroxychloroquine for prophylaxis of covid-19 landscape analysis of candidate therapeutics for covid-19 efficacy and safety of corticosteroids in covid-19 clinical recommendations from an observational study on mers: glucocorticoids was benefit in treating sars patients effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients update on therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) use of convalescent plasma therapy in sars patients in hong kong challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience the convalescent sera option for containing covid-19 treatment with convalescent plasma for critically ill patients with sars-cov-2 infection treatment of 5 critically ill patients with covid-19 with convalescent plasma sars: systematic review of treatment effects global plasma leaders collaborate to accelerate development of potential covid-19 hyperimmune therapy global plasma leaders collaborate on potential covid-19 hyperimmune therapy tocilizumab treatment in covid-19: a single center experience reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19). medrxiv effective treatment of severe covid-19 patients with tocilizumab tocilizumab in covid-19 pneumonia favipiravir combined with tocilizumab in the treatment of novel coronavirus pneumonia (covid-19) -a multicenter, randomized application of chinese medicine in acute and critical medical conditions chinese herbs combined with western medicine for severe acute respiratory syndrome (sars) glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds virtual screening for finding natural inhibitor against cathepsin-l for sars therapy traditional chinese medicine in the treatment of patients infected with 2019-new coronavirus (sars-cov-2): a review and perspective potential treatments for covid-19; a narrative literature review clinical characteristics and treatment of patients infected with covid-19 in shishou clinical characteristics of 51 patients discharged from hospital with covid-19 in chongqing,china. medrxiv characteristics of funding of clinical trials: cross-sectional survey and proposed guidance review -coronavirus disease 2019 (covid-19): latest developments in potential treatments drugsincontext world health organization. 2019 novel coronavirus global research and innovation forum: towards a research roadmap the covid-19 vaccine development landscape can chinese medicine be used for prevention of corona virus disease 2019 (covid-19)? a review of historical classics, research evidence and current prevention programs the facemask in public and healthcare workers: a need, not a belief using the world's most powerful supercomputers to tackle covid-19 landscape analysis of therapeutics as 21st a trial of remdesivir in adults with mild and moderate covid-19 a prospective, randomized controlled clinical study of antiviral therapy in the 2019-ncov pneumonia outcomes related to covid-19 treated with hydroxychloroquine among in-patients with symptomatic disease the impact of camostat mesilate on covid-19 infection (camoco-19 various combination of protease inhibitors, oseltamivir, favipiravir, and hydroxychloroquine for treatment of covid-19 : a randomized control trial overview of antiretroviral agents used to a systematic review of side effects of nucleoside and nucleotide drugs used for treatment of chronic hepatitis b favipiravir (united states: not commercially available; refer to prescribing and access restrictions): drug information oseltamivir: drug information glucocorticoid therapy for covid-19 critically ill patients with severe acute espiratory failure overview: the history and pediatric perspectives of severe acute respiratory syndromes: novel or just like sars none. key: cord-305582-3hmsknon authors: li, lei; li, ranran; wu, zhixiong; yang, xianghong; zhao, mingyan; liu, jiao; chen, dechang title: therapeutic strategies for critically ill patients with covid-19 date: 2020-04-20 journal: ann intensive care doi: 10.1186/s13613-020-00661-z sha: doc_id: 305582 cord_uid: 3hmsknon since the 2019 novel coronavirus disease (covid-19) outbreak originated from wuhan, hubei province, china, at the end of 2019, it has become a clinical threat to the general population worldwide. among people infected with the novel coronavirus (2019-ncov), the intensive management of the critically ill patients in intensive care unit (icu) needs substantial medical resource. in the present article, we have summarized the promising drugs, adjunctive agents, respiratory supportive strategies, as well as circulation management, multiple organ function monitoring and appropriate nutritional strategies for the treatment of covid-19 in the icu based on the previous experience of treating other viral infections and influenza. these treatments are referable before the vaccine and specific drugs are available for covid-19. in late december 2019, a group of patients with pneumonia of unknown cause were confirmed to be infected with a novel coronavirus (2019-ncov) in wuhan, china. the 2019-ncov has now infected tens of thousands of people in china and has spread rapidly around the globe [1] . the world health organization (who) has declared coronavirus disease 2019 (covid-19) as a public health emergency of international concern and released interim guidelines on patient management [2] . due to the severity and the spreading of covid-19 (novel coronavirus pneumonia, ncp), the chinese government and the medical institutions have executed strict strategies to control the influence of this epidemic [3] . until the end of february, the epidemic has been controlled to a great extent nationally. in wuhan, the situation tends to be stable while a high proportion of critically ill patients are still under treatment of intensive care. coronaviruses (covs) are enveloped viruses with a single positive-stranded rna genome (~ 26-32 kb in length). covs mainly cause respiratory tract infections and some strains have high infectivity and mortality as well as heavy damage on public health, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). the 2019-ncov is a β-cov of group 2b with over 70% similarity in genetic sequence with sars-ncov [4, 5] . the latest version of diagnosis and treatment plan pointed out that the main transmission route is droplet transmission and close contact transmission. in addition, there are risks of airborne spread of 2019-ncov during aerosol-generating medical procedures in specific circumstances [6, 7] . for the positive nucleic acid in oropharyngeal swabs. asymptomatic cases also have the risk, although weak, of transmission. respiratory viral infection can cause severe illness, especially in the elderly and persons with co-morbidities [9] . according to the latest version of diagnosis and treatment guidelines, confirmed cases infected with 2019-ncov are classified to have severe illness once complying with one of the following symptoms: (1) anhelation, respiratory rate ≥ 30 times/min; (2) oxygen saturation at rest ≤ 93%; (3) pao2/fio2 ≤ 300 mmhg; and classified to be the critical/life-threatening illness once complying with one of the following symptoms: (1) respiratory failure, mechanical ventilation needed; (2) shock; (3) other organ dysfunction syndrome and requirement of intensive care unit admission. the progress of the severe illness with covid-19 is usually rapid and there is no clear separation between the severe illness and the critical illness. therefore, patients of these two classes are combined to be the critical illness, which is helpful for health care workers to diagnose and treat patients with intensive care and resources at the early stage of the critical illness. the diagnostic evidences for icu admission according to the previous experience in the treatment of sars include old age (> 60 years old), presence of co-morbidities (particularly, diabetes mellitus, hepatic or cardiac disease), and elevated lactate dehydrogenase levels on admission to hospitals [10, 11] . 2019-ncov invades through the respiratory mucosa and infects other cells, inducing cytokine storm systemically [12] . some patients may progress rapidly with ards, disseminated intravascular coagulation (dic), septic shock, and eventually multiple organ failure [13] . therefore, early identification and timely treatment of critical cases is of crucial importance. evidence-based therapy and supportive care in icu is the mainstay for the management of severe and life-threatening illness of covid-19. the severe and critical illnesses with covid-19 should be treated in icu in the hospital with nosocomial infection control. strict volume management, multi-organ function evaluation, critical care of the nutritional assessment/appropriate nutritional support are essential for these patients in icu. in addition, attention should be paid to bedbound patients to prevent deep vein thrombosis. at present, there is no antiviral treatment with confirmed effectiveness for covid-19. available drug options that come from the clinical experience of treating sars, mers and other previous influenza virus have been used for the treatment of covid-19 patients. although these antiviral drugs are promising in the treatment of covid-19, it should be kept in mind that: (1) the adverse effects of the drugs need to be monitored in clinic, (2) the effects of these drugs on critically ill patients still need to be clarified, (3) the potential mutation of the coronavirus may lead to the drug resistance of the virus. nucleoside analogs have a broad-spectrum antiviral effect via the mechanisms of lethal mutagenesis, chain termination, and inhibition of nucleotide biosynthesis. fabiravir and ribavirin are representative drugs of nucleoside analogs and exhibit the antiviral effect by inhibiting nucleotide biosynthesis. it has been demonstrated that the combination of fabiravir and oseltamivir in the treatment of severe influenza may accelerate clinical recovery than oseltamivir alone [14] . in addition, it has been reported that the combination of ribavirin and interferon alpha (ifn-⍺) significantly reduced the 14-day mortality of critically ill patients infected with mers, although the 28-day mortality was not affected [15] . ribavirin and ifn-⍺ were also used in the treatment for sars. however, ribavirin might have side effects such as anemia and liver injury, and ifn-⍺ may not improve the patients' outcome [16] . therefore, the use of ribavirin and ifn-⍺ in the treatment of covid-19 needs to be further elucidated by clinical studies. lopinavir/ritonavir is a protease inhibitor in the treatment of hiv infection. lopinavir/ritonavir showed the antiviral activity by inhibiting the replication of coronavirus in vitro. it has been reported that the combination of lopinavir/ritonavir with ribavirin could lower the risk of ards compared with ribavirin alone [17] . most recently, the randomized clinical trial of lopinavir/ritonavir (400 mg/100 mg, twice-daily for 14 days) in the treatment of covid-19 by cao et al. has shown that in hospitalized adult patients with severe covid-19, no beneficial effect was observed with lopinavir/ritonavir treatment compared with standard care group [18] . the adverse effects of lopinavir/ritonavir treatment include anorexia, nausea, abdominal discomfort, diarrhea, or acute gastritis. moreover, the risk of hepatic injury, pancreatitis, more severe cutaneous eruptions, as well as the drug interactions due to cyp3a inhibition has been observed in the clinical trial, which arouses concern about the use of higher or prolonged dose regimens for outcome improvement [18] . in addition, serious complications such as acute kidney injury and secondary infection were fewer than in those not receiving treatment. future trials with severe illness might help to elucidate the possibility of benefit of lopinavir/ritonavir treatment. remdesivir (gs-5734) is a new nucleoside analog and has been recognized as a potential and promising antiviral drug against a wide array of rna viruses, including sars/mers-cov. it is currently under clinical development for the treatment of ebola virus infection [19] . remdesivir potentially inhibits the rna-dependent rna polymerase from mers-cov, reduces virus replication, decreases the virus titer in mouse lungs infected with mers-cov, and improves the lung tissue damage [20, 21] . the antiviral activity of remdesivir and ifn-beta was found to be superior to that of the combination of lopinavir/ritonavir and ifn-beta against mers-cov [22] . a randomized, controlled trial has reported that the prolonged use of remdesivir in the treatment of ebola virus disease (evd) is safe [19] , and no adverse events have been observed [23] . as a candidate drug that has not been approved, information about the side effects of remdesivir has not been reported yet. at present, two randomized, controlled, double-blind clinical trials are ongoing to evaluate the efficacy and safety of remdesivir (200 mg loading dose on day 1, followed by 100 mg i.v. once-daily maintenance dose for 9 days) in hospitalized patients with mild/moderate or severe covid-19 respiratory disease [24, 25] . the results of these clinical trials may open the window for effective antiviral therapy for such an epidemic infectious disease. arbidol is a small indole-derivative molecule and is approved for the prophylaxis and treatment of influenza and other respiratory viral infections [26] . it also showed inhibitory activity against other viruses, enveloped or not, responsible for emerging or globally prevalent infectious diseases such as hepatitis b and c [27] . in addition, arbidol has been reported to have antiviral activity against the pathogen of sars, and the effect of arbidol mesylate-a derivative of arbidol, was almost five times higher than arbidol in reducing the reproduction of sars in cells in vitro [28] . it has been claimed that arbidol was effective against 2019-ncov in vitro [29] . a randomized multicenter controlled clinical trial of arbidol in patients with 2019-ncov is in progress in china [30] . it is known that angiotensin-converting enzyme-2 (ace2) as a membrane protein is a functional receptor of sars-cov and it can facilitate virus entry into the cells by binding to the spike (s) protein of the virus, which mediates the fusion of viral and host membranes [31] [32] [33] . therefore, it may be of importance to block the binding of s protein to ace2 to treat viral infection, such as sars-cov [34] . chloroquine is a 9-aminoquinoline known since 1934, the sulfate and phosphate salts of which have both been commercialized as widely used antimalarial and autoimmune disease drugs. chloroquine also shows broad-spectrum antiviral effects [35] . it was found to be a potent inhibitor of sars-cov infection due to its inhibitory effect on ace2 [36] . it has been demonstrated that 2019-ncov enter the epithelial cells of oral mucosa via the essential receptor ace2 [37] , and chloroquine can function at both entry and post-entry stages of 2019-ncov infection [38] . besides the antiviral activity, chloroquine has an immune-modulating activity, which may synergistically enhance its antiviral effect in vivo. recently, wang et al. have demonstrated that chloroquine is highly effective in the control of 2019-ncov infection in vitro and is suggested to be assessed in human patients suffering from covid-19 [38] . in addition, the results from more than 100 covid-19 patients have indicated that chloroquine phosphate is superior to the control treatment in inhibiting the exacerbation of pneumonia, improving lung imaging, promoting virus negative conversion, and shortening the disease course [39] . however, attention should be paid to the potential detrimental effects of chloroquine observed in previous attempts to treat viral diseases. at present, the clinical trials to evaluate the efficacy and safety of chloroquine in the treatment of covid-19 is ongoing [40] . the use of chloroquine in the treatment of covid-19 should refer to the most recent announcements if any. in addition, hydroxychloroquine is a 4-aminoquinoline derivative antimalarial drug. hydroxychloroquine is an immunosuppressive drug with mature clinical application in the treatment of rheumatic immune diseases such as rheumatoid arthritis and systemic lupus erythematosus [41, 42] . it has been found to be more potent than chloroquine in inhibiting 2019-ncov in vitro. hydroxychloroquine sulfate 400 mg given twice daily for 1 day, followed by 200 mg twice daily for another 4 days is recommended in the treatment covid-19 [43] . at present, the clinical evaluation of hydroxychloroquine in the treatment of covid-19 is in progress [44] , which might shortly provide preliminary results about the effectiveness of hydroxychloroquine. patients with pneumonia, especially in severe condition, may encounter with co-infection or cross-infection of bacterial pathogens, for instance staphylococcus aureus, during medical treatment in the hospital. considering the high incidence of bacterial infection for critically ill patients with covid-19, it is essential to test the kinetics of procalcitonin (pct) and c-reaction protein (crp) in covid-19 patients for timely diagnosis and intervention of bacterial infection. according to the recent 2019 ats/idsa clinical practice guidelines, besides antiviral treatment for patients with viral-infected pneumonia, clinicians should empirically give antibacterial therapy to patients that initially have severe diseases (extensive pneumonia, respiratory failure, hypotension, and fever), or deteriorate after initial improvement, or fail to improve after 3 to 5 days of antiviral treatment [45] . thus, antibiotic treatment is recommended in the treatment of covid-19 patients based on the evidence of bacterial infection. the blind and inappropriate use of antibiotics, especially the broad-spectrum antibiotics, should be avoided. immune disorders have been observed in the treatment of patients with covid-19. the virus infection activates the immune cells, leading to cytokine storm which is associated with disease severity [46] . on the other hand, the critical illness with covid-19 mainly affect elders or people with chronic diseases, some of whom have very low number of lymphocytes, especially cd4+ t cells, implying deficiency of immune system. therefore, to modulate the immune responses, a variety of pharmacologic agents have been proposed [47, 48] . the use of corticosteroids in the treatment of ards is controversial. observational data in sars suggest that immunomodulation with regimens of high-dose methylprednisolone might be helpful in modulating inflammatory responses and lung damage [49, 50] . on the other hand, other studies showed that use of steroids is associated with increased risk for bacterial infection, increased mortality, and even antiviral resistance in influenza-associated pneumonia or ards [51] [52] [53] [54] . moreover, multiple studies have reported that corticosteroid treatment is associated with delayed viral shedding in hospitalized patients without significant change in 90-day mortality [55] [56] [57] . corticosteroid therapy in patients with mers was shown to be not associated with a difference in mortality, but associated with delayed mers coronavirus rna clearance [57] .the early use of parenteral glucocorticoids therapy for fever reduction and pneumonia prevention has been shown to increase the risk for critical disease or death from h1n1 infection [58] . the currently limited clinical research does not support the use of corticosteroids in the treatment of ards in covid-19 patients to improve the outcome of patients. the who recommended that corticosteroids should not be used in the treatment of viral pneumonia or ards. there is no convincing proof for the therapeutic benefits of corticosteroids in the treatment of covid-19, which still need to be demonstrated in clinical research. thymosin alpha-1 is a thymic peptide hormone with significant benefits in restoring the homeostasis of the host immune system [59] . it is chemically synthesized and used in diseases with impaired immune system [60] . it has been reported that the low lymphocyte count is associated with the poor prognosis of septic patients. the use of thymosin alpha-1 therapy in combination with conventional medical therapies was effective in improving clinical outcomes and reducing mortality in severe sepsis [61] . in addition, thymosin alpha-1 can enhance the immune responses of sars patients and help to limit the spreading of sars [62] . therefore, although there is no clinical evidence showing the beneficial effects of thymosin alpha-1 in covid-2019, it has been recommended to be used for some patients to enhance cellular immunity for the resistance of viral infection. cyclosporine a is widely used in transplantation and autoimmune disorders due to its immunosuppressive effect. cyclophilin a as a key member of immunophilins is the cellular receptor for cyclosporine a [63] . the inhibition of cyclophilins by cyclosporine a could block the replication of coronavirus, including sars-cov [64] . therefore, non-immunosuppressive derivatives of cyclosporine a might serve as broad-range cov inhibitors applicable against emerging virus like 2019-ncov, which still needs to be confirmed by clinical studies in the future. there are two types of interferons (ifns), type i ifns and type ii ifns. it has been demonstrated that type i ifns can inhibit the replication of both sars and mers-cov [65, 66] . kuri et al. have reported that ifn transcription was blocked in tissue cells infected with sars-cov and cells infected with sars-cov were able to partially restore their innate immune responsiveness to sars-cov after priming with small amounts of ifns [67] . moreover, in patients with severe mers-cov infection, the combination of ifn-alpha-2a with ribavirin was shown to improve survival [66] . recently, the combination of remdesivir and ifn-beta was shown to have significant antiviral activity [22] . intravenous gammaglobulin is considered as the safest immunomodulating drug available for the treatment of severe infection and sepsis. it has high titers of neutralizing antibodies against broad-spectrum virus, bacteria, and other pathogens, and can modulate the host immune responses in several ways. however, a large-scale multicenter randomized placebo-controlled trial did not show improved survival with intravenous gammaglobulin in severe sepsis [68] . moreover, a cochrane review showed that intravenous gammaglobulin did not reduce the mortality of septic patients [69] . therefore, there is no convincing argument to recommend intravenous gammaglobulin in the treatment of 2019-ncov. one of the most important mechanism underlying the deterioration of covid-19 is cytokine storm characterized by elevated levels of il6, ifn-and other cytokines, which will lead to ards or even multiple organ failure [70] . tocilizumab is a recombinant humanized monoclonal antibody binding to il6 receptor and inhibiting its signal transduction. tocilizumab has been used in the treatment for rheumatoid arthritis (ra) [71] . moreover, tocilizumab has been reported to be effective against cytokine release syndrome induced by car-t cell infusion against b cell acute lymphoblastic leukemia [72] . in diagnosis and treatment guidelines of ncp (trial version 7.0) [73] , tocilizumab is recommended for the immunotherapy of patients with extensive lung lesions and severe cases that show an increased level of il6 in laboratory testing. the efficacy of tocilizumab in covid-19 patients still needs to be investigated. in 2003, chinese traditional medicine was used to prevent and treat sars [74] . in 2009, during the pandemic of h1n1 influenza, the traditional chinese medicine of china issued a chinese traditional medicine prevention program, which included several chinese herbal medicine formulae for the prevention of infection of adults and children. shufengjiedu capsules and lianhuaqingwen capsules have also played a role in the prevention and treatment of new respiratory infectious diseases such as influenza a (h1n1) [75, 76] . some studies have confirmed that yupingfeng powder has antiviral, antiinflammatory and immunoregulatory effects [77] . a multicenter, large-scale, randomized trial found that yinqiao powder plus another heat-clearing formula could reduce time for fever resolution in patients with the h1n1 influenza virus infection [78] . it is suggested that high-risk populations exposed to covid-19 patients, including medical staff, family members, and other people who are in close contact with covid-19 patients, as well as residents living in covid-19 outbreak areas, might benefit from taking chinese traditional medicine formulae for prevention. however, the efficacy and safety of these chinese traditional medicine formulae in covid-19 need to be further confirmed by clinical trials. the convalescent plasma derived from the patients with antibodies against 2019-ncov can be effective in reducing the mortality rate of critically ill patients with infectious disease [79] . convalescent plasma has been found to have an immunotherapeutic potential for the treatment of mers, sars and ebola virus disease [80] [81] [82] . the explanation for the efficacy of convalescent plasma therapy is that antibodies from convalescent plasma might suppress viremia via free viral clearance, blockade of new infection, as well as the acceleration of infected cell clearance [83] . in addition, the use of high-titer mers serum from camel could significantly improve the histology of lung damage and increase the clearance of mers-cov in mice [84] . moreover, the use of convalescent plasma or serum was also suggested by who under blood regulators network when vaccines and antiviral drug was unavailable for an emerging virus. evidence shows that convalescent plasma therapy is not associated with the occurrence of severe adverse events [85] . convalescent plasma, if available, can be used for the treatment of critically ill patients with covid-19 after the evaluation of the valence of antibody. it is worthwhile to test the efficacy and safety of convalescent plasma transfusion in covid-19 patients. a meta-analysis showed that among patients with 2019-ncov infection, the incidence of ards is approximately 15% [12] . moreover, between 50% to 85% of patients admitted to icu have hypoxemia/or development of respiratory exhaustion [86] . therefore, timely and effective respiratory support can contribute to reduce complications and improve the survival of such critically ill patients. oxygen therapy, high-flow nasal cannula, and non-invasive ventilation may reduce the need of endotracheal intubation and decrease ventilator-associated complications and mortality. however, several studies have reported that the failure of non-invasive ventilation was up to 85% in which invasive ventilation was ultimately required in the treatment of severe influenza a (h1n1) in canada [87] . non-invasive ventilation may be effective and safe for some patients, whereas it might increase virus transmission to health care workers because of risk for infected aerosol generation. therefore, for the treatment of 2019-ncov, non-invasive ventilation may be used in selected patients in early stages with milder acute hypoxemic respiratory failure [88] . while for critically ill patients, the effectiveness of transitionally oxygen therapy, such as respiratory status and oxygen index (po2/ fio2), needs to be closely monitored and it should be switched to mechanical ventilation when necessary. high-flow nasal cannula has emerged as an alternative to non-invasive ventilation to prevent intubation and reduce mortality in patients with acute hypoxemic respiratory failure [89] . high-flow nasal cannula has been reported to significantly reduce 90-day mortality of community-acquired pneumonia compared with standard oxygen or non-invasive ventilation [89] . hui et al. have shown that the breath dispersion distance is limited therefore lowering the risk of air transmission. however, the loose connection of the cannula with nasal obstruction can significantly increase the dispersion distance [90] . wearing masks (particularly n95) can effectively reduce the breath dispersion distance during high-flow nasal ventilation to prevent nosocomial transmission [91, 92] . in addition, high-flow nasal cannula might increase virus transmission risk due to aerosol generation. therefore, staff protection of health care workers is critical. mechanical ventilation for patients with severe ards should be managed with lung-protective strategies to minimize ventilator-associated lung injury and to improve survival. the approach to minimize ventilatorassociated lung injury and to improve survival includes: ventilation with low tidal volumes (4 to 8 ml/kg of predicted body weight), targeting plateau pressure (< 30 cmh 2 o) [93] , and minimizing the inspired oxygen concentration to decrease oxygen toxicity. high positive endexpiratory pressure (peep) can reduce the need for high fio2 by improving gas exchange and lung compliance, whereas too high peep may lead to lung overdistension and hemodynamic instability. optimal peep which can be titrated by pressure-volume curve, oxygenation, stress index, electrical impedance tomography (eit), ultrasound, and some other clinical parameters is associated with improved survival rate among severe ards patients [9] . lung recruitment needs to be evaluated for mechanically ventilated patients when uncorrectable hypoxemia occurs, and lung recruitment should be executed for patients whose lungs can restore aeration. ct, eit, ultrasound, and other bedside techniques should be used to evaluate lung recruitability before lung recruitment. for critically ill patients managed with mechanical ventilation, excessive spontaneous breathing due to stretching may lead to lung injury. therefore, neuromuscular relaxant may be used to control the spontaneous breathing and protect the lung. prone positioning ventilation is a technique that often improves oxygenation in ards, possibly through improvements in ventilation-perfusion matching, the uniformity of ventilation, and gravity-related atelectasis. prone ventilation was used in mechanically ventilated sars patients without enough data to draw any conclusion with regard to its efficacy [86] . although prone ventilation showed no improvement in survival or organ dysfunction overall, it might be beneficial for patients with severe ards. a multicenter rct demonstrated that early application of prone positioning in patients with severe ards resulted in decreased mortality [94] . in addition, the use of prone positioning in patients with h7n9 influenza-induced severe ards was shown to be related with improved oxygenation, sustained after returning to a supine position, and with decreased carbon dioxide retention [95] . generally speaking, prone positioning ventilation for no less than 12 h daily is a relatively safe procedure that rarely worsens a patient's respiratory status. it can be thus recommended for the treatment of 2019-ncov-induced severe ards. ecmo has become the important life-support strategy for the standardized treatment of ards patients. ecmo should be considered as early as possible when the lung recruitment and prone positioning ventilation show to be ineffective. observational studies have reported that patients with ards induced by h1n1 influenza showed lower hospital mortality with transfer to an ecmo center compared with matched non-ecmo-supported patients [96] . the use of ecmo also showed survival benefit in patients with severe mers [97] . ecmo tends to improve patient outcomes when used among those with limited organ failures and good pre-morbid functional status [9] . substantial proportion of critically ill patients with covid-19 appear to have developed cardiac arrhythmias or shock [13] , and may need ecmo support. however, for those who will develop septic shock or refractory multiple organ failure, ecmo is not suggested due to its less benefit. ecmo is a resource-intensive, highly specialized and expensive form of life support with potential for significant complications such as hemorrhage and nosocomial infection. therefore, the use of ecmo should be strictly limited in the treatment of covid-19. moreover, since the number of critically ill patients is still increasing and the resource of ecmo is finite, judgment is needed to decide when ecmo may be worthwhile and when it may not. support with ecmo is supposed to be for the most critically ill patients in regions with extensive resources for this therapy [98] . for patients with ards, restrictive and timely fluid resuscitation is associated with better oxygenation and lower mortality. aggressive fluid administration may worsen oxygenation and ventricular dysfunction, which may result in longer duration of mechanical ventilation and even mortality. therefore, it is necessary to assess fluid responsiveness and to evaluate ventricular function during fluid resuscitation. conservative fluid administration while maintaining adequate mean arterial pressure and organ perfusion with the appropriate use of diuretics and vasopressors is of importance [99] . according to the latest epidemiological report, the incidence for the critically ill patients to develop multiple organ dysfunction syndrome is up to 11% [48] . covid-19 may be combined with other organ injuries, including liver injury, cardiac dysfunction, coagulopathy, which may need the routine functional support for critically ill patients in icu. moreover, all the critically ill patients with covid-19 admitted into icu have negative nitrogen balance and malnutrition [12, 46] , which has been considered as a contributing factor to the emergence of viral infectious diseases. therefore, appropriate nutritional strategy is pivotal for the treatment of critical illnesses when necessary. there are no specific antiviral drugs or vaccines for 2019-ncov at present. therefore, it is important to enhance the host immune response against the infection with 2019-ncov. all of the drug options are based on the experience treating sars, mers or some other previous influenza viruses. the efficacy of existing drugs as well as adjunctive pharmacologic interventions in the treatment of critical ill patients with covid-19 warrants further verification in clinical research. to completely stop the epidemic spreading of covid-19, a vaccine for 2019-ncov is urgently needed. besides enhancing the host immune responses against viral infection, appropriately respiratory supportive strategies, monitoring and support of multiple organ function, modulating the immune status and inflammatory responses individually, as well as the prophylaxis and treatment of complications are all important guarantee for the recovery of critically ill patients with covid-19. for a better understanding of this novel virus, more research needs to be done to get optimal strategies for the treatment of covid-19. a novel coronavirus from patients with pneumonia in china organization wh. clinical-management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected-interim guidance intensive care during the coronavirus epidemic the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest, novel coronavirus outbreak in wuhan, china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding consideration and prevention for the aerosol transmission of 2019 novel coronavirus aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment critical care management of adults with community-acquired severe respiratory viral infection. intens care med clinical features and shortterm outcomes of 144 patients with sars in the greater toronto area a major outbreak of severe acute respiratory syndrome in hong kong clinical characteristics of 50466 hospitalized patients with 2019-ncov infection clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china comparative effectiveness of combined favipiravir and oseltamivir therapy versus oseltamivir monotherapy in critically ill patients with influenza virus infection combination therapy with lopinavir/ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 a randomized, controlled trial of ebola virus disease therapeutics the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov first case of 2019 novel coronavirus in the united states mild/moderate 2019-ncov remdesivir rct-full text view clinicaltrials severe 2019-ncov remdesivir rct-full text view-clinicaltrials. gov arbidol as a broad-spectrum antiviral: an update arbidol: a broad-spectrum antiviral compound that blocks viral fusion antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures drug treatment options for the 2019-new coronavirus (2019-ncov). biosci trends. 2020 clinical study of arbidol hydrochloride tablets in the treatment of pneumonia caused by novel coronavirus angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus the secret life of ace2 as a receptor for the sars virus characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry severe acute respiratory syndrome coronavirus entry into host cells: opportunities for therapeutic intervention anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model chloroquine is a potent inhibitor of sars coronavirus infection and spread high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies chloroquine prevention of coronavirus disease (covid-19) in the healthcare setting (copcov) hydroxychloroquine in systemic lupus erythematosus (sle) mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine treatment for severe covid-19 pulmonary infection (hydra trial) (hydra) clinical practice guidelines by the infectious diseases society of america: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenza clinical features of patients infected with 2019 novel coronavirus in wuhan advances in respiratory virus therapeutics-a meeting report from the 6th isirv antiviral group conference epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome glucocorticoids and acute lung injury the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and metaanalysis corticosteroids for the treatment of human infection with influenza virus: a systematic review and metaanalysis use of early corticosteroid therapy on icu admission in patients affected by severe pandemic (h1n1)v influenza a infection prolonged viral shedding in pandemic influenza a(h1n1): clinical significance and viral load analysis in hospitalized patients factors associated with prolonged viral shedding in patients with avian influenza a(h7n9) virus infection corticosteroid therapy for critically ill patients with middle east respiratory syndrome early use of glucocorticoids was a risk factor for critical disease and death from ph1n1 infection thymosin alpha 1 and hiv-1: recent advances and future perspectives serum thymosin alpha 1 levels in normal and pathological conditions the efficacy of thymosin alpha 1 for severe sepsis (etass): a multicenter, single-blind, randomized and controlled trial clinical investigation of outbreak of nosocomial severe acute respiratory syndrome cyclophilin a: a key factor in virus replication and potential target for anti-viral therapy the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review interferon priming enables cells to partially overturn the sars coronavirus-induced block in innate immune activation score-based immunoglobulin g therapy of patients with sepsis: the sbits study intravenous immunoglobulin for treating sepsis, severe sepsis and septic shock. cochrane database syst rev advances in the research of cytokine storm mechanism induced by corona virus disease 2019 and the corresponding immunotherapies effects of tocilizumab, an anti-interleukin-6 receptor antibody, on serum lipid and adipokine levels in patients with rheumatoid arthritis fda approval summary: tocilizumab for treatment of chimeric antigen receptor t cell-induced severe or life-threatening cytokine release syndrome national health commission & state administration of traditional chinese medicine chinese herbal medicine for severe acute respiratory syndrome: a systematic review and meta-analysis unique synergistic antiviral effects of shufeng jiedu capsule and oseltamivir in influenza a viral-induced acute exacerbation of chronic obstructive pulmonary disease the chinese prescription lianhuaqingwen capsule exerts anti-influenza activity through the inhibition of viral propagation and impacts immune function yu ping feng san, an ancient chinese herbal decoction, induces gene expression of anti-viral proteins and inhibits neuraminidase activity oseltamivir compared with the chinese traditional therapy maxingshigan-yinqiaosan in the treatment of h1n1 influenza: a randomized trial convalescent plasma: new evidence for an old therapeutic tool? convalescent plasma for ebola virus disease retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients use of convalescent plasma therapy in sars patients in hong kong enhanced clearance of hiv-1-infected cells by broadly neutralizing antibodies against hiv-1 in vivo passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection convalescent plasma as a potential therapy for covid-19 critically ill patients with severe acute respiratory syndrome critically ill patients with 2009 influenza a(h1n1) infection in canada official ers/ats clinical practice guidelines: noninvasive ventilation for acute respiratory failure high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure exhaled air dispersion during high-flow nasal cannula therapy versus cpap via different masks exhaled air dispersion during coughing with and without wearing a surgical or n95 mask expert consensus on preventing nosocomial transmission during respiratory care for critically ill patients infected by 2019 novel coronavirus pneumonia surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19). intensive care med prone positioning in severe acute respiratory distress syndrome a multicenter retrospective review of prone position ventilation (ppv) in treatment of severe human h7n9 avian flu referral to an extracorporeal membrane oxygenation center and mortality among patients with severe 2009 influenza a(h1n1) extracorporeal membrane oxygenation for severe middle east respiratory syndrome coronavirus preparing for the most critically ill patients with covid-19: the potential role of extracorporeal membrane oxygenation management of critically ill adults with covid-19 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to express their appreciation for all of the health care workers and other hospital staff for their efforts in combating the outbreak of covid-19. all the authors have participated in literature retrieval and viewpoint discussion. ll and rl have written this article under the supervision of dc and jl. all authors read and approved the final manuscript. not applicable. not applicable. not applicable. the authors declare that they agree for the publication of this article. the authors declare that they have no competing interests. key: cord-320238-qbjrlog1 authors: okba, nisreen m. a.; widjaja, ivy; van dieren, brenda; aebischer, andrea; van amerongen, geert; de waal, leon; stittelaar, koert j.; schipper, debby; martina, byron; van den brand, judith m. a.; beer, martin; bosch, berend-jan; haagmans, bart l. title: particulate multivalent presentation of the receptor binding domain induces protective immune responses against mers-cov date: 2020-05-29 journal: emerging microbes & infections doi: 10.1080/22221751.2020.1760735 sha: doc_id: 320238 cord_uid: qbjrlog1 middle east respiratory syndrome coronavirus (mers-cov) is a who priority pathogen for which vaccines are urgently needed. using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the mers-cov spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (rbd) ─ and tested their immunogenicity and protective capacity in rabbits. using a “plug-and-display” spytag/spycatcher system, we coupled rbd to lumazine synthase (ls) particles producing multimeric rbd-presenting particles (rbd-ls). rbd-ls vaccination induced antibody responses of high magnitude and quality (avidity, mers-cov neutralizing capacity, and mucosal immunity) with cross-clade neutralization. the antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against mers-cov infection in rabbits. this arrayed multivalent presentation of the viral rbd using the antigen-spytag/ls-spycatcher is a promising mers-cov vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as sars-cov-2. emerging zoonotic viruses, such as severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have been able to cross the species barrier posing a threat to the human population. mers-cov causes severe respiratory disease and fatalities in humans [1, 2] , and the virus is continuously introduced into the human population through infected dromedary camels, the viral reservoir with resulting outbreaks [3] . the wide geographical distribution of this viral reservoir, the high case-fatality rate in humans (35%), and the lack of treatment and licensed vaccines, make the virus a threat to the human population. this has put mers-cov on the recent who list of diseases having an epidemic or even pandemic potential for which countermeasures are lacking and are urgently needed [4] . vaccination is potentially one of the most effective ways to prevent the ongoing mers-cov outbreaks. several mers-cov vaccine candidates have been developed using different platforms including inactivated, live-attenuated, and subunit vaccines [5] . compared to other vaccine production platforms, recombinant subunit proteins have a higher safety profile, are relatively faster and easier to produce, and can be scaled-up in a more cost-effective manner; nonetheless, they tend to induce lower levels of protective immunity [6] . the use of self-assembling multimeric protein scaffold particles (mpsp) to present antigens in a multivalent virus-mimicking manner (size, repetitiveness, and geometry), has been shown to enhance vaccine-induced immune responses [7] [8] [9] [10] [11] , and to offer advantages over other multimeric antigen presentation platforms (reviewed in [12] ). both lumazine synthase (ls) and i3-01 (i3) can self-assemble into 60-meric particles, which can be expressed in e. coli and have been used as scaffolds for development of multimeric vaccines with improved immune responses compared to monomeric forms [13] [14] [15] . an ls-based hiv vaccine, (eod-gt8), has recently advanced to a phase i human clinical trial (nct03547245). linking of antigens to these mpsp can be achieved through several mechanisms; as e.g. genetic fusion or the syptag-spycatcher (st/sc) system [16] . while the former requires the antigen and scaffold to be produced in the same expression system, the latter allows each to be expressed in its suitable system harnessing a rapid post-translational "plug-andplay" assembly. this is advantageous, allowing scaffold-sc to be produced at scalable levels in e. coli and spytagged glycosylated antigens such as viral surface proteins to be produced in its optimal system, such as mammalian or insect cells. the antigen-st can then be multivalently displayed on the surface of the scscaffolds through the spontaneous formation of a stable isopeptide bond. this can be a platform for rapid vaccine manufacturing in case of epidemics or pandemics, to create optimized vaccines at reduced costs and also with reduced development times. the mers-cov spike (s) protein is the main target for subunit vaccine development [5] it assembles as a homotrimer and consists of an n-terminal head (s1 subunit) and a c-terminal stalk (s2 subunit). the s1 subunit mediates virus attachment and entry through its n-terminal s1 a domain and its c-terminal receptor binding domain (rbd), respectively [17, 18] . the s1 a domain binds sialic acids, a viral attachment factor, while the rbd binds to the viral receptor, dipeptidyl peptidase 4 (dpp4). following attachment and entry, the s2 subunit mediates viral fusion to the host cell through its fusion machinery; comprised of the fusion peptide (fp) and the two heptad repeats -hr1 and hr2 [19] . mers-cov neutralizing antibodies (abs) mainly recognize epitopes in the rbd of the spike head s1 subunit; and to a lower extent, epitopes in the sialic acid binding domain and the fusion-mediating more conserved s stalk (s2). nonetheless, antibodies directed against the sialic acid binding s1 a domain or the more conserved s2 subunit, although subdominant, may protect against mers-cov [20, 21] . immune focusing can enhance immune responses to subdominant regions [22] . in the current study, using ls and i3 self-assembling particles, we evaluated whether immune focusing and multivalent presentation can induce immune responses to the more sequence-conserved s2 regions: fp and hr2. furthermore, using a syptag/spycatcher system and ls particles, we tested whether immune focusing with/ without multivalent presentation of the viral rbd can lead to enhanced protection against a mers-cov challenge in rabbits. expression constructs were cloned using standard pcr methods. the gene encoding the 6,7-dimethyl-8-ribityllumazine synthase (ls; genbank accession no. wp_010880027.1) of a. aeolicus was synthesized using human-preferred codons obtained from gen-script usa, inc, as described previously [17] . the cysteine at position 37 and asparagine at position 102 of ls were mutated to alanine and glutamine, respectively. the gene encoding i3-01 (i3; pdb 5kp9, amino acid residues 19-222) derived from thermotoga maritima was synthesized using human-preferred codons obtained from genscript usa. the gene fragments encoding the δn1spycatcher (sc; uniprot accession no. afd50637.1; amino acid residues 48-139; [23] ) and spytag (st; uniprot accession no. wp_129284416.1; amino acid residues 981-994) based on the cna b-type domain-containing protein of streptococcus pyogenes were synthesized using human-preferred codons obtained from genscript usa, inc. the ls and i3 gene constructs were cloned into the pgex-2 t bacterial expression vector (sigma aldrich). to generate the hr2-ls expression vector, the hr2 region (amino acid residues 1215-1287) encoding sequence of the mers-cov s gene (accession no. nc_019843) was ligated in-frame with an n-terminal sequence encoding a cd5 signal sequence and streptag tag purification tag, and with a c-terminal sequence encoding the ls via a linker, and subsequent cloned into the pcaggs mammalian expression vector. to generate the i3-hr2 expression vector, the heptad repeat 2 encoding region (hr2, amino acid residues 1215-1287) of the mers-cov s gene was ligated in-frame with an n-terminal sequence encoding the i3-01 and a c-terminal streptag purification tag interspaced with a linker, and subsequent cloned into the pgex-2 t bacterial expression vector (sigma aldrich). to generate the fp-i3 and fp-ls expression vectors, the fusion peptide (fp; amino acid residues 884-898) encoding sequence of the mers-cov s gene was ligated in-frame with an n-terminal sequence encoding the i3-01 or ls, and a c-terminal streptag purification tag and subsequently cloned into the pgex-2 t bacterial expression vector (sigma aldrich). to generate the rbd-st expression vector, the mers-rbd (amino acid residues 358-588) encoding sequence of the mers-cov s gene was ligated inframe with an n-terminal sequence encoding a cd5 signal sequence and with a c-terminal sequence encoding the st followed by a double streptag, and subsequently cloned into the pcaggs mammalian expression vector. to generate the ls-sc expression vector, the codon optimized sc sequence equipped with an n-terminal flag-tag (dykddddk) was cloned to the n-terminus of the ls sequence in the pet15b bacterial expression vector (novagen). all protein sequences are provided in supplementary figures s1 and s2. mammalian expression of the hr2-ls and rbd-st constructs was done, as described previously [17] . in short, expression plasmids were polyethylenimine (pei)-transfected into 60% confluent hek-293 t cells for 6 h, after which transfections were removed and medium was replaced with 293 sfm ii-based expression medium (gibco life technologies) and incubated at 37°c in 5% co 2 . tissue culture supernatants were harvested 5-6 d post transfection, and expressed proteins were purified using streptactin sepharose beads (iba) according to the manufacturer's instruction. bl21 cells (novagen) were transformed with pgex-2 t expression vectors and grown in 2× yeast-tryptone medium to log phase (od 600 ∼1.0) and subsequently induced by adding iptg (isopropyl-β-d-thiogalactopyranoside) (gibco brl) to a final concentration of 1 mm. two hours later, the cells were pelleted, resuspended in 1/25 volume of 10 mm tris (ph 8.0)-10 mm edta-1 mm phenylmethylsulfonyl fluoride, and sonicated on ice (five times, 2 min each). the cell homogenates were centrifuged at 20,000 × g for 60 min at 4°c. proteins were purified from the cell lysate supernatant using streptactin sepharose beads (iba) according to the manufacturer's instruction. all purified proteins were analyzed on a 12% sds/ page gel under reducing conditions and stained with gelcodeblue stain reagent (thermo scientific). purified proteins were stored at 4°c until further use. expression of the flag-ls-sc was performed as described above with the following modifications: 1) cells were treated with 1 mg/ml lysozyme in lysis buffer (50 mm tris-hcl, 150 mm nacl, 1% triton x-100) for 1 h at room temperature prior to sonification on ice. 2) purification was performed using anti-flag® m2 affinity gel (sigma aldrich) as recommended by the manufacturer. purified proteins were dialyzed against 1x tbs buffer (50 mm tris-hcl, 150 mm nacl, ph 7.4) and stored at −80°c until further use. rabbit immunizations and challenge were carried out at viroclinics bioscience b.v. under permit no. avd277002015283-wp03, using bsl-3 containment facilities. female new zealand white rabbits (envigo, venray, the netherlands) of 11 weeks age were assigned to six groups (i-vi) of five animals each. immunizations were performed intramuscularly with either i) hr2-ls, ii) fp-ls, iii) ls, at day 0 and boosted with either i) hr2-i3, ii) fp-i3, iii) i3 on day28 or iv) pbs, v) rbd + ls, vi) rbd-ls on days 0 and 28. each animal received each time 15 µg of antigen adjuvanted with adjuplex (5%; sigma-aldrich, zwijndrecht, the netherlands) in a total volume of 500 µl. three weeks after the last vaccination (day 49 of the study), all animals were challenged intranasally under anesthesia with mers-cov (10 6 50% tissue culture infectious dose (tcid 50 ) mers-cov emc strain (accession no. nc_019843) in a volume of 1 ml divided over both nostrils). the animals were euthanized on day 4 postchallenge (day 53 of the study). serum samples were collected on days 0, 28, and 46. nasal swabs were collected on day 46 (pre-challenge) and on days 1 through 4 post-challenge. following euthanasia, lungs were examined for gross pathology and lung tissue samples were collected for virus detection, and in 10% formalin histopathology and immunohistochemistry. antigen-binding and anti-ls (scaffold) antibodies produced after vaccination were tested in the sera collected at different time points as well as in pre-challenge nasal swabs using elisa. costar high-binding 96-well elisa plates were coated overnight at 4°c with 1 µg/ ml of either recombinant ls, mers-cov s1 or s2 proteins in pbs. the plates were washed with pbs and blocked for 1 hr using 1%bsa/0.5%tween-20/pbs. following blocking, diluted samples (1:100 or serially diluted) were added and further incubated for 1 hr. the plates were then washed and and probed with an hrp-labeled goat anti-rabbit ig (1:2000, dako) secondary antibody. tmb was used for signal development and the absorbance of each sample was measured at 450 nm (od 450 ). antibody avidity was assessed using an ammonium thiocyanate (nh 4 scn)-displacement elisa. this was carried out as described above using serum dilutions containing same level of s1 absorbance units added in triplicates. following serum incubation and washing, nh 4 scn (0-5 m) was added to the wells for 15 min. the plates were then washed and further developed as described above. the concentration of nh 4 scn resulting in a 50% reduction in signal was taken as the avidity index (ic 50 ). to confirm the antigenicity of the rbd-ls particles, we tested its binding to well-characterized monoclonal antibodies binding conformational rbd epitopes [20] . human monoclonal antibodies 7.7g6, 1.6f9, 1.2g5, 1.8e5, 4.6e10 targeting the receptor binding domain of the mers-cov spike protein were produced and purified as described earlier [20] . nunc maxisorp plates (thermo scientific) were coated with the rbd-ls antigen at 100 ng /well at 4°c overnight. plates were washed three times with pbs containing 0.05% tween-20 and blocked with pbs with 5% protifar in pbs containing 0.1% tween-20 at room temperature for 2 h. four-folds serial dilutions of mabs starting at 10 µg/ml (diluted in blocking buffer) were added and plates were incubated for 1 h at room temperature. plates were washed three times and incubated with hrp-conjugated goat anti-human secondary antibody (itk southern biotech) diluted 1:2000 in blocking buffer for one hour at room temperature. hrp activity was measured at 450 nm using tetramethylbenzidine substrate (biofx) and an elisa plate reader (el-808, biotek). the presence of mers-cov neutralizing antibodies in the sera and nasal swabs of vaccinated animals was tested using a plaque reduction neutralization assay (prnt). heat -inactivated two-fold serially diluted samples (starting 1:10) were mixed 1:1 with 400 pfu of mers-cov (emc/2012) and incubated for one hour. the mix was then overlaid on huh-7 cells in 96-well plates. following one hour of incubation, the mix was removed and the cells were incubated for 8 hr. the cells were then fixed, permeabilized and stained using a mouse anti-mers-cov n protein monoclonal antibody (sino biological) followed by an hrp-labelled goat anti-mouse igg1 (southernbiotech). the signal was developed using a precipitate forming peroxidase substrate (true blue, kpl). the immunospot® image analyzer (ctl europe gmbh) was used to count the number of infected cells per well. the neutralization titre of each serum sample was determined as the reciprocal of the highest dilution resulting in a ≥50% (prnt 50 ) or ≥90% (prnt 90 ) reduction in the number of infected cells. a titre of ≥ 20 was considered to be positive. to evaluate the protective efficacy of vaccination against mers-cov challenge, nasal swabs, and homogenated lung tissues were tested for the presence of mers-cov rna using rt-qpcr for and for the presence of infectious virus by virus titration. the presence of viral rna in nasal swabs and lung tissues was tested using upe rt-qpcr as previously described [24] . rna was extracted from samples using magnapure lc total nucleic acid isolation kit (roche). rna amplification and quantification were carried out using a 7500 real-time pcr system (applied biosystems). samples with a c t value <40 were considered positive. rna dilutions extracted from a mers-cov stock of known titre was used to generate a standard curve in order to calculate the tcid 50 equivalent of rna detected in samples. concentrations of viral rna in lung tissue are expressed in as tcid 50 equivalents per gram tissue (tcid 50 eq/ g), and in the nasal swabs as tcid 50 eq/ml. the presence of mers-cov infectious viral particles in respiratory tract samples (nasal swabs and lung tissue homogenates) was detected by titration on vero cells as described previously [24] . briefly, 10-fold serially diluted samples (starting undiluted) were overlaid on vero cells and the plates were incubated for five days at 37°c and the cytopathic effect was recorded. infectious virus titres in lung tissue are expressed as tcid 50 per gram tissue (tcid 50 /g), and infectious virus titre in nose swabs are expressed as tcid 50 /ml. lung tissue samples were collected in formalin and embedded in paraffin for pathological analysis. hematoxylin-eosin staining was carried out for histopathological analysis. the presence of mers-cov nucleoprotein was detected by immunohistochemistry as previously published [24] . statistical analyses were performed using prism 7 (graphpad software inc, usa). data were compared using mann-whitney u test or student's t-test. pvalues < 0.05 were considered significant. all data are available within the article and its supplementary information or available from the authors on request. particulate multivalent antigen display can enhance immunogenicity through different mechanisms, allowing for induction of immune responses against otherwise weakly immunogenic antigens [7, 25] . we sought to design antigens capable of inducing strong immune responses against critical parts of the viral entry and fusion machinery within the mers-cov spike protein through immune focusing and multivalent presentation on self-assembling particles (figure 1 ). within the s1 subunit, the rbd is the main target for the induction of neutralizing antibodies and has been used to develop several vaccine candidates for mers-cov [5, 26] . indeed, the immunogenicity of rbd can be enhanced by its presentation on ferritin nanoparticles [27] . likewise, the fusion peptide (fp) and the hr2, which show a high degree of sequence conservation among covs relative to the rbd, play crucial roles in the cov spike-mediated fusion machinery, and can be targets for cov protective antibodies [28] [29] [30] [31] [32] . genetic fusion was chosen for fp and hr2, due to their small size, whereas the st/sc system was used for rbd display on particles to ensure correct folding of the protein. two 60-meric hyperstable self-assembling particles with icosahedral symmetry were used for multivalent display of mers-cov domains. the lumazine synthase (ls) particle, an icosahedron with a diameter of 15 nm (pmid: 23539181) and the i3-01 (i3) particle, a dodecahedron with a diameter of 25 nm (pmid: 27309817). the n-and c-termini of both scaffolds are surface exposed, providing a platform to multivalently present (antigenic) domains. two functional segments of the s2 subunit of the mers-cov spike protein were genetically fused to these nanoparticles; the fusion peptide containing region (amino acid residues 884-898) and the hr2 containing region (amino acid residues 1215-1287) ( figure 1b, supplementary figure s1 ). chimeric nanoparticles were purified after expression in eukaryotic (mammalian) or prokaryotic systems (figure 1 ). in addition, we used the spytag/spycatcher system to multivalently display the mers-cov rbd on ls nanoparticle via covalent bonding [8] . for this purpose, the spycatcher (sc) was genetically fused to ls and expressed and purified from e. coli. the spytag (st) was genetically fused to the mers-rbd (amino acid residues 358-588) and expressed and purified from hek-293 t cells ( figure 1c ). rbd-st was incubated with ls-sc in different molar ratios to assess the optimal coupling of both components. a 1:2 molar ratio of rbd-st and ls-sc allowed the optimal coupling of all of the provided rbd-st antigens to the sc-ls particles ( figure 1d ). the resulting conjugation products were used for immunization. in order to assess the effect of the particle-based multivalent antigen display on immunogenicity, a mixture of non-coupled rbd-st and ls (without sc) was taken along for immunization in the same molar ratio. all particulate preparations displaying mers-s antigenic domains (genetically fused or sc/st coupled) were analyzed by sds-page ( figure 1e, supplementary table s1 ), confirming their molecular integrity. we further confirmed the antigenicity of the rbd-ls particles by testing their capacity to bind monoclonal antibodies directed against conformational epitopes on the rbd [20] using elisa. all antibodies bound to rbd-ls in a dose dependant manner ( figure s3 ) indicating that the rbd is correctly folded confirming its antigenicity. we then evaluated the immunogenicity of the multimeric spike antigens using six groups of rabbits (n = 5 per group), which were intramuscularly immunized twice at a 4-week interval (figure 2a ). the ls/i3 and pbs immunized groups served as controls. after the first immunization, we detected antibody responses against the corresponding s subunit (s1 or s2) in the vaccinated rabbits. while the control groups remained negative ( figure 2b -e). endpoint antibody titres for the vaccinated groups are shown as geometric mean titres (gmt) in supplementary table s2 . the antibody responses were further boosted after the second immunization in all groups, while no responses were detected in the control groups, confirming the immunogenicity of the tested antigens in rabbits. anti-s2 antibody responses were detected in the hr2 and fp vaccinated groups with weak to no mers-cov neutralizing capacity ( figure 2b,c) . only hr2 vaccination induced low levels of mers-cov neutralizing antibodies (prnt 90 titres: 20 -40) in 4/ 5 rabbits; all 5 had mers-cov neutralizing antibodies at a 50% cut-off (data not shown). likewise, both the monomeric rbd (rbd + ls) and the multimeric rbd-ls were immunogenic and elicited high s1-specific antibody titres which were further boosted after the second immunization. the rbd-ls-induced s1 antibody titres were significantly higher than those induced by the monomeric rbd following the prime-as well as booster-vaccination (p = 0.0397 and p = 0.0317, respectively by mann-whitney u test) ( figure 2d ). multimeric rbd-ls vaccination elicited higher mers-cov neutralizing antibodies, a main correlate of protection, than the monomeric rbd + ls when tested for live virus neutralization using prnt 90 assay (p = 0.0109, and p = 0.0079, post-prime and boost, respectively by mann-whitney u test) ( figure 2e ). the vaccine induced antibodies were able to neutralize clade a (emc/2012 strain; figure 2e ) as well as the more recently circulating clade b (qatar15/2015 strain; supplementary figure s4 ) viruses. the spike protein of the former strain differs from the clade a emc/2012 strain in two positions; t95s and q1020r. following a single immunization, binding antibody titres were four-fold higher and neutralizing antibodies were eleven-fold higher in the coupled multimeric rbd-ls group than in the uncoupled monomeric rbd + ls (supplementary table s2 ). three weeks after the boost, binding antibody responses were seven-fold higher (p = 0.0079, mann-whitney u test) and neutralizing antibodies were ten-fold higher (p = 0.0079, mann-whitney u test) in the coupled rbd-ls group than in the uncoupled rbd + ls ( figure 2d , e supplementary table s2 ). additionally, we tested for vaccine induced mucosal immunity in the respiratory tract of vaccinated rabbits pre-challenge (day 49) using elisa. mers-cov specific antibodies were only detected in the nasal swabs of the groups vaccinated with conjugated or non-conjugated rbd (figure 2 f,g) . antibody responses detected in the rbd-ls vaccinated group were higher than those in the rbd + ls vaccinated group (p = 0.0357, student's t-test). this demonstrates that rbd-ls induces improved local mucosal immune responses compared to the monomeric rbd. thus, vaccination with the newly produced rbd-ls mers-cov mpsp vaccines induce a robust immune response. the avidity of mers-cov spike-specific antibodies in the monomeric versus the multimeric rbd vaccinated groups was analyzed at days 28 (4 weeks after prime) and 46 (3 weeks after boost) using an ammonium thiocyanate (nh 4 scn)-displacement elisa [33] . the avidity index ic 50 was determined for each vaccinated rabbit and compared between the two groups. the avidity of the s1-specific antibody responses was higher following rbd-ls vaccination compared to the monomeric rbd + ls vaccination (p < 0.0001, student's ttest) (figure 3 ), indicating that a multimeric rbd-ls vaccine can induce antibody responses of both higher quantity and quality ( figures 2d,e and 3) . in addition to evaluating anti-s (antigen) responses, we also tested for the induction on ls-specific (scaffold) antibodies. antibody responses were elicited against the ls-particle in all groups except the pbs group, indicating that the particle was accessible and not sterically hidden by antigens displayed on its surface; even when rbd was displayed on its surface using spytag:spy-catcher linkage (figure 4 ). despite that, antigenspecific responses were not adversely affected by the presence of these anti-scaffold antibodies, as demonstrated by the booster effect after the second immunization ( figure 2d,e) . nonetheless, we tested whether a heterologous scaffold boost could help in minimizing such anti-scaffold responses using an ls/i3 primeboost scheme. using this approach, we found no significant increase in anti-scaffold antibody responses compared to the homologous prime-boost scheme ( figure 4c ). this indicates that a heterologous scaffold prime-boost approach could be advantageous for limiting unnecessary anti-scaffold responses. to evaluate the protective efficacy of the immune responses induced by the different mers-cov spike mpsp vaccines, rabbits were challenged intranasally with 10 6 tcid 50 of mers-cov (strain hcov-emc/2012) and nasal swabs were collected up to 4 days post inoculation (pi) (figure 2a ). on day 4 pi, the animals were euthanized, and lung tissue samples were collected. consistent with earlier reports [34, 35] , none of the rabbits in the control group developed any clinical signs of infection upon mers-cov inoculation, and titration of infectious virus from lung tissues and nasal swabs was variable. thus, to evaluate protection, we tested for mers-cov rna by qrt-pcr, for mers-cov infectious virus by virus titration, and for mers-cov antigen (n protein) in lung tissues by immunohistochemistry (ihc). except for the rbd-ls vaccinated group, viral rna was detected in all vaccinated groups from day 1 through day 4 postchallenge at levels similar to control groups ( figures 5 and 6 ). viral rna titres were significantly reduced in the nasal swabs of the rbd-ls vaccinated groups as early as day 1 post-challenge and were undetectable by day 4, in line with the absence of detectable infectious virus particles ( figure 5 ). viral rna was also reduced in the lungs of rbd-ls-vaccinated rabbits ( figure 6 ). consistently, ihc revealed no viral antigen in the lungs of the rbd-ls vaccinated rabbits ( figure 6c ), and antigen was also not detected a, b) the percentage of serum antibodies bound following the addition of different concentration of scn was used to determine (c) the avidity index (ic 50 ). the difference in serum avidity between both groups was tested for statistical significance using a student's t-test, with asterisks indicating the level of significance. ***p ≤ 0.001, ****p ≤ 0.0001. error bars indicate mean ± s.e.m. in the rbd + ls vaccinated rabbits. overall, in contrast to the monomeric form, the antigen-focused multimeric rbd-ls vaccine was able to block mers-cov replication significantly in the nose and lungs of the infected rabbits. the efficacy of rbd-ls immunization in protecting against a mers-cov challenge, makes it a potential vaccine candidate. however, for production at industrial scale, unnecessary sequences (e.g. tags) need to be removed, preparations have to be further structurally and biochemically characterized. recombinant subunit proteins provide advantages regarding safety, costs, and speed of vaccine production, making them very attractive platforms for the development of vaccines for emerging viruses. multivalent antigen display allows for virus-mimicking presentation of antigens and has been shown to induce antibodies of high avidity and magnitude [7, 10, 11, 27, 36] ; with non-viral self-assembling mpsp providing advantages over other multimeric antigen presentation platforms [8, 12] . among the mers-cov vaccine candidates developed so far, the latter approach has been used to design two candidates, both are based on the receptor-binding domain [27, 37] , the main target for mers-cov protective antibodies [26] . one used self-assembling ferritin nanoparticles [27] and the second used canine parvovirus (cpv) vp2 structural protein forming virus like particles [37] as scaffolds. both vaccine candidates were able to induce humoral and cellular immune responses in mice, nonetheless none has been tested for its protective capacity in a viral-challenge animal model. in our study, using an immune-focusing approach to target protective epitopes and domains along with multivalent presentation on self-assembling ls particles using a spontaneous covalent linker (spy-tag/spycatcher). we report for the first time the invivo protective capacity of a multimeric mers-cov rbd particle vaccine. we used self-assembling ls and i3 particles to generate chimeric multimeric protein scaffold particle displaying critical domains in the mers-cov spike protein and evaluated their immunogenicity and protective efficacy in rabbits. multimeric fp and hr2 vaccinations induced high levels of anti-s2 antibodies, nonetheless, with low to undetectable virus neutralizing capacities and couldn't protect rabbits against virus challenge. meanwhile, multimeric rbd-ls vaccination was highly immunogenic and induced robust antibody responses of high magnitude, avidity and neutralizing capacity. following a live virus challenge, it protected upper and lower respiratory tract of rabbits as detected by decrease in viral rna titres, with an 6 ). despite producing strong antibody responses, the monomeric rbd failed to protect rabbits against mers-cov following an intranasal challenge. the presence of ls did not seem to influence the outcome, as it was included in the formulation of the monomeric form (rbd + ls), indicating that the coupling and the multimeric presentation are responsible for the enhanced response seen with the multimeric rbd-ls vaccine. the "plug-and-display" spytag/spycatcher system [8] used to generate these multimeric rbd-ls particles allows for rapid and robust production of vaccines in a cost-effective manner. this enables the development of vaccines in a timely manner, which is crucial to prevent global public health consequences of evolving, emerging and re-emerging viruses. the efficacy of rbd-ls immunization in protecting against a mers-cov challenge, makes it a potential vaccine candidate for further development. nonetheless, in case of production at an industrial scale, unnecessary sequences (e.g. tags) need to be removed, preparations have to be further structurally and biochemically characterized. when using scaffolds as antigen carriers, anti-scaffold antibody responses need to be considered to avoid their potential to compromise the targeted antigen-induced responses or to induce potential auto-antibodies against human antigens. antibody responses were induced against the ls protein scaffold used in this study. however, antigen-specific responses were boosted following the second immunization and were not adversely affected by the presence of these anti-scaffold antibodies (figure 4 ), similar to other reports [38] . since the sequence of the ls protein does not show any similarity to any human sequences, it is unlikely that they will induce unwanted auto-(antihuman) antibodies. an ls-based vaccine for hiv, in a current phase 1 clinical trial (nct03547245), can provide further evidence for the safety of this platform. nonetheless, we developed a heterologous scaffold prime-boost using ls and i3 which can help in reducing anti-scaffold responses. a challenge facing mers-cov vaccine development is the limited number of appropriate animal models for testing protection against clinical virus isolates. rabbits provide some advantages as an animal model for mers-cov. by having the mers-cov receptor dpp4 expressed in both the upper and lower respiratory tract epithelium [24] , the rabbits can be naturally infected. this allows the evaluation of both upper and lower respiratory tract mers-cov infection and in turn protection using natural field virus isolates rather than adapted strains. however, the animals are not able to develop severe infection such as that seen in severe human cases [34] . nonetheless, severe infection, thus far, has not been established consistently in any of the other animal models without genetic modification and/or virus adaptation, except for marmosets [39] . in addition to the aforementioned, rabbits are readily available and easier to handle compared to other species that can be naturally infected such as non-human primates. following the addition of mers-cov as a priority pathogen in the who r&d blueprint for action to prevent epidemics, a target product profile was developed which called for three types of mers-cov vaccines [40] . these include one for camels to prevent virus shedding and transmission, and two for humans: a two-dose vaccine for long-term protection of those at continuous high risk such as camel handlers and health-care workers, and a single-dose vaccine for rapid onset of immune responses to protect those at acute risk in outbreak settings. the rbd-ls can be used to develop the two-dose vaccine required to protect the high-risk populations, and can be further optimized using the heterologous scaffold prime/boost scheme developed in this study. nonetheless, evaluating the longevity of the induced immune responses is warranted. following the prime, rbd-ls vaccination induced antibody responses of high avidity and mers-cov neutralizing capacity. owing to the robust immune responses induced after one dose, the rbd-ls can be a candidate for developing a rapid single-dose vaccine for mers-cov, which is required for reactive use in outbreak situations [40] . additionally, this vaccine candidate was able to block mers-cov replication in the upper respiratory tract of infected rabbit, thus it could potentially be of use as a dromedary vaccine to block mers-cov transmission. however, both approaches need to be further validated. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation list of blueprint priority diseases middle east respiratory syndrome coronavirus vaccines: current status and novel approaches influenza vaccines: from whole virus preparations to recombinant protein technology nanoparticle vaccines adopting virus-like features for enhanced immune potentiation plug-and-display: decoration of virus-like particles via isopeptide bonds for modular immunization. sci rep a sweeter approach to vaccine design innate immune recognition of glycans targets hiv nanoparticle immunogens to germinal centers induction of potent neutralizing antibody responses by a designed protein nanoparticle vaccine for respiratory syncytial virus selfassembling protein nanoparticles in the design of vaccines rational hiv immunogen design to target specific germline b cell receptors design of a hyperstable 60-subunit protein dodecahedron engineering a rugged nanoscaffold to enhance plugand-display vaccination new routes and opportunities for modular construction of particulate vaccines: stick, click, and glue identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the mers-coronavirus spike glycoprotein. emerg microbes infect importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on the middle east respiratory syndrome coronavirus spike glycoprotein to avoid neutralization escape application of built-in adjuvants for epitope-based vaccines structural analysis and optimization of the covalent association between spycatcher and a peptide tag lack of middle east respiratory syndrome coronavirus transmission in rabbits. viruses vaccine delivery: a matter of size, geometry, kinetics and molecular patterns advances in mers-cov vaccines and therapeutics based on the receptor-binding domain. viruses chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles identification of an immunodominant linear neutralization domain on the s2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure analysis of murine coronavirus surface glycoprotein functions by using monoclonal antibodies human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing characterization of neutralizing monoclonal antibodies recognizing a 15-residues epitope on the spike protein hr2 region of severe acute respiratory syndrome coronavirus (sars-cov) monoclonal antibodies targeting the hr2 domain and the region immediately upstream of the hr2 of the s protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus antibody avidity determination by elisa using thiocyanate elution asymptomatic middle east respiratory syndrome coronavirus infection in rabbits enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody nanoassembly routes stimulate conflicting antibody quantity and quality for transmission-blocking malaria vaccines. sci rep novel chimeric viruslike particles vaccine displaying mers-cov receptorbinding domain induce specific humoral and cellular immune response in mice self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies infection with mers-cov causes lethal pneumonia in the common marmoset a roadmap for mers-cov research and product development: report from a world health organization consultation we thank the technical staff of the preclinical department of viroclinics biosciences b.v. for their excellent technical support. key: cord-312434-yx24golq authors: deng, ziqin; chen, junsheng; wang, ting title: bibliometric and visualization analysis of human coronaviruses: prospects and implications for covid-19 research date: 2020-09-23 journal: front cell infect microbiol doi: 10.3389/fcimb.2020.581404 sha: doc_id: 312434 cord_uid: yx24golq human coronaviruses, which can cause a range of infectious diseases, have been studied for nearly 60 years. the field has gained renewed interest from researchers around the world due to the covid-19 outbreak in late 2019. despite a large amount of research, little is known about the knowledge structure and developing trends of this topic. here, we apply bibliometric analysis along with visualization tools to analyze 15,207 publications related to human coronavirus from the scopus database, using indicators on publication and citation, journal, country or territory, affiliation and international cooperation, author, and keyword co-occurrence cluster. the results show that research on human coronavirus is dominated by sars-cov. although there have been many publications, only 626 publications (4.1% of total) have more than 100 citations. the top 20 journals with most publications account for 20.6% of total publications and 41% of total citations. in addition to the united states and some european countries, many asian and african countries are involved in this research, with china holding an important position in this area. leading researchers from various fields of human coronavirus research are listed to facilitate collaboration and promote effective disease prevention and control. the keywords co-occurrence analysis reveals that the research focus on virology, public health, drugs and other hotspot fields, and uncovers changes in the direction of coronavirus research. the research map on human coronavirus obtained by our analysis are expected to help researchers to efficiently and effectively explore covid-19. coronaviruses, which were discovered in the 1930s (estola, 1970) and cause a range of respiratory and intestinal infections in animals and humans (geller et al., 2012) , are enveloped viruses with positive-sense single-strand rna which belong to the nidovirales order, the coronaviridae family and the coronavirinae subfamily (gonzalez et al., 2003) . the name "coronavirus" is derived from the latin corona, meaning crown or halo, and refers to the virus's characteristic appearance under an electron microscope, which appears like a crown or solar corona (almeida and tyrrell, 1967) . there are four genera contained in the coronavirinae subfamily: the alpha-, beta-, gamma-, and deltacoronavairus. these viruses can infect not only birds (gamma-and deltacoronaviruses), but also a range of mammalian species (mainly alpha-and betacoronaviruses), including humans (corman et al., 2018) . coronaviruses have been known for more than 80 years (saif, 2004) , while only seven of them have been proven to infect humans so far. plenty of evidence has strongly suggested that human coronaviruses exist with or origin from livestock or some wild animals (corman et al., 2018) . in this case, diseases caused by coronaviruses can also be defined as a kind of zoonosis. human coronavirus (hcov) had not been considered as a highly pathogenic virus to humans until the severe acute respiratory syndrome (sars) outbreak in 2002 (peiris et al., 2003) and the middle east respiratory syndrome (mers) outbreak in 2012 (zumla et al., 2015) . recently, another coronavirus disease, the covid-19, caused by a novel coronavirus, sars-cov-2 (ciotti et al., 2019; tan et al., 2020) , has evoked these painful memories and made people more intensely aware of the pathogenic potential of these microorganisms (cui et al., 2019) . this is the seventh coronavirus identified so far to infect humans, with the others being hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, sars-cov, and mers-cov (geller et al., 2012) . hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku1 are able to cause common cold in humans and majority of these infections only manifest mild symptoms in respiratory system (mackay et al., 2012; owusu et al., 2014; annan et al., 2016) . however, sars-cov and mers-cov are more serious and responsible for high case fatality rates, both of whom belong to genus beta. once the case-fatality rate of sars was ∼10% (cheng et al., 2007) , while that of mers ranged around 35% 1 . similar to sars-cov and mers-cov, sars-cov-2 is also one of the betacoronaviruses, but with quite unique qualities zhu et al., 2020) . the clinical casefatality rate is not as severe as that of sars-cov and mers-cov, but it is more infectious than either virus (chan et al., 2020; huang et al., 2020) . as of 3:05 pm cest, 2 july 2020, there have been 10,533,779 confirmed cases of covid-19, including 512,842 deaths, reported to who 2 . and the virus had also spread to 213 countries and territories around the world and 2 international conveyances 3 by august 19, 2020, 09:28 gmt. scientists have been studying the human coronavirus since its discovery in the 1960s (hamre and procknow, 1966; bradburne et al., 1967; mcintosh et al., 1967) , and more than 15,000 papers related to coronavirus have been published (according to our search) (supplementary figure 1) . however, researchers need to devote a significant amount of time to reading and identifying relevant work in related fields due to the long research interval, large amount of data, uneven quality of scientific research papers, the presence of unnecessary duplications, and the differences between human coronaviruses and emerging viruses. therefore, it is particularly urgent to sort out important, effective and meaningful information from large databases in order to guide the scientific research, and promote the proper prevention, control, diagnosis and treatment of human coronavirus. bibliometrics, together with novel visualization methods of scientific information, have been reported to be helpful to identify emerging outbreaks of infectious disease. this is particularly true in the current age, where thousands of reports can be easily exchanged between public health specialists and healthcare providers across the internet (takahashi-omoe and omoe, 2012; unkel et al., 2012) . bibliometrics is also recognized as an essential tool and is widely used in a variety of fields to measure and evaluate scientific research quantitatively and qualitatively (aggarwal et al., 2016; romero and portillo-salido, 2019; deng et al., 2020) . therefore, in order to accurately, effectively and systematically reveal connections within the human coronavirus field, our study applied bibliometrics and visualization methods to analyze human coronaviruses-related publications and citations, countries and affiliations, as well as journal performance, author impact and keyword cooccurrence cluster. this study seeks to serve as a valuable reference and guidance for virologists, pharmacists, clinicians and epidemiologists studying the emerging human coronavirus, and to provide novel ideas for finding effective control measures, as well as drugs and vaccines, as soon as possible. our analysis was conducted using the online database scopus (https://www.scopus.com/), which provides a comprehensive collection of different types of scientific peer-reviewed literature (romero and portillo-salido, 2019; bonilla-aldana et al., 2020) . the document search was performed on february 15, 2020. in order to cover as many target documents as possible, we had selected terms that might be used by most scientific publications before the search formula was designed: besides "human coronavirus, " "novel coronavirus" and their abbreviation forms "hcov" and "ncov, " we have the terms "human coronavirus 229e" ("hcov-229e"), "human coronavirus oc43" ("hcov-oc43"), "human coronavirus nl63" ("hcov-nl63"), and "human coronavirus hku1" ("hcov-hku1") to search for publications related to these four currently known nonseverely pathogenic human coronavirus; for sars related publications, we have "severe acute respiratory syndrome-related coronavirus" ("sarsr-cov" or "sars-cov") and "severe acute respiratory syndrome" ("sars"); for mers related publications, we have "middle east respiratory syndrome-related coronavirus" ("mers-cov") and "middle east respiratory syndrome" ("mers") as well as "mers coronavirus erasmus medical center/2012" ("mers coronavirus emc/2012, " "hcov-emc/2012, " or "emc/2012"), "novel coronavirus-2012" ("2012-ncov") and "camel flu"; as for the covid-19 related publications, we have "severe acute respiratory syndrome coronavirus-2" ("sars-cov-2"), "coronavirus disease-2019" ("covid-19"), "2019-novel coronavirus" ("2019-ncov"), "2019-ncov acute respiratory disease, " "novel coronavirus pneumonia" and "novel coronavirus-infected pneumonia." several writing formats that are not generally accepted, like "corona virus" and "hcov229e, " were also taken into consideration after we had found them been used in some publications. notably, when directly using the abbreviation "sars" and "mers" to search, we were provided with a massive number of unrelated results in other fields. we decided to add qualifiers as limitation to these terms to have the accuracy of search optimized ( table 1) . and finally, we conducted the search with the following formula, designed according to the search rules of scopus (deng et al., 2020) : title-abs-key ("human coronavirus" or "human corona virus" or hcov or "novel coronavirus" or "novel corona virus" or ncov or "severe acute respiratory syndrome" or ({sars} and " * cov" or * virus or crisis or outbreak or epidemic or pandemic) or "sarsr-cov" or "middle east respiratory syndrome" or ({mers} and " * cov" or * virus or crisis or outbreak or epidemic or pandemic) or "camel flu" or "emc/2012" or "coronavirus disease 2019" or "covid-19" or (coronavirus or "corona virus" or " * cov" and 229e or oc43 or nl63 or hku1) or hcov229e or hcovoc43 or hcovnl63 or hcovhku1) ( table 1) . all search results, data and information that were essential for our analysis, including the count of citations and some indicators, were extracted from scopus on february 15, 2020. complete document lists were exported as csv files, which were then imported into microsoft excel 2016 for ranking and counting. bar charts were made by graphpad prism 8, and vosviewer 1.6.12 was used to generate the visualization maps. in the analysis of country (or territory), affiliation and author, every co-author was counted equally. in this case, documents with too many authors are not comparable to documents with fewer authors. since only documents with a maximum of 15 authors were counted by scopus, we followed the same strategy when conducting the visualization analysis with vosviewer. our search yielded 15,979 results, based on their titles, abstracts and key words. then we limited our results to publications in english and chinese, which yielded 15,207 documents in total, including 9,182 articles, 2,170 reviews and 3,855 documents of other types (supplementary figure 1) . as expected, the annual publications count was naturally divided into four sections, due to three notable epidemic events in history. before the outbreak of sars, the annual publication amount remained low, reaching a maximum of 29 in 1998. it was not until the sars crisis occurred in 2002 that the scientific publications in this field experienced explosive growth. the annual publication number rose suddenly to 1,729 in 2003 and continued to rise the following year, when it reached 1,754 publications. for the next few years, the publication count declined gradually, reaching a low of 488 publications in 2011. it was the first outbreak of mers in the middle east that led to renewed interest in coronavirus and an increase in publications. this lasted for 4 years, during which time the annual number reached another peak, 838 publications in 2015, coinciding with the second outbreak of mers in south korea. notably, the annual publication number has continued to decline since then. for the publications on human coronavirus in 2020, only publications released prior to february 15 were counted. interestingly, the trend for summed citation of annual publications closely aligned with annual publication production prior to 2013 (figure 1 ). for our citation analysis, we counted the number of publications with different citation amounts. we found that more than half of the publications, up to 9,383 (61.7%, 4,991 articles, 1,075 reviews), had been cited no more than 10 times, including 3,336 that had gone uncited (21.9%, 1,373 articles, 308 reviews). only 4.1% of publications (626 publications, 432 articles, 161 reviews) had more than 100 citations, and only 206 publications (1.4%) and 46 publications (0.3%) had been cited more than 200 and 500 times, respectively (supplementary figure 2) . the top 20 most cited articles and non-articles are listed separately in table 2 and supplementary table 1 . among the top 20 articles, six were published in the new england journal of medicine, four in science, three in nature, two in the lancet and the remaining five articles were published in five other journals. in total, these 20 articles were published in nine different journals. most of them were published after 2002, corresponding to the time of the sars outbreak. the research areas of the top 20 articles involve public health, preventive medicine, epidemiology, clinical reports and virological study, including the identification, isolation, and analysis of natural hosts of these coronaviruses. all english and chinese documents came from 3,443 different journals. the 21 journals with the most publications on human coronavirus are listed in table 3 . this list includes four journals that published articles that were in the top 20 article list (table 2) (new england journal of medicine, science, nature and lancet). these 21 journals produced 20.6% (3,126) of all publications analyzed here and 41.0% of the total citations (140,450 of 342,451). among them, the new england journal of medicine had the highest values in two indicators, citations per document and citescore (2018). also, notably, contributions made by chinese journals like hong kong medical journal and chinese medical journal cannot be ignored. figuring out core journals in a specific research field is of considerable importance. this can not only help academic achievements be known and used as much as possible, but also provide those who need the information with more concentrated access. the united states has dominated this field with 143,960 citations on its 4,225 published documents. china ranked second with a total of 49,316 citations for its 2,720 documents from researchers in mainland china, 48,828 total citations on 1,411 documents from researchers in china hong kong and 13,408 total citations on 646 documents from researchers in china taiwan. the united kingdom and canada have also contributed significantly to this research area ( in chan, leo l.m. poon and guan yi, which have all garnered high citation counts, cannot be ignored. the studies of those active and emerging researchers with unique ideas rather deserves our attention. it inspired us that more comprehensive and detailed evaluation of researchers will help to identify their specific areas of expertise, and therefore make their work more valued when applicable. a co-occurrence relationship is formed between two keywords when they both occurred in the same paper. keywords with strong co-occurrence relationship can reveal research hotspots more precisely than a single keyword. in our visual representation, strongly connected keywords were colored the same, indicating that they might share something in common. keywords shown in red are roughly connected to public health, preventive medicine and epidemiology. according to these keywords, human coronavirus diseases like "sars, " "mers" and covid-19 may have something worthwhile for comparison with other "infectious diseases" like "influenza" in their epidemiological characteristics; "healthcare workers, " "transmission, " "surveillance, " "quarantine, " or "isolation" may be the focuses of these studies, which can help to promote current disease control and prevention measures. keywords in blue and yellow are related to virus detection and clinical diagnosis. among them, "serology" and "rt-pcr" are likely to be the methods, the research objects are mainly some kinds of "respiratory viruses, " and the research purposes are mostly on "evolution, " "phylogeny, " and "diagnosis." the words in green are mainly virology-related and also include some immunological and pharmaceutical research: "spike protein, " "nucleocapsid protein, " "receptor-binding domain, " "ace-2" and "apoptosis" are mainly on "pathogenesis, " while "epitope, " "antibody, " "vaccine, " "inhibitor, " "interferon, " "ribavirin, " and "antiviral activity" are mainly about antiviral solutions (figure 4) . with keyword co-occurrence analysis, we can not only figure out the hotspots, but also discover the limitations, and even come up with new ideas. with the outbreaks of sars in 2002 and mers in 2012, along with current covid-19 pandemic (harapan et al., 2020) , the world has battled three serious human coronavirus outbreaks during the twenty first century. in particular, the covid-19 outbreak has surpassed the previous two ones in terms of its transmission scale, largely due to the strong infectivity and long asymptomatic incubation period of the emerging pathogen (epidemiology working group for ncip epidemic response, chinese center for disease control and prevention, 2020). human coronavirus has been an area of concern for many years, with the first study conducted almost 60 years ago and plenty of scientific documents published in the decades since. however, when the novel virus emerged, controlling the spread of this virus effectively and promptly remained a significant challenge. this is partially due to the unclear knowledge map of human coronavirus research and an inadequate understanding of the present research status, hotspots and development trends. this can result in a large amount of repetitive, insignificant or inefficient research work, which can prevent effective analysis of this virus and further delay the development of targeted prevention and control measures. therefore, we applied an innovative bibliometric and visualization analysis to sort and analyze more than 15,000 human coronavirus-related scientific publications spanning 60 years, with the purpose of mapping and managing previous research in this field. the trends in the annual number of publications and summed citation of annual publications on human coronaviruses (figure 1) reflect the interest and development of this area over the years (durieux and gevenois, 2010) . from 1965 to 2002, the number of publications was low and remained at a stable amount. strikingly, two explosive growth peaks appear separately in 2003 and 2012, matching the onset of the sars and mers outbreaks. this sharp growth seen at these times is reflective of the severe impact of emerging coronaviruses on human health. the number of publications is directly proportional to the extent and spread of the infective disease outbreak (hurtado et al., 2018) . the peak that emerged during the sars crisis is significantly higher than the one that emerged during the mers outbreak, which is due to the more widespread outbreak and more extensive impact of sars. considering the severity of the covid-19 outbreak, we can predict that this trend will continue and a large amount of publications will follow. meanwhile, after the short mers outbreak, the summed citation of annual publications dropped quickly and became out of sync with the number of annual publications, which might reflect a gradual declining interest in human coronavirus research. however, it is believed that the covid-19 outbreak caused by the emerging sars-cov-2 will bring the research on human coronavirus back to forefront. just as we predicted, the number of literatures on sars-cov-2 and covid-19 has doubled in the past 6 months, according to our latest online-searching in august. this has proven that the consequences of covid-19 are so catastrophic and far-reaching that it has attracted attention worldwide. the citation number of an article represents the extent of its dissemination and influence, and thus partly reflects its quality as well (muniz et al., 2018) . although there have been over 15,000 publications on human coronavirus, only 626 (4.1%) have more than 100 citations, while 9,383 (61.7%) have <10 citations (supplementary figure 2) . this indicates that although there has been much research on human coronavirus, there might be only a small percentage of these studies that are of high quality. it also indicates that the research field is wide but not deep, implying that much remains for researchers to study about this virus, and in-depth exploration in some specialized areas could shift the focus and direction of this field for the future. among the top 20 articles listed in table 2 , six were published in the new england journal of medicine, four in science, three in nature, two in the lancet and the other five articles were published in five different journals. these nine different journals are very responsive to novel emerging viruses and related diseases. moreover, 18 of these top articles are related to the sars outbreak and only one is connected to the mers outbreak. for the top 20 non-article publications listed in supplementary table 1 , there were up to 18 different journals in the list, indicating the attention received by these publications, all of which are related to sars research, from other sources. therefore, sars-cov plays an important and enlightening role in analyzing the research on human coronavirus. despite the fact that all english and chinese publications were obtained from 3,443 different sources, the top 20 journals with the most published documents account for 20.6% of all publications and 41.0% of all citations. this is reflective of the authority of these journals, as well as their high degree of interest in research related to human coronaviruses. due to the limitations of scopus, journals can only be sorted by the total number of published documents. however, as shown in table 3 , the new england journal of medicine, proceedings of the national academy of sciences of the united states of america, science, nature and the lancet rank in the top five based on citations per document, which is consistent with the results in table 2 . popular journals and their research trends in a particular field provide researchers with reliable references. in addition, core journals provide researchers with faster search routes and can serve as an important publication guide (zhuang et al., 2014; wang et al., 2019) . the united states, the united kingdom, germany and other european countries are usually the most active countries at the forefront of scientific research (sweileh, 2017) . however, as can be seen from figure 2a , the situation is quite different in the human coronavirus field. the united states remains dominant in this field, while some asian and african countries, such as china, singapore, saudi arabia, south korea, japan, india and egypt ranked in the top 20 for human coronavirus-related research. this implies that the research on emerging viruses is no longer limited within those developed countries with more developing countries getting involved. one possible factor lies in the wide and fast spread of the diseases, which has enabled more and more people have come to realize the urgency of taking timely actions and having a clear understanding of emerging infectious diseases. additionally, advancements in science and technology, as well as increased funding support from national policies in these regions have been instrumental in discovering and analyzing emerging viral pathogens. in particular, the top 20 affiliations with the highest number of documents published (figure 3, supplementary table 3 ) reflect chinese scientists' contributions to the human coronavirus field, with nearly a half of the top 20 affiliations coming from china. similarly, a recent article from stanley perlman also praised the contribution of chinese scientists to the study of novel human coronaviruses (perlman, 2020) . here, we generated a map to show the contributions of different countries to the human coronavirus field and display the cooperative relationship between countries (or territories) intuitively ( figure 2b ). this map indicates that international cooperation efforts between coronavirus researchers are led by the united states, china, china hong kong, the united kingdom and canada. these results can provide significant guidance for initiating collaborative projects, project applications, and academic exchanges, as well as providing information that can be used in the political sphere in different countries, territories and affiliations concerned with the impact of human coronavirus (fiala, 2012) . as for the covid-19 pandemic, the lack of information sharing and collaborative efforts is very detrimental to the prevention of diseases. countries and affiliations are supposed to take on the responsibilities to strengthen mutual trust and international cooperation, so that we can fight against this fatal virus with more joint efforts. analyses of author can help to understand a research field in a more comprehensive manner, and to evaluate the contributions of researchers, as well as their research level and academic status in this field, objectively (podsakoff et al., 2008) . in our study, the top 20 authors with the most publications were used to screen out those active researchers in this area. as shown in table 4 , yuen kwok-yung has produced the largest number of documents, guan yi has the highest number of citations per document, and joseph sung jao-yiu currently has the highest hindex. these data and indicators provide different perspectives on the research level and academic authority of each researcher. interestingly, consistent with figure 3 , table 4 reveals half of the top 20 researchers are from china, which also reflects the more active state of chinese researchers on human coronaviruses. it should be noted that among all the authors, some are researchers from research institutions at universities, some are doctors from hospitals, and some are experts from the centers for disease control and prevention (cdc), etc. with the current covid-19 outbreak, it is vital for scientists, clinicians and cdc experts to share and exchange information, and to develop a united approach in emerging viral disease outbreak responses and control (wang et al., 2020a) . in addition, we have learned from the covid-19 outbreak that at the beginning of a sudden outbreak, the allocation of emergency expert personnel and the expertise, authority and experience of these experts both have a significant impact on the spread of the epidemic. therefore, it is necessary to consult and analyze the literature in order to identify these leaders ahead of another novel human coronavirus emergency. our study is able to supplement and complement these efforts. a large amount of meaningful information can be obtained from keyword co-occurrence analysis, which could enable the identification of hotspots and trends, and guide researchers to related topics in their field (romero and portillo-salido, 2019) . in figure 4 , four clear research fields within human coronavirus can be seen, which mainly involve aspects of public health, preventive medicine and epidemiology, clinical work and pharmaceutical research. by contrast, the studies on tracing, evolution and animal carriers of human coronavirus account for only a small proportion of the studies performed, suggesting that the research in these areas is still insufficient and there is room for growth. in our study, it is demonstrated that current research on sars-cov-2 and covid-19 are mainly focused on prevention and treatment. although there is former experience of sars and mers to learn from, people are still likely to get overwhelmed at first in face of global public health emergencies like this, for its rapid spread on a grand scale (peeri et al., 2020) . promoting the correct use of protective masks in public places, encouraging a safe social distance and avoiding crowd gathering are all effective alternatives to prevent large-scale public transmission (gasmi et al., 2020) . and enhancing medical stuff with reasonable allocation of medical resources (emanuel et al., 2020) , separating different diagnosis and treatment areas, and providing centralized isolation areas for mild patients if necessary (hellewell et al., 2020) can all help to ease the shortage of medical resources and avoid nosocomial infections. with present absence of specific antiviral drugs or vaccines to covid-19, one vital step for now is to prevent the spread of sars-cov-2. based on reported data, the effective reproductive number (r o ) of the sars-cov-2 was estimated to be 2.2 approximately , which means that each infected individual can transmit the infection to more than two healthy individuals. it has been indicated that sars-cov-2 may attach the angiotensin converting enzyme 2 (ace2) receptor with its spike protein, in the way similar to sars-cov, to enter target cells (lu et al., 2020; walls et al., 2020) . however, it is still not sufficient enough to explain the high infective efficiency. further research has revealed some possible reasons. on one hand, the case fatality rate of covid-19 is much lower than that of sars ; on the other hand, x-ray crystal diffraction has implied that the combination between s protein of sars-cov-2 and ace2 is a little stronger than that of sars-cov (lan et al., 2020; shang et al., 2020) . in fact, different from sars-cov, significant mutation in the s protein of sars-cov-2 has been identified by genomics analysis, and a specific furin-like protease also plays an essential role in recognition, entry, stability and transmission of the coronavirus (coutard et al., 2020; drak alsibai, 2020) . in addition, neutralizing antibody against the virus has also been a hotspot in human coronavirus research nowadays. as for to sars-cov and mers-cov, several types of representative neutralizing antibodies such as monoclonal antibodies, their functional antigen-binding fragment and the single-chain variable region etc., has been found to block the binding between receptor-binding domain(rbd) region of s protein with ace2 receptor and then to inhibit the infection (jiang et al., 2020) . this inspires us that the rbd region may be noteworthy when studying the neutralizing antibodies induced by viruses or vaccines. a recent study has reported a human monoclonal antibody 47d11 which targets a conserved epitope of the sars-cov-2 s-s1b domain, and might play a role in the prevention and treatment (wang et al., 2020b) . convalescent sera have been applied to treating covid-19, but attention needs to be paid to the phenomenon of antibody-dependent enhancement (ade) caused by non-neutralizing antibodies which target in non-rbd regions (du et al., 2009) . although there are a range of studies on the origin and transmission of human coronaviruses, especially sars-cov, mers-cov, and sars-cov-2, current evidences are still not so convincing enough. despite of this, it is widely accepted so far that these three coronaviruses are able to transmit from person to person. they seem to origin from bats and need some intermediate host to facilitate replication, evolution, and variation, and thus can infect human directly. civet cats and dromedary camels are supposed to act as intermediate hosts for sars-cov and mers-cov, respectively azhar et al., 2014) . according to genome sequencing and evolutionary analysis, bats are also speculated to be the natural host of sars-cov-2 (guo et al., 2020) , while turtles, pangolin and snakes are alternatively possible to serve as the intermediate host for its transmission from animal to human (liu et al., 2020) . study on the origin of the human coronavirus is extremely significant for better understanding, prevention and control of relevant diseases. as it may become the focus of research on emerging human coronaviruses in the near future, more experts, material and financial resources may be urgently needed. in general, previous research on sars-cov and mers-cov can provide important guidance for the study of sars-cov-2 and speed up the development in therapeutic drugs and vaccines. although the keyword co-occurrence analysis shows the hotspots and trends within human coronavirus research, it also indicates that some fields remain unexplored or underexplored. for example, the relationship between human coronavirus and immune metabolism, the application of rna-seq and single cell sequencing technology in coronavirus research, and the possibility of cocktail therapy in viral treatment have not been studied extensively. due to the short duration of the recent covid-19 outbreak, studies on sars-cov-2 were insufficient in various fields. keyword co-occurrence analysis could help researchers studying sars-cov-2 by providing abundant potential directions in similar fields with other human coronaviruses and revealing the cross-disciplinary exploration potential. today we are experiencing an information data explosion. because researchers have been studying coronavirus for 60 years, there is a massive and complicated array of data. thus, being able to benefit from such an unprecedented amount of data without being overwhelmed poses a significant obstacle for researchers, especially when facing the emergency of a novel infectious disease outbreak, such as covid-19. a bibliometric analysis combined with data visualization is critical for exploring and communicating information effectively, and helping researchers to continue to progress (wong, 2012) . for this reason, we applied bibliometric and visualization methods to analyze 15,207 documents of human coronavirus-related studies from the scopus database using various indicators. we hope that our study will indicate potential directions for scientists to explore, promote cooperation with other human coronavirus researchers across disciplines, guide emerging researchers toward specialities that have yet to be fully developed, enable the development and use of new technologies in this field, and provide valuable ideas for the prevention, diagnosis and treatment of covid-19. datasets generated for this study will be made available by the authors, to any qualified researcher upon request. the state of lung cancer research: a global analysis the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture similar virus spectra and seasonality in paediatric patients with acute respiratory disease, ghana and germany evidence for camel-to-human transmission of mers coronavirus sars-cov, mers-cov and now the 2019-novel cov: have we investigated enough about coronaviruses? -a bibliometric analysis effects of a "new" human respiratory virus in volunteers a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection covid-19 outbreak: an overview hosts and sources of endemic human coronaviruses the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade origin and evolution of pathogenic coronaviruses bibliometric analysis of dendritic epidermal t cell (detc) research from 1983 to 2019 expression of angiotensin-converting enzyme 2 and proteases in covid-19 patients: a potential role of cellular furin in the pathogenesis of sars-cov-2 the spike protein of sars-cov-a target for vaccine and therapeutic development bibliometric indicators: quality measurements of scientific publication fair allocation of scarce medical resources in the time of covid-19 the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china coronaviruses, a new group of animal rna viruses bibliometric analysis of citeseer data for countries individual risk management strategy and potential therapeutic options for the covid-19 pandemic human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies a comparative sequence analysis to revise the current taxonomy of the family coronaviridae isolation and characterization of viruses related to the sars coronavirus from animals in southern china the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status a new virus isolated from the human respiratory tract coronavirus disease 2019 (covid-19): a literature review feasibility of controlling covid-19 outbreaks by isolation of cases and contacts clinical features of patients infected with 2019 novel coronavirus in wuhan evaluating the frequency of operational research conducted during the 2014-2016 west africa ebola epidemic neutralizing antibodies against sars-cov-2 and other human coronaviruses structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 recovery in tracheal organ cultures of novel viruses from patients with respiratory disease citation analysis and trends in review articles in dentistry human coronaviruses associated with upper respiratory tract infections in three rural areas of ghana the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? coronavirus as a possible cause of severe acute respiratory syndrome another decade, another coronavirus scholarly influence in the field of management: a bibliometric analysis of the determinants of university and author impact in the management literature in the past quarter century trends in sigma-1 receptor research: a 25-year bibliometric analysis animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? structural basis of receptor recognition by sars-cov-2 global research trends of world health organization's top eight emerging pathogens worlwide trends in infectious disease research revealed by a new bibliometric method a novel coronavirus genome identified in a cluster of pneumonia cases -wuhan statistical methods for the prospective detection of infectious disease outbreaks: a review structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a human monoclonal antibody blocking sars-cov-2 infection from hendra to wuhan: what has been learned in responding to emerging zoonotic viruses tracking knowledge evolution, hotspots and future directions of emerging technologies in cancers research: a bibliometrics review points of view: visualizing biological data a novel coronavirus from patients with pneumonia in china efficient and robust large medical image retrieval in mobile cloud computing environment middle east respiratory syndrome tw designed the study. zd and jc performed the search. tw, zd, and jc analyzed the data and wrote the manuscript. all authors contributed to the article and approved the submitted version. this work was supported by the national natural science foundation of china (81000738 to tw) and the national undergraduate innovation funding of huazhong university of science and technology (201910487117 to tw, 2019a0199 to tw). the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2020.581404/full#supplementary-material the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 deng, chen and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-338538-uea9kwge authors: shehata, mahmoud m.; mostafa, ahmed; teubner, lisa; mahmoud, sara h.; kandeil, ahmed; elshesheny, rabeh; boubak, thamer a.; frantz, renate; pietra, luigi la; pleschka, stephan; osman, ahmed; kayali, ghazi; chakraborty, trinad; ali, mohamed a.; mraheil, mobarak abu title: bacterial outer membrane vesicles (omvs)-based dual vaccine for influenza a h1n1 virus and mers-cov date: 2019-05-28 journal: vaccines (basel) doi: 10.3390/vaccines7020046 sha: doc_id: 338538 cord_uid: uea9kwge vaccination is the most functional medical intervention to prophylactically control severe diseases caused by human-to-human or animal-to-human transmissible viral pathogens. annually, seasonal influenza epidemics attack human populations leading to 290–650 thousand deaths/year worldwide. recently, a novel middle east respiratory syndrome coronavirus emerged. together, those two viruses present a significant public health burden in areas where they circulate. herein, we generated a bacterial outer membrane vesicles (omvs)-based vaccine presenting the antigenic stable chimeric fusion protein of the h1-type haemagglutinin (ha) of the pandemic influenza a virus (h1n1) strain from 2009 (h1n1pdm09) and the receptor binding domain (rbd) of the middle east respiratory syndrome coronavirus (mers-cov) (omvs-h1/rbd). our results showed that the chimeric antigen could induce specific neutralizing antibodies against both strains leading to protection of immunized mice against h1n1pdm09 and efficient neutralization of mers-cov. this study demonstrate that omvs-based vaccines presenting viral antigens provide a safe and reliable approach to protect against two different viral infections. acute respiratory infections are among the leading causes of disease and mortality in developing and developed countries [1, 2] . the severity of these acute infections is usually potentiated following the dissemination of the infection throughout the lower respiratory tract, leading to millions of human deaths worldwide each year [3] . annually, seasonal influenza epidemics attack 10-20% of the human population leading to 290-650 thousand deaths/year worldwide [4] . beside these epidemics, the world is confronted every 10-40 years with antigenically distinct pandemic influenza virus strains of wide geographical distribution and considerable human-to-human transmissibility resulting in high mortality rates [5] . in recent years, the world has been challenged with newly emerging influenza a virus (iav) infections, which have the potential to cause sporadic fatalities in the human population within limited epidemics. for instance, highly pathogenic avian influenza viruses (hpaiv) of the h5n1-subtype and low pathogenic avian influenza viruses (lpaiv) of the h7n9-, h10n8-and h6n1-subtypes [6] [7] [8] [9] have caused sporadic human infections. in addition, other iavs caused global pandemic outbreaks, such as the 2009 swine-origin h1n1 influenza virus (h1n1pdm09) [10, 11] . in 2012, a novel middle east respiratory syndrome coronavirus (mers-cov) emerged. by february 2019, a total of 2374 laboratory-confirmed human cases, including 823 associated deaths, were reported globally in 27 countries (case-fatality rate: 35.4%) [12] . the majority of these cases were reported from the arabian peninsula, specifically saudi arabia (cases = 1896; deaths = 732; case-fatality rate = 38.6%) [12] . to combat iav and mers-cov infections, vaccination represents an affordable and a facile way to protect against devastating epidemics and occasional pandemics. however, despite significant efforts to develop a safe and effective vaccine [13] , there are no approved vaccines for mers-cov till now. recent reports have also demonstrated that replication of recombinant iav vaccine strains in either embryonated eggs or in cell-culture systems allows viral adaptation, which may affect the antigenicity of the vaccine [14] [15] [16] . therefore, genetically and phenotypically stable vaccines represent a promising alternative to control iav and mers-cov infections [14] . outer membrane vesicles (omvs) are natural, spherical nanoparticles (50-250 nm) derived from gram-negative bacteria. omvs are released from both pathogenic and non-pathogenic bacteria and are highly immunogenic due to their components, including lipopolysaccharides (lps), bacterial outer membrane (om) proteins, lipids, immunogenic toxins, dna/rna and other periplasmatic and cytoplasmatic proteins [17, 18] . omvs from pathogenic bacteria have been commercially used to induce specific antibodies against different bacterial strains, including neisseria meningitidis serogroup b [19] . the composition of the omvs can be adapted and used as a vaccine platform via incorporation of heterologous antigens into the vesicles [20] . this engineering approach is advantageous because (i) it retains the antigens in their native conformation, (ii) it enables the omvs to target specific immune responses, and (iii) it provides multiple and commensurate protein antigens in a single production process [17] . however, bacteria-based vaccines are not well explored to deliver viral antigens. therefore, we engineered a stable omvs-based dual vaccine against h1n1pdm09 and mers-cov by producing omvs with a chimeric hemagglutinin (ha) comprising of both ha1 and ha2 from the h1n1pdm09 and the receptor binding domain (rbd) of mers-cov. mers-cov strain was isolated and grown in vero-e6 cells. the two viruses were used for preparation of the omvs-based dual vaccine and inactivated vaccines were used as positive control. influenza to construct the pmp-h1/rbd plasmid, three pcr fragments (f1, f2, and f3) encompassing (i) 5'-ncr and signal peptide of ha from cal-h1n1pdm2009, (ii) rbd of mers-cov and a 7-amino acid/peptide linker (gsagsag), (iii) the coding sequence and 3'-ncr of ha were amplified with sequence-specific primers (table 1) and phusion high-fidelity pcr master mix with hf buffer (invitrogen, carlsbad, ca) and then simultaneously ligated into linearized pmpccdb vector [21] . briefly, for the pcr amplification of each fragment, 25 µl of 2× phusion master mix, 2.5 µl of forward and reverse primers (10 µm/µl), and 50 ng of the according template dna were mixed and the reaction was then brought to a total volume of 50 µl using rnase-/dnase-free ddh 2 o. the plasmid pmp-ha-gi [21] encoding the ha of cal-h1n1pdm2009 was used as template dna for f1 and f3, while the plasmid pcdna3.1-spike-mers-cov encoding the spike protein from the isolate mers-cov/camel/egypt/hku-nrce-270/2013 was used as a template for f2. the pcr settings were: 95 • c for 1 min then 3 steps of 40 cycles (95 • c for 10 s, 56 • c for 30 s, and 72 • c for 2 min), with a final extension step at 72 • c for 10 min. the three amplified pcr fragments were then loaded onto a 1% agarose-gel for electrophoresis. separation and purification of the three specific fragments was done by using qiaquick gel purification kit according to manufacturer's (qiagen, germany) instructions. after purification the three fragments were digested by corresponding restriction enzymes, shown in table 1 . ligation of the three fragments and the linearized vector was performed using t4 dna ligase (promega, madison, wi, usa) by adding 5 µl of each purified fragment (20 ng/µl) to 2 µl 10× buffer, 2 µl t4 dna ligase, and 1 µl linearized vector (20 ng/µl). the mixture was then incubated overnight at 4 • c. transformation of escherichia coli dh5-α competent cells was performed by mixing 5 µl of the ligation reaction with 50 µl bacterial suspension (invitrogen, ca, usa) and subsequent incubation on ice for 30 min. the bacterial cells were then subjected to heat shock at 42 • c for 30 s in a water bath and were then chilled on ice for 2 min before adding 250 µl of soc media (invitrogen, ca, usa). the reaction tubes were then rotated at 250 rpm in a shaking incubator at 37 • c for 1 h. after an incubation time of 1 h, 100 µl of the transformed bacterial suspension was spread on ampicillin containing luria-bertani (lb) agar plate and incubated for 16 h at 37 • c. single colonies were then selected and incubated in 5 ml liquid lb for 16 h for subsequent plasmid isolation and the correct e. coli dh10ß competent bacteria (invitrogen, ca, usa) were transformed with pmp-h1/rbd according to manufacturer's instructions as described above with 20 ng of pmp-h1/rbd plasmid. individual colonies from ampicillin containing lb agar plate were picked and incubated in 5 ml liquid lb for 16 h. these cultures were then used to inoculate the large-volume cultures in the next step. omvs are typically purified from supernatants of transformed e. coli dh10ß cells. to this point, 2 liters (4 × 500 ml) of dh10ß cultures (inoculated with 5 ml of the starter culture) were grown in lb broth at 37 • c in an orbital shaking incubator at 180 rpm until reaching the exponential phase (od 600nm 1.0). the grown bacteria were pelleted at 6000× g for 15 min and the supernatant was sterile-filtered (millipore express plus membrane filter, pes, 0.22 µm) to remove residual bacteria. afterwards, the bacteria free supernatant was concentrated by ultrafiltration using krosflo research ii tff and a 100 kda hollow fiber membrane (spectrum labs, germany) to a final volume of 30 ml. the resulting filtrate (30 ml) was subjected to further ultracentrifugation at 150,000× g for 3 h and 4 • c in a sw41 ti rotor (beckman, ga, usa) to separate the omvs fraction. subsequently, the omvs containing pellet was resuspended in 300 µl pbs (dulbecco's phosphate buffered saline, biochrom gmbh), sterile filtered (millex-gv syringe filter unit, pvdf, 0.22 µm) and stored at −80 • c until use. the amount of isolated omvs was quantified by protein concentration measurement using bradford protein assay. enriched omvs (5 µg) from cultured dh10ß, either transformed with empty vector (control) or with pmp-h1/rbd, were mixed with 10 µl 4× sds sample buffer (40% glycerol, 240 mm tris/hcl (ph 6.8), 8% sds, 0.04% bromophenol blue, and 5% β-mercaptoethanol) and incubated for 5 min at 95 • c. the omvs samples were then separated on precast gradient nupage ® novex ® 4-12% bis-tris protein gels (invitrogen, usa) and subsequently transferred onto immobilon-fl polyvinylidene fluoride (pvdf) membranes (merck millipore). following protein transfer, the pvdf membrane was blocked using blocking buffer (1× tbs (20 mm tris-hcl, ph 7.6, 140 mm nacl) containing 5% non-fat dry milk) for 1 h at room temperature (rt). the membrane was washed once for 5 min using washing buffer (1× tbs-tween (20 mm tris-hcl, ph 7.6, 140 mm nacl, 0.05% tween20)). afterwards, detection of the viral ha1 protein was achieved using rabbit monoclonal antibodies recognizing influenza a virus hemagglutinin (abcam), diluted 1:1000 in blocking buffer. 1 h later, the membrane was washed three times for 5 min with washing buffer. next, the membranes were incubated in the dark for 1 h with the corresponding goat anti-rabbit irdye (li-cor, nebraska, usa), diluted 1:10,000 in blocking buffer containing a 1:1000 dilution of 10% sds. after three washing steps (5 min each), twice with washing buffer and once with 1× tbs, the proteins were visualized using an odyssey infrared imaging system and application software package (li-cor, nebraska, usa). the propagated virus was inactivated using 0.1% formaldehyde in 4 • c for 24 h. to ensure that there are no active viral particles following inactivation process of the inactivated control vaccines, mdck and vero e6 cells were inoculated with 100 µl of the inactivated strains. about 72 h post-inoculation, the cell-culture supernatant was then tested using either ha assay (for h1n1) or plaque assay (for mers-cov). a volume of 15 ml of the inactivated viral harvest was then carefully layered with 6 ml of 20% sucrose in an ultra-centrifugation tube and centrifuged in a sorvall mtx 150 ultracentrifuge (thermo scientific, ca, usa) at 28,000 rpm for 2 h at 4 • c. the pellets were further resuspended in 500 µl 1× pbs. the required amounts of viral antigen (µg) of each virus were mixed with imject alum adjuvant (invitrogen, ca, usa) in a ratio of 1:1 (v/v). the final antigen/adjuvant combination was continuously mixed for 30 min under cooling conditions to effectively adsorb the antigen into the surface of the adjuvant and generate optimal vaccine formulation. female balb/c mice (6-8 week-old) were reared and supplied from the animal house at the national research centre (nrc), egypt. mice were divided into 5 groups (7 mice/group). two groups of mice were intramuscularly injected with 5 µg of omvs-h1/rbd and omvs-empty. three other groups were used as controls including negative control group that was injected with sterile pbs and two positive control groups that were injected either with inactivated h1n1pdm09 or inactivated mers-cov. all animals received booster immunizations after 3 weeks. serum samples were collected at 0, 2, 4, 6, and 8 weeks after prime immunization. all mice sera were separated and stored at −20 • c until used. sera collected from immunized/control mice were treated with receptor-destroying enzyme (rde) from vibrio cholerae (denka seiken, tokyo, japan) and kept overnight at 37 • c. the rde was then inactivated by incubation at 56 • c for 1 h. diluted sera were incubated with four ha units of h1n1pdm and a 1.0% suspension of chicken red blood cells, incubated for 1 h at rt. hai titer is the reciprocal value of the dilution at which no agglutination was observed. titers <1:10 were considered as negative. plaque-reduction neutralization test (prnt) assay was performed to determine the efficacy of stimulated antibodies in sera from vaccinated/control balb/c mice to neutralize mers-cov. briefly, sera were inactivated by heating at 56 • c in a water bath for 30 min. sera were diluted two-fold serial dilution from 1:20 to 1:160 dilution in 40 µl of dmem/2% fbs. an equal amount of plaque forming unit in 40 µl dmem/2% fbs was added over sera dilutions. the serum/virus dilutions were then incubated at 37 • c for 1 h in a humidified incubator with 5% co 2 . afterwards, 50 µl of each dilution were inoculated into individual wells of 12-well tissue culture plates with confluent vero-e6 cell monolayers and incubated at 37 • c for 1 h. the plates were periodically undulated every 20 min to avoid cell drying. after 1 h of virus adsorption, inoculum was removed gently from the infected monolayer cells, washed with 1× pbs and covered with an overlay containing 1× mem media, 1% agar, 1% penicillin/streptomycin (pen/strep). the plates were left to solidify and incubated at 37 • c with 5% co 2 upside down until the formation of viral plaques were visible (3 days). the cell monolayers were then fixed with 3.4% formaldehyde solution for 1 h at rt, stained with 1% crystal violet solution (in 20% methanol) for 30 min at rt, and washed with water to visualize the plaques. the percent (%) of inhibition is calculated as following: % of plaque reduction = (virus control plaques count-sample plaques count)/(virus control plaques count) × 100 the prnt 50 is defined as the reciprocal of the antibody dilution required to reduce the number of mers-cov plaques in vero-e6 cells by 50% relative to the control wells. eight weeks after prime immunization, mice were anesthetized by intra-peritoneal injection with ketamine solution with doses adjusted to their individual body weight (2 µg/g). an infectious dose of 10 5.5 tcid 50 of influenza virus a/california/04/2009 (h1n1) wild type, was administered intra-nasally to all vaccinated and control groups. to ensure full separation between the groups and the absence of natural infection, an additional control group of five mice was added and not subjected to infection. body weight was monitored daily and mice showing a weight loss of more than 30% of their initial vaccines 2019, 7, 46 6 of 13 body weight were euthanized and recorded as dead. mice were kept under specific pathogen free (spf) conditions at the national research centre facility unit, egypt. all animal trials were conducted in accordance with the recommendations and guidelines of the egyptian animal welfare legislation. the ethics committee of the national research centre, egypt, approved the animal trial in mice (approval code: . all experiments with infectious virus were performed according to egyptian regulations for the propagation of influenza viruses. all experiments involving low pathogenic and highly pathogenic avian influenza a viruses were performed in biosafety level 2 and 3 (bsl2, 3) containment cabinets, respectively, approved for such use by the local authorities. as schematically represented in figure 1a , the pmp-h1/rbd construct comprises of the 3'-non-coding region (ncr) and the signal peptide (sp) of the ha gene from a/giessen/06/2009 (h1n1pdm2009), followed by the receptor-binding domain (rbd) of the mers-cov spike gene (rbd: 1099-1818 nt = 367-606 amino acids (aa)), a 7-aa flexible linker peptide (lp: gsagsag, [22] ) and the coding sequence plus the 5'-ncr of the ha gene. the pmp-h1/rbd is designed to link the rbd and ha0 fragments into a single polypeptide chain. the final fusion protein contains amino acid (aa) residues 1-240 of the rbd, aa residues 1-7 of the lp and aa residues 1-566 of the ha0. vaccines 2019, 7, x for peer review 6 of 13 their initial body weight were euthanized and recorded as dead. mice were kept under specific pathogen free (spf) conditions at the national research centre facility unit, egypt. all animal trials were conducted in accordance with the recommendations and guidelines of the egyptian animal welfare legislation. the ethics committee of the national research centre, egypt, approved the animal trial in mice (approval code: . all experiments with infectious virus were performed according to egyptian regulations for the propagation of influenza viruses. all experiments involving low pathogenic and highly pathogenic avian influenza a viruses were performed in biosafety level 2 and 3 (bsl2, 3) containment cabinets, respectively, approved for such use by the local authorities. as schematically represented in figure 1a , the pmp-h1/rbd construct comprises of the 3`-non-coding region (ncr) and the signal peptide (sp) of the ha gene from a/giessen/06/2009 (h1n1pdm2009), followed by the receptor-binding domain (rbd) of the mers-cov spike gene (rbd: 1099-1818 nt = 367-606 amino acids (aa)), a 7-aa flexible linker peptide (lp: gsagsag, [22] ) and the coding sequence plus the 5'-ncr of the ha gene. the pmp-h1/rbd is designed to link the rbd and ha0 fragments into a single polypeptide chain. the final fusion protein contains amino acid (aa) residues 1-240 of the rbd, aa residues 1-7 of the lp and aa residues 1-566 of the ha0. to produce outer-membrane vesicles (omvs), comprising an expressed mers-cov rbd and h1n1pdm2009 ha (omvs-h1/rbd) hybrid protein, the e. coli strain dh10ß was transformed with pmp-h1/rbd plasmid. the presence of the rbd from mers-cov and the h1-ha from h1n1pdm2009 in the omvs particles was examined by immunoblot analysis using 5 µg of purified omvs-h1/rbd. the blotting pattern confirmed the presence of rbd-linked ha viral protein; corresponding to ha0-rbd (77 kda + 25 kda equal 102 kda). in contrast, omvs isolated from untransformed dh10ß (omvs-empty) did not show any cross-reacting proteins (figure 1b) . to produce outer-membrane vesicles (omvs), comprising an expressed mers-cov rbd and h1n1pdm2009 ha (omvs-h1/rbd) hybrid protein, the e. coli strain dh10ß was transformed with pmp-h1/rbd plasmid. the presence of the rbd from mers-cov and the h1-ha from h1n1pdm2009 in the omvs particles was examined by immunoblot analysis using 5 µg of purified omvs-h1/rbd. the blotting pattern confirmed the presence of rbd-linked ha viral protein; corresponding to ha0-rbd (77 kda + 25 kda equal 102 kda). in contrast, omvs isolated from untransformed dh10ß (omvs-empty) did not show any cross-reacting proteins (figure 1b) . to assess the immunogenicity of the bivalent omvs-h1/rbd preparations, female balb/c mice were immunized with 5 µg/mouse of omvs-h1/rbd and omvs-empty in comparison with inactivated h1n1pdm2009 and inactivated mers-cov as a positive controls and pbs as a negative control. mice received a booster dose three weeks after prime immunization (figure 2a ). sera were collected every two weeks from week two to eight after prime immunization. ncr: non-coding region, sp: signal peptide, rbd: receptor binding domain (mers-cov), l: nucleotide sequence of peptide linker (ggtagcgccggtagcgccgga), ha1: hemagglutinin 1, and ha2: hemagglutinin 2. (b) immunoblotting pattern of omvs, extracted either from various control/non-transformed omvs (c1, c2, and c3) (empty omvs) or pmp-h1/rbd-transformed (omvs-h1/rbd) dh10ß (s1-s6), against antiserum of swine ha1 antibody. to assess the immunogenicity of the bivalent omvs-h1/rbd preparations, female balb/c mice were immunized with 5 µg/mouse of omvs-h1/rbd and omvs-empty in comparison with inactivated h1n1pdm2009 and inactivated mers-cov as a positive controls and pbs as a negative control. mice received a booster dose three weeks after prime immunization (figure 2a ). sera were collected every two weeks from week two to eight after prime immunization. at two weeks post-vaccination, mice vaccinated with inactivated h1n1pdm2009 virus or omvs-h1/rbd revealed a 2-3 × log2 increase in hai antibody titer as compared to the control and omvs-empty groups (figure 2b ). four weeks after vaccination we observed a drop in the hai titers in these mice due to the booster vaccination at week three. interestingly, the two groups vaccinated with omvs-h1/rbd or inactivated h1n1pdm2009 showed a significant increase in geometric mean hai antibody titers to 145.4 (7.2 log2) and 420 (8.7 log2) at week six, and at week eight, the geometric mean hai antibody titers decreased to 70.7 (6.1 log2) and 210 (7.7 log2), respectively. these results revealed that the vaccinated mice had developed a strong immunogenic response against the h1n1pdm2009 virus. in the negative pbs control group and in the omvs-empty group hai titers remained low, reflecting that all animals were indeed housed under influenza-free conditions (no natural infection). at two weeks post-vaccination, mice vaccinated with inactivated h1n1pdm2009 virus or omvs-h1/rbd revealed a 2-3 × log2 increase in hai antibody titer as compared to the control and omvs-empty groups (figure 2b ). four weeks after vaccination we observed a drop in the hai titers in these mice due to the booster vaccination at week three. interestingly, the two groups vaccinated with omvs-h1/rbd or inactivated h1n1pdm2009 showed a significant increase in geometric mean hai antibody titers to 145.4 (7.2 log2) and 420 (8.7 log2) at week six, and at week eight, the geometric mean hai antibody titers decreased to 70.7 (6.1 log2) and 210 (7.7 log2), respectively. these results revealed that the vaccinated mice had developed a strong immunogenic response against the h1n1pdm2009 virus. in the negative pbs control group and in the omvs-empty group hai titers remained low, reflecting that all animals were indeed housed under influenza-free conditions (no natural infection). in addition, the omvs-h1/rbd vaccinated mice showed a significant increase of neutralizing antibodies against the mers-cov strain hku-nrce-270 at week 2 and reached the highest neutralizing titer 160 (7.3 log2) at week eight compared to the control group (p < 0.001) (figures 2c and 3a) . control (1× pbs), inactivated h1n1pdm2009 and omvs-empty groups showed no neutralizing antibodies against mers-cov during the eight weeks of infection (figure 3b-d) . vaccines 2019, 7, x for peer review 8 of 13 in addition, the omvs-h1/rbd vaccinated mice showed a significant increase of neutralizing antibodies against the mers-cov strain hku-nrce-270 at week 2 and reached the highest neutralizing titer 160 (7.3 log2) at week eight compared to the control group (p < 0.001) (figures 2c and 3a) . control (1× pbs), inactivated h1n1pdm2009 and omvs-empty groups showed no neutralizing antibodies against mers-cov during the eight weeks of infection (figure 3b-d) . on the other hand, a plaque reduction neutralization test (prnt50) using sera from mice vaccinated with inactivated mers-cov showed complete neutralization (prnt50 titer, approximately >1:160) after eight weeks of first immunization of active mers-cov (figure 3e) . to investigate the protection level of vaccinated mice against h1n1pdm09 infection, vaccinatedand control groups of balb/c mice (8 weeks post-vaccination) (figure 2a) were infected with wild type cal-h1n1pdm2009 virus. to ensure full separation between the groups and the absence of natural infection, an additional control group of five mice was added and not subjected to infection. both groups, vaccinated either with inactivated h1n1pdm2009 or omvs-h1/rbd, showed no weight losses till 14 days p.i. (post infection) in comparison to the infected pbs control group. interestingly, these results showed that the vaccinated groups with omvs-h1/rbd and inactivated h1n1pdm2009 virus protected all mice from body weight loss (bwl) (figure 4a ) and mortality up to 14 days post challenge infection (figure 4b ). vaccines 2019, 7, x for peer review 9 of 13 interestingly, these results showed that the vaccinated groups with omvs-h1/rbd and inactivated h1n1pdm2009 virus protected all mice from body weight loss (bwl) (figure 4a ) and mortality up to 14 days post challenge infection (figure 4b ). in contrast, the control group of pbs-treated mice infected with cal-h1n1pdm2009 100% exhibited a bwl of more than 30% from day four to six p.i. resulting in euthanasia (figure 4a ). the mortality rate in this control group was 29% four days p.i. and increased gradually to 85% at day 5 p.i., and 100% at 6 days p.i. (figure 4b) . mortality in the omvs-empty group reached 57% at 13 days p.i. and resulted in euthanization of 4 mice (bwl ≥ 30%) (figure 4b ). the continuous evolution of h1n1pdm09 in swine and human populations, and the recent emergence of mers-cov infections with high mortality rate in humans has raised awareness of both viruses as serious emergent global health topics [23, 24] . since vaccination is the most important strategy to combat emerging human viral infections, an effective vaccine remains a necessity, particularly for the mers-cov. the mers-cov spike (s) protein plays an essential role during virus entry through the binding of its antigenic rbd region to the dpp4 host cell receptor [25] . the rbd is recognized as a major antigenic glycoprotein fragment for inducing a potent humoral and cellular neutralizing antibody (nab) immune responses [26] [27] [28] [29] [30] [31] . traditionally, influenza vaccines are produced by generating a natural or recombinant reassortant iav expressing the immunogenic ha antigen [14, 32] . the iav comprises of two subunits ha1 and ha2, hosting the antigenic sites to which specific and neutralizing antibodies are elicited to combat iavs strains during vaccination or natural infection [14, 33] . however, it was reported that the specificity of the vaccine produced in cell-culture and embryonated eggs is occasionally impaired by amino acid (aa) changes, due to seed strain adaptation, with a drastic impact on vaccine effectiveness [14] [15] [16] . the vaccine platform presented in this study depends on plasmid-based bacterial expression of recombinant viral antigen(s). these plasmids, encoding viral antigen(s), can be easily and quickly modified to insert non-synonymous changes in the encoding region of the antigen(s). additionally, the bacterial expression has lower mutation rates than eukaryotes [34] . this platform can be also a base for incorporating combinations of different viral antigens to address additional vaccines needed to combat seasonal h1n1, h3n2 and influenza b viruses. omvs had been introduced as a part of novel vaccine formulations carrying antigenic proteins eliciting protective responses in animal models from diverse microorganisms such as n. meningitis b, vibrio cholera, salmonella typhimurium, pseudomonas aeruginosa, gallibacterium anatis, acinetobacter baumannii, chlamydia trachomatis, shigella spp., and mycobacterium tuberculosis [35] [36] [37] [38] [39] [40] [41] . lps in the outer surface of omvs acts as a self-adjuvant that induces humoral and cellular immunity. therefore, omvs vaccines may be used without extra adjuvant to increase the immunogenicity and produce antiviral innate immune responses against various influenza virus infections via activation of macrophages [42] [43] [44] . despite that the exact role of lps in the context of omvs vaccines requires in contrast, the control group of pbs-treated mice infected with cal-h1n1pdm2009 100% exhibited a bwl of more than 30% from day four to six p.i. resulting in euthanasia (figure 4a ). the mortality rate in this control group was 29% four days p.i. and increased gradually to 85% at day 5 p.i., and 100% at 6 days p.i. (figure 4b) . mortality in the omvs-empty group reached 57% at 13 days p.i. and resulted in euthanization of 4 mice (bwl ≥ 30%) (figure 4b ). the continuous evolution of h1n1pdm09 in swine and human populations, and the recent emergence of mers-cov infections with high mortality rate in humans has raised awareness of both viruses as serious emergent global health topics [23, 24] . since vaccination is the most important strategy to combat emerging human viral infections, an effective vaccine remains a necessity, particularly for the mers-cov. the mers-cov spike (s) protein plays an essential role during virus entry through the binding of its antigenic rbd region to the dpp4 host cell receptor [25] . the rbd is recognized as a major antigenic glycoprotein fragment for inducing a potent humoral and cellular neutralizing antibody (nab) immune responses [26] [27] [28] [29] [30] [31] . traditionally, influenza vaccines are produced by generating a natural or recombinant reassortant iav expressing the immunogenic ha antigen [14, 32] . the iav comprises of two subunits ha1 and ha2, hosting the antigenic sites to which specific and neutralizing antibodies are elicited to combat iavs strains during vaccination or natural infection [14, 33] . however, it was reported that the specificity of the vaccine produced in cell-culture and embryonated eggs is occasionally impaired by amino acid (aa) changes, due to seed strain adaptation, with a drastic impact on vaccine effectiveness [14] [15] [16] . the vaccine platform presented in this study depends on plasmid-based bacterial expression of recombinant viral antigen(s). these plasmids, encoding viral antigen(s), can be easily and quickly modified to insert non-synonymous changes in the encoding region of the antigen(s). additionally, the bacterial expression has lower mutation rates than eukaryotes [34] . this platform can be also a base for incorporating combinations of different viral antigens to address additional vaccines needed to combat seasonal h1n1, h3n2 and influenza b viruses. omvs had been introduced as a part of novel vaccine formulations carrying antigenic proteins eliciting protective responses in animal models from diverse microorganisms such as n. meningitis b, vibrio cholera, salmonella typhimurium, pseudomonas aeruginosa, gallibacterium anatis, acinetobacter baumannii, chlamydia trachomatis, shigella spp., and mycobacterium tuberculosis [35] [36] [37] [38] [39] [40] [41] . lps in the outer surface of omvs acts as a self-adjuvant that induces humoral and cellular immunity. therefore, omvs vaccines may be used without extra adjuvant to increase the immunogenicity and produce antiviral innate immune responses against various influenza virus infections via activation of macrophages [42] [43] [44] . despite that the exact role of lps in the context of omvs vaccines requires further investigations, high amounts of lps could be a drawback due to its known endotoxicity and ability to induce excessive secretions of pro-inflammatory cytokines [45] . therefore, several ongoing investigations aim to produce genetically detoxified and less reactogenic lps to improve omv safety [42, 46, 47] . additionally, modified bacterial strains such as clearcoli™ bl21(de3), which do not trigger lps-related immune response, can be applied for omv production [48] . based on these observations we engineered the expression of antigenically-stable and immunogenic (omvs)-based bivalent vaccine that elicits protective antibodies (abs) following immunization to control infections with h1n1pdm09 and mers-cov. a recombinant construct comprising the ha of h1n1pdm09 fused to the rbd of the mers-cov s protein is expressed in an e. coli bacterial strain. the expressed bivalent antigens were incorporated within the released omvs (omvs-h1/rbd). this novel chimeric omvs-h1/rbd produced high levels of a neutralizing abs titer against influenza h1n1 virus at 8 weeks post immunization. stimulated neutralizing abs (humoral immunity) together with lps-induced cellular immunity could fully protect immunized mice after challenge with h1n1pdm09 without significant loss in body weight. surprisingly, the induced non-specific cellular immunity induced by omvs-empty could partially protect the mice. this emphasize the synergistic effect of humoral and cellular immunities secreted upon vaccination with the chimeric omvs-h1/rbd formulation. serum transfer experiments would be able to further elucidate the role of humoral immunity independent of cellular immunity [49] . additionally, omvs-h1/rbd-vaccinated mice demonstrated a significant increase in the neutralizing abs titer against mers-cov (1:160) at week 8 in comparison to the control group as in figure 2c . these findings ensured that omv vaccination platform can provide a protection by efficient neutralization of invading h1n1pdm09 and mers-cov. the data presented in this study are consistent with recent reports describing the potential of omvs as biologically active, stable and highly immunogenic vaccines to protect against iavs. a newly developed recombinant omvs bearing the conserved m2e protein (omvs-m2e) from iavs efficiently protected mice from an h1n1-and h3n2-type iav challenge [50, 51] . our study represents an extension of these studies and suggests the generation of omvs that incorporate combinations of different viral antigens to generate safe and efficient vaccines in animal husbandry and for humans. in summary, the results show that the generated (omvs-h1/rbd)-based vaccine presenting the antigenic stable chimeric fusion protein of h1-type ha of the pandemic influenza a virus (h1n1) strain and rbd of mers-cov induces specific neutralizing antibodies against h1n1pdm09 and mers-cov leading to protection of immunized mice against both viruses. these results demonstrate that omvs-based vaccines presenting viral antigens have the potential to be a vaccine platform that provides simultaneous protection against two different viral infections. bacterial and viral pathogen spectra of acute respiratory infections in under-5 children in hospital settings in dhaka city estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study a history of influenza h7n9 avian flu infects humans for the first time characterization of an avian influenza virus h5n1 egyptian isolate clinical and epidemiological characteristics of a fatal case of avian influenza a h10n8 virus infection: a descriptive study origin and molecular characterization of the human-infecting h6n1 influenza virus in taiwan outbreak of 2009 pandemic influenza a (h1n1) at a new york city school emergence and pandemic potential of swine-origin h1n1 influenza virus understanding the latest human coronavirus threat. viruses influenza h3n2 vaccines: recent challenges contemporary h3n2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains a structural explanation for the low effectiveness of the seasonal influenza h3n2 vaccine bioengineering bacterial outer membrane vesicles as vaccine platform bacterial outer membrane vesicles: new insights and applications meningococcal serogroup b vaccines: will they live up to expectations? antibody-mediated immunity induced by engineered escherichia coli omvs carrying heterologous antigens in their lumen improved dual promotor-driven reverse genetics system for influenza viruses design of an ha2-based escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge high prevalence of mers-cov infection in camel workers in saudi arabia cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein novel chimeric virus-like particles vaccine displaying mers-cov receptor-binding domain induce specific humoral and cellular immune response in mice receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov middle east respiratory syndrome coronavirus: a comprehensive review the pb1 segment of an influenza a virus h1n1 2009pdm isolate enhances the replication efficiency of specific influenza vaccine strains in cell culture and embryonated eggs protective immunity based on the conserved hemagglutinin stalk domain and its prospects for universal influenza vaccine development evolution of the mutation rate comparison of intranasal outer membrane vesicles with cholera toxin and injected mf59c.1 as adjuvants for malaria transmission blocking antigens anapn1 and pfs48/45 immunogenicity of vibrio cholerae outer membrane vesicles secreted at various environmental conditions overexpression of mica induces production of ompc-enriched outer membrane vesicles that protect against salmonella challenge immunogenicity and protective efficacy of vibrio cholerae outer membrane vesicles in rabbit model evaluation of intranasal and subcutaneous route of immunization in neonatal mice using dodab-bf as adjuvant with outer membrane vesicles of neisseria meningitis b vaccination with outer membrane vesicles and the fimbrial protein flfa offers improved protection against lesions following challenge with gallibacterium anatis immunization with pseudomonas aeruginosa outer membrane vesicles stimulates protective immunity in mice outer membrane vesicles as platform vaccine technology engineered outer membrane vesicle is potent to elicit hpv16e7-specific cellular immunity in a mouse model of tc-1 graft tumor bacterial outer membrane vesicles provide broad-spectrum protection against influenza virus infection via recruitment and activation of macrophages lipopolysaccharide endotoxins next-generation outer membrane vesicle vaccines against neisseria meningitidis based on nontoxic lps mutants detoxifying escherichia coli for endotoxin-free production of recombinant proteins. microbial endotoxin-free protein production-clearcoli™ technology (application note) outer-membrane-vesicle-associated o antigen, a crucial component for protecting against bordetella parapertussis infection recombinant m2e outer membrane vesicle vaccines protect against lethal influenza a challenge in balb/c mice safe recombinant outer membrane vesicles that display m2e elicit heterologous influenza protection key: cord-318954-pj5lsvsa authors: arabi, yaseen; balkhy, hanan; hajeer, ali h.; bouchama, abderrezak; hayden, frederick g.; al-omari, awad; al-hameed, fahad m.; taha, yusri; shindo, nahoko; whitehead, john; merson, laura; aljohani, sameera; al-khairy, khalid; carson, gail; luke, thomas c.; hensley, lisa; al-dawood, abdulaziz; al-qahtani, saad; modjarrad, kayvon; sadat, musharaf; rohde, gernot; leport, catherine; fowler, robert title: feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol date: 2015-11-19 journal: springerplus doi: 10.1186/s40064-015-1490-9 sha: doc_id: 318954 cord_uid: pj5lsvsa as of september 30, 2015, a total of 1589 laboratory-confirmed cases of infection with the middle east respiratory syndrome coronavirus (mers-cov) have been reported to the world health organization (who). at present there is no effective specific therapy against mers-cov. the use of convalescent plasma (cp) has been suggested as a potential therapy based on existing evidence from other viral infections. we aim to study the feasibility of cp therapy as well as its safety and clinical and laboratory effects in critically ill patients with mers-cov infection. we will also examine the pharmacokinetics of the mers-cov antibody response and viral load over the course of mers-cov infection. this study will inform a future randomized controlled trial that will examine the efficacy of cp therapy for mers-cov infection. in the cp collection phase, potential donors will be tested by the enzyme linked immunosorbent assay (elisa) and the indirect fluorescent antibody (ifa) techniques for the presence of anti-mers-cov antibodies. subjects with anti-mers-cov ifa titer of ≥1:160 and no clinical or laboratory evidence of mers-cov infection will be screened for eligibility for plasma donation according to standard donation criteria. in the cp therapy phase, 20 consecutive critically ill patients admitted to intensive care unit with laboratory-confirmed mers-cov infection will be enrolled and each will receive 2 units of cp. post enrollment, patients will be followed for clinical and laboratory outcomes that include anti-mers-cov antibodies and viral load. this protocol was developed collaboratively by king abdullah international medical research center (kaimrc), gulf cooperation council (gcc) infection control center group and the world health organization—international severe acute respiratory and emerging infection consortium (isaric-who) mers-cov working group. it was approved in june 2014 by the ministry of the national guard health affairs institutional review board (irb). a data safety monitoring board (dsmb) was formulated. the study is registered at http://www.clinicaltrials.gov (nct02190799). the middle east respiratory syndrome coronavirus (mers-cov) was initially identified in september 2012 from samples obtained from a saudi arabian patient who developed severe acute respiratory infection and subsequent acute renal failure leading to death (zaki et al. 2012) . as of september 30, 2015, a total of 1589 cases have been identified with 567 related deaths (world health orgnization 2015) . to date, there is no specific treatment of proven effect for mers-cov infection. public health england and the international severe acute respiratory and emerging infection consortium (isaric) have published a decision support tool for clinicians managing cases of mers-cov infection. the document suggests that current evidence is strongest for testing convalescent plasma (cp) or other therapeutics which contain neutralizing antibodies (such as hyperimmune immunoglobulin) for treatment of serious mers-cov illness (public health england 2015) . prior experience in sars and severe influenza suggest that cp may be considered for patients who are deteriorating (despite other specific and supportive therapy) and in whom the virus remains detectable (hung et al. 2011; luke et al. 2006; cheng et al. 2005; kong and zhou 2006; yeh et al. 2005) . a recent systematic review of 32 reports from sars and severe influenza concluded that cp therapy appears safe and may reduce mortality, especially if administered early in the illness (mair-jenkins et al. 2015 ). an exploratory post hoc meta-analysis showed a statistically significant reduction in the pooled odds of mortality following treatment compared to placebo or no therapy (odds ratio 0.25; 95 % confidence interval 0.14-0.45; i 2 = 0 %) (mair-jenkins et al. 2015) . citing case series, the authors commented that (1) patients with severe presentations appeared to demonstrate temporal clinical improvements after treatment with cp and (2) administration as early as possible in the diseases course appears to be associated with greatest potential clinical effect. one randomized clinical trial (rct) in critically ill influenza a (h1n1pdm09)-infected patients found a survival benefit when hyperimmune globulin was administered within 5 days of symptom onset (hung et al. 2013) . however, there are no data at present to support the efficacy of cp treatment in mers-cov infection; therefore, it has been recommended to administer cp only in the context of a clinical trial. while an rct will be required to evaluate effectiveness, evaluating effectiveness on clinical endpoints such as mortality will likely require several hundred to several thousand seriously ill mers-cov patients in order to achieve sufficient statistical power, anticipating reasonable potential effect sizes. additionally, cp from different mers-cov survivors will likely contain differing levels of neutralizing anti-mers-cov antibodies. since seriously ill mers-cov-infected patients may have detectable viral rna in various locations that can be sampled (for example lower respiratory tract secretions) for prolonged periods, it might be possible to first determine the relationship between neutralizing antibody dose and antiviral effects on clinical and laboratory features in a small open-label study. this information would be very helpful to design of an rct and in determining the most appropriate neutralizing antibody dose, or dosing range for the study. this may also inform dose selection for follow-on anti-mers-cov antibody preparations currently in preclinical development (for example, neutralizing human monoclonal antibodies, polyclonal human neutralizing immunoglobulin derived from transchromosomic cattle (personal communication, thomas c. luke). therefore, we plan to conduct a 2-phase study. in the first phase (cp collection phase), we will explore the feasibility of collection of cp from donors who have significant titers of anti-mers-cov antibodies. in the second phase, patients with mers-cov infection will be treated with cp. if the protocol is feasible, safe, and associated with temporal changes in viral load and illness, this pilot study will inform a larger concealed intervention, placebo-controlled rct that is powered to evaluate efficacy of cp on relevant clinical outcomes. the inclusion criteria for screening potential cp donors include individuals from the following cohorts: (1) healthcare workers (hcws) who had documented exposure to mers-cov, (2) recovering patients from confirmed or suspected mers-cov infection, (3) household contacts of known mers-cov infected patients and (4) other subjects who are willing to donate plasma. females with prior pregnancy will not be included for donation. we will screen consecutive critically ill patients admitted to the intensive care unit or other areas of the hospital where critically ill patients receive care for the following criteria: inclusion criteria 1. critical illness as defined by one or more of the following: admission to an icu; current receipt of mechanical invasive or non-invasive ventilation; partial pressure of oxygen to fraction of inspired oxygen ratio (pao2:fio2) of <300 mmhg; current receipt of intravenous vasoactive medications to maintain mean arterial pressure >65 mmhg; new-onset (since development of mers-cov symptoms) receipt of renal replacement therapy or extra-corporeal life support. 2. laboratory-confirmed mers-cov infection (by realtime reverse-transcription polymerase chain reaction rrt-pcr). 3. age of more than or equal to 14 years. 1. symptomatic illness exceeding two weeks (14 days) at time of enrollment. 2. negative rrt-pcr from respiratory secretions or blood within 48 h prior to assessment of eligibility. 3. history of allergic reaction to blood or plasma products (as judged by the investigator). 4. known iga deficiency. 5. medical conditions in which receipt of 500 ml intravascular volume may be detrimental to the patient (e.g., actively decompensated congestive heart failure). the research coordinator and/or physician investigator will explain the objectives of this study and its potential risks and benefits to the donor or patient (or to his/her surrogate decision maker) and will obtain the following consent forms in and as appropriate: cp collection phase 1. consent for mers-cov serologic testing and mers-cov rt-pcr for donors. 2. consent for cp donation for those who have elevated anti-mers-cov titers as described below. cp therapy phase 1. consent for enrollment in the cp therapy phase. 2. consent for enrollment in the observational study where no intervention will be received, but participants will still have blood (and possibly respiratory) samples taken-for participants not receiving the intervention. for the cp collection phase 1. eligible candidates for cp donation (as per the inclusion and exclusion criteria above) will be approached to have their blood tested for anti-mers-cov serology (see laboratory methods). subjects who are seropositive will be screened subsequently for mers-cov rrt-pcr to exclude active infection. 1. critically ill mers-cov patients who meet the above patient eligibility criteria will be approached for consent. 2. patients will have their blood type determined. cp must be abo compatible with the recipient's blood type. 3. the trial intervention include the administration of 2 units of cp. each unit of plasma will be given over 2 h with an interval of 1 h between the two units. plasma transfusion will be done in accordance with the standard policies for administration of blood products. the clinical team will have full, independent control of patient management and as such, management other than cp therapy will not be influenced by the intervention or study team. co-interventions, including corticosteroids, ribavirin, intravenous immunoglobulin and interferon, will be documented on the study case report forms. co-enrollment in another study is permissible as long as the enrollment in the other study would not be at moderate to high risk of biologically or analytically confounding the results of this study, as judged by the study management committee and as per the published guidelines. clinical and laboratory data will be collected at baseline, 30 min after first dose, 30 min after second dose, study days 1, 3, 5, 7, 14, and 28. we will explore the feasibility of the study intervention, as measured by ability to screen potential plasma donors, and derive sufficient plasma to enrol 20 patients in a 12 months period. we will also qualitatively describe logistical challenges experienced through the conduct of this study, including ethical, administrative and regulatory challenges. 1. we will establish safety of the study intervention, as measured by number of serious adverse events related to study intervention (adverse events include development of complications of intravascular volume overload and clinical pulmonary edema by temporally related-shortness of breath, chest radiograph findings and change in oxygenation requirements; development of transfusion-related acute lung injury (trali) or substantial allergy or anaphylaxis). these serious events will be adjudicated by a committee of 3 investigators. 2. clinical outcomes we will measure (1) sequential organ failure assessment (sofa) scores on study days 1, 3, 5, 7, 14, and 28 (2) requirement for organ support (oxygen and ventilation; dialysis; vasopressors) after enrollment; (3) length of stay in icu defined as the number of calendar days between admission and final discharge from icu for the same icu admission of enrollment; and duration of mechanical ventilation, defined as the number of calendar days between start and final liberation from mechanical ventilation for the same icu admission of enrollment and hospital length of stay as defined as the number of calendar days between admission to hospital and final discharge from hospital for the same hospital admission; and (4) vital outcome (mortality) in icu, hospital and at 28 days. 3. other clinical outcomes include "icu-free days", defined as the number of days that patients are not in icu in the first 28 days after enrollment. patients who die within 28 days will be counted separately, and not categorised by icu-free days. similarly, "ven-tilator-free days" is defined as the number of days that patients do not receive mechanical ventilation in the first 28 days after enrollment. "renal replacement therapy-free days" and "vasopressor-free days" are defined in a similar way. serial chest radiograph findings, as obtained by the clinical team will also be recording as per case report form, graded as unilateral or bilateral infiltrates, in 1-4 quadrants. 4. laboratory outcomes we will measure the following laboratory outcomes: (a) the serum level of anti-mers-cov antibodies before and after administration of cp. (b) mers-cov viral load (the primary laboratory outcome is viral clearance from all sampled sites by day 3 after administration of cp). mers-cov antibodies will be tested first by the enzyme linked immunosorbent assay (elisa) as a screening test (drosten et al. 2014; müller et al. 2015) according to manufacturer's instructions (euroimmun ag, lübeck, germany). results will be reported as the optic density (od) ratio, which is calculated as the od value of the patient's sample divided by the calibrator od value. we will use the cut-off values recommended by the manufacturer: a ratio of <0.8 is considered negative, >0.8 and <1.1 borderline and a ratio of >1.1 is considered positive. confirmation will be done by the indirect fluorescent antibody (ifa, euroimmun ag, lübeck, germany) according to manufacturer's instructions. samples with ≥1:10 will be considered reactive according to the manufacturer's instructions, subjects will be considered candidate for plasma donation if they have titers of ≥1:160; which is a similar threshold to what has been used in a convalescent plasma trial for h1n1 influenza (hung et al. 2011) . the primary coordinating study center is the intensive care department at king saud bin abdulaziz university for health sciences (ksauhs) in riyadh, saudi arabia. the study will be conducted in accordance with the ethical principles of the declaration of helsinki and the international conference on harmonization-good clinical practice (ich-gcp) guidelines. several measures will be taken to ensure optimal compliance with the study protocols. before launching the study, icu physicians and nurses will attend the training sessions with special emphasis on any adverse events noted during the intervention. the steering committee, led by the principal investigator, will be responsible for overseeing the conduct of the trial, for upholding or modifying study procedures as needed, addressing challenges with protocol implementation, formulating the analysis plan, reviewing and interpreting the data and preparing the manuscript. the study also has an independent data safety monitoring board (dsmb) which is responsible for reviewing reports submitted to the regarding safety of study patients, protocol adherence and may making recommendations to continue or terminate the study based on safety analysis results. the dsmb, composed of 5 members (who are named at the end of this document) will meet at the beginning of phase ii of the study followed by 6-monthly or as needed. in the event of an acute transfusion reaction, the transfusion will be stopped immediately and must be reported to the blood bank the principal investigator immediately as well as to the study management committee. all the serious adverse events (sae) adjudicated as related to the study intervention will be reported to the institutional research ethics board and the dsmb. this is an exploratory study, aimed at rectifying the current lack of information on the use of cp to treat mers-cov infection. due to the exploratory nature of this study and the paucity of sequential data on viral rna levels in respiratory tract and blood samples from mers-covinfected patients, and on their clinical progress, the sample size is fixed at 20, which is a realistic target for a study of 12 months duration. the sample size of 20 is sufficient to reach a conclusion that the 28-day survival rate significantly exceeds 60 % (p = 0.032, 2-sided) if 17 or more patients survive for the 28 days of follow-up. this would represent promising evidence to motivate a full-scale comparative clinical trial. 1. serial mers-cov viral load measurements will be displayed as box and whisker plots for the 20 treated patients against time. 2. the probability of a patient having an undetectable viral load from all sampled sites by day 3 after administration of therapy will be estimated by the proportion of the 20 treated patients for whom this occurs. an exact, conservative, two-sided confidence interval for this probability will be calculated using the method of clopper and pearson (1934) . 3. the relationship between log viral load at day 3 and the neutralizing antibody dose received will be characterised by fitting a regression model to the data from the 20 treated patients. the log viral load at baseline will be included in this model. 4. the relationship between the probability of a patient having an undetectable viral load by day 3 and the neutralizing antibody dose received will be characterised by fitting a log-logistic regression model to the data from the 20 treated patients. the log viral load at baseline will be included in this model. 1. the sofa score and indicators of whether the patient requires organ support via oxygen and ventilation, dialysis or vasopressors will be plotted against time. 2. the relationships between the sofa score at day 3 and the neutralizing antibody dose received, and between receipt of any type of organ support during the 28 days of observation and the neutralizing antibody dose received, will be characterised by fitting a logistic regression model to the data from the 20 treated patients. the log viral load at baseline will be included in these models. 3. the vital status (alive or dead) of each patient will be recorded for all days 0-28. the proportion alive will be plotted against time. 4. the relationship between the hazard of death and the neutralizing antibody dose received will be characterised by fitting a cox proportional hazards regression model to the data from the 20 treated patients. the log viral load at baseline will be included in this model. 5. the probability of a patient dying on or before 28 days will be estimated by the proportion of the 20 treated patients for whom this occurs. an exact, conservative, two-sided confidence interval for this probability will be calculated using the method of clopper and pearson (1934) . 6. the time from infection/exposure and sample collection in days, duration from infection/exposure to cp therapy, length of stay in icu; the number of icu-free days; the duration of mechanical ventilation; the numbers of ventilator-free days, of renal replacement therapy-free days, and of vasopressor-free days; and the length of stay in hospital will be presented as histograms, and suitable summary statistics will be computed. we will conduct exploratory stratified analyes based on (1) the time between symptom onset and cp therapy initiation, (2) comorbidities, (3) co-intervention; and (4) baseline severity (sofa scores) at treatment initiation. the sas system for windows version 9.3 (sas institute, inc., cary, north carolina) and r will be used for all analyses. this protocol was developed collaboratively by king abdullah international medical research center (kaimrc), gulf cooperation council (gcc) infection control center group and the world health organization-international severe acute respiratory and emerging infection consortium (isaric-who) mers-cov working group. it was approved by the ministry of the national guard health affairs institutional review board (irb) (approval number irbc/13/244, 5th june 18, 2014) and has been registered at clinicaltrials.gov (nct02190799). if proven effective, cp therapy is an attractive therapeutic option for mers-cov infection. besides the biologic plausibility of this therapy, it is easy to obtain and administer, relatively inexpensive, and is likely to be acceptable to patients and treating teams. side effects are unlikely to differ from those of transfusion of any other fresh frozen plasma. we believe this study protocol sets the stage to a large efficacy trial. the strengths and weaknesses of the study protocol should be noted. in the cp collection phase subjects will be enrolled from 4 different cohorts, in order to explore all potential donors. it is unknown, at this point, which subjects are likely to have high antibody titers and therefore be cp donors. we are hoping that this feasibility study will help identifying a group of superdonors who have very high titers. by identifying the characteristics of such individuals, a more focused approach for donation can be followed. the cp therapy phase is not designed to establish efficacy; such objective requires an adequately powered randomized controlled trial. however, we believe performing this feasibility study is an essential step to examine the safety, clinical and laboratory effects and the pharmacokinetics of the mers-cov antibody response. the study involves giving critically ill patients this therapy in a controlled monitored setting. however, a recent systematic review suggested that early treatment with cp is likely to be more effective than late treatment (mair-jenkins et al. 2015) . therefore, if the feasibility study shows that cp is safe and feasible, the next step should be a randomized controlled trial that is sufficiently powered to detect effect on mortality and enrolls patients early in the course of the disease. our study is anticipated to provide information about the feasibility of collecting convalescent plasma in large quantities for therapeutic use in a large numbers of mers-cov patients. the data is anticipated to inform about the relation between the antibody titers in the cp and viral clearance and other laboratory and clinical endpoints. this data will be critical in planning a larger rct to examine the efficacy of cp on patients with mers-cov infection. use of convalescent plasma therapy in sars patients in hong kong ) the use of confidence or fiducial limits illustrated in the case of the binomial transmission of mers-coronavirus in household contacts convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a(h1n1) infection successful treatment of avian influenza with convalescent plasma meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study middle east respiratory syndrome coronavirus (mers-cov): clinical management and guidance guidelines on assessing donor suitability for blood donation who blood regulators network (brn) (2015) position paper on collection and use of convalescent plasma or serum as an element in middle east respiratory syndrome coronavirus response middle east respiratory syndrome coronavirus experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital isolation of a novel coronavirus from a man with pneumonia in saudi arabia we would like to thank the dsmb members: the authors declare that they have no competing interests. this trial is funded by kaimrc/ksauhs, riyadh, saudi arabia. the principal investigator (dr. yaseen arabi) declares that the sponsor had no influence on the design of protocol, patient recruitment or data generation and will not have any impact on the analysis of the results or writing of the manuscript. the views expressed by t. c. luke do not necessarily reflect the official policy or position of the department of the navy, department of defense, or the usa government. he is an employee of the us government, and this work was prepared as part of his official duties. title 17 usc. §105 provides that 'copyright protection under this title is not available for any work of the united states government. title 17. t. c. key: cord-312740-2ro2p77q authors: babadaei, mohammad mahdi nejadi; hasan, anwarul; vahdani, yasaman; bloukh, samir haj; sharifi, majid; kachooei, ehsan; haghighat, setareh; falahati, mojtaba title: development of remdesivir repositioning as a nucleotide analog against covid-19 rna dependent rna polymerase date: 2020-05-20 journal: j biomol struct dyn doi: 10.1080/07391102.2020.1767210 sha: doc_id: 312740 cord_uid: 2ro2p77q severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the causative representative of a severe respiratory illness resulted in widespread human infections and deaths in nearly all of the countries since late 2019. there is no therapeutic fda-approved drug against sars-cov-2 infection, although a combination of anti-viral drugs is directly being practiced in some countries. a broad-spectrum of antiviral agents are being currently evaluated in clinical trials, and in this review, we specifically focus on the application of remdesivir (rvd) as a potential anti-viral compound against middle east respiratory syndrome (mers) -cov, sars-cov and sars-cov-2. first, we overview the general information about sars-cov-2, followed by application of rdv as a nucleotide analogue which can potentially inhibits rna-dependent rna polymerase of covs. afterwards, we discussed the kinetics of sarsor mers-cov proliferation in animal models which is significantly different compared to that in humans. finally, some ongoing challenges and future perspective on the application of rdv either alone or in combination with other anti-viral agents against covs infection were surveyed to determine the efficiency of rdv in preclinical trials. as a result, this paper provides crucial evidence of the potency of rdv to prevent sars-cov-2 infections. communicated by ramaswamy h. sarma the new coronavirus (cov), known as severe acute respiratory syndrome (sars)-cov-2, which is currently spreading around the world, can lead to a respiratory illness that can be exacerbated novel, 2020; . the disease known as cov diseases-19 (covid-19) appears to induce a mortality rate of less than 2%, which is lesser that of most epidemics that have ever become global headlines pan et al., 2020) . covs are very similar to influenza viruses and show almost identical symptoms (heymann & shindo, 2020; rothan & byrareddy, 2020) . two recent outbreaks of the new cov, including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) have originated from animals (gretebeck & subbarao, 2015; song et al., 2019; yao et al., 2014) . the diseases caused by these viruses were extremely fatal to humans, and very few cases were reported as mild or asymptomatic. however, it has been reported that the mortality rate of covid-19 has been more than that of sars and mers (mahase, 2020; peeri et al., 2020) . in the past days, it has been repeatedly reported that a vaccine for the sars-cov-2 has been discovered, but vaccines, like other medicines, require a lengthy testing process to be approved for medical applications (ahmed et al., 2020; prompetchara et al., 2020) . one strategy to date is the replication of a piece of virus rna that could one day serve as a vaccine (enayatkhani et al., 2020) . although, well-developed dna sequencing devises has let to rapid genetic sequencing, vaccine development is costly, complex, and time intensive (plotkin et al., 2017) . this includes finding a viral sequence that, while providing memory to the immune system, does not lead to an acute inflammatory reaction. achieving this goal requires laboratory experimentation on animal models before subjecting humans. in addition, once the vaccine is discovered, it is not possible to dispatch the sample quickly and easily worldwide. therefore, pharmaceutical companies have generally found it more profitable to invest in drugs that are used for chronic medical conditions. the cov, as in case of influenza, may undertake mutations and therefore would require continuous vaccine development. with having a considerable number of people worldwide infected with covid-19, scientists have identified a number of cases of broad-spectrum antiviral agents (bsaas) that could serve as potential candidates for the treatment of the viral diseases (andersen et al., 2020; ianevski et al., 2018) . indeed, re-purposing of current approved anti-viral drugs could be a solution to treat new viral infections (guo, 2020; kouznetsova et al., 2014; mercorelli et al., 2018; xu et al., 2016) . drug re-purposing means that by examining existing drugs, they will find therapeutic effects on new diseases (aggarwal et al., 2020; khan, jha, et al., 2020; senathilake et al., 2020) . bsaas are small molecules that can inhibit different infections by blocking the viral replication (pant et al., 2020; xiong et al., 2020; . these drugs block the virus or host-related factors and thus prevent the virus from proliferating, then lowering the level of the virus in the body to an extent that the immune system can inhibit their infection (cui et al., 2020; ji & li, 2020) . bsaa has received special attention with the emergence of numerous new viral diseases. re-purposing existing drugs, or even rejected drugs, for viral diseases increases the possibility of market success as well as reducing the costs and time required to launch it. the benefit of drug re-purposing is that drug details, such as the stages of chemical synthesis, mass production processes, various stages of clinical trials and many more have been identified beforehand (aanouz et al., 2020; gupta et al., 2020) . there is currently no drug or vaccine to prevent the sars-cov-2 infection, but the use of widespread antivirals can be effective against the prophylaxis of this virus (boopathi et al., 2020; elmezayen et al., 2020) . for example, chloroquine and remdesivir (rdv, gs-5734) are two drugs that in vitro studies have suggested that they can inhibit the viral replication . teicoplanin, oritavancin, dalbavancin, and monensin antibiotics also prevent viral replication (andersen et al., 2020) . currently, the bsaas are treatment or pyrophylaxis candidates against sars-cov-2 (senanayake, 2020) . furthermore, a combination of bsaas can be applied against a wider range of viruses, such as those that are not yet well recognized or drug-resistant viruses . potential clinical trials are currently being conducted on these drugs and their results will be published soon (li & de clercq, 2020; senanayake, 2020) . perhaps in a near future, bsaas will be used to treat covid-19 patients. although a number of bs have been reported to date, in this review we focused on the rdv as a potential compound that has been assessed on the animal models and reaches the clinical phase against mers -cov, sars-cov and sars-cov-2. covs are a large family of viruses ranging from the common cold virus to the cause of sars (cascella et al., 2020; paules et al., 2020) . the structure of cov has a common rna genome and is classified as enveloped viruses (mackie, 2003) . sars-cov-2 originating in china and the city of wuhan is believed to be a member of the corona family, which has infected many people to date . despite the emergence of the virus in china, it is spreading rapidly to other parts of the world zhu et al., 2020) . similar cases have been reported in other countries such as thailand, south korea, japan, taiwan, australia, and the united states, and even more recently in iran (velavan & meyer, 2020; zhu et al., 2020) . the disease can be spread through close contact with the infected person, handling contaminated equipment and airborne outbreaks (velavan & meyer, 2020) . most people with hypertension, diabetes, respiratory problems, and weak immune systems are at the higher risk to infection and the likely death from it (fang et al., 2020) . covs have four types of proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) (hasan et al., 2020) . s protein is attached to the virus membrane and play an important role in binding and entry into the host cell; hence, targeting this protein with various drugs and inhibitors is one potential approach to combat these types of viruses (chatterjee, 2020; lai & cavanagh, 1997; woo et al., 2010) . sars-cov-2 has proteins on its surface that mediate viral infection by binding to angiotensin-converting enzyme 2 (ace2) receptor (batlle et al., 2020; chen & hao, 2020) . therefore, one promising way to stop infection by sars-cov-2 is to find a compound that blocks the receptor, and consequently prevent the infection by preventing the interaction of s protein with ace2 receptor (andersen et al., 2020; joshi et al., 2020) . the cov also has 16 unstructured protein (nsp 1-16) encoded by orf1a/1b which act as important co-factors for activation of viral replication enzymes (figure 1) . although a recent research in china suggests bat as the potential source of the virus, there are ongoing researches to clarify the exact origin of the virus (guo et al., 2020) ( figure 1 ). researches into the origin of sars outbreaks have led to the discovery of many bat viruses. sars-cov-2 belongs to this category of sars-related viruses (fung et al., 2020) . the two genome sequences of rhinolophus sinicus show 80% similarity with sars-cov-2. the third genome of the rhinolophus affinis virus, ratg13, resembles 96% similarity with sars-cov-2 (andersen et al., 2020) . to have better sense of this variation, it is similar to the rate of mutation observed over ten years in the human h3n2 influenza virus strain . rdv (gs-5734) as a nucleotide analogue was originally developed to treat ebola (tchesnokov et al., 2019) . the laboratory assessments has shown that rdv is effective against sars-cov (ju et al., 2020) and mers-cov (gordon et al., 2020) viruses, therefore it can be used as a potential anti-viral agent against sars-cov-2 . the mechanism of rdv's anti-viral function is based on the blockage of viral rna transcription as revealed in molecular examinations using different recombinant viral polymerases sarma et al., 2020; tchesnokov et al., 2019; warren et al., 2016) . siegel et al. (2017) reported that gs-5734 can be used as a potential candidate for the treatment of ebola and emerging cov. agostini et al. (2018) reported that cov is susceptible to the rdv targeting the viral polymerase and the nsp14 exoribonuclease (exon). they compared the sensitivity of wt and exon (-) virus to rdv, which exon (-) virus showed a greater decrease in viral titer in the presence of gs-5734 relative to wt virus and the determined ec 50 value for exon (-) virus was around 0.019 m, whereas the ec 50 value for to the wt was determined to be 0.087 m (figure 2a(i) ). this increased inhibition of exon (-) virus by gs-5734 (figure 2a (ii)) indicated that gs-5734 is integrated into viral genome and can be excluded by exon (agostini et al., 2018) . also, it was shown that the type of cov, concentration of antiviral drug, type of anti-viral drug, and incubation time can play an important role on the inhibition of virus infection ( figure 2b figure 4) . furthermore, they found that rdv was the only therapeutic agent which remarkably decreased pulmonary infection. furthermore, gordon et al. (2020) and de wit et al. (2020) revealed that rdv derives anti-viral effects against mers-cov though inhibition of rna polymerase. as with sars-cov investigations treated by rdv, similar outcomes were observed for mers-cov along with limited weight loss, increased pulmonary activity and decreased virus replication (sheahan et al., 2017) . the kinetics of sars-or mers-cov proliferation in animal models is significantly different compared to that in humans. in animal, sars-cov or mers-cov proliferation in the lung tissue reaches to the maximum at 2 dpi and mortality is stimulated at 7-10 dpi (douglas et al., 2018; sheahan et al., 2017) . thus, the therapeutic window to control infected animal model prior to the peak of cov proliferation is less than 2 days. in human, mers-cov replication in the lung tissue reaches the maximum at 7-10 days after the onset of infections and the disease severity increases to death within 21 days oh et al., 2016) . thus, the time for therapeutic handling is substantially divergent in humans and experimentally infected animal models. although, applying rdv led to a reduction in mers-cov pathogenesis and pronounced decrease in viral dose, therapeutic handling did not thoroughly reduce infection. furthermore, at high levels of cov, rdv is unable to sustain viral viability and pulmonary cells functionality, despite of remarkable decrease in viral loads. these outcomes are the same as those reported for sars-cov, where therapeutic platforms were launched after the peak of virus titer and lung injury did not show any progress in resultant outcomes (sheahan et al., 2017) . since sars-and mers-cov infections are controlled by both covs and host immune system modulators, therefore early handling of antiviral therapeutics either solely or in combination with other therapeutic drugs, and based on the stage of the disorder progression, can inhibit virus proliferation and immunopathology, switch on repairing systems, or control the pulmonary homeostasis. arising viral disorders have resulted in meaningful catastrophic pandemics. genetic exploration in animal models have shown a pronounced mutation of viral genome in the case of cov, and have even pointed out some viruses indistinguishable to ongoing and past pathogenic strains in animals (ge et al., 2013; woo et al., 2006) . therefore, in the absence of fda-approved therapeutics for reducing the human cov infection, useful broad-spectrum therapeutic platforms against well-known epidemic and zoonotic strains probably pave a way for diminishing the current epidemic diseases. in the case of cov, patients were received off-label antiviral drugs as well as immunomodulators, either solely or combined, to reduce severe disease symptoms (zumla et al., 2016) . however, due to the lack of patient, therapeutic consistency, and verified standard efficiency measurement is a complicated process. although interferon does not result in improved clinical consequence in mers-cov patients (morra et al., 2018) showed that rdv stimulated superior anti-viral function against mers-cov both in vitro and in vivo in comparison with lopinavir/ritonavir-interferon b. nucleoside/nucleotide analogues inhibit virus replication by blocking the activity of the polymerase enzyme in the virus (chhikara et al., 2020; men endez-arias et al., 2014) . the usage of nucleoside/nucleotide analogues is a major step in the treatment of patients infected with covs due to the appropriate antiviral response (chhikara et al., 2020) . however, the application of these drugs may lead to genetic variation and subsequent mutation emergence. hence, the safety of rdv and its broad-spectrum anti-viral activity should be considered before suggesting them as potential alternative candidates for clinical development. over the recent years, animal model progression of rdv seems to orient primarily on cov respiratory infections (sheahan et al., 2020) . clinical trials in selective patient populations with cov or cov-like diseases are needed to examine the efficacy of the developed drugs. regarding safety data for rdv, some necessary studies in covid-19 patients should be conducted to proceed the clinical trials. studies over covlike diseases will probably require the enrollment of a large number of infected patients. there is currently no approved antiviral drug against sars-cov-2 to treat hospitalized patients. moreover, clinical trials over covid-19 patients seems to be complicated due to several factors such as inability to apply a placebo, underlying diseases, and evaluating anti-viral drug efficiency. if the synergistic activity of rdv and other anti-viral agents in cell cultures is approved by the current phase 3 clinical trials in patients with sars-cov-2, the outcome may propose a way for developing and performing clinical trials of the relative integration to rdv monotherapy and other anti-viral drug monotherapy for treating patients hospitalized with covid-19. ongoing and future perspectives are trying to determine the resistance of different covs to rdv both in vitro and in vivo, and to elucidate whether the mutational strains behave in a same way as wild types. due to the emergence of a new respiratory infections such as the sars-cov-2, progression of animal studies and subsequent preclinical and clinical trials are required to explore the activity of rdv. some preclinical explorations are ongoing to examine the potential of the rdv against the sars-cov-2. given application history of rdv in treating a wide range of infections, as well as the outcomes from clinical trials in patients with sars-and mers-cov, reinforces the rationale for additional trials of rdv against a wider range of infectious including covid-19. the ongoing studies in sars-cov-2 supported by the who is expected to furnish potential data corresponding to rdv application in treating covid-19. other similar investigations may be envisioned with respect to the increasing identifications of the importance of covs as a driving force of covid-19. apart from the potential progression of rdv for treating sars-and mers-covs, the emergence of other viral illnesses may pave the way for clinical trials of rdv derivatives. the authors declare no conflict of interest. (sheahan et al., 2020) . moroccan medicinal plants as inhibitors of covid-19: computational investigations repurposing papaverine as an antiviral agent against influenza viruses and paramyxoviruses coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies discovery and development of safe-in-man broad-spectrum antiviral agents treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-b1b (miracle trial): study protocol for a randomized controlled trial soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase features, evaluation and treatment coronavirus (covid-19) understanding the nature of variations in structural sequences coding for coronavirus spike, envelope, membrane and nucleocapsid proteins of sars-cov-2 the role of angiotensin converting enzyme 2 in coronaviruses/influenza viruses and cardiovascular disease the sars-cov-2 vaccine pipeline: an overview corona virus sars-cov-2 disease covid-19: infection, prevention and clinical advances of the prospective chemical drug therapeutics identification of cellular microrna mir-188-3p with broad-spectrum anti-influenza a virus activity prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection an interactive web-based dashboard to track covid-19 in real time adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease drug repurposing for coronavirus (covid-19): in silico screening of known drugs against coronavirus 3cl hydrolase and protease enzymes reverse vaccinology approach to design a novel multi-epitope vaccine candidate against covid-19: an in silico study are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? a tug-ofwar between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor the antiviral compound remdesivir potently inhibits rnadependent rna polymerase from middle east respiratory syndrome coronavirus animal models for sars and mers coronaviruses. current opinion in virology old weapon for new enemy: drug repurposing for treatment of newly emerging viral diseases the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin covid-19: what is next for public health? the lancet novel activities of safe-in-human broad-spectrum antiviral agents medicinal chemistry strategies toward host targeting antiviral agents initiation, extension, and termination of rna synthesis by a paramyxovirus polymerase discovery of potential multi-target-directed ligands by targeting host-specific sars-cov-2 structurally conserved main protease$ nucleotide analogues as inhibitors of sars-cov polymerase targeting sars-cov-2: a systematic drug repurposing approach to identify promising inhibitors against 3c-like proteinase and 2'-o-ribosemethyltransferase identification of chymotrypsin-like protease inhibitors of sars-cov-2 via integrated computational approach identification of 53 compounds that block ebola virus-like particle entry via a repurposing screen of approved drugs severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and corona virus disease-2019 (covid-19): the epidemic and the challenges the molecular biology of coronaviruses therapeutic options for the 2019 novel coronavirus (2019-ncov) the reproductive number of covid-19 is higher compared to sars coronavirus remdesivir (gs-5734) protects african green monkeys from nipah virus challenge the classification of viruses infecting the respiratory tract nucleoside/nucleotide analog inhibitors of hepatitis b virus polymerase: mechanism of action and resistance drug repurposing for viral infectious diseases: how far are we? clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta-analysis computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china viral load kinetics of mers coronavirus infection time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia. radiology peptide-like and small-molecule inhibitors against covid-19 coronavirus infections-more than just the common cold the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? the complexity and cost of vaccine manufacturing -an overview immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak in-silico homology assisted identification of inhibitor of rna binding against 2019-ncov n-protein (n terminal domain) drug repurposing strategies for covid-19 virtual screening of inhibitors against spike glycoprotein of 2019 novel corona virus: a drug repurposing approach. preprints, 1-15 broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov discovery and synthesis of a phosphoramidate prodrug of a pyrrolo [2, 1-f][triazin-4-amino] adenine c-nucleoside (gs-5734) for the treatment of ebola and emerging viruses from sars to mers, thrusting coronaviruses into the spotlight mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir the covid-19 epidemic remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys coronavirus genomics and bioinformatics analysis. viruses molecular diversity of coronaviruses in bats novel and potent inhibitors targeting dhodh, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against rna viruses including newly emerged coronavirus sars-cov-2 broad spectrum antiviral agent niclosamide and its therapeutic potential identification of small-molecule inhibitors of zika virus infection and induced neural cell death via a drug repurposing screen pathological findings of covid-19 associated with acute respiratory distress syndrome an animal model of mers produced by infection of rhesus macaques with mers coronavirus epidemic trend of corona virus disease 2019 (covid-19) in mainland china. zhonghua yu fang yi xue za zhi coronaviruses -drug discovery and therapeutic options key: cord-323125-qtlevnbt authors: al hosani, farida ismail; kim, lindsay; khudhair, ahmed; pham, huong; al mulla, mariam; al bandar, zyad; pradeep, krishna; elkheir, kheir abou; weber, stefan; khoury, mary; donnelly, george; younis, naima; el saleh, feda; abdalla, muna; imambaccus, hala; haynes, lia m; thornburg, natalie j; harcourt, jennifer l; miao, congrong; tamin, azaibi; hall, aron j; russell, elizabeth s; harris, aaron m; kiebler, craig; mir, roger a; pringle, kimberly; alami, negar n; abedi, glen r; gerber, susan i title: serologic follow-up of middle east respiratory syndrome coronavirus cases and contacts—abu dhabi, united arab emirates date: 2019-02-01 journal: clin infect dis doi: 10.1093/cid/ciy503 sha: doc_id: 323125 cord_uid: qtlevnbt background: although there is evidence of person-to-person transmission of middle east respiratory syndrome coronavirus (mers-cov) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. methods: a seroepidemiological investigation was conducted among mers-cov case patients (cases) and their household contacts to investigate transmission risk in abu dhabi, united arab emirates. cases diagnosed between 1 january 2013 and 9 may 2014 and their household contacts were approached for enrollment. demographic, clinical, and exposure history data were collected. sera were screened by mers-cov nucleocapsid protein enzyme-linked immunosorbent assay and indirect immunofluorescence, with results confirmed by microneutralization assay. results: thirty-one of 34 (91%) case patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. mers-cov antibodies were detected in 13 of 24 (54%) case patients with available sera, including 1 severely symptomatic, 9 mildly symptomatic, and 3 asymptomatic case patients. no serologic evidence of mers-cov transmission was found among 105 household contacts with available sera. conclusions: transmission of mers-cov was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. these results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission. although there is evidence of person-to-person transmission in household and healthcare settings [10] [11] [12] [13] [14] , more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. these data would be of importance to the public health response given that approximately 25% of confirmed mers-cov cases reported to the world health organization have been described as mildly symptomatic or asymptomatic [15] . during 1 january 2013-9 may 2014, the department of health-abu dhabi (doh) investigated 65 laboratory-confirmed cases and conducted extensive contact investigations in both household and healthcare settings [5] . through these investigations, 72% of the laboratory-confirmed cases reported no symptoms or mild illness [5] . contacts of case patients were tested by diagnostic pcr assays; however, results could include false negatives due to the 14-day incubation period. in this investigation, we use serological detection of mers-cov antibodies to evaluate if asymptomatic or mildly ill case patients had detectable mers-cov antibodies, estimate transmission rates from known cases to their household contacts, and identify potential risk factors. this investigation occurred in the emirate of abu dhabi, which occupies >80% of the uae's total area [16] and is comprised of 3 regions: abu dhabi (capital city), al ain region, and al dhafra. the emirate of abu dhabi has a population of 2.8 million (2015 estimate) [17] . the al ain region borders oman and saudi arabia and houses the second largest city in the emirate, al ain city. while al ain city is an oasis, the rest of the region primarily consists of desert and mountains. the al dhafra region is mainly desert and rural with approximately 285 000 residents and a population density of 8 residents/km 2 [18] . all laboratory-confirmed mers-cov cases (n = 65) in the emirate of abu dhabi diagnosed between 1 january 2013 and 9 may 2014 and their household contacts (n = 452) were eligible for the investigation. these cases were a convenience sample during the ongoing mers-cov outbreak. two of the 431 (0.5%) household contacts tested for mers-cov during initial contact investigations were pcr positive and eligible to be enrolled as cases for our investigation (figure 1 ). the enrolled case was a healthcare worker who might have been exposed by another coworker, who also lived in the case's household; therefore, the enrolled case was a result of either household or healthcare transmission prior to this investigation's initiation. the case not enrolled in this investigation was exposed in the household. household contacts were defined as any person who stayed at least 1 night at the same location as the case patient during the 14 days prior to the case patient's symptom onset or the date of first positive specimen if the case patient was asymptomatic. excluded cases included palace workers and other high-level officials; their associated household contacts were also excluded. for each mers-cov case identified in the investigation, clinical information, including symptoms, was collected using the international severe acute respiratory and emerging infection consortium form, which was filled out in real time by healthcare providers and subsequently verified by retrospective chart review. in abu dhabi during this time period, all individuals who tested positive for mers-cov were admitted to a healthcare facility for observation and infection control regardless of symptom status. the same definitions for case severity were used as in al hosani et al [5] including the following: asymptomatic cases reported no symptoms at the time of a positive test as recorded by a healthcare provider in the medical chart; mildly symptomatic cases reported symptoms, such as pharyngitis, rhinorrhea, or cough, and did not require oxygen during their hospitalization; and severely symptomatic cases required supplemental oxygenation during their hospitalization, ranging from nasal cannula to mechanical ventilation. using data collected from doh's surveillance of mers-cov cases, households with mers-cov case patients were approached. household contacts who were eligible for the investigation included those that had been identified through contact investigations associated with the case patient performed by doh officials within 24 hours' notification. three attempts were made to contact each household. if no response was received after 3 attempts, the household was not enrolled. households that agreed to be enrolled were given an appointment at the local disease prevention and screening center for questionnaire administration and serum collection. questionnaires were administered in english, arabic, or, if an interpreter was available, the participant's native language. data collected included demographics; residence/household description; exposure history to other mers-cov cases, healthcare settings, and animals; travel history; and medical history, including any long-term effects reported by case patients. for deceased case patients, a proxy completed the case patient questionnaire using recall. the real-time reverse-transcription pcr (rrt-pcr) results were obtained from the doh surveillance data. upper (ie, nasopharyngeal, oropharyngeal) and lower respiratory tract specimens (ie, sputum, bronchoalveolar lavage fluid, tracheal aspirates) were analyzed using rrt-pcr in the sheikh khalifa medical center laboratory. additional laboratory result verifications were performed in a random sample of 23 specimens using nucleocapsid-based rrt-pcr [5] . serum samples were inactivated using 2 × 10 6 rads gamma irradiation and stored at ≤ -70°c until use. screening of serum specimens by mers-cov nucleocapsid enzyme-linked immunosorbent assay (elisa) was performed at the sheik khalifa medical city in abu dhabi, uae and the centers for disease control and prevention (cdc), atlanta, georgia. titers of ≥1:400 were reported as positive. recombinant full length mers-cov nucleocapsid protein indirect elisa was used to screen serum specimens as described by al-abdallat et al [19] . serum samples were tested for the presence of neutralizing antibodies to mers-cov using a microneutralization assay (mnt) [19] . the neutralization titer was measured as the reciprocal of the highest serum dilution that completely inhibited vero cell lysis in at least 1 of the 3 triplicate wells. positive and negative controls were included for each mnt performed and included back-titration and mock-infected cells. titers of ≥1:20 were reported as positive. all work with live mers-cov was done in biosafety level 3 containment at the cdc. immunofluorescence assays (ifas) were performed by screening sera at a dilution of 1:50 and 1:100 on paraformaldehyde-fixed, acetone-methanol permeabilized mers-cov (strain mers-cov hu/england-n1/2012) infected or uninfected control vero cells. antihuman immunoglobulin g, m, and a fluorescein isothiocyanate conjugate was used to detect anti-mers-cov antibodies in human serum, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole to allow identification of individual mers-cov-infected cells. fluorescence was detected using a zeiss axioimager fluorescence microscope. the positive control for the assay is a serum sample from a patient infected with mers-cov hu/ england-n1/2012. a positive result was scored when these 3 conditions were met: cells were evenly stained (instead of punctate staining); fluorescence intensity was higher than that of the negative controls; and signal intensity declined with serial dilution. a minimum of 2 negative controls were included with each ifa. approximately 10% of specimens negative by nucleocapsid elisa were screened by both ifa and mnt to confirm the negative result. mers-cov antibody positivity was defined as one of the following: (1) 2 of 3 tests (ie, mers-cov nucleocapsid elisa, mers-cov mnt, and ifa) were positive; or (2) mers co-v mnt was the only positive test. household survey data were entered into electronic forms in epi info 7 version 7.1 (cdc). quality control and assurance were performed through epi info 7 intelligent codes programmed into the forms. household survey data were merged with the laboratory results, and descriptive analysis was completed. differences in proportions were compared using the mantel-haenszel χ 2 test, while differences in continuous variables were compared using the student t test. p < .05 was considered statistically significant. data analysis of the merged dataset was conducted with sas version 9.3 software (sas institute, cary, north carolina). following local customs, informed consent was obtained from the head of the household, who provided consent for all members of a household; however, each individual was still able to decline participation. this investigation was determined by doh and cdc to be part of a public health response, not research, and therefore not subject to institutional review board review. thirty-four case patients' households were included (supplementary table 1 ). household residences ranged in size from 7 m 2 to 1100 m 2 (interquartile range [iqr], 70-200 m 2 ). a median of 4 individuals (range, 1-30) lived in the households 14 days prior to the diagnosis of a mers-cov household case patient. more than half of mers-cov case patients shared a bathroom with others in the household. all households reported having air conditioning. thirty-four cases of 65 (52%) and 124 household contacts of 452 (27%) participated (table 1) . females comprised a higher proportion of case patients compared with household contacts (70.6% vs 53.2%), and case patients were older compared with household contacts (median, 42 years vs 31 years). most case patients and contacts were from the al ain region of the abu dhabi emirate. seventy-one percent (n = 24) of case patients reported working in a healthcare setting 14 days prior to diagnosis, with nurses being most represented (24%, n = 8) ( table 1) ; only 24% (n = 30) of household contacts worked in a healthcare setting 14 days prior to a case patient's diagnosis. compared with household contacts, case patients less frequently reported visiting or owning a farm (12% vs 14%), but reported camel exposure more frequently (12% vs 7%). household contacts reported the following frequent exposures to mers-cov case patients: hugging (54%, n = 67), using the same bathroom (51%, n = 63), sharing meals (49%, n = 61), and kissing or nose-kissing (ie, rubbing tips of noses against one another) (48%, n = 60). case patients reported a higher proportion of underlying medical conditions than household contacts, including diabetes (18% vs 5%, p = .01), hypertension (27% vs 7%, p < .001), kidney failure (6% vs 2%, p = .03), and heart failure (6% vs 0%, p < .01) ( table 1 ). case patients also reported taking medications for any illness more frequently than contacts (38% vs 17%). three case patients (9%) reported limitation to activities due to mers-cov with a median duration of 5 days (iqr, 4-24 days) ( table 1) . normal activities were resumed at a median of 5 days (iqr, 3-7 days). of 34 case patients, 17 (50%) reported being asymptomatic, 14 (41%) reported being mildly symptomatic, and 3 (9%) were severely symptomatic. age and proportion having underlying medical conditions increased with symptom severity ( table 2) . symptom duration did not have any noticeable trend with symptom severity (data not shown). all severe case patients were treated in the intensive care unit, as well as 1 asymptomatic case, who had underlying diabetes, hypertension, and kidney disease. the median days hospitalized increased with symptom severity ( table 2) . sera were obtained from 24 of 34 (71%) case patients and 105 of 124 (85%) household contacts. among the 24 case patients with available sera (table 3) among the 13 case patients with detectable antibodies against mers-cov, all of them were aged <60 years, with a median age of 43 years, compared to a median of 32 years for case patients without detectable antibodies (table 4 , p = .04). number of days of pcr positivity was notably higher among those who had detectable antibodies compared to those who did not (median, 15 days vs 2 days, p = .01). we describe the results of follow-up of 34 mers-cov case patients and 124 of their household contacts from the emirate of abu dhabi during 2013-2014. notably, serologic testing did not find any evidence of mers-cov transmission in the households of mers-cov case patients in our investigation, suggesting that viral transmission from asymptomatic or mildly symptomatic individuals to household contacts does not readily occur. sera were tested with a combination of 3 different laboratory assays (nucleocapsid elisa, ifa, and mnt); we feel confident that individuals identified as "negative" did not seroconvert. although there was clear evidence of household transmission in 1 household not enrolled in this investigation, our investigation's results did not show evidence of additional household transmission. overall, our findings support current recommendations that home isolation may be appropriate for asymptomatic cases and close contacts who are ill and do not require hospitalization in consultation with local public health departments [20, 21] . because this investigation occurred during may-june 2014, many case patients were recruited from the april 2014 healthcare-associated outbreak at an al ain region hospital [22] . a kingdom of saudi arabia study found that while healthcare personnel were at high risk for infection, most illness was relatively mild and could be unrecognized, highlighting potential undetected transmission of the virus to others [23] . in our investigation, case patients tended to be younger (30-59 years) , and most reported working in a healthcare setting 14 days prior to their diagnosis where they were exposed to a similar to previous studies, case patients with severe disease had higher frequency of comorbid conditions and required intensive care, including intubation [24] [25] [26] . in a recent investigation from south korea, patients with a higher host infectivity, which included evaluation of pcr cycle threshold values, along with higher numbers of contacts, were more likely to transmit mers-cov [27] . it is likely that most of the primary case patients in this investigation had lower host infectivity. while our investigation found that some asymptomatic or mildly symptomatic case patients had detectable antibodies, we did not find any detectable antibodies in 11 asymptomatic and mildly symptomatic case patients. other studies also did not find detectable antibodies in some asymptomatic and mildly ill cases [6, 28] . if seroconversion is to occur in case patients, studies have demonstrated that this usually occurs within the first month of illness [28] [29] [30] . for the majority of case patients with detectable antibodies, we found persistence of antibody response for several months after the initial diagnosis, even close to a year. additionally, these case patients had a longer duration of mers-cov pcr positivity than those who did not have detectable antibodies, indicating a potential relationship between longer viral shedding and seroconversion. previous studies have demonstrated that asymptomatic and mildly symptomatic case patients can test pcr positive >2 weeks from lower respiratory tract specimens [5, 31] . our investigation's serology results do not provide additional evidence of transmission to household contacts, though there is evidence from other settings to suggest limited household transmission [11] . also, very low rates of household transmission have been reported during hospital-based outbreaks [19, 32] . more robust transmission studies involving larger numbers of case patients representing a range of clinical and demographic characteristics and their contacts are needed to further investigate risk exposures. there are several limitations to this investigation. first, serum samples were collected at varying intervals after illness onset for each case patient, potentially affecting serology results. the duration of antibody response is unknown. second, recall bias might have led to the misclassification of symptom severity among household contacts; however, for case patients, to minimize this bias, we relied upon retrospective medical chart review, though this also might not be as complete since it depended on the initial healthcare provider's history and physical. third, these case patients were immediately isolated in hospitals after pcrpositive results were discovered. the removal from the household setting might have reduced exposure to household contacts although the case patients were residing with household contacts at the time of the contact investigations. last, because our investigation did not detect household transmission, we cannot comment on any behaviors or exposures that would increase risk among household contacts of case patients. in summary, we did not document additional household transmission in this investigation that included a preponderance of asymptomatic and mildly symptomatic confirmed mers-cov case patients. our investigation findings support the recommendation to consider home isolation for asymptomatic and mildly ill cases that do not require hospitalization while using proper precautions, including face masks, frequent hand washing, and minimizing exposure to the case patient in the household [20, 21, 33] . while no vaccines or antivirals against mers-cov are currently available, reducing transmission through effective infection control management remains a major priority. understanding transmission risk for different mers-cov-infected patients who live in different settings will be important data that must be factored into prevention strategies. further studies on human-to-human transmission in different settings should be conducted to inform mers-cov prevention and control guidelines. supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov): current situation 3 years after the virus was first identified world health organization. who mers-cov global summary and risk assessment risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia response to emergence of middle east respiratory syndrome coronavirus antibody response and disease severity in healthcare worker mers survivors mers-cov antibody responses 1 year after symptom onset, south korea persistence of antibodies against middle east respiratory syndrome coronavirus comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity mers-cov outbreak in jeddah-a link to health care facilities transmission of mers-coronavirus in household contacts hospital outbreak of middle east respiratory syndrome coronavirus family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus transmission in extended family, saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-update: disease outbreak news abu dhabi emirate: facts and figures statistical yearbook of abu dhabi hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance implementing home care and isolation or quarantine of people not requiring hospitalization for mers-cov transmission of middle east respiratory syndrome coronavirus infections in healthcare settings risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea serologic responses of 42 mers-coronavirusinfected patients according to the disease severity viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection kinetics of serologic responses to mers coronavirus infection in humans a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker health care worker contact with mers patient, saudi arabia interim guidance for healthcare professionals acknowledgments. the authors gratefully thank the participants; the teams from the disease prevention and screening center-al ain, the disease prevention and screening center-abu dhabi, ghayathi hospital, and silla hospital that supported this investigation; and suvang trivedi and seyhan boyoglu-barnum at the centers for disease control and prevention (cdc) for technical support.disclaimer. the conclusions, findings, and opinions expressed herein are those of the authors and do not necessarily reflect the official position of the us department of health and human services, the us public health service, the cdc, or the authors' affiliated institutions.financial support. this work was supported by the cdc. potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-341620-nmrkhx5t authors: chirico, francesco; sacco, angelo; bragazzi, nicola luigi; magnavita, nicola title: can air-conditioning systems contribute to the spread of sars/mers/covid-19 infection? insights from a rapid review of the literature date: 2020-08-20 journal: int j environ res public health doi: 10.3390/ijerph17176052 sha: doc_id: 341620 cord_uid: nmrkhx5t the airborne transmission of sars-cov-2 is still debated. the aim of this rapid review is to evaluate the covid-19 risk associated with the presence of air-conditioning systems. original studies (both observational and experimental researches) written in english and with no limit on time, on the airborne transmission of sars-cov, mers-cov, and sars-cov-2 coronaviruses that were associated with outbreaks, were included. searches were made on pubmed/medline, pubmed central (pmc), google scholar databases, and medrxiv. a snowball strategy was adopted to extend the search. fourteen studies reporting outbreaks of coronavirus infection associated with the air-conditioning systems were included. all studies were carried out in the far east. in six out the seven studies on sars, the role of heating, ventilation, and air conditioning (hvac) in the outbreak was indirectly proven by the spatial and temporal pattern of cases, or by airflow-dynamics models. in one report on mers, the contamination of hvac by viral particles was demonstrated. in four out of the six studies on sars-cov-2, the diffusion of viral particles through hvac was suspected or supported by computer simulation. in conclusion, there is sufficient evidence of the airborne transmission of coronaviruses in previous asian outbreaks, and this has been taken into account in the guidelines released by organizations and international agencies for controlling the spread of sars-cov-2 in indoor environments. however, the technological differences in hvac systems prevent the generalization of the results on a worldwide basis. the few covid-19 investigations available do not provide sufficient evidence that the sars-cov-2 virus can be transmitted by hvac systems. many countries have responded to the "coronavirus disease 2019" (covid-19) pandemic caused by the emerging infectious agent termed as "severe acute respiratory coronavirus type 2" (sars-cov-2) by adopting stringent containment measures, such as the so-called lockdown. while these behavioral, non-pharmacological interventions have been proven effective in curbing the spread of the virus [1, 2] , they are having, on the other hand, significant economic and social consequences [3, 4] and cannot, by their nature, be implemented and enforced indefinitely [5] . several governments have started gradually easing and relaxing restrictions, by reopening selected businesses and production activities. the resumption of daily activities is now taking place in some countries, during the summer season, when the use of air-conditioning systems in indoor environments is necessary to ensure comfortable working conditions. the question therefore arises whether the use of heating, ventilation, and air conditioning (hvac) systems can contribute to the further spread of the microorganisms in the workplace. being a novel virus, the precise mode and routes of transmission of sars-cov-2 are still debated. sars-cov-2 is thought to primarily spread from human to human, by close contact transmission, as well as through respiratory droplets of >5-10 µm in diameter, which are generated when an infected person coughs, sneezes, talks, or sings at short distances, generally <1-2 m [6, 7] . the "airborne" transmission of droplet nuclei ("aerosolized particles of <5 µm in diameter"), remaining infectious when suspended in the air over long distances and time, is still controversial [7] . indeed, it was found that some individuals behave as "speech super-emitters," releasing sars-cov-2 particles of ≤1 µm in diameter during normal breathing and speech [8] , coughing and talking [9, 10] . some airborne "superspreading" events have occurred in the past with other coronaviruses, e.g., in 2003, a sars-cov-1 outbreak initiated in hong kong and spread to other countries, and in 2012, a "middle eastern respiratory syndrome" (mers)-cov outbreak in the kingdom of saudi arabia and in 2015 in south korea [11] [12] [13] . moreover, the airborne transmission of sars-cov-2 can occur during medical procedures that generate aerosols ("aerosol-generating procedures") [14] . many aerosol-generating surgical procedures have been identified [15] [16] [17] [18] [19] [20] , and special preventive measures have been proposed [21, 22] . the transmission capacity, and therefore the infectious power of the aerosols, also depends on the presence of airborne particulates. one feature of all particulates is that they can convey and release toxic molecules, as well as microorganisms, including viruses [23] . some studies have associated the spread of covid-19 cases with pm2.5 microparticle pollution levels [24] [25] [26] . in italy and other european countries, technical guidelines have been released to limit the operation of hvac systems, such as the recirculation of exhausted air [27] [28] [29] . the different methods of air distribution have different limitations and solutions [30] . all safety measures, including those on the use of air-conditioning, have costs, which must be balanced by the benefits. the limitation of the use of air conditioners could create thermal discomfort, and negatively affect productivity and health outcomes in the workplace-where people spend one-third of their lifetime-and other indoor environments. the assessment of the risk of infection associated with the use of air-conditioning is essential for making correct decisions. therefore, to evaluate the covid-19 risk associated with the presence of air-conditioning systems, we conducted a rapid review of the literature concerning outbreaks of coronaviruses (sars-cov-1, mers-cov, and sars-cov-2) in indoor environments. due to time constraints related to the urgency of the ongoing pandemic, we carried out a rapid review [31] , thus streamlining the rules that characterize a systematic review. we utilized the participants-exposure-comparisons-outcome (pecos) criteria, and we defined them according to evidence-based practice [32] -p (participants) is human subjects residing in indoor environments, e (exposure) is exposed to air-conditioning systems (hvac), c (comparisons) is any comparison between the pathogens under study, and o (outcome) is respiratory infection outbreaks caused by sars-cov-1, mers-cov, or sars cov-2. only original studies (both observational and experimental researches) written in english were retrieved, with no limit on time. narrative reviews, opinions, and commentaries were excluded. we also excluded experimental studies on the airborne transmission of coronaviruses that were not associated with outbreaks. articles indexed in pubmed/medline, pubmed central (pmc), google scholar databases, and medrxiv (a pre-print server for health sciences), were retrieved on july 11, 2020. a snowball strategy was then adopted to extend the search, including extensive cross-referencing, and using subject headings or keywords to search for similar relevant articles. keyword checking was performed by the authors independently. the electronic search strategy used keywords related to the topic under investigation ("air-conditioning" or "hvac") in combination with "covid-19," "virus," "sars," "mers," "sars-cov-2," and "coronavirus," properly combined by boolean operators. after independently reviewing all titles/abstracts to identify potentially relevant articles, an author (fc) selected studies for full-text review [32] . data concerning the country of study, the study design, and the study setting, as well as the study's outcomes, were extracted. the results of the studies retained in the present rapid review were synthesized qualitatively. in our review, we found 14 studies reporting outbreaks of coronavirus infection in indoor environments (table 1) . seven studies concerned sars-cov-1, and six sars-cov-2, respectively, while only one regarded mers-cov. seven studies were carried out in hong kong, one in south korea, three in japan, and three in china. the studies were conceived as cross-sectional, in two cases with a retrospective collection of data, and in six cases complemented by an airflow modelling method or environmental sampling. in the sars-cov-1 epidemic, studies focused mainly on two outbreaks, which occurred in the prince of wales hospital, a regional and teaching government hospital, and in amoy gardens, a private housing estate in hong kong, which were the most seriously affected locations during the 2003 outbreak of severe acute respiratory syndrome (sars). both were served by air-conditioning systems. the episodes occurred in 2003. in the first one, in march 2003, 156 patients in the prince of wales hospital in hong kong were infected by a patient treated with a jet nebulizer [33] . in this episode, which affected healthcare workers, medical students, and patients, the proximity of the patients may have allowed the infection to spread through nuclei droplets, although the production of aerosols during treatment was evident. this outbreak was also studied by wong et al. [34] , with a retrospective observational study on 66 medical students who visited the index patient's ward. the authors showed that the flow rate was highest in the air supply diffuser in the index patient's cubicle, and lowest in the corresponding exhaust grille. this imbalance would have favored the spread of aerosols. the computed concentration contours of aerosols, in fact, matched epidemiologic data. a retrospective study of on outbreak involving 74 patients in the same hospital indicated that the rapid evaporation of the droplets produced by coughing in a relatively dry, air-conditioned environment, could also induce virus-laden aerosol, which was probably responsible for spreading the infection to patients who were not in the same room [35] . the ward, however, was air-conditioned by a fan coil system with a separate fresh air supply. the exhaust air from this ward was discharged to the outside and not recirculated to other wards. a modelling study conducted by chen et al. [36] on ward 8a of the same hospital in hong kong, where patients and staff were infected, demonstrated the direct responsibility of hvac systems in the diffusion of the disease. the air exchange owing to temperature difference played a significant role in sars transmission during the nosocomial outbreak in ward 8a, and the two-way airflow effect at the openings could have played an important role in bioaerosol transmission [36] . the sars outbreak that occurred in 2003 in the isolation ward of an infectious disease hospital in hong kong also had a spatial infection pattern compatible with airborne transmission. some design defects in the air-distribution system, with unbalanced flow rates in supply diffusers and exhaust grilles, could have played a role in infection spreading. computational fluid dynamics simulations confirmed this hypothesis [37] . in the same year, more than 300 residents of a private high-rise housing estate were infected with sars cov-1 within a short period, and 42 died. the case index was a tenant receiving haemodialysis. virus-containing aerosol could have escaped into the narrow lightwell between the buildings and spread in rising air currents. the association between the predicted bioaerosol concentration and the spatial infection pattern suggested an airborne transmission route, and the aerosol distribution patterns coincided with the spatial and temporal distribution of infections [38, 39] . unfortunately, none of the investigations were completed with the isolation of microorganisms in the environment. in 2015, the spread of mers was associated with aerosol transmission in three patients from two hospitals in south korea [40] . environmental sampling from the air exhaust damper revealed the presence of viral particles of mers-cov. the potential airborne transmission of mers was supported by the disclosure of viral particles in the hospital environment including air, fomites, and environmental surfaces. in summary, six of the seven sars studies suspected that the air-conditioning system had played a role in the spread of the infection. in the only study available on mers, contamination of hvac by viral particles was demonstrated. data concerning the new coronavirus sars-cov-2 are obviously scarce. the analysis of 318 outbreaks involving, in different settings, 1245 infected individuals in 120 cities in china from 29 december to 11 february 2020, prompted quian et al. to assert that long-range aerosol transmission had occurred in crowded spaces with poor ventilation. detection of infectious particles in the air and on inaccessible surfaces suggested that the virus might be transmitted via airborne routes, and not only from droplet exposure [41] . three studies were devoted to the outbreak that occurred in the diamond princess cruise ship, which affected 355 people in japan. according to zhang et al., the main route for transmission was from person to person, but other routes, including aerosol transmission via central air supply or drainage systems, could not be excluded [42] . the studies of xu et al. [43] and mizumoto and chowell [44] on the same outbreak, led to the opposite conclusion. most of the cases originated by passenger-to-passenger transmission through close contact and fomites. after the adoption of quarantine measures, new cases among passengers were limited to those who stayed in the same room with an infected passenger. therefore, the ship's central air-conditioning system would not have played a role, i.e., the long-range airborne route was not relevant in the outbreak. another outbreak occurred in a chinese restaurant from january 26 to february 10, 2020. ten persons from three families were infected with sars-cov-2. the restaurant had an air-conditioned, fifth-floor building without windows. each floor had its own air conditioner. according to lu et al. [45] , droplet transmission was likely prompted by air-conditioned ventilation. the key factor for the outbreak was the direction of the airflow. studying the same outbreak with computer simulations, li et al. [46] postulated that the infection distribution was consistent with a spread pattern representative of exhaled virus-laden aerosols. in summary, in four out of the six studies on sars-cov-2, the diffusion of viral particles through hvac was suspected or supported by computer simulation, while in the other two studies, it was excluded based on epidemiological considerations. the few covid-19 investigations available do not provide sufficient evidence that hvac systems can play an important role in the indoor outbreaks reported to date. demonstration of the contamination of hvac systems by sars-cov-2 viral particles is currently lacking. this evidence, in fact, was collected only during the mers epidemic, and it is still unknown if the sars-cov-2 virus has the same potential transmission and spreading diffusion as the previous one. furthermore, modelling studies on the distribution of viral particles for covid-19, which in the first sars epidemic strongly supported the possibility that sars-cov-1 aerosols could be captured and re-emitted by hvac, are not yet available. nevertheless, the findings obtained from the previous coronavirus epidemics, in which the hvac systems were often held responsible for the spreading of the virus, do not allow us to believe that they are now totally risk-free. furthermore, we know that certain medical procedures may produce aerosols, and that they have been responsible for the spread of coronavirus in healthcare settings [47] . environmental investigations have shown the presence of sars-cov-2 viral particles on surfaces that cannot be reached by droplets, and this is indirect evidence that the transmission of aerosols has taken place [48, 49] . recent covid-19 outbreaks reported during choir practice [50] , in a call-center [51] and in fitness classes [52] , have suggested the role of aerosol transmission in combination with droplet transmission, particularly in crowded and inadequately ventilated indoor spaces. experimental studies in a laboratory-controlled environment demonstrated that aerosolized sars-cov-2 particles (<5 µm) remained suspended in the air for at least 3 h, viable in air for at least 1 h, and on surfaces for up to days [53] [54] [55] . the half-life of viral particles could be different in relation to meteorological conditions-such as temperature, relative humidity, and ultraviolet radiation-that could degrade and weaken the virus [56] [57] [58] . ventilation systems have been already reported as a way of transmitting/spreading infectious diseases such as measles, chickenpox, flu, smallpox [59, 60] , and the 2009 influenza a (h1n1) pandemic [61] . the possibility that the sars-cov-2 virus remains suspended in the air, in free micro-drops or attached to particulates, and therefore can be picked up by the air-conditioning systems and put back into circulation, poses an evident safety problem for indoor environments [62, 63] . the characteristics of hvac systems, therefore, are very important for safety purposes. unfortunately, the available studies refer exclusively to outbreaks that occurred in the far east, in environmental and plant conditions that cannot be considered representative of other parts of the world. building types, air-conditioning systems, regulations, and maintenance requirements may differ between countries, so the available studies may not be generalizable. on the other hand, the accumulated knowledge about the novel sars-cov-2 indicates that it has higher aerosol and surface stability than sars-cov-1 and can remain viable and infectious in aerosol for hours [64] . patients affected by covid-19 could have a high viral load, which correlates with transmission risk and disease severity [65] . when dealing with a new disease, particular caution is required. the aerosol model of sars-cov-2 transmission has several practical implications, and this is confirmed by the analysis of the most important guidelines released by organizations and international agencies [27] [28] [29] , which should be based on the best available scientific evidence. most of the existing recommendations, which suggest deactivating air recirculation mode in all indoor environments, seem to be especially based on previous evidence of airborne transmission of sars-cov-1 and other airborne viruses, as research on sars-cov-2 is still limited. only future studies will clarify whether hvac implants played a role in favoring the spread of viruses in covid-19. in the meanwhile, occupational preventive measures should be based on the best possible scientific evidence by balancing costs and effectiveness, yet according to the principles of a precautionary approach. limiting recirculating air may not be technically possible in all workplaces, may give rise to additional costs, and could not balance cooling and heating functions in summer and winter, with appropriate levels of relative humidity and air velocity [66] . moreover, too high, or too low temperatures, and low ambient humidity, are known to enhance viral transmission [67, 68] , and thus could harm the elderly and workers with "fragilities." therefore, employers, assisted by their consultants, must conduct a careful assessment of the risks and benefits associated with the use of air-conditioning systems. the choice to keep the systems in operation, guaranteeing comfortable working conditions, must always be accompanied by a continuous and scrupulous maintenance of the systems, with the use, where possible, of high-efficiency total filters. the technical and engineering characteristics of hvac are of the utmost importance in the diffusion of pollutants. undoubtedly the most effective methods to reduce pollution are the increase in ventilation (increase in air flow rates) and the minimization of air recirculation, with the introduction and treatment of external air. however, these methods are very expensive, and often not very efficient. all air intake systems should be designed so as to avoid the presence of air currents in the respiratory area of workers or occupants; this undoubtedly contributes to avoiding the long-distance transport of droplets and aerosols. the efficiency of hvac systems filters could be increased by nanomaterials [69] . in healthcare settings, environmental engineering controls, consisting of physical engineering elements such as negative pressure rooms, dilution ventilation, high-efficiency particulate air filtration, ultraviolet lights, and scavenging devices, can reduce the aerosol diffusion [70] . in other types of environment, proposed alternatives to hvac include adopting natural ventilation and personalized ventilation-personalized exhaust systems (pv-pe) for microenvironments [61] . in the risk assessment process, it is of primary importance to estimate the probability that an infectious person is inside the premises. this probability can be inferred from the trend of the epidemic in the region where the building is located, and from the consideration that covid-19 can spread in many people in an entirely asymptomatic way, making screening at the entrance ineffective [71] . it was observed that oligosymptomatic patients with a sars-cov-2 infection can have a greater viral load than that of patients hospitalized with severe forms [72] , and therefore represent a real threat. therefore, the risk assessment and the decision to keep the air-conditioning system active must be dynamic and based on the epidemiological evolution of the pandemic, as well as on the verification of the characteristics of the system and its efficiency. this rapid review aimed to synthesize the available information for policy-makers, employers, technicians, and workers concerning the aerosol transmission of sars, mers and covid-19 infections in air-conditioned indoor environments, in order to give a guide to the best choices for workers' health protection. the weaknesses of our study are mainly in the data retrieved. studies included in this review were few, often anecdotal, and published in non-peer-reviewed journals. the lack of quantitative data did not allow us to extract meta-analytical information. the "rapid" nature of this review, and finally the type of studies included, prevented us from making an appropriate evaluation of their quality. in conclusion, this short review is a warning for health and safety consultants, and a stimulus for further studies on the relationship between sars-cov-2 and air-conditioning systems. the studies available to date are not sufficient to support the conclusion that air-conditioning systems favor the spread of the sars-cov-2 infection in office and indoor community environments. however, in previous coronavirus epidemics, hvac systems not specifically designed to accommodate infectious patients were suspected of facilitating the spread of sars-cov-1 and mers-cov in hospital and community settings. the guidelines released by organizations and international agencies for controlling the spread of sars-cov-2 in indoor environments are based on these studies. the formation of aerosols, which is possible in many medical activities, and the great variability in the viral load in patients, make it necessary to adapt the safety of the hvac systems to the specific control needs. prevention in workplaces must be personalized, with the adoption of more stringent measures when epidemiological circumstances (e.g., increase in the number of cases, increase in hospital admissions) require it. the precautionary principle requires the utmost attention in the design and management of air-conditioning systems, while waiting for further evidence on the effectiveness of safety measures proposed by international guidelines. only strict quarantine measures can curb the coronavirus disease (covid-19) outbreak in italy quarantine alone or in combination with other public health measures to control covid-19: a rapid review shielding from covid-19 should be stratified by risk covid-19: the critical balance between appropriate governmental restrictions and expected economic, psychological and social consequences in italy. are we going in the right direction? joint european roadmap towards lifting covid-19 containment turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid-19 who. modes of transmission of virus causing covid-19: implications for ipc precaution recommendations aerosol emission and superemission during human speech increase with voice loudness small droplet aerosols in poorly ventilated spaces and sars-cov-2 transmission violent expiratory events: on coughing and sneezing the role of super-spreaders in infectious disease understanding and modeling the super-spreading events of the middle east respiratory syndrome outbreak in korea analysis of the healthcare mers-cov outbreak in king abdulaziz medical center who. transmission of sars-cov-2: implications for infection prevention precautions: scientific brief aerosol-generating otolaryngology procedures and the need for enhanced ppe during the covid-19 pandemic: a literature review covid-19 dentistry-related aspects: a literature overview urologic surgery and covid-19: how the pandemic is changing the way we operate covid-19 coronavirus: recommended personal protective equipment for the orthopaedic and trauma surgeon covid 19 pandemic and gynaecological laparoscopic surgery: knowns and unknowns. facts views vis. obgyn covid-19-what should anaethesiologists and intensivists know about it? anaesthesiol who. advice on the use of masks in the context of covid-19: interim guidance first aid during the covid-19 pandemic. (editorial) air pollution and respiratory viral infection exposure to air pollution and covid-19 mortality in the united states spatial correlation of particulate matter pollution and death rate of covid-19 can atmospheric pollution be considered a co-factor in extremely high level of sars-cov-2 lethality in northern italy? who. modes of transmission of virus causing covid-19: implications for ipc precaution recommendations: scientific brief ashrae position document on infectious aerosols climatizzazione in strutture comunitarie non sanitarie e in ambienti domestici in relazione alla diffusione del virus sars-cov-2. versione del 25 maggio 2020 a review of advanced air distribution methods-theory, practice, limitations and solutions national collaborating centre for methods and tools. rapid review guidebook steps for conducting a rapid review a scoping review of rapid review methods a major outbreak of severe acute respiratory syndrome in hong kong outbreak study group. cluster of sars among medical students exposed to single patient temporal-spatial analysis of sars among hospital inpatients role of two-way airflow owing to temperature difference in severe acute respiratory syndrome transmission: revisiting the largest nosocomial severe acute respiratory syndrome outbreak in hong kong role of air distribution in sars transmission during the largest nosocomial outbreak in hong kong evidence of airborne transmission of the severe acute respiratory syndrome virus multi-zone modeling of probable sars virus transmission by airflow between flats in block e extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards indoor transmission of sars-cov-2. medrxiv 2020 estimation of the reproductive number of novel coronavirus (covid-19) and the probable outbreak size on the diamond princess cruise ship: a data-driven analysis transmission routes of covid-19 virus in the diamond princess cruise ship transmission potential of the novel coronavirus (covid-19) onboard the diamond princess cruises ship covid-19 outbreak associated with air conditioning in restaurant evidence for probable aerosol transmission of sars-cov-2 in a poorly ventilated restaurant aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards transmission characteristics of mers and sars in the healthcare setting: a comparative study recognition of aerosol transmission of infectious agents: a commentary high sars-cov-2 attack rate following exposure at a choir practice coronavirus disease outbreak in call center cluster of coronavirus disease associated with fitness dance classes aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 persistence of severe acute respiratory syndrome coronavirus 2 in aerosol suspensions persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents effect of ultraviolet germicidal irradiation on viral aerosols effects of air temperature and relative humidity on coronavirus survival on surfaces the effects of temperature and relative humidity on the viability of the sars coronavirus airborne transmission of a small round structured virus airborne transmission of nosocomial varicella from localized zoster ventilation control for airborne transmission of human exhaled bio-aerosols in buildings airborne route and bad use of ventilation systems as non-negligible factors in sars-cov-2 transmission sampling and detection of corona viruses in air: a mini review airborne transmission route of covid-19: why 2 m/6 feet of inter-personal distance could not be enough sars-cov-2 viral load predicts covid-19 mortality strategy and methods for the risk assessment of thermal comfort in the workplace mechanisms by which ambient humidity may affect viruses in aerosols rapid expert consultation on sars-cov-2 survival in relation to temperature and humidity and potential for seasonality for the covid-19 pandemic can nanotechnology and materials science help the fight against sars-cov-2? nanomaterials possible sars coronavirus transmission during cardiopulmonary resuscitation hospital infection and covid-19: do not put all your eggs on the "swab" tests association of initial viral load in sars-cov-2 patients with outcome and symptoms this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-316013-7dckgg6b authors: wang, lili; xu, jiyan; kong, yu; liang, ruiying; li, wei; li, jinyao; lu, jun; dimitrov, dimiter s.; yu, fei; wu, yanling; ying, tianlei title: engineering a novel antibody-peptide bispecific fusion protein against mers-cov date: 2019-11-04 journal: antibodies (basel) doi: 10.3390/antib8040053 sha: doc_id: 316013 cord_uid: 7dckgg6b in recent years, tremendous efforts have been made in the engineering of bispecific or multi-specific antibody-based therapeutics by combining two or more functional antigen-recognizing elements into a single construct. however, to the best of our knowledge there has been no reported cases of effective antiviral antibody-peptide bispecific fusion proteins. we previously developed potent fully human monoclonal antibodies and inhibitory peptides against middle east respiratory syndrome coronavirus (mers-cov), a novel coronavirus that causes severe acute respiratory illness with high mortality. here, we describe the generation of antibody-peptide bispecific fusion proteins, each of which contains an anti-mers-cov single-chain antibody m336 (or normal human igg1 ch3 domain as a control) linked with, or without, a mers-cov fusion inhibitory peptide hr2p. we found that one of these fusion proteins, designated as m336 diabody-pep, exhibited more potent inhibitory activity than the antibody or the peptide alone against pseudotyped mers-cov infection and mers-cov s protein-mediated cell-cell fusion, suggesting its potential to be developed as an effective bispecific immunotherapeutic for clinical use. the antibody-based therapeutic modalities have shown clinical success in the treatment of many diseases [1] [2] [3] [4] . in recent years, tremendous efforts have been made in the engineering of bispecific or multi-specific antibodies by combining two or more functional antigen-recognizing elements into a single construct [5, 6] . such novel antibodies, or antibody-based fusion proteins, could be particularly beneficial for the treatment of viral infections, which typically require potent and multi-functional therapeutics to prevent the frequent incidence of viral escape mutants [7] . for instance, we previously engineered a bispecific and multivalent anti-hiv-1 fusion protein, by incorporating the hiv-1 neutralizing antibody and the engineered single-domain cd4 into a single antibody-like molecule, four constructs, m336 scfv, m336 scfv-pep, m336 diabody-pep, and ch3-pep were generated. the m336 scfv was generated by linking the m336 variable region heavy and variable region light chains with a 15 amino acids (g 4 s) 3 linker. fusion protein m336 scfv-pep was prepared by linking m336 scfv with mers-cov-derived hr2p peptide (ltqinttlldltyemlslqqvvkalnesyidlkel) using another (g 4 s) 3 linker as the spacer. fusion protein m336 diabody-pep was generated by replacing the (g 4 s) 3 linker between vh and vl of m336 scfv-pep with a 5 amino acids ggggs linker, so that two vhs and vls can pair together to form the dimeric diabody. the ch3-pep was also generated by fusing hr2p peptide with igg1 ch3 domain. coding fragments were synthesized by genewiz biotech co., and inserted between two sfii restriction sites in the pcomb3x vector with flag-tag (dykddddk) and his 6 -tag on c-termini. the sequences of the constructs were verified by direct dna sequencing. all four constructs were expressed as soluble proteins in e. coli and purified on ni-nta column. briefly, the expression vectors were transformed into e. coli strain hb2151 competent cells. a single fresh colony was inoculated into 3 ml of 2yt medium containing 100 µg/ml ampicillin and incubated at 37 • c, 250 rpm overnight. the incubated culture was transferred to 200 ml of fresh 2yt medium with 100 µg/ml ampicillin for large-scale protein production and 4-6 h growth at 37 • c. then, 1 mm isopropyl-β-d-thiogalactoside (iptg) was applied to induce protein overexpression, and the cells were grown overnight at 30 • c before harvesting by centrifugation. protein fragments were harvested from the bacterial cell pellet and the precipitate was re-suspended with 50 ml pbs with 0.5 m nacl. then, 0.2 ml 1 m polymyxin b was added to lyse bacteria and the sample was rotated 30 min at room temperature, 250 rpm. the cultures were centrifuged at 12,000 rpm for 15 min at 4 • c. supernatant was collected and loaded onto a ni-nta superflow (qiagen, redwood city, ca, usa). after the impurities were removed, target proteins were eluted with elution buffer (250 mm imidazole in pbs). the proteins were resolved with sds-page and the purity was estimated as >90%. the protein purity was further confirmed by size exclusion chromatography using an fplc akta system (ge healthcare, chicago, il, usa) with a superdex 200 10/300 gl column (ge healthcare). the protein concentration was measured spectrophotometrically (nanovue, ge healthcare). the experiments were performed using the proteon xrp36 system (bio-rad, hercules, ca, usa) to measure the binding kinetics of m336 scfv, m336 scfv-pep, m336 diabody-pep, and ch3-pep to mers-cov s protein (amino acid 18-725). the s protein was immobilized on a proteon glm biosensor chip using standard amine coupling chemistry (300 nm in 10 mm sodium acetate buffer, ph 5.0), and~3000 resonance units were immobilized. the surface of the sensor chip was activated with 200 mm 1-ethyl-3-dimethyl aminopropylcarbodiimide hydrochloride and 50 mm n-hydroxysulfosuccinimide. m336 scfv, m336 scfv-pep, m336 diabody-pep, and ch3-pep were prepared in pbs, ph 7.4, containing 0.005% tween-20 (pbs-t) and injected (50 µl/min for 120 s, 1:3 dilution from 200 nm). the dissociation phase was followed for 600 s and chip surfaces were regenerated by injecting 10 mm glycine hcl, ph2.0, 100 µl/min for 18 s. data were analyzed using proteon manager 3.1 software and fitted to the 1:1 interaction model [42] . mers-cov neutralization assay was performed as previously described [43, 44] . briefly, 293t cells in 10 cm 2 dishes were transiently co-transfected with a pcdna3.1-mers-cov-s plasmid and a pnl4-3.luc.re plasmid encoding an env-defective luciferase-expressing hiv-1genome. after 48 h post-transfection, the produced pseudovirus was harvested from the supernatant, and filtered through 0.45 µm sterilized membrane. the mers-cov pseudovirus was incubated with four inhibitors at 37 • c for 30 min, and then pseudovirus and inhibitors were added to dpp4-expressing huh-7 cells (10 4 /well) preplated in 96 well tissue culture plates for 6 h. after 12 h, fresh medium was added to the plates and incubated for another 48 h. cells were lysed with lysis reagent (promega, madison, wi, usa) and lysates were transferred into 96-well costar flat-bottom luminometer plates (corning, corning, ny, usa). luciferase substrate was added and the readings were recorded with an ultra 384 microplate reader (tecan, männedorf, switzerland). to further compare the inhibitory effects of fusion proteins, the cell-cell fusion assay was performed as mers-cov s protein, which was expressed on the cell surface, can mediate cell fusion with neighboring cells [43] . first, 293t cells were transiently transfected with paa-ires-mers-egfp (293t/mers/egfp, effector cells) encoding the mers-cov proteins or paa-ires-egfp (293t/egfp) as a negative control. cells were cultured in dmem containing 10% fbs at 37 • c for 48 h. preplated huh-7 cells (10 4 , target cells), which express the dpp4 mers-cov receptor, were cultured in 96 well plates at 37 • c for 5 h. after addition of 293t/mers/egfp or 293t/egfp cells with 2-fold diluted m336 scfv, m336 scfv-pep, m336 diabody-pep, or ch3-pep from 2.5 µg/ml, the mixture and huh-7 cells (target cells) were further cultures at 37 • c for 4 h, and fused or unfused cells were visualized under an inverted fluorescent microscope (nikon eclipse ti-s100, nikon, tokyo, japan) [45] . to generate the bispecific antibody-peptide inhibitor, the mers-cov s2-derived peptide hr2p was fused to m336 scfv using a flexible (g 4 s) 3 linker. furthermore, a dimeric format, namely m336 diabody-pep, was also generated by replacing the (g 4 s) 3 linker between variable region heavy (vh) and variable region light (vl) of m336 scfv with a 5 amino acids ggggs linker. the shorter linker can result in the pairing of vh and vl from two different chains and thus the formation of dimeric diabody with two vhs and vls. a dimeric peptide-only control, namely ch3-pep, was also generated by linking hr2p peptide and igg1 ch3 domain with the flexible (g 4 s) 3 linker. this fusion protein possesses two peptides and comparable molecular weight compared to other constructs ( figure 1a ). plates at 37 °c for 5 h. after addition of 293t/mers/egfp or 293t/egfp cells with 2-fold diluted m336 scfv, m336 scfv-pep, m336 diabody-pep, or ch3-pep from 2.5 μg/ml, the mixture and huh-7 cells (target cells) were further cultures at 37 °c for 4 h, and fused or unfused cells were visualized under an inverted fluorescent microscope (nikon eclipse ti-s100, nikon, tokyo, japan) [45] . to generate the bispecific antibody-peptide inhibitor, the mers-cov s2-derived peptide hr2p was fused to m336 scfv using a flexible (g4s)3 linker. furthermore, a dimeric format, namely m336 diabody-pep, was also generated by replacing the (g4s)3 linker between variable region heavy (vh) and variable region light (vl) of m336 scfv with a 5 amino acids ggggs linker. the shorter linker can result in the pairing of vh and vl from two different chains and thus the formation of dimeric diabody with two vhs and vls. a dimeric peptide-only control, namely ch3-pep, was also generated by linking hr2p peptide and igg1 ch3 domain with the flexible (g4s)3 linker. this fusion protein possesses two peptides and comparable molecular weight compared to other constructs ( figure 1a) . the four proteins, m336 scfv, m336 scfv-pep, m336 diabody-pep, and ch3-pep were soluble expressed in e. coli cells with high efficiency. purified proteins were obtained with yields of 15-30 mg/l bacterial culture. the m336 diabody-pep migrated as a monomer under the denaturing conditions of the sds-page, and thus displayed similar molecular weight (~30 kda) with m336 scfv and m336 scfv-pep ( figure 1b ). all proteins were monomeric as demonstrated by size exclusion chromatography (data not shown). to measure the binding kinetics of the fusion proteins with mers-cov s protein, the surface plasmon resonance (spr) assay was performed by using a proteon system spr biosensor instrument ( figure 2 ). we have previously demonstrated that m336 bound specifically to mers-cov s protein, but not other unrelated proteins [35] . as shown in table 1 , all three m336-based fusion proteins (m336 scfv, m336 scfv-pep, and m336 diabody-pep) exhibited potent binding to s protein with very similar binding pattern. the equilibrium dissociation constant (kd) of m336 scfv for s protein was 0.8 nm with on-rate (kon) of 4.7 × 10 5 m −1 s −1 and off-rate (koff) of 3.8 × 10 −4 s −1 . the m336 diabody-pep displayed similar binding kinetics to that of m336 scfv (kon 1.1 × 10 6 m −1 s −1 , koff 1.2 × 10 −3 s −1 , kd 1.1 nm). the m336 the four proteins, m336 scfv, m336 scfv-pep, m336 diabody-pep, and ch3-pep were soluble expressed in e. coli cells with high efficiency. purified proteins were obtained with yields of 15-30 mg/l bacterial culture. the m336 diabody-pep migrated as a monomer under the denaturing conditions of the sds-page, and thus displayed similar molecular weight (~30 kda) with m336 scfv and m336 scfv-pep ( figure 1b) . all proteins were monomeric as demonstrated by size exclusion chromatography (data not shown). to measure the binding kinetics of the fusion proteins with mers-cov s protein, the surface plasmon resonance (spr) assay was performed by using a proteon system spr biosensor instrument ( figure 2) . we have previously demonstrated that m336 bound specifically to mers-cov s protein, but not other unrelated proteins [35] . as shown in table 1 , all three m336-based fusion proteins (m336 scfv, m336 scfv-pep, and m336 diabody-pep) exhibited potent binding to s protein with very similar binding pattern. the equilibrium dissociation constant (k d ) of m336 scfv for s protein was 0.8 nm with on-rate (k on ) of 4.7 × 10 5 m −1 s −1 and off-rate (k off ) of 3.8 × 10 −4 s −1 . the m336 diabody-pep displayed similar binding kinetics to that of m336 scfv (k on 1.1 × 10 6 m −1 s −1 , k off 1.2 × 10 −3 s −1 , k d 1.1 nm). the m336 scfv-pep had slightly more potent binding affinity (k d 0.2 nm) with slower dissociation rate constant (k off 1.2 × 10 −4 s −1 ), compared to the other two fusion proteins. in contrast, the ch3-peptide displayed much lower affinity, compared to the antibody m336-based fusion proteins (k d 1.1 m). next, we measured the neutralization activity of m336 scfv, m336 scfv-pep, m336 diabody-pep, and ch3-pep at graded concentrations against pseudotyped mers-cov infection. as shown in figure 3 , m336-scfv, m336 scfv-pep and m336 diabody-pep exhibited potent neutralization activity. among them, m336 scfv-pep exhibited the most potent neutralization capability, with 50% inhibitory concentration (ic50) of 0.21 ± 0.06 nm. although, displaying comparable ic50 (m336 diabody-pep, 0.25 ± 0.07 nm; m336 scfv, 0.69 ± 0.03 nm), interestingly, m336 diabody-pep inhibited the infection more potent than that of m336 scfv at low protein concentrations (<0.03 nm). in a previous study, igg1 m336 displayed exceptional neutralization against pseudotyped mers-cov infection with ic50 of 0.03 nm [35] . the control fusion protein ch3-pep could not inhibit the infection. next, we measured the neutralization activity of m336 scfv, m336 scfv-pep, m336 diabody-pep, and ch3-pep at graded concentrations against pseudotyped mers-cov infection. as shown in figure 3 , m336-scfv, m336 scfv-pep and m336 diabody-pep exhibited potent neutralization activity. among them, m336 scfv-pep exhibited the most potent neutralization capability, with 50% inhibitory concentration (ic 50 ) of 0.21 ± 0.06 nm. although, displaying comparable ic 50 (m336 diabody-pep, 0.25 ± 0.07 nm; m336 scfv, 0.69 ± 0.03 nm), interestingly, m336 diabody-pep inhibited the infection more potent than that of m336 scfv at low protein concentrations (<0.03 nm). in a previous study, igg1 m336 displayed exceptional neutralization against pseudotyped mers-cov infection with ic 50 of 0.03 nm [35] . the control fusion protein ch3-pep could not inhibit the infection. we used a well-established mers-cov s protein-mediated cell-cell fusion assay to determine whether the fusion proteins have the ability to inhibit mers-cov fusion with the target cells [40, 46] . in such assay, the 293t cells that express mers-cov s protein and egfp were used as the effector cells, and huh-7 cells that express dpp4 receptor were used as the target cells. the ch3-pep showed no appreciable activity at a concentration of 83 nm, which is consistent with the previous report that the hr2p peptide did not have significant inhibitory effect at concentrations lower than 100 nm [40] . notably, we found that m336 diabody-pep was able to block the fusion between 293t/mers/egfp cells and huh-7 cells at a concentration as low as 0.5 nm (figure 4) . the calculated ic50 for m336 diabody-pep was 0.81 ± 0.02 nm. as shown in figure 4 , the m336 diabody-pep was significantly more potent than m336 scfv in inhibiting cell-cell fusion. the m336 scfv-pep also showed evidently more potent inhibitory activity than m336 scfv at low concentrations, with slightly lower ic50 (m336 scfvpep, 5.25 ± 0.03 nm; m336 scfv, 7.76 ± 0.02 nm). these results indicate that antibody-peptide bispecific fusion proteins, especially the m336 diabody-pep, have the potential to be developed as potent mers-cov inhibitors that could, not only neutralize mers-cov infection, but also inhibit mers-cov s protein-mediated cell-cell fusion. we used a well-established mers-cov s protein-mediated cell-cell fusion assay to determine whether the fusion proteins have the ability to inhibit mers-cov fusion with the target cells [40, 46] . in such assay, the 293t cells that express mers-cov s protein and egfp were used as the effector cells, and huh-7 cells that express dpp4 receptor were used as the target cells. the ch3-pep showed no appreciable activity at a concentration of 83 nm, which is consistent with the previous report that the hr2p peptide did not have significant inhibitory effect at concentrations lower than 100 nm [40] . notably, we found that m336 diabody-pep was able to block the fusion between 293t/mers/egfp cells and huh-7 cells at a concentration as low as 0.5 nm (figure 4 ). the calculated ic 50 for m336 diabody-pep was 0.81 ± 0.02 nm. as shown in figure 4 , the m336 diabody-pep was significantly more potent than m336 scfv in inhibiting cell-cell fusion. the m336 scfv-pep also showed evidently more potent inhibitory activity than m336 scfv at low concentrations, with slightly lower ic 50 (m336 scfv-pep, 5.25 ± 0.03 nm; m336 scfv, 7.76 ± 0.02 nm). these results indicate that antibody-peptide bispecific fusion proteins, especially the m336 diabody-pep, have the potential to be developed as potent mers-cov inhibitors that could, not only neutralize mers-cov infection, but also inhibit mers-cov s protein-mediated cell-cell fusion. we used a well-established mers-cov s protein-mediated cell-cell fusion assay to determine whether the fusion proteins have the ability to inhibit mers-cov fusion with the target cells [40, 46] . in such assay, the 293t cells that express mers-cov s protein and egfp were used as the effector cells, and huh-7 cells that express dpp4 receptor were used as the target cells. the ch3-pep showed no appreciable activity at a concentration of 83 nm, which is consistent with the previous report that the hr2p peptide did not have significant inhibitory effect at concentrations lower than 100 nm [40] . notably, we found that m336 diabody-pep was able to block the fusion between 293t/mers/egfp cells and huh-7 cells at a concentration as low as 0.5 nm (figure 4) . the calculated ic50 for m336 diabody-pep was 0.81 ± 0.02 nm. as shown in figure 4 , the m336 diabody-pep was significantly more potent than m336 scfv in inhibiting cell-cell fusion. the m336 scfv-pep also showed evidently more potent inhibitory activity than m336 scfv at low concentrations, with slightly lower ic50 (m336 scfvpep, 5.25 ± 0.03 nm; m336 scfv, 7.76 ± 0.02 nm). these results indicate that antibody-peptide bispecific fusion proteins, especially the m336 diabody-pep, have the potential to be developed as potent mers-cov inhibitors that could, not only neutralize mers-cov infection, but also inhibit mers-cov s protein-mediated cell-cell fusion. monoclonal antibodies represent one of the most promising immunotherapeutic agents for the treatment of cancers and infectious diseases [7, [47] [48] [49] [50] [51] [52] . previously, we used rbd of mers-cov s protein to screen a non-immune phage-displayed fab library and identified a highly potent human neutralizing mab m336 and a panel of other mabs against mers-cov [34, 36, 37] . jiang et al. also used mers-cov s rbd to screen a non-immune yeast-displayed scfv library and identified two potent mers-cov neutralizing mabs, mers-4 and mers-27 [53] . tang et al. used a full-length s protein to screen a non-immune phage-display scfv library and identified a potent mers-cov neutralizing mab 3b11 [54] . all these reported mabs inhibit the binding of virus to cellular reporter dpp4 by recognizing the different epitopes of rbd, thus having the potential to be developed as anti-mers-cov therapeutics. however, further engineering is still needed to address the concerns in using such mabs in clinics, e.g., the high production costs, etc. in this study, we generated two bispecific anti-mers-cov fusion proteins, m336 scfv-pep and m336 diabody-pep. compared with the antibody or peptide inhibitors that block only one site of the spike protein, the bispecific inhibitors showed greater potency. the m336 scfv-pep showed the highest binding affinity to mers-cov s protein, as well as the most potent neutralizing activity, while the m336 diabody-pep was the most potent in blocking mers-cov s-mediated cell-cell fusion. these results confirmed the improved potency of bispecific antibody-peptide inhibitors compared to antibody or peptide alone. the m336-scfv interferes with viral attachment to the dpp4 receptor on human cells [35] , while the hr2p peptide inhibits the formation of the 6-helix-bundle fusion core and thus interrupts viral fusion with the target cell membrane [40] . it is probably that the antibody-peptide fusion proteins, which are composed of both antiviral components, linked by flexible (g 4 s) 3 linker, can act at both steps during viral infection. interestingly, the m336 diabody-pep was found to potently block mers-cov s-mediated cell-cell fusion even at concentrations lower than 0.5 nm. this phenomenon may be attributed to the difference in steric hindrance of the fusion proteins. compared to the m336 scfv-pep, the m336 diabody-pep has a much larger (twice) molecular weight and size, and thus, inhibits the viral fusion to target cells by competitive inhibition and steric hindrance, leading to the much greater inhibitory activity than that of m336 scfv-pep. although, having largely improved potency in inhibiting cell-cell fusion, m336 diabody-pep did not show evidently increased neutralization activity compared to m336 scfv. further investigations are required to explore the mechanism for the disparate performance of m336 diabody-pep in different assays. as shown in figures 3 and 4 , the ic 50 of m336 diabody-pep was 0.25 ± 0.07 nm in the neutralization assay, and 0.81 ± 0.02 nm in the cell-cell fusion assay. importantly, m336 diabody-pep was much more potent than m336 scfv at concentrations higher than 5 nm in the cell-cell fusion assay. at these concentrations, all the fusion proteins can result in high percentage of inhibition (>70%) in the neutralization assay, and therefore, may explain the little difference among difference groups. all the m336-based fusion proteins could be easily produced in e. coli in large amount and low production cost. their sizes are larger than m336 scfv or hr2p peptides, suggesting that they would possess longer in vivo half-life. still, their half-life would be shorter than full-size igg due to the lack of fc region. therefore, their prophylactic and therapeutic efficacy against mers-cov should be assessed carefully using different approaches and doses in vivo. however, the rbd of mers-cov specifically targets on human dpp4 [29] , and most small animal models are not susceptible to mers-cov infection [55] , which pose a significant barrier to the development of anti-mers-cov inhibitors. fortunately, researchers have constructed several animal models recently that simulate the morbidity and mortality of human infections, of which nonhuman primates (nhp) models and human dpp4-expressing mouse model are considered to be ideal candidates [56] and the latter is promising to be utilized in our following studies. in conclusion, our study indicated that the bispecific inhibitors have increased the efficacy against mers-cov, compared to the neutralizing antibody or polypeptide alone. such inhibitors with the advantage of multiple biologics have the potential to be further developed as effective prophylactic and therapeutic agents, and may find wide application in treating viral diseases. potent neutralization of hendra and nipah viruses by human monoclonal antibodies a neutralizing human monoclonal antibody protects african green monkeys from hendra virus challenge reversion of advanced ebola virus disease in nonhuman primates with zmapp a broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain iii the making of bispecific antibodies a native-like bispecific antibody suppresses the inflammatory cytokine response by simultaneously neutralizing tumor necrosis factor-alpha and interleukin-17a human monoclonal antibodies as candidate therapeutics against emerging viruses potent in vivo nk cell-mediated elimination of hiv-1-infected cells mobilized by a gp120-bispecific and hexavalent broadly neutralizing fusion protein a defucosylated bispecific multivalent molecule exhibits broad hiv-1-neutralizing activity and enhanced antibody-dependent cellular cytotoxicity against reactivated hiv-1 latently infected cells rapid elimination of broadly neutralizing antibodies correlates with treatment failure in the acute phase of shiv infection development of protein-and peptide-based hiv entry inhibitors targeting gp120 or gp41 isolation of a novel coronavirus from a man with pneumonia in saudi arabia comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? camel exposure and knowledge about mers-cov among australian hajj pilgrims in 2014 coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol evidence for camel-to-human transmission of mers coronavirus evolutionary insights into the ecology of coronaviruses human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study polyphyletic origin of mers coronaviruses and isolation of a novel clade a strain from dromedary camels in the united arab emirates the emergence of the middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus in bats, saudi arabia urgent development of effective therapeutic and prophylactic agents to control the emerging threat of middle east respiratory syndrome (mers) adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc engineered soluble monomeric igg1 ch3 domain: generation, mechanisms of function, and implications for design of biological therapeutics the multifunctional or moonlighting protein cd26/dppiv a predicted receptor-binding and critical neutralizing domain in s protein of the novel human coronavirus hcov-emc receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor combining a fusion inhibitory peptide targeting the mers-cov s2 protein hr1 domain and a neutralizing antibody specific for the s1 protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd mapping of anti-human il-9 supernatants a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines peptide-based membrane fusion inhibitors targeting hcov-229e spike protein hr1 and hr2 domains middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein engineered antibody domains with significantly increased transcytosis and half-life in macaques mediated by fcrn engineered fc based antibody domains and fragments as novel scaffolds monomeric igg1 fc molecules displaying unique fc receptor interactions that are exploitable to treat inflammation-mediated diseases a potent germline-like human monoclonal antibody targets a ph-sensitive epitope on h7n9 influenza hemagglutinin recent progress on neutralizing antibodies against hepatitis b virus and its implications single-domain antibodies as therapeutics against human viral diseases potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection animal models of middle east respiratory syndrome coronavirus infection this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflicts of interest. the funding sponsors had no role in the writing of the manuscript or the decision to publish this article. key: cord-339762-lh8czr0a authors: ng, dianna l.; al hosani, farida; keating, m. kelly; gerber, susan i.; jones, tara l.; metcalfe, maureen g.; tong, suxiang; tao, ying; alami, negar n.; haynes, lia m.; mutei, mowafaq ali; abdel-wareth, laila; uyeki, timothy m.; swerdlow, david l.; barakat, maha; zaki, sherif r. title: clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates, april 2014 date: 2016-03-31 journal: the american journal of pathology doi: 10.1016/j.ajpath.2015.10.024 sha: doc_id: 339762 cord_uid: lh8czr0a middle east respiratory syndrome coronavirus (mers-cov) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal mers-cov infection is unknown. we describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of mers-cov in the world, which was related to a hospital outbreak in the united arab emirates in april 2014. the main histopathologic finding in the lungs was diffuse alveolar damage. evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. double staining immunoassays that used anti–mers-cov antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of mers-cov antigen; double immunostaining with dipeptidyl peptidase 4 showed colocalization in scattered pneumocytes and syncytial cells. no evidence of extrapulmonary mers-cov antigens were detected, including the kidney. these results provide critical insights into the pathogenesis of mers-cov in humans. middle east respiratory syndrome coronavirus (mers-cov) infection causes an acute respiratory illness and is associated with a high case fatality rate; however, the pathogenesis of severe and fatal mers-cov infection is unknown. we describe the histopathologic, immunohistochemical, and ultrastructural findings from the first autopsy performed on a fatal case of mers-cov in the world, which was related to a hospital outbreak in the united arab emirates in april 2014. the main histopathologic finding in the lungs was diffuse alveolar damage. evidence of chronic disease, including severe peripheral vascular disease, patchy cardiac fibrosis, and hepatic steatosis, was noted in the other organs. double staining immunoassays that used antiemers-cov antibodies paired with immunohistochemistry for cytokeratin and surfactant identified pneumocytes and epithelial syncytial cells as important targets of mers-cov antigen; double immunostaining with dipeptidyl peptidase 4 showed colocalization in scattered pneumocytes and syncytial cells. middle east respiratory syndrome coronavirus (mers-cov) was initially isolated from a sputum specimen of a patient who died of respiratory and renal failure in saudi arabia in 2012. 1 recent data have indicated that dromedary camels are likely to transmit mers-cov to humans. 2, 3 human-to-human mers-cov transmission is well documented, particularly in the setting of nosocomial outbreaks. 4, 5 as of july 7, 2015, the world health organization (who) was notified of 1368 laboratory-confirmed cases of mers-cov infection with 487 reported deaths (35.6%) from 26 countries, primarily in men with a median age of 50 years. 6 most cases (>75%) were reported from the kingdom of saudi arabia. 6 mers-cov infection can result in a wide clinical spectrum from asymptomatic infection, upper respiratory tract illness, to severe pneumonia and multiorgan failure. 7e9 mers-cov binds to dipeptidyl supported by cdc operational funds. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the cdc. disclosures: none reported. current address of m.g.m., national cancer instituteeshady grove, rockville, md. peptidase 4 (dpp4) receptors that are primarily in the lower respiratory tract but also distributed in other tissues. 10 although there have been numerous cases and fatalities, the pathologic changes and viral distribution in humans associated with severe mers-cov illness are unknown, and knowledge of pathogenesis remains limited. this report provides the first autopsy, clinicopathologic, immunohistochemical (ihc), and ultrastructural description of a fatal case of mers-cov. this patient, who had multiple close contacts, was identified as part of an epidemiologic investigation of a large cluster of mers-cov infections at hospital in the united arab emirates (uae) in april 2014. this outbreak occurred in the context of a surge of cases in the arabian peninsula in which 515 mers-cov cases were identified in saudi arabia from april 11 to june 9, 2014 (world health organization, http://www.who.int/csr/don/2014_06_13_ mers/en, last accessed october 9, 2015). tissues obtained at autopsy were fixed in 10% buffered formalin, paraffin-embedded, sectioned at 4 mm, and stained by routine hematoxylin and eosin. an immunostaining protocol was applied with use of several mouse antibodies and one human antibody to mers-cov as described previously. 11 ihc assays that used mouse anti-mers antibodies ( table 1) lung tissue samples were excised from a paraffin block with the use of a 2-mm punch and processed for electron microscopy examination as previously described. 12 full genome sequencing by the sanger method with the use of direct genome walking pcr and next-generation sequencing (illumina miseq; illumina, san diego, ca) were performed as previously described on confirmed positive lung tissue, 13 named as abu dhabi_uae_8_2014 and deposited into genbank (http://www.ncbi.nlm.nih.gov; genbank accession number kp209306). clustalw was used to align the full genome with the respective available complete or near complete mers-cov genomes in the american journal of pathologyajp.amjpathol.org the patient was a 45-year-old, nonsmoking, obese (body mass index, 30.5 kg/m 2 ) filipino man with no relevant past medical history, recent travel, or exposure to sick contacts or farm animals. he worked in a storage room at a paramedic station with no patient care duties. he shared housing with five roommates from the paramedic department. he presented to an emergency department in abu dhabi, uae, on april 2, 2014, with a 4-day history of fever, rhinorrhea, and productive cough. at evaluation, he was afebrile with an oxygen saturation of 99% on room air, and a chest x-ray showed a left-sided opacity ( figure 1a) . the patient was diagnosed as acute bronchitis, prescribed 20 mg prednisolone daily for 5 days and paracetamol, and discharged. he returned to the emergency department on april 6, 2014, with persistent cough and shortness of breath; a chest x-ray showed a left-sided opacity and air bronchograms, and the patient was diagnosed with pneumonia, given a prescription for levofloxacin, and discharged ( figure 1b) . he returned to the emergency department the same day, with worsening shortness of breath and was admitted. he had a temperature of 38.7 c, pulse of 113 beats per minute, a respiratory rate of 24 breaths per minute, blood pressure of 123/73, an oxygen saturation of 93% on room air, and left basal crackles and rales. an arterial blood gas on room air showed ph of 7.49, partial pressure of carbon dioxide of 27.6 mm hg, partial pressure of oxygen of 52.4 mm hg, and bicarbonate of 20.7 meq/l, consistent with respiratory alkalosis. laboratory evaluation showed a normal white blood cell count with lymphopenia (0.5 k/ml) and creatinine of 0.9 mg/dl (supplemental table s1 ). blood, sputum, and urine cultures were obtained, and a nasopharyngeal swab specimen was sent for mers-cov testing. on april 7, day 1 of admission, oseltamivir, ceftriaxone, and azithromycin were started empirically. the day after admission he was transferred to the intensive care unit for tachypnea and respiratory distress and was intubated for mechanical ventilation; a portable chest x-ray showed multiple patchy airspace opacities ( figure 1c ). the patient became hypotensive, requiring inotropic support, and developed acute kidney injury and renal failure, requiring dialysis. on april 8, rt-pcr assay of the nasopharyngeal swab specimen collected on april 7 was reported to be positive for mers-cov for upe and orf1a gene targets. 16 bacterial cultures of blood, sputum, and urine specimens obtained at admission after antibiotic administration were negative. on april 9, he was given 100 mg hydrocortisone intravenously every 8 hours and started on nitric oxide at 16 ppm. the patient's condition continued to deteriorate, and he died april 10. the body was kept refrigerated at 4 c, and an autopsy was performed 10 days after death. notable findings included massive pleural effusion (5 l), substantial pericardial effusion (150 ml), and abdominal effusion; edematous and consolidated lungs; and generalized congestion. the predominant pulmonary histologic pattern was exudative-phase diffuse alveolar damage with denuding of bronchiolar epithelium, prominent hyaline membranes, alveolar fibrin deposits, type 2 pneumocyte hyperplasia, rare multinucleated syncytial cells, and alveolar septa involved by edema and lymphocytes with fewer plasma cells, neutrophils, and macrophages (figure 2, aec) . dispersed foci of necrotic debris were seen both subpleurally and within alveoli. no viral inclusions were seen, and moderate anthracosis was present. all four antibodies (1510/1513, sections of trachea and bronchi showed mild-to-moderate lymphocytic mucosal and submucosal inflammation with a few neutrophils and plasma cells ( figure 2g ). occasional clusters of noninvasive and extracellular candida yeast and hyphae were seen in septa, alveolar spaces, and overlying respiratory epithelium, suggesting postmortem overgrowth. bronchial submucosal glands were focally necrotic ( figure 2h ). immunostaining for mers-cov antigens was identified in both unremarkable and necrotic bronchial submucosal glands ( figure 2i ). multiple double immunostaining immunoassays were performed to assess for colocalization of mers-cov and cells labeling for cytokeratin, cd68, surfactant, and dpp4. double staining with cytokeratin confirmed the presence of viral antigens within pneumocytes and epithelial syncytial cells, whereas double staining with cd68 showed two distinct populations with no colocalization of mers-cov and macrophages (figure 3, a and b) . surfactant double staining revealed type 2 pneumocytes and syncytial cells with intracytoplasmic viral antigens ( figure 3c ). dpp4 antigens were detected in pneumocytes, syncytial cells, mononuclear leukocytes, and vascular endothelium, although colocalization of mers-cov and dpp4 was observed in scattered pneumocytes and syncytial cells ( figure 3d ). electron microscopy showed infected and degenerated pneumocytes encased between hyaline membranes, composed of fibrin and basement membranes ( figure 3e ). clusters or individualized, predominately spherical, 50 to 150 nm in diameter, viral particles were observed in membrane-bound vesicles ( figure 3f) . the kidney showed increased globally sclerotic glomeruli (5% to 10% of glomeruli), thickened bowman capsules, severe atherosclerosis and hyaline arteriolosclerosis, patchy interstitial inflammation, and intratubular proteinaceous and granular casts. diminished lymphoid follicles and a robust interfollicular proliferation of pleomorphic immunoblasts intermixed with a polymorphous population of reactive lymphocytes were seen in multiple lymph nodes; the spleen also contained numerous immunoblasts and reactive lymphocytes. the bone marrow was normocellular with maturing trilineage hematopoiesis and left-shifted the american journal of pathologyajp.amjpathol.org granulopoiesis. the heart revealed diffuse myocyte hypertrophy, moderate coronary atherosclerosis, and patchy fibrosis. moderate steatosis, scattered calcifications, and mild portal tract and lobular lymphocytic inflammation were identified in the liver. the sections of the cerebrum and cerebellum were unremarkable. mers-cov ihc was negative in multiple specimens from different organs, including kidney, liver, spleen, several lymph nodes, bone marrow, small intestine, and colon. overall the histology was well-preserved; although the sections of brain showed minimal-to-mild autolysis and kidney showed moderate autolysis. the genome sequence is similar (>99%) to other known mers-covs and clustered closely with camel-derived mers-cov strains (kj650295 to kj650297) obtained in al-hasa, saudi arabia, in 2013, suggesting recent origin in camels, although the patient had no known camel exposures. as indicated from the phylogenetic tree (supplemental figure s1 ), the sequences from this case and close contacts cluster most closely in the same clade, further supporting their transmission link between these cases. this report provides invaluable insights as the first description in the published literature of the clinicopathologic, ihc, and ultrastructural findings of a fatal case of mers-cov infection. the histopathologic pattern observed in the lungs was diffuse alveolar damage. mers-cov ihc and double staining techniques showed viral antigens were predominantly localized to type 2 pneumocytes and epithelial syncytial cells. although the pathogenesis of severe and fatal mers-cov infection is unknown, these postmortem findings provide critical insights, including evidence that pneumocytes are important targets, suggesting that direct cytopathic effects contribute to mers-cov respiratory symptoms. an ex vivo study that examined mers-coveinfected human lung tissue found evidence of pneumocyte damage by electron microscopy, including detachment of type 2 pneumocytes, and membrane blebbing, suggestive of apoptosis. 17 however, ihc staining for mers-cov was patchy, implying that other causes such as immune dysfunction may be relevant. acute renal failure is commonly observed in critically ill mers-cov patients 7e9,18 and mers-cov rna was detected in urine 18, 19 ; however, no evidence of extrapulmonary mers-cov dissemination was observed, suggesting that acute renal failure in this patient was not caused by direct renal infection but likely by other factors, such as hypoperfusion or cytokine dysregulation. the presence of mers-cov antigens in submucosal glands provides a mechanism by which the virus enters respiratory secretions and becomes transmissible. the pathologic features of mers-cov are shared by other similar respiratory illnesses, such as severe acute respiratory syndrome (sars)-cov. several reports that evaluated fatal cases of sars-cov describe diffuse alveolar damage in various stages as the most characteristic feature, 20 with ihc staining of sars-cov antigen primarily in alveolar epithelial cells. 20 syncytial cells may also be seen with other coronavirus infections, including sars-cov and paramyxovirus infections. 20 mers-cov entry is mediated by dpp4, which is expressed throughout the lower respiratory tract 17 but also numerous other organs, including the kidney. 10 double staining with mers-cov and dpp4 ihc was seen in pneumocytes and syncytial cells, consistent with the identification of these cells as targets of mers-cov infection. in addition, ex vivo models have detected mers-cov antigen in bronchial epithelial cells, pneumocytes, and endothelium and dpp4 in bronchiolar epithelium, endothelium, pneumocytes, and alveolar macrophages, 17, 21 correlating with our findings. in contrast, the functional receptor for sars-cov is angiotensin-converting enzyme 2. 22 similar to ddp4, angiotensin-converting enzyme 2 has a broad distribution, with heavy expression in alveolar epithelial cells, enterocytes, and endothelial cells. 23 although evidence shows that sars-cov also infects type 2 pneumocytes, 24,25 sars-cov was detected in several extrapulmonary sites by in situ hybridization and electron microscopy, including circulating lymphocytes, lymphoid tissues, renal distal tubules, and small intestinal mucosa. 26 because mers-cov rna was detected in blood, 19 and dpp4 is widely distributed in different tissues, 10 extrapulmonary dissemination could be possible, but we did not detect any evidence of mers-cov spread outside the respiratory tract. pulmonary findings of consolidation, diffuse alveolar damage, and pleural effusions with no evidence of bacterial pneumonia are consistent with the clinical features reported in other critically ill adults with mers-cov infection. 7e9 pathologic evidence of several chronic diseases in this patient, including myocardial fibrosis, atherosclerosis, and hepatic steatosis, correlates with reports of individuals with underlying comorbidities, such as end-stage renal disease, diabetes mellitus, hypertension, or cardiac disease, having an increased risk of developing severe mers-cov infection. 4, 7 the impact of low-dose oral prednisolone before admission and intravenous hydrocortisone on the clinical course and fatal outcome of this mers-cov patient is unknown. follicular depletion in lymph nodes, consistent with corticosteroid use, was noted, and no bacterial coinfections were identified. the hydrocortisone dosing of 100 mg every 8 hours in the intensive care unit before death was higher than recommended for refractory septic shock by the surviving sepsis campaign guidance, 27 but the patient only received a few doses. a systematic review of high-dose corticosteroids for treatment of sars-cov reported evidence of harm without survival benefit. 28 in patients with influenza a(h1n1)pdm09 virus infection, corticosteroid treatment was associated with increased mortality, 29 although further studies are needed. therefore, until further evidence is available, high-dose corticosteroid treatment for mers-cov pulmonary disease should be avoided. 30 current clinical management of mers-cov patients is based on supportive care of complications and adherence to recommended infection prevention and control measures (cdc, http://www.cdc.gov/coronavirus/mers/infection-preventioncontrol.html, last accessed october 9, 2015). our findings provide invaluable and unprecedented insights into the histologic changes, pathogenesis, and viral dissemination of mers-cov in humans. further studies, including additional postmortem examinations, evaluation of the host immune response, and identification of sites of viral replication, are necessary to strengthen knowledge on pathogenesis, transmission patterns, and effective treatment strategies for optimal clinical management and infection control practices. isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation ksa mers-cov investigation team: hospital outbreak of middle east respiratory syndrome coronavirus investigation team: hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description world health organization: middle east respiratory syndrome coronavirus (mers-covesummary of current situation. lit update risk assess 7 epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study albarrak am: clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia al raiy b: clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc evaluation of pigtail macaques as a model for the effects of copper intrauterine devices on hiv infection pathology and pathogenesis of fatal bordetella pertussis infection in infants fullgenome deep sequencing and phylogenetic analysis of novel human betacoronavirus mrbayes: bayesian inference of phylogenetic trees realtime reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs kinetics and pattern of viral excretion in biological specimens of two mers-cov cases clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan multiple organ infection and the pathogenesis of sars surviving sepsis campaign guidelines committee including the pediatric subgroup: surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock sars: systematic review of treatment effects do corticosteroids reduce the mortality of influenza a (h1n1) infection? a meta-analysis world health organization: clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance 2 we thank cynthia goldsmith for reviewing the electron microscopy and azaibi tamin for providing the mers-coveinfected vero cell cultures. supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2015.10.024. key: cord-293871-hzes7mwt authors: mcguinness, sarah l.; wu, henry m. title: pretravel considerations for non-vaccine-preventable travel infections date: 2018-11-26 journal: travel medicine doi: 10.1016/b978-0-323-54696-6.00007-0 sha: doc_id: 293871 cord_uid: hzes7mwt pretravel advice should be tailored to the individual following a thorough review of his or her itinerary, planned activities, and host characteristics. in addition to vaccinations and malaria chemoprophylaxis, a pretravel consultation should include advice on regionally endemic or emerging non–vaccine-preventable infections that can cause severe illness or chronic morbidity. these include mosquito-borne infections such as dengue, chikungunya, and zika, and regionally endemic severe respiratory infections such as middle east respiratory syndrome (mers) and some strains of avian influenza. zika virus is notable given its capacity for sexual transmission and association with congenital birth defects. preventive advice for other potentially relevant infections associated with specific exposures or activities (e.g., schistosomiasis and leptospirosis from freshwater exposure) should be provided where relevant. understanding the epidemiology and prevention of these infections is crucial to providing a comprehensive pretravel consultation. to provide optimal advice, travel health providers should be able to educate the traveler on preventive measures against key travel-related infections, including those for which no vaccine is available. tailoring this advice for an individual requires a thorough review of the travelers' itinerary and planned activities, consideration of the travelers' host characteristics, and a working knowledge of the epidemiology of relevant diseases. travelers play an important role in the global epidemiology of infectious diseases; therefore ensuring that travelers are aware of specific preventive measures not only protects the health of the individual but has the potential to protect the health of their communities. in this chapter, pretravel considerations for major non-vaccine-preventable infectious diseases are covered, including specific advice for dengue, chikungunya, zika, middle east respiratory syndrome coronavirus (mers-cov), and avian influenza. dengue virus (denv), chikungunya virus (chikv), and zika virus (zikv) are globally important mosquito-borne viruses spread via aedes aegypti and a. albopictus. the public health impact of these viruses has increased dramatically over the last 50 years, with epidemics of increasing size, geographic reach, and severity recorded. with factors such as population growth, urbanization, globalization, travel, and climate change facilitating increased transmission, travel medicine practitioners in temperate countries are increasingly likely to see returned travelers with these infections. furthermore, since the ranges of aedes vectors extend into temperate areas, infected returned travelers can precipitate outbreaks of these viruses in nonendemic regions. aedes mosquitoes are typically daytime biters and have a preference for the morning and late afternoon hours (crepuscular periods). 1 a. aegypti, the primary mosquito vector for dengue, chikungunya, and zika, is found in tropical, subtropical, and some temperate climates and has adapted to cohabit with humans in both urban and rural environments. 2 a. aegypti typically lays eggs in manmade or artificial containers in or around the home and can bite indoors. 2 a. albopictus (the asian tiger mosquito) can live in a broader temperature range and at cooler temperatures than a. aegypti and thus has a wider geographic distribution, extending into temperate regions. a. albopictus feeds on animals as well as humans, prefers natural habitats, usually bites outdoors, and is generally considered a less efficient vector of human disease than a. aegypti. 1 aedes mosquitoes can be found in temperate areas, including southern europe (a. albopictus), northern queensland in australia (a. aegypti), and southeastern regions of the united states (both species) 2,3 ( fig. 7.1 ). denv is a flavivirus that is the most common and arguably most important arbovirus globally. originating in africa, denv is now endemic in more than 100 countries across africa, southeast asia, the americas, the western pacific, and the eastern mediterranean regions. 4, 5 estimates suggest 390 million infections occur worldwide annually, with 70% of cases occurring in asia. 6 denv has four distinct serotypes (denv 1-4), with most endemic countries reporting circulation of all four serotypes. 7 primary infection provides lifelong serotype-specific protection but only short-lived cross-protection to other serotypes. 4, 8 broadly neutralizing antibodies are produced following a second dengue infection, and symptomatic disease is rarely seen with subsequent infections. 9 pretravel advice should be tailored to the individual following a thorough review of his or her itinerary, planned activities, and host characteristics. in addition to vaccinations and malaria chemoprophylaxis, a pretravel consultation should include advice on regionally endemic or emerging non-vaccine-preventable infections that can cause severe illness or chronic morbidity. these include mosquito-borne infections such as dengue, chikungunya, and zika, and regionally endemic severe respiratory infections such as middle east respiratory syndrome (mers) and some strains of avian influenza. zika virus is notable given its capacity for sexual transmission and association with congenital birth defects. preventive advice for other potentially relevant infections associated with specific exposures or activities (e.g., schistosomiasis and leptospirosis from freshwater exposure) should be provided where relevant. understanding the epidemiology and prevention of these infections is crucial to providing a comprehensive pretravel consultation. between nonhuman primates, small mammals, and aedes mosquitoes. 14 however, in outbreaks chikv can spread without the need for animal reservoirs. 14 among populations with no prior immunity, chikv outbreaks can be explosive, and attack rates as high as 70% have been documented. 15 introduction of chikv into asia occurred during or before the 1950s and led to outbreaks in india and southeast asia. 16 reemergence of chikv from africa in 2004 resulted in major outbreaks involving millions of people across the islands of the indian ocean in 2005 (including the comoros islands, la reunion, and mauritius) and india in 2005-2006. 16 furthermore, introduction of chikv to temperate areas in this period resulted in autochthonous transmission in italy and france. 17, 18 the first report of local transmission of chikv in the americas occurred in 2013 in saint martin, with subsequent spread to >40 countries and territories across north, central, and south america. 15 the unprecedented magnitude of chikv outbreaks in recent years is probably attributable to several factors including increased urbanization, global travel, and a series of adaptive mutations in the virus which have resulted in enhanced transmission by a. albopictus. 16 the incubation period of chikungunya is typically 2-4 days (range 1-14 days). 14 most chikungunya infections are symptomatic, with more than 85% of people with serologic evidence of infection reporting a history of symptoms. 16 chikungunya infection is characterized by sudden onset of fever and severe, potentially disabling arthralgia. notably, the name chikungunya is derived from a makonde word describing the bent posture that can be seen with severe arthralgia. 16 the arthralgia/ arthritis is usually symmetric and affects multiple joints, with fingers, wrists, ankles, elbows, toes, and knees the most often affected. 14 additional symptoms include headache, myalgia, conjunctivitis, and rash. the case fatality rate of chikungunya is <1%, but disease can be associated with significant acute and long-term morbidity secondary to debilitating polyarthralgia/arthritis. 15 although self-limiting in most individuals, some of those affected develop chronic joint pain that may last for months to years, with older people (>35-45 years) more predisposed. 14, 18 unlike dengue, in which nonsteroidal antiinflammatory drugs (nsaids) are contraindicated, antiinflammatory drugs are indicated for symptomatic management of chikungunya infections. zikv is a flavivirus that was first isolated in 1947 from a rhesus monkey in the zika forest of uganda, with the first human cases detected in uganda and tanzania in 1952. 19,19a following its discovery, the virus remained in relative obscurity for over 50 years, with only 14 cases reported until 2007, when an explosive outbreak infected approximately three-quarters of the population of yap, federated states of micronesia. 20 subsequent outbreaks occurred across the pacific islands from 2013 to 2016; spread of the virus to brazil in march 2015 preceded subsequent transmission throughout latin america, the caribbean, mexico, and florida and texas in the united states. 21, 22 as of february 2018, 86 countries, territories, or subnational areas have reported evidence of vectorborne zikv transmission. 23 unlike denv and chikv, there is now substantial evidence that direct person-to-person transmission of zikv is possible-both horizontally through sexual transmission, and vertically from the mother to the fetus during pregnancy. 22 transmission through blood transfusion has been reported, 22 as well as a single case report of transmission probably resulting from close contact with bodily fluids from an infected patient. 24 the incubation period is thought to be similar to other mosquito-borne flaviviruses 21 with an estimated range from 3-14 days. 25 male-to-female, male-to-male, and femaleto-male transmission to unprotected sexual contacts of returning following a short incubation period, with symptoms typically beginning 4-7 days (range 3-14 days) after exposure, dengue can present with a wide spectrum of illnesses, from asymptomatic infection to severe and fatal disease. 5 most infections are asymptomatic or subclinical; symptomatic infections occur in approximately one-third of cases. 4 patients who recover after a self-limited febrile illness, typically characterized by fever, headache, retroorbital pain, arthralgia, and myalgia, are classified as having dengue. 5 the small proportion who progress to capillary (plasma) leakage with or without bleeding, circulatory collapse, or severe end organ impairment are designated as having severe dengue. 5 epidemiologic risk factors for severe dengue include young age, secondary infection with a different serotype, and infection with a more virulent strain of virus. 4, 10 severe dengue occurs in approximately 1%-3% of dengue cases, with case fatality rates ranging from <1%-5%; the greatest burden of severe dengue occurs in children and infants in endemic countries. 11 dengue is a common diagnosis in travelers, accounting for >3% of presentations to geosentinel surveillance clinics. most infections are acquired in asia, followed by the americas, with only a small proportion acquired in africa. 12 the incidence of dengue infection in travelers ranges from 10.2-30 infections per 1000 person months, and varies according to travel destination, duration, and season of travel. 11 regionspecific peaks of travel-related dengue infections have been demonstrated for southeast asia (june and september), south central asia (october), and south america (march). 13 viraemic travelers can introduce dengue into new areas, with autochthonous transmission documented in the continental united states, europe, and australia. 11, 12 some travelers with dengue may require hospitalization or even evacuation. 10 studies of dengue in travelers have reported a dengue hemorrhagic fever prevalence of 0.9%-3%, though this is likely an overestimate as patients experiencing more severe symptoms are more likely to seek medical attention. 11 epidemiologic studies in endemic settings have shown that the risk of severe disease is significantly higher during a second denv infection than during a primary infection. 5 however, a lack of consensus exists regarding risk factors for severe disease in travelers. 11 results of one study in travelers suggest that severe dengue may occur at similar rates among cases with primary and secondary infections. 9 given that most dengue infections are asymptomatic, and that severe dengue in travelers is rare, travelers with a history of dengue infection need not avoid known dengue areas but rather should be advised to use the personal protection strategies outlined in box 7.1 to prevent subsequent infection. chikv is a mosquito-borne alphavirus first isolated in tanzania in 1952. 14 in africa, chikv exists in an enzootic sylvatic transmission cycle • wear an insect repellent containing an active ingredient such as deet or picaridin, particularly during daylight hours when the mosquitoes are most active. • wear long-sleeved shirts and long pants to help protect yourself from bites. light-colored clothes are best. • treat clothes and shoes with an insecticide such as permethrin or purchase pretreated clothing. • use mosquito coils, plug-in mosquito repellent devices, or insecticide surface sprays inside your accommodation; or stay in screened or air-conditioned accommodation. 29 testing may also be considered in asymptomatic potentially exposed pregnant women after considering risk of infection, patient preferences, and clinical judgment. 29 nonpregnant individuals and couples traveling to zika-affected areas should also be counseled on measures to prevent sexual transmission and congenital infection. due to the risk of prolonged viral shedding in semen, public health authorities advise men with risk of zika exposure to wait 3 months from the last possible exposure to zika (or after onset of symptoms following symptomatic infection) before attempting procreation. 22 most authorities advise women to wait 8 weeks from the last possible exposure to zika (or after onset of symptoms following symptomatic infection) before attempting to conceive 22 ; one exception is the who, which advises women to wait 6 months before attempting conception. 30 because many pregnancies are unplanned, all sexually active travelers and female partners of travelers should also practice measures to prevent congenital zika infection. readers are encouraged to review the most updated recommendations for prevention of sexual transmission of zikv and congenital zika infection from public health authorities including who and the us centers for disease control and prevention (cdc) ( the 2003 severe acute respiratory syndrome (sars) outbreak highlighted the potential for travelers to introduce novel respiratory infections into their home countries. more recently, concern has focused most on the emergence of middle eastern respiratory syndrome (mers) and certain strains of avian influenza. travelers has been reported with the former felt to be most prominent. sexual transmission from patients with both symptomatic and asymptomatic disease has been described. 22 currently it appears that zika can remain in semen longer than in other body fluids (including cervical mucus, vaginal fluids, urine, and blood). 26 in semen, zikv rna has been detected as long as 188 days after the onset of symptoms, and infectious virus has been cultured up to 69 days after symptom onset. 22 most zikv infections are asymptomatic, with serosurvey studies indicating that only 19% of those infected report clinical illness. 20 in symptomatic cases, illness is generally mild and self-limiting with symptoms including fever, rash, pruritus, arthralgia, myalgia, conjunctivitis, and headache. 21 zikv has been associated with neurologic complications including guillain-barré syndrome (gbs) and adverse fetal outcomes including congenital microcephaly. an association with gbs was first reported in 2013-2014 during the french polynesian outbreak. more than 20 countries have now reported an increased incidence of gbs and/or laboratory confirmation of a zikv infection among gbs cases. 23 in february 2016, following reports from brazil of microcephaly in babies whose mothers had been exposed to zika during pregnancy, the world health organization (who) declared that zika constituted a public health emergency of international concern (pheic). 19 microcephaly is one of several neurologic and musculoskeletal birth defects described in congenital infection; this constellation of findings is now known as congenital zika syndrome. 27 although a live attenuated tetravalent dengue vaccine (cyd-tdv; dengvaxia) has been registered in several countries, and several other dengue vaccine candidates are in clinical development, 8 at this time no dengue vaccine is licensed for travelers. likewise no vaccines are licensed for chikungunya or zika; therefore prevention of these viruses largely relies on personal protection strategies that limit contact between humans and aedes mosquitoes (see box 7.1 and chapter 6; insect protection) as well as avoidance of travel during peak transmission or outbreak periods. travelers returning to nonendemic areas with aedes mosquitos (see fig. 7 .1) should also be advised to avoid mosquito bites on their return to prevent local transmission. symptomatic travellers should seek medical evaluation immediately. infection with chikv is thought to result in lifelong protective immunity. 14 duration of zikv immunity following infection is currently unknown. in contrast, due to the multiple serotypes, individuals can be infected with dengue up to four times, and travelers with a history of infection should be educated about the potential risks of subsequent infections. due to the high prevalence of asymptomatic infections and the risk of sexual and vertical transmission, zika-specific preventative advice is important for those traveling to zika affected areas. pregnant women who do not reside in zika transmission risk areas should be advised not to travel to areas with risk; if travel cannot be avoided, advice to prevent mosquito bites and sexual transmission should be given. 28 measures to prevent sexual transmission include abstaining from sexual activity or use of condoms during sexual activity (including vaginal, anal, and oral sex, and sharing of sex toys) during the entire pregnancy. 28 pregnant women possibly exposed to zikv due to travel or sexual contact should discuss the potential exposure with their although no vaccines are available at this time for prevention of mers and avian influenza (h5n1 and h7n9) in travelers, providers should routinely review the most recent epidemiology of severe respiratory infections reported by authorities such as the who and cdc (see table 7 .1) and promote general hygiene and other preventative measures to travelers to these areas (box 7.2). while seasonal influenza vaccination does not protect against avian influenza or mers, vaccine-related prevention of seasonal influenza may reduce the chance of coinfections and overall risk of respiratory infections. travelers should also be advised to inform their health care providers of their travel history whenever seeking medical care for respiratory (and other illnesses) acquired during or soon after travel (see chapter 59). travel medicine providers should also be familiar with risk areas and specific preventive advice for other non-vaccine-preventable infectious diseases with regional distributions. some of the more important of these infections are presented in table 7 .2, along with specific preventive advice, such as insect bite avoidance (e.g., for prevention of african trypanosomiasis) or freshwater contact avoidance (e.g., for prevention of schistosomiasis or leptospirosis). mers is a respiratory infection caused by mers coronavirus (mers-cov). first described in 2012, 31 mers is an endemic infection in the arabian peninsula with epidemic potential in health care and travel settings. following an incubation period of 2-14 days, initial symptoms of mers are similar to many common viral respiratory infections and include fever, rhinorrhea, sore throat, and muscle aches. rapid progression to acute respiratory distress syndrome may follow, but mild and asymptomatic infections have also been described. 32, 33 nausea, vomiting, or diarrhea, and acute kidney injury can also occur. 32 risk factors for severe mers include age >50 years and comorbid conditions such as hypertension, diabetes, heart disease, end stage renal disease, chronic lung disease, cancer, or those receiving immunosuppressive therapy. 32 among confirmed cases reported to who up to july 2017, 35% have been fatal. 33a seroepidemiologic studies indicate that mers-cov circulates in dromedary camels in the middle east and africa, and direct contact with camels has been described in 33% of primary cases without known exposure to mers cases or health care settings. 34 the route of transmission from dromedaries to humans is unclear, but contact with infectious bodily secretions and fluids are suspect, and consumption of raw dromedary products has also raised some concern. 32 person-to-person transmission is primarily described in health care settings, although transmission among household close contacts has been described. 35 all cases of mers reported outside of the arabian peninsula have occurred in returned travelers, or as a result of secondary transmission from a patient with recent travel to the arabian peninsula, as was the case for the 2015 health care-associated outbreak in south korea that resulted in 186 cases and 36 deaths. 32, 36, 37 in this outbreak, delayed mers diagnoses and inadequate infection control precautions led to multiple generations of infections affecting other patients, visitors, and health care workers. 37, 38 the risk of traveler-initiated health care-associated outbreaks with mers mirrors the global experience of sars. like mers, the viral agent of sars is a coronavirus that emerged in southern china in 2002 and subsequently caused over 8000 infections and 774 deaths in more than 20 countries. 38, 39 the reservoir of sars coronavirus (sars-cov) is unknown, but some cases appear to have resulted from contact with animals used for human consumption such as civet cats. 40 although no cases of sars have been reported since 2004, the potential for reemergence is possible. 39 while seasonal influenza is a common infection among travelers, other influenza types including certain strains of avian influenza can also pose a risk to travelers. although avian influenza typically affects birds, human cases and outbreaks occur sporadically. avian influenza strains associated with severe respiratory infections with high mortality rates in humans include the highly pathogenic avian influenza a h5n1, 41 and more recently, novel avian influenza a h7n9. 42 although the majority of human infections caused by avian influenza are linked to direct contact with infected birds (primarily poultry), unsustained person-to-person transmission has been reported for h5n1 and h7n9. 41, 43 furthermore cocirculation of different influenza a viruses in humans and animals raises the concern of reassortment leading to new strains that spread more readily from person to person. 42 h5n1, first described in southern china in 1996, has since been documented in over 60 countries in asia and africa. 41 long-term sequelae of chikungunya virus disease: a systematic review zika: the origin and spread of a mosquito-borne virus zika virus: history of a newly emerging arbovirus zika virus outbreak on yap island, federated states of micronesia zika virus epidemiology, prevention, and potential future treatments of sexually transmitted zika virus infection zika virus classification tables fatal zika virus infection with secondary nonsexual transmission estimated incubation period for zika virus disease persistence of zika virus in body fluids-preliminary report congenital zika virus infection beyond neonatal microcephaly update: interim guidance for health care providers caring for pregnant women with possible zika virus exposure-united states (including us territories) world health organization. prevention of sexual transmission of zika virus-interim guidance update isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome asymptomatic mers-cov infection in humans possibly linked to infected dromedaries references the efficacy of repellents against aedes, anopheles, culex and ixodes spp.-a literature review the dengue vector aedes aegypti: what comes next the global distribution of the arbovirus vectors aedes aegypti and ae. albopictus the global distribution and burden of dengue global spread of dengue virus types: mapping the 70 year history dengue vaccine: who position paper a travel medicine view of dengue and dengue hemorrhagic fever geographic expansion of dengue: the impact of international travel dengue fever and international travel geosentinel surveillance network. geosentinel surveillance of illness in returned travelers geosentinel surveillance network. seasonality, annual trends, and characteristics of dengue among ill returned travelers chikungunya: a re-emerging virus epidemiology of chikungunya in the americas chikungunya virus infections infection with chikungunya virus in italy: an outbreak in a temperate region arrival of chikungunya virus in the new world: prospects for spread and impact on public health increase in human infections with avian influenza a(h7n9) virus during the fifth epidemic-china avian influenza a (h5n1) infection with respiratory failure and meningoencephalitis in a canadian traveller avian influenza a(h7n9) virus infection in 2 travelers returning from china to canada human african trypanosomiasis leishmaniasis in travelers: a literature review histoplasmosis infections worldwide: thinking outside of the ohio river valley leptospirosis: an emerging disease in travelers rickettsial infections in the tropics and in the traveler myiasis in travelers schistosomiasis in travelers and migrants multicenter geosentinel analysis of rickettsial diseases in international travelers mers-cov global summary and assessment of risk risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia transmission of mers-coronavirus in household contacts the clinical and virological features of the first imported case causing mers-cov outbreak in south korea preliminary epidemiological assessment of mers-cov outbreak in south korea ii, 21163 transmission characteristics of mers and sars in the healthcare setting: a comparative study who guidelines for the global surveillance of severe acute respiratory syndrome (sars) lessons from the severe acute respiratory syndrome outbreak in hong kong global epidemiology of avian influenza a h5n1 virus infection in humans, 1997-2015: a systematic review of individual case data epidemiology of avian influenza a h7n9 virus in human beings across five epidemics in mainland china key: cord-325261-bdumhy5b authors: clemente, valentino; d’arcy, padraig; bazzaro, martina title: deubiquitinating enzymes in coronaviruses and possible therapeutic opportunities for covid-19 date: 2020-05-15 journal: int j mol sci doi: 10.3390/ijms21103492 sha: doc_id: 325261 cord_uid: bdumhy5b following the outbreak of novel severe acute respiratory syndrome (sars)-coronavirus (cov)2, the majority of nations are struggling with countermeasures to fight infection, prevent spread and improve patient survival. considering that the pandemic is a recent event, no large clinical trials have been possible and since coronavirus specific drug are not yet available, there is no strong consensus on how to treat the coronavirus disease 2019 (covid-19) associated viral pneumonia. coronaviruses code for an important multifunctional enzyme named papain-like protease (plp), that has many roles in pathogenesis. first, plp is one of the two viral cysteine proteases, along with 3-chymotripsin-like protease, that is responsible for the production of the replicase proteins required for viral replication. second, its intrinsic deubiquitinating and deisgylating activities serve to antagonize the host’s immune response that would otherwise hinder infection. both deubiquitinating and deisgylating functions involve the removal of the small regulatory polypeptides, ubiquitin and isg15, respectively, from target proteins. ubiquitin modifications can regulate the innate immune response by affecting regulatory proteins, either by altering their stability via the ubiquitin proteasome pathway or by directly regulating their activity. isg15 is a ubiquitin-like modifier with pleiotropic effects, typically expressed during the host cell immune response. plp inhibitors have been evaluated during past coronavirus epidemics, and have showed promising results as an antiviral therapy in vitro. in this review, we recapitulate the roles of plps in coronavirus infections, report a list of plp inhibitors and suggest possible therapeutic strategies for covid-19 treatment, using both clinical and preclinical drugs. a pneumonia of unknown cause was detected in wuhan, people's republic of china and first reported to the world health organization (who) country office on 31 december 2019. the pathogen was subsequently discovered to be a novel coronavirus (cov) and named severe acute respiratory syndrome (sars)-cov2, based on sequence similarity with members of the β-coronavirus family, such as sars-cov and mers-cov, two highly pathogenic coronaviruses responsible for previous epidemics of severe acute respiratory syndrome and middle-east respiratory syndrome, respectively [1] [2] [3] [4] . genomes of coronaviruses are typically organized with a certain degree of similarity, suggesting analogous pathogenic mechanisms. other human coronaviruses include the nl63, 229e, oc43 and hku1, which are usually responsible for common colds and/or other respiratory infections in children and older or immunocompromised individuals [5, 6] . on 11 february 2020, the who named the new coronavirus disease: covid-19, alias corona-virus disease 2019. two months later, following a large spread outside china, with epidemic foci in south korea and japan and a massive outbreak in italy, the who declared covid-19 a pandemic [7] . thus far, this is the first highly pathogenic coronavirus epidemic to reach these proportions. mortality data and severity assessments made available by the chinese government and the who indicate that about 80% of affected individuals have symptoms similar to those of mild seasonal influenza. the remaining 20%, however, develop viral interstitial pneumonia, requiring hospitalization. in approximately 5% of these patients, the pneumonia is critical and requires intensive care. overall mortality ranges between 0.5% and 5%, with a clear positive correlation with age and other comorbidities [8] . based on these numbers, the majority of the world's health care systems are ill-equipped to deal with the surge of patients requiring hospitalization without taking additional measures to prevent system collapse. there is an immediate concern that the mortality rate could increase further due to a lack of available intensive care unit beds required to treat severe cases. furthermore, since no large clinical trial has been possible considering the timescale of the pandemic, there is no consensus on how to treat severe cases of covid-19 viral pneumonia. thus far, all evidence of treatment options are derived from a few case experiences during the sars and mers epidemics, from administrations made in special cases based on analogy with other diseases, intuitions and experimental therapy of some pathological features/symptoms [4, [9] [10] [11] [12] . as we are in the rising part of an epidemic time/contagion graph, with a significant proportion of the world's population under restrictions or quarantine, the identification of therapeutic options for treating severe cases of covid-19 is of paramount importance. the ubiquitin proteasome system (ups) is a key regulator of protein homeostasis. the pathway to proteasomal degradation consists of a group of enzymes called ubiquitin (ub) ligases, that attach ubiquitin moieties to target proteins. the addition of ubiquitin serves as a highly specific destruction tag, where ubiquitinated proteins are trafficked to the proteasome for degradation. a reverse pathway involving ubiquitin removal also exists and is mediated by the action of deubiquitinating enzymes (dubs) that catalyze the removal of ub from tagged proteins [13] . while proteasome targeting tends to involve poly-ub chains containing ub moieties linked via isopeptide bonds at lysine residue 48 (k48) of ub, other ub linkages (particularly k63) and ub-like modifications (i.e., sumoylation, neddylation and isgylation) are involved in signal transduction and the regulation of immune responses. it is of no surprise that viruses, including coronaviruses, often use modulation of ubiquitin and ubiquitin-like modifiers to evade the host cell immune response [14, 15] . each coronaviridae family member codes for dubs, named viral papain-like proteases (plps), which remove ubiquitin from target proteins and alter cellular pathways important for infection. some members encode two, but, sars, mers covs and the novel sars-cov2 [3] only encode one, named sars-cov plp, mers-cov plp and sars-cov2 plp respectively. for many coronaviruses, viral plps have been studied extensively and shown to play a crucial role during viral infection of the host cell. these enzymes are multifunctional and in addition to their dub activity, also containing intrinsic cysteine protease and deisgylating activity that are required for viral replication and the evasion of host responses [5, 6] . the deisgylating activity of plps is similar to dub activity and involves deconjugating interferon (ifn)-stimulated gene (isg)-15 moieties from tagged proteins. isg15 is a small ub-like peptide that can be covalently attached to target proteins in a mechanism similar to ub, resulting in a large number of regulatory effects. isg15 is largely stimulated during antiviral responses, and although its broad functions are not fully elucidated, it acts as an effector and a regulator of the host cells innate immune response during viral infections [16, 17] . since viral plps are used by coronaviruses to both replicate and to antagonize the innate immune response, they are considered important therapeutic targets for coronavirus infections and thus may be of interest for future covid-19 treatment strategies. in this review, we report an up-to-date description of coronaviral plps functions and their inhibitors, and provide possible therapeutic strategies for covid-19 treatment, using both clinically approved and preclinical drugs. the following keywords: "dubs in coronavirus" "dubs in sars-cov" "sars-cov plp role" "plp activity" "plp inhibitors" "plp in sars-cov2" and "sars therapy", were used in a literature search of the pubmed database. the cut off dates were 2005 for the pathogenesis dissertation and 2013 for novel drugs. viral plps are highly conserved among the nidovirales order members [5] and the structure of some relevant coronaviruses plps has been elucidated using crystallography and the enzymatic assays [18] [19] [20] [21] [22] [23] [24] [25] . the multifunctional activities of plps, namely as cysteine proteases, dubs and deisgylating enzymes, play two important roles in coronavirus pathogenesis: the first involves the production of non-structural proteins (nsp) required for the replication process and the second consists of blocking the innate immune system of the infected host cell. plps play their first role during the early replicative phase of coronavirus infection. after the virus enters the host cell, a replication/transcription complex (rtc) is required to orchestrate the replication of the viral units in the cytoplasm. here, the plps' cysteine protease activity is essential for the cleavage of the n-terminal segment of the rtc polyprotein (pp). specifically, the rtc is coded by two open reading frames (1a and 1b), that, with a ribosomal frame shift mechanism, lead to the transcription of two polyproteins: pp1a, that features the nsps from 1 to 11, and the larger pp1ab, that, in addition, contains nsps 12 to 16. the pps need to be processed correctly into the nsps, which are the active elements of the rtc. plps are encoded within nsp3 and free the n-terminal nsps of the rtc, while the remaining units are processed by the three chymotrypsin-like cysteine protease (3clp alias main protease), which may also serve as an attractive therapeutic target [5, 6, 26] . sars and mers covs are known to adhere to this model and a visual description of the sars-cov plp role as a cysteine protease is given in figure 1 . the host cell innate immune response to viruses begins once the viral nucleic acids are sensed by pattern recognition receptors such as toll-like receptors (tlr) and retinoid inducible gene i (rig-i). this event triggers signal cascades, including kinases such as tank-binding kinase-1 (tbk1) and inhibitor-kb kinases (ikks), leading to activation of transcription factors such as interferonregulatory factor-3 (irf3) and the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb). these proteins translocate into the nucleus and serve as transcription factors by binding to promoter elements in target genes, leading to the expression of several cytokines, including type i ifns (as α and β ifns), which in turn will trigger a huge number of intra and extra cellular processes leading to activation of the innate immune response [27] . coronaviruses infections, such as sars, generally include a dysregulation of the immune system in their pathogenic features at systemic level [28, 29] . indeed, early research has shown that active sars-cov replication does not stimulate infs production in cell culture [30] . since it is known that cellular antiviral pathways include (de)ubiquitination within their regulatory mechanisms [14, 15] , plps are believed to contribute to infection pathogenesis by using their intrinsic dub and deisgylating activities to antagonize the activation of the host cell innate immune response [5] . specifically, plps use their dub activity to interfere with the proteins that mediate the intracellular sensing and signaling of viral infection, therefore leading to a dysregulation of the immune pathways, such as the irf3 and nf-kb pathways, that in turn, results in a decrease in the antiviral response. the dub activity of plps is usually broad spectrum and not selective for specific ubiquitin linkage types, such as k48 or k63 [31] [32] [33] . since dub activity was first hypothesized as an important component of the host cell innate immune response to viruses begins once the viral nucleic acids are sensed by pattern recognition receptors such as toll-like receptors (tlr) and retinoid inducible gene i (rig-i). this event triggers signal cascades, including kinases such as tank-binding kinase-1 (tbk1) and inhibitor-kb kinases (ikks), leading to activation of transcription factors such as interferon-regulatory factor-3 (irf3) and the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb). these proteins translocate into the nucleus and serve as transcription factors by binding to promoter elements in target genes, leading to the expression of several cytokines, including type i ifns (as α and β ifns), which in turn will trigger a huge number of intra and extra cellular processes leading to activation of the innate immune response [27] . coronaviruses infections, such as sars, generally include a dysregulation of the immune system in their pathogenic features at systemic level [28, 29] . indeed, early research has shown that active sars-cov replication does not stimulate infs production in cell culture [30] . since it is known that cellular antiviral pathways include (de)ubiquitination within their regulatory mechanisms [14, 15] , plps are believed to contribute to infection pathogenesis by using their intrinsic dub and deisgylating activities to antagonize the activation of the host cell innate immune response [5] . specifically, plps use their dub activity to interfere with the proteins that mediate the intracellular sensing and signaling of viral infection, therefore leading to a dysregulation of the immune pathways, such as the irf3 and nf-kb pathways, that in turn, results in a decrease in the antiviral response. the dub activity of plps is usually broad spectrum and not selective for specific ubiquitin linkage types, such as k48 or k63 [31] [32] [33] . since dub activity was first hypothesized as an important component of sars-cov plp in 2005 [34] , a number of studies investigating the dub activity of coronaviruses plps and their effects on host cell innate immunity have been performed [35] [36] [37] [38] . a map of the pathways that will be discussed in the next paragraph is given in figure 2 . [34] , a number of studies investigating the dub activity of coronaviruses plps and their effects on host cell innate immunity have been performed [35] [36] [37] [38] . a map of the pathways that will be discussed in the next paragraph is given in figure 2 . once the virus is detected by pathogen recognition receptors as rig-i, the signal is transduced via mavs to the activating kinases of the transcription factors: irf3 and nf-kb. tbk1 and ikki phosphorylate irf3 and thus trigger its dimerization and nuclear translocation. ikkα-γ free nf-kb, that moves to the nucleus, by phosphorylating its inhibitor, ikbα. activated sting and traf3 form complexes with irf3 upstream regulators and thus increase the activation state. finally, irf3 and nf-kb promote the activation of the type i inf antiviral response. the plp dub activity antagonizes these pathways at multiple steps, resulting in a global antagonism of the signaling and lower activation of inf-β. for a detailed description of the interplays between the plp and the proteins, the reader should refer to the text. while the mers-cov plp has been reported to have analogous effects [31, 39] , the sars-cov plp dub activity is the best characterized and has been demonstrated to antagonize the irf3 pathway at many steps. this plp was first reported to inhibit the rig-i and tlr3 induction of the ifn-β [32, 40] , to block the phosphorylation and dimerization of irf3 by interacting with its upstream regulators [40, 41] and to directly deubiquitinate irf3and thus disrupt its ifn-β promoter activity [42] . however, recent studies, highlight a k63 ub dub dependent broad interaction with various, if not all, components of the irf3 pathway, such as sting, traf3/6, mavs and tbk1 [43] [44] [45] . another important pathway influenced by the sars-cov plp is nf-kb. here, the dub activity is known to be exerted upon ikbα, an inhibitor of nf-kb [41] , and on the tnfα activation of nf-kb [21, 45] , also resulting in inf-β antagonism. considering that this plp has more than one site to bind ub [6] and a study showing that inhibition of plp protease activity results in reduced nf-kb (but not irf3) antagonism [32] , it could suggest that irf3 antagonism depends on dub activity towards k63 linked ub chains and nf-kb antagonism instead on dub activity against k48 linked ub chains [21, 45] ; a type of linkage that in vitro seems to be preferred by this plp [18] . in summary, we suggest that plps antagonism of the antiviral response is generalized upon the pathways, rather than focused on their single elements, and counteracts various ub modifications. once the virus is detected by pathogen recognition receptors as rig-i, the signal is transduced via mavs to the activating kinases of the transcription factors: irf3 and nf-kb. tbk1 and ikki phosphorylate irf3 and thus trigger its dimerization and nuclear translocation. ikkα-γ free nf-kb, that moves to the nucleus, by phosphorylating its inhibitor, ikbα. activated sting and traf3 form complexes with irf3 upstream regulators and thus increase the activation state. finally, irf3 and nf-kb promote the activation of the type i inf antiviral response. the plp dub activity antagonizes these pathways at multiple steps, resulting in a global antagonism of the signaling and lower activation of inf-β. for a detailed description of the interplays between the plp and the proteins, the reader should refer to the text. while the mers-cov plp has been reported to have analogous effects [31, 39] , the sars-cov plp dub activity is the best characterized and has been demonstrated to antagonize the irf3 pathway at many steps. this plp was first reported to inhibit the rig-i and tlr3 induction of the ifn-β [32, 40] , to block the phosphorylation and dimerization of irf3 by interacting with its upstream regulators [40, 41] and to directly deubiquitinate irf3and thus disrupt its ifn-β promoter activity [42] . however, recent studies, highlight a k63 ub dub dependent broad interaction with various, if not all, components of the irf3 pathway, such as sting, traf3/6, mavs and tbk1 [43] [44] [45] . another important pathway influenced by the sars-cov plp is nf-kb. here, the dub activity is known to be exerted upon ikbα, an inhibitor of nf-kb [41] , and on the tnfα activation of nf-kb [21, 45] , also resulting in inf-β antagonism. considering that this plp has more than one site to bind ub [6] and a study showing that inhibition of plp protease activity results in reduced nf-kb (but not irf3) antagonism [32] , it could suggest that irf3 antagonism depends on dub activity towards k63 linked ub chains and nf-kb antagonism instead on dub activity against k48 linked ub chains [21, 45] ; a type of linkage that in vitro seems to be preferred by this plp [18] . in summary, we suggest that plps antagonism of the antiviral response is generalized upon the pathways, rather than focused on their single elements, and counteracts various ub modifications. although additional studies may be required to fully elucidate the exact interactions through which each coronaviral plp promotes viral infections, there are supporting evidences that the dub activity of plps results in a reduction of the ifn mediated anti-viral response and in a broad spectrum downregulation of proinflammatory cytokines, i.e., ccl5 or cxcl10 [39] . once translated, type i ifns activate the jak-stat pathway through the ifns receptors via an autocrine and paracrine mechanism. this subsequently leads to the expression of a plethora of isgs, either effectors, regulators or both, that embody the antiviral response [46] . isg15 is a small ub-like peptide that can be covalently attached to proteins by a three enzymes process, similar to ub conjugation. during viral infections, isgylation seems to target a high number of proteins at the translational level, either as an activating or inhibitory signal, while free isg15 can also act as a cytokine [16] . the large number of potential targets in multiple signaling cascades makes its role difficult to delineate, however it is clear that it acts as an effector and a modulator of the antiviral response [17] . isgylation can antagonize the formation of viral molecular complexes required for replication, activate molecules involved in immune pathways and act as a negative feedback for upstream factors. interestingly, isg15 deficiency in mice leads to increased and often deadly viral infections, but in humans seems to involve increased susceptibility to mycobacteria and dysregulated immune responses rather than viral infections [16] . there are well established evidence that coronaviral plps are able to deconjugate isg15 moieties from proteins [19, 21, 24, 25, 39] and that this enzymatic activity is conserved among the species [5] , but, since the detailed mechanisms through which isg15 exerts its antiviral activity are still not completely elucidated, the exact consequences of plps deisgylating activity in coronavirus infections are still poorly understood. however, two mouse models demonstrate in vivo that isg15 has antiviral properties against a murine hepatitis coronavirus [47] and that plps may disrupt them [48] . this does suggest that the deisgylating activity of plps is an important mechanism used by coronaviruses to counteract the host's antiviral response. recent reviews highlight some reciprocal interplays between the irf3 and nf-kb pathways and the isgs [46, 49] , while previous studies have also suggested an activating effect of isg15 on irf3 [50] . we would thus propose that the global effects of plps on innate immune responses could result from a crosstalk and additive action of both their dub and deisgylating activities. in addition to the above-described roles of plps enzymatic activities, other authors have demonstrated further effects on the infection pathogenesis. the sars-cov plp has been shown to complex with sud (sars-unique domain) and stabilize the p53 e3 ligase rchy1, leading to subsequent ubiquitination and the degradation of p53, thus resulting in a decrease of p53 mediated antiviral activity, similar to other human covs plps. [51] . another report demonstrates a downregulation of erk1 by the proteasome inhibitor mg132, as a way to suppress the inf-α stimulation by this plp [52] . last, but not least, this plp was shown to induce an upregulation of the transcription and translation of transforming-growth-factor (tgf)-β1 through erk, which was inhibited by treatment with mg132. noticeably, the treatment with the erk1/2 inhibitor u0126 inhibited the activation of tgf-β1 activated genes, with an 8.4-fold reduction observed for type i collagen [53] . moreover, two recent papers have further investigated these connections. the results of these studies contributed to the understanding of the molecular mechanisms underlying this feature of sars-cov plp, confirmed the effects seen on human samples, and showed pro-fibrotic features induced in lung histology by this plp in a mouse model. taken together, these considerations suggest the connection of plps with collagen expression via tgf-β1 signaling and a pathogenic role of sars-cov plp in inducing lung fibrosis [54, 55] . furthermore, in a previous study, mg132 was also shown to improve the lung histology in a sars murine model [56] and removing the dub activity of plp2 in a murine hepatitis virus also resulted in a better liver histology if compared to controls [57] , adding strength to this hypothesis (see below). although many additional structural and non-structural coronavirus proteins may contribute to suppress the cellular responses to viral infections [58] [59] [60] , the role of plps in the production of the replicase proteins and their location at the center of numerous signaling nodes suggest validity in targeting them as an anti-viral strategy. collaterally, considering these effects of plps and other viral proteins on dysregulating the innate immune response, one may hypothesize that they could be at the origins of a mechanism that underlies the potential development of a cytokine storm [28, 29, 61] . while the activities of plps are usually conserved among the nidovirales order members [5] , to which coronaviruses belong, an enhanced plp antagonism of innate immunity seems to be a peculiar characteristic of zoonotic coronaviruses such as sars-cov [62] and to be conserved among the highly pathogenic β-coronaviruses [39] . since the plp present in novel sars-cov2 shows a high degree of similarity to the better-known sars-cov [2] [3] [4] , we speculate they might have similar roles in pathogenesis. crystallographic analysis might clarify the mechanisms underlying the covid-19 pathogenesis and provide a therapeutic target for the identification of inhibitors through high throughput molecular screening. based on this, we propose that plp inhibitors should be evaluated as a possible therapeutic option for the treatment of covid-19. several in vitro studies support the idea that plp inhibitors may be effective in reducing coronaviruses infection. for instance, a recent work evaluated the effects on sars-cov by substituting its native plp with the one derived from a bat sars-related cov. interestingly, the obtained sars-cov replicated 10.3-fold less than the wild type in human airway epithelial cells, but showed no differences in non ifn competent cells [62] . meanwhile, several past independent studies showed that small molecule plp inhibitors can inhibit sars-cov replication in vero e6 cells without toxic effects on the host [63] [64] [65] [66] . remarkably, in a 2017 work, its authors developed an innovative therapeutic strategy, using ub variants, to inhibit mers-cov plp in vero cells. these protein-based inhibitors were shown to be highly selective for this plp and to disrupt all of its enzymatic functions (pp processing, dub and deisgylating activity), leading to a four orders of magnitude decrease in viral titer [67] . as the authors themselves recognize, the only questionable feature in this therapeutic strategy was the requirement of sophisticated protein engineering techniques. however, such a suggestion should not be rejected, and may indicate that other bio-pharmacological techniques, such as monoclonal antibodies designed against sars-cov2 plp or proteolysis targeting chimeras (protacs), capable of mediating the ubiquitin-proteasome mediated degradation of sars-cov2 plp, may have merit. despite promising results shown in mice with proteasomal inhibition in a sars model [56] , only two main studies on murine models specifically consider plp inhibition in vivo. first, in a 2014 study, ifn-receptor knockout mice that lack isgs, and are not able to survive sindbis virus infections, were infected with chimeric sindbis viruses, engineered to express isg15 and either sars-cov/mers-cov plps or their inactive mutants. mice infected with the mutated plp virus showed a 76% survival, attributable to isg15 restoration, while the wild type plp virus infection resulted in more than 80% mortality [48] . the treatment, with a drug developed in the authors' laboratory and displayed in another report [66] , was specific for plp and induced an increase in isgylation, that led to an increase in survival to the wild type plp virus. unfortunately, there was no significative efficacy in the sars-cov mouse model [48] . an additional more recent work reported similar results. here, a usp18 (a cellular dub with physiological deisgylating activity) knockout mouse model, displaying increased level of isgylation, was infected with a murine hepatitis coronavirus. higher isgylation levels were positively correlated with a delay in coronavirus replication, and, instead, negatively with the plp2 levels in vivo. although, plp2 inhibition was tested in vitro, lower viral titers were observed [47] . the latest, and second main, plp inhibition model in vivo is an engineered murine hepatitis coronavirus. the dub, and not the protease activity, of a murine hepatitis coronavirus plp2 was disrupted through structure guided mutagenesis. the obtained mutant coronavirus was tested in macrophages for its ability to elicit cells' innate immunity and showed an increased activation of ifn. however, inoculation in mice only resulted in a mild attenuation of virulence, despite an improvement in histological features [57] . as an explanation, the authors hypothesized that the dub activity of this plp may not be necessary in vivo, or may only be required in some tissue-specific/environmental contexts. remarkably, the authors reported difficulties in isolating a mutant virus without plp dub activity and claim this was due to the difficulty in separating it from other activities that resulted in no replication if disrupted. this difficulty, per se, may have more meaning than just a technical issue, and suggests that plps may be essential for viral replication. in summary, these three mouse models may confirm in vivo the previously described individual roles of plps enzymatic activities in the pathogenesis of coronaviruses infections and give some promising insights on targeting plps as an antiviral strategy. however, thus far, the evidence of the therapeutic efficacy of targeting plp in vivo remains questionable. the first study gave positive results in a sars-adapted mouse model, but only a single compound was investigated, while the second did not consider the inhibition of plp in vivo and gave insights on its pathogenic role. the third study was specifically aimed to investigate the role of the dub activity of plps, rather than to evaluate the therapeutic efficacy of targeting plps. as far as we are aware, the number of in vivo studies on plps is limited. therefore, in vivo, therapeutic-oriented, studies in more reliable models, such as those developed for sars and mers, are strongly recommended [68] . in vivo studies, thus far, do not support the evidence of antiviral efficacy in targeting plps. however, since we suggest in vivo studies, on the basis of the plps pivotal role in pathogenesis, of the positive results in vitro against coronaviruses replication and of some in vivo features, plp inhibitors might deserve at least a further discussion. as previously mentioned, a number of drugs are already used or under clinical evaluation for the treatment of covid-19. this includes the anti-malaria agent hydroxychloroquine, several antiretrovirals, as well as interferon and tocilizumab [4, [9] [10] [11] [12] . importantly, because the structure of the sars-cov and mers-cov plps has been solved using crystallography, this has allowed for high throughput molecular screenings for the identification of a number of plp inhibitors, that showed activity against viral replication in cells [63] [64] [65] [66] . many active compounds against sars-cov plp have been previously reviewed [69] . in a recent report, molecular modeling and high-throughput screening methods of 30,000 molecules were used to identify inhibitors against the mers-cov plp. the research conducted to compound 6 and zt626, which were found to be competitive inhibitors in silico [70] . importantly, sequence analysis has shown a high degree of similarity between sars-cov plp and sars-cov2 plp [3, 4] . this has prompted computational screening, which has led to a number of predicted sars-cov2 plp inhibitors, both clinical drugs and natural derivatives, with pre-clinical and clinical potential [3] . here we report a series of plp inhibitors evaluated for other coronaviruses, that could be considered for the novel sars-cov2. a brief overview is given in table 1 . [71] . the evaluation of derivatives from angelica keiskei leaves led to the isolation of 9 chalcones and 4 coumarins, with inhibitory activity against both proteases of the same coronavirus. of these, compound 6 (xanthoangelol e) has an ic 50 of 1.2 µm against the plp and 11.4 µm against 3clp (a chymotrypsin-like protease, also proposed as a molecular target against coronaviruses). angelica keiskei derivatives were also identified as inhibitors of plp dub and deisgylating activity [72] . park et al., who proposed many natural derivatives against coronaviruses, recently reported similar results from broussonetia papyrifera root derivatives, including the mers-cov proteases [73] . lastly, the seeds of psoralea corylifolia have been another source of lead compounds, such as isobavachalcone and psoralidin, with activity towards the sars-cov plp [74] . unfortunately, all of these molecules have thus far only been evaluated biochemically in vitro and not at more advanced pre-clinical development stages, but, encouragingly, the extracts of strobilanthes cusia leaves demonstrated plp2 inhibitory properties and positive antiviral results against the human coronavirus nl63 in cells [75] . therefore, we suggest that further research should be performed. in addition, a recent meta-analysis demonstrates a protective effect of flavonoids against upper respiratory tract infections [80] and resveratrol has been found effective against mers-cov infection in vero e6 cells [81] . this could indicate that the use of plants extracts such as cystus052, a polyphenols rich medication already clinically tested in preventing influenza symptoms [82, 83] , could possibly be helpful in coronavirus infections, without having significant side effects. cystatin-c, a small endogenous polypeptide, that physiologically acts as a cysteine protease inhibitor and is used as a biomarker of kidney [84] or brain [85] injury, has antiviral properties [86] [87] [88] and was found to be reduced in vero e6 cells infected with a porcine epidemic diarrhea coronavirus [89] . furthermore, it has been used at slightly supraphysiological levels in a previous study to reduce viral titers of two human coronaviruses in cell cultures, where the authors hypothesized a plp as target, [79] and internalization of cystatin c in cells has been demonstrated [90] . on the basis of these considerations, we would propose further studies on cystatin c antiviral properties against highly pathogenic coronaviruses. i.e., investigate the outcome of covid-19 in patients with high levels of cystatin c. within the category of clinically available drugs, thiopurine analogs, used to treat diseases such as cancer or autoimmune diseases, may have been overlooked. they have previously been shown to display activity as sars-cov plp inhibitors in vitro [76, 91] and with similar activities, either as monotherapy or in association with mycophenolic acid, been shown against the mers-cov plp [77] . due to their well described mechanism of action, the authors proposed in vivo studies, that have not been performed thus far [76, 77] . in addition, as the thiocarbonyl group has been judged to be the active element of these compounds, they may also serve as models to develop safer and more effective molecules [76] . indeed, since such drugs have immunomodulatory actions and are known to depress the immune system, we would suggest a degree of caution in their use in infectious diseases. however, no evidence indicating a higher risk of covid-19 in patients under chronic treatment with immunosuppressive drugs has been reported, although several physicians have expressed concern [92, 93] . we agree with the need of further investigation and propose a retrospective analysis to be performed as soon as possible on covid-19 patients databases, to evaluate both the risk in individuals on immunosuppressive agents and, possibly, the effects of thiopurine analogs (or rather their prodrugs as azathioprine) on the outcome. moreover, other similar therapeutic strategies have been proposed in vitro, i.e., alpha-interferon and cyclosporin [94] . severe and advanced cases of covid-19 pneumonias often involve immune system mediated damage, a so-called cytokine storm, and immunosuppression has been proposed for this context [95] . furthermore, an il6 selective monoclonal antibody is currently being tested on humans for this specific purpose [11, 12] . on the basis of these elements, rather than in upfront therapy, we would consider thiopurine analogs and their associated drugs for the treatment of the cytokine storm in covid-19, that could eventually result in an antiviral and a double immunomodulatory efficacy: inhibiting the viral replication, suppressing hyperinflammation damage and enhancing the endocellular innate immune response against sars-cov2. we again recommend, extreme prudence in considering these drugs, as their side effects might be a danger. in addition, we would like to highlight that a recent report demonstrated that disulfiram (a clinically approved alcohol abuse antagonist, now repurposed as an anticancer drug) can competitively and noncompetitively inhibit sars and mers plps, respectively, and this activity is synergic with 6-thioguanine (a thiopurine) and mycophenolic acid against mers, but not sars, plp [78] . these recent findings in vitro, together with a relatively good risk/benefit ratio, may make disulfiram a clinically evaluable drug against covid-19. however, as a hepatic acetaldehyde dehydrogenase inhibitor, its pharmacological interactions might be considered. the proteasome inhibitor mg132 has been shown to reduce cytokine levels and improve survival and lung histology in a sars murine model [56] . the authors did not propose a strong explanation for this effect of mg132, but the reported anti-inflammatory [96] and anti-viral internalization effects [97] of the proteasome inhibitors may be the mechanisms underlying it. interestingly, mg132 has been used in recent studies to counteract the effects induced by plp [52] [53] [54] [55] . this, taken together with the results in the recentmurine model of dub-mutated plp2 in murine hepatitis [57] , suggests an involvement of plp. however, a mechanism has been proposed by other authors, who exclude proteasome inhibition and link mg132 antiviral activity to m-calpain (a cysteine protease) inhibition, suggesting that viral cysteine proteases, such as plp, are possible targets of this drug. even more promising results were obtained in vitro when the calpain specific inhibitor mdl28170 was used [98] , and these data align with previous in vitro studies on calpain inhibitors in sars [99] . in summary, coronaviruses plps are multifunctional enzymes with cysteine protease, dub and deisgylating activities that contribute significantly to infection pathogenesis. the efficacy of targeting plps as an antiviral therapy is demonstrated, at least in vitro, with further in vivo studies strongly recommended. several compounds, both clinical and preclinical, have documented actions against these proteins, and thus may provide an attractive therapeutic strategy for covid-19. on the basis of these considerations, we propose four main suggestions towards the development of plp inhibitors with the potential to treat covid-19. the first is to consolidate our knowledge on the structure and function of sars-cov2 plp in order to highlight its role in pathogenesis and facilitate drug design. secondly, we claim that the plps effects may be strongly interconnected between all their enzymatic activities. antagonizing one of these functions alone may be less effective than inhibiting the function of the whole protein. thus, we suggest developing and testing molecules with such capability [67] , rather than focusing on each of the plps' functions alone [57] . however, since the plp protease activity seems to be critical for virus replication, inhibiting at least this activity could be effective. third, the number and the specificity of in vivo plp inhibition models must be increased. lastly, given the importance of a new coronavirus-specific drug in the fight against the current pandemic, the hypothesis of testing the proposed drugs in covid-19 should not be underestimated. in detail: • drugs reviewed in the past [69] and novel plant derivatives should progress to the next step in pharmacological development, hopefully being evaluated in vitro against sars-cov2 and, subsequently, in animal models; • recently proposed sars-cov2 plp inhibitors [3] and other drugs proposed in our review should also undergo further evaluations; • effects of dietary integration with plant compounds such as polyphenols on covid-19 prevention should be investigated clinically, in order to demonstrate if this can be a relatively safe and effective health measure; • a clinical study on the effects of high levels of cystatin c (as uremia) on the outcome of patients with covid-19 should be performed. trialing the exogenous administration of this peptide, i.e., via aerosol, may then be considered in humans; • a similar strategy should be used for thiopurine analogs and disulfiram. however, the evaluation of thiopurine analogs may need to be retrospective and their administration reasoned cautiously. disulfiram may be tested more safely, paying attention to its pharmacological interactions and any alcohol consumption. the authors declare no conflict of interest. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deisgylating activities nsp3 of coronaviruses: structures and functions of a large multi-domain protein who declares covid-19 a pandemic novel coronavirus pneumonia emergency response epidemiology team drug treatment options for the 2019-new coronavirus (2019-ncov) a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 advances in the research of cytokine storm mechanism induced by corona virus disease updated approaches against sars-cov-2 proteostasis regulation by the ubiquitin system regulation of cellular innate antiviral signaling by ubiquitin modification pleiotropic roles of the ubiquitin-proteasome system during viral propagation isg15: it's complicated the antiviral activities of isg15 recognition of lys48-linked di-ubiquitin and deubiquitinating activities of the sars coronavirus papain-like protease decoupling deisgylating and deubiquitinating activities of the mers virus papain-like protease crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease x-ray structure and enzymatic activity profile of a core papain-like protease of mers coronavirus with utility for structure-based drug design structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product 15 from different species structurally guided removal of deisgylase biochemical activity from papain-like protease originating from middle east respiratory syndrome coronavirus identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity post-translational regulation of antiviral innate signaling pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases the papain-like protease of avian infectious bronchitis virus has deubiquitinating activity deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? transmissible gastroenteritis virus papain-like protease 1 antagonizes production of interferon-β through its deubiquitinase activity the papain-like protease of porcine epidemic diarrhea virus negatively regulates type i interferon pathway by acting as a viral deubiquitinase plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production plp2 of mouse hepatitis virus a59 (mhv-a59) targets tbk1 to negatively regulate cellular type i interferon signaling pathway mers-cov papain-like protease has deisgylating and deubiquitinating activities regulation of irf-3 dependent innate immunity by the papain-like protease domain of the sars coronavirus severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling the sars coronavirus papain like protease can inhibit irf3 at a post activation step that requires deubiquitination activity coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 transcriptional regulation of antiviral interferon-stimulated genes protein interferon-stimulated gene 15 conjugation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis a chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses interferon-stimulated genes as enhancers of antiviral innate immune signaling positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification p53 down-regulates sars coronavirus replication and is targeted by the sars-unique domain and plpro via e3 ubiquitin ligase rchy1 severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferon-induced responses through downregulation of extracellular signal-regulated kinase 1-mediated signalling pathways correlation between tgf-β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain-like protease sars coronavirus papain-like protease induces egr-1-dependent up-regulation of tgf-β1 via ros/p38 mapk/stat3 pathway sars coronavirus papain-like protease up-regulates the collagen expression through non-samd tgf-β1 signaling proteasome inhibition in vivo promotes survival in a lethal murine model of severe acute respiratory syndrome structure-guided mutagenesis alters deubiquitinating activity and attenuates pathogenesis of a murine coronavirus coronavirus endoribonuclease and deubiquitinating interferon antagonists differentially modulate the host response during replication in macrophages the sars-cov nucleocapsid protein: a protein with multifarious activities severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists human intracellular isg15 prevents interferon-α/β over-amplification and auto-inflammation the papain-like protease determines a virulence trait that varies among members of the sars-coronavirus species a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication structure-based design, synthesis, and biological evaluation of a series of novel and reversible inhibitors for the severe acute respiratory syndrome−coronavirus papain-like protease severe acute respiratory syndrome-coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand x-ray structure and biological evaluation x-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases potent and selective inhibition of pathogenic viruses by engineered ubiquitin variants animal models for sars and mers coronaviruses the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds identification and design of novel small molecule inhibitors against mers-cov papain-like protease via high-throughput screening and molecular modeling papain-like protease (plpro) inhibitory effects of cinnamic amides from tribulus terrestris fruits chalcones isolated from angelica keiskei inhibit cysteine proteases of sars-cov evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes inhibitory effects of recombinant human cystatin c on human coronaviruses effect of flavonoids on upper respiratory tract infections and immune function: a systematic review and meta-analysis effective inhibition of mers-cov infection by resveratrol effect of cystus052 and green tea on subjective symptoms in patients with infection of the upper respiratory tract cistus incanus (cystus052) for treating patients with infection of the upper respiratory tract. a prospective, randomised, placebo-controlled clinical study examining the utility of cystatin c as a confirmatory test of chronic kidney disease across the age range in middle-aged and older community-dwelling adults predictive value of serum creatinine/cystatin c in neurocritically ill patients cystatin c, a human proteinase inhibitor, blocks replication of herpes simplex virus evaluation of cystatin c activities against hiv enhanced anti-rotavirus action of human cystatin c by site-specific glycosylation in yeast analysis of protein expression changes of the vero e6 cells infected with classic pedv strain cv777 by using quantitative proteomic technique internalization of cystatin c in human cell lines thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme are patients with inflammatory bowel disease at increased risk for covid-19 infection? coronaviruses and immunosuppressed patients. the facts during the third epidemic effect of interferon alpha and cyclosporine treatment separately and in combination on middle east respiratory syndrome coronavirus (mers-cov) replication in a human in-vitro and ex-vivo culture model covid-19: consider cytokine storm syndromes and immunosuppression the proteasome inhibitor bortezomib drastically affects inflammation and bone disease in adjuvant-induced arthritis in rats the ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain inhibition of severe acute respiratory syndrome-associated coronavirus (sarscov) by calpain inhibitors and β-d-n4-hydroxycytidine key: cord-341698-k5leys8j authors: park, seung won; jang, hye won; choe, yon ho; lee, kyung soo; ahn, yong chan; chung, myung jin; lee, kyu-sung; lee, kyunghoon; han, taehee title: avoiding student infection during a middle east respiratory syndrome (mers) outbreak: a single medical school experience date: 2016-05-27 journal: korean j med educ doi: 10.3946/kjme.2016.30 sha: doc_id: 341698 cord_uid: k5leys8j purpose: in outbreaks of infectious disease, medical students are easily overlooked in the management of healthcare personnel protection although they serve in clinical clerkships in hospitals. in the early summer of 2015, middle east respiratory syndrome (mers) struck south korea, and students of sungkyunkwan university school of medicine (skkusom) were at risk of contracting the disease. the purpose of this report is to share skkusom’s experience against the mers outbreak and provide suggestions for medical schools to consider in the face of similar challenges. methods: through a process of reflection-on-action, we examined skkusom’s efforts to avoid student infection during the mers outbreak and derived a few practical guidelines that medical schools can adopt to ensure student safety in outbreaks of infectious disease. results: the school leadership conducted ongoing risk assessment and developed contingency plans to balance student safety and continuity in medical education. they rearranged the clerkships to another hospital and offered distant lectures and tutorials. five suggestions are extracted for medical schools to consider in infection outbreaks: instant cessation of clinical clerkships; rational decision making on a school closure; use of information technology; constant communication with hospitals; and open communication with faculty, staff, and students. conclusion: medical schools need to take the initiative and actively seek countermeasures against student infection. it is essential that medical schools keep constant communication with their index hospitals and the involved personnel. in order to assure student learning, medical schools may consider offering distant education with online technology. in outbreaks of infectious disease, healthcare personnel (hcp) are at increased risk of contracting emerging infections in the process of patient care [1, 2] . the rate of hcp-related infection with middle east respiratory syndrome corona virus, ebola virus, and severe acute respiratory syndrome (sars) virus has been reported to be around 1% to 27%, 2.5% to 12%, and 11% to 57% of total cases, respectively [3] . it has been suggested that early and rapid detection of suspected infected patients with contagious diseases along with adequate infection control practice, education, and global and national preparation guidelines could help prevent disease transmission to hcp [3] . due to their participation in clinical clerkships, medical students should be considered to be at-risk hcp during infectious disease outbreaks. when sars struck hong kong in 2003, a number of medical students contracted the disease as a result of exposure to sars patients [4] . despite the fact that protecting medical students from infection exposure is important, the literature on infection control practices for hcp appears to focus mostly on healthcare professionals other than students [3, 5] . during the mers outbreak, mainly students in year 4, 5, and 6 were exposed to risk of contracting mers. besides performing clinical clerkships, students attended lectures held in classrooms located at the main teaching hospital (smc). thus, skkusom students faced a greater risk than those of other medical schools with a school building separate from their teaching hospital. in this study, we examined skkusom's efforts to avoid student infection during the mers outbreak and scrutinized the rationale behind the decisions made. through a process of reflection-on-action [6] , we identified the necessary actions taken to ensure student safety and derived practical guidelines that medical schools can take to protect students in the face of outbreaks of infectious disease. three principles guided the decision-making processes: (1) ensuring student safety, (2) minimizing loss of student learning, and (3) reducing anxiety and concern on the part of staff and students. details of skkusom's actions are discussed below. at the end of may 2015, the first korean patient with mers was reported at the smc, and several nearby patients were subsequently identified as having mers. one doctor also acquired the disease from the index patient. skkusom was immediately notified of these cases. out of concern for the safety of our year 5 and home. because many classes were taught in small group-based learning formats such as problem-based learning, students had closer contact with instructors and peer students than they would in large lecture classes. in addition, given the fact that many medical students spend a great amount of time and share living spaces (e.g., living in a dormitory) with their peers, infection of one student could readily spread to multiple students. with continuous assessment of all these risk factors, the school leadership admitted an imperative need to completely restrict student access to the hospital, which amounted to closure of the school. while agreeing to the closure of the school, the faculty council expressed a concern that students would lose legitimate learning opportunities owing to the cancellation of classes. in order to minimize such loss for the students, the emergency committee declared the school closure period (4 weeks) to be the summer break and to continue the remaining courses after the break. this strategy was particularly practical because the regular summer break at skkusom was scheduled to begin only 3 weeks ahead after the mers outbreak occurred. by moving up the summer break, although interruption in the regular curriculum was inevitable, the courses could still be carried out as originally planned, and most class times were retained. with concern about possible spread of the disease at the smc after the break, the school also prepared remote lectures and classes so that students could continue their education without the risk of exposure to infection. considering the need to balance student safety and continuity in medical education, the school declared an early summer break and closed the school temporarily. one alternative to closure of the school is the use of in an outbreak of infectious disease, the risk of transmitting a disease to hcp is unavoidable [14] . medical students are also at risk of becoming infected mainly due to clinical clerkship rotations at affiliated hospitals, but they are often overlooked in plans to prevent hcp infection [15] . although students are not yet professionals, they should be considered as hcp and middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment-as of 5 world health organization world health organization. ebola situation report [internet]. world health organization risks to healthcare workers with emerging diseases: lessons from mers-cov, ebola, sars, and avian flu a major outbreak of severe acute respiratory syndrome in hong kong bc interdisciplinary respiratory protection study group. protecting health care workers from sars and other respiratory pathogens: organizational and individual factors that affect adherence to infection control guidelines the reflective practitioner: how professionals think in action sars and its effect on medical education in hong kong medical school on bypass during the sars outbreak limited knowledge and practice of chinese medical students regarding health-care associated infections the challenges of "continuing medical education" in a pandemic era medical students and pandemic influenza severe acute respiratory syndrome and the delivery of continuing medical education: case study from toronto using synchronous technology to enrich student learning lack of transmission among close contacts of patient with case of middle east respiratory syndrome imported into the united states student vaccination requirements of u.s. health professional schools: a national survey simulation suggests that rapid activation of social distancing can arrest epidemic development due to a novel strain of influenza funding: none.conflicts of interest: none. key: cord-318585-cp76qr9f authors: matsuyama, ryota; nishiura, hiroshi; kutsuna, satoshi; hayakawa, kayoko; ohmagari, norio title: clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis date: 2016-11-29 journal: bmc public health doi: 10.1186/s12889-016-3881-4 sha: doc_id: 318585 cord_uid: cp76qr9f background: while the risk of severe complications of middle east respiratory syndrome (mers) and its determinants have been explored in previous studies, a systematic analysis of published articles with different designs and populations has yet to be conducted. the present study aimed to systematically review the risk of death associated with mers as well as risk factors for associated complications. methods: pubmed and web of science databases were searched for clinical and epidemiological studies on confirmed cases of mers. eligible articles reported clinical outcomes, especially severe complications or death associated with mers. risks of admission to intensive care unit (icu), mechanical ventilation and death were estimated. subsequently, potential associations between mers-associated death and age, sex, underlying medical conditions and study design were explored. results: a total of 25 eligible articles were identified. the case fatality risk ranged from 14.5 to 100%, with the pooled estimate at 39.1%. the risks of icu admission and mechanical ventilation ranged from 44.4 to 100% and from 25.0 to 100%, with pooled estimates at 78.2 and 73.0%, respectively. these risks showed a substantial heterogeneity among the identified studies, and appeared to be the highest in case studies focusing on icu cases. we identified older age, male sex and underlying medical conditions, including diabetes mellitus, renal disease, respiratory disease, heart disease and hypertension, as clinical predictors of death associated with mers. in icu case studies, the expected odds ratios (or) of death among patients with underlying heart disease or renal disease to patients without such comorbidities were 0.6 (95% confidence interval (ci): 0.1, 4.3) and 0.6 (95% ci: 0.0, 2.1), respectively, while the ors were 3.8 (95% ci: 3.4, 4.2) and 2.4 (95% ci: 2.0, 2.9), respectively, in studies with other types of designs. conclusions: the heterogeneity for the risk of death and severe manifestations was substantially high among the studies, and varying study designs was one of the underlying reasons for this heterogeneity. a statistical estimation of the risk of mers death and identification of risk factors must be conducted, particularly considering the study design and potential biases associated with case detection and diagnosis. electronic supplementary material: the online version of this article (doi:10.1186/s12889-016-3881-4) contains supplementary material, which is available to authorized users. cases of middle east respiratory syndrome (mers), caused by mers-associated coronavirus (mers-cov), have continuously been reported since june 2012. as of 29 june 2016, the total number of laboratory-confirmed cases notified to the world health organization (who) reached 1,768 cases, including 630 deaths [1] . particularly large outbreaks of mers-cov infection have been reported in the kingdom of saudi arabia (ksa) and the republic of korea (rok), while smaller outbreaks and importation events have been reported in other 25 countries [1] . of these, 10 countries are located in the middle east, 7 countries in europe, 3 countries in africa, 3 countries in southeast and east asia, and 1 in north america (the united states of america) [2] [3] [4] . because of the regular reporting of mers cases in the middle east, countries across the world are now facing a continuous threat of mers outbreak. to understand the clinical burden of mers, it is necessary to quantify the risk of developing severe clinical manifestations. the case fatality risk (cfr) is a measure of the risk of death among those who satisfy the case condition [5] , while risks of admission to an intensive care unit (icu) and that of requiring mechanical ventilation are also useful to measure the extent of developing severe mers complications. however, it is not only necessary to estimate such risks, but it is also critically important to identify epidemiological determinants of those risks to then predict the risk of severe complications for each patient before the onset of disease exacerbation [6] . in previous studies, the risk of death among secondary cases was estimated based on statistical modelling and was found to range from 20 to 22%, approximately [7] [8] [9] [10] [11] . meanwhile, among the primary cases, the risk of death was estimated to be greater at approximately 40%, perhaps because of biases associated with case detection and diagnosis [6] [7] [8] . as for epidemiological determinants of mers death, elderly patients with underlying comorbidities have been identified as the most susceptible population with a high risk of death [6, 9, 11] . despite our further understanding of the risk of developing severe mers, the abovementioned estimates are mostly based on a subset of mers cases; for instance, some of the risk estimates are a result of the analysis of cases diagnosed in 2015 in the rok or ksa alone. published articles with different study designs and populations have yielded different estimates and effect sizes associated with mers death. because of this variability, it is valuable to comprehensively and systematically analyze published mers studies that have recorded the clinical prognoses of cases. a systematic review is a highly informative review method that combines published results from different studies, thereby merging and contrasting results across multiple studies and answering study questions using the pooled estimates [12] . thus, we aimed to perform a systematic review to assess risks of death and other severe complications and determine the risk factors for mers-associated death and contrast these results by study population and study design. the present study was a systematic review conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (prisma) statement [13] . pico statement: our study question is focused on laboratory confirmed cases of mers regardless of their treatment status, and thus, involves only retrospective observational studies, measuring their risks of admission to intensive care unit (icu) and death and comparing those risks by age, gender and underlying comorbidities. our systematic review protocol is summarized as additional file 1. published studies that referred to the clinical prognosis of mers cases were retrieved from medline (pubmed) and web of science electronic databases on 16 may 2016. the following search terms were used in "all fields" to identify relevant published articles: 1. "mers" or "middle east respiratory syndrome" or "novel coronavirus" or "novel coronavirus 2012" 2. "sever*"or "fatal*"or "death" or "mortalit*" 3. "hospitalization" or "intensive care" or "icu" 4. 1 and 2 and 3 we limited the search to articles published between april 2012 (i.e., after the first mers case was reported) and june 2016. additional studies reporting associated outcomes that were not identified by the abovementioned search strategy were manually retrieved by tracking the references of included articles (i.e., ancestry and discordancy approach). we restricted ourselves to publications written in english. all titles identified by the abovementioned search strategy were independently screened by two authors (rm and hn). abstracts of potentially relevant articles were subsequently reviewed for eligibility, and if a description of severe or lethal mers was available, articles were selected for closer examination of the full text. to be eligible for inclusion, published studies were required to meet the following characteristics: (i) studies focused on patients infected with mers-cov and (ii) explicitly documenting clinical outcomes (i.e., prognosis) and characteristics of both surviving and deceased patients. studies that allowed us to stratify the risk of severe or fatal mers by demographic or medical condition were preferred, but this was not an essential inclusion criterion. to calculate the risk of severe mers or mers death, we excluded case reports that documented only one or two cases (i.e., case reports with a sample size n ≥ 3 were eligible). included studies were further classified into five groups based on the study design and population studied: (i) case reports comprising published studies that described the clinical course of individual patients including mild cases; (ii) studies including only icu cases (hereafter referred to as icu studies): case reports or retrospective studies that reported outcomes of patients admitted to the icu only; (iii) hospital studies: retrospective or descriptive studies that aimed to document the outbreak in a hospital or healthcare-associated facility; (iv) retrospective studies: published studies that retrospectively analyzed the series of mers cases that were registered in the patient database or tracked medical records; and (v) surveillance studies: published studies that extracted data from a database of cases, systematically gathering epidemiological data, as coordinated by a country or who. the primary data extracted were the proportions of deceased mers patients, patients admitted to the icu and patients undergoing mechanical ventilation. all of these outcomes were dealt with as dichotomous variables, and thus, we calculated the 95% confidence interval (ci) for each included study using the binomial distribution. whenever possible, we stratified the risk of death by age, sex, underlying medical condition and study design. for the analysis of the effect of each covariate on the outcome, the odds ratio (or) for death among those with underlying conditions was calculated and compared with those without comorbidities. stratified analysis could not have been made for the proportions of icu admission and mechanical ventilation because the dataset of such covariates was not commonly available for these two outcomes. we employed a fixed effects inverse variance weighted model. weighted means (i.e. pooled estimate) of the abovementioned proportions and the or for death by each covariate were calculated using the inverse of variance estimates from each study. the heterogeneity among identified studies was statistically assessed by the i 2 statistic. to explore the possible sources of heterogeneity, we stratified pooled estimates by study design. a forest plot was used to illustrate the distribution of the outcome and effect size obtained from each published study. the flow diagram of the search and study selection process is shown in fig. 1 . among a total of 599 potentially relevant articles, 575 and 13 articles were excluded by screening of the titles and abstracts, respectively. one article was excluded by full-text screening. following the same process for 23 additional manually identified articles, a total of 25 articles were selected as eligible articles [8, and all were subject to meta-analysis. of these, four studies were classified as case reports, four as reports of icu cases, four as hospital outbreak studies, eight as retrospective studies and five as surveillance study. the majority of included articles were reported either from the ksa or the rok, except for one study conducted in jordan [22] and the who the estimated cfr was reported in 25 articles, ranging from 14.5 to 100% (fig. 2) . the pooled cfr was 39.1% (95% ci: 37.2, 41.1), but the i2 was as large as 92.4%. the sample size of case reports ranged from 3 to 12, while studies with other designs tended to have larger samples, with 10 or more cases, except for one icu study, one retrospective study and one hospital outbreak study. the proportions of icu admission and mechanical ventilation among all cases were available in 12 and 16 articles, respectively. the proportion of icu admission ranged from 44.4 to 100% with the pooled estimate at 78.2% (95% ci: 73.5, 82.9) and an i 2 value of 78.2%. the proportion of mechanical ventilation ranged from 25.0 to 100% with the pooled estimate at 73.0% (95% ci: 68.5, 77.5) and an i 2 value of 68.0%. [19] age and sex distributions are shown in relation to the risk of death by mers in fig. 3 . in the majority of the studies (except for a study from jordan), survivors were younger than those who died of mers. although not generally, infected men tended to die more often than women, and the pooled or of death among men compared with women was 1.4 (95% ci: 1.1, 1.6 ). the i 2 value of the sex difference for the risk of death was 48.6%. the risks of death, icu admission and mechanical ventilation were stratified by study design and are shown in fig. 4 , in which the pooled estimate for each study design was compared. the risk of death in the hospital outbreak and surveillance studies was significantly smaller than in icu case and retrospective studies. risks of icu admission and mechanical ventilation were the highest among icu case studies, followed by case report and retrospective studies. hospital outbreak studies yielded the smallest pooled risks of icu admission and mechanical ventilation. when comparing surveillancebased data between ksa and rok (fig. 2) , the risk of death in rok (i.e., 14.5-22.0% [8, 33, 34] ) tended to be lower than that in ksa (i.e., 46.0% by alsahafi and cheng [35] ), perhaps reflecting the presence of the contact tracing effort in the rok. figure 5 shows the possible association between five selected underlying medical conditions and the risk of death by mers. pooled estimates of the or were greater than the value of 1 for all five comorbidities, including diabetes mellitus (n = 8 studies), renal disease (6 studies), respiratory disease (5 studies), heart disease (5 studies) and hypertension (5 studies). among a total of five predictors, heart disease yielded the greatest or value at 3.5 (95% ci: 3.1, 4.8) followed by respiratory disease with an or of 3.1 (95% ci: 2.6, 4.2). figure 6 shows the potential association between the risk of death by mers and potential predictors, including sex, heart disease and renal disease. men from icu studies tended to yield a greater or for death compared with other study designs. conversely, expected values of ors for death among those with heart disease and renal disease compared with those without appeared to be lower than the value of 1.0. the present study systematically reviewed the risk of severe manifestations and death by mers by systematically searching and analyzing published articles from the ksa and the rok and calculating not only the cfr but [16] . icu represents intensive care unit also the risks of icu admission and requiring mechanical ventilation. several clinical predictors of death were identified including older age, male sex and underlying medical conditions, including diabetes mellitus, renal disease, respiratory disease, heart disease and hypertension. the risk estimate appeared to vary by study design. in particular, studies focusing on patients in the icu yielded the greatest estimates, while the cfrs for surveillance and hospital outbreak studies were smaller. these findings indicate that ascertainment biases in surveillance and hospital outbreak studies, frequently involving case finding effort, were smaller than in other types of studies. the importance of case finding effort is likely reflected in the different cfr estimates based on surveillance data between ksa and rok. although the presently identified clinical predictors are in line with previously published studies [6, 7, 11] , the present study is the first to systematically analyze published studies, including clinical research studies, and extract findings that echo those of published articles. as was observed in this study, systematic search and analysis of the transmission characteristics [38] and spatial spreading patterns of mers [39] have been successful. an important contribution of the present study is that we demonstrated that the risk of death or severe manifestations is highly heterogeneous for various reasons, including different study designs. it is recognized that mers involves asymptomatic infection [9] , and thus, studies must be clear as to how the risk is estimated, including the definition and diagnostic methods used to identify infected individuals. depending on the study design, the clinical predictors of death also differed. for fig. 4 estimated risks associated with middle east respiratory syndrome (mers) by study design. panels show the risk estimates by study outcome: (a) risk of death, (b) risk of admission to intensive care unit (icu) and (c) risk of mechanical ventilation. cfr represents the case fatality risk. the estimate for each study design represents the pooled risk of death calculated using the inverse variance of the risk of death in each published study. the size of the diamonds reflects the sample size, and the whiskers extend to the lower and upper values of the 95% confidence interval (ci). the diamond without fill represents the pooled estimate using the inverse variance of the risk of death. i 2 measures the extent of the heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity example, renal and heart diseases might not predict the risk of death in an icu setting, but they may be critically important in other settings that involve milder cases. not only studies in icu settings, but also retrospective studies yielded relatively high risk estimates for severe manifestations and death. our finding raises concerns regarding the retrospective analysis of confirmed cases in registered databases without referring to biases associated with case detection and diagnosis, which could yield a biased risk estimate of mers severity. in fact, that could explain why the cfr of confirmed cases among registered cases in patients' database has been as high as 40%, while the cfr of secondary cases in the presence of contact tracing has been estimated at about 20% [6] [7] [8] [9] [10] . the comorbidities identified in our study are in line with those already identified elsewhere [6, 37] . the identification of comorbidities is not only stressed based on previous and present findings [11] , but it is critically important to understand the underlying pathophysiological mechanisms. high representation of men among deceased cases may reflect the interaction of factors related to sex-specific lifestyle (e.g., smoking habits in the middle east). older age might reflect the greater likelihood of having underlying medical conditions. diabetes, renal and respiratory diseases could predispose patients to be immunologically vulnerable and heart disease could induce water retention (e.g., secondary aldosteronism), both exacerbating the systemic condition. hypertension could have been confounded by some other explanatory factor (s), for example, obesity could have likely led to both hypertension and mers death. nevertheless, identified predictors are accompanied by reasonable biological explanations. the present study is not free from limitations. the biggest concern is, given the absence of identifying information, the included articles most likely referred to the same cases multiple times, potentially overestimating the . the vertical dashed line shows the threshold value of or = 1. the diamond without fill represents the pooled estimate using the inverse variance of the or. i 2 measures the extent of heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity number of cases. in fact, the total number of diagnosed and reported cases of mers as of june 2016 is approximately 1,768 cases, but our systematic review included as many as 2,081 cases. thus, it is likely that multiple reports from rok (e.g., cowling et al. [8] , kcdcp [33] and majumder et al. [34] ) reported on the same cases multiple times. rather, we did not avoid any overlap of cases in datasets because that adjustment forced us to adjust the overlap among the cases from the ksa in a similar manner. for this reason, the pooled estimate would never represent the actual pooled outcome data because the same case was counted multiple times. if we remove cowling et al. [8] and majumder et al. [34] from our analysis and include kcdcp [33] , which had the largest sample size, the pooled estimate of the cfr would be increased to 45.4% (95% ci: 43.2, 47.7). this is understandable owing to the diminished impact of the extensive contact tracing effort in the rok. despite these overlaps, we conducted this systematic review to demonstrate that ascertainment biases likely act as a key factor that characterizes differential mortality across countries. to avoid any overlap of cases and better identify risk factors of icu admission and death, it is advised to set up a common case registration system across countries and allocate identity number for each individual case. as the second technical limitation to remember, it should be noted that the access to individual data was not achieved, and thus, for instance the age-related analysis did not rest on individual age data, and similarly, we have had limitations in the precision of the majority of outcome evaluations. third, clinical predictors of death have been classified only at organ level, and moreover, individual behavioral factors or habitat [40] have not been examined in relation to the risk of mers death. fourth, non-english language manuscripts have been missed, and they include at least a few publications in korea and one from jordan. despite these problems, we cannot help but consider that the present study successfully and systematically . odds ratio (or) represents the odds ratio of death among men with underlying medical condition compared with women without comorbidities, respectively. the size of the diamonds reflects the sample size, and the whiskers extend to the lower and upper values of the 95% confidence interval (ci). the diamond without fill represents the pooled estimate using the inverse variance of the risk of death. i 2 measures the extent of heterogeneity, representing the proportion of variance in a meta-analysis that is attributable to study heterogeneity evaluated the risk of severe manifestations and death by mers by collecting published information on clinical predictors of the risk of death. an important consideration is that the associated risk estimation and identification of risk factors of mers call for particular care in terms of study design, especially in aiming to eliminate biases associated with detection and diagnosis. heterogeneity in risks of death and severe manifestations secondary to mers was substantial. differential study design was one of underlying reasons for the large heterogeneity. statistical estimation of the risk of mers death and identification of risk factors must be conducted with particular careful attention paid to study design, especially accounting for biases associated with case detection and diagnosis. additional file middle east respiratory syndrome coronavirus (mers-cov). geneva: world health organization clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus (mers-cov) into the united states first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission case fatality: rate, ratio, or risk? middle east respiratory syndrome middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility preliminary epidemiologic assessment of mers-cov outbreak in south korea estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea systematic reviews and meta analysis of published studies: an overview and best practices preferred reporting items for systematic reviews and meta-analyses: the prisma statement a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case family cluster of middle east respiratory syndrome coronavirus infections ribavirin and interferon-α2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological investigation of mers-cov spread in a single hospital in south korea acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study association of higher mers-cov virus load with severe disease and death, saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus outbreak in the republic of korea mortality risk factors for middle east respiratory syndrome outbreak, south korea the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study hospital outbreak of middle east respiratory syndrome coronavirus risk of mers importation and onward transmission: a systematic review and analysis of cases reported to who predicting the international spread of middle east respiratory syndrome (mers) identifying determinants of heterogeneous transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, 2015: a retrospective epidemiological analysis funding hn received funding support from the japan agency for medical research and development, the japanese society for the promotion of science (jsps) kakenhi grant numbers 16kt0130, 16 k15356 and 26700028, the japan science and technology agency (jst) crest program and ristex program for science of science, technology and innovation policy. no received funding support from the ministry of health, labor, and welfare, japan (h27-shinkogyosei -shitei-006). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. collected datasheet is available from the authors (rm) upon request. authors' contributions hn conceived the systematic review. rm and hn implemented systematic search. rm and hn performed statistical analyses. rm and hn drafted the early version of the manuscript and hn substantially rewrote the text. sk, kh and no further revised the manuscript. all other authors gave comments on the revised manuscript and approved the final version of the manuscript. the authors are experts with interest in infectious disease epidemiology and also in clinical infectious diseases, and the team of lead author is led by professor from hokkaido university graduate school of medicine. the authors declare that they have no competing interests. not applicable.ethics approval and consent to participate not applicable.author details 1 graduate school of medicine, hokkaido university, kita 15 jo nishi 7 chome, kita-ku, sapporo 060-8638, japan. 2 crest, japan science and technology agency, 4-1-8, honcho, kawaguchi-shi, saitama 332-0012, japan.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-330315-upcf15q5 authors: oudshoorn, diede; rijs, kevin; limpens, ronald w. a. l.; groen, kevin; koster, abraham j.; snijder, eric j.; kikkert, marjolein; bárcena, montserrat title: expression and cleavage of middle east respiratory syndrome coronavirus nsp3-4 polyprotein induce the formation of double-membrane vesicles that mimic those associated with coronaviral rna replication date: 2017-11-21 journal: mbio doi: 10.1128/mbio.01658-17 sha: doc_id: 330315 cord_uid: upcf15q5 betacoronaviruses, such as middle east respiratory syndrome coronavirus (mers-cov), are important pathogens causing potentially lethal infections in humans and animals. coronavirus rna synthesis is thought to be associated with replication organelles (ros) consisting of modified endoplasmic reticulum (er) membranes. these are transformed into double-membrane vesicles (dmvs) containing viral double-stranded rna and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. previous evidence suggested that the nonstructural proteins (nsp’s) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (sars-cov), which contain transmembrane domains, would all be required for dmv formation. we have now expressed mers-cov replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either mers-cov or sars-cov, to characterize the membrane structures induced. using electron tomography, we demonstrate that for both mers-cov and sars-cov coexpression of nsp3 and nsp4 is required and sufficient to induce dmvs. coexpression of mers-cov nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar dmvs, and in both setups we observed proliferation of zippered er that appeared to wrap into nascent dmvs. moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for mers-cov dmv formation. addition of the third mers-cov transmembrane protein, nsp6, did not noticeably affect dmv formation. these findings provide important insight into the biogenesis of coronavirus dmvs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral dmv formation. in the first luminal loop of nsp4 (34, 37, 40) . when both these sites were mutated in mhv nsp4 (38, 40) , the virus was attenuated in cell culture and dmv formation was impaired, suggesting that nsp4 plays a critical role in coronaviral ro formation. the combined membrane-spanning regions of these proteins (i.e., including all luminal loops and flanking transmembrane domains) are commonly referred to as tm1, tm2, and tm3, respectively. nsp3, nsp4, and nsp6 are nonconventional transmembrane proteins in the sense that they are derived from a polyprotein and do not contain n-terminal signal sequences for cotranslational membrane insertion. it is currently unknown how their membrane insertion is accomplished and whether polyprotein cleavage precedes (or is perhaps required for) translocation across the er membrane. to a certain extent, nsp2, nsp3, and nsp5 of the distantly related arteriviruses (also members of the order nidovirales) can be considered equivalent to coronavirus nsp3, nsp4, and nsp6, in terms of their relative position in the replicase polyprotein and their membrane-spanning properties. for arteriviruses, expression of nsp2 and nsp3 alone was necessary and sufficient for the formation of double-membrane structures strikingly resembling the dmvs observed in infected cells (41) . coexpression of nsp5 reduced the size of the induced dmvs but did not change their overall architecture (18) . in the case of coronaviruses, it was recently reported that the transient coexpression of sars-cov nsp3, nsp4, and nsp6 led to the formation of dmvs (42) . cells coexpressing (21) , and nsp4 was detected with anti-v5 monoclonal antibody. (c) constructs expressing mers-cov nsp3 or nsp4 or a gfp control were transfected into 293t cells, which were metabolically labeled with [ 35 s]methionine-cysteine from 4 to 20 h posttransfection. lysates were immunoprecipitated with the indicated antibodies, separated on an sds-page gel, and visualized using phosphorimaging. bands not corresponding to expected protein size in the western blot are indicated with asterisks. the~130-kda band in the nsp3 ip was also observed in the western blot. nsp4 bands in ip were fuzzy likely due to the relatively high hydrophobicity of the protein. (d) huh-7 cells were transfected with the indicated plasmids, and localization of mers-cov nsp3 and nsp4 was analyzed using immunofluorescence labeling and confocal microscopy at 24 h posttransfection. nsp3 was detected with anti-sars-cov-nsp3 serum, and nsp4 was detected with anti-v5 monoclonal antibody. nsp3 and nsp4 alone contained so-called maze-like bodies (mlbs), consisting of paired er membranes (zippered er) and some circular profiles that were interpreted as cross sections of double-membrane tubules. therefore, it was concluded that nsp6 is essential for the biogenesis of sars-cov dmvs, whereas nsp3 and nsp4 can mediate the pairing of membranes that are likely an intermediate in dmv formation (42) . in the current study, we examined the role of mers-cov nsp's in betacoronavirus ro biogenesis. using em and et, we found that mers-cov nsp3 and nsp4, either coexpressed from separate plasmids or expressed as a self-cleaving polyprotein fragment (nsp3-4), are essential and sufficient for the formation of dmvs that assemble into an rvn. addition of the third transmembrane subunit of the mers-cov replicase, nsp6, did not alter the overall morphology of the induced dmvs. when nsp3-4 polyprotein processing was prevented by mutagenesis, this blocked the formation of dmvs while membrane pairing did still occur, strongly suggesting that proteolytic processing coordinates dmv formation in time and/or space. to compare our results for mers-cov with the previous work on sars-cov (42) , we used et to analyze the three-dimensional (3d) structure of the maze-like bodies induced upon coexpression of sars-cov nsp3 and nsp4 and were thus able to conclude that the circular profiles observed in that setting in fact correspond to dmvs rather than tubules. this established that, also in the case of sars-cov, coexpression of nsp3 and nsp4 suffices to induce dmv formation. together, our results provide important new insights regarding the biogenesis of coronavirus ros and demonstrate the conservation of certain principles underlying ro formation, both among the coronaviruses and in comparison to more distantly related members of the order nidovirales. mers-cov nsp3 and nsp4 colocalize in the perinuclear region of the cell. to study whether the transmembrane nsp's of mers-cov are able to induce dmv formation, we expressed nsp3 and nsp4 from a cag promoter (43) either by cotransfection of cells with plasmids encoding individual proteins or by transfection with a single plasmid encoding a self-cleaving nsp3-4 polyprotein fragment ( fig. 1a ; table s1 ). constructs were codon optimized for expression in human cells, potential splice sites were eliminated, and the encoded proteins were equipped with hemagglutinin (ha), myc, or v5 tags at their termini. the constructs were transfected into 293t cells to verify protein expression and processing (fig. 1b) . the wild-type nsp3-4 polyprotein was fully cleaved into mature nsp3 and nsp4, as was previously described (44) . as a control, a mutant in which the nsp3/nsp4 cleavage site was inactivated (g2739a/g2740a; ggͼaa) (45) was included to generate the noncleaved precursor. interactions between nsp3 and nsp4 were previously shown to occur for mhv and sars-cov (46, 47) , and we assessed whether this was also the case for the corresponding mers-cov proteins. to this end, 293t cells were transfected with a construct expressing ha-nsp3-myc or nsp4-v5 or cotransfected with both constructs. expression products were labeled metabolically with [ 35 s]methionine and [ 35 s]cysteine and subsequently immunoprecipitated with either ha-or v5-specific antibodies (fig. 1c ). upon immunoprecipitation with the ha-specific antiserum, nsp4-v5 was brought down when ha-nsp3 was present (left panel). conversely, when using the v5-specific antibody, ha-nsp3 was coimmunoprecipitated when nsp4-v5 was present (right panel). these findings demonstrated that these two mers-cov proteins interact and further supported the notion that this is a common feature of coronaviruses. when using immunofluorescence microscopy, separate expression of nsp3 or nsp4 in huh-7 cells yielded a reticular labeling pattern, with some more-intense foci in the perinuclear region of the cell, suggesting that-in the absence of the other-either protein localized at least partially to the er (fig. 1d ). this reticular pattern (but without the foci) has been described previously upon transient expression of mhv and sars-cov nsp4 (34, 48) , whereas full-length sars-cov nsp3 was reported to localize to foci similar to those that we observed (42) . when coexpressing mers-cov nsp3 and nsp4 or when expressing the self-cleaving nsp3-4 polyprotein, the reticular pattern was much less pronounced and the two proteins mainly colocalized in foci in the perinuclear region (fig. 1d , lower panels). this was in agreement with the finding that mers-cov nsp3 and nsp4 interact and suggested that this interaction strongly promotes their recruitment to the foci in the perinuclear region. mers-cov nsp3 and nsp4 are required and sufficient to induce dmv formation. the next step was to determine whether nsp3 and nsp4 could induce the formation of double-membrane structures similar to those observed during infection. as a reference, mers-cov-infected huh-7 cells were analyzed by em. the membrane structures that were previously described in high-pressure frozen and freeze-substituted vero cells infected with mers-cov (21) were readily apparent at 10 h postinfection (p.i.) in chemically fixed huh-7 cells ( fig. 2a) . numerous dmvs were found (red asterisks), often adjacent to areas containing cm. the dmv interior appeared electron translucent, a difference from cryofixed samples (21) that can likely be attributed to the different sample preparation method, as the contents of cov-induced dmvs are easily lost upon chemical fixation (22, 24, 28) . occasionally some smaller circular profiles were observed that seemed similar in size to the spherules recently described for the gammacoronavirus infectious bronchitis virus (ibv) (red arrows) (28) . none of these structures was found in mock-infected control samples (fig. 2b) . when huh-7 cells expressed either nsp3 or nsp4, areas containing modified membranes were observed, which likely corresponded to the foci observed in fluorescence microscopy (fig. 1d) . in nsp3-expressing cells (fig. 2c) , we detected large regions, usually several micrometers in diameter, of disordered membrane bodies (dmbs), which were similar to those previously observed after sars-cov nsp3 expression (42) . the membrane structures clustering in these dmbs were reminiscent of the surrounding er cisternae, with which they were frequently connected, suggesting that dmbs consisted of clustered er-derived membranes. upon expression of mers-cov nsp4, large clusters of modified single membranes (msm) were observed (fig. 2d ), but these structures seemed more irregular than those induced by nsp3 (fig. 2c) . the expression of sars-cov nsp4 did not result in changes in intracellular membrane morphology (42) , in contrast with our present observations following mers-cov nsp4 expression. whether this reflects differences between the experimental setups used or an actual difference between these viral proteins remains to be determined. when mers-cov nsp3 and nsp4 were expressed in the same cell, either by cotransfection or by expression of the self-cleaving nsp3-4 polyprotein, a remarkably different set of membrane structures was observed ( fig. 2e and f) . a combination of circular double-membrane profiles (red asterisks) and paired membranes (red arrows) was present in both cases, suggesting that the combined expression of mers-cov nsp3 and nsp4 is sufficient to induce dmv formation. there was no apparent difference between the structures resulting from coexpression of nsp3 and nsp4 and those resulting from expression of the self-cleaving nsp3-4 polyprotein ( fig. 2e and f), but in both cases the circular profiles were significantly smaller than the ones observed in mers-cov-infected cells (average diameters of 146 and 148 nm, respectively, versus 252 nm in infection) (see fig. s1 in the supplemental material). these membrane modifications were frequently found in all the samples analyzed. in order to further investigate how the frequency of em-positive cells compared to the transfection efficiency, a quantitative analysis was carried out on samples of cells expressing mers-cov nsp3-4. immunofluorescence microscopy showed that approximately 40% of the transfected cells were positive for expression of mers-cov nsp3 and nsp4 (n ϭ 174), while around 19% of the cell sections (n ϭ 288) contained doublemembrane structures. both dmvs and zippered er clustered together in all the em positive cell sections, although at slightly different ratios (see fig. s2 for a gallery). as the em analysis was based on one random section per cell (~100 nm thick) that may not always capture the region with membrane modifications, it was not surprising that the fraction of positive cells observed in em was smaller than that observed in whole cells using light microscopy. the numbers above in fact strongly suggest that the formation of dmvs and zippered er is induced in most, if not all, cells expressing mers-cov nsp3 and nsp4. while the circular profiles observed were suggestive of double-membrane vesicle formation, they could also correspond to cross sections of double-membrane tubular structures. to resolve this issue, we obtained 3d reconstructions of these membrane structures using et ( fig. 3 ; movies s1 and s2), which confirmed that genuine dmvs were indeed formed upon expression of either nsp3 plus nsp4 or nsp3-4 of mers-cov. the distinctive feature that unambiguously identifies a vesicle in a tomogram is a circular profile that is largest at the vesicle's equator and decreases in diameter when moving up or down from that plane through successive tomographic slices until, if the vesicle is fully contained in the section, it disappears. indeed, many profiles like this were observed in the tomograms (fig. 3a , red asterisks; movies s1 and s2, green dots). we found no openings connecting the dmv interior and the cytosol, similar to what was observed previously upon tomographic analysis of coronavirus-infected cells (19, 28) . the tomograms corroborated the structural similarity between the membrane structures induced by cotransfection with nsp3 and nsp4 constructs and by expression of the self-cleaving nsp3-4 polyprotein. the electron density of the dmv interior seemed similar to that of the surrounding cytoplasm, and in this sense, it was different from that of dmvs in mers-cov-infected cells (compare to fig. 3a ), which is likely due to the absence of other viral proteins and double-stranded rna. in some cases, dmvs appeared to be contained in a larger double-membrane structure (fig. 3b , red asterisks). such structures have not been observed in coronavirus-infected cells. the paired membranes were often continuous with er cisternae (fig. 3b ) and resembled the so-called zippered er that has also been observed in ibv-infected cells (28) , although they have not been documented so far for betacoronavirus-infected cells. these paired membranes may represent an intermediate of dmv biogenesis. further supporting this explanation, structures in which the zippered er seemed to transform into a nascent dmv could readily be observed in the tomograms (fig. 3b , red arrows). we also observed dmv-dmv, dmv-zippered er, and dmv-er connections (fig. 3c , red arrows), whereas completely isolated dmvs were in fact rare. in summary, while the described differences between nsp3-4-expressing and mers-cov-infected cells suggest that other viral components may modulate the process of dmv formation and would be required to form the full array of membrane structures observed during infection, our results establish that mers-cov nsp3 and nsp4 are sufficient to trigger all the membrane-remodeling steps required for inducing dmv formation, likely through the transformation of er membranes into an rvn consisting of dmvs and modified er. mers-cov nsp6 does not alter dmv morphology. the dmvs induced by expression of mers-cov nsp3 and nsp4 largely mimicked those observed during infection. overviews of reconstructed tomograms (available as movies s1 and s2, respectively) for both conditions. some of the fully reconstructed closed dmvs are indicated with red asterisks. (b) zippered er curving into putative intermediates during dmv biogenesis (indicated with red arrows) is shown. two dmvs that are enclosed within other dmvs are indicated with red asterisks. (c) examples of connections between dmvs and (zippered) er (indicated with red arrows). all the images are virtual 5-nm-thick slices from the reconstructed tomograms. bars, 250 nm. mers-cov nsp3 and nsp4 suffice to induce dmv formation ® however, the additional rvn elements that have been observed in this and previous studies of coronavirus-infected cells (cm and spherules) were not detected. to investigate whether nsp6, the third transmembrane subunit of the coronavirus replicase, plays a role in their formation or affects dmv formation, we aimed to extend the expressed polyprotein fragment to include nsp5 and nsp6. in addition to pl pro cleaving the nsp3/nsp4 site, this should lead to processing of the nsp4/nsp5 and nsp5/nsp6 junctions by the nsp5-based m pro , an assumption based on sequence conservation and studies performed with other coronaviruses (3), as the kinetics of mers-cov polyprotein processing in cell-based assays have not been documented in any detail. remarkably, however, when the "regular" nsp3-6 polyprotein was expressed, efficient processing of the nsp3/4 site was achieved, but nsp's located downstream of this junction were retained in processing intermediates due to poor cleavage of the nsp4/5 and nsp5/6 junctions, as observed by immunoprecipitation (ip) analysis ( fig. 4a and b) . this prompted us to design a set of alternative polyprotein constructs to investigate and optimize the proteolytic autoprocessing of the nsp3-6 region (see table s1 ). efficient cleavage at all sites was observed only for an engineered polyprotein (nsp3-5-gfp-6) in which green fluorescent protein (gfp) had been inserted between two copies of the nsp5/6 cleavage site (fig. 4a ). immunoprecipitation analysis established that this nsp3-5-gfp-6 polyprotein was processed into four separate nsp's and gfp (fig. 4b) . consequently, this construct could be used to evaluate the effect of expressing nsp6 in addition to nsp3 and nsp4. when huh-7 cells expressed the "regular" nsp3-6 polyprotein, which was barely cleaved at the nsp5/6 junction (fig. 4b) , we no longer detected the dmvs previously observed upon nsp3-4 expression. instead, large areas of highly organized and curved membrane structures were seen (fig. 4c) , which were connected to surrounding er cisternae (fig. 4c, red arrows) . in contrast to the large single-membrane clusters observed in nsp3-or nsp4-expressing cells (fig. 2c and d) , they consisted of double membranes (fig. 4c , black arrow in the inset). the geometric pattern in these large areas containing double-membrane structures is typical of cubic membranes (49) , which can result from overexpression and/or misfolding of er proteins, leading to protein and membrane aggregation. in contrast, when huh-7 cells expressed the engineered nsp3-5-gfp-6 polyprotein, which was almost fully processed (see above), cubic membranes were not observed and we found instead putative dmvs together with zippered er (fig. 4d) , structures very similar to the ones found in cells expressing just nsp3 and nsp4 (compare with fig. 2e and f) . also, the average size of these dmvs (146 nm) was comparable to that of dmvs induced by nsp3-4 expression (148 nm) (fig. s1 ). circular profiles (putative dmvs) were detected in 33 out of 642 cell sections analyzed; however, none of these regions contained cm or spherules. this suggests that, while nsp3 and nsp4 are necessary and sufficient to induce the rearrangement of intracellular membranes into dmvs, the presence of (cleaved) nsp6 does not suffice to trigger the formation of the additional membrane structures typical of mers-cov infection. other viral components that are present during mers-cov replication, such as viral rna or other viral proteins, might thus be required for the formation of convoluted membranes and spherules. cleavage of the mers-cov nsp3/nsp4 junction is essential for dmv formation. to gain more insight into the biogenesis of coronavirus dmvs, we set out to determine the role of the nsp3/nsp4 cleavage event. we surmised that the membrane modifications induced by an uncleaved nsp3-4 polyprotein could differ from those triggered by the (cleaved or coexpressed) nsp3 and nsp4 subunits. we transfected huh-7 cells with plasmids encoding nsp3-4 carrying either a mutated nsp3/nsp4 cleavage site (ggͼaa) or a catalytic site mutation in the nsp3 pl pro domain (c1592a) that inactivates the protease (50) . in both cases, only the uncleaved nsp3-4 precursor was observed (fig. 5a) . interestingly, dmvs were no longer found and instead we detected concentric structures consisting of zippered er that mostly lacked the pronounced curvature present in dmvs ( fig. 5b and c) . cotransfection of the cells with a plasmid encoding the active pl pro domain restored the nsp3/nsp4 cleavage in the nsp3-4 c1592a mutant polyprotein but not in the nsp3-4 polyprotein with the mutated cleavage site (fig. 5a) . accordingly, expression of pl pro together with the nsp3-4 cleavage site mutant (fig. 5d ) did not alter the structures observed. in contrast, when transcleavage of the nsp3/nsp4 site, by coexpression of pl pro with the nsp3-4 c1592a polyprotein, was achieved, dmv formation was at least partially restored and resulted in a mixture of abundant dmv and zippered er profiles (fig. 5e ), as observed before (fig. 2e and f) . expression of pl pro by itself did not have a membrane-remodeling effect. these results clearly showed that the nsp3-4 precursor is able to induce the membrane pairing required to form zippered er but that cleavage of the nsp3/nsp4 junction is essential for the formation of dmvs. sars-cov nsp3 and nsp4 are also sufficient to induce dmv formation. recently, angelini et al. reported that, in the case of sars-cov, nsp3, nsp4, and nsp6 are all required for the formation of dmvs when these proteins are transiently expressed as individual subunits (42) . in their two-dimensional (2d) imaging study, coexpression of sars-cov nsp3 and nsp4, in the absence of nsp6, led to the formation of so-called maze-like bodies (mlbs), large clusters of double-membrane structures that were interpreted as closely packed double-membrane tubules, not vesicles. since our mers-cov tomography data (fig. 3) established that dmv formation can be triggered just by coexpression of nsp3 and nsp4, the interpretation of angelini et al. suggested that these subunits of mers-cov and sars-cov differ in their ability to induce dmv formation in the absence of nsp6. to address this issue, we coexpressed nsp3 and nsp4 of either virus, using the same experimental setup previously used for sars-cov by angelini et al. (293t cells trans-fected using lipofection), and employed et for a comparative analysis in 3d. coexpression of sars-cov nsp3 and nsp4 led to the formation of mlbs very similar to those observed by angelini et al. (42) , with areas of zippered er, often clustered as regularly spaced profiles, and circular double-membrane profiles ( fig. 6a and b) . the latter were postulated to be cross sections of double-walled tubules, of which the regularly spaced zippered er profiles would then represent longitudinal sections (42) . the fact that the spacing between clustered zippered er profiles roughly coincided with the diameter of the circular profiles supported this interpretation; however, angelini et al. also acknowledged that et would be required for its validation. to determine whether the circular profiles in the mlbs represented tubular or vesicular structures, we now used et to analyze several mlbs, two of which are shown in fig. 6a and b. in one of those images, zippered er is the dominant structure ( fig. 6a; movie s3) , whereas the other mainly contained circular double-membrane profiles ( fig. 6b; movie s4) . in both tomograms, we could detect multiple double-membrane profiles that increase and decrease in diameter when progressing through the tomogram and ultimately disappear (marked with green dots in the tomogram movies), indicating that they represent vesicles rather than tubules. in fact, no tubular structures were observed in the tomograms. the presumed longitudinal views of tubular structures turned out to consist of zippered er winding through the mlb. for mers-cov, coexpression of nsp3 and nsp4 in 293t cells led to the formation of numerous circular double-membrane profiles together with some zippered er (fig. 6c and d) , which strongly resembled what we had observed in huh-7 cells previously. taken together, our et results make it clear that, in the case of sars-cov as well, coexpression of nsp3 and nsp4 suffices for the induction of dmv formation and strongly suggest that this is a common feature among betacoronaviruses. the generation of membranous organelles that support their replication machinery is a universal mechanism among positive stranded rna viruses infecting eukaryotes. the formation of these ros is induced by viral proteins (32, 51, 52) , which are largely uncharacterized in most instances, and appears to be also reliant on host factors, some of which have been identified as important players (53, 54) . in this study, focusing on betacoronaviruses, we sought to identify the viral proteins required to induce the formation of dmvs, the most prominent membrane structure formed during coronavirus infection. using et, we found that coexpression of nsp3 and nsp4 of either sars-cov or mers-cov was required and sufficient to trigger the formation of erderived dmvs. moreover, the 3d architecture of these membrane structures was similar to what has been observed during betacoronavirus infection (19) . the dmvs formed upon coexpression of nsp3 and nsp4 were closed, with no detectable opening connecting the dmv interior and the surrounding cytosol, whereas their outer membrane generally was continuous with those of other dmvs and/or with (modified) er. our data importantly alter the conclusions of an earlier sars-cov study (42) , which was based on the transient coexpression of sars-cov nsp3 and nsp4 from separate plasmids and the 2d imaging of the resulting membrane structures. the observation of maze-like bodies and circular double-membrane profiles, which were interpreted to represent tubular structures, led these authors to conclude that coexpression of sars-cov nsp3 and nsp4 was not sufficient for dmv formation. using et, we could now show that the circular profiles observed in these maze-like bodies are in fact dmvs (fig. 6a and b) , suggesting that the basic capability of nsp3 and nsp4 to induce dmv formation probably is a common feature of betacoronaviruses. these findings also highlight the importance of 3d analysis as a tool to ascertain and characterize the ultrastructure of membranous viral ros. interestingly, in the case of the arterivirus equine arteritis virus (eav), nsp2 and nsp3 mers-cov nsp3 and nsp4 suffice to induce dmv formation ® were found to be required and sufficient for dmv formation (18, 41) . at least to a certain extent, these proteins can be considered the functional equivalents of coronavirus nsp3 and nsp4, respectively, as they share a number of features like the presence of multiple membrane-spanning domains and a papain-like protease in the upstream protein that cleaves the junction between the two subunits. they also occupy comparable positions in the replicase polyproteins, suggesting that there may also be similarities in terms of the relative order in which these subunits are synthesized, released, and targeted to the membranes that they transform (17) . these functional similarities and the potential to trigger dmv formation may thus be shared by these proteins of all coronaviruses and arteriviruses and possibly extend to other branches of the order nidovirales, like the poorly studied ronivirus and mesonivirus families. our findings also shed more light on dmv biogenesis, for which two models have been proposed that are not mutually exclusive. the first has been termed "double budding," where a vesicle would first bud into the er lumen and then bud out again to acquire a second membrane. the alternative model is based on "wrapping": membranes would first pair or "zipper" and then curve and finally form a closed dmv after a membrane fission event (18, 55) . the frequent observation of zippered er after coexpression of nsp3 and nsp4 and multiple em images in which zippered er seemed to wrap into a dmv (e.g., fig. 3b ) suggest that this structure is a dmv precursor. interestingly, whereas the uncleaved nsp3-4 precursor was able to induce the pairing of er membranes, dmv formation occurred only upon cleavage of the nsp3/nsp4 junction, which strongly suggests that membrane pairing is an early step in dmv formation. our findings contrast with what was observed previously for arteriviruses, where cleavage of the nsp2/3 junction was not required for the formation of dmvs in an expression system (56) . together, our observations favor the wrapping model for dmv formation, and even though the existence in parallel of a double budding mechanism cannot be formally ruled out, the current data add to the mounting evidence pointing toward double-membrane wrapping as the central mechanism for dmv formation. for the distantly related arteriviruses (18, 57) and the unrelated picornaviruses (30, 31) , putative wrapping intermediates have been also described, indicating that this might be a common mechanism of dmv biogenesis among positive stranded rna viruses. several steps are required for dmv biogenesis: pairing of membranes, membrane curvature (both positive and negative), and fission (18, 55) . in the wrapping model for dmv biogenesis, membrane pairing is an early step that may be mediated directly by interactions between the viral proteins inducing dmv formation. the interaction(s) between nsp3 and nsp4 that we described here for mers-cov may be sufficient to facilitate membrane pairing. similar observations have been made for sars-cov and mhv (42, 48, 58) . the most likely candidate regions for this kind of interactions are the luminal loops of nsp3 and nsp4 (33, 35) that are located in the tm1 and tm2 regions, respectively, as these could interact with their counterparts on the opposite side of the er cisterna, thus inducing membrane pairing. this view is partly supported by studies on mhv and sars-cov for which a truncated nsp3 lacking the region upstream of tm1 coexpressed with nsp4 was sufficient to induce membrane pairing but not the formation of dmvs (48, 58) . this suggests that, although the cytosolic n-terminal region of nsp3 is required for complete dmv formation, the tm1 region (together with nsp4) may be sufficient to induce membrane pairing. in principle, the liberation (by pl pro -mediated cleavage of the nsp3/nsp4 junction) and presumed membrane insertion of the hydrophobic n-terminal domain of nsp4 may be an important determinant of the ultimate transmembrane configuration of this protein, potentially with direct implications for the transformation of zippered er into dmvs. however, we found no major differences between dmvs induced by expression of self-cleaving mers-cov nsp3-4 and those induced by coexpression of nsp3 and nsp4, suggesting that nsp4 is properly inserted in the membrane when individually expressed. the concentric zippered er observed after expression of the uncleaved nsp3-4 polyprotein could then reflect an intermediate stage in which the lack of nsp3/nsp4 cleavage prevents proper membrane remodeling. the proximal (and largest) luminal loop of nsp4 contains an n-linked glycosylation site (n2985 in mers-cov), similar to the glycosylation site(s) in sars-cov and mhv (34, 37, 40) . analysis of the use of that site in proteolytically processed nsp4 and the uncleaved nsp3-4 precursor could provide insight into the sequence of events leading to the membrane insertion of mers-cov nsp4. although our data establish that expression of nsp3 and nsp4 suffices for coronaviral dmv formation, the precise role in this process-if any-of the nsp6 transmembrane subunit remains unclear. our mers-cov data suggest that, compared to cells expressing nsp3 and nsp4 only, coexpression of cleaved nsp6 does not affect dmv formation, nor does it lead to the formation of additional structures like cm or spherules. however, coexpression of sars-cov nsp3, nsp4, and nsp6 was previously reported to induce cm formation in addition to dmvs (42) . additionally, expression of nsp6 alone resulted in the formation of small single-membrane vesicles near the microtubule organizing center (42) , which suggested that nsp6 may have membrane proliferation and vesiculation abilities that could play a role in ro formation. when mers-cov nsp6 was retained in an unprocessed nsp4-6 precursor, dmvs were no longer formed and membrane clusters that resemble cubic membranes appeared (fig. 4c ). it has been proposed that the cm formed by coronaviruses are in fact a form of cubic membranes (59) . this might be related to observations that, compared to dmvs, cm are mostly formed relatively late in infection (19, 21, 24) when viral proteins or polyprotein fragments accumulate. it is conceivable that such accumulation could lead to aggregation, misfolding, and/or impaired polyprotein processing resulting in the formation of cubic membranes. in other words, there could be a link between the status of polyprotein processing in the nsp4-6 region and the membrane structures formed. along the same lines, the observation that blocking the cleavage at the nsp3/4 junction impedes dmv formation (see above) directly implicates polyprotein processing in the control of membrane remodeling, possibly by facilitating conformational changes required for specific interactions with the membrane and/or between nsp3 and nsp4. for an unrelated dmv-forming virus, hepatitis c virus (hcv), it was recently shown that dmv formation became less efficient when the proteolytic cleavage of the ns4b/5a site in the viral polyprotein was accelerated, which similarly suggests a role for the polyprotein processing in dmv formation (60) . the existence of different proteolytic processing intermediates containing nsp5 is well documented for arterivirus infection (61) , although the role of the different precursors in dmv formation has not been studied in depth so far. unfortunately, the kinetics of polyprotein processing by m pro in mers-cov and sars-cov are still largely unknown. m pro 's enzymatic activity has mainly been assessed using recombinant nsp5 and peptide substrates in vitro (9, 11, 13, 50) , but an analysis of the kinetics in a large (or larger) polyprotein setting is mostly lacking. most information on the processing of the coronavirus nsp4-to-nsp10 region is derived from studies on other coronaviruses, such as mhv, ibv, and human coronavirus 229e (hcov-229e) (the latter two being a gamma-and an alphacoronavirus, respectively) (9, 62, 63) . an in-depth analysis of the kinetics of polyprotein maturation during coronavirus infection, the identification of nsp6-containing processing intermediates, and the investigation of their possibly distinct roles in membrane remodeling could help to further unravel the mechanisms underlying the formation of the coronavirus ros. cells, viruses, and antibodies. huh-7 cells (kindly provided by ralf bartenschlager, heidelberg university) were grown in dulbecco's modified eagle's medium (dmem; lonza) supplemented with 8% (vol/vol) fetal calf serum (fcs; bodinco), 2 mm l-glutamine (paa laboratories), and nonessential amino acids (paa laboratories). 293t cells (kindly provided by the virgin lab, washington university school of medicine in st. louis, mo) were cultured in dmem with 10% (vol/vol) fcs. all cell culture media contained 100 u/ml penicillin and 100 g/ml streptomycin. infection of huh-7 cells with mers-cov (emc/2012 strain kindly provided by ron fouchier, erasmus medical center, the netherlands [3, 4] ) was performed as previously described (21) . primary antibodies used were mouse anti-ha (clone ha.c5; abcam), mouse anti-␤-actin (clone ac-74; sigma), and mouse anti-v5 (clone 2f11f7; thermo fisher). a rabbit serum recognizing sars-cov-nsp3 that cross-reacts with mers-cov nsp3 has been previously described (21, 22) . a polyclonal rabbit serum was used against a combination of two mers-cov nsp5 peptides, sglvkmshpsgdveac (amino acids 3248 to 3263 of pp1a) and cpadqlsdpnydalli (amino acids 3291 to 3306), which was produced by eurogentec. plasmid construction and transfection. human codon-optimized coding sequences of mers-cov nsp3-6 were designed using geneart, ordered from thermo fisher in four fragments, and subsequently assembled in low-copy-number vector pacnr1180 (64) using conventional cloning. the precise parts of mers-cov pp1a used for polyprotein constructs are outlined in table s1 in the supplemental material. the nsp4 construct included the 21 c-terminal aa of nsp3 to prevent the n-terminal hydrophobic region of nsp4 from acting as a signal sequence, which could result in improper membrane insertion. in all constructs with a c-terminal myc or v5 tag, the c-terminal glutamine of the viral sequence was omitted to prevent the removal of the tag by m pro . the sars-cov nsp3 gene (frankfurt 1 strain, pp1a amino acids 819 to 2740) was synthesized by bio basic inc. (ontario, canada). coding sequences were transferred to the pcaggs expression vector (addgene) for expression. pcaggs-sars-nsp4 was described previously (42) . 293t cells were transfected using lipofectamine 2000 (thermo fisher) according to the manufacturer's instructions. huh-7 cells were transfected using a nucleofector 2b device (lonza) with nucleofector kit t (lonza) in 6 ϫ 10 6 cells and 12 g of plasmid dna per transfection. cotransfections were carried out with equimolar amounts of plasmids. western blotting. cells were lysed in 2ϫ laemmli sample buffer (50 mm tris-hcl, ph 6.8, 20% [vol/vol] glycerol, 4% [wt/vol] sodium dodecyl sulfate [sds], 20 mm dithiothreitol, 0.02 mg/ml bromophenol blue) and separated by electrophoresis on sds-polyacrylamide gels. proteins were transferred to polyvinylidene fluoride membranes (amersham) using a trans-blot turbo transfer system (bio-rad). blots were blocked with 5% (wt/vol) elk skimmed milk powder (campina) in phosphate-buffered saline (pbs) supplemented with 0.05% (vol/vol) tween 20. secondary horseradish peroxidase (hrp)-conjugated antibodies (dako) and ecl plus western blotting substrate (thermo fisher) were used to visualize protein signal. immunofluorescence microscopy. after electroporation, huh-7 cells were seeded on coverslips and fixed 24 h later with 3% (wt/vol) paraformaldehyde in pbs. samples were permeabilized with 0.2% (vol/vol) triton x-100 and incubated with antibodies, including fluorescent conjugates, diluted in 5% (wt/vol) bovine serum albumin (bsa) in pbs. nuclei were stained with 1 g/ml hoechst 33258. after embedding with prolong gold (thermo fisher), samples were analyzed with a leica tcs sp8 confocal laser scanning microscope, which was equipped with a 63ϫ objective (numerical aperture [na] 1.40; 1 airy unit) and a leica hyd hybrid detector. and incubated with antibody overnight. antibody-protein complexes were then pulled down using protein a and protein g sepharose beads (ge healthcare), which were first blocked with 2% (wt/vol) bsa in pbs, and incubated for several hours. after repeated washing of the beads with ip buffer, proteins were eluted by heating in 2ϫ laemmli sample buffer. after separation on large 10% polyacrylamide gels and gel drying, signal was visualized using an imaging screen-k (bio-rad) and a typhoon 9410 scanner (ge healthcare). electron microscopy. transfected huh-7 or 293t cells were fixed 24 h posttransfection in 1.5% (wt/vol) glutaraldehyde in 0.10 m cacodylate buffer (ph 7.4) for 1 h at room temperature. after washing in 0.14 m cacodylate buffer, samples were postfixed and stained at 4°c with 1% (wt/vol) osmium tetroxide in 0.10 m cacodylate buffer for 1 h. after washing with 0.14 m cacodylate and milli-q water, cells were scraped and stained with 1% (wt/vol) tannic acid in milli-q water on a 3d rotator for 1 h at room temperature. following washing with milli-q water, cells were spun down in heated 3% (wt/vol) agar in pbs, and after solidification, pellets were excised, cut into small blocks, and dehydrated in increasing concentrations of ethanol. samples were embedded in epoxy resin (lx-112; ladd research), and after polymerization, 100-nm sections were placed on mesh-100 copper em grids covered with a carboncoated pioloform layer. following poststaining with 7% (wt/vol) uranyl acetate and reynolds lead citrate, samples were analyzed on an fei tecnai 12 biotwin microscope equipped with an eagle cooled slow-scan charge-coupled device (ccd) camera (fei) and operated at 120 kv. measurements of circular profiles from 2d em images were done with imagej software and aperio imagescope software (leica). circular profiles were measured over their longest and shortest axes, and the geometric mean of those values was used as the diameter. one hundred circular profiles were measured for each condition. electron tomography. sections of 150-nm thickness were cut from the resin-embedded blocks of transfected huh-7 or 293t cells prepared as described above. prior to poststaining, colloidal gold particles of 10 nm were applied to both sides of the em grid to serve later as fiducial markers for alignment. tomography data were recorded on an eagle ccd camera (fei) in an fei tecnai 12 biotwin (huh-7 samples) or a twin (293t samples) electron microscope operated at 120 kv, with the grids mounted on a 2040 fischione tomography holder. dual-axis tilt series of the regions of interest were collected using xplore3d software (fei) at magnifications that resulted in a pixel size of 1.7 nm (biotwin data) or 1.4 nm (twin data). the angular coverage for each single-axis tilt series was 130°sampled in identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronaviruses post-sars: update on replication and pathogenesis sars and mers: recent insights into emerging coronaviruses nidovirales: evolving the largest rna virus genome the nonstructural proteins directing coronavirus rna synthesis and processing virus-encoded proteinases and proteolytic processing in the nidovirales the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deisgylating activities recent advances in targeting viral proteases for the discovery of novel antivirals modification of intracellular membrane structures for virus replication cytoplasmic viral replication complexes endoplasmic reticulum: the favorite intracellular niche for viral replication and assembly untangling membrane rearrangement in the nidovirales biogenesis and architecture of arterivirus replication organelles sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum rna replication of mouse hepatitis virus takes place at double-membrane vesicles mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex ultrastructural characterization of sars coronavirus qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus morphogenesis of coronavirus hcov-nl63 in cell culture: a transmission electron microscopic study targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus ultrastructural characterization of membranous torovirus replication factories infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses complex dynamic development of poliovirus membranous replication complexes the transformation of enterovirus replication structures: a three-dimensional study of single-and doublemembrane compartments three-dimensional architecture and biogenesis of membrane structures associated with hepatitis c virus replication bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning detection of nonstructural protein 6 in murine coronavirus-infected cells and analysis of the transmembrane topology by using bioinformatics and molecular approaches mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications genetic analysis of murine hepatitis virus nsp4 in virus replication murine hepatitis virus nonstructural protein 4 regulates virus-induced membrane modifications and replication complex function non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5 crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease mobility and interactions of coronavirus nonstructural protein 4 genome-wide analysis of protein-protein interactions and involvement of viral proteins in sars-cov replication membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4 cubic membranes: a legend beyond the flatland* of cell membrane organization assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles a positive-strand rna virus replication complex parallels form and function of retrovirus capsids 2012. (ϩ)rna viruses rewire cellular pathways to build replication organelles the dependence of viral rna replication on co-opted host factors open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex formation of the arterivirus replication/ transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication do viruses subvert cholesterol homeostasis to induce host cubic membranes? ns5a domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis c virus replication alternative proteolytic processing of the arterivirus replicase orf1a polyprotein: evidence that nsp2 acts as a cofactor for the nsp4 serine protease mouse hepatitis virus 3c-like protease cleaves a 22-kilodalton protein from the open reading frame 1a polyprotein in virus-infected cells and in vitro processing of the human coronavirus 229e replicase polyproteins by the virus-encoded 3c-like proteinase: identification of proteolytic products and cleavage sites common to pp1a and pp1ab nucleotide sequence of classical swine fever virus strain alfort/187 and transcription of infectious rna from stably cloned full-length cdna computer visualization of three-dimensional image data using imod key: cord-334628-axon4jdc authors: lee, saemi; jo, seong-deok; son, kidong; an, injung; jeong, jipseol; wang, seung-jun; kim, yongkwan; jheong, weonhwa; oem, jae-ku title: genetic characteristics of coronaviruses from korean bats in 2016 date: 2017-07-19 journal: microb ecol doi: 10.1007/s00248-017-1033-8 sha: doc_id: 334628 cord_uid: axon4jdc bats have increasingly been recognized as the natural reservoir of severe acute respiratory syndrome (sars), coronavirus, and other coronaviruses found in mammals. however, little research has been conducted on bat coronaviruses in south korea. in this study, bat samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 natural bat habitat sites in south korea. rt-pcr and sequencing were performed for specific coronavirus genes to identify the bat coronaviruses in different bat samples. coronaviruses were detected in 2.7% (18/672) of the samples: 13 oral swabs from one species of the family rhinolophidae, and four fecal samples and one carcass (intestine) from three species of the family vespertiliodae. to determine the genetic relationships of the 18 sequences obtained in this study and previously known coronaviruses, the nucleotide sequences of a 392-nt region of the rna-dependent rna polymerase (rdrp) gene were analyzed phylogenetically. thirteen sequences belonging to sars-like betacoronaviruses showed the highest nucleotide identity (97.1–99.7%) with bat-cov-jtmc15 reported in china. the other five sequences were most similar to mers-like betacoronaviruses. four nucleotide sequences displayed the highest identity (94.1–95.1%) with bat-cov-hku5 from hong kong. the one sequence from a carcass showed the highest nucleotide identity (99%) with bat-cov-sc2013 from china. these results suggest that careful surveillance of coronaviruses from bats should be continued, because animal and human infections may result from the genetic variants present in bat coronavirus reservoirs. coronaviruses are large, pleomorphic, enveloped viruses containing a single linear, positive-sense single-stranded rna molecule. coronavirus genomes are approximately 27-32 kb in length, the largest continuous rna genomes among mammalian viruses [1] . coronaviruses are classified into four genera: alpha, beta, gamma, and delta [2] . coronaviruses are the second most prevalent cause of the common cold in humans [3] . in livestock and poultry, coronaviruses are recognized causes of enteric and respiratory infections that are often fatal in young animals [4] . an outbreak of severe acute respiratory syndrome (sars) in 2002 and 2003 resulted in infection of 8096 people worldwide, with 774 (9.5%) of them dying from this novel human coronavirus [5] . sars coronavirus is believed to have been acquired from an animal species, and the chinese horseshoe bat may have been the original source of sars infection [6] [7] [8] [9] . the emergent middle east respiratory syndrome coronavirus (mers-cov) may also have originated in bats, with fragments of mers-cov genes being identified in bats from both saudi arabia [10] and africa [11] . more than 200 viruses have been isolated from and detected in bats [12] . the ability of bats to fly and migrate, as well as the large sizes of their social groups, predisposes bats to acquire and maintain viruses [13] . given the import of mers into south korea [14] and the presence of sars in the relatively close geographic location of china [9] (fig. 3) , together with the fact that bats are a reservoir for coronaviruses, the prevalence of coronavirus infection in korean bat species should provide valuable information. although sars-like and mers-like bat coronaviruses have already been reported in the feces of korean bats [14] , the prevalence and presence of coronaviruses in other bat samples have not been described. therefore, we investigated the prevalence of coronaviruses in korean bat species using various kinds of samples, including oral swabs, fecal and urine samples, and carcasses. the coronaviruses found were characterized for their relationships to each other and to coronaviruses isolated in south korea and in other countries. a total of 672 samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 sites of natural bat habitat from january 2016 to september 2016 (table 1 ). bats were captured by net for collection of oral swabs, fecal samples, and urine samples, and released immediately after sampling. fecal samples were also collected from guano. the bat carcasses collected were naturally dead bats found at the site where the other samples were taken. swab samples were kept in viral transport medium at 4°c, and organs collected from carcasses and other samples were stored at −80°c before processing. all fecal and tissue samples were resuspended in 1% antibiotic-antimycotic solution (corning, usa) diluted in phosphate-buffered saline (pbs), and clarified by centrifugation at 3500×g for 10 min. rna from 200 μl sample was extracted with the qiaamp® viral rna mini kit (qiagen, germany) and eluted in 60 μl rnase-free water. cdna was synthesized by primescript first strand cdna synthesis kit (takara, japan) following the manufacturer's instructions. bat-cov screening was performed by a pancoronavirus pcr method based on primers used by poon et al. (corona 1 forward, 5′-ggttgggactatcc taagtgtga-3′, and corona 2 reverse, 5′-ccat catcagatagaatcatcata-3′) [15] , followed by sequencing of the amplified product to confirm bat-cov identification. the pan-coronavirus primers were used to amplify and sequence a 440-bp segment of the highly conserved rna-dependent rna polymerase (rdrp) gene. cycle sequencing reactions were performed using the bigdye® terminator cycle sequencing kit version 1.1 (applied biosystems, foster city, ca, usa). reactions were purified using sigmaspin™ post-reaction clean-up columns (sigma, st. louis, mo, usa) and sequenced on an abi prism 3130 automated capillary dna sequencer (applied biosystems) according to the manufacturer's instructions. primer sequences were excluded from the raw sequence data, and the sequences we analyzed were 392bp long. all sequences obtained in this study were submitted to genbank (accession numbers ky432454-432471). the nucleotide sequences were aligned and compared to 52 selected human and animal cov sequences available from the genbank database using clustalw software implemented in bioedit version 7.0.9.0. the phylogenetic trees were drawn using the neighbor joining method using the maximum composite likelihood model with mega 7 software. coronaviruses were detected in 13 oral swabs, one carcass (intestine), and four fecal samples from a total of 672 samples, giving an overall detection rate of 2.7% (18/672). four of the 14 bat species tested were found to harbor coronavirus. thirteen positive oral swabs were detected from one species of the family rhinolophidae, which accounted for 24.3% (163/672) of the samples, and the other positive samples were from vespertilionidae bats that comprised 55.5% (373/672) of the samples. the 13 coronavirus-positive oral swabs were collected from bats at an abandoned mine in jeonbuk province ( fig. 1 ). oral swabs and other samples (n = 60) were obtained from three species of bats, rhinolophus ferrumequinum, miniopterus schreibersii, and myotis macrodactylus, but coronaviruses were only detected in samples from r. ferrumequinum (table 2 ). one carcass was collected from a cave in chungbuk province (fig. 1 ). in total, 94 samples were obtained from six bat species (m. schreibersii, myotis petax, vespertilio sinensis, murina leucogaster, r. ferrumequinum, and m. macrodactylus), and coronavirus was detected from a v. sinensis sample. four coronaviruspositive fecal samples were collected in four different regions, in gyeongbuk province (andong, yeongdeok, and gyeongju) and in the metropolitan city gwangju (fig. 1 ). in the forest of andong, 43 samples in total were collected from three species of bats (eptesicus serotinus, m. petax, and pipistrelus abramus), and coronavirus was detected from one p. abramus bat. in yeongdeok, a total of five samples was collected from p. abramus living in the forest, and one sample was positive for coronavirus. in the forest of gyeongju, five samples in total were collected only from p. abramus bats, and one of the samples was positive for coronavirus. in the forest of gwangju, 18 samples in total were collected from two species of bats (e. serotinus and myotis aurascens) and coronavirus was detected from one e. serotinus bat ( table 2) . to determine the genetic relationships between the 18 bat coronaviruses obtained in this study and previously known coronaviruses, the 392-nt rdrp sequences were analyzed phylogenetically. all the sequences obtained in this study belonged to the betacoronavirus genus (fig. 2) . the sequences (fig. 2) . the 13 sequences from oral swabs were 97.1-100.0% identical with each other, and the closest known cov strain, with 97.1-99.7% nucleotide identity, was bat-cov-jtmc15 that was recently isolated from r. ferrumequinum in china. the nucleotide identity with sars-cov was only 86.7-89.0%. one sequence from a carcass showed the highest identity (99%) with bat-cov-sc2013 that was isolated from vespertilio superans (synonym of v. sinensis), also from china [16] . the nucleotide identity with mers-cov was 83.9%. four sequences from fecal samples shared 96.9-100.0% identity with each other and showed 94.1-95.1% identity with bat-cov hku5 [17] . the nucleotide identity with mers-cov was 84.1-84.4%. due to shorter reported sequences from kim et al. [14] , another phylogenetic tree was constructed from the 355-bp rdrp sequences of korean bat coronaviruses obtained by kim et al. [14] and in this study to analyze the genetic relation between bat-covs in south korea (fig. 3) . thirteen sequences from oral swabs were clustered with bat-cov b15-21, which was detected in fecal bat samples collected from an abandoned mine in gangwon province. the major bat species present there was r. ferrumequinum, but the coronaviruspositive bat species were not specified [14] . nucleotide identity between bat-cov b15-21 and the 13 clustered sequences was 97.7-100.0%. the other three korean bat coronaviruses (bat-cov b15-8, b15-40, and b15-41) belonged to the alphacoronavirus genus, but we did not detect coronaviruses belonging to this group in our study. there are approximately 24 species of bats in south korea, and most are insectivorous and relatively small [18] . bats in south korea usually do not migrate over distances greater than 1000 km [19], but apart from two species found only in south korea, they are generally distributed in neighboring countries such as china, east siberia, japan, and taiwan. although it is not clear if bats go between china and other countries, some species that migrate over 500 km can carry pathogens to south korea. the fact that the 18 bat coronaviruses (bat-covs) detected in this study were all in this study, the prevalence of coronavirus in korean bat samples was 2.7% overall. the prevalence by sample type was 5.4% (13/332) for oral swabs, 1.8% (1/57) for carcasses, 1.6% (4/245) for fecal samples, and 0.0% (0/38) for urine samples. reported rates from other countries for coronavirus prevalence in bats were 6.5% (64/985) and 5.3% (50/951) for china, 8.2% (16/195) for italy, and 5.3% (32/606) for mexico [20] [21] [22] [23] , not greatly different from our figure of 2.7% for prevalence in south korea. fourteen out of 24 korean bat species were screened for covs in this study. the species positive for bat-covs were r. ferrumequinum, v. sinensis, p. abramus, and e. serotinus. the bat-covs that showed the highest nucleotide identity with our isolates were obtained from the same species that we identified as positive, except for e. serotinus. to our knowledge, this is the first time a bat-cov has been detected from e. serotinus in asia. in europe (italy), a lineage c betacoronavirus was detected from e. serotinus in 2014 [24] . bat-covs were usually detected at higher rates in alimentary samples (fecal samples, rectal swabs, and intestine) than in respiratory samples (oral and throat swabs) [7, 15, 17, 22, 25, 26] . we collected alimentary and respiratory samples and urine samples. however, bat-covs were not detected in urine samples. animal coronaviruses usually produce either enteric or respiratory infections. animal models show similar clinical features to sars and mers patients; the majority of whom presented with respiratory symptoms but in some cases also suffered from enteric complications [27] . unlike other human coronavirus infections, a number of mers cases were associated with acute renal failure. mers-cov replication in the human kidney suggests the potential of shedding viruses in urine [28] . bat-cov isolate bat sl-cov-wiv1 (kf367457) showed infectivity to rhinolophus sinicus kidney cell lines, which means it may be possible to detect bat-cov in urine [6] . the current study revealed that korean bats have sarsand mers-related bat-covs, consistent with earlier work [ 1 4 ] . t h e p r e v i o u s s t u d y d e t e c t e d a l p h a -a n d betacoronaviruses only from fecal samples in four regions, two in gangwon, one in chungbuk, and one in gyeongbuk [14] . the only betacoronavirus rdrp sequence bat-cov b15-21 (ku528590.1) was clustered with 13 bat-covs β-cov lineage b β-cov lineage c α-cov fig. 3 phylogenetic tree of korean bat coronaviruses detected by kim et al. [13] and in this study, based on 355-nt sequences. phylogenetic trees were constructed by using the neighbor joining method and bootstrap values were determined by 3000 replicates. scale bar the estimated genetic distance of these viruses. black circle sequences from oral swabs. black up-pointing triangle sequences from fecal samples. white uppointing triangle sequences from carcass (16bo122, 129, 132, 133, 134, 135, 139, 141, 143, 145, 150 , 154, and 155) detected from danyang, chungbuk province. the identity between the 14 sequences was very high at 97.7-100%, even though the sample types and collection sites were different. the species of bat infected with bat-cov b15-21 was not specified in the paper, but the major bat species of the sample collection site was r. ferrumequinum; the same species in which we detected 13 bat-covs. the same bat-covs were detected from fecal samples in the prior study [14] and from oral swabs in our study. spike proteins, which are translated from spike genes, define viral tropism by receptor specificity and by membrane fusion activity during entry into cells [29] . although the spike genes were not identified in this study, full spike gene analysis of bat-cov b15-21 suggested a low potential for human emergence. one mers-like betacoronavirus, bat-cov b15-1-6 (ku528586.1, ku528585.1), was identified by kim et al. [14] , but the similarity of this isolate to our isolates could not be analyzed because the rdrp sequence was not obtained. high nucleotide identity between bat-cov b15-1-6 and bat-cov-sc2013 (kj473821) suggests that bat-cov b15-1-6 and 16bt3 might be very similar. bat-covs have been identified in many countries including the australasian region [10, 11, 14-16, 20-26, 30, 31] . but bat-covs identified in korea were genetically close to bat-covs from china, a close neighbor of korea. according to the results of bat-cov research in china, the degree of host restriction for coronaviruses in bat populations was high. for example, similar viruses were detected in the same species of bat in different regions, approximately 1600 km apart, while two different bat species in the same habitat had different coronaviruses [22] . in our results, three bat-covs (16bf109, 211, and 244) from p. abramus were clustered with each other, even though samples were collected from different habitats in different regions (andong, yeongdeok, and gyeongju). the bat-cov detected from e. serotinus bat in the metropolitan city gwangju (16bf104) was also clustered with these three bat-covs mentioned above. however, there was no matching virus for 16bf104 found in the same species of bat in other locations, nor in other species in the region where samples were collected. our data could support the possibility of relationships between genetic variation, geographic locations where bat-covs were isolated, and bat host species, but more data on bat-covs in korea would be needed to address this. in summary, the prevalence of coronavirus in korean bats was determined and found to be comparable to or lower than the rates in other countries. we detected sars-related and mers-like bat-covs that were genetically very similar to bat-covs identified in china. for the study of bat-cov seasonal occurrence and transmission, continuous monitoring during all seasons is required, and ideally alimentary, respiratory, and other samples need to be collected from each bat. future work to determine the complete genome sequences of bat-covs from south korea would give a more complete picture of their pathogenicity and the possibility of infectious spread to other animals and humans. bat cov-jtmc15(ku182964) bat cov-273(dq648856) sars bat cov-lingbao(kf294456) sars bat cov-rf1(dq412042) bat cov-rp(jx993987) bat cov hku3-1(dq022035) bat cov-rp3(dq071615) bat cov rsshc014(kc881005) sars cov-ma15(jf292915) sars cov-tor2(jx163928) sars cov-urbani(ay278741) sars-cov(nc004718) bat cov bm48-31/bgr/2008(gu190251) bat cov slo1a0082(gq404797) phev(nc007732) mhv(nc001846) hcov-hku-1(nc006577) bat cov-hku9(nc009021) tcv(nc010800) ibv(nc001451) bat cov-hku4(nc009019) kbat cov-16bf104 kbat cov-16bf109 kbat cov-16bf221 kbat cov-16bf244 bat cov hku5-1(dq249217) bat cov-sc2013(kj473821) kbat cov-16bt3 bat cov(kc243390) bat cov btcov/ukr-g17(kc243392) mers-cov(nc019843) human beta-cov(jx869059) human beta-cov(kc667074) human beta-cov(kc776174) coronavirus genome structure and replication a comparative sequence analysis to revise the current taxonomy of the family medical microbiology animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? ecology, evolution and classification of bat coronaviruses in the aftermath of isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor bats are natural reservoirs of sarslike coronaviruses science veterinary microbiology and microbial disease a review of studies on animal reservoirs of the sars coronavirus middle east respiratory syndrome coronavirus in bats close relative of human middle east respiratory syndrome coronavirus in bat bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? bats: important reservoir hosts of emerging viruses detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats identification of a novel coronavirus in bats mers-related betacoronavirus in vespertilio superans bats genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus bats. moonji publishing, seoul 19 detection of coronaviruses in bats of various species in italy viruses prevalence and genetic diversity of coronaviruses in bats from detection and characterization of diverse alpha-and betacoronaviruses from bats in alpha and lineage c betacov infections in italian bats coronaviruses in bent-winged bats (miniopterus spp detection of alpha and betacoronaviruses in multiple iberian bat species microbiology, ecology, and natural history of coronaviruses in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection coronavirus spike proteins in viral entry and pathogenesis coronavirus infection and diversity in bats in the australasian region ecohealth detection of group 1 coronaviruses in bats in north america emerg acknowledgements we thank dr. c.w. jeong and his colleagues for their efforts in the collection of wild bat carcasses and samples. this research was supported by grant no. 2016-01-01-033 from the nier of the republic of korea. the funders had no roles in the study design, data collection and analysis, decision to publish, or the preparation of the manuscript. key: cord-337499-jzpgtkai authors: yong choi, sung; shin, joongbo; park, woori; choi, nayeon; sei kim, jong; i choi, chan; ko, jae-hoon; ryang chung, chi; son, young-ik; jeong, han-sin title: safe surgical tracheostomy during the covid-19 pandemic: a protocol based on experiences with middle east respiratory syndrome and covid-19 outbreaks in south korea date: 2020-06-17 journal: oral oncol doi: 10.1016/j.oraloncology.2020.104861 sha: doc_id: 337499 cord_uid: jzpgtkai background: a subset of patients with covid-19 require intensive respiratory care and tracheostomy. several guidelines on tracheostomy procedures and care of tracheostomized patients have been introduced. in addition to these guidelines, further details of the procedure and perioperative care would be helpful. the purpose of this study is to describe our experience and tracheostomy protocol for patients with mers or covid-19. materials and methods: thirteen patients with mers were admitted to the icu, 9 (69.2%) of whom underwent surgical tracheostomy. during the covid-19 outbreak, surgical tracheostomy was performed in one of seven patients with covid-19. we reviewed related documents and collected information through interviews with healthcare workers who had participated in designing a tracheostomy protocol. results: compared with previous guidelines, our protocol consisted of enhanced ppe, simplified procedures (no limitation in the use of electrocautery and wound suction, no stay suture, and delayed cannula change) and a validated screening strategy for healthcare workers. our protocol allowed for all associated healthcare workers to continue their routine clinical work and daily life. it guaranteed safe return to general patient care without any related complications or nosocomial transmission during the mers and covid-19 outbreaks. conclusion: our protocol and experience with tracheostomies for mers and covid-19 may be helpful to other healthcare workers in building an institutional protocol optimized for their own covid-19 situation. in december 2019, a local outbreak of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) occurred in wuhan (hubei, china). the coronavirus disease 2019 (covid19) was highly infectious from the early stage and rapidly spread to several countries. as of may 16, 2020, covid-19 has been reported in 185 countries, with more than 4,486,990 cases and more than 306,306 deaths. [1] since south korea recorded its first case of covid-19 on january 20, 2020, the total number of confirmed cases stands at 11,037, which is concentrated mainly in daegu and gyeongsangbuk-do (74.6% of all confirmed cases) and the number of the virus-associated deaths has reached 262 people. [2] most patients are projected to have mild symptoms (81%) and the mortality rate in covid-19 is relatively low (2.3%). [3] compared with mortality rates of 10% for severe acute respiratory syndrome (sars) [4] and 37% for middle east respiratory syndrome coronavirus (mers) [5] . however, some infected patients are classified as severe or critical cases, and often require intubation and mechanical ventilation (9.8%-15.2%). [3, 6] critically ill patients with prolonged intubation ultimately need tracheostomy for proper airway management and lung care. tracheostomy is a routine surgical procedure, and there has been a debate on the optimal time for tracheostomy in critically ill patients requiring intensive respiratory care. [7] in general, a timely tracheostomy within seven to ten days after intubation is preferred in terms of minimizing mechanical ventilation time, length of stay in the intensive care unit (icu) and mortality. [8] however, in this epidemic situation, the risks of exposure and transmission from patients to healthcare workers should be carefully considered when the tracheostomy is planned. it is essential that surgeons and icu staff stay current on the protocols and guidelines for infection prevention during the tracheostomy, and these should be based on real experience and the best available evidence on this topic. in 2015, we experienced the largest in-hospital mers outbreak with 92 laboratory-confirmed mers cases. [9] although all surgical procedures for mers patients were delayed as long as possible according to our institutional policy, nine cases inevitably required surgical tracheostomy. thus, we developed our own institutional protocol for safe tracheostomy in patients with mers. five years later, as the covid-19 pandemic rapidly spread, we revised and modified our tracheostomy protocol to prepare for the covid-19 situation. we applied and tested this protocol in a patient with covid-19 patient for whom tracheostomy was indicated in march 2020. here we describe our experience and protocol for surgical tracheostomy in patients with covid-19 in our hospital. this study was a retrospective analysis using clinical and pathological data from patients with mers and covid-19 who underwent surgical tracheostomy. the study protocol was approved by our institutional review board (no. 2020-04-178) and the electronic medical records and interviews of medical staff who cared for patients with mers and covid-19 who underwent surgical tracheostomy were used for the study. all data were de-identified. the study population included nine patients with mers who had undergone surgical tracheostomy at our institution from may to july 2015 (mers outbreak). on the basis of hospital closing date (june 13), we defined the early phase of the outbreak (before june 13) as phase 1 (two tracheostomies) and the middle phase of the outbreak (after june 13) as phase 2 (seven tracheostomies). [10, 11] one covid-19 patient who had undergone surgical tracheostomy at our institution was also included in this study. for mers-cov and sars-cov-2 pcr tests, either sputum or nasopharyngeal swab samples were collected using a sterile, leak-proof, screw-capped sputum collection container and nasopharyngeal swabs were collected with an eswab (482 c, copan diagnostics inc., murrieta, ca, usa). mers samples were tested by rrt-pcr with amplification targeting the upstream e region (upe) and confirmed by subsequent amplification of the open reading frame (orf)1a using powercheck™ mers real-time pcr kits (kogene biotech, seoul, korea). [9] covid-19 samples were screened by rrt-pcr with amplification targeting the envelope gene (e) and confirmed by subsequent amplification of the rna-dependent rna polymerase gene (rdrp) using powercheck™ sars-cov-2 real-time pcr kits (kogene biotech, seoul, korea). for serologic surveillance, we used commercial anti-mers-cov enzyme-linked immunosorbent assay (elisa) igg kits (euroimmun, lübeck, germany) to detect antibody response. we used automated fluorescent immunoassay system (afias) covid-19 ab assay kit for sars-cov-2 antibody detection (bodi-tech med inc., chuncheon, korea). the perioperative tracheostomy protocol for mers and covid-19 patients was developed and revised through multidisciplinary discussions led by our in-hospital infection control team during the mers and covid-19 outbreaks. a multidisciplinary discussion among icu, ent and infection control departments is essential in the decision to perform tracheostomy in an infected patient. when a tracheostomy was planned for a patient with mers, an open surgical tracheostomy was preferred to a percutaneous dilatational tracheostomy (pdt) due to decreased potential for aerosolization. thirteen patients with mers were admitted to the icu, and nine (69.2%) of them required surgical tracheostomy. tracheostomy was necessary is one of the seven patients with covid-19 in our hospital. surgical tracheostomy was also performed in this case not only because the open surgical tracheostomy is considered lower risk in terms of aerosol-generation compared to pdt, but also because a high-riding brachiocephalic (innominate) artery was noted on preoperative computed tomography (ct). thus, preoperative evaluation of neck anatomy is also important to determine the optimal procedure and reduce surgical complications. level of personal protective equipment (ppe) during tracheostomy during phase 1 of the mers outbreak (before june 13), two surgical tracheostomies were performed and standard personal protective equipment (ppe) comprising surgical gloves, surgical gowns, eye shields, and n95 respirators was used by health care workers on the tracheostomy teams. there was no tracheostomy-related mers transmission with this level of ppe, suggesting that standard ppe without papr could be appropriate depending on the situation. however, there were four cases of mers in healthcare workers involved in other procedures in patients with high viral loads (sputum pcr cycle threshold value <16) despite use of this level of ppe. as a result, the infection control department at our institution increased the level of recommended protection, and all members of the tracheostomy team used enhanced ppe, which included coverall clothes including a head cover, shoe covers, two pairs of surgical gloves, powered air purifying respirators (paprs) and n95 respirators. in addition to enhanced ppe, primary surgeons and surgical assistants used an outer surgical gown and gloves, resulting in double gowning and triple gloving. all members of the tracheostomy team remained free of disease, during and after performing a total of nine tracheostomies for patients with mers, suggesting these protections were successful and safe. thus, enhanced ppe including papr was also used with the patient with covid-19 (cycle thresholds 30.5 for e gene and 30.44 for rdrp gene from trans-tracheal aspirates) (supplementary figure 1) and there was no perioperative covid-19 transmission ( table 1) . as strict donning and doffing procedures are crucial to prevent operator contamination, institutional training, and education on the proper use of ppe was provided to the surgical teams before they cared for covid-19 patients ( figure 2 ). on the day of tracheostomy, surgical teams were carefully assisted and closely supervised by skilled nurses in the designated donning and doffing location in the icu ( figures 3a and 3b ). during the mers outbreak, we had no permanent negative-pressure icu rooms, and two patients inevitably underwent surgical tracheostomy in an isolated icu created for mers patients. because a negative pressure icu is ideal for surgical procedures to minimize airborne viral spread, isolated icus were temporarily converted to comprise negative-pressure icu rooms to facilitate performing surgical procedures in mers patients. [11] we performed seven surgical tracheostomies on patients with mers after this icu conversion was completed. based on lessons learned from the 2015 mers outbreak, two negative pressure icus with anterooms and 15 negative pressure isolation wards were separately constructed outside the main hospital in 2016. during the covid-19 pandemic, at the request of the government, a critically ill covid-19 patient with prolonged intubation was transferred directly to the negative-pressure icu at our hospital in march 2020. one week later, surgical tracheostomy was performed at the bedside in the icu in a negative-pressure room. our institution could not limit the number of team members involved in the tracheostomy procedure and post-operative management at the time of the mers outbreak. two surgeons comprising a primary surgeon and surgical assistant took turns with the icu specialist assisted by a standby nurse in performing tracheostomies. in contrast, the surgical tracheostomy for the covid-19 patient was performed by one dedicated head and neck surgeon and icu medical staff (two intensivists and one senior nurse), who worked only in the negative pressure room for covid-19, and assisted with all procedures (supplementary figure 1) . general principles for minimizing aerosolization and surgery time were applied during the tracheostomies. these included complete paralysis to prevent cough and movement, lower positioning, and hyper-inflation of the endotracheal tube cuff, holding ventilation before tracheal incision, and prompt cannula insertion and cuff inflation while withdrawing the endotracheal tube to just above the window. [12] [13] [14] [15] [16] performing a tracheostomy with enhanced ppe was not easy. enhanced ppe limited manual tactile sensation (multiple gloves), free surgical motion (double gowns), illumination and visualization. thus, we typically made a relatively wide incision (4-5 cm) to ensure a clear surgical field and visualization even if additional skin sutures were needed at the end of the procedure. a surgical light was also required for optimal visualization during the procedure. a wearable headlight or headlamp was used in all cases. however, the headlight did not fit a surgeon's head because of the enhanced ppe head cover. instead, surgical assistants (first and second) wore the headlamp and were in charge of illuminating the surgical field ( figure 3e ). different from many recommendations for avoiding diathermy and suction, we generally used electrical devices including bipolar and monopolar diathermy for hemostasis and to save time and we did not limit suctioning throughout the surgical tracheostomy procedure ( figure 3d ). nevertheless, there was no transmission caused by using diathermy and suction, suggesting that the possibility of transmission through diathermy producing vapor plumes or suction-related aerosolization is extremely low in the setting of enhanced ppe in a negative pressure room. we did not place stay sutures or a björk flap for any of the mers or covid-19 patients. instead, we made an oval-shaped tracheal window by removing the tracheal cartilage, which prevented forceful insertion and avoided tracheal damage or false passage. we prepared various sized non-fenestrated cuffed tubes and adjustable tubes on the surgical table to reduce the possibility of a poorly fitted cannula. portex ® "vocalaid" cuffed blue line ® tracheostomy tubes (id 7.5) were used in six mers patients and vocal aid cuffed mera ® sofit clear tubes (id 7.5) were used in two mers patients. a portex ® "vocalaid" cuffed blue line® tracheostomy tube (id 7.0) was used in the covid-19 patient. these were no accidental decannulation events. after tracheostomy and the associated procedures (e.g., tube insertion, balloon inflation, circuit connection, ventilation resumption and endotracheal removal), peristomal dressing and skin suture using 4-0 vicryl (absorbable) performed to minimize the need for tube and dressing changes ( figure 3e ). during the mers outbreak, the tracheostomy wound was dressed daily by trained icu nurses with enhanced ppe. a tracheostomy tube change was performed three days after the operation, and a subsequent change was performed ten days postoperatively by ent surgeons wearing enhanced ppe. there were no cannula-related complications, including stomal infection and cannula occlusion with a mucous plug (table 1) . we subsequently revised the tube management protocols based on other guidelines and experience in our icu system. these revisions included no dressing changes unless there were signs of infection and delaying the first tube change until covid-19 patients tested negative for viral rna. the first cannula change for the covid-19 patient was performed by the same surgeon with enhanced ppe at 13 days because that patient had three consecutive negative sars-cov-2 pcr tests 11 days after tracheostomy. the stoma site and tube lumen were noted to be clean despite the delay. the patients stayed in the negative-pressure icu for an additional three weeks to minimize the risk of nosocomial transmission, and was then transferred to an isolated icu, where decannulation without down-sizing and corking were performed four days after transfer. the patient was transferred to the general ward seven days after decannulation. during the mers outbreak, health care workers involved in tracheostomy and related procedures continued to work with monitoring and were removed immediately from duty if symptoms developed. however, at the end of the mers outbreak in our hospital, all healthcare workers who participated in procedures for the last mers patient were placed in home quarantine for 14 days from the last day of exposure and their sputum was tested by rrt-pcr as a screening test before they returned to general patient care. the pcr results for all associated staff were negative and serologic testing for mers-cov antibody was also negative. [17] during the covid-19 pandemic, all members of the team who participated in tracheostomy for the covid-19 patient were put under active monitoring (checking temperature and symptoms twice a day) while working (table 1) . at the end of patient care, icu staff were also placed on seven days of home quarantine and underwent screening by sputum rrt-pcr, and additional pcr screening was performed before they returned to work. the pcr results were all negative. although there was no pcr screening and no quarantine for the primary surgeon, serologic testing was negative for the anti-sars-cov-2 antibody. several studies related to guidelines or recommendations on surgical tracheostomy for covid-19 patients have been published. however, the detailed context of the procedure seems inconsistent and varies by the developing group, specialty, hospital and national health care systems. there is a limited number of protocols or recommendations based on real experience on this topic. fortunately, we have clinical experience with tracheostomies for both mers and covid-19 patients, and we thought it would be helpful to share our experience and protocol with readers. there has been a debate on whether pdt spreads more virus-containing aerosols than surgical tracheostomy. surgical tracheostomy is usually recommended over pdt in most guidelines. [14] [15] [16] 18] preoperative evaluation of individual anatomy and patient functional status is critical. this includes particular attention to anatomical variations (a high-riding major artery in our case), obesity, un-extended or short neck, bleeding tendency, or ventilator dependency. in addition to the possibility of aerosol dissemination, surgeons should consider these factors in determining the most appropriate tracheostomy procedure and to reduce surgical complications. some guidelines recommend a double-lumen cannula comprising a non-fenestrated cuffed outer with a disposable inner cannula. [16] however, the interface between the inner and outer cannulas can vary by manufacturer and ventilation setting, thereby increasing the chance of air leakage. [19] furthermore, double lumen cannulas tend to be rigid, which can cause mucosal irritation or injury. thus, we prefer to use single lumen non-fenestrated cuffed tubes with or without an adjustable function. this minimizes the risk of viral transmission through air leakage, particularly for infected patients receiving positive pressure ventilation. b virus (hbv) have reported that the plume originating from diathermy contains viable infectious particles that can be transmitted to the upper respiratory tract through inhalation of surgical smoke. [20, 21] in this context, some guidelines recommend avoiding or limiting the use of electrocautery to reduce exposure to the surgical plume. [14] [15] [16] however, although the possibility of disease transmission through electrocautery-induced surgical plumes has been recognized, only hpv transmission has been reported in rare cases [22] ; no prior study has demonstrated that brief exposure to electrosurgical smoke alone causes viral infection. there has been no evidence to indicate that covid-19 is transmissible through surgical plumes. [23] additionally, one study reported that none of the blood samples from covid-19 patients tested positive for rna from sars-cov-2, suggesting that the virus may not be present within the smoke produced by electrocautery. [24] consistent with our study, 10 surgical tracheostomies for covid-19 patients were preformed using an electrocautery device without any cases of transmission in a recent study. [25] therefore, we consider the clinical benefits of electrocautery, including reduced operation time, surgical view, and easy bleeding control, to exceed the risk of potential viral transmission. aerosol-generating procedures have highlighted the risk of nosocomial transmission of emerging viruses such as sars-cov. [26] many medical procedures including bronchoscopy, cardiopulmonary resuscitation (cpr), ventilation, surgery, nebulizers, and suction have been considered potential aerosol-generating procedures. based on these findings, use of suction during tracheostomy is not recommended in recent guidelines. during the sars-cov outbreak, only direct airway-stimulating procedures such as bronchoscopy, cpr, ventilation, and intubation have been reported to be potentially associated with sars-cov transmission. [27] [28] [29] during surgical tracheostomy, exposure of the tracheal lumen is very short and suction can be used to evacuate the diathermy-producing plume. furthermore, enhanced ppe in a negative pressure room minimizes exposure to aerosols and electrocautery-inducing smoke. therefore, we did not limit suction or diathermy in our institutional tracheostomy protocol for mers and covid-19 patients. complete hemostasis achieved by electrocautery and suction of blood or sputum in surgical fields could contribute to rapid and safe tracheostomy with fewer complications. a stay suture technique, suturing the anterior tracheal wall to the skin after making a tracheal window, facilitates insertion and prevents false passage in accidental decannulation. placing stay sutures or making a björk flap may lead to direct exposure to tracheal secretions through an opened tracheal window in infected patients, thereby increasing the chance of viral particle transmission. thus, we did not use a stay suture or björk flap during surgical tracheostomy in mers and covid-19 patients. instead, we made a round opening on the tracheal cartilage directly beneath the skin wound. fortunately, our patients did not suffer from false lumen formation or accidental decannulation, even without the stay sutures. one of the major modifications in the covid-19 tracheostomy protocol at our institution was postoperative management including dressing and cannula changes. during the mers outbreak, there was no difference in cannula dressing and change intervals between infected and non-infected cases. in preparing the covid-19 tracheostomy protocol, we agreed that daily cannula dressing seems unnecessary and the first cannula change can be delayed until the patient no longer tests positive. additionally, delaying the tube change allows maturation of the skin-to-trachea tract to avoid false passage without a suture or björk flap. our data and recent reports revealed that the rate of negative conversion within 21 days was 91.2% [30] and the median time from onset of symptoms to mechanical ventilation was 10.5 days in covid-19 patients. [6] thus, the modified time to cannula change should be within 14 days after tracheostomy. in our patient, the first tracheostomy cannula change was on postoperative day 13, which was two days after the patient had three negative tests. ultimately, decannulation was possible on day 28 after the first cannula change without any complications. decannulation is a critical process for weaning patients from the tracheostomy. [31] however, the process includes many aerosol-generating procedures, such as down-sizing, cannula type changes, balloon deflation, airway evaluation, active coughing to prevent aspiration, and repeated capping/uncapping. thus, we chose the abrupt tube removal method for covid-19 patients to decrease the potential risk of exposures. in response to reports of multiple cases testing positive for sars-cov-2 after having recovered, the patient stayed for an additional seven days in an isolated icu for close monitoring and to allow the stoma to seal, but this later proved unnecessary as no evidence has suggested that re-positive cases are infective. another stark difference in our revised protocol is the creation of a designated covid tracheostomy team comprised of one highly experienced head and neck surgeon, two attending icu specialist (one to manage ventilator/endotracheal tube, one to assist with the procedures) and a senior icu nurse. during the mers outbreak in 2015, we had to perform eight mersrelated tracheostomies in a short period between june 15 and june 29 without a dedicated team because of limited resources at our institution. as our institution is a tertiary referral center, we are prepared to care for severe cases of covid-19 requiring intensive medical support. thus, we were able to focus on critically ill covid-19 patients by preparing medical resources and creating a dedicated team in advance, without any limitations to accessibility or safety for non-covid-19 patients (figure 2 ). however, if team members in the icu need to be kept to the minimum critical number, an additional icu nurse could be omitted from the tracheostomy team. therefore, the optimal number and composition of covid-19 tracheostomy teams could vary depending on the medical resources available for each center, region, and country. in addition, we prepared a highly organized infection control system including a negative pressure icu with double anterooms and a validated screening strategy for healthcare workers. as shown in figure 3a , designated space in a negative pressure icu was created for procedures to minimize potential risk of exposures. it consisted of space for donning ppe and material equipment, one anteroom for entering, a second anteroom for doffing ppe, and a fitting and shower room for personnel protection. every step was guided and supervised by a senior icu nurse ( figure 3a-e) . we also confirmed the appropriateness of our screening and monitoring strategy (active monitoring and quarantine followed by sputum rrt-pcr) for involved healthcare workers by serologic investigation after the end of the mers outbreak, in which none of the tested sera were positive for mers-cov antibody. [17] these screening protocols were applied to assigned icu staff (icu specialists and nurses) in the covid-19 pandemic. however, pcr screening and quarantine for the primary surgeon was omitted as they wear enhanced ppe and are exposed only for a short period of time during the tracheostomy procedure and first cannula change. we had no transmission among healthcare workers who used enhanced ppe during the mers outbreak. [10] serum collected from the primary surgeon was negative for anti-sars-cov-2 antibody at the end of our hospital's care of covid-19 patients, implying that our screening protocol based on clinical situation is effective and practical. these facilities and screening systems for covid-19 allowed for all associated medical staff to continue their routine clinical work and daily life. to date, we have no cases of transmission from covid-19 patients to healthcare workers. here we presented our experience with tracheostomy in patients with mers and covid-19. the covid-19 pandemic has escalated and poses a global threat, therefore most hospitals should prepare for performing tracheostomy and perioperative management in patients with covid-19. our modified protocol and experience from the mers outbreak and covid-19 pandemic could serve as one reference to inform the design of protocols unique to other institutions' own covid-19 situation. there are no conflicts of interest. figures figure 1 . cross-sectional ct image of a covid-19 patient with tracheostomy. ct scans showed a high-riding innominate artery to the right of the trachea just below the thyroid. supplementary figure 1 . dedicated team for covid-19 tracheostomy. tracheostomy was performed by one experienced head and neck surgeon and two attending intensivists (one to the manage the ventilator/endotracheal tube, one to assist with the procedure). one icu nurse assisted with the procedure outside the surgical field. all team members used powered air purifying respirators (paprs). table 1 . details of tracheostomies for mers and covid-19 patients. phase 1 # phase 2 # no. of tracheostomies performing tracheostomy and perioperative management in patients with covid-19 should be based on real experience and the best available evidence on this topic. our protocol allowed for all associated healthcare workers to continue their routine clinical work and daily life. our protocol guaranteed safe return to general patient care without any related complications or nosocomial transmission during the mers and covid-19 outbreaks. coronavirus disease (covid-19) pandemic korean center for disease control and prevention. coronavirus disease-19 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention summary of probable sars cases with onset of illness from 1 middle east respiratory syndrome coronavirus (mers-cov) clinical features of patients infected with 2019 novel coronavirus in wuhan early versus late tracheostomy for critically ill patients timing of tracheostomy in patients with prolonged endotracheal intubation: a systematic review mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea infection prevention measures for surgical procedures during a middle east respiratory syndrome outbreak in a tertiary care hospital in south korea safe tracheostomy for patients with severe acute respiratory syndrome elective and emergency surgery in patients with severe acute respiratory syndrome (sars) corona-steps for tracheotomy in covid-19 patients: a staff-safe method for airway management surgical considerations for tracheostomy during the covid-19 pandemic: lessons learned from the severe acute respiratory syndrome outbreak a framework for open tracheostomy in covid-19 patients serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak elective tracheostomy in mechanically ventilated patients affected by covid-19: preliminary case-series from lombardy leakage characteristics of dual-cannula fenestrated tracheostomy tubes during positive pressure ventilation: a bench study surgical plume and its implications: a review of the risk and barriers to a safe work place the dangers of electrosurgical smoke to operating room personnel: a review surgical smoke exposure in operating room personnel: a review safe management of surgical smoke in the age of covid-19 virological assessment of hospitalized patients with covid-2019 safety and prognosis in percutaneous vs surgical tracheostomy in 27 patients with covid-19 nosocomial transmission of emerging viruses via aerosol-generating medical procedures aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review transmission of severe acute respiratory syndrome during intubation and mechanical ventilation possible sars coronavirus transmission during cardiopulmonary resuscitation factors associated with negative conversion of viral rna in patients hospitalized with covid-19 the practice of tracheostomy decannulation-a systematic review none sung yong choi a , joongbo shin a , woori park a , nayeon choi a , jong sei kim a , chan i choi key: cord-327863-6cw9f7qu authors: majumder, maimuna s.; kluberg, sheryl a.; mekaru, sumiko r.; brownstein, john s. title: mortality risk factors for middle east respiratory syndrome outbreak, south korea, 2015 date: 2015-11-17 journal: emerg infect dis doi: 10.3201/eid2111.151231 sha: doc_id: 327863 cord_uid: 6cw9f7qu as of july 15, 2015, the south korean ministry of health and welfare had reported 186 case-patients with middle east respiratory syndrome in south korea. for 159 case-patients with known outcomes and complete case histories, we found that older age and preexisting concurrent health conditions were risk factors for death. five potential covariates were analyzed: sex, age, concurrent health condition status, health care worker status, and time from onset to diagnosis. for time-to-event analyses, patients were categorized into outbreak weeks by date of onset. we tested the cox proportional hazards assumption by using schoenfeld residuals and included an interaction term for predictor and follow-up time. of the 159 case-patients analyzed, 94 (59%) were men. per who definitions, 25 (16%) had concurrent health conditions and 22 (14%) were health care workers. age was normally distributed (range 16-87 years, mean [sd] 55 [15.9] years). all deaths occurred in patients >48 years of age. time from onset to diagnosis was positively skewed: median 4 days (interquartile range [iqr] 2-7 days). median time from diagnosis to death and from diagnosis to discharge were 13 (iqr 17-25.3) and 22 (iqr 9-16.5) days, respectively. as of july 15, a total of 35/159 cases analyzed were considered fatal, which yielded an estimated case-fatality rate (cfr) of 22%. univariate logistic regression models for each risk factor showed that older age and having a concurrent health condition were associated with death (both p<0.001); both variables remained significant after we adjusted for all 5 variables in a multivariate logistic regression model (table) . the model estimated that odds of dying were 7 times higher for persons with concurrent health conditions than for persons without these conditions (odds ratio 7.14, 95% ci 2.27-22.41). furthermore, for every 1-year increase in age, odds of dying increased by 12% (odds ratio 1.12, 95% ci 1.07-1.17). time from onset to diagnosis decreased from a median of 10 days during outbreak week 2 (iqr 8.0-12.0 days) to 2 days during week 7 (iqr 1.0-2.0 days). there was a 43.7% average increase in hazard of diagnosis per week by a univariate cox proportional hazards model (p<0.001). separate univariate cox models showed that no recorded risk factors were associated with this change. time from onset to discharge for patients who survived decreased from a median of 27 days during outbreak week 2 (iqr 22.0-32.0 days) to 19 days during week 7 (iqr 17.0-23.0). univariate cox proportional hazards analyses estimated a 34% average increase in the hazard of discharge per week (p<0.001), a 63% increase for health care workers (p = 0.046), and an 8% decrease for every 1-day increase in time-to-diagnosis. multivariate analysis controlling for all risk factors showed that the increase in author hazard by week decreased to 26% (p = 0.032); no other covariates remained significant. we found that older age and preexisting concurrent health conditions were associated with an increase in odds of death from mers. although being a health care worker appears to be protective, the association is not significant, probably because only 1 health care worker had died of mers as of july 15, 2015. time from onset to diagnosis was not an indicator for death, which suggests that the rapidity with which a patient receives supportive care may be of marginal consequence. similarly, although being a male patient seems to increase odds of death, this relationship was not significant. on the basis of case-patients who had known outcomes through july 15, the ongoing mers outbreak in south korea had an estimated cfr (22%) that was half the cfr (44%) for all known case-patients with mers in saudi arabia (3), but a cfr similar to that calculated for patients with only nonsporadic illness (21%) (4). because 19 (10%) of 186 case-patients reported remain hospitalized, the final cfr of the outbreak might be higher than our current estimate. however, the proportion of patients who died (18%-19%) has been fairly stable since june 27 (figure 2) , which might indicate an asymptotic approach toward the final outbreak-specified cfr (5-7). if so, the final cfr associated with the mers outbreak in south korea during 2015 might be <22%. if all remaining hospitalized case-patients died, the final outbreak cfr would be 29%, which provides an upper limit for our current estimate of 22% excluding additional cases. a total of 16 (64%) case-patients with mers in south korea who had concurrent health conditions died, compared with 19 (14%) case-patients without concurrent health conditions. this finding is comparable to that in a mers study in saudi arabia, which reported a 60% cfr for a study population in which 45 (96%) patients had concurrent health conditions (8) . although only 25 (16%) case-patients had documented concurrent health conditions, the mers outbreak in south korean during 2015 has been largely nosocomial in nature. this finding suggests that observed differences between average cfrs in south korea and saudi arabia might be driven in part by differential rates of concurrent health conditions for susceptible persons. use of publicly available data poses unique challenges. although such data enable preliminary epidemiologic research during an ongoing outbreak, case information is stringently restricted to protect patient privacy. because of this limitation, a follow-up analysis will be conducted pending availability of additional covariate data on potentially relevant biometrics (e.g., blood pressure) and behaviors (e.g., tobacco use), as well as outcomes for patients still hospitalized. despite these limitations, we found that risk factors for death among patients with mers in south korea who had known outcomes (age and concurrent health conditions) were similar to those identified for mers case-patients in saudi arabia (8) (9) (10) . given these epidemiologic similarities and assuming that inherent virulence of mers coronavirus is not context specific, the cfr difference might be caused not only by differential prevalence of risk factors but also by treatment or surveillance disparities. time to diagnosis decreased during the first 7 outbreak weeks, which probably contributed to the reduced length of hospitalization for patients who recovered and indicates that supportive care in south korea might be highly adaptive. furthermore, as reported by cowling et al. (11) , intensive case-finding activities might have produced more comprehensive diagnosis and reporting, thereby capturing less severe cases. in either event, given the frequency of importation events (12) and propensity for super-spreading (13), these findings provide information about mers and mers coronavirus in south korea that might be useful in improving early case detection and preventing death. south korea ministry of health and welfare. press releases world health organization. mers-cov cases in the republic of korea as of 6/26/2015 saudi arabia ministry of health. moh: 1 new confirmed corona cases recorded middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility early epidemiological assessment of the virulence of emerging infectious diseases: a case study of an influenza pandemic severe acute respiratory syndrome: temporal stability and geographic variation in case-fatality rates and doubling times methods for estimating the case fatality ratio for a novel, emerging infectious disease epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus preliminary epidemiological assessment of mers-cov outbreak in south korea global map of countries with confirmed cases of mers-cov the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission this study was supported by the national library of medicine, national institutes of health (grant r01lm010812).ms. majumder is an engineering systems doctoral student at the massachusetts institute of technology, cambridge, massachusetts, and computational epidemiology research fellow at boston children's hospital, boston, massachusetts.her research interests involve probabilistic modeling, bayesian statistics, and systems epidemiology in the context of emerging infectious diseases. key: cord-312038-g76cpjp7 authors: brunaugh, ashlee d.; seo, hyojong; warnken, zachary; ding, li; seo, sang heui; smyth, hugh d.c. title: broad-spectrum, patient-adaptable inhaled niclosamide-lysozyme particles are efficacious against coronaviruses in lethal murine infection models date: 2020-10-07 journal: biorxiv doi: 10.1101/2020.09.24.310490 sha: doc_id: 312038 cord_uid: g76cpjp7 niclosamide (nic) has demonstrated promising in vitro antiviral efficacy against sars-cov-2, the causative agent of the covid-19 pandemic. though nic is already fda-approved, the oral formulation produces systemic drug levels that are too low to inhibit sars-cov-2. as an alternative, direct delivery of nic to the respiratory tract as an aerosol could target the primary site of for sars-cov-2 acquisition and spread. we have developed a niclosamide powder suitable for delivery via dry powder inhaler, nebulizer, and nasal spray through the incorporation of human lysozyme (hlys) as a carrier molecule. this novel formulation exhibits potent in vitro and in vivo activity against mers-cov and sars-cov-2 and may protect against methicillin-resistance staphylococcus aureus pneumonia and inflammatory lung damage occurring secondary to cov infections. the suitability of the formulation for all stages of the disease and low-cost development approach will ensure wide-spread utilization in terms of pharmaceutical development, a major limitation of nic as an antiviral therapy stems from its poor solubility in water, which is reported at 1.6 mg/l (30) . this makes systemic absorption of the drug by the oral route of administration at therapeutically relevant concentrations difficult. administration of the existing fda-approved chewable tablet formulation was found to result in inadequate systemic concentrations for inhibiting sars-cov-2 replication. (31, 32) as an alternative, direct delivery of niclosamide to the lung could overcome the limitations of the oral nic formulation by generating high drug concentrations at the site of infection. for use against p. aeruginosa, costabile et al previously developed an inhalable dry powder consisting of nic nanocrystals embedded in mannitol particles. (33) however, these particles required a high amount of polysorbate 80 to ensure production of a stable suspension (10% w/w to nic) which is beyond what is in currently fda approved inhaled products (34). furthermore, the utilization of mannitol as the carrier system may induce bronchospasm and cough (35) (36) (37) which may contribute to increased risk of spread of sars-cov-2 through generation of aerosolized respiratory droplets from infected individuals. an additional considerable challenge in developing therapies for covid-19 is the variable presentation of illness. patients may act as asymptomatic carriers of the virus or develop severe pneumonias and acute respiratory disease (38) , which can result in the requirement for mechanical ventilation. in the case of ventilated patients, a nebulizer is often used to delivery aerosolized drug to the lungs. however, for treatment of asymptomatic carriers or in regions of developing economies with reduced access to clean water sources, nebulizer-based aqueous products may present an undue burden and reduce therapy compliance. for these populations, dry powder inhaler (dpi) or nasal spray systems, or a combination of both, would be the preferred option based upon the rapid administration time and ease of use and could also potentially be utilized as a prophylactic therapy in high risk populations such as healthcare workers and first responders. utilizing repurposed nic, and with the goal of developing a therapeutically effective, rapidly scalable and globally distributable antiviral therapy to reduce the spread of sars-cov-2, we describe an inhalable nic formulation that can be administered using three major models or respiratory tract delivery systems: dpi, nasal spray and nebulizer. to achieve these aims, we utilized human lysozyme (hlys), an endogenous protein in the upper and lower respiratory tracts, as a therapeutically active matrix material for the delivery of nic to the airways, based upon its known anti-inflammatory (39) , antiviral (40) (41) (42) (43) , and anti-bacterial activity (44) (45) (46) as well as its surface active properties. the antiviral, antibacterial, and anti-inflammatory efficacy of the nic-hlys powders were evaluated in vitro and in vivo in mers-cov and sars-cov-2 infection mouse models. the composition of the nic-hlys formulation was optimized using a constrained mixtures design of experiments (doe) achieve particles with a size appropriate for inhalation both in the dry powder state and when reconstituted in aqueous media. physicochemical characterization of the optimized powder was performed and nasal, dpi, and nebulizer systems were developed and tested. to determine the utility of hlys as a carrier molecule for the nasal and pulmonary delivery of nic, we assessed the in vitro antiviral activity of our novel formulation against a lysozyme-free nic suspension (nic-m). nic-hlys particles (0.7% w/w nic) were administered at varying doses (based upon nic content) to vero e6 cells infected with mers-cov or sars-cov-2, and the ec50 was calculated based upon observed cytopathic effect (cpe). the addition of hlys to the nic formulation resulted in improved antiviral activity based upon reductions in the ec50 dose for mers-cov (0.016 µg/ml nic to 0.0625 µg/ml nic) and sars-cov-2 (0.030 µg/ml to 0.008 µg/ml) (fig 1a and 1b) . we assessed the antiviral dose-response of nic-hlys particles in separate assay utilizing qpcr quantification of viral rna collected from infected cells. at the highest dose tested (0.125 µg/ml nic), vero cells with an established mers-cov infection exhibited an 82.2% ± 0.8% decrease in viral load compared to untreated controls after 24-hours of exposure to nic-hlys particles ( fig 1d) . this effect was sustained at 48 hours post-drug exposure (92.7% ± 2.0%; mean ± sd). a similar dose-response profile was achieved in vero cells with an established sars-cov-2 infection ( fig 1e) . a dose 0.125 µg/ml nic resulted in a 24-hour inhibition of 92.7% ± 0.5% relative to untreated controls. however, in contrast to the mers-cov response, inhibition dropped to 67.5% ± 2.3% at 48 hours post-exposure in the sars-cov-2 infected cells. a separately conducted trypan blue viability assay in uninfected vero e6 cells determined that the highest dose of nic-hlys utilized (0.125 µg/ml) had no effect on cell viability versus untreated controls (98.3% viability in treated cells). interestingly, hlys alone appeared to exhibit activity against sars-cov-2 (fig 1f) , which has not been previously reported. though the inhibitory activity was not as potent as that of nic, it may contribute to observed increase in potency of nic-hlys in the cpe-based ec50 assay. nic-hlys, nic-m and nic dissolved in dmso (nic-dmso) were compared for their inhibitory activity against sars-cov-2 at a nic dose of 0.125 µg/ml. a two-way anova with tukey multiple comparisons test revealed statistically significant increase in viral inhibition of the nic-hlys particles versus nic-dmso at 24 hours (p < 0.0001) and 48 hours (p = 0.0001), though no significant differences were noted in the viral inhibition of nic-hlys and nic-m formulations at the tested dose ( fig 1c) , thus, an improvement in solubility does not appear to be the mechanism for the increased activity of nic-hlys. , where an initial drop in activity is preceded by a sharp rise in activity. this profile was also noted in inhibitory assays for s. aureus. hlys alone exhibits some antiviral activity which was more pronounced at 48 hours post dosing than 24 hours (f). data presented as mean + sem (n = 3). ***p < 0.001, ****p < 0.0001 using two-way anova with tukey's multiple comparisons test. the in vivo efficacy of nic-hlys particles was assessed in lethal infection models for both mers-cov and sars-cov-2. hddp4 transgenic mice were inoculated intranasally with 1 × 10 5 pfu mers-cov and rested for 24 hours, after which once daily treatment with varying doses of intranasal nic-hlys (dosed based nic component) was initiated (fig 2a) . in this initial efficacy study, animals were sacrificed at day 6 to determine viral titres in brain and lung tissue compared to untreated controls. notable decreases in brain viral titres was observed at the 120 µg/kg dose although these differences were not statistically significant (two-way anova with dunnet's multiple comparisons test, p = 0.1724) (fig 2c) . in a follow-up survival study utilizing lethal inoculum (1 × 10 5 pfu), mers-cov-infected hddp4 mice were dosed at 240 µg/kg nic-hlys daily by the intranasal route. by day 10 (study endpoint), 43% of treated mice had survived compared to 0% of untreated controls (fig 2b) . surviving mice were left untreated for an additional 3 days, at which point they were sacrificed. during this period of no treatment, the survival percentage remained at 43%. a statistically significant decrease in lung viral titres was noted in these surviving mice compared to the day 6 untreated controls from the earlier efficacy study (two-way anova with dunnett's multiple comparisons test, p = 0.011,). while brain viral titres did not exhibit further reduction from levels noted in the preliminary efficacy study, the inoculation of vero e6 cells with viral particles obtained from lung and brain homogenates of surviving animals resulted in no observation of cpe at any of the inoculum concentrations tested, which indicates that remaining viral particles were not active. thus, in the 43% of surviving animals it appears that the lethal mers-cov infection was essentially cured. serological assays revealed that surviving animals expressed anti-mers cov igg antibodies. though notable in a lethal infection model, survival probability differences in treated and untreated mice were not statistically significant (mantel-cox test, p = 0.2984). in a similar study, the efficacy of nic-hlys particles was assessed in hace2 transgenic mice infected intranasally with a lethal dose of sars-cov-2 (1 × 10 4 pfu) (fig. 3a) . after a 24-hour rest period, daily intranasal treatment with 0.9% sodium chloride (untreated control) or nic-hlys (240 µg/kg nic) was initiated. a portion of mice from each group (n = 3) were sacrificed at day 6 p.i. to determine viral loads in brain, kidney and lung tissues, while remaining animals were utilized in a 10-day survival study. non-significant changes in day 6 viral titers were observed (two-way anova with dunnet's multiple comparisons test) (fig. 3c) . by the day 10 study endpoint, 30% of treated mice had survived, compared to 0% in the untreated arm ( fig 3b) . similar to the mers-cov study, these surviving mice were left untreated for an additional 3 days, at which point they were sacrificed (day 14 p.i.). the surviving mice exhibited a statistically significant decrease in viral loads in lung tissue compared to day 6 p.i. untreated controls (two-way anova with dunnett's multiple comparisons test), p = 0.023,), and no virus particles were detected in brain and kidney tissue using qpcr (fig. 3c ). inoculation of vero e6 cells with 10-fold dilutions of tissue homogenates resulted in no observed cpe, and surviving animals were sera positive for anti-sars-cov-2 antibodies. similar to the mers-cov model, differences in survival probability between treated and untreated were not significant (mantel-cox test, p = 0.2959). in both mers-cov and sars-cov-2 infection models, the lung tissue of infected and nic-hlys-treated mice ( fig. 2f and 3f) showed lower levels of interstitial pneumonia than that of infected and non-treated mice ( fig. 2e and 3e) on day 6 p.i.. inflammation was further reduced in the treated/infected groups on day 14 p.i. (fig. 2f and 3f ) and was more comparable to the mock-infected mice ( fig. 2d and 3d ), which showed no signs of interstitial pneumonia. . the viral particles obtained from lung and brain homogenates of surviving animals did not produce cpe when administered to vero e6 cells, indicated that they were no longer active. compared to lung tissue of uninfected mice (d), infection with sars-cov-2 resulted in the development of interstitial pneumonia without treatment (e). by day 6 of treatment with 240 µg/kg nic, interstitial pneumonia was notably reduced (f) and further resolved by day 14 (f) . data are presented as mean + sem (n = 3). *p < 0.05, using two-way anova with dunnet's multiple comparisons test. covid-19 patients may be at-risk for secondary bacterial pneumonias and severe inflammatory lung damage. the efficacy of nic-hlys in the treatment of these important covid-19 sequalae was therefore assessed. a resazurin-based microbroth dilution assay was performed to determine the inhibitory activity of several nic formulations (nic-hlys, nic-bsa, nic-m, and nic-dmso). compared to the other nic formulations, nic-hlys reached an mic50 at lower levels of nic (0.0625 µg/ml), and 100% inhibition was noted at a concentration of 0.125 µg/ml (fig 4a-c) . no inhibitory activity was observed for hlys alone. interestingly, all nic formulations exhibited a similar dose-response profile where a sharp dip in activity preceded the concentrations at which 100% inhibition was achieved. this same pattern was also noted in the dose-response profiles for anti-mers-cov and anti-sars-cov-2 activity (fig 1d and e) . plating of the wells with 100% inhibition noted resulted in the growth of colonies at all drug concentrations tested, which indicates that the antimicrobial activity of nic may be bacteriostatic rather than bactericidal. a feared consequence of sars-cov-2 infection is the development of ards, which is a major contributor to morbidity and mortality and dramatically increases the burden on healthcare systems. ards is caused by the massive release of inflammatory cytokines in the lungs, which occurs in some patients in response to pathogenic infiltration. both nic and hlys are known to exhibit anti-inflammatory activity. the anti-inflammatory activity of two nic formulations, nic-bsa and nic-hlys, was assessed using an acute macrophage inflammation model. nic-hlys and nic-bsa exhibited similar suppression of the inflammatory cytokines il-6 and tnf-a ( fig 4d and e) . the similarities in the levels of these two cytokines across the two formulations tested indicates that the suppression may be related to the activity of nic rather than hlys. suppression of il-1b was not observed for either formulation, and the highest concentration of nic-hlys tested (18 µg/ml total powder) resulted in statistically significant increase in compared to both the untreated, lps-stimulated control and an equivalent dose of the nic-bsa formulation ( fig 4f) . of, note this is an equivalent powder concentration to the dose exhibiting the highest efficacy in the in vitro antiviral assays. . data presented as mean + sem (n = 6), **p < 0.01,****p < 0.0001, using two-way anova with dunnet's multiple comparisons test. nic-hlys significantly reduced production of the inflammatory cytokines il-6 (d) and tnf-a (g) in thp-1 macrophages stimulated with 10 ng/ml lipopolysaccharide (lps), though a significant increase in il-1b production was noted at the highest dose tested compared to both the untreated control and nic-bsa formulation, which may point towards the role of hlys in inducing production of this cytokine. data presented as mean + sem (n =3), *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001 using one-way anova with sidak's multiple comparisons test. targeted delivery of antivirals to the respiratory tract carries substantive benefits for the treatment of covid-19, particularly for compounds with limited oral bioavailability like nic. however, the wide range of symptoms and disease severity associated with covid-19 makes development of a broadly applicable therapy difficult. the clinical applicability of our novel nic-hlys formulation was assessed by determining the delivery efficiency of the drug using three commercially available respiratory drug delivery platforms: a disposable dpi (twincaps®), a vibrating mesh nebulizer (aerogen solo®) and a nasal spray (vp7 aptar®). the composition of spray dried nic-hlys particles was optimized using a constrained-mixtures design of experiments (doe) to achieve respirable dry particles (geometric median diameter ≤ 5 µm) that could be easily reconstituted as suspension suitable for nasal spray or nebulizer-based administration. the doe generated several promising powder formulations (supplementary table 1 ), of which formulation 8 (f8) was selected for further characterization. for ambulatory patients, dpis provide a convenient treatment option for lung-targeted delivery. the rapid administration time for the device as well as the compact size improves patient acceptability and compliance. a disposable dpi is likely to be preferred in the treatment of covid-19, given the currently unknown risks regarding re-infectability. a commercially available disposable dpi, the twincaps® (hovione) was therefore selected as an exemplary delivery platform. nic-hlys powder inhalation was successfully delivered using the twincaps dpi, with a 136.0 ± 7.4 µg nic fine particle dose (i.e., recovered drug mass with less than 5 µm aerodynamic diameter) achieved per 60 mg total powder actuation (0.7% nic content) when using inhalation flow rate conditions reflective of a healthy patient (fig 5h) . given the potential for shortness of breath and reduced inspiratory abilities that may occur in covid-19, the delivery efficiency was also examined using inhalation flow-rate conditions reflective of a patient with reduced lung capacity due to illness or age. similar fine particle doses (121.1 ± 7.7 µg) were achieved in these reduced inhalation flow rate conditions, which indicates a minimal dependence on inspiratory flow rate to achieve successful delivery of the drug to the peripheral lung regions. covid-19 may result in the need for mechanical ventilation for continued patient survival. the delivery efficiency of reconstituted nic-hlys particles was assessed using an aerogen solo vibrating mesh nebulizer, which can be utilized aerosol drug delivery in-line with a ventilator circuit. nic-hlys powder reconstituted in 0.45% sodium chloride to a 25 mg/ml concentration (equivalent to 175 µg/ml nic) resulted in the delivery of a fine particle dose of 62.3 ± 6.4 µg nic after a 2-minute run time ( fig 5h) . furthermore, a range of concentrations (10 to 100 mg/ml) could be successfully emitted using the aerogen solo device (fig 5g) . by altering the reconstitution concentration, the dose of nic-hlys could therefore be adjusted if required for pediatric patients, or those with hepatic or renal insufficiencies. the zeta potential of the reconstituted nic-hlys powder was determined to be +1.8, in contrast to the poorly performing nic-bsa particles, which exhibited a zeta potential of -10.9 when reconstituted in water. epithelial cells of the upper respiratory tract (i.e., nasal passages) exhibit significantly higher expression of ace2 receptors than those of the lower respiratory tract, which indicates these tissues may be more prone to infection with sars-cov-2(47). as such, the feasibility of administration of the nic-hlys formulation using a nasal spray was assessed using spray pattern and plume geometry analysis. nic-hlys powders were reconstituted in 0.45% sodium chloride at concentrations ranging from 10 to 50 mg/ml and actuated using a vp7 aptar® nasal spray device. suitable plume angles and uniform spray patterns for nasal administration were achieved for all tested concentrations (fig 5i) . an increase in plume angle was noted at increasing powder concentrations which was inversely related to changes in suspension viscosity (supp table 2 ) and may be reflective of the decreased surface tension resulting from the presence of hlys and other surface-active stabilizers. the utilization of a slightly hypotonic reconstitution medium (supp table 3 ) was selected and used in the in vivo studies to improve absorption and potentially assist nose-to-brain penetration of nic-hlys. the decreases in brain and kidney viral titers noted in vivo when nic-hlys was administered intranasally may reflect an achievement of therapeutic drug concentrations outside the respiratory passageways, though this will be evaluated further in a future pharmacokinetic study. nic is a poorly water-soluble drug, which renders the commercially available oral formulation ineffective against respiratory diseases due to the limited absorption of the drug from the gastrointestinal tract. direct delivery to the airways represents a promising alternative to oral delivery, as it would enable achievement of high levels of drug at the site of disease. however, limited solubility and delayed dissolution of niclosamide particles in the upper respiratory tract could result in rapid clearance of the particles by the mucocilliary escalator or through alveolar macrophage uptake. hlys exhibits surface active properties which could enhance the dissolution rate of poorly water soluble nic particles. to test this hypothesis, the dissolution rate of nic-hlys particles exhibiting an aerodynamic diameter of ~2 µm was compared against hlys-free nic particles in simulated lung fluid medium. though the deposited particles exhibited equivalent sizes and surface areas, the inclusion of hlys as a formulation component resulted in a slightly faster rate of dissolution, with 82.1% the deposited dose of nic dissolved by 6 hours (fig 5f) . . this novel system was developed as an alternative to traditional lactose-based carrier systems (c) and enabled the targeted respiratory delivery of nic as a powder via dpi or a reconstituted suspension via nebulizer or nasal spray. the optimized formulation exhibited a size distribution that was appropriate for inhalation (i.e., geometric median diameter < 5 µm) in both the dry powder state as well as when reconstituted using water or 0.45% nacl (d). similar effects could not be achieved when a negatively charged protein, bovine serum albumin, was substituted in the formulation for the positively charged hlys (e). though hlys is surface active, it appeared to only slightly enhance the dissolution of nic compared to nic particles blended with lactose (f). a respirable droplet size distribution could be achieved with multiple different reconstituted concentrations when nebulized using the aerogen solo (g). these concentrations resulted in no aggregation to the lysozyme component. efficient aerosol delivery was achieved with both the nebulizer and disposable dpi, with ~50% of the emitted dose being of an appropriate size for lung deposition. this was significantly improved compared to a traditional lactose carrier particle system (h). reproducible plume geometry could be achieved using a variety of reconstituted concentrations when actuated using the aptar device (i). data is presented as mean + sem (n = 3). *p < 0.05, using two-way anova with tukey's multiple comparisons test (comparisons of dpis presented only). protein aggregation may result in a loss of therapeutic activity or an immunogenic response (48) (49) (50) . we have previously demonstrated that hen egg white lysozyme is robust to processinduced aggregation using typical particle engineering techniques (51) , which provided part of the rationale for its selection as a therapeutically active carrier for the aerosol delivery of nic particles. using size exclusion chromatography (sec), we investigated the formation of higher molecular weight hlys aggregates before and after spray drying. no further increases in higher molecular weight aggregates were noted after spray drying (supp fig 1b) or after nebulization at varying reconstituted concentrations (supp fig 1ctable 4 ). interestingly, a decrease in the percentage of solubilized aggregates was noted in the spray dried hlys formulations compared to the initial unprocessed hlys product, which may be explained by the shift in secondary structure from towards a higher percentage of parallel b-sheet structure upon spray drying, whereas the unprocessed hlys had a higher percentage of anti-parallel b-sheet structure (supp table 5 ). the glass transition temperature (tg) measured for the nic-hlys powder was 79.4°c, which makes it suitable for storage in ambient conditions without risk of further protein degradation. the water content of the spray dried powder was determined to be 8.8% based upon karl fisher coulometric titration, which is similar to literature reported values for the water content of lysozyme.(52) the co-formulation of the cationic, endogenous protein hlys with micronized nic produced a 4fold increase in potency against covs and a 2-fold increase in potency against mrsa compared to nic particles alone. though the inclusion of hlys did slightly increase the dissolution rate of micronized nic particles, this alone cannot explain the increased potency, as solubilized nic exhibited lower antiviral activity at the 0.125 µg/ml dose than both nic-m and nic-hlys. hlys plays an important role in the innate immune system, and it is found in abundance at the mucosal surfaces of the respiratory tract as well as in the lysosomal granules of neutrophils and macrophages. (53) the antibacterial efficacy of hlys stems from both its enzymatic activity, which results in the hydrolysis of the glycosidic bonds linking peptidoglycan monomers in bacterial cells walls, as well as its cationic activity, which enables insertion of the protein and formation of pores in negatively charged bacterial membranes.(46) to our knowledge, the activity of lysozyme against coronaviruses has not been previously reported. a peptide (hl9) located within the helix-loop-helix motif of hlys has been previously demonstrated to block hiv-1 viral infection and replication, whereas mutants of hl9 did not. (43) the location of this peptide was deemed to be separate from the hydrolytic site. it is possible that this peptide sequence, or another, is responsible for the anti-coronavirus activity of hlys. cationic peptides have been hypothesized to exhibit antiviral activity due to a disruption of the viral particle membrane (54) . hlys may also disrupt various signaling pathways (tgfb, p53, nfkb, protein kinase c, and hedgehog signaling) which affect host cell susceptibility to viral infection. (43) an immunomodulatory response may explain the notable delay in anti-sars-cov-2 activity at lower doses when hlys was used alone. additionally, a unique up-regulation in il-1b activity was noted for the nic-hlys formulation in a macrophage model of inflammatory stimulation, while expression of other inflammatory cytokines (il-6, tnf-a) was decreased. il-1 family cytokines have been previously associated with the induction of antiviral transcriptional responses in fibroblasts and epithelial cells. (55) in sars-cov infected african green monkeys, significantly lower levels of il-1b were noted in the lungs of aged monkeys compared to juvenile monkeys.(56) similarly, elderly mice infected with influenza exhibited lower levels of 1l-1b, and administration of il-1b augmenting compound improved morbidity and mortality.(57) thus, it is possible that a similar immune-mediated mechanism is contributing to the increased activity of nic-hlys compared to other nic formulations, though this requires further investigation. at physiological ph, hlys exhibits a positive charge while nic exhibits a negative charge. this attraction may contribute to the improved dispersibility of nic suspensions when hlys is utilized as the carrier protein, as demonstrated by the notable aggregation observed when a negatively charged protein, bsa, is used in the formulation. this charge-based interaction may have additional benefits when nic is in the solubilized state. nic has two substituted aromatic rings, the electronegativity of which has been deemed critical for its activity against other viruses (9) . non-covalent interactions between aromatic compounds is a known phenomenon, which occurs as a result of the overlap of the p-orbital of the two electron clouds of the aromatic rings (58, 59) and p-p stacking of nic is observed in the crystalline form. (60) though not investigated in this study, it's possible that self-association of nic molecules may disrupt the availability of the strongly electro-negative groups necessary for antiviral activity. this may explain why a decrease in antiviral activity was noted with nic solubilized in dmso compared to nic particles. interactions between nic and hlys in the microenvironment may serve as a mechanism to disrupt this self-association and ensure availability of the electronegative functional groups, as lysozyme has been previously demonstrated to complex with negatively charged molecules (61) and a hydrophobic drug (62) . given the physicochemical properties of nic, both charged-based and hydrophobic interactions with hlys may be important. future studies will examine the interactions and mechanisms of complexation between nic and hlys to further elucidate how the molecular interactions may contribute to improved antiviral activity. at present, there are limited treatment options for covid-19. in august 2020, the fda approved remdesivir for the treatment of all hospitalized adult and pediatric patients with covid-19, irrespective of the severity of disease.(63) this approval is based upon a statistically significant reduction in median time to recovery for patients treated with remdesivir in a recent clinical trial.(64) however, remdesivir is currently not recommended for use in patients with acute or chronic kidney disease (gfr < 30 ml/min), which may limit its utility in severe covid-19 infection. this contraindication stems from the incorporation of sulfobutylether-β-cyclodextrin (sbecd), which is utilized as a vehicle for the poorly water soluble remdesivir in the intravenous product, and which can accumulate in cases of renal failure. clinical trials examining inhalationbased delivery of remdesivir are currently under way(65, 66), though details of this aerosol formulation and whether or not it contains sbecd are not provided. a pre-print of a powder aerosol formulation of remdesivir has also been recently published (67) . these studies overall indicate the clinical interest in the development of an inhalation-based treatment for covid-19. based upon our data, nic may be a promising alternative or adjunt therapy to remdesivir for the treatment of covid-19. a previous study examining the efficacy of remdesivir for the treatment of a lethal mers-cov infection (5 × 10 5 pfu) followed by treatment 24 hours p.i with remdesivir resulted in ~2.5-3 log reduction in viral particles in the lung tissue (68) , which is similar to the reduction noted in our study using intranasal nic-hlys. at the day 6 p.i. endpoint of the remdesivir mers-cov study equivalent survival (50%) was noted in the treated and untreated groups. in comparison, at day 7 p.i., we noted 71.4% survival in treated animals versus 60% in untreated controls, and at day 10 p.i. 43% of treated animals had survived while 0% of untreated animals had survived. notably, these animals continued to survive after treatment was ceased, which was reproduced in the lethal sars-cov-2 infection model. while it is difficult to make comparisons between the two drugs on survival improvement given differences in study length and inoculum concentration, it appears survival with nic-hlys treatment is comparable if not improved compared to remdesivir treatment in lethal mers-cov infections. expansion of the dosing range or frequency in future studies, as well as the incorporation of prophylactic dosing may enable a significant improvement in survival to be achieved, similar to what was noted with remdesivir when used prophylactically in vivo. furthermore, we have demonstrated in vitro efficacy of nic-hlys reducing complications related to covid-19, most notably secondary bacterial pneumonia by mrsa and dampening (but not complete suppression) of tnf-a and il-6, which are known markers for covid-19 disease severity (69) . this broad-spectrum activity may provide a unique advantage for nic-hlys compared to other leading covid-19 candidates as an early report on covid-19 reported 50% of patients that died had a secondary infection (70) . though nic appears to be primarily renally cleared (71) , limited data is available regarding the effects that hepatic or kidney failure may have on the toxicity profile of the drug. future studies will evaluate the pharmacokinetic distribution of nic-hlys delivered via the intranasal or pulmonary routes. we have successfully demonstrated nic-hlys is effective in vitro and in vivo for the treatment of covid-19, and importantly, scalable inhaled drug delivery systems can be developed based upon the formulation which will enable rapid availability to a global patient population. historically, it has been observed that variations in patient inspiratory force can have profound effects on the magnitude and region of dose delivery to the airways from dpis.(72) one of the most common symptoms of covid-19 is shortness of breath, reportedly occurring in 50.8% of patients.(73) moreover, patients with severe infection were significantly more likely to have shortness of breath than patients with non-severe infection.(73) nic-hlys powder exhibited efficient aerosol performance using a disposable dpi device even at reduce inspiratory efforts which will ensure reproducible dose delivery throughout disease progression, as well as in pediatric patients. the powder can be reconstituted at the point of care to form a stable suspension appropriate for nebulization, thus enabling treatment of both ambulatory and severely ill or mechanically ventilated patients. lastly, nic-hlys suspension could be reproducibly actuated using a commercially available nasal spray device and has demonstrated promising antiviral activity in vivo when administered to infected mice via the intranasal route. the utilization of a slightly hypotonic carrier may improve systemic penetration from the nasal route and promote distribution in the brain (74, 75) . sars-cov-2 has been found to exhibit neuroinvasive properties, and severely affected covid-19 patients appear to be more likely to develop neurological symptoms compared to those with mild disease (76, 77) . we observed that the brain tissue of surviving, nic-hlys treated animals with sars-cov-2 infection exhibited no detectable viral particles. nic-hlys delivered by the intranasal route may therefore be effective in preventing viral invasion of brain tissue or reducing viral replication within brain tissue. we acknowledge limitations within our study, namely the lack of pharmacokinetic data which may explain the mechanism of viral load reduction in brain, kidney, and lung tissue, as well as the utilization of only one dosing level of nic-hlys in the sars-cov-2 model. in our study, we utilized a "worst-case scenario" of efficacy evaluation by administering lethal inoculums of covs and initiating treatment 24 hours after the infection was established (78) . the effect of the timing of treatment initiation, i.e., prophylactic versus 24 or 48 hours p.i., was not examined. incorporation of prophylactic dosing may result in the increased efficacy of nic-hlys, as the antiviral mechanism of action of nic may be related to the prevention of viral entry into host cells.(9) future studies will examine these variables, based upon the notable activity of the novel nic-hlys formulation in these initial proof-of-concept efficacy studies. in conclusion, a novel formulation of nic-hlys optimized for delivery to the upper and lower respiratory tracts as a powder or stable suspension was developed. in vitro, the incorporation of hlys into the formulation was noted to improve potency against mers-cov and sars-cov-2, as well as mrsa, which is an important causative agent for secondary bacterial pneumonias associated with covid-19. the nic-hlys particles exhibited suppression of the inflammatory cytokines tnf-a and il-6, which have been implicated in the development of more severe covid-19, while elevating the production of the inflammatory cytokine il-1b, which may contribute to enhanced antiviral activity. intranasal administration of nic-hlys particles improved survival and reduced viral tissue loads in vivo in two lethal cov infections at a level that was comparable to the leading covid-19 treatment candidate, remdesivir. thus, nic-hlys is likely to not only have efficacy in the treatment of the current sars-cov-2 pandemic but could be utilized as a treatment in future cov pandemics. niclosamide (nic) was obtained from shenzhen neconn pharmtechs ltd. (shenzhen, china) and micronized in-house using an model 00 jet-o-mizer air jet mill (fluid energy processing and equipment co, telford, pa, usa) using a grind pressure of 75 psi and a feed pressure of 65 psi for a total of three milling cycles to achieve an x50 diameter of 2.2 µm and an x90 diameter of 4.1 µm. to generate a powder formulation of nic suitable for dpi-based delivery as well as suspension-based nebulizer and nasal spray delivery, micronized nic particles were embedded in a matrix of recombinant human lysozyme (hlys) (invitria, junction city, ks, usa), sucrose (sigma-aldrich, darmstadt, germany), polysorbate 80 (sigma-aldrich) and histidine (sigma-aldrich) using spray drying. our group has previously identified that histidine (buffering agent), sucrose (lyoprotectant agent), and polysorbate 80 (surface active agent) can be used to generate stable and dispersible formulations of lysozyme for delivery via dpi (51, 79) . preliminary screening experiments indicated that spray drying with a feed solid content greater than 1% w/v resulted in a dry particle size distribution (psd) that was greater than the respirable size (typically less than 5 µm particle diameter). a constrained mixtures doe was therefore utilized to determine the optimal ratio of micronized niclosamide, human lysozyme, sucrose, and polysorbate 80 in the 1% w/v feed to generate powder with both a dry and reconstituted psd suitable for oral or nasal inhalation (supplementary table 6 ). the upper constraint of polysorbate 80 was selected to match the maximum fda approved level of the excipient for the inhalation route.(34) the lower level of human lysozyme was set at 60% w/w, based upon previously published results indicating that a respirable powder cannot be generated below this level (80) . sucrose and polysorbate 80 lower level constraints were set at conservative values, as their inclusion is necessary to ensure stability of lysozyme during the drying process, but the lowest level needed for stability has not been defined. the micronized niclosamide lower and upper constraints were selected to be around the point at which the average number of particles is excepted to be one, based upon equation 1 (81) . where ̅ is the average number of particles per droplet, ! is the volume fraction of particles of diameter # , " is the diameter of the particles (assumed to be equivalent to the x90 diameter of the micronized niclosamide particles) and # is the diameter of the droplets (assumed to be equivalent to the x90 diameter of the atomized droplets at conditions used in the experiment). a constrained mixtures doe was generated using the rsm package (82) in the opensource software r (83) . of these 25 mixtures, a 12-run d-optimal subset (supplementary table 7 ) was generated and prepared using spray drying to enable fitting of the results to the scheffé quadratic model. the dry components of the formulations were mixed using a process of geometric dilution and wetted and suspended using polysorbate 80 followed by incremental additions of 0.174 mg/ml histidine buffer. all suspensions were spray dried with a buchi b-290 mini-spray dryer (buchi corporation, new castle, de, usa) coupled to a syringe pump (kd scientific inc, holliston, ma, usa) set at a feed rate of 1 ml/min. a 2-fluid pneumatic atomizer nozzle (0.7 mm with 1.5 mm cap) was used to atomize the suspension, and house air was used as the atomization gas. the cleaning needle of the nozzle was removed to prevent disruptions to the feed flow rate.(51) the spray dryer was set at an inlet temperature of 130°c, which corresponded to an outlet temperature of ~70°c. for all runs, no settling of the feed suspensions was noted during processing. formulations were evaluated on the basis of dry powder psd and reconstituted suspension psd, and the composition exhibiting the most promising characteristics was selected for further evaluation. comparative powders were generated for the purposes of evaluation of physicochemical characteristics, aerosol performance, and efficacy. a nic-free hlys spray dried powder was generated using the optimized formulation composition identified in the doe, minus the addition of micronized nic. to compare the novel nic-hlys powders against a traditional lactose-based carrier system, micronized nic was blended with crystalline lactose particles (lactohale 100; dfe pharma) using geometric dilution followed by mixing in a turbula powder blender. the concentration nic in the niclosamide-lactose blend (nic-lac) was set to match that in the nic-hlys powder (0.7%). lastly, a powder was generated in which bovine serum albumin (bsa) was substituted for the hlys in the optimized nic formulation, in order to assess the effect of the protein on formulation characteristics. particle size distribution (psd) of nic-hlys powders was measured using a rodos disperser coupled to a sympatec laser diffractor unit (sympatec gmbh, clausthal-zellerfeld, germany). dispersion pressure was set at 3.0 bar and feed table rotation was set at 20%. time slices of the plume exhibiting an optical concentration between 5-25% were averaged to generate the psd. the psd of the reconstituted powders was determined in both ½ normal saline (ns) and di water using the cuvette attachment for the laser diffraction. a spin bar was set to rotate at 2000 rpm, and the dry powders were added directly to the solvent in the cuvette until an optical concentration exceeding 5% was reached. three measurements were taken and averaged. zeta potential of nic-hlys suspensions before and after spray drying was determined using a zetasizer nanozs (malvern panalytical ltd, malvern, uk) and compared against a nic-bsa suspension. the morphology of nic-hlys powders was observed using scanning electron microscopy (sem). samples were mounted onto aluminum stubs using double-side carbon tape and sputter coated with 15 nm of platinum/palladium (pt/pd) under argon using a cressington sputter coater 208 hr (cressington scientific instruments ltd, watford, uk). images were obtained using a zeiss supra 40vp sem (carl zeiss microscopy gmbh, jena, germany). glass transition temperature (tg) and crystallinity of nic-hlys powder was determined using modulated dsc. powder samples were loaded into tzero pans with hermetically sealed lids, and a hole was pierced to prevent pan deformation. a scan was performed on a q20 dsc (ta instruments, new castle, de, usa) by ramping 10°c/min to -40°c, followed by a 2°c/min ramp from -40°c to 280°c with a modulation cycle of ±0.5°c every 40 seconds. data was processed using ta universal analysis. the dissolution profile of the aerosolized nic-hlys and nic-lac powders was evaluated based upon previously published methods (84, 85) using a composition of simulated lung fluid (slf) adapted from hassoun et al (86) . briefly, whatman gf/c glass microfiber filters (diameter = 24 mm) were placed in a stage 4 of a next generation impactor, which corresponded to an aerodynamic size cut off of 2.01 µm at the 40 l/min flow rate used in the experiment. powders were dispersed using a disposable twincaps (hovione) dpi. the filters were transferred to a modified transwell system (membrane removed) to enable contact of the bottom of the filter with a basal compartment containing 1.5 ml slf. the apical side of the filters were wetted with 0.1 ml of slf. the dissolution results are presented in table 3 . the transwell system was placed in a 37°c isothermal chamber and 0.1 ml samples were removed from the basal compartment at various timepoints and replaced with fresh slf. effects of processing and nebulization on the aggregation of hlys was assessed using size exclusion chromatography (sec) based upon a previously published method (80) . the effect of processing on the secondary structure of hlys was determined using a niclolet is50 fourier transform infrared spectrophotometer with attenuated total reflectance (ftir-atr) (thermo scientific). spectra were acquired using omnic software from a wavelength of 700 cm -1 to 4000 cm -1 with 64 acquisitions in total. an atmospheric background scan was collected and subtracted from all powder spectra. secondary structure analysis was performed in originpro (orginlab corporation) using the second derivative of the amide i band region (1580-1720 nm) and the peak analyzer function. the region of interest was first baseline-corrected, and the second derivative of the spectra was smoothed using the savitzky-golay method with a polynomial order of 2 and 50 points in the smoothing window. peaks identified from the second derivative minimums were iteratively removed to assess the effect on the model fit. the aerosol performance of the spray-dried composite nic-hlys powder was assessed using a disposable twincaps dpi. performance was assessed at both a 4kpa and 2kpa pressure drop through the device to determine the effects of inspiratory flow rate on emitted and fine particle dose. for comparative purposes, the performance of traditional lactose carrier-based dry powder, nic-lac, was also assessed. a 4 kpa pressure drop was generated through the twincaps dpi using an inspiratory flow rate of 40 l/min. a 6.5 second actuation time was used to pull 4 l of air through the ngi. a 2 kpa pressure drop was generated using an inspiratory flow rate of 28.3 l/min, and an 8 second actuation time was used. for all experiments, 60 mg of powder was loaded into the device. after actuation of nic-hlys powders, niclosamide was collected by dissolving the deposited powder using a 50-50 water:acetonitrile mix. an aliquot was taken, and an additional volume of acetonitrile was added to bring the final ratio to 20:80 water:acetonitrile. to induce phase separation, a 2m solution of ammonium acetate was added to this mixture at a volume that was 20% of the water:acetonitrile mix. niclosamide was assayed from the upper organic layer by measuring absorbance at 331nm using a plate reader. for the niclosamide-lactose blend, the deposited powder was collected by dissolving it in 20:80 water:acetonitrile, centrifuging, and then measuring absorbance at 331nm. delivery of the reconstitued nic-hlys suspension was assessed using the disposable aerogen® solo vibrating mesh nebulizer (aerogen). preliminary screening experiments indicated that a reconstituting 25 mg/ml of nic-hlys powder in 0.45% w/v sodium chloride (1/2 ns) reduced the changes in nebulizer concentration during therapy compared to higher concentrations; therefore, this concentration was utilized for further analysis. the inspiratory flow rate was set for 15 l/min and the apparatus was chilled to 4°c as specified by the united states pharmacopeia (usp) (87) . nebulization was performed for two minutes to ensure sufficient deposition of drug in the stages for analysis. after drug collection, aerosol performance was evaluated on the basis of emitted fraction or dose (niclosamide mass emitted from the device as a percentage of the total recovered powder) and fine particle fraction (niclosamide mass with a size cut off of less than 5 µm aerodynamic diameter or 3 µm aerodynamic diameter, as a percentage of the emitted dose). ngi stage cutoffs were determined for the flow rate utilized based upon eq. 2, while the moc cut-off diameter was determined using eq. 3. where %&,( is the cut-off diameter at the flow rate , %&,() is the cut-off diameter at the archival reference values of qn = 60 l/min, and the values for the exponent, x, are those obtained from the archival ngi stage cut size-flow rate calculations determined by marple et al (88) . aerosol performance was evaluated on the basis of emitted fraction (ef), which is defined as the cumulative mass emitted from the device as a fraction of the recovered mass, fine particle fraction less than 5 µm (fpf<5µm), defined as the mass less than 5 µm aerodynamic diameter as a fraction of the emitted dose, and the fine particle fraction less than 3 µm (fpf<3µm) the fpf<5µm and fpf<3µm values were interpolated from a graph plotting the cumulative percentage of nic deposited in a stage against the cut-off values of the stage. to evaluate the utility of the optimized nic-hlys powders for nasal administration, suspensions of varying concentrations (10 mg/ml, 25 mg/ml, and 50 mg/ml) were prepared in 1/2 ns and placed in a vp7 pump aptar® pump meter spray device. spray patterns and plume geometries were evaluated using laser-assisted high speed imaging based on methods previously reported by warnken et al.(89) briefly, the loaded spray devices were actuated using a mightyrunt automated actuator (innovasystems, inc) set at parameters that mimic those of an average adult user (90) . a laser-sheet was oriented either parallel or perpendicular to the actuated spray at distances of 2 and 5 cm from the nozzle tip in order to assess the plume geometry and spray pattern, respectively. the actuation was conducted in a light-free environment in order to isolate the portions of the spray photographed by the high-speed camera (thorlabs, inc.) from those illuminated in the plane of the laser. image analysis of the plume geometry and spray pattern were permed in fiji. (91) for the plume geometry, the outline of the observed plume was traced and the slope of each side of the plume was determined. this was used to calculate the angle formed at the intersection of the two lines. the spray pattern characteristics including maximum and minimum diameters were determined using the software's measurement function to determine the feret diameters. the ability of the optimized nic-hlys formulation to dampen inflammatory response was evaluated using lipopolysaccharide (lps) stimulation in a thp-1 macrophage model. thp-1 monocytes were seeded in 6-well plates at a concentration of 4 x 10 5 cells/ml (5 ml total) in rpmi 1640 media supplemented with 10% fbs, 1% penicillin/streptomycin, and 15 ng/ml phorbol 12-myristate (pma) to induce differentiation into mature macrophages. the cells were incubated in the presence of pma for 48 hours, after the media was replaced with pma-free media and cells were rested for 24 hours. nic-hlys and nic-bsa powders were suspended in rmpi 1640 media at varying concentrations (2 to 18 µg/ml, based on total powder content) and added to the cells simultaneously with 10 ng/ml lps. the cells were then incubated for 6 hours to achieve peak cytokine expression (92) . following incubation, supernatants were collected, and cytokine concentrations were quantified using elisa (duoset, r&d systems) and compared against untreated controls. all antiviral efficacy experiments were performed using vero-e6 cells obtained from american type culture collection (manassas, virginia, usa). vero-e6 cells were maintained in minimal essential medium (mem) supplemented with 10% fetal bovine serum (fbs) and 1x antibioticantimycotic solution (sigma, st. louis, usa) (i.e., mem complete). cells were infected with either mers-cov (emc2012 strain) or sars-cov-2 (sars-cov-2/human/korea/cnuhv03/2020 strain). all experimental procedures involving potential contact with mers-cov or sars-cov-2 were conducted in a biosafety level 3 laboratory of chungnam national university, which was certified by the korean government. vero-e6 cells (2 x10 4 /ml) were seeded in the wells of 6-well tissue culture plates. after a 3-day incubation period, cells were washed with warm pbs (ph 7.4) twice and were infected with sars-cov-2 (1.7 x 10 3 pfu) or mers-cov (2 x 10 4 pfu) diluted in mem with 2% fbs, which was followed by a 24-hour rest period. the media was then replaced with mem complete containing various concentrations of the investigational formulations (prepared as suspensions). for the assessment of solubilized nic, stock solutions were prepared by dissolving the drug in dmso, and the diluting in mem-complete, with the resulting media not containing more than 1% dmso. drug concentrations and formulations were assessed in triplicate. for each 6-well plate, 1 well was utilized as untreated control. cells were incubated for 24 or 48 hours, at which point viral rna from samples was isolated using rneasy mini kit (qiagen, hilden, germany). viral rna was quantified with taqman real time fluorescent pcr (rtqpcr) using a topreal tm one-step rt qpcr kit (enzynomics, daejeon, korea) and sars-cov-2 and mers-cov primers and probe (supplementary table 8 ). real-time amplification was performed using a rotor-gene 6000 (qiagen, hilden, germany). an initial incubation was performed at 50°c for 30 minutes and at 95°c for 10 minutes, after which 45 cycles of a 5 second hold at 95°c and a 30 second hold at 60°c were performed. cycle threshold (ct) values were converted to plaque forming units (pfu) using a standard curve generated from data using stock viruses with known pfu titers by plaque assay. vero-e6 cells grown in tissue culture flasks were detached by treatment with trypsin-edta and were seeded in 96-well tissue culture plates. when confluent, cells were washed with warm pbs (ph 7.4) and infected with mers-cov or sars-cov-2. the half maximal effective concentration (ec50) of the formulations was assessed by dosing infected vero e6 cells plated in 96-wells with nic-hlys suspensions with nic content ranging from 0.25 µg/ml to 0.004 µg/ml once daily over the course of 72 hours. cell viability was determined on day 4 by observing cytopathic effects (cpe) under microscope. the ec50 was calculated as the concentration of nic resulting in no observable cpe in 50% of the wells. for comparative purposes, the ec50 of micronized nic without the inclusion of hlys was also evaluated. the inhibitory activity of various nic formulations (nic-hlys, nic-m, nic-bsa, and nic-dmso) against methicillin-resistant s. aureus strain mu50 was assessed using a resazurinbased 96-well plate microdilution assay (93) . varying concentrations of the nic formulations (: 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.0156, 0.008 µg/ml, dosed based on nic content) were plated with a 5 x 10 5 cfu/ml inoculum of s. aureus (n = 6 per dose). one column of the plate was used as growth control, i.e., no antibiotics were added, while another column was used as a sterile control, i.e., no bacteria added. the plates were incubated for 24 hours at 37°c with 150 rpm shaking, after which point 30 µl a 0.015% resazurin sodium solution was added. the plates were incubated for an additional 2 hours to allow color change to occur, and the fluorescence of the wells was read at 530 nm excitation/590 nm emission. the fluorescence of the sterile wells was subtracted from the fluorescence of all treated wells, and a decrease in fluorescence of the treated wells versus the growth control was noted as inhibitory activity. the content of wells exhibiting 100% inhibition were plated on tryptic soy agar plates and incubated overnight to determine the mean bactericidal concentration (mbc). hdpp-4 transgenic mice were kindly provided by dr. paul b. mccray jr (university of iowa). mers-cov infection was initiated in anaesthetized mice by intranasal (i.n.) administration of 50 µl (1 × 10 5 pfu) of mers-cov (emc2012 strain), which was kindly drs bart haagmans and ron fouchier (erasmus medical center). efficacy was initially established using a dose-finding study, in which treatments were initiated 1-day post-infection (p.i.) and daily for 6 days, at which point animals (n = 3 from each group) were sacrificed. nic-hlys powder was reconstituted in 0.45% sodium chloride to achieve a dose of 60 or 120 µg/kg nic (n = 7 per group). the suspensions were administered i.n. in a volume of 50 µl, and 50 µl of 0.9% sodium chloride was administered as a control (n = 6). though a second timepoint was intended for day 9, death due to illness or as a result of treatment administration prevented obtainment of these data. a separate study was conducted to compare the survival of mers-cov-infected mice treated with 240 µg/kg nic-hlys (n = 7) and placebo (n = 6). in this study, mice were dosed intranasally for 10 days, at which point treatment was terminated. surviving mice were rested without treatment for an additional 3 days, and sacrifice was performed day 14 p.i. to obtain tissues (lung and brain) for viral titres and tissue pathology. the weight of mice was recorded daily. tissues (0.1 g per sample) were homogenised using a beadblaster homogeniser (benchmark scientific, edison, new jersey, usa) in 1 ml of pbs (ph 7.4) to measure virus titres by rt-qpcr. the remaining portions of tissues were used for histopathology. mice were lightly anaesthetized with isoflurane usp (gujarat, india) prior to all viral inoculation and dosing procedures. hace-2 transgenic mice (k18-hace2 mice) (the jackson laboratory, usa) were lightly anaesthetized with isoflurane usp (gujarat, india) and inoculated intranasally (i.n.) with 50 µl (1 × 10 4 pfu) of sars-cov-2/human/korea/cnuhv03/2020. animals were rested for 24-hours, after which daily treatment was initiated with i.n. nic-hlys reconstituted in 0.45% sodium chloride (240 µg/kg nic) (n = 13) or 0.9% sodium chloride as a placebo (n = 8). all treatments were performed on anaesthetized mice. on day 6 post-infection, 3 mice per group were euthanized, and lung, brain and kidney tissues were collected for viral titres and tissue pathology. treamtne was performed until 10 days p.i., at which point surviving animals were left untreated for 3 days, and then sacrificed on day 14 p.i. to obtain tissues for viral titres and pathology. tissues (0.1 g per sample) were homogenised using a beadblaster homogeniser (benchmark scientific, edison, new jersey, usa) in 1 ml of pbs (ph 7.4) to measure virus titres by rt-qpcr. the remaining portions of tissues were used for histopathology. mouse tissues were fixed in 10% neutral buffered formalin (10%) and then embedded in paraffin. the lung tissue was cut into 5 μm sections, which were stained with haematoxylin (h) solution for 4 min. the stained tissue sections were washed with tap water for 10 min and then stained with eosin (e) solution. the stained sections were visualised under an olympus dp70 microscope and photographed (olympus corporation, tokyo, japan). to determine whether measured viral particles in lung and brain tissue were dead or alive, the log10tcid50/ml was determined. vero-e6 cells grown in tissue culture flasks were detached by treatment with trypsin-edta and were seeded in 96-well tissue culture plates with mem containing 10% fbs and 1× antibiotic-antimycotic solution. when confluent, the cells were washed with warm pbs (ph 7.4) and infected with virus samples, which were 10-fold diluted in mem with 2% fbs. the cells in four wells were infected with the diluted virus samples for 4 days in a humidified incubator at 37°c. the cells were observed for cpe under microscope. the presence of igg antibody specific for mers-cov or sars-cov-2 in the sera of infected and treated animals was determined using enzyme-linked immunosorbent assays (elisa). the purified and inactivated mers-cov or sars-cov-2 antigen was diluted to final concentration of 100μg/ml in coating buffer (carbonate-bicarbonate buffer, ph 9.6). the diluted antigen (100μl) was coated to the wells of a nunc-immuno ™ microwell ™ 96 well solid plates (sigma-aldrich, mo, usa) and was incubated overnight at 4°c. after removing the coating buffer, the plate was washed twice by filling the wells with 400μl of washing buffer (0.05% tween 20 pbs (ph 7.4) the buffer was removed, and sera (100μl diluted in 1:64 in pbs) collected from treated mice on 14 days post treatment were added to the plate and incubated for 1hr at room temperature. the plate was washed 4 times with washing buffer. goat anti-mouse igg cross-adsorbed secondary antibody hrp (invitrogen, ma, usa) was diluted (1:5000) in blocking buffer, and 100 µl was added to each well and incubated for 1hr at room temperature. after washing the plate 4 times with the washing buffer, 100 µl of the tmb elisa substrate (mabtech, nacka strand, sweden) was dispensed into the wells and incubated for 30min at 4℃. abts® peroxidase stop solution -2 lethal infection models were evaluated for statistical significance using the mantel-cox test. statistical analysis on tissue homogenate viral titres was performed using a two-way anova with dunnet's multiple comparisons test to evaluate post-hoc differences between groups. alpha was set at 0.05. statistical analysis was performed using prism8 (graphpad software all the studies were approved and were conducted in accordance with the relevant legal guidelines and regulations prescribed by cnu, republic of korea. references 1. who director-general's opening remarks at the mission briefing on estimated demand for us hospital inpatient and intensive care unit beds for patients with covid-19 based on comparisons with wuhan and guangzhou, china disease and healthcare burden of covid-19 in the united states structure, function, and antigenicity of the sars-cov-2 spike glycoprotein structural basis for human coronavirus attachment to sialic acid receptors projecting the transmission dynamics of sars-cov-2 through the postpandemic period identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects broad spectrum antiviral agent niclosamide and its therapeutic potential multi-targeted therapy of cancer by niclosamide: a new application for an old drug beyond an antihelminthic drug host-targeted niclosamide inhibits c. difficile virulence and prevents disease in mice without disrupting the gut microbiota structure-activity analysis of niclosamide reveals potential role for cytoplasmic ph in control of mammalian target of rapamycin complex 1 (mtorc1) signaling a novel high content imaging-based screen identifies the anti-helminthic niclosamide as an inhibitor of lysosome anterograde trafficking and prostate cancer cell invasion extracellular ph modulates neuroendocrine prostate cancer cell metabolism and susceptibility to the mitochondrial inhibitor niclosamide structure-activity studies of wnt/beta-catenin inhibition in the niclosamide chemotype: identification of derivatives with improved drug exposure repurposing the anthelmintic drug niclosamide to combat helicobacter pylori repurposing salicylanilide anthelmintic drugs to combat drug resistant staphylococcus aureus repurposing niclosamide as a versatile antimicrobial surface coating against device-associated, hospitalacquired bacterial infections screening a commercial library of pharmacologically active small molecules against staphylococcus aureus biofilms the anthelmintic drug niclosamide synergizes with colistin and reverses colistin resistance in gram-negative bacilli toward repositioning niclosamide for antivirulence therapy of pseudomonas aeruginosa lung infections: development of inhalable formulations through nanosuspension technology new life for an old drug: the anthelmintic drug niclosamide inhibits pseudomonas aeruginosa quorum sensing synergistic activity of niclosamide in combination with colistin against colistin-susceptible and colistin-resistant acinetobacter baumannii and klebsiella pneumoniae antituberculosis activity of certain antifungal and antihelmintic drugs activities of drug combinations against <span class="named-content genus-species" id="named-content-1">mycobacterium tuberculosis</span> grown in aerobic and hypoxic acidic conditions niclosamide as a promising antibiofilm agent niclosamide repurposed for the treatment of inflammatory airway disease identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs a phase i study of niclosamide in combination with enzalutamide in men with castration-resistant prostate cancer toward repositioning niclosamide for antivirulence therapy of pseudomonas aeruginosa lung infections: development of inhalable formulations through nanosuspension technology nebulized mannitol, particle distribution, and cough in idiopathic pulmonary fibrosis dissociation in the effect of nedocromil on mannitol-induced cough or bronchoconstriction in asthmatic subjects* post-inhalation cough with therapeutic aerosols: formulation considerations. adv drug deliv rev asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths anti-inflammatory effects of lysozyme against hmgb1 in human endothelial cells and in mice antiviral activity of lysozyme antiviral effects of nisin, lysozyme, lactoferrin and their mixtures against bovine viral diarrhoea virus study on antimicrobial and antiviral activities of lysozyme from marine strain s-12-86 in vitro structural and functional modeling of human lysozyme reveals a unique nonapeptide, hl9, with anti-hiv activity human lysozyme possesses novel antimicrobial peptides within its n-terminal domain that target bacterial respiration activity of abundant antimicrobials of the human airway. american journal of respiratory cell and molecular biology from bacterial killing to immune modulation: recent insights into the functions of lysozyme reverse genetics reveals a variable infection gradient in the respiratory tract immunogenicity of protein aggregates--concerns and realities immunogenicity of therapeutic proteins: influence of aggregation immunogenicity of therapeutic protein aggregates effect of particle formation process on characteristics and aerosol performance of respirable protein powders integrity of crystalline lysozyme exceeds that of a spray-dried form lysozymes in the animal kingdom antibacterial peptides: basic facts and emerging concepts severe acute respiratory syndrome-coronavirus infection in aged nonhuman primates is associated with modulated pulmonary and systemic immune responses rethinking the term "pi-stacking an in vitro study of aromatic stacking of drug molecules pharmaceutical cocrystals of niclosamide complexation of lysozyme with sodium poly(styrenesulfonate) via the two-state and non-two-state unfoldings of lysozyme covid-19 update: fda broadens emergency use authorization for veklury (remdesivir) to include all hospitalized patients for treatment of covid-19 study to evaluate the safety and antiviral activity of remdesivir (gs-5734™) in participants with moderate coronavirus disease (covid-19) compared to standard of care treatment development of remdesivir as a dry powder for inhalation by thin film freezing comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov an inflammatory cytokine signature predicts covid-19 severity and survival clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study. the lancet the biology and toxicology of molluscicides, bayluscide the impact of inspiratory flow rate on drug delivery to the lungs with dry powder inhalers dysregulation of immune response in patients with covid-19 in wuhan, china brain delivery of vasoactive intestinal peptide (vip) following nasal administration to rats ciclesonide, a hypotonic intranasal corticosteroid neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study nervous system involvement after infection with covid-19 and other coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov excipient-free pulmonary delivery and macrophage targeting of clofazimine via air jet micronization influence of formulation factors on the aerosol performance and stability of lysozyme powders: a systematic approach aerosol technology: properties, behavior, and measurement of airborne particles package 'rsm' r: a language and environment for statistical computing in vitro aqueous fluid-capacity-limited dissolution testing of respirable aerosol drug particles generated from inhaler products evaluation of the transwell system for characterization of dissolution behavior of inhalation drugs: effects of membrane and surfactant design and development of a biorelevant simulated human lung fluid overcoming sink limitations in dissolution testing: a review of traditional methods and the potential utility of biphasic systems next generation pharmaceutical impactor (a new impactor for pharmaceutical inhaler testing). part ii: archival calibration personalized medicine in nasal delivery: the use of patient-specific administration parameters to improve nasal drug targeting using 3d printed nasal replica casts automated actuation of nasal spray products: determination and comparison of adult and pediatric settings fiji: an opensource platform for biological-image analysis transcription profiles of lpsstimulated thp-1 monocytes and macrophages: a tool to study inflammation modulating effects of foodderived compounds resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants the authors wish to acknowledge the valuable contributions of miguel orlando jara gonzalez (university of texas at austin, college of pharmacy) in the obtainment and review of background literature to support the premise of this work. key: cord-323093-u3ozc9ry authors: rathnayake, athri d.; zheng, jian; kim, yunjeong; perera, krishani dinali; mackin, samantha; meyerholz, david k.; kashipathy, maithri m.; battaile, kevin p.; lovell, scott; perlman, stanley; groutas, william c.; chang, kyeong-ok title: 3c-like protease inhibitors block coronavirus replication in vitro and improve survival in mers-cov–infected mice date: 2020-08-19 journal: sci transl med doi: 10.1126/scitranslmed.abc5332 sha: doc_id: 323093 cord_uid: u3ozc9ry pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and the newly emerged sars-cov-2, the causative agent of coronavirus disease 2019 (covid-19). we describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3c-like protease (3clpro), an enzyme essential for viral replication. the optimized compounds were effective against several human coronaviruses including mers-cov, sars-cov, and sars-cov-2 in an enzyme assay and in cell-based assays using huh-7 and vero e6 cell lines. two selected compounds showed antiviral effects against sars-cov-2 in cultured primary human airway epithelial cells. in a mouse model of mers-cov infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. these results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses. coronaviruses are a large group of viruses that can cause a wide variety of diseases in humans and animals (1) . human coronaviruses generally cause the common cold, a mild upper respiratory illness. however, global outbreaks of new human coronavirus infections with severe respiratory disease have periodically emerged from animal reservoirs, including severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and, most recently, sars-cov-2, the causative agent of coronavirus disease 2019 . sars-cov-2 emerged in china in december 2019 and subsequently rapidly spread throughout the world. genetic analysis of sars-cov-2 revealed that it is closely related to sars-like betacoronaviruses of bat origin, bat-sl-covzc45 and bat-sl-covzxc21 (2) . despite the periodic emergence of new coronaviruses capable of infecting humans, there are currently no licensed vaccines or antiviral drugs against any coronaviruses, underscoring the urgent need for the development of preventive and therapeutic measures against pathogenic coronaviruses. the coronavirus genome contains two overlapping open reading frames (orf1a and orf1b) at the 5′ end terminal, which encode polyproteins pp1a and pp1ab. the polyproteins are processed by a 3c-like protease [3clpro or main protease (mpro)] (11 cleavage sites) and a papain-like protease (plpro) (3 cleavage sites), resulting in 16 mature nonstructural proteins, including an rna-dependent rna polymerase (rdrp). both 3clpro and plpro are essential for viral replication, making them attractive targets for drug development (3) (4) (5) (6) (7) . coronavirus 3clpro is a cysteine protease that has two n-terminal domains containing two -barrel chymotrypsin-like folds (8) (9) (10) . the active site of 3clpro is located in the cleft between the two domains and is characterized by a catalytic cys-his dyad. we have developed broad-spectrum inhibitors of an array of viruses, including coronaviruses and noroviruses (11) (12) (13) (14) (15) (16) (17) (18) that use 3clpro for viral replication and picornaviruses that use 3c protease (19) . we have shown efficacy of the coronavirus 3clpro inhibitor gc376 (currently in clinical development) in animal models of coronavirus infection (20, 21) . specifically, administration of gc376 to cats with feline infectious peritonitis (fip), a coronavirus-induced systemic disease that is 100% fatal, reversed the progression of fip and resulted in clinical remission (20, 21) . we have recently reported the results of exploratory in vitro studies using a dipeptidyl series of mers-cov 3clpro inhibitors that embody a piperidine moiety as a new design element, as well as pertinent structural and biochemical studies (17) . here, we report the development of 3clpro inhibitors against multiple coronaviruses, including sars-cov-2, and demonstrate in vivo efficacy against mers-cov in a mouse model. the synthesis scheme for compound series 6a to 6k and 7a to 7k is shown in fig. 1 and described in supplementary materials and methods. the activity of compounds 6a to 6k and 7a to 7k against the 3clpro enzymes of mers-cov, sars-cov, and sars-cov-2 was evaluated in a fluorescence resonance energy transfer (fret) enzyme assay (table 1 and table s1 ). several compounds in this series (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j, and 7j) were also tested in cell-based assays ( table 2) . table 1 and table s1 show 50% inhibitory concentration (ic 50 ) values in a fret enzyme assay for select compounds (6a, 7a, 6c, 7c, 6e, 7e, 6h, 7h, 6j, and 7j) and gc376. fifty percent inhibitory concentration (ec 50 ) values and 50% cytotoxic concentration (cc 50 ) values for select compounds and gc376 were measured in cell culture assays (table 2) . cell culture assays included huh-7 cells infected with mers-cov, vero e6 cells infected with sars-cov-2, the crandell-rees feline kidney (crfk) cells infected with fip virus (fipv), and l929 cells infected with mouse hepatitis virus (mhv) ( table 2 ). inhibitors with a p 2 leucine (leu) residue were more potent than those with a cyclohexylalanine against mers-cov 3clpro (compounds 6h and 7h versus 6i and 7i), with submicromolar ic 50 values (table 1 and table s1 ). the compounds tested against mers-cov in cell culture (7a, 6c, 7e, 7h, and 6j) also displayed submicromolar ec 50 values. among these compounds, 6j showed the most potent antiviral activity against mers-cov with an ec 50 value of 0.04 m. gc376 with a p 2 leu residue and a nonfluorinated benzyl cap exhibited 20-fold lower potency against mers-cov in cell culture compared to compound 6j ( table 2) . the compounds were also effective against sars-cov-2 with ec 50 values ranging from 0.15 to 0.9 m in vero e6 cells (table 2) . these compounds were also found to be potent against fipv and mhv, with ec 50 values ranging from 0.07 to 0.22 m. in the fret enzyme assay, these compounds were active against the 3clpro of sars-cov and sars-cov-2 ( table 1 ). the ic 50 values of these compounds against sars-cov-2 3clpro ranged from 0.17 to 0.82 m. among these compounds, 6e showed the most potent antiviral ac-tivity against sars-cov-2 in the enzyme assay (ic 50 , 0.17 m) and cell-based assay (ic 50 , 0.15 m) (tables 1 and 2) . gc376 also exhibited activity against the 3clpro of sars-cov-2 with an ic 50 value of 0.62 m in the enzyme assay ( table 1) . the antiviral effects of compounds 6j and 6e against sars-cov-2 were confirmed in cultured primary human airway epithelial cells from three donors. in the absence of a 3clpro inhibitor, viral titers in the infected cultured primary human airway epithelial cells reached 10 7.3 (donor 1), 10 7.1 (donor 2), and 10 8.4 (donor 3) plaque-forming units (pfu) per milliliter of culture medium. in the presence of compound concentrations that were about two-to threefold higher than the ec 50 values obtained in vero e6 cells (2 m, 6j or 0.5 m, 6e), viral titers were reduced to 10 6.4 (donor 1), 10 6.1 (donor 2), and 10 6.3 (donor 3) pfu/ml for compound 6j or 10 6.1 (donor 1), 10 6.5 (donor 2), and 10 8.1 (donor 3) pfu/ml for compound 6e (table 3) . although there was some variation in viral replication among infected cells from the three donors (especially donor 3), the antiviral effects of both 6j and 6e were evident at the tested concentrations. for infected cells from donors 1 and 2, both compounds inhibited viral replication about 10-fold at the tested concentrations. for infected cells from donor 3, 6e inhibited virus replication about 50%, whereas 6j inhibited virus replication 100-fold at the tested concentrations. we determined multiple high-resolution cocrystal structures of compounds 6b, 6d, 6g, 6h, 7i, or 7j with the 3clpro of mers-cov (fig. 2 , a to f, and figs. s1 to s3). these inhibitors bound to the active site of mers-cov 3clpro, demonstrating that the vicinity of the s 4 pocket is encompassed by an array of primarily hydrophobic residues, including phe 188 , val 193 , ala 171 , and leu 170 (fig. 2 , c and f, and fig. s3 ). hydrophobic and hydrogen-bonding functionalities were incorporated into the 3clpro inhibitors to capture additional interactions, and the position of the cyclohexyl moiety was also examined using appropriate congeners. the bisulfite adducts reverted to the corresponding aldehydes, which subsequently reacted with cys 148 to form nearly identical covalent complexes with a tetrahedral arrangement at the newly formed stereocenter (fig. 2 , a to f, and figs. s1 to s3). the backbone of compound 6h (table 1 and fig. 2 , a to c) engaged in h-bond interactions with amino acid residues gln 192 , gln 167 , and glu 169 . three additional side-chain h-bonds between the -lactam ring and his 166 , phe 143 , and glu 169 also were clearly evident (fig. 2b) fig. 1 . synthesis scheme for compound series 6a to 6k and 7a to 7k. stepwise compound synthesis with intermediate compounds is shown for 3c-like protease (3clpro) inhibitors of the 6a to 6k and 7a to 7k series. the alcohol inputs were reacted with (l) leucine isocyanate methyl ester or (l) cyclohexylalanine isocyanate methyl ester to yield products, which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. subsequent coupling of the acids to glutamine surrogate methyl ester "8" furnished compounds "4". lithium borohydride reduction yielded alcohols "5", which were then oxidized to the corresponding aldehydes "6" with dess-martin periodinane reagent. the bisulfite adducts "7" were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate. a amino acid methyl ester isocyanate/tea/ch 3 was ensconced in the hydrophobic s 2 pocket (fig. 2c) . the extra methylene group in compound 7j, which was converted to an aldehyde and thus became identical to 6j, resulted in the reorientation of the difluorocyclohexyl group and the formation of three h-bonds between gln 195 and ala 171 and the fluorine atoms, with concomitant loss of one of the gln 192 hydrogen bonds and the displacement of phe 143 (fig. 2e ). the substitution of the p 2 leu with p 2 cyclohexylalanine (compound 7i; table s1) resulted in the loss of an h-bond with gln 192 but otherwise adopted the same interactions as observed for compound 6h (fig. s2a ). the electron density, hydrogen bond interactions, and electrostatic surface representations for mers-cov 3clpro in complex with compounds 6b, 6g, and 6d are shown in figs. s1 to s3. next, compound 7j was cocrystallized with sars-cov or sars-cov-2 3clpro, and the structures were compared to that for mers-cov 3clpro and 7j. in the sars-cov 3clpro-7j complex (fig. 2 , g to i), the backbone of compound 7j formed direct h-bonds with cys 145 , his 163 , his 164 , glu 166 , and gln 189 . compound 7j also formed an additional h-bond with his 41 and a water-mediated contact with gly 143 (fig. 2h ). however, there was a loss of the three h-bonds between gln 195 and ala 171 and the fluorine atoms, compared to the cocrystal structure of 7j with mers-cov 3clpro. the electron density map was consistent with both possible enantiomers at the new stereocenter formed by covalent attachment of the s atom of cys 145 in the cocrystal structure of sars-cov 3clpro with 7j. the electron density map for compound 7j in complex with sars-cov-2 3clpro was most consistent with a single enantiomer, although it adopted a similar binding mode and hydrogen bond interactions as observed in the sars-cov 3clpro-7j cocrystal structure (fig. 2 , j to l). superposition of compound 7j with mers-cov 3clpro, sars-cov 3clpro, and sars-cov-2 3clpro ( fig. s4 ) revealed a very similar binding mode for 7j among all three viral proteases. the most potent compound of the series, 6j, was identified in a cellbased assay and had an ec 50 value of 0.04 m against mers-cov (fig. 3a) . we determined the efficacy of compounds 6j and 6h in transgenic hdpp4-ki mice expressing human dipeptidylpeptidase 4, a model of mers-cov infection. first, hdpp4-ki mice were infected with the mouse-adapted mers-cov (mers ma -cov) virus strain and then were treated with compounds 6h and 6j (50 mg/kg per day, once a day) or vehicle as a control starting 1 day post virus infection (1 dpi) and continuing until 10 dpi. all mice treated with vehicle control died by 8 dpi (fig. 3b ). in contrast, 40% of mice treated with compound 6h survived, and all mice treated with compound 6j were alive at the end of the study (15 dpi) (fig. 3b ). the survival of mice treated with compound 6j or 6h was increased compared to the vehicle control (p < 0.05), and the 6j-treated mice had an improved survival rate compared to 6h-treated mice (p < 0.05). all mice treated with compound 6j rapidly recovered from body weight loss starting at 3 dpi (fig. 3c ). the mice that survived after 6h treatment continued to lose body weight until 6 dpi but then started to gain weight from 9 dpi (fig. 3c ). after we observed that treatment with compound 6j resulted in the survival of mers ma -cov-infected hdpp4-ki mice, we conducted another study by delaying treatment initiation until 3 dpi. similar to the first study, no untreated mice or mice given vehicle (control) survived, and there was no statistical difference between these two groups (0% survival) (fig. 3d ). when 6j treatment was started on 1 dpi, four of the five mice survived (80% survival), and there was a statistically significant increased survival in mice treated starting at 1 dpi compared to untreated or vehicle control-treated mice (p < 0.05; fig. 3d ). when 6j treatment was delayed by one additional day (2 dpi), the survival of mice treated with 6j decreased to 40%, but this was still higher than the 0% survival for untreated or vehicle controltreated mice. however, there was no statistical difference between the 6j treatment group starting at 2 dpi and the untreated or vehicle control-treated groups (fig. 3d ). treatment with 6j starting at 3 dpi also failed to improve the survival of mice compared to the untreated or vehicle control-treated groups (fig. 3d ). all mice lost body weight after virus infection, but surviving mice treated with 6j regained the lost weight by 15 dpi (fig. 3e ). recovery of body weight was faster in mice treated with 6j starting at 1 dpi than table 3 . antiviral effects of compounds 6j and 6e against sars-cov-2 in cultured primary human airway epithelial cells. the study was a single experiment, and viral titers were measured in duplicate with a plaque-forming assay. vehicle control at 2 dpi (fig. 3e) . these results show that survival of mice markedly increased when 6j was given at 1 dpi. the lung pathology caused by mers ma -cov infection of hdpp4-ki mice resembles that seen in severe cases of human mers-cov infection, with diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22) . a group of hdpp4-ki mice were infected with mers ma -cov and treated with compound 6j or vehicle as a control starting at 1 dpi. mouse lungs were collected for determination of virus load at 3 and 5 dpi and for histopathology at 6 dpi. lung virus titers decreased in the 6j-treated mice compared to control mice at both 3 and 5 dpi (p < 0.01) (fig. 4a) . edema in the lungs of the treated mice was reduced compared to control mice (p < 0.01) (fig. 4b) . scores for hyaline membrane formation were reduced in 6j-treated mice but were not statistically different from control mice. mers ma -cov-infected hdpp4-ki mice treated with vehicle showed patches in lung tissue variably composed of cellular inflammation, vascular congestion, and atelectasis (fig. 4 , c and e). the airways of these animals were generally intact, with only scattered, uncommon sloughed cells (fig. 4 , c and e). in some lungs from these mice, lymphatic vessels were filled with degenerative cells and cellular debris (fig. 4 , c and e). alveolar edema was detected in some lung tissue sections (fig. 4 , c and e). in contrast, there were few observed lesions in the lungs of mers ma -cov-infected hdpp4-ki mice treated with compound 6j starting at 1 dpi (fig. 4 , d and f). there are currently no approved vaccines or small-molecule therapeutics for the treatment of mers-cov, sars-cov, or sars-cov-2 infection. however, numerous preventive and therapeutic options are under development (3) (4) (5) (6) (7) . the most clinically advanced antiviral compound with a broad spectrum of activity is remdesivir (gs-5734). this nucleoside analog was originally developed as an antiviral drug against ebola virus and has been shown to be effective against both mers-cov and sars-cov in cell culture assays and in animal models of coronavirus infection (23) (24) (25) (26) . prophylactic treatment or early therapeutic treatment of infected mice with remdesivir reduced mers-cov-or sars-cov-mediated weight loss and decreased lung virus titers and lung injury scores compared to those of vehicle-treated animals (23, 26) . remdesvir also showed potent activity against sars-cov-2 in cell culture assays and animal models (27) and was recently issued an emergency use authorization by the u.s. food and drug administration as an investigational antiviral drug for covid-19. another nucleoside analog, eidd-2801, which is a broad-spectrum inhibitor against multiple viruses including influenza viruses, was also shown to be effective against mers-cov and sars-cov in mouse models (28) . our group has been engaged in the discovery of broad-spectrum inhibitors targeting the 3clpro of multiple human and animal coronaviruses. we initially generated dipeptidyl and tripeptidyl series of compounds (29) and observed that the dipeptidyl compound series had superior pharmacokinetic (pk) profiles compared to the tripeptidyl compound series (20) . a representative compound of the dipeptidyl series is gc376, which is currently in clinical development for fip in cats and covid-19. the pk characteristics of multiple dipeptidyl compounds similar to compound 6j, including gc376, were examined through an intraperitoneal or subcutaneous administration in animals, including mice. it was determined that their maximum plasma concentration (c max ) values were >100-fold of the ec 50 for the target virus and that the elimination half-life (t 1/2 ) was 3 to 5 hours. the in vivo efficacy of gc376 against mice infected with mhv or murine norovirus has been demonstrated (11, 15) . inspection of previously obtained crystal structures of the dipeptidyl compounds in a complex with coronavirus 3clpro (17, 19) revealed the potential to achieve enhanced binding interactions with the s 4 subsite by introducing diverse functionalities at the cap position in the inhibitors. in the current study, a new dipeptidyl series focusing on the design of structural variants in the cap substructure were synthesized and evaluated for their activity against coronavirus 3clpro. the ec 50 of gc376 against mers-cov was determined to be ~1 m. one of our goals was to generate compounds with near or below 0.1 m potency against mers-cov and other target coronaviruses. all synthesized compounds displayed varying degrees of inhibitory activity against multiple coronaviruses in the fret enzyme assay and cell-based assays. among these compounds, 6e showed the most potent antiviral activity against sars-cov-2 3clpro in a fret enzyme assay (ic 50 , 0.17 m) and cell-based assays (ec 50 , 0.15 m), and 6j showed the most potent antiviral activity against mers-cov with an ec 50 value of 0.04 m (table 2) . it was previously demonstrated that optimal potency is attained when the p 1 and p 2 residues are a glutamine surrogate and leu, respectively, and that replacement of the p 2 leu with a cyclohexylalanine is inimical to potency (17, 18) . this is clearly evident when comparing the relative potencies of 6h and 7h versus 6i and 7i (table 1 and table s1 ). furthermore, compounds with leu at the p 2 position showed higher cc 50 values compared to those with cyclohexylalanine at p 2 (table s1). x-ray crystallography confirmed the mechanism of action of the inhibitors, which involves formation of a covalent bond between the active site cysteine and the carbonyl carbon of the aldehyde. x-ray crystallography also identified the structural determinants associated with binding, accounting for the observed differences in potency. the high-resolution cocrystal structures of 3clpro inhibitors 7j and 7i with mers-cov 3clpro also confirmed that the difference in activity arose from the loss of a h-bond with gln 192 and the loss of two additional h-bonds from the displacement of gln 167 and phe 143 with 7i ( fig. s2a ). the nature of the interaction of 7j with the s 4 subsite is unique among the compounds examined and provides strong support for our approach, vis-à-vis our focus on the cap position for enhancing binding affinity and potency. compound 7j was cocrystallized with mers-cov, sars-cov, and sars-cov-2 3clpro. superposition of compound 7j with these 3clpro enzymes revealed a similar binding mode among all three proteases. however, a key difference lies with the different conformation adopted by the difluorocyclohexyl ring in the mers-cov 3clpro s 4 subsite, enabling it to engage in additional h-bond binding interactions (fig. 2e and fig. s4 ). compound 7j had a moderately lower potency against sars-cov 3clpro and sars-cov-2 3clpro compared to mers-cov 3clpro in the fret enzyme assay, suggesting that moieties forming h-bonds that were accommodated at the s 4 subsite had an important impact on potency. the barrier to the development of drug resistance increases when an inhibitor engages in h-bond interactions with the backbone of the 3clpro. we used a robust mouse model of mers-cov infection (30-32) to evaluate the efficacy of compounds 6j and 6h. hdpp4-ki mice expressing human dipeptidylpeptidase 4 were infected with a mouseadapted mers-cov virus (mers ma -cov). the infected mice develop fatal lung disease with severe inflammation and weight loss (32) . furthermore, the lung pathology caused by mers ma -cov infection of the hdpp4-ki mice closely resembles that of severe human mers-cov infection and is characterized by diffuse alveolar damage, pulmonary edema, hyaline membrane formation, and infiltration of lymphocytes into the alveolar septa (22) . in the current study, we demonstrated that survival rates in this mouse model were higher with 6j treatment compared to 6h treatment (fig. 3b) . compounds 6j and 6h share a near-identical structure except for the extra methylene group present in compound 6j. compound 6h showed potent anti-3clpro activity, whereas the antiviral activity of compound 6h in cell culture was lower than that of compound 6j (tables 1 and 2) , which may have been the reason for its diminished therapeutic efficacy in the mouse model (fig. 3b) . our findings indicate that therapeutic treatment of infected mice with compound 6j was associated with a reduction in lung viral load and lung pathology (fig. 4) . moreover, treatment of mice with 6j at 1 dpi resulted in the survival of infected mice, whereas delaying treatment initiation until 3 dpi resulted in decreased survival. overall, mouse survival was markedly increased only when 6j was given to mice at 1 dpi (p < 0.05; fig. 3 , d and e). treatment with compound 6j starting at 2 dpi resulted in moderately increased survival of infected mice, but this was not statistically significant (p > 0.05). these results emphasize the importance of early therapeutic intervention in attaining a positive clinical outcome. earlier studies from our group showed that gc376 can cure fatal feline coronavirus disease in cats (20, 21) , demonstrating that a specific coronavirus protease inhibitor can be effective therapeutically against coronavirus disease in a natural host. the mers-cov mouse model used here provides proof of principle regarding the therapeutic potential of our protease inhibitors for treating severe human respiratory coronavirus disease. limitations of the current study include differences in host receptor usage, mortality, and transmissibility between mers-cov and sars-cov-2. thus, further evaluation of our protease inhibitors in mice, hamsters, or nonhuman primates experimentally infected with sars-cov-2 will be crucial to assess these inhibitors as potential therapeutic options for covid-19. our study showed that these compounds were broadly active against the 3clpro of several coronaviruses, with compound 6j displaying the highest activity against mers-cov and compound 6e displaying the highest activity against sars-cov-2. clinical efficacy is influenced by many factors, including drug bioavailability, pk, metabolism, and the chemical stability of a compound. this poses a major challenge with respect to reliably predicting whether the difference in potency against different coronaviruses in assays in vitro can be translated to differences in clinical efficacy. therefore, further research is needed to establish whether one protease inhibitor can be an effective therapeutic for both mers-cov and sars-cov-2 infections in humans. the results from our previous and current studies suggest that the dipeptidyl compound series can serve as a platform suitable for the structure-guided design of one or more inhibitors against highly virulent human coronaviruses. we have generated potent inhibitors of the 3clpro of several coronaviruses, including sars-cov-2, and tested their efficacy in cultured cells and primary human airway epithelial cells. furthermore, we have demonstrated proof-of-concept therapeutic efficacy for one 3clpro inhibitor 6j in hdpp4-ki mice infected with mers ma -cov. our study lays the foundation for advancing this compound series further along the drug development pipeline. the goal of this study was to evaluate the efficacy of 3clpro inhibitors against human coronaviruses, including sars-cov-2, in a fret enzyme assay and cell culture assays, as well as in a mouse model of mers-cov infection. initial antiviral screening was performed with recombinant 3clpro from sars-cov, mers-cov, and sars-cov-2 in the fret enzyme assay. antiviral activity was then assessed in cultured huh-7 cells infected with mers-cov and vero e6 cells infected with sars-cov-2. selected 3clpro inhibitors were further examined using x-ray cocrystallization with mers-cov, sars-cov, and sars-cov-2 3clpro to elucidate the mechanism of action and identify the structural determinants of potency. last, two selected compounds were evaluated for in vivo efficacy in a mouse model of mers-cov infection (hdpp4-ki mice expressing human dipeptidylpeptidase 4 infected with a mouse-adapted mers-cov). age-and sex-matched mice were randomly assigned into various groups for virus infection and treatment studies. microscopic analysis of lung lesions was conducted in a blinded manner; other experiments were not blinded. no mice were excluded from analysis. in vivo studies were performed in animal biosafety level 3 facilities at the university of iowa. all experiments were conducted under protocols approved by the institutional animal care and use committee at the university of iowa according to guidelines set by the association for the assessment and accreditation of laboratory animal care and the u.s. department of agriculture. the studies with mers-cov and sars-cov-2 were performed in biosafety level 3 facilities at the university of iowa. all experiments were conducted under protocols approved by the institutional biosafety committee at the university of iowa according to guidelines set by the biosafety in microbiological and biomedical laboratories, the u.s. department of health and human services, the u.s. public health service, the u.s. centers for disease control and prevention, and the national institutes of health. compounds 6a to 6k and 7a to 7k were readily synthesized as illustrated in fig. 1 and are listed in table 1 and table s1 . briefly, the alcohol inputs were reacted with l-leucine isocyanate methyl ester or l-cyclohexylalanine isocyanate methyl ester to yield dipeptides "2", which were then hydrolyzed to the corresponding acids with lithium hydroxide in aqueous tetrahydrofuran. the subsequent coupling of the acids to glutamine surrogate methyl ester "8" (33, 34) furnished compounds "4". lithium borohydride reduction yielded alcohols "5", which were then oxidized to the corresponding aldehydes "6" with dess-martin periodinane reagent. the bisulfite adducts "7" were generated by treatment with sodium bisulfite in aqueous ethanol and ethyl acetate (35) . the expression and purification of the 3clpro of mers-cov and sars-cov were performed by a standard method described previously by our lab (11, 19, 20) . we also cloned and expressed the 3clpro of sars-cov-2. the codon-optimized complementary dna (cdna) of the full length of 3clpro of sars-cov-2 (genbank accession number mn908947.3) fused with sequences encoding six histidine at the n terminus was synthesized by integrated dna (coralville, ia). the synthesized gene was subcloned into the pet-28a(+) vector. the expression and purification of sars-cov-2 3clpro were conducted after a standard procedure described by our lab (19) . briefly, stock solutions of compounds 6a to 6k and 7a to 7k were prepared in dimethyl sulfoxide (dmso) and diluted in assay buffer, which was composed of 20 mm hepes buffer (ph 8) containing nacl (200 mm), edta (0.4 mm), glycerol (60%), and 6 mm dithiothreitol (dtt). the protease (3clpro of mers-cov, sars-cov, or sars-cov-2) was mixed with serial dilutions of each compound or with dmso in 25 l of assay buffer and incubated at 37°c for 30 min (mers-cov) or at room temperature for 1 hour (sars-cov and sars-cov-2), followed by the addition of 25 l of assay buffer containing substrate (fam-savlq/sg-qxl 520, anaspec, fremont, ca). the substrate was derived from the cleavage sites on the viral polyproteins of sars-cov. fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (flx800, biotek, winooski, vt) 1 hour after the addition of substrate. relative fluorescence units (rfu) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously (19) . the dosedependent fret inhibition curves were fitted with a variable slope by using graphpad prism software (graphpad, la jolla, ca) to determine the ic 50 values of the compounds. some compounds in the 6a to 6k and 7a to 7k series were also investigated for their antiviral activity against the replication of mers-cov, fipv, or mhv-1 in huh-7, crfk, or l929 cells, respectively (19) . briefly, medium containing dmso (<0.1%) or each compound (up to 100 m) was added to confluent cells, which were immediately infected with viruses at a multiplicity of infection (moi) of 0.01. after incubation of the cells at 37°c for 24 hours, viral titers were determined with the median tissure culture infectious dose (tcid 50 ) method (fipv or mhv) with the crfk or l929 cells or plaque assay with vero81 cells (mers-cov). for sars-cov-2, confluent vero e6 cells were inoculated with ~50 to 100 pfu per well, and medium containing various concentrations of each compound and agar was applied to the cells. after 48 to 72 hours, plaques in each well were counted. ec 50 values were determined by graph-pad prism software using a variable slope (graphpad, la jolla, ca) (19) . to confirm that these inhibitors also inhibit sars-cov-2 in primary human cells, differentiated human airway epithelial cells from three donors were used as previously described (36, 37) . two compounds (6j and 6e) were tested for their antiviral effects against sars-cov-2. briefly, airway epithelial cells were washed with phosphate-buffered saline (pbs), and sars-cov-2 was inoculated at a moi of 0.1 for 1 hour. after the inoculum was removed, media containing 6j (2 m) or 6e (0.5 m) was added. after 48 hours, cells were subjected to a freeze/thaw cycle, and virus titers were determined by plaque assay on vero e6 cells. the 50% cytotoxic concentration (cc 50 ) for compounds 6a to 6k and 7a to 7k was determined in huh-7, crfk, or l929 cells. confluent cells grown in 96-well plates were incubated with various concentrations (1 to 100 m) of each compound for 72 hours. cell cytotoxicity was measured by a cytotox 96 nonradioactive cytotoxicity assay kit (promega, madison, wi), and the cc 50 values were calculated using a variable slope by graphpad prism software. the in vitro therapeutic index was calculated by dividing the cc 50 by the ec 50. protein purification, crystallization, and data collection in x-ray crystallographic studies mers-cov 3clpro and sars-cov 3clpro were purified as described previously (17, 19) . an escherichia coli codon-optimized construct encoding residues ser 3264 to phe 3568 of the orf1ab polyprotein (sars-cov-2 3clpro, genebank accession number qhd43415.1) was cloned into a pet his6 sumo tev lic cloning vector (2s-t, addgene). expression and initial ni-column purification were performed as described for mers-cov 3clpro and sars-cov 3clpro. the small ubiquitin-like modifier protein (sumo) fusion elution fractions of sars-cov-2 were dialyzed against 20 mm tris (ph 8.0) and 100 mm nacl and treated with the tobacco etch virus (tev) protease (1:10, w/w) overnight. this mixture was loaded onto a 5-ml histrap hp column (ge healthcare) equilibrated with 20 mm tris (ph 8.0) and 100 mm nacl and eluted with 20 mm tris (ph 8.0), 100 mm nacl, and 500 mm imidazole using an äkta pure fast protein liquid chromatography (fplc). the flow-through fractions containing sars-cov-2 3clpro without the sumo fusion were loaded onto a superdex 75 10/300 gl size exclusion column equilibrated with 20 mm tris (ph 8.0) and 100 mm nacl. the fractions were pooled and concentrated to 9.6 mg/ml for crystallization screening. note that four residues from cloning (ser-asn-ile-gly) remain at the n terminus after treatment with tev protease. purified mers-cov 3clpro, sars-cov 3clpro, and sars-cov-2 3clpro in 100 mm nacl and 20 mm tris (ph 8.0) were concentrated to 10.6 mg/ml (0.3 mm), 22 mg/ml (0.64 mm), and 9.6 mg/ml (0.28 mm), respectively, for crystallization screening. all crystallization experiments were set up using an nt8 drop-setting robot (formulatrix inc.) and uvxpo mrc (molecular dimensions) sitting-drop vapor diffusion plates at 18°c. one hundred nanoliters of protein and 100 nl of crystallization solution were dispensed and equilibrated against 50 l of the latter. stock solutions (100 mm) of compounds 6b, 6d, 6g, 6h, 7i, and 7j were prepared in dmso, and complexes were generated by mixing 1 l of the ligand (2 mm) with 49 l (0.29 mm) of the protease and incubating on ice for 1 hour. crystals of the mers-cov 3clpro inhibitor complexes were obtained from the following conditions: compounds 6b, 6d, 6g intensities were integrated using xds (38, 39) using autoproc (40) , and the laue class analysis and data scaling were performed with aimless (41) . structure solution was conducted by molecular replacement with phaser (42) (46), respectively. disordered side chains were truncated to the point for which electron density could be observed. structure validation was conducted with molprobity (47) , and figures were prepared using the ccp4mg package (48) . crystallographic data are provided in table s2. the two best compounds (6j and 6h) in the series were examined for their in vivo efficacy using 10-week-old male hdpp4-ki mice infected with mers ma -cov (30) . in the first study, animals were divided into three groups (n = 5 to 6) and were lightly anesthetized with ketamine/xylazine and infected with 50 l of 750 pfu mers ma -cov via intranasal inoculation. compound 6j or 6h were formulated in 10% ethanol and 90% peg400 and given to mice from 1 to 10 dpi at 50 mg/kg per day (once per day) via intraperitoneal administration. the control mice received vehicle. animals were weighed daily and monitored for 15 days. animals were euthanized when an animal lost 30% of initial weight or at 15 dpi. in the next study, treatment with compound 6j was delayed up to 3 dpi to determine the impact of delayed treatment on mouse survival. animals were divided into five groups (n = 5), and compound 6j (50 mg/kg per day, once per day) was administered to mice starting at 1, 2, or 3 days after virus challenge (1, 2, or 3 dpi, respectively) until 10 dpi. mice were monitored for weight loss and survival as described above for 15 days after virus challenge. as controls, vehicle (10% ethanol and 90% peg400) was administered equivalently to the experimental compound, or animals received no treatment (untreated). the third study was conducted to assess the effects of therapeutic treatment of compound 6j in the lungs. for lung harvest and virus titration, animals were divided into three groups (n = 4) of mice, and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi until euthanasia. animals were euthanized at 3 or 5 dpi, and lungs were removed aseptically, disassociated with a manual homogenizer in 1× pbs, briefly centrifuged, and supernatants removed. samples were titered on vero81 cells as reported elsewhere (49) . for lung histopathology analyses, animals were divided into two groups (n = 5), and compound 6j (50 mg/kg per day, once per day) or vehicle was administered to mice starting at 1 dpi for 5 days. mice were euthanized at 6 dpi, lungs were fixed with 10% formalin, and hematoxylin and eosinstained tissues were examined by a veterinary pathologist using the postexamination method of masking (50) . briefly, tissues were scored in an ordinal manner for edema and hyaline membrane formation using the following scale: 0, none; 1, rare (<5 alveoli); 2, <33% of lung fields; 3, 34 to 66% lung fields; and 4, >66% lung fields (30) . the analysis of survival curves in groups was performed using a log-rank (mantel-cox) test and a gehan-breslow-wilcoxon test using graphpad prism software (san diego, ca). log-transformed viral titers in the lungs and lung edema and hyaline membrane formation in groups of mice were analyzed with multiple t tests using graphpad prism software. stm.sciencemag.org/cgi/content/full/12/557/eabc5332/dc1 materials and methods table s1 . compound ic50 in the fret enzyme assay and cc50 in a cell culture assay for mers-cov 3clpro. table s2 . cocrystal structure data for compounds with the 3clpro of mers cov and sars cov. data file s1. individual-level data for all figures and tables. references (51) (52) (53) (54) (55) view/request a protocol for this paper from bio-protocol. genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding coronaviruses -drug discovery and therapeutic options broad-spectrum coronavirus antiviral drug discovery recent aspects on the pathogenesis mechanism, animal models and novel therapeutic interventions for middle east respiratory syndrome coronavirus infections middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies crystal structure of sars-cov-2 main protease provides a basis for design of improved alpha-ketoamide inhibitors structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus broad-spectrum inhibitors against 3c-like proteases of feline coronaviruses and feline caliciviruses inhibition of norovirus 3cl protease by bisulfite adducts of transition state inhibitors potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3c-like protease potent inhibition of norovirus 3cl protease by peptidyl alpha-ketoamides and alpha-ketoheterocycles structure-guided design and optimization of dipeptidyl inhibitors of norovirus 3cl protease. structure-activity relationships and biochemical, x-ray crystallographic, cell-based, and in vivo studies structure-based exploration and exploitation of the s4 subsite of norovirus 3cl protease in the design of potent and permeable inhibitors structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element protease inhibitors broadly effective against feline, ferret and mink coronaviruses broad-spectrum antivirals against 3c or 3c-like proteases of picornaviruses, noroviruses, and coronaviruses reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor efficacy of a 3c-like protease inhibitor in treating various forms of acquired feline infectious peritonitis clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses respiratory disease in rhesus macaques inoculated with sars-cov-2 an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice design, synthesis, and bioevaluation of viral 3c and 3c-like protease inhibitors middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 4. incorporation of p1 lactam moieties as l-glutamine replacements an efficient synthesis of a key intermediate for the preparation of the rhinovirus protease inhibitor ag7088 via asymmetric dianionic cyanomethylation of n-boc-l-(+)-glutamic acid dimethyl ester a novel, nonaqueous method for regeneration of aldehydes from bisulfite adducts an in vitro model of differentiated human airway epithelia. methods for establishing primary cultures ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia automatic indexing of rotation diffraction patterns data processing and analysis with the autoproc toolbox an introduction to data reduction: space-group determination, scaling and intensity statistics phaser crystallographic software n y times web china novel coronavirus investigating and research team, a novel coronavirus from patients with pneumonia in china towards automated crystallographic structure refinement with phenix.refine features and development of coot molprobity: all-atom structure validation for macromolecular crystallography developments in the ccp4 molecular-graphics project rapid generation of a mouse model for middle east respiratory syndrome principles and approaches for reproducible scoring of tissue stains in research scaling and assessment of data quality improved r-factors for diffraction data analysis in macromolecular crystallography global indicators of x-ray data quality resolving some old problems in protein crystallography linking crystallographic model and data quality we thank rudragouda channappanavar for performing the initial assays demonstrating drug efficacy against mers-cov. we thank d. george for technical assistance. key: cord-315316-w7cn9iqp authors: earnest, james t.; hantak, michael p.; li, kun; mccray, paul b.; perlman, stanley; gallagher, tom title: the tetraspanin cd9 facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases date: 2017-07-31 journal: plos pathog doi: 10.1371/journal.ppat.1006546 sha: doc_id: 315316 cord_uid: w7cn9iqp infection by enveloped coronaviruses (covs) initiates with viral spike (s) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound s proteins, which prompts s protein-mediated virus-cell membrane fusion. infection therefore requires close proximity of receptors and proteases. we considered whether tetraspanins, scaffolding proteins known to facilitate cov infections, hold receptors and proteases together on cell membranes. using knockout cell lines, we found that the tetraspanin cd9, but not the tetraspanin cd81, formed cell-surface complexes of dipeptidyl peptidase 4 (dpp4), the mers-cov receptor, and the type ii transmembrane serine protease (ttsp) member tmprss2, a cov-activating protease. this cd9-facilitated condensation of receptors and proteases allowed mers-cov pseudoviruses to enter cells rapidly and efficiently. without cd9, mers-cov viruses were not activated by ttsps, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. thus, we identified dpp4:cd9:ttsp as the protein complexes necessary for early, efficient mers-cov entry. to evaluate the importance of these complexes in an in vivo cov infection model, we used recombinant adenovirus 5 (rad5) vectors to express human dpp4 in mouse lungs, thereby sensitizing the animals to mers-cov infection. when the rad5-hdpp4 vectors co-expressed small rnas silencing cd9 or tmprss2, the animals were significantly less susceptible, indicating that cd9 and tmprss2 facilitated robust in vivo mers-cov infection of mouse lungs. furthermore, the s proteins of virulent mouse-adapted mers-covs acquired a cd9-dependent cell entry character, suggesting that cd9 is a selective agent in the evolution of cov virulence. introduction enveloped virus-cell entry requires glycoprotein-catalyzed fusion of viral and host cell membranes. these viral fusion glycoproteins are catalytically-inactive on virus particles and become triggered to mediate membrane mergers only in response to cellular and environmental factors. this triggering process ensures that virus-cell entry occurs at the appropriate time and place. the triggering factors include host cell receptors, endosomal acids, and proteases. many viruses require a single, soluble trigger, for example, influenza a virus fusion proteins are triggered by protons within the target-cell endosome [1] . other viruses require two triggering agents, for example, avian sarcoma leukosis virus fusion proteins are partially advanced into fusion-catalyzing forms after binding to host cell receptors, and then fully execute fusion after being exposed to endosomal protons [2] . most covs also require two triggering agents, receptor binding and proteolytic cleavage, with the proteolysis taking place on receptor-bound viral ligands [3] . as many of the cov-cleaving proteases are transmembrane-anchored, it follows that cov-susceptible cells might have the two triggering agents, receptors and proteases, in close proximity. here we considered whether the two cov entry factors are coalesced on cell surfaces to facilitate infection, and whether particular host cell features are required to juxtapose the two entry factors. the cov receptors are all transmembrane glycoproteins. their presence is a defining feature of host cell susceptibility to infection [4] [5] [6] [7] . proteases, the second required determinants of host susceptibility, are variable in type and subcellular location [8] , with proteases in the type ii transmembrane serine protease (ttsp) family figuring prominently [8] [9] [10] . ttsp family members, most notably the transmembrane protease serine type 2 (tmprss2), can cleave cov fusion glycoproteins (termed spike [s] proteins), into unlocked, fusion-catalyzing forms [8, 9, 11] at the cell surface and facilitate a rapid, "early" entry. studies examining hiv [12] and influenza [13] glycoproteins indicate that multiple adjacent fusion glycoproteins must be activated in order to successfully complete the fusion reaction. assuming similar requirements for cov fusion, it is likely that multiple s proteins need simultaneous receptor engagement and sufficient proteolytic cleavage to form an activated cluster that can pull opposing membranes together. thus, fusion likely occurs in regions of the cell membrane with a relatively high local concentration of these entry factors. recent studies have confirmed that ttsps are concentrated into punctate locations on the cell surface, in association with tetraspanin scaffolding proteins [14] . tetraspanins comprise a family of proteins with four transmembrane spans and two extracellular loops [15] . tetraspanins interact with other tetraspanins [16] and with other membrane-associated proteins [17, 18] , including transmembrane proteases [19, 20] , to form "webs" of interacting proteins [15] . there is evidence that these tetraspanin webs are locus points for cov-cell entry, as tetraspanin-specific antibodies protect several cell types from cov infection [14] . however, it remains unclear if individual tetraspanin proteins facilitate cov entry and what function they have in determining viral entry routes. as there are demonstrations that the tetraspanin cd9 interacts with the mers--cov receptor dipeptidyl peptidase 4 (dpp4) [21, 22] and hints of similar cd9 interactions with the hcov-229e receptor aminopeptidase n (apn) [23] , we hypothesized that cd9 is necessary to bring these virus receptors to ttsp-enriched regions on the cell surface. no study to date has determined the relative importance of individual tetraspanins and ttsps to mers-cov infection in the lung environment. indeed, there are 34 human tetraspanins and at least 17 members of the ttsp protease family [24] as well as several soluble extracellular proteases, such as elastases [25] , that may be expressed in the lung parenchyma. while studies suggest that tmprss2 can trigger mers-cov in cell culture [9, 25] , it is unclear whether cd9 or tmprss2 stand out in vivo as single proviral members of their respective protein families. therefore, we set out to determine whether, and to what extent, mers-cov utilizes cd9 and tmprss2 during in vivo infection. to this end, we established a mouse model in which virus-resistant mice are rendered susceptible to mers-cov infection by expression of human dpp4 (hdpp4). the system utilizes a recombinant adenovirus type 5 (rad5) to transduce the hdpp4 gene, thereby sensitizing only the ad5-transduced lung cells to subsequent mers-cov infection [26] . the rad5-hdpp4 vectors were engineered to include additional genes encoding the potential virus-promoting factor human tmprss2 [9] or potential virus-restricting factors, in the form of shrnas targeting murine tmprss2 and cd9. we considered the rad5-hdpp4 system to be especially valuable, as mers-cov infection can only occur in cells expressing hdpp4 and, thus, only in cells simultaneously expressing the putative virus-promoting or virus-restricting factors. using the dual-expressing rad5 vectors, as well as tetraspanin knock-out cell lines, we evaluated the roles for cd9 and another related tetraspanin, cd81, in dictating receptor clustering with proteases and in promoting cov infection. our results indicate that a cov-cell entry portal is a multipartite complex that minimally includes the virus receptor, a virus-activating protease, and one or more tetraspanins. these complexes are responsible for the majority of mers-cov entry in lung cells. furthermore, cd9 facilitated cell entry by mers-cov spikes that were adapted for lung virulence, but cd9 provided no support to cell culture-derived, avirulent spike-mediated cell entry. these data establish tetraspanins as factors controlling early entry events in pathogenic cov infections. tetraspanins cd9 and cd81 are known to influence enveloped virus entry [14, 27, 28] . therefore, we used crispr/cas9 technology [29] to eliminate these tetraspanins from cells, with the expectation that this would affect cell susceptibility to covs. 293t and hela cells were transfected with cas9/guide rnas targeting cd9 or cd81, selected for puromycin resistance, and cloned by endpoint dilution. all ko cell lines grew equivalent to parallel "wt" control clones, and the only observable distinctions were with the cd9ko cells, which adhered less tightly to plastic than wt or cd81ko cells. western blot analyses of the wt and ko clones confirmed the absence of cd9 or cd81, with maintenance of a control tetraspanin cd63 (fig 1a) . interestingly, cd81 levels were highest in cd9ko cells and cd9 levels were low in cd81ko cells, possibly due to heterotypic cd9-cd81 interactions influencing tetraspanin stability. lower-resolution immunofluorescent assays (ifas) of umpermeabilized cells showed similar cell-surface cd9 in wt and cd81ko cells, confirmed the absence of the respective tetraspanins in ko cells, and demonstrated that cd63 distribution remained unchanged in all cell lines ( fig 1b) . to determine whether the tetraspanins cd9 or cd81 operate in cov entry, we utilized hiv pseudoparticle (pp) transduction methodologies, which allow for a specific focus on the viruscell entry stage. we first sensitized the cells to transduction by overexpressing cov receptors, then transduced cells with the respective covpps. relative to wt cells, cd9ko cells were 94% less susceptible to mers (emc strain) pp transduction (fig 1c) , and 80% less susceptible to 229epp transduction ( fig 1d) . however, cd9ko cells remained fully susceptible to sarspp or mhvpp transduction (fig 1e and 1f ). cd9 complementation restored susceptibility to merspp and 229epp transductions (fig 1c and 1d ). cd81 ko cells were fully susceptible to all four of the covpps (s1 fig) . these data identify an individual tetraspanin, cd9, as an entry factor for a cov. to determine whether receptor overexpression might have contributed to cd9 dependence, merspps were also transduced into cells containing endogenous cov receptor levels. consistently, cd9 was necessary to fully sensitize cells to merspps (s2 fig), indicating that cd9 proviral activity was independent of hdpp4 receptor levels. however, cd9 was not necessary for merspp transduction into cells overexpressing tmprss2, a mers-cov activating protease (s2 fig). the fact that tmprss2 obviated the cd9 requirement indicated a role for cd9 in proteolytic activation of cov entry. the observation that a single tetraspanin family member, cd9, promoted cell entry for some, but not all covs, suggested that cd9 interacts with one or more mers-cov and 229e-cov entry factors. we considered whether cd9 associates with dpp4 and apn, the mers-cov and 229e-cov receptors, or with tmprss2. furthermore, we considered whether cd9 does not interact with ace2 and ceacam, the receptors for cd9-independent sars and mhv-covs. this was first investigated through biochemical isolation of tetraspanin-enriched membrane fractions, and detection of tetraspanin-associated receptors and proteases. to this end, cd9 or cd81 ko cells overexpressing cov receptors or tmprss2 were surface-biotinylated, and tetraspanins were liberated from cells using zwitterionic chaps detergent, which solubilizes cell membranes while leaving tetraspanin-mediated protein interactions largely intact [30] . low-density (ld) fractions, with ρ<1.13 g/ml, were then separated from high-density (hd) chaps-solubilized proteins on sucrose density gradients [31] . as evaluated by streptavidin pull-down and western immunoblotting, the ld sucrose gradient fractions from chapssolubilized cells contained nearly 100% of cell-surface tetraspanins (s3 fig) , but only~20% of the surface-biotinylated plasma membrane proteins [14] , indicating efficient tetraspanin segregation into ld fractions. strikingly, the ld fractions from wt control cells contained~60% of cell-surface dpp4, while ld fractions from cd9 ko cells completely lacked dpp4 (fig 2a, rows 1 and 2 ). complementing cd9 back into cd9ko cells restored ld-associated dpp4 (fig 2a, row 3) . the presence or absence of cd81 had no effect on dpp4 distribution between hd and ld fractions (fig 2a, rows 4 and 5). similar results were observed with the 229e receptor apn (fig 2b) . by contrast, cd9 and cd81 expression had little effect on the distribution of ace2, ceacam, or tmprss2, all of which distributed about equally between ld and hd fractions (fig 2c-2e ). these data indicated that dpp4 and apn positioning into tetraspanin-enriched membranes required cd9. the fact that cd9 repositioned dpp4 and apn, but not ace2 or ceacam, correlated with the fact that cd9 promoted the entry of dpp4and apn-utilizing mers and 229e viruses, but not ace2-or ceacam-utilizing sars and mhv viruses (fig 1) . the evaluations of membrane fractions suggested that cd9 might localize the mers-cov and 229e-cov receptors close to virus-activating tmprss2. to determine whether cd9 facilitates specific interactions between dpp4 and tmprss2, we analyzed intact tetraspanin microdomains in situ. we performed proximity ligation assays (plas), which can determine whether two or more transmembrane proteins are adjacent [32] . in plas, antibodies differentially tagged with oligonucleotide probes are applied to cells, and their close spacing (<40 nm) allows for probe hybridization into dna polymerization templates, which provide a locus point for fluorescent dna synthesis [33] . plas have been used to identify interactions between tetraspanins and their partner proteins [34, 35] and we used this method to analyze clustering of two tetraspanin partner proteins. hela cells were chosen for plas because their relatively flat morphology facilitated quantification of fluorescent foci, and because our quantitative reverse transcriptase-pcr measurements revealed endogenous expression of cd9, dpp4 and tmprss2 (s1 table) . notably, cd9 transcripts were plentiful in the hela cells (~10 times more abundant than the reporter gene hprt), while dpp4 and tmprss2 were scarce (~50 times less abundant than hprt, and 5-to 100 times less than that found in several human airway epithelia-derived cell cultures (see s1 table) ). thus, we presumed that, with hela cells, we could readily detect a cd9-directed coalescence of sparse dpp4 and tmprss2. we performed plas on unpermeabilized cd9ko hela cells, using primary antibodies to cd9, dpp4, and/or tmprss2. following secondary antibody incubation and amplification of ligated oligonucleotide templates, punctate fluorescent dnas were detected by confocal microscopy and counted using imaris version 6.3.1 software. using hdpp4 and htmprss2 antibodies, fluorescent foci were rarely observed on the hela-cd9ko cells (fig 3a) , and the cells were only modestly susceptible to merspp transduction ( fig 3h, leftmost bar) . when cd9 was replenished in the cd9ko cells, foci were~10-fold more abundant (fig 3d) , and these increased foci correlated with a greater cell susceptibility to merspp transduction ( fig 3h) . these findings argue that cd9 sensitizes cells to mers-cov entry by bringing dpp4 and tmprss2 into proximity. we considered whether this role for cd9 applied only when dpp4 and tmprss2 levels were low, i.e., at endogenous hela-cell levels. thus, hdpp4 and htmprss2 were forcibly overexpressed; with overexpression,~30 foci/ cell were observed (fig 3e) , and this increased to~80 foci/cell in the presence of cd9 ( fig 3f) . merspp entry into cells correlated with the number of foci present, at least for values up to3 0 foci/cell ( fig 3h) . overall, these results indicated that cd9 connects dpp4 and tmprss2 entry factors, and is necessary for their proximity when they are sparse on cell surfaces. the cd9:dpp4:tmprss2 complexes then function as mers-cov entry portals. cd9 also helped to connect overexpressed dpp4 and tmprss2 together, but in this overexpression condition, cd9 did not increase merspp transduction, perhaps because other tetraspanins come in to bridge the abundant receptors and proteases. these results also revealed cd9-directed dpp4: tmprss2 complexes on intact cells in the absence of virus, suggesting that the covs infect through pre-existing complexes. because cd9 brought dpp4 in proximity with ttsps, we hypothesized that cd9 facilitates ttsp-mediated early cell entry at or near plasma membranes, but does nothing to support the late, endosomal route that is enabled by cathepsin proteases. to test this, we inactivated cellular ttsps using camostat [25] and found that camostat suppressed merspp transduction into wt cells by~50%, but did not affect transduction into cd9ko cells ( fig 4a) . cd9 complementation modestly restored merspp sensitivity to camostat. furthermore, cd81 had no effect, as merspp entry into cd81ko and cd81-positive cells were equally suppressed by camostat ( fig 4a) . these data were consistent with cd9 specifically enabling ttsp-directed, early virus entry. without cd9, the merspp entry route may be directed to a late, endosomal stage in which cathepsins provide fusion-activating triggers. to test this, we blocked late entry in wt and cd9ko with 100 μm bafilomycin a (baf), an inhibitor of endosome acidification, or with 10 μm e64d, a cysteine protease inhibitor. in wt cells, baf did not significantly decrease merspp entry, while e64d decreased entry~4-fold ( fig 4b) . however, in cd9ko cells, these inhibitors were far more antiviral, decreasing entry 20-and 100-fold, respectively. complementing cd9 back into the cd9ko cells restored the wt phenotype in which the inhibitors were only weakly antiviral (fig 4b) . these differential effects of the inhibitors were not observed in cd81ko or cd81-overexpressing cells (fig 4b) . we conclude that cd9 is necessary for ttsp-mediated mers early entry. we advanced to evaluating mers-cov entry factors in vivo. of note, a previous study has demonstrated that camostat inhibits sars-cov spread in mouse lungs [36] , suggesting that the virus exhibits dependence on serine proteases, probably ttsps, for its entry in vivo. however, the importance of specific ttsps, or for tetraspanins, is unknown for any in vivo cov infection. here we established infections in the mouse lung under conditions in which putative cov entry factors were reduced. to do this, we developed dual-expressing recombinant adenovirus 5 (rad5) vectors expressing both human dpp4, which sensitizes mouse cells to mers-cov infection [26, 37, 38] , and shrnas that knock down tmprss2 or cd9 mrnas. in initial experiments, these rad5 vectors were transduced into mouse lung epithelial type 1 (let-1) cells, a line derived from c57/bl6 mouse alveolar type 1 cells [39] . after 3-days, the cells were analyzed for the presence of hdpp4, tmprss2, and cd9 by western blot (fig 5a) . relative to the control rad5-gfp transductions, all single and dual-expressing rad5-hdpp4 transductants contained recognizable dpp4 and tmprss2, and those ad5 vectors expressing shrnas reduced the levels of endogenous cd9 proteins (fig 5a) . due to endogenous tmprss2 protein levels being too low for detection on immunoblots, we used qrt-pcr to quantify tmprss2 transcripts. let-1 cells transduced with rad5-hdpp4-shtmprss2 had only 25% of the transcripts of cells transduced with rad5-hdpp4-empty vector (fig 5b) . this level of tmprss2 transcripts indicated an efficient knockdown of tmprss2 in the approximately 75% of cells that were successfully transduced. these results indicate that the different rad5 vectors, transduced into cells derived from mouse alveolar epithelia, consistently express equivalent levels of hdpp4, while simultaneously increasing or decreasing tmprss2 or cd9. to determine whether the rad5-transduced let-1 cells were susceptible to mers-cov s protein-directed virus entry, the cells were inoculated with recombinant vsvs encoding firefly luciferase [40] and pseudotyped with mers-cov s proteins. as expected, hdpp4 expression established susceptibility to vsv-merspp transduction (fig 5c) . tmprss2 co-expression from the ad5 vectors increased susceptibility to merspps by~4-fold, while shtmprss2 and shcd9 both restricted merspps by~3 fold (fig 5c) . these results indicated that cd9 and tmprss2 act as entry factors in mouse lung-derived let-1 cells, and suggested that the dualexpressing ad5 vectors might be effective tools for identifying viral entry factors in the mouse lung. to identify the role of cd9 and tmprss2 in vivo, the ad5 vectors were instilled intranasally into mice which were, after 5 days, challenged with mers-cov. lungs were harvested 2 days post-infection (d.p.i.) and mers-cov titers were measured as pfu/gram of tissue. relative to mers-cov titers in rad5-hdpp4 transduced animals, the mers-cov titers in rad5-hdpp4-shcd9 transduced animals were~20-fold lower (fig 5d) . furthermore, the mers-cov titers in rad5-hdpp4-shtmprss2 transduced mice were reduced by~10-fold. interestingly, overexpression of tmprss2 by the rad5-hdpp4-tmprss2 vector had no effect on mers-cov titers in the lungs, presumably because the lung environment has sufficient endogenous murine tmprss2 to facilitate efficient mers-cov infection. these data indicate that cd9 and tmprss2 act as mers-cov susceptibility factors in the lung parenchyma and that their role in entry is slightly more pronounced in vivo than in in vitro let-1 mouse alveolar cell cultures. indeed, these data show that cd9 and tmprss2 are responsible for~90% of mers-cov titers in vivo. virulent mers viruses utilize cd9-dependent early entry mers-cov, a camel and human virus [41, 42] , has recently been adapted for robust growth and virulence in hdpp4 + mouse lungs [43, 44] . this adaptation process was initiated by intranasally infecting mice with avirulent, vero cell culture-adapted (cca) mers-covs and then serially passaging viruses through hdpp4 + mouse lungs. relative to cca mers-covs, the mouse-adapted (ma) viruses have distinct s protein changes [44] (s2 table) . we considered whether these ma changes fixed into s proteins adapt viruses to utilize cd9-facilitated early entry. to address this question, we produced vsv-based merspps, pseudotyped with the cca or ma s proteins. these cca and ma merspps were transduced into cd9-replete or cd9-knocked down (cd9kd) let-1 cells. the cd9-replete and cd9kd cells were equally susceptible to cca s-mediated pp entry. however, the same cd9kd cells had 90% and >95% reduced susceptibility to ma1 and ma2 s-driven pp entry, respectively (fig 6a) . thus, it appears that in vivo passage in mouse lungs adapts mers-covs to a cd9-facilitated cell entry pathway. the ma viruses' utilization of cd9 for entry correlated with their relatively rapid entry kinetics [44] . furthermore, cd9-facilitated entry correlates with ttsp utilization (fig 4) , and ttsp utilization correlates with rapid cov entry into cells [45] . therefore, we hypothesized that cd9 is a determining factor in cov-cell entry kinetics. to test this, cca and ma merspps were transduced into cd9-replete or cd9kd let-1 cells. to measure pp entry kinetics, the transduction process was abruptly halted at defined time points with a nontoxic protease inhibitor cocktail that prevents s-directed fusion, but has no effect on transduced reporter gene expression [45] . this strategy allows fluc reporter accumulations to indicate the extents of virus entry taking place within timed intervals. we found that cd9 had no influence on the rate of cca s-directed virus entry. in both cd9-replete and cd9-kd cells, half-maximal entry was complete within 45 min (fig 6b) . a 30-45 min half time for virus entry is found for several viruses requiring endocytosis prior to genome delivery [46] . however, cd9 strongly influenced ma s-mediated virus entry. the ma1 and ma2 pps reached 50% entry in cd9-replete cells in 20 and 19 minutes, but were delayed to 34 and 30 minutes, respectively, in cd9kd cells (fig 6c and 6d) . these data were corroborated with the tetraspanin ko cell lines (s4 fig). thus, we conclude that cd9 utilization and rapid cell entry correlate with mouse adaptation and mers virulence in mouse lung infections. in this study, we demonstrated that the mers coronavirus enters cells using an entry complex that includes a receptor, a protease and a tetraspanin. the tetraspanin operates by bringing the receptor and protease(s) into proximity, such that viral spikes, once attached to receptors, are quickly and efficiently cleaved into fusion-activated forms. these ternary complexes pre-exist on virus-target cells and can theoretically have highly variable subunit composition. presently, there are three known human cov receptors, 17 human transmembrane serine proteases, and 34 human tetraspanins, and therefore there are thousands of potential combinations of receptor, protease and tetraspanin that might provide a coronavirus entry platform. for the mers coronavirus, a particularly effective complex includes the hdpp4 receptor, the protease tmprss2, and the tetraspanin cd9. this was discovered, in large part, through creative use of recombinant adenoviruses (rads). the approaches used here extended from the finding that a transducing rad5 expressing the mers-cov receptor hdpp4 sensitized laboratory mice to mers-cov infection [26] . by incorporating rna silencing genes into rad5-hdpp4, we were able to simultaneously establish mers-cov susceptibility, through hdpp4 expression, and potentially restrict mers-cov, through shrna-mediated suppression of candidate proviral factors. thus, the dual-expressing rad5 vectors revealed cd9 and tmprss2 as relevant proviral factors, operating to support the primary hdpp4 susceptibility factor. it is notable that dual-expressing adenovirus vectors can potentially be utilized to identify any mers-cov host factor. indeed, they can be utilized to identify host factors in any virus-host system that requires an exogenously supplied susceptibility determinant. furthermore, the adenovirus transduction process bypasses the need to establish partially humanized mice for studies of human coronavirus infections, and actually has distinct advantages over transgenic animals, in that shrnas reduce pro-or anti-viral factors solely in coronavirusinfectable cells, making for reliable measurements of changes in virus susceptibility. we expect that dual-expressing rad5 vectors will be excellent general tools to rapidly identify in vivo proand anti-viral host factors. the hdpp4:cd9:tmprss2 complexes promoted an "early" mers-cov entry. cov s proteins require simultaneous receptor engagement and proteolysis before catalyzing virus-cell membrane fusion [3, 47, 48] , a process demanding that tmprss2 be closely juxtaposed to hdpp4. we suggest that cd9 tetraspanins position tmprss2 next to the receptor-bound s proteins, perhaps in association with cholesterol, a lipid having profound effects on both tetraspanin structural interactions [49] and cov entry [50] [51] [52] [53] [54] . precisely how tmprss2 abuts against hdpp4 to access s proteins is not clear, although the structures of three cov s proteins [55, 56] and hdpp4 [57] indicate that the proteolytic cleavage sites would be displayed at the outer edges of each s trimer. furthermore, it is likely that proteolytic cleavage of several adjacent s proteins is needed to activate membrane fusion, as cooperative "pulling" by several viral fusion proteins is frequently required for virus entry processes [12, 13, 58] . therefore, the tetraspanin-enriched environment, in which dpp4 and tmprss2 are collected together, likely permits rapid and simultaneous cleavage of multiple, closely-spaced virion s proteins, generating clusters of activated s proteins that drive the membrane fusion process. without cd9, the hdpp4 and tmprss2 are not held closely together (fig 3) . in this condition, mers-cov still infects hdpp4-positive target cells (fig 3) , but it takes a slower "late" endosomal route, which we and others find to be around 90% less efficient than early entry (figs 1, 5 and 6 ) [25] . in the late entry route, virus-associated s proteins are first endocytosed and then cleavage-activated by furin proprotein convertases [59, 60] , cathepsin l [8, 25, 61, 62] , and/or cathepsin b [63] . however, the protease-enriched endo-lysosomal environment [64] can also generate inactivating cov s protein cleavages, as evidenced by c-terminal s protein fragments, 40 kda and smaller, that must be inactivated fusion domain fragments [48, 65] . therefore, in the late entry route, there may be a short time span between a cathepsin-activated fusogenic state and a permanently inactivated, excessively proteolyzed state, accounting for inefficient entry. inefficient late entry may also be explained by differences in lysosomal and plasma membranes, which have unique lipid profiles [66] and therefore may be differentially susceptible to s -catalyzed fusion. finally, late entry is restricted by interferon-induced gene products, notably interferon-induced transmembrane (ifitm) proteins [67, 68] , but early ttsp-facilitated entry is not [8] . all of these virus-restricting conditions may combine in vivo to make cd9-facilitated "early" cell entry the predominant route for mers-cov infections. that the ttsp-facilitated entry route is predominant in vivo is supported by the recent finding that serine protease inhibitors reduce sars-cov infection in mouse lungs [36] . additionally, clinical hcov-229e isolates use a rapid ttsp-facilitated entry route, unlike labadapted hcov-229es [45] . more recently, similar patterns were observed for mers-covs. mouse lung-adapted mers-covs take a rapid tmprss2-mediated cell entry, while cell culture-adapted (cca) mers-covs are avirulent and enter cells through the slower and less efficient endocytic route [44] . here we demonstrated that the virulent ma mers-cov s proteins utilized cd9 during cell entry, while avirulent cca viruses did not. this new finding suggests that cov receptors and proteases alone are not the selective agents in cov adaptation. rather, the covs adapt to the ternary receptor-tetraspanin-protease complexes. in the case of mers--cov, the key adaptive s mutation facilitating the usage of the ternary complexes was at position 1015 (s2 table) . in the mers-cov s protein cryo-em structure [55, 56, 69] , this residue 1015 is part of a peptide that connects two of the helices comprising the fusion domain. the change from n1015 to t may ease restrictions to conformational change in the s trimer, thereby exposing cleavage sites to the nearby cd9-associated transmembrane protease, with cleaved spikes then converting to fusion-active forms. finally, these findings may shed light on general roles for tetraspanins in virology. four cov receptors (dpp4, apn, ace2, and ceacam) were found in tetraspanin-rich membrane fractions (fig 4) , and our previous report indicated that tetraspanin antibodies block several cov infections by interfering with receptor-associated cov access to surface proteases [14] . even antibodies binding to cd81 suppressed mers s-mediated entry [14] , indicating that several tetraspanins, including those that are not required per se for clustering hdpp4 and tmprss2, organize into cell-surface "webs" [15] and enclose the cov entry factors. here, there may be parallels with several tetraspanin-facilitated viruses, including influenza a (iav) [28] and canine distemper (cdv) [70] ; the retroviruses hiv [71, 72] , feline immunodeficiency virus (fiv) [73] , and human t-lymphocytic virus 1 (htlv-1) [74] ; herpes simplex virus 1 (hsv-1) [75] ; hepatitis c virus [76] ; and several human papillomaviruses (hpvs) [77] . for these viruses, tetraspanins facilitate viral entry (covs, iavs, hcv, hpvs) syncytia formation (cdv, hiv, fiv, htlv-1), or promote viral exit (iavs, hsv-1 and hiv), by unclear mechanisms. conceivably, a common mechanism may involve tetraspanin-mediated clustering of host factors. for example, tetraspanin cd81 is both an hcv receptor [78] and a linker of the hcv co-receptors scavenger receptor class b i (sr-bi) [76] and claudin-1 [79] , whose complexing promotes viral endocytosis (reviewed in [80] ). another example is with the tetraspanins cd151 and cd63, which do not directly interact with hpvs, but rather hold several coreceptors together to permit hpv binding and endocytosis (reviewed in [81] . therefore, many of the proviral activities ascribed to tetraspanins may relate to their ability to cluster transmembrane proteins, as we have found for the pro-mers-cov activity of cd9. given that several viruses depend on tetraspanin webs, it may be useful to consider ways to target entry-blocking drugs to these locations and thereby increase their antiviral efficacy. c57bl/6 mice were purchased from the national cancer institute and housed in the animal care facility at the university of iowa. the mers-cov (emc2012 strain) was provided by drs. bart haagmans and ron fouchier (erasmus medical center). hek293t and hela cells were obtained from dr. edward campbell (loyola university chicago) and maintained in dulbecco's modified eagle media (dmem) supplemented with 10% fetal bovine serum (fbs, atlanta biologicals), 10 mm hepes, 100 mm sodium pyruvate, 0.1 mm non-essential amino acids, 100 u/ml penicillin g, and 100 μg/ml streptomycin. let-1 cells were obtained from bei resources and were maintained in dmem supplemented with 10% fbs, 100 u/ml penicillin g, and 100 μg/ml streptomycin. cells were maintained in a humidified environment at 37˚c and 5% co 2 . hae cultures were isolated and maintained as described previously [48] . this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. animal experiments were approved by the institutional animal care and use committee at the university of iowa (protocol #4041009). codon-optimized mers-cov s containing a c9 tag was purchased from genscript and subsequently cloned into pcdna3.1+ between the ecori and noti restriction sites. pcdna3.1-229e-spike-c9 and pcdna3.1-hapn plasmids were provided by dr. fang li, (university of minnesota). pcdna3.1-sars-spike-c9 and pcdna3.1-ace2-c9 plasmids were provided by dr. michael farzan (scripps research institute). c-terminal flag-tagged human dpp4 plasmid pcmv6-entry-hdpp4 (ncbi reference sequence nm_001935) was purchased from ori-gene. pcaggs-tmprss2-flag was previously constructed [82] . the pnl4.3-hivluc plasmid was provided by the nih aids research and reference library. pcmvsport6-hcd9 was purchased from open biosystems. pspcas9-bb-2a-puro was a gift from feng zhang (addgene plasmid # 52961). monoclonal mouse antibodies against cd9 (clone m-l13), cd63 (clone h5c6), and cd81 (clone js-81) were obtained from bd pharmingen. rabbit anti-flag was obtained from sigma aldrich. mouse anti-rhodopsin antibodies were obtained from millipore. rabbit anti-cd13 (apn) antibodies were obtained from abcam. mouse anti-cd26 (clone m-a261) was obtained from bd biosciences. rabbit anti-tmprss2 (clone epr3681) was obtained from abcam. secondary antibodies were purchased from invitrogen and include goat-anti-rabbit-alexafluor 488, goat-anti-mouse-alexafluor 488, and goat-anti-mouse-alexafluor 568. donkey-anti-goat, goat-anti-mouse, and goat anti-rabbit hrp conjugated antibodies were purchased from thermo scientific. recombinant adenovirus vectors were produced as previously described by the university of iowa gene transfer vector core [83] . to generate tmprss2-expressing adenoviruses, htmprss2 containing a c-terminal flag tag was cloned into the pad5cmv shuttle vector between xhoi and ecori restriction sites. to generate shrna-expressing adenoviruses, gene blocks containing an shrna targeting either the coding region of cd9 (target sequence: ccgattcgactctca gaccaa) or the 3' utr of tmprss2 (target sequence: acactagagtggatgaatgt ctgga), flanked by the u6 promoter and rnapoliii termination sequence, were purchased from genscript. these gene blocks were subcloned into the pacad5k-npa e1 shuttle vector between the kpni and ecori restriction sites. shuttle vectors were linearized and transfected into hek 293 cells along with a linearized ad5 backbone containing an rsv promoter -driven hdpp4 in the e3 region. homologous recombination in hek 293 cells yielded recombinant adenovirus encoding both the shrna and hdpp4. titers of purified recombinant adenoviruses ranged from 10 10 −10 11 pfu/ml. isoflurane-anesthetized mice were transduced intranasally with 2.5 x 10 8 pfu of the indicated ad5 virus in 75 μl of dmem. 5 days posttransduction, mice were infected intranasally with 10 5 pfu of mers-cov in a total volume of 50 μl dmem. at 2 d.p.i., mice were euthanized by isoflurate inhalation followed by cervical dislocation. lungs were removed into pbs and manually homogenized. virus was plaqued on vero81 cells. cells were fixed with 10% formaldehyde and stained with crystal violet 3 d.p.i. all work was performed in the university of iowa biosafety level 3 (bsl3) laboratory. hiv pseudoviruses were produced as previously described [84] . briefly, 293t cells were cotransfected with pnl4.3-hiv-luc and pcdnas encoding appropriate glycoproteins. after two days, supernatants were collected, centrifuged at 10,000 x g at 4˚c for 10 minutes to remove debris, and stored in aliquots at -80˚c. vsv pseudoviruses were produced by the methods of whitt, 2010 [40] . briefly, 293t cells were transfected with plasmids encoding viral glycoproteins. two days later, cells were inoculated for 2h with vsvδg-luciferase [40] , rinsed extensively and incubated for one day. supernatants were collected, centrifuged at 10,000 x g at 4˚c for 10 minutes to remove cellular debris, and stored in aliquots at -80˚c. pseudovirus transductions were carried out by incubating target cells with pseudoviruses for 1h at 37˚c. following initial incubation, unadsorbed viruses were removed by washing thrice with pbs. complete media was placed on the cells and incubated for 18h for vsv or 48h for hiv at 37˚c. at the end of transduction periods, cells were dissolved into cell culture lysis buffer (25 mm tris-phosphate [ph 7.8], 2 mm dtt, 2 mm 1,2-diaminocyclohexane-n, n,n 0 ,n 0 -tetraacetic acid, 10% glycerol, 1% triton x-100) and luciferase levels were measured by addition of firefly luciferase substrate (1 mm d-luciferin, 3 pspcas9-bb-2a-puro was digested with esp3i (fermentas) for 4h at 37˚c. the digested plasmid was purified and ligated with annealed guide dnas specific for cd9 or cd81. tetraspanin-specific pspcas9-bb-2a-puro plasmids were transfected into 293t cells. after 72h, cells were selected with 4 μg/ml puromycin for 96h. selected cells were serially-diluted to isolate clonal populations and clones were selected by western blot. adherent 293t cells (~10 5 / cm 2 ) were rinsed with ice-cold pbs, incubated for 30 min at 4˚c with pbs-1 mg/ml ez-link sulfo-nhs-lc-biotin (pierce), then for 20 min at 4˚c with pbs-100 mm glycine. cells were rinsed with pbs, then incubated for 20 min at 4˚c in mes buffer (25 mm mes [ph 6.0], 125 mm nacl, 1 mm cacl 2 , 1 mm mgcl 2 ) containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (chaps) detergent (calbiochem cat # 220201) or 1% triton x-100 detergent (sigma). cell lysates (10 7 /ml) were removed from plates and emulsified by 20 cycles of extrusion through 27g needles. nuclei were removed by centrifugation, lysates mixed with equal volumes of 80% w/v sucrose in mes buffer, placed into beckman sw60 tubes, and overlaid with 3 ml of 30% w/v sucrose, then with 0.5-ml of 5% w/v sucrose, both in mes buffer. samples were centrifuged with a beckman sw60 rotor at 370 k x g for 18 h at 4˚c. fractions were collected from air-gradient interfaces. biotinylated proteins in gradient fractions were bound to streptavidin agarose beads (pierce). non-reducing western-blotting procedures were used to identify the distributions of proteins in gradient fractions, as described previously [38] . hela cells were transfected with indicated plasmid dnas and a gfp reporter, incubated for two days, and then lifted from tissue culture plates using 0.05% trypsin. cells were transferred to microscope coverslips coated with fibronectin. cells were allowed to adhere for 24h. cells were then fixed for 30 minute at 37˚c with 3.7% paraformaldehyde in 0.1 m piperazine-n,n 0bis(2-ethanesulfonic acid) buffer (ph 6.8). coverslips were washed with pbs and pla was performed using duolink proximity ligation assay (sigma-aldrich) using primary antibodies against tmprss2 and cd26. images were captured using a deltavision microscope (applied precision) equipped with a digital camera (coolsnap hq; photometrics), using a 1.4-numerical aperture 60x objective lens. images were deconvoluted with softworx deconvolution software (applied precision). pla foci were detected and quantified using imaris version 6.3.1 (bitplane scientific solutions). 293t cells were transfected with dpp4 and either an empty vector or complementing tetraspanin. 24h after transfection, cells were plated in a 96-well plate. merspps were added to cells at 4˚c for 1 hour to allow viral binding. media was removed and replaced with 37˚c media and the plates were moved to an incubator. at sequential time points following the shift to 37˚c, a protease inhibitor cocktail was added to cells such that the final concentration was 100 μm camostat, 10 μm proprotein convertase inhibitor, and 10 μm e64d. these drugs were left on cells overnight before cells were lysed and luciferase was measured as described above. luciferase levels were compared to that of cells treated only with dmso control. 293t cells were transfected with dpp4 and an empty vector or the complementing tetraspanin. cells were pre-treated for 1h with 100 μm camostat, 100 μm bafilomycin, or 10 μm e64d before transduction with merspps in the presence of the inhibitors. after 2h, cells were washed to remove drugs and unadsorbed virus. luciferase assays were performed as described above. quantitative reverse transcriptase-pcr cellular rna was isolated using the rneasy mini kit (qiagen) and 100 or 500 ng was reverse transcribed using an iscript cdna synthesis kit (bio-rad). quantitative pcr was performed using power sybr green (thermo fisher) and primers specific to human cd9, dpp4, tmprss2, or hprt. statistical comparisons were made by two-tailed student's t-test. error bars in the figures indicate the standard error of the data. non-linear regression analysis was used to fit a curve to the entry kinetics data and obtain the time of 50% infection. this analysis was performed using minitab 17 software. cleavage of influenza virus hemagglutinin by airway proteases tmprss2 and hat differs in subcellular localization and susceptibility to protease inhibitors retroviral entry mediated by receptor priming and low ph triggering of an envelope glycoprotein two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis cloning of the mouse hepatitis virus (mhv) receptor: expression in human and hamster cell lines confers susceptibility to mhv angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human aminopeptidase n is a receptor for human coronavirus 229e dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc tmprss2 activates the human coronavirus 229e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease estimating the stoichiometry of human immunodeficiency virus entry influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains lateral organization of membrane proteins: tetraspanins spin their web. the biochemical journal functional domains in tetraspanin proteins. trends in biochemical sciences ewi-2 and ewi-f link the tetraspanin web to the actin cytoskeleton through their direct association with ezrin-radixinmoesin proteins direct extracellular contact between integrin α3β1 and tm4sf protein cd151 functional interplay between tetraspanins and proteases. cellular and molecular life sciences: cmls the sheddase activity of adam17/tace is regulated by the tetraspanin cd9. cellular and molecular life sciences: cmls proteomic analysis of the tetraspanin web using lc-esi-ms/ms and maldi-fticr-ms cd9 negatively regulates cd26 expression and inhibits cd26-mediated enhancement of invasive potential of malignant mesothelioma cells tetraspanins: push and pull in suppressing and promoting metastasis type ii transmembrane serine proteases middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 rapid generation of a mouse model for middle east respiratory syndrome interacting regions of cd81 and two of its partners, ewi-2 and ewi-2wint, and their effect on hepatitis c virus infection dual function of cd81 in influenza virus uncoating and budding genome engineering using the crispr-cas9 system tetraspanin functions and associated microdomains evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts direct observation of individual endogenous protein complexes in situ by proximity ligation two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements hepatitis c virus infection propagates through interactions between syndecan-1 and cd81 and impacts the hepatocyte glycocalyx plasma membrane tetraspanin cd81 complexes with proprotein convertase subtilisin/kexin type 9 (pcsk9) and low density lipoprotein receptor (ldlr), and its levels are reduced by pcsk9 protease inhibitors targeting coronavirus and filovirus entry mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection characterization of innate responses to influenza virus infection in a novel lung type i epithelial cell model. the journal of general virology generation of vsv pseudotypes using recombinant deltag-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines evidence for camel-to-human transmission of mers coronavirus a mouse model for mers coronavirus-induced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice clinical isolates of human coronavirus 229e bypass the endosome for cell entry high-content analysis of sequential events during the early phase of influenza a virus infection inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism differential effect of cholesterol on type i and ii feline coronavirus infection requirements for ceacams and cholesterol during murine coronavirus cell entry importance of cholesterol-rich membrane microdomains in the interaction of the s protein of sars-coronavirus with the cellular receptor angiotensin-converting enzyme 2 murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer pre-fusion structure of a human coronavirus spike protein structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 modeling how many envelope glycoprotein trimers per virion participate in human immunodeficiency virus infectivity and its neutralization by antibody host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry proteases and proteolysis in the lysosome the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies membrane lipids: where they are and how they behave ifitm proteins inhibit entry driven by the mers-coronavirus spike protein: evidence for cholesterol-independent mechanisms distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cd9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus assembly differentially alters dynamics and partitioning of tetraspanins and raft components formation of syncytia is repressed by tetraspanins in human immunodeficiency virus type 1-producing cells inhibition of feline immunodeficiency virus infection by cd9 antibody operates after virus entry and is independent of virus tropism interaction of cd82 tetraspanin proteins with htlv-1 envelope glycoproteins inhibits cell-to-cell fusion and virus transmission egress of hsv-1 capsid requires the interaction of vp26 and a cellular tetraspanin membrane protein cell entry of hepatitis c virus requires a set of co-receptors that include the cd81 tetraspanin and the sr-b1 scavenger receptor tetraspanin cd151 mediates papillomavirus type 16 endocytosis binding of hepatitis c virus to cd81 inhibition of hepatitis c virus infection by anti-claudin-1 antibodies is mediated by neutralization of e2-cd81-claudin-1 associations the tetraspanin cd151 in papillomavirus infection a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry a simple method for the rapid generation of recombinant adenovirus vectors labeling hiv-1 virions with two fluorescent proteins allows identification of virions that have productively entered the target cell the authors thank drs. bart haagmans and ron fouchier for providing mers-cov; drs. lijun rong, fang li, and michael farzan for plasmid dnas; and dr. michael whitt for recombinant vsvs. the authors acknowledge the expertise of the university of iowa gene transfer vector core for ad5 production. we thank dr. edward campbell for providing expertise with confocal microscopy. we thank dr. tim o'brien for performing statistical analysis of our data, especially the non-linear regression analysis. key: cord-334960-l5q5wc06 authors: park, su eun title: epidemiology, virology, and clinical features of severe acute respiratory syndrome -coronavirus-2 (sars-cov-2; coronavirus disease-19) date: 2020-04-02 journal: clin exp pediatr doi: 10.3345/cep.2020.00493 sha: doc_id: 334960 cord_uid: l5q5wc06 a cluster of severe pneumonia of unknown etiology in wuhan city, hubei province in china emerged in december 2019. a novel coronavirus named severe acute respiratory syndrome coronavirus-2 (sars-cov-2) was isolated from lower respiratory tract sample as the causative agent. the current outbreak of infections with sars-cov-2 is termed coronavirus disease 2019 (covid-19) by the world health organization (who). covid-19 rapidly spread into at least 114 countries and killed more than 4,000 people by march 11 2020. who officially declared covid-19 a pandemic on march 11, 2020. there have been 2 novel coronavirus outbreaks in the past 2 decades. the outbreak of severe acute respiratory syndrome (sars) in 2002–2003 caused by sars-cov had a case fatality rate of around 10% (8,098 confirmed cases and 774 deaths), while middle east respiratory syndrome (mers) caused by mers-cov killed 861 people out of a total 2,502 confirmed cases between 2012 and 2019. the purpose of this review is to summarize known-to-date information about sars-cov-2, transmission of sars-cov-2, and clinical features. in december 2019, a cluster of patients with pneumonia of unknown origin was identified in wuhan, hubei province, china. most patients witnessed a history of visiting huanan seafood wholesale market. on december 31, 2019, the chinese center for disease control and prevention (china cdc) and wuhan city health authorities reported an outbreak of pneumonia of unknown causes in wuhan city. 1,2) on january 7, 2020, the china cdc identified a novel coronavirus from the lower respiratory tract samples of the patients with pneumonia and disclosed the genomic sequence on january 11. 3, 4) this novel coronavirus was later named severe acute respiratory syndrome coronavirus-2 (sars-cov-2). the world health organization (who) named this infection, caused by sars-cov-2 identified in 2019, coronavirus disease 2019 . 5) despite the effort to stop the transmission of covid-19, the infection spread throughout mainland china, and in january 2020, cases were reported in thailand, japan, and south korea. 6, 7) within less than 3 months since the discovery of the unknown pathogen, the infection spread to at least 114 countries and caused more than 4,000 deaths. on march 11, the who announced covid-19 outbreak a pandemic. 8) in south korea, the first case was identified on january 20, 2020 7) , and as of march 26, a total of 9,241 confirmed cases leading to 131 deaths (1.42% fatality rate) has been reported. 9) two novel strains of coronavirus have jumped species from animal to human, spread by human-to-human transmission, and caused severe acute respiratory syndrome leading to high fatality rate in the past 2 decades. 10) severe acute respiratory syndrome-associated virus (sars-cov), previously unknown coronavirus traced to horseshoe bats in southern china, caused 8,096 confirmed cases and 774 deaths (9.6% fatality rate) in 29 countries from november 2002 to july 2003. 11) in south korea, the first suspected case was reported in april 3, 2003. as of june 15, 2003, three probable cases and 17 suspected cases were identified without any fatality. 12) globally, no sars-cov infection has been reported since 2004. 10) in 2012, another novel coronavirus was first identified in a patient with acute severe respiratory syndrome and multiple organ failure in saudi arabia. 13) this pathogen, later named mers-cov, associated with pneumonia and acute respiratory syndrome (middle east respiratory syndrome), leading to an outbreak in arabian peninsula. between april 2012 and december 2019, a total of 2,502 confirmed cases and 861 deaths (34.4% fatality rate) were reported in 27 countries. 14) in may 2015, a returned traveler from saudi arabia was linked to 186 confirmed mers-cov infections and 38 deaths (20.4% fatality rate) in south korea. 15) this article reviews characteristics, pattern of transmission, and clinical features of covid-19. we also briefly describe % identity with sars-cov and mers-cov, respectively and it is closely related to bat-origin sars-like coronavirus (bat-sl-covzc45) with 87.6%-89% identity. 19, 20) the virus was initially called 2019-novel coronavirus (2019-ncov) upon its emergence, until the coronaviridae study group of international committee on taxonomy of viruses named the virus severe acute respiratory syndrome coronavirus-2 (sars-cov-2) based on the phylogenetic analysis, on february 11, 2020. 21) on the same day, the who named the disease caused by the novel coronavirus coronavirus disease 2019 , in alignment with who best practices for naming of new human infectious disease. 5) based on the virus genome sequencing data, bats are assumed to be the reservoir of sars-cov-2, but the intermediate hosts are yet to be known. data indicate sars-cov evolved from bat coronavirus in horseshoe bats through civet cats or other intermediated animal hosts. mers-cov also likely evolved from bat coronavirus, with dromedary camels as intermediate hosts. [22] [23] [24] [25] sars-cov-2 gains entry to a host cell through binding its spike proteins, which determine host tropism, to host cell receptors. 26) preliminary researches had suggested that sars-cov-2 might share a host cell receptor with sars-cov, because the 2 strains have similar receptor-binding protein structures. successive studies showed that sars-cov-2 binds to angiotensin converting enzyme 2 (ace2) as sars-cov does. 27, 28) hcov nl63 also binds to ace2, but with less avidity than sars-cov; hcov 229e binds to aminopeptidase n (cd-13); receptor for hcov oc43 is still unknown; mers-cov binds to dipeptidyl peptidase-4. 28) sars-cov-2 was first isolated from a bronchoalveolar lavage sample, 18) and rna of the virus was also detected in nasopharyngeal and throat swabs as well as blood, stool, urine, and saliva. 29, 30) when covid-19 first emerged in wuhan, china, no infection of healthcare personnel had been reported, and human-tohuman transmission of the virus was considered unlikely until distinct features of sars-cov-2 in comparison to sars-cov and mers-cov (table 1 ). coronaviruses are enveloped positive sense single-stranded rna viruses sized 80-220 nm in diameter. the envelop bears crown-like, 20-nm in length spikes that resemble corona of the sun under electron microscopy, hence given its name coronavirus. the virus can cause disease both in animal and human. it carries the largest genome among the currently known rna viruses. 16) coronaviruses are members of the subfamily coronavirinae in the family coronaviridae and the order nidovirales. this subfamily divides into 4 genera -alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. so far, 6 coronaviruses are known to cause human diseases. four coronaviruses are endemic in humans. these are human coronaviruses (hcov) 229e, oc43, nl63, and hku1. two epidemic human corona viruses are sars-cov and mers-cov. alphacoronavirus includes hcov 229e and nl63, and betacoronavirus includes hcov oc43, hku1, sars-cov, and mers-cov. the gammacoronavirus includes avian infectious bronchitis virus and several other coronaviruses, and deltacoronavirus includes several re cently discovered avian coronavirus. within the coronavirus particle, a nucleoprotein (n) wraps the rna genome to form a coiled tubular structure together. the viral envelop (e) surround this helical nucleocapsid. two or 3 structural proteins are associated with viral envelop. the matrix protein (m) embedded in envelop. the spike structural protein (s) anchored in envelop is target of neutralizing antibody. the hemagglutinin esterase is found in several of the betacoronaviruses. 17) the coronaviruses have 5 essential genes which are for 4 structural proteins (n, e, m, s) and for viral replication/ transcription (rna dependent rna polymerase, rdrp). the genome organization is 5'-rdrp-s-e-m-n-3'. this gene order of coronaviruses is highly conserved. 17) whole genome sequencing of sars-cov-2 reveals that it is a novel betacoronavirus distinct from sars-cov. 18) the nucleotide sequence of sars-cov-2 showed 79.0% and 51.8 patients unrelated to the seafood market began to appear. as community transmission cases and outbreaks outside china were reported, human-to-human transmission became apparent. 20, 31) mode of transmission for hcov 229e, oc43, nl63, and hku1 are not clearly known, but as with other respiratory viruses, human coronavirus transmission occurs via droplets, indirect or direct contacts. 16) possibility of aerosol transmission remains to be investigated. mers-cov spreads mostly through droplet and direct contact, but transmission through indirect contact, aerosol, fecal-to-oral route is also possible. 10, 16) sars-cov primarily spreads via droplet and direct contact. medical procedures that induce production of aerosol, such as nebulizer treatment or intubation, are reported to increase the risk of transmission. fecal-to-oral route may be possible, but little evidence supports it. 10) sars-cov-2 seems to share the mode of transmission with sars-cov and mers-cov, as it spreads mainly through respiratory secretion or droplet. recent studies showed that sars-cov-2 can remain viable on various surfaces such as stainless steel, plastic, glass, and cardboard at least several hours. 32, 33) this indicates that transmission of sars-cov-2 through contaminated surfaces might be possible. aerosol is currently not considered as primary mode of transmission since no airborne transmission has been reported yet, although it seems possible in hospital settings where aerosol-producing procedures occur. sars-cov-2 rna has been detected in stool, whole blood and urine of covid-19 patients, but whether transmission via such medium is possible is still unknown. 29) sars-cov-2 is more rapidly spreading across the globe than sars-cov did. within 3 months since the initial report of outbreak in wuhan, 80,555 confirmed cases in china and 17,821 confirmed cases in 90 countries outside of china had occurred by march 5, 2020. on march 11, covid-19 had spread to at least 114 countries around the globe and had killed more than 4,000 lives. on the same day, the who declared covid-19 a pandemic. 8) it is notable that sars-cov infection was not declared a pandemic despite of 8,096 confirmed cases leading to 774 deaths during the 8-month period since the first report of sars case on november 16, 2002 . meanwhile, in april 2009, when h1n1pdm09 virus spread rapidly around the globe and caused more than 30,000 cases in 74 countries, the who announced h1n1pdm09 influenza virus infection a pandemic. 34) the transmissibility of sars-cov-2 is not precisely known. a meta-analysis of 12 studies published between january 1 and february 7, 2020 suggests that basic reproduction number (r0) of sars-cov-2 ranged from 1.4 to 6.49 (mean, 3.23; median, 2.79). 35) accumulation of more epidemiologic data may modify this value, but r0 is expected to be around between 2 and 3. sars-cov transmission occurs several days after the onset of symptoms, in alignment with the period of peak respiratory viral load. 10) sars-cov was effectively contained by isolating the patient soon after the onset of illness, since secondary attack rate was greatly reduced by isolating the index case within 5 days. on the other hand, the point of transmission for sars-cov-2 remains unknown. in general, respiratory viruses have the highest transmissibility when the patient has symptoms, therefore we assume the same for covid-19. according to a study that followed respiratory viral load of 18 covid-19 pati ents in china, viral load peaks shortly after the onset of illness and declines there on. 36) the viral nucleic acid shedding pattern of patients infected with sars-cov-2 resembles that of patients with influenza virus. the study results suggested that transmission of covid-19 may occur during first few days after the onset of symptoms. moreover, the number of virus rna copies from one asymptomatic case was comparable to that of the rest of symptomatic cases. in addition, a case of transmission from an asymptomatic family member was recently reported. 37) therefore, we can assume that transmission of covid-19 may occur with the illness onset and even with mild or no symptoms. however, further research is needed on viral transmission from an asymptomatic patient because there is a possibility that the patient may not have been aware of his or her symptoms or may not have been recognized by caregivers due to old age or underlying illness of the patient. the pattern of transmission observed in covid-19 is distinctive from that of sars-cov; such pattern indicates that the spread may not be effectively controlled by isolating the patient after the illness onset. trans missibility of the virus during incubation period is yet to be established. little is known about vertical transmission of covid-19. in a study of 12 sars-cov infected pregnant women during outbreak in 2002-2003, 3 women (25% case fatality rate) died and 4 had spontaneous miscarriage. 38) five mothers delivered; 4 by caesarean section and 1 by spontaneous vaginal delivery. gestational ages ranged 26-37 weeks. all 5 newborns did not show evidence of sars-cov infection, but 2 newborns out of 5 had intrauterine growth retardation. another study analyzed several reports about pregnancy and perinatal outcome of mers-cov infected pregnant women 39) ; of the total 9, 5 mothers received intensive care, and 3 out of 5 them died (33.3% case fatality rate). two women had spontaneous miscarriage, and 5 mothers delivered at gestational age between 32 to term; none of the newborns had signs of mers-cov infection. chen et al. 40) analyzed 9 pregnant women who had been diagnosed with covid-19 in their third trimester and had caesarean section. clinical features of mothers and their newborns were recorded, along with sars-cov-2 detection in amniotic fluid, cord blood, breastmilk, and nasopharyngeal swab of the newborn. none of the 9 mothers developed severe pneumonia, died or had miscarriage. all the amniotic fluid, cord blood, breastmilk, and nasopharyngeal swab of the newborn tested negative for sars-cov-2. this study suggested that there is currently no evidence of vertical transmission of sars-cov-2 in women who developed pneumonia during 3rd trimester of pregnancy. however, the number of pregnant women of the study is very small and only the infection in the late of pregnancy is included, so it cannot be concluded at the moment on the impact of the infection especially in the early or mid-term pregnancy on pregnant women and fetuses. the median incubation period was 5.1 days, 41) ranged from 2-14 days. 31) an analysis of household transmissions revealed that fever and respiratory symptoms appeared 3-7 days after exposure to the virus. fever, dry cough, and fatigue were more commonly reported, whereas nasal congestion, rhinorrhea, sore throat, and myalgia were relatively rare. 42) occasionally, nonrespiratory symptoms such as palpitation, diarrhea, or headache preceded respiratory symptoms. some patients were initially afebrile. clinical spectrum of covid-19 ranged from asymptomatic to fatal pneumonia. the rate of asymptomatic infection is yet to be defined, since most initially asymptomatic infections eventually turned symptomatic. based on the data from the chinese national reporting system, as of february 20, 2020, the median age of the confirmed cases was 51 years (2 days-100 years), of which 77.8% were 30-69 years. 43) a total of 51.1% were male. of the reported confirmed cases, 80% were without pneumonia, or had mild to mode rate pneumonia; approximately 15% had severe pneumonia; approximately 6% were under intensive care due to respiratory failure, shock, or multiple organ failure. fatality rate of covid-19 in china is 3.8%; the fatality rate in wuhan city is 5.8%, in comparison to that of 0.7% in the rest of mainland china. risk factors of severe pneumonia or death include ages 60 or older, and medical comorbidity such as hypertension, diabetes mellitus, cardiovascular disease, chronic pulmonary disease or malignancy. laboratory tests of the confirmed covid-19 cases showed leukopenia, lymphopenia, mild elevated c-reactive pro tein. but the patients with severe pneumonia had elevations in leukocytes, neutrophils, and creatinine kinase. computed tomo graphy of the chest revealed ground glass appearance, interstitial infiltration, or multiple patchy consolidations in both lung fields. 44) a group of researchers in wuhan city analyzed 138 hospitalized patients based on the severity of pneumonia. 45) among the 138 patients with pneumonia, 36 required intensive care. the median age of the intensive care group was 66 years, while that of the other group was 51 years. the intensive care group was more likely to have underlying conditions including hypertension and diabetes mellitus. the median time from onset of symptoms to dyspnea was 5 days and to acute respiratory syndrome was 8 days. at the point of publication, 6 patients had died with overall mortality of 4.3%. in fatal cases, elevations in total leukocyte count, neutrophil ratio, and decrease in lymphocyte were observed 7 days after the onset of symptoms. meanwhile, sars-cov infection most commonly presents with fever. the infection begins with systemic complaints including myalgia, chills, or fatigue, followed by dry cough and dyspnea after a few days to a week. symptoms of upper respiratory tract infection such as rhinorrhea or sore throat are rare. watery diarrhea may be accompanied in 10%-25% patients in later course of the disease. intensive care was required in 20%-30% of patients, with 10% fatality rate. in ages older than 60 years, fatality rate was 50%. death mostly occurred in the third week from the onset of symptoms. in mers-cov infections, the disease course is especially severe in patients with preexisting conditions such as renal failure. cough and dyspnea appear a few days after the onset of symptoms; plain chest radiographs reveal infiltrations in unilateral or bilateral lung fields; the symptoms aggravate rapidly, and ventilator care ensues. fatality rate was 35%. vomiting, diarrhea, or abdominal pain was accompanied in approximately 25% of cases. 10) conclusion within 3 months since the discovery of a novel coronavirus in patients with pneumonia of unknown origin in wuhan city, china, covid-19 has spread rapidly throughout the world and is beating sars-cov and mers-cov in the number of confirmed cases and deaths. modern medical knowledge and technology enabled us to promptly identify the previously unknown pathogen, share the genomic information of the virus, develop diagnostic tools, detect the disease, isolate patients, and provide appropriate medical care. however, several unanswered questions remain to be addressed: the reservoir and intermediate hosts of sars-cov-2, the peak periods of transmission, the duration of transmission, and pathogenesis of severe pneumonia. the world needs to not only strive to slow the spread but also invest in development of treatment and vaccines for covid-19. no potential conflict of interest relevant to this article was reported. outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle world health organization. novel coronavirus (2019-ncov), situation report-1 world health organization gisaid database. 2020 coronavirus gisaid database full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event world health organization. who director-general's remarks at the media briefing on world health organization return of the coronavirus: 2019-ncov. viruses the first case of 2019 novel coronavirus pneumonia imported into korea from wuhan, china: implication for infection prevention and control measures world health organization. who director-general's opening remarks at the media briefing on covid-19 -11 world health organization updates on covid-19 in republic of korea report of the committee on infectious diseases world health organization. summary of probable sars cases with onset of illness from 1 world health organization severe acute respiratory syndrome (sars) isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. mers situation update world health organization middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea human coronaviruses, including middle east respiratory syndrome coronavirus coronavirus genome structure and replication a novel coronavirus from patients with pneumonia in china identification of a novel coronavirus causing severe pneumonia in human: a descriptive study a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus evidence for camel-to-human transmission of mers coronavirus mechanisms of coronavirus cell entry mediated by the viral spike protein functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses angiotensinconverting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mecha nisms and potential therapeutic target epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore consistent detection of 2019 novel coronavirus in saliva early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 world health organization. world now at the start of 2009 influenza pandemic world health organization the reproductive number of covid-19 is higher compared to sars coronavirus sars-cov-2 viral load in upper respiratory specimens of infected patients presumed asymptomatic carrier transmission of covid-19 pregnancy and perinatal outcomes of women with severe acute respiratory syndrome mers-cov infection in a pregnant woman in korea clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures report of the who-china joint mission on coronavirus disease world health organization relation between chest ct findings and clinical conditions of coronavirus disease (covid-19) pneumonia: a multicenter study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses epidemic and emerging coronaviruses (severe acute respiratory syndrome and middle east respiratory syndrome) acknowledgments i would like to thank all the members of the committee on infectious diseases of the korean pediatric society and the korean society of pediatric infectious diseases for their efforts in searching and analyzing the data related with sars-cov-2.this review article is published jointly by the clinical and experimental pediatrics and the pediatric infection and vaccine. key: cord-325902-33pxylb3 authors: hemida, maged gomaa title: middle east respiratory syndrome coronavirus and the one health concept date: 2019-08-22 journal: peerj doi: 10.7717/peerj.7556 sha: doc_id: 325902 cord_uid: 33pxylb3 middle east respiratory syndrome coronavirus (mers-cov) is one of the major threats to the healthcare systems in some countries, especially in the arabian peninsula. mers-cov is considered an ideal example of the one health concept. this is due to the animals, especially dromedary camels, play important roles in the transmission and sustainability of the virus, and the virus can be transmitted through aerosols of infected patients into the environment. however, there is some debate regarding the origin of mers-cov either from bats or other unknown reservoirs. the dromedary camel is the only identified animal reservoir to date. these animals play important roles in sustaining the virus in certain communities and may act as an amplifier of the virus by secreting it in their body fluids, especially in nasal and rectal discharges. mers-cov has been detected in the nasal and rectal secretions of infected camels, and mers-cov of this origin has full capacity to infect human airway epithelium in both in vitro and in vivo models. other evidence confirms the direct transmission of mers-cov from camels to humans, though the role of camel meat and milk products has yet to be well studied. human-to-human transmission is well documented through contact with an active infected patient or some silently infected persons. furthermore, there are some significant risk factors of individuals in close contact with a positive mers-cov patient, including sleeping in the same patient room, removing patient waste (urine, stool, and sputum), and touching respiratory secretions from the index case. outbreaks within family clusters have been reported, whereby some blood relative patients were infected through their wives in the same house were not infected. some predisposing genetic factors favor mers-cov infection in some patients, which is worth investigating in the near future. the presence of other comorbidities may be another factor. overall, there are many unknown/confirmed aspects of the virus/human/animal network. here, the most recent advances in this context are discussed, and the possible reasons behind the emergence and sustainability of mers-cov in certain regions are presented. identification of the exact mechanism of transmission of mers-cov from camels to humans and searching for new reservoir/s are of high priority. this will reduce the shedding of the virus into the environment, and thus the risk of human infection can be mitigated. the main reason behind developing this article is to summarize the current understanding about mers-cov in the context of the one health concept. in this article, i highlight the known information about the mers-cov infection and its pathogenesis in humans, the patterns of mers-cov in dromedary camels, the potential roles of other animals in the transmission cycle of mers-cov, and the interaction of mers-cov/humans/animals. i elaborate on how some strategies can be used to stop or reduce the frequencies of mers-cov outbreaks based on the one health concept, identified some gaps in the literature, and drew conclusions. the one health concept established to ensure the good health and well beings of the human, animal and the environment. human health is mainly affected by environment health as well as animal health (lerner & berg, 2015) . for this review article, i conducted a literature search of the most up-to-date published articles on mers-cov in the past 7 years. first, i focused the introduction section on the historical background of coronaviruses and the one health concept. then, i highlighted the most up-to-date literature from pubmed central, google scholar and researchgate on the interaction of mers-cov/humans/animals. i identified some important gaps in the research dealing with mers-cov/human/environment in the context of the one health concept. i also summarized the current acceptable theories on the emergence and evolution of mers-cov. finally, i highlighted progress to date in the control of mers-cov. historically, mers-cov was first identified in saudi arabia in a patient suffered from severe pneumonia and shortening of breath. the virus was called the novel coronavirus at that time (zaki et al., 2012) . another retrospective study conducted in jordan early 2012 revealed the detection of this novel coronavirus in 11 patients. eight out of them were from the health care workers (hijawi et al., 2013) . coronaviruses are a large group of viruses causing many health problems (respiratory, enteric, and nervous syndromes) in various species of animals and humans. six human coronaviruses that have been identified to date (hcov-229e, hcov-oc43, hcov-nl-63, hcov-huk-1, sars-cov, and mers-cov). two out of them emerged in the past 15 years (lau & chan, 2015) , namely, the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). sars-cov emerged in 2003 in china and spread to many countries throughout the world (peiris et al., 2003) . approximately 8,000 people were infected, and 10% of them died (aronin & sadigh, 2004) . only 9 years later, mers-cov emerged in saudi arabia (zaki et al., 2012) . this is a relatively short period for the emergence of a new coronavirus. one of the main reasons behind the rapid emergence of new coronaviruses is the poor proofreading capability of their rna polymerases (hofer, 2013) . this is in addition to the possibility of the recombination of different coronaviruses (makino et al., 1986) , and it will not be surprising if new coronaviruses emerge in the near future. mers-cov continues to pose great challenges to the healthcare system of some countries in the middle east and arabian peninsula. since its discovery late in 2012 (zaki et al., 2012) , there are ongoing reports to the world health organization (who) from some countries in the middle east, especially the arabian peninsula, with spread to other countries around the globe. according to the latest who statistics, there have been a total of 2,428 laboratory-confirmed cases of mers-cov infection including at least 838 deaths (reported case fatality rate of 35.0%) (who, 2018) . the continuous ongoing reports on mers-cov suggesting the presence of some factors favor its sustainability in certain regions. there are many uncertain aspects of the virus evolution, pathogenesis, and transmission cycle. unfortunately, recently, there was some decline in the rate of research on the virus from different aspects (hemida et al., 2017b) . this hampered the production of new data about the mers-cov from different aspects. below, i summarize the current understanding of the virus in the context of the one health concept. the one health concept is an interesting concept outlining the close interaction among humans, animals and the environment (destoumieux-garzon et al., 2018) . currently, there are two coronaviruses candidates representing the one health concept, sars-cov and mers-cov. animals play important roles in the transmission cycle of both viruses (alshukairi et al., 2018; wang et al., 2005) . both viruses were proven to be of zoonotic origin (gao et al., 2016) . the palm civet cat played important role in the transmission cycle of sars-cov (wang et al., 2005) . some patients proved to visit one restaurant serving the civet cats as a meal (wang et al., 2005) . culling of the civet cats resulted in marked decline in the reported sars-cov cases and now become extinct. many studies made a direct link between the exposure to camel and its meat and milk products and mers-cov human cases . several studies reported the presence of mers-cov specific antibodies in sera of human came in close contact with camels reusken et al., 2016) . meanwhile, mers-cov was isolated from air pf positive dromedary camel herds in saudi arabia (azhar et al., 2014) . mers-cov may infect a wide group of people ranging from very young ages, even infants less than one year of age, to 109 years of age (cdc, 2016) . however, children are less likely to be infected with mers-cov when compared to adults and, if infected, they tend to have asymptomatic or mild disease (arwady et al., 2016) . the reason for this is still not entirely clear and requires further study. the case fatality rate is always very high in case of the immunocompromised infected patients especially those who are suffering from chronic diseases such as cancer, diabetes, blood pressure, kidney problems, etc. (arwady et al., 2016) . human-to-human transmission is reported in many cases. mers-cov replicates efficiently in various in vitro and ex vivo models (chan et al., 2014) . moreover, many family clusters and hospital outbreaks were reported in the past 5 years (arwady et al., 2016; drosten et al., 2014; memish et al., 2013) . this confirms the potential spread of mers-cov among those who are in close contact in the population (mollers et al., 2015) . the most at-risk groups are healthcare workers including nurses, medical doctors and other hospital staff and the elderly with underlying chronic diseases (arabi et al., 2014) . the prevalence rate of mers-cov in primary cases among males is relatively higher than that of females (darling et al., 2017) , which may be because exposure to infected dromedary camels is much higher in males than in females. mers-cov infection triggers some unique interferons and cytokine gene expression profiles. the virus seems to be a poor interferon inducer (chan et al., 2014) . this suggests the potential immune evasion strategies triggered by the virus to hijack the host immune system and may be responsible for the high fatality rate, at least in part. viral spreading among people seems to not yet be very efficient. those in close contact are among the at-risk groups for infection (drosten et al., 2014) , as observed in many hospital outbreaks as well as family clusters (alfaraj et al., 2018; choi et al., 2017; xiao et al., 2018) . this suggests that transmission of the virus among people requires exposure to a high viral load, which will sometimes produce active infection in people who are in close contact. several mers-cov family clusters have been reported (drosten et al., 2014) . interestingly, mers-cov is reported in the dromedary camels in many african countries (egypt, nigeria, tunisia, and ethiopia), but no primary human cases have been reported in these countries to date (ali et al., 2017; roess et al., 2016; van doremalen et al., 2017) , which may be related to some variation in the circulating asian and african strains of mers-cov. some important deletions in the mers-cov currently circulating in dromedary camels from africa were recently reported (chu et al., 2018) . these deletions may explain at least in part the reason behind the variations in the pathogenesis among the asian and african strains of mers-cov. another potential reason behind the absence of human cases in the african countries is the diverse cultural habits among people in africa and the arabian peninsula (fao, 2016) . people in arabian peninsula get in more close touch with camels during the camel show, sports, trade than in africa. this make the human risk of exposure much higher in the ap than africa. mers-cov infection varies from severe respiratory illness accompanied by a high fever and respiratory distress to mild asymptomatic cases. patients are usually admitted to the intensive care unit (icu) and provided with a source of oxygen. most cases result in pneumonia, which is fatal in almost 40% of the affected patients (hong et al., 2017; rubio et al., 2018) . some patients may develop renal failure [13] . several mers-cov travel-associated infections were in many cases associated with the middle east (bayrakdar et al., 2015; rubio et al., 2018) . among these reported was the korean outbreak in early 2015 (choi et al., 2017; kim, andrew & jung, 2017; xiao et al., 2018) . one korean citizen visited some countries in the middle east and then returned home ill. this person visited several healthcare facilities in korea. this resulted in the largest mers-cov human outbreak outside the arabian peninsula (ap) (xiao et al., 2018) . this outbreak confirmed the human-to-human transmission. during this outbreak, mers-cov was isolated from air samples from the hallways of the healthcare facilities close to the hospitalized patients (xiao et al., 2018) . this at least explains in part the rapid development of mers-cov hospital outbreaks. since the discovery of mers-cov late in 2012 (zaki et al., 2012) , many research groups have searched for its potential animal reservoir/s. dromedary camels are the only currently proven reservoir for mers-cov (hemida et al., 2014; hemida et al., 2017b; reusken et al., 2014; reusken et al., 2016) . interestingly, others were able to trace the virus back 30 years ago in dromedary camel specimens in retrospective studies (corman et al., 2014; hemida et al., 2014; reusken et al., 2014) . all these data suggest that the virus has been circulating for decades without being recognized. although the actual and typical clinical features of the mers-cov natural infection in dromedary camels is not well documented to date, very few studies reported these patterns under experimental infection approaches (adney et al., 2014) . based on these findings, camels do not show any pathognomonic signs despite a subtle fever and mild nasal discharge for up to 6 days post-infection (dpi) (hemida et al., 2014) . meanwhile, shedding of the infectious virus was reported in the experimentally infected camels started at 2 dpi up to the 7th dpi (adney et al., 2014) . interestingly, viral rna was still detected at 35 dpi (adney et al., 2014) , though it is not known whether the viral rnas may act as potential sources of infection similar to some other positive-sense rna viruses. no viral shedding in the oral secretions, rectal swabs, urine, or sera of these animals has been reported (adney et al., 2014) , in contrast to the detection of the virus in the fecal specimens and swabs under natural viral field infection (hemida et al., 2014) . these finding suggesting differential patterns of mers-cov infection between natural and experimental approaches. further studies are required to understand the natural mers-cov infection in dromedary camels, which may be achieved by conducting long-term longitudinal studies as well as careful monitoring of the virus infection in large populations of camels. on necropsy examination of mers-cov, experimentally infected dromedary camels revealed only mildto-moderate inflammatory reactions in the upper respiratory tract (khalafalla et al., 2015) . detection of the viral antigens in the tissue sections of the turbinate bone and the upper respiratory tract was reported (adney et al., 2014) . interestingly, seroconversion of the inoculated animals was reported to begin at 14 dpi (hemida et al., 2014) , indicating that mers-cov induces a robust humoral immune response after infection. more recently, one longitudinal study reported the possibility of mers-cov infection in seropositive animals. this raises concern about the role of the antibodies in the protection of the mers-cov infection (hemida et al., 2017a) . it seems that all the members of the family camelidae (dromedary, alpaca, and llamas) are susceptible to mers-cov infection (corman et al., 2014; vergara-alert et al., 2017) . david et al. (2018) reported the presence of antibodies against mers-cov in some alpacas and llamas in israel but only used commercial elisa kits, and they did not address the possibility of cross-reactivity with other coronaviruses especially bcov. it had been previously showed that there is clear cross-reactivity between mers-cov and bcov in dromedary camels (david et al., 2018) . interestingly, one study showed an absence of any detectable antibodies of mers-cov in the sera of bactrian camels (chan et al., 2015) , though this is the only study to report this finding concerning the seronegativity of bactrian camels to mers-cov. it is not well known whether the absence of detectable mers-cov antibodies in the sera of bactrians camels is due to the geographical location of the tested animals in mongolia, far from the middle east and africa. this may be supported by similar findings in dromedary camels in australia and the canary islands (crameri et al., 2015) . another possibility is that this might be due to some genetic factors, which contribute to the resistance of bactrians to mers-cov infections; this point is worthy of further investigation. experimental mers-cov infection in both alpacas and llamas showed a similar pattern to that of dromedary camels (crameri et al., 2016; vergara-alert et al., 2017) , which suggested that both animals might act as a model animal for the study of mers-cov in vivo. however, the experimental infection of pigs with mers-cov did not reveal as much infection as that reported in alpacas and llamas (vergara-alert et al., 2017) . active mers-cov particles were neither retrieved from the experimentally infected animals nor from the close contact non-infected animals during the duration of this study (vergara-alert et al., 2017) . this result suggested that pigs might not play an active role in the transmission of mers-cov. although bats are considered the main reservoir for many coronaviruses, their roles in the mers-cov still need further clarifications. one study reported the presence of mers-cov in one specimen collected from bats in saudi arabia (memish et al., 2013) . the genome sequence of this particular virus showed almost 100% identity to a mers-cov index case (memish et al., 2013) . more recently, jamaican fruit bats were found to be permissible for mers-cov infection (munster et al., 2016) . however, mers-cov-infected bats did not show any apparent clinical signs; however, viral shedding was reported in the swabs from bats up to 9 dpi. the clinical profiles and viral shedding curve during the course of the mers-cov infection in these bats look similar to that of dromedary camels (munster et al., 2016) , yet the amount of infectious viral shedding in bats is less compared to that in dromedary camels. this species of bat is not the most relevant for mers-cov infection, but this study offers some insights into the molecular pathogenesis of mers-cov in bats. interestingly, another study revealed the expression of mers-cov receptors (dipeptidyl peptidase-4, dpp4) in the respiratory and digestive tracts of some insectivorous bats ). an interesting study tested the potential roles of other species in the transmission of mers-cov such as cattle, sheep, goats, donkeys, buffaloes, mules, and horses from egypt, tunisia and senegal (kandeil et al., 2019) . this study revealed the presence of neutralizing antibodies in the sera of some sheep and goats. meanwhile, viral rna was detected in swabs collected from some sheep, goats and donkeys from these countries (kandeil et al., 2019) . several attempts were made to identify an appropriate experimental animal model for mers-cov. the syrian hamster was non-permissive to mers-cov infection (de wit et al., 2013) . experimental infection of this animal neither develops any clinical signs or pathology nor produces any cytokines after infection (de wit et al., 2013) . this was in contrast to new zealand white rabbits, which showed active infection after inoculation with the mers-cov (monchatreleroy et al., 2017) . furthermore, both rhesus macaques and common marmosets supported mers-cov infection (yu et al., 2017) . additionally, both the transgenic and the transduced mice expressing the dipeptidyl peptidase 4 human receptors worked as a model for mers-cov studies (zhao et al., 2015) . interestingly, a new study reported the seropositivity of some sheep and goat to mers-cov from tunisia, senegal and egypt (kandeil et al., 2019) . same study reported the detection of the viral rnas in samples from cow, sheep, goat and donkeys from egypt (kandeil et al., 2019) . this highlights the importance of continuous surveillance and searching for new reservoir/s for the mers-cov transmission cycle. it is now well accepted that human exposure to mers-cov-infected dromedary camels is a predisposing factor to human infection, particularly in immunocompromised people (zumla et al., 2015) . based on the latest who reports, the prognosis of mers-cov infection is poor for elderly patients who have chronic diseases such as cancer, diabetes, kidney failure, etc. (arabi et al., 2014) . transmission of mers-cov from dromedary camels to humans has been proven indirectly in some previous reports (azhar et al., 2014) . one study showed strong evidence of direct transmission of mers-cov from an infected camel to its owner, which was confirmed by comparing the viral genome sequencing of the virus isolated from the infected dromedary camel to that isolated from its owner. both viruses were almost a 100% match (azhar et al., 2014) . meanwhile, this study reported the detection of mers-cov nucleic acid in air samples from the indicated dromedary camel barn during the active course of the viral infection (azhar et al., 2014) . there is a debate about the role of the dromedary camel's milk and meat products and by-products in the transmission of mers-cov. experimental introduction of mers-cov to raw milk revealed little difference between the virus stock in milk and that kept in dmem (van doremalen et al., 2014) . due to their culture, some people in the middle east would drink raw camel milk in efforts to seek treatment for some diseases such as diabetes. the authors acknowledge that mers-cov was introduced into the dromedary camel milk at a high dose and that the viral rna was detected in a limited concentration in the camel's milk (van doremalen et al., 2017) . thus, drinking the raw camel milk poses a great risk to the people consuming this milk without any heat treatment or pasteurization (van doremalen et al. 2014; (zhou et al., 2017) . one study connected the infection of some people to the drinking of the milk from one infected camel (memish et al., 2015) . however, another study was conducted in qatar to assess the possibility of acquiring the infection from contaminated teats and udders of infected she-camels during the process of milking , though no active mers-cov shedding in milk has yet to be reported. further studies are encouraged to come to a conclusion about the potential role of raw camel milk in the transmission of mers-cov. nonetheless, the role of camel meat in the transmission of mers-cov has not been studied carefully to date. thus, special attention should be paid to the efficient cooking of camel meat and its products as well as thorough boiling of camel milk. it is suggested that people not drink raw camel milk to avoid any risk of infection not only with mers-cov but also with other pathogens such as brucellosis (garcell et al., 2016) . some studies reported that mers-cov is one of the occupational zoonotic viral diseases, as was claimed based on some studies investigating the seroconversion of some at-risk group of people to mers-cov. this study reported the presence of specific mers-cov antibodies in approximately 3% of the workers in some slaughterhouses in qatar (farag et al., 2015) . on the other hand, some studies reported the absence of any detectable antibodies in the sera of some herdsmen, veterinarians, and slaughterhouses in saudi arabia (hemida et al., 2015) . one possible explanation for the variations between the two studies is the difference in the sensitivity of the techniques used. those two studies used two different techniques to report the presence/absence of the mers-cov antibodies in the sera of at-risk people (farag et al., 2015; hemida et al., 2015) . regardless, further investigation on a large-scale basis is required to solidify this conclusion about mers-cov. there is more to be known about the molecular biology of mers-cov. identification of the dpp-4 as viral receptors does not rule out the presence of other co-receptors or transcription/translation factors that favor the virus infection in a certain host. there are many immune evasion strategies triggered by mers-cov to hijack the host immune responses, and the mechanisms of such strategies have not been well studied. moreover, there are many unknown aspects especially in the context of the mers-cov/human/animal interaction. meanwhile, some studies were conducted on a small scale or with low numbers of animals/specimens and reported some important conclusion. these studies need further confirmation, and refinement of some of these observations is urgently needed in the near future. here, we highlight some gaps in the research regarding the evolution and transmission of mers-cov. presumably, there might be an unidentified reservoir in the transmission cycle of mers-cov. although respiratory infection still is the main route of mers-cov infection, the exact mechanism of transmission of mers-cov from dromedary camels to humans is still not well understood. the possibility of another reservoir in the transmission cycle of mers-cov has not been ruled out. thus, there might be a missing link in the chain of the human/camel interaction. meanwhile, the exact modes of transmission of mers-cov from dromedary camels to humans have not been well clarified, and the typical pattern of the natural mers-cov infection in dromedary camels has not been well studied. additionally, the potential role of most camel secretions and excretions has not been fully understood. the seroprevalence of mers-cov was reported in the dromedary camels from different countries in africa and asia (ali et al., 2017; hemida et al., 2014) , though feral camels in australia and the canary islands were found to be seronegative (crameri et al., 2015) . the reason behind this phenomenon may be due to these regions being isolated lands and away from the mena region as described above; it may also be due to the absence of an active camel movement between the middle east and africa and these regions of the world. very few studies reported the cross-reactivity between mers-cov and other coronaviruses such as the bovine coronavirus (bcov) that might infect dromedary camels. it is unknown if this might be the reason behind the high seroprevalence of mers-cov among the dromedary camel population. this may be due to the high frequency of exposure to the mers-cov infection during the camel's life, the cross-reactivity of other coronaviruses, or an unknown mechanism related to the dromedary camel's immune system. these considerations require further studies. there is ongoing demand for the development of novel diagnostic assays for coronaviruses, and special interest should be paid to those techniques that enable the simultaneous detection of the viral nucleic acids and those that can simultaneously distinguish between the antibodies for several coronaviruses. furthermore, it is not well understood why only the bactrian camels among the family camelidae did not seroconvert to mers-cov infection (chan et al., 2015) . the genetic susceptibility of certain human populations, especially blood-related people, is not clear in the context of mers-cov infection. there are several levels of human exposure to dromedary camels, such as camel attendants, workers in camel abattoirs, veterinarians inspecting their carcasses, and camel owners. those groups of people are in close contact with camels for various amounts of time and are considered to be a high-risk group of people due to the long time they spend in close contact with the dromedary camels. meanwhile, there is an urgent need to develop a risk scoring system for human exposure to the dromedary camels. it is believed that there is some unidentified reservoirs in the context of mers-cov transmission presenting the virus to the community. this virus is able to infect dromedary camels, which act as an amplifier host for the virus, favoring the circulation of the virus in some camel herds. the virus has the ability to circulate among the animals in the same herd and the camel herds in close proximity to them (fig. 1) . mers-cov in camels has the full potential to infect the human especially immunocompromised persons. once the virus infects a human, there is always a possibility of infecting other people, especially closelyrelated individuals (fig. 1) , including household relatives and workers plus healthcare workers such as doctors and nurses. infection depends on the level of exposure to the infected person, and mers-cov infection in humans ranges from very severe cases of pneumonia to death. currently available data indicate that severely infected individuals can shed the infectious virus into the environment (kim et al., 2016) , though there are few data regarding the capacity of mildly infected individuals to transmit the virus. asymptomatic individuals, however, are unlikely to transmit the virus (moon & son, 2017) . there are many factors behind the emergence, sustainability and spread of mers-cov. the presence of an unidentified mers-cov reservoir in the transmission cycle is still considered, and this unknown reservoir may contribute substantially to the suitability of the virus in certain regions. dromedary camels remain the amplifier of the virus; the close contact of these animals to the human population in certain regions of africa and asia may pose a great risk for human infection and indirectly contribute to the spread of the virus. additionally, public animal markets, especially for dromedary camels, may act as an amplifier of the virus. this poses a great risk to the surrounding community. interesting study addressed the mapping of mers-cov cases in association with the environmental conditions and camel exposure (reeves, samy & peterson, 2015) . this study revealed that the virus is transmitted to dromedary camels through an unknown mechanism. the dromedary camels act as amplifying hosts for the virus. mers-cov is transmitted from dromedary camels to humans through the respiratory aerosols and some other unknown mechanisms. the virus is then transmitted among the human population through respiratory routes. the human-to-human transmission has been confirmed. the human-to-camel transmission still needs further clarification. question marks indicate the non-confirmed phenomenon. full-size doi: 10.7717/peerj.7556/ fig-1 camel exposure is a key predisposing factor for some of mers-cov human cases (reeves, samy & peterson, 2015) . the lack of active surveillance programs for respiratory viruses, especially coronaviruses, may result in many subclinical or mild cases of mers-cov being missed in a certain population. these patients may shed the virus in their secretions and may act as a source of infection to other persons in close contact with them. although many mers-cov vaccine and drug candidates are being researched, none are available yet. all these factors may favor the sustainability of mers-cov in certain regions. interestingly, the case fatality rate of mers-cov among the affected population dropped from almost 50% in 2012 to 34% early 2019 (who, 2018), and we may relate this progress in the control of mers-cov over the past 7 years to many factors. first, identification of the main reservoir of the virus, namely, the dromedary camel (hemida et al., 2014) . second, continuous molecular and serological surveillance of mers-cov among the dromedary camel population in the arabian peninsula and africa (corman et al., 2014; farag et al., 2015; hemida et al., 2017a; hemida et al., 2017b; khalafalla et al., 2015; reusken et al., 2014) . currently, testing the population of camels in regional camel markets is associated with shutting down of the market in case of positive shedding of mers-cov by the animals. i believe this will substantially minimize the risk of community-acquired infections through these positive populations. third, vaccination of dromedary camels, especially animals under two years of age, will have a great impact on the reduction of the viral shedding from these animals to the surrounding community. this will also have a great positive impact on the reduction of the number of reported human infections. fourth, there has been progress in the current understanding of viral tropism, pathogenesis, and mode of transmission in the past five years (chan et al., 2014; widagdo et al., 2017) . fifth, new strategies have been adopted to reduce the spread of infection in health care units (rajakaruna et al., 2017) . sixth, some therapeutic and control approaches for mers-cov such as cyclosporine, ribavirin and interferon show promising trends for the treatment of mers-cov-infected patients (al-tawfiq et al., 2014; de wilde et al., 2013) . meanwhile, good progress has been made in screening large numbers of drugs/therapies for the treatment of mers-cov (han et al., 2018; he et al., 2019; niu et al., 2018; totura & bavari, 2019) . this may lead to the development of some effective novel drugs against mers-cov infection in the near future. to stop mers-cov outbreaks, there are several strategies to be adopted in the context of the one health concept. some strategies are related to the animal, while others are related to human health. the main objective is to minimize or stop the viral shedding from dromedary camels to the environment (fig. 2) . this may be achieved in many ways including regular monitoring of the population of dromedary camels. active animal shedders need to be identified, and quarantine measures should applied until they stop shedding the virus. vaccination of young dromedary camel calves should occur during their first 6 months of life, which will minimize the chances of these animals becoming infected and actively passing the virus to older animals and then to the environment. reorganization and reshaping of the camel industry includes allocating the camel markets away from the cities. global awareness concerning the necessity of thorough boiling and cooking of the camel milk and meat products, respectively, should take place. animal abattoirs should be established far away from large cities. they should not use mixed-animal platforms, and each platform should deal with one species of animal. thorough decontamination of animals' biological wastes in abattoirs should occur using the appropriate standard protocols. regular surveillance of mers-cov among the population especially during the active peak of virus shedding by the animals should occur during november to april every year. people who are in close contact with the camels should wear proper personal protective equipment at all times. at almost 7 years after its emergence, there are ongoing reports of mers-cov infection from time to time. this may be related to many unknown aspects of the viral evolution and pathogenesis. some of these unknown aspects are the following. this work was funded by a grant from the king abdul-aziz city for science and technology (kacst), through the mers-cov research grant program (number 20-0004), which is part of the targeted research program (trp). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the following grant information was disclosed by the author: king abdul-aziz city for science and technology (kacst). mers-cov research grant program: 20-0004. targeted research program (trp). syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters the one health concept: 10 years old and a long road ahead transmission of merscoronavirus in household contacts high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases from sars to mers: evidence and speculation outbreaks of brucellosis related to the consumption of unpasteurized camel milk neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection enhanced ability of oligomeric nanobodies targeting mers coronavirus receptor-binding domain lack of middle east respiratory syndrome coronavirus transmission from infected camels high-frequency rna recombination of murine coronaviruses middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases middle east respiratory syndrome coronavirus in bats, saudi arabia follow-up of contacts of middle east respiratory syndrome coronavirus-infected returning travelers, the netherlands identification of alpha and beta coronavirus in wildlife species in france: bats, rodents, rabbits, and hedgehogs infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus infection replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) a novel human mab (mers-gd27) provides prophylactic and postexposure efficacy in mers-cov susceptible mice coronavirus as a possible cause of severe acute respiratory syndrome strategy and technology to prevent hospital-acquired infections: lessons from sars, ebola, and mers in asia and west africa mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels mers-cov infection of alpaca in a region where mers-cov is endemic camels, mers-cov, and other emerging infections in east africa definitive diagnosis in suspected middle east respiratory syndrome coronavirus cases broad-spectrum coronavirus antiviral drug discovery stability of middle east respiratory syndrome coronavirus in milk high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan livestock susceptibility to infection with middle east respiratory syndrome coronavirus sars-cov infection in a restaurant from palm civet tissue distribution of the merscoronavirus receptor in bats middle east respiratory syndrome cornavirus (mers-cov) a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of korea comparative pathology of rhesus macaque and common marmoset animal models with middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus host-directed therapies for improving poor treatment outcomes associated with the middle east respiratory syndrome coronavirus infections the author declares there are no competing interests. â�¢ maged gomaa hemida conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. the following information was supplied regarding data availability: this is a literature review; there is no raw data. key: cord-329876-4cgrjnjo authors: lei, jian; hilgenfeld, rolf title: structural and mutational analysis of the interaction between the middle-east respiratory syndrome coronavirus (mers-cov) papain-like protease and human ubiquitin date: 2016-05-30 journal: virol sin doi: 10.1007/s12250-016-3742-4 sha: doc_id: 329876 cord_uid: 4cgrjnjo the papain-like protease (pl(pro)) of middle-east respiratory syndrome coronavirus (mers-cov) has proteolytic, deubiquitinating, and deisgylating activities. the latter two are involved in the suppression of the antiviral innate immune response of the host cell. to contribute to an understanding of this process, we present here the x-ray crystal structure of a complex between mers-cov pl(pro) and human ubiquitin (ub) that is devoid of any covalent linkage between the two proteins. five regions of the pl(pro) bind to two areas of the ub. the c-terminal five residues of ub, rlrgg, are similar to the p5–p1 residues of the polyprotein substrates of the pl(pro) and are responsible for the major part of the interaction between the two macromolecules. through sitedirected mutagenesis, we demonstrate that conserved asp165 and non-conserved asp164 are important for the catalytic activities of mers-cov pl(pro). the enzyme appears not to be optimized for catalytic efficiency; thus, replacement of phe269 by tyr leads to increased peptidolytic and deubiquitinating activities. ubiquitin binding by mers-cov pl(pro) involves remarkable differences compared to the corresponding complex with sars-cov pl(pro). the structure and the mutational study help understand common and unique features of the deubiquitinating activity of mers-cov pl(pro). electronic supplementary material: supplementary material is available for this article at 10.1007/s12250-016-3742-4 and is accessible for authorized users. to date, six coronaviruses infecting humans have been characterized. infections with human coronaviruses (hcovs) 229e (hamre & procknow, 1966) , oc43 (mcintosh et al., 1967) , nl63 (van der hoek et al., 2004) , and hku1 (woo et al., 2005) cause relatively mild symptoms in most cases, whereas severe acute res-piratory syndrome coronavirus (sars-cov; ksiazek et al., 2003; kuiken et al., 2003; peiris et al., 2003) and middle-east respiratory syndrome coronavirus (mers-cov; zaki et al., 2012) are connected with severe respiratory-tract infection and, in particular in case of mers-cov, acute renal failure (eckerle et al., 2013) , leading to high case-fatality rates of ~10 and 35%, respectively. in spite of 13 years of research on sars-cov (hilgenfeld & peiris, 2013) , no approved drugs or vaccines are available for the treatment or prevention of coronavirus infection (wang et al., 2016) . this is mainly due to the fact that although these emerging viruses have devastating effects on those infected, the absolute numbers of cases (~8000 for sars, 1733 so far for mers; (http://www.who.int)) imply that the de-velopment of specific antivirals is very likely not commercially viable. on the other hand, the global risk posed by mers-cov must not be underestimated. since its discovery in september 2012, the number of mers cases reported has been rising steadily, with some intermittent peaks connected to hospital outbreaks in saudi arabia (assiri et al., 2013) . man-to-man transmission of mers-cov has also been impressively demonstrated by the recent outbreak of mers in south korea, which was traced back to a single traveller from the arab peninsula (butler, 2015) . therefore, it is imperative that academic laboratories help increase the preparedness against a possible mers-cov pandemic by characterizing antiviral drug targets and by identifying lead compounds interfering with them. in order to successfully infect humans, a virus has to meet at least two conditions: 1), it should maintain a sufficiently correct replication of its genetic material; 2), it should inhibit the host antiviral response. the papainlike protease (pl pro ) of mers-cov (or sars-cov) is involved in both of these tasks (yang et al., 2013; barretto et al, 2005) . the pl pro is a domain located in the middle part of the largest non-structural protein, nsp3, of mers-cov (or sars-cov) . it is responsible for releasing nsp1, nsp2, and nsp3 from the polyproteins 1a (pp1a) and 1ab (pp1ab), an essential step of replication (harcourt et al., 2004) . like its sars-cov counterpart, the mers-cov pl pro also has deubiquitinating (dub) and deisgylating activities in vivo as well as in vitro (yang et al., 2013; mielech et al., 2014; lei et al., 2014; baez-santos et al., 2014b) . k48-and k63-polyubiquitin poly (ub) and isg15 (interferon-stimulated gene 15)conjugated targets are usually involved in host innate immune regulation (liu et al., 2005; maringer & fernandez-sesma, 2014) . the pl pro has the ability to digest k48-and k63-linked polyub chains and to remove isg15 from isg15-linked proteins (baez-santos et al., 2014b) , thereby interrupting the signalling pathways leading to the innate immune response. thus, the pl pro can block the activation of ifn regulatory factor 3 (irf3) (yang et al., 2013) and subsequently the production of interferon β (ifnβ) (mielech et al., 2014) . interestingly, mers-cov pl pro shows a similar cleavage rate for k48-and k63-linked polyub chains, while the sars-cov enzyme prefers k48-over k63-linked chains (baez-santos et al., 2014b) . the former enzyme degrades a polyub chain by removing mono-ubs, whereas the latter cleaves di-ub units off the polyub chain (bekes et al., 2015) . we have reported the first crystal structure of the mers-cov pl pro (lei et al., 2014) . later, two other groups also described the structure of this enzyme (bailey-elkin et al., 2014; lee et al., 2015) . the structure of pl pro can be divided into two parts: a ubiquitin-like (ubl) domain and a catalytic domain with thumb, palm, and fingers subdomains. the overall fold of mers-cov pl pro is not only similar to that of sars-cov pl pro , but also to that of several human ubiquitinspecific proteases (usps) (hu et al., 2005) . in 2014, the x-ray structure of the complex of sars-cov pl pro with ubiquitin has been reported (chou et al., 2014; ratia et al., 2014) . several key residues (such as glu168 or tyr265) of sars-cov pl pro that are important for ubiquitin recognition (chou et al., 2014; ratia et al., 2014) , are not conserved in mers-cov pl pro . bailey-elkin et al. (2014) described the structure of an artificially linked, covalent complex between ubiquitin and mers-cov pl pro . here, we present the crystal structure of a noncovalent complex between the two proteins and a mutational study of the interactions involved. for these studies, we used the cys111ser active-site variant of mers-cov pl pro . the pl pro of mers-cov (strain 2c emc/2012; gen-bank no. afv09327.1) contains 320 residues, from gln1482 to asp1801 of pp1a/1ab. for simplification, gln1482 was renumbered into gln1 here. the dna plasmid coding for mers-cov pl pro was produced earlier (lei et al., 2014) . the mers-cov pl pro c111s, d164e, d164a, d165e, d165a, and f269y variants were produced using the same strategy that we described before (lei et al., 2014) . all primers for these variants are listed in supplemental table s1 . all dna plasmids coding for the altered pl pro were verified by sequencing. genes coding for wild-type (wt) mers-cov pl pro and for its variants were expressed and the corresponding proteins were purified according to our previous description (lei et al., 2014) . performed according to the procedure described for mers-cov pl pro (lei et al., 2014) . purified mers-cov pl pro (wt) and a variant that had the active-site cys111 replaced by ser (pl pro (c111s)) were both concentrated to ~24 mg/ml in 20 mmol/l tris-hcl, 150 mmol/l nacl, ph 8.8, 10 mmol/l β-mercaptoethanol (bme). human ubiquitin (bostonbiochem) was dissolved to 6 mg/ml in 20 mmol/l tris-hcl, 150 mmol/l nacl, ph 8.8. 500 μl pl pro (wt) or pl pro (c111s) were mixed with 500 μl ubiquitin (~1:1 molar ratio) at 4°c overnight. (c111s)-ub were observed under condition no. 9 of md1-01, whereas no crystal of pl pro (wt)-ub was obtained. optimized crystals of pl pro (c111s)-ub were subsequently obtained within two days under the condition: 22% w/v peg 4000, 15% v/v 2-propanol, 0.1 mol/l tri-sodium citrate ph 4.8, and 10% glycerol. 2 μl of protein and 2.5 μl of reservoir were mixed to equilibrate against 500 μl reservoir. crystals were placed in a nitrogen-gas stream (100 k). a 3.16-å dataset was collected at wavelength 0.91841 å at beamline 14.2 of bessy, berlin (mueller et al., 2012) . the program xds (kabsch, 2010) was used to process the diffraction data. the space group was found to be p6 3 , with unit-cell parameters a = b =138.14 å, c = 57.59 å, γ = 120°. diffraction data statistics are shown in table 1 . the structure of the mers-cov pl pro (c111s)-ub complex was solved by molecular replacement using molrep (vagin & teplyakov, 2010) . the program selected the mers-cov pl pro (protein data bank (pdb) entry 4p16, lei et al., 2014) as the first search model. human ubiquitin (pdb entry: 1ubq, vijaykumar et al., 1987) was used as the second search model. the model of the complex was inspected and rebuilt using coot (emsley et al., 2010) , and refined using phenix.refine (headd et al., 2012) . the refinement statistics are shown in table 1 kinetic assays of purified pl pro s all enzymatic assays were performed using a 96-well microtiter plate and the reaction buffer 20 mmol/l tris-hcl, 150 mmol/l nacl, ph 7.9, 2 mmol/l dithiothreitol (dtt). the fluorogenic substrates cbz-arg-leu-arg-gly-gly-7-amino-4-methylcoumarin (z-rlrgg-amc) (bachem), z-lrgg-amc (bostonbiochem), and ubiquitin-amc (ub-amc) (bostonbiochem) were used. the fluorescence of free amc with different concentrations (5 nmol/l-2.5 μmol/l) in reaction buffer was measured to generate a calibration curve, in order to convert the change of fluorescence intensity per unit of time, δ(afu) /s, into the amount of hydrolyzed substrate in μmol/l/s. the enzymatic cleavage reactions were run with an flx800 fluorescence spectrophotometer (biotek), to measure the increased fluorescence signal (λ ex : 360 nm; λ em : 460 nm) resulting from amc release. reactions were initiated by addition of the proteases to the reaction system. the peptide-hydrolysis kinetic assays were performed with the following conditions: 1 μmol/l mers-cov pl pro variant (d164e, d164a, d165e, f269y), or 10 μmol/l d165a, or 0.1 μmol/l sars-cov pl pro , with different concentrations (10, 20, 40, 80, 100 μmol/l) of z-rlrgg-amc or z-lrgg-amc in a final volume of 100 μl at 25 °c. the kinetic curves for the proteases and their variants with the substrates z-rlrgg-amc or z-lrgg-amc were linear and the initial velocities also increased linearly with substrate concentration. no saturation was observed. therefore, the data were fitted to the equation v/[e] tot. = k app [s] , where k app approximates k cat /k m , as described previously (barretto et al., 2005; wojdyla et al., 2010) . the deubiquitinating kinetic assays were performed under the following conditions: 0.1 μmol/l mers-cov pl pro wild-type or its variants d164e, d164a, d165e, f269y, or 0.5 μmol/l d165a, or 0.025 μmol/l sars-cov pl pro were incubated with increasing concentrations (1, 2, 4, 8 μmol/l) of ub-amc in a final volume of 50 μl, at 25 °c. although pl pro actually cleaves the isopeptide bond between the carboxyl group of the c-terminal gly in ub and the ε-amino group of lys in ubiquitinated targets in vivo, we used the hydrolysis of ub-amc here to test the deubiquitinating activity in vitro. the kinetic curves of proteases and variants with the substrate ub-amc were hyperbolic and the initial velocities were not linear over the concentration of substrate. however, saturation was still not observed within a reasonable time. only when the ratio of protein to substrate was 1:1 or larger, were we able to achieve saturation within a limited time (data not shown). as the initial velocities did not increase in a strictly linear fashion with substrate concentration, application of the equation v/[e] tot. = k app [s] to mimic k cat /k m would lead to large standard errors. we were however able to fit the data to the michaelis-menten equation using the graphpad prism program (graphpad software), even though saturation could not be reached (supplemental figure 1 ). all assays were performed in duplicates. the substrate-binding site of mers-cov pl pro features significant differences from those of the corresponding sars-cov enzyme and human ubiquitin-specific proteases (usps, such as, usp14) (hu et al., 2005; chou et al., 2014; ratia et al., 2014) . it is therefore of interest to determine the crystal structure of the complex between mers-cov pl pro and its substrate, human ubiquitin. hence, we crystallized the ubiquitin (ub) complex of a mers-cov pl pro variant that had the active-site cys111 replaced by serine (c111s) and determined the structure at 3.16 å ( figure 1a ). there is one pl pro (c111s)-ub complex per asymmetric unit. using the pdbepisa server (krissinel & henrick, 2007) , the total interface region of mers-cov pl pro (c111s)-ub was determined as 813 å 2 , close to the 915 å 2 interface of sars-cov pl pro -ubal (ubiquitin aldehyde; pdb entry: 4mm3, ratia et al., 2014) and the 999 å 2 of sars-cov pl pro (c112s)-ub (pdb: 4m0w, chou et al., 2014) , but less than the 1503 å 2 observed for usp14-ubal (pdb: 2ayo, hu et al., 2005) . the overall structure of mers-cov pl pro (c111s) is very similar to that of the substrate-free pl pro (lei et al., 2014) , with a root-mean-square deviation (rmsd) of 0.91 å for corresponding cα atoms between these two structures. nevertheless, two differences are immediately visible: 1), the zinc-finger motif has moved and closed in onto the ub, compared to the free pl pro . the zinc ion position has shifted by about 4 å and the largest deviation between the two structures is ~6 å for cys228 of the zinc-finger region ( figure 1b) ; 2) the mobile loop 271 gietavg 277 , also named "bl2 loop", is defined by clear main-chain electron density ( figure 1c ). this loop is disordered in substrate-free pl pro (lei et al., 2014; bailey-elkin et al., 2014) . compared to mers-cov pl pro , the bl2 loop 267 gnyqcg 272 is shorter by one residue in sars-cov pl pro . at the time when we deposited in the pdb the coordinates for the crystal structure of the non-covalent complex mers-cov (c111s)-ub (pdb: 4wur), bailey-elkin et al. (2014) described two crystal structures for a covalent complex mers-cov pl pro -ub, in which an alkyl bromide group introduced at the c-terminus of ub had formed a thioether with the active-site cys111 of mers-cov pl pro . these structures were in space groups p6 5 22 and p6 3 and were named "closed" and "open" pl pro -ub complexes (pdb: 4rf0 and 4rf1), respectively (bailey-elkin et al., 2014) . even though the zinc-finger motif of pl pro in the former complex (space group p6 5 22) is closer to the ub than in the p6 3 complex, both the "closed" and "open" complex show almost the same interactions between pl pro and ub (bailey-elkin et al., 2014). the differences may be caused by crystal packing. our non-covalent complex is similar to the "open" form of the covalent pl pro -ub complex (rmsd = 0.58 å for the pl pro and 0.85 å for ub, based on all cα atoms). all parts of the pl pro except for the ubiquitin-like (ubl) domain interact with ub. most interactions in-volve five surface regions of the pl pro and two regions of ub ( figure 2a ). these five regions of the pl pro are labelled by roman numbers: i, leu106-tyr112; ii, ala162-arg168; iii, cys208-val210; iv, gly248-pro250; v, phe269-tyr279. i and ii are situated in the thumb domain; iii is in the fingers domain; and iv and v are in the palm domain ( figures 1a and 2a ). in the following and in the figure labels, residues of ub are indicated in italics to distinguish them from residues of pl pro . the two interacting regions of ub are: a, arg42-gln49; b, arg72-gly76 (figure 2a ). region a of ub consists of β3, β4, and the loop between β3 and structure of mers-cov pl pro -ubiquitin complex β4 ( figure 2b ). regions ii and iii of pl pro interact with region a of ub. region b comprises the five c-terminal residues, rlrgg, and is in contact with regions i, ii, iv, and v of pl pro ( figure 2c ). the c-terminal rlrgg motif contributes the majority of the interactions with the pl pro ; the buried surface between ub region b and pl pro is 477 å 2 (out of a total of 813 å 2 ). in order to make these interactions clear, we describe here in some detail the contacts between the pl pro and regions a and b of ub. region a (arg42-gln49) of ub inserts into the space between the thumb and fingers domains of pl pro (figures 2a-b) . residues ile44, ala46, and gly47 engage in hydrophobic interactions with tyr209 and val210 of region iii of pl pro ( figure 2b ). the hydrophobic patch (ile44, ala46, and gly47) is a common interaction region utilized by ub-binding proteins (dikic et al., 2009) . in particular, the interaction of ile44 with val210 of pl pro is important for the deubiquitinase (dub) but not for the protease activity. the variant v210r shows dramatically reduced dub activity, as demonstrated by bailey-elkin et al. (2014) (according to the numbering scheme of these authors, v210 is v1691). a salt-bridge exists between region a of ub and the pl pro ( figure 2b ), namely between the side-chains of arg42 and asp165 (region ii in pl pro ). in addition, a hydrogen bond is formed between the main-chain o atom of gly47 and the main-chain amide of val210 (region iii). in the sars-cov pl pro (c112s)-ub complex (chou et al., 2014) , arg42 forms a salt-bridge with the negatively charged glu168, a residue which is replaced by the positively charged arg168 in mers-cov pl pro . consequently, the same salt-bridge cannot be formed in the mers-cov pl pro (c111s) -ub complex. instead, mers-cov pl pro has asp165 interacting with arg42. in our deubiquitinating (dub) kinetic assays, the d165a variant shows a dramatically reduced dub activity; the k cat /k m is about 78-fold decreased compared to that of the wild-type ( table 2 ). the k m value of the d165a variant is about 4-fold higher than that of the wild-type mers-cov pl pro , suggesting that this amino-acid replacement reduces the ub binding affinity. meanwhile, the d165e amino-acid replacement shows a catalytic efficiency comparable to wild-type towards ub-7-amino-4methylcoumarin (ub-amc) ( table 2 ). however, we noticed that the k m value of d165e for the dub activity is about 2-fold larger than for the wt enzyme. although glu165 can mimic asp165 here, we propose that the longer side-chain of glu may fit less perfectly compared to asp. these results indicate that the salt-bridge between asp165 and arg42 could be important for the pl pro 's dub activity, in addition to the interaction between asp165 and the p4-amide group (see below). region b of ub comprises the five c-terminal residues, rlrgg. these five residues bind to the narrow activesite channel between the thumb and palm domains of the pl pro ( figure 2c ). they mainly interact with regions i, ii, iv, and v of the protease. residues rlrgg are compatible with the pl pro cleavage motif, (r/k) (l/i) xgg (p5-p1), in the mers-cov polyproteins; their interactions with the pl pro are discussed here in terms of subsites s1 to s5. s1 and s2 subsites. pro163 (region ii of pl pro ) and the side-chains of asn109 and tyr112 (located in region i) form a space-restricted s1 site to accommodate gly76 (p1). the carbonyl oxygen atom of gly277 (region v) accepts a hydrogen bond from the amide of gly76 (figure 3) . the side-chains of tyr112 (region i) and phe269, val276 as well as tyr279 (region v), and gly277 form a restricted space for gly75 (p2). two hydrogen bonds link the main-chain at asp164 of the pl pro and gly75 (figure 3) . the s1 and s2 sites of the protease are too small to accommodate any other residue but glycine. s3 subsite. the main-chain o atom of arg74 (p3) accepts a hydrogen bond from the gly277 amide. in the complexes sars-cov pl pro (c112s)-ub or sars-cov pl pro -ubal, the main chain at arg74 forms two hydrogen bonds (chou et al., 2014; ratia et al., 2014) , namely with the amide of gly272 and with the hydroxyl group of tyr265. the former hydrogen bond is conserved in the mers-cov pl pro (c111s)-ub complex, but the latter is not. tyr265 of sars-cov is replaced by phe269 in mers-cov, which lacks the ability to form a hydrogen bond with the main-chain amide of arg74. in agreement with this difference, the dub activity of sars-cov pl pro is 2.5-fold higher than that of mers-cov pl pro in our kinetic assay; furthermore, the mers-cov pl pro f269y amino-acid replacement leads to enhancements by about 1.5-, 1.4-, and 1.9-fold of the hydrolytic activities towards carbobenzoxy-arg-leu-arg-gly-gly-7amino-4-methylcoumarin (z-rlrgg-amc), z-lrgg-amc, and ub-amc, respectively ( table 2) . the side-chain of arg74 is exposed to the solvent in our mers-cov pl pro (c111s)-ub complex. this sidechain shows remarkable variability in the interactions it makes in the different complexes. in the "open" but not in the "closed" covalent mers-cov pl pro -ub complex (bailey-elkin et al., 2014) , it donates a hydrogen bond to the main-chain carbonyl oxygen of thr1755 of the bl2 loop (corresponding to thr274 in our numbering scheme). in sars-cov pl pro -ubal (ratia et al., 2014) but not in the sars-cov pl pro (c112s)-ub complex (chou et al., 2014) , the side-chain of arg74 forms a hydrogen bond with the main-chain carbonyl oxygen of gln270. instead, arg74 is involved in a relatively weak salt-bridge with glu162 in the sars-cov pl pro (c112s)-ub complex (chou et al., 2014) . none of these interactions exist in our mers-cov pl pro -ub complex. s4 subsite. the main-chain amide of leu73 (p4) donates a hydrogen bond to the side-chain of asp165 (region ii) (figure 3 ). this hydrogen bond is conserved in all the complexes compared here. in our peptide-cleavage assay, the d165a variant shows about 16-fold and 10-fold lower activities towards substrates z-rlrgg-amc and z-lrgg-amc, respectively, indicating that asp165 is not only important for interacting with arg42 of ub (see above). the side-chain of leu73 is embedded in a hydrophobic pocket which is formed by the cβ atom of asp165, the side-chain of pro250 (region iv), phe269, as well as the cβ and cγ atoms of glu273 (region v). asp165 and pro250 are conserved in sars-cov pl pro (asp165 and pro249). phe269 is replaced by tyr265, and glu273, situated in the bl2 loop, is replaced by tyr269. however, the side-chains of all these non-conserved residues possess the ability to provide a hydrophobic environment to accommodate leu73. s5 subsite. the side-chain of arg72 (p5) is located between the pl pro thumb domain and region a of ubiquitin. it forms a salt-bridge with the side-chain of asp164 in mers-cov pl pro . in addition, the guanidinum group of arg72 may be involved in a π-π interaction with that of arg168 (figure 3 ). these interactions have also been described for the covalent complex of mers-cov pl pro with ub (bailey-elkin et al., 2014) . arg72 is not subject to strict space limitations; accordingly, this residue displays different binding patterns in the two sars-cov residues are shown in purple in the ball-and-stick style, and they are labeled in red. for clarity, the 2f o -f c electron density (blue; 1.0 σ) of the rlrgg main chain is shown. pl pro residues are displayed in green in the balland-stick style, and they are labeled in black. hydrogen bonds are displayed as black dashed lines, and the salt-bridge between r72 and d164 is depicted as two red dashed lines. structure of mers-cov pl pro -ubiquitin complex pl pro -ub complexes. in the covalent sars-cov pl pro -ubal complex (ratia et al., 2014) , arg72 forms a saltbridge with glu168. in the non-covalent sars-cov pl pro (c112s)-ub complex (chou et al., 2014) , arg72 is exposed to the solvent and does not interact with glu168 (arg42 instead forms a salt-bridge with glu168, as mentioned above). in our kinetic assay, the d164a variant of mers-cov pl pro displays a ~4.5-fold and a ~3.5-fold reduced activity, respectively, for z-rlrgg-amc and ub-amc (table 2) . for z-lrgg-amc, the activity is decreased just a little (by about ~1.5-fold), because there is no p5-arg in this substrate (table 2 ). these data demonstrate that asp164 is important for the interaction with arg72. in summary, the main-chain heteroatoms of p5-p1 form a hydrogen-bonding network with pl pro . the binding characteristics of p1, p2, and p4 are conserved in all mers-cov and sars-cov pl pro -ub complexes. however, p3-arg and p5-arg assume binding patterns that differ between the various mers-cov and sars-cov pl pro -ub complexes. viral proteins are likely to possess multiple functions, as exemplified by non-structural protein 1 (ns1) of influenza a viruses (hale et al., 2008) , the nucleocapsid (n) protein of coronaviruses (chang et al., 2014) , or the nsp14 exonuclease-guanyl-7-methyltransferase of coronaviruses (chen et al., 2009) . exhibiting dub and proteolytic activities, the papain-like proteases of coronaviruses are no exception here. although the overall folds are conserved, the enzyme activity and substrate-binding modes of cov pl pro s differ in detail. therefore, no coronavirus pl pro can be considered a general model for all its homologues (baez-santos et al., 2014b) . we have previously noticed that the oxyanion hole of the mers-cov pl pro appears to be deficient (lei et al., 2014 ; also see the discussion below). similarly, the recognition of ubiquitin by the enzyme appears to be suboptimal. thus, the main-chain amide of the p3-arg residue has no hydrogen-bonding partner on the mers-cov pl pro , because the near-by side-chain of phe269 is incapable of accepting an h-bond. the corresponding residue is tyr265 in the sars-cov pl pro , which is perfectly positioned to accept the hydrogen bond from the p3-amide. indeed, when we replaced phe269 by tyr in mers-cov pl pro , the dub activity of the enzyme increased by a factor of almost 2 and the peptidolytic activity by ~1.5. the evolution of viral enzymes is not neces-sarily driven by optimization of catalytic efficiency. this is particularly true for viral proteases that have to ensure the availability of non-structural proteins in the correct temporal order when they cleave them out of the viral polyproteins; in fact, too rapid a polyprotein processing might be counterproductive. on the other hand, a more efficient dub activity should help the virus in counteracting the innate immune response of the host cell. as the binding of the p5-p1 residues of ubiquitin and of the polyprotein cleavage sites obviously influences both the dub and proteolytic activities of the pl pro , the suboptimal catalytic efficiency that we observe may be a consequence of a compromise between the requirements of the two activities. with regard to the oxyanion hole, we previously proposed that the backbone amide of asn109 (located in a β-turn connecting β7 and α4, figure 4 ) may contribute to the stabilization of the oxyanion intermediate in pl pro catalysis, along with the main-chain amide of the active site cys111, although this may require a slight rearrangement of this β-turn (lei et al., 2014) . in our non-covalent mers-cov pl pro -ub complex, we do not see any rearrangement of the β-turn. more or less in agreement with our suggestion, bailey-elkin et al. (2014) proposed on the basis of their covalent mers-cov pl pro -ub complex structure that the main-chain amides of asn1590, asn1591, and cys1592 (corresponding to asn109, asn110, and cys111 in our numbering scheme) form the oxyanion hole. on the other hand, lee et al. (2015) argued that the side-chain of asn109 could contribute to the oxyanion hole. they found that the n109a replacement completely abolished enzyme activity. as we reported earlier (lei et al., 2014) , the side-chain amide of asn109 makes a strong hydrogen bond with the conserved gly161 (figure 4 ). any reorientation of the asn109 side-chain towards the oxyanion would require a disruption of this strong interaction; this is not very likely. in conclusion, lacking a side-chain in the proper spatial orientation and capable of donating a hydrogen bond to the oxyanion (such as trp107 in sars-cov pl pro ), the oxyanion hole of mers-cov pl pro seems to be deficient. apart from the less than optimum binding of the p3 residue, there are other differences in the way mers-cov pl pro and sars-cov pl pro recognize human ubiquitin. the formation of a salt-bridge between arg72, the p5 side-chain of ub, and asp164 is unique for mers-cov pl pro . the same interaction exists in the two covalent mers-cov pl pro -ub complexes (bailey-elkin et al., 2014) . this binding mode is very different from the glu168 -arg72 salt-bridge in the sars-cov pl pro -ubal complex (ratia et al., 2014) . glu168 is conserved in hcov-nl63 pl2 pro and replaced by asp in the hcov-229e, hcov-oc43, and hcov-hku1 pl2 pro s (for sequence alignments, see barretto et al., 2005; baez-santos et al., 2014b) , so the same type of interaction is likely to be realized in the ub complexes of these enzymes. however, the corresponding residue in mers-cov pl pro is arg168; hence, asp164 is used instead to bind arg72. asp164 is in fact unique in mers-cov. it is replaced by gly in sars-cov pl pro and the pl2 pro s of hcov nl63 and hcov 229e, by ala in hcov-oc43 pl2 pro , and by ser in hcov-hku1 pl2 pro . our kinetic results for the d164a replacement (see results) emphasize the importance of the unique asp164 residue. in our pl pro (c111s)-ub complex, the ubl domain of pl pro shows no interaction with ubiquitin. the relative orientation of the ubl domain in the substrate-bound pl pro is the same as in substrate-free pl pro . the ubl domain of mers-cov pl pro is not required for the ifn antagonism activities (baez-santos et al., 2014b), but it is required in case of sars-cov pl pro (frieman et al., 2009) . recently, mielech et al. (2015) reported that the ubl domain of mouse hepatitis virus (mhv) is an important modulator of pl pro stability and viral pathogenesis. although the ubl domain shows variable effects in different covs, the high degree of conservation of the domain throughout the family suggests that it may play a common biological role. one possible function is that the ubl might be involved in protein-protein interactions. ubiquitin-like domains are known to function as binding modules in such interactions. for example, the kinase raf contains a ubl domain for interaction with human ras (fetics et al., 2015) . also, human ubiquitin-specific protease (usp) 7 includes five ubl (1-5) domains, of which the second is bound by the herpes simplex virus-1 immediate-early protein icp0 to counteract the intrinsic antiviral response of the host cell (pfoh et al., 2015) . the bl2 loop of sars-cov pl pro is important for binding inhibitors (baez-santos et al., 2014a; lee et al., 2015) . this loop is variable in different cov pl pro s. a potent inhibitor of the sars-cov pl pro , n-[(3-fluorophenyl) methyl]-1-[(1r)-1-naphthalen-1-ylethyl] piperidine-4-carboxamide (compound 3k, ic 50 = 0.15 ± 0.01 μmol/l) was found to have no effect on the mers-cov enzyme (baez-santos et al., 2014b) . tyr269 and gln270 of sars-cov pl pro , which are important for binding this inhibitor (baez-santos et al., 2014a) , are replaced by glu273 and ala275 in the mers-cov protease. it seems that this structural difference in the bl2 loop has a remarkable impact on the effectiveness of the inhibitor. the structure of the mers-cov pl pro (c111s)-ub complex presented here will facilitate virtual screening of chemical libraries for specific anti -mers-cov pl pro inhibitors (hilgenfeld, 2014) . the di-ub site of pl pro sars-cov and mers-cov pl pro s can digest k48-and k63-linked (poly)ubiquitin chains and remove isg15 from proteins covalently linked to it (ratia et al., 2014 , baez-santos et al., 2014b . sars-cov pl pro prefers binding of k48-ub 2 and isg15 over mono-ubiquitin (ratia et al., 2014) . all evidence suggests that on the pl pro , at least two ubiquitin-binding sites (or a binding site for diubiquitin-like molecules such as isg15) exist. ratia et al. (2014) proposed hypothetic models for complexes of sars-cov pl pro with k48-ub 2 and isg15. these authors identified two major hydrophobic binding sites on the pl pro for the first ub (ub1) and the second ub (ub2). the binding site for ub1 comprises met209, pro248, and pro249. this hydrophobic patch is conserved in mers-cov pl pro , although the residues are not exactly the same. met209 of sars-cov pl pro is replaced by val210 (region iii) in mers-cov. pro248 is replaced by thr249, whereas pro249 is conserved (pro250; region v) ( figure 5 ). the hypothetic second binding site for ub2 (also named "ridge" region, ratia et al., 2014) is located to the first α helix (α2) in the thumb domain, including residues phe70, his74, and leu76 of sars-cov pl pro . according to a structural alignment of the sars-cov and mers-cov pl pro s, phe70, his74, and leu76 are changed to lys69, gly73, and val75, respectively ( figure 5 ). in sars-cov pl pro , the f70s and f70a replacements lost the affinity to k48-ub 2 and isg15 in vitro (ratia et al., 2014) ; therefore, the presence of lys69 in mers-cov pl pro instead of phe70 strongly suggests that this enzyme should bind ub2 in a different way. bailey-elkin et al. (2014) predicted asn1673 and val1674 of the fingers subdomain (corresponding to asn192 and val193 in our pl pro ) as the distal ub site of k63 di-ub, but they found that their dub activity data do not support their prediction for the ub2 binding site. the structure of the mers-cov pl pro -ub complex reported here reveals the exact ub1 binding site on the pl pro , but the ub2 binding site should be identified by crystallizing the pl pro in complex with poly-or di-ubiquitin. in summary, the crystal structure of the mers-cov pl pro -ub complex provides valuable information that helps understand the multiple functions of coronavirus papain-like proteases. mutational studies additionally highlight features of the mers-cov pl pro . the different substrate-binding patterns should be kept in mind when designing inhibitors for pl pro s of covs, even though the overall structures of these enzymes are conserved. furthermore, it would be helpful to obtain the structure of pl pro in complex with di-ub or isg15 in the future. ing sites are depicted as purple dots. the n and c termini of pl pro are marked by underlined letters. all residues related to the two ub binding sites are labeled. the catalytic triad cys111-his278-asp293 is indicated by yellow, blue, and red spheres. ksa mers-cov investigation team. 2013. hospital outbreak of middle east respiratory syndrome coronavirus xray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases catalytic function and substrate specificity of the plpro domain of nsp3 from the middle east respiratory syndrome coronavirus (mers-cov) crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity sars hcov papain-like protease is a unique lys 48 linkage-specific di-distributive deubiquitinating enzyme south korean mers outbreak spotlights lack of research the sars coronavirus nucleocapsid protein-forms and functions molprobity: all-atom structure validation for macromolecular crystallography functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-binding domains-from structures to functions identification of a novel coronavirus in patients with severe acute respiratory syndrome in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection features and development of coot allosteric effects of the oncogenic rasq61l mutant on raf-rbd severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling the multifunctional ns1 protein of influenza a viruses a new virus isolated from the human respiratory tract identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design from sars to mers: 10 years of research on highly pathogenic human coronaviruses structure and mechanisms of the proteasome-associated deubiquitinating enzyme usp14 inference of macromolecular assemblies from crystalline state a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov crystal structure of the papain-like protease of merscoronavirus reveals unusual, potentially druggable active-site features immunity by ubiquitylation: a reversible process of modification message in a bottle: lessons learned from antagonism of sting signalling during rna virus infection growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease mers-cov papain-like protease has delsgylating and deubiquitinating activities murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis facilities for macromolecular crystallography at the helmholtz-zentrum berlin coronavirus as a possible cause of severe acute respiratory syndrome crystal structure of usp7 ubiquitin-like domains with an icp0 peptide reveals a novel mechanism used by viral and cellular proteins to target usp7 structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease identification of a new human coronavirus structure of ubiquitin refined at 1.8 å resolution recent progress in the discovery of inhibitors targeting coronavirus proteases on the use of the merging r factor as a quality indicator for x-ray data papain-like protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease isolation of a novel coronavirus from a man with pneumonia in saudi arabia technical assistance by susanne zoske is gratefully acknowledged. we thank stefan anemüller for discussion. we also acknowledge access to beamline bl14.2 operated by the helmholtz-zentrum berlin at the bessy ii electron storage ring (berlin-adlershof, germany). this work was supported by the european commission through its "silver" project (contract no. health-f3-2010-260644) and by the german center for infection research (dzif). rh acknowledges support by the dfg cluster of excellence "inflammation at interfaces" (exc 306) this article does not contain any studies with human or animal subjects. both authors declare no competing interest. jl and rh designed all experiments. jl performed the experiments. jl and rh analyzed all data. jl and rh wrote the manuscript.supplementary figure and table are available on the websites of virologica sinica: www.virosin.org; link. springer.com/ journal/12250. key: cord-313684-61hkogdh authors: samaddar, arghadip; grover, malika; nag, vijaya lakshmi title: pathophysiology and potential therapeutic candidates for covid-19: a poorly understood arena date: 2020-09-17 journal: front pharmacol doi: 10.3389/fphar.2020.585888 sha: doc_id: 313684 cord_uid: 61hkogdh coronavirus disease 2019 (covid-19), an acute onset pneumonia caused by a novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), emerged in the wuhan city of china in december 2019 and evolved into a global pandemic. to date, there are no proven drugs or vaccines against this virus. hence, the situation demands an urgent need to explore all potential therapeutic strategies that can be made available to prevent the disease progression and improve patient outcomes. in absence of clinically proven treatment guidelines, several repurposed drugs and investigational agents are currently being evaluated in clinical trials for their probable benefits in the treatment of covid-19. these include antivirals (remdesivir, lopinavir/ritonavir, umifenovir, and favipiravir), interferon, antimalarials (chloroquine/hydroxychloroquine), antiparasitic drugs (ivermectin and nitazoxanide), biologics (monoclonal antibodies and interleukin receptor antagonist), cellular therapies (mesenchymal stem cells and natural killer cells), convalescent plasma, and cytokine adsorber. though several observational studies have claimed many of these agents to be effective based on their in vitro activities and extrapolated evidence from sars and middle east respiratory syndrome (mers) epidemics, the currently available data remains inconclusive because of ill-defined patient selection criteria, small sample size, lack of concurrent controls, and use of intermediary outcomes instead of patient-relevant outcomes. moreover, there is a need to clearly define the patient populations who warrant therapy and also the timing of initiation of treatment. understanding the disease pathology responsible for the clinical manifestations of covid-19 is imperative to identify the potential targets for drug development. this review explains the pathophysiology of covid-19 and summarizes the potential treatment candidates, which can provide guidance in developing effective therapeutic strategies. in december 2019, a cluster of cases of unexplained acute pneumonia was reported from the wuhan city of china's hubei province. as the causative agent could not be identified, these initial cases were classified as "pneumonia of unknown etiology." later on, the cause of this illness was attributed to a novel betacoronavirus, which was designated as 2019-novel coronavirus (2019-ncov) by the world health organization (who) (cascella et al., 2020) . on january 30, 2020, as per the international health regulations (ihr, 2005) , the outbreak was declared a public health emergency of international concern (pheic) by the who. on february 11, 2020, the disease was renamed as coronavirus disease 2019 , and on the same day, the coronavirus study group (csg) of the international committee on taxonomy of viruses designated 2019-ncov as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) due to its phylogenetic similarity with severe acute respiratory syndrome coronavirus (sars-cov) (cascella et al., 2020) . considering its potential to evolve into a pandemic, the who raised the threat to the epidemic to the "very high" level on february 28, 2020. 1 with the alarming increase in the number of covid-19 cases outside china, affecting thousands of people across several countries, the who declared covid-19 a pandemic on march 11, 2020. 2 as of august 16, 2020, covid-19 has affected more than 21 million people across 216 countries and territories, with 761,779 deaths. 3 usa accounts for the maximum number of cases, followed by brazil, india, and russia. coronaviruses (covs) are a group of enveloped viruses with positive sense single-stranded rna genome and having a crownlike appearance under an electron microscope. they belong to the order nidovirales, family coronaviridae, and subfamily orthocoronavirinae. based on genetic and antigenic criteria, covs are classified into four genera: alphacoronavirus (a-cov), betacoronavirus (b-cov), deltacoronavirus (d-cov), and gammacoronavirus (g-cov). to date, seven covs capable of infecting humans (hcovs) have been identified (cascella et al., 2020) . according to an estimate, 2% of the population are healthy carriers of covs and they account for 5 to 10% of acute respiratory infections . the common hcovs, hcov-oc43 and hcov-hku1 (b-covs) and hcov-229e and hcov-nl63 (a-covs), cause mild self-limiting respiratory tract infections. other human covs, sars-cov, sars-cov-2, and middle east respiratory syndrome coronavirus (mers-cov) (b-covs), cause epidemics with variable clinical severity featuring respiratory and extrarespiratory manifestations. sars-cov and mers-cov infections possess pandemic potential and can cause life-threatening disease with mortality rates up to 10% and 35%, respectively (cascella et al., 2020) . the g-covs infect avian species, while d-covs tend to infect both mammals and birds (zumla et al., 2016; de wilde et al., 2018) . phylogenetic analysis has placed sars-cov-2 under the subgenus sarbecovirus of the genus betacoronavirus. next generation sequencing data has revealed that the genome of sars-cov-2 bears 96.2% sequence homology with a bat coronavirus ratg13 and shares 79.5% identity with sars-cov (zheng, 2020) . based on phylogenetic and evolutionary analyses, it has been proposed that both bat-cov ratg13 and sars-cov-2 might have evolved from a common ancestor, and sars-cov-2 might have jumped from bats to humans via some unknown intermediate hosts (perrotta et al., 2020) . covid-19 is an acute respiratory disease with a clinical spectrum ranging from mild and moderate disease (80%) to severe (15%) and critical illness (5%), with an overall case fatality rate (cfr) of 0.5-2.8%. 4 the severe and critical illness categories (nearly 20% of all infections) are of special concern in elderly population and those with underlying comorbidities, as the severity and cfr are particularly high in these groups (perrotta et al., 2020) . several risk factors related to disease severity have been outlined by the united states centers for disease control and prevention (cdc). advanced age, male sex, and smoking have been reported as independent risk factors for disease progression, severity, and mortality. it was observed that 20% of the patients in italy over 80 years of age succumbed to the disease (livingston and bucher, 2020) , and as per cdc reports, 31-70% of the patients above the age of 85 in the united states required hospitalization (vishnevetsky and levy, 2020) . a weekly surveillance report by the who regional office for europe reported that over 95% of all deaths due to covid-19 were people aged 60 years or above, and more than 50% were people aged 80 years or older. 5 it has been hypothesized that there occurs an age-related decline in the clearance of inhaled particles in small airways, possibly due to decrease in the number of cilia and ciliated epithelial cells in the airways (svartengren et al., 2005) . an age-dependent increase in nasal-cavity volume coupled with decreased nasal resistance and upper airway size are other contributory factors (levitzky, 1984) . as age advances, there occurs a disruption of the innate and adaptive arms of the immune system with an impairment of both effector memory t cell and competent b cell functions, along with continuous production of inflammatory mediators and cytokines (inflammaging) (aw et al., 2007) . in healthy state, angiotensin converting enzyme 2 (ace2) catalyzes the conversion of angiotensin 2 to angiotensin 1−7 and thus, maintains a homeostasis between inflammatory and anti-inflammatory pathways. ace2 levels have been found to decrease in old age causing elevated angiotensin-2, which increases pulmonary vascular permeability and inflammation, thereby worsening lung injury due to covid-19 in such patients (dhochak et al., 2020) . moreover, in the elderly, there occurs an age-related decrease in the vital capacity of lungs and perfusion of vital organs, such as, heart, lungs, and kidneys. sars-cov-2 causes a much more severe pneumonia in the aged than younger individuals. it has been observed that the incidence of acute respiratory distress syndrome (ards) is higher in the elderly and those with heart, liver and kidney ailments (perrotta et al., 2020; wang l. et al., 2020) . also, older age is a surrogate for comorbid illnesses, such as, respiratory and cardiovascular disorders, morbid obesity (i.e., body mass index of ≥40), hypertension, diabetes mellitus, and significant renal and hepatic impairment (preskorn, 2020) . all these risk factors have been linked to higher rates of intensive care unit (icu) admission, greater disease severity, and poor prognosis. it has been observed that 90% of the patients who require hospitalization, icu admission, or succumb to the disease have one or more comorbid conditions, irrespective of age (garg et al., 2020) . therefore, in the elderly, immunosenescence and underlying comorbidities are likely to be the major contributory factors for life-threatening respiratory failure and multisystemic involvement associated with covid-19. on the contrary, children develop milder symptoms, rarely require hospitalization, and have an overall better prognosis when compared to adults (ludvigsson, 2020) . a systematic review and meta-analysis including 7780 children with covid-19 from 26 countries reported milder self-limiting symptoms in majority of the cases, with 0.14% being critical and seven deaths. unlike adults, children rarely progressed to severe disease requiring icu admission (hoang et al., 2020) . this can be attributed to the immature immune system in pediatric population, cross-protection from related coronaviruses and other rna viruses to which they get exposed early in life, competitive inhibition of sars-cov-2 by other respiratory viruses simultaneously invading the airways and the lungs, trained non-specific immunity due to childhood immunization (e.g., bacillus calmette-guerin vaccine and mumps measles rubella vaccine), good regenerative capacity of lungs in children, absence of immunosenescence and age-related comorbidities, and high ace2 expression causing increased metabolism of angiotensin 2 (dhochak et al., 2020) . despite overwhelming global efforts, covid-19 remains a poorly understood disease with limited success in the field of drug development. understanding the disease pathogenesis is crucial for choosing effective drug targets. this review explains the pathophysiology of covid-19 and summarizes the potential treatment candidates, which can provide guidance in developing efficient therapeutic strategies. sars-cov-2 is a positive sense, single-stranded rna virus belonging to the genus betacoronavirus (subgenus sarbecovirus, subfamily orthocoronavirinae). the genomic mrna has a 5´-cap and a 3´-poly (a) tail and can act as an mrna for translation of the viral polyproteins. in addition, both 5´and 3´ends of the genomic rna contain a highly structured untranslated region (utr) that plays an important role in the regulation of rna replication and transcription. the sars-cov-2 genome contains 14 open reading frames (orfs), preceded by transcriptional regulatory sequences (trss). the two main transcriptional units, orf1a and orf1ab, comprise two-thirds of the viral genome and encode two major polyproteins: pp1a (~500 kda) and pp1ab (~800 kda), respectively. the synthesis of pp1ab involves programmed ribosomal frame shifting during translation of orf1a. these polypeptides are cleaved by virally encoded chymotrypsin-like protease (3clpro), main protease (mpro), and papain-like protease (plpro) into 16 non-structural proteins (nsp1-nsp16), which assemble to form the replicationtranscription complex (rtc) involved in genome transcription and replication (naqvi et al., 2020; romano et al., 2020) . pp1a is cleaved into 11 non-structural proteins (nsp1-nsp11) and pp1ab into five (nsp12-nsp16). the non-structural proteins play an important role in the pathogenesis of covid-19. nsp3 and nsp5 encode plpro and 3clpro, respectively, which help in peptide cleaving and host innate immune antagonism. nsp12 and nsp15 encode rna-dependent rna polymerase (rdrp) and rna helicase, respectively. other orfs at the 3´end of the viral genome encode four structural proteins: the spike surface glycoprotein (s), membrane (m), envelope (e), and the nucleocapsid (n) proteins, which are the major components of the virus playing a crucial role in structural integrity and pathogenesis (romano et al., 2020; . the s protein is a homotrimeric transmembrane glycoprotein that determines diversity to coronaviruses and host tropism. it has two functional subunits: s1, responsible for binding to the host ace2 receptors and s2, for the fusion of the virion and cellular membranes. the m protein helps in transport of nutrients across the cell membrane, bud release, and the formation of viral envelope. the e protein plays a significant role in viral morphogenesis and assembly. the n protein plays an important role in packaging of viral rna into ribonucleocapsid and also helps in immune evasion by attenuating host immune responses (astuti, 2020; naqvi et al., 2020) . besides these structural proteins, the 3´end also contains eight putative orfs for accessory proteins: 3a, 3b, p6, 7a, 7b, 8b, 9b, and orf14. the structural and accessory proteins are translated from a set of nested sub-genomic rnas (sgrnas) . the life cycle of sars-cov-2 consists of five steps: attachment, penetration, biosynthesis, maturation, and release. entry of the virus into the host cells is facilitated by interactions between the s protein and its receptors, ace2, which are found in various organs such as heart, lungs, kidneys, and gastrointestinal tract. the s protein binds to ace2 through the receptor binding domain (rbd) region of the s1 subunit, which consists of a core and a receptor binding motif (rbm). rbm specifically recognises human ace2 as its receptor (yuki et al., 2020) . the s protein/ace2 interaction (attachment) is the primary determinant to infect a host species and also controls tissue tropism. ace2 mediates human-to-human transmission, and also acts as a receptor for sars-cov and respiratory coronavirus nl63 (astuti, 2020) . following binding of the virus to the host ace2 receptors, the s protein undergoes a two-step sequential proteolytic cleavage, one at s1/s2 cleavage site for priming and another at s2ˊsite for activation. the latter acts as a viral fusion peptide that inserts into the membrane, followed by the joining of two heptad repeats in s2 forming a six-helix bundle. the formation of this bundle results in fusion and entry of virus into the host cell (penetration) (tang et al., 2020) . another receptor, which has found to be of importance in viral invasion, is cluster of differentiation 147 (cd147), also known as extracellular matrix metalloproteinase inducer (emmprin) or basigin . a characteristic unique to sars-cov-2 is the existence of a novel furin cleavage site (prrars) at s1/ s2, which confers the ability to infect organs and tissues where furin is ubiquitously expressed such as the brain, lung, liver, gastrointestinal tract, and pancreas . other proteases that may play a role in virus entry are transmembrane protease serine 2 (tmprss2) and cathepsin l. following internalization, there is uncoating and release of viral ssrna in the host cell cytoplasm, which then gets attached to the ribosomes and is translated into two large polyproteins, pp1a and pp1ab. these polyproteins are cleaved by virus-encoded proteinases into 16 nsps. many of these non-structural proteins congregate to form the rtc in double-membrane vesicles (dmvs), which are mainly an assembly of rdrp-and helicase-containing subunits (astuti, 2020; . synthesis of genomic rna follows the translation and assembly of viral replicase complexes. rtc is responsible for rna replication and transcription of the sgrnas. the latter serve as mrnas for the translation of structural and accessory proteins (biosynthesis). following translation, the s, e, and m proteins are transported to the endoplasmic reticulum where they move along the secretory pathway into the endoplasmic reticulum-golgi intermediate compartment (ergic). in the compartment, the viral genomes are encapsidated by the n protein, which then bud into the membrane resulting in formation of the mature virus (maturation) (fehr and perlman, 2015) . the m protein regulates most of the protein-protein interactions required for virus assembly. however, virus-like particles (vlps) can only be formed when m protein is co-expressed with e protein, suggesting the role of these two proteins for production of viral envelope. following assembly, the virions are transported to the cell surface in vesicles and released by exocytosis (release) (fehr and perlman, 2015; astuti, 2020) . host immune response to sars-cov-2 the entry of virus into the host cells triggers stimulation of innate immune response via antigen presenting cells (apcs) like dendritic cells and macrophages, which represent the first line of defence against viruses. apcs have pattern recognition receptors (prrs), such as, toll-like receptors (tlrs), nod-like receptors (nlrs), rig-i-like receptors (rlrs), and melanoma differentiationassociated protein 5 (mda5) present at various locations like plasma membrane, endosomal membrane, lysosomes, and cytosol . they recognize various structural components of the virus, such as, nucleic acids, carbohydrate moieties, glycoproteins, lipoproteins, and dsrna and induce a signaling cascade to produce the immune system effectors. the apcs present the viral antigenic peptides to the cd8+ t cells in association with major histocompatibility complex (mhc) class i. the cd8+ t cells get activated, undergo clonal expansion and develop into virus-specific effector and memory t cells. with their perforin and granzymes, cd8+ t cells lyse the virusinfected cells and induce apoptosis. in addition, there occurs an upregulation of natural killer (nk) cell activation and production of pro-inflammatory cytokines via the nuclear factor kappa b (nf-kb) and interferon regulatory factor 3 (irf3) signaling pathways. this leads to further recruitment of neutrophils and monocytes to the site of infection and activation of several other pro-inflammatory cytokines (astuti, 2020; li et al., 2020) . during an infection, activation and priming of innate and adaptive immune responses result in pathogen clearance and recovery. however, sars-cov-2 causes suppression of host's innate immune response by inhibiting certain signaling pathways and thus, evades detection by the immune system, leading to a more severe disease and fatal outcomes (felsenstein et al., 2020) . it has been postulated that sars-cov-2, like sars-cov, alters the ubiquitination and degradation of rna sensors (rig-i and mda5) and inhibits the activation of mitochondrial antiviral-signaling protein (mavs), thereby preventing the activation and nuclear translocation of irf3 in response to activated rna sensors. moreover, sars-cov2 inhibits tumor necrosis factor (tnf) receptor-associated factors (traf) 3 and 6, which are crucial for the induction of irf-3/7 in response to tlr3/7 and nfkb signaling pathways. it also inhibits the phosphorylation of janus kinase/signal transducers and activators of transcription (jak/stat) transcription factor and blocks type i/iii interferon (ifn) signaling pathways (kindler et al., 2016) . these mechanisms allow the virus to replicate evading the innate antiviral responses and induce the production of cytokines required for recruitment of adaptive immune cells. the transition between innate and adaptive immune responses is critical for the clinical course of covid-19. this phase determines whether the immune regulatory events will culminate in protective immunity or an exacerbated immune response. the protective immunity is t cell mediated, with cd8+ t cells eliminating the virus-infected cells and cd4+ t cells helping the b cells to produce neutralizing antibodies and orchestrating the response of other immune cells. the t cells account for 80% of the infiltrating cells in sars-cov-2 infection. however, a dysregulated t cell response can result in immunopathology leading to exaggerated cytokine release and a cytokine storm (cao, 2020; tay et al., 2020) . this condition is characterized by increased secretion of pro-inflammatory cytokines, such as interleukin (il)-1b, il-2, il-6, il-7, il-8, il-9, il-10, and il-17; granulocyte-macrophage colony stimulating factor (gm-csf); tnf-a, ifn-g and ifn-g inducible protein 10 (ip10); monocyte chemoattractant protein 1 (mcp-1); macrophage inflammatory protein-1 alpha and -1 beta (mip-1a and -1b); chemokines like cc chemokine ligand 2 (ccl2), ccl3, and ccl5; and c-x-c motif chemokine ligand 8 (cxcl8), cxcl9, and cxcl10. the cytokine storm induces a hyperinflammatory state causing acute lung injury and various complications like ards, respiratory failure, shock, disseminated intravascular coagulation, multiorgan failure and death (qin et al., 2020; xu z. et al., 2020) . this complex cascade of inflammatory response triggers platelet activation, endothelial dysfunction, and vascular stasis. recent studies suggest that covid-19 induces a hypercoagulable state that may predispose to venous and arterial thromboembolic events and worsened outcomes (abou-ismail et al., 2020) . the humoral immune response is critical for virus clearance and preventing reinfection. sars-cov-2 elicits a robust b cell response, as evidenced by detection of virus-specific neutralizing antibodies in most cases following infection. seroconversion occurs between 7 and 14 days after symptomonset, and antibody titers persist in the weeks following virus clearance (vabret et al., 2020) . the rbd of s protein is highly immunogenic, and antibodies against this domain can block virus interaction with the host ace2 receptors and thus, prevent virus entry (ju et al., 2020) . the subepithelial dendritic cells and macrophages recognize the viral proteins and present them to cd4+ t cells in association with mhc class ii, which induces differentiation of these t cells into th1, th17, and memory t follicular helper (t fh ) subsets. the t fh cells induce the conversion of b cells to plasma cells and promote the production of virus-specific igm, iga, and igg antibodies (cao, 2020) . like other viral infections, the initial antibody response in covid-19 is predominantly igm, which is transient and shortlived and soon gets replaced by igg antibodies. the latter has a longer half-life and lower molecular weight, which enable it to confer long-term protection and effective tissue penetration. however, different patterns of igm and igg seroconversion have been observed, such as synchronous seroconversion, igm seroconversion preceding that of igg, and igm seroconversion later than that of igg. secretory iga plays a crucial role in mucosal immunity by neutralizing the virus and preventing its attachment to the mucosal epithelium (long et al., 2020) . the proposed host immune response to sars-cov-2 has been shown in figure 1 . currently, there are no clinically proven antiviral drugs or biologics for the treatment of covid-19 patients. a protocol issued by national health commission of the people's republic of china states that optimized symptomatic management, together with respiratory support should be the mainstay of treatment . most existing data on antiviral therapy for covid-19 are derived from related coronaviruses, such as, sars-cov (2003) , and mers-cov (2012) and noncoronaviruses such as ebola virus. how well these data can be extrapolated to sars-cov-2 remains unclear. moreover, a lack of pharmacokinetic/pharmacodynamic or clinical data comparing achievable exposures with treatment outcomes further questions the clinical relevance of in vitro activity of antiviral drugs, which may vary widely and therefore, should be compared cautiously. since the onset of this pandemic, several studies emphasizing the therapeutic benefits of a wide range of antiviral drugs and biologics have been published in medical literature. however, a thorough analysis of these drugs is warranted to ascertain whether the existing evidence supports the currently proposed management strategies. an overview of various repurposed and investigational drugs undergoing clinical trials against covid-19 has been depicted in figure 2 (tu et al., 2020) . there are more than 300 ongoing clinical trials evaluating the safety and efficacy of these drugs. the major proposed therapeutic candidates that seem promising for the treatment of covid-19 are summarized in table 1 . remdesivir (veklury; gilead sciences, inc.) is an analog of adenosine triphosphate, which incorporates into the nascent viral rna chains and results in delayed chain termination during replication of viral rna. it has broad-spectrum antiviral activity against several rna viruses including ebola, marburg, mers-cov, sars-cov, respiratory syncytial virus (rsv), nipah virus, and hendra virus and has demonstrated prophylactic and therapeutic efficacy against coronaviruses (gordon et al., 2020) . use of remdesivir in sars-covinfected mice resulted in reduced viral loads and improved disease outcomes. recently, the drug has been shown to possess in vitro activity against sars-cov-2. remdesivir seems to possess a favorable safety profile, as evidenced in 500 participants, including healthy volunteers and patients who received remdesivir for ebola virus disease (mulangu et al., 2019) . its prophylactic and therapeutic efficacy was demonstrated in a rhesus macaque model of mers-cov infection, in which prophylactic administration of remdesivir 24 hours prior to mers-cov inoculation completely prevented clinical disease, inhibited viral replication, and prevented the development of pulmonary lesions. therapeutic administration of the drug 12 hours post-inoculation reduced the severity of clinical symptoms, attenuated viral replication, and decreased the pulmonary lesions (de wit et al., 2020) . gilead sciences, in a recent case series, considered compassionate-use of remdesivir in 53 covid-19 patients with severe disease and reported that 68% of the cases showed clinical improvement after a median followup of 18 days, with mortality of 13% and a favorable safety profile (grein et al., 2020) . the findings were, however, not compared with a control group that received only standard care. at present, there are six ongoing clinical trials evaluating the safety and efficacy of remdesivir in adult patients diagnosed with covid-19 (moderate/severe disease): two initiated by gilead sciences, one by national institute of allergy and infectious diseases (niaid), one by inserm (france), and two by china-japan friendship hospital. all these clinical trials are currently in phase iii. formal recommendations regarding the use of remdesivir can be made once these trials come up with some conclusive evidence. lopinavir/ritonavir (lpv/r; kaletra) is a combination of protease inhibitors used for the treatment of hiv infection. ritonavir is also a potent inhibitor of cytochrome p450, a class of enzymes responsible for metabolism of lopinavir, and the co-administration augments the plasma levels of lopinavir, improving its antiviral activity (molla et al., 2002) . lpv/r has demonstrated in-vitro antiviral activity against sars-cov and mers-cov. since this combination was not specifically formulated for treatment of coronavirus infections, this alone may not demonstrate a significant advantage over placebo in reducing viral load (yao et al., 2020) . a clinical trial involving 199 patients with laboratory-confirmed sars-cov-2 infection reported that lpv/r combination did not offer any clinical benefit over the standard management . there are several ongoing clinical trials comparing the efficacy of lpv/r alone and in combination with other drugs like umifenovir, carrimycin, danoprevir/ritonavir, interferon, xiyanping, and traditional chinese medicines. lpv/r in combination with ifn-b1b reduced mers-cov viral load and improved lung pathology in a marmoset model (yao et al., 2020) . however, sheahan et al. (2020) reported that combining lpv/r with ifn-b did not significantly augment the antiviral activity of the latter against mers-cov. in an open label clinical trial involving hospitalized sars patients, lpv/r in combination with ribavirin was found to decrease the mortality rate and requirement of ventilator support compared to the control group (median, 6 days versus 11 days; 95% ci, −9-0) . thus, considering the therapeutic benefits in the treatment of sars and mers, the safety and efficacy of lpv/r based combination regimen in the treatment of covid-19 needs to be evaluated. figure 1 | host response to sars-cov-2. the virus attaches to ace2 receptors and enters the target cell by membrane fusion. upon entry, the virus is recognized by innate immune receptors tlr7/8, cytosolic rna sensors rig-i/mda-5, and the inflammasome sensor nlr family pyrin domain-containing-3 (nlrp3). this leads to the activation of nf-кb and irf3/7 and the subsequent production of pro-inflammatory cytokines (e.g., il-1b, il-6, and tnf-a) and type i ifns, respectively. cytokines released by infected cells modulate the adaptive immune response by causing recruitment and activation of macrophages, b cells, and t cells which facilitate elimination of the virus. however, an unbalanced immune response can cause massive release of pro-inflammatory cytokines, leading to a cytokine storm which is responsible for the severe clinical manifestations of covid-19. umifenovir (arbidol, pharmstandard ltd.) is a fusion inhibitor that interacts with viral hemagglutinin and prevents the fusion of viral envelope with host cell membrane. the drug is currently licensed for use only in russia and china for the treatment and prophylaxis of influenza and other respiratory viral infections. umifenovir has a broad-spectrum antiviral activity due to its dual action as direct-acting antiviral and host-targeting agent. it has been found to be active against several enveloped and nonenveloped rna and dna viruses, including chikungunya virus, zika virus, foot-and-mouth disease virus, lassa virus, ebola virus, hsv, hbv, hcv, chikungunya virus, reovirus, hantaan virus, and coxsackie virus b5 (blaising et al., 2014; kadam and wilson, 2017) . it also inhibits clathrin-mediated exocytosis and intracellular trafficking by interacting with the cell membrane (blaising et al., 2013) . considering its unique mechanism of action, umifenovir alone and in combination with antiretroviral drugs is currently being investigated for treatment and prophylaxis of covid-19. however, a retrospective study by lian et al., patients showed that umifenovir did not shorten the sars-cov-2 negativity time or improve the prognosis in non-icu patients compared to the supportive treatment . there are currently four ongoing clinical trials of umifenovir for covid-19 treatment: one in comparison with the basic treatment 6 , and the other three comparing the effects in combination with oseltamivir 7 , lopinavir/ ritonavir 8 , and carrimycin. 9 , 2017) . besides influenza a and b, it has been found to be effective against avian influenza. it has also been investigated for the treatment of infections caused by ebola virus, lassa virus, and now, sars-cov-2 . favipiravir is a prodrug that gets metabolized to an active form favipiravirribofuranosyl-5′-triphosphate (favipiravir-rtp), which selectively binds to rdrp and inhibits viral replication. in contrast to the existing antivirals against influenza that primarily block the entry and exit of the virus from cells, favipiravir's novel mechanism of action allows its active form to get incorporated into the nascent rna strand, thus preventing strand elongation and viral proliferation. the drug has an oral bioavailability of 97.6 and is 54% plasma protein-bound with an elimination half-life of 2-5 hours . the rdrp gene of sars-cov-2 is structurally similar to that of sars-cov and mers-cov, as revealed by genome sequencing (cascella et al., 2020) . a clinical trial (chictr2000029600) conducted in shenzhen, china reported that covid-19 patients who received favipiravir demonstrated significantly shorter viral clearance time and higher improvement in chest imaging, compared to the control group (4 days, 91.4% versus 11 days, 62%) (cai et al., 2020) . in another multi-centre randomized trial (chictr2000030254), treatment with favipiravir was found to be beneficial for covid-19 patients with diabetes and/or hypertension as evidenced by decreased time-to-relief for fever and cough. also, seven days clinical recovery rate increased from 55.9 to 71.4% . these studies indicate that favipiravir can be a safe and effective treatment option for covid-19. the drug is currently undergoing phase iii clinical trial, which is expected to be completed by july 2020. ifns are a family of inducible cytokines produced by various cell types in response to viral infections. ifns exert their actions through pattern recognition receptors (prrs), which are largely species specific. of particular interest are the type 1 ifns (viral ifns), which are secreted by the plasmacytoid dendritic cells and are among the first cytokines produced during a viral infection. ifn-i comprises of several subtypes (a, b, ϵ, w, and k) (samuel, 2001) , which exert their actions after binding with interferon-a/b receptor (ifnar). ligand binding induces phosphorylation of the receptor and activation of signal transducers and several transcriptional factors such as stat1 and stat2. these form complexes that are translocated to the nucleus, where they activate interferon-stimulated genes (isg). isgs include prrs, irfs, and members of the jak-stat signaling pathway, which sensitize the cell to pathogens, and play a prominent role in inflammation, antiviral innate signaling, immunomodulation, and interfere with several steps of viral replication (schneider et al., 2014) . thus, ifn-i plays a vital role in antiviral immunity. because of their immunomodulatory and antiviral properties, they are often evaluated for the treatment of several emerging viral infections. sars-cov-2 bears a close resemblance with other members of the coronaviridae family such as mers-cov and sars-cov and exhibits similar properties, despite differences in their epidemiology, pathology, and several of their structural proteins. numerous in vivo and in vitro studies have evaluated the role of ifn-i in the treatment of mers-cov and sars-cov, either alone or in combination with lopinavir/ritonavir (chan et al., 2015) , ribavirin (omrani et al., 2014) , remdesivir, corticosteroids, and ifn-g (sainz et al., 2004) . though both ifn-a and-b have demonstrated efficacy in vitro and succeeded in certain animal models, they failed to improve the disease in humans. such difference in therapeutic responses could be attributed to ifn signaling pathway used by the viruses, limited number of study subjects, varied experimental settings or clinical conditions, and ifn-subtype diversity. studies have shown that ifnb, particularly the b1 subtype (ifnb1b or ifnb1a), is a more potent inhibitor of coronaviruses than ifna and thus appears to be more relevant in the treatment coronavirus infections (stockman et al., 2006) . in the lungs, ifnb1 stimulates the secretion of anti-inflammatory adenosine and promotes maintenance of endothelial barrier function by up-regulating cd73 in pulmonary endothelial cells. this can be a possible explanation to the reduction of vascular leakage in ards with ifnb1a treatment (bellingan et al., 2014) . the timing of ifn-i administration plays a critical role, with positive effects being observed early in the course of infection while delayed administration failed to inhibit viral replication (channappanavar et al., 2019) . based on previous knowledge, it has been hypothesized that sars-cov and mers-cov are able to disrupt the interferon signaling pathway probably through involvement of orf6 and orf3b (kopecky-bromberg et al., 2007) . however, due to the truncated nature of orf6 and orf3b proteins in sars-cov-2, they may have lost their antiinterferon activities. this could be a possible explanation for sars-cov-2 displaying substantial in vitro sensitivity to ifn-i. thus, ifn-i is expected to be more promising for the treatment of covid-19 than for sars (lokugamage et al., 2020) . the assumption is further supported by the fact that ifna2b sprays minimise the infection rate of sars-cov-2 and can be used prophylactically against the virus administration of 5 million units of ifna by vapor inhalation twice a day, in combination with ribavirin (dong et al., 2020) . vapor inhalation offers the advantage of specifically targeting the respiratory tract. the efficacy of ifn-i can be further improved if given in combination with lopinavir/ritonavir, ribavirin, or remdesivir because of the efficacy of such combinations observed in vitro against other coronaviruses (sheahan et al., 2020) . further research on ifn-based treatment is expected in near future, which should give more accurate information on the efficacy of this therapy and possible outcomes. ivermectin (stromectol; merck & co., inc.) is a broad spectrum anthelmintic agent belonging to class of avermectins and is derived from the soil bacterium streptomyces avermitilis. it's selective and high affinity binding with glutamate-gated chloride channels in nerve and muscle cells of nematode, increases the permeability of the cell membrane to chloride ions, resulting in hyperpolarization of cells and paralysis and death of the parasite. it is 93.2% plasma protein-bound and has a half-life of 18 hours following oral administration. the drug was originally launched by merck laboratories in 1987 for use against onchocerciasis (river blindness) as a part of the onchocerciasis control programme in west africa. subsequently, the drug was approved for the treatment of a number of human parasitic infections including strongyloidiasis, ascariasis, trichuriasis, enterobiasis, lymphatic filariasis, and scabies in several countries (australia, france, japan, the netherlands, usa, etc) (ikeda, 2003) . besides its anti-parasitic action, several studies have demonstrated the potent antiviral activity of ivermectin against a broad range of viruses in vitro (caly et al., 2020) . it has been shown to inhibit the interaction between the hiv-1 integrase protein (in) and the importin (imp) a/b1 heterodimer, causing inhibition of hiv-1 replication (wagstaff, 2012) . ivermectin has also been reported to limit infections caused by several rna viruses (dengue viruses 1-4, west nile virus, venezuelan equine encephalitis virus, and influenza virus) and dna virus (pseudorabies virus) (wagstaff, 2012; caly et al., 2020) . studies have found that host cell division might be affected during sars-cov infection, due to a signal-dependent nucleocytoplasmic shutting of the viral nucleocapsid protein involving impa/b1 (timani, 2005; wulan, 2015) . the antiviral activity of the stat1 transcription factor is blocked by sars-cov accessory protein orf6, which causes sequestration of impa/b1 on the rough endoplasmic reticulum/golgi membrane (frieman, 2007) . considering ivermectin's inhibitory action on impa/b1-mediated nuclear import, it is presumed to be effective against sars-cov-2. caly et al. (2020) studied the antiviral activity of ivermectin against sars-cov-2 and observed that a single treatment with ivermectin was able to cause ∼5000-fold reduction of virus titre at 48 h in vero/hslam cell culture. ivermectin has a favorable safety profile in humans with high dose therapy considered as safe as the standard low-dose regimen. however, the therapeutic benefits from multiple drug dosing need to be evaluated in covid-19 patients. an effective antiviral drug given early in the course of infection can help reduce the viral load and prevent disease progression while limiting person-person transmission. ivermectin's unique antiviral action combined with a favorable safety profile allows it for further consideration as a possible treatment option in covid-19. hydroxychloroquine (hcq) (plaquenil; sanofi-synthelabo inc.) is an aminoquinoline like chloroquine and is indicated for the treatment of uncomplicated malaria, prophylaxis of malaria in places without chloroquine resistance, chronic discoid lupus erythematosus, systemic lupus erythematosus, and rheumatoid arthritis. in addition, hcq has been found to be effective against intracellular bacteria such as coxiella burnetii (raoult et al., 1990) and tropheryma whipplei (boulos et al., 2004) . hcq has also been shown to possess antiviral properties and is already being used in clinical trials for the treatment of hiv infection. it increases endosomal ph which prevents viral fusion and entry into the host cells, inhibits antigen processing and presentation, blocks dimerization of major histocompatibility complex (mhc) class ii, and reduces host inflammatory response by decreasing the release of cytokines like il-1 and tnf-a. hcq inhibits terminal glycosylation of ace2 receptor, the main portal of entry for sars-cov and sars-cov-2. non-glycosylated ace2 interacts less efficiently with the viral spike protein, thus preventing viral entry (colson et al., 2020) . several studies have proposed that repurposing of approved drugs such as chloroquine, hcq, azithromycin, metformin, losartan, and simvastatin could be useful in the treatment of covid-19. clinical trials from china have shown the efficacy of chloroquine in the treatment of covid-19 patients, as evidenced by subsidence of fever, improvement of radiological findings, and delay in disease progression. azithromycin (az) is a macrolide antibiotic that has demonstrated in vitro activity against zika and ebola viruses (bosseboeuf et al., 2018) . several authors have mentioned a synergistic effect of hcq/az combination in the treatment of covid-19. an open label non-randomized clinical trial from france showed that covid-19 patients treated with 600 mg of hcq daily had a significant reduction in viral carriage at day 6 post-inclusion, with 70% of the patients having a negative pcr test result compared to only 12.5% in the untreated control group. moreover, patients who were treated with a combination of hcq and az (500 mg on day 1, followed by 250 mg daily for the next four days) showed complete virological cure at day 6 postinclusion compared to 57.1% in the group that received hcq alone (gautret et al., 2020a) . another study from france claimed that patients who received a combination of hcq and az had a significant clinical improvement as evidenced by a rapid fall in viral load, with 83% tested negative by quantitative pcr on day 7 and 93% on day 8. virus cultures of respiratory samples were negative in 97.5% patients on day 5 (gautret et al., 2020b) . however, the apparent beneficial effects of hcq in the treatment of covid-19 have been completely negated by a pilot study from china, where no significant differences in outcomes were observed between hcq-treated group and the control group . a large observational study in hospitalized covid-19 patients in the us also showed that treatment with hcq was not associated with significant clinical benefits and has no influence on intubation or death (geleris et al., 2020) . furthermore, the use of hcq alone or in combination with az is not free from hazards. both these drugs are associated with an increased risk of qt c prolongation, torsades de pointes, ventricular tachycardias, and gastrointestinal side effects. it has been observed that patients receiving a five-day course of az had an increased risk of sudden cardiac death with a hazard ratio of 2.71 (ray et al., 2012) . considering the cumulative adverse effects of hcq and az on cardiac conduction, it is advised to have baseline and follow-up ecg monitoring, along with careful consideration for other concomitant medications known to prolong the qt c interval, if this combination has to be used. guidelines published by the infectious disease society of america mentioned that despite a higher proportion of clinical improvement in the hcq group, the beneficial effect of hcq on viral clearance or disease progression cannot be judged by the currently available evidence due to certain drawbacks such as small sample sizes, ill-defined patient selection criteria, cointerventions, and methodological limitations (bhimraj et al., 2020) . moreover, none of the studies have addressed patientrelevant outcomes like mortality, rate of disease progression to ards, and need for mechanical ventilation. also, the mortality rate among patients receiving hcq/az combination was not compared with an untreated cohort. though studies have claimed that patients receiving hcq and az experienced less virologic failure (43% pooled virologic failure) as compared to historical controls (100% virologic failure) (gautret et al., 2020b; molina et al., 2020) , such comparison lacks certainty because of unmeasured confounding and selection bias. furthermore, these studies have relied mainly on intermediary outcomes such as reduction in development of pneumonia, and less hospital or icu admission to ascertain therapeutic benefits, which raise question on their precision and feasibility. therefore, a rct should be the ideal approach for determining the therapeutic effects of hcq in covid-19 patients. the leading cause of mortality in covid-19 is respiratory failure from ards. a cytokine profile resembling secondary hemophagocytic lymphohistiocytosis (hlh), characterized by a fulminant and fatal hypercytokinemia with multiorgan failure is associated with covid-19. there is a massive and uncontrolled release of pro-inflammatory cytokines like il-2, il-6, g-csf, ip10, mcp-1, mip-1-a and tnf-a (mehta et al., 2020; xu z. et al., 2020) . a recent retrospective study involving 150 confirmed covid-19 cases from wuhan, china, revealed that elevated levels of serum ferritin and il-6 were independent predictors of fatality, probably due to virally driven hyperinflammation (ruan et al., 2020) . tocilizumab (actemra, roche) is a humanized monoclonal antibody against the interleukin-6 receptor (il-6r) approved for the treatment of seriously ill covid-19 patients with elevated il-6 by the national health commission of china. xu x. l. et al. (2020) observed the effects of tocilizumab in 21 covid-19 patients with severe disease, in addition to routine therapy, and reported significant therapeutic benefits as evidenced by subsidence of fever and other symptoms within a few days and improvement of oxygen saturation in 75% of patients. there were no obvious treatment-related adverse reactions. in another report from china, a case of covid-19 with pre-existing multiple myeloma was successfully treated with tocilizumab, highlighting its potential therapeutic benefits in the treatment of covid-19 patients . on march 26, 2020, the drug entered phase iii clinical trial for the treatment of covid-19 pneumonia. the main contributory factors for increased mortality in covid-19 patients are acute lung injury (ali) and ards, brought about by a cytokine-mediated hyperinflammatory response. pulmonary edema is the key detrimental feature of ali/ards. covid-19 is associated with more exaggerated pulmonary mucus exudation than sars as revealed by autopsy . pulmonary imaging and histopathological examination also support similar findings. however, specific pharmacotherapy to combat this pathology is lacking. vascular endothelial growth factor (vegf) is one of the most potent inducers of increased vascular permeability in covid-19affected lungs, causing fluid extravasation and pulmonary edema. expression of vegf is induced by hypoxia through activation of prolyl hydrolases (phd)-hypoxia inducible factor (hif)-1 pathway, which upregulates transcription of vegf. therefore, blockade of vegf signaling pathway might help in reducing inflammation and improving tissue perfusion in patients with severe covid-19. bevacizumab (avastin; genentech ltd.) is a recombinant humanized monoclonal antibody targeted against vegf and is currently recommended for the treatment of malignancies (colorectal, lung, breast, renal, brain, and ovarian), age-related macular degeneration, and diabetic retinopathy. it acts by reducing the elevated vegf levels secondary to hypoxia and severe inflammation, thereby improving tissue perfusion. (wang et al., 2004) . this might help in subsidence of pulmonary edema in covid-19 patients. qilu hospital of shandong university, china is conducting two clinical trials of bevacizumab, both of which are expected to be over by may 2020. thus, bevacizumab holds promise as a potential therapeutic option in the treatment of severe covid-19 patients. studies till date recognize angiotensin converting enzyme 2 (ace2) as the major entry portal for sars-cov-2. however, a novel route of viral invasion through direct interaction between the sars-cov-2 spike protein and cd147, also known as emmprin, expressed on epithelial cells has been recently described by wang k. et al. (2020) meplazumab (ketantin, jiangsu pacific meinuoke biopharmaceutical co. ltd.) is a humanized igg2 monoclonal antibody against cd147 that has demonstrated dose-dependent inhibitory action on sars-cov-2 replication and virus-induced cytopathic effect in vitro (bian et al., 2020) . cd147 binds to cyclophilin a (cypa), a proinflammatory cytokine up-regulated in viral infection, and regulates cytokine secretion and leukocyte chemotaxis. meplazumab is a monoclonal anti-cd147 antibody that inhibits cypa-induced t cell chemotaxis and thus reduces local inflammation. bian et al. (2020) studied the effects of meplazumab in 17 hospitalized patients with covid-19 at tangdu hospital, china, and reported that meplazumab treatment significantly improved the clinical outcomes in severely ill patients. also, the time to virus negativity in the meplazumab group was shortened compared to the control group. these evidences suggest that meplazumab therapy improves the recovery of patients with sars-cov-2 pneumonia and has a favorable safety profile. the drug is currently in phase ii clinical trial, which is expected to be completed by december 2020. itolizumab (alzumab, biocon ltd.) is a humanized anti-cd6 igg1 monoclonal antibody that was introduced in india in 2013 for the treatment of chronic plaque psoriasis. it binds specifically to domain 1 of cd6 and modulates the activation and proliferation of t cells by cd6 co-stimulation, without interfering with the interaction between cd6 and activated leukocyte-cell adhesion molecule. it inhibits intracellular phosphoproteins like mitogen-activated protein kinase (mapk) and stat3 and interferes with cd6mediated intracellular signaling pathways and th17 development. itolizumab downregulates the transcription of pro-inflammatory cytokine genes and thus leads to decreased levels of ifn-g, il-6, and tnf-a, causing attenuation of cytokine storm and t cell infiltration (menon and david, 2015) . considering its unique mechanism of action, the drug has been repurposed for the treatment of crs, which is the leading cause of death in covid-19. a prospective, multi-centric, randomized phase ii study conducted on 30 severely ill covid-19 patients (20 cases and 10 controls) in india showed significant improvement in blood oxygen levels with reduced levels of proinflammatory cytokines and reduced mortality rate in patients who received itolizumab. a similar trial conducted in cuba also indicated positive results with 79.2% of the patients discharged from icu after 2 weeks of treatment. 10 itolizumab has been approved by drugs controller general of india for the treatment of crs in moderate to severe ards patients with covid-19. anakinra (kineret; amgen inc.) is a recombinant human il-1 receptor antagonist that competitively inhibits the binding of il-1a and il-1b to the high-affinity il-1 receptor. it is the first biological agent approved for the treatment of rheumatoid arthritis. it is administered through subcutaneous route and has an absolute bioavailability of 95% (cvetkovic and keating, 2002) . in covid-19 patients, halting the disease progression from manageable hypoxia to frank respiratory failure and ards can have a significant impact on patient management and outcomes. therefore, a therapy directed at intercepting the cytokine storm may be beneficial in this regard. there is an ongoing prospective, randomized, interventional trial comparing the therapeutic effects of individual and simultaneous blockade of il-6 and il-1 versus standard care in covid-19 patients. the trial will include 342 participants whose clinical status after 15 days of treatment will be assessed to measure the effectiveness of anakinra alone and in combination with tocilizumab and siltuximab in restoring lung homeostasis. 11 the study is estimated to be completed in december 2020. considering the role of il-1 in the pathogenesis of acute lung injury in covid-19, anakinra seems to be a promising therapeutic option in the management of such patients. several studies have recognized the potential benefits of cellbased therapies in a number of disease processes including pulmonary, cardiovascular, hepatic, renal, metabolic, and mulculoskeletal disorders. a guideline published by the italian college of anesthesia, analgesia, resuscitation and intensive care has mentioned that stem cells have the potential to decrease icu admission and curtail the number of icu days in covid-19 (vergano et al., 2020) . currently, usfda recommends autologous bone marrow stem cells as the only candidate for stem cell therapy. mesenchymal stem cells (mscs) have shown benefit in the treatment of musculoskeletal disorders such as lowback pain and spinal injuries. the other stem cells that can be considered for clinical use include adipose, amniotic, and umbilical cord stem cells. among these, umbilical cord stem cells seem to be the more attractive as unlike bone marrow, umbilical cord (wharton jelly) has a high concentration of mscs that can be extracted noninvasively (arutyunyan et al., 2016) . moreover, they have fast doubling times, more plasticity, greater potency, and can be efficiently be expanded in the laboratory to cater the large number of expected coronavirus patients (nagamura-inoue and he, 2014). despite being allogenic, mscs can evade the host immune system as they express low levels of mhc i, mhc ii, and t cell co-stimulatory molecules, cd80 and cd86, on their surface. at a cellular level, mscs demonstrate powerful immunomodulatory activity through secretion of anti-inflammatory molecules by paracrine effect and direct interaction with t and b lymphocytes, dendritic cells, macrophages, and nk cells. all these may help in attenuating the cytokine storm (tipnis et al., 2010) . they suppress the hyperactive immune system and promote endogenous repair by improving the cellular microenvironment. multiple studies have demonstrated the beneficial effects of mscs in the settings of ali and ards. when given intravenously, mscs accumulate in the lungs and improve lung function by decreasing inflammation, reducing pulmonary endothelial permeability, facilitating alveolar fluid transport, preventing pulmonary fibrosis, and promoting tissue repair. several clinical trials have documented the safety and efficacy of mscs in immune-mediated inflammatory diseases, such as graft versushost disease (gvhd) and autoimmune disorders (li et al., 2016; atluri et al., 2020; behnke et al., 2020) . mscs secrete antimicrobial peptides and proteins (amps) such as cathelicidin ll-37, human beta-defensin-2 (hbd-2), hepcidin, and lipocalin-2 (lcn2) and anti-inflammatory molecules such as indoleamine 2,3dioxygenase (ido) and interleukin (il)-17. amps cause disruption of membrane integrity, inhibition of protein and nucleic acid synthesis, and blockade of interaction with intracellular targets (alcayaga-miranda et al., 2017) . mscs regulate the host immune response by maintaining a dynamic equilibrium between pro-and anti-inflammatory cytokines. there was a concern that sars-cov-2 can infect the stem cells and render them ineffective. however, a study of seven covid-19 patients (one critically ill, four serious and two mild) in beijing revealed that sars-cov-2 was not able to infect the injected umbilical cord mscs. all patients who received single dose of stem cell therapy recovered during the 14 days follow-up period, while two out of three patients (with serious disease) who did not receive stem cell therapy (control group) had unfavorable outcomes (one died and one developed ards). there was gradual normalization of oxygen saturation and levels of inflammatory biomarkers like crp, aspartic aminotransferase, creatine kinase, and myoglobin in the treated group with no treatment-related adverse events. follow-up ct scan of lungs showed significant radiological improvement (leng et al., 2020) . thus, mscs can be a safe and effective treatment option for patients with covid-19 pneumonia. natural killer (nk) cells (large granular lymphocytes) are innate lymphocyte subsets that constitute the frontline defence system against virus infected and tumor cells. they originate in the bone marrow and represent up to 15% of peripheral blood mononuclear cells. nk cells are phenotypically defined by expression of cd56 and absence of cd3 and do not require prior stimulation to perform their effector functions. nk cells display a diverse range of biological activities that are controlled by several inhibitory and activating receptors. the inhibitory receptors recognize self-mhc class i and prevent nk cell activation. in viral infections, there is upregulation of activating receptors and downregulation of mhc class i expression, which causes activation of nk cells. the major activating receptors include cytotoxicity receptors (nkp46 and nkp44), c-type lectin receptors, and immunoglobulinlike receptors. among the inhibitory receptors, the killer immunoglobulin-like receptors and leukocyte inhibitory receptors have prominent role in defence against viral infections. nk cells lack antigen-specific receptors and kill virus-infected cells through the production of cytokines (tnf-a, gm-csf, ccl5/rantes, and ifn-g), perforin-granzyme-mediated cellular destruction, and death receptor-mediated cytolysis (cooper et al., 2001) . perforin, a pore forming protein, increases the cell permeability, which allows granzymes, a family of serine proteases, to enter into the cell and disrupt cell cycle progression, inflict dna damage, and promote karyolysis (vivier et al., 2008) . they also cause recruitment and activation of other effector cells, including cd8+ t cells and cd4+ th 1 cells. patients with deficient nk cell response are predisposed to recurrent viral infections (jost and altfeld, 2013) . currently, the role of nk cells for immunotherapy in infectious diseases is being explored and results seem to be promising. as hunt for new therapeutic options in the treatment of covid-19 continue to expand, focus has been on the potential benefits of nk cell-based therapy. on april 3, 2020, usfda approved the use of cynk-001, the only cryo-preserved allogeneic nk cell therapy, derived from placental hematopoietic stem cells, in adults with covid-19. the agent's manufacturer celularity, a new jersey-based therapeutic company, in collaboration with sorrento therapeutics is about to launch a phase i/ii clinical trial on cynk-001, involving 86 covid-19 patients. 12 the therapy is already being tested in patients with acute myeloid leukemia, multiple myeloma, and various solid tumors. in january 2020, celularity's cynk-001 was approved by usfda for treatment of glioblastoma multiforme. thus, considering the potent antiviral and immunomodulatory properties of nk cells, their efficacy in the treatment of covid-19 seems promising and needs to be evaluated in clinical trials. convalescent plasma therapy (cpt) is a passive immunization strategy that has been used for the prevention and treatment of several infectious diseases for more than a century. cpt has been successfully used in the treatment of sars (cheng et al., 2005) , mers (ko et al., 2018) , and influenza a h1n1 (hung et al., 2011) , with satisfactory efficacy and safety profile. a protocol for the use of convalescent plasma (cp) in the treatment of mers was established in 2015. cpt is associated with a significant reduction in viral load and pooled mortality as revealed in a large meta-analysis on sars and severe influenza (mair-jenkins et al., 2015) . patients with a high titer of nab, after having recovered from covid-19 may be a valuable donor for cp. it has been observed that the nabs titers in covid-19 patients remain low for the first 10 days following disease-onset and tends to increase thereafter, reaching a peak in 12 to 15 days after the onset . usfda has laid down eligibility criteria for covid-19 cp donors which include: i) evidence of confirmed covid-19 documented by a positive nasopharyngeal pcr at the time of illness or a positive sars-cov-2 antibody test after recovery, ii) complete resolution of symptoms at least 28 days prior to donation or at least 14 days prior to donation and negative results for covid-19, either from a nasopharyngeal swab specimen or by a molecular diagnostic test from blood, iii) male/female donors tested negative for hla antibodies, and iv) sars-cov-2 neutralizing antibody titers of ≥1:160. 14 in a study from china, cpt supplemented with supportive care and antiviral agents was associated with significant clinical and radiological improvement with a rise in neutralizing antibody titers and a fall in c-reactive protein levels within 7 days of initiation of treatment. no treatment-related adverse effects were observed (duan et al., 2020) . similar findings were reported by . a systematic review on cpt for the treatment of covid-19 revealed that cpt is safe, effective, and reduces mortality in critically ill patients (rajendran et al., 2020) . a clinical trial evaluating the benefits of cp in the treatment of covid-19 is being conducted by universidad del rosario, colombia (nct04332380), the results of which are expected to be declared by december 2020. cytosorb (cytosorbents corp.) is an extracorporeal cytokine adsorber that acts by removing the circulating cytokines and redirecting the activated neutrophils to the site of infection. this may help in ameliorating cytokine storm that can otherwise trigger uncontrolled systemic inflammatory response, organ failure, and death. cytosorb offers significant survival benefits in septic shock as observed in several studies. it has been safely used in over 80,000 cases worldwide, primarily in the treatment of several immune-mediated life-threatening conditions such as septic shock, influenza, ards, secondary hlh, liver failure, and pancreatitis. cytosorb helps in protecting endothelial tight junctions, thus reducing capillary leak syndrome. it also modulates pulmonary metabolism, edema formation, and cellmediated infiltration and injury to the lungs. 15 on april 10, 2020, the usfda approved emergency use of cytosorb for the treatment of adult covid-19 patients admitted to icu with features of respiratory failure. 16 sars-cov-2 can induce a sepsis-like syndrome, and in such cases, since pharmacological approaches fail to give promising results, removal of proinflammatory cytokines by hemoadsorption through cytosorb should be considered. to date, more than 200 critically ill patients with covid-19 infection have been treated with cytosorb across various centers in italy, china, and germany. based on positive results in italy, the brescia renal covid task force has formally recommended the use of cytosorb in severe covid-19 patients with stage 3 acute kidney injury, receiving continuous renal replacement therapy (crrt). cytosorb therapy has also been recommended by the national guidelines for the care of adult patients covid-19, panama. in addition, the handbook of covid-19 prevention and treatment, issued by zhejiang university school of medicine, china is also recommending cytosorb therapy for the management of cytokine storm in critically ill covid-19 patients. 15 currently, an ongoing clinical trial (nct04324528) is investigating the efficacy of cytosorb in the treatment of patients with severe covid-19 disease. 17 it is expected to be completed by november 2020. formulating appropriate treatment strategies for covid-19 poses a considerable challenge. during pandemics, in absence of clinically proven treatment guidelines, the tendency is to repurpose drugs based on their antiviral and immunomodulatory activities, as evidenced through observational studies. however, such studies have certain drawbacks like lack of concurrent controls, ill-defined patient selection criteria, small sample size without randomization, and use of intermediary outcomes like viral clearance rather than patient-relevant outcomes. though several repurposed drugs have shown promising results, and their potential clinical benefits appear to outweigh the relatively minor risk of adverse events, conclusive evidence is lacking. there is a need to clearly define the patient populations who warrant therapy and the timing of initiation of treatment. since viral loads are highest early in the course of infection and the disease progression can occur rapidly in stable patients, it is rational to consider rapid initiation of therapy in highrisk populations (old age, hospitalized patients, those with underlying diseases and comorbidities), ideally in the context of a well-controlled, randomized clinical trial. moreover, the demand for unproven therapies can cause shortages of medications that are otherwise indicated for more prevalent diseases like hiv, malaria, hypertension, and diabetes mellitus. the idsa guidelines for treatment of patients with covid-19 raise concern upon these aspects. in an attempt to generate and disseminate clinical data on an urgent basis, a phenomenal increase in fast-track publications related to covid-19 has been observed. however, caution should be exercised because the bulk of the available clinical data are often uncontrolled, not peer reviewed, and subject to publication bias (with an intention to publish outstanding results, there may be a tendency to publish positive outcomes and disregard the negative findings). there are several ongoing clinical trials, some with versatile designs that can reasonably explain the therapeutic benefits offered by these drugs in the management of covid-19. given the plethora of uncertainties concerning the reliability of existing data and the safety and efficacy of the proposed treatments, it would be wise to wait for the results of clinical trials than to adopt clinically unproven therapies. this article has been released as a pre-print at authorea (samaddar et al., 2020) . food and drug administration covid-19 and cytosorb therapy. available at fda has authorized the emergency use of cytosorb 300 ml device for emergency treatment of covid-19 cytokine adsorption in severe covid-19 pneumonia requiring extracorporeal membrane oxygenation (cycov) the hypercoagulable state in covid-19: incidence, pathophysiology, and management antimicrobial activity of mesenchymal stem cells: current status and new perspectives of antimicrobial peptide-based therapies umbilical cord as prospective source for mesenchymal stem cell-based therapy severe acute respiratory syndrome coronavirus 2 (sars-cov-2): an overview of viral structure and host response expanded umbilical cord mesenchymal stem cells (uc-mscs) as a therapeutic strategy in managing critically ill covid-19 patients: the case for compassionate use immunosenescence: emerging challenges for an ageing population msc-based therapies-new perspectives for the injured lung the effect of intravenous interferon-beta-1a (fp-1201) on lung cd73 expression and on acute respiratory distress syndrome mortality: an open-label study infectious diseases society of america guidelines on the treatment and management of patients with covid-19 meplazumab treats covid-19 pneumonia: an open-labelled, concurrent controlled add-on clinical trial arbidol inhibits viral entry by interfering with clathrin-dependent trafficking arbidol as a broad-spectrum antiviral: an update azithromycin inhibits the replication of zika virus antibiotic susceptibility of tropheryma whipplei in mrc5 cells experimental treatment with favipiravir for covid-19: an open-label control study the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 covid-19: immunopathology and its implications for therapy features, evaluation and treatment coronavirus (covid-19) statpearls [internet] (treasure island (fl: statpearls publishing). available at treatment with lopinavir/ritonavir or interferon-b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes emerging coronaviruses: genome structure, replication, and pathogenesis favipiravir versus arbidol for covid-19: a randomized clinical trial. medrxiv a pilot study of hydroxychloroquine in treatment of patients with common coronavirus disease-19 (covid-19). zhejiang da xue xue bao yi xue ban use of convalescent plasma therapy in sars patients in hong kong chloroquine for the 2019 novel coronavirus sars-cov-2 the biology of human natural killer-cell subsets host factors in coronavirus replication prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection pathophysiology of covid-19: why children fare better than adults? discovering drugs to treat coronavirus disease 2019 (covid-19). drug discovery ther favipiravir: pharmacokinetics and concerns about clinical trials for 2019-ncov infection effectiveness of convalescent plasma therapy in severe covid-19 patients coronaviruses: an overview of their replication and pathogenesis covid-19: immunology and treatment options severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease 2019-covid-net, 14 states hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study observational study of hydroxychloroquine in hospitalized patients with covid-19 the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus compassionate use of remdesivir for patients with severe covid-19 the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status covid-19 in 7780 pediatric patients: a systematic review convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection pharmacological effects of ivermectin, an antiparasitic agent for intestinal strongyloidiasis: its mode of action and clinical efficacy control of human viral infections by natural killer cells human neutralizing antibodies elicited by sars-cov-2 infection structural basis of influenza virus fusion inhibition by the antiviral drug arbidol interaction of sars and mers coronaviruses with the antiviral interferon response challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists transplantation of ace2 mesenchymal stem cells improves the outcomes of patients with covid-19 pneumonia effects of aging on the respiratory system low levels of tgf-b1 enhance human umbilical cord-derived mesenchymal stem cell fibronectin production and extend survival time in a rat model of lipopolysaccharideinduced acute lung injury coronavirus infections and immune responses umifenovir treatment is not associated with improved outcomes in patients with coronavirus disease 2019: a retrospective study coronavirus disease 2019 (covid-19) in italy type i interferon susceptibility distinguishes sars-cov-2 from sars-cov. biorxiv antibody responses to sars-cov-2 in patients with covid-19 systematic review of covid-19 in children shows milder cases and a better prognosis than adults the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis covid-19: consider cytokine storm syndromes and immunosuppression itolizumab -a humanized anti-cd6 monoclonal antibody with a better side effects profile for the treatment of psoriasis no evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe covid-19 in vitro antiviral interaction of lopinavir with other protease inhibitors a randomized, controlled trial of ebola virus disease therapeutics umbilical cord-derived mesenchymal stem cells: their advantages and potential clinical utility insights into sars-cov-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study covid-19 and the elderly: insights into pathogenesis and clinical decision-making the 5% of the population at high risk for severe covid-19 infection is identifiable and needs to be taken into account when reopening the economy dysregulation of immune response in patients with coronavirus 2019 (covid-19) in wuhan, china convalescent plasma transfusion for the treatment of covid-19: systematic review bactericidal effect of doxycycline associated with lysosomotropic agents on coxiella burnetii in p388d1 cells azithromycin and the risk of cardiovascular death a structural view of sars-cov-2 rna replication machinery: rna synthesis, proofreading and final capping clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) pathophysiology and potential therapeutic candidates for covid-19: a poorly understood arena antiviral actions of interferons interferonstimulated genes: a complex web of host defenses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov diagnosis and treatment of 2019 novel coronavirus infection in children: a pressing issue treatment of 5 critically ill patients with covid-19 with convalescent plasma sars: systematic review of treatment effects long-term clearance from small airways decreases with age coronavirus membrane fusion mechanism offers a potential target for antiviral development the trinity of covid-19: immunity, inflammation and intervention nuclear/nucleolar localization properties of c-terminal nucleocapsid protein of sars coronavirus immunosuppressive properties of human umbilical cord-derived mesenchymal stem cells: role of b7-h1 and ido a review of sars-cov-2 and the ongoing clinical trials immunology of covid-19: current state of the science siaarti: raccomandazioni di etica clinica per l'ammissione a trattamenti intensivi e per la loro sospensione, in condizioni eccezionali di squilibrio tra necessità e risorse disponibili -versione 01 rethinking high-risk groups in covid-19 functions of natural killer cells ivermectin is a specific inhibitor of importin alpha/betamediated nuclear import able to inhibit replication of hiv-1 and dengue virus biological activity of bevacizumab, a humanized anti-vegf antibody in vitro coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on 4-week follow-up sars-cov-2 invades host cells via a novel route: cd147-spike protein a unique protease cleavage site predicted in the spike protein of the novel pneumonia coronavirus (2019-ncov) potentially related to viral transmissibility genome composition and divergence of the novel coronavirus (2019-ncov) originating in china neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications nucleocytoplasmic transport of nucleocapsid proteins of enveloped rna viruses pathological findings of covid-19 associated with acute respiratory distress syndrome effective treatment of severe covid-19 patients with tocilizumab a systematic review of lopinavir therapy for sars coronavirus and mers coronavirus-a possible reference for coronavirus disease-19 treatment option covid-19 pathophysiology: a review first case of covid-19 in a patient with multiple myeloma successfully treated with tocilizumab sars-cov-2: an emerging coronavirus that causes a global threat coronaviruses-drug discovery and therapeutic options the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 samaddar, grover and nag. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-320928-flsaa1wx authors: aldohyan, meshal; al-rawashdeh, nedal; sakr, farouk m.; rahman, saeed; alfarhan, ali i.; salam, mahmoud title: the perceived effectiveness of mers-cov educational programs and knowledge transfer among primary healthcare workers: a cross-sectional survey date: 2019-03-21 journal: bmc infect dis doi: 10.1186/s12879-019-3898-2 sha: doc_id: 320928 cord_uid: flsaa1wx background: knowledge transfer of middle east respiratory syndrome coronavirus (mers-cov) involves the dissemination of created/acquired information on mers-cov in hospitals, making this information accessible to all healthcare workers (hcws). this study evaluated the perceived effectiveness of mers-cov educational programs and knowledge transfer among primary care hcws at a hospital in saudi arabia that witnessed the largest outbreak of confirmed mers-cov cases in this country. methods: a survey was distributed among primary care hcws at five clinics in saudi arabia in 2016. those with non-direct patient care responsibilities were excluded. their knowledge was evaluated against facts published by mayo clinic foundation, and its percentage mean score (pms) ± standard deviation was calculated. hcws’ perceived effectiveness of educational programs and knowledge transfer was classified as negative or positive. results: sample comprised of 404 hcws, of which 64% were females and 36% were males. almost 26% were ≤ 30 years old, and 42% had > 10 years of work experience. almost 46.5% were nurses, 23.0% physicians, 18.1% were pharmacists, and 12.4% were technical staff. pms for knowledge was 71.1 ± 19.4. the prevalence of negative perceptions towards educational programs was 22.5% and of knowledge transfer was 20.8%. older(> 40 years of age) and more experienced(> 10 years) hcws had the highest pms for knowledge(73.4 ± 18.9,p = 0.005 and 76.9 ± 15.7,p < 0.001 respectively). negative perceptions of educational programs (49.4 ± 20.7; p < 0.001) and knowledge transfer (46.0 ± 19.7; p = 0.001) were associated with a lower knowledge pms. males were 2.4[95% confidence interval 1.4–4.2] times and 2.0[1.1–3.5] times more likely to have negative perceptions of educational programs and knowledge transfer (adjusted (adj.)p = 0.001 and adj. p = 0.023, respectively). physicians/pharmacists were 1.8[1.03–3.11] and 2.8[1.6–5.0] times more likely to have negative perceptions of both outcomes (adj. p = 0.038 and adj. p = 0.001, respectively). less experienced hcws were 2.1[1.3–3.5] times and 4.9[2.6–9.2] times more likely to exhibit negative perceptions of the two outcomes (adj. p < 0.001 each). conclusions: a negative perception of the effectiveness of mers-cov knowledge transfer was associated with poorer knowledge and was more prevalent among male hcws, physicians/pharmacists and less experienced hcws. hospitals should always refer to efficient knowledge sharing and educational strategies that render beneficial outcomes to patients, hcws, and the public community. electronic supplementary material: the online version of this article (10.1186/s12879-019-3898-2) contains supplementary material, which is available to authorized users. middle east respiratory syndrome coronavirus (mers-cov) has created an epidemiological and clinical crisis within 27 countries in north africa, europe, asia and the usa but mainly in the middle east (kingdom of saudi arabia) [1] . it is a viral respiratory illness, initially discovered in 2012 and speculated to have originated from camels or bats in saudi arabia, with subsequent spread to humans and across borders [2] . since 2012, a minimum of 1415 laboratory-confirmed cases have been reported in saudi arabia, of which 610 patients have died, 833 have recovered and two were under treatment [3] . high-risk groups were those in close contact with camels, geriatric persons, pregnant women, healthcare workers (hcws) and those with pre-existing comorbidities [1] . mers-cov infection ranged from asymptomatic or mild respiratory symptoms to severe acute respiratory disease and even death, which was reported in three to four out of every 10 reported mers-cov cases [4] . biologic samples of subjects with a suspected mers-cov infection (based on clinical symptoms) and of those exposed to reported mers-cov cases are tested using real-time reverse transcription polymerase chain reaction (rrt-pcr) assays. serology, such as an enzyme-linked immunosorbent assay and immunofluorescence assay, is also used to confirm mers-cov by the presence of antibodies [5] . in saudi arabia, a series of modifications was applied to the patient pathways while visiting the emergency departments or admitted as in-patients. this included segregating patients during triage based on prioritizing the inflow of patients by their chief complaints, bed availability and screening of flu symptoms/history of exposure. the infrastructure of the medical facility, particularly the ventilation system and isolation capacity of rooms, was subject to changes. some hospital wards and staff (especially nurses) were dedicated specifically to confirmed mers-cov cases to limit the chance of cross-contamination across wards and hcws. the infection prevention and control department (ipcd) at smng-ha, in particular, was on high alert for such mers-cov outbreaks, especially with the evident transmission of viral infections between patients and hcws at smng-ha. crisis management required a rapid implementation of adequate infection prevention, control procedures and case isolation, in addition to collaboration and coordination with local and international consultations. exceptional efforts have been made by the ipcd to apply the latest and most effective means of universal standard precautions throughout the mers-cov crisis. rules and regulations pertinent to infection control and prevention have been revisited and environmental surveillance has been carried out regularly to ensure that all wards are equipped with suitable protection and precautionary gear. numerous seminars, workshops and awareness campaigns have been launched for hcws of all disciplines to boost their knowledge on mers-cov, as well as their morale, to maintain a high-quality, safe and dedicated service for the patients. the latest updates issued on mers-cov from the world health organization (who), the centers for disease control and prevention (cdc), collaborative task forces (local and regional) and researchers have been circulated regularly among all hcws and across all managerial levels. numerous research studies have been conducted and published on the perception, knowledge and attitude of hcws towards mers-cov. it is rare to find a hcw who has not attended an educational program on mers-cov in saudi arabia. dissemination of mers-cov information/updates or knowledge transfer within a healthcare organization is a process in which this information is created, generated or acquired, and then organized and distributed within the system to ensure it is accessible to all hcws. one of the mechanisms of knowledge transfer is personalization whereby knowledge is transferred from one individual to another, while the other is codification where knowledge is converted into products such as documents, images and videos [6] . the need to transfer efficiently the precautionary regulations and updates about mers-cov to large numbers of hcws necessitates the mechanism of codification [7] . in addition, knowledge transfer or information sharing was found to be positively associated with job satisfaction [8] . authors hypothesized that although the dissemination of knowledge and updates on mers-cov among hcws has been given full consideration, these hcws might have reservations on the effectiveness and quality of mers-cov related educational offerings. therefore, there was an emerging need to evaluate the perceived effectiveness of mers-cov educational programs and knowledge transfer from the hcw's perspective, in a setting that witnessed the largest outbreak of confirmed mers-cov cases in saudi arabia. this was a cross-sectional study, based on an anonymous survey in english distributed among hcws at the primary healthcare centers in smng-ha medical centers, in riyadh, saudi arabia, between october and december 2016. the smng-ha is the second-largest healthcare sector in the country, second only to the saudi ministry of health, and provides healthcare services to the community of national guards, their dependents and employees [9] . the targeted primary healthcare centers were five randomly selected clinics out of 11 clinics that employ physicians, pharmacists, technicians and nurses. these clinics serve a population of 60,000 registrants, with a rate of four visits per registrant annually. eligible primary care hcws were targeted as being in frontline contact with potential confirmed cases of mers-cov. those occupying positions of management, education or non-direct patient care were excluded. medical and nursing students were also excluded. this study was ethically approved by the institutional review board at king abdullah international medical research center, king saud bin abdul-aziz university for health sciences, sp16/191 . the provisioned sample size in this study was calculated based on a reported level of knowledge between 43.3 and 57.4% by alkot et al. among hcws in the western region of saudi arabia. assuming an expected level of knowledge of 57%, with a 95% confidence limit (z = 1.96), and a margin of error 5%, the estimated sample size for this study was 376. for convenience, all eligible hcws at the targeted setting were invited to participate in this survey, to overcome a 25% nonresponse rate. the survey was provided in a sealed envelope with a cover letter that described the objectives of the study. the survey was in english language, as the targeted study participants were english literate and the educational offerings provided at the targeted setting were also in english. participants who agreed to enroll in this study hand-signed an agreement statement at the end of an informed consent, with no need for any personal identifier. the data collection tool comprised the characteristics of the hcw, principally gender, age category (years), job position and experience (years) [10] . the knowledge of hcws was measured using 16 statements based on undisputed facts published in the literature and issued by the mayo foundation for medical education and research in 2018 [11] . correct answers were scored "1", while wrong/don't know answers were scored "0". the percentage mean score (pms) of knowledge was calculated by adding the correct responses of the 16 statements, dividing the score by 16 and multiplying it by 100 (range of score 0 to 100). the perceived effectiveness of the mers-cov educational programs was measured using one statement: "prevalence of mers can be reduced by active participation of healthcare workers in the hospital infection control program", while the perception of knowledge transfer was measured by one statement: "any related information about mers should be disseminated among healthcare workers". both statements were rated on a four-point likert scale (strongly disagree, disagree, agree and strongly agree). those who responded by disagree or strongly disagree were classified as having a negative perception, while those who responded agree or strongly agree were classified as having a positive perception. the negative perception rate was calculated by dividing the number of participants who had negative responses over the total number of respondents. in addition, participants were asked about the source of mers-cov information. the survey was piloted on a group of five hcws, and their subjective comments were considered. the internal consistency or reliability (cronbach's alpha) of the knowledge domain measured 0.67 (additional file 1). data were analyzed using the statistical package for social studies (spss 25; ibm corp., new york, ny, usa). hcw characteristics, perceptions (negative vs positive) and incorrect knowledge response statements were presented in terms of frequencies and percentages, while the pms of knowledge was presented as the mean ± standard deviation. missing data were replaced by the average of the total, and outliers were dropped out. pearson's chi-square test was used to test categorical outcomes across hcw characteristics, while a mann-whitney test and a kruskal-wallis test were used to test the non-normally distributed pms of knowledge scores. two binary logistic regression models were constructed to determine the factors significantly associated with negatively perceived effectiveness of mers-cov educational programs and knowledge transfer. due to the small subgroup size of job positions, nurses were grouped with technicians, while pharmacists were grouped with physicians. these two subgroups had job positions comparable in terms of the educational levels, scope of practice and nature of patient care. the adjusted odds ratios [95% confidence interval] were calculated, and statistical significance was set at a value of p < 0.05. initially, 600 surveys were distributed among hcws; 404 participants agreed to enroll and completed the survey (response rate 67.3%). those who did not participate were mainly either off duty or busy with their workload. females constituted 64.1% of the sample, while males comprised 35.9%. almost 26% were ≤ 30 years old, 29% were 30-40 years old and 45% were > 40 years old. the majority (46.5%) were nurses, followed by physicians (23.0%), pharmacists (18.1%) and technical staff (12.4%). most hcws (42.1%) had accumulated > 10 years of work experience, with 29.2% having < 5 years of experience and 28.7% having 5-10 years (table 1) . overall, 36.1% of respondents claimed that their main source of information was the internet, while 47.8% reported more than one source, including research studies, books, media and others. the pms of knowledge score was 71.1 ± 19.4. the most common incorrect response to the statements (83.4%) was that for "incubation time for virus", followed by 43.6% with an incorrect response to the statement that "antibiotics are the first-line treatment for the management of mers-cov". other incorrect responses to statements are listed in chronological order in table 2 . overall, 22.5% of participants reported a negative perceived effectiveness of mers-cov educational programs, while 20.8% had a negative perception of knowledge transfer. with regard to the perceived effectiveness of mers-cov educational programs, male hcws had significantly a more negative perception than female hcws (n = 52, 35.9%, vs n = 39, 15.1%, respectively; p < 0.001). pharmacists (n = 24, 32.9%) and physicians (n = 30, 32.3%) reported more negative perceptions than technical staff (n = 9, 18.0%) and nurses (n = 28, 14.9%) (p = 0.001). hcws with work experience of < 5 years had the most negative perceptions in comparison with the other groups (p = 0.001). a number of factors were associated with a negative perception of knowledge transfer of mers-cov information. male hcws had a greater negative perception than females (n = 47, 32.4%, vs n = 37, 14.3%, respectively; p < 0.001). physicians (n = 31, 33.3%) and pharmacists (n = 23, 31.5%) had more negative perceptions of knowledge transfer in comparison with technical staff (n = 9, 18.0%) and nurses (n = 21, 11.2%) (p < 0.001). junior hcws with work experience of < 5 years (35.4%) had the highest rate of negative perception of knowledge transfer (p < 0.001) ( table 3) . hcws > 40 years old (pms 73.4 ± 18.9) had the highest knowledge scores in comparison with the other age groups (p = 0.005). more experienced hcws (> 10 years) also had the highest knowledge scores (pms 76.9 ± 15.7; p < 0.001). those who had a negative perception of the effectiveness of mers-cov educational programs (pms 49.4 ± 20.7) and of knowledge transfer of mers-cov updates (pms 46.0 ± 19.7) both had lower knowledge scores in comparison with the positive-perception group (p < 0.001 and p = 0.001, respectively), table 4 . logistic regression analyses showed that male hcws table 5 . mers-cov educational programs at healthcare institutions are a formal and reliable channel to deliver essential knowledge to hcws. for the sake of personal safety, job satisfaction and work morale, hcws should not pass up any opportunity to increase their theoretical knowledge and practical skills. hospital administrators do not necessarily face the challenge of producing new information, as an immense amount of valuable information already exists in the literature. the problem arises from the fact that current knowledge is either poorly structured or inaccessible to hcws [12] . for example, advanced practice nurses are observed to be "knowledge brokers" in a sense that they act as disseminators of knowledge among the nursing body. furthermore, health educators retrieve different types of evidence, synthesize it in different forms, translate it by evaluation, interpret it and then distribute it among nurses [13] . health education can improve levels of awareness and perception among hcws towards mers-cov infections [14] , and these higher levels of knowledge can aid in the control n frequency, % percentage of disease outbreaks [15] . however, published evidence in saudi arabia has shown that there is limited knowledge on mers-cov (both microbiological and virological aspects) among hcws in southern saudi arabia [16] . another study also claimed that knowledge about emerging infectious diseases was poor, and that infection control practices were suboptimal and also seemed to be overestimated [17] . the association between younger age and less experience on one hand and lower knowledge scores on the other was a reasonable finding. similar to literature findings, the knowledge of hcws in this setting was suboptimal and gaps remain that should direct the focus towards the mechanisms and quality of knowledge transfer. dissemination of mers-cov updates using e-mail, the internet, institutional announcements, employee meetings, the media and even personal communications are all methods of knowledge transfer. hcws can experience knowledge transfer both passively, absorbing information unconsciously, and actively. investigators in this study were curious to know how hcws perceived the transfer of knowledge about mers-cov and why this would be of concern to hospital administrators. for instance, knowledge transfer has been adopted with regard to smoking as a health hazard, hiv transmission as a sexual risk and seat belts in motor vehicles as a safety measure. people are exposed almost daily to precautionary advice by a variety of methods but unfortunately still undertake high-risk activities and are exposed to these hazards. this occurs regardless of the duration, frequency and quality of awareness campaigns. therefore, it is an aggravating concern that the repetitive exposure of hcws to mers-cov campaigns might have created some sort of "tolerance". hcws might disremember or take lightly the acquisition of current or new updates about mers-cov precautions due to routine attendance of educational programs or repetitive circulation of e-mails. knowledge and skills must be passed on in a systematic way from expert to novice employees in a way that makes sense [18] . managers who support work-empowering environments are actually boosting the engagement of participations in terms of knowledge transfer [19] . in fact, one of the key elements in seeking accreditation or managing crises such as mers-cov is knowledge communication, in the sense that effective communication ensures a purposeful exchange of information, thus allowing a more thorough understanding of the outbreak [20, 21] . interactive workshops remain highly recommended for the sharing and transferring of knowledge among hcws. however, one study noted that, although those who attended such workshops valued the expert input and discussions, after few months their sustainability of attendance was lost [22] . some barriers to mers-cov knowledge transfer could be the inability of hcws to recognize and articulate the instructions, personal opposition or resistance to change, the quality of the communication technologies, the absence of visual representations, language and cultural differences, deficiency in expertise, the work environment, a lack of job incentive/motivation, the organizational culture and others [23, 24] . current efforts to manage the mers-cov crisis are directed towards developing educational programs that target both the community and hcws [25] . a negative perception of mers-cov educational programs in this setting might result in outdated knowledge among hcws, which jeopardizes their compliance with disease precautionary and control measures. a mers-cov task force committee pointed out that the saudi arabian ministry of health has posted updates on mers-cov through videos, posters, handouts, posters and an official website. resilience against mers-cov increases with enriched education and awareness [25] . a saudi arabian study reported that hcws were unaware of the availability of mers-cov information at their work areas; they did not feel they had sufficient training and were not confident about infection control guidelines. these factors may also contribute to having a negative perception of mers-cov-related educational programs [26] . one study reported that the interest in following disease updates among hcws improved significantly after the implementation of a mers-cov educational program [14] . these programs improved the attitude of the hcws towards governmental measures taken regarding the crisis [14] . hcws often grasp their mers-cov educational information primarily from watching tv reports, or from the internet. a negative perception of knowledge transfer might be due to a pre-existing lack of trust in the media or in websites that might, to some degree, lack scientific credibility in comparison with educational programs provided in healthcare centers [27] . knowledge itself is complex, and its transfer process within healthcare institutions carries many challenges [28] . one way to overcome these challenges is to determine the characteristics of hcws who might be more likely to exhibit negative perceptions of knowledge transfer for significant mers-cov updates. in the literature, knowledge transfer has been investigated more frequently in manufacturing industries and firms, or among the public community. it has been seldom evaluated among hcws [29] , and never in a middle eastern setting or related to a mers-cov outbreak. a crossnational study suggested that organizational culture was a significant influence on the capacity of hcws to engage in knowledge transfer [30] . a systematic review paper study stated that knowledge transfer could streamline productivity and coordinate the use of resources more efficiently [29] . this review paper claimed to be the first to review published research focused on the perceptions of hcws about knowledge management [29] . knowledge management was defined as having an efficient idea or new practice accepted and adopted by an individual or a group through communication channels (successful diffusion of ideas) [31] . this definition also applies to the dissemination of updated regulations on the outbreak of mers-cov. this information, once absorbed by people, should be sustained for as long as it is useful, and not decay over time [32] . accordingly, a negative perception of the importance of knowledge transfer could be a warning sign of an interruption in this sustainability of retained information about mers-cov. signs of information decay were evident among hcws in this study, as those who had negative perceptions had lower knowledge scores about mers-cov in comparison with those who had positive perceptions. one of the key goals of knowledge transfer is to educate and train the less experienced and/or the newly hired hcws [33] . this phase of staff development is crucial yet stressful for novice hcws, who are expected to acquire skills and competencies rapidly to ensure that a safe and quality service flow is maintained at the institution. this explains why hcws with less work experience (< 5 years) had significantly more negative perceptions of knowledge transfer and the perceived effectiveness of mers-cov educational programs. as they gain more work experience, this perception improves as they realize the importance of education not only for their patients but also for their career advancement. the level of knowledge on mers-cov among hcws in primary healthcare clinics in this setting was found to be less than optimal. as the frontline in the battle of disease prevention and control, hcws are expected to be equipped with the relevant theoretical updates about mers-cov. special consideration should be paid to younger and less experienced hcws whose knowledge on mers-cov was moderately low. a negative perception of the knowledge transfer of mers-cov information and educational programs was associated with poorer knowledge. this negative perception was more prevalent among male hcws, physicians/pharmacists and less experienced hcws. this study has been conducted at one setting, yet the struggle against mers-cov has not ended and will continue against future emerging strains of viruses and bacteria causing communicable diseases in other settings too. knowledge is a valuable asset, and its holders within any healthcare institution should be retained and motivated so that they continue to spread their benefit among other hcws. all healthcare institutions should always identify and refer to reliable sources of knowledge. for instance, the center for disease control and prevention is a leading national public health institute and accountable for disseminating up-to-dates on various infectious topics. in saudi arabia, the ministry for public health has designated communication channels to release updates on mers-cov on their websites, through scientific arenas, memorandums and helpdesks. knowledge sharing and management strategies in the healthcare sector can render beneficial outcomes to patients, hcws, the organization and the public community [29] . in addition to the attendance of seminars or workshops, other methods of knowledge dissemination might involve launching of journal clubs among hcws to discuss updates on mers cov. audiovisuals at hospitals, such as educational videos on tv screens in lobbies or corridors, constantly enlighten hcws. deeper understanding of the negativity in the perception towards the quality or method of knowledge transfer necessitates a qualitative methodological approach, as face to face interviews with hcws aid in determining the underlying reasons and at a more personal level. furthermore, the execution of these strategies needs to be routinely monitored and evaluated so that the transfer of knowledge is time efficient and effective. optimal theoretical knowledge and practical competence are two main indicators of successful knowledge transfer among hcws. last but not least, a number of key points can be noted: as well as their morale, to maintain a high-quality, safe and dedicated service for patients. -the perceived effectiveness of mers-cov educational programs and knowledge transfer among health workers in this high risk setting was evaluated. -primary health workers were expected to be aware of the most recent updates on mers-cov, yet younger and less experienced hcws had moderate knowledge. -a negative perception of the effectiveness of mers-cov knowledge transfer was associated with poorer knowledge, and was more prevalent among male hcws, physicians/pharmacists and less experienced hcws. global summary and risk assessment middle east respiratory syndrome effects of educational program on mers-coronavirus among nurses students at jazan university 1439-2017 an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia laboratory testing for middle east respiratory syndrome coronavirus (mers-cov). cdc what's your strategy for managing knowledge? knowledge management research & practice. journal article does perception of knowledge sharing ,transfer and recognition have an impact on job satisfaction? an empirical study in saudi arabia smoking cessation advice: the selfreported attitudes and practice of primary health care physicians in a military community, central saudi arabia predictors of attitude and intention to use knowledge management system among korean nurses what is mers-cov, and what should i do? : mayo clinic implementing knowledge management practices in hospital-in-the-home units the role of advanced practice nurses in knowledge brokering as a means of promoting evidence-based practice among clinical nurses knowledge, attitude, and practice toward mers-cov among primary health-care workers in makkah al-mukarramah: an intervention study knowledge and perception of health practitioners towards mers-cov in hail region, kingdom of saudi arabia knowledge and attitude towards the middle east respiratory syndrome coronavirus among healthcare personnel in the southern region of saudi arabia knowledge, attitudes and behaviours of healthcare workers in the kingdom of saudi arabia to mers coronavirus and other emerging infectious diseases sense making and knowledge transfer: capturing the knowledge and wisdom of nursing leaders nurses' participation in personal knowledge transfer: the role of leader-member exchange (lmx) and structural empowerment knowledge communication: a key to successful crisis management l'accréditation, source de connaissance et d'enrichissement using interactive workshops to prompt knowledge exchange: a realist evaluation of a knowledge to action initiative culture as an issue in knowledge sharing: a means of competitive advantage academic conferences limited understanding change and change management processes: a case study an educational programme for nursing college staff and students during a mers-coronavirus outbreak in saudi arabia questionnaire-based analysis of infection prevention and control in healthcare facilities in saudi arabia in regards to middle east respiratory syndrome middle east respiratory syndrome-related knowledge, preventive behaviours and risk perception among nursing students during outbreak intra-firm knowledge transfer-a qualitative case study of knowledge transfer and its implications in a soft service firm knowledge management practices in healthcare settings: a systematic review the importance of knowledge transfer between specialist and generic services in improving health care: a cross-national study of dementia care in england and the netherlands diffusion of innovations impaired memory retrieval correlates with individual differences in cortisol response but not autonomic response expatriate knowledge transfer, subsidiary absorptive capacity, and subsidiary performance this study was approved and monitored by king abdullah international medical research center, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia. the authors would like to thank the research office and the institutional review board for their tremendous support. none to declare. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. all authors conceptualized and designed the study. md, fs, smr and aaf supervised the conduct of the study and data collection. md, smr and aaf undertook the recruitment of subjects and managed the data. fs, smr and aaf were accounted for the quality control. nar and ms provided statistical advice on study design, data analysis and responded to reviewers' comments. all authors drafted the manuscript, and contributed substantially to its revision as submitted and agree to be accountable for all aspects of the work. ethics approval and consent to participate a self-explanatory letter of invitation to participate was presented to each of the participants. all participants had given written informed consents for their participation in the research presented in this manuscript with full knowledge of the possible risks and benefits of participation. participants consented by ticking "agree", indicating their agreement to provide their feedback for this research study. study was approved by the institutional review board of the saudi ministry of national guard health affairs, riyadh, saudi arabia (protocol # sp16/191). this study followed the recommendations of the international conference on harmonization for good clinical practice (ich-gcp). not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.author details key: cord-331022-tek4u751 authors: sinderewicz, emilia; czelejewska, wioleta; jezierska-wozniak, katarzyna; staszkiewicz-chodor, joanna; maksymowicz, wojciech title: immune response to covid-19: can we benefit from the sars-cov and mers-cov pandemic experience? date: 2020-09-09 journal: pathogens doi: 10.3390/pathogens9090739 sha: doc_id: 331022 cord_uid: tek4u751 the global range and high fatality rate of the newest human coronavirus (hcov) pandemic has made sars-cov-2 the focus of the scientific world. next-generation sequencing of the viral genome and a phylogenetic analysis have shown the high homology of sars-cov-2 to other hcovs that have led to local epidemics in the past. the experience acquired in sars and mers epidemics may prove useful in understanding the sars-cov-2 pathomechanism and lead to effective treatment and potential vaccine development. this study summarizes the immune response to sars-cov, mers-cov, and sars-cov-2 and focuses on t cell response, humoral immunity, and complement system activation in different stages of hcovs infections. the study also presents the quantity and frequency of t cell responses, particularly cd4(+) and cd8(+); the profile of cytokine production and secretion; and its relation to t cell type, disease severity, and utility in prognostics of the course of sars, mers, and covid-19 outbreaks. the role of interferons in the therapy of these infections is also discussed. moreover, the kinetics of specific antibody production, the correlation between humoral and cellular immune response and the immunogenicity of the structural hcovs proteins and their utility in the development of a vaccine against sars, mers, and covid-19 has been updated. coronaviruses (covs) were discovered in the 1930s as zoonotic spherical pathogens causing mostly respiratory or enteric diseases [1, 2] . coronaviruses vary in size and are enveloped with club-shaped spikes on their surface [3] [4] [5] . a helically symmetrical nucleocapsid comprising positive-sense single-stranded rna is one of the largest virus genomes, ranging from 26 to 32 kilobases in length [6] . although covs are distributed mainly among mammals and birds, over the last 20 years some cov infections have resulted in lethal epidemics in humans. since 1960, when the first human coronavirus (hcov) was identified, seven hcovs species have been described [7] . four of them, hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1, lead to mild diseases such as the common cold, while the sars-cov, mers-cov, and sars-cov-2 caused severe disorders, manifesting acute respiratory system failures and fatalities [8] . the first identified hcov, sars-cov, originated from southern china in 2003 and induced an epidemic of severe acute respiratory syndrome (sars) with a mortality rate of 10-15% [9] [10] [11] . the first case of mers-cov, it was also found that the total number of t lymphocytes, cd3 + , cd4 + , and naive cd4 + t cells was still lower one year post-sars-cov infection compared to the control value [41] . only cd8 + t cells returned to normal level in the recovery period, probably as the effect of recirculation between blood and organs [41] . the ability of the mers-cov to infect t cells and the activation of extrinsic and intrinsic apoptosis pathways in t cells was also proven [42] . similar to sars-cov infection, effective transmission of mers-cov resulted in downregulation of th2 and high frequencies of reactive cd8 + t cells in the first phase of the disease, but not in the convalescent phase [43, 44] . furthermore, a correlation between th1 and th2 downregulation and the fatality rate of mers-cov and sars-cov infection was found [40, [43] [44] [45] . the newest reports have also documented an assessment of the t cell numbers in covid-19 patients. similar to sars and mers, the number of total t cells, cd4 + , and cd8 + t cells was significantly diminished in covid-19 patients in comparison to healthy controls and positively correlated with the severity of the disease [46, 47] . moreover, an age-dependent reduction in t cell numbers was observed in covid-19, with the lowest t cells numbers detected in individuals older than 60 years old, indicating an enhanced susceptibility to sars-cov-2 infection in elderly patients. furthermore, besides the decreasing number of t cells, the limited function of these cells has been described as a result of enhanced expression of immune-inhibitory factors, such as programmed death receptor 1 (pd1) or hepatitis a virus cellular receptor 2 (tim-3) [47] . a flow cytometry analysis illustrated significantly greater expression of the pd1 and tim-3 on t cell surfaces isolated from covid-19 patients in comparison to healthy controls [47] . growing pd1 and tim-3 levels were found as patients progressed from prodromal to overtly symptomatic stages, suggesting that the surviving t cells lost their functionality, particularly in patients requiring intensive care unit care. moreover, stimulation of peripheral blood mononuclear cells (pbmcs) from group of severe covid-19 patients with peptides covering all viral proteins activated both cd4 + and cd8 + sars-cov-2-specific t cells [48] . furthermore, a greater cd4 + :cd8 + ratio in covid-19 patients was observed in comparison with control individuals [48] . similar to sars-cov, sars-cov-2-specific cd4 + t cells were identified as central memory t cells, based on cd45ra and ccr7 expression. a mixed phenotype of cd8 + t cells in covid-19 patients was also documented [48] . a low content of cd3 + t cells in peripheral blood of covid-19 patients and a negative relationship between viral and cd4 + titers were also reported [48] . moreover, sars-cov-2-specific cd8 + and cd4 + t cells were detected in~70% and 100% of covid-19 convalescents, respectively [49] . the response of cd4 + t cell to sars-cov-2 s protein was also correlated with the specific igg and iga titers in patients who recovered from covid-19 [49] . li et al. [40] established detailed maps of t cell immune responses to sars-cov using pbmcs from sars convalescents. the 55 new t cell epitopes were identified which induced a response to eight of fourteen sars proteins: replicase, orf3, orf4, orf13, spike (s), envelope (e), membrane (m), and nucleocapsid (n). almost 70% of the responses were focused on structural proteins (s, e, m, and n), principally on s protein (41%), whereas the most abundant in sars-cov proteome replicase was much less immunogenic [31, 40] . immune-informatic tools were used to identify significant cytotoxic t lymphocyte (ctl) and b cell epitopes in the sars-cov-2 surface glycoprotein [50] . ahmed et al. [51] documented s and n sars-cov-2 protein-derived epitopes, which were comparable to the sars-cov map b cell and t cell epitopes. the surface glycoprotein of sars-cov-2 was found to have 76.3% identity and 87.3% similarity to sars-cov [50] . moreover, five ctl epitopes, three sequential b cell epitopes, and five discontinuous b cell epitopes in the viral surface glycoprotein were detected and described [50] . despite their high similarity to sars-cov, 12 of 13 identified sequential ctl and b cell epitopes were at least partially unique to sars-cov-2 compared with bt-cov, sars-cov, and mers-cov. a molecular dynamics stimulation showed that all ctl epitopes bind strongly to the peptide-binding groove of corresponding mhc class i molecules [50] . these features of ctl epitopes suggested their potential in induction of immune responses and, thereby, utility in a vaccine against sars-cov-2. the t cell responses against the s and n proteins were documented as the most long-term reaction in sars-cov-infection [52] . similar results, showing strong specific t cell response against structural proteins, including s, n, m, and e proteins, were noted in mers-cov infections [53] [54] [55] [56] . specific sars-cov-2 s and n proteins were also documented as the most immunogenic and greatly expressed during covid-19 [48, 50, 51] . although cd4 + t cell activation was reported against s, m, and n proteins, as well as against nsp3, nsp4, orf3a, and orf8, only s protein induced a robust response [49] . activation of cd8 + t cells was detected against sars-cov-2 s and m proteins and at least eight orfs [49] . similar results were presented by le bert et al. [57] , who proved the reactivity of both cd4 + and cd8 + t cells to the n protein and non-structural (nsp7 and nsp13 of orf1) proteins of sars-cov-2 in covid-19 convalescents. it was also shown that sars convalescents responded to the n protein of sars-cov-2 [57] . uninfected individuals also revealed sars-cov-2-specific cd4 + , indicating possibility of cross-reactive t cell stimulation with the other hcovs [49, 57] . interestingly, sars-cov-2-specific t cells in controls expressed a different pattern of immunodominance in comparison to sars and covid-19 convalescents [57] . patients who recovered from sars or covid-19 responded mainly to n protein, whereas the control group revealed dominant reactivity to both n protein and orf1-encoded proteins [57] . cytokines, produced mainly by immune cells like macrophages, b and t lymphocytes, and mast cells, modulate the balance between humoral and cell-based immune responses [58] . their concentration in biological fluids may be an important marker of immune system activity and disease progress. cytokines include several protein groups which vary in function, cell secretion, and target action. the current study reviewed the role of interleukins (ils) with tumor necrosis factors (tnfs), chemokines and interferons (ifns) in the immune response to hcovs. a comparison of the content of proinflammatory th1 and th2 cytokines in the serum of sars patients with healthy controls documented a significantly greater concentration of tnf-α, il-6, il-8, il-10, and il-12 in the early stage of the sars-cov infection [32, 40] . decreasing levels of these cytokines were correlated with the course of recovery from sars-induced pneumonia. furthermore, significantly greater contents of il-4, il-5, and il-10 were reported in fatal sars cases [40] . the enhanced secretion of il-1α, il-1β, il-6, il-8 il-12, and ifn-γ as an antiviral and inflammatory response to mers-cov was also documented [43, 44, 59, 60] . moreover, il-8 and il-12 were produced in a greater amount in response to mers-cov than sars-cov [59] . interestingly, in vitro studies showed that enhanced il-6 and il-8 levels in sars and mers patients were observed exclusively in the presence of s protein [32, 60] . among the cytokines involved in the immune response against hcovs, several have been proposed as potential predictors of disease cause and progression. it has been documented that increased il-6 concentration in plasma of sars patients was significantly increased in severe cases, but not in convalescent or control subjects, suggesting a positive correlation between serum il-6 level and disease severity [61] . inversely, il-8 and tgf-β concentrations were significantly reduced in sars patients with a severe course of the disease [61] . tnf-α was considered a predictor of disease progression due to its greatest level in the early stage of recovery [32] . moreover, a decreased content of il-4 and increased level of il-10 were only found in convalescent patients [61] . it was also proven that the il-8 profile in patient's serum indicated the cause of pneumonia-a significantly lower il-8 concentration was detected in sars patients compared to others. a detailed analysis showed that in a group of sars patients with severe symptoms, cytokine secretion was varied among different t cell subpopulations. it was shown that ifn-γ and tnf-α were produced both by cd8 + and cd4 + t cells, whereas the production of il-2 was typical exclusively for cd4 + [40] . moreover, in this group of patients the number of polyfunctional memory cd4 + t cells producing more than one cytokine was significantly higher compared to sars patients with a mild or moderate course of disease [40] . a similar effect was not observed for cd8 + t cell responses, although intensified degranulation was observed in severe course of sars via cd107a activation on cd8 + t cells surface [40] . stimulation of pbmcs from recovered sars patients with peptides overlapping the entire e protein, a membrane component of sars-cov, resulted in cytokine production by both cd4 + and cd8 + t cells [31] . similar observations have been reported in studies concerning covid-19. the lack of expression of the receptor for sars-cov-2 on t cells, ace2, suggested that the limited t cell number in covid-19 patients was likely caused by the influence of cytokine signaling and not by the direct infection of t cells [47, 62] . the stimulation of pbmcs from covid-19 patients with s protein peptides resulted in production of effector or th1 cytokines (ifn-γ, tnf-α, and il-2) and, to a lesser extent, th2 (il-5, il-13, il-9, and il-10) and th17 (il-17a, il-17f, and il-22) cytokines [48] . however, among the numerous serum cytokines, only tnf-α, il-1, il-6, and il-10 levels were significantly increased in sars-cov-2 infected patients [46, 47, 63] . these changes were characteristic of severe progression of the disease, supporting the hypothesis that covid-19 is driven by proinflammatory cytokines, which are responsible for histological changes and clinically full-blown cases of the disease. among detected cytokines, il-6 appears to be the most significantly involved in covid-19 progress. chen et al. [46] detected an enhanced level of il-2r in severe cases of covid-19, although no significant differences among examined and control groups were detected in il-2 [47] . the presence of il-4, greatly expressed in fatal sars cases, was also not detected in the plasma of covid-19 patients [47] . moreover, the concentration of tnf-α, il-6, and il-10 was negatively correlated with amounts of total t cells, cd4 + t cells, and cd8 + t cells, respectively. furthermore, serum concentrations of il-10, il-6, and tnf-α were significantly lower in patients in the disease resolution in comparison to the disease period, whereas the total number of t cells, cd4 + t cells, and cd8 + t cells was restored during the decline period of covid-19. these results suggested that in sars-cov-2 infections, a high serum concentration of tnf-α, il-6, and il-10 negatively regulated t cell survival and/or proliferation [47] . interestingly, the production of cytokines by cd4 + (mainly il-2 and ifn-γ and trace amounts of il-4, il-5, il-13, or il-17α) was also reported in covid-19 convalescents [49] . thus, the functional response of cd4 + against sars-cov-2 was suggested in recovered patients. chemokines are essential in determining immune cell localization [64] , and some of them act as factors involved in response to hcovs. enhanced contents of ip-10/cxcl-10, mcp-1/ccl-2, mip-1α/ccl-3, and rantes/ccl-5 were identified in the lungs and peripheral blood of sars and mers patients [45, 59, 65, 66] . moreover, both the production and secretion of these molecules were greater in response to mers-cov in comparison to sars-cov [59] . the upregulation of cxcl-10 at both transcriptional and translational levels was proven in murine epithelial cells, lung fibroblast cells, monocyte-derived macrophages, and dendritic cells as a result of the overexpression of mers-cov n protein [33] . high secretion and a persistent increase of cxcl-10 in mers-cov patients were associated with disease severity [60] . mers-cov infection also resulted in cxcl-8 chemokine production by th1 cells [43, 44] . the presence of chemokines and their action has not been reported in covid-19, although the number of studies concerning sars-cov-2 infection is still limited. among crucial elements of the immediate antiviral response, interferons (ifns) are pivotal for limiting viral replication and spread. therefore, ifns were extensively studied as potential therapeutic tools of sars-cov and mers-cov infections. the presence of an enhanced level of ifn-γ was documented in sera of sars-cov-and mers-cov-infected patients [40, 59] . similar to the other cytokines, the ifn-γ profile was correlated with the cause of pneumonia. ifn-γ production was significantly greater in sars patients compared to others [61] . on the other hand, further studies documented relatively low ifn-γ production in response to sars-cov infection. zhou et al. [59] found greater levels of ifn-γ in sera of mers patients compared to sars-cov-infected patients. moreover, scagnolari et al. [67] showed that ifn-γ production in response to sars-cov was significantly lower compared to well-established ifn-inducing viruses, such as vesicular stomatitis (vsv) and newcastle viruses (ndv), suggesting a limited role of ifns in early host defense against sars-cov infection. the lack of the antiviral ifn response to sars-cov with simultaneous enhanced secretion of several proinflammatory cyto-and chemokines suggested that the virus suppresses the induction of ifn production [65] . the natural host defense based on ifn action may be restricted because of the documented inhibition of ifns type i and cytokines production in toll-like receptor (tlr) 3, tlr7, and retinoic acid-inducible gene 1 (rif-i) pathways in response to sars-cov infection. this limitation occurs via suppressing the activation of transcription factors, such as interferon regulatory factor 3 (irf3), nuclear factor (nf)-κb, and adaptor related protein complex 1 (ap1) and downregulation of tnf receptor associated factor (traf) 3 and traf6 [68] . it was also documented that mers-cov n and m proteins inhibited the gene expression of ifns type i and iii, resulting in the host antiviral response impairment [55, 56] . similar results were documented for sars-cov-2 infection. chen et al. [46] showed the production of ifn-γ by cd4 + t cells in response to sars-cov-2 tended to be lower in severe (14.1%) than in moderate (22.8%) cases of covid-19. however, among the very few reports concerning the role of ifns in covid-19 disease was a study documenting a lack of ifn-γ in the serum of patients infected with sars-cov-2 [47] . on the other hand, further studies documented the utility of ifns in the treatment of sars-cov infection. chen et al. [65] confirmed virus susceptibility to exogenous type i ifn. it was also shown that early administration of ifns-i decreased immunopathological changes via downregulation of the expression of factors inducing apoptosis; upregulation of hypoxia/hyperoxia-related genes and the regulation of tlr, cytokine, and chemokine signaling; and expression of mhc-, lysosome-, and fibrosis-related genes [69, 70] . however, high-level virus replication resulted in retardation of ifns-i signaling, which promoted the cumulation of pathogenic inflammatory monocyte macrophages and resulted in increased cytokine and chemokine levels in lungs, vascular leakage, and reduced virus-specific t cell responses, and thereby strong lung pathology. animal models showed that genetic ablation of ifn-α/β receptor (ifnar) depletion protected from lethal infection, without affecting viral load [70] , suggesting that ifns therapy may be effective mainly in the early stage of infection. it was also shown that ifns-i and tlr agonists were the most effective in sars and mers therapy, which activates interferons [66] . the best results were observed for ifn-β1a, which reduced mortality by 20% in comparison to patients, who received ifn-α2a. the efficacy of ifns was lower in older patients [68] . the ability to induce ifns mrna accumulation by sars-cov in pbmcs from healthy donors was also investigated by castilletti et al. [71] , who proved that combination of ifn-α and ifn-γ strongly inhibited virus replication, while single cytokines were much less effective. an analysis of molecular mechanisms of the immune response to hcovs showed that effective cytokine production correlated to the availability of functional hcovs receptors. the effective increase in il-8 level was similar to concentration observed for s protein binding to sars-cov functional receptor, ace2, or to neutralizing monoclonal antibody. it was documented that il-8 production also depended on nf-κb activation and translocation and was suppressed by an nf-κb inhibitor [32] . moreover, the latest studies suggested that protein s could activate pbmcs via the tlr2 ligand. it was demonstrated that a lack of functional tlr3, tlr4, and tlr adaptor molecule 2 (tram) enhanced the possibility of sars-cov infection, reduced lung function and increased lung pathology and mortality. the suppression of tlr adaptor molecule 1 (trif) in mice infected with sars-cov resulted in changes in inflammation and positively correlated with acute respiratory distress syndrome [72] . on the other hand, infection of macrophages with mers-cov resulted in a reduced capacity to produce tnf-α and il-6 and enhanced the il-10 secretion [73] . the role of mers-cov s protein in upregulation of the irak-m expression, which is a negative regulator of tlr signaling, as well as expression of the transcriptional repressor ppar-γ was documented. moreover, it was documented that the immunosuppressive effect was mediated by dipeptidyl peptidase 4 (dpp4), which competitively inhibits mers-cov via binding to common for mers-cov and dpp4 functional receptor, dpp4r [73, 74] . in human dendritic cells (dc), the induction of c-c motif chemokine receptor (ccr) 1, ccr3, and ccr5 in the presence of sars-cov was detected [75] . the sars-cov infection induced also significant upregulation of tnf-related apoptosis-inducing ligand (trail) gene expression in dcs [75] . it was demonstrated that, in mers-cov infection, c-type leptin receptor (clr) was also upregulated and a retinoic acid-inducible-i-like receptor (rlr) pathway was activated [76] . the main aspects of t cell response in hcovs infections are shown in figure 1 . the animal model of sars-cov infection documented activation of the complement cascade in the lungs and showed that absence of complement significantly reduced the pathological changes in the respiratory tract, even though the viral load is the same. in the lungs of the transgenic mice deficient in c3 (c3 −/− ), which is the central component of the complement system, significantly lower neutrophils and inflammatory monocytes were presented than in infected controls [128] . moreover, diminished cytokine and chemokine contents in both the lungs and serum of c3 −/− mice were humoral immune response restrains the infection via neutralizing antibodies production and prevents reinfection in the future [77] . in sars, the presence of igg, igm, and iga antibody responses was detected during both the infection and convalescent phases, although with variable dynamics [78] [79] [80] [81] . the presence of specific igg and igm antibodies was also documented in the first two weeks of the sars-cov infection (59.1% and 36%, respectively) [78, 80, 82, 83] . the levels of igg and igm increased during the next two weeks to 97% and 82%, respectively. the serum samples examined 25 days after the onset of disease were positive only for sars-specific igg [78, 83] . a study analyzing the kinetics of specific antibody contents in plasma of sars patients presented by mo et al. [84] also showed a further significant increase in igg antibody levels. the highest concentration of igg was documented on day 60, remaining at the same level until day 180. then, the igg content gradually decreases until day 540. the igm antibody level peaked shortly after its detection and, in contrast to previous studies, declined until day 180 when igm was undetectable [84] . similar results were found by chen et al. [79] , who suggested that sars-cov-specific igg antibody, persisting for a longer time than specific igm and iga antibodies, was the primary humoral immune response against sars. however, a significantly lower level of igg was detected in severe than in mild or recovering sars patients, which may be a result of some kind of immune system dysfunction in long-suffering acute patients. however, li et al. [40] reported that strong t cell responses correlated significantly with a higher level of neutralizing antibody activity. in contrast to memory t cell responses, ensuring long-term protection, the antibody response was transient in convalescent sars patients [85] . cao et al. [86] documented the presence of specific antibodies within three years from the onset of sars symptoms in 94.7% of examined samples. however, six years post-infection, sars-cov-specific igg and ag-specific memory b cells were undetectable in sars convalescents, whereas memory t cell responses to a pool of sars-cov s peptides were revealed in 60% of convalescents. the most recent study reported presence of long-lasting memory t cells responding to sars-cov n protein in sars convalescents 17 years after the sars pandemic [57] . moreover, memory t cell response was stronger in former patients, who revealed severe clinical manifestations during sars [85] . similar to sars-cov infection, igm and igg levels increased during the first week after sars-cov-2 infection. the greatest concentration of igm was detected in the second week, after which its content was reduced to initial level in most patients, whereas the igg level remained at a high level for a long period [87] . interestingly, the igm and igg antibody levels were not significantly different among mild, severe, and critical disease groups [87] . however, the levels of igg, iga, and ige were greater in covid-19 fatalities in comparison to survivors [88] . igm and igg against n and s proteins of sars-cov-2 were also detected in covid-19 convalescents [89] . the igg titer remained high for at least 14 days post-discharge, whereas igm was detected only in newly recovered patients [89] . moreover, negative correlation between viral and igg titers [48] and a significant positive correlation between the content of neutralizing igg and the number of n protein-specific t cells was observed, suggesting interdependence between humoral and cellular immunity in sars-cov-2 infection [48, 89] . the kinetics of specific igg and igm antibodies were also analyzed in the serum of mers patients. robust serological responses were detected in most patients during the second or third week after symptom onset [90] [91] [92] . specific igm antibodies were detectable at the same time or slightly later than igg [93] . interestingly, the whole group of survivors, and only half of all fatalities, produced igg and neutralizing antibodies [91] . although the mers-cov antibody response in the early phase of infection correlated with reduction of the disease severity [90] , the presence of antibodies did not allow to the virus removal from the lower respiratory tract [91] . mers-cov-specific igg was also detectable one year post-infection in all severe disease survivors [94] . on the other hand, alhetheel et al. [95] found a very low rate of mers-cov-igg positive patients and a lack of correlation between nucleic acid and serological analysis [95] . the presence of specific iga in serum and respiratory tract secretions of mers patients was also confirmed. moreover, the correlation between iga and igg concentration in serum of mers-cov-infected individuals was proven [96, 97] . however, as the majority of studies concerning humoral response in mers used a limited number of patients, using serological analysis is not recommended as a tool to determine disease severity or prognosis. animal models showed that the main antibody responses were induced by the most exposed s protein of sars-cov [98] [99] [100] . mice immunization with a vector encoding a transmembrane domain of s protein resulted in neutralizing antibody production and action. in consequence, viral replication in the lungs of mice was significantly reduced and immune defense was provided by a humoral but not a t cell-dependent immune mechanism [98] . however, deming et al. [99] showed that the efficacy of the humoral response to sars-cov s protein depended on the homology of the virus strain. vaccines including a virus replicon expressing sars-cov s protein ensured complete shortand long-term protection against homologous strain challenge in young and senescent mice. on the contrary, the implementation of a construct encoding a synthetic s protein gene of the most genetically different human strain resulted in complete short-term protection in vaccinated young mice and limited protection in senescent animals [99] . high tolerance for the vaccine encoding the sars-cov s protein and its high immunogenicity has also been documented in humans, with specific antibodies being detected in 80% of subjects [86, 101] . moreover, sars-cov-specific cd4 + t cell responses were observed in all vaccinated patients and cd8 + t cell responses in 20% of individuals [101] . neutralizing b cell antibody responses to the sars-cov s protein were also major in sars convalescents, suggesting that a spike-based vaccine can be sufficient for a preventive vaccine, as it was previously demonstrated in animal models [40] . as was mentioned above, the strongest response against sars-cov s protein was shown by the cd4 + t cells. the possible cooperation of cd4 + t cell and b cells in neutralizing ab producing was described previously by mitchison et al. [102] , and the possibility of the enhanced reaction of plasma b cells, stimulated by cd4 + t cells, specific to the same protein, has also been suggested [40] . several studies have documented the presence of antibodies generated against the n protein of sars-cov [103, 104] and the high affinity of epitopic sites located in the n protein for forming peptide-antibody complexes in the serum of sars patients, particularly 8 to 14 days after the onset of infection. interestingly, vaccines containing sars-cov n protein failed to protect from homologous and heterologous challenges. in consequence, in the lungs of sars-cov-infected mice, the eosinophilic infiltrates were promoted and increased immunopathology was observed [99] . the strongest humoral responses against s and n proteins were also detected in mers. although it was proven that mers-n-specific antibodies occurred later than s-specific antibodies [93] , the vaccines containing mers-specific antibodies are still unknown. the main features of the humoral response in hcovs infections are presented in figure 1 . despite the high range of sars-cov-2 infection, the severe course of the disease has mainly affected elderly patients, with children excluded from the risk group [105] . moreover, despite the high rates of seropositivity of anti-receptor-binding domain (rbd) igg and igm (100% and 94%, respectively) and slightly lower rates of anti-n protein igg and igm measured after 14 days of symptom onset, the disease was still active and clinical symptoms severe [106] . to explain this phenomenon, antibody-dependent enhancement (ade) after previous exposure to other hcovs with a wide range of affinities has been proposed. in ade, infection is promoted through a virus binding to non-neutralizing antibodies from previous exposures to similar antigens. the virus-antibody immune complex binds to fc receptor (fcr) or complement receptors on the host cell surface, facilitating entry of the virus and sometimes enhancing its replication [107, 108] . the results of ade are enhanced inflammatory process, overexpression and release of cytokines (cytokine storm) and multi-organ failure as a consequence of these processes. immune-mediated covs infections have been widely described. the vaccine against feline cov aggravated future disease via induction of infection-enhancing antibodies [109, 110] . although the full-length sars-cov s protein stimulated protective immune response action in rodents, in human b cell lines it induced infection [111] . in vitro studies have demonstrated, that anti-spike immune serum enhanced the infection of immune cells by sars-cov spike-pseudotyped lentiviral particles, as well as replication-competent sars-cov, via fcγ receptor ii (fcγrii), but not ace2 [111, 112] . similarly, yip et al. [113] documented that human macrophages may be infected by sars-cov via fcγrii. however, binding of an immune complex to fcγrii was not sufficient for ade induction, indicating that activation of the other signaling pathways downstream binding to fcγrii receptors is required [113] . in a sars-cov infection of the promonocytic cell line expressing both fcγrii and ace2, a high concentration of antibodies neutralized the virus, whereas a low content of antibodies induced ade [114] . immunization of rhesus monkeys with a full length sars-cov s protein resulted in enhanced disease severity, with a dominant proinflammatory m1-like macrophage profile in the lung tissue, increasing lung injury [110] . moreover, macrophages produced a significantly greater amount of cytokines in the presence of deceased patients' serum and sars-cov in comparison with the virus alone [115] . enhanced cytokine production was reduced after fcr blockade. on the contrary, in sars-cov the greatest neutralizing antibody titer was observed earlier in deceased patients in comparison with convalescents [116] . however, a recent study showed a new mechanism for ade by engaging neutralizing antibodies. monoclonal neutralizing antibody (mab) binding to the mers-cov s protein induced changes in the s protein structure and mediated viral entry to the host cell via igg fcr [117, 118] . moreover, ade of mers-cov admission depended on the mab dosage as well as the fcr expression on the cell surface [118] . in humans, besides the immune cells (including monocytes) infiltrating lungs during pneumonia, epithelial cells of the lower respiratory tract also significantly expressed fcγr [119] . in a severe course of sars and covid-19 substantial lung opacity was observed, indicating infiltration by monocytes [120, 121] . the infection of human monocytes and macrophages by sars-cov-2 was also proven [122] . furthermore, an early humoral response to sars-cov-2 and greater antibody titer were positively correlated with a delay in the viral clearance and, in consequence, with the severity of the disease [123] . as mentioned above, great sequence identity and the presence of cross-reactive epitopes of sars-cov-2 and other hcovs were also documented [36, 37, 124] . monocyte migration to the lungs and the presence of cross-reactive memory antibodies potentially promote the receptivity of elderly individuals to sars-cov-2. the lack of immune memory of closely related hcovs (and the consequent inability of ade activation) might be responsible for the absence of clinical symptoms, as well as for the great frequency of undocumented sars-cov-2 infection, particularly in children [124] . however, upregulation of ace2 which is significant component of the renin-angiotensin system (ras) has also been suggested as a cause of severe courses of hcovs infections. animal models demonstrated that angiotensin ii receptor type i (at2r1) antagonists or ace inhibitors upregulated ace2 expression [125, 126] . thus, the medicaments widely used in cardiac and hypertensive patients potentially promote the virus binding to the host cells. according to the above, an unequivocal assessment of ade presence in sars-cov-2 infection seems to be crucial in the vaccine development and antibody-based drug therapy. besides the application of specific anti-sars-cov-2, the use of anti-ace2 with anti-fcγrii monoclonal antibodies to block ade activation and plasmapheresis for restraining cytokine storm elements in plasma has also been proposed as potentially the most effective method of covid-19 treatment [127] . the animal model of sars-cov infection documented activation of the complement cascade in the lungs and showed that absence of complement significantly reduced the pathological changes in the respiratory tract, even though the viral load is the same. in the lungs of the transgenic mice deficient in c3 (c3 −/− ), which is the central component of the complement system, significantly lower neutrophils and inflammatory monocytes were presented than in infected controls [128] . moreover, diminished cytokine and chemokine contents in both the lungs and serum of c3 −/− mice were detected, suggesting that inhibition of complement signaling might be an efficient therapeutic tool in the treatment of sars-cov infection. similarly, the complement system was inordinately activated in mers-cov-infected transgenic mice with human cd26/dipeptidyl peptidase 4 (hdpp4), which is a functional receptor for mers-cov. in response to mers-cov infection, enhanced contents of c5a and c5b-9 complement activation products in serum and lung tissues of hdpp4-tg mice, respectively, were observed [129] . inhibiting c5a production by blocking its receptor (c5ar) reduced lung damage and inflammatory responses [129] . interestingly, the covid-19 non-survivors presented lower levels of c3 and c4 proteins at admission in comparison to patients who recovered [88] . level of c3 protein was suggested as a predictor of mortality of covid-19 patients. recent research showed that mechanisms of responses against hcovs may also be enhanced by other elements of innate immunity. activation of human β-defensin 2 (hbd 2) resulted in the conjugation of this protein with the rbd of the mers-cov s protein (s rbd) [130, 131] . in consequence, expression of chemokines able to recruit leukocytes (comprising monocytes, macrophages, natural killer cells, granulocytes, t cells, and dendritic cells) was promoted. moreover, enhanced expression of primary immune-inducing molecules (nod2, tnf-α, il-1β, and il-6) and antiviral factors (such as ifn-β, ifn-γ, mxa, pkr, and rnasel) were also detected, compared to treatment with s rbd alone. immunization of mice with hbd 2-conjugated s rbd enhanced the immunogenicity of s rbd and induced a stronger s rbd-specific neutralizing antibody response [130] . the s rbd-hbd 2 treatment also increased phosphorylation and activation of receptor-interacting serine/threonine-protein kinase 2 and ifn regulatory factor 3. moreover, hbd 2 promoted ccr2-mediated nod2 signaling, inducing the production of type i ifns and an inflammatory response [131] . although recognition of the relationship between sars-cov-2 infection and complement system activation is extremely important in defining the best path of treatment, the effect of sars-cov-2 on complement cascades is still unknown. li et al. [40] analyzed the association between the cytokine pattern in acute infection and death in sars and suggested that the quality of immune response, rather than magnitude, may be critical in the progress of the disease. the investigation of innate, humoral, and t cell responses during the critical first 2-3 weeks may indicate whether they require immunosuppressing therapy or not. sars-cov-2 has induced the most widespread pandemic in recent decades. the presence of sars-cov-2 has resulted in almost 8.5 million covid-19 patients in 188 countries and territories, including 454,000 fatalities (reported: 18 june 2020 by john hopkins university). the differing severity of the covid-19 outbreak has affected different parts of the world for reasons which are still unclear. the epidemiological studies of a previous pandemic, sars, suggested that human-to-human transmission may enhance the immunogenicity of the virus and its virulence [40] . as sars-cov-2 is the first hcov being transmitted directly among humans, it is highly probable that the wide range of the pandemic is a result of the way of transmission. although the phylogenetic similarity of sars-cov-2 concerned only sars-cov and mers-cov, but not common hcovs inducing mild infections of the respiratory system, the potential cross-reactivity of t cells and antibodies between these viruses and its potential impact on total immune responses and clinical outcomes cannot be excluded. moreover, the presence of mutations in the viral genome is also possible. the next danger is relatively late symptoms occurring in infected people and, in some cases, an asymptomatic course of infection, resulting in a lack of isolation in the early stage of the infection. on the other hand, the genetic similarity of sars-cov-2, sars-cov, and to a lesser extent to mers-cov, the similarities between the structure of the epitopes and receptors, the course of the disease and the effects of the infection, allow undertaking a strategy against covid-19 based on experience gained during the previous pandemics until the mechanisms of covid-19 are better understood. the first studies related to covid-19 suggested a protective role of both cell-dependent and humoral immune responses in humans. similar to sars-cov and mers-cov, the sars-cov-2 infection primarily affected t lymphocytes, particularly cd4 + and cd8 + t cells, resulting in a reduction in their numbers and changes in cytokines secretion, including enhanced ifn-γ production by cd4 + t cells. several studies have also shown the diagnostic utility of serology in sars, mers, and covid-19 investigation. moreover, the correlation between the severity of the disease and potential immunological markers was documented, which may be a useful prognostic tool of the disease progression, and thereby, in the further course of the pandemic. based on previous experience, immune-informatic tools were used to define the structure of cytotoxic t lymphocyte and b cell epitopes. however, since sars-cov-2 antibody persistence and re-exposure occurrence are still unknown, further studies and a better understanding of the molecular mechanisms of immune responses to sars-cov-2 are essential in the new therapeutics development and evaluation of the efficiency of potential vaccines against sars-cov-2. the viruses and their replication history and recent advances in coronavirus discovery the molecular biology of coronaviruses supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion coronavirus: how a large rna viral genome is replicated and transcribed properties of coronavirus and sars-cov-2 an overview of their replication and pathogenesis world-health-organization update 49-sars case fatality ratio, incubation period functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a new coronavirus associated with human respiratory disease in china the species severe acute eespiratory syndrome related coronavirus: classifying 2019-ncov and naming it sars-cov-2 coronaviruses post-sars: update on replication and pathogenesis early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia active monitoring of persons exposed to patients with confirmed covid-19 -united states community transmission of severe acute respiratory syndrome coronavirus 2 the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application presymptomatic transmission of sars-cov-2-singapore quasipecies of bovine enteric and respiratory coronaviruses based on complete genome sequences and genetic changes after tissue culture adaptation origin and evolution of pathogenic coronaviruses cross-species transmission of the newly identified coronavirus 2019-ncov coronavirus host divergence and novel coronavirus (sars-cov-2) outbreak efficacy of various disinfectants against sars coronavirus sars coronavirus spike protein-induced innate immune response occurs via activation of the nf-kappab pathway in human monocyte macrophages in vitro overexpression of the nucleocapsid protein of middle east respiratory syndrome coronavirus up-regulates cxcl10 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 the novel coronavirus 2019 (2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome rapid loss of both cd4+ and cd8+ t lymphocyte subsets during the acute phase of severe acute respiratory syndrome cell responses to whole sars coronavirus in humans dynamic changes of t lymphocyte subsets in the long-term follow-up of severe acute respiratory syndrome patients middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways immune responses to middle east respiratory syndrome coronavirus during the acute and convalescent phases of human infection mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract clinical and immunological features of severe and moderate coronavirus disease reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19). front phenotype and kinetics of sars-cov-2-specific t cells in covid-19 patients with acute respiratory distress syndrome targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection characteristics of traveler with middle east respiratory syndrome cells responding to the middle east respiratory syndrome coronavirus nucleocapsid protein delivered by vaccinia virus mva in mice middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 middle east respiratory syndrome coronavirus nucleocapsid protein suppresses type i and type iii interferon induction by targeting rig-i signaling sars-cov-2-specific t cell immunity in cases of covid-19 and sars, and uninfected controls cytokines, inflammation and pain active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in chemokines: a new classification system and their role in immunity the immunobiology of sars sensitivity of sars/mers cov to interferons and other drugs based on achievable serum concentrations in humans severe acute respiratory syndrome coronavirus elicits a weak interferon response compared to traditional interferon-inducing viruses sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 sars-cov regulates immune function-related gene expression in human monocytic cells dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice coordinate induction of ifn-alpha and -gamma by sars-cov also in the absence of virus replication toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection middle east respiratory syndrome corona virus spike glycoprotein suppresses macrophage responses via dpp4-mediated induction of irak-m and pparγ reduction of soluble dipeptidyl peptidase 4 levels in plasma of patients infected with middle east respiratory syndrome coronavirus toll-like receptors, chemokine receptors and death receptor ligands responses in sars coronavirus infected human monocyte derived dendritic cells activation of c-type lectin receptor and (rig)-i-like receptors contributes to proinflammatory response in middle east respiratory syndrome coronavirus-infected macrophages antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus-associated illnesses profile of specific antibodies to the sars-associated coronavirus antibody response and viraemia during the course of severe acute respiratory syndrome (sars)-associated coronavirus infection kinetics of severe acute respiratory syndrome (sars) coronavirus-specific antibodies in 271 laboratory-confirmed cases of sars chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus a novel coronavirus associated with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study potent and persistent antibody responses against the receptor-binding domain of sars-cov spike protein in recovered patients detection of igm and igg antibodies in patients with coronavirus disease 2019 abnormal immunity of non-survivors with covid-19: predictors of mortality detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals kinetics of serologic responses to mers coronavirus infection in humans viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection comparative serological study for the prevalence of anti-mers coronavirus antibodies in high-and low-risk groups in qatar characterization of anti-mers-cov antibodies against various recombinant structural antigens of mers-cov in an imported case in china mers-cov antibody responses 1 year after symptom onset assessing the detection of middle east respiratory syndrome coronavirus igg in suspected and proven cases of middle east respiratory syndrome coronavirus infection infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia suggested new breakpoints of anti-mers-cov antibody elisa titers: performance analysis of serologic tests a dna vaccine induces sars coronavirus neutralization and protective immunity in mice vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants induction of specific immune responses by severe acute respiratory syndrome coronavirus spike dna vaccine with or without interleukin-2 immunization using different vaccination routes in mice a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial t-cell-b-cell cooperation assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (sars)-associated coronavirus in probable sars patients susceptibility of the elderly to sars-cov-2 infection: ace-2 overexpression, shedding, and antibody-dependent enhancement (ade) temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study antibody-dependent enhancement of virus infection and disease antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications early death after feline infectious peritonitis virus challenge due to recombinant vaccinia virus immunization antibody-dependent enhancement of feline infectious peritonitis virus infection in feline alveolar macrophages and human monocyte cell line u937 by serum of cats experimentally or naturally infected with feline coronavirus antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcγr pathway antibody-dependent infection of human macrophages by severe acute respiratory syndrome coronavirus immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection antibody responses against sars coronavirus are correlated with disease outcome of infected individuals a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein molecular mechanism for antibody-dependent enhancement of coronavirus entry the protein cell atlas webpage clinical characteristics of coronavirus disease 2019 in china sars: radiological features host-viral infection maps reveal signatures of severe covid-19 patients viral kinetics and antibody responses in patients with covid-19 antibody dependent enhancement due to original antigenic sin and the development of sars. front. immunol. 2020, 11, 1120 effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 upregulation of angiotensin-converting enzyme (ace) 2 in hepatic fibrosis by ace inhibitors anti-ace2 and anti-fcγrii monoclonal antibodies: a possible treatment for severe cases of covid-19. drug des. dev blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov human β-defensin 2 plays a regulatory role in innate antiviral immunity and is capable of potentiating the induction of antigen-specific immunity human β-defensin 2 is involved in ccr2-mediated nod2 signal transduction, leading to activation of the innate immune response in macrophages funding: this research received no external funding. the authors declare no conflict of interest. key: cord-341056-iwu428pk authors: alnaeem, abdelmohsen; kasem, samy; qasim, ibrahim; al-doweriej, ali; refaat, mohamed; al-shabebi, abdulkareem; hemida, maged gomaa title: the dipeptidyl peptidase-4 expression in some mers-cov naturally infected dromedary camels in saudi arabia 2018–2019 date: 2020-05-06 journal: virusdisease doi: 10.1007/s13337-020-00586-y sha: doc_id: 341056 cord_uid: iwu428pk mers-cov usually causes respiratory and renal failure in some patients, which may be the underlying cause of death. dromedary camels are the only known reservoir of the virus until now. they shed the virus in their body secretions thus potentiate a risk for human infection. mers-cov tropism and replication is mainly affected by the presence of certain receptor ligands on the target tissues. the dipeptidyl peptidase-4 (dpp-4) is believed to act as receptors for mers-cov. the main objective of this study was to determine the expression levels of the dpp-4 in various organs of some naturally infected camels. we conducted a surveillance study to identify some positive mers-cov infected camels. three positive animals identified by the real time pcr. our results are clearly showing the high level of expression of the dpp-4 in various organs of these animals’ particularly nasal turbinate, trachea, and lungs. the expression level may explain at least in part the pathogenesis of mers-cov in these organs. these findings confirm the pivotal roles of the dpp4 in the context of the mer-cov infection in dromedary camels. further studies are needed for a better understanding of the molecular pathogenesis of mer-cov infection. showing the high level of expression of the dpp-4 in various organs of these animals' particularly nasal turbinate, trachea, and lungs. the expression level may explain at least in part the pathogenesis of mers-cov in these organs. these findings confirm the pivotal roles of the dpp4 in the context of the mer-cov infection in dromedary camels. further studies are needed for a better understanding of the molecular pathogenesis of mer-cov infection. keywords mers-cov á natural á infection á dpp4 expression á pcr á immunohistochemistry mers-cov is one of the respiratory coronaviruses of human. as of feb 2020, there are 2494 laboratory-confirmed human cases reported from 27 countries across the globe. about 858 were passed away. the majority of the infection and fatalities were reported in the arabian peninsula [1] . mers-cov is belonging to the lineage c of betacoronaviruses. it is a positive sense single stranded rna virus. the virus replicates in the cytoplasm of the infected cells. the virus replication cycle occurs in several steps starting with the adsorption of the virus to the cell surface. this step requires the presence of some specific viral receptors on the target cells. the dpp-4 was reported to be one of the main viral receptors utilized by the virus to facilitate the virus entry to the target cells in addition to the sialic acid in transgenic mice model [2] . some studies proved the upregulation of the dpp4 in heavy smokers in the context of mers-cov infection which contributed to severe lung complications and finally death of the affected patients [3] . interestingly, glycosylation of the dpp-4 protein resulted in marked inhibition of mers-cov replication in mouse model [4] . some recent studies reported the correlation between the expression of dpp-4 and the success of mers-cov infection both in vitro in illama and pigs [5] . in similar manner, experimental infection of dromedary camels resulted in extensive ciliary loss of the respiratory tract as well as severe loss of the dpp-4 in the respiratory tract of the infected camels [6] . however, few studies were conducted to explore the roles of dpp-4 in the context of natural mers-cov infection in dromedary camels. we conducted this study to show the expression levels of the dpp-4 during the active natural mers-cov infection in dromedary camels in saudi arabia. we conducted this study according to the guidelines of the animal ethics protocols and the national committee of bio-ethics, king abdul-aziz city of science and technology, royal decree no. m/59 (http://www.kfsh.med.sa/ kfsh_website/usersuploadedfiles%5cncbe%20regula tions%20english.pdf). meanwhile, sampling protocol approved by the ethics committee of the ministry of environment. this study was conducted as a part of a molecular surveillance of mers-cov among dromedary from march to april 2018 at the ministry of environment, water and agriculture (meowa), riyadh, in collaboration with the king faisal university, saudi arabia. a total of 75 dromedary camels were examined in the south riyadh slaughterhouse. the target animal population was less than 2 years old. two nasal swabs were collected per each animal, the first was collected on the buffer of rapid mers-cov ag detection kit while the second was collected on the viral transport media (copan italia, italy), and tested by the (real time pcr technique (rtrt-pcr). all collected swabs were transferred to the riyadh veterinary laboratory within 2-h after collection to confirm the presence of the mers-cov-rna by rt-pcr. three positive as well as other two negative mers-cov animals were selected for further testing. tissues from upper and lower respiratory tract, and kidney were fixed in 10% neutral-buffered formalin for 5 days. tissues were processed and paraffin sections were stained with haematoxylin and eosin (h&e) for histopathological examination as previously described [6] . tissue sections from trachea, turbinate bones, lungs and kidneys were screened for the presence of cell surface receptor dipeptidyl peptidase 4 (dpp4 by immunohistochemistry. we used the polyclonal rabbit anti dpp4 (abin213568, antibodies-online.com, germany), to detect the dpp4, proteins. the technique was carried out as per the manufactures instructions as well as previously described [6] . several dromedary camels were kept in isolation yard in the slaughterhouse for testing (fig. 1a) . we identified three animals to be slaughtered for further processing. before slaughtering, these animals were physically inspected to identify any obvious clinical signs (fig. 1b) . we collected some nasal swabs from these animals to be tested by the quick mers-cov latex agglutination test to identify some positive animals. positive animals showed double bands on the strips while the negative animals showed only one band (fig. 1c) . three out of the tested positive animals were subjected to necropsy examination after slaughtering. immunohistochemical results of the examined tissues elucidated detectable signals of the cell surface receptor di-peptidyl peptidase 4 (dpp4) in different organs ( table 1 ). the dpp4 receptor signals was detected in the all examined tissues (nasal turbinate, trachea, lung, and kidneys) with moderate to strong signals ( table 1 ). the evaluation of the signals depended on both the distribution as well as the strength of the reaction in the examined tissues. most of the reactions were obviously detected in both the apical epithelial layers and submucosal glands of the turbinate bones, trachea and bronchi. in the lungs, the reaction of dpp4 receptors and was detected in the alveolar walls (fig. 2) . viral replication and tropism is mainly governed by many factors including the availability of specific viral receptors and transcription factors. it is believed that mers-cov utilizes the dpp-4 as main receptors and sialic acid as accessory receptors [7] . mers-cov infection usually starts with the attachment of the virus by its spike glycoproteins to the target cell dpp4 ligands [7] . one study have found that dpp4 was detected on cells in the cortical apical proximal tubular epithelium and arteriolar smooth muscle of kidney of camel [8] . our study considered natural infected animals and others studies done under experimental conditions using different animals (rhesus macaques, common marmosets, and dromedary camels) are confirming the importance of the 14 amino acids within the dpp4 which binds to the rbd of the mers-cov in the successful viral infections [9, 10] . extensive ciliary loss in the respiratory organs of the dromedary camels was recently shown under experimental infection using the recombinant mers-cov/vaccinia virus-ankara (mva-s) [6] . our results showed high level of dpp4 expression levels in nasal turbinate and kidney. this may explains the multi-organs failure in some mers-cov patients particularly lung and kidneys [11] . our results is very much consistent with the pathogenesis of mers-cov infection in human as well as the experimental infection of mers-cov in dromedary camels and other members of family camelade [12] . silencing of the dpp4 could be a novel fig. 2 results of the dpp4 expression in various organs of mers-cov naturally infected dromedary camels. results of the immunohistochemistry testing of some organs from mers-cov naturally infected dromedary camels. positive signals were detected at various levels in various organs including nasal turbinate, trachea, lung and kidney. this in comparison to the similar organs from non-infected camels the dipeptidyl peptidase-4 expression in some mers-cov naturally infected dromedary camels… approach to reduce the risk of mers-cov infection in the future. this direction worth investigation in the future. the dpp4 is highly expressed in the respiratory system as well as in the kidney of the naturally infected mers-cov dromedary camels. this is confirming their potential roles as receptors for the mers-cov as well as highlighting their roles in the molecular pathogenesis of the virus. middle east respiratory syndrome coronavirus (mers-cov)-the kingdom of saudi arabia acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with middle east respiratory syndrome coronavirus dpp4, the middle east respiratory syndrome coronavirus receptor, is upregulated in lungs of smokers and chronic obstructive pulmonary disease patients glycosylation of mouse dpp4 plays a role in inhibiting middle east respiratory syndrome coronavirus infection co-localization of middle east respiratory syndrome coronavirus (mers-cov) and dipeptidyl peptidase-4 in the respiratory tract and lymphoid tissues of pigs and llamas experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4 species-specific colocalization of middle east respiratory syndrome coronavirus attachment and entry receptors the pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models a comparative review of animal models of middle east respiratory syndrome coronavirus infection infection with mers-cov causes lethal pneumonia in the common marmoset clinical outcomes among hospital patients with middle east respiratory syndrome coronavirus (mers-cov) infection mers-cov infection of alpaca in a region where mers-cov is endemic acknowledgements we wish to thank the king abdul-aziz city for science and technology (kacst) for their generous funding through the mers-cov research grant program (number 20-0004), which is a part of the targeted research program (trp). data availability statement data will be available upon request. conflict of interest all authors declares no conflict of interest. key: cord-321259-wio2b49i authors: carmona-gutierrez, didac; bauer, maria a.; zimmermann, andreas; kainz, katharina; hofer, sebastian j.; kroemer, guido; madeo, frank title: digesting the crisis: autophagy and coronaviruses date: 2020-05-04 journal: microbial cell doi: 10.15698/mic2020.05.715 sha: doc_id: 321259 cord_uid: wio2b49i autophagy is a catabolic pathway with multifaceted roles in cellular homeostasis. this process is also involved in the antiviral response at multiple levels, including the direct elimination of intruding viruses (virophagy), the presentation of viral antigens, the fitness of immune cells, and the inhibition of excessive inflammatory reactions. in line with its central role in immunity, viruses have evolved mechanisms to interfere with or to evade the autophagic process, and in some cases, even to harness autophagy or constituents of the autophagic machinery for their replication. given the devastating consequences of the current covid-19 pandemic, the question arises whether manipulating autophagy might be an expedient approach to fight the novel coronavirus sars-cov-2. in this piece, we provide a short overview of the evidence linking autophagy to coronaviruses and discuss whether such links may provide actionable targets for therapeutic interventions. the ongoing outbreak of the respiratory disease covid-19 (coronavirus disease 2019) is endangering individuals, governments and societies around the world. indeed, this pandemic has unprecedented global consequences at the economic and social levels as it challenges the medical and research communities. the first case of covid-19 was reported in december 2019 in wuhan (china) [1] and, despite increasingly drastic efforts to contain the disease, the infection rapidly spread across the planet, leading the who to declare it, first, a global health emergency (in january 2020) and eventually a pandemic (on march 11, 2020) [2, 3] . as of april 30 th 2020, more than 200 countries and territories have reported covid-19 cases with a total of over three million confirmed cases and more than 220.000 confirmed deaths [4] . in the current absence of effective treatments and vaccines, these numbers will continue to grow inexorably. the main clinical features of given the devastating scenario that covid-19 has already generated, a multitude of potential approaches against sars-cov-2 are currently being conceived. in that sense, we think that the manipulation of autophagy might be worth exploring. macroautophagy (hereafter referring to autophagy) is an intracellular catabolic mechanism that regulates the elimination of unwanted, superfluous or defective cell components [17] . in this process, the targeted molecules/organelles are enclosed in double-membrane vesicles called autophagosomes, which eventually fuse with lysosomes for degradation of the delivered contents. this process is induced upon energy deprivation and/or physical activity in order to secure sufficient nutrient supply and is thus often referred to as a cellular self-digestion system. the compounds resulting from lysosomal degradation (e.g., fatty acids, amino acids) may be released into the cytoplasm for the generation of new macromolecules and/or bioenergetics reactions. autophagy, therefore, represents a recycling mechanism under circumstances of low resource availability [17] . the digestion of damaged and thus potentially detrimental cargo also increases the fitness at the cellular and consequently at the organismal level. accordingly, the induction of autophagy by dietary interventions (e.g., caloric restriction, fasting regimens), behavioral cues (e.g., exercise) or pharmacological agents (e.g., caloric restriction mimetics like spermidine, rapamycin, resveratrol or specific chalcones [18] [19] [20] [21] [22] ) has been consistently linked to beneficial effects on health [23] . in turn, reduced autophagic capacity is connected to aging progression and a number of pathologies including cardiovascular disease, cancer and neurogeneration [24] . the autophagic machinery can retrieve its cargo in bulk, but it can also selectively target distinct intracellular components and organelles via specific adapter proteins that interact with both the individual substrates and the autophagosomes [25] . specifically, these autophagy adaptors (e.g., p62/sqstm1, optineurin) contain both a ubiquitin-binding domain (ubd) and an lc3-interacting region (lir). the ubd recognizes ubiquitin tags decorating the specific target, while the lir allows binding of lc3 proteins attached to nascent phagophores, the structures that eventually close to form autophagosomes [26] . accordingly, autophagic subforms have been defined that describe, for example, the specific removal of damaged mitochondria (mitophagy), the disposal of protein aggregates (aggrephagy), the digestion of lipid droplets (lipophagy), etc [27] . yet another selective autophagy subroutine is xenophagy, the specialized elimination of intracellular pathogens, including fungi, bacteria and viruses [28] . the xenophagic disposal of viruses (sometimes also termed virophagy) has been described for different viral pathogens, including human immunodeficiency virus-1 (hiv-1) [29, 30] and herpes simplex virus-1 (hsv-1) [31, 32] . however, many viruses (including hiv-1 and hsv-1) and other pathogens have also evolved strategies to escape or inhibit autophagy and sometimes even to manipulate the autophagic machinery for their replicative benefit [33] . thus, autophagy emerges as a crucial element in the evolutionary-driven arms race of host against pathogen. this further underlines the significance of xenophagy as a central antimicrobial mechanism, but also limits the potential of autophagy induction to fight infections. the question whether autophagy induction might be beneficial to specifically counteract sars-cov-2 infection cannot be answered at this point. however, the existing data on other covs suggest that -despite some conflicting results -autophagy induction might be a valid approach that should be subjected to further evaluation. the autophagylysosomal system does indeed seem to play a central role during the infection with different covs, including sars-covs [34] [35] [36] [37] . nevertheless, the diverse putative roles of autophagy during viral infection [38] require that two opposite aspects be covered by this overview: (i) is there evidence for autophagy being used for the survival or replication of covs (proviral effects)? (ii) can autophagy induction reduce cellular infection, and does the virus actively inhibit autophagy (suggesting antiviral effects for autophagy)? of note, cellular manipulation of autophagic levels during infection may also reflect desperate attempts of the cell to reestablish homeostasis, either through restriction of viral entry by actively shunting endocytosis/endosomal trafficking (possibly resulting in autophagy reduction as a sideeffect) [39] or to counteract virally induced cell death by increasing cytoprotective autophagy. as a third major as-open access | www.microbialcell.com pect, (iii) the autophagic effects on antiviral immunity must be taken into consideration. over the last 15 years, a number of studies have addressed these issues in the context of the "old" covs, those that historically precede sars-cov-2. covs rely on the formation of replication complexes at double membrane vesicles (dmvs), where viral genome replication and transcription occurs [40, 41] . as it may be at least partly the case for autophagosomes, dmvs probably depend on er-derived membranes for their generation [40, 42] . therefore, it was initially suspected that covs might usurp the autophagosomal machinery for dmv formation. in fact, an early study using atg5 knockout cells implied that autophagy may promote mouse hepatitis virus (mhv) replication via formation of dmvs [43] . in a follow-up work, the same authors reported the co-localization of specific replication proteins (nsp2, nsp3, nsp8) with endogenous lc3, suggesting a connection between replication and autophagosome formation [44] . other studies reported that the porcine covs transmissible gastroenteritis virus (tgev) [45] and porcine epidemic diarrhea virus (pedv) [46] may induce autophagy to promote replication. however, the link between autophagy and replication of covs has been challenged by a number of different reports, showing that deletion of the essential autophagy genes atg5 or atg7 does not affect the replication of mhv [47, 48] or sars-cov [49] . furthermore, the co-localization of lc3 (or gfp-lc3) with the sars-cov rna replication complex could not be confirmed [50] . a study using pharmacological induction (rapamycin) or genetic inhibition (knockdown of lc3, atg5, or atg7) of autophagy even demonstrated that tgev replication is negatively regulated by autophagy [51] . in addition, rapamycin reduced infectivity of pedv [52] . altogether, although some conflicting results exist, autophagy may not be primarily engaged in promoting cov replication. however, single components of the autophagic machinery may be hijacked independently of their activity in autophagic processing. non-lipidated lc3, for instance, is used for membrane derivation for dmvs in covs, and downregulation of lc3 but not inactivation of autophagy, counteracts cov infection [47] . notably, some components of the canonical autophagy machinery can target invading pathogens by promoting phagosome-to-lysosome fusion [53] . during lc3-associated phagocytosis (lap), for instance, which does not involve sequestration in autophagosomes, lc3 is recruited to phagosome membranes to facilitate lysosomal fusion and degradation [54] . in addition, it has been proposed that non-canonical autophagy and intruding viruses, both of which can be inhibited by the fungal compound brefeldin a, may converge in the retrieval of membranes [55] . non-canonical autophagy includes atg5-and atg7-independent as well as beclin-1independent autophagy [56, 57] . altogether, these observations add further layers of possible modulation of autophagic processes in trying to attenuate viral infection (figure 1 ). intriguingly, a body of evidence indicates that covs may actually inhibit autophagy, which in turn teleologically suggests an antiviral role for autophagy. accordingly, a number of studies have shown that increasing autophagic capacity may be beneficial against infection (figure 1) . a temporal kinome analysis of mers-cov-infected hepatocytes demonstrated selective activation of the erk/mapk and pi3k/akt/mtor signaling responses, both of which have inhibitory effects on autophagy [58] . accordingly, pharmacological inhibition of these pathways was able to inhibit mers-cov infection [58] . moreover, although one of the central replicase proteins (nsp6) of avian infectious bronchitis virus (ibv) seems to promote autophagosome formation at the omegasome level [59] , nsp6 also limits further autophagosomal expansion [60] , thus compromising autophagic delivery of viral components to lysosomes. interestingly, nsp6 is also present in other covs, including sars-cov-2 [61] . overexpression of the protease plp2 of sars-cov and mers-cov in different cell lines inhibited autophagosomelysosome fusion and autophagic flux [62] . a recent report corroborated the autophagy-inhibitory activity of mers-cov and showed that induction of autophagy can reduce mers-cov replication [37] . specifically, the authors found that pharmacological inhibition of the ubiquitin ligase skp2 increases the levels of beclin-1, a central regulator of phagophore formation [37] . skp2 executes lysine-48-linked poly-ubiquitination of beclin-1, targeting it for proteasomal degradation, the inhibition of which promoted autophagy and efficiently reduced mers-cov replication. this work also shows that ectopic expression of at least three viral proteins (nsp6, p4b, p5) restricts autophagy [37] . while nsp6 may limit autophagosome expansion (see above), p4b inhibits rnase l activation (a pro-autophagic event [63] ), and p5 has been connected to the blockage of ifn-β induction [63, 64] , which itself may be linked to autophagy [65] . accordingly, deletion of p4b or p5 resulted in reduced mers-cov growth, although disputing some previous observations, which the authors explain by methodological differences [37] . thus, the group-specific accessory proteins, which by definition are not essential for viral replication but are involved in the modulation of host cells and immune evasion [66, 67] , may represent targets for reducing the autophagy-inhibitory effects of covs. the fda-approved anti-malarial drugs chloroquine and hydroxychloroquine have been suggested to be repurposed for the treatment of covid-19 [68] [69] [70] , but this remains widely controversial [71] [72] [73] . although chloroquine is a lysosomotropic agent that blocks autophagic degradation, possibly by impairing autophagosome fusion with lysosomes [74] , the putative effects on autophagy may not be necessarily causal for the antiviral activity. in fact, endosomal acidification after endocytosis is critical for sars-cov-2 entry [75] , and chloroquine inhibits this acidification [76] . in addition, chloroquine limits terminal glycosylation of the metallopeptidase ace2, the functional receptor for sars-cov and sars-cov-2 cell entry [68, 75, 77] . nonglycosylated ace2 seems to interact less efficiently with open access | www.microbialcell.com the sars-cov spike protein, thus reducing viral entry [78] . these modes of action would target the virus upstream of autophagy, making it unlikely that autophagy modulation contributes to the outcome of chloroquine treatment at that point. additionally, chloroquine has been shown to induce autophagy-independent effects, for instance, golgi disorganization [74] and pulmonary vasodilation [79] that may contribute to its controversial clinical activity. interestingly, the net effects on autophagy may differ depending on parameters like cell type and dose: for instance, chloroquine-mediated lysosomal stress may promote the nuclear translocation of the pro-autophagic transcription factors tfeb and tfe3 [80, 81] . altogether, further clinical work must clarify if chloroquine and hydroxychloroquine have major effects on covid-19 and at which (early?) phase of the disease these drugs should be administered. if chloroquine or its derivative turned out to be clinically useful against covid-19, it will be important to understand to which extent such effects are connected to the modulation of autophagy. indeed, repurposing of known (especially currently fda-approved) drugs might offer a welcome shortcut to rapidly developing treatments against covid-19. obviously, even in the present period of desperate search for pharmacological treatments, the efficacy (and lack of sideeffects) of anti-covid-19 treatments needs to be proven by clinical studies subjected to rigorous scientific scrutiny. while data regarding such attempts remain preliminary at this stage, several currently available preprints allow the speculation of autophagy as a possible druggable target. for instance, a recent preprint mapping the sars-cov-2 interactome in human host cells identifies several host factors connected to autophagy [82] . amongst others, the authors find viral-human interactions with proteins directly modulated by mammalian target of rapamycin complex 1 (mtorc1) [82] , a master regulator of cell proliferation and autophagy known to be affected by other viruses [83, 84] . intriguingly, another recent preprint presents in vitro data showing that sars-cov-2 infection restricts autophagy and that, in turn, pro-autophagic compounds -including spermidine -may inhibit viral propagation [85] . admittedly, open access | www.microbialcell.com spermidine has a plethora of physiological functions, including regulation of rna-to-protein translation, as well as amelioration of rna and dna stability [86] , and these effects need further investigation, specifically in relation with a possible autophagy induction. in fact, the role of spermidine during infection may be pleiotropic. on the one hand, spermidine is an autophagy inducer [18, [87] [88] [89] [90] and may promote viral xenophagy; also, it may contribute to endosomal ph buffering [91] and thus block viral entry. on the other hand, polyamines are essential for viral replication [92] , and the overexpression of spermidine/spermine-n(1)acetyltransferase by the host, which drives spermidine (and spermine) depletion, is a common cellular response to viral infections, including that of rna viruses [93] . this underlines once more that any pharmacological candidate for the fight against sars-cov-2 should be examined for its (possible) pleiotropy during infection. autophagy directly impacts immune signaling, and autophagy induction hence may counteract covs by promoting immunological defense mechanisms (figure 1) . for instance, autophagy regulates, and can be regulated by, socalled pattern recognition receptors (prrs), which sense molecules specifically derived from microorganisms (pathogen-associated molecular patterns, pamps) [94, 95] . prrs are present in various cells of the innate immune system and many epithelial cells [95] . there, they are distributed at diverse locations (either at the cell surface or in the cytoplasm), providing a recognition system for different life cycle phases of a given pathogen [95] . pamp-induced signaling triggers a multitude of pathways, including proinflammatory responses, that result in the synthesis of immunostimulatory molecules including cytokines, chemokines and immunoreceptors [94] . autophagy further assists immune responses by processing and supporting the presentation of antigens on major histocompatibility complex (mhc) class ii, responsible for loading extracellular antigens [96, 97] . in addition, autophagy can deliver exogenous viral antigens into mhc-i molecules for cross-presentation (the mhc-i pathway is usually employed for loading endogenous antigens) [97, 98] . moreover, autophagy directly impacts the activation, proliferation and differentiation of immune cells [99] . amongst others, autophagy has been connected to the differentiation of cd8 + t-cells into cytotoxic t lymphocytes, to dendritic cell and b cell development, to plasma cell differentiation, and to macrophage differentiation, therefore covering multiple instances of the innate and adaptive immune systems [99] . of note, autophagy activation by caloric restriction mimetics (including hydroxycitric acid and spermidine) also improves anticancer immunosurveillance [100] . in addition, the general cellular pro-survival effects of autophagy extend to several immune cell types. pharmacological autophagy induction by spermidine [87] , for instance, has been demonstrated to promote the maintenance and survival of memory cd8 + t cells [101] as well as to counteract b lymphocyte senescence [102] . the interplay of autophagy and inflammation is complex, since it does not only encompass positive but also negative regulatory mechanisms. for example, autophagy can digest the interleukin precursors produced by inflammasomes (e.g. pro-il-1), but also directly target inflammasome components (e.g. nlrp3, aim2 and asc) [103] . in sum, autophagy ensures acute inflammatory responses while preventing excessive and prolonged hyperinflammation. finally, it should be noted that autophagy may have paracrine functions in the form of unconventional secretion [104] , thus adding yet another layer of non-cell autonomous effects. in the context of viral infections, this mechanism may be used to induce protective responses in cells neighboring an infection site [105, 106] . in primary human placental trophoblasts, for instance, autophagy induction promotes packaging and exosome-dependent delivery of specific mirnas, which induce autophagy in non-placental recipient cells, conferring resistance to a variety of viruses [105] . this may be (one of) the mechanism(s) through which placental and maternal cells optimize their defense against viral infections during pregnancy. the existing evidence allows the speculation that autophagy induction might counteract cov infection at different levels, although more specific data are certainly required. as mentioned above, the restriction of calorie intake is the most robust method to induce autophagy. however, the fight against acute infections also requires sufficient energy supply, suggesting that autophagy induction via caloric restriction or fasting regimens may be counterproductive, at least in the short-term during (as well as shortly before) infection. therefore, caloric restriction mimetics [107, 108] , natural or synthetic compounds with the ability to induce autophagy, may circumvent this problem. irrespectively of whether autophagy modulation will eventually be part of the strategies against covid-19, the current pandemic outbreak is a shocking reminder that emerging (and re-emerging) infectious pathogens are (and will be) a major challenge. in view of the exposed vulnerability of our medical structures and socioeconomic wellbeing, this pandemic underlines how essential it is to further establish and secure global healthcare as well as to promote and extend robust research against infectious diseases. open access | www.microbialcell.com project "epiage". gk is supported by the ligue contre le cancer (équipe labellisée); agence national de la recherche (anr) -projets blancs; anr under the frame of e-rare-2, the era-net for research on rare diseases; ammica us23/cnrs ums3655; association pour la recherche sur le cancer (arc); association "le cancer du sein, parlons-en!"; cancéropôle ile-de-france; chancelerie des universités de paris d.c-g., g.k. and f.m. are the scientific co-founders of samsara therapeutics, a company that develops novel pharmacological autophagy inducers. a.z. has equity interests in samsara therapeutics. f.m. and d.c-g. have equity interests in tll (the longevity labs), a company that develops natural food extracts. covid-19: what has been learned and to be learned about the novel coronavirus disease potential treatments for covid-19; a narrative literature review a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical characteristics of coronavirus disease 2019 in china the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study identification of a novel coronavirus causing severe pneumonia in human: a descriptive study the covid-19 epidemic coronavirus envelope protein: current knowledge molecular evolution of human coronavirus genomes epidemiology, genetic recombination, and pathogenesis of coronaviruses coronavirus infections: epidemiological, clinical and immunological features and hypotheses sars and mers: recent insights into emerging coronaviruses zoonotic origins of human coronaviruses autophagy: machinery and regulation induction of autophagy by spermidine promotes longevity autophagy in the pathogenesis of disease a diversity of selective autophagy receptors determines the specificity of the autophagy pathway cargo recognition and trafficking in selective autophagy cargo recognition and degradation by selective autophagy autophagy restricts hiv-1 infection by selectively degrading tat in cd4+ t lymphocytes autophagy plays an important role in the containment of hiv-1 in nonprogressor-infected patients trim23 mediates virus-induced autophagy via activation of tbk1 a neuron-specific role for autophagy in antiviral defense against herpes simplex virus autophagy during viral infection -a double-edged sword the interaction between nidovirales and autophagy components sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes sars-coronavirus open reading frame-3a drives multimodal necrotic cell death skp2 attenuates autophagy through beclin1-ubiquitination and its inhibition reduces mers-coronavirus infection viruses and the autophagy pathway autophagy and the endo/exosomal pathways in health and disease rna replication of mouse hepatitis virus takes place at doublemembrane vesicles targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus sarscoronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum coronavirus replication complex formation utilizes components of cellular autophagy identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins mitophagy in tgev infection counteracts oxidative stress and apoptosis porcine epidemic diarrhea virus induces autophagy to benefit its replication coronaviruses hijack the lc3-i-positive edemosomes, er-derived vesicles exporting short-lived erad regulators, for replication coronavirus replication does not require the autophagy gene atg5 severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasomeindependent inhibition of m-calpain ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex rapamycin-induced autophagy restricts porcine epidemic diarrhea virus infectivity in porcine intestinal epithelial cells autophagy and viruses: adversaries or allies? lc3-associated phagocytosis at a glance alternative autophagy, brefeldin a and viral trafficking pathways discovery of atg5/atg7-independent alternative macroautophagy unsaturated fatty acids induce non-canonical autophagy antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate coronavirus nsp6 restricts autophagosome expansion genomic characterization of the 2019 novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan coronavirus membrane-associated papain-like proteases induce autophagy through interacting with beclin1 to negatively regulate antiviral innate immunity new advances in our understanding of the "unique" rnase l in host pathogen interaction and immune signaling the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists ifnb1/interferon-β-induced autophagy in mcf-7 breast cancer cells counteracts its proapoptotic function attenuation of replication by a 29 nucleotide deletion in sarscoronavirus acquired during the early stages of human-to-human transmission accessory proteins of sars-cov and other coronaviruses chloroquine is a potent inhibitor of sars coronavirus infection and spread hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro regulators split on antimalarials for covid-19 chloroquine and hydroxychloroquine in covid-19 should chloroquine and hydroxychloroquine be used to treat covid-19? a rapid review chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases structure, function, and antigenicity of the sars-cov-2 spike glycoprotein inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus nl63 spike pro-microbial cell tein-mediated entry by altering the glycan processing of angiotensin iconverting enzyme 2 chloroquine is a potent pulmonary vasodilator that attenuates hypoxia-induced pulmonary hypertension the nutrient-responsive transcription factor tfe3, promotes autophagy, lysosomal biogenesis, and clearance of cellular debris the transcription factor tfeb links mtorc1 signaling to transcriptional control of lysosome homeostasis a sars-cov-2-human protein-protein interaction map reveals drug targets and potential drug-repurposing. biorxiv adapting the stress response: viral subversion of the mtor signaling pathway comprehending a killer: the akt/mtor signaling pathways are temporally high-jacked by the highly pathogenic 1918 influenza virus analysis of sars-cov-2-controlled autophagy reveals spermidine, mk-2206, and niclosamide as putative antiviral therapeutics version 1 functions of polyamines in mammals spermidine in health and disease cardioprotection and lifespan extension by the natural polyamine spermidine spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner chloride accumulation and swelling in endosomes enhances dna transfer by polyamine-dna polyplexes polyamines and their role in virus infection interferon-induced spermidine-spermine acetyltransferase and polyamine depletion restrict zika and chikungunya viruses the interplay between pattern recognition receptors and autophagy in inflammation pathogen recognition and innate immunity autophagy promotes mhc class ii presentation of peptides from intracellular source proteins autophagy beyond intracellular mhc class ii antigen presentation autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv-1 infection the relationship between autophagy and the immune system and its applications for tumor immunotherapy caloric restriction mimetics enhance anticancer immunosurveillance autophagy is a critical regulator of memory cd8+ t cell formation polyamines control eif5a autophagy and inflammasomes secretory autophagy human placental trophoblasts confer viral resistance to recipient cells human tank-binding kinase 1 is required for early autophagy induction upon herpes simplex virus 1 infection a discovery platform for the identification of caloric restriction mimetics with broad health-improving effects caloric restriction mimetics against age-associated disease: targets, mechanisms, and therapeutic potential the authors are grateful to the austrian science fund fwf (sfblipotox f3007&f3012, w1226, p29203, p29262, p27893) and the austrian federal ministry of education, science and research and the university of graz for grants "unkonventionelle forschung" and "flysleep" (bmwfw-80.109/0001-wf/v/3b/2015).the authors also acknowledge the funding of dk metabolic and cardiovascular disease (fwf) and the doctoral college "metabolic and cardiovascular disease" (fwfw1226) as well as support from nawi graz and the biotechmed-graz flagship open access | www.microbialcell.com key: cord-321131-f8qeytxc authors: zhou, yanchen; vedantham, punitha; lu, kai; agudelo, juliet; carrion, ricardo; nunneley, jerritt w.; barnard, dale; pöhlmann, stefan; mckerrow, james h.; renslo, adam r.; simmons, graham title: protease inhibitors targeting coronavirus and filovirus entry date: 2015-04-30 journal: antiviral research doi: 10.1016/j.antiviral.2015.01.011 sha: doc_id: 321131 cord_uid: f8qeytxc abstract in order to gain entry into cells, diverse viruses, including ebola virus, sars-coronavirus and the emerging mers-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. the respective enzymes are thus excellent targets for antiviral intervention. in cell culture, activation of ebola virus, as well as sarsand mers-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin l (ctsl) and cathepsin b (ctsb). in addition, sarsand mers-coronavirus can use serine proteases localized at the cell surface, for their activation. however, it is currently unclear which protease(s) facilitate viral spread in the infected host. we report here that the cysteine protease inhibitor k11777, ((2s)-n-[(1e,3s)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(e)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. k11777 is already in advanced stages of development for a number of parasitic diseases, such as chagas disease, and has proven to be safe and effective in a range of animal models. k11777 inhibition of sars-cov and ebola virus entry was observed in the sub-nanomolar range. in order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized k11777-derivative with that of camostat, an inhibitor of tmprss2 and related serine proteases. employing a pathogenic animal model of sars-cov infection, we demonstrated that viral spread and pathogenesis of sars-cov is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of sars and potentially mers, while vinyl sulfone-based inhibitors are excellent lead candidates for ebola virus therapeutics. in order to gain entry into cells, diverse viruses, including ebola virus, sars-coronavirus and the emerging mers-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. the respective enzymes are thus excellent targets for antiviral intervention. in cell culture, activation of ebola virus, as well as sars-and mers-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin l (ctsl) and cathepsin b (ctsb). in addition, sars-and mers-coronavirus can use serine proteases localized at the cell surface, for their activation. however, it is currently unclear which protease(s) facilitate viral spread in the infected host. we report here that the cysteine protease inhibitor k11777, ((2s)-n-[(1e,3s)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(e)-4-methylpiperazine-1-carbonyl] amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. k11777 is already in advanced stages of development for a number of parasitic diseases, such as chagas disease, and has proven to be safe and effective in a range of animal models. k11777 inhibition of sars-cov and ebola virus entry was observed in the sub-nanomolar range. in order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized k11777-derivative with that of camostat, an inhibitor of tmprss2 and related serine proteases. employing a pathogenic animal model of sars-cov infection, we demonstrated that viral spread and pathogenesis of sars-cov is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of sars and potentially mers, while vinyl sulfone-based inhibitors are excellent lead candidates for ebola virus therapeutics. ó 2015 published by elsevier b.v. emerging viral diseases pose a unique risk to public health. ebola virus, severe acute respiratory syndrome coronavirus (sars-cov) and members of the henipavirus genus of paramyxoviruses are all highly pathogenic viruses that have arisen in the past 40 years and caused, or threaten to cause, major outbreaks. new viral threats continue to emerge, most recently demonstrated by a novel beta-coronavirus, middle east respiratory syndrome coronavirus (mers-cov), which was identified in 2012 (zaki et al., 2012; memish et al., 2013; de groot et al., 2013) . there are currently no approved vaccines or therapeutics for many of the highly pathogenic viruses potentially dependent on cathepsins, including ebola virus, nipah virus (niv), mers-cov and sars-cov. broadspectrum antiviral drugs, with overlapping therapeutic indications, would facilitate rapid responses to new or changing pandemic threats, potentially even without precise identification of the agent. targeting host factors involved in viral entry provides an excellent avenue for such drug development, due to the limited number of pathways involved the glycoproteins of corona-, filo-and paramyxoviruses facilitate viral entry into target cells by binding to receptors and by driving fusion of viral and host cell membranes. however, the glycoproteins are synthesized as inactive precursors and depend on activation by host cell proteases to acquire a fusion active form. as a consequence, the respective enzymes are potential targets for broad-spectrum antiviral intervention. cell culture studies demonstrated that endosomal cysteine proteases, in particular cathepsin b (ctsb) and/or l (ctsl), can activate the glycoproteins of filoviruses, sars-cov, other coronaviruses, and niv and hendra (hev) viruses to facilitate entry into certain cell lines. in addition, activation of coronaviruses can also be accomplished by tmprss2, or other serine proteases located at the cell surface, or secreted into the extracellular space (simmons et al., 2013) . however, the respective roles of endosomal and cell surface proteases in viral spread in the infected host is unknown. the development of protease inhibitors able to inhibit ctsl, ctsb and related proteases would be an excellent starting point for development of broad-spectrum antiviral therapies . we describe here the discovery of k11777 and its related compounds, as broad-spectrum antivirals targeting endosomal proteases involved in viral entry. k11777, a cysteine protease inhibitor, blocked infection when viral entry did not require activating serine proteases, as is the case with ebolavirus (ebov). k11777 also fully inhibited coronavirus infection, but only when target cell lines lacking activating serine proteases were used. if cells expressed cell-surface serine proteases known to activate coronaviruses, both k11777 and a serine protease inhibitor, such as camostat were required for full inhibition. thus, both compounds were deployed to examine which activation pathway is predominant in vivo. camostat displayed antiviral activity in a pathogenic animal model for sars-cov infection, indicating that serine protease inhibitors are suitable for treatment of sars and potentially mers. the predicted effect of k11777 and related cysteine protease inhibitors versus ebola virus in vivo must await studies in approved biocontainment facilities. the cysteine protease inhibitor library screened in this work has been described elsewhere (ang et al., 2011) . briefly, the library includes $2100 electrophilic cysteine protease inhibitors of various chemotype (glycine nitriles, ketobenzoxazoles, ketooxadiazoles, vinylsulfones, etc.), which were synthesized during the course of industrial drug discovery programs targeting human cathepsins (palmer et al., 1995 (palmer et al., , 2005 (palmer et al., , 2006 rydzewski et al., 2002) . camostat mesylate, leupeptin, bafilomycin a1, ammonium chloride, and chloroquine were purchased from sigma-aldrich. k11777 and novel p3 derivatives were synthesized according to the general approach described previously (jaishankar et al., 2008) and as illustrated here (scheme 1). the n-substituted piperazines were obtained from commercial sources or (for r = cyclopentyl and cyclopropylmethyl) were prepared by reductive amination of boc-protected piperazine followed by treatment with hcl in dioxane (51-53% over two steps). we find that the final coupling of p3/p2 carboxylic acid to vinylsulfone amine is best accomplished via the mixed anhydride, as this minimized epimerization of the phenylalanine side chain. final vinylsulfone analogs were >95% pure as judged by lc/ms analysis. characterization data for final analogs is provided below. 156.6, 145.2, 140.1, 139.89, 136.4, 133.14, 130.14, 129.0, 128.9, 128.49, 128.29, 128.09, 127.3, 126.8, 125.9, 76.7, 76.4, 55.7, 54.2, 48.8, 47.8, 43.8, 37.8, 35.4, 31.4, 18. human embryonic kidney 293 cells, 293t cells, clone 17 (293t/ 17), and vero cells were obtained from american type culture collection (atcc), while the huh7.5 cell line was a gift from apath llc. all cells were grown in dulbecco's modified eagle medium (dmem; invitrogen) supplemented with 10% fbs and penicillin and streptomycin (10 u/ml). 293t/17 stably expressing ace2 (293t/ace2) were established by transfecting 293t/17 cells with pcdna6 (invitrogen) encoding the ace2 gene and selecting for stable transformants using blasticidin s (2.5 lg/ml). 293 stably expressing human cd13 [also called aminopeptidase n (apn)] (293/cd13) were established by transfecting cells with pcdna3 (invitrogen) encoding the cd13 gene and selecting for stable transformants using geneticin 418 (2.0 mg/ml). expression of cd13 was measured with flow cytometric analysis. lentiviral pseudotypes were generated from two plasmids, one encoding the envelope and the second an envelope-deficient hiv reporter construct -either pnl4-3 luc-r -e -(pnl-luc) or pnl4-3.ren.r-econnor et al., 1995) . plasmids encoding spike (s) proteins from human coronaviruses sars-cov, nl63 and 229e, mers-cov, glycoproteins (gp) from filoviruses ebov (formerly known as ebolavirus zaire), sudan ebolavirus (sudv), tai forest ebolavirus (tafv), reston ebolavirus (restv), and marburg (marv), as well as niv f and g, lassa virus gp, vesicular stomatitis virus (vsv) g protein, chikungunya virus (chikv) e1/e2, and mlv envelope, have been described simmons et al., 2005 simmons et al., , 2004 simmons et al., , 2002 salvador et al., 2013 salvador et al., , 2009 ). bundibugyo ebolavirus (bebov) gp was a gift from edward wright (university of westminster). hcv e1e2 was synthesized (genscript, ca) while junin virus g protein was a gift from sean amberg (siga technologies, or). plasmids encoding cellular type ii transmembrane serine proteases (ttsp) tmprss2 were previously described . lentiviral pseudovirions were produced essentially as previously described (zhou et al., 2010) . briefly, 293t/17 cells were transfected with up to 30 lg of viral envelope encoding plasmid and 10 lg of pnl4-3 reporter backbone per 10-cm dish by calcium phosphate transfection. the next day, expression was induced with sodium butyrate (10 mm) for 6 h before washing once. forty hours after transfection, supernatant was filtered through a 0.45 lmpore-size filter and frozen at à80°c. virus was titrated essentially as it would be used in the screening assay. if required, virions were purified and concentrated by ultracentrifugation (28,000 rpm in a sw28 rotor, beckman) over a 20% sucrose cushion, resuspended in hank's balanced salt solution (hbss) and stored at à80°c as aliquots. pseudoviruses were normalized for equal infectivity by transduction of target cells with serially diluted stock followed 48 h later by determination of luciferase activity in cell lysates according to the manufacturer's instruction (promega). vsv-based pseudotypes bearing junin virus g were produced essentially as described (steffen et al., 2013) by transfecting 293t cells with 16 lg of junin g plasmid and then infecting the cells with recombinant vsvdg-gfp(vsv-g). progeny vsvdg-gfp(junin-g) virus was then collected, titrated and used for inhibition studies. in the case of niv, vsvdg-gfp(niv f/g) viruses were produced via calcium phosphate transfection of 293t cells with 10 ug of niv f and 10 lg of niv g. transfected cells were left for 16 h before an initial medium change; then infected with recombinant vsvdg-gfp(vsv-g) (moi 0.1-0.3) after five additional hours. media alone or compound of interest were then added at the desired concentration and cells were incubated overnight before supernatant was harvested and filtered. to assay for inhibition, production of entry-competent virus was examined. target cells were pre-plated at 25,000 cells/ 50 ll in 96 well plates and allowed to attach overnight. 50 ll of undiluted vsvdg-gfp niv f/g made in the presence or absence of inhibitor was added. cells were incubated at 37°c with 5% co2 for two days, and then washed and fixed with 2% paraformaldehyde and gfp fluorescence determined by flow cytometry using a becton dickinson lsrii cytometer and flowjo software. 100% infection was determined with samples infected with pseudovirons produced from cells with no compound exposure. high-throughput screens for viral entry inhibitors were performed in 384-well plates using the dual-envelope pseudotype (dep) assay . briefly, compounds and controls were diluted in dmem with 10% fbs to 50 lm (5% dmso) and 10 ll were transferred to 384-well white tissue culture plates (nunc) using a biomek fx-p (beckman-coulter). a mixture of the target virus [e.g., (hiv-luc(sars-cov s)) and the control virus [hiv-ren(lassa gp) or hiv-ren(mlv env)] was made, with the concentration and ratio derived empirically to give similar robust levels of reporter expression. 10 ll of reporter virus mix was added to each well using a matrix well-mate (thermo scientific). 30 ll of cells (170,000 cells per milliliter) were then added to all wells. plates were incubated for two days at 37°c/5% co 2 and firefly and renilla luciferase reporter expression was determined using the dual-glo luciferase assay substrate (promega). assays for dose response curves were performed in 96-well white tissue culture plates (nunc). target cells were pretreated with test compounds or inhibitors serially diluted in medium, followed by either a single virus or a two reporter virus mixture, depending on the purpose of the assay. the env/reporter combinations were reversed in order to demonstrate inhibition was not directed at the backbone or reporter enzyme rather than entry. plates were incubated for two days at 37°c/5% co 2 and firefly and renilla luciferase reporter expression was determined using the dual-glo luciferase substrate (promega), or detection of firefly luciferase reporter expression using the bright-glo™ luciferase substrate (promega). the infectivity for pseudotyped vsvs with niv f/g was analyzed by measuring the number of gfp expressing cells by flow cytometric analysis. either caco2 or 293-cd13 cells transiently expressing tmprss2 were pretreated with serially diluted k11777, a combination of serially diluted k11777 and camostat mesylate at 1 or 10 lm or a combination of serially diluted camostat mesylate and k11777 at 2.5 lm for 60 min at 37°c and then incubated with infectivity-normalized pseudoviruses in the presence of the inhibitors. the cells were then cultured at 37°c/5% co 2 for two days and luciferase expression was measured. antiviral replication with urbani and toronto-2 strains of live sars-cov, as well as cytotoxicity of selected compounds was investigated using three in vitro assays, cytopathic effect (cpe) inhibition assay, neutral red (nr) uptake assay, and virus yield reduction assay as described in kumaki et al. (2011) . for cell viability assays, cells were seeded in 96-well black tissue culture plates (costar) and treated with compounds with final concentration of 1% dmso. the quantity of the atp present in metabolically active cells was determined with celltiter-glo ò luminescent cell viability assay kits (promega, madison, wi). smdc256160 (50 mg/kg), camostat (30 mg/kg) alone, smdc256160 (50 mg/kg) combined with camostat (30 mg/kg), or negative control (water) were administrated into 6-8 week old female balb/c mice by oral gavage twice a day for 9 days beginning 10 h prior to virus exposure. ten mice were assigned to each group. the texas biomedical research institute's institutional (texas biomed) animal care and use committee approved all animal protocols. live virus assays were performed at the absl-4 facility at texas biomed using a mouse adapted strain of sars-cov (ma15) kindly provided by ralph baric (university of north carolina). mice were infected by administering 10,000 pfu of virus by intranasal instillation. statistical calculations were performed in excel (microsoft, seattle, wa). compounds from the primary screens were considered inhibitory when the luciferase readings of sars-cov, but not the internal control pseudotyped viruses, fell below the predefined cut-off, mean-3 â sd (m-3sd). ic 50 (50% inhibitory concentration) and cc 50 (50% cell cytotoxic concentration) values were calculated using non-linear regression analysis based on the sigmoidal dose response equation using prism 6 (graphpad software inc) (applied to the percent inhibition and concentration data. a selectivity index (si) was calculated using the formula si = cc 50 /ic 50 . we recently developed an internally-controlled dual virus hts assay for identification of inhibitors of viral entry . using sars-cov entry assays, we screened a library of $2100 cysteine protease inhibitors with confirmed activity against human cathepsins. unsurprisingly, a large number of hits were identified. upon validation of the hits, the most robust activity was observed for k11777 ((2s)-n-[(1e,3s)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(e)-4-methylpiperazine-1-carbonyl] amino}-3-phenylpropanamide) (fig. 1a) , a compound known to inhibit cruzain, a cathepsin-l like protease from the protozoan parasite trypanosoma cruzi (engel et al., 1998) . in addition, k11777 inhibits a variety of cysteine proteases, including human cathepsins (choy et al., 2013) and cathepsin-like proteases from several other parasites (ndao et al., 2013; abdulla et al., 2007) . to determine whether k11777 can inhibit entry driven by other viral envelope proteins, hiv-based pseudotypes bearing spikes from coronaviruses (sars-cov, hcov-229e, nl63, mers-cov) or glycoproteins from filoviruses (ebov, sudv, tafv, restv, bebov and marv) were examined together with control pseudotypes. we also tested the ability of k11777 to prevent activation and hence infectivity during production of vsv-based pseudotypes (salvador et al., 2009) bearing niv f and g. k11777 was active against all the major enveloped viruses previously known to require cathepsin-mediated proteolysis, including a variety of coronaviruses and filoviruses, especially ebov ( fig. 1b; table 1 ). k11777 inhibited sars-cov pseudovirus entry with an ic 50 of 0.68 nm (fig. 1b , table 1 ) while no toxicity was observed, cc 50 > 10 lm (data not shown). mers-cov and nl63 envelope required higher concentrations of k11777 for inhibition, likely due to less reliance on ctsl hofmann et al., 2006) . nevertheless, the ic 50 s were very low: 46 nm for mers-cov and <7 nm for nl63. in contrast, 100 nm k11777 did not inhibit infection mediated by envelope glycoproteins from an alphavirus (chikv), a rhabdovirus (vsv), a flavivirus (hcv), the retroviruses mlv-a and xmrv or two arenaviruses, lassa and junin virus. coronaviruses including sars-cov, human coronavirus 229e (hcov-229e) and mers-cov use two distinct pathways for cell entry: (i) the endosomal pathway, in which spike activation is facilitated by the ph-dependent endosomal protease ctsl; or (ii) entry at the plasma membrane, which relies on spike activation by secreted or surface proteases -such as trypsin and type ii transmembrane serine proteases hat (human airway trypsin-like protease) or tmprss2 bertram et al., 2011 bertram et al., , 2013 . the serine protease inhibitor camostat mesylate (camostat) inhibits the enzymatic activity of tmprss2 and other cell-surface proteases involved in coronavirus activation (kawase et al., 2012) . we therefore assessed whether k11777 displays antiviral activity in tmprss2 expressing cells. for this, we incubated target cells with camostat, k11777, or a combination of k11777 and camostat and then infected with pseudoviruses bearing 229e-s. k11777 alone demonstrated up to $70% inhibition of 229e-s-mediated transduction. simultaneous treatment with camostat and k11777 increased inhibition to $90% (fig. 2a, left panel) . similar inhibition patterns were obtained using the human intestinal epithelial cell line caco-2, which express endogenous tmprss2 and cathepsins (fig. 2b) . in contrast, k11777 alone fully blocked ebola pseudovirus infection, while camostat had no impact on viral infection (fig. 2a, middle panel) . finally, treatment of cells with k11777, camostat or both, had no impact on vsv-g driven viral entry (fig. 2a, right panel) , which is known to be independent of cysteine and serine protease activity. these results indicate that both serine and cysteine proteases can activate 229e-s for viral entry, as expected, while ebov-gp exclusively relies on cysteine proteases for activation. we next synthesized a series of k11777 analogs to further explore the antiviral activity of vinylsulfone-class protease inhibitors ( table 2 ). given that the piperazine ring in k11777 is basic (pka $7.8 for the conjugate acid) we considered that the compound might accumulate in the acidic (lysosomal and endosomal) compartments where target proteases such as ctsl and ctsb are abundant. to explore this notion and to more generally evaluate structure-activity trends, we synthesized new vinylsulfone analogs in which the substituent on the piperazine ring nitrogen atom was modified systematically. while the majority of these analogs (table 2 ) retain a basic piperazine ring, the n-phenyl analog smdc-256158 is only weakly basic (pka $3.42 for the conjugate acid) and thus will be neutral at physiological ph and would not be expected to exhibit lysosomotropic behavior. nearly all of the new analogs possessed potency comparable or superior to k11777 against sars-cov and ebov (table 2) , the most potent analogs being smdc256122 (sars-cov ic 50 = 0.04 nm; ebov ic 50 = 0.12 nm), smdc256159 (sars-cov ic 50 = 0.07 nm; ebov ic 50 = 0.16 nm) and smdc256160 (sars-cov ic 50 = 0.08 nm; ebov ic 50 = 0.11 nm). flaviviridae ssrna(+) huh7.5 >100 a ic 50 (inhibitory concentration) values are the concentrations required to inhibit the infectivity of the pseudotyped viruses on cells by 50%, which were determined from dose response curves. all envelopes apart from nipah and junin were used to make hiv-based pseudotypes. target cells (293t, 293t expressing ace2 or cd13, or vero cells) were then pretreated with serial dilutions of k11777 and exposed to virus. vsv-based pseudotypes were made by transfecting cells with nipah f and g plasmids, or junin envelope, and transducing with vsvdg(gfp)-g. progeny virus was then collected and titered on target cells. a non-linear regression analysis based on the sigmoidal dose response equation was applied to the percent inhibition and concentration data. data is shown as means of triplicate measurements ± standard deviation. values are representative of at least two independent experiments. of particular note from the structure-activity data is that the weakly basic analog smdc-256158 was 10-100-fold less potent than the other basic and protonatable vinylsulfone analogs ( table 2 ). the reduced potency of smdc-256158 is likely not related to the size of the phenyl substituent, since even larger, biaryl p3 substituents are known to be well tolerated in cathepsin-l like proteases such as cruzain (beaulieu et al., 2010) . also consistent with this interpretation, we find that other bulky tertbutyl and cyclopentyl groups are tolerated in analogs like smdc-256160 and smdc-256161. therefore, the most likely explanation is that as a weak base and the only analog expected not to be protonated at physiological ph, smdc-256158 does not accumulate in the lysosome to the same extent that the other, more basic, analogs do. conversely, k11777 and the other basic analogs accumulate in acidic endosomal compartments where target cysteine proteases such as ctsl and ctsb are located. to further verify the antiviral effects of three of the most efficient drug candidates, inhibition assays were carried out with two strains (urbani and toronto-2) of replication competent sars-cov, and using two separate readouts of replication (summarized (si, cc 50 /ic 50 ) ranged from 94.5 (smdc256159 inhibition against the toronto-2 strain) to over 1000. thus, these compounds were identified as ideal tools to determine whether cysteine or serine proteases promote sars-cov spread in the host. the pharmacokinetics and bioavailability of smdc256159 and smdc256160 in male and female sprague-dawley rats were determined following a single i.v. or p.o. dose administration (data not shown) and demonstrated similar profiles to k11777 (jacobsen et al., 2000) . in initial experiments, the antiviral efficacy of low-dose (1-10 mg/kg) smdc256160 was examined in a lethal sars-cov mouse model (day et al., 2009) . while there was a trend toward protection, there was no statistically significant reduction in mortality or disease severity (data not shown). experiments were therefore repeated at higher doses of cysteine protease inhibitor (50 mg/kg), either alone or in combination with the serine protease inhibitor, camostat (30 mg/kg) (fig. 3) . smdc-256160 alone was no more effective than vehicle treated controls (fig. 3) . in contrast, camostat was effective in protecting mice against death due to a lethal infection by sars-cov, with a survival rate of $60%. combining both classes of inhibitors did not significantly improve survival versus camostat alone. thus, sars-cov depends on serine protease activity for viral spread in vivo. viral entry is a multi-step process and an attractive target for antivirals (zhou and simmons, 2012) . the fact that disparate pathogenic viruses such as sars-cov, ebov and niv all utilize a common host factor for entry -ctsl -suggested that inhibitors of cathepsins might have broad applicability. cysteine proteases have proved to be druggable targets and their inhibitors are generally of low toxicity. we screened a library of drug-like compounds with established activity against ctsl and ctsb for activity against sars-cov and filoviruses, including ebov. we describe here the confirmation that protease inhibitors, such as k11777 and related compounds, are broad-spectrum antiviral drug candidates targeting viral entry. a number of additional vinylsulfone analogs were synthesized, some of which exhibited enhanced potency compared to k11777. most notably, potent antiviral activity was correlated with the presence of a basic piperazine ring at the p3 position, a finding that is consistent with accumulation in endosomal (acidic) compartments where the target cysteine proteases required for viral entry are located. the vinylsulfones described herein were broadly active against viral entry for three viral families: the corona-, filo-and paramyxoviruses, and are very well tolerated in vivo (barr et al., 2005) . the notion that coronaviruses, including sars-cov, use both a cathepsin-dependent endosomal pathway and a direct cell-surface serine protease-mediated pathway for entry (simmons et al., 2013) is supported by our finding that the combination of k11777 and camostat was superior to either compound alone. in contrast, ebov infection was effectively inhibited by k11777, but not by camostat. while unidentified additional proteases have been reported to mediate infection by other filoviruses, such as marv (gnirss fig. 3 . effects of per os administered smdc256160 and/or camostat on survival of balb/c mice infected with a lethal sars-cov. ten mice per group were dosed twice a day by oral gavage with smdc256160 and/or camostat or diluent alone (sterile water) for 9 days beginning 10 h prior to infection with 10,000 pfu of mouseadapted sars-cov. et al., 2012) and sudv (misasi et al., 2012) , efficient inhibition by the vinylsulfone analogs suggests that the unidentified proteases are cysteine proteases related to ctsb and l. activation of niv and hev appears to be fully dependent on ctsl and/or ctsb (pager et al., 2006; diederich et al., 2008 diederich et al., , 2012 . thus, vinylsulfones are promising antiviral lead compounds for further optimization as potent inhibitors of these two important groups of pathogenic emerging viruses, including ebov. previous reports showed that compound k11777 and analogs have satisfactory safety and pharmacokinetic profiles in rodents, dogs and primates (abdulla et al., 2007) . the fact that k11777, as a vinylsulfone, is an irreversible and not highly selective cysteine protease inhibitor does not appear to be a liability, at least if it is used as a short course antiviral. indeed, in the case of filoviruses, the lack of target selectivity is likely a boon -increasing effectiveness by also inhibiting secondary proteases (gnirss et al., 2012; misasi et al., 2012) . the availability of a novel, highly potent and largely non-toxic cysteine protease inhibitor, smdc256160, afforded the opportunity to assess whether the activity of cysteine or serine proteases is required for viral spread in vivo. for this, a mouse model for lethal sars-cov infection was employed. notably, only inhibition of serine proteases mitigated sars-cov pathogenesis in vivo. thus, future development of anti-coronavirus therapeutics should focus on inhibiting serine rather than cysteine proteases, with camostat being an excellent starting candidate. indeed, in japan camostat is used clinically, particularly to treat chronic pancreatitis (ikeda et al., 1988; sai et al., 2010) , with a reasonable safety profile (fiopan ò tablets, 2009 ). our results showed that targeting viral entry, and more specifically, the endosomal proteolysis step of entry, is an attractive strategy to discover new antiviral agents -particularly for filoviruses, like ebov, and some paramyxoviruses. although endosomal and cell-surface proteases can facilitate coronavirus entry in cultured cells, only the activity of serine proteases is required for viral spread in the infected host. nevertheless, the highly potent cysteine protease inhibitors identified here might be excellent starting points for the development of highly effective inhibitors of ebola virus and paramyxovirus entry, and constitute excellent research tools for dissecting the molecular mechanisms of viral entry. schistosomiasis mansoni: novel chemotherapy using a cysteine protease inhibitor mining a cathepsin inhibitor library for new antiparasitic drug leads a cysteine protease inhibitor protects dogs from cardiac damage during infection by trypanosoma cruzi identification of potent and reversible cruzipain inhibitors for the treatment of chagas disease cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease tmprss2 activates the human coronavirus 229e for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium chemical-biological characterization of a cruzain inhibitor reveals a second target and a mammalian off-target vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes a new mouseadapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group role of endocytosis and cathepsinmediated activation in nipah virus entry activation of the nipah virus fusion protein in mdck cells is mediated by cathepsin b within the endosome-recycling compartment cysteine protease inhibitors cure an experimental trypanosoma cruzi infection the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response cathepsins b and l activate ebola but not marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of tmprss2 expression highly conserved regions within the spike proteins of human coronaviruses 229e and nl63 determine recognition of their respective cellular receptors protease inhibitor therapy for recessive dystrophic epidermolysis bullosa. in vitro effect and clinical trial with camostat mesylate in vitro evaluation of the disposition of a novel cysteine protease inhibitor potency and selectivity of p2/p3-modified inhibitors of cysteine proteases from trypanosomes simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry inhibition of severe acute respiratory syndrome coronavirus replication in a lethal sars-cov balb/c mouse model by stinging nettle lectin, urtica dioica agglutinin family cluster of middle east respiratory syndrome coronavirus infections filoviruses require endosomal cysteine proteases for entry but exhibit distinct protease preferences a cysteine protease inhibitor rescues mice from a lethal cryptosporidium parvum infection a mature and fusogenic form of the nipah virus fusion protein requires proteolytic processing by cathepsin l vinyl sulfones as mechanismbased cysteine protease inhibitors design and synthesis of tri-ring p3 benzamide-containing aminonitriles as potent, selective, orally effective inhibitors of cathepsin k keto-1,3,4-oxadiazoles as cathepsin k inhibitors peptidic 1-cyanopyrrolidines: synthesis and sar of a series of potent, selective cathepsin inhibitors efficacy of camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease characterization of chikungunya pseudotyped viruses: identification of refractory cell lines and demonstration of cellular tropism differences mediated by mutations in e1 glycoprotein filoviruses utilize glycosaminoglycans for their attachment to target cells ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research characterization of the bas-congo virus glycoprotein and its function in pseudotyped viruses isolation of a novel coronavirus from a man with pneumonia in saudi arabia development of novel entry inhibitors targeting emerging viruses a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms inhibitors of sars-cov entry -identification using an internally-controlled dual envelope pseudovirion assay development and application of a high-throughput microneutralization assay: lack of xenotropic murine leukemia virus-related virus and/or murine leukemia virus detection in blood donors this work was supported by grants r01ai074986 and r21ai107165 from the national institute of allergy and infectious diseases (to g.s.) and funding from the sandler foundation (to a.r.r.). preliminary animal studies were supported by niaid contracts hhsn266200600011c (sri international) and hhsn272201000039i, pi. john morrey, phd (to d.b.). key: cord-327685-fymfqvp3 authors: channappanavar, rudragouda; perlman, stanley title: pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology date: 2017-05-02 journal: semin immunopathol doi: 10.1007/s00281-017-0629-x sha: doc_id: 327685 cord_uid: fymfqvp3 human coronaviruses (hcovs) can be divided into low pathogenic and highly pathogenic coronaviruses. the low pathogenic covs infect the upper respiratory tract and cause mild, cold-like respiratory illness. in contrast, highly pathogenic hcovs such as severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) predominantly infect lower airways and cause fatal pneumonia. severe pneumonia caused by pathogenic hcovs is often associated with rapid virus replication, massive inflammatory cell infiltration and elevated pro-inflammatory cytokine/chemokine responses resulting in acute lung injury (ali), and acute respiratory distress syndrome (ards). recent studies in experimentally infected animal strongly suggest a crucial role for virus-induced immunopathological events in causing fatal pneumonia after hcov infections. here we review the current understanding of how a dysregulated immune response may cause lung immunopathology leading to deleterious clinical manifestations after pathogenic hcov infections. coronaviruses belong to the virus family coronaviridae and are enveloped, positive-sense rna viruses. the coronavirus genome is approximately 31 kb, making these viruses the largest known rna viruses [1, 2] . coronaviruses infect a variety of host species, including humans and several other vertebrates. these viruses predominantly cause respiratory and intestinal tract infections and induce a wide range of clinical manifestations [3, 4] . coronaviruses infecting the respiratory tract have long been recognized as significant pathogens in domestic and companion animals and as the cause of mild and severe respiratory illness in humans [4, 5] . in general, coronaviruses infecting humans can be classified into low pathogenic hcovs, which include hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku and highly pathogenic covs such as severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) [6, 7] . low pathogenic hcov infect upper airways and cause seasonal mild to moderate cold-like respiratory illnesses in healthy individuals. in contrast, the highly pathogenic hcovs (pathogenic hcov or hcov hereafter) infect the lower respiratory tract and cause severe pneumonia, which sometimes leads to fatal acute lung injury (ali) and acute respiratory distress syndrome (ards), resulting in high morbidity and mortality [8] [9] [10] [11] [12] . highly pathogenic hcovs pose a substantial threat to public health. during the 2002-2003 epidemic, sars-cov infected approximately 8400 individuals with a 9.6% overall mortality rate [13, 14] . more recently, mers-cov crossed species to infect 1936 individuals resulting in 690 deaths (∼36% mortality rate) as of april 5, 2017 [15, 16] . recent identification of sars-like coronaviruses in bats and mers-cov in domesticated camels makes it likely that these viruses will continue to cross species barriers and cause additional outbreaks in human populations [17] [18] [19] [20] . these highly pathogenic hcovs cause a wide spectrum of clinical manifestations in humans, with a large fraction of patients developing short period of moderate clinical illness and a small but a substantial number of patients experiencing severe disease characterized by ali and ards [21] [22] [23] 10] . thus, there are basically two groups of patients, those developing milder disease, which resolved and those with severe disease, which was commonly fatal. the disease severity in pathogenic hcov infections was also influenced by several factors such as initial viral titers in the airways and age and comorbid conditions of the infected individual. while younger individuals below 18 years experience mild-moderate clinical illness, elderly individuals exhibit worse outcomes after infection with sars-cov or mers-cov [22, 10, 24] . additionally, individuals with comorbid conditions such as diabetes, obesity, heart failure, and renal failure among others experience severe disease, particularly after mers-cov infection [25, 26] . despite several years of research, specific factors causing the unusually high morbidity and mortality following pathogenic hcovs are incompletely understood. virus-induced direct cytopathic effects and viral evasion of host immune responses are believed to play major roles in disease severity. however, studies from humans who died of sars and more recent studies in animal models suggested that a dysregulated immune response occurred, resulting in an exuberant inflammation and lethal disease. in this review, we discuss recent advances in our understanding of hcov pathogenesis, with a special emphasis on cytokine storm and immunopathology as causes for deleterious consequences during hcov infections. sars-cov infection in humans resulted in an acute respiratory illness that varied from mild febrile illness to ali and in some cases ards and death [27, 10] . the clinical course of sars presents in three distinct phases. the initial phase was characterized by robust virus replication accompanied by fever, cough, and other symptoms, all of which subsided in a few days. the second clinical phase was associated with high fever, hypoxemia, and progression to pneumonia-like symptoms, despite a progressive decline in virus titers towards the end of this phase [28] . during the third phase, ∼20% of patients progressed to ards, which often resulted in death [29, 30] . because of a progressive decline in virus titers, the third phase is thought to have resulted from exuberant host inflammatory responses. the most common clinical manifestations of mers include flu-like symptoms such as fever, sore throat, nonproductive cough, myalgia, shortness of breath, and dyspnea, which rapidly progress to pneumonia [25, 21] . other atypical presentations include mild respiratory illness without fever, chills, wheezing, and palpitations. mers-cov in humans also causes gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea. the majority of mers patients with dyspnea progress to develop severe pneumonia and require admission to an intensive care unit (icu). although most healthy individuals present with mild-moderate respiratory illness, immunocompromised and individuals with comorbid conditions experience severe respiratory illness, which often progressed to ards [21] . overall, mers-cov caused severe disease in primary index cases, immunocompromised individuals and in patients with comorbid conditions, but secondary cases of household contacts or healthcare workers were mostly asymptomatic or showed mild respiratory illness. gross and microscopic pathology of sars typically, analyses of lungs from patients who succumbed to sars showed lung consolidation and edema with pleural effusions, focal hemorrhages, and mucopurulent material in the tracheobronchial tree. diffuse alveolar damage (dad) was a prominent histological feature in sars lungs [31, 32] . other changes included hyaline membrane formation, alveolar hemorrhage, and fibrin exudation in alveolar spaces with septal and alveolar fibrosis observed during later stages [32, 33] . staining for viral antigen revealed infection of airway and alveolar epithelial cells, vascular endothelial cells, and macrophages [31, 32] . furthermore, sars-cov viral particles and viral genome were also detected in monocytes and lymphocytes [31] . in addition to these changes, histological examination of lungs from patients who died of sars revealed extensive cellular infiltrates in the interstitium and alveoli. these cellular infiltrates included neutrophils and macrophages with macrophages being the predominant cell type [31, 32] . these results correlated with increased numbers of neutrophils and monocytes and lower cd4 and cd8 t cell counts in the peripheral blood samples of patients with fatal sars [34-36]. despite numerous laboratory-confirmed cases and deaths due to mers-cov infection in several countries, only one autopsy report of mers in humans is available. analysis of lung tissue from this patient showed pleural, pericardial, and abdominal effusions associated with generalized congestion, edema, and consolidation of lungs [37] . similar to sars-cov infection, dad was a prominent feature in the lungs. additionally, epithelial cell necrosis, sloughing of bronchiolar epithelium, alveolar edema, and thickening of alveolar septa were also noted. immunohistochemical examination showed that mers-cov predominantly infected airways and alveolar epithelial cells, and endothelial cells and macrophages. the severity of lung lesions correlated with extensive infiltration of neutrophils and macrophages in the lungs and higher numbers of these cells in the peripheral blood of mers patients [37] . cytokines and chemokines have long been thought to play an important role in immunity and immunopathology during virus infections. a rapid and well-coordinated innate immune response is the first line of defense against viral infections, but dysregulated and excessive immune responses may cause immunopathology [38] [39] [40] . although there is no direct evidence for the involvement of pro-inflammatory cytokines and chemokines in lung pathology during sars and mers, correlative evidence from patients with severe disease suggests a role for hyper-inflammatory responses in hcov pathogenesis. additionally, sars-cov-infected airway epithelial cells (aecs) also produce large amounts of ccl3, ccl5, ccl2, and cxcl10 [43]. the delayed but excessive production of these cytokines and chemokines is thought to induce a dysregulated innate immune response to sars-cov infection. high serum levels of pro-inflammatory cytokines (ifn-γ, il-1, il-6, il-12, and tgfβ) and chemokines (ccl2, cxcl10, cxcl9, and il-8) were found in sars patients with severe disease compared to individuals with uncomplicated sars [44] [45] [46] [47] . conversely, sars patients with severe disease had very low levels of the anti-inflammatory cytokine, il10 [44] . in addition to pro-inflammatory cytokines and chemokines, individuals with lethal sars showed elevated levels of ifn (ifn-α and ifn-γ) and ifn-stimulated genes (isgs) (cxcl10 and ccl-2) compared to healthy controls or individuals with mild-moderate disease [48-51]. these results were the first to suggest a possible role for ifns and isgs in the immunopathogenesis of sars in humans. thus, it appears from these studies that dysregulated and/or exaggerated cytokine and chemokine responses by sars-cov-infected aecs, dcs, and macrophages could play an important role in sars pathogenesis. similar to sars, mers-cov infection of human airway epithelial cells induces significant but delayed ifn and proinflammatory cytokine (il-1β, il-6, and il-8) responses [52] . while mers-cov replicates both in naïve and activated human monocyte-macrophages and dcs, only activated t cells support mers-cov replication [53] [54] [55] . this is in contrast to sars-cov, which abortively infected monocyte-macrophages, dcs, and t cells. mers-cov infection of thp-1 cells, a monocyte cell line, and human peripheral blood monocyte-derived macrophages and dendritic cells induced delayed but elevated levels of pro-inflammatory cytokines and chemokines such as ccl-2, ccl-3, ccl-5, il-2, and il-8 [54, 55]. however, induction of ifn-α/β by monocytemacrophages and dcs was not substantial except for plasmacytoid dendritic cells, which produced copious amounts of ifns upon mers-cov infection [56] . recent studies showed elevated levels of serum pro-inflammatory cytokines (il-6 and ifn-α) and chemokines (il-8, cxcl-10, and ccl5) in individuals with severe mers compared to those with mild to moderate disease [57, 58] . high serum cytokine and chemokine levels in mers patients correlated with increased neutrophil and monocyte numbers in lungs and in the peripheral blood, suggesting a possible role for these cells in lung pathology [57, 58, 37 ]. several inbred mouse strains have been evaluated to study sars-cov pathogenesis. mice infected with the human strain of sars-cov (sars-cov-urbani) were permissive to virus replication but developed only mild lung pathology and clinical illness [59] . subsequently, isolation of mouseadapted strains of sars-cov (e.g., sars-cov-ma15) allowed studies of lethal sars [60] [61] [62] . ma15 infects airway and alveolar epithelial cells and epithelial cells of other organs [62] . young mice of many strains (e.g., c57bl/6, 129) support ma15 replication in the lungs but are resistant to developing significant clinical disease [63, 64] . in contrast, young balb/c mice infected with ma15 develop lethal disease characterized by diffuse alveolar damage, enhanced monocyte/macrophage and neutrophil accumulation, pulmonary edema, and hyaline membrane formation [62] . furthermore, aged mice of all strains develop lethal clinical disease and succumb to infection [65, 66, 64] . in addition to mouse models, sars-cov infection of aged rhesus macaques resulted in significantly more pathology than young adult animals [67] . these animal models replicated several key features of sars-cov infection in humans and were thus useful for investigating sars pathogenesis. studies in animal models have been particularly useful in elucidating the role of cytokines and chemokines in mediating lung immunopathology following hcov infections. infection of non-human primates (nhps) with sars-cov induced a dysregulated immune response resulting in increased disease severity in aged but not young nhps, despite similar viral titers in the airways [67] . since enhanced expression of genes regulating inflammation but not virus titers correlated with disease severity, an exaggerated immune response is thought to induce lethal disease in aged nhps [67] . similarly, in sars-cov-infected balb/c mice, disease severity in aged mice correlated with early and disproportionately strong upregulation of ards-associated inflammatory gene signatures [66] . in a recent study, we identified a pathogenic role for ifn-i in mice infected with ma15. our results showed that rapid sars-cov replication in balb/c mice induced a delayed ifn-α/β response accompanied by an excessive influx of pathogenic inflammatory monocyte-macrophages (imms) [38] . the accumulating imms themselves produced additional levels of monocyte chemo-attractants such as ccl2, ccl7, and ccl12 (through ifn-α/β receptor stimulation), resulting in further accumulation of pathogenic imms, which in turn enhanced disease severity. these infiltrating imms produced elevated levels of pro-inflammatory cytokines such as tnf, il-6, il1-β, and inos. blocking ifn signaling, depleting imms, or neutralizing a single inflammatory cytokine, tnf, protected mice from lethal sars-cov infection. additionally, ifn-α/β or imm-derived pro-inflammatory cytokines sensitized t cells to undergo apoptosis, further impeding virus clearance [38] . in another study of sars-cov infection, loss of tir-domain-containing adapter-inducing interferon-β (trif), an adapter molecule for tlr3 and tlr4 signaling, resulted in a distinct inflammatory signature characterized by neutrophil and other inflammatory cell infiltration [68] . a dysregulated immune response to sars-cov in trif-deficient mice was associated with aberrant antiviral ifn (ifn-α and ifnβ), pro-inflammatory cytokine and chemokine (il-6, tnf, ifn-γ, and ccl5), and interferonstimulated gene (rsad2, ifit1, and cxcl10) responses. notably, virus titers were significantly higher in tlr3 −/− and trif −/− mice compared to their wt controls [68] . although the viral factors regulating the pro-inflammatory response of neutrophils and monocyte-macrophages remain to be identified, the e protein of sars-cov has been shown to enhance pro-inflammatory cytokine and chemokine and inflammasome activity via its ion channel activity [69] [70] [71] . these results support the notion that higher virus titers and dysregulated cytokine/chemokine responses cause a bcytokine storm^with lung immunopathological changes following sars-cov infection. animal models employed to study mers include rhesus macaques, rabbits, marmosets, and mice among others. mers-cov challenged rhesus macaques developed mild to moderate disease [72] . similarly, mers-cov-infected rabbits displayed mild clinical disease with mild-moderate perivascular, peribronchiolar infiltration, and to a lesser extent lung interstitial inflammation [73, 74] . in contrast, marmosets displayed moderate-severe respiratory disease characterized by broncho-interstitial pneumonia, alveolar edema, and fibrin deposition [75] . marmosets with severe disease showed increased neutrophil and macrophage infiltration in alveoli and interstitial septa, although whether marmosets develop severe disease remains controversial [75, 76] . although gross and histological lesions and inflammatory cell infiltration in mers-cov infected marmosets resemble human disease, there are no data available describing cytokine and chemokine responses in these animals. small laboratory animals, particularly rodents, do not support mers-cov replication due to inability of mers-cov-spike protein to bind to human dpp4 (hdpp4) orthologs in these animals [77] . the first mouse model to study mers was generated by intranasal transduction of adenovirus encoding hdpp4. these mice developed mild to moderate pneumonia, especially in immunodeficient mice [78] . several hdpp4 transgenic mouse models developed thereafter exhibited variable organ tropism and disease severity, depending on the promoter driving the hdpp4 expression [79, 80] . more recently, hdpp4 knock-in mice in which hdpp4 is expressed under the mouse hdpp4 promoter have also been described. these mice also developed moderate clinical disease after infection with human isolates of mers-cov [81] . we and others recently developed a similar mouse model and showed that serial passage of human isolate of mers-cov resulted in mouse adaptation. mice infected with this adapted virus caused lethal respiratory illness and will be useful for studies of pathogenesis [82, 83] . overall, delayed and aberrant antiviral and proinflammatory cytokine production in mers-cov-infected human macrophages and dendritic cells and high serum proinflammatory cytokine levels in patients with severe disease compared to mild-moderate clinical disease suggesting that possible dysregulated and enhanced cytokine responses promote lung pathology following mers-cov infection. to counter innate antiviral cytokine responses, sars-cov and mers-cov encode several structural and non-structural proteins (nsps) that antagonize antiviral immune response. sars-cov encoded nsp1, nsp3-macrodomain, nsp3deubiquitinase (dub), and orf3b, orf6, and orf9b subvert antiviral response by antagonizing ifn and isg responses [84] [85] [86] [87] [88] [89] . while nsp3 impairs ifn responses by unknown mechanism, nsp1 inhibits ifn responses by blocking stat1 phosphorylation [90, 91] . additionally, structural proteins such as the membrane (m) and nucleocapsid (n) proteins dampen ifn signaling by inhibiting tbk1/ikke and by unknown mechanisms, respectively [92] [93] [94] [95] . similarly, mers-cov structural proteins m and n and accessory proteins orf3, orf4a, and orf4b antagonize ifn responses [85, 96, 97] . it should be noted that most if not all of these putative antiviral mechanisms were demonstrated in transient expression assays and whether they are actually important in the context of infectious virus remains to be determined. structural and nonstructural protein antagonism of ifn responses further amplifies inflammatory responses by promoting unrestrained virus replication resulting in increased viral pamps that further dampen ifn signaling and stimulate prrs to induce an aberrant inflammatory response. lack of ifn signaling also leads to an excessive accumulation of ly6c low monocytes and neutrophils. despite several years of research studying sars and mers pathogenesis, specific host factors that drive lung pathology following hcov infections are relatively unknown. however, a careful review of the literature related to sars-cov and mers-cov pathogenesis in humans and animal models highlights several key factors that may play a crucial role in the initiation and progression of an exacerbated inflammatory responses. studies from animal models, especially mouse models, provide correlative evidence for differential disease outcome if the viruses predominantly infect airway epithelial cells versus both airway and alveolar epithelial (type i and type ii pneumocytes) cells. in b6 and 129 strains, both of which are permissive to virus replication but resistant to developing clinical disease, viral antigen is predominantly located in airway epithelial cells early after infection. in contrast, in highly susceptible balb/c mice, virus antigen is detected in the lung airways and in alveolar type i and ii pneumocytes (fig. 1) . these results suggest a critical role for hcov-infected type i and ii pneumocytes in mediating lung pathology and host susceptibility. cov-specific t cells are crucial for virus clearance and limit further damage to host [64, 104] . additionally, t cell responses also dampen overactive innate immune responses [105, 106] . exuberant inflammatory responses caused by pathogenic hcov diminish the t cell response, in the case of sars-cov infection via tnf-mediated t cell apoptosis, thus resulting in uncontrolled inflammatory response. 3. accumulation of alternatively activated macrophages and altered tissue homeostasis: in some sars patients with extended duration of disease, dad was accompanied by fibrosis of interstitial and alveolar spaces and hyperplasia of pneumocytes. similar histological features were noticed in lungs of sars-cov-challenged stat −/− mice on b6 and 129 backgrounds. lungs from these mice revealed an enhanced perivascular infiltration of alternatively activated macrophages, neutrophils, and fibroblasts accompanied by extensive fibrin deposition and alveolar collapse, features observed during end stage ali and ards in humans [63, 107] . further studies revealed that abrogation of stat1 signaling, specifically in myeloid cells, resulted in alternative activation of macrophages [108] . in addition, a delicate balance between host coagulation and fibrinolysis processes regulates tissue remodeling and ali [109] . 4. ards: inflammatory mediators play a key role in the pathogenesis of ards, a primary cause of death in patients infected with sars-cov or mers-cov [110, 111] . several pro-inflammatory cytokines, including il-6, il-8, il-1β, and gm-csf, reactive oxygen species, and chemokines such as ccl2, ccl-5, ip-10, and ccl3 contribute to ards [48, 112, 113] . additionally, uncontrolled epithelial cell proliferation and impaired tissue remodeling during later stages induce ards leading to pulmonary fibrosis and death. a summary of causes and consequences of cytokine storm and immunopathology to hcov pathogenesis is demonstrated in fig. 2 . high virus titers and subsequent exuberant inflammatory cytokine and chemokine responses correlate with high morbidity and mortality observed during pathogenic hcov infections. a systematic review of therapeutic effects of several commonly used antiviral and immunomodulatory agents used during sars outbreak showed inconclusive results [114] . similarly, therapeutic interventions aimed towards reducing viral load were somewhat beneficial when administered early but not during later stages of mers-cov infection [115] [116] [117] . these results suggest that besides controlling viral load, novel strategies directed at attenuating inflammatory responses will likely improve clinical outcomes. here, we describe agents that have the potential to mitigate hcov-induced inflammation. corticosteroid therapy corticosteroids are a class of steroidal hormones that exert anti-inflammatory functions and are generally used to suppress inflammatory conditions. during the 2003 sars epidemic, corticosteroids were the mainstay of immunomodulatory therapy. the timely administration of corticosteroids often leads to early improvement in terms of reduced fever, resolution of radiographic lung infiltrates, and better oxygenation [118] [119] [120] . however, while some studies showed no beneficial effect, other demonstrated adverse outcomes following corticosteroid therapy during sars-cov infection in humans. early treatment of corticosteroids in sars patients enhanced plasma viral load in non-icu patients, thus leading to exacerbated disease [118] . overall, these results show that the timing, dosage, and duration of corticosteroid therapy are critical if this intervention is to be beneficial in hcov infections. in general, corticosteroid therapy is not recommended for treatment of hcov respiratory infections. interferons pegylated and non-pegylated interferons have been under investigation for therapeutic purposes in hcovinfected individuals. however, therapeutic use of these agents produced mixed results both in humans and animal models of hcov infections. early administration of ifn was marginally beneficial in reducing viral load and resulted in moderate improvement in clinical manifestations. in contrast, delayed administration of ifn did not have any advantage compared to placebo controls. similarly, early administration of combination of ifn and ribavirin modestly ameliorated disease severity but did not affect mortality [115, 121, 117, 122] . other possible therapeutics ifn-αβ inhibitors and ifn-λ ifn-αβ restrict virus replication through induction of isgs. however, ifn-αβ can also exacerbate disease by enhancing recruitment and function of imms and other innate immune cells. while an early interferon response was protective in sars-cov-infected mice, delayed ifn-αβ signaling dysregulated the anti-sars-cov immune response suggesting that timing of ifn therapy is critical in determining the disease outcome. based on these results, the administration of ifn-αβ receptor blockers or antagonists should be considered as an option to prevent exuberant inflammatory responses during later stages of severe disease, particularly during sars [38] . in contrast to ifn-αβ, ifn-λ mainly activates epithelial cells and lacks monocytemacrophage-mediated pro-inflammatory activity of ifn-αβ [123] . additionally, ifn-λ suppresses neutrophil recruitment to the site of inflammation [124] . since sars-cov and mers-cov predominantly infect aecs and ifn-λ stimulates antiviral gene in epithelial cells without over-stimulating the immune system, use of ifn-λ may be an ideal therapeutic option. suppression of oxidized phospholipids oxidized phospholipids (oxpl) have been shown to promote ali by increasing lung macrophage cytokine/chemokine production via tlr4-trif signaling in influenza a virus (iav)-infected mice [125] . in a recent study, therapeutic administration of the tlr4 antagonist, eritoran, protected mice from lethal iav infection by reducing the levels of oxpl and inflammatory cytokines and chemokines [126] . despite potent immunomodulatory functions, eritoran has no direct antiviral activity, suggesting its use in the amelioration of inflammatory responses. since pathogenic human coronaviruses cause acute lung injury and promote oxpl production in the lungs [125] , strategies to suppress oxpl either by using eritoran or other similar compounds could be of value in dampening hcovinduced inflammation. sphingosine-1-phosphate receptor 1 agonist therapy in mice infected with iav, sphingosine-1-phosphate receptor 1 fig. 2 schematic representation of protective versus pathogenic inflammatory responses to pathogenic hcov infections (s1p1) signaling in endothelial cells was shown to orchestrate pathogenic inflammatory responses [127] . targeted s1p1 agonism restrained excessive inflammatory cell recruitment, suppressed pro-inflammatory cytokines and chemokines, and reduced iav induced morbidity and mortality [127, 128] . , so that sars-cov infection of endothelial cells may drive s1p1-mediated inflammatory cytokine/chemokine responses and neutrophil and macrophage accumulation. therefore, s1p1 agonism could be a potential therapeutic agent in hcov patients to dampen pathogenic cytokine and chemokine responses, if a role for an excessive immune response by these cells is demonstrated. inhibitors of monocyte recruitment and function studies in animal models demonstrate pathogenic roles for imms during lethal hcov infections. in a mouse model of cardiac inflammation, systemic delivery of optimized lipid nanoparticles containing a ccr2-silencing short interfering rna (sirna) efficiently degraded ccr2 mrna and impaired imm recruitment to sites of inflammation thus resulting in improved disease outcome [129, 130] . since hcovs are single-stranded rna (ssrna) viruses and stimulation of imms with the tlr7 agonist, r837 (a synthetic ssrna mimic), induces strong inflammatory responses, it is possible that immspecific tlr-7 signaling promotes excessive inflammation in response to hcov infection. thus, a tlr7 antagonisttargeted approach to mitigate inflammation could prove beneficial. other immunomodulatory agents several other immunomodulatory agents that could ameliorate inflammatory responses following pathogenic hcov infections include cytokine/chemokine inhibitors and danger-associated molecular pattern (damp) antagonists [131] . studies from animal models show a significant contribution of tnf to acute lung injury and impaired t cell responses in sars-covchallenged mice. in vivo neutralization of tnf activity or infection of mice lacking tnfr provides protection against sars-cov-induced morbidity and mortality [38, 132] . however, it is to be noted that tnf was not detected in the serum of sars patients at least during later stages of infection. inflammation is an indispensable part of an effective immune response, without which successful elimination of an infectious agent is difficult. the inflammatory response begins with the initial recognition of a pathogen, which then mediates immune cell recruitment, eliminates pathogens, and ultimately results in tissue repair and return to homeostasis. however, certain viruses such as highly pathogenic covs, iav, and ebola viruses induce excessive and prolonged cytokine/ chemokine response known as bcytokine storms,^which results in high morbidity and mortality due to immunopathology. although studies reviewed in this manuscript provide evidence that bcytokine storms^and immunopathology can occur during pathogenic hcov infections, we do not yet have a sufficient understanding of the specific factor/s responsible for exuberant inflammatory responses. studies from human autopsies and animal models strongly suggest a pathogenic role for inflammatory cytokines/chemokines derived from imm and neutrophils. therefore, therapeutic interventions targeting these pro-inflammatory cytokines and chemokines could prove beneficial in ameliorating undesirable inflammatory responses. additionally, since high virus titers at early and later stages of infection strongly correlate with disease severity in humans, strategies directed at controlling viral load as well as attenuating the inflammatory response might prove beneficial. therefore, future studies should focus on identification of specific signaling pathways that mediate inflammatory responses in hcov-infected patients and animals. (9) coronaviridae coronaviruses, toroviruses, and arteriviruses coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives epidemiology, genetic recombination, and pathogenesis of coronaviruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus coronavirus-associated pneumonia in previously healthy children clinical disease in children associated with newly described coronavirus subtypes identification of a novel coronavirus in patients with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans cytokines and chemokines in human macrophages: implications for pathogenesis high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism age-related increases in pgd(2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection exacerbated innate host response to sars-cov in aged non-human primates toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp3 inflammasome middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques asymptomatic middle east respiratory syndrome coronavirus infection in rabbits prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection infection with mers-cov causes lethal pneumonia in the common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/ 2012 isolates does not result in lethal disease receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection rapid generation of a mouse model for middle east respiratory syndrome animal models for sars and mers coronaviruses animal models of middle east respiratory syndrome coronavirus infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a mouse model for mers coronavirusinduced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane interaction of sars and mers coronaviruses with the antiviral interferon response severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling interferon and cytokine responses to sars-coronavirus infection sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain the conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists initial viral load and the outcomes of sars proliferative growth of sars coronavirus in vero e6 cells viral load kinetics of mers coronavirus infection lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed tnf-related apoptosis-inducing ligand macrophage-expressed ifn-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia cellular inhibitor of apoptosis protein ciap2 protects against pulmonary tissue necrosis during influenza virus infection to promote host survival t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice adaptive immune cells temper initial innate responses not so fast: adaptive suppression of innate immunity transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in stat1 knockout mice during severe acute respiratory syndrome coronavirus infection induction of alternatively activated macrophages enhances pathogenesis during severe acute respiratory syndrome coronavirus infection genome wide identification of sars-cov susceptibility loci using the collaborative cross clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome characterization of cytokine/chemokine profiles of severe acute respiratory syndrome expression profile of immune response genes in patients with severe acute respiratory syndrome sars: systematic review of treatment effects ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome corticosteroid treatment of severe acute respiratory syndrome in hong kong pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques coronaviruses-drug discovery and therapeutic options ifnlambda is a potent anti-influenza therapeutic without the inflammatory side effects of ifnalpha treatment ifn-lambda resolves inflammation via suppression of neutrophil infiltration and il-1beta production identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury the tlr4 antagonist eritoran protects mice from lethal influenza infection endothelial cells are central orchestrators of cytokine amplification during influenza virus infection suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus silencing of ccr2 in myocarditis therapeutic sirna silencing in inflammatory monocytes in mice immunomodulatory therapy for severe influenza the effect of inhibition of pp1 and tnfalpha signaling on pathogenesis of sars coronavirus acknowledgements we thank dr. anthony fehr for careful review of this manuscript. this work was supported in part by grants from the n.i.h. (po1 ai060699, ro1 ai091322). key: cord-346331-d0s028wl authors: lackey, kimberly a.; pace, ryan m.; williams, janet e.; bode, lars; donovan, sharon m.; järvinen, kirsi m.; seppo, antti e.; raiten, daniel j.; meehan, courtney l.; mcguire, mark a.; mcguire, michelle k. title: sars‐cov‐2 and human milk: what is the evidence? date: 2020-05-30 journal: matern child nutr doi: 10.1111/mcn.13032 sha: doc_id: 346331 cord_uid: d0s028wl the novel coronavirus sars‐cov‐2 has emerged as one of the most compelling and concerning public health challenges of our time. to address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical and public health communities has rapidly engaged to collectively find answers and solutions. one area of active inquiry is understanding the mode(s) of sars‐cov‐2 transmission. although respiratory droplets are a known mechanism of transmission, other mechanisms are likely. of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. however, there is limited published literature related to vertical transmission of any human coronaviruses (including sars‐cov‐2) via human milk and/or breastfeeding. results of the literature search reported here (finalized on 17 april 2020) revealed a single study providing some evidence of vertical transmission of human coronavirus 229e; a single study evaluating presence of sars‐cov in human milk (it was negative); and no published data on mers‐cov and human milk. we identified 13 studies reporting human milk tested for sars‐cov‐2; one study (a non‐peer‐reviewed preprint) detected the virus in one milk sample, and another study detected sars‐cov‐2 specific igg in milk. importantly, none of the studies on coronaviruses and human milk report validation of their collection and analytical methods for use in human milk. these reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses (in particular, sars‐cov‐2) during breastfeeding are discussed. the global pandemic caused by the sars-cov-2 virus is one of the most compelling and concerning global health crises of our time. fortunately, this pandemic has rapidly mobilized the full range of expertise represented by researchers, clinicians and public health officials. although our understanding of the biology, clinical implications and strategies for mitigation continues to evolve, one issue that has received limited attention is the implication of this pandemic for infant feeding practices. this lack of attention has resulted in mixed messages regarding guidance about optimal infant feeding practices (e.g., american academy of pediatrics, 2020; centers for disease control and prevention, 2020a; world health organization, 2020a; united nations children's fund [unicef] , 2020) and a consequent lack of confidence about best approaches to infant feeding in the face of this growing pandemic. even when a mother is positive for covid-19, the world health organization (who) recommends breastfeeding be initiated within 1 h of birth, exclusive breastfeeding be continued for 6 months and breastfeeding be continued for up to 2 years. they suggest use of appropriate respiratory hygiene, hand hygiene and environmental cleaning precautions. the unicef recommends that covid-19-positive mothers continue breastfeeding while applying precautions, such as wearing a mask and handwashing before and after feeding (unicef, 2020) . the u.s. centers for disease control and prevention (cdc) neither recommends nor discourages breastfeeding but advises that decisions be made by the mother and family in consultation with their health care providers (centers for disease control and prevention, 2020a) . they recommend that during temporary separation (should that occur), mothers who intend to breastfeed should express their milk using proper hand hygiene and that the expressed milk should be fed to the newborn by a healthy caregiver. further, if a mother and newborn do room-in and the mother wishes to feed at the breast, the cdc recommends that she should wear a facemask and practice hand hygiene before each feeding. it is well established that viral transmission through human milk can occur (jones, 2001; lawrence & lawrence, 2004) . notable examples include human immunodeficiency virus (hiv; black, 1996; ziegler, johnson, cooper, & gold, 1985) , cytomegalovirus (cmv; stagno & cloud, 1994) and human t-cell lymphotropic virus type 1 (htlv-1; boostani, sadeghi, sabouri, & ghabeli-juibary, 2018) . perhaps the most prominent example of mother-to-child viral transmission via breastfeeding is hiv infection, during which higher milk and serum viral loads are associated with an increased risk of transmission (davis et al., 2016; semba et al., 1999; willumsen et al., 2003) . the risk of postnatal infection for breastfed infants of hiv+ mothers is ≈10-20% over the first 2 years of life without the use of antiretroviral therapies (art; dunn, newell, ades, & peckham, 1992; nduati et al., 2001) . however, compared with mixed feeding, exclusive breastfeeding is associated with lower risk of transmission of hiv infection to infants (coutsoudis et al., 2001; iliff et al., 2005) . in many high-income nations, breastfeeding is contraindicated in the case of maternal hiv infection with or without maternal art (e.g., american academy of pediatrics, 2012; centers for disease control and prevention, 2020b) . conversely, in low-and-middle-income nations, infant mortality from malnutrition and infectious disease may outweigh the risk of acquiring hiv via vertical transmission during breastfeeding. as such, breastfeeding is recommended (world health organization, 2016) . with respect to cmv, it is estimated that 60-70% of breastfed infants of cmv-seropositive mothers become infected with cmv (dworsky, yow, stagno, pass, & alford, 1983; minamishima et al., 1994) . the risk of cmv infection in neonates is highest in preterm and very low birthweight (less than 1,500 g) infants (hamprecht & goelz, 2017; lanzieri, dollard, josephson, schmid, & bialek, 2013) . a small percentage of infected infants develop a severe complication known as cmv sepsis-like syndrome, which can be fatal (fischer et al., 2010) . nonetheless, breastfeeding is not contraindicated in cmv-seropositive women with healthy, term infants for htlv-1, breastfeeding is considered the major route of infection for infants (moriuchi, masuzaki, doi, & katamine, 2013) . htlv-1 infection is lifelong, and although most infected individuals remain asymptomatic, approximately 10% develop severe disease, including adult t-cell leukaemia, a highly aggressive and usually fatal malignancy (rosadas & taylor, 2019) . some organizations and agencies list maternal htlv-1 as a contraindication for breastfeeding (american academy of pediatrics, 2012; centers for disease control and prevention, 2019a), whereas others do not (world health organization, 2009 ). human coronaviruses are enveloped, positive-sense, singlestranded rna viruses first described in 1965 (tyrrell & bynoe, 1965) . there are seven identified strains known to infect humans. four of the strains (alphacoronaviruses 229e, nl63 and oc43 and betacoronavirus hku1) are ubiquitous in humans and cause the common cold. there is limited evidence that one of these (229e) may be vertically transmitted from mothers to infants, although the mechanism remains unclear (gagneur et al., 2008) . the presence of 229e in neonatal gastric samples suggests that one possible mechanism for infection is through human milk, although this study did not evaluate human milk specifically (gagneur et al., 2008) . in light of the emergence of the novel coronavirus sars-cov-2, several issues related to human milk and coronavirus infection demand immediate attention, the first and foremost being whether or • very little is known about coronaviruses in human milk and whether breastfeeding is a possible mode of vertical transmission. • limited, weak evidence suggests that some coronaviruses (including sars-cov-2) may be present in human milk, but these studies do not report methods of sample collection and validation of reverse transcription polymerase chain reaction (rt-pcr) assays for human milk. • nothing is known about the timing of the antibody response in human milk to sars-cov-2 infection. • future research should utilize validated methods and focus on both potential risks and protective effects of breastfeeding. not the virus is present in human milk produced by infected or exposed women. of particular interest in this context are (1) the potential role that breastfeeding could play in vertical transmission of sars-cov-2 from women to infants via human milk and (2) the potential protective effects of targeted antibodies and other immunoprotective components in human milk against covid-19. the goal of this review was to evaluate the published evidence regarding the presence of this and other human coronaviruses in human milk. we used a variety of databases to identify relevant literature published as of 17 april 2020, and the list of databases and search terms used can be found in table 1 . it is noteworthy that in addition to using standard scientific databases (e.g., pubmed), we also used a general google search and a search of preprint servers to identify reports that had not yet been published in refereed journals (i.e., grey literature). any research in which human milk was collected and tested for a human coronavirus was included in this review. see addendum in the supporting information online. this review does not constitute human subject research, and as such, ethical approval was not required. the deadliest of the human coronaviruses to date is mers-cov, which emerged in saudi arabia in 2012. the disease caused by mers-cov, middle eastern respiratory syndrome (mers), is characterized by severe respiratory illness with symptoms of fever, cough and shortness of breath. mers-cov is a betacoronavirus, and the case fatality rate of mers is 34% (mahase, 2020) . there are no reports of the presence or absence of mers-cov in human milk. however, there are reports of the presence of mers-cov in the milk of dromedary camels (camelus dromedaries; conzade et al., 2018; hemida et al., 2015; reusken et al., 2014) , and there is one report of a human likely infected through the consumption of raw (unpasteurized) camel milk (memish et al., 2014) . in camel milk samples spiked with currently, there is one report in which human milk was tested for sars-cov (robertson et al., 2004) and two reports of human milk being tested for sars-cov antibodies (robertson et al., 2004; stockman, lowther, coy, saw, & parashar, 2004 ). robertson and colleagues described a woman infected during the second trimester of pregnancy (19 weeks). a single milk sample was collected 131 days after the onset of symptoms, but no additional detail on the collection methodologies was provided. milk was submitted to the cdc, where it was analysed using reverse transcription polymerase chain reaction (rtsearch terms, databases and preprint servers used to identify existing literature reporting the possibility of vertical transmission of coronaviruses from mother to infant during breastfeeding as of 17 april 2020 the infant in this study was never tested for sars-cov infection. the first trimester of pregnancy (7 weeks the novel coronavirus sars-cov-2 was named after sars-cov due to its shared sequence homology (77.9%; kim et al., 2020) and similar clinical characteristics. the first reported cases of sars-cov-2 infection emerged in late 2019 in china. although the current estimated case fatality rate for covid-19 (the disease caused by the sars-cov-2 virus) is much lower than those of sars and mers at roughly 2% (mahase, 2020) , the spread of this pathogen has been much more rapid and extensive. at the time of writing, there were 13 studies (seven case studies and six case series; three of which were preprints or preliminary reports that had not been formally peer-reviewed; table 2 ) reporting direct testing of milk produced by women infected with sars-cov-2 dong et al., 2020; fan et al., 2020; liu, wang, li, et al., 2020; liu, wang, zhang, et al., 2020; salvatori et al., 2020; wang et al., 2020; yu et al., 2020; wu et al., 2020) or by women whose infants were infected kam et al., 2020; yuehua et al., 2020) . in total, 48 milk samples produced by 32 women had been tested; all but one sample (wu et al., 2020; non-peer-reviewed preprint) were negative for the presence of the virus. two milk samples produced by a single woman were tested for sars-cov-2 specific antibodies; igg but not igm was identified in both samples non-peer-reviewed preprint) . a description of the relevant characteristics for the women and infants in these studies can be found in table 2 . investigators conducting nine of the 13 studies analysed milk samples collected at birth or shortly thereafter, reporting only findings in colostrum or transitional milk. those same nine studies reported on the milk produced by women who were infected during the third trimester of pregnancy, whereas the other four reported findings from milk produced by mothers of infants infected at 1.5, 3, 6 and 13 months of age kam et al., 2020; yu et al., 2020; yuehua et al., 2020) . for the infants born to women infected during pregnancy, most were immediately separated from their mothers post-delivery and were not breastfed for the duration of the period observed in their respective reports. fourteen of the 32 infants described in these reports were born via caesarean section; only two were specified as vaginal births. repeated milk samples, collected up to 27 days apart, were analysed for eight of the women. all the studies were conducted in china cui et al., 2020; dong et al., 2020; fan et al., 2020; liu, wang, li, et al., 2020; liu, wang, zhang, et al., 2020; wang et al., 2020; yuehua et al., 2020 wu et al., 2020) , singapore (kam et al., 2020) or italy (salvatori et al., 2020) . pneumonia were collected after their first lactation' and that milk was collected following who guidelines, but they did not provide a citation for this collection method. all milk tested negative for the virus, but no information was provided on the methods used for analysis. in a report by liu, wang, zhang, et al. (2020) , milk produced by two women was tested. one woman was 34 years old and at in a case series by liu, wang, li, et al. (2020) , milk produced by 10 women infected during late pregnancy was tested via rt-pcr for sars-cov-2; all samples tested negative. it is noteworthy that this report included data from 19 women, but milk was collected from only 10 of them. the authors did not specify for which of the women milk was collected. none of the 19 infants reported in this study tested positive for sars-cov-2 via rt-pcr. the only detail available on collection or testing methods is that rt-pcr was used to test the samples and that 'milk was collected after the first lactation' . despite their results, the authors concluded that delivery should occur in an isolation room and that infants be separated from infected mothers. li et al. (2020) described a 30-year-old woman at 35-week gestation who was positive for sars-cov-2 and who delivered a male infant via emergency caesarean section. the infant was tested immediately upon delivery via oropharyngeal swab, which was negative. after delivery, the infant was kept in isolation away from his mother. milk was collected immediately after delivery and on days 2 and 3 post-partum; all samples were negative. again, no information on the collection or testing methods for the milk sample is available in this report. the infant's breastfeeding status was not specified in the report, but it is presumed that he was at least partially breastfed as the mother was producing milk at 6-month post-partum. d study presented data from three women but only presented data on the milk produced by two women. e study presented data from 19 women but only presented data on the milk produced by 10 women. f study presented data from 13 women but only presented data on the milk produced by three women. while the previous reports focused on infected women, there are also three case studies focused on infected infants. in these studies, milk produced by the infants' mothers was tested for sars-cov-2. the youngest of these infants was reported by cui et al. (2020) . after being exposed to infected family members, the 55-day-old female was admitted to the hospital with symptoms of covid-19 and diagnosed based on clinical data and exposure history. the infant was 'mixed fed'. her mother's milk was collected on the first three consecutive days of her hospitalization; all were negative for sars-cov-2. no information on the collection or testing methods for the milk sample is included in this report. yuehua et al. (2020) reported on a 3-month-old, breastfed female who was hospitalized and tested via throat swab for sars-cov-2; the swab was positive. a single milk sample was collected from the infant's mother; it tested negative. the authors provided no information on the collection or testing methods for the milk. importantly, this infant developed symptoms of covid-19 7 days before her parents became ill. as such, one possibility is that she was infected first and passed the infection to them. another case report on a mature milk sample comes from singapore (kam et al., 2020) . this report is particularly interesting as the infant had no symptoms but was hospitalized and tested because his caregivers were all hospitalized with covid-19, and there was no one to care for him. the infant was 6 months old and presumably at least partially human milk fed as a sample of milk was successfully collected from his mother. despite being asymptomatic, a nasopharyngeal swab taken from the infant was positive for sars-cov-2. the authors reported that milk produced by the mother on a single day tested negative for the virus but did not specify how many samples were taken. this report provided no data on the methods used for the collection and analysis of these sample(s). cov and sars-cov-2, there remains much to be learned about their modes of transmission. respiratory droplets are a documented source of the virus (world health organization, 2020b), but other sources such as breastfeeding and/or human milk may exist. the primary purpose of this review was to examine the evidence (or lack, thereof) for the vertical transmission of sars-cov-2 from mother to infant via breastfeeding considering what is known about other human coronaviruses. we also examined the evidence presented in the same reports related to maternal/infant antibody production to the virus. in total, we identified 14 studies that had tested human milk for human coronaviruses directly. thirteen of these studies were newly published reports on sars-cov-2 and human milk, which collectively encompassed 48 milk samples. all but one of these samples tested negative for sars-cov-2, and that result was reported in a non-peerreviewed, online preprint. we identified no comparable data for mers; a single case report for sars, which yielded a negative result for the presence of the virus but positive results for antibodies specific to sars-cov; and no reports of human milk tested for other human coronaviruses. there was one report of antibody tests in milk specific to sars-cov-2, which identified igg but not igm . the single milk sample that was supposedly positive for sars-cov-2 (wu et al., 2020) is clearly anomalous to the larger body of evidence. importantly, since no methodologies were provided and the study has not been published in a refereed journal, any interpretation of this result must be made with extreme caution. this underscores the fact that it is critical for the research community to focus its efforts on analysing appropriately collected human milk using laboratory methods that have been validated and optimized for the human milk matrix. in addition, results should be submitted to high-quality, refereed journals for peer review. only then can methods and results be rigorously assessed by scientists with the knowledge base needed to judge them. the dearth of high-quality evidence substantially compromises the ability to effectively respond to this pandemic and provide guidance to some of the most vulnerable individuals: pregnant and lactating women and infants. limited and weak data suggest mers may be present in camel milk, but the relevance to sars-cov-2 in human milk is unclear. notably, reusken et al. (2014) reported that milk analysed in the camelid studies was not collected aseptically; rather, samples were obtained according to local milking customs. as such, it is possible that the presence of mers-cov in camel milk could be due to contamination from the milker, the calf or the environment, rather than milk representing an endogenous source of the virus. this is likely an issue with all the studies on sars-cov-2, where only one reported cleaning of the breast prior to sample collection (wu et al., 2020) . however, the limited data available on all three of these viruses (and human coronaviruses, in general) leave many questions unanswered with respect to the role, if any, of human milk in vertical transmission of coronaviruses. one possible reason that most of the rt-pcr results for the milk samples tested were negative is that the methods used were neither designed nor validated for human milk. milk is a complex matrix containing substantial fat, dnases (babina, kanyshkova, buneva, & nevinsky, 2004) , rnases (das, padhy, koshy, sirsat, & rich, 1976; mccormick, larson, & rich, 1974; ramaswamy, swamy, & das, 1993) and other pcr inhibitors (abu al-soud, jonsson, & radstrom, 2000; al-soud & radstrom, 2001; schrader, schielke, ellerbroek, & johne, 2012) . of note is the fact that commonly used silica columnbased rna isolation methods are designed for a limited sample volume, and as such are not suitable for more voluminous liquid samples. thus, validation of methods using human milk is needed (see box 1). in addition, other than general statements about the timing of collection (e.g., 'milk was collected after the first lactation') and brief descriptions of the rt-pcr assays used for nasal and throat swabs, none of the studies to date has described the methods of collection or how the milk was handled and stored in any detail. in addition, nothing is known about stability of sars-cov-2, if present, in human milk and how quickly (or at what temperature) it must be frozen to preserve fidelity. information on sample collection, handling and storage is critical to evaluating whether the negative results described in these studies could be due to inadequate methods used. some factors to consider when validating methods for human milk testing of coronaviruses. • method of milk collection: use of manual milk expression versus electric pump, cleaning procedures of breast and pump, partial versus full breast expression and foremilk versus hindmilk. • sample handling and storage: container material, temperature and duration of refrigeration/freezing. • assay validation: nucleic acid extraction protocols, amplification protocols, reagent selection, proper positive and negative controls and fresh versus frozen milk. • viral quantification and viability: infectious dose and biologically relevant concentrations. another possibility is that there is low abundance of the virus in human milk, and it is often not captured in the limited samples tested so far. for example, in the report on other human coronaviruses by gagneur et al. (2008) , 159 maternal-infant dyads were tested (including 161 infants, two sets of twins). in this report, 229e was present in both maternal and infant samples in only two dyads. additionally, in the milk of dromedary camels, mers-cov appears to be present at very low abundance (reusken et al., 2014) . this suggests the possibility that a very low viral load in milk might also lead to an inflation of false negatives. limited evidence from two patients suggests that sars-cov-2 shedding in respiratory samples peaks at 6 days after onset of symptoms (pan, zhang, yang, poon, & wang, 2020) , indicating that timing of sample collection also plays an important role in virus detection. only one study has investigated antibodies in milk specific to sars-cov-2 non-peer-reviewed preprint) , and these researchers identified igg in one milk sample produced by a woman at 13-month post-partum. although limited to a single study, this finding combined with a large body of literature documenting targeted antibodies in human milk indicates that there may be a protective effect of breastfeeding when the mother is covid-19 positive. the infant in this study was older than all the other infants described here, was likely not exclusively breastfed (based on reported age) and likely had a more mature immune system than the youngest infants described in other reports. still, further investigation into this finding is a critical next step in understanding how breastfeeding and/or the infant's con(robertson et al., 2004) . together, these observations suggest that infant infection may occur in utero but that the virus may simply be absent from the upper respiratory tract immediately after birth and therefore undetectable on pharyngeal swabs. very recent work has demonstrated that, like sars-cov and human coronavirus nl63 (hofmann et al., 2005) , angiotensinconverting enzyme 2 (ace2) is one of the receptors used by sars-cov-2 to enter host cells (w. h. li et al., 2003; yan et al., 2020) . ace2 is expressed across many body sites and tissue types, including the oral cavity (e.g., tongue and oral mucosa) and in mammary tissue . if mammary epithelial cells express this receptor, then it follows that viable virus could exist in milk. if it does, then the introduction of virus-containing human milk could represent a mechanism of entry for sars-cov-2 and covid-19 infection for infants. another observation worth considering is that, in at least one of the reports (yuehua et al., 2020) , the infant was infected and symptomatic 7 days prior to the infant's parents. this suggests the possibility that a 'reverse' vertical transmission from infant to mother could occur, a phenomenon that has been observed for other pathogens, such as hiv (belitsky, 1989; little et al., 2012) and ebola virus (sissoko et al., 2016) . one possible mechanism for maternal infection in this case is through retrograde flow, where milk and saliva move back into the mammary gland from the infant's mouth during suckling (ramsay, kent, owens, & hartmann, 2004) . although this mechanism is speculative, it represents a possible route whereby an infant could theoretically transfer a pathogen it has encountered in the environment to the mother. it is also possible that maternal infection could occur through other mechanisms, such as infant respiratory droplets (world health organization, 2020a) or via faecal matter . to date, all reports on sars-cov-2 and human milk have originated in asia (china and singapore) or europe (italy). although this limited geography makes sense given the fact that the initial epicentre of this pandemic was in asia and this was followed by a large outbreak throughout italy, studies from other globally representative populations are needed to make definitive conclusions regarding the possible presence and/or role of sars-cov-2 in human milk. additionally, the importance of a coordinated, international effort by scientists, clinicians and public health officials to elucidate answers to the many remaining questions related to sars-cov-2 and breastfeeding cannot be overemphasized. human milk is the gold standard for infant feeding. however, confidence as to its safety and best practices around breastfeeding during maternal covid-19 infection has been compromised by the lack of rigorous evidence as to whether sars-cov-2 can be vertically transmitted in milk and/or during breastfeeding. as such, there exists an immediate need to rapidly generate rigorous evidence for the role substantial interdisciplinary research on this topic is required and should be performed rigorously and rapidly to best inform policies regarding early feeding choices and clinical management of breastfeeding mothers infected with sars-cov-2 and their infants. to understand the role of human milk and sars-cov-2 infection, the following points must be rapidly addressed. • optimization of human milk collection and storage protocols for sars-cov-2 research. • validation of assays for identification of sars-cov-2 rna and sars-cov-2-specific immune components in human milk. • multinational population studies documenting presence or absence of sars-cov-2 virus and immune factors (including antibodies) in milk produced by infected women, women with infected infants and women who have been exposed to sars-cov-2; if the virus is identified in milk, its viability must be verified. • multinational population studies documenting (or not documenting) risk of covid-19 infections in breastfed versus nonbreastfed infants whose mothers are covid-19 positive. • research delineating implications of skin-to-skin breastfeeding versus consumption of pumped human milk. identification and characterization of immunoglobulin g in blood as a major inhibitor of diagnostic pcr purification and characterization of pcr-inhibitory components in blood cells policy statement: breastfeeding and the use of human milk initial guidance: management of infants born to mothers with covid-19 lactoferrin is the major deoxyribonuclease of human milk children infect mothers in aids outbreak at a soviet hospital transmission of hiv-1 in the breast-feeding process human t-lymphotropic virus type i and breastfeeding; systematic review and meta-analysis of the literature cytomegalovirus (cmv) and congenital cmv infection full breastfeeding duration and associated decrease in respiratory tract infection in us children clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases method of feeding and transmission of hiv-1 from mothers to children by 15 months of age: prospective cohort study from durban a 55-day-old female infant infected with 2019 novel coronavirus disease: presenting with pneumonia, liver injury, and heart damage human milk samples from different ethnic-groups contain rnase that inhibits, and plasma-membrane that stimulates, reverse transcription maternal and breastmilk viral load: impacts of adherence on peripartum hiv infections averted-the breastfeeding, antiretrovirals, and nutrition study possible vertical transmission of sars-cov-2 from an infected mother to her newborn prolonged and exclusive breastfeeding reduces the risk of infectious diseases in infancy risk of human immunodeficiency virus type 1 transmission through breastfeeding. the lancet cytomegalovirus infection of breast milk and transmission in infancy perinatal transmission of covid-19 associated sars-cov-2: should we worry? severe postnatally acquired cytomegalovirus infection presenting with colitis, pneumonitis and sepsis-like syndrome in an extremely low birthweight infant materno-fetal transmission of human coronaviruses: a prospective pilot study postnatal cytomegalovirus infection through human milk in preterm infants: transmission, clinical presentation, and prevention dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov). transboundary and emerging diseases human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry early exclusive breastfeeding reduces the risk of postnatal hiv-1 transmission and increases hivfree survival mers-cov infection in a pregnant woman in korea maternal transmission of infectious pathogens in breast milk a well infant with coronavirus disease 2019 (covid-19) with high viral load identification of coronavirus isolated from a patient in korea with covid-19. osong public health and research perspectives breast milk-acquired cytomegalovirus infection and disease in vlbw and premature infants breast milk and infection angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus lack of vertical transmission of severe acute respiratory syndrome coronavirus 2, china a review of evidence for transmission of hiv from children to breastfeeding women and implications for prevention coronavirus disease 2019 (covid-19) during pregnancy: a case series clinical characteristics of 19 neonates born to mothers with covid-19 coronavirus covid-19 has killed more people than sars and mers combined, despite lower case fatality rate rnase inhibition of reverse-transcriptase activity in human milk human infection with mers coronavirus after exposure to infected camels, saudi arabia role of breast-milk in acquisition of cytomegalovirus-infection mother-to-child transmission of human t-cell lymphotropic virus type 1 effect of breastfeeding on mortality among hiv-1 infected women: a randomized trial viral load of sars-cov-2 in clinical samples. the lancet infectious diseases purification and characterization of a high-molecular-weight ribonuclease from human-milk ultrasound imaging of milk ejection in the breast of lactating women middle east respiratory syndrome coronavirus (mers-cov) rna and neutralizing antibodies in milk collected according to local customs from dromedary camels sars and pregnancy: a case report mother-to-child htlv-1 transmission: unmet research needs managing covid-19-positive maternal-infant dyads: an italian experience pcr inhibitors-occurrence, properties and removal potential maternal and infant outcomes from coronavirus 2019-ncov (sars-cov-2) infecting pregnant women: lessons from sars, mers, and other human coronavirus infections human immunodeficiency virus load in breast milk, mastitis, and mother-to-child transmission of human immunodeficiency virus type 1. the journal of infectious diseases ebola virus persistence in breast milk after no reported illness: a likely source of virus transmission from mother to child working parents-the impact of day-care and breast-feeding on cytomegalovirus infections in offspring sars during pregnancy, united states. emerging infectious diseases cultivation of a novel type of commoncold virus in organ cultures stability of middle east respiratory syndrome coronavirus in milk a case report of neonatal covid-19 infection in china breastmilk rna viral load in hiv-infected south african women: effects of subclinical mastitis and infant feeding guideline: updates on hiv and infant feeding. the duration of breastfeeding, and support from health services to improve feeding practices among mothers living with hiv who mers-cov global summary and assessment of risk clinical-management-of-severe-acuterespiratory-infection-when-novel-coronavirus-(ncov)-infection-issuspected world health organization. (2020b). modes of transmission of virus causing covid-19: implications for ipc precaution recommendations viral shedding of covid-19 in pregnant women high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding structural basis for the recognition of sars-cov-2 by full-length human ace2 breast milk-fed infant of covid-19 pneumonia mother: a case report a case of three-month-old infant with new coronavirus infection antibodies in infants born to mothers with covid-19 pneumonia postnatal transmission of aids-associated retrovirus from mother to infant sars-cov-2 and human milk: what is the evidence? matern child nutr the authors acknowledge funding for other covid-19 and human milk related projects, including the generous gift by the family larsson-rosenquist foundation (flrf) in support of uc san diego's momi core; the bill & melinda gates foundation; the national instikey: cord-340836-eb5a9ln3 authors: aghazadeh-attari, javad; mohebbi, iraj; mansorian, behnam; ahmadzadeh, jamal; mirza-aghazadeh-attari, mohammad; mobaraki, kazhal; oshnouei, sima title: epidemiological factors and worldwide pattern of middle east respiratory syndrome coronavirus from 2013 to 2016 date: 2018-04-06 journal: int j gen med doi: 10.2147/ijgm.s160741 sha: doc_id: 340836 cord_uid: eb5a9ln3 background: middle east respiratory syndrome coronavirus (mers-cov) is an emerging threat to global health security with high intensity and lethality. this study was conducted to investigate epidemiological factors and patterns related to this disease. methods: full details of mers-cov cases available on the disease outbreak news section of the world health organization official website from january 2013 to november 2016 were retrieved; demographic and clinical information, global distribution status, potential contacts, and probable risk factors for the mortality of laboratory-confirmed mers-cov cases were extracted and analyzed by following standard statistical methods. results: details of 1,094 laboratory-confirmed cases were recorded, including 421 related deaths. significant differences were observed in the presentation of the disease from year to year, and all studied parameters differed during the years under study (all p-values <0.05). evaluation of the effects of various potential risk factors of the final outcome (dead/survived) revealed that two factors, namely, the morbid case being native and travel history, are significant based on a unifactorial analysis (p <0.05). from 2013 to 2016, these factors remained important. however, factors that were significant in predicting mortality varied in different years. conclusion: these findings point to interesting potential dimensions in the dynamic of this disease. furthermore, effective national and international preparedness plans and actions are essential to prevent, control, and predict such viral outbreaks; improve patient management; and ensure global health security. middle east respiratory syndrome coronavirus (mers-cov) is an emerging threat to global health security. this infection is caused by a zoonotic single-stranded, positivesense rna virus of the β-coronavirus family that causes epidemics or even pandemics with substantial morbidity and mortality. 1, 2 this emerging disease was first detected in september 2012 in a patient with fatal pneumonia in jeddah, saudi arabia, a country where a large number of muslims from all over the world gather to attend the hajj pilgrimage (one of the largest islamic rituals ) each year. 2, 3 following the initial case, some cases in europe, africa, south east asia, the united states, and several arabian countries were reported to be sporadic. the majority of infected patients (85%) are from saudi arabia. world health organization (who) travel advisory has not issued travel and trade restrictions or screening in saudi arabia. however, the health ministry of saudi arabia recommends aghazadeh-attari et al pilgrims over 65 years; those with chronic diseases, such as heart, kidney, and respiratory diseases, diabetes, and immune deficiency; pregnant women; and children under 12 years, who are planning to attend the hajj, to postpone their visit. 4, 5 the case fatality rate (cfr) of mers-cov differed but remained high 6 : 36%, 7,8 40%, and 67%. 9 incubation period varied from 2 to 14 days. 2,10-12 as a result, who and the us centers for disease control and prevention have recommended that individuals who are returning from the arabian peninsula and other affected countries should be monitored for mers-cov infection for at least 14 days. 7 several studies indicate that health care staff, including physicians, nurses, and laboratory personnel, has a higher risk of contracting, developing, and transmitting this infection. for this reason, the health ministry of saudi arabia developed and implemented standard guidelines for the prevention, infection control, and rapid response to mers-cov outbreaks according to the who recommendations. 6, 10, 13 the transmission mechanisms of mers-cov are still unknown. however, previous studies suggest that humanto-human transmission through sneezing, coughing, contact with contaminated items, and consumption of raw camel milk may play a major role in the transmission of infection. 16 the risk factors of mers-cov are not fully understood, and a definite risk factor in the initial human-to-human or animal-to-human transmission has not been confirmed by epidemiological studies yet. 6 globally, awareness of mers-cov is low. the disease has high intensity and lethality with unknown epidemiological factor and pattern. every year, millions of muslims travel to the epicenter (saudi arabia) of this infection to attend hajj. upon returning home, pilgrims hold ceremonies, which are attended by family and friends. long-standing traditions of sharing and hospitality on such occasions increase the likelihood of mers-cov transmission to others. therefore, unknown epidemiological patterns and the probable risk factors of mers-cov should be investigated to prevent its spread and devise effective interventions. authorization from who and the ethical committee of urmia university of medical sciences authorities was obtained. by census method, data related to laboratory-confirmed mers-cov cases between september 23, 2012, and november 11, 2016, were extracted from the disease outbreak news on mers-cov section of the who website. from september 23, 2012, to november 11, 2016, the occurrence of 1,879 laboratory-confirmed cases of mers-cov infection, including 659 deaths, was reported to who by the national ihr focal points of 27 countries in europe, north africa, the middle east, the united states of america, and asia. due to the small number of cases in 2012 (n=9), to avoid random bias, we merged 2012 occurred cases with 2013. details for 1,094 laboratory-confirmed cases of infection with mers, including 421 related deaths, were recorded in the disease outbreak news section of the who website for this time. extracted data comprised demographic information, such as age, gender, nationality; and country of origin; clinical data on the year of morbidity, day/month of the onset of symptom; day/month of admission to the hospital and icu or negative pressure isolation room; potentially hazardous contacts, including background of contact with morbid cases at home or hospital and health care facilities 1-14 days prior to the onset of symptoms and background of direct or indirect contact with a camel; and consumption of raw camel products. other probable risk factors included travel history, initial or secondary case, and comorbidity. the authors would like to confirm that all data of mers-cov cases retrieved on the who website were anonymous or de-identified. statistical analysis was performed using spss version 20. quantitative and qualitative measures were expressed as absolute frequencies and percentages. logistic regression was used to assess the potential relationship between probable risk factors and the final outcome (dead/survived) of the laboratory-confirmed mers-cov cases. p-values <0.05 were considered statistically significant, and for the model, a value of 0.1 was considered significant. table 1 shows the characteristics of laboratory-confirmed cases of infection with mers. in this table, we see that the cases of the disease are increasing year by year, and all studied parameters related to the cases differ for the years under study (all p-values <0.05). table 2 displays and compare the effects of various potential risk factors on the final outcome (dead/survived) of laboratory-confirmed mers-cov cases worldwide. this table shows that among the possible variables under consideration in this study, some factors such as nativity and travel history (p=0.011 and p=0.038, respectively) that are important in predicting the disease mortality have been identified. there was no significant difference between the two groups (dead/ survived) of laboratory-confirmed mers-cov in aspects of other probable factors investigated in this study. table 3 presents that the effects of probable risk factors on the mortality of laboratory-confirmed mers-cov cases this report stated that the chance of death due to mers-cov infection in native morbid case was 1.60 times higher than non-native cases. there were statistically significant differences between an infected individual who had travel history and in those who had not, for dying from mers-cov infection. the chance of death due to mers-cov infection in an individual who had travel history was 1.68 (95% ci: 1.04-2.73) times higher than those who had not. the occurrence of a large number of mers-cov cases and its death related in the world indicates that this disease must be considered as a threat to public health. results of the present study show the outbreaks of mers-cov infection in recent years. because of the occurrence of a large number of mers-cov cases in the world and deaths related to it, this disease was considered as a threat to public health. 14 our results support the high fatality of this disease, and the cfr (38.5%) of mers-cov infection should be a major health concern at the global scale; thus, the characteristics of this disease and potential factors contributing to its fatality should be comprehensively studied to effectively know its health risk. evidence of person-to-person transmission (particularly during close contact) has already been emphasized in the study by perlman and mccray. 15 in our study, of 1,094 cases, 38.5% involved close contact with laboratory-confirmed cases of infection with mers-cov 14 days prior to the onset of symptoms. this finding highlights the notion that early cases have enormous potential for disseminating mers-cov among health care workers and other community members. furthermore, who recommendations on surveillance and control should be practiced with great vigilance, and hospitals must utilize negative pressure isolation rooms for morbid cases of mers-cov. the analysis of the global distribution status of mers-cov in the world suggests that outbreaks of this disease emanate primarily from saudi arabia and coincides with the largest annual mass gathering of muslims from around the world in this country to perform hajj and umrah rituals. 2, [14] [15] [16] [17] in addition, the tradition of consuming raw camel milk is observed in arab countries with high occurrence rates of mers-cov infection; several studies have also addressed this point. 16, 17 as demonstrated in this study (table 1) , the presentation of the disease from year to year has a significant difference, and all studied parameters related to the cases differ for the years under study (all p-values are <0.05). these parameters include demographic factors, such as gender, mean age, nationality of the patients, and salient characteristics, and features related to the disease, i.e., travel history, background of contact with camel or camel milk 14 days prior to the onset of symptoms and contact with morbid case at home or hospital 14 days prior to the onset of symptoms, admission to a hospital, need for admission to a negative pressure isolation room or icu, and the final outcome during or after occurrence (dead/survived). in table 1 we also see that in all years, the proportion of males was higher than in females, more of them had the saudi nationality, and more number of mers-cov cases occurred approximately at age near and up to 50 years. one of the reason for this is the people near and up to 50 years of age are at a greater risk of complications resulting from mers-cov or viral infections than other people in low and middle ages. on the other hand, men in comparison to women are likely to spend more time outdoors and therefore have a higher risk of exposure to a source of infection. the comparison of characteristics of the cases and the effect of various potential risk factors on the final outcome (dead/survived) of laboratory-confirmed mers-cov cases in the world (table 2) reveal that two factors, namely, morbid case being native and travel history, are considered significant in a unifactorial analysis (p-values are <0.05) and with the potential of bearing on the dynamics of the disease. as we follow the dynamics of the disease from 2013 to 2016 (table 3) , these factors remain important. however, some interesting factors such as nativity and travel history that are significant in predicting mortality varied in different years. with attention to this fact, the majority of mers-cov cases that occurred outside of the middle east had travel history to countries in or near the arabian peninsula within 14 days before symptom onset. therefore, this can be an evidence for the human-tohuman transmission of mers-cov infection. for nativity, there is a need for further studies on genetic features of the culprit virus. some limitations were recognized in our study. first, the design of the study was cross-sectional so that no causal inferences could be made. further prospective studies are necessary to explain the effects of all potential risk factors investigated in this study for the outcome (dead/survived) in individuals with laboratory-confirmed mers-cov the international journal of general medicine is an international, peer-reviewed open-access journal that focuses on general and internal medicine, pathogenesis, epidemiology, diagnosis, monitoring and treatment protocols. the journal is characterized by the rapid reporting of reviews, original research and clinical studies across all disease areas. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. 125 epidemiological factors of mers-cov infection. second, possible misclassification in the categorization of cases may be due to the respondents, declaration such as background of contact with morbid case at home or hospital 14 days prior to the onset of symptoms household, comorbidity, and background of contact with camel or camel milk 14 days prior to the onset of symptoms, which potentially may occur as a result of the measurement bias. third, it should be mentioned that from 186 mers-cov cases occurred in republic of south korea, only details related to 57 cases were published in the disease outbreak news on the who website. this might introduce a negligible selection bias in the results. in summary, these findings point to interesting and potential dimensions of the dynamic evolution of this disease, and the need for further studies on genetic features of the culprit virus and the epidemiological parameters and risk factors of mers-cov mortality is also emphasized. furthermore, effective national and international preparedness plans and actions are essential to prevent, control, and predict such viral outbreaks; improve patient management; and ensure global health security. ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus hadj ritual and risk of a pandemic transmission of mers-coronavirus in household contacts fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea the knowledge and attitude about hiv/aids among jordanian dental students: (clinical versus pre clinical students) at the university of jordan risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia middle east respiratory syndrome coronavirus: a comprehensive review middle east respiratory syndrome and severe acute respiratory syndrome transmission of middle east respiratory syndrome coronavirus infections in healthcare settings epidemiologic parameters of the middle east respiratory syndrome outbreak in korea abbas zadeh bazzi m. evaluation the effect of health education on knowledge, attitude and practices of zabol's women barbers about aids in 2008 middle east respiratory syndrome: a new global threat middle east respiratory syndrome coronavirus during pregnancy mers-cov scenario and modeling working group. estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia person-to-person spread of the mers coronavirus-an evolving picture evidence for camel-to-human transmission of mers coronavirus evidence for camel-to-human transmission of mers coronavirus this work was funded by the urmia university of medical sciences (grant number 2122). this research is dedicated to dr. m. m. dilar for her sincerity and honesty and for her endeavor in motivating us to do the research. the authors would like to thank rana sidani, senior communication officer in the who regional office for the eastern mediterranean (cairo, egypt) for guidance and help. all authors contributed toward data analysis, drafting and critically revising the paper, and agreed to be accountable for all aspects of the work. the authors report no conflicts of interest in this work. key: cord-344330-zsx7wfyj authors: su, shuo; wong, gary; shi, weifeng; liu, jun; lai, alexander c.k.; zhou, jiyong; liu, wenjun; bi, yuhai; gao, george f. title: epidemiology, genetic recombination, and pathogenesis of coronaviruses date: 2016-03-21 journal: trends microbiol doi: 10.1016/j.tim.2016.03.003 sha: doc_id: 344330 cord_uid: zsx7wfyj human coronaviruses (hcovs) were first described in the 1960s for patients with the common cold. since then, more hcovs have been discovered, including those that cause severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), two pathogens that, upon infection, can cause fatal respiratory disease in humans. it was recently discovered that dromedary camels in saudi arabia harbor three different hcov species, including a dominant mers hcov lineage that was responsible for the outbreaks in the middle east and south korea during 2015. in this review we aim to compare and contrast the different hcovs with regard to epidemiology and pathogenesis, in addition to the virus evolution and recombination events which have, on occasion, resulted in outbreaks amongst humans. gastrointestinal symptoms. for instance, transmissible gastroenteritis coronavirus (tgev) [9, 10] , bovine coronavirus (bcv) [11] , feline coronavirus (fcov) [12] , canine coronavirus (ccov) [13] , and turkey coronavirus (tcv) [14] are known to cause enteritis in their respective hosts [1] . in humans, cov infections primarily involve the upper respiratory tract and the gastrointestinal tract, and vary from mild, self-limiting disease, such as the common cold, to more severe manifestations, such as bronchitis and pneumonia with renal involvement [15] . the first human coronavirus (hcov) was isolated during 1965 from the nasal discharge of patients with the common cold and termed b814 [16] . currently, six different cov strains are known to infect humans. these include: hcov-229e (229e), hcov-oc43 (oc43), severe acute respiratory syndrome coronavirus (sars-cov), hcov-nl63 (nl63), hcov-hku1 (hku1), and middle east respiratory syndrome coronavirus (mers-cov) [17] . 229e and oc43 are the prototype viruses from the two main hcov lineages (alpha and beta, respectively) that cause 15-29% of all common colds, and are the best characterized [18] . sars-cov is the aetiological agent that was behind an outbreak of severe respiratory disease through china during 2002-2003 [19] , and mers-cov is the pathogen responsible for an ongoing outbreak of severe respiratory disease centered in the middle east since 2012 [20] . in this review, we first compare and contrast the epidemiology and pathogenesis of the known covs infecting humans, in which covs that cause mild to severe disease in humans are newly emerged from a zoonotic source. second, we describe the ecology of covs, and highlight evidence for viral recombination between the different covs within animal populations, which may result in the generation of novel covs that are transmissible and lethal to humans. finally, we summarize the phylogeny, evolution, and genetic recombination of covs in detail. the epidemiology and pathogenesis of each hcov are discussed within the following sections and summarized in table 1 and figure 1 , respectively. in general, 229e, oc43, and nl63 are distributed globally ( figure 1a ) and tend to be transmitted predominantly during the winter season in temperate-climate countries [21] , while nl63 showed a spring-summer peak of activity from a study in hong kong [22] . human coronavirus 229e (alphacoronavirus) strain 229e was discovered during 1966, when researchers were characterizing five novel agents that were isolated from the respiratory tract of humans who had contracted the common cold [23] . 229e was adapted to grow in wi-38 lung fibroblast cell lines, and was later shown to be morphologically identical to ibv and mhv [24] . symptoms of 229e infection include general malaise, headache, nasal discharge, sneezing, and a sore throat [25] . a small portion of patients (10-20%) will also exhibit fever and cough. the incubation time is approximately 2-5 days, followed by illness lasting between 2 and 18 days, and clinically indistinguishable from respiratory tract infections caused by other pathogens, such as rhinovirus and influenza a [18] . 229e is distributed globally ( figure 1a ). 5 to 7 days later, which may result in death (http://www.who.int/csr/sars/en/whoconsensus. pdf?ua=1). other notable features in some cases include infection of the gastrointestinal tract, liver, kidney, and brain. diffuse alveolar damage, epithelial cell proliferation, and an increase in macrophages is seen in sars-cov infection of the lung. lymphopenia, hemophagocytosis in the lung, in addition to white-pulp atrophy of the spleen observed in sars patients, are similar to fatal h5n1 influenza virus infections [1] . diarrhea is observed in approximately 30-40% of sars infections (http://www.cdc.gov/sars/about/faq.html). figure 1b ). an eventual 8273 cases were reported, with 775 deaths for a case fatality rate (cfr) of 9%, and the majority of cases and deaths occurred in mainland china and in hong kong [19] . the elderly were more susceptible to sars disease, with a mortality rate of over 50% (http://www.cdc.gov/sars/surveillance/absence.html). key: human coronavirus nl63 (alphacoronavirus) nl63 is primarily associated with young children, the elderly, and immunocompromised patients with respiratory illnesses [26] . nl63 was a novel hcov isolated from a 7-month-old child with coryza, conjunctivitis, fever, and bronchiolitis in the netherlands during late 2004 [26] , and an independent investigation described the isolation of virtually the same virus from a nasal sample collected from an 8-month-old boy suffering from pneumonia in the netherlands [27] . nl63 was also detected in new haven, usa, during 2005 amongst 79 of 895 children, and was initially called hcov-nh (nh), but genetic and phylogenetic analyses showed that nh and hku1 likely are the same species [26, 28] . infections with nl63 typically result in mild respiratory disease similar to the common cold, characterized by cough, rhinorrhea, tachypnea, fever, and hypoxia, and resolve on their own [29] . obstructive laryngitis, also known as croup, is frequently observed with nl63 infections. a study estimated that nl63 accounts for an estimated 4.7% of common respiratory diseases [26] . nl63 is distributed globally ( figure 1a ). human coronavirus hku1 (betacoronavirus, lineage a) hku1 was first discovered during january 2005 in a 71-year-old patient from hong kong who had been hospitalized with pneumonia and bronchiolitis [30] . the symptoms of hku1 respiratory tract infections are not able to be separated from those caused by other respiratory viruses. most patients present with fever, running nose, and cough for infections in the upper respiratory tract, whereas a fever, productive cough, and dyspnea are the common symptoms presenting for infections in the lower respiratory tract [31] . most hku1 infections are self-limiting, with only two deaths reported in patients with pneumonia due to hku1 [32] . although hku1 is clinically relatively mild in children, hku1 infection is associated with a high incidence of seizures, and was found in a patient with meningitis [33, 34] . hku1 cases outside asia were detected in new haven, usa, in 2 out of 851 children [35] , and also in australia [36] , france [37] , and brazil [38] , indicating a global distribution for the virus ( figure 1a ). mers-cov was first isolated from the lungs of a 60-year-old patient who had died from a severe respiratory illness in jeddah, saudi arabia, 2012 [39] . clinical manifestations of mers-cov infection range from asymptomatic to severe pneumonia with acute respiratory distress, septic shock, and renal failure resulting in death [40] . a typical disease course begins with fever, cough, chills, sore throat, myalgia, and arthralgia, followed by dyspnea and rapid progression to pneumonia [40] [41] [42] [43] . approximately one-third of patients present with gastrointestinal symptoms, such as diarrhea and vomiting. acute renal impairment was the most striking feature of disease caused by mers-cov, which is thus far unique for human cov infections [40, 44] . seventy-five percent of patients with mers disease also had at least one other comorbidity, and patients who died were more likely to have a pre-existing/underlying condition [40] . countries around the arabian peninsula are known to be endemic for mers-cov, and saudi arabia has reported the most cases, but since its discovery in 2012, cases have been occasionally exported to other countries through travel, sometimes causing clusters of secondary outbreaks ( figure 1b ) [45] . as of december 31, 2015, a total of 1621 laboratory-confirmed infections have been reported, with 584 deaths (cfr = 36.0%) over 26 countries (http://www.who.int/csr/ disease/coronavirus_infections/en), making mers-cov one of the most dangerous viruses known to humans. based on currently available evidence, 229e, oc43, nl63, and hku1 are well adapted to humans, and the viruses widely circulate in the human population ( figure 2) , with most cases causing mild disease in immunocompetent adults, and none of these viruses have been found to be maintained within an animal reservoir. however, in the case of sars-cov and mers-cov, the viruses are not as well adapted to being maintained in humans, and thus are likely spread mainly in zoonotic reservoir(s), with occasional spillover into the susceptible human population, possibly via an intermediate host species. regarding sars-cov, epidemiology data implicated masked palm civets (paguma larvata) from live animal markets (lam) in guangdong province, china, as a route of exposure to sars-cov [46] . however, masked palm civets from the wild or from farms without lam exposure were largely negative for sars-cov [47] . this suggests that palm civets were an intermediate host, but not a reservoir for sars-cov [48] . subsequent studies have shown that wild horseshoe bats (rhinolophidae family), which can also be found in lam in china and served in some chinese restaurants in guangdong, china, have detectable levels of antibodies against sars-cov and a sars-cov-like virus (sarsr-rh-bat cov) [49, 50] , suggesting a bat origin for sars-cov. an evolutionary relationship between coronaviruses and bats was proposed, in which the ancestor for sars-cov first spread to bats of the hipposideridae family, then rhinolophidae, then masked palm civets and eventually humans [51] . recently, two new sars-like covs were isolated in horseshoe bats, and these viruses showed the highest relationship to sars-cov from all known bat coronaviruses [52] . orf8 analysis of the sars-like covs in bats suggests that chinese horseshoe bats are the natural reservoirs of sars-cov [52, 53] , and that intermediate hosts might not be needed for direct human infection, particularly for some bat sars-cov-like viruses [52] . in the case of mers-cov, studies in oman, saudi arabia, qatar, united arab emirates, and jordan have shown that dromedary camels are seropositive for neutralizing antibodies against mers-cov [54] , as well as camels of middle east origin in africa, including egypt, kenya, nigeria, ethiopia, tunisia, somalia, and sudan [55] . subsequent studies on dromedary camels in saudi arabia [56] , qatar [57] , and egypt [58] show that live mers-cov can be isolated primarily from the nasal swabs of camels, showing that camels are potential source of mers-cov infection. however, many confirmed cases lack contact history with camels [59] , suggesting direct human-to-human transmission, or through contact with a yet-to-be-identified animal species that is maintaining mers-cov. studies on hku4, a coronavirus of bat origin and the most phylogenically closely related to mers-cov, had shown that hku4 is able to utilize the cd26 receptor for virus entry [60] . as cd26 is a known receptor for mers-cov [61] , the similarity in receptor specificity of these two covs supports the hypothesis that mers-covs is also a bat-originated cov. however, live mers-cov has not been isolated from wild bats, nor have viral sequences been detected. a recent investigation discovered that multiple hcov species, including mers-cov, beta-cov group a, and a 229e-like virus, circulate amongst dromedary camels in saudi arabia [62] . furthermore, multiple lineages of circulating mers-cov in these camels had resulted in a dominant, recombinant mers-cov strain that was responsible for the mers-cov outbreaks during 2015 [62] . these results show that various covs of human and animal origin are currently circulating in the wild, providing ample opportunity for covs to undergo evolution and genetic recombination, thereby resulting in recombinant covs that may potentially be more deadly to humans. in the following sections, we summarize the evolution and genetic recombination of the current hcovs. the phylogenetic tree for the covs of zoonotic and human origin was constructed based on the full genome. it can be observed that 229e and nl63 belong to the alpha-cov genus and are grouped together with porcine epidemic diarrhea virus (pedv), tgev, porcine respiratory coronavirus (prcv), feline infectious peritonitis virus (fipv), and some bat-derived coronaviruses ( figure 3 ). the topology of the phylogeny is similar to the tree constructed based on the rnadependent rna polymerase gene [55] . oc43, hku1, sars-cov, and mers-cov belong to the beta-cov genus. lineage a includes oc43 and hku1, mhv, porcine hemagglutinating encephalomyelitis virus (phev), in addition to equine, rabbit, camel, bovine, antelope-derived animal coronaviruses. lineage b includes sars-cov, sars-like viruses of bat and palm civet origin, and some bat-derived covs. lineage c includes mers-cov and some bat-derived viruses. lineage d contains only bat-derived coronavirus (figure 3) . in contrast to alpha-and beta-covs, gamma-covs consist mainly of avian coronaviruses (such as ibv), as well as covs isolated from aquatic animals, whales, and dolphins. covs of wild-bird origin are clustered into the delta-cov, which also contains some swine-derived covs (figure 3 ). in general, alpha-and beta-covs primarily include covs from mammals, whereas gamma-and delta-covs mostly include covs of avian origin. notably, all currently known hcovs belong to either the alpha-or betacoronavirus genera, which also contain covs of mainly bat origin. the estimated mutation rates in cov are moderate to high compared to other single-stranded rna (ssrna) viruses, and the average substitution rate for covs was $10 à4 substitutions per year per site [63] . the nucleotide mutation rate of ibv for the hypervariable region in the s gene was estimated to be 0.3-0.6 â 10 -2 per site per year [64] . the s gene of oc43 possessed an average rate of 6.410.58 â 10 -4 substitution rates per site per year [65] . additionally, the s gene of 229e possessed a rate of $3 â 10 à 4 substitutions per site per year [63] . for sars-cov, the mutation rate in the whole genome was estimated to be 0.80-2.38 â 1 -3 nucleotide substitutions per site per year, and the nonsynonymous and synonymous substitution rates were estimated to be 1.16-3.30 â 10 -3 and 1.67-4.67 â 10 -3 per site per year, respectively, which is similar to other rna viruses [66] . the evolutionary rate for mers-cov genomes was estimated as 1.12 â 10 à3 substitutions per site per year (95% credible interval [95% ci], 8.76 â 10 à4 ; 1.37 â 10 à3 ) [67] . furthermore, the large rna genome in cov allows for extra plasticity in genome modification by mutations and recombinations, thereby increasing the probability for intraspecies variability, interspecies 'host jump', and novel covs to emerge under the right conditions [68] [69] [70] . the major reason for recombination may be at the replication step in the virus life cycle. during replication, a set of subgenomic rnas is generated, increasing the homologous recombination rate among closely related genes from different lineages of covs or other viruses by template switching [33] . concurrently, circulating covs in multiple host species likely contribute to increases in the rate of recombination events. however, the exact mechanism of genetic recombination in covs remains unclear: recombination site 'breakpoints' in the viral genome the crossing point of the recombinant genes between two distinct viral strains or genotypesappear to be random, as different recombinant strains have different breakpoints. genetic recombination has been previously documented for animal covs, for instance: mhv [71] , tgev [72] , as well as feline and canine coronaviruses [73, 74] . genetic recombination for other hcovs including oc43, nl63, hku1, sars-cov [51] , and mers-cov have also been documented and are discussed in detail below. oc43 has diverged into five distinct genotypes (a to e). genotype a is typified by the prototype vr759 strain that was isolated in 1967 [75] . genotypes b and c are two naturally circulating viruses. genotype d emerged via recombination between genotypes b and c viruses at the 14490-14523, 15210-15238, 19206-19253, 19396-19420 15210-15253, 15417-15457, 17088-17125, 19102-19132, 20712-20747 14986-15055, 15417-15457, 17088-17125, 19102-19132, 20712-20747 17688-17714, 19548-19588 15210-15232, 18558-18577, 20797-20827 14808-14835, 15062-15093, 15624-15670, 17783-17809, 19113-19132 breakpoint of 2500-5000 bp in the nsp2-nsp3 gene and 15500 bp-3 0 end in nsp12-n genes [75] (figure 4 , key figure) . genotype e, identified in 2014, emerged by recombination amongst the genotype b, c, and d viruses with different breakpoints in the genes compared to the generation of genotype d [76] (figure 4) . a study in france, between 2001 and 2013, demonstrated that oc43 strains have more recombination patterns in lower normandy, and circulating oc43 viruses have a high genetic diversity [17] . nl63 also has the potential for genetic recombination, and one such example is between the amsterdam 1 and 496 strains. recombination site breakpoints were identified in two parts of the s gene [63] (figure 4 ). recombination events have been observed for different genotypes of hku1, in which recombination breakpoints were found on genotypes b and c in the nsp6-nsp7 and nsp16-he genes, respectively [70] (figure 4) . hku1 was also shown to be able to recombine with other animal beta-covs, such as mhv, at the nsp3, nsp4, nsp6, nsp7, nps9, and nsp10 genes [77] (figure 4 ). sars-cov has a possible recombinant history with lineages of alpha-and gamma-cov [78] . many specific breakpoints and a large number of smaller recombinant regions had been identified in the rna-dependent rna polymerase (rdrp, nsp12 gene) at the following locations: nucleotides 13 392-13 610, 15 259-15 342, and 15 974-16 108 based on the sequence of the sars-cov tor2 strain [78] . recombination locations were also identified in the nsp9 and most of nsp10 gene located at 12 613-13 344, and parts of nsp14 (18 117-18 980) [78] . another study analyzing the genome of the sars-cov cv7 strain had shown that seven putative recombination locations were found between sars-cov and six other coronaviruses: pedv, tgev, bcv, 229e, mhv, and ibv ( figure 4 ). this further suggests that sars-cov emerged via serial horizontal transmission and by genetic recombination, enhancing the adaptation process to its new host [79] . recombination was also detected between the sars-related bat-associated cov (sarsr-bat covs), in which recombination between the rp3 strain from guangxi province and rf1 strain from hubei province, china, generated a new strain (sarsr-civet cov sz3) in civets, with a breakpoint at the nsp16/spike and s2 region [51] (figure 4) . a recent study showed that the epidemic mers-cov experienced recombination events between the different lineages, which occurred in dromedary camels in saudi arabia [62] . the result from a high frequency of recombination events in covs is the generation of novel viruses with a high genetic diversity, with unpredictable changes in virulence during human infections. with multiple species of covs circulating in the wild amongst different animal species that may constantly interact with one another, it is likely not a matter of if, but when, the next recombinant cov will emerge and cause another outbreak in the human population. as such, some crucial future areas of investigation include: (i) the prevalence of hcovs already circulating within the animal population, (ii) the commonality of coronavirus recombination in animals, (iii) animals which may potentially serve as mixing vessels for the generation of novel recombinant how common is coronavirus recombination in animals? can coronavirus undergo recombination in animal hosts other than camels? is there a way to predict the potential emergence of a novel recombinant coronavirus in the human population? covs, and (iv) a surveillance network to monitor and predict the potential emergence of a highly virulent, recombinant cov from animals (see outstanding questions). furthermore, lessons from the sars-cov and mers-cov outbreaks must be urgently learned in advance to effectively prepare for the next cov outbreak. shuo su and gary wong contributed equally to this manuscript. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol coronaviridae. in fields virology the molecular biology of coronaviruses attachment of mouse hepatitis virus to o-acetylated sialic acid is mediated by hemagglutinin-esterase and not by the spike protein rotavirus and coronavirus associated diarrhoea in domestic animals coronavirus avian infectious bronchitis virus detection of a group 2 coronavirus in dogs with canine infectious respiratory disease pathogenesis of demyelination induced by a mouse hepatitis transmissible gastroenteritis of swine: virus-intestinal cell interactions. ii. electron microscopy of the epithelium in isolated jejunal loops transmissible gastroenteritis of swine: virus-intestinal cell interactions. i. immunofluorescence, histopathology and virus production in the small intestine through the course of infection replication of an enteric bovine coronavirus in intestinal organ cultures pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683 recovery and characterization of a coronavirus from military dogs with diarrhea pathogenicity of turkey coronavirus in turkeys and chickens recently discovered human coronaviruses cultivation of a novel type of common-cold virus in organ cultures genomic analysis of 15 human coronaviruses oc43 (hcov-oc43s) circulating in france from 2001 to 2013 reveals a high intra-specific diversity with new recombinant genotypes medical reviews coronavirus as a possible cause of severe acute respiratory syndrome mers: emergence of a novel human coronavirus coronavirus infections in working adults. eight-year study with 229 e and oc 43 human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease signs and symptoms in common colds human coronavirus nl63, a new respiratory virus a previously undescribed coronavirus associated with respiratory disease in humans evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children understanding human coronavirus hcov-nl63 characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia more and more coronaviruses: human coronavirus hku1 clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia coronavirus hku1 and other coronavirus infections in hong kong epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method coronavirus hku1 infection in the united states evidence of human coronavirus hku1 and human bocavirus in australian children detection of the new human coronavirus hku1: a report of 6 cases coronavirus hku1 in children, brazil isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection mers in south korea and china: a potential outbreak threat? isolation and characterization of viruses related to the sars coronavirus from animals in southern china identification of a novel coronavirus in bats coronaviruses post-sars: update on replication and pathogenesis antibodies to sars coronavirus in civets molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronaviruslike virus in palm civets at an animal market and on farms ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events isolation and characterization of a bat sarslike coronavirus that uses the ace2 receptor orf8-related genetic evidence for chinese horseshoe bats as the source of human severe acute respiratory syndrome coronavirus middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease mers coronavirus in dromedary camel herd, saudi arabia isolation of mers coronavirus from a dromedary camel mers coronaviruses in dromedary camels concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia mosaic structure of human coronavirus nl63, one thousand years of evolution does ibv change slowly despite the capacity of the spike protein to vary greatly? genetic drift of human coronavirus oc43 spike gene during adaptive evolution moderate mutation rate in the sars coronavirus genome and its implications spread, circulation, and evolution of the middle east respiratory syndrome coronavirus coronavirus diversity, phylogeny and interspecies jumping infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 recombination between nonsegmented rna genomes of murine coronaviruses evidence of recombinant strains of porcine epidemic diarrhea virus, united states emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination genotype shift in human coronavirus oc43 and emergence of a novel genotype by natural recombination phylogenetic and recombination analysis of coronavirus hku1, a novel coronavirus from patients with pneumonia evidence from the evolutionary analysis of nucleotide sequences for a recombinant history of sars-cov testing the hypothesis of a recombinant origin of the sars-associated coronavirus origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis coronavirus 229e-related pneumonia in immunocompromised patients coronaviruses in children epidemiological and clinical features of human coronavirus infections among different subsets of patients isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism severe acute respiratory syndrome (sars) multiple organ infection and the pathogenesis of sars a cluster of cases of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl63 in children hospitalized with respiratory tract infections in belgium human coronavirus nl63 infection in canada croup is associated with the novel coronavirus nl63 human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong the common cold: a review of the literature detection of viruses identified recently in children with acute wheezing state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection key: cord-323087-3cxyogor authors: widagdo, w.; begeman, lineke; schipper, debby; run, peter r. van; cunningham, andrew a.; kley, nils; reusken, chantal b.; haagmans, bart l.; van den brand, judith m. a. title: tissue distribution of the mers-coronavirus receptor in bats date: 2017-04-26 journal: sci rep doi: 10.1038/s41598-017-01290-6 sha: doc_id: 323087 cord_uid: 3cxyogor middle east respiratory syndrome coronavirus (mers-cov) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase-4 (dpp4) as its receptor. the distribution of dpp4 in the respiratory tract tissues of humans and camels reflects mers-cov tropism. apart from dromedary camels, insectivorous bats are suggested as another natural reservoir for mers-like-covs. in order to gain insight on the tropism of these viruses in bats, we studied the dpp4 distribution in the respiratory and extra-respiratory tissues of two frugivorous bat species (epomophorus gambianus and rousettus aegyptiacus) and two insectivorous bat species (pipistrellus pipistrellus and eptesicus serotinus). in the frugivorous bats, dpp4 was present in epithelial cells of both the respiratory and the intestinal tract, similar to what has been reported for camels and humans. in the insectivorous bats, however, dpp4 expression in epithelial cells of the respiratory tract was almost absent. the preferential expression of dpp4 in the intestinal tract of insectivorous bats, suggests that transmission of mers-like-covs mainly occurs via the fecal-oral route. our results highlight differences in the distribution of dpp4 expression among mers-cov susceptible species, which might influence variability in virus tropism, pathogenesis and transmission route. immunohistochemistry to detect dpp4 was performed on nose, lung, intestine, kidney, salivary gland, and liver tissues of different bat species: common pipistrelle bat, serotine bat, gambian epauletted fruit bat (further referred as gambian fruit bat), and egyptian fruit bat. the assay was replicated two-three times for each tissue. we have used the same technique to map dpp4 localization in the respiratory tract tissues of human, dromedary camel, sheep, horse, pig, and llama 9, 26 . the antibody used in this study recognizes bat dpp4 as was demonstrated in transfection experiments using cloned pipistrelle bat dpp4 22 . hematoxylin and eosin staining on subsequent slides of the same tissues from the bats did not show significant histological changes. dpp4 was not found in the nasal olfactory epithelial cells of common pipistrelle bat, serotine bat, gambian fruit bat, or egyptian fruit bat (fig. 1 ). in the nasal tissues of common pipistrelle bat, dpp4 was not detected in the respiratory epithelial cells lining the nasal cavity, but was detected in the epithelial cells lining the ducts of the submucosal glands in this species. in the serotine bat and gambian fruit bat, multifocal dpp4 expression was detected in a limited number of nasal respiratory epithelial cells. in contrast, in the nasal tissues of the egyptian fruit bat, dpp4 was prominently detected at the apical surface of the respiratory epithelial cells lining the nasal cavity as well as in glandular and ductular epithelial cells of the submucosal glands. in the lungs of the common pipistrelle and serotine bat, dpp4 was found in the endothelial cells of the capillaries but not in the bronchial, bronchiolar or alveolar epithelial cells. in the gambian and egyptian fruit bat, dpp4 was detected in the bronchial, bronchiolar and alveolar epithelial cells as well as in endothelial cells of small blood vessels (fig. 1 ). in the intestinal tissues of all four bat species, dpp4 was prominently expressed on the apical surface of both small and large intestinal epithelial cells (fig. 2 ). in the kidney of all four bat species, dpp4 was found in glomerular cells, parietal squamous epithelial cells of the bowman's capsule, and in the proximal tubular epithelial cells. in the salivary gland of common pipistrelle bat, dpp4 was only detected in the ductular epithelial cells, while in the serotine bat, it was detected in a limited number of glandular epithelial cells. in the gambian and egyptian fruit bat, it was detected in both the glandular and ductular epithelial cells of the salivary gland. in the liver of the common pipistrelle bat and serotine bat, dpp4 was present in a limited number of endothelial cells lining the sinusoids. in contrast, in the liver of the gambian and egyptian fruit bat, dpp4 was detected in the bile duct epithelial cells, in the endothelial cells of the hepatic arteries, and in the endothelial cells of the sinusoids (fig. 2) . variation in dpp4 signal and localization were occasionally observed between animals within the same species. in one common pipistrelle bat, the paranasal sinus and pharynx were examined and showed a limited number of dpp4 positive epithelial cells. the results of the dpp4 immunohistochemistry staining were scored qualitatively and summarized in table 1 . in general, our results showed that dpp4 was prominently expressed in the intestine and the respiratory tract tissues of the frugivorous bats, i.e. the gambian and the egyptian fruit bat. however, it is limitedly expressed in the respiratory tract tissues of the insectivorous bats, i.e. the common pipistrelle bat and the serotine bat. in the common pipistrelle bat, dpp4 was not detected in the nasal respiratory, nasal olfactory, bronchiolar, or alveolar epithelium, but was abundant on the apical surface of the epithelium lining the small and large intestine. we compared these findings to our previous results on dromedary camel and human tissues 26 . in dromedary camels, dpp4 is strongly detected in the nasal respiratory, tracheal, and bronchial epithelium, while there is limited expression in the alveolar epithelium (fig. 3 ). in humans, it is not found in the nasal epithelium and is present mainly in the alveolar epithelium. additionally, we performed dpp4 staining on intestinal tissues of dromedary camels obtained from a previous study 8 . we found that dpp4 was expressed mainly on the apical surface of the small intestinal epithelium (data not shown), similar to what has been reported for humans [32] [33] [34] [35] (fig. 3 ). the tissue distribution of the mers-cov receptor, dpp4, has previously been studied in humans, dromedary camels, and other livestock animals 9, 26 . here, we show that dpp4 is differentially expressed among bat species, especially between insectivorous and frugivorous bats. it is strongly detected in the intestine of the common pipistrelle bat, the serotine bat, the gambian fruit bat and the egyptian fruit bat. it is also prominent in the respiratory tract epithelium of the gambian and egyptian fruit bat, but expression is limited in that of the common pipistrelle and serotine bat. given the essential role of dpp4 in the entry of mers-cov into cells, these results suggest that mers-like-covs are not likely able to replicate in the respiratory tract in these two insectivorous bats. this is in line with our previous report on mers-cov infection experiment in sheep, showing that the lack of dpp4 in the respiratory tract of the sheep was associated with restricted mers-cov replication in these animals upon intranasal inoculation 9 . rather, in these two insectivorous bats, mers-like-covs may preferentially replicate in the gastrointestinal tract. this is partly supported by the fact that viral genomes of mers-like-covs were mainly obtained from faecal and intestinal tissue samples of insectivorous bats [14] [15] [16] [17] [18] [19] [20] 36 . this intestinal tropism indicates that these viruses transmit mainly through the fecal-oral route. therefore, future screening of mers-like-covs from insectivorous bats, particularly the common pipistrelle bat, might focus on fecal material, rectal swabs, or intestinal tissues, rather than throat or nasal swabs. prominent dpp4 expression in both respiratory tract and intestinal epithelium of the gambian fruit bat and the egyptian fruit bat suggests that mers-cov is able to replicate in both the respiratory tract and intestine of the fruit bats. these results are in line with the fact that mers-cov was able to replicate in the lungs of jamaican fruit bat (artibeus jamaicensis) upon intranasal and intraperitoneal inoculation 37 be detected in the rectal swabs of these animals up to day 9 p.i. and infectious virus was also isolated in the duodenum of one of the bats at day 28 p.i. 37 . these data suggest that mers-cov infects and replicates in the intestine of these bats, not only in the respiratory tract. mers-cov infection in these bats is likely mediated by dpp4, since hamster bhk cells, a non-susceptible cell line, could be infected by mers-cov when modified to express jamaican fruit bat's dpp4 37 . dpp4 expression in the intestine and respiratory tract of these jamaican fruit bats, however, was not investigated. since the jamaican fruit bat is a new world fruit bat, unlike the gambian fruit bat and the egyptian fruit bat, which are old world fruit bats, their genetic difference might influence the variation in dpp4 expression among these species. in contrast to the fruit bats, where dpp4 is expressed throughout the respiratory tract, dpp4 is rarely detected in the respiratory tract tissues of insectivorous bats. this limited dpp4 expression in insectivorous bats might significantly restrict the replication of mers-like-covs in these tissues and minimize the possibility of transmission of these viruses from the respiratory tract. the limited dpp4 expression in the respiratory tract of the two insectivorous bat species, particularly the common pipistrelle bat, is different from what has been reported for dromedary camels and humans. in humans, dpp4 is merely expressed in the lower respiratory tract, while in the dromedary camels, it is detected in the upper respiratory tract epithelium 26 . this renders humans to develop pneumonia upon mers-cov infection, while camels develop upper respiratory tract infection 2, 38, 39 . in the intestine of both dromedary camels and humans, dpp4 is mainly present in the apical surface of the small intestine epithelium [32] [33] [34] [35] . mers-cov has been isolated from faecal samples of a naturally infected dromedary camel, which suggests that this virus is able to replicate in the intestinal tract of this species 40 . however, in dromedary camels, the chance of detecting mers-cov rna in faecal samples is much lower than from nasal swabs 40 . we also observed that low amounts of viral rna are detectable in rectal swabs taken from mers-cov-inoculated dromedary camels 2 . while mers-cov has not yet been isolated from human faecal samples, low amounts of viral rna could be detected in stool samples of mers patients 41 , and several mers patients have also been reported to suffer from diarrhoea [42] [43] [44] . these observations suggest that mers-cov replicates in the intestine of both dromedary camels and humans although only to a limited extent. it is currently unclear what factors restrain mers-cov replication in the intestinal tract of dromedary camels and humans. the human intestinal tract is protected by a mucus layer, commensal microorganisms, multiple innate and adaptive immune cells 45 . also, adenosine deaminase (ada), a natural antagonist of dpp4 that can inhibit mers-cov infection in vitro 10 , has also been found in the human intestine. the amount of ada in the human intestine is four times higher compared to that in the lung 46 . the presence of dpp4 in the intestinal tract of bats suggests an intestinal tropism of mers-like-covs. we also detected dpp4 in the salivary glands and kidneys in all of the bats. in vitro, mers-cov has also been shown to replicate in primary kidney cell culture derived from common pipistrelle bat 13 . however, there has been no further evidence supporting the susceptibility of these two tissues in vivo, nor have there been any reports of mers-like-covs isolated from these two tissues or from bat urine samples. whether these viruses are transmitted through bat saliva or urine, therefore, is currently unclear. in general, our study describes the variation in dpp4 distribution among four bat species, with notable differences between insectivorous and frugivorous bats. more importantly, the tissue distribution of dpp4 in insectivorous bats, believed to be one of the natural hosts for mers-like-covs, is different to that in dromedary camels and humans. our results indicate intestinal tropism of mers-like-covs in the insectivorous bats we examined. the existence of a co-receptor that might influence mers-like-covs tropism and replication in these bats, however, could not be disregarded. ceacam5 is recently reported as an attachment factor that facilitates entry of mers-cov in-vitro 47 . whether ceacam5 plays an important role in-vivo, particularly in bats, remains to be investigated. in-vivo infection experiments are necessary to confirm our findings, but such studies are ethically and technically challenging. nevertheless, our data are relevant for future monitoring and surveillance of mers-like-covs in insectivorous bats, particularly in the common pipistrelle bat 14, 17 , as well as for future efforts to better understand the pathogenesis and transmission of mers-like-covs in their natural host. common pipistrelle and serotine bats were found stranded and severely wounded on different occasions, and admitted to an official local bat shelter in the netherlands. the animals were euthanized by veterinarians due to ethical reasons using officially approved methods. the gambian fruit bats and three of four egyptian fruit bats used in this study originated from free-ranging populations in ghana. the bats were sampled for an unrelated study and this study was approved by the ethics committee of the zoological society of london (ref. wle715) and the council for scientific and industrial research in accra, ghana. one of the egyptian fruit bats was obtained from the captive colony at the friederich loeffler institute, riems, germany. it had been euthanized due to reasons not related to this study. all methods were performed in accordance with the relevant guidelines and regulations. after euthanasia, the bats were necropsied and tissues were collected. parts of the lung, intestine, salivary gland, liver, and kidney were obtained from nine common pipistrelle bats, seven serotine bats, three gambian fruit bats, and four egyptian fruit bats. parts of the noses were obtained from five common pipistrelle bats, six serotine bats, three gambian fruit bats, and three egyptian fruit bats. these tissues were all fixed in 10% formalin and embedded in paraffin. the noses were decalcified in 10% edta for 9 days before being embedded in paraffin. the formalin fixed paraffin embedded tissues were sectioned (4 μm), deparaffinized, and subsequently hydrated. citric acid buffer 10 mm ph 6 was used to retrieve antigens. blocking with normal rabbit serum 5% was performed prior to staining with polyclonal goat igg anti-dpp4 (r&d systems, abingdon, uk) in 5 µg/ml concentration. normal goat serum (mp biomedicals, santa ana, ca, usa) in equal concentration was used as negative control. dpp4 staining was performed at 4 °c overnight. secondary antibody rabbit anti-goat igg labeled with peroxidase were applied subsequently in 1:200 dilution for 1 hour at room temperature (dako, glostrup, denmark). the red signal was revealed with 3-amino-9-ethyl-carbazole (sigma-aldrich, st. louis, missouri, usa) before counterstaining with hematoxylin. dromedary camel intestinal tissues were obtained from three different animals sacrificed at day 14 post infection with mers-cov in a previous mers-cov vaccination experiment 2 . two of these animals were vaccinated beforehand, while one was not. mers-cov was not detected in the intestinal tissues of these animals using pcr, virus titration or immunohistochemistry detecting nucleoprotein of mers-cov. information on dpp4 expression in human respiratory and intestinal tissues was derived from the previous studies 26, [33] [34] [35] . middle east respiratory syndrome an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan antibodies against mers coronavirus in dromedary camels risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation livestock susceptibility to infection with middle east respiratory syndrome coronavirus adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus mers-cov infection of alpaca in a region where mers-cov is endemic cd26/dpp4 cell-surface expression in bat cells correlates with bat cell susceptibility to middle east respiratory syndrome coronavirus (mers-cov) infection and evolution of persistent infection replicative capacity of mers coronavirus in livestock cell lines circulation of group 2 coronaviruses in a bat species common to urban areas in western europe middle east respiratory syndrome coronavirus in bats, saudi arabia close relative of human middle east respiratory syndrome coronavirus in bat human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe mers-related betacoronavirus in vespertilio superans bats group c betacoronavirus in bat guano fertilizer detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus differential expression of the mers-coronavirus receptor in the upper respiratory tract of humans and dromedary camels state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans eptesicus serotinus pipistrellus pipistrellus proteomics. tissue-based map of the human proteome expression of sucrase-isomaltase and dipeptidylpeptidase iv in human small intestine and colon expression and different polarity of aminopeptidase n in normal human colonic mucosa and colonic tumors regional expression of epithelial dipeptidyl peptidase iv in the human intestines rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs mers coronavirus in dromedary camel herd, saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection a case of imported middle east respiratory syndrome coronavirus infection and public health response epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission intestinal epithelial cells: regulators of barrier function and immune homeostasis adenosine deaminase isozymes in human tissues carcinoembryonic antigen-related cell adhesion molecule 5 is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus this study is supported by a top project grant (91213066) and by the zoonoses in the night project (5o-52200-98-308) both funded by zonmw. the e. gambianus and three of four r. aegyptiacus used in this study originated from a study in ghana in collaboration with richard suu-ire, from the forestry commission, accra. we would like to thank anne buschmann-balkema for her assistance in preparing the r. aegyptiacus tissues that come from the friederich loeffler institute, riems, germany. we would like to thank stichting vleermuisopvang oss for providing the tissues of p. pipistrellus. we thank brigitta m laksono for her advice on the schematic figure. competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-334738-k6002qzb authors: shalhoub, s.; abdraboh, s.; palma, r.; alsharif, h.; assiri, n. title: mers-cov in a healthcare worker in jeddah, saudi arabia: an index case investigation date: 2016-04-16 journal: j hosp infect doi: 10.1016/j.jhin.2016.04.002 sha: doc_id: 334738 cord_uid: k6002qzb in september 2015, a confirmed case of middle east respiratory syndrome (mers) was diagnosed in a healthcare worker in jeddah, saudi arabia. given the absence of confirmed mers cases in jeddah at the time, an epidemiological index case investigation took place. the investigation identified a probable source of an index case who had been in hospital in jordan in august 2015 while there was an ongoing mers outbreak and who then subsequently sought medical care in jeddah. since the discovery of the middle east respiratory syndrome coronavirus (mers-cov) in 2012, saudi arabia has faced several mers outbreaks in both hospitals and communities. 1e3 the most recent outbreak has been in riyadh. 4 on september 11th, 2015, a laboratory-confirmed mers case was diagnosed in a tertiary care hospital in jeddah. this report describes the index case investigation that took place. king fahad armed forces hospital is a 480-bed tertiary care hospital that serves military personnel, their families, and other community members in the western region of saudi arabia. there are 68 beds in the emergency department (ed) where admitted medical patients sometimes have extended stays due to bed limitations in the medical ward. patients nursed by patient a (a nurse) were identified using ed records and those who remained in hospital were screened for mers-cov. diagnosis of mers was confirmed in the same centre where the investigation took place using a reverse transcriptionepolymerase chain reaction (pcr) diagnostic kit (mers-coronavirus emc orf1a and sa1 upstream e-gene, light mix modular assays; roche, mannheim, germany). results were obtained within 6e8 h. patient a was a 42-year-old nurse, with no significant medical history, who worked in the ed of a tertiary care hospital. she travelled back from a 28-day-long vacation in the philippines on august 28th, 2015, and worked three ed shifts on august 30th, 31st, and september 1st. patient a exhibited fever and dry cough on september 4th. she sought medical advice in the ed, she was initially given antipyretics, and she was thought to have a transient viral upper respiratory tract infection. chest x-ray initially revealed no infiltrates. she was later referred to an internal medicine clinic on september 7th and was discharged home on antipyretic medications. she was admitted to the hospital on september 10th with fever and epistaxis. her laboratory investigations revealed a reduced white blood cell count of 3.7â10 9 /l and platelet counts of 143â10 9 /l, and she was therefore admitted with a provisional diagnosis of dengue fever. chest radiography revealed lobar infiltrates, and, due to a working diagnosis of viral pneumonia, she was placed in a negative pressure isolation room in ed. two nasopharyngeal swab (np) samples taken for mers-cov reverse transcriptionepolymerase chain reaction (rtepcr) were negative. a subsequent sputum sample obtained on september 11th tested positive for mers-cov. due to lack of vacant beds in the medical unit, initially patient a remained in the ed in a neutral room for 6 h, and was later transferred to a negative pressure isolation room. once the mers-cov diagnosis was confirmed, patient a was moved to a negative pressure isolation room in the intensive care unit (icu). both rooms in the ed were terminally disinfected. infection control measures were taken as follows: all patients present in ed on the day of patient a's diagnosis were screened for mers-cov using lower respiratory tract samples or np samples using rtepcr. contacts of patient a were divided between close contacts (defined as unprotected exposure within a 1.5 m distance for >10 min) or non-close contacts through personal interviews by infection control practitioners. 5 fifteen nurses (including one room-mate and two flat mates) and five physicians were deemed to be close contacts. all were asymptomatic and screening np samples were negative for mers-cov. they were monitored for symptoms for 14 days from the last day of unprotected exposure. the patients nursed by patient a prior to the onset of her illness on august 30th, 31st, and september 1st were identified as follows: two patients in icu2 on mechanical ventilators, one patient in main icu on mechanical ventilator, three patients in ed [one in c bay, one in b bay (patient b) on a mechanical ventilator and one in code room 2] (figure 1 ), three deceased patients (all deceased patients had been taken for burial), one patient in medical ward ii, and five discharged patients. the discharged patients were contacted; three tested negative and the remaining two had no symptoms and remained well for 14 days after the last day of exposure. two tracheal aspirate samples from the ventilated patients and two sputum samples from non-ventilated patients were tested for mers-cov rtepcr. initial np samples taken from patient b on september 11th and 12th tested negative for mers-cov. a tracheal aspirate sample obtained on september 15th tested positive for mers-cov. patient b was a 72-year-old woman, with a background of diabetes mellitus and right lower limb ischaemia. she was admitted on august 30th with shortness of breath and right foot gangrene. the patient had travelled to amman, jordan for femoralepopliteal artery bypass surgery on august 18th due to right lower limb ischaemia. on admission, the patient required oxygen 2 l/min through nasal prongs. examination findings included a conscious patient with a temperature of 38.2 c, infected right groin wound and a right gangrenous foot. respiratory tract examination revealed coarse crackles at lung bases. screening for mers-cov on september 2nd through rtepcr using np swab was negative and the patient was not producing any sputum. her fever resolved on september 5th following treatment with broad-spectrum antibiotics for the infected groin wound and a urinary tract infection. however, due to respiratory failure and increased oxygen requirements she required endotracheal intubation and mechanical ventilation on september 11th. a tracheal aspirate sample obtained after intubation tested mers-cov rtepcr positive on september 15th. case b was initially admitted for three days to the critical care area (code room) in the ed. she was then moved to the holding bay for three days and finally to b bay (figure 1 ). names and medical record numbers of patients who had been admitted to these areas on the same days were collected. of those, 11 remained in the hospital and were screened for mers-cov using a minimum of two samples including tracheal aspirates, sputum, or, if neither unavailable, np swabs. patients who had fever, cough, leucopenia, hypo-oxygenation or infiltrates on chest x-ray were considered highly suspected mers-cov cases and were therefore moved to negative pressure isolation rooms while screening was undertaken. highly suspected mers-cov cases were kept in negative pressure isolation rooms until there were three negative respiratory samples from three consecutive days. a total of 23 patients were screened. a patient who died an hour after confirmation of the mers-cov diagnosis in patient b was screened for mers-cov using a tracheal aspirate sample, and tested negative, before the body was released for burial. patients who had been discharged were contacted and symptoms elicited. screening for mers-cov with rtepcr was done at least once if symptoms were absent, and more frequently in the presence of symptoms. as of october 20th, 2015, a total of 151 contacts had been screened for mers-cov rtepcr, 11 of whom were close contacts of case b (i.e. exposure for >10 min at a distance of <1.5 m), including five nurses, four physicians, and two respiratory therapists. all screened contacts were negative. 5 patient a had not worn personal protective equipment when nursing for patient b because at the time of admission to ed, patient b was not suspected of having mers-cov infection. however, patient a reported complying with good hand hygiene practice. patient a recovered with no complications. she resumed working in the ed two weeks after recovery. patient b was transferred to a designated hospital to manage confirmed mers cases and was then transferred back to the primary hospital after being cleared for mers-cov. she was kept in isolation until clearance of mers-cov was confirmed. however, the patient later died due to complications related to her underlying illnesses. transmission of respiratory viral infections across countries has been described in several reports. 6, 7 there have been no recent mers-cov cases reported in the philippines; it was assumed that the nurse had contracted the infection from one of the patients she attended to during her shifts between august 30th and september 1st. based on that assumption, an index case investigation was carried out. the systematic screening of contacts to confirmed mers cases is in line with saudi arabian ministry of health recommendations. 5 the city of amman had been experiencing a nosocomial outbreak of mers-cov where patient b was admitted for the femoralepopliteal bypass. 8 she had no history of travelling to any other city in saudi arabia that had been dealing with mers-cov cases. the patient's diagnosis was probably delayed due to an initial negative mers-cov np swab sample, which was obtained because the patient had no cough and was unable to produce sputum. following admission and negative np result, infection control precautions were lifted because the patient's main complaint was an infected gangrenous foot with minimal respiratory symptoms and oxygen requirements. however, had the link between recent hospitalization of the patient in jordan and the mers-cov outbreak in jordan been noticed, more than one sample would have been obtained prior to lifting isolation precautions. moreover, patients who are highly suspected to have mers-cov infections are screened at least twice from sputum or tracheal aspirate samples, or, if unavailable, three times from np samples due to previously reported reduced sensitivity of np samples compared with lower respiratory tract samples. 9, 10 it was only after intubation that tracheal aspirate samples were obtained, testing positive for mers-cov. case b was cared for by a number of health professionals and it remains unclear why only one nurse became infected with mers-cov. case a was in contact with case b during a 12 h shift on august 30th, and performed standard nursing care that included inserting a peripheral intravenous access, taking vital signs, accompanying the patient to the radiology department and inserting a urinary catheter. case a was a smoker but had no known medical comorbidities. in conclusion, although transmission of mers-cov from saudi arabia to other countries has been reported before, this article describes the importation of mers-cov from a neighbouring country. we have highlighted the importance of detailed history-taking, especially an epidemiological history. moreover, maintaining a high index of suspicion to ensure the correct identification of all suspected cases and the strict employment of infection control measures remain crucial in preventing spread of mers-cov. isolation of a novel coronavirus from a man with pneumonia in saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah e a link to health care facilities ministry of health. command and control center. press releases. statistics. available at infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection preliminary epidemiological assessment of mers-cov outbreak in south korea first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission saudi arabia, jordan, who. archive number: 20151002.3680716. 2 october 2015. 05:37:52. international society for infectious diseases ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome the authors would like to thank d. abu al saod, s. fallatah, and a. jamal for their excellent technical assistance with processing and running the samples. none declared. none. key: cord-336775-d4hi9myk authors: kirtipal, nikhil; bharadwaj, shiv; kang, sang gu title: from sars to sars-cov-2, insights on structure, pathogenicity and immunity aspects of pandemic human coronaviruses date: 2020-08-13 journal: infection, genetics and evolution doi: 10.1016/j.meegid.2020.104502 sha: doc_id: 336775 cord_uid: d4hi9myk abstract human coronaviruses (hcov), periodically emerging across the world, are potential threat to humans such as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) – diseases termed as covid-19. current sars-cov-2 outbreak have fueled ongoing efforts to exploit various viral target proteins for therapy, but strategies aimed at blocking the viral proteins as in drug and vaccine development have largely failed. in fact, evidence has now shown that coronaviruses undergoes repaid recombination to generate new strains of altered virulence; additionally, escaped the host antiviral defense system and target humoral immune system which further results in severe deterioration of the body such as by cytokine storm. this demands the understanding of phenotypic and genotypic classification, and pathogenesis of sars-cov-2 for the production of potential therapy. in lack of clear clinical evidences for the pathogenesis of covid-19, comparative analysis of previous pandemic hcovs associated immunological responses can provide insights into covid-19 pathogenesis. in this review, we summarize the possible origin and transmission mode of covs and the current understanding on the viral genome integrity of known pandemic virus against sars-cov-2. we also consider the host immune response and viral evasion based on available clinical evidences which would be helpful to remodel covid-19 pathogenesis; and hence, development of therapeutic against broad spectrum of coronaviruses. previously, the coronaviridae study group (csg) identified sars-cov and mers-cov strains into a new species under the new informal subgroup of betacov genus (van boheemen et al., 2012) . however, recent introduction of subgenus rank in virus taxonomy have established the two informal subgroups of sars-cov and mers-cov as subgenera sarbecovirus and merbecovirus (de groot et al., 2013; gorbalenya et al., 2004) , respectively. remarkably, newly emerged sars-cov-2 differs from previously reported zoonotic pandemic viruses, viz. sars-cov and mers-cov; and hence, taxonomic position of sars-cov-2 under subgenera sarbecovirus may be tentative to change based on further evidences (gorbalenya et al., 2020) ( fig. 1) . phylogenetic tree analysis of covs constructed based on s gene using molecular evolutionary genetics analysis 6 software under neighbor-joining method and 1000 bootstrap values (biswas et al., 2020) . before the emergence of sars-cov, construction of phylogenetic tree for covs was based on pol (polymerase) or nucleocapsid (n) gene as standard practice. initially, this method proposed sars-cov as a member of gammacov (rota et al., 2003) . however, further evidences on amino-terminal domain of sars-cov spike protein discovered that 19 out of 20 cysteine residues were structurally conserved with in betacov group while only 5 conserved residues were matched within alphacov and gammacov group (rota et al., 2003) . subsequently, whole genome-based phylogenetic analysis indicated sars-cov as member of betacov lineage b. likewise, mers-cov was also identified as novel betacov lineage c with high pathogenicity . remarkably, rna-dependent rna polymerase (rdrp) and spike (s) animal covs have been known since the late 1930s like transmissible gastroenteritis virus of swine (tgev), bovine coronavirus (bcov), feline infectious peritonitis virus (fipv), and infectious bronchitis virus (ibv) transmissible (saif, 2004) . recent studies associated hcovs evolution with expedited urbanization and poultry farming; these practices permitted frequent interchange of species and simplified crossing of species barrier and genomic recombination in these viruses (jones et al., 2013) . bats harbored a great diversity of covs; viral sampling conducted by the ecohealth alliance in china alone identified about 400 new strains of covs . besides, study of covs diversity harbored by bats in eastern thailand revealed forty-seven covs (wacharapluesadee et al., 2015) . moreover, bats were documented with unique immune systems that allowed them to cope with a variety of viruses by comparison to other terrestrial mammal (xie et al., 2018) . also, a high diversity of zoonotic alphacovs and betacovs were also detected in the circulating bats of western europe (ar gouilh et al., 2018) . generally, bats harbored rna viruses but can be infected by dna viruses (xie et al., 2018) . although, clear transmission of covs from bats to humans is not yet fully discovered but transmission by intermediary host to humans via direct contact has been widely suggested as one of the probable modes of transmission. the first sars-cov case was documented in november 2002 in foshan, china (ge et al., 2015) . it became an epidemic, affected 28 countries around the world with 8,096 cases and 774 deaths (ge et al., 2015) . during the sars epidemic, the first indication for the source of sars-cov viral strain was detected in masked palm civets (paguma larvata) and raccoon dog (nyctereutes procyonoides). later discovery of antibodies for virus in chinese ferret badgers (melogale moschata) in a live-animal market in shenzhen, china were suspected as source of human infection (drexler et al., 2014; guan et al., 2003; song et al., 2005) . however, later findings predicted these animals merely as incidental hosts in absence of concrete results to support the motion of sars-cov-like viruses in palm civets among the wild or in the breeding factors endorsing bats as animal reservoirs of mammalian viruses were also defined in terms of their densely packed colonies, longevity, close social interaction, and ability to fly (calisher et al., 2006; luis et al., 2013) . likewise, first human case of mers was reported in june 2012 in jeddah, saudi arabia (ge et al., 2015) . as of november 2019, 2,494 cases of mers have been reported in 27 countries, resulting in 858 fatalities (who, 2020) . initially, search for mers-cov reservoir was focused on bats; however, serological survey in dromedary camels (camelus dromedarius) from oman and the canary islands exhibited a significant prevalence of mers-cov-neutralizing antibodies (reusken et al., 2013) . additionally, dromedary camels were detected with mers-cov rna at a farm in qatar related to two human cases of mers; virus particles were detected from dromedary camels in qatar and saudi arabia (hemida et al., 2014; raj et al., 2014) . serological evidence supported that dromedary camels has been harboring mers-cov-like virus in eastern africa and northern africa, and middle east, dating back as far as 1983 (muller et al., 2014) . additionally, in saudi arabia, dromedary camels were detected with various viral genetic lineages (sabir et al., 2016) , including which crossed the species barrier and caused the outbreaks in humans (omrani et al., 2015) . collectively, these evidences strongly suggested the contribution of dromedary camels as considerable reservoir to mers-cov; the ancestral virus were predicted to cross the species barrier into dromedary camels from bats more than ≥30 years ago as supported by serological evidences (fig. 2) . recent sars-cov-2 outbreak highlights the hidden wild zoonotic reservoir of deadly viruses and possible threat of spillover zoonoses (malik et al., 2020) . in 2019, a food market that sold live in wuhan, china was linked to the outbreak of sars-cov-2. through genetic analyses, bats were again suggested as native host of sars-cov-2 as it shared 96% genome to two sars-like covs, viz. bat-sl-covzx45 and bat-sl-covzx2 from bats zhou et al., 2020b) . however, intermediate host and route which assisted viral transmission to humans is yet fully understood. initially, ji, et al., proposed snakes as an intermediate host of sars-cov-2 transmission from bats to humans which involved homologous recombination within the viral spike (s) protein (ji et al., 2020) . later, pangolins (manis spp.) were suggested as potential intermediate host for sars-cov-2 as it shared 99% genetic homology with covs in pangolins ( fig. 2) (yi et al., 2020) . however, 1% variance all over the two-genome sequence was considered as significant difference; and thus, conclusive and concrete evidences are still under j o u r n a l p r e -p r o o f journal pre-proof investigation (yi et al., 2020) . moreover, scientists also showed concerned about its validation genetics technique (codon usage bias) (callaway and cyranoski, 2020) . hence, diversity of covs in bat population needs further investigation in detail along with critical surveillance and monitoring of bats to prevent future outbreaks in animals and public. after infection in human, hcovs are transmitted among the human population by close personto-person contact. for example, close contact with infected person under in 3 feet distance has been concluded as major factor for cov transmission . the pandemic viruses, sars-cov, mers-cov and sars-cov-2, transmitted between humans mostly by nosocomial transmission; supported by evidences that health care workers and patients had higher frequency of infection against their relatives (de wit et al., 2016) . the collected data predicted that only 22-39% of sars and 13-21% of mers transmission occurred between family members. also, sars-cov transmission from infected patients to health care workers was very frequent (33-42%) and mers-cov transmission between patients was recorded as the most common course of infection (62-79% of cases) (chowell et al., 2015) . such a predominance of nosocomial transmission was predicted due to significant virus shedding that occurred following the onset of infection when most patients were already under medical care (cowling et al., 2015; peiris et al., 2003) . a study on hospital surfaces following treatment of mers-cov infected patients detected with viral rna for several days even after patients no longer detected for viral infection (bin et al., 2016) . moreover, many patients with sars or mers were infected through super spreaders (korea centers for disease control and prevention, 2015) (chowell et al., 2015; kucharski and althaus, 2015) . in addition, sars-cov was also suspected for transmission through air (airborne spread) or some other unkown transmission routes . (e), nucleocapsid (n) proteins, and hemagglutinin (ha), where s, m, and e proteins are embedded in viral envelop while n protein protects viral rna genome located as core of virus ( fig. 3 ) (zhou et al., 2020b) . s protein is characterized as heavily glycosylated protein and contained the receptor binding domain (rbd), which is the most variable structure in covs (zhou et al., 2020b) ; mediates viral entry into host cells (bosch et al., 2003) . the two different types of spike proteins have been discovered in covs: spike glycoprotein trimmer (s) that can be found in all covs, and hemagglutinin-esterase (he) that found in specific covs (belouzard et al., 2012; cui et al., 2019) . some covs, including sars-cov and sars-cov-2, contain polybasic cleavage site (rrar/s) at the junction of two subunits (s1 and s2) of s protein. this allowed digestion by host furin-like protease during viral replication (bosch et al., 2003) and contributed in determination of viral infectivity towards host range (nao et al., 2017) . although function of polybasic cleavage site in sars-cov-2 is not yet discovered; experimental studies with sars-cov and mers-cov have determined that insertion of a furin cleavage site (fcs) at s1-s2 junction enhanced the cell-cell fusion without affecting viral entry (follis et al., 2006) and efficient digestion enabled mers-cov like covs from bats to infect human cells (menachery et al., 2020) . furthermore, m protein exists in higher quantities against e protein in virion structure (nal et al., 2005) and critically required in orchestrating viral shape, assembly and in generation of mature viral envelopes (siu et al., 2008) . moreover, it also functions in intracellular virion formation without the requirement of s protein. in some occasions, such as in presence of tunicamycin, covs were reported to generate noninfectious virions without spike but contained m protein in envelop (de haan et al., 1998) . likewise, structural protein, e protein assisted in mature virions secretion from host cells, in addition to other functions like ion channel activity, inhibit the host cell stress response and implicate pathogenesis in host cell through virus (ruch and machamer, 2012) . n protein essentially assisted in genomic rna packing during viral particle assembly (chang et al., 2006; hurst et al., 2009) . whereas, he, which is a hemagglutinin similar to influenza virus hemagglutinin, conducts the acetyl-esterase activity (klausegger et al., 1999) . recently, role of he was also logged to catalyst viral invasion across host cell membrane and in pathogenesis (ashour et al., 2020) . hcovs, including sars-cov, mers-cov, and sars-cov-2, contained largest nonsegmented positive-sense single-stranded rna genome of size between 26.2 and 31.7 kb with varied g+c contents of 32% to 43% (mousavizadeh et al., 2020; yang et al., 2006) . the genome organization for a cov is 5′-leader-utr-replicase/transcriptase-spike (s)-envelope (e)membrane (m)-nucleocapsid (n)-3′utr-poly (a) tail (fig. 4) . the 5′utr (untranslated region) and 3′utr are involved in interand intramolecular interactions, essentially required in rna-rna interactions and for binding of viral and cellular proteins (yang and leibowitz, 2015) . the orf1a and orf1b occupied first two-thirds of rna genome and produced replicase/transcriptase polyprotein which undergoes autoproteolytic activity with the aid of papain-like proteinase (plpro) and 3c-like main protease (3clpro or m pro ) to form 16 nonstructural proteins (nsps) . plpro conducts cleavage at n-terminus of replicase polyprotein to produce nsp1, nsp2, and nsp3, required for viral replication (harcourt et al., 2004) . however, m pro is first protein which autoproteolytically separates polyprotein replicase 1ab (~790 kda, recognition sequence located at leu-gln↓ser,ala,gly; where ↓marks stand for cleavage site) to produced mature viral enzymes and further cleaved downstream nsps at 11 sites to release nsp4-nsp16 ( fig. 4) zhang et al., 2020) . these events indicated the direct role of m pro to mediate maturation of nsps, which are basically required for virus life cycle in host cells. hence, plpro and mpro have been suggested as potential targets in drug development against hcovs. the later reading frames on the genome encoded the four major structural proteins; s, e, m, and n, which are common proteins among all covs . all the viral structural and accessory proteins are decoded by subgenomic (sg) rnas of covs where accessory proteins are interspersed between orfs stands but their number and functional activities are restricted to cov strains . specifically, sars-cov genome translated 8 accessory gene (3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b), mers-cov encoded 5 accessory genes (3, 4a, 4b, 5, and 8b), and though exact number of functional proteins remains to be established in sars-cov-2 but based on previous study on sars-cov, sars-cov-2 has been predicted with translation of 4 structural proteins (yoshimoto, 2020 ) and at least 6 or 9 accessory proteins (3, 6, 7a, 7b, 8, 9b, 10b, 13, 14) (liu et al., 2014; wu et al., 2020c; zhou et al., 2020b) . also, orf1ab position in sars-cov-2 genome (251-21541 nt) was noticed with change at starting codon position by comparison to sars-cov j o u r n a l p r e -p r o o f (265-21486 nt) and mers-cov (279-21514 nt) . interestingly, conserved amino acid substitution at positions 439, 501, 493, 485 and 486 were also logged in rbd domain of sars-cov-2 s protein, which was considered as important in sars-cov . it is important to add that genomic material of hcovs is highly susceptible to frequent recombination process which have been directly associated with establishment of altered virulence in new hcov strains (hilgenfeld, 2014) . all the sars-like covs exhibits typical streadgy for replication and translation following infection in host cell. this section will briefly summarize sars-cov, mers-cov and sars-cov-2, their comparative lifecycle in host cell, which began on attachment of virus with host cell receptor and finished with release of newly generated progeny from infected cells. the binding of hcov to host-cell receptor is the initial step in viral infection which determined the severity of infection and pathogenesis. a better understanding of coupling of viral structural proteins with targeted receptor on host cells and other involved proteins, such as host protease, can help to predict the specific zoonotic covs infection in humans. hcovs infection begins with attachment of viral particle on host cell surface by densely glycosylated s protein, which is a trimeric class i fusion protein and consist of two major subunits; a receptor binding domain (s1; also known as rbd) and a second domain (s2) mediated viral fusion with host cell membrane. the fusion process of viral membrane with host cell membrane is triggered when s1 domain binds with host-cell receptor; for example, angiotensin-converting enzyme 2 (ace2) for sars-cov and sars-cov-2 (wu et al., 2020b, c) , also as an alternative receptor cd209l (a c-type lectin, also called l-sign) with low affinity for sars-cov (jeffers et al., 2004) , dipeptidyl peptidase-4 (dpp4, also known as cd26) for mers-cov . thus, cleavage of spike glycoprotein is performed by host cell proteases enables interaction of s2 domain for virus entry into the cell (de wit et al., 2016) . in the respiratory tract, ace2 receptor is widely distributed on the epithelial cells of trachea, bronchi, bronchial serous glands, and alveoli , as well as alveolar monocytes and macrophages ; sars-cov attacked these cells and mature virions are later released to infect other new target cells (zhang et al., 2004) . ace2 is also diffusely expressed on endothelial cells of arteries and veins, cerebral neurons and immune cells, tubular epithelial cells of kidneys, mucosal cells of intestines, and epithelial cells of renal tubules, presented as a variety of susceptible targets to sars-cov infection (gu and korteweg, 2007; guo et al., 2008) . whereas, mers-cov is known to target respiratory tract, liver small intestines, kidney, prostate and activated leukocytes (widagdo et al., 2016) . remarkably, unlike sars-cov in vitro studies, mers-cov was found to infect human dendritic cells and macrophages ; therefore, virus disrupted the immune system. besides, t (lymphocyte) cells are another potential target for mers-cov due to presence of high amounts of cd26 . hence, this cov was predicted to dysregulate antiviral t-cell responses because of stimulation of t-cell apoptosis yeung et al., 2016) . recent studies suggested that sars-cov-2 employs ace2 as main receptor as in sars-cov infection with higher affinity, suggesting the likelihood of same group of host-cells being targeted and infected (zhou et al., 2020b; zou et al., 2020) . after attachment to host-cell surface, virus entry into cell has been deciphered by two different paths based on availability of host cell protease to activate receptor-attached spike protein (fig. 5 ) (simmons et al., 2013) . in first path, covs invaded host cell as an endosome which is mediated by clathrin-dependent and-independent endocytosis (fig. 5) (kuba et al., 2010; wang et al., 2008a) . this phenomenon induced conformational changes in viral particle which subsequently fused viral envelope with endosomal wall (simmons et al., 2013) . alternatively, in second pathway, direct invasion of virus particles into host cell are mediated via proteolytic cleavage of receptor-attached spike protein by host's transmembrane serine protease 2 (tmprss2) or transmembrane serine protease 11d (tmprss11d) on the cell surface (heurich et al., 2014; zumla et al., 2016) . herein, s2 domain of spike protein accomplished direct membrane fusion between virus and plasma membrane as initially observed in sars-cov after virus and host cell membrane fusion event, virus released the nucleocapsid packed genomic rna into cellular cytoplasm under the influence of induced structural conformation changes . then, viral genome acted as a mrna and cell's ribosome translates two-thirds of this rna, crossponds to orf1a and orf1b into two large overlapping polyproteins (pp): pp1a and pp1ab. the larger polyprotein pp1ab translated from a -1 ribosomal frame shift triggered by slippery sequence (uuuaaac) and downstream rna pseudo knot at end of orf1a (masters, 2006) . this ribosomal frameshift enabled continuous translation of orf1a followed by orf1b . the encoded polyproteins possess the proteases; pl pro and m pro which assisted in generation of 16 nsps (nsp1-nsp16) from polyprotein pp1ab, including several replication proteins such as rdrp, rna helicase, and exoribonuclease (exon) . moreover, multifunction and enzyme activities for specific nsps in the previously reported hcov, i.e. sars-cov and mers-cov have been discovered over the past years and summarized elsewhere (masters, 2006; ziebuhr, 2005) . most of the newly translated nsps together with structural protein, i.e. n protein, formed the multi-protein replicase-transcriptase complex (rtc) which further conducted the viral genome replication and transcription . at the site of replicative organelles (knoops et al., 2008) , rtc complex contained rdrp as main replicase-transcriptase protein for the synthesis of negative-sense subgenomic (sg) rna strands from viral rna and transcription of negative-sense subgenomic rna molecules from corresponding positive-sense mrnas . the newly produced minus strands of genomic and sgrnas are subsequently employed as templates for production of positive sense strands (mrnas), j o u r n a l p r e -p r o o f specifically in generation of genomic rna (genome replication) and sg mrnas (transcription). these newly synthesized rna strands acted as genome for generated new viral progeny. also, several smaller mrnas are produced from viral last third of genome which tracks reading frames orf1a and orf1b, interpreted into viral four structural proteins (s, e, m, and n) and along with accessory proteins (orf3a to orf9b) become part of viral progeny . besides, other nsps in rtc complex also assist in viral replication and transcription process . for instance, nsp15 protein, a 3'-5' exoribonuclease, provides an extra fidelity to rtc complex via proofreading function in rna replication, which lacked in rdrp. likewise, nsp7 and nsp8 proteins generated a hexadecameric sliding clamp for rtc complex to catalyst the processivity of rdrp . this increased fidelity and processivity is essentially required in covs during rna synthesis because of their relatively bulky rna genome by comparison to other rna viruses (sexton et al., 2016) . the viral rna translation process occurred inside host cells' endoplasmic reticulum lead to formation of structural proteins, viz. s, e and m, which moved along the secretory pathway into golgi intermediate compartment. herein, n protein packed the newly produced rna genome and thereby shaped into a helical nucleocapsid. following, virion assembly is triggered by m protein via multiple protein-protein interactions which assisted in incorporation of nucleocapsid, envelope, and spike proteins into virus particles . later, viral progeny germinated into endoplasmic reticulum-golgi intermediate compartment (ergic) and released as secretory vesicles which fused with plasma membrane and secrete from host cell by exocytosis (fig. 6) lim et al., 2016) . generally, human population lacks immunity against covs and hence, susceptible to novel cov infections such as sars-cov-2. since, immune response against sars-cov-2 is not fully j o u r n a l p r e -p r o o f deciphered, recent studies suggested to refer the previous reports on other hcovs, especially sars-cov and mers-cov (fig.7) (yi et al., 2020) . thus, tlrs mainly mediated the detection of viral rna in mdcs (such as tlrs3 and some tlr7) and pdcs by tlr7 (and tlr8 in human pdcs) (schreibelt et al., 2010) . based on a specific prr, various-partially cross-talking-signaling pathways resulted in promoters transactivation of antiviral genes (o'neill et al., 2013) . for instance, a prototypical pamp relevant for covs is dsrna, a by-product from genome replication and transcription (weber et al., 2006; zielecki et al., 2013) . dsrna can also be detected by tlr3 in the endosome while sensed by rna helicases rig-i, mda5 and kinase pkr in the cytoplasm (rasmussen et al., 2009; yim and williams, 2014; yoneyama et al., 2016) . also, specific ssrnas are presented as pamps, either if they displayed particular features or in the wrong location; for example, tlr7 detects gu-rich ssrna in the endosome (heil et al., 2004) . thus, numerous types of prrs are regularly surveying the intracellular and extracellular space to sense virus infections in a sensitive and timely manner. the detection of viral infection triggers complex signaling cascades such as myd88 that induced manufacture of type i ifns and stimulation of transcription factor nuclear factor-κb (nf-κb). in turn, active nf-κb induced transcription of pro-inflammatory cytokines (fig.7) . moreover, type i ifns signal by ifnα/β receptor (ifnar) and following downstream signal activators and transducers of transcription (stat) proteins triggered the manufacture of antiviral proteins that are programmed by interferon-stimulated genes (isgs) like ifn-induced protein j o u r n a l p r e -p r o o f with tetratricopeptide repeats 1 (ifit1). altogether, this launches an antiviral immune response that restricts viral replication in infected and in neighbouring cells (de wit et al., 2016) . however, studies have showed that family of covs can significantly suppressed human immune responses against viral infection by evading immune detection machinery (kikkert, 2020) . for instance, papain-like protease (plpro) domain of nsp3 of sars-cov interrelated with ifn regulatory factor 3 (irf3) and inhibited its activation (devaraj et al., 2007; frieman et al., 2009) . besides, plpro was reported to cause deubiquitination (or inhibit ubiquitination) of tankbinding kinase 1 (tbk1), rig-i, and irf3 (clementz et al., 2010; devaraj et al., 2007; frieman et al., 2009; sun et al., 2012) . the several functions and mechanisms adopted to counteract ifn induction, and strategies to develop resistance against ifn by previously pandemic hcovs are summarized earlier (kindler et al., 2016) . remarkably, sars-cov and mers-cov were reported to induced production of double-membrane vesicles that lack prrs, replicated in these vesicles, and thereby escaped host detection system for viral dsrna (de wilde et al., 2013; knoops et al., 2008) . moreover, covs induced very little ifn production in most of cell types (kindler et al., 2016) . thus, high levels of dsrna, which are generated during viral replication cycle (weber et al., 2006; zielecki et al., 2013) , do not caused an adequate induction of ifn system. moreover, antigen presentation subsequently stimulated body's humoral and cellular immunity on viral invasion, which are mediated by virus-specific b and t cells. t lymphocytes, including cluster of differentiation 4 (cd4 + ) and cluster of differentiation 8 (cd8 + ) t cells, play a central role against viral infection, stimulated b cells to produce virus-specific antibodies while cd8 + t cells directly kills virus-infected cells. also, humoral immunity, including complements such as c3a, c5a and antibodies, are critical in fighting viral infection (fig. 7) (mathern and heeger, 2015; traggiai et al., 2004) . recent flow cytometric analysis of peripheral blood from sars-cov-2-infected patients showed substantially reduced cd4 + and cd8 + t cells number while their status was excessively activated as evident from high proportions of human leukocyte antigen -dr isotype (hla-dr) (cd4 3.47%) and cd38 (cd8 39.4%) double positive fractions . similarly, acute phase response in patients with sars-cov was associated with severe decrease of cd4 + t and cd8 + t cells. even if there is no antigen, cd4 + and cd8 + memory t cells are known to persist for four years as observed in sars-cov recovered individuals and were able to perform t cell proliferation, delayed-type hypersensitivity (dth) j o u r n a l p r e -p r o o f response and production of ifn-γ (fan et al., 2009) . also, six years after sars-cov infection, specific t-cell memory response to sars-cov s peptide library was identified in 14 of 23 recovered sars patients (tang et al., 2011) . the specific cd8 + t cells were documented to exhibit a similar effect on mers-cov clearance in mice (zhao et al., 2014) . these discoveries could be helpful to deduce a valuable information for the rational design of vaccines against sars-cov-2. for example, antibodies collected from a recovered patient were reported to neutralized mers-cov infection (niu et al., 2018) . moreover, t helper cells also produced proinflammatory cytokines to assist the defending cells. however, cov (sars-cov and mers-cov) were established to halt t cell function and induced their apoptosis mathern and heeger, 2015; yeung et al., 2016) . besides, sars-cov-2 assumed to attach with ace2 as in sars-cov infection, target and infect the same set of host cells zhou et al., 2020b; zou et al., 2020) . additionally, ace2-associated lung injury was documented in sars-cov infection kuba et al., 2005) ; s protein of sars-cov downregulates ace2 (glowacka et al., 2010; wang et al., 2008b) and induce detachment of catalytically active ace2 ectodomain (haga et al., 2008; jia et al., 2009) . this results in nonfunctional pulmonary ace2 receptor which has been advised to be linked with acute lung injury (imai et al., 2008; kuba et al., 2006) ; decrease activity of ace2 further triggered reninangiotensin system (ras) dysfunction and caused enhance inflammation and vascular permeability (gheblawi et al., 2020) . in animal studies with murine acute respiratory distress (ard) model showed association of reduced ace2 function with an increased lung edema, neutrophil accumulation, enhanced vascular permeability, and diminished lung function . also, in human airway epithelia, constitutive shedding of ace2 receptor by activity of a metalloprotease 17 (adam17), also called as tumour necrosis factor alpha (tnf-α) converting enzyme (tace), and disintegrin secrete functionally active soluble ace2 (sace2) (lambert et al., 2005) . hcovs infection and inflammatory cytokines like interleukin 1 beta (il-1β) and tnf-α were related with enhance ace2 shedding (haga et al., 2008; jia et al., 2009) . notably, in vitro studies presented close association of sars-cov s protein-induced ace2 shedding with tnf-α production (haga et al., 2008) . although, biological function of sace2 is yet unknown, however, recent studies have suggested direct involvement of sace2 in inflammatory responses against sars-cov, and possibly also connected with sars-cov-2 (fu et al., 2020) . additionally, apoptosis of type 2 alveolar cells caused release of specific j o u r n a l p r e -p r o o f inflammatory mediators which further instigate macrophages to secrete three essential immune substances; interleukin-1 (il-1), interleukin-6 (il-6), and tnf-α; also called as cytokines. these immune substances in the bloodstream produced infection symptoms linked to hcovs . recent in vitro cell experiments showed that delay release of cytokines and chemokines occurred in respiratory epithelial cells, dendritic cells (dcs), and macrophages at the early stage of sars-cov infection (choi and joo, 2020) . like sars, mers-cov also infected human airway epithelial cells, thp-1 cells (a monocyte cell line), human peripheral blood monocyte-derived macrophages and dcs, and induced late but elevated levels of proinflammatory cytokines and chemokines (tynell et al., 2016; zhou et al., 2014) . notably, sars-cov-2 infection is suspected to cause pyroptosis in lymphocytes and macrophages. it was observed that lymphocytopenia is often seen in severe sars-cov-2 patients and cytokine release syndrome (crs) induced by sars-cov-2 was also considered to be facilitate by leukocytes other than t cells . a vast majority of patients (82.1%) infected with sars-cov-2 have been logged for peripheral blood lymphopenia (guan et al., 2020) , supported the pulmonary infiltration of lymphocytes and/or cell damage via pyroptosis or apoptosis . previous pandemics triggered by mers-cov and sars-cov were also reported with massive production of chemokines and cytokines; sars-cov exhibited ccl2, ccl3, ccl5, cxcl8, cxcl9, cxcl10, etc.; and il-12, il-18, il-6, il-1beta, il-33, ifn-alpha, ifn-gamma, tnf-α and transforming growth factor (tgf)-beta. likewise, mers-cov was reported for elevated expression of cytokines ifn-α, il-6, and chemokine (cxcl-10, ccl-5, and cxcl-8) . hence, acute respiratory distress syndrome (ards) is caused by cytokine storm that triggers a destruction in host cells via immune system and subsequently results into multiple organs failure or death as stated in case of sars-cov-2 outbreak; similar observations were noted in case of sars-cov infection (kumar et al., 2020) . albeit, there is no direct data is available to corelate the participation of pro-inflammatory cytokines and chemokines in lung pathology through sars-cov and mers-cov, correlative observations and recorded from patients with severe disease suggested a role of hyper-inflammatory responses in hcov pathogenesis (fig.7) (channappanavar and perlman, 2017) . for instance, in mers-cov infection, plasmacytoid dendritic cells, but not mononuclear macrophages and dcs (scheuplein et al., 2015) , were induced to produce a large amount of ifns (choi and joo, 2020) . (yi et al., 2020) . despite of similar virus titers in respiratory tract, sars-cov-infected old nonhuman primates are more likely to develop immune dysregulation than infected young primates, leading to more severe disease manifestations (smits et al., 2010) . it seems that excessive inflammatory response rather than virus titer is more relevant to the death of the old nonhuman primates (smits et al., 2010) . similarly, in bagg albino/c (balb/c) mice infected with sars-cov, disease severity in old mice was related to early and disproportionately strong upregulation of ards-related inflammatory gene signals (rockx et al., 2009) . the rapid replication of sars-cov in balb/c mice induces delayed release of ifn-α/β, which was accompanied by influx of many pathogenic inflammatory mononuclear macrophages (channappanavar et al., 2016) . depleting inflammatory monocyte-macrophages or neutralizing inflammatory cytokine tnf protected the mice from fatal sars-cov infection. thus, inflammatory mediators contributes an vital role in pathogenesis of ards, which was estimated as an leading cause of death in patients infected with sars-cov or mers-cov lew et al., 2003) . it is now known that several proinflammatory cytokines consequences of cytokine storm contribute to the occurrence of ards (channappanavar and perlman, 2017; reghunathan et al., 2005) . these mechanisms also lead to activation of coagulation pathways. massive inflammation can impaired all the three control mechanisms like antithrombin iii, tissue factor pathway inhibitor, and protein c system (jose et al., 2014) ; with reduced anticoagulant concentrations due to reduced production and increasing consumption. this defective procoagulant-anticoagulant balance predisposes in the development of microthrombosis, disseminated intravascular coagulation, and multiorgan failure; as evident from severe sars-cov-2 pneumonia with raised d-dimer concentrations being a poor prognostic feature and disseminated intravascular coagulation common in nonsurvivors zhou et al., 2020a) . in this over all proposed mechanism, proteinase-activated receptor-1(par-1) acts as main thrombin receptor and mediates thrombininduced platelet aggregation, and interplay between coagulation, inflammatory, and fibrotic responses; all of these factors are an important aspects of pathophysiology of fibroproliferative lung disease (jose et al., 2014) , such as seen in sars-cov-2. on the other hand, the proposed events of cytokine storms in case of sars-cov-2 was suggested to increased inflammationrelated biomarkers, including c-reactive protein (crp), ferroprotein, erythrocyte sedimentation j o u r n a l p r e -p r o o f rate (esr) and il-6 huang et al., 2020) . in conclusion, based on previous hcovs infection, severity and pathogenesis of sarscov-2 was suggested to divided into three stages; stage-1, an asymptomatic incubation time with or without detectable virus; stage-2, nonsevere symptomatic period and detection of viral particles; stage-3, severe respiratory symptomatic stage and detection of high viral load . furthermore, based on previous studies of sars-cov, the inflammatory responses in sars-cov-2 infection can be classified into primary and secondary responses . initially, primary inflammatory responses produced subsequently viral infection, prior to arrival of neutralizing antibodies (nab). the activation of these factors is primarily driven by viralmediated ace2 downregulation and shedding, vigorous viral replication, and host antiviral defense system. secondary inflammatory responses are generated by the adaptive immunity and nab. moreover, virus-nab complex was also suggested to triggered fc receptor (fcr)-mediated inflammatory responses and acute lung injury that results in persistent viral replication and inflammatory responses from host macrophages (fig. 8) (fu et al., 2020) . it was observed that patients could survive in primary inflammatory responses but secondary inflammatory responses could be fatal. secondary inflammatory responses in which virus-nab complex formed was considered as targeted to develop potential therapeutic strategy along with anti-inflammatory drugs (fu et al., 2020) . accumulating data on hcovs exhibits the importance of understanding the zoonotic viral transmission and pathogenesis in humans. recent studies on virus have established the viral proteins as prime target in the drug designing and vaccine development; however, this review have highlighted the utmost importance of intermediate host for the viral transmission to human. future approaches, therefore, should considered the strains from intermediate hosts in the therapeutic development pipelines to better understand the severity of virulence via genome recombination process in covs. we also concluded major damaged caused by the failure of host j o u r n a l p r e -p r o o f immune system against hcov infection and further caused by insurgence of cytokines which lead to severe mutilation of the host body. synergistic and/or multi-target strategies that check viral growth and malfunctioned immune system of the host cells simultaneously might also be successful in battle against hcovs. nevertheless, synergistic approaches must take into account crossponding to the complementary target pathways. when developing novel therapeutic strategies to check the immunoregulatory cytokines such as tnfβ and il6, investigation should be considered on the viral strain and targeted organ specificity; for example, sars-cov-2 has more affinity to ace2 which are scattering on different organs like lung and kidney while mers-like cov can even infect t-cells. in addition, we should be cognizant of the feedback responses that produce with any treatment and consider the possibility to leveraged in a combinatorial fashion, taking into consideration the outcomes of preceding standard care and/or target immunoregulatory molecules will affect the efficacy and incidence of virulence. another utmost point to be consider is how to generalize the therapeutic decision under the different immunological responses to the viral infection; for instance, susceptibility of some persons and with asymptomatic symptoms or sever immune response to the infection. given the relatively high emergence of hcovs and potential threat to humans, future therapeutic strategies should involve deep immune profiling of the infected individuals and personalization of therapeutics when feasible to accelerate progress towards more effective treatment approaches. this goal is ever closer to becoming a reality as multiplexed imaging, immunophenotyping and mutational analysis tools are increasing the high-throughput process. although, many trials have failed before, but given the progress in our understanding on the interaction between host immune responses and viral escape plans, and the emerging sophisticated strategies we have reasons to be hopeful for the future successful treatment against hcovs. authors declares no conflict of interest. insights into the recent 2019 novel coronavirus (sars-cov-2) in light of past human coronavirus outbreaks cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites mechanisms of coronavirus cell entry mediated by the viral spike protein modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the a(2b) receptor environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea emergence of novel coronavirus and covid-19: whether to stay or die out? the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex bats: important reservoir hosts of emerging viruses why snakes probably aren't spreading the new china virus modular organization of sars coronavirus nucleocapsid protein dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study preparedness and proactive infection control measures against the emerging novel coronavirus in china the pathogenesis and alternative treatment of sars-cov2 transmission characteristics of mers and sars in the healthcare setting: a comparative study middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases new coronavirus threat galvanizes scientists preliminary epidemiological assessment of mers-cov outbreak in south korea origin and evolution of pathogenic coronaviruses this scientist hopes to test coronavirus drugs on animals in locked-down wuhan middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group coronavirus particle assembly: primary structure requirements of the membrane protein mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment sars and mers: recent insights into emerging coronaviruses regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus ecology, evolution and classification of bat coronaviruses in the aftermath of sars clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection characterization of sars-cov-specific memory t cells from recovered individuals 4 years after infection the nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis coronaviruses: an overview of their replication and pathogenesis furin cleavage of the sars coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappa b signaling understanding sars-cov-2-mediated inflammatory responses: from mechanisms to potential therapeutic tools bat coronaviruses. bats and viruses angiotensin-converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2 differential downregulation of ace2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl63 the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 severe acute respiratory syndrome coronavirus phylogeny: toward consensus pathology and pathogenesis of severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china pathogenetic mechanisms of severe acute respiratory syndrome modulation of tnf-alpha-converting enzyme by the spike protein of sars-cov and ace2 induces tnf-alpha production and facilitates viral entry a new virus isolated from the human respiratory tract identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 mers coronavirus in dromedary camel herd, saudi arabia tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein the discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice angiotensin-converting enzyme 2 protects from severe acute lung failure human coronavirus oc43 infection induces chronic encephalitis leading to disabilities in balb/c mice cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus cross-species transmission of the newly identified coronavirus 2019-ncov ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia zoonosis emergence linked to agricultural intensification and environmental change proteinase-activated receptors in fibroproliferative lung disease human coronavirus-hku1 infection among adults in cleveland innate immune evasion by human respiratory rna viruses interaction of sars and mers coronaviruses with the antiviral interferon response identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza c virus and bovine coronavirus sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum trilogy of ace2: a peptidase in the reninangiotensin system, a sars receptor, and a partner for amino acid transporters angiotensin-converting enzyme 2 in lung diseases a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission. euro surveillance : bulletin europeen sur les maladies transmissibles host immune response and immunobiology of human sars-cov-2 infection tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats a major outbreak of severe acute respiratory syndrome in hong kong fruit bats as reservoirs of ebola virus acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus bats are natural reservoirs of sars-like coronaviruses human coronaviruses: a review of virus-host interactions accessory proteins of sars-cov and other coronaviruses epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques drug treatment options for the 2019-new coronavirus (2019-ncov) genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? emerging novel coronavirus (2019-ncov)-current scenario, evolutionary perspective based on genome analysis and recent developments the molecular biology of coronaviruses molecules great and small: the complement system recovery in tracheal organ cultures of novel viruses from patients with respiratory disease middle east respiratory syndrome coronavirus in bats, saudi arabia trypsin treatment unlocks barrier for zoonotic bat coronavirus infection host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein immunology, infection, 2020. genotype and phenotype of covid-19: their roles in pathogenesis mers coronavirus neutralizing antibodies in camels differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e genetic predisposition to acquire a polybasic cleavage site for highly pathogenic avian influenza virus hemagglutinin supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient the history of toll-like receptors -redefining innate immunity are bats really 'special' as viral reservoirs? what we know and need to know. bats and viruses middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study coronavirus 229e-related pneumonia in immunocompromised patients isolation of mers coronavirus from a dromedary camel dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc innate recognition of intracellular pathogens: detection and activation of the first line of defense middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection the coronavirus e protein: assembly and beyond co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? revue scientifique et technique-office international des épizooties 23 high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy homology-based identification of a mutation in the coronavirus rna-dependent rna polymerase that confers resistance to multiple mutagens covid-19 infection: the perspectives on immune responses additional changes to taxonomy ratified in a special vote by the international committee on taxonomy of viruses proteolytic activation of the sarscoronavirus spike protein: cutting enzymes at the cutting edge of antiviral research the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles exacerbated innate host response to sars-cov in aged non-human primates human coronavirus nl63 infections in infants hospitalised with acute respiratory tract infections in south africa unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage crosshost evolution of severe acute respiratory syndrome coronavirus in palm civet and human from sars to mers, thrusting coronaviruses into the spotlight coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus middle east respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. mbio 3. van der hoek development of one-step, real-time, quantitative reverse transcriptase pcr assays for absolute quantitation of human coronaviruses oc43 and 229e clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway review of bats and sars endocytosis of the receptorbinding domain of sars-cov spike protein together with virus receptor ace2 double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negativestrand rna viruses differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods a new coronavirus associated with human respiratory disease in china 2020c. a new coronavirus associated with human respiratory disease in china dampened sting-dependent interferon activation in bats pathological findings of covid-19 associated with acute respiratory distress syndrome the structure and functions of coronavirus genomic 3 ' and 5 ' ends drug design targeting the main protease, the achilles' heel of coronaviruses design of wide-spectrum inhibitors targeting coronavirus main proteases mers coronavirus induces apoptosis in kidney and lung by upregulating smad7 and fgf2 covid-19: what has been learned and to be learned about the novel coronavirus disease protein kinase r and the inflammasome regulation of antiviral innate immune signaling by stressinduced rna granules the proteins of severe acute respiratory syndrome coronavirus-2 (sars cov-2 or n-cov19), the cause of covid-19 crystal structure of sars-cov-2 main protease provides a basis for design of improved alphaketoamide inhibitors the life cycle of sars coronavirus in vero e6 cells rapid generation of a mouse model for middle east respiratory syndrome single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov. biorxiv elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis a novel coronavirus from patients with pneumonia in china coronavirus replication and reverse genetics human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection coronaviruses -drug discovery and therapeutic options middle east respiratory syndrome key: cord-318935-xsfolppr authors: yang, jieun; park, eun-cheol; lee, sang ah; lee, sang gyu title: associations between hand hygiene education and self-reported hand-washing behaviors among korean adults during mers-cov outbreak date: 2018-07-16 journal: health educ behav doi: 10.1177/1090198118783829 sha: doc_id: 318935 cord_uid: xsfolppr background. hand washing is an effective way to prevent transmission of infectious diseases. education and promotional materials about hand washing may change individuals’ awareness toward hand washing. infectious disease outbreak may also affect individuals’ awareness. aims. our study aimed to examine associations between hand-washing education and self-reported hand-washing behaviors among korean adults during the year of the middle east respiratory syndrome (mers) outbreak. methods. data from the 2015 community health survey were used for this study. the total study population comprised 222,599 individuals who were older than 20 years of age. a multiple linear regression model was used to investigate associations between hand hygiene education and self-reported hand-washing behaviors. subgroup analyses stratified by age, sex, income, and mers outbreak regions were also performed. results. individuals who received hand-washing education or saw promotional materials related to hand washing had significantly higher scores for self-reported use of soap or sanitizer (β = 0.177, p < .0001) and self-reported frequency of hand washing (β = 0.481, p < .0001) than those who did not have such experiences. the effect of hand-washing education on self-reported behavior change was greater among older adults, women, and lower income earners. the effect of hand hygiene education on self-reported use of soap or sanitizer was similar regardless of whether the participants lived in mers regions. conclusion. our findings emphasize the importance of education or promotions encouraging hand washing, especially for older adults, women, and lower income earners. in addition, mers outbreak itself affected individuals’ awareness of hand-washing behaviors. well-organized campaigns that consider these factors are needed to prevent infectious diseases. vaccine against this worldwide disease, whose fatality rate is approximately 35% (world health organization, 2017) . proper hand washing is considered the first and most crucial means of preventing mers infection (kcdc, 2017b) . therefore, the mers outbreak may have raised koreans' awareness of the importance of hand hygiene and changed hand-washing behavior. previous studies in korea have focused on hand hygiene performance rates among health care workers (oh, 2015) , elementary school to high school students (yang et al., 2014) , and adults (y.-h. lee et al., 2016) . other studies have investigated the relationship between hand hygiene education and the behaviors of targeted individuals, including nurses (kim & choi, 2002) , undergraduate students (choi, jang, & choi, 2014) , and adolescents (min & chang, 2014) . additionally, one study focused on the national handwashing survey administered to korean adults from 2006 to 2014 (m.-s. lee, hong, & kim, 2015) ; however, this study was conducted before the 2015 mers outbreak in korea. therefore, the present study aimed to examine associations of hand-washing education with self-reported handwashing frequency and self-reported use of soap or hand sanitizer among korean adults during the year of the mers outbreak in korea using a nationally representative survey conducted 3 months after the first confirmed case occurred. we also performed subgroup analyses to evaluate associations of hand hygiene education or promotion with selfreported hand hygiene frequency and self-reported use of soap or hand sanitizer according to age group, sex, income, and mers outbreak region. this study analyzed data from the community health survey (chs) conducted by the kcdc. the chs has been conducted annually since 2008 among individuals aged 19 years and older across the country. we used data from the 2015 chs. the study's population was limited to adults ≥20 years old, and we excluded individuals who were younger than 20 years old (n = 2,616) and individuals with missing data (n = 3,343). therefore, the present study evaluated data from 222,599 individuals. the chs received ethical approval from the institutional review board of the kcdc (irb no. 2014-08exp-09-4c-a), and written consent was obtained from all survey participants. the main variable of interest was hand-washing education and promotion, which was assessed using self-reported data based on responses to the chs question, "did you receive any education on correct hand washing or see promotional materials related to hand washing during the past year?" possible responses were "yes" and "no." the dependent variables were self-reported hand-washing method and frequency. these were evaluated using selfreported data based on responses to the chs questionnaire. to evaluate hand-washing methods, we used the question, "how often do you usually wash your hands with soap or hand sanitizer?" the possible responses were "always," "often," "sometimes," "rarely," and "never." hand-washing method was scored on a 5-point scale from 1 (never) to 5 (always). to evaluate hand-washing frequency, we used three questions: "how often did you wash your hands before having a meal during the past week?" "how often did you wash your hands after using the toilet during the past week?" "how often did you wash your hands after returning from outside during the past week?" each question had four possible responses: "always," "often," "sometimes," and "rarely." hand-washing frequency in each situation was scored on a 4-point scale from 1 (rarely) to 4 (always). the total score for hand-washing frequency was the sum of the three questions; thus, total scores ranged from 3 to 12. the analyses were adjusted for participants' demographic, socioeconomic, and health-related characteristics. the demographic characteristics considered were age group (20-29, 30-39, 40-49, 50-59, 60-69, 70-79, or ≥80 years), sex (male or female), and marital status (married and cohabitating, married and non-cohabitating, or single). the socioeconomic characteristics were income level (low, lower middle, upper middle, or high), education (elementary school and less, middle school, high school, or college and higher), and occupation (white collar [managerial, professional, or clerical] , pink collar [sales and services], blue collar [manual labor], or nonworking). the health-related factor was perceived health status (good, average, or poor). we also adjusted for residence in a mers outbreak region in korea (yes or no), as mers occurred in 43 of the 254 regions where the 2015 chs was conducted ("list of hospitals," 2017). all covariates were treated as categorical variables. the general characteristics of the study participants were examined using t tests and analysis of variance. a multiple linear regression model was used to investigate associations of hand hygiene education with self-reported hand-washing behaviors, including both methods and frequency, among korean adults after adjusting for demographic, socioeconomic, and health-related characteristics. subgroup analyses were also performed, with participants stratified by age, sex, income, and mers outbreak regions. differences were considered statistically significant at p values <.05. effect sizes were calculated using partial eta squared, which refers to the proportion of variance explained. all statistical analyses were performed using sas software (version 9.4, sas institute, cary, nc). table 1 shows the general characteristics of the study population. among the 222,599 participants, 169,662 (76.2%) indicated that they had received education on correct hand washing or had seen hand-washing-related promotional materials within the past year, whereas 52,937 participants (23.8%) reported no exposure to hand-washing education or promotional materials on hand washing. the means and standard deviations (sds) for hand washing with soap or hand sanitizer were 4.23 ± 0.90 among those who had and 3.87 ± 1.04 among those who had not experienced handwashing education, respectively, and the means and sds for hand-washing frequency were 10.36 ± 1.81 and 9.54 ± 2.14, respectively. table 2 shows the factors associated with self-reported hand-washing methods and frequency. the results demonstrate that exposure to hand-washing education or promotional materials was significantly associated with self-reported handwashing behaviors. individuals who had received education on correct hand washing or seen promotional materials related to hand washing within the previous year had significantly higher scores for both self-reported hand-washing methods (β = 0.177, p < .0001) and self-reported hand-washing frequency (β = 0.481, p < .0001) than those who had not had such educational opportunities. men exhibited significantly lower scores for both self-reported hand-washing methods (β = −0.113, p < .0001) and self-reported hand-washing frequency (β = −0.900, p < .0001) than women. with the highest income level set as the reference group, scores for self-reported hand-washing methods (β = −0.030, p < .0001; β = −0.051, p < .0001; β = −0.131, p < .0001) and self-reported frequency (β = −0.063, p < .0001; β = −0.083, p < .0001; β = −0.122, p < .0001) were progressively lower in the following order: upper middle > lower middle > low income, respectively. individuals who lived in mers outbreak regions showed significantly higher scores for both self-reported hand-washing methods (β = 0.081, p < .0001) and self-reported hand-washing frequency (β = 0.154, p < .0001) than did those who lived elsewhere. table 3 shows the results of subgroup analyses stratified by age group, sex, income, and mers outbreak region. the subgroup analyses showed significant differences in each group, although modifying effects of sex on self-reported use of soap or sanitizer and mers outbreak region on both dependent variables were not significant in the tests for interaction. participants in the 30 to 39, 40 to 49, 50 to 59, 60 to 69, 70 to 79, and ≥80 years age groups who had received education on correct hand washing or had seen related promotional materials exhibited higher scores with age for both self-reported hand-washing methods (β = 0.090, p < .0001; β = 0.119, p < .0001; β = 0.161, p < .0001; β = 0.198, p < .0001; β = 0.239, p < .0001; and β = 0.319, p < .0001, respectively) and selfreported hand-washing frequency (β = 0.267, p < .0001; β = 0.387, p < .0001; β = 0.439, p < .0001; β = 0.471, p < .0001; β = 0.603, p < .0001; and β = 0.830, p < .0001, respectively). women who received education on correct hand washing or saw promotional materials related to hand washing showed higher scores in self-reported hand-washing methods (β = 0.211, p < .0001) than did men. women who received education on hand washing or saw promotional materials showed a trend toward a greater magnitude of self-reported hand-washing frequency scores than men. as income level decreased from high to upper middle, lower middle, and low, individuals who reported exposure to hand-washing education or promotional materials exhibited progressively higher scores in both self-reported hand-washing methods (β = 0.111, p < .0001; β = 0.131, p < .0001; β = 0.169, p < .0001; β = 0.247, p < .0001, respectively) and self-reported hand-washing frequency (β = 0.373, p < .0001; β = 0.397, p < .0001; β = 0.480, p < .0001; β = 0.586, p < .0001, respectively). individuals who lived in mers outbreak regions and who had exposure to hand-washing education or promotional materials showed a trend toward a slightly greater magnitude of self-reported hand-washing frequency scores than individuals who did not live in those regions (β = 0.528, p < .0001; β = 0.472, p < .0001, respectively). the results of the present study revealed that self-reported behaviors related to hand-washing methods and frequency were significantly associated with exposure to hand-washing education and promotion. individuals who had received education on correct hand washing or saw related promotional materials within the previous year had higher scores in both self-reported hand-washing methods and self-reported handwashing frequency than did those who did not have such experiences. men, single people, individuals with lower incomes, and individuals who had less education had significantly lower scores for both self-reported hand-washing methods and self-reported hand-washing frequency than did their counterparts. the associations reported here are similar to those in previous studies on hand-washing behaviors in several countries, including associations with hand hygiene education (pittet et al., 2000) , sex (van de mortel, bourke, mcloughlin, nonu, & reis, 2001 ), income, and educational level (y.-h. lee et al., 2016) . interestingly, in our study, individuals who lived in mers outbreak regions showed significantly higher scores for both self-reported hand-washing methods and self-reported hand-washing frequency than did those who lived elsewhere. in korea, the first confirmed mers patient occurred on may 20, 2015, and the number of confirmed patients rapidly increased in june 2015. as of july 4, 2015, no more confirmed cases were reported (ministry of health and welfare, 2016). meanwhile, the 2015 chs that we used in this study was performed from august 31, 2015 , to november 8, 2015 (kcdc, 2017a . since the survey was conducted only a few months after the severe outbreak, the responses to the survey questions were probably affected by individuals' motivation to prevent the disease. a major outbreak of severe acute respiratory syndrome (sars), which is mainly transmitted by respiratory droplets, occurred in 2002 in hong kong. the sars fatality rate was high, and it served to remind individuals living in hong kong of the importance of hand washing; indeed, their self-reported hand hygiene compliance increased during the sars outbreak and remained high nearly 2 years later (fung & cairncross, 2007) . higher self-reported compliance in regions of korea where mers occurred in 2015 might be interpreted as similar to that during the 2002 sars outbreak in hong kong. to avoid contracting mers, individuals who lived in mers outbreak regions were more aware of using soap or hand sanitizer and frequent hand washing than were those who lived outside mers areas. the subgroup analyses of hand-washing education revealed that age, sex, income level, and mers outbreak regions were significantly associated with self-reported hand-washing methods and frequency, although the modifying effects of sex and mers outbreak regions were not significant. regarding age, the effect of hand-washing education or promotion on self-reported hand-washing methods and frequency became stronger with increasing age. generally, older adults have less opportunity to receive education on hand hygiene than do younger generations. thus, once older adults receive education related to hand hygiene or are exposed to hand hygiene campaigns, their awareness of the importance of hand hygiene may increase. the provision of hand hygiene education or promotion in senior centers might be an effective way to raise older adults' hand hygiene awareness. regarding gender, women who received education about correct hand washing or saw promotional materials related to hand hygiene obtained higher scores for both self-reported use of soap or sanitizer and self-reported hand-washing frequency than did men. generally, women are more compliant than are men (lindahl & heimann, 1997) , and they may follow newly learned guidance received through hand hygiene education or promotional materials more easily. thus, the effect on women of hand-washing education or promotion could be stronger than that on men. regarding income level, the effect of hand-washing education or promotion increased as income level decreased. individuals with relatively lower income have less opportunity to receive education on hand hygiene or be exposed to hand hygiene promotions than do those with higher income. greater opportunity to receive education on correct hand washing would likely increase their hand hygiene awareness. regarding mers outbreak regions, individuals who lived in mers outbreak regions and experienced hand-washing education or promotion showed slightly higher scores for self-reported hand-washing frequency than those who did not live in these regions. additionally, the effect of hand hygiene education or promotion on self-reported use of soap or hand sanitizer when washing hands was similar in individuals who lived in mers outbreak regions and those who did not. because the mers outbreak was a seriously disturbing event, individuals who lived in mers regions might already have been aware of the importance of washing with soap or hand sanitizer and might have been more likely to do so to avoid getting the disease. thus, their self-reported behaviors might not have been greatly influenced by hand hygiene education or promotional materials. the present study has several strengths. first, we used chs data gathered by a national institution; these data are more statistically reliable than are those from surveys conducted by private survey institutions. second, our study revealed how specific demographic and socioeconomic factors affected individuals' hand hygiene behaviors. the present study also has several limitations. first, the chs is a cross-sectional survey, and we therefore could not establish a causal relationship between hand-washing education and self-reported hand-washing methods and frequency. second, we used self-reported data to identify hand-washing behaviors, which might have resulted in recall bias. third, selfreported data of socially desirable behavior such as correct hand washing might have resulted in social desirability bias (biran et al., 2008; manun'ebo et al., 1997) . fourth, careful interpretation of statistical significance is needed due to the very large sample size of this study. fifth, we could not distinguish between individuals who received hand hygiene education and those who saw promotional materials related to hand washing because the chs questionnaire asked about both items in a single question. if a later version of the questionnaire could distinguish between hand-washing education and promotion, it might improve our findings regarding the association between hand-washing education and selfreported hand-washing behaviors among korean adults. the present study has several implications. our study indicated that hand-washing education or promotion is important to strengthen individuals' awareness of correct hand-washing behaviors. moreover, targeted hand-washing education or promotion could increase individuals' awareness of hand-washing compliance effectively. thus, wellorganized hand hygiene educational programs or promotional campaigns that address associated demographic and socioeconomic factors we have investigated are needed to prevent infectious diseases. in addition, serious 2015 mers outbreak in korea affected individuals' awareness of proper hand-washing behaviors. regarding these results, further studies on how long the 2015 mers effects on hand washing would be sustained and how to keep these positive effects longer are needed to prevent infectious diseases. comparing the performance of indicators of hand-washing practices in rural indian households effect of a behaviour-change intervention on handwashing with soap in india (superamma): a cluster-randomised trial the effect of handwashing with water or soap on bacterial contamination of hands the effect of an educational hand washing program on knowledge, attitude and performance of hand washing in undergraduates the impact of alcohol hand sanitizer use on infection rates in an extended care facility how often do you wash your hands? a review of studies of hand-washing practices in the community during and after the sars outbreak in 2003 effects on nurses' hand washing behavior and reduction of respiratory isolation rate of mrsa of the hand washing education introduction to community health survey middle east respiratory syndrome handwashing with soap and national handwashing projects in korea related factors to handwashing with soap in korean adults social proximity in early mother-infant interactions: implications for gender differences? infant and child development list of hospitals where confirmed mers patients either stayed or visited effect of handwashing on child health: a randomised controlled trial measuring hygiene practices: a comparison of questionnaires with direct observations in rural zaire educating healthcare workers to optimal hand hygiene practices: addressing the need an experience of personal hygiene education and hand-washing practices among adolescents in the korean youth risk behavior web-based survey the 2015 mers outbreak in the republic of korea: learning from mers analysis of hand hygiene practices of health care personnel a close look at alcohol gel as an antimicrobial sanitizing agent effectiveness of a hospital-wide programme to improve compliance with hand hygiene hand hygiene compliance in healthcare workers effect of education and performance feedback on handwashing: the benefit of administrative support in argentinean hospitals a randomized, controlled trial of a multifaceted intervention including alcoholbased hand sanitizer and hand-hygiene education to reduce illness transmission in the home gender influences handwashing rates in the critical care unit middle east respiratory syndrome coronavirus related factors of handwashing with soap and its practices by students in south korea handwashing with soap or alcoholic solutions? a randomized clinical trial of its effectiveness the authors declared no potential conflicts of interests with respect to the research, authorship, and/or publication of this article. the authors received no financial support for the research, authorship, and/or publication of this article. jieun yang https://orcid.org/0000-0002-6065-0940 eun-cheol park https://orcid.org/0000-0002-2306-5398 key: cord-324165-afdmsbw2 authors: joo, heesoo; henry, ronald e.; lee, yeon-kyeng; berro, andre d.; maskery, brian a. title: the effects of past sars experience and proximity on declines in numbers of travelers to the republic of korea during the 2015 mers outbreak: a retrospective study date: 2019-08-31 journal: travel medicine and infectious disease doi: 10.1016/j.tmaid.2019.05.009 sha: doc_id: 324165 cord_uid: afdmsbw2 abstract background the experience of previous sizable outbreaks may affect travelers’ decisions to travel to an area with an ongoing outbreak. methods we estimated changes in monthly numbers of visitors to the republic of korea (rok) in 2015 compared to projected values by selected areas. we tested whether areas’ experience of a previous sars outbreak of ≥100 cases or distance to the rok had a significant effect on travel to the rok during the mers outbreak using t-tests and regression models. results the percentage changes in visitors from areas with a previous sars outbreak of ≥100 cases decreased more than the percentage changes in visitors from their counterparts in june (52.4% vs. 23.3%) and july (60.0% vs. 31.4%) during the 2015 mers outbreak. the percentage changes in visitors from the close and intermediate categories decreased more than the far category. the results from regression models and sensitivity analyses demonstrated that areas with ≥100 sars cases and closer proximity to the rok had significantly larger percentage decreases in traveler volumes during the outbreak. conclusions during the 2015 mers outbreak, areas with a previous sizable sars outbreak and areas near the rok showed greater decreases in percentage changes in visitors to the rok. the 2015 middle east respiratory syndrome (mers) outbreak in the republic of korea (rok) was initiated by the arrival of a single infected international traveler who visited the middle east [1] . the first case was confirmed on may 20, 2015 , and the outbreak resulted in 186 cases and 38 deaths before the government of the rok and the world health organization (who) officially announced the end of the outbreak on december 23, 2015 [2] . during the 2015 mers outbreak, the rok government conducted entry screening and active monitoring of all travelers from the middle east region to avoid additional importations of mers cases [2] . in addition, the rok government restricted international departures by confirmed or suspected mers patients or their contacts to prevent the global spread of mers [2] . although who did not recommend any travel restrictions or entry screening for mers coronavirus (mers-cov) [3] , the numbers of international travelers visiting the rok decreased significantly during the 2015 mers outbreak [4, 5] . fear-induced behavioral changes during novel infectious disease outbreaks have been reported to depend on an individual's perception of risk rather than the actual risk [6] . one factor that could affect an individual's perception of the risk of an infectious disease outbreak is the individual's previous experience of a similar infectious disease outbreak. governments of countries that had a similar sizable outbreak, as well as individuals from those countries, may have responded differently to the rok mers outbreak than governments or individuals who had less exposure to a similar sizable outbreak. this evaluation estimates the changes in numbers of non-citizen short-term visitor arrivals from selected areas to the rok during the 2015 mers outbreak and examines the correlation between travel volume declines and previous experience of the most similar sizable outbreak, the 2003 severe acute respiratory syndrome (sars) outbreak. we also examined the correlation between decrease in travel volume and proximity to the rok. organization [7] . the numbers of arrivals reported in this analysis are limited to the numbers of non-citizen short-term visitor arrivals. each area includes one country or political unit. note that area information in the data set is based on travelers' passports rather than the areas from which travelers departed. first, we selected areas comprising more than 0.5% of total non-citizen visitor arrivals, i.e. more than 70 000 visitors to the rok during 2014. selected areas in descending order of numbers of arrivals to the rok during 2014 are china (mainland china excluding hong kong special administrative region (sar), macau, and taiwan), japan, united states (usa), taiwan-china, hong kong sar-china, thailand, philippines, malaysia, russia, indonesia, singapore, india, canada, vietnam, australia, united kingdom (uk), germany, and france. they combined to contribute 94% of the total number of arrivals to the rok during 2014. monthly actual numbers of arrivals in 2015 by area from tourgo are shown in appendix table 1 . where "i" stands for each area, while "j" stands for each month during the rok mers outbreak from june 2015 to december 2015. if the actual value is smaller than the projected value in a given area-month, i.e. decreasing arrivals associated with the mers outbreak, the negative value y ij corresponds to the estimated percentage decrease in visitor arrivals. the number of travelers for each area in each month were treated as a single observation. we assumed that any changes of numbers of arrivals caused by the mers outbreak would be observed starting in june because the first mers case was confirmed on may 20, 2015. we chose two main variables that could affect individuals' decisions to travel to an outbreak area. the first variable was whether an area had a sizable outbreak of limited duration of a novel viral respiratory disease before the 2015 mers outbreak. the 2003 sars outbreak was chosen because it was sizable including local transmission and its duration was limited [8] . during the 2003 sars outbreak, more than 8000 individuals were infected worldwide with a 9.6% average case fatality rate [9] . in addition, sars and mers are both viral respiratory infections caused by coronaviruses, although the case fatality rates reported for sars were lower than for mers [10] . community transmission of sars was more commonly reported, while mers transmission in the rok occurred primarily in healthcare settings or through close contact, e.g. caring for or living with an infected person [10, 11] . the 2009 h1n1 influenza pandemic was also caused by viral respiratory infection; however, country-specific h1n1 attack rates were orders of magnitude higher and its case fatality rate was much lower compared to the limited-duration outbreaks of sars and mers [10, 12, 13] . using who data for reported numbers of probable sars cases by area during the 2003 outbreak [9] , we hypothesized that individuals from areas with ≥100 sars cases during the 2003 outbreak may have been significantly more or less likely to visit the rok during the 2015 mers outbreak. china, taiwan-china, singapore, canada, and hong kong sar-china each had ≥100 sars cases during the 2003 outbreak, and all of those areas were included in the current analyses. although the threshold was set at 100 probable sars cases, each of the five areas experienced at least 238 probable sars cases, while the country/area with next highest number of probable sars cases, vietnam, had 64 cases (appendix figure 3 ) [9] . thus, our results would be consistent with any threshold set between 65 cases and 237 who-reported probable cases. the second variable we considered was travel distance to the rok from each area. since travel time cost is likely to be correlated to travel distance between two areas, the opportunity cost to change travel plans from a more distant place to the rok would be expected to be higher. thus, individuals from more distant areas might be less likely to cancel trips to the rok during the outbreak period. we subdivided selected areas into three groups based on distance to the rok: (1) close (china, japan, taiwan-china, and hong kong sar-china), (2) intermediate (thailand, philippines, malaysia, indonesia, singapore, india, and vietnam), and (3) far (usa, russia, canada, australia, uk, germany, and france). more details about how we defined distance categories for analyses are shown in appendix tables 2 and 3 for both variables, we performed welch's t-tests for two samples with unequal variances [14] to examine whether the monthly average percentage decreases in numbers of arrivals from each category differed significantly. for the sars variable, the null hypothesis is that the simple average percentage change of arrivals from areas with < 100 sars cases (reference) was the same as the percentage change of arrivals for areas with ≥100 sars cases. the alternative hypothesis is that the percentage decreases for arrivals from areas with ≥100 sars cases were larger than for areas with < 100 sars cases. for the distance categories, the null hypothesis is that the simple average percentage change of arrivals from the far category (reference) was the same as the percentage change of arrivals from the intermediate or the close category. the alternative hypothesis is that the percentage decreases for arrivals from areas in the far category were smaller than for arrivals from the areas in the intermediate or the close categories. when p-values from welch's t-tests are < 0.05, we reject the null hypotheses in favor of the alternative hypotheses. we used ordinary least squares (ols) regression models to examine the impact of each factor on percentage changes in numbers of visitor arrivals for each month during the rok mers outbreak (june-december 2015). the following equation was used in an ols regression model. is a dependent variable that shows percentage changes in numbers of arrivals by area-month. "sars i " is a dummy variable, which is one an area "i" reported ≥100 sars cases during the 2003 outbreak, otherwise zero. "close i " is a dummy variable, which is one when an area "i" is in the "close" category, otherwise zero. "intermediate i " is an additional distance dummy variable. when an area "i" is in the "intermediate" category, the variable is one, otherwise zero. in summary, the ols model included two independent variables: 1) a binary variable to identify areas with ≥100 sars cases during the 2003 outbreak (reference category: < 100 sars cases during the 2003 outbreak), and 2) a categorical variable summarizing the distance between an area and the rok that was transformed into two binary variables (reference category: "far"). we conducted sensitivity analyses using alternative projected values. the baseline model used the average numbers of arrivals in 2013 and 2014 by area to project the expected numbers of arrivals to the rok in the absence of the outbreak. the baseline model, however, may not account for year-to-year increasing or decreasing trends in the numbers of arrivals to the rok. that omission could result in underestimated or overestimated projected values in the absence of the mers outbreak, causing biased estimates from regression models. thus, we examined two alternative models. for alternative 2, we again started with the monthly average numbers of arrivals in 2013 and 2014. since the rok mers outbreak was not expected to affect travel volumes during the pre-outbreak period in 2015, we compared the arrivals from january to may 2015 to the average number of arrivals from january to may in 2013 and 2014 to calculate an area-specific adjustment to the projected numbers of arrivals from june to december 2015 in the absence of the outbreak. alternative 2 used the same method that was used to compare projected and actual numbers of arrivals in mexico during the 2009 h1n1 pandemic [15] . to avoid bias, we conducted additional sensitivity analyses by excluding outliers. potential outliers were flagged if any area-month (y ij ) observation was ≥100% or ≤ -100%. we only identified one observation that met these outlier criteria. we excluded the observation for india in august 2015, when an exceptional increase in the number of crewmembers arriving at seaports [16] resulted in an increase of 106.4% relative to the average of arrivals for india in august 2013 and 2014. decreases in numbers of arrivals to the rok were observed during june and july 2015 across all examined areas compared to the average numbers of arrivals in 2013 and 2014 (table 1 ). in june, the number of arrivals decreased by 7.5% from the least affected area and 74.2% from the most affected area. the simple average decrease across 17 areas was 31.4%. in july, the simple average decrease in numbers of arrivals was 39.3% (range 4.8%-82.0% across areas). although travel volumes declined across all areas during june and july, actual traveler volumes for some areas exceeded projected values starting in august and continuing through december 2015 (appendix figures 1 and 2) . table 2 shows that the percentage decrease in numbers of arrivals from areas with ≥100 sars cases during the 2003 sars outbreak was significantly greater than the decrease from areas with < 100 sars cases in june (52.4% vs. 23.3%), july (60.0% vs. 31.4%), and august 2015 (28.5% vs. 3.8%). in comparison, during the pre-outbreak january to may period, the simple averages from both groups showed higher numbers of arrivals than the averages for 2013 and 2014 ( fig. 1 ). the numbers of arrivals from areas with a previous sars outbreak with ≥100 cases were 21.7%-38.5% higher in january-may 2015 than in january-may averages for 2013 and 2014, while the simple averages from areas with < 100 sars cases were only 1.7%-15.4% higher in january-may 2015 ( table 2) . the percentage changes in arrivals from areas in the close category decreased more than the percentage changes in arrivals in the far category in june (56.1% vs. 19.5%), july (69.9% vs. 17.1%), and august table 1 percentage changes between 2015 actual and projected (average of 2013 and 2014) monthly non-citizen arrivals to the republic of korea (rok) by area (%). . 2 and table 3 ). in addition, the percentage changes in arrivals in the intermediate category decreased more than the percentage changes in arrivals in the far category in july (44.0% vs. 17.1%). changes in all other months did not show significant differences across distance categories. the results from baseline ols regression models also identified statistically significant correlations between the percentage decrease of numbers of arrivals and previous sars outbreak of ≥100 cases (table 4) . areas with a previous sars outbreak of ≥100 cases showed significant correlations in june (p-value = 0.032). also, the correlation between the percentage decrease of numbers of arrivals and distance from the rok was statistically significant. the significant percentage decreases in numbers of visitor arrivals occurred in areas in the close category in june (p-value = 0.012) and july (p-value≤ 0.001), and in the intermediate category in july (p-value≤ 0.001) . results from sensitivity analyses were consistent with the baseline analysis. for baseline and alternatives 1, the percentage changes of arrivals from areas with a previous sars outbreak of ≥100 cases showed greater decreases than the percentage changes in arrivals from their counterparts in june, july, and august 2015 (appendix table 4 ). for alternative 2, the differences between areas with and without a sars outbreak of ≥100 cases were significant in every month from june through december 2015, except for september. for both alternatives, declines in numbers of arrivals from areas in the close category were larger than for areas in the far category from june to august 2015 (appendix table 5 ). for alternative 1, the declines in arrivals in the intermediate category were significantly larger than the declines in arrivals from the far category from april through december 2015, except for august. however, if we instead used alternative 2, the decline in numbers of arrivals from the intermediate distance category was statistically significantly greater than the far category only in april, june, and july 2015. the results from sensitivity analyses of regression models were also consistent with the results from baseline models. using all three projected values, the percentage decrease of arrivals in june and july showed some significant correlations with proximity and previous sars outbreaks of ≥100 cases (appendix table 6 ). when excluding outliers, the observation for india in august 2015, the difference in the percentage changes in arrivals between the intermediate and far categories was significant; however, when the india, august 2015 observation was included in the analysis (i.e. not excluded), the difference between the intermediate and far categories was no longer significant (appendix table 7 ). results from the baseline and sensitivity analyses found that the 2015 mers outbreak in the rok appeared to have a greater effect on travel volumes from areas with ≥100 sars cases in 2003, especially in june and july 2015. most of the rok mers cases were confirmed during those 2 months, and the rok government declared a de facto end to the outbreak on july 28, 2015 by which time no individuals were quarantined because of mers, nor were there any newly confirmed cases [2] . the decrease of arrivals during the 2015 mers outbreak is underscored when we compare the trends of numbers of arrivals before and during the 2015 mers outbreak. positive values in tables 1-3 (i.e. increases in arrivals) before the 2015 mers outbreak reflect the general trend that the number of arrivals to the rok would be increasing without the outbreak. visitors from areas with a previous sars outbreak of ≥100 cases may have been more likely to change their travel plans because of similarities between sars and mers, which are both caused by coronaviruses and capable of causing serious respiratory impairment. in addition, the governments in areas that experienced previous sars outbreaks may have had stronger restrictions or recommendations about travel to the rok during the mers outbreak period. although who did not recommend any travel restrictions to the rok during the 2015 mers outbreak [17] , who noted that raising awareness about table 4 monthly marginal percentage change between actual (2015) and baseline projected (average of 2013 and 2014) non-citizen arrivals to the republic of korea (rok) associated with areas that experienced ≥100 probable severe acute respiratory syndrome (sars) cases and distance to the republic of korea. notes: non-citizen arrivals are limited to non-citizen short-term visitor arrivals. boldface indicates statistical significance (p < 0.05). the results were from baseline ordinary least squares (ols) regression models using percentage changes between 2015 actual and the average of 2013 and 2014 monthly arrivals as dependent variables. the reference category for the 2003 sars outbreak areas with < 100 probable sars cases, and the reference for distance is the far category. mers among those traveling to affected areas was good public health practice [18] . some areas issued travel notices to inform travelers about the potential risks and risk mitigation strategies. although the intent of notices was not to restrict travel, such notices may have raised additional concern among travelers, who may then have decided to cancel their travel plans. for instance, hong kong sar, which showed the biggest percentage difference between projected and actual arrivals to the rok, had experienced a sizable sars outbreak in 2003 with 1755 cases [9] . hong kong sar issued a red outbound travel alert (ota) for the rok on june 9, 2015 [19] . hong kong's red ota recommends to adjust travel plans or avoid non-essential travel and is the second highest level travel notice [20] . the alert was lifted on august 1, 2015 [21] . in addition, the travel industry council of hong kong canceled all tours, excluding cruises, to the rok that were scheduled to depart from june 9 to the end of june in 2015 [22] . since only about 2% of travelers from hong kong sar to the rok arrive via cruise [7] , most tourists from hong kong sar were affected by the cancellation. in addition, some local governments in mainland china (sichuan and shandong provinces, and guangzhou, the capital of guangdong province) issued travel notices to the rok because of the 2015 mers outbreak [23] . also, some flights between china and jeju international airport in the rok were cancelled [24] . taiwan also experienced a sizable sars outbreak in 2003, and the taiwan centers for disease control (cdc) issued a level 2 travel notice (alert) for seoul and a level 1 travel notice (watch) for all other areas in the rok on june 2, 2015 [25] . the level 2 travel notice was extended to the entire rok on june 9, 2015 [26] . the notice included a recommendation that travelers avoid unnecessary hospital visits in the rok [26] . the taiwan cdc lowered its travel notice to level 1 on july 7, 2015 [27] , then removed the notice on july 28, 2015 [28] . the taiwan ministry of foreign affairs also issued a travel alert for mers to the rok [29] . the experience with sars outbreaks in 2003 is only one potential reason that governments with previous sars outbreak experiences may have issued stronger restrictions or recommendations about travel to the rok during the mers outbreak than governments without previous sars outbreak experiences. for example, a korean traveler who was experiencing symptoms consistent with mers pushed ahead with his plan to travel to guangdong, china with a layover in hong kong sar [17] . when this traveler was diagnosed with mers after arrival in china, a public health response was required in china [2] . although there was no further mers transmission in china or hong kong sar, public awareness of this imported case may have exacerbated fear among individuals in hong kong sar and guangdong as well as their local governments. in taiwan, a patient with suspected mers was reported on may 30, 2015, although this patient was later confirmed to have influenza b virus infection [30] . this suspected case was one of 17 suspected mers cases reported in taiwan between september 2012 and may 2015 [30] . the 17th suspected case was in a person who was neither a rok citizen nor had visited the rok [30] . however, the patient could have increased public awareness of mers in taiwan. although countries issued varied levels of travel notices, no countries enforced travel bans or strict quarantine requirements for travelers from the rok as some did for travelers from countries with ebola during the west african epidemic [31] . since the 2015 mers outbreak ended, the rok government has continuously made efforts to prevent future mers outbreaks in the rok. the korea centers for disease control & prevention updated its national prevention and control guidelines for mers in april 2016 [32] . these guidelines include response plans at airports, hospitals, and local health departments to expedite the identification of individuals suspected of mers-cov infection after arriving from mers-affected areas [32] . following the guidelines, virological testing for mers-cov and other respiratory viruses has been implemented to confirm whether individuals with recent travel to mers-affected areas and symptoms consistent with mers are infected with mers-cov or not [32, 33] . also, the rok's amended quarantine act specifies mers as a quarantinable infectious disease [34] . these efforts may decrease the risk of future mers outbreaks in the rok. this analysis has some limitations. first, we assumed that the nationality of an individual was a proxy for the area of departure for that individual. for instance, a japanese national may live in the united states before visiting the rok but would be counted as if traveling from japan (close category) for our analysis. such discrepancies between citizenship and residency might cause some bias in examining the effect of distance on travel volumes to the rok during the mers outbreak. next, we were limited in our ability to assess the mechanism by which a previous sizable outbreak experience would affect travel to the rok. as we discussed, issuance of travel notices may correlate with past outbreak experience in that governments in countries or localities that previously experienced outbreaks may be more likely to issue stronger notices. in addition, stronger travel notices in some countries may have correlated with greater decreases in travel volumes during the mers outbreak in the rok. however, we were unable to create categorical variables for issuance of travel notices because each country has its own process and terminology for travel notices. although many countries have similar structures for level of travel notices, they are not easily comparable. in addition to level, each travel notice contains outbreakspecific recommendations. recommendations, such as avoiding unnecessary hospital visits while in the rok, further complicate comparisons of the intensity of travel health notices among countries. thus, we were unable to quantify differences between stronger and weaker travel notices across countries. also, there could be other country-or area-specific factors, which could affect travel volumes during a disease outbreak, but were not considered in the current analyses. last, we chose only one previous outbreak, the 2003 sars outbreak as an indicator for previous outbreak experiences. while sars was also caused by a novel coronavirus resulting in respiratory disease similar to mers, exposure to other sizable outbreaks, such as the 2009 h1n1 pandemic or the 2013-2015 ebola epidemic, may also have affected governments' and individuals' perceptions of risk from the 2015 mers outbreak. although there was a large difference between the top five areas with at least 238 probable sars cases for each and the next highest country with 63 probable sars cases during the 2013 outbreak (appendix figure 3) , a single threshold of ≥100 sars cases were used. also, a threshold of ≥100 sars cases was used without considering other potentially critical factors, such as population size, the extent of geographic area, the time duration of the sars outbreak, or extent of unlinked community transmission. for instance, both canada and singapore had ≥100 sars cases in 2003. however, more than 95% of probable sars cases in canada (n = 247) were observed in the greater toronto area [35] , a small part of canada, while the sars outbreak in singapore (n = 238) was over the entire country [9] . thus, canadian governments' and individuals' perceptions of risk from the 2015 mers outbreak associated with their sars outbreak experience could be different from the perception of risk from the 2015 mers outbreak in singapore. during the 2015 mers outbreak, the numbers of arrivals to the rok from around the world decreased. countries with a previous sizable sars outbreak and countries closer to the rok had the greatest percentage decreases in travel volumes to the rok. this is the first analysis that has examined area-specific impacts on travel volume to an outbreak area as we know. future research using different outbreak situations may improve our understanding of the effects of outbreaks on travel volume. appendix table 6 monthly marginal percentage change of non-citizen arrivals to the republic of korea (rok) during 2015 from areas associated with ≥100 probable severe acute respiratory syndrome (sars) cases and by distance to the rok based on projected values from regression models-sensitivity analyses using different models to project values. notes: non-citizen arrivals are limited to non-citizen short-term visitor arrivals. boldface indicates statistical significance (p-value < 0.05). middle east respiratory syndrome coronavirus outbreak in the republic of korea korean ministry of health and welfare. the 2015 mers outbreak in the republic of korea: learning from mers middle east respiratory syndrome coronavirus (mers-cov) fact sheet trends in non-resident arrivals, citizen departure, and tourism revenue and expenditure in economic impact of the 2015 mers outbreak on the republic of korea's tourism-related industries responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management tourgo (tourism knowledge and information system) summary table of areas that experienced local transmission of sars during the outbreak period from 1 world health organization. summary of probable sars cases with onset of illness from 1 severe acute respiratory syndrome vs. the middle east respiratory syndrome transmission characteristics of mers and sars in the healthcare setting: a comparative study estimating age-specific cumulative incidence for the 2009 influenza pandemic: a meta-analysis of a(h1n1)pdm09 serological studies from 19 countries estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study the generalisation of student's problems when several different population variances are involved the economic impact of h1n1 on mexico's tourist and pork sectors trends in non-resident arrivals, citizen departure, and tourism revenue and expenditure in world health organization. summary and risk assessment of current situation in republic of korea and china world health organization. who statement on the ninth meeting on the ihr emergency committee regarding mers-cov the government of the hong kong special administrative region. press releases: government issues red outbound travel alert on korea the government of the hong kong special administrative region security bureau. outbound travel alert the government of the hong kong special administrative region. press releases: government removes red outbound travel alert for korea hong kong issues 'red alert' against south korea travel due to mers travel warnings to the republic of korea due to the mers outbreak from all foreign countries are lifted: response activities, achievements, and future plans for the mers outbreak from ministry of foreign affairs airlines cut flights to south korea as china steps up mers alert in light of current mers-cov outbreak in south korea, taiwan cdc issues travel notice level for seoul to level 2: alert for mers-cov and plans five major response strategies travel notice of level 2: alert for mers-cov to entire south korea issued in response to growing mers outbreak in south korea; public urged to avoid unnecessary hospital visits in south korea taiwan cdc lowers travel notice level for south korea to level 1: watch for mers and advises travelers visiting china to take precautions against tick bites taiwan cdc removes mers travel notice for south korea and bhrain as outbreak in south korea brought under control foreign ministry raises travel advisory for seoul test results of taipei suspected mers-cov case came back negative countries' response to who's travel recommendations during the 2013-2016 ebola outbreak prevention and control guideline for middle east respiratory synderome coronavirus surveillance and public health response for travelers returning from mers-cov affected countries to gyeonggi province statutes of the republic of korea, quarantine act, chapter i general provisions, article 2 (definition) coordinated response to sars appendix fig. 3 . numbers of probable severe acute respiratory syndrome (sars) cases by area (november 1, 2002-july 31, 2003) . notes: numbers of probable sars cases by area are from the world health organization (https://www.who.int/csr/sars/country/table2004_04_21/en/). appendix table 1 actual numbers of monthly non-citizen arrivals in the republic of korea in 2015 by area table 4 monthly percentage changes between actual and projected non-citizen arrivals to the republic of korea (rok) during 2015 by whether an area had 100 or more severe acute respiratory syndrome (sars) cases during the 2003 outbreak-sensitivity analyses using different projected values for the number of expected travelers in the absence of a middle east respiratory syndrome (mers) outbreak appendix table 5 monthly key: cord-318315-r6wqywwe authors: memish, ziad a.; almasri, malak; turkestani, abdulhafeez; al-shangiti, ali m.; yezli, saber title: etiology of severe community-acquired pneumonia during the 2013 hajj—part of the mers-cov surveillance program date: 2014-06-23 journal: int j infect dis doi: 10.1016/j.ijid.2014.06.003 sha: doc_id: 318315 cord_uid: r6wqywwe background: pneumonia is the leading cause of hospital admission during the annual islamic pilgrimage (hajj). the etiology of severe pneumonia is complex and includes the newly emerged middle east respiratory syndrome coronavirus (mers-cov). since 2012, the saudi ministry of health (moh) has required screening for mers-cov for all cases of severe pneumonia requiring hospitalization. we aimed to screen hajj pilgrims admitted to healthcare facilities in 2013 with severe community-acquired pneumonia (cap) for mers-cov and to determine other etiologies. methods: sputum samples were collected from all pilgrims admitted to 15 healthcare facilities in the cities of makkah and medina, saudi arabia, who were diagnosed with severe cap on admission, presenting with bilateral pneumonia. the medical records were reviewed to collect information on age, gender, nationality, and patient outcome. samples were screened for mers-cov by pcr, and a respiratory multiplex array was used to detect up to 22 other viral and bacterial respiratory pathogens. results: thirty-eight patients met the inclusion criteria; they were predominantly elderly (mean age 58.6 years, range 25–83 years) and male (68.4%), and all were from developing countries. fourteen of the 38 patients died (36.8%). mers-cov was not detected in any of the samples. other respiratory pathogens were detected in 26 (68.4%) samples. of these, bacterial pathogens were detected in 84.6% (22/26) and viruses in 80.7% (21/26). twenty-one (80.7%) samples were positive for more than one respiratory pathogen and 17 (65.3%) were positive for both bacteria and viruses. the most common respiratory virus was human rhinovirus, detected in 57.7% of the positive samples, followed by influenza a virus (23.1%) and human coronaviruses (19.2%). haemophilus influenzae and streptococcus pneumoniae were the predominant bacteria, detected in 57.7% and 53.8%, respectively, of the positive samples, followed by moraxella catarrhalis (36.4%). conclusions: mers-cov was not the cause of severe cap in any of the hospitalized pilgrims investigated. however we identified a variety of other respiratory pathogens in the sputum of this small number of patients. this indicates that the etiology of severe cap in hajj is complex with implications regarding its management. severe community-acquired pneumonia (cap) is not uncommon during hajj. mandourah and colleagues investigated all critically ill patients, who were of over 40 nationalities, admitted to 15 hospitals in two cities in the 2009 and 2010 hajj seasons. 3 pneumonia was the primary cause of critical illness in 27.2% (123 cases) of all icu admissions and occurred most commonly in the second week of hajj, corresponding to the period of greatest pilgrim density. severe cap accounted for 18.1% of all icu admissions. worldwide, pneumonia is a common illness that is potentially life-threatening, especially in older adults and those with comorbid diseases. 7, 8 the etiology of pneumonia differs between and within countries depending on regional differences in prevalence and types of microorganism, and other factors such as the frequency of use of antibiotics, environmental pollution, awareness of the disease, and life-expectancy of the population. 9 the etiology may also differ depending on whether the pneumonia is community-or hospital-acquired. 7, 8 in this context, the hajj is a special case as it brings a large number of people, many elderly with underlying diseases, from various regions of the world, into close proximity to perform physically exhausting religious rights. these factors, combined with the common use of antibiotics among pilgrims, make the etiology of pneumonia during hajj complex, and hence standard guidelines for the management of the disease may not always work during this mass-gathering event. although many pathogens have been associated with pneumonia, a small range of key pathogens are usually the cause of most cases. 7, 10 in recent years, the middle east respiratory syndrome coronavirus (mers-cov) has also emerged as a cause of serious illness including severe pneumonia. 11 since the first reported case of mers in saudi arabia in 2012, 12 the saudi ministry of health (moh) has set up an ongoing mers-cov surveillance system. as part of this surveillance, it is required that all cases of severe cap with bilateral pneumonia requiring hospitalization are investigated for mers-cov. hence, we used molecular techniques to screen the sputum of hajj pilgrims diagnosed with severe cap requiring hospitalization in 2013 for the presence of mers-cov. other etiologies were also investigated using a respiratory multiplex array to detect bacterial and viral respiratory pathogens. all pilgrims attending the 2013 hajj who were admitted to 15 healthcare facilities in the cities of makkah and medina, saudi arabia, and diagnosed on admission with bilateral pneumonia, were included in the study. the medical records of the patients were reviewed to collect information on age, gender, nationality, and patient outcome. during the period 26 september to 2 november 2013, sputum samples were collected from each patient on admission, prior to any antibiotic therapy. samples were kept refrigerated until processing. mers-cov was detected in the samples using reverse transcriptase polymerase chain reactions (rt-pcr) targeting the region upstream of the e gene (upe) and the open reading frame (orf) 1a (nsp6 protein), as described previously. 13, 14 briefly, nucleic acid was purified from a 200-ml volume of sample using the magna pure lc nucleic acid extraction kit (roche, in, usa). each sample was independently tested with the two rt-pcr assays in a 25-ml reaction containing 5 ml of rna, 12.5 ml of 2x buffer (superscript iii one-step rt-pcr with platinum taq (invitrogen, ny, usa)), 0.4 ml of mgcl 2 (50 mm), 1 ml of forward primer (10 mm), 1 ml of reverse primer (10 mm), 1 ml of probe (5 mm), 3 we collected sputum samples from all pilgrims hospitalized in 15 hospitals of two cities in saudi arabia who were diagnosed with severe cap during the 2013 hajj season. thirty-eight patients fulfilled the inclusion criteria; they were predominantly elderly (mean age 58.6 years, range 25-83 years) and male (68.4%). all patients were from developing countries, the majority of whom (78.3%) were from asia. the nationalities most represented were indonesia (32.4%), pakistan (18.9%), and india (10.8%). of the 38 patients, 30 (78.9%) required icu admission. fourteen (36.8%) patients died, while the remaining patients recovered and were discharged. the mortality rate among those admitted to the icus was 46.6%. mers-cov was not detected in any of the sputum samples. other respiratory pathogens were detected in 26 (68.4%) of the 38 samples, while the remaining samples were negative for the 22 respiratory pathogens in the testing panel (table 1 ). of the positive samples, bacterial pathogens were detected in 84.6% (22/26) and viruses in 80.7% (21/26). twenty-one (80.7%) samples were positive for more than one respiratory pathogen and 17 (65.3%) were positive for both bacteria and viruses. the most common respiratory virus was human rhinovirus, which was detected in 57.7% of the positive amples, followed by influenza a virus (23.1%) and human coronaviruses (19.2%). h. influenzae and s. pneumoniae were the predominant bacteria, detected in 57.7% and 53.8%, respectively, of the positive samples, followed by m. catarrhalis (36.4%). respiratory tract infections are common illnesses during the hajj, 15 and pneumonia is the leading cause of hospital admission, including admission to the icu, during the pilgrimage. [1] [2] [3] [4] 16 for instance, a study of hospital admissions in makkah and mina during the 2002 hajj reported that 39% of hospitalizations were for pneumonia. 16 in the current study, as part of the saudi moh mers-cov surveillance, we investigated the etiology of severe cap in pilgrims attending the 2013 hajj requiring hospitalization. most of the 38 patients were elderly, with a large proportion of males, and all were from developing countries. these observations are similar to those of previous reports investigating pneumonia during hajj. 17, 18 for example, alzeer and colleagues investigated 64 patients admitted with pneumonia to hospitals in the 1994 hajj season. 17 nearly all patients were from developing countries; their mean age was 63 years (range 21-91 years) and 75% were males. the overall mortality rate among the patients we investigated was 36.8%, and among those admitted to icus was 46.6%. internationally, the reported mortality of patients with severe cap requiring icu admission is over 30% and the long-term mortality of cap is between 35.8% and 39.1% at 5 years. 8, 19, 20 our results are in agreement with these figures. a few investigations have reported the mortality rates from pneumonia during hajj. one study 5 during the 1986 hajj season reported a pneumonia case fatality rate of 34%, while another 17 reported a mortality rate of 17% among 64 patients admitted to hospitals in the 1994 hajj season. mandourah and colleagues investigated severe pneumonia during the 2009 and 2010 hajj seasons. 3 pneumonia was community (hajj)-acquired in 66.7% of cases and the overall short-term mortality (during the 3 weeks of hajj) was 19.5%. most patients with diagnosed cap are treated empirically and the role of microbiological testing for patients with cap is still a matter of debate. 7 however there is a clear rationale for establishing the causative agent to allow the optimal selection of agents against a specific pathogen and to limit the misuse of antibiotics and its consequences; it is also important to identify pathogens associated with notifiable diseases such as legionnaires' disease and tuberculosis. 21 the possible involvement of mers-cov is an additional, current, reason. knowledge of the etiological agent of pneumonia-related illness is a challenging step in the management of pneumonia in hajj. [1] [2] [3] 17 in general, the identification of the etiology of cap remains difficult in any setting despite advances in microbiological and serological methods. molecular diagnostic tests for common and atypical causative pathogens of cap are now available and have increased the diagnostic yield and decreased the time required to render results dramatically. [22] [23] [24] although many pathogens have been associated with cap, a small range of key pathogens are the cause of most cases. internationally, the predominant pathogen in cap is s. pneumoniae. 7,10 other causative agents include, but are not limited to, h. influenzae, m. pneumoniae, c. pneumoniae, legionella spp, chlamydia psittaci, coxiella burnetii, enteric gram-negative bacteria (enterobacteriaceae), pseudomonas aeruginosa, staphylococcus aureus, anaerobes, and respiratory viruses (influenza virus, adenovirus, respiratory syncytial virus, parainfluenza virus, coronavirus). 7, 8 the frequencies of other causes, such as mycobacterium tuberculosis, c. psittaci, c. burnetii, francisella tularensis, and endemic fungi (histoplasmosis, coccidioidomycosis, blastomycosis) vary between epidemiological settings. 7 recently, mers-cov has also emerged as a cause of serious illnesses including pneumonia and is the subject of worldwide concern. 11 the primary objective of the study was to determine if mers-cov was the cause of the severe pneumonia in the hospitalized patients. our results indicate that mers-cov was not the etiological agent of the illness. these results support previous reports suggesting that mers-cov has not so far been problematic during hajj. a study conducted during the 2013 hajj, the same year as our study, found no evidence of mers-cov nasal carriage among 5235 hajj pilgrims screened. two reports on french pilgrims during the 2012 and 2013 hajj seasons also reported a lack of mers-cov nasal carriage among the pilgrims screened despite a high rate of respiratory symptoms. 25, 26 we found s. pneumoniae to be prevalent in the sputum samples. this is in accordance with many international reports. 7,10 studies performed during previous hajj seasons have reported the organism as a cause of respiratory tract infections including penumonia. 3, 17, 18 for example, among 395 sputum samples collected from hajjis with respiratory tract infections in 1991 and 1992, s. pneumoniae was detected in 4.8% and 12.3%, respectively. 27 among the 64 patients with pneumonia admitted to two tertiary hospitals in makkah during the 1994 hajj, s. pneumoniae was detected in 9.4% of the cases. 17 3 in addition to s. pneumoniae, other common pathogens identified in our sputum samples were h. influenzae, m. catarrhalis, and viral agents, in particular human rhinovirus, influenza a virus, and human coronaviruses. studies from the gulf corporation council (gcc) states have found similar results. the common pathogens causing cap in gcc states were found to be s. pneumoniae, h. influenzae, and m. catarrhalis. 28, 29 in addition, the importance of atypical pathogens including m. pneumoniae, c. pneumoniae, and l. pneumophila in the etiology of cap in the gcc region has been documented. 28 other etiologies, particularly influenza viruses, varicella zoster virus, and m. tuberculosis, are increasingly recognized as causative pathogens of cap within the region. 28 in the context of hajj, in addition to s. pneumoniae, a number of other organisms have been reported as the cause of pneumonia. these include influenza a (h1n1), 3 m. tuberculosis, 1,3,17,18 s. aureus, 3 fungi such as candida albicans, 3, 18 and gram-negative organisms including p. aeruginosa, l. pneumophila, acinetobacter sp, and members of the enterobacteriaceae family. 3, 17, 18 some, however, have dismissed many of these organisms as more likely to be respiratory tract colonizers rather than the causative agents. 30 in our study, respiratory pathogens were detected in 68.4% of sputum samples (26/38) and 80.7% (21/26) of these were positive for more than one pathogen. this is a higher proportion than that reported previously by asghar et al., who isolated more than one pathogen in only 16.3% of the samples from 76 patients with confirmed cap in the 2005 hajj. 18 in another study, a higher percentage (35%) was reported. 24 the differences in detection rates may reflect the differences in identification methods used in the various studies. our study has some limitations. in addition to mers-cov, our test panel detects a specific set of 22 bacterial and viral respiratory pathogens. this means that other respiratory pathogens including fungi and other viruses and bacteria not included in the panel could have been missed. this may be of importance, as organisms not included in the panel such as m. tuberculosis, enterobacteriaceae, p. aeruginosa, and fungi, have been reported as causative agents of pneumonia during hajj. 3, 17, 18 also, we only used sputum samples for identification, and no microbiological investigations of other samples (e.g. blood) were performed to confirm the cause of pneumonia. finally, some of the organisms identified may have been respiratory tract colonizers and not the causative agents. in this context, a strength of our study is that the sputum samples were obtained on admission and before the start of antibiotic therapy. collecting sputum samples after the start of antibiotic treatment would have been of little value as it would have detected mainly respiratory tract colonizers. in conclusion, we investigated the etiology of severe cap in 38 hospitalized hajj pilgrims. mers-cov was not the cause of pneumonia in any of the patients. however, we detected a variety of pathogens in sputum samples of the patients, with most samples containing more than one agent. this observation, along with previous reports on cap in hajj, indicates that typical pneumonia treatment regimens may not work well during the hajj season due to the wide variety of organisms that may be involved. this may necessitate more active investigations into the causes of pneumonia for identification and sensitivity testing in order to provide optimal treatment and a good outcome. molecular methods can be a quick and sensitive means to determine the possible causative agents. pneumonia is a significant illness during hajj and interventions to reduce its burden during the pilgrimage should be adopted. measures to reduce respiratory tract infections during hajj are already in place. 31 other strategies may include improved respiratory tract infection surveillance and optimization and dissemination of recommendations for adult vaccination. 32, 33 continuous surveillance for mers-cov during hajj and outside the pilgrimage season is crucial to monitor the mers-cov situation in saudi arabia. conflict of interest: no conflict of interest to declare. causes of admission to intensive care units in the hajj period of the islamic year 1424 causes of hospitalization of pilgrims in the hajj season of the islamic year clinical and temporal patterns of severe pneumonia causing critical illness during hajj severe sepsis and septic shock at the hajj: etiologies and outcomes health hazards and risk factors in the 1406 h (1986 g) hajj season comparison of mortality and morbidity rates among iranian pilgrims in hajj community-acquired pneumonia bts guidelines for the management of community acquired pneumonia in adults: update bacteriological and clinical profile of community acquired pneumonia in hospitalized patients the bacterial aetiology of adult community-acquired pneumonia in asia: a systematic review state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans isolation of a novel coronavirus from a man with pneumonia in saudi arabia detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections respiratory tract infection during hajj pattern of admission to hospitals during muslim pilgrimage (hajj) tuberculosis is the commonest cause of pneumonia requiring hospitalization during hajj (pilgrimage to makkah) profile of bacterial pneumonia during hajj empiric antibiotic therapy and mortality among medicare pneumonia inpatients in 10 western states patients with community acquired pneumonia admitted to european intensive care units: an epidemiological survey of the genosept cohort practice guidelines for the management of community-acquired pneumonia in adults. infectious diseases society of america diagnostic tests for agents of community-acquired pneumonia molecular diagnostics for detection of bacterial and viral pathogens in community-acquired pneumonia etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms lack of mers coronavirus but prevalence of influenza virus in french pilgrims after bacteria and viruses that cause respiratory tract infections during the pilgrimage (haj) season in makkah, saudi arabia microbiology of community-acquired pneumonia in the gulf corporation council states demographics and microbiological profile of pneumonia in united arab emirates aetiological agents of community acquired pneumonia respiratory tract infections during the annual hajj: potential risks and mitigation strategies pneumococcal disease in the arabian gulf: recognizing the challenge and moving toward a solution the potential for pneumococcal vaccination in hajj pilgrims: expert opinion key: cord-337066-pztrwvib authors: choi, won suk; kang, cheol-in; kim, yonjae; choi, jae-phil; joh, joon sung; shin, hyoung-shik; kim, gayeon; peck, kyong ran; chung, doo ryeon; kim, hye ok; song, sook hee; kim, yang ree; sohn, kyung mok; jung, younghee; bang, ji hwan; kim, nam joong; lee, kkot sil; jeong, hye won; rhee, ji-young; kim, eu suk; woo, heungjeong; oh, won sup; huh, kyungmin; lee, young hyun; song, joon young; lee, jacob; lee, chang-seop; kim, baek-nam; choi, young hwa; jeong, su jin; lee, jin-soo; yoon, ji hyun; wi, yu mi; joung, mi kyong; park, seong yeon; lee, sun hee; jung, sook-in; kim, shin-woo; lee, jae hoon; lee, hyuck; ki, hyun kyun; kim, yeon-sook title: clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea date: 2016-06-30 journal: infect chemother doi: 10.3947/ic.2016.48.2.118 sha: doc_id: 337066 cord_uid: pztrwvib background: from may to july 2015, the republic of korea experienced the largest outbreak of middle east respiratory syndrome (mers) outside the arabian peninsula. a total of 186 patients, including 36 deaths, had been diagnosed with mers-coronavirus (mers-cov) infection as of september 30th, 2015. materials and methods: we obtained information of patients who were confirmed to have mers-cov infection. mers-cov infection was diagnosed using real-time reverse-transcriptase polymerase chain reaction assay. results: the median age of the patients was 55 years (range, 16 to 86). a total of 55.4% of the patients had one or more coexisting medical conditions. the most common symptom was fever (95.2%). at admission, leukopenia (42.6%), thrombocytopenia (46.6%), and elevation of aspartate aminotransferase (42.7%) were observed. pneumonia was detected in 68.3% of patients at admission and developed in 80.8% during the disease course. antiviral agents were used for 74.7% of patients. mechanical ventilation, extracorporeal membrane oxygenation, and convalescent serum were employed for 24.5%, 7.1%, and 3.8% of patients, respectively. older age, presence of coexisting medical conditions including diabetes or chronic lung disease, presence of dyspnea, hypotension, and leukocytosis at admission, and the use of mechanical ventilation were revealed to be independent predictors of death. conclusion: the clinical features of mers-cov infection in the republic of korea were similar to those of previous outbreaks in the middle east. however, the overall mortality rate (20.4%) was lower than that in previous reports. enhanced surveillance and active management of patients during the outbreak may have resulted in improved outcomes. middle east respiratory syndrome (mers), which is caused by β-coronavirus of the c lineage (mers-cov), ranges in severity from asymptomatic to a severe respiratory illness with rapid progression to respiratory failure [1] . since the identification of the first case in saudi arabia, the world health organization (who) has been notified of 1,638 laboratory-confirmed cases, including 587 fatal cases, in 26 countries as of february 26th, 2016. these confirmed cases have been directly or indirectly linked to countries in the arabian peninsula [2] . although the exact mode of transmission remains unknown, primary mers-cov infections are presumably associated with community zoonotic exposure and may be passed to family members via limited secondary transmission [3] . human-to-human spread of mers-cov is assumed to occur through close contact, such as caring for or living with an infected person [4, 5] . as infected people have spread mers-cov to others in hospitals, the importance of vigilant surveillance and appropriate infection control measures must be emphasised to prevent transmission in healthcare settings. from may to july 2015, the republic of korea experienced the largest outbreak of mers outside the arabian peninsula. infection was confirmed in a 68-year-old businessman returning from the middle east on may 20th, 2015, when he presented with atypical pneumonia and he failed to response to anti-microbial therapy for community-acquired pneumonia. prior to the confirmation of mers-cov infection, he had visited three hospitals where more than 600 people (including other patients, care givers, and healthcare workers) were exposed, resulting in 36 cases of mers-cov infection [6] . as of december 5th, 2015, 16,693 individuals had been quarantined and 186 cases of mers-cov had been confirmed. all confirmed cases were associated with a total of 17 healthcare facilities, and approximately 80% of the confirmed cases were caused by 'super-spreading' events that originated from five patients in four hospitals [6] . we aimed to determine the clinical features and outcomes of korean patients confirmed to be infected with mers-cov, and the risk factors contributing to mortality. this retrospective observational study focused on the clinical characteristics of confirmed cases of mers-cov infection in the republic of korea. approval for this study and a waiver for requiring informed consent were obtained from institutional review board of chungnam national university hospital. all patients with laboratory-confirmed mers-cov infection were identified during the outbreak in the republic of korea, during which confirmatory tests were performed only for suspected cases. suspected cases were defined as follows: 1) fever and pneumonia or acute respiratory distress syndrome (based on clinical or radiologic evidence) and either a history of travel from countries in or near the arabian peninsula within 14 days before symptom onset or close contact with a symptomatic traveller who developed fever and acute respiratory illness (not necessarily pneumonia) within 14 days after traveling from countries in the arabian peninsula. 2) fever and symptoms of respiratory illness (e.g., cough, shortness of breath) and presence in a healthcare facility (as a patient, worker, or visitor) within 14 days before symptom onset in a country in or near the arabian peninsula. 3) fever or symptoms of respiratory illness (e.g. cough, shortness of breath) and close contact with a confirmed symptomatic mers case [7] . since june 4th, 2015, symptomatic people who visited a healthcare facility within 14 days before the onset of symp-toms in two or more confirmed healthcare-associated mers cases were also included as suspected cases. one of the hospitals that treated patients with laboratory-confirmed mers-cov infection also performed active surveillance of asymptomatic health care workers who were involved in the care of confirmed cases. in the republic of korea, real-time reverse-transcriptase polymerase chain reaction (rrt-pcr) assays for mers-cov diagnosis were performed exclusively at the korea centre for disease control and prevention until may 28th, 2015. medical centres and referral laboratories began to perform rrt-pcr assays for mers-cov on may 28th and june 3rd, 2015, respectively. these rrt-pcr tests targeted the upstream e protein (up-e) and open reading frame 1a (orf1a) gene segments of mers-cov, as described in the who guideline [8] . patient information was collected using a standardized case-report form. gathering of data was carried out by the physicians who cared for the mers-cov-infected patients of each hospital. a major means of obtaining data was review of medical records, including doctors' and nurses' charts, imaging findings, and laboratory results. in those cases where additional information was necessary, re-interview of patients was performed. collected data were categorized with respect to demographics, clinical symptoms, comorbidities, laboratory test results, image findings, treatments performed, and clinical outcomes. we carried out descriptive analysis for the aforementioned categories. continuous variables were reported as means with standard deviations or medians with ranges. for categorical variables, the proportion of patients was calculated for each variable. comparison analysis for subgroups was performed using student's t-test, pearson's chi-square test, or fisher's exact test, and cox-regression analysis was also performed to evaluate risk factors of mortality. for cox-regression analysis, the data was censored at september 30, 2015 for survivors. a p-value <0.05 was considered to indicate statistical significance. all analysis was performed with spss software (spss version 20.0, spss inc., chicago, il, usa) for windows. we described the clinical characteristics of all 186 patients with confirmed mers-cov infection during the outbreak in the republic of korea (table 1) . we also compared the clinical characteristics of survivors and deceased at 28 days after symptom onset. the median age of the patients was 55 years (range, 16 to 86); 52.2% of the patients were 55 years of age or older (table 1) . male patients predominated in number over female patients, with a sex ratio of approximately 3:2. a total of 55.4% of the patients had one or more coexisting medical conditions. hypertension, diabetes, and solid organ malignancy were the most common coexisting medical conditions. thirty-nine patients (21.0%) were health care workers (hcws), of which nurses were most common (38.6%), and two cases (1.1%) were asymptomatic. compared with survivors, the deceased were older and more frequently had coexisting medical conditions. one hundred fifty-two patients (81.7%) manifested fever and more than 50% had developed cough at admission. almost all of the patients (95.2%) developed fever during the course of disease. unstable vital signs were observed in approximately a quarter of the patients at admission. compared with survivors, the deceased more frequently presented with dyspnea, hypotension, and tachypnea at admission. at admission, 178 (95.7%) of the 186 patients underwent one or more blood tests (table 1) including complete blood counts in 178 (95.7%), c-reactive protein (crp) analyses in 168 (90.3%), and liver function and blood chemistry tests in 173 (93.0%). among those, 42.1% had leukopenia (<4,000 cells/mm 3 ), 4.5% had leukocytosis (>10,000/mm 3 ), 32.0% had anemia (<12 g/dl), and 46.6% had thrombocytopenia (<150,000 /mm 3 ). crp was elevated (>3 mg/dl) in 47.6% of patients. elevated level of aspartate aminotransferase (ast) was observed in 43.4%, whereas elevated creatinine was observed in only 5.8% of patients. among the 103 patients who underwent urinalysis, 44(42.7%) had proteinuria and 36(35.0%) had hematuria. forty-four patients underwent arterial blood gas analysis, which revealed a decreased pao 2 :fio 2 ratio (<300) in 56.8% of patients. compared with survivors, the deceased more frequently exhibited leucocytosis, hypoalbuminaemia, elevated serum creatinine and crp levels, and hypoxemia (pao2:fio2 ratio <300). abnormalities in chest radiography were detected in 123 of 180 patients (68.3%) who underwent chest radiography at admission and 147 (80.8%) of the 182 patients manifested pneumonia during the course of disease. the most common features of the abnormalities were ground glass opacity or consolidation. the abnormal findings appeared in peripheral (78.9%), focal (57.1%), and unilateral (62.6%) patterns. multifocal or bilateral location was relatively rare (42.9% and 37.4%, respectively). the abnormalities had disappeared in only onethird of patients by the time of discharge. antiviral therapy was administered to 139 patients (74.7%; table 2 ). the median time from the onset of illness to the initiation of antiviral therapy was 6 days (range, 1-20 days), although 14.0% of patients received antiviral therapy within 48 hours of symptom onset. the most commonly prescribed antiviral regimen was a combination of interferon (ifn), ribavirin, and lopinavir/ritonavir. one hundred thirty-eight patients (75.0%) received antibiotic therapy. in addition, 45 patients (24.5%) were treated with mechanical ventilation, 15 (8.2%) with haemodialysis, and 13 (7.1%) with extracorporeal membrane oxygenation (ecmo). seven (3.8%) patients were treated with convalescent serum. acute respiratory distress syndrome, acute kidney injury, and shock occurred in 20.7%, 14.0%, and 13.4% of patients, respectively. among the 186 patients, a total of 38 (20.4%) died in hospitals. from the day of symptom onset, seven patients (3.8%) died within 7 days and 33 (17.7%) died within 28 days. the median interval from symptom onset to death was 14 days (range, 1-174 days). two patients (1.1%) died before confirmation of mers. among cured patients, the median interval from symptom onset to discharge was 21 days (range, 7-187 days), the median duration of fever was 8 days (range, 0-54 days), and the median time to a negative conversion of virus as determined via rrt-pcr analysis of sputum was 17 days (range, 4-45 days). in univariate analysis, the deceased were older than the survivors (median age, 69 vs. 51 years), and included a smaller portion of hcws than did the survivors (2.8 vs. 25.3%). the deceased had fever, dyspnea, or decreased consciousness more frequently at admission. they had coexisting medical conditions more frequently, especially diabetes, chronic heart disease, chronic lung disease, and solid organ malignancy. hypotension and tachypnea were observed more frequently in the deceased. laboratory abnormalities, namely leukocytosis, thrombocytopenia, hypoalbuminemia, elevated serum creatinine, and elevated crp, were reported more frequently in the deceased at admission and during the course of disease. however, leukopenia was reported more frequently in survivors. in multivariate cox-regression analysis, age ≥55 years, occurrence of dyspnea during the disease course, presence of coexisting medical conditions including diabetes or chronic lung disease, systolic blood pressure <90 mm hg at admission, leukocytosis at admission, and the use of mechanical ventilation were found to be independent predictors of death (table 3 ). the use of mechanical ventilation was assumed to be an independent indicator of severe disease. among the 45 patients with mechanical ventilation, 28 (55.6%) died. the deceased among patients with mechanical ventilation were older than the survivors, and the proportion of hcws was smaller than among the survivors (table 4) . they also had underlying diseases more frequently, especially chronic lung disease and solid organ malignancy. elevation of serum creatinine was observed more frequently in the deceased. in contrast, the occurrence of diarrhea and the elevation of alt were observed more frequently in survivors. ecmo or convalescent serum was employed more frequently among survivors. since its initial identification in saudi arabia in 2012, 1,638 cases of laboratory-confirmed mers-cov infection have been reported as of february 26th, 2016. in that time interval, another multi-facility outbreak in an endemic area has occurred since the first healthcare facility-related outbreak was reported by assiri, et al [9] . here, we describe the clinical findings of 186 mers patients in the republic of korea from may through september 2015. to the best of our knowledge, this is the largest report of an outbreak outside of the middle east. in this study of the mers outbreak in the republic of korea, the reported data suggest that mers-cov is most likely to be transmitted as a healthcare-associated infection. no community-associated infections lacking any history of contact with mers-cov were found in this outbreak. even though the overall transmissibility of mers-cov was relatively low, there were several so-called 'super-spreading' events posing a serious threat to public health. rapid transmission and high attack rates in dialysis units were noted, in a previous report, in saudi arabia [5] . however, no additional cases in dialysis units were reported in this korean outbreak, even though many patients undergoing hemodialysis were exposed to mers in dialysis units. this apparent heterogeneity in transmission, with many infected patients not transmitting disease at all and several patients transmitting disease to others, may be characteristic of mers-cov infections, similar to sars [4, 5, 10, 11] . the clinical features of patients with mers-cov infection in the republic of korea were generally similar to those reported in saudi arabia [4, 5, 10] . asymptomatic patients were rare in this outbreak, presumably because confirmatory mers-cov tests were performed only for symptomatic patients, although active surveillance of asymptomatic hcws involved in patient care was conducted at one hospital. in a previous outbreak in jeddah, 25.1% of laboratory-confirmed cases were reported to have been asymptomatic; however, a telephone survey revealed that about 80% of those who were reached by telephone had experienced at least one symptom [4] . the proportions of patients with underlying medical conditions such as diabetes (18.8%), chronic lung disease (10.2%), or chronic kidney disease (4.8%) were smaller in this study, which might explain why cases with complicating respiratory or renal failure were relatively uncommon in the outbreak in the republic of korea, compared to those reported in saudi arabia. the incidence of pneumonia (80.8%) in this outbreak is similar to that reported in saudi arabia, although the proportion of patients requiring mechanical ventilation was smaller (24.2% in the republic of korea vs. 56-78% in saudi arabia) [5, 9] . in addition to the reduced presence of comorbidities, many patients in this study were diagnosed relatively early because rrt-pcr tests for mers-cov were actively performed for those with an epidemiological linkage as soon as fever or respiratory symptoms occurred. this early diagnosis might have led to early treatment initiation and thus prevented disease progression to a severe status. the case fatality rate observed in this study was lower than that reported in other studies of previous mers outbreaks (20.4% vs. 36.5-65%) [4, 5, 10] . this low case fatality rate could be attributed to the application of aggressive treatment measures, including antiviral agents, mechanical ventilation, or ecmo. antiviral treatments were administered to 74.5% of the patients, although clinical decisions for their use were made by attending physicians. during the mers-cov epidemic in the republic of korea, the korean society of infectious diseases and the korean society for chemotherapy collaborated to generate and distribute recommendations for the use of antiviral treatments based on existing available data from experiences with sars and mers [12, 13] . we were not able to assess the clinical impact of antiviral therapy on outcomes, as most patients with severe pneumonia received combination antiviral therapy along with comprehensive supportive care. the therapeutic efficacy of combination antiviral therapy should be evaluated in further studies. our study has several limitations. first, clinical data were retrospectively collected through electronic medical records and chart review, although cases were enrolled prospectively by means of active surveillance of outbreaks. the present study was observational, and thus unknown risk factors and bias might have been unequally distributed between the two groups in the survival analysis. the possibility of limitations that preclude accurate comparisons should be kept in mind given the observational nature of this study. clinical judgments regarding management of patients were made by attending physicians, not by researchers. second, we have presented 4 months of clinical data after control of the outbreak, and thus the data might represent acute complications of mers-cov-infected patients. only 36.2% of patients showed resolution of abnormal findings on chest radiologic evaluation, and there are concerns regarding chronic complications such as pulmonary fibrosis. further follow-up prospective cohort studies are warranted in the future. third, although this study was performed with a nationwide database, a relatively small number of patients with mechanical ventilation were included, with limited statistical power. the impact of ecmo and convalescent serum therapy on survival may not be fully adjusted for in the survival analysis. finally, this study was conducted mainly at large referral centers in korea, and our findings may not be generalizable to other institutions outside of korea. in conclusion, a mers-cov outbreak in the republic of korea was initiated by a patient recently returned from saudi arabia, and transmitted as a healthcare-associated infection. although clinical features did not differ significantly from those of previous outbreaks in the middle east, the overall mortality rate was 20.4%, which was lower than that reported in other countries. the early detection of cases with mers-cov infection and active management of patients during this outbreak may have improved patient outcomes. our data sug-gest that mers-cov infections pose an important public health threat in regions outside the middle east, and that more information on the emergence and dissemination of this virus is necessary in order to prevent its further spread. isolation of a novel coronavirus from a man with pneumonia in saudi arabia spread, circulation, and evolution of the middle east respiratory syndrome coronavirus family cluster of middle east respiratory syndrome coronavirus infections mers-cov outbreak in jeddah--a link to health care facilities hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus outbreak in the republic of korea response guidelines for middle east respiratory syndrome (the 3rd revision) laboratory testing for middle east respiratory syndrome coronavirus -interim recommendations (revised) multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome rapid response team. antiviral treatment guidelines for middle east respiratory syndrome ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study this work was supported by a grant (2015p2600100) from the research of korea centers for disease control and prevention (kcdc). we express the deepest thanks to physicians, field epidemiologists, and the officials of kcdc, who did not spare the dedicated efforts for the control of the mers outbreak in korea. http://orcid.org/0000-0003-1142-5488 jae-phil choi http://orcid.org/0000-0003-4805-7930 key: cord-322354-x61eqaca authors: young lee, jun; sup shin, young; lee, jihye; kwon, sunoh; jin, young-hee; seong jang, min; kim, seungtaek; hwan song, jong; rae kim, hyoung; min park, chul title: identification of 4-anilino-6-aminoquinazoline derivatives as potential mers-cov inhibitors date: 2020-08-08 journal: bioorg med chem lett doi: 10.1016/j.bmcl.2020.127472 sha: doc_id: 322354 cord_uid: x61eqaca new therapies for treating coronaviruses are urgently needed. a series of 4-anilino-6-aminoquinazoline derivatives were synthesized and evaluated to show high anti-mers-cov activities. n(4)-(3-chloro-4-fluorophenyl)-n(6)-(3-methoxybenzyl)quinazoline-4,6-diamine (1) has been identified in a random screen as a hit compound for inhibiting mers-cov infection. throughout optimization process, compound 20 was found to exhibit high inhibitory effect (ic(50) = 0.157 μm, si = 25) with no cytotoxicity and moderate in vivo pk properties. severe respiratory diseases in a broad range of animal species, including humans. [1] [2] [3] in 2003, one of the novel coronaviruses, severe acute respiratory syndrome cov (sars-cov), caused a total of 8,422 cases of sars with 916 deaths. 4 the other novel coronavirus, middle east respiratory syndrome cov (mers-cov), has emerged in april 2012 and posed a serious threat to public health. as of 4 april 2020, a total of 2,494 human mers-cov infections with 858 deaths had been reported from 27 countries. 5 although mers-cov can cause primary infections from direct contact with animal reservoirs like camels, 6 person-to-person transmission of this virus has mainly occurred in health-care facilities and family clusters. [7] [8] [9] recently outbreak of covid-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), emerged in china and has spread to several other countries. drug repositioning for an fda-approved compound library found that numerous compounds inhibited mers-cov infection. however, there are still no approved drugs for coronaviruses. 10 through high content screening (hcs) platform of institut pasteur korea (ipk) using the korea chemical bank (kcb), 11 we found several novel compounds that can inhibit mers-cov infection. 12 we found that n 4 -(3-chloro-4-fluorophenyl)-n 6 -(3methoxybenzyl)quinazoline-4,6-diamine 1 was effective for inhibiting mers-cov infection. quinazoline derivatives have previously been reported as potent inhibitors of the protein kinase of epidermal growth factor receptor (egfr). 13 most of those compounds were approved as anticancer drugs, including gefitinib, 14 lapatinib, 15 erlotinib, 16 and afatinib 17 . we thought that quinazoline compounds can exhibit good bioavailability and be easily extended to treatment of mers-cov infection. here we report on the synthesis and biological effects of 4-anilino-6-aminoquinazoline derivatives. the general synthetic route for 4-aminoquinazoline derivatives is described in scheme 1. our preliminary structure activity relationship (sar) studies surrounding 1 were carried out by introducing halide groups on phenyl ring of 4-anilino group (table 1) . 4-bromo substituent (6) maintained the potency. 4-fluoro (7), 3-chloro (8), 4-cyano (9) , and 3-acetyl (10) reduced potency compared to 1. interestingly, 4-trifluoromethyl (11) can slightly improve the potency. in the next phase optimization, we observed the effects of the substituents of 6 position of quinazoline ring, having fixed with 3-chloro-4-fluoro and 4-trifluoromethyl substituents on phenyl ring at 4 position ( table 2 ). considering activity and cytotoxicity, 20 was thought to be the best compounds for anti-mers drug and evaluated for its herg, metabolic stability, cytotoxicity (table 3) and oral (p.o.) routes at 2 and 5 mg/kg, respectively (table 4) . 20 showed reasonable oral bioavailability (21 %) summary table of sars cases by country middle east respiratory syndrome coronavirus (mers-cov) the chemical library used in this study was kindly provided by korea chemical bank key: cord-312533-4u3bmb0e authors: shen, li wen; mao, hui juan; wu, yan ling; tanaka, yoshimasa; zhang, wen title: tmprss2: a potential target for treatment of influenza virus and coronavirus infections date: 2017-08-01 journal: biochimie doi: 10.1016/j.biochi.2017.07.016 sha: doc_id: 312533 cord_uid: 4u3bmb0e influenza virus and coronavirus epidemics or pandemics have occurred in succession worldwide throughout the early 21st century. these epidemics or pandemics pose a major threat to human health. here, we outline a critical role of the host cell protease tmprss2 in influenza virus and coronavirus infections and highlight an antiviral therapeutic strategy targeting tmprss2. pathogenic microorganisms have always been a major threat to human health. the black death, etiologically caused by yersinia pestis, is estimated to have killed 30e60% of europe's total population in the 14th century [1] ; the 1918e1920 h1n1 influenza pandemic killed approximately 50 million people worldwide [2] . today, many pathogenic microorganisms that once posed great threat to human health, such as smallpox and yersinia pestis, have become extinct or are now under control due to advances in medical technology and the public health system [3, 4] . for other etiological agents such as influenza virus and coronavirus, however, man have not yet found an effective control method. in the 21st century alone, four large-scale respiratory virus epidemics or pandemics have occurred worldwide: the viruses responsible are sars-cov [5] , 2009 h1n1 pandemic influenza a virus [6] , mers-cov [7] and asian h7n9 influenza a virus [8] . for example, in 2017, h7n9 influenza a virus emerged in china again [9, 10] . mers swept through saudi arabia in 2012 and continues to spread there even now, and there have been more than 186 mers cases in south korea in 2015 [11e14] . vaccination has been the most effective means in controlling pandemic, but genetic mutations could make vaccines ineffective via inducing nonprotective responses to newly emerged viruses [15] ; this would leave even vaccinated populations highly vulnerable. antiviral drugs targeting viral proteins will also eventually lose their effectiveness as viral mutation occurs; virus strains resistant to amantadine and oseltamivir have already emerged among 2013 asian h7n9 influenza virus and 2009 pandemic h1n1 influenza virus [16e18] . the emergence of drugresistant strains highlights the need for novel antiviral therapeutic approaches. recently, a great deal of evidence has suggested that a transmembrane protease, serine 2 (tmprss2), a type ii transmembrane serine protease (ttsp), plays a critical role in sars and abbreviations: are, androgen receptor element; aebsf, 4-(2-aminomethyl) benzenesulfonyl fluoride hydrochloride; bhh, bromhexine hydrochloride; cov, coronavirus; desc1, serine protease desc1; est, (2s,3s)-trans-epoxysuccinyl-lleucylamido-3-methylbutane ethyl ester; fda, food and drug administration; hat, human airway trypsin-like protease; hai-2, hepatocyte growth factor activator inhibitor 2; hgf, hepatocyte growth factor; ifitm, interferon-induced transmembrane protein; mmp-2, matrix metalloproteinase-2; mspl, transmembrane protease, serine 13; pai-1, plasminogen activator inhibitor 1; par-2, protease activated receptor 2; ppmo, peptide-conjugated phosphorodiamidate morpholino oligomer; rbs, receptor binding subdomain; the, human tracheal epithelial; tmprss2, transmembrane protease, serine 2; tmprss4, transmembrane protease serine 4; ttsp, type ii transmembrane serine protease; vrnps, viral ribonucleoproteins. mers coronavirus (cov) and in 2013 asian h7n9 influenza virus and several h1n1 subtype influenza a viruses infections, indicating that targeting tmprss2 could be a novel antiviral strategy to treat coronavirus and some low pathogenic influenza virus infections [19e28]. tmprss2 gene is located on human chromosome 21: 41, 464, 551-41, 531, 116 (fig. 1) . a significant feature of the tmprss2 gene is that several androgen receptor elements (ares) are located upstream of the transcription start site and the first intron [29, 30] . as shown in fig. 2 , the tmprss2 gene encodes a predicted protein of 492 amino acids which anchors to the plasma membrane. it converts to its form through autocatalytic cleavage between arg255 and ile256. after cleavage, the mature proteases are mostly membranebound, yet a noticeable portion of them can be liberated into the extracellular milieu. the protease catalytic domain contains a catalytic triad consisting of the amino acid residues his296, asp345 and ser441, corresponding to his57, asp102 and ser195 of chymotrypsinogen [29, 30] . tmprss2 is predominantly expressed in prostate, with relatively lower level of expression in lungs, colon, liver, kidneys and pancreas. in lung cancer cell line a549 and prostate cancer cell line lncap, tmprss2 is expressed in an androgen-dependent manner [30, 31] . it has been demonstrated that tmprss2 activates protease activated receptor 2 (par-2), a g-protein coupled receptor, and that the activation of par-2 causes the upregulation of matrix metalloproteinase-2 (mmp-2) and mmp-9, both of which are key proteases in the metastasis of tumor cells [32] . furthermore, tmprss2-activated hepatocyte growth factor (hgf) promotes c-met receptor tyrosine kinase signaling and induces a pro-invasive epithelial-mesenchymal transition phenotype in prostate cancer cells [32] . a recent study suggested that tmprss2 plays a role as a cell membrane-anchored mediator in cancer pain and pain in general [33] . however, tmprss2-deficient mice showed no obvious phenotypic abnormality such as death, infertility or visible sickness, and the exact physiological function of tmprss2 in vivo remains unknown. it is speculated that tmprss2 may contribute to a specialized but nonvital function that is apparent only under certain conditions [34] . viral entry is the first step in the viral replication cycle. the entry of enveloped viruses into host cells in most cases requires virions binding to cell surface receptors and fusing to host-cell membrane. both processes are controlled by viral envelope glycoproteins. virus entry is a coordinated receptor binding process which involves numerous conformational changes in the viral envelope glycoproteins [35, 36] . influenza virus ha is a class i viral fusion protein which has two functional subunits, ha1 and ha2 (fig. 3 ). ha is synthesized as a fusion-inactive precursor ha0. after proper proteolytic cleavage, disulfide bound subunits ha1 and ha2 form the homotrimer. in the structure, the three ha2 chains are seen to form a stem, embraced by the n-and c-terminal segments of ha1. the main central portion of each ha1 chain forms a globular 'head' domain that sits at top the stem. a fusion peptide, consisting mainly of apolar residues, is located in the n-terminus of ha2 and buried at the hydrophobic pocket formed partially by the fusion domain of ha1 [37e42]. proteolytic cleavage of ha into ha1 and ha2 fragments is a critical step for virus infectivity, because it endows the ha fusion competent. precursor ha cleavage liberates the fusion peptide, which is inserted into target membranes during fusion. further, cleavage allows the ha being in a metastable conformation that can be triggered by the acidic ph of endosomes to undergo the structural rearrangements required for fusion. after proteolytic cleavage, the receptor binding subdomain of ha1 attaches to sialic receptors at the target membrane surface of host cells. this triggers internalization of the virion by endocytic pathway. during maturation of the endosome, the acidic environment of the endosome triggers rearrangment of ha1 which further provokes full exposure and release of fusion peptide from inner pocket. subsequently, low phtriggered ha2 reconformation causes fusion peptide insertion into the target endosomal membrane, forming an intermediate structure called the prehairpin. several prehairpins undergo a further structural rearrangements, bending back at a hinge point to drive viral and cellular membranes close proximity, then induces hemifusion, complete fusion, ultimately generates an fusion pore that allows for viral genetic material escaping into the cytosol (fig. 4) [37e42]. as a class i viral fusion protein, coronavirus spike glycoprotein (s) shares many structural and mechanistic features of influenza virus ha. coronavirus s protein contains two functional domains, s1 and s2 (fig. 3) . s1 harbors the receptor-binding domain, s2 contains functional elements involved in membrane fusion. a distinctive feature of the coronavirus s protein is that it can harbor more than one proteolytic cleavage site. the first identified cleavage site is located at the s1/s2 boundary and another is within s2 upstream of the putative fusion peptide, which is called s2'. after cleavage of spike glycoprotein, s1 and s2 domains remain associated by noncovalently, but not disulfide bonds. this is an important fig. 1 . a schematic diagram of tmprss2 genomic location. distinction from influenza virus ha. since the two domains are not held covalently, the s1 domain may be shed from the s2 stalk domain of the protein [43e45]. some studies have suggested that tmprss2, hat and other ttsps such as transmembrane protease serine 4 (tmprss4), homo sapiens serine protease desc1 (desc1) and homo sapiens transmembrane protease, serine 13 (mspl) can cleave human and avian influenza virus ha proteins with an arginine residue in cleavage site [21,22,46e48] . as shown in fig. 5 , hat cleaves newly synthesized ha during the release of the progeny virions and ha of incoming viruses before they are incorporated into the host cells. tmprss2 cleaves only nascent ha within the host cells and is not involved in the proteolytic activation for ha of incoming virions [40, 47] . it is speculated that the soluble form of tmprss2 possesses only minimal enzyme activity, which is insufficient to support cleavage of ha. in animal models, infection of wild-type mice with h7n9 influenza virus (a/anhui/1/13) and h1n1 influenza virus (a/ pr/8/34) led to severe disease with mortality rates of 100% and 20%, respectively; whereas in tmprss2-deficient mice, these viruses were a pathogenic [20] . for example, tmprss2 knockout mice were highly tolerant of challenge infection by h7n9 influenza virus (a/ anhui/1/13) and mouse-adapted h1n1 influenza virus a/california/ 04/09 (maca04) with 1000 50% lethal doses (ld50) for wt mice [19] . these results demonstrate the essential role of tmprss2 in the pneumotropism and pathogenicity of h7n9 influenza virus and some h1n1 subtype influenza a viruses in vivo. the sars-cov s protein can be cleaved by a wide variety of host proteases, such as tmprss2, hat, mspl, desc1, factor xa and cathepsin l/b [49e51]. it has been shown that sars-cov enters into cells via two distinct pathways: one is mediated by tmprss2 at the cell surface and the other done by cathepsin l/b in the endosome (fig. 6) [43, 44, 50, 51] . the serine protease inhibitor camostat can effectively protect mice infected with the otherwise lethal sars-cov from death, but treatment with both serine and cathepsin inhibitors failed to improve survival significantly over that achieved with camostat alone [52] , indicating that sars-cov propagation and pathogenesis is mediated by tmprss2 rather than cathepsin in vivo. kawase et al. found that sars-cov entry increased 2.6-fold in the presence of tmprss2; conversely, sirna targeting tmprss2 caused a five-fold decrease in sars-cov entry into calu-3 cells [53] . moreover, the levels of sars-cov rna are nine-fold higher in cells expressing active tmprss2 than in cells expressing enzymatically inactive tmprss2 (s441a) [54] . western blot analysis revealed that sars-cov s is cleaved into several fragments upon expression of tmprss2 (cis-cleavage) in the infected cells as well as upon contact between sars-cov s-expressing cells and tmprss2-expressing cells (trans-cleavage). cis-cleavage results in the release of sars-cov s fragments into the cellular supernatant, which may interfere with antibody-mediated neutralization. trans-cleavage activates sars-cov s on the target cell, allowing for efficient sars-cov s-driven viral fusion [55] . in addition, the activation of sars-cov by tmprss2 interferes with the inhibition of sars-coa s by interferon-induced transmembrane proteins (ifitms), a class of interferon-induced host cell proteins that inhibit the entry of several enveloped viruses. collectively, the obtained evidence suggests that tmprss2 plays an important role in sars-cov infection [56e58]. the host proteases involved in the priming of mers-cov include tmprss2 [22] , hat [59] , mspl, desc1 [46] , furin [60] and endosomal cathepsin b/l [44] . similar to sars-cov infection, mers-cov infection is largely dependent on the activity of endogenous ttsps [59e61]. camostat treatment alone is as effective as camostat plus est, a cathepsin inhibitor, in the inhibition of virus entry into calu-3 cells, indicating a dominant role of tmprss2 in mers-cov entry. indeed, tmprss2 augments mers-cov infection by up to 100-fold in vero cells [22] . a non-catalytic tmprss2 (s441a) significantly abrogated the entry of mers-cov compared to normal tmprss2 [62] . although other host proteases such as hat, mspl and desc1 have been suggested to be involved in the priming of mers-cov, their expression levels in lung tissue are considerably lower than that of tmprss2, indicating a limited role of these proteases in viral propagation in host lung tissue [63, 64] . these observations demonstrate that mers-cov infection is largely dependent on the activity of endogenous tmprss2. the details are shown in fig. 6 . the redundant physiological functions along with its critical role in h7n9 influenza virus (a/anhui/1/13) and h1n1 influenza virus (a/pr/8/34) and sars-and mers-cov infections suggest that tmprss2 can be a promising target for drug development. one possible drug is aprotinin (pdb id: 1bpi), a polypeptide consisting of 58 amino acid residues purified from bovine lung. this polypeptide can inhibit serine proteases and suppress the cleavage of influenza virus ha, thus limiting the reproduction of the influenza virus [65] . aerosol inhalation of aprotinin has been observed to have a therapeutic effect against influenza and parainfluenza in mice and humans [66] . a small-particle aerosol of aprotinin has been approved in russia for the treatment of patients with mild-tomoderate influenza infections [65, 66] . camostat is a serine protease inhibitor used in the treatment of cancer, pancreatitis and liver fibrosis [67e71]. when human tracheal epithelial (hte) cells are treated with camostat at a concentration of 10 mg/ml, h1n1 influenza virus (a/pr/8/34) titers in the culture supernatant are significantly reduced. furthermore, camostat administration protects mice from infection with h1n1 influenza virus (a/taiwan/1/86 and a/pr/8/34) [72, 73] . a recent study demonstrated that the inhibition of tmprss2 with camostat led to a 10-fold reduction in sars-cov titers in calu-3 cells [54] . the drug is also effective at protecting mice from sars-cov infection, with a survival rate of 60% [52] . in addition, camostat at a concentration of 10 mm impairs mers-cov entry 15-fold in vero-tmprss2 cells, and the amount of viral rna in the culture supernatant of calu-3 cells is reduced 270-fold in the presence of 100 mm camostat at three days post mers-cov infection [22] . more recently, nafamostat, a serine protease inhibitor, was reported, to block mers-cov infection in vitro via inhabiting the activity of tmprss2, and reduced viral entry by 100-fold at a concentration of as low as 1 nm, which is more effective than camostat [74] . in addition, bromhexine hydrochloride (bhh) is an ingredient in a mucolytic cough suppressant that also effectively attenuates prostate cancer metastasis in mice. furthermore, it shows specific inhibition of tmprss2 (ic50 ¼ 0.75 mm). given that bhh is an fdaapproved drug with no significant adverse effects, it could be used for treatment of influenza virus and coronavirus infections as an inhibitor of tmprss2 [32] . other fda-approved serine protease inhibitors such as 4-(2-aminomethyl) benzenesulfonyl fluoride hydrochloride (aebsf) and leupeptin also exhibited varying degrees of anti-viral activities [74e76] . pretreatment of c57bl/6j mice with aebsf prior to h1n1 influenza virus (a/pr/8/34) infection significantly reduced weight loss and resulted in a faster recovery compared to that seen in untreated mice [73] . ovomucoid (pdb id: 2gkt), a trypsin inhibitor, is more effective than aprotinin at suppressing the propagation of h1n1 influenza virus (a/memphis/14/96) at the concentration of 50 mm [75] . a recent study demonstrated that 3-amidinophenylalanylderived compounds could inhibit tmprss2 in a nanomolar range. the most potent derivative inhibited tmprss2 with a ki value of 0.9 nm and suppressed h1n1 influenza virus (a/hamburg/5/2009) propagation in human airway epithelial cells in a dose-dependent manner [77] . it was also reported that three benzamidine-derived peptide mimetic compounds suppressed h1n1 influenza virus (a/ memphis/14/96 and a/hamburg/5/2009) infection to varying degrees in tmprss2-and hat-expressed cells [47] . dittmann et al. reported that plasminogen activator inhibitor 1 (pai-1, pdb id: 4u32) efficiently inhibited trypsin-and tmprss2mediated cleavage of ha and suppressed h1n1 influenza virus (a/wsn/33) propagation ex vivo and in vivo [78] . in addition, hepatocyte growth factor activator inhibitor 2 (hai-2, pdb id: 3q02) has been reported to efficiently inhibit ha-cleaving proteases such as tmprss2 and tmprss4 [64] . hai-2 has also been shown to inhibit h1n1 influenza virus (a/pr/8/34) infection in vitro and to protect animals from h1n1 influenza virus (a/pr/8/34) infections in a mouse model [79] . these results suggest that localized administration of pai-1 or hai-2 in the respiratory tract might be a new therapeutic approach for the treatment of influenza virus, coronavirus or other respiratory virus infections that require host protease-driven maturation. it is noteworthy that none of the compounds or proteins described above is a tmprss2-specific inhibitor. the non-specific nature of those inhibitors may cause unpredictable side effects. for instance, the food and drug administration (fda) has found that aprotinin increases the risks of kidney failure, heart disease and stroke, resulting in banned or restricted status of the drug in some countries [80] . therefore, it is necessary to develop inhibitors that specifically target tmprss2. b€ ottcher-friebertsh€ auser et al. have designed and synthesized a single-stranded dna-like antisense agent with low toxicity called a peptide-conjugated phosphorodiamidate morpholino oligomer (ppmo, fig. s1 ), which can readily enter into cells and alter the splicing pattern of pre-mrna. in cells treated with ppmo, tmprss2 was expressed in an incomplete and enzymatically inactive form. consequently, the cleavage of ha was impaired and h1n1 influenza virus (a/memphis/14/96) titers can be reduced 100e1000 fold in calu-3 cells [81] . [32, 97] . devan et al. have developed a polyamide compound which can interact with 5 0 -wgwwcw-3 0 in the consensus are in an attempt to inhibit the growth of prostate cancer cells. the study showed that the polyamide moderately inhibited the expression of tmprss2 by binding to the are in the tmprss2 promoter and suppressing rna transcription [98] . the are is most likely to be functionally important in stabilizing juxtaposed enhancer-target gene promoter loops to enable optimal gene expression [99, 100] . this indicates that a cell membrane-permeable polyamide can specifically interact with a particular are of tmprss2 which controls or regulates tmprss2 expression. it is thus possible that polyamide compounds targeting to tmprss2 would specifically suppress asian h7n9 influenza a virus and some h1n1 subtype influenza a viruses and sars and mers-cov infections. structures of some small molecular inhibitors over-mentioned are summarized in fig. 7 . one of the most direct impact of the recent increase in population mobility and globalization is the spread of infectious disease. since the turn of the 21st century, influenza and coronavirus epidemics have occurred worldwide. the emergence of drug-resistant viruses and new viruses highlight the need for novel antiviral approaches and strategies. targeting cellular factors is a relatively new antiviral strategy that may reduce or avoid the emergence of escape mutants. tmprss2 is one of the host factors that is essential for pneumotropism and pathogenicity of asian h7n9 influenza a virus and several h1n1 subtype influenza a viruses infections; this enzyme also plays an important role in sars-cov and mers-cov infections, indicating that it is a promising drug target for the treatment of these viral infections. although fda-approved inhibitors that specifically inhibit tmprss2 are not yet available, some drugs such as camostat and nafamostat that have inhibitory activity against a variety of serine proteases have been approved for the treatment of other diseases and also suppress influenza virus and coronavirus infections. because these drugs inhibit tmprss2 in vitro and in vivo, drug repositioning may help in developing novel measures to inhibit efficient viral entry and replication. taken together, the available evidence suggests that tmprss2 is quite likely to be a target for new therapeutics against influenza virus and coronavirus infections. the authors declare that there is no conflict of interests. this work was supported by national natural science foundation of china (21572207), program of international s & t supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2017.07.016. the black death and the origins of the 'great divergence' across europe, 1300e1600 what's old is new: 1918 virus matches 2009 h1n1 strain should remaining stockpiles of smallpox virus (variola) be destroyed? emerg yersinia pestis and plague-an update advisors of expert sars group of hospital authority, effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) preparing for 2009 h1n1 influenza middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group human infection with a novel avian-origin influenza a (h7n9) virus human infection with avian influenza a(h7n9) virus-china human infection with avian influenza a(h7n9) virus-china middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-republic of korea middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia novel insights into proteolytic cleavage of influenza virus hemagglutinin emergence of oseltamivir-resistant pandemic h1n1 virus during prophylaxis emergence of a novel drug resistant h7n9 influenza virus: evidence based clinical potential of a natural ifn-a for infection control and treatment flipping in the pore: discovery of dual inhibitors that bind in different orientations to the wild-type versus the amantadine-resistant s31n mutant of the influenza a virus m2 proton channel the host protease tmprss2 plays a major role in in vivo replication of emerging h7n9 and seasonal influenza viruses tmprss2 is a host factor that is essential for pneumotropism and pathogenicity of h7n9 influenza a virus in mice efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium al-tawfiq, therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)epossible lessons from a systematic review of sars-cov therapy coronaviruses-drug discovery and therapeutic options potential cellular targets for anti-influenza drug development serine proteases and their inhibitors in human airway epithelial cells: effects on influenza virus replication and airway inflammation identification of tmprss2 as a susceptibility gene for severe 2009 pandemic a (h1n1) influenza and a (h7n9) influenza tmprss2 (transmembrane protease, serine 2) prostate-localized and androgen-regulated expression of the membrane-bound serine protease tmprss2 androgen receptor and androgen-dependent gene expression in lung the androgen-regulated protease tmprss2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis tmprss2, a novel membrane-anchored mediator in cancer pain phenotypic analysis of mice lacking the tmprss2-encoded protease the structural biology of type i viral membrane fusion viral membrane fusion vare ckov a, the role of fusion activity of influenza a viruses in their biological properties cleavage activation of the influenza virus hemagglutinin and its role in pathogenesis influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion the hemagglutinin: a determinant of pathogenicity, influenza pathogenesis and control receptor binding and ph stability-how influenza a virus hemagglutinin affects host-specific virus infection composition and functions of the influenza fusion peptide host cell proteases: critical determinants of coronavirus tropism and pathogenesis mechanisms of coronavirus cell entry mediated by the viral spike protein a structural view of coronavirusereceptor interactions desc1 and mspl activate influenza a viruses and emerging coronaviruses for host cell entry cleavage of influenza virus hemagglutinin by airway proteases tmprss2 and hat differs in subcellular localization and susceptibility to protease inhibitors proteolytic activation of the 1918 influenza virus hemagglutinin inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research cleavage of spike protein of sars coronavirus by protease factor xa is associated with viral infectivity protease inhibitors targeting coronavirus and filovirus entry simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus human coronaviruses: a review of virusehost interactions tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection influenza and sars-coronavirus activating proteases tmprss2 and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts functional analysis of a missense mutation in the serine protease inhibitor spint2 associated with congenital sodium diarrhea aprotinin aerosol treatment of influenza and paramyxovirus bronchopneumonia of mice aprotinin and similar protease inhibitors as drugs against influenza the serine protease inhibitor camostat inhibits influenza virus replication and cytokine production in primary cultures of human tracheal epithelial cells retinoids in liver fibrosis and cancer antiproteases in the treatment of chronic pancreatitis proteolytic activation of the epithelial sodium channel and therapeutic application of a serine protease inhibitor for the treatment of salt-sensitive hypertension the serine protease inhibitor camostat mesilate attenuates the progression of chronic kidney disease through its antioxidant effects evaluation of anti-influenza effects of camostat in mice infected with non-adapted human influenza viruses inhibition of lung serine proteases in mice: a potentially new approach to control influenza infection identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay mdck cells that express proteases tmprss2 and hat provide a cell system to propagate influenza viruses in the absence of trypsin and to study cleavage of ha and its inhibition different host cell proteases activate the sars-coronavirus spike-protein for cellecell and virusecell fusion identification of the first synthetic inhibitors of the type ii transmembrane serine protease tmprss2 suitable for inhibition of influenza virus activation a serpin shapes the extracellular environment to prevent influenza a virus maturation inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor hai-2 the current place of aprotinin in the management of bleeding inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease tmprss2 comparison of patients hospitalized with influenza a subtypes h7n9, h5n1, and 2009 pandemic h1n1 prognosis and survival of 128 patients with severe avian influenza a (h7n9) infection in zhejiang province age-specific and sex-specific morbidity and mortality from avian influenza a (h7n9) sex-and age-related differences in morbidity rates of 2009 pandemic influenza a h1n1 virus of swine origin in japan national influenza a pandemic (h1n1) 2009 clinical investigation group of china, clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china effects of early oseltamivir therapy on viral shedding in 2009 pandemic influenza a (h1n1) virus infection clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in an university hospital of morocco do men have a higher case fatality rate of severe acute respiratory syndrome than women do? case fatality of sars in mainland china and associated risk factors a comparative epidemiologic analysis of sars in hong kong epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study ailan, on behalf the world health organization regional office for the western pacific mers event management team, sex mattersea preliminary analysis of middle east respiratory syndrome in the republic of korea update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques androgen receptor (ar) differential roles in hormone-related tumors including prostate, bladder, kidney, lung, breast and liver suppression of androgen receptor-mediated gene expression by a sequence-specific dna-binding polyamide enhancer rnas and regulated transcriptional programs ligand-dependent enhancer activation regulated by topoisomerase-i activity key: cord-330913-8aezw81h authors: albahri, a. s.; hamid, rula a.; alwan, jwan k.; al-qays, z.t.; zaidan, a. a.; zaidan, b. b.; albahri, a o. s.; alamoodi, a. h.; khlaf, jamal mawlood; almahdi, e. m.; thabet, eman; hadi, suha m.; mohammed, k i.; alsalem, m. a.; al-obaidi, jameel r.; madhloom, h.t. title: role of biological data mining and machine learning techniques in detecting and diagnosing the novel coronavirus (covid-19): a systematic review date: 2020-05-25 journal: j med syst doi: 10.1007/s10916-020-01582-x sha: doc_id: 330913 cord_uid: 8aezw81h coronaviruses (covs) are a large family of viruses that are common in many animal species, including camels, cattle, cats and bats. animal covs, such as middle east respiratory syndrome-cov, severe acute respiratory syndrome (sars)-cov, and the new virus named sars-cov-2, rarely infect and spread among humans. on january 30, 2020, the international health regulations emergency committee of the world health organisation declared the outbreak of the resulting disease from this new cov called ‘covid-19’, as a ‘public health emergency of international concern’. this global pandemic has affected almost the whole planet and caused the death of more than 315,131 patients as of the date of this article. in this context, publishers, journals and researchers are urged to research different domains and stop the spread of this deadly virus. the increasing interest in developing artificial intelligence (ai) applications has addressed several medical problems. however, such applications remain insufficient given the high potential threat posed by this virus to global public health. this systematic review addresses automated ai applications based on data mining and machine learning (ml) algorithms for detecting and diagnosing covid-19. we aimed to obtain an overview of this critical virus, address the limitations of utilising data mining and ml algorithms, and provide the health sector with the benefits of this technique. we used five databases, namely, ieee xplore, web of science, pubmed, sciencedirect and scopus and performed three sequences of search queries between 2010 and 2020. accurate exclusion criteria and selection strategy were applied to screen the obtained 1305 articles. only eight articles were fully evaluated and included in this review, and this number only emphasised the insufficiency of research in this important area. after analysing all included studies, the results were distributed following the year of publication and the commonly used data mining and ml algorithms. the results found in all papers were discussed to find the gaps in all reviewed papers. characteristics, such as motivations, challenges, limitations, recommendations, case studies, and features and classes used, were analysed in detail. this study reviewed the state-of-the-art techniques for cov prediction algorithms based on data mining and ml assessment. the reliability and acceptability of extracted information and datasets from implemented technologies in the literature were considered. findings showed that researchers must proceed with insights they gain, focus on identifying solutions for cov problems, and introduce new improvements. the growing emphasis on data mining and ml techniques in medical fields can provide the right environment for change and improvement. the outbreak of new coronavirus (covid-19) infections has caused worldwide concern because this disease has caused illness, including illness resulting in death and sustained person-to-person spread in many countries [1, 2] . covs are a large family of viruses, including the middle east respiratory syndrome (mers)-cov, severe acute respiratory syndrome (sars)-cov [3, 4] , and the new virus named sars-cov-2 [1] . in 2012, saudi arabia experienced the outbreak of mers-cov, which is responsible for causing mild to moderate colds. infection with mers-cov can lead to fatal complications. mers-cov is responsible for causing severe acute respiratory illness that leads to death in many cases. according to al-turaiki and his group [4] , mers-cov symptoms include cough, fever, nose congestion, breath shortness and sometimes diarrhoea. unfortunately, information on how the virus spreads and how patients are affected is limited [4] . fifteen years after the first highly pathogenic human cov caused the sars-cov outbreak, another severe acute diarrhoea syndrome-cov devastated livestock production by causing fatal diseases in pigs. the two outbreaks began in china and were caused by covs of bat origin [5, 6] . on february 11, 2020, the world health organisation named the ensuing disease 'covid-19' [1] . chinese health officials have reported tens of thousands of cases of covid-19 in china, with the virus reportedly spreading from person-toperson in several parts of the country. covid-19 illnesses, where most of them are associated with travel from wuhan, have been reported in a growing number of international locations, including the united states [1] . artificial intelligence (ai) is gradually changing medical practice. with the recent progress in digitised data acquisition, machine learning (ml) and computing infrastructure, ai applications are expanding into areas that are previously believed to be only the area of human experts [7] . various types of data mining methods have been applied by a few researchers with real cov datasets (e.g. mers-cov) based on several types of ml classifiers [8] . providing prediction systems that can accurately anticipate and diagnose such virus remains challenging. the growth of ai-driven techniques to identify epidemiologic risks in advance will be the key to improving the prediction, prevention and detection of future global health risks [9] . the main contributions of this study are the exploration of the cov family by reviewing articles on data mining and ml algorithms, the acquisition of a clear understanding of its enhancements, and how previous research has addressed prediction, regression, and classification methods. this study also aims to collect various information from the literature that are relevant to ml, such as application nature, the use of ml and data mining algorithms, and evaluation methods and accuracy. the datasets utilised in the literature are constructed and presented with url sources. the motivations, challenges, and limitations of this approach are examined, and recommendations on improving the approach efficiency are provided. other important information collected, especially on the types of case study used, features and classes for cov, are explained in separate tables. the rest of this paper is organised as follows. the second section describes the research methods used in the selected literature. the third section presents the literature review. the fourth section discusses the results, motivations, challenges, recommendations and limitations, case study used, and features and classes of cov. the fifth section provides a conclusion. this study followed the literature review style recommended by the preferred reporting items for systematic reviews and meta-analyses (prisma) statement [10] . five digital databases, namely, sciencedirect (sd), ieee xplore, web of science (wos), pubmed and scopus, were selected. sd provides access to a highly reliable journal in the field of science and technology. ieee explore contains updated research papers in the field of computer science, electronic engineering, and the applications of engineering and computer technology in medical applications. wos is an extremely reliable resource on social sciences, engineering, science, arts, humanities and cross-disciplinary studies. pubmed is considered the optimal database for medical and biomedical engineering research. scopus is a dependable resource in different areas of research, such as medical, health, science, technology and engineering. the five databases cover all academic aspects of covid-19. the outcome of this literature review can help save lives by providing deep insights into this disease, existing medical diagnosis systems for this virus, and recommended solutions for developing reliable medical systems. a comprehensive literature search was conducted in the five mentioned databases for english language citations published from 2010 to 2020. the selection of these indices was because of their sufficient coverage of studies related to our research considering that identified novel cov requires greater attention than other infections. this study presented and conducted a three boolean search strategy using various keywords related to pervasive 'coronavirus' (e.g. 'cov' or 'coronaviridae' or 'coronavirus') and keywords related to the detection, diagnosis and classification of cov under the concept of ai and ml. we used these query techniques to strengthen our search of different ai and ml systems and application studies for cov. 1. the article is an english journal or conference paper. 2. the main focus is on the development of different artificial intelligence and ml applications, systems, algorithms, methods and techniques. 3. the development only focuses on the detection, diagnosis and classification of adaptive cov. table 1 summarises the sequences of the boolean search query used in this paper and the results. this process was initiated by removing duplicated articles and screening nonduplicated articles by their titles and abstract to check their compatibility with our inclusion and exclusion criteria. the relevant articles were subjected to a full reading process for collecting and extracting research data and constructing the review article. in all research articles, the entire research process was monitored and supervised by a senior author (corresponding author) to ensure the production of a highly reliable and beneficial research paper. given the multidisciplinary topic of this systematic review, data extraction and classification of the selected studies, including data concerning cov with ai applications (especially ml techniques), were conducted to evaluate the efficacy of this virus in terms of detection, diagnosis and classification throughout ai enhancements. data elements were extracted from academic literature and included authors' nationalities, date of publication, number of articles per year and number of articles per database. for conferring a comprehensive viewpoint of cov, this study discussed the cov and analysed the growth scale of the worldwide epidemic in the context of ai using various data mining and ml algorithms, such as classification, regression and prediction. for each study in the literature, this study extracted the important feature names, evaluation methods used, and state of accuracy for each method. brief motivation, challenges, limitation and recommendation were extracted from the reviewed papers to address the serious public health concern for cov. the results of search queries conducted in this study are presented in figure 1 . three search queries were accomplished to encompass all databases and their search engine mechanisms during data collection. the first result comprised 1305 articles from all five databases. the number of duplicated articles in all databases was 66, and the results were 1239. the next process was screening the articles based on the title and abstract followed by the mapping of inclusion and exclusion criteria, and the results were 249 articles. the final process was the full reading of all articles, and the outcome was only 8 articles that met the inclusion and exclusion criteria. our understanding of the purpose/aim of these studies inspired us to analyse each study depending on two related sequences within the search query that was conducted in this systematic review. the first sequence is the article should identify the cov, and the second is the utilisation of ml. the findings of academic literature search showed that various algorithms are used by previous researchers. figure 2 summarises these algorithms and methods. the decision tree (j48) algorithm was the most frequently used (five times). naive bayes and support vector machine (svm) algorithms were each used four times. k-nearest neighbour (k-nn) was utilised two times, and the others were each used once. table 2 illustrates the state-of-the-art cov prediction algorithms. table 3 presents the cov dataset descriptions with available sources. eligibility included figure 1 schematic of the approach to identify, screen and include relevant studies. the analysis indicated many important points that are discussed to identify the research gaps. in [11] , three ml techniques were applied to the mers-cov dataset to identify the best classification model for binary class and multiclass labels. the results showed that the k-nn classifier is the best model for the two-class problems, and the decision tree and naïve bayes are the best models for multiclass problems. in [4] , two experiments are applied to the mers-cov infection dataset, and the decision tree classifier shows higher prediction proficiency than the other models. the experimental results indicated that age and symptoms are the two dominant features for the prediction model and that healthcare staff are likely to survive. in [12] , a study was conducted in saudi arabia to identify the dominant factors that influence human infection using statistical methods, such as univariate and multivariate regression methods. the results indicated four dominant features, namely, disease severity, patient age, the patient job as a healthcare staff or not and history of chronic disease. interaction with camels does not have a high impact on recovery. in [8] , a study was conducted in saudi arabia between 2013 and 2017 to improve medical diagnosis systems for binary and multiclass problems in mers-cov datasets. the experimental results showed that the decision tree classifier achieves the best accuracy for the multiclass labels, and the svm classifier obtains the highest accuracy for the two binary class labels in the mers-cov dataset. in [13] , an emotional recognition system based on ml technique was proposed to understand human reactions to a widespread epidemic of transferrable diseases, such as mers. the study was conducted using a dataset collected in korea in 2015, and the results indicated the impact of lightening excessive panic in reducing infection. in [14] , the author investigated people over 50 years old using three ml methods to predict mers cov. the results showed that elderly people are more likely to be infected than others. in [15] , an svm classifier based on sigmoid, normal and polynomial iterations was used to analyse the mers and sars proteins. the results showed their behaviour similarity and approximate dissimilarities. in [16] , data mining based on statistical methods was utilised to develop a cloud-based medical system with a high prediction accuracy to prevent mers-cov spread within different regions. the dataset comprises the following attributes: drug, patient and cloud-based user medical record. the role of ai in healthcare for enhancing the detection and prediction of numerous viruses and diseases has been previously discussed [9] . in this review, we aimed to obtain a large number and extensive contributions of published articles regarding the utilisation of ai for the detection and clinical diagnosis of mers-cov and sars-cov. however, the lack of studies on the recent outbreak of covid-19 indicates the need and opportunity to apply ai for predicting such outbreaks. ml technique based on supervised and unsupervised learning provides the opportunity to develop a medical diagnosis system. in supervised learning, the target class of each sample in the dataset, where mers-cov and sars-cov classes are previously identified and the developed system can be adapted to a new disease. although mers-cov and sars-cov have similarity within the same cluster, they are dissimilar to the objects in other clusters. thus, the clustering technique based on unsupervised learning is considered an efficient method to cluster the collected datasets, as presented in table 3 . consequently, detection and diagnosis can be remarkably enhanced. all studies in this review reported the use of ai techniques, such as case-based reasoning and rule-based systems. however, none of the studies utilised other classification methods, such as neural networks, reinforcement learning and hybrid classification. none of the studies utilised and integrated optimisation techniques, such as genetic algorithms and particle swarm optimisation, to their systems. data mining and ml algorithms used in diagnostic operations primarily rely on classification algorithms, including decision tree, svm and naive bayes classifiers ( figure 2 and table 2 ). notably, no study in the literature exploited clustering algorithms for the detection and diagnosis of the cov family. this technique can be used as a pre-processing step before feeding data into the classification model to gain valuable insights into the case study data by understanding which groups of disease symptoms fall into when applying these algorithms. in this review, the sample sizes representing the observations in each dataset are displayed in table 3 . in [11] , the sample size includes all mers-cov patients in saudi arabia in the second half of 2016, and in [8] , the sample size includes the cases from 2013 to 2017. the sample size in [4] represents 1082 records of cases reported from 2013 to 2015 distributed as 633 new case records, 231 recovery records and 218 death records. the study in [12] includes 836 patient records, and 52 patients are reported as dead and only 784 cases are used. in [13] , the sample size is represented by articles collected from the internet and reported by 153 news media outlets in korea and the comments associated with these articles from day 1 (first confirmed case on may 20, 2015) to the day 70. in [15] , the dataset contains 322 records, 92 infected cases and 230 uninfected cases. in [16] , synthetic data are generated for 0.2 million users. most of the available datasets are related to mers and sars infection cases, but no dataset is found for covid-19 because of its novelty. four aspects are presented in the following subsections. data were collected through an academic literature review of cov. these aspects are provided as follows: the motivations of using different ml and data mining algorithms to explore meaningful algorithms for the cov family; the potential challenges in predicting cov infections in humans that should be overcome to realise their potential; recommendations to alleviate challenges related to ml algorithms for recovery from cov infection; and case study dataset types of covs to assess to date, research areas in the field of ai, such as data mining and ml technique-based applications have been rapidly developed because of their large impact on human life in terms of social, scientific, medical and engineering-based applications. accordingly, this section presents the motivation of studies conducted on the cov problem to save lives. data mining for medical diagnosis systems is efficient and can be utilised to control the spread of mers-cov and protect humans [8] . data mining can also be used to estimate and predict the recovery rates from cov infections [4] . ml technique can be used to identify and predict the dominant factors affecting the recovery from mers-cov [12, 13] . thus, studies conducted on identifying the best model can help minimise the effects of epidemic diseases [11] . the distinction between sars and mers viruses can be considered a challenging task because of the similarities in their symptoms, such as breathing problems and high fever [15] . medical diagnosis systems play a significant role in identifying patient health conditions with mers-cov symptoms [14] . studies, such as [16] , integrated the gps with their medical diagnosis systems to cluster the population of infected people based on their geographical area. covid-19 has spread worldwide and threatened human life. accordingly, several studies have been conducted to develop an intelligent medical diagnosis system using ai technique to control the effects of this virus. however, numerous challenges and research limitations have been indicated in the academic literature and need to be addressed in the future [8] . some of these challenges are related to mers-cov nature and behaviour because understanding how the virus spreads and how people can be infected caused by the complexity of this epidemic disease is extremely difficult. the lack of a large dataset in the academic literature for mers-cov is considered a challenging task for ai researchers because it hinders the understanding of viral patterns and features [4, 12] . the demand to construct a dataset that can be understood -the analysed data were collected from the control and command centre. -a total of 836 patient records were used for analysis. fifty-two patients out of the 836 cases were initially reported as dead. hence, those cases were removed from the dataset, and 784 cases were used in the study. ministry of health website of the kingdom of saudi arabia. -synthetic data were generated for 0.2 million users. -raw information from users was collected using body-worn sensors and manually recorded data using the mobile application. n/a j med syst (2020) 44:122 by ml algorithms has increased because the current dataset involves infographic data [11] . other challenges are correlated with people and government responses to mers-cov that requires more new monitoring approaches and additional efforts compared with the traditional approach for controlling epidemic diseases [13] . another challenge with mers-cov is the large variation in symptoms that are mostly similar to common cold symptoms, with many other variations of diseases that may occur in cases but not in others. some patients have unique symptoms, and others have no symptoms at all. activists have generated huge and complex volumes of data that render its analysis impractical and difficult to predict using linear classifiers [14, 15] . the protection of citizens by the government and health agencies is a significant challenge because no specific vaccine exists for this virus to date and requesting people to undergo medical checkups is difficult [16] . this study aimed to mitigate some of the challenges that have been addressed in the academic literature with their recommended solution for future studies. studies, such as [8] , suggested a pre-processing method to solve the missing-value problem that directly impacts the classification accuracy for the mers virus. they also suggested the use of an ensemble technique by combining the cosine method with k-nn ml algorithm to improve the classification accuracy to >50%. another study [4] recommended increasing the number of samples for the cov dataset and collecting data from patients within the same geographical area by directly communicating with dedicated hospitals and health agencies [13] . [11] proposed the use of svm classifier for the binary class problem and conducted an empirical study for multiclass problems in mers-cov. [17] recommended the use of the r language because of its efficiency and high functionality in supporting ai algorithms that can enable the development of an effective intelligent medical diagnosis system for cov. another study [14] indicated the special medical issues related to the female status, such as whether she is pregnant, which need to be considered in the treatment of cov-infected patients [15] . [16] suggested the utilisation of the internet of things (iot) technology for developing a highly dependable medical diagnosis system for covid-19. based on the discussion, analysis and details in tables 2 and 3 , a case study found in the literature review can be divided into two types, namely, real and analysis datasets (table 4 ). real datasets consist of a number of real cases of infected and healthy patients within a specific period. four studies provided real datasets for patients affected by cov [4, 8, 11, 12] , and most datasets in other studies are published and redistributed by the ministry of health website of the kingdom of saudi arabia. in analysis datasets, standardisation is intended to increase the consistency of review and assessment analysis for mers-cov and sars-cov. an organised collection of data is found in four studies for the two viruses. in [13] , the massive media outlet data were collected during the nationwide outbreak of mers-cov in korea in 2015. in [14] , mers-cov cases were recorded from several medical analytical papers focused on the early symptoms of this virus. in [15] , spike glycol protein sequence data of sars (dq412574.1) and mers (kp236092.1) were obtained. [16] utilised the gps to represent each mers-cov user on google maps where 5000 users are adopted in r studio through the 'bnlearn' package. two attributes, namely, (1) personal patient information attributes and (2) cov attributes, were recognised in the collected studies to explore the effects of cov features and classes on case study datasets used with ml algorithms. as shown in table 5 , only four studies focused on 'personal patient information attributes', only two studies considered 'cov attributes', and two studies considered the two attributes. table 5 shows that the details mostly encompass mers-cov and sars-cov attributes and classes. age attribute is considered the most important and dangerous factor in the infected patients because people over the age of 50 are more likely to be at risk and to have this type of virus than others [4, 8, 12, 14, 16] . gender attribute is an important predictor in four out of eight studies [4, 8, 11, 12] . the city was used in three studies [4, 8, 11] . other related attributes are less frequently associated with personal patient information attributes, including address, sex and name. although sob is mentioned in only two studies, it is considered the most important concern with mers-cov and sars-cov attributes because the two studies focused on patients affected by the two viruses. from a specialised medical perspective, the features and classes of the cov family are similar to one another. in this context, the new epidemic of covid-19 depends on the same features and classes of mers-cov and sars-cov. thus, extensive research has been conducted to prove a new ai pathway. as proof for the above discussion, several reliable reports and government news have mentioned that age is the most important feature of patients with covid-19. patients over the age of 50 are susceptible to contract the disease and be exposed to its risks and complications. the gender and city of an infected patient are the next concerns. the medical team has reported that sob is the most important symptom attribute because it carries a high specificity for covid-19. this systematic review provided an exhaustive overview of integrated ai based on data mining and ml algorithms with the cov family. state-of-the-art cov prediction algorithms were presented. distinct information, such as application nature, ml used, the evaluation conducted for each study, and extracted features and classes with accuracy percentages for the utilised ml algorithms were indicated. a set of propositions for the risk recovery of this virus was established to serve as a guide for future research in the context of data mining algorithms. despite the increasing rates of death and the number of people affected with cov, developments based on ml algorithms to improve cov datasets remain at a redefinition stage, especially for covid-19. the shortage of studies in the literature is a real concern and may have serious implications for detecting and minimising the spread of this virus. an emerging, rapidly evolving situation for the virus must be considered in the viewpoint of ai applications, and researchers should provide updated contributions because they are necessary. supportive information, such as new datasets, must be provided, and many complex features and classes must be added. close cooperation among researchers in the biomedical engineering field and the medical community is necessary to stop the growing public health threat posed by the 2019 cov. the goal is to conduct new studies that can guide governments and communities in the early control of the impact of this virus by utilising the features and classes collected in the literature. the need for integrated sensor technologies specifically for outdoor scenarios is highly recommended. this process is only possible when the technique is interlinked with iot technologies, focusing on evaluating and improving ml algorithms for cov datasets with increased efficiency. in this context, ai software developers in healthcare can develop different software packages to remotely help analyse the extracted features and classes for patients with covid-19. conflict of interest the authors declare no conflict of interest. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent informed consent was obtained from all individual participants included in the study coronavirus disease 2019 (covid-19) situation summary the sars-cov-2 outbreak: what we know bat coronaviruses in china building predictive models for mers-cov infections using data mining techniques identification of a novel coronavirus in patients with severe acute respiratory syndrome fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin artificial intelligence in healthcare selecting accurate classifier models for a mers-cov dataset the role of augmented intelligence (ai) in detecting and preventing the spread of novel coronavirus preferred reporting items for systematic reviews and meta-analyses: the prisma statement identifying accurate classifier models for a text-based mers-cov dataset main factors influencing recovery in mers co-v patients using machine learning large-scale machine learning of media outlets for understanding public reactions to nation-wide viral infection outbreaks mers-cov disease estimation (mde) a study to estimate a mers-cov by classification algorithms comparison between sars cov and mers cov using apriori algorithm, decision tree, svm an intelligent system for predicting and preventing mers-cov infection outbreak a systematic analysis of performance measures for classification tasks publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-344954-gpb25fga authors: hashem, anwar m; algaissi, abdullah; agrawal, anurodh shankar; al-amri, sawsan s; alhabbab, rowa y; sohrab, sayed s; s. almasoud, abdulrahman; alharbi, naif khalaf; peng, bi-hung; russell, marsha; li, xuguang; tseng, chien-te k title: a highly immunogenic, protective, and safe adenovirus-based vaccine expressing middle east respiratory syndrome coronavirus s1-cd40l fusion protein in a transgenic human dipeptidyl peptidase 4 mouse model date: 2019-11-15 journal: j infect dis doi: 10.1093/infdis/jiz137 sha: doc_id: 344954 cord_uid: gpb25fga background: infection control measures have played a major role in limiting human/camel-to-human transmission of middle east respiratory syndrome coronavirus (mers-cov); however, development of effective and safe human or camel vaccines is warranted. methods: we extended and optimized our previous recombinant adenovirus 5 (rad5)–based vaccine platform characterized by in vivo amplified and cd40-mediated specific responses to generate mers-cov s1 subunit-based vaccine. we generated rad5 constructs expressing cd40-targeted s1 fusion protein (rad5-s1/f/cd40l), untargeted s1 (rad5-s1), and green fluorescent protein (rad5-gfp), and evaluated their efficacy and safety in human dipeptidyl peptidase 4 transgenic (hdpp4 tg(+)) mice. results: immunization of hdpp4 tg(+) mice with a single dose of rad5-s1/f/cd40l elicited as robust and significant specific immunoglobulin g and neutralizing antibodies as those induced with 2 doses of rad5-s1. after mers-cov challenge, both vaccines conferred complete protection against morbidity and mortality, as evidenced by significantly undetectable/reduced pulmonary viral loads compared to the control group. however, rad5-s1– but not rad5-s1/f/cd40l–immunized mice exhibited marked pulmonary perivascular hemorrhage post–mers-cov challenge despite the observed protection. conclusions: incorporation of cd40l into rad5-based mers-cov s1 vaccine targeting molecule and molecular adjuvants not only enhances immunogenicity and efficacy but also prevents inadvertent pulmonary pathology after viral challenge, thereby offering a promising strategy to enhance safety and potency of vaccines. the middle east respiratory syndrome coronavirus (mers-cov) is a novel zoonotic virus that was first isolated from a fatal human case in saudi arabia in 2012 [1] . the virus can cause severe acute and fatal respiratory symptoms associated with systemic infection and occasional multiorgan failure [1] . as of october 2018, mers-cov has caused >2250 laboratoryconfirmed infections with an approximately 35% mortality rate in 27 countries, with saudi arabia being the most affected country [2] . most global cases are linked to the arabian peninsula, where mers-cov continues to be endemic for >6 years with the potential to spread globally, as seen in the 2015 outbreak in south korea [3] . the spike (s) glycoprotein of mers-cov (1353 aa) is a type i trimeric membrane protein expressed on the virus surface, and binds to dipeptidyl peptidase 4 (dpp4) on target cells [4] . it is comprised of an n-terminal globular s1 subunit (aa 1-751) containing the receptor-binding domain (rbd) and a membraneproximal s2 subunit (aa 752-1353) consisting of a transmembrane domain and an intracellular cytoplasmic domain (cd) involved in viral fusion with target cells [4, 5] . given its critical role in viral replication, the s protein has been the focus for mers-cov vaccine development similar to severe acute respiratory syndrome coronavirus (sars-cov), where it has been the main target for vaccines that led to robust induction of neutralizing antibody (nab)-mediated protection in immunized animals [6] [7] [8] . several vaccine candidates based on full-length or truncated s protein including vectored, dna, and nanoparticle vaccines, as well as s or rbd protein-based subunit vaccines, have been developed and investigated in animal models [9] [10] [11] [12] [13] [14] [15] [16] [17] . although some of these candidates can induce high levels of nabs in vaccinated animals and have entered phase 1 clinical trials [15] [16] [17] , most of these candidates are associated with low immunogenicity requiring multiple doses [18] . importantly, vaccine research on other members of the coronaviruses including sars-cov [19] [20] [21] [22] [23] [24] [25] [26] [27] revealed several safety concerns associated with the use of s-based vaccines, including inflammatory and immunopathological effects such as pulmonary eosinophilic infiltration and antibody (ab)-mediated disease enhancement following subsequent viral challenge of vaccinated animals. recently, we also showed that whole inactivated mers-cov vaccine (wiv) is associated with significantly increased pulmonary eosinophilic infiltration accompanied by elevated levels of th2 cytokines in vaccinated human dpp4 transgenic (hdpp4 tg + ) mice in response to virus challenge [28] . given these previous observations, a mers-cov vaccine based on the full-length s protein could pose potential safety concerns similar to those associated with sars-cov vaccines. while the neutralizing epitope-rich s1 region could be considered as an alternative target for an effective and safe mers vaccine, s1-based protection could clearly be enhanced through appropriate immunological modulation. cd40 ligand (cd40l), a type ii membrane protein, is a key co-stimulatory molecule and an essential regulator of the immune system. it is expressed transiently on activated cd4 + t cells mainly [29, 30] . cd40l and its receptor cd40, which is constitutively expressed on all antigen-presenting cells (apcs) [29] [30] [31] , represent a crucial link between innate and adaptive immunity [31, 32] . the potential of cd40l as a molecular adjuvant has been investigated by several groups using multiple strategies [33] [34] [35] [36] . we have previously developed a nonreplicating recombinant adenovirus 5 (rad5) vectored prototype vaccine in which secreted viral proteins are targeted to cd40-expressing apcs using cd40l [37, 38] . such studies provided a proof of concept for a vaccine platform characterized by enhanced durability, breadth, potency, and universal protection against influenza viruses in different mouse models. here, we further extended and optimized our previous platform using rad5 expressing a secreted and cd40-targeted fusion protein comprised of head globular ectodomains of both mers-cov s1 protein and murine cd40l (rad5-s1/f/cd40l) using a trimerization motif to express the fusion protein as a trimer and a nonpolar linker to provide flexibility. the vaccine was then subjected to systematic analyses of protection and safety in the highly mers-cov permissive hdpp4 tg + mouse model. the fusion gene was designed and codon-optimized to express a secreted and cd40-targeted consensus mers-cov s1 subunit (amino acids 1-747). in brief, all available mers-cov s sequences were downloaded from the genbank database, and the dataset was filtered by removing sequences containing ambiguous amino acid codes (bjouxz). the final dataset was multiply aligned using clustalw, the shannon entropy for each position was determined, and the consensus s1 subunit sequence was obtained. the signal peptide from the mers-cov s protein was maintained at the n-terminus to secrete the fusion protein from rad5-infected cells. the synthetic fusion gene was synthesized by linking the coding region of mers-cov s1 to a histidine tag coding sequence, followed by coding region for the fibritin trimerization motif [37] connected via a nonpolar amino acid linker to the ectodomain of cd40l (amino acids 117-260) coding region to express the fusion protein s1/f/cd40l. the fusion gene was then used to generate the proposed rad5 construct (rad5-s1/f/cd40l) in addition to rad5 vaccines expressing secreted and consensus s1 protein alone (rad5-s1) and a vector control expressing green fluorescent protein (rad5-gfp) as shown in figure 1 . vero e6 and huh7.5 cells were cultured and maintained in complete dulbecco's modified eagle's medium (dmem) supplemented with 10% heat-inactivated fetal bovine serum. mers-cov-emc/2012 was provided by heinz feldmann (national institutes poly a p s1 f tag cd40l l poly a p s1 poly a p gfp rad5-s1/f/cd40l rad5-s1 rad5-gfp figure 1 . schematic representation of middle east respiratory syndrome coronavirus vaccine candidates. the proposed recombinant adenovirus 5 (rad5) construct (rad5-s1/f/cd40l) was engineered to express a fusion protein with the s1 subunit containing the s protein signal peptide at the n-terminal, followed by a trimerization motif derived from t4 bacteriophage fibritin (f) fused with the ectodomain of murine cd40 ligand at the c-terminus (cd40l). thus, the s1 and cd40l are expressed as a trimerized fusion protein via f (s1/f/cd40l). the construct was placed under the control of the cytomegalovirus promoter (cmv) and in front of the bovine growth hormone-poly a site (poly a). other control constructs included rad5-s1 and a control rad5 vector expressing green fluorescent protein (rad5-gfp). 50 ) assay as previously described [11] . all work involving infectious mers-cov was conducted within approved biosafety level 3 (bsl-3) at the galveston national laboratory (gnl) at the university of texas medical branch (utmb) in galveston. the immunogenicity and the protective efficacy of the rad5based vaccine candidates were evaluated against mers-cov infection in the hdpp4 tg + mouse model. a total of 45 hdpp4 tg + mice aged 4-5 months were used (15 mice/group). mice were intramuscularly immunized twice, 28 days apart, with 10 9 of rad5 vaccine candidates ( figure 2 ). blood samples were collected 3 weeks after each immunization via retro-orbital bleeding. four weeks after the second immunization, mice were intranasally challenged with 10 3 tcid 50 (~100 median lethal dose [ld 50 ]) of mers-cov. on days 3 and 6 postinfection, 4 mice from each group were killed to assess the virus titer and lung pathology. the remaining mice were monitored for morbidity and mortality on a daily basis for up to 18 days. all animal studies involving infectious mers-cov were conducted within approved animal bsl-3 laboratories at gnl in accordance with the guide for the care and use of laboratory animals of the nih and association for assessment and accreditation of laboratory animal care, and with institutional animal protocol approval from utmb. animals were housed in on-site animal facilities under a 12:12 light:dark cycle with room temperature and humidity kept between 21°c and 25°c and 31%-47%, respectively, with ad libitum access to food and water. at day 3 and 6 after challenge, 4 mice in each group were euthanized and their lungs were collected and examined for pathological changes after immunization and viral challenge. in brief, lung tissues were fixed in 10% buffered formalin for 72 hours, transferred to 70% ethanol, and later paraffin embedded. histopathological evaluation was performed on deparaffinized sections stained by routine hematoxylin and eosin staining. evaluations for histopathology were done by pathologists who were blinded to each specimen source. numeric scores were assigned to assess the extent of pathological damage as follows: 0, no observed pathology (undetectable infiltration); 1, mild pathology (up to 5% infiltration); 2, moderate pathology (up to 20% infiltration); 3, severe pathology (>20% infiltration). lungs collected from challenged mice on day 3 postchallenge were used to determine viral titer by tcid 50 . in brief, pieces of collected lung tissue were weighed and homogenized in phosphate-buffered saline containing 2% fetal calf serum with a tissuelyser (qiagen). after clarification of the cellular and tissue debris by centrifugation, the titers of infectious virus in the suspensions of infected tissues were determined using tcid 50 assay. the virus titers of individual samples were expressed as log 10 tcid 50 per gram of tissue and the minimal detectable level of virus was 2.5 log 10 tcid 50 as determined by lung size. lung samples from each group of mice (n = 4 per group) were transferred to individual vials containing rnalater solution and subsequently homogenized and subjected to total rna isolation using trizol reagent. to determine the viral titer in the lung, mers-cov-specific upstream e gene (upe) and endogenous control gene (mouse β-actin) were quantified using 1-step reverse-transcription quantitative polymerase chain reaction (rt-qpcr monitoring for morbidity and mortality upe mrna expression value was calculated for each replicate and expressed as the equivalent of log 10 equivalents per gram (tcid eq/g) of the tissue by the standard ct (∆∆ct) method using bio-rad cfx manager 3.0 software. the end-point titers of anti-s1 total immunoglobulin g (igg) as well as its isotypes including igg1, igg2a, and igg2b from immunized mice were determined by enzyme-linked immunosorbent assay as described previously [11] . serum samples were tested in a 2-fold serial dilution starting from 1:100. end-point titers were determined and expressed as the reciprocals of the highest dilution that gave an optical density signal higher than the cutoff value, defined as the mean of prebleed samples plus 3 standard deviations (sd). mers-pseudotyped viral particles (merspp) were produced and titrated using the huh7.5 cell line as described previously [39] . plasmids for generating merspp were kindly provided by dr nigel temperton at the university of kent, united kingdom. heat-inactivated serum samples were prepared in a 3-fold serial dilution starting from 1:20 and tested in duplicates in 96-well nunc white plates. a standard amount of merspp (~200 000 relative light units) was added to each well and plates were incubated for 1 hour at 37°c. then, approximately 10 000 huh7.5 cells were added to each well and incubated for 48 hours. cells only and cells with merspp only were included in quadruplicate as controls in all plates. following incubation, cells were lysed and luciferase activity was developed and measured using the bright-glo luciferase assay system (promega) according to the manufacturer's instructions. log 10 median inhibitory concentration (ic 50 ) neutralization titers were calculated for each serum sample using graphpad prism software. the standard live virus microneutralization (mn) assay was used as previously described [11] . in brief, starting at a dilution of 1:10, 60 μl volumes of serial 2-fold dilutions of heat-inactivated sera were mixed with equal volume of media containing 120 tcid 50 of mers-cov. each dilution was tested in duplicate in 96-well plates. the antibody-virus mixtures were incubated for 1 hour at 37°c before transfer of 100 μl into confluent vero e6 cell monolayers in 96-well plates. vero e6 cells cultured with dmem medium with or without virus were included as positive and negative controls, respectively. after 72 hours of incubation, the nab titer of each serum sample was determined as the reciprocal of the highest dilution capable of completely preventing virus-induced cytopathic effect in 50% of the wells (mn 50 ). statistical analysis was conducted using 1-way or 2-way analysis of variance with bonferroni posttest to adjust for multiple comparisons between groups using graphpad prism software. to evaluate the immunogenicity of the generated rad5 vectorbased mers vaccines, we immunized mice intramuscularly with 2 doses of individual vaccines and measured the levels of circulating s1-specific igg and its isotypes before and at 3 weeks after each immunization. here, we observed that both rad5-s1/f/cd40l and rad5-s1 induced s1-specific abs from all isotypes in hdpp4 tg + mice after primary or secondary immunization ( figure 3 ). as expected, control vector (rad5-gfp) failed to elicit any s1-specific ab response. notably, a single dose or 2 doses of rad5-s1/f/cd40l consistently elicited significantly higher levels of total igg as well as igg1, igg2a, and igg2b isotypes compared with the control group immunized with rad5-gfp (figure 3 ), whereas immunization with a single dose of rad5-s1 induced significant titers of total igg, igg1, and igg2a but not igg2b compared to the control group, with a second dose of rad5-s1 eventually inducing significantly higher levels of igg2b compared to the control group. interestingly, immunizing mice with a second dose of rad5-s1/f/cd40l enhanced the levels of circulatory igg and all tested isotypes compared with the rad5-s1-vaccinated group. furthermore, 2 doses of rad5-s1 only enhanced igg2b significantly compared to 1 dose, with mean igg1:igg2a ratios being 3.8 (sd, 3.2) and 3.9 (sd, 3.2) after priming and boosting, respectively. in contrast, boosting with a second dose of rad5-s1/f/cd40l resulted in a significant increase in total igg, particularly in igg2a and igg2b isotypes but not igg1, compared with a single dose of rad5-s1/f/cd40l, suggesting a bias toward th1 response as demonstrated by mean igg1:igg2a ratios of 3.4 (sd, 2.4) and 1.8 (sd, 1.4) after priming and boosting, respectively. together, these data showed that targeting the secreted s1 to cd40-expressing apcs via cd40l (rad5-s1/f/ cd40l) not only enhanced s1-specific igg abs levels but also boosted th1 responses, as demonstrated by markedly elevated titers of s1-specific igg2a and igg2b antibodies, especially after boosting. to extend our analysis and to evaluate the effector function of induced abs, we measured their neutralizing activities before and after each immunization. as expected, all immunized mice from all groups showed no detectable levels of nabs in samples collected before immunization. as shown in figure 4 , a single dose of either rad5-s1/f/cd40l or rad5-s1 elicited high levels of nabs compared to the control group (rad5-gfp), with rad5-s1/f/cd40l, but not rad5-s1, being consistently capable of inducing significantly higher levels of nabs against both live and pseudotyped mers-cov virus, suggesting that rad5-s1/f/cd40l is better than rad-s1 vaccine in promoting strong humoral response and neutralizing activity against mers-cov. however, after boosting once, all rad5-s1/f/ cd40l-and rad5-s1-immunized animals elicited robust and significant levels of nabs, indicating that at least 2 doses of rad-s1 are required to induce nab levels similar to those obtained by a single dose of rad5-s1/f/cd40l. these findings clearly confirm that incorporation of cd40l as molecular adjuvant could effectively enhance the immunogenicity of s1-based vaccines and may represent a very promising vaccine platform to induce protective immunity with a single dose. having demonstrated that cd40-targeted vaccine was superior in eliciting immune responses in hdpp4 tg + mice, we next investigated if these s1-based vaccines could effectively protect these highly mers-cov permissive mice from viral challenge. to this end, the vaccinated mice were challenged with 100 ld 50 of mers-cov and subsequently monitored for 3 weeks. it was clear that mice immunized twice with either rad5-s1 or rad5-s1/f/cd40l were completely protected based on clinical signs of disease (weight loss) and mortality ( figure 5 ), whereas rad5-gfp-immunized animals expectedly succumbed to lethal infection within days, likely due to encephalitis [28, 40] . these findings suggest that both rad5-s1 and rad5-s1/f/cd40l vaccines are protective in this mouse model. to further confirm that immunized hdpp4 tg + mice were protected from mers-cov infection after challenge, we measured lung viral titer, both infectious progeny virus and viral rna, on day 3 postchallenge by vero e6-based infectivity assay and rt-qpcr, respectively. analysis of infectious viral titer revealed that immunization with both vaccines significantly protected against viral replication to undetectable levels compared with rad5-gfp-immunized and -challenged mice ( figure 6 ). these results were further confirmed by an average of 3-to 4-log reduction in pulmonary viral rna, compared to that of the rad5-gfp-immunized group ( figure 6 ). it should be mentioned that, albeit not statistically significant, rad5-s1/f/cd40l-immunized animals had a viral rna load lower by 1 log compared with the rad5-s1 group, consistent with the overall stronger immune response we observed in this group. finally, investigations were carried out to assess safety of these vaccine candidates to rule out any vaccine-associated pulmonary injuries as observed with some vaccines against other coronaviruses. evaluation of lung pathology in immunized and challenged mice showed up to 15% multiple monocytic and lymphocytic infiltrations (moderate pathology) in the lungs of the rad5-gfp group 3 and 6 days postchallenge compared to other groups, which showed minimal to no inflammatory infiltration ( figure 7) . surprisingly, although rad5-s1 immunization protected animals from death and weight loss and prevented the lung infiltration similar to rad5-s1/f/cd40l, we observed perivascular hemorrhage in the whole lung of all examined mice immunized with rad5-s1 but not those vaccinated with rad5-s1/f/cd40l (figure 7) . together, these results suggest that although s1 alone could protect the animals from viral infection, it may inadvertently induce lung pathology, and that using cd40l as targeting molecule and molecular adjuvant does not only enhance immunogenicity and protective efficacy but also prevents vaccine-associated pulmonary pathology. the rapid spread and persistence of mers-cov in the arabian peninsula, in addition to the associated high mortality rates, represent a serious global public health concern, particularly that the elimination of the zoonotic reservoir is extremely unlikely at the current setting. this threat is further complicated by the absence of prophylactic or therapeutic measures. therefore, development of an effective and safe vaccine is urgently needed to prevent or contain mers-cov. several groups have investigated various mers-cov vaccine platforms in several animal models [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 28] . however, serious safety concerns are associated with vaccines for several coronaviruses including mers-cov and need to be elucidated and better understood [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . we showed in this study that although rad5 expressing s1 or cd40-targeted s1 were both capable of inducing significant levels of igg and nabs specific to mers-cov in immunized mice, incorporation of cd40l substantially enhances the immunogenicity of s1, as demonstrated by the effectiveness of a single immunization dose, which was sufficient to elicit stronger and robust immune responses compared to control groups, consistent with our previous reports [37, 38] . importantly, both s1 and cd40-targeted s1 vaccines provided complete protection, prevented virus replication significantly, and prevented monocytic and lymphocytic lung infiltration as compared to control group. however, the non-cd40-targeted vaccine (rad5-s1) uniformly induced varying degrees of severe perivascular hemorrhage within the lungs of all examined mice following viral challenge, regardless of the exhibited full protection against lethal mers-cov challenge. while these data suggest that there might be yet to be explained lung pathology associated with rad5 vector-and/or mers-cov s1-based vaccines, our results clearly show that including cd40l as a fusion protein of s1-based mers-cov vaccine could prevent or at least mitigate the safety concern of undesirable immunopathology while affording complete protection in hdpp4 tg + mice. at least 3 groups have developed different rad-vectored mers-cov vaccines [14, 41, 42] . however, these studies have mainly focused on the immunogenicity and/or the protective efficacy, and may have overlooked any possible vaccine-associated pathology, especially after viral challenge. our finding that rad5-s1 immunized animals were completely protected despite the observed pathology is reminiscent of the immunopathology documented using wiv for mers-cov, sars-cov, and respiratory syncytial virus after viral challenge [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [43] [44] [45] . it is of note that lung pathology was also observed in some previous studies involving rhesus macaques immunized with high doses of dna vaccines expressing mers-cov s protein [16] , as well as in mouse models vaccinated with rbd or vectored vaccines encoding s protein [46] [47] [48] . nevertheless, this is the first report of vaccine-associated severe pulmonary perivascular hemorrhage after viral challenge. although the exact molecular mechanisms underlying such vaccine-induced pulmonary injury associated with rad5-s1 vaccination remain to be determined, several factors could have been involved. specifically, the likely higher levels of residual infectious viruses in rad5-s1-immunized mice as detected by rt-qpcr, and the quantitative and perhaps qualitative difference of specific antibody and t-cell responses, as evidenced by the lower levels of ig2a and igg2b found in this group, could have played a role. . pulmonary viral load. viral load was determined in the lungs of immunized and challenged mice with middle east respiratory syndrome coronavirus (mers-cov). viral load was determined as median tissue culture infectious dose per gram (tcid 50 /g) and tcid 50 equivalent per gram (tcid 50 eq/g) by tcid 50 and reverse-transcription quantitative polymerase chain reaction (rt-qpcr) assays, respectively. limit of detection in the tcid 50 assay was 2.5 log 10 (dashed line) . tcid 50 eq/g was determined using a standard curve generated from dilutions of rna from a mers-cov with known virus titer. titers are shown as mean with standard deviation from 4 mice per group from 1 experiment. ***p < .0002 (1-way analysis of variance with bonferroni posttest); ns, not significant. see the figure 1 legend for explanation of the recombinant adenovirus 5 (rad5) constructs. rad5-gfp rad5-s1 rad5-s1/f/cd40l 100 μm therefore, future studies should include systematic analyses of t-cell responses as we have previously conducted [37, 38] , to fully explore the mechanisms of immunization-induced hemorrhage of the lungs. collectively, these previous data, along with our report here, indicate that viral components (ie, within the s protein) could be involved in inadvertent adverse reactions similar to those associated with the different s-based sars vaccines [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] . our study suggests that although rad5-s1 could be a potential vaccine candidate, it might be associated with severe lung pathology upon viral challenge or infection. these findings are of great importance in order to develop safe and effective human mers-cov vaccine. moreover, there is a huge gap in our understanding of the correlates of protection in camels, and the waning nature of nabs in these animals [49, 50] might hinder the "one health" approach in tackling the mers epidemic. therefore, it is critical to better understand and elucidate any safety concerns that might be associated with mers-cov vaccine to develop a safe and effective human vaccine. our previous studies have mainly focused on the development of candidate universal influenza vaccines using vectored vaccines by targeting antigens to cd40-expressing cells via cd40l. this platform was modified, optimized, and utilized to generate an immunogenic, protective, and safe mers-cov vaccine based on the s1 subunit of the mers-cov s protein, thus representing a promising platform for vaccine development against a broad range of pathogens. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia disease outbreak news outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine identification and characterization of novel neutralizing epitopes in the receptor-binding domain of sars-cov spike protein: revealing the critical antigenic determinants in inactivated sars-cov vaccine the spike protein of sars-cov-a target for vaccine and therapeutic development evaluation of candidate vaccine approaches for mers-cov purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice immunogenicity of candidate mers-cov dna vaccines based on the spike protein searching for an ideal vaccine candidate among different mers coronavirus receptorbinding fragments-the importance of immunofocusing in subunit vaccine design middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates chadox1 and mva based vaccine candidates against 1566 • jid 2019:220 (15 november) • hashem et al mers-cov elicit neutralising antibodies and cellular immune responses in mice prospects for a mers-cov spike vaccine immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses evaluation of modified vaccinia virus ankara based recombinant sars vaccine in ferrets vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants sars cov subunit vaccine: antibody-mediated neutralisation and enhancement monoclonal antibodies to the spike protein of feline infectious peritonitis virus mediate antibody-dependent enhancement of infection of feline macrophages antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus the role of cd40 and cd154/cd40l in dendritic cells the cd40-cd154 interaction in b cell-t cell liaisons cd40 ligand gene defects responsible for x-linked hyper-igm syndrome cd40 ligand expressed in adenovirus can improve the immunogenicity of the gp3 and gp5 of porcine reproductive and respiratory syndrome virus in swine multimeric soluble cd40 ligand (scd40l) efficiently enhances hiv specific cellular immune responses during dna prime and boost with attenuated poxvirus vectors mva and nyvac expressing hiv antigens significant alterations of biodistribution and immune responses in balb/c mice administered with adenovirus targeted to cd40(+) cells immunogenicity and protective efficacy of a dna vaccine encoding a chimeric protein of avian influenza hemagglutinin subtype h5 fused to cd154 (cd40l) in pekin ducks cd40 ligand preferentially modulates immune response and enhances protection against influenza virus targeting the ha2 subunit of influenza a virus hemagglutinin via cd40l provides universal protection against diverse subtypes an optimised method for the production of mers-cov spike expressing viral pseudotypes characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp4 mouse model il-13 is sufficient for respiratory syncytial virus g glycoproteininduced eosinophilia after respiratory syncytial virus challenge an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study key: cord-346787-uo8k6qic authors: jorgensen, sarah cj; kebriaei, razieh; dresser, linda d title: remdesivir: review of pharmacology, pre‐clinical data and emerging clinical experience for covid‐19 date: 2020-05-23 journal: pharmacotherapy doi: 10.1002/phar.2429 sha: doc_id: 346787 cord_uid: uo8k6qic the global pandemic of novel coronavirus disease 2019 (covid‐19) caused by severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) has created an urgent need for effective antivirals. remdesivir (formerly gs‐5734) is a nucleoside analogue pro‐drug currently being evaluated in covid‐19 clinical trials. its unique structural features allow high concentrations of the active triphosphate metabolite to be delivered intracellularly and it evades proofreading to successfully inhibit viral rna synthesis. in pre‐clinical models, remdesivir has demonstrated potent antiviral activity against diverse human and zoonotic β‐coronaviruses, including sars‐cov‐2. in this article we critically review available data on remdesivir with an emphasis on biochemistry, pharmacology, pharmacokinetics and in vitro activity against coronaviruses as well as clinical experience and current progress in covid‐19 clinical trials. the global pandemic of novel coronavirus disease 2019 (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has created an urgent need for effective antivirals. 1, 2 a wide variety of novel and repurposed agents are currently being evaluated in clinical trials and anecdotal reports of off-label and compassionate use of a number of these agents have emerged. 3, 4 remdesivir (formerly gs-5734) is a monophosphoramidate nucleoside analogue prodrug that was originally developed in response to the 2014 -2016 ebola outbreak in west africa. 5, 6 ribavirin against coronaviruses, for example, has been attributed to its removal by exon. 11 remdesivir is more active against viruses lacking exon but is able to partly evade proofreading and maintain potent antiviral activity in the presence of exon. this was nicely illustrated in a βcoronavirus murine hepatitis virus (mhv) model. 11 compared to wild-type mhv (ec 50 0.087 μm), viruses lacking exon were approximately 4-fold more sensitive to remdesivir inhibition (ec 50 0.019 μm). the reason remdesivir's activity is only modestly decreased by exon relates to two unique properties. first, remdesivir is incorporated into replicating rna more efficiently than natural nucleotides. 10, 12, 13 this was shown in a series of kinetic studies that determined the selectivity of βcoronaviruses for remdesivir vs. natural nucleotides. 10, 12 in this context selectivity was defined as the ratio of the parameters vmax (reflecting the maximal velocity of nucleotide incorporation) to km interestingly, selectivity values were higher for ebola virus (4) and for other nucleotide analogues with sars-cov-2 (favipiravir = 570 and ribavirin >> 1000). 10 the second reason that remdesivir is able to partly evade exon is because it functions as a nonobligate or delayed rna chain terminator. 10, 12, 13 delayed chain termination occurs when a nucleotide analogue has a free 3'-oh group required for the addition of natural nucleotides. the incorporation of the delayed chain terminator however perturbs the rna structure and synthesis is halted at some point downstream. 13 in sars-cov-1, sars-cov-2 and mers-cov, remdesivir (gs-443902) incorporation consistently results in chain termination after three additional nucleotides are added. 10, 12 it is thought that these nucleotides provide protection from exon excision. 10, 12 as described in the chemistry and pharmacology section, remdesivir is a pro-drug; concentrations decline rapidly after iv administration (plasma half-life, t ½ ~ 1 hour), followed by the sequential appearance of the intermediate alanine metabolite gs-704277 and the nucleoside monophosphate metabolite gs-441524 (plasma t ½ 24.5 hours) ( figure 1 ). inside cells, gs-441524 is rapidly converted to the pharmacologically active triphosphate analogue, gs-443902 which has a prolonged intracellular t ½ (peripheral blood mononuclear cell, pbmc t ½ ~ 40 hours). both remdesivir and gs-441524 exhibit linear pk following single doses between 3 mg and 225 mg and no remdesivir accepted article accumulation was observed following once daily dosing for up to 5 days. by contrast, gs-441524 reaches steady state around day 4 and accumulates by ~ 2-fold after multiple once daily dosing. 3 the remdesivir dosing regimen being evaluated in clinical trials (200 mg iv on day 1, then 100 mg iv on days 2 through 5 or 10) was substantiated by in vitro data and bridging the pk with the rhesus monkey experience to humans. 7, 8, 14 table 1 summarizes pertinent pk parameters of remdesivir and metabolite gs-441524, which were derived from single and multiple-dose studies in healthy human adult volunteers. 14 as shown, remdesivir c max values are many fold above concentrations required in vitro to inhibit sars-cov-2 replication by 50% and 90% (ec 50 0.137 -0.77 μm, ec 90 1.76 μm, see microbiology section). 14, 15 due to the near complete first pass effect of phosphoramidates, remdesivir is expected to have poor oral bioavailability. 6 plasma protein binding for remdesivir is moderate (free fraction of 12.1%). by contrast metabolites gs-704277 and gs-441524 exhibit low plasma protein binding with mean free fractions ≥ 85%. 14 in cynomolgus monkeys, radiolabeled remdesivir or its metabolites were detectable in the testes, epididymis, eyes and brain 4 hours after a 10 mg/kg dose (equivalent to 200 mg in humans). 16 levels in the brain were significantly lower than other tissues but accumulated over time. 16 distribution studies in humans have not yet been reported. remdesivir is a substrate of several cytochrome p450 enzymes in vitro (see drug interactions section), however clinical implications are unclear since the pro-drug is rapidly metabolized by plasma hydrolases. 14 by similar reasoning, the effect of hepatic impairment on remdesivir plasma levels should be low although specific studies have not been conducted in patients with hepatic impairment and the drug is contraindicated in patients with severe hepatic impairment. 14 metabolism of metabolites gs-704277, gs-441524 and gs-443902 has not been characterized. remdesivir exhibits low renal excretion (< 10%). however, 49% of a radiolabeled dose was recovered as gs-441524 in urine. 14 theoretically, plasma exposure of gs-441524 may be increased in patients with renal impairment. remdesivir formulations contain sulfobutylether β-cyclodextrin sodium (sbecd) as a solubility enhancer. 14 formulations containing sbecd have historically been cautioned against in patients with renal impairment although clinical data suggest sbecd accumulation does not increase the risk of acute kidney injury. 17 there are no recommendations for dose adjustments in patients with mild to moderate renal impairment at this time. under the fda emergency use authorization guidance, remdesivir is not recommended in patients with an estimated accepted article glomerular filtration rate (egfr) < 30 ml/min or serum creatinine ≥ 1 mg/dl unless the potential benefit outweighs the potential risk. 18 patients with an egfr < 30 ml/min and those who are receiving hemodialysis or hemofiltration are excluded from the ema compassionate use program. 14 there are no pk data available for children or women who are pregnant or breast feeding. remdesivir has demonstrated broad-spectrum activity against a diverse panel of zoonotic and clinically relevant human coronaviruses including sars-cov-1, sars-cov-2 and mers-cov with micromolar ec 50 or ic 50 values in multiple in vitro systems. 3, 9, 11, 19, 20 in human airway epithelial cell cultures for example, remdesivir inhibited sars-cov-1 and mers-cov replication with ic 50 values of 0.069 and 0.074 μm, respectively. 9 emerging data suggests remdesivir also exhibits potent activity against sars-cov-2. 14, 15, 21 in testing performed by the chinese cdc in collaboration with the manufacturer, gilead sciences, inc., using vero e6 cells (a cell line from green monkey kidney epithelial cells that is commonly used to evaluate antiviral activity), the ec 50 value was 0.137 μm against sars-cov-2. 14 14, 15 in this study viral load was fitted in logarithm scales (log 10 tcid 50 /ml and log 10 viral rna copies/ml). using this scale ec 50 values were many fold higher at 23.15 μm and 26.90 μm, respectively. 21 it is uncertain which method is most reflective of activity in vivo. it should also be remembered that ec 50 and ic 50 values represent a single point on the dose-response curve and the slope of the curve may be more important when evaluating potency. 22 this relationship is generally neglected when assessing in vitro antiviral activity. furthermore, clinical outcomes may depend on whether > 90% inhibition is achieved but ec 90 values are reported in a minority of studies. it is reassuring that the ec 90 value reported by wang and colleagues (1.76 μm) is achievable with the dosing regimen under evaluation (see pk section). 14, 15 the development of remdesivir resistance in coronaviruses has been assessed by cell culture in mhv which has similar ec 50 values to sars-cov-1, sars-cov-2 and mers-cov. 11 following > 20 accepted article in vitro passages, two nonsynonymous mutations were selected in the nsp12 rdrp: f476l and v553l. neither of the mutations directly altered rdrp's catalytic site or substrate binding pocket but they did cause minor structural alterations that are thought to impact rdrp's fidelity checking step before catalysis. 11, 13 compared to wild-type virus, these mutations conferred 2.4-fold and 5-fold reduced susceptibility to remdesivir, respectively while the double mutant showed 5.6-fold reduced susceptibility in vitro. 11 the ec 50 values of the mutants (0.057 -0.13 μm) however, remained below achievable human drug exposures. 3, 11, 13 furthermore, the mutations appear to confer a fitness cost, with wild-type virus rapidly outcompeting the mutants in the absence of remdesivir. of concern however, the affected residues are conserved across coronaviruses raising the possibility of a common pathway to resistance. in fact, substitutions at homologous sars-cov-1 residues conferred a 6-fold decrease in susceptibility to remdesivir (ec 50 0.01 μm → 0.06 μm). 11 no data specific to sars-cov-2 remdesivir resistance have been published at the time of writing. remdesivir's efficacy against respiratory diseases caused by β-coronaviruses has been evaluated in both rodent and rhesus monkey models. 7-9, 14, 19 before summarizing these studies the reader should be aware that rodent models are not ideal for evaluating remdesivir efficacy against β-coronaviruses. first, high levels of serum esterases rapidly degrade the pro-drug (t ½ ~ 5 minutes). 9, 16 investigators have attempted to overcome this by using esterase knock-out mice. 9 this improves plasma stability (t ½ ~25 minutes), but tissue levels of active metabolites are still ~ 10-fold lower. 9 second, the active triphosphate metabolite (gs-443902) has a significantly shorter t ½ in the mouse lung (3 hours) compared to human lung cells and nonhuman primate lungs (t ½ 20 -40 hours). 9, 14 finally, mice are naturally resistant to infection by mers-cov due to the inability of the spike protein binding domain to interact with the mouse dpp4 receptor. therefore transgenic mice harboring a modified humanized dpp4 must be used. 19 these limitations notwithstanding, sheahan and colleagues evaluated prophylactic and therapeutic remdesivir against sars-cov-1 in an esterase-deficient mouse model. 9 compared to control mice, prophylactic administration of remdesivir (24 hours before viral inoculation) resulted in less weight loss, lower viral lung titers and reduced sars-cov-1-induced lung pathology. 9 similarly, therapeutic remdesivir (administered 1 day post-inoculation) significantly diminished weight loss and viral load and improved pulmonary function, although to a lesser degree than the prophylactic regimen. when this article is protected by copyright. all rights reserved remdesivir was initiated 2 days post-infection however, it did not improve disease outcomes even though viral lung titers were reduced. the disease course in mice is accelerated compared to humans with viral titers peaking around day 2 concurrent with maximal damage to lungs. the same group of investigators conducted a similar study evaluating remdesivir and lopinavir/ritonavir +/-interferon-β against mers-cov in an esterase-deficient, humanized ddp4 mouse model. 19 prophylactic remdesivir significantly improved mers-cov-induced weight loss, decreased viral lung titers, and diminished features of acute lung injury compared to control mice. it also prevented mortality in mice administered a lethal dose of virus. in contrast, prophylactic lopinavir/ritonavir +/-interferon-β slightly reduced viral loads but had no impact on other disease outcomes. therapeutic remdesivir also led to improved disease outcomes and lower viral loads, but again, the effect size was diminished compared to prophylactic administration. remdesivir administered after a lethal viral dose was given (as opposed to before with prophylaxis), did not prevent mortality. 19 the prophylactic and/or therapeutic efficacy of remdesivir has been evaluated in mers-cov-infected and sars-cov-2-infected rhesus monkeys. these models more accurately recapitulate the lung disease observed in humans with mild-moderate mers-cov and sars-cov-2 infection compared to rodent models. 23, 24 dosing and pharmacokinetic analyses can also serve as a bridge to human dosing regimens. 7 however, due to the acute and accelerated infection course in monkeys, it is difficult to directly translate the timing of drug initiation to corresponding disease stages in humans. 23, 24 furthermore, neither model replicates many of the extrapulmonary disease manifestation seen in humans. 23, 24 in the mers-cov study, monkeys were administered remdesivir (5mg/kg which approximates drug exposures equivalent to 100mg in humans) 24 hours before mers-cov inoculation (prophylaxis) or 12 hours post-inoculation (treatment). 8 viral titers peak shortly after 12 hours in this model. for both groups, remdesivir was continued once daily until 6 days post-inoculation. prophylactic and therapeutic remdesivir treatment significantly reduced mers-cov-induced clinical signs, viral titers in respiratory specimens and the severity of lung lesions compared to control animals. these effects were most pronounced in the prophylactic group. 8 a similar prophylactic study was conducted using a higher remdesivir dose (10mg/kg which approximates drug exposures equivalent to 200mg in humans). 14 clinical signs and viral loads were significantly reduced vs. controls. increases in serum accepted article creatinine and blood urea nitrogen suggestive of altered renal function were only observed in remdesivir treated animals. 14 in the sars-cov-2 study, remdesivir was again initiated shortly before viral titers are expected to peak at 12 hours post-inoculation and a dosing regimen equivalent to the regimen being tested in human covid-19 clinical trials was used (10 mg/kg load ~ 200 mg in humans, then 5 mg/kg daily ~ 100mg daily in humans x 6 days). 7 compared to vehicle-treated monkeys, remdesivir-treated monkeys had improved clinical and radiographic outcomes. viral titers were reduced in lower respiratory tract specimens and undetectable at 3 days post inoculation. viral titers were not reduced in upper respiratory tract specimens nor in rectal swabs however. 7 if translated in humans, the absence of a reduction in viral shedding in these sites may have implications for potential transmission risk following clinical improvement. 7 initial experience with remdesivir in patients with covid-19 emerged in the form of case reports and uncontrolled case series as detailed in table 2 . 3, [25] [26] [27] [28] [29] all patients in these reports received remdesivir through compassionate use or expanded access programs. 14, 30 the largest report included 61 patients from centers in north america, europe and japan. 3 all patients were hospitalized with microbiologically confirmed covid-19 and had an oxygen saturation ≤ 94% on room air or required oxygen support. those with severe renal impairment, elevated hepatic enzymes, or receiving another investigational agent at baseline were excluded. of the 53 patients with short-term follow-up data available, 30 (56%) were receiving mechanical ventilation at baseline and a further 4 (8%) required extracorporeal membrane oxygenation (ecmo). remdesivir was started at a median of 12 days following symptom onset. after a median follow-up of 18 days, 7 (13%) patients died including 6 (18%) who were receiving invasive ventilation. adverse events were reported in 32 (60%) patients, including 12 (23%) who experienced at least one serious adverse event. the most frequent adverse events were hepatic enzyme elevations, diarrhea, rash, renal impairment, and hypotension. the publication of this report generated a great deal of controversy. the authors rightly acknowledge many of its limitations including the small sample size, the short duration of follow-up, missing data and the absence of a control group. 3 the lack of post day 1 data on 7 patients is particularly concerning and not adequately addressed in the publication. in addition, a target sample size was not reported nor was a rationale given for why the data were analyzed and reported at the point they were. it is unclear how many physicians applied for compassionate use for their patients but were denied. it would be interesting to know how these patients differed from the small number whose request for remdesivir was approved. finally, preclinical studies suggest that remdesivir has little benefit when administered after the peak in viral replication has occurred. 7-9, 14, 19 although the precise timing of peak viral loads was not accessed in these patients, it may be problematic to attribute favorable outcomes to the remdesivir when initiation was delayed beyond 12 days for many patients. against this backdrop, the first randomized, double-blind, placebo-controlled study evaluating remdesivir for covid-19 was recently published. 31 in this study 237 hospitalized adults with severe covid-19 were enrolled at 10 centers in wuhan, hubei, china and randomized (2:1) to remdesivir or matching placebo for a planned 10-day treatment course (200mg iv day 1, 100mg iv daily on days 2 to 10). severe covid-19 was defined as a sars-cov-2 positive respiratory specimen by rt-pcr, pneumonia confirmed by chest imaging and an oxygen saturation ≤ 94% on room air or a ratio of arterial oxygen partial pressure to fractional inspired oxygen ≤ 300mmhg. the primary outcome was time to clinical improvement defined as discharge alive from hospital or a 2-point improvement on a 6-point ordinal scale adapted from the world health organization's 8-point illness severity scale. 32 the study was stopped before reaching its target sample size of 453 due to slow enrollment. baseline characteristics were similar between groups: the median duration of illness before enrollment was 10 days and most patients (82%) required only low-flow supplemental oxygen at baseline. 31 use of antibiotics (91%) and corticosteroids (66%) were high in both groups. in addition, 28% and 32% of patients received lopinavir/ritonavir and/or interferon-α2β, respectively during the course of their illness. with regards to the primary outcome, there was no significant difference in time to clinical improvement between the remdesivir and placebo groups [ this article is protected by copyright. all rights reserved adequately powered clinical trials or meta-analyses. considering remdesivir's potent in vitro activity against sars-cov-2 and impressive results in pre-clinical models, one of the most unexpected findings in this study was that remdesivir had no impact on viral load. it is plausible that the delayed time to administration may have played a role. on the other hand, in vitro activity and animal data often do not translate into meaningful benefit for patients. 5, 33, 34 strengths of this study include its randomized, double-blind, placebo-controlled design, high protocol adherence and low loss to follow-up. 31 premature termination however leaves an underpowered study with inconclusive results. although cautious of over-interpretation, the point estimate for the primary outcome (hr 1.23) suggests any benefit of remdesivir may be more modest than hoped for (hr 1.40) and the small difference in favor of remdesivir for the composite primary outcome appeared to be driven largely by change in oxygenation status rather than the more clinically meaningful hospital discharge. as discussed in the safety section, this study did give valuable data to help better characterize the adverse effect profile of remdesivir. on the same day that the peer-reviewed publication of the study by wang and colleagues was released, partial results of the national institute of allergy and infectious diseases sponsored adaptive covid-19 trial (actt-1) were made public in a press release. 31 actt-1 was a randomized, double-blind, placebo-control study evaluating remdesivir (200mg iv day 1, then 100mg iv days 2 to 10) in hospitalized adult patients with covid-19. the primary endpoint, (which was changed 9 days before the independent data safety monitoring board meet to review the results announced in the press release) was time to recovery within 28 days after randomization using a 3point ordinal scale. after 606 recovery events, the median time to recovery was 11 days in the remdesivir group vs. 15 days in the placebo group (hr 1.31; 95% ci 1.12 to 1.54). mortality was numerically, but not statistically lower, in the remdesivir group (8.0% vs. 11.6%; p = 0.059). 31 in a second statement, gilead announced that similar outcomes had been demonstrated in their open-label, multicenter clinical trial of 5 vs. 10 days of remdesivir in patients with covid-19 not requiring invasive mechanical ventilation or ecmo. 35 more comprehensive data about these studies is anticipated. as of 5 may 2020, there are 10 clinical studies underway and registered on clinicaltrials.gov, including 6 randomized-controlled trials, the full results of which are eagerly awaited. 36 (table 3) this article is protected by copyright. all rights reserved information on the safety profile of remdesivir is rapidly evolving. until recently, most clinical experience has been in patients with ebola virus infection which has very different clinical manifestations compared to covid-19 making extrapolation of drug safety across populations problematic. 5, 14 in the palm study, 9 of 175 patients who received remdesivir for ebola virus infection experienced serious adverse events judged by trial investigators to be potentially related to remdesivir. 5 the most serious of these was hypotension during the loading dose rapidly followed by cardiac arrest and death. 5 among 38 ebola virus infection survivors who were enrolled in the single arm phase ii prevail iv study, 1 patient required a remdesivir dose reduction due to transaminase elevations. 14 safety data from four phase 1 pk studies in healthy volunteers has also been partly reported. 3, 23 in these studies, subjects received single remdesivir doses of up to 225mg or multiple doses of 150mg once daily for 7 or 14 days or 200mg once followed by 100mg daily for a total of 5 or 10 days. the most common adverse events, recorded in at least 5 subjects, were phlebitis, constipation, headache, ecchymosis, nausea and extremity pain. 3 transient asymptomatic grade 1 or 2 alanine aminotransferase (alt) elevations were observed in most subjects (exact frequency not reported) in the multi-dose pk studies including 1 individual with alt values > 10 x baseline. 3, 23 transaminase increases have also been reported in covid-19 patients treated with compassionate use remdesivir (table 2) 3, 23, 26, 28 in the report detailing the first 12 covid-19 cases in the us, all 3 patients who received remdesivir experienced transient transaminase elevations and gastrointestinal symptoms. 26 in addition one patient in the case series from france experienced alt elevation to 3 times the upper limit of normal and a maculopapular rash leading to remdesivir discontinuation 4 days following the first dose. 28 the fda reports that among 163 patients enrolled in the compassionate use program, the overall incidence of liver function test abnormalities was 11.7%. 23 seven cases were considered serious. the time to onset from first dose ranged from 1 to 16 days. with the exception of 1 case of seriously elevated bilirubin, none of the other cases had associated hyperbilirubinemia or symptoms of hepatitis. 23 in the rct reported by wang and colleagues, adverse effects were recorded in 66% of patients randomized to remdesivir of which 8% were grade 3 or 4 (thrombocytopenia n =4, hypokalemia n=2, hyperkalemia n = 2, anemia n=1, increased total bilirubin n = 1) . 31 the incidence and distribution of adverse effects was similar in the placebo group. aspartate aminotransferase (ast) elevations were observed in 5% of patients in the remdesivir arm compared to 12% in the placebo arm. no patients in accepted article either arm experienced grade 3 or 4 transaminase elevations. more patients in the remdesivir discontinued treatment prematurely (12% vs. 5%) with respiratory failure or acute respiratory distress syndrome being the most common event leading to drug discontinuation in the remdesivir group. in a summary of safety data reported by the fda from the a remdesivir clinical trial comparing 5 and 10day treatment courses in patients with covid-19, grade 3 and 4 alt and/or ast elevations occurred in 7% patients. elevations in bilirubin were uncommon (1.3%). 23 at this time, it is unclear if the liver enzyme abnormalities seen in patients receiving remdesivir for covid-19 are a component of the infectious process or due to the drug. the fact that abnormalities, albeit less severe, were seen in healthy volunteers suggests remdesivir is at least partly culpable. whether asymptomatic abnormalities are harbingers of more serious liver toxicity is also unknown. other approved nucleoside analogues including those used to treat hiv, hepatitis b and cytomegalovirus, are known to cause liver injury by a variety of mechanisms. 24 the most common involves inhibition of mitochondrial dna synthesis leading to mitochondria depletion or dysfunction. first generation hiv reverse transcriptase inhibitors are thought to cause hepatic injury by this mechanism. mitochondrial dysfunction can affect multiple tissues manifesting as myopathy, neuropathy, pancreatitis, bone marrow suppression and/or hepatic injury. 24 extra-hepatic manifestations of mitochondrial dysfunction have not been reported in patients exposed to remdesivir to date. nucleoside analogues may also cause liver injury through acute hypersensitivity reactions or the production of toxic intermediates. 24 these reactions tend to be idiosyncratic and uncommon whereas transaminase elevations are consistently observed in a minority of remdesivir treated patients. a fulsome safety assessment of remdesivir will require thorough review of data from recently completed and ongoing studies in addition to post-marketing surveillance and real-world experience. at the time of writing, no in vivo drug interaction studies of remdesivir have been published but the ability of remdesivir to inhibit or induce cytochrome p450 (cyp450) enzymes and transporters has been tested in vitro. 14 importantly however, as a pro-drug, remdesivir is rapidly degraded in vivo so the potential for clinically significant drug interactions is likely limited. 14 data on the potential for remdesivir metabolites to perpetrate drug interactions is even scarcer. in in vitro studies, remdesivir was a weak inhibitor of cyp1a2, cyp2c9, cyp2c19 and cyp2d6. 14 remdesivir's ic 50 for cyp3a was 1.6 μm suggesting inhibition may occur briefly with standard human exposures. inhibition of accepted article cyp450 enzymes by metabolites was not investigated. 14 tests of remdesivir cyp450 induction have been inconsistent; it may induce cyp1a2 and cyp2b6. 14 again the clinical importance of this is questionable. gs-441524 and gs-704277 demonstrated no cyp450 induction in these studies. remdesivir was found to be a substrate (oatp1b, p-glycoprotein) or inhibitor (oat1b1, oat1b3) of several drug transporters. 14 there are no exclusion criteria related to drug-drug interactions in current remdesivir clinical studies (table 3) . remdesivir is available in 2 bioequivalent formulations: a concentrated solution (5mg/ml) and a lyophilized powder formulation. 14, 18 vials contain 100mg of remdesivir and are preservative-free. 14, 18 readers are referred to the fda fact sheet for full storage, preparation and administration instructions. 18 for adults and children weighing ≥ 40kg requiring invasive mechanical ventilation or ecmo, the recommended dose is 200mg iv on day 1 followed by 100mg iv once daily on days 2 to 10. for those not requiring invasive mechanical ventilation or ecmo, a 5 day regimen is recommended. doses should be administered over 30 minutes to 2 hours. readers are referred to the fda fact sheet for full pediatric dosing recommendations. 18 there is no information on direct iv push, intramuscular or subcutaneous administration at this time. at this time there are no therapies that have been scientifically proven to improve mortality in covid19 . current management is largely focused on supportive care and prevention of complications. 37, 38 efficacious and safe antiviral agents are therefore urgently needed to relieve the burden on healthcare systems. as detailed in this review, remdesivir is a nucleoside analogue pro-drug with unique structural features that allow high concentrations of the active triphosphate metabolite to be delivered intracellularly. 6 it evades proofreading to successfully inhibit viral rna synthesis and has demonstrated potent antiviral activity against β-coronaviruses, including sars-cov-2 both in vitro and in animal models. 7-12, 15, 19, 20 these data, coupled with early safety data from clinical experience in ebola virus infection 5 , provide strong rationale for prioritizing testing of remdesivir in covid-19 clinical trials. the unpredictable nature of a pandemic however poses many challenges to researchers attempting to conduct clinical trials. 39 the first randomized controlled trial evaluating remdesivir for covid-19 was conducted a multiple sites in the initial outbreak epicenter yet it failed this article is protected by copyright. all rights reserved to meet its target sample size due to slow enrollment after the surge in cases diminished and it produced inconclusive results. when patient enrollment in clinical trials is not available, many clinicians face intense pressure to offer unproven therapies based on compelling pre-clinical data. as of april 30, 2020, more than 2000 patients with covid-19 have received remdesivir through compassionate use or expanded access programs. 35 it is impossible to know if these patients benefited or were harmed but we do know these programs do little to advance science. efforts must focus on ensuring the necessary infrastructure is in place to expand patient access to pragmatic clinical trials and make it simple for clinicians to enroll them. this could obviate the need for compassionate use programs. with at least 6 remdesivir randomized-controlled trials currently underway worldwide, there is reason to be optimistic that we will accumulate good data and learn if remdesivir provides a meaningful benefit to covid-19 patients. infectious diseases society of america. april 2020. https://www.idsociety.org/practice this article is protected by copyright. all rights reserved pharmacologic treatments for coronavirus disease 2019 (covid-19): a review characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention compassionate use of remdesivir for patients with severe covid-19 coronavirus disease 2019 treatment: a review of early and emerging options controlled trial of ebola virus disease therapeutics discovery and synthesis of a phosphoramidate prodrug of a adenine c-nucleoside (gs-5734) for the treatment of ebola and emerging viruses clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses remdesivir is a direct-acting antiviral that inhibits rna-dependent rna polymerase from severe acute respiratory syndrome coronavirus 2 with high potency coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus accepted article this article is protected by copyright. all rights reserved remdesivir and sars-cov-2: structural requirements at both nsp12 rdrp and nsp14 exonuclease active-sites remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys evaluation of sulfobutylether-beta-cyclodextrin (sbecd) accumulation and voriconazole pharmacokinetics in critically ill patients undergoing continuous renal replacement therapy broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro the antiviral activity of approved and novel drugs against hiv-1 mutations evaluated under the consideration of dose-response curve slope pneumonia from human coronavirus in a macaque model respiratory disease and virus shedding in rhesus macaques inoculated with sars-cov-2 delayed initiation of remdesivir in a covid-19 positive patient all rights reserved 26. team c-i. clinical and virologic characteristics of the first 12 patients with coronavirus disease 2019 (covid-19) in the united states covid-19 in solid organ transplant recipients: initial report from the us epicenter clinical and virological data of the first cases of covid-19 in europe: a case series a community transmitted case of severe acute respiratory distress syndrome due to sars cov2 in the united states. clin infect dis 2020. 30. emergency access to remdesivir outside of clinical trials remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial world health organization. covid-19 therapeutic trial synopsis nonhuman primates and translational research: progress, opportunities, and challenges hydroxychloroquine treatment of patients with human immunodeficiency virus type 1 earnings results clinical management of patients with moderate to severe covid-19 -interim guidance. public health agency of canada/canadian critical care society/association of medical microbiology and infectious disease canada infectious diseases society of america guidelines on the treatment and management of patients with covid-19 accepted article key: cord-328298-tm7gds8h authors: gardner, lauren m.; chughtai, abrar a.; macintyre, c. raina title: risk of global spread of middle east respiratory syndrome coronavirus (mers-cov) via the air transport network date: 2016-09-05 journal: j travel med doi: 10.1093/jtm/taw063 sha: doc_id: 328298 cord_uid: tm7gds8h background: middle east respiratory syndrome coronavirus (mers-cov) emerged from the kingdom of saudi arabia (ksa) in 2012 and has since spread to 26 countries. all cases reported so far have either been in the middle east or linked to the region through passenger air travel, with the largest outbreak outside ksa occurring in south korea. further international spread is likely due to the high travel volumes of global travel, as well as the occurrence of large annual mass gathering such as the haj and umrah pilgrimages that take place in the region. methods: in this study, a transport network modelling framework was used to quantify the risk of mers-cov spreading internationally via air travellers. all regions connected to mers-cov affected countries via air travel are considered, and the countries at highest risk of travel-related importations of mers-cov were identified, ranked and compared with actual spread of mers cases. results: the model identifies all countries that have previously reported a travel acquired case to be in the top 50 at-risk countries. india, pakistan and bangladesh are the highest risk countries which have yet to report a case, and should be prepared for the possibility of (pilgrims and general) travellers returning infected with mers-cov. in addition, the uk, egypt, turkey and the usa are at risk of more cases. conclusions: we have demonstrated a risk-analysis approach, using travel patterns, to prioritize countries at highest risk for mers-cov importations. in order to prevent global outbreaks such as the one seen in south korea, it is critical for high-risk countries to be prepared and have appropriate screening and triage protocols in place to identify travel-related cases of mers-cov. the results from the model can be used by countries to prioritize their airport and hospital screening and triage protocols. a novel coronavirus, middle east respiratory syndrome coronavirus (mers-cov) emerged from the kingdom of saudi arabia (ksa) in 2012 and has since spread to 26 countries. 1 as of march, 2016 around 1700 laboratory confirmed cases of mers-cov have been reported to the world health organization (who), with a case fatality rate of cfr 36%. 2 the vast majority of cases in middle east have been reported from ksa, followed by united arab emirate (uae), jordan and qatar. all cases of mers-cov reported so far have either been reported in the middle east or can be linked to the region through passenger travel, with the largest outbreak outside of the middle east occurring in south korea, following an imported case. in that instance a failure in identification, hospital triage and infection control resulted in 186 cases (185 in the republic of korea and 1 in china). 3 the large number of highly travelled airports in mers-cov affected countries poses a risk of further international spread through infected air passenger travellers. 4 in addition, ksa hosts annual mass gathering events such as the haj and umrah pilgrimages, during which millions of pilgrims from around the world travel to and congregate in regions where mers-cov is actively being reported. to further complicate the risk of spread, the origin of mers-cov is unknown, and there are still many uncertainties surrounding the sporadic epidemic patterns and transmission mechanisms. [5] [6] [7] mers-cov is phylogenetically related to bat coronaviruses, 8 and known to have been circulating for decades in dromedary camels in northeast africa. 9 serological surveys in camels during the recent outbreaks also report presence of anti-mers-cov antibodies in camels in egypt, kenya, oman, saudi arabia and uae. [10] [11] [12] [13] previous studies have also identified dromedary camel exposure to be a risk factor for mers-cov 14 and transmission from a dromedary camel to human has been confirmed. 15 it is generally believed that the mers-cov entered human populations from direct and indirect contact with camels or camel-related products. however, details of camel-to-person transmission are unclear, many cases have not reported camel contact or epidemiologic links, and large studies of handlers of infected camels fail to show transmission to humans. 16 with the exception of south korea, large epidemics have not seeded outside of the middle east. travel-related cases have been reported from the uk, germany, austria, france, turkey, greece, netherlands, malaysia, philippines, china, tunisia, algeria and the usa, with the number of secondary cases in all locations very small. 17 south korea represents the largest outbreak outside of the middle east, resulting in 186 cases and 36 deaths (cfr: 19%). 3 all secondary cases in south korea were linked to a single chain of transmission and associated with health care facilities. as was the case in south korea, human-to-human transmission has so far been mainly nosocomial, and modelling studies have revealed a 4-fold higher risk of transmission in healthcare setting compared with community settings. 18 yet, there is still no evidence of sustained human-to-human transmission. without a clear understanding of local transmission combined with ongoing cases in the middle east, the risk of global spread will continue. as such, risk analysis to identify countries at high risk of imported cases is critical for disease control. network models have been used previously to quantify the risk of disease transmission posed by the global air traffic system, [19] [20] [21] [22] [23] and airline travel data have been previously used to model the potential spread of mers-cov through air traffic network. 24, 25 khan et al. 24 utilized the flight itinerary and historic haj pilgrim data to predict the spread of mers-cov to other countries during haj season. the study-based risk on 2012 travel volumes departing all airports in four countries, saudi arabia, jordan, qatar and the uae. in addition, poletto et al. 25 assessed the risk of mers-cov, where the focus was estimating local level transmission parameters, and the global level risk analysis was simply based on the capacity of traffic volumes between the middle east and other countries. this study, similarly, proposes a global transport network modelling framework that accounts for international air travel originating in mers-cov-affected regions to quantify the importation risk of mers-cov posed to other countries via air travel passengers, but defines the risk more precisely, and at a more spatially disaggregate scale than the previous studies. the countries at highest risk of travel-related importations of mers-cov are identified, and the relative risk posed to each is quantified. the results provide a country level ranking and corresponding expected relative risk, which can be used by public health authorities in each country to ensure the appropriate screening and triage protocols are in place to identify travel-related cases of mers-coronavirus. the proposed model quantifies the relative risk of disease spread by mers-cov-infected travellers departing from the middle east and arriving at any given world airport. in the modelled network structure, airports are represented as nodes, and the links in the network represent directed air travel routes between airports (with and without stopovers). the risk of mers-cov spread posed by air travel between an origin airport i and destination airport j is r ij , and defined in equation (1): equation (1) is specific to the origin-destination (od) pair (i,j), and is dependent on the origin being in an active region (i.e. the virus is assumed to be in circulation and/or in the environment, and therefore this region poses a risk of local infection), the outbreak intensity at the origin (x i ), and the total passenger volume (v ij ) travelling between (i,j). the variable, a i , is a binary variable which indicates whether mers-cov is active in a given region. if a region is assumed to be active then the status of a i is set to one for all airports in that region, otherwise it is set to 0. active airports therefore pose the risk of exporting infected travellers. the outbreak intensity, x i , is equal to the relative outbreak size at the origin, and is normalized to the largest outbreak size across all active regions. this variable is assumed to be correlated with the outgoing travel risk posed by a region, and thus inflates the risk per outgoing traveller at a constant relative rate. the passenger flow variable, v ij , or the total passenger volume originating at airport i and travelling to airport j, captures the potential dispersal for the disease, and includes travel on both direct routes and indirect routes with stopovers between airport i and airport j. the od level travel risk can be aggregated across all origin airports, i, which are connected via travel routes to destination airport j, to quantify the risk posed to a destination airport j. the aggregated risk posed to destination j is defined by equation (2): the destination level risks are then normalized (as shown in equation 3) by dividing by the highest value computed over all destinations, j; thus what is being estimated is the expected relative risk posed to a destination airport j from all incoming travel: finally, the country-level risk is computed by aggregating the risk posed to all airports in a given country. similar to the airport-level destination risk, the country-level risk is normalized by dividing by the highest country-level risk across all countries. the final outcome is the expected relative risk of mers-cov-infected passengers arriving in each country. similar measures for importation risk have been used previously in the context of vector borne diseases. 19 the model requires passenger air travel data and case report data for mers-cov. passenger air travel data were purchased from the international air transport association (iata), 26 and includes the calibrated passenger travel volumes for all international air travel routes, where a route is defined by the origin, destination and stopover airports. the route-specific passenger travel volumes supplied by iata were calibrated based on data from 240 airlines comprising 84% of global air traffic, and includes over 9000 airports (iata). the passenger volumes were available at a monthly temporal resolution, which thus determined the temporal resolution of the model. the analysis was conducted using travel volumes from january to august, 2015. this data are used for the passenger flow variable, v ij . the mers-cov case data used in the model were collected from flutrackers. 27 detail of each case was sought from various sources including world health organization (who) 28 and european centre for disease prevention and control (ecdc). 17, 27 . the location and number of cases reported in each city in ksa between january and august 26 of 2015 was provided by ecdc. 17 these data define the outbreak intensity variable for each region, x i . the complete set of cities where mers-cov has been reported since 2012 was collected from who, 28 which is used to determine the status of region, a i . two case studies are evaluated to quantify the global risk posed by travellers departing from two different specified regions (denoted as scenarios 1 and 2). thus, the scenarios differ by the set of active regions specified in the model. scenario 1 quantifies the global risk posed by travellers departing mers-cov affected cities in ksa. in scenario 1 all cities in ksa which have reported cases in 2015 are assumed to be an active transmission region (a i ¼1), and the outbreak intensity variable, x i , is set equal to the number of reported cases for each city between january and august 26 of 2015. while this estimate is likely an underestimation of the actual number of people infected with mers in a given city, the value acts as a proxy for the number of infected individuals in a given city and inflates the outgoing (per person) travel risk proportionally. in scenario 1, all regions outside ksa are designated as inactive, and considered potentially at risk of imported cases. the input data for scenario 1, including the list of cities in ksa with confirmed cases, corresponding number of cases and assigned airport for each city are listed in supplementary table s1 . the airports were selected by identifying the closest (in terms of vehicle travel distance) major airport to each active city. this analysis is of particular relevance for events such as the haj and umrah, which attract millions of pilgrims to ksa, and more importantly, to cities where mers-cov is known to be in circulation. after congregating in masses at these events, the pilgrims return to their countries of origin. the results from scenario 1 can be used to help the home countries be better prepared for the return of potentially infected pilgrims post such mass gatherings in ksa. the relative risk posed to each country is computed, allowing those countries at highest risk to be identified and targeted for increased surveillance. scenario 2 increases the set of active regions identified in scenario 1 to include all regions that have reported non-travelrelated cases since mers-cov was first diagnosed in humans in 2012. scenario 2 is based on the assumption that the cases in these regions could have been contracted from either an infected human or alternatively, an animal or environmental source. thus our active regions are assumed to still pose a risk because the virus may still be in circulation in the environment, even if there has not been a recently reported case. this assumption is further supported by the evidence of mers circulating in animal populations in these active regions, which was noted previously. in efforts to conduct a conservative risk analysis, we consider all such regions potential sources of infection for travellers. the set of active transmission regions in scenario 2 includes a more comprehensive list of cities in ksa (listed in supplementary table s2) in addition to cities in jordan, kuwait, yemen, qatar, oman, uae, lebanon and iran. this scenario excludes south korea as an active transmission region because all cases in the south korea outbreak were traceable to the original human source, thus knowingly not contracted from the local environment. our list of active transmission regions is restricted to those in which cases were locally acquired and the source of infection is unknown (i.e. not from an infected traveller). supplementary table s3 lists the set of countries defined as active (in addition to ksa) in scenario 2 and the corresponding airports assumed to pose an outgoing risk. because it is impossible to accurately estimate the number of infected individuals in each active region at any given time, the outbreak intensity variable is set to a constant for each active region in scenario 2. therefore, in scenario 2, each active region is considered equally likely to have an infected traveller departing the city. while this is a major simplifying assumption of the model, it is still able to capture the risk as a direct function of the relative connectivity to regions with confirmed local cases, and the method is illustrated to predict the likelihood of imported infection cases accurately. for each scenario evaluated, the results are presented in both tables and figures. tables 1 and 2 list the top 50 countries atrisk of importing a mers-cov infected traveller from an active region, and corresponding relative expected risk for scenarios 1 and 2, respectively. figures 1 and 2 present the same information on a global map. to validate the model the results are compared with the set of confirmed travel imported cases that have been reported. the set of travel-related cases reported since 2012 are listed in supplementary table s4 . this list excludes cases that were reported in jordan, kuwait, yemen, qatar, oman, uae, lebanon and iran because the existence of locally acquired cases in these regions makes it impossible to confirm whether the reported cases in patients with travel histories acquired the virus locally or abroad. the airport-level risks which were aggregated to generate the country level risk are provided in supplementary tables s5 and s6 for scenarios 1 and 2, respectively. the tables include the top 50 airports at-risk of importing a mers-cov-infected traveller from an active region, and their corresponding relative expected risk. the country-level results are presented in tables 1 and 2 and figures 1 and 2 for scenarios 1 and 2, respectively. each table includes the top 50 at-risk countries, their respective ranking in terms of the relative risk posed, and the last column specifies if the country has previously reported a travel related mers-cov case (from the specified set of active regions which is scenariospecific), and if so how many. in scenario 1, where ksa is the only source of infected travellers considered in the model, india is identified to be at the highest risk, which has surprisingly not yet reported a case. the uae and egypt rank 2nd and 3rd, respectively, and both have reported travel-acquired cases from ksa. in total there have been 23 travel-imported cases confirmed from ksa in 15 different countries; 9 of the countries were included in the top 20 atrisk countries, and all were captured in the top 50. of the top 10 at-risk countries in scenario 1, 6 (60%) have previously reported cases from ksa. additional countries in the top 10 for scenario 1 that have not yet reported imported cases from ksa included pakistan, sudan and bangladesh. in scenario 2, where the set of mers-cov source regions is expanded to include all middle east countries that have previously reported locally acquired cases, 9 of the 22 travelimported cases reported outside the middle east (41%) were reported in one of the countries falling in the top 10 of our list, and 6 of the top 8 ranked countries (75%) have previously reported travel acquired cases. india is again identified as the most at-risk country, and along with pakistan which is ranked third, have yet to report mers-cov cases. countries such as uae and jordon, which were identified as high-risk in scenario 1, are no longer included in table 2 because they are treated as high-risk travel origins (rather than considered potentially atrisk destinations). in addition, because scenario 2 considers a more comprehensive set of travel sources, the risk is increased to highly connected regions such as the uk and germany, which both appear in the top 10 at-risk countries. the proposed model identifies the set of countries at greatest risk of importing mers-cov-infected travellers. two scenarios are evaluated, which differ by the set regions from which infected travellers are assumed to depart from. scenario 1 limits the outgoing travel risk to be from ksa only, while scenario 2 considers all regions which have reported locally acquired cases as potential sources. scenario 2 results are more likely to represent the expected risk posed globally by mers-cov. scenario 1 may be more appropriate for evaluating the risk during and immediately after major mass gatherings in ksa. for both scenarios, the quantified relative risk and ranking can be useful for informing public health authorities on the optimal locations for airport screening and travel protocols. for both scenarios, india is identified as the highest at-risk country, while pakistan and bangladesh are also included in the top 10 in both scenarios. critically, none of these countries have reported mers-cov cases as of june 2016, and may therefore be unprepared to diagnose and treat a case were one to arise. furthermore, each of these countries has substantial muslim populations who may travel to ksa for religious pilgrimages, and should be prepared for the possibility of (pilgrims and general) travellers returning infected with mers-cov. 29 in addition to the pilgrimage, a large number of people from these countries work in the middle east, and travel back and forth regularly. although these countries are already preparing for mers outbreaks and have issued policy documents, actual capacity to diagnose and treat cases as they arrive is questionable. it is likely that mers-cov cases may have already been imported to these countries, but the cases were not picked up by local surveillance due to mild disease, lack of diagnostic capacity and reporting. lack of proper diagnosis can pose significant harm to a country, as was illustrated by the episode in south korea, where the index case visited four hospitals before properly diagnosed, by which time he had spread infection to many people. surveillance systems should be improved to identify cases, and travellers from high risk areas should be screened and monitored for associated symptoms. a rapid response system should be developed accordingly, and healthcare workers should be trained to identify symptoms and manage the cases safely. in contrast to india, pakistan and bangladesh, the remaining seven of the top 10 at-risk countries across the two scenarios, have previously reported travel-imported cases, and these countries are likely to remain at-risk of importing infected travellers, and should continue surveillance and public travel health awareness campaigns, especially for religious pilgrims. for both scenarios all countries that have previously reported travel acquired cases were identified in the top 50, with the majority of countries reporting travel imported cases identified in the top 20. these results suggest that the model is able to capture the risk posed to most countries for importing mersinfected travellers. as further validation, the model results are consistent with previous travel-related studies. poletto et al. 25 reported the highest air traffic from middle east was to india (11.7%), bahrain (8.7%), pakistan (8.6%), uk (8.4%), oman (5.8%) and egypt (5.2%). in addition, khan et al. 24 found india, egypt, pakistan, uk, kuwait, bangladesh, iran and bahrain to be the highest risk countries of importing mers-cov cases. each of these countries were identified as high-risk in scenario 1 ranking as well, but in scenario 2 iran and kuwait were included as potential sources of infection in our model (which was not the case in 2012). while our country-level rankings are similar to khan et al., the variation is due to three main factors, (i) our model incorporates a weighting for each travel origin based on the outbreak intensity variable (included only in scenario 1) which serves to differentiate the outgoing traveller infection risk posed by different regions in the middle east where mers-cov has been reported, (ii) outgoing infected travellers departures are restricted to airports nearest the set of regions with reported locally acquired cases, rather than the entire middle east and (iii) more current air travel data are used. since 2012, when khan et al. conducted their study, the regions where mers-cov has been locally acquired have grown, and the travel patterns within the middle east have also changed. thus, the results in this study should be more accurate estimates of importation risk to countries connected to the middle east via the air traffic network. it should be noted, however, that several high-risk countries identified by the model have not had an imported case as of yet, whilst lower risk countries have. this outcome has substantial implications: (i) countries with lower risk should not be complacent, as south korea was ranked below the top 30 countries, yet experienced a large epidemic, and (ii) countries such as india and pakistan, identified at highest risk but yet to report any cases, should be prepared for potential imported cases. this model is subject to various limitations. the first limitation results from the uncertainty surrounding mers-cov transmission. the analysis quantifies the relative expected risk of mers-cov-infected (air travel) passengers arriving at airports based on a set of active transmission regions, the outbreak size at each and travel patterns; the model does not include the potential importation of infected intermediary hosts or intermediary host by-products since the influence of that possibility is yet to be established. second, an estimate of the true number of cases in each region based on the confirmed reported case count is not included, and the reported number of cases is instead used as a proxy. third, the potential harm posed to a region by local transmission (if successfully introduced into the population by an infected traveller) is not accounted for in the risk assessment. fourth, the model is limited by the available travel data. air travel reliably captures human mobility patterns at large spatial scales such as travel between countries and across large bodies of water; however, it does not fully capture travel patterns within countries due to the availability of alternative modes of travel such as road and rail. because the proposed model is solely based on air travel data, the intra-country mobility patterns are not fully captured. for this reason the risk is modelled and validated at the country level rather than a more disaggregate spatial scale, such as the city level, which cannot be reliably quantified without a more comprehensive multi-modal data set. the same issue regarding travel via alternative modes can also arise at the intercountry level, especially for smaller neighbouring countries. these limitations may all be factors in why several high-risk countries identified by the model have not had an imported case as yet, whilst lower risk countries have. other modelling approaches which focus on intra-country transmission are needed to inform internal disease control policy for a particular country. in summary, mers-cov has persisted in human populations for more than 4 years. 25, 30 despite uncertainty about transmission, a mixed pattern of both sporadic and epidemic spread continue to be observed, 6 suggesting the virus is still present in the environment in various regions of the middle east. the risk of travel-related cases will therefore remain as long as mers-cov transmission persists in the region. furthermore, the haj pilgrimage to mecca in ksa and other regional mass gatherings pose an ongoing risk of spread by international travellers. although travel cases were not reported after haj in 2013-2015 despite increase likelihood of secondary infection during such mass gathering, 5 several travel acquired cases of mers-cov were reported in travellers who had returned from umrah, a minor pilgrimage, in 2014. 31 luckily, the numbers of secondary cases resulting from these umrah travellers was not high, and the number of secondary cases resulting from infected travellers is typically very low. a recent modelling study analysed the data of 36 travel-related cases and estimated 22.7% risk of secondary transmission and 10.5% risk of tertiary transmission in event of importation of a mers case from middle east. 32 none the less, the potential harm posed by a single mers-cov-infected traveller to an unprepared country can be significant, as was exemplified by the south korean epidemic, in which 186 cases resulted from a single index case. in this instance, failure to recognize mers coronavirus infection at the hospital and lack of appropriate, timely triage and infection control procedures resulted in a large epidemic. for this reason, it is critical for all countries to be prepared and have appropriate screening and triage protocols in place to identify travel-related cases of mers-cov, and for risk stratification to be utilized to prioritize awareness and preparedness for mers-cov. countries higher on the risk scale, such as india and pakistan could invest more in preparedness and review existing protocols and policies. middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus outbreaks and emergencies. mers-cov in republic of korea at a glance as of 29 air passenger market analysis a scenario-based evaluation of the middle east respiratory syndrome coronavirus and the hajj the discrepant epidemiology of middle east respiratory syndrome coronavirus (mers-cov) unanswered questions about the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease mers coronavirus neutralizing antibodies in camels middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study mers coronavirus in dromedary camel herd, saudi arabia mers coronaviruses in dromedary camels antibodies against mers coronavirus in dromedary camels risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia evidence for camel-to-human transmission of mers coronavirus lack of middle east respiratory syndrome coronavirus transmission from infected camels severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission a global airport-based risk model for the spread of dengue infection via the air transport network a predictive spatial model to quantify the risk of air-travel-associated dengue importation into the united states and europe the risk of dengue spread from the philippines after typhoon haiyan inferring infection-spreading links in an air traffic network assessment of the potential for international dissemination of ebola virus via commercial air travel during the 2014 west african outbreak potential for the international spread of middle east respiratory syndrome in association with mass gatherings in saudi arabia assessment of the middle east respiratory syndrome coronavirus (mers-cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach passenger intelligence services (paxis) 2012-2015 case list of moh/who novel coronavirus mers ncov announced cases world health organisation (who) mers may not be sars; but india is still vulnerable interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response assessing the risk of observing multiple generations of middle east respiratory syndrome (mers) cases given an imported case conflicts of interest: none declared. key: cord-321080-pgxxkfc0 authors: wang, cong; hua, chen; xia, shuai; li, weihua; lu, lu; jiang, shibo title: combining a fusion inhibitory peptide targeting the mers-cov s2 protein hr1 domain and a neutralizing antibody specific for the s1 protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd date: 2019-01-06 journal: viruses doi: 10.3390/v11010031 sha: doc_id: 321080 cord_uid: pgxxkfc0 middle east respiratory syndrome coronavirus (mers-cov) has continuously posed a threat to public health worldwide, yet no therapeutics or vaccines are currently available to prevent or treat mers-cov infection. we previously identified a fusion inhibitory peptide (hr2p-m2) targeting the mers-cov s2 protein hr1 domain and a highly potent neutralizing monoclonal antibody (m336) specific to the s1 spike protein receptor-binding domain (rbd). however, m336 was found to have reduced efficacy against mers-cov strains with mutations in rbd, and hr2p-m2 showed low potency, thus limiting the clinical application of each when administered separately. however, we herein report that the combination of m336 and hr2p-m2 exhibited potent synergism in inhibiting mers-cov s protein-mediated cell–cell fusion and infection by mers-cov pseudoviruses with or without mutations in the rbd, resulting in the enhancement of antiviral activity in contrast to either one administered alone. thus, this combinatorial strategy could be used in clinics for the urgent treatment of mers-cov-infected patients. middle east respiratory syndrome (mers) coronavirus (mers-cov), a lineage c beta-coronavirus, was reported to cause severe respiratory tract infection [1, 2] . to date, 2266 laboratory-confirmed cases of infection with mers-cov, including 804 mers-cov associated deaths, have been reported to the world health organization (who) from 27 countries. currently, no effective therapeutics or vaccines are available to treat or prevent mers-cov infection. the spike (s) protein of mers-cov plays important roles in virus attachment, fusion, and entry into the target cell [3] [4] [5] . similar to other coronaviruses, the s protein of mers-cov consists of s1 and s2 subunits. the s1 subunit is responsible for the binding of the virion by its receptor binding domain (rbd) to the cellular receptor, dipeptidyl peptidase-4 (dpp4), while the s2 subunit mediates the fusion the 293t cell line was obtained from atcc (manassas, va, usa), and the huh-7 cell line was from the cell bank of the chinese academy of sciences (shanghai, china). these two cell lines were propagated in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum (fbs). peptide hr2p-m2 was synthesized by solid phase peptide synthesis at syn inc. (shanghai, china), and human mab m336 was provided by prof. tianlei ying at fudan university, shanghai, china. recombinant plasmids encoding the mers-cov s protein with d509g, d510g, q522h, or i529t mutations were kindly provided by dr. lanying du at the new york blood center, ny, usa. mers-cov pseudoviruses were constructed as described previously [19, 20] . briefly, 293t cells were plated in a t175 tissue culture flask and incubated at 37 • c for 16 h. cells were cotransfected with plasmids pnl4-3.luc.re encoding env-defective, luciferase-expressing hiv-1 and pcdna3.1-mers-cov-s encoding s protein with or without mutation in rbd at mass ratio of 1:1 using vigofect (vigorous biotechnology, beijing, china), according to the manufacturer's recommendation. the supernatant was replaced with fresh dmem at 8-10 h post-transfection and harvested after incubation for an additional 72 h. in order to remove cell debris, the supernatant was centrifuged at viruses 2019, 11, 31 3 of 12 3000 rpm for 10 min, followed by filtration through a 0.45 µm filter. mers-cov pseudovirus in the supernatant was quantified by testing p24 content in the product of mers-cov pseudovirus. a mers-cov pseudovirus inhibition assay was performed as previously described [5, 15, 21] . briefly, huh-7 cells were seeded (10 4 cells/well) into a 96-well plate and incubated overnight at 37 • c. mers-cov pseudovirus was incubated with a serially diluted inhibitor for 30 min at 37 • c, followed by the addition of huh-7 cells. the cells were incubated with or without pseudovirus as virus control and cell control, respectively. the culture was replaced with fresh medium 12 h post-infection and incubated for an additional 72 h. cells were lysed using lysis reagent (promega, madison, wi, usa), and cell lysates were transferred to a 96-well costar flat-bottom luminometer plate (corning costar, new york, ny, usa), followed by the addition of luciferase substrate (promega) to measure luminescence using an infinite m200 pro (tecan, grödig, austria). mers-cov s protein-mediated cell-cell fusion was performed as previously described [5] . briefly, plasmid paav-ires-mers-egfp encoding the mers-cov s protein was transfected into 293t cells (293t/mers/egfp) using the transfection reagent, vigofect (vigorous biotechnology, beijing, china). the target huh-7 cells expressing dpp4were incubated at 2 × 10 4 cells/well in wells of a 96-well plate for 12 h. the effector 293t/mers/egfp cells that express mers-cov s protein and egfp or the control 293t/egfp cells that express egfp only were preincubated at 10 4 cells/well with an inhibitor at the indicated concentration or phosphate buffered saline (pbs) as control at 37 • c for 30 min. the mixture of 293t/mers/egfp cells and an inhibitor or pbs were added to huh-7 cells in the wells, followed by a co-culture at 37 • c for 2 h. the 293t/mers/egfp cells fused or unfused with huh-7 cells were fixed with 4% pfa and counted under an inverted fluorescence microscope (nikon, tokyo, japan). the fused cell showed much larger size and weaker fluorescence intensity than the unfused cell because of the diffusion of egfp from one cell to more cells (figure 1 ). almost no fused cells could be observed in the groups of negative control (pbs+293t/egfp+huh-7) or peptide treatment (hr2p-m2+293t/mers/egfp+huh-7) (figure 1 ). the concentration for 50% inhibition (ic 50 ) was calculated using calcusyn software kindly provided by dr. t.c. chou [22] . viruses 2019, 11, x for peer review 3 of 12 was centrifuged at 3000 rpm for 10 min, followed by filtration through a 0.45 µm filter. mers-cov pseudovirus in the supernatant was quantified by testing p24 content in the product of mers-cov pseudovirus. a mers-cov pseudovirus inhibition assay was performed as previously described [5, 15, 21] . briefly, huh-7 cells were seeded (10 4 cells/well) into a 96-well plate and incubated overnight at 37 °c. mers-cov pseudovirus was incubated with a serially diluted inhibitor for 30 min at 37 °c, followed by the addition of huh-7 cells. the cells were incubated with or without pseudovirus as virus control and cell control, respectively. the culture was replaced with fresh medium 12 h post-infection and incubated for an additional 72 h. cells were lysed using lysis reagent (promega, madison, wi, usa), and cell lysates were transferred to a 96-well costar flat-bottom luminometer plate (corning costar, new york, ny, usa), followed by the addition of luciferase substrate (promega) to measure luminescence using an infinite m200 pro (tecan, grödig, austria). mers-cov s protein-mediated cell-cell fusion was performed as previously described [5] . briefly, plasmid paav-ires-mers-egfp encoding the mers-cov s protein was transfected into 293t cells (293t/mers/egfp) using the transfection reagent, vigofect (vigorous biotechnology, beijing, china). the target huh-7 cells expressing dpp4were incubated at 2 × 10 4 cells/well in wells of a 96-well plate for 12 h. the effector 293t/mers/egfp cells that express mers-cov s protein and egfp or the control 293t/egfp cells that express egfp only were preincubated at 10 4 cells/well with an inhibitor at the indicated concentration or phosphate buffered saline (pbs) as control at 37 °c for 30 min. the mixture of 293t/mers/egfp cells and an inhibitor or pbs were added to huh-7 cells in the wells, followed by a co-culture at 37 °c for 2 h. the 293t/mers/egfp cells fused or unfused with huh-7 cells were fixed with 4% pfa and counted under an inverted fluorescence microscope (nikon, tokyo, japan). the fused cell showed much larger size and weaker fluorescence intensity than the unfused cell because of the diffusion of egfp from one cell to more cells ( figure 1 ). almost no fused cells could be observed in the groups of negative control (pbs+293t/egfp+huh-7) or peptide treatment (hr2p-m2+293t/mers/egfp+huh-7) (figure 1 ). the concentration for 50% inhibition (ic50) was calculated using calcusyn software kindly provided by dr. t.c. chou [22] . six-week-old female specific-pathogen-free (spf) balb/c mice (bodyweight about 20 g) were divided into 3 groups of 3 mice each. mice in group 1, 2, and 3 were intraperitoneally (i.p.) injected with m336 (0.01 mg in 100 µl pbs) alone, hr2p-m2 (1 mg in 100 µl pbs) alone, and the combination of m336 (0.01 mg in 100 µl pbs) and hr2p-m2 (1 mg in 100 µl pbs), respectively. mice were sedated with nembutal (100 mg/kg body weight) before and 2 h after injection of the inhibitors, respectively, and bled retro-orbitally. the blood was centrifuged at 6000 rpm for 10 min after standing at room temperature for 3 h. the sera were collected and heat-inactivated at 56 • c for 30 min. inhibitory activity of the inhibitors on mers-cov pseudovirus was evaluated in serum as described above. to assess the potential synergistic effect, hr2p-m2 and m336 were mixed at the indicated molar concentration ratio, while hr2p-m2 alone and m336 alone were included as controls. the mixtures were serially diluted and tested for their inhibitory activity on mers-cov pseudovirus infection as described above. each sample was tested in triplicate, and data were analyzed for synergistic effect by calculating the combination index (ci), using the calcusyn program. ci values of <1 and >1 indicate synergy and antagonism, respectively, and synergy was divided into different strengths, according to ci values, as follows: <0.1 indicates very strong synergism; 0.1-0.3 indicates strong synergism; 0.3-0.7 indicates synergism; 0.7-0.85 indicates moderate synergism; and 0.85-0.90 indicates slight synergism [23, 24] . fold of potency enhancement was calculated with the ratio of concentrations of inhibitor testing alone and in combination. to determine the significance of difference in sensitivity between wild-type and mutant viruses to inhibitors and the inhibitory activity detected in sera from balb/c mice treated with inhibitors alone or combination, statistical analyses were performed using a two-tailed unpaired student's t-test, using graphpad prism, version 5.0. values with p < 0.05 and p < 0.01 were considered statistically significant and very significant, respectively. we first investigated the potential cooperative effects of combining hr2p-m2 with m336 on mers-cov pseudovirus infection. in our preliminary study, we found that ic 50 values of hr2p-m2 and m336 for inhibiting mers-cov pseudovirus infection were about 600 nm and 0.06 nm, respectively. therefore, we tested the inhibitory activity of hr2p-m2 alone, m336 alone, and hr2p-m2/m336 in combination at a molar concentration ratio of 10,000:1, respectively. as shown in figure 2 and table 1 , combining hr2p-m2 and m336 resulted in strong synergistic inhibitory activity against mers-cov pseudovirus infection with ci values of 0.13-0.20 for 50-90% inhibition, including potency enhancement of 12.9-to 18.9-fold for m336 and 8.4-to 12.9-fold for hr2p-m2. this result suggested that the mers-cov fusion inhibitory peptide hr2p-m2 and the mers-cov neutralizing mab m336 could be used in combination to enhance anti-mers-cov activity. note: each sample was tested in triplicate, and the mean values are presented. ratio of molar concentration of hr2p-m2 and m336 in combination is 10,000:1. next, we tested the potential synergistic activity of the hr2p-m2/m336 combination on mers-cov s protein-mediated cell-cell fusion. we adjusted the molar concentration ratio of hr2p-m2 and m336 in the combination to 4500:1, since the ic50 values of hr2p-m2 and m336 for inhibiting mers-cov s protein-mediated cell-cell fusion in our preliminary studies were about 700 nm and 0.15 nm, respectively. as shown in figure 3 and table 2 , the combination also exhibited strong synergism against mers-cov s protein-mediated cell-cell fusion (ci = 0.27) with enhancement of 18-fold for m336 and 4-fold for hr2p-m2. this result confirms that combining hr2p-m2, a mers-cov fusion inhibitor, with m336, a human neutralizing mab, results in strong synergism on s protein-mediated membrane fusion because they target the different stages of mers-cov fusion and entry processes. the blue curves represent inhibitors used alone, and the red curves represent each inhibitor used in combination. the width between two curves represents the fold of enhancement between an inhibitor used alone and in combination. note: each sample was tested in triplicate, and the mean values are presented. ratio of molar concentration of hr2p-m2 and m336 in combination is 10,000:1. next, we tested the potential synergistic activity of the hr2p-m2/m336 combination on mers-cov s protein-mediated cell-cell fusion. we adjusted the molar concentration ratio of hr2p-m2 and m336 in the combination to 4500:1, since the ic 50 values of hr2p-m2 and m336 for inhibiting mers-cov s protein-mediated cell-cell fusion in our preliminary studies were about 700 nm and 0.15 nm, respectively. as shown in figure 3 and table 2 , the combination also exhibited strong synergism against mers-cov s protein-mediated cell-cell fusion (ci = 0.27) with enhancement of 18-fold for m336 and 4-fold for hr2p-m2. this result confirms that combining hr2p-m2, a mers-cov fusion inhibitor, with m336, a human neutralizing mab, results in strong synergism on s protein-mediated membrane fusion because they target the different stages of mers-cov fusion and entry processes. note: each sample was tested in triplicate, and the mean values are presented. the molar concentration ratio of hr2p-m2 and m336 in combination is 4500:1. du et al. have previously shown that mers-cov pseudoviruses with mutations in rbd, such as d509g and d510g detected in some mers-cov strains isolated from different regions and at different times [17, 18] , are resistant to the neutralizing activity of an rbd-specific mouse mab mersmab1 [16] . in the present study, the sensitivity of pseudotyped mers-cov strains with key mutations in rbd, as identified in some mers-cov mutants isolated during the 2012-2015 outbreaks [17] , including d509g, d510g, q522h, and i529t, along with wild-type mers-cov, was compared between the inhibitory activity of hr2p-m2 peptide alone and m336 neutralizing mab alone. as shown in table 3 , the resistance of mers-cov mutants to the neutralizing activity of m336 is about 2-to 8-fold, whereas the pseudoviruses with or without mutations were equally sensitive to fusion inhibitory activity of hr2p-m2. this result suggested that use of mab m336 alone is unable to control the infection by mers-cov strains with mutations in rbd. table 3 . sensitivity of mers-cov pseudoviruses with or without mutations in the receptor-binding domain (rbd) to the inhibitory activity of m336 (nm) and hr2p-m2 (µm) separately. [17, 18] , are resistant to the neutralizing activity of an rbd-specific mouse mab mersmab1 [16] . in the present study, the sensitivity of pseudotyped mers-cov strains with key mutations in rbd, as identified in some mers-cov mutants isolated during the 2012-2015 outbreaks [17] , including d509g, d510g, q522h, and i529t, along with wild-type mers-cov, was compared between the inhibitory activity of hr2p-m2 peptide alone and m336 neutralizing mab alone. as shown in table 3 , the resistance of mers-cov mutants to the neutralizing activity of m336 is about 2-to 8-fold, whereas the pseudoviruses with or without mutations were equally sensitive to fusion inhibitory activity of hr2p-m2. this result suggested that use of mab m336 alone is unable to control the infection by mers-cov strains with mutations in rbd. table 3 . sensitivity of mers-cov pseudoviruses with or without mutations in the receptor-binding domain (rbd) to the inhibitory activity of m336 (nm) and hr2p-m2 (µm) separately. to determine whether the combination of hr2p-m2 and m336 also exhibited synergistic antiviral activity against infection of mers-cov strains with mutations in rbd or in the hr1 domain, we constructed pseudoviruses bearing mers-cov s protein with mutations in rbd, including d509g, d510g, q522h, or i529t, and those in the hr1 domain, including q1020h and q1020r [25, 26] . we then tested their sensitivity to the inhibition of hr2p-m2 alone, m336 alone, and the hr2p-m2/m336 combination. as shown in table 4 , combining m336 with hr2p-m2 exhibited strong synergism against infection by pseudotyped mers-cov strains with or without mutations in the rbd or hr1 domain with ci value less than 0.3 and potency enhancement in the range of 6-to 25-fold, suggesting that this combinational therapy has potential to be further developed for treatment of patients infected by different mers-cov strains, including those with resistance to rbd-specific neutralizing antibodies. table 4 . combination index and fold of enhancement for inhibiting mers-cov pseudoviruses with or without mutations in rbd in s1 subunit and hr1 in s2 subunit of mers-cov s protein by hr2p-m2 and m336. to determine whether the hr2p-m2/m336 combination could sustain its efficacy in vivo compared to hr2p-m2 or m336 alone, we tested the anti-mers-cov pseudovirus activity of the inhibitors in sera of mice treated with i.p. injection of hr2p-m2, m336, and the hr2p-m2/m336 combination, respectively. as shown in figure 4 , the inhibitory activity detected in sera from mice treated with hr2p-m2 or m336 alone was significantly higher than that detected in sera from mice before inhibition of any inhibitor. on the other hand, the anti-mers-cov activity detected in sera from mice treated with the hr2p-m2/m336 combination was significantly more potent than that detected in sera of mice administered with hr2p-m2 or m336 alone. this result confirms that combining hr2p-m2 with m336 affords synergism against mers-cov s infection, both in vitro and in vivo. note: the molar concentration ratio of hr2p-m2 and m336 in combination against wildtype virus, viruses with mutations in rbd, and those in hr1 is 10,000:1, 4500:1, and 10,000:1, respectively. to determine whether the hr2p-m2/m336 combination could sustain its efficacy in vivo compared to hr2p-m2 or m336 alone, we tested the anti-mers-cov pseudovirus activity of the inhibitors in sera of mice treated with i.p. injection of hr2p-m2, m336, and the hr2p-m2/m336 combination, respectively. as shown in figure 4 , the inhibitory activity detected in sera from mice treated with hr2p-m2 or m336 alone was significantly higher than that detected in sera from mice before inhibition of any inhibitor. on the other hand, the anti-mers-cov activity detected in sera from mice treated with the hr2p-m2/m336 combination was significantly more potent than that detected in sera of mice administered with hr2p-m2 or m336 alone. this result confirms that combining hr2p-m2 with m336 affords synergism against mers-cov s infection, both in vitro and in vivo. the high mortality of mers-cov-infected patients [27] [28] [29] calls for the development of highly effective anti-mers-cov therapeutics. although we and others have previously identified a mers-cov fusion inhibitory peptide (hr2p-m2) targeting the mers-cov s2 protein hr1 domain and a highly potent human neutralizing mab (m336) targeting the mers-cov s1 protein rbd data are presented as means ± sd. **, and *** represent p < 0.01, and p < 0.001, respectively. the high mortality of mers-cov-infected patients [27] [28] [29] calls for the development of highly effective anti-mers-cov therapeutics. although we and others have previously identified a mers-cov fusion inhibitory peptide (hr2p-m2) targeting the mers-cov s2 protein hr1 domain and a highly potent human neutralizing mab (m336) targeting the mers-cov s1 protein rbd [10, 26, 30] , their further development is limited by low potency in the case of hr2p-m2 and low efficacy to neutralize mers-cov strains with rbd mutations in the case of m336 [10, [31] [32] [33] . the combinatorial use of drugs with different mechanisms of action, i.e., cocktail regimen, has been widely applied in clinics [22] . for example, the combinatorial use of hiv reverse transcriptase (rt) inhibitors and protease inhibitors, known as highly active anti-retrovirus therapy (haart), has shown significant synergism in inhibiting hiv-1 infection, reducing adverse effects and delaying the emergence of drug resistance, thus extending the lifespan of millions of hiv/aids patients [34] [35] [36] . moreover, we previously showed that combining hiv-1 attachment inhibitors with rt inhibitors, or combining the 1st, 2nd, and/or 3rd generation hiv fusion inhibitors that target different sites in the hiv-1 gp41 hr1 domain, exhibited synergistic and complementary effect against infection by a broad spectrum of hiv-1 strains, including those resistant to hiv attachment inhibitors, fusion inhibitors, and rt inhibitors [37] [38] [39] . in this study, we compared the anti-mers-cov activity of hr2p-m2 alone and m336 alone with that of hr2p-m2/m336 in combination and found that the inhibitory activity of the hr2p-m2/m336 combination was significantly more potent than either one administered alone against mers-cov s protein-mediated cell-cell fusion and mers-cov pseudovirus infection, suggesting synergistic activity based on the dual mechanisms of action whereby hr2p-m2 targets the s2 subunit hr1 domain for inhibiting s2-mediated virus-cell or cell-cell fusion [5] and m336 targets the s1 subunit rbd for inhibiting virus-cell binding or virus attachment [10] . it has been well known that drug synergism can be expected when drugs that act by different mechanisms of action are mixed together [22] . while mers-cov pseudoviruses with mutations in rbd were resistant to the rbd-specific mab m336, they were equally sensitive to hr1-targeting peptide hr2p-m2. notably, however, the hr2p-m2/m336 combination exhibited strong synergistic antiviral activity against all pseudotyped mers-cov strains, including those with mutations in rbd of s protein, which are even resistant to an rbd-specific mouse mab mersmab1 [16] . we also demonstrated that sera from mice treated with the hr2p-m2/m336 combination revealed significant efficacy in inhibiting mers-cov pseudovirus infection compared to hr2p-m2 or m336 alone. collectively, these results suggest that the combinatorial strategy overcomes the weaknesses of hr2p-m2 peptide and m336 mab, while, at the same time, takes advantage of the unique mechanism of action of each to provide, by the sum of both, much more effective inhibitory activity against mers-cov infection than either peptide or mab used alone. the strong synergy of the combination is expected to reduce the dosage of the individual inhibitor in such combinational therapy, resulting in decreased cost and toxicity, thus making the final product more affordable and safer. therefore, this combinational therapy shows promise for further clinical development. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group mechanisms of coronavirus cell entry mediated by the viral spike protein structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov naadp-dependent ca(2+) signaling regulates middle east respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies anti-hiv antibody and drug combinations exhibit synergistic activity against drug-resistant hiv-1 strains synergistic effect resulting from combinations of a bifunctional hiv-1 antagonist with antiretroviral drugs protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans hospital outbreak of middle east respiratory syndrome coronavirus urgent development of effective therapeutic and prophylactic agents to control the emerging threat of middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov mutations in the spike protein of mers-cov transmitted in korea increase resistance towards antibody-mediated neutralization quantifying residual hiv-1 replication in patients receiving combination antiretroviral therapy antiviral effect of double and triple drug combinations amongst hiv-infected adults: lessons from the implementation of viral load-driven antiretroviral therapy the challenge of finding a cure for hiv infection combination of candidate microbicides cellulose acetate 1,2-benzenedicarboxylate and uc781 has synergistic and complementary effects against human immunodeficiency virus type 1 infection combinations of the first and next generations of human immunodeficiency virus (hiv) fusion inhibitors exhibit a highly potent synergistic effect against enfuvirtidesensitive and -resistant hiv type 1 strains synergistic efficacy of combination of enfuvirtide and sifuvirtide, the firstand next-generation hiv-fusion inhibitors we thank lanying du at the new york blood center for providing the plasmid encoding mers-cov s protein with mutation in rbd and tianlei ying at fudan university for providing mab m336. the authors declare no competing financial interests.viruses 2019, 11, 31 key: cord-343302-g9vcchrh authors: agrawal, anurodh shankar; ying, tianlei; tao, xinrong; garron, tania; algaissi, abdullah; wang, yanping; wang, lili; peng, bi-hung; jiang, shibo; dimitrov, dimiter s.; tseng, chien-te k. title: passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection date: 2016-08-19 journal: sci rep doi: 10.1038/srep31629 sha: doc_id: 343302 cord_uid: g9vcchrh middle east respiratory syndrome coronavirus (mers-cov) has repeatedly caused outbreaks in the arabian peninsula. to date, no approved medical countermeasures (mcm) are available to combat mers-cov infections. several neutralizing human monoclonal antibodies (mabs), including m336, a germline-like human mab, have been chosen as promising mcm for mers-cov. however, their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections. we assessed the prophylactic and therapeutic efficacy of m336 by using well-characterized transgenic mice shown to be highly sensitive to mers-cov infection and disease. we found that mice treated with m336 prior to or post lethal mers-cov challenging were fully protected, compared to control mice which sufferered from profound weight loss and uniform death within days after infection. taken together, these results support further development of m336 and other human monoclonal antibodies as potential therapeutics for mers-cov infection. scientific reports | 6:31629 | doi: 10.1038/srep31629 and long-term threat to people, particularly those who interact closely with camels in the arabian peninsula. even though mers-cov presently has limited human-to-human transmission 2, 16 , the high mortality rate of this virus and limited information on the mechanism able to confer increased human-to-human transmission have raised concerns of a potential mers pandemic. indeed, the recent outbreaks in korea and the appearance of super-spreading events indicate that mers-cov has the ability to cause large outbreaks outside of the arabian peninsula [17] [18] [19] . currently, no approved vaccines or drugs are available to treat this viral infection. these facts highlight an urgent need to develop potent prophylactic and therapeutic agents to fight this lethal virus. similar to other coronaviruses, mers-cov uses the envelope spike (s) glycoprotein, a class i transmembrane protein, for interaction with its cellular receptor for binding, fusion and entry into the target cell 20 . the receptor binding domain (rbd) located in the s1 domain of the mers-cov spike is responsible for binding to the well-characterized cellular receptor identified as dpp4 (cd26) and is, therefore, critical for binding and entry of the virus [20] [21] [22] . therefore, neutralizing antibodies capable of blocking such interaction could be promising preventive and/or therapeutic candidates. recently, human monoclonal antibodies (mabs) capable of neutralizing mers-cov have been identified and characterized by several research groups [23] [24] [25] [26] [27] [28] . these antibodies have been isolated from naive human antibody libraries, from transgenic "humanized" mice, or from b cells of an infected individual, and they recognize different epitopes on mers-cov rbd. one of the most potent mabs, m336, is a germline-like antibody identified from a very large (~10 11 size) phage-displayed antibody library derived from b cells of healthy donors. this mab exhibits exceptionally potent neutralizing activity (ic 50 = 0.005 μ g/ml) in vitro 23 . moreover, because its epitope almost completely (~90%) overlaps with the receptor-binding site of dpp4 on mers-cov rbd, as is evident by its recently solved crystal structure 29 , the probability of generation of resistant mutants may be absent or very low. notably, although the functions of these mabs have been extensively characterized in vitro, their further clinical development has been hindered by the lack of an effective animal model of mers-cov infection. mers-cov cannot infect small laboratory animals (e.g., mice, hamsters and ferrets) as a consequence of species-specific differences in dpp4, while only causing mild-to-moderate symptoms in rhesus macaques. marmosets, which are more susceptible to mers-cov, developed a moderate-to-severe disease, but limited availability and high cost have hampered their use 30 . rabbits can be infected, but the infectious virus is challenging to detect 31, 32 . it was found that the expression of human dpp4 could overcome the lack of susceptibility in normal mice. with prior transduction of adenoviral human dpp4-expressing vectors, mice became susceptible to mers-cov infection without revealing any measurable clinical manifestations 33 . in contrast, transgenic (tg) mice with the human dpp4 gene integrated into the genome readily developed acute morbidity (weight loss), and uniform death occurred within a week 34, 35 , making it an ideal preclinical model for the development of vaccines and treatments against mers. some of the aforementioned human neutralizing monoclonal antibodies have been shown to protect engineered human dpp4-expressing mice and the naturally permissive rabbits, entirely based on their ability to inhibit mers-cov infection and/or alleviate histopathology of the lungs 27, 28, 36, 37 . to further verify the protective efficacy of these human monoclonal antibodies, particularly m336, against mers-cov infection, it is highly desirable to use the well-characterized human dpp4 tg mice known to result in acute disease (weight loss) and death 34, 35 . by using this highly permissive tg mouse model, we evaluated the prophylactic and therapeutic efficacy of m336 mab in vivo. we report in this study for the first time that treatment of tg mice with a single-dose of m336 antibody prior to or after challenging with 1,000 ld 50 of mers-cov protected mice from the lethality in a dose-dependent manner, thereby representing the first antibody tested for its protective efficacy against lethal mers-cov infection. prophylactic efficacy of mers-cov rbd-specific human monoclonal antibody, m336. we established a tg mouse model which is profoundly sensitive and susceptible to mers-cov infection, as determined by high viral titers in the lungs, as well as a high rate of morbidity and mortality 34, 38 . equipped with this small animal model of human mers-cov, we investigated the protective efficacy of mab m336. to accomplish this, each group (n = 6) of mice was treated via the intraperitoneal (i.p.) route with two different doses: 0.1 mg and 1 mg per mouse diluted in 100 μ l pbs, and challenged intranasally (i.n.) at 12 h post treatment with 10 4 tcid 50 (i.e., 1,000 ld 50 ) of mers-cov in a volume of 60 μ l 38 . challenged mice were monitored daily for clinical manifestations (weight loss) and mortality. as shown in fig. 1 , the group treated with 1 mg mab survived viral infection without showing any clinical symptoms. these mice initially showed either no weight loss or recovered from mild weight loss within three days (fig. 1a) . on the other hand, the group treated with 0.1 mg mab showed a gradual weight loss (15-20%) until day 13 just before starting to recover (fig. 1a ). all surviving mice (one died on day 13 in mice treated with 0.1 mg of m336) continued to recover and appeared well up to 21 dpi when the experiment was terminated (fig. 1b) . all mers-cov-challenged mice pretreated with a high dose (1 mg) of irrelevant mab m102.4 exhibited profound weight loss (> 15%) and succumbed to infection with 100% mortality by day 8 p.i. (fig. 1a,b ). therapeutic efficacy of mers-cov rbd-specific human monoclonal antibody, m336. to determine the therapeutic potential of this human monoclonal m336 antibody, groups of mice (n = 6 per group) were challenged (i.n.) with 10 4 tcid 50 of mers-cov (i.e., 1,000 ld 50 ) in a volume of 60 μ l and then treated (i.p.) 12 hours later with a single dose of either 1 mg or 0.1 mg of m336 or 1 mg of m102.4 antibody (control) in 100 μ l per mouse, followed by monitoring daily for wellbeing (weight loss and other clinical manifestations) and mortality of mice. we noted that whereas treatment with 1 mg of m336 antibodies was effective in the protection against the lethality caused by mers-cov infection, it failed to protect mice fully from the onset of clinical illness (weight loss). specifically, all of the challenged mice treated with 1 mg of m336 antibody suffered an attenuated (< 10%), and transient weight loss until day 9, and gradually recovered to day 21 when the experiment was terminated (fig. 2) . similarly, challenged mice treated with a low dose of 0.1 mg of m336 antibodies suffered from attenuated and transient weight loss until day 7 p.i. and gradually recovered. however, we noted a single death at day 9 in this low dose treatment group (fig. 2) . as expected, all mice treated with a single dose of 1 mg of control m102.4 antibody exhibited profound weight loss (> 15%) and succumbed to mers-cov infection with 100% mortality by day 8 p.i. (fig. 2) . taken together, these results indicate that this mers-cov rbd-specific human m336 antibody can be highly effective as prophylactic or therapeutic modalities in protecting highly permissive transgenic mice against mers-cov infection and disease. we also investigated the protective mechanism of m336 against mers-cov by determining the lung virus titers in challenged mice at day 2 after treatment. specifically, we sacrificed two mice (out of 6) in each group, as described above for figs 1 and 2 and their lung specimens were harvested for determining viral titers by using via vero e6 cell-based infectivity assay and quantitative pcr (q-pcr)-based assay targeting the upstream e gene of mers-cov. as shown in fig. 3a we were unable to recover infectious virus from any mouse treated with 1 mg of m336 antibody either before or after challenge with mers-cov. however, we were able to detect a barely detectable infectious virus, with the limit of detection (lod) of 2.3 log ticd 50 /g, from a single mouse receiving 0.1 mg of m336 prior to viral challenge. these results indicated that mab m336 most likely confers protection from lethal challenge by restricting viral replication within the lungs, thereby preventing viral infection in the brains and other organs. titers of viral rna copy number, as shown by qrt-pcr assays, were also compared among groups having different doses of mabs. lungs of infected mice were harvested on day 2 post-and pre-virus challenge group. all groups exhibited detectable viral rna. titers were significantly lower than those in the control group in all m336-treated groups. in the pretreatment group, mice treated with 1 mg of m336 showed a 2-log reduction in viral rna detection, while a ~1 log reduction in viral numbers was seen in mice treated within 0.1 mg m336 when compared to mice receiving control mab m102.4. in the post-treatment group, a smaller (~1 log) difference in viral rna copy number (compared to that in the pretreatment group) was observed between mice treated with 1 mg antibody compared with those receiving control antibody, while a more than 1 log reduction in viral rna number was seen in mice treated with 0.1 mg m336 when compared to mice receiving control mab (fig. 3b) . these data indicate that m336 confers significant protection to mice when administered pre-or post-viral challenge. taken together, these results suggest to us that the epitope targeted by this exceptionally potent rbd-specific m336 antibody has a great potential for further development as a potent preventive and therapeutic agent in the future. treatment with m336 attenuates lung pathology associated with mers-cov infection. the effect of m336 antibody treatment on the pulmonary pathology associated with mers-cov infection was evaluated by using formalin-fixed, paraffin embedded, and hematoxylin/eosin (h&e)-stained lung specimens harvested at day 2 p.i. pulmonary pathology was noted in all mice that were treated with different doses of m336 or control m102.4 antibodies either before or after viral infection. on a severity scale of 0 to 3 (none, mild, moderate, severe), h&e-stained samples from mice pretreated with 1 mg and 0.1 mg of m336 antibody were graded 0 and 1, respectively, for perivascular and intra-alveolar infiltration of mononuclear cells, including lymphocytes, macrophages/monocytes (fig. 4, middle panel) , whereas those obtained from mice that received post-infection lung specimens collected at day 2 after viral challenge were processed for assessing the viral titers by using both vero e6-based infectivity assay and qrt-pcr targeting upstream e gene of mers-cov, and expressed as log 10 tcid 50 /gram and log 10 tcid 50 equivalent (eq.)/gram, respectively. (a) prophylactic and therapeutic efficacy of human m336 antibody treatment in reducing the lung titers of infectious virus. (b) prophylactic and therapeutic efficacy of human m336 antibody in reducing the titers of viral rna. the data shown are representative of at least two independently conducted assays using the same samples. data is presented as mean ± standard error (se). * * * p < 0.001 as determined by using student's t test. scientific reports | 6:31629 | doi: 10.1038/srep31629 treatment with either dose of m336 were graded 1 (fig. 4, right panel) , compared to the grade 2 assigned to mice received control antibody treatment prior to infection (fig. 4, left panel) . mers-cov has attracted significant basic research and clinical studies since it was first discovered in early 2012. even though the transmissibility of mers-cov among humans remains low at present, as a mutation-prone rna virus, it could eventually evolve into a highly communicable and more virulent human pathogens. this emphasizes the urgent need for the development of an effective antiviral therapy which could restrict the spread of this deadly disease. in other viral infections, neutralizing antibodies have been shown to protect the host from disease progression and/or reduce the severity of clinical symptoms. passive immunotherapy for prophylaxis and treatment of infectious viral diseases has been widely used for many decades [39] [40] [41] [42] [43] . passive transfer of neutralizing antibodies is also a promising strategy for both prophylaxis and treatment against mers-cov infection. to this end, we and others have successfully demonstrated the protective efficacy of specific human neutralizing monoclonal antibodies in animal models of mers-cov infection 23, 24, 26, 28 . among a panel of mers-cov-specific mabs generated by using a vast phage display library 23 , we identified three mabs which specifically bind to the mers-cov rbd with very high affinity. among these three identified, we noted that mab m336 exhibited the highest potency in neutralizing live mers-cov. here, we further characterized this novel human mab in our tg mouse model of mers-cov infection and showed prophylactic and therapeutic protection of mice treated with m336 before and after a lethal challenge with the virus, respectively. thus, mab m336 is highly promising as a potent inhibitor for urgent prophylaxis in adjunctive treatment for patients infected with mers-cov. in our studies, we noted that passively transferred with 1 mg and 0.1 mg of m336 monoclonal antibodies to individual mice 12 h prior to challenge with 1,000 ld 50 of mers-cov resulted in 100% and 75% protection against lethality, respectively (fig. 1) , suggesting that using 0.1 mg m336/mouse as a prophylaxis is suboptimal to completely neutralize viral infection, thereby allowing residual viruses to replicate within lungs during the course of infection. these data demonstrate that m336 confers a dose-dependent reduction of mers-cov infection, corroborating lower viral rna levels and live virus isolation determined for these mice when compared to control mice. our study also confirmed the therapeutic efficacy of m336 in a dose-dependent manner. similar to the prophylactic studies, administration of a single-dose of m336 antibody at a concentration of either 1 or 0.1 mg per mouse at 12 h after mers-cov challenge provided 100% and 75% protection, respectively, against infection-induced lethality, accompanied by reduced viral loads (both infectious virus and viral rna) within the lungs. however, we also noted the recovery of bodyweight loss and the reduction of viral loads in mice treated with 1 mg of m336 at 12 hrs after infection were slower than those treated with 0.1 mg of m336, as shown in figs 2a and 3b, respectively. while there is no clear evidence showing an adverse impact on the overall wellbeing of mice imposed upon treatment with 1 mg of m336 antibody before mers-cov challenge (fig. 1) , it is difficult to completely rule out the existence of subtle "yet-to-be investigated" high-dose drug toxicity. we speculate that such a subtle high-dose drug toxicity in the phase of acute and dynamic mers-cov infection initiated at 12 hrs before treatment with 1 mg of m336 could exacerbate drug toxicity, resulting in reduction of appetite and antiviral capacity. however, such a negative impact imposed upon high-dose treatment of virally infected mice appeared to be transient and did not irreversibly alter the final outcome of infection, as judged by the mortality (fig. 2b) . additional studies, especially the pharmacokinetics and the dosing frequency of m336 are warranted in the future to optimize preventive and therapeutic strategies with this promising antibody. the transgenic mice that we used for evaluating the prophylactic and, especially, the therapeutic efficacy of this m336 antibody are extremely sensitive to mers-cov infection and disease, with ld 50 and id 50 of 4.5 and 0.4 tcid 50 of mers-cov, respectively (data not shown), titers which are lower than our original estimations 38 . such a striking ability of this m336 antibody, as a prophylactic or therapeutic agent, to significantly protect these transgenic mice against challenge with 1000 ld 50 of mers-cov is highly impressive. the rbd of the mers-cov, targeted by this m336 antibody, is highly conserved among various clinical isolates and the mutation rate of this rbd appears to be extremely low, compared to that of other rna viruses 23, 28 , thereby making the development of escape mutants to m336 unlikely. however, a combination treatment with multiple neutralizing mabs targeted at different epitopes or the mers-cov-specific hr2p fusion inhibitor targeting the hr1 domain of the s2 subunit of the mers-cov s protein 38,44 could be desirable. by immunizing mice with rbd of mers-cov s protein, li, y. et al. recently developed a humanized mab, named 4c2h, that exhibited strong neutralizing activity with nd 50 of ~0.71 and ~6.25 μ g/ml against the pseudotyped and live mers-cov, respectively 36 , which are about 100-fold less potent than m336 (nd 50 = 0.005 and 0.07 μ g/ml against the pseudotyped and live mers-cov, respectively) 23 . using ad5-hcd26-transduced mouse model 33 , they demonstrated that intravenous administration of a single dose of 4c2h one day before or after the mers-cov challenge resulted in reduction of viral titer by 2 log at 3 dpi. however, intraperitoneal administration of m336 to our hdpp4 tg mice lead to the reduction of viral titer as high as 4 log at 2 dpi. since mers-cov challenged ad5-hcd26-transduced mice showed no severe disease, the effect of 4c2h on the weight loss and mortality in these mice is unavailable. additionally, unlike hdpp4 transgenic mice that we used in this study with well-defined hdpp4 expression as well as 50% lethal dose (ld 50 ) and infectious dose (id 50 ), the intensities of hcd26 expression among the ad5-hcd26-transduecd mice are variable, ranging from undetectable to a high level 45 . although both 4c2h and m336 bind to the rbd of mers-cov s protein, some of the critical amino acid residues recognized by these two mabs are different 29, 36 . the epitope of m336 overlaps extensively with the dpp4-binding site, which is composed of mers-cov rbd residues n501-k502, s504, f506, d510, e513, w535-r542, w553, v555, s557 and s559 29 . the epitope of mab 4c2, the parental mouse mab of 4c2h, only overlaps with partial of dpp4-binding site, which is composed of five rbd residues, w535-e536 and d539-r542. most of other rbd amino acids recognized by 4c2, including y397-n398, k400, l495-k496, p525 and v527-s532, are not located on the dpp4-binding site, indicating that the neutralization efficacy of 4c2h is largely attributed to the steric hindrance created by its binding with mers-cov rbd 36 .these results suggest that combinational use of 4c2h and m336 may exhibit synergistic antiviral effect against both wild-type strains and escape mutants (if any) of mers-cov. taken together, these results suggest to us that the mers-cov rbd protein-specific m336 mab is an excellent candidate for passive immunotherapy to provide immediate and effective protection to individuals who may be exposed to mers-cov and to treat patients who have been exposed. testing in humans is needed for its potential use as a therapeutic for the treatment of mers-cov-infected patients. monoclonal antibody production. for expression of m336 igg1, the previously described m336 igg1 vector 23 was used to infect cho-k1 cells (atcc, manassas, va) with polyfect transfection reagent (qiagen, valencia, ca). after screening of 960 clones for antibody productivity by elisa and subsequent characterization, a stable cell line was generated and inoculated into a bioflo 410 bioreactor (new brunswick scientific, nj) for large-scale production of m336 igg1. purification was carried out by using a protein g column (ge healthcare), and endotoxin was removed by detoxi-gel endotoxin removing columns (thermo scientific) according to the manufacturer's instructions. transgenic mice expressing human dpp4 established by us were used throughout the study. animals were housed in on-site animal facilities at galveston national laboratory under a 12:12 light/dark cycle with room temperature and humidity kept between 21-25 °c and 31-47%, respectively, and with ad libitum access to food and water. all experiments were performed in accordance with the guide of nih and aaalac and were approved by the institutional animal care and use committee at the university of texas medical branch, as described previously 34 . briefly, groups of 6-8-weeks tg mice were challenged intranasally (in) with 10 4 tcid 50 /ml (~1,000 ld 50 ) of mers-cov-emc/2012, originally provided by heinz feldmann (nih, niaid rocky mountain laboratories, hamilton, mt) and ron a. fouchier (erasmus medical center, rotterdam, netherlands). the titers of individual virus stocks, stored at − 80 °c, were determined by using vero e6-based infectivity assays and expressed as 50% tissue culture infectious doses (tcid 50 )/ml. scientific reports | 6:31629 | doi: 10.1038/srep31629 viral infections and isolation. all of the animal studies involving infectious mers-cov were conducted within approved animal biosafety level 3 (absl-3) at the galveston national laboratory. experimental designs and strategies in different tg mouse groups involving intranasal challenge with live mers-cov were described in individual experiments in the results section. for live virus isolation, lung tissues were collected at day 2 post mers-cov challenge, weighed, and homogenized in phosphate-buffered saline (pbs) containing 10% fetal calf serum (fcs) by using tissuelyser (qiagen, retsch, haan, germany), as previously described 34 . the resulting suspensions of infected tissues were tittered in the standard vero e6 cell-based infectivity assays to quantify yields of infectious virus expressed as log 10 tcid 50 per gram (g) of tissue. rna extraction and viral titers determination by real-time q-pcr. lung tissue samples from each group of mice were transferred to individual vials having rna later solution (qiagen) and subsequently homogenized and subjected to total rna isolation, by using trizol reagent (life technologies), to assess mers-cov-specific genome targeting of virus-specific upstream e gene (upe) and endogenous control gene (mouse β -actin) by using a one-step rt-pcr kit (invitrogen), as previously described 34 . ct values for each sample were analyzed against ct values generated in our lab from the standard curve of mers-cov mrna copy number. relative mers-cov upe mrna expression value was calculated for each replicate and expressed as the equivalent of log10 tcid 50 per gram (g) of the tissue by the standard threshold cycle (∆∆ct) method. ct value analysis was done by using bio-rad cfx manager 3.0 software. is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sarslike pandemic? hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria mers coronaviruses in dromedary camels co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia family cluster of middle east respiratory syndrome coronavirus infections the first case of the 2015 korean middle east respiratory syndrome outbreak middle east respiratory syndrome coronavirus outbreak in the republic of korea environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germlinelike antibody infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits prophylaxis with a mers-cov-specific human monoclonal antibody protects rabbits from mers-cov infection rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease animal models of middle east respiratory syndrome coronavirus infection a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo characterization and demonstration of the value of a lethal mouse model of middle east respiratory syndrome coronavirus infection and disease the growth and potential of human antiviral monoclonal antibody therapeutics the spike protein of sars-cov-a target for vaccine and therapeutic development passive immunity in prevention and treatment of infectious diseases prophylaxis and therapy for chikungunya virus infection post-exposure treatment of ebola virus using passive immunotherapy: proposal for a new strategy structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor we thank dr. heinz feldmann, national institute of health at hamilton, montana, and dr. ron a. fouchier, erasmus medical center at rotterdam, the netherlands for the mers-cov. this research was supported in part by a national institutes of health grant, r21ai113206-01 (to c-t.k.t), and pilot grants from the center for biodefense and emerging infectious diseases and from the galveston national laboratory (grant number: 5uc7ai094660-05. project title: national biocontainment laboratories (nbls) operations support), university of texas medical branch, galveston, tx (to c-t.k.t.), intramural funding of nci (to dsd), and the national natural science foundation of china (#31570936 to sj, #31570936 to ty). t.g was supported in part by a t32 biodefense training program (5 t32 ai 60549-12) awarded to utmb by nih. histopathology. mice were necropsied, lung tissues were inflated and fixed in 10% neutral buffered formalin for 3 days before paraffin-embedded and processed for routine hematoxylin and eosin stain (h&e) for assessing the histopathology 46 . key: cord-335567-ssnvr6nj authors: berry, michael; gamieldien, junaid; fielding, burtram c. title: identification of new respiratory viruses in the new millennium date: 2015-03-06 journal: viruses doi: 10.3390/v7030996 sha: doc_id: 335567 cord_uid: ssnvr6nj the rapid advancement of molecular tools in the past 15 years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. the inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. in 2001, this led to the discovery of human metapneumovirus (hmpv) and soon following that the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (hcov) nl63 and hcov-hku1. human bocavirus, with its four separate lineages, discovered in 2005, has been linked to acute respiratory tract infections and gastrointestinal complications. middle east respiratory syndrome coronavirus (mers-cov) represents the most recent outbreak of a completely novel respiratory virus, which occurred in saudi arabia in 2012 and presents a significant threat to human health. this review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since 2001. viral infections of the upper and lower respiratory tract are among the most common illness in humans. children and infants bear the major burden of infection, typically presenting with 5 to 6 episodes annually [1] . these infections are often associated with significant patient morbidity and related mortality. for this reason, urtis and lrtis represents the leading cause of death in children younger than five years of age worldwide [2, 3] ; this accounts for approximately 4 million deaths annually [4] . acute respiratory tract disease is the leading cause of hospitalization in children and febrile episodes in infants younger than three months of age [5, 6] . bacteria only represent approximately 10% of all upper respiratory tract infections with the subsequent 90% of infections caused by respiratory viruses [7] . despite the viral aetiological origin of most respiratory infections, antibiotics are often prescribed in the treatment of such diseases [8] , exacerbating antibiotic abuse. the morbidity and fiscal implications associated with respiratory infections are significant, with approximately 500 million cases reported in the united states alone each year with subsequent direct and indirect costs to the us economy estimated at $40 billion annually [2] . the burden of respiratory tract infections is increased in patients with chronic comorbidities or clinical risk factors including asthma [9] , chronic obstructive pulmonary disease (copd) [10] , young, elderly [11] and immunocompromised [12, 13] . the viruses primarily associated with upper respiratory tract infections commonly include rhinoviruses, enteroviruses, adenoviruses, parainfluenza viruses (piv), influenza viruses, respiratory syncytial viruses (rsv) and coronaviruses [3, 8, 14, 15] . in recent years six new human respiratory viruses have been reported including human metapneumovirus (hmpv) [16] , bocavirus and four new human coronaviruses including severe acute respiratory syndrome coronavirus (sars-cov), human coronavirus nl63 (hcov-nl63), hcov-hku1 and middle east respiratory syndrome coronavirus (mers-cov). this review will detail these newly discovered and emerging respiratory viruses. coronaviruses affect a diverse group of animal hosts, and cause a plethora of diseases in animals including progressive peritonitis, acute and chronic hepatitis, gastroenteritis, nephritis, and encephalitis [17] . in humans coronavirus infection results in respiratory tract complications with varying degree of severity and have been associated with gastroenteritis. four human coronaviruses (hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1) are endemic in the human population and are mainly associated with mild, self-limiting respiratory illnesses. another two human coronaviruses, namely sars-cov and mers-cov cause severe respiratory syndromes and present a significant threat with their high fatality rates. the first human coronaviruses were identified in the 1960s by tyrell and bynoe who passaged a virus, named b814, in human embryonic tracheal organ cultures. when this virus was inoculated intranasally into human volunteers, common cold-like symptoms were produced [18, 19] . hamre and procknow (1966) later isolated a virus, which they were able to grow in tissue culture, from subjects presenting with symptoms of the common cold. the virus was later named human coronavirus 229e (hcov-229e) [20] . seroepidemiological studies have shown that 40% of wild animal traders and 20% of people responsible for the slaughtering of animals in the region where human sars was thought to originate, were seropositive for sars-cov, although all cases were asymptomatic. this indicates that these people were previously exposed to a sars-like-cov, which resulted in asymptomatic infection [38] . sars presents as an atypical pneumonia [42, 43] , with pneumocytes being the primary target of infection. infection results in haemorrhagic inflammation in most pulmonary alveoli with alveolar thickening, diffuse alveolar damage, desquamation of pneumocytes, formation of hyaline membranes and multinucleated pneumocytes with capillary engorgement and microthrombosis [44] . approximately 60% of patients deteriorated in the second week of infection, presenting with persistent fever, dyspnoea and oxygen desaturation [29] . approximately 20%-30% of patients were subsequently admitted to intensive care, where mechanical ventilation was necessary [45] . a surprising finding with the sars outbreak was that it was not as great a threat to infants and children [33, 46] . clinical presentation was less severe in infants and no children aged between 1 and 12 required intensive care or mechanical ventilation [47, 48] . this is in sharp contrast to the age related burden of other respiratory infections and the underlying biological mechanism remains unclear [49] . a family of viruses that were previously understood to cause mild, self-limiting upper respiratory tract infections was showcased by the sars-cov outbreak to be a significant threat to global public health. the economic losses brought on by the sars pandemic was estimated to be in the region 40 billion dollars [50] with hong kong bearing a significant proportion of the losses. tourism, entertainment and restaurant industries in the area recorded up to 80% loss in business. the pressure on healthcare facilities in affected areas was substantially exacerbated by the spread to healthcare workers. several hospitals were forced to close to new admissions as large numbers of staff became infected with sars-cov. to view this as an isolated incidence would be naï ve and the potential for the emergence and re-emergence of new and existing infectious agents poses a probable risk. understanding the sars-cov outbreak has provided immense knowledge and an excellent model to replicate in the event of further outbreaks of communicable diseases. in 2003 a 7 month old child presenting with bronchiolitis and conjunctivitis was screened for several respiratory viruses to identify the causative agent, with all diagnostics yielding negative results. the group led by lia van der hoek then used a modified cdna amplified restriction fragment-length polymorphism (cdna-aflp) technique (virus-discovery-cdna-aflp or vidisca), to identify the causative agent. briefly, the technique utilizes reverse transcription-pcr of viral rna with subsequent restriction digest of the cdna using frequently cutting restriction enzymes. since the restriction sites are selected and therefore known, the resultant "sticky ends" can be ligated into anchors for amplification and sequencing with specific primers. the results showed highest sequence similarities with known coronaviruses, but with significant sequence divergence indicating the discovery of a new coronavirus species, later named human coronavirus nl63 [51] . at about the same time, two other independent groups identified essentially the same virus [52, 53] . shortly after the van der hoek paper [51] , a novel coronavirus that replicated efficiently in tertiary monkey kidney and vero cells, was retrospectively isolated from a nose swab sample collected in 1988 from an 8 month-old boy presenting with pneumonia. this virus was reported to be similar to hcov-nl63 and named hcov-nl [52] . in 2005, also reported the identification of a novel coronavirus isolated in new haven, connecticut, which was named hcov-nh. this novel virus was identified by pcr which was adapted to amplify a conserved region within the replicase 1a or pol gene [53] . subsequent sequence analysis showed that these three viruses were essentially the same virus or variants thereof [53, 54] . it is obvious that hcov-nl63 has been circulating in the human population since before 1988 [52] . in fact, molecular clock analyses have shown that hcov-nl63 and hcov-229e diverged from a most recent common ancestor, in a zoonotic event, approximately 563 to 822 years ago [55] . hcov-nl63 later diverged into two lineages with subsequent recombination of the two lineages during co-infection. this frequent recombination has given hcov-nl63 a mosaic structured genome with multiple recombination sites [56] . a 71 year old male patient from china was admitted to hospital with pneumonia in january 2004. viral cultures, rt-pcr and direct antigen detection from nasopharyngeal aspirate were all negative for respiratory viruses. a pan-coronavirus rt-pcr targeting a conserved region of the pol gene confirmed the presence of a coronavirus however attempts to culture the causative agent were all unsuccessful. sequencing the gene segment amplified by the pan-coronavirus assay indicated a high homology to other viruses of the βcov genus including hcov-oc43 but of novel origin. the human coronavirus, termed hcov-hku1, was later isolated from another female patient. efforts to culture the virus had posed complicated and the complete genome was isolated, amplified and sequenced directly from rna extracted from a nasopharyngeal aspirate [57] . successful propagation of the virus was achieved recently in human ciliated airway epithelial cells [58] but culturing of hcov-hku1 still remains a daunting task. the presence of the he gene further characterizes hcov-hku1 as belonging to the βcov genus. since the discovery of hcov-hku1, its prevalence has shown a global distribution and retrospective analysis on stored nasopharyngeal aspirates have confirmed its existence since 1995 [59] ; however, phylogenetic analysis suggests a much earlier divergence. june 2012 saw the most recent emergence of a completely novel strain of human coronavirus. a sputum sample was collected from a 60 year old male patient presenting with severe respiratory disease in the dr. soliman fakeeh hospital in jeddah, saudi arabia. viral assays frequently used could not identify any aetiological agent responsible for the disease. the sputum sample was sent to dr. ron fouchier at the erasmus medical college in rotterdam, netherlands, where the virus was identified as a novel coronavirus, provisionally termed hcov-emc (human coronavirus erasmus medical college). the patient later succumbed to the disease with acute pneumonia and subsequent renal failure [60] . a retrospective study further traced the virus back to april 2012 where an outbreak of pneumonia, resulting in two fatalities, occurred in health care workers in an intensive care unit in zarqa, jordan [61] . since the initial discovery, several new isolates were identified and described in scientific literature, databases and media under various names. this provoked the convening of the coronavirus study group to introduce a naming convention and avoid confusion within the research field, health care authorities, governments and general public. the name middle east respiratory syndrome coronavirus (mers-cov) was coined and has been widely accepted by the discoverers of the virus and pioneers of the field, the who and saudi ministry of health [61] . primary infections, to which all cases were linked directly or indirectly, occurred in middle eastern countries including saudi arabia, qatar, jordan, oman and united arab emirates and subsequently spread to united kingdom, tunisia, france, italy and germany with egypt and the united states recently reporting their first laboratory confirmed cases [62, 63] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [64] and only reported in close, sustained contact [65] such as within families [66] , healthcare workers [67] and as nosocomial transmission [68] , especially if the patient represents with comorbidities. there has been no evidence to suggest sustained community transmission [62] . the ever increasing case numbers and countries affected by the disease however suggests mers-cov does represent potential for widespread, global outbreak [69] . from the first reported case in june 2012, until 7 february 2014, a total of 182 cases had been reported with 79 fatalities. according to the latest figures available from the who, as of 11 june 2014, the virus had resulted in a total of 699 laboratory confirmed cases with a total of 209 fatalities [70] . these statistics indicate that within a 4 month period case numbers had more than trebled suggesting the virus is far from controlled. the fatality rate of 30% is also substantially increased in patients with comorbidities, with a large number of reported cases being identified in immunocompromised patients or those with underlying disease [68, 71] and a severe threat of nosocomial transmission has been demonstrated [72] . clinical presentation is similar to sars and includes a spectrum of respiratory diseases with the most common symptoms including cough, fever and gastrointestinal symptoms [69] before progressing to pneumonia [68] . acute respiratory distress syndrome (ards), renal failure, pericarditis and disseminated intravascular coagulation have also been reported [69] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [64] and only reported in close, sustained contact [65] such as within families [66] , healthcare workers [67] and as nosocomial transmission [68] , especially if the patient presents with comorbidities. there has been no evidence to suggest sustained community transmission [62] . the ever increasing case numbers and countries affected by the disease however suggests that mers-cov does represent potential for widespread, global outbreak [69] . the inefficient spread between infected patients strongly suggests zoonotic transmission, however over two years on from the initial discovery of mers-cov it is still unclear where it originated from and how it infects humans. during the outbreak of sars-cov the identification of the wet markets of china as the probable source greatly contributed to the control of the disease [38] . this highlights the importance in identifying the origins of disease outbreak. phylogenetic analysis places it in the same genus as sars-cov but within a new lineage, 2c. the closest detectable relatives suggests origins from bat coronavirus species with relative high sequence homology between bat-cov hku4 and hku5 in china [73] , vm314 in the netherlands [74] and a recently discovered isolate from south africa [75] . assuming that bats also form the immediate source is improbable and as with sars-cov an intermediate animal host species for transmission to humans is of greater likelihood [61] . high levels of neutralizing antibodies, viral rna and infectious viruses have been discovered in dromedary camels suggesting a potential for camels to form the source for human transmission [76] [77] [78] [79] . a recent study identified identical single nucleotide polymorphisms in a sequence of mers-cov isolated from an infected patient and camel, which was cared for by the patient. phylogenetic analysis of these two sequences supported the conclusion that transmission occurred between patient and camel, however the direction of transmission could not be confirmed [80] . it has since been proven that a patient who succumbed to mers-cov infection obtained the virus directly from his camel herd. the direction of transmission was confirmed as camel-to-human by serological analysis [81] . these facts strongly contribute to an increasingly popular hypothesis that mers-cov infections in humans are directly acquired from camels, however very few patients have had reported contact with camels. many residents of the arabian peninsula frequently consume unpasteurized camel milk and this has been suggested as a potential source as the virus has been detected and can remain viable for prolonged periods in milk [82, 83] . the who, saudi arabia and qatar have recently issued warnings and recommended against consuming unpasteurized camel milk and to be cautious when interacting with dromedary camels [84] . the outbreak of sars-cov in 2003 and mers-cov less than 10 years later highlights the significant threat of coronaviruses to humans and confirms that the sars outbreak was not an isolated incident. with the ever increasing diversity of animal coronavirus species, especially within bats, the likelihood of recombination leading to future outbreaks is high and the threat of potential pandemics is real as highly pathogenic coronaviruses continue to spill over from zoonotic sources into the human population. misdiagnosis of these outbreaks pose a further substantial threat to healthcare workers with nosocomial spread to other patients putting further pressure on an already strained healthcare system. understanding the dynamics and molecular characteristics of human coronaviruses currently in circulation and how they emerge, infect and cause disease, we can be better prepared for future pandemics allowing for improved response, management and treatment of related conditions. the clinical presentation of human coronaviruses clearly follows two distinct lines of progression. sars-cov and mers-cov present with severe respiratory complications and often multisystem involvement with renal failure and enteric symptoms. the high fatality rates associated with these infections is not reflected in the remaining four human coronaviruses, hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1. the clinical importance of sars-cov and mers-cov are evident and discussed in detail in previous sections allowing for the presentation of the involvement of the remaining non-severe human coronaviruses and there implications in human health. the clinical presentation of these four non-severe human coronaviruses is largely identical and indistinguishable symptomatically, commonly presenting with rhinorrhoea, sore throat, cough and fever [85, 86] . majority of infections are associated with self-limiting upper respiratory tract disease or "the common cold" but can also present with high morbidity outcomes of the lower respiratory tract including bronchiolitis, pneumonia, [87] [88] [89] , asthmatic exacerbations [9] acute exacerbations of chronic obstructive pulmonary disease (copd) [10] and croup in hcov-nl63 infected patients [90] . a report by esper et al. found a correlation between hcov-nl63 infections and kawasaki disease [91] , although other studies could not replicate the association [92] [93] [94] . febrile seizures have been reported for most human coronaviruses but the significance of hcov-hku1 is alarming with one study indicating that 50% of patients infected with hcov-hku1 experience febrile seizures [9] . human coronaviruses affect all age groups [85, 86] but elicit more serious disease in young, elderly and immunecompromized [87, 95, 96] , frequently resulting in hospitalization. reports on the prevalence of human coronaviruses and their association with upper and lower respiratory tract disease vary but range between 3.3% and 16% [85, 86, 95, 97] . over 70% of the general public has seroconverted towards all four non-severe human coronaviruses with primary infection shown to occur in childhood [98] and reinfection occurring throughout life [95] . given the high prevalence of respiratory infections, human coronaviruses represent a substantial disease burden which is exacerbated by the high implications of healthcare workers in coronavirus outbreaks [97] . high rates of co-infection with other respiratory viruses are commonly reported [85, 86, 95, 99] . viruses frequently associated with co-infection include enterovirus, rhinovirus and piv [100] however reports of co-infection with two human coronaviruses are limited. dijkman et al. recently demonstrated that hcov-oc43 and hcov-nl63 may elicit immunity that protects against hcov-hku1 and hcov-229e, respectively [101] . clinical progression and outcomes of disease in patients presenting with co-infection are however similar to patients presenting with mono-infection [85, 95] . there is also no substantial difference in coronaviral load between co-infected and mono-infected patients. no substantial difference in disease progression in co-infected versus mono-infected patients has been reported and therefore understood to have little impact; however, the role in facilitation of infection of one respiratory virus by another is still speculative [95] . all four human coronaviruses are endemic worldwide but the prevalence, regional distribution and pathogenicity of individual human coronaviruses is unclear and highly subjective on study conducted with parameters including population sampled, respiratory sample collected, sensitivity of diagnostic assay used, region where study was conducted and duration of the study playing a substantial role in epidemiological findings. a common finding in majority of studies conducted is the increased prevalence of human coronaviruses during winter months in temperate climates [95, 97] . however even this has shown to be geographically dependent with a spring/summer predominance in subtropical and tropical climates [9, 102] . biennial outbreaks are frequently reported [85, 95, 99, 100] for all strains of non-severe human coronaviruses. in 2001 a previously undiscovered virus was identified in 28 epidemiologically distinct patients in the netherlands. patient symptoms were similar to those infected with rsv and, several patients required hospitalization and mechanical ventilation. viral isolates were cultured in tertiary monkey kidney (tmk) cells and cytopathic effects caused by the virus were largely identical to those caused by rsv. electron microscopy of infected cell supernatants revealed paramyxovirus-like particles; however, rt-pcr assays to detect known paramyxoviruses were all negative. the low stringency of the assays used indicated a currently unknown, genetically distinct virus. a rap-pcr assay was then utilized to obtain sequence information of the unknown virus and fragments amplified by the rap-pcr allowed for further sequencing of the 3'-end of the genome. based on the sequence homology and gene organization, the unidentified virus displayed closest homology with avian pneumovirus, but to be a tentative new member of the metapneumovirus genus and the first virus in the genus to infect humans, provisionally termed human metapneumovirus (hmpv) [16] . symptomatic differentiation between hmpv and other respiratory viruses cannot be made as there is a significant overlap in clinical presentation [103, 104] . the most common presentation of hmpv in children includes complications of the upper respiratory tract with rhinorrhoea, cough and fever [105] . acute otitis media is also frequently reported [106, 107] and conjunctivitis, rash, diarrhea and vomiting are reported but infrequently [103] . bronchiolitis, pneumonia, croup and asthmatic exacerbations are the most frequently associated lower respiratory tract complications [108] and viral load is directly associated with disease severity [109] . hmpv infection in the young and elderly frequently requires hospitalization and fatalities have been reported in the elderly [110, 111] . an increased morbidity in elderly patients with a delayed clearance of symptoms has been reported and is likely related to the age related impairment of the innate and adaptive immunity [103] or an over stimulated immune response leading to inflammation [112] . elderly patients requiring hospitalization most frequently present with acute bronchitis, copd exacerbations, pneumonia and congestive heart failure [113] . in healthy adults asymptomatic infections or cold-and flu-like symptoms are the most prevalent presentation [114] . the pathogenesis of hmpv infection is strongly affected by bacterial coinfections with pneumococcus. one study has shown that administration of a conjugate pneumococcal vaccine is sufficient to reduce the incidence of hmpv infection of the lower respiratory tract and the incidence of clinical pneumonia in both hiv positive and negative patients [115] . these finding suggest that the incidence of hospitalizations in hmpv infections may be decreased by vaccination with a conjugate pneumococcal vaccine. another case report of severe respiratory failure was found to be caused by coinfection with hmpv and streptococcus pneumonia in a 64 year old patient [116] . both in vitro and in vivo studies have shown that infection with hmpv facilitates adhesion of pneumococcal bacteria, which may provide an explanation for the coinfection with pneumococcal strains and hmpv [117] . viral coinfections between hmpv and rsv have been reported, but remain a contentious issue. the typical seasonal overlap of the two viruses has been suggested to promote viral coinfection. one study reported a 10-fold increase in risk of admission to an intensive care unit in pediatric patients coinfected with rsv and hmpv and associated the dual infection as capable of augmenting severe bronchiolitis [118] . other studies do not support this finding and further report a decreased correlation between hmpv-rsv coinfections and hospitalization and additionally lists dual infection, along with breastfeeding, as having protective effects [119] . although hmpv was only discovered in 2001, it has been shown by phylogenetic analysis to have been in existence for approximately 50 years [120, 121] . soon after the discovery of hmpv, it was evident that two lineages, a and b, existed. these two lineages were further subdivided into two sublineages per lineage, a1-a2 and b1-b2 [16] . a recent report analyzing sequence divergence of the attachment and fusion surface glycoproteins indicates the presence of five sublineages, namely a1, a2a, a2b, b1 and b2 [122] . from long term retrospective studies it was evident that these lineages are not restricted to certain locations or times and that multiple lineages can exist in the same location and period [108, 123] . it has also become evident that old sublineages may be replaced by new variants [103] . disease progression or varying clinical outcomes related to different lineages of hmpv has become a contentious issue. several studies have reported that lineage a presents with more severe clinical outcomes [124] [125] [126] where the same is reported for lineage b by other groups [127, 128] . it has been further reported that there is no difference in disease outcomes related to the two lineages [108, 129, 130] . between 7% and 19% of all cases of respiratory infections in children are caused by hmpv, in both hospitalized and outpatients [108, 131, 132] and has been reported to be the second most frequently identified virus in respiratory tract infections [133] . extrapolation of consensus data suggests a total of 20 000 hospital and one million clinic visits annually in the us among children younger than 5 [131] . children hospitalized with hmpv infections are also more likely to present with pneumonia or asthma and required longer stay in intensive care units with supplemental oxygen, when compared to other respiratory viruses [131] . seroprevalence studies indicates that 100% of young adults are seropositive for hmpv with stable neutralizing titres, which further suggests that reinfection occurs throughout life [16] , with a potential for genetic variation between clades promoting reinfection [108] . hmpv has a worldwide distribution and affects all age groups but predominantly affects young, elderly and immunocompromised patients [111] , with children younger than five years of age being most susceptible to infection [134] . children and adults with underlying or chronic conditions such as asthma, chronic lung disease, congenital heart disease, cancer or copd are more likely to be hospitalized with hmpv infection [131] . infection with hmpv occurs throughout the year but seasonal prevalence in late winter and spring has been observed and coincides with the peak of rsv infection [131, [135] [136] [137] . the first human bocavirus (hbov) was discovered in 2005 from nasopharyngeal aspirates of 282 patients with unresolved lower respiratory tract infections in sweden. researchers utilized a novel technique which included steps of dnase treatment to exclude contaminating, or non-viral, nucleic acids followed by pcr amplification by nonspecific primers. the pcr-products were subsequently cloned with large-scale sequencing of the clones. bioinformatic analysis of generated sequence data yielded the discovery of a new parvovirus with a high homology to bovine and canine minute parvoviruses. the genus name bocavirus was in fact derived from the species infected by the known virus strains, namely bovine and canine. the new virus was named hbov1 and was the first virus to be discovered by molecular virus screening [138] . three additional species of hbov were later discovered in 2010 and added to the genus; these were named hbov2, hbov3 and hbov4 [139] [140] [141] . hbov1 is a respiratory pathogen affecting all regions of the globe and is associated with approximately 2%-19% of all upper and lower respiratory tract conditions [142] [143] [144] . hbov1 productively infects human airway epithelium cell cultures and leads to damage of airway epithelial cells [145] [146] [147] , which supports clinical observations that infection does result in respiratory disease. in contrast, hbov2-4, are found in the gastrointestinal tract and hbov2, and possibly hbov3, are associated with gastroenteritis [139, [148] [149] [150] . interestingly, hbov2 is the only enteric bocavirus to be isolated from nasopharygeal aspirates and may, therefore, also be associated with respiratory disease [151, 152] . hbov1 is detected in all age groups, but predominantly in young children between the ages of 6-24 months [143, 153, 154] and is rarely detected in adults [155] [156] [157] [158] [159] . transmission and infection occurs throughout the year, but predominantly during winter and spring months [158, [160] [161] [162] . seroprevalence studies suggest that maternal antibodies, which provide protection, are present in infants younger than 2 months of age [163, 164] , after which seropositivity decreases with low levels of detection until 6-12 months. virtually 100% of children aged 6 are seroconverted for hbov1 and as reinfection occurs throughout life this remains into adulthood [143, [163] [164] [165] [166] . the presence of the three enteric bocaviruses does however complicate the findings of seroconversion as cross-reactivity does exist [166] . as with many respiratory viruses, clinical differentiation with hbov1 infection is not possible by symptomatic presentation [8] . common features of infection of the upper respiratory tract include common cold-like symptoms with cough, rhinorrhoea and acute otitis media [167] . infection of the lower respiratory tract in children is associated with pneumonia, acute wheezing, asthmatic exacerbations and bronchiolitis [160, 162, [168] [169] [170] , but life-threatening complications are rare with hbov1 infection [143] . although hbov1 has been isolated from stool samples, there is no statistical evidence to associate hbov1 with gastrointestinal disease [139] . hbov1 has not only been found in the upper and lower respiratory tract and gastrointestinal specimens, but also in urine samples, serum, saliva, and tonsils [143] . rather than having a role in disease pathogenesis, this viraemia and systemic spread may be a feature common to all parvoviruses as they require proliferating host cells for replication [138] . interestingly, hbov appears to be more than just a respiratory or gastrointestinal virus. in a recent study, hbov was identified in 18.3% of lung (n = 11/60) and 20.5% of colorectal (n = 9/44) tumors screened. unfortunately, the study did not investigate whether the hbov genomes were in fact incorporated into the host genome as reported for other known parvoviruses. therefore, based on their observations as well as previous studies on other parvoviruses, the authors speculate that hbov could contribute to the development of some lung and colorectal tumors. however, they do also acknowledge that these tumors could simply be providing an optimal environment for hbov replication and more conclusive studies are required to resolve this issue [171] . hbov1 has been associated with a prolonged period of persistence in the mucosa of the respiratory tract. this prolonged presence has possibly led to a high frequency of coinfection found with hbov1 infections of the both the upper and lower respiratory tract [142, 157, 158] . the high rate of detection of multiple respiratory viruses within up to 83% of respiratory specimens, and the presence of asymptomatic hbov1 infections, does complicate the determination of the actual pathogenic role of hbov [143] . high viral load is statistically associated with symptoms [172] and this may therefore be a better indication of coinfections which are related to disease severity or symptomatic presentation. it has been further suggested that patients presenting with viraemia are better candidates to assess the symptoms of disease when compared to investigations of respiratory secretions [172] . the effects and mechanisms of latency, persistence, reactivation and reinfection are however poorly understood and therefore its effects on coinfection and its contribution to active disease cannot be accurately stated [173] . the etiological agents of 12%-39% of lower respiratory tract infections still remain to be identified [138, 158] . these results may vary significantly depending on the sensitivity of the diagnostic assay used, respiratory site sampled and even geographical location of the study but it does suggest that many uncharacterized respiratory pathogens could still remain elusive awaiting discovery. the vast improvements in molecular techniques within the past decade have however led to the discovery of four previously circulating respiratory viruses and also the rapid characterization of two completely novel viruses, namely sars-cov and mers-cov. all these viruses have varying but significant impact on human health and the potential for outbreak of completely novel, emergent respiratory viruses, seen with sars and mers, poses their own unique threats. lessons learned from these viruses, and others currently in circulation, provide health care authorities and scientists with suitable expertise and knowledge to rapidly identify and combat novel respiratory viruses and as our techniques improve we will be in a position to characterize those viruses that are currently difficult to isolate and identify. burtram c. fielding receives funding from the national research foundation, south africa. any opinion, findings and conclusions or recommendations expressed in this material are those of the author and therefore the nrf does not accept any liability in regard thereto. mb wrote the earlier drafts of the manuscript. jg edited and reviewed the early drafts of the manuscript. bcf conceptualized the paper and wrote the submission draft of the manuscript. the authors declare no conflict of interest. viral upper respiratory tract infection and otitis media complication in young children the economic burden of non-influenza-related viral respiratory tract infection in the united states the clinical impact of human respiratory virus infections the global burden of disease center for population and development studies: massachusetts serious bacterial infections in febrile infants 1 to 90 days old with and without viral infections children with multiple viral respiratory infections are older than those with single viruses systematic review of the treatment of upper respiratory tract infection viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis coronavirus hku1 and other coronavirus infections in hong kong human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden respiratory viral infections in immunocompetent and immunocompromised persons community respiratory virus infections in immunocompromised patients with cancer epidemiology of respiratory viruses in bronchoalveolar lavage samples in a tertiary hospital impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses. influenza respir. viruses a newly discovered human pneumovirus isolated from young children with respiratory tract disease coronaviruses in the limelight cultivation of a novel type of common-cold virus in organ cultures cultivation of viruses from a high proportion of patients with colds a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease antigenic relationships amongst coronaviruses antigenic relationships among the coronaviruses of man and between human and animal coronaviruses a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation the genome sequence of the sars-associated coronavirus cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human sars molecular epidemiology: a chinese fairy tale of controlling an emerging zoonotic disease in the genomics era severe acute respiratory syndrome (sars) clinical management and infection control of sars: lessons learned the novel human coronaviruses nl63 and hku1 ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection analysis of multimerization of the sars coronavirus nucleocapsid protein detection of antibodies against sars-coronavirus using recombinant truncated nucleocapsid proteins by elisa the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors lung pathology of fatal severe acute respiratory syndrome history and recent advances in coronavirus discovery clinical presentations and outcome of severe acute respiratory syndrome in children severe acute respiratory syndrome among children severe acute respiratory syndrome learning from sars: preparing for the next disease outbreak-workshop summary identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children one virus, three names, three claims evidence supporting a zoonotic origin of human coronavirus strain nl63 mosaic structure of human coronavirus nl63, one thousand years of evolution characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures coronavirus hku1 in children, brazil isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group advancing priority research on the middle east respiratory syndrome coronavirus world health organisation. global alert and response (gar), coronavirus infections interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk coronaviruses: important emerging human pathogens family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus infections in health care workers hospital outbreak of middle east respiratory syndrome coronavirus data gaps for laboratory preparedness mers-cov summary updates epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans close relative of human middle east respiratory syndrome coronavirus in bat mers coronavirus in dromedary camel herd, saudi arabia geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from dromedary camel human infection with mers coronavirus after exposure to infected camels evidence for camel-to-human transmission of mers coronavirus stability of middle east respiratory syndrome coronavirus in milk middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels reply to "concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers characterization of human coronavirus etiology in chinese adults with acute upper respiratory tract infection by real-time rt-pcr assays clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations coronavirus 229e-related pneumonia in immunocompromised patients clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia an outbreak of coronavirus oc43 respiratory infection in normandy, france croup is associated with the novel coronavirus nl63 association between a novel human coronavirus and kawasaki disease lack of association between infection with a novel human coronavirus (hcov), hcov-nh, and kawasaki disease in taiwan kawasaki disease lacks association with human coronavirus nl63 and human bocavirus human coronavirus-nl63 infection is not associated with acute kawasaki disease epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients epidemiological and clinical features of human coronavirus infections among different subsets of patients. influenza respir. viruses first infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood high incidence but low burden of coronaviruses and preferential associations between respiratory viruses prevalence of human coronaviruses in adults with acute respiratory tract infections in beijing the dominance of human coronavirus oc43 and nl63 infections in infants human coronaviruses hcov-nl63 and hcov-hku1 in hospitalized children with acute respiratory infections in beijing, china human metapneumovirus: lessons learned over the first decade ten years of human metapneumovirus research comparison of clinical features of pediatric respiratory syncytial virus and human metapneumovirus infections frequency of human metapneumovirus in the upper respiratory tract of children with symptoms of an acute otitis media detection of human metapneumovirus from children with acute otitis media human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children human metapneumovirus viral load is an important risk factor for disease severity in young children an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups aging promotes neutrophil-induced mortality by augmenting il-17 production during viral infection human metapneumovirus infections in adults: another piece of the puzzle human metapneumovirus: a new player among respiratory viruses pneumococcal coinfection with human metapneumovirus severe respiratory failure due to co-infection with human metapneumovirus and streptococcus pneumoniae preceding human metapneumovirus infection increases adherence of streptococcus pneumoniae and severity of murine pneumococcal pneumonia human metapneumovirus in severe respiratory syncytial virus bronchiolitis comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children evolutionary dynamics of human and avian metapneumoviruses genetic diversity and evolution of human metapneumovirus fusion protein over twenty years genetic diversity and molecular evolution of the major human metapneumovirus surface glycoproteins over a decade the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience genetic variability of human metapneumovirus amongst an all ages population in cambodia between seasonal distribution and phylogenetic analysis of human metapneumovirus among children in osaka city differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children human metapneumovirus genotypes and severity of disease in young children (n = 100) during a 7-year study in dijon hospital, france a 1-year experience with human metapneumovirus in children aged <5 years genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness comparison of human metapneumovirus genotypes from the province of bolzano in northern italy with strains from surrounding regions in italy and austria new vaccine surveillance, n. burden of human metapneumovirus infection in young children dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients clinical impact and diagnosis of human metapneumovirus infection emerging respiratory agents: new viruses for old diseases? dé ry, p. human metapneumovirus infections in hospitalized children human metapneumovirus in the community cloning of a human parvovirus by molecular screening of respiratory tract samples a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study human bocavirus-the first 5 years human bocavirus, a respiratory and enteric virus establishment of a reverse genetics system for studying human bocavirus in human airway epithelia human bocavirus 1 infects commercially available primary human airway epithelium cultures productively human bocavirus can be cultured in differentiated human airway epithelial cells newly recognized bocaviruses (hbov, hbov2) in children and adults with gastrointestinal illness in the united states detection of human bocavirus-2 in children with acute gastroenteritis in south korea human bocavirus species 2 and 3 in brazil novel human bocavirus in children with acute respiratory tract infection human bocavirus 2 in children swiss paediatric respiratory research group. isolation of human bocavirus from swiss infants with respiratory infections human bocavirus: passenger or pathogen in acute respiratory tract infections? human bocavirus infection severe pneumonia and human bocavirus in adult human bocavirus epidemiological profile and clinical associations of human bocavirus and other human parvoviruses human bocavirus infections in hospitalized children and adults human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus seroepidemiology of human bocavirus in hokkaido prefecture seroepidemiology of human bocavirus defined using recombinant virus-like particles correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection seroepidemiology of human bocaviruses 1-4 the human bocaviruses: a review and discussion of their role in infection serodiagnosis of human bocavirus infection detection of human bocavirus in hospitalised children human bocavirus in children suffering from acute lower respiratory tract infection in beijing children's hospital the human bocavirus is associated with some lung and colorectal cancers and persists in solid tumors human bocavirus and acute wheezing in children human bocavirus infections key: cord-333882-zrdsr3nh authors: beigel, john h; voell, jocelyn; kumar, parag; raviprakash, kanakatte; wu, hua; jiao, jin-an; sullivan, eddie; luke, thomas; davey, richard t title: safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study date: 2018-04-30 journal: the lancet infectious diseases doi: 10.1016/s1473-3099(18)30002-1 sha: doc_id: 333882 cord_uid: zrdsr3nh summary background middle east respiratory syndrome (mers) is a severe respiratory illness with an overall mortality of 35%. there is no licensed or proven treatment. passive immunotherapy approaches are being developed to prevent and treat several human medical conditions where alternative therapeutic options are absent. we report the safety of a fully human polyclonal igg antibody (sab-301) produced from the hyperimmune plasma of transchromosomic cattle immunised with a mers coronavirus vaccine. methods we did a phase 1 double-blind, placebo-controlled, single-dose escalation trial at the national institutes of health clinical center. we recruited healthy participants aged 18–60 years who had normal laboratory parameters at enrolment, a body-mass index of 19–32 kg/m2, and a creatinine clearance of 70 ml/min or more, and who did not have any chronic medical problems that required daily oral medications, a positive rheumatoid factor (≥15 iu/ml), iga deficiency (<7 mg/dl), or history of allergy to intravenous immunoglobulin or human blood products. participants were randomly assigned by a computer-generated table, made by a masked pharmacist, to one of six cohorts (containing between three and ten participants each). cohorts 1 and 2 had three participants, randomly assigned 2:1 to receive active drug sab-301 versus normal saline placebo; cohorts 3 and 4 had six participants randomised 2:1; and cohorts 5 and 6 had ten participants, randomised 4:1. participants received 1 mg/kg, 2·5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 50 mg/kg of sab-301, or equivalent volume placebo (saline control), on day 0, and were followed up by clinical, laboratory, and pharmacokinetic assessments on days 1, 3, 7, 21, 42, and 90. the primary outcome was safety, and immunogenicity was a secondary outcome. we analysed the intention-to-treat population. this trial is registered with clinicaltrials.gov, number nct02788188. findings between june 2, 2016, and jan 4, 2017, we screened 43 participants, of whom 38 were eligible and randomly assigned to receive sab-301 (n=28) or placebo (n=10). 97 adverse events were reported: 64 adverse events occurred in 23 (82%) of 28 participants receiving sab-301 (mean 2·3 adverse events per participant). 33 adverse events occurred in all ten participants receiving placebo (mean 3·3 adverse events per participant). the most common adverse events were headache (n=6 [21%] in participants who received sab-301 and n=2 [20%] in those receiving placebo), albuminuria (n=5 [18%] vs n=2 [20%]), myalgia (n=3 [11%] vs n=1 [10%]), increased creatine kinase (n=3 [11%] vs 1 [10%]), and common cold (n=3 [11%] vs n=2 [20%]). there was one serious adverse event (hospital admission for suicide attempt) in one participant who received 50 mg/kg of sab-301. the area under the concentration–time curve (auc) in the 50 mg/kg dose (27 498 μg × days per ml) is comparable to the auc that was associated with efficacy in a preclinical model. interpretation single infusions of sab-301 up to 50 mg/kg appear to be safe and well tolerated in healthy participants. human immunoglobulin derived from transchromosomic cattle could offer a new platform technology to produce fully human polyclonal igg antibodies for other medical conditions. funding national institute of allergy and infectious diseases, national institutes of health, and biomedical advanced research and development authority. middle east respiratory syndrome (mers) is a severe respiratory illness caused by a novel coronavirus (cov). the spectrum of clinical illness ranges from asymptomatic illness to a severe acute respiratory disease requiring intensive care and mechanical ventilation. mers has an overall mortality of 35% and there is no licensed or proven treatment. 1 the use of immune anti-mers coronavirus (mers-cov) plasma has been suggested as one potential therapeutic approach. 2 however, it is difficult to collect sufficient anti-mers-cov plasma from infected patients to implement this strategy. 3 the use of passive immune therapy, either in the form of direct plasma infusion or intravenous immunoglobulin, is often invoked both for emerging infectious diseases, such as ebola virus disease, 4 as well as more common diseases with high morbidity (despite the availability of antiviral therapeutics), such as severe influenza. 5 however, the use of immune plasma in all of these conditions has similar constraints. consistent production of large quantities of anti-pathogen-neutralising human plasma or immunoglobulin requires collection of plasma from convalescent or vaccinated human volunteers. the limited plasma supply restricts wide implementation of these therapeutics. one novel alternative method of manufacturing neutralising intravenous antibodies of consistently high affinity and avidity is to use the hyperimmune plasma of transchromosomic cattle, which produce highly potent and antigen-specific, fully human polyclonal igg de novo, and which mount a robust antibody immune response after vaccination. to develop the transchromosomic cattle, a human artificial chromosome was constructed that comprised the entire human immunoglobulin gene repertoire (human immunoglobulin heavy chain [igh] and human κ light chain [igk] ), which resides on two different chromosomes in human beings, specifically the igh locus from chromosome 14 and the igk locus from chromosome 2. 6 the immunoglobulin gene repertoires from the human artificial chromosome provide the large diversity of human polyclonal antibodies. to avoid possible human-bovine interspecies incompatibility between the human immunoglobulin-μ-chain protein (higm) and bovine transmembrane α and β immunoglobulins (bigα and bigβ) in the pre-b-cell-receptor complex, the higm constant domain was partly replaced with the counterpart of bovine igm (bigm). furthermore, the dna regulatory elements iγ1 and sγ1 were bovinised on the human artificial chromosome to facilitate dna-protein interactions in the bovine b cell. this human artificial chromosome is designated as iskchac∆, and is transferred to bovine fibroblasts that are homozygous triple knockouts of the two bovine immunoglobulin-μ heavy-chain loci (bighm and bighml1) and the bovine λ light-chain locus (bigl). the triple-knockout bovine fibroblasts carrying the human artificial chromosome were used as nuclear donors to clone transchromosomic cattle. the mers-cov spike protein attaches to the host-cell receptor cd26 (also known as dipeptidyl peptidase 4 or dpp4), and mediates subsequent cell fusion. 7 vaccination of mice with the plasmid vaccines expressing the spike glycoprotein showed the generation of neutralising antibodies through blocking both the receptor binding and non-receptor-binding domains. 8 seroconversion to the spike protein (measured by igg) was associated with resolution of clinical illness in patients with mers-cov infection, and the levels of anti-spike igg were inversely correlated with lower respiratory tract viral loads. 9 for these reasons, purified al-hasa strain mers-cov spike protein nanoparticles 10 (a clade b strain, manufactured by novavax inc, gaithersburg, md, usa) were chosen as the immunogen to generate an anti-mers-cov intravenous immunoglobulin in this transchromosomic bovine system (thereafter known as sab-301). 11 the purpose of this phase 1 study was to evaluate the safety, tolerability, and pharmacokinetics of sab-301 after a single infusion of sab-301 in healthy adults. this trial represents the first-in-human clinical study using a fully human antibody derived from transchromosomic cattle. we did this double-blind, placebo-controlled, single-dose escalation, phase 1 randomised controlled trial at the national institutes of health (nih) clinical center (bethesda, md). the study was conducted in accordance with the applicable regulatory and international conference on harmonization-good clinical practice requirements. the study protocol was approved by the national institute of allergy and infectious diseases institutional review board (protocol 16-i-0119). evidence before this study we searched pubmed on oct 4, 2017, for studies using these search terms in the title or abstract: ("middle east respiratory syndrome" or "mers") and ("antivirals" or "treatment"). we restricted the article type to clinical trials and the species to human, and there were no language restrictions. we found no articles. after removing the search restriction of title or abstract for these terms, removing the species restrictions to human beings, and excluding studies that did not assess mers therapeutics (rather they mentioned it in the background or discussion), we found no previous publications of mers therapeutics in human beings. additionally, we searched for studies using the search terms ("intravenous immunoglobulin" or "igiv"), and ("bovine" or "cow"), and restricted the article type to clinical trials and the species to human. after excluding studies that did not assess bovine intravenous immunoglobulin, we identified no previous publications of mers therapeutics in humans. to our knowledge, this is the first study to show the safety, tolerability, and pharmacokinetics of intravenous immunoglobulin derived from transchromosomic cattle, and the first study to describe the safety of a putative therapeutic specifically for mers. the evidence from this study shows that single infusions of sab-301 up to 50 mg/kg appear to be safe and well tolerated in healthy participants, and should be considered for further investigation in the treatment of mers. eligible participants were healthy adult men or women aged 18-60 years, with a body-mass index of 19-32 kg/m and a creatinine clearance of 70 ml/min or more (calculated using the chronic kidney disease epidemiology collaboration formula). all participants were required to have normal laboratory parameters at enrolment, and could not have any chronic medical problem that required daily oral medications, a positive rheumatoid factor (defined as a rheumatoid factor ≥15 iu/ml), or iga deficiency (defined as iga <7 mg/dl), nor any history of an allergy to intravenous immunoglobulin or human blood products. all participants were required to use two effective forms of contraception, at least one of which had to be a barrier method. a full list of exclusion criteria is included in the appendix. participants provided written informed consent before screening. an unmasked pharmacist at the nih clinical center pharmacy generated a blinded randomisation scheme before study initiation. after screening, study participants were enrolled sequentially in one of six cohorts. on the day of study drug dosing, the unmasked pharmacist determined treatment allocation by referring to the next position on the randomisation scheme. cohorts 1 and 2 had three participants, randomly assigned 2:1 to receive active drug sab-301 versus normal saline placebo; cohorts 3 and 4 had six participants randomly assigned 2:1; and cohorts 5 and 6 had ten participants, randomly assigned 4:1. study participants and study team members remained masked throughout the entire study. the study treatment was provided in an infusion bag, the contents of which were obscured by an opaque covering over the bag and tubing. sab-301 was manufactured by sab biotherapeutics inc (sioux falls, sd, usa). two transchromosomic bovines were hyperimmunised with purified al-hasa strain mers-cov s protein nanoparticles (novavax inc; 2-5 mg/kg) formulated with sab's proprietary adjuvant formulation (sab-adj-1). the transchromosomic cattle were vaccinated five times at 3-4-week intervals. hyperimmune plasma (up to 2·1% of bodyweight) was collected from each transchromosomic cow on days 8, 11, and 14 days after the third, fourth, and fifth vaccination. plasma was stored at -20°c until use. additional sab-301 manufacturing methods are detailed in the appendix. sab-301 was supplied in vials containing 672 mg per 9 ml. the lot of sab-301 we used had an elisa binding titre of 109 236 u/mg. we assayed sab-301 for its ability to neutralise mers-cov in vitro using a fluorescencereduction neutralising assay in vero e6 cells. the concentration of sab-301 at which 50% inhibition of relative fluorescence intensity was observed was 2·69 µg/ml. the doses used in the six cohorts were: 1 mg/kg (cohort 1; prepared at a concentration of 1 mg/ml in normal saline), 2·5 mg/kg (cohort 2; prepared at 1 mg/ml), 5 mg/kg (cohort 3; prepared at 4 mg/ml), 10 mg/kg (cohort 4; prepared at 4 mg/ml), 20 mg/kg (cohort 5; prepared at 20 mg/ml), and 50 mg/kg (cohort 6; prepared at 20 mg/ml). participants randomly assigned to placebo received a normal saline control at a volume equivalent to receiving active drug above. the infusions were started at a rate of 0·5 ml/kg per h, escalating by 0·5 ml/kg per h increments every 15 min to a maximum rate of 1-3 ml/kg per h, depending on cohort. in the highest dose cohort at the maximum infusion rate of 40 mg/kg per h, this infusion was less than a tenth of the maximum rate of standard intravenous immunoglobulin as per administration guidelines at the nih clinical center (maximum 480 mg/kg per h). the starting dose of 1 mg/kg was 370-times lower than the maximum preclinical animal toxicity study dose of 370 mg/kg. the target dose of 50 mg/kg exceeds the effective doses in preclinical models, 11 and is 7·4-times lower than the maximum nonclinical dose (sab biotherapeutics inc, unpublished). participants were screened up to 4 weeks before dosing. vital signs were obtained before the start of infusion, and then approximately 15 and 30 min after the start of the infusion, every 30 min thereafter until the end of the infusion, and then every 1 h for 6 h after infusion. participants were seen on days 1, 3, 7, 21, 42, and 90. blood samples for the pharmacokinetic analysis were obtained at baseline, 1 h after the end of infusion, 6 h after the end of infusion, and on days 1, 3, 7, 21, 42, and 90. at each study visit, participants were questioned about new symptoms, their medical condition, and new medications. all new symptoms or abnormal laboratory results were captured as adverse events. we tested serum for sab-301 pharmacokinetics using an elisa assay and functional inhibition (pharmacodynamics) using a mers micro-neutralisation assay. we assessed immunogenicity by testing for anti-igg antibodies (using rheumatoid factor), sab-301 antidrug antibodies, anti-bovine κ antibodies, and anti-camelid antibodies (an antibody used in the manufacturing process; appendix). the primary outcome was safety-ie, the type and frequency of adverse events experienced by participants receiving sab-301 compared with placebo. secondary outcomes were the pharmacokinetic profile, mers virus neutralisation assay over time (pharmacodynamics), and immunogenicity. we determined the study sample size to detect an adverse event with a true rate of 20% and a probability of 0·83 in the cohorts with larger doses (cohort 5 and 6), whereas see online for appendix rarer events with a true rate of 1% could be detected with a probability of 0·06. we did the analysis in the intention-to-treat population. we did not impute missing data. we analysed pharmacokinetic data using phoenix winnonlin software (version 7.0; cetara, st louis, mo) for both non-compartmental analysis and compartmental modelling. for the drug in the serum, key non-compartmental analysis pharmacokinetic parameters of interest included the maximum observed concentration (c max ); apparent elimination rate constant (λ z , determined by calculating the absolute value of the slope of the log-linear regression plasma concentration-time plot, with a minimum of three concentrations along the terminal phase); the elimination half-life (t ½ , calculated as 0·693/λ z ); the area under the concentration-time curve (auc), from 0 h to the last pharmacokinetic sample post-dose (auc last ) and to infinity (auc 0-∞ ), calculated with the linear-up, log-down trapezoidal rule; clearance calculated as dose/auc 0-∞ ; and volume of distribution, calculated as dose/(auc last × λ z ). we calculated geometric mean ratios and 90% cis to compare pharmacokinetic parameters between cohorts for assessment of dose linearity and proportionality. we tested structural models of one, two, and three compartments. to determine goodness of fit of the compartmental models, we visually inspected plots of observed and predicted concentrations versus time, and weighted residuals versus predicted concentrations, pharmacokinetic parameter coefficient of variation, and akaike information criteria. the two-compartment model that we selected was parameterised in terms of central compartment clearance, central compartment volume of distribution, inter-compartmental clearance, peripheral volume of distribution, and secondary parameters of distribution elimination rate and distributional half-life. we plotted the reciprocal of the highest 50% neutralisation titres over time and used them to calculate the auc last . we assessed the correlation of neutralisation titre auc last with sab-301 auc last using linear regression. this trial is registered with clinicaltrials.gov, number nct02788188. employees of the sponsor of the study were involved with study design, data collection, data analysis, data interpretation, and writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. between june 2, 2016, and jan 4, 2017, we screened 43 participants for this study, of whom 38 were sequentially randomly assigned to six cohorts (figure 1). the study ended after being fully enrolled. after allocation to study groups, all participants completed the planned follow-up. baseline characteristics of the randomly assigned participants are shown in table 1. 97 adverse events were reported: 64 occurred in 23 (82%) of 28 participants receiving sab-301 (mean 2·3 adverse events per participant), and 33 adverse events occurred in all ten participants receiving placebo (mean 3·3 adverse events per participant; table 2). there was one grade 3 adverse event of depression, and one grade 4 adverse event of suicide attempt, both occurring in the same participant. there was one serious adverse event of hospital admission for a suicide attempt by the same participant who had received 50 mg/kg of sab-301, which, as the other two adverse events in this participant, was judged to be unrelated to study intervention. this participant had a history of depression for several years that was not disclosed at the time of screening, and the participant was not receiving medical care or treatment at the time of enrolment. the most common adverse events were headache, albuminuria, increased creatine kinase, common cold, myalgia, and low serum bicarbonate. most adverse events were noted in similar proportions in those receiving sab-301 and placebo (table 2). there were two hypotensive events that occurred during study drug administration, both in cohort 6 (50 mg/kg of sab-301): one in a participant who received sab-301 and the other in one who received placebo. regarding the hypotensive event in the participant receiving sab-301, 30 min after the start of the infusion, the participant complained of feeling warm and slightly lethargic. blood pressure at the time was 96/58 (about 20 mm hg lower than pre-infusion), and there was no increase in heart rate. the infusion was stopped for 15 min with no other intervention, and the hypotension resolved. the infusion was restarted with no further episodes of hypotension. low serum bicarbonate was seen only in participants receiving sab-301 (three [11%] of 28), and were only noted in cohorts 5 (20 mg/kg of sab-301) and 6 (50 mg/kg of sab-301). two vasovagal reactions occurred in participants assigned to receive sab-301, dose-proportional increases in the parameters of c max and auc 0-∞ over the 20-fold range of doses from 2·5 mg/kg to 50 mg/kg (table 3) . we did not use pharmacokinetic data from the 1 mg/kg cohort because a terminal phase could not be identified because of multiple concentrations that were below the limit of assay detection (15·63 µg/ml). mean concentration versus time profiles of the sab-301 cohorts at each dose are shown in figure 2a . sab-301 concentrations after infusion appeared to follow a bi-exponential decline and best fit a two-compartmental pharmacokinetic model. results for key pharmacokinetic parameters from non-compartmental analysis are shown in table 4 and parameters obtained from the two-compartmental model are presented in the appendix. the average terminal elimination half-life (t 1/2 ) was within the range of a typical antibody in human beings (approximately 28 days) across all dose cohorts, excluding the 1 mg/kg cohort. both uncorrected and baselineadjusted serum immunoglobulin concentrations over time did not appear to correlate with dose of sab-301 administered or sab-301 pharmacokinetic concentrations (data not shown). we determined anti-mers neutralising antibody titres using the jordan-n3/2012 strain of mers-cov (a clade a strain). microneutralisation titres correlated with sab-301 concentrations in serum ( figure 2b) . the functional neutralisation of the mers virus seems to follow the pharmacokinetic exposure of sab-301 described previously (r²=0·845, p<0·0001). pharmacodynamic analysis using the microneutralisation titres showed similar results, with geometric mean titres in the 50 mg/kg cohort of 1/1240 at 7 days and 1/600 at 21 days (appendix). we investigated the immunogenicity of sab-301 in several ways. we assessed the presence of anti-igg antibodies using rheumatoid factor. one participant in cohort 5 (20 mg/kg of sab-301) became rheumatoidfactor positive on day 21, with a maximum rheumatoid factor of 17 iu/ml. this positive rhematoid factor persisted throughout the study, and there were no associated symptoms with this finding (the only adverse event reported in this participant was albuminuria). we also assessed anti-drug antibody (anti-sab-301). three participants had anti-drug antibody present at baseline (before study drug administration), which persisted and did not change throughout the study (one participant in the 10 mg/kg of sab-301 cohort and two in the 50 mg/kg of sab-301 cohort). the pharmacokinetic parameters for these three participants were compared with the other participants in their respective cohorts, and there was no significant difference in t ½ , c max , or auc. all participants with preexisting anti-sab-301 had higher results for t ½ and auc than the cohort geometric mean. no new anti-drug antibodies were developed after administration of sab-301. we assessed the presence of anti-bovine κ light chain, representing residual bovine proteins that might be present in the final intravenous immunoglobulin product. seven participants had antibodies to the bovine κ light chain detected at baseline. there was no development of new anti-bovine κ-light-chain antibodies after administration of sab-301. we also assessed whether anti-camelid antibody was present, representing immunogenicity towards a llama anti-human κ-light-chain antibody used in the manufacturing process to purify sab-301. no participants had anti-camelid antibody at baseline, or developed these antibodies during the study. sab-301 is a novel anti-mers-cov intravenous immunoglobulin manufactured from the hyperimmune plasma of transchromosomic cattle that produce fully human polyclonal igg. the use of passive immunotherapy, either as immune plasma or intravenous immunoglobulin, has been recommended for multiple severe and emerging infectious diseases such as severe seasonal influenza, 5 pandemic influenza, 12 severe acute respiratory syndrome, 13 mers, 2 and ebola. 4 there are often limitations in collecting sufficient human plasma for production. 3, 5 albeit novel, the transchromosomic bovine production system is, more importantly, rapid and scalable. in transchromosomic cattle, new antigen-specific and high-titre human intravenous immunoglobulin can be generated within 3 months of a vaccine being available. therefore, our study is noteworthy not only by advancing a potential therapeutic for mers, but also by showing the potential safety of a novel production platform that can quickly generate a new putative drug candidate to an emerging infectious disease. the results of this first-in-human phase 1 study suggest that sab-301 appears to be safe and well tolerated at single doses up to 50 mg/kg. the adverse events seen in participants given sab-301 were largely comparable to those given placebo. in events occurring in more than one participant given sab-301, low bicarbonate, fatigue, loose stools, sore throat, vasovagal reaction, and aspartate aminotransferase increase did not have any corresponding events in the placebo group. both vasovagal reactions occurred before study drug administration. only low blood bicarbonate and sore throat seemed to occur more commonly in the higher dose cohort. with a small number of participants (as is typical in a phase 1 study), it is difficult to discern with certainty the attribution of adverse events to the study drug. these potential events will need to be investigated closely in any future studies. the pharmacokinetic profile of sab-301 is similar to what would be expected for human polyclonal or monoclonal antibodies with no human targets. in the critically ill population, the clinical illness of mers (defined by duration of hospital stay) is a median 19 days (iqr 10-35), and mers-cov shedding (as detected by pcr) is median 20 days (95% ci 17-26) in survivors and can exceed 37 days in those that die from mers. 14 the pharmacodynamic profile of sab-301, with a neutralisation titre of 1/600 at 21 days and 1/240 at 35 days for 50 mg/kg, suggests good viral neutralisation activity for a period that exceeds the clinical illness and viral shedding after administration of a single dose. the maximum concentration occurred in the initial sample (1 h after the end of infusion), and sab-301 concentrations remained detectable at the end of 90 days following single 10 mg/kg, 20 mg/kg, and 50 mg/kg doses. of course, these might be different in an infected patient, in light of antibody-virus binding and subsequent alteration in pharmacokinetics or pharmacodynamics. the efficacy of sab-301 has previously been shown in an adenovirus human dipeptidyl peptidase 4 (ad5-hdpp4)-transduced balb/c mouse model. 11 ad5-hdpp4 transduced balb/c mice were intranasally infected with mers-cov and given sab-301 at 24 or 48 h after infection. for mice given 500 μg (25 mg/kg, assuming 20 g body weight for mice) of sab-301 at 24 h post infection, mers-cov titres were below the level of detection at day 5 (p<0·0001). when sab-301 was administered 48 h after infection, viral titres were detectable but reduced by about 1000-fold by day 5 compared with the untreated control (p<0·0001). given that animal models of mers-cov challenge do not replicate human disease effectively, the relevance of this model to extrapolate human efficacy is not known. the mouse dose of 500 μg (equivalent to 25 mg/kg) equates to a human dose of approximately 2 mg/kg based on body surface area conversion. in view of the high mortality rate in people with mers-cov infection and the difficulties extrapolating preclinical models to human disease, we suggest the highest tolerated dose in this phase 1 trial (50 mg/kg) should be used initially in human efficacy trials. the vaccine used for immunising transchromosomic cattle to generate sab-301 is the spike protein nanoparticle vaccine of the al-hasa strain, which belongs to a clade b of mers-cov, and the pharmacokinetic data show high antibody titres towards this clade b strain. sab-301 was previously shown to neutralise the jordan (jordan-n3/2012) and erasmus medical center 2012 (emc/2012) strains of mers-cov, both of which are clade a. 11 therefore sab-301 is anticipated to have naturalising capacity to current clade a and b strains of mer-cov. to our knowledge, this is the first study to show the safety, tolerability, and pharmacokinetics of a novel therapeutic for mers, as well as for this new source of fully human igg produced in transchromosomic cattle. the data attained in this study suggest that sab-301 is safe and well tolerated at potentially therapeutic exposures. further clinical investigation of sab-301 for the treatment of mers is warranted. contributors jhb, jv, es, tl, and rtd were responsible for initial study design. jhb, jv, and rtd were responsible for study implementation and enrolment of participants. kr was responsible for the micro-neutralisation assay. hw, j-aj, and es were responsible for the pharmacokinetic assay and the immunogenicity assays. jhb, jv, pk, and rtd analysed and interpreted the data and wrote the first draft of the report. all authors had opportunity to review the data and edited the final report. hw, j-aj, and es have financial interests in sab biotherapeutics inc. all other authors declare no competing interests. who. middle east respiratory syndrome coronavirus middle east respiratory syndrome feasibility of using convalescent plasma immunotherapy for mers-covinfection, saudi arabia the use of convalescent plasma to treat emerging infectious diseases: focus on ebola virus disease immune plasma for the treatment of severe influenza: an open-label, multicentre, phase 2 randomised study species-specific chromosome engineering greatly improves fully human polyclonal antibody production profile in cattle molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 evaluation of candidate vaccine approaches for mers-cov viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study the authors thank the volunteers who participated in this research study, without whom the study would not have been possible. we also thank the physicians, nurses, and other health-care personnel at the national institutes of health who provided invaluable services and support for this study. this project was funded in part with federal funds from intramural research programmes of the national institute of key: cord-319447-xanewi59 authors: sun, jiya; ye, fei; wu, aiping; yang, ren; pan, mei; sheng, jie; zhu, wenjie; mao, longfei; wang, ming; huang, baoying; tan, wenjie; jiang, taijiao title: comparative transcriptome analysis reveals the intensive early-stage responses of host cells to sars-cov-2 infection date: 2020-05-01 journal: biorxiv doi: 10.1101/2020.04.30.071274 sha: doc_id: 319447 cord_uid: xanewi59 severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has caused a widespread outbreak of highly pathogenic covid-19. it is therefore important and timely to characterize interactions between the virus and host cell at the molecular level to understand its disease pathogenesis. to gain insights, we performed high-throughput sequencing that generated time-series data simultaneously for bioinformatics analysis of virus genomes and host transcriptomes implicated in sars-cov-2 infection. our analysis results showed that the rapid growth of the virus was accompanied by an early intensive response of host genes. we also systematically compared the molecular footprints of the host cells in response to sars-cov-2, sars-cov and mers-cov. upon infection, sars-cov-2 induced hundreds of up-regulated host genes hallmarked by a significant cytokine production followed by virus-specific host antiviral responses. while the cytokine and antiviral responses triggered by sars-cov and mers-cov were only observed during the late stage of infection, the host antiviral responses during the sars-cov-2 infection were gradually enhanced lagging behind the production of cytokine. the early rapid host responses were potentially attributed to the high efficiency of sars-cov-2 entry into host cells, underscored by evidence of a remarkably up-regulated gene expression of tprmss2 soon after infection. taken together, our findings provide novel molecular insights into the mechanisms underlying the infectivity and pathogenicity of sars-cov-2. coronavirus disease 2019 (covid-19) triggered by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is currently affecting global health. the sars-cov-2 is the third highly pathogenic coronavirus following sars-cov and mers-cov that cause severe accurate respiratory symptoms in humans. since december 2019, this virus has caused more than 80 thousand covid-19 cases in china. nowadays, the number of infections in countries outside china is growing rapidly. the most remarkable feature of the sars-cov-2 incidences and epidemiology is its great capacity for human-to-human transmission [1] . clinically, the majority of covid-19 patients have mild and moderate symptoms, and the elderly appear to have severe symptoms [2] . based on the analysis of china data, the covid-19 case-fatality rate was estimated as around 4.0% (3,341deaths over 82,249 confirmed cases of sars-cov-2 infection) [3] , lower than those of sars and mers [4] . however, due to the large-scale infected population, the sars-cov-2 has already caused more than ninety thousand deaths as of april 11 th 2020, sowing great social panic around the world. while recent efforts have been focused on transcriptome analysis of host responses to sars-cov-2 infection at a certain time point in certain cell lines [5, 6] , the transcriptional dynamics of host responses to the virus infection has remained largely unexplored. generally, once the virus enters the cell, the host innate immune responses, such as the interferon-mediated antiviral responses and cytokine production, have a pivotal role in suppressing the virus replication, which, if inadequate, might contribute to the viral pathogenesis. this hypothesis has been supported by our previous study, which has shown that the high pathogenicity of avian influenza virus is associated with abnormal coordination between interferon-mediated antiviral responses and cytokine production in host cells [7] . similar to both sars-cov and mers-cov, which induce the overactivation of cytokines [8] , increased cytokine levels are also observed in patients hospitalized with covid-19 [9] . transcriptome analysis of in vitro host cells shows that sars-cov and mers-cov elicit distinct responses to the expression of the host genes [10] . until now, the time-series gene-expression profiling of the host response to sars-cov-2 remains unknown and thus is urgently needed uncovering its pathogenesis. in this study, we used the sars-cov-2 strain isolated from patients [11] to infect in vitro calu-3 cells, and performed rna sequencing to determine the time-series transcriptome profiling data of the host. we established the host response patterns for sars-cov-2 by comprehensive analysis of the transcriptomic profiles from sars-cov-2, sars-cov and mers-cov. these results provide profound new insights into the pathogenesis and progression of the covid-19 disease caused by sars-cov-2, illuminating new strategies for the prevention and control of sars-cov-2 transmission and eventually leading to a cure of the covid-19 disease. cells and virus. calu-3 human airway epithelial cells(atcc, htb-55) were cultured in minimum essential media (mem, hyclone) supplemented with 10% fetal bovine serum(fbs), 1% mem neaa, and 100 u/ml penicillin-streptomycin solution (gibco, grand island, usa) at 37 °c in a humidified atmosphere of 5% (v/v) co2. vero cells (atcc, ccl-81) were cultured at 37 °c in dulbecco's modified eagle's medium (dmem, gibco) supplemented with 10% fbs (gibco) in the atmosphere with 5% co2. sars-cov-2 strain betacov/wuhan/ivdc-hb-01/2019 (c-tan-hb01, gisaid accession no. epi_isl_402119) was isolated from a human patient [11] . viruses were harvested and viral titrations were performed in vero cells using plaque assay. [12] . the cleaned data were mapped to the human grch38 reference genome using star aligner (v2.7.2a) [13] . the htseq-count command was used to count reads mapped to each gene [14] . the r package deseq2 was applied to further identify differentially expressed genes (degs) (fdr<0.05, |log2fc|>=1) [15] . the unmapped reads against the entire human genome were further aligned to the reference genome of sars-cov-2 (epi_isl_402119). virus genome annotation was based on our previous work [16] . go enrichment analysis was performed by fisher's exact test with the 19932 human protein-coding genes as a background in r. for analysis of microarray data of sars-cov and mers-cov, normalization and identification of degs (fdr<0.05, |log2fc|>=1) were conducted using the r package limma [17] . we carried out in time-course experiments to identify dynamic changes in transcripts in response to sars-cov-2 based on the infected and mock-infected groups across four time points (0, 7, 12 and 24 hpi), in which three biologically independent replicates for each treatment group were used for constructing cdna libraries. the calu-3 human airway epithelial cell line, a model of human respiratory disease [16] , was used as the host cell of sars-cov-2, subjected to the same moi and host cell used in the previous analyses of sars-cov and mers-cov infections. after the total rna isolation and sequencing, we obtained the host transcriptomes, as well as the genomes and transcripts of viruses. the high-throughput sequencing resulted in an average of 49 million pairedend reads per sample, and the sequencing quality was high with a mean mapping rate of unique reads at approximately 72% among mock samples (supplementary table 1 ). the quality control of all samples was assessed by the pca analysis based on normalized counts from deseq2, which indicated that high quality was achieved given that the majority of samples were well clustered except only one sample from the infection group at 24 hpi that was removed before further analysis ( figure 1a ). to evaluate the growth rate of sars-cov-2, we calculated the rna level of the virus represented by unique reads mapping rates at different time points. our results showed that in general the virus reads increased sharply from 1.4% to 61.2% while reads mapped to the host genome dropped rapidly from 67.2% to 11.4% ( figure 1b) , suggesting a rapid replication of the virus within 24 hours. from the results, at the earliest time point (0 hpi), virus produced high-levels of viral genome rna as evidenced by relatively even coverage depth across the whole genome ( figure 1c ). interestingly, we found that there was a significantly active transcription of the 3' end of sars-cov-2 at 7 hpi, especially for the m, 6, 7a, 7b, 8b and n genes ( figure 1c) which could play important roles in the antagonism with host immune response [18, 19] . after that, the relatively even depth distribution of reads along viral genome was again observed at panels of 12 and 24 hpi. this time-dependent patterns of virus replication and transcription was most likely to play critical roles in the pathology of sars-cov-2. to elucidate the global changes of host gene expression along with virus growth, we identified the overall up-and down-regulated degs during sars-cov-2 infection ( figure 1d and supplementary table 2 ). as shown in figure 1d , during the early stage of infection before 7 hpi, there were many more up-regulated genes than down to investigate specific host responses during sars-cov-2 infection, we performed a comparative transcriptome analysis by integrating two public host transcriptomes of sars-cov (gse33267) [20] and mers-cov (gse45042) [10] figure 3b ). in addition, a list of the virus-specific antiviral-related genes was identified, including parp10 [21] and cmpk2 [22] for sars-cov-2, bst2 [23] , ititm1 and usp41 for sars, and parp4 [24] for mers-cov ( figure 3b ). secondly, we further used a set of 113 human cytokines to quantify host inflammation responses between three viruses. the 113 cytokines from the cytoreg database were often cited by various publications and play a primary role in the immune system [25] . our results showed that, for sars-cov-2, the level of cytokine production was highly induced at 0 hpi, decreased at 7 hpi, and then slowly recovered thereafter ( figure 3c ). and mers-cov. our analysis also provided evidence that sars-cov-2 had more cytokines in common with sars-cov than with mers-cov ( figure 3d next, to gain possible explanations for the distinct patterns in host antiviral capacity and cytokine production during sars-cov-2 infection, dynamic expression of four types of key genes were evaluated, including virus receptors for cell entry, pathogen recognition receptors (prrs) for an innate immune startup, regulator genes for induction of antiviral-related genes and interferon production (figure 4) . for the three cell entry related genes (ace2 as the receptor of sars-cov and sars-cov-2 [28, 29] , dpp4 as the receptor of mers-cov [30] and protease tmprss2 for s protein priming of sars-cov-2 [27] ), we observed the dramatic changes in tmprss2 expression with very early induction during sars-cov-2 infection, and the slightly down-regulated expression of ace2 in cells infected with sars-cov-2 and sars-cov, whereas dpp4 was more up-regulated in mers-cov (figure 4 ). for the two prrs, ddx58 is a canonical rig-i-like receptor for rna virus recognition [31] , and tlr3 is a toll-like receptor playing important roles in initiating a protective innate immune response to highly pathogenic coronavirus infections [32] . we observed that all three viruses had a notably up-regulated expression of ddx58 while only mers-cov had a suppressed tlr3 at the 24 hpi (figure 4) , which is consistent with the fact that decreased expression of tlr3 contributes to the pathology of highly pathogenic coronavirus infections [32] . among the four regulator genes, irf7 is responsible for the expression of most ifn-α subtypes and the type i ifn amplification loop [33] , and irf9, stat1 and stat2 form the isgf3 complex that binds to interferon-stimulated response elements and thereby induces the expression of interferon-stimulated genes [34] . as expected, gradually up-regulation of the four primary regulator genes was observed for all three viruses (figure 4 ). at last, we found a significant difference in the expression of ifnb1 between sars-cov-2, sars-cov and mers-cov, indicating that ifnb1 likely accounted for the observed variations of the host antiviral capacities among three viruses (figure 4) . taken together, early induction of tmprss2 and gradually increased expression level of ifnb1 were likely responsible for the distinct host immune response patterns of sars-cov-2 infection. using time-series profiling of the virus genome and host transcriptome at the same time during sars-cov-2 infection coupled with comparative transcriptome analysis, we found that, compared to sars-cov and mers-cov, sars-cov-2 induces strong host cell responses at the very early stage of infection that not only favor its high infectivity to host cells but also restrict its pathogenesis. here we sequenced the transcriptomes of sars-cov-2 and virus-infected host cells simultaneously during the early stages of infection, providing a robust reference dataset to speculate the antagonistic pattern between pathogen and host cells. to summarize, our findings showed that sars-cov-2 induced the significantly high expression of the cellular serine protease tmprss2 at 0 hpi to help the entry of viral particles into cells [28] (figure 4 ). at the same time, host cell initiated an immediate response for the invasion of sars-cov-2 virus ( figure 1d ). then, the virus successfully suppressed the acute response of host cells for fast proliferation by increasing the transcripts of its 3' genome end, including m, 6, 7a, 7b, 8 and n genes which were consistent with their reported regulations to host immune response [18, 19] . as a response from hosts cell, a number of antiviral pathways and cytokine productions were up-regulated to resist the virus infection (figures 2 and 3 ). in particular, several metabolism-associated pathways were down-regulated at 12hpi and 24 hpi (figure 2 ). after the antagonistic cycle, a dramatic proliferation of viral particles was detected in the early infection of host cells ( figure 1b) , which could possibly be an explanation for the fast spread of sars-cov-2 in humans. as sars-cov-2 was reported a relatively low risk of mortality [3] compared to the other two serious human coronaviruses, sars-cov and mers-cov, we compared and contrasted the host transcriptomes in response to the viral infections. we found that some cytokines in sars-cov-2-infected cells were markedly up-regulated at a very early stage, which was not observed for sars-cov and mers-cov and even less frequently observed for other viruses. the unusual high expression of cytokines at 0 hpi possibly explains why patients with severe clinical symptoms rapidly deteriorated. although the number of infected cases was very high, the majority of infections displayed mild symptoms which are partly explained by a gradual increase in host antiviral capability from 7 to 24 hpi. in contrast to sars-cov-2, both sars-cov and mers-cov were able to inhibit the antiviral capability of the host significantly, which could explain their observed relatively high mortalities. mers was associated with a higher mortality than sars, which could be in part attributed to the higher expression of cytokines suppressing the antiviral responses. recently, blanco-melo et al. [5] have published transcriptome data of host responses to sars-cov-2 from in vitro cell lines including a549 (moi of 0.2) and nhbe (moi of 2) at 24 hpi. this previously published data is complemented by our study designed to investigate the early response phase of cell lines infected with sars-cov-2. while the previous work did not observe the elevated levels of ifnb1, ifnl1 and ifnl3, our findings show that not only ifnb1 but also ifnl1 and ifnl3 expressions are upregulated between 7 and 24 hpi ( figure 3d ). also, they did not detect gene expression of ace2 and tmprss2 at 24 hpi, while we observed that ace2 is down-regulated at 24 hpi and tmprss2 is only up-regulated at 0 hpi before returning to the normal levels ( figure 4) . our time-series sampling revealed distinct early-response features of sars-cov-2, which provided a possible explanation for some clinical observations. for example, a recent clinical study [35] found that sars-cov-2 could replicate effectively in upper respiratory tract tissues, and that the viral loads appeared earlier (before day 5) and were substantially more than expected. findings from the present study have confirmed that, at 7 hpi, the 3' end of sars-cov-2 genome start to express densely, reducing the effectiveness of host immune surveillance, which possibly enables the rapid replication of sars-cov-2 in upper respiratory tract tissues. in spite of the fact that several studies have already demonstrated a consistent correlation between gene expression measured by rna-seq and by microarray [36] [37] [38] , we still need to exclude the possibility of bias resulting from different methodologies. first, because rna-seq can potentially detect more genes than microarrays, we only early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of coronavirus disease 2019 in china estimates of the severity of coronavirus disease 2019: a model-based analysis from sars to mers, thrusting coronaviruses into the spotlight. viruses sars-cov-2 launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients regulation of early host immune responses shapes the pathogenicity of avian influenza a virus clinical features of patients infected with 2019 novel coronavirus in wuhan cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus. mbio a novel coronavirus from patients with pneumonia in china trimmomatic: a flexible trimmer for illumina sequence data star: ultrafast universal rna-seq aligner htseq--a python framework to work with highthroughput sequencing data moderated estimation of fold change and dispersion for rna-seq data with deseq2 genome composition and divergence of the novel coronavirus (2019-ncov) originating in china limma powers differential expression analyses for rna-sequencing and microarray studies human coronaviruses: a review of virus-host interactions. diseases human coronavirus: host-pathogen interaction release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells the interaction between the parp10 protein and the ns1 protein of h5n1 aiv and its effect on virus replication cmpk2 and bcl-g are associated with type 1 interferon-induced hiv restriction in humans tetherin inhibits hiv-1 release by directly tethering virions to cells rapid evolution of parp genes suggests a broad role for adpribosylation in host-virus conflicts global landscape of mouse and human cytokine transcriptional regulation pathological findings of covid-19 associated with acute respiratory distress syndrome dysregulation of immune response in patients with covid-19 in wuhan, china sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc rig-i in rna virus recognition toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection. mbio irf-3, irf-5, and irf-7 coordinately regulate the type i ifn response in myeloid dendritic cells downstream of mavs signaling the molecular basis for functional plasticity in type i interferon signaling virological assessment of hospitalized patients with covid-2019 a comprehensive comparison of rna-seq-based transcriptome analysis from reads to differential gene expression and cross-comparison with microarrays: a case study in saccharomyces cerevisiae correlation between rna-seq and microarrays results using tcga data large scale comparison of gene expression levels by microarrays and rnaseq using tcga data the sequencing data from this study have been submitted to the national genomics data center (https://bigd.big.ac.cn/) with the accession number prjca002617. jiang, tan and huang devised the experiment and wrote the paper; sun, wu, sheng and zhu conducted bioinformatics analysis; ye, huang and yang prepared samples; wang provided calu-3 cell; pan run rna sequencing; and all authors revised the manuscript. the authors declare that they have no conflict of interest. key: cord-317688-mr851682 authors: oh, myoung-don; park, wan beom; park, sang-won; choe, pyoeng gyun; bang, ji hwan; song, kyoung-ho; kim, eu suk; kim, hong bin; kim, nam joong title: middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea date: 2018-02-27 journal: korean j intern med doi: 10.3904/kjim.2018.031 sha: doc_id: 317688 cord_uid: mr851682 middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in 2012. the 2015 korea outbreak of merscov involved 186 cases, including 38 fatalities. a total of 83% of transmission events were due to five superspreaders, and 44% of the 186 mers cases were the patients who had been exposed in nosocomial transmission at 16 hospitals. the epidemic lasted for 2 months and the government quarantined 16,993 individuals for 14 days to control the outbreak. this outbreak provides a unique opportunity to fill the gap in our knowledge of mers-cov infection. therefore, in this paper, we review the literature on epidemiology, virology, clinical features, and prevention of mers-cov, which were acquired from the 2015 korea outbreak of merscov. middle east respiratory syndrome coronavirus (mers-cov) was first isolated from a patient with severe pneumonia in september 2012 [1] . since then, as of 31 january 2018, the world health organization (who) has been notified of 2,143 laboratory-confirmed cases of mers-cov infection, including at least 750 deaths, from 27 countries (fig. 1) [2] . most of mers-cov human infections have occurred in the arabian peninsula. saudi arabia reported 1,783 cases, including 726 deaths (fatality rate, 40.7%) [3] . a recent article summarized the current knowledge on the epidemiology, virology, human pathogenesis, clinical management, and prevention of mers-cov infection [4] . another comprehensive review article on risk factors and determinants of transmission of mers-cov from human to human in the community and hospital settings has been published recently [5] . public health england also published an extensive review of literature for clinical decision-making support for treatment of mers-cov patients [6] . the 2015 korea outbreak of mers-cov is the largest outside the arabian peninsula and provides a unique opportunity to fill the gap in our knowledge about mers-cov infection. since it has now been almost 3 years since the 2015 mers outbreak in the republic of korea, we reviewed the literature to summarize the lessons we have learned from the korea outbreak. the korean journal of internal medicine vol. 33, no. 2, march 2018 fields. as of 31 january 2018, a total of 221 articles were retrieved, and 216 of them were written in english. the full texts of the english articles were reviewed and critically assessed. the 2015 korea outbreak of mers-cov resulted in 186 cases including 38 fatalities [7] . the index case was a returning traveler from the middle east. the infection had spread within the hospital, and subsequently to other hospitals because of patient movement, resulting in nosocomial transmission at 16 clinics and hospitals. the epidemic lasted for 2 months, with the government declaring a "virtual" end to the epidemic on july 6, 2015. in order to control the outbreak, the government quarantined 16,993 individuals for 14 days, and the economic loss was estimated at 9.311 trillion korean won (8.5 billion us dollars) [8] . the first patient (index case) with mers-cov infection was a 68-year-old korean man returning from the middle east. he ran a commercial greenhouse business in bahrain. he flew from seoul to bahrain on april 24, 2015 , and traveled through the united arab emirate and the kingdom of saudi arabia, and came back to seoul on may 4, 2015. during his 10-day business trip, he did not visit a camel farm or a hospital. he did not eat camel meat or any other camel products. he did not remember meeting any persons with respiratory symptoms [9] [10] [11] [12] . seven days after returning home, on may 11, he developed fever and myalgia. he visited a local clinic on may 12, 14, and 15. his symptoms, however, did not improve and a new non-productive cough developed on 15th may. he visited pyeongtaek st. mary's (ptsm) hospital (65 km south from seoul), a secondary hospital in gyeonggi province. his chest x-ray showed a consolidation in the upper zone of right lung, and he was admitted to the hospital for pneumonia on may 15. ceftriaxone and amikacin were started, but his pneumonia progressed despite the antibiotic treatment. he was transferred to 2,103 reported to who as of november 17, 2017 republic of korea other countries saudi arabia 12 18 24 30 36 42 48 01 07 13 19 25 31 37 43 49 03 09 15 21 27 33 39 45 51 05 11 17 23 29 35 41 47 01 07 13 19 25 31 37 43 49 03 09 15 21 27 the chain of transmission was identified in all 186 mers patients [13, 14] . of the 86 healthcare institutions that had taken in mers-cov-infected patients, nosocomial transmission occurred in 12 hospitals and four clinics (plus two ambulances) [13, 15, 16] . the epidemiological characteristics are summarized in table 1 [17] . the epidemic curve is shown in fig. 2 . during his admission from may 15 to 17 at ptsm hospital, the first patient was unknowingly spreading mers-cov. patients, visitors, and healthcare workers (hcws) were exposed to mers-cov. therefore, by may 20, when the korea cdc noticed the importation of mers-cov into the republic of korea, individuals staying on the same floor as the first case had already been exposed to mers-cov. as a result, a total of 36 mers cases occurred at ptsm hospital. of them, 26 patients developed symptoms within 14 days after exposure, suggesting they were first-generation cases; the remaining 10 patients may be second-generation cases [7, 10] . the mode of transmission of mers-cov at ptsm hospital remains uncertain. in addition to family members of the first patient, only one patient shared the same room (#8104) with him; all the other cases stayed in other rooms, which were located > 2 m apart from room #8104. all patient rooms were equipped with an air ventilating system except for room #8104. a recent study using a multi-agent modeling framework suggested the transmission occurred via a long-range airborne route or via a combination of a long-range airborne route and direct contact transmission [18] . a chest computed tomography (ct) scan taken in the early stages (day 1 or 2) of illness showed very small, ground glass nodules in the periphery of the lungs, suggesting inhalation of airborne particles [19] . many individuals had been exposed to the superspreader-1 at the er, and contact tracing identified 675 patients, an estimated 683 visitors, and 218 hcws as contacts of superspreader-1. of them, 278 were quarantined and 617 were under active monitoring. as a result of exposure to superspreader-1, 82 had laboratory-confirmed mers-cov infection, and an additional 10 were secondarily infected from them [20, 21] . the index case (superspreader-2) for the daejeon (100 km south from pyeontaek city) outbreak was infected at ptsm hospital. he developed symptoms on may 20 and was admitted to daechung hospital on may 22. as his pneumonia progressed despite the treatments, he was transferred to geonyang hospital on may 28. a total of 25 secondary cases (14 in daechung hospital and 11 in geonyang hospital) were detected. although superspreader-2 caused the two hospital outbreaks, the me11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 dian time of incubation (8.8 days vs. 4.6 days), secondary attack rates (4.7% vs. 3.0%), and case fatality rate (28.6% vs. 63.6%) were different between the two outbreaks. the high fatality rate was associated with pre-existing pneumonia or poor underlying pulmonary function [22] [23] [24] . a 44-year-old male, who had been exposed to the first index case on may 16, developed back pain on may 21. he is a son of the third mers case. his sister had also been exposed to mers epidemiological characteristics have been reported by several groups (table 2) [7, 16, 20, 21, 23, [27] [28] [29] [30] . the defining epidemiological characteristics were superspreading events at hospitals [30] [31] [32] . early superspreading events generated a disproportionately large number of second-ary infections, and the transmission potential decreased sharply in subsequent generations [30, 33] . a total of 83% of transmission events were due to five superspreaders, and 44% of the 186 mers cases were in-patients who had been exposed within the hospitals [7] . the spreaders transmitted mers-cov from days 1 to 11 of their illness (median, 7 days), and the number of patients infected by each spreader ranged from 1 to 84 (interquartile range, 1 to 12) [27] . the median incubation period and serial interval was 7 days and 12.5 days, respectively [7, 34] . r0 ranged from 2.5 to 7.2, higher than the previous estimate of < 1 [35] . it was estimated at 4.04 for the outbreak cluster in ptsm hospital, and 5.0 for the outbreak in smc [36] . a mathematical modeling study demonstrated that although r0 < 1 overall, cluster sizes of over 150 cases are not unexpected for mers-cov infection [37] . compared with the non-spreaders, the spreaders had a higher frequency of fever (≥ 38.5°c) and chest infiltrates in more than three lung zones, and longer non-isolated in-hospital days [27] . the spreaders had higher viral load in sputum samples with the cycle thresholds for the upe and orf1a genes of 22.7 vs. 27.2 and 23.7 vs. 27.9, respectively. the spreaders with more than three transmissions had higher numbers of contacts and er visits [38] . a brief exposure of a 10-minute stay and 2 minutes of talking was enough for the transmission of mers-cov [39] . the whole genome sequences of mers-cov isolated from the korea outbreak were reported [26, [40] [41] [42] [43] . the virus most similar to the korean isolates was a camel virus (camel/riyadh/ry159/2015) that belonged to lineage 5, a recombinant of lineage 3 and 4 [44, 45] . viruses from lineage 5 had been predominant in saudi arabian camels since november 2014, but human viruses of this lineage were reported from february 2015 [44] . a phylogenetic study of spike glycoprotein genes also showed the korean strains were closely related to the viral strains from riyadh, saudi arabia [46] . in a molecular study, 11 of 13 mers-cov genome had an i529t mutation and one d510g mutation in the receptor binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, cd26 [47] . heterogeneity analysis revealed the combined frequency of i529t and d510g was high (87.7%), although the frequency of both mutations varied greatly among specimens [48] . another genome sequence study also reported deletions of 414 and 419 nucleotides between orf5 and the e protein, suggesting that the virus might be defective in its ability to package mers cov [49] . a microevolution study found no evidence of changes in the evolutionary rate [43] . however, whether the transmissibility and virulence of the korean isolates are different from those of the previous isolates from the kingdom of saudi arabia remains to be determined by phenotypic assays. a viral shedding study showed that the copies of mers-cov rna detected by real-time reverse transcription polymerase chain reaction (rrt-pcr) in respiratory samples peaked during week 2, and the median value was 7.21 log 10 in the severe group and 5.54 log 10 in mild cases [50] . the study also showed that viral titers were higher in throat samples than in nasopharyngeal samples. another study also demonstrated higher viral loads were associated with severity and mortality [51] . after mers-cov infection, antibody responses developed by the third week of illness in most patients [52] . seroconversion rates increased with the increase of dis-ease severity. in a serologic study of 42 mers patients, the seroconversion rate was 0% in asymptomatic infection, 60.0% in symptomatic infection without pneumonia, 93.8% in pneumonia without respiratory failure, and 100% in pneumonia with respiratory failure [53] . the serological response remained detectable for > 12 months in all survivors (11/11) who had severe disease. antibody titers were not detectable in four of six patients who had mild pneumonia, suggesting that mers-cov seroepidemiological studies may underestimate the extent of mild and asymptomatic infection [54] . the korean society of infectious diseases compiled the clinical data of the 186 mers patients [55] . the median age was 55 years (range, 16 to 86). the male to female ratio was 3:2. the most common coexisting medical conditions were hypertension (31.7%), diabetes (18.8%), solid organ malignancy (13.4%), and chronic lung disease (10.2%). patients with mers-cov infection developed a spectrum of illnesses, ranging from asymptomatic to fulminant pneumonia with fatal outcome. the respiratory symptoms were similar to other acute viral respiratory infections. at the time of presentation, fever had developed in 81.7%, cough in 56.7%, and sputum in 39.8% of patients. symptoms of upper respiratory-tract infections were infrequent: sore throat was present in 9.1% and rhinorrhea in 1.6% of patients. gastrointestinal symptoms were also observed: diarrhea was present in 19.4%, nausea or vomiting in 14.0%, and abdominal pain in 8.1% of patients. diarrhea may be due to the side effect of lopinavir/ritonavir, as 87% of the patients received antiviral drugs for mers-cov treatment [55] . in the early stages of illness, cough and sputum were not prominent even in patients who later developed overt pneumonia [56] . at admission, 68% (123/180) of patients had abnormalities on chest radiographs, while 80.8% (147/182) of them developed the abnormalities during the course of the disease [55] . sudden progression of pneumonia occurred around day 7 of illness [50, 57] . predictive factors for development of pneumonia included older age, high fever, thrombocytopenia, lymphopenia, c-reactive protein (crp) ≥ 2 mg/dl, and a high viral load www.kjim.org https://doi.org/10.3904/kjim.2018.031 in sputum (threshold cycle value of rrt-pcr < 28.5) [58] . forty-five patients (24.5%) were treated with mechanical ventilation and 15 patients (8.2%) needed extracorporeal membrane oxygenation [55] . the typical course of pneumonia is shown in fig. 3 . acute kidney injury (aki) developed in 14.0% of the patients, 42.7% (44/103) had proteinuria, and 35.0% (36/103) had hematuria. fifteen patients were treated with renal replacement therapy [55] . old age is an independent risk factor for the occurrence of aki [59] . neuromuscular manifestations were not uncommon; hypersomnolence, weakness and tingling in the extremities were reported during the treatment of mers, suggesting guillain-barré syndrome or virus-related sensory neuropathy [60] . serial changes in chest radiographs was reported in five patients [56] . chest ct scans revealed rapidly developed multifocal nodular consolidations with ground-glass opacity halo and mixed consolidation, mainly in the dependent and peripheral areas [61] . there are few laboratory findings specific to mers-cov infection, although monocytosis with normal white blood cell count and low crp level were more common in mers patients at initial presentation [57, 62] . the guidelines for the molecular diagnosis of mers-cov infection have been published by the korean society for laboratory medicine [63, 64] . specimen type and quality is important in the laboratory diagnosis of mers-cov infection. lower respiratory-tract samples, such as sputum and tracheal aspirates, have higher viral loads than upper respiratory-tract samples [50] . however, mers patients may not shed the virus during the early stage of their illness. therefore, initial negative results should not rule out the possibility of mers [65] , and patients suspected of having mers-cov infection should be retested using a lower respiratory-tract sample [63, 66] . when sputum cannot be obtained, throat swabs may be an alternative source of diagnostic samples [50] . special attention should be given to diagnosing mers-cov infection in immunocompromised patients, as they may present with atypical features, such as a longer incubation period, a longer period from initial pcr positivity to symptom onset, and persistent viral shedding [67] . an antiviral treatment guideline has been published by the writing committee with support from the korean society of infectious diseases and the korean society for chemotherapy [68] . the guideline recommended a triple combination regimen of type 1 interferon, ribavirin, and lopinavir/ritonavir for 10 to 14 days. however, the guideline was mostly based on expert opinions and further study for the optimal antiviral treatment in mers is warranted. the median duration of fever was 8 days. the median time to negative conversion of mers-cov in sputum samples by rrt-pcr was 17 days (range, 4 to 45) [55] . the median time from symptom onset to death was 14 days. the case fatality rate was 20.4% (38/186) (fig. 4) . the in-hospital mortality, 7-day mortality, and 28-day (from symptom onset) mortality were 19.4% (36/186), 3.8% (7/186), and 17.7% (33/186), respectively [55] . host factors associated with mortality were old age (> 60 years), smoking history, pre-existing pneumonia, abnormal renal function, and comorbid conditions [24, [69] [70] [71] . low albumin, altered mentality, and high pneumonia severity index score at admission were risk factors for mortality [69] . mers-cov rna in blood samples was detected in 33% (7/21) of patients at presentation, and it was associated with a worse clinical outcome [72] . a shorter incubation period was also associated with an increased risk of death [24, 73] . a pregnant woman infected with mers-cov in her 35 weeks of gestational age developed dyspnea and her chest radiograph showed diffuse infiltrates in the left lower lung zone. she recovered from the infection and delivered a healthy full-term baby without vertical mers-cov transmission [74] . a patient developed organizing pneumonia 7 days after the resolution of mers-cov infection. he was successfully treated with corticosteroids [75] . in a mental health study, 7.6% of 1,656 patients who were quarantined showed symptoms of anxiety, and 6.4% reported feelings of anger during the 2 weeks of quarantine [76] . mental health support, accurate information, and appropriate supplies, including food, clothes, and accommodation, should be provided to isolated persons [76] . a case of possible transfusion-related acute lung injury following transfusion of convalescent plasma was also reported [77] . a comprehensive "mers infection prevention and control guideline for healthcare facilities" was drafted by the guideline development committee with generous support from korean academic societies [78] . the guideline included practical aspects of infection control and prevention based on the experience from the korea outbreak, such as the composition of members for the mers emergency committee, a space plan for an anteroom for donning and doffing, isolation of in-patients and hcws in a hospital affected by an outbreak, and management of the deceased and autopsy [78] . the hemodialysis unit may become an epicenter for mers-cov outbreak because hemodialysis patients must receive renal replacement therapy even during the outbreak, and the risk of exposure to mers-cov continues. during the korea outbreak, a hemodialysis unit was found to have a mers patient, and a total of 104 patients and 18 hcws were exposed to mers-cov. with support from the korean society of nephrology, the dialysis unit could continue to operate while the exposed patients were isolated in the unit. fortunately, no further transmission occurred at the unit [79] . after this successful experience, the society published a clinical practice guideline for hemodialysis facilities dealing with mers patients [80] . the korea outbreak was driven by the superspreaders who visited multiple healthcare facilities; thus, generating a large number of secondary cases. limiting unnecessary contacts with patients with respiratory symptoms in healthcare settings, especially in emergency departments, is of critical importance [33] . mers-cov could survive for longer than 48 hours at 20°c and 40% of relative humidity, suggesting contact or fomite transmission might occur in healthcare settings [81] . mers-cov was detected by rrt-pcr in specimens taken from the medical equipment [82] [83] [84] . mers-cov was also isolated by cell culture of the air and swab samples taken from a mers isolation unit, suggesting extensive contamination of the isolation unit [85, 86] . of the 186 cases, 23% were infected by undocumented contact between cases (i.e., indirect transmission of mers-cov via environmental contact) [87] . therefore, fomites with possible mers-cov contamination should be sanitized, and a minimum room ventilation rate of six air changes per hour should be implemented to minimize recirculation of pathogen-bearing droplets. meticulous environmental cleaning may be important for preventing transmission in healthcare settings. there was a case of a 39-year-old female nurse who was infected with mers-cov during a cardiopulmonary resuscitation lasting 1 hour for a mers patient who had pneumonia and hemoptysis [88] . serological surveillance conducted after the mers outbreak for asymptomatic infection among hcws involved in the direct care of mers patients showed that 0.3% (2/737) of them were mers-cov igg positive by an indirect immunofluorescent assay. among the hcws who did not use appropriate personal protective equipment (ppe), seropositivity was 0.7% (2/294) compared with 0% (0/443) in hcws with appropriate ppe use [89] . another serological survey also showed that none of the 285 hcws were positive for mers-cov immunoglobulin g, although 38.2% (109/285) of the hcws reported experiencing mers-like symptoms while caring for the mers patients [90] . in a third study, 0 of 189 hcws showed seroconversion by a plaque reduction neutralization test, although 20% to 25% of hcws reported mers-like symptoms [91] . during the outbreak in smc, all hcws assigned to mers patients were screened for mers-cov, regardless of the presence or absence of symptoms. of the 591 hcws, three (0.5%) asymptomatic hcws (two nurses and one physician) were found to be mers-cov (+), but they did not transmit the virus to others [21] . in another study, an asymptomatic nurse without ppe contacted 82 hcws, including 33 close contacts who were exposed within 2 m from the index nurse and were not using ppe. none of the exposed hcws were infected [92] . however, the potential for transmission from asymptomatic rrt-pcr positive individuals is still unknown. therefore, asymptomatic hcws who are rrt-pcr-positive for mers-cov should be isolated and should not return to work until two consecutive respiratory-tract samples test negative on rrt-pcr [93] . the government requested the korean society of infectious diseases to participate in the control of the mers outbreak. on june 8, 2015, the society organized the rapid response team, consisting of 15 infectious-disease doctors and two infection-control professionals [94] . critical suggestions to prevent future epidemics were made regarding a rapid alerting system for index cases, provision of a sufficient airborne infection isolathe korean journal of internal medicine vol. 33, no. 2, march 2018 tion facility, education and training of hcws for important infectious diseases, and overcrowding and visitor control in the hospital [95] . the 2015 korea outbreak was the largest outbreak outside of the middle east. researchers had a unique opportunity to compare the nature of mers-cov infection with the middle east experience. the outbreak unveiled the weak points of infrastructure in our medical system, especially of preparedness for emerging global infectious diseases. nosocomial transmission was one of the core features of mers-cov infection. the introspection for loose medical referral systems, overcrowding at the er, a lack of expert resources and infection control infrastructure, and lack of organized preparedness for medical crises prompted the government to reform the healthcare system, and healthcare sectors to invest further in infectious diseases and infection control. although the improvement is still ongoing, the speed and content are still currently insufficient. international cooperation to prepare for and defeat emerging infectious diseases should also be emphasized. lessons we learned from the outbreak are summarized in table 3 . a single, missed case may trigger a huge, nationwide outbreak the first line of defense is not the thermal scanner at the airport. it is doctors in the community clinics/hospitals. due to sudden deterioration of pneumonia around day 7 of illness, patients may visit emergency department, or be admitted to intensive care unit. superspreading events may occur in healthcare settings, especially at the emergency department. patients may transmit mers-cov as early as 2 days after symptom onset. early detection and isolation is of critical importance. aggressive strategy for quarantine maybe necessary, especially when large number of individuals are exposed in the healthcare settings. huge socioeconomic impact with an estimated 8.5 billion us dollars isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) [internet]. geneva: world health organization ministry of health kingdom of saudi arabia. mers-cov daily update ministry of health kingdom of saudi arabia, c2014 middle east respiratory syndrome middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients uk): public health england middle east respiratory syndrome coronavirus outbreak in the republic of korea the korean middle east respiratory syndrome coronavirus outbreak and our responsibility to the global scientific community the clinical and virological features of the first imported case causing mers-cov outbreak in south korea epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital middle east respiratory syndrome in 3 persons, south korea the first case of the korean middle east respiratory syndrome outbreak south korea coronavirus mers case list: including imported and exported cases. ministry of health & who confirmed data only fl): flutrackers, c2018 mers-cov infection: current status-interactive and infographics probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications ministry of health and welfare. the 2015 mers outbreak in the republic of korea: learning from mers (the white paper) a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of middle east respiratory syndrome coronavirus superspreading event involving 81 persons, korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon hospital outbreaks of middle east respiratory syndrome high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea imported case of mers-cov infection identified in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea epidemiologic parameters of the middle east respiratory syndrome outbreak in korea epidemiological investigation of mers-cov spread in a single hospital in south korea transmission characteristics of mers and sars in the healthcare setting: a comparative study understanding and modeling the super-spreading events of the middle east respiratory syndrome outbreak in korea mers outbreak in korea: hospital-to-hospital 244 www transmission identifying determinants of heterogeneous transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, 2015: a retrospective epidemiological analysis comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia estimating and modelling the transmissibility of middle east respiratory syndrome coronavirus during the 2015 outbreak in the republic of korea high reproduction number of middle east respiratory syndrome coronavirus in nosocomial outbreaks: mathematical modelling in the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea transmission among healthcare worker contacts with a middle east respiratory syndrome patient in a single korean centre complete genome sequence of middle east respiratory syndrome coronavirus kor/knih/002_05_2015, isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china isolation of middle east respiratory syndrome coronavirus from a patient of the 2015 korean outbreak microevolution of outbreak-associated middle east respiratory syndrome coronavirus, south korea co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia the recent ancestry of middle east respiratory syndrome coronavirus in korea has been shaped by recombination variations in spike glycoprotein gene of mers-cov spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak analysis of intrapatient heterogeneity uncovers the microevolution of middle east respiratory syndrome coronavirus two deletion variants of middle east respiratory syndrome coronavirus found in a patient with characteristic symptoms viral load kinetics of mers coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity kinetics of serologic responses to mers coronavirus infection in humans serologic responses of 42 mers-coronavirus-infected patients according to the disease severity mers-cov antibody responses 1 year after symptom onset, south korea clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea clinical implications of 5 cases of middle east respiratory syndrome coronavirus infection in a south korean outbreak clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients renal complications and their prognosis in korean patients with middle east respiratory syndrome-coronavirus from the central mers-cov designated hospital neurological complications during treatment of middle east respiratory syndrome middle east respiratory syndrome-coronavirus infection: a case report of serial computed tomographic findings in a young male patient differential cell count and crp level in blood as predictors for middle east respiratory syndrome coronavirus infection in acute febrile patients during nosocomial outbreak guidelines for the laboratory diagnosis of middle east respiratory syndrome coronavirus in korea korean society for laboratory medicine practice guidelines for the molecular diagnosis of middle east respiratory syndrome during an outbreak in korea in 2015 importance of specimen type and quality in diagnosing middle east respiratory syndrome an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) atypical presentations of mers-cov infection in immunocompromised hosts antiviral treatment guidelines for middle east respiratory syndrome predictors of mortality in middle east respiratory syndrome (mers) mortality risk factors for middle east respiratory syndrome outbreak, south korea real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection association between severity of mers-cov infection and incubation period mers-cov infection in a pregnant woman in korea successful treatment of suspected organizing pneumonia in a patient with middle east respiratory syndrome coronavirus infection: a case report mental health status of people isolated due to middle east respiratory syndrome possible transfusionrelated acute lung injury following convalescent plasma transfusion in a patient with middle east respiratory syndrome middle east respiratory syndrome infection control and prevention guideline for healthcare facilities middle east respiratory syndrome coronavirus transmission in dialysis unit and infection control interventions in korea middle east respiratory syndrome clinical practice guideline for hemodialysis facilities stability of middle east respiratory syndrome coronavirus (mers-246 www cov) under different environmental conditions environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea environmental contamination and viral shedding in mers patients viral shedding and environmental cleaning in middle east respiratory syndrome coronavirus infection extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards transmissibility of middle east respiratory syndrome by the airborne route nosocomial amplification of merscoronavirus in south korea healthcare worker infected with middle east respiratory syndrome during cardiopulmonary resuscitation in korea surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity seroprevalence of middle east respiratory syndrome coronavirus among healthcare personnel caring for patients with middle east respiratory syndrome in south korea serologic evaluation of mers screening strategy for healthcare personnel during a hospital-associated outbreak middle east respiratory syndrome coronavirus transmission in dialysis unit and infection control interventions in korea management of asymptomatic persons who are rt-pcr positive for middle east respiratory syndrome coronavirus (mers-cov): interim guidance [internet]. geneva: world health organization, c2018 collaborative intervention of middle east respiratory syndrome: rapid response team institutional preparedness to prevent future middle east respiratory syndrome coronaviruslike outbreaks in republic of korea no potential conflict of interest relevant to this article was reported. key: cord-314651-e4uaw5fy authors: zhao, guangyu; jiang, yuting; qiu, hongjie; gao, tongtong; zeng, yang; guo, yan; yu, hong; li, junfeng; kou, zhihua; du, lanying; tan, wenjie; jiang, shibo; sun, shihui; zhou, yusen title: multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus date: 2015-12-23 journal: plos one doi: 10.1371/journal.pone.0145561 sha: doc_id: 314651 cord_uid: e4uaw5fy the middle east respiratory syndrome coronavirus (mers-cov) causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. for the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of mers-cov infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against mers-cov infection. in this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hdpp4), the receptor for mers-cov. after intranasal inoculation with mers-cov, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. in addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. taken together, this study characterizes the tropism of mers-cov upon infection. importantly, this hdpp4-expressing transgenic mouse model will be applicable for studying the pathogenesis of mers-cov infection and investigating the efficacy of vaccines and antiviral agents designed to combat mers-cov infection. all procedures involving animals were approved by the laboratory animal center, state key laboratory of pathogen and biosecurity, beijing institute of microbiology and epidemiology iacuc's (the permit number is bime 2014-0017). animal studies were carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals. all experimental operations to mice were performed under sodium pentobarbital anesthesia, and mice were euthanized with overdose inhalational carbon dioxide. all efforts were made to minimize suffering of animals. considering the species differences, the hdpp4-coding sequence was codon-optimized and synthesized (genscript, nanjing, china) according to the mrna sequence (accession number nm_001935.3) and cloned into the multiple cloning site (mcs) of a pcaggs plasmid, leading to strong expression of the gene. the recombinant plasmid pcaggs-hdpp4 contained the hcmv ie enhancer (hcmviee), the cag promoter (chicken β-actin promoter/intron), a codon-optimized open reading frame (orf) of hdpp4 and rabbit β-globin polya ( fig 1a) . expression of the codon-optimized hdpp4 was achieved by transfection of the pcaggs-hdpp4 plasmid into cos-7 cells, followed by confirmation of expression using anti-human cd26 monoclonal antibody (r&d systems, minneapolis, mn, usa). to confirm the binding of hdpp4 to the mers-cov receptor-binding domain (rbd), transfected cos-7 cells were incubated with a recombinant protein expressing the rbd of mers-cov fused to human igg fc (rbd-fc) [18] , followed by addition of dylight 549-conjugated goat anti-human igg to detect binding. the purified 5861bp fragment generated from apal1 digestion of pcaggs-hdpp4 was used as the transgene for injection into the pronuclei of fertilized f1 (c57bl/6 × c57bl/6) mouse eggs to generate transgenic embryos. the mice used in this studywere backcrossed two to three times onto a c57bl/6 background. all animal studies were performed in strict accordance with the recommendations outlined in the guide for the care and use of laboratory animals. genomic dna from each transgenic founder line and from wild-type c57bl/6 mice was isolated from the liver tissue using dnazol reagent (qiagen, valencia, ca, usa) according to the manufacturer`s instructions. the hdpp4 dna copy numbers were determined by quantitative pcr using power sybr 1 green pcr master mix (life technologies, carlsbad, ca, usa). total hdpp4 dna was normalized using a single-copy reference gene (pkd1) as an endogenous control. the primers specific for hdpp4 were forward 5' ctacagctcctggg caacgtgctg 3' and reverse 5' agatgtcgtaactggcggtgtaag 3'. the primers specific for mpkd1 were forward 5' ggctgctgagcgtctggta 3' and reverse 5' ccaggtcctgcgtgtctga 3'. to detect the expression of optimized hdpp4 in the tissues of our transgenic mice model, samples were harvested and stored immediately in rnalater rna stabilization reagent (qiagen, valencia, ca, usa) until total rna was extracted and purified using an rneasy extraction kit (qiagen, valencia, ca, usa). for each sample, 2 μg of total rna was used as template for first-strand cdna synthesis. the resulting cdna was subjected to quantitative pcr using power sybr 1 green pcr master mix (life technologies, carlsbad, ca, usa) to determine the relative abundances of hdpp4 in the tissues. the forward and reverse primers for hdpp4 amplicons were as the same as those noted above. the relative amount of hdpp4 in different tissues was obtained by normalizing mrna expression to that of the endogenous control gene gapdh [19] . mers-cov (hcov-emc/2012 strain) was propagated and titered on vero cells in an approved biosafety level 3 laboratory. following intraperitoneal anesthetization with sodium pentobarbital (5 mg/kg of body weight), mice under virus infection were intranasally inoculated with mers-cov (10 4.3 tcid 50 ) in 20 μl dulbecco's modified eagle's medium (dmem), and mice in sham group were treated with the same volume of dmem. mice were monitored for weight loss and survival for 12 days. we have taken special precautions and followed standard guidelines outlined in the guide for the care and use of laboratory animals to ensure that ample food and water as well as sanitary cage conditions with enrichment devices were present in order to maximize the animal's comfort. humane endpoints were used during the survival experiments. after virus infection, mice were closely monitored by daily observation and weighing of trained animal caretaker, and the monitoring will raise to twice per day once mice lost more than 10% of their initial body weight, or exhibited lethargy, ruffled hair coat, or hunched posture. animals were deemed gravely ill and were euthanized by overdose inhalation of carbon dioxide if they lost more than 25% of their weight, or lost ability to ambulate and access food or water. sera were collected on day 5 and inactivated at 56°c for 30 min before the levels of cytokines and chemokines were measured. to assess viral replication and histopathologic damage following mers-cov infection, mice were euthanized with overdose inhalational carbon dioxide, and tissues included lungs, kidneys, livers, spleens, intestines and brains were harvested on indicated time points. the viral rna copies in tissues were determined by qrt-pcr according to the protocol described elsewhere [20] . briefly, total rna was extracted from 20mg of collected tissues using rneasy extraction kits (qiagen, valencia, ca, usa) following the manufacturer's instructions. mers-cov rna was quantified in a 25 μl mixture containing 5 μl rna using the invitrogen superscript™ iii one-step rt-pcr system with platinum 1 taq (life technologies, carlsbad, ca, usa). the primers and probes specific for the upe envelope gene of the mers-cov virus were as follows: forward 5' gcaacgcgcgattcagtt 3'; reverse 5' gcctctac acgggacccata 3'; fluorescence probe /56-fam/ ctcttcacataatcgccccgagctcg cg/36-tamsp/ [21] . mouse gapdh was used as a housekeeping gene control. a standard curve was generated for pcr reaction using 10-10 7 copies of quantified rna transcripts to calculate copy numbers for each reaction. the results were considered positive at c t values below 35 for primer and probe set, and were calculated as viral rna copies per gram of tissues. the tissues of infected mice were harvested aseptically at indicated time points and homogenized in minimal essential medium (mem) plus antibiotics to produce 10% (w/v) suspensions. tissue homogenates were centrifuged and titered on the monolayer of vero cells. the cytopathic effects (cpe) were daily observed under phase-contrast microscropy for 3 days. the viral titer was determined as tcid 50 by cpe-based assay, and calculated using the reed and muench method. the viral titer in tissue was expressed as log 10 tcid 50 /g of tissue. collected tissue samples were immediately fixed in 10% neutral buffered formalin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (h&e) to examine histopathology as described previously [22] . the expression of virus antigens and infiltration of neutrophils and macrophages in organs after mers-cov infection was detected by immunohistochemistry. briefly, formalin-fixed, paraffin-embedded lung sections were deparaffinized and hydrated using graded alcohols. expression of virus antigen and inflammatory cell infiltration was assessed using rabbit polyclonal antibody to mers-cov nucleoprotein (np) (sino biological inc., beijing, china), neutrophil marker nimp-14, (santa cruz biotechnology, paso robles, ca, usa) and cd68 (abcam, cambridge, ma, usa). the reaction was detected using a standard streptavidin-biotin detection system (beijing zhongshan biotechnology co., ltd., beijing, china) and visualized using dab with hematoxylin counterstaining. cytokines and chemokines in mouse sera were quantified using a commercial milliplex mouse cytokine/chemokine magnetic panel kit (merck millipore, usa.). a panel of inflammatory cytokines and chemokines (ifn-γ, ip-10, il-1β, il-17, il-6, tnf-α, gm-csf, kc, mcp-1, mip-1ɑ, mip-1β and rantes) was measured according to the manufacturer's protocols. statistical analyses were performed using graphpad prism version 5.01. to compare the groups in terms of hdpp4 copies, inflammatory cytokine/chemokine levels, and tissue viral rna copies, student's t test with welch's correction was used. the significance between survival curves was analyzed by kaplan-meier survival analysis with log-rank test. p values lower than 0.05 were considered statistically significant. dpp4 is constitutively expressed in human parenchyma cells in tissues including the liver, intestines and kidneys as well as in t and b cells [7] . we developed transgenic mice in which hdpp4 expression was driven by the chicken β-actin promotor in the pcaggs plasmid ( fig 1a) . expression of hdpp4 in vitro was achieved by transfection of the pcaggs-hdpp4 plasmid into cos-7 cells and detected by immunofluorescence analysis. the results showed that hdpp4 was expressed in cos-7 cells and that it effectively bound mers-cov (fig 1b-1e ). after microinjection of linearized dna, founder lines that transferred the hdpp4 gene to their progeny were established. the levels of transgenic dna in the founder lines ranged from 2 to 6 copies per genome, as determined by qpcr (fig 1f) . in addition, the mrna levels of hdpp4 in the trachea, lung, liver, spleen, kidney, intestine and brain in two of the founder lines were measured by qrt-pcr. the data showed that hdpp4 expression was higher in the trachea, lung, spleen, kidney and brain in both lines, and the expressions were relative higher in trachea and kidney in line 4 compared with that in line 2 ( fig 1g) . in order to determine the effective infection of mers-cov in different lines of transgenic mice, 6 mice in each line were infected with mers-cov and the changes of body weight were monitored. the results showed that all mice in line 1, 2 and 3 survived with no significant changes in body weight but all mice in line 4 died by day 10 following mers-cov inoculation (fig 1h) . in order to confirm the virus replications in transgenic mice, three mice of each line were sacrificed on day 5 after mers-cov infection for the detection of viral titer and viral antigen expression in lungs. as expected, compared with high level of lung viral titers in line 4 mice, no viral titer were detected (fig 1i) in lungs of line 1, 2 and 3 mice, which also confirmed by the negative results of viral antigen expression detected by immunohistochemistry in lung (data not shown). wild-type mice, such as balb/c, 129/svev and stat1 mice, are not permissive to mers-cov infection, and syrian hamsters do not support replication of mers-cov [9] [10] [11] [12] . macaques have been confirmed to develop mild to marked interstitial pneumonia upon mers-cov infection [23] , but fail to progress to the extent observed in human subjects. marmoset model, which has the same interaction characteristic between its dpp4 and mers-cov spike protein, was partially lethal and developed a progressive severe pneumonia after mers-cov infection [14] . in this study, we found that hdpp4 transgenic mice infected with mers-cov exhibited decreased activity and significant weight loss from day 6 after infection, and all of the infected mice died by day 10 (fig 2a and 2b) . importantly, symptoms of neural defects with paralysis were observed on day 9 in the infected transgenic mice. compared to the sham-infected group (fig 3a, 3d, 3g and 3j) , the histopathological analysis of the tissues in transgenic mice on days 5 and 9 after mers-cov infection showed the presence of inflammatory tissue damage in the kidney, liver and spleen, with mild inflammatory responses in the lungs but no significant changes in the intestines. on day 5 after inoculation, the hdpp4 transgenic mice exhibited mild inflammation in the lungs with focal exudation and hemorrhage (fig 3b) , and by day 9, the damage was more severe, with evidence of diffused alveolar damage, alveolar septal thickening, hemorrhage and activated macrophage infiltration (fig 3c) . in the kidneys, mild interstitial inflammation with inflammatory cell infiltration was observed in the interstitium and exudates of renal tubules on day 5 (fig 3e) . by day 9, more renal tubular epithelial cells were injured, with evidence of focal hemorrhage in the renal interstitium (fig 3f) . in the liver, mild to moderate liver damage was seen in the transgenic mice infected with mers-cov on day 5, including scattered hepatocyte necrosis and numerous activated kupffer cells and macrophage infiltrates in hepatic sinusoid (fig 3h) , and on day 9, fatty change in hepatocytes with less hepatocyte necrosis was observed (fig 3i) . in spleens, necrosis of splenic cells and increased reticulum cells in red pulp with significant hemosiderin deposition was observed on both day 5 and day 9 (fig 3k and 3l) . typical characteristics of viral encephalitis were observed in the brain at day 9, with perivascular cuffs ( fig 3m) and neuronal cell necrosis in the cerebral cortex (fig 3n) , including hippocampal neurons (fig 3o) . there was no apparent damage in the intestines after mers-cov infection on day 5 or day 9 (data not shown). to evaluate mers-cov infection in the hdpp4 transgenic mice, distribution of the viral antigen np protein was assessed in mouse tissues by immunohistochemical staining. the results showed that type i and type ii pneumocytes in the lungs and renal tubular epithelial cells in the kidneys expressed viral antigen on day 5 after mers-cov infection and that the expression was more abundant on day 9, and no viral antigen was detected in sham-infected group ( fig 4a-4f) . strong viral antigen expression was evident not only in neuronal cell bodies, but also in dendrites, axons and some microglia in the cerebral cortex (fig 4h) including the hippocampus (fig 4i) . no viral antigen expression was observed in the brains of mice in the shaminfected group (fig 4g) . viral rna copies in lung and brain were detectable on day 5 postinfection and increased to a higher level on day 9 (fig 4j) . lower level of viral rna copies can be also detected in kidney collected on day 9. in order to further confirm the effective replication of mers-cov, the dynamic changes of viral titer in organs were detected. the results showed that on day 3 after infection, only lower viral titer was detected in lung and not detectable in other organs. however, on day 5, viral titer was also detected in brain. on day 7 and day 9, increased viral titers were detected in both lung and brain, and lower level viral titer in kidney was detectable (fig 4k) . these results indicate that the transgenic mice had been successfully infected and that mers-cov exhibited cell and tissue tropism especially for pneumocytes and neurons. furthermore, synapses may be one of the structures by which viruses diffuse through the brain after mers-cov infection. to date, only limited data on the immune response of mers patients are available [24] . because of the clinical similarity between the symptoms of mers-cov and those of patients infected with sars-cov [1] , the aberrant immune response may be related to the pathogenesis of mers-cov infection. in our mouse model, an aberrant inflammatory response was confirmed by infiltration of neutrophils in the lung, liver and spleen (fig 5a-5f ) and macrophages in the lung, liver and kidney (fig 5g-5l ). in addition, deposition of hemosiderin in the spleen (fig 3k and 3l ) indicated an increase in activated macrophages, and a systemic inflammatory response after virus infection was demonstrated by a significant increase in cytokines such as ifn-γ, ip-10, and il-17 in the serum on day 5 after mers-cov infection (fig 5m) . although several species, such as rhesus macaques and common marmosets are reported to be sensitive to mers-cov infection and to develop mild to marked broncho-interstitial pneumonia, small animal models are urgently needed to study the pathogenesis of this disease and evaluate the effects of vaccines and antiviral agents. a mers-cov-infection mouse model was generated by transducing mice with a recombinant non-replicating adenovirus expressing hdpp4; however, while the mice exhibit viral antigen expression and progress to interstitial pneumonia, there is no mortality after virus infection [15] . although a transgenic mouse model expressing human dpp4 was also established, and its immune response was studied after infection with mers-cov [16] , the transgenic mice in the study died on day 6 with only progressive pneumonia and mild perivascular cuffing in brain, and no neurological disorder or other multi-organ damage was observed. different from the aforementioned transgenic mice, our hdpp4 transgenic mice experienced a longer incubation period post-infection and developed progressive pneumonia and neurological disorders accompanied by histological damage to the lungs, brain, spleen and liver, which more closely resembles the clinical cases. the results from this study indicate that mers-cov infection was lethal in hdpp4 transgenic mice with higher transgene copy number, while lines with lower levels of hdpp4 expressed especially in trachea had no significant clinical symptoms (data not shown), suggesting that the copy number of the transgene was closely related to the efficiency of mers-cov infection. although the expression level of optimized hdpp4 was not highest in the brains, the tissues had higher viral titers following mers-cov infection, revealing the tropism of mers-cov for both lungs and brains, tissues in which pneumocytes and neurons may be the main target cells (fig 4) . however, mers-cov infection in brain via blood or olfactory nerves needs to be further studied due to the strong expression of viral antigens in dendrites and axons. although few autopsy reports exist on fatal mers-cov cases and atypical pneumonia and respiratory failure are suspected the causes of death [24] , other types of extrapulmonary organ dysfunction have also been documented in mers critically ill patients with abnormal clinical manifestations [25] [26] [27] . for example, acute renal failure was described in a number of mers cases [28] [29] [30] [31] [32] . in addition, renal epithelial cells may produce almost 1000-fold more infectious mers-cov progeny than bronchial epithelial cells [33] . more recently, severe neurologic syndrome including altered level of consciousness from confusion to coma, ataxia and focal motor deficit was also reported in three critically cases by balkhy h et al [4] . according to the neurologic manifestations and distinct imaging patterns, an important question was raised on the pathogenic mechanisms that underlie the occurrence of neurologic injury in patients. it has been studied that corona virus such as sars-cov are generally known for causing respiratory illness, and the strong tropism of sars-cov to cns and causing neuronal injury had also been demonstrated both in clinical and experimental studies [34] [35] [36] [37] [38] , which suggested the possible relations of mers-cov infection with brain injury. as to the extensive expression of hdpp4 in the lung, kidney, placenta, liver, skeletal muscle, brain, endothelium, pancreasour and t cell in human, our globally expressed hdpp4 transgenic mouse manifested with multiorgan damage infected with mers-cov could be a suitable model for the pathogenic mechanisms study. systemic inflammation is believed to be a primary reason for the severe outcome in mers--cov infections [14, 24] . although the transgenic mice model died after mers-cov infection with multi-organ damages, it is still uncertain whether these mice are mainly dying from lung damage. in our transgenic mice, aberrant immune response such as elevated ip-10, ifn-γ and il-17, which are closely related to acute virally-mediated lung injury [39] [40] [41] [42] , was also a specific manifestation after mers-cov infection. so, as to the mechanism of lethal which was due to the virus replication directly or induced by the aberrant inflammatory response need to be further studied. the mechanism of mers-cov infection caused death is complex and complicated. aberrant immune response after mers-cov infection may be one of main reasons which need to be further studied. we have added more discussions on this issue in the revised manuscript. in summary, the transgenic mouse model developed in this study was efficiently infected by mers-cov and exhibited severe acute respiratory injury and considerable extrapulmonary organ damage. this model will be useful for studying the pathogenesis of mers-cov infection and evaluating the efficacy of mers vaccines and therapeutic agents. molecular pathology of emerging coronavirus infections state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans hospital outbreak of middle east respiratory syndrome coronavirus severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the multifunctional or moonlighting protein cd26/dppiv middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction inhibition of complement activation alleviates acute lung injury induced by highly pathogenic avian influenza h5n1 virus infection treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques pathogenesis of middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia family cluster of middle east respiratory syndrome coronavirus infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection latest outbreak news from promed-mail: novel coronavirus-middle east clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection possible central nervous system infection by sars coronavirus detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis multiple organ infection and the pathogenesis of sars pathology and pathogenesis of severe acute respiratory syndrome susceptibility of human and rat neural cell lines to infection by sars-coronavirus early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus severe acute respiratory syndrome in children interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome therapy of h7n9 pneumonia: current perspectives key: cord-327867-1wkbjtji authors: da'ar, omar b.; ahmed, anwar e. title: underlying trend, seasonality, prediction, forecasting and the contribution of risk factors: an analysis of globally reported cases of middle east respiratory syndrome coronavirus date: 2018-06-11 journal: epidemiol infect doi: 10.1017/s0950268818001541 sha: doc_id: 327867 cord_uid: 1wkbjtji this study set out to identify and analyse trends and seasonal variations of monthly global reported cases of the middle east respiratory syndrome coronavirus (mers-cov). it also made a prediction based on the reported and extrapolated into the future by forecasting the trend. finally, the study assessed contributions of various risk factors in the reported cases. the motivation for this study is that mers-cov remains among the list of blueprint priority and potential pandemic diseases globally. yet, there is a paucity of empirical literature examining trends and seasonality as the available evidence is generally descriptive and anecdotal. the study is a time series analysis using monthly global reported cases of mers-cov by the world health organisation between january 2015 and january 2018. we decomposed the series into seasonal, irregular and trend components and identified patterns, smoothened series, generated predictions and employed forecasting techniques based on linear regression. we assessed contributions of various risk factors in mers-cov cases over time. successive months of the mers-cov cases suggest a significant decreasing trend (p = 0.026 for monthly series and p = 0.047 for quarterly series). the mers-cov cases are forecast to wane by end 2018. seasonality component of the cases oscillated below or above the baseline (the centred moving average), but no association with the series over time was noted. the results revealed contributions of risk factors such as camel contact, male, old age and being from saudi arabia and middle east regions to the overall reported cases of mers-cov. the trend component and several risk factors for global mers-cov cases, including camel contact, male, age and geography/region significantly affected the series. our statistical models appear to suggest significant predictive capacity and the findings may well inform healthcare practitioners and policymakers about the underlying dynamics that produced the globally reported mers-cov cases. this study set out to identify trends and seasonal variations; made a prediction based on the globally reported cases of the middle east respiratory syndrome coronavirus (mers-cov), extrapolated into the future by forecasting the trend and assessed contributions of various risk factors for the mers-cov cases. specifically, we consider the questions: (1) what is the underlying growth or trend of the globally reported mers-cov cases? (2) are there seasonal variations present in globally reported mers-cov cases over time and how do they affect the series? (3) what are the contributions of various risk factors for mers-cov cases? the motivation for this study is that to date, the world health organisation (who) places the mers-cov among the list of blueprint priority diseases. although a survey of the literature shows a rapid increase in research activities related to mers-cov [1] , yet there is still a general paucity of empirical literature examining trends and seasonality. to the best of our knowledge, there is a single study, which anecdotally examined seasonality and time series patterns of mers-cov to date [2] . given mers-cov remains a potential pandemic disease globally, it is important to understand the dynamics of the underlying growth or trend of the globally reported cases. the motivation for this study also comes from the aetiology of mers-cov, especially its causes, spread, the complexity of its diagnosis and mortality. mers-cov is a virus that causes severe viral pneumonia in humans, known to have a high mortality rate [3] [4] [5] [6] [7] and has clinical symptoms similar to severe acute respiratory syndrome coronavirus [8, 9] . it was first reported in saudi arabia [10] and after that, the virus exhibited outbreaks in several regions of the world, particularly saudi arabia and the republic of korea [10, 11] . additionally, the complexity of mers-cov and its diagnosis of infection have been acknowledged in the literature [12, 13] . according to who, 2160 laboratory-confirmed cases of mers-cov were reported at the end of january 2018, including 773 associated deaths (case-fatality rate: 35.8%) that were reported globally [14] . the majority of these cases were reported in saudi arabia. this is a time series analysis using publicly reported mers-cov monthly global cases. the who receives confirmed mers-cov cases from countries across the world. to date, new cases continue to be identified and reported to the who, specifically from the middle east region. these data are available at http:// www.who.int/csr/don/archive/disease/coronavirus_infections/en/. the latest report included one case from malaysia on 8 january 2018. we used time series of mers-cov cases reported between january 2015 and january 2018, where who began using standard case report. a research assistant retrieved data from who webpage and reviewed for quality by the second study author. the data retrieved include patient and clinical data such as age, gender, healthcare worker, comorbidity, the source of infection and geographical regions. the main outcome was the number of cases reported on a monthly basis from january 2015 to january 2018. the analysis was performed using stata 12 (stata corp., texas, usa) and microsoft excel 10. using the classical multiplicative time series model, we decomposed the original series into seasonal, irregular and trend components and examined their effects. additionally, we identified patterns, smoothened series, generated predictions and employed forecasting techniques based on linear regression. finally, we assessed contributions of various risk factors in mers-cov cases over time. p-values <0.05 were considered statistically significant. figure 1 shows that although cases of mers-cov are decreasing in the range period selected, the series exhibits peaks and spikes. figure 1 also reveals a negative trend of mers-cov series from january 2015 to january 2018. we investigated direction and significance of this trend, as well as stationarity of the series. while a unit root test for nonstationarity confirmed the mers-cov cases had a negative and statistically significant trend, the series was found to be stationary. the negative and statistically significant trend was also confirmed by subsequent regressions. we collapsed the monthly series into quarters and then smoothened out the series using a centred moving average in order to understand underlying growth component. we assumed the classical multiplicative time series model by decomposing original series into seasonal, irregular, and trend components ( table 1) our decomposition of the mers-cov cases shows that in 2016q2, the seasonality and irregularity components of the series were 8% below the baseline (the centred moving average). decomposing further, seasonality component was 11% above the baseline in 2016q2, while it was 41% below the baseline in 2016q4. our analysis also shows the de-seasonalised series of the original mers-cov by removing seasonality and irregularity components. using a linear regression, we then estimated the effect of time on the deseasonalised series to capture the underlying growth or trend component using a linear regression in order to make predictions. since the last available data was in january 2018, we also made a forecast of three more quarters (2018q2, q3, q4), an additional 9 months into the future. the forecast of the series revealed that mers-cov cases would approach zero by end of 2018 or beginning of 2019, making further extrapolation into the horizon infeasible. figure 2 shows decomposition of mers-cov series of the original series, centred moving average (smoothed series), forecast omar b. da'ar and anwar e. ahmed and linear forecast. together, after accounting for irregular and trend components of the series, seasonality was found to range between 41% below the baseline i.e. the centred moving average (of order 4) in some quarters and 11% above the baseline in other quarters. the average seasonality component was found to be 14% below the baseline. however, regression estimation revealed that, unlike the trend component, seasonality was not statistically significant, a fact also backed by our statistical test, which showed the monthly global mers-cov cases series were stationary. regressions table 2 shows results of the effect of trend and seasonal on mers-cov cases. we compared the seasonal dummy variables, interpreted by comparing them with quarter 4 (q4) (base season) while holding time constant. time, in this analysis, was represented by successive months and was interpreted as the effect of the linear trend on mers-cov cases over time, holding the effect of the seasons constant. the regression results revealed that after accounting for the trend, mers-cov cases quarter 1 (q1) each year averaged about 14 cases more than q4 cases, although the effect was not found to be statistically significant. similarly, after adjusting for the trend, cases in quarter 2 (q2) averaged around 16 cases more than q4 cases. quarter 3 (q3) cases averaged 19 cases more than q4 cases after accounting for the trend component time. it is important to note that the effects of seasonality were not statistically significant. however, what was revealed statistically significant is the negative trend. consistent with the negative trend shown in figure 1 , the regression results revealed that each additional quarter registered approximately an average decrease of one case, after adjusting for the season. in other words, the mers-cov cases decreased, on average, by four (4 × −0.8667627) per quarter year to year. the model as a whole appears to suggest statistically significant predictive capacity. we analysed fluctuations of reported mers-cov cases from period to another by graphing the residuals (generated from the regression of trend and seasonality on mers-cov cases) against time, as is the convention with time series analysis. the results indicated no clear patterns, suggesting that correlated errors are not a problem with this model. other diagnostic tests also revealed neither violation of the classical linear assumptions nor correlation between the reported mers-cov cases in each month with cases reported in earlier months. we further examined the effects of various risk factors of mers-cov cases such as camel contact, healthcare worker contact, exposure, gender and region. table 3 shows regression that adjusts for these factors. the results reveal camel contact, saudi table 3 ). the model in this estimation also appears to suggest statistically significant predictive capacity. using linear time series models and their application to the modelling and prediction of the globally reported mers-cov data, the present study identified trends, analysed seasonality, predicted and forecast evolution of mers-cov cases and assessed the contribution of various risk factors. the decomposition of the time series of mers-cov cases into trend and seasonality components and making predictions have not hitherto been studied in the context of mers-cov pandemic. in this study, we set out to understand the dynamics of its growth over time. the results of our time series analysis of globally reported mers-cov cases suggest a significant negative trend that is forecast to be eradicated in the near future unless something unexpected happens. our study showed that although seasonality oscillated below or above the baseline i.e. the centred moving average (of order 4) over time, the average seasonality component was found to be 14% below the baseline. even then, our analysis showed that, unlike the trend component, seasonality did not affect the series over time. many risk factors are associated with mers-cov cases, mortalities, or complications. our results indicate those aged under 30 years (reference category) are less likely to be a mers-cov case than those aged over 30, consistent with several studies that associated mers-cov with elderly patients [15, 16] . surprisingly, comorbidity did not show a statistically significant contribution to mers-cov cases. however, there are studies that showed mers-cov cases were associated with patients with comorbidities [17] [18] [19] . a recent systematic study, for example, suggests the prevalence of comorbidities of mers-cov cases, diabetes, hypertension and cardiac diseases [20] . while our analysis suggests males contribute to the global reported mers-cov cases, gender was reported to have a mixed effect on mers-cov mortalities in the literature. some studies showed men as high risk [7, 18] and mers-cov infects more males than females [5, [21] [22] [23] . other literature indicated that the frequency of deaths was less in men [24] . the literature showed that mers-cov can be spread via human-human [25] [26] [27] [28] [29] , or healthcare facilities [23, [30] [31] [32] . other studies revealed animal to human [33, 34] as the primary culprit of mers-cov virus transmission. specifically, the literature showed camels act as a direct source of human mers-cov infection [31, 35] , while healthcare workers were reported to be at higher risk [7, 16, 19, 24] . the results of our study in this regard were mixed. while our study indicated that the effect of camel cases on overall mers-cov reported cases are positive and significant, the contribution of healthcare workers was not. our analysis also showed evidence of geographical contributions to mers-cov cases such as saudi arabia and greater middle east compared with south korea. this can be seen as somewhat consistent with earlier studies that demonstrated a link between mortality associated with mers-cov and geography [15] . this finding is also intuitive in that saudi arabia and middle east, in general, remain the global epicentre of mers-cov, hence the name. the contribution of our study is that it adduces empirical evidence by making inferences and predictions based on the globally reported cases of mers-cov and extrapolated into the future by forecasting the trend. unlike previous studies that descriptively analysed seasonality patterns of mers-cov and influenza in the middle east [2] , our study presents statistically significant results of trends of global mers-cov cases, consistent with regularities underlying the empirical dynamics and classical time series analysis. however, there are limitations of this study. first, the data used for this study comprised 37 months (january 2015 to january 2018). while this was just enough for several years' worth of monthly observations to appropriately model seasonality, time series analysis can be sensitive to the number of observations. hence, sufficiently large number of observations might have provided a better fit and results. additionally, the analysis utilised who open source globally reported data, which may lack harmonisation from the various country sources. this study contributes to the time series analysis of mers-cov literature. in particular, our analysis of trends and seasonality components the series, the prediction based on the globally reported cases of mers-cov and extrapolation into the future by forecasting the trend is envisaged to help in understanding the dynamics of the reported cases over time. the study findings suggest a significant negative trend of the monthly and quarterly data from 2015 to 2018. however, a further extrapolation into the future reveals that the mers-cov cases are forecast to be zero by end 2018 or beginning of 2019 unless something unexpected happens. seasonality component of the series oscillated below or above the baseline, i.e. the centred moving average but did not affect the series over time. the results demonstrated that camel contact, exposure, gender, age and geography/region significantly contributed to the overall global reported mers-cov cases. the findings may well inform healthcare practitioners and policymakers about the underlying dynamics that produced the globally reported mers-cov cases. data. these dataset used and/or analysed are available at http://www.who.int/ csr/don/archive/disease/coronavirus_infections/en/. global research trends of middle east respiratory syndrome coronavirus: a bibliometric analysis differences in the seasonality of mers-cov and influenza in the middle east the predictors of 3-and 30-day mortality in 660 mers-cov patients development of a risk-prediction model for middle east respiratory syndrome coronavirus infection in dialysis patients epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data severe acute respiratory syndrome vs. the middle east respiratory syndrome viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov). fact sheet middle east respiratory syndrome corona virus alert verification in mers-cov diagnosis: an update comparative evaluation of three homogenization methods for isolating middle east respiratory syndrome coronavirus nucleic acids from sputum samples for real-time reverse transcription pcr epidemic and pandemic-prone diseases. mers situation update estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study building predictive models for mers-cov infections using data mining techniques clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia risks of death and severe disease in patients with middle east respiratory syndrome coronavirus prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hospital outbreak of middle east respiratory syndrome coronavirus treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia evidence of person-to-person transmission within a family cluster of novel coronavirus infections the epidemiology of middle east respiratory syndrome coronavirus in the kingdom of saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections mers-cov outbreak in jeddah-a link to health care facilities human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection early identification of pneumonia patients at increased risk of middle east respiratory syndrome coronavirus infection in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia acknowledgements. the authors are grateful for the collegiality and research support at the college of public health and health informatics, king saud bin abdulaziz university for health sciences. the information and opinions contained in this work do not necessarily reflect the views or policy of these institutions. this research is supported by king abdullah international medical research centre (kaimrc), king saud bin abdulaziz university for health sciences, national guard health affairs, riyadh, saudi arabia.author contributions. obd analysed the data and wrote manuscript. aea retrieved the data from the who /registry website. aea reviewed analysis and manuscript. all authors approved final manuscript for submission. ethical standards. not applicable. key: cord-328175-4i3cz20j authors: van doremalen, neeltje; falzarano, darryl; ying, tianlei; de wit, emmie; bushmaker, trenton; feldmann, friederike; okumura, atsushi; wang, yanping; scott, dana p.; hanley, patrick w.; feldmann, heinz; dimitrov, dimiter s.; munster, vincent j. title: efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets date: 2017-07-31 journal: antiviral research doi: 10.1016/j.antiviral.2017.03.025 sha: doc_id: 328175 cord_uid: 4i3cz20j abstract cases of middle east respiratory syndrome coronavirus (mers-cov) continue to be identified and with a lack of effective clinical treatment and no preventative strategies, treatment using convalescent plasma or monoclonal antibodies (mabs) is a potential quick route to an intervention. passive immunotherapy via either convalescent plasma or mabs has proven to be effective for other infectious agents. following infection with mers-cov, common marmosets were treated with high titer hyperimmune plasma or the mab m336, at 6 and 48 h post inoculation. both treatments reduced signs of clinical disease, but reduction in viral loads in the respiratory tract were only found in the hyperimmune plasma group. a decrease in gross pathology was found only in the mab-treated group, but no histological differences were observed between treated and control animals. while both hyperimmune plasma and the m336 treatments reduced the severity of disease in the common marmoset, neither treatment resulted in full protection against disease. middle east respiratory syndrome coronavirus (mers-cov) was first detected in 2012 in a resident of saudi arabia, and has since resulted in > 1900 cases with a case fatality rate of 36% (who, 2015) . the severity and the epidemic potential of mers-cov highlights the importance of the development of treatment options. as of yet, no specific vaccine or antiviral treatment against mers-cov is available. few studies have been published investigating the effectiveness of existing antiviral treatments, and no treatments have been thoroughly assessed in clinical trials as of yet. convalescent plasma has been identified by the world health organization (who) and the international severe acute respiratory and emerging infection consortium (isaric) as a potential treatment against mers-cov to reduce clinical consequences of mers-cov infection (2013 , who, 2014 and recently a study protocol was developed to investigate the feasibility of convalescent plasma treatment in mers patients (arabi et al., 2015) . in vivo, the administration of convalescent sera obtained from dromedary camels resulted in dose-dependent decreased lung viral titers and disease severity in an adenovirus-hdpp4 mouse model . several monoclonal antibodies (mabs) have been developed against mers-cov, which show neutralizing capacity in vitro (jiang et al., 2014; tang et al., 2014; ying et al., 2014) . efficacy of mabs has been assessed in several mers-cov mouse models generally showing reduction in virus replication (corti et al., 2015; li et al., 2015; pascal et al., 2015; luke et al., 2016) . these studies suggest that mabs have potential as mers-cov treatment. the mab m336, identified from a large phage-displayed antibody library panned against recombinant mers-cov spike protein receptor binding domain, inhibited 90% mers-cov pseudovirus infection at a concentration of 0.039 mg/ml (ying et al., 2014) . m336 was shown to almost completely overlap with the binding site of dpp4 and mimic critical interactions between dpp4 and the mers-cov spike protein (modjarrad et al., 2016) . it has therefore been speculated that the potential for viral escape mutants might be limited by the requirement of the spike protein to bind to dpp4 (ying et al., 2015) . prophylactic treatment with m336 resulted in significantly reduced viral titer in rabbit lung tissue (houser et al., 2016) and both prophylactic and therapeutic treatment with m336 protected mice against lethality by mers-cov infection (agrawal et al., 2016) . here we assess the effect of treatment with marmoset-derived hyperimmune plasma as well as the human mab m336 on disease outcome in the recently developed marmoset mers-cov infection model, which recapitulates severe respiratory disease (falzarano et al., 2014) . approval of animal experiments was obtained from the institutional animal care and use committee at rocky mountain laboratories. all experiments were performed in an association for assessment and accreditation of laboratory animal care-approved facility by certified staff, following the guidelines and basic principles in the united states public health service policy on humane care and use of laboratory animals, the nih guide for the care and use of laboratory animals and the animal welfare act, united states department of agriculture. the institutional biosafety committee (ibc) approved work with infectious mers-cov strains under bsl3 conditions. sample inactivation was performed according to ibc-approved standard operating procedures for removal of specimens from high containment. hyperimmune plasma was obtained from a convalescent common marmoset (callithrix jacchus) from a previous experiment (falzarano et al., 2014) inoculated with 5.2 â 10 6 tcid 50 mers-cov via the intratracheal, intranasal, ocular and oral route, then inoculated with 5.2 â 10 6 tcid 50 mers-cov on 20 dpi via the intratracheal route and finally inoculated with 5.2 â 10 6 tcid 50 mers-cov adjuvated with titermax gold (sigma aldrich) on 41 dpi via the intramuscular route. the final virus neutralizing (vn) titer was 3840. this method was chosen as sera collected after the initial infection did not contain sufficient neutralizing antibodies (vn titer ¼ 40). as a control, plasma was obtained from an uninfected common marmoset (internal collection), vn titer <20. the common marmoset mers-cov infection model was used; mers-cov infection results in the development of more severe respiratory disease than observed in the rhesus macaque model (de wit et al., 2013; munster et al., 2013; falzarano et al., 2014) . common marmosets were procured from an usda-approved source (worldwide primates inc). animals were monitored for the presence of disease by clinical observation and serology for the presence of disease at worldwide primates inc. additionally, when animals arrived at rocky mountain laboratories they were placed in quarantine and clinically evaluated by serum chemistry, complete blood counts and thoracic radiography to confirm absence of previous infection. three different groups were created; the hyperimmune plasma group (h), the monoclonal antibody group (m), and the control group (c). three animals were randomly assigned per group and inoculated as described previously (falzarano et al., 2014) . briefly, inoculation with mers-cov strain emc/2012 was performed intranasally (100 ml per nare), orally (500 ml), intratracheally (500 ml) and ocular (50 ml per eye) with dmem containing 4 â 10 6 tcid 50 mers-cov/ml (total dose 5.2 â 10 6 tcid 50 ). the hyperimmune plasma and monoclonal antibody groups, consisting of one female and two male common marmosets each, received 1 ml hyperimmune plasma or m336 diluted in pbs (5 mg/ml) intravenous (i.v.) at 6 hpi, and 1 ml hyperimmune plasma or m336 subcutaneous (s.c.) at 2 dpi (marmosets h1-3, m1-3). two out of three animals in the control group (all male common marmosets), received 1 ml control plasma i.v. 6 hpi, and 1 ml control plasma s.c. 2 dpi (marmosets c1-2). the third animal received 1 ml of pbs (diluent of the mab) via the same routes (marmoset c3). the animals were observed twice daily for clinical signs of disease, using a scoring system as described previously (falzarano et al., 2014) . breathing was scored as normal (<60/minute), increased (60e100/ minute), or severely increased (>100/minute). based on the scoring sheet, euthanasia was indicated at a clinical score of 35 or more. clinical exams were performed on 0, 2, 5, and 7 dpi on anaesthetized animals using isoflurane and ketamine. x-rays were taken and nasal, oral, fecal, and urogenital swabs were collected in 1 ml dmem with 50 u/ml penicillin and 50 mg/ml streptomycin. blood samples were collected on à5, 2, and 7 dpi and examined using the piccolo xpress chemistry analyzer (abaxis). the blood sample collected on 2 dpi was obtained before treatment was administered. temperature was monitored with iptt-300 temperature probes (bmds) that were injected interscapularly prior to the start of the experiment. all animals were euthanized at 7 dpi (fig. 1a) . terminal blood samples were obtained and samples of the following tissues were collected: conjunctiva, nasal mucosa, tonsil, trachea, four lung lobes, mediastinal lymph node, liver, spleen, kidney, and bladder. gross pathology (surface area of the lung which was either consolidated and/or hyperemic) per lung lobe was documented as percentage area affected by lesions. radiographic images acquired included ventrodorsal, right lateral and left lateral thoracic images. thoracic radiographs were obtained using a mobile digital radiography unit with a flat panel digital detector (sound technologies tru/dr, sound-eklin carlsbad, ca). each set of radiographs was graded according to a published scoring paradigm (brining et al., 2010) as follows: 0, normal examination; 1, mild interstitial pulmonary infiltrates; 2, moderate interstitial infiltrates, perhaps with partial cardiac border effacement and small areas of pulmonary consolidation (alveolar patterns and air bronchograms); 3, pulmonary consolidation as the primary lung pathology, seen as a progression from grade 2 lung pathology. grading per animal was done independently and blinded by two veterinarians. hcov-emc/2012 was provided by the erasmus medical center, rotterdam, the netherlands. virus propagation was performed in veroe6 cells (provided by the bowen laboratory, colorado state university) in dmem supplemented with 2% fetal calf serum, 1 mm l-glutamine, 50 u/ml penicillin and 50 mg/ml streptomycin (2% dmem). veroe6 cells were maintained in dmem supplemented with 10% fetal calf serum, 1 mm l glutamine, 50 u/ml penicillin and 50 mg/ml streptomycin. marmoset tissues were evaluated for pathology and the presence of viral antigen. all tissues were fixed for a minimum of 7 days in 10% neutral-buffered formalin and subsequently embedded in paraffin. lungs were perfused with 10% formalin and processed for histologic review. the lung is divided into right upper, right lower, left upper, and left lower lobe. each of these four sections are then sampled at the hilus, at mid-lobe and at the periphery of the lobe for a minimum of 12 sections per animal. this method is used for all non-human primate studies at rocky mountain laboratories. hereafter, tissue sections were stained with hematoxylin and eosin. for the detection of viral antigen immunohistochemistry was performed using an in-house produced rabbit polyclonal antiserum against hcov-emc/2012 (1:1000). grading was done blinded by a board-certified veterinary pathologist. to obtain morphometrical data of immunohistochemistry staining, stained sections were scanned with an aperio scanscope xt (aperio technologies, inc., vista, ca) and analyzed using the imagescope positive pixel count algorithm (version 9.1). between 30 and 105 mm squared were evaluated at 2â magnification. the default parameters of the positive pixel count (hue of 0.1 and width of 0.5) detected antigen adequately. tissue for rna analysis was collected in triplicate. lung tissue was obtained from the hilar and mid-lobe region of the lung. samples were analyzed independently in duplicate. tissues (30 mg) were homogenized in rlt buffer and rna was extracted using the rneasy method (qiagen) according to the manufacturer's instructions. rna was extracted from swabs using the qiaamp viral rna kit on the qiaxtractor. the upe mers-cov detection assay was used for the detection of mers-cov viral rna (corman et al., 2012) . 5 ml rna was tested with the rotor-genetm probe kit (qiagen) according to instructions of the manufacturer. dilutions of mers-cov with known titer were run in parallel. dilutions of in vitro transcribed upe mers-cov rna were run on the digital droplet pcr (biorad) in quadruplicate to determine genome copies. hereafter, dilutions were run on the rotor-genetm in quadruplicate. the last dilution to give a ct-value in all replicates was defined as the limit of detection (lod) in genome copies. finally, the number of genome copies was determined in the mers-cov dilutions with known titer, and lod was calculated in tcid 50 equivalent. small tissue samples (up to 100 mg) in 1 ml of 2% dmem were homogenized. hereafter, mers-cov was titrated in quadruplicate in veroe6 cells; cells were inoculated with ten-fold serial dilutions of tissue homogenate, incubated 1 h at 37 c, washed twice with pbs, and scored for cytopathic effect 5 days later. tcid 50 was adjusted for tissue weight and calculated by the method of spearman-karber. two-fold serial dilutions of heat-inactivated (30 min, 56 c) marmoset sera were prepared in 2% dmem, after which 100 tcid 50 of mers-cov virus was added. after 1hr incubation at 37 c, virus was added to veroe6 cells. at 5 dpi, cytopathic effect was scored. the virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum, which still inhibited mers-cov virus replication. student's t-test (unpaired, one-tailed) was used to test for significance. p values of <0.05 were considered significant. all values are reported as mean ± sd. the final vn titer of hyperimmune plasma was 3840, control plasma was <20 and m336 was 491530. animals were inoculated with 5.2 â 10 6 tcid 50 mers-cov strain emc/2012 and treated iv 6 hpi and sc 48 hpi. these two different administration routes were chosen to achieve high systemic bioavailability; i.v. administration immediately results in high circulating neutralizing antibody titers whereas s.c. administration results in a slower systemic distribution of antibodies, therefore allowing a longer bioavailability (wohlrab, 2015) . animals treated with hyperimmune plasma reached a neutralizing titer between 40 and 160 at 2 dpi and maintained these levels throughout the experiment. animals treated with m336 reached neutralizing titers between 10,240 and 20,480 at 2 dpi, which decreased to 3840e7680 at 7 dpi. in contrast, serum obtained from animals treated with control plasma or pbs did not contain detectable levels of neutralizing antibodies at any time point (fig. 1b) . upon inoculation with mers-cov strain emc/2012, all animals developed clinical disease. clinical scores of control group animals were found to be higher than clinical scores of treated animals post inoculation ( fig. 2a) . no changes in body temperature were observed. all animals showed loss of appetite and decreased activity, often seen combined with a hunched posture. no changes in respiratory rate were reported for treated animals m1 and m2. increased respiration rate (>60 breaths/minute) was observed in all other animals, and progressed to labored breathing (>100 breaths/ minute) in all three control animals (c1-c3) and one treated animal (h1). this was accompanied by open mouth breathing in all control animals, but not h1. all radiographs were independently and blindly graded by clinical veterinarians (brining et al., 2010) ) and in all views were normal (score ¼ 0) prior to inoculation on day 0. within the control group, all three animals were graded at 2 at 5 dpi with severe interstitial infiltrates seen early that progressed to pulmonary consolidation within the caudal and middle lung lobes on 7 dpi (score ¼ 3). both the hyperimmune plasma treatment and the mab treatment group on average had lower graded radiographs in comparison to the control group. on 7 dpi, one hyperimmune plasma-treated animal was characterized as having grade 1 (h2, mild interstitial pulmonary infiltrates in the left caudal lung lobes), while the remaining two hyperimmune plasma-treated animals had either moderate (h1, grade 2) or severe (h3, grade 3) radiographic signs. one mab-treated animal had normal radiographic findings (m2, grade 0) throughout the duration of the study, whereas the other two animals (m1, m3) were scored as presenting with mild radiographic findings (grade 1) ( fig. 2b and c and fig. s1 ). blood chemistry values were investigated using the piccolo xpress chemistry analyzer on à5, 2, and 7 days post inoculation. overall, no apparent differences in measured clinical chemistries were noted between the control and the treated groups. the values of the investigated parameters (bun, creatinine, alt, ast, alp, ggt, total protein, glucose and calcium) were found to be within the normal ranges for marmosets, and no consistent patterns or trends were noted between groups (fig. s2 ). gross pathology of the lungs was scored by a board-certified veterinary pathologist for each lung lobe, both dorsal and ventral. for the control animals, the median percent lesions was 32.5% (c3), 55% (c1) and 67.5% (c2). no abnormal pathological findings were observed in one of the hyperimmune plasma-treated animals (h2), whereas the median of gross pathology of the lungs in the other two treated animals was 25% (h1) and 77.5% (h3). animals treated with m336 showed relatively little gross pathology (median ¼ 0% (m1), 2.5% (m2) and 22.5% (m3)). this difference between control and treated animals was found to be significantly different using an unpaired one-tail student's t-test (hyperimmune plasma-treated p ¼ 0.0352; mab-treated p ¼ 0.0007) (figs. 2c and 3a) . as a measure of pulmonary edema and inflammation, lung to body weight ratios were calculated for each animals. no significant differences were observed between the control and hyperimmune plasmatreated group, however the mab-treated group had a significantly lower lung to body weight ratio than the control group (p ¼ 0.0039) (fig. 3b) . no histological differences were observed in the severity or nature of the pneumonia or distribution of viral antigen between the control and treated animals. all marmosets, with the exception of one treated marmoset (h2) which developed no lung pathology, developed multifocal to coalescing, moderate to marked subacute bronchointerstitial pneumonia with type ii pneumocyte hyperplasia. the adjacent alveolar interstitium was thickened by congestion, edema and fibrin and moderate numbers of macrophages and neutrophils. alveolar spaces contained moderate to marked numbers of pulmonary macrophages and neutrophils. immunohistochemistry demonstrated small numbers of pneumocytes and macrophages positive for viral antigen. these cells were predominantly associated with areas of pneumonia (fig. 3c ). no differences in the amount of viral antigen between groups could be found using morphometrical analysis. on 0, 2, 5, and 7 dpi nasal, oropharyngeal, urogenital and fecal swabs were collected and the presence of viral rna was examined via quantitative reverse transcription polymerase chain reaction (qrt-pcr). viral rna presence was often below detection limit, except for two and one nasal swabs for hyperimmune plasmatreated animals and control animals, respectively, and one oral and fecal swab for one control animal on 7 dpi. in all instances, urogenital swabs were negative (fig. s3) . upon necropsy of the animals on 7 dpi, 13 tissue samples were collected and the presence of viral rna in these tissues was analyzed using qrt-pcr. as observed in previous infection of marmosets (falzarano et al., 2014) , the highest viral loads were found in the lower respiratory tract of the animals (fig. 4a ). viral loads in lung tissues from hyperimmune plasma-treated compared to control animals were found to be significantly lower using a onetailed unpaired student's t-test (average of 4.0 â 10 4 and 1.2 â 10 6 tcid 50 equivalent/gram, respectively, p-value ¼ 0.008). this difference was found to be significant even when negative values from animal h2 were excluded from analysis (average of 5.9 â 10 4 tcid 50 equivalent/gram, p-value ¼ 0.0263). however, treatment of marmosets with m336 did not significantly reduce viral load in lung tissue (average of 5.4 â 10 5 and 1.2 â 10 6 tcid 50 equivalent/gram, respectively, p-value ¼ 0.0864) (fig. 4b) . viral rna was found in the nasal turbinates, trachea, conjunctiva, tonsils and mediastinal lymph nodes of some but not all animals with no pattern related to treatment. viral rna in the liver, spleen, kidney and bladder was below the limit of detection (fig. 4c) . no infectious virus could be found in any tissue samples. currently no specific antivirals or vaccines are approved for mers-cov treatment, and limited information is available on the efficacy of potential treatment options in vivo. a potential ventral-dorsal and lateral thoracic radiographs as well as gross pathology images of marmosets taken 7 dpi. shown are animals h3, m1 and c1. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) treatment for mers-cov would be the use of neutralizing antibodies, either via convalescent plasma or monoclonal antibodies. severity of disease has been associated with a delayed adaptive immune response (park et al., 2015) , and thus antibody-based therapy might be beneficial. administration of neutralizing antibodies early in disease onset when patients were infected with respiratory pathogens such as sars-cov and influenza a virus was reported to be beneficial (mair-jenkins et al., 2014) . treatment of sars-cov-infected patients with convalescent plasma early in disease progression resulted in a higher likelihood of disease remission and survival (cheng et al., 2005) and administering convalescent plasma in two sars-cov-infected healthcare workers resulted in complete recovery (yeh et al., 2005) . convalescent plasma treatment of patients with severe a(h1n1)pdm09 influenza virus infection resulted in reduced mortality and respiratory tract viral load (hung et al., 2011) . meta-analysis of studies on the treatment of spanish influenza h1n1 1918 with convalescent plasma showed an absolute reduction of 21% in case-fatality rate (luke et al., 2006) . a recent study suggests that the availability of donors with sufficiently high mers-cov antibody titers might be limited; only 12 out of 443 tested sera obtained from patients, healthcare workers and household contacts were positive on elisa (arabi et al., 2016) . furthermore, it has been suggested that severity of disease is linked to serological response; in patients who developed severe disease upon infection with mers-cov higher neutralizing antibody levels were detected, whereas in patients with mild or subclinical disease lower and potentially short-lived neutralizing antibody levels were detected (drosten et al., 2014; park et al., 2015) . it has been well-established that severity of disease is linked to the prevalence of comorbidities (badawi and ryoo, 2016) , the existence of which might contradict the collection of convalescent serum. thus, the pool of healthy subjects with sufficient neutralizing antibody titer in convalescent plasma might be very limited. finally, the hyperimmune plasma used in this study had a high neutralizing antibody titer of 3840, whereas at best human convalescent plasma has neutralizing antibody titers of 320e800 (park et al., 2015; arabi et al., 2016) (these studies use different methods to measure neutralizing antibodies). it is unclear whether convalescent plasma with lower neutralizing titers would have a similar effect on disease progression, as lower neutralizing antibody titers in the circulation will likely be reached. monoclonal antibodies could provide an alternative; mab treatment of healthy volunteers inoculated with influenza a virus h3n2 resulted in a reduction in median viral load in nasal swabs and a 35% reduction in median total symptoms (ramos et al., 2015) . as of yet, very few studies have been done to evaluate the efficacy of convalescent plasma treatment of mers-cov. administration of camel sera to mice sensitized to mers-cov infection resulted in a decrease in viral titer in lungs. in addition, when repeating the study with type i interferon receptor-deficient (ifnar à/-) mice, which lose weight upon inoculation with mers-cov, a decrease in weight loss as well as less severe histological changes in lung tissue was observed . so far, only one documented mers-cov case has received convalescent plasma treatment and this patient was still hospitalized on day 77 of illness (park et al., 2015) . the effect of mabs against mers-cov has been shown both in vitro (jiang et al., 2014; tang et al., 2014; ying et al., 2014) and prophylactic and therapeutic administration of mabs to mers-cov mouse models has been shown to decrease the viral titer and load found in lung tissue (corti et al., 2015; li et al., 2015; pascal et al., 2015; luke et al., 2016) . prophylactic treatment with m336 via the i.v. or i.n. route resulted in significantly reduced viral titer (40e9000 fold) in rabbit lung tissue. in contrast, administration of m336 1 dpi via the same routes did not reduce viral rna titers in the lungs of rabbits (houser et al., 2016) . reduced mortality and morbidity was observed in a mers-cov mouse model upon intraperitoneal prophylactic and therapeutic treatment with m336, and therapeutic treatment resulted in a decrease in viral titer as well as viral rna in lung tissue (agrawal et al., 2016) . it can be argued that neither the rabbit nor the mouse model have a high predictive value for potential mers therapies in humans. rabbits remain asymptomatic upon inoculation with mers-cov and infection appears to be more prominent in the upper respiratory tract, which suggests that disease progression in rabbits differs considerably from that observed in patients who will require antiviral therapy most (haagmans et al., 2015) . the mouse as a model is valuable as an initial validation method of a therapy, but the predictive value of mouse models for therapeutic applications in humans is relatively limited as opposed to non-human primate models (seok et al., 2013) . in this study, hyperimmune plasma treatment of marmosets inoculated with mers-cov resulted in a small (0.5e1 log) but significant reduction in respiratory tract viral loads, as well as reduced disease severity such as observed with radiographs, compared to marmosets treated with non-convalescent plasma or pbs. however, the observed differences were relatively minimal and no differences in histopathology were observed. in contrast, treatment with m336 in the common marmoset did not result in a significant decrease in respiratory tract viral loads compared to control animals, however a significant reduction in disease severity was observed. this was most pronounced when comparing diseaseassociated signs. only one animal treated with m336 showed increased respiratory rates (>60 breaths/minute), and all animals had no evidence of infiltration radiographically, showed decreased levels of gross pathology and the lowest lung to body weight ratio. however, no changes in histology were observed compared to control animals. qrt-pcr and in situ staining for mers-cov were negative in the lungs of one marmoset treated with hyperimmune plasma, although mers-cov viral loads and associated pathology were detected in the conjunctiva of this animal. it is possible that in this case, inoculation with mers-cov did not result in a successful infection of the respiratory tract. the remaining two marmosets treated with hyperimmune plasma were found to have viral loads of up to 1.1 â 10 5 tcid 50 equivalent per gram lung tissue. the marmosets utilized in this study are outbred, and the significance of genetic factors or differences in immunology cannot be excluded. importantly, when statistical tests were performed excluding the marmoset with no viral load in the respiratory tract, differences in tissue of marmosets 7 dpi. mean values ± sd were calculated. statistical significance was calculated using a one-tailed unpaired student's t-test; p-values: * > 0.05. (c) viral load in extra-respiratory tissues from marmosets 7 dpi. mean values ± sd were calculated. red ¼ hyperimmune plasma-treated marmosets; blue ¼ mab-treated marmosets; green ¼ control marmosets. dotted line ¼ limit of detection. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) viral load in the lobes of the lower respiratory tract between control and hyperimmune plasma-treated animals were still significant (p ¼ 0.0263). we show that treatment of mers-cov infected animals with hyperimmune plasma or m336 results in lower clinical scores. treatment with mabs reduced the occurrence of severe respiratory symptoms further than hyperimmune plasma did, combined with less gross pathology. mabs might therefore be a better therapy if the goal is to elevate symptoms associated with severe mers disease. hyperimmune plasma reduced viral titers, but if a reduction in viral lung load does not result in less severe symptoms, the question is raised of what the importance is of such a change. regardless, the observed differences are small and most treated animals showed mild-to-moderate respiratory symptoms, suggesting that the effect of both treatments is limited. passive transfer of a germlinelike neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and metaanalysis thoracic radiography as a refinement methodology for the study of h1n1 influenza in cynomologus macaques (macaca fascicularis) use of convalescent plasma therapy in sars patients in hong kong detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques transmission of mers-coronavirus in household contacts infection with mers-cov causes lethal pneumonia in the common marmoset asymptomatic middle east respiratory syndrome coronavirus infection in rabbits prophylaxis with a mers-cov-specific human monoclonal antibody protects rabbits from mers-cov infection convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis a roadmap for mers-cov research and product development: report from a world health organization consultation pneumonia from human coronavirus in a macaque model kinetics of serologic responses to mers coronavirus infection in humans pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza genomic responses in mouse models poorly mimic human inflammatory diseases identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution coronavirus infections from who-isaric joint mers-cov outbreak readiness workshop: clinical management and potential use of convalescent plasma 10e12 pharmacokinetic characteristics of therapeutic antibodies experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection the authors would like to thank drs. bart supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.antiviral.2017.03.025. key: cord-343107-oj1re34k authors: zhou, haixia; chen, yingzhu; zhang, shuyuan; niu, peihua; qin, kun; jia, wenxu; huang, baoying; zhang, senyan; lan, jun; zhang, linqi; tan, wenjie; wang, xinquan title: structural definition of a neutralization epitope on the n-terminal domain of mers-cov spike glycoprotein date: 2019-07-11 journal: nat commun doi: 10.1038/s41467-019-10897-4 sha: doc_id: 343107 cord_uid: oj1re34k most neutralizing antibodies against middle east respiratory syndrome coronavirus (mers-cov) target the receptor-binding domain (rbd) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (dpp4). the epitopes and mechanisms of mabs targeting non-rbd regions have not been well characterized yet. here we report the monoclonal antibody 7d10 that binds to the n-terminal domain (ntd) of the spike glycoprotein and inhibits the cell entry of mers-cov with high potency. structure determination and mutagenesis experiments reveal the epitope and critical residues on the ntd for 7d10 binding and neutralization. further experiments indicate that the neutralization by 7d10 is not solely dependent on the inhibition of dpp4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. these properties give 7d10 a wide neutralization breadth and help explain its synergistic effects with several rbd-targeting antibodies. m iddle east respiratory syndrome coronavirus (mers-cov), a novel lethal human virus in the family of coronaviridae, was first identified in saudi arabia in june 2012 1 . infection by this pathogen causes an acute respiratory disease designated as mers, with symptoms that are very similar to those of sars 2 . globally, mers-cov infections have been confirmed in 27 countries causing 803 deaths (http://www.who. int/emergencies/mers-cov/en/). interspecies transmission from dromedary camels to humans is considered to be one major route of transmission in the middle east region 3, 4 . however, many infected patients without camel exposure and a recent mers outbreak in korea demonstrated that large-scale human-tohuman transmissions can occur through close contacts 5 . due to its potential for mutating toward efficient human-to-human transmission and causing a pandemic, mers-cov was listed as a category c priority pathogen by the us national institute of allergy and infectious diseases. monoclonal antibodies (mabs) with potent neutralizing activity have become promising candidates for both prophylactic and therapeutic interventions against viral infections 6 . on coronaviruses, the component primarily targeted by mabs is the homotrimeric spike (s) glycoprotein of the virion. as a typical class i fusion glycoprotein, the s trimer of highly pathogenic coronaviruses such as mers-cov and sars-cov, which mediates receptor recognition and membrane fusion during viral entry [7] [8] [9] [10] [11] [12] , undergoes protease cleavage into the s1 and s2 subunits, positional change of the receptor-binding domain (rbd) in the s1 subunit for receptor binding, dissociation of the s1-receptor complex, and finally formation of a six-helix bundle by the s2 subunits. a series of rbd-targeting antibodies against mers-cov, which block the binding of the s trimer to the cellular receptor dpp4, have been reported and characterized [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . these antibodies exhibited high potency in inhibiting the infectivity of pseudotyped and live mers-cov in cells and animal models. the neutralizing epitopes and mechanisms of antibodies including 4c2, d12, m336, mers-27, jc57-14, cdc-c2, mers-4, and mers-gd27 were further elucidated at the atomic level by structural and functional studies [19] [20] [21] [22] [23] [24] [25] . sequence comparisons of different mers-cov strains have shown that most naturally occurring mutations of the s glycoprotein are located on the rbd of the s1 subunit and the s2 subunit. considering the rapid evolution and high genome variation of rna viruses, more mutations on the rbd may enable the new strains to escape neutralization by currently known rbd-targeting antibodies. therefore, new mabs targeting other functional regions of the mers-cov s glycoprotein and/or neutralizing by different mechanisms are important for developing effective prophylactic and therapeutic interventions against mers-cov infection. although several mabs targeting non-rbd regions have recently been reported, their neutralizing epitopes and mechanisms remain unclear 20, 21, 26 . in this study, we isolated and characterized the mouse mab 7d10 by combining structural, biochemical, and functional studies. the 7d10 antibody recognizes the ntd of mers-cov s glycoprotein and neutralizes the infectivity of pseudotyped and live virus with a potency comparable to those of the most active rbd-targeting antibodies. we also found that the epitope and mechanism of 7d10, which are different from those of rbdtargeting antibodies, enable it to have a better neutralizing breadth and to work synergistically with other antibodies against different mers-cov strains. all these results indicate that 7d10 is a very promising candidate for the future combined use of different antibodies in our battle against mers-cov. characterization of neutralizing mab 7d10 targeting the ntd. to generate mers-cov neutralizing mabs with epitopes outside the rbd, mice were immunized with recombinant mers-cov s protein (residues 1-1297). subsequently, the spleenocytes were harvested and fused with sp2/0 myeloma cells, and the hybridoma cell lines were screened for positive clones by elisa with the s protein 27 . the positive clones were further tested for their reactivity to different s fragments, including the s1 subunit ntd (residues 18-353), rbd (residues 367-606), and the s2 subunit (residues 726-1297). one ntd-specific mab, named as 7d10, was finally isolated with an ec 50 of approximately 0.31 μg ml −1 in elisa (fig. 1a) . it exhibited no crossreactivity with the rbd at a concentration of 4 μg ml −1 (fig. 1b) . we further assessed the potential of 7d10, in the form of crude extracts from mouse ascites, for inhibiting mers-cov entry into susceptible huh7 cells and vero e6 cells with either pseudotyped or infectious viruses. as expected, 7d10 was able to neutralize the infectivity of pseudotyped and live mers-cov (fig. 1c, d) . the neutralizing activity of 7d10 was dose-dependent, with an ic 50 of approximately 0.18 μg ml −1 against pseudotyped virus and practically the same ic 50 of approximately 0.2 μg ml −1 against live virus (emc strain) (fig. 1c, d) . images illustrating the reduced pfu formation, corresponding to the rate of neutralization of live mers-cov, are shown in fig. 1e . antibody isotyping showed that 7d10 belongs to the igg1 subtype. sequencing further determined that the heavy chain germline v and j segments are ighv1-12*01 and ighj2*03, while those of light chain are igkv03-12*01, igkj1*01, and igkj1*02, respectively (supplementary table 1) . we also generated a chimeric version of 7d10 (7d10-h) by combining the v segments of 7d10 with the human igg1 backbone, which was efficiently expressed and purified in freestyle 293-f cells ( supplementary fig. 1a ). the bio-layer interferometry (bli) experiment showed that the affinity constant of the binding between 7d10-h and ntd was approximately 25 nm (table 1 and supplementary fig. 1b) . the ic 50 of the purified 7d10-h against cell entry by pseudotyped mers-cov was approximately 0.06 μg ml −1 (supplementary fig. 1c ). we also investigated the protective efficacy of 7d10-h against infection of pseudotyped mers-cov using r26-hdpp4 mice model with a human dpp4 inserted into the rosa26 locus by crispr/cas9, which could also been productively infected by high-titer mers-cov pseudovirus, with effects comparable to the authentic infection 28 . bioluminescence of the fluc reporter showed that the pseudovirus infection in the mice was clearly prevented by 7d10-h and rbd-specific mab mers-4 when both antibodies were administered by the intraperitoneal injection with a dose of 200 μg per mouse ( supplementary fig. 1d ). the recombinant chimeric 7d10-h, which retained the activities as the mouse 7d10 and protected r26-hdpp4 mice against challenge of pseudotyped mers-cov, was utilized in subsequent binding and neutralization experiments. overall structure of the 7d10 scfv bound to the ntd. to structurally characterize the 7d10 and its binding to the spike protein, we determined the crystal structure of the antibody scfv (7d10-scfv) in complex with the ntd at a resolution of 3.0 å with a final r work of 0.188 and r free of 0.224. statistics of diffraction data collection, processing, and structure refinement are listed in table 2 . there were three complexes of 7d10-scfv bound to ntd per asymmetric unit. the refined model contains residues tyr18 to ser353 of mers-cov ntd, glu1 to ser120 of the v h and asp26 to lys136 of the v l . n-linked glycans attached to asn66, asn104, asn125, asn155, asn166, asn222, asn236, and asn244 of the ntd are also included in the model. it has been previously shown that the mers-cov ntd folds into a galectin-like structure, which can be separated into top, core and bottom subdomains (fig. 2a) . upon binding, the 7d10-scfv contacts the top subdomain of the ntd and the asn222-linked glycans with its heavy and light chains ( fig. 2a and supplementary fig. 2 ). all three cdrs of the heavy chain and the cdr1 and cdr3 of the light chain participate in the binding (fig. 2a) . the buried surface between the 7d10-scfv and the ntd encompasses approximately 551 å 2 for the heavy chain and 320 å 2 for the light chain. structural features of the interface between 7d10 and ntd. the binding interface between 7d10-scfv and ntd consists of 12 residues and asn222-linked glycans from the ntd, as well as 15 residues from all 6 cdrs except for lcdr2 (fig. 2b, c) . the interacting residues from the ntd are tyr18, asp20, pro23, asp24, val26, ser28, glu188, ser191, asn226, lue234, arg235, and asn236. together with the asn22-linked nag508, nag509 and man519, they form the conformational epitope recognized by 7d10 (fig. 2b) . the residues recognizing 7d10 are ser31, tyr32, asn33 from the hcdr1, tyr52, asn55, and ser59 from the hcdr2, arg98, tyr99, asn101, tyr102, and tyr105 from the hcdr3, tyr59, and tyr61 from the lcdr1, and arg121 and asp122 from the lcdr3 (fig. 2c) . specifically, 7d10 hcdr1 residues ser31, tyr32, and asn33 interact with pro23 and asp24 from the ntd, and a formed hydrogen bond is from 7d10 asn33 to ntd asp24 ( fig. 2d and supplementary fig. 2 crystal structure of 7d10-scfv bound to ntd and the binding interface. a an overall structure of the ntd/7d10-scfv complex in which the ntd, n222-linked glycans on the ntd, 7d10 v l , and 7d10 v h are colored in blue, gray, magenta, and cyan, respectively. b epitope on the ntd recognized by 7d10. the ntd is represented as blue surface, on which the protein region bound by 7d10 is displayed in orange and the n222-linked glycans are displayed as gray sticks. c 7d10 residues that are involved in the binding. the v l and v h are colored in magenta and cyan, respectively, and the residues interacting with 7d10 are displayed in orange. d interactions between the 7d10 v h residues and the corresponding residues of ntd. e interactions between the 7d10 v l residues and the corresponding residues of ntd. f zoom-in view of interactions between n222-linked glycans and 7d10 interacting with tyr18, asp20, pro23, asp24, and arg235 of the ntd (fig. 2d ). tyr99 and asn101 of 7d10 form two hydrogenbonding interactions with asp24 of the ntd (supplementary table 2 ). for the light chain, the lcdr1 and lcdr3 residues tyr59, tyr61, arg121, and asp122 interact with glu188, ser191, arg235, and asn236 of the ntd, and a salt bridge is formed between arg121 of lcdr3 and glu188 of the ntd (fig. 2e and supplementary table 2) . a prominent feature at the interface is the extensive recognition of asn222-linked glycans by all three heavy chain cdrs ( fig. 2f and supplementary table 3 ). specific hydrogen-bonding interactions occur between tyr58 and arg98 of 7d10 and the nag508 and man519 glycans, respectively ( fig. 2f and supplementary table 2 ). confirmation of the neutralizing epitope. to confirm the epitope and its critical residues, we performed a mutagenesis study by introducing single mutations to all 13 ntd recognized residues including trp18, asp20, pro23, asp24, val26, ser28, glu188, ser191, asn222, asn226, lue234, arg235, and asn236. we first examined the effects of these ntd mutations on the binding by 7d10-h. the 7d10-h bound the wild-type ntd with an affinity of approximately 25 nm (table 1 and supplementary fig. 3 ). by contrast, the d24a and r235a mutations dramatically reduced the binding, to a level that was undetectable by bli experiment (table 1 and supplementary fig. 2 ). the e188a and n222q mutations reduced by the binding affinity by 148-fold to 3.7 μm and 112-fold to 2.8 μm, respectively (table 1 and supplementary fig. 3 ). all the other nine mutations had variant unequal effects on the binding by reducing the affinity in the range of 2-to 15-fold (table 1 and supplementary fig. 2 ). the effects of these mutations on the neutralizing activity of 7d10-h were in consistent with the changes of binding affinity. pseudotyped mers-cov bearing d24a, e188a, or r235a mutation in the spike glycoprotein escaped the neutralization by 7d10-h (table 1 and supplementary fig. 4 ). the ic 50 values of 7d10 against pseudotyped mers-cov bearing d20a, v26a, or n222q mutation were increased approximately by 60-, 181-, and 50-fold (table 1 and supplementary fig. 4 ). the binding and neutralization assays collectively revealed that asp24, val26, glu188, arg235, and asn222-linked glycans are critical for recognition and neutralization of mers-cov by 7d10. sequencing of multiple clinical isolates had revealed that the mers-cov s glycoprotein is evolving at an average rate of 1.12 × 10 −3 substitutions per site per year 29 . alignments of the deposited sequences in the ncbi identified 22 naturally changing residues from the prototype emc sequence including v26f, v26i, v26a, d158y, l411f, t424i, a482y, l506f, d509g, v530l, v534a, e536k, d537e, v810i, q833r, q914h, r1020h, r1020q, a1193s, t1202i, g1224s, and v1314a, which are located in the ntd (residues 18-353) and rbd (residues 367-606) of the s1 subunit, and the s2 subunit (residues 752-1297). several residue changes on the rbd, such as those occurring on d506, d509, and e536, indeed enabled the mers-cov to escape the neutralization of antibodies targeting the rbd 20, 21 . considering that most of the mutations are outside the ntd, we speculated that 7d10-h would have a better tolerance for these naturally occurring mutations. we generated pseudotyped mers-cov bearing the emc strain s glycoproteins and its mutants harboring all the 22 listed residue changes. the neutralization assays showed that 7d10-h showed effective neutralizing activity against almost all pseudotyped mers-cov variants. only the two mutations v26f and v26a on the ntd increased the ic 50 value of 7d10-h by more than 150-fold and significantly reduced its neutralization activity (fig. 3a, b) , which confirmed the results of the structural and biochemical studies of the binding interface. all other naturally occurring mutations, most of them on the rbd and the s2 subunit did not affect the neutralization capability of 7d10-h (fig. 3a, b) , indicating that 7d10 would have a wide neutralization breadth against different variants of mers-cov. combination of 7d10 with other rbd-targeting antibodies. the current available mers-cov antibody epitopes with solved structures are all on the rbd, which can be grouped into three categories: (1) epitope of mers-4; (2) epitopes of mers-27, d12, 4c2, and jc57-14; and (3) epitopes of m336, mca1, cdc-c2, and the newly reported mers-gd27 ( supplementary fig. 5 ) 25 . in our study of the rbd-specific mab mers-4, we also found synergism with the ntd-targeting mab 5f9 25 . thus, the elucidation of the epitope targeted by 7d10, which added a category outside the rbd ( supplementary fig. 5 ), prompted us to study the combined effect of 7d10 together with the three representative antibodies mers-4, mers-27, and mers-gd27 in the neutralization of pseudotyped mers-cov by titrating the neutralizing potency of an equimolar mixture of the two antibodies and comparing the dose response with that observed in neutralization assays performed with the individual antibody alone. as shown in the fig. 4 , the combination index (ci) values of mers-gd27 combined with 7d10 at fa values of effective dose 50%, 75%, 90%, and 95% (ed 50 , ed 75 , ed 90 , and ed 95 , respectively) were 0.26, 0.25, 0.24, and 0.24, respectively. as a ci value of 1 indicates an additive effect, <1 indicates synergism, and >1 indicates antagonism, the combination of 7d10 and mers-gd27 worked in a clearly synergistic manner. meanwhile, the combination index (ci) values of combined mers-4 with 7d10 at fa values of effective dose 50%, 75%, 90%, and 95% (ed 50 , ed 75 , ed 90 , and ed 95 ) were 0.25, 0.27, 0.30, and 0.33, respectively. thus, the combination of mers-4 and 7d10 also demonstrated synergism, in particular at relatively lower concentrations. however, the percent neutralization obtained using combined mers-27 and 7d10 showed no obvious difference of half maximal inhibitory concentration (ic 50 ) compared with that of 7d10 alone. the combination index (ci) values of combined mers-27 and 7d10 at fa values of effective dose 50%, 75%, 90%, and 95% (ed 50 , ed 75 , ed 90 , and ed 95 ) were 0.82, 0.87, 0.94, and 1.00, respectively. it indicated that the combination of 7d10 with mers-27 exhibited neither synergy nor antagonism. mechanism of 7d10 neutralization. a major reported mers-cov neutralization mechanism relies on inhibiting the binding of the s trimer with the cellular receptor dpp4. the epitopes of these reported antibodies all reside in the rbd responsible for receptor binding. the fact that the 7d10 epitope is outside the rbd indicated that it may have a different neutralizing mechanism. we first examined if 7d10 is still able to inhibit the receptor binding by the s trimer. the facs analysis of cellsurface staining showed that the scfv and fab fragments of 7d10-h did not inhibit the staining of huh7 cells by the s trimer, while the 7d10-h slightly reduced the staining (fig. 5a , supplementary table 4 and supplementary fig. 6 ). by contrast, the rbdtargeting mab mers-4 was much more potent than 7d10-h in inhibiting the binding of the s trimer to huh7 cells. moreover, the fab and scfv fragments of mers-4 retained nearly the same potency in the inhibition ( fig. 5a and supplementary table 4 ). surface plasmon resonance (spr) analysis confirmed these conclusions by showing that 7d10-h, and not its fab or scfv fragments, could interfere with the binding of the s trimer to chipcoupled dpp4 in a dose-dependent manner ( supplementary fig. 7) , while the igg, fab, and scfv of mers-4 all inhibited the binding ( supplementary fig. 7) . to investigate why the igg, fab, and scfv of 7d10 inhibit receptor binding differently, we constructed models of their binding to the s trimer. the mers-cov s trimer structure was determined by cryo-em with the rbd in standing or lying positions, and only the standing rbd could bind to the dpp4 receptor. after superimposing the ntd/7d10-scfv crystal structure onto the s trimer, we observed no steric clashes between three ntd-bound scfv fragments and one or two rbdbound dpp4 receptors ( fig. 5b and supplementary fig. 8 ). the s trimer with three rbd-bound receptors was not considered because the cryo-em study of the mers-cov s trimer only revealed conformations with one or two standing rbds. when the scfv was replaced with the fab, there were also no steric clashes between the fab and dpp4 receptor (fig. 5c ). it is more complicated to model the binding of 7d10-h to the s trimer, considering that the igg form has two binding sites and the intrinsic flexibility. we found that binding of the 7d10-h igg to the ntd in certain orientations could inhibit the binding of dpp4 due to steric clashes, while there were still no steric clashes with the 7d10-h bound in some other orientations (fig. 5d, e) . these results provided a structural explanation for the inability of 7d10-h scfv and fab to inhibit the binding of the s trimer to the dpp4 receptor. they may also explain why the 7d10-h igg form is not as potent as the mers-4 igg, fab, and scfv which all directly bind to the rbd. in parallel with biochemical studies, we also examined the neutralizing activities of 7d10-h igg, fab, and scfv. the 7d10-h fab and scfv did not interfere with the binding of the s trimer to the dpp4 receptor. however, they were still able to inhibit the cell entry of pseudotyped mers-cov with ic 50 value of 0.26 μg ml −1 and 0.28 μg ml −1 , respectively (fig. 6a) . although the 7d10-h fab and scfv are less active than the igg in infection inhibition, they were still comparable to the fab or scfv fragments of several reported rbd-targeting antibodies such as mers-4 fab (ic 50 : 1.49 μg ml −1 ) and mers-4 scfv (ic 50 : 0.55 μg ml −1 ) ( supplementary fig. 9a ). these results collectively indicated that neutralization by 7d10-h involves other mechanism besides interfering with the initial receptor binding. we tested and compared the neutralizing activity of 7d10 in pre-attachment and post-attachment settings. after the cell attachment, 7d10 was still able to inhibit infection by pseudotyped mers-cov with an ic 50 of 0.55 μg ml −1 (fig. 6b) . in comparison, mers-4, which is more potent than 7d10 in inhibiting receptor binding, exhibited very weak neutralization after receptor binding (supplementary fig. 9b) . the above results, especially the retaining activity of 7d10 after viral attachment indicated that 7d10 would also interfere with the prefusion to postfusion conformational transition of the s glycoprotein required for membrane fusion. this transition 30, 31 . we showed that the mers-cov s glycoprotein in the prefusion state is sensitive to the digestion of proteinase k (fig. 6c) . previous studies have demonstrated that cleavage at the s1/s2 site by trypsin and the binding with cellular receptor greatly enhanced the prefusion to postfusion transition of the spike glycoprotein 31 . consistently, the amount of a 50 kda and proteinase-k-resistant band of the s glycoprotein representing the postfusion six-helix bundle was at the maximum level in the presence of trypsin and dpp4 (fig. 6c) . and the addition of 7d10-h fab obviously reduced the intensity of the band (fig. 6c) . meanwhile, we analyzed the full-length mers-cov s trimer embedded in the membrane of pseudotyped virus and the trigger we used to induce the conformational transition was the incubation with huh 7 cells that endogenously expressing dpp4 receptor. after incubating the pseudotyped virus with huh 7 cells for 1 h at 37°c, a proteinase-k resistant band on the sds-page gel appeared and the addition of 7d10-h, 7d10-h fab, or 7d10 scfv all clearly decreased the intensity of this band ( supplementary fig. 10) . thus, these biochemical results strongly suggest that 7d10 could also exert its neutralizing activity in the postattachment stage after receptor-binding by inhibiting the conformational transition of the s glycoprotein required for membrane fusion (fig. 6d) . since dpp4, which is a critical step for viral cell attachment. in this study, we first isolated the neutralizing mouse antibody 7d10 targeting the ntd of the s glycoprotein. neutralization assays showed that 7d10 is highly potent and its activity is comparable to that of the most potent rbd-targeting antibodies. structural determination of 7d10 scfv bound to the ntd and mutagenesis studies revealed the epitope and key residues on the ntd for binding and neutralization at atomic level. comparisons of 7d10 scfv, fab, and igg forms in dpp4-binding competition and neutralization assays indicated that its activity is not solely dependent on the inhibition of dpp4 binding. further experiments indicated that the neutralizing activity of 7d10 after cell attachment is through the inhibition of prefusion to postfusion conformational transition of the s glycoprotein trimer, which mediates the fusion of viral and cell membranes. we also showed that 7d10 has a wide neutralization breadth against mers-cov variants bearing naturally occurring mutations and exhibited synergistic effects with several rbd-targeting antibodies. these results collectively revealed an antibody epitope and neutralization mechanism on the s glycoprotein, which would contribute to the global efforts to control mers-cov infection and transmission by providing alternatives for mers-cov immunotherapy. similar the ntds of the s protein of other betacoronaviruses such as mhv, bcov and hku1, that of mers-cov also folds into a galectin-like structure. although the galectin domain is a typical carbohydrate-recognition domain, the betacoronavirus ntds can include structural variations that enable more diverse functions in viral infection. the examples, include the ntd of bcov that retains the glycan-binding activity recognizing 5-n-acetyl-9-oacetylneuraminic acid (neu5,9ac2) and the ntd of mhv that evolved specific protein-protein interactions with its cellular receptor ceacam1, and both interactions are important for the viral cell attachment 36, 37 . however, there is still no report on the glycan or protein-binding activities of the mers-cov ntd. in fact, crystallographic structure determination showed that the glycanbinding site on the mers-cov ntd is occupied by a short helix (residues 222-231) and the asn222-linked glycan, indicating that it is not able to bind glycans in the same way as the ntd of bcov 11 . notably, the asn222-linked glycan is involved in the recognition by 7d10, whereby nag508 and man519 undergoes specific hydrogen-bonding interactions with tyr58 and arg98 of 7d10, respectively. the ntd n222q mutation also dramatically reduced the binding and neutralization by 7d10, but did not dramatically affect the cell infection of pseudotyped mers-cov ( supplementary fig. 11) . therefore, the asn222-linked glycan serves as an important anchor point for the binding of 7d10 to the mers-cov ntd. as the largest class i viral fusion protein, the coronavirus s glycoprotein is expected to undergo a prefusion to postfusion conformational transition to mediate the interaction between viral and cellular membrane proteins, although structural studies just began to shed light on this recently. the s glycoprotein of betacoronaviruses mhv and hku1, whose structures have been determined by the cryo-em method, all adopt a similar prefusion homotrimeric architecture 38, 39 . interestingly, in the prefusion architecture of the s trimer of highly pathogenic mers-cov and sars-cov, two major conformational states were observed. a 7d10-h igg was tested for neutralizing activity against pseudotyped mers-cov before or after receptor binding. vrc01 mab was used as unrelated control. c the effect of 7d10-h fab on the conformational change of the mers-cov s trimer was probed by western blotting using an anti-mers-cov s2 polyclonal antibody. refolding to the postfusion conformation was detected by the appearance of a proteinase-k resistant band. trypsin was used at 5 μg ml −1 and proteinase k at 10 μg ml −1 . digestion experiments and western blots were performed in triplicates, and a representative result is shown for each of them. d a cartoon representation designed by us showing the neutralization mechanism by which 7d10 blocks mers-cov entry. on the one hand, some virus particles can not bind to dpp4 due to steric hindrance caused by 7d10 binding. on the other hand, 7d10 still recognizes the particles when the up receptor-binding domain (rbd) binds to dpp4, and may inhibit the prefusion to postfusion transition of the s2 subunit and the initiation of membrane fusion. source data are provided as a source data file major difference between them is the change of the rbd in the s1 subunit from a down to an up position, which was proposed to be a prerequisite for the binding of the s trimer to their respective cellular receptor dpp4 and ace2 9 . this proposal was recently confirmed by our cryo-em study of the sars-cov s trimer in complex with ace2, and we also showed that ace2-binding could induce the dissociation of the s1 subunit, which results in the falling apart of the prefusion s trimer and the transition to the prefusion state of the s2 subunit 12 . a major neutralization mechanism of antibodies against mers-cov is to directly or indirectly compete with the cellular receptor dpp4 for binding to the rbd. in theory, antibodies that interfere with the coronavirus membrane fusion process other than receptor binding would also have a neutralizing activity, and the 7d10 mab targeting the ntd we studied is one such example. here, we showed that 7d10 neutralization is not solely dependent on dpp4-binding competition, and its inhibition of the s trimer conformational transition after cell attachment also plays a significant role in the neutralization. we suggested that the binding of 7d10 may stabilize the prefusion architecture of the s trimer, even after the binding of dpp4 receptor. the stabilization of viral fusion protein at one conformational state for neutralization has also been observed and studied in other viruses such as hiv. a recent study revealed that the hiv env trimer is intrinsically dynamic with three major and distinct prefusion conformations 40 . among them, the closed, ground-state conformation is dominant and could be remodeled to another two conformations by cd4 receptor binding, which is essential for the subsequent prefusion to postfusion transition 40 . the binding of neutralizing antibodies, whether inhibiting the binding of the cd4 receptor (such as vrc01) or not (such as 2g12 and pgt145) all resulted in the stabilization of the ground-state conformation of the env, which finally disfavors its prefusion to postfusion state transition required for viral entry 40, 41 . to the best of our knowledge, our study offers the first structural definition of the neutralizing epitope of an antibody targeting the s ntd of mers-cov. as we summarized in supplementary table 5 , a total of six anti-ntd mabs have been reported 20, 21, 26, 42 . all of them neutralize the infection of pseudotyped mers-cov emc strain with high potency except for mab 1.10f3. the mab 5f9 and our 7d10 showed the same neutralizing activity against live mers-cov in plaque reduction neutralization testing. notably, the mouse mab g2 can greatly relieve the symptom of dpp4-transgenic mice infected following mers-cov infection and our 7d10-h can inhibit the infection of pseudotyped mers-cov in r26-hdpp4 mice. however, the specific neutralizing epitopes and mechanisms of 5f9, g2, jc57-13, and fib-h1 are largely unknown. in addition, the combination of different antibodies is supposed to be an effective strategy to combat mers-cov infection as it continues to spread among multiple animal species and to probe and adapt to the human population [43] [44] [45] . an effective combination would require the candidate antibodies to bind to disparate epitopes or with distinct mechanisms and hence display additive or synergistic effects, as the mabs mers-4 and 5f9 we mentioned before 25 . although the exact mechanism that leads to the synergy or additive is uncertain, our 7d10-h with mers-gd27 or mers-4 antibodies demonstrated a synergy in inhibiting the infectivity of pseudotyped mers-cov, while 7d10-h and mers-27 antibodies together had an additive effect. consequently, 7d10 is currently the most comprehensively studied ntd-targeting mab with a different epitope and working mechanism, which makes it an excellent candidate, in combination with other rbd-targeting neutralizing antibodies or alone, in our battle against mers-cov infection. three weeks after the initial immunization, these mice were boosted twice at 2-week intervals. cells collected from the spleens of sacrificed animals were fused with cultured sp2/0 cells at a 10:1 ratio in the presence of peg1450 (sigma). hat selection medium was used for the fused hybridoma cultures. after 2-weeks of incubation, the positive hybridomas were selected via s-coated elisa, and the positive clones were subjected to limited dilutions and downstream validation. for large-scale mab production, ascites fluid from mice inoculated with the hybridomas was collected and purified by the caprylic acid-ammonium sulfate precipitation method. protein expression and purification. the coding sequence of the mers-cov spike glycoprotein ectodomain (emc strain, spike residues 1-1290) was ligated into the pfastbac-dual vector (invitrogen) with a c-terminal t4 fibritin trimerization domain and a hexa-his-strep tap tag to facilitate further purification processes. briefly, the protein was prepared using the bac-to-bac baculovirus expression system, purified by sequentially applying strep-tactin and superose 6 column (ge healthcare) with hbs buffer (10 mm hepes, ph 7.2, 150 mm nacl). fractions containing mers-cov s glycoprotein were pooled and concentrated for subsequent biochemical analyses. the sequence encoding mers-cov s1 ntd (residues 18-353) with a c-terminal hexa-his tag was inserted into the eukaryotic expression vector pvax. freestyle 293-f cells were transfected with the plasmid using polyethylenimine (pei) (sigma). after 72 h, the supernatant was collected and the ntd was purified using nta sepharose (ge heathcare) and superdex 200 high performance column (ge healthcare) with hbs buffer (10 mm hepes, ph 7.2, 150 mm nacl). the sequence encoding the 7d10 v l and v h were separately cloned into the backbone of antibody expression vectors containing the constant regions of human igg1. the chimeric antibody 7d10-h was expressed in freestyle 293-f cells by transient transfection and purified by affinity chromatography using protein a sepharose and gel-filtration chromatography. the purified 7d10-h was exchanged into phosphate-buffered saline (pbs), and was digested with papain protease (sigma) over night at 37°c. the digested antibody was then passed back over protein a sepharose to remove the fc fragment, and the unbound fab in the flow through was additionally purified using a superdex 200 high performance column (ge healthcare). the gene encoding the 7d10 v l followed by v h with a connecting triple gggs linker and a c-terminal hexa-his tag was synthesized and cloned into the eukaryotic expression vector pvrc8400. freestyle 293-f cells were transfected the plasmid in the presence of pei (sigma). the cell-culture supernatant was collected 72 h after the transfection, and the 7d10 scfv was collected and captured on nta sepharose (ge healthcare). the bound 7d10 scfv was eluted with hbs buffer containing 500 mm imidazole and was then further purified by gel-filtration chromatography using a superdex 200 high performance column (ge healthcare). complex preparation and crystallization. the mers-cov ntd and the scfv fragment of 7d10 were mixed at a molar ratio of 1:1.2, incubated for 2 h at 4°c and further purified by gel-filtration chromatography. the purified complex concentrated to approximately 10 mg ml −1 in hbs buffer (10 mm hepes, ph 7.2, data collection and structure determination. to collect the diffraction data, all crystals were flash-cooled in liquid nitrogen after being incubated in reservoir solution containing 20% (v/v) glycerol. the diffraction images were collected on the bl17u beamline at the shanghai synchrotron research facility (ssrf) 46 with the wavelength of 0.9796 å. all images were processed with hkl2000 47 . the structure was solved by molecular replacement using phaser from the ccp4 suite 48 . the search models were the mers-cov ntd structure (pdb id: 5vyh) and the structures of the variable domain of the heavy and light chains available in the pdb with the highest sequence identities. subsequent model building and refinement were performed using coot and phenix, respectively 49, 50 . there are 94% of most favored, 5.3% of allowed and 0.3% of disallowed ramachandran plot in the final refinement model. all structural figures were generated using pymol 51 . neutralizing assay of pseudotyped mers-cov. 293t cells cultured in 100 mm dish were co-transfected with 6 μg of pcdna3.1-mers-spike or its mutants and 24 μg of pnl4-3.luc.re. the supernatants containing sufficient pseudotyped mers-cov were harvested 48-72 h post-transfection. subsequently, the 50% tissue culture infectious dose (tcid 50 ) was determined by infection of huh7 cells. for the neutralization assay, 100 tcid 50 per well of pseudoytped virus were incubated with 16 or 8 serial 1:3 dilutions of purified antibodies, fabs or scfvs for 1 h at 37°c, after which huh7 cells (about 1.5 × 10 4 per well) were added. after incubation for 72 h at 37°c, the neutralizing activities of antibodies were determined by the luciferase activity and presented as ic 50 , calculated using the dose-response inhibition function in graphpad prism 5 (graphpad software inc.) cell entry of pseudotyped virus. the concentration of the harvested pseudotyped virions was normalized by p24 elisa kit (beijing quantobio biotechnology co., ltd., china) before infecting the target huh7 cells. the infected huh7 cells were lysed at 48 h after infection and viral entry efficiency was quantified by comparing the luciferase activity between pseudotyped viruses bearing the mutant-and wildtype mers-cov spike glycoproteins. postattachment neutralization assay. for the postattachment pseudotyped virus neutralization assay, huh7 cells, upon reaching a density of 1.5 × 10 4 per well in a 96-well plate, were incubated with 100 tcid 50 per well of pseudotyped virus at 4°c for 1 h. after removing the supernatant, 200 μl of pbs was added twice to each well to wash the un-bond pseudotyped viruses. a total of 16 serial 1:3 dilutions of purified antibodies in dmem (10% fbs) were then added to the huh 7 cells with attached pseudotyped viruses, as well as dmem (10% fbs) alone as control. neutralization activities were determined based on the luciferase activity after incubation for 72 h at 37°c and also presented as ic 50 , calculated using the dose-response inhibition function in graphpad prism 5 (graphpad software inc.) cooperativity of mabs for neutralization. synergistic, additive, and antagonistic interaction between 7d10 and mers-gd27, 7d10, and mers-27, as well as 7d10 and mers-4 for virus neutralization were evaluated by the median effect analysis method using compusyn software as previously reported 52, 53 . the measured neutralization values were input to the program as fractional effects (fa) ranging between 0.01 and 0.99 for each of the two antibodies and for both in combination. ci values were calculated in relation to fa values. a logarithmic ci value of 0 indicates an additive effect, <0 indicates synergism, and >0 indicates antagonism. live mers-cov neutralization assay. the neutralizing activity of the mabs against live mers-cov was also determined in dpp4-expressing vero e6 cells. upon reaching a density of 5 × 10 4 per well in a 12-well plate, cell monolayers were infected with 30-35 plaque-forming units (pfu) of live virus in the presence or absence of the mab. after three days of incubation at 37°c, the inhibitory capacity of the mabs was assessed by determining the numbers of plaques compared with the potent mers-cov anti-rbd and anti-n9 mabs. murine model of mers-cov pseudovirus infection. the mers-cov susceptible animal model hdpp4-knockin mouse, which was established by inserting human dipeptidyl peptidase 4 (hdpp4) into the rosa26 locus using crispr/cas9, resulting in global expression of the transgene in a genetically stable mouse line 28 , was used in this experiment. mice (n = 5) were challenged by intraperitoneal injection (i.p.) with doses of 1.27 × 10 7.5 tcid 50 of pseudotyped mers-cov. 7d10-h and mers-4 were administered i.p. to r26-hdpp4 mice at a dose of 200 μg per mouse prior to challenge with pseudovirus. mice (n = 4 for the pbs group and n = 3 for the 3c11 group) were also administered pbs or control mab 3c11 (mab of anti-na of h5n1, at a dose of 400 μg per mouse) and challenged using the same i.p. dose of pseudovirus. the ivis-lumina ii imaging system (xenogen, baltimore, md, usa) was used to detect bioluminescence. prior to measuring luminescence, the mice were anesthetized using an i.p. injection of sodium pentobarbital (240 mg kg −1 ). the exposure time was 60 s, and fluorescence intensity in regions of interest was analyzed using living image software (caliper life sciences, baltimore, md, usa). different wavelengths were used for detecting pseudovirus and tdtomato fluorescence. the substrate, d-luciferin (50 mg kg −1 , xenogen-caliper corp., alameda, ca, usa), was injected i.p. and imaging was conducted 10 min later. the relative intensities of emitted light were represented as colors ranging from red (intense) to blue (weak) and quantitatively presented as photon flux in photons s −1 cm −2 sr −1 . binding studies using bli. binding kinetics of mers-cov ntd and its mutants with 7d10 were studied using a fortébio octet htx instrument. assays with agitation set to 1000 rpm in hbs buffer (10 mm hepes, ph 7.2, 150 mm nacl) supplemented with 0.01% (v/v) tween 20 were performed at 25°c in solid black tilted-bottom 96-well plates (greiner bio-one). 7d10 (20 μg ml −1 ) was used to load anti-human igg fc capture probes for 300 s to capture levels of 0.5-1 nm. biosensor tips were then equilibrated for 300 s in hbs buffer supplemented with 0.01% (v/v) tween 20 prior to binding assessment with different concentrations of wild-type or mutant mers-cov ntd for 120 s, followed by dissociation for 240 s. data analysis and curve fitting were performed using octet software, version 9.0. binding competition assays by spr. real-time binding and analysis by spr were conducted on a biacore t200 instrument with cm5 chips (ge healthcare) at room temperature. for all the analyses, hbs buffer consisting of 10 mm hepes, ph 7.2, 150 mm nacl and 0.005% (v/v) tween 20 was used, and all proteins were exchanged to the same buffer. the blank channel of the chip was used as the negative control. dpp4 (20 μg ml −1 ) was immobilized on the chip at about 100 response units. soluble mers-cov spike trimer (s) at the same gradient in the present or absence of the concentration gradient of iggs, fabs, or scfvs was flowed over the chip surface. after each cycle, the sensor surface was regenerated with 7.5 mm naoh. data were analyzed using the biacore t200 evaluation software by fitting to a 1:1 langmuir binding model. facs analysis of cell-surface staining. the binding between recombinant soluble mers-cov spike trimer (s) and human dpp4 expressed on the surface of huh7 cells was measured using fluorescence-activated cell sorting (facs). all cellsurface staining experiments were performed at room temperature. soluble mers-cov spike trimer (s) with strep-tag (1 μg) was incubated with monoclonal antibodies (mabs) in advance at molar ratios of 1:1, 1:3, 1:9, and 1:27 for 1 h. huh7 cells were trypsinized and then incubated with s or s and mabs mixtures for 1 h. after washing the un-bound s with pbs 3 times, the huh7 cells were then stained with streptavidin apc (bd ebioscience) for another 45 min. cells were subsequently washed with pbs 5 times and analyzed by flow cytometry on a facs aria iii machine (bd ebiosciences). western blots. totally, 10 μl pseudotyped mers-cov was thawed and mixed with 2 μg of antibodies (igg, fab or scfv) for 1 h. the virus alone or the mixture was incubated with 20 μl of huh7 cell suspension for another 1 h at 37°c. an equal volume of buffer and proteinase-k (final concentration of 10 μg ml −1 ; thermo_fisher) was then added and incubated 1 h at 4°c. for the soluble s, 1 μg of the s trimer was incubated with 3 μg of the dpp4 ectodomain or 3 μg of 7d10 fab for 1 h on ice. trypsin (final concentration of 5 μg ml −1 ; thermo_-fisher) was then added to these samples and incubated 30 min at 37°c. subsequently, the samples were supplemented with 10 μg ml −1 proteinase-k and incubated 30 min at 4°c. 6× sds-page loading buffer was then added to all samples prior to boiling at 100°c. samples were run on a 4-12% gradient tris-mops-gel (genscript) and transferred to polyvinylidene fluoride membranes. an anti-s2 mers-cov s polyclonal antibody (1:2000 dilution; thermo_fisher; cat#pa5-81788) and an hrp-conjugated goat anti-rabbit secondary antibody (1:500 dilution; huaxingbio; cat#hx2027) were used for western blotting. ai600 was used to develop images. reporting summary. further information on research design is available in the nature research reporting summary linked to this article. the source data underlying figs. 1a-d, 3, 4, and 6a-c and supplementary figs. 1a-c, 3, 4, 7, 9-11 are provided as a source data file. crystal structures presented in this work has been deposited in the protein data bank (pdb) and are available with accession code 6j11. isolation of a novel coronavirus from a man with pneumonia in saudi arabia the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea history of passive antibody administration for prevention and treatment of infectious diseases structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein evaluation of candidate vaccine approaches for mers-cov importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein a novel neutralizing monoclonal antibody targeting the nterminal domain of the mers-cov spike protein receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov spread, circulation, and evolution of the middle east respiratory syndrome coronavirus two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis unexpected receptor functional mimicry elucidates activation of coronavirus fusion protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 crystal structure of bovine coronavirus spike protein lectin domain crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor pre-fusion structure of a human coronavirus spike protein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer conformational dynamics of single hiv-1 envelope trimers on the surface of native virions broadly neutralizing antibodies and the search for an hiv-1 vaccine: the end of the beginning towards a solution to mers: protective human monoclonal antibodies targeting different domains and functions of the merscoronavirus spike glycoprotein hiv therapy by a combination of broadly neutralizing antibodies in humanized mice human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing improving neutralization potency and breadth by combining broadly reactive hiv-1 antibodies targeting major neutralization epitopes automatic crystal centring procedure at the ssrf macromolecular crystallography beamline processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software coot: model-building tools for molecular graphics phenix: building new software for automated crystallographic structure determination pymod 2.0: improvements in protein sequence-structure analysis and homology modeling within pymol quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors drug combination studies and their synergy quantification using the chou-talalay method we would like thank dr. changfa fan (division of animal model research, institute for laboratory animal resources, national institutes for food and drug control) for help in providing the r26-hdpp4 mouse model and experimental method. we thank dr. jianhua he and the staff scientists at the ssrf bl17u beam line, as well as dr. shilong fan at the x-ray crystallography platform of the tsinghua university technology center for assistance in diffraction data collection. this work was supported by the national key plan for scientific research and development of china (grants 2016yfd0500307 and 2016yfd0500301), the national natural science foundation of china (grants 31470751 and u1405228), and the national major project for control and prevention of infectious disease in china (2016zx10004001-003). h.z., w.t., l.z. and x.w. designed the experiments. y.c., k.q. and w.t. isolated the antibody 7d10 and sequenced the corresponding v l and v h genes. b.h. carried out the neutralizing assay with live mers-cov. h.z. and s.z. expressed, purified, and crystallized the protein, and h.z. carried out the bli and spr analysis. h.z. conducted all the neutralizing assays based on pseudotyped mers-cov with the help of w.j. h.z. conducted dpp4-competition assays and the western blots analysis. p.n. performed the protection assay in mice. h.z. and j.l. collected the diffraction data. h.z. and x.w. processed the diffraction data, determined, and analyzed the structure. h.z. and x.w. wrote the paper with contributions from l.z. and w.t. supplementary information accompanies this paper at https://doi.org/10.1038/s41467-019-10897-4.competing interests: the authors declare no competing interests.reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ peer review information: nature communications thanks the anonymous reviewers for their contribution to the peer review of this work. peer reviewer reports are available.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-349661-ppw80s0l authors: al ghobain, mohammed; aldrees, turki; alenezi, abdullah; alqaryan, saleh; aldabeeb, dana; alotaibi, najed; aldhabib, abdulrahman; alghalibi, shaker; alharethy, sami title: perception and attitude of emergency room resident physicians toward middle east respiratory syndrome outbreak date: 2017-04-10 journal: emerg med int doi: 10.1155/2017/6978256 sha: doc_id: 349661 cord_uid: ppw80s0l introduction. middle east respiratory syndrome (mers) outbreaks have had a considerable negative impact on health systems in saudi arabia. we aimed to study the psychological impact of a middle east respiratory syndrome coronavirus (mers-cov) outbreak on emergency room resident physicians (errps). methods. we assessed the mers-related psychological impact and concerns of errps using a self-report questionnaire. results. the majority (91%) of the errps agreed that their work put them at risk of infection, but most (65%) did not agree that they should not be looking after patients infected with mers. despite that, 54% of errps reported being afraid of contracting the infection from infected patients and only 4.2% of them were willing to change their current job. the majority of the errps (85%) felt that their job would expose their families to risk of infection. conclusions. our study demonstrated the considerable psychological impact of mers outbreaks on errps. the errps' concerns and the psychological impact of mers outbreaks should be considered in greater detail by hospital policymakers. middle east respiratory syndrome (mers) is a deadly and contagious disease caused by a novel coronavirus (mers-cov) that has had an intensive impact on the healthcare system. the mers-cov can be transmitted in different ways, including direct contact with dromedaries, living with an infected person, or contact with infected persons inside healthcare facilities. the disease has a high mortality rate of up to 36% [1] . according to the world health organization (who), mers is a threat to the entire world and thus needs prompt attention and action [1] . mers was first identified in saudi arabia in 2012 [2] . apart from saudi arabia, nearly 22 countries including kuwait, qatar, indonesia, uk, uae, south korea, and usa have reported mers cases. the who has reported 1,621 laboratory-confirmed cases of infection with mers-cov, and there have been nearly 584 deaths related to mers-cov since september 2012 [3] . dromedary camels are the primary animal host for mers-cov [4, 5] . people with mers have had a history of close contact with camels and drinking camel milk. however, human-to-human transmission has also been documented [6] . the majority of patients who become infected with the virus in the community seek their initial medical help from primary healthcare centers or from the emergency department in which emergency staff, including emergency 2 emergency medicine international physicians and nurses, are the first-line providers of care for suspected and unscheduled patients that require immediate attention. in addition, the emergency room resident physicians (errps) are at risk of falling ill from exposure to mers patients and the possibility of spreading the illness to their family members, which could affect their overall effectiveness and willingness to work during a mers outbreak. therefore, their anxiety and fears must be taken seriously and strategies should be incorporated to manage them. the mers outbreak in saudi arabia was a very good opportunity to explore these concerns. to the best of our knowledge, no study has explored errps' perceptions and attitude toward mers or the psychological impact of mers on errps in hospitals. thus, the present study aimed to assess the perceptions, attitude, and psychological impact of mers on errps. a cross-sectional study was conducted using a self-administered questionnaire. the study was conducted in the emergency department after obtaining ethical approval from the king saud university research center. all residents were informed regarding the purpose of the study and their verbal consent was given. the study was conducted in four tertiary care hospitals in riyadh, saudi arabia: king khalid university hospital, king fahad medical city hospital, king abdulaziz medical city hospital, and king faisal specialist. we included all errps working in these four hospitals between november 2015 and december 2015 after a major outbreak of coronavirus (mers-cov) in saudi arabia. data were collected using a self-administered questionnaire. the questionnaires were distributed and collected during the regular weekly residents' education activities. the questionnaire contained a brief description of the study's objectives and statements assuring participants of their anonymity and right to stop participating of their own volition. the questionnaire was adapted and modified, based on a review of the literature, from one used in a previous study by wong et al. [7] . the questionnaire comprised two parts. the first part contained questions about participants' background information, including age, marital status, training level, and knowledge of mers. the second part comprised four main sections that included an assessment of (1) work-related concerns, (2) non-work-related concerns, (3) the perceived impact of mers on errps, and (4) their preparedness for a mers outbreak. all of the descriptive data were summarized as percentages and frequencies. categorical variables were compared using the chi-square test. all statistical analyses were performed using ibm spss statistics 21 (ibm inc., armonk, ny). the total sample size of this study was 94 errps (out of 137 errps; response rate 69%). the errps had a mean age of 27 years, 65% were male, and 37% were married. fifty-six percent of the errps rated their knowledge about mers-cov as inadequate while 63% had had direct contact with at least one patient infected with mers-cov (table 1) . the majority of the errps (91%) agreed that their work put them at risk of infection, but most (65%) did not agree that they should not be looking after patients infected with mers. despite that, 54% of errps were afraid of contracting the infection from infected patients and only 4.2% of them were willing to change their current job. the majority felt that their job would expose their families (85%), parents (83%), and close friends (77%) to risk of infection (table 2) . table 3 summarizes the perceived impact of mers-cov and readiness for an outbreak. of the errps, 48% would be afraid of telling their families about the risk of exposure, 42% agreed that there would be inadequate staff in the workplace to handle the increased demand, and 42% would feel more stressed at work. furthermore, 57% of errps had received infection control training and 55% reported that their hospitals have enough infection control staff to deal with such an outbreak. there were no significant differences in level of concern by rotation except that first year training levels residents were more likely to be afraid of being infected than other training levels ( < 0.003). there were also no significant differences in terms of knowledge of mers but respondents who had previously provided care for mers patients were more likely to agree that the job put them at risk and were more afraid of being infected ( = 0.016 and = 0.040, respectively) (data not shown in tables). our study highlights the perceptions, attitude, and psychological impact of mers outbreaks among errps in saudi arabia. as mers was a new outbreak that began in saudi emergency medicine international 3 arabia, to our knowledge, this was the first study to address the impact of a mers outbreak among errps. the findings of our study showed that the majority of errps believed that their work put them at risk of infection and they were afraid of contracting mers. the majority of the errps felt that their job would expose their families, parents, and close friends to risk of infection. a small pilot study conducted in a single center in jeddah, saudi arabia, in which the majority of respondents were nurses from the philippines, did address the emotions and coping strategies of the healthcare workers who faced a mers-cov outbreak. it was found that the main sentiments centered upon fear for personal safety and the well-being of colleagues and family. their fear was alleviated by positive attitudes in the workplace, clinical improvement of infected colleagues, and stoppage of disease transmission among healthcare workers after adopting strict protective measures. the most important emotion that drove them to continue working was their ethical and professional obligation toward their profession [8] . our findings are similar to those of other non-mers viral outbreaks reported from different parts of the world. for instance, during the severe acute respiratory syndrome outbreak (sars) in 2003, approximately two-thirds of healthcare workers in toronto experienced concern for their own or their family's health [9] while 68% of healthcare workers in hong kong reported a high level of stress and 57% were found to have experienced psychological distress [10] . a study of the psychological impact of the 2003 outbreak of sars on hospital employees in beijing, china, found that about 10% of the respondents had experienced high levels of posttraumatic stress symptoms [11] . an interesting finding of our study was that most errps did not agree that they should not be looking after patients infected with mers. we think that these finding are reflections of the high standards of ethical values of errps and their professional obligation toward their jobs and patients. in contrast, a study conducted, in australia, anticipated a high rate of absenteeism among hospital staff, reaching up to 36% in response to influenza pandemic hypothesized survey scenario [12] . more than half of errps had attended infection control training, and they reported that their hospitals had enough 4 emergency medicine international infection control staff to deal with such an outbreak and their hospitals have a clear plan to handle a mers outbreak. this is a positive aspect and a positive learning experience from a mers outbreak as it reflects good preparation and a high level of awareness of dealing with such an outbreak. how hospitals plan to handle a mers outbreak was included but not limited to cancellation of outpatient clinics, visitor restrictions, mandatory wearing of protective measures (e.g., masks), limited hospital entrance, mandatory screening of suspect infected visitors, and public awareness and education. the limitations of our study include it having a crosssectional design, possible reporting bias, and being conducted in a single city in saudi arabia with a small sample size limited to a single profession (i.e., errps). however, the study was conducted in four major hospitals in saudi arabia that all treated and handled patients with mers infection, which justifies the generalization of our findings. we limited our study to errps, as we believe that they are the healthcare workers at the highest risk of exposure because they deal with suspected cases before the diagnosis is established. the sample size was representative and reflects the real number of errps working in these hospitals during the outbreak. our study demonstrated the considerable psychological impact of mers outbreaks on errps. the errps' concerns and the psychological impact of mers outbreaks should be considered in greater detail by hospital policymakers. no financial support was obtained for this study. middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) evidence for camel-to-human transmission of mers coronavirus mers coronaviruses in dromedary camels spread of mers to south korea and china concerns, perceived impact and preparedness in an avian influenza pandemic-a comparative study between healthcare workers in primary and tertiary care healthcare workers emotions, perceived stressors and coping strategies during a mers-cov outbreak psychosocial effects of sars on hospital staff: survey of a large tertiary care institution severe acute respiratory syndrome (sars) in hong kong in 2003: stress and psychological impact among frontline healthcare workers the psychological impact of the sars epidemic on hospital employees in china: exposure, risk perception, and altruistic acceptance of risk how would australian hospital staff react to an avian influenza admission, or an influenza pandemic? this study was supported by the college of medicine research center, deanship of scientific research, king saud university, kingdom of saudi arabia. key: cord-331558-6rqd3fmj authors: sun, chuan-bin; wang, yue-ye; liu, geng-hao; liu, zhe title: role of the eye in transmitting human coronavirus: what we know and what we do not know date: 2020-04-24 journal: front public health doi: 10.3389/fpubh.2020.00155 sha: doc_id: 331558 cord_uid: 6rqd3fmj the outbreak of the current 2019 novel coronavirus (2019-ncov, now named sars-cov-2) infection has become a worldwide health threat. currently, more information is needed so as to further understand the transmission and clinical characteristics of 2019-ncov infection and the infection control procedures required. recently, the role of the eye in transmitting 2019-ncov has been intensively discussed. previous investigations of other highly infectious human covs, that is, severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov), may provide useful information. in this review, we describe the genomics and morphology of human covs, the epidemiology, systemic and ophthalmic manifestations, and mechanisms of human cov infection, and recommendations for infection control procedures. the role of the eye in the transmission of 2019-ncov is discussed in detail. although the conjunctiva is directly exposed to extraocular pathogens, and the mucosa of the ocular surface and upper respiratory tract are connected by the nasolacrimal duct and share the same entry receptors for some respiratory viruses, the eye is rarely involved in human cov infection, conjunctivitis is quite rare in patients with 2019-ncov infection, and the cov rna positive rate by rt-pcr test in tears and conjunctival secretions from patients with 2019-ncov and sars-cov infection is also extremely low. this suggests that the eye is neither a preferred organ of human cov infection nor a preferred gateway of entry for human covs for infecting the respiratory tract. however, pathogens that the ocular surface is exposed to might be transported to nasal and nasopharyngeal mucosa by constant tear rinsing through the lacrimal duct system and then cause respiratory tract infection. considering that close doctor-patient contact is quite common in ophthalmic practice and is apt to transmit human covs by droplets and fomites, strict hand hygiene and proper personal protection are highly recommended for health care workers to avoid hospital-related viral transmission during ophthalmic practice. coronavirus (cov) is an enveloped single-stranded positive-sense rna virus that typically causes respiratory and enteric infections affecting both human and wild animals (1) (2) (3) . since first being identified in the 1960s, human covs were considered relatively benign and usually caused mild upper respiratory tract infections (the common cold) until the emergence of the severe acute respiratory syndrome coronavirus (sars-cov) in 2002 and, later, the middle east respiratory syndrome coronavirus (mers-cov) in 2012 (4) . the latter two covs can result in severe lower respiratory tract infection, rapidly proceeding to pneumonia, and have caused thousands of cases of infection and hundreds of deaths in about 30 countries, respectively (2, 4) . in december 2019, another outbreak of highly infectious pneumonia caused by a novel coronavirus (2019-ncov, now named as sars-cov-2) emerged in wuhan, china, and soon became a major global health threat (2, 3) . currently, more detailed information about the transmission of 2019-ncov is urgently needed to prevent its pandemic spread. human covs mostly spread through respiratory droplets expelled by infected individuals and direct contact with viruscontaminated fomites (4) . anatomically, the conjunctiva of the eye is easily exposed to infectious droplets and fomites during close contact with infected individuals and contaminated hands. some respiratory viruses such as human adenovirus (species d) and avian influenza virus (h7) frequently cause highly infectious conjunctivitis or keratoconjunctivitis. hence, conjunctiva is postulated to be an important portal of entry for respiratory viruses, while tear and conjunctival secretions may contain virus and spread viral infection (4, 5) . however, the role of the eye in the transmission of human covs is still under discussion, as considerable controversy exists. this review presents the genomics and morphology of human covs, the epidemiology, systemic and ophthalmic manifestations, and mechanisms of human cov infection, and the role of the eye in the transmission of human covs. infection control procedures and personal protective equipment against human cov transmission in ophthalmic practice are also reviewed. covs have an enveloped single positive-strand rna genome with a 5 ′ -terminal cap structure and a poly(a) sequence at the 3 ′ end. cov genome is approximately 30 kb (27∼32 kb) long and is the largest rna genome known so far (1, 4, 6) . covs belong to the family coronaviridae and the order nidovirales and are classified into four genera: α-cov, β-cov, γ-cov, and δ-cov (1, 6, 7) . until now, a total of seven human covs have been identified, namely hcov-229e, hcov-nl63, hcov-oc43, hcov-hku1, sars-cov, mers-cov, and, recently, 2019-ncov (1-3, 6-8). the former two human covs belong to the genus α-cov, and the latter five human covs belong to the genus β-cov. three recently identified human covs, that is, sars-cov, mers-cov, and 2019-ncov, have been recognized as zoonotic viruses, which transmit between animals and human. recent studies revealed that sars-cov was transmitted from civet cats to humans, mers-cov from dromedary camel, and 2019-ncov (probably) from pangolin (1, 2, (6) (7) (8) (9) . recent investigations indicated that bats were most probably the natural reservoir of sars-cov, mers-cov, and 2019-ncov (1, 6, (9) (10) (11) . genome sequence analysis revealed that 2019-ncov was distinct from sars-cov (about 79% identity) and mers-cov (about 50% identity) yet more closely related to sars-like-covs (about 88% identity) in bats (10, 11) . cov particles have a spherical or elliptical shape with a diameter of about 100 nm (50∼200 nm). they carry three major structural proteins in the envelope and contain a helical nucleocapsid formed by the viral genomic rna and the viral nucleoprotein. the viral spike protein has receptor-binding and fusogenic functions and is essential for initiation of cov infection (1, 8, (12) (13) (14) . further three-dimensional structure analyses suggest that the spike protein is composed of two subunits: s1, which mediates sars-cov binding to receptors on host cell membranes, and s2, which triggers the membrane fusion between the virus and host cells (11, 13) . four human covs, that is, hcov-229e, hcov-nl63, hcov-oc43, and hcov-hku1, are usually low in infectiousness and primarily infect the upper respiratory tract, causing mild respiratory symptoms (the common cold), whereas the other three human covs, sars-cov, mers-cov, and 2019-ncov, are zoonotic and highly infectious and predominantly cause severe lower respiratory tract infection which can rapidly proceed to pneumonia (1-3, 8, 15, 16) . the outbreak of sars in 2002 in china resulted in 8,098 cases and 774 deaths (case-fatality rate, 9.6%) in 37 countries, and the outbreak of mers in 2012 in middle east countries led to 2,494 cases and 858 deaths (case-fatality rate, 34%) in 27 countries (2) . as of february 24, 2020, 2019-ncov has caused 77,262 cases and 2,595 deaths in china, and 2,069 cases and 23 deaths in 29 other countries (total case-fatality rate, 3.3%) (15) (16) (17) . hence, although 2019-ncov can cause a severe respiratory disease like sars and mers, it appears to be less pathogenic than sars-cov and much less so than mers-cov. however, the number of 2019-ncov infected patients in the first two months was nearly 10 times that of sars patients in total, which indicated that 2019-ncov is more transmissible than sars-cov and mers-cov (16) . human covs primarily spread by virus-containing droplets or aerosols expelled by infected individuals when patients cough, talk loudly, or sneeze. direct contact with virus-contaminated fomites is also a route of human cov transmission (4, 8, (18) (19) (20) . recently, sars-cov and 2019-ncov have also been detected in stool and urine samples from patients by rt-pcr assay and have been isolated from the mucous membranes of gastrointestinal tract in a few cases (9, 16, 21) . hence, fecal-oral route may also be a route of transmission for sars-cov and 2019-ncov. the clinical features of coronavirus disease 2019 (covid-19) are similar to those of sars and mers. most patients present with fever, dry cough, dyspnoea, and bilateral ground-glass opacities on chest ct scans (2, 3, (22) (23) (24) . however, covid-19 rarely results in notable symptoms of upper respiratory tract infection (e.g., rhinorrhea, sneezing, or sore throat), which are commonly manifested in sars and mers. some covid-19 patients even manifest no apparent respiratory symptoms at onset, which never occurred in sars and mers (24) (25) (26) . mathematical models have revealed that the 2019-ncov virus may replicate very slowly in the first days after infection and that it could be below detection levels during the first four days post infection (26) . recent investigations have also revealed that covid-19 occasionally manifests as enteric infection symptoms such as diarrhea, whereas about 20∼25% of patients with mers or sars had diarrhea (25) . moreover, more than 80% of covid-19 has manifested as mild or moderate pneumonia, and the severe covid-19 has mostly occurred in the patients of over 60 years old, usually accompanied by at least one underlying disorder, for example, cardiovascular disorders, diabetes, chronic obstructive pulmonary disease, and hypertension (24) . the eye is rarely involved in human cov infection. until now, conjunctivitis has been reported in only five cases with 2019-ncov infection, and in four cases with hcov-nl63 infection, whereas no conjunctivitis or other ocular complications have been reported in patients with sars-cov and mers-cov infection (4, (27) (28) (29) (30) (31) . recently, human cov rna in tears and conjunctival scraping samples were tested by reverse transcription-polymerase chain reaction (rt-pcr) assay in patients with sars and covid-19, yet the positive rate of the rt-pcr test was extremely low (4, 30-37). loon and colleagues detected sars-cov in tear samples from 36 consecutive sars suspects (eight patients were laboratoryconfirmed later) by rt-pcr (32). sars-cov was positive only in three of the eight sars cases. three patients whose tears were sars-cov positive were sampled in the early phase of their illness (on days 3, 4, and 9 after onset of fever, respectively), whereas the other five sars cases, whose tears were sars-cov negative, were sampled in the later phase (mean 19.4 days) of their illness (32) . nearly at the same time, chan and colleagues reported their negative results of sars-cov testing in tear and conjunctival scraping samples from 20 probable sars patients (17 patients were laboratory-confirmed later) by rt-pcr and virus culture (33) . among 17 confirmed sars patients, 6, 8, and 3 cases were recruited during the first, second, and third week of their diseases, respectively. sars-cov rna was not detected by rt-pcr and sars-cov was not isolated in virus culture in any of the tear and conjunctival scraping samples (33) . leong and colleagues tested for sars-cov in 126 conjunctival specimens from 64 sars patients in the convalescent phase by rt-pcr but did not detect sars-cov in any of the patients' conjunctival samples (22) . on january 22, 2020, a chinese respiratory specialist who visited wuhan as a member of the national expert panel on pneumonia claimed that he was infected by 2019-ncov despite being fully gowned with a protective suit and n95 respirator (34) . his first clinical manifestation was unilateral conjunctivitis, followed by fever and catarrhal symptoms 2 or 3 h later. he postulated that 2019-ncov probably first infected the conjunctiva, then spread and cause viral pneumonia (34) . soon after his report, health care personnel in china were urged to use eye protection when they were in close contact with covid-19 patients or suspected patients. however, zhou and colleagues, in a preprint posted at medrxiv, reported that conjunctivitis was identified only in one patient out of 63 covid-19 cases and 4 suspected covid-19 cases (27) . conjunctivitis was also the first symptom of 2019-ncov infection in this patient. however, 2019-ncov rna tested by rt-pcr was positive and probably positive in conjunctival swab samples from only one and two covid-19 cases without conjunctivitis, respectively. none of the above three patients had ocular symptoms. 2019-ncov rna was not detected in conjunctival swab samples from the covid-9 patient complicated by conjunctivitis, who was an anesthesiologist. her ocular symptoms occurred soon after performing tracheal intubation for a patient who was confirmed as having covid-19 later, and this was followed by fever and cough. unfortunately, the personal protections used by this anesthesiologist during the tracheal intubation procedures were only a surgical mask, cap, and gloves, without a gown, face shield or goggles. her five colleagues were also infected by the same patient, yet none of them exhibited any ocular complications (27) . more recently, two investigative groups from china simultaneously reported conjunctivitis and 2019-ncov rnapositive tests in conjunctival swab samples from covid-19 patients (28, 31) . zhang and colleagues, in a preprint posted at medrxiv, reported conjunctivitis in two patients out of 72 laboratory-confirmed covid-19 cases; however, 2019-ncov was detected in conjunctival swab samples by rt-pcr in only one patient who was a nurse working in the emergency department (28) . this patient presented with excessive tearing and redness in both eyes, which were typical ocular manifestations of viral conjunctivitis, accompanied by a moderate fever of 38.2 • c that occurred 1 day earlier. 2019-ncov rt-pcr tests for the conjunctival and oropharyngeal swabs sampled 2 days after the onset of fever was positive, but for those sampled 9, 18, and 20 days after the onset of fever were all negative (28) . xia and colleagues reported unilateral conjunctivitis in one patient out of 30 confirmed covid-19 cases; conjunctival swabs sampled from this patient 3 and 5 days after the onset of covid-19 were both positive for 2019-ncov by rt-pcr, whereas 58 conjunctival swab samples from the other 29 covid-19 patients were all negative for 2019-ncov (31) . however, 2019-ncov was not isolated and cultured in the conjunctival swab samples from the covid-19 patient with conjunctivitis. in contrast, 55 of the 60 sputum samples from 30 covid-19 cases showed positive pcr results for 2019-ncov (31) . although tears have been reported by the world health organization in 2003 to be one of the body fluids that might contain sars-cov, the infectivity and clinical importance is not yet understood (35) . recent investigations have revealed that highly infectious human covs (mainly sars-cov and 2019-ncov) are rarely detected by rt-pcr and never isolated by virus culture in tears and conjunctival secretions from sars and covid-19 patients (27) (28) (29) (30) (31) (32) (33) (34) 36) . hence, it is hard to assess the infectivity of tears and conjunctival secretions and their roles in virus transmission. the extremely low positive rate of human cov rna test by rt-pcr in tears and conjunctival secretions from patients with sars and covid-19 may have several interpretations. firstly, the sensitivity of rt-pcr testing still needs to be improved. previous reports on the sensitivity of rt-pcr in excretions reported a range from 50% to 60% (33, 37) . in current clinical practice, some suspected 2019-ncov cases often had 2∼3 repeated tests of nasopharyngeal swabs before the positive results were obtained (28) . the need remains for a highly sensitive and specific pcr test to diagnose human cov infections. secondly, the samples were not collected at the right time. recent evidence indicated that human cov rna-positive cases were all sampled in the early part of the disease course, whereas human cov rna-negative cases sampled in the later or convalescent phase of their illness (33) . de wit and colleagues demonstrated that, based on their rhesus macaque model study, mers-cov rna could be detected in the conjunctiva only within 6 days post infection (38) . hence, it is reasonable to postulate that human cov may present in tears only for a short period during the early phase of the disease. thirdly, the contribution of antimicrobial agents, including lactoferrin and secretory iga, in tears and constant tear rinsing, which continuously eliminates the virus on the ocular surface into the nasal cavity through the nasolacrimal duct (37, 39, 40) , should be considered. lactoferrin can inhibit the binding of sars-cov to its entry receptor, angiotensin-converting enzyme 2 (ace2), by preventing the adhesion of sars-cov to its attachment receptor, heparan sulfate proteoglycans (hspgs) (41) . secretory iga is another important antimicrobial agent in tears that helps to kill both bacteria and viruses. the host immune system can be activated and result in a significant increase in lactoferrin and secretory iga levels in tears and circulating igm level in plasm on the 3rd to 5th day and circulating igg level in plasm on the 10th to 15th day after cov infection or inoculation (39, 41) , which may contribute to why cov rna presents only in the early phase of the disease. fourthly, the collection technique may not appropriate. the world health organization highly recommends the use of only synthetic fiber swabs with plastic shafts rather than calcium alginate swabs or swabs with wooden shafts for specimen sampling, as the latter two types of swabs may contain substances that inactivate some viruses and inhibit pcr testing (40) . topical anesthesia is also not recommended for tear and conjunctival scraping sampling, for a topical anesthetic agent maybe also have a negative influence on the viability of viruses (40) . moreover, the volume of tears collected when sampling may also have some influence on the positivity of the rt-pcr test. anatomically, the mucosa of the ocular surface (i.e., conjunctival and corneal epithelia) and the upper respiratory tract are connected by the nasolacrimal duct (4). when dropped into the eye, liquid is partially absorbed by the cornea and conjunctiva but mostly drained into the nasal cavity through the nasolacrimal duct and then transported toward the lower part of the respiratory tract, including the nasopharynx and trachea, or swallowed into the gastrointestinal tract (37) . this allows pathogens to which the eye is exposed to be transported to respiratory and gastrointestinal tract mucosa. moreover, previous investigations have revealed that the mucosa of the ocular surface and respiratory tract share the same receptors for some respiratory viruses (4, (42) (43) (44) . ace2, the entry receptor of sars-cov, hcov-nl63, and 2019-ncov, is highly expressed on human lung alveolar epithelial cells, enterocytes of the small intestine, and the proximal tubular cells of the kidney (4, 42) . positive expression of ace2 was also detected in human conjunctival and corneal epithelial cells; however, ace2 expression in human ocular surface is much lower than in human lung and kidney tissues (43) . the binding capability of ace2 protein on conjunctival epithelial cells to sars-cov spike protein is much lower than that on vero e6 cells and that in lung tissues (44) . the efficacy of virus entry into host cells depends on three points: the invasiveness of the virus, viral receptors on host cell membrane, and the immune conditions of the host. the virus binding to host cell membrane by its receptors is the first and key step for viral invasion. ace2, a metallopeptidase, also the entry receptor of sars-cov, hcov-nl63, and 2019-ncov, is responsible for binding to spike protein on the sars-cov and hcov-nl63 surface and mediating sars-cov and hcov-nl63 entry into host cells (4, 11, (42) (43) (44) (45) , while mers-cov and most α-covs have been identified to utilize dipeptidyl peptidase 4 and aminopeptidase n as an entry receptor of their host cells, respectively (46) . further investigations have revealed that the invasion of sars-cov and hcov-nl63 into host cells not only relies on the presence of ace2 on host cell membrane as an entry receptor but also is modulated by other factors on host cell membranes such as hspgs, which serve as attachment receptors (40, 45, 47) . at present, the mechanism of human cov invasion into host cells is still not clear. lang and milewska described the possible mechanism of ace2-mediated host cell entry for sars-cov and hcov-nl63 virus (41, 45, 47) . first, the virus docked and bound to host cells by the interaction between the spike protein on viral surface and heparan sulfate chains of hspgs on host cell membrane. this action facilitated further binding of spike protein on viral surface to its entry receptor, ace2, on host cell surface. then, the binding of spike protein of the virus to ace2 protein of host cell membrane triggered the recruitment of clathrin, followed by clathrin-mediated dynamindependent endocytosis of viral particles, which required actin cortex remodeling (39, 45, 47) . considering the 2019-ncov has similar spike protein to sars-cov, and also uses ace2 as its entry receptor to infect host cells, it is reasonable to presume that 2019-ncov has the same invasive strategy for host cell entry as sars-cov and that hspgs may also act as attachment receptors during the entry of 2019-ncov into its host cells. patients infected by 2019-ncov, similar to sars cases, mostly present with non-specific symptoms such as fever, dry cough, and dyspnoea, or, in some cases, no evident symptoms, at the early phase of the disease (9, 16, (23) (24) (25) (26) (27) 48) . hence, it is a challenging task for health care professionals in the northern hemisphere to distinguish early 2019-ncov infection from influenza and other respiratory viral infections in the seasons of winter and spring when respiratory diseases frequently break out (48) . hospitalrelated viral transmission, especially transmissions between patients and health-care workers, is frequently reported just before the outbreak of a highly infectious novel respiratory virus such as sars-cov and 2019-ncov (8) . previous investigations have revealed that patients infected by a novel virus never identified before can easily transmit the pathogen to health personnel without enough personal protection; the latter getting infected will further become a source of spread and soon cause hospital-related viral transmission (8, (49) (50) (51) (52) (53) . in fact, 386 of 1,755 patients (21.9%) and eight deaths were health-care workers during the sars outbreak (49) . as of february 11, 2020, a total of 3019 medical health workers have been infected by 2019-ncov in china, among whom 1,716 cases were laboratoryconfirmed covid-19, and five cases passed away, including an ophthalmologist named wenliang li, the whistleblower of 2019-ncov infection in china (9, 16) . at present, the physicochemical properties of 2019-ncov are not yet clear. based on previous experience in sars-cov and mers-cov infection control, it is postulated that 2019-ncov is sensitive to ultraviolet irradiation and heating. sterilization can be achieved by heating at 56 • c for 30 min and by lipid solvents including 75% ethanol, chlorine-containing disinfectant, peroxyacetic acid, and chloroform but not by chlorhexidine (50) (51) (52) . many ophthalmic instruments, i.e., probes for atype and b-type ultrasound, ocular contact lenses such as the goldmann three-mirrored lens and gonioscope, trial frames, slitlamp microscope, direct ophthalmoscope, automatic perimeter, and fundus camera, are frequently used by direct or close contact with patients and may act as media for virus spread. a non-contact tonometer may create an aerosol when measuring intraocular pressure by punching air onto the cornea of patients; hence, it may also facilitate virus spread by aerosol transmission. therefore, complete sterilization by 75% ethanol or hydrogen peroxide cleaning or immersion should be performed soon after each use of above the ophthalmic instruments (50) (51) (52) . complete sterilization using chlorine-containing disinfectant, peroxyacetic acid, and hydrogen peroxide is mandatory for clinics and operating rooms. hand washing, preferably with the use of chlorhexidine alcoholic hand rub, after each ophthalmic examination or therapeutic procedure is highly recommended for the prevention of cross-infection. routine ophthalmic examinations such as slit-lamp examination and direct ophthalmoscopy are all performed by close contact, which means that the ophthalmologists can easily be exposed to the droplets and tears or ocular secretions from, or to the ophthalmic instruments contaminated by, patients with or suspected of having sars, mers, or covid-19. hence, strict hand hygiene and proper personal protection equipment, including masks, gowns, gloves, and goggles, are highly recommended to avoid hospital-related viral transmission during ophthalmic practice (49) (50) (51) (52) (53) . when an ophthalmologist examines general ophthalmic outpatients, primary personal protection with disposable cap, surgical mask, and gown is recommended. when high-risk procedures are performed on these patients, for example, direct ophthalmoscopy, lacrimal irrigation and probing, intraocular pressure measurement with non-contact tonometry, ophthalmic laser therapy, and ophthalmic surgeries, n95 respirator, gloves, and goggles or face shield, are highly recommended (50) . for patients with confirmed or suspect sars, mers, or covid-19, any ophthalmic consultation should be completed within the quarantine ward to avoid cross-infection. personal protective equipment, including disposable caps, n95 respirator, goggles, face-shields, gloves, top and pants, and protective gowns, should be worn at all times (51, 53) . moreover, hand washing, preferably with the use of a chlorhexidine alcoholic hand rub, and gloves changed after each high-risk procedure are mandatory to prevent crossinfection. ophthalmic personnel are also recommended not to touch their goggles, face shield, surgical/n95 mask, eye, head, and neck region before the handwashing procedure is completed (51, 53) . non-urgent ophthalmic operations and interventions, for example, cataract operations, ophthalmic plastic surgeries, squint extraocular muscle surgeries, intravitreal anti-vegf injection, retinal photocoagulation, and yag: nd laser capsulotomy should be delayed if possible (50) (51) (52) (53) . ophthalmic emergencies such as acute angle-closure glaucoma and severe ocular injury should be operated upon immediately, but the operating theater should be regarded a high-risk area, and the use of proper personal protection equipment (i.e., disposable caps, n95 respirators, face shields, goggles, surgical gowns, and gloves) should be practiced strictly. when ophthalmic emergency surgeries are performed on patients with confirmed or suspected sars, mers, or covid-19, the recommended personal protection equipment are similar to those for ophthalmic consultation of these patients. to avoid aerosol transmission during tracheal intubation, local ophthalmic anesthesia is highly recommended rather than general anesthesia, and patients should wear n95 respirators during ophthalmic surgeries under local anesthesia (51, 53) . the outbreak of the current 2019-ncov infection has become a worldwide health threat. although respiratory droplets and direct contact have been identified as the main routes of transmission for human covs, the role of the eye in transmitting human covs is still under discussion. considering that the conjunctiva of the eye is directly exposed to infectious droplets and fomites during close contact with infected individuals and contaminated hands and that the mucosa of the ocular surface and the upper respiratory tract are connected by the nasolacrimal duct and share the same entry receptors for some respiratory viruses, it is reasonable to postulate three roles that the eye may play in human cov infection. firstly, it may be a target organ for human covs. secondly, the conjunctiva may be a portal of entry for or a transporter of human covs to infect the respiratory tract. thirdly, tears and conjunctival secretions may act as media that spread human covs. however, the eye is rarely involved in sars-cov, mers-cov, and 2019-ncov infection; conjunctivitis has been reported in only five cases with covid-19 but never in sars and mers patients. this suggests that the eye is neither a preferred organ for human cov infection nor a preferred gateway of entry that enables human covs to infect the respiratory tract. although it is quite rare, the possibility cannot be excluded that pathogens exposed to the eye might be transported to nasal and nasopharyngeal mucosa by constant tear rinsing through the lacrimal duct system and then cause respiratory tract infection, since mild to moderate symptomatic sars can be developed in a cynomolgus macaques model by nasal and conjunctival sars-cov inoculation as well as by nasal and bronchial sars-cov inoculation (4, 54) . moreover, the extremely low positive rate of human cov rna tests by rt-pcr in tears and conjunctival secretions from patients with sars and covid-19 may be related to the relatively low sensitivity of the current rt-pcr technique, later timing sample collection, and the activation of the host immune system and significant increases in lactoferrin and secretory iga levels in tears and in circulating igm and igg levels in plasm. hence, current negative rt-pcr results cannot exclude the possibility of the presence of sars-cov and 2019-ncov in tears and conjunctival secretions. considering that close doctor-patient contact is quite common in ophthalmic practice and is apt to transmit human covs via droplets and fomites, strict hand hygiene and proper personal protection are highly recommended for health care workers to avoid hospital-related viral transmission during ophthalmic practice. emerging coronaviruses: genome structure, replication, and pathogenesis preparation for possible sustained transmission of 2019 novel coronavirus: lessons from previous epidemics a new coronavirus associated with human respiratory disease in china ocular tropism of respiratory viruses viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects emerging threats from zoonotic coronavirusesfrom sars and mers to 2019-ncov structure, function, and evolution of coronavirus spike proteins the novel coronavirus: a bird's eye view special expert group for control of the epidemic of novel coronavirus pneumonia of the chinese preventive medicine association. an update on the epidemiological characteristics of novel coronavirus pneumonia(covid-19) a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding viral and cellular mrna translation in coronavirus-infected cells ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence the molecular biology of coronaviruses tracking the epidemic the novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china covid-19) situation report-35 what to do next to control the 2019-ncov epidemic? respiratory virus shedding in exhaled breath and efficacy of face masks infection and rapid transmission of sars-cov-2 in ferrets virus-specific rna and antibody from convalescent-phase sars patients discharged from hospital clinical characteristics of 138 hospitalized patients with epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan inhost modelling of covid-19 kinetics in humans ophthalmologic evidence against the interpersonal transmission of 2019 novel coronavirus through conjunctiva. medrxiv the evidence of sars-cov-2 infection on ocular surface human coronavirus nl63, france identification of a new human coronavirus evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov-2 infection the severe acute respiratory syndrome coronavirus in tears tears and conjunctival scrapings for coronavirus in patients with sars peking university hospital wang guangfa disclosed treatment status on weibo and suspected infection without wearing goggles. xinjing newpaper update 27 -one month into the global sars outbreak: status of the outbreak and lessons for the immediate future conjunctiva-upper respiratory tract irrigation for early diagnosis of severe acute respiratory syndrome sars and mers: recent insights into emerging coronaviruses immunoglobulin a as an early humoral responder after mucosal avian coronavirus vaccination centers for disease control and prevention. interim guidelines for collecting, handling, and testing clinical specimens from persons under investigation (puis) for coronavirus disease inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis expression of sars coronavirus s proteinfunctional receptor-angiotensin-converting enzyme 2 in human cornea and conjunctiva mechanism of the action between the sars-cov s240 protein and the ace2 receptor in eyes entry of human coronavirus nl63 into the cell dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc human coronavirus nl63 utilizes heparan sulfate proteoglycans for attachment to target cells guidelines for the diagnosis and treatment of novel coronavirus (2019-ncov) infection by the national health commission (trial version 7 world health organization. world health organization summary of probable sars cases with onset of illness from 1 suggestions for disinfection of ophthalmic examination equipment and protection of ophthalmologist against 2019 novel coronavirus infection precautions in ophthalmic practice in a hospital with a major acute sars outbreak: an experience from hong kong chinese preventive medicine association, beijing ophthalmological society and youth committee of beijing ophthalmological society. suggestions from ophthalmic experts on eye protection during the novel coronavirus pneumonia epidemic novel coronavirus disease (2019). (covid-19): the importance of recognising possible early ocular manifestation and using protective eyewear cynomolgus macaque as an animal model for severe acute respiratory syndrome this study was supported by the ophthalmology star program (qmx2019-01-001). the funding organization did not have any role in the design or conduct of this study. key: cord-348467-a2e3f161 authors: alqahtani, amani salem; wiley, kerrie elizabeth; tashani, mohamed; heywood, anita elizabeth; willaby, harold wayne; bindhim, nasser fahad; booy, robert; rashid, harunor title: camel exposure and knowledge about mers-cov among australian hajj pilgrims in 2014 date: 2016-01-18 journal: virol sin doi: 10.1007/s12250-015-3669-1 sha: doc_id: 348467 cord_uid: a2e3f161 [image: see text] more chronic diseases: 8% had diabetes, 7% asthma, 7% hypercholesterolemia and 5% hypertension. most pilgrims (82%) were travelling to hajj for the first time and planned to stay in saudi arabia for a median of 27 (range: 7 to 40) days. about two-thirds of respondents (64%) received 'travel health advice' before hajj, including from general practitioners (gps) (81%), travel clinics (24%) and specialist websites (e.g. saudi ministry of health [moh] website) (12%). many pilgrims consulted other individuals or sources: for example, 67% consulted hajj tour group leaders, 46% consulted their friends and family members who had previously attended hajj, and 17% browsed 'general websites'. about half of the participants (49%) knew about mers-cov before travelling to saudi arabia; of these, 70% reported mass media as the main information source. demographic factors significantly associated with mers-cov knowledge were female gender, age > 40 years, and middle eastern origin ( table 1) . receipt of pre-travel advice from their gps (odds ratio [or] 1.6, 95% confidence interval [ci] 1.0-2.4, p = 0.03) or from hajj tour group leaders (or 1.6, 95% ci 1.0-2.5, p = 0.04) were also significantly associated with mers-cov awareness (table 1) . of those aware of the disease, 77% answered correctly that the virus affects the respiratory system whereas 60% thought the disease was serious only for the elderly. furthermore, 48% of pilgrims who knew about the disease reported that they were either "not concerned" or "slightly concerned" about contracting the disease during hajj. half (51%) of the respondents were aware of the saudi moh advice for 'at risk' individuals to postpone hajj that year; however, none of these 'at risk' individuals adhered to this recommendation (table 2) . participants were acceptable with using preventive measures during hajj for protection from mers-cov infection: 79% were willing to wash hands frequently, 73% planned to avoid contact with sick people showing symptoms of a respiratory infection, 65% were willing to use facemasks, and another 65% planned to avoid touching their eyes, nose, and mouth. most departing pilgrims (62%) were aware of a mod-erate to high infection risk from raw camel milk consumption, yet 21% of participants were willing to drink it. additionally, 27% of participants were willing to visit a camel farm during hajj, or at least stated the possibility of doing so. pilgrims who knew about mers-cov were significantly more aware of the risks of mers-cov from drinking unpasteurized camel milk compared to those who did not know (43% vs. 23%, p < 0.01). nevertheless, among those who were aware of mers-cov, 27% did not fully realize the risk of catching the disease from unpasteurized camel milk, 15% were willing to drink raw camel milk, and 23% were keen to visit camel farm in saudi arabia (table 3) . for the second survey, 150 returning pilgrims aged 18-72 (median 41) years were recruited after hajj; 47% were men, and 16% reported having one or more chronic diseases: 6% had asthma, 3% diabetes mellitus, 2% malignancies, and 5% had other conditions. most participants (71%) had up to high school education level, 65% were employed, and 77% went for hajj for the first time; the median duration of stay at hajj was 25 (range: 7 to 35) days. of 150 pilgrims, 7% reported having actual contact with camels (e.g. taking photographs with camels), or consuming camel products, including meat (5%) and unpasteurized milk (1.3%). although this was the third year of mers-cov circulating in saudi arabia, only 50% australian departing pilgrims knew about the disease. however, this was a clear improvement compared to the findings of another australian survey conducted a few months earlier (only 35% pilgrims were aware of mers-cov in saudi arabia) (tashani et al., 2014) . compared to the ebola outbreak in 2014 about which 90% australian hajj pilgrims were aware (alqahtani et al., 2015b) , fewer pilgrims were aware of mers-cov. the ebola outbreak severity with consequent wider media coverage probably played a role in increasing awareness. for instance, ebola has affected over 28,640 people with a relatively higher fatality rate (40%) compared to fewer mers-cov cases (about 1,621) and a slightly lower fatality rate (36%) (who, 2015) . in this study, 62% of departing pilgrims were aware of the risk of contracting the disease from drinking raw camel milk while 21% were willing to drink camel milk if offered at hajj. this contrasts with the findings of gautret et al. who showed that only 14% french pilgrims knew about the disease risk from drinking raw camel milk but 40% of them were willing to drink camel milk if offered during hajj (gautret et al., 2013a) , indicating that pilgrims' awareness influenced their attitude. a unique finding to emerge from our study was that departing pilgrims with knowledge about mers-cov were significantly more aware of the risk of drinking raw camel milk (43% vs. 23%, p < 0.01), yet a considerable proportions of them were willingness to drink raw milk and visit camel farms (respectively 15% and 23%). this possibly indicates that despite being aware of mers-cov epidemic in saudi arabia, some individuals were not fully informed about the high risk behaviors (al-mohrej et al., 2015) . this could be addressed by public awareness campaigns. providing direct health education to hajj pilgrims at entry points (e.g. airport) in saudi arabia could improve their understanding about the possible modes of transmission of mers-cov and how to avoid exposure risks (turkestani et al., 2013) . in practice, about 7% of returning pilgrims reported actually coming in close contact with camels and 1.3% drank unpasteurized camel milk. this is unsurprising considering that about 8% french hajj pilgrims had a history of consuming raw camel milk in north africa and saudi arabia (gautret et al., 2013a ). an iphone appbased small survey conducted parallel to this survey revealed that 16% of international hajj pilgrims reported contact with camels during hajj (alqahtani et al., 2015a) . consumption of raw camel products (milk, liver, meat, or urine) can lead to outbreaks of other infections such as brucellosis, plague, and rift valley fever (bin saeed et al., 2005; davies, 2006; kalimuddin et al., 2010) . therefore, pilgrims who consume raw milk or other products are at risk of other zoonotic diseases if not mers-cov, and therefore, could benefit from appropriate health education. the current mers-cov guidelines by saudi moh stated that any person with close contact with camels (in the last 14 days), including drinking unpasteurized camel milk and showing respiratory symptoms, is suspected with mers-cov (ccc, 2015) . the recent outbreak in south korea, worsened by the initial failure of heath care providers to recognize the infection, shows the importance of early recognition of symptoms of mers-cov (kim and lee, 2015) . to prevent virus introduction to their lands, some countries (e.g. the uk) have issued a set of guidelines for managing suspected mers-cov cases (kumar et al., 2015) , which is exemplary (phe, 2015) . given that 7% people reported having close contact with camels, we suggest development of a public health strategy to better communicate the risk to pilgrims and information about when to seek medical help should symptoms arise upon return. over half of the departing pilgrims were aware of the saudi moh advice to postpone hajj; nevertheless, none of the high-risk pilgrims decided to cancel their trip. this is consistent with the findings of french researchers who also discovered that none of the at-risk pilgrims had planned to postpone their trip (gautret et al., 2013b) . as mass media was the main source of mers-cov knowledge, we believe mainstream media outlets (e.g. tv) should be used to propagate heath messages immediately before, during, and after the hajj season. tour group leaders can also play an important role in disseminating health advice, as shown here and previously (alqahtani et al., 2015b) . (16) travel agents 11 (6) saudi moh 8 (5) 'smarttraveller' website** 8 (5) other sources 3 (2) very serious 100 (60) moderately serious 43 (25) slightly serious 14 (8) not serious 11 (6) for pilgrims with chronic diseases*** very serious 86 (51) moderately serious 48 (28) slightly serious 15 (9) not serious 20 (12) very serious 47 (27) moderately serious 43 (25) slightly serious 47 (27) not serious 36 (21) very concerned 47 (27) moderately concerned 43 (25) slightly concerned 47 (27) not concerned 36 ( digestive tract 12 (7) brain 11 (6) note: *some pilgrims obtained the information from more than one sources, so the percentage is >100; **smartraveller.gov. au; ***missing data have been excluded. in conclusion, many australian hajj pilgrims are not aware of mers-cov in saudi arabia, and some of them engage in activities that may put them at risk of mers-cov; therefore, there is a need for improved awareness among hajj pilgrims and other travelers to the middle east regarding mers-cov. infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection vector borne zoonotic dis mers, influenza and respiratory illness in travellers returning from the hajj key: cord-324324-8ybfiz8f authors: decaro, nicola; lorusso, alessio title: novel human coronavirus (sars-cov-2): a lesson from animal coronaviruses date: 2020-04-14 journal: vet microbiol doi: 10.1016/j.vetmic.2020.108693 sha: doc_id: 324324 cord_uid: 8ybfiz8f the recent pandemic caused by the novel human coronavirus, referrred to as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), not only is having a great impact on the health care systems and economies in all continents but it is also causing radical changes of common habits and life styles. the novel coronavirus (cov) recognises, with high probability, a zoonotic origin but the role of animals in the sars-cov-2 epidemiology is still largely unknown. however, covs have been known in animals since several decades, so that veterinary coronavirologists have a great expertise on how to face cov infections in animals, which could represent a model for sars-cov-2 infection in humans. in the present paper, we provide an up-to-date review of the literature currently available on animal covs, focusing on the molecular mechanisms that are responsible for the emergence of novel cov strains with different antigenic, biologic and/or pathogenetic features. a full comprehension of the mechanisms driving the evolution of animal covs will help better understand the emergence, spreading, and evolution of sars-cov-2. eighteen years after the emergence of severe acute respiratory syndrome (sars) in china and 8 years after the emergence of middle east respiratory syndrome (mers) in saudi arabia, a novel coronavirus (cov) epidemic, recently classified as pandemic by the who, is threatening the human population worldwide (zhou et al., 2020) . the disease, now referred to as coronavirus disease 2019 , is caused by a novel human cov, which was initially denominated 2019 novel coronavirus (2019-ncov) and later renamed as sars coronavirus 2 (sars-cov-2) the by coronavirus study group of the international committee on taxonomy of viruses (gorbalenya et al., 2020) . covid-19 emerged in december 2019 in wuhan city, hubei province, china, in humans exposed to wildlife at the huanan seafood wholesale market, which is the largest seafood market in central china, and where different species of farm and wild animals are commonly sold (lorusso et al., 2020) . the epidemic has then expanded not only to neighbouring asian countries, but also to other continents (https://www.who.int/ docs/default-source/coronaviruse/situation-reports/20200415-sitrep-86-covid-19.pdf?sfvrsn=c615ea20_2). . a list of human covs is showed in table 1 . historically, only two human covs (hcovs) had been known before the sars emergence, namely hcov-229e, an alphacoronavirus originated in bats and transmitted to humans through alpacas, and hcov-oc43, a betacoronavirus which had passed from rodents to humans through cattle (corman et al., 2015 (corman et al., , 2018 . after 2002-2003 sars epidemic, the renovated interest in hcovs allowed the discovery of two additional viruses, the alphacoronavirus hcov-nl63 and the betacoronavirus hcov-hku1, derived from bats and rodents, respectively (tao et al., 2017) . all these four viruses are usually responsible for mild respiratory symptoms in immunocompetent patients. sars-cov and mers-cov are two unrelated betacoronaviruses originated in bats and transmitted to humans by wild carnivores and dromedary camels, respectively. in contrast to other hcovs, these two viruses displayed an increased virulence, causing severe pneumonia and even the death of affected people, with mortality rates of about 10 % and 30 %, respectively (guarner, 2020) . the occurrence of three highly pathogenic covs with a zoonotic origin in less than two decades, highlights the role of animals in generating covs with increased virulence that can adapt to humans, causing epidemics (and eventually pandemics) with high impact on human health. indeed, cov infections of veterinary interest have been known since almost a century (cavanagh, 2007; pedersen, 2014; decaro et al., 2020) , so that animal covs are paradigmatic of how this large family of viruses evolves, generating strains with different biological properties. in addition, the efforts done in veterinary medicine https://doi.org/10.1016/j.vetmic.2020.108693 received 10 march 2020; received in revised form 10 april 2020; accepted 10 april 2020 evolve rapidly, changing their antigenic profile, tissue tropism or host range by means of two distinct mechanisms. the viral replicase (an rna dependent-rna polymerase) does not possess a good proof reading activity, therefore the incorporation of wrong nucleotides at each replication cycle and the consequent accumulation of mutations in the viral genome lead to a progressive differentiation of the viral progeny from the parental strain. this mechanism, which is well known for influenza viruses being responsible for the so called antigenic drift, may cause the progressive adaptation of the viral surface proteins to the cell receptors of new animal species, increasing the viral fitness. in addition, the particular replicating machinery of covs facilitates recombination events due to the presence of consensus sequences upstream each gene. therefore, in the case of coinfection by more than one cov strain, the rna polymerase can jump from the rna of a strain to that of the other one, synthetizing a hybrid rna containing sequences from both viruses. recombination can occur not only with genomic sequences of other covs (homologous recombination), but also with rnas of different viruses and other organisms (heterologous recombination) (luytjes et al., 1988; banner and lai, 1991; lai, 1996; zeng et al., 2008; huang et al., 2016) . recombination is an alternative mechanism that let covs acquire novel biological properties in terms of virulence, host range and tissue tropism, so that cov strains, which are non-pathogenic or lowpathogenic in the original host, may increase their pathogenicity in the same species or adapt to different species spreading in the new host with exceptional rapidity (banner and lai, 1991) . the occurrence of three human cov epidemics in less than 20 years, along with the emergence of less pathogenic human covs, arises some questions on how these viruses that have their reservoirs in bats and rodents may overcome the species barriers jumping to humans. the animal-to-human transmission of viruses has been already occurred in the past, but it seems that its frequency has been increased in the last decades, involving in a short time span not only covs, but also a plethora of genetically and biologically different viruses with zoonotic potential, such as ebola virus, influenza viruses, flaviviruses, hendra and nipah viruses (mcmahon et al., 2018) . climate changes that are intensifying in this first quarter of the 21st century are favouring the spread of vector-borne diseases through increasing the proliferation of vectors and predisposing to their occupation of new ecological niches. the emergence in temperate climate areas such as europe of vectorborne diseases caused by viruses considered exotic until few years ago (west nile virus, usutu virus, chikungunya virus) accounts for a progressive geographic expansion of tropical diseases thanks to the ongoing phenomenon of tropicalisation (mcmahon et al., 2018) . deforestation and urbanization are other major factors that facilitate the spill-over of zoonotic agents to humans by reducing the habitat of wildlife and increasing the chances of contacts between wild animals (like bats, rodents and birds) and human beings (beena and saikumar, 2019; lorusso et al., 2020) . this could be the case of ebola virus, hendra and nipah viruses, hantavirus and coronavirus infections. in addition, the close contact between human beings and different animal species sold at the wet markets of east asia represents the optimal situation for the host species jump and adaptation to humans of potentially zoonotic agents like covs. it is not a coincidence that two of the most severe zoonoses of the last two decades (highly pathogenic h5n1 avian influenza and sars) have emerged in the same chinese province of guangdong where the contact between humans and animals is closer (lorusso et al., 2020) . table 2 reports the most important avian cov species recognised so far and their associated diseases. the number of avian species in which covs have been detected in the last years is humongous. since the emergence of sars-cov in 2002, there has been increased interest in table 2 main coronaviruses in domestic and domesticated avian species. schalk and hawn (1931); beach and schalm (1936) ; beaudette and hudson (1937) turkey ( respiratory and kidney disease spackman et al. (1983) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 covs in other species, including birds. prior to that time, our knowledge of covs in avian species was limited largely to three birds of the order galliformes, i.e., domestic fowl (gallus gallus), turkeys (genus meleagris) and pheasants (phasianidae), with their infectious bronchitis virus (ibv), turkey coronavirus (tcov), and pheasant coronavirus (phcov), respectively. these three viruses were considered for a long-time different species for several reasons such as the diverse pathotype (enterotropic or respirotropic), host range and genetic relatedness of the s protein (cavanagh, 2007) . this scenario radically changed after the discovery of several novel covs with high genetic diversity from different avian species and the novel rules for species designation of the coronavirus study group (csg https://talk.ictvonline.org/ictv-reports/ ictv_9th_report/positive-sense-rna-viruses-2011/w/posrna_viruses/ 222/coronaviridae). all these viruses as well as analogous ibv-like covs detected in other birds including penguins, pigeons, peafowl, parrots, waterfowl, teal, quail, duck and whooper swan (cavanagh et al., 2002; circella et al., 2007; domanska-blicharz et al., 2014; torres et al., 2013; hughes et al., 2009; liu et al., 2005; wille et al., 2016; jordan et al., 2015; bande et al., 2016; suryaman et al., 2019) have been assigned to the same viral species known as avian coronavirus (acov) within the subgenus igacovirus of genus gammacoronavirus. ibv and ibv-like strains are commonly detected in both gallinaceous and non-gallinaceous birds, also asymptomatically (cavanagh, 2005) . this might suggest that these species would act as wild reservoirs, spreading ibv strains over the world (de wit et al., 2011) . as for the huge economic impact of the disease it causes, ibv is one of the most studied covs over the last decades. ibv causes the infectious bronchitis (ib), a term adopted in 1931 for describing the main clinical characteristics of a transmissible respiratory disease of poultry detected for the first time in north dakota (usa). ib has been now diagnosed worldwide and is one of the most important viral diseases of poultry characterised by respiratory signs, but it can also affect the kidneys and reproductive tract following viremia with a severity that differs depending on the involved viral strain (cavanagh and gelb, 2008) . the disease also affects wild and ornamental birds chen et al., 2013) . ib control has been hampered by the intricate ibv evolution, which has been entailed, over the years, by the emergence of many different antigenic or genotypic types, commonly referred to as variants, with divergent molecular, biological, and antigenic properties. being a cov, ibv has, indeed, a considerable ability to change both by mutation and by homologous recombination events, which may cause, along with replicase stuttering or slippage, also insertions and deletions in the genome (cavanagh and gelb, 2008) . if these mechanisms involve the hypervariable region s1, they frequently result in the emergence of new ibv variants. although many new variants are not successful, a few may emerge, spread, causing devastating disease either worldwide or in limited geographic areas. currently 32 lineages have been recognized, categorized into six genotypes (gi to gvi) (valastro et al., 2016) . through their s protein, ibv and ibv-like viruses recognise as cellular host receptor the α2,3-linked sialic acid glycan, widely distributed in the respiratory tract and in several other host tissues, factor which may explain the tropism also for several organs of the infected host (winter et al., 2006 (winter et al., , 2008 shahwan et al., 2013; ambepitiya wickramasinghe et al., 2011) . extensive use of vaccines has greatly contributed to the high variability of ibv strains thorough recombination between vaccine and field viruses and viral selection pressure resulting from vaccination and presence of partially immune birds (gandon and day, 2008; gandon et al., 2001; bande et al., 2017) . important ibv-like strains are tcov, responsible for enteritis in turkey (also known as bluecomb disease), guinea fowl coronavirus (gfcov) and quail coronavirus (qcov) responsible for fulminating enteric disease in guinea fowl and quail, respectively cavanagh, 2005; liais et al., 2014) . tcov, gfcov and qcov are evolutionarily distant from acov based on the s protein. while ibv is a primarily respiratory pathogen, tcov causes gastrointestinal disease (liais et al., 2014; guy, 2008) . enterotropism has also been observed for some ibv serotypes; however, all ibv strains infect primarily the respiratory tract, resulting in mild to severe inflammation of the nasal and tracheal epithelia (cavanagh, 2005 (cavanagh, , 2007 . the s1 domain of the s protein is highly variable, with the amino acid sequences of ibv and tcov from the usa sharing < 25 % sequence identity. phylogenetic analysis of the s1 gene shows, indeed, grouping of ibv and ibv-like viruses on the one hand and tcov-us, gfcov and qcov on the other hand (ambepitiya wickramasinghe et al., 2015a) . accordingly, the emergence of covs in turkeys in the usa was proposed to have resulted from recombination events involving ibvs and an as-yet-unidentified cov donating a novel s gene. this switch contributed largely to determine the in vivo tissue tropism of tcov and related viruses. intriguingly, the s protein of these covs requires nonsialylated type 2 poly-lacnac structures on n-glycan cores for binding. this is in marked contrast to the α2,3-linked sialic acid glycan binding of ibv and ibv-like viruses (ambepitiya wickramasinghe et al., 2015b) . the s1 subdomain of a tcov isolate from france in 2008 (tcov-fr) had only 42 % sequence identity to that of the tcov-us strain (maurel et al., 2011) . this diversity was biologically evident by the prominent tropism for the epithelium of the bursa of fabricius and only mild tropism for the small intestine of turkey. tcov-fr s1 protein did not show, indeed, affinity for nonsialylated type 2 poly-lacnac (ambepitiya wickramasinghe et al., 2015a) . this genetic diversity between tcovs is in accordance with several recombination events involving ibvs on different continents with several unknown covs. on the one hand, the s genes of gfcov/fr/2011 (isolated in france in 2011) and tcov-us share significant genetic relationships, and thus these viruses must have acquired their s gene from a common ancestor. on the other hand, gfcov/fr/2011 and fr tcov have a very similar genetic background in other genes. two recombination events may be responsible for the genesis of tcov-us and fr tcov. a first event occurred between an ibv eu recipient strain and an unknown acov donor, resulting in a virus with a new s gene, whose evolution would have resulted in fr tcov and gfcov/fr/2011. a second recombination event involving a us ibv recipient and gfcov/fr/2011 would have generated us tcov viruses, which share a stronger s gene similarity with gfcov/fr/2011 than with fr tcov . additional covs distinct from acovs and mainly circulating in ducks (duck coronavirus, dcov), pigeons (pigeon coronavirus, pcov), or geese (goose coronavirus, gcov) have been identified (cheng et al., 2013; jonassen et al., 2005; muradrasoli et al., 2010; kim and oem, 2014; zhuang et al., 2015; papineau et al., 2019) . although their genome seems to fulfill the official ictv criteria required to distinguish a new species within the gammacoronavirus genus, ictv approval is still pending. historically, covs of birds were all included in the gammacoronavirus genus and, in turn, all covs belonging to this genus were identified only in birds. however, this suggestion was rebutted by the evidence of a cov belonging to the gammacoronavirus genus in a beluga whale first discovered in 2008 (viral species beluga whale coronavirus sw1 species, subgenus cegacovirus, genus gammacoronavirus) (mihindukulasuriya et al., 2008) , and of three novel covs, bucov hku1, thcov hku12, and mucov hku13 in birds of the order passeriformes, namely bulbuls (pycnonotus jocosus), thrushes (turdidae) and munias (lonchura punctulate), respectively, which did not cluster phylogenetically with extant covs identified in birds. these latter three viruses were distinct from known covs forming a unique cluster in the phylogenetic tree, which was the basis for generation of the deltacoronavirus genus (woo et al., 2009) . importantly, additional novel viruses belonging to this novel genus were detected in wild birds chu et al., 2011; durães-carvalho et al., 2015; torres et al., 2016) . these viruses cluster with previously unclassified covs detected in various asian carnivores, i.e., the asian leopard cat (prionailurus bengalensis) and chines ferret badger (nyctereutes procyonoides) (dong n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 et al., 2007) . covs belonging to the betacoronavirus genus, which are strictly related to mouse hepatitis virus (mhv), were also described in wild birds, including parrots, in brazil (durães-carvalho et al., 2015) . interestingly, this was not the first detection of viruses belonging to the betacoronavirus genus in birds. often overlooked is the discovery over 38 years ago of a cov from the manx shearwater (puffinus puffinus), a bird that visits the shores of britain in summer (nuttall and harrap, 1982; cavanagh et al., 2007) . this virus was also related to mhv. however, at that time, considering the unusual finding and that the virus was isolated by passage of shearwater material in the brains of mice, it was speculated that the detected virus was an mhv strain already present in the mice before inoculation (cavanagh, 2007) . bats are an ancient and heterogeneous group of ecologically important mammals, representing nearly a quarter of all mammalian diversity on earth. they belong to the order chiroptera and further classified in two suborders yinpterochiroptera and yangochiroptera. the first includes the non-echolocating pteropodidae family (megabats) and five echolocating rhinolophoidea microbat superfamilies. yangochiroptera contain thirteen echolocating microbat families (tsagkogeorga et al., 2013) . bats are thought to host a large plethora of viruses. these include, amongst the others, lyssaviruses, filoviruses, henipaviruses, and reoviruses (calisher et al., 2006) . before sars-cov epidemic, bats were not known to host covs. indeed, the first evidence of a bat cov was published in 2005 . after the sars epidemic, there was a boost in interest regarding searching for novel covs in various animals, including bats. to date, over 200 novel covs have been identified in bats and approximately 35 % of the bat virome sequenced to date is composed of covs (chen et al., 2014) . this data has been made available following the massive surveillance, coupled with the advent of next-generation sequencing (ngs) technology, which has been performed in wild animals banerjee et al., 2019) . just a small portion of these covs have been officially recognised by the ictv; many others are still pending for official designation. cov species detected in bats and officially recognised by the ictv are listed in table 3 and the following chapter reasonably discusses only officially recognized bat cov species. bats can carry and transmit covs into local bat populations via migration even though little is known about the migratory patterns of these animals. closely related covs can be detected in the same bat species living at locations separated by thousands of miles (drexler et al., 2010) and different cov species or genera can be found in different bat species living at the same roosting sites. however, some covs have been shown to be species-specific. accordingly, regional patterns of bat cov outbreaks at species level can be deduced from the population distribution of their respective bat hosts. although bats seem to develop clinical diseases induced by several viruses and bacteria (mühldorfer et al., 2011) , generally covs do not cause apparently overt disease in these mammals, also experimentally. this phenomenon seems to be related with peculiar characteristics of their immune system (ahn et al., 2019; brook et al., 2020) . based upon genomic data available so far, it is widely accepted that while birds represent the reservoir for covs belonging to genera gammacoronavirus and deltacoronavirus, bats are the natural reservoir for alpha-and betacoronaviruses. however, only betacoronaviruses of subgenera sarbecovirus, merbecovirus, nobecovirus and hibecovirus have been detected in bats so far. given that several betacoronaviruses from the subgenus embecovirus have been discovered in rodents, it was speculated that rodent covs may be the ancestors of currently circulating viruses belonging to this subgenus . covs have been detected at high frequency in bats in all continents, with alphacoronaviruses being more widespread than betacoronaviruses . subgenus colacovirus (genus alphacoronavirus) officially comprises the viral species bat coronavirus cdphe15, so far composed by two bat covs strains named cdphe15/usa/2006 and myotis lucifugus cov (myl-cov), which share a 98.2 % nucleotide identity across the whole genome. both strains have been detected in myotis lucifugus bats (vespertilionidae) also known as the northern american little brown bats. the former was detected in 2006 in colorado (genbank acc. no. kf430219), while the latter was reported in 2010 in canada. this virus was identified in the intestines and lungs and associated with minimal pathology or inflammation (subudhi et al., 2017) . subgenus decacovirus (genus alphacoronavirus) comprises the species rhinolophus ferrumequinum alphacoronavirus hub-2013 composed so far by btms-al-phacov/gs2013 and btrf-alphacov/hub2013 strains discovered in china in myotis spp. and rhinolophus ferrumequinum bats, respectively. these two viruses share very high sequence identities (higher than 98 %), which dramatically decrease in the s genes (only 85% nucleotide identity) (wu et al., 2016) . woo et al., 2006) shares an 86 % sequence identity with severe acute diarrhoea syndrome-coronavirus (sads-cov) of pigs . these two viruses are now included in the same viral species rhinolophus bat coronavirus hku2 (subgenus rhinacovirus, genus alphacoronavirus). viral strains btkynl63-9a, btkynl63-9b, btkynl63-15 and btkynl63-9a, identified in 2010 in triaenops afer bats from kenya, form the viral species nl63-related bat coronavirus strain btkynl63-9b that is part of the subgenus setracovirus (genus alphacoronavirus) along with human coronavirus nl63 (tao et al., 2017) . in this regard, a bat origin has been strongly suggested for two of the less-pathogenic hcovs causing mild respiratory symptoms in immunocompetent people, namely hcov-229e and hcov-nl63, both belonging to the alphacoronavirus genus. whereas hcov-229e (subgenus duvinacovirus) recognises as direct ancestor an alphacoronavirus from alpacas, which in turn derives from 229e-related covs identified in hipposiderid bats (corman et al., 2015) , hcov-nl63 is likely a recombinant virus originating from the distantly related 229e-related covs associated with hipposiderid bats and covs associated with triaenops afer bats (tao et al., 2017) (table 1 ). the s protein of hcov-nl63 is more closely related to that of 229e-related covs, whereas the rest of the genome with covs included in the nl63-related bat coronavirus strain btkynl63-9b species (tao et al., 2017) . different from the bovine coronavirus (bcov)-like viruses that cause enteric disease, in 2007 a novel alpaca cov was associated to respiratory disease in california, usa. full-length genome analysis showed that this respiratory alpaca cov was closely related to the alphacoronavirus hcov-229e (subgenus duvinacovirus) (crossley et al., 2012) . more recently, close relatives of hcov-229e were detected in african hipposiderid bats. interestingly, both bat and alpaca viruses displayed an intact accessory gene orf8 located at the genomic 3' end, while hcov-229e retained only a conserved trs preceding remnants of this orf, suggesting its loss after acquisition of a 229e-related cov by humans. therefore, hcov-229 is likely a descendant of the alpaca alphacoronavirus (corman et al., 2015) . strains forming the viral species bat hp-betacoronavirus zhejiang2013 (subgenus hibecovirus, genus betacoronavirus) were discovered in hipposideros pratti bats from china in 2013 (wu et al., 2016) . strain ro-batcov gccdc1 356 was identified from stools of rousettus leschenaultii, a species of fruit bats (pteropodidae) of southern asia, which were collected in yunnan province, china, in 2014 (huang et al., 2016) . ro-batcov gccdc1 356 shows a small intact orf of 276 nucleotides embedded between the n and ns7a genes. this orf has no homology to any known coronavirus, and the encoded protein exhibited 54.9 % amino acid identity with the p10 protein encoded by the first orf of segment s1 of bat fusogenic orthoreoviruses (genus orthoreovirus, species nelson bay orthoreovirus, also known as pteropine orthoreovirus). these viruses are double-stranded segmented rna viruses, belonging to the family reoviridae, and are able to cause severe pneumonia in humans (chua et al., 2007; lorusso et al., 2015) . ro-batcov gccdc1 356 is included in the viral species rousettus bat coronavirus gccdc1 within the subgenus nobecovirus, genus betacoronavirus. rousettus bat coronavirus hku9, belonging to subgenus nobecovirus, was also identified in rousettus leschenaultii and in other bat species (mendenhall et al., 2017) . this virus was first detected in 2007 in guangdong province in china (woo et al., 2007) . subsequent studies suggested that the virus was widely distributed and is circulating in different bat species (ge et al., 2012) . covs from the bthku9-like cluster were also detected in hipposidereos commersoni and rousettus aegyptiacus bats in kenya (tong et al., 2009) . being a fruit bat, rousettus leschenaultii has a wider flying range than most of the insectivorous bats in china, thus it may carry viruses over long distances. a comparison of the reported hku9-cov sequences showed a high genetic diversity within this viral species (luo et al., 2018a, b; lau et al., 2010; ge et al., 2012) . when mers-cov was first isolated in the middle east in 2012 and its genome sequenced, it was found that it was most closely related to ty-batcov hku4 discovered in tylonycteris pachypus and pi-batcov hku5 discovered in pipistrellus abramus, which were the only known members of subgenus merbecovirus at that time. these two viruses are now the prototype strains of tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 viral species, respectively, within subgenus merbecovirus, genus betacoronavirus. although mers related covs (mers-rcovs) were lately discovered, mers-cov was much closer in the s1 region to hku4-cov than to mers-rcov or hku5-cov. indeed, dipeptidyl peptidase 4 (dpp4), the receptor for mers-cov, is also the receptor for hku4, but neither for hku5 nor for early discovered mers-rcovs. however, hku4 prefers bat dpp4 over human dpp4, whereas mers-cov shows the opposite trend . so far, hku4-covs are only carried by tylonycteris spp. bats (t. pachypus and t. robustula) and are relatively conserved; hku5-covs are found in different pipistrellus spp. bats, including p. abramus, p. pipistrellus and p. minus . due to the current sars-cov-2 pandemic, attention should be given to the viral species severe acute respiratory syndrome-related coronavirus (sars-rcov,subgenus sarbecovirus, genus betacoronavirus) and middle east respiratory syndrome-related coronavirus (mers-rcov, subgenus merbecovirus, genus betacoronavirus), which enclose sars-cov and mers-cov, the first two highly pathogenic covs that were discovered in humans. in 2002, at the beginning of the sars epidemic, almost all early human index patients had animal exposure in a market place, in guangdong province, before developing disease. after sars-cov was identified, its rna and/or specific antibodies were found in masked palm civets (paguma larvata) and animal handlers in a market place. however, later investigations of farmed and wild-caught civets revealed that sars-cov strains found in market civets were transmitted to them by other wild animals (tu et al., 2004; kan et al., 2005) . subsequently, novel covs related to human sars-cov (sars-rcovs) were discovered in horseshoe bats (genus rhinolophus) in china and hong kong lau et al., 2005) . these sars-rcovs showed genome sequence identity of 88-90 % among themselves and 87-92 % identity to human or civet sars-cov isolates. sars-rcovs were detected in rhinolophus spp. bats of other regions of china (tang et al., 2006; woo et al., 2006; yuan et al., 2010; ge et al., 2013) . sars-rcovs with higher genetic diversity with respect to chinese strains were also detected in rhinolophid bats from slovenia, bulgaria and italy in europe (drexler et al., 2010; rihtaric et al., 2010; balboni et al., 2011) . covs related to sars-rcov were also detected in hipposideros spp. and chaerophon spp. bats from ghana, kenya and nigeria (hu et al., 2015) . these evidences suggested that bats may be the natural hosts for sars-cov and that wild carnivores were only intermediate hosts. although these sars-rcovs showed high sequence identity to sars-cov, they were demonstrated to be unable to bind to the human cell angiotensin converting enzyme ii (ace2) receptor, the receptor of sars-cov, as a consequence of deletions in their s protein (ren et al., 2008) . besides, the theory of bat origin of sars-cov lacked a powerful support due to the failure of direct isolation of this virus from bats. thus, considering that no direct progenitor of sars-cov was found in bats and that rna recombination is the fuel for cov evolution, it has been proposed that sars-cov emerged through recombination of bat sars-rcovs. this hypothesis was made after the evidence of a single bat cave in yunnan, china, with very high covs diversity and considering that, within the identified covs, all genetic elements needed to form sars-cov have been identified in that single cave (ge et al., 2013) . recombination analysis also strongly supported the hypothesis that the civet sars-cov strain sz3 originated following a recombination event of two existing bat strains, wiv16 and rf4092 (hu et al., 2017) . moreover, wiv1, the closest relative to sars-cov that has been found in bats so far (more than 95 % nucleotide identity, higher than that of any other bat sars-rcovs (76-92 %)), likely arose through recombination of two other prevalent bat sars-rcov strains. the most frequent recombination breakpoints were within the s gene and upstream of orf8, which encodes an accessory protein. these genes were also involved in the crucial adaptation pathways of sars-cov from bats to wild carnivores, from wild carnivores to humans, and from human to human (cui et al., 2019) . wiv1 has been shown to have the capacity to bind to the human, civet and bat cell ace2 receptor (ge et al., 2013) . the isolation in cellculture of a highly related sars-cov strain, coupled with the evidence of a functional s protein capable of using the same ace2 receptor, provided robust and conclusive evidence for the bat origin of sars-cov. an additional sars-rcov strain has been shown, by reverse genetics studies, to have the capacity to bind to the human ace2 receptor (menachery et al., 2015) . quite the opposite, a direct bat cov highly related to mers-cov of humans was never detected. indeed, the genome sequences of mers-cov in human and dromedaries possess only around 65-80 % nucleotide identities to those of the other members of subgenus merbecovirus from different bats. human mers-covs were instead almost identical to mers-covs identified in dromedary camels (camelus dromedaries). lately, genomic sequence analyses indicated that covs now belonging to the mers-rcov species were found in several bat species from two bat families, vespertilionidae and nycteridae (lelli et al., 2013; de benedictis et al., 2014; corman et al., 2014a, b; anthony et al., 2017; moreno et al., 2017; wong et al., 2019) . however, none of these mers-rcovs is a direct progenitor of mers-cov, as their s proteins differ substantially from that of the human virus. the closest relative to mers-cov of humans and dromedary camels is mers-rcov strain neoromicia/5038 isolated from neoromicia capensis bats in south africa (geldenhuys et al., 2018, table 1 ). a short sequence (around 200 nucleotides) of viral rna identical to that of mers-cov was also detected in a taphozous perforates bat in saudi arabia (memish et al., 2013) . overall, although it is widely accepted that mers-cov ancestor is in bats, further studies are warranted in order to discover the precise mechanisms of its emergence in dromedary camels and humans. it was suggested that mers-cov ancestors had been circulating in bats for very long time. mers-cov has evolved to adapt to use human receptor and the dpp4-recognising bat coronaviruses like hku4 may follow up, thereby posing a serious risk to human health. recent mers-rcovs were shown to have the capacity to bind to the dpp4 as entry cell receptor as they acquired the s1 through recombination with hku4-like viruses (luo et al., 2018a, b) . as for the recent and threatening covid-19 outbreak in humans, we certainly know that sars-cov-2 belongs to the species sars-rcov together with sars-cov from humans and sars-rcovs from wild carnivores and horseshoe bats (genus rhinolophus) (gorbalenya et al., 2020; zhou et al., 2020; wu et al., 2020) . epidemiological investigations revealed that many initial patients were exposed to wildlife at the huanan seafood wholesale market (south china seafood market), which is the largest seafood market in central china (lorusso et al., 2020) . sars-cov-2 has been assigned to an existing species of hundreds of known viruses largely isolated from bats. these viruses have names derived from sars-cov, but only the viral isolates originating from the 2002-2003 outbreak have been confirmed to cause sars in humans (gorbalenya et al., 2020) . importantly, it has also been confirmed that sars-cov-2 uses the ace2 receptor through the receptor binding domain (rbd) of the s protein (hoffmann et al., 2020; zhou et al., 2020) . likely, also sars-cov-2 has a bat origin. according to genome sequences available so far, the most closely related virus (96.2 % of nucleotide sequence identity) to sars-cov-2 is strain batcovratg13 identified from a bat, rhinolophus affinis, from yunnan province, china, followed by sars-rcovs identified from pangolins (tang et al., 2020) . the receptor-binding spike protein of sars-cov-2 is highly divergent from other covs with less than 75 % nucleotide sequence identity to all previously described sars-rcovs, except for a 93.1 % nucleotide identity to batcovratg13 (zhou et al., 2020) . although sars-cov-2 uses the ace2 receptor, five out six critical amino acid residues in rbd were different between sars-cov-2 and sars-cov; the same residues were instead identical to those of pangolin sars-rcovs and, in turn, only one of these residues was identical to those of batcovratg13 (tang et al., 2020) , although this latter shows the highest nucleotide sequence identity with sars-cov-2 along the whole genome. thus, it was tempting to speculate that sars-cov-2 rbd region might have originated from recent recombination event in pangolins or that sars-cov-2 and sars-rcovs of pangolins represent the result of coincidental evolution (lam et al., 2020; tang et al., 2020) . overall, it remains to be solved whether also sars-cov-2 needed an intermediate (and amplification) host before being able to infect humans as it was the case for sars-cov and other hcovs. since a mammal reservoir has not yet been identified, a prudent use of specific antigens is strongly recommended for serological diagnosis of sars-cov-2 in animals as cross-reactions with viruses of the alphacoronavirus genus, widespread in animals, might occur (sun and meng, 2004) . analogously to bats, but with a lesser extent, also rodents have been recently demonstrated to play a significant role in the evolution of cov, in particular of those belonging to subgenus embecovirus of genus betacoronavirus. rodentia (rodents) is the largest order of mammals with more than 2000 species worldwide, representing a major source of zoonotic infectious diseases (han et al., 2015) . for decades, only one species of coronavirus, murine coronavirus (subgenus embecovirus, genus betacoronavirus), has been associated with rodents. the prototype virus, which was named mouse hepatitis virus (mhv), was first isolated in mice in 1949 (cheever et al., 1949) . a mhv variant was lately identified in rats in 1970 (parker et al., 1970) . rat coronavirus (rcov) causes epidemics of respiratory disease in laboratory rat colonies. the two prototype strains of rcov are sialodacryoadenitis virus (sdav) and parker's rcov (rcov-p) (bhatt et al., 1972; parker et al., 1970) . both strains infect the respiratory tract, and sdav can also infect the eye, salivary and lacrimal glands. young rats are especially susceptible to rcov with the infection occurring in the lower respiratory tract and developing into interstitial pneumonia (parker et al., 1970) . together with feline infectious peritonitis virus (fipv) and ibv, mhv has been one of the most strictly animal cov studied ever. mhv is a natural pathogen of mice, normally infecting the liver, gastrointestinal tract, and central nervous system, causing a wide range of disease, including hepatitis, gastroenteritis, and acute and chronic encephalomyelitis. importantly, it served as model for cov replication and pathogenesis, with emphasis for neuro-invasion and neurovirulence (weiss and navas-martin, 2005) . as for the additional structural protein he, some strains (such as jhm) of mhv contain the he protein, while others (such as a59) do not yokomori et al., 1989) . the role of rodents in the evolution of covs belonging to embecoviruses has been recently highlighted by means of the discovery of a novel betacoronavirus in norway rats (rattus norvegicus) in china. this virus forms a separate species named china rattus coronavirus hku24 (chrcov hku24) within the embecovirus subgenus. although designated as a novel species, this virus possessed genome characteristics that resemble to those of both betacoronavirus-1 and murine coronavirus, suggesting that chrcov hku24 represents the murine origin of betacoronavirus-1, with interspecies transmission from rodents to other mammals having occurred centuries ago (lau et al., 2015) . genus betacoronavirus consists of five subgenera, with bat covs being including in all but one of subgenus embecovirus, where rodent, human and bovine covs are included (https://talk.ictvonline.org/ taxonomy/). this supports the hypothesis that rodent covs were the ancestors of embecoviruses of other animals, while bats are the natural reservoirs for all other betacoronaviruses. importantly, rodent covs are not restricted to genus betacoronavirus. a deep virological screening was performed in 1465 rodents sampled in zhejiang province, china, during 2011-2013, with nearly 2% of rodents testing positive for cov . in particular, covs were detected in 10 striped field mice (apodemus agrarius), 4 norway rats, 14 lesser ricefield rats (rattus losea), 1 asian house rat (rattus tanezumi) and 1 chinese white-bellied rat (niviventer confucianus). amplicons of the replicase gene sequences were recovered from 21 (70 %) of the cov rna positive rodent samples described above and whole genome or nearly whole genome sequences (> 98 %) were recovered from 1 and 4 cov positive samples, respectively. by means of whole genome sequence analysis, authors were able to identify a divergent alphacoronavirus, which was lately officially designated as species lucheng rn rat coronavirus (lrnv) within the subgenus luchacovirus, and two novel betacoronaviruses termed longquan aa mouse coronavirus (lamv) and longquan rl rat coronavirus (lrlv) and assigned to the two established species betacoronavirus-1 and murine coronavirus, respectively . moreover, lrnv seems to be a recombinant virus as its n protein gene is more closely related to those of the genus betacoronavirus. overall, the discovery of rodent-associated covs belonging to subgenera that are distinct from those including bat covs warrants further investigations upon the role played by rodents in the evolution and emergence of these viruses. sars-cov replication has been studied in mice, syrian golden and chinese hamsters. the most severe symptoms of sars were observed in aged animals. indeed, aged mouse model of sars-cov has been generated (gretebeck and subbarao, 2015) . transgenic mice expressing human ace2 were also developed to closely mimic sars-cov infection in humans. some animal models have been tested and analysed on the genomic and proteomic level to study the pathogenesis of sars-cov. therefore, we have reason to believe that such models would work also for sars-cov-2. quite the opposite, studies have demonstrated that mice, guinea pigs and hamsters are not susceptible to experimental mers-cov infection, mainly because their homologous dpp4 molecules table 4 coronaviruses in domestic swine and associated diseases. do not function as receptors for mers-cov entry (cockrell et al., 2014) . the first mouse model of mers infection reported in 2014 involved transducing animals with recombinant adenovirus 5 encoding human dpp4 (hdpp4) molecules intranasally, and this resulted in replication of mers-cov in the lungs. this mouse model also showed clinical symptoms of interstitial pneumonia, including inflammatory cell infiltration, and thickened alveolar and mild oedema (song et al., 2019) . currently, six covs are circulating in swine (table 4 ). these include four alphacoronaviruses, transmissible gastroenteritis virus of swine (tgev) and its derivative porcine respiratory coronavirus (prcov) (subgenus tegacovirus), porcine epidemic diarrhoea virus (pedv) (subgenus pedacovirus) and sads-cov (subgenus rhinacovirus), one betacoronavirus, porcine haemagglutinating encephalomyelitis virus (phev) (subgenus embecovirus), and one deltacoronavirus, porcine deltacoronavirus (pdcov) (subgenus buldecovirus). tgev, pedv, sads-cov and pdcov are responsible for acute gastroenteritis in swine, with fatal infections in piglets born to seronegative sows, prcov causes a mild respiratory disease and phev is the causative agent of neurological and/or digestive disease in pigs (mora-díaz et al., 2019; wang et al., 2019). tgev was first described in uk in 1950s, representing the oldest known swine cov. tgev and prcov are closely related to canine coronavirus (ccov) and feline coronavirus (fcov) forming with these carnivore covs a unique species, referred to as alphacoronavirus-1. based on the analysis of the accessory protein gene orf3, it has been postulated that tgev has originated from ccov type ii (ccov-ii), since while ccov type i (ccov-i) exhibits an intact gene, both ccov-ii and tgev, which are strictly related in the s gene, have only remnants of orf3 (lorusso et al., 2008) . prcov, in turn, has derived from tgev through the deletion of ≈600 nucleotides at the 5' end of the s gene (corresponding to ≈200 amino acids at the n-terminus of the spike protein) and consequent change of the major tissue tropism from the enteric to the respiratory epithelium. this large deletion caused the loss of sialic acid binding activity that allows the attachment to mucins and mucin-type glycoproteins, so that tgev but not prcov is able overcome the intestinal mucus barrier, having access to the gut mucosa . prcov shares some epitopes for neutralising antibodies with tgev, so that its extensive circulation in swine herds has resulted in a drastic reduction of tge outbreaks worldwide. pedv was introduced in the pig population in the 1970s, likely as a consequence of a spillover event from bats. the virus was first described in europe and had been primarily maintained as an endemic pathogen in european and asian swine populations until its introduction into north america in 2013. pedv is more strictly related to a scotophilus bat coronavirus 512 than to other known alphacoronaviruses, including tgev and human alphacoronaviruses hcov-229e and hcov-nl63. therefore, pedv and btcov/512/2005 likely have a common evolutionary precursor and a cov cross-species transmission may have occurred between bats and pigs (banerjee et al., 2019) . accordingly, pedv contains signature motifs at the 5′-untranslated region that are shared by bat covs, thus providing further support of the evolutionary origin of pedv from bats and potential cross-species transmission (huang et al., 2013) . currently, different pedv genotypes are described based on the s gene: i) g1a pedv, including classical european and asian strains with moderate virulence; ii) g2 pedv, also called "original us pedv", comprising highly virulent strains that originated in asia and are now widespread in the usa; iii) g1b pedv, which is represented by the so-called s-indel strains, i.e., strains presenting insertions and deletions in the s gene that are associated with mild clinical outbreaks. these strains are natural recombinant pedvs with a g2-like genomic backbone carrying an s1 region of g1a strains; iv) s1 n-terminal domain-deletion (ntd-del) strains that are g2-like strains containing a 194 to 216-aa deletion within the n-terminal domain of the s1 subunit, also associated to mild clinical forms (hou and wang, 2019) . recombinant strains between pedv and tgev have been also reported in europe (akimkin et al., 2016; belsham et al., 2016; boniotti et al., 2016) . sadv-cov, now referred to as swine enteric alphacoronavirus (seacov), is another virulent swine enteric alphacoronavirus that originated from bats, sharing an 86 % sequence identity with a bat alphacoronavirus hku2-cov. since viruses displaying a 96-98 % sequence identity to sads-cov were detected in rhinolophus spp. bats, sads-cov and hku2-cov likely descend from a common ancestor . accordingly, both viruses now belong to the unique species rhinolophus bat coronavirus hku2. in contrast, phev, which was first described in 1957 in nursery pigs with encephalomyelitis in ontario, canada, has not derived from bat covs, but its evolutionary history is tightly intermingled with other two closely related betacoronavirus, hcov-oc43 and the oldest known bcov, with which phev may have common ancestors (vijgen et al., 2006) and is included in the same viral species, betacoronavirus-1 (corman et al., 2018) . most probably, hcov-oc43 and phev descend from a rodent betacoronavirus through preliminary adaptation to bcov, from which they may have emerged in the context of a pandemic recorded historically at the end of the 19th century (corman et al., 2018) . pdcov was recently detected in 2012 in hong kong during cov molecular surveillance in avian and mammalian species. this swine deltacoronavirus seems to recognise another different ancestor, likely emerging from a host-switching event between avian and mammal covs. the most closely related pdcov relative has been identified in quail deltacoronavrus uae-hku30 and the virus has been proposed to be a recombinant between other two avian deltacoronaviruses, sparrow cov hku15 and bulbul cov hku11. all these deltacoronavirus are now members of the same species coronavirus hku15 (lau et al., 2018) . pigs were found to be susceptible to experimental infection with the betacoronavirus mers-cov (vergara-alert et al., 2017), while sars-cov rna was detected in pigs and wild boars wang et al., 2005) . in contrast, a recent experimental infection demonstrated that pigs are not susceptible to sars-cov-2 . few studies have been carried out to assess the circulation of covs in farmed or free-ranging wild boars (sus scrofa). antibodies against tgev/prcov were detected in some animals in slovenia (vengust et al., 2006) and croatia (roic et al., 2012) and pedv rna was demonstrated in south korea (lee et al., 2016) . a wild boar sold at a live animal market of guangzhou, china, was positive for sars-cov rna . the main covs infecting ruminants are reported in table 5 . the oldest known ruminant cov is bcov, which is also the prototype of the species betacoronavirus-1 (subgenus embecovirus, genus betacoronavirus). this virus is able to cause a variety of clinical forms, including enteric disease with high mortality rates in neonate calves, winter disease (a severe enteric form) in lactating cows (decaro et al., 2008b) , and a respiratory disease, also known as shipping fever, in cattle of all ages, with a higher prevalence in 2-3 month-old calves (decaro et al., 2008a) . it was postulated that the presence of genetic signatures differentiates enteric and respiratory bcovs (hasoksuz et al., 1999) , but it was ultimately evident that the same virus strain could be responsible for simultaneous appearance of enteric and respiratory disease in the same animals (chouljenko et al., 2001) . it has been postulated that bcov originated from a rodent cov (corman et al., 2018) . very recently, a novel cov, representing a new viral species, referred to as china rattus coronavirus hku24 (chrcov-hku24), was detected in norway rats in china. this virus was phylogenetically distinct from mhv and hcov-hku1 and displayed genome features that were intermediate between bcov and mhv. therefore, chrcov hku24 may represent the murine origin of bcov and rodents are likely an important reservoir for ancestors of subgenus embecovirus (lau et al., 2015) . bcov is paradigmatic of how covs are able to cross the interspecies barriers, establishing its derivatives as separate viral lineages affecting the respiratory and/or enteric tract of humans (hcov-oc43), swine (phev), horses (equine coronavirus, ecov), and dogs (canine respiratory coronavirus, crcov). a number of bcov-related viruses, all currently included in the unique species betacoronavirus-1, have been detected in the enteric and/or respiratory tract of domestic and wild ruminants. these bcov-like covs include viruses of domestic and domesticated ruminants that were reported in sheep and goats (reinhardt et al., 1995; yang et al., 2008) , water buffalo (bubalus bubalis) (decaro et al., 2008c) , llamas (lama lama) and alpacas (vicugna pacos) (cebra et al., 2003; jin et al., 2007) . in the wild, bcov-like covs were demonstrated in six species of the cervidae family, which are caribou/ reindeer (rangifer tarandus caribou), elk/wapiti (cervus elephus), samber deer (cervus unicolor), white-tailed deer (odocoileus virginianus), sika deer (cervus nippon yesoensis) and water deer (hydropotes inermis) (amer, 2018) . similar viruses were also found to circulate in the giraffe (giraffa camelopardalis) (hasoksuz et al., 2007) , several species of antelopes (alekseev et al., 2008; chung et al., 2011) , wisent (bison bonasus), himalayan tahr (hemitragus jemlahicus) (chung et al., 2011) , and dromedary camels (camelus dromedarius) (woo et al., 2014) . the last strain, detected in the united arab emirates and consequently named dromedary camel coronavirus uae-hku-23 (dccov uae-hku23), was slightly divergent from other bcov-like viruses (woo et al., 2014) . dromedary camels are susceptible to mers-cov infection, developing asymptomatic infections or mild upper respiratory disease, so that they are considered the natural host of mers-cov, with adult animals in many countries in the middle east as well as in north and east africa showing > 90 % seroprevalence to the virus (hemida et al., 2017b) . although human-to-human transmission has occurred outside middle east due to travel-associated patients with mers and has caused large clusters of human cases within healthcare facilities in saudi arabia, jordan and united arab emirates, it remains inefficient and sustained community transmission has not being documented so far, thus suggesting multiple virus introduction into the human population by infected dromedaries (hemida et al., 2017b) . more recently, a phylogenetic study of 173 mers-cov full-genome sequences revealed recombination signatures that defined five major phylogenetically stable lineages, all of which contained human and camel mers-cov sequences (sabir et al., 2016) . in the same study, an alphacoronavirus strictly related to hcov-229e was found in the respiratory tract of dromedary camels of saudi arabia (sabir et al., 2016) . although some studies ruled out the susceptibility of other domestic ruminants to mers-cov (reusken et al., 2013; adney et al., 2016) , a recent study detected specific antibodies and rna in sera and nasal secretions, respectively, of domestic ruminants raised in africa, including sheep, goats and cattle (kandeil et al., 2019) . llamas were found to be susceptible to experimental infections with mers-cov (vergara-alert et al., 2017). the only cov that has been so far known in horses is ecov, which is a bcov-descendant betacoronavirus (subgenus embecovirus). ecov was first isolated from the faeces of a diarrhoeic foal in 1999 (ecov-nc99) in north carolina, usa (guy et al., 2000) , and was initially believed to only affect foals. since 2010, the virus has been recognised in japan, europe and the usa as a new, clinically important, enteric virus of adult horses (pusterla et al., 2018) . despite mers-cov was successfully adapted to the in-vitro growth in equine cell lines (meyer et al., 2015) , serological and molecular table 5 coronaviruses in domestic and domesticated ruminants and associated diseases. sabir et al. (2016) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 investigations have demonstrated that horses are not naturally infected by mers-cov (meyer et al., 2015; hemida et al., 2017a) , nor they are susceptible to experimental infection (adney et al., 2016; vergara-alert et al., 2017) . however, surprisingly, mers-cov rna was detected in respiratory specimens of three donkeys of 42 from egypt (kandeil et al., 2019) , a finding that requires further confirmation. a molecular survey aimed to assess cov circulation in horses in saudi arabia and oman has detected two dccov uae-hku23 strains in enteric samples of horses (hemida et al., 2017a) . scarce data are available about cov circulation in donkeys. these equids are susceptible to ecov infection since positive rt-pcr results were obtained from a donkey in ireland (nemoto et al., 2019) . in addition, three donkeys (7.1 %) of 42 from egypt tested positive for mers-cov rna in their nasal secretions (kandeil et al., 2019) . covs of carnivores are listed in table 6 . three covs are known in dogs, i.e., two alphacoronaviruses of the subgenus tegacovirus, namely ccov-i and ccov-ii, and one betacoronavirus of the subgenus embecovirus, namely crcov. ccovs (species alphacoronav-irus-1) are commonly responsible for mild, self-limiting enteritis in pups . although they are neglected viruses and vaccination is not recommended due to the absence of an effective challenge model, two independent studies have demonstrated their significant involvement in the onset of acute canine enteritis (duijvestijn et al., 2016; dowgier et al., 2017) . the evolutionary history of ccovs is tightly intermingled with that of tgev and fcovs. ccov-i possesses a divergent spike protein and the intact form of an additional gene, orf3, whose remnants are present in ccov-ii and, at a lesser extent, in tgev. therefore, ccov-ii has likely emerged as a consequence of recombination between the original ccov-i and an unknown cov in the s gene and of progressive loss of orf3 (lorusso et al., 2008) . a further recombination occurred in the very 5' end of the s gene between ccov-ii and tgev, giving rise to back recombinant ccov-ii strains, also known as tgevlike ccovs, having a spike protein n-terminus of tgev in a ccov-ii backbone (decaro et al., , 2010 . consequently, the ccov taxonomy was revised, with classical and tgev-like strains being referred to as ccov-iia and ccov-iib, respectively. while ccovs are usually involved in mild forms of diarrhoea, there are some hypervirulent strains that are associated to severe, haemorrhagic, sometimes fatal gastroenteritis. in addition, ccov-iia strains, designated pantropic ccov, that are able to spread systemically and cause severe disease and the death of infected dogs have been reported in italy (buonavoglia et al., 2006; alfano et al., 2020) , other european countries (decaro et al., 2013) and south america (pinto et al., 2014) . genomic sequences from pantropic ccovs were analysed, but no obvious genetic signatures that may have caused the switch in pathogenicity were found (decaro and buonavoglia, 2011; decaro et al., 2013) . different from ccov-i and ccov-ii, the betacoronavirus crcov is associated with mild respiratory signs and has been proposed as an etiological agent of canine infectious respiratory disease (cird) together with other viral and bacterial agents . the virus was first detected firstly in uk in 2003 (erles et al., 2003) and subsequently in other european and extra-european countries (decaro et al., 2007 (decaro et al., , 2016 mitchell et al., 2017; maboni et al., 2019; piewbang et al., 2019; more et al., 2020) . being a bcov derivative, crcov possesses the same genomic organisation, with some differences in accessory orfs located between the s and e protein genes. in particular, while some crcovs possess a unique 8.8 kda protein gene directly downstream of the s protein gene, other canine bcov-like covs display the canonical set of bcov accessory genes but with truncated forms of the 4.8 kda protein gene . in cats, two alphacornavirus-1 genotypes are known, namely fcov type i (fcov-i) and fcov type ii (fcov-ii), the latter being generated as table 6 coronaviruses in domestic and domesticated carnivores and associated diseases. jakob, 1914jacob (1914 , pedersen et al. (1984) cat ( erles et al. (2003) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 a consequence of recombination events between ccov-ii and fcov-i that generated viruses with a ccov-ii genomic region, encompassing orf1b, orf2 (s gene), orf3abc, orf4 (e gene), and partial orf5 (m gene), in the context of an fcov-i backbone (pedersen, 2014) . both genotypes are involved in the development of feline infectious peritonitis (fip), a perivascular pyogranulomatosis of cats that may occur in two clinical forms, effusive and non-effusive fip, which are characterised by prevalence of effusions in the body cavities and of pyogranulomatous lesion in organs, respectively. fip occurs as a consequence of a change in tissue tropism of an enteric fcov strain (feline enteric coronavirus, fecv), infecting enterocytes of the intestinal villi, that acquires the ability to infect monocytes/macrophages switching to the more virulent fipv, which is responsible for systemic infections and dysregulation of the proinflammatory cytokines (addie et al., 2009) . the changes responsible for the pathogenetic shift have been investigated for many decades, being suggested to be variably represented by point mutations located in the s gene (rottier et al., 2005) , deletion/insertion in the group-specific genes 3c (vennema et al., 1998; chang et al., 2010) , 7b (vennema et al., 1998) or 7a (kennedy et al., 2001a) . however, none of these differences appeared to consistently correlate with disease phenotype. more recent studies have identified specific genetic signatures in the s gene of fcov-i that are implicated in monocyte/macrophage tropism. two amino acid substitutions, m1058 l and/or s1060a, corresponding to nucleotide mutations a23531 t/c and t23537 g, respectively, in the viral genome, together distinguished fcovs found in the tissues of fip cats from those found in the faeces of healthy cats without fip in > 95 % of cases (chang et al., 2012) . however, subsequent studies concluded that these mutations are likely to be markers of systemic fcov infection rather than fip per se (porter et al., 2014; barker et al., 2017) . two alphacoronaviruses, both belonging to subgenus minacovirus, are currently known in mustelids, namely mink coronavirus 1 (mcov-1) and ferret coronavirus (frcov). mcov-1 has been recently identified as the etiological agent of mink epizootic catarrhal gastroenteritis (ecg), an infectious disease of farmed american (neovison vison) and european (mustela lutreola) mink first described in 1975 (larsen and gorham, 1975) and later affecting several million mink in different countries (vlasova et al., 2011) . the disease is observed at greater frequency in mink of ≥4 months and is characterised by seasonality, high morbidity (approaching 100 %) and low mortality (< 5 %). recent full-genome analysis demonstrated that mcov-1 is phylogenetically distant from ccovs and fcovs, being closely related to frcov (vlasova et al., 2011) . presently, the two viruses are considered separate species within subgenus minacovirus (https://talk.ictvonline.org/taxonomy/). frcov has been recognised as the causative agent of epizootic catarrhal enteritis (ece), first described in 1993 in domestic ferrets (mustela putorius furo) in the eastern part of the usa (williams et al., 2000) and subsequently reported in domestic and laboratory ferrets throughout the world (murray et al., 2010) . analogous to fcov, frcov exists in two different pathotypes: i) ferret enteric coronavirus (frecv) is associated to ece, a highly contagious diarrhoeal disease also known as green slime disease, which affects mainly young ferrets with morbidity and mortality rates similar to those of ecg; ii) ferret systemic coronavirus (frscv) is responsible for a systemic diseases of ferrets, which is characterised by pyogranulomatous perivasculitis and peritonitis resembling to those of fip (murray et al., 2010) . similar to fip, wise et al. (2010) have shown that frecv and frscv differ significantly in spike protein and that deletions in frcov 3c may also correlate with the severe pathotype of frscv. recombination in the s, 3c and e genes between different frcov has been also reported (lamers et al., 2016) . different covs were found to circulate in wild carnivores. ccovs were detected in wolves (canis lupus), red foxes (vulpes vulpes), eurasian otters (lutra lutra), common genets (genetta genetta) (alfano et al., 2019; rosa et al., 2020) . ccov-like viruses were also found in african wild carnivores, including spotted hyenas (crocuta crocuta) and silver-backed jackals (canis mesomelas) (goller et al., 2013) . fcovs have a wide circulation in non-domestic felids (kennedy et al., 2002 (kennedy et al., , 2003 , with fip cases being reported in servals (felis serval) (juan-salles et al., 1997) , cheetah (acinonyx jubatus) (kennedy et al., 2001b) , mountain lion (puma concolor) (stephenson et al., 2013) , and european wildcat (felis silvestris) (watt et al., 1993) . divergent alphacoronavirus-1 viruses were detected in chinese ferret badger (nyctereutes procyonoides) and raccoon dog (melogale moschata) (dong et al., 2007) . the same study reported the identification in asian leopard cat (prionailurus bengalensis) and chinese ferret badger of an unclassified cov, which was closely related to gammacoronaviruses in most parts of the genome, whereas the s gene displayed the highest sequence identity to alphacoronaviruses (dong et al., 2007) . with the discovery of deltacoronaviruses, these viruses were later included in this novel genus along with avian and porcine strains (woo et al., 2009; wang et al., 2014) . some domestic and wild carnivores are also susceptible to sars-cov infection. while the potential natural reservoirs are horseshoe bats, sars-like cov strains were found to be widespread in masked palm civets (paguma larvata) and raccoon dogs, which were suspected to be intermediate hosts (guan et al., 2003) . full-genomic comparative analysis has shown that sars-like covs isolated from palm civets are under strong selective pressure and are genetically most closely related to sars-cov strains infecting humans early in the outbreaks (song et al., 2005) . sequence analysis of the sars-cov-like virus in masked palm civets indicated that they were highly homologous to human sars-cov with nucleotide identity over 99.6 %, indicating the virus has not been circulating in the population of masked palm civets for a very long time (shi and hu, 2008) . a chinese ferret-badger (melogale moschata) was found to have neutralising antibodies against sars-cov (guan et al., 2003) , whereas sars-cov rna was detected in naturally infected cats and red foxes (vulpes vulpes), but not in domestic dogs . there was, however, a single dog testing positive for sars-cov (https://apps.who.int/iris/bitstream/handle/10665/ 70863/who_cds_csr_gar_2003.11_eng.pdf). among carnivores, sars-cov-2 is able to infect cats, ferrets and, at a lesser extent, dogs . in 2008, a highly divergent cov, tentatively named sw1, was discovered a deceased beluga wale (delphinapterus leucas) with pneumonia and hepatic necrosis (mihindukulasuriya et al., 2008) . the virus was only distantly related to ibv, so that it now represents the prototype of the single mammalian cov species belonging to the genus gammacoronavirus, namely beluga wale coronavirus sw1 (bwcov-sw1) (subgenus cegacovirus). few years later, related gammacoronaviruses were retrieved from faecal samples of three indo-pacific bottlenose dolphins (tursiops aduncus), which were named bottlenose dolphin cov (bdcov) hku22. comparative genome analysis showed that bdcov-hku22 and bwcov-sw1 have similar genome characteristics and structures, displaying a 98 % nucleotide sequence identity each to other (woo et al., 2014) . a novel betacoronavirus distantly related to mers-cov was detected in the faeces of european hedgehogs (erinaceus europaeus), an insectivorous mammal belonging to a related order of chiroptera, from germany. the virus was tentatively referred to as erinaceus cov (ericov) (corman et al., 2014b) and covs found in hedgehogs in france, england and italy had an identity from 92% to 98 % with the ericov (monchatreleroy et al., 2017; saldanha et al., 2019; delogu et al., 2020) . these hedgehog covs are are now included in a unique species, hedgehog coronavirus 1 (subgenus merbecovirus). the virus was not associated to any form of disease, so that western european hedgehog is a reservoir host of ericov in the absence of apparent disease, suggesting that hedgehogs in addition to bats may contribute to the evolution of merbecovirus (saldanha et al., 2019) . a slightly divergent merbecovirus was later found in amur hedgehogs (erinaceus amurensis) in china and was poposed as a prototype of a separate species, namely erinaceus amurensis hedgehog coronavirus hku31 (ea-hedcov hku31) . a novel coronavirus, named wénchéng shrew coronavirus (wesv) was detected in shrews (suncus murinus) in china . wesv is highly divergent from other alphacoronaviruses, exhibiting less than 71.1 % amino acid similarity to any known members of the genus alphacoronavirus in the coronavirus-wide conserved domains of the replicase polyprotein pp1ab and less than 61.3 % amino acid similarity to the other three coronavirus genera. however, taking into account the current ictv criteria, wesv is sufficiently divergent to be considered a distinct member of the genus alphacoronavirus, but not a new genus of the subfamily orthocornavirinae . covs have been known in veterinary medicine since many decades; some of these viruses, such as ibv, swine enteric covs, bcov and mustelid covs, can cause diseases that have a great impact on the farm industry. other covs, namely fipv, frscv and mhv, cause severe disease in companion (cats, ferrets) or laboratory (mice) animals. animal covs are paradigmatic on how covs evolve through accumulation of point mutations and homologous (and heterologous) recombination, generating different genotypes and pathotypes. these virus variants may have different antigenic properties, escaping the host immunity induced by vaccines, as is the case of ibv. alternatively, they may have a different tissue tropism in the same host that can increase or decrease the virus pathogenicity, as observed for the virus pairs fecv/fipv or frecv/frscv and tgev/prcov, respectively. in other circumstances, the cov evolution may result in the switch of the host range from one animal species to another one or from animals to humans. the former event is well documented in veterinary medicine, with a plethora of viruses being originated from ibv and bcov that adapted to different animal species. however, the most interesting scenario is the jumping and further adaptation of an animal cov to humans. there is increasing evidence that all hcovs currently known recognise an animal origin, with bat or rodent covs being the most probable ancestors. in most instances, it was suggested that other mammals served as intermediate hosts prior to final adaptation to humans, i.e., alpacas and cattle for the low-pathogenic hcov-229e and hcov-oc43, respectively, and wild carnivores and dromedary camels for the high-pathogenic sars-cov and mers-cov, respectively. other two hcovs, namely hcov-nl63 and hcov-hku1, were likely derived from bats and rodents, respectively, but whether this transmission required an intermediate mammalian host is presently unknown. the origin of sars-cov-2 should be zoonotic, since highly related sequences were detected in bats, but a definitive intermediate host has been not identified so far. what should we expect from the current pandemic? when hcov-oc43 crossed the species barrier to infect humans from domestic livestock around 1890, an epidemic of respiratory infection was recorded. even though, several years later, influenza was suspected to be the cause of it, in that pandemic involvement of central nervous system was more pronounced than in other influenza outbreaks. this evidence is further supported by molecular studies claiming that the most recent common ancestor of bcov and hcov-oc43 emerged around 1890 (vijgen et al., 2005) and by the fact that hcov-oc43 can be neuroinvasive (arbour et al., 2000) . likely, hcov-oc43 crossed species to infect dogs becoming established in this species as crcov . a similar scenario could be observed with sars-cov-2 with dogs and, at a greater extent, cats. apparently, cats represent, within the domestic animals which have been experimentally infected, the host, together with ferrets, which is able to sustain more efficiently sars-cov-2 replication . furthermore, based on structural studies and biochemical experiments, sars-cov-2 seems to have an rbd that binds with high affinity to ace2 also from ferrets and cats (andersen et al., 2020) . reasonably, a full comprehension of the animal cov molecular evolution, host range and pathobiology is beneficial to better understand the mechanism driving the emergence and adaptation to humans of zoonotic covs. the present review has highlighted that in the last 18 years, also thanks to the availability of novel sequencing technologies, we have witnessed a large number of novel covs being discovered in a large number of animals. truth to be told, it was difficult for us to summarise, in this single review, all covs detected in animals and the tight interaction existing between them and human covs. among animals, it is evident that bats are the group of mammals that harbor the largest number of covs and that many other animal covs recognise their ancestors in bat covs. in an excellent review (cui et al., 2019) written by the group coordinated by dr. zheng-li shi of the wuhan institute of virology, hubei, (china), city infamously known for being the epicenter and origin of the covid-19 outbreak, authors stated that "...given the prevalence and great genetic diversity of bat sars-rcovs, their close coexistence and the frequent recombination of covs, it is expected that novel variants will emerge in the future". this forecasting statement was not surprising to coronavirologists and it was not, importantly, surprising to those scientists that daily deal with the plethora of viruses existing at the human/animal health interface. although scientists were well aware of this hazard, no substantial actions were taken forward the limitations of strict and repeated contacts between humans and wildlife. indeed, whereas biological mechanisms underlying viral evolution are not under human control, social and cultural habits can be modified accordingly through a deep and pounding informative campaigns. if to the human habits we sum the impact of modern agricultural practices and urbanization and the decrease of vital space for wildlife, it is quite easy to understand that, if countermeasures are not taken, we will face novel serious health emergencies of animal origin in the following years with tremendous social and economic impact on our lives. as clearly demonstrated by the sars-cov-2 emergence, covs are the main characters of this intricate puzzle characterised by the interactions of viral biological mechanisms and human habits. our review was reasonably prepared also to highlight (once more!) how covs originate, evolve, jump, mutate and infect their host. could have the current covid-19 outbreak been avoided? answering this question is not relevant now, but actions to avoid the next viral spillover from animals to humans is certainly a priority. this task needs to be coupled with massive genomic surveillance in wild animals not limited to covs. massive sequencing of sars-cov-2 strains detected in humans and covs of wildlife will help further assess the origin of this novel human pandemic and plan future measures able to reduce the risk of emergence of new cov spillover events. however, additional tasks should be provisionally addressed in order to reduce the risk of future cov pandemic like the current one. these include: i) prevention of animal-to-human infections through a ban of the wet markets and a more friendly management of the environment; ii) studies on cov-host interactions to be performed both in vitro (cell cultures, ex-vivo explants of the respiratory tract) and in vivo (animals susceptible to sars-cov-2 infection); iii) development of new anticoronaviral drugs and evaluation of their efficacy in cell cultures and animal models. feline infectious peritonitis. abcd guidelines on prevention and management inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host new chimeric porcine coronavirus in swine feces bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences dentification of pantropic canine coronavirus in a wolf (canis lupus italicus) in italy circulation of pantropic canine coronavirus in autochthonous and imported dogs binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity novel receptor specificity of avian gammacoronaviruses that cause enteritis host tissue and glycan binding specificities of avian viral attachment proteins using novel avian tissue microarrays bovine-like coronaviruses in domestic and wild ruminants the proximal origin of sars-cov-2 further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus neuroinvasion by human respiratory coronaviruses detection of a virus related to betacoronaviruses in italian greater horseshoe bats isolation and metagenomic identification of avian leukosis virus associated with mortality in broiler chicken global distributions and strain diversity of avian infectious bronchitis virus: a review random nature of coronavirus rna recombination in the absence of selection pressure bats and coronaviruses limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis cultivation of the virus of infectious bronchitis emerging horizon for bat borne viral zoonoses. virus dis characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 characterization of the virus of sialodacryoadenitis of rats: a member of the coronavirus group recovery and characterization of a coronavirus from military dogs with diarrhea porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus coronavirus genome structure and replication accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence first complete genome sequence of european turkey coronavirus suggests complex recombination history related with us turkey and guinea fowl coronaviruses canine coronavirus highly pathogenic for dogs bats: important reservoir hosts of emerging viruses coronaviruses in poultry and other birds coronavirus avian infectious bronchitis virus infectious bronchitis coronaviruses from pheasants (phasianus colchicus) are genetically closely related to coronaviruses of domestic fowl (infectious bronchitis virus) and turkeys potential pathogens in feces from unweaned llamas and alpacas with diarrhea feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene spike protein fusion peptide and feline coronavirus virulence a murine virus jhm causing disseminated encephalomyelitis with extensive destruction of myelin. isolation and biological properties of the virus sars-associated coronavirus transmitted from human to pig identification and survey of a novel avian coronavirus in ducks dbatvir: the database of bat-associated viruses detection of specific antibodies to the nucleocapsid protein fragments of severe acute respiratory syndrome-coronavirus and scotophilus bat coronavirus-512 in three insectivorous bat species comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia coronaviruses in bent-winged bats (miniopterus spp genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats avian coronavirus in wild aquatic birds a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans detection and characterization of bovine-like coronaviruses from four species of zoo ruminants coronavirus associated with an enteric syndrome on a quail farm mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs evidence for an ancestral association of human coronavirus 229e with bats hosts and sources of endemic human coronaviruses identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229e origin and evolution of pathogenic coronaviruses infectious bronchitis virus variants: a review of the history, current situation and control measures alpha and lineage c betacov infections in italian bats canine coronavirus: not only an enteric pathogen serological and molecular evidence that canine respiratory coronavirus is circulating in italy respiratory disease associated with bovine coronavirus infection in cattle herds in southern italy severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (bubalus bubalis) calves recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs an update on canine coronaviruses: viral evolution and pathobiology recombinant canine coronaviruses in dogs european surveillance for pantropic canine coronavirus molecular surveillance of traditional and emerging pathogens associated with canine infectious respiratory disease eco-virological preliminary study of potentially emerging pathogens in hedgehogs (erinaceus europaeus) recovered at a wildlife treatment and a transmissible gastroenteritis in pigs covid-19 from veterinary medicine and one health perspectives: what animal coronaviruses have taught us detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations detection of a novel and highly divergent coronavirus from asian leopard cats and chinese ferret badgers in southern a molecular survey for selected viral enteropathogens revealed a limited role of canine circovirus in the development of canine acute gastroenteritis genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences enteropathogen infections in canine puppies: (co-)occurrence, clinical relevance and risk factors coronaviruses detected in brazilian wild birds reveal close evolutionary relationships with beta-and deltacoronaviruses isolated from mammals detection of a group 2 coronavirus in dogs with canine infectious respiratory disease bat coronaviruses in china a previously undescribed coronavirus associated with respiratory disease in humans evidences of parasite evolution after vaccination imperfect vaccines and the evolution of pathogen virulence a hemagglutinating virus producing encephalomyelitis in baby pigs metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in china isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor a metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in neoromicia bats within south africa coronavirus genotype diversity and prevalence of infection in wild carnivores in the serengeti national park the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. coronaviridae study group of the international committee on taxonomy of viruses animal models for sars and mers coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china three emerging coronaviruses in two decades turkey coronavirus enteritis characterization of a coronavirus isolated from a diarrheic foal a new virus isolated from the human respiratory tract middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation rodent reservoirs of future zoonotic diseases isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe coronavirus infections in horses in saudi arabia and oman dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor emerging highly virulent porcine epidemic diarrhea virus: molecular mechanisms of attenuation and rational design of live attenuated vaccines bat origin of human coronaviruses discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus origin, evolution, and genotyping of emergent porcine epidemic diarrhea virus strains in the united states a bat-derived putative cross-family recombinant coronavirus with a reovirus gene genetically diverse coronaviruses in wild bird populations of northern england studies on broiler's ibv and ib-like virus from guinea fowl therapeutische, kasuistische und statistische mitteilungen aus der klinik für kleine haustiere an der reichstierarzneischule in utrecht (holland) analysis of the genome sequence of an alpaca coronavirus molecular identification and characterization of novel coronaviruses infecting graylag geese anser anser feral pigeons columbia livia and mallards anas platyrhynchos identification of avian coronavirus in wild aquatic birds of the central and eastern usa an outbreak of feline infectious peritonitis in captive servals (felis serval): clinical, pathological, and immunohistochemical findings calf diarrhea coronavirus. the lancet molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals deletions in the 7a orf of feline coronavirus associated with an epidemic of feline infectious peritonitis detection of feline coronavirus infection in captive cheetahs (acinonyx jubatus) by polymerase chain reaction detection of feline coronavirus in captive felidae in the usa detection of feline coronavirus infection in southern african nondomestic felids surveillance of avian coronaviruses in wild bird populations of korea a novel coronavirus associated with severe acute respiratory syndrome identifying sars-cov-2 related coronaviruses in malayan pangolins naturally occurring recombination in ferret coronaviruses revealed by complete genome characterization a new mink enteritis: an initial report severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coexistence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku9 belonging to a novel betacoronavirus subgroup recent transmission of a novel alphacoronavirus, bat coronavirus hku10, from leschenault's rousettes to pomona leaf-nosed bats: first evidence of interspecies transmission of coronavirus between bats of different suborders discovery of a novel coronavirus, china rattus coronavirus hku24, from norway rats supports the murine origin of betacoronavirus 1 and has implications for the ancestor of betacoronavirus lineage a discovery and sequence analysis of four deltacoronaviruses from birds in the middle east reveal interspecies jumping with recombination as a potential mechanism for avian-to-avian and avian-to-mammalian transmission identification of a novel betacoronavirus (merbecovirus) in amur hedgehogs from china. viruses 11 wild boars harbouring porcine epidemic diarrhea virus (pedv) may play an important role as a pedv reservoir detection of coronaviruses in bats of various species in italy novel avian coronavirus and fulminating disease in guinea fowl isolation of avian infectious bronchitis coronavirus from domestic peafowl pavo cristatus and teal anas gain, preservation, and loss of a group 1a coronavirus accessory glycoprotein molecular characterization of a canine respiratory coronavirus strain detected in italy a new member of the pteropine orthoreovirus species isolated from fruit bats imported to italy novel coronavirus (sars-cov-2) epidemic: a veterinary perspective longitudinal surveillance of betacoronaviruses in fruit bats in yunnan province discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus sequence of mouse hepatitis virus a59 mrna 2: indications for rna recombination between coronaviruses and influenza c virus canine infectious respiratory disease: new insights into the etiology and epidemiology of associated pathogens first full-length sequences of the s gene of european isolates reveal further diversity among turkey coronaviruses recovery in tracheal organ cultures of novel viruses from patients with respiratory disease ecosystem change and zoonoses in the anthropocene middle east respiratory syndrome coronavirus in bats, saudi arabia a sars-like cluster of circulating bat coronaviruses shows potential for human emergence identification of a lineage d betacoronavirus in cave nectar bats (eonycteris spelaea) in singapore and an overview of lineage d reservoir ecology in se asian bats serologic assessment of possibility for mers-cov infection in equids identification of a novel coronavirus from a beluga whale by using a panviral microarray european surveillance of emerging pathogens associated with canine infectious respiratory disease identification of alpha and beta coronavirus in wildlife species in france: bats, rodents, rabbits, and hedgehogs porcine hemagglutinating encephalomyelitis virus: a review a serological survey of canine respiratory coronavirus in new zealand detection and full genome characterization of two beta cov viruses related to middle east respiratory syndrome from bats in italy diseases and causes of death in european bats: dynamics in disease susceptibility and infection rates role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain prevalence and phylogeny of coronaviruses in wild birds from the bering strait area (beringia) ferret coronavirus-associated diseases the first detection of equine coronavirus in adult horses and foals in ireland isolation of a coronavirus during studies on puffinosis, a disease of the manx shearwater (puffinus puffinus) isolation and characterization of viruses associated with transmissible enteritis (bluecomb) of turkeys genome organization of canada goose coronavirus, a novel species identified in a mass die-off of canada geese rat coronavirus (rcv): a prevalent, naturally occurring pneumotropic virus of rats isolamento di un coron-avirus simile da quaglie giapponesi (coturnixcoturnix japonica) con sindrome respiratoria:prima descrizione della malattia ed isolamentodel virus an update on feline infectious peritonitis: virology and immunopathogenesis cross-sectional investigation and risk factor analysis of community-acquired and hospital-associated canine viral infectious respiratory disease complex characterization of pantropic canine coronavirus from brazil pathogenic differences between various feline coronavirus isolates isolation of a porcine respiratory, nonenteric coronavirus related to transmissible gastroenteritis identification of a novel coronavirus in bats amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis enteric coronavirus infection in adult horses two genotypes of canine coronavirus simultaneously detected in the fecal samples of dogs with diarrhea the behaviour of recent isolates of human respiratory coronavirus in vitro and in volunteers: evidence of heterogeneity among 229e-related strains diagnosis of coronavirus in sheep in valdivia province difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan identification of sars-like coronaviruses in horseshoe bats (rhinolophus hipposideros) in slovenia electron microscopy of coronavirus-like particles characteristic of turkey bluecomb disease prevalence of antibodies to selected viral pathogens in wild boars (sus scrofa) in croatia in 2005-06 and 2009-10 unveiling patterns of viral pathogen infection in free-ranging carnivores of northern portugal using a complementary methodological approach acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia extension of the known distribution of a novel clade c betacoronavirus in a wildlife host an apparently new respiratory disease of baby chicks transmissible gastroenteritis virus infection: a vanishing specter sialic acid binding properties of soluble coronavirus spike (s1) proteins: differences between infectious bronchitis virus and transmissible gastroenteritis virus a review of studies on animal reservoirs of the sars coronavirus susceptibility of ferrets, cats, dogs, and different domestic animals to sars-coronavirus-2 identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus rna genome cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human from sars to mers, thrusting coronaviruses into the spotlight isolation of infectiousbronchitis virus from pheasants feline infectious peritonitis in a mountain lion a persistently infecting coronavirus in hibernating myotis lucifugus, the north american little brown bat antigenic cross-reactivity between the nucleocapsid protein of severe acute respiratory syndrome (sars) coronavirus and polyclonal antisera of antigenic group i animal coronaviruses: implication for sars diagnosis isolation and characterization of avian coronavirus from healthy eclectus parrots (eclectus roratus) from indonesia prevalence and genetic diversity of coronaviruses in bats from on the origin and continuing evolution of sars-cov-2 surveillance of bat coronaviruses in kenya identifies relatives of human coronaviruses nl63 and 229e and their recombination history detection of novel sars-like and other coronaviruses in bats from kenya an avian coronavirus in quail with respiratory and reproductive signs gammacoronavirus and deltacoronavirus in quail phylogenomic analyses elucidate the evolutionary relationships of bats enteric coronavirus-like coronavirus particles in sheep s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification a serological survey of selected pathogens in wild boar in slovenia feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses livestock susceptibility to infection with middle east respiratory syndrome coronavirus complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event evolutionary history of the closely related group 2 coronaviruses: porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, and human coronavirus oc43 molecular characterization of a new species in the genus alphacoronavirus associated with mink epizootic catarrhal gastroenteritis surveillance on severe acute respiratory syndrome associated coronavirus in animals at a live animal market of guangzhou in 2004 detection and genetic characterization of deltacoronavirus in pigs discovery, diversity and evolution of novel coronaviruses sampled from rodents in china discovery of a highly divergent coronavirus in the asian house shrew from china illuminates the origin of the alphacoronaviruses emerging and re-emerging coronaviruses in pigs an extended outbreak of infectious peritonitis in a closed colony of european wildcats (felis silvestris) coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol high prevalence and putative lineage maintenance of avian coronaviruses in scandinavian waterfowl coronavirus-associated epizootic catarrhal enteritis in ferrets sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent comparative sequence analysis of the distal one-third of the genomes of a systemic and an enteric ferret coronavirus an apparently new syndrome of porcine epidemic diarrhoea global epidemiology of bat coronaviruses characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia molecular diversity of coronaviruses in bats comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features comparative analysis of complete genome sequences of three avian coronaviruses reveals a novel group 3c coronavirus coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases a new coronavirus associated with human respiratory disease in china serosurveillance of viral diseases in korean native goats (capra hircus) receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans structure of coronavirus hemagglutinin-esterase offers insight in corona and influenza virus evolution isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic analysis and surveillance of the coronavirus dominant in ducks in china funding were provided by the italian ministry of health, izs am 08/19 rc , ricerca corrente 2019 "ngs e diagnostica molecolare in sanità animale: fast d2", recipient alessio lorusso. supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2020.108693. key: cord-331980-m6dflwmm authors: alqahtani, amani s.; wiley, kerrie e.; mushta, sami m.; yamazaki, kaoruko; bindhim, nasser f.; heywood, anita e.; booy, robert; rashid, harunor title: association between australian hajj pilgrims’ awareness of mers-cov, and their compliance with preventive measures and exposure to camels date: 2016-07-18 journal: j travel med doi: 10.1093/jtm/taw046 sha: doc_id: 331980 cord_uid: m6dflwmm through a prospective cohort study the relationship between travellers’ awareness of mers-cov, and compliance with preventive measures and exposure to camels was evaluated among australian hajj pilgrims who attended hajj in 2015. only 28% of australian hajj pilgrims were aware of mers-cov in saudi arabia. those who were aware of mers-cov were more likely to receive recommended vaccines [odds ratio (or) 3.1, 95% confidence interval (ci): 1.5–5.9, p < 0.01], but there was no significant difference in avoiding camels or their raw products during hajj between those who were aware of mers-cov and those who were not (or 1.2, 95% ci: 0.3–5.2, p = 0.7). hajj pilgrims’ awareness is reflected in some of their practices but not in all. as of 22 june 2016, middle east respiratory syndrome coronavirus (mers-cov) infection has been identified in 1762 people in 27 countries with 36% mortality. 1 recent studies have demonstrated the potential role of camels, the only known zoonotic source of mers-cov, in the transmission of mers-cov to humans. 2, 3 saudi arabia, the epicentre of mers-cov (with >80% of global mers-cov burden), 1 hosts the hajj pilgrimage in mecca, where 2-3 million people assemble annually. millions also visit mecca and medina throughout the year on a minor pilgrimage called umrah. although no mers-cov cases have been reported in relation to hajj, several imported cases have been recorded among returning umrah pilgrims in the uk, malaysia, tunisia, algeria and the netherlands. [4] [5] [6] potential sources of infection are exposure to camels or their products, other mers patients and hospital visits. 5 previous studies assessing awareness of mers-cov among french, australian and turkish hajj pilgrims found that between 35 and 65% pilgrims were unaware of the presence of the disease in saudi arabia. [7] [8] [9] however, no study has attempted to investigate the association between hajj pilgrims' awareness of mers-cov, and their compliance with preventive measures and exposure to camels. therefore, we conducted a prospective cohort study among australian travelers who attended hajj pilgrimage in 2015. brief communication cruited participants were then followed up during hajj, and again after their return from hajj. before hajj, between mid-august and the first week of september 2015, the researchers attended pre-hajj seminars run by eleven specialist hajj travel agents in sydney. most prospective pilgrims attend these seminars as part of their preparations for travel to mecca, making it an ideal place to recruit a relatively representative and generalizable sample. all pilgrims attending these seminars were approached and invited to participate in the study. upon consent, data on socio-demographic characteristics, details of their travel itinerary and receipt of pre-travel health advice from medical (such as a general practitioner [gp]) and nonmedical sources (tour operator) were collected in a selfadministered questionnaire. the respondents' knowledge, attitudes and perceptions regarding mers-cov, their willingness to use preventive measures while at hajj, and their understanding of the risk of mers-cov infection from exposure to camels, were descriptively analyzed using a three-point likert scale. during hajj, we endeavored to meet all the same participants in mina, saudi arabia and followed them daily throughout the peak days of hajj (from 21 to 26th of september 2015). a separate questionnaire, in the form of diary card, was administered to determine their actual use of preventive measures such as facemask and hand hygiene, and document development of symptoms suggestive of acute respiratory infection retrospectively for three days before arriving in mina, then daily for seven consecutive days. after hajj, the participants took part in a computer aided telephone interview (cati) 7 to 10 days after their return to australia (until 26th of december, 2015). pilgrims were asked about the history of their contact with camels and consumption of camel products (e.g. milk and meat) at hajj and development of respiratory symptoms after their return from hajj. data collected before, during and after hajj were linked by a unique barcode. a non-random sampling plan was used to gather a sample which was representative of pilgrims residing in nsw. the sample size calculation was based on a previous study 11 where a sample of 10% of the target population (3500 australian pilgrims in 2015) was deemed sufficient. we estimated that about 35% australian pilgrims would be aware of mers-cov, 7 and considering an error margin of 2.5%, a sample of 350 was deemed to be sufficient for this study; with compliance adjustment of 20% a total of 420 participants were approached. statistical analysis was performed using the statistical package for social sciences (spss) v.23.0 (spss, inc., chicago, il, usa). pearson correlation coefficient and chi-square test were used to assess variables and determine associations and correlations. univariate factors with p values < 0.25 were entered into multivariate regression models. two-tailed p values < 0.05 were considered statistically significant in multivariate analyses. this study was reviewed and approved by the human research ethics committee (hrec) at the university of sydney (project no: 2014/599). a total of 421 pilgrims were enrolled in the study; their demographics are described in table 1 . their median duration of stay in saudi arabia was 25 (range: 10-45) days. the majority (78%) of participants received one or more recommended vaccines: 76% (319/421) received influenza vaccine and 25% (106/ 421) additionally received pneumococcal vaccine. fifty eight percent (245/421) of pilgrims received pre-travel medical advice before hajj; many consulted other sources as shown in table 1 . of the 421 participants enrolled, 391 (93%) were followed during hajj, and 300 (71%) could be reached by cati after their return home. twenty eight percent (117/421) of respondents were aware of mers-cov before travelling to saudi arabia. of those who were aware, 44% (51/117) answered correctly that the virus affects the respiratory tract, and 26% (31/117) were 'very concerned' of contracting mers-cov at hajj ( table 2) . none of the demographic factors were associated with mers-cov awareness. pilgrims who were aware of mers-cov were more likely to receive recommended vaccines than those who were not [odds ratio (or) 3.1, 95% confidence interval (ci): 1.5-5.9, p < 0.01]. overall, 39% (165/421) believed that there was a moderate to high risk of contracting disease from consumption of raw camel milk, 12% (50/421) thought the risk was low or nil, while about 49% (207/421) did not know of the risk. pilgrims who were aware of mers-cov were significantly more likely to consider it a risk to drink unpasteurized camel milk as high than those who were unaware (74% vs 39%, p < 0.01). the acceptability of preventive measures, and participant's actual compliance are presented in table 2 . those who were aware of mers-cov were more likely to intend to avoid contact with camels (or 2.1, 95% ci: 1.1-4.0, p ¼ 0.01) and consume their raw products (or 2.2, 95% ci: 1.2-3.9, p ¼ 0.01) than those who were unaware. moreover, participants who were concerned with catching mers-cov during hajj were more likely to accept to use facemask compared with those who were not concerned (or 3.6, 95% ci: 1.6-8.3, p < 0.01). however, except for vaccination, no significant difference was observed in the actual uptake of preventive measures between those who were aware or concerned of mers-cov and those who were not. seven pilgrims (2%) reported actually coming in contact with camels and/or consuming their products during hajj; five (1%) were unaware of mers-cov before travel. the type of exposure to camels included taking photographs [1.3% (1/150)] with camels, consuming boiled [1% (3/300)] or raw milk [0.6% (2/300)] and meat [0.6% (2/300)]. out of seven pilgrims who came in contact with camels four (1%) developed respiratory symptoms during and/or immediately after hajj. no significant differences were found in avoiding camels or their raw products during hajj between those who were aware (or 1.2, 95% ci: 0.3-5.2, p ¼ 0.7) or concerned (or 0.9, 95% ci: 0.1-10.9, p ¼ 0.9) of mers-cov and those who were not. this study shows that only 28% of australian pilgrims were aware of mers-cov before attending the hajj 2015, with some engaging in high risk behaviours such as exposure to camels (2%), and non-compliance with preventive measures. awareness was lower compared with previous surveys of similar australian studies showing that up to 49% of pilgrims were aware of mers-cov; 7,12 other studies have shown 45% of turkish and 65% of french pilgrims to be aware. 8, 9 importantly, this study showed that pilgrims who were aware of mers-cov were twice more likely to intend to avoid contact with camels and consume their raw products during hajj than those who were unaware. similarly, those who were concerned of catching mers during hajj were approximately four times more likely to intend to use facemask during hajj. this may indicate that pilgrims' awareness of infectious diseases is reflected in how acceptable they find using some preventive measures and is consistent with a previous study by our team that showed that pilgrims who were aware of mers-cov were also significantly more aware of its risks from drinking unpasteurized camel milk compared with those who were unaware (43% vs 23%, p < 0.01). 12 conversely, this study highlights that while the knowledge and perceived susceptibility to the disease were associated with their intention to use preventive measures, these factors were absent in their actual use. theoretically, the frame work of "precaution adoption model" sets forward stages of health behavior change in an individual and the factors that lead people to move from one stage to another such as awareness, perceived severity and susceptibility, and cues to action. 13 however, in this study we observed that perceived severity and susceptibility did not seem to play much role in motivating the pilgrims to comply with preventive measures (figure 1 ). although majority of pilgrims who were concerned with contacting mers-cov at hajj planned to use preventive measures, in practice few applied the measures and no significant difference was observed between those who were concerned and those who were not. this may indicate that among hajj pilgrims, there may be unique factors and barriers that affect their compliance with preventive measures against the infectious diseases which needs further investigation. other studies have documented the pilgrims' willingness to comply with mers-cov preventive measures, 8, 9 in practice many participants might not have adhered to those as seen in this study. two percent (n ¼ 7) of pilgrims actually came into contact with camels or consumed their products during their pilgrimage, which is an improvement compared with the previous year when between 7 and 16% pilgrims were reported to be exposed to camels. 12, 14 this apparent decline may be attributed to the saudi arabian authorities' banning of bringing and sacrificing camels into hajj sites in 2015. 15 to our knowledge, this is the first cohort study which has assessed pilgrims' awareness about mers-cov and their actual practice of risk avoidance, especially their contact with camels at hajj; however, the study findings have limited generalizability outside of australia. additionally, the pilgrims' participation in the pre-travel survey may have increased their awareness of the recommended preventive health measures, leading to higher reported usage (known as 'the hawthorne effect'). although this is certainly a theoretical consideration, research into the hawthorne effect on practice-based research suggests that its impact is minimal. 16 moreover, the exact site and time of camel exposure were not explored, and data collection relied on self-report. in conclusion, this study showed that many australian pilgrims are unaware of the risk of mers-cov in saudi arabia; and some engaged in high-risk practices such as coming in contact with camels. pilgrims' awareness of the risk is reflected in their vaccine uptake but not in their avoidance of camel exposure. middle east respiratory syndrome coronavirus co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia middle east respiratory syndrome coronavirus: current situation and travel-associated concerns imported cases of middle east respiratory syndrome: an update middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands australian hajj pilgrims' knowledge about mers-cov and other respiratory infections hajj pilgrims knowledge about middle east respiratory syndrome coronavirus attitudes and practices concerning middle east respiratory syndrome among umrah and hajj pilgrims in samsun exploring barriers to and facilitators of preventive measures against infectious diseases among australian hajj pilgrims: cross-sectional studies before and after hajj camel exposure and knowledge about mers-cov among australian hajj pilgrims in 2014 a model of the precaution adoption process: evidence from home radon testing pilot use of a novel smartphone application to track traveller health behaviour and collect infectious disease data during a mass gathering: hajj pilgrimage 2014 saudi arabia, animal reservoir, camels, hajj. archive number: 20150912. 3641457 an assessment of the hawthorne effect in practice-based research professor robert booy has received funding from baxter, csl, gsk, merck, novartis, pfizer, roche, romark and sanofi pasteur for conducting this research, travel to conferences or consultancy work; all funding received is directed to research accounts at the children's hospital at westmead. dr a.e.h. has received grant funding from gsk and sanofi pasteur for investigator-driven research. dr h.r. has received fees from pfizer and novartis for consulting or serving on an advisory board.conflict of interest: none declared. key: cord-338980-pygykil7 authors: rahaman, jordon; siltberg-liberles, jessica title: avoiding regions symptomatic of conformational and functional flexibility to identify antiviral targets in current and future coronaviruses date: 2016-11-09 journal: genome biol evol doi: 10.1093/gbe/evw246 sha: doc_id: 338980 cord_uid: pygykil7 within the last 15 years, two related coronaviruses (severe acute respiratory syndrome [sars]-cov and middle east respiratory syndrome [mers]-cov) expanded their host range to include humans, with increased virulence in their new host. coronaviruses were recently found to have little intrinsic disorder compared with many other virus families. because intrinsically disordered regions have been proposed to be important for rewiring interactions between virus and host, we investigated the conservation of intrinsic disorder and secondary structure in coronaviruses in an evolutionary context. we found that regions of intrinsic disorder are rarely conserved among different coronavirus protein families, with the primary exception of the nucleocapsid. also, secondary structure predictions are only conserved across 50–80% of sites for most protein families, with the implication that 20–50% of sites do not have conserved secondary structure prediction. furthermore, nonconserved structure sites are significantly less constrained in sequence divergence than either sites conserved in the secondary structure or sites conserved in loop. avoiding regions symptomatic of conformational flexibility such as disordered sites and sites with nonconserved secondary structure to identify potential broad-specificity antiviral targets, only one sequence motif (five residues or longer) remains from the >10,000 starting sites across all coronaviruses in this study. the identified sequence motif is found within the nonstructural protein (nsp) 12 and constitutes an antiviral target potentially effective against the present day and future coronaviruses. on shorter evolutionary timescales, the sars and mers clades have more sequence motifs fulfilling the criteria applied. interestingly, many motifs map to nsp12 making this a prime target for coronavirus antivirals. severe acute respiratory syndrome (sars)-cov and middle east respiratory syndrome (mers)-cov are two closely related zoonotic coronaviruses. both have successfully crossed the species barrier to allow animal-to-human transmission, and further to allow human-to-human transmission (song et al. 2005; reusken et al. 2016) . the sars outbreak in 2003 had a mortality rate of 10% (anderson et al. 2010) , and sars-cov was considered the most aggressive coronavirus compared to other human coronaviruses that commonly cause mild to moderate infection in their hosts (van der hoek 2007) . mers-cov is the cause of an ongoing outbreak of the respiratory illness mers (de groot et al. 2013) . at the time of writing, 1791 mers cases have been confirmed with a mortality rate of approximately 35% (world health organization 2016). both mers and sars have higher mortality rates in elderly and immunosuppressed populations (gralinski and baric 2015) . the host changes by mers-cov and sars-cov suggest that other coronaviruses can potentially cross the species barrier, become zoonotic, and enable human-to-human transmission, ultimately causing high morbidity and mortality. sars-cov and mers-cov exploited mechanistically different approaches to overcome the human species barrier, but these two viruses have a lot in common (lu et al. 2015) . here, we aim to identify the vulnerable regions in the proteomes of coronaviruses that neither sars-cov nor mers-cov nor their contemporary and forthcoming relatives can proliferate without, and address how to mobilize a defense against the present and future coronaviruses by targeting these regions. sars-cov and mers-cov are positive (+)-strand rna viruses encoding approximately 25 protein products. the mers-cov proteome is primarily composed of two polyproteins, orf1a and orf1ab; the latter is generated by a -1 ribosomal slippage frameshift. these proteins are cleaved into 16 nonstructural proteins (nsps). nsps 1-10 are products of both polyproteins, whereas nsps 12-16 are only yielded by orf1ab. nsp11 is unique to orf1a ( van boheemen et al. 2012) . structural proteins envelope (e), spike (s), membrane (m), and nucleocapsid (n) are elements of the physical structure that encloses the viral genome and come from distinct reading frames, unlike orf1a and orf1ab, which come from overlapping reading frames. additionally, the structural proteins are the product of subgenomic mrnas that are joined during discontinuous negative rna strand synthesis (van boheemen et al. 2012) . finally, ns3 protein (ns3), ns4a protein (ns4a), ns4b protein (ns4b), ns5 protein (ns5), and orf8b protein encompass the remainder of the proteome and also arise from distinct reading frames (van boheemen et al. 2012) . our approach utilizes genomic sequence data, which is readily available for viruses known to cause disease. however, because most viruses pose no major threat to their host, they pass by unnoticed leaving the majority of virus genome space uncharted. with the availability of costefficient genome sequencing technology, and recent developments in the field of viral metagenomics, large-scale identification of viral genome space is on the rise (rosario and breitbart 2011; mokili et al. 2012) . by exploring viral diversity, critical components constituting a viral genus' fitness can be evaluated. examples such as the common influenza virus illustrate the rapidity of viral gene mutation and in order to maintain immune protection, an annual flu vaccination is recommended. underway efforts aim to generate broadly neutralizing vaccines whose design accounts for the genomic sequences of multiple types of influenza virus to eliminate frequent re-vaccination against the flu ross 2011, 2012) . development of broadly neutralizing vaccines often relies on the consensus or ancestral sequences of extant viral sequences in order to provide greater coverage for related viruses (kesturu et al. 2006) . unfortunately, consensus sequences can be misleading, and ancestral sequence reconstruction is error-prone for quickly diverging sequences (mccloskey et al. 2014 ). in addition, viruses with compact genomes often express proteins with structural disorder that may undergo structural transformations. although these transformer proteins, like vp40 in ebola, are masters at changing their structure, and thus expanding their functional repertoire as needed for the life cycle of the virus (bornholdt et al. 2013) , flexible regions are potentially important in rewiring protein-protein interactions between the virus and its host (le breton et al. 2011; ortiz et al. 2013; gitlin et al. 2014) . the flexibility trait of many viral proteins is a complicating factor in vaccine development. for instance, dengue virus exhibits serotype-specific antibody affinity that causes antibodydependent enhancement, an obstacle in the development of dengue vaccines that protects against all four serotypes (flipse and smit 2015) . to overcome the hurdle posed by structural flexibility, we propose an additional screening step in identifying potential vaccine or antiviral targets that considers the structural flexibility of the viral proteins. the structural genomics initiatives increased their success rate by excluding proteins predicted to be structurally disordered (slabinski et al. 2007) . a similar approach can perhaps benefit vaccine development. furthermore, to make this approach robust to potential mutations, minimizing loss in efficacy or resistance, the evolutionary context of sequence and structure must be considered. thus, we suggest expanding the concept of broadly neutralizing vaccines/antivirals by increasing the diversity of viruses considered if possible. sites conserved for sequence, structure, and with low disorder propensity among diverse virus protein homologs are very likely to be constrained from 1) changing sequence on evolutionary time scales and 2) undergoing real-time structural transitions. these sites have potential as targets for broad-specificity antivirals or vaccines because conservation makes them broad-specificity and low dynamics avoids targeting a conformational ensemble, which is not only difficult (yu et al. 2016) , but that may change as the sequence diverges (siltberg-liberles et al. 2011) . a recent large-scale study of structural disorder in >2,000 viral genomes in 41 viral families found the amount of disorder in different virus families varying from 2.9% to 23.1% (pushker et al. 2013) . it was reported that coronaviridae has very low disorder content (mean disorder 3.68%) (pushker et al. 2013) . coronaviridae contains two subfamilies: coronavirinae and torovirinae. sars-cov and mers-cov are part the coronavirinae subfamily, from here on referred to as coronavirus (cov). the lack of disorder is intriguing because it may be important for rewiring interactions between viral proteins and host proteins (ortiz et al. 2013 ) and providing opportunities to acquire novel functional sequence motifs (gitlin et al. 2014) . structural disorder has also been proposed to be important for viral viability, enabling multifunctionality and vigor in response to changes in the environment (xue et al. 2014) . given the low fraction of structural disorder reported across coronaviridae, we set out to investigate the conservation of structural disorder and secondary structure across cov. sites identified as conserved for structure and lacking disorder can be considered to be vulnerable and druggable in the proteomes of coronaviruses. the structural divergence capacity of these regions is limited, leaving a wider range of the present and emergent coronaviruses susceptible to the effects of potential broadly neutralizing anti-cov therapies targeting these sites. we will refer to these sites as target sites. protein sequences were identified by individual blast searches with mers-cov (taxonomy id: 1335626) proteins orf1ab (yp_009047202.1; polyprotein), s protein (yp_009047204.1), m protein (yp_009047210.1), e protein (yp_009047209.1), and n protein (yp_009047211.1) against coronaviruses. blast searches of the orf1ab protein were performed, using start and end positions as detailed in the orf1ab ncbi reference sequence file, against the refseq_protein database. the sequences retrieved from the blast output maintained the following cutoff: >30% sequence identity and >50% coverage relative to mers-cov sequence query. the 30% sequence identity and 50% query coverage cutoff strikes a balance between alignment quality and at least 10 sequences for most protein families. nsp1 (yp_009047202.1; 1-193), nsp2 (yp_009047202.1; 194-853), ns3 (yp_009047205.1), ns4a (yp_009047206.1), ns4b (yp_009047207.1), ns5 (yp_009047208.1), orf8b protein (yp_009047212.1), and nsp11 (yp_009047203.1; 4378-4391) are not included in this study due to <10 blast hits. multiple sequence alignments were constructed for the selected blast hits using mafft (katoh et al. 2002) . phylogenetic trees were constructed using mrbayes 3.2.2 with a four category gamma distribution and the mixed model for amino acid substitution (huelsenbeck and ronquist 2001; ronquist and huelsenbeck 2003) . each tree ran for five million generations, with a sample frequency of 100. the final tree was constructed from the last 75% of samples, discarding the first 25% of samples as the default burnin, and using the half-compatible parameter, to avoid weakly supported nodes (i.e., with a posterior probability <0.5). all trees were midpoint rooted. for every protein family, the amino acid substitution rate per site in its multiple sequence alignment was calculated using empirical bayesian estimation as implemented in rate4site (mayrose et al. 2004) . substitution rates were calculated using 16 gamma categories, the jtt substitution matrix (jones et al. 1992) , and the reconstructed phylogenies. the rates were normalized per protein family with an average across all sites equal to zero and sd equal to 1. this means that sites with a rate <0 are evolving slower than average, whereas sites with a rate >0 are evolving faster than average. intrinsic disorder propensity was inferred using two different predictors: iupred (default settings; "long" option) (dosztá nyi et al. 2005a (dosztá nyi et al. , 2005b and disopred2 (ward et al. 2004 ) for all proteins. for iupred, the site-specific continuous disorder propensities for each protein were mapped onto their corresponding position in the multiple sequence alignment as raw disorder propensities and as binary states, order or disorder, using two cutoffs of 0.4 and 0.5. disorder propensities below the cutoff were assigned order and disorder propensities at the cutoff or above were assigned disorder. for the disopred2 predictions that were inferred using the nr database, the continuous disorder propensities for every site in a protein were mapped onto their corresponding position in the multiple sequence alignment as raw disorder propensities and as binary states, order or disorder, using a cutoff of 5. consequently, for every protein family (a multiple sequence alignment and its corresponding phylogenetic tree), two continuous matrices and three binary matrices resulted: iupred 0.4, iupred 0.5, and disopred2. an additional matrix was generated to indicate sites where the binary order and disorder assignments differ between iupred 0.4 and disopred2. a similar methodology was employed to analyze secondary structure predicted by psipred (mcguffin et al. 2000) and jpred (drozdetskiy et al. 2015) . for both predictors, the uniref90 database was used and sites were classified as loops, alpha helices, or beta strands and mapped back onto their corresponding sites in the multiple sequence alignment. this resulted in two three-state matrices for each protein family alignment, one for each predictor, and two binary matrices displaying secondary structure elements (alpha helix and beta strand) or loops. an additional matrix was generated to indicate sites where the secondary structure assignments differ between psipred and jpred. for every protein family, the binary matrices resulting from the different disorder predictions and from the different secondary structure predictions were analyzed in the corresponding evolutionary context using gloome. gloome (gain-loss mapping engine) analyzes binary presence and absence patterns in a phylogenetic context . in this study, the rate4site option in gloome was used to analyze the binary matrices (iupred 0.4, iupred 0.5, disopred2, psipred, and jpred) with the corresponding phylogenetic trees to map change of state across sites in each individual protein phylogeny . gloome was run with 16 gamma categories and a substitution matrix set to equal rates within each state and transitions between states treated equally. from the binary disorder and order matrices, transition rates between disorder and order or vice versa (dot) were estimated. from the binary structure and loop matrices, transition rates between structure and loop or vice versa (slt) were estimated. similar to rate4site, the rates were normalized per protein family with an average across all sites equal to zero and sd equal to 1. this means that sites with a rate <0 are evolving slower than average, while sites with a rate >0 are evolving faster than average. protein families were visualized in an integrative manner with a phylogenetic tree, any matrix (multiple sequence alignment or predictor based) displayed as a heatmap, and site-specific sequence transition rates using python packages ete3 (huerta-cepas et al. 2016) and matplotlib (hunter 2007) . amino acid evolutionary rates (seq) for all sites across all alignments were aggregated and binned into four possible categories characterized by the distribution of psipred predicted secondary structure at each site. sites predicted to have a loop across all sequences are "conserved loops; c(l)" and sites predicted to have a helix across all sequences or a strand across all sequences are "conserved helix-strand; c(hs)" (table 3) . sites predicted to have all three states (helix, strand, and loop) or any combination of loop and one other state are "non-conserved helix, loop, strand; nc(hls)" and sites predicted to have a mixture of helix and strand are "nonconserved helix-strand; nc(hs)" (table 3) . in all cases, gaps were ignored when classifying combinations of secondary structure at a site or if secondary structure conservation exists at a particular site. phylogenies were built for all protein products encoded in the mers-cov single-stranded rna genome, except for nsp1, nsp2, ns3, ns4a, ns4b, orf8b protein, and nsp11, all of which had insufficient sequence data (<10 sequence hits with blast). nsp12 is often used as a measure for newly identified coronaviruses. according to the international committee of taxonomy of viruses, a major criterion in determining if a coronavirus is considered novel is pairwise sequence identity below 90% for nsp12 in all comparisons to previously known coronaviruses (bermingham et al. 2012) . four main clades, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus ( fig. 1) , are identified in agreement with the taxonomic classifications described by the ictv (international committee on taxonomy of viruses 2015). coronaviruses not listed by the ictv are assumed to be a part of the clade in which representatives with known classifications are situated in our nsp12 phylogeny. the mers clade and sars clade are sister clades in the nsp12 phylogeny. the hku1 clade and equ clade are also sister clades. together these four clades form the betacoronavirus clade, in accordance with the ictv classification (international committee on taxonomy of viruses 2015). betacoronavirus is represented in all phylogenies although the order of the individual subclades varies. alphacoronavirus is often found as the sister clade or outgroup to betacoronavirus. deltacoronavirus or gammacoronavirus are the most distantly related to the betacoronavirus. in the nucleocapsid phylogeny, gammacoronavirus is the first outgroup clade to betacoronavirus, and alphacoronavirus is the most distant outgroup. most nsp trees exhibit some unresolved nodes at junctures immediately preceding terminal nodes. as an effect of the 50% majority rule, most of the 546 resolved nodes are well supported with posterior probability >0.9 for 82% and >0.99 for 68% (supplementary fig. s1 , supplementary material online). most trees follow the nsp12 topology for the main clades, with minor clade rearrangements. it should be noted that for nsp5, the entire alphacoronavirus clade is placed within the betacoronavirus clade, as a sister clade to the mers clade (supplementary fig. s1 , supplementary material online). this may be due to increased sequence divergence rates or due to recombination. recombination events are rather frequent in coronaviruses (su et al. 2016) , and the mers clade potentially underwent multiple recombination events as part of the host change (zhang et al. 2016) . the phylogenies for membrane protein, spike protein, nsp5, and nsp8-nsp16 demonstrate (with the given blast cutoffs) recoverable protein homologs such that all coronaviruses are represented (i.e., all coronaviruses represented in the nsp12 phylogeny). nucleocapsid, nsp4, and nsp7 have recoverable homologs in all clades except deltacoronavirus. nsp3 and nsp6 homologs are too divergent in deltacoronavirus and/or gammacoronavirus relative to mers-cov. envelope appears specific to betacoronavirus ( fig. 1) , but it is a short protein that has been found to diverge rapidly and is likely present outside betacoronavirus (fehr and perlman 2015) . because different protein families yield slightly different phylogenies, for the remaining evolutionary analyses, every protein family was analyzed in the context of its own phylogeny. for all protein families, structural disorder propensities were predicted using iupred (dosztá nyi et al. 2005a (dosztá nyi et al. , 2005b and disopred2 (ward et al. 2004) . to verify the robustness of the binary iupred and disopred2 predictions, the binary assignments were compared on a site-by-site basis (table 1) . when converted to binary (i.e., two states per site disordered or ordered) iupred 0.4 and iupred 0.5 are in good agreement with the larger differences seen for nsp8, nsp9, and nucleocapsid (7.5%, 6.5%, and 19.0%, respectively) (table 1). comparing iupred 0.4 or iupred 0.5 to disopred2, large differences are in particular seen for nucleocapsid (38.7% and 29.7% respectively) and nsp8 (23.5% and 25.9%, respectively) (table 1) . for nucleocapsid, regions that are found to be disordered by iupred 0.4 are found to be ordered by iupred 0.5 and disopred2 ( fig. 2 and supplementary fig. s2 , supplementary material online). for nsp8, regions that are only slightly disordered in a few sequences according to iupred 0.4 and iupred 0.5, disopred2 predicts disorder to be conserved for all sequences (fig. 3) . to quantify the fraction of disordered sites per protein family, we report the iupred 0.4 results only for simplicity (table 1). in general, iupred 0.4 predicts more disorder than disopred2, but several protein families have almost no disordered sites. nsp3 and nsp8-10 have some variation in disorder content for different viruses. based on the fraction of disorder, nucleocapsid is the only highly disordered protein among the covs in this study, even if nsps 8-10 have outliers that are >20% disordered. to compare the disorder-to-order transition rates (dot) for all protein families where the binary matrices of disorder and order include both states, the quadrant count ratio (qcr) was estimated as a measure of association in assigning slower than average vs. faster than average transition rates. for iupred 0.4 vs. iupred 0.5, for iupred 0.5 vs disopred2, and for iupred 0.4 vs. disopred2, the qcrs (table 2) . for nucleocapsid and nsp8, the positive associations are weaker, suggesting that many sites have iupred disorder propensity in the 0.4 to 0.5 range and large differences between iupred and disopred2, in accordance with the large disagreement between the binary assignment of these predictors (tables 1 and 2). for all protein families, secondary structure elements were predicted using psipred (mcguffin et al. 2000) and jpred (drozdetskiy et al. 2015) . for most protein families, the disagreement between secondary structure predictors is greater than for the disorder predictors (table 1). in fact, 15 of the 17 protein families compared disagree at more than 10% of alignment sites, and two of these disagree at more than 20% of sites. to compare the binary structureto-loop transitions (slt), qcr was estimated as a measure of association for slt based on the different predictors. in general, there is a moderate positive association between slt for psipred vs. slt for jpred that is weaker than for the different dot comparisons (table 2) . it should be noted that slt does not differentiate between alpha helix and beta strand, but considers both as "structure." this is a correct assumption if protein structure is conserved and consistently predicted, but for some protein families that is not the case. four protein families (nsp3, nsp12, nsp13, and spike) have more than 40% of their sites found within the nc(hls) category with non-conserved helix, strand, and loop (two or three states present at the same site) (table 3). for nsp13, jpred predicts 72% of all sites to be a mixture of helix, strand, and loop, or any combination of loop and one other structural element ( fig. 4) . envelope and nsp6 have 13% and 12% of their respective sites in the nc(hs) category. considering only the psipred predictions, the nc(hs) category has 245 sites across all 17 protein families. that is one-tenth the size of the next smallest set which is c(hs) with 2275 sites. next, c(l) has 3344 sites, and the largest category is nc(hls) with 4257 sites. comparing the evolutionary sequence rates for the sites in the different categories, based on psipred predictions only, reveals that sites in the c(hs) category are evolving at a slower rate than all other categories. nc(hs) is only just significantly different (p = 4.62eà03) from c(hs), and is not significantly different from nc(hls) and c(l) (p = 1.85eà02 and p = 8.33eà01, respectively). however, nc(hls) and c(l) are significantly different from each other, and both are significantly different from c(hs) (p = 1.82eà46 and p = 2.33eà21, respectively) ( fig. 5 ). for regions with five or more consecutive sites that were 100% conserved in sequence across 1) all cov or 2) across the mers and sars clades, the information of structural disorder prediction from iupred and disopred2 was used to identify all ungapped sites that were consistently predicted to have 100% conserved order. next, the information of secondary structure prediction from psipred and jpred was used to narrow down this list further by only including sites that are not changing their predicted secondary structure state for both predictors. applying the aforementioned filters to the initial 10,000 sites resulted in one (1) region of five residues or more conserved across all cov within the n-terminal domain of nsp12: dnqdl (table 4) . interestingly, this region is in the vicinity of sites found important for nucleotidylating activity across the order nidovirales (lehmann et al. 2015) . considering only the sequences in the sars and mers clades, 21 sequence regions of five residues or more were found in seven protein families (table 4) . for nsp5, nsp7, and nsp14, experimentally determined structures show that most regions are surface accessible ( fig. 6) . some of the identified target sites are known for their functional importance. for instance, c145 in the middle of gscgs in nsp5 is part of the catalytic dyad in the nsp5 protease (yang et al. 2003) . for nsp12 and nsp13, which have the majority of all sites, no structures are available. the sites adjacent to dnqdl are also conserved in the sars and mers clades, and five additional target sites, conserved for the sars and mers clades, are found in the c-terminal direction relative to the dnqdl motif (table 4) . continuing into the rna-dependent rna polymerase domain (rdrp) in nsp12, four additional regions of target sites are found, and the last three regions are found in the c-terminal part. importantly, in rdrp and in the c-terminal part are sites that are also conserved across all covs in this study. nsp13 has four regions of target sites distributed across the protein. 3. -the evolutionary context of intrinsic disorder in nsp8. the phylogenetic tree was built using the multiple sequence alignments for nsp8. (a) the multiple sequence alignment is colored by amino acid according to scale, arranged based on top-idp disorder promoting propensity of the amino acids (campen et al. 2008) , and gray denotes gaps. (b) iupred disorder propensity per site in the multiple sequence alignment. blue-to-white-to-red shows disorder propensity according to the scale for iupred 0.4. (c) iupred disorder propensity per site in the multiple sequence alignment. blue-to-white-to-red shows disorder propensity according to the scale for iupred 0.5. (d) disopred2 disorder propensity per site in the multiple sequence alignment. blue-to-white-tored shows disorder propensity according to the scale. above the multiple sequence alignment, the normalized evolutionary rates per site for amino acid substitution (seq) and the dot for the binary transformations of b-d are shown. heat maps visualized with the python packages ete3 (huerta-cepas et al. 2016) and matplotlib (hunter 2007) . see supplementary figures s2 and s4, supplementary material online for additional graphics for every protein family. we have analyzed the protein evolution of the genetic components that make up the mers-cov proteome. as previously established, mers-cov has the same genomic makeup as hku4-cov and hku5-cov in the mers clade (woo et al. 2012) . some protein products are only found in the mers clade, and these were excluded from this study due to insufficient data. furthermore, for other protein products, some clades may not be represented in our protein families if their proteins were too divergent. this was an important factor in determining the applied blast hit cutoffs, as relaxing cutoffs produced alignments with more gaps and increasing stringency reduced the representative pool. because alignment quality is important due to the sensitivity of both rate4site and for phylogenetic reconstruction, the chosen cutoffs are suitable. we note some clade-specific differences in recoverable homologs between different cov, but many components are shared among them ( fig. 1 ). viral proteins often possess multifunctionality, mediated by a conformational change in response to environment-specific factors (xue et al. 2014) . although conformational flexibility is important for function, it also offers flexibility in what sequence motifs are on display. if these sequences are rapidly diverging, different sequence motifs will be displayed, reinforcing the notion that flexible regions are potentially important in rewiring protein-protein interactions between virus and host (gitlin et al. 2014) . although most cov proteins have almost no intrinsic disorder, several cov protein families have homologous sites that display loop in some sequences, helix in others and strands in some (table 3, supplementary fig. s3 , supplementary material online). these sites are not necessarily disordered but they may be conformationally flexible in realtime (with secondary structure transitions in the same sequence, making them difficult to predict) or on evolutionary time-scales (so that different secondary structure elements actually are present in different sequences). the c(hs) and c(l) sites make up approximately 50-80% of most multiple sequence alignments. with the common expectation that protein structure is more conserved than sequence these numbers are surprisingly low. neither psipred nor jpred consistently predicts the same state for 20-50% of all sites in these multiple sequence alignments. the accuracy of psipred and jpred's secondary structure predictions are about 80% (bryson et al. 2005; drozdetskiy et al. 2015) . psipred has been found to rarely predict an alpha helix instead of a beta strand and vice versa, and most of the psipred errors are due to secondary structure not being predicted (li et al. 2014) . when secondary structure is not conserved for the same site in a multiple sequence alignment, it suggests that the secondary structure prediction may be 1) inaccurate, 2) not predicted with high confidence, or 3) the regions are indeed metamorphic; they can transition from one element to another. although (1) is difficult to address without experimentally determined structures for all sequences, (2) and (3) are not necessarily incompatible interpretations because low confidence secondary structure prediction could indicate metamorphic secondary structure regions. metamorphic secondary structure regions have interesting consequences for conformational and functional flexibility. it should be noted that, despite the low amount of disordered sites in most cov proteins, several regions are not conserved in disorder propensity across all sequences, but sometimes the different predictors disagree as in the case of nsp8. clade-specific disordered regions resulting from indel events suggest that they are not essential to the critical functions of the protein, but could cause gain-and-loss of interactions with its hosts. however, when disorder propensity is only mildly fading for a region that is present across the protein family, it may be important for the fundamental function of the protein. the virus structural proteins that interact to form the virion commonly include an envelope protein, a membrane protein, and a capsid protein that together form the machinery that encases, transports, and releases the virus. the interactions between the structural proteins are often regulated by conformational changes like vp40 in ebola (bornholdt et al. 2013 ) and envelope protein from dengue virus (zheng et al. 2014 ). conformational changes to another. it should also be noted that two mers clade specific inserts around position 241 and toward the c-terminal are consistently predicted to be highly disordered. with inserts and changing structural dynamics between clades or viruses, the questions become 1) which sequence motif are displayed and 2) to what extent are these sequence motifs displayed? furthermore, based on the inconsistent prediction of secondary structure elements, the possibility that covs are more conformationally flexible than their intrinsic disorder content implies is noteworthy. altogether, this suggests that various mechanisms for rewiring conformational and functional space are operating in the coronaviruses studied here. if regions symptomatic of conformational and functional flexibility can be avoided in order to identify broad-specificity antiviral targets with potential to be effective against coronaviruses of today and in the future, coronaviruses as a group may become more attractive drug targets for the pharmaceutical industry in the event an additional coronavirus changes host to include humans or increase its virulence. supplementary tables s1 and s2 and figures s1-s5 are available at genome biology and evolution online (http://www. gbe. oxfordjournals.org/). update on sars research and other possibly zoonotic coronaviruses the protein data bank severe respiratory illness caused by a novel coronavirus structural rearrangement of ebola virus vp40 begets multiple functions in the virus life cycle flavivirus ns3 and ns5 proteins interaction network: a high-throughput yeast two-hybrid screen protein structure prediction servers at university college london top-idp-scale: a new amino acid scale measuring propensity for intrinsic disorder gloome: gain loss mapping engine inference and characterization of horizontally transferred gene families using stochastic mapping middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group iupred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content the pairwise energy content estimated from amino acid composition discriminates between folded and intrinsically unstructured proteins jpred4: a protein secondary structure prediction server coronaviruses: an overview of their replication and pathogenesis the complexity of a dengue vaccine: a review of the human antibody response target sites shown in 3d context. (a) nsp5 dimer, based on pdb id 1uk4 (yang et al. 2003). (b) nsp7, based on pdb id 5f22 (unpublished). (c) nsp14, based on pdb id 5c8t a computationally optimized broadly reactive antigen (cobra) based h5n1 vlp vaccine elicits broadly reactive antibodies in mice and ferrets computationally optimized antigens to overcome influenza viral diversity rapid evolution of virus sequences in intrinsically disordered protein regions molecular pathology of emerging coronavirus infections mrbayes: bayesian inference of phylogenetic trees ete 3: reconstruction, analysis, and visualization of phylogenomic data virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses the rapid generation of mutation data matrices from protein sequences mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform minimization of genetic distances by the consensus, ancestral, and center-of-tree (cot) sequences for hiv-1 variants within an infected individual and the design of reagents to test immune reactivity discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the rna polymerase-containing protein of all nidoviruses bayesian model of protein primary sequence for secondary structure prediction bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex comparison of site-specific rate-inference methods for protein sequences: empirical bayesian methods are superior an evaluation of phylogenetic methods for reconstructing transmitted hiv variants using longitudinal clonal hiv sequence data the psipred protein structure prediction server metagenomics and future perspectives in virus discovery rapid evolutionary dynamics of structural disorder as a potential driving force for biological divergence in flaviviruses marked variability in the extent of protein disorder within and between viral families cross host transmission in the emergence of mers coronavirus mrbayes 3: bayesian phylogenetic inference under mixed models exploring the viral world through metagenomics the evolution of protein structures and structural ensembles under functional constraint the challenge of protein structure determination-lessons from structural genomics cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human epidemiology, genetic recombination, and pathogenesis of coronaviruses genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans the disopred server for the prediction of protein disorder genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 world health organization. 2016. who j middle east respiratory syndrome coronavirus structural disorder in viral proteins the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor structure-based inhibitor design for the intrinsically disordered protein c-myc evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission a toggle switch controls the low ph-triggered rearrangement and maturation of the dengue virus envelope proteins we thank joseph ahrens, janelle nunez-castilla, and helena gomes dos santos for assistance in the lab and for helpful discussions. the authors would also like to acknowledge the instructional & research computing center (ircc) at florida international university for providing hpc computing resources that have contributed to the research results reported within this article, web: http://ircc.fiu.edu. key: cord-338057-ycmr9prw authors: lee, jae hoon; lee, chang-seop; lee, heung-bum title: an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) date: 2015-07-15 journal: j korean med sci doi: 10.3346/jkms.2015.30.8.1207 sha: doc_id: 338057 cord_uid: ycmr9prw nan the editorial, "better understanding on mers coronavirus (mers-cov) outbreak in korea, " was previously published by lee (1) . he briefly summarized the ongoing status of the middle east respiratory syndrome (mers) outbreak and emphasized close monitoring of medical staffs, patients, and visitors, and timely well-designed briefings to mass media. he pointed out critical aspects to control the mers-cov outbreak in korea. the present opinion proposes a topic on mers by focusing on early diagnosis of patients via appropriate specimen collection. as on july 2, 2015, there have been 183 confirmed cases of mers and 33 fatalities reported in the republic of korea (2). this national outbreak of mers has not been controlled yet but there is a rapid decrease in the number of current infections and fatalities. all patients in korea acquired their disease in hospital settings where they came in direct or indirect contact with mers patients. the ministry of health and welfare and the korea centers for disease control and prevention have carried out strong enforcement measures including quarantine care for up to 14 days (the maximum incubation period of the disease) in cases of symptomatic travelers from the arabian peninsula who arrived in korea within the past 2 weeks and individuals who came in close contact with confirmed cases of mers (2) . suspicious cases are transferred to an assigned hospital if symptoms occur during the observation period. real-time reversetranscription polymerase chain reaction (rt-pcr) assay conducted in 48-hour-intervals using respiratory samples is the standard diagnostic tool for mers. a 63-year-old man developed fever, chills, and myalgia on june 10, 2015. on may 27, he visited the emergency department of a hospital that had previously reported several cases of mers. during this time, he stayed in close contact with a laboratoryconfirmed mers patient. he was quarantined on may 27, 2015 at his home and he developed fever and chills, nausea, anorexia, and myalgia after the quarantine isolation. he was then moved to an institutional isolation care unit where real-time rt-pcr was conducted twice on sputum samples in 48-hour-intervals and resulted in negative findings. he was afebrile but his gastrointestinal discomfort and myalgia continued, and the isola-tion location was changed to his home on june 2, 2015 because he was improving. on june 8, he became febrile with symptoms of cough, dyspnea, and myalgia, which gradually deteriorated. he was tested again and the results returned positive for mers-cov on june 10. radiographic findings revealed far-progressed bilateral peripheral radio-opacities. some reasons that could be considered for the delayed diagnosis of mers include: 1) low initial viral load and shedding, and 2) poor sample collection in patients with "no cough" or dry cough. rapid and correct diagnosis of infection and control measures are critical in preventing the possible spread of mers-cov. however, the early symptoms of mers are non-specific and are the same as those of common pneumonia. in this context, it is very important to conduct thorough and careful patient interviews with a high interest of mers in mind and to obtain optimal samples for diagnosis. in the clinical findings of patients infected with mers-cov, patients with dry cough are more common than patients with productive sputum (3, 4) . therefore, optimal sample collection is frequently limited, particularly in patients with no cough or dry cough. dipeptidyl peptidase 4 (dpp4) has been identified as the receptor for mers-cov (5). dpp4 is expressed in type i and ii alveolar cells, ciliated or non-ciliated bronchial epithelium, and bronchial submucosal glands (5, 6) . this corresponds with viral tropism in ex vivo human lung cultures (5) (6) (7) . in studies with rhesus macaques, intra-tracheally inoculated virus was present in the lungs but neither in the upper respiratory tracts, trachea, nor other organs (8, 9) . all of marmosets infected with a high dose of mers-cov via various routes showed multifocal to coalescing, moderate to marked acute broncho-interstitial pneumonia centered on small calibre and terminal bronchioles which further extended into the adjacent pulmonary parenchyma. in summary, in vitro and in vivo studies concluded that mers-cov had adherence to the lower respiratory tract and high viral loads were mainly detected in distal respiratory tissues. therefore, lower respiratory tract specimens such as bronchoalveolar lavage fluid, deep tracheal aspirates, and induced sputum contain the highest viral loads which are optimal for increasing diagnostic accuracy (10) (11) (12) (13) . in re-evaluating the patient's diagnostic history, his viral load could have been low due to the early phase of disease and/or could have been falsely negative due to inadequate dry coughlinked respiratory samples. in any occasion, his diagnosis was quite delayed. delayed diagnosis of patients is inevitably linked to delayed quarantine care of persons with contact history. to obtain an accurate diagnosis, the circumstances in the early phase of mers-cov infection should be considered and at least two repeated diagnostic tests during the late incubation period should be considered for patients with less severity but persistent symptoms. the nationwide pneumonia census has been conducted to identify hidden mers-cov infections. however, inadequate sputum specimens must have resulted in false negatives. for accurate surveying, appropriate specimens should have been obtained by collecting sputum from the lungs or bronchi and not saliva. in suspicious patients who are unable to produce sputum for examination, aerosol administration of a hypertonic saline solution may be used to increase the flow of secretions and stimulate coughing. however, during the procedure of induced specimen collection, clinicians must consider the high risk of contamination of the surroundings because the procedure would create a large amount of aerosols and increase the risk of transmitting the virus to other individuals. the optimum time for collection of a sputum specimen is in the early morning before eating or drinking. at this time secretions accumulated in the bronchi through the night are more readily available. in conclusion, to control the mers-cov infection completely, delicate history taking related with mers is very important, and appropriate specimen collection is essential as well. moreover, even two real-time rt-pcr tests in negative results during the early stage of the disease process cannot rule out silent mers-cov infections. clinicians should consider the possibility of false negative real-time rt-pcr findings resulting from inappropriate sample collection. finally, a sufficient period and strict quarantine of suspected cases are critical for control of the mers outbreak in korea. better understanding on mers corona virus outbreak in korea middle east respiratory syndome information middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs pathogenesis of middle east respiratory syndrome coronavirus an animal model of mers produced by infection of rhesus macaques with mers coronavirus middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome kinetics and pattern of viral excretion in biological specimens of two mers-cov cases first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities key: cord-337089-ksh62ni0 authors: salajegheh tazerji, sina; magalhães duarte, phelipe; rahimi, parastoo; shahabinejad, fatemeh; dhakal, santosh; singh malik, yashpal; shehata, awad a.; lama, juan; klein, jörn; safdar, muhammad; rahman, md. tanvir; filipiak, krzysztof j.; rodríguez-morales, alfonso j.; sobur, md. abdus; kabir, farrokhreza; vazir, bita; mboera, leonard; caporale, marco; islam, md. saiful; amuasi, john h.; gharieb, rasha; roncada, paola; musaad, sahar; tilocca, bruno; koohi, mohammad kazem; taghipour, ali; sait, ahmet; subbaram, kannan; jahandideh, alireza; mortazavi, pejman; abedini, mohammad amin; hokey, david a.; hogan, unarose; shaheen, mohamed n. f.; elaswad, ahmed; elhaig, mahmoud m.; fawzy, mohamed title: transmission of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) to animals: an updated review date: 2020-09-21 journal: j transl med doi: 10.1186/s12967-020-02534-2 sha: doc_id: 337089 cord_uid: ksh62ni0 covid-19 caused by a novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) originated in wuhan (hubei province, china) during late 2019. it has spread across the globe affecting nearly 21 million people with a toll of 0.75 million deaths and restricting the movement of most of the world population during the past 6 months. covid-19 became the leading health, economic, and humanitarian challenge of the twenty-first century. in addition to the considerable covid-19 cases, hospitalizations, and deaths in humans, several cases of sars-cov-2 infections in animal hosts (dog, cat, tiger, lion, and mink) have been reported. thus, the concern of pet owners is increasing. moreover, the dynamics of the disease requires further explanation, mainly concerning the transmission of the virus from humans to animals and vice versa. therefore, this study aimed to gather information about the reported cases of covid-19 transmission in animals through a literary review of works published in scientific journals and perform genomic and phylogenetic analyses of sars-cov-2 isolated from animal hosts. although many instances of transmission of the sars-cov-2 have been reported, caution and further studies are necessary to avoid the occurrence of maltreatment in animals, and to achieve a better understanding of the dynamics of the disease in the environment, humans, and animals. future research in the animal–human interface can help formulate and implement preventive measures to combat the further transmission of covid-19. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the disease was referred to as coronavirus disease-2019 (covid-19) [3] . the sars-cov-2 is the third coronavirus that has emerged in the last two decades. others are the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov), which emerged during 2002 and 2012, respectively [2, 4] . compared to sars-cov and mers-cov, sars-cov-2 is responsible for the most significant economic loss and the highest number of infections and deaths [5] . until 13 august 2020 around 21 million covid-19 cases and over 0.75 million deaths have been reported. sars-cov-2 belongs to the order nidovirales, suborder coronavirineae, and family coronaviridae. the family coronaviridae contains two subfamilies named lentovirinae and orthocoronavirinae. the latter is further classified into four genera, namely alphacoronavirus (αcov), betacoronavirus (βcov), gammacoronavirus (γcov), and deltacoronavirus (δcov), as shown in fig. 1 . the γcovs and δcovs cause diseases in birds while αcovs and βcovs are mainly found in mammals such as bats, rodents, civets, pigs, horses, cattle and humans [6] [7] [8] [9] . sars-cov-2 clusters with lineage βcov together with sars-cov and mers-cov [8] ; both originating from bats [1, 10, 11] . coronaviruses are enveloped viruses with singlestranded positive-sense rna (+ss) and genome size between 26 and 32 kb in length [12, 13] . these viruses possess four essential structural proteins: spike glycoprotein (s), matrix protein (m), envelope protein (e), and nucleocapsid protein (n) [13] [14] [15] 22] . according to the genomic analysis, sars-cov-2 shares a 96.2% identity with the genome of bat cov; ratg13, indicating a possible origin of the virus in bats [3, 16] . several studies suggested that the pangolin could be the potential intermediate host involved in the evolution of the virus because of the unique receptor-binding domain configuration [17] [18] [19] 22] . among the global fear due to the rapid spread of the covid-19 and absence of specific treatment or vaccine, the first case of human-to-animal transmission was recorded in hong kong, where a 17-years-old pomeranian dog was affected [20] . this case raised concerns about the possibility of sars-cov-2 transmission from humans to animals and vice versa, which would represent the difficulties in fighting the virus significantly. however, based on recently published findings, other authors hypothesized that an immunological cross-protection between sars-cov-2 and canine respiratory coronavirus (crcov) exists due to the high homology between the spike protein epitopes of the two taxonomicallyrelated coronaviruses [21] . the objective of the present study was to gather, present, and discuss information on the reported cases of covid-19 in animals focusing on the virus transmission cases in pets and perform genomic and phylogenetic analyses of sars-cov-2 isolated from animal hosts. further studies on the dynamics of the disease are essential to adopt suitable control measures to reduce transmission of the virus. to discuss the origin of sars-cov-2, it is necessary to analyze the source of other coronaviruses such as sars-cov and mers-cov, as shown in fig. 2 [22] . sars-cov emerged in guangdong province, southern china, in november 2002 and was characterized as a contagious respiratory disease of humans [22, 23] . records showed 8422 registered cases and 916 deaths in humans, spreading to 29 countries during the epidemic with a case fatality rate (cfr) of 9.6% [23] . studies revealed the presence of several coronaviruses in species of horseshoe bats (genus rhinolophus), which are evolutionarily related to sars-cov in their genome organization and sequence [24, 25] . in the further investigations of the initial sars outbreak of 2002 in hong kong, it was recognized that cats (felis domesticus) and ferrets (mustela furo) could be infected with sars-cov [26] . in 2012, another coronavirus causing severe acute respiratory syndrome in humans was first reported in saudi arabia and was later called the middle east respiratory syndrome coronavirus (mers-cov) [27] . mers-cov is βcov of the c lineage with a genotype very similar to that of bats of the same line, such as btcov-hku4 and btcovhku5 [28] . the transmission of mers-cov to dromedaries was identified through the detection of specific antibodies against the virus in these animals [29] . according to corman et al. [30] , mers-cov has been circulating in dromedaries for more than 20 years [30] . therefore, it can be assumed that bats are reservoirs for several coronaviruses, including sars-cov, mers-cov, and sars-cov-2 [25, 31, 32] . according to reports sars-cov-2 is closely related to sars-cov [33, 34] and the similarity between the genomes was about 80% [3, 16, 35] . though sars-cov-2 has most likely originated from bats, it is not yet clear which animal served as an intermediate host and contributed towards the evolution of the virus before the spillover to humans occurred [22] . in another study, it was demonstrated that sars-cov-2 was a chimeric virus between a bat coronavirus and a coronavirus of unknown origin [36] . in a similar research, it was reported a novel bat-derived coronavirus, denoted rmyn02, identified from the metagenomic analysis of samples from 227 bats collected from yunnan province in china between may and october 2019 [37] . notably, rmyn02 shares a 93.3% nucleotide identity with sars-cov-2 at the scale of the complete genome and 97.2% identity in the orf1ab gene. it could, therefore, be considered the closest relative of sars-cov-2 reported to date. in contrast, rmyn02 showed low sequence identity (61.3%) with sars-cov-2 in the receptor-binding domain (rbd), suggesting that rmyn02 might not bind to angiotensin-converting enzyme 2 (ace2). critically, and in a similar manner to sars-cov-2, rmyn02 was characterized by the insertion of multiple amino acids at the junction site of the s1 and s2 subunits of the spike (s) protein. this provides strong evidence that such insertion events can occur naturally in animal βcov [37, 39] . it can be seen that tiger sars-cov-2/usa/ ny-040420 epi_isl_420293 shares 99.96% nucleotide identity with human sars-cov-2 reference genome at the complete genome-scale, followed by mink sars-cov-2/nb04 epi_isl_447634 (99.90%), mouse sars-cov-2/hrb-26 m epi_isl_459910 (99.87%), cat sars-cov-2 epi_isl_437349 (99.85%), and dog sars-cov-2/hkg/20-03695/2020 (99.51%) demonstrating a high level of nucleotide identity between sars-cov-2 isolates from domestic animals and humans (table 1) . also, noteworthy is the shared identity between tiger sars-cov-2/usa/ny-040420 epi_isl_420293 and mink sars-cov-2/nb04 epi_isl_447634 (99.89%), cat sars-cov-2 epi_isl_437349 and tiger sars-cov-2 usa ny-040420 epi_isl_420293 (99.84%), cat sars-cov-2 epi_isl_437349 and mink sars-cov-2/ nb04 epi_isl_447634 (99.78), cat sars-cov-2 epi_ isl_43734 and dog sars-cov-2/hkg/20-03695/2020 (99.39%), tiger sars-cov-2/usa/ny/040420 epi_ isl_420293 and dog sars-cov-2/hkg/20-03695/2020 (99, 54%), and dog sars-cov-2/hkg/20-03695/2020 and mink sars-cov-2/nb04 epi_isl_447634 (99.53%) ( table 1) . regarding the percentage of nucleotide identity of the spike protein, dog sars-cov-2/hkg/20-03695/2020, tiger sars-cov-2 usa ny-040420 epi_isl_420293, and cat sars-cov-2 epi_isl_437349 share 99.97% with human sars-cov-2 ( table 2 ).the amino acid identity of the spike protein between dog sars-cov-2/hkg/20-03695/2020, tiger sars-cov-2 usa ny-040420 epi_isl_42029 and cat sars-cov-2 epi_ isl_437349 with sars-cov-2 is 99.92% (table 3) . however, the spike protein amino acid and nucleotide identity among tiger sars-cov-2/usa/ny/040420 epi_isl_42029, dog sars-cov-2/hkg/20-03695/2020, and cat sars-cov-2 epi_isl_437349 was 100% (tables 2 and 3) . 18:358 this result seems to be related to animal species that have been infected by sars-cov-2 through humans (dog, cat, and tiger, at first). transmission to humans does not seem so likely, however, given the identity between nucleotides and amino acids of the spike protein, transmission between animals seem possible. because it has a similar genome to other animal coronaviruses, sars-cov-2 may have undergone nucleotide mutation when transmitted to animals, expressing amino acids that increased its pathogenicity in animals, especially those related to spike protein [38, 39] . the rbd of sars-cov-2 spike protein, which lies in the s1 domain, is a critical element for determining the susceptibility of the new host species. the studies performed on the interaction between the viral rbd with host cellular receptor (ace2) revealed snakes, pangolins, and turtles as the potential intermediate hosts [40] . turtles, along with other animal species, are favored animals in the huanan seafood wholesale market. however, extended studies are needed to prove their associations scientifically [11] . one of the probable intermediate hosts for sars-cov-2 is a pangolin. pangolin-cov has 91.02% and 90.55% identity to sars-cov-2 and batcov ratg13, respectively [33, 35] . furthermore, the sars-cov-2 spike proteins rbd resembles closely malayan pangolin cov (pangolin-cov) [40] . these findings suggested that pangolins can be the intermediate host for sars-cov-2 transmission. further research is necessary to confirm the origin and transmission dynamics of sars-cov-2. the possible protective effect of pet-ownership against coronaviruses warrants consideration where coronavirus prevalence is high across the pet population. canine respiratory coronaviruses often occur among dogs. ownership of an infected pet can lead to the transmission of viruses from animals to humans. although possible protection caused by the possession of a pet has not yet been found, the frequent occurrence of coronavirus in canines could help the human immune system develop a better response against sars-cov-2 [41] . at the beginning of the sars-cov-2 outbreak, it was thought that pets were not susceptible to the sars-cov-2. however, a natural infection of a cat was reported in belgium with traces of the virus identified in the collected samples by pcr. this cat exhibited respiratory difficulty, vomiting, and diarrhea, which may indicate active replication of the virus inside the animal [39, 41, 42] . however, the animal was not examined by a veterinarian, so further evaluation such as serology was necessary. in another study, shi et al. showed that cats could be not only naturally infected with sars-cov-2 but also that adolescent cats artificially inoculated with the virus presented severe histological lesions and died [18] . however, in further studies, dogs exhibited seroconversion, but the virus could not be isolated. the susceptibility of cats and ferrets to sars-cov-2 could be attributed to the ace2 (sars-cov-2 receptors) [3, 43] . these receptors are expressed in type ii pneumocytes serous epithelial cells of tracheobronchial submucosal glands in ferrets [44] . moreover, the sars-cov-2 spike-contacting regions of ace2 are similar in both ferrets and cats, differing by only two amino acids [5] . the previous reports have shown that sars-cov can infect ferrets and cats [26] , which implies that ferrets and cats may also be susceptible to sars-cov-2. this possibility might be associated with cases of sars-cov-2 transmission to animals. in addition to probably sharing the same origin, bats have significant similarities among their genotypes [3, 25, 31, 32, 35, 36] . based on sars-cov epidemiology, the covid-19 pandemic raises the alarm that animals may become infected and become potential transmitters to humans. the fear of possible transmission of animals to humans and the fact that many residents were forced to leave their animals behind due to evacuation and quarantine, thinking that they would return soon, generated a large number of animal abandonment [45] . authorities in hunan and zhejiang provinces of china also announced that they would start killing pets found in public to prevent the transmission of the virus. this concern intensified in late february 2020, when dogs in hong kong tested positive for the new coronavirus, being considered the first known case of transmission of covid-19 from humans to animals later, felines [20, 39, 46, 47] . in a recent study, it was investigated the possible protection conferred by previous exposure of individuals to naturally infected animals with coronaviruses taxonomically related to the circulating sars-cov-2 [21, 41, [48] [49] [50] . the animals were experimentally infected by sars-cov-2 via the intranasal route through the receptor ace2 [51] . several studies report the use of ace2 by the new coronavirus as its receptor for cell entry [3, 43] . dogs were also inoculated, although seroconversion was observed, no virus could be isolated from the inoculated and uninoculated contacts. further, ferrets showed equal susceptibility to cats, while pigs, chicken, and ducks were found referent to vulnerability [5] . until now, studies based on rbd domain analysis ruled out the probability of mice, rats, and rabbit's involvement in the sars-cov-2 cycle [52] . the findings on ferrets, orangutans, and monkeys showed a higher affinity of ace2 with the rbd domain of sars-cov-2 s protein [53] . a codon-usage based analysis pointed to snakes as a probable host, although these findings were contradicted by subsequent studies [54] . some researchers revealed that cats could naturally be infected with other coronaviruses such as feline coronavirus (fcov) just as canines can be infected with canine coronavirus (ccov) [55, 56] . these animals probably get infection once the virus binds to the receptor, ace2 [51] . another research group conducted a retrospective serological survey on cats infected with sars-cov-2 in wuhan, china. the authors collected 102 samples after the emergence of covid-19 and included 39 samples collected before the outbreak. fifteen (14.7%) of 102 serum samples collected after the outbreak were positive for antibodies against the sars-cov-2 receptor-binding domain (rbd) by elisa test. among the positive samples, 11 had neutralizing antibodies to sars-cov-2 with a titer ranging from 1/20 to 1/1080. twenty-nine out of the 39 serum samples collected before the outbreak were negative. however, no cat that tested positive on serological tests showed any clinical signs. no serological cross-reactivity was detected between sars-cov-2 and type i or ii of the feline infectious peritonitis virus (fipv). as the authors suggested, the cat population studied in wuhan was infected with sars-cov-2 after the beginning of the outbreak [57] . in france, a group of 18 veterinary students investigated the spread of the new coronavirus in 21 pets (9 cats and 12 dogs). eleven cases showed symptoms compatible with covid-19, while only two confirmed positive for the new coronavirus. although three of these cats showed clinical signs of respiratory or gastrointestinal disease, no animal was considered positive by rt-pcr or by the presence of specific antibodies to sars-cov-2 [51] . in a separate study, the researchers demonstrated that ferrets, cats, and dogs could be experimentally infected by sars-cov-2 via the intranasal route [40] . several studies reported that the sars-cov-2 uses the same receptor, ace2, to enter the respiratory mucosa [3, 5, 43] . this probably indicates the possibility of transmission of sars-cov-2 from humans to animals. in a recent experimental study, it was observed that cats infected with sars-cov-2 could transmit the virus to naïve cats that come into contact with them [58] . however, whether they can transmit the virus to humans or else, humans can transmit the virus to pets, or other animals are not yet fully understood. also, the first non-domesticated animal case of sars-cov-2 transmission was from a big cat, nadia, and a 4-year-old malayan tiger infected from the covid-19 positive workers at the bronx zoo, new york, united states. this was the first known animal infection in the us and a tiger anywhere in the world, and was confirmed by the us department of agriculture (usda), the us centers for disease control and prevention (cdc), national veterinary services laboratories, and the wildlife conservation society (wcs) [54, 59, 60] . consequently, covid-19 was detected in four tigers and three lions. mild infection of respiratory distress was also noted in two pet cats in the usa [61] . a new case was reported in moskva, moskovskaya oblast, russia, where a 5-year-old cat tested positive for sars-cov-2. samples (throat and nasal swabs) were taken from the suspect cat to detect sars-cov-2. the lab tests were performed using rt-pcr in real-time with electrophoretic detection of amplification products. the obtained amplification reaction product was sequenced using selected specific primers flanking a 232 bp n gene fragment (orf1ab) of the sars-cov-2. the tests showed 100% identity of the analyzed fragment of the n gene orf1ab of the sars-cov-2. the animals were quarantined [62] . it was previously shown that sars-cov does not infect or cause disease in poultry. because the covid-19 virus belongs to the same group as sars-cov and uses the same ace2 host cell receptor, it is highly unlikely that poultry is susceptible to covid-19. still, it remains to be scientifically proven [63] . for the phylogenetic analysis, complete genome sequences of the viruses falling in the betacoronavirus genus were retrieved from ncbi genbank. the sequences involve sars-cov and sars-cov-2 of sarbecovirus subgenus and few of the sequences from different animal species including the dog, mink, rabbit, rat, pangolin, hedgehog, camel, bats, and wild and domestic felines comprising subgenuses merbecovirus, embecovirus, hibecovirus and nobecovirus within the genus betacoronavirus (fig. 3) . the sequence alignment was done with clustalw in mega 6.02 software, and phylogeny was constructed using the gtr + g + i substitution model applying the maximum likelihood method. in the analysis, canine and feline covs of alphacoronavirus were taken as the outgroup for constructing a phylogenetic tree of betacoronavirus. all the covs of genus betacoronavirus clustered in their respective subgenus clades. notably, the mink and tiger virus isolates showed 99.6-99.9% homology with human sars-cov-2 isolates from different parts of the world. contrarily, the canine betacoronavirus (crcov) from subgenus embecovirus showed 45.8-46.2% similarity with sars-cov-2. furthermore, pangolin cov, sars-like bat covs, and bat covs (ratg13 strain) showed homology between 86.6 and 96.3% with sars-cov-2. the camel isolate of mers-cov showed a 51% similarity with sars-cov-2 at the nucleotide level (fig. 3) . our analysis demonstrated a high divergence between animal origin covs and sars-cov-2 except for a few strains which were isolated from mink and tiger as were the cases of covid-19 infected persons. though the exact ways of transmission of sars-cov-2 from infected humans to animals are vaguely understood, the possible and promising transmission may occur through touching their noses or mouth by infected hands defiled with respiratory droplets or saliva [64] . at the time of sneezing, coughing, or even talking, infected humans can disseminate respiratory droplets, which have a pivotal role in the transmission of the virus to animals [65] . however, the transmission of the virus from affected people can be facilitated by some favorable risk factors, e.g., kissing, petting, licking, or hugging pet animals [66] . according to the government of the netherlands, through a letter issued by the ministry of agriculture, nature and food quality, it is possible that an employee who worked on a mink farm infected with sars-cov-2 contracted the virus, having already been recovered from the disease. also, research shows that minks can be asymptomatic and that cats play an important role in the potential transfer of viruses between investigated farms [67]. there are multiple studies on the origin of coronaviruses and their zoonotic potential. some coronaviruses that infect animals can sometimes be spread to humans and then spread between people as happened in the case of mers and sars. this is also what happened with the virus that caused the current outbreak of covid-19. however, the exact origins of this virus are still unclear. the scientific community has put significant effort into identifying the source of sars-cov-2, and to date, genetic evidence suggests that it was likely acquired from bats. the first infections were linked to a live animal market in wuhan, suggesting a zoonotic origin. the virus is now spreading from person to person and caused one of the most significant pandemics of the recent era. infection with sars-cov-2 has been reported in several animals through serological and molecular analyses and experimental inoculations. however, it is not proved that animals can transmit sars-cov-2 to humans and to what extent humans can transmit this virus to animal species. veterinary public health officials and designated public health officials should work to determine whether animals should be tested for sars-cov-2 when the animals are in the same environment as infected owners using a one health approach. available information suggests that the risk of sars-cov-2 spread from animals to humans is low. even though human-to-animal transmission or vice versa is possible, caution and effective communication with the pet owners are necessary to prevent the abandonment and death of animals indiscriminately. although a case of sars-cov-2 transmission from animals to humans has already been reported, this is only a report, so further research is needed before declaring that animals can transmit the new coronavirus to humans. geographic information system (gis) based on computer and information technology would be useful in analyzing the disease pattern. the collaborative global effort, such as "one-human-environmental-animal health, " is required to reduce the global risk of zoonotic diseases. further studies are essential to understand better the origin of the virus and transmission dynamics, which will help educate people and avoid unnecessary discrimination to animals. preventive measures that have proven to be effective should be encouraged, such as correct hygiene, social distance, and, if necessary, social isolation or quarantine. good practices such as the use of emerging and vector-borne diseases program, sacids foundation for one health 25 virology department, pendik veterinary control institute, ministry of food and forestry, 34890 pendik-istanbul dokki, giza 12622, egypt. 31 department of animal wealth development, faculty of veterinary medicine genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a novel coronavirus outbreak of global health concern severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges a novel coronavirus emerging in china-key questions for impact assessment susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 discovery of a novel coronavirus, china rattus coronavirus hku24, from norway rats supports the murine origin of betacoronavirus 1 and has implications for the ancestor of betacoronavirus lineage a the covid-19 pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus emerging coronaviruses: genome structure, replication, and pathogenesis composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 epidemiology, genetic recombination, and pathogenesis of coronaviruses coronavirus infection in equines: a review efficient assembly and release of sars coronavirus-like particles by a heterologous expression system mers-cov virus-like particles produced in insect cells induce specific humoural and cellular immunity in rhesus macaques emerging novel coronavirus (2019-ncov)-current scenario, evolutionary perspective based on genome analysis and recent developments evolutionary trajectory for the emergence of novel coronavirus sars-cov-2 the phyre2 web portal for protein modeling, prediction and analysis the proximal origin of sars-cov-2 low-level of infection whit covid-19 in pet dog molecular basis of covid-19 relationships in different species: a one health perspective novel sars-cov-2/covid-19: origin, pathogenesis, genes and genetic variations, immune responses and phylogenetic analysis world health organization who: summary table of sars cases by country severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses sars virus infection of cats and ferrets isolation of a novel coronavirus from a man with pneumonia in saudi arabia genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe bats may be sars reservoir infection and rapid transmission of sars-cov-2 in ferrets covid-19, sars and mers: are they closely related? probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human a novel bat coronavirus closely related to sars-cov-2 contains natural insertions at the s1/s2 cleavage site of the spike protein receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus genomic characterization of a novel sars-cov-2 clinical manifestations and outcome of sars-cov-2 infection during pregnancy do pets protect their owners in the covid-19 era? med hypotheses belgian cat infected by owner. the brussels times sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor pathology of experimental sars coronavirus infection in cats and ferrets cats and dogs abandoned at the start of the coronavirus outbreak are now starving or being killed coronavirus can infect cats-dogs, not so much world organization for animal health oie. immediate notification novel human coronavirus (sars-cov-2): a lesson from animal coronaviruses immunoinformatic analysis of the sars-cov-2 envelope protein as a strategy to assess cross-protection against covid-19 comparative computational analysis of sars-cov-2 nucleocapsid protein epitopes in taxonomically related coronaviruses a mouse model of sars-cov-2 infection and pathogenesis covid-19 preclinical models: human angiotensin-converting enzyme 2 transgenic mice receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars cross-species transmission of the newly identified coronavirus 2019-ncov canine coronavirus highly pathogenic for dogs a tale of two viruses: the distinct spike glycoproteins of feline coronaviruses sars-cov-2 neutralizing serum antibodies in cats: a serological investigation transmission of sars-cov-2 in domestic cats a tiger at the bronx zoo has tested positive for coronavirus centers for disease control and prevention cdc. coronavirus disease 2019 (covid-19). if you have animals confirmation of covid-19 in two pet cats in new york world organization for animal health oie what we know about avian coronavirus infectious bronchitis virus (ibv) in poultry and how that knowledge relates to the virus causing covid-19 in humans pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses practical measures to prevent covid-19: a mini-review the risk of sars-cov-2 transmission to pets and other wild and domestic animals strongly mandates a one-health strategy to control the covid-19 pandemic. one health publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank dr. amir ghorbani of the ohio state university and joão inácio magalhães duarte, for his critical comments on this paper. we gratefully acknowledge the authors, originating, and submitting laboratories of the sequences from the gisaid's epicov ™ database, on which part of the phylogenetic tree reconstruction is based. the initial writing was carried out by sst and pmd; the other authors contributed to the complementation, critical review, and final of the manuscript. all authors read and approved the final manuscript. not applicable. all data analyzed or generated during this study are included in this publication and its additional files. not applicable. not applicable. the authors declare that they have no competing interests. key: cord-323428-jd91k19z authors: ababneh, mustafa; alrwashdeh, mu’men; khalifeh, mohammad title: recombinant adenoviral vaccine encoding the spike 1 subunit of the middle east respiratory syndrome coronavirus elicits strong humoral and cellular immune responses in mice date: 2019-10-11 journal: vet world doi: 10.14202/vetworld.2019.1554-1562 sha: doc_id: 323428 cord_uid: jd91k19z background and aim: middle east respiratory syndrome coronavirus (mers-cov) has rapidly spread throughout the middle east since its discovery in 2012. the virus poses a significant global public health threat with potentially devastating effects. in this study, a recombinant adenoviral-based vaccine encoding the spike 1 (s1) subunit of the mers-cov genome was constructed, and its humoral, and cellular immune responses were evaluated in mice. materials and methods: mice were immunized initially by intramuscular injection and boosted 3 weeks later by intranasal application. expression of the s1 protein in the lungs and kidneys was detected using conventional polymerase chain reaction (pcr) and immunohistochemistry (ihc) targeting specific regions within the s1 subunit at weeks 3, 4, 5, and 6 after the first vaccination. antigen-specific humoral and cellular immune responses were evaluated in serum and in cell culture following in vitro stimulation with a specific 9-mer epitope within the s1 protein (cysslildy). results: s1 protein expression was only detected by ihc in the kidneys of the ad-mers-s1 group at week 6 from first immunization, and in both lungs and kidneys of ad-mers-s1 group by conventional pcr at weeks 3 and 5 post-prime. the vaccine elicited a specific s1-immunoglobulin g antibody response, which was detected in the sera of the vaccinated mice at weeks 4 and 6 from the onset of the first immunization. there was a significant increase in the amount of th1-related cytokines (interferon-γ and interleukin [il] 12), and a significant decrease in the th2-related cytokine il-4 in splenocyte cell culture of the vaccinated group compared with the control groups. conclusion: the results of this study suggest that this recombinant adenovirus vaccine encoding the s1 subunit of mers-cov elicits potentially protective antigen-specific humoral and cellular immune responses in mice. this study demonstrates a promising vaccine for the control and/or prevention of mers-cov infection in humans. middle east respiratory syndrome coronavirus (mers-cov) is a newly emerging human coronavirus that was discovered in 2012 in a 60-year-old saudi arabian man [1] . following its discovery, many cases were identified in different regions of the arabian peninsula and worldwide thereafter [2, 3] . the most recent outbreak occurred in june 2015 in south korea and was linked to a south korean man who had recently traveled to the middle east [3] . the infection then rapidly spread to 26 persons through close contact in a hospital. within a few months, many cases (n=186) were reported in hospitalized and non-hospitalized persons in south korea [3] . the disease showed a high mortality rate that reached up to 40%, which was higher than that of the severe acute respiratory syndrome coronavirus (sars-cov) outbreak in 2002-2003 (10%) [4] . coronaviruses belong to the subfamily coronavirinae within the family coronaviridae of the order nidovirales [5] . five human coronaviruses were identified (229e, oc43, nl63, hku1, and sars-cov) before mers-cov. lineage c of betacoronaviruses includes bat coronaviruses, which give a primitive impression regarding the origin of the virus [6] . the detection of mers-cov and its neutralizing antibodies in the sera of dromedary camels has shed light on the role of the camel as a possible animal reservoir, which may transmit the virus to humans [7] [8] [9] [10] . indeed, researchers isolated the same mers-cov strain from both a camel "in a barn" and its infected owner in saudi arabia, thus providing further evidence of the potential airborne and direct contact transmission of the virus between camels and humans [11] . there have been several attempts to develop a protective vaccine against mers-cov [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . researchers around the world are focused on the spike protein as the main target for vaccine development against mers-cov. the spike protein of mers-cov attaches to the host dipeptidyl peptidase-4 (dpp4) receptor, which is expressed on several types of human cells [24] . many studies published since 2012 suggesting vaccine models were constructed based on the spike protein and its receptor-binding domain (rbd) region to elicit a strong and protective immune response in vitro and in vivo [25] . recombinant adenoviral vector vaccines are one of the most effective vaccines and showed interesting results during sars-cov outbreaks [12, 26, 27] . since 2013, several studies were published, in which different viral vectors (e.g., adenoviruses and vaccinia virus) were used to develop recombinant vaccine candidates based on full spike gene or part of it and tested their ability to produce protective immunity against mers-cov infection [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] . however, further investigations are needed on these suggested vaccines including testing their ability to elicit neutralizing antibodies in different animal models, stimulation of both innate and adaptive immune responses and their corresponding cytokine and chemokine profiles, distribution within the host, and their safety and duration profiles. in this study, a recombinant adenoviral-based vaccine encoding the spike 1 (s1) subunit of the mers-cov genome was constructed, and its humoral, and cellular immune responses were evaluated in mice. all procedures performed in this study were approved by the jordan university of science and technology animal use and care committee. the recombinant human adenovirus type 5 (de1/e3) vaccine was designed to express the s1 domain of the spike protein of mers-cov isolate hu/ jordan-20140010168/2014 (genbank accession number kt861627) under the control of the cytomegalovirus (cmv) promoter. this vaccine was constructed in the laboratories of vector biolabs (the gene delivery company, pa, usa) [28] . six to eight week-old male balb/c mice (n=168) were purchased from animal house at jordan university of science and technology (irbid-jordan) and distributed into three groups in a replicate manner (56 each). animals in the first group were immunized with the ad-cmv-mers-cov-spike1 "ad-mers-s1." animals in the second group received the vector-only "ad-cmv," and animals in the last group served as a control and received 1× phosphate-buffered saline (pbs). for the first immunization, the ad-mers-s1 vaccine was injected intramuscularly at a dose of 0.1 ml containing 1×10 7 pfu/ dose/mouse. the same volume of ad-cmv or pbs was used for the other two groups. the booster dose was carried out 3 weeks later by intranasal route at a dose of 0.1 ml containing 1×10 7 pfu/dose/mouse. all experimental procedures used in this study were approved by the animal care and use committee at the faculty of veterinary medicine (irbid-jordan). lung, kidney, and spleen tissue samples were harvested from each mouse in all groups after 3, 4, 5, and 6 weeks from the first immunization. parts of lung and kidneys were preserved in 10% formalin solution for 24-h, then embedded in paraffin blocks for immunohistochemical analysis, while the other parts were stored at −80°c for polymerase chain reaction (pcr) analysis. spleen tissues were harvested and stored at −80°c for cell culture analysis. blood samples were drawn through facial vein route, left 30 min at room temperature, and centrifuged at 2000 rpm for 20 min. collected sera were stored at −20°c for subsequent analysis. to study the distribution of the recombinant vaccine, total of 50-100 mg of lung and kidney tissues at weeks 3 and 5 post-prime immunization was cut and homogenized in 1 ml of diethyl pyrocarbonate-treated water. homogenized samples were then centrifuged at 800 rpm for 5 min, and 150 µl of the supernatant was used for the extraction. the extraction was performed using the viral gene-spin rna extraction kit (intron biotechnology, korea) according to the manufacturer's instructions. the extracted rna was used for cdna synthesis through a reverse transcription system (promega, madison, wi, usa) according to the manufacturer's instructions. the cdna was kept at −20°c until pcr analysis. the dna template of the constructed vaccine was also purified and used as a positive control in this experiment. heminested pcr was carried out using specific primers targeting specific sequences within the s1 region, as shown in table1 [29] . the first pcr targeted a 1172 bp sequence. pcr mixture contained 0.4 µm of each primer and using 3 µl of the cdna template. touchdown pcr assay was programmed on the mycycler thermal cycler (bio-rad laboratories, hercules, ca, usa) as the following conditions: 95°c for 10 min, followed by 10 cycles of 95°c for 35 s, 63-54°c for 40 s, and 72°c for 1 min and 15 s, then 35 cycles of 95°c for 40 s, 56°c for 40 s, 72°c for 1 min and 15 s, and a final extension step at 72°c for 5 min. two microliters from the first pcr product were used as a template for the second (heminested) pcr to amplify the 1142 bp product following the first pcr conditions. the bands were visualized in 1.5% agarose gels using an ultra-violet transilluminator. localization of s1 protein expression was investigated in both kidney and lung tissues from all groups at week 6 after the first vaccination. ihc using rabbit-specific hrp/dab (abc) detection ihc kit (abcam, cambridge, uk) was adapted, with using antinovel coronavirus spike protein s1 polyclonal antibodies (1:1000; sino-biological, pa, usa). a 5-µm thick section of formalin was fixed, and paraffin-embedded tissues were cut and loaded on specially coated slides. the prepared slides were put in an oven at 70°c for 30 min followed by 15 min in a xylene solution for deparaffinization. the sections were dried and surrounded by liquid blocker (dako pen). the antigen retrieval (pretreatment) step was executed by immersing the sections in 1× reveal decloaker reagent (biocare medical, pacheco, ca, usa) and heating in a microwave several times for 30 min then cool at room temperature for 15-20 min. after that, staining procedure was carried out by following the manufacturer's instructions of the abc kit. to eliminate the effect of internal renal biotin, 0.05% avidin was applied (sigma-aldrich, st. louis, mo, usa) only on kidney sections for 15 min at room temperature [30, 31] . the immunoreactivity of the expressed s1 protein was visualized using a light microscope at different magnifications. spleen tissues from each group were collected, pooled, and homogenized to obtain single-cell suspensions. the cells were centrifuged for 10 min at 800 rpm, and the pellet was re-suspended in roswell park memorial institute medium (rpmi) (gibco, grand island, ny, usa), supplemented with 10% fetal calf serum, 20 mm hepes, 10 µg/ml penicillin/streptomycin, 2 mm l-glutamine, 50 µm 2-mercaptoethanol, and 500 ng/ml amphiostat b (complete rpmi) [32] . the cell suspension was washed again by centrifugation as described earlier. spleen cells were re-suspended in red blood cell lysis buffer (trisbuffered nh 4 cl) to remove erythrocytes. cells from each group (1×10 5 ) were re-suspended in 1 ml of complete rpmi and plated in 24-well plates in triplicate in parallel, adjacent wells. the first set of samples was stimulated with 10 µg of specific peptide "epitope" cysslildy per well. this antigen was defined as the highest potential t-cell epitope from a total of eight potential epitopes within the rbd region of the s1 subunit of the spike protein of mers-cov genome and was expected to display interactions with the maximum number of major histocompatibility complex (mhc) class i molecules, and to show the highest peak in the b-cell antigenicity plot [33] . the second set of samples was stimulated with 50 µg of non-specific stimulant phytohemagglutinin to confirm the viability and productivity of the cultured cells. the third set of samples was not stimulated as a control set. cells were incubated in round-bottom 24-well microtiter plates at 37°c in 5% co 2 . the cultured media were collected after 96 h, and the samples were centrifuged for 20 min at 2000 rpm. the supernatants were stored at −20°c before cytokine analysis, while the pellets were stored at −80°c until real-time (rt)-pcr analysis. detection and measurement of specific mers-s1 igg were achieved using a semi-quantitative anti-mers-cov elisa (igg) (euroimmun ag, luebeck, germany) with a slight modification using universal enzyme conjugate, anti-mouse iggconjugated hrp, to be able to detect mouse igg antibodies instead of human igg antibodies. microtiter plates were pre-coated with purified s1 antigen of mers-cov. mouse diluted serum samples (1:100) were incubated in each well from the three experimental groups (ad-mers-s1, ad-cmv, and pbs) for 30 min at room temperature by following the manufacturer's instructions. the color intensity was measured using an elisa plate reader (biotek, winooski, vt, usa). cell culture supernatants from stimulated and corresponding non-stimulated samples of the three groups were tested for the level of mouse cytokines at weeks 4 and 6 post-prime using a quantitative sandwich enzyme immunoassay technique (quantikine elisa mouse cytokines immunoassay, minneapolis, mn, usa) according to the manufacturer's instructions. relative quantification of mouse ifn-γ and il-4 was normalized to the housekeeping gene β-actin in the cell culture yield of stimulated groups. the collected pellets from cell culture were used to extract total rna using the sv total rna isolation system (promega) according to the manufacturer's instructions; this extraction kit has a dnase incubation step to eliminate any dna in the sample. isolated rna (40 µl) was stored at −80°c before rt-pcr analysis. rna concentration was adjusted to 10 ng/µl using te buffer. purified rna (10 µl) was used as a template to produce 20 µl of cdna using the reverse transcription system (promega) kit. relative rt-pcr assay was performed to determine the mrna expression levels of mouse ifn-γ and il-4 in the cell culture. the fold changes were calculated using the 2 −δδct method normalized to β-actin [34] . specific primer sets were used, as shown in table2 [35] . extracted rna (2 µl) was used as a template in the pcr reaction mixture (20 µl), which was composed of 0.5 µm each of the forward and reverse primers, 10 µl of powerup sypr green master mix (applied biosystems, foster city, ca, usa), and 6 µl of nuclease-free water. rt-pcr conditions were programmed in rotor gene-q (qiagen, hilden, germany) as follows: 50°c for 2 min, 95°c for 10 min, followed by 40 cycles of 95°c s, 60°c for 1 min, and a melting step at 55-99°c for 30 s. data for elisa and fold changes were expressed at mean ± standard deviation. one-way anova and t-test were used to compare different values in all groups using openepi software (emory university, usa). parameter differences were considered statistically significant at p<0.05. parameter differences were indicated by small letters labeled within each group; different letters indicated significant differences at p<0.05. distribution and expression of the s1 subunit of the mers-cov spike protein in mice tissues were detected at weeks 3 and 5 post first immunization in the kidneys and lungs of the vaccinated group but not in control groups using conventional pcr (figure-1 ). using ihc, s1 protein expression was detected in the cytoplasm of proximal tubule epithelium in the vaccinated mice at week 6 post first immunization but not in the control groups (figure-2) . s1 expression was not detected in lung tissues in either the vaccinated or the control groups (figure-2) . there was a significant level (p<0.05) of mers-s1 specific igg antibodies detected in the mice sera of the vaccinated group at weeks 4 and 6 post-prime. the combined results of weeks 4 and 6 revealed that the igg antibody response was significantly higher (p<0.05) in the vaccinated group than in the control groups (figure-3) . available at www.veterinaryworld.org/vol.12/october-2019/7.pdf the level of mouse cytokines at weeks 4 and 6 after the first vaccination in the cell culture supernatants of stimulated and corresponding non-stimulated samples of the three groups was tested by sandwich elisa assay. mice cytokines (ifn-γ, il-12, and il-4) production in the ad-cmv and pbs groups were combined and expressed collectively as one group (control groups) due to the non-significant differences shown across these time points between these two groups (figure-4) . therefore, the levels of each cytokine at the two tested time points (weeks 4 and 6 after the first vaccination) were also combined and expressed as one unit. regardless of in vitro stimulation, production of ifn-γ was significantly (p<0.05) higher in the vaccinated group compared with the control groups (ad-cmv and pbs) at all tested time points (figure-4) . in terms of il-12 production, in vitro stimulation with the mers-specific peptide "cysslildy" resulted in a significant (p<0.05) upregulation in production when compared with the antigen-stimulated and non-stimulated cell culture in the vaccinated group only (figure-4) . although there was no significant difference in il-4 production between the control groups and the vaccinated group in non-stimulated cell culture, antigen-specific stimulation clearly revealed a higher production of this cytokine in the control groups above the vaccinated group level of production (p<0.05). in addition, in vitro stimulation with the mers specific peptide "cysslildy" resulted in a significant upregulation in this production of this cytokine in the control groups while the same antigen stimulation resulted in a significant decrease in il-4 production in the vaccinated group (p<0.05) (figure-4) . at week 4 post first immunization, the fold change in ifn-γ gene expression was significantly higher than that in the control groups (ad-cmv and pbs) (p<0.05). however, there was a prominent increase in ifn-γ gene expression at week 6 postprime in both the vaccinated and ad-cmv groups compared to that in week 4 (p<0.05). the expression of ifn-γ was slightly higher but not statistically significant in the vaccinated group compared to that in the ad-cmv group at week 6 (p>0.05) (figure-5) . the only significant change (p<0.05) in il-4 gene expression was observed in the ad-cmv group at week 6 (figure-5) while no significant changes were observed in the vaccinated group at any time point. in this study, potentially protective humoral and cellular immune responses were elicited in mice by immunization using a recombinant adenoviral-based vaccine encoding the s1 subunit of the mers-cov spike gene. several studies have been published that constructed similar vaccines against mers-cov and tested their ability to induce production of neutralizing antibodies and other immune system components [13, 14, 17, [19] [20] [21] [22] [23] . the results of these studies were encouraging and showed that 12] , and il-4) in vitro. regardless of in vitro stimulation, production of ifn-γ was significantly (p<0.05) higher in the vaccinated group with ad-middle east respiratory syndrome (mers)-s1 compared with the control groups (ad-cytomegalovirus and phosphate-buffered saline) at all tested time points. in vitro, antigen-specific stimulation resulted in a significant upregulation of il-12 production when compared with the antigen-stimulated and non-stimulated cell culture in the vaccinated group only. in vitro stimulation using the mers-specific peptide "cysslildy" resulted in a significant upregulation of il-4 production in the control groups while the same antigen stimulation resulted in a significant decrease in il-4 production in the vaccinated group. ns: non-antigen stimulated, ag: antigen-stimulated. different letters mean significantly different at p<0.05. available at www.veterinaryworld.org/vol.12/october-2019/7.pdf such vaccines may be promising to prevent the global threat of mers-cov infection [13, 14, 17, [19] [20] [21] [22] [23] . however, the safety and efficacy of these vaccines still to be confirmed. it was confirmed that the s protein is responsible for the production of neutralizing antibodies [22, 31] . the s1 subunit contains the rbd, which binds to its host receptor dpp4 (also known as cd26) and induces production of specific neutralizing antibodies. several studies have supported the role of this domain to elicit protective immunity against mers-cov challenge [25, 36, 37] . in this study, we were able to demonstrate the distribution and expression of the s1 protein in the vaccinated group but not in the control groups (ad-cmv and pbs) by detection of the truncated protein-encoding segment in kidney and lung tissues following vaccination using conventional pcr and ihc. these findings support the ability of this vaccine candidate to distribute effectively within host tissues and express the target protein. it has been established that dpp4 is expressed on multiple cell types in the human body including cells in the kidneys and lungs [24] . however, the n glycoprotein, but not the s glycoprotein, of mers was the target of investigation of mers-cov localization using ihc [38] . the n glycoprotein was clearly present in lung tissues following mers exposure. in fact, the current study can be considered pioneering given the use of the s1 glycoprotein to express viral distribution in tissues. therefore, the expression of s1 protein in kidney tissues and not in lung might be used to explain previous in vitro experiments, which found a cytopathic effect of the mers-cov infection inside primary human kidney cells but not in primary human bronchial epithelial cells [39] . in a recent study, the adenoviral vector with enhanced green fluorescent protein but not the adenoviral vector with mers in a hdpp4 mice experiment after intramuscular or intranasal injection [21] . this supporting our finding that this construct was able to reach the lung tissues, but the s1 protein was not expressed in lung tissues. however, s1 gene detection in lung tissues by conventional pcr and not by ihc indicates further investigation is necessary regarding the level and intensity of s1 expression in this tissue. additional studies are also needed to explore s1 protein expression in other tissues such as the liver and intestine given the expression of dpp4 in these tissues [40] . in this study, there was a significant serum level of the s1-specific igg antibody in the vaccinated group but not in the control groups (ad-cmv and pbs). similar results have been shown previously: the sars-cov vaccine had a strong ability to elicit the s1-specific igg antibody response [26] . the results presented in this study also supported the findings of recent publications applying a similar vector vaccine construction [13, 14, 17, [19] [20] [21] [22] [23] . the t helper 1 immune response is known as the dominant immune response in the case of intracellular pathogens, such as viruses and bacteria, while the t helper 2 immune response is the dominant immune response in cases of extracellular pathogens and allergic reactions [41] . many pathogens, especially viruses, shift the host immune response toward th2 dominance over the th1 response to evade cellular immunity. in this study, ad-mers-s1 was able to provoke pro-inflammatory but not anti-inflammatory cytokine release. both th1 and th2 responses were elicited following immunization. cytokines such as ifn-γ and il-12, which represent the th-1 response, were upregulated, while il-4, which represents the th-2 response, was downregulated. the ad-mers-s1 was able to induce production of a significant amount of ifn-γ regardless of antigen-specific stimulation. a similar increase in il-12 was apparent in vaccinated animals in response to specific in vitro antigen stimulation. the mrna expression of ifn-γ was detected as early as 4 weeks after the first immunization. in contrast, il-4 production in cell culture showed a significant increase in control animals after antigen stimulation, while the production of this cytokine was significantly decreased in the at week 4, the fold change in ifn-γ gene expression was significantly higher in the ad-middle east respiratory syndrome (mers)-s1 group than that of both control groups (ad-cytomegalovirus [cmv] and phosphate-buffered saline). however, there was a prominent increase in ifn-γ gene expression at week 6 in both ad-mers-s1 and ad-cmv groups compared to that in week 4. the only significant change in il-4 gene expression was in the ad-cmv group at week 6, while no significant changes were observed in the ad-mers-s1 group at any time point. different letters indicate statistically significant differences at p<0.05. vaccinated group. the results demonstrated neither positive nor negative impact on il-4 expression in mice vaccinated with the ad-mers-s1 or mice that received only pbs. interestingly, there was a significant increase in il-4 and ifn-γ gene expression in antigen-stimulated cell culture obtained from mice vaccinated with ad-cmv at week 6. this might be due to the ability of adenoviruses to elicit strong humoral and cellular immune responses [42, 43] . therefore, it can be concluded that the presence of the s1 protein gene in the adenovirus vector genome disturbs the immune stimulation ability of the adenovirus vector to elicit il-4 cytokine. two studies have shed light on the ability of the s1 subunit, and specifically, the ability of the rbd domain, to shift the host immune response toward th1 [14, 44] . it was demonstrated in these two studies that igg1, which is produced mainly during the th2 immune response, was decreased, while igg2a, which is produced mainly during the th1 immune response, was upregulated following vaccination with their vaccine candidate. the truncated rbd fused with the fc portion of human igg was able to elicit both isotypes with a relatively higher amount of the igg2a isotype (th1 response) [44] . recombinant adenovirus vector carrying the s1 subunit or the whole s gene was also able to induce both types of the immune response. the igg2a level was detected earlier and higher in mers-s than mers-s1 at week 2 post-vaccination but not after week 3 [14] . accordingly, these findings lead to the dominance toward the th1 cellular immunity. several in vivo and in vitro studies have revealed the ability of mers-cov to evade the host innate immune response through downregulating the antigen-presenting pathway and proinflammatory cytokines such as ifn-γ and il-12 [38, [45] [46] [47] . several mers-cov proteins were previously identified as potent ifn antagonists, including the m structural protein and the orf 4a, orf 4b, and orf 5 accessory proteins [46] . in addition, a comparison of the immune response of two mers-infected patients [48] , one of whom with a poor outcome (died) that had elevated levels of il-17 and il-10 which promote the th2 immune response, while the other patient who overcame the infection had increased levels of ifn-α, ifn-γ, and il-12, which promote the th1 immune response [48] . in general, it is crucial to induce early and strong innate immune responses against mers-cov infection to save lives. our vaccine candidate was able to induce production of the key cytokines of activated t lymphocytes toward cd4+ th1 cells, which is also an indication of upregulation of the antigen-presenting pathway. the vaccine also did not trigger the production of il-4, which is involved in the th2 response, which may be referred to the counter effect of the produced ifn-γ in il-4 producing cells [41] . finally, it is important to clarify that the in vitro antigen stimulation was done using the specific 9-mer peptide "cysslildy," this peptide is located at position 437-445 within the conserved region of the s glycoprotein among all identified mers-cov strains. this was selected in a computerized simulation that showed that this sequence has the highest b cell antigenicity plot and has the ability to form the greatest number of interactions with mhc-class i alleles [33] . although this epitope was not able to increase ifn-γ production level after in vitro stimulation, it resulted in a significant increase in il-12 production and a significant decrease in il-4 production in the vaccinated group. in addition, this epitope was able to increase il-4 production after in vitro stimulation in ad-cmv group. however, these findings were not enough to prove the claim that this epitope maybe has a crucial role in mers-cov pathogenesis. hence, further studies (such as in vivo or knockout experiments) should be conducted to address the role of this epitope in mers-cov pathogenesis. overall, the adenoviral vector encoding the s1 subunit of the mers-cov genome is a promising vaccine candidate against mers-cov infection. it was clearly demonstrated that this recombinant vaccine is distributed in host tissues. in addition, this construct could induce production of a specific anti-s1 igg antibody response as well as the th1 immune response, which was evident from the increased levels of pro-inflammatory cytokines in the vaccinated animals. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution mers: emergence of a novel human coronavirus middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov--are we on the verge of a pandemic? middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation antibodies against mers coronavirus in dromedary camels detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein heterologous prime-boost vaccination with adenoviral vector and protein nanoparticles induces both th 1 and th 2 responses against middle east respiratory syndrome coronavirus distribution and absence of generalized lesions in mice following single-dose intramuscular inoculation of the vaccine candidate mva-mers-s immunogenicity of different forms of middle east respiratory syndrome s glycoprotein chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp4 mouse model single intranasal immunization with chimpanzee adenovirus-based vaccine induces sustained and protective immunity against mers-cov infection a highly immunogenic, protective and safe adenovirus-based vaccine expressing mers-cov s1-cd40l fusion protein in transgenic human dpp4 mouse model dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the receptor-binding domain of mers-cov: the dawn of vaccine and treatment development adenoviral expression of a truncated s1 subunit of sars-cov spike protein results in specific humoral immune responses against sars-cov in rats available at www.veterinaryworld.org comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus the gene delivery company): recombinant adenovirus construction middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia endogenous biotin expression in renal and testicular tumors and literature review retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry effects of gamma interferon, interleukin-10, and transforming growth factor beta on the survival of mycobacterium avium subsp. paratuberculosis in monocyte-derived macrophages from naturally infected cattle computer-aided prediction and identification of potential epitopes in the receptor-binding domain (rbd) of spike (s) glycoprotein of mers-cov analysis of relative gene expression data using real-time quantitative pcr and the 2(-delta delta c(t)) method comparison of intranasal and transcutaneous immunization for induction of protective immunity against chlamydia muridarum respiratory tract infection structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv th 1 /th 2 balance: the hypothesis, its limitations, and implications for health and disease adenoviruses as vaccine vectors replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? this study was supported by the deanship of research at jordan university of science and technology (grant no. 275/2014). ma and mua designed the adenovirus vaccine for the mers-s1 vaccine. ma and mk designed the mice experiments. mua, ma, and mk took the different tissue samples from the mice. mua performed the molecular assays and the immunohistochemical staining. ma and mua prepared the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. veterinary world remains neutral with regard to jurisdictional claims in published institutional affiliation. key: cord-348821-2u6ki9dv authors: xu, ping; sun, guo-dong; li, zhi-zhong title: clinical characteristics of two human to human transmitted coronaviruses: corona virus disease 2019 versus middle east respiratory syndrome coronavirus. date: 2020-03-10 journal: nan doi: 10.1101/2020.03.08.20032821 sha: doc_id: 348821 cord_uid: 2u6ki9dv after the outbreak of the middle east respiratory syndrome (mers) worldwide in 2012. currently, a novel human coronavirus has caused a major disease outbreak, and named corona virus disease 2019 (covid-19). the emergency of mres-cov and covid-19 has caused global panic and threatened health security. unfortunately, the similarities and differences between the two coronavirus diseases remain to be unknown. the aim of this study, therefore, is to perform a systematic review to compare epidemiological, clinical and laboratory features of covid-19 and mers-cov population. we searched pubmed, embase and cochrane register of controlled trials database to identify potential studies reported covid-19 or mers-cov. epidemiological, clinical and laboratory outcomes, the admission rate of intensive cure unit (icu), discharge rate and fatality rate were evaluated using graphpad prism software. thirty-two studies involving 3770 patients (covid-19 = 1062, mers-cov = 2708) were included in this study. the present study revealed that compared with covid-19 population, mers-cov population had a higher rate of icu admission, discharge and fatality and longer incubation time. it pointed out that fever, cough and generalised weakness and myalgia were main clinical manifestations of both covid-19 and mers-cov, whereas ards was main complication. the most effective drug for mers-cov is ribavirin and interferon. coronaviruses are rna viruses with envelope and non-segmented positive-sense, causing respiratory and intestinal tract infections in humans and other mammals [1] . despite . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 4 although many previous studies have reported clinical characteristics of covid-19 or mers-cov diseases [8] [9] [10] [11] , systematic comparison of clinical features between covid-19 and mers-cov diseases has yet been published. thus, the purpose of this study is to perform a systematic review of epidemiological, clinical and laboratory characteristics of patients infected by covid-19 or mers-cov disease, and to compare covid-19 and mers-cov in the context of their incubation, laboratory features, admission rate of intensive cure unit (icu) and rate of discharge and fatality, which will provide a comprehensive reference for clinical physicians in treatment of coronavirus diseases. a comprehensive and systematic search was performed using pubmed, embase and or (2019 novel coronavirus)). if necessary, we also contacted corresponding author to obtain accurate data. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 5 the study that met following criteria were included: (1) reporting clinical characteristics of covid-19 or mers-cov disease, (2) minimum sample size of five, (3) confirmed covid-19 or mers-cov disease, (4) english literature. studies were excluded as following criteria: duplicate publications, case report, meta-analysis, letter, review, technology report, commentary, animal trial, correspondence, predictive study, guidence, radiograph study and meeting report. at the beginning, 4743 potential publications were identified. we removed 678 duplicates and reviewed the titles and abstracts of remaining 4065 publications. 4015 publications were excluded for following reasons: not involved research point (n = 1378), review (n = 569), no english (n = 33), case report (n = 316), meta-analysis (n = 1), letter (n = 127), technology report (n = 196), commentaries (n = 61), animal study (n = 1107), correspondence (n = 29), predictive study (n = 110), guidence (n = 28), meeting report ( n = 16) and radiograph (n = 44). then, a comprehensive review of full-text was conducted for remaining 50 publications. two reviewers independently screened eligible literature, and any argument was solved by discussion with a third reviewer. finally, thirty-two studies were included in this study . the process was shown in figure 1 . two reviewers extracted independently common, clinical and laboratory characteristics of included studies, with disagreements were solved by discussion with a third reviewer. the extracted data included incubation time, white blood cell (wbc) count, lymphocyte count, creactive protein (crp), alamine aminotransferase (alt), aspartate aminotransferase (ast), . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 6 creatinine, creatine kinase (ck), the admission rate of intensive cure unit (icu), rate of discharge and fatality, symptom, comorbidity, complications and cure rate of drug. for normality distribution data, outcomes were extracted directly. for skewness distribution data, the outcomes was extracted after being converted as specific formula [40] . the quality assessment of included studies was performed through the newcastle-ottawa quality assessment scale (nos), as recommended by the cochrane non-randomized studies [41] . the nos includes three parts for risk of bias, with nine points in total: (1) selection of research groups (four points); (2) inter-group comparability (two points); and (3) ascertainment of exposure and outcomes (three points) for case-control and cohort studies, respectively. study that scored 6 or more was qualified for systematic review [42] . the assessment process was completed by two reviewers independently. all debates were solved by discussion with a third reviewer. all statistical analyses and graphs were generated and plotted using graphpad prism version 7.00 software (graphpad software inc). the p value < 0.05 suggests significant difference. all included studies were retrospective study . among ten studies reported covid-19, one trial was performed in netherlands [12] and remaining nine trials were conducted in china [8-9, 13-16, 37-39] . the sample size ranged from 6 to 425, and had a total . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint [24-26, 31, 33] , one trial was performed in iran [19] and one trial was conducted in japan [10] . the sample size ranged from 5 to 883, and had a total of 2708 patients. the year of publications ranged from 2013 to 2020. clinical and laboratory characteristics of included study were shown in table 1 and table 2 respectively. among thirty-two included studies, four studies obtained 6 points of nos [26, 28, 32, 35] , and remaining twenty-eight studies obtained 7 points of nos or more [8-25, 27, 29-31, 33-34, 36-39] . the result of quality assessment was presented in table 3 . for covid-19 population, the number of patients with fever was 480 (45.2%), cough was 373 (35.1%), generalised weakness and myalgia was 318 (29.9%), stuffy or rhinorrhoea was 6 (0.6%), pharyngalgia was 32 (3%), chest pain was 10 (0.9%), diarrhoea or anorexia was 123 (11.6%), dyspnoea was 140 (13.2%) and dizziness or headache was 60 (5.6%). for mers-cov population, the amount of patients with fever was 404 (14.9%), cough was 424 (15.7%), generalised weakness and myalgia was 337 (12.4%), stuffy or rhinorrhoea was 17 (0.6%), pharyngalgia was 47 (1.7%), chest pain was 27 (1%), diarrhoea or anorexia was 128 (4.7%), dyspnoea was 271 (10%) and dizziness or headache was 131 (4.8%). the above results were shown in table 4 . for covid-19 population, the main complications included shock, arrhythmia, acute respiratory distress syndrome (ards), acute cardiac injury, acute kidney injury and acute . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint liver injury, and the amount of which was 17 (1.6%), 28 (2.6%), 51 (4.8%), 11 (1%), 10 (0.9%) and 2 (0.2%) respectively. for mers-cov population, the number of individuals presented shock was 22 (0.8%), arrhythmia was 11 (0.4%), ards was 83 (3.1%), acute cardiac injury 10 (0.4%), acute kidney injury was 30 (1.1%), acute liver injury was 22 (0.8%) and neurological symptoms was 4 (0.1%). the results were shown in table 5 . table 6 . systematic review was performed for clinical and laboratory outcomes of coronavirus disease. there was no significant difference in age (50.9 ± 2 vs. 53.6 ± 1.5, p = 0.3), wbc table 7 . . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint the higher admission rate of icu was found in mers-cov population than in covid-19 population ( 43.6% vs. 22.4%, figure 2 ). there was a higher discharge rate in mers-cov populations compared with covid-19 population ( 59.9% vs. 33.5%, figure 3 ). the lower fatality rate was found in covid-19 population compared with mers-cov population (6.8% vs. 34.1%, figure 4 ). coronavirus is an important pathogen causing respiratory and intestinal infection. of seven identified coronaviruses, the two very pathogenic viruses, sars-cov and mers-cov, cause severe ards and even acute respiratory failure. with a mortality rate of more than 10% and more than 35% respectively [43] [44] . the four other human coronaviruses in addition, we found that the number of males is more than that of females in either covid-19 or mers-cov population. the possible reason of reduced susceptibility of females to viral infection is that females have a lot of x chromosome and estrogen that are vital components in development of innate and adaptive immunity [47] . meanwhile, numbers of patients with covid-19 infection had chronic comorbidities, mainly hypertension, diabetes and cardiovascular disease, which is similar to mers-cov population. those results indicate that older adult males with chronic underlying disease might be more susceptibility to covid-19 or mers-cov. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 1 1 in terms of laboratory testing, reduced lymphocytes and increased crp were found in both covid-19 and mers-cov patients. this result indicates that covid-19 might be associated with cellular immune response, mainly act on lymphocytes like mers-cov does [48] . the cells infected by viruses induce the release of numbers of pro-inflammatory cytokines and inflammation storm in the body. moreover, increased cytokines might make damage to related organs such as liver [49] . our results demonstrated that abnormal value of ast was found in mers-cov population, but not in covid-19 population. the possible reason is that the follow-up time of covid-19 population was too short, and the liver might remain to be in compensatory stage. for this result, a long follow-up time study is urgently needed. on the other hand, our results suggested that mers-cov population had a higher rate of icu admission and fatality than covid-19 population, indicating that compared with mers-cov, covid-19 has less toxic and more easily cured. however, lower discharge rate was found in covid population than in mers-cov population. the possible explanation is that most of covid-19 patients remained to be hospitalized at the time of manuscript submission, and data on those patients could not be obtained in time. thus, careful interpretation is urged for this result. up to now, no effective strategy has been found for treatment of covid-19 infection [50] . currently, the measure to covid-19 is to control the source of the infection; taking personal protective works to reduce the risk of transmission; and early diagnosing, isolating and supportive treating for confirmed patients. in the present study, our systematic results of cure rate of drug for mers-cov infection indicated that ribavirin and interferon, oseltamivir, antivirals and intravenous immunoglobulin all had been effective for mers-cov infection, with the cure rate of those drugs was 74.2%, 69.2%, 67.1% and 62.5% respectively. thus, we . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint 1 2 assume that those drugs might also be effective at covid-19 infection. however, further studies are needed to confirm this idea. this study has several limitations. first, many patients infected by covid-19 remained to be hospitalized at the time of manuscript submission, leading to the unavailable of some data. second, the follow-up time of covid-19 population is too short to get related data from long-term observations of this disease. third, finding of statistical tests and p values between covid-19 and mers-cov populations should be interpreted with caution. fourth, the number of mers-cov patients treated by drugs is small, careful understanding is needed for the cure rate of drug for this disease. finally, as covid-19 is still developing around the world and remains to have many unknowns, the results of this study are staged and need to be carefully understood. more large-sample, multicentre, high-quality research should be performed to update this study. our systematic review reveals that main clinical manifestations of both covid-19 and mers-cov populations are fever, cough and generalised weakness and myalgia. ards is main complication of both two populations. covid-19 population has a shorter incubation time and lower rate of icu admission, discharge and fatality compared with mres-cov population. this study was supported by grants from the national natural science foundation of china (nos.31970862) and guangzhou municipal science and technology project (nos. . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint . cc-by-nc-nd 4.0 international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/10.1101/2020.03.08.20032821 doi: medrxiv preprint clinical virology sars and other coronaviruses as causes of pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical and laboratory findings of middle east respiratory syndrome coronavirus infection characteristics and outcome of viral pneumonia caused by influenza and middle east respiratory syndrome-coronavirus infections: a 4-year experience from a tertiary care center incubation period of 2019 novel coronavirus (2019-ncov) infections among travellers from wuhan, china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster emerging coronavirus 2019-ncov pneumonia clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical predictors of mortality of middle east respiratory syndrome coronavirus (mers-cov) infection: a cohort study ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study epidemiological status of the middle east respiratory syndrome coronavirus in 2019: an update from clinical outcomes among hospital patients with middle east respiratory syndrome coronavirus (mers-cov) infection estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study mortality rate of icu patients with the middle east respiratory syndrome -coronavirus infection at king fahad hospital, jeddah, saudi arabia critically ill healthcare workers with the middle east respiratory syndrome (mers): a multicenter study middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications clinical and epidemiologic characteristics of spreaders of middle east respiratory syndrome coronavirus during the 2015 outbreak in korea neurological complications during treatment of middle east respiratory syndrome a comparative study of clinical presentation and risk factors for adverse outcome in patients hospitalised with acute respiratory disease due to mers coronavirus or other causes characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards patient characteristics infected with middle east respiratory syndrome coronavirus infection in a tertiary hospital clinical presentation and outcomes of middle east respiratory syndrome in the republic of korea mers-cov outbreak in jeddah--a link to health care facilities renal complications and their prognosis in korean patients with middle east respiratory syndrome-coronavirus from the central mers-cov designated hospital clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical characteristics of 140 patients infected with sars-cov-2 in wuhan, china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study estimating the mean and variance from the median, range, and the size of a sample the newcastle-ottawa scale (nos) for assessing the quality of nonrandomised studies in meta-analyses. ottawa hospital research institute is diabetes mellitus an independent risk factor for colon cancer and rectal cancer ? sars and other coronaviruses as causes of pneumonia author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint from sars to mers, thrusting coronaviruses into the spotlight origin and evolution of pathogenic coronaviruses epidemiology, genetic recombination, and pathogenesis of coronaviruses sexual dimorphism in innate immunity prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis liver manipulation during liver surgery in humans is associated with hepatocellular damage and hepatic inflammation sars and mers: recent insights into emerging coronaviruses author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint key: cord-334667-0cah15lg authors: arabi, yaseen m.; asiri, ayed y.; assiri, abdullah m.; aziz jokhdar, hani a.; alothman, adel; balkhy, hanan h.; aljohani, sameera; al harbi, shmeylan; kojan, suleiman; al jeraisy, majed; deeb, ahmad m.; memish, ziad a.; ghazal, sameeh; al faraj, sarah; al-hameed, fahad; alsaedi, asim; mandourah, yasser; al mekhlafi, ghaleb a.; sherbeeni, nisreen murad; elzein, fatehi elnour; almotairi, abdullah; al bshabshe, ali; kharaba, ayman; jose, jesna; al harthy, abdulrahman; al sulaiman, mohammed; mady, ahmed; fowler, robert a.; hayden, frederick g.; al-dawood, abdulaziz; abdelzaher, mohamed; bajhmom, wail; hussein, mohamed a. title: treatment of middle east respiratory syndrome with a combination of lopinavir/ritonavir and interferon-β1b (miracle trial): statistical analysis plan for a recursive two-stage group sequential randomized controlled trial date: 2020-01-03 journal: trials doi: 10.1186/s13063-019-3846-x sha: doc_id: 334667 cord_uid: 0cah15lg abstract: the miracle trial (mers-cov infection treated with a combination of lopinavir/ritonavir and interferon-β1b) investigates the efficacy of a combination therapy of lopinavir/ritonavir and recombinant interferon-β1b provided with standard supportive care, compared to placebo provided with standard supportive care, in hospitalized patients with laboratory-confirmed mers. the miracle trial is designed as a recursive, two-stage, group sequential, multicenter, placebo-controlled, double-blind randomized controlled trial. the aim of this article is to describe the statistical analysis plan for the miracle trial. the primary outcome is 90-day mortality. the primary analysis will follow the intention-to-treat principle. the miracle trial is the first randomized controlled trial for mers treatment. trial registration: clinicaltrials.gov, nct02845843. registered on 27 july 2016. middle east respiratory syndrome (mers) is a viral respiratory disease caused by the middle east respiratory syndrome coronavirus (mers-cov). mers cases continue to occur and are often associated with respiratory and multiorgan failure [1] . there is no antiviral treatment with proven efficacy at present [1, 2] . the miracle trial (mers-cov infection treated with a combination of lopinavir/ritonavir and interferon-β1b) is the first randomized controlled trial for mers treatment. the study protocol has been previously published [3] . there are several challenges in a trial for treatment of a disease like mers: (1) there is not enough information on the effect size of the lopinavir/ritonavir and interferon-β1b provided with standard supportive care compared to placebo provided with standard supportive care to conduct adequate planning for the study sample size; (2) mers is a sporadic, unpredictable, and rare disease, which makes it difficult to plan a separate pilot study to collect the necessary information needed for the planning of the main trial. to overcome these challenges, we designed the miracle trial as a recursive two-stage adaptive trial, which is a relatively new method for group sequential trials [4] [5] [6] [7] . the approach is based on the conditional error principle, which allows for flexible and continuous adjustment of the trial parameters using data observed during prior stages without inflation of the type i error [8] . another advantage of this method is the flexibility in setting the timing and the number of needed interim analyses. such flexibility is necessary in a situation where recruitment rate is unpredictable and a sudden flux in recruitment of patients could happen at any time. finally, the design takes advantage of the accumulated information throughout the trial from every single recruited patient as opposed to a traditional two-study approach (pilot followed by the main trial). in this article, we describe the miracle trial statistical analysis plan (sap) in advance of trial completion. we identify the procedures to be followed for the primary and secondary analyses for the trial. the sap was written by the study steering committee members led by the principal investigator, who remains blinded to both group allocation and to study results until after completing patient recruitment, patient follow-up, and completion and locking of the database. the final study report will follow the guidelines of the consolidated standards of reporting trials (consort) for reporting randomized controlled trials [9, 10] . the trial is being conducted according to the standard requirements of good clinical practice e6 [11] . the sap was developed in accordance with the international council for harmonisation guidelines (e9 statistical principles for clinical trials and e3 clinical study reports guidelines) [12, 13] and with the guidelines for the content of statistical analysis plans in clinical trials [14] . the miracle trial is a recursive, two-stage, group sequential, multicenter, randomized, placebo-controlled, double-blind trial. the trial includes hospitalized patients who are 18 years old or older with laboratoryconfirmed mers in addition to evidence of acute organ dysfunction that is judged related to mers. inclusion and exclusion criteria have been detailed in a previously published protocol manuscript [3] . patients are randomized to receive lopinavir/ritonavir and recombinant interferon-β1b or placebo. randomization is stratified according to center and according to whether the patients require mechanical ventilation (invasive or noninvasive) at the time of enrollment, as mechanical ventilation is a major, but pragmatic, surrogate for severity of illness. the study interventions continue for 14 days or until hospital discharge. patients are followed up daily until day 28 or hospital discharge and then at day 90. a consort flow diagram of the trial progress will be constructed (fig. 1 ). the number of randomized patients to each group will be reported as well as the number of randomized patients who received the interventions. we will also report the number of screened patients (defined as all hospitalized patients with mers) who met the eligibility criteria but were not enrolled and the reasons for non-enrollment. the intention-to-treat population consists of all enrolled patients whether or not they received the allocated intervention, and will be used for the primary analysis. a per-protocol analysis will be conducted for patients who received the allocated interventions (defined by any dose of the study intervention). baseline characteristics will be presented for the two study groups (additional file 1: table s1 ) including age, sex, and body mass index, the presence of co-infections, nosocomial versus community-acquired mers infection, acute physiology and chronic health evaluation (apa-che) ii scores, sequential organ failure assessment scores, and the karnofsky performance status scale score [3] . we will report comorbidities and the interventions received before randomization for the patients in each group. we will report baseline laboratory values (international normalization ratio, platelet count, hemoglobin, white blood cell count, lymphocyte count, liver enzymes, glucose, serum amylase, blood urea nitrogen, creatinine, creatine kinase, lactate) and respiratory and vital parameters in addition to the location of the patient at time of randomization. for each group we will report the time of hospital admission to randomization and the time of randomization to the first dose received of the study drugs. we will report the received study intervention and its duration for each group, in addition to the missing or incomplete doses and protocol violations (additional file 1: table s2 and table s8 ). we will compare any use of vasopressors, renal replacement therapy, neuromuscular blockade, mechanical ventilation, extracorporeal membrane oxygenation (ecmo), nitric oxide, prone ventilation, and tracheostomy. we will also compare the use of intravenous immunoglobulin, antiviral therapy, antibiotics, corticosteroids, and statins (additional file 1: table s2 ). the primary outcome is 90-day mortality (additional file 1: table s3 ). the primary outcome is defined as all-cause mortality after enrollment in the trial within 90 days, as either an inpatient or outpatient. secondary outcomes and subgroups are defined as presented in table 1 and additional file 1: table s4 , and s5). in addition, we will compare the physiological parameters among patients treated in the treatment group and the control group. all analyses will be performed using sas 9.4 with specially written code for the analysis of the primary supplemental oxygen-free days number of days within the first 28 days after enrollment when patients do not receive of supplemental oxygen. patients who die within 28 days will be assigned the value "0" renal replacement therapy-free days number of days within the first 28 days after enrollment when patients do not receive of renal replacement therapy. patients who die within 28 days will be assigned the value "0" vasopressor-free days number of days within the first 28 days after enrollment when patients do not receive of vasopressors. patients who die within 28 days will be assigned the value "0" invasive or non-invasive mechanical ventilation-free days number of days within the first 28 days after enrollment when patients do not receive of mechanical ventilation. patients who die within 28 days will be assigned the value "0" organ support-free days number of days within the first 28 days after enrollment when patients do not receive of invasive mechanical ventilation, renal replacement therapy and vasopressor. patients who die within 28 days will be assigned the value "0" extracorporeal circulation support-free days number of days within the first 28 days in which patients are not receiving extracorporeal circulation support. patients who die within 28 days will be assigned the value "0" icu-free days number of days in which patients are not being cared for in the icu during the first 28 days after enrollment. patients who die within 28 days will be assigned the value "0" post-randomization hospital length of stay number of days between randomization and discharge from the hospital. because of the competing risk effect of death on length of stay, length of stay will be also reported for survivors alone the number and percentage of reported serious adverse events any time during the study period. these saes include: acute pancreatitis, severe elevation of alanine aminotransferase (alt) to more than five-fold the upper normal limit, anaphylaxis, bleeding diathesis and others the number and percentage of adverse events graded using the common terminology criteria for adverse events, at any time within 28 days after enrollment. the adverse drug reactions include: allergic reactions, gastrointestinal, general nervous system and others. see also table s6 functional outcomes karnofsky score karnofsky performance status scale for functional impairment, which is a scale from 100 (indicating "normal," no complaints; no evidence of disease) to 0 (indicating death) at day 90 outcome that accounts for the recursive design, as described in chang [4] . a detailed interim analysis plan is reported in the mir-acle protocol [3] . the trial is designed as a recursive, two-stage, group sequential randomized trial. the first interim analysis will be conducted when 34 subjects (17 per group) have completed 90 days of follow-up. this is about 17.5% of the total sample size needed for the classical design (a classic two-group design requires a total of 194 subjects (97 subjects per group) to have an 80% power at a significance level of 5% using a one-sided z test for difference in proportion to detect 20% absolute risk reduction in 90 days mortality among subjects receiving treatment (20%) compared to a control group (40%)). a data and safety monitoring board (dsmb) will be convened to review the unblinded data (efficacy and safety) and advise on continuation or termination of the trial. the determination of the stopping boundaries in the first two-stage design was calculated using the conditional power method based on the summing stagewise p values. at the first interim analysis, the dsmb will determine whether the trial should be terminated for futility or not using the following boundaries and their corresponding decisions ( table 2) . we will summarize and report the demographics and baseline clinical characteristics using descriptive statistics. as appropriate, the chi-square test or fisher's exact test will be used to compare the categorical variables, which will be reported as numbers and percentages. student's t test or the mann-whitney u test will be used as appropriate to compare the continuous variables, which will be reported as means and standard deviations or as medians and interquartile ranges. all adverse events will be grouped using common terminology criteria for adverse events (ctcae) version 4 of the national institutes of health (nih) (additional file 1: table s6 ). adverse events will be grouped into aggregate groups and reported for the entire study period (additional file 1: table s7 ). all results will be summarized in terms of frequency and percentage and will be compared across study arms using fisher's exact test. all results will be declared statistically significant with a p value < 0.05. let k be the number of stages of the current clinical trial needed to complete the trial and i ∈ {1, 2} be the index for the two-stage design in the k th stage. let r 1ki and r 2ki be the proportions of 90 days mortality in the standard of care and treatment group respectively. then the z test statistic for the difference in proportion can be calculated as follows: where n ki is the sample size per group for the i th twostage design of the k th stage. in the interim analysis (i.e., at each i = 1 of the k th two-stage), the primary outcome will be evaluated, and the trial sample size will be reestimated for the subsequent stage based on the observed effect size using the following formula assuming a conditional power of 80% (pc = 0.8) to decide if the trial should continue: here α k, 2 is the precalculated rejection boundary for efficacy at the second stage of the two-stage design at the k th stage, and p k, 1 is the raw table probability corresponding to the z ki statistic. at the first interim analysis, should the data suggest that another stage of the twostage steps is required, we will recalculate the conditional error and new boundaries will be calculated for k = 2. let β k + 1, 1 , α k + 1, 1 be the rejection boundaries for futility and efficacy for the first (i = 1) of the twostage step of the k th + 1 stage. then the conditional error is: efficacy stopping boundary (α 2 ) 0.2250 stop trial for efficacy at the second stage or recalculate based on conditional power at first interim analysis α1 is the maximum probability threshold under which the trial will be terminated early for efficacy. β1 is the maximum probability threshold above which the trial will be terminated for futility. α2 is the maximum probability threshold (the sum of the stage-wise p-values), above which the study will be declared as met its endpoint where a(p 0, 1 ) is the type i error, which is set to 0.05. the new α k + 1, 2 boundary for the k th + 1 stage for pre chosen β k + 1, 1 , α k + 1, 1 will be calculated as follows: at the end of the trial, the treatment will be declared efficacious if the calculated stage-wise ordered p value p k, 2 is less than α k, 2 . the adjusted p value will be obtained using backward recursion as follows: where k 0 is the total number of two-stage stages, and t is the sum of stage-wise raw p values. finally, the adjusted overall 95% one-sided confidence interval will be calculated by: where δ i, 1 and δ k 0 ;2 are the stage-wise and the last stage of the kth two-stage design confidence interval bound. the last stage confidence bound δ k 0 ;2 can be found by solving the following equation numerically for δ k 0 ;2 : where n k0, 1 and n k0, 2 are the sample sizes for the first and second stage of the last k th two-stage design, and p k0, 1 , p k0, 2 are the stage-wise adjusted p values. in order to stay consistent with the method that was used in calculating the boundaries for the trial, we will not account for stratification in the primary outcome analysis. in general, this approach is acceptable and it preserves both type i and type ii errors as long as the weighted average of the effect size stays close to the hypothesized effect size [15] . furthermore, as long as the sample size re-estimation at the interim analysis was based on the weighted average of the effect size, the overall power of the trial will be preserved. with the exception of the analysis of the primary outcome, all other analyses will be tested using regular statistical methods and will be two-sided. a secondary adjusted analysis will be conducted using multiple logistic regression analysis, in which death within 90 days will be modeled as the dependent variable, and a set of baseline variables that are strongly believed to affect the outcome of mers will be included as independent variables. those variables will include at minimum the following: age, community-acquired versus hospital-acquired infection, mechanical ventilation, center, and sequential organ failure assessment score. ninety-day median survival time will be summarized and reported using kaplan-meier curves and will be compared between the study groups using the log-rank test (additional file 1: figure s1 ). analysis of secondary outcomes will be compared in the intention-to-treat cohort only. subgroup analyses will be conducted if patient numbers permit (e.g., no fewer than five patients in subgroups of interest) in a priori defined subgroups (additional file 1: table s5 ). multivariable logistic regression will be used to report the results of tests of interactions for these subgroups. all missing data will be reviewed and characterized in terms of their pattern (e.g., missing completely at random, missing at random, etc.). for missing completely at random, all analyses will be based on a list-wise deletion approach where only observations with complete values will be considered for analysis. for variables with values missing at random, multiple imputation techniques will be utilized to impute the missing values, as suggested by rubin [16] . to adjust for multiple testing, we will use the false discovery rate (fdr) as described by benjamini and hochberg [17] . in this procedure all hypothesis tests will be sorted in ascending order based on their calculated p value. all hypothesis tests below an index k will be rejected, where k is calculated as follows: where i = m, … ,1, m is the total number of tested hypotheses, and q = 0.05. additional details about the sap are available in additional file 2. the miracle trial investigates the efficacy of a combination therapy of lopinavir/ritonavir and recombinant interferon-β1b provided with standard supportive care, compared to placebo provided with standard supportive care, in hospitalized patients with laboratory-confirmed mers. the first patient was enrolled in november 2016. at present, 14 sites are actively screening for eligible patients. the recruitment rate in the miracle trial has been slow mainly related to the decline in the number of mers cases in saudi arabia. due to the uncertainty of the efficacy level of the treatment and the recruitment rate, the trial is designed to be a recursive, two-stage, group sequential randomized trial [4] . several methods could be utilized to build an adaptive trial. however, most of these methods would require one to specify a priori the time and type of adjustments that need to take place in the trial. for a disease such as mers there are many factors that could limit the ability to specify a priori those elements; thus, the recursive two-stage design is a natural choice. this type of design provides enough flexibility to introduce different adjustments while learning from the observed data without inflating the type i error. reporting of the sap to the miracle trial in advance of trial completion will enhance evaluation of the clinical data and support confidence in the final results and the conclusion. prior specification of the statistical methods and outcomes analysis will facilitate unbiased analyses of these important clinical data. recruitment started in november 2016 and is currently ongoing. supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-019-3846-x. additional file 1: table s1 . baseline characteristics of intention-to-treat (itt) population. table s2 . summary of interventions and cointerventions. table s3 . primary outcome: 90-day mortality. table s4 . secondary outcomes. table s5 . subgroup analyses. table s6 . classification of adverse events in the miracle trial (mers-cov infection treated with a combination of lopinavir/ritonavir and interferon-β1b) using the nih common terminology criteria for adverse events (ctcae), version 4.0. table s7 . summary of adverse events by severity. table s8 . summary of protocol violations. middle east respiratory syndrome saudi critical care trials group. ribavirin and interferon therapy for critically ill patients with the middle east respiratory syndrome: a multicenter observational study treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-beta1b (miracle trial): study protocol for a randomized controlled trial adaptive design theory and implementation using sas and r benefit-risk evaluation of multi-stage adaptive designs draft guidance for industry on enrichment strategies for clinical trials to support approval of human drugs and biological products; availability flexible interim analyses in clinical trials using multistage adaptive test designs modification of the sample-size and the schedule of interim analyses in survival trials based on data inspections explanation and elaboration: updated guidelines for reporting parallel group randomised trials statement: updated guidelines for reporting parallel group randomised trials harmonisation of technical requirements for registration of pharmaceuticals for human use: good clinical practice (gcp) guideline the international council for harmonisation of technical requirements for pharmaceuticals for human use (ich): statistical principles for clinical trials international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use: e3 -structure and content of clinical study reports guidelines for the content of statistical analysis plans in clinical trials robustness of an odds-ratio test in a stratified group sequential trial with a binary outcome measure multiple imputation after 18+ years controlling the false discovery rate: a practical and powerful approach to multiple testing publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to acknowledge the following data safety monitoring board (dsmb) chair the miracle trial is funded by king abdullah international medical research center, riyadh, kingdom of saudi arabia. the study sponsor does not have any role in the study design, collection, management, analysis or interpretation of the data, or in writing the report. the datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. the miracle study is approved by the scientific committee and the institutional review board at the national guard health affairs, riyadh, saudi arabia (rc15/142) and all participating sites and registered at the saudi food and drug authority (sfda), riyadh, saudi arabia. patients who meet the eligibility criteria or substitute decision-makers (for patients lacking decisionmaking capacity) of eligible patients will be approached to obtain informed consent for enrollment. not applicable. the authors declare that they have no competing interests. ya and fgh are unpaid consultants on antivirals active for mers for gilead sciences, sab biotherapeutics, and regeneron. key: cord-351685-n70tkf38 authors: altamimi, asmaa; abu-saris, raghib; el-metwally, ashraf; alaifan, taghreed; alamri, aref title: demographic variations of mers-cov infection among suspected and confirmed cases: an epidemiological analysis of laboratory-based data from riyadh regional laboratory date: 2020-02-19 journal: biomed res int doi: 10.1155/2020/9629747 sha: doc_id: 351685 cord_uid: n70tkf38 introduction. middle east respiratory syndrome coronavirus was first recognized in september 2012 in saudi arabia. the clinical presentations of mers and non-mers sari are often similar. therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of mers-cov is essential. however, the real challenge is to flag these patients through some demographic markers. the nature of these markers has not previously been investigated in saudi arabia, and hence, this study aims to identify them. methods: it was a surveillance system-based study, for which data from a total of 23,646 suspected patients in riyadh and al qassim regions were analyzed from january 2017 until december 2017 to estimate the prevalence of mers-cov among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. results: of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. these confirmed cases (67.2% of which were males) had a mean age of 43.23 years (sd ± 22.8). around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. the study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. conclusion: the study provides evidence that the mers-cov epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. future studies should aim to confirm such findings in other regions of saudi arabia as well and explore potential preventable risk factors. a respiratory viral disease caused by the middle east respiratory syndrome coronavirus (mers-cov) was first isolated in 2012, in a 60-year-old man who died in jeddah, ksa due to severe acute pneumonia and multiple organ failure [1] . since then, 27 countries have reported the presence of this virus, including the 12 countries of the eastern mediterranean region. several outbreaks have occurred in multiple countries including saudi arabia, the united arab emirates and the republic of korea [2] . recent fatality rate (cfr) of 21% [5, 6] . very limited evidence is available for exploring the epidemiology of this virus among the pediatric population [7] . e literature shows that mers-cov infects males more than females [8, 9] . e casefatality rate of men (52%) is higher than that of women (23%) [10] . males with a history of serious medical conditions are highly susceptible to this infection. moreover, the mean age of infection in adults is 60 years [10] . e mode of transmission is not entirely understood yet [2] ; however, human-to-human [11] and zoonotic sources of transmission [12] have been documented in many studies. dromedary camels are the major animal source of mers-cov transmission to humans. interhuman transmission of the virus did not occur easily, but it is seen mainly in patients' families and healthcare settings [2] . clinical pictures of this infection varied from asymptomatic to mild respiratory symptoms to severe respiratory distress and death [2] . severe ailment can often cause respiratory catastrophes that need mechanical ventilation and support in icus across different healthcare settings [4] . studies have suggested an incubation period of 16 days with a mean of 5-6 days [12, 13] , while the median time until death is 11-13 days (range 5-27 days) among severely ill patients [13] . e gold standard test for the detection of this virus is real-time reverse-transcription polymerase chain reaction (rrt-pcr) assays [14] . ere is no specific treatment for mers-cov. like most viral infections, the treatment options are supportive and symptomatic [2] . at present, no vaccine exists for preventing the infections of mers-cov. e cdc indicated that preventative actions should be taken for any type of respiratory illness [4] . such actions include washing hands with water and soap for around 20 seconds or using hand sanitizers with alcohol if no water is available. one must cover their nose and mouth during instances of sneezing and coughing with a tissue and avoid touching the mouth, nose, or eyes with their hands until washed properly. repeatedly touched surfaces, such as door knobs, should be disinfected and cleaned regularly. intimate personal contact, e.g., kissing, and sharing cups or eating utensils must also be avoided [15] . many studies have been conducted in recent years in saudi arabia to combat this deadly disease. a large multicentre study showed that it is nearly impossible to differentiate between patients of mers-cov and non-mers-cov just on the basis of clinical presentation [16] . another cohort study, which was hospital-based (17 cases vs. 82 controls), found that there were statistically significant differences in terms of gender, clinical, and radiographic presentations [17] . similarly, two more single-centre case control studies reported that the presenting symptoms of mers-cov infection were not specific [18, 19] . physicians and public health practitioners need to identify suspected cases which have higher chances of diagnosis as confirmed cases prior to laboratory testing (which usually takes between 12 and 24 hours). identification of a confirmed case is necessary to implement preventive strategies to combat the spread of the disease to family members and hospital healthcare workers [20] . mild symptomatic cases, which result in a positive pcr, may be isolated at home. severe to moderate cases should be admitted to and isolated in a hospital until they improve and then be discharged for isolation at home for an extended period. both mild and severe cases are retested after 7 days, and the test is subsequently repeated after every 3 days until a negative result is obtained [20] . identifying suspected cases which may have higher chances of getting diagnosed as a confirmed case and implementing strict procedures on them might offer the best solution. e challenge is to flag these patients by some demographic markers, as the clinical presentation of mers-cov infected patients were non-specific. erefore, we aimed to identify some demographic markers specific to confirmed cases of mers-cov. e nature of these markers has not been investigated in saudi arabia, and hence this study aims to identify them. a cross-sectional study was conducted at the regional laboratory and blood bank, located at shumaisi hospital in riyadh, ksa. e laboratory has received the central blood banks and reference laboratories accreditation program saudi central board for accreditation of healthcare institution (cbahi) 2018 [21] . technique. data were collected during the period of january 2017 to december 2017. all patients in riyadh and al-qassim regions who had their samples tested at riyadh regional lab during the study period were considered as suspected cases. e study had two aims: descriptive and analytical. for the descriptive aim, we estimated the prevalence of mers-cov. for the analytical aim, a binary logistic regression model was developed. in this model, we included the risk factors of gender, age, seasons, nationality, healthcare status (yes/no), hospitals, and area of residence. data were cross-checked with a labcomputerized database. further data were collected on demographic characteristics (age and sex), underlying nationality, and health care status. we collected data from 25,400 cases, of which 23,646 suspected cases of mers-cov were included in the final analysis. data were cleaned, entered, stored, and managed with an excel database and ibm spss version 25. e statistical analyses consisted of descriptive counts and percentages. for those continuously scaled items, nonparametric statistics (medians, interquartile ranges, minimum, and maximum) were used to describe the distribution. a logistic regression analysis was used to identify predictors of confirmation of infection within the suspected cases groups. at first, univariate analyses were conducted to estimate the unadjusted contribution and to determine the significant risk factors. is was followed by a multivariate logistic regression analysis to estimate the independent contribution of each covariate. to determine significant factors, a p value below 0.05 and a 95% confidence interval were considered. a confirmed case is defined as a suspected case with laboratory confirmation of mers-cov infection [20] . a total of 23,646 of mers-cov suspected cases were included in this study, of which 52.3% were males (n � 12376) and 47.7% were females (n � 11270). e age of individuals with suspected cases ranged between 0 to 92 years with a mean age of 43. 23 e adjusted odds of mers-cov remained significant among different age groups; the odds of patients aged between 20-40 years increased threefold (a.or: 3.11, 95% ci: 1.104-8.76, p value � 0.032), whereas in the age group of 41-60 years, it increased further to a risk that was six times higher is cross-sectional study about the epidemiological analysis of mers-cov infection laboratory-based data was conducted in riyadh over a one-year period (2017). a total of 23,646 suspected cases were included in the results. of the total suspected cases, 119 cases had been confirmed via laboratory results. all the confirmed cases are reported to moh through hesn (health electronic surveillance networks) and to the world health organization (who) through the international health regulations (ihr), national focal point of saudi arabia. we found that mers-cov infection was found significantly in people aged between 41 and 60 years and was reported most commonly during the summer season. e odds of infection among males were found to be twice as high as that of females with suspected cases. during the study period, i.e., the year 2017, only 119 confirmed cases were reported, which means that the number of mers-cov infection cases has decreased in riyadh and al-qassim regions in comparison to that of the last three years. from 2015 to 2016, there was a 25.4% decrease, whereas from 2016 to 2017, it decreased by 48.7%, which translates into a 50% decrease between the two periods. is also complements the findings reported by of da'ar and ahmed in their paper [23] . e predominance of infection in males was also observed in another study pwefromed in ksa (2015), which reported the percentage of confirmed cases among males to be 66%, compared with 34% among females [24] . it is worth mentioning that saudi arabia defines age categories differently from the who (children: 0-14, adult: otherwise) [20] . however, unlike the classification used in saudi arabia, we have followed the who categorization of age to differentiate between children/adolescents (0 to 19 years) and adults (20 years and older) as indicated in who reports for age-standardized population and in infectious diseases [25] . is categorization was also followed by aly and his collaborators in their recent paper published in 2017 [14] . adults were further subcategorized into three groups according to the age distribution of the study population using the following two cutoff points (age of 41 and age of 60) [14] . ese data agreed with a previous surveillance study, which stated that the majority of confirmed cases of mers-cov were reported among people aged 40 and above [24] . in 2016, only 9 of 552 cases (1.6%) of mers-cov infection were found among pediatric patients. moreover, the study which was conducted in king fahad medical city in riyadh (kfmc) between january 2012 and december 2013 did not report any mers-cov cases among children [26] . e study which was conducted across the gulf countries for four years by mahmoud aly et al. between 2012 and 2016 suggests that the prevalence and distribution of mers-cov were the highest-risk in elderly aged 60 years or above [14] . similar to our results, this study also reported the highest number of confirmed cases during the summer season [14] . among confirmed cases, only 25.2% were healthcare workers, whereas around 75% were non-healthcare workers. is is in agreement with the study done by ahmad to estimate the survival rate in mers-cov globally prior to 26 january 2017; 86.9% were not health-care workers compared with 13.1% confirmed cases of healthcare workers [27] . similarly, other studies also reported a lower prevalence in healthcare workers [28] [29] [30] . our data reported a higher prevalence of infection among saudi nationals as compared with non-saudi. another study also showed similar results but with a much higher percentage among saudis, which may be due to the fact that it included saudis from all regions [29] . ere is no finding basis for comparison as such, because our study was focused on the riyadh and al qassim regions only. in our study, we detected a low prevalence (0.5%). e low positive predictive value of our lab results is not related to the low sensitivity and specificity of the lab assay. e estimated analytical sensitivity and specificity of the real star kit from altona was reported to be 100% with no cross reactivity with other respiratory pathogens [31] . moreover, this low predictive value in the lab results is related to the high burden of false positive cases referred to the lab. in fact, this research is just the starting point to shed the light on more factors that might help in putting more descriptive criteria to lower the financial and human resources burden. to the best of our knowledge, no one has developed a logistic regression that focuses on demographic risk factors such as sex, age, and seasons prior to our study. however, it is worth mentioning that ahmed et al. developed a risk prediction model that encompasses risk factors such as chest pain, leukopenia, and elevated aspartate aminotransferase (ast) [21] . however, further investigations are needed to confirm our findings. one of the major strengths of our study is that it is a comprehensive regional study which included all the suspected cases of mers-cov in the riyadh and al-qassim regions. secondly, the external validity of our study is also expected to be high, as it covers the two regions completely, meaning that the records of all suspected cases in these two main regions in saudi arabia were included. irdly, the quality of the data is considered to be high, given that the contagious and life-threatening nature of this disease has led to strict obedience to rules which are enforced in a timely manner, thus ensuring accurate reporting of suspected cases. in addition to this, quality assurance policies are implemented at hesn in order to maintain the highest level of validity and reliability of the data collection process. e variables available for suspected cases were limited to demographics, which limited the scope of our research, but they provided valuable information to form a basis for future studies of a broader scope. variables such as primary/secondary infections are vital pieces of information, but due the limitation of the data available, we could not determine their effects. according to our knowledge, this is one of the few studies that have specifically investigated mers-cov risk factors in the riyadh and al-qassim areas (two major regions in ksa). given that all suspected and confirmed cases were included in this study, we assume that our results are generalizable for both the regions with confidence. it must be noted that the comparative group of this study is different from that of the previous ones, as we compared those with confirmed mers-cov with those with suspected mers-cov who have passed all stages of screening at the hospital, whereas other studies were hospital but not lab-based with an aim of identifying factors that help in suspecting rather than confirming cases. is might be the reason why we have found some significant demographic factors unlike other reports. in conclusion, this research is about predictors for the confirmation of diagnosis among suspected cases only, meaning that the factors we found can help in identifying suspected cases that may have a higher chance of testing positive. is will help primary healthcare professionals to develop a better screening tool for suspected cases, as currently only a small minority of suspected cases are confirmed positive via lab results, consequently resulting in a lot of resources being spent to test thousands of samples, just for the identification of a few cases. e three factors we identified are important because, for example, a female, aged 18, presenting in winter will be less likely to be diagnosed than a male, aged 45, presenting in the summer, or, to give another example, a 60-year-old male who is presenting mers-cov signs with a negative lab result may need retesting. our study covered two main regions in saudi arabia and provides evidence that the mers-cov epidemic in these two regions has specific characteristics that might help future plans for prevention and management of such contagious diseases. our results showed that only a minority of suspected cases are actually diagnosed with the disease, meaning that the procedures being implemented seemed to be highly sensitive but not highly specific. e majority of confirmed cases were male, aged 41 to 60 years, and presented to healthcare facilities in the summer. future studies should aim to confirm such findings in other regions in saudi arabia, to explore potential preventable risk factors and go deeper to know the underlying factors that make male aged 41-60 more susceptible than others. e laboratory data used to support the findings of this study were provided by riyadh regional laboratory under license and are not freely available. however, access to data will be considered from the corresponding author upon request. e authors declare that they have no competing interests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who, middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome (mers), who cov outbreak largest outside kingdom of saudi arabia middle east respiratory syndrome coronavirus in children hospital outbreak of middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection e pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update an epidemic whirlwind critically ill patients with the middle east respiratory syndrome middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia a comparative study of clinical presentation and risk factors for adverse outcome in patients hospitalised with acute respiratory disease due to mers coronavirus or other causes development of a risk-prediction model for middle east respiratory syndrome coronavirus infection in dialysis patients mers-cov diagnosis: an update underlying trend, seasonality, prediction, forecasting and the contribution of risk factors: an analysis of globally reported cases of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): impact on saudi arabia 2000-2025) standard-standard populations-seer datasets [internet], acute myeloid leukemia-cancer stat facts acute viral respiratory infections among children in mers-endemic riyadh estimating survival rates in mers-cov patients 14 and 45 days after experiencing symptoms and determining the differences in survival rates by demographic data, disease characteristics and regions: a worldwide study diagnostic delays in 537 symptomatic cases of middle east respiratory syndrome coronavirus infection in saudi arabia e predictors of 3-and 30-day mortality in 660 mers-cov patients risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of middle east respiratory syndrome coronavirus from upper respiratory tract specimens acknowledgments e authors would like to thank dr. waleed alsalem, dr. ahmed hakawi, and dr. mutaz mohammed from ministry of health, saudi arabia, dr. kamel al-dossari from riyadh regional lab for their help in this research, hatim al-mutairi for data cleaning, dima zailaey for structuring, and dr. munazza jawed from dow university of health sciences, karachi, for proofreading. e authors would also like to thank miss laila mohamed ghoneim from the american university cairo for english-language editing. all authors contributed to the writing of the manuscript and had access to the data. all authors read and approved the final manuscript. key: cord-321185-kj67rd7g authors: eckerle, isabella; corman, victor m.; müller, marcel a.; lenk, matthias; ulrich, rainer g.; drosten, christian title: replicative capacity of mers coronavirus in livestock cell lines date: 2014-02-17 journal: emerg infect dis doi: 10.3201/eid2002.131182 sha: doc_id: 321185 cord_uid: kj67rd7g replicative capacity of middle east respiratory syndrome coronavirus (mers-cov) was assessed in cell lines derived from livestock and peridomestic small mammals on the arabian peninsula. only cell lines originating from goats and camels showed efficient replication of mers-cov. these results provide direction in the search for the intermediate host of mers-cov. and representative peridomestic small mammals on the arabian peninsula. to estimate mers-cov permissiveness of cell cultures derived from these animals, we compared mers-cov replication and infectious virus production with that in bat-and primate-derived cells known to be permissive for mers-cov. the mers-cov receptor dipeptidyl peptidase 4 (dpp-4) is expressed in epithelial cells of the lung and kidney, and patients with mers-cov consistently show severe involvement of both organs; thus, we focused on lung and kidney cells in potential animal hosts (10, 11) . using enhanced respiratory personal protection equipment in a biosafety level 3 facility, we cultivated, in parallel, cell lines from goats, sheep, cattle, camelids (dromedary and alpaca), rodents, insectivores, bats, and human and nonhuman primates (table) . cells were checked to ensure the absence of mycoplasma contamination and genotyped for their species of origin by sequencing of the mitochondrial cytochrome c subunit oxidase i gene (12) . all cells expressed dpp-4, as determined by immunofluorescence staining (figure 1 ) and western blot analysis (data not shown). because several of the ungulate cell lines had not previously been used for viral infection experiments, we determined permissiveness of all cell lines for rift valley fever virus (rvfv) clone 13, a virus mutant known to be attenuated yet broadly infectious for ungulate cell lines (13) . triplicate infections with multiplicities of infection (mois) of 0.5 infectious units/cell resulted in highly consistent levels between cells (maximal variation 3.2-fold) ( figure 2 , panel a). in addition, to demonstrate the ability of the cells to support cov replication, we infected all cell lines in triplicate (mois of 0.5) with bovine cov strain nebraska. the strain replicated to high levels in all cell lines; replication varied by <52.9fold, which constitutes small relative variations in light of the overall levels of replication ( figure 2, panel b) . we conducted mers-cov infections under the same conditions and with mois of 0.5. in addition to livestock cell lines, we used rodent, insectivore, bat, and primate cell lines in the experiments (table) . bat and primate cells known to be permissive for mers-cov served as controls. mers-cov-inoculated cells were incubated for 1 h and then washed twice before supernatant was harvested (0 h after incubation) ( figure 2 , panel c). we quantified virus replication by using the upe assay (14), a mers-cov real-time reverse transcription pcr that screens upstream of the e gene. replication was seen in lung and kidney cell lines derived from goats (capra hircus) and in umbilical cord and kidney cells from camelids (camelus dromedarius and llama pacos) ( genome equivalents/ml of cell culture supernatant) was seen in the goat kidney cells; this replication level was similar to that in vero e6 cells (interferon-deficient primate kidney cells). goat lung cells, alpaca kidney cells, and dromedary umbilical cord cells also showed strong replication, but virus did not replicate in sheep, cattle, rodent, or insectivore cells. in addition, expression of the receptor, dpp-4, was confirmed in all cells, including the nonpermissive sheep, cattle, rodent, and insectivore cells. after following virus growth in all permissive cells for another 20 h, we harvested supernatants and, to confirm the production of infectious virus particles, we titrated the supernatants by using a plaque assay in vero cells. mers-cov replication was seen in all permissive cells except tt-r.b (figure 2 , panel d), and all permissive cells showed cytopathic effects. the highest production of virus particles was in goat lung and kidney cells (1.0 × 10 7 and 2.7 × 10 6 pfu/ml, respectively). this level of replication was comparable to that in human lung cells (a549) and vero e6 (figure 2, panel d) . transmission of mers-cov between humans is still limited, and the identification of an intermediate animal host could enable the development of public health measures to prevent future spread of the virus among humans. although mers-cov neutralizing antibodies have been detected in camels from oman, spain, and egypt, the virus has not previously been detected in camels (8, 9) . an informed focusing of investigations on a select group of species, such as camels, could benefit epidemiologic investigations. to identify potential intermediate host species of mers-cov, we used in vitro testing to determine virus permissiveness in select cell culture models. in general, cell lines cannot depict the full pathogenicity of in vivo infection because infection is influenced by epitheliumspecific differentiation of target cells and the presence of immune cells. however, for viruses such as covs, whose tropism is believed to be determined mainly by the availability of an appropriate entry receptor (10) , epithelial cell cultures could indeed constitute valid surrogates of virus †parents and the calf phenotypically resembled c. bactrianus. the most likely explanation is cross-breeding between the 2 species with mitochondria stemming from a c. dromedarius mother in one of the former generations. permissiveness in vivo. with these limitations in mind, our results are in concordance with the findings of mers-cov neutralizing antibodies in camels and with information regarding patient contact with animals in reports of 2 human cases of mers-cov infection (11, 15) . one of the patients owned a farm on which camels and goats were kept. before onset of his own illness, the patient reported illness in several goats on his farm. the patient did not have direct contact with animals, but he reported having eaten goat meat and having had contact with one of the animal caretakers, who suffered from respiratory disease (15) . the second patient reported direct contact with a diseased camel shortly before onset of his symptoms (11) . in our study, production of infectious virus particles was seen in goat lung and kidney cells and in camelid kidney cells. excretion patterns indicative of kidney infection should be investigated once further clues to the identity of the mers-cov animal reservoir become available. our preliminary findings suggest that ungulates, such as goats and camels, are a possible intermediate host of mers-cov; thus, exposure to urine and feces from these animals might constitute a source of human infection. moreover, food products derived from these animals (e.g., meat and milk) should be tested for their potential to transmit mers-cov. the results of our study suggest that investigations into the mers-cov animal reservoir and intermediate host should focus on caprid (e.g., goats) and camelid hosts, and we identified several new cell lines for use in virus isolation studies. coronavirus diversity, phylogeny and interspecies jumping identification of a novel coronavirus in patients with severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe close relative of human middle east respiratorysyndrome coronavirus in bat seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection disentangling vector-borne transmission networks: a universal dna barcoding method to identify vertebrate hosts from arthropod bloodmeals characterization of clone 13, a naturally attenuated avirulent isolate of rift valley fever virus, which is altered in the small segment detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction contact investigation of a case of human novel coronavirus infection treated in a german hospital we thank benjamin meyer, stephan kallies, and jan-erik mittler for excellent technical assistance; sandra junglen, marco marklewitz, and valerie biewener for providing insectivore samples; ulrike m. rosenfeld for providing rodent samples for the generation of cell lines; friedemann weber for providing rift valley fever virus clone 13; and martin beer and malte dauber for providing bovine coronavirus.this study was supported by the european commission under project antigone (contract no. 278976) and the german centre for infection research.dr eckerle is a virologist at the institute of virology in bonn, germany. her primary research interest is characterization of novel and emerging zoonotic viruses. key: cord-319780-rfj9t99r authors: alexander, s.p.h.; armstrong, j.; davenport, a.p.; davies, j.; faccenda, e.; harding, s.d.; levi‐schaffer, f.; maguire, j.j.; pawson, a.j.; southan, c.; spedding, m.j. title: a rational roadmap for sars‐cov‐2/covid‐19 pharmacotherapeutic research and development. iuphar review 29 date: 2020-05-01 journal: br j pharmacol doi: 10.1111/bph.15094 sha: doc_id: 319780 cord_uid: rfj9t99r in this review, we identify opportunities for drug discovery in the treatment of covid‐19 and in so doing, provide a rational roadmap whereby pharmacology and pharmacologists can mitigate against the global pandemic. we assess the scope for targetting key host and viral targets in the mid‐term, by first screening these targets against drugs already licensed; an agenda for drug re‐purposing, which should allow rapid translation to clinical trials. a simultaneous, multi‐pronged approach using conventional drug discovery methodologies aimed at discovering novel chemical and biological means targetting a short‐list of host and viral entities should extend the arsenal of anti‐sars‐cov‐2 agents. this longer‐term strategy would provide a deeper pool of drug choices for future‐proofing against acquired drug resistance. second, there will be further viral threats, which will inevitably evade existing vaccines. this will require a coherent therapeutic strategy which pharmacology and pharmacologists are best placed to provide. pubmed has already accumulated a vast repository of information on sars-cov-2/covid-19, which increases on a daily basis (on 2020-03-23, there were 1369 hits for covid-19; this number more than doubled in the space of two weeks, so that by 2020-04-06 there were 2780 hits in pubmed for . clearly, there is a need to summarise this information critically and prioritise the elements which are constructive and useful for each individual sector. this document suggests priorities for how drug discovery and development might be rationally focussed for the rapid identification and successful translation of therapeutic agents to treat covid-19. given the urgency of the current situation, clearly initial drug discovery should focus on repurposing licensed drugs, as dosage and safety information are largely to hand. unfortunately, there is controversy over proof of efficacy for essentially all the potential repurposed agents for which preliminary, and, in many cases, non-peer reviewed data have surfaced. some of this controversy is addressed below but efforts are underway from both who and nih to coordinate larger, higher powered and better controlled studies in an attempt to demonstrate efficacy unequivocally. as a 'second wave', de novo discovery focussing on novel agents may allow future refinement and capacity to treat patients who are unable to be treated by, or are unresponsive to, the repurposed agents, but it would be very unlikely to have these new drugs available to treat the current crisis. the iuphar/bps guide to pharmacology (gtopdb) is an open-access database, developed by the international union of basic and clinical pharmacology (iuphar) and the british pharmacological society (bps). it provides expert-curated descriptions of almost 3,000 human proteins and over 10,000 ligands, including more than 1400 approved drugs. management of the new resource is the responsibility of the nomenclature and standards committee of iuphar (nc-iuphar), which acts as the scientific advisory and editorial board. the committee has an international network of over 700 expert volunteers organized into ∼60 subcommittees dealing with individual target families. the database is notably enhanced through the continued linking of relevant pharmacology with key immunological data types as part of the iuphar guide to immunopharmacology (supported by the wellcome trust) and by a major new extension, the iuphar/mmv guide to malaria pharmacology (in partnership with the medicines for malaria venture). the gtopdb team centred at the university of edinburgh have constructed a resource (faccenda et al.) , which provides a precis of the current understanding about the virus and potential associated drug targets and drugs. as with the other databases, the emphasis of the curation process is on stringent provenancing of the information provided, although inevitably the current situation limits the capacity for triangulation of data. sequencing analysis of the novel virus has identified a high level of similarity with the virus identified to cause the severe acute respiratory syndrome (sars) outbreak in china in 2002/03/04, which was known as the sars coronavirus or sars-cov. provisionally named as 2019-ncov, the virus has been renamed sarscov-2 (viruses, 2020) . for the purposes of this document, the virus is described as sars-cov-2, while the infectious disease is named as covid-19 (world health organization, 2020) . one of the positive aspects of the emergence of sars-cov-2 and covid-19 is the rapidity at which aspects like genome sequencing (for example, wu et al., 2020) and 3d structures (for example, yan et al., 2020) have been described. protein targets and drugs in the current review follow nomenclature as presented on the guidetopharmacology.org website (alexander, ball & tsoleridis. sars-cov-2 proteins, accessed on 2020-04-24) and the concise guide to pharmacology 2019/20 (alexander et al., 2019) . for general reviews of the coronaviruses, see masters, 2006; fehr and perlman, 2015; de wit et al., 2016; zumla et al., 2016; cui et al., 2019; desforges et al., 2019; song et al., 2019 . sars-cov-2 is a betacoronavirus; a lipid-enveloped, single-stranded, positive sense rna virus. other human coronaviruses include alphacoronaviruses, such as human coronavirus-229e (hcov-229e), and betacoronaviruses, such as sars-cov and mers-cov (responsible for the middle east respiratory syndrome) (for review, see zumla et al., 2016; corman et al., 2018; pillaiyar et al., 2020) . more than 200 viral types have been associated with the common cold, of which 50% of infections are rhinovirus, but also include respiratory syncytial virus, influenza and coronaviruses, particularly hcov-229e. although hcov-229e is regarded as 'relatively benign' since monocytes are much more resistant to infection, it does rapidly kill dendritic cells (mesel-lemoine et al., 2012) . classically, the viral lifecycle can be divided into six elements: cell attachment; cell entry; viral uncoating; nucleotide replication; viral assembly, and release (see figure 1 ). positive-stranded rna viruses replicate in the cytoplasm of infected cells, in close contact with intracellular membranes. this organization allows a concentration of viral and host factors to enable virus production and to evade innate immune responses (reviewed by yager and konan, 2019) . the sars-cov-2 coronavirus 30 kb genome encodes 29 proteins has 15 open reading frames, two of which encode viral polyproteins that generate 16 non-structural proteins (see below) (wu et al., 2020) . historically, therapeutic benefit has been gained through exploitation of the differences between viral and host proteins that subserve superficially similar functions (proteases and nucleotide polymerases, for example). the rapidity with which structural elements of the sars-cov-2 proteome have been identified provides hope that drug discovery approaches will soon provide agents to target the virus selectively, with minimal impact on the host. based on the evidence from orthologous proteins from other betacoronaviruses and the information currently available on sars-cov-2 (some of it not yet from peer-reviewed sources), we propose here the priority targets for pharmacological investigation. that should not be taken to mean that research should be limited to these targets, since there are undoubtedly a number of functions of the viral proteins still to be ascertained. it would be remiss not to conduct a thorough examination of all the viral proteome, both in isolation and in combination. the strategies we learn from investigation of the host:viral interaction from sars-cov-2 will stand us in good stead for future viral threats. coronavirus binds to cell surface proteins on target cells and, following proteinase priming of spike proteins on the virus surface, the virus is internalized into endosomal fractions that are subsequently acidified, or accumulates through a non-endosomal route (fehr and perlman, 2015) (figure 1 ). the endosomal route appears to involve clathrin (inoue et al., 2007) , but there are contradictory reports of the importance of the intracellular c-terminus of ace2 in this mechanism (inoue et al., 2007; haga et al., 2008) . a fusion domain permits insertion of a key protein (s, see below), which then allows mixing of the viral and cellular membranes and subsequent release of the coronaviral genome into the cytoplasm. following entry into the host cell cytoplasm and viral uncoating, the replicase gene of the viral rna is translated. the genome of coronaviruses consists of a single, continuous, linear, ssrna, capped at the 5' end and with a 3'-polya tail (fehr and perlman, 2015) . translation occurs from open reading frame (orf) 1a and 1b at the 5' terminus, with a ribosomal frameshifting mechanism allowing the overlap between orf1a and orf1b to generate the two polyproteins pp1a and pp1ab (fehr and perlman, 2015; perlman and netland, 2009; snijder et al., 2003; thiel et al., 2003) . in sars-cov-2, the polyproteins are long, 4405 and 7096 aa, respectively. encoded within the polyproteins of betacoronaviruses are two proteinases: papain-like proteinase, plpro, and chymotrypsin-like proteinase, 3clpro. in sars-cov, plpro, derived from the polyproteins, has three endoproteinase target sites, which release nsp1-3 . 3clpro has 11 cleavage sites to release the remaining non-structural proteins. in the coronavirus family, these proteinases process the polyproteins to generate 16 functional non-structural proteins identified as nsp1-16 (anand et al., 2003; thiel et al., 2003; ziebuhr et al., 2007; kindler et al., 2016; cui et al., 2019) . downstream of the orf1a and 1b are genes encoding four structural proteins (spike, envelope, membrane and nucleocapsid) (figure 2 ) and a short series (described as at least 13 in total, srinivasan et al., 2020) of other proteins (see below). once sufficient protein and rna accumulate, coronavirus assembly takes place, centred on the structural proteins. the release of coronavirus particles involves the secretory pathway of the endoplasmic reticulum and golgi apparatus and vesicular exocytosis (for review, see de haan and rottier, 2005; fehr and perlman, 2015) , and it is likely, but as yet unconfirmed, that sars-cov-2 adopts this mechanism also. to date, there is more evidence about the molecular detail involved in (and the possibilities to modify) viral recognition, entry and replication compared to uncoating, assembly and release, hence the attention paid here to the former three mechanisms. the cell-surface anchor -ace2 among the coronaviruses, the spike protein interacts with proteinases to anchor on host cell surfaces. the cell-surface anchoring point for the alphacoronavirus hcov-229e is aminopeptidase n (also known as cd13, yeager et al., 1992) . for the betacoronavirus mers-cov, dipeptidylpeptidase 4 (also known as cd26, raj et al., 2013) is an anchor. analysis of the co-crystal structure suggested that the sars spike protein binds to the active site of angiotensin converting enzyme 2 (ace2, li et al., 2005) . binding of sars-cov spike to ace2 seems to require cholesterol-rich rafts in the host cells (glende et al., 2008) 00. recent evidence points to the spike protein of sars-cov-2 also binding to ace2. both sars-cov (li et al., 2003) and sars-cov-2 (hoffmann et al., 2020; letko et al., 2020) have been described to require ace2 to enter cells ( figure 1) . a particular domain of the spike protein of sars-cov-2, a so-called receptor-binding domain (rbd), has been shown to facilitate binding to ace2 (hoffmann et al., 2020) . the ace2 peptidase active site is located remotely from the cell membrane (li et al., 2005; wrapp et al., 2020; yan et al., 2020) , into which the spike protein binds. the rbd of the spike protein is located in the s1 ectodomain, approximately a third of the way along the protein. ace2 is a carboxypeptidase, which means it removes the terminal amino acid from oligopeptides, and so it seems unlikely that the spike protein is a substrate for ace2. in sars-cov-infected mouse lung, ace2 protein expression was downregulated compared to uninfected mice . following sars-cov spike protein administration to mice, angiotensin ii was increased in the lungs . these observations led to the suggestion that this was the molecular mechanism for the frequent development of acute respiratory distress syndrome (ards) during sars-cov infections kuba et al., 2005) . ace2 activity has been reported to be released from plasma membranes by proteolysis, thought to be through the action of tnfα convertase (adam17, a disintegrin and metalloproteinase domain containing protein 17, lambert et al., 2005) (figure 1) . the activity of adam17 can be increased by g protein-coupled receptor activation, including the at1 angiotensin receptor (schafer et al., 2004) . ace2, and ace, activity can be measured in human plasma (ocaranza et al., 2006; herath et al., 2007; lew et al., 2008) . human plasma ace2 activity is reported to be 'masked' by the presence of endogenous inhibitors (lew et al., 2008) , which don't yet appear to have been precisely defined. blood ace2 activity can be altered in pathology; for example, serum ace2 was found to be decreased in patients following acute ischemic stroke (bennion et al., 2016) . the expression of ace2 mrna and enzyme activity in cardiac tissues were increased following repeated oral administration of the at1 angiotensin ii receptor antagonist losartan, while oral administration of an ace inhibitor lisinopril only increased cardiac mrna expression, but not enzyme activity (ferrario et al., 2005) . studies using disruption of the ace2 gene in mice indicated an increase in circulating angiotensin ii levels and a severe cardiac contractility defect, which could be 'rescued' with simultaneous genetic disruption of ace (crackower et al., 2002) . an early investigation of ace2 polymorphisms in man failed to show an association with hypertension (benjafield et al., 2004) . a study of sars victims and ace2 polymorphisms failed to find a correlation with patient outcomes (chiu et al., 2004) . the spike protein is the largest viral structural protein (~1200-1400 aa) and is heavily glycosylated, forming extended trimeric structures providing the characteristic 'crown' feature of coronaviruses (belouzard et al., 2012 ) (see figure 2 ). the ectodomain is divided into the s1 domain responsible for binding to ace2, whereas the s2 domain is responsible for the fusion machinery. following binding of the s1 domain to ace2, a deformation of the pre-fusion trimer results (wrapp et al., 2020) . surface plasmon resonance of the binding of human ace2 to the immobilized sars-cov-2 indicated an affinity (kd value) of 15 nm, an order of magnitude larger than sars-cov binding to ace2 (wrapp et al., 2020) . using a related label-free technique, biolayer interferometry, affinities of 5 and 1.2 nm for binding of sars-cov and sars-cov-2 spike protein, respectively, to human ace2 has been reported (walls et al., 2020) . although a proteolytic cleavage site at the s1/s2 boundary of the sars-cov spike protein is the best characterised, a second site upstream of the fusion peptide in the s2 domain, called s2' has also been described (belouzard et al., 2009) . this raises the possibility that multiple other proteases might be targetted to influence coronavirus activation (millet and whittaker, 2015) . a key difference between the spike proteins in sars-cov and sars-cov-2 is the presence in the latter of a site at the s1/s2 boundary predicted to be sensitive to the proteinase furin, and which may be targetted during viral assembly and maturation (walls et al., 2020) . the sars-cov s2 domain has a pair of α-helices, which may participate in coiled:coil structures during membrane fusion (petit et al., 2005) . the host complex of zdhhc9 (link to uniprot) with golga7 (link to uniprot), a palmitoyltransferase, which modifies the low molecular weight g proteins nras and hras (swarthout et al., 2005) , also palmitoylates the cysteine-rich s2 endodomain of the sars-cov to facilitate membrane fusion (petit et al., 2007) . very recently, in a comparison of the s2 domains of sars-cov and sars-cov-2, an enhanced capacity of the novel virus' s2 domain for membrane fusion was observed and suggested to result from eight differing amino acids . using a series of oligopeptides conjugated to lipid entities, high affinity (ic50 values in the nanomolar range) inhibitors of cell fusion were identified. given that the spike protein binds to the active site of ace2 (li et al., 2005) , in theory, any alteration in the availability of the active site should influence the binding of the spike protein and, hence, interfere with sars-cov-2 infection. one option would be to provide an excess of an endogenous peptide substrate, or more conventionally to apply a selective enzyme inhibitor. endogenous substrates of ace2 ace2, discovered in 2000 (donoghue et al., 2000) , shares 40% sequence similarity to ace within the n-terminal domain and is a type i transmembrane metallopeptidase. unlike ace, it functions as a zinc carboxypeptidase to cleave single c-terminal amino acids from peptides, particularly hydrolysing pro-phe residues in angiotensin-(1-8) to angiotensin-(1-7), [pyr 1 ]-apelin 13 to [pyr 1 ]-apelin-(1-12) and [des-arg 9 ]-bradykinin to bradykinin-(1-8) with high efficiency. it may also cleave other peptides less effectively (vickers et al., 2002) , shown below: angiotensin i  angiotensin-(1-9) + leu angiotensin ii  angiotensin-(1-7) + phe apelin-(1-13)  qrprlshkgpmp + phe apelin-(1-36)  ….qrprlshkgpmp + phe [des-arg 9 ]-bradykinin  rppgfsp + phe dynorphin a-(1-13)  yggflrrirpkl + lys 115 other peptides were not hydrolysed by ace2 including adrenocorticotrophic hormone, calcitonin, cholecystokinin, met-enkephalin, glucagon, glucagon-like peptide-1, melaninconcentrating hormone, pituitary adenylyl cyclase-activating polypeptide, somatastatin-14, urocortin or vasoactive polypeptide (vickers et al., 2002) . in humans, levels of mrna encoding ace2, together with immunoreactive peptide, are highest in the gastrointestinal tract, followed by heart, kidney, testes and gall bladder and other tissues (uhlen et al., 2015) . within organs, ace2 immunoreactivity was predominantly localised to epithelial (for example, in the lungs) and endothelial cells from all vascular beds examined (yang et al., 2017) . importantly, the ace2 antisera used in this study for immunocytochemistry was the same as that employed in the study described in the section below "using biopharmaceutical/antibody approaches to target ace2:spike interactions" (hoffmann et al., 2020) , to block entry of the virus in cell culture. the epitope of this antisera would be a rational starting point for the development of selective therapeutic antibodies. the presence of ace2 on airway epithelial cells is consistent with the isolation of sars-cov-2 from broncho-alveolar lavage of patients with covid19 and the infection of cultured airway epithelial cells . in humans, levels of ace2 immunoreactivity tend to be low. however, in addition to being upregulated by ace inhibitors and angiotensin receptor antagonists (see above), ace2 expression has been reported to be increased in human cardiovascular disease, for example, in the cardiomyopathic heart (zisman et al., 2003) . since ace2 is critical for viral entry, it may be one explanation for the high incidence of co-morbidity of covid-19 patients with cardiovascular disease. manipulation of ace2 activity by synthetic agents assays employing fluorogenic surrogate substrates to screen for inhibitors of ace2 activity are wellestablished, for example using methoxycoumarin-rppgfsafk(dnp)-oh (ocaranza et al., 2006; bennion et al., 2016) , or methoxycoumarin-apk(dnp)oh (herath et al., 2007; lew et al., 2008; mores et al., 2008) . detailed protocols for the use of methoxycoumarin-apk(dnp)oh have been described for fret-based high throughput screening xiao and burns, 2017) . this style of assay identified that ace2 was not inhibited in the presence of 10 µm lisinopril, enalaprilat, or captopril, inhibitors of angiotensin-converting enzyme (tipnis et al., 2000) . there are no licensed drugs described to inhibit ace2 activity. however, dx600 is a peptide-based ace2 inhibitor (huang et al., 2003) , while mln4760 and compound 28 are described as sub-nanomolar potency ace2 inhibitors (mores et al., 2008) . there is evidence for allosteric regulation of ace2 activity, in that a xanthenone derivative (xnt) was observed to enhance ace2, but not ace, activity in vitro with a potency of 20 µm (hernandez prada et al., 2008 ). an in silico study later identified a binding site in an allosteric hinge region of ace2, distinct from the proteinase active site, against which 1217 fda-approved drugs were screened (kulemina and ostrov, 2011) . a subsequent kinetic assay with the recombinant enzyme and a fluorigenic substrate identified labetalol and diminazene as agents able to double the maximal velocity of ace2 enzyme activity. whether any of these compounds alter the binding of the spike protein from either sars-cov or sars-cov-2 or viral infection in general does not appear to have been examined yet. a speculative area that should be explored further is the concept of enhancing the activity of the serine proteinase adam17 to increase cleavage and release of membrane bound ace2. peptides such as angiotensin ii are reported in animal models to cause release ('shedding') following binding to at1 receptors (xu et al., 2017) . although angiotensin ii is licensed by the federal drug administration to treat sepsis (known as giapreza, davenport et al., 2020) , it would be inadvisable as a treatment for covid-19 given the detrimental action of angiotensin ii on the lungs. in contrast, the investigational agent [pyr 1 ]-apelin-13 is currently used in clinical studies (davenport et al., 2020) and may also interact with its cognate receptor to downregulate membrane-expressed ace2. this peptide also has beneficial effects on the heart, including an increase in cardiac output (japp et al., 2010) . using biopharmaceutical/antibody approaches to target ace2:spike interactions an alternative approach to the small molecule manipulation of the ace2 enzyme would be to target the spike or ace2 proteins with selective antibodies. antibodies directed against ace2 led to a reduction in sars-cov-2 virus entry into target cells (hoffmann et al., 2020) , although this is likely to be some distance away from a therapeutic application. a truncated version of human recombinant ace2, lacking the transmembrane domain, mitigated against sars-cov infection of cells (li et al., 2003) and has been used in animal models to reduce symptoms of severe acute lung failure , diabetic nephropathy (oudit et al., 2010) and cardiac hypertrophy and fibrosis . treating sars-cov-2 victims with a soluble form of ace2 (batlle et al., 2020) or a fusion protein of the spike-binding portion of ace2 combined with the fc portion of human igg (lei et al., 2020) has been suggested. apeiron biologics has approval to conduct a phase ii clinical trial of apn01 (human recombinant ace2) for the treatment of covid-19 in three european countries (austria, germany and denmark) (nct04335136). this recombinant version of ace2 was originally licensed to glaxosmithkline and previously tested as gsk2586881 in a phase 2 multicentre trial (nct01597635) in patients with lung injury or ards, both features of sars and mers (and now covid-19). the study tested the hypothesis that cleavage of angiotensin ii (which causes lung injury -vasoconstriction, inflammation, fibrosis, vascular leak, and sodium absorption) to angiotensin-(1-7), would have counter regulatory beneficial action and reduce long term injury. gsk2586881 was well-tolerated in patients with ards, and the rapid modulation of peptides of the renin-angiotensin system demonstrated target engagement, in that levels of angiotensin ii decreased rapidly whereas angiotensin-(1-7) levels increased and remained elevated for 48 h, although the study was not powered to detect changes in acute physiology or clinical outcomes (khan et al., 2017) . sera from convalescent sars-cov patients prevented the cell entry of sars-cov-2 (hoffmann et al., 2020) and this approach has been used with some success in the sars, mers and covid-19 outbreaks (for review, see bloch et al., 2020) . the difficulty in identifying the precise molecular mechanism/s of convalescent sera action and issues with collection, standardization and scaling-up will be a challenge (bloch et al., 2020) . a bacterial equivalent of ace2 (based on 3d structure rather than primary sequence) termed b38-cap has been described, which is reported to reduce hypertension and limit cardiac dysfunction in an animal model (minato et al., 2020) . whether this agent might provide a decoy anchor to 'chelate' viral particles prior to cell entry has not been investigated. in a preliminary (as yet, not peer reviewed) study, a conformational change in the s1 rbd of the sars-cov-2 spike protein in the presence of heparin was noted (mycroft-west et al., 2020) . cellsurface heparan sulphate glycosaminoglycans have previously been suggested to be a lactoferrinsensitive alternative attachment point for the sars-cov virus (lang et al., 2011) . these observations suggest further routes for pharmacological targetting of viral infection and propagation. the cell-surface priming mechanism -tmprss2 tmprss2 is a single transmembrane domain protein with an extracellular serine protease domain, which appears to cleave substrates preferentially at basic residues (arg/lys), with a calcium-binding ldl receptor class a domain (paoloni-giacobino et al., 1997) . the tmprss2 gene encodes a cell-surface proteinase (transmembrane serine protease 2, tmprss2) and is located at chromosomal locus 21q22.3 in close proximity to erg, a gene encoding an ets transcription factor (link to uniprot, paoloni-giacobino et al., 1997) . (erg fusion with ews leads to ewing's sarcoma) fusion of the tmprss2 and erg (or the related etv1) genes has been reported to occur in the majority of prostate cancers and is suggested to lead to an androgen-dependent amplification of ets-regulated genes (tomlins et al., 2005) . tmprss2 expression is androgen-regulated (lin et al., 1999; chen et al., 2019) ; it is expressed highly in prostate cancer (lin et al., 1999; lucas et al., 2008) (for review, see tanabe and list, 2017) and loss of tmprss2 in the prostate is associated with reduced metastatic potential (lucas et al., 2014) . in aggressive versions of prostate cancer, tmprss2 undergoes autocatalytic proteolysis at arg 255 -ile 256 (afar et al., 2001) , where the two chains may remain in combination due to interchain disulphide bridges (chen et al., 2010) or the catalytic moiety may be secreted (chen et al., 2010) . in lncap human prostate cancer cells, the pparα agonist fenofibrate was able to mitigate against the androgen receptor agonist-evoked increase in tmprss2 expression (zhao et al., 2013) . following binding of the s protein to ace2, tmprss2 'primes' the spike protein to facilitate entry of the virus into the target cell (hoffmann et al., 2020; matsuyama et al., 2020) . pathogenesis of two strains of influenza virus has been reported to be markedly diminished by gene disruption of tmprss2 in mice (hatesuer et al., 2013; tarnow et al., 2014) , inferring that targeting this enzyme may have antiviral potential. interfering with the tmprss2:spike interaction using immunohistochemical analysis (bertram et al., 2012) and, very recently, using single nuclei and single cell rna sequencing (lukassen et al., 2020) , as yet not peer reviewed) of lung samples from otherwise healthy subjects, ace2 and tmprss2 were shown to be co-expressed in human bronchial epithelial cells, which could be a nexus for primary infection. a similar approach identified coexpression of ace2 and tmprss2 in nasal goblet cells, lung type ii pneumocytes and small intestine absorptive epithelia (ziegler et al., 2020) . in the same study, human primary nasal epithelial cells showed an upregulation in ace2 expression following 12 h incubation with interferon-α2 and interferon-γ, which suggests the potential for a feed-forward mechanism whereby the virus interacts preferentially with 'activated' cells to suppress the innate immune response (see below) (ziegler et al., 2020) . by analogy with the previous consideration of ace2 (above), alternatives to manipulate tmprss2 activity would be to provide endogenous substrates or synthetic inhibitors. however, the potential to make use of endogenous substrates seems limited. thus, although tmprss2 has been described to hydrolyse and activate the cell-surface g protein-coupled receptor proteinase-activated receptor 2 (wilson et al., 2005) , mice lacking tmprss2 failed to display an overt phenotype (kim et al., 2006) . as with ace2, there are no reports of licensed drugs which inhibit tmprss2 activity. cbz-ggraminomethylcoumarin has been described as a surrogate fluorogenic substrate suitable for highthroughput screening (paszti-gere et al., 2016) , although it is also a substrate for other proteinases, such as chymotrypsin. i432, a 3-amidinophenylalanine, has been described as a high affinity selective inhibitor (compound 92, ki of 0.9 nm) of tmprss2 (meyer et al., 2013) . in ipec-j2 pig jejunal epithelial cells, 10-50 µm i432 reduced tmprss2-derived product in cell media (paszti-gere et al., 2016) . in an investigation of sars-cov entry into hela cells expressing recombinant ace2 and tmprss2, a number of serine proteinase inhibitors (benzamidine, aprotinin, gabexate, tosyl lysyl chloromethyl ketone and camostat) were tested (mostly) at 10 µm for 30 min before exposure to pseudotyped viruses. only camostat was effective at reducing viral entry (kawase et al., 2012) , and further experiment suggested that 1 µm camostat was also effective, but only when tmprss2 was expressed. at 10 and 50 µm, camostat inhibited cell entry of the sars-cov and sars-cov-2 spike protein (hoffmann et al., 2020) . a direct inhibition of tmprss2 activity appears not to have been reported for camostat. potential ancillary proteins for virus entry -b 0 at1/slc6a19 and b 0 at3/slc6a18 the slc6 family of transporters includes the well-characterised net, sert and dat monoamine transporters, as well as the less well-exploited neutral amino acid transporter subfamily. b 0 at1/slc6a19 and b 0 at3/slc6a18 allow sodium-and chloride-dependent accumulation of neutral, aliphatic amino acids at the apical membranes of epithelial cells in the small intestine (b 0 at1/slc6a19) and kidney (b 0 at1/slc6a19 and b 0 at3/slc6a18) (for review, see broer and gether, 2012) . b 0 at3/slc6a18 is also highly expressed in the gi tract and gall bladder (protein atlas) and may play a role in the faecal:oral transmission of coronavirus (yeo et al., 2020) . the cell-surface expression of these neutral amino acid transporters is dependent on co-expression of ace2 (kowalczuk et al., 2008; fairweather et al., 2012) , aminopeptidase n (fairweather et al., 2012) or collectrin (an adaptor protein, which has high homology to the transmembrane region of ace2, camargo et al., 2009, link to uniprot) , in an apparently tissue-dependent manner (kuba et al., 2010) . a recent cryo-em structure suggested that ace2 and b 0 at1/slc6a19 form a heterodimer which pairs up through interfaces between the two ace2 partners (figure 1) , with the rbd of sars-cov-2 spike protein binding to the peptidase active site of ace2 suggesting that b 0 at1/slc6a19 may facilitate entry of the novel coronavirus. in the small intestine, absorptive epithelial cells were identified to co-express mrnas encoding for ace2 and tmprss2 (ziegler et al., 2020) . although it is not yet tested, it would be attractive to speculate that the colocalized expression of these targets may play a role in the faecal:oral transmission of coronavirus (yeo et al., 2020) . assays for b 0 at1/slc6a19 and b 0 at3/slc6a18 tend to be traditional accumulation of amino acids labelled with ionising or stable isotopes. recently, a primary screen using a membrane potentialsensitive fluorescence-based assay was used and followed up with a stable isotope accumulation assay to identify a novel inhibitor, cinromide, which exhibited modest potency (0.3-0.4 µm) for inhibiting b 0 at1/slc6a19 in cell-based assays (danthi et al., 2019) . once inside the cell, the endosomal cysteine proteases cathepsin b and cathepsin l have been described to process sars-cov (simmons et al., 2005 ) and this appears to be maintained for sars-cov-2 (hoffmann et al., 2020) although the significance of such intracellular proteinase activity is unclear. potent inhibitors for these two proteinases have been reported as pharmacological probes, but there are no licensed drugs identified to target them. following entry into the cell, many viruses accumulate in acidified lysosome-like vesicles, and so weak bases (including ammonium chloride and chloroquine) which target the lysosome have been used in vitro to target this mechanism. ammonium chloride (at 20 mm) has been described as a nonspecific inhibitor of viral replication in vitro, targeting viral uncoating (mizzen et al., 1985) and, at 50 mm, ammonium chloride inhibited cell entry of both sars-cov and ssars-cov-2 (hoffmann et al., 2020) . chloroquine was also observed to reduce infection of l cells by mouse hepatitis virus 3 (krzystyniak and dupuy, 1984) . following entry into the cell, the virus subverts nucleotide, protein, lipid and carbohydrate turnover of the host cell to produce multiple copies of itself. the viral rna is translated into multiple proteins to produce the replication machinery. as protein translation from the viral genome occurs, the two polyproteins are the first to be synthesised, with the two intrinsic proteases able to cleave the polyproteins into their constituent products. targetting the viral proteinases the low sequence similarities between mammalian and viral proteases has allowed successful drug targetting of viral diseases, including both hiv/aids and hcv/hepatitis c. the genome of sars-cov-2 contains two proteinases intrinsic to the polyproteins, plpro and 3clpro. the more n-terminally-located plpro is the larger (~2000 aa) of the two proteins (for review, see baez-santos et al., 2015; lei et al., 2018) , and, in sars-cov, is a membrane-associated, polyfunctional entity (harcourt et al., 2004) . sequence modelling of sars-cov-2 plpro suggested the presence of 6tm domains towards the c terminus, with the majority of the protein extending into the cell cytoplasm (angeletti et al., 2020) . in other coronaviruses, the enzyme is also capable of hydrolysing ubiquitin from protein substrates (barretto et al., 2005; ratia et al., 2006) , as well as removing the ubiquitin-like protein interferon-stimulated gene 15 (isg, link to uniprot) from isgconjugated proteins (yang et al., 2014) . using the orthologous proteinase from the mouse hepatitis coronavirus, analysis of three distinct structural domains suggested that the papain-like proteinase domain coincided with the deubiquitinylating and deisgylating functions (chen et al., 2015) . in sars-cov, the plpro also contains an adrp functional phosphatase domain directed at adp-ribose-1''-phosphates, although the functional significance of the hydrolase activity may be less impactful than the capacity to bind adp-ribose, at least for the enzyme from hcov-229e (putics et al., 2005) . this domain is thought to contribute to processing of the viral subgenomic rnas and the suppression of the innate immune system through reducing interferon production (lei et al., 2018) . investigating the peptidase activity of sars-cov plpro suggested a preference for larger proteins (ubiquitinated or isgylated) rather than simpler fluorescent-tagged oligopeptide substrates (lindner et al., 2005; lindner et al., 2007; baez-santos et al., 2014; ratia et al., 2014) making screening more complicated. the chymotrypsin-like proteinase, 3clpro the smaller proteinase from sars-cov-2 is 3clpro (sometimes called the main prote(in)ase, mpro). in silico docking models of sars-cov-2 3clpro has led to suggestions that particular existing antiviral agents, including velpatasvir and ledipasvir (licensed agents for treating hepatitis c when combined with sofosbuvir in the uk), should be screened for functional activity . a recent screen of ~10 000 compounds including approved drugs, candidate drugs and natural products used a substrate derived from the n-terminal autocleavage site of the sars-cov-2 3clpro which was modified (methylcoumarinylacetyl-avlqsgfr-lys(dnp)-lys-nh2) to allow a fret-based assay . the same substrate was used in a screen of the equivalent enzyme from the another coronavirus, hcov-hku1, which transferred to humans (zhao et al., 2008) . a number of inhibitors of the sars-cov 3clpro proteinase have been described (lu et al., 2006; yang et al., 2006; goetz et al., 2007) , without progressing into the clinic. recently, an in silico approach using orthologues of the sars-cov 3clpro from other coronaviruses and enteroviruses allowed production and testing in vitro of a series of α-ketoamides . one compound (11r) exhibited submicromolar potency against sars-cov 3clpro in a cell-free fret-based assay, and micromolar potency in a cell infection assay with sars-cov . in a preliminary (not yet peer-reviewed) report, the sars-cov-2 3clpro expressed in hek293 cells was found to interact with histone deacetylase 2 (hdac2) by affinity purification/mass spectrometry (gordon et al., 2020b) . a number of approved drugs target hdac2 in the treatment of various t cell lymphomas, including romidepsin, belinostat, and vorinostat with nanomolar potency (bradner et al., 2010) . targetting nucleotide turnover a relatively large proportion of the viral genome is inevitably devoted to nucleotide turnover. for sars-cov-2, this includes nsp7/nsp8/nsp12 as an rna-dependent rna polymerase; nsp13 as a helicase; nsp10/nsp14 as an 3'-to-5' exonuclease complex; nsp15 as an endoribonuclease and nsp16 as a 2'-o-ribose methyltransferase. remdesivir (currently in clinical trials to treat covid-19), is described as a non-selective inhibitor of multiple rna viruses, and has shown some efficacy in mers-cov and sars-cov infection of monkeys (de wit et al., 2020) . in in vitro investigations, the triphosphate analogue of remdesivir inhibited rna synthesis of mers-cov rna-dependent rna polymerase (primarily nsp8/nsp12 complexes derived from co-expression in insect cells of a construct containing nsp5, nsp7, nsp8 and nsp12) with an ic50 value of 32 nm when nucleotide levels were low, increasing to 690 nm at higher nucleotide concentrations (gordon et al., 2020a) . in silico modelling identified that remdesivir, as well as the approved antiviral drugs ribavirin, sofosbuvir and tenofovir could bind tightly to the active site of nsp12 from sars-cov-2, based on the crystal structure of sars-cov (elfiky, 2020) . however, ribavirin alone had no significant effect in a clinical trial with sars patients, although a combination of ribavirin with lopinavir-ritonavir and corticosterone had lower rating of ards and death (for review, see zumla et al., 2016) . in-depth analysis has not been completed with mers patients, although an ongoing phase 2 clinical trial for mers uses a combination therapy of lopinavir/ritonavir and interferon β1b (arabi et al., 2020) . nsp13 is a helicase, which enables unwinding of duplex rna. the exoribonuclease activity of nsp14 sets the coronaviruses apart (snijder et al., 2003) , as the enzyme is suggested to remove damaging mutations from the genome (eckerle et al., 2010; sevajol et al., 2014) . in other coronaviruses, the endoribonuclease nsp15 has some selectivity for hydrolysing polyu sequences (hackbart et al., 2020) . this enables the virus to delay or minimise initiation of the innate immune system by hydrolysing negative sense polyu nucleotides, which activate the mda5 system to evoke interferon production (discussed further below). nsp16 is a methyltransferase, which uses s-adenosyl-lmethionine as a co-substrate to assist in cap formation (decroly et al., 2008) . protein: protein interactions in recombinant expression in a preliminary (not yet peer reviewed) report, a series of tagged recombinant proteins from sars-cov-2 were expressed in hek293 cells and then protein partners were identified by affinity purification/mass spectrometry (gordon et al., 2020b) . for nsp12 (rna-dependent rna polymerase) and nsp14 (3'-5'-exonuclease) of sars-cov-2, interactions with receptor interacting protein kinase 1 (ripk1) and inosine monophosphate dehydrogenase 2 (impdh2), respectively, were identified. for these two targets, there are established approved drugs. thus, ponatinib, which is used to treat acute myelogenous leukemia or chronic myelogenous leukemia (philadelphia chromosome), targets multiple protein kinases, inhibiting ripk1 with an ic50 value of 12 nm (najjar et al., 2015) . mycophenolic acid and ribavirin are impdh2 inhibitors with ic50 values of 20 nm (nelson et al., 1990) and 1-3 µm (wittine et al., 2012) ranges, respectively, with clinical uses in organ transplantation and antiviral therapy, respectively. reservations about the use of ribavirin have already been noted above. mycophenolic acid as a monotherapy was examined in a mer-cov-infected non-human primate model, where the authors concluded it actually worsened the condition . nsp13 (helicase) and nsp15 (endoribonuclease) have been described to bind to centrosomeassociated protein 250 (cep250) and rnf41 (also known as nrdp1, link to uniprot), respectively, in a preliminary report of recombinant expression (gordon et al., 2020b) . cep250 is suggested to influence centrosome cohesion during interphase (de castro-miro et al., 2016) and to be elevated in peripheral t cell lymphomas (cooper et al., 2011) . the functional relevance of nsp13 interaction with cep250 is not yet clear. rnf41 is an e3 ubiquitin ligase, which polyubiquitinates myeloid differentiating primary response gene 88 (myd88, link to uniprot), an adaptor protein for toll-like receptors, which allows activation of tbk1 and irf3 (see below) and thereby increases type i interferon production (wang et al., 2009) . the lipid profile of viruses appears to be important in terms of viral entry into the cell, either as sites for anchoring or for endocytosis (for review, see heaton and randall, 2011; mazzon and mercer, 2014) . replication of sars-cov is reported to take place associated with the endoplasmic reticulum in 'replicative organelles' incorporating convoluted membranes and interconnected doublemembrane vesicles, inferring a capacity for the virus to induce extensive reorganization of intracellular phospholipid membranes (knoops et al., 2008) . three non-structural proteins from sars-cov with transmembrane domains, nsp3 plpro (see above), nsp4 and nsp6 when co-expressed in model cells prompted the formation of these double-membrane vesicles (angelini et al., 2013) , although it is unclear whether specific catalytic activities are necessary for this action. the lipidome of influenza virus (also a positive strand rna virus) consists of glycerophospholipids, sterols (mainly cholesterol) and sphingolipids, with sphingolipids and cholesterol enriched compared to the host cell membrane (gerl et al., 2012) , but there does not yet appear to be a parallel investigation of sars-cov. cytosolic phospholipase a2α, cpla2α, hydrolyses phospholipid to produce lysophospholipids and free fatty acids. using alphacoronavirus hcov-229e-infected huh-7 cells, inhibition of cpla2α using pyrrolidine-2 at higher concentrations (20 µm) evoked an inhibition of viral titre (muller et al., 2018) . arachidonoyl trifluoromethylketone, a non-selective inhibitor of multiple eicosanoid-metabolising enzymes including pla2 isoforms, also inhibited viral titres at higher concentrations (muller et al., 2018) . transmission electron microscopy suggested that cpla2α inhibition reduced the density of double-membrane vesicles (muller et al., 2018) . analysis of lipid metabolites indicated that hcov-229e-infected huh-7 cells showed increases in levels of ceramides, lysophospholipids and phosphatidylglycerols, with decreases in phosphatidic acids (muller et al., 2018) . 20 µm pyrrolidine-2 inhibited the elevations in lysophospholipids and phosphatidylglycerols, but not the ceramides. intriguingly, some selectivity of the involvement of pla2α was suggested as pyrrolidine-2 also displayed antiviral activities against other members of the coronaviridae (and togaviridae) families, while members of the picornaviridae family were not affected. although speculative, there is the possibility that some of the benefits of glucocorticoid administration in the clinic might be the up-regulation of annexins, and the subsequent binding and concealment of membrane phospholipid from further metabolism (for review, see lemmon, 2008) . while clearly some distance from a validated target, since phospholipids are an essential component of enveloped viral proliferation, targeting the host availability of key structural lipids, particularly sphingolipids, has been proposed to be a useful strategy in preventing propagation of enveloped human rna viruses, including influenza, hiv and hepatitis c (yager and konan, 2019) . currently, however, assays to screen inhibitors of cpla2α are relatively limited. targetting carbohydrate turnover given that a number of the viral proteins, including the two structural proteins spike and membrane, are glycoproteins, there is clearly a diversion of sugars from the host. it is unclear as yet, whether specific sugars are involved and whether specific host glycosyltransferases are involved in the processing of coronavirus glycoproteins and might, therefore, form further tractable targets for drug discovery. notably, in studies using site-directed mutagenesis of the spike protein from sars-cov, glycosylation was identified at three glutamine residues within the s1 region, with no loss of binding to ace2-expressing cell of mutated (non-glycosylated) fragments (chakraborti et al., 2005) . the e envelope protein the envelope proteins of sars-cov, hcov229e and mers are small (<100 aa) single transmembrane domain proteins (see figure 2 ) and constitute ion channels with selectivity for monovalent cations over monovalent anions (wilson et al., 2004; zhang et al., 2014) apparently forming homopentamers in model membranes (pervushin et al., 2009; surya et al., 2015) . infecting or transfecting the coronavirus e message into cells results in accumulation of protein in the golgi region (ruch and machamer, 2012) . conserved cys residues proximal to the transmembrane domain internally within the virus are palmitoylated (lopez et al., 2008) , a post-translational modification suggested to allow an internal inflexion point in the protein (ruch and machamer, 2012) . hexamethylene-amiloride has been described as an inhibitor of the hiv-1 virus vpu ion channel (ewart et al., 2002) and to reduce virus proliferation in human macrophages in culture . hexamethylene-amiloride, but not the clinically-used amiloride, inhibited the sars-cov envelope protein-associated ion channel activity when expressed in hek293 cells (pervushin et al., 2009) . amantadine has had multiple uses clinically, including in the therapy of parkinson's disease (for review, see vanle et al., 2018) . it has been used to treat influenza a infection through targeting the m2 ion channel (pinto et al., 1992; wang et al., 1993; holsinger et al., 1994) , although it is no longer recommended in the uk or us because of drug resistance (for review, see li et al., 2015) . amantadine at higher concentrations (100 µm) was found to inhibit the sars-cov e protein expressed in model membranes (torres et al., 2007) . sars-cov e protein was identified as being pro-apoptotic upon transfection into vero e6 monkey epithelial cells, where it localized to both plasma membrane and punctate cytoplasmic locations (chan et al., 2009) . indeed, the sars-cov e protein's ion channel function has been linked to calcium entry into endoplasmic reticulum/golgi membrane complexes and the subsequent activation of the nlrp3 inflammasome, leading to interleukin-β (il-1β) production (nieto-torres et al., 2015) . sirna targeting of the envelope protein of sars-cov reduced virus release in culture media, without altering n and p gene expression in frhk-4 monkey kidney epithelial cells (lu et al., 2006) . a similar observation was reported for the orf4a protein (derived from the orf4a gene) of hcov229e (zhang et al., 2014) . infecting mice with sars-cov in which the e protein ion channel function was disrupted showed unchanged viral proliferation but reduced il-1β and oedema levels in the lungs and better survival over 10 days post-infection (nieto-torres et al., 2014) . in a preliminary (as yet, unreviewed) report, the e protein of sars-cov-2 has been reported to interact with brd2/brd4 bet family bromodomain kinases when expressed in hek293 cells (gordon et al., 2020b) . jq1 and rvx208 are brd2/4 inhibitors with ic50 values with 40-120 and 50-1800 nm ranges, respectively. the membrane protein is usually regarded as the most abundant protein in the coronavirus envelope (see figure 2 ) and is of intermediate size in sars-cov-2 (222 aa). it is thought to assist in viral assembly by collating the other surface structural proteins (ruch and machamer, 2012) . the n protein is of moderate size in sars-cov-2 (419 aa), highly basic and binds the viral rna as a dimeric entity (fan et al., 2005) into nucleocapsids (see figure 2) , which afford protection for the viral genome, while also providing access for replication at appropriate times (for review, see mcbride et al., 2014) . in a preliminary (not yet peer reviewed) report, the n protein of sars-cov-2 was tagged and expressed in hek293 cells and then protein partners were identified by affinity purification/mass spectrometry (gordon et al., 2020b) . the n protein was suggested to interact with casein kinase 2 (ck2), la-related protein 1 (larp1, link to uniprot) and stress granule protein ras gtpase-activating protein-binding protein 1 (g3bp1, link to uniprot). ck2 phosphorylates a broad range of cellular targets, mostly in the nucleus, to regulate development and differentiation (for review, see gotz and montenarh, 2017) . although not in use clinically, two inhibitors are described to target ck2 with high affinity. silmitasertib is a ck2 inhibitor with an ic50 value of 1 nm (pierre et al., 2011) , while tmcb has a ki value of 21 nm (janeczko et al., 2012) . larp1 is an rna-binding protein, which releases rna when phosphorylated by mtorc1 (fonseca et al., 2015; hong et al., 2017) . larp1 seems to preferentially bind 5'-terminal oligopyrimidines with an as-yet unclear cellular role (philippe et al., 2020) . of the three targets suggested to associate with sars-cov-2 n phosphoprotein, g3bp1 seems a relevant focus for therapy against covid-19. g3bp1 regulates the innate immune response liu et al., 2019; wiser et al., 2019; yang et al., 2019) and stress granules reduce the replication of mers-cov (nakagawa et al., 2018) , so there is a potential for targetted drug discovery. interactions with the host innate immune system sars-cov produces proteins that interfere with interferon pathways (nsp1, nsp3, nsp16, orf3b, orf6, orf9b, m and n proteins, wong et al., 2016) and nlrp3 inflammasome activators (e, orf3a, orf8b) which are closely related to orthologues found in sars-cov-2. fung et al (2020) have recently reviewed the molecular aspects whereby sars-cov and, by inference, sars-cov-2, evades immune surveillance, activates the inflammasome and causes pyroptosis. other coronaviruses may give an indication as to how this is happening. hcov-229e rapidly kills dendritic cells, while monocytes are much more resistant. the rapid invasion of, and replication in, dendritic cells kills them within a few hours of infection (mesel-lemoine et al., 2012) . dendritic cells are sentinel cells in the respiratory tract, and plasmacytoid dendritic cells are a crucial antiviral defence via interferon production, and by modifying antibody production. thus, these viruses can impair control of viral dissemination and the formation of long-lasting immune memory. penetration of sars-cov-2 infection deep into the lungs, and eventually the alveolae, results in the 'cytokine storm' which accompanies pneumonia and lung fibrosis and is probably a major determinant of the necessity for intubation, and also mortality (shi et al., 2020b) . it is currently not known what specific factor/s differentiate the patients who develop this; although mortality among younger health workers may indicate that initial viral load may play a role. immunological agents which can prevent or control the 'cytokine storm' could therefore have a major effect on necessity to intubate and mortality. tocilizumab is a monoclonal antibody targeting interleukin-6 receptors, as a means to interfere with the effects of chronic autoimmune disorders such as rheumatoid arthritis. the chinese clinical trials registry has two studies that are designed to evaluate tocilizumab efficacy in patients with severe covid-19 pneumonia (registration numbers chictr2000029765 and chictr2000030442). similarly, anakinra, which is a slightly modified version of an endogenous antagonist of interleukin-1 receptors, is being investigated in clinical trials in multiple locations in patients with covid-19 infection (nct04324021, nct04330638 and nct02735707). it has been reported that in stage iii of covid-19, a critical point with a high viral load and severe respiratory involvement, lungs of patients appear with 'ground-glass' patches in ct scans, while autopsy reports indicate that the lungs are filled with a 'clear liquid jelly' (shi et al., 2020c; xu et al., 2020) , similar to an observation in drowning victims. on the hypothesis that inflammation-driven hyaluronan production (via hyaluronan synthase 2, has2, link to uniprot), and associated water retention may be critical; a recent study proposed therapy via administration of recombinant hyaluronidase or inhibitors of has2 (shi et al., 2020c) . the interaction between the virus and the innate immune system is complex and multifactorial, with temporal intricacies. it is beyond the scope of this review to identify all the multiple components and so we discuss here those pathways we consider most tractable. the positive sense rna of coronaviruses is translated to produce the replication machinery, which allows complementary negative sense rna to be synthesised, which itself is the template for the synthesis of positive strand rna. as a consequence, double-stranded rna is produced, which act as a pathogen-associated molecular pattern (pamp) targetting mda5 (interferon induced with helicase c domain i, also known as melanoma differentiation antigen 5, kato et al., 2006) from the rig-1-like receptor family of cytoplasmic pattern recognition receptors (for reviews, see schlee, 2013; bryant et al., 2015) . mda5 differs from rig-1 (dexd/h-box helicase 58, also known as retinoic acid-inducible gene 1) in recognising longer dsrna (kato et al., 2006; goubau et al., 2014) , and it has been proposed this differentiates the sensing of positive-stranded viruses by mda5 compared to negative strand virus sensing by rig-i (kato et al., 2006; goubau et al., 2013) . rig-1-like receptors have an nterminal caspase activation and recruitment domain (card), which shows ligand-dependent interaction with cards from other proteins, such as mitochondrial antiviral signalling protein (mavs, link to uniprot). mavs activates ikk family kinases, such as tank binding kinase (tbk1) and ikk-ε, leading to the phosphorylation of interferon regulatory factors, such as irf3 (link to uniprot) and irf7 (link to uniprot). this induces the transcription of type i interferon genes, such as interferon-β and ccl5 (also known as rantes) (doyle et al., 2002; fitzgerald et al., 2003; sharma et al., 2003) . mavs present in peroxisomes is also able to recruit short-acting, interferon-independent defense factors (dixit et al., 2010) . the orf9b protein from sars-cov has also been reported to target mitochondrial mavs to limit the interferon response, as well as triggering proteolysis of dynamin-like protein 1 (link to uniprot) thereby prompting the formation of mitochondria-associate autophagosomes claimed to create 'havoc' in energy production in infected cells (shi et al., 2014) . in a preliminary (as yet, unreviewed) report, orf9b of sars-cov-2 has been reported to interact with translocases of outer membrane 70 (tom70, link to uniprot) when expressed in hek293 cells (gordon et al., 2020b) tom70 activates mitochondrial irf3 and so this is a potential locus for pharmacological intervention, but as yet with no inhibitors described in the literature. a number of other coronavirus proteins have been identified to influence the irf3 pathway to restrict interferon production. this includes the mers-cov plpro proteinase (yang et al., 2014) , as well as the orf6 and nucleocapsid proteins from sars-cov (kopecky-bromberg et al., 2007) . the orf6 protein of sars-cov has also been described to reduce the activity of a series of karyopherindependent host transcription factors (sims et al., 2013) . karyopherin is an importin, which traffics proteins between the cytoplasm and the nucleus (for review, see kosyna and depping, 2018; . translocases of outer membrane 70 (tom70, link to uniprot) activates mitochondrial irf3 . the orf9b protein of sars-cov-2 has been reported to interact with tom70 when expressed in hek293 cells (gordon et al., 2020b) . clearly, the induction and suppression of interferon production are central to numerous human diseases and have been extensively studied; the 'trick' to treat covid-19 will be to identify a novel angle for therapeutic exploitation. working with sars-cov (not sars-cov-2), pfefferle and colleagues used yeast two-hybrid screens to identify interactions between the viral and human proteomes (pfefferle et al., 2011) . they identified an interesting interaction between viral nsp1 and a group of host peptidyl-prolyl cis-transisomerases (ppia, ppig, ppih and fkbp1a, fkbp1b), all of which modulate the calcineurin/nfat pathway important in immune activation (reviewed by hogan et al., 2003) . the nsp1 protein acts on these to activate nfat signalling and immune activation. cyclosporine a, an inhibitor of this pathway, has used for several decades to control transplant rejection and some autoimmune diseases and, in a simple in vitro assay, cyclosporine inhibited sars-cov transcription/replication in (non-immune-system) cells (pfefferle et al., 2011) . sars-cov-2 has an nsp1 protein closely related to that of sars-cov (dong et al., 2020; srinivasan et al., 2020) , though its effect on the nfat pathway seems not to have been reported. nevertheless, cyclosporine has been shown to inhibit sars-cov2 in an in vitro vero cell-based assay in a preliminary report (as yet not peer-reviewed, jeon et al., 2020) . it has therefore been suggested as a drug target (see, for example, li and de clercq, 2020) . it may seem paradoxical to suggest an inhibitor of immune activation as a treatment for viral disease, but for the subgroup of patients that might suffer cytokine storms (mehta et al., 2020) , the doubleaction might be useful. the orf3a protein of sars-cov appears to bind calcium in a cytoplasmic domain (minakshi et al., 2014) and to elicit a response from the innate immune system by enhancing the ubiquitination of apoptosis-associated speck-like protein containing a card (asc, link to uniprot), which in turn activates the nlrp3 inflammasome and caspase 1 (siu et al., 2019) . the potential for targetting asc and the nlrp3 inflammasome for therapeutic benefit in inflammatory conditions has recently been reviewed (mangan et al., 2018) , although there are no inhibitors in the clinic as yet. in sars-cov, the orf8a and orf8b genes became separated as the disease progressed by a 29nucleotide deletion (chinese sars molecular epidemiology consortium, 2004; oostra et al., 2007) . the orf8a gene of sars-cov encodes a short (31 aa, 1 tm, link to uniprot) protein, which forms a cation channel of predicted pentameric structure (chen et al., 2011) . in sars-cov-2 and a batderived coronavirus, in contrast to the sars-cov-2 genome, orf8 encodes a continuous 121 aa orf8 protein (cagliani et al., 2020) . given that sequence analysis of different strains of sars-cov-2 suggests that the orf8 locus displays only limited evidence of positive selection (cagliani et al., 2020) , it seems germane to investigate the profile of orf8 protein in more depth. sequence comparisons led to prediction of secondary structure composed of an α-helix and a β-sheet containing six strands , but there appears not to be any literature as to whether this entity is a functional ion channel. in a preliminary (as yet, unreviewed) report, the orf14 protein (link to uniprot) of sars-cov-2 has been reported to interact with nod-like receptor x1 (nlrx1), proteinase-activated receptor 2 (par2/f2rl1) and nedd4 family-interacting protein 2 (ndfip2, impdh2 link to uniprot), among other proteins of the iκb/nfκb pathway, when expressed in hek293 cells (gordon et al., 2020b) . at the moment, there are no approved drugs targeting par2, although az3451 (link to gtop) acts as a negative allosteric modulator with pic50 values of 5-23 nm (cheng et al., 2017) . there is a limited insight into the roles or potential exploitability of the remaining range of other viral proteins (nsp2; nsp9; nsp11, proteins orf3b; orf6; orf7a; orf7b; orf10). the spike glycoproteins in sars-cov and mers-cov are crucial for host specificity and jumping between species, e.g. from bats to humans (lu et al., 2015) , and from dromedary camels to humans (mers-cov) and also the recent cross-over of a hku2-related coronavirus to pigs as a swine acute diarrhoea syndrome (sads-cov) (zhou et al., 2018) . sads-cov appears to influence the innate immune system by reducing interferon-β production evoked through ips-1 and rig-i pathways, but not through irf3, tbk1 and ikkε (zhou et al., 2020) . ace2, as an anchoring point for the spike glycoprotein, is present throughout the animal kingdom, but small structural differences are critical for interaction with the spike protein (li et al., 2020b; luan et al., 2020) . key sequences of the spike protein from sars-cov and sars-cov-2 are responsible for binding to ace2. luan et al. (2020) found that the key residues in s protein, from sars-cov and sars-cov-2, recognised in ace2 from dog, cat, pangolin and circetidae mammals (simulated through homology modelling) were broadly similar. mouse ace2 is inefficient in prompting entry of both sars-cov and sars-cov-2 (fung et al., 2020) . cats and dogs suffer from their own specific coronavirus infections (e.g. canine respiratory coronavirus, feline coronavirus) without significant cross-over to humans. a preliminary (as yet lacking peer review) very recent report has suggested that cats and ferrets are sensitive to sars-cov-2, but dogs, pigs, chickens and ducks are much less sensitive (shi et al., 2020a) . ferrets, which have previously been used as models for respiratory tract infections,and retained the sars-cov-2 virus in the respiratory tract, while. shi et al. (2020) showed that the infection was transmitted between cats by aerosol (which may have implications for confinement); infected cats subsequently produced antibodies (shi et al., 2020a) . the syrian hamster has been used as a model for sars-cov (roberts et al., 2005; roberts et al., 2006; de wit et al., 2013) and studies with mice and syrian hamsters are ongoing with sars-cov-2. a preliminary report (as yet not peer-reviewed) suggests that monkeys can be infected and show signs of sickness similar to covid-19, producing antibodies which minimize the signs of subsequent infection (). in a small study where three juvenile (3-5 years old) and two mature (15 years old) rhesus macaques were infected intratracheally with the sars-cov-2 virus, all the monkeys showed symptoms of inflammation and interstitial pneumonia, with a greater apparent severity in the older animals . thus, while there is intensive research in animal models, a clearly validated model is still not apparent. given the similarities in the viruses and their symptoms, there is clearly a value to comparing the profiles of sufferers from the original sars and subsequent mers outbreaks with covid-19 to evaluate the risk factors associated with each event individually and collectively. a detailed consideration is beyond the scope of this review, but there are some obvious questions to ask (not in an order of priority). 1. what factor/s determine resistance to infection? it is apparent that many individuals who test positive for sars-cov-2 infection only experience 'mild' symptoms, others suffer a level of debilitation requiring hospitalization with limited supervision, and a third group require assisted breathing. there is preliminary evidence (as yet, not peer-reviewed) suggesting that people with type a blood might be more at risk of covid-19 than those with other blood types one potential explanation for the relatively high proportion of male victims has been suggested to be previous smoking history (cai, 2020; olds and kabbani, 2020; vardavas and nikitara, 2020) , clearly a general risk factor for many diseases. is there evidence from the sars and mers outbreaks to suggest a commonality of susceptibility? 6. what is the impact of contracting the virus on individuals with other underlying conditions? for example, what are the mechanism/s underlying why some sufferers of hypertension and/or diabetes might be at higher risk (https://www.immunopaedia.org.za/breakingnews/why-are-hypertension-and-diabetes-patients-at-high-risk-of-severe-covid-19/)? is there evidence that patients on ace inhibitors or angiotensin receptor blockers were at higher risk with sars-cov and mers-cov infections and, currently, for sars-cov-2 infection? 7. how will the evolution of the virus alter rates of infection and the severity of symptoms? some level of mutation is to be expected, and indeed has been noted for the sars-cov-2. at the moment, it is too early to identify the significance of any influence of these mutations on the course of covid-19. some of these questions are more tractable since the sars and mers outbreaks because of the strides being made in sophisticated molecular biological techniques (e.g. nextgen sequencing). an additional distinction compared to the previous outbreaks is the major increase in patient numbers associated with covid-19, allowing greater comparisons to be made in many more geographical locations. inevitably other questions will form as greater detail becomes available. this review has concentrated on the prevailing hypothesis that an essential first step in infection is sars-cov-2 binding to ace2 and for tmprss2 to prime the viral spike protein. we further hypothesise that both proteins must be expressed on a target cell for the virus to gain entry. tmpess2 has an extensive cellular expression profile, whereas ace2 is more limited and is usually at low levels, unless increased by risk factors such being sex, age, and smoking history, so is likely to be rate-limiting. other potential target proteins such as cathepsin l or b 0 at1 may also prove important. currently, although there are no drugs approved for the treatment of patients with covid-19, the pandemic has triggered a stampede into clinical trials with both approved and investigational agents. the pharmacological rationale for these trials is sometimes obscure, but there is a logic to focus on viral entry and replication, as well as limiting the host immune response. for the immediate term, the highest priority would be to investigate known antivirals to mitigate effects of covid-19. for the longer term, a vaccine (for review, see amanat and krammer, 2020) seems to hold the most promise to reduce covid-19 damage. there is also a role in the mid-term, however, for drug discovery conducted in mainstream pharmacology labs. the goal here would be an international co-ordinated approach to drug re-purposing; examining the spectrum of licensed drugs (likely to be less than 2000, varying dependent on jurisdictions). these would ideally be screened in a co-ordinated, blinded fashion in multiple labs simultaneously to account for any minor methodological differences. this requires the re-opening of screening and protein biosynthesis labs closed at the start of the pandemic, while ensuring that workers are kept safe. if one were to write a target product profile for a drug to treat covid-19, several parallel profiles can be identified. there are clear considerations, which may be identified as desirable pharmacodynamic, screening methodologies, drug metabolism and pharmacokinetic and formulation profiles. from a pharmacodynamic perspective, a priority would be to screen the proteinases identified in this review (ace2, tmprss2, adam17, cathepsin l, cathepsin b, plpro and 3clpro). a second parallel stream would assess inhibitors of the viral rna polymerase and endoribonuclease complexes, as well as the ion channel functions of the viral envelope (and potentially the orf8 protein). clearly there are multiple other targets, which might bear fruit, and so further studies should assess the tractability of b 0 at1/slc6a19, b 0 at3/slc6a18, impdh2 and has2. further, the molecular mechanism of action of ivermectin should be assessed, since it has recently been shown to inhibit in vitro sars-cov-2 replication (caly et al., 2020) . this agent is used clinically as an anthelmintic, probably through blocking invertebrate glutamate receptors although it also inhibits mammalian glycine receptors and acts as a positive allosteric modulator of other mammalian ligand-gated ion channels. from a screening aspect, biophysical and biochemical screens would probably take a matter of daysto-weeks. following mass availability of the recombinant proteins involved, the capacity for inhibition should be assessed using a library of already approved drugs. biophysical methods can be applied, such as surface plasmon resonance or biolayer interferometry, to monitor the affinity of interaction between host ace2 and viral spike glycoprotein in the presence of these agents, as well as the relevant proteins where multimerization is critical, such as the trimeric spike glycoprotein. assessing the remainder of the targets would likely adopt standard, fluorescent-based pharmacological methodologies. if the assay involves the use of viral proteins, the constructs should acknowledge the inevitable mutations which the viral genome has/will undergo. an overarching priority for the in vitro screening would be to recognise and replicate, as much as possible, relevant features of the virus and its lifecycle. this would include post-translational modifications of the viral proteins, such as glycosylation of the spike and membrane proteins. additionally, while the high throughput screens described above for identifying inhibitors associated with components of the viral entry system, such as ace2, should be confirmed in more translational assays, such as have been described for hiv cell entry in an automatable format (bradley et al., 2004) . a desirable element would also be to minimise adverse effects on the cardiovascular and respiratory system, given the high incidence of damage described associated with those systems (esler and esler, 2020; li et al., 2020a; lippi et al., 2020) . candidate drugs should also not increase activity of the il-6 (or any other pro-inflammatory cytokine) pathway to avoid provoking a cytokine storm. if a similar approach were taken to the ways in which targetted therapy is applied for certain types of cancer, there would be an increased benefit in a multimodal strategy. thus, in cancers where egf/egf receptors are involved, it is possible to target the ligand using chelating antibodies, to antagonise the receptor using blocking antibodies, to use specific antibodies to prevent dimerization of the receptor and to inhibit the catalytic activity of the receptor with small molecular inhibitors. it should be possible to reproduce this approach by simultaneously targetting several steps in the viral cycle (while naturally being cognisant of the potential for phenomena of drug:drug interactions, for instance in terms of convergent pathways of drug metabolism). this approach, enacted for the treatment of hepatitis c and human immunodeficiency viruses, for example, should also show benefit in reducing the capacity for drug-driven mutation in the enzyme. from a dmpk perspective, a beneficial profile for any agent would avoid drug:drug interactions by not converging on key metabolic enzymes and/or transporters. ideally, a once-daily treatment regimen would be optimal, but if more frequent administration were needed, there is likely to be good patient adherence, given the public response to 'spatial distancing'. from a formulation perspective, prophylactic use or for treatment of mild symptoms, an orally-administered or inhaled formulation would be appropriate. for more severe cases, where breathing is significantly impaired, an inhaled aerosolised version may be difficult to administer effectively; in this circumstance, a soluble version to be applied intravenously is likely to be useful. micro-organisms, such as viruses and bacteria, continue to evolve to evade our immune systems and previous pandemics contributed to the decline and fall of civilizations. there is a widespread hope that the current pandemic will be controlled by the rapid development of a safe and efficacious vaccine. clearly, there are major successes with vaccines targetting viral disease, but, to date no vaccine has been successfully produced to protect against human betacoronaviruses such as those causing sars and mers. on the contrary, multiple viral diseases have been successfully controlled by pharmacological agents. hiv-aids became more widespread in the last century and was associated with high morbidity and mortality. as a result of the discovery of novel pharmacological treatments, including specific antivirals, it is now a chronic condition and a cure has been effected in at least two individuals. similarly, the highly variable hepatitis c virus has resisted vaccines, but can be treated with direct antiviral agents allowing elimination of the virus in a very high proportion of those treated. this gives us hope that the roadmap outlined in this review may provide some relief from covid-19 (and indeed for viral threats yet to come). the novel virus uses angiotensin converting enzyme 2 (ace2) to attach to target cells, including epithelial and endothelial cells, particularly in the lungs. sars-cov-2 requires the camostat-sensitive serine proteinase tmprss2 to prime the spike protein for fusion and internalization. thereafter, host cellular processes are exploited for viral replication and release from the cell. ace2 is also expressed in high levels in the gi tract where it is associated with b 0 at1/slc6a19 that actively transports neutral amino acids across the apical membrane of epithelial cells. the serine proteinase adam17, present on cell surfaces, cleaves ace2 to release an ectodomain of ace2, including the active site, the circulation. this circulating form of ace2 may also bind sars-cov-2, but this complex is predicted not to internalize and therefore could be exploited as a beneficial viral decoy. recombinant ace2 (gsk2586881) has been tested in phase 2 clinical trials for the potential treatment of acute respiratory distress syndrome but it is not yet established if the compound will reduce viral load by acting as a decoy. catalytic cleavage of the androgenregulated tmprss2 protease results in its secretion by prostate and prostate cancer epithelia the concise guide to pharmacology 2019/20: introduction and other protein targets sars-cov-2 vaccines: status report coronavirus main proteinase (3cl pro ) structure: basis for design of anti-sars drugs covid-2019: the role of the nsp2 and nsp3 in its pathogenesis severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles treatment of middle east respiratory syndrome with a combination of lopinavir/ritonavir and interferon-1b (miracle trial): statistical analysis plan for a recursive two-stage group sequential randomized controlled trial catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the middle east respiratory syndrome coronavirus the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites mechanisms of coronavirus cell entry mediated by the viral spike protein no association of angiotensin-converting enzyme 2 gene (ace2) polymorphisms with essential hypertension this article is protected by copyright. all rights reserved serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke influenza and sars-coronavirus activating proteases tmprss2 and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts deployment of convalescent plasma for the prevention and treatment of covid-19 development and automation of a 384-well cell fusion assay to identify inhibitors of ccr5/cd4-mediated hiv virus entry chemical phylogenetics of histone deacetylases the solute carrier 6 family of transporters international union of basic and clinical pharmacology. xcvi. pattern recognition receptors in health and disease computational inference of selection underlying the evolution of the novel coronavirus, sars-cov-2 sex difference and smoking predisposition in patients with covid-19 the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro tissue-specific amino acid transporter partners ace2 and collectrin differentially interact with hartnup mutations the sars coronavirus s glycoprotein receptor binding domain: fine mapping and functional characterization the ion channel activity of the sarscoronavirus 3a protein is linked to its pro-apoptotic function treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan this article is protected by copyright. all rights reserved orf8a of sars-cov forms an ion channel: experiments and molecular dynamics simulations x-ray structural and functional studies of the three tandemly linked domains of non-structural protein 3 (nsp3) from murine hepatitis virus reveal conserved functions tmprss2, a serine protease expressed in the prostate on the apical surface of luminal epithelial cells and released into semen in prostasomes, is misregulated in prostate cancer cells prediction of the sars-cov-2 (2019-ncov) 3c-like protease (3cl (pro)) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates androgen receptor-activated enhancers simultaneously regulate oncogene tmprss2 and lncrna prcat38 in prostate cancer structural insight into allosteric modulation of protease-activated receptor 2 ace2 gene polymorphisms do not affect outcome of severe acute respiratory syndrome molecular evolution of the sars coronavirus during the course of the sars epidemic in china identification and characterization of peripheral t-cell lymphoma-associated serex antigens hosts and sources of endemic human coronaviruses angiotensin-converting enzyme 2 is an essential regulator of heart function origin and evolution of pathogenic coronaviruses identification and characterization of inhibitors of a neutral amino acid transporter, slc6a19, using two functional cell-based assays novel candidate genes and a wide spectrum of structural and point mutations responsible for inherited retinal dystrophies revealed by exome sequencing molecular interactions in the assembly of coronaviruses the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters sars and mers: recent insights into emerging coronaviruses prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'o)-methyltransferase activity the host's angiotensin-converting enzyme polymorphism may explain epidemiological findings in covid-19 infections human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? peroxisomes are signaling platforms for antiviral innate immunity a guideline for homology modeling of the proteins from newly discovered betacoronavirus, 2019 novel coronavirus (2019-ncov) a novel angiotensinconverting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1 irf3 mediates a tlr3/tlr4-specific antiviral gene program infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing ribavirin, remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov-2 rna dependent rna polymerase (rdrp): a molecular docking study can angiotensin receptor-blocking drugs perhaps be harmful in the covid-19 pandemic? amiloride derivatives block ion channel activity and enhancement of virus-like particle budding caused by hiv-1 protein vpu potential new anti-human immunodeficiency virus type 1 compounds depress virus replication in cultured human macrophages intestinal peptidases form functional complexes with the neutral amino acid transporter b(0)at1 the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties coronaviruses: an overview of their replication and pathogenesis effect of angiotensinconverting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensinconverting enzyme 2 ikk and tbk1 are essential components of the irf3 signaling pathway la-related protein 1 (larp1) represses terminal oligopyrimidine (top) mrna translation downstream of mtor complex 1 (mtorc1) a tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses quantitative analysis of the lipidomes of the influenza virus envelope and mdck cell apical membrane importance of cholesterol-rich membrane microdomains in the interaction of the s protein of sarscoronavirus with the cellular receptor angiotensin-converting enzyme 2 substrate specificity profiling and identification of a new class of inhibitor for the major protease of the sars coronavirus the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus a sars-cov-2-human protein-protein interaction map reveals drug targets and potential drug repurposing protein kinase ck2 in development and differentiation cytosolic sensing of viruses antiviral immunity via rig-imediated recognition of rna bearing 5'-diphosphates therapeutic dissolution of aberrant phases by nuclear-import receptors this article is protected by copyright. all rights reserved coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors modulation of tnf--converting enzyme by the spike protein of sars-cov and ace2 induces tnf-production and facilitates viral entry identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity tmprss2 is essential for influenza h1n1 virus pathogenesis in mice multifaceted roles for lipids in viral infection upregulation of hepatic angiotensinconverting enzyme 2 (ace2) and angiotensin-(1-7) levels in experimental biliary fibrosis structure-based identification of small-molecule angiotensin-converting enzyme 2 activators as novel antihypertensive agents sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor transcriptional regulation by calcium, calcineurin, and nfat influenza a virus m2 ion channel protein: a structure-function analysis larp1 functions as a molecular switch for mtorc1-mediated translation of an essential class of mrnas novel peptide inhibitors of angiotensin-converting enzyme 2 angiotensin-converting enzyme 2 protects from severe acute lung failure clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted ck2 and ck2' subunits differ in their sensitivity to 4,5,6,7-tetrabromo-and 4,5,6,7-tetraiodo-1h-benzimidazole derivatives acute cardiovascular effects of apelin in humans: potential role in patients with chronic heart failure identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs structure of m(pro) from covid-19 virus and discovery of its inhibitors differential roles of mda5 and rig-i helicases in the recognition of rna viruses simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome the stress granule protein g3bp1 binds viral dsrna and rig-i to enhance interferon- response phenotypic analysis of mice lacking the tmprss2-encoded protease interaction of sars and mers coronaviruses with the antiviral interferon response sarscoronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists controlling the gatekeeper: therapeutic targeting of nuclear transport a protein complex in the brush-border membrane explains a hartnup disorder allele entry of mouse hepatitis virus 3 into cells a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury trilogy of ace2: a peptidase in the reninangiotensin system, a sars receptor, and a partner for amino acid transporters this article is protected by copyright. all rights reserved prediction of off-target effects on angiotensin-converting enzyme 2 tumor necrosis factor- convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) inhibition of sars pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig nsp3 of coronaviruses: structures and functions of a large multi-domain protein membrane recognition by phospholipid-binding domains functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses angiotensin-converting enzyme 2 catalytic activity in human plasma is masked by an endogenous inhibitor prevalence and impact of cardiovascular metabolic diseases on covid-19 in china structure of sars coronavirus spike receptor-binding domain complexed with receptor therapeutic options for the 2019 novel coronavirus (2019-ncov) analysis of angiotensin-converting enzyme 2 (ace2) from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-ncov clinical implications of antiviral resistance in influenza angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus prostate-localized and androgenregulated expression of the membrane-bound serine protease tmprss2 the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme selectivity in isg15 and ubiquitin recognition by the sars coronavirus papain-like protease cardiac troponin i in patients with coronavirus disease 2019 (covid-19): evidence from a meta-analysis tom70 mediates activation of interferon regulatory factor 3 on mitochondria g3bp1 promotes dna binding and activation of cgas importance of conserved cysteine residues in the coronavirus envelope protein bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release spike protein recognition of mammalian ace2 predicts the host range and an optimized ace2 for sars-cov-2 infection the androgen-regulated type ii serine protease tmprss2 is differentially expressed and mislocalized in prostate adenocarcinoma the androgen-regulated protease tmprss2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis sars-cov-2 receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells targeting the nlrp3 inflammasome in inflammatory diseases the molecular biology of coronaviruses enhanced isolation of sars-cov-2 by tmprss2-expressing cells lipid interactions during virus entry and infection the coronavirus nucleocapsid is a multifunctional protein covid-19: consider cytokine storm syndromes and immunosuppression a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes identification of the first synthetic inhibitors of the type ii transmembrane serine protease tmprss2 suitable for inhibition of influenza virus activation host cell proteases: critical determinants of coronavirus tropism and pathogenesis the sars coronavirus 3a protein binds calcium in its cytoplasmic domain b38-cap is a bacteria-derived ace2-like enzyme that suppresses hypertension and cardiac dysfunction attenuation of murine coronavirus infection by ammonium chloride development of potent and selective phosphinic peptide inhibitors of angiotensin-converting enzyme 2 inhibition of cytosolic phospholipase a2alpha impairs an early step of coronavirus replication in cell culture the 2019 coronavirus (sars-cov-2) surface protein (spike) s1 receptor binding domain undergoes conformational change upon heparin binding structure guided design of potent and selective ponatinib-based hybrid inhibitors for ripk1 the endonucleolytic rna cleavage function of nsp1 of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines synthesis and immunosuppressive activity of some side-chain variants of mycophenolic acid severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp3 inflammasome enalapril attenuates downregulation of angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat is nicotine exposure linked to cardiopulmonary vulnerability to covid-19 in the general population? the 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8 who technical guidance human recombinant ace2 reduces the progression of diabetic nephropathy cloning of the tmprss2 gene, which encodes a novel serine protease with transmembrane, ldlra, and srcr domains and maps to 21q22 in vitro characterization of tmprss2 inhibition in ipec-j2 cells structure and inhibition of the sars coronavirus envelope protein ion channel genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion palmitoylation of the cysteinerich endodomain of the sars-coronavirus spike glycoprotein is important for spike-mediated cell fusion the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors global analysis of larp1 translation targets reveals tunable and dynamic features of 5' top motifs discovery and sar of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (cx-4945), the first clinical stage inhibitor of protein kinase ck2 for the treatment of cancer recent discovery and development of inhibitors targeting coronaviruses influenza virus m2 protein has ion channel activity adp-ribose-1"-monophosphatase: a conserved coronavirus enzyme that is dispensable for viral replication in tissue culture dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme structural basis for the ubiquitin-linkage specificity and deisgylating activity of sars-cov papain-like protease severe acute respiratory syndrome coronavirus infection of golden syrian hamsters therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters the coronavirus e protein: assembly and beyond distinct adam metalloproteinases regulate g protein-coupled receptor-induced cell proliferation and survival master sensors of pathogenic rna -rig-i like receptors insights into rna synthesis, capping, and proofreading mechanisms of sars-coronavirus triggering the interferon antiviral response through an ikk-related pathway sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 susceptibility of ferrets, cats, dogs, and different domestic animals to sars-coronavirus-2 covid-19 infection: the perspectives on immune responses inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage from sars to mers, thrusting coronaviruses into the spotlight structural genomics of sars-cov-2 indicates evolutionary conserved functional regions of viral proteins determining the enzymatic activity of angiotensin-converting enzyme 2 (ace2) in brain tissue and cerebrospinal fluid using a quenched fluorescent substrate mers coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels dhhc9 and gcp16 constitute a human protein fatty acyltransferase with specificity for h-and n-ras the role of type ii transmembrane serine protease-mediated signaling in cancer tmprss2 is a host factor that is essential for pneumotropism and pathogenicity of h7n9 influenza a virus in mice mechanisms and enzymes involved in sars coronavirus genome expression a human homolog of angiotensinconverting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase recurrent fusion of tmprss2 and ets transcription factor genes in prostate cancer conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of sars coronavirus envelope protein proteomics. tissue-based map of the human proteome nmda antagonists for treating the non-motor symptoms in parkinson's disease covid-19 and smoking: a systematic review of the evidence hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 structure, function, and antigenicity of the sars-cov-2 spike glycoprotein ion channel activity of influenza a virus m2 protein: characterization of the amantadine block the e3 ubiquitin ligase nrdp1 'preferentially' promotes tlr-mediated production of type i interferon sars coronavirus e protein forms cation-selective ion channels the membrane-anchored serine protease, tmprss2, activates par-2 in prostate cancer cells g3bp1 enhances cytoplasmic dna pattern recognition novel 1,2,4-triazole and imidazole derivatives of l-ascorbic and imino-ascorbic acid: synthesis, anti-hcv and antitumor activity evaluations a molecular arms race between host innate antiviral response and emerging human coronaviruses cryo-em structure of the 2019-ncov spike in the prefusion conformation a new coronavirus associated with human respiratory disease in china inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion this article is protected by copyright. all rights reserved measurement of angiotensin converting enzyme 2 activity in biological fluid (ace2) clinical relevance and role of neuronal at1 receptors in adam17-mediated ace2 shedding in neurogenic hypertension pathological findings of covid-19 associated with acute respiratory distress syndrome sphingolipids as potential therapeutic targets against enveloped human rna viruses structural basis for the recognition of sars-cov-2 by full-length human ace2 synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent sars coronavirus 3cl protease inhibitor g3bp1 inhibits rna virus replication by positively regulating rig-i-mediated cellular antiviral response proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease human aminopeptidase n is a receptor for human coronavirus 229e enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov-2 possible? age-related rhesus macaque models of covid-19 -ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication: structure-based design, synthesis, and activity assessment the orf4a protein of human coronavirus 229e functions as a viroporin that regulates viral production fenofibrate down-regulates the expressions of androgen receptor (ar) and ar target genes and induces oxidative stress in the prostate cancer cell line lncap relationship between the abo blood group and the covid-19 susceptibility structure of the main protease from a global infectious human coronavirus, hcov-hku1 angiotensin-converting enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin swine acute diarrhea syndrome coronavirus (sads-cov) antagonizes interferon-beta production via blocking ips-1 and rig-i a novel coronavirus from patients with pneumonia in china human coronavirus 229e papain-like proteases have overlapping specificities but distinct functions in viral replication sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues increased angiotensin-(1-7)-forming activity in failing human heart ventricles: evidence for upregulation of the angiotensinconverting enzyme homologue ace2 coronaviruses -drug discovery and therapeutic options this article is protected by copyright. all rights reserved.we would like to thank our colleagues in the universities of cambridge, edinburgh and nottingham for helpful discussions and wellcome trust 107715/z/15/z (to a.p.d., j.j.m.). this article is protected by copyright. all rights reserved. key: cord-345591-zwh1xj5u authors: al-dorzi, hasan m.; aldawood, abdulaziz s.; khan, raymond; baharoon, salim; alchin, john d.; matroud, amal a.; al johany, sameera m.; balkhy, hanan h.; arabi, yaseen m. title: the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study date: 2016-10-24 journal: ann intensive care doi: 10.1186/s13613-016-0203-z sha: doc_id: 345591 cord_uid: zwh1xj5u background: middle east respiratory syndrome coronavirus (mers-cov) has caused several hospital outbreaks, including a major outbreak at king abdulaziz medical city, a 940-bed tertiary-care hospital in riyadh, saudi arabia (august–september 2015). to learn from our experience, we described the critical care response to the outbreak. methods: this observational study was conducted at the intensive care department which covered 5 icus with 60 single-bedded rooms. we described qualitatively and, as applicable, quantitatively the response of intensive care services to the outbreak. the clinical course and outcomes of healthcare workers (hcws) who had mers were noted. results: sixty-three mers patients were admitted to 3 mers-designated icus during the outbreak (peak census = 27 patients on august 25, 2015, and the last new case on september 13, 2015). most patients had multiorgan failure. eight hcws had mers requiring icu admission (median stay = 28 days): seven developed acute respiratory distress syndrome, four were treated with prone positioning, four needed continuous renal replacement therapy and one had extracorporeal membrane oxygenation. the hospital mortality of icu mers patients was 63.4 % (0 % for the hcws). in response to the outbreak, the number of negative-pressure rooms was increased from 14 to 38 rooms in 3 mers-designated icus. patients were managed with a nurse-to-patient ratio of 1:0.8. infection prevention practices were intensified. as a surrogate, surface disinfectant and hand hygiene gel consumption increased by ~30 % and 17 n95 masks were used per patient/day on average. family visits were restricted to 2 h/day. although most icu staff expressed concerns about acquiring mers, all reported to work normally. during the outbreak, 27.0 % of nurses and 18.4 % of physicians working in the mers-designated icus reported upper respiratory symptoms, and were tested for mers-cov. only 2/196 (1.0 %) icu nurses and 1/80 (1.3 %) physician tested positive, had mild disease and recovered fully. the total sick leave duration was 138 days for nurses and 30 days for physicians. conclusions: our hospital outbreak of mers resulted in 63 patients requiring organ support and prolonged icu stay with a high mortality rate. the icu response required careful facility and staff management and proper infection control and prevention practices. the middle east respiratory syndrome (mers) coronavirus is a recently identified virus that is closely related to the severe acute respiratory syndrome coronavirus (sars-cov) [1] , causes severe hypoxemic respiratory failure with multiorgan failure and frequently requires admission to the intensive care unit (icu) [2, 3] . as of september 27, 2016 , the world health organization (who) reported 1806 laboratory-confirmed cases, including 643 related deaths [4] . the majority (~80 %) of mers cases occurred in saudi arabia [4] , where several hospital outbreaks happened [5, 6] with 45 % of all cases taking place within healthcare facilities [7] . the outbreak in the republic of korea illustrated the global threat of this disease. it started in may 2015 and resulted from a case with travel history to the arabian peninsula [4] . human-to-human transmission occurred to close contacts [family members, other patients and healthcare workers (hcws)] and led to 186 cases of mers-cov infections with 19 % fatality rate. in our hospital, king abdulaziz medical city-riyadh, an outbreak of mers disease occurred in august to september 2015 [4] , led to significant disruption of hospital functions and resulted in 130 mers cases [8] with 63 patients requiring icu admission. the outbreak was attributed to crowding, movement of infected but undiagnosed patients and breaches in infection prevention and control practices [4] . details of the hospital outbreak have been published elsewhere [8] . most of the medical literature on mers has focused on describing the characteristics and outcomes of affected patients. preparedness and the response of the healthcare system at its different levels are crucial to contain this disease and manage its associated outbreaks. nevertheless, little is published about how the healthcare system responded to the disease and hospital outbreaks. the objective of this study was to describe the response of the icu to a hospital mers outbreak, the associated changes in its workflow and the impact on its hcws. this study was an observational study conducted at the intensive care department of king abdulaziz medical city, a 940-bed tertiary-care referral hospital in riyadh, saudi arabia, that was accredited by the joint commission international. the hospital had an infection prevention and control department. the intensive care department covered 5 units: an 8-bed trauma icu (unit a), a 21-bed medical-surgical intensive care unit (unit b), a 9-bed surgical icu (unit c), an 8-bed neurologic icu (unit d) and a 14-bed stepdown unit (unit e). the department also provided coverage to boarding patients in the 15-bed resuscitation area in the emergency department (ed). the hospital had also an 8-bed burn icu. the icus were operated as closed units with 24-h, 7-day onsite coverage by boardcertified critical care intensivists [9] . normally, 5 medical teams covered the units during the day with each team consisting of one intensivist consultant, one registrar/ fellow and 1-3 residents. the nurse-to-patient ratio in all the icus was mostly 1:1. one certified respiratory therapist covered a maximum of six ventilated patients. additionally, the department had a rapid response team, which covered the hospital wards and was activated according to predefined criteria and is covered by a sixth separate team [10] . the department has had several ongoing quality improvement projects and indicators. infection prevention and control practices, such as hand hygiene and the ventilator care bundle, were monitored by multidisciplinary icu teams and the infection prevention and control department [11] . for intubated patients, ventilator care was provided by specialized respiratory therapists and included using closed endotracheal suctioning systems which were changed every 72 h or as clinically indicated [12] , changing the ventilator circuits in between patients or if they became soiled or damaged [12] , and using heat and moisture exchangers which were changed every 7 days or when visibly soiled [12] . due to the high prevalence of multidrug-resistant organisms and prior cases of influenza and mers infection in the hospital, droplet precautions were applied to all icu patients since february 2012 [11] . sporadic cases of mers cases have been managed in our icu since february 2013. the characteristics, management and outcomes of the initial mers cases managed in our unit were described previously [3] . all hcws were required to undergo fit testing for the n95 respirators (table 1 ). an infectious disease epidemic plan (idep) was initially released in may 2014 and was revised in march 2015. table 1 describes selected plan elements. according to the idep, one unit (unit a) was designated as the primary receiving unit for mers patients, because of its geographic location being away from main hospital traffic and because 7 of its 8 rooms were negative-pressure airborne infection isolation rooms (aiir). the plan was not explicit about which units would be used if the number of cases exceeded the capacity of this unit. however, unit b had 4 negative-pressure rooms. in our hospital, mers was suspected based on clinical presentation and confirmed by laboratory testing as recommended by the who and the saudi ministry of health [4, 13] . in our icu, the workup of patients having lower respiratory tract infections was standardized to include bacterial gram stain and culture, mers-cov polymerase chain reaction (pcr), h1n1 pcr, bacterial and viral multiplex pcr on respiratory samples, mycoplasma, chlamydia and legionella serology and, if suspected, tuberculosis workup. the respiratory samples were routinely obtained by blind deep tracheal aspirate in intubated patients. in the hospital biosafety level 2 laboratory, mers-cov screening was performed by real-time reverse-transcription (rrt) pcr on respiratory samples by checking for the upstream e protein genome (roche modular dx coronavirus) and infection confirmation by detecting the open reading frame 1a genome (mers-cov kit from tib molbiol) [14] . laboratory workers were fit-tested and applied n95 respirators while handing respiratory samples. positive samples were sent to the saudi ministry of health reference laboratory for confirmation. viral culture was not performed as biosafety level 3 is needed. we noted mers cases admitted to the icu from july 1 to october 21, 2015, and collected data on the clinical characteristics, management and outcomes of the affected hcws. we also obtained data about the rrt-pcr performed for icu patients. we identified the physicians and nurses who reported sick leave for respiratory illnesses. as surrogate for infection control practices, we obtained data on the related consumables for units a and b before and during the outbreak (aprilseptember 30, 2015) . we also collected qualitatively our own observations and those of other hcws on the icu response during the outbreak using interviews and open discussions. descriptive data were presented as means and standard deviations or frequencies and percentages, as appropriate. the infection prevention and control consumables were compared in the 4 months before (april to july) and the 2 months during (august and september) the outbreak. the plan will be activated by the chair of the outbreak response committee based on the phase definition phase i 0-5 cases of suspected or confirmed in the hospital phase ii 6-30 cases of suspected or confirmed in the hospital phase iii >30 cases of suspected or confirmed in the hospital phase i confirmed mers-cov cases requiring intubation will be assigned a negative-pressure room and cohorted in one icu confirmed cases that have been diagnosed with mers-cov in any icu other than the trauma icu (unit a), shall be transferred to the trauma icu (unit a) as soon as possible. phase ii all mers-cov patients will be cohorted in one unit. if the number of patients exceeds its capacity, then other units are identified to receive the additional cases closure of all nonessential hospital functions phase i all services run without interruptions except for certain precautions for mers patients phase ii all elective surgery shall be canceled to free more icu beds phase iii all elective cardiac surgery shall be canceled outpatient clinic visits shall be limited to urgent visits only healthcare worker (hcw) management all hcws shall be aware of 1. relevant infection prevention and control policies and procedures 2. their annual influenza immunization status. if not vaccinated, please contact the employee health clinic to arrange for an appointment 3. their n95 fit check/test status. if have not been fit-tested, please contact the employee health clinic to arrange for an appointment hcws exposed to a confirmed mers-cov case shall be assessed according to a predetermined protocol hcws requiring isolation at home and happen to share a room with another hcw will be provided a room in the designated accommodation for isolation till cleared by the infection prevention and control department an infection prevention and control officer is available 24 h per day, 7 days per week in july 2015, five cases of acute respiratory failure were referred to the icu team from the ed and wards and were diagnosed to have mers pneumonia. as the number of mers patients increased, the idep was activated on august 2, and included strict implementation of infection control measures, including airborne and contact isolation for confirmed and probable mers cases, and droplet and contact isolation for suspected cases [8] . on august 18, phase iii of the idep was activated (table 1) , which included ed closure, elective surgical procedure cancelation and outpatient clinic suspension [8] . meanwhile, the icu maintained full operations. figure 1 suspected mers cases had rrt-pcr on nasopharyngeal swabs in nonintubated patients and on deep tracheal aspirates in intubated patients. fiberoptic bronchoscopy was not performed for the diagnostic workup of mers or ventilator-associated pneumonia. repeated testing was frequently needed to make the diagnosis. among our 63 critically ill mers patients, the initial mers-cov testing was performed on nasopharyngeal swabs in 29 and deep tracheal aspirates in the rest (n = 34). in the first sample, mers-cov was detected in only 6/29 (20.7 %) nasopharyngeal samples and 26/34 (76.5 %) deep tracheal aspirates. after initial negative or equivocal nasopharyngeal swabs (n = 23), a second nasopharyngeal swab was performed in 16 patients (positive in 37.5 %) and deep tracheal aspirate in 5 (positive in 100 %). our microbiology laboratory extended its working hours and prioritized testing samples coming from the icu and the rest of the hospital. the number of mers-cov tests performed on icu patients went up from an average of 1.5 before to 5.3 per day during the outbreak with a maximum of 19 tests on september 4, 2015. in patients with suspected mers and negative rrt-pcr, the test was repeated after 1-2 days. there was a general consensus among our intensivists that three negative lower respiratory samples and low clinical pretest probability were needed to exclude mers-cov infection. for patients with confirmed mers, the test was repeated twice weekly until 3 consecutive tests were negative. the mean age of the 63 patients was 57.9 ± 18.6 years with the majority (69.8 %) being males. eight hospital workers required icu admission after acquiring mers. table 2 summarizes their characteristics. half of them did not have direct contact with patients. one of them was a pregnant nurse that worked in the ed. all but one required intubation. the medical management of mers patients was largely supportive. most (80.9 %) mers patients required endotracheal intubation, which was performed by the most experienced available physician with airborne precautions. lung-protective ventilation was implemented for acute respiratory distress syndrome with tidal volumes (4-6 ml/kg of ideal body weight). to reduce the airborne generating procedures, we discouraged the use of noninvasive ventilation for suspected mers cases. nevertheless, it was used in the initial management of 12 (19.0 %) patients. these patients were either suspected to have concomitant cardiogenic pulmonary edema or had milder disease. intubation was needed for 10 patients. highflow oxygen therapy was not used as it was unavailable. when needed, bronchodilators were used via metered dose inhalers rather than nebulizers. most (58.7 %) mers patients required vasopressors. renal replacement therapy was provided for 37 (58.7 %) patients. for the hospital workers acquiring mers (n = 8), cisatracurium infusion was used in 7 (87.5 %), early prone positioning in 4 (50 %), continuous renal replacement therapy in 4 (50 %) and extracorporeal membrane oxygenation in 1 ( table 2) . none of the patients received ribavirin, interferon therapy or high-dose steroids. the hospital mortality of mers patients was 63.4 % with all deaths occurring in the icu. all hospital workers who had mers survived and were discharged to home. the icu and hospital length of stay were prolonged (15.0 ± 17.0 and 30.6 ± 23.1 days, respectively). during the outbreak, our hospital established a command center, which met twice daily, and oversaw all interventions in accordance to the idep. the intensive care department chairman was a member of the command center and presented daily the number of suspected and confirmed cases in the icu, bed and staff management issues and any challenges. the department chairman attended all morning handover meetings in the icu where he received input from the icu teams and provided feedback from the command center. the hospital provided an intranet page that had educational material on mers, mers management guidelines and proper infection control practices. the page was regularly updated. additionally, the hospital frequently informed staff about the mers outbreak status through emails. staff could communicate with the command center regarding any outbreak-related concern or question. the intensive care department communicated with the medical staff about the saudi ministry of health and who practice guidelines. before and during the outbreak, the who interim guidance for the management of suspected and confirmed mers-cov infection [15] was circulated to our icu staff. moreover, a letter expressing gratitude and encouragement was sent from the department chairman to all hcws. family visiting to mers patients was restricted from an open visiting policy to 2 h per day during the evening with visitors not allowed to enter patient rooms. visiting outside these hours was allowed in selected cases if the clinical condition required. to update the patients' families, the icu consultant contacted and updated the next of kin by phone every day and addressed the family concerns. a nurse was assigned to screen all staff and visitors entering each unit by asking for symptoms of acute respiratory infection and measuring temperature. staff and family members with symptoms of acute respiratory illness or fever were not allowed to enter. the initial mers cases were admitted to the designated mers unit (unit a). as the number of patients increased, the icu leadership identified other icus as potential placement units. the hospital clinical engineers converted a total of 24 standard rooms in unit b and unit c to negative-pressure rooms by increasing air exhaust more than supply by 50 cubic feet per minute. as the number of suspected and confirmed mers patients increased, unit b and then unit c were used. as the number of our mers patients increased beyond unit a capacity, patients without mers were transferred to other units or hospitals to increase bed capacity. the old pediatric icu, which was recently vacated in june 2015 after the opening of a new pediatric hospital, was used to care for stable and long-term patients. during the outbreak, 13 and 7 patients from units a and b, respectively, were transferred to the other icus (units c-e and the old pediatric icu), and 1 patient to another hospital. the care for mers patients was demanding. for example, 4 of the 8 affected hcws required prone positioning for the management of acute respiratory distress syndrome ( table 2 ). also 4 of them required continuous table 2) . the care was also associated with significant exposure risk. this can be reflected in the duration of mechanical ventilation for the hcws who required intubation (7-57 days) and length of icu stay (7-63 days) ( table 2) . during the outbreak, the nurse-to-patient ratio was mostly 1:1 except for one patient on ecmo (2:1). additionally, 1-2 additional nurses were deployed in each unit to assist in procedures such as prone positioning and to monitor and correct infection control practices. in unit a, for instance, the nurse-to-patient ratio was 1:1.2 before the outbreak and became 1:0.8 during the outbreak. we restricted medical management to the attending physicians, senior registrars and critical care fellows. rotating residents were not allowed to work in the icu during the outbreak. entry of nonclinical staff, such as research coordinators, was restricted and the ongoing clinical trials were put on hold, except for mers studies, to reduce staff exposure. during the outbreak, concerns among icu staff were raised about acquiring mers and transmitting the virus to their families; a concern that was substantiated by seeing hospital workers infected and developing critical illness. however, many felt privileged to be part icu team managing the outbreak and taking care of mers patients; none refused to report to work as per schedule. two pregnant icu nurses were redeployed to low-risk units. staff members who developed fever, respiratory symptoms or gastrointestinal illness were asked not to present to work, but rather to report to the ed or the employee health clinic depending on their illness severity. of the 152 bedside nurses covering units a and b, 41 (27.0 %) nurses had symptoms of acute respiratory infection during the outbreak and consequently had nasopharyngeal swabs obtained for mers-cov; all tested negative. their total sick leave duration was 138 days (range: 1-15 days per nurse). in comparison, in the 2 months before the outbreak, 26 (17.1 %) nurses had sick leaves for a total of 33 days (range 1-2 days per nurse). of the 49 nonresident icu physicians, 9 (18.4 %) physicians received sick leave for a total of 30 days (range 3-4 days per physician). in unit c, two nurses (1.0 %) of 196 nurses who worked in mers units (units a, b and c) and one rotating resident (1.3 %) out of 80 physicians covering the icus tested positive for mers-cov. the resident and one of the nurses were symptomatic and required hospitalization in a mers ward for approximately 1 week and both recovered fully. in february 2012, long before the mers outbreak, droplet precautions had been added to the standard precautions for all icu patients, mainly to prevent the transmission of influenza. during the outbreak, airborne precautions were added for all confirmed and suspected mers cases. although all staff were required to be fit-tested for the n95 respirators before the outbreak (table 1) , we discovered that many icu staff were not tested. during the outbreak, a clinic was emergently opened to fit test hcws and the results were documented. specific policies and procedures were developed or updated for donning and doffing personal protective equipment (ppe). related visual instructions were provided inside each icu room. outside patient rooms, carts containing ppe were organized to facilitate donning in the correct sequence. during the outbreak, additional training on hand hygiene techniques and ppe application was provided. housekeepers were also retrained on proper cleaning techniques and ppe use. the intensive care department worked closely with the infection prevention and control department on all aspects of infection control. the implementation of such infection control measures required having adequate ppe supplies, such as respirators, goggles, face shields and gowns. table 3 describes the consumption of surface disinfectants, antiseptic alcohol for hand hygiene, n95 masks and other ppe before and during the mers-cov outbreak. during the outbreak, the consumption of detergent surface disinfectant and ethyl alcohol for alcohol-based hand rub increased by almost 30 % and the use of n95 masks increased by >15 times compared to the preceding 4 months. the number of examination gowns per patient per day decreased during the outbreak probably due to staff avoiding unnecessary exposure. twenty-four powered air-purifying respirators were made available to staff who failed the n95 respirator fit test. they were used by 8 physicians, 7 nurses and 14 respiratory therapists. training sessions on their application were conducted. in this report, we described how the icu responded to a mers-cov outbreak at a tertiary-care hospital. the outbreak led to 63 mers patients requiring prolonged icu care and most received invasive mechanical ventilation, vasopressors and renal replacement therapy. the overall mortality was 63 %, but all affected hospital workers survived. the outbreak management included almost tripling the icu capacity of negative-pressure rooms and intensifying infection prevention and control practices. even though icu staff had significant exposure risk, very small number acquired mers-cov. response to incidents such as an infectious hospital outbreak requires a robust hospital-wide command and control structure that is able to make rapid informed decisions across an institution. as it was the case in our hospital, the control of outbreak may require major interventions such as closing the ed, suspending elective surgeries, preventing inter-facility patient transfers, canceling ambulatory clinics and outpatient diagnostic procedures, preventing hospital staff from working at other institutions and restricting hospital visitors [16] . our hospital had a preexisting idep, which facilitated managing and containing the mers-cov outbreak. such a plan is mandatory for every hospital. mers infection is associated with several challenges. its presenting symptoms overlap with those of other severe acute respiratory illnesses and include fever (71 %), cough (68 %), dyspnea (66 %) and diarrhea (32 %) [17] , often in older adults with preexisting chronic comorbidities [17, 18] . common laboratory abnormalities include leukopenia, lymphocytopenia, thrombocytopenia and elevated serum creatinine, lactate dehydrogenase, and liver enzymes [17] . the initial chest radiographs show minimal abnormality to extensive bilateral infiltrates [17] . unfortunately, many frontline physicians are unfamiliar with the mers case definition, probably because cases are sporadic, leading to delayed or even missed diagnosis. delayed recognition may lead to exposing many other patients, visitors and hcws to the infection as it was the case in our hospital. mers-cov nosocomial transmission is thought to be via respiratory droplets, and contact spread is suspected [19] . in korea, the delayed diagnosis of an infected traveler to the arabian peninsula led to 186 mers cases and resulted in intraand inter-hospital transmission [20] . in saudi arabia, 45 % of mers cases were acquired in the healthcare setting with 12 % of all cases being hcws [7] . therefore, taking a detailed history, knowing mers case definitions, standardizing pneumonia workup, obtaining lower respiratory tract specimens [21] and implementing droplet isolation for suspected cases are crucial interventions to break the transmission chain in the healthcare setting. admitting mers patients in single-bedded negativepressure rooms and cohorting them in selected units are recommended to facilitate providing care and monitoring [22] . during an outbreak, clinical engineering should have expedient plans to convert standard rooms. retrofitting the rooms with externally exhausted hepa filters may be a solution [16] . outbreaks can lead to significant increase in the need for icu beds, but may simultaneously reduce the available beds. the sars outbreak in toronto led to 10-day closures of 35 icu beds, which represented 38 % of the tertiary-care university medical-surgical icu beds in toronto [16] . hence, hospitals should always have plans to augment icu bed capacity, such as by transforming general wards. the ability of any hospital to deal with an infectious outbreak is decided by the availability of icu beds [23] . caring for mers patients represents a substantial exposure risk for icu staff because of three reasons: high exposure dose, long daily contact hours and prolonged icu stay with viral shedding. mers-cov patients requiring icu admission have higher viral load than other mers patients [24] . aerosol-generating procedures, such as noninvasive ventilation, suctioning and bronchoscopy, add to the exposure and transmission risk [25] . extended bedside care is needed for mers patients due to the requirement of organ support such as mechanical ventilation, vasopressor therapy, continuous renal replacement therapy, prone positioning and extracorporeal membrane oxygenation [2, 3] . in this study, we observed an increase in the nurse-to-patient ratio from 1:1.2 to approximately 1:0.8 during the outbreak. the sars epidemic was also associated with increases in the nurse-topatient ratio [26] . stay in the icu can last for weeks [3] , which we observed. additionally, mers-cov shedding can be prolonged and may last for >30 days [27] . the exposure risk to mers-cov can exert significant psychosocial stress on hcws. death has occurred in young hcws who acquired mers-cov infection [28] , which adds to this fear. during the sars outbreak in toronto, a survey of hcws found that >60 % of respondents reported sars-related concern for their own or their family's health [29] . moreover, 29 % of respondents had probable emotional distress [29] . during our outbreak, many icu staff expressed concerns about acquiring mers. staff safety should be a primary goal in a hospital infectious outbreak. pregnant and immunocompromised staff should be redeployed to lower-risk areas [30] , which we did. proper exposure management should be pre-planned, which was determined in our idep. n95 respirator fit testing should be performed at hiring of new staff and done for all other staff before any outbreak. although fit testing was required for all staff, many did not have fit testing done. however, in response to the outbreak, fit testing was performed to all staff and powered air-purifying respirators were provided to those who failed fit testing. strict infection prevention and control practices should be implemented and audited. this was performed in our units. repetitive training is recommended [31] . despite intensive infection prevention practices, 3 of our icu staff had mers-cov infection during the outbreak likely due to suboptimal ppe use while intubating yet undiagnosed patients. mers management was supportive and largely adhered to the who recommendations [15] . it included intubation, early prone positioning and neuromuscular blockade for moderate-to-severe acute respiratory distress syndrome as these interventions have been shown to improve the outcomes of ards patients [32, 33] . although noninvasive ventilation use was discouraged, it was used in 12 patients. the who considers noninvasive ventilation an option in selected mers cases [15] . it should be used as a short trial without delaying intubation if unsuccessful [15] . moreover, high-flow oxygen by nasal cannula may be another option [15, 34] ; however, the associated transmission risk as a result of aerosol generation is unknown. systematic corticosteroids, ribavirin and interferon were avoided as they have no proven benefit [15, 35] . our mers patients had high mortality (63 %). the previously reported mortality of mers patients who had critical illness ranged from 58 to 84 % [2, 3, 18] . none of our hcws who developed mers died, which was gratifying to our staff. our mers-cov hospital outbreak stressed our system to unprecedented limits. we learned many lessons from it ( table 4 ). the successful management of outbreak required integrating icu functions with the hospitalwide plans, having preparedness plans, implementing proper infection control practices and managing staffing and staff exposure. every hospital should have an infectious disease epidemic plan that should govern the response to an infectious disease outbreak. the response should cover organizing patient services, implementing infection control, managing employee exposure and communicating with national health services and with hospital staff hospital leaders should be prepared to increase the capacity of negative-pressure airborne infection isolation rooms in the case of an infectious disease outbreak all healthcare workers should receive training on proper hand hygiene and personal protective equipment application. hand hygiene and personal protective equipment practices should be monitored. education should be repeated periodically all healthcare workers should be fit-tested for n95 respirators on hire with the result documented in their files. periodic audit of this requirement should be done hospitals should make plans to acutely increase personal protective equipment supplies as consumption increases tremendously during an infectious disease outbreak hospital and icu leaders should have plans to cover healthcare workers who are exposed or become symptomatic to avoid potential staff shortage severe acute respiratory syndrome vs. the middle east respiratory syndrome clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities the national command control center; ministry of health-kingdom of saudi arabia. mers-cov in ksa notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia weekend and weeknight admissions have the same outcome of weekday admissions to an intensive care unit with onsite intensivist coverage impact of an intensivist-led multidisciplinary extended rapid response team on hospital-wide cardiopulmonary arrests and mortality a multifaceted approach to improve hand hygiene practices in the adult intensive care unit of a tertiary-care center comprehensive evidence-based clinical practice guidelines for ventilator-associated pneumonia: diagnosis and treatment ministry of health kingdom of saudi arabia. infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection assays for laboratory confirmation of novel human coronavirus (hcovemc) infections clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected-interim guidance identification and containment of an outbreak of sars in a community hospital middle east respiratory syndrome: knowledge to date middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions mers outbreak in korea: hospital-to-hospital transmission an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus hospital preparedness and sars association of higher mers-cov virus load with severe disease and death, saudi arabia aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review clinical review: sars-lessons in disaster management middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications middle east respiratory syndrome coronavirus infections in health care workers psychosocial effects of sars on hospital staff: survey of a large tertiary care institution sars and hospital priority setting: a qualitative case study and evaluation infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease prone positioning in severe acute respiratory distress syndrome neuromuscular blockers in early acute respiratory distress syndrome high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study none. the authors declare that they have no competing interests. written informed consent was not obtained for publication of these data. the institutional review board of the ministry of national guard health affairs approved the retrospective clinical data collection on mers patients, and no consents were required. abbreviations ed: emergency department; hcw: healthcare worker; icu: intensive care unit; idep: infectious disease epidemic plan; mers: middle east respiratory syndrome; ppe: personal protective equipment; rrt-pcr: real-time reversetranscription polymerase chain reaction; who: world health organization. hmd was involved in conception and design, data collection, statistical analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content and approval of the final version to be published. asa contributed to the analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. rk helped in data collection, analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. sb contributed to the analysis and interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. jda helped in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. aam was involved in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. smj helped in data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. hhb contributed to data collection, interpretation of data, critical revision of the manuscript for important intellectual content and approval of the final version to be published. yma helped in conception and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content and approval of the final version to be published. all authors read and approved the final manuscript.author details 1 icu2 and ticu, intensive care department, king abdulaziz medical city, king key: cord-319707-j8y9gt2o authors: kato, verstrepen; laure, baisier; harald, de cauwer title: neurological manifestations of covid-19, sars and mers date: 2020-06-19 journal: acta neurol belg doi: 10.1007/s13760-020-01412-4 sha: doc_id: 319707 cord_uid: j8y9gt2o since december 2019, the world is affected by an outbreak of a new disease named covid-19, which is an acronym of ‘coronavirus disease 2019’. coronaviruses (cov) were assumed to be associated with mild upper respiratory tract infections, such as common cold. this perception changed in time due to occurrence of the severe acute respiratory syndrome (sars) caused by sars-cov in 2002 and the middle east respiratory syndrome (mers) caused by mers-cov in 2012, both inducing an epidemic severe viral pneumonia with potentially respiratory failure and numerous extra-pulmonary manifestations. the novel coronavirus, sars-cov-2, is likewise a causative pathogen for severe viral pneumonia with the risk of progression to respiratory failure and systemic manifestations. in this review, we will give a summary of the neurological manifestations due to sars and mers, as those might predict the neurological outcome in the novel covid-19. additionally, we provide an overview of the current knowledge concerning neurological manifestations associated with covid-19, to the extent that literature is already available as the pandemic is still ongoing. viruses of the coronaviridae family are positive-sensed, single-stranded rna viruses. they are broadly distributed in different animal species including avian host, cats, dogs, bats, camels, cattle and mice. among these viruses, some are pathogenic to human [1] [2] [3] . in humans, cov infections were primarily associated with upper respiratory tract and gastrointestinal tract infections. however, the last 2 decades the world was affected by several viral epidemics, such as severe acute respiratory syndrome coronavirus (sars-cov) in 2002−2003 and middle east respiratory syndrome coronavirus (mers-cov) in 2012, both resulting in high mortality rate, respectively, 10% and 35%. since december 2019, the world is affected by an outbreak of a new disease named covid-19, which is an acronym of 'coronavirus disease 2019'. it is caused by a novel coronavirus (cov), named sars-cov-2, due to similarities with the severe acute respiratory syndrome coronavirus (sars-cov) [1] . all three infections show a broad spectrum of clinical manifestation, varying from asymptomatic or mild disease to severe illness with risk of progress to respiratory failure due to viral pulmonary infection [4, 5] . it is known that human coronaviruses can reach the central nervous system (cns) and that they could be associated with neurological symptoms [6] . several cases of neurological involvement during sars and mers and the potential mechanisms have already been described in literature [4] [5] [6] [7] . conversely, despite the current global outbreak with many more patients affected, little is known about neurological manifestations in covid-19 after 6 months. in this review, we will give an overview of these neurological manifestations reported due to sars and mers as this might be of great importance in dealing with the novel covid-19. additionally, we present a summary of the current knowledge-still evolving in literature-on neurological manifestations associated with sars-cov-2-infection. the authors searched pubmed/medline databases in march 2020. articles related to the topic were identified by following terms: "severe acute respiratory syndrome", "middle east respiratory syndrome", coronavirus disease 2019", "neurology", "mers", "sars", "covid-19", "stroke", "epilepsy", "guillain-barré syndrome", "encephalitis", "myelitis", "meningitis", "neurological sequels", "polyneuropathy" and "carotid dissection". we used a date restriction ranging from the 1 st of january 2002 until present. there were limited linguistic restrictions (articles in english, dutch, french and german were eligible for inclusion). "middle east respiratory syndrome" and "neurology" identified 53 articles, of which 20 articles were retained based on review of title and abstract to select material for potential review. "severe acute respiratory syndrome" and "neurology" revealed 102 articles, "coronavirus disease 2019 and neurology" revealed 1 article, "mers" and "neurology" 109 articles, "sars" and "neurology" 25 articles, "covid-19" and "neurology" 5 articles, ("sars" or "mers" or "covid-19") and "stroke" 17 articles, ("sars" or "mers" or "covid-19") and "epilepsy" 15 articles, ("sars" or "mers" or "covid-19") and guillain-barré syndrome" 3 articles, ("sars" or "mers" or "covid-19") and "myelitis" 23 articles, ("sars" or "mers" or "covid-19") and "carotid dissection" 1 articles, but after reviewing the titles and abstracts, no additional articles were retained. ("sars" or "mers" or "covid-19") and "encephalitis" revealed 252 articles, of which 6 articles were selected for the review based on title and abstract. ("sars" or "mers" or "covid-19") and "meningitis revealed 45 articles, of which 1 article was a potential result for the review. however, this article was only accessible in danish and was not retained for this review. ("sars" or "mers" or "covid-19") and "neurological sequels" revealed 47 articles, of which 3 were selected for the review. ("sars" or "mers" or "covid-19") and "polyneuropathy" delivered 7 results, of which 1 was retained. the manuscripts that were considered as suitable for the review were evaluated via full text review. interesting articles for our review noticed in the references of these articles, were used for additional information. human coronaviruses (hcov) are known to have neurotropic and neuro-invasive capabilities. desforges et al. hypothesize human coronaviruses are neurovirulent, as they could contribute in short-and long-term neurological disorders such as encephalomyelitis and multiple sclerosis [6] [7] [8] . the presence of hcov rna in the human cns confirms these properties [9] . viruses, in general, may enter the brain and spinal cord via hematogenous or retrograde neuronal distribution. it is already known that hcov can also spread from the respiratory tract to the central nervous system (cns) through transneuronal and hematogenous routes [10] . hcov may persist in human brains, so it is assumed that long-term sequelae can develop related to emergence or aggravation of chronic neurological diseases, as it is already described for the human coronaviruses hcov-229e and hcov-oc43. the latter have been detected in various neurological diseases, including multiple sclerosis [9] . several recent articles report associated cases of encephalitis, acute flaccid paralysis and other neurological symptoms, such as guillain-barré syndrome or adem, as possible complications of a hcov infection [6] . in this article, we will focus on the neurological manifestations described in sars, mers, and covid-19. sars may present with non-specific symptoms such as persistent fever, non-productive cough, myalgia, dyspnoea and headache. in roughly 20% of the patients, the initial presentation got complicated with acute respiratory distress syndrome [6] . aside from the respiratory tract, sars-cov infection may also target intestinal mucosal cells, renal tubular epithelial cells and neurons as well as cells from the lymphoid and reticuloendothelial system. as a consequence, extrapulmonary features including watery diarrhoea and vomiting may also be part of the clinical presentation. furthermore, dizziness, nausea, decrease in general well-being and confusion have also been reported [5] . the clinical presentation of mers may also be nonspecific. runny nose, sore throat, low-grade fever and myalgia can present before the viremia becomes detectable [4, 11] . in case of severe disease, progression to acute respiratory distress may occur. extrapulmonary manifestations including gastrointestinal symptoms and acute kidney failure have been reported in case of severe illness, as well as neurological manifestations [11] . common clinical features of covid-19 include fever, cough, sore throat, headache, fatigue, myalgia, anosmia and dyspnoea. the infection may evolve into pneumonia, respiratory failure, multiple organ failure and death due to extreme rise in inflammatory cytokines [1, 12] . current data, mainly from chinese brain autopsy studies, confirm cns invasion by sars-cov [13, 14] . cases described in the articles mentioned below concerning neurological manifestations reported during sars-covinfection are rendered in table 1 [3] . another impact of the sars-epidemic was the adverse effect on seizure control due to withdrawal of anti-epileptic drug treatment as many patients avoided hospital visits [16] . neuromuscular disorders in sars are predominantly lateonset sequelae and include critical-illness polyneuropathy and myopathy [17] . muscle weakness and elevated serum creatine kinase levels occur in more than 30% of patients with sars [18] . in patients with fatal sars, leung et al. examined post-mortem histological muscle samples and described a myopathy, either resulting from critical illness myopathy or from the immune response against sars-cov [19] . critical illness myopathy and critical illness polyneuropathy can both occur in a context entitled the systemic inflammatory response syndrome (sirs). sirs is a term comprising sepsis and multiple organ failure, which can both be aspects of sars-cov-infections and be related to excessive production of tumour necrosis factor alfa and nitric oxide in the macrophages [20] [21] [22] rhabdomyolysis has previously been associated with many infectious diseases, but it is rarely seen in sars-covinfection [23] . in a case series, rhabdomyolysis correlated with renal failure and was considered to be a severe manifestation of sars. therefore, creatine kinase levels of sarspatients should be monitored thoroughly [24] . in contrast to frequent occurrence of anosmia in early stages of covid-19 (see below), it is anecdotal in sars and only described in late stage of the disease. nevertheless, olfactory function test should be part of routine work-up for patients with sars [25] . five cases of large artery ischaemic stroke were described in sars. it is suggested that a pro-coagulant state could be present in patients with sars [26, 27] . this could contribute to large cerebral arterial thromboembolism, together with factors as systemic hypotension and cardiac dysfunction. however, stroke is not uncommon in critically ill patients with co-morbidity. furthermore, it is believed that the use of intravenous immunoglobulins, used to treat sars, could play a role in sars-related stroke. hyperviscosity induced by intravenous immunoglobulins in patients already prone to hypercoagulable state due to viral infection and the inflammatory response, could play a key role in stroke [26, 27] . chronic post-sars syndrome was described by moldofsky et al. the syndrome is characterized by persistent fatigue, diffuse myalgia, weakness, depression, nonrestorative sleep with associated rem-related apneas/hypopneas, an elevated sleep eeg cyclical alternating pattern and alpha eeg sleep anomaly [28] . other authors suggest that prolonged fatigue and malaise may be linked to peripheral and autonomic nervous system dysfunction. chronic fatigue may be present months after recovery from acute illness. one study shows autonomic dysfunction to be present in 50% of recovered sarspatients. subclinical orthostatic hemodynamic disturbances would lead to fatigue and dizziness [29, 30] . cases described in the articles mentioned below concerning neurological manifestations reported during mers-covinfection are rendered in table 2 . although it is exceptional, the cns could be involved by mers-cov infection, mostly due to auto-immune reaction through autoreactive t-cells which recognize viral and myelin antigens as similar molecules, rather than to viral infection itself [31] [32] [33] . a retrospective study in saudi arabia stated that about 25% of mers patients developed confusion and 8.6% experienced a seizure [34] . another case series of three patients with altered level of consciousness varying from confusion to coma, ataxia and focal motor deficit, describes diffuse cns disease with mri showing new onset, widespread, bilateral hyperintense lesions on t2-weighted imaging within white matter and subcortical areas of different lobes, basal ganglia, corpus callosum, pons, cerebellum and upper cervical cord with only non-specific increased protein level in csf. final working diagnoses in the three patients were respectively acute disseminated encephalomyelitis, acute bilateral non-occlusive stroke due to mers-cov vasculopathy and encephalitis [35] . the negative csf for mers-cov rt-pcr might be due to the timing of the test, the lack of meningeal involvement as shown on mri, or the virus being located intra-neuronally as reported with sars-cov [36] . intracerebral haemorrhage as result of thrombocytopenia, disseminated intravascular coagulation and platelet dysfunction, is very rare [32] . in 2007, al-hameed et al. described the case of a female health care worker who developed a sudden-onset diabetes insipidus and spontaneous massive intracranial haemorrhage with intra-ventricular extension and tonsillar herniation. her platelet count and coagulation profile did not show any abnormalities. as mentioned in the table, the woman was obese. the severity of mers in obese patients can be explained as dipeptidyl peptidase 4 (dpp4), a transmembrane protein involved in cell entry of viruses such as mers-cov, is overexpressed in obese patients [37, 38] . only one case of a 28-year-old male, an orthopaedic resident, was reported. after a long stay in intensive care, he suffered from weakness in both legs and inability to walk, along with numbness and tingling in stocking distribution. he was diagnosed with critical illness polyneuropathy [32] . kim et al. published a case report of four patients who developed neurological manifestations during treatment of table 3 [39] . interferon alpha-2a is a possible causative drug for peripheral neuropathy, sensory neuropathy, vasculitic neuropathy, bell's palsy, guillain barré syndrome, chronic inflammatory demyelinating polyneuropathy and autonomic polyneuropathy [40] [41] [42] . lopinavir/ritonavir is another possible causative drug for peripheral neuropathy [43] . ribavirine is not associated with peripheral neuropathy [42] . similar to sars-cov, sars-cov-2 profits from the angiotensin converting enzyme 2 receptor (ace2-r) to infiltrate in the intracellular space. it has been described that the brain expresses ace2-r, which have been detected in glial cells and neurons as well as endothelial cells and smooth muscle cells. therefore, these cells and neurons are potential targets of covid-19 [44] covid-19 may reach the central nervous system via the systemic circulation or via the cribriform plate of the ethmoid bone during an early or later phase of the infection as has been reported in sars-cov [45] once the neuronal tissues are reached, the interaction between sars-cov-2 and ace2-r can initiate a cycle of viral growth with co-occurrence of neuronal damage without substantial inflammation as has been seen with cases of sars-cov [45] . what does this mean in the clinical setting? these are similar to sars and mers and merely non-specific including dizziness, nausea, vomiting, and headache [46, 47] . hypogeusia and hyposmia are frequently reported and have become a cardinal factor in the early diagnosis of covid-19 [46] . most probably, this is caused by direct invasion of the olfactory bulb when sars-cov-2 makes its entry into the skull via the cribriform plate [44] . a recent german report states that it seems to be well distinguishable from a post-infectious olfactory disorder following rhinitis or another upper respiratory tract infection: most of the patients describe a rather sudden, almost complete loss of odour (i.e. mostly anosmia, less often hyposmia). covid-19 patients are less likely to have other nasal [48] . by the end of april 2020, after 4 months of evolving pandemic, guillain barré syndrome (gbs) was reported in less than ten covid-19-patients, but more cases are in press. most of the cases occurred in the early stage of covid-19 in patients presenting with only minor respiratory symptoms at onset of gbs. although it is notable that the disease severity for both the gbs and pneumonia can deteriorate simultaneously [49] [50] [51] . one report described gbs in a patient without any respiratory symptoms, but with loss of smell and taste preceding gbs [52] . a variety of cerebrovascular disorders has been recently reported, probably due to endothelial dysfunction, or hypercoagulability in covid-19 patients. these disorders include ischemic stroke, intracerebral hemorrhage, and cerebral venous sinus thrombosis [44, [54] [55] [56] . cerebrovascular disease was associated with poor outcome (more respiratory distress, more multi-organ dysfunction, and higher mortality) of covid-19 patients [56] . the three major outbreaks of corona viruses (sars-cov, mers-cov, and sars-cov-2) show similarities in neurological manifestations, but also some differences. due to the hospital emergency plan and the splitting of hospitals in hot zones versus cold zones to prevent hospital clusters of corona-infection, neurological work up is limited to bedside clinical evaluation, spinal fluid analysis and ct scan. mri imaging is mostly located in the cold zone, thus limited available for infected patients. a surveillance of neurological manifestations (guillain-barré syndrome, meningoencephalitis, stroke…) in these patients is warranted as is surveillance for thromboembolic sequelae (pulmonary embolism, stroke). many patients present with fever, respiratory symptoms, but also headache, anosmia, dizziness. however, spinal tap is not routinely performed and literature on spinal fluid analysis is very sparse. an explanation for the lack of csf-data would be that there is currently no validated test to detect sars-cov-2 in csf. a multidisciplinary approach and more neurology consultations in the emergency department and covid-19-wards could prevent under-diagnosing of neurological complications and thus insufficient treatment. rapid diagnosis and proper treatment of guillain-barré syndrome, critical illness polyneuropathy, stroke, and other neurological disorders could have a positive impact on outcome. as the covid-19 pandemic is evolving, our knowledge on the neurological manifestations of covid-19 is also evolving. although the current pandemic is of a much higher scale than sars and mers, and far more neurological cases could be expected, the thusfar available literature about neurological manifestation of covid-19 is rather limited. this review will hopefully contribute to increased alertness for both neurologists and non-neurologists involved in the care of covid-19 patients. conflict of interest all the authors report no disclosure nor conflict of interest relevant to the manuscript. all authors report no financial disclosure. ethical approval this manuscript does not contain any studies with human participants or animals performed by any of the authors. di napoli r (2020) features, evaluation and treatment coronavirus (covid-19) middle east respiratory syndrome (mers): a new zoonotic viral pneumonia genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding emerging respiratory infections: the infectious disease pathology of sars, mers, pandemic influenza, and legionella severe acute respiratory syndrome: historical, epidemiologic, and clinical features human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system possible central nervous system infection by sars coronavirus detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis neuroinvasion by human respiratory coronaviruses human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis middle east respiratory syndrome a review of coronavirus disease-2019 (covid-19). indian journal of pediatrics multiple organ infection and the pathogenesis of sars detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome the impact of sars on epilepsy: the experience of drug withdrawal in epileptic patients neuromuscular disorders in severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong myopathic changes associated with severe acute respiratory syndrome: a postmortem case series the neuronal death induced by endotoxic shock but not that induced by excitatory amino acids requires tnf-alpha local expression of inducible nitric oxide synthase in an animal model of neuropathic pain infectious etiologies of rhabdomyolysis: three case reports and review rhabdomyolysis associated with probable sars olfactory neuropathy in severe acute respiratory syndrome: report of a case strokes, thromboembolic events, and ivig: rare incidents blemish an excellent safety record large artery ischaemic stroke in severe acute respiratory syndrome (sars) chronic widespread musculoskeletal pain, fatigue, depression and disordered sleep in chronic post-sars syndrome; a case-controlled study autonomic dysfunction in recovered severe acute respiratory syndrome patients outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong spontaneous intracranial hemorrhage in a patient with middle east respiratory syndrome corona virus neurological complications of middle east respiratory syndrome coronavirus: a report of two cases and review of the literature neurologic syndrome due to mers: is there a possibility that the virus can cross the blood-brain barrier to cause a neurological problem clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) pathology and pathogenesis of severe acute respiratory syndrome dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc dipeptidyl peptidase 4 is a novel adipokine potentially linking obesity to the metabolic syndrome neurological complications during treatment of middle east respiratory syndrome peripheral neurotoxicity of pegylated interferon alpha. a prospective study in patients with hcv chronic inflammatory demyelinating polyneuropathy after treatment with interferon-alpha acute inflammatory demyelinating polyneuropathy associated with pegylated interferon alpha 2a therapy for chronic hepatitis c virus infection human immunodeficiency virus protease inhibitors and risk for peripheral neuropathy evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients guntinas-lichius o (2020) the covid-19 pandemic and otolaryngology: what it comes down to? guillain-barré syndrome associated with sars-cov-2 infection: causality or coincidence? covid-19 and guillain-barré syndrome: more than a coincidence! revue neurologique early guillain-barré syndrome in coronavirus disease 2019 (covid-19): a case report from an italian covidhospital guillain-barre syndrome during sars-cov-2 pandemic: a case report and review of recent literature cerebrovascular disease in covid-19 covid-19-related stroke cerebral venous sinus thrombosis as a presentation of covid-19 impact of cerebrovascular and cardiovascular diseases on mortality and severity of covid-19 -systematic review, meta-analysis, and meta-regression key: cord-321851-ku4z34lu authors: alosaimi, bandar; hamed, maaweya e.; naeem, asif; alsharef, ali a.; alqahtani, saeed y.; aldosari, kamel m.; alamri, aref a.; al-eisa, kholoud; khojah, taghreed; assiri, abdullah m.; enani, mushira a. title: mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract date: 2020-02-29 journal: cytokine doi: 10.1016/j.cyto.2019.154895 sha: doc_id: 321851 cord_uid: ku4z34lu abstract mers-cov, a highly pathogenic virus in humans, is associated with high morbidity and case fatality. inflammatory responses have a significant impact on mers-cov pathogenesis and disease outcome. however, cd4+ t-cell induced immune responses during acute mers-cov infection are barely detectable, with potent inhibition of effector t cells and downregulation of antigen presentation. the local pulmonary immune response, particularly the th1 and th2-related immune response during acute severe mers-cov infection is not fully understood. in this study, we offer the first insights into the pulmonary gene expression profile of th1 and th2-related cytokines/chemokines (th1 & th2 responses) during acute mers-cov infection using rt2 profiler pcr arrays. we also quantified the expression level of primary inflammatory cytokines/chemokines. our results showed a downregulation of th2, inadequate (partial) th1 immune response and high expression levels of inflammatory cytokines il-1α and il-1β and the neutrophil chemoattractant chemokine il-8 (cxcl8) in the lower respiratory tract of mers-cov infected patients. moreover, we identified a high viral load in all included patients. we also observed a correlation between inflammatory cytokines, th1, and th2 downregulation and the case fatality rate. th1 and th2 response downregulation, high expression of inflammatory cytokines, and high viral load may contribute to lung inflammation, severe infection, the evolution of pneumonia and ards, and a higher case fatality rate. further study of the molecular mechanisms underlying the th1 and th2 regulatory pathways will be vital for active vaccine development and the identification of novel therapeutic strategies. middle east respiratory syndrome (mers) is a novel viral respiratory illness caused by the mers-cov that was first described in june 2012 in a patient who was hospitalized with signs of severe respiratory tract infection. the patient later died from respiratory and renal failure [1] [2] [3] . according to the statistics provided by the world health organization (who) on june 10, 2019, a total of 2428 laboratory-confirmed cases of human mers-cov infection have been reported, with an estimated 838 deaths in 27 countries. the accumulated evidence suggested that mers-cov infections are characterized by high levels of systemic inflammatory cytokines/chemokines as well as immunopathology [4] [5] [6] [7] [8] [9] [10] [11] . high inflammatory cytokines and chemokines levels have been strongly correlated with poor disease outcomes, immunopathology and massive infiltration of the immune inflammatory cells into the lungs [10, 12] . moreover, the stimulation of various cytokines, chemokines and innate immune cells has been associated with the appearance of cytokine storms, which occur during infections with pathogenic coronavirus (cov) and influenza virus [6, 13, 14] . in addition, infection, viral bronchiolitis pathogenesis, severe immunopathology, and disease enhancement during respiratory syncytial virus (hrsv) infection [10, 12, [15] [16] [17] . likewise, increased levels of inflammatory il-1β and il-1α cytokines have been associated with tissue damage and acute inflammatory responses leading to mortality and severe pathogenesis, as well as induction of the inflammatory loop [14, 18, 19] . mers-cov evades and antagonizes the antiviral immune response, as well as the nuclear factor-κb (nf-κb) signaling pathway and the ifn signaling cascade [20] [21] [22] . the disease pathogenesis of mers-cov infection is complex, with various factors involved in the onset of severe pulmonary damage and dissemination of the virus to other organs. th1 cells cytokines ifn-γ, tnf-α, tnf-β and il-2 play a key role in antiviral immunity by enhancing the toxic effects of cd8 t cells, stimulate nk cells and contribute to the regulation of cellular immunity [9, [23] [24] [25] [26] [27] [28] [29] . th2 cells secrete il-4, il-5, il-6, il-9, il-10, and il-13 which mediate humoral immune response and stimulate antibody production [9, [23] [24] [25] [26] [27] [28] [29] . imbalanced th1/th2 cytokines/chemokines can contribute to the pathological change, inefficient clearing infections, and host susceptibility to immune-mediated diseases. in vivo data highlights the important roles of t cells in controlling and reducing the pathogenesis and decreasing mers-cov titers [30] . there have been no reports describing the complete cellular immune response of the lower respiratory tract to mers-cov. therefore, it is still unclear whether the cd4 + t-helper (th1/th2)-induced cytokines/chemokines are beneficial or harmful for mers-cov-infected patients. moreover, the host immune responses which are activated in the lower respiratory tract during a mers-cov infection also remain unclear. in this study, we offer the first insight into the pulmonary molecular gene expression profile of th1 and th2 cytokines/chemokines (th1 & th2 responses) during acute mers-cov infection using rt 2 profiler pcr. we also describe the expression level of primary inflammatory cytokines and chemokines in the lower respiratory tract. the institutional review board at king fahad medical city reviewed and approved the study protocol (irb register number 17-182). the handling of respiratory samples, as well as aliquoting and viral and cellular rna extraction were executed using appropriate personal protective equipment in a biosafety level 3 laboratory (riyadh regional laboratory, ministry of health, riyadh, saudi arabia) the lower respiratory samples were collected from 39 mers-cov positive patients and 30 healthy non-infected individuals. to exclude effects of antiviral therapy on the expression of cytokines/chemokines understudy, the samples were collected before the initiation of any antiviral treatment for mers-cov. for the sputum sample collections, the patients were asked to rinse their mouth and gargle with water twice immediately before obtaining the sample and instructed not to expectorate saliva or postnasal discharge into the container during collection to reduce the possibility of contamination with upper respiratory tract fluids. subsequently, the patient was requested to breathe and expectorate deep cough sputum directly into a sterile sputum cup (screw cap). tracheal aspirate (ta) was collected via the endotracheal tube as reported elsewhere [31, 32] . bronchoalveolar lavage (bal) was collected by following a standardized protocol (technique, sampling, and procedure) as previously described [33] [34] [35] . the first bal fluid aliquots recovered were discarded to reduce cross-contamination with upper respiratory tract fluids. in order to exclude the effect of viral co-infection with influenza a h1n1 on cytokines/chemokines expression levels, all mers-cov positive patients were screened for h1n1 by genexpert flu (cepheid, sunnyvale, ca) according to the manufacturer′s instructions. samples were centrifuged at 1000 rpm at 4°c for 5-10 min. the cell-free supernatants were frozen at −80°c until they were used for subsequent analysis of the inflammatory cytokine and chemokine by elisarray, as well as for the mers-cov viral load test. the pellets were used for total cellular rna extraction using the rneasy mini kit (qiagen, valencia, ca) to detect mers-cov rna concentrations in the lower respiratory tract, viral rna was isolated from 140 µl of lower respiratory specimens using the qiaamp viral rna extraction mini kit (qiagen) according to the manufacturer′s instructions. the rna was dissolved in 60 µl of elution buffer and stored in separate aliquots at −80°c. an internal control (ic) was included as a control for the procedure used for sample preparation (viral rna purification). the extracted viral rna was tested by real-time rt-pcr duplex assays targeting the upe and orf1a regions of the mers-cov genome, using the realstar® mers-cov rt-pcr kit 1.0 (rt-pcr duplex assay). this system involves two independent assays, one targeting and quantifying a region upstream of the e gene (upe) and the other targeting the open reading frame regions 1a (orf1a) of the mers-cov genome. the analysis was performed using an abi prism® 7500 (applied biosystems). the main 12 human proinflammatory cytokines and chemokines (il-1α, il-1β, il-2, il-4, il-6, il-8, il-10, il-12, il-17a, ifn-γ, tnf-α, and gm-csf) were measured in lower respiratory samples from both mers-cov infected patients and the healthy non-infected control group, using multi-analyte elisarray (qiagen, germantown, md, usa) according to the manufacturer′s protocol. the absorbance of the obtained products after 30 min was measured at 450 nm, and cytokine/ chemokine levels were determined by comparing them to the negative (assay buffer) and positive control (cocktail containing all standard 12 cytokines or chemokines, respectively). the presence of a determined cytokine/chemokine was based on an absorbance value above the negative control. the mean absorbance of the samples and all standards was calculated as replicates. the concentrations were calculated using standard curves. cytokine/chemokines levels were expressed as pg/ml. pulmonary expression genes involved in the th1 and th2 response was assessed. cellular rna was isolated from lower respiratory tract samples using the rneasy mini kit (qiagen, valencia, ca). the concentration (ng/μl) and purity (a260/a280) of the rna was assessed using a nanodrop2000 (thermo-scientific). cdna synthesis and genomic dna elimination were performed using an rt 2 first-strand synthesis kit (qiagen, germantown, md, usa), using 1.5 µl (0.5 μg) of rna. mrna expression levels of th1/th2 immune genes were measured using an rt 2 -pcr profiler array kit (qiagen, germantown, md, usa) on an abi prism 7500 system (applied biosystems) with the rt 2 sybr green rox qpcr master mix (qiagen, germantown, md, usa). briefly, the cdna template was combined with the rt 2 real-time sybr green master mix and rnase-free water. a final reaction volume of 25 μl was added to each well of the rt 2 -pcr profiler array. cyclethreshold (ct) values were obtained using a constant baseline for all real-time rt-pcr runs as previously described [36] . five endogenous control genes provided by the array (actb, b2m, gapdh, gusb, and hsp90ab1) were used to calculate the arithmetic mean, which was then set as the ct value for normalization. the online rt 2 -pcr profiler data pcr array analysis software was used to identify the most appropriate reference genes to use for the normalization of the rt-pcr array gene expression data. real-time rt-pcr array results were analyzed using the online rt 2 -pcr profiler data pcr array analysis software (https://dataanalysis.qiagen.com/pcr/arrayanalysis.php). each sample was assessed for reverse transcription efficiency, pcr array reproducibility, and genomic dna contamination using the quality control function of the software. experimental gene expression was quantified as δδct and calculations were normalized to the housekeeping genes. statistical analysis was performed using spss version 16 (spss, inc., chicago, il). variables were compared between mers cov infected patients and healthy non-infected controls. the t-test and one-way anova analysis were used as appropriate. the data were presented as mean ( ± standard deviation) and counts (percentages). a p-value of < 0.05 was considered statistically significant. the demographic data, outcomes, preexisting conditions, underlying chronic diseases, clinical characteristics of mers cov infected patients and disease outcomes are presented in table 1 . the real-time rt-pcr results showed high viral loads in all mers-cov patients regardless of the type of specimen. the mean mers-cov rna ct values and standard deviations were 25.30 ± 5.1 and 26.3 ± 5.02 for upe and orf1a, respectively ( table 3) . the viral load of mers-cov and the expression levels of inflammatory cytokines/ chemokines were found to be significantly correlated with the case fatality of mers-cov infected patients (p < 0.002). furthermore, we found a significant correlation (p < 0.0001) between mers-cov viral load and expression levels of inflammatory cytokines il-1α, il-1β and neutrophils chemoattractant chemokines il-8 (cxcl8). no influenza a h1n1 co-infections were detected among mers-cov positive patients. the lower respiratory tract samples from mers-cov infected patients and healthy non-infected controls were used to quantify expression levels of the main 12 human pro-inflammatory cytokines and chemokines (il-1α, il-1β, il-2, il-4, il-6, il-8, il-10, il-12, il-17a, ifn-γ, tnf-α, and gm-csf). among the panel of 12 inflammatory cytokines/chemokines, the mean expression levels of il-1α (1148 pg/ml) (p < 0.001) and il1-β (1230 pg/ml) (p < 0.001) were significantly higher in the lower respiratory tract of mers-cov infected patients when compared to healthy non-infected controls ( table 2) . furthermore, our results showed a significant correlation between il and 1α (p < 0.035) and il-1β (p < 0.013) levels and the case fatality of mers-cov infected patients. ifn-γ expression levels were detected in only four patients and tnf-α expression levels were increased only in five patients. the expression of il-8 (cxcl8) was found to be significantly increased (1956 pg/ml) (p < 0.001) in all mers-cov patients when compared to healthy controls (table 3) . our results showed a significant correlation between the expression level of il-8 and the case fatality of mers-cov infected patients (p < 0.003). in order to study the pulmonary th1 and th2 responses in mers-cov infected patients, molecular cytokine/chemokine profiles were assessed using a rt 2 -pcr array. only genes with at least a two-fold change in expression when compared to the housekeeping gene were selected for further analysis. a side-by-side analysis of the data derived from the rt 2 -pcr profiling of pulmonary th1/th2 responses showed that genes encoding th1 and th2-related cytokines and chemokines were largely downregulated in the lower respiratory tract of mers-cov infected patients. a total of 26 genes involved in regulating the th1 and th2 immune response were downregulated in mers-cov infected patients ( table 4 ). the mrna expression levels of 10 th1-related cytokines/chemokines were downregulated. in contrast, the expression of only 6 th1 cytokine/chemokine mrnas, namely il-18, il-18r1, socs5, ccr2, cd4, and cxcr3, were upregulated (table 4) . with regards to the th2 immune response, we identified 15 cytokines/ chemokines mrnas expression levels were downregulated in mers-cov infected patients (table 4) . we did not observe any modulation or differences in th1/th2 responses and inflammatory cytokines/chemokines expression levels between mers-cov infected patients with underlying chronic medical conditions and mers-cov infected patients without underlying chronic medical conditions. the disease outcome, as well as the immunopathology and pathogenesis of mers-cov, sars, and other respiratory viral infections in humans and other animals are strongly linked to the expression levels of inflammatory cytokines and chemokines [16, 37, 38] . various cytokines, chemokines, and innate immune cells have been associated with the immunopathology during coronavirus (cov) and influenza virus infection [5, 6, 13, 14] . this study offers the first insight into the lung molecular gene expression profile of th1 and th2-related cytokines/ chemokines (th1 & th2 responses) during acute mers-cov infection. previous studies have shown that dysregulated and excessive immune responses may cause immunopathology and fatal disease [6, [39] [40] [41] . as such, direct cytopathic effects, high virus titers, and viral evasion of host immune responses are believed to play a major role in the severity of the diseases resulting from sars-cov and mers-cov infections [6, 39, 40] . our results showed high viral load (ct) values in all mers-cov infected patients regardless of the specimen type (table 3) . several pro-inflammatory cytokines, such as il-8 and il-1β, contribute to ards pathogenesis [6, 15, [42] [43] [44] . ards was shown to be the primary cause of death among patients with sars-cov or mers-cov infections [6, 45] . in this study, the majority of mers-cov infected patients progressed to ards, subsequently developing pneumonia and requiring admission to an intensive care unit (icu). in vitro studies have revealed that mers-cov induces significant but delayed modifications in the expression of both ifn and pro-inflammatory cytokines, including il-1β, il-6, il-8, and other chemokines [7, 10, 46] . our results showed high expression levels of il-1α, il-1β and il-8 (cxcl8) in the lower respiratory tract of mers-cov infected patients. evidence from studies investigating sars and respiratory syncytial virus (hrsv) has revealed that il-8 was associated with acute sars infection, bronchiolitis, immunopathology and disease enhancement during hrsv infections [10, 12, [15] [16] [17] . il-8 (cxcl8) is a critical chemokine involved in neutrophil recruitment, activation, and local neutrophil accumulation, and was shown to induce the formation of neutrophil extracellular traps (nets) [44, 47] . nets are highly immunogenic and extremely toxic to the host tissue, being able to directly cause inflammation, pathological changes, and epithelial and endothelial cell death [44, 48] . as such, lung nets can increase inflammation by stimulating il-8 expression, which leads to the recruitment of more neutrophils [44, 47] . we hypothesize that high il-8 expression levels may cause netosis, which results in severe mers-cov infection and increases immunopathology. il-1β has been associated with tissue damage, neutrophil infiltration, acute inflammatory responses, higher case fatality and severe respiratory viral infection [10, 14, 19, 49, 50] . in this study, high expression levels of il-1β were measured in the lower respiratory tracts of mers-cov infected patients. previous studies have shown that the expression of il-1β during influenza a h1n1 infection increased lung inflammation, and subsequent treatment with an il-1β antagonist significantly reduced both inflammation and lung tissue damage, suggesting that il-1β is a critical cytokine that contributes to lung inflammation [51, 52] . likewise, another study has shown that il-1β mrna levels were upregulated in calu-3 cells infected with mers-cov and/or sars-cov [10] . il-1α is an inflammatory cytokine that can be rapidly induced and quickly reaches high levels in response to a range of stimulants, such as il-1β and pathogenic agents [18, 53] . in mouse models, il-1α was shown to be a key player in triggering neutrophilic inflammation [18, 54] . likewise, our results revealed high il-1α expression levels in mers-cov infected patients. therefore, we think that high levels of il-8, il-1α, and il-1β may play a critical role in the immunopathology, severity, and case fatality of mers-cov infected patients. il-1α and il-1β establish an inflammatory loop which activates downstream signaling and enables the il-1α-il-1r1 interaction, leading to further il-1α and il-1β production. thus, a loop of continued and self-perpetuating inflammation occurs, resulting in extensive tissue damage and pathological changes [18] . as such, elevated il-1β and il-1α levels during acute mers-cov infection may cause an inflammatory loop that contributes to the extensive tissue damage and pathological changes associated with this disease, it is important to note that the il-1α and il-1β expression levels were not timely monitored at different intervals in this study. thus, the proposed inflammatory loop may not be generalizable to cases with severe disease or patients treated by antiinflammatory/antiviral drugs. it has been shown that effective control of host inflammatory response and antiviral treatment were associated with a reduction in inflammatory cytokine levels, steady improvement in disease condition, and pulmonary function [55] [56] [57] [58] [59] . consequently, studying il-1α and il-1β expression levels at different intervals could assist in a better understanding of mers-cov immunopathology. we speculate that, monitoring of il-1α and il-1β expression levels at different time points during mers-cov infection may alter the proposed inflammatory loop. tnf-α is an important innate and antiviral cytokine. high levels of tnf-α were detected in an in vitro study of sars-cov and mers-cov at both 24 and 30 h [10] . in our study, the induction of tnf-α was largely absent. similarly, in a recent study, tnf-α expression was not detected in most patients with mers-cov infections in the acute or convalescent stages [11] . this may indicate the limited early in vivo tnf-α response during mers-cov infections. one explanation for the differences between in vitro and in vivo studies could be the different kinetics of the mers-cov responses in the in vitro model, where there is a gradual increase in gene expression over time. however, a more likely explanation is that the complex interplay of target cell infection, mers-cov replication, viral load and time of sample collection. we measured cytokines/chemokines levels and viral load at a single time point in the early phase of infection; assessing cytokines/chemokines concentrations and viral load at different time points during mers-cov infection to create a kinetic profile of cytokines/chemokines might yield additional information. overall, the distinct tnf-α responses in vitro and in vivo might impact on the in vivo pathogenesis and viral amplification. cd4 + t-cell immune responses during respiratory viral infections characterized by the production of signature cytokines, ifn-γ, tnf-α, and il-2 for th1 cells and il-4, il-5, il-6, il-9, il-10, and il-13 for th2 cells [9, 28, 29] . the balance between the th1 and th2 responses is critical for the outcome of viral infections [60, 61] . to our surprise, we found that the expression of genes encoding th1 and th2 cytokines/chemokines were largely downregulated in the lower respiratory tract of mers-cov infected patients ( table 4 ). the pulmonary th1 and th2 responses showed the downregulation of 26 genes encoding th1 and th2 cytokines/chemokines in mers-cov infected patients. ifnγ plays an important role in early immunity, inducing the apoptosis of infected cells and stimulating both cd8 + and natural killer (nk) cells [62, 63] . in this study, both ifnγ and main th1associated cytokine mrna expression levels were downregulated in the lower respiratory tract of mers-cov infected patients. on the other hand, only 6 th1-related cytokines and chemokines (il-18, il-18r1, socs5, ccr2, cd4, and cxcr3) were overexpressed in the lungs of mers-cov infected patients (table 4 ). in regards to th2-induced immune responses, 15 th2 cytokines were downregulated. th2 cytokines play an important role in the humoral immune response and promote antibody production. mers-cov infection completely downregulated the th2 response in this study. we could not detect the mrnas for the specific antiviral immune response in mers-cov infected patients. reghunathan et al. also found a strong inflammatory response in acute sars despite complete downregulation of the mrna expression of genes involved in specific antiviral immune response [15] . downregulation of the major th1/th2 cytokines/chemokines can contribute to the pathological change, inefficient clearing infections, and host susceptibility to immune-mediated diseases. the upregulated th1 cytokines/chemokines (il-18, socs5, ccr2, and cxcr3) have been shown to play an important role in virus-induced immunopathology and usually associated with acute lung inflammation. il-18 was associated with acute lung inflammation [64] . in addition, il-18 was shown to enhance the severity of hrsv disease [65] . socs proteins affect antiviral signaling pathways, allowing viruses to evade the host immune response, and facilitate viral replication. moreover, socs5 has been suggested to shape the presentation of viral disease [66, 67] . previous data showed that, socs5 mrna was significantly upregulated in response to h1n1, h3n2, h5n1, and h11n9. suppressing socs signals improved influenza infection and inhibited hrsv replication [68, 69] . ccr2 mrna upregulation was correlated with hrsv-disease severity [70] . ccr2 antagonism reduced pathology, morbidity, and mortality during influenza a (h1n1) infection [71] . during hrsv infection increased cxcr3 was associated with the underlying pathology [72] . in addition, it has been shown that blocking cxcr3 reduced immunopathology during respiratory virus infections [73] . therefore, we think il-18, socs5, ccr2, and cxcr3 overexpression may play a critical role in immunopathology, severity, and case fatality of mers-cov infected patients. factors contribute to cd4 molecules upregulation during mers-cov infection in this study need further study. previous data showed that, cd4 and cd8 expression on t cells were not altered by mers-cov infection [74] . mers-cov could persistently induce the expression of pro-inflammatory cytokines/chemokines such as il-8, which would up-regulate cd4 molecules to enhance helper t cell infection. expression of high levels of il-8 and the net formation could have important effects on the cd4 expression. we propose a mechanism, which can explain the role of lower respiratory tract cytokines/ chemokines during acute mers-cov infection. il-1β and il-8 (cxcl8) recruit and activate neutrophils. subsequently, activated neutrophils undergo degranulation and netosis, which contributes to pathological changes and leads to the recruitment of more inflammatory. elevation of il-1β and il-1α during acute mers-cov infection may cause an inflammatory loop that contributes to extensive tissue damage and pathological changes. il-8 and il-1β are essential mediators in the development and pathogenesis of ards [6, 15, [42] [43] [44] . moreover, overexpression of il-1α, il-1β, il-8 (cxcl8), il-18, cxcr3, socs5, and ccr2 may play a vital role in the severity, immunopathology, and case fatality of mers-cov infected patients. it is important to note that the samples in this study were screened only for h1n1. we were not able to screen other respiratory viruses. viral co-infections could result in specific virus-virus interactions [75] . however, the inflammatory response induced by one virus may not be significantly changed by the infection of a second or third virus [76] . we did not examine the influences of co-infections with other respiratory viruses on cytokines/chemokines expression levels. any changes in immune profiles in mers-cov infected patients due to other respiratory pathogens are unknown. however, there is a possibility that other respiratory viral infections, which were not screened in our study, might skew immune profiles in mers-cov infected patients. we think that simultaneous study of other variables (ie, the interplay between mers-cov and other respiratory viruses), which could potentially skew the data is likely required to assess any alterations of the immune profiles in mers-cov infected patients. four limitations of this study should be noted. first, we measured cytokines/chemokines expression levels and viral load at a single time point in the early phase of infection; assessing cytokines/chemokines expression levels and viral replication at different time points during mers-cov infection to create a kinetic profile might yield additional information. second, in our study design, we did not include controls from patients infected with other respiratory viral pathogens. third, we have screened mers-cov positive samples only for h1n1, we did not screen our samples for other respiratory viruses. fourth, we were not able to recruit health non-infected individuals with similar mers-cov infected patients underlying diseases/pre-existing conditions. future study designs to close these gaps need to be considered. taken together, the high expression of inflammatory cytokines/ chemokines il-1 α and il-1β, il-8 (cxcl8), il-18, cxcr3, socs5, and ccr2 in the lower respiratory tracts of mers-cov infected patients confirmed the lung immunopathology [46, [77] [78] [79] [80] . therefore, the high expression of inflammatory cytokines and downregulation of the th1 and th2 immune responses in the lower respiratory tracts of mers-cov infected patients may contribute to a more severe infection, higher case fatality, lung inflammation, and immunopathology. furthermore, high levels of inflammatory cytokines/chemokines could be a key point in the subsequent development of pneumonia and ards in mers-cov infected patients. as such, the characterization of pulmonary th1/th2 response in mers-cov infection could be valuable for effective vaccine development and novel therapeutic strategies. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. pathogenesis of middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus-two years into the epidemic interaction of sars and mers coronaviruses with the antiviral interferon response cytokine mers-cov infection in humans is associated with a pro-in fl ammatory th1 and th17 cytokine profile pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology middle east respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis molecular pathology of emerging coronavirus infections delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment immune responses to middle east respiratory syndrome coronavirus during the acute and convalescent phases of human infection innate immune dysfunction is associated with enhanced disease severity in infants with severe respiratory syncytial virus bronchiolitis cytokine storms in infectious diseases new fronts emerge in the influenza cytokine storm expression profile of immune response genes in patients with severe acute respiratory syndrome analysis of serum cytokines in patients with severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory interleukin 1α and the inflammatory process reassessing the role of the nlrp3 inflammasome during pathogenic influenza a virus infection via temporal inhibition mers-cov accessory orfs play key role for infection and pathogenesis antagonism of dsrna-induced innate immune pathways by ns4a and ns4b accessory proteins during mers coronavirus infection mers-cov 4b protein interferes with the nf-kappab-dependent innate immune response during infection cytokines and transcription factors that regulate t helper cell differentiation: new players and new insights regulation of the t cell response t-cell subset regulation in atopy effects of various radiation doses on induced t-helper cell differentiation and related cytokine secretion mrna expression of interleukins and th1/th2 imbalance in patients with pulmonary embolism t cell-mediated immune response to respiratory coronaviruses modulation of the immune response by middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome collection of tracheal aspirate: safety and microbiological concordance between two techniques should lower respiratory tract secretions from intensive care patients be systematically screened for influenza virus during the influenza season ? a simple method of reducing complications of pediatric nonbronchoscopic bronchoalveolar lavage respiratory viruses in bronchoalveolar lavage: a hospitalbased cohort study in adults cough suppression during flexible bronchoscopy using combined sedation with midazolam and hydrocodone: a randomised, double blind, placebo controlled trial naked dna immunization with full-length attachment gene of human respiratory syncytial virus induces safe and protective immune response fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice disease-promoting effects of type i interferons in viral, bacterial, and coinfections age-dependent dysregulation of innate immunity characterization of cytokine/chemokine profiles of seven acute respiratory syndrome human immunopathogenesis of severe acute respiratory syndrome (sars) net balancing: a problem in inflammatory lung diseases severe acute respiratory syndrome (sars) active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis induction of neutrophil extracellular dna lattices by placental microparticles and il-8 and their presence in preeclampsia neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones is il-1 a good therapeutic target in the treatment of arthritis? evidence that cytokines play a role in rheumatoid arthritis interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection induction of interleukin-1 beta (il-1beta) is a critical component of lung inflammation during influenza a (h1n1) virus infection complexity of inflammatory responses in endothelial cells and vascular smooth muscle cells determined by microarray analysis cytokine endogenous il-1 a is a chromatin-associated protein in mouse macrophages cytokine inhibition in the treatment of copd nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus infections and the inflammatory response in acute respiratory distress syndrome dynamic changes in clinical features and cytokine/ chemokine responses in sars patients treated with interferon alfacon-1 plus corticosteroids plasma and sal cytokine response to corticosteroid rescue treatment in late ards th1/th2 balance: the hypothesis, its limitations, and implications for health and disease effects of strenuous exercise on th1/th2 gene expression from human peripheral blood mononuclear cells of marathon participants nk cells controlling virus-specific t cells: rheostats for acute vs. persistent infections distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? role of il-18 in acute lung inflammation interleukin 18 coexpression during respiratory syncytial virus infection results in enhanced disease mediated by natural killer cells hsv-1-induced socs-1 expression in keratinocytes: use of a socs-1 antagonist to block a novel mechanism of viral immune evasion viral exploitation of host socs protein functions rsv replication is attenuated by counteracting expression of the suppressor of cytokine signaling (socs) molecules suppressor of cytokine signaling (socs) 5 ameliorates influenza infection via inhibition of egfr signaling chemokine-receptor upregulation and disease severity in respiratory syncytial virus infection ccr2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection production of chemokines in the lungs of infants with severe respiratory syncytial virus bronchiolitis cxcr3 directs antigen-specific effector cd4 + t cell migration to the lung during parainfluenza virus infection middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways quasispecies diversity determines pathogenesis through cooperative interactions within a viral population respiratory and gastrointestinal epithelial modulation of the immune response during viral infection, innate immun immunopathogenesis of coronavirus infections: implications for sars ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection the authors thank the research center at king fahad medical city for funding this study (grant no 017-066). we also thank the deanship of scientific research and rssu at king saud university for their technical support. key: cord-328361-hyrke6j2 authors: ithete, ndapewa laudika; stoffberg, samantha; corman, victor max; cottontail, veronika m.; richards, leigh rosanne; schoeman, m. corrie; drosten, christian; drexler, jan felix; preiser, wolfgang title: close relative of human middle east respiratory syndrome coronavirus in bat, south africa date: 2013-10-17 journal: emerg infect dis doi: 10.3201/eid1910.130946 sha: doc_id: 328361 cord_uid: hyrke6j2 nan respiratory syndrome coronavirus in bat, south africa to the editor: the severe acute respiratory syndrome (sars) outbreak of 2002-03 and the subsequent implication of bats as reservoir hosts of the causative agent, a coronavirus (cov), prompted numerous studies of bats and the viruses they harbor. a novel clade 2c betacoronavirus, termed middle east respiratory syndrome (mers)-cov, was recently identified as the causative agent of a severe respiratory disease that is mainly affecting humans on the arabian peninsula (1) . extending on previous work (2), we described european pipistrellus bat-derived covs that are closely related to mers-cov (3) . we now report the identification of a south africa bat derived cov that has an even closer phylogenetic relationship with mers-cov. during 2011-2012, fecal pellets were collected from 62 bats representing 13 different species in the kwazulu-natal and western cape provinces of south africa and stored in rnalater solution (life technologies, carlsbad, ca, usa). details about the bat sample are available in the online technical appendix table ( wwwnc. cdc.gov/eid/article/19/10/13-0946-techapp1.pdf). rna was extracted by using the qiaamp viral rna mini kit (qiagen, hilden, germany). screening for covs was done by nested reverse transcription pcr using broadly reactive oligonucleotide primers targeting a conserved region in the rna-dependent rna polymerase (rdrp) gene (online technical appendix). pcr results were positive for 5 (8%) of the 62 specimens. pcr amplicons for 4 positive specimens yielded alphacoronavirus sequences related to recently described bat alphacoronaviruses from south africa (4) . the other positive specimen, termed pml/2011, was from an adult female neoromicia cf. zuluensis bat sampled in 2011; the specimen yielded a novel betacoronavirus (genbank accession no. kc869678). online technical appendix figure 1 shows the distribution of this bat species. to obtain better phylogenetic resolution, we extended the 398-nt rdrp fragment generated by the screening pcr to 816 nt, as described (5). pml/2011 differed from mers-cov by only 1 aa exchange (0.3%) in the translated 816-nt rdrp gene fragment. thus, pml/2011 was much more related to mers-cov than any other known virus. the amino acid sequence of the next closest known relatives of mers-cov, from european pipistrellus bats (3), differed from mers-cov by 1.8%. the amino acid sequences of viruses from nycteris bats in ghana (3) and the 2c prototype bat covs, hku4 and hku5, from china (6) differed by 5.5%-7.7% from mers-cov. the smaller 152to 396-nt rdrp fragments of 2c bat covs from a hypsugo savii bat in spain (7), bat guano in thailand (8), and a nyctinomops bat in mexico (9) showed no or only partial overlap with the 816-nt fragment generated in this study; thus, a direct comparison could not be done. however, in their respective rdrp fragments, these covs yielded amino acid sequence distances of 3.5%-8.0% and were thus probably more distant from mers-cov than the virus described here. a bayesian phylogenetic analysis of the 816-nt rdrp sequence confirmed the close relationship between pml/2011 and mers-cov (figure) . their phylogenetic relatedness was as close as that of sars-cov and the most closely related bat coronavirus known, rs672 from a rhinolophus sinicus bat (figure) . like pml/2011 and mers-cov, rs672 and sars-cov showed only 1 aa exchange in the translated 816-nt rdrp fragment. to confirm this relatedness, we amplified and sequenced a short 269-nt sequence encompassing the 3′-terminus of the spike gene for pml/2011 (oligonucleotide primers available upon request from the authors). a partial spike gene-based phylogeny using this sequence yielded the same topology as that using the partial rdrp sequence (online technical appendix figure 2 ). again, pml/2011 was most closely related to mers-cov, showing only a 10.9% aa sequence distance in this gene, which encodes the glycoprotein responsible for cov attachment and cellular entry. this distance was less than the 13.3% aa sequence distance between mers-cov and the european pipistrellus covs (3) and less than the 20.5%-27.3% aa sequence distance between mers-cov and hku5 and between mers-cov and hku4 (6) in the same sequence fragment. our results further support the hypothesis that, like human cov-229e and sars-cov, ancestors of mers-cov might exist in old world insectivorous bats belonging to the family vespertilionidae, to which the genera to the editor: extraintestinal pathogenic escherichai coli (expec) bacteria have the ability to cause diverse and serious diseases, such as urinary tract infections (utis) and bacteremia (1-3); incidence of bacteremia is increasing globally (4) . the emergence of multidrug resistance in e. coli is also becoming a global concern, with particular emphasis on e. coli sequence type (st) 131, which is being increasingly reported in utis. drug resistance is mediated by extended-spectrum β-lactamases (es-bls), mainly of the ctx-m family, particularly ctx-m-15 and 14, and less frequently of the shv and oxa families (5, 6) . few studies are available regarding the characterization of e. coli strains causing bacteremia. we characterized 140 e. coli isolates from bacteremia patients treated at nottingham university hospital (nottingham, uk) over a 5-month period, with the aim of developing an epidemiologic profile of the population of expec that causes bacteremia. for context, we compared the isolates with 125 e. coli isolates from urine samples collected during the same period. cases were selected to include isolates from a diverse patient group: patient ages ranged from 1 month to 90 years; patient sex was evenly divided between male and female; infections were community-and hospitalassociated; and suspected sources of infection varied. antimicrobial drug susceptibility tests, pcr detection of esbl genes , multilocus sequence typing using the achtman scheme (http:// mlst.ucc.ie/mlst/dbs/ecoli), and virulence-associated gene (vag) carriage screening by pcr were performed on isolates as described (7). significantly more bacteremia e. coli isolates than urine e. coli isolates were resistant to ciprofloxacin (25.7% isolation of a novel coronavirus from a man with pneumonia in saudi arabia circulation of group 2 coronaviruses in a bat species common to urban areas in western europe human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe coronaviruses in south african bats genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features detection of alpha and betacoronaviruses in multiple iberian bat species group c betacoronavirus in bat guano fertilizer coronaviruses in bats from mexico complete genome analysis of 33 ecologically and biologically diverse rift valley fever virus strains reveals widespread virus movement and low genetic diversity due to recent common ancestry we thank tobias bleicker, sebastian brünink, and monika eschbach-bludau for technical assistance; thomas seifert, sonja matthee, and conrad mathee for invaluable help; and anna-marie corman for assistance with geographic information processing. key: cord-345081-15s2i6f0 authors: al-sehaibany, fares s. title: middle east respiratory syndrome in children: dental considerations date: 2017-04-17 journal: saudi med j doi: 10.15537/smj.2017.4.15777 sha: doc_id: 345081 cord_uid: 15s2i6f0 as of january 2016, 1,633 laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection and 587 mers-related deaths have been reported by the world health organization globally. middle east respiratory syndrome coronavirus may occur sporadically in communities or may be transmitted within families or hospitals. the number of confirmed mers-cov cases among healthcare workers has been increasing. middle east respiratory syndrome coronavirus may also spread through aerosols generated during various dental treatments, resulting in transmission between patients and dentists. as mers-cov cases have also been reported among children, pediatric dentists are at risk of mers-cov infection. this review discusses mers-cov infection in children and healthcare workers, especially pediatric dentists, and considerations pertaining to pediatric dentistry. although no cases of mers-cov transmission between a patient and a dentist have yet been reported, the risk of mers-cov transmission from an infected patient may be high due to the unique work environment of dentists (aerosol generation). middle east respiratory syndrome coronavirus is a novel beta coronavirus of the coronaviridae family that causes a severe respiratory disease with a high fatality rate. [3] [4] [5] [6] as of january 2016, 1633 laboratoryconfirmed cases of mers-cov infection and 587 related deaths have been reported by the world health organization (who) globally. 7 the male-to-female ratio of the affected patients was 2:1, with a median age of 49 years. 8 the incubation period for human-tohuman transmission ranges from 2-15 days. 9 the symptoms of mers-cov infection range from being asymptomatic to severe pneumonia, acute respiratory distress syndrome, septic shock, and multi-organ failure, leading to death. the symptomatic patients may have fever, cough, chills, throat soreness, myalgia, arthralgia, vomiting, or diarrhea. 10 furthermore, men over 45 years of age, patients with comorbidities, and healthcare workers have been reported to be high-risk groups for mers-cov infection. 11 the virus has been detected in respiratory, gastrointestinal and other bodily secretions, 12, 13 as well as in air samples, which indicates the possibility for airborne transmission. 14 several studies have reported mers-cov infections among healthcare workers 4,6,15,16 and children. 1, 17 viral infections, such as severe acute respiratory syndrome saudi med j 2017; vol. 38 (4) www.smj.org.sa (sars-cov), may be transmitted to healthcare workers from infected patients through aerosols. 18 considering that several types of dental equipment, such as handpieces, air-water syringes and ultrasonic scalers, produce considerable amounts of aerosols, the potential for the spread of infections from patients to dentists or dental assistants is high. 19 this review is an attempt to discuss mers-cov infection among children and those providing dental treatment to them, including precautions and considerations pertaining to the practice of pediatric dentistry. mers-cov infection in healthcare workers. the 3 patterns of mers-cov transmission suggested in the literature include the following: a) sporadic cases occurring in communities; b) transmission within families; and c) nosocomial transmission. 20 healthcarerelated mers-cov transmission is associated with high morbidity, extended use of mechanical ventilation and fatality rates of up to 65%; this type of transmission can be attributed to shortcomings in observing stringent infection control protocols. 4, 6, 15, 21 furthermore, mers-cov has been reported to be viable in hospitallike environments for up to 48 hours with a stability that is unaltered during aerosolization. 22 among 200 healthcare workers' contacts identified in al-hassa, saudi arabia, mers-cov infection was confirmed in 2 cases. 4 a saudi study 20 compared healthcare worker and family contact with laboratory-confirmed mers-cov patients and reported a lower rate (1.12%) of infection among the healthcare workers than among the families (3.4%). healthcare workers could be infected with mers-cov through exposure in the community or at their workplace, 1,6,20 they could be diagnosed late, 1,6 and they could remain asymptomatic or mildly symptomatic. 6, 8, 16 furthermore, unsuspected cases entering healthcare facilities have been considered the main source of mers-cov. 4 considering these aspects and that healthcare workers may continue to work regardless of being symptomatic, 15 the possibility of transmitting the infection to their patients is also high. memish et al 1 reported that the age of 11 saudi pediatric patients who presented clinically with symptoms of mers-cov infection ranged from 2-16 years with a female-to-male ratio of 2.7 to 1. of the 11 cases, 2 were symptomatic, and 9 were asymptomatic with no underlying comorbidities. another report by thabet et al 17 concluded that although mers-cov infection presents with a wide range of clinical manifestations, the mortality rate in children is lower than that in adults. a review on the effects of coronavirus infection in children concluded that human coronavirus infections may be associated with respiratory symptoms and may also involve central nervous system. the authors suggested that the clinical and genetic traits of human coronavirus infection among children should be monitored closely for early prevention. 23 dental considerations. bioaerosols in dental practice. bioaerosols are defined as a suspension of biological particles in gaseous media. 24 apart from radiation exposures, dermatitis, eye injuries, and musculoskeletal and respiratory disorders, other occupational health risks exist in dentistry. these risks include percutaneous exposure incidents and exposure to infectious diseases, such as those that may be present in bioaerosols. 25 subgingival scaling of periodontally involved teeth with ultrasonic scalers may produce aerosols containing blood. 26 one study reported that ultrasonic scalers and tips produced significantly more aerosol and splatter than a handheld curette, regardless of the scaler type used. 27 miller et al 28 studied the characteristics of blood-containing aerosols generated by common powered dental instruments. the author concluded that all the recovered particles could contain the hepatitis b virus and be inhaled and retained (20-100%) in the human respiratory system. the repeated and chronic exposure to bioaerosols generated during certain dental procedures and the relatively small particle size contribute to an increased risk of infection among dental professionals. 29 furthermore, the protection provided by surgical masks worn by dental professionals may be low for small particles; in addition, these masks may not fit perfectly in clinical practice. 30 a study reported that 15-83% of plasma aerosol particles ranging from 0.06-2.5 microns in size passed through the filters of 9 makes of surgical masks used by dentists. 28 these factors may play a role in the airborne route of viral infections among dentists and clinical dental auxiliaries. the likelihood of detecting, reporting, documenting and publishing dentistry-associated infections is relatively less. 31 although no definitive evidence of an extensive public health hazard from exposure to dental unit waterlines has been reported. 32 few case reports have suggested a definitive link between exposure to contaminated dental unit waterlines and legionella infection. 33, 34 disclosure. authors have no conflict of interest, and the work was not supported or funded by any drug company. infection control in dental practice. a review by scully and samaranayake 35 on the emerging and fluctuating viral diseases in the new millennium concluded that infection control plays an equally important role in the practice of dentistry as do the understanding of oral manifestations and the diagnosis and management of viral infections. while differences exist in the virology, epidemiology, and clinical outcomes of mers-cov and sars-cov infections, 36 the clinical symptoms of mers-cov resemble those of sars-cov, apart from acute renal failure. 37 although these results cannot be directly interpreted for a definitive conclusion, the management protocols for different mers-cov infection-associated with clinical scenarios for dental professionals may be similar to those of sars-cov. the implications of sars-cov for general dental practitioners, the significance of droplets and aerosols in disease transmission, the recommended management protocols for sars-cov infection and specific infection control measures have been well described by li et al. 38 a comprehensive review on universal and standard precautions has been published elsewhere 39 and is beyond the scope of this article, infection control measures pertaining only to pediatric dental practice in the context of mers-cov infection are discussed here. in pediatric dental practice, effective infection control measures for the prevention or minimization of viral infection transmission can be implemented by a) controlling the gag or cough reflex; b) reducing aerosol/ splatter generation; c) managing contaminated air and; d) improving personal protection. the gag or cough reflex may be stimulated by certain procedures, such as posterior intraoral and bite-wing radiographs and taking impressions. orthopantomographs or oblique lateral views may be considered instead of intraoral radiographs for screening, 40 whereas oral mucosa in very sensitive patients may be anesthetized before taking impressions. 41 sedation may also be considered to control gag reflex. 42 varying levels of physical, intellectual, emotional and social development of children and adolescents are major challenges for dentists especially attending to their psychological needs within adequate infection control regimen. toys provided for pediatric patients may be a potential source of cross-infection. soft toys are more likely to be contaminated, difficult to disinfect and may re-contaminate quickly compared to hardsurfaced toys. furthermore, restraining devices used to control movements of pediatric patients such as velcro fasteners may also be contaminated and should be disinfected accordingly. 43 extra-oral evacuation devices and special aerosol reduction devices may be used in combination with ultrasonic scalers to reduce the amount of aerosol produced. 44, 45 in high-risk cases, chemomechanical caries removal 46 or the atraumatic restorative technique 47, 48 may be utilized to prevent or reduce aerosol and splatter generation. in addition, high-volume evacuation removes infectious droplets at the source as they are emitted; thereby, minimizing, or preventing their dispersion in the air. to maintain their efficacy, the filters in the suction apparatus should be cleaned every day, and the exhaust air should be vented outside to prevent the recirculation of contaminated air. 38 contaminated air can be managed by improving dental clinic ventilation and/or by disinfecting the air. an ideal airflow pattern combined with a minimum of 3 air changes per hour has been recommended for dental clinics. [49] [50] [51] moreover, although its use in dental clinics is unconfirmed, ultraviolet germicidal irradiation may be installed and is effective against fungi, viruses, and bacteria, namely, tubercle bacilli and anthrax. 38, 49 on the other hand, measures of improving personal protection include washing hands frequently before and after treatment, using disposable barriers, dispensing instruments and materials just before treatment (thereby preventing particles from settling on the surfaces), and sterilizing soiled instruments. 38 after each patient visit, surfaces may be disinfected using hospital-grade disinfectants, which are effective against coronavirus. 52 personal protective equipment, such as gowns, hair covers, masks, gloves, shielded face masks and shoe covers, should be used as appropriate. 51 a higher level of respiratory protection should be considered, especially with aerosol-generating procedures. the use of n95 respirators offers a certain level of protection against the airborne transmission of sars-cov, although the exact level of protection offered by these respirators for individuals may vary. 53 examples of personal protective equipment such as face mask with plastic shield (figure 1 ) and n95 respirator (figure 2) . these respirators may also provide some level of protection against mers-cov and may be utilized instead of surgical masks in dental clinics. apart from these measures, vaccination of pediatric dentists and staffs working in pediatric dental clinics against measles, mumps, varicella and rubella may be necessary for their protection from these viral infections. 54 in conclusions, considering the unique work environment of dentists, which involves close patient contact and aerosol production, the risk of mers-cov transmission from an infected patient is high. children are also prone to mers-cov infection. as the number of mers-cov cases may increase in future, pediatric dentists should be well informed and educated about not only the signs and symptoms of the condition but also how to follow stringent infection control measures in these cases. middle east respiratory syndrome coronavirus disease in children isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov). update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of middle east respiratory syndrome (mers): a new zoonotic viral pneumonia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mers-cov outbreak in jeddah--a link to health care facilities a scenario-based evaluation of the middle east respiratory syndrome coronavirus and the hajj molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome middle east respiratory syndrome coronavirus in children factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises aerosols and splatter in dentistry: a brief review of the literature and infection control implications screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions effects of coronavirus infections in children cultivation of bacteria and fungi occupational health problems in modern dentistry: a review blood contamination of the aerosols produced by in vivo use of ultrasonic scalers aerosol and splatter contamination from the operative site during ultrasonic scaling characteristics of blood-containing aerosols generated by common powered dental instruments measurement of airborne bacteria and endotoxin generated during dental cleaning efficacy of three face masks in preventing inhalation of airborne contaminants in dental practice infected health care workers and patient safety: a double standard risk assessment of dental unit waterline contamination a survey of methods used to detect nosocomial legionellosis among participants in the national nosocomial infections surveillance system legionella contamination of dental-unit water emerging and changing viral diseases in the new millennium middle east respiratory syndrome coronavirus "mers-cov": current knowledge gaps in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection severe acute respiratory syndrome (sars) and the gdp. part ii: implications for gdps guidelines for infection control in dental health-care settings essentials of dental radiography and radiography oral bioscience. london (uk): churchill-livingstone sedation in dentistry. part 2: management of the gagging patient infection control issues related to pediatric dentistry reduction of bacteria-containing spray produced during ultrasonic scaling the usefulness of the modified extra-oral vacuum aspirator (eova) from household vacuum cleaner in reducing bacteria in dental aerosols chemochemical caries removal: a review of the techniques and latest developments the atraumatic restorative treatment (art) technique: does it have a place in everyday practice? atraumatic restorative treatment (art): rationale, technique, and development the application of ultraviolet germicidal irradiation to control transmission of airborne disease: bioterrorism countermeasure infection control for the dental team. copenhagen: munksgaard additional precautions for tuberculosis and a self-assessment checklist managing sars amidst uncertainty possible sars coronavirus transmission during cardiopulmonary resuscitation safety management and infection control in pediatric dentistry key: cord-326851-0jxdnm1l authors: lee, sang m.; lee, donhee title: lessons learned from battling covid-19: the korean experience date: 2020-10-16 journal: int j environ res public health doi: 10.3390/ijerph17207548 sha: doc_id: 326851 cord_uid: 0jxdnm1l background: the covid-19 pandemic has swept the world like a gigantic tsunami, turning social and economic activities upside down. methods: this paper presents some of the innovative response strategies implemented by the public health system, healthcare facilities, and government in south korea, which has been hailed as the model country for its success in containing covid-19. korea reinvented its public health infrastructure with a sense of urgency. results: korea’s success rests on its readiness, with the capacity for massive testing and obtaining prompt test results, effective contact tracing based on its world-leading mobile technologies, timely provision of personal protective equipment (ppe) to first responders, effective treatment of infected patients, and invoking citizens’ community and civic conscience for the shared goal of defeating the pandemic. the lessons learned from korea’s response in countering the onslaught of covid-19 provide unique implications for public healthcare administrators and operations management practitioners. conclusion: since many epidemic experts warn of a second wave of covid-19, the lessons learned from the first wave will be a valuable resource for responding to the resurgence of the virus. the recent spread of the coronavirus (covid-19) has thrown the world into total chaos. covid-19 has caused not only a health and social crisis of immense proportions forcing people to deal with the fear of infection and the physical, emotional, and financial damage from government recommended physical distancing, but it has also caused a global economic turmoil [1, 2] . the virus started spreading in wuhan, china in late december 2019, and became a shocking pandemic by mid-march 2020; by this time, it had already caused several hundred deaths, disrupted the global economy, and forced countries to close their doors to visitors [3] [4] [5] . the world health organization (who) defined covid-19 as "an infectious disease caused by a newly discovered coronavirus" [6] . who declared the novel coronavirus outbreak a global pandemic, the highest category for infectious diseases, on 11 march 2020, officially notifying the global community that a health crisis is upon us [3, 7] . major infectious diseases that have occurred during the past 20 years include severe acute respiratory syndrome (sars: 2003), novel influenza virus (h1n1: 2009), middle eastern respiratory syndrome (mers: 2012), avian influenza (2014 and 2017), and covid-19 (2019), most of which were originally reported in china. particularly, covid-19 has potent infectivity, implying that it spreads quicker and its mutations are more complex than other infectious diseases. as the numbers of confirmed cases and deaths began to rise exponentially, halting the coronavirus became a global issue. in addition to the immediate health concerns, the covid-19 pandemic disrupts and destroys global supply chains along the associated systems of purchasing, manufacturing, logistics, and sales [8] . this study attempts to present directions for potential changes in the crisis response systems of public healthcare worldwide, by analyzing covid-19 pandemic response cases, both successes and failures, in korea. more specifically, this study has the following objectives: (1) to analyze korean experiences with cases where healthcare facilities failed to prevent previous infectious diseases from spreading, and how these failures served the government in devising effective approaches to encounter the covid-19 pandemic, (2) to dissect cases that showed innovative and successful response measures to deal with the covid-19 pandemic, and (3) to elaborate on suggestions for crisis management based on the lessons learned from these covid-19 response cases in korea. the rest of this paper is structured as follows. in section 2, we present a review of relevant literature on global infectious diseases and covid-19 as well as several real cases of korean healthcare providers in managing the covid-19 pandemic. section 3 presents the innovative response strategies deployed in korea (k-response model) to fight the pandemic. in section 4, we summarize the important lessons learned from the korean experience. we conclude the study in section 5 by discussing implications of the study results, limitations of the study, and future research needs. infectious diseases refer to those "caused by pathogenic microorganisms, such as bacteria, viruses, parasites, or fungi; the diseases can spread, directly or indirectly, from one person to another" [20] . these diseases can infect people by contact with other humans, animals, or other reservoirs infected by a pathogen or toxic substance. additionally, communicable diseases refer to those that directly or indirectly spread between humans or between humans and animals. thus, an infectious disease pandemic is an epidemic that has the potential to be easily transmitted and to affect the global population due to its highly infectious nature [21] . since the second world war, the world has seen many innovative developments in vaccines and antibiotics; such advances have ensured that communicable and infectious diseases are reasonably controlled [11] . an important study by the institute of medicine, "emerging infections: microbial threats to health in the united states," shed some light on the issues involved with novel infectious diseases [22] . soon thereafter, the who passed the resolution "global health security: epidemic alert and response" in 2001 [23] , which enabled the collection of information and enforcement of actions through cooperative work by inter-governmental agencies, non-governmental institutions, private organizations, and governments around the world. in 2003, the sars outbreak in china quickly spread fear of a pandemic that could cross borders and affect countries worldwide. particularly, china failed to promptly and transparently disclose epidemic information. the chinese government reported the outbreak to the who several months after the first confirmed case of sars, thus delaying effective response measures by world organizations. immediately, the who raised the alert that the sars outbreak was of high risk, subsequently issuing a travel advisory notice (e.g., advising a travel ban to places where the epidemic had occurred) aimed at suppressing the spread of the disease. as a result of this experience with sars, many countries worldwide recognized the importance of a global infectious disease governance system, which should stretch beyond the governance of each country [24] . in 2005, the international health regulations were revised and expanded to include not only communicable diseases but also other possible threats (i.e., biological terrorism and events that induce international public health crises). nevertheless, other highly contagious diseases have continued to emerge throughout the last two decades. in the presence of healthcare emergencies, such as the infectious and coronavirus outbreaks discussed above, public healthcare should be available throughout the country not only with rapid response but also based on an equitable basis [5, 19, 25] . the sense of equity involves a person's perception of the input and output relationship which should not create tension or displeasure as a result of cognitive dissonance [26] . rousseau [27] defined equity as a function of customer's perception in a service encounter experience. equity is realized when a person believes his/her outcome concerning resources invested is in harmony with that of others [28] . equity theory has become the theoretical foundation for service recovery as it helps create possible recovery approaches for service failures through recognition, procedures, and mutual interaction involving customer complaints [29, 30] . it is important that people perceive that a service is being provided equitably. especially in a crisis such as the covid-19 pandemic, it is imperative for people to perceive that urgent public healthcare is being provided equitably [5] . such perceived equity inspires people to be transparent about their activities (e.g., infection status, contacts, self-quarantine, etc.) that are the first line defense against the disease. this study examines the successes and failures of the korean healthcare organizations in their efforts to contain covid-19, from an equity perspective relating to healthcare services. korea has a history of responding poorly to infectious diseases (e.g., sars, h1n1, and mers). in 2012, saudi arabia was the first country to experience the mers outbreak. korea was the most detrimentally affected country by the virus as many korean global firms have operations in saudi arabia. in korea, the first mers case was confirmed in 2015 and its rapid spread resulted in a significant number of casualties which heightened anxiety that swept throughout the country. furthermore, the serious blow caused to the national economy clearly revealed the weakness of korea's infection crisis management system [10, 31] . the korean government's limited response capacity regarding mers and its poor communication to its citizens weakened people's trust in the government's infection crisis management policies to the point where many started believing that the national epidemic prevention system could easily collapse [31] . there were 185 confirmed cases of mers among those who traveled to the middle east, 38 of whom died in 2015. the causes of the high mortality rate could be attributed to the limited capacity of the healthcare delivery system for handling the new virus, shortage of epidemic prevention equipment for medical first responders, and the moral hazard among patients [10, 31] . after painful experiences in dealing with past diseases, the korean government was determined to establish an effective infrastructure to deal with future epidemic emergencies, with kcdc as the control tower. the new infrastructure includes an increased number of negative-pressure isolation wards, real-time systems for data and transparent information collection and analysis, and modernization of the healthcare system. since the mers crisis, the korean government has reinvented a national healthcare delivery system equipped with advanced digital technologies and expanded the facilities specifically designed to deal with infectious diseases (e.g., the creation of negative pressure wards) [10, 31] . thus, kcdc was well prepared to respond effectively to epidemic emergencies when the covid-19 crisis occurred. when covid-19 began to spread, the korean government raised the response level to serious (the highest) on 23 february 2020 and promptly established the central disaster and safety countermeasure headquarters, headed by the prime minister to bolster government-wide responses to the virus with kcdc as the command center [32] . according to the korea economic daily [33] , the rapid spread of covid-19 around the world, especially in china, italy, and in the united states, and the subsequent spike in the number of deaths, has brought global attention to the prevention model and early response operational strategy implemented by daegu city, the epicenter in korea. daegu took aggressive actions with speed to prevent the collapse of its healthcare system without placing the city in a lockdown [34] . the city government performed aggressive screening, testing, and quarantining of patients in the communities that were confirmed to have, or suspected of having, infected citizens. according to kcdc [32] , daegu city did not implement this approach at the onset of the covid-19 outbreak in korea (18 february 2020). daegu and the north gyeongsang province (where daegu is located) were heavily criticized for the exponential growth rate of infected patients as ground zero. this region accounted for 70% of the confirmed cases in korea, caused primarily by the shincheonji church gatherings (worship services where people sat on the floor shoulder-to-shoulder) and the mass infections that occurred among the first medical responders while providing care services. the city government performed screening tests of the entire congregation of the shincheonji church, isolated severely ill patients, and secured enough quarantine beds for those in need of treatment and isolation. to achieve this, daegu operated a public-private partnership (ppp) network (composed of the emergency response advisory group, the daegu medical association, and three infectious disease management support groups), which served as the control team for the covid-19 epidemic [35] . the ppp collaboration network deployed several response strategies against covid-19. first, private hospitals were converted into isolation hospitals. a group chatroom for the control team was created, through which experts held discussions about the situation throughout the night. through these discussions, the daegu dongsan hospital and the ministry of defense were contacted and asked to secure as many beds as possible at the daegu armed forces hospital and daejeon hospital. at that time (18) (19) (20) (21) (22) (23) (24) (25) , there were only about 30 available negative pressure wards in daegu, and some confirmed patients died while waiting to be admitted into a hospital. second, the entire congregation of the shincheonji church was tested, and those who had symptoms were identified. on the night of 18 february 2020, 70% of confirmed cases of covid-19 were members of the shincheonji church. the daegu secured information on the 3000 members of the church, identified them, and ordered 544 symptomatic patients to remain in self-quarantine for two weeks. a leading physician at the kyungbook national university stated that "the rate of confirmed cases reached 80% among patients who showed symptoms; so, if we had not prompted early isolation of those shincheonji church members who showed symptoms, daegu might have been in the same situation as europe or the united states" [33] . third, members of the daegu medical association provided care to those patients in self-quarantine via video calls using 100 outgoing-call-only smartphones provided by daegu city, hence eliminating the previously existing void in the response system. these response activities represent innovative strategies implemented in the initial stage of the covid-19 invasion (e.g., aggressive testing, almost immediate test results, contact tracing of infected persons, and prompt treatment of severely ill patients). the mortality rates in new york, usa (6.44%) and in madrid, spain (12.62%) are much higher than that in daegu, korea (2.76%) as of 1 june 2020 (see table 1 ). these high mortality rates indicate that their patient monitoring and healthcare facilities operations were not systematic. it is evident that "the most potent operational strategy amid the lack of a cure is not search and destroy, but identification and isolation of symptomatic citizens" [33] . fourth, drive-through screening centers were developed for the first time in the world, supported by a world-leading ict infrastructure [36] . the yeungnam university medical center, in the vicinity of daegu, had admitted a covid-19 patient on 19 february 2020, which resulted in the closing of the emergency room (er) and prompted self-isolation of its medical staff. based on this experience, the medical center decided to establish a drive-through screening center, which eliminated the risk of shutting down er and self-quarantining first responder medical staff. moreover, the existing screening center was inefficient in handling the large crowd of people needing testing in a small space. thus, this was another innovation in need that led to the strategy of developing drive-through testing centers. laura bicker, a bbc correspondent in seoul, referred to the drive-through testing centers as "such a clever idea and so quickly set up". sam kim, an economics reporter at bloomberg, mentioned that korea "once again proved to be among the world's innovative nations," and ian bremmer, president of the eurasia group, a think tank in the us, stated that "innovation drives resilience" [37] . innovative operation strategies are critical in fighting such a formidable global pandemic as covid-19. finally, creative applications of the national ict infrastructure and rapid development of mobile apps by young entrepreneurs have helped analyze the details about confirmed patients and their contacts (e.g., locations, people contacted, and travel patterns before their infection confirmation). kcdc [32] collected and released relevant information (e.g., regions, pockets of high infection density, and places visited) on covid-19 patients in real-time. such transparency regarding the handling of covid-19 patients encouraged citizens to voluntarily participate in physical distancing and personal hygiene. this is another strategy that has helped korea effectively manage the crisis when compared to other nations such as italy and the us. in korea, after kcdc disclosed the movements of the first covid-19 patient in daegu on 18 february, all the places the patient had visited were immediately shut down and disinfected; moreover, the government analyzed security footage (e.g., cctv from the entrance of the church) to help identify and isolate anyone who might have come into contact with the patient. local governments sent out emergency alert text messages to provide real-time updated information so that the population of other provinces and cities could be advised not to visit the infected locations. table 1 summarizes the current (as of 1 june 2020) state of the cities with the greatest number of confirmed cases among countries most affected by covid-19, and the innovative operational strategies implemented by daegu to respond to the covid-19 pandemic. daegu, the epicenter of covid-19 cases, is currently in the process of being transformed into a smart city. it is noteworthy that the city/area where an explosive outbreak of covid-19 cases occurred had higher mortality rates than that of the country as a whole. table 2 summarizes statistics of the number of confirmed covid-19 infection cases, deaths, and mortality rates among the top 10% of 169 countries, including italy, china, and south korea, as of 7 september 2020. the table also indicates average statistics for the entire 169 countries. figure 1 shows the mortality rates of the top 10 countries for covid-19 infection, including south korea, as of 7 september 2020 (including the average for the entire 169 countries as a group). the bars in the figure show the number of deaths per 100,000 population. in the early phase of the coronavirus spread, south korea recorded the second highest number of infected persons after china. however, as shown in tables 1 and 2 and figure 1 , korea initiated aggressive strategies for testing and contact tracing based on its well-established public health infrastructure. thus, the country has been able to flatten the curve of infected cases which resulted in relatively low rates of deaths/infected cases and mortality (deaths/100,000 population) [44] . source [45] . the crisis caused by a pandemic can lead to issues of equity in the public healthcare service [46] . particularly, failures in providing equitable public healthcare and in community participation in the decision making process should not be repeated. thus, it is necessary to analyze the successes and failures experienced in the current situation (i.e., the first wave) as a preparation for the possible mortality: deaths/100k population (7 september 2020) the crisis caused by a pandemic can lead to issues of equity in the public healthcare service [46] . particularly, failures in providing equitable public healthcare and in community participation in the decision making process should not be repeated. thus, it is necessary to analyze the successes and failures experienced in the current situation (i.e., the first wave) as a preparation for the possible onslaught of the second wave of the pandemic. to conduct an in-depth analysis of the causes and consequences of the covid-19 virus spread in korea, we examine the operational procedures and strategies implemented by several healthcare facilities. many seriously ill patients with the virus were diagnosed or infected in er. even in a normal day, er operates at the disaster level [47] . the covid-19 pandemic severely tested the agility, flexibility, and resilience of er operations at every hospital. the following five cases are investigated based on the information released by kcdc [32] . hospital a is an 808-bed tertiary general hospital located in seoul with 2000 employees. the hospital provides care to an average of 600 inpatients and 2000 outpatients daily. the first covid-19 case in this hospital was confirmed on 21 february 2020, and was consequently quarantined for two weeks until 5 march. a careful contact tracing of the patient resulted in 14 additional confirmed cases within the hospital. the first confirmed case in the hospital was a patient aide who helped patients move from the ward to the lab. prior to the diagnosis, it was found that this aide had helped 207 patients. after the diagnosis of the first confirmed case, the hospital's er and outpatient clinics were closed and quarantined. hospital b is a 251-bed general hospital with 644 employees located in seoul. it was closed for two weeks, from 8 to 22 march, due to a patient's dishonesty. the patient was a resident of daegu city but falsified his home address when hospitalized. after being diagnosed with covid-19, the patient was isolated. the hospital shut down its outpatient center, some wards, and er. the particular concern which this patient caused was the fact that the person had visited the artificial kidney unit, contacting many high-risk patients. although there was no additional positive case found, the hospital was quarantined for two weeks. hospital c is a psychiatric hospital (housed on floors 8-11 in a high-rise building) in daegu with 286 inpatients and 72 employees. the first positive diagnosis of covid-19 was made on 26 march, after which the number of confirmed cases increased at an alarming rate. this case happened because the first patient was originally at a convalescent hospital (located on floors 3-7 in the same building) where the first positive case was tested on 20 march and spread quickly to133 patients. when the first case was confirmed at the convalescent hospital, the building management firm failed to disinfect the entire building and consequently the virus spread to hospital c. hospital c conducted virus tests on its own employees but did not screen all patients because all the employees were tested negative. there were 196 infected patients as of 20 april and the hospital was closed. kcdc conducted a thorough investigation of hospital c and found that the mass infection occurred because of the failure to screen all patients [32] . further, due to the nature of the psychiatric hospital (i.e., closed wards in confined spaces), the infection spread rapidly, and subsequently the number of infected cases increased at an accelerated rate. hospital d is a 700-bed general hospital located in seongnam city, in the vicinity of seoul, with 1400 employees, including 140 specialists in 26 specialty areas. the hospital serves an average of 5000 patients daily. one patient was discharged after treatment in the hospital and returned to receive outpatient care. the same patient became very ill and was brought to er and diagnosed with covid-19 infection on 5 march. consequently, a mass infection of the virus occurred among healthcare providers within the hospital (43 confirmed cases). hence, the hospital was closed from 6 march to 12 april (38 days). hospital d provided incomplete information to kcdc, as a person in need of isolation was omitted from the list [32] . hospital e implemented proactive measures to suppress the spread of covid-19. all patients and their respective caregivers were required to fill out a paper-based health questionnaire upon admission. however, there were concerns about having the patients complete the questionnaire, which might take too much time while they were crowded in a limited space. in order to reduce crowding and minimize hospital-acquired virus infections, a mobile health questionnaire was delivered by the hospital. the questionnaire asked patients to precisely list all travel to foreign countries, visits to regions or facilities with confirmed cases, and any symptoms of fever or respiratory difficulties. hospital e reported that (https://www.yuhs.or.kr/en/), between 12 and 19 march, an average of 6136 people submitted the mobile health questionnaire each day. on average, the questionnaire took 1 min 29 s to complete (8.9 s for each of 10 items). further, hospital e sent the mobile health questionnaire to visitors scheduled for outpatient services or testing at 6 am on the day of the appointment via kakaotalk (korean sns) or text message. once the patient had completed the questionnaire, a quick response (qr) code was generated. if the patient had self-reported covid-19-related flagging, a red qr code was assigned, and no flagging was noted with a black qr code. only visitors who had the red qr code were issued with proper stickers and allowed to enter the hospital. the patients with the red qr code were required to undergo an additional evaluation at the hospital entrance. based on this evaluation, they were either directed to a designated safe care facility or were not allowed to enter the hospital for treatment. moreover, visitors who were not able to use a mobile questionnaire or were not aware of this requirement were provided with the paper-based questionnaire at the entrance. the implementation of this mobile questionnaire was an effective measure to reduce hospital-acquired infections, among the patients and between employees and patients (hospital press release; https://www.yuhs.or.kr/en/). table 3 presents a summary of problem causes and response strategies employed by the sample hospitals. during a pandemic crisis, the government should encourage people to take preventive measures for their own safety, and share information transparently, including that on the risk involved. after failing to effectively manage the mers outbreak in the past, korea improved its public health infrastructure and expanded its relevant medical facilities. this experience enabled the korean government and healthcare providers to develop innovative response strategies to covid-19. drive-through and walk-through testing centers were implemented for the first time in the world. these systems have been heralded as a creative response model to the pandemic, and they have been benchmarked and copied by countries worldwide [14, 36, 48] . the compelling motivation for these systems was rooted in the challenging problems experienced by some screening centers. these centers were overwhelmed by an onslaught of patients with suspected covid-19 symptoms. the average waiting time for a screening was as high as six hours or even longer in some cases, consequently raising serious cross-infection concerns [32] . a drive-through screening center can process suspected patients through several steps (e.g., completion of the patient questionnaire, physician's examination, sample collection, and education) without them getting out of their automobiles, hence shortening the testing duration to less than 10 min per patient [32] . these testing systems lower the risk of infection, minimize contact between test recipients and healthcare providers, and save time. in a regular screening center, the room must be disinfected and ventilated after treating each patient. drive-through centers, on the other hand, do not require such procedures, thus not only diminishing the risk of contamination but also greatly increasing the number of daily cases handled. for example, a regular screening center could process two cases per hour (20 daily cases), whereas a drive-through center can process up to six cases per hour (60 daily screenings) [32] . furthermore, the people being tested preferred the drive-through system because the waiting time for the test is far less and the queue time is spent in the comfort and safety of their own cars [48] . the most challenging task in controlling the spread of covid-19 is making sure incoming international travelers are not carriers of the virus. thus, kcdc set up 16 walk-through outdoor screening booths (eight for each of the two international terminals). all incoming passengers are tested as they walk through the station at the rate of 5 min per person, screening more than 2000 passengers per day. one of the most important aspects of suppressing the spread of the pandemic is the battle against time. performing aggressive testing in a short period of time to identify and isolate confirmed patients is the most critical operational strategy for flattening the curve of infection cases [49] . the screening systems discussed here represent innovations developed due to the desperate need to contain covid-19 at the most critical point of time, the beginning of the rapid spread stage [32] . on 10 march 2020, the korean government established residential treatment centers to treat confirmed covid-19 patients while maintaining their quarantine. in this residential system, each room is occupied by one person as a rule, and all residents must conduct self-monitoring twice daily and are on constant monitoring and treatment by health professionals residing in these centers. such centers' primary aim is to triage and categorize patients into four levels (mild, moderate, severe, critical), and the secondary aim is to manage patients with mild conditions. this system helps ensure that hospital beds are provided for covid-19 patients needing critical inpatient care. the resident treatment centers are managed independently by each local government. the primary goal of these centers is to prevent the spread of the virus by treating, isolating, and medically monitoring infected patients. while it is not necessary to send patients with mild symptoms to hospitals, it is still necessary to isolate them as they have the potential to widen the spread of the disease. thus, these patients are the major targets of residential centers. to achieve their strategic objectives, these residential centers need to make their operations flexible and dichotomous, encompassing both hospitals and centers. hence, their provision requires accurate triage of confirmed patients, active participation by residents, and strict compliance with the care policies and regulations within the center. these residential treatment centers represent an innovative model of care that has helped prevent the spread of the infection to the community while addressing the shortage of hospital beds. "untact" means no human-to-human contact in service encounters [17] . during the covid-19 pandemic, korea has been able to provide untact services to its population based on innovative applications of digital technologies. this operational strategy has contributed a great deal to suppressing the spread of covid-19 through an advanced healthcare system (e.g., drive-through screening centers). one of the best examples of such untact services is the self-diagnosis app and the self-quarantine protection app [32] . on 10 february 2020, the central disaster management headquarters of korea announced that it was going to use a self-diagnosis app to monitor the virus infection status of all incoming travelers through a special entry procedure in order to strengthen infection management. this app monitors the symptoms once a day and provides a quick consultation by medical staff for everyone who enters the country. after this app became mandatory, permission to enter korea has been granted to only those (citizens and foreigners alike) who provided their personal information (e.g., name, passport information, nationality, and actual address) in the app. anyone entering korea must consent to use the app and download it themselves as a special entry procedure. the results of the self-diagnosis feature (i.e., fever, cough, and/or sore throat) are submitted to the corresponding public health center and kcdc. moreover, this app provides important information to the user about screening centers (i.e., the closest available area/location for inspection and contact information), the sns channel of kcdc, and 1339 consultation call centers. untact technology-based approaches are currently making a substantial contribution to address the challenges regarding patient management, shortage of healthcare staff, and lack of resources needed to provide a diagnosis. additionally, aimed at healthcare providers, a dashboard was developed by the korea university medical center and softnet to automatically send information about patients' (self-assessed) body temperature, symptoms recorded on the app, pulse and blood pressure (taken with a bluetooth blood pressure cuff) to healthcare providers [50] . the application of such innovative apps involves a trade-off between the concern of staying safe from the virus and personal privacy [50] . nonetheless, these measures have proven to be essential for preventing the spread of the pandemic through prompt and convenient reporting of symptoms among all travelers to korea. the crisis caused by a pandemic requires a robust response system to prevent global health, economic, and social disasters. the key lesson learned from the korean experience in managing the covid-19 crisis is the importance of innovative response strategies at the onset of the pandemic. based on the review of some of the experiences of sample hospitals and the practices that have been proven effective, an innovative pandemic management system should include the following strategies. the pandemic response cannot be made by one independent organization. there should be a collaborative public-private partnership that can utilize the synergistic capabilities of governments, private enterprises, healthcare institutions, university research centers, and volunteer organizations (e.g., daegu's collaborative network: emergency response advisory group + daegu medical association + daegu center for infectious disease control and prevention). such a collaborative innovation partnership is imperative to generate creative strategies, operational plans, and detailed work procedures. when a collaborative system is established, especially with many volunteer entities, it is difficult to seamlessly execute strategies and actions in a timely fashion in the face of the rapidly spreading pandemic. thus, an explicitly designed governance system with clearly defined roles, responsibilities, and authorities is as important as the strategic plans. in the exponential increase stage of the pandemic infection, healthcare facilities in the ground zero area could be overwhelmed by severely ill patients, causing the collapse of the system. for effective response management, healthcare facilities in the affected area (cities and surrounding counties) should be integrated as an emergency response system. then, the various healthcare facilities can be dichotomized based on their scale and core competencies so as to designate some as intensive care facilities where severely ill patients are quickly assigned for treatment and others as safe facilities where patients with non-virus related illnesses are treated [4] . fighting a pandemic requires much more than simply testing and treating infected patients. it is essential to secure a sufficient quantity and quality of medical supplies, protection of frontline health professionals, and secure supply chains. it is imperative to develop an effective logistics system for timely delivery of medical supplies (e.g., testing kits, medications, ventilators, additional er facilities, ambulances, helicopters, etc.). the healthcare delivery system is bound to collapse if frontline healthcare providers are infected because of insufficient or ineffective personal protective equipment (ppe) such as face shields, masks, and body covers (for head, hands, shoes, and body). in addition, stable and emergency supply chains are necessary to ensure timely supply of all medical and other supplies in order to provide urgent care to the patients on an on-demand basis in the face of the pandemic crisis [8] . the spread of a pandemic is not even throughout a country or region. while a national pandemic management center is necessary (e.g., kcdc) to develop nation-wide guidelines and strategies, each region of the country has its own unique patterns of virus infection. thus, each local area government or virus control center should be allowed to establish its own strategies to control the spread of the pandemic. as the virus spreads throughout the community at a rapid pace, the need for mature civic consciousness usually grows proportionately. the current physical distancing campaign is a good example. many countries incurred enormous damage due to citizens not abiding to recommended guidelines to prevent the spread of the pandemic (e.g., social distancing, shelter-in-place, personal hygiene, etc.), awakening the need for social co-consciousness and citizen engagement [51] . therefore, policymakers of the central government should develop definite policies regarding the devolution of control and direction, which are required of all citizens during the critical phase of a pandemic. the covid-19 pandemic has brought a total upheaval to the way people live, businesses operate, and governments function. the pandemic has infected more than 27 million people, brought profound sadness to people who lost their loved ones (881,464 deaths as of 7 september 2020), and hundreds of millions lost their jobs around the world in a matter of several months. we must learn from our experience of failure, and some successes, so that we can be better prepared to prevent and manage future pandemics. there are several lessons that have now been learned and identified as important elements for managing a pandemic from the covid-19 experience in korea. at the time of the mers outbreak, the ministry of public safety and security of korea sent out tips to the entire population on how to prevent the virus via an emergency text message. however, this only occurred during the early days of the outbreak, and the government's crisis management system-which should have directed efforts to manage the epidemic-failed to function properly [10] . consequently, despite having a response system for infectious diseases, the government failed to effectively execute early responses and was criticized for aggravating the damage caused by mers. this inefficacy of early responses resulted in human casualties, public anxiety, and substantial economic damage, all of which revealed the weakness of the government's infection crisis management system [52] . this failure not only made the korean government recognize the vulnerability of the national epidemic prevention system but also provided an opportunity to learn from the failure [31] . leadership was recognized as a key contributor to the successful containment of the sars outbreak in korea in 2003. previous studies show that leadership has played a significant role in the effective implementation of proactive measures and facilitation of inter-agency communication in the face of an epidemic [53] . more specifically, during the current pandemic outbreak, the national crisis management capacity which was strengthened due to the lessons learned from mers helped execute necessary response steps at the critical early stage of the pandemic spread. an emergency leadership team should be established with experts in the relevant fields to develop specific strategies for prevention, response measures, and medical treatment procedures to contain the pandemic. the international media have been reporting korean strategies and practices for controlling covid-19 as a model [44, 54] . most korean provincial governments have been effectively containing the spread of the pandemic without shutting down business operations such as restaurants, coffee shops, retail stores, and even golf courses [48, 54] . particularly, the international media praised korea's testing capacity with its innovative drive-through and walk-through stations, rapid processing for results (less than 4 min), and the korean government's information management that did not require a lockdown enforcement like wuhan, china. the new york times [55] reported that, if south korea succeeds in containing the infection as it has, it will set an example for the world to follow. johnson et al. [56] also noted that korea's advanced diagnostic capability was proven to be the enabler of its massive testing of more than 35,000 a day, while the u.s. was testing a mere 426 persons (reported on 25 february 2020). moreover, health korea news [57] reported that the mortality rate of covid-19 patients was only 0.7% among 10,000 infected patients during the early stage of covid-19 spread. it is evident that the korean government's quick response capacity, information disclosure transparency, and implementation of innovative models to protect patients and healthcare providers were the key factors for early success in containing the pandemic. korea had a bitter experience with mers which helped the government to learn from failures and then establish a proven infrastructure to handle the spread of pandemics. a set of best practices, what is now known as "the k-response model," is based on standardized proven systems that can be applied by all healthcare providers, including testing procedures, contact tracing, isolation by self-quarantine, hospitalization, treatment procedures of severely ill patients, and release after being cured. the control tower in central government and in each local government should proclaim a standardized preventive system (e.g., closing all large audience events such as sports, theaters, shopping centers or educational institutions; limiting the number of people in any group gathering to 10 or less, with social distancing of 6 feet or more; the stay-at-home policy; closing all business operations for 2-4 weeks, including all personal services such as hair salons, massage parlors, physical therapy centers, dental offices, etc.) [32] . the operating systems and strategies that the korean government implemented to contain covid-19 have proven to be effective. the ebola outbreak in west africa in 2014 caused more than 10,000 deaths with a 40% mortality rate. the affected governments' approaches to combatting the virus failed to foster trust among the citizens, causing fear, and inaccurate information led to a considerable amount of time spent on tracing the actual movements of those infected [58] . a previous study analyzed the outbreak of novel infectious diseases since 1990, focusing on global pandemic management systems [11] . this study stressed the importance of transnational monitoring and information sharing about the spread of diseases (e.g., disease symptoms and infected areas). another study investigated the outbreak of sars and pinpointed the chinese government's failure to provide effective early response to the pandemic, either concealing or underreporting, as the reason for the global spread of the disease [59] . the korean government, in response to the current covid-19 pandemic, has been reporting the number of confirmed cases, the number of deaths, and the actual movements of confirmed patients in real-time. such government efforts for transparency and urgency regarding the pandemic have gained the public's attention regarding the risk involved. such government efforts also gained the trust of citizens and helped people comply with issued guidelines. the government also announced disinfection measures and schedules for locations where confirmed patients made contact with others on a daily basis. this information indirectly advised the general public not to visit those hot spots. in addition, local governments sent out text messages to all citizens upon confirmation of a new infection case in their region and other helpful safety tips about the virus. such real-time information sharing about the pandemic has been possible because of the advanced mobile technology infrastructure and the public's high mobile device usage in korea. as covid-19 began to spread throughout the country in february 2020, various apps were developed rapidly by young entrepreneurs (e.g., apps showing confirmed patients' locations, the closest place where masks and gloves can be purchased, assistance tips for self-quarantine, etc.). currently, several european countries (e.g., uk and italy) are also attempting to utilize a gps tracking system to locate confirmed patients and to inform disinfection and prevention activity areas using smartphone apps. a team of medical researchers at oxford university in england published a report suggesting that communicable diseases can be controlled effectively if many utilize digital contact tracing [60] . based on the current covid-19 situation in korea, while the government's response capacity is crucial to prevent the disease from spreading, mature civic consciousness is essential to ensure social compliance with the imposed measures. in korea, people did not engage in panic buying during the early days of the pandemic outbreak. for example, when there was a shortage of masks, instead of the profiteering behavior of some hoarders, many people began to donate some of their daily allotted masks to the community for those in need. mass media also encouraged beneficence through public emotions showing a strong spirit of community and unity. people recorded their daily lives in sns, including health status and places visited, to help prevent the infection from spreading to others around them. some people even traveled only on foot to prevent spreading the disease in the community. furthermore, people complied with the five-day rotating purchase system for masks, which was instituted by the government to ensure a fair distribution to everyone in the face of mask shortages. the global media has praised koreans for their voluntary civic engagement and compliance with government guidelines to contain covid-19. the washington post [61] reported that korean citizens canceled major events and most religious services were held online at the outset of the pandemic breakout. daegu, the epicenter of a massive spread of the virus, was able to manage the situation without a lockdown, as people in other parts of the country voluntarily refrained from visiting the city. moreover, the bbc news [62] stated that south korea was able to manage the spread of covid-19 without implementing a complete lockdown or strict measures against people's movement. koreans voluntarily wore masks everywhere outside of their homes and were tested for covid-19, demonstrating mature community and civic consciousness. responding together and responsibly to the threat of covid-19 have now become a battle cry for koreans [63] . contrastingly, there were cases where civic duty was not practiced. there were incidents where people lied about their addresses, pretending to be from an area of mass infection, to receive priority care. there were also cases where people under the required self-quarantine violated the isolation guidelines and roamed around the community restaurants and coffee shops, thus spreading the infection [32] . there was a major relapse of the virus infection after two weeks of almost no daily infection had been reported in korea. during 24 april-6 may 2020, over 5500 young party goes visited several night clubs in seoul during the social distancing enforcement period. these clubs are known for their loud music, dancing, and drinking in a rather confined space. these clubs often restrict entry only to young people, enforced by a reverse carding system (usually only under 40 years of age). among those who visited the clubs, more than 270 infected people were identified by june 1. however, there was a social stigma issue (regarding the sexual orientation of many regular customers of the clubs) involved which made contact tracing difficult for those who visited the clubs. it was reported that many club goers falsified their names or addresses (e.g., cell phone numbers). to suppress the spread of a dangerous pandemic, a spirit of unity and shared purpose is required. korean people realized the potentially devastating chain of infection that could sweep through their communities and the country as a result of the misguided actions of a single person. in addition to the government's control measures, the public's strict adherence to government guidelines and voluntary participation in implementing certain rules (e.g., mask rationing) based on a sense of community have played a major role in suppressing the spread of covid-19 [32, [62] [63] [64] . in response to the covid-19 pandemic, physical distancing has been encouraged based on the recommendation of the who [2, 32, 64] . as people refrain from engaging in outside activities, many businesses (e.g., restaurants) start to experience financial difficulties. the drive-through covid-19 screening model has been applied to other businesses, and a new drive-through shopping model emerged. for example, south korea's large seafood markets are utilizing their parking lots to provide drive-through services, where customers can order sushi meals from their cars as they approach the market and vendors fulfill the order immediately. department stores are delivering pre-ordered products to customers at the valet parking service lot. in addition, services such as drive-through book-lending and agricultural product sales have flourished [65] . most of package and food delivery services have transitioned from personal service to the "untact" method that minimizes direct human-to-human contacts. classes and lectures in elementary, middle, and high schools and colleges have transitioned to online platforms. video conferences and home-offices have also become the common method of running operations. automobile repair services now provide a "special pick-up and delivery" option to ensure that their services are untact, helping those customers who have hesitated to visit a service center due to covid-19. diebner et al. [66] pp. 3-4 stated that digital delivery has become a necessity for "most customers who are confined at home" and "that app downloads and new sign-ups have grown between 80-250%" during the covid-19 pandemic. as covid-19 has been reported to infect people via contact with infected people's droplets [32, 60] , untact services utilizing innovative technology applications have flourished and are expected to expand continuously. when a pandemic outbreak occurs, identifying the source of infection and suppressing its spread are the most important steps. amidst the covid-19 crisis, healthcare institutions are like battlefield military units that are fighting an enemy with necessary weapons, albeit in the form of much-needed medical supplies. in response to the pandemic emergency, many organizations have shifted to remote-working to ensure operational continuity and employee safety. however, many business firms that cannot operate remotely (e.g., manufacturing plants, construction sites, sports events, etc.) had to completely shut down business. enterprises are scrambling to make fast adjustments to their disrupted supply chains [8] . educational institutions were ordered to shift the teaching mode from the classroom to the online educational environment. these are "new normal" in the covid-19 crisis [2] . this study reviewed the cases of innovative responses, as well as failures, to the explosive spread of covid-19 in korea since the first confirmed case on 18 february 2020. based on the review of these cases, we summarized the lessons learned from korea's covid-19 experiences. the knowledge gained from the struggle against the virus provides new insights about required strategies for managing the pandemic. our study suggests that the healthcare policy makers and related organizations must be transparent in demonstrating to the citizens that emergency healthcare services are being provided on an equitable basis throughout the country. based on these experiences, policy makers should develop strategies that include the government's response capacity, information sharing, mature sense of unity and community, and application of advanced technologies in the time of urgency. the results of this study provide several theoretical and practical implications. first, the covid-19 outbreak taught the world that massive and rapid testing is essential to identify infected patients and infection clusters to prevent the pandemic from spreading. the identified patients can be either treated promptly (severely ill cases) or quarantined. second, we showed that the spread of an epidemic can be effectively suppressed only through well prepared public health infrastructure; coordinated and exhaustive efforts of the central/local governments, disinfection and prevention authorities and healthcare providers; and the spirit of unity and community of citizens (e.g., adhering to the government guidelines regarding social distancing, stay-at-home, avoiding large gatherings of people, etc.). third, innovative operational strategies should be established based on past experiences (e.g., the mers failures) in order to ensure success in managing the pandemic. in korea, the primary cause of the early spread of covid-19 was related to a mass gathering within a confined indoor space (e.g., worship services of religious organizations). the koreans learned quickly about the perils of such undisciplined activities and their consequences in terms of the uncontrollable spread of the pandemic [32] . moreover, the korean government enforced an aggressive covid-19 screening program to promptly identify and trace contacts made by infected people and treat seriously ill patients while strictly isolating them from the general population. furthermore, these measures cannot be implemented successfully without active cooperation of the citizens. from the outset, the government asked all citizens to refrain from participating in group gatherings or events, both indoors and outdoors, that could pose a threat to others, and strongly encouraged the practice of physical distancing. moreover, the government also placed a legal liability on agents who proceeded with non-recommended events. a mature civic consciousness is needed to voluntarily comply with government guidelines. a crisis is said to be a combination of danger and opportunity. president john f. kennedy analyzed the word "crisis" in chinese and pointed out that the word consists of two characters, one representing danger and the other representing opportunity [67] . winston churchill's famous quote was also in the same vein, "a pessimist sees the difficulty in every opportunity; an optimist sees the opportunity in every difficulty" [68] . these quotes serve as reminders that every crisis encompasses opportunities for creating a better future. the koreans have learned this lesson from their experience with the covid-19 pandemic. therefore, we might view the crisis from the perspective of "crisis = danger + opportunity" based on response efforts. the covid-19 crisis shows how each country organizes the delicate balance between achieving efficient results (avoiding high rate of mortality) and intrusion on personal privacy and economic security. this means that there is a trade-off relationship between two important factors in life: health and economy. for example, much of the offline education system will most likely transition to the online environment, causing a trade-off relationship between students' face-to-face education needs and a safer/cheaper mode of delivery. the measures undertaken by the korean government to avoid repeating the same mistakes incurred during the mers outbreak (i.e., re-organization of the kcdc, the healthcare delivery system, and disinfection and prevention systems, as well as the expansion of healthcare facilities) were shown to have a significant impact on the effectiveness of the implemented response strategies in the face of the covid-19 pandemic. therefore, developing an effective public healthcare infrastructure and new operational strategies based on past experiences could turn a crisis into an opportunity for preventing such virus infections [5] . we are confident that the fear of covid-19 that is currently sweeping the globe will soon be overcome and hope that this costly experience will serve the world well in preparing for the next pandemic. this study has reviewed the response strategies of korea in dealing with the covid-19 pandemic outbreak. korea's pandemic management approach, known as the k-response strategy, has been effective in containing covid-19 as the country learned a bitter lesson from the pains of mers and reinvented its public health infrastructure as a preparation for the next pandemic. we do hope that the operational strategies of korea discussed in this study would help prepare effective crisis management systems in other nations. this study, however, has some limitations. first, the scope and experience of the covid-19 cases discussed in this study are specific to korea. thus, the results of the study have limited generalizability to other situations or countries with different cultures and social systems. future research that includes various cases from around the world would further reinforce the findings of our study. second, the covid-19 pandemic is still causing havoc around the world and its end is difficult to predict. furthermore, the world will surely encounter new coronavirus pandemics in the future. the results of our study are limited to the discussion of covid-19 that we are battling today. thus, new pandemics will require different research approaches, although the lessons learned from the current pandemic will certainly be of much value. covid-19 and commercial pharma: navigating an uneven recovery the path to the next normal who declares covid-19 a pandemic sustainable covid-19 mitigation: wuhan lockdowns, health inequities, and patient evacuation covid-19 pandemic: what can the west learn from the east world health organization (who). coronavirus united nations office for the coordination of humanitarian affairs) reimagining medtech for a covid-19 world a study on improvement of national disaster management system through the mers outbreak in korea changes of global infectious disease governance in 2000s: rise of global health security and transformation of infectious disease control system in south korea the covid-19 crisis" as an opportunity for introspection: a multi-level rreflection on values, needs, trust and leadership in the future testing on the move south korea's rapid response to the covid-19 pandemic. transportation research interdisciplinary perspectives korea's foreign minister explains how the country contained covid-19 feeling connected after experiencing digital nature: a survey study untact": a new customer service strategy in the digital age hospitals consider universal do-not-resuscitate orders for coronavirus patients. the washington post developing an environmental health sciences covid-19 research agenda: results from the niehs disaster research response (dr2) work group's modified delphi method who. infectious diseases. 2020. available online what is a pandemic? emerging infections: microbial threats to health in the united states epidemic alert and response governance, and global public health in the wake of sars maintaining places of social inclusion: ebola and the emergency department a theory of cognitive dissonance psychological contracts in organizations: understanding written and unwritten agreements inequality in social exchange a model of customer satisfaction with service encounters involving failure and rrecovery customer evaluations of service complaint experiences: implications for relationship marketing a study on the national crisis management system in the case of the new infection diseases: focusing on school infection prevention activities available online the korea economic daily. daegu, the initial response was fast. it was different from south korea's health minister on how his country is beating coronavirus without a lockdown daegu operated a public-private partnership network drive-through screening center for covid-19: a safe and efficient screening system against massive community outbreak corona 19 'drive through' topic covid-19 pandemic data sources covid-19 pandemic in mainland china covid-19 pandemic in italy. available online covid-19 pandemic in spain. available online covid-19 pandemic in south korea covid-19 pandemic in the united states special report: how korea trounced u.s. in race to test people for coronavirus. reuter covid-19 pandemic data sources healthcare service justice and community engagement in crisis situation: focusing on failure cases in response to covid-19 emergency department resilience to disaster-level overcrowding: a component resilience framework for analysis and predictive modeling south korea's drive-through testing for coronavirus is fast-and free. national public radio needs to do today to follow south korea's model for fighting coronavirus time community treatment center. weekly dong-a 2020 civic capital and social distancing: evidence from italians' response to covid-19. vox cepr policy portal uncertainties of international standards in the mers cov outbreak in korea: multiplicity of uncertainties factors influencing the response to infectious diseases: focusing on the case of sars and mers in south korea south korea controlled its coronavirus outbreak in just 20 days. here are the highlights from its 90-page playbook for flattening the curve worldwide confirmed coronavirus cases top 2 million a faulty cdc coronavirus test delays monitoring of disease's spread. the philadelphia inquirer korean government's response to corona 19 addressing contact tracing challenges-critical to halting ebola virus disease transmission implications of sars epidemic for china's public health infrastructure and political system quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing south korea shows that democracies can succeed against the coronavirus coronavirus: what could the west learn from asia? lessons from south korea's covid-19 outbreak: the good, bad, and ugly. the diplomat effectiveness of workplace social distancing measures in reducing influenza transmission drive-thrus booming in korean society amid virus scare adapting customer experience in the time of coronavirus kennedy presidential residential; library and museum author contributions: all authors have conceptualization, writing, and read of the manuscript. all authors have read and agreed to the published version of the manuscript.funding: this research received no external funding. the authors declare no conflict of interest. key: cord-319877-izn315hb authors: de wit, emmie; van doremalen, neeltje; falzarano, darryl; munster, vincent j. title: sars and mers: recent insights into emerging coronaviruses date: 2016-06-27 journal: nat rev microbiol doi: 10.1038/nrmicro.2016.81 sha: doc_id: 319877 cord_uid: izn315hb the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. the continuing introductions of mers-cov from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. scientific advancements since the 2002–2003 severe acute respiratory syndrome coronavirus (sars-cov) pandemic allowed for rapid progress in our understanding of the epidemiology and pathogenesis of mers-cov and the development of therapeutics. in this review, we detail our present understanding of the transmission and pathogenesis of sars-cov and mers-cov, and discuss the current state of development of measures to combat emerging coronaviruses. supplementary information: the online version of this article (doi:10.1038/nrmicro.2016.81) contains supplementary material, which is available to authorized users. mers 12 , and a cluster of three cases of mers in the uk was identified in september 2012 (ref. 13 ). mers-cov continued to emerge and spread to countries outside of the arabian peninsula as a result of travel of infected persons; often, these imported mers cases resulted in nosocomial transmission. in may 2015, a single person returning from the middle east started a nosocomial outbreak of mers in south korea that involved 16 hospitals and 186 patients 14 . as of 26 april 2016, there have been 1,728 confirmed cases of mers, including 624 deaths in 27 countries 15 . this review highlights the pandemic and epidemic potential of emerging coronaviruses and discusses our current knowledge of the biology of sars-cov and mers-cov, including their transmission, their pathogenesis and the development of medical countermeasures. key features of these viruses are the dominance of nosocomial transmission, and pathogenesis that is driven by a combination of viral replication in the lower respiratory tract and an aberrant host immune response. several potential treatments for sars and mers have been identified in animal and in vitro models, including small-molecule protease inhibitors, neutralizing antibodies and inhibitors of the host immune response. however, efficacy data from human clinical trials are lacking but are needed to move these potential countermeasures forward. respectively (fig. 1a) . similarly to all viruses in the order nidovirales, sars-cov and mers-cov have a unique coding strategy: two-thirds of the viral rna is translated into two large polyproteins, and the remainder of the viral genome is transcribed into a nested set of subgenomic mrnas 16, 17 (fig. 1b) . the two polyproteins, pp1a and pp1ab, encode 16 non-structural proteins (nsp1-nsp16) 18 that make up the viral replicase-transcriptase complex. the polyproteins are cleaved by two proteases, papainlike protease (plpro; corresponding to nsp3) and a main protease, 3c-like protease (3clpro; corresponding to nsp5). the nsps rearrange membranes that are derived from the rough endoplasmic reticulum (rer) into double-membrane vesicles, in which viral replication and transcription occur 19 . one unique feature of coronaviruses is the exoribonuclease (exon) function of nsp14 (ref. 20) , which provides the proofreading capability required to maintain a large rna genome without the accumulation of detrimental mutations 21, 22 . sars-cov and mers-cov transcribe 12 and 9 subgenomic rnas, er-golgi intermediate compartment (ergic) . a cellular compartment that facilitates transport between the endoplasmic reticulum (er) and the golgi complex. infected individuals who each infect a disproportionately large number of secondary cases. respectively, and these encode the four structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as several accessory proteins that are not involved in viral replication but interfere with the host innate immune response or are of unknown or poorly understood function. the envelope spike glycoprotein binds to its cellular receptor, angiotensin-converting enzyme 2 (ace2) for sars-cov and dipeptidyl peptidase 4 (dpp4) for mers-cov 23 . after membrane fusion, either directly with the host cell membrane or with the endosome membrane, the viral rna genome is released into the cytoplasm, and the rna is uncoated to allow translation of the two polyproteins, transcription of the subgenomic rnas and replication of the viral genome (fig. 1b) . newly formed envelope glycoproteins are inserted in the rer or golgi membranes; genomic rna and nucleocapsid proteins combine to form nucleocapsids, and the viral particles bud into the er-golgi intermediate compartment (ergic). virion-containing vesicles subsequently fuse with the plasma membrane to release the virus 24 . the first indication of the source of sars-cov was the detection of the virus in masked palm civets and a raccoon dog and the detection of antibodies against the virus in chinese ferret badgers in a live-animal market in shenzhen, china 25 . however, these animals were only incidental hosts, as there was no evidence for the circulation of sars-cov-like viruses in palm civets in the wild or in breeding facilities 26 . rather, bats are the reservoir of a wide variety of coronaviruses, including sars-cov-like and mers-cov-like viruses 27 (fig. 2) . thus, the search for the reservoir of mers-cov initially focused on bats, but a serological survey in dromedary camels from oman and the canary islands showed a high prevalence of mers-cov-neutralizing antibodies in these animals 28 . in addition, mers-cov rna was detected in swabs that were collected from dromedary camels at a farm in qatar that was linked to two human cases of mers, and infectious virus was isolated from dromedary camels in saudi arabia and qatar [29] [30] [31] [32] . serological evidence for the circulation of a mers-cov-like virus in dromedary camels has been obtained in the middle east, eastern africa and northern africa, dating back as far as 1983 (ref. 33 ). dromedary camels in saudi arabia harbour several viral genetic lineages 34 , including those that have caused human outbreaks. taken together, these data strongly point to the role of dromedary camels as a reservoir for mers-cov. the ubiquity of infected dromedary camels close to humans and the resulting continuing zoonotic transmission may explain why mers-cov continues to cause infections in humans, whereas sars-cov, without the continuing presence of an infected intermediate host and with relatively infrequent human-bat interactions, has caused no more infections in humans. human-to-human transmission of sars-cov and mers-cov occurs mainly through nosocomial transmission; 43.5-100% of mers cases in individual outbreaks were linked to hospitals, and very similar observations were made for some of the sars clusters 35, 36 . transmission between family members occurred in only 13-21% of mers cases and 22-39% of sars cases. transmission of mers-cov between patients was the most common route of infection (62-79% of cases), whereas for sars-cov, infection of health care workers by infected patients was very frequent (33-42%) 35 . the predominance of nosocomial transmission is probably due to the fact that substantial virus shedding occurs only after the onset of symptoms 37, 38 , when most patients are already seeking medical care 39 . an analysis of hospital surfaces after the treatment of patients with mers showed the ubiquitous presence of viral rna in the environment for several days after patients no longer tested positive 40 . moreover, many patients with sars or mers were infected through super spreaders 14, 35, 37, [41] [42] [43] . the clinical courses of sars and mers are remarkably similar, although there are subtle differences (box 1) . owing to the current sparsity of data on human mers-cov infections 44 , the pathogenesis of this virus is poorly understood; however, similar mechanisms may underlie the pathogenesis of both mers and sars. the binding of spike protein to ace2 and the subsequent downregulation of this receptor contribute to lung injury during sars 45 . although it seems counterintuitive that receptor downregulation would increase pathology, it has been shown that ace2 can protect against acute lung injury. the downregulation of ace2 results in the excessive production of angiotensin ii by the related enzyme ace, and it has been suggested that the stimulation of type 1a angiotensin ii receptor and middle east respiratory syndrome coronavirus (mers-cov) encode two large polyproteins, pp1a and pp1ab, which are proteolytically cleaved into 16 non-structural proteins (nsps), including papain-like protease (plpro), 3c-like protease (3clpro), rna-dependent rna polymerase (rdrp), helicase (hel) and exonuclease (exon). an additional 9-12 orfs are encoded through the transcription of a nested set of subgenomic rnas. sars-cov and mers-cov form spherical particles that consist of four structural proteins. the envelope glycoprotein spike (s) forms a layer of glycoproteins that protrude from the envelope. two additional transmembrane glycoproteins are incorporated in the virion: envelope (e) and membrane (m). inside the viral envelope resides the helical nucleocapsid, which consists of the viral positive-sense rna ((+)rna) genome encapsidated by protein nucleocapsid (n). b | following entry of the virus into the host cell, the viral rna is uncoated in the cytoplasm. orf1a and orf1ab are translated to produce pp1a and pp1ab, which are cleaved by the proteases that are encoded by orf1a to yield 16 nsps that form the rna replicase-transcriptase complex. this complex localizes to modified intracellular membranes that are derived from the rough endoplasmic reticulum (er) in the perinuclear region, and it drives the production of negative-sense rnas ((-)rnas) through both replication and transcription. during replication, full-length (-)rna copies of the genome are produced and used as templates for full-length (+)rna genomes. during transcription, a subset of 7-9 subgenomic rnas, including those encoding all structural proteins, is produced through discontinuous transcription. in this process, subgenomic (-)rnas are synthesized by combining varying lengths of the 3′end of the genome with the 5′ leader sequence necessary for translation. these subgenomic (-)rnas are then transcribed into subgenomic (+)mrnas. although the different subgenomic mrnas may contain several orfs, only the first orf (that closest to the 5′end) is translated. the resulting structural proteins are assembled into the nucleocapsid and viral envelope at the er-golgi intermediate compartment (ergic), followed by release of the nascent virion from the infected cell. one of a panel of recombinant inbred mouse strains derived from a genetically diverse set of founder strains and designed for the analysis of complex traits. the aggregation of leukocytes around blood vessels. (agtr1a) increases pulmonary vascular permeability, thus potentially explaining the increased lung pathology when the expression of ace2 is decreased 46 . immunopathology. the immune response is essential for the resolution of an infection, but it can also result in immunopathogenesis. one indication that immunopathogenesis may contribute to sars was the observation that viral loads were found to be decreasing while disease severity increased 39, 47 . it is unclear whether a similar trend applies to mers 48, 49 . moreover, progression to acute respiratory distress syndrome (ards) is associated with the upregulation of pro-inflammatory cytokines and chemokines, particularly interleukin-1β (il-1β), il-8, il-6, cxc-chemokine ligand 10 (cxcl10) and cc-chemokine ligand 2 (ccl2) 50, 51 ; increased plasma levels of these molecules have been detected in patients with sars [52] [53] [54] [55] . retrospective longitudinal studies in patients who recovered from sars versus those who succumbed to the disease have shown an early expression of interferon-α (ifnα), ifnγ, cxcl10, ccl2 and proteins that are encoded by ifn-stimulated genes (isgs) in all patients, but only patients who survived then had gene expression profiles that are indicative of the development of an adaptive immune response. by contrast, patients who succumbed maintained high levels of cxcl10, ccl2 and isg-encoded proteins, whereas spike-specific antibodies were present at low levels or were absent 56 , which suggests that severe disease is related to the lack of a switch from an innate immune response to an adaptive immune response. experiments using collaborative cross mouse lines and mouse-adapted sars-cov identified one host gene, trim55, as important for sars pathogenesis. although there was no difference in clinical signs or viral replication in trim55 −/− mice compared with wild-type mice, perivascular cuffing and the number of inflammatory cells in the lungs were reduced in the trim55 −/− mice 57 . a biological process in which small rna molecules induce the degradation of specific mrna molecules, thereby inhibiting gene expression. systems in which a dna molecule is produced that contains the viral leader and trailer sequences, with an assayable reporter replacing the viral orfs. when combined with the expression of viral proteins in trans, this system can be used to model the viral life cycle without the necessity of using infectious virus. the involvement of the host immune response in the pathogenesis of sars, and most likely also that of mers, suggests that drugs which inhibit viral replication will need to be combined with treatments that control detrimental immune responses. immune evasion. sars-cov and mers-cov use several strategies to avoid the innate immune response. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna (dsrna) or uncapped mrna, are detected by pattern recognition receptors (prrs), such as retinoic acid-inducible gene i protein (rig-i; also known as ddx58) or melanoma differentiation-associated protein 5 (mda5; also known as ifih1) 58 . this triggers complex signalling cascades involving myd88 that lead to the production of type i ifns and the activation of the transcription factor nuclear factor-κb (nf-κb). in turn, active nf-κb induces the transcription of pro-inflammatory cytokines (fig. 3a) . type i ifns signal through ifnα/β receptor (ifnar) and downstream molecules such as signal transducer and activator of transcription (stat) proteins to stimulate the production of antiviral proteins that are encoded by isgs, such as ifn-induced protein with tetratricopeptide repeats 1 (ifit1 ; fig. 3b ). collectively, this establishes an antiviral immune response that limits viral replication in infected and in neighbouring cells (fig. 3) . infection of knockout mice revealed the importance of innate immunity. infection of myd88 −/− and stat1 −/− mice, but not mice that were deficient in ifn receptors, with a mouse-adapted strain of sars-cov resulted in more severe disease than infection with a non-adapted sars-cov strain 59, 60 . moreover, mers-cov infection of wild-type mice that were transduced with human dpp4 caused mild disease, but symptoms were more severe in myd88 −/− mice and ifnar1 −/− mice 61 . sars-cov and mers-cov avoid host detection of their dsrna by replicating in virus-induced doublemembrane vesicles that lack prrs 19, 62, 63 . moreover, the recognition of sars-cov mrnas, for example, by mda5 and ifit1 is prevented by capping of the viral mrnas by nsp14 and the nsp10-nsp16 complex 64 . recombinant sars-cov that lacks the methylation activity of nsp16 is attenuated and exhibits increased sensitivity to type i ifns. this effect is dependent on ifit1 or mda5, as the same virus is not attenuated in mice that are deficient in either molecule 65 . although mrna capping has not yet been shown for mers-cov, structural similarity between the mers-cov nsp10-nsp16 complex and the sars-cov nsp10-nsp16 complex suggests that a similar mechanism exists to avoid host recognition of mers-cov mrnas by cytosolic prrs 66 . sars-cov encodes at least eight proteins that interact with the signalling cascades downstream of prrs; in mers-cov, several proteins have been identified with similar functions (fig. 3) . the nucleocapsid protein of sars-cov has been associated with the suppression of rnai in mammalian cells 67 . furthermore, this protein antagonizes ifn induction, probably early in the signalling cascade, as downstream signalling molecules relieve the inhibition 68 . mers-cov orf4a has a similar ifn-antagonistic function, involving the binding of dsrna and subsequent inhibition of mda5 activation 69 , potentially through interaction with ifn-inducible dsrna-dependent protein kinase activator a (prkra; also known as pact), which interacts with mda5 and rig-i 70 . moreover, mers-cov orf4a, orf4b, orf5 and membrane protein inhibit the nuclear trafficking of ifn-regulatory factor 3 (irf3) and activation of the ifnb promoter 71 . these viral proteins, except for orf5, also inhibit the expression of genes that are under the control of an ifn-stimulated response element (isre), and orf4a reduces the expression of genes that are stimulated by nf-κb 71 . finally, mers-cov orf4b interacts with tbk1 and inhibitor of nf-κb kinase-ε (ikkε), thereby suppressing the interaction between ikkε and mitochondrial antiviral-signalling protein (mavs) and inhibiting the phosphorylation of irf3 (ref. 72 ). the membrane protein of sars-cov inhibits the formation of a signalling complex that contains ikkε, thus repressing the activation of irf3 and irf7 and their induction of type i ifn expression. the membrane protein of mers-cov inhibits irf3 function and the expression of genes that are regulated by an isre, including ifnβ 71 , but whether this occurs through a mechanism similar to that of sars-cov is unclear. sars-cov plpro disrupts nf-κb signalling 73 and blocks the phosphorylation of irf3 indirectly 73, 74 . furthermore, sars-cov plpro inhibits the induction of type i ifns, potentially through the deubiquitylation of phosphorylated irf3 (refs 73, 75) . similar functions have been described for mers-cov plpro 76 . experiments involving recombinantly expressed proteins, in vitro translation, protein overexpression and minireplicon systems have shown that nsp1 of sars-cov blocks the ifn response through the inhibition of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have an incubation period of ~5 days, and 95% of patients develop disease within 13 days of exposure 14, 38, [144] [145] [146] . common early symptoms are fever, chills, coughing, malaise, myalgia and headache, and less common symptoms include diarrhoea, vomiting and nausea 2, 6, 39, 89, 90, 92, 95, 144, [146] [147] [148] . upper respiratory tract symptoms and viral shedding are rare, which explains difficulties in obtaining a laboratory diagnosis from nasal or nasopharyngeal swabs 149 . abnormal chest x-rays are more common in patients with mers (90-100%) 144,148 than in those with sars (60-100%) 39, 89 . accordingly, only 20-30% of patients with sars require intensive care and subsequent mechanical ventilation, whereas 50-89% of patients with mers require intensive care 2, 39, 89, 90, 95, 144, 147, 148 . the higher incidence of acute respiratory distress syndrome (ards) in individuals with mers is reflected in the case fatality rate: this is ~36% for mers compared with ~10% for sars 15, 145 . comorbidities have an important role in both sars and mers. several risk factors are associated with poor disease outcome, especially advanced age and male sex 2, 14, 39, 144, 146, 148, 150, 151 . for mers, additional risk factors for a poor outcome include diabetes mellitus, hypertension, cancer, renal and lung disease, and co-infections 14, 144, 146, 148, 150, 151 . health care settings seem to increase the risk of viral transmission owing to aerosol-generating procedures such as intubation and bronchoscopy. appropriate hospital hygiene practices and awareness are crucial to limit future nosocomial outbreaks. both nsp7 and nsp15 from sars-cov were also suggested to be ifn antagonists, but the underlying mechanism is unknown 73 . nsp15 is an inhibitor of mavsinduced apoptosis; however, this occurs through an ifn-independent mechanism 84 . finally, transcriptomic and proteomic analysis of human airway cell cultures showed that mers-cov but not sars-cov induces repressive histone modifications that downregulate the expression of certain isgs 85 . it should be noted that most of the interactions of sars-cov and mers-cov proteins with innate immune pathways were established in in vitro systems, which rely on the overexpression of viral and, . following prr-mediated detection of a pamp, the resulting interaction of prrs with mitochondrial antiviral-signalling protein (mavs) activates nuclear factor-κb (nf-κb) through a signalling cascade involving several kinases. activated nf-κb translocates to the nucleus, where it induces the transcription of pro-inflammatory cytokines. the kinases also phosphorylate (p) ifn-regulatory factor 3 (irf3) and irf7, which form homodimers and heterodimers and enter the nucleus to initiate the transcription of type i interferons (type i ifns). both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have developed mechanisms to interfere with these signalling pathways, as shown; these subversion strategies involve both structural proteins (membrane (m) and nucleocapsid (n)) and non-structural proteins (nsp1, nsp3b, nsp4a, nsp4b, nsp5, nsp6 and papain-like protease (plpro); indicated in the figure by just their nsp numbers and letters). b | binding of type i ifns to their dimeric receptor, ifnα/β receptor (ifnar), activates the janus kinase (jak)-signal transducer and activator of transcription (stat) signalling pathway, in which jak1 and tyk2 kinases phosphorylate stat1 and stat2, which form complexes with irf9. these complexes move into the nucleus to initiate the transcription of ifn-stimulated genes (isgs) under the control of promoters that contain an ifn-stimulated response element (isre). collectively, the expression of cytokines, ifns and isgs establishes an antiviral innate immune response that limits viral replication in infected and in neighbouring cells. again, viral proteins have been shown to inhibit these host signalling pathways to evade this immune response. iκbα, nf-κb inhibitor-α. a broadly active antiviral nucleoside analogue with several direct and indirect mechanisms of action; mainly used for the treatment of hepatitis c, in combination with interferon. having polyethylene glycol (peg) attached, to a drug for example; this moiety improves the solubility, decreases the immunogenicity and increases the stability, of the drug of interest, thereby allowing a reduced dosing frequency to be used. (table 1) and sars-cov, including the use of antibodies, ifns, inhibitors of viral and host proteases, and host-directed therapies. in the absence of a clinically proven effective antiviral therapy against sars-cov and mers-cov, patients mainly receive supportive care, which is often supplemented by different combinations of drugs. ribavirin 86 and various types of ifn have been given to patients with mers in saudi arabia 87 and china 88 , typically in combination with a broad-spectrum antibiotic and oxygen. the efficacy of treatments for sars-cov and mers-cov infection currently remains unclear. in addition, treatment for mers is typically started only in a late disease stage, when immunopathology predominates and antiviral drugs are likely to provide little benefit. ribavirin was used most frequently during the sars outbreak, often in combination with corticosteroids, which have an anti-inflammatory effect 2,89-92 . ifnα was also given, usually in combination with immunoglobulins or thymosins, which stimulate the development of t cells, and in a small number of cases in combination with ribavirin 93, 94 . none of these treatments was tested in a clinical trial, which makes it difficult to assess their efficacy. in fact, retrospective analysis did not yield a treatment combination that was clearly effective. moreover, the data from patients are contradictory about whether ribavirin, when used alone, provided a benefit or was possibly even detrimental 89, 90, 92, 95 . in vitro coronaviruses have a lower sensitivity to ribavirin than other viruses. deletion of the nsp14-encoding sequence increases the sensitivity of coronaviruses to ribavirin; however, the underlying mechanism is unclear and is not related to the proofreading function of nsp14 (ref. 96 ). therefore, ribavirin should be considered only in combination with other antiviral treatments. although ifns are effective against mers-cov in vitro [97] [98] [99] , their effect in humans has yet to be proved. the effectiveness of ifn is increased in vitro if ribavirin is added 98, 100 , and a combined use of the two drugs reduces disease severity in a rhesus macaque model of mers 101 . the potential side effects of these treatments, such as fatigue, depression and anaemia, have inhibited their use as a first-line treatment for mers, and they are generally administered only after a patient's condition starts to deteriorate. for example, one study of five patients who were infected with mers-cov indicated no survival following ribavirin and ifnα2b therapy; however, therapy was not started until 10 days after admission 87 . a separate study found an improvement in survival 14 days after mers diagnosis and the start of treatment, but not 28 days after 102 . in a third study, a combination of ifnα2a and ribavirin or ifnβ1a and ribavirin did not improve survival; however, some of the patients were more than 50 years old and had preexisting renal failure 103 . in a single case in which ribavirin and ifnα2b were started shortly after admission to hospital, the patient started to improve on day 6 after admission and made a complete recovery 104 . ifnβ1b is a more potent inhibitor of mers-cov replication in vitro than other types of ifn 97, 99 , and an improved outcome of disease was observed in common marmosets after challenge with mers-cov 105 . thus, the type of ifn that is used for treatment in humans should be reconsidered (usually, ifnα is used). furthermore, ribavirin and/or ifns should be tested in clinical trials to determine their efficacy in mers treatment and to establish treatment protocols. additional antiviral treatments. the protease inhibitors lopinavir and ritonavir, which are used in combination to treat infection with hiv, improved the outcome of patients with sars when combined with ribavirin, compared with patients who were treated with ribavirin alone 106, 107 . lopinavir showed no clear antiviral activity against mers-cov in vitro 97 , and it is thus rarely used in patients with mers. however, lopinavir and ritonavir improve the outcome in common marmosets when treatment is initiated 6 hours after infection with mers-cov 105 . thus, the testing of lopinavir and ritonavir in clinical trials in patients with mers should be reconsidered. one patient who received pegylated ifnα, ribavirin, lopinavir and ritonavir in combination had undetectable levels of mers-cov in the blood 2 days after the initiation of therapy; however, this patient did not survive 108 . the combination of ifnα, ribavirin, lopinavir and ritonavir was also used for mers treatment in south korea, but efficacy data are not yet available. however, three case reports indicate recovery in five out of seven patients who were treated with this combination [109] [110] [111] . as 3clpro and plpro are essential for cleavage of the viral polyproteins and are distinct from cellular proteases, they are ideal drug targets, in particular plpro, compounds that mimic biologically active peptides or proteins. a complement-activated molecule that is important for the recruitment to and activation of inflammatory cells in the lungs. which is involved in both viral replication and ifn antagonism. indeed, most antiviral drug-like molecules have been developed against 3clpro and plpro, which was aided by the rapid report of crystal structures of these proteases 112 . plpro was initially identified as a drugable target for sars-cov; recently, it has been noted that some of the compounds that target plpro from sars-cov are also active against plpro from mers-cov. for example, both 6-mercaptopurine and 6-thioguanine inhibit mers-cov and sars-cov in vitro 113 ; however, the efficacy of these molecules has not yet been tested in vivo. mycophenolic acid also inhibits the replication of mers-cov in vitro 97, 99 through the inhibition of plpro 113 , but it had no effect in marmosets 105 . as new coronaviruses are likely to emerge from bats, protease inhibitors were designed against bat coronaviruses with the goal of developing a universal antiviral compound against emerging zoonotic coronaviruses. this approach yielded an inhibitor of tylonycteris bat coronavirus hku4 (hku4-cov), which is closely related to mers-cov 11 . this inhibitor, named sg85, indeed inhibits mers-cov replication in vitro 112 . similarly, peptidomimetics that target and inhibit 3clpro of mers-cov, hku4-cov and pipistrellus bat coronavirus hku5 (hku5-cov) have also been identified, but have not yet progressed beyond the in vitro stage 114 . several other drugs that were approved for use in humans were shown to inhibit the replication of mers-cov in vitro, notably chloroquine, chlorpromazine, loperamide and cyclosporine a 62, 99, 113, 115 , although their mechanisms of action are unknown, and the benefit of cyclosporine a in patients is debatable owing to the immunosuppressive effect of the drug. although cyclophilin inhibitors that do not result in immunosuppression are available, their activity against mers-cov has not yet been tested. antibody and plasma therapy. plasma from convalescent patients and/or antibody therapies have been the leading proposed treatment for mers so far 116 . there are several potential advantages to this approach. for example, as case numbers increase, the pool of survivors becomes larger; provided these individuals have sufficiently high antibody titres and are willing and able to donate plasma, this is a low-tech, reasonably safe treatment option. furthermore, generation of monoclonal antibodies for use in humans is well established, with a fairly straightforward path to safety and efficacy testing. however, to date, there are very few reports on the use of convalescent plasma and none on the use of monoclonal antibodies as treatments for acute, severe respiratory disease in humans. a post hoc meta-analysis of 32 studies of either sars or severe influenza found a significant reduction in the pooled odds of mortality when convalescent plasma was used 117 . however, study design was rated as low or very low quality, as there were generally a lack of control groups and a moderate-to-high risk of bias, which suggests that a properly designed clinical trial of convalescent plasma use in severe respiratory infections is needed 117 . potent monoclonal antibodies that neutralize the mers-cov spike protein in vitro have been developed [118] [119] [120] [121] . however, with a few exceptions, in vivo data relating to the use of convalescent plasma or monoclonal antibodies in the treatment of mers are currently lacking. serum from high-titre dromedary camels decreased mers-cov loads in the lungs of mice that had been transduced with human dpp4 (ref. 122 ). human polyclonal antibodies against the spike protein were generated by vaccinating transchromosomic bovines, and treatment with these antibodies reduced viral titres in the lungs of dpp4-transduced mice when treatment was administered 24 or 48 hours after challenge with mers-cov 123 . dpp4-transduced mice were also treated with humanized neutralizing monoclonal antibody 4c2h, which is directed against the receptor-binding domain of the mers-cov spike protein, 1 day after mers-cov challenge, and this treatment also decreased viral titres in the lungs 124 , as did the neutralizing antibody lca60, which was obtained from a convalescent patient and produced recombinantly 125 . human neutralizing monoclonal antibodies regn3048 and regn3051 also provided a benefit in mice that expressed human dpp4 and were challenged with mers-cov 126 . the human neutralizing monoclonal antibody m332 reduced mers-cov replication in the lungs of rabbits following prophylactic, but not therapeutic, treatment 127 . treatment of rhesus macaques with the human monoclonal antibody 311b-n1 day after challenge resulted in reduced lung pathology 128 . in all of these studies, viral replication was not completely inhibited, and there were some pathological alterations to the lungs, despite the therapy. furthermore, none of the studies addressed the potential emergence of escape mutants in vivo. alternatively, antibodies that target the region of dpp4 that binds to the spike protein could be used to prevent entry of mers-cov; this approach was successful in vitro 129 . however, whether such a treatment would be feasible and would not have substantial adverse effects in humans remains to be determined. host-directed strategies can also limit viral replication. for example, the spike protein of sars-cov is cleaved by cathepsin b and cathepsin l, transmembrane protease serine 2 (tmprss2) and possibly other host proteases 130, 131 . inhibition of host serine proteases by camostat reduced the entry of sars-cov and increased survival in a mouse model 132 . however, the targeting of host proteases is more likely to result in undesirable side effects than the targeting of viral proteases. another underappreciated strategy is attenuation of detrimental host responses. the development of such treatments would require a thorough understanding of the host responses that are involved in acute lung injury and ards, processes that are unfortunately poorly understood at the moment. nonetheless, in vitro studies and limited studies in animal models with other respiratory viruses have shown that anaphylatoxin c5a is important for the development of acute lung injury, and blocking anaphylatoxin c5a can reduce lung pathology 133 . vaccines that contain immunogenic parts of a pathogen rather than the entire pathogen. vaccines based on the direct introduction of a plasmid encoding an antigen; following in situ production of this antigen, an immune response is mounted against it. changes in gene expression during in vitro mers-cov infection were used to predict potential effective drugs. one of the drugs with predicted efficacy, the kinase inhibitor sb203580, modestly inhibited sars-cov and mers-cov replication following the treatment of cells prior to infection, but treatment after infection inhibited replication of only sars-cov and not mers-cov 134 . vaccines. vaccination could be used to prevent infection or to reduce disease severity, viral shedding and thereby transmission, thus helping to control mers outbreaks. several vaccination strategies were developed against sars-cov and tested in animals, such as an inactivated virus, a live-attenuated virus, viral vectors, subunit vaccines, recombinant proteins and dna vaccines 135, 136 . similar approaches have been used for the development of experimental mers-cov vaccines 137 . to date, three mers-cov vaccines have been evaluated in non-human primates. in one study, rhesus macaques were primed with dna encoding the spike protein, followed by boosts with spike dna and with recombinant protein consisting of the spike subunit containing the receptor-binding domain, or primed and boosted once with the subunit protein. both approaches reduced pathological changes in lung function in animals that were infected with mers-cov 19 weeks after the last vaccination 138 . moreover, three vaccinations with a recombinantly expressed protein that contains the receptor-binding domain of the spike protein reduced viral loads and lung pathology in rhesus macaques that were infected 2 weeks after the last vaccination 139 . three dna vaccinations with a construct encoding the full-length spike sequence reduced viral loads and pathology in the lungs after challenge with mers-cov 5 weeks after the last vaccination 140 . one concern of vaccination in humans is vaccinemediated enhancement of disease, a process in which the disease following infection is more severe in vaccinated individuals than in unvaccinated individuals. although this was observed in only a small subset of vaccine studies that were carried out for sars-cov 136 and has not yet been observed in any of the published mers-cov vaccine studies, it is an important concern. moreover, it is unclear who to vaccinate against mers-cov, as healthy individuals seem to be at little risk of severe disease. older patients or patients with underlying disease, who have the highest risk of severe mers, would be important target populations. however, vaccination in such patients can be problematic owing to their poor immune responses, as has been established for influenza virus 141 . in addition, vaccination of people with a high risk of exposure to mers-cov, such as health care workers, slaughterhouse workers and camel herders, is advisable 142 . as our understanding of the pathogenesis of emerging coronaviruses increases, so will the opportunities for the rational design of therapeutics that target viral replication or immunopathology. the rational design of new drugs and the repurposing of existing compounds have already resulted in the development of plpro inhibitors and the identification of kinase inhibitors that inhibit the replication of sars-cov and mers-cov in vitro. however, only a few potential treatments have progressed past the identification of an effect in vitro, and in vivo studies to select the most promising treatment options are required. the development of several mouse models of mers is thus an important step forward (box 2) . owing to the acute nature of mers and the important role of immunopathology, combination therapies aimed at simultaneously inhibiting viral replication, limiting viral dissemination and dampening the host response are likely to yield the best results. furthermore, treatment should be started as early as possible, rather than waiting until the patient has already developed extensive lung damage. the development of therapies against sars and mers needs to focus on testing in humans, in properly controlled clinical trials. the current non-standardized, uncontrolled approach to treatment is not informative and may not be beneficial to the patient. the recent ebola outbreak has demonstrated that rapid clinical trial design and approval are possible and that exceptional situations call for deviations from normal procedures . while treatments are being developed and evaluated, the prevention of viral transmission is key to reducing the burden of mers. the large proportion of nosocomial mers-cov infections indicates that preventive measures in hospitals are currently either not fully implemented or insufficient. prevention of zoonotic transmission from dromedary camels is another possibility to decrease the number of mers cases. the most of our understanding of the pathogenesis of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) comes from animal studies. ideally, these models recapitulate all or specific aspects of human disease. several mouse models have been established, for example by using mouse-adapted sars coronavirus (sars-cov) or expressing human receptors in mice 152 . although it has been recognized that mice might poorly mimic specific human responses to infection, the availability of knockout and transgenic mice enables the targeted study of virus-host interactions. several non-human primate models have been developed for sars-cov and mers coronavirus (mers-cov) 152 . the close relationship of non-human primates to humans often allows faithful recapitulation of a disease and the host response. however, these benefits are countered by the need for specialized husbandry, the sometimes limited availability of the animals and reagents, and high costs. the pathogenesis of sars-cov and mers-cov in their respective reservoir hosts is not nearly as well studied as that in humans. currently, only one experimental-infection study has been carried out in bats with mers-cov 153 , and none has been carried out for other coronaviruses. thus, data are mostly limited to the detection of coronaviruses in naturally infected bats. the detection of coronaviruses mainly in faecal samples from bats and not in oral swabs suggests that replication in bats occurs predominantly in the gastrointestinal tract 9,154,155 . by contrast, a combination of intratracheal and intranasal inoculation of masked palm civets with sars-cov resulted in interstitial pneumonia, with oral and rectal viral shedding 156 . the pathogenesis of mers-cov in dromedary camels has been studied experimentally in a limited number of animals. these animals developed transient mild disease; however, large quantities of mers-cov were shed from the upper respiratory tract, in line with the predominant replication of mers-cov in the nasal turbinates and larynx in these animals, which explains the frequent zoonotic transmission 157 . first vaccines against mers-cov have been tested in dromedary camels 140, 143 ; indeed, when camels were vaccinated with a modified vaccinia virus that expresses the mers-cov spike protein, subsequent challenge of these animals with mers-cov resulted in less viral shedding than in unvaccinated animals 143 , thereby potentially limiting the transmission to naive animals or to humans. certainly, there has been progress in the development of vaccines and therapies against emerging coronaviruses, but more research and rigorous testing is required if we are to successfully combat these novel pathogens. when the severe acute respiratory syndrome (sars) outbreak developed into the first pandemic of the twenty-first century, it became clear that the medical and scientific communities were not adequately prepared for the emergence of highly pathogenic viruses. whereas several months elapsed and several thousand cases of sars were observed before the causative agent was identified as sars coronavirus (sars-cov) 4-6 , subsequent advances in molecular diagnostic tools, such as next generation sequencing, meant that middle east respiratory syndrome coronavirus (mers-cov) was identified before it caused a large outbreak of mers 11 . the availability of the full-length genome of mers-cov enabled the rapid development and distribution of diagnostic assays. the first animal models of disease, several treatment efficacy studies and the identification of the reservoir followed soon after. unfortunately, the sars pandemic did not yield solid clinical data on the efficacy of treatment regimens. these data are urgently needed for the treatment of mers, as well as to prepare for novel coronaviruses that may emerge. several studies have used synthetic biology to study the zoonotic transmission potential of sars-cov-like viruses from bats 9,10,158,159 . the ebola virus outbreak in west africa has highlighted the need for fast-tracking of potential treatments, as several clinical trials were started only towards the end of the outbreak. the combined experiences of the outbreaks of sars, mers and ebola provide a blueprint for the response to emerging pathogens: after the identification of the causative agent, diagnostic assays need to be developed and distributed rapidly, and simultaneously, awareness of the new syndrome and reporting of (suspected) cases must be increased. in addition, infection control measures in health care facilities are essential. research needs to focus on understanding the epidemiology, including pathogen transmission and identification of the reservoir and/or intermediate hosts. animal models need to be developed, as well as therapeutic and prophylactic measures. finally, promising treatments need to be fast-tracked into clinical trials. epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china a major outbreak of severe acute respiratory syndrome in hong kong molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome summary of probably sars cases with onset of illness from 1 sars-cov infection in a restaurant from palm civet the isolation of a bat sars-cov-like virus that uses the human ace2 as a receptor without prior adaptation a sars-like cluster of circulating bat coronaviruses shows potential for human emergence the first identification of mers-cov as the cause of severe lower respiratory disease in humans patient with new strain of coronavirus is treated in intensive care at london hospital middle east respiratory syndrome coronavirus outbreak in the republic of korea who. coronavirus infections: disease outbreak news. who nidovirus transcription: how to make sense…? coronaviruses post-sars: update on replication and pathogenesis coronaviruses: an overview of their replication and pathogenesis sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing insights into rna synthesis, capping, and proofreading mechanisms of sars-coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc isolation and characterization of viruses related to the sars coronavirus from animals in southern china ecology, evolution and classification of bat coronaviruses in the aftermath of sars middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation evidence for camel-to-human transmission of mers coronavirus mers coronavirus in dromedary camel herd, saudi arabia isolation of mers coronavirus from a dromedary camel co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia transmission characteristics of mers and sars in the healthcare setting: a comparative study an analysis of the predominant role for nosocomial transmission in the epidemiology of both sars and mers transmission of middle east respiratory syndrome coronavirus infections in healthcare settings epidemiology, transmission dynamics and control of sars: the 2002-2003 epidemic preliminary epidemiological assessment of mers-cov outbreak in south korea clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study a description of the clinical representation of sars-cov respiratory disease in patients from hong kong environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea evidence that infectious mers-cov can be detected on common hospital surfaces during an outbreak, which highlights the potential for nosocomial transmission and stresses the need for infection control the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission middle east respiratory syndrome coronavirus superspreading event involving 81 persons the role of super-spreaders in infectious disease clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure temporal relationship of viral load, ribavirin, interleukin (il)-6, il-8, and clinical progression in patients with severe acute respiratory syndrome clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases biomarkers in acute respiratory distress syndrome the mercurial nature of neutrophils: still an enigma in ards? sars-cov virus-host interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalin-fixed paraffin-embedded tissues distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients early enhanced expression of interferon-inducible protein-10 (cxcl-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome genome wide identification of sars-cov susceptibility loci using the collaborative cross sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism myd88 is required for protection from lethal infection with a mouse-adapted sars-cov rapid generation of a mouse model for middle east respiratory syndrome a study in which the dpp4-based host restriction is overcome in mice by expression of the human variant of dpp4, leading to the development of several transgenic mouse models mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex in vitro reconstitution of sars-coronavirus mrna cap methylation attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2′-o-methyltransferase activity coronavirus non-structural protein 16: evasion, attenuation, and possible treatments the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells sars-cov nucleocapsid protein antagonizes ifn-β response by targeting initial step of ifn-β induction pathway, and its c-terminal region is critical for the antagonism middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus the sars coronavirus papain like protease can inhibit irf3 at a post activation step that requires deubiquitination activity crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mrna severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists mavs-mediated apoptosis and its inhibition by viral proteins pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses mechanisms of action of ribavirin against distinct viruses ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study clinical analysis of the first patient with imported middle east respiratory syndrome in china clinical features and short-term outcomes of 144 patients with sars in the greater toronto area identification of severe acute respiratory syndrome in canada development of a standard treatment protocol for severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays ribavirin and interferon-β synergistically inhibit sars-associated coronavirus replication in animal and human cell lines treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques the first application of a potential treatment option for mers through the repurposing of ifnα2b and ribavirin in a non-human primate model ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study ribavirin and interferon-α2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a non-human primate model of common marmoset treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome-coronavirus infection: a case report of serial computed tomographic findings in a young male patient combination therapy with lopinavir/ ritonavir, ribavirin and interferon-α for middle east respiratory syndrome: a case report clinical implications of five cases of middle east respiratory syndrome coronavirus infection in south korea outbreak from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design thiopurine analogs and mycophenolic acid synergistically inhibit the papainlike protease of middle east respiratory syndrome coronavirus ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3cl pro ): implications for nsp5 regulation and the development of antivirals screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture international severe acute respiratory & emerging infection consortium. treatment of mers-cov: decision support tool. international severe acute respiratory & emerging infection consortium the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis a conformation-dependent neutralizing monoclonal antibody specifically targeting receptorbinding domain in middle east respiratory syndrome coronavirus spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylaxis with a mers-covspecific human monoclonal antibody protects rabbits from mers-cov infection 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody identification of a broadspectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response protease inhibitors targeting coronavirus and filovirus entry the role of c5a in acute lung injury induced by highly pathogenic viral infections cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus a decade after sars: strategies for controlling emerging coronaviruses sars vaccines: where are we? middle east respiratory syndrome: current status and future prospects for vaccine development evaluation of candidate vaccine approaches for mers-cov recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates immune efficacy of first and repeat trivalent influenza vaccine in healthy subjects and hemodialysis patients presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study the finding that vaccination of dromedary camels reduces mers-cov shedding on infection, which provides a proof-of-principle for the vaccination of dromedary camels to block zoonotic transmission epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study a report of the clinical presentation of mers in patients in saudi arabia the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients middle east respiratory syndrome hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome association of higher mers-cov virus load with severe disease and death mortality risk factors for middle east respiratory syndrome outbreak, south korea animal models for sars and mers coronaviruses replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates the first description of mers-cov replication and shedding in the respiratory tract of dromedary camels synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice sars-like wiv1-cov poised for human emergence the work was supported by the intramural research program of the national institute of allergy and infectious diseases (niaid), us national institutes of health. the authors declare no competing interests. key: cord-343184-kptkmgdm authors: crameri, gary; durr, peter a.; klein, reuben; foord, adam; yu, meng; riddell, sarah; haining, jessica; johnson, dayna; hemida, maged g.; barr, jennifer; peiris, malik; middleton, deborah; wang, lin-fa title: experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus date: 2016-06-17 journal: emerg infect dis doi: 10.3201/eid2206.160007 sha: doc_id: 343184 cord_uid: kptkmgdm we conducted a challenge/rechallenge trial in which 3 alpacas were infected with middle east respiratory syndrome coronavirus. the alpacas shed virus at challenge but were refractory to further shedding at rechallenge on day 21. the trial indicates that alpacas may be suitable models for infection and shedding dynamics of this virus. (mers-cov) was first reported in september 2012 (1); since then, >1,600 confirmed cases have been reported to the world health organization (http://www.who.int/csr/ don/29-february-2016-mers-saudi-arabia/en). the role of domestic animals as an intermediate host for humans was initially suggested by case histories of infected patients who had visited farms or tended sick animals shortly before onset of infection (2) . this suggestion was given credence by a study of camel serum samples that showed high levels of neutralizing antibodies in disparate camel populations (3) ; the findings were subsequently confirmed by virus detection and sequencing (4) . infection trials in camels have been limited (5, 6) , mainly because of difficulties in housing and handling the animals in a high-containment facility, which is necessary because the virus has a biosafety level 3 classification (7) . however, the alpaca, a close relative within the camelidae family, may provide a temperamentally suitable and valuable animal model for mers-cov infection, particularly for developing and testing vaccine candidates for camels. we sought to assess whether alpacas could be infected by means of a natural (oronasal) route, to determine whether viral shedding occurred after reinfection, and to evaluate the development of serologic markers of protection. we obtained 3 adult female alpacas (vicugna pacos) from a commercial supplier in victoria, australia, and housed them in the biosafety level 3 containment facility at the csiro australian animal health laboratory. before experiments, the alpacas were allowed to acclimatize for 6 days; during this time, intrauterine temperature data loggers were implanted according to a previously published procedure (8) . we found no previous mers-cov challenge trial reported in alpacas, so we chose a preliminary dose and rechallenge time on the basis of our experience with other virus infection trials for other emerging infectious diseases (8) . we used a camel isolate of mers-cov (dromedary_ mers-cov_al-hasa_kfu-hku13/2013; genbank accession nos. kj650295-kj650297) for infection; the isolate was prepared in vero cells as described (9) . the 3 alpacas were exposed oronasally to a 10 6 50% tissue culture infective dose of mers-cov in 5 ml of phosphate-buffered saline. the animals were monitored for 21 days, reexposed to a replicate challenge of mers-cov, and observed for 14 more days. clinical samples of blood (in edta for obtaining serum) and swabs (deep and superficial nasal, oral, rectal, and urogenital) were collected immediately before inoculation and thereafter on days 3, 5, 7, 10, 12, 14, 21, 26, 28, 31, 33, and 35. alpacas were electively euthanized, 1 on day 33 and the others on day 35. the animals remained clinically healthy except for a reduced condition score that occurred by day 18 in 1 animal (alpaca 2); no signs of upper or lower respiratory tract disease appeared in any animal. increased temperature was noted in alpaca 2 during days 17-20, but fever (rectal temperature >39°c) was not recorded. gross abnormalities at postmortem examination were found only in alpaca 2 and comprised extensive adhesions of the caudal sac of compartment 1 of the stomach to the umbilicus; clinical findings in this animal were attributed to this lesion. rna extraction and real-time pcr were performed by following specimen-handling procedures established for hendra virus (8) and were used to identify shedding patterns after each challenge. after initial challenge, (table 1) . virus isolation was undertaken with vero cells by using published protocols (9) and was successful for all 3 animals from all types of samples. virus recovery was successful from oral and superficial nasal swab samples through day 12; deep nasal swab samples were positive only through day 10. all urogenital and rectal swab samples were negative by both real-time pcr and virus isolation. after rechallenge, viral rna was not detected with confidence from any sample (figure) . serum samples were assessed for immunologic responses by using a virus neutralization test (vnt) and a luminex bead assay to the nucleocapsid protein. we used in-house assays modeled after those previously developed to assess the serologic status of feral camels in central australia (10) . all animals were seronegative by both luminex and vnt before challenge. antibody was first detected by luminex on day 10 or day 12 in each animal ( table 2) ; neutralizing antibody titers were 1:20 to 1:40 in alpaca 2 from day 10. neutralizing antibody titers of 1:10 to 1:20 were detected in alpaca 1 from day 21 on but not in alpaca 3 at any time during the study. for controls, we used mers-cov positive and negative serum samples from egypt and australia (online technical appendix table, http://wwwnc.cdc.gov/eid/article/22/6/16-0007-techapp1.pdf). our study confirms that alpacas are susceptible to mers-cov infection; this finding is consistent with a previous report showing that alpaca kidney cell lines possessing the dipeptidyl peptidase-4 receptor could be infected in vitro (11) . our challenge/rechallenge trial was planned as a first stage in the assessment of the alpaca as a potential surrogate for camels for mers-cov vaccine testing. consequently, the trial was not designed for direct comparison with 2 previous mers-cov challenge trials reported in camels (5,6). our trial used a lower challenge dose and a different timeframe for observation; nevertheless, some preliminary comparative observations may be useful. in the previous studies, as in ours, the animals were inoculated by the oronasal route, and live virus was detected through day 7 postinfection. similarly, neutralizing antibodies were detected beginning 7-8 days postinfection. however, findings in the trials with camels differed considerably from findings in our trial. the trials with camels detected live virus from nasal washes at days 1-3, a nasal discharge, and transient temperature rises; viral rna was detected by real-time pcr for an extended period. furthermore, the vnt titers for camels were much higher than those for the alpacas in our study. these differences possibly represent underlying dissimilarities in immune responses to mers-cov for the 2 species but may also result from the higher infecting dose (10 7 50% tissue culture infective dose) used in the camel studies. our study showed that alpacas secreted live virus after oronasal infection and that the immune response to the initial infection prevented further excretion following reinfection. an underlying assumption in our trial is that the initial infection equates to natural vaccination and that the lack of viral excretion thus follows an induced immune memory response. however, our results indicate that this immunologic response is complex; although a strong serologic response developed in only 1 alpaca, all 3 alpacas were refractory to reinfection. this study has several limitations. first, it was a preliminary study with only 3 animals and functioned more as proof of concept than a definitive study of the use of alpacas as a model for studying infection dynamics of mers-cov in camelids. second, our observation period of 21 days before rechallenge is informative but does not provide complete information on duration of protective immunity. future studies should have a larger sample and a longer period of study postinoculation. third, our study did not seek to understand the pathogenesis of infection; we did not conduct histopathology or immunohistochemistry to understand the site of initial viral replication and the role of mucosal immunity in mounting an effective immune response upon infection. notwithstanding these limitations, we believe that the alpaca might be a useful model that could greatly facilitate the development and testing of vaccine candidates. we recommend further research and trials to substantiate this potential. experimental infection of alpacas with mers-cov isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels inactivation and safety testing of middle east respiratory syndrome coronavirus hendra virus vaccine, a one health approach to protecting horse, human, and environmental health mers coronavirus in dromedary camel herd, saudi arabia absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread replicative capacity of mers coronavirus in livestock cell lines infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas we thank kaylene selleck, leah frazer, jean payne, rachel arkinstall, and mahen perera for providing technical assistance and support for this study. mr. crameri is a virologist with the csiro australian animal health laboratory. he has pioneered work in the high biocontainment facility at csiro and worked on hendra, nipah, sars, mers, ebola, and many other viruses with high impact to human and animal health.note added in proof: adney et al. also report infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas in this issue of emerging infectious diseases (12) . key: cord-338973-73a7uvyz authors: xu, jiabao; zhao, shizhe; teng, tieshan; abdalla, abualgasim elgaili; zhu, wan; xie, longxiang; wang, yunlong; guo, xiangqian title: systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov date: 2020-02-22 journal: viruses doi: 10.3390/v12020244 sha: doc_id: 338973 cord_uid: 73a7uvyz after the outbreak of the severe acute respiratory syndrome (sars) in the world in 2003, human coronaviruses (hcovs) have been reported as pathogens that cause severe symptoms in respiratory tract infections. recently, a new emerged hcov isolated from the respiratory epithelium of unexplained pneumonia patients in the wuhan seafood market caused a major disease outbreak and has been named the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this virus causes acute lung symptoms, leading to a condition that has been named as “coronavirus disease 2019” (covid-19). the emergence of sars-cov-2 and of sars-cov caused widespread fear and concern and has threatened global health security. there are some similarities and differences in the epidemiology and clinical features between these two viruses and diseases that are caused by these viruses. the goal of this work is to systematically review and compare between sars-cov and sars-cov-2 in the context of their virus incubation, originations, diagnosis and treatment methods, genomic and proteomic sequences, and pathogenic mechanisms. coronaviruses (covs) are a group of viruses that co-infect humans and other vertebrate animals. cov infections affect the respiratory, gastrointestinal, liver, and central nervous systems of humans, livestock, birds, bats, mice, and many other wild animals [1] [2] [3] . for example, severe acute respiratory syndrome (sars) in 2002 and the middle east respiratory syndrome (mers) in 2012 were both coronaviruses that transmitted from animals to humans [4, 5] . the source of unexplained pneumonia was first discovered in wuhan in dec, 2019, and sars-cov-2, a new coronavirus, was isolated from the respiratory epithelium of patients. it belongs to a new evolutionary branch within the cov. on feb. 11th, 2020, the new coronavirus was officially renamed "sars-cov-2" from "2019-ncov" [6] . the disease caused by sars-cov-2 was called "coronavirus disease 2019" (covid-19) [7] . according to on nov. 27th, 2002, a respiratory illness erupted in guangdong province, china [19] . in feb, 2003, the chinese ministry of health announced that this acute respiratory syndrome had thus far resulted in 305 cases and five deaths [20] . the following month, there were clusters of atypical pneumonia reported in other parts of mainland china, hong kong [21] , canada [22] , and singapore [23] . in jul, 2003, sars-cov spread across 26 countries in six continents, and caused a cumulative 8,096 cases and 774 deaths (9.6%) [24] . in particular, a higher mortality (21%) was found in hospital personnel [25, 26] . on dec. 29th, 2019, the health departments of hubei province received a report that four employees of the south china seafood wholesale market were diagnosed with unknown-caused pneumonia in a local hospital, which was the first report of sars-cov-2 [27] . on dec. 31st, 2019, the national health commission of people republic of china and chinese center for disease control and prevention (china cdc) participated in the investigation and case-searching work [27] . on the same day, the government of wuhan released information about the disease outbreaks to society [28] . nowadays, the number of patients infected with sars-cov-2 continues to climb worldwide. by the date of this paper's submission, a cumulative 67,081 cases and 1,526 deaths (2.1%) were reported worldwide. in wuhan, china, the number is 37,914. the main timeline of sars and covid-19 epidemic development were shown in figure 1a ,b, respectively. glucocorticoid and interferon lopinavir/ritonavir (in testing) on nov. 27th, 2002, a respiratory illness erupted in guangdong province, china [19] . in feb, 2003, the chinese ministry of health announced that this acute respiratory syndrome had thus far resulted in 305 cases and five deaths [20] . the following month, there were clusters of atypical pneumonia reported in other parts of mainland china, hong kong [21] , canada [22] , and singapore [23] . in jul, 2003, sars-cov spread across 26 countries in six continents, and caused a cumulative 8,096 cases and 774 deaths (9.6%) [24] . in particular, a higher mortality (21%) was found in hospital personnel [25, 26] . on dec. 29th, 2019, the health departments of hubei province received a report that four employees of the south china seafood wholesale market were diagnosed with unknown-caused pneumonia in a local hospital, which was the first report of sars-cov-2 [27] . on dec. 31st, 2019, the national health commission of people republic of china and chinese center for disease control and prevention (china cdc) participated in the investigation and case-searching work [27] . on the same day, the government of wuhan released information about the disease outbreaks to society [28] . nowadays, the number of patients infected with sars-cov-2 continues to climb worldwide. by the date of this paper's submission, a cumulative 67,081 cases and 1,526 deaths (2.1%) were the initial symptoms of sars patients were fever (100%), cough (61.8%), myalgia (48.7%), dyspnea (40.8%), and diarrhea (31.6%) [29] , and the prognosis of patients was associated with host characteristics (including age, gender, etc.) [30] . during hospitalization, respiratory distress occurred in 90.8% of sars patients [29] . the duration from disease onset to severe respiratory distress was an average of 9.8 ± 3.0 days [29] . during the disease course, some patients developed leukopenia, lymphopenia, and thrombocytopenia with an upregulation of aspartate transaminase (ast), alanine aminotransferase (alt), lactic dehydrogenase (ldh), and c-reactive protein (crp) [29] . in comparison, covid-19 showed similar trends with sars patients [28] . fever, fatigue, and dry cough are the main manifestations of the patients, while nasal congestion, runny nose, and other symptoms of the upper respiratory tract are rare. beijing centers for diseases control and prevention indicated that the typical case of covid-19 has a progressive aggravation process. covid-19 can be classified into light, normal, severe, and critical types based on the severity of the disease [31] : (1) mild cases-the clinical symptoms were mild, and no pneumonia was found on the chest computed tomography (ct); (2) normal cases-fever, respiratory symptoms, and patients found to have imaging manifestations of pneumonia; (3) severe cases-one of the following three conditions: respiratory distress, respiratory rate ≥ 30 times/min (in resting state, refers to oxygen saturation ≤ 93%), partial arterial oxygen pressure (pao2)/oxygen absorption concentration (fio2) ≤ 300 mmhg (1 mmhg = 0.133 kpa); (4) critical cases-one of the following three conditions: respiratory failure and the need for mechanical ventilation, shock, or the associated failure of other organs requiring the intensive care unit [32] . the current clinical data shows that the majority of the deaths occurred in the older patients. however, severe cases have been documented in young adults who have unique factors, particularly those with chronic diseases, such as diabetes or hepatitis b. those with a long-term use of hormones or immunosuppressants, and decreased immune function, are likely to get severely infected. the incubation period of sars is 1-4 days [33] . however, in a small number of patients, the incubation period may be longer than 10 days [34] . it has been demonstrated that the latency of covid-19 varies from 3-7 days on average, for up to 14 days [35] . during this incubation period, patients are contagious, and it has been reported that each case infected on average 3.77 other people (uncertainty range 2.23-4.82) [36] . by comparison, we found that the average latency of covid-19 is slightly longer than that of sars. according to the demographic information of sars patients, infection occurred in all age groups (the average age was dyspnea (40.8%), and diarrhea (31.6%) [29] , and the prognosis of patients was associated with host characteristics (including age, gender, etc.) [30] . during hospitalization, respiratory distress occurred in 90.8% of sars patients [29] . the duration from disease onset to severe respiratory distress was an average of 9.8 ± 3.0 days [29] . during the disease course, some patients developed leukopenia, lymphopenia, and thrombocytopenia with an upregulation of aspartate transaminase (ast), alanine aminotransferase (alt), lactic dehydrogenase (ldh), and c-reactive protein (crp) [29] . in comparison, covid-19 showed similar trends with sars patients [28] . fever, fatigue, and dry cough are the main manifestations of the patients, while nasal congestion, runny nose, and other symptoms of the upper respiratory tract are rare. beijing centers for diseases control and prevention indicated that the typical case of covid-19 has a progressive aggravation process. covid-19 can be classified into light, normal, severe, and critical types based on the severity of the disease [31] : (1) mild cases-the clinical symptoms were mild, and no pneumonia was found on the chest computed tomography (ct); (2) normal cases-fever, respiratory symptoms, and patients found to have imaging manifestations of pneumonia; (3) severe cases-one of the following three conditions: respiratory distress, respiratory rate ≥ 30 times / min (in resting state, refers to oxygen saturation ≤ 93%), partial arterial oxygen pressure (pao2)/oxygen absorption concentration (fio2) ≤ 300 mmhg (1 mmhg = 0.133 kpa); (4) critical cases-one of the following three conditions: respiratory failure and the need for mechanical ventilation, shock, or the associated failure of other organs requiring the intensive care unit [32] . the current clinical data shows that the majority of the deaths occurred in the older patients. however, severe cases have been documented in young adults who have unique factors, particularly those with chronic diseases, such as diabetes or hepatitis b. those with a long-term use of hormones or immunosuppressants, and decreased immune function, are likely to get severely infected. the incubation period of sars is 1-4 days [33] . however, in a small number of patients, the incubation period may be longer than 10 days [34] . it has been demonstrated that the latency of covid-19 varies from 3-7 days on average, for up to 14 days [35] . during this incubation period, patients are contagious, and it has been reported that each case infected on average 3.77 other people (uncertainty range 2.23-4.82) [36] . by comparison, we found that the average latency of covid-19 is slightly longer than that of sars. according to the demographic information of sars patients, infection occurred in all age groups (the average age was ≦45) [37] . there was a proportional difference between male and female (female predominance) [37] , with a male-to-female ratio of 1:1.25 [38] . in addition, hospital staff had a higher risk due to the proximal interactions with large numbers from the infected population. for example, hospital staff accounted for 22% of all cases in hong kong and 22.8% in guangdong [37] . the mortality caused by sars increased with age (> 64 years) [37] , and the overall mortality rate during the outbreak of sars was estimated at 9.6% [39, 40] . li et al. reported that people who have not been exposed to sars-cov-2 are all susceptible to covid-19 [41] . among the 8,866 patients who have been confirmed with covid-19, nearly half of the patients have been aged 50 years or older (47.7%) [36] . the male-to-female ratio is about 2.7:1 [38] and the average incubation period is 5.2 days [42] . however, severe covid-19 cases and deaths have mostly been in the middle-aged adults and the elderly with long smoking histories or other 45) [37] . there was a proportional difference between male and female (female predominance) [37] , with a male-to-female ratio of 1:1.25 [38] . in addition, hospital staff had a higher risk due to the proximal interactions with large numbers from the infected population. for example, hospital staff accounted for 22% of all cases in hong kong and 22.8% in guangdong [37] . the mortality caused by sars increased with age (> 64 years) [37] , and the overall mortality rate during the outbreak of sars was estimated at 9.6% [39, 40] . li et al. reported that people who have not been exposed to sars-cov-2 are all susceptible to covid-19 [41] . among the 8,866 patients who have been confirmed with covid-19, nearly half of the patients have been aged 50 years or older (47.7%) [36] . the male-to-female ratio is about 2.7:1 [38] and the average incubation period is 5.2 days [42] . however, severe covid-19 cases and deaths have mostly been in the middle-aged adults and the elderly with long smoking histories or other basic diseases, such as heart disease and hypertension [43, 44] . at the time that this paper was been submitted, covid-19 patients mortality rate was 2.1% [45] . in 2010, shi et al. isolated a sars-like coronavirus which was highly homologous to the sars-cov, confirming that rhinolophus sinicus (chinese rufous horseshoe bat) was a natural host of the sars-cov [46] . bats are known to be hosts for 30 coronaviruses based on complete genomic sequences analysis [47] . epidemiological investigations have shown that civet cats in the wildlife market were the direct source of sars-cov [48] . among the four patients with sars discovered during the winter of 2003-2004, two were waitresses at a restaurant in guangzhou, china, and one was a customer who viruses 2020, 12, 244 5 of 17 ate in the restaurant a short distance from a civet cage. all six civets in this cage were tested positive for sars-cov [48] . the new emerging sars-cov-2 shares about 80% of the gene sequence of sars-cov, released by the military medical research institute of nanjing military region in 2003 [28] . recently, shi et al. reported that the sequence similarity of coronavirus between sars-cov-2 and the coronavirus isolated from rhinolophus affinis is 96.2%, and suggested that bats may be the source of the virus [49] . so far, the intermediate hosts of sars-cov-2 are elusive and have been reported to be snakes, minks, or variable others [50, 51] . recently, a research group of south china agricultural university reported that pangolins may be one of the intermediate hosts for sars-cov-2, by analyzing more than 1,000 metagenomic samples, because they found that 70% of pangolins are positive for the coronavirus. moreover, the virus isolate from pangolin shared 99% sequence similarity with the current infected human strain sars-cov-2 [52] . taking this recent research into consideration, we agreed that pangolin is more likely to be one of intermediate hosts of sars-cov-2. according to the who data on jul. 31th, 2003 [24] , a total of 8,096 clinically diagnosed cases of sars were reported worldwide, with 774 deaths and 26 countries and regions affected (figure 2a ). most cases were in asia, europe, and america. the main countries in asia were china (including mainland, macao, hong kong, and taiwan), singapore, and so on. the total number of cases in mainland china was 5,327, with 349 deaths [53] . the cases were mainly concentrated in beijing, guangdong, and shanxi ( figure 2b ) [54] . in total, 2,102 patients were from hong kong, macao, and taiwan, with 336 deaths [24] . viruses 2020, 12, x for peer review 5 of 18 basic diseases, such as heart disease and hypertension [43, 44] . at the time that this paper was been submitted, covid-19 patients mortality rate was 2.1% [45] . in 2010, shi et al. isolated a sars-like coronavirus which was highly homologous to the sars-cov, confirming that rhinolophus sinicus (chinese rufous horseshoe bat) was a natural host of the sars-cov [46] . bats are known to be hosts for 30 coronaviruses based on complete genomic sequences analysis [47] . epidemiological investigations have shown that civet cats in the wildlife market were the direct source of sars-cov [48] . among the four patients with sars discovered during the winter of 2003-2004, two were waitresses at a restaurant in guangzhou, china, and one was a customer who ate in the restaurant a short distance from a civet cage. all six civets in this cage were tested positive for sars-cov [48] . the new emerging sars-cov-2 shares about 80% of the gene sequence of sars-cov, released by the military medical research institute of nanjing military region in 2003 [28] . recently, shi et al. reported that the sequence similarity of coronavirus between sars-cov-2 and the coronavirus isolated from rhinolophus affinis is 96.2%, and suggested that bats may be the source of the virus [49] . so far, the intermediate hosts of sars-cov-2 are elusive and have been reported to be snakes, minks, or variable others [50, 51] . recently, a research group of south china agricultural university reported that pangolins may be one of the intermediate hosts for sars-cov-2, by analyzing more than 1,000 metagenomic samples, because they found that 70% of pangolins are positive for the coronavirus. moreover, the virus isolate from pangolin shared 99% sequence similarity with the current infected human strain sars-cov-2 [52] . taking this recent research into consideration, we agreed that pangolin is more likely to be one of intermediate hosts of sars-cov-2. according to the latest data on feb. 14th, 2020 [55, 56] , there have been a total of 67,081 clinically diagnosed cases of covid-19 in worldwide, with 1,526 deaths. a total of 25 countries and regions have infected people. due to the spring festival transportation peak, the disease has been spread more rapidly across china (figure 2c ). as the origin area of covid-19, hubei province has been the most severely infected area, with 54,406 cumulative diagnosis cases. wuhan city has 37,914 cases. guangdong, henan, and zhejiang province have 1,294 cases, 1,212 cases, and 1,162 cases, respectively (figure 2d ). at present, the covid-19 outbreak has been spread to all parts of china and around the world, including the united states, thailand, and japan. it has been noticed that most of these patients have ever been to wuhan or contacted with people who had been in wuhan. the distribution of covid-2019 patients in china (including hong kong, macao and taiwan) and hubei province is shown in figure 3 . sars were reported worldwide, with 774 deaths and 26 countries and regions affected (figure 2a) . most cases were in asia, europe, and america. the main countries in asia were china (including mainland, macao, hong kong, and taiwan), singapore, and so on. the total number of cases in mainland china was 5,327, with 349 deaths [53] . the cases were mainly concentrated in beijing, guangdong, and shanxi (figure 2b ) [54] . in total, 2,102 patients were from hong kong, macao, and taiwan, with 336 deaths [24] . according to the latest data on feb. 14th, 2020 [55, 56] , there have been a total of 67,081 clinically diagnosed cases of covid-19 in worldwide, with 1,526 deaths. a total of 25 countries and regions have infected people. due to the spring festival transportation peak, the disease has been spread more rapidly across china (figure 2c ). as the origin area of covid-19, hubei province has been the most severely infected area, with 54,406 cumulative diagnosis cases. wuhan city has 37,914 cases. guangdong, henan, and zhejiang province have 1,294 cases, 1,212 cases, and 1,162 cases, respectively (figure 2d ). at present, the covid-19 outbreak has been spread to all parts of china and around the world, including the united states, thailand, and japan. it has been noticed that most of these patients have ever been to wuhan or contacted with people who had been in wuhan. the distribution of covid-2019 patients in china (including hong kong, macao and taiwan) and hubei province is shown in figure 3 . as the number of covid-19 patients in china has been growing rapidly, preventing the spread of sars-cov-2 is the most important and urgent task [57] . it was shown that human-to-human transmission of sars-cov-2 has spread via droplets or close contacts [58, 59] , but aerosol and fecal-oral transmission still need further study [60, 61] . to reduce virus transmission, early detection and isolation are essential. in addition, close monitoring in crowded places is also important [62] . the possible pathogens of sars and covid-19 are both derived from wild animals [63] . therefore, hunting, selling, and eating wild animals not only seriously damage the ecosystem, but also lead to the spread of epidemic diseases [64] . thus, banning all wildlife trade is an effective measure to prevent viral prevalence. wearing level-d protective clothing can protect medical staff from infection of respiratory viruses [65] . a vaccine against sars-cov has not been described in any published articles [66] . however, on jan. 26th, 2020, the china cdc started to develop a new vaccine for sars-cov-2. the virus has been successfully isolated and seed strains have been screened [67] . the early symptoms of sars and covid-19 are very similar to winter influenza, and the most important way to distinguish flu and pneumonia is to take throat swabs for viral testing [68] . current diagnostic tests for coronavirus include rt-pcr, real-time reverse transcription pcr (rrt-pcr), reverse transcription loop-mediated isothermal amplification, as well as real-time rt-lamp [69] [70] [71] [72] . national medical products administration has approved seven new nucleic acid test reagents for coronavirus, which were developed based on fluorescence pcr by feb. 1st, 2020 [73] . suspected infections can be detected accurately and quickly for timely isolation and treatment to avoid infecting others by using these test reagents. both sars-cov and sars-cov-2 are covs; hence, the treatment strategies of sars could be relevant for covid-19 [74] . in 2003, sars was mainly treated by isolation of the patients, hormones treatment, antiviral and symptomatic treatments, and many drugs such as glucocorticoid [29] and interferon [75] . now, isolation, antiviral, and symptomatic treatments are still mainly adopted for covid-19 treatment. as effective drugs for sars, hormones and interferons can also be used to treat covid-19 [74] . lopinavir is one kind of protease inhibitor used to treat hiv infection, with ritonavir as a booster. lopinavir and/or ritonavir has anti coronavirus activity in vitro. hong kong scholars found that, compared with ribavirin alone, patients treated with lopinavir/ritonavir and ribavirin had lower risk of acute respiratory distress syndrome (ards) or death caused by sars-cov [76, 77] . lopinavir/ritonavir has also been clinically tested in treatment of covid-19, and showed wonderfully effective treatment for some patients, but the general clinical effect has not been determined [78] . more effective treatments are still under continuing exploration: on jan. 25th, 2020, a joint research team from the shanghai institute of materia medica, chinese academy of sciences, and shanghai tech university screened and identified 30 potential drugs that are reported to be effective against sars-cov-2 [79] . a high-resolution crystal structure of sars-cov-2 coronavirus 3cl hydrolase (mpro) was announced after the outbreak of covid-19 in the world [80] , and human coronaviruses (hcovs) have been treated as severe pathogens in respiratory tract infections. nelfinavir was predicted to be a potential inhibitor of sars-cov-2 main protease [81] . the first patient in the us had been trial-treated with intravenous remdesivir (a novel nucleotide analogue prodrug in development) due to a severe infection [82, 83] . no adverse reactions were observed during the administration, and the patient's condition was effectively improved [84] . clinical trials of remdesivir for treatment of covid-19 just started on feb. 5th and 12th, 2020 in wuhan and beijing, respectively, and the experimental results remain unclear [85, 86] . covs are rna viruses and contain the largest genomes of all rna viruses [87] . covs belong to the subfamily coronavirinae in the family of coronaviridae of the order nidovirales, and this subfamily includes four genera: α-coronavirus, β-coronavirus, γ-coronavirus, and δ-coronavirus [88] . both sars-cov-2 and sars-cov are in the coronavirus family, β-coronavirus genera [89] . the genome of sars-cov-2 is more than 85% similar to the genome of the sars-like virus zc45 (bat-sl-covzc45, mg772933.1), and together these types of viruses form a unique orthocoronavirinae subfamily with another sars-like virus zxc21 in the sarbecovirus subgenus [35] . all the three viruses show typical β-coronavirus gene structure. human sars-cov and a genetically similar bat coronavirus (bat-sl-covzxc21, mg772934) from southwest of china have formed another clade within the sarbecovirus [35] . we also performed comparative genomic analyses of sars-cov-2 and sars-cov by zpicture. the results showed that the genomic sequences of sars-cov-2 and sars-cov have extremely high homology at the nucleotide level (figure 4a,b) . there are six regions of difference (rd) in the genome sequence between sars-cov and sars-cov-2, and the rds are named according to the order of discovery. rd1, rd2, and rd3 (448nt, 55nt, and 278nt, respectively) are partial coding sequences of the orf lab gene; rd4 and rd5 (315nt and 80nt, respectively) are partial coding sequences of the s gene; rd6 is 214nt in size and is part of the coding sequence of the orf7b and orf8 genes. these rds may sequences of the orf lab gene; rd4 and rd5 (315nt and 80nt, respectively) are partial coding sequences of the s gene; rd6 is 214nt in size and is part of the coding sequence of the orf7b and orf8 genes. these rds may provide new molecular markers for the identification of sars-cov-2 and sars-cov, and also help to develop new drugs against sars-cov-2. to analyze the homogeneity of sars-cov, mers-cov, and sars-cov-2, an evolutionary tree was constructed based on the genomes of 35 sars-cov-2 strains from different data submission units, one sars-cov strain, and two mers-cov strains ( figure s1 ). phylogenetic analysis showed that the distance of sars-cov (ay274119) is closer to sars-cov-2 strains than mers-cov (kc164505, jx869059). to analyze the homogeneity of sars-cov, mers-cov, and sars-cov-2, an evolutionary tree was constructed based on the genomes of 35 sars-cov-2 strains from different data submission units, one sars-cov strain, and two mers-cov strains ( figure s1 ). phylogenetic analysis showed that the distance of sars-cov (ay274119) is closer to sars-cov-2 strains than mers-cov (kc164505, jx869059). to further explore whether all encoded proteins of sars-cov-2 are homologous to that of sars-cov, we performed a protein sequence alignment analysis using blastp. the results showed that most of sars-cov-2 proteins are highly homologous (95%-100%) to the proteins of sars-cov virus, indicating the evolutionary similarity between sars-cov and sars-cov-2 (table 2) . however, two proteins (orf8 and orf10) in sars-cov-2 have no homologous proteins in sars-cov. the amino acid sequence of orf8 in sars-cov-2 is different from sequences of conserved orf8 or orf8b derived from human sars-cov [11] . orf8 protein of sars-cov-2 does not contain known functional domain or motif. an aggregation motif vlvvl (amino acid 75-79) has been found in sars-cov orf8b which was shown to trigger intracellular stress pathways and activate nod-like receptor family pyrin domain-containing-3 (nlrp3) inflammasomes [90] . therefore, it will be clinically meaningful to analyze the biological function of these two specific proteins (orf8 and orf10) in sars-cov-2. as the most abundant protein in covs, nucleocapsid (n) protein is highly conserved across covs [91] . the n protein in sars-cov-2 shares~90% amino acid identity with that in sars-cov [28] , which indicates that antibodies against the n protein of sars-cov would likely recognize and bind the n protein of sars-cov-2 as well. n antibodies do not provide immunity to sars-cov-2 infection, but the antibodies have a cross reactivity with sars-cov n protein viruses, which would allow a serum-based assay to identify the asymptomatic sars-cov-2 infected-cases [28] . although previous studies have found serum reactivity to group sars-cov n proteins in chinese populations [92] , exposure to sars-cov-2 should increase the dilution factor if the infection had occurred. this information has important implications for preventing the spread of asymptomatic infections. in addition, spike stalk s2 in sars-cov-2 is highly conserved and shares 99% identity with those of the two bat sars-like covs (bat-sl-covzxc21 and bat-sl-covzc45) and human sars-cov [11] . thus, the broad spectrum antiviral peptides against s2 has the potential to be effective treatment [93] . many studies have been performed to study the pathogenesis of sars-cov [94] [95] [96] . the spike (s) protein and n protein confer stability to the viral particle [97] . the n protein is a structural protein involved in virion assembly, and plays a pivotal role in virus transcription and assembly efficiency [97] . s protein can bind to the cellular receptors of sensitive cells and mediate infection of their target cells, after which it begins to replicate in the cytoplasm [98] . sars-cov mainly targets the lungs, immune organs, and small systemic blood vessels and causes systemic vasculitis and decrease of immune function [99] . more seriously, the infection leads to extensive pulmonary consolidation, diffuse alveolar damage, and the formation of a transparent membrane, finally deteriorating to respiratory distress [100] . cov can enter host cells through the interaction between cov s protein and its host receptor angiotensin-converting enzyme 2 (ace2), which is isolated from sars-cov-permissive vero-e6 cells. the receptor-binding motif (rbm) of s protein can directly contact ace2 [47, 101, 102] . aec2 have been identified to be key binding residues and functional receptor for sars-cov, and it can also protect alveolar cells [103] . the binding of spike protein to ace2 and the subsequent downregulation of this receptor contribute to severe alveolar injury during sars [49] . the downregulation of ace2 results in the excessive production of angiotensin ii by the related enzyme ace, and the stimulation of type 1a angiotensin ii receptor (agtr1a) can lead to the increase of pulmonary vascular permeability, which potentially explains the increased lung pathology when the expression of ace2 is decreased [104] . ace2-transfected t cells form multinucleated syncytia with cells expressing s protein. the virus was shown to replicate effectively in ace2-transfected, but not in mock-transfected t cells. antibodies targeting ace2 can block viral replication in vero-e6 cells [105] . recently, ji et al. demonstrated that the receptor binding domain of sars-cov-2 was capable of binding ace2 in the context of the sars-cov spike protein [51] . among sras-cov spike protein's fourteen residues predicted to interact directly with human ace2 as the receptor for sars-cov, eight amino acids are well conserved in homology sars-cov-2 spike protein [26, 102] . at the same time, wan et al. showed that sars-cov-2 uses ace2 receptors to infect humans, bats, civets, monkeys, and swine, but not mice [106] . compared to previously reported sars-cov strains, sars-cov-2 uses ace2 receptors more efficiently than human sars-cov (year 2003), but less efficiently than human sars-cov (year 2002) [106] . the mutation of proteins determines two important characteristics of the sars-cov-2: a higher ability to infect and enhanced pathogenicity than the bat-like sars-cov, but a lower pathogenicity than sars-cov [43] . as a large number of people have left wuhan, the control of the epidemic situation is extremely urgent, and the treatments of covid-19 are imminent. on feb. 14th, 2020, there were more than 54,000 confirmed patients in hubei province, china [55] . due to the lack of effective antiviral drugs, the prognosis of patients solely depends on their age and physical condition [74] . although it was reported that the clinically recovered patients exceed the number of dead, the majority of the patients are still not cured in hospital. in addition, the potential adaptive mutation of sars-cov-2 makes it difficult for vaccine development. therefore, it is urgent for us to develop more sensitive inspection methods and effective drugs. seven type of covs have been identified to cause human disease [107] . the two highly pathogenic viruses, sars-cov and mers-cov, cause severe respiratory syndrome in humans. the other four human covs (hcov-nl63, hcov-229e, hcov-oc43 and hku1) induce only mild upper respiratory diseases, although some of them can cause severe infections in infants, young children, and elderly individuals [4, 108] . the latest one is sars-cov-2. it has been reported that sars-cov-2 shared almost 80% of the genome with sars-cov [11] . our results also showed that almost all encoded proteins of sars-cov-2 are homologous to sars-cov proteins ( table 2) . hence, clinical drugs and therapies for treating sars may be used as a reference for covid-19 treatment [74] . in addition to the well-known sars-cov, mers-cov, as one merbecovirus subgenus of β-covs, is also extremely invasive. mers-cov is the pathogen of the middle east respiratory syndrome, which can infect both humans and animals, and can be transmitted through camels [109] . it mainly occurs in saudi arabia and has a high mortality rate [66] . studies had demonstrated that the clinical course of sars and mers was highly similar, and sars and mers may have similar pathogenesis [66] . the genome sequence of sars-cov-2 also shows some similarities to that of mers-cov. it will be very interesting to study the relationship among sars-cov, mers-cov, and sars-cov-2 that may be exploited for future developing broad-spectrum antiviral therapies. although more and more studies for sars-cov-2 have sprung up since the outbreak of this epidemic covid-19, based on our comparison, we propose some key questions to be clarified in future studies (table 3 ). in-depth understanding the underlying pathogenic mechanisms of sars-cov-2 will reveal more targets for better therapy of covid-19. table 3 . proposed questions to study sars-cov-2 for future studies. what is the effect of the surface epitope and receptor binding domain of s protein of sars-cov-2 on the virus' infectivity? is there any effect of sars-cov vaccine designed according to s protein on sars-cov-2? does sars-cov-2 orf8 and orf10 proteins, which have no homology proteins in sars-cov, play roles in the infectivity and pathogenicity of sars-cov-2? can the susceptibility of asymptomatic carriers be judged by detecting the serum reactivity level of n protein? apart from droplet transmission and contact transmission, are there other methods to transmit sars-cov-2? what is the percentage of covid-19 patients have been infected with sars and produced antibodies? is there an effective specific anti-sars-cov-2 solution? does traditional chinese medicine have any effect on the treatment of covid-19 caused by sars-cov-2? do ethnic differences affect the transmissibility and pathogenicity of sars-cov-2? do any environmental factors, such as regional conditions or climate, affect sars-cov-2 transmission? supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/2/244/s1, figure s1 : phylogeny of sars-cov-2, sars-cov and mers-cov. unrooted phylogenetic relationships are represented by constructing neighbor-joining tree. table s1 : the genomic information of latest sars-cov-2 strains author contributions: conceptualization, l.x., y.w. and x.g.; methodology, j.x. and w.z.; software, s.z. and w.z.; validation, j.x., s.z. and t.t.; formal analysis, j.x. and s.z.; writing-original draft preparation, j.x. and s.z.; writing-review and editing, l.x., y.w., w.z. and x.g.; visualization, j.x., s.z., and a.e.a.; funding acquisition, l.x., y.w. and x.g. all authors have read and agreed to the published version of the manuscript. isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor molecular mechanisms of coronavirus rna capping and methylation origin and evolution of pathogenic coronaviruses transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group who named the new pneumonia "covid-19 isolation and characterization of viruses related to the sars coronavirus from animals in southern china mers outbreak in korea: hospital-to-hospital transmission evidence for camel-to-human transmission of mers coronavirus genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan ncbi database. 2019-ncov. available online gsaid database. 2020 coronavirus. available online mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets prospects for inferring very large phylogenies by using the neighbor-joining method severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada severe acute respiratory syndrome-singapore summary of probable sars cases with onset of illness from 1 the sars wake-up call travel implications of emerging coronaviruses: sars and mers-cov chinese center for disease control and prevention. epidemic update and risk assessment of 2019 novel coronavirus return of the coronavirus: 2019-ncov. viruses clinical manifestations, laboratory findings, and treatment outcomes of sars patients middle east respiratory syndrome: emergence of a pathogenic human coronavirus diagnosis and treatment of pneumonia caused by 2019-ncov (trial version 4) pulmonary rehabilitation guidelines in the principle of 4s for patients infected with 2019 novel coronavirus (2019-ncov) incubation periods of acute respiratory viral infections: a systematic review multiple contact dates and sars incubation periods a novel coronavirus from patients with pneumonia in china a novel coronavirus outbreak of global health concern the severe acute respiratory syndrome the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients infectious disease expert li lanjuan responded to six questions of 2019-ncov early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical features of patients infected with 2019 novel coronavirus in epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study press conference of the national health commission of the people's republic of china on 3 intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans novel coronavirus: from discovery to clinical diagnostics sars-cov infection in a restaurant from palm civet discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin host and infectivity prediction of wuhan 2019 novel coronavirus using deep learning algorithm homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human pangolin may be a potential intermediate host of new coronavirus. available online model parameters and outbreak control for sars a comparative epidemiologic analysis of sars in hong kong, beijing and taiwan chinese center for disease control and prevention. distribution of pneumonia infected by 2019-ncov real-time updates of covid-19 epidemic in china the extent of transmission of novel coronavirus in wuhan, china, 2020 potential of large 'first generation' human-to-human transmission of 2019-ncov national health commission of the people's republic of china. the role of fecal-oral transmission in all transmission still need further observation and research there is no evidence that the new coronavirus can be transmitted through aerosol sars transmission, risk factors, and prevention in hong kong outbreak of pneumonia of unknown etiology in wuhan china: the mystery and the miracle wild animal mortality monitoring and human ebola outbreaks the fight against the 2019-ncov outbreak: an arduous march has just begun recent insights into emerging coronaviruses chinese center for disease control and prevention. the first new coronavirus species information was published by the national pathogen microorganism resource bank the utility of preemptive mass influenza vaccination in controlling a sars outbreak during flu season characterization of a new member of alphacoronavirus with unique genomic features in rhinolophus bats real-time sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in wuhan, china, as at 22 national medical products administration. national medical products administration examined and approved the new nucleic acid test reagent of coronavirus again emerging coronaviruses: genome structure, replication, and pathogenesis treatment of sars with human interferons role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings drug treatment options for the 2019-new coronavirus (2019-ncov) explanation on the situation that drugs for aids can be tried to treat pneumonia caused by 2019-ncov infection chinese academy of sciences, and shanghaitech university discovered a batch of old drugs and traditional chinese medicines with therapeutic potential for covid-19 high-resolution crystal structure of 2019-ncov coronavirus 3cl hydrolase (mpro) was announced by the joint research team of shanghai institute of materia medica nelfinavir was predicted to be a potential inhibitor of 2019 ncov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a randomized, controlled trial of ebola virus disease therapeutics first case of 2019 novel coronavirus in the united states research progress on novel coronavirus (2019-ncov) related drugs in vitro/vivo friendship hospital: clinical experimental research work is being carried out on remdesivir recombination in large rna viruses discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes jumping species-a mechanism for coronavirus persistence and survival serological evidence of bat sars-related coronavirus infection in humans clinical virology and pathogenesis expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis coronaviruses post-sars: update on replication and pathogenesis the clinical pathology of severe acute respiratory syndrome (sars): a report from organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways sars: clinical features and diagnosis host cell proteases: critical determinants of coronavirus tropism and pathogenesis a pneumonia outbreak associated with a new coronavirus of probable bat origin a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars the novel chinese coronavirus (2019-ncov) infections: challenges for fighting the storm epidemiology, genetic recombination, and pathogenesis of coronaviruses acknowledgments: all the genomic data of sars-cov-2 is from gisaid, ncbi/genbank, nmdc, cngb/cngbdb and 2019ncovr (table s1 ). here, we would like to express our gratitude to all units and individuals who are responsible for the sample collection and data submission. the authors declare no conflict of interest. key: cord-352492-6ihyiwgb authors: eickmann, markus; gravemann, ute; handke, wiebke; tolksdorf, frank; reichenberg, stefan; müller, thomas h.; seltsam, axel title: inactivation of ebola virus and middle east respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet c light and methylene blue plus visible light, respectively date: 2018-05-06 journal: transfusion doi: 10.1111/trf.14652 sha: doc_id: 352492 cord_uid: 6ihyiwgb background: ebola virus (ebov) and middle east respiratory syndrome coronavirus (mers‐cov) have been identified as potential threats to blood safety. this study investigated the efficacy of the theraflex uv‐platelets and theraflex mb‐plasma pathogen inactivation systems to inactivate ebov and mers‐cov in platelet concentrates (pcs) and plasma, respectively. study design and methods: pcs and plasma were spiked with high titers of cell culture–derived ebov and mers‐cov, treated with various light doses of ultraviolet c (uvc; theraflex uv‐platelets) or methylene blue (mb) plus visible light (mb/light; theraflex mb‐plasma), and assessed for residual viral infectivity. results: uvc reduced ebov (≥4.5 log) and mers‐cov (≥3.7 log) infectivity in pcs to the limit of detection, and mb/light decreased ebov (≥4.6 log) and mers‐cov (≥3.3 log) titers in plasma to nondetectable levels. conclusions: both theraflex uv‐platelets (uvc) and theraflex mb‐plasma (mb/light) effectively reduce ebov and mers‐cov infectivity in platelets and plasma, respectively. has been transmitted through the transfusion of blood products. however, the fact that the mortality rate is high (>30%) and the transmission mechanisms are still not completely understood highlight the need for increased awareness of the potential threat to blood safety by mers-cov. mers-cov is an enveloped, positive-sense single-stranded rna coronavirus. interestingly, viral rna was detected in the sera of asymptomatic patients during an outbreak of severe acute respiratory syndrome, which is caused by another coronavirus, severe acute respiratory syndrome-cov. consequently, there might be an asymptomatic viremic phase that could theoretically lead to transmission via blood transfusion. however, blood transfusions have not been implicated in the transmission of mers-cov to date. 5 ebov, a member of the filoviridae family, is an enveloped pathogen containing negative-sense single-stranded rna as its genetic material. ebov disease is extremely fatal and is known to cause death rates of up to 90%. recent outbreaks of ebov disease in west africa have devastated guinea, liberia, and sierra leone, killing more than 11,000 out of nearly 30,000 persons affected. 6 ebov is transmitted to humans from animals and spreads through human-to-human transmission, that is, via direct contact with infected blood and/or secretions. different pathogen inactivation (pi) techniques can effectively inactivate or incapacitate such viral agents, rendering them unable to replicate in blood components or products. 7 theraflex uv-platelets (macopharma), a novel ultraviolet c (uvc)-based pi system for platelet concentrates (pcs), is currently undergoing clinical efficacy and safety testing. [8] [9] [10] in contrast to other pi technologies, it works without exogenously added photoactive substances. shortwave uvc light (254 nm) interacts with nucleic acids directly, inducing the formation of cyclobutane pyrimidine and pyrimidine pyrimidone dimers that prevent the elongation of nucleic acid transcripts. 11 the theraflex mb-plasma (macopharma) is a photodynamic pi procedure for single units of fresh-frozen plasma. this system, based on the administration of methylene blue (mb) in combination with visible light (mb/light), was developed to increase the viral safety of plasma transfusions. 12, 13 more than 5 million units have been treated by this method and safely transfused to patients for more than 10 years. both systems have been shown to effectively inactivate a broad range of different dna and rna viruses, including arboviruses such as west nile virus, chikungunya virus, and zika virus; recently, large outbreaks of the latter viruses have occurred outside of their previously known geographic ranges, seriously affecting thousands of people. 9, [13] [14] [15] [16] [17] [18] this study aimed to investigate the efficacy of theraflex uv-platelets and theraflex mb-plasma to inactivate mers-cov and ebov in pcs and plasma, respectively. all components were prepared from whole blood (500 ml) collected from screened, unpaid volunteers and stored in 70 ml of cpd according to german guidelines. 19 blood was separated into red blood cells (rbcs), plasma, and buffy coat by standard blood banking procedures. plasma units were leukoreduced by filtration (plasmaflex, macopharma), pooled, split again, and stored at -208c until further use. plasma-reduced pcs were prepared from pools of five buffy coats as described by eriksson and colleagues. 20 the pools were mixed with 280 ml of pas ssp1 (macopharma), which is equivalent to pas-e. 21 the suspension was centrifuged for 7.5 minutes at 527 3 g. pcs were isolated, leukoreduced with a high-efficiency filter system (autostop bc, haemonetics), and stored under agitation at 22 6 28c. the platelet (plt) concentration was approximately 10 9 /ml, the plasma content approximately 35%, and the residual white blood cell (wbc) content was not more than 10 6 per unit. air was routinely removed from all plasma units and pcs. the anti-mers-cov enzyme-linked immunosorbent assay (igg; euroimmun) was used to screen pcs and plasma units for the presence of antibodies against mers-cov. only products tested for antibodies to mers-cov and confirmed to be negative were used in the inactivation experiments. plts were treated using theraflex uv-platelets, a pi system consisting of a uvc illumination device (maco-tronic, macopharma) and a disposable set (ref xuv 4005 xu, macopharma). briefly, the pcs were irradiated with uvc light at a wavelength of 254 nm to a total dose of 0.2 j/cm 2 , with vigorous agitation to ensure the uniform treatment of all compartments. 9 the amount of uvc energy delivered was expressed in j/cm 2 . 9 plasma units were mb/light-treated using theraflex mb-plasma (macopharma), a pi system consisting of a theraflex mb-plasma kit and the macotronic b2 ledbased illumination device. its closed-bag system contains a mb pill that delivers 85 mg of mb per unit of plasma or approximately 1 mmol/l per plasma unit. the standard treatment cycle includes initial filtration for leukoreduction (plasmaflex filter) followed by a second postillumination filtration step for the removal of mb and its photoproducts (blueflex filter). 12, 22 plasma processing for pi efficacy testing deviated from the standard procedure in that the removal of mb and photoproducts (blueflex filtration) was not performed so as to exclusively determine the virus inactivation effects of illumination. the amount of led-based light energy delivered was expressed in j/cm 2 . the total illumination dose was set at 120 j/cm 2 , as is routinely recommended for this system. 12 endpoint titration assays for microtiter plates were employed to measure virus titers in eight serial 1-in-3 dilutions per parallel sample. vero e6 cells were used as the indicator cells. sample titration was performed at the initial dilution at which vero cells exhibited no cytotoxicity. the plates were incubated at 378c in a humid atmosphere with 5% co 2 . after 5 to 7 days of incubation, the cell layers were assessed for virus-induced changes in morphology (cytopathology). the 50% tissue culture infectious dose (tcid 50 ) was calculated according to the spearman-k€ arber method and was expressed as log tcid 50 . 30, 31 the effectiveness of each individual process step for each virus was calculated as the log reduction factor (rf) using the formula rf 5 loga 0 -loga n , where r is the reduction factor, a 0 is the total virus load after spiking, and a n is the total virus load in the treated sample. the overall reduction factor was expressed as the sum of rfs for all steps. the limit of detection of the assay was defined as the lowest tcid 50 achievable at noncytotoxic sample concentrations. vero e6 cells (atcc ccl-22) together with the zaire ebolavirus strain (mayinga-76) were grown to approximately 80% confluence in dulbecco's modified eagle's medium (dmem) with 2% fetal bovine serum (fbs), 2 mmol/l l-glutamine, 100 mg/ml streptomycin, and 100 iu/ml penicillin. on day 6, the viral supernatant was collected, centrifuged, aliquoted, and frozen at -808c until further use (spiking experiments). vero e6 cells (atcc ccl-22) together with mers-cov (emc/2012), obtained from ron a. fouchier, were grown to approximately 80% confluence in dmem with 2% fbs, 2 mmol/l l-glutamine, 100 mg/ml streptomycin, and 100 iu/ml penicillin. on days 3 to 5, the viral supernatant was collected, centrifuged, aliquoted, and frozen at -808c until further use (spiking experiments). for each virus, two bags of pcs (volume, 374 ml) and plasma (volume, 315 ml) were spiked with 10% viral supernatant and treated as described with uvc or mb/light, respectively. each treatment was delivered incrementally up to the full light dose (0.2 j/cm 2 for uvc and 120 j/cm 2 for mb/light). at different process steps, samples were collected and virus titrations were performed. reference samples were taken from each bag before treatment, stored at room temperature, and tested at the end of the experiments to account for any intrinsic virus inactivation by the blood product. virus titers after spiking were below the expected dilution factor in six of the eight blood units used. in one pc and in one plasma unit used for ebov inactivation and in all four blood units used for mers-cov inactivation, the virus titers before pi treatment were up to 1 log lower than expected from a 1in-10 dilution. however, no additional reduction in virus titers was observed during the course of the experiment. the results of the infectivity assay demonstrated that uvc irradiation dose-dependently inactivated ebov and mers-cov in plasma-reduced pcs (table 1 ). at 0.15 j/cm 2 (75% of the full uvc dose), ebov and mers-cov infectivity levels were below the detection limit, resulting in virus reduction factors of greater than 4.5 for ebov and of greater than 3.7 for mers-cov. table 2 , light doses as low as 30 j/cm 2 , or 25% of the standard full light dose of 120 j/cm 2 for led-based illumination, inactivated ebov and mers-cov in plasma to levels below the detection limit. these results correspond to at least 4.6 and at least 3.3 log reductions of ebov and mers-cov, respectively. the theraflex uv-platelets and theraflex mb-plasma systems are designed to inactivate or remove pathogens in plt and plasma products, as has been demonstrated in a wide range of pathogenic agents. 9, 12, 17, 18, [23] [24] [25] the mechanism of theraflex uv-platelets is that shortwave uvc light penetrates blood fluids and cell membranes, causing direct covalent damage to the nucleic acids of pathogens and wbcs. this mechanism of action is broadly effective against extracellular and intracellular transfusion-transmitted dna/rna-containing pathogens, such as viruses, bacteria, and protozoa. theraflex mb-plasma combines mb-a phenothiazine compound that intercalates into viral nucleic acid-with visible light. the illumination of mb-treated plasma results in the generation of singlet oxygen, which leads to the destruction of viral nucleic acids and thus prevents viral replication. 12 because mb only partially penetrates cell membranes, mb/light treatment is less effective against intracellular viruses. this is why two filters are integrated in the process chain. although the second filter mainly removes mb and its photoactivated products after illumination, both filters have multiple layers of small-meshed membranes capable of removing residual wbcs, rbcs, and plts that may carry intracellular pathogens. it was recently shown that the two filters efficiently remove high levels (up to 5.9 log colonyforming units per milliliter) of viable bacteria and bacterial spores. 25 it can be assumed that parasites of similar sizes can also be removed by the theraflex mb-plasma procedure. a previous investigation showed that the filtration step before the addition of mb and visible light illumination was critical to achieving the complete and reproducible elimination of trypanosoma cruzi, the causative agent of chagas disease. 26 it is estimated that there are more than 1400 known human pathogens, 13% of which are considered emerging or reemerging. 27 the ability to provide proactive protection against emerging infectious agents complementary to the existing blood screening programs is a major additional benefit of pi systems for blood safety. thus, it is imperative that manufacturers continuously challenge their pi systems with new infectious agents. to our knowledge, this is the first study investigating the efficacy of uvc and mb/light against mers-cov and ebov or any other member of the coronaviridae and filoviridae families. our results showed that these two pi systems reduced mers-cov and ebov titers to below the limit of detection in the two blood units tested. the maximum viral titer reduction detectable in a cell culture infectivity assay is based on the starting viral concentration of the blood product and the detection limit of the assay which, in turn, is determined by the susceptibility of the cell culture to plasma and plt suspensions. as in a previous study, the observed reduction of ebov titers and mers-cov titers in the present study was beyond the expected dilution factor in pcs and plasma. 28 this phenomenon may occur due to the presence of nonspecific immune mediators that neutralize viruses in plasma. 28, 29 the mean log reduction factors of at least 3.7 (mers-cov) and at least 4.5 (ebov) in pcs treated with uvc and of at least 3.3 (mers-cov) and at least 4.6 (ebov) in plasma treated with mb/light suggest that both pi technologies used in this study are considerably effective against these two virus types. this study had a number of limitations. for example, the number of replicates was limited by safety constraints, as experiments with mers-cov and ebov are subject to the highest biosafety level. in addition, large-volume plating could not be performed, although this would have improved the detection limit and enabled more exact determination of log reduction factors. although plasma viral rna loads in mers-cov patients may be up to 6 log genome copies/ml, and median viral rna loads of up to 6.68 log genome copies/ml have been detected in the blood of ebola patients, 30, 31 virus titers in the plasma of asymptomatic infected or convalescent individuals recruited to donate blood may be significantly lower. as it is known for other infectious diseases, ebov viral titers expressed in genome copies per milliliter do not necessarily reflect the infectivity titer. while viral titers measured by quantitative polymerase chain reaction are based on the detection of a small fragment of the viral genome, infectivity titers describe the number of intact, functional viral units required for replication and disease transmission. although ebov is highly infectious and low doses of less than 10 plaque-forming units of the virus are sufficient to cause disease, 32 the ability of the uvc-and mb/ light-based systems to reduce mers-cov and ebov infectivity in blood products by several log steps may be sufficient to eliminate or significantly reduce the risk of transmission via the transfusion of pcs or plasma. in conclusion, this study further expands the list of pathogens that theraflex uv-platelets and theraflex mb-plasma can effectively inactivate in pcs and plasma, respectively. emerging infectious disease agents and their potential threat to transfusion safety current perspectives in transfusion-transmitted infectious diseases: emerging and re-emerging infections middle east respiratory syndrome coronavirus: five years later update on the epidemiology of middle east respiratory syndrome coronavirus cov) infection, and guidance for the public, clinicians, and public health authorities identification of a novel coronavirus in patients with severe acute respiratory syndrome ebola situation report 12 update on the use of pathogenreduced human plasma and platelet concentrates a novel approach to pathogen reduction in platelet concentrates using shortwave ultraviolet light uvc irradiation for pathogen reduction of platelet concentrates and plasma mitochondrial dna multiplex real-time polymerase chain reaction inhibition assay for quality control of pathogen inactivation by ultraviolet c light in platelet concentrates inter-strand photoproducts are produced in high yield within a-dna exposed to uvc radiation main properties of the theraflex mb-plasma system for pathogen reduction methylene bluetreated fresh-frozen plasma: what is its contribution to blood safety? photodynamic virus inactivation of blood components updates on pathogen inactivation of plasma using theraflex methylene blue system virus inactivation in blood components by photoactive phenothiazine dyes inactivation of dengue, chikungunya, and ross river viruses in platelet concentrates after treatment with ultraviolet c light reduction of zika virus infectivity in platelet concentrates after treatment with ultraviolet c light and in plasma after treatment with methylene blue and visible light month 2018 transfusion 5 2206 transfusion richtlinien zur gewinnung von blut und blutbestandteilen und zur anwendung von blutprodukten (h€ amotherapie) k€ oln: deutscher € arzte-verlag platelet concentrates in an additive solution prepared from pooled buffy coats. in vivo studies storage of platelets in additive solutions: effects of phosphate two pathogen reduction technologies-methylene blue plus light and shortwave ultraviolet light-effectively inactivate hepatitis c virus in blood products the effectiveness of uvc pathogen inactivation system on reducing the trypansosoma cruzi and leishmania infantum burden in platelets the efficacy of the ultraviolet c pathogen inactivation system in the reduction of babesia divergens in pooled buffy coat platelets challenge study of the pathogen reduction capacity of the theraflex mb-plasma technology the efficacy of photochemical treatment with methylene blue and light for the reduction of trypanosoma cruzi in infected plasma emerging pathogens: the epidemiology and evolution of species jumps treatment of blood with a pathogen reduction technology using ultraviolet light and riboflavin inactivates ebola virus in vitro mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complementmediated virus neutralization viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection age and ebola viral load correlate with mortality and survival time in 288 ebola virus disease patients clinical recognition and management of patients exposed to biological warfare agents key: cord-351852-ilxaurgt authors: jung, heeja; jung, sun young; lee, mi hyang; kim, mi sun title: assessing the presence of post-traumatic stress and turnover intention among nurses post–middle east respiratory syndrome outbreak: the importance of supervisor support date: 2020-03-09 journal: workplace health saf doi: 10.1177/2165079919897693 sha: doc_id: 351852 cord_uid: ilxaurgt background: south korea faced the middle east respiratory syndrome (mers) outbreak for the first time in 2015, which resulted in 186 infected patients and 39 deaths. this study investigated the level of post-traumatic stress disorder (ptsd) and turnover intention, the relationship between ptsd and turnover intention, and the buffering effect of supervisor support among nurses post-mers outbreak. methods: in total, 300 nurses from three of 15 isolation hospitals in south korea were invited to participate. we collected data pertaining to ptsd, turnover intention, supervisor support, work-related factors, and socio-demographic factors through a structured survey distributed to the nurses at the hospitals after the outbreak. for the statistical analyses, descriptive statistics and multiple regression were employed. findings: of the 147 participants, 33.3% were involved in the direct care of the infected patients, whereas 66.7% were involved in the direct care of the suspected patients. more than half (57.1%) of the nurses experienced ptsd, with 25.1% experienced full ptsd and 32.0% with moderate or some level of ptsd. the mean score of turnover intention was 16.3, with the score range of 4 to 20. the multiple regression analysis revealed that ptsd was positively associated with turnover intention, and supervisor support had a strong buffering effect. conclusion/application to practice: these findings confirmed that after a fatal infectious disease outbreak like mers, nurses experience high level of ptsd and show high intention to leave. organizational strategies to help nurses to cope with stress and to prevent turnover intention, especially using supervisor support, would be beneficial. among the 186 total infected patients, 44.1% (n = 82), 34.9% (n = 65), and 21.0% (n = 39) were pre-admitted patients, family members or visitors, and health care professionals, respectively. out of the 39 infected health care professionals, 15 were nurses. due to the lack of vaccine or special treatment along with a high mortality rate (36%), nurses directly involved in the care of mers patients showed high levels of stress and fear (khalid et al., 2016; who, 2018) . similarly, taiwanese nurses who were directly involved with the care of patients of severe acute respiratory syndrome (sars) in 2003 reported more severe post-traumatic stress compared with the nurses who were not involved in the direct care (chen et al., 2005) . post-traumatic stress disorder (ptsd) is associated with exposure to traumatic events resulting in a state of psychological unbalance (blake et al., 1995) . medical staff, including nurses, are more likely to be in environments that increase their sensitivity to ptsd (tang et al., 2017; wu et al., 2009) . another consistent issue of the nursing industry is the workforce shortage and high turnover rates. particularly in korea, less than 50% of registered nurses are actively working, and the average turnover rate is 12.4%, with a rate of 34% for newly graduated nurses (ministry of ). prior studies on the relationship between posttraumatic stress and turnover intention reported strong associations among nurses working in emergency rooms (han & lee, 2013; maeng & sung, 2015) . many other factors, including socio-demographic factors, organizational commitment, perceived advancement opportunities, job and career satisfaction, salary level, and workplace relationships and support, have also been found to influence turnover intention (ayalew et al., 2015; brunetto et al., 2013; y. kim & kang, 2015; laschinger, 2012; oyeleye et al., 2013; takase et al., 2016) . however, workplace relationships and support, especially supervisor support, have been proven to be strong buffers on work-related traumatic stress, work-related stress, and turnover (larocco et al., 1980; stephens & long, 2000) ; supervisor support has shown to be positively associated with job and career satisfaction (nissly et al., 2005) . to date, there is a lack of studies investigating the relationship between post-traumatic stress and turnover intention among nurses in the event of outbreak epidemics, such as mers. the purpose of this study was to examine the levels of ptsd and intention to leave among a sample of korean nurses who were directly involved in the care during the mers outbreak, as well as the buffering effects of supervisor support on this relationship. we conducted a cross-sectional study, in which quantitative data were collected through a survey from october 1 through november 30, 2015, shortly after the mers epidemic ended. during the 2015 mers outbreak, 15 hospitals were designated as cohort isolation facilities. we contacted all hospitals and three agreed to participate in the study. these included two hospitals in daejeon city that were isolated for infection control from june 9 through june 23, and the third hospital in gyeonggi province which was isolated from may 31 through june 15 (kcdc, 2018). the kcdc (2018) announced the end of the cohort isolations on july 26. we conducted the surveys approximately 2 months after the announcement; we considered that the nurses have recovered to normal working conditions by this time. the nurses were asked to recall during the period of the mers outbreak and answer the survey questionnaires based on that prior time period. a total of 300 nurses from three general hospitals, who provided either direct or indirect care for mers, were invited to participate in the study. the participants included in the final analysis were those who provided direct care during the mers outbreak. this study was conducted after receiving the institutional review board approval from the konyang university hospital (irb kyuh 2015-09-009-001) as well as informed consent from the participants. post-traumatic stress disorder was assessed by using the impact of event scale-revised korean version (ies-r-k; eun et al., 2005) . the original impact of event scale-revised (ies-r; weiss & marmar, 1997) was composed of three subdimensions (avoidance, intrusion, and hyperarousal) consisting of 22 questions, and the ies-r-k is composed of five subdimensions (avoidance, intrusion, hyperarousal, and sleep problems and numbness). the ies-r-k consists of 22 questions including six items on hyperarousal (score range of 0-24), six items on avoidance (score range of 0-24), five items on intrusion (score range of 0-20), and five items on sleep problems and numbness (score range of 0-20). the 5-point likert-type scale (0-4 points) consists of the total sum of scores for the four sub-dimensions ranging from 0 to 88, with higher scores indicating higher level of post-traumatic stress. eun et al. (2005) presented the total ptsd score of 25 as the cutoff point for experiencing full ptsd, and the score of 18 to 24 indicating moderate or some level of ptsd. the cronbach's alpha of the original ies-r was .79, and that of the ies-r-k was .93 (eun et al., 2005; weiss & marmar, 1997) . the cronbach's alpha of the ies-r-k in this study was .95. the nurses were asked to recall their feelings and emotions associated with the mers outbreak time period. supervisor support was measured using the korean version of the job content questionnaire (k-jcq; eum et al., 2007) . the k-jcq consists of three sub-domains (autonomy of work, job demands, and social support of the workplace), with a total of 45 items. the social support domain of the k-jcq includes supervisor support, co-worker support, and job instability. in this study, we used four questions regarding the supervisor support, including questions 34 ("my boss is interested in the welfare of his or her subordinates"), 35 ("my boss is interested in my opinion and listens carefully."), 37 ("my boss helps me to accomplish my tasks."), and 38 ("my boss is good at making people work together"). a 4-point likert-type scale (1 = strongly disagree, 2 = disagree, 3 = agree, 4 = strongly agree) was used. the total possible score ranges between 4 and 19, with higher scores indicating higher supervisor support. cronbach's alpha was .84 at the time of development of the among 147 nurses who were involved with the direct care of either infected or suspected middle east respiratory syndrome (mers) patients, 25.1% experienced full post-traumatic stress disorder (ptsd) and 32.0% experienced moderate or some level of ptsd. turnover intention was also high among this cohort. high ptsd scores were positively associated with high turnover intention, and supervisor support was proven to have a strong buffering effect. these findings suggest that supervisor support is a successful management strategy to lower turnover intention post epidemic outbreak. organizational strategies to help nurses to cope with stress and to prevent turnover intention, especially using supervisor support, would be beneficial. measurement (karasek et al., 1998) and that of the k-jcq (eum et al., 2007) was .71; the cronbach's alpha in this study was .93. turnover intention was estimated by the turnover intention measurement (h. s. park, 2002) , which is the korean version of the scale originally developed by lawler (1983) . the measurement consists of four items, each of which is a 5-point likert-type scale (1 = very unlikely, 2 = unlikely, 3 = neutral, 4 = likely, 5 = very likely). the possible range of scores is from 4 to 20, with higher scores indicating higher intention to leave. the cronbach's alpha for the original measurement was .88 (h. s. park, 2002) and that of the current study was .84. the general health questionnaire (ghq) was used to measure current self-reported mental health. we used the ghq-12, a reliable and sensitive short form which is ideal for research studies (liang et al., 2016) . the scale consists of 12 items and is a 4-point likert-type scale (0 = much less than usual, 1 = same as usual, 2 = more than usual, 3 = much more than usual), with a total score ranging from 1 to 36. the cutoff point in the scores of ghq-12 is 24, indicating the presence of mental health problems (makowska et al., 2002) . the ghq was included as one of the confounding variables. to measure the difference of stress levels between the period of the mers outbreak and after the outbreak, nurses were asked to recall their stress levels during the mers outbreak and compare it with their current day-to-day stress levels after the outbreak. a question asking the stress level during the outbreak in a 5-point likert-type scale and another question asking the stress level after the outbreak were included in the survey. the authors, then, calculated the difference between the two levels of the stress. data were analyzed using the statistical package for social sciences, version 20.0 (spss, inc., 2011). the descriptive statistics were calculated using frequency and percentage, as well as means and standard deviation. cross-tabulation, including t test and f test, was conducted to examine the characteristics of participants according to ptsd, supervisor support, and turnover intention measures. the relationship between ptsd (independent variable) and turnover intention (dependent variable), as well as the buffering effect of supervisor support as an effect modifier, was examined by multiple regression analysis. work experience, work position, shift work, department, marital status, level of education, annual income, mental health status, difference in stress levels, and level of involvement in the care of mers patients were considered as covariates (jeong et al., 2008; mosadeghrad, 2013; yang & kim, 2016) . table 1 describes the general characteristics of the study participants. of the 300 invited participants, 152 responded with 147 providing usable data (response rate: 49%). all of the participants were female, of which 52.4% had work experience of 1 to 4 years, 18.4% with less than 1 year, 19.0% with 5 to 9 years, and 10.2% with over 10 years of work experience. the majority were staff nurses (91.2%) who worked in rotating shifts (98%). approximately 37.0% of the nurses worked in either intensive care or in emergency services, whereas 62.6% of them worked in general medicine departments. the majority of the nurses were single (85.7%) and completed 4 years of college or higher (65.3%), and earned an annual salary of more than us$30,000 (62.6%). of the 147 nurses, 33.3% of them were directly involved with the treatment of confirmed infected patients, whereas 66.7% of them were directly involved with the treatment of suspected patients. the mental health of the nurses, measured by ghq, showed the mean score of 28.2 (of 36), indicating problematic self-rated mental health. the difference of stress levels between every day and the days during the outbreak was 1.34, which showed that the stress level during the outbreak was higher. a total of 57.1% (n = 84) of the nurses experienced ptsd. considering the score of 25 and above as experiencing full ptsd, 25.1% (n = 37) experienced full ptsd and 32.0% (n = 47) experienced some level of ptsd, with scores of 18 to 24. the mean score of supervisor support was 10.8 with the range of 4 to 19, which indicated moderate supervisor support. the turnover intention score ranged from 4 to 20, and the mean turnover intention in this study was 16.3, which was high. table 2 shows the characteristics of participants according to the ptsd, supervisor support, and turnover intention measures. department (p < .05), mental health (p < .01), and the level of involvement during the mers outbreak (p < .05) were associated with post-traumatic stress. mental health was negatively associated with supervisor support (p < .01). years of work experience (p < .01), shift work (p < .05), marital status (p < .05), annual income (p < .01), and self-reported mental health (negative association, p < .01) were associated with turnover intention. table 3 shows the results from the multiple regression analysis in which we observed that nurses with work experience of between 1 and 4 years (β = .313), direct involvement with the treatment of a suspected patient (β = .224), and high score of ptsd (β = .188) were positively associated with higher intention to leave. higher supervisor support (β = −.395) was, on the contrary, associated with lower turnover intention. the interaction between ptsd and supervisor support was significant (β = .177) with intention to leave, illustrating the buffering effect of supervisor support. the purpose of this study was to examine the levels of ptsd and turnover intention of nurses after the mers outbreak in korea. the result showed that 57.1% of the nurses who were involved with the direct care of either infected or suspected mers patients experienced ptsd. in particular, 25.1% of the nurses experienced full level of ptsd, which is higher than 20.4% of nurses experiencing full level of ptsd using the same ies-r-k from a study on nurses working at emergency departments (han & lee, 2013) . another study on japanese firefighters using the original ies-r measure showed that only 9.7% of the participants experienced full level of ptsd (saijo et al., 2012) . a study of medical staff involved with patient care during the sars outbreak found that 10% of the respondents had experienced high levels of ptsd (wu et al., 2009) . another study found that around 20% of participating doctors and nurses showed ptsd symptoms that were involved in the care of avian influenza a (h7n9) patients during the h7n9 influenza epidemic in 2015 to 2016 (tang et al., 2017) . the average turnover intention of nurses in this study was 16.29 when the total score range is from 4 to 20, showing high turnover intention. a previous study that used the same measurement revealed average score of 15.41 among nurses working at emergency department (lee & ahn, 2015) . other studies also showed lower scores compared with that of our research, which were 13.75 among nurses working at general hospitals (c.-h. kim et al., 2009 ) and 9.72 among nurse managers (k. o. park et al., 2012) . additional aim of this study was to investigate the relationship between ptsd and turnover intention. the result of this study validated significant and positive relationship between ptsd and turnover intention among nurses who were involved with the care during the mers outbreak. prior studies on nurses have presented the same results. a study on korean nurses working at emergency departments revealed significant and positive association between ptsd and turnover intention (han & lee, 2013) . also, after a traumatic incident, more than 20% of nurses working at emergency departments displayed high turnover intention. the final investigation was to see whether supervisor support has buffering effect between ptsd and turnover intention, which was proven to be legitimate. social support of workplace, including supervisor support, has been known to act as a moderator against the impact of high stress on well-being and related health outcomes on the buffering hypothesis of cohen and wills (1985) . other study has investigated the buffering effect of social support and higher level of organizational stress as well as turnover intention (nissly et al., 2005) . however, majority of studies on buffering effect of social support studies were done on the relationship between general or job stress and turnover intention, so future studies should be specified on the relationship between ptsd and turnover intention during fetal epidemic events as well as the buffering effect of social support, such as supervisor support. despite the fact that this is one of the few studies on nurses who were involved with the direct patient care during the mers outbreak in korea, it has some limitations. fifteen hospitals were cohort isolated during the mers breakouts; however, only three hospitals agreed to participated in the study. the small sample size limits our ability to generalize the findings beyond the study hospitals. in addition, the three hospitals that agreed to participate are those that were praised by the media for their successful response to the pandemic. therefore, the levels of ptsd and the buffering effect of supervisor support might have different results if all of 12 hospitals participated in the research. in addition, data were collected after the outbreak due to facility isolation during the outbreak. post-traumatic stress disorder, turnover intention, and supervisor supports were all asked as recall questions 2 months after the outbreak, introducing possible recall bias. the results of this study are significant and relevant because the participants were nurses who were involved with direct patient care during the mers outbreak. more than half (57.1%) of the nurses experienced ptsd-25.1% with full level and 32.0% with some level of ptsd. also, ptsd and turnover intention were significantly and positively related. in addition, the results of this study suggest that social support, particularly supervisor support, should be provided to reduce the impact of ptsd on nurses' turnover intentions in the case of serious infectious diseases. support system should be discussed at departmental, organizational, and national levels. infectious disease and occupational health professionals should consider developing and implementing coping management strategies to reduce ptsd and turnover intention related to epidemic outbreaks as well as providing educational programs to supervisors to provide adequate support to nurses. this study was approved by the konyang university hospital institutional review board (irb kyuh 2015-09-009-001). the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the author(s) received no financial support for the research, authorship, and/or publication of this article. mi sun kim https://orcid.org/0000-0003-3970-901x factors affecting turnover intention among nurses in ethiopia the development of a clinicianadministered ptsd scale the importance of supervisor-nurse relationships, teamwork, wellbeing, affective commitment and retention of north american nurses psychological distress of nurses in taiwan who worked during the outbreak of sars stress, social support, and the buffering hypothesis psychometric properties of the korean version of the job content questionnaire: data from health care workers a study on reliability and validity of the korean version of impact of event scale-revised the relationship of post-traumatic stress, job stress and turnover intention in emergency department nurses the risk factors influencing turnover intention of nurses the job content questionnaire (jcq): an instrument for internationally comparative assessments of psychosocial job characteristics healthcare workers emotions, perceived stressors and coping strategies during a mers-cov outbreak a structural equation model of nurses' turnover intention effects of self-efficacy, career plateau, job embeddedness, and organizational commitment on the turnover intention of nurses mers white paper social support, occupational stress, and health job and career satisfaction and turnover intentions of newly graduated nurses satisfaction and behavior impact of job stress on turnover intention among emergency room nurses the factor structure of the 12-item general health questionnaire (ghq-12) in young chinese civil servants influencing factors on turnover intention of nurses in emergency department the validity of general health questionnaires, ghq-12 and ghq-28, in mental health studies of working people open data portal occupational stress and turnover intention: implications for nursing management stress, social support, and workers' intentions to leave their jobs in public child welfare relationship of workplace incivility, stress, and burnout on nurses' turnover intentions and psychological empowerment relationship between perceived nursing care role orientation, job characteristics, and turnover among nurses a model on turnover intention of chief nurse officers post-traumatic stress disorder and job stress among firefighters of urban japan spss version 20.0 for windows communication with police supervisors and peers as a buffer of work-related traumatic stress retaining the nursing workforce: factors contributing to the reduction of nurses' turnover intention in japan prevalence and related factors of post-traumatic stress disorder among medical staff members exposed to h7n9 patients the impact of event scale-revised middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia the psychological impact of the sars epidemic on hospital employees in china: exposure, risk perception, and altruistic acceptance of risk factors influencing turnover intention in clinical nurses: compassion fatigue, coping, social support, and job satisfaction heeja jung, rn, phd, key: cord-319689-33h22ikl authors: srivastava, sukrit; kamthania, mohit; singh, soni; saxena, ajay k; sharma, nishi title: structural basis of development of multi-epitope vaccine against middle east respiratory syndrome using in silico approach date: 2018-11-21 journal: infect drug resist doi: 10.2147/idr.s175114 sha: doc_id: 319689 cord_uid: 33h22ikl background: middle east respiratory syndrome (mers) is caused by mers coronavirus (mers-cov). thus far, mers outbreaks have been reported from saudi arabia (2013 and 2014) and south korea (2015). no specific vaccine has yet been reported against mers. purpose: to address the urgent need for an mers vaccine, in the present study, we have designed two multi-epitope vaccines (mevs) against mers utilizing several in silico methods and tools. methods: the design of both the multi-epitope vaccines (mevs) are composed of cytotoxic t lymphocyte (ctl) and helper t lymphocyte (htl) epitopes, screened form thirteen different proteins of mers-cov. both the mevs also carry potential b-cell linear epitope regions, b-cell discontinuous epitopes as well as interferon-γ-inducing epitopes. human β-defensin-2 and β-defensin-3 were used as adjuvants to enhance the immune response of mevs. to design the mevs, short peptide molecular linkers were utilized to link screened most potential ctl epitopes, htl epitopes and the adjuvants. tertiary models for both the mevs were generated, refined, and further studied for their molecular interaction with toll-like receptor 3. the cdnas of both mevs were generated and analyzed in silico for their expression in a mammalian host cell line (human). results: screened ctl and htl epitopes were found to have high propensity for stable molecular interaction with hla alleles molecules. ctl epitopes were also found to have favorable molecular interaction within the cavity of transporter associated with antigen processing. the selected ctl and htl epitopes jointly cover upto 94.0% of worldwide human population. both the ctl and htl mevs molecular models have shown to have stable binding and complex formation propensity with toll-like receptor 3. the cdna analysis of both the mevs have shown high expression tendency in mammalian host cell line (human). conclusion: after multistage in silico analysis, both the mevs are predicted to elicit humoral as well as cell mediated immune response. epitopes of the designed mevs are predicted to cover large human population worldwide. hence both the designed mevs could be tried in vivo as potential vaccine candidates against mers. middle east respiratory syndrome (mers) is a respiratory disease caused by middle east respiratory syndrome coronavirus (mers-cov). mers involves high fever, cough, difficulty in breathing, chills, chest pain, body aches, sore throat, diarrhea, nausea/vomiting, running nose, renal failure, and pneumonia. the first case of mers in humans was reported in saudi arabia in 2012. after 2012, within a span of only 4 years, mers-cov infection was reported from 27 countries. 1 three mers outbreaks have already occurred in saudi arabia (2013 and 2014) and south korea (2015). [2] [3] [4] the outbreak reported in south korea also involved spread of mers through hospital-to-hospital transit of patients and had the fatality rate as high as 40%. high attack rate and easy spread of mers indicate an epidemic risk. [5] [6] [7] to date, no specific vaccine is available for mers. the steep increase in mers cases and its high mortality rate demand an urgent need for specific and safe mers vaccine. thus far, only little information is known about the pathogenesis of mers-cov. hence, an immunoinformatics approach to thoroughly study and screen immunogenic proteins from the available proteome sequence data of mers-cov is essential for vaccine design. the proteome of mers-cov consists of several important vaccine candidate and drug target proteins. these proteins are involved in infection and pathogenesis of mers-cov to human host cells. spike (s) glycoprotein, in particular, its receptor-binding domain, is involved in virus and host cell interaction. 8 envelope (e) protein plays an important role in host cell recognition. 9 nucleocapsid (n) protein is involved in rna binding during ribonucleocapsid formation by mers-cov. 10 membrane (m) protein has interferon (ifn)-antagonizing properties, thus reducing ifn levels in infected patients. 11 open reading frame (orf) proteins also play critical roles in viral infection and pathogenesis. a mutation study of orfs (orf3, orf4a, orf4b, and orf5) indicated that orfs have major implications in viral infection involving disruption of host cell processes, dysregulated ifn pathway activation, and abrupt inflammation. 12 proteins orf1a (4p16) and orf1ab (4wur) are papain-like proteases (pl(pro)) involved in viral infection and are potential targets for the development of antiviral drugs. 13 pl(pro) facilitate infection by their proteolytic, deubiquitinating, and deisgylating activities suppressing innate immune response from the host cell. protein orf1a (4rsp), a protease (3clpro), facilitates proteolytic activity during viral infection and replication. 14, 15 protein orf1ab (5wwp) is a helicase protein of mers-cov, and it is one of the most conserved proteins among nidoviruses. protein orf8b is a highly conserved protein, and during infection, it induces various immune responses. 16 in the present study, we propose two multi-epitope vaccine (mev) designs for mers. the proposed mevs are composed of cytotoxic t lymphocyte (ctl) and helper t lymphocyte (htl) epitopes. both ctl and htl mevs contain overlapping regions of linear b-cell epitopes. both mevs also contain human β-defensin-2 (hbd-2) and human β-defensin-3 (hbd-3) as adjuvants to enhance the immunogenic response. 17, 18 methodology to design the mevs, different in silico tools were used to screen potential ctl, htl, and b-cell epitopes from 13 mers-cov proteins that are involved in virus-host interaction and viral proliferation. all three types of epitopes were studied for overlapping consensus regions. ctl and htl epitopes showing partial or complete overlapping regions with all or any of the three kinds of epitopes and the epitopes with the highest number of human leukocyte antigen (hla) allele binders were selected for detailed analysis. the selected ctl and htl epitopes were further validated for their molecular interaction with their hla allele binders. furthermore, the selected ctl epitopes were validated for their molecular interaction with cavity of transporter associated with antigen processing (tap) to analyze their smooth passage from the cytoplasm to the endoplasmic reticulum (er) lumen. 19 three-dimensional (3d) structure model for both mevs was generated, refined, validated, and analyzed for different physiochemical properties. both ctl and htl mev models were further screened to have several b-cell discontinuous epitopes and ifn-γ-inducing epitopes. because the immune system broadly recognizes pathogens partially due to the involvement of toll-like receptor 3 (tlr-3) and its signaling cascade, both ctl and htl mevs were further studied for their molecular interaction with tlr-3. 20,21 moreover, the cdnas of both mevs were analyzed and were predicted to be highly expressing in a mammalian host cell line (human) ( figure s1 ). for vaccine development, proteins critically involved in virus-host cell interaction, viral proliferation, and host cell disruption were chosen. thirteen proteins of mers-cov proteome chosen for epitope screening included the following: s glycoprotein; e protein; n protein; m protein; orfs (orf3, orf4a, orf4b, and orf5); orf1a (4p16), a papain-like protease (pl(pro)); orf1a (4rsp), a protease structural basis of development of multi-epitope vaccine against mers (3clpro); orf1ab (4wur), a pl(pro); orf1ab (5wwp); and orf8b. for sequence-based epitope screening, fulllength protein sequences of the abovementioned mers-cov proteins were retrieved from the ncbi database (national center for biotechnology information, https://www.ncbi. nlm.nih.gov/protein). a total of 2,499 amino acid sequences belonging to different proteins of mers-cov of different strains and origins were retrieved. for structure-based epitope screening, available 3d structures of mers-cov proteins were retrieved from protein data bank (pdb, http://www.rcsb.org/pdb/home/home.do). for the proteins with no structure available in pdb, homology modeling was performed using the swiss-model, (http://swissmodel. expasy.org/) (table s1 ). 22 to screen ctl epitopes, the immune epitope database (iedb) tool "proteasomal cleavage/tap transport/mhc class i combined predictor" (http://tools.iedb.org/processing/) was used. [23] [24] [25] the total score generated by the tool is a combined score of proteasome, tap (n-terminal interaction), major histocompatibility complex (mhc), and processing analysis scores. the score is generated using the combination of six methods, namely, consensus, nn-align, smm-align, combinatorial library, sturniolo, and netmhciipan. the ic50 (nm) value was also obtained using this iedb tool. epitopes with high, intermediate, and least affinity of binding to an hla allele have ic50 values <50 nm, <500 nm, and <5,000 nm, respectively. immunogenicity of the screened ctl epitopes was also obtained by the "mhc i immunogenicity" tool of iedb (http://tools.iedb.org/ immunogenicity/) with all parameters set to default (first, second, and c-terminus amino acids). 26 the tool predicts immunogenicity of a peptide mhc complex on the basis of physiochemical properties of amino acids and their position within the short peptide sequence. to screen htl epitopes, the iedb tool "mhc-ii binding predictions" (http://tools.iedb.org/mhcii/) was used. the percentile rank for each peptide is generated by the combination of three methods (combinatorial library, smm-align, and sturniolo) by comparing the score of peptide against the scores of other random five million 15-mer peptides from the swissprot database. [27] [28] [29] [30] the rank for the consensus method was generated by the median percentile rank of the three methods. the "population coverage" tool of iedb (http://tools. iedb.org/population/) was used to analyze the world human population coverage by the shortlisted 28 ctl and 28 htl epitopes. 31 use of multi-epitopes involving both ctl and htl epitopes would have the higher probability of larger human population coverage worldwide. b-cell epitope prediction sequence-based b-cell epitope prediction protein sequence-based linear b-cell epitopes were predicted by six different prediction methods available at "b cell epitope prediction tools" tool of iedb server (http://tools. iedb.org/bcell/); these tools are based on the propensity scale method and physicochemical properties of the antigenic sequence. these methods include "bepipred linear epitope prediction" (propensity scale method such as hidden markov model), "chou & fasman beta-turn prediction", "emini surface accessibility prediction", "karplus & schulz flexibility prediction", "kolaskar & tongaonkar antigenicity", and "parker hydrophilicity prediction". [32] [33] [34] [35] [36] [37] structure-based b-cell epitope prediction two structure-based b-cell epitope prediction methods, namely, discotope 2.0 (structure-based antibody prediction tool; http://tools.iedb.org/discotope/) and ellipro (antibody epitope prediction tool; http://tools.iedb.org/ellipro/), available in iedb, were used for linear and discontinuous b-cell epitope prediction. 38, 39 the ellipro method is based on the location of the residue in the protein's 3d structure. the residues lying outside of the ellipsoid covering 90% of the inner core residues of the protein score highest protrusion index (pi) of 0.9 and so on. discontinuous epitopes predicted by ellipro are clustered on the basis of distance r in å between two residues' centers of mass lying outside the largest possible ellipsoid. the larger the value of r, the larger will be the number of discontinuous epitopes clustered. discotope 2.0 is based on the number of contacts of the residues' cα carbon atom with other cα carbon atoms in the 3d structure within the 10 å distance and the propensity of a residue to be a part of an epitope. toxicity assessment of ctl and htl epitopes and b-cell epitopes was performed by toxinpred (http://crdd.osdd.net/ raghava/toxinpred/multi_submit.php) analysis. this analysis allows to identify highly toxic or nontoxic peptides. the analysis was performed by the "support vector machine (svm) (swissprot)-based" method with all the parameters set to default. 41 overlapping residue analysis multiple sequence alignment (msa) analysis using the clustal omega tool (https://www.ebi.ac.uk/tools/ msa/clustalo/) of european bioinformatics institute was performed for all the shortlisted ctl, htl, and b-cell epitopes from 13 mers-cov proteins. 42 msa by clustal omega virtually aligns any number of protein sequences and delivers accurate alignments. epitope selected for molecular interaction study with hla allele and tap ctl and htl epitopes were shortlisted for further in silico analysis on the basis of their overlapping sequence regions among all three types of epitopes (ctl, htl, and b-cell) or complete overlap among any two types of epitopes or the highest number of hla allele binders. tertiary structure modeling of hla alleles and selected t-cell epitopes template-based homology modeling for hla class i and ii allele binders of the shortlisted epitopes was performed by the swiss-model. 22 protein sequences of hla class i and ii alleles were retrieved from immuno polymorphism database (ipd-imgt/hla) (https://www.ebi.ac.uk/ipd/ imgt/hla/allele.html). 69 template with high sequence identity was chosen for modeling, and the models thus generated were validated for quality by the qualitative model energy analysis (qmean) analysis. the qmean value is a composite (both global and local [ie, per residue] structure) quality estimate of the generated model. 43 models with an acceptable qmean value with a cutoff of −4.0 were chosen for further studies (table s2) . "natural peptides module for beginners" module of the pepstrmod tool (http://osddlinux.osdd.net/raghava/ pepstrmod/nat_ss.php) was used to generate tertiary structures of the selected epitopes. 44 prediction was carried out with a simulation time window of 100 ps, and the peptide environment was set to vacuum. autodock tool 4.2 and autodock vina were used for molecular docking study of the selected epitopes and their respective hla allele binders. 45, 46 further, the docked complexes were subjected to md simulation study by gromacs 5.1.4 using optimized potentials for liquid simulations -all atom (opls-aa) force field. 47, 48 molecular interaction analysis (docking) of the selected ctl epitopes with tap molecular docking study of the shortlisted ctl epitopes with tap receptor was performed by autodock vina. 45, 46 for more accurate prediction, instead of homology modeling, the cryo-em structure of tap (pdb id: 5u1d) was used. 49 for docking, the antigen from the tap cavity of the original structure was removed. from the screened and shortlisted ctl and htl epitopes, two mevs were designed using eaaak and ggggs as short peptide rigid and flexible linkers, respectively ( figure 1a and b). to enhance the immune response, hbd-2 (pdb id: 1fd3, sequence: gigdpvtclksgaichpvfcprrykqigtcg lpgtkcckkp) and hbd-3 (pdb id: 1kj6, sequence: gii ntlqkyycrvrggrcavlsclpkeeqigkcstrgrk ccrrkk) were used as adjuvants for both mevs at n and c terminals, respectively. 17, 18, [50] [51] [52] [53] upon lung infection, the expression of hbd-2 and hbd-3 was found to be increased. β-defensins are involved in chemotactic activity for memory t cells, monocytes, and immature dendritic cells as well as in degranulation of mast cells. thus, hbds enhance innate and adaptive immunity and therefore were chosen here as adjuvants for the design of mevs. ifn-γ-inducing epitope prediction ifn-γ epitopes with potential to induce the release of ifn-γ from cd4+ t cells from both mevs were predicted by "ifnepitope" server (http://crdd.osdd.net/raghava/ ifnepitope/scan.php) using the "motif and svm hybrid" (merci: motif-emerging and with classes-identification, and svm) approach. the tool generates overlapping sequences from the query protein/antigen and uses it for ifn-γ-inducing epitope prediction. the prediction is based on a dataset of ifn-γ-inducing and ifn-γ-noninducing mhc class ii binders. 54 for both mevs, algpred was used for allergenicity prediction (http://crdd.osdd.net/raghava/algpred/submission.html). 56 the algpred prediction of allergens is based on similarity of the known epitope with any region of the submitted protein. vaxijen (http://www.ddg-pharmfac.net/vaxijen/ vaxijen/vaxijen.html) was used to analyze the probability of antigenicity of both mevs. 57 the vaxijen analysis applies an alignment-free approach that is solely based on the physicochemical properties of the sequences of the submitted proteins. to analyze the physiochemical properties of ctl and htl mevs, protparam (https://web.expasy.org/protparam/) was used. 58 protparam analysis performs empirical investigation for a given protein amino acid sequence. both the ctl and htl mevs were further subjected to 3d structure modeling by raptorx structure prediction server (http://raptorx.uchicago.edu/structureprediction/predict/). 59 the quality of the generated model is indicated by its p-value. the p-value is the probability of a predicted model being worse than the best. it indicates relative quality in terms of modeling error by combining global distance test (gdt) and un-normalized gdt, that is, error at each residue. the smaller the p-value, the higher is the quality of the predicted model. the generated mev models were further refined by modrefiner and galaxyrefin. 60, 70 galaxyrefine performs repeated structure perturbation along with subsequent structural relaxation by dynamics simulation to refine a protein structure. to avoid breaks in model structures, galaxyrefine uses the triaxial loop closure method. the refined 3d models of both mevs were then subjected to rampage analysis for generating a ramachandran plot (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php). 61 the ramachandran plot shows the residues that form energetically allowed and disallowed dihedral angles psi (ψ) and phi (ϕ), which are calculated on the basis of van der waal radius of the amino acid side chain. both the designed ctl and htl mevs were subjected to discontinuous b-cell epitope prediction to analyze structurebased humoral immunogenic potential of both mevs using the ellipro tool. 39 molecular docking and md simulation study of mevs and the immunological receptor tlr-3 to study the molecular interaction, the refined models of ctl and htl mevs were docked with tlr-3 by patchdock server (http://bioinfo3d.cs.tau.ac.il/patchdock/). [62] [63] [64] the 3d structure of human tlr-3 ectodomain (ecd) was retrieved optimized cdnas for both mevs were generated by the codon usage wrangler tool with the option of a mammalian host cell line (human) as the expression system (http://www. mrc-lmb.cam.ac.uk/ms/methods/codon.html). further, the genscript rare codon analysis tool (https://www.genscript. com/tools/rare-codon-analysis) was used to analyze the cdnas of both mevs. the tool provides gc content, codon adaptation index (cai), and tandem rare codon frequency for cdna. [65] [66] [67] results and discussion screening of potential epitopes t-cell epitope prediction screening of ctl epitope on the basis of "total score" and acceptably low ic50 (nm) value of epitope-hla class i allele pairs, 75 cd8+ ctl epitopes were chosen. later, 28 epitopes were shortlisted with high scoring and larger number of hla class i allele binders for further studies. immunogenicity of the screened ctl epitopes was also determined. the higher the immunogenicity score, the greater is the immunogenicity of the epitope (tables s3 and s4) . screening of htl epitopes screening of htl epitopes was performed on the basis of "percentile rank". small percentile rank shows the high affinity of the peptide for its respective hla allele. initially, from the 13 mers-cov proteins, 70 cd4+ t-cell epitopes with the highest percentile rank were screened, and 28 epitopes with high percentile rank and the highest number of hla class ii allele binders were then shortlisted (tables s5 and s6 ). several ctl and htl epitopes predicted in the present study show overlapping regions with the epitopes detected to induce t-cell responses in previous studies done using peripheral blood mononuclear cells from infected patients. 68 hence, epitopes screened and reported in the present study could be predicted to originate from the highly immunogenic stretch of mers-cov protein sequences. in this study, most geographical regions of the world, and in particular, the countries of middle east, south asia, east asia, and northeast asia, were included. this study indicates that the combined use of all shortlisted ctl and htl epitopes would have an average worldwide population coverage of 94.0%, with an sd of 20.19 (table s7 ). sequence-based b-cell epitope prediction initially, a total of 144 b-cell epitopes were screened from 13 mers-cov proteins, with epitope length of four or more than four amino acids by the bepipred linear epitope prediction method. the epitopes predicted by five other methods based on different physicochemical properties showed a significant consensus overlap of amino acid sequences with that of bepipred linear epitope prediction. from the 144 b-cell epitopes, 12 with the length of 4-19 amino acids were shortlisted (table s8 and figure 2 ). discontinuous and linear epitopes predicted by discotope 2.0 and ellipro methods showed significant consensus overlap of amino acid sequence with the linear epitopes predicted by the bepipred linear epitope method (table s8 and figure 2 ). this result confirms that the shortlisted bepipred linear epitopes are highly immunogenic b-cell epitopes. the conservation analysis of shortlisted 28 ctl, 28 htl, and 12 b-cell epitopes shows that amino acid sequence conservancy of ctl epitopes varied from 72.7% to 100%, that of htl epitopes varied from (50% for two epitopes) 68.18% to 100%, and that of b-cell epitopes varied from 85.71% to 99.26% (tables s3, s5 , and s8). this result indicates high conservancy nature of the shortlisted ctl, htl, and b-cell epitopes. all the shortlisted ctl, htl, and b-cell epitopes analyzed by the toxinpred tool were predicted to be nontoxic (tables s3, s5 , and s8). the analysis was based on the toxinpred main dataset consisting of 1,805 toxic peptides for its prediction. msa analysis of all the screened ctl, htl, and b-cell epitopes from the 13 mers-cov protein candidates revealed that several epitopes of ctl, htl, and b-cell have overlapping amino acid sequence regions. epitopes with two or more than two residues in the overlapping region are shown in figure 3 . overlapping ctl, htl, and b-cell epitopes. notes: overlapping ctl (red), htl (blue), and b-cell epitopes (green) were sorted out by msa using clustal omega at ebi server. the ringed clusters of epitopes involve all three types of epitopes, epitopes with full sequence overlap, and the epitopes with highest number of hla allele binders. abbreviations: ctl, cytotoxic t lymphocyte; ebi, european bioinformatics institute; hla, human leukocyte antigen; htl, helper t lymphocyte; msa, multiple sequence alignment. figure 2 overlapping regions of the bepipred linear b-cell epitopes and the epitopes predicted by other methods. notes: this analysis shows a strong consensus between the bepipred linear b-cell epitopes and the epitopes predicted by other sequence-based and structure-based epitope prediction methods. different colors are used to highlight overlapping regions of epitopes predicted by the different methods. epitope selected for molecular interaction study with hla allele and tap overlapping ctl and htl epitopes clustering with all three (ctl, htl, and b-cell) types of epitopes or with complete epitope sequence overlap or with the highest number of hla allele binders were identified and selected for their molecular interaction analysis with hla alleles and tap ( figure 3 ). these include ctl epitopes: 47 molecular interaction analysis of the selected epitopes with hla allele molecular docking and md simulation study of the selected epitopes with hla alleles molecular docking study of all the selected epitopes with their respective hla allele binders revealed a significantly favorable molecular interaction. docking complexes thus formed have significantly negative binding energy, and several amino acid residues of epitopes and hla alleles were involved in hydrogen bond formation ( figure 4 ). to analyze the stability of binding, docking complexes were further subjected to md simulation study with an analysis time window of 0.1 ns at the reasonably invariable temperature (~300 k) and pressure (~1 bar). the results of md simulation for all the epitope-hla allele complexes showed a very convincing reasonably invariant root mean square deviation (rmsd) value between ~0.1 and 0.2 nm, thus indicating stable complex formation ( figure 5) . moreover, the reasonably invariant radius of gyration (rg) of the complexes ( figure s2 ) and rms fluctuation (rmsf) for all the atoms in the complexes ( figure s3 ) indicate that the epitope-hla complexes remain very stable in their folded form. b-factor of the epitope and hla allele complexes is shown in the rainbow color presentation in figure s4 . most of the regions of the complexes are stable (blue) with a very small region being acceptably fluctuating (yellow and orange). molecular docking results show a favorable molecular interaction between the selected ctl epitopes and the tap cavity. the result reveals several molecular interaction sites for the selected epitopes within the tap cavity. two sites of interaction-one close to the cytoplasm and another close to the er lumen-are shown in figure 6 . all the interactions showed a significantly negative binding energy with one or more than one hydrogen bond formation at both sites of interaction. from this study, we may predict a smooth passage for selected ctl epitopes from the cytoplasm to the er lumen throughout the entire passage of the tap cavity ( figure 6 ). ifn-γ-inducing epitope prediction ifn-γ is involved in both adaptive and innate immune responses. it stimulates macrophages and natural killer cells. ifn-γ-inducing 15-mer peptide epitopes were predicted from (table s9 and figure 7c and g). the algpred analysis of ctl and htl mevs revealed that both mevs are nonallergens with the score of −0.95415008 and −0.8647714, respectively, with sensitivity and specificity of 88.87% and 81.86%, respectively, while the default threshold value was −0.4. the vaxijen analysis indicated both mevs to be probable antigens with prediction score of 0.5302 and 0.5097 for ctl mevs and htl mevs, respectively, while the default threshold value was 0.4 for viral proteins. hence, both mevs are predicted to be nonallergic and potentially antigenic in nature. protparam analysis showed that ctl mevs have 500 amino acids, 50.4 kda molecular weight, and 9.72 theoretical pi. the half-life in mammalian reticulocytes, yeast, and escherichia coli was 30 hours, 20 hours, and 10 hours, respectively; aliphatic index was 61.68, and grand average of hydropathicity (gravy) was −0.020, indicating globular and hydrophilic nature of ctl mevs; the instability index was 50.70, indicating that ctl mevs are theoretically close to stable in nature. protparam study showed htl mev has 657 amino acids, 67.583 kda molecular weight, and 9.29 theoretical pi. the half-life in mammalian reticulocytes, yeast, and e. coli was 30 hours, 20 hours, and 10 hours, respectively; aliphatic index was 94.22, and grand average of hydropathicity (gravy) was 0.337, both indicating globular and hydrophilic nature of htl mev; the instability index was 37.66, indicating theoretically stable nature of htl mev. the model generated by raptorx for ctl mev has 4% helix, 37% β-sheet, and 58% coil; the model is 26% exposed, 41% medium, and 31% buried. it has three domains: first domain (1-46 aa, template-1fd3:a); second domain (47-444 aa, template-4dou:a, 4zch:a); and third domain (445-500 aa, template-1kj6:a) ( figure 7a ). the model generated by raptorx for htl mev has 35% helix, 20% β-sheet, and 44% coil; the model is 26% exposed, 35% medium, and 37% buried. it has four domains: first domain (1-46 aa, template-1fd3:a); second domain (47-546 aa, templates-4ufca, 2eaba, and 2rdya); third domain (547-601 aa, template-1tue:b); and fourth domain (602-657 aa, template-1kj6:a) ( figure 7e ). the p-values of the best template of ctl and htl mev models are 3.98e-05 and 5.41e-04, respectively. good quality alpha proteins and good quality beta proteins should have p-value of less than 10 -3 and 10 -4 respectively. hence, both the models of ctl and htl mevs generated by raptorx are of good quality. because the ctl and htl epitopes used for the design of both mevs show overlapping with b-cell linear epitopes (figure 3 ), both the ctl and htl mev models also carry overlapping regions of b-cell linear epitopes ( figure 7b and f) . the structural accuracy of initial model including joining of the gaps was performed by modrefiner refinment. galaxy-refine was used to further refine the 3d models of ctl and htl mevs. for both mevs, refined model 1 was chosen on the basis of best scorings. for ctl mev refinement, the score of model 1 was as follows: rama favored was 94.2%, gdt-ha was 0.9500, rmsd was 0.433, molprobity was 2.211, clash score was 18.3, and poor rotamers was 1.2. for htl mev refinement, the score of model 1 was as follows: rama favored was 93.4%, gdt-ha was 0.9502, rmsd was 0.412, molprobity was 2.262, clash score was 21.9, and poor rotamers was 0.6. here, molprobity is the log-weighted combination of clash score, percentage ramachandran not favored, and percentage bad side-chain rotamers. after refinement, all the mentioned parameters improved significantly as compared to those of the initial model (table s10 ). the rampage analysis showed that the refined ctl mev model has 94% residues in the favored region, 3.8% residues in the allowed region, and only 1.4% residues in the outlier region, while the refined htl mev model has 94% of residues in the favored region, 5.2% residues in the allowed region, and only 0.8% residues in the outlier region ( figure 7d and h). discontinuous b-cell epitope prediction from both mevs was performed by the ellipro tool. the screening revealed ctl mev to have six discontinuous epitopes and htl mev to have five discontinuous epitopes. the pi score for ctl mev epitopes ranged from 0.549 to 0.906 and that for htl mev ranged from 0.56 to 0.729 (table s11 and figure 7c and g). the higher the score, the greater is the potential of the b-cell discontinuous epitope. the refined models of ctl and htl mevs were further analyzed for their interaction with the ecd of human tlr-3 by molecular docking using the patchdock tool. docking conformation chosen for the ctl and htl mevs showed scores of 18,096 and 23,690, respectively, which were highest among all docked complexes. the highest score indicates the best geometric shape complementarity fitting of ligand and receptor predicted by the tool. the docking complex shows a fitting confirmation of both the mevs within the ecd of tlr-3 ( figure 8a and e). further, the md simulation analysis of the docked ctl-tlr-3 and htl-tlr-3 complexes showed a very convincing reasonably stable rmsd value between ~0.4 and 0.5 nm for a given time window of 6 ns at the reasonably invariable temperature (~300 k) and pressure (~1 bar). these results indicate a stable complex formation for both mevs with tlr-3 ( figure 8b and f). the reasonably invariant rg of mev-tlr-3 complexes ( figure 8c and g) and rmsf for all the atoms in the complexes ( figure 8d and h) indicate that both the mev-tlr-3 complexes are reasonably stable and are properly folded. the b-factor of ctl and htl mev complexes with the tlr-3 receptor is shown by rainbow color presentation in figure 8a and optimized cdnas for both ctl and htl mevs were generated by the codon usage wrangler tool with a mammalian host cell line (human) as the choice of the expression system. the genscript rare codon analysis tool showed that the gc content of optimized ctl mev cdna was 69.74% and cai score was 1.0 with 0% tandem rare codons. likewise, the gc content of optimized htl mev cdna was 66.63%, and the cai score was 1.0 with 0% tandem rare codons. ideally, the gc content should be 30%-70%, the cai score indicating the possibility of cdna expression in the chosen expression system should be between 0.8 and 1.0, and tandem rare codon frequency indicating the percentage of low-frequency codons present in cdna for the chosen expression system should be <30%. tandem rare codons may hinder the expression of cdna or even interrupt the translational machinery. hence, the optimized cdnas of both mevs are predicted to be highly expressing in the mammalian host cell line (human). in this study, we propose the design of two mevs against mers-cov that consisted of ctl and htl epitopes. the selected ctl and htl epitopes were validated by in silico methods for their molecular interaction with hla alleles and tap (for ctl epitopes). the population coverage by the designed mevs is as high as 94% of the world population. both mevs were found to contain ifn-γ epitopes and b-cell discontinuous epitopes. moreover, they also showed stable interaction with the immunoreceptor tlr-3. on the basis of the design and in silico validation of both the ctl and htl mevs, their joint administration is predicted to induce humoral and cell-mediated immune response. codon-based cdnas of both mevs are predicted to have high expression in the mammalian host cell line (human); hence, they could be cloned and expressed at the laboratory level for in vivo trials on humanized hla-expressing mice for further study. available from: www.who.int/emergencies/mers-cov/mers-summary-2016 ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah -a link to health care facilities hospital outbreaks of middle east respiratory syndrome epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital mers outbreak in korea: hospital-to-hospital transmission reponse: coronavirus infections evaluation of candidate vaccine approaches for mers-cov analysis of the genome sequence and prediction of b-cell epitopes of the envelope protein of middle east respiratory syndrome-coronavirus crystallographic analysis of the n-terminal domain of middle east respiratory syndrome coronavirus nucleocapsid protein middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 mers-cov accessory orfs play key role for infection and pathogenesis crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals structural and mutational analysis of the interaction between the middle-east respiratory syndrome coronavirus (mers-cov) papain-like protease and human ubiquitin sequencing and phylogenetic analyses of structural and accessory proteins of middle east respiratory syndrome coronavirus from the first imported case in china antiviral mechanisms of human defensins rhinovirus increases human β-defensin-2 and-3 mrna expression in cultured bronchial epithelial cells. pathogens and disease assembly and export of mhc class i peptide ligands innate immunity: structure and function of tlrs toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection the swiss-model workspace: a web-based environment for protein structure homology modelling modeling the mhc class i pathway by combining predictions of proteasomal cleavage, tap transport and mhc class i binding identifying mhc class i epitopes by predicting the tap transport efficiency of epitope precursors netmhcpan, a method for mhc class i binding prediction beyond humans properties of mhc class i presented peptides that enhance immunogenicity peptide binding predictions for hla dr, dp and dq molecules quantitative peptide binding motifs for 19 human and mouse mhc class i molecules derived using positional scanning combinatorial peptide libraries prediction of mhc class ii binding affinity using smm-align, a novel stabilization matrix alignment method generation of tissue-specific and promiscuous hla ligand databases using dna microarrays and virtual hla class ii matrices predicting population coverage of t-cell epitope-based diagnostics and vaccines improved method for predicting linear b-cell epitopes prediction of the secondary structure of proteins from their amino acid sequence induction of hepatitis a virus-neutralizing antibody by a virus-specific synthetic peptide prediction of chain flexibility in proteins a semi-empirical method for prediction of antigenic determinants on protein antigens new hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and x-rayderived accessible sites reliable b cell epitope predictions: impacts of method development and improved benchmarking ellipro: a new structure-based tool for the prediction of antibody epitopes development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines in silico approach for predicting toxicity of peptides and proteins fast, scalable generation of highquality protein multiple sequence alignments using clustal omega qmean: a comprehensive scoring function for model quality assessment pepstrmod: structure prediction of peptides containing natural, non-natural and modified residues autodock4 and autodocktools4: automated docking with selective receptor flexibility autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading high performance molecular simulations through multi-level parallelism from laptops to supercomputers development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids structure of the transporter associated with antigen processing trapped by herpes simplex virus a flexible peptide linker enhances the immunoreactivity of two copies hbsag pres1 (21-47) fusion protein the structure of human beta-defensin-2 shows evidence of higher order oligomerization immunoinformatics analysis and in silico designing of a novel multi-epitope peptide vaccine against staphylococcus aureus fusion protein linkers: property, design and functionality vaccineda: prediction, design and genome-wide screening of oligodeoxynucleotide-based vaccine adjuvants designing of interferon-gamma inducing mhc class-ii binders algpred: prediction of allergenic proteins and mapping of ige epitopes vaxijen: a server for prediction of protective antigens, tumour antigens and subunit vaccines protein identification and analysis tools on the expasy server template-based protein structure modeling using the raptorx web server galaxyweb server for protein structure prediction and refinement structure validation by calpha geometry: phi,psi and cbeta deviation the molecular structure of the tolllike receptor 3 ligand-binding domain structural basis of development of multi-epitope vaccine against mers efficient unbound docking of rigid molecules patchdock and symmdock: servers for rigid and symmetric docking designing an efficient multi-epitope oral vaccine against helicobacter pylori using immunoinformatics and structural vaccinology approaches synonymous codon usage pattern in glycoprotein gene of rabies virus computational identification of rare codons of escherichia coli based on codon pairs preference recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses the ipd and imgt/hla database: allele variant databases improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization the authors acknowledge the advance instrumentation research facility (airf) at jawaharlal nehru university, new delhi, for providing advance computational facility to conduct experiments. the current research was also supported by dst-purse grant to jawaharlal nehru university. s srivastava and mk designed the protocol and performed the experiments. s srivastava, mk, s singh, aks, and ns contributed toward data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work. the authors report no conflicts of interest in this work. infection and drug resistance is an international, peer-reviewed openaccess journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. the journal is specifically concerned with the epidemiology of antibiotic resistance and the mechanisms of resistance development and diffusion in both hospitals and the community. the manuscript management system is completely online and includes a very quick and fair peerreview system, which is all easy to use. visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors. key: cord-333606-5z3kumu9 authors: lee, sangjoon; channappanavar, rudragouda; kanneganti, thirumala-devi title: coronaviruses: innate immunity, inflammasome activation, inflammatory cell death, and cytokines date: 2020-10-15 journal: trends immunol doi: 10.1016/j.it.2020.10.005 sha: doc_id: 333606 cord_uid: 5z3kumu9 the innate immune system acts as the first line of defense against pathogens, including coronaviruses. sars-cov and mers-cov are epidemic zoonotic coronaviruses that emerged at the beginning of the 21st century. the recently emerged virus sars-cov-2 is a novel strain of coronavirus that has caused the covid-19 pandemic. scientific advancements made by studying the sars-cov and mers-cov outbreaks have provided a foundation for understanding pathogenesis and innate immunity against sars-cov-2. in this review, we focus on our present understanding of innate immune responses, inflammasome activation, inflammatory cell death pathways, and cytokine secretion during sars-cov, mers-cov, and sars-cov-2 infection. we also discuss how the pathogenesis of these viruses influences these biological processes. cov, as infection with this virus recapitulates several aspects of the severe human disease, including pulmonary pathology, morbidity, and mortality [19] . for infections with mers-cov and sars-cov-2, human dpp4 transgenic mice with a mouse-adapted strain of mers-cov and human ace2 (hace2) transgenic mice with sars-cov-2 have been used to replicate several features of severe human disease, including pulmonary pathology and mortality [20, 21] . limitations with these systems have been noted. in human dpp4 transgenic mice, the lung disease that develops in response to the mouse-adapted strain of mers-cov infection may depend on species-specific mutations in the new host, which may not recapitulate all aspects of the human infection [20] . a potential disadvantage of studying sars-cov-2 in hace2 transgenic mice is that the brain infection observed in mice that succumb to disease may not reflect the pathogenesis of sars-cov-2 in humans [21] . additionally, hace2 is expressed under the control of the hfh4/foxj1 promoter in these mice, which is largely specific to epithelial cells [21] . consequently, hematopoietic cells from hace2 transgenic mice cannot be infected with sars-cov-2 [21] . despite these limitations, significant work has been done to molecularly characterize the innate immune pathways involved in detecting and controlling cov infections. patients with severe or critical covid-19 also found that reduced amounts of type i ifns in the blood during sars-cov-2 infection were associated with increased viral load in the blood, and exacerbation of the inflammatory response [38] . furthermore, data from whole genome sequencing of patients with life-in driving or contributing to infection outcomes, and certainly warrant further investigation. innate immune cells play a major role in anti-viral immunity, inflammatory signaling, and cytokine production. among the proinflammatory cytokines, il-1 pyroptosis is an inflammasome-mediated form of inflammatory cell death [3] . inflammasome assembly leads to the activation of inflammatory caspases table 2) . table 2) , and additional work is needed to determine whether this is occurring. pyroptosis and necroptosis are similar in that they are lytic forms of cell death driven by the gsdmd pore and mlkl channel, respectively, that release proinflammatory cytokines and other cellular factors to alert the surrounding cells of danger and to recruit innate and adaptive inflammatory cells [54, 55]. apoptosis was originally thought to be a less inflammatory form of cell death that disassembled the cell via membrane blebbing to avoid directly releasing cellular contents [58]. however, more recent studies have suggested that it is not always immunologically silent, as there is crosstalk between apoptotic proteins and lytic cell death executioners [58] . apoptosis is driven by initiator caspase-8, -9, and -10 cleavage of executioner caspase-3 and -7. the apoptotic caspase-3 has been reported to activate gasdermin e (gsdme) to induce a lytic form of cell death table 2) . overt activation of inflammatory cell death pathways may lead to critical tissue one of the goals of the innate immune response is to use prrs to recognize pathogenshere, viral agentsand ultimately trigger an inflammatory response and programmed cell death to limit viral replication and promote clearance. in another study also showed that bats have a mutation in an essential and highly inflammation [113] , suggesting that sex-based differences might affect inflammation and susceptibility to cov infection. these results suggest that innate host protection may play an important role in directing cov disease resistance. we have highlighted the sensing of different covs by the  optimal nlrp3 inflammasome activation is beneficial for the host but aberrant activation may lead to detrimental cov infection outcomes.  specific cov infections can activate inflammatory cell death (panoptosis), thereby inducing cytokine release.  cov disease tolerance occurs in age-, species-, and sex-dependent manners.  more studies are needed to define the innate immune response, specifically during sars-cov-2 infection, and to inform the development of candidate therapeutics. j o u r n a l p r e -p r o o f molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases gasdermin d is an executor of pyroptosis and required for interleukin-1beta secretion cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death points of control in inflammation bat origin of human coronaviruses coronaviruses: an overview of their replication and pathogenesis a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 effective treatment of severe covid-19 patients with tocilizumab compassionate use of remdesivir for patients with severe covid-19 e393-e400 heart acute myocardial injury and chronic damage to the cardiovascular system [11, 135] liver steatosis and abnormal liver function with ast and alt intestine gastrointestinal symptoms e.g. diarrhea and severe acute ulcerative colitis cov viral proteins can function as cytosolic pathogen-associated molecular patterns (pamps) and stimulate inflammasome assembly, resulting in activation of caspase-1. active caspase-1 then cleaves pro-il-1pro-il-18, and gasdermin d (gsdmd). the n-terminal fragment of gsdmd may then oligomerize within membranes to form membrane pores and execute pyroptosis sars-cov-2, and mhv infection initiate a signaling cascade mediated by caspase-8 activation. caspase-8 activates caspase-3 to drive apoptosis hcov-oc43 and mhv infection initiate necroptosis, a receptor-interacting serine/threonine-protein kinase 1 (ripk1) and ripk3 complex-dependent form of inflammatory cell death that depends on activation of the protein mixed lineage kinase domain-like pseudokinase (mlkl) to form channels in the membrane we apologize to our colleagues in the field whose work could not be cited due to space limitations. we thank all the members of the kanneganti laboratory for their comments and suggestions and rebecca tweedell, phd, for scientific editing and writing support. work from our laboratory is supported by the us national institutes of health (ai101935, ai124346, ar056296, and ca253095 to key: cord-342691-8jcfzexy authors: ochsner, scott a.; pillich, rudolf t.; mckenna, neil j. title: consensus transcriptional regulatory networks of coronavirus-infected human cells date: 2020-09-22 journal: sci data doi: 10.1038/s41597-020-00628-6 sha: doc_id: 342691 cord_uid: 8jcfzexy establishing consensus around the transcriptional interface between coronavirus (cov) infection and human cellular signaling pathways can catalyze the development of novel anti-cov therapeutics. here, we used publicly archived transcriptomic datasets to compute consensus regulatory signatures, or consensomes, that rank human genes based on their rates of differential expression in mers-cov (mers), sars-cov-1 (sars1) and sars-cov-2 (sars2)-infected cells. validating the cov consensomes, we show that high confidence transcriptional targets (hcts) of mers, sars1 and sars2 infection intersect with hcts of signaling pathway nodes with known roles in cov infection. among a series of novel use cases, we gather evidence for hypotheses that sars2 infection efficiently represses e2f family hcts encoding key drivers of dna replication and the cell cycle; that progesterone receptor signaling antagonizes sars2-induced inflammatory signaling in the airway epithelium; and that sars2 hcts are enriched for genes involved in epithelial to mesenchymal transition. the cov infection consensomes and hct intersection analyses are freely accessible through the signaling pathways project knowledgebase, and as cytoscape-style networks in the network data exchange repository. infection of humans by coronaviruses (cov) represents a major current global public health concern. signaling within and between airway epithelial and immune cells in response to infections by cov and other viruses is coordinated by a complex network of signaling pathway nodes. these include chemokine and cytokine-activated receptors, signaling enzymes and transcription factors, and the transcriptional targets encoding their downstream effectors [1] [2] [3] . placing the transcriptional events resulting from cov infection in context with those associated with host signaling systems has the potential to catalyze the development of novel therapeutic approaches. the cov research community has been active in generating and archiving transcriptomic datasets documenting the transcriptional response of human cells to infection by the three major cov strains, namely, middle east respiratory syndrome coronavirus (mers-cov, or mers) and severe acute respiratory syndrome coronaviruses 1 (sars-cov-1, or sars1) and 2 (sars-cov-2, or sars2) [4] [5] [6] [7] [8] [9] . to date however the field has lacked a resource that fully capitalizes on these datasets by, firstly, using them to identify human genes that are most consistently transcriptionally responsive to cov infection and secondly, contextualizing these transcriptional responses by integrating them with 'omics data points relevant to host cellular signaling pathways. we recently described the signaling pathways project (spp) 10 , an integrated 'omics knowledgebase designed to assist bench researchers in leveraging publically archived transcriptomic and chip-seq datasets to generate research hypotheses. a unique aspect of spp is its collection of consensus regulatory signatures, or consensomes, which rank genes based on the frequency of their significant differential expression across transcriptomic experiments mapped to a specific signaling pathway node or node family. by surveying across multiple independent datasets, we have shown that consensomes recapitulate regulatory relationships between signaling pathway nodes and their transcriptional targets to a high confidence level 10 . here, as a service to the research community to catalyze the development of novel cov therapeutics, we generated consensomes for infection of human cells by mers, sars1 and sars2 covs. computing the cov consensomes against those for a broad range of cellular signaling pathway nodes, we discovered robust intersections between genes with high rankings in the cov consensomes and those of nodes with known roles in the response to cov infection. integration of the cov table 1 . doi-driven links to consensomes and hct intersection networks. spp dois point to the tabular web version of the consensome, which can be downloaded as an excel file. ndex consensome dois point to the full consensome network; for ease and clarity of display, only the top 5% of the consensome is shown in the initial graphic display; in addition, a subset of the data corresponding only to the top 5% of the consensome can be reached via a link in the "description". ndex virus node family hct intersection dois point to networks containing all node families; ndex virus node hct intersection dois point to the full hct intersection network; for ease and clarity of display, only the top 5% of the hct intersection network is shown in the initial graphic display; in addition, a subset of the data corresponding only to the top 5% of the hct intersection network can be reached via a link in the "description". isgs were assigned elevated rankings across the four viral consensomes. the mean percentile of the isgs was however appreciably higher in the iav (98.7 th percentile) and sars1 (98.5 th percentile; p = 6e-1, t-test iav vs sars1) consensomes than in the sars2 (92 nd percentile, p = 5e-2, t-test iav v sars2) and mers (82 nd percentile; p = 7e-5, t-test iav v mers) consensomes. this is consistent with previous reports of an appreciable divergence between iav and sars2 infection with respect to the interferon transcriptional response 8 . other genes with known critical roles in the response to viral infection have high rankings in the cov consensomes, including ncoa7 14 (percentiles: sars1, 98 th ; sars2, 97 th ; mers, 89 th ; iav, 99 th ), stat1 15 (percentiles: sars1, 99 th ; sars2, 98 th ; mers, 89 th ; iav, 99 th ) and tap1 16 (percentiles: sars1, 99 th ; sars2, 94 th ; mers, 83 rd ; iav, 99 th ). in addition to the appropriate elevated rankings for these known viral response effectors, the cov consensomes assign similarly elevated rankings to transcripts that are largely or completely uncharacterized in the context of viral infection. examples of such genes include psmb9, encoding a proteasome 20s subunit (percentiles: sars1, 98 th ; sars2, 97 th ; mers, 98 th ; iav, 98 th ); csrnp1, encoding a cysteine and serine rich nuclear protein (percentiles: sars1, 99 th ; sars2, 94 th ; mers, 98 th ; iav, 94 th ); and ccnl1, encoding a member of the cell cycle-regulatory cyclin family (percentiles: sars1, 99 th ; sars2, 94 th ; mers, 99 th ; iav, 97 th ). finally, a preprint of a crispr/cas9 study described validation of 27 human genes as critical modulators of the host response to sars2 infection of human cells 17 . corroborating our analysis, 16 of these genes have significant (q < 0.05) rankings in the sars2 consensome, including ace2 and dyrk1a (both 97 th percentile), ctsl (96 th percentile), kdm6a, atrx, pias1 (all 94 th percentile), rad54l2 and smad3 (90 th percentile). to gather evidence for human signaling pathway nodes orchestrating the transcriptional response to cov infection, we next compared transcripts with elevated rankings in the cov consensomes with those that have predicted high confidence regulatory relationships with cellular signaling pathway nodes. we generated four lists of genes corresponding to the mers, sars1, sars2 and iav transcriptomic consensome 95 th percentiles. we then retrieved genes in the 95 th percentiles of available spp human transcriptomic (n = 25) consensomes and chip-seq (n = 864) pathway node consensomes 10 . for convenience we will refer from hereon to genes in the 95 th percentile of a viral infection, node (chip-seq) or node family (transcriptomic) consensome as high confidence transcriptional targets (hcts). we then used the r geneoverlap package 18 to compute the extent and significance of intersections between cov hcts and those of the pathway nodes or node families. we interpreted the extent and significance of intersections between hcts for covs and pathway node or node families as evidence for a biological relationship between loss or gain of function of that node (or node family) and the transcriptional response to infection by a specific virus. results of viral infection and signaling node hct intersection analyses are shown in fig. 2 ifit1 ifih1 ddx58 ifi6 ifit3 mx1 isg15 irf9 ifit2 isg20 ifitm3 ifitm1 ifi35 ifi35 irf7 ifit5 ifi35 irf7 ifit5 ifitm2 ifi16 ifi30 sars2 consensome percentile consensome q-value sars1 0 10 20 30 40 50 60 70 80 90 100 1e-55 1e-47 1e-39 1e-31 1e-23 1e-15 1e-07 ifit1 ifi6 ifi35 ifit2 mx1 ifitm1 ddx58 isg15 oas1 irf9 ifit5 ifih1 irf7 ifitm3 isg20 ifit3 ifitm2 ifi27 ifi16 ifi30 0 10 20 iav ifit2 ifi27 mx1 ifitm1 ifitm3 irf9 ifit1 ifit3 isg15 oas1 ddx58 irf7 ifih1 ifi35 ifit5 isg20 ifitm2 ifi6 ifi16 ifi30 a. b. c. d. www.nature.com/scientificdata www.nature.com/scientificdata/ transcription factors and enzymes, respectively) and figshare file f2 12 (based on chip-seq consensomes for selected co-nodes). figshare file f1 12 , sections 6 (node family transcriptomic hct intersection analysis) and 7 (node chip-seq hct intersection analysis) contain the full underlying numerical data. please refer also to table 1 for links to virus-node family and virus-node hct intersection networks in the ndex repositorysee section "visualization of the cov transcriptional regulatory networks in the signaling pathways project knowledgebase and network data exchange repository" below and the instructional youtube video (http://tiny. cc/2i56rz; strategy 6) for instructions on navigating these resources. we surveyed q < 0.05 hct intersections to identify (i) canonical inflammatory signaling pathway nodes with characterized roles in the response to cov infection, thereby validating the consensome approach in this context; and (ii) evidence for nodes whose role in the transcriptional biology of cov infection is previously uncharacterized, but consistent with their roles in the response to other viral infections. in the following sections all q-values refer to those obtained using the geneoverlap analysis package in r 18 . receptors reflecting their well-documented roles in the response to cov infection [19] [20] [21] [22] , we observed appreciable significant intersections between cov hcts and those of the toll-like (tlrs; q-values: sars1, 3e-85; sars2, 5e-49; mers, 2e-33), interferon (ifnr; q-values: sars1, 1e-109; sars2, 6e-53; mers, 1e-24) and tumor necrosis factor (tnfr; q-values: sars1, 1e-48; sars2, 1e-35; mers, 5e-32) receptor families (fig. 2) . indeed, anti-tnf therapy has been recently mooted as a potential clinical approach to sars2 infection 23 . hct intersections between cov infection and receptor systems with previously uncharacterized connections to cov infection, including epidermal growth factor receptors (egfr; q-values: sars1, 4e-21; sars2, 3e-48; mers, 1e-35), and notch receptor signaling (q-values: sars1, 6e-24; sars2, 2e-33; mers, 2e-29; fig. 2 ), are consistent with their known role in the context of other viral infections [24] [25] [26] [27] [28] . the notch receptor hct intersection points to a possible mechanistic basis for the potential of notch pathway modulation in the treatment of sars2 29 . the strong hct intersection between cov infection and xenobiotic receptors (q-values: sars1, 1e-30; sars2, 1e-44; mers, 5e-32; fig. 2 ) reflects work describing a role for pregnane x receptor in innate immunity 30 and points to a potential role for members of this family in the response to cov infection. in addition, the robust intersection between hcts for sars2 infection and vitamin d receptor (q = 2e-35) is interesting in light of epidemiological studies suggesting a link between risk of sars2 infection and vitamin d deficiency 31, 32 . consistent with a robust signature for the glucocorticoid receptor across all covs (gr; q-values: sars1, 3e-35; sars2, 1e-35; mers, 7e-32), recent studies have shown the gr agonist dexamethasone is a successful therapeutic for sars2 infection 33 . finally, independent in vitro analyses confirm our predictions of the modulation by sars2 infection of erbb/egfr 22, 34 and tgfbr 17,34 signaling systems (fig. 2) . transcription factors not unexpectedly -and speaking again to validation of the consensomes -the strongest and most significant cov hct intersections were observed for hcts for known transcription factor mediators of the transcriptional response to cov infection, including members of the nfκb (q-value ranges: sars1, 1e-7-1e-9; sars2, 9e-3-2e-3; mers, 1e-3-1e-4) [35] [36] [37] , irf (q-value ranges: sars1, 2e-2-1e-31; sars2, 2e-4-1e-17; mers, 9e-4-7e-5) 38 and stat (q-value ranges: sars1, 1e-7-1e-55; sars2, 2e-3-3e-29; mers, 5e-2-3e-5) [39] [40] [41] transcription factor families (fig. 3) . consistent with the similarity between sars1 and iav consensomes with respect to elevated rankings of isgs (fig. 2a,d) , the irf1 hct intersection was strongest with the sars1 (q = 2e-34) and iav (q = 3e-49) hcts. corroborating our finding of a strong intersection between stat2 and sars2 infection hcts (q = 3e-29), a recent preprint has shown that stat2 plays a prominent role in the response to sars2 infection of syrian hamsters 42 . hct intersections for nodes originally characterized as having a general role in rna pol ii transcription, including tbp (q-values: sars1, 2e-10; sars2, 6e-23; mers, 3e-16), gtf2b/ www.nature.com/scientificdata www.nature.com/scientificdata/ tfiib (q-values: sars1, 7e-10; sars2, 3e-23; mers, 9e-14) and gtf2f1 (q-values: sars1, 2e-4; sars2, 2e-13; mers, 5e-5) were strong across all covs, and particularly noteworthy in the case of sars2. in the case of gtf2b, these data are consistent with previous evidence identifying it as a specific target for orthomyxovirus 43 , and the herpes simplex 44 and hepatitis b 45 viruses. moreover, a recent preprint has identified a high confidence interaction between gtf2f2 and the sars2 nsp9 replicase 34 . white cells represent q > 5e-2 intersections. due to space constraints some class and family names may differ slightly from those in the spp knowledgebase. all q-values refer to those obtained using the geneoverlap analysis package in r 18 . full numerical data are provided in figshare file f1 12 , section 7; see also . this is likely due to the fact that chip-seq consensomes are based on direct promoter binding by a specific node antigen, whereas transcriptomic consensomes encompass both direct and indirect targets of specific receptor and enzyme node families. enzymes compared to the roles of receptors and transcription factors in the response to viral infection, the roles of signaling enzymes are less well illuminated -indeed, in the context of cov infection, they are entirely intersections. due to space constraints some class and family names may differ slightly from those in the spp knowledgebase. all q-values refer to those obtained using the geneoverlap analysis package in r 18 . full numerical data are provided in figshare file f1 12 , section 7; see also table 1 for links to virus-node hct intersection networks in the ndex repository. www.nature.com/scientificdata www.nature.com/scientificdata/ unstudied. through their regulation of cell cycle transitions, cyclin-dependent kinases (cdks) play important roles in the orchestration of dna replication and cell division, processes that are critical in the viral life cycle. cdk6, which has been suggested to be a critical g1 phase kinase 46, 47 , has been shown to be targeted by a number of viral infections, including kaposi's sarcoma-associated herpesvirus 48 and hiv-1 49 . consistent with this common role across distinct viral infections, we observed robust intersection between the cdk family hcts (q-values: sars1, 8e-23; sars2, 2e-31; mers, 1e-30; fig. 2 ) and the cdk6 hcts (q-values: sars1, 1e-7; sars2, 8e-8; mers, 3e-4; fig. 4 ) and those of all viral hcts. as with the tlrs, ifnrs and tnfrs, which are known to signal through cdk6 50,51 , intersection with the cdk6 hcts was particularly strong in the case of the sars2 hcts (fig. 4) . again, the subsequent proteomic analysis we alluded to earlier 34 independently corroborated our prediction of a role for cdk6 in the response to sars2 infection. consistent with a recent study 52 , the intersection of hcts for the lysine demethylase kmt2a was much stronger with iav (q-value = 2e-9) than with any of the covs (q-values: sars1, 7e-2; sars2, 2e-3; mers, 1e-2). ccnt2 is another member of the cyclin family that, along with cdk9, is a component of the viral-targeted p-tefb complex 53 . reflecting a potential general role in viral infection, appreciable intersections were observed between the ccnt2 hcts and all viral hcts (q-values: sars1, 4e-4; sars2, 6e-3; mers, 7e-5; fig. 4 ). finally in the context of enzymes, the dna topoisomerases have been shown to be required for efficient replication of simian virus 40 54 and ebola 55 viruses. the prominent intersections between dna topoisomerase-dependent hcts and the cov hcts (q-values: sars1, 3e-15; sars2, 6e-21; mers, 1e-26; fig. 2 ) suggest that it may play a similar role in facilitating the replication of these covs. hypothesis generation use cases. we next wished to show how the cov consensomes and hct intersection networks, supported by existing canonical literature knowledge, enable the user to generate novel hypotheses around the transcriptional interface between cov infection and human cellular signaling pathways. given the current interest in sars2, we have focused our use cases on that virus. figshare file f2 12 contains an additional use case linking the telomerase catalytic subunit tert to cov infection that was omitted from the main text due to space constraints. unless otherwise stated, all q-values below were obtained using the geneoverlap analysis package in r 18 . we stress that all use cases represent preliminary in silico evidence only, and require rigorous pressure-testing at the bench for full validation. hypothesis generation use case 1: transcriptional regulation of the sars2 receptor gene, ace2. ace2, encoding membrane-bound angiotensin converting enzyme 2, has gained prominence as the target for cellular entry by sars1 56 and sars2 57 . an important component in the development of ace2-centric therapeutic responses is an understanding of its transcriptional responsiveness to cov infection. interestingly, based on our cov consensome analysis, ace2 is more consistently transcriptionally responsive to infection by sars covs (sars1: 98 th percentile, consensome q value (cqv) 10 = 1e-25; sars2: 97 th percentile, cqv = 4e-7) than by iav (78 th percentile, cqv = 3e-8) or mers (49 th percentile, cqv = 2e-16; figshare file f1 12 , sections 2-5). the data points underlying the cov consensomes indicate evidence for tissue-specific differences in the nature of the regulatory relationship between ace2 and viral infection. in response to sars1 infection, for example, ace2 is induced in pulmonary cells but repressed in kidney cells (fig. 5) . on the other hand, in response to sars2 infection, ace2 is repressed in pulmonary cells -a finding corroborated by other studies 58,59 -but inducible in gastrointestinal cells (fig. 5) . these data may relate to the selective transcriptional response of ace2 to signaling by ifnrs (92 nd percentile; figshare file f1 12 , section 8) rather than tlrs (48 th percentile; figshare file f1 12 , section 9) or tnfrs (13 th percentile, figshare file f1 12 , section 10). these findings are consistent with a recent study confirming repression of induction of ace2 by interferon stimulation and by iav infection 60 . our data reflect a complex transcriptional relationship between ace2 and viral infection that may be illuminated in part by future single cell rna-seq analysis in the context of clinical or animal models of sars2 infection. hypothesis generation use case 2: evidence for antagonistic cross-talk between progesterone receptor and interferon receptor signaling in the airway epithelium. a lack of clinical data has so far prevented a definitive evaluation of the connection between pregnancy and susceptibility to sars2 infection in covid-19. that said, sars2 infection is associated with an increased incidence of pre-term deliveries 61 , and pregnancy has been previously associated with the incidence of viral infectious diseases, particularly respiratory infections 62, 63 . we were therefore interested to observe consistent intersections between the progesterone receptor (pgr) hcts and cov infection hcts (q-values: sars1, 3e-35; sars2, 5e-41; mers 5e-28), with the intersection being particularly evident in the case of the sars2 hcts ( fig. 2; figshare file f1 12 , section 6). to investigate the specific nature of the crosstalk implied by this transcriptional intersection in the context of the airway epithelium, we first identified a set of 12 genes that were hcts for both sars2 infection and pgr. interestingly, many of these genes encode members of the classic interferon-stimulated gene (isg) response pathway 13 . we then retrieved two spp experiments involving treatment of a549 airway epithelial cells with the pgr full antagonist ru486 (ru), alone or in combination with the gr agonist dexamethasone (dex). as shown in fig. 6 , there was unanimous correlation in the direction of regulation of all 12 genes in response to cov infection and pgr loss of function. these data are consistent with the reported pro-inflammatory effects of ru486 in a mouse model of allergic pulmonary inflammation 64 . interestingly, sars2-infected pregnant women are often asymptomatic 65, 66 . based on our data, it can be reasonably hypothesized that suppression of the interferon response to sars2 infection by elevated circulating progesterone during pregnancy may contribute to the asymptomatic clinical course. indeed, crosstalk between progesterone and inflammatory signaling is well characterized in the reproductive system, most notably in the establishment of uterine receptivity 67 as well as in ovulation 68 . consistent with our hypothesis, a recently launched clinical trial is evaluating the potential of progesterone for treatment of covid-19 in hospitalized men 69 . interestingly, the recently reported inhibition by progesterone of sars2 replication in www.nature.com/scientificdata www.nature.com/scientificdata/ vero 6 cells 70 indicates an additional mechanism, distinct from its potential crosstalk with the interferon response, by which progesterone signaling may impact sars2 infection. hypothesis generation use case 3: association of an epithelial to mesenchymal transition transcriptional signature with sars2 infection. epithelial to mesenchymal transition (emt) is the process by which epithelial cells lose their polarity and adhesive properties and acquire the migratory and invasive characteristics of mesenchymal stem cells 71 . emt is known to contribute to pulmonary fibrosis 72 , acute interstitial pneumonia 73 and acute respiratory distress syndrome (ards) 74 , all of which have been reported in connection with sars2 infection in covid-19 [75] [76] [77] . we were interested to note therefore that significant hct intersections for three well characterized emt-promoting transcription factors were specific to sars2 infection (q-values: snai2/slug 78 , 2e-2; epas1/hif2α 79 , 9e-9; lef1 80 , 1e-3; fig. 3 , bold symbols; figshare file f1 12 , section 7). consistent with this, intersections between hcts for tgfbrs, smad2 and smad3, known regulators of emt transcriptional programs 81 -were stronger with hcts for sars2 (q-values: tgfbrs, 2e-31; smad2, 2e-7; smad3, 5e-17) than with those of sars1 (q-values: tgfbrs, 6e-29; smad2, 2e-2; smad3, 3e-9) and mers (q-values: tgfbrs, 1e-16; smad2, 3e-3; smad3, 2e-12) -see also figs. 2 and 3 and figshare file f1 12 , sections 6 and 7). moreover, a recent crispr/cas9 screen identified a requirement for both tgfbr signaling and smad3 in mediating sars2 infection 17 . to investigate the connection between sars2 infection and emt implied by these hct intersections, we then computed intersections between the individual viral hcts and a list of 335 genes manually curated from the research literature as emt markers 82 , see also figshare file f1 12 , section 11. in agreement with the hct intersection analysis, we observed significant enrichment of members of this gene set within the sars2 hcts (q = 4e-14), but not the sars1 or mers (both q = 2e-1) hcts (fig. 7a) . consistent with previous reports of a potential link between emt and iav infection 83 , we observed significant intersection between the emt signature and the iav hcts (q = 1e-4). one possible explanation for the selective intersection between the literature emt signature and the sars2 hcts relative to sars1 and mers was the fact that the sars2 consensome was exclusively comprised of epithelial cell lines, whereas the sars1 and mers consensomes included non-epithelial cell biosamples (figshare www.nature.com/scientificdata www.nature.com/scientificdata/ file f1 12 , section 1). to exclude this possibility therefore, we next calculated airway epithelial cell-specific consensomes for sars1, sars2 and mers and computed intersections between their hcts and the emt signature. we found that significant intersection of the emt signature with the cov hcts remained specific to sars2 (q-values: sars1 & mers, 2e-1; sars2, 1e-8) in the lung epithelium-specific cov consensomes. we next retrieved the canonical emt genes in the sars2 hcts and compared their percentile rankings with the other cov consensomes. although some emt genes, such as cxcl2 and irf9, had elevated rankings across all four viral consensomes, the collective emt gene signature had a significantly higher mean percentile value in the sars2 consensome than in each of the other viral consensomes ( fig. 7b ; sars2 mean percentile = 97.5; sars1 mean percentile = 86, p = 1e-5, t-test sars2 v sars1; mers mean percentile = 63, p = 1e-9, t-test sars2 v mers; iav mean percentile = 76, p = 2e-7, t-test sars2 v iav)). a column named "emt" in figshare file f1 12 , sections 2 (sars1), 3 (sars2), 4 (mers) and 5 (iav) identifies the ranking of the emt genes in each of the viral consensomes. given that emt has been linked to ards 74 , we speculated that the evidence connecting emt and sars2 acquired through our analysis might be reflected in the relatively strong intersection between ards markers in sars2 hcts compared to other viral hcts. to test this hypothesis we carried out a pubmed search to identify a set of 88 expression biomarkers of ards or its associated pathology, acute lung injury (ali). a column named "ali/ards" in figshare file f1 12 , sections 2 (sars1), 3 (sars2) 4 (mers) and 5 (iav) identifies the expression biomarker genes using the pubmed identifiers for the original studies in which they were identified. consistent with our hypothesis, we observed appreciable intersections between this gene set and the hcts of all four viruses (sars1 odds ratio (or) = 7, q = 5e-9; sars2 or = 10.4, q = 1e-9; mers, or = 4.2, q = 2e-5; iav or = 6.8; q = 9e-8) with a particularly strong intersection evident in the sars2 hcts. although emt has been associated with infection by transmissible gastroenteritis virus 84 and iav 83 , this is to our knowledge the first evidence connecting cov infection, and specifically sars2 infection, to an emt signature. interestingly, lipotoxin a4 has been shown to attenuate lipopolysaccharide-induced lung injury by reducing emt 85 . moreover, several members of the group of sars2-induced emt genes have been associated with signature pulmonary comorbidities of cov infection, including adar 86 , cldn1 87 and sod2 88 . of note in the context of these data is the fact that signaling through two sars2 cellular receptors, ace2/at2 and cd147/basigin, has been linked to emt in the context of organ fibrosis [89] [90] [91] . finally, a recent preprint has described emt-like transcriptional and metabolic changes in response to sars2 infection 92 . collectively, our data indicate that emt warrants further investigation as a sars2-specific pathological mechanism. . based on these data, we speculated that sars2 infection might impact the expression of e2f-regulated cell cycle genes more efficiently than other covs. to investigate this we retrieved a set of sars2 hcts that were also hcts for at least three of e2f1, e2f3, e2f4 and tfdp1 (figshare file f1 12 , section 3, columns e2f1, e2f3, e2f4, tfdp1 & 95th 3/4). consistent with the role of e2f/dp-1 nodes in the regulation of the cell cycle, many of these genes -notably cdk1, pcna, cdc6, cenpf and nusap1 -are critical positive regulators of dna replication and cell cycle progression [97] [98] [99] [100] [101] and are known to be transcriptionally induced by e2fs 102-105 . strikingly, with the exception of e2f3, all were consistently repressed in response to sars2 infection (fig. 8a ). to gain insight into the relative efficiency with which the four viruses impacted expression of the e2f/dp-1 hct signature, we compared their mean percentile values across the viral consensomes. consistent with efficient repression of the e2f/dp-1 hcts by sars2 infection relative to other viruses, their mean percentile ranking was appreciably higher in the sars2 consensome (97 th percentile) than in the sars1 (76 th percentile; p = 6e-12, t-test sars2 v sars1), mers (71.2 percentile; p = 9e-6, t-test sars2 v mers) and iav (71.2 percentile; p = 2e-5, t-test sars2 v iav) consensomes (fig. 8b) . although manipulation of the host cell cycle and evasion of detection through deregulation of cell cycle checkpoints has been described for other viruses [106] [107] [108] , this represents the first evidence for the profound impact of sars2 infection on host cell cycle regulatory genes, potentially through disruption of e2f mediated signaling pathways. the sars2 infection-mediated induction of e2f3 (fig. 8a) may represent a compensatory response to transcriptional repression of other e2f family members, as has been previously observed for this family in other contexts 109, 110 . consistent with our prediction in this use case, while this paper was in revision, a study appeared showing that infection by sars2 results in cell cycle arrest 111 . our results represent evidence that efficient modulation by sars2 of e2f signaling, resulting in repression of cell cycle regulatory genes, may contribute to its unique pathological impact. to enable researchers to routinely generate mechanistic hypotheses around the interface between cov infection human cell signaling, we next made the consensomes and accompanying hct intersection analyses freely available to the research community in the spp knowledgebase and the network data exchange (ndex) repository. table 1 contains digital object identifier (doi)-driven links to the consensome networks in spp and ndex, and to the virus-node and virus-node family hct intersection networks in ndex. we have previously described the spp biocuration pipeline, database and web application interface 10 . figure 9 shows the strategy for consensome data mining on the spp website. the individual cov consensomes can be accessed by configuring the spp ominer query form as shown, in this example for the sars2 consensome (fig. 9a) . figure 9b shows the layout of the consensomes, showing gene symbol, name, percentile ranking and www.nature.com/scientificdata www.nature.com/scientificdata/ other essential information. genes in the 90 th percentile of each consensome are accessible via the user interface, with the full consensomes available for download in a tab delimited text file. target gene symbols in the consensome link to the spp regulation report, filtered to show only experimental data points that contributed to that specific consensome (fig. 9c ). this view gives insights into the influence of tissue and cell type context on the regulatory relationship. these filtered reports can be readily converted to default reports that show evidence for regulation of a specific gene by other signaling pathway nodes. as previously described, pop-up windows in the report provide experimental details, in addition to links to the parent dataset (fig. 9d) , curated accordingly to our previously described protocol 10 . per fair data best practice, cov infection datasets -like all spp datasetsare associated with detailed descriptions, assigned a doi, and linked to the associated article to place the dataset in its original experimental context (fig. 9d) . the full list of datasets is available for browsing in the spp dataset listing (https://www.signalingpathways.org/index.jsf). the ndex repository facilitates collaborative publication of biological networks, as well as visualization of these networks in web or desktop versions of the popular and intuitive cytoscape platform [112] [113] [114] . figure 10 shows examples of consensome and hct intersection network visualizations within the ndex user interface, in which transcripts or nodes, respectively, are organized using spp's category-class-family classification 10 . for ease of viewing, the initial rendering of the full sars2 (fig. 10a ) and other consensome networks shows a sample (fig. 10a, red arrow 1) containing only the top 5% of regulated transcripts; the full data can be explored using the "neighborhood query" feature available at the bottom of the page (red arrow 2). the integration in ndex of the popular cytoscape desktop application enables any network to be seamlessly be imported in cytoscape for additional analysis (red arrow 3). zooming in on a subset of the sars2 consensome (orange box) affords an appreciation of the diversity of molecular classes that are transcriptionally regulated in response to sars2 infection (fig. 10b) . transcript size is proportional to rank percentile, and edge weight is proportional to the transcript geometric mean fold change (gmfc) value. selecting a transcript allows the associated consensome data, such as rank, gmfc and family, to be examined in detail using the info table (fig. 10b, right panel) . highlighted to exemplify this feature is il6, an inflammatory ligand that has been previously linked to sars2 infection-related www.nature.com/scientificdata www.nature.com/scientificdata/ pathology 8, 115 . consensome gmfcs are signless with respect to direction of regulation 10 . researchers can therefore follow the spp link in the info table (fig. 10b, red arrow 4) to view the individual underlying viral infection data points on the spp site (fig. 9c shows the example for ifi27) . a network of the top 20 ranked transcripts in the sars2 consensome (fig. 10c) includes genes with known (oas1, mx1 116 ) and previously uncharacterized (pdzkip1, sat1, tm4sf4) transcriptional responses to sars2 infection. finally, to afford insight into pathway nodes whose gain or loss of function contributes to sars2 infection-induced signaling, fig. 10d shows the top 5% ranked nodes in the sars2 node hct chip-seq intersection network (see figshare file f1 12 , section 7; see also figs. 2 and 3 and accompanying discussion above). in this, as with all hct intersection networks, node size is proportional to the q-value, such that the larger the circle, the lower the q-value, and the higher the confidence that a particular node or node family is involved in the transcriptional response to viral infection. the ndex interface leverages the spp classification system to provide visual insights into the impact of cov infection on human cell signaling that are not readily appreciated in the current spp interface. for example, it is readily apparent from the ndex sars2 consensome network ( fig. 10c; table 1 ) that the single largest class of sars2 hcts encodes immunomodulatory ligands (or = 4.6, p = 3.8 e-24, hypergeometric test), many of which are members of the cytokine and chemokine superfamilies. in contrast, although still overabundant (or = 1.58, p = 6.8e-4, hypergeometric test), inflammatory ligands comprise a considerably smaller proportion of the sars1 hcts (table 1) . these data represent evidence that compared to sars1, sars2 infection may be relatively efficient in modulating a transcriptional inflammatory response in host cells. clicking on a data point opens a fold change information window that links to the spp curated version of the original archived dataset (d). like all spp datasets, cov infection datasets are comprehensively aligned with fair data best practice and feature human-readable names and descriptions, a doi, one-click addition to citation managers, and machine-readable downloadable data files. for a walk-through of cov consensome data mining options in spp, please refer to the accompanying youtube video (http://tiny.cc/2i56rz). www.nature.com/scientificdata www.nature.com/scientificdata/ fig. 10 visualization of viral consensomes and hct intersection networks in the ndex repository. in all panels, transcripts (consensome networks; panels a-c) and nodes (hct intersection network; panel d) are color-coded according to their category as follows: receptors (orange), enzymes (blue), transcription factors (green), ion channels (mustard) and co-nodes (grey). additional contextual information is available in the description of each network on the ndex site. red arrows are explained in the text. www.nature.com/scientificdata www.nature.com/scientificdata/ an effective research community response to the impact of cov infection on human health demands systematic exploration of the transcriptional interface between cov infection and human cell signaling systems. it also demands routine access to computational analysis of existing datasets that is unhindered either by paywalls or by lack of the informatics training required to manipulate archived datasets in their unprocessed state. moreover, the substantial logistical obstacles to high containment laboratory certification emphasize the need for fullest possible access to, and re-usability of, existing cov infection datasets to focus and refine hypotheses prior to carrying out in vivo cov infection experiments. meta-analysis of existing datasets represents a powerful approach to establishing consensus transcriptional signatures -consensomes -which identify those human genes whose expression is most consistently and reproducibly impacted by cov infection. moreover, integrating these consensus transcriptional signatures with existing consensomes for cellular signaling pathway nodes can illuminate transcriptional convergence between cov infection and human cell signaling nodes. to this end, we generated a set of cov infection consensomes that rank human genes by the reproducibility of their differential expression (p < 0.05) in response to infection of human cells by covs. using hct intersection analysis, we then computed the cov consensomes against high confidence transcriptional signatures for a broad range of cellular signaling pathway nodes, affording investigators with a broad range of signaling interests an entrez into the study of cov infection of human cells. although other enrichment based pathway analysis tools exist 117 , hct intersection analysis differs from these by computing against only genes that have the closest predicted regulatory relationships with upstream pathway nodes (i.e. hcts). the use cases described here represent illustrative examples of the types of hct-based analyses that users are empowered to carry out to illuminate principles of cov infection signaling. previous network analyses across independent viral infection transcriptomic datasets, although valuable, have been limited to stand-alone studies 118, 119 . here, to enable access to the cov consensomes and their >3,000,000 underlying data points by the broadest possible audience, we have integrated them into the spp knowledgebase and ndex repository to create a unique, federated environment for generating hypotheses around the impact of cov infection on human cell signaling. ndex provides users with the familiar look and feel of cytoscape to reduce barriers of accessibility, and provides for intuitive click-and-drag data mining strategies. incorporation of the cov data points into spp places them in the context of millions more existing spp data points documenting transcriptional regulatory relationships between human pathway nodes and their transcriptional targets. in doing so, we provide users with evidence for signaling nodes whose gain or loss of function in response to cov infection gives rise to these transcriptional patterns. the transcriptional impact of viral infection is known to be an amalgam of host antiviral responses and co-option by viruses of the host signaling machinery in furtherance of its life cycle. it is hoped that dissection of these two distinct modalities in the context of cov infection will be facilitated by the availability of the cov consensomes in the spp and ndex knowledgebases. the cov consensomes have a number of limitations. primarily, since they are predicated specifically on transcriptional regulatory technologies, they will assign low rankings to transcripts that may not be transcriptionally responsive to cov infection, but whose encoded proteins nevertheless play a role in the cellular response. for example, masp2, which encodes an important node in the response to cov infection 120 , has either a very low consensome ranking (sars1, mers and iav), or is absent entirely (sars2), indicating that it is transcriptionally unresponsive to viral infection and likely activated at the protein level in response to upstream signals. this and similar instances therefore represent "false negatives" in the context of the impact of cov infection on human cells. another limitation of the transcriptional focus of the datasets is the absence of information on specific protein interactions and post-translational modifications, either viral-human or human-human, that give rise to the observed transcriptional responses. although these can be inferred to some extent, integration of existing 34, 70, 111 and future proteomic and kinomic datasets will facilitate modeling of the specific signal transduction events giving rise to the downstream transcriptional responses. finally, although detailed metadata are readily available on the underlying data points, the consensomes do not directly reflect the impact of variables such as tissue context or duration of infection on differential gene expression. as additional suitable archived datasets become available, we will be better positioned to generate more specific consensomes of this nature. the human cov and iav consensomes and their underlying datasets are intended as "living" resources in spp and ndex that will be updated and versioned with appropriate datasets as resources permit. this will be particularly important in the case of sars2, given the expanded budget that worldwide funding agencies are likely to allocate to research into the impact of this virus on human health. incorporation of future datasets will allow for clarification of observations that are intriguing, but whose significance is currently unclear, such as the intersection between the cov hcts and those of the telomerase catalytic subunit (figshare file f2 12 ), as well as the enrichment of emt genes among those with elevated rankings in the sars2 consensome (fig. 7) . although they are currently available on the spp website, distribution of the cov consensome data points via the spp restful api 10 will be essential for the research community to fully capitalize on this work. for example, several co-morbidities of sars2 infection, including renal and gastrointestinal disorders, are within the mission of the national institute of diabetes, digestive and kidney diseases. in an ongoing collaboration with the niddk information network (dknet) 121 , the spp api will make the cov consensome data points available in a hypothesis generation environment that will enable users to model intersections of cov infection-modulated host signaling with their own research areas of interest. we welcome feedback and suggestions from the research community for the future development of the cov infection consensomes and hct node intersection networks. consistent with emerging nih mandates on rigor and reproducibility, we have used the research resource identifier (rrid) standard 122 to identify key research resources of relevance to our study. (2020) 7:314 | https://doi.org/10.1038/s41597-020-00628-6 www.nature.com/scientificdata www.nature.com/scientificdata/ dataset biocuration. datasets from gene expression omnibus (rrid: scr_005012) and array express (rrid: scr_002964) were biocurated as previously described, with the incorporation of an additional classification of peptide ligands 123 to supplement the existing mappings derived from the international union of pharmacology guide to pharmacology (rrid: scr_013077). dataset processing and consensome analysis. array data processing. to process microarray expression data, we utilized the log2 summarized and normalized array feature expression intensities provided by the investigator and housed in geo. these data are available in the corresponding "series matrix files(s)". the full set of summarized and normalized sample expression values were extracted and processed in the statistical program r. to calculate differential gene expression for investigator-defined experimental contrasts, we used the linear modeling functions from the bioconductor limma analysis package 124 . initially, a linear model was fitted to a group-means parameterization design matrix defining each experimental variable. subsequently, we fitted a contrast matrix that recapitulated the sample contrasts of interest, in this case viral infection vs mock infection, producing fold-change and significance values for each array feature present on the array. the current bioconductor array annotation library was used for annotation of array identifiers. p values obtained from limma analysis were not corrected for multiple comparisons. rna-seq data processing. to process rna-seq expression data, we utilized the aligned, un-normalized, gene summarized read count data provided by the investigator and housed in geo. these data are available in the corresponding "supplementary file" section of the geo record. the full set of raw aligned gene read count values were extracted and processed in the statistical program r using the limma 124 and edger analysis 125 packages. read count values were initially filtered to remove genes with low read counts. gene read count values were passed to downstream analysis if all replicate samples from at least one experimental condition had cpm >1. sequence library normalization factors were calculated to apply scale normalization to the raw aligned read counts using the tmm normalization method implemented in the edger package followed by the voom function 126 to convert the gene read count values to log2-cpm. the log2-cpm values were initially fit to a group-means parameterization design matrix defining each experimental variable. this was subsequently fit to a contrast design matrix that recapitulates the sample contrasts of interest, in this case viral infection vs mock infection, producing fold-change and significance values for each aligned sequenced gene. if necessary, the current bioconductor human organism annotation library was used for annotation of investigator-provided gene identifiers. p values obtained from limma analysis were not corrected for multiple comparisons. differential expression values were committed to the consensome analysis pipeline as previously described 10 . briefly, the consensome algorithm surveys each experiment across all datasets and ranks genes according to the frequency with which they are significantly differentially expressed. for each transcript, we counted the number of experiments where the significance for differential expression was ≤0.05, and then generated the binomial probability, referred to as the consensome p-value (cpv), of observing that many or more nominally significant experiments out of the number of experiments in which the transcript was assayed, given a true probability of 0.05. genes were ranked firstly by cpv, then by geometric mean fold change (gmfc). a more detailed description of the transcriptomic consensome algorithm is available in a previous publication 10 . the consensomes and underlying datasets were loaded into an oracle 15c database and made available on the spp user interface as previously described 10 . statistical analysis. high confidence transcriptional target intersection analysis was performed using the bioconductor geneoverlap analysis package 18 (rrid: scr_018419) implemented in r. briefly, given a whole set i of ids and two sets a ∈ i and b ∈ i, and s = a ∩ b, geneoverlap calculates the significance of obtaining s. the problem is formulated as a hypergeometric distribution or contingency table, which is solved by fisher's exact test. p-values were adjusted for multiple testing by using the method of benjamini & hochberg to control the false discovery rate as implemented with the p.adjust function in r, to generate q-values. the universe for the intersection was set at a conservative estimate of the total number of transcribed (protein and non protein-coding) genes in the human genome (25,000) 127 . r code for analyzing the intersection between an investigator gene set and cov consensome hcts has been deposited in the spp github account. a two tailed two sample t-test assuming equal variance was used to compare the mean percentile ranking of the emt (12 degrees of freedom) and e2f (14 degrees of freedom) signatures in the mers, sars1, sars2 and iav consensomes using the prism software package v 7.0 (rrid: scr_005375). the procedure for generating transcriptomic consensomes has been previously described 10 . to generate the chip-seq consensomes, we first retrieved processed gene lists from chip-atlas 128 (rrid: scr_015511), in which human genes are ranked based upon their average macs2 occupancy across all publically archived datasets in which a given pathway node is the ip antigen. of the three stringency levels available (10, 5 and 1 kb from the transcription start site), we selected the most stringent (1 kb). according to spp convention 10 , we then mapped the ip antigen to its pathway node category, class and family, and the experimental cell line to its appropriate biosample physiological system and organ. we then organized the ranked lists into percentiles to generate the node chip-seq consensomes. the 95 th percentiles of all consensomes (hcts, high confidence transcriptional targets) was used as the input for the hct intersection analysis. spp web application. the spp knowledgebase (rrid: scr_018412) is a gene-centric java enterprise edition 6, web-based application around which other gene, mrna, protein and bsm data from external databases such as ncbi are collected. after undergoing semiautomated processing and biocuration as described above, the data and annotations are stored in spp's oracle 15c database. restful web services exposing spp data, which are served to responsively designed views in the user interface, were created using a flat ui toolkit with a combination of javascript, d3.js, ajax, html5, and css3. javaserver faces and primefaces are the primary www.nature.com/scientificdata www.nature.com/scientificdata/ technologies behind the user interface. spp has been optimized for firefox 24+, chrome 30+, safari 5.1.9+, and internet explorer 9+, with validations performed in browserstack and load testing in loaduiweb. xml describing each dataset and experiment is generated and submitted to crossref (rrid: scr_003217) to mint dois 10 . important note on data availability: this paper refers to the first versions of the consensomes and hct intersection networks based on the datasets available at the time of publication. as additional cov infection datasets are archived over time, we will make updated versions of the consensomes and hct intersection analyses accessible in future releases. the entire set of experimental metadata is available in figshare file f1 12 , section 1. consensome data points are in figshare file f1 12 , sections 2-5. spp spp mers 129 , sars1 130 , sars2 131 and iav 132 consensomes, their underlying data points and metadata, as well as original datasets, are freely accessible at https://ww.signalingpathways.org. programmatic access to all underlying data points and their associated metadata are supported by a restful api at https://www.signalingpathways.org/docs/. all spp datasets are biocurated versions of publically archived datasets, are formatted according to the recommendations of the force11 joint declaration on data citation principles, and are made available under a creative commons cc by 4.0 license. the original datasets are available are linked to from the corresponding spp datasets using the original repository accession identifiers. these identifiers are for transcriptomic datasets, the gene expression omnibus (geo) series (gse); and for cistromic/chip-seq datasets, the ncbi sequence read archive (sra) study identifier (srp). spp consensomes are assigned dois as shown in table 1 . ndex ndex versions of consensomes (mers 133 , sars1 134 , sars2 135 and iav 136 ) and node family (mers 137 , sars1 138 , sars2 139 and iav 140 ) and node (mers 141 , sars1 142 , sars2 143 and iav 144 ) hct intersection networks are freely available in the ndex repository and assigned dois as shown in table 1 . ndex is a recommended repository for scientific data, springer nature and the plos family of journals and is registered on fairsharing. org; for additional info and documentation, please visit http://ndexbio.org. the official spp account in ndex is available at: https://bit.ly/30nn129. spp source code is available in the spp github account under a creative commons cc by 4.0 license at https:// github.com/signaling-pathways-project. toll-like receptors how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndromeassociated coronavirus infection imbalanced host response to sars-cov-2 drives development of covid-19 sars-cov-2 productively infects human gut enterocytes the signaling pathways project, an integrated'omics knowledgebase for mammalian cellular signaling pathways a comprehensive collection of systems biology data characterizing the host response to viral infection a transcriptional regulatory atlas of coronavirus infection of human cells interferon-stimulated genes: a complex web of host defenses the interferon-inducible isoform of ncoa7 inhibits endosome-mediated viral entry a partial form of recessive stat1 deficiency in humans tap, the human homolog of mex67p, mediates cte-dependent rna export from the nucleus genome-wide crispr screen reveals host genes that regulate sars-cov-2 infection test and visualize gene overlaps toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection interferon-beta 1a and sars coronavirus replication dysregulation in mtor/hif-1 signaling identified by proteo-transcriptomics of sars-cov-2 infected cells up-regulation of il-6 and tnf-alpha induced by sars-coronavirus spike protein in murine macrophages via nf-kappab pathway trials of anti-tumour necrosis factor therapy for covid-19 are urgently needed viruses exploit the function of epidermal growth factor receptor viral infection increases glucocorticoid-induced interleukin-10 production through erk-mediated phosphorylation of the glucocorticoid receptor in dendritic cells: potential clinical implications the glucocorticoid receptor (gr) stimulates herpes simplex virus 1 productive infection, in part because the infected cell protein 0 (icp0) promoter is cooperatively transactivated by the gr and kruppel-like transcription factor 15 viral interactions with the notch pathway the critical role of notch ligand delta-like 1 in the pathogenesis of influenza a virus (h1n1) infection covid-19 in the heart and the lungs: could we 'notch' the inflammatory storm? xenobiotic pregnane x receptor (pxr) regulates innate immunity via activation of nlrp3 inflammasome in vascular endothelial cells does vitamin d status impact mortality from sars-cov-2 infection? med. drug discov editorial: low population mortality from covid-19 in countries south of latitude 35 degrees north supports vitamin d as a factor determining severity coronavirus breakthrough: dexamethasone is first drug shown to save lives multi-level proteomics reveals host-perturbation strategies of sars-cov-2 and sars-cov the nf-kappab-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229e-infected cells influenza viruses and the nf-kappab signaling pathway -towards a novel concept of antiviral therapy the ns1 protein of influenza a virus blocks rig-i-mediated activation of the noncanonical nf-kappab pathway and p52/relb-dependent gene expression in lung epithelial cells the molecular basis of viral inhibition of irf-and stat-dependent immune responses severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane the role of interferon in influenza virus tissue tropism the unique role of stat2 in constitutive and ifn-induced transcription and antiviral responses stat2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in sars-cov-2 infected hamsters viral targeting of tfiib impairs de novo polymerase ii recruitment and affects antiviral immunity a new paradigm for transcription factor tfiib functionality hepatitis b virus px targets tfiib in transcription coactivation sumo1 modification stabilizes cdk6 protein and drives the cell cycle and glioblastoma progression beyond the cell cycle: a new role for cdk6 in differentiation cak-independent activation of cdk6 by a viral cyclin cell cycle control and hiv-1 susceptibility are linked by cdk6-dependent cdk2 phosphorylation of samhd1 in myeloid and lymphoid cells cyclin-dependent kinase activity is required for type i interferon production toll-like receptor-4 (tlr4) down-regulates microrna-107, increasing macrophage adhesion via cyclin-dependent kinase 6 multiomics investigation revealing the characteristics of hiv-1-infected cells in vivo p-tefb goes viral in vitro replication of dna containing either the sv40 or the polyoma origin dna topoisomerase 1 facilitates the transcription and replication of the ebola virus genome angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor the pivotal link between ace2 deficiency and sars-cov-2 infection a transcription regulatory network within the ace2 locus may promote a pro-viral environment for sars-cov-2 by modulating expression of host factors sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia pregnancy and susceptibility to infectious diseases pandemic 2009 influenza a(h1n1) virus illness among pregnant women in the united states ru486 blocks the anti-inflammatory effects of exercise in a murine model of allergen-induced pulmonary inflammation covid-19 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of new york city hospitals universal screening for sars-cov-2 in women admitted for delivery interferons and progesterone for establishment and maintenance of pregnancy: interactions among novel cell signaling pathways ovulation: new dimensions and new regulators of the inflammatory-like response progesterone for the treatment of covid-19 in hospitalized men a sars-cov-2 protein interaction map reveals targets for drug repurposing molecular mechanisms of epithelial-mesenchymal transition epithelial-mesenchymal transition contributes to pulmonary fibrosis via aberrant epithelial/fibroblastic cross-talk alveolar epithelial cells undergo epithelial-mesenchymal transition in acute interstitial pneumonia: a case report inflammatory and fibrinolytic system in acute respiratory distress syndrome pulmonary fibrosis and covid-19: the potential role for antifibrotic therapy chest ct findings of early and progressive phase covid-19 infection from a us patient a pneumonia outbreak associated with a new coronavirus of probable bat origin the transcription factor slug represses e-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with snail and e47 repressors hif-2α promotes epithelial-mesenchymal transition through regulating twist2 binding to the promoter of e-cadherin in pancreatic cancer the transcription factor lef-1 induces an epithelial-mesenchymal transition in mdck cells independent of β-catenin tgf-beta-induced epithelial to mesenchymal transition dbemt: an epithelial-mesenchymal transition associated gene resource influenza a virus infection dysregulates the expression of microrna-22 and its targets; cd147 and hdac4, in epithelium of asthmatics persistent transmissible gastroenteritis virus infection enhances enterotoxigenic escherichia coli k88 adhesion by promoting epithelial-mesenchymal transition in intestinal epithelial cells lipoxin a(4) ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial-mesenchymal transition the role of adar1 and adar2 in the regulation of mirna-21 in idiopathic pulmonary fibrosis identification and validation of differentially expressed transcripts by rna-sequencing of formalin-fixed, paraffin-embedded (ffpe) lung tissue from patients with idiopathic pulmonary fibrosis extracellular superoxide dismutase in pulmonary fibrosis basigin/cd147 promotes renal fibrosis after unilateral ureteral obstruction angiotensin ii as a morphogenic cytokine stimulating renal fibrogenesis cd147 induces epithelial-to-mesenchymal transition by disassembling cellular apoptosis susceptibility protein/ e-cadherin/beta-catenin complex in human endometriosis sars-cov-2 infection induces emt-like molecular changes, including zeb1-mediated repression of the viral receptor ace2, in lung cancer models the molecular basis of e2f-1/dp-1-induced s-phase entry and apoptosis the rb/e2f pathway: expanding roles and emerging paradigms e2f3a is critically involved in epidermal growth factor receptor-directed proliferation in ovarian cancer the retinoblastoma tumour suppressor in development and cancer cyclin-dependent kinase 1 (cdk1) is essential for cell division and suppression of dna re-replication but not for liver regeneration proliferating cell nuclear antigen is required for dna excision repair cdc6: from dna replication to cell cycle checkpoints and oncogenesis silencing cenp-f weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint nusap is essential for chromatin-induced spindle formation during early embryogenesis cell cycle arrest through indirect transcriptional repression by p53: i have a dream increased expression of rrm2 by human papillomavirus e7 oncoprotein promotes angiogenesis in cervical cancer an e2f site in the 5′-promoter region contributes to serum-dependent up-regulation of the human proliferating cell nuclear antigen gene the role of cellular transcription factor e2f in the regulation of cdc2 mrna expression and cell cycle control of human hematopoietic cells sars coronavirus 7a protein blocks cell cycle progression at g0/g1 phase via the cyclin d3/prb pathway breaking bad: how viruses subvert the cell cycle the dna damage response arouses the immune system compensation and specificity of function within the e2f family transcription factor compensation during mammary gland development in e2f knockout mice the global phosphorylation landscape of sars-cov-2 infection ndex: a community resource for sharing and publishing of biological networks ndex 2.0: a clearinghouse for research on cancer pathways cytoscape: a software environment for integrated models of biomolecular interaction networks detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes a review of pathway-based analysis tools that visualize genetic variants a network integration approach to predict conserved regulators related to pathogenicity of influenza and sars-cov respiratory viruses a dual controllability analysis of influenza virus-host protein-protein interaction networks for antiviral drug target discovery coronaviruses hijack the complement system the niddk information network: a community portal for finding data, materials, and tools for researchers studying diabetes, digestive, and kidney diseases a simple step toward improving reproducibility through rigor and transparency of experimental methods a draft network of ligand-receptor-mediated multicellular signalling in human limma powers differential expression analyses for rna-sequencing and microarray studies edger: a bioconductor package for differential expression analysis of digital gene expression data voom: precision weights unlock linear model analysis tools for rna-seq read counts chess: a new human gene catalog curated from thousands of large-scale rna sequencing experiments reveals extensive transcriptional noise chip-atlas: a data-mining suite powered by full integration of public chip-seq data middle east respiratory syndrome coronavirus (mers-cov) transcriptomic consensome, version 1. signaling pathways project sudden acute respiratory syndrome coronavirus 1 (sars-cov-1) transcriptomic consensome, version 1. signaling pathways project sudden acute respiratory syndrome coronavirus 2 (sars-cov-2) transcriptomic consensome, version 1. signaling pathways project influenza a virus (iav) transcriptomic consensome, version 1. signaling pathways project mers-cov transcriptomic consensome -full. the network data exchange sars-cov-1 transcriptomic consensome -full. the network data exchange sars-cov-2 transcriptomic consensome -full. the network data exchange iav transcriptomic consensome -full. the network data exchange -ndex https mers-cov vs pathway node family (transcriptomic) -all families. the network data exchange sars-cov-1 vs pathway node family (transcriptomic) -all families. the network data exchange sars-cov-2 vs pathway node family (transcriptomic) -all families. the network data exchange hct overlap: iav vs pathway node family (transcriptomic) -all families. the network data exchange mers-cov vs pathway node (chip-seq) -full. the network data exchange sars-cov-1 vs pathway node (chip-seq) -full. the network data exchange sars-cov-2 vs pathway node (chip-seq) -full. the network data exchange hct overlap: iav vs pathway node (chip-seq) -full. the network data exchange analysis of the dexamethasone (dex)-dependent transcriptome in a549 lung adenocarcinoma cells this work was supported by the national institute of diabetes, digestive and kidney diseases niddk information network (dk097748), the national cancer institute (ca125123, ca184427) and by the brockman medical research foundation. we acknowledge the assistance of apollo mcowiti, shijing qu and michael dehart in making the datasets and consensomes available in the spp knowledgebase. we thank all investigators who archive their datasets, without whom spp would not be possible. the authors declare no competing interests. correspondence and requests for materials should be addressed to n.j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-347460-9vechh4x authors: chang, feng-yee; chen, hsiang-cheng; chen, pei-jer; ho, mei-shang; hsieh, shie-liang; lin, jung-chung; liu, fu-tong; sytwu, huey-kang title: immunologic aspects of characteristics, diagnosis, and treatment of coronavirus disease 2019 (covid-19) date: 2020-06-04 journal: j biomed sci doi: 10.1186/s12929-020-00663-w sha: doc_id: 347460 cord_uid: 9vechh4x on march 11, 2020, the world health organization declared the worldwide spread of the infectious disease covid-19, caused by a new strain of coronavirus, sars-cov-2, as a pandemic. like in all other infectious diseases, the host immune system plays a key role in our defense against sars-cov-2 infection. however, viruses are able to evade the immune attack and proliferate and, in susceptible individuals, cause severe inflammatory response known as cytokine storm, particularly in the lungs. the advancement in our understanding of the mechanisms underlying the host immune responses promises to facilitate the development of approaches for prevention or treatment of diseases. components of immune system, such as antibodies, can also be used to develop sensitive and specific diagnostic methods as well as novel therapeutic agents. in this review, we summarize our knowledge about how the host mounts immune responses to infection by sars-cov-2. we also describe the diagnostic methods being used for covid-19 identification and summarize the current status of various therapeutic strategies, including vaccination, being considered for treatment of the disease. on december 31, 2019, a cluster of cases of pneumonia was announced in wuhan, hubei province, china. subsequently, on january 7, 2020, the chinese health authorities confirmed that this cluster was associated with a novel coronavirus, ncov, which was later named as sars-cov-2, and the ensuing disease was named covid-19. the covid-19 outbreak by the new coronavirus strain was recognized as a pandemic by the world health organization (who) on march 11, 2020. throughout history, there have been a number of pandemic diseases; the more notable and recent ones caused by viruses include the influenza pandemic (spanish flu) in 1918 and another by the influenza virus h1n1 in 2009. the immune system clearly plays a key role in the host defense against the infectious agents during these pandemics. the host is able to mount immune responses upon infection by viruses, as well as other microbes, and control the spread of these pathogens within the body. however, some viral strains are capable of evading the immune attack and proliferate in the body, as well as elicit inflammatory responses, in particular in the lungs, resulting in pneumonia. more importantly, in susceptible individuals, viruses can cause massive inflammatory responses, known as "cytokine storm", resulting in a severe pathological consequence. the advancement in our understanding of the mechanisms of the host immune response are crucial to development of approaches for prevention and treatment of these fast spreading and devastating infectious diseases. the components derived from our immune systems, such as antibodies, can be used to develop sensitive and specific methods for the diagnosis of infectious diseases, as well as novel therapeutic modalities. in this review, we briefly summarize our knowledge about the host immune response upon infection by sars-cov-2. we also discuss the epidemiological aspects of the outbreak, and the potential mechanism of the severe host response, such as cytokine storm. we also describe the antibody-based approaches for diagnosis of covid-19 infection and summarize the current status of various preventive and therapeutic modalities for treatment of the infection. coronaviruses are single-stranded enveloped rna viruses that cause diseases in mammals and birds. in humans, the low pathogenicity strains, including hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku, infect the upper respiratory tract and cause mild to moderate common cold-like symptoms in healthy individuals. they are responsible for 15-30% of all common cold cases. the highly pathogenic strains, including those causing severe acute respiratory syndrome [sars-cov] , middle east respiratory syndrome [mers-cov] , and covid-19 [new sars-cov-2], infect the lower respiratory tract and can cause severe pneumonia [1] . in addition to their rna genetic material, coronaviruses are composed of nucleocapsid (n) and spike (s) proteins, which participate in viral genome assembly, transcription and replication, or mediate viral entry and cause cytopathic effect [2, 3] . the s protein mediates the fusion of viral and host membrane [4] and contains a receptor-binding domain (rbd) that attaches to cells during viral entry. angiotensin-converting enzyme 2 (ace-2) is the receptor for both sars-cov and sars-cov-2 [5] . notably, the four human coronaviruses that cause common cold like symptoms show limited sequence homology in their n (30-67%) and s proteins (9-57%) compared with those of sars-cov-2 [6] . mers-cov also exhibits more distal relationship to sars-cov and sars-cov-2. however, the latter two are more closely related, with their n and s proteins sharing high homology (70-90%) . in 2003, the sars-cov infection, which started in southern china, led to an epidemic; in total, over 8400 cases were reported, which included close to 900 deaths with a case fatality rate of 11% [7] . in 2012, the first case of mers took place in saudi arabia. from that moment on, close to 2500 cases have been reported globally, which included close to 860 deaths, with an estimated case fatality rate of approximately 34% [8] . evidence is mounting that covid-19 spreads via human-to-human transmission of the virus [9] . after exposure to sars-cov-2, the majority of patients recover with little or mild symptoms that include cough and fever [10] . however, it is estimated that approximately 20% of infected individuals develop severe disease, including acute respiratory distress syndrome (ards). according to who, as of april 22, 2020, a total of 2,503, 072 confirmed cases of covid-19 have been detected and 171,791 deaths resulting from the infection have been confirmed worldwide. the case fatality rates in wuhan and worldwide were approximately 4.5 and 6.9%, respectively. the numbers are lower in some countries, for example approximately 5.4% in the united states, 2.2% in south korea, 3.3% in germany, and 1.4% in taiwan. these numbers are likely affected by the extent of screening, thereby implying that covid-19 cases might be underdiagnosed in many other countries. the availability and infrastructure of medical facilities in afflicted countries, especially those seriously affected ones, also likely affect the overall death rates. using public and published information, wu et al. estimated that the overall "symptomatic case fatality risk" (the probability of dying after developing symptoms) associated with covid-19 was 1.4% [11] . the rate of development of severe symptoms and death are clearly associated with the age. it is to be noted this is based on all tested and confirmed cases of covid19, and the true fatality risk is likely lower than 1.4%, since many mildly symptomatic/asymptomatic people might have never got tested. nevertheless, the risk is still higher than that associated with seasonal influenza virus, which is approximately 0.1%. with regard to the spectrum of the severity of the disease, chinese center for disease control and prevention reported of the 44,672 confirmed cases (with the age distribution of > 80 years: 3%; 30-79 years: 87%; 20-29 years: 8%; 10-19 years: 1%; < 10 years: 1%), the spectrum of disease were: mild: 81%; severe: 14%, critical: 5%; and case fatality rate: 2.3% [12] . finally, the basic reproduction number (ro) of the virus has been estimated to be between 1.4 and 3.9, meaning each infection from the virus can result in 1.4 to 3.9 new infections, when no members of the community are immune and no preventative measures, such as vaccination, are taken. by comparison, the median ro value for sars-cov was in the range of 2 to 4 and for 1918 influenza was 1.80. the outcome of clinical infection likely largely depends on the capacity in mounting effective antiviral immune responses in time, to control viral spreading, to limit organ injuries and to speed up recovery. here we summarize the immune response induced by cov. three components are crucial for sars-cov induced diseases: 1) the role of cd8+ t cells in defense against the virus, which causes apoptosis in the infected cells, 2) interactions of the virus with macrophages and dendritic cells, which initiate the early innate and subsequent adaptive immune responses, and 3) type i interferon (ifn) system, an innate response against viral infections, which can inhibit virus replication in the early phase. firstly, the central part of the body's anti-viral immunity is based on the interaction between antigen and antigen presentation cells (apc) when the virus enters the cells. the infected cells are recognized by virus-specific cytotoxic t lymphocytes (ctls) via viral peptides as the antigen presented by major histocompatibility complex (mhc). the antigen presentation of virus mostly depends on mhc i molecules, but mhc ii also has its contribution in some cases. the mhc i molecules display pieces of virus proteins on the surface of infected cells, which creates a signal to activate nearby cd8+ t cells to induce apoptosis in the infected cells. there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov [13] [14] [15] , but little is known about this association in covid-19. such information could provide beneficial aspects of personalized medicine for treatment or prevention of covid-19. secondly, dendritic cells and macrophages are other first patrolling components of innate immune network, which play important roles in driving both innate and adaptive immune responses to the viral pathogens [16] . the invasion of viruses can be recognized by innate immune cells via pathogen-associated molecular patterns (pamps). in the case of cov, pamps are viral genomic rna, which are recognized by endosomal rna receptors such as tlr3, tr7, tr8, and tlr7 [17] . this can cause rapid responses of the innate immune cells to viruses, resulting in production of a large amount of type i ifn with antiviral functions. lastly, efficient innate immune responses against viruses also depend on type i ifn responses and downstream cascade. type i ifn, by directly interfering with the viruses' replication ability, can prevent reproduction of viruses in infected cells. by mounting type i ifn responses successfully, viral replication and dissemination in an early stage are suppressed. sars-cov and mers-cov use several strategies to avoid the innate immune response, these are probably also employed by sars-cov-2. these include the inhibition of type i ifn recognition and signaling, as well as downregulation of mhc class i and class ii molecules in infected macrophages or dendritic cells, resulting in impaired antigen presentation and diminished t cell activation. moreover, some proteins encoded by sars-cov can interact with the signaling cascades downstream of the pattern recognition receptors. after exposure to sars-cov-2, patients respond to the virus by generating specific igm antibodies within a few days, followed by specific igg production within a week [3, 6, 18] . in the case of sars-cov infection, although the serum anti-viral igm antibody levels decline in a few months, the antiviral igg antibody titers can persist for years. among the many structural and non-structural proteins encoded by sars-cov-2, the n and s proteins are the most immunogenic antigens. antibodies against the n protein are the first to appear and thus can serve as an early and reliable serum marker for virus exposure, whereas antibodies against the s protein develop later and can bind to the viral envelope. recent studies indicated that the convalescent serum contains antibodies that can neutralize sars-cov-2 in cell cultures [2, 3] . therefore, igg against the s protein is both a marker for viral exposure and an indicator of recovery. the potential risk of disease exacerbation by ade, a phenomenon in which pre-existing poorly neutralizing antibodies lead to enhanced infection, has been a serious concern for vaccine development and antibody-based therapeutic strategy. compared to the ade in dengue viral infections, which was supported by a great deal of epidemiological and clinical evidence in the past four decades [19] , this phenomenon in coronaviruses has mainly been observed in cell-based experimental models [20, 21] . to illustrate this further by also using dengue virus as an example: while more severe symptoms, such as dengue hemorrhagic fever (dhf)/dengue shock syndrome (dss), can be observed during primary infection, they are much more frequently developed following a secondary infection with a different serotype (out of the four existing serotypes) [19] . furthermore, it has been well documented that the high level of virus replication seen during secondary infection with a heterotypic virus is a direct consequence of ade of viral replication. this is mediated primarily by the pre-existing, non-neutralizing, or sub-neutralizing antibodies to the virion surface antigens, resulting in enhanced access to target cells, through binding of the virion-antibody complexes to igg fc receptors (fcγr) on these cells [19] . this common underlying theme of ade-based magnification of virus replication also indicates that severe disease is not merely attributable to inherent virulence of virus [22] . interestingly, as described in a later section, several available evidence from the use of convalescent sera in patients with sars, mers [23] and 245 cases with covid-19 [24] suggest the feasibility and safety of convalescent serum trials. here, caution and vigilance to identify any evidence of enhanced coronavirus infection by ade will be required. despite the fact epidemiological and clinical observations supportive of existence of ade in coronavirus infection is not available, a molecular mechanism behind ade of coronavirus has currently been provided [21] . the authors demonstrated that a neutralizing antibody binds to the s protein of coronaviruses like a viral receptor, triggering a conformational change of the spike and mediating a viral entry into fcγr -expressing cells through canonical viralreceptor-dependent pathways. however, an enhanced entry of these pseudovirus-based approaches does not support directly the magnification of viral replication in these cells. compared to dengue viruses, these fcγrbearing cells such as dendritic cells, monocytes and macrophages, even if infected through ade process, would presumably not be so "permissive" for coronaviruses, in terms of their replication and assembly. however, these viewpoints need further investigation. apparently, many host factors could also exacerbate disease during secondary infection. these host factors need to be identified by combined epidemiological and genetic analyses of appropriate patients, and the contribution of underlying host factors to the control of coronavirus replication needs to be determined. for example, ade of replication can possibly occur with the vaccine strains of viruses in the endemic populations, such as attenuated or recombinant coronavirus vaccines. however, the level of replication will likely remain low, and thus this small enhancement of replication will probably not augment the disease. in addition, this could result in heightened vaccine immunogenicity due to a small increase in the virus load. however, further investigation on correlations between immunological responses and disease outcome and the validation of these findings in vaccine trials will be invaluable for developing safe and effective sars-cov2 vaccines (see below). while sars-cov can evade innate immune system, they can also induce intensive inflammatory reactions through innate immune cells. in fact, sars-cov and sars-cov-2 infections are known to activate a massive over-production of cytokines by the host immune systema phenomenon known as "cytokine storm", which usually occurs a few days after the onset of the illness. this also results in increased local and systemic vascular permeability in major organs. cytokine storm is reported in many viral infections, and contributes significantly to the pathogenesis and severity of acute viral infections. the tissue tropism of each virus determines the cytokine profiles of virus-induced cytokine storm (reviewed in [25] ). for example, macrophages produce a higher amount of proinflammatory cytokines than endothelial cells, while virus-infected endothelial cells are the major source of chemokines. however, this may not be generalizable, and it is crucial to elucidate the tropism of sars-cov-2 in order to interpret the data. most proinflammatory cytokines are released from macrophages and severe acute infections are usually associated with the activation of macrophages by enveloped viruses. in addition, activation of neutrophils may also be involved. as mentioned above, viral nucleic acids can induce the production of interferons and proinflammatory cytokines, through engaging endosomal tlrs. these intracellular nucleic acid receptors/sensors have been defined as "protective host factors", as they are critical for host defense against viral infections. however, the identity and contribution of "pathogenic host factors" to virus-induced severe inflammatory reactions and lethality, and how different viruses cause distinct clinical symptoms, remain unclear. some available information related to the cytokine storm induced by sars-cov and mers-cov is summarized below. it is to be noted that while both sars-cov and sars-cov-2 utilize ace-2 as their receptors, mers-cov binds to the receptor dipeptidyl peptidase 4 (dpp4/cd26). the differences in receptor usage may account for the differences in disease patterns, including the organs involved and the extent of the cytokine storm induced. [26] [27] [28] . the clinical course of this infection has three phases: 1) robust virus replication accompanied by fever in the first few days; 2) high fever and pneumonia with progressive decline of virus titers; 3) ards resulting from active host immune responses in the absence of detectable viruses [1] . in addition to infecting and proliferating in the airways and alveolar epithelial cells, sars-cov can also infect dendritic cells, monocytes, and macrophages, without undergoing proliferation (i.e., abortive infection) [29] . sars-cov-infected epithelial cells produce high levels of chemokines such as ccl2, ccl3, ccl5, and cxcl10. in addition, sars-cov-infected dendritic cells [30, 31] and macrophages [29] secrete high levels of proinflammatory cytokines tnf and il-6, and significant amounts of chemokines. it is interesting to note that higher levels of il-1, il-12, ifn-gamma, il-8, and cxcl9, in addition to the cytokines and chemokines mentioned above, were also observed in sars patients with severe diseases. this suggests that other cell types also contribute to sars-cov-induced cytokine storm. the typical pathological changes in the lungs include focal hemorrhage and mucopurulent materials in bronchial trees with diffuse alveolar damage. histological examination shows extensive macrophage and neutrophil infiltration with lower levels of t lymphocytes. existing information suggests that the sars-cov-infected airways and alveolar epithelial cells secrete abundant chemokines to attract immune cell infiltrations to the lungs, including macrophages and neutrophils, thereby causing damage due to high levels of proinflammatory cytokines and other mediators secreted by these cell types. in addition to the airway epithelial cells, mers-cov can also replicate in human monocytes, macrophages, dendritic cells, and activated t cells. the typical lung pathological changes caused by mers-cov is diffuse alveolar damage. in addition, pleural and pericardial effusions associated with generalized congestion and consolidation of lungs have been noted [32] , and the severity of lung lesions were noted to be correlated with extensive infiltration of neutrophils and macrophages [32] . similar to sars-cov, mers-cov can induce high levels of proinflammatory cytokines and chemokines in human monocyte-derived macrophages and dendritic cells. mers-cov infection was also reported to induce increased concentrations of proinflammatory cytokines (ifn-γ, tnf-α, il15, and il17), [33] . the high serum cytokine and chemokine levels in mers patients were correlated with increased infiltration of neutrophil and monocytes along with severe tissue damage in the lungs [32, 34, 35] . thus, the pathological change in the lungs is similar between sars-cov and mers-cov. whereas, the higher mortality rate in mers-cov-infected patients may be due to the higher incidence of pericarditis in infected patients. the first autopsy of covid-19 victims along with immuno-histological staining revealed the presence of sars-cov-2 in the airway epithelia and macrophages, suggesting that the virus can infect both epithelial cells and macrophages [36] . the majority of infiltrating cells are macrophages and monocytes with moderate amounts of multinucleated giant cells and neutrophils. increased levels of cytokines and chemokines, including il-2, il-7, g-csf, m-csf, ifn-γ, ip-10, mcp-1, mip-1α, and tnf-α, were detected in the plasma of covid-19 patients [37] . the most significant predictors of mortality in these patients are serum ferritin level and il-6, suggesting that mortality is due to virus-induced hyperinflammation and cytokine storm during viral infection [38, 39] . compared to the low pathogenic coronaviruses, the common features of high pathogenic coronaviruses include extensive infiltration of leukocytes, which secrete abundant proinflammatory cytokines and other chemical mediators to cause diffuse alveolar damage. also, high pathogenic viruses are associated with abortive infection; for example, in contrast to the less pathogenic strain of influenza virus h1n1, the highly pathogenic influenza virus h5n1 does not replicate in macrophages; the latter is also a more potent inducer of the chemokine cxcl10 [40] the key innate immunity receptors/sensors responsible for high pathogenic coronavirus-induced proinflammatory cytokines are still unclear. finally, notably, more deaths from covid-19 have been caused by multiple organ dysfunction syndrome rather than respiratory failure, which is different from infections caused by sars-cov and mers-cov; the basis for this remains unknown. detection of viral rna in the secretions from the respiratory tract of infected patients by reverse transcription-polymerase chain reaction (rt-pcr) test is currently the standard method for diagnosis of covid-19. they have some limitations, including long turnaround time (typically 2-4 h) and the requirement of specialized facilities. scientists around the world have been devoting effort to developing improved nucleic acid-based, simpler and faster methods. the us fda issued an emergency-use authorization to cepheid's xpert xpress sars-cov-2 test, which became the first pointof-care covid-19 diagnostic test to receive this designation in the us. the test is designed to use the company's automated genexpert systems and has a turnaround time of approximately 45 min. another prominent example is a method for detection of sars-cov-2 by using crispr technologies [41] . these newly developed platforms will clearly require clinical testing before approval for routine use. tests based on antibodies are obviously another option for diagnosis and screening. immediately after infections, viral genome and proteins start to increase, becoming the earliest markers for diagnosis within days. as the host immune responses gear up to confront and reduce viral replications, the viral rna or antigen level declines, but the antiviral igm and igg titers rise up. as patients usually present themselves with cough, fever or shortness of breath, and are already beyond the early stage of infection [10] ; here, nucleic acid tests are expected to pick up only a proportion of patients. a complementary serological test for specific igm or igg will help identify the rest. one report from shenzhen studied 173 patients within 7 days of illness, who were later diagnosed with covid-19 [42] ; in this study rt-pcr could detect two-thirds of the patients, but only 45% tested positive even after 15 days. in contrast, although the serological positive rate within 1 week was less than 40%, the rate increased to 100% after day 15 of disease onset. a combination of nucleic acid and serological test significantly increased the diagnosis rate from 66 to 78% even within 1 week of illness [42] . if these results can be validated, such a combination may become a standard clinical practice in the future. in this regard, li et al., developed an immunoassay that can detect igm and igg antibodies against sars-cov-2 in human blood within 15 min. they tested samples from close to 400 confirmed patients and over 100 negatively-tested patients at 8 different clinical sites and reported a sensitivity of over 88% and specificity of over 90% [43] . in the early stage (containment) of covid-19 pandemic, there was a strong interest for rapid diagnosis and thus prototypes of rapid viral antigen or antibody tests were being developed for point-of-care use. these platforms have the advantage of convenience and a fast turnaround time, but suffer from inadequate sensitivity and specificity, as compared with standard rt-pcr. hence, their results need to be interpreted with caution. preparation of high-quality antibodies and antigens requires years in perfecting such point-of-care tests, judging from the experience in developing such tests for influenza viruses. eventually, the more stringent criteria for a definitive diagnosis will need paired serum samples to demonstrate a true seroconversion [6] . finally, the issue of antibody cross-reactivity with other human coronaviruses warrants discussion. as mentioned above, serological assays usually adopt viral n or s protein as antigens and the protein components from all four human coronaviruses that cause common cold show very limited sequence homology with those of sars-cov-2. thus, despite the fact the majority of the populations have been exposed to the four low pathogenic human coronaviruses, their sera do not react positively in sars-cov-2 elisa [6, 18] . while mers-cov is also more distally related and presents no concerns, the situation for sars-cov-exposed patients is different. as the n and s proteins of the sars-cov strain share high homology (70-90%) with those of sars-cov-2, serum from sars patients can actually cross-react with sars-cov-2 n or s protein in immunoblot or viral neutralization assays [2, 3] . however, as sars-cov epidemic was only transient with a very small proportion of the populations being exposed, this cross-reactivity should not be an important issue. as with many vaccine-preventable viral diseases like measles and chicken pox, the newly emerged sars-cov-2 infection assumes an epidemiological characteristic capable of evading containment measures and facilitating pandemic potential. thus, a high proportion of undetected infections with mild or no symptoms can efficiently sustain viral transmission [44] . while containment and lockdown can serve as temporary control measures, effective vaccines or therapeutic agents are much needed for the ultimate control of the disease. the vaccine research and development thus far have progressed at an unprecedented speed; the first dose of rna-based sars-cov-2 vaccine was administered to test its safety in humans on march 16, 2020, only 2 months after the new virus was first identified. such rapid progress was facilitated by a combination of multiple factors, including advances in vaccine research on sars and mers, as recently reviewed [45] , progress in a number of vaccine technology platforms to the early stage of human trial [46] [47] [48] , and readily available support from the well-orchestrated international collective effort of the coalition for epidemic preparedness innovations (cepi) [49] . the aforementioned innovative aspects that could potentially drive sars-cov-2 vaccine development to market launch within a year or two are summarized below. the coronavirus s protein is a critical target for antiviral neutralizing antibodies and functions to mediate entry into mammalian cell expressing the viral receptor ace2 [3, 50] . moreover, the target neutralizing epitope of sars-cov was further narrowed to a smaller fragment of the s protein, later termed receptor binding domain (rbd) [51] . building on these paradigmatic scientific advances that the s protein is the putative target antigen, sars-cov-2 vaccine candidates are designed to include full or various lengths of the s protein focusing on the rbd. vaccine based on the whole virus may be less preferred due to its association with eosinophilic pulmonary pathology [52, 53] . a number of novel vaccine platforms, including vector-, dna-, and rna-based vaccines, are being developed or improved with innovative technology specifically to combat pandemic-prone outbreaks and have been recently reviewed [54] . nucleic acid vaccines, including both dna and rna, offer the potential to accurately express any protein antigen in host cells and to present the antigen closely resembling antigen expression and presentation during viral infection. dna vaccines against mers and rna vaccine against h7n9 have completed phase i trials that showed these platforms to be safe [46] [47] [48] . the nucleic acid vaccine can be completely synthetic and formulated within a few weeks at sufficient quantities to support clinical trialsa valuable feature when facing potential pandemic. vector-based vaccine consists of a target antigen inserted into a viral genome to render faithful antigen generation, targeting and processing in vivo after vaccination. the first ebola vaccine approved by us fda is a vector-based vaccine using vesicular stomatitis virus expressing a surface glycoprotein of ebola [55] , thus supporting the applicability of this platform in combating emerging infectious diseases. these platforms offer versatile adaptation for antigen of new diseases, and the process development for production is relatively simple and quick, indicating the value of these platforms for the urgent response to new diseases. the rapid infusion of funding from and coordination by cepi in january 2020 was the major driver for the speed of sars-cov-2 vaccine r&d progress (fig. 1) . with its mission being "to stimulate, finance, and coordinate the development of vaccines for epidemic diseases" especially aiming to drive vaccine innovation for highpriority public health threats, cepi has supported a number of "technology platforms". these included a vaccine printer, molecular clamp technology for protein production, and a self-amplifying rna vaccine platform since 2017 (cepi web page https://cepi.net/research_ dev/technology/). an innovated vaccine platform technology pertains to a system that uses the same basic core technological components as a backbone and can be adapted by inserting new genetic or protein sequences to target newly emerging pathogens. it is with the application of this concept that an rna-based sars-cov-2 vaccine, built on the avian flu vaccine platform [46] , is expected to be developed and quickly proceed to human trial within only a few months since covid-19 became an epidemic, and a dna-based vaccine candidate modeling that of mers is soon to follow [46] . the cepi coordinated effort encompassing a wide range of available technologies from industry and research institutes globally has shown preliminary success toward an urgent response to control a pandemic. while coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines also present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology [53, 56] ; this seems to be either exacerbated or eliminated by the formulation of adjuvant selection depending on the th1/th2 bias and induction of durable ifn-γ responses, respectively [57] . in addition, ade, described above, was seen in the lungs of macaques after administration of inactivated sars-cov vaccine or vaccine composed of certain s antigen fragments [58] . these studies highlight the importance of designing the target antigen and selection of adjuvants to ensure both efficacy and safety. considering the novel nature of sars-cov-2 and that an animal model has yet to be established for testing of the vaccine to especially focus on the immunopathological perspectives, the safety concern is anticipated to present most of the hurdles in the development process. herd immunity refers to a state when sufficient proportion of a population becomes immune to sars-cov-2, via natural infection or vaccination, so as to eventually halt further spread of disease, and thus individuals not immune to the virus are protected. in the case of covid-19, the herd immunity was estimated to be 60% of the population. before vaccines are available for mass immunization, the strategy of achieving herd immunity via natural infection has been considered and deemed not advisable when a number of factors were considered. thus, self-isolation and social distancing remain crucial in combating this pandemic so that the initial pressure on our healthcare systems is reduced, and more time is given to us to develop vaccines or effective therapies. the disease spectrum of covid-19 can be divided into mild infection, pneumonia, ards, and even multiple organ failure [37] . after a decade of research on coronavirus, unfortunately, still there are no licensed vaccines, effective specific antivirals, nor drug combinations supported by high-level evidence to treat the infection, especially for newly emerging strains such as sars-cov-2 [59] . several strategies are being considered for the treatment of covid-19, including the use of antimicrobial agents, immunotherapy with virus-specific antibodies in convalescent plasma, monoclonal and polyclonal antibodies produced in vitro or genetically modified antibodies, and interferons. here we focus on immunebased therapies, but for the sake of completeness, we also include therapies using antimicrobial agents as 7supplementary information. the potential interventions for sars-cov infection are summarized in table 1 . biologic drugs composed of monoclonal antibodies (mabs) have been developed for treatment of a variety of diseases. it is thus not surprising that this approach is being considered for the treatment of sars-cov infection and shows promise. a human igg1 mab, cr3022, that binds to sars-cov s protein has been developed [70] . sui et al. found one recombinant human mab (single-chain variable region fragment, scfv, 80r) against the s1 domain of s protein of sars-cov from two nonimmune human antibody libraries. the mab could efficiently neutralize sars-cov and inhibit syncytia formation between cells expressing s protein and those expressing the sars-cov receptor ace2 [71] . a human igg1 mab, cr3014, has been generated and found to be able to neutralize sars-cov and shown to be able to prevent sars-cov infection in ferrets [72] . more recently, ju et al. reported the isolation and characterization of 206 rbd-specific mabs derived from single b cells of eight sars-cov-2 infected individuals [79] . for clones from one patient they demonstrated their ability to neutralize live sars-cov-2. none of these antibodies cross-reacted with rbds from either sars-cov or mers-cov, although the patient plasma exhibited such cross-reactivity. these neutralizing antibodies have the potential to be used for prophylaxis for or treatment of sars-cov-2 infection. agents that directly block the binding of s protein to the functional receptor ace2 also have the potential to be used for prevention of covid-19. guillon et al. demonstrated that binding of sars-cov s protein to ace2 could be inhibited by anti-histo-blood group antibodies, presumably because the virus carries histo-blood group antigen structures of the host [4] . while whether this approach can be developed into effective treatment strategies is uncertain, the findings have a bearing on the effect of the naturally occurring anti-histo-blood group antibodies on the individual variations in susceptibility to sars-cov infection. convalescent plasma can be employed for passive immunotherapy and is usually chosen when there are no specific vaccines or drugs available for emerging infection-related diseases. yeh et al. reported a favorable outcome in the use of convalescent plasma for treatment of sars-cov-infected healthcare workers [73] . arabi et al. tested the feasibility of convalescent plasma therapy as well as its safety and clinical efficacy in critically ill patients suffering from mers-cov infection [74, 80] . if available, convalescent plasma could certainly be considered for the treatment of sars-cov-2-infected critically ill patients. interferons (ifns), including ifn-α and ifn-β, are produced during the innate immune response to virus infection and they are able to inhibit the replication of virus in vitro [75, 81] . as mentioned above, ifn transcription was blocked in tissue cells infected with sars-cov. recombinant ifn-α given on 3 days before the infection could reduce viral replication and lung damage, as compared with the control in monkeys and in a pilot clinical trial [82] . ifn-α inhalation can also be considered. combination of interferon-α-2a with ribavirin was used in treatment of patients with severe mers-cov infection and the survival of these patients was improved [76, 83] . these findings suggest that these fda-approved ifn's could be used for the treatment of covid-19. as mentioned above, cytokine storm is the major underlying pathology in severe cases of covid-19. thus, neutralization of some of the major cytokines are considered as a novel approach for treatment of severely ill cases and reducing morbidity and mortality. huang c et al. reported that increased il-1ß, ifn-γ, ip-10, and mcp-1 in sars-cov-2 infection and higher concentrations of g-csf, ip-10, mcp-1, mip-1a, and tnf-α were found in patients requiring treatment at icu than those not treated at icu [37] . they also noted that cytokine storm was associated with disease severity. conti p et al. reported that pro-inflammatory cytokines of interleukin (il)-1β and il-6 in mild and acute respiratory syndrome are associated with development of lung fibrosis in covid-19 [77] . thus, suppression of proinflammatory il-1 family members and il-6 might have a potential therapeutic effect. il-37, an immunosuppressive cytokine, acts on mtor and increases the activity of adenosine monophosphate kinase, which inhibits protease inhibitors nelfinavir cell a selective post-translational inhibitor [65] nucleotide analog prodrug remdesivir cell and clinical use (first case of covid-19 in the united states) possible inhibitor of rna replication [66, 67] indole-derivative molecule arbidol cell inhibits fusion between viral envelope and cellular membranes [68] immunosuppressive agent cyclosporine a cell block replication via inhibition of nucleocapsid protein [69] monoclonal antibody cr3022 cell and clinical use potently binds the receptor binding domain of s protein [70] monoclonal antibody single-chain variable region fragments, scfv, 80r cell acts against the s1 domain of s protein [71] monoclonal antibody cr3014 cell neutralization of viral infectivity [72] immunotherapeutic potential convalescent plasma cell and clinical use neutralization of viral infectivity [73, 74] interferons ifn-α and ifn-β cell induction of interferon-stimulated genes to suppress viral replication [75, 76] cytokine blocker cytokine il-37 cell inhibits inflammation, by acting on mtor and increasing the activity of adenosine monophosphate kinase [77] cytokine blocker lianhuaqingwen cell anti-inflammation; inhibits il-6 receptor [78] cytokine blocker antibody against il-6 receptor clinical use anti-inflammation; inhibits il-6 receptor inflammation by suppressing production of multiple cytokines downstream of myd88, including il-1β, il-6, tnf and ccl2. il-37 might be a potential therapeutic cytokine for inhibition of inflammation in covid-19 [77] . runfeng l et al. demonstrated that lianhuaqingwen, a traditional chinese medicine, significantly inhibited sars-cov-2 replication by suppressing mrna of il-6 and other pro-inflammatory cytokines in vero e6 cells [78] . cytokine blocker that target interleukin 6 receptor in covid-19 could be potentially developed as therapeutic agents in future. in fact, fda approved mab against il-6 receptor (il-6r) is available for treatment of rheumatoid arthritis. the society for immunotherapy of cancer has issued a statement on access to il-6-targeting therapies for covid-19. it is encouraging that pharmaceutical companies have in fact initiated clinical trials of anti-il-6r for treatment of patients with severe covid-19. there are still a large number of unanswered questions. how fast sars-cov-2 would mutate and would the mutated virus become more infectious or invasive. according to andersen et al. [84] , viruses constantly mutate, but those mutations do not typically make the virus more virulent or cause more serious disease. in fact, most mutations are detrimental to the virus or have no effect. there was a study of the sars-cov in primate cells suggesting that a mutation in this viral strain acquired during the 2003 sars outbreak probably reduced virulence of the virus [85] . another issue is whether sars-cov-2, unlike sars-cov and mers-cov will continue to cause epidemic or even behave like seasonal flu. in fact, we have already witnessed the second wave of outbreak occurring in north america and europe, after the first wave that occurred in asia, and covid-19 may bounce back-and-forth between north and south hemispheres, as influenza virus does each year. finally, factors that determine the individual susceptibility to covid-19 remain to be elucidated. as mentioned above, there are many reports on the relationship between various mhc polymorphisms and the susceptibility to sars-cov. also, what governs the development of severe illness, including cytokine storm, besides the pre-existence of certain diseases and age factor, awaits clarification. despite these uncertainties, scientists in academia and industry around the world have moved at an unprecedented speed to develop improved methods for detection of the virus and treatment of the disease. advancement in immunology over the years has certainly facilitated many of these developments. we shall witness some of the recent advancements in development of vaccines and biologics for treatment of various other serious illnesses being used for fighting against covid-19. we are hopeful these efforts will be sustained even after the pandemic is over, allowing us to be even more ready in the unfortunate event that another epidemic or pandemic, like covid-19, takes place in the future. supplementary information accompanies this paper at https://doi.org/10. 1186/s12929-020-00663-w. additional file 1. supplemental information. pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a pneumonia outbreak associated with a new coronavirus of probable bat origin inhibition of the interaction between the sars-cov spike protein and its cellular receptor by anti-histo-blood group antibodies structural basis for the recognition of the sars-cov-2 by full-length human ace2 serological assays for emerging coronaviruses: challenges and pitfalls update 49 -sars case fatality ratio, incubation period middle east respiratory syndrome coronavirus (mers-cov) a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster china novel coronavirus i. and research t. a novel coronavirus from patients with pneumonia in china estimating clinical severity of covid-19 from the transmission dynamics in wuhan characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention human-leukocyte antigen class i cw 1502 and class ii dr 0301 genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection association of human leukocyte antigen class ii alleles with severe acute respiratory syndrome in the vietnamese population association of human leukocyte antigen class ii alleles with severe middle east respiratory syndrome-coronavirus infection plasmacytoid dendritic cell precursors/type i interferon-producing cells sense viral infection by toll-like receptor (tlr) 7 and tlr9 recognition of pathogen-associated molecular patterns by tlr family evaluation of antibody responses against sars coronaviral nucleocapsid or spike proteins by immunoblotting or elisa immune response to dengue virus and prospects for a vaccine the convalescent sera option for containing covid-19 molecular mechanism for antibody-dependent enhancement of coronavirus entry pathogenesis of dengue: challenges to molecular biology convalescent plasma study g. the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis china puts 245 covid-19 patients on convalescent plasma therapy cytokine determinants of viral tropism identification of a novel coronavirus in patients with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis chemokine up-regulation in sars-coronavirus-infected, monocytederived human dendritic cells severe acute respiratory syndrome coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity a pathological report of three covid-19 cases by minimally invasive autopsies clinical features of patients infected with 2019 novel coronavirus in wuhan the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan proinflammatory cytokine response and viral replication in mouse bone marrow derived macrophages infected with influenza h1n1 and h5n1 viruses a protocol for detection of covid-19 using crispr diagnostics antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019. medrxiv development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic preclinical and clinical demonstration of immunogenicity by mrna vaccines against h10n8 and h7n9 influenza viruses a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial cepi: driving progress towards epidemic preparedness and response angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge new vaccine technologies to combat outbreak situations ebola vaccine vsv-ebov rapidly protects macaques against infection with the 2014/15 ebola virus outbreak strain severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology gold nanoparticle-adjuvanted s protein induces a strong antigen-specific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor evaluation of antiviral activities of houttuynia cordata thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection discovering drugs to treat coronavirus disease 2019 (covid-19) covid-19: an update on the epidemiological, clinical, preventive and therapeutic evidence and guidelines of integrative chinese-western medicine for the management of 2019 novel coronavirus disease ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines synergistic antiviral effect of galanthus nivalis agglutinin and nelfinavir against feline coronavirus washington state -nco v.c.i.t. first case of 2019 novel coronavirus in the united states remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro drug treatment options for the 2019-new coronavirus (2019-ncov) effect of interferon alpha and cyclosporine treatment separately and in combination on middle east respiratory syndrome coronavirus (mers-cov) replication in a human in-vitro and ex-vivo culture model potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association human monoclonal antibody combination against sars coronavirus: synergy and coverage of escape mutants experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital convalescent plasma as a potential therapy for covid-19 immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling middle east respiratory syndrome coronavirusencoded orf8b strongly antagonizes ifn-beta promoter activation: its implication for vaccine design induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies lianhuaqingwen exerts anti-viral and antiinflammatory activity against novel coronavirus (sars-cov-2) potent human neutralizing antibodies elicited by sars-cov-2 infection feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol prevention of experimental coronavirus colds with intranasal alpha-2b interferon interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review the proximal origin of sars-cov-2 attenuation of replication by a 29 nucleotide deletion in sars-coronavirus acquired during the early stages of human-to-human transmission publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank dr. ming-hsiang hong for his assistance in preparation of the manuscript. authors' information none. not applicable.availability of data and materials not applicable. consent for publication not applicable. the authors declare that they have no competing interests. key: cord-342052-v4y1xc90 authors: horigan, verity; gale, paul; kosmider, rowena d.; minnis, christopher; snary, emma l.; breed, andrew c.; simons, robin r.l. title: application of a quantitative entry assessment model to compare the relative risk of incursion of zoonotic bat-borne viruses into european union member states date: 2017-10-02 journal: microb risk anal doi: 10.1016/j.mran.2017.09.002 sha: doc_id: 342052 cord_uid: v4y1xc90 this paper presents a quantitative assessment model for the risk of entry of zoonotic bat-borne viruses into the european union (eu). the model considers four routes of introduction: human travel, legal trade of products, live animal imports and illegal import of bushmeat and was applied to five virus outbreak scenarios. two scenarios were considered for zaire ebolavirus (webov, cebov) and other scenarios for hendra virus, marburg virus (marv) and middle east respiratory syndrome coronavirus (mers-cov). the use of the same framework and generic data sources for all eu member states (ms) allows for a relative comparison of the probability of virus introduction and of the importance of the routes of introduction among mss. according to the model webov posed the highest risk of an introduction event within the eu, followed by marv and mers-cov. however, the main route of introduction differed, with webov and mers-cov most likely through human travel and marv through legal trade of foodstuffs. the relative risks to eu mss as entry points also varied between outbreak scenarios, highlighting the heterogeneity in global trade and travel to the eu mss. the model has the capability to allow for a continual updating of the risk estimate using new data as, and when, it becomes available. the model provides an horizon scanning tool for use when available data are limited and, therefore, the absolute risk estimates often have high uncertainty. sensitivity analysis suggested virus prevalence in bats has a large influence on the results; a 90% reduction in prevalence reduced the risk of introduction considerably and resulted in the relative ranking of marv falling below that for mers-cov, due to this parameter disproportionately affecting the risk of introduction from the trade route over human travel. bats are natural reservoir hosts for many viruses which are recognised as serious potential threats to human and/or animal health (calisher et al., 2006) . the bat-borne viruses emerging in the african, asian and australian continents have come to the fore more recently with regards to their threat to human health and pandemic potential. since 2003 there have been a number of large-scale human outbreaks of bat-borne diseases e.g. zaire ebolavirus (ebov) and severe acute respiratory syndrome (sars) in western africa and asia respectively, whilst a significant number of human cases of nipah virus (niv) are reported in bangladesh every year (iedcr, 2014) . pteropid bats are known to be the natural host of hendra virus (hev) (halpin et al., 2000) , a member of the same genus (henipavirus) as nipah virus. since 1994 hev has been responsible for seven human cases in australia, four of which were fatal (smith et al., 2016) . bats have also been linked with marburg virus (marv) , and, more tenuously, with the emerging middle east respiratory syndrome coronavirus (mers-cov) (memish et al., 2013) . within the european union (eu), zoonotic incidents of bat-borne viruses have been restricted to european bat lyssavirus types 1 and 2 which have been responsible for less than 10 human cases since 1977 (fooks et al., 2003) . to date, there have only been a few isolated reports of introduction of bat-borne viruses (e.g. marv, sars, mers-cov) from outside the eu mainly through entry of infected humans (puzelli et al., 2013; desenclos et al., 2004; who, 2008; reuss et al., 2014) . however, these incidents illustrate that introduction can occur and support the need for some level of surveillance activity to assess the probability of when and where further incursions may take place. for most bat-borne zoonotic diseases, primary transmission routes for human infection include direct or indirect contact with bat bodily fluids (leroy et al., 2009; luby et al., 2006) , or via intermediate animal hosts (parashar et al., 2000; hanna et al., 2006) . onward transmission of disease is then possible via human-to-human contact or contact with contaminated fomites or the environment, with nosocomial infections being particularly important in some instances (baron et al., 1983; chowell et al., 2014) . disease introduction into the eu could therefore potentially occur from a number of routes, including human travel, illegal and legal importation of food products and transport of live animals. these routes have previously been associated with incursion of other viruses into the eu. for example, human travel and immigration are thought to be the primary reasons why individual member states (mss) have a high prevalence of the same human immunodeficiency virus (hiv) subtypes as their historical african colonies (faria et al., 2012) . classical rabies has been detected in imported domestic pets (suárez-rodríguez et al., 2013; mcquiston et al., 2008) and avian influenza (h5n1 type a) has been detected in illegal imports of crested hawk eagles from thailand into belgium (van borm et al., 2005) . in the case of trade, illegal importation of food products has been a suggested route of origin for the foot and mouth disease epidemic in the uk in 2001 (defra, 2001) , whilst legal trade in fresh produce, such as fruit and vegetables, has been associated with norovirus (hjertqvist et al., 2006) and hepatitis a outbreaks (dentinger et al., 2001) . virus specific transmission characteristics may influence the relative importance of these potential routes of disease introduction. in terms of government financial resource allocation, it is important to develop methods to assist in efficient targeting of surveillance activities e.g. to inform which pathogen(s) are most likely to enter the eu, where they are most likely to enter and what scenarios would have the most impact with regards to human or animal health and welfare or trade implications. to address these issues, a number of relative risk ranking tools have previously been developed, such as the eu wide discontools (2016) and the uk specific d2r2 (gibbens et al., 2016) . however, these tools are qualitative and are generally based on chosen criteria rather than a defined quantitative assessment. there is, therefore, benefit in a quantitative model that can utilises freely available numerical data from datasets on trade and human travel such as eurostat (2014) and faostat (2014) . to address this need, a generic quantitative risk assessment framework for the entry of bat-borne zoonotic viruses to the eu was developed (simons et al., 2016) , considering the pathways: human travel, live animal movement, legal trade of food products and illegal trade of bushmeat. using current knowledge of virus characteristics such as environmental survival and host incubation periods, the framework was parameterised for niv, to provide an assessment of the relative risks of transmission through the known pathways of introduction into the eu. in this paper the model framework is parameterised for a number of other virus outbreak scenarios (marv, ebov, hev and mers-cov) and the relative probabilities of introduction to eu mss are compared and discussed. the impact of uncertainty in the parameter estimates is also investigated though scenario analyses. the entry assessment model parameterised for niv (simons et al., 2016) , was re-parameterised for five outbreak scenarios (marv, ebov, hev and mers-cov), to compare and assess the relative risk of introduction to the 28 eu mss for these 5 viruses of concern. for ebov two different outbreak scenarios were considered: 1) disease geographically distributed in western africa, where the human cases are on a similar scale to that observed in the 2014 west africa outbreak (webov) (i.e. epidemic situation), 2) disease geographically distributed in central africa, where human outbreaks have previously been relatively limited (cebov) (i.e. non-epidemic situation). it is acknowledged that the link between bats and mers-cov is more tenuous than initially thought when it first emerged (memish et al., 2013) , but the virus was included here to provide an example of a respiratory coronavirus circulating within the middle east area. recent evidence of replication and shedding of mers-cov in experimentally infected jamaican fruit bats (artibeus jamaicensis) (munster et al., 2016) and discovery of closely related mers-like cov (anthony et al., 2017) further support the hypothesis that bats are ancestral reservoirs for mers-cov. as neither live ebov nor mers-cov virus has been isolated from bats a very low prevalence of infection in bats was assumed. different prevalence values for all the viruses were considered in the scenario analysis. the assessment was conducted following the world organisation for animal health (oie) code for import risk analysis (oie, 2004) . under traditional oie guidelines, there are three components of risk assessment: entry, exposure and consequence. this model only considered the entry assessment (i.e. it ceases at the point at which virus is released into the eu) and did not consider subsequent potential exposure of the virus to humans, livestock or wildlife on entry to the eu. the model has been discussed in detail by simons et al. (2016) . briefly, the main outputs of the model were a relative estimate of the annual probability of at least one introduction event into each eu ms, j, p v (j), and an overall estimate of the probability of at least one introduction event for the eu as a whole. this estimate took into account factors such as the probability an individual unit is infected (or contaminated) in the exporting country, the survival of the virus over the duration of the journey, whether or not the animal/human displays clinical signs and the annual volume of products being imported. the model equations for the various routes are described by simons et al. (2016) . the model was coded in the r software package and is deterministic; as such no stochastic variability of specific parameters was considered in the baseline model. the relative risk estimate was derived by combining the probability of at least one introduction event from each of the routes included in the model (fig. 1 ) from all the potential exporting countries to produce an overall probability for each ms: where r is the total number of routes considered for the virus (human travel (r = 1), live animal movement (r = 2), legal trade of 'at risk' products (r = 3), illegal trade of bushmeat (r = 4)) and p r (j) is the probability of at least one introduction event via route r to ms j per year. the average number of years to an introduction event was calculated, y v (j) = 1/p v (j). in addition the 28 eu mss were ranked according to their probability of disease introduction, z v (j) = {1:28} by comparing the average number of years, y v (j), to an introduction event for each route and ms and ranking the mss from 1 to 28 accordingly. this provided an indication of where in the eu an introduction event is more likely. note that the average number of years to an introduction event is based on the input data provided and so does not account for subsequent changes in future years of model factors such as trade patterns or disease prevalence; e.g. for webov it would be assumed that the same number of cases will occur in western africa every year as in 2014. the model considered the four primary routes of introduction based on extensive literature reviews (simons et al., 2014) . while other potential routes exist such as direct exposure from bats through natural migration or accidental exposure via aeroplane strikes, they were not considered here for the viruses of concern. however, as the model framework is adaptable, and eq. (1) is multiplicative with respect to the routes, the choice of routes can be amended and these pathways can be considered in the future, as and when appropriate data become available. the model was parameterised for hev, marv, mers-cov, webov and cebov. the genus ebolavirus includes five species, each with a single member virus (kuhn et al., 2013) . due to the potential differences in parameters for the different viruses only ebola virus (from species zaire ebolavirus) was parameterised here. the model considered the probability of introduction to eu mss from 'exporting countries', that is, those in which virus was strongly expected to be circulating in humans, livestock or wildlife (fig. 2) . this was determined from peer-reviewed publications of where the viruses had been reported. for livestock and wildlife, including bats, only positive test results for isolation of live virus or detection of viral rna were considered (active bat infection). countries that had reported positive seroprevalence or those which had reported a human case known to have arisen from recent travel to another country were not considered as an 'exporting country'. the full details of the generic model parameters are presented in simons et al. (2016) . in this section an overview of the data sources used to parameterise each route is presented. human travel: passenger travel data from exporting countries to eu mss were obtained from the eurostat dataset aviapaexcc (eurostat, 2014) . for ebov, marv and hev for which outbreaks are sporadic, n hinf (k) was estimated using the average number of cases per outbreak over a 15 year period. this value was assumed by the authors to encompass all relevant historical data. however, as human cases of mers-cov have been reported regularly since march 2012, n hinf (k) was calculated by dividing the number of reported cases by the number of reporting years assuming a constant rate per year. to account for differences in prevalence between passenger types e.g. business, visiting family, and tourist etc., the baseline prevalence of infection in the exporting country was weighted by the average passenger duration of stay (days) in the exporting country. the ratio of passenger types was assumed to be the same for each exporting country. the sub-clinically infected population was estimated by multiplying the prevalence of infection in passenger type i, θ(i,k) by the incubation period of the virus. passenger detail such as healthcare employees potentially exposed to infected patients or eco-tourists with the intention of visiting bat caves was not accounted for here although it is acknowledged that these factors could influence the risk outcome as has been documented (who, 2008) . legal trade: to determine whether a product was considered contaminated, the concentration of virus on the product on arrival to an eu ms was estimated where c min is a threshold viral load upon arrival at the eu ms, below which the product was considered not to be contaminated. note, that this value was set to 1 log 10 tcid 50 for all viruses as a worst case scenario. the model considered the prevalence of contamination in at risk raw products (see table 1 for definition), the initial concentration of virus on a raw product in the exporting country and any reduction in viral load between initial contamination of the raw product and arrival in the eu ms, including the effect of processing. the default estimate for the prevalence of contamination in raw products, pgraw(k), was based on the estimated prevalence of active virus v. horigan et al. microbial risk analysis 7 (2017) 8-28 shedding in bats, pbinf(k), the contact rate of the bat with the product, pbcontact(k) and seasonality of virus shedding, i.e. the proportion of the year that bats can shed the virus. data on volume of trade from exporting countries to eu mss were obtained from faostat (2014). bushmeat: in this model bushmeat was assumed to enter an eu ms via aircraft passenger luggage. the volume of contaminated bushmeat entering the eu was estimated by combining the probability of a passenger of type i bringing in bushmeat from exporting country k, p bm (i,j,k), and the probability that a consignment of bushmeat was contaminated. the actual number of bushmeat consignments entering the eu from country k was estimated based on the number of bushmeat consignments seized in the eu ms, nseized(i,j,k), and an under-reporting factor accounting for the proportion of passengers luggage that were searched. the under-reporting factor was estimated to be 0.5% based on literature (falk et al., 2013) and assuming targeted testing of passengers occurs (simons et al., 2016) .the model did not account for any virus reduction that may occur from processing bushmeat such as smoking or salting and assumed that the possibility of luggage being searched for bushmeat was the same for each exporting country. live animals: this route considered the number of animals of species s arriving from an exporting country k in one year and the prevalence of live animal infection of species s in the exporting country to give the probability that at least one infected animal entered a ms. numbers of live animal exports from exporting countries to eu mss were obtained from the trans-european trade control and expert system (traces) database which provides data on the number of animals that are brought into the eu and issued with a common veterinary entry document (traces, 2014) . virus specific parameter estimates used in the model are given in table 1 . estimates for niv are also provided for comparison (simons et al., 2016) . further information (including references) on these estimates is provided in appendix a. the model developed here is deterministic for ease of use in an outbreak situation where rapid parameterisation and data availability for all eu mss are key requirements. uncertainty and variability in the model were previously considered for niv by implementing a series of analyses using alternative parameter values (simons et al., 2016) . it was found that while some scenarios had an impact on the absolute values of probability of introduction of niv, the relative rankings, of both routes and mss were more robust. however, the estimate for the prevalence of niv in bats had considerable impact on the average number of years to an eu introduction of niv relative to the baseline model and much lower estimates for this prevalence were the only scenarios to have an impact on the relative ranking between the routes. given this, and the complexity involved in assessing multiple uncertainties between multiple scenarios, the scenarios considered here were a 90% and 99% reduction in the virus prevalence in bats as these reductions both had considerable impact in the previous model for niv; smaller reductions in virus prevalence had little impact. at the eu level the probability of viral introduction was ranked highest for webov with an overall average prediction of at least one introduction event occurring in one year (table 2) , primarily via human travel and associated illegal importation of bushmeat. in relative terms, and given the uncertainties in the absolute value estimates, marv and mers-cov were of a comparable risk whilst the overall probability of introduction was lowest for hev. a 90% or 99% reduction in virus prevalence in the exporting country bat population only affected the risk estimates for the legal trade and bushmeat routes. consequently, for ebov and mers-cov, which had a relatively high probability of introduction from human travel, the decrease in risk from trade and bushmeat was not sufficient to affect the overall probability of disease introduction. for hev and niv, however, where the legal trade and bushmeat routes posed the highest risk in the baseline model the decrease in overall risk was substantial. human travel replaced legal trade and bushmeat as the route with the highest associated probability for hev, marv and niv introduction when the virus prevalence in bats was reduced by 99% (90% for marv). the number of imports of live animals was low for all exporting countries resulting in a relatively low probability of introduction via this route for all viruses (table 2) . only dromedary camels (camelus dromedaries) have been shown to be a risk factor for mers-cov transmission (azhar et al., 2014) , but as there is no legal trade of live camelids to the eu from countries reporting cases of mers-cov, the risk from live animals was considered to be negligible. within the eu, individual mss demonstrated different relative probabilities for the various pathogens when the probabilities for all the routes of introduction were combined for each ms (fig. 3) . the probability of introduction for mers-cov was quite high across most of the eu mss, but for other viruses it was mainly focussed in a few mss, usually in western europe with the probability of introduction for mss from eastern europe and scandinavia generally being much lower (fig. 3) . overall, the probability of introduction was highest for individual viruses in those mss with strong historical links to relevant exporting countries, e.g. the united kingdom (uk) for niv and france for cebov. such links usually correspond to a relatively large volume of human travel or legal trade movements between the countries. it should be noted that this analysis does not consider movement within the eu after the initial entry. the countries are ranked according to the probability of introduction for each virus in table 3 . overall there was a relatively wide variation in the relative ranking of many of the mss between the different viruses. different distributions of risk scores were observed between routes but considering the relative ranking of the mss (1-28), the uk, france, germany and the netherlands generally have the highest probability of introduction for all viruses considered here. the entry assessment described here shows the potential for application of a quantitative model framework for any pathogens, using zoonotic bat-borne viruses as an example. although a scarcity of data for virus specific parameters resulted in a high degree of uncertainty in the absolute risk values presented, the main strengths of this model lie in the estimates of relative risks between routes of entry and those mss which are at greater risk of virus introduction. the model has the capability to allow for a continual updating of the risk estimate using new research data as, and when, it becomes available. any increase in the model risk estimate output would allow the stakeholder to consider employing suitable risk reduction strategies or heightened surveillance providing a rapid and cost-effective response. of particular value was the model's ability to illustrate the relative importance of the different routes of entry between viruses; legal trade of foodstuffs was more important for hev, niv and marv while human travel was more important for mers-cov and both ebov scenarios. these differences could be partly attributed to the virus specific parameters. for example rapid decay of mers-cov influenced the relative risk of the transmission pathways; the half-life of mers-cov is very short compared to the other viruses (48 min at pre-harvest temperatures (van doremalen et al., 2013) so it is unlikely to persist in high numbers on any produce imported via legal trade or in contaminated bushmeat. the probability of introduction to the eu via the pathways under consideration here varies across the eu at ms level; the uk, france, germany and the netherlands often had the highest probability of table 2 the expected number of years to eu entry for different viruses, by individual route and all routes combined for the baseline model. results for 90% and 99% reduction in virus prevalence in bats are shown in brackets respectively for legal trade, bushmeat and all routes (the model assumes no effect on human travel and live animal routes). human travel introduction for all viruses considered. in general those countries which ranked the highest, with regard to probability of introduction (table 3) corresponded to those with the highest population and the highest 'disposable income' (calculated as gross domestic product (gdp) derived from purchasing power parity) (see appendix b). other contributory factors could include immigration population densities and trade partner characteristics, both of which frequently have a historical basis. the netherlands was an exception to this in that it ranked highly in the probability of virus introduction yet only 8th for population density and 7th for gdp (see appendix b). one explanation for this could be that the netherlands is serving as a hub for travellers and trade entering the eu and that a reasonable proportion entering the netherlands are going onto other european countries. it is also possible that more dutch people, compared to other eu mss, travel to countries with these viruses. data from uganda suggest that in 2012 the netherlands was the 15th most popular country of origin for tourist arrivals, the only european countries with more arrivals were the united kingdom and germany, but dutch tourists represented a higher proportion of their population (republic of uganda, 2013). freely available statistical data on trade and human travel for western europe were, in general, more complete than for eastern european mss. data for countries such as estonia, latvia and lithuania were lacking for some routes resulting in a low ranking for these countries which may not be a true reflection of the actual risk. it is difficult to determine whether this is a true data gap or if the route genuinely has a low probability of introduction for these countries. this was a particular problem for cebov where twelve countries were lacking data for specific routes and, therefore, equally ranked as 22.5. the generic parameters for which eu wide datasets exist have a relatively high degree of completeness although there is a concern that potentially high risk low volume trade products e.g. camel milk may be under-recorded therefore underestimating the risk via these trade products. virus specific parameters depend more upon focussed research studies and peer reviewed literature and rely upon detection of pathogens in reporting countries. uncertainty in these virus parameters, in particular, the prevalence of infection in bats in the exporting country, and viral persistence during processing and storage may limit the application of the model by introducing considerable uncertainty. the sensitivity analysis of virus prevalence in bats demonstrated that the results for the relative importance of the routes for ebov and mers were quite robust with human travel remaining the route with the highest probability of introduction. with regards to hev, niv and marv, however the sensitivity to the variation in prevalence indicates that further data for this particular parameter would strengthen the model results; this is particularly true of marv where a 90% reduction in virus prevalence changed the risk ranking order of the routes of introduction. note that this analysis is to consider the uncertainty about the true prevalence in bats, as such it has no impact on the parameterisation of the human prevalence, which is based on human outbreak data. it should be noted that the parameterisation of this model uses the best available data at the current time. some parameters are subject to high uncertainty and the probability of introduction of different viruses will be dynamic, changing over time if a virus spreads amongst different animal species populations or if new human outbreaks occur. simons et al. previously demonstrated that changes in the exporting country (e.g. if china were to get niv in the future) or 'at risk' product types can have a large effect on the model outputs (simons et al., 2016) . we have demonstrated here that changes in the virus prevalence in bats in the exporting country can have an impact on the average number of years to eu entry for the different viruses and on the relative ranking of the individual routes of entry. we have also highlighted the differences in probabilities for two ebola scenarios; a relatively small non-epidemic human outbreak and a large epidemic scale outbreak. it is acknowledged that the viruses considered here could have differing sensitivity to stochastic variability of specific parameters given the complex dynamics between the routes and viruses. alternative scenarios could, therefore, be considered in the future. all risk pathways were given equal weight within the model as the model predicts probability of introduction not risk of human/animal exposure and consequence as stated in the oie risk assessment (oie, 2004) . for example, the model results suggest that the legal trade (fruit) route has a high probability of introduction for hev, although human infection from consumption of contaminated fruit is not a proven transmission route for this virus. this route was considered in the model based on the knowledge that fruit bats are known to consume raw fruit in orchards (eby and lunney, 2002) and date palm sap is a known route of transmission for niv (another henipavirus) (luby et al., 2006; khan et al., 2012; nahar et al., 2014) . similarly, there is currently no evidence of human-to-human transmission of hev but as this has occurred for the bangladesh strain of niv (gurley et al., 2007) it is plausible that this could occur with or without mutation and adaptation of currently identified strains. real-time application of the model would allow for removal or addition of pathways if future scientific work provides suitable input data or if trade patterns between third countries and the eu alter. thus, all pathways were assessed for completeness according to the dogma 'absence of evidence is not evidence of absence'. whilst eu wide trade controls are implicitly accounted for within the model parameters, risk mitigation procedures put in place by individual mss such as targeted sampling are not taken into account. it is possible that there have already been introduction events of the diseases under consideration here within the eu, but these have remained undetected due to lack of subsequent human/animal infection and/or onward transmission within the individual ms. for example, although the importation of mers-cov cases to the eu remains possible, an ecdc risk assessment determined that the risk of sustained human-tohuman transmission is low (ecdc, 2015a). however, the outbreak of mers-cov in south korea demonstrated that the potential exists for a serious risk of onward human spread, with >185 cases arising from the importation of 1 human index case (su et al., 2015) . validation of such a model presented here is difficult as there are few independent resources for which to compare the results. however, it is of relevance that the five mss suggested to have the highest probability of introduction of mers-cov by the model (germany, uk, italy, france and the netherlands) have already had imported human cases of this pathogen (ecdc, 2014) . the model results are also consistent with other reports which predict more imported cases of mers-cov to arrive into the eu (ecdc, 2014; who, 2014a; bialek et al., 2014; poletto et al., 2014) . all cases reported outside of the middle east have had a recent travel history to the middle east or contact with a case that had travelled from this region (su et al., 2015) . this is in line with the highest probability of introduction for mers-cov predicted by this model to be via human travel (table 2) . overall, the approach developed here provides a high-level horizon scanning tool for the probability of introduction of bat-borne zoonotic viruses into the eu. the virus scenario with the highest probability was the webov scenario with an overall average prediction of just under one introduction event per year, primarily via human travel. due to the wide scope of the model, which necessitated using global datasets sometimes with incomplete data, there was a high degree of uncertainty in the absolute risk values presented. a general lack of data on virus specific parameters also contributed to this uncertainty. thus, the main strengths of this model lie in the comparison of the relative risks between viruses and routes of entry. whilst there have been several risk assessments carried out for the introduction of individual pathogens into the eu (rolin et al., 2013; durand et al., 2013; mur et al., 2014; snary et al., 2012) this model was able to assess a range of viruses and could be adapted for other pathogens, as it has the advantage of easy access to a number of relevant databases. the model also allows for a continual updating of the risk estimate enabling the stakeholder to respond in a rapid and risk appropriate manner, for example, by implementing risk-based surveillance and control strategies. (table a1 ). the beginning of 2014 saw the largest recorded outbreak of ebola emerge in western africa with sierra leone, liberia and guinea being the countries most affected. due to the potential differences in parameters for the different viruses only ebola virus (from species zaire ebolavirus), ebov, is parameterised here. the number of cases per outbreak estimated over a 15 year period was used for n hinf (k).this is estimated at 16,125 for west africa, 75 for the drc, 65 for gabon and 79 for roc. time to clinical signs, t ip (k). the recognised incubation period for ebov disease is 2-21 days (del rio et al., 2014) . a review of epidemiological parameters from ebola outbreaks including incubation period has recently been published (van kerkhove et al., 2015) . using a sum of all the estimated means divided by the number of studies the time to clinical signs used is 8.82 for ebov only (table a2) . bat infection prevalence in exporting country, p binf (k). evidence of ebov infection in bats (epomops franqueti, hypsignathus monstrosus, and myonycteris torquata) is currently based on seroprevalence and presence of viral rna. table a3 shows the relevant papers that have attempted isolation of ebov. combined, 1033 bats were tested with 0 testing positive for viral shedding of ebola. as such the prevalence of ebov in bats for this model was assumed to be 0.1% . product types (l): the potential for ebov to act as a foodborne pathogen has been addressed (bausch, 2011) . parallels have been drawn between the emergence of the reston strain of ebov in domestic pigs in the philippines in 2008 and the 1998-1999 outbreak of niv in malaysia and singapore. both imported fruit products and live pigs/pig products were therefore considered as potential risk factors for the introduction of ebov into the eu. proportion of the year bats may shed active ebov virus, p season (k). seasonal climate has been found to be associated with human ebov outbreaks (ng et al., 2014) . increased great ape mortality has frequently been reported during the dry seasons of july and december (pourrut et al., 2007) and the biannual birthing periods of the bat species identified as potential natural reservoirs also occur during the dry seasons when fruit is scarce (langevin and barclay, 1990) . in the absence of more definitive data the proportion of the year in which bats are presumed to be infectious is estimated as 0.5. initial viral load on raw product, c 0 (x). the initial concentration of ebov on the raw product is assumed to be equivalent to that shed by bats. as there have been no successful virus isolation attempts from bats, values are extrapolated from experimental evidence. nasal washes and oral and rectal swabs from pigs challenged via mucosal exposure with 1 × 10 6 pfu of the zaire strain of ebov had infectious titres ranging from 1 × 10 2 to 1 × 10 3 tcid 50 /ml (kobinger et al., 2011) . infectivity of mucosal wash fluids obtained from monkeys experimentally infected with the reston strain of ebov ranged from <0.7 log 10 pfu/ml at initial infection to a maximum of 2.9 log 10 pfu/ml in terminal animals (jahrling et al., 1996) . based on these data the initial viral load used here follows a log normal distribution with mean 3 log 10 tcid 50 /ml (variance = 1 log 10 tcid 50 /ml in the absence of any other data). virus decay in the environment and during transport, c hlenv (j,k), c hltrans (j,k). in a study on the survival of filoviruses in liquids and on solid substrates the half-life of ebov was calculated to range from 6.6 to 11.5 days at +4°c in tissue culture media and sera respectively and 3 days at room temperature (piercy et al., 2010) . no virus could be recovered from any solid substrate stored at room temperature but at +4°c the virus had a half-life of ∼5.5 days on glass and ∼7 days on plastic substrates. from the data available, a mean half-life of 7 days (168 h) is used as an estimate for virus reduction during transport at +4°c and virus reduction pre-harvesting is estimated at 3 days (72 h) using liquid media data. minimum viral load to consider product contaminated in eu ms, c min . experimental infection of bats with the zaire strain of ebov was achieved with an inoculation dose of 10 4.6 ffu (fluorescent focus forming units) (swanepoel et al., 1996) . non-human primates (nhp) have been shown to be uniformly susceptible to intramuscular inoculation of 1000 pfu of the zaire strain of ebov (geisbert et al., 2003; smith and wang, 2013) . however, doses as low as 50 pfu were sufficient to cause infection in rhesus macaques (kortepeter et al., 2011) and 100 pfu of a guinea pig adapted strain of the virus was used to experimentally infect baboons (ignatiev et al., 2000) . johnson and colleagues (johnson et al., 1995) reported using a dose of 400 pfus to infect rhesus monkeys with ebov by inhalation. in another experiment, three out of four orally inoculated rhesus monkeys were infected when using a dose of 5.2 log 10 of ebov (jaax et al., 1995) . rhesus macaques were aerosol challenged with calculated doses between 743 and 274,000 pfu of ebov delivered as a small-particle aerosol (twenhafel et al., 2013) whilst a lethal dose of 100 ld 50 in african green monkeys and 20-50 ld 50 in baboons has been demonstrated (ryabchikova et al., 1999) . as a worst case scenario it is assumed that c min = 1 log 10 tcid 50 . species of animal, s. detection of ebov by serology and pcr in animals has been recently summarised by pigott et al. (2014) . dogs and pigs are, so far, the only domestic animals identified as species that can be infected with ebov (weingartl et al., 2013; allela et al., 2005) . although dogs can be asymptomatically infected, they may excrete infectious viral particles in urine, faeces, and saliva for a short period before virus clearance. pigs have been shown to be susceptible to both the reston and zaire strains of ebola. conversely, the zaire strain is also capable of transmission from pigs to cynomolgus macaques without direct contact (weingartl et al., 2012) . pigs, challenged with ebov via mucosal exposure, replicated the virus to high titres mainly in the respiratory tract with shedding observed from oronasal mucosa up to 14 days post-exposure. transmission to cohabiting naïve pigs was also observed (kobinger et al., 2011) . animal species in which evidence of natural/experimental ebov infection has been demonstrated and considered as potentially capable of introducing ebov into the eu include: pig, domestic dog, lord derby's scaly-tailed squirrel, duiker, non-human primates, small rodents and the shrew (morvan et al., 1999 (falk et al., 2013 , chaber et al., 2010 . a study on the species of bushmeat items confiscated at us ports of entry between 2005 and 2010, suggested that bats accounted for around 1.5% of all bushmeat (bair-brake et al., 2013) . thus, in the absence of other information, we assume that 1.5% of bushmeat is bats. the same study suggested that around 6% of bushmeat is derived from nhp and 50% from rodents. rodents and blue duikers made up 75% of the total number of bushmeat carcasses detected at paris roissy-charles de gaulle airport from sub-saharan africa (chaber et al., 2010) . despite being herbivorous, duikers have been known to eat the flesh of decomposing carcasses and could become infected with ebov via this transmission route (rouquet et al., 2005) . number of human infection in exporting country, n hinf (k). human cases of hev have been restricted to the state of queensland in australia (table a4) . hendra virus was first described in 1994 since which time 7 human infections have occurred in 45 separate outbreaks with a 57% case fatality rate. no human cases have been reported for the last 6 years (since 2009) which is attributed to horse keepers/vets greater awareness of the disease and an equine vaccination programme. the number of human infections in exporting country k in one year is assumed to be 1. time to clinical signs, t ip (k). estimates of average time to clinical signs in humans can be seen in table a5 . taking an average of all the estimates available, a value of 12.8 days was used for the average time to clinical symptoms of hev. bat infection prevalence in exporting country, p binf (k). in 2011 hev rna was detected in up to two-thirds of pooled-urine samples from bats near hev cases in horses (plowright et al., 2015) . variable virus excretion has been reported in urine, with prevalence in pooled urine samples collected under roosting flying-foxes ranging from 3-33% in the one-in-four sampling events that yielded positive results. subsequent studies have detected excretion spikes as high as 60% on rare occasions (field et al., 2011) . based on documented evidence of virus isolation (table a6 ) the prevalence of hev in bats was assumed to be 0.47%. product types, l. whilst there is currently no evidence of human hev infection as a result of contaminated fruit consumption it is plausible that this could occur with or without mutation and adaptation of currently identified strains. this framework models only the possibility that fruit products contaminated with hev could enter the eu. all products in the faostat database recorded under section 8 -fruits and derived products, are therefore included. proportion of the year bats may shed active hev virus, p season (k). flying foxes appear to excrete hev at any time of year and spillover in horses can occur in any month but the majority of equine cases (94 confirmed or possible cases as of december 2015 (smith et al., 2016) ) have occurred from june to september suggesting that there is a greater risk of infection at this time (field et al., 2011) . there was an initial coincidence of hev outbreaks with birthing seasons of australian fruit bat species and the isolation of hev from the uterine fluid and aborted foetus of a p. poliocephalus bat indicated that this may be a significant route of infection for horses (fogarty et al., 2008) . however, there now appears to be a temporal clustering of spillovers during the winter period with 35/51 spillovers to june 2014 occurring in june, july and august (goldspink et al., 2015) . the proportion of the year that bats are assumed to shed active virus is therefore estimated to be ∼4 months or 0.33 of 1 year. initial viral load on raw product, c 0 (x): the initial concentration of hev on the raw product is assumed to be equivalent to that shed by bats. virus has been detected in the urine, faeces, saliva and birthing fluids of experimentally infected flying-foxes (williamson et al., 2000 (williamson et al., , 1998 halpin et al., 2011) , and in the urine, uterine fluid and foetal tissue of naturally infected free-living flying-foxes (halpin et al., 2000; field et al., 2011) . whilst these studies report on prevalence of virus there are no data on quantification of virus in bats available. however, in experimental inoculations of pigs virus titres of 4.6 log 10 tcid 50 /ml were found in nasal swab samples (li et al., 2010a) . based on these data the initial viral load on the raw product follows a log normal distribution with mean 4.6 log 10 tcid 50 /ml (variance = 1 log 10 tcid 50 /ml in the absence of any other data). virus decay in the environment and during transport, c env (j,k), c trans (j,k): using an exponential decay model the half-life of hendra virus under laboratory conditions was calculated to be 1.85 min, 50.2 and 308 h. for 56, 22 and 4°c respectively (scanlan et al., 2014) . more recent modelling predictions using the same data calculated half-lives of 3.5 s, 2.9 h and 268 h using a weibull distribution (martin et al., 2015) . when incubated in p. alecto urine (ph ∼7) hev had a half-life of 19 h. at 22°c and 3 h at 37°c (fogarty et al., 2008) . the half-life in mango flesh ranged from 0.3 h. at ph3 to 22 h. at ph5 at 22°c (fogarty et al., 2008) . the calculation of 2.9 h at 22°c was assumed to be the most accurate for this scenario and this value was therefore used for virus decay in the environment; a value of 268 h was used for decay during transport consistent with the modelling prediction using weibull distribution. minimum viral load to consider product contaminated in eu ms, c min uniform disease occurred with an inoculation value of 3.4 × 10 6 pfu of hev for guinea pigs and 6.6 × 10 7 pfu for landrace pigs whereas an inoculate dose of 2 × 10 7 pfu in minipigs did not cause uniform fatality (li et al., 2010b) . horses in a vaccine efficacy study were challenged oronasally with 2 × 10 6 tcid 50 in experimental infection with hendra (marsh et al., 2011 , middleton et al., 2014 while cats orally challenged with 5 × 10 3 tcid 50 succumbed to disease after a 9 day incubation period (hooper et al., 1997) . as a worst case scenario it is assumed that c min = 1 log 10 tcid 50 . species of animal, s. horses are moved internationally for competition, breeding, slaughter and as companion animals. all horses being permanently exported to europe from australia must complete a 30 day pre-export isolation at an approved quarantine stable in australia. in horses, the incubation period is estimated to be 5-16 days although the incubation period in one horse may have been 31 days (doh, 2012) . evidence suggests that horses have the potential to excrete virus in nasal secretions up to 2 days before showing signs of infection (kung et al., 2013) and should be considered as potentially infectious from 72 h prior to onset of clinical signs of disease. virus is recoverable from infected horse's urine and saliva for at least 21 days. transmission of hev or niv via semen has not been investigated, although the likelihood of a stallion being infected, clinically healthy and having semen collected for export is considered remote (maf, 2000) . experimental inoculation of pigs has indicated that they could be a potential host for hev (li et al., 2010b) . in a serological survey of 100 swine herds in queensland, australia (black et al., 2001) no hev antibodies were found in the 500 tested serum samples. two dogs have tested positive for hev antibodies on properties where horses developed hev infection in july 2011 and july 2013. although the source of exposure for the dogs cannot be definitively ascertained, horse-to-dog transmission is the most plausible scenario. experimental hendra virus infections have been performed in horses, cats, ferrets, hamsters, african green monkeys and guinea pigs all of which developed fatal diseases. cats from australia are prohibited from entering the uk unless they are accompanied by a certificate from the australian veterinary authorities confirming that they had not been on a holding where hev has been confirmed during the 60 days prior to export. pigs, dogs, cats and horses are all considered in the model. species of bushmeat, p bmsp (s). bushmeat is part of the traditional diet of indigenous australian people whilst australian game meat plays a part in (goldspink et al., 2015) a samples are the same reported in different articles. (kissling et al., 1970 (kissling et al., ) 1975 south africa 3 1 unknown -possibly from zimbabwe visited sinoia caves 8-9 days prior to onset of symptoms yes (gear et al., 1975) 1980 kenya 2 1 kitum cave (<70 miles from lake kyoga where 1967 monkeys originated) yes (smith et al., 1982) 1987 kenya 1 1 kitum cave (<70 miles from lake kyoga where 1967 monkeys from) no (johnson et al., 1996) 1998 modern australian cuisine. some of the animals that were traditionally hunted for meat are now endangered and protected including the flying foxes (in new south wales and queensland) although aboriginal people are excluded from protection laws and have the legal right to hunt native animals for their own consumption. a study reported on the species of bushmeat items confiscated at us ports of entry between 2005 and 2010, suggested that bats accounted for around 1.5% of all bushmeat (bair-brake et al., 2013) . thus, in the absence of other information, we assume that 1.5% of bushmeat is bats. a.3.1. human travelnumber of human infections in exporting country, n hinf (k). since 1980 marv outbreaks have originated in kenya, the drc, angola and uganda (table a7) . time to clinical signs, t ip (k). data from secondary cases of marv in kenya place the median incubation period at 9 days. however the incubation period in index cases following exposure to a reservoir source is calculated as having a mean of 9.8 days (timen et al., 2009; amman et al., 2012) with a range of 3-15 days. based on documented cases of exposure and subsequent infection marv was calculated to have an incubation period ranging from 3 to 21 days (typically 5-10 days) the range being modulated by factors such as infectious dose and possibly by route of infection (brauburger et al., 2012) . using data available from historical marburg cases with precise exposure dates (n = 18), the median incubation rate for marburg was calculated by pavlin to be 7 days with no significant difference between primary and secondary cases (pavlin, 2014) . the value of 7 days is used in the model. bat infection prevalence in exporting country, p binf (k). the estimate for the prevalence of marv in bats is based on published information from peer reviewed publications on the isolation of active marv (table a8 ). the number of bats actively shedding virus is taken as a percentage of the entire pool tested as it is assumed that if no rna is detectable then virus isolation would be highly unlikely as a direct correlation between rna levels and the ability to isolate virus has been demonstrated (towner et al., 2009) . due to the uncertainty surrounding this parameter, the prevalence of active virus shedding in bats was assumed to be p binfw (k) = 0.29% as a worst case scenario. product types, l. marv has been isolated from rousettus aegypticus, a fruit bat which is known to discard hard particles in their food at foraging sites (herzig-straschil and robinson, 1978) . they are known to consume various fruit crops produced for human consumption such as date, fig, apricot and peach. a recent paper succeeded in isolating marv from both oral and rectal secretions of r. aegyptiacus experimentally infected with virus demonstrating potential avenues for viral shedding (amman et al., 2015) . thus all products in the faostat database recorded under section 8 -fruits and derived products, are included. proportion of the year bats may shed active marv virus, p season (k). retrospective analysis of historical human infections found there was a temporal clustering of infections coinciding with the seasonal pulses in virus circulation in r. aegyptiacus covering 6 months of the year in total (amman et al., 2012) . the observation of these distinct pulses of virus infection in older juvenile bats appears to coincide with the peak biannual birthing seasons. thus the proportion of the year that bats are presumed to be infectious with marv is 0.5. initial viral load on raw product, c 0 (x): the initial concentration of marv on the raw product is assumed to be equivalent to that shed by bats. successful isolation of marv roughly correlated with tissue samples that had rt-pcr ct values of 30 or less (>2000 tcid 50 /ml) (amman et al., 2012) . the highest rna level, measured using ct values, corresponded to an approximate infectious titre of 1 × 10 5 pfu/ml (towner et al., 2009) . measurements of concentrations of marv in experimentally microbial risk analysis 7 (2017) 8-28 infected guinea pig saliva, urine and faeces showed a virus concentration of 2.3-3.3 log ld 50 (median lethal dose) (chupurnova et al., 2000) . the ld 50 was calculated to be 5 × 10 −2 tcid 50 of virus for wild type mice (qiu et al., 2014) . however, it should be noted that this will be a rodent adapted strain of the virus. the virus has also been found to be excreted at high levels of up to 10 6 guinea pig infectious doses in urine of experimentally challenged monkeys (simpson, 1969) . marburg virus was isolated from oral secretions of r. aegyptiacus experimentally infected with virus from a naturally infected bat of the same species (amman et al., 2015) . viral loads were measured by qrt-pcr analysis of viral rna and reported as mean tcid 50 equivalents. marburg virus positive oral swabs were obtained on day 4-14 post infection with highest viral loads detected on day 8 (1.32 × 10 3 tcid 50 /ml equivalents) and cleared from oral secretions by day 14. based on these data the initial viral load on the raw product follows a log normal distribution with mean 3.12 log 10 tcid 50 /ml (variance = 1 log 10 tcid 50 /ml in the absence of any other data). virus decay in the environment and during transport, c env (j,k), c trans (j,k): in a study on the survival of filoviruses in liquids and on solid substrates the half-life of marburg virus in liquid media was calculated to be between 5.1 and 6.6 days at +4°c and ∼3 days at room temperature (piercy et al., 2010) . no virus could be recovered from any solid substrate stored at room temperature but at +4°c the virus had a half-life of ∼4.5-5.5 days on glass and ∼4.5-10 days on plastic substrates depending on the media in question. from the data available, a mean half-life of 6 days (144 h) is used as an estimate for virus reduction during transport at + 4°c and virus reduction pre-harvesting is estimated at 3 days (72 h) using liquid media data. minimum viral load to consider product contaminated in eu ms, c min : evidence of experimental infection in rodents is not considered here as these models generally use adapted viruses obtained through sequential passage in the rodent species as the wild-type virus does not cause uniform lethality (bray, 2001) . a single intramuscular injection of a common marmoset with as little as 10 pfu of virus has resulted in fatal haemorrhagic disease (carrion et al., 2011) whilst 1000 pfu has proven to be a uniformly lethal dose of virus (thi et al., 2014; geisbert et al., 2007; hensley et al., 2011; smith et al., 2013) . however doses as low as 2-14 pfu and 99-705 pfu have been reported as causing disease by viral inhalation. the doses were equally fatal but symptoms were delayed by one day in the lower dose group (alves et al., 2010) . when bats were experimentally infected by subcutaneous inoculation with a dose of 10 4 tcid 50 marv there was evidence of infection in all bats although no clinical signs were observed. as a worst case scenario it is assumed that c min = 1 log 10 tcid 50 . species of animals (s): all species of nhps were considered as susceptible to marv due to previous research (simpson, 1969 (chaber et al., 2010; falk et al., 2013) . a study reported on the species of bushmeat items confiscated at us ports of entry between 2005 and 2010, suggested that bats accounted for around 1.5% of all bushmeat (bair-brake et al., 2013) . thus, in the absence of other information, we assume that 1.5% of bushmeat is bats. the same study suggested that around 6% of bushmeat is derived from nhp which is of relevance here as marv has been previously transmitted from nhp to humans. a.4.1. human travel number of human infections in exporting country, n hinf (k). sporadic cases have occurred in europe and the rest of the world but the index case of these outbreaks has always recently travelled to the middle east. a large number of cases reported in saudi arabia have been nosocomial; the recent outbreak of mers-cov in south korea illustrates the role the hospital environment can play in the spread of pathogens (park et al., 2015) . the number of human infections (table a9) were taken as the total number (as at november 18th 2016) divided by the period of time over which the infections have been recorded. time to clinical signs, t ip (k). the incubation period of mers-cov has been estimated using data from exposure of secondary cases to the index case in a hospital outbreak (assiri et al., 2013) and using traveller-related clusters (cauchemez et al., 2014) . the median incubation period for confirmed cases in the hospital outbreak was 5.2 days (95% ci 1.9 -14.7), and 5.5 days (95% ci 3.6-10.2) for travel related clusters in the uk, france, italy and tunisia (fisher-hoch, 2005) . a figure of 5.3 days is used in this model. bat infection prevalence in exporting country, p binf (k). growing serological and molecular evidence suggests that the dromedary camel (camelus dromedarius) is an intermediate species for mers-cov (ecdc, 2015b) but the virus is hypothesised to have originated from bats. a mers-cov sequence identical to that of the virus isolated from an index-case patient was detected in a faecal pellet from a taphozous perforatus bat sample (table a10 ) collected near the home of an index case in an area which is an important date palm production area in saudi arabia (memish et al., 2013) . as yet no evidence has been found of actual infection in chiropteran populations. product types, l. some human cases of mers-cov infection have had gastrointestinal symptoms including diarrhoea and vomiting. coronaviruses have been demonstrated to survive on fresh produce over a period of days (yepiz-gomez et al., 2013; mullis et al., 2012) . thus, it is possible that fruit and vegetables contaminated with coronavirus may be potential vehicles for transmission to humans. infected bats (or other animals) contaminating raw fruit via saliva or urine whilst foraging for food is considered a risk and thus all products in the faostat database recorded under section 8 -fruits and derived products, are included. other legal imports considered as potential risk commodities are camel meat and camel milk. mers-cov rna and antibodies have been detected in milk collected from actively shedding dromedary camels in qatar providing evidence of the potential for virus transmission (reusken et al., 2014a) . camel milk imports from uae to the uk began in 2014 and are transported by plane as a chilled product. the operation is stated to have obtained iso 2000 certification for both the farm and dairy processing facilities to fulfil the eu import requirements. there have been 6 consignments in 2014 comprising of: proportion of the year bats may shed active mers-cov virus, p season (k). although there is no evidence of viral shedding in bats to observe any seasonal fluctuations, analysis of human infections since the first reporting of mers-cov found there was a temporal clustering of infections with a sharp rise in the number of cases in april and may and a smaller peak in august-september. the birthing period for taphozous perforates is in april and may. in line with the peak in human infections in saudi arabia the proportion of the year that the source is presumed to be infectious with mers-cov is 4 months or 0.33. initial viral load on raw product, c 0 (x): experimental infection of common marmosets with 5.2 × 10 6 tcid 50 mers-cov exhibited viral loads of ∼6 log 10 tcid 50 eq/g in nasal mucosa 6 days after challenge (falzarano et al., 2014) . respiratory samples gave higher values than blood or other tissues. camels inoculated with doses of 10 7 tcid 50 gave viral titre of maximum of 6 log 10 tcid 50 eq/ml of nasal swab samples (adney et al., 2014) . based on these data the initial viral load on the raw product follows a log normal distribution with mean 5 log 10 tcid 50 eq/ml (variance = 1 log 10 tcid 50 /ml in the absence of any other data). virus decay in the environment and during transport, c env (j,k), c trans (j,k): the stability of mers-cov (isolate hcov-emc/2012) was evaluated under three different environmental conditions (van doremalen et al., 2013) . the mean half-life was calculated as: 0.947 (h) at 20°c 40% humidity and 0.708 h at 30°c 30% humidity indicating that temperature influences the environmental decay of the virus. in liquid matrices mers-cov was found to have a half-life of ∼10 h in milk at +22°c and 72 h at +4°c (van doremalen et al., 2014) . the mean half-life for mers-cov in the environment was taken to be 0.773 h, as it was assumed to most accurately mimic a real-life situation whereby fruit contaminated by either infected bat saliva or urine would then be exposed to temperatures of ∼30°c prior to harvesting (assuming a peak shedding prevalence in april/may and aug/sep time). however, after harvest when the fruit is assumed to be kept at +4°c during transport the average half-life time of 72 h, as calculated from milk, is used as virus survival time is assumed to be more similar between solid and liquid microbial risk analysis 7 (2017) 8-28 matrices at this temperature than at 30°c. minimum viral load to consider product contaminated in eu ms, c min . the only animal models found to be naturally permissive to infection are nonhuman primates. rhesus macaques challenged with 1 × 10 7 tcid 50 (de wit et al., 2013) and 6.5 × 10 7 tcid 50 (yao et al., 2014) showed mers-cov related pathology and had detectable levels of viral rna. challenge with a total viral dose of 5.2 × 10 6 tcid 50 caused a severe partially lethal disease in the common marmoset (callithrix jacchus) (falzarano et al., 2014) . camels inoculated with doses of 10 7 tcid 50 gave viral titre of maximum of 6 log 10 tcid 50 eq/ml of nasal swab samples (adney et al., 2014) . as a worst case scenario it is assumed that c min = 1 log 10 tcid 50 . species of animal, (s). there has been documented evidence of mers-cov viral rna (chu et al., 2014) , virus specific antibodies (alagaili et al., 2014; briese et al., 2014; corman et al., 2014; meyer et al., 2014; reusken et al., 2014b) and virus isolation (raj et al., 2014; hemida et al., 1993) in dromedary camels, both from countries in the arabian peninsula and the african continent. according to the ec (commission regulation (ec) no 206/2010) no camel imports are allowed from the arabian peninsula or countries in africa where antibodies to the mers-cov has been detected in camel sera. however, there is no evidence of camels from approved 3rd countries being tested for the mers-cov and, as this virus is suspected of being circulating in camels for some time, it is not impossible that they could be harbouring the coronavirus too. only dromedary camels were considered as 'at risk' for live animal imports to the eu. species of bushmeat, pbmsp(s): bats, or camel meat, illegally imported as bushmeat from the arabian peninsula and africa could be of potential risk in the transmission of the mers-cov. as has previously been stated, in the absence of other information, we assume that 1.5% of bushmeat is bats. it is possible that camel meat could be present in generic 'red meat' illegally imported as bushmeat from the arabian peninsula. replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia ebola virus antibody prevalence in dogs and human risk ebola virus disease outbreak in nigeria: transmission dynamics and rapid control aerosol exposure to the angola strain of marburg virus causes lethal viral hemorrhagic fever in cynomolgus macaques seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection marburgvirus resurgence in kitaka mine bat population after extermination attempts oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) hospital outbreak of middle east respiratory syndrome coronavirus progress audit of biosecurity queensland response activities at cawarral in evidence for camel-to-human transmission of mers coronavirus is that a rodent in your luggage? a mixed method approach to describe bushmeat importation into the united states ebola virus disease in southern sudan: hospital dissemination and intrafamilial spread marburg hemorrhagic fever associated with multiple genetic lineages of virus ebola virus as a foodborne pathogen? cause for consideration, but not panic first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities serological examination for evidence of infection with hendra and nipah viruses in queensland piggeries forty-five years of marburg virus research the role of the type i interferon response in the resistance of mice to filovirus infection a search for ebola virus in animals in the democratic republic of the congo and cameroon: ecologic, virologic, and serologic surveys, 1979-1980 middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia ebola hemorrhagic fever in kikwit, democratic republic of the congo: clinical observations in 103 patients bats: important reservoir hosts of emerging viruses potential for large outbreaks of ebola virus disease a small nonhuman primate model for filovirus-induced disease middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility the scale of illegal meat importation from africa to europe via paris the basic reproductive number of ebola and the effects of public health measures: the cases of congo and uganda synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission mers coronaviruses in dromedary camels assay for level of marburg virus in blood and isolates from experimentally infected animals ebola haemorrhagic fever in zaire antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques origin of the uk foot and mouth disease epidemic ebola hemorrhagic fever in 2014: the tale of an evolving epidemic an outbreak of hepatitis a associated with green onions introduction of sars in france hendra virus. national guidelines for public health units transmission of ebola hemorrhagic fever: a study of risk factors in family members identification of hotspots in the european union for the introduction of four zoonotic arboviroses by live animal trade managing the grey-headed flying-fox as a threatened species in nsw severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov). ninth update ecdc, severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) 21st update incubation period of ebola hemorrhagic virus subtype zaire statistics database illegal import of bushmeat and other meat products into switzerland on commercial passenger flights infection with mers-cov causes lethal pneumonia in the common marmoset trade data phylogeographical footprint of colonial history in the global dispersal of human immunodeficiency virus type 2 group a chains of transmission and control of ebola virus disease in conakry, guinea, in 2014: an observational study hendra virus infection dynamics in australian fruit bats lessons from nosocomial viral haemorrhagic fever outbreaks henipavirus susceptibility to environmental variables european bat lyssaviruses: an emerging zoonosis ebola hemorrhagic fever transmission and risk factors of contacts outbreak of marburg virus disease in johannesburg pathogenesis of ebola hemorrhagic fever in cynomolgus macaques -evidence that dendritic cells are early and sustained targets of infection marburg virus angola infection of rhesus macaques: pathogenesis and treatment with recombinant nematode anticoagulant protein c2 collection of mammals and arthropods during the epidemic of haemorrhagic fever in zaire d2r2: an evidencebased decision support tool to aid prioritisation of animal health issues for government funding natural hendra virus infection in flying-foxes -tissue tropism and risk factors person-to-person transmission of nipah virus in a bangladeshi community isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission identification of likely natural hosts for equine morbillivirus hendra virus infection in a veterinarian long-term survival of an urban fruit bat seropositive for ebola and lagos bat viruses demography of straw-colored fruit bats in ghana seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity pathogenesis of marburg hemorrhagic fever in cynomolgus macaques on the ecology of the fruit bat, rousettus aegyptiacus leachi (a. smith, 1829) in the tsitsikama coastal national park four outbreaks of norovirus gastroenteritis after consuming raspberries the lesions of experimental equine morbillivirus disease in cats and guinea pigs nipah situation in 2014 immune and pathophysiological processes in baboons experimentally infected with ebola virus adapted to guinea pigs transmission of ebola virus (zaire strain) to uninfected control monkeys in a biocontainment laboratory experimental infection of cynomolgus macaques with ebola-reston filoviruses from the 1989-1990 us epizootic lethal experimental infections of rhesus-monkeys by aerosolized ebola-virus characterization of a new marburg virus isolated from a 1987 fatal case in kenya a randomized controlled trial of interventions to impede date palm sap contamination by bats to prevent nipah virus transmission in bangladesh marburg virus replication, pathogenicity, shedding, and transmission of zaire ebolavirus in pigs real-time monitoring of cardiovascular function in rhesus macaques infected with zaire ebolavirus virus nomenclature below the species level: a standardized nomenclature for laboratory animal-adapted strains and variants of viruses assigned to the family filoviridae hendra virus and horse owners -risk perception and management marburg virus in fruit bat hyspignathus monstrosus search for the ebola virus reservoir in kikwit, democratic republic of the congo: reflections on a vertebrate collection statistical inference in a stochastic epidemic seir model with control intervention: ebola as a case study fruit bats as reservoirs of ebola virus human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo dissemination of cefotaxime-m-producing escherichia coli isolates in poultry farms, but not swine farms, in china experimental inoculation study indicates swine as a potential host for hendra virus foodborne transmission of nipah virus proportion of deaths and clinical features in bundibugyo ebola virus infection biosecurity authority, ministry of agriculture and forestry is marburg virus enzootic in gabon? experimental infection of horses with hendra virus/australia/ horse/2008/redlands hendra virus survival does not explain spillover patterns and implicates relatively direct transmission routes from flying foxes to horses importation of dogs into the united states: risks from rabies and other zoonotic diseases middle east respiratory syndrome coronavirus in bats, saudi arabia antibodies against mers coronavirus in dromedaries hendra virus vaccine, a one health approach to protecting horse, human, and environmental health identification of ebola virus sequences present as rna or dna in organs of terrestrial small mammals of the central african republic stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) modular framework to assess the risk of african swine fever virus entry into the european union piloting the promotion of bamboo skirt barriers to prevent nipah virus transmission through date palm sap in bangladesh epidemiologic and clinical aspects of the ebola virus epidemic in mosango, democratic republic of the congo association between temperature, humidity and ebolavirus disease outbreaks in africa handbook on import risk analysis for animals and animal products an outbreak of ebola in uganda ebola virus antibodies in fruit bats case-control study of risk factors for human infection with a new zoonotic paramyxovirus, nipah virus, during a 1998-1999 outbreak of severe encephalitis in malaysia epidemiological investigation of mers-cov spread in a single hospital in south korea calculation of incubation period and serial interval from multiple outbreaks of marburg virus disease the survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol mapping the zoonotic niche of ebola virus disease in africa human hendra virus encephalitis associated with equine outbreak ecological dynamics of emerging bat virus spillover assessment of the middle east respiratory syndrome coronavirus (mers-cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach spatial and temporal patterns of zaire ebolavirus antibody prevalence in the possible reservoir bat species large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in establishment and characterization of a lethal mouse model for the angola strain of marburg virus isolation of mers coronavirus from a dromedary camel ministry of tourism, wildlife and antiquities 2013 sector statistical abstract middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels geographic distribution of mers coronavirus among dromedary camels contact investigation for imported case of middle east respiratory syndrome modeling the impact of interventions on an epidemic of ebola in sierra leone and liberia the risk of rift valley fever virus introduction and establishment in the united states and european union wild animal mortality monitoring and human ebola outbreaks, gabon and republic of congo an analysis of features of pathogenesis in two animal models of ebola virus infection survival of hendra virus in the environment: modelling the effect of temperature infection of humans and horses by a newly described morbillivirus a generic quantitative risk assessment framework for the entry of bat-borne zoonotic viruses into the european union potential for introduction of bat-borne zoonotic viruses into the eu: a review marburg agent disease: in monkeys twenty years of hendra virus: laboratory submission trends and risk factors for infection in horses marburg-virus disease in kenya identifying hendra virus diversity in pteropid bats bats and their virome: an important source of emerging viruses capable of infecting humans interferon-beta therapy prolongs survival in rhesus macaque models of ebola and marburg hemorrhagic fever qualitative release assessment to estimate the likelihood of henipavirus entering the united kingdom mers in south korea and china: a potential outbreak threat? temporary loss of rabies free certificate experimental inoculation of plants and animals with ebola virus studies of reservoir hosts for marburg virus ebola virus disease in west africa-the first 9 months of the epidemic and forward projections west african ebola epidemic after one year-slowing but not yet under control marburg virus infection in nonhuman primates: therapeutic treatment by lipid-encapsulated sirna response to imported case of marburg hemorrhagic fever, the netherlands marburg virus infection detected in a common african bat isolation of genetically diverse marburg viruses from egyptian fruit bats trade control and expert system pathology of experimental aerosol zaire ebolavirus infection in rhesus macaques highly pathogenic h5n1 influenza virus in smuggled thai eagles stability of middle east respiratory syndrome coronavirus in milk stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions a review of epidemiological parameters from ebola outbreaks to inform early public health decision-making ebola hemorrhagic fever associated with novel virus strain transmission of ebola virus from pigs to non-human primates review of ebola virus infections in domestic animals case of marburg haemorrhagic fever imported into the netherlands from uganda who. mers-cov summary updates transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guineapigs and fruit bats (pteropus poliocephalus) an animal model of mers produced by infection of rhesus macaques with mers coronavirus survival of respiratory viruses on fresh produce serological evidence of ebolavirus infection in bats key: cord-349680-rz2ep5jf authors: lee, jacob title: better understanding on mers corona virus outbreak in korea date: 2015-06-10 journal: j korean med sci doi: 10.3346/jkms.2015.30.7.835 sha: doc_id: 349680 cord_uid: rz2ep5jf nan in june 2012, the first human with middle east respiratory syndrome coronavirus (mers-cov) infection was found in saudi arabia. the one was a 60-year old man. he visited a hospital due to pneumonia; it was later found out that renal failure developed (1) . he eventually died due to his illnesses. from 1 june 2015, 1,154 patients were infected with the virus; 431 patients died (who http://www.who.int/csr/don/archive/disease/coro-navirus_infections/en/). mers is suspected to spread from animals to humans like severe acute respiratory syndrome coronavirus (sars-cov). it was from a similar coronavirus with respiratory infection syndromes and it is highly transmitted. additionally, since the virus is related to a high rate of fatality, it has become a big issue for public health doctors and officials over the world. the latency period of mers-cov is known to be between 2 to 14 days (median 5.4 days). from the development of the disease to the patient`s admission, it takes 4 days and the period that people die from the disease takes 11.5 days (2) . in the first stage, flu like symptoms such as fever, coughing, chilling, myalgia, and arthralgia are observed. after this, respiratory difficulty is added. this quickly progresses to pneumonia (3). a part (30%) of the patients complain of bowel symptoms like vomiting and diarrhea (1). cases of mers-cov can be found in countries like america, uk, france, tunisia, italy, malaysia, the philippines, greece, egypt, the netherlands, algeria, austria, turkey, etc. whose citizens have travelled to the middle east. on may 20 2015, a man at the age of 68 was the first to be diagnosed with mers-cov in korea. he travelled to bahrain, saudi arabia, and qatar for 16 days. on may 4 2015, this patient entered in korea, and febrile sense and respiratory symptoms appeared on may 11. he visited clinic a on the day and was admitted to hospital b from may 15 to 17. since the symptoms got worsened, he visited clinic c on may 18, and finally he was transferred to a university hospital in seoul on may 18. on may 20, it was confirmed that he was suffering from mers-cov. after finding out about the disease, his family members and medical staffs who had been exposed to the virus were isolated. by june 9, 2015, 2 medical staffs in the clinic a and c, one medical staff in the hospital b, one patient and her wife who was together with the index case in the same room and 35 of admitted patients in the same ward and their family members visiting same ward with the index case in the hospital b were confirmed to have been infected with mers-cov. after then, several tertiary cases were identified in the hospital b or other hospitals that secondary patients were transferred from the hospital b. a total of 108 people were infected, and 9 (8.3%) of them died by june 10, 2015. one person among the 36 patients exposed in the hospital b left for china through hong kong, because the korea center for disease control and prevention (cdc) could not confirm the exposure to the index case. china cdc is performing isolated treatments. hong kong and china cdc started to manage people who are suspected to be exposed. at present, the outbreak pattern in korea has been progressing similarly to the hospital outbreak occurred in the middle east. the secondary infection developed in people who had a close contact with the person who was initially infected. medical staffs who were involved in treating some of the patients with mers-cov were also infected (2) . the secondary ones to be infected like the patients and medical staffs were not as severe as the first infected patients and mortality was lower than the index case (1). if korea also follows the outbreak pattern of the middle east, i expect more tertiary infection will be developed. the secondary infection occurred from the index case before the correct diagnosis, which was inevitable. at present, the korea cdc should focus on close monitoring of medical staffs and patients or visitors who have been exposed to the index, secondary, and tertiary cases in hospitals. it is very important to make nationwide effort to cope with this outbreak more actively and aggressively by organizing an emergency team with medical experts. also correct and timely well-designed briefing to mass media is necessary in order to prevent further spread, to calm down public panic, and to lessen its untoward impacts in the society. further imported cases of other variable noble infectious diseases may occur within the foreseeable future. health officials and infectious disease researchers have to be prepared about further challenges of these new infectious diseases. severe acute respiratory syndrome vs. the middle east respiratory syndrome ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation key: cord-349300-x50tvq3a authors: de wit, emmie; feldmann, friederike; cronin, jacqueline; jordan, robert; okumura, atsushi; thomas, tina; scott, dana; cihlar, tomas; feldmann, heinz title: prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection date: 2020-03-24 journal: proc natl acad sci u s a doi: 10.1073/pnas.1922083117 sha: doc_id: 349300 cord_uid: x50tvq3a the continued emergence of middle east respiratory syndrome (mers) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. remdesivir (gs-5734) effectively inhibited mers coronavirus (mers-cov) replication in vitro, and showed efficacy against severe acute respiratory syndrome (sars)-cov in a mouse model. here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of mers-cov infection, the rhesus macaque. prophylactic remdesivir treatment initiated 24 h prior to inoculation completely prevented mers-cov−induced clinical disease, strongly inhibited mers-cov replication in respiratory tissues, and prevented the formation of lung lesions. therapeutic remdesivir treatment initiated 12 h postinoculation also provided a clear clinical benefit, with a reduction in clinical signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. the data presented here support testing of the efficacy of remdesivir treatment in the context of a mers clinical trial. it may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus 2019-ncov. the continued emergence of middle east respiratory syndrome (mers) cases with a high case fatality rate stresses the need for the availability of effective antiviral treatments. remdesivir (gs-5734) effectively inhibited mers coronavirus (mers-cov) replication in vitro, and showed efficacy against severe acute respiratory syndrome (sars)-cov in a mouse model. here, we tested the efficacy of prophylactic and therapeutic remdesivir treatment in a nonhuman primate model of mers-cov infection, the rhesus macaque. prophylactic remdesivir treatment initiated 24 h prior to inoculation completely prevented mers-cov−induced clinical disease, strongly inhibited mers-cov replication in respiratory tissues, and prevented the formation of lung lesions. therapeutic remdesivir treatment initiated 12 h postinoculation also provided a clear clinical benefit, with a reduction in clinical signs, reduced virus replication in the lungs, and decreased presence and severity of lung lesions. the data presented here support testing of the efficacy of remdesivir treatment in the context of a mers clinical trial. it may also be considered for a wider range of coronaviruses, including the currently emerging novel coronavirus 2019-ncov. mers-cov | antiviral | animal model | remdesivir | therapy s ince its discovery in 2012, cases of middle east respiratory syndrome coronavirus (mers-cov) have continued to emerge, with the current case count close to 2,500 cases, and a case fatality rate ∼35% (1). this continuous emergence of mers-cov infections in saudi arabia and its ability to spread through humanto-human transmission has prompted the world health organization to include mers on their list of emerging diseases likely to cause major epidemics and for which countermeasures are urgently needed (2) . through the coalition for epidemic preparedness innovations, mers-cov vaccines are going to advance through preclinical and clinical trials (3), but, despite the urgent need, a similar initiative does not exist for the development and clinical testing of antivirals effective against mers-cov. remdesivir (gs-5734) is a nucleotide prodrug that has broad antiviral activity against viruses from different families in vitro (4) , and therapeutic efficacy in nonhuman primate models of lethal ebola virus and nipah virus infection (5, 6) . studies in human airway epithelial cells showed that remdesivir also inhibits replication of a wide range of coronaviruses, including mers-cov (7) . efficacy studies in mice showed that remdesivir had therapeutic efficacy against severe acute respiratory syndrome (sars)-cov and mers-cov in ces1c −/− mice, deficient in a secreted carboxylesterase responsible for poor pharmacokinetics profile of remdesivir in mice, when administered before the peak of virus replication (7, 8) . in vitro studies with mouse hepatitis virus showed that remdesivir inhibits coronavirus replication through interference with the viral polymerase, despite the presence of a viral proofreading exoribonuclease (9) . importantly, coronaviruses partially resistant to inhibition by remdesivir, obtained in vitro following >20 passages in the presence of gs-441524, a parent nucleoside that is metabolized into the same active triphosphate metabolite, were still sensitive to higher concentrations of remdesivir, and fitness was impaired in the resistant viruses as compared to wild-type mers-cov (9) . with these promising data in mind, we tested the prophylactic and therapeutic efficacy of remdesivir treatment in a nonhuman primate model of mers-cov infection, the rhesus macaque (10) . and therapeutic treatment. to assess the efficacy of remdesivir to alleviate clinical signs of mers-cov infection, 18 rhesus macaques were randomly assigned to three groups of six animals. three animals in the control group were treated with 1 ml/kg vehicle solution 24 h before mers-cov inoculation, and three animals were treated at 12 h post mers-cov inoculation. another group of six rhesus macaques was treated prophylactically 24 h before mers-cov inoculation with 5 mg/kg remdesivir, and one group of six animals was treated therapeutically at 12 h postinoculation with mers-cov with 5 mg/kg remdesivir. treatment middle east respiratory syndrome, caused by the mers coronavirus (mers-cov), continues to cause severe respiratory disease with a high case fatality rate. to date, potential antiviral treatments for mers-cov have shown limited efficacy in animal studies. here, we tested the efficacy of the broad-acting antiviral remdesivir in the rhesus macaque model of mers-cov infection. remdesivir reduced the severity of disease, virus replication, and damage to the lungs when administered either before or after animals were infected with mers-cov. our data show that remdesivir is a promising antiviral treatment against mers that could be considered for implementation in clinical trials. it may also have utility for related coronaviruses such as the novel coronavirus 2019-ncov emerging from wuhan, china. data deposition: all data discussed here will be made available to readers upon request. 1 to whom correspondence may be addressed. email was continued once daily until 6 d postinoculation (dpi), when animals were euthanized and necropsied (fig. 1 ). after inoculation with mers-cov on day 0, all animals were closely observed for signs of disease, and clinical scores were assigned according to a previously determined scoring sheet. all vehicle-treated animals displayed signs of disease, starting as early as 1 dpi, such as decreased appetite and ruffled fur; all vehicle-treated animals had respiratory signs such as increased respiration for 4 (n = 1) or 5 (n = 5) d after inoculation. the animals treated prophylactically with remdesivir did not show any respiratory signs of disease, but decreased appetite, possibly due to daily anesthesia, was noted in five of six animals. the animals treated therapeutically with remdesivir all displayed reduced appetites, and five out of six animals had increased respiration rates at 2 (n = 2), 3 (n = 2), or 4 (n = 1) d after inoculation. these observations are reflected in the clinical scores of the animals, with clinical scores in the prophylactically treated animals being statistically significantly lower than in vehicletreated control animals at 2 to 6 dpi, and in the therapeutically treated animals at 2 to 4 dpi ( fig. 2a) . on days 0, 1, 3, 5, and 6, clinical examinations were performed on the animals, and respiration rates were determined on anesthetized animals. there was a clear increase in respiration rates in the vehicle-treated animals ( fig. 2b ), while respiration rates in prophylactically treated animals remained normal throughout the study. although respiration rate was increased in therapeutically treated animals at 1 dpi, respiration was statistically significantly lower than in vehicle-treated controls at 3 and 6 dpi (fig. 2b ). on examination days, radiographs were collected from all animals and analyzed for the presence of infiltrates; from 3 dpi onward, lung infiltrates became visible on x-ray (si appendix, fig. s1 ). at 6 dpi, there was statistically significantly less infiltration in the lungs of animals treated both prophylactically and therapeutically with remdesivir as compared to vehicle-treated control animals (fig. 2c ). at 6 dpi, all animals were euthanized, and respiratory tissues were collected for quantitative analysis of the levels of viral rna by qrt-pcr. compared to vehicle-treated control animals, prophylactic remdesivir treatment resulted in significantly lower levels of mers-cov replication in the lungs, with lung viral loads 2.5 to 4 logs lower in each lung lobe (fig. 3a) . although lung viral loads were, on average, lower in individual lung lobes after therapeutic treatment, this was statistically significant in only a few lung lobes, due to larger variation between animals in the therapeutically treated group (fig. 3a) . however, when all lung lobes were combined, the lung viral load in therapeutically treated animals was clearly lower than in vehicle-treated animals (fig. 3b ). additionally, viral loads were significantly lower in trachea, bronchi, tonsils, and mediastinal lymph nodes of animals treated prophylactically and therapeutically with remdesivir than in vehicle-treated control animals ( fig. 3c and si appendix, fig. s2 ); viral rna was not detected in kidney tissue samples (si appendix, fig. s2 ). treatment. upon necropsy, the area of each lung lobe affected by gross lesions was estimated by a board-certified veterinary pathologist. gross lung lesions were present in several lung lobes of all of the vehicle-treated control animals (fig. 4a ). in contrast, gross lung lesions were completely absent in the lungs of animals that received prophylactic remdesivir treatment. in animals treated therapeutically with remdesivir, there were obvious gross lesions present in five out of six animals; however, the total area of lungs affected by gross lesions was statistically significantly smaller than in vehicle-treated control animals (fig. 4a ). in addition, the severity of histologic lung lesions was assessed by assigning a score for each lung lobe. the resulting cumulative lung histology score was compared between treatment groups to assess differences in the severity of histologic lesions. cumulative lung histology scores were significantly lower in animals treated prophylactically with remdesivir (fig. 4b) . the large variation between animals in the therapeutically treated group meant that the lower average histology score did not reach statistical significance (fig. 4b) . histologically, all of the vehicle-treated control animals developed some degree of pulmonary pathology when inoculated with mers-cov. lesions were multifocal, frequently centered on terminal bronchioles, and consisted of minimal to marked, interstitial pneumonia, characterized by thickening of alveolar septae by edema fluid and fibrin and small to moderate numbers of macrophages and fewer neutrophils. alveoli contained moderate numbers of pulmonary macrophages and neutrophils. in areas with moderate to marked changes, there was abundant alveolar edema and fibrin with multifocal formation of hyaline membranes, as well as abundant type ii pneumocyte hyperplasia. perivascular infiltrates of inflammatory cells multifocally within and adjacent to affected areas of the lung were also observed (fig. 4c ). in contrast, all animals treated prophylactically with remdesivir had essentially normal pulmonary tissue with no evidence of coronavirus infection (fig. 4c ). animals treated with remdesivir therapeutically demonstrated various levels of severity of coronaviral pneumonia. in two out of six animals, no histologic evidence of pneumonia was detected. in three animals, multifocal, minimal to moderate interstitial pneumonia was observed like that described for the control animals; however, the lesions were less severe than in the controls and not as widely distributed throughout the lung lobes. only one out of six animals had moderate interstitial pneumonia that was indistinguishable from the vehicle-treated control animals in severity and distribution. immunohistochemical analysis for the presence of mers-cov antigen showed small numbers of antigen-positive type i pneumocytes in all vehicle-treated control animals and in five out of six animals treated therapeutically with remdesivir; there was no difference in number or distribution of antigen-positive cells in animals where antigen was detected. mers-cov antigen could not be detected in any of the animals treated prophylactically with remdesivir (fig. 4d ). prophylactic remdesivir treatment prevented mers-cov−induced clinical disease and lung lesions in rhesus macaques inoculated with mers-cov, and strongly inhibited mers-cov replication in respiratory tissues. since nosocomial transmission accounts for approximately one-third of mers-cov cases (11) , prophylactic remdesivir treatment of patients, contacts of patients, and healthcare personnel with high-risk exposure to a diagnosed mers patient and at high risk of developing severe mers due to underlying conditions (12) could be considered. therapeutic remdesivir treatment also provided a clear clinical benefit, with a reduction in clinical signs and virus replication, and the absence of lung lesions in two out of six remdesivir-treated animals and a reduction in lesion severity in three additional animals. absence of histologic lung lesions, as seen in two out of the six animals with therapeutic remdesivir treatment, has so far rarely been observed in studies testing the efficacy of mers-cov antivirals in nonhuman primate models (13) (14) (15) (16) ; it has only been shown once before in one out of three common marmosets treated with hyperimmune plasma at 6 h after inoculation (17) . thus, although it is hard to compare different studies due to the fact that different species were used and treatment was initiated at different time points after inoculation, remdesivir appears to be one of the most promising antiviral treatments tested in a nonhuman primate model to date. therapeutic remdesivir treatment was administered at 12 h after inoculation with mers-cov, and, although this may seem relatively early after inoculation, it is close to the peak of mers-cov replication in the rhesus macaque model (10) . a drug that inhibits virus replication may be of little use once virus replication has reached its peak, as was shown in vitro (9) . however, in a considerable number of severe cases of mers, viral rna and infectious virus can still be detected in respiratory tract samples several weeks after the onset of symptoms (18, 19) , with this prolonged virus replication most likely due to the presence of underlying conditions such as diabetes mellitus (18) . likewise, an increase in virus replication over a longer period of time was observed in immunocompromised rhesus macaques (20) . thus, remdesivir treatment could not only be of benefit to patients diagnosed with mers early after symptom onset but may also improve recovery in those patients with severe cases of mers where prolonged virus replication occurs. human safety data are available for remdesivir. it has been used on a compassionate basis in several unique cases of ebola virus disease (21, 22) , as well as on a large scale in the ongoing ebola virus outbreak in the democratic republic of congo (23), with around 400 treated patients. in addition, its efficacy is currently being tested in a clinical trial in ebola virus disease survivors with prolonged virus shedding (24, 25) . although the efficacy of remdesivir was lower in the ebola virus trial than that of the different antibody treatments tested, survival was increased as compared to overall survival rate in this outbreak. taken together, the data presented here on the efficacy of remdesivir in prophylactic and therapeutic treatment regimens, the difficulty of coronaviruses to acquire resistance to remdesivir (9) , and the availability of human safety data warrant testing of the efficacy of remdesivir treatment in the context of a mers clinical trial. our results, together with replication inhibition by fig. 2 . clinical findings in rhesus macaques inoculated with mers-cov and treated with remdesivir. three groups of six rhesus macaques were inoculated with mers-cov strain hcov-emc/2012; one group was i.v.-administered 1 ml/kg vehicle solution (vehicle control; gray circles), one group was administered 5 mg/kg remdesivir starting at 24 h before inoculation (prophylactic remdesivir; black squares), and one group was administered 5 mg/kg remdesivir starting at 12 h after inoculation (therapeutic remdesivir; red triangles). after inoculation, the animals were observed twice daily for clinical signs of disease and scored using a predetermined clinical scoring system (a). on 0, 1, 3, 5 and 6 dpi, clinical examinations were performed during which respiration rate was determined (b), and radiographs were taken. radiographs were used to score individual lung lobes for severity of pulmonary infiltrates by a clinical veterinarian according to a standard scoring system (0: normal; 1: mild interstitial pulmonary infiltrates; 2: moderate pulmonary infiltrates perhaps with partial cardiac border effacement and small areas of pulmonary consolidation; 3: serious interstitial infiltrates, alveolar patterns and air bronchograms); the cumulative x-ray score is the sum of the scores of the six individual lung lobes per animal; scores shown are from 6 dpi (c). asterisks indicate statistically significant difference in a two-way (a and b) or one-way (c) anova with dunnett's multiple comparisons; black asterisks indicate statistical significance between the vehicle control and prophylactic remdesivir groups, and red asterisks indicate statistical significance between the vehicle control and therapeutic remdesivir groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ethics and biosafety statement. all animal experiments were approved by the institutional animal care and use committee of rocky mountain laboratories, nih and carried out by certified staff in an association for assessment and accreditation of laboratory animal care international accredited facility, according to the institution's guidelines for animal use, and followed the guidelines and basic principles in the united states public health service policy on humane care and use of laboratory animals, and the guide for the care and use of laboratory animals. rhesus macaques were housed in adjacent individual primate cages allowing social interactions, in a climate-controlled room with a fixed light−dark cycle (12-h light/12-h dark). animals were monitored at least twice daily throughout the experiment. commercial monkey chow, treats, and fruit were provided twice daily by trained personnel. water was available ad libitum. environmental enrichment consisted of a variety of human interaction, commercial toys, videos, and music. the institutional biosafety committee (ibc) approved work with infectious mers-cov strains under bsl3 conditions. sample inactivation was performed according to ibc-approved standard operating procedures for removal of specimens from high containment. study design. to evaluate the effect of remdesivir treatment on mers-cov disease outcome, we used the rhesus macaque model of mers-cov infection that results in transient lower respiratory tract disease (10) . rhesus macaques were chosen because of the requirement of daily anesthesia and intravenous (i.v.) injections that were perceived to be problematic in the alternative nonhuman primate model of mers-cov infection, the common marmoset (27), due to their small size. all animals were randomly assigned to groups and inoculated as described previously with a total dose of 7 × 10 6 tcid50 of mers-cov strain hcov-emc/2012 via intranasal, oral, ocular (1 × 10 6 tcid50 each), and intratracheal (4 × 10 6 tcid50) routes (10) . in the first experiment, the efficacy of prophylactic remdesivir treatment was tested in one group of six rhesus macaques (all males; female rhesus macaques were not available from the supplier at the time of this study) treated with 5 mg/kg remdesivir in vehicle solution (5 mg/ml 12% sulfobutylether-β-cyclodextrin in water and hydrochloric acid, ph3.5) and three control rhesus macaques (all males) who received the same volume (1 ml/kg) of vehicle solution. this 5 mg/kg dosing in rhesus macaques is roughly equivalent to the 100-mg daily dosing used in humans in the ebola virus clinical trials. treatment was initiated at 24 h before virus inoculation and continued once daily until 6 dpi. after observing good efficacy of remdesivir upon prophylactic treatment, a second experiment was performed to assess its therapeutic efficacy. one group of six rhesus macaques (all males) was treated with 5 mg/kg remdesivir, and three control rhesus macaques (all males) received the same volume of vehicle solution. due to the acute nature of the mers-cov model in rhesus macaques, therapeutic treatment was initiated at 12 h after inoculation with mers-cov and continued once daily until 6 dpi. treatment was delivered as a slow i.v. bolus injection (total dose delivered over ∼5 min) administered alternatingly in the left or right cephalic and saphenous veins. the animals were observed twice daily for clinical signs of disease, using a standardized scoring sheet as described previously (28); the same person, who was blinded to the group assignment of the animals, assessed the animals throughout the study. the predetermined endpoint for this experiment was 6 dpi. clinical examinations were performed at 0, 1, 3, 5, and 6 dpi on anesthetized animals. on examination days, clinical parameters such as body weight and respiration rate were collected, as well as dorsal−ventral and lateral chest radiographs. chest radiographs were analyzed by a board-certified clinical veterinarian blinded to the group assignment of the animals. after euthanasia at 6 dpi, necropsies were performed. the percentage of gross lung lesions were scored by a board-certified veterinary pathologist blinded to the group assignment of the animals, and samples of the following tissues were collected: conjunctiva, nasal mucosa, mandibular lymph node, tonsil, pharynx, trachea, all six lung lobes, mediastinal lymph node, liver, spleen, kidney, and bladder. histopathological analysis of tissue slides was performed by a board-certified veterinary pathologist blinded to the group assignment of the animals. -administered 1 ml/kg vehicle solution (vehicle control; gray circles), one group was administered 5 mg/kg remdesivir starting at 24 h before inoculation (prophylactic remdesivir; black squares), and one group was administered 5 mg/kg remdesivir starting at 12 h after inoculation (therapeutic remdesivir; red triangles). treatment was continued once daily until 6 dpi, when all animals were euthanized and necropsies were performed. at necropsy, tissue samples were collected from all six lung lobes, rna was extracted, and viral load was determined as tcid50 equivalents per gram tissue. individual animals and lung lobes are indicated (a), and averages and sds per group (b). similarly, viral loads were determined in additional tissues from the respiratory tract of each animal (c). r: right; l: left. asterisks indicate statistically significant differences in a two-way anova with dunnett's multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. qpcr. tissues (30 mg) were homogenized in rlt buffer, and rna was extracted using the rneasy kit (qiagen) according to the manufacturer's instructions. for detection of viral rna, 5 μl of rna was used in a one-step real-time rt-pcr upe assay (29) using the rotor-gene probe kit (qiagen) according to instructions of the manufacturer. in each run, standard dilutions of a titered virus stock were run in parallel, to calculate tcid50 equivalents in the samples. histopathology and immunohistochemistry. histopathology and immunohistochemistry were performed on rhesus macaque tissues. after fixation for 7 d in 10% neutral-buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (h&e). to detect hcov-emc/2012 antigen, immunohistochemistry was performed using an in-house rabbit polyclonal antiserum against hcov-emc/2012 (1:1,000) as a primary antibody. stained slides were analyzed by a board-certified veterinary pathologist blinded to the group assignment of the animals. statistical analysis. statistical analyses were performed using graphpad prism software version 7.04. for analysis, the three vehicle control animals from the first and second experiment were combined to form one group of six animals. data availability statement. all data discussed here will be made available to readers upon request. fig. 4 . pathological findings in the lungs of rhesus macaques inoculated with mers-cov and treated with remdesivir. three groups of six rhesus macaques were inoculated with mers-cov strain hcov-emc/2012; one group was i.v.-administered 1 ml/kg vehicle solution (vehicle control; gray circles), one group was administered 5 mg/kg remdesivir starting at 24 h before inoculation (prophylactic remdesivir; black squares), and one group was administered 5 mg/kg remdesivir starting at 12 h after inoculation (therapeutic remdesivir; red triangles). treatment was continued once daily until 6 dpi, when all animals were euthanized and necropsies were performed. at necropsy, the percentage of each lung lobe affected by gross lesions was estimated by a board-certified veterinary pathologist (a). lung samples were collected and stained with h&e and analyzed for the presence of lesions by a board-certified veterinary pathologist. each lung was given a score from 0 to 4 based on the abundance of lesions; the cumulative histology score is the sum of the scores of the six individual lung lobes per animal (b). one representative h&e image was chosen for each group (magnification: 100×) (c). lung samples were also stained with a polyclonal α-mers-cov antibody; one representative image was chosen for each group (magnification: 200×) (d). images in c and d were chosen as representative images of lung lesions and antigen expression, respectively, rather than being images from consecutive tissue slides. asterisks indicate statistically significant differences in a two-way anova with dunnett's multiple comparisons. **p < 0.01; ****p < 0.0001. world health organization, coronavirus infections a roadmap for mers-cov research and product development: report from a world health organization consultation cepi-a new global r&d organisation for epidemic preparedness and response gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys remdesivir (gs-5734) protects african green monkeys from nipah virus challenge broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset prophylactic and therapeutic efficacy of mab treatment against mers-cov in common marmosets treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets middle east respiratory syndrome coronavirus infection dynamics and antibody responses among clinically diverse patients, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea pathogenicity and viral shedding of mers-cov in immunocompromised rhesus macaques first newborn baby to receive experimental therapies survives ebola virus disease late ebola virus relapse causing meningoencephalitis: a case report palm writing group; palm consortium study team, a randomized, controlled trial of ebola virus disease therapeutics gs-5734 to assess the antiviral activity, long-term clearance of ebola virus and safety in male ebola survivors with evidence of ebola virus persistence in semen investigational therapeutics for the treatment of people with ebola virus disease infection with mers-cov causes lethal pneumonia in the common marmoset thoracic radiography as a refinement methodology for the study of h1n1 influenza in cynomologus macaques (macaca fascicularis) detection of a novel human coronavirus by real-time reversetranscription polymerase chain reaction key: cord-342739-iy9vjpuh authors: schwartz, david a.; graham, ashley l. title: potential maternal and infant outcomes from coronavirus 2019-ncov (sars-cov-2) infecting pregnant women: lessons from sars, mers, and other human coronavirus infections date: 2020-02-10 journal: viruses doi: 10.3390/v12020194 sha: doc_id: 342739 cord_uid: iy9vjpuh in early december 2019 a cluster of cases of pneumonia of unknown cause was identified in wuhan, a city of 11 million persons in the people’s republic of china. further investigation revealed these cases to result from infection with a newly identified coronavirus, initially termed 2019-ncov and subsequently sars-cov-2. the infection moved rapidly through china, spread to thailand and japan, extended into adjacent countries through infected persons travelling by air, eventually reaching multiple countries and continents. similar to such other coronaviruses as those causing the middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars), the new coronavirus was reported to spread via natural aerosols from human-to-human. in the early stages of this epidemic the case fatality rate is estimated to be approximately 2%, with the majority of deaths occurring in special populations. unfortunately, there is limited experience with coronavirus infections during pregnancy, and it now appears certain that pregnant women have become infected during the present 2019-ncov epidemic. in order to assess the potential of the wuhan 2019-ncov to cause maternal, fetal and neonatal morbidity and other poor obstetrical outcomes, this communication reviews the published data addressing the epidemiological and clinical effects of sars, mers, and other coronavirus infections on pregnant women and their infants. recommendations are also made for the consideration of pregnant women in the design, clinical trials, and implementation of future 2019-ncov vaccines. coronaviruses are spherical, enveloped, and the largest of positive-strand rna viruses. they have a wide host range, including birds, farm animals, pets, camels, and bats, in which they primarily cause respiratory and gastrointestinal disease. belonging to the order nidovirales, family coronaviridae, and the subfamily orthocoronaviridae there are four genera of coronaviruses-alphacoronavirus, betacoronavirus, deltacorona virus, and gammacoronavirus [1] [2] [3] [4] . in humans, they are a cause of mild illnesses including the common colds occurring in children and adults, and were believed to be of modest medical importance. however, two zoonotic coronaviruses-including the severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov)-can produce severe lower respiratory in the beginning of december 2019, a cluster of persons with a pneumonia of unknown cause was identified in wuhan, the capital of hubei province and a large city of approximately 11 million persons located in the central region of the people's republic of china [7, 8] . between 8 and 18 december 2019 there were 7 cases of pneumonia identified whose clinical features resembled that of a viral pneumonia. the outbreak was initially believed to be linked to the wuhan huanan (south china) seafood wholesale market. this market, termed a "wet" market, sells a variety of seafood, cuts of meat, and both live and dead animals in over one thousand stalls in constant close contact; however, whether this market was the origin of the outbreak remains unknown [9] . on 31 december 2019, the chinese center for disease control and prevention (china cdc) sent a rapid response team to hubei to work alongside health personnel from the provincial and wuhan city health departments to conduct an epidemiologic investigation. as the disease was spreading through secondary and tertiary cases, the world health organization (who) china country office was informed on 31 december 2019 of the occurrence of these cases of pneumonia of unknown etiology. during the period from 31 december 2019 to 3 january 2020, 44 patients with pneumonia of unknown etiology were reported by the chinese authorities to the who. on 7 january 2020 investigators in china identified the etiological agent of the epidemic as a previously unknown coronavirus, and it was given the designation 2019-ncov (for 2019 novel coronavirus) [8] . analysis of the clinical features of 41 hospitalized patients with laboratory-confirmed 2019-ncov infection revealed that 30 were men (73%); less than one-half had underlying co-morbid conditions (13; 32%) which included diabetes (8, 20%) , hypertension (6, 15%), and cardiovascular disease (6; 15%); and the average age was 49.0 years old. the most common symptoms at the beginning of their illness included fever (40, 98%) , cough (31, 76%) , and fatigue or myalgia (18, 44%) , sputum production (11, 28%) , and headache (3, 8%) [10] . among these 41 initial cases of 2019-ncov infection there were 12 patients (32%) who developed acute respiratory distress syndrome (ards), 13 (32%) required intensive care and 6 (15%) died. during the first weeks of january the infection spread rapidly through china and extended to adjacent countries where cases began to appear-13 january in thailand, 15 january in japan, 20 january in the republic of korea, and taiwan and the united states on 21 january [11] . infected travelers, mostly via commercial air travel, are known to have been responsible for introducing the virus outside of wuhan. the new coronavirus continued to spread throughout multiple countries and continents, and by 9 february 2020 the who reported 37,251 confirmed cases in china that resulted in 812 deaths, surpassing the number of deaths that occurred during the 2002-2003 sars epidemic. an additional 307 cases of 2019-ncov infection have occurred among 24 other countries outside of china [12] . (figure 1 ) at the meeting of the emergency committee of the who on 30 january, the novel coronavirus 2019 epidemic was declared a public health emergency of international concern (pheic) [11, 13] . viruses 2020, 12, 194 3 of 16 epidemic. an additional 307 cases of 2019-ncov infection have occurred among 24 other countries outside of china [12] . (figure 1 ) at the meeting of the emergency committee of the who on 30 january, the novel coronavirus 2019 epidemic was declared a public health emergency of international concern (pheic) [11, 13] . this newly recognized coronavirus, producing a disease that has been termed covid-19, is rapidly spreading throughout china, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. it is certain that 2019-ncov will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with sars-cov and mers-cov. in order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. it is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . in one study pneumonia was the 3rd most common cause of indirect maternal death [17] . approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . as with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . this has been evident historically during previous epidemics. the case fatality rate (cfr) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . during the 1957-1958 asian flu epidemic, 10% of all deaths occurred in pregnant women, and their cfr was twice as high as that of infected women who were not pregnant [24] . the most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include this newly recognized coronavirus, producing a disease that has been termed covid-19, is rapidly spreading throughout china, has crossed international borders to infect persons in neighboring countries, and humans infected by the virus are travelling via commercial airlines to other continents. it is certain that 2019-ncov will infect women who are pregnant, leaving the question open as to whether the novel coronavirus will have a similar or different effect on them compared with sars-cov and mers-cov. in order to address the potential obstetrical outcomes of infection to both mother and infant, the present communication describes the current state of knowledge regarding the effects of other coronavirus infections in pregnancy. pneumonia arising from any infectious etiology is an important cause of morbidity and mortality among pregnant women. it is the most prevalent non-obstetric infectious condition that occurs during pregnancy [14] [15] [16] . in one study pneumonia was the 3rd most common cause of indirect maternal death [17] . approximately 25 percent of pregnant women who develop pneumonia will need to be hospitalized in critical care units and require ventilatory support [16] . although bacterial pneumonia is a serious disease when it occurs in pregnant women, even when the agent(s) are susceptible to antibiotics, viral pneumonia has even higher levels of morbidity and mortality during pregnancy [18] . as with other infectious diseases, the normal maternal physiologic changes that accompany pregnancy-including altered cell-mediated immunity [19] and changes in pulmonary function-have been hypothesized to affect both susceptibility to and clinical severity of pneumonia [20] [21] [22] . this has been evident historically during previous epidemics. the case fatality rate (cfr) for pregnant women infected with influenza during the 1918-1919 pandemic was 27%-even higher when exposure occurred during the 3rd trimester and upwards of 50% if pneumonia supervened [23] . during the 1957-1958 asian flu epidemic, 10% of all deaths occurred in pregnant women, and their cfr was twice as high as that of infected women who were not pregnant [24] . the most common adverse obstetrical outcomes associated with maternal pneumonias from all causes include premature rupture of membranes (prom) and preterm labor (ptl), intrauterine fetal demise (iufd), intrauterine growth restriction (iugr), and neonatal death [14] [15] [16] . the sars epidemic began quietly at the turn of the 21st century. in november 2002, a cook in guangdong province, china, died from an unidentified illness. he had worked at a restaurant in which meat from wild animals was served. on 27 november 2002 chinese-language media and internet reports were picked up by canada's global public health intelligence network (gphin) that indicated a flu-like illness was occurring in china [25, 26] . unfortunately, the reports were not translated, and china failed to report the occurrence of this illness to the world health organization (who) until february 2003. the disease spread to other countries where it primarily infected healthcare workers. one of these was dr. carlo urbani, a who physician investigating a patient with the new disease in hanoi. he recognized that the pneumonia was probably caused by a new, highly infectious agent, and rapidly notified the who. he contracted the sars-cov while there, became febrile and later died after traveling to thailand to attend a conference. on 12 march 2003, who issued a global alert regarding the disease that was occurring primarily among health care workers in hanoi, vietnam and hong kong. the disease continued to spread, and by 31 july 2003 there were 8422 probable cases, leading to 916 deaths in 29 countries, with the majority of cases occurring in mainland china and hong kong. approximately 30% of infections occurred in healthcare workers. by the termination of the epidemic the global cfr was 11% [27] . although there were relatively few documented cases of sars occurring during pregnancy, several case reports and small clinical studies have described the clinical effects in pregnant women and their infants. in reviewing these reports describing pregnant women with sars in china it is possible, and perhaps even probable, that some of the same patients were included in more than one publication. however, even if this is the case, there is no doubt that sars coronavirus infection was found to be associated with severe maternal illness, maternal death, and spontaneous abortion [19, [28] [29] [30] [31] . martha anker, an expert in statistics formerly with the who and the university of massachusetts, estimated that more than 100 cases of sars-cov infection occurred in pregnant women, which warrants closer inspection [27] . the clinical outcomes among pregnant women with sars in hong kong were worse than those occurring in infected women who were not pregnant [32] . wong et al. [29] evaluated the obstetrical outcomes from a cohort of pregnant women who developed sars in hong kong during the period of 1 february to 31 july 2003. four of the 7 women (57%) that presented during the 1st trimester sustained spontaneous miscarriages, likely a result of the hypoxia that was caused by sars-related acute respiratory distress. among the 5 women who presented after 24 weeks gestation, 4 had preterm deliveries (80%). a case-control study to determine the effects of sars on pregnancy compared 10 pregnant and 40 non-pregnant women with the infection at the princess margaret hospital in hong kong [27, 33] . there were 3 deaths among the pregnant women with sars (maternal mortality rate of 30%) and no deaths in the non-pregnant group of infected women (p = 0.006). renal failure (p = 0.006) and disseminated intravascular coagulopathy (p = 0.006) developed more frequently in pregnant sars patients when compared with the non-pregnant sars group. six pregnant women with sars required admission to the intensive care unit (icu) (60%) and 4 required endotracheal intubation (40%), compared with a 12.5% intubation rate (p = 0.065) and 17.5% icu admission rate (p = 0.012) in the non-pregnant group. maxwell et al. [32] reported 7 pregnant women infected with sars-cov who were followed at a designated sars unit-2 of the 7 died (cfr of 28%), and 4 (57%) required icu hospitalization and mechanical ventilation. in contrast, the mortality rate was less than 10% and mechanical ventilation rate less than 20% among non-pregnant, age-matched counterparts who were not infected with sars-cov. two women with sars recovered and maintained their pregnancy but had infants with iugr. among the live newborn infants, none had clinical or laboratory evidence for sars-cov infection. the new mothers who had developed sars were advised not to breastfeed to prevent possible vertical transmission of the virus. zhang et al. [34] described sars-cov infections in 5 primagravidas from guangzhou, china at the height of the sars epidemic. two of the mothers became infected in the 2nd trimester, and 3 developed infection in the 3rd trimester. two of the pregnant women had hospital-acquired sars infections, and the other 3 were community-acquired. all 5 pregnant women had fever and abnormal chest radiographs; 4 had cough; 4 developed hypoalbuminemia; 3 had elevated alanine aminotransferase levels (alt), 3 had chills or rigor, 2 had decreased lymphocytes, and 2 had decreased platelets. one pregnant woman required intensive care, but all recovered and there were no maternal deaths. the 5 infants were clinically evaluated, and none had evidence of sars. two pregnant women with sars were reported from the united states. in a detailed case report, robertson et al. [35] described a 36-year-old pregnant woman with an intermittent cough of approximately 10 days duration and no fever. while travelling in hong kong during the 2003 epidemic, she was exposed at her hotel to a person subsequently known to be infected with sars-cov. at 19 weeks gestation she developed fever, anorexia, headache, increasing cough, weakness, and shortness of breath. upon returning to the united states she was hospitalized with pneumonia. obstetrical ultrasounds revealed a low-lying placenta (placenta previa) but were otherwise normal. following her discharge home and clinical recovery, she was found to have antibodies to sars-cov. she underwent cesarean section at 38 weeks gestation because of the placenta previa and a healthy baby girl was delivered [35, 36] . the placenta was interpreted as being normal. at 130 days post-maternal illness, maternal serum and whole blood, swabs from maternal nasopharynx and rectum, post-delivery placenta, umbilical cord blood, amniotic fluid, and breast milk were collected for analysis-no viral rna was detected in specimens tested by reverse transcriptase polymerase chain reaction (rt-pcr). antibodies to sars-cov were detected from maternal serum, umbilical cord blood, and breast milk by enzyme immunoassay (eia) and indirect immunofluorescence assay. no clinical specimens (except for cord blood) were available for testing from the infant. the second case in the usa occurred in a 38-year-old woman who had travelled to hong kong at 7 weeks gestation where she was exposed to sars-cov in the same hotel as the aforementioned american woman [37] . following her return to the united states, her husband developed the clinical onset of sars, and 6 days later she became ill with fever, myalgia, chills, headache, coryza, and a productive cough with shortness of breath and wheezing. following her hospitalization for sars she recovered, serum samples taken on days 28 and 64 post-onset of illness were positive for antibodies to sars-cov by enzyme immunoassay and immunofluorescent assays. her pregnancy continued and was unremarkable except for developing elevated glucose levels. a cesarean section that was performed at 36 weeks gestation due to preterm rupture of membranes and fetal distress resulted in a healthy baby boy. at the time of delivery, the mother's serum samples were positive for antibodies to sars-cov, but samples taken of umbilical cord blood and placenta were negative. breast milk sampled 12 and 30 days after delivery were also negative for sars-cov antibodies. specimens evaluated from maternal blood, stool, and nasopharynx samples, as well as umbilical cord blood of the infant, were all negative for coronavirus rna by rt-pcr. neonatal stool samples obtained on days-of-life 12 and 30 were also negative for viral rna. from canada, yudin et al. [38] reported a 33-year-old pregnant woman who was admitted to the hospital at 31 weeks gestation with a fever, dry cough, and abnormal chest radiograph demonstrating patchy infiltrates. she had acquired sars from contact with an infected family member. following a 21-day stay in the hospital, during which she did not require ventilatory support, her convalescent antibody titers were positive for coronavirus infection. she had a normal labor and delivery and her newborn girl had no evidence of infection. in a study of 5 liveborn neonates who were delivered to women infected with sars-cov during the hong kong epidemic, results from multiple tests-including serial rt-pcr assays, viral culture, and paired neonatal serological titers-were negative for sars-cov [39] . none of the 5 neonates developed any clinical signs or symptoms of respiratory infection or compromise. fortunately, there were no cases of vertical transmission identified among pregnant women infected with sars-cov during the 2002-2003 asian epidemic [27, 30, 31, 39, 40] , and with the exception of a small cluster of cases that recurred in late 2003, no new cases of sars have occurred. in the only reported study of the placental pathology of mothers with sars, ng et al. [41] reported the findings from 7 pregnant women infected with sars-cov. in the case of 2 women who were convalescing from sars-cov infection during the 1st trimester of pregnancy, the placentas were found to be normal. three placentas were delivered from pregnancies in which the mothers had acute sars-cov infection-these were abnormal and demonstrated increased subchorionic and intervillous fibrin, a finding that can be associated with abnormal maternal blood flow to the placenta. in the placentas of 2 women who were convalescing from sars-cov infection in the 3rd trimester of pregnancy the placentas were highly abnormal. they showed extensive fetal thrombotic vasculopathy with areas of avascular chorionic villi-chronic findings of fetal vascular malperfusion. these 2 pregnancies also were complicated by oligohydramnios and had poor obstetrical outcomes-both infants had developed iugr. it is interesting that villitis, the microscopic finding of inflammation of the chorionic villi that is the histologic hallmark of many maternal hematogenous infections that are transmitted through the placenta to the fetus, was not identified in any of these placentas. similar to other coronavirus infections, sars-cov is easily spread from person-to-person via respiratory droplets and secretions as well as through nosocomial contacts [42, 43] . in addition to transmission of sars-cov through natural aerosols from infected patients, it was found that in hong kong the sars-cov could also be transmitted by mechanical aerosols [44] . environmental factors had an important role when it was discovered that during the amoy gardens housing estate outbreak as many as two-thirds of infected persons had diarrhea, sars-cov was excreted in their stools, and that aerosols arising from the flushing of toilets could transmit the virus [44] . healthcare facilities were also an important source of new sars infections during the 2002-2003 epidemic, and healthcare workers were also at high risk for acquiring the infection. in order to address the safety issues for the obstetrical management and delivery of pregnant women with sars, guidelines were prepared by the canadian task force on preventive health care and the society of obstetricians and gynaecologists of canada [45] . these recommendations include: 1. "all hospitals should have infection control systems in place to ensure that alerts regarding changes in exposure risk factors for sars or other potentially serious communicable diseases are conveyed promptly to clinical units, including the labour and delivery unit. at times of sars outbreaks, all pregnant patients being assessed or admitted to the hospital should be screened for symptoms of and risk factors for sars. upon arrival in the labour triage unit, pregnant patients with suspected and probable sars should be placed in a negative pressure isolation room with at least 6 air exchanges per hour. all labour and delivery units caring for suspected and probable sars should have available at least one room in which patients can safely labour and deliver while in need of airborne isolation. if possible, labour and delivery (including operative delivery or caesarean section) should be managed in a designated negative pressure isolation room, by designated personnel with specialized infection control preparation and protective gear. 5. either regional or general anaesthesia may be appropriate for delivery of patients with sars. neonates of mothers with sars should be isolated in a designated unit until the infant has been well for 10 days, or until the mother's period of isolation is complete. the mother should not breastfeed during this period. 7. a multidisciplinary team, consisting of obstetricians, nurses, pediatricians, infection control specialists, respiratory therapists, and anaesthesiologists, should be identified in each unit and be responsible for the unit organization and implementation of sars management protocols. 8. staff caring for pregnant sars patients should not care for other pregnant patients. staff caring for pregnant sars patients should be actively monitored for fever and other symptoms of sars. such individuals should not work in the presence of any sars symptoms within 10 days of exposure to a sars patient. 9. all health care personnel, trainees, and support staff should be trained in infection control management and containment to prevent spread of the sars virus. 10. regional health authorities in conjunction with hospital staff should consider designating specific facilities or health care units, including primary, secondary, or tertiary health care centers, to care for patients with sars or similar illnesses." middle east respiratory syndrome (mers) was first reported in september 2012 in saudi arabia, following isolation of mers-cov from a male patient who died months earlier from severe pneumonia and multiple organ failure [1] . in the 8 years since then, there have been more than 2494 confirmed cases of mers resulting in upwards of 858 deaths globally [46] . while 27 countries have reported cases of mers, approximately 80% of confirmed cases originated in saudi arabia [47] . to date, all known cases of mers can be linked to travel or residence in countries along the arabian peninsula-that is, bahrain; iraq; iran; israel, the west bank, and gaza; jordan; kuwait; lebanon; oman; qatar, saudi arabia; syria; the united arab emirates (uae); and yemen [48] . the largest documented outbreak outside of this region occurred in 2015 in the republic of korea, in which 186 infections occurred, resulting in 38 deaths [49] . the index case in this outbreak reportedly returned from the arabian peninsula just prior to onset of illness [50] . mers-cov is characterized by sporadic zoonotic transmission events as well as spread between infected patients and close contacts (i.e., intra-familial transmission) [51] . nosocomial outbreaks in health care settings-the result of poor infection control and prevention-are widely recognized as the hallmark of mers [1] . superspreading events have been recorded in healthcare settings in jordan, al hasa, jeddah, abu dhabi and south korea [47, [52] [53] [54] [55] . like other coronaviruses, mers-cov can be spread through person-to-person contact, likely via infected respiratory secretions [48] . transmission dynamics, however, are otherwise poorly understood [1] . bats are believed to be the natural reservoir of mers-cov, and dromedary camels can have the virus and have been suggested as possible intermediary hosts as well as a source of infection to humans [2, 56, 57] . there are no clinical or serological reports of perinatal transmission of mers, though vertical transmission has been reported for non-coronavirus respiratory viruses including influenza and respiratory syncytial virus (rsv) [58] . researchers have not yet discovered ongoing transmission of mers-cov within communities outside of health care settings. the clinical presentation of mers varies from asymptomatic to severe pneumonia with acute respiratory distress syndrome (ards), septic shock, and multiple organ failure, often resulting in death. most patients with mers develop severe acute respiratory illness accompanied by fever, cough, and shortness of breath [50] . progression to pneumonia is swift-usually within the first week -and at least one-third of patients also present with gastrointestinal symptoms [1] . mers progresses much more rapidly to respiratory failure and has a higher case fatality rate than sars [1] . unlike sars, however, infection with mers-cov is generally mild in healthy individuals but more severe in immunocompromised patients and people with underlying comorbidities [1] . the overall cfr of mers is approximately 34.4% [46] . most fatalities have been associated with pre-existing medical conditions like chronic lung disease, diabetes, and renal failure, as well as weakened immune systems [59] , making such individuals high risk. as a result of the immunological changes that occur during pregnancy, women who are pregnant are included in this high-risk group. pregnant women may develop severe disease and fatal maternal and/or fetal outcomes as a result of mers-cov infection; however, little is known of the pathophysiology of this infection during pregnancy. limited data exists on the prevalence and clinical features of mers during pregnancy, birth, and the postnatal period. it is likely, however, that the immunological changes that normally occur in pregnancy may alter susceptibility to the mers-cov and the severity of clinical illness [60] . pregnant women infected with sars-cov, a related coronavirus, appear to have increased morbidity and mortality when compared to non-pregnant women, suggesting that mers-cov could also lead to severe clinical outcomes in pregnancy. to date, however, very few pregnancy-associated cases (n = 11) have been documented, with 91% having adverse clinical outcomes. between november 2012 and february 2016, there were 1308 cases of mers reported by the saudi arabia ministry of health (moh). of these, 5 patients were pregnant, according to a retrospective study by assiri et al. [47] , and all resulted in adverse outcomes. patient ages ranged from 27 to 34 years, with occurrence of exposure in either the 2nd or 3rd trimester. all 5 cases received intensive care. two women died and there were 2 cases of perinatal death-1 stillbirth and 1 neonatal death shortly after emergency cesarean section. these instances of severe maternal and perinatal outcomes are consistent with other reports of mers-cov infection in pregnant women, as well as outcomes associated with sars-cov infection. the authors of the retrospectives study concede that unreported cases of mers in pregnancy are likely due to lack of routine pregnancy testing [47] . they conclude that pregnancy testing for women of reproductive age should be considered for those who test positive for mers-cov, to contribute to overall understanding of pathogenesis and epidemiological risk. additionally, 2 of the 5 patients were healthcare workers, which corresponds with existing knowledge of higher risk of exposure to mers-cov in healthcare settings. in a separate case report of mers occurring in pregnancy, alserehi et al. [58] described a 33-year-old critical care nurse who became infected during the 3rd trimester in the midst of a large hospital outbreak. in the days following hospital admission, she developed respiratory failure necessitating mechanical ventilation and administration of dexamethasone as prophylaxis for the fetus. following an emergency cesarean section at 32 weeks gestation, she was transferred to the intensive care unit (icu) and later recovered. the preterm but otherwise healthy infant was kept in the neonatal unit for observation and later released along with his mother. in contrast to other reported cases, this patient had a successful outcome, perhaps due to the timing of mers-cov exposure, her young age, the use of steroids, and differences in immune response. alfaraj et al. [61] described 2 cases of maternal infection with mers-cov at the prince mohammed bin abdulaziz hospital (pmah) in saudi arabia. maternal infection in both cases was confirmed by nasopharyngeal swab testing by rt-pcr. one patient was a 29-year-old woman at 6 weeks gestation with no underlying medical conditions. the second patient, a 39-year-old at 24 weeks gestation, had several comorbidities, including end stage renal disease, hypertension, and hemodialysis. this woman presented to the hospital after contact with a mers-cov-infected person during an active outbreak. both patients later tested negative for mers-cov and were subsequently discharged. the younger patient delivered a healthy, full-term infant. the status of the other delivery is unknown. neither fetus was tested for mers-cov. according to payne et al. [62] , epidemiologic investigation of the 2012 mers outbreak in zarqa, jordan, revealed that a 2nd trimester stillbirth (5 months gestational age) had occurred as a result of maternal exposure to mers-cov. the mother experienced fever, fatigue, headache and cough, concurrently with vaginal bleeding and abdominal pain. on the 7th day of symptoms, she had a fetal death. the mother was confirmed to have antibody to mers-cov, and she self-reported having had unprotected contact with family members who later tested positive for the virus. this was the first documented occurrence of stillbirth during maternal infection with mers-cov. on 24 november 2013, a 32-year-old pregnant woman in the united arab emirates (uae) developed ards following admission to the icu after suspected community-acquired pneumonia advanced to respiratory failure and hypotension [60] . later that day, her baby was delivered by caesarean section and subsequent apgar scores were within healthy range. the next day, rt-pcr evaluation revealed that the mother was positive for mers-cov. despite rigorous intervention, including oral ribavirin-peginterferon-α therapy and ventilator support, the woman continued to deteriorate, developed septic shock, and died. while the outcome for this mother was fatal, malik et al. noted that virus shedding ceased during therapy with ribavirin and peginterferon-α and radiographic evidence indicated clinical improvement before her death [58] . more research is needed to determine safety, efficacy, and dosage of these therapies in the general population but also in pregnant women. while few data exist on the effects of these treatments in pregnant humans, ribavirin is generally contraindicated during pregnancy [58] . outside of the middle east the only confirmed case of mers in pregnancy occurred in 2015 in south korea. jeong et al. [49] reported that a 39-year-old patient was exposed during the 3rd trimester following contact with a patient having mers. despite abrupt vaginal bleeding and rupture of membranes, the patient recovered fully and delivered a healthy infant at 37 weeks and 5 days gestation. subsequent testing of the infant's blood did not detect any igg, igm, or iga antibodies to mers-cov. the mean maternal age of the 11 confirmed maternal sars cases described above was 33.2 years, with a mean gestational age of 26.3 weeks. the source of infection in 2 of the cases was attributed to contact with family members who tested positive for mers-cov, unknown in 3 cases, likely due to animal exposure in 1 case, and 6 were healthcare-associated (2 of these patients were healthcare workers). six patients required intensive care and 3 died. of those who died, 2 were exposed to mers-cov in the 3rd trimester, and 1 was exposed during the 2nd trimester. the infant death rate for all 11 cases was 27%. fetal survival did not appear to correlate with the timing of maternal infection and gestational age; however, more data are needed to draw conclusions about this relationship. according to alfaraj et al. [61] , the cfr for the 11 infected women-also 27%-was not statistically different from the overall cfr of mers in the general population (35%) (p = 0.75). only 1 case resulted in both maternal and fetal death. similar to sars in pregnancy, more research is needed to understand the pathogenesis and epidemiology of mers in pregnancy including the relationship between the timing of maternal infection, gestational age of the fetus, the effects of comorbid factors, and the occurrence of adverse outcomes. few studies documented the presence of mers-cov antibodies in the umbilical cord or neonatal blood, making it difficult to assess perinatal transmission. as such, future studies should involve the collection of samples from relevant specimens including amniotic fluid, placenta, and umbilical cord [49] . mers prevention should be high priority for high-risk exposures such as healthcare workers, pregnant women and individuals working with camels, camel meat-milk processors and in abattoirs [57] . since 2013, the saudi arabia moh has recommended that pregnant women postpone travel to saudi arabia for the hajj and umrah [47] . to further reduce risk of exposure among pregnant women, additional measures such as avoiding contact with camels and sick persons-particularly in healthcare settings-are also recommended. pregnant women who present with symptoms of pneumonia, influenza-like illness (ili), or sepsis on the arabian peninsula may also benefit from mers-cov screening to expedite early diagnosis and improve disease management [60] . while multiple agents have been used to treat mers, none have been tested in large clinical studies. available data are limited to the use of combination therapies of interferon and other agents in case reports and case series [63] . a prospective or randomized study may prove difficult given the sporadic nature of mers-cov outbreaks. due to a gap in research on the treatment of mers in pregnancy, there are no therapeutic options currently recommended for pregnant women [58] . therapies under development and testing may be considered inappropriate for pregnant women due to the unknown potential for teratogenic effects. for example, during the 2003 sars outbreak, ribavirin was administered to pregnant women with severe cases of the disease, but ribavirin therapy has been documented to increase the risk of teratogenic effects in newborns [58] . the alphacoronaviruses hcov 229e and nl63, as well as the betacoronaviruses hku 1 and oc43, can infect humans and cause the common cold. in order to investigate the potential maternal-fetal transmission of human coronaviruses during pregnancy, gagneur et al. [64, 65] evaluated 3 types of maternal-infant paired specimens that included maternal vaginal and respiratory specimens that were obtained during labor, as well as gastric samples from the newborn infants. these specimens were evaluated for the presence of hcov 229e, oc-43, nl63 and hku 1 using rt-pcr methodology. between the period from july 2003 to august 2005 the authors examined 159 mother-infant dyads. human coronaviruses were identified in 12 samples (hcov 229e: 11; hku 1 : 1) from 7 mother-child pairs. in 3 mother-infant dyads only maternal respiratory samples were positive; in 2 other pairs all 3 of the samples tested positive for human coronavirus; in 1 case only the maternal vaginal and newborn gastric samples were positive; and in another case the maternal vaginal sample alone was positive. there were no signs of clinical infection in any of the 3 neonates that had positive gastric samples for human coronavirus. it is beyond the scope of this communication to discuss the various technical challenges inherent in developing a safe and efficacious vaccine for coronavirus infections in humans. there are clearly challenges to this endeavor-protective antibodies to coronaviruses are not long-lasting, tissue damage has been reported to occur as a result of exposure to sars-cov, development of animal models that closely resemble human infection are limited, and the extensive time and expense necessary to perform clinical trials in humans, to name a few [66] [67] [68] . it is vitally important that pregnant women be considered in the design, clinical trial, and implementation of vaccine candidates for 2019-ncov. in examining the history of vaccine design, it is clear that the needs of pregnant women have rarely been prioritized in either the preclinical development or the clinical trial phases of production. today, pregnant women are usually excluded from experimental trial of drugs and vaccines that do not target obstetric conditions [69] . excluding pregnant women and their infants from participation in vaccine development and implementation undermines ethical principles of justice-fairness, equity, and maximization of benefit-and potentially places their health at risk during outbreaks and other health emergencies [69] [70] [71] . on 23 january 2020 the coalition for epidemic preparedness innovations (cepi) announced three programs to develop a vaccine against the novel wuhan coronavirus. the chief executive officer of cepi, richard hatchett, said [72] : "given the rapid global spread of the ncov-2019 virus the world needs to act quickly and in unity to tackle this disease. our intention with this work is to leverage our work on the mers coronavirus and rapid response platforms to speed up vaccine development." the novel coronavirus is the first epidemic disease to emerge since the formation of cepi in davos in 2017. cepi was created with the express intent to enable speedy research and development of vaccines against emerging pathogens. in may 2017, who released the target product profile (tpp) for mers-cov vaccines, following the prioritization of mers-cov as one of eight priority pathogens for prevention of epidemics [73] . cepi and partners aim to use existing platforms-that is, the existing "backbone" that can be adapted for use against new pathogens-that are currently in preclinical development for mers-cov vaccine candidates. following the who declaration on 30 january that the current 2019-ncov outbreak is a public health emergency of international concern (pheic), global health organizations and researchers will be further mobilized-bolstered by new mechanisms for action and greater resources-to stop the spread of disease. a critical question that must be answered at this stage-with a clear view of the potential deleterious effects of a new coronavirus in pregnancy-is will maternal immunization be a priority in research and development? as of the pheic declaration, 12 groups have announced that they are developing new vaccines against 2019-ncov and seven others announced initiatives to develop new therapies [74] . safe testing of experimental vaccines in a pregnant population is difficult and, as a result, vaccines are not typically developed with pregnant women in mind. to date, very few clinical trials for vaccines have proactively included pregnant women [75] , and the exclusion of pregnant and lactating women from receiving the rvsv-zebov vaccine through 3 ebola virus epidemics serves as a recent example [69] [70] [71] . given the potential severity in pregnancy, as demonstrated by this review of maternal infections of sars and mers, women who are pregnant should be considered a priority population in all efforts to prepare for and prevent infection by novel coronaviruses. on 5 february 2020 it was reported by multiple media outlets that a newborn infant delivered during the epidemic in wuhan had tested positive for 2019-ncov at the wuhan children's hospital in hubei province 30 hours following its birth. according to the official xinhua news agency, the infant was delivered on 2 february to a mother who had tested positive for the virus. reports have stated that the infant had stable vital signs, no fever or cough, but had shortness of breath together with abnormal chest radiographs and abnormalities of liver function [76] [77] [78] . dr. zeng lingkong, chief physician at the neonatal medicine department of the hospital, said [78] , "this reminds us to pay attention to mother-to-child being a possible route of coronavirus transmission" the hospital also provided information about a previous case of a baby that had been delivered on 13 january 2020. following its birth, the infant's nanny was diagnosed with 2019-ncov, and the mother was diagnosed days later [76] . on 29 january the baby began to develop symptoms. according to dr. zeng lingkong [76] , "whether it was the baby's nanny who passed the virus to the mother who passed it to the baby, we cannot be sure at the moment. but we can confirm that the baby was in close contact with patients infected with the new coronavirus, which says newborns can also be infected" in considering whether these and future cases of neonatal infection are acquired prior to delivery, it is important to remember that newborn infants can acquire an infection in other ways beyond intrauterine maternal-fetal transmission. in some cases, viral infection can be acquired when the infant passes through the birth canal during a vaginal delivery or through post-partum breast feeding, although these mechanisms would be highly unusual for a respiratory virus. neonatal infection from respiratory viruses can occur after delivery through such mechanisms as inhalation of the agent through aerosols produced by coughing from the mother, relatives or healthcare workers or other sources in the hospital environment. based upon past experience with pregnant women who developed mers and sars, and realizing that the numbers are limited, there has never been confirmed intrauterine coronavirus transmission from mother to fetus. discussing the most recent baby to be diagnosed with the 2019-ncov infection, dr. stephen morse, an epidemiologist at the mailman school of public health at columbia university stated [77] , "it's more likely that the baby contracted the virus from the hospital environment, the same way healthcare workers get infected by the patients they treat," "it's quite possible that the baby picked it up very conventionally-by inhaling virus droplets that came from the mother coughing." and according to dr. paul hunter, professor of medicine at the university of east anglia [79] , "as far as i am aware there is currently no evidence that the novel coronavirus can be transmitted in the womb. when a baby is born vaginally it is exposed to the mother's gut microbiome, therefore if a baby does get infected with coronavirus a few days after birth we currently cannot tell if the baby was infected in the womb or during birth." there is limited knowledge regarding coronavirus infections that occur during pregnancy-what is known has, for the most part, been the result of epidemics resulting from two different diseases, sars and mers. these previous experiences with coronavirus infections in pregnancy indicates that these agents are capable of causing adverse clinical outcomes including life-threatening maternal disease that in some cases requires hospitalization, intensive care and ventilatory support. both of these coronaviruses can result in maternal death in a small but significant number of cases, but the specific risk factors for a fatal outcome during pregnancy have not been clarified. coronaviruses can also result in adverse outcomes for the fetus and infant including intrauterine growth restriction, preterm delivery, admission to the icu, spontaneous abortion and perinatal death. unlike some viral infections, notably ebola virus [70] and zika virus [80] , the likelihood of intrauterine maternal-fetal transmission of coronaviruses is low-there have been no documented cases of vertical transmission occurring with either sars or mers. it remains to be seen during the current wuhan 2019-ncov epidemic how this newly-emergent coronavirus affects pregnant women and their infants, as well as which factors may modulate obstetrical disease and outcomes including the timing of maternal coronavirus exposure by gestational age, the effects of medications or other treatment regimens, differences in host immune responses, occurrence of coexisting medical and obstetrical conditions, and other covariables. however, pregnant women should be considered to be at high risk for developing severe infection during this current outbreak of 2019-ncov. additional clinical research on the treatment of sars, mers, and the new coronavirus 2019-ncov is necessary if we are to understand the potential risks and benefits of novel therapies and new vaccines in pregnancy. this research will be critical in improving the care, and even saving the lives, of pregnant women in the current as well as future outbreaks. epidemic and emerging coronaviruses (severe acute respiratory syndrome and middle east respiratory syndrome) origin and evolution of pathogenic coronaviruses coronaviridae. available online another decade, another coronavirus severe acute respiratory syndrome: historical, epidemiologic, and clinical features early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china wuhan seafood market may not be source of novel virus spreading globally clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet available online: https://www. who.int/news-room/detail/30-01-2020-statement-on-the-second-meeting-of-the-international-healthregulations-(2005)-emergency-committee-regarding-the-outbreak situation report-20 declares global emergency as wuhan coronavirus spreads antepartum pneumonia in pregnancy risk factors associated with the increasing prevalence of pneumonia during pregnancy pneumonia during pregnancy: has modern technology improved maternal and fetal outcome? indirect obstetric deaths in the state of michigan 1960-1968 pneumonia during pregnancy emerging infections and pregnancy immunobiologic adaptations of pregnancy acute respiratory failure in pregnancy due to staphylococcal pneumonia pregnancy and the lung influenza occurring in pregnant women; a statistical study of thirteen hundred and fifty cases observations on excess mortality associated with epidemic influenza learning from sars: renewal of public health in canada-sars in canada: anatomy of an outbreak reporting incidence of severe acute respiratory syndrome (sars) appendix a: chronology consensus document on the epidemiology of severe acute respiratory syndrome (sars) severe acute respiratory syndrome and pregnancy pregnancy and perinatal outcomes of women with severe acute respiratory syndrome infection control for sars in a tertiary neonatal centre 225-management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) a case-controlled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome clinical analysis of pregnancy in second and third trimesters complicated severe acute respiratory syndrome sars during pregnancy, united states severe acute respiratory syndrome in pregnancy infants born to mothers with severe acute respiratory syndrome severe acute respiratory syndrome (sars) in neonates and children the placentas of patients with severe acute respiratory syndrome: a pathophysiological evaluation identification of severe acute respiratory syndrome in canada advisors of expert sars group of hospital authority. effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome the sars epidemic in hong kong: what lessons have we learned? maternal fetal medicine committee; infectious disease committee. management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia middle east respiratory syndrome (mers) fact sheet mers-cov infection in pregnant woman in korea family cluster of middle east respiratory syndrome coronavirus infections epidemiological findings from a retrospective investigation mers-cov outbreak in jeddah-a link to health care facilities transmission of middle east respiratory syndrome coronavirus infections in healthcare settings middle east respiratory syndrome: what we learned from the 2015 outbreak in the republic of korea middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome an overview of the latest infectious diseases around the world middle east respiratory syndrome coronavirus during pregnancy middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of literature stillbirth during infection with middle east respiratory syndrome coronavirus update on therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) vertical transmission of human coronavirus. prospective pilot study materno-fetal transmission of human coronaviruses: a prospective pilot study emerging respiratory viruses: challenges and vaccine strategies duration of antibody responses after severe acute respiratory syndrome immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus clinical trials and administration of zika virus vaccine in pregnant women: lessons (that should have been) learned from excluding immunization with the ebola vaccine during pregnancy and lactation being pregnant during the kivu ebola virus outbreak in dr congo: the rvsv-zebov vaccine and its accessibility by mothers and infants during humanitarian crises and in conflict areas maternal and infant death and the rvsv-zebov vaccine through three recent ebola virus epidemics-west africa, drc équateur and drc kivu: 4 years of excluding pregnant and lactating women and their infants from immunization coalition for epidemic preparedness innovations. cepi to fund three programmes to develop vaccines against the novel coronavirus a dozen vaccine programs underway as who declares coronavirus public health emergency. biocentury who. who target product profiles for mers-cov vaccines when is it acceptable to vaccinate pregnant women? risk, ethics, and politics of governance in epidemic crises chinese baby tests positive for coronavirus 30 hours after birth a pregnant mother infected with the coronavirus gave birth, and her baby tested positive 30 hours later coronavirus: doctors fear pregnant women can pass on illness after newborn baby is diagnosed expert reaction to newborn baby testing positive for coronavirus in wuhan zika virus infection in pregnancy, microcephaly and maternal and fetal health-what we think, what we know, and what we think we know this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-332268-x30svp5y authors: bearden, donna m.; aiken, patricia b.; cheng, yu hsin; mai, emily; peters, timothy m. title: covid-19: a primer for healthcare providers date: 2020-05-20 journal: wien klin wochenschr doi: 10.1007/s00508-020-01678-x sha: doc_id: 332268 cord_uid: x30svp5y according to the world health organization (who) the china office was first notified of cases of atypical pneumonia in wuhan city on 31 december 2019. a viral genome sequence of a novel coronavirus, currently termed sars-cov‑2, with a disease process called covid-19 was released 1 week later via online resources to obtain public health support in control of spread. since then, the virus rapidly evolved into a global pandemic. therefore, healthcare providers need to be familiar with the clinical presentation of infected patients and measures to quickly isolate them. the prevention of nosocomial spread is paramount to proper control of covid-19 and is reviewed. currently, treatment is supportive. researchers are working to develop vaccines and identify effective antiviral interventions. those recently discussed in the literature are briefly reviewed. coronaviruses include a large number of viruses found in animals. human coronaviruses were first identified in the 1960s, isolated from patients with mild upper respiratory infections [1] . since then, additional human coronaviruses have been discovered, including the causative agents of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). the first human coronaviruses upper respiratory infections in humans. in immunocompromised hosts and children, however, they can result in severe pneumonia and bronchiolitis [1] . the sars represented the first pandemic of a coronavirus. it started in guangdong province in china in 2002 [2] . severe atypical cases of pneumonia emerged and quickly spread worldwide. investigation by epidemiologists suggested that animal to human transmission occurred at live game markets in guangdong. small mammals such as racoon dogs and palm civets likely transmitted the disease to humans. the natural hosts are bats [2] . eventually, public health systems and governments were able to contain the outbreak and bring it to a close. the world health organization (who) reported 8422 cases of sars worldwide, spread out among 32 countries. they reported 916 deaths from the virus, with a fatality rate of 10-15% [1] . a recent report in jama also suggested a 10% fatality rate and estimated that 20-30% of infected patients required mechanical ventilation [2] . as expected, the fatality rate was higher among the aged and those with comorbidities. perhaps most importantly, the sars outbreak showed that animal coronaviruses could spread to humans. the coronavirus has also demonstrated the ability to emerge into new hosts and then cause novel severe disease. in 2012, the mers coronavirus was discovered in saudi arabia, isolated from the sputum of a man who died from a severe respiratory syndrome. the illness was subsequently termed mers and since then 2494 cases have been reported, resulting in 858 deaths. this yields a higher case fatality rate than sars, at 36%, with mechanical ventilation needed in 50-89% of patients [2] . the mers is en-demic in camels, and in the middle east and east africa it continues to cause human infections [3] . to date, mers has spread to 20 countries by air travel [4] . in 2018, a single patient caused an outbreak of 186 cases in south korea, while a more recent case identified in an emergency department patient, was contained due to rapid implementation of public health interventions [4, 5] . at outbreak onset, the chinese government rapidly initiated broad isolation measures in an attempt to contain the virus. first the city of wuhan was quarantined, then the entire hubei province, stranding 35 million residents [6] . currently, however, the disease continues to spread. a retrospective study suggested travelling by train in china yielded the highest proportion of imported cases to new provinces (69%), followed by flying (19%) and car (12%) [7] . several factors contribute to continued spread. qun et al. reported that delays between onset of illness and seeking medical attention are usually short, with 27% seeking attention within 2 days of onset; however, delays to hospitalization were longer, with 89% of patients not hospitalized until at least 5 days of illness, during which time multiple other persons are exposed to the virus [8] . they suggest "committing considerable resources to testing in outpatient clinics and emergency departments for proactive case finding, both as part of the containment strategy" [8] . qun et al.'s preliminary estimate of the incubation period supported a 14-day quarantine for exposed persons, but was based on only 10 cases [8] . subsequent reports on quarantined patients have demonstrated the disease can occasionally manifest past the observed 14-day isolation period. furthermore, it has been demonstrated that the virus can spread during the incubation period, when patients are asymptomatic [9] . it has also been detected in patients during the convalescent period. additionally, the first person in the usa with coronavirus infection shed the virus in loose stool specimens, yielding concerns for fecal-oral transmission [10] . to clean public surface areas after potential contamination, one group of researchers in china reported that previous research on sars and mers suggested that liquid solvents, such 75% ethanol, disinfectants containing chlorine , peroxyacetic acid and chloroform have the potential to inactivate sars-cov-2. they also reported ultraviolet radiation and heating to 56°c for 30 min may inactivate the virus. chlorhexidine is not recommended as a disinfectant [11] . human to human transmission was realized early in the sars pandemic. primary symptoms included fever, cough and dyspnea [2] . outbreaks were reported in hotel and housing complexes. healthcare facilities served as a reservoir for the virus, with many healthcare workers sustaining infections. it is theorized that nosocomial spread occurred because the human receptor for the sars spike glycoprotein occurs in the lower respiratory tract. hence, most patients present with pneumonia rather than upper respiratory tract symptoms. viral shedding occurs about 10 days after initial infection, when most patients were already hospitalized. then, interventions such as nebulizer treatments, oxygen, intubation and mechanical ventilation contributed to aerosolization of the virus, with increased risk of iatrogenic infection [2] . the mers also causes a severe atypical pneumonia, but patients frequently have accompanying gastrointestinal symptoms and acute renal failure. like sars, the human receptors for the mers virus are located in the lower respiratory tract, but additional receptors are found in the gastrointestinal tract and kidneys [2] . it is most important, however, to glean data from patients infected with sars-cov-2. several papers have reported on clinical presentation and characteristics of early cases in china and 1 of the latest publications studied the first 425 confirmed cases in wuhan. their median age was 59 years and 56% were male. none of the cases reported were in children below the age of 15 years [8] ; however, an increasing number of pediatric cases are now being reported from china [11] . the adult patients in the wuhan study had a mean incubation period of 5.2 days, with the 95th percentile of the distribution at 12.5 days. at onset, the epidemic grew exponentially, doubling in size every 7.4 days. the basic reproductive number was estimated to be 2.2, meaning "that on average, each patient has been spreading infection to 2.2 other people" [8] . the authors report that as long as the basic reproductive number is greater than 1, the epidemic will continue to spread. control measures are designed to decrease the reproductive number to less than 1. perhaps the most detailed study to date, shedding light on how patients may present and progress, is an analysis of the first 99 cases of confirmed novel corona pneumonia in wuhan [12] . infections were confirmed by obtaining throat swab specimens from all patients on admission and testing for the virus using real-time polymerase chain reaction protocols previously described [13] . of the cases studied, 68% were men and 32% were women, with a mean age of 55.5 years. only a slight majority of patients (51%) had chronic diseases, the majority being cardiovascular or cerebrovascular diseases [12] . covid-19: a primer for healthcare providers k main topic presenting symptoms were fever in 83% of patients and cough in 82%. shortness of breath on admission was present in 31%. other presenting symptoms in 4% or more of admitted patients included: myalgia, headache, confusion, sore throat and rhinorrhea. only 1-2% of patients reported nausea and vomiting, or chest pain or diarrhea and 90% of patients had more than one sign or symptom present on admittance [12] . an analysis of laboratory values of infected patients showed the erythrocyte sedimentation rate was elevated in 85% of patients on admittance and the c-reactive protein was elevated in 86%. surprisingly, the procalcitonin level was only increased in 6% of patients with confirmed covid-19 pneumonia. a low albumin was identified in 98% of patients admitted, an elevated lactate dehydrogenase in 76% of patients and 51% had a decreased hemoglobin [12] . based on chest x-ray and computed tomography (ct) results, 75% of patients had bilateral pneumonia at presentation, and 25% had unilateral pneumonia. during hospitalization, 76% of patients required oxygen therapy, 13% required noninvasive mechanical ventilation and 4% required invasive mechanical ventilation. at the time the study was released, 31% of patients had been discharged, and 11% had died. all other patients were still hospitalized [12] . discharge criteria suggested by pediatricians in china include children with a normal body temperature for at least 3 days, significant improvement in respiratory symptoms and completion of two consecutive negative tests of respiratory respiratory pathogenic ribonucleic acid. they suggested sampling intervals of at least 1 day. if patients meet these criteria, they are considered safe for discharge. if needed, home isolation for 14 days can be advised [11] . a retrospective study from china analyzed chest ct scans from 21 symptomatic patients with confirmed covid-19 [14] . consistent with clinical findings among infected a patients, radiographic finding demonstrated most patients (76%) had bilateral involvement on ct and in 71% more than 2 lung lobes were involved. the most frequent ct findings included bilateral pulmonary parenchymal groundglass and consolidative pulmonary opacities (57%). lung consolidation was observed in 29%. lung cavitation, discrete pulmonary nodules, pleural effusions and lymphadenopathy were absent in all of the scans. follow-up imaging in eight patients during the study period demonstrated mild or moderate progression of disease [14] . other clinicians in china also studied chest ct scans and confirmed typical multilobar involvement on presentation, but summarized "when one or two lobes are involved, the effect on lung function is not serious, and the symptoms of shortness of breath and dyspnea are not severe" [15] . if the disease progresses, however, the patient will develop a "white lung" with diffuse alveolar damage involving multiple lobes [15] . the clinical characteristics associated with the development of acute respiratory distress syndrome (ards) and death after admission for covid-19 were recently described in a retrospective cohort study [16] . older age, hypertension and development of fever greater than 39°c was associated with ards, but higher fever was also associated with better outcomes. increasingly, it is recognized that other organ systems, besides the pulmonary system, can be adversely affected by infection with sars-cov-2. a retrospective, observational analysis of 214 confirmed cases of covid-19, specifically studied those with severe pneumonia, using criteria established by the american thoracic society [17] . of those patients with severe pneumonia, 36.4% had neurological manifestations of disease, such as acute cerebrovascular disease (5.7%), impaired consciousness (14.8%) and skeletal muscle injury (19.3%). coagulation disorders and cardiac manifestations of covid-19 infection have also been reported [18, 19] . a recent manuscript out of canada provided an extensive review of measures designed to prevent the nosocomial spread of the disease [20] . they stressed the importance of identifying and isolating patients infected with sars-cov-2 early. currently, suspicion requires fever and symptoms of respiratory illness. with recent community spread, identifiable links to the virus may be difficult to discern and a high index of suspicion should be maintained. failure to identify infected patients has led to preventable dissemination in prior pandemics [21] . the primary method of transmission for sars-cov-2 is via contact/droplet spread, related to respiratory conditions; however, as with sars, airborne transmission can occur. there are at least two reports of sars-cov-2 being isolated from stool specimens, so fecal-oral transmission is considered possible as well [10, 22] . currently, the canadian public health agency advises placing patients with confirmed covid-19 or patients with suspected infection who are ill, in airborne isolation. anterooms with space to put on and remove personal protective equipment should be adjacent to these rooms. if an airborne isolation room is not available, the patient should be placed in a single room with closed doors [23] . in the care of critically ill patients, where airborne transmission is possible, recommended personable protective gear includes fluid-resistant gowns, gloves, eye protection, full face shield and fit-tested n-95 respirators. hair covers or hoods should also be worn. longer sleeved gloves are preferred to prevent wrist exposure in the case of glove slippage. if necessary, vertical tape strips can be applied to keep gloves in place. circumferential taping is not advised as it may make glove and gown removal more difficult. full face protection with a shield is preferred. if not available, goggles or side shields are needed. scrubs or full coveralls should be worn under the personal protective gear. shoes worn should be impermeable to fluids and capable of being decontaminated. shoe covers can increase the risk of self-contamination during removal. strict hand hygiene must be performed after removal of protective equipment. wax and christian advised that infection control coaches should be used in some circumstances of equipment removal, such as after a "code blue." the coaches go through a checklist as the equipment is removed, to reduce the risk of self-contamination [20] . in fact, during the sars outbreak the "protected code blue" was developed and published to guide healthcare workers in proper resuscitation techniques for infected patients. online demonstrations of the procedure have been cited by candadian researchers [20] . it is advised that team members entering an infected patient's room be restricted to four, with team members bringing the defibrillator, medications and equipment needed in modular packs, rather than an entire cart [20] . healthcare personnel arriving at the patient's room prior to the specialized response team can assist the patient with resuscitation interventions that have a low risk of viral transmission, such as placing an oral airway, placing an oxygen mask with an exhalation filter, chest compressions, defibrillation and cardioversion, obtaining intravenous or intraosseous access and administering drugs. higher risk interventions which may generate aerosol and increase risk for infection to staff and nosocomial spread include high flow nasal cannula, bag-mask ventilation, cpap/bipap (continuous positive airway pressure/ bilevel positive airway pressure), and endotracheal intubation [20] . the authors also note that during the sars outbreak, there were "case reports of considerable sars transmission risk with the use of bipap to many patients over extended periods" [20] . they concluded, in general cpap/bipap should be avoided in patients with covid-19 and should never be used outside of airborne/droplet isolation [20] . they also note that high flow nasal cannula delivery systems may cause an increased risk of viral spread and practitioners should consider avoidance of humidified oxygen in covid-19 patients as well. bronchodilators should preferably be administered by metered dose inhalers [20] . pediatricians in china have also provided recommendations for personal protective gear [11] . they stated: 1. all medical personnel are required to wear surgical masks during medical activities. 2. those in triage areas wear medical overalls, caps and surgical masks. 3. in the emergency department and clinics where exposure is likely: medical overalls, caps, disposable clothing, surgical masks and goggles or face shields for daily rounding. when collecting body fluid samples, latex gloves, impermeable clothing and respiratory hood should be used when needed to prevent contamination by aerosolization or splash. 4. all personal protective equipment should be worn and removed with a strict on-off procedure, and personnel should not leave the ward with contaminated equipment. 5. patients and their accompanying family members are required to wear surgical masks [11] . currently, no effective vaccines or drugs have been approved for clinical use, although inhibitors designed to treat infections in vivo are being developed. in order to increase the speed of development of potential treatments available, two approaches are generally employed. firstly, testing of current antiviral drugs and medications used to treat other infections or other disease states. the second approach is to develop novel agents based on current information and understanding of the particular coronavirus targeted [1] . several publications have recently identified associations with decreased in vitro activity against sars and mers viruses. neurotransmitter inhibitors, such as promethazine were among such agents [1] . pillaiyar et al. list additional compounds with antiviral activity in their extensive review [1] . nowak and walkowiak, in a recently released review of five in vitro studies reporting on the effect of lithium in coronavirus infections, concluded that the drug does have antiviral activity and should be explored as a potential treatment or prophylaxis for covid-19 [24] . currently, the most widely studied antiviral agents against coronaviruses are remdesivir, ribavirin, lopinavir, ritonavir, and interferon beta. ribavirin, used alone, has had no demonstrated effect against sars. when combined with lopinavir, plus rotinavir and a corticosteroid and given to patients infected with sars, those treated were less likely to develop ards, and death rates were lower than those treated with ribavirin and a corticosteroid [1] . most studies published have not shown a benefit when corticosteroids are used in coronavirus infections. in fact, the use of methylprednisolone as an intervention for sars patients was associated with a higher 30-day mortality rate [1] . a randomized, placebo-controlled study of sars patients suggested that those given steroids early in the infection developed prolonged viremia. finally, patients with mers who were treated with methylprednisolone, with or without antiviral agents or interferons, showed no improved outcomes [1] . covid-19: a primer for healthcare providers perhaps remdesivir, a broad-spectrum antiviral drug, shows the most promise for human coronavirus infections. it has superior activity against mers-cov in vitro, when tested against existing antivirals. in mouse models, when used for prophylaxis and treatment, it showed improved pulmonary lung function values, reduced lung viral loads and lung pathology findings on post-mortem examination [3] . the authors concluded "our work suggests that remdesivir may improve disease outcomes in coronavirus patients, serve to protect health care workers in area with endemic mers-cov and prove valuable in preventing future epidemics " [3] . another recent report, by a different group of researchers, also supported the use of remdesivir against coronaviruses. this group showed that remdesivir inhibited virus infection efficiently in a human cell line sensitive to sars-cov-2 [6] . the same authors tested chloroquine and concluded "remdesivir and chloroquine are highly effective in the control of covid-19 infection in vitro." they further suggested both should be assessed in human patients with covid-19 [6] . finally, neuraminidase inhibitors, such as oral oseltamivir, inhaled zanamivir and intravenous peramivir are approved antiviral treatments for influenza and mers infections; however, oral oseltamivir has been used in patients with covid-19 infections in china, but to date, there is no convincing evidence of its' effectiveness [25] . interferon has been studied in vitro against coronaviruses, but there is no evidence it will be effective against covid-19. pediatricians in china have suggested nebulized interferon be considered in infected children [11] ; however, they reported there is no data to support its' use and publications from canada advise against routine nebulizer use, noting the potential for spreading of the virus by aerosolization [20] . vaccines against covid-19 are being pursued. researchers report that the timeline has been compressed to 3.25 months for a phase i trial, using messenger rna technologies [2] . other groups are attempting to construct vaccines using viral vectors and vaccines directed at subunits [2] . despite worldwide efforts to identify methods to prevent and treat covid-19, a recent comprehensive review concluded "no therapies have been shown effective to date" [26] . currently, covid-19 continues to spread. latest estimates of the replication number remain well above two, so proliferation will continue. healthcare providers will play a major role in the early identification and isolation of infected patients, important measures to prevent dissemination. measures to prevent nosocomial spread are also paramount to control. finally, while treatment is largely supportive, the scientific community is working ceaselessly to de-velop interventions and vaccines to end this outbreak. health professionals need to stay abreast of all these developments. recent discovery and development of inhibitors targeting corona viruses corona virus infections-more than just the common cold comparativetherapeuticefficacy of remdesivir and combination lopinavir,ritonavir, and interferon beta against mers-cov middle east respiratory syndrome coronavirus (mers-cov) mers-cov outbreak following a single patient exposure in an emergency department in south korea: an epidemiological outbreak study remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro the association between domestictraintransportationandnovel coronavirus(2019-ncov) outbreak in china from 2019 to 2020: a data driven correlation report early transmission dynamics in wuhan, china of novel corona-infected pneumonia transmission of 2019-ncov infection from an asymptomatic contact in germany first case of 2019 novel coronavirus in the unites states diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus epidemiological and clinical characteristicsof 99 casesof 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan ct imaging features of 2019 novel coronavirus (2019-ncov) imaging changes in patients with 2019-ncov risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china neurological manifestations of hospitalized patients with coronacirus disease coagulopathy andantiphospholipid antibodies in patients with covid-19 cardiac involvement in a patient with coronavirus disease 2019 (covid-19) practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019ncov) patients 22. xinhuanet. novel coronavirus may spread via digestive system: experts. english.news.cn. 2020 interim national case definition: novel coronavirus (2019-ncov) is lithium a potential treatment for the novel wuhan (2019-ncov) coronavirus? a scoping review drug treatment options for the 2019-new coronavirus (2019-ncov) pharmacologic treatments for coronavirus disease 2019 (covid-19) a review key: cord-349643-jtx7ni9b authors: uyeki, timothy m.; erlandson, karl j.; korch, george; o’hara, michael; wathen, michael; hu-primmer, jean; hojvat, sally; stemmy, erik j.; donabedian, armen title: development of medical countermeasures to middle east respiratory syndrome coronavirus date: 2016-07-17 journal: emerg infect dis doi: 10.3201/eid2207.160022 sha: doc_id: 349643 cord_uid: jtx7ni9b preclinical development of and research on potential middle east respiratory syndrome coronavirus (mers-cov) medical countermeasures remain preliminary; advancements are needed before most countermeasures are ready to be tested in human clinical trials. research priorities include standardization of animal models and virus stocks for studying disease pathogenesis and efficacy of medical countermeasures; development of mers-cov diagnostics; improved access to nonhuman primates to support preclinical research; studies to better understand and control mers-cov disease, including vaccination studies in camels; and development of a standardized clinical trial protocol. partnering with clinical trial networks in affected countries to evaluate safety and efficacy of investigational therapeutics will strengthen efforts to identify successful medical countermeasures. f rom september 2012 through april 27, 2016, a total of 1,728 laboratory-confirmed middle east respiratory syndrome coronavirus (mers-cov) infections, leading to 624 deaths (36% case-fatality proportion), had been reported to the world health organization (who) (1) . most infections (75%) have been identified in saudi arabia (2) . zoonotic transmission from exposure to mers-cov-infected arabian camels, known as dromedaries, or their raw milk and limited, nonsustained human-to-human transmission have been reported, including large outbreaks in healthcare facilities (3) (4) (5) . the recovery of infectious mers-cov in virus cultures of specimens from bed sheets, bedrails, intravenous fluid hangers, and radiograph equipment indicates the potential for fomite transmission of the virus in hospitals providing care for mers-cov patients (6) . however, sustained human-to-human transmission has not been documented, and some case-patients have no identified source of exposure to mers-cov. as of april 2016, a total of 26 countries had reported locally acquired or exported cases from the arabian peninsula, including 2 cases in the united states identified during may 2014 in healthcare personnel who became ill after working in saudi arabia (7, 8) . a traveler who visited saudi arabia, qatar, the united arab emirates, and bahrain and then returned to south korea infected with mers-cov in mid-2015 triggered 184 mers-cov cases, resulting in 38 deaths in multiple health facilities and 1 additional case in a person who traveled to china (9, 10) . human infections with mers-cov are expected to continue to occur on the arabian peninsula because of the prevalence of mers-cov in dromedaries and the cultural importance of these camels (i.e., for food, milk, and racing purposes) in the region. during the 2003 outbreak of severe acute respiratory syndrome (sars) in china, civet cats, the suspected reservoir of sars coronavirus (sars-cov), were culled aggressively; no outbreaks were identified after 2004. in contrast, culling of camels is culturally impractical in the middle east, and mers-cov zoonotic infections of humans have continued since 2012. the potential for emergence of mers-cov mutations that could facilitate sustained community transmission and global dissemination cannot be predicted. no vaccines against or specific treatments for human infection with sars-cov, mers-cov, or other coronaviruses have been approved. since 2013, efforts have focused on furthering development of animal models, vaccines, and therapies against mers-cov (11,12). in this report, we update the current state of development for mers-cov medical countermeasures, including regulatory challenges in the united states, and draw attention to areas in immediate need of increased infrastructure support for development of these countermeasures. persons at higher risk for more severe disease, including persons >65 years of age and those with chronic medical conditions. therapeutic drugs with specific activity against mers-cov (e.g., antiviral drugs, immunotherapeutic treatments) or that target the host immune response could be used for treatment of human illness caused by mers-cov infection or for pre-or postexposure prophylaxis. before human clinical trials of potential mers-cov medical countermeasures are started, proof-of-concept data must be obtained from in vivo studies of experimentally infected animals. such data may indicate a product's potential efficacy and provide a mechanism for selection of available medical countermeasure candidates. in addition, mers-cov vaccines could be developed for animals and used for vaccination of dromedaries on the arabian peninsula and in source countries for camel imports to the horn of africa to reduce mers-cov transmission among camels and possibly from camels to humans. preclinical development of mers-cov medical countermeasures has been hindered by several factors, including limited data on the natural history of mers-cov infection in humans; the lack of a small animal model that is naturally susceptible to mers-cov; and the inability to consistently replicate severe human disease in mers-cov-infected nonhuman primates (nhps). another factor is limited access to clinical samples and recent virus isolates; for example, a mers-cov strain isolated from a patient in 2012, rather than a more recently isolated strain, is currently used by most investigators worldwide. small animal and nhp models are useful for testing potential medical countermeasures for efficacy (table 1) . studies in mice, both dipeptidyl peptidase-4 (dpp4 or cluster differentiation 26) transduced and transgenic, and in rabbits, hamsters, and ferrets have been reviewed elsewhere (16, 20, 21) . these small animal models have been used for screening potential mers-cov medical countermeasures (13, 14, 22) . the major nhp models under development include rhesus macaques and common marmosets (17, 18, 23) . overall, common marmosets appear to be better suited than rhesus macaques for therapeutic studies designed to target severe disease because marmosets show slightly slower onset of illness and longer duration and severity of disease and their small size requires lower doses of therapeutic drugs. however, the marmoset model has not been standardized and is not consistent between laboratories (18, 24, 25) . furthermore, the size of marmosets substantially limits sequential blood sampling for virologic or pharmacokinetic testing. challenges to the development of nhp models include determination and standardization of the optimal mers-cov challenge dose and of the volume and route of exposure, as well as the limited availability of nhps, especially marmosets. large animal models in development include camels and camelids such as alpacas (19, 26, 27) . these models may be vital in understanding the virology and immunology of mers-cov infection in dromedaries, a natural host. in addition, serologic evidence of mers-cov infection in alpacas has been reported in qatar (28) . major gaps for all animal models include a lack of consensus and availability of the optimal animal model to replicate severe human illness from mers-cov infection; limited availability of currently or recently circulating mers-cov strains; the lack of understanding of clinically relevant symptoms that can be incorporated into clinical scores or used as a signal to begin treatment in animal models; and competition for funding, laboratory space, availability of animals, and expertise with other emerging or reemerging infectious diseases, such as ebola virus disease and zika virus disease. (29, 30) . the secretary of the us department of health and human services declared a potential public health emergency on may 29, 2013, regarding mers-cov infection that could have a high potential to affect national security or the health and security of us citizens living abroad. the us food and drug administration (fda) subsequently issued an emergency use authorization to the centers for diseases control and prevention (cdc) for an in vitro molecular diagnostic test to diagnose mers-cov infection in multiple types of clinical specimens from symptomatic patients. the use of this test was later expanded to include the ability to test asymptomatic contacts of a person infected with mers-cov who traveled from saudi arabia to the united states. the cdc made this test available to multiple us public health laboratories, the us department of defense, and who laboratories worldwide. although the test has been distributed extensively, it is limited in terms of the cdc's ability to scale up the supply of reagents to support a surge in mers-cov cases in the united states and in other countries where the test has been made available. therefore, an emergency use authorization was issued on july 17, 2015, for the commercially developed realstar mers-cov rt-pcr kit u.s. (altona diagnostics gmbh, hamburg, germany) for use in the in vitro qualitative detection of mers-cov rna in tracheal aspirate or tracheal secretion samples (31) . although this commercial assay is a first step in bridging the diagnostic test availability gap in case of a surge scenario, the current coverage, at least in the united states, is insufficient until alternative, fda-cleared commercial tests are available (table 2) . a worldwide gap exists in the lack of readily available, simple, rapid, and accurate diagnostic tests for use in outpatient and inpatient clinical settings where the ability of the facility to use currently available, higher complexity molecular tests is limited. the lack of commercial development of mers-cov assays may be partially related to the limited availability of clinical specimens and mers-cov isolates from infected patients. availability of serum specimens from rt-pcrconfirmed mers-cov patients who survived can help facilitate development of serologic tests. if paired acute and convalescent serum samples are available, serologic tests can be used to confirm mers-cov infection when viral shedding is not detectable, and for surveillance purposes such as measuring population exposures and immunity to mers-cov infection. no investigational therapeutic drugs have been evaluated for treatment of mers-cov patients in prospective randomized controlled clinical trials. potential therapeutic drugs for mers-cov patients include available approved drugs with nonspecific properties, such as immunomodulators, small-molecule drugs with broad antiviral activity, repurposed fda-approved small-molecule drugs that show activity against mers-cov in vitro (table 3 ) (34, 35) , and newly developed monoclonal or polyclonal antibody therapies with specific activity against mers-cov (table 4 ) (54). one promising approach has been to investigate libraries of drugs approved by the fda and the european medicines agency. considering development times and manufacturing requirements for new products, repurposing of existing drugs might potentially facilitate a rapid response to outbreaks of emerging viruses (see regulatory section for a discussion on repurposing). other early-stage work on mers-cov therapeutics includes studies focusing on the essential viral replication steps of fusion, proteolysis, and rna polymerization (table 3 ) (54) . immunotherapeutics under evaluation consist of convalescent plasma and monoclonal and polyclonal antibodies. most of the monoclonal antibodies in development have specific neutralizing activity against the mers-cov spike protein (55, 56) . platforms are being developed to rapidly discover monoclonal antibodies, either from fully human convalescent blood or from transgenic animals, which can be manufactured on a large scale and are likely to have a good safety profile. the most advanced immunotherapeutic for mers-cov uses a transchromosomal bovine production system to produce fully human polyclonal mers-cov antibodies; a phase i study of this product was recently implemented (57; https://clinicaltrials.gov/ct2/ show/nct02788188). preliminary results from immunoprophylaxis or treatment studies have shown efficacy of fully human monoclonal or polyclonal antibodies in mers-cov-infected mice and nhps ( profile and a defined set of preclinical toxicology studies, challenges to development of immunotherapeutics include ensuring the absence of antibody-dependent enhancement of disease and reducing the risk for generation of escape mutant viruses that would be resistant to treatment. development of mers-cov candidate vaccines was initiated by the national institute for allergy and infectious diseases at the national institutes of health, academic investigators, and several companies (table 5 ). most candidate vaccines are still being evaluated in animal models. they have generally targeted the spike protein of mers-cov and are recombinant virus, subunit, dna, or virus-like vector vaccines (60,63-67). one live-attenuated mers-cov candidate vaccine is in early development (66) . preliminary studies for several other mers-cov vaccine candidates have been initiated, and early results demonstrate immunogenicity; 2 have progressed to nhp challenge, and a phase 1 clinical study in adults of 3 different doses of a dna plasmid vaccine that expresses the mers-cov spike protein was started in january 2016 (61) . ongoing assessment of antigenic evolution of circulating mers-cov strains is essential for informing vaccine development (68) . a concern that must be addressed in the development of mers-cov vaccines is the potential for causing antibody-dependent enhancement of disease upon virus challenge, such as what was observed with a sars-cov candidate vaccine upon sars-cov challenge (69) . the lack of a precedent of coronavirus vaccines for humans poses another challenge for the evaluation of mers-cov vaccines for humans, although vaccines against other animal coronaviruses are safe and in use in animals. considering the cultural importance of dromedaries on the arabian peninsula for meat, milk, and racing, prevention of camel-to-camel mers-cov transmission and reduction of spread from dromedaries to humans by camel vaccination is being investigated by government, academic, and commercial investigators (table 6 ). young camels appear to be at high risk for mers-cov infection and could be a priority group for vaccination (73, 74) ; the loss of maternal mers-cov antibodies ≈5-6 months after birth suggests a short time window for vaccination (75) . a major challenge to this approach is that dromedaries can be reinfected with mers-cov; a study by farag et al. found no correlation between mers-cov rna levels and neutralizing antibodies in camels (76) , suggesting that antibodies may not be protective against infection. because older camels can be reinfected, a camel vaccination strategy may require multiple dosing and booster vaccination to increase effectivein the united states and overseas (19) . in addition, 3 doses of a dna vaccine containing the mers-cov spike protein induced humoral immunity in dromedaries (60) . in a recent study, a modified vaccinia virus ankara vaccine that expresses the mers-cov spike protein was administered intranasally and intramuscularly to dromedaries; when challenged intranasally with mers-cov, vaccinated dromedaries had fewer signs of respiratory infection and lower mers-cov titers in the upper respiratory tract compared with unvaccinated dromedaries (77) . alpacas (new world camelids) are being investigated as a suitable proxy for camels because of the lack of available dromedaries in the united states, the high cost of acquiring dromedaries, and the relatively smaller size of alpacas (26, 27) . regulatory considerations for mers-cov medical countermeasures in the united states are focused on a pathway to human clinical trials for drugs and vaccines through submission of investigational new drug applications. investigational new drug submissions must adhere to requirements set forth in the code of federal regulations, title 21, part 312 (21 cfr 312; http://www.ecfr.gov/cgibin/text-idx?tpl=/ecfrbrowse/title21/21cfr312_main_02. tpl). several guidance documents exist on the fda website related to virology, microbiology, pharmacology and toxicology, and clinical and medical considerations (78). the most appropriate approval pathway is likely to be product-specific and will require consideration of existing product data, proposed intended use and population for use, and validated endpoints for efficacy predictive of clinical benefit, if any. likewise, data needed for consideration of an emergency use authorization, including dose finding and dose ranging, duration, and safety, can be obtained through sources such as investigational new drug clinical trials. repurposing of drugs approved by the fda for other illnesses for a mers-cov indication can potentially be expedited or accelerated if 1) the mechanism of action for antiviral activity is defined, 2) there is no change to the approved final drug form and route of administration, 3) dosing does not exceed the currently approved dose and duration for the currently indicated population and adequate pharmacokinetics data support this dosing, and 4) the risk-benefit profile is acceptable for the intended population and indication. for example, the risk-benefit profile for an approved drug with an oncology indication may be unacceptable if the drug is repurposed for administration to a healthy population for mers-cov postexposure prophylaxis. however, data requirements to initiate human trials will depend on the characteristics of the drug product and its intended use against mers-cov. as such, sponsors should consider prioritizing drug development on the basis of the totality of scientific evidence and merit of the drug alone, not on whether the drug has been previously approved. in the absence of a standardized and accepted animal model that simulates human disease from mers-cov infection, it is unclear how the fda may be able to expedite licensure or approval when data are lacking. the best approach may be collection of preclinical safety data and implementation of adaptive human clinical trials. this approach was taken for medical countermeasures in response to the 2013-2016 ebola virus disease outbreak. for diagnostic devices, the current emergency use authorization pathway serves as a fast approach to make products available for emergency public health purposes. after an emergency has been terminated, premarket notifications for these products should be submitted to fda for a more thorough evaluation as 510(k)s (http://www.fda.gov/ e6 emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 7, july 2016 medicaldevices/productsandmedicalprocedures/deviceap provalsandclearances/510kclearances/default.htm). the overarching goal for clinical research of mers-cov patients is to optimize clinical management and to identify effective therapies to improve survival. although clinical data on some mers-cov patients have been published in case series (58, 79, 80) , there is a need for much more epidemiologic, clinical, virologic, and immunologic data to improve the limited understanding of the pathogenesis of mers-cov infection in humans. gaps include information on viral load and duration of viral shedding in blood, urine, respiratory, and other clinical specimens from infected persons; understanding of the innate and adaptive immune response to mers-cov infection; pathology data on the distribution of mers-cov in respiratory and extrapulmonary tissues in fatal cases; information from autopsies of persons who died of mers-cov; and an overall improved understanding of the pathogenesis of mers-cov in humans. only 1 study has investigated mers-cov infection in autopsy tissues of a patient who died from the disease (81) . collaborations are especially needed to pool and systematically collect serial clinical specimens from mers-cov patients for virologic, immunologic, and biomarker analyses to correlate with clinical illness, and to conduct long-term follow-up of survivors of severe disease (82) (83) (84) . detailed understanding of host factors and cofactors associated with disease severity from asymptomatic infection to fatal illness is needed. efforts to promote international sharing of clinical specimens and mers-cov isolates are needed to foster development of diagnostics, therapeutics, and vaccines. use of standardized clinical data collection instruments and common biologic sampling protocols for serial prospective data collection will facilitate data pooling from mers-cov cases and comparisons across clinical sites and countries. global collaborations among clinical networks are also needed to implement clinical trials, preferably randomized controlled clinical trials, of mers-cov investigational therapeutics (82) (83) (84) (85) . without an international agreement on protocols and systematic standardization of case reporting and data collection methods, haphazard or anecdotal reporting and analysis of disease course and outcome may continue. who and the international severe acute respiratory and emerging infection consortium are collaborating in adapting standardized protocols for controlled clinical trials for mers-cov (83) . prospective controlled clinical trials (ideally randomized clinical trials) of potential mers-cov therapies and vaccines in humans are needed urgently; however, there is uncertainty in estimating timelines for the development of potential mers-cov medical countermeasures because of the need to further characterize existing and new animal models, the unpredictability of demonstrating a favorable risk-benefit outcome during preclinical testing, and competition for resources with other emerging infectious diseases. in addition, the risk for antibody-dependent enhancement of disease may interrupt the timeline for conducting human clinical trials of mers cov vaccines and immunotherapeutics. researchers of all potential mers-cov medical countermeasures should have preclinical toxicology data available before initiating human clinical trials. although animal efficacy data are not technically required before implementing human clinical trials of potential countermeasures, such data are considered important for identifying the most promising medical countermeasure candidates, justifying risk in human volunteers, and informing the design of future clinical studies. timeframes for the production of specimen panels and repositories to aid commercial diagnostic development are also contingent on obtaining adequate funding and clinical samples. although preclinical development and research on potential mers-cov medical countermeasures has achieved appreciable progress to date, such development is preliminary, and substantive challenges must be overcome before most potential countermeasures are ready for human emerging infectious diseases • www.cdc.gov/eid • vol. 22, no. 7, july 2016 e7 results of substantial progress in establishing the infrastructure and platforms for preclinical and advanced clinical development of countermeasures can serve as a model to enable more timely response to other emerging infectious diseases of global public health concern in the future. world health organization. emergencies preparedness, response: middle east respiratory syndrome coronavirus saudi arabia world health organization. summary of current situation, literature update and risk assessment hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah-a link to health care facilities risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome coronavirus (mers-cov)-republic of korea world health organization. middle east respiratory syndrome coronavirus (mers-cov)-china rapid medical countermeasure response to infectious diseases: enabling sustainable capabilities through ongoing public and private-sector partnerships rapid generation of a mouse model for middle east respiratory syndrome generation of transgenic mouse model of middle east respiratory syndrome-coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques infection with mers-cov causes lethal pneumonia in the common marmoset replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels animal models for sars and mers coronaviruses a comparative review of animal models of middle east respiratory syndrome coronavirus infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection animal models of middle east respiratory syndrome coronavirus infection treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a non-human primate model of common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas. emerg infect dis experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus. emerg infect dis mers-cov infection of alpaca in a region where mers-cov is endemic viral shedding and environmental cleaning in middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications regulatory and policy framework-emergency use authorization; middle east respiratory syndrome coronavirus (mers-vov) eus information assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections first international external quality assessment of molecular diagnostics for mers-cov screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study public summary of opinion on orphan designation. interferon alfa-n3 for the treatment of middle east respiratory syndrome systematic review of treatment effects protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 evaluation of ssya10-001 as a replication inhibitor of sars, mhv and mers coronaviruses anti-infective immunoadhesins from plants protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors anti-mers-cov convalescent plasma therapy cows with human chromosomes enlisted to fight hantavirus exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution a conformationdependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in mers-cov spike protein inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus coronaviruses-drug discovery and therapeutic options 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 development of human neutralizing monoclonal antibodies for prevention and therapy of mers-cov infections human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection evaluation of candidate vaccine approaches for mers-cov a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates phase i, open label dose ranging safety study of gls-5300 in healthy volunteers middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus camels emit dangerous mers virus, csu confirms mers surges again, but pandemic jitters ease immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice mers-cov in upper respiratory tract and lungs of dromedary camels mers coronavirus in dromedary camel herd, saudi arabia time course of mers-cov infection and immunity in dromedary camels high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in united arab emirates middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways international severe acute respiratory and emerging infection consortium. severe acute respiratory infection data tools open source clinical science for emerging infections clinical management of severe acute respiratory infection when middle east respiratory syndrome coronavirus (mers-cov) infection is suspected. interim guidance we thank john beigel, wendy carter, david cho, su-young choi, eric donaldson, heinz feldmann, barney graham, lisa hensley, cynthia kelley, peter miele, vincent munster, david spiro, kanta subbarao, and melissa willis for participating in foundational discussions and for help assessing potential therapeutic drugs and methods; vaccines under development or investigation; timelines for animal studies; scale-up of medical countermeasures; when human trials are to be started; regulatory issues for investigational new drug (ind) and emergency use authorization (eua) applications, and clinical studies; and safety issues and prioritization for clinical trials. we also thank dean erdman for insightful discussions on mers-cov diagnostic assay development, rick bright, tom dreier, and byron rippke for helpful commentary, and thuy phan for organizational assistance.dr. uyeki is the chief medical officer for the influenza division, national center for immunization and respiratory diseases, cdc. his research interests are in the epidemiology and clinical management of influenza and emerging infectious diseases. key: cord-334530-krclgmc4 authors: abroug, fekri; slim, amine; ouanes-besbes, lamia; kacem, mohamed-ali hadj; dachraoui, fahmi; ouanes, islem; lu, xiaoyan; tao, ying; paden, clinton; caidi, hayat; miao, congrong; al-hajri, mohammed mohammed; zorraga, mokhtar; ghaouar, wissem; bensalah, afif; gerber, susan i. title: family cluster of middle east respiratory syndrome coronavirus infections, tunisia, 2013 date: 2014-09-17 journal: emerg infect dis doi: 10.3201/eid2009.140378 sha: doc_id: 334530 cord_uid: krclgmc4 in 2013 in tunisia, 3 persons in 1 family were infected with middle east respiratory syndrome coronavirus (mers-cov). the index case-patient’s respiratory tract samples were negative for mers-cov by reverse transcription pcr, but diagnosis was retrospectively confirmed by pcr of serum. sequences clustered with those from saudi arabia and united arab emirates. a s of may 23, 2014, a total of 635 laboratory-confirmed human cases of middle east respiratory syndrome coronavirus (mers-cov) infections had been reported to the world health organization; the epidemic has subsequently accelerated (1) . of these patients, 193 (30%) died. this new virus causes disease similar to that caused by severe acute respiratory syndrome coronavirus, but mers-cov is genetically distinct (2) . we investigated a cluster of 3 mers-cov cases in 1 family in tunisia. patient 1, the index case-patient, was a 66-year-old tunisian man with a 4-year history of untreated diabetes mellitus. during march 20-april 27, 2013, he visited his daughter (patient 2) in qatar for 5 weeks (figure 1 ), 1 week of which they spent on a muslim pilgrimage to mecca, kingdom of saudi arabia. on april 18, results of a physical examination (including chest radiograph) for a visa extension in qatar were unremarkable. on the day of arrival back in tunisia (april 28), the patient experienced chills, followed by arthralgia, dry cough, and fever. the daughter reported that her father had had no direct contact with camels during his stay in qatar or saudi arabia. one of his children (patient 3, a nurse) gave him acetaminophen and aspirin for 3 days and then intravenously administered dexamethasone (4 mg) twice a day for 2 days. on may 6, patient 1 experienced worsened dyspnea and he sought care at the centre hospitalier-universitaire fattouma bourguiba hospital (monastir, tunisia) emergency department, where he received a fifth injection of dexamethasone. chest radiograph showed left lower lobe infiltrate (online technical appendix figure, http:// wwwnc.cdc.gov/eid/article/20/9/14-0378-techapp1. pdf). the patient was first admitted to the pulmonary ward, where he received amoxicillin-clavulanate (1 g) 3 times daily; however, on may 8, respiratory failure and peripheral signs of shock necessitated admission to the intensive care unit (icu), where he was positioned prone and given noradrenalin infusion and mechanical ventilation with additional nitric oxide. mini (<10 ml fluid injected) bronchoalveolar lavage recovered a liquid of low cellularity; cultures for bacteria and fungi were negative. serologic tests for common respiratory viruses were negative. the patient was first given amoxicillin-clavulanate, ciprofloxacin, and rifampin. on his second day in icu, oseltamivir was added. the lavage fluid was then tested in the tunisia national reference on august 5, 2013, the centers for disease control and prevention (cdc) tested a serum sample collected from the index-patient on may 9. independent rrt-pcrs were positive for mers-cov (5); targets were upe (cycle threshold [c t ] 30.27) and nucleocapsid protein (n)2 (c t 30.46). sequences of the full n and spike (s) protein coding regions were submitted to genbank (accession nos. kf811035 and kf811036, respectively). nucleotide/predicted amino acid sequence identities with published mers-cov sequences for the n and s gene coding regions ranged from 99.2%-100% to 99.0%-100% and from 99.4%-99.9% to 99.4%-99.8%, respectively. phylogenetic relationships between this virus (designated tunisia-qatar_2013) and other published mers-cov sequences showed clustering with geographically diverse sequences from saudi arabia and the united arab emirates (figure 2 ). patient 2 was the 30-year-old daughter who had accompanied the index case-patient to mecca. she remained in qatar until she attended her father's funeral in tunisia on may 11, 2013, when she reported sore throat, cough, and fever. on may 13, a chest radiograph showed bronchial thickening. a nasopharyngeal swab sample collected on may 16 was positive for mers-cov by rrt-pcr performed at the tnrl: upe c t 27.5, orf1a c t 27.46, and orf1b c t 37.55. testing at cdc detected a c t of 28.46 for upe and negative results for n2 and n3 (5) . a few days after she received oseltamivir, the patient's symptoms resolved. patient 3 was the 34-year-old son of the index casepatient, a nurse in the icu where his father had been admitted. he had not traveled outside the country during the incubation period, and his first contact with the index casepatient was after his father's return to tunisia and illness onset. he cared for his father at home during the initial phase of illness and thereafter in the pulmonology department and icu. patient 3 reported a sore throat on the day after his father's funeral. a nasopharyngeal swab sample obtained on may 16 was positive for mers-cov by rrt-pcr performed at tnrl: upe c t 21.56, orf1a c t 27.6, and orf1b c t 31.39. at cdc, the nasopharyngeal swab sample was positive for mers-cov by 3 independent rrt-pcrs (5): c t 21.67 for upe, 34.51 for n2, and 32.32 for n3. patient 3 recovered without treatment. contact tracing involved the 4 remaining family members. nasopharyngeal and/or throat swab samples were family cluster of mers-cov infections, tunisia collected a mean of 5 weeks after contact from the other 2 (not ill) children of patient 1, his spouse, and the spouse of patient 3. health care workers who had been in contact with the index case-patient in the pulmonology ward (n = 2) or icu (n = 6) and who had reported sore throat, hyperthermia, and/or diarrhea (1 worker) were also investigated. all respiratory samples from contacts were negative for mers-cov by rrt-pcr. the fact that the diagnosis for the index case-patient was made by pcr of a serum sample collected 10 days after symptom onset and tested several weeks later highlights the value of testing serum samples for mers-cov rna. this finding also provides valuable information about viremia in mers cov-infected patients, contributing to our understanding of the natural history of mers-cov infection and kinetics of virus shedding (7) . given the incubation period of the disease (up to 15 days), the father most likely acquired his infection in qatar (8, 9) . patient 3, who had not traveled outside tunisia, could have been exposed during the 11 days he cared for his father at home and in the hospital. the history of patient 2 is less clear; she might have acquired the virus from the same source as her father in qatar, or she might have been secondarily infected by contact with him before he left qatar, given that her illness began almost 12 days after her father's. patient 1 was severely ill at the time of icu admission; in <3 days, his condition rapidly evolved to multiple organ system failure and death. although we cannot account for the diabetes or corticosteroid contributions to his disease severity, we can speculate that they might have worsened his outcome. other mers cov patients who have died had concurrent conditions (2) , and corticosteroids are thought to worsen the outcomes for patients with influenza a(h1n1) virus infection (10) . the contact tracing results shed light on the potential for person-to-person transmissibility of mers-cov. only 2 family members who had been in close and prolonged contact with the index case-patient became infected. infection was not acquired by the case-patient's wife, his 2 children who did not live with him, or the icu workers who had short-term close contact with him. however, these results should be interpreted cautiously because only nasopharyngeal swab samples obtained 5 weeks after contact with the index case-patient were tested. in addition, serologic testing, which was not performed in the present investigation, could have shed more light on person-to-person mers-cov transmissibility. middle east respiratory syndrome coronavirus (mers-cov)-update state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans detection of a novel human coronavirus by realtime reverse-transcription polymerase chain reaction world health organization. mers-cov summary and literature update-as of 31 real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission mers coronavirus: data gaps for laboratory preparedness epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk early corticosteroids in severe influenza a/h1n1 pneumonia and acute respiratory distress syndrome we thank christian brun-buisson for helpful discussions and critical revision of earlier versions of the manuscript and azaibi tamin, jennifer l. harcourt, and suvang trivedi for technical assistance with the mers-cov serologic testing.dr abroug is professor of intensive care medicine at the faculté de médecine de monastir and head of the icu at centre hospitalier-universitaire fattouma bourguiba, monastir. his areas of research include scorpion envenomation, nosocomial and emerging infections (west nile virus), and exacerbation of chronic obstructive pulmonary disorder. key: cord-329190-kv9n2qj3 authors: rabaan, ali a.; alahmed, shamsah h.; bazzi, ali m.; alhani, hatem m. title: a review of candidate therapies for middle east respiratory syndrome from a molecular perspective date: 2017-09-01 journal: journal of medical microbiology doi: 10.1099/jmm.0.000565 sha: doc_id: 329190 cord_uid: kv9n2qj3 there have been 2040 laboratory-confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) in 27 countries, with a mortality rate of 34.9 %. there is no specific therapy. the current therapies have mainly been adapted from severe acute respiratory syndrome (sars-cov) treatments, including broad-spectrum antibiotics, corticosteroids, interferons, ribavirin, lopinavir–ritonavir or mycophenolate mofetil, and have not been subject to well-organized clinical trials. the development of specific therapies and vaccines is therefore urgently required. we examine existing and potential therapies and vaccines from a molecular perspective. these include viral s protein targeting; inhibitors of host proteases, including tmprss2, cathepsin l and furin protease, and of viral m(pro) and the pl(pro) proteases; convalescent plasma; and vaccine candidates. the medline database was searched using combinations and variations of terms, including ‘middle east respiratory syndrome coronavirus’, ‘mers-cov’, ‘sars’, ‘therapy’, ‘molecular’, ‘vaccine’, ‘prophylactic’, ‘s protein’, ‘dpp4’, ‘heptad repeat’, ‘protease’, ‘inhibitor’, ‘anti-viral’, ‘broad-spectrum’, ‘interferon’, ‘convalescent plasma’, ‘lopinavir ritonavir’, ‘antibodies’, ‘antiviral peptides’ and ‘live attenuated viruses’. there are many options for the development of mers-cov-specific therapies. currently, mers-cov is not considered to have pandemic potential. however, the high mortality rate and potential for mutations that could increase transmissibility give urgency to the search for direct, effective therapies. well-designed and controlled clinical trials are needed, both for existing therapies and for prospective direct therapies. middle east respiratory syndrome coronavirus overview middle east respiratory syndrome coronavirus (mers-cov) was first isolated in jeddah in the kingdom of saudi arabia (ksa) from a 60-year-old male hospital patient, who died 24 june 2012, 11 days after presenting with acute pneumonia and subsequent renal failure [1] . since then, the who have been notified of 2040 laboratory-confirmed cases, including 712 deaths [2] . while most cases have arisen in the middle east, cases have also emerged in 27 countries worldwide in travellers from the middle east and/ or in their contacts [2] . most human mers-cov infections are considered to be the result of multiple zoonotic transfers. bats are the most likely mers-cov natural reservoir, as with other mammalian coronaviruses (covs), while camels are likely to be the major zoonotic source for human infections [3] [4] [5] . secondary human-to-human transmission is considered to be limited, occurring mainly within family and healthcare settings. the first cluster of cases in humans was retrospectively identified to have occurred in a public hospital in jordan in april 2012 [6] . multiple healthcare facility-associated outbreaks have since occurred in the middle east, most notably in ksa, often linked to deficiencies in infection control procedures [7] [8] [9] [10] [11] [12] [13] . although cases outside the middle east have mainly been isolated, a large outbreak occurred in korea in june 2015, in which human-human transmission resulted in 186 cases and 36 deaths [14] . increased vulnerability to either cross-species or trans-human transmission could result from viral adaptations [15] . mers-cov infection is often accompanied by acute viral pneumonia, and sometimes gastrointestinal symptoms. clinical severity varies from asymptomatic to death, and the extent of asymptomatic spread is unclear. the high mortality rate is mainly accounted for by acute respiratory distress syndrome (ards) [7, 15, 16] . higher mortality is observed among vulnerable patients, such as older individuals and those suffering from comorbid illness, and is also associated with high viral load [7, 11, 15, 17] . in one study in a ksa hospital, intensive care unit (icu) admission among mers-cov patients was associated with a mortality rate of 74.2 % [11] . while mers-cov is not currently considered to have pandemic potential, it is clear that human-human transmission does occur. the exact mechanisms by which mers-cov is transmitted from animals to humans have not been fully elucidated. in the south korean outbreak, the virus emerged in second-and third-generation contacts, resulting in the first human case to be imported into china [18] . this raised concern that viral mutations were contributing to humanhuman transmission. given its high mortality and poor outcomes for vulnerable patients, and the potential for viral mutations, there is no room for complacency in the search for therapeutic options for mers-cov. there is currently no specific therapy. many of the therapeutic options used have been adapted from approaches used to treat severe acute respiratory syndrome (sars-cov) during the outbreak of 2003, and/or the h1n1 influenza virus during the outbreak of 2009 [19] . however, while mers-cov and sars-cov are phylogenetically related betacoronaviruses, they differ in many important respects. mers-cov utilizes human dipeptidyl peptidase 4 (dpp4; cd26) receptors, with binding mediated by the viral spike (s) protein, not the angiotensin-converting enzyme 2 (ace-2) receptors used by sars [20] [21] [22] [23] [24] . mers-cov also has a wider cellular tropism [24] [25] [26] . therapies currently used include broad-spectrum antibiotics, corticosteroids and anti-viral treatments, such as interferons (ifn), ribavirin, lopinavir-ritonavir, or mycophenolate mofetil [19, 22, [27] [28] [29] [30] [31] . however, the efficacy and/or safety of many of these therapies is unclear, and none are specific to mers-cov. ribavirin monotherapy, for example, is associated with multiple side-effects in the treatment of other viral illnesses, including sars-cov, has uncertain efficacy, and has not been tested in animal studies or randomized control trials for mers-cov [22, 31, 32] . corticosteroids have not been successful in the treatment of respiratory distress or lung fibrosis in mers-cov [31, 32] . meanwhile, studies in sars-cov and h1n1 patients suggest that corticosteroid use may in fact increase viral replication in airways, and sars patient and animal studies indicate that it contributes to immunosuppression [33] [34] [35] . mycophenolate mofetil has been associated with fatal disease and high viral loads in a marmoset model of mers-cov infection [36] . ifn therapy, alone or in combination with ribavirin or lopinavir-ritonavir, has shown greater promise in in vitro, animal and human studies [37] [38] [39] [40] . however, clinical studies on ifns vary with respect to factors such as time of administration and type of patient [19, 22, 40] . overall, there is a lack of randomized control trials (rcts) designed to test the safety and efficacy of any potential therapies specific to mers-cov, and much of the information available for existing therapies is based on in vitro and/or animal studies [22, 40, 41] . a position paper on the evidence base for specific mers-cov therapies, published by public health england (phe) and the world health organization-international severe acute respiratory and emerging infection consortium (isaric-who), suggested that benefit was likely to exceed risk for convalescent plasma, lopinavir-ritonavir, ifns and monoclonal/polyclonal antibodies, while, by contrast, for ribavirin monotherapy and corticosteroids it was considered that the risks would outweigh the benefits [42] . for interferon/ribavirin combination therapy, nitazoxanide and chloroquine, the available data were considered to be inadequate for assessment [42] . in this review, we consider potentially effective mers-cov therapies, including ifns, lopinavir-ritonavir and inhibitors of proteases, including tmprss2 and cathepsin l, as well as mers-cov-specific potential therapies, including convalescent plasma, monoclonal antibodies (mabs), antiviral peptides and candidate vaccines. these therapies will be considered from a molecular perspective, in the context of the infection and replication mechanisms of mers-cov. the therapies are summarized in table 1 . mers-cov lineage and structure mers-cov is a betacoronavirus belonging to clade c (lineage 3) of the betacoronaviruses [43] . its closest known coronavirus relatives are the prototypic clade c betacoronaviruses, tylonycteris bat virus hku4, pipistrellus bat hku5 virus and neoromicia zuluensis bat pml/2011 (neocov) virus [1, [43] [44] [45] [46] [47] . in common with other coronaviruses, the genome of mers-cov is a single, positive-stranded rna of over 30 000 nucleotides. it encodes 10 predicted open reading frames (orfs) and genes for 4 structural proteins, namely the spike (s), nucleocapsid (n), membrane (m) and envelope (e) proteins (figs 1 and 2) [48] [49] [50] . orf 1a and 1b encode virus replication-related proteins (pp1a, pp1ab), which are cleaved to give 16 non-structural proteins (nsps) involved in synthesis of viral rna and recombination ( fig. 2) [48] [49] [50] . these include nsp-14, which contains a 39-to-59 exoribonuclease (exon) domain that is important in viral proofreading and in determining the sensitivity of rna viruses to mutagens. thus small-molecule inhibitors references s1/dpp4 binding antibody (mouse): s1 rbd in vitro [76] antibody (human): s1 rbd m336, m337, m338 in vitro/in vivo (mouse, rabbit -m336) [77] [78] [79] antibody (human): s1 rbd in vitro [80, 81] antibody (mouse-humanized): s1 rbd in vitro/in vivo (mouse) prophylactic and therapeutic [82] antibody (mouse-humanized): s1 rbd hms-1 in vitro/in vivo (mouse) [83] antibody (human): s1 rbd in vitro/in vivo (mouse) potential for more mers-specific agents [61] of exon activity could be candidates for mers-cov and other coronavirus therapies [51] . as with other coronaviruses, the mers-cov s protein is critical to host cell receptor binding and cell entry, and is considered to have been under strong positive selection pressure when the virus was transmitted to humans [52, 53] . hence the s protein is a major target for potential anti-mers-cov therapies [53] . the s protein of mers-cov is composed of s1 and s2 subunits ( fig. 2 ) [53] . in common with other coronaviruses, entry into host cells depends on the s1 subunit, which contains a receptor-binding domain (rbd) comprising a core subdomain and a receptor-binding motif (rbm). the mers-cov rbm differs from that of sars-cov and dictates that mers-cov uses the dpp4 receptor, as opposed to the ace-2 receptor [20, 21] . the infection process is shown in fig. 2 . dpp4, which is widely expressed in tissues, including the lung and kidneys, is critical in the species tropism of mers-cov infection; bat, human, camel, non-human primate and swine cells, for example, are permissive for mers-cov infection, whereas mouse, hamster and ferret are not [54, 55] . host species restriction has been attributed to differences in five amino acids involved in dpp4-rbd binding, with glycosylation of the mouse dpp4 also identified as being important in the inhibition of mers-cov infection [54] [55] [56] . the human dpp4 receptor is therefore a potential target for mers-covspecific therapeutics, in particular anti-dpp4 mabs (fig. 2 , table 1 ) [53] [54] [55] [56] . adenosine deaminase (ada), which is a dpp4-binding protein, competes with mers-cov for dpp4 binding and hence is a natural mers-cov antagonist; this gives potential insights for the development of therapeutic antagonists [55] . mers-cov binds to the permissive host cell dpp4 via the rbd of the s1 domain, one of the major targets for potential mers-cov therapies [53] . similarly to other coronaviruses, mers-cov then uses the s2 subunit for virus-host membrane fusion (fig. 2) . fusion results in cleavage of the s protein at the s1/s2 boundary by host proteases [57] . the s2 subunit contains the fusion peptide, two heptad repeat domains termed hr1 and hr2, and a transmembrane (tm) domain ( fig. 2 ) [57] . membrane fusion requires conformational rearrangement of s2, the formation of a sixhelix bundle (6hb) fusion core, of which hr1 and hr2 are essential elements, and exposure of the fusion peptide, which inserts itself into the host cell membrane [52, 57, 58] . hr2-derived peptides have been identified as potentially effective anti-viral agents for treatment of mers-cov [52] ( fig. 2 ; table 1 ). the availability of host cell proteases is essential for mers-cov entry into cells [23, 53] . the host proteases responsible for s protein cleavage at the s1/s2 boundary include the serine protease tmprss2, endosomal cathepsins such as cathepsin l, and furin protease [23, 53, [59] [60] [61] . in vitro studies suggest that uncleaved mers pseudovirus can enter host cells by cathepsin l-dependent endocytosis, but that cleavage of virus during maturation by host proteases such as tmprss2 results in viral entry at neutral ph and the formation of massive syncytia [58] . host cell proteases are therefore potential molecular therapeutic targets for mers-cov prophylaxis and/or treatment (fig. 2 , table 1 ). the tmprss2 inhibitor camostat, for example, has been identified as a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [59] . following host cell entry, mers-cov pp1a and pp1ab are synthesized and then cleaved by two viral proteases, the main protease (mpro/3clpro) and the papain-like protease (plpro) (fig. 2) [57, 63] . thus viral proteases represent further potential molecular targets for therapy ( table 1) . the recently described mers-cov mpro crystal structure resembles other coronavirus mpro proteases [60] . the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg), can inhibit mers cov protease activity in vitro, as can the immunosuppressant drug mycophenolic acid [61] . however, caution is required, as the results of studies on marmosets have associated use of mycophenolate mofetil with fatal disease and high viral loads [36] . other mers-cov proteins involved in helping the virus to circumvent the immune system also present potential molecular targets (fig. 2) . for example, the accessory protein products of orf 4a, 4b and 5 are interferon (ifn) antagonists [62, 63] . the orf 4a protein both inhibits type i ifn production via cytoplasmic and nuclear mechanisms, and interferes with the ifn-mediated interferon-stimulated response element (isre) promoter element signalling pathways [62, 63] . this gives a molecular level rationale for the use of ifn as a therapeutic option in mers-cov treatment. mers-cov can also infect dendritic cells and macrophages [26, 64, 65] . endosomal uptake of mers-cov by dendritic cells following binding via dpp4 prompts these cells to produce abundant amounts of type i and iii ifns [65] . this gives context to the ifn antagonism exhibited by mers-cov accessory proteins. recently, mers-cov has also been shown to infect t cells, which are rich in dpp4, both in vitro and in marmoset spleen [66] . this results in t cell apoptosis and could contribute significantly to viral pathogenesis and further emphasizes the potential therapeutic utility of molecular targeting of dpp4 and/or the mers-cov s protein. both convalescent plasma containing virus-specific antibodies and the use of specific mabs provide options for targeting mers-cov infection at a molecular level. the use of convalescent plasma [or hyperimmune iv immunoglobulin (hvig) from the plasma of convalescent donors] for infectious disease treatments has a long history, including in the treatment of respiratory diseases [67] [68] [69] [70] . for influenza and sars-cov infection, early convalescent plasma treatment within 4-5 days of symptoms is associated with decreased viral load and reduction in mortality [67] [68] [69] [70] . however, for sars-cov the quality of studies has been inconsistent and the results have been inconclusive, with a lack of adequate clinical trials [69, 70] . according to the phe and isaric-who position paper, convalescent plasma (or high neutralizing antibody titre products) is indicated for the treatment of serious mers-cov infection [42] . one rct on 35 critically ill patients with h1n1 infection identified a significant reduction in viral load and mortality in patients who received hvig within 5 days of the onset of symptoms [68] . to date, no rcts have been completed on the use of convalescent plasma/hvig in mers-cov patients. in the light of results from sars and influenza patients, an ongoing clinical trial on the safety and efficacy of convalescent plasma treatment for critically ill mers-cov patients was initiated in may 2014 and is due to report in june 2017 [75; clinicaltrials.gov identifier: nct02190799]. this trial is being carried out in ksa. however, as is common for convalescent plasma therapies, the trial has been affected by logistical and technical issues, including the availability of sufficient donors [71, 72] . issues can also arise in the collection of convalescent plasma that has sufficient levels of mers-cov antibodies, particularly outside the middle east [22, 72] . while there are two case reports in which intravenous immunoglobulin (ivig) was used in treatment of mers-cov, it is uncertain as to whether this contributed to patient recovery [73, 74] . thus, while convalescent plasma is a promising potential therapy for mers-cov, the available clinical evidence is very limited and the results of the ongoing clinical trial will be vital in guiding any future use [71] . focused development of neutralizing monoclonal antibodies targeted against specific mers-cov proteins has meanwhile yielded promising in vitro and/or in vivo results. monoclonal antibodies: s1-dpp4 binding a number of mouse and human neutralizing mabs against the s1 region of mers-cov have been developed and tested in vitro and/or in animal models [52] . targeting of s protein for therapeutic purposes was recently comprehensively reviewed by du et al. [53] [52] . in particular, the s1 rbd is a popular target, as mabs directed against this region have the most potent neutralizing capacity. however, in terms of vaccine development, neutralizing antibodies raised by immunization with full-length s or s1 protein expression vectors may produce a more effective immunogenicity through the targeting of multiple epitopes and the reduction of the possibility of escape mutations [75] . nevertheless, many mouse and human mabs targeting the s1 rbd have given promising results in vitro and in mouse models [19] . (table 1) . a mouse monoclonal antibody, mersmab1, blocks mers pseudovirus cell entry in vitro by binding to the rbd and preventing s1 binding to dpp4 [76] . meanwhile, the human monoclonal antibodies m336, m337 and m338, which target overlapping epitopes in the rbd, all potently neutralize pseudovirus and live mers-cov in vitro [77] . significantly, intraperitoneal injection of m336 has also been shown to have both prophylactic and therapeutic protective effects against mers-cov infection in a wellestablished human dpp4 (hdpp4)-expressing transgenic mouse model, and in rabbits [78, 79] . other anti-rbd human antibodies, mers-4 and mers-27, which recognize distinct rbd regions and block binding to dpp4, likewise have potent in vitro neutralizing activity against pseudovirus and live virus infection, and they also act synergistically [80, 81] . mers-4 has anti-syncytia formation activity [80] . the crystal structure of mers-27 bound to the dpp4 receptor revealed two critical rbd residues [81] . the crystal structure of another anti-rbd antibody 4c2, which was raised in mice, has also been elucidated. this has allowed the identification of an epitope that partially overlaps the rbd receptor binding unit [82] . 4c2 was consequently humanized to give an antibody with prophylactic and therapeutic properties, shown by a reduction of mers-cov lung viral titres in an ad5-hcd26 (hdpp4) transgenic mouse model [82] . another humanized anti-rbd antibody, hms-1, similarly has potent in vivo protective properties against fatal mers-cov infection in a transgenic hdpp4 mouse model [83] . the human antibody lca60 targets both the n-terminal domain (ntd) and the rbd of the s1 region, and was isolated from b cells of a mers-cov-infected human donor before being used to rapidly establish a stable cho cell line that can be used to reliably produce clinical grade antibody [84] . this is a promising candidate for clinical development, given the antibody's potent prophylactic and therapeutic activities against mers-cov infection in ad5/hdpp4 transgenic mice and type i interferon receptor (ifnar)-ko mice [84] . the human anti-rbd antibody 3b11-n is another promising candidate that prophylactically reduces lung pathology in rhesus monkeys infected with mers-cov [85] . targeting the s1-dpp4 interaction from the host side through the development of anti-dpp4 (cd26) antibodies is another possible therapeutic option. the anti-cd26 antibodies 2f9, 1f7 and ys110 target the mers-cov entry into cells in vitro [86] . the 2f9 epitope maps close to the binding site of ada, a natural dpp4 binding protein and mers-cov antagonist, while the 1f7 and ys110 epitopes lie outside this region [55, 86] . thus, targeting of the s1-dpp4 interaction by use of mabs is a promising strategy for the clinical development of molecular therapeutics against mers-cov. another molecular approach involves targeting of the s2-mediated mers-cov-host cell fusion element of the mers-cov infection cycle by use of antiviral peptides. the role of hr1 and hr2 in viral fusion makes them potentially effective molecular therapeutic targets. this has been borne out by in vitro and in vivo results obtained using hr2 peptides (table 1 ). hr1 peptides are ineffective antivirals as they aggregate in physiological solutions [87] [88] [89] . a peptide named hr2p, which spans residues 1251-1286 of hr2, effectively inhibits viral replication and s proteinmediated cell fusion in vitro [87] . a hr2p analogue named hr2p-m2 is an even more potent fusion blocker in vitro, and inhibits mers cov-expressing pseudovirus infection [90] . hr2p-m2 interacts with an hr1 peptide to effectively block 6hb bundle formation. in vivo hr2p-m2 intranasal administration to ad5/hdpp4 transgenic mice protected them from mers-cov infection, as evidenced by the reduction of the lung viral titres by more than 1000-fold [90] . the addition of ifn-b along with hr2p-m2 enhanced the protective effect [90] . thus, s2 hr2 peptides have potential as mers-cov intranasal antiviral treatments. the s protein is also the focus of a number of candidate vaccines (table 1 ) [75, [91] [92] [93] . a fusion product combining truncated rbd and the fc portion of human igg can bind human dpp4 and inhibit mers-cov infection in an in vitro cell culture model [91] . importantly, this rbd-igg fusion product can induce a humoral response in mice vaccinated by subcutaneous injection, hence blocking rbd-dpp4 binding and inhibiting mers-cov infection [91] . further in vivo studies have indicated that intranasal administration to mice induces similar long-term igg humoral responses to those achieved with subcutaneous injection, but superior cellular immune responses and local mucosal responses in lungs [92, 93] . this suggests that this type of construct is both potentially effective and readily deliverable by intranasal means. use of an adjuvant, particularly mf59, significantly improves the humoral and t cell immunogenicity of the rbd s377-588-fc igg fusion construct in subcutaneously immunized mice [94] . the possibility of using the s1 rbd as a vaccine molecular target for a range of divergent mers-cov strains and escape mutants has also been explored recently [95] . the use of five recombinant rbds with mutations observed in different mers-cov outbreaks or in camel strains induced potent neutralizing antibody responses against several mers-cov pseudoviruses [95] . however, while the rbd of the s1 subunit is a logical and promising target for mers-cov vaccine development, the epitope scope is relatively limited and full-length s protein may be a preferable option [75] . technical difficulties in stably expressing abundant quantities of full-length s protein have presented a barrier. however, studies on delivery options, including the use of adjuvants and nanoparticles, may help in overcoming such issues. one study undertaken by novavax (gaithersburg, maryland, usa) showed that the inoculation of mice by intramuscular injection with fulllength s protein proprietary nanoparticles produced a relatively low neutralizing antibody response after 21 days [96] . however, the addition of the adjuvants alum or matrix m1 resulted in a robust and sustained anti-mers-cov neutralizing antibody response [96] . [98] . these viruses are expected to enter clinical trials as a proposed prophylactic mers-cov vaccine. likewise, intramuscular injection of ad5 or ad41 expressing full-length s protein induces both antigen-specific t cell and neutralizing antibody responses in mice [99] . finally, intraperitoneal injection of measles virus expressing either membraneanchored, full-length s protein or soluble s protein lacking the tm domain induces robust mers-cov antigen-specific neutralizing antibody and cytotoxic t lymphocyte in interferon-a/b receptor (ifnar)-deficient mice [100] . recently, an mva-based vaccine expressing s protein has been shown to induce mucosal immunity in mers-cov-infected dromedary camels, with a reduction in excreted virus and viral transcripts [101] . this has potential for veterinary use and the reduction of cross-species infection of humans by camels [101] . dna plasmids gls-5300 is a dna-plasmid vaccine that encodes mers-cov s protein (table 1 ) [102, 103] . it was co-developed by inovio, geneone life science, inc. and the walter reed army institute of research, and has become the first potential mers-cov vaccine to enter human testing [102, 103] . a phase i clinical trial in healthy volunteers commenced in 2016 for the evaluation of its safety and ability to generate antibody and cellular immune responses over a 1-year period, using one of three dosages in a three-injection regimen [102] . the vaccine has already undergone pre-clinical trials in mice, camels and macaques [103] . it induced robust and antigen-specific cytotoxic t lymphocyte and neutralizing antibody responses, which effectively protected animals against viral infection [103] . gls-5300 and other potential vaccine candidates provide an opportunity to develop a prophylactic mers-cov vaccine. however, the barriers to development of a prophylactic vaccine include the current relatively low mers-cov incidence in humans, as well as sourcing suitable small animal models [75, 97, 104] . these factors complicate the definition of a target population for mass prophylactic vaccination and pre-clinical demonstration of vaccine efficacy [104] . in this context, the monoclonal antibodies described above may be invaluable resources in an outbreak situation [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] . in vitro and animal studies while mers-cov-specific therapies are offering promising pre-clinical results, and gls-5300 has entered clinical trials, there is as yet no specific evidence-based therapy or vaccine clinically available for mers-cov. as described in the mers-cov infection and replication section, mers-cov accessory protein products are ifn antagonists [62, 63] . attenuation of the ifn response is an important mers-cov immune response circumvention mechanism [105] . the orf4a in particular inhibits ifn-b production via the inhibition of interferon regulatory transcription factor (irf)-3 and nuclear factor (nf)-kb actions, and thus irf-3-activating small molecules, for example, may be potential therapeutic agents for restoring ifn responses [62, 63] . toll-like receptor-3 (tlr-3) is also involved in the immune response of mice to sars-cov and mers-cov, recognizing viral molecular patterns and initiating the innate response that leads to ifn production (fig. 2 ) [106] . thus, tlr-3 agonists are another possible candidate for mers-cov-specific anti-viral agents [106] . therapeutically, ifn itself is particularly useful prophylactically or during the early days of viral exposure, including for coronaviruses [105, 107] . in vitro and animal studies have confirmed the potential efficacy of ifns in mers-cov therapy, in particular in combination with other therapeutic agents such as ribavirin and/or lopinavir. in vitro, mers-cov was substantially more susceptible to ifn-a than sars-cov [107] . while mers-cov in vero or llc-mk2 cells was sensitive to both ifn-a2b and ribavirin separately, relatively high concentrations were required to reduce viral replication [108] . however, combination therapy allowed the concentrations of each to be substantially reduced [108] . combination therapy of ifn-a2b and ribavirin in macaques administered 8 hours after mers-cov infection reduced systemic and local viral effects, and reduced viral genome copy number and gene expression levels [109] . bioinformatics data from microarray analysis recently showed that ifn-a2b and ribavirin treatment impacts on mers-cov gene expression in 10 different pathways, including genes involved in recognition of pathogens, immune responses and release of cytokines [110] . both ifn-b1b and lopinavir treatment, alone or in combination, also protected marmosets from the adverse clinical, radiological and pathological effects of mers-cov infection [111] . clinically, the use of ifn monotherapy, or ifn therapy in combination with ribavirin and/or lopinavir/ritonavir, has shown some promise (table 1 ) [37] [38] [39] [40] . however, the interpretation of clinical studies has been complicated by variability in factors such as the stage of infection at which therapy was administered. the available data are limited to case studies and retrospective cohort studies [22, 40] . in one case study on a patient who died in a greek hospital, pegylated ifn along with ribavirin and lopinavir was administered as part of the treatment regime, but not until the thirteenth day of the illness [39] . by contrast, in another preliminary study on two patients, the first patient was treated with ifn-a2b and ribavirin within a day of admission prior to mers-cov diagnosis, but he was also being treated with antibiotics, steroids and non-invasive ventilation [37] . patient 2, the wife of patient 1, was treated prophylactically after developing a low-grade fever and poorly defined lung infiltrates, but a diagnosis of mers-cov was not formally made [37] . thus, while patient 1 survived and patient 2 had only a mild course of illness, it is difficult to draw any firm conclusions regarding the efficacy of the treatment. in another case study on a patient in korea, administration of pegylated ifn-a2a along with ribavirin and lopinavir 4 days after hospital admission was deemed to have been effective in viral clearance and patient survival [38] . these case studies do not overall provide firm evidence for the efficacy or otherwise of ifn combination therapy for mers-cov. in one case involving a series of five patients who were critically ill with mers-cov infection and on mechanical ventilation and corticosteroids, ifn-a2b and ribavirin was administered on average 19 days after admission [27] . all five patients died, but they may not have benefited, as they were treated late in their illness and were already critically ill [27] . the benefit of earlier treatment in less vulnerable patients was suggested in another series of six patients in which three who received ifn-a2b and ribavirin early in the illness survived, while three other patients who were older and had comorbid conditions received the combination treatment later and all died [112] . however, in another study in which 20 mechanically ventilated patients with severe mers-cov infection who received pegylated ifn-a 2a and ribavirin early in treatment were compared to 24 patients who did not receive the combination therapy, the 14-day survival rate was significantly higher in the treatment group, but the 28-day survival rate was equivalently low in the two groups [113] . in another retrospective analysis of results from a series of 32 patients who received either ifn-a2a or ifn-b1a in combination with ribavirin, no significant difference in outcome between the two types of ifn was shown, and there was no survival benefit due to use of either ifn [29] . however, most of the patients in this study were aged more than 50 years and some had comorbid conditions, including end-stage renal disease [29] . thus the retrospective studies that have been carried out are heterogeneous in terms of type of patient, stage of disease and type of ifn used, including whether or not it was pegylated or short-acting. there is an urgent need for well-controlled clinical trials for ifn combination therapy in mers-cov, preferably early in the illness, as ifns are routinely available agents whose safety and efficacy is established for other viral illnesses and whose use has a sound molecular basis for mers-cov treatment. another type of therapy with a logical molecular basis for mers-cov treatment is the targeting of proteases, both host and viral (table 1 ; fig. 2 ) [19, 23, 53, 59, 60, [114] [115] [116] [117] . camostat, an inhibitor of tmprss2, is a potential therapeutic agent for coronaviruses such as sars-cov and mers-cov [59] . in a pathogenic mouse model of sars-cov infection, viral spread and pathogenesis was effectively blocked by camostat, and it is likely that it would have a similar impact on mers-cov [59] . as camostat is already in clinical use for the treatment of chronic pancreatitis, it represents a potentially safe and effective therapeutic option. recently, another tmprss2 inhibitor, nafamostat, was identified in a split protein-based cell-cell fusion assay as a potent inhibitor of mers-cov s protein-mediated hostviral membrane fusion in vitro [118] . nafamostat is already clinically approved for use by the us food and drug administration (fda) and is used as an anticoagulant [118] . the cathepsin l inhibitor teicoplanin, a glycopeptide antibiotic, was recently shown, via high throughput screening of fda-approved drugs, to block entry of mers-cov, sars-cov and ebola pseudoviruses into the cytoplasm [119] . teicoplanin is currently used clinically as an antibiotic in both prophylaxis and the treatment of serious grampositive bacterial infections. it also has derivatives, including dalbavancin, oritavancin and telavancin, all of which also block viral entry. the viral proteases, mpro (3clpro) and pl(pro), also represent potential molecular therapeutic targets [57, 60] . as well as its role in viral maturation, the mers-cov pl(pro) causes deubiquitination of ifn regulatory factor 3 (irf-3), and hence suppression of ifn b production, which contributes to viral suppression of the innate immune response (fig. 2) [120, 121] . the x-ray 3d crystal structure of mers-cov pl(pro) is similar to that of sars-cov, and includes ubiquitin-like and catalytic core domains [120] . thus the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg), can inhibit mers cov protease activity in vitro [61] . however, the mers-cov pl pro crystal structure also has unique aspects, including the oxyanion hole, and s3 and s5 subsites, which may be viable molecular targets for antivirals specifically designed against mers-cov [120] . a commercial compound termed compound 4 (commercial code f2124-0890,life chemicals) has been identified as an inhibitor of mers-cov and sars-cov plpro activity [122, 123] . the critical binding interactions and mode of inhibition differ between the two viral proteases, with the compound acting as a competitive inhibitor against mers-cov pl(pro), but an allosteric inhibitor of sars-cov pl[pro) [122] . however, f2124-0890 may lose potency in physiological reducing environments [123] . lopinavir is a protease inhibitor with activity against the sars-cov main protease m pro [124] . in a screen of a library of 348 fda-approved drugs to identify anti-mers-cov activity in cell culture, lopinavir emerged as one of four compounds that inhibited viral activity in a low micromolar range [125] . however, the clinical efficacy of lopinavir in mers-cov treatment has not yet been fully established. as mentioned above, it has usually been used clinically in combination with ifn and data are only available from case studies and series. however, notably, lopinavir-ritonavir treatment resulted in better clinical, radiological and pathological outcomes and reduced mortality in marmosets infected with mers-cov [36] . lopinavir has also been identified in a position paper from phe and isaric-who as a potential mers-cov therapy whose benefits are likely to exceed its risks [47] . thus far, mers-cov has not been considered to have pandemic potential. most cases have occurred in the middle east, particularly in ksa. outbreaks have been primarily linked to healthcare institutions, and shortcomings in infection control and prevention procedures [6] [7] [8] [9] [10] [11] [12] [13] [14] . however, potential viral mutations could facilitate expanded viral host range and enhance cross-species and human-human transmission [20, 58, 114] . the outbreak in korea resulted in mers-cov emergence in second-and third-generation contacts, highlighting the potential for mutational changes that could increase the likelihood of human-human transmission [14, 18] . mers-cov also exacts a high mortality rate, mainly due to the development of ards [15] [16] [17] . these factors emphasize the importance of developing targeted therapies and/or vaccines. the most promising advances in the development of specific molecular mers-cov therapies relate to targeting of the viral s protein by means of anti-s1monoclonal antibodies, hr-targeted antiviral peptides and viruses or plasmids bearing s protein as potential vaccine candidates [52, 55, 58, [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] . the use of ifns, usually in combination with other therapies such as ribavirin or lopinavir, also has a logical molecular basis given that ifn antagonism is an important mechanism by which the virus circumvents the innate immune system [62, 63, 105, 107] . targeting of host and viral proteases is also a sound molecular approach, as host proteases are important in viral-host membrane fusion, while viral proteases are key to viral maturation and are also involved in targeting ifns [23, 53, [59] [60] [61] [114] [115] [116] [117] . the therapies currently used for mers-cov have mainly been extrapolated from those used for sars-cov treatment, regardless of the important differences in receptor usage and cellular tropism between the viruses [20] [21] [22] [23] [24] [25] [26] . none of these therapies have been subject to well-controlled trials, and in some cases the risks are likely to outweigh any poorly defined benefits [19, 22, [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] . in general, the clinical research response to mers-cov may have been too slow [126] . thus, while there are many promising lines of research in terms of specific molecular targeting of mers-cov, no potential therapies have yet been subject to welldesigned clinical trials, and none have been approved for clinical use, apart from the gls-5300 dna-plasmid vaccine [102, 105] . continuing outbreaks of mers-cov, with possible increases in human-human transmission, are likely to galvanize the research community to push ahead with the design and performance of clinical trials for some of the available monoclonal antibodies and/or antiviral peptides for use in outbreak situations. there are various challenges inherent in the development of specific mers-cov therapies. these include the difficulty of identifying a target population for potential prophylactic vaccines, limited small animal model availability and dependence on transgenic mouse models, and the current relatively low incidence of infection, which complicates the performance of adequate clinical trials [75, 96, 97, 104] . for example, one currently ongoing trial on convalescent plasma therapy has been affected by logistical and technical issues, including insufficient available donors and difficulty in collecting convalescent plasma containing sufficient mers-cov antibody levels [71, 72] . thus, while numerous monoclonal antibodies have been raised with anti-mers activity, in particular against the s protein [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] , and promising antiviral hr2 peptides have been synthesized [87, 90] , the available data are thus far limited to in vitro and animal studies. despite these issues, there is cause for optimism, given the many candidate antibody and peptide therapies. there is also some promising in vitro and animal model evidence suggesting that use of ifns, which are well-established therapies in other viral illnesses, may be of benefit if used sufficiently early in mers-cov treatment, or as a prophylactic, especially in combination with other therapies, including ribavirin or lopinavir-ritonavir [108] [109] [110] [111] . likewise, other drugs that are currently in clinical use for other conditions have been shown to be potentially useful for mers-cov treatment, including camostat and nafamostat; teicoplanin and its derivatives dalbavancin, oritavancin and telavancin; and the sars-cov pl(pro) inhibitors, 6-mercaptopurine (6mp) and 6-thioguanine (6tg) [59, 61, [118] [119] [120] [121] . these drugs have already been shown to be safe and well-tolerated by humans. repurposing of existing drugs may therefore prove to be the most viable option in mers-cov therapy. for example, 1 screen of 290 approved drugs uncovered 27 candidates with in vitro activity against both mers-cov and sars-cov, including oestrogen receptor inhibitors and dopamine receptor inhibitors [127] . thus, there are many options available on a molecular level for the development of new mers-cov-specific therapies, as well as the adoption of drugs that are currently in use for other purposes, which should assist in more effective and reliable prevention and treatment of this virus. isolation of a novel coronavirus from a man with pneumonia in saudi arabia ecology, evolution and classification of bat coronaviruses in the aftermath of sars middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study epidemiological findings from a retrospective investigation/infections par le nouveau coronavirus en jordanie, avril 2012: r esultats epid emiologiques d'une etude r etrospective hospital outbreak of middle east respiratory syndrome coronavirus mers-cov outbreak in jeddah -a link to health care facilities molecular epidemiology of hospital outbreak of middle east respiratory syndrome an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital -riyadh, saudi arabia preventing healthcare-associated transmission of the middle east respiratory syndrome (mers): our achilles heel middle east respiratory syndrome coronavirus (mers-cov). mers-cov in republic of korea at a glance middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis coronavirusesdrug discovery and therapeutic options dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 a review of treatment modalities for middle east respiratory syndrome middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines tropism and replication of middle east respiratory syndrome coronavirus from dromedary camels in the human respiratory tract: an invitro and ex-vivo study active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) -possible lessons from a systematic review of sars-cov therapy ifn-a2a or ifn-b1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans prolonged disturbances of in vitro cytokine production in patients with severe acute respiratory syndrome (sars) treated with ribavirin and steroids cytokine responses in porcine respiratory coronavirus-infected pigs treated with corticosteroids as a model for severe acute respiratory syndrome effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients corticosteroid treatment in critically ill patients with pandemic influenza a/ h1n1 2009 infection: analytic strategy using propensity scores treatment with lopinavir/ritonavir or interferon-b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset ribavirin and interferon-a2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases combination therapy with lopinavir/ritonavir, ribavirin and interferon-a for middle east respiratory syndrome virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen sensitivity of sars/mers cov to interferons and other drugs based on achievable serum concentrations in humans update on therapeutic options for middle east respiratory syndrome coronavirus treatment of mers-cov; information for clinicians. clinical decision-making support for treatment of mers-cov patients. www.google.ie/?gws_rd= ssl#q=public+health+england+treatment+mers-cov virus taxonomy: classification and nomenclature of viruses. ninth report of the international committee on taxonomy of viruses cross host transmission in the emergence of mers coronavirus rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat close relative of human middle east respiratory syndrome coronavirus in bat mers-coronavirus molecular epidemiology and genetic analysis -origin and evolution mers coronavirus: diagnostics, epidemiology and transmission genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission mers-cov spike protein: a key target for antivirals host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus glycosylation of mouse dpp4 plays a role in inhibiting middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission protease inhibitors targeting coronavirus and filovirus entry critical assessment of the important residues involved in the dimerization and catalysis of mers coronavirus main protease thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways adjunctive therapies and immunomodulating agents for severe influenza hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a(h1n1) infection the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis sars: systematic review of treatment effects feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol treatment strategies for middle east respiratory syndrome coronavirus clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states evaluation of candidate vaccine approaches for mers-cov a conformationdependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection rapid generation of a human monoclonal antibody to combat middle east respiratory syndrome 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeatderived peptides protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection a truncated receptorbinding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines receptor-binding domain-based subunit vaccines against mers-cov identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice toward developing a preventive mers-cov vaccine-report from a workshop organized by the saudi arabia ministry of health and the international vaccine institute protective efficacy of recombinant modified vaccinia virus ankara delivering middle east respiratory syndrome coronavirus spike glycoprotein systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus a highly immunogenic and protective middle east respiratory syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels gls-5300 syncon® immunotherapy targeting middle east respiratory syndrome. www.inovio.com/products/ infectious-disease-vaccines/mers a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates mers-cov vaccine candidates in development: the current landscape delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-a treatment inhibition of novel b coronavirus replication by a combination of interferon-a2b and ribavirin treatment with interferon-a2b and ribavirin improves outcome in mers-cov-infected rhesus macaques bioinformatics analysis on molecular mechanism of ribavirin and interferon-a in treating mers-cov treatment with lopinavir/ritonavir or interferon-b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein the heptad repeat region is a major selection target in mers-cov and related coronaviruses identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov x-ray structure and enzymatic activity profile of a core papain-like protease of mers coronavirus with utility for structure-based drug design small molecules targeting severe acute respiratory syndrome human coronavirus screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture towards improving clinical management of middle east respiratory syndrome coronavirus infection repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection mechanisms of coronavirus cell entry mediated by the viral spike protein the authors received no specific grant from any funding agency. the authors declare that there are no conflicts of interest. this is a review article and no experimental work with humans was performed. five reasons to publish your next article with a microbiology society journal 1. the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer reviewaverage time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord-319784-lpmsalux authors: alqahtani, amani s.; bindhim, nasser f.; tashani, mohamed; willaby, harold w.; wiley, kerrie e.; heywood, anita e.; booy, robert; rashid, harunor title: pilot use of a novel smartphone application to track traveller health behaviour and collect infectious disease data during a mass gathering: hajj pilgrimage 2014 date: 2015-08-13 journal: j epidemiol glob health doi: 10.1016/j.jegh.2015.07.005 sha: doc_id: 319784 cord_uid: lpmsalux this study examines the feasibility of using a smartphone application (app) to conduct surveys among travellers during the hajj pilgrimage, where the use of apps has not been evaluated for infectious disease surveillance. a longitudinal study was conducted among pilgrims at the hajj 2014 using an iphone app with separate questionnaires for three study phases covering before, during, and after hajj. forty-eight pilgrims from 13 countries downloaded the app. respondents were aged between 21 and 61 (median 36) years and 58.5% (24/41) were male. of these, 85% (41/48) completed the first phase, 52% (25/41) completed both the second and third phases, and 25 of these reported meningococcal vaccination, with 36% (9/25) receiving other vaccines. all (25) reported hand hygiene use and 64% (16/25) wore a facemask at some point during the pilgrimage. four (6%) reported close contact with camels. respiratory symptoms commenced from the 4th day of hajj, with sore throat (20%) and cough (12%) being the most common. three participants (12%) reported respiratory symptoms after returning home. conducting a prospective survey using a smartphone app to collect data on travel-associated infections and traveller compliance to prevention is feasible at mass gatherings and can provide useful data associated with health-related behaviour. pilot use of a novel smartphone application to track traveller health behaviour and collect infectious disease data during a mass gathering: hajj pilgrimage 2014 1 the annual hajj pilgrimage to mecca, saudi arabia, is a striking example of intensely crowded human activity where 2-3 million pilgrims assemble from over 180 countries. incidence of acute respiratory tract infections (ari) is high [1, 2] . moreover, emergence of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and other countries poses a new public health challenge [3] . in order to reduce the risk of ari among hajj pilgrims, the saudi arabian health authority recommends a range of infection control measures [4] , however, compliance to these measures is highly variable [5] [6] [7] [8] . several studies have addressed pilgrim knowledge, attitude, and practice (kap) towards preventive measures and infectious diseases, finding that their understanding about the potential severity of respiratory infection and the need for protective measures was inadequate [8] [9] [10] [11] [12] [13] . gautret et al. [9] found that <50% of french pilgrims were aware of social distancing, available treatment options, and facemask use as precautionary measures against the spread of respiratory infections. other studies assessed pilgrim knowledge of emerging infections, such as mers-cov and ebola, and found that 60% of pilgrims were not aware of mers-cov circulation in arabia and about 40% had no accurate knowledge of ebola transmission. however, longitudinal studies examining these questions before, during, and after travel are lacking. conducting longitudinal studies among travellers during mass gatherings involves many challenges, including requirements of a large sample size and high response rate, as well as continuous follow up throughout the course of travel with real-time data capturing. conducting such studies using conventional ''pen and paper"-based methods requires significant time and resources. smartphones are increasingly becoming an integral part of modern life, making it possible to conduct prospective surveys among hajj pilgrims through their use. several studies have demonstrated their usefulness in conveying health messages in a variety of contexts and audiences, with high response and retention rates and fewer dataentry errors during descriptive studies and randomised controlled trials [14, 15] . thus, smartphones may provide better platforms to conduct prospective surveys among hajj pilgrims than conventional ''pen and paper"-based methods [14] [15] [16] [17] [18] . additional advantages include constant internet connection, location-detection services, and user proximity making it an ideal tool for collecting infectious disease data during mass gatherings. data concerning smartphone usefulness in infectious disease research at mass gatherings are very limited [19] . therefore, we conducted a pilot study using a smartphone app to examine its feasibility to track not only hajj pilgrim kap regarding preventive measures, but also symptom onset and participation in high-risk activities before, during, and after hajj 2014. a prospective cohort study was conducted among hajj pilgrims at three time points, including before, during, and after hajj 2014 (between september 5th and october 30th). this involved using three sets of questionnaires in english, including a pre-hajj questionnaire composed of 23 questions, seven identical pages of hajj questionnaires (containing five questions per questionnaire) each to be completed daily over a week during the peak hajj period, and a post-hajj questionnaire composed of six questions. we developed an iphone application (app) called 'hajj health diary', utilising the 'health monitor' app template [20] and released it in the apple app store on september 5, 2014. users started by registering their device in our online secure research database and were assigned a unique identifier for their device. this method was used successfully in previous studies [15, 16, 21] . the app determined user location through the smartphone location service and recorded it in our research database each time the participant used it. this study and the materials described below were approved by the human research ethics committee at the university of sydney (project no: 2014/599). the terms and conditions of downloading the app were communicated before obtaining participant consent. participants aged 18 years and older and participating in the 2014 hajj pilgrimage in mecca were included. attendance was confirmed by tracking their location during hajj. participants aged less than 18 years or whose stay in mecca during the hajj period could not be confirmed through location-tracking services were excluded from the study. the app consists of three main screens ( fig. 1) and an 'about' screen, which includes the participant information sheet and consent form. the first screen (first phase) is the pre-hajj questionnaire, including data on participant demographics, pre-existing chronic diseases, vaccinations received before travel, factors influencing vaccination decision and uptake, perception of the risk of respiratory infection during hajj, willingness to participate in highrisk activities, such as drinking unpasteurised milk, and awareness of official health recommendations provided by saudi arabian authorities. this phase lasted from september 5 to 30, 2014. the second screen (second phase) included the hajj questionnaire, which consisted of seven pages of identical questionnaires, one page for each day of the 'peak' hajj period. these questionnaires asked about development of respiratory symptoms and their adherence to preventive measures. given that the 'peak' hajj period in 2014 lasted from october 1 to 7, the app pushed a local notification each day to the user to complete the questionnaire. if the user did not complete it on the same day, they were reminded to do it the next time they opened the app. therefore, the first default questionnaire needed to be completed first before going to the next. the participant could not start completing the hajj questionnaire before completing the pre-hajj questionnaire and not before october 1, 2014. the third screen (third phase) includes a post-hajj questionnaire, including questions about participant use of infection prevention methods, involvement in high-risk activities, and development of ari symptoms 1 week after the conclusion of hajj. as the hajj pilgrims usually spend up to 2 weeks in saudi arabia before returning to their home countries, the app pushed a daily reminder to complete the post-hajj questionnaire from october 15 to 30, 2014. if the participant did not have an internet connection, data were stored locally on the device and automatically transferred to our database as soon as an internet connection was available. to understand usage behaviour, we also collected data entry date and time and transferred it to the database. participants had to complete all questions before submitting any of the questionnaires and could not submit any questionnaire more than once, even if they deleted and re-installed the app. we used both active and passive recruitment strategies. first, we recruited australian pilgrims through distribution of brochures during pre-hajj travel seminars in sydney. second, we released the app to the apple app store globally (and exclusively), expecting that some users searching for hajjrelated apps would find it. this was likely given that our app was the only one that would appear under the search term 'hajj health' in that outlet. in the pretravel questionnaire, we included a question on how the participants heard about the app. we identified a priori that there might be potential challenges, including loss of internet connection, app de-installation and re-installation, advertent or inadvertent omission of survey questions, and failure to follow the recommended order while completing the surveys. to minimise these pitfalls, the following measures were taken: if the participant lost the internet connection, the data would be stored locally on the device and transferred to our database as soon as the internet connection became available. if the participant de-installed the app and re-installed it, the app would not allow resubmission of the same questionnaire, requiring the participant to start from where they finished before de-installing the app. this would also maintain the sequence of questionnaire completion. to avoid any omission or delay in completing the questionnaires, the app would push a daily reminder to complete the current survey. the app was downloaded by 48 pilgrims from 13 countries (table 1) . of them, 85% (41/48) completed the first phase (pre-hajj questionnaire) and of those, 61% (25/41) completed all three study phases (fig. 2) . of the 41 participants who completed the pre-hajj questionnaire, 28 (68.3%) opened the app in saudi arabia at least once while having internet connection. the respondents were aged between 21 and 61 (median 36) years and 58.5% (24/41) were male. a large portion (46.3%, 19/41) was university educated and 80.5% (33/41) were employed. sixteen (39%) participants had pre-existing chronic diseases, including five with diabetes (12.2%), three each with hypertension, bronchial asthma, and hypercholesterolemia (7.3%), and one with heart disease (2.4%). the participants stayed in mecca for a median of 14 days (range, 9-30 days), 70.7% (29/41) attended hajj for the first time, and only 9.8% (4/41) were aware of annual saudi arabian health recommendations for hajj pilgrims. in terms of how the participants heard about the app, 41.5% (17/41) reported first seeing it in the apple app store, 19.5% (8/41) heard about it in pre-hajj seminars, 12.2% (5/41) from the study researchers, and 26.8% (11/41) from other sources. regarding convenience of using the app, 53.6% (22/41) found it very convenient, 31.7% (13/41) found it slightly convenient, 9.8% (4/41) found it a little inconvenient, and 4.9% (2/41) found it very inconvenient. those who found it inconvenient (to any degree) left the survey incomplete. concerning usage behaviour, the number of participants who completed the hajj questionnaire on the day the reminder was pushed, i.e., the 1st day, was 31, but dropped to 27 on the 2nd day and remained at 25 from the 3rd day onward (fig. 2) . those who did not complete the hajj questionnaire on the specified day completed it within the next 1-6 days. all participants who completed the hajj questionnaire subsequently also completed the post-hajj questionnaire. of those who completed the study phases, all reported receiving the compulsory meningococcal vaccine. the main factors driving meningococcal vaccine uptake were severity of the disease 80% (20/25) and effectiveness of the vaccine 76% (19/25). only 9 (36%) pilgrims received other vaccines before hajj (8 had influenza vaccine and one pneumococcal vaccine). three of these had comorbidities. forty-four percent (11/25) of participants were unconcerned about catching influenza while at hajj and 76% were unconcerned about developing a cough. forty percent (10/25) of respondents expressed concern about contracting mers-cov during hajj, at least to a modest extent, while the rest did not. however, 36% (9/25) of participants were willing to visit a camel farm during hajj and 16% (4/25) were willing to drink unpasteurised camel milk if offered in saudi arabia. in practice, 16% (4/25) of pilgrims (2 american, 1 canadian, and 1 australian) actually reported coming into contact with camels, including visiting a camel farm, taking photographs with camels, and drinking their milk (2) . the onset of respiratory tract symptoms began from the 4th day of the peak hajj period and continued over the next several days. sore throat (20%) and cough (12%) were the most frequently reported symptoms (fig. 3) . after returning home from hajj, 12% (3/25) of participants reported developing a cough and sore throat within 1 week and, among these, one pilgrim from australia reported having had contact with camels during hajj. sixty-four percent (16/25) reported wearing a facemask during hajj, with uptake highest on day 4 ( table 2 ). protection from infectious agents and air pollutants was the main reason for mask use. difficulty in breathing and a feeling of suffocation were commonly cited as barriers to the use of facemasks. on the other hand, all participants practiced hand hygiene at some point (mostly during the 1st 4 days) during hajj. respondents stated that hand hygiene was easy to implement, convenient, and believed it to be effective in preventing infections. overall, this pilot study indicates that conducting a prospective survey using a smartphone app to collect data on travel-associated infections and traveller compliance to prevention is feasible, given that the response rate was >50%. this survey also demonstrates that many pilgrims partake in activities that may increase risk of acquiring emerging infections. of the 48 people who downloaded the app, 41 (85%) participated in the first survey and of these, 25 (61%) went on to complete it. previous paperbased cross-sectional surveys reported response rates ranging between <20% and >80% [7, 22, 23] . the studies with high response rates involved recruitment with continuous follow up of worshippers throughout their pilgrimage. this approach requires significant resources and greater investment of time and cost [22, 23] . other paper-based surveys where pilgrims were not followed up continuously had response rates as low as 19.7% [7] . the strengths of our study include its low cost, ability to reach far and wide to allow real-time analyses and longitudinal follow-up, and ability to capture data daily during the peak hajj period, something not accomplished in other studies. this pilot survey reveals that all pilgrims complied with hand hygiene. this is supported by a review by benkouiten et al. [24] , which found that hand hygiene was the most popular nonpharmaceutical preventive measure among hajj pilgrims. ease of use and participant belief regarding its effectiveness against infection were important driving factors. in this study, we found that respiratory symptoms commenced on the 4th day of tent stay during hajj and continued thereafter, with cough and sore throat being the most commonly reported symptoms. this is likely because the incubation period of most commonly circulating respiratory viruses is about 1-4 days [25] . used in combination with a geographic information system as a tool for syndromic surveillance, this novel method can be used to detect real-time clusters of respiratory infections at hajj and other mass gatherings. although mers-cov has been circulating in saudi arabia since 2012, no case of hajj-associated mers-cov has been reported [26] . evidence suggests that mers-cov can be transmitted to humans through close contact with an infected camel [27] . interestingly, our study identified that some pilgrims had close contact with camels, including visiting camel farms, photo opportunities with camels, and drinking their raw milk. through this survey, we identified one australian pilgrim who had close contact with camels and subsequently developed respiratory symptoms within 1 week of returning home. further follow up of the case was not possible, however, given that no mers-cov case was reported in australia, it is highly unlikely that the person had mers-cov. therefore, this pilot study suggests that smartphones could help detect patients with potential emerging infectious diseases. electronic surveillance to identify outbreaks of infectious diseases at a mass gathering has been attempted previously [28] . for instance, surveillance using electronic medical records deployed during the 2002 winter olympic games helped detect an influenza outbreak, which was subsequently described with the aid of laboratory diagnosis [29, 30] . digital interfaces, including smartphones, were applied at the 2012 london olympics to identify illnesses and injuries among athletes [31] . our study, the first of its kind at hajj, demonstrates the feasibility of a smartphone app in a prospective survey of pilgrim illness and adherence to preventive measures throughout the course of travel. owing to delays in development, testing, and app store approval, the app was only released a few weeks before hajj, limiting the amount of time it was available to respondents prior to their journey. we speculate that earlier app release will result in greater numbers of participants. because this survey was conducted only in english, multilingual applications could have expanded participation into diverse language groups. data on pilgrim demographics show that 60-80% of pilgrims are older than 40 years of age [8, 32] , while most smartphone users are aged 25-44 years [33] , which might have impacted study outcomes. however, since smartphone use is gradually becoming ubiquitous, respondent demographics are likely to be less important in the future. finally, we designed the app only for iphone users, thus excluding users of other smartphone platforms, such as android or windows mobile. extending availability of this app to other platforms will likely increase participation rates. in conclusion, this pilot study demonstrates that smartphone apps can be used to conduct surveys to prospectively gather data concerning onset and progression of symptoms and location information during mass gatherings. such data collection can potentially reinforce education associated with disease prevention behaviours, thus improving public health. a larger study with multilingual apps for both iphone and android smartphones is planned for hajj 2015. mass gathering medicine: 2014 hajj and umra preparation as a leading example prevention of influenza at hajj: applications for mass gatherings travel implications of emerging coronaviruses: sars and mers-cov health conditions for travellers to saudi arabia for the umra and pilgrimage to mecca (hajj) vaccinations against respiratory tract infections at hajj protective practices and respiratory illness among us travelers to the 2009 hajj the prevalence of acute respiratory symptoms and role of protective measures among malaysian hajj pilgrims detection of respiratory viruses among pilgrims in saudi arabia during the time of a declared influenza a (h1n1) pandemic hajj pilgrims' knowledge about acute respiratory infections australian hajj pilgrims' infection control beliefs and practices: insight with implications for public health approaches australian hajj pilgrims' knowledge, attitude and perception about ebola french hajj pilgrims' experience with pneumococcal infection and vaccination: a knowledge, attitudes and practice (kap) evaluation health knowledge, attitude and practice among iranian pilgrims pro-smoking apps for smartphones: the latest vehicle for the tobacco industry? depression screening via a smartphone app: cross-country user characteristics and feasibility confirming the one-item question likert scale to measure anxiety adherence to a smartphone application for weight loss compared to website and paper diary: pilot randomized controlled trial smartphone versus pen-and-paper data collection of infant feeding practices in rural china the world's first application of participatory surveillance at a mass gathering: fifa world cup health monitor project who uses smoking cessation apps? a feasibility study across three countries via smartphones circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage protective measures against acute respiratory symptoms in french pilgrims participating in the hajj of non-pharmaceutical interventions for the prevention of respiratory tract infections during hajj pilgrimage incubation periods of acute respiratory viral infections: a systematic review has hajj-associated middle east respiratory syndrome coronavirus transmission occurred? the case for effective post-hajj surveillance for infection evidence for camel-to-human transmission of mers coronavirus new digital technologies for the surveillance of infectious diseases at mass gathering events hospital electronic medical recordbased public health surveillance system deployed during the 2002 winter olympic games illness and injury in athletes during the competition period at the london 2012 paralympic games: development and implementation of a web-based surveillance system (web-iiss) for team medical staff causes of mortality for indonesian hajj pilgrims: comparison between routine death certificate and verbal autopsy findings digital industry association for australia. australian mobile phone lifestyle index professor robert booy has received funding from baxter, csl, gsk, merck, novartis, pfizer, roche, romark, and sanofi pasteur for the conduct of sponsored research and travel to present at conferences or consultancy work. all funding received is directed to research accounts at the children's hospital at westmead. dr. anita e. heywood has received grant funding for investigator-driven research from gsk and sanofi pasteur. dr. harunor rashid received fees from pfizer and novartis for consulting or serving on an advisory board. the other authors have no competing interests to declare. key: cord-349812-nw1nlc1y authors: jang, won mo; jang, deok hyun; lee, jin yong title: social distancing and transmission-reducing practices during the 2019 coronavirus disease and 2015 middle east respiratory syndrome coronavirus outbreaks in korea date: 2020-06-09 journal: j korean med sci doi: 10.3346/jkms.2020.35.e220 sha: doc_id: 349812 cord_uid: nw1nlc1y background: the absence of effective antiviral medications and vaccines increased the focus on non-pharmaceutical preventive behaviors for mitigating against the coronavirus disease 2019 (covid-19) pandemic. to examine the current status of non-pharmaceutical preventive behaviors practiced during the covid-19 outbreak and factors affecting behavioral activities, we compared to the 2015 middle east respiratory syndrome coronavirus (mers-cov) outbreak in korea. methods: this was a serial cross-sectional population-based study in korea with four surveys conducted on june 2 and 25, 2015 (mers-cov surveys), and february 4, and april 2, 2020 (covid-19 surveys). of 25,711 participants selected using random digit dialing numbers, 4,011 participants (aged ≥ 18 years) were successfully interviewed, for the 2020 covid-19 (n = 2,002) and 2015 mers-cov (n = 2,009) epidemics were included. participants were selected post-stratification by sex, age, and province. the total number of weighted cases in this survey equaled the total number of unweighted cases at the national level. we measured the levels of preventive behaviors (social distancing [avoiding physical contact with others]), and practicing transmission-reducing behaviors such as wearing face mask and handwashing. results: between the surveys, respondents who reported practicing social distancing increased from 41.9%–58.2% (mers-cov) to 83.4%–92.3% (covid-19). the response rate for the four surveys ranged between 13.7% and 17.7%. practicing transmission-reducing behaviors (wearing face masks and handwashing) at least once during covid-19 (78.8%, 80.2%) also increased compared to that during mers-cov (15.5%, 60.3%). the higher affective risk perception groups were more likely to practice transmission-reducing measures (adjusted odds ratio, 3.24–4.81; 95 confidence interval, 1.76–6.96) during both covid-19 and mers-cov. conclusion: the study findings suggest markedly increased proportions of non-pharmaceutical behavioral practices evenly across all subgroups during the two different novel virus outbreaks in korea. strategic interventions are needed to attempt based on preventive behavior works. many countries are battling with the coronavirus disease 2019 (covid-19) pandemic because of the absence of effective antiviral medications and vaccines. to control the spread of covid-19, social distancing, wearing of face masks, and washing of hands, which are transmission-reducing behaviors, are being recommended as some of the most important measures. 1-3 because early transmission of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is caused by pre-symptomatic-or pauci-symptomatic-infected individuals, these non-pharmaceutical preventive behaviors are getting more attention for the containment of the covid-19 pandemic. 4-6 these attempts at behavioral change are aimed at slowing down the spread of emerging infectious diseases and to flatten the epidemic curve. thus, the healthcare system resource capacity can be conserved while allowing time for the development of drugs and vaccines. social distancing (or spatial distancing), including avoidance of outdoor activities, public transportation, healthcare facilities, and crowded places with potential for physical contact between individuals, reduced the number of infections. 2, 3 wearing of face masks and washing of hands are also associated with reduced propensity of transmission. [5] [6] [7] [8] periodically, korea has experienced outbreaks including the severe acute respiratory syndrome (sars), influenza h1n1, middle east respiratory syndrome coronavirus (mers-cov), and covid-19 outbreaks in 2003, 2009, 2015 , and 2020, respectively. 9-11 differences occurred in epidemiologic outcomes (number of cases, fatality rates) of these novel infectious disease outbreaks in korea. only 3 confirmed cases and no death during sars, more than 100,000 confirmed cases and 260 deaths during influenza h1n1, and 185 confirmed cases and 36 deaths during mers-cov occurred. 9 since diagnosing the first covid-19 case in korea on 20 january, 2020, 10,738 cases have been confirmed, 243 deaths occurred, and 3,246 patients were isolated until 27 april, 2020. 12 in february 2020, korea became the worst affected country, aside from china, for a while, and had several surges in the number of cases. 13 however, the korea epidemic curve flattened without coercive restrictions following rapid interventions beginning in march 2020. 14 one possible explanation for the mitigation of the surge was the strong efforts implemented by the korean government and the citizens of korea to practice social distancing and transmission-reducing behaviors. resurgence in the emerging infectious disease provides opportunity for comparing individual's levels of practicing non-pharmaceutical preventive behaviors. however, no study has compared the proportion of practicing non-pharmaceutical preventive behaviors between covid-19 and 2015 mers-cov outbreaks. 2,3,7,15-18 the current study aimed to quantify and compare the individuals' adherence to social distancing and transmission-reducing behavioral practices during the covid-19 and mers-cov outbreaks in korea. factors influencing these practices were also determined. we hypothesized that there would be differences in the rate of adherence to the non-pharmaceutical preventive behaviors and the factors affecting these behaviors between the 2020 covid-19 and 2015 mers-cov outbreaks. the 95% confidence interval (ci), 0.025 desired margin of error, 0.2 behavioral response proportion of population. the 4,011 participants included 2,009 participants older than 19 years who were monitored during the mers-cov outbreak between june 2 and 25, 2015; and 2,002 participants, older than 18 years, who were investigated during covid-19 outbreak between february 4 and april 2, 2020. all surveys were conducted using mobile (85%) or landline (15%) random digit dialing numbers in 8 regions (nationally representative). participants were selected post-stratification by sex, age, and province and chosen independently by each survey. the total number of weighted cases in this survey equaled the total number of unweighted cases at the national level. the weights were normalized to calculate the proportions and ratios but not for estimating the subtotal populations. trained interviewers conducted all interviews using computer assisted telephone interviewing. surveys 3 and 4 began approximately 2 weeks after the index case occurred, while surveys 1 and 2 were conducted approximately a month after surveys 3 and 4. survey 1 was conducted just 10 days before the last confirmed patient of mers-cov on july 4, 2015. however, survey 2 was conducted when there were more than 100 confirmed cases. the surveys were conducted by gallup korea, an affiliation of gallup international. details, including period, number of respondents successfully interviewed, and response rate for each of the four surveys are provided in table 1 . sex, age, occupation, self-reported household economic status, residential area, presidential job approval rating, party identification, and affective risk perception as participants' characteristics, were investigated to identify factors influencing non-pharmaceutical preventive behaviors. age was classified into 5 levels (19-29, 30s, 40s, 50s, and 60 years and older). occupation was classified into five levels (unemployed, self-employed including farming/ forestry/fishery, blue-collar worker, white-collar worker, and full-time homemaker or student). self-reported household economic status was classified into five levels (lower, lower middle, middle, upper middle, and upper). participants were classified as either metropolitan or non-metropolitan residents. presidential job approval rating was assessed using the following options: "approval," "disapproval," or "no opinion." support for party identification was assessed based on alignment either with the ruling party, opposition party, or no opinion. affective risk perception was assessed using the options "worried" or "not worried." 19 the interviews were conducted on the two aspects of the non-pharmaceutical preventive behaviors, which are social distancing measures and transmission-reducing practices ( supplementary data 1 and 2) . social distancing was assessed using the following four questions: 1) "did you reduce or avoid outdoor activities or attend meetings this week because of mers-cov or covid-19?"; 2) "did you reduce or avoid using public transportation such as the bus or the subway this week because of mers-cov or covid-19?"; 3) "did you reduce or avoid using healthcare facilities such as the hospitals or public health centers this week because of mers-cov or covid-19?"; and 4) "did you reduce or avoid visiting crowded markets, departmental stores, or large discount stores this week because of mers-cov or covid-19?" transmission-reducing practice was assessed using the following two questions: 1) "do you wash your hands more often than usual because of mers-cov or covid-19?" and 2) "have you ever worn a face mask because of mers-cov or covid-19?" all the questions about the non-pharmaceutical preventive behaviors required "yes/no" responses. the development of the questionnaires on preventive behaviors had not gone through a validity procedure due to the urgency of the outbreak. we also imposed the survey items on existing questionnaire developed by gallup korea, an affiliation of gallup international. response rates according to preventive behaviors were calculated according to participants' characteristics. univariable analyses using χ 2 test were performed in the four surveys, entirely and respectively, to identify the relationships between practicing preventive behaviors and each demographic variable. missing values of any outcome variable were ≤ 3.6%. multivariable logistic regression analysis was performed to explore factors influencing preventive behaviors in the four surveys, entirely and respectively. we performed multivariable logistic regression model adjusted for sex, age, occupation, selfreported household economic status, residential area, presidential job approval, and party identification. 15 affective risk perception was excluded from survey 1 and survey 2 logistic regression models to attain comparability because no data existed for it in survey 2. on the avoidance of outdoors activities, extremely large number of events made the odds ratios (ors) in survey 2 logistic regression model unstable; therefore, surveys 1 and 2 logistic models were not reported. using logistic regression analysis for transmission-reducing measures and social distancing measures, "y = 1" was used when "yes" for preventive behaviors, otherwise "y = 0" was used. we analyzed with a 2-sided p value of less than 0.05 considered significant using sas version 9.4 (sas institute inc, cary, nc, usa). this study was reviewed and approved by the institutional review board (irb) of seoul metropolitan government-seoul national university boramae medical center (irb no. 20200403/07 -2020 -12/043). the need for informed consent was waived by the board. differences in participants' general characteristics between surveys 1 and 2 are shown in table 2 . overall, the practice rate of avoiding outdoor activities in survey 2 increased 1.7-fold compared to that in survey 1. depending on the general characteristic, avoiding outdoor activities' practice rate differed by as little as 28.2% (upper economic status) and as much as 54.6% (presidential job approval). overall, avoiding public transportation practice rate in survey 2 increased 2.1-fold compared to that in survey 1. depending on the general characteristic, avoiding public transportation' practice rate differed by as little as 29.3% (aged 30-39 years) and as much as 57.6% (no opinion of presidential job approval). overall, avoiding healthcare facilities' practice rate in survey 2 increased 1.6-fold compared to that in survey 1. depending on the general characteristic, avoiding healthcare facilities' practice rate differed by as little as 22.7% (opposition party identification) and as much as 49.9% (presidential job approval). overall avoiding crowded places' practice rate in survey 2 increased 1.8-fold compared to that in survey 1. depending on the general characteristic, avoiding crowded places' practice rate differed by as little as 4.4% (upper economic status) and as much as 49.3% (presidential job approval). there were no statistically significant differences between surveys with participants' characteristics except with occupation, self-reported household economic status, presidential job approval rating, and party identification. with occupation, higher proportions occurred in the unemployed and blue-collar workers in survey 2, while lower proportions occurred in white-collar workers and home makers or students. of the self-reported household economic status, survey 4 had higher proportions in the 'upper middle' and 'middle' status, while lower proportions occurred in 'low middle' and 'lower' status. with respect to the presidential job approval rating, the percentage of participants who reported obtaining 'approval' increased in survey 2 compared to survey 1. of the party identification, the proportion in the 'ruling party' in survey 2 were higher than that in survey 1. comparison of the general characteristics of the participants between surveys 3 and 4 are shown in table 3 . overall, wearing of face mask rate in survey 4 had increased by more than 5-fold compared to that in survey 3. depending on the general characteristic, the wearing of face mask rate differed by as little as 39.2% (upper economic status) and as much as 71.3% low middle economic status). overall, the washing of hands rate in survey 4 increased by 1.3fold compared to that in survey 3. depending on the general characteristic, washing of hands rate differed by as little as 12.0% (aged 19-29 years) and as much as 37.5% (upper economic status). there were definitively, statistically significant differences in the wearing of face masks rate in all subgroups between surveys 3 and 4. no significant differences occurred in participants' proportions between surveys, except with occupation, self-reported household economic status, presidential job approval rating, and party identification. with occupation, survey 4 had higher proportions of the unemployed and blue-collar workers, while had lower proportions of the self-employed and home makers or students. of the self-reported 6/11 https://jkms.org https://doi.org/10.3346/jkms.2020.35.e220 household economic status, survey 4 had a higher proportion of those in the 'middle' status, while had a lower proportion of those in the 'low middle.' with respect to presidential job approval rating, the percentage of participants who reported 'approval' increased when survey 4 was compared with survey 3. of the party identification, the proportion in the 'ruling party' in survey 4 were higher than that in survey 3. table 4 reports the association between variables and non-pharmaceutical preventive behaviors, social distancing and transmission-reducing behaviors. of social distancing behaviors, generally, none of the factors (characteristics) consistently affected any kinds of social distancing behaviors (avoiding public transportation, healthcare facilities, and crowded places) in both surveys 1 and 2. the results showed that sex, presidential job approval rating, and party identification were significantly associated with social distancing behaviors in survey 1, but not in survey 2. those aged 30 years and older were more likely to avoid public transportation in both surveys 1 and 2. participants aged 30-39 years were more likely to avoid healthcare facilities in survey 2 only. those aged 30-49 years were more likely to avoid crowded places in survey 1 only. only in survey 2 were residents of metropolitan cities identified to have practiced avoidance of public transportation behaviors less. in transmission-reducing behaviors, both surveys 3 and 4 reported that females were more likely to practice preventive behaviors (adjusted or [aor], 1.57-2.43; 95% ci, 1.04-3.78), which tended to be stronger in survey 4. the association of affective risk perception with transmission-reducing behaviors was also observed in both surveys 3 and 4. participants who reported being 'worried' were more likely to practice both the wearing of face masks and handwashing (aor, 3.24-4.81; 95% ci, 1.76-6.96). those living in metropolitan cities more frequently wore face masks in both surveys 3 and 4. participants aged 50 years and older practiced less wearing of face masks in survey 4 only. the results showed that presidential job approval rating and washing of hands were significant in survey 3, but not in survey 4. possibly, the current study is the first to explore changes in individuals' non-pharmaceutical preventive behaviors during two different consecutive emerging infectious disease outbreaks in korea. 2,3,7,15-17,20-22 first, our study showed a marked increase in non-pharmaceutical preventive behaviors such as social distancing, wearing of face masks, and washing of hands, evenly, across all subgroups during covid-19 compared to 2015 mers-cov. during the previous 2003 sars outbreak in hong kong, the level of preventive behavioral practice increased over time, but differences in level was not compared between outbreaks. 23 a possible explanation for the increase of preventive behavioral practices during covid-19 in korea could be due to previous experience of emerging infectious disease epidemic, intensifying the practice. 24 additional study is needed to examine why the preventive behavioral practice increased during covid-19 outbreak, and to understand how differences in preventive behavioral practices affected the transmission during repeated and different emerging infectious disease outbreaks. 25 however, there is need to investigate for further understanding, the association between risk perception (affective and cognitive) and preventive behaviors. 19 third, our results, showing that low level of trust in the president and identification of opposition party influenced preventive behavioral practice during 2015 mers-cov but not during 2020 covid-19 are inconsistent with those of previous studies. 15,17 differences in the korean government's responses to the two different emerging infectious disease epidemics could have affected how the public perceived the image of the president and the ruling party, as well as the trust in the government. 9,14,19,26-32 further research is needed to understand the conditions of trust in the president and identification of a party that could affect preventive behavioral practices. however, this study has some limitations. first, that this study used surveys on self-reported practices could mean that the data could be different from those obtained through observed practices. therefore, there could have been measurement errors. (i.e., social desirability bias, 'yes-saying' bias) however, surveys of observed practices are difficult to conduct during health crisis. second, this study used a cross-sectional design; hence, it could not establish causal relations. third, risk perception (affective and cognitive reactions) was not fully surveyed during the outbreaks, limiting the interpretation of findings. fourth, the current study could not evaluate the intensity of the preventive behaviors. finally, because of the unexpected rapidly evolving outbreak, this study could not examine the validity of the questionnaire using a test-retest design. in conclusion, the present study suggests, for the first time, the level of the practice rate of non-pharmaceutical preventive behaviors and influencing factors during 2020 covid-19 and 2015 mers-cov in korea. affective risk perception can increase practicing reducing transmission measures and it can be used to prevent the failure of preventive behavior management. to understand the mechanism of behavioral immunity, further exertions are needed behind the citizens, the governmental public health sector, as well as the academic society. strategic interventions to suppress the spread of infectious diseases based on preventive behaviors works through cooperation of individuals with regulations and will be a salient contribution to a quick end to covid-19 pandemic. thus, policies to guide such strategic interventions need to be developed. basic protective measures against the new coronavirus impact assessment of nonpharmaceutical interventions against coronavirus disease 2019 and influenza in hong kong: an observational study the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study virological assessment of hospitalized patients with covid-2019 social distancing against covid-19: implication for the control of influenza seventy-two hours, targeting time from first covid-19 symptom onset to hospitalization effectiveness of handwashing in preventing sars: a review mathematical modeling of the effectiveness of facemasks in reducing the spread of novel influenza a (h1n1) central government reform to improve national disease control severe acute respiratory syndrome epidemiological characteristics of imported influenza a (h1n1) cases during the 2009 pandemic in korea covid-19: surge in cases in italy and south korea makes pandemic look more likely korean society of infectious diseases; korean society of pediatric infectious diseases report on the epidemiological features of coronavirus disease 2019 (covid-19) outbreak in the republic of korea from preventive behavioral responses to the 2015 middle east respiratory syndrome coronavirus outbreak in korea preventive behaviors by the level of perceived infection sensitivity during the korea outbreak of middle east respiratory syndrome in 2015 social distancing and transmission-reducing practices during covid-19 outbreak in korea 17. lee km, jung k. factors influencing the response to infectious diseases: focusing on the case of sars and mers in south korea adoption of personal protective measures by ordinary citizens during the covid-19 outbreak in japan influence of trust on two different risk perceptions as an affective and cognitive dimension during middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea: serial cross-sectional surveys perceived risk, anxiety, and behavioural responses of the general public during the early phase of the influenza a (h1n1) pandemic in the netherlands: results of three consecutive online surveys risk perception, preventive behaviors, and vaccination coverage in the korean population during the 2009-2010 pandemic influenza a (h1n1): comparison between high-risk group and non-high-risk group community-based risk communication survey: risk prevention behaviors in communities during the h1n1 crisis monitoring community responses to the sars epidemic in hong kong: from day 10 to day 62 social distance and sars memory: impact on the public awareness of 2019 novel coronavirus (covid-19) outbreak. medrxiv. forthcoming early assessment of anxiety and behavioral response to novel swine-origin influenza a(h1n1) institutional trust and misinformation in the response to the 2018-19 ebola outbreak in north kivu, dr congo: a population-based survey perception of hazards: the role of social trust and knowledge public health crisis response and establishment of a crisis communication system in south korea: lessons learned from the mers outbreak mers tarnishes korean president's image as leader middle east respiratory syndrome in south korea during 2015: risk-related perceptions and quarantine attitudes coronavirus in south korea: how 'trace, test and treat' may be saving lives how south korea flattened the curve we would like to thank gallup korea, an affiliation of gallup international, for supporting surveys and data collection for this manuscript. questionnaire (korean). key: cord-353121-ot7jsx20 authors: choi, jun yong; oh, jin ok; ahn, jin young; choi, heun; kim, jung ho; seong, hye; jeong, su jin; ku, nam su; yeom, joon-sup; choi, jae-phil title: absence of neutralizing activity in serum 1 year after successful treatment with antivirals and recovery from mers in south korea date: 2019-01-31 journal: clin exp vaccine res doi: 10.7774/cevr.2019.8.1.86 sha: doc_id: 353121 cord_uid: ot7jsx20 we evaluated the neutralizing activity in serum from three patients >1 year after recovery from middle east respiratory syndrome (mers) associated with mild pneumonia treated with antivirals during the mers outbreak in south korea at 2015. the neutralizing activity in serum was measured by pseudovirus inhibition assays. three-fold diluted serum of subjects showed only 9.7%, 10.3%, and 2.2% reductions in relative light units. so, significant neutralizing activity was not demonstrated in any sera of three patients with mild pneumonia >1 year after being successfully treated with antiviral agents and recovering from mers coronavirus infection. no potential conflict of interest relevant to this article was reported. this work was supported by a faculty research grant of yonsei university college of medicine for (6-2015-0153) . we evaluated the neutralizing activity in serum from three patients >1 year after recovery from middle east respiratory syndrome (mers) associated with mild pneumonia treated with antivirals during the mers outbreak in south korea at 2015. the neutralizing activity in serum was measured by pseudovirus inhibition assays. three-fold diluted serum of subjects showed only 9.7%, 10.3%, and 2.2% reductions in relative light units. so, significant neutralizing activity was not demonstrated in any sera of three patients with mild pneumonia >1 year after being successfully treated with antiviral agents and recovering from mers coronavirus infection. institutes of health) in a t175 tissue culture flask, using the calcium phosphate method. the cells were changed into fresh dulbecco's modified eagle's medium 8 hours later. supernatants were harvested 72-hour post-transfection and used as a source of pseudoviruses for single-cycle infection. a pseudovirus inhibition assay was established to detect neutralizing activity in patient sera [4, 5] . pseudovirus-containing supernatants were incubated with serially diluted patient serum at 37°c for 1 hour before addition to target cells (vero cells) pre-plated in 96-well culture plates (10 4 cells/well). the media was replaced after 24 hours, followed by lysis of the cells 72 hours later using cell lysis buffer (perkin-elmer, waltham, ma, usa), with the lysates transferred into 96-well luminometer plates. luciferase substrate (perkin-elmer) was added to the plates, and the relative luciferase activity was determined. the inhibition of mers-cov pseudovirus was presented as percentage inhibition of relative light units (rlu). control serum was collected from a healthy volunteer. subject 1 was a 38-year-old man diagnosed with mers-cov infection by real time polymerase chain reaction (pcr) of nasopharyngeal aspirate on 13 june 2015. he was an ambulance driver exposed to a mers-cov infected patient during patient transfer on 5-6 june 2015. symptoms including fever, myalgia, and headache appeared after 10 june 2015. he did not have a cough, sputum production, dyspnea, or other respiratory symptoms, but chest radiography performed on 13 june 2015 showed pneumonic consolidation on the right lower lung field. he was treated with combination of interferon-α2a (180 µg/wk), ribavirin (2,000 mg loading followed by 1,200 mg every 8 hours for 4 days and 600 mg every 8 hours), and lopinavir/ritonavir (400 mg/100 mg orally every 12 hours) from 15-24 june 2015. repeated mers-cov pcr tests showed negative results on 23 and 24 june 2015. full recovery was achieved without any complications, and a blood sample was collected on 1 july 2016. subject 2 was a 33-year-old woman diagnosed with mers-cov infection on 9 june 2015. she was indirectly exposed to mers-cov between 25 and 29 may 2015 at another hospital, where mers-cov infections were diagnosed in a multi-bed room. diarrhea commenced 5 days prior to diagnosis, with fever, myalgia, sore throat, cough, sputum production, and mild dyspnea commencing 4 days before diagnosis. chest radiography showed peribronchial pneumonic consolidation in the right middle lung field. between 9 and 12 june 2015, the patient was treated with a combination of interferon-α2a, ribavirin, and lopinavir/ritonavir. from 13 june 2015, lopina-vir/ritonavir was discontinued and the ribavirin dose was reduced to 10 mg/kg every 8 hours due to jaundice. two consecutive negative results of mers-cov pcr were confirmed on 23 and 24 june 2015. full recovery was achieved without any complications, and a blood sample was collected on 1 july 2016. subject 3 was a 45-year-old man diagnosed with mers-cov infection on 14 june 2015. he was exposed to a mers-cov infected patient on 6 june 2015 and high fever, myalgia, and coughing commenced on 13 june 2015. chest radiography showed pneumonic consolidation on the left middle and right lower lung fields. he was treated with a combination of interferon-α2a, ribavirin, and lopinavir/ritonavir from 16-28 june 2015. two consecutive negative results of mers-cov pcr were confirmed on 27 and 28 june 2015. the fever subsided 3 days after initiation of treatment associated with improvement on chest radiography. full recovery was achieved without any complications, and a blood sample was collected on 29 september 2016. all subjects had no underlying comorbidities. a western blot to identify the incorporation of mers-cov s protein in the packaged mers-cov pseudovirus showed a clear band corresponding to the mers-cov s protein. as shown in fig. 1 , three-fold diluted serum of subjects 1 and 2 showed only 9.7% and 10.3% reductions in rlu, respectively, while that of subject 3 showed only a 2.2% reduction. the findings of this study are inconsistent with previous observations. a previous study on severe acute respiratory syndrome coronavirus (sars-cov) infection found that 93.88% and 89.58% of patients were igg positive at 1-and 2-year postsymptom onset, respectively [6] . however, 3 years later, only 53.57% of the population had sars-cov specific igg. a study on antibodies against mers-cov, including neutralizing antibodies, showed antibody persistence in six of seven individuals (86%), 34 months after the 2012 mers-cov outbreak in jordan [7] . all seven subjects had respiratory symptoms and few underlying diseases, and five had substantial pneumonia. mild or asymptomatic mers-cov infections could potentially be associated with development of lower levels of mers-cov neutralizing antibodies over time [8] . patients from the above study might not have been treated with antivirals. subjects in our study were also young and healthy, with mild pneumonic consolidations at admission. moreover, all patients in our study were treated with interferon-α, ribavirin, and lopinavir/ritonavir. antiviral treatment for mers associated with mild pneumonia might decrease viral replication and reduce antibody responses in the early stages of the diseases, resulting in the identified low-level neutralizing activity within convalescent serum. the small number of subjects is an important limitation of this study. furthermore, we could not evaluate paired sera comparing the early stage of illness and 1-year post-recovery. the viral load at early infection time may be associated with the neutralizing antibody and the disease outcome, but we could not collect data on the viral load of subjects. in addition, we could not validate the pseudovirus inhibition assay with positive control, and subjects without treatment could not be enrolled for comparisons. in conclusion, neutralizing activity was not demonstrated in any sera of three patients with mild pneumonia >1 year after being successfully treated with antiviral agents and recovering from mers-cov infection. an outbreak of middle east respiratory syndrome coronavirus infection in south korea rapid response team. antiviral treatment guidelines for middle east respiratory syndrome development of a safe and convenient neutralization assay for rapid screening of influenza ha-specific neutralizing monoclonal antibodies a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov development and evaluation of a pseudovirus-luciferase assay for rapid and quantitative detection of neutralizing antibodies against enterovirus 71 duration of antibody responses after severe acute respiratory syndrome persistence of antibodies against middle east respiratory syndrome coronavirus transmission of mers-coronavirus in household contacts key: cord-320746-iuzfexig authors: rasmussen, sonja a.; gerber, susan i.; swerdlow, david l. title: middle east respiratory syndrome coronavirus: update for clinicians date: 2015-02-20 journal: clinical infectious diseases doi: 10.1093/cid/civ118 sha: doc_id: 320746 cord_uid: iuzfexig although much recent focus has been on the recognition of ebola virus disease among travelers from west africa, cases of middle east respiratory syndrome coronavirus (mers-cov), including travel-associated cases, continue to be reported. us clinicians need to be familiar with recommendations regarding when to suspect mers-cov, how to make a diagnosis, and what infection control measures need to be instituted when a case is suspected. infection control is especially critical, given that most cases have been healthcare-associated. two cases of mers-cov were identified in the united states in may 2014; because these cases were detected promptly and appropriate control measures were put in place quickly, no secondary cases occurred. this paper summarizes information that us clinicians need to know to prevent secondary cases of mers-cov from occurring in the united states. although much recent focus has been appropriately placed on the recognition of ebola virus disease in travelers returning from west africa, the recent increase in cases of middle east respiratory syndrome coronavirus (mers-cov) infection (including travelassociated cases) is also of concern [1, 2] . mers-cov is a novel virus first reported in saudi arabia in 2012. as of 25 february 2015, a total of 1026 laboratoryconfirmed cases of mers-cov infection, including at least 376 deaths (36.7%), have been confirmed by the world health organization (who) (figure 1 ) [2] . between 1 august 2014 and 25 february 2015, there have been 172 cases (and 69 deaths) confirmed by who, with an additional 16 cases and 12 deaths pending who confirmation. all cases reported thus far have had a direct or indirect link through travel or residence to 9 countries in or near the arabian peninsula (saudi arabia, united arab emirates, qatar, oman, jordan, kuwait, iran, lebanon, and yemen); at least 19 countries (including the united states) have had travel-associated cases ( figure 1 ). most mers-cov patients have had fever, cough, and shortness of breath, often leading to severe respiratory complications (eg, pneumonia or acute respiratory distress syndrome) [3, 4] ; some cases with mild or no symptoms (often tested as part of a contact investigation) have also been reported [5, 6] . other reported symptoms include chills/rigors, headache, cough, dyspnea, myalgia, sore throat, coryza, dizziness, nausea/vomiting, diarrhea, and abdominal pain. laboratory findings seen in some patients include leukopenia, lymphopenia, thrombocytopenia, thrombocytosis, and elevated levels of lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase [3, 4] . in some mers-cov patients, coinfection with other respiratory pathogens has been reported [7, 8] ; thus, identification of an alternative respiratory diagnosis should not rule out the possibility of mers-cov. the median age of persons with laboratory-confirmed mers-cov infection is 50 years, approximately 63% have been males, and approximately 18% have been healthcare workers [2] . early on in the outbreak, a predominance of male patients and those with comorbidities was noted, and most cases had severe illness. however, as additional data are collected, the full spectrum of illness is becoming more clear, with a decrease in the percentage of severe cases and increase in asymptomatic cases, as well as a decline in the male-to-female ratio, median age, and case-fatality rate [9] . mers-cov transmission is not fully understood; however, many cases have had exposure(s) to healthcare settings during the 14 days before symptom onset (the median incubation period is approximately 5 days, with a range of 2-14 days). recent data highlight the likely role of camels as a source of human infection in some cases (eg, mers-cov has been found in camels in the region; mers-cov gene sequences in camels are similar to those in humans; camels have had antibodies to mers-cov or a mers-cov-like virus, suggesting previous mers-cov infection; and full gene sequencing has linked mers-cov identified in a camel to a patient who died of mers-cov) [10] [11] [12] [13] [14] [15] . on 1 may and 11 may 2014, 2 cases of mers-cov infection were identified in indiana and florida, respectively [16, 17] . both us cases were healthcare workers in saudi arabia and presented to hospitals in the united states with mild to moderate, nonspecific findings, where they were identified as having mers-cov by astute clinicians sensitized to the need to identify and test returning travelers with respiratory illnesses. both patients were appropriately isolated and no secondary cases were identified despite extensive follow-up. these cases emphasize the importance of us clinicians' becoming familiar with the clinical and epidemiologic features and actions needed to detect and manage mers-cov patients. the number of mers cases has continued to increase; therefore, the risk of exportation of cases to the united states mandates that clinicians need to continue to be vigilant. the centers for disease control and prevention (cdc) recommends evaluation for mers-cov infection for the following patients [18] : 1. patients with fever and clinical or radiographic evidence of pneumonia or acute respiratory distress syndrome and either a history of travel from countries in or near the arabian peninsula (bahrain; iraq; iran; israel, the west bank, and gaza; jordan; kuwait; lebanon; oman; qatar; saudi arabia; syria; the united arab emirates; and yemen) within 14 days before symptom onset, or close contact with a symptomatic traveler who developed fever and acute respiratory illness within 14 days after traveling from countries in the region. 2. patients who are a member of a cluster of patients with severe acute respiratory illness (eg, fever and pneumonia requiring hospitalization) of unknown etiology in which mers-cov is being evaluated, in consultation with state and local health departments. 3. patients with fever and symptoms of respiratory illness (eg, cough, shortness of breath) who have a history of being in a healthcare facility (as a patient, worker, or visitor) within 14 days before symptom onset in a country or territory in or near the arabian peninsula in which recent healthcare-associated cases of mers-cov have been identified. laboratory testing for suspected mers-cov cases in the united states can be performed using the cdc's real-time reverse transcription polymerase chain reaction assay [19] . testing of multiple specimens from different sites (ie, nasopharyngeal swab, oropharyngeal swab, sputum, serum, and stool/rectal swab) at different times after symptom onset is recommended to maximize the probability of mers-cov detection. lower respiratory tract specimens (eg, sputum or bronchoalveolar lavage) and serum should be tested when possible because mers-cov infection has sometimes been detected in lower respiratory specimens or serum when upper respiratory specimens had tested negative, including in the 2 us cases [16, 17] . most state health department laboratories in the united states are approved for mers-cov testing; clinicians should contact their state or local health department when evaluating a patient for mers-cov infection. if testing is not available through coordination with state and local health departments, samples can be sent to the cdc for testing [18] . serologic testing, which is useful for making a retrospective diagnosis, is only available in the united states at the cdc. healthcare-associated infections have played a major role in the transmission of mers-cov [4, 9, [20] [21] [22] [23] . based on the experience with severe acute respiratory syndrome (sars) coronavirus patients and early data from mers-cov, strict infection control practices prevent spread of infection [24] . thus, the cdc recommends implementation of screening and triage procedures for early recognition of potentially infected patients and prompt institution of infection control measures, including standard, contact, and airborne precautions (airborne precautions include care in an airborne infection isolation room, eye protection with goggles or face shield, and respiratory protection at least as protective as a fit-tested n95 filtering facepiece respirator that has been national institute for occupational safety and health certified), to prevent secondary cases in healthcare workers or other patients [18] . healthcare workers exposed to aerosol-generating procedures (eg, sputum induction, intubation, and airway suctioning) appear to be at particularly high risk [23] . a high degree of respiratory protection (ie, including airborne precautions) is currently recommended by the cdc because data on mers-cov transmission are limited; these guidelines are similar to what was recommended during the sars response and will be updated as additional information becomes available. no specific vaccine or treatment is currently available for mers-cov. many hospitalized patients become severely ill, with respiratory failure followed by multiorgan failure [8] . clinical management focuses on supportive care [8, 16, 18] . in a small retrospective cohort study, 20 severely ill patients with mers-cov were treated with a combination of ribavirin and interferon alfa-2a, vs 24 patients in a comparison group. improved survival in treated patients was noted at 14 days, but the results at 28 days were not statistically significant. a randomized controlled trial will be needed to determine if this treatment is of benefit [25] . no restrictions on travel to or from the arabian peninsula are recommended [26] . however, the cdc recommends that travelers protect themselves from mers-cov exposure by washing their hands frequently and avoiding close contact with ill persons. given the emerging data on camels as a reservoir, the world health organization and the cdc recommend general hygiene measures (including regular handwashing before and after touching animals and avoiding contact with sick animals) for persons visiting farms, markets, or other places where animal contact is possible [26] . consumption of raw or undercooked animal products, including camel milk, should also be avoided. additional precautions that are recommended for persons at high risk for severe mers ( people with diabetes, kidney failure, chronic lung disease, or weakened immune systems) include avoiding contact with camels as well as consumption of raw camel milk and urine. in addition, people who are traveling to the region to provide healthcare services should review the cdc's infection control guidelines for mers [26] . travelers from the region with onset of fever or respiratory symptoms during their trip or within 14 days of leaving the region should seek medical care. before presenting for care, patients should inform the healthcare facility of their recent travel so appropriate isolation measures can be implemented. to prevent further introduction of cases of mers-cov into the united states, the cdc has developed guidance for us travelers to countries in or near the arabian peninsula about mers-cov, including information aimed at those attending mass gatherings, such as the hajj and umrah [26] , and public health messages about mers have been posted at us airports. the cdc has also conducted mers outreach with community-based organizations, travel agencies, institutes of higher learning, and businesses for travelers to the arabian peninsula. the cdc has also shared guidance to rapidly identify mers-cov cases among travelers to the united states with airlines, us customs and border protection, and the us transportation security administration. the cdc is working with international partners to conduct studies aimed at better understanding mers-cov transmission and risk factors for illness in community and hospital settings. as new information becomes available, the cdc will update its recommendations [18] . mers-cov and ebola virus disease remind us all of the interconnectedness of the united states to the rest of the world. with the increase in global travel, we must remain alert for emerging infectious diseases to protect us residents and the world from these public health threats. disclaimer. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention (cdc). financial support. all authors completed this work as part of their official duties at the cdc. potential conflicts of interest. all authors: no potential conflicts of interest. update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities world health organization. coronavirus infections-disease oubreak news epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome coronavirus disease in children state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans enhanced mers coronavirus surveillance of travelers from the middle east to england clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus: epidemiology and disease control measures isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia evidence for camel-to-human transmission of mers coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states middle east respiratory syndrome (mers): information for health providers real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus an observational, laboratorybased study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia middle east respiratory syndrome coronavirus: implications for health care facilities hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infections in health care workers hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study acknowledgments. we acknowledge huong pham, jessica rudd, aaron curns, and rebecca dahl for their assistance with collection and compilation of epidemiologic data on middle east respiratory syndrome cases. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-324671-7xdnmms9 authors: seo, yae eun; kim, hyun chung; yoo, so young; lee, kang uk; lee, hae woo; lee, so hee title: factors associated with burnout among healthcare workers during an outbreak of mers date: 2020-07-08 journal: psychiatry investig doi: 10.30773/pi.2020.0056 sha: doc_id: 324671 cord_uid: 7xdnmms9 objective: although healthcare workers (hcws) experienced significant stress during the 2015 outbreak of middle east respiratory syndrome (mers), the factors associated with this stress remain unknown. thus, the present study assessed burnout among hcws during the mers outbreak to identify the influential factors involved in this process. methods: this study was a retrospective chart review of the psychological tests and questionnaires completed by 171 hospital employees from two general hospitals that treated mers patients. the tests included the oldenburg burnout inventory, positive resources test, the questionnaires assessed exposure to the mers outbreak event and perceptions about mers. results: of the 171 hcws, 112 (65.5%) experienced disengagement and 136 (79.5%) suffered from exhaustion. disengagement was associated with lower levels of purpose and hope, a higher perception of job risk, and exposure to the media. exhaustion was associated with lower levels of purpose and hope, a higher perception of little control of the infection, a higher perception of job risk, prior experience related to infections, and being female. conclusion: our results revealed the risk and protective factors associated with burnout among hcws during an outbreak of mers. these findings should be considered when determining interventional strategies aimed at ameliorating burnout among hcws. after a mers outbreak. kim and choi 13 examined burnout after mers in terms of job stress caused by mers, fear of mers infection, hospital resources for the treatment of mers, and support from family and friends. although they found that job stress, poor hospital resources, and poor support from family and friends explained 47.3% of burnout, they did not examine whether personal strength or coping could prevent burnout or how experiences at the time of the incident and perceptions about epidemic disease impact it. thus, we invested the proportion of hcws that suffered from burnout (i.e., engagement and exhaustion) during the 2015 outbreak of mers and the protective and/or risk factors associated with it (i.e., engagement and exhaustion). the present study was conducted between june 2015 and june 2016 during the mers outbreak. a retrospective chart review of psychological tests completed by 171 hospital employees from two general hospitals that treated mers patients was conducted; the sample consisted of 32 doctors, 77 nurses, and 61 others (i.e., pharmacists, technician, officers, and so forth). approval was obtained from the institutional review board of national medical center in korea (no. h-1507-056-004). burnout was measured with the oldenburg burnout inventory (olbi), which was validated in korean by na. 14, 15 this scale includes 16 items: 8 measure exhaustion (e.g., "there are days when i feel tired before i arrive at work") and 8 measure disengagement (e.g., "it happens more and more often that i talk about my work in a negative way"). each of the subscales includes four positively worded items and four negatively worded items and each item is scored on a scale ranging from 1 (strongly agree) to 4 (strongly disagree). as described in a previous study, the following cutoff scores were defined as high: ≥16.8 for disengagement and ≥18 for exhaustion. 16 in the present study, the cronbach's alpha values were 0.70 for disengagement and 0.82 for exhaustion. positive resources were measured with the positive resources test, which was developed and validated in korean by huh et al. 17 this measure includes 23 items scored on a five-point scale ranging from 1 (strongly disagree) to 5 (strongly agree). scores are generated for five subscales: optimism, purpose and hope, self-regulation, social support, and caring and serving. their cronbach's alpha values were 0.79, 0.78, 0.55, 0.70, and 0.81, respectively. participants also answered the following questions about their exposure to mers: "have you been within 2 meters of a mers patient while not wearing personal protective equipment (ppe)?, " "have you treated mers patients in person while wearing ppe?, " "have you experiences mers-like symptoms after contact with mers patients?, " "have you ever been quarantined?, " "have your hospital or employees been exposed to media reports about the mers outbreak, such as tv, internet, radio and so on?. " all questions were answered with either "yes" or "no. " perceptions about mers were assessed with a 10-item measure similar to the one described by chong et al. 18 questions addressed the following: perceived job risk, acceptance, fear of falling ill with mers, lack of control over infection, chance of survival if infected, fear of infecting others with mers, concern about infecting family and friends, and other's avoidance of their family because of their work. each item was answered on a 5-point scale ranging from 1 (strongly disagree) to 5 (strongly agree). because items were heterogeneous, we treated each item as a separate variable (table 1 ). all data were analyzed using spss for windows version 22.0 (ibm corp., armonk, ny, usa). the means and standard deviations (sd) were calculated for burnout, positive resources, and mers-related recognition. categorical data were summarized using frequencies and percentages of occurrence (sex, job title, and so forth). the psychological effects of the mers outbreak on employees were assessed based on scores on items measuring burnout and factors that might explain the variance in burnout were identified using between-group differences and correlation analyses. between-group differences in parastepwise regression analyses were performed to performed to derive predictive model for burnout. all factors identified as significant by the difference tests and correlation analyses were included as predictor variables and burnout (exhaustion, disengagement) was treated as the criterion variable. statistical significance was set at α=0.05 (two-tailed). table 2 presents the characteristics of the total sample. of the 171 participants, the mean age was 34.2 years (sd=9.8), 66.1% were female, 45.3% were nurses, 18.8% were doctors, 17.65% were pharmacists or other healthcare workers, 18.24% were technicians or office workers, and 88.7% had received mers education. in addition, 3.5% were exposed to one or more mers patients within 2 m without personal protective equipment (ppe), 44% had cared for mers patients in person with ppe, 7.3% experienced mers-like symptoms, 3% were quarantined, and 78.6% responded that their hospital or employees were exposed to mers-related media. the mean score for disengagement was 17.6 and the mean score for exhaustion was 20.7 (table 3 ). more than 65.8% of participants experienced disengagement and more than 79.5% experienced exhaustion. to identify the factors that influenced burnout, betweengroup analyses of demographic variables, medical history, traumatic experiences, work-related characteristics, and exposure to mers were conducted ( table 4 ). the level of disengagement significantly differed according to sex, work experience, providing care while using ppe, and exposure to media. the level of exhaustion significantly differed according to sex, prior traumatic events, prior experience related to infection, job type, providing care while using ppe, and exposure to media. correlation analyses were performed to assess positive resources and perceptions about mers ( table 5 ). the level of disengagement was significantly correlated with optimism, purpose and hope, self-regulation, caring and serving, perceived job risk, fear of falling ill with mers, lack of control over infection, little chance of survival if infected, fear of passing mers to others, worry of family and friends getting infected through oneself, and avoiding one's family because of one's work. the level of exhaustion was significantly correlated with optimism, purpose and hope, self-regulation, social support, perceived job risk, acceptance, fear of falling ill with mers, lack of control over infection, little chance of survival if infected, fear of passing mers to others, worry of family and friends getting infected through oneself, and avoiding one's family because of one's work. stepwise regression analyses were conducted to determine which variables accounted for significant variance in burnout ( table 6 ); variables that were significant (p<0.05) in previous analyses were retained. purpose and hope, perceived job risk, and exposure to media explained 33% of disengagement and purpose and hope, lack of control over infection, perceived job risk, prior experience related to infection, and sex explained 38% of exhaustion. we found that 65.5% of hcws reported disengagement and 79.5% reported exhaustion during the 2015 mers outbreak. in addition, disengagement was associated with lower levels of purpose and hope, the perception of higher job risk, and ex*mann-whitney u test posure to media whereas exhaustion was associated with lower levels of purpose and hope, a lower level of perceived control, and the perception of higher job risk. however, receiving mers education did not have a significant effect. it is important to note that the majority of participants in the present study experienced burnout during the outbreak. the rate of burnout was similar to that observed in emergency nurses after the outbreak although the present sample included various job types, including doctors, pharmacists, and technicians. 13 we also found that, among these groups, nurses reported the highest level of burnout and that women had a higher level of burnout than men. this is in contrast to the findings of maslach and jackson, 19 who reported that men are slightly more vulnerable to burnout than women. it is possible that there were more female nurses than females in the other groups and thus future studies should determine whether high levels of burnout are more affected by sex or job. nonetheless, our findings suggest that nurses should be a priority of interventions aimed at reducing burnout. of the personal strength variables, purpose and hope were the most powerful protective factors against both exhaustion and disengagement. this suggests that people who have purpose and hope can bear hardships and cope well with disastrous situations, which is consistent with previous findings. for example, youssef found that employees with hope report higher levels of job satisfaction, work happiness, and actual performance. 20 similarly, sherwin et al. 21 examined occupational burnout among nurses in chronic care rehabilitation units and found that a higher level of hope is significantly associated with lower burnout. therefore, during an outbreak of epidemic disease, setting attainable purposes and committing to those purposes could aid in the prevention of burnout in hcws. we also found that perceptions about mers were an important variable predicting hcw burnout, particularly in terms of perception of job risk and lack of control. the subjective perception of job risk was a risk factor for both exhaustion and disengagement and lack of control over infection was a risk fac22, 23 it is possible that one's perception of job risk and feelings of lack of control could result in feelings of helplessness, which in turn would result in hcws detaching themselves from work and feeling exhausted. preparedness can increase confidence in the ability to do a job as well as psychological well-being. 24 however, our results indicate that receiving mers education was not protective against burnout, which is inconsistent with previous findings showing that perceived adequacy of training is a significant predictive factor. 12 taken together, these findings suggest that mers education programs should be revised to strengthen safety and have tougher anti-infection measures to better protect against burnout in hcws. in terms of experiences at the time of infection, we found that exposure to media was one of the most important factors. when exposed to media, individuals may feel like they are under constant surveillance and could be subjected to negative comments by anonymous people, which may lead to the experience of stress and withdrawal from work. for example, when mers broke out in korea, it was common to observe a social stigma against patients who recovered from the infection, the families of infected people, and hcws. in this type of environment, exposure to media might be stressful and leave hcws feeling alienated. volpone and avery 25 reported that perceived discrimination based on race, sex, age, family obligation, and/ or sexual orientation is related to psychological withdrawal that leads to physical issues such as lateness, absenteeism, and the intent to quit. our findings suggest that discrimination in society as well as in the workplace could lead to burnout among hcws. thus, social campaigns and practical measures aimed at decreasing excessive public fear and rumors toward hcws may be helpful. on the other hand, we found that experience of mers-like symptoms, quarantine, and exposure without the use of ppe were not significantly associated with burnout. it is possible that the number of hcws who experienced these situations was insufficient to produce significant results. we also found that prior work experience with infections was related to high levels of exhaustion, which indicates that experienced workers were more likely to feel tired. it is possible that prior work experience with treating infected patients could be traumatic for workers. for example, workers with repeated indirect traumatic experiences are more likely to experience burnout, and thus the identification and treatment of individuals with traumatic work experience may be effective for reducing future burnout in hcws. 26 it is also possible that experienced workers might be overloaded and have significant responsibilities. a high work load is related to dissatisfaction at work and differences in quality of care, and thus it may be helpful to properly distribute work responsibilities and focus on experienced workers when intervening to prevent burnout in hcws. 27, 28 the present study had several limitations that should be considered. first, we included hcws from only two hospitals, which may limit the generalizability of the results. second, although we targeted individuals who performed certain tasks related to mers, we did not assess the exact tasks that they did at that time, and thus it was not possible to determine which specific tasks or experiences were more likely to lead to burnout. regardless, our findings suggest that hcws in risky work situations were more vulnerable to burnout. third, perceptions about mers and exposure to mers were not measured using validated scales. although the subjective perception of infections is a very important factor related to psychological problems, an appropriate scale has yet to be developed. thus, it will be necessary to develop and validate instruments that can measure subjective attitudes and perceptions about infections. fourth, the numbers of hcws who experienced mers-like symptoms, were quarantined, and who were exposed to mers without ppe were insufficient to produce significant results. future studies should include a sufficient number of individuals with these experiences. the present findings are meaningful in several ways. first, we found that a majority of hcws in hospitals that treated mers patients during the outbreak experienced burnout. second, nurses, women, and experienced workers are more vulnerable to burnout. third, our findings expand on previous findings by showing that personal strength and perceptions about infections are important factors that predict burnout in hcws, which emphasizes the importance of personal strength factors, such as purpose and hope, in protecting workers against burnout. on the other hand, perception of high job risk and a sense of a lack of control are risk factors of burnout. finally, our findings suggest practical ways to reduce burnout. for example, exposure to media is an important factor in burnout, which suggests that guidelines for the media coverage of epidemic diseases should be set. isolation of a novel coronavirus from a man with pneumonia in saudi arabia an outbreak of middle east respiratory syndrome coronavirus infection in south korea job burnout: new directions in research and intervention the relationship of organizational culture, stress, satisfaction, and burnout with physicianreported error and suboptimal patient care: results from the memo study organizational stress: a review and critique of theory, research, and applications. thousand oaks: sage the oldenburg burnout inventory: a good alternative to measure burnout and engagement nurse staffing, burnout, and health care-associated infection stress and burnout in community mental health nursing: a review of the literature determinants and prevalence of burnout in emergency nurses: a systematic review of 25 years of research burnout' , absence and turnover amongst british nursing staff factors that influence the development of compassion fatigue, burnout, and compassion satisfaction in emergency department nurses long-term psychological and occupational effects of providing hospital healthcare during sars outbreak factors influencing emergency nurses' burnout during an outbreak of middle east respiratory syndrome coronavirus in korea the job demands-resources model of burnout suwon: the graduate school of ajou university work characteristics and sickness absence in burnout and nonburnout groups: a study of swedish health care workers development of clinical short-form positive resources test psychological impact of severe acute respiratory syndrome on health workers in a tertiary hospital the role of sex and family variables in burnout positive organizational behavior in the workplace: the impact of hope, optimism, and resilience negotiating the reality of caregiving: hope, burnout and nursing hospital worker's psychological resilience after the 2015 middle east respiratory syndrome outbreak perceived control, depressive symptomatology, and professional burnout: a review of the evidence a systematic, thematic review of social and occupational factors associated with psychological outcomes in healthcare employees during an infectious disease outbreak it's self defense: how perceived discrimination promotes employee withdrawal a meta-analysis of the relationship between job burnout and secondary traumatic stress among workers with indirect exposure to trauma stress, work overload, burnout, and satisfaction among paramedics in israel overload, autonomy, and burnout as predictors of physicians' quality of care this study was supported by a grant from the korean mental health technology r&d project, ministry of health & welfare, republic of korea (hl19c0007). key: cord-330583-ltkpt80u authors: lee, kyu-myoung; jung, kyujin title: factors influencing the response to infectious diseases: focusing on the case of sars and mers in south korea date: 2019-04-22 journal: int j environ res public health doi: 10.3390/ijerph16081432 sha: doc_id: 330583 cord_uid: ltkpt80u following the 2003 the severe acute respiratory syndrome (sars) and the 2015 middle east respiratory syndrome (mers) outbreak in south korea, this research aims to explore and examine the factors influencing the response to infectious diseases, which encompasses both communicable and non-communicable diseases. through a qualitative research method, this research categorizes the factors as inputs, processes and outputs and applies them into the 2003 sars and mers outbreak in south korea. as the results conducted meta-analyses to comprehensively analyze the correlations of factors influencing disaster response from a korean context, the findings show that the legislative factor had direct and indirect influence on the overall process of infectious disease response and that leadership of the central government, establishment of an intergovernmental response system, the need for communication, information sharing and disclosure and onsite response were identified as key factors influencing effective infectious disease response. recently, a wide array of disasters, including earthquakes, forest fires, various infectious disease outbreaks and marine accidents have occurred in korea. accordingly, the importance of disaster response has drawn more attention than it ever has in korea, which was once considered a safe zone from various disasters. however, following the recent and sudden increase in the frequency of disasters, sufficient learning and legislature, as well as policy-making processes in korea have exposed limitations in the disaster management system, relative to other countries and regions where disasters have occurred more frequently. the recent efforts to change the disaster response system after certain disasters has exposed many limitations. under such circumstances, it is difficult to respond appropriately to disasters and there are limitations to avoiding or reducing social and economic losses caused by disasters. after the sinking of mv sewol, also called the sewol ferry disaster, in 16 april 2014, the korea coast guard was dissolved and the ministry of public safety and security (mpss) took on the role of a control tower. every episode of failed disaster response was always blamed on the lack of a control tower. therefore, the introduction of mpss was expected to be a panacea for effective disaster response. however, the initial response still failed during subsequent disasters, including in the middle east respiratory syndrome (mers) outbreak, the geyongju earthquake and various marine accidents, which confirmed that the disaster response was still not being effectively executed. when president the present study conducted an in-depth review of the concept of infectious disease and infectious disease response, which are the subjects of its theoretical review. in the past, the terms "contagious" or "communicable disease" were generally used. however, because the term "communicable diseases" implied diseases that were transmitted from one person to another, which further implied difficulties in controlling them, the term was changed to "infectious diseases," which encompasses both communicable and non-communicable diseases. for effective prevention and management of infectious diseases, the existing "parasitic disease prevention act" and "communicable disease prevention act" were merged. according to the "infectious disease control and prevention act," infectious diseases include class 1-5 of infectious diseases, designated infectious diseases, who-monitored infectious diseases, bioterror infectious diseases, sexually transmitted diseases, zoonotic infectious diseases and healthcare-associated infections. korea has experienced outbreaks of diseases that were traditionally regarded as "diseases that occur in developing countries," such as hepatitis a, tuberculosis, chicken pox and malaria, while cholera patients were identified for the first time in 15 years in 2016. moreover, despite continued outbreaks of emerging infectious diseases since the sars outbreak, there have been no noticeable changes in prevention and response measures. with the subsequent occurrence of the mers and zika virus in korea and the re-emergence of cholera, an infectious disease that had not been experienced for a while, public anxiety about health safety is growing. according to the infectious diseases surveillance yearbook published by the korea center for disease control and prevention (kcdc), the level of imported infectious diseases has been increasing every year, with 300-400 newly reported cases every year since 2010. experts have warned that this is only the beginning of the war against infectious diseases, due to the following reasons. with the changing global environment and increased human migration, prevention of emergence or re-emergence of infectious diseases is fundamentally impossible. moreover, infectious diseases tend to evolve along with advances in medical technology and emerging infectious diseases are difficult to handle since they spread quickly and have no readily available treatment. in particular, knowledge about the characteristics, route of infection and control measures of emerging infectious diseases are lacking or uncertain and it is difficult to predict when, where and how these diseases may emerge. moreover, globalization has brought with it an increased level of international trade and travel. therefore, korea, which has a high foreign trade dependency, is constantly exposed to the risk of imported infectious diseases. despite the growing anxiety and concerns about infectious diseases, studies on infectious disease response and control are lacking. after the mers outbreak, numerous studies on the response to this outbreak were conducted . however, most of the studies focused mostly on medicine and communication, with relatively fewer studies focusing on administrative fields [38] [39] [40] [41] [42] [43] [44] [45] [46] . studies in the medical field must precede the response to infectious diseases, so that information and knowledge about the infectious disease can be applied in response measures. however, if the national infectious disease response system is not ready when an actual infectious disease outbreak occurs, then medical determination and response, as well as crisis management and communication cannot be executed properly. this is because medical response, crisis management and communication are sub-elements in such a national-scale system. therefore, it is important to conduct studies on infectious diseases and responses in every specialty. however, there is also need for comprehensive discussions that include the establishment of laws; regulations; resources; information on infectious disease response from administrative and policy perspectives; information sharing system; and the establishment of an international cooperation system and national response system involving the central government, the regional government, private organizations and the public for effective response when an actual infectious disease outbreak occurs. in addition to infectious diseases being difficult to handle, the mers outbreak in 2015 also revealed that even if prevention and response measures are in place, a failed initial response can lead to an unanticipated increase in the rate and scope of infection transmission. moreover, disaster responses do not always pan out as planned and uncertainties and complexities that emerge after the disaster must be handled. therefore, it is necessary to identify government-level responses and make effort to improve the response system. however, previous studies on responses to infectious diseases are still lacking despite the importance of this issue. accordingly, the present study was conducted with the consciousness of the need to analyze response systems based on past response experiences in order to effectively respond to future infectious diseases, which are a threat. the present study analyzed two cases of infectious disease outbreaks based on the factors that influence response to disaster, as identified through existing studies and theories and aimed to derive factors that have a strong influence on the effectiveness of actual disaster response. in the following section, the factors that influence disaster response will be categorized from a system theory perspective to form a categorization framework. factors that influence disaster response have been identified through numerous studies over several years. most of the studies on disaster response analyzed actual cases by applying analytical tools based on major variables presented in existing studies and theories [14, [47] [48] [49] [50] [51] [52] [53] [54] or they analyzed the factors that influence disaster response by administering questionnaire surveys to members of agencies associated with disaster response [12, 55, 56] . therefore, instead of comprehensively examining the factors influencing disaster response, these studies handled the subject at a macroscopic level, focusing on the major variables. the present study aimed to organize factors and variables that influence the entire process of disaster response from a comprehensive and systematic perspective and categorize these factors and variables based on a system theory perspective in order to present an analytical framework. this concept and context are similar to those of perry [57] , who claimed that influencing factors of disaster response that are derived without classifying according to disaster types do not need different analytical frameworks since they can be described and analyzed according to the factors presented and they only differ in intensity according to the type of disaster. the present study reviewed existing studies, focusing on those with "disaster response" and "effective disaster response" as the outcome variables. the factors that influence disaster response can be broadly categorized into financial resources, human resources, physical resources, information, education and training, leadership, intergovernmental relationships, onsite response, information sharing, environmental context, characteristics of disaster and the legal/institutional environment. resources that influence disaster response can be categorized into financial, human and physical resources. financial resources include the government's budget for disaster response, funding to support processes involved in disaster response and support from the government or community [50] [51] [52] [53] . therefore, financial resources can be regarded as the disaster-related budget, the disaster management fund and financial support measures for processes entailed in korea's disaster response system. human resources included disaster response-related organizations and agencies, education and training of relevant organizations and the general public and utilization of specialists. existing studies have pointed out that the establishment of disaster response-related organizations or crisis management centers and the securement of specialists have a very significant influence on disaster response [49, 51, 52, 55, 58, 59] . this is because identification of disaster response-related organizations and agencies must come first to allow effective communication about disaster response and secure accountability in disaster response [55] . moreover, education and training for disaster response organizations and their members has been mentioned as an element that allows effective disaster response [49, 51] . lastly, physical resources refer to securement of disaster management-related resources and establishment of disaster management facilities. as indicated in the study by lindell et al. [51] , securement of disaster response and management resources within the organization allows timely and accurate disaster response, which was expressed as disaster response equipment in the study by jung [56] . such physical resources can be viewed as disaster management resources and facilities and whether the expansion of negative-pressure units and emergency isolation units have occurred also influences the response to infectious diseases. other influencing factors besides resource-related factors include education and training. knowledge can be explained as a collection of disaster-related information and information sharing in advance, where information about different types of disasters must be collected before the occurrence of a disaster. factors associated with disaster information have a positive effect on disaster response, as indicated in the study by kim [60] , which reported that when a disaster information system is established the recognition of the importance of information quality and higher information quality had a positive effect on achieving disaster management duties. next, leadership, intergovernmental relationships and communication, onsite response and information sharing have been identified as factors that influence disaster response. first, leadership in the context of this paper refers to leadership in the central government, which can be divided into leadership from the president and leadership from central organizations and agencies. the president's level of interest in disaster management and response and the governance style in running an organization, were analyzed as factors that have significant influence on disaster response [61] . moreover, onsite leadership by the heads of organizations and agencies must be demonstrated in a timely manner, especially in initial disaster response, in order to prevent the disaster from spreading [50, 53, 56, 59, 62] . second, intergovernmental relationships and communication are also factors that influence effective disaster response [50, 52, 53, 63, 64] . in addition, network was also pointed out as a factor that influences disaster response. existing studies tended to use the concepts of network and inter-organizational cooperation without clearly differentiating them but both network and inter-organizational cooperation were analyzed as factors that positively influence disaster response [54, 63, 64] . one of the reasons inter-organizational communication and coordination, network and cooperation have been identified as important influencing factors is that appropriate allocation and utilization of disaster response-related resources are essential for effective disaster response. accordingly, the present study analyzed intergovernmental relationships in order to comprehensively examine intergovernmental and inter-organizational relationships, communication and cooperation. information sharing has also been identified as an important factor in disaster response. information sharing between organizations and with the general public after a disaster was found to have a positive influence on actual disaster response. in particular, a study by hyun [6] found that information disclosure-related legislation, the organization's budget, personal awareness and attitude and information quality influenced the effectiveness of disaster management. among various factors influencing disaster management and response, factors associated with disaster-related information disclosure and sharing were tested for their influence on the effectiveness of disaster management. the results showed that greater information quality in information disclosure and sharing and greater personal awareness and attitude positively influenced the effectiveness of disaster management. effective disaster response may comprise sub-variables from its outcome aspect. in a study by denise [65] , the effectiveness of disaster response was determined by measuring life loss, property damage, satisfaction of stakeholders, society's resilience, operational efficiency and budget maximization. moreover, a study by byun (2014) examined fire service organizations and thus effectiveness was determined by evaluating fire containment and rescue, while efficiency was represented by reduction in damage and cost-effectiveness. other factors influencing disaster response include environmental factors [55, 56, 58, 59] , disaster characteristics [51, 55, 59] and legislative factors [3, 51, 55, 66 ]. sars stands for severe acute respiratory syndrome. it has a latency period of 10 days, after which the victim experiences high fever (â�¥38â°c), coughing and respiratory distress. it is transmitted by respiratory routes to medical staff and family members who come in close contact with the patient. complete cure is possible if treatment is administered early, where approximately 90% of infected patients recover easily within one week. however, sars may rapidly become severe for elderly or frail patients or for patients with chronic illness, yielding a mortality rate of approximately 3.5%. sars became known worldwide on 11 february 2003, when the chinese health authority announced that 305 patients with sars had been identified in china between november 16, 2002 and 9 february 2003, five of whom had died. who, which had strengthened its surveillance activities in the asian region after identifying the likelihood of the emergence of influenza, issued a worldwide warning on 12 march 2003. according to the official statistics released by who in november 2003, between november 2002 and july 2003, a total of 8098 suspected sars cases from a total of 28 countries were identified and a total of 774 sars related deaths were reported. consequently, sars emerged as the first new disease in the 21st century that was highly contagious and severe and its transmission through international air travel received special attention. since february 2003, when sars became known for the first time, korea continued to monitor sars outbreak trends through who data and recognized the need for national quarantine measures. accordingly, guidelines for strengthening nationwide sars quarantine measures were passed down on 12 february 2003 and on march 16 the korean government issued a sars alert, in keeping with the announcement of the global sars alert by who and established a quarantine system. with nih playing a central role, all healthcare institutions, including 12 national quarantine stations and 242 health centers, maintained a 24-hour emergency operation-ready status as part of the emergency sars quarantine measures. moreover, a quarantine system was established by designating 41 hospitals as isolation treatment hospitals according to regions. in addition, the policy of measuring people's body temperature as they entered the country through airports and sea ports was implemented in order to detect sars inflow from overseas and to prevent the disease from spreading. furthermore, follow-up investigations were conducted on people who entered korea from sars-risk regions and a system through which patients suspected of being infected could be transported immediately for isolation and treatment was put in place. moreover, additional isolation measures were implemented for people who came in contact with infected patients to prevent the disease from spreading further, in addition to preventing the import of sars. recommendations were made to refrain from traveling to high-risk regions to further prevent the import of sars into korea and to encourage precautions during travel. during the response process to the outbreak, a meeting for city and provincial quarantine officials and experts was held on 3 april 2003. on 23 april 2003, a discussion was held on measures of blocking the importation of sars and preventing its spread. the decision to implement government-wide response measures by setting up a central sars measures headquarter, led by the minister of health and welfare and satellite stations at city and province levels was made. subsequently on 28 april 2003, a government-wide comprehensive sars situation room was introduced and prime minister kun goh released a general public statement, urging active cooperation from the general public regarding the response measures taken by the government. eventually, who declared on 17 june 2003 that korea had won the war against sars, effectively subduing sars in less than 100 days after the global sars alert was issued [67] . mers stands for middle east respiratory syndrome. the outbreak of mers coronavirus started on 24 april 2015 following its introduction into korea by a 68-year-old male, who worked in floriculture and was returning home to korea after a visit to the middle east. the patient was treated at a clinic for a fever he developed seven days after arriving in korea but his condition did not improve. after his visit to the clinic, he received inpatient treatment for three days at pyeongtaek st. mary's hospital and was subsequently discharged. because of continued symptoms of high fever and respiratory distress, he visited another clinic and was eventually admitted to a single-bed unit at the samsung medical center in seoul on 18 may 2015. the staff at samsung medical center had learned that the patient had visited the middle east and based on a suspicion of mers, the doctor in charge requested kcdc to perform further testing on the patient the following day (19 may 2015) . the diagnostic test performed by nih detected middle east respiratory syndrome coronavirus: (mers-cov) genes in the patient. following the announcement of these findings on 20 may 2015, identification of the first mers patient in korea was officially reported [68] . as shown in figure 1 , the first mers patient in korea visited multiple clinics and large hospitals for treatment over a 10-day period since the symptoms first appeared, during which time he came in contact with family members, other patients and medical staff, resulting in many cases of secondary infection. [68] . as shown in figure 1 , the first mers patient in korea visited multiple clinics and large hospitals for treatment over a 10-day period since the symptoms first appeared, during which time he came in contact with family members, other patients and medical staff, resulting in many cases of secondary infection. as shown in the graph above, the highest number of confirmed mers cases outside of the middle east region was found in korea. the response to mers completely exposed contradictions in the national quarantine system, as well the healthcare system. kcdc and local government entities all proved inadequate in their ability to respond to the public health crisis caused by this infectious disease. there was confusion due to lack of clarity in the delegation of roles between the central and regional governments and the cooperation system between health authorities and medical institutions did not operate smoothly either. most medical institutions, including general hospitals, small-to-medium-sized hospitals and clinics, were not prepared to deal with healthcare-associated infections and as a result the infection continued to spread among patients and medical staff. in addition, problems in the transport and referral system for confirmed or suspected patients were discovered, while compensation for medical institutions and research and development of emerging infectious diseases became points of contention. moreover, medical staff who participated in the isolation and treatment of mers patients complained about job-related burden and stress. the mers outbreak exposed fundamental problems in the public healthcare system and vulnerabilities in the national quarantine system but the solutions to these problems have not been clearly identified to date. the mers outbreak caused restrictions in korean citizens' day to day lives and significantly impacted the national economy. the socioeconomic impact of mers has still not as shown in the graph above, the highest number of confirmed mers cases outside of the middle east region was found in korea. the response to mers completely exposed contradictions in the national quarantine system, as well the healthcare system. kcdc and local government entities all proved inadequate in their ability to respond to the public health crisis caused by this infectious disease. there was confusion due to lack of clarity in the delegation of roles between the central and regional governments and the cooperation system between health authorities and medical institutions did not operate smoothly either. most medical institutions, including general hospitals, small-to-medium-sized hospitals and clinics, were not prepared to deal with healthcare-associated infections and as a result the infection continued to spread among patients and medical staff. in addition, problems in the transport and referral system for confirmed or suspected patients were discovered, while compensation for medical institutions and research and development of emerging infectious diseases became points of contention. moreover, medical staff who participated in the isolation and treatment of mers patients complained about job-related burden and stress. the mers outbreak exposed fundamental problems in the public healthcare system and vulnerabilities in the national quarantine system but the solutions to these problems have not been clearly identified to date. the mers outbreak caused restrictions in korean citizens' day to day lives and significantly impacted the national economy. the socioeconomic impact of mers has still not been accurately assessed. what is clear at this point is that the entire korean society has become more interested in infectious diseases and that infectious diseases have become an agenda directly linked to public safety. moreover, people recognized that in order to respond to emerging infectious diseases it is necessary to continually assess and monitor infectious diseases that occur worldwide and establish manuals based on up-to-date knowledge through research, specialists and timely crisis analysis during the response process. in addition, the need to establish an infectious disease response network and partnerships between medical institutions and local government, as well as a central government, was also presented. the objective of this study was to inductively explore the factors that influence response based on studies related to disaster and infectious disease response. for this, a meta-analysis method called successive approximation was used [69] . prior to inductive exploration of the factors that influence disaster response, sample articles were used to categorize these factors. the study also aimed to perform successive meta-analyses to present a model based on detailed explanation and revision of the previously established factors influencing disaster response. accordingly, the present study used a rough model based on the categorization of the factors influencing disaster response presented in existing studies to perform meta-analyses on sars and mers cases. in summary, after establishing the initial model, several rounds of meta-analyses were performed to refine the model. accordingly, the incomplete preliminary theoretical framework, which can be viewed as the initial model, represented simplification and categorization of major factors through existing disaster response-related studies. the study aimed to conduct successive analyses based on the incomplete framework to present a refined model by revising the factors and the relationships between them. accordingly, precedent studies previously examined in chapter 2 were used to derive the factors influencing disaster response from a system perspective. on a review of numerous studies, it was discovered that the duties assigned to various organizations and agencies and the factors that actually influence disaster response show regularity [47, 70, 71] . therefore, based on such regularity, the study aimed to categorize these factors according to timelines from a system theory perspective. first, the studies that presented communication, coordination, cooperation and network from the process level as the mediating variables for effective disaster response included those by [3, 50, 55, 71] . other studies selected the process level variable as one variable among many independent variables in analyzing the influence on effective disaster response. it was commonly pointed out that resources related to disaster response influenced the outcome of disaster response through the interactions and coordination between organizations and agencies in the response process. kapucu [71] analyzed the influence of the system, organizational environment, tools for organizational capability and cooperation and the decision-making process of actors in the entire process on effective disaster response. the system was a variable that included organizational structure, culture and goals, while the environment included time pressure, uncertainty and complexity of the situation. capability was a factor that involved decision-making support, communication tools, previous cooperation experience, flexibility in responding to disaster and immediate response capability, while actors included the number of stakeholders, experts, interdependence and trust. the study examined whether these independent variables influenced effective disaster response through cooperative decision-making processes, meaning open and honest exchange of opinions, shared model construction, negotiation and utilization of relevant knowledge and information. the proposed research model was used to conduct social network analysis through content analysis, in addition to in-depth case analysis on countries that were victims of terrorist attacks, including the us, indonesia, turkey, spain and the uk. moreover, a study by lindell et al. [51] also revealed that various resources influenced disaster planning and such process had a significant influence on the efficiency of disaster response. on the basis of these studies, a framework consisting of independent variables having an influence on the effectiveness of disaster response through the disaster response processes was constructed. even in studies that do not present process variables as mediating factors, the majority of process variables were selected as independent variables for analysis and thus it is necessary to reorganize and categorize the variables that were presented in previous studies. as shown in figure 2 the present study selected the factors influencing disaster response presented in existing studies and categorized them largely into environment, inputs and process based on system theory. this model was presented because disasters act as a single system that includes the aforementioned factors, regardless of their type (natural or social disaster) [54, [72] [73] [74] . the strength of the influencing factors may vary depending on the type of disaster but because they were described and analyzed by the factors that are presented below, analyzing or describing social and natural disasters using different frameworks is unnecessary [57] . based on the categorization of the influencing factors shown in the figure below, the study aimed to conduct future meta-analyses to explore detailed factors and identify the relationships between these factors. had a significant influence on the efficiency of disaster response. on the basis of these studies, a framework consisting of independent variables having an influence on the effectiveness of disaster response through the disaster response processes was constructed. even in studies that do not present process variables as mediating factors, the majority of process variables were selected as independent variables for analysis and thus it is necessary to reorganize and categorize the variables that were presented in previous studies. as shown in figure 2 the present study selected the factors influencing disaster response presented in existing studies and categorized them largely into environment, inputs and process based on system theory. this model was presented because disasters act as a single system that includes the aforementioned factors, regardless of their type (natural or social disaster) [54] , [72] , [73] [74] . the strength of the influencing factors may vary depending on the type of disaster but because they were described and analyzed by the factors that are presented below, analyzing or describing social and natural disasters using different frameworks is unnecessary [57] . based on the categorization of the influencing factors shown in the figure below, the study aimed to conduct future meta-analyses to explore detailed factors and identify the relationships between these factors. for inductive exploration of the factors influencing infectious disease response, meta-analyses were performed based on the aforementioned factor categorization framework and in-depth interviews were conducted for testing and supplementation. the present study used previous studies on disaster response to compile a list of the factors influencing disaster response and presented a theoretical framework. moreover, the factors influencing disaster response were explored through case review, while qualitative meta-analysis was performed to identify the correlations between the factors. qualitative meta-analysis was conducted to allow a comprehensive analysis of qualitative studies [60] . this method of analysis is different from meta-analysis which integrates results from existing empirical studies using a quantitative method [75] , [76] . the approach in qualitative meta-analysis involves interpretive analysis, which strives to include major concepts that appeared in individual qualitative studies but at the same time, generate a higher-level concept that can connect these concepts to a higher dimensional theoretical structure to allow for comprehensive understanding of the phenomenon and possibility of new interpretation and theoretical creation [77] . therefore, the major goal of qualitative meta-analysis is to contribute to knowledge. from this perspective, schreier et al. [78] listed theory building, theory explication and theory development as the three overlapping goals of qualitative meta-analysis. in the present study, the factors influencing disaster response were reviewed from existing studies in the theory building process and organized from a system theory level. subsequently, meta-analysis was performed to explore factors through for inductive exploration of the factors influencing infectious disease response, meta-analyses were performed based on the aforementioned factor categorization framework and in-depth interviews were conducted for testing and supplementation. the present study used previous studies on disaster response to compile a list of the factors influencing disaster response and presented a theoretical framework. moreover, the factors influencing disaster response were explored through case review, while qualitative meta-analysis was performed to identify the correlations between the factors. qualitative meta-analysis was conducted to allow a comprehensive analysis of qualitative studies [60] . this method of analysis is different from meta-analysis which integrates results from existing empirical studies using a quantitative method [75, 76] . the approach in qualitative meta-analysis involves interpretive analysis, which strives to include major concepts that appeared in individual qualitative studies but at the same time, generate a higher-level concept that can connect these concepts to a higher dimensional theoretical structure to allow for comprehensive understanding of the phenomenon and possibility of new interpretation and theoretical creation [77] . therefore, the major goal of qualitative meta-analysis is to contribute to knowledge. from this perspective, schreier et al. [78] listed theory building, theory explication and theory development as the three overlapping goals of qualitative meta-analysis. in the present study, the factors influencing disaster response were reviewed from existing studies in the theory building process and organized from a system theory level. subsequently, meta-analysis was performed to explore factors through korean studies and articles and interviews. the protocol was constructed through data collection and analysis and effort was made to ensure reliability and validity of the study [79] . using this approach, the study was conducted systematically, from the data collection stage to the final analysis. after comprehensively collecting data, including domestic research articles, media reports and audit reports from the board of audit and inspection (bai) of korea related to sars and mers cases, data to be analyzed were selected on the basis of the inclusion and exclusion criteria. the data collection method will be discussed in more detail in the data collection section. the collected data were codified and categorized on the basis of the meta-analysis framework consisting of the factors influencing disaster response extracted from existing studies and theories. as shown in figure 3 , for the influencing factors identified from the data, the factor and source were recorded, and evidence of the correlation was identified. the evidence included statistical data, media reports, interviews with experts and claims made by authors. with this coding process, consistency of the results when the same analysis is performed by different researchers can be maintained and this can be used to ensure reliability. data for meta-analysis were collected from various sources, including listed academic journals, articles from daily and weekly periodicals and audit reports from bai. duplicate items were eliminated based on search results and data that met the objective of the present study were selected through in-depth reviews and discussions with fellow researchers. academic articles were limited to those published in journals listed in the national research foundation of korea, while duplicate articles and articles with low correlation to the research question were excluded. the period of data of academic articles was from 2003 (sars outbreak) to 2017 (at the time of the research). all searched articles were tallied and data were selected through validity testing and unanimity with fellow researchers. media reports were collected from daily and weekly periodicals to provide information that was not covered in academic articles. the search process used the naver news site and the official home pages of each newspaper. the search keywords were disaster case names: sars, mers and different variations of these terms in korean. additionally, data that mentioned a disaster name along with the term "response" were reviewed. to eliminate political bias, chosun ilbo, donga ilbo, kyunghyang shinmun and hankyoreh were selected from daily periodicals, while weekly chosun, weekly donga, weekly kyunghyang and hankyoreh 21 were selected from weekly periodicals. the period included in data collection was set to one year to include the infection outbreak and response period between january and december 2003 for sars and between january and december 2015 for mers. lastly, the homepage of bai was searched for bai audit reports on sars and mers but since audit reports for sars did not exist only mers cases were analyzed. among the 59 cases that appeared as search results for mers, the results that were unrelated to the selected cases were excluded. as a result, a total of 38 cases of audit reports for various organizations were selected for the analysis (table 1) . the results of factors coded and explored based on aforementioned media reports, bai audit reports, academic articles and in-depth interviews are provided here. to effectively demonstrate the results of exploring the factors influencing disaster response, analysis was performed by identifying how each factor, as an independent factor, influenced other factors; and by gathering evidence of the relationship between time, cause and outcomes. the present study underwent the process of identifying and testing correlations through a meta-analysis of the factors influencing infectious disease response. the analysis results on the factors influencing infectious disease response were as follows. legislation, sociocultural factors and disaster characteristics were identified as the environmental factors influencing disaster response. with respect to sars, although there was no legislative system for disaster response and management, some respiratory transmission diseases, including sars, were temporarily designated as "infectious diseases subject to quarantine and surveillance" for onsite response. enactment and amendment of laws have procedural and time requirements and thus quarantine or isolation was made possible by presenting them as subjects of quarantine and surveillance following the decree of the minister of health and welfare, which actually had a positive influence on onsite response. moreover, while legislation for mers was in place, it was incomplete and not detailed enough. this caused confusion in the response process because of the possibility of arbitrary decisions and because it contained inaccurate information about infectious diseases, it had a negative influence on onsite response. consequently, mers spread to other patients, leading to a failed initial response. moreover, unlike the sars outbreak, when international public health crisis was declared, there was no announcement of an international public health crisis with mers, which caused a lack of awareness on the importance of prevention and response. financial resources, human resources, physical resources, information and education and training were identified as the input factors influencing disaster response. human resources also acted as a mediating factor in the relationship between legislation and the effectiveness of response to infectious diseases. in the processes of responding to sars and mers, problems related to human resources, especially epidemiologists, were identified. this was also very apparent in the correlations. although epidemiological investigation in infectious disease response is very important for preventing the spread of infectious diseases and for timely response, an insufficient number of epidemiologists made it impossible to keep up with the rate at which the disease was spreading and since public health physicians were mostly responsible for epidemiological investigation, a lack of specialization was also a serious problem. moreover, budget, the proportion of public healthcare and infection control infrastructure, such as negative-pressure units, were also found to be insufficient during both sars and mers outbreaks. one of the factors that was identified as being important in the correlation analysis was education and training. since everyone may experience an actual disaster, simulated training according to given scenarios and education for response personnel are very important. leadership, intergovernmental relationships, information sharing and onsite response were identified as the process factors influencing response to infectious disease outbreak, while information sharing was found to influence stakeholder satisfaction. with respect to leadership, as mentioned earlier, the role of the prime minister and the president was an important factor in the implementation of timely and effective disaster response. during the sars outbreak, prime minister kun goh was at the forefront, urging the public and departments to cooperate. on the other hand, during the process of responding to mers, the control tower changed at least twice and the president made it clear through the spokesperson that the blue house was not the control tower. during this process, the intergovernmental relationship was not smooth either. moreover, poor information sharing and communication between departments and between the central and local government caused confusion and increased the level of distrust among the general public. among the process factors, intergovernmental relationships, information sharing and onsite response were independent variables that influenced the outcome and acted as mediating factors between legislation and outcome. lastly, although not presented in existing analytical frameworks, the factors identified through meta-analysis and interviews were interest and cooperation from the private sector (volunteerism). with respect to interest, an analysis of sars cases showed that the interest of local citizens, meaning regional self-centeredness, caused the designation of sars quarantine hospitals to be nullified, acting as a factor that interfered with infectious disease response. these factors were confirmed in the interview results. the interest of the agency in charge of the control tower emerged as a factor that interfered with the infectious disease response, albeit at a different level than the interest of local citizens. the agency in charge of determining the disclosure of information was the mohw and because the same agency was responsible for both promoting actual related projects and managing disaster, conflict of interest did not allow immediate response measures to be implemented. cooperation from the private sector (volunteerism) was a factor that did not appear in the meta-analysis but was identified in interviews with workers. its correlations were not analyzed in the meta-analysis data of the present study and existing studies did not discuss the role of volunteers in infectious disease response either. however, resource support for self-quarantine patients in actual infectious disease response was lacking but active participation by volunteers played a major role in helping to slow the spread of mers and to successfully implement self-quarantine. moreover, the hidden context of correlations identified through interviews, which was not identified in existing articles, was education and training. education and training was analyzed as a factor influencing infectious disease response, while the interview results revealed that education and training not only had a direct influence on response but also had an impact on the relationship between the people in charge of disaster response. timely response was made possible by relationships built between people in charge of disaster response through continued training, which may be attributed to the uniqueness of korean culture. as shown earlier, the factors influencing infectious disease response in korea were very diverse and they became more refined and detailed when compared to categorization of factors presented in the introduction of this paper. these were factors identified through meta-analyses and in-depth interviews and should be considered in the improvement of the infectious disease response system in korea. a comprehensive model that summarizes the aforementioned exploration of the influencing factors is shown in figure 4 . factor influencing infectious disease response, while the interview results revealed that education and training not only had a direct influence on response but also had an impact on the relationship between the people in charge of disaster response. timely response was made possible by relationships built between people in charge of disaster response through continued training, which may be attributed to the uniqueness of korean culture. as shown earlier, the factors influencing infectious disease response in korea were very diverse and they became more refined and detailed when compared to categorization of factors presented in the introduction of this paper. these were factors identified through meta-analyses and in-depth interviews and should be considered in the improvement of the infectious disease response system in korea. a comprehensive model that summarizes the aforementioned exploration of the influencing factors is shown in figure 4 . the present study conducted meta-analyses to comprehensively analyze the correlations of factors influencing disaster response from a korean context. for inductive exploration of the factors influencing infectious disease response in korea, the present study collected and selected reliable data from academic research on infectious disease response conducted in korea, newspaper articles and audit reports from bai. the reason for limiting the studies to those conducted within korea was based on the determination that it was necessary to review how well domestic studies and articles explained domestic cases. the objective was to use the findings to point out the limitations of infectious disease-related studies in korea and to present factors influencing infectious disease response within a korean context. the analysis results confirmed that, overall, the studies korea focused on factors from the process aspect when analyzing the factors influencing infectious disease response. a summary of other major findings are as follows: first, among environmental factors, the legislative factor had direct and indirect influence on the overall process of infectious disease response. other environmental factors were regarded as factors influencing disaster response based on their correlations but the legislative factor was considered especially important. disaster-related legislation enacted in various forms including basic laws, manuals and code of conduct should be systematic and exhibit high integrity to allow timely and accurate response in crisis situations. however, owing to insufficiencies in many aspects, it had a negative influence throughout the entire response process. the present study conducted meta-analyses to comprehensively analyze the correlations of factors influencing disaster response from a korean context. for inductive exploration of the factors influencing infectious disease response in korea, the present study collected and selected reliable data from academic research on infectious disease response conducted in korea, newspaper articles and audit reports from bai. the reason for limiting the studies to those conducted within korea was based on the determination that it was necessary to review how well domestic studies and articles explained domestic cases. the objective was to use the findings to point out the limitations of infectious disease-related studies in korea and to present factors influencing infectious disease response within a korean context. the analysis results confirmed that, overall, the studies korea focused on factors from the process aspect when analyzing the factors influencing infectious disease response. a summary of other major findings are as follows: first, among environmental factors, the legislative factor had direct and indirect influence on the overall process of infectious disease response. other environmental factors were regarded as factors influencing disaster response based on their correlations but the legislative factor was considered especially important. disaster-related legislation enacted in various forms including basic laws, manuals and code of conduct should be systematic and exhibit high integrity to allow timely and accurate response in crisis situations. however, owing to insufficiencies in many aspects, it had a negative influence throughout the entire response process. the legislative factor indirectly influenced disaster response, making it an important factor that influences the overall disaster response process. in other words, human resource was identified as the mediating factor in the relationship between the legislative factor, human resources and onsite response. on the other hand, intergovernmental relationships, information sharing and onsite response were identified as the mediating factors in the relationships between the legislative factor, intergovernmental relationships and the effectiveness of disaster response; the relationship between the legislative factor, information sharing and the effectiveness of disaster response and the relationship between the legislative factor, onsite response and the effectiveness of disaster response, respectively. along with the determination of mediating factors, the study also found that the establishment of legislation had an overall impact on infectious disease response. second, the results showed that most input factors, including physical resources, human resources and information were insufficient. within a korean context, it is believed that this problem stemmed from the lack of a disaster response system or many studies related to disaster response, as indicated by the fact that basic laws about disaster management were implemented in korea from 2004. considering that the systematization of disaster response following the passing of basic related laws was relatively recent, more detailed issues, such as securement of resources, did not draw attention until a disaster actually occurred, leading to gradual improvement. therefore, factors related to these resources showed insufficiencies no matter which case was reviewed. however, considering the differences in the timeframes of the cases raises concern on whether the experience gained from the disaster response system is actually being used as an asset to improve the disaster response system in korea. third, major findings regarding process factors were as follows. leadership of the central government, establishment of an intergovernmental response system, the need for communication, information sharing and disclosure and onsite response were identified as key factors influencing effective infectious disease response. existing studies have found that information sharing occurred top-down, from the central government to local government [80] . even so, information sharing was correlated with process factors. nondisclosure of hospital names by the government had an impact on the spread of infectious diseases and on failed initial response. further, the general public voluntarily shared information and made the effort to share accurate information, such as creating a mers map and sharing information on websites. in addition, the interests of local citizens and departments also acted as a factor that interfered with effective infectious disease response. by analyzing the factors influencing infectious disease response within a korean context, the present study presents the following theoretical and policy implications. theoretically, the study established a model of factors influencing infectious disease response by performing inductive exploration on the factors influencing infectious disease response in korea, which was utilized for comprehensive analysis. policy-wise, the study aimed to emphasize the need for improvement of infectious disease response-related legislation, strengthening the authority of kcdc, which currently serves as the control tower, qualitative and quantitative supplementation of disaster response-related human resources, legal grounds for the authority of response personnel and preparation of protective measures. lastly, since the importance of information collection and sharing and cooperation between agencies was demonstrated, it is necessary to establish a system for information sharing and disclosure, as well as a cooperation system involving the central government, the local government, health centers and medical institutions. author contributions: k.m. designed study and collected data; k.j. gave an important advice on analyzing data; and k.m. and k.j. wrote the paper. interorganizational coordination in dynamic context-networks in emergency response management interorganizational coordination in complex environments of disasters-the evolution of intergovernmental disaster response systems government response to disaster: the conflict between bureaucratic procedures and emergent norms the dynamics of performance management: constructing information and reform the core and periphery of emergency management networks a comparative study on emergency system of local government: focus on the marine environmental damage by oil pollution in south korea and japan management of emergent networks during disasters-a meta synthesis relationship between effectiveness of disaster management and cooperative factors of agencies in disaster management mers isolation patient-a public enemy? issue diagn a study on the implementation factors and collaborative governance of disaster management policy: focusing on the recognition of local governments officials building the governance system for the effective disaster management of local government: focusing on buchon city an empirical study on the factors of effective disaster management: focused on the disclosure and sharing of information of the local government the study on the factors affecting the effectiveness of disaster management in governance-focused recognition of fire officials improvement of administrative response system of infectious diseases: an information-process perspective dealing with vulnerability: balancing prevention with resilience as a mehod of governance a study on urban railway passenger's safety strategy for emergency: a focus on the mers case uncertainties of international standards in the mers cov outbreak in korea: multiplicity of uncertainties a reflection on korean mers crisis in 2015: socio-medical approach the study on behavior intention of health protection affected by news media and news usage a study on the impact of the epidemic disease on the number of books checked out of the public libraries: based on the middle east respiratory syndrome coronavirus a social analysis of the limitation of governmental mers risk communication a study on the use of twitter by local government for coping with disaster mers: 70 days of records the effects of exposure to mers information and issue involvement on perceived information influence, disease prevention and information sharing effects of risk information seeking and processing on mers preventive behaviors and moderating roles of sns use during 2015 mers outbreak in korea impacts of the outbreak and proliferation of the middle east respiratory syndrome on rail transit ridership in the seoul metropolitan the age of infectious diseases, where is the reporting standing contents and quality of online news articles on preventive food for mers during mers outbreak in south korea the roles of interpersonal communication between exposure to mass media and mers-preventive behavioral intentions: the moderating and mediating effects of face-to-face and online communication the study of effectiveness of mers on the law and remaining task semantic network analysis of government's crisis communication messages during the mers outbreak policy pr and crisis management communication: the case of seoul metropolitan government on mers how did mers outbreak become a political matter?: how political ideology moderates the effect of multidimensional health locus of control beliefs on citizens' attribution process of mers outbreak news frames about political leaders of different genders in national crises: a comparative analysis of a(h1n1) in 2009 and mers in 2015 an analysis of risk communication-a case study of mers-cov in korea turnover intention of nurses that were cohort quarantined during the middle east respiratory syndrome (mers) outbreak estimating economic loss from mers-cov geography of mers outbreak and politics of bio-power. space environ risk society and bureaucratic responsibility: a comparative analysis between sewol ferry disaster and mers-cov directions for crisis management: lessons from mers in south korea an analysis of blame avoidance behaviors from the failure of initial governmental responses to mers a review on the korea's national public health crisis system future issues and the direction of healthcare system reform in korea brought on by mers crisis status and task of infection prevention by regional local government as viewed through the mers outbreak four problems and improvement measures of public-private cooperation system for infection prevention viewed from mers outbreak the study on medical privatization policy change through acf and policy network model managing the emergency response tight-coupling and reliability: connecting normal accidents theory and high reliability theory catastrophe and the public service: a case study of the government response to the destruction of the world trade center interagency communication networks during emergencies: boundary spanners in multiagency coordination fundamentals of emergency management all the best laid plans conditions impeding proper emergency response causes and management strategies of natural disasters: three cases and ahp analysis comparative study on disaster response systems of local government with reference to network approach a study of crisis management policy in korea: an analysis of multi-organizational relationships in the implementation structure uniformed rank system and its effects on organizational performance: effectiveness and efficiency for disaster response of the fire service organizations in korea comprehensive emergency management: evacuating threatened populations analysis on determinants of efficiency in disaster management system in regional governments a study on factors affecting efficiency of disaster management system a study on the effects of the quality of information on disaster response performance the effect of presidential leadership on crisis management performance: focused on the cases of national crisis over the infectious respiratory disease a study on establishing national crisis management system-ahp approach an impact of interorganizational relationship characteristics on the cooperation and performance in disaster management a study on disaster management governance system building effectiveness in multi-state disaster management systems: the case of the caribbean disaster and emergency response agency the analysis about the potential effects of local overnment's disaster control type on the efficiency of its disaster control system collaborative networks for the infrequent emergency management: a comparative analysis on the case of controlling mers collaborative governance in theory and practice organized behavior in disaster facing the unexpected: disaster preparedness and response in the united states the network governance in response to acts of terrorism: comparative analyses; routledge: abingdon-on-thames a study on the establishment of integrated disaster management system by local governments a study of semantic network analysis of newspaper articles on mers situation: comparing conservative and progressive news media qualitative meta-synthesis on training and workplace experiences of individuals with disabilities: focusing on practical issues of applying qualitative meta-synthesis secondary and meta-analysis of research focus on qualitative methods qualitative metasynthesis: issues and techniques public health network structure and collaboration effectiveness during the 2015 mers outbreak in south korea: an institutional collective action framework the authors declare no conflict of interest. key: cord-353965-0bb729sp authors: halim, ashraf abdel; alsayed, badr; embarak, sameh; yaseen, taha; dabbous, salwa title: clinical characteristics and outcome of icu admitted mers corona virus infected patients date: 2016-01-31 journal: egyptian journal of chest diseases and tuberculosis doi: 10.1016/j.ejcdt.2015.11.011 sha: doc_id: 353965 cord_uid: 0bb729sp abstract middle east respiratory syndrome (mers) is a novel respiratory illness firstly reported in saudi arabia in 2012. it is caused by a new corona virus, called mers corona virus (mers-cov). most people who have mers-cov infection developed severe acute respiratory illness. aim of the work this work is done to determine the clinical characteristics and the outcome of intensive care unit (icu) admitted patients with confirmed mers-cov infection. patients and methods this study included 32 laboratory confirmed mers corona virus infected patients who were admitted into icu. it included 20 (62.50%) males and 12 (37.50%) females. the mean age was 43.99±13.03years. diagnosis was done by real-time reverse transcription polymerase chain reaction (rrt-pcr) test for corona virus on throat swab, sputum, tracheal aspirate, or bronchoalveolar lavage specimens. clinical characteristics, co-morbidities and outcome were reported for all subjects. results the main symptoms among the included patients were: fever (96.87%), cough (93.75%), dyspnea (90.62%), sore throat (75%), runny nose (75%), sputum (50%), headache (43.75%), myalgia (40.62%), chest pain (37.50%), hemoptysis (37.50%), nausea and vomiting (34.37%), abdominal pain (21.87%) and diarrhea (15.62%). the presence of abdominal symptoms is significantly (p <0.05) associated with bad prognosis. out of the included 32 patients, 18 patients (56.25%) survived and 14 patients (43.75%) expired. there was a statistically significant difference in the duration of symptoms before hospitalization, mechanical ventilation and icu and total hospital stay between the expired group and survivors (p <0.01). current smoking and smoking severity were statistically significantly (p <0.01) higher in the expired group compared to survivors. also, there was a statistically (p <0.05) significant positive correlation between mortality and smoking severity (r =0.640). most of the expired patients presented with bilateral pulmonary infiltrates or unilateral infiltrates, but most of the survivors presented with normal radiology or increased bronchovascular markings, and this difference in the results was statistically highly significant (p <0.01). there were statistically highly significant (p <0.01) differences in the mean values of apache ii score (21.11±3.70 vs 24.21±3.82), sofa score (5.83±2.64 vs 8.85±2.17) and cpis (7.55±1.14 vs 8.64±1.39) between the expired group and survivors respectively. also, there was a statistically significant decrease in ph, pao2, o2 saturation and hco3 (p <0.05) among the expired group in comparison with the survivors, but no statistical difference regarding paco2 (p >0.05). there was a statistically significant positive correlation between mortality and old age (r =0.633), obesity (r =0.712), diabetes mellitus (r =0.685), renal failure (r =0.705), chronic heart diseases (0.591), copd (r =0.523), malignancy (r =0.692), kidney transplantation (r =0.644) and liver cirrhosis (r =0.525) (p <0.05). there was a statistically (p <0.05) positive correlation between the number of associated co-morbidities and mortality (r =0.735). conclusions most mers corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. the presence of abdominal symptoms may indicate bad prognosis. prolonged duration of symptoms before patients’ hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, copd, malignancy, renal failure, renal transplantation and liver cirrhosis are associated with a poor outcome of icu admitted mers corona virus infected patients. organization reported the first case of pneumonia caused by mers-cov in saudi arabia. all cases of mers have been linked to countries in and near the arabian peninsula. this virus has spread from ill people to others through close contact, such as caring for or living with an infected person. also, contact with the camels may be a potential source. however, there is no evidence of sustained spread in community settings. most mers patients developed a severe acute respiratory illness [1, 2] . this work is done to determine the clinical characteristics and the outcome of icu admitted patients with confirmed mers-cov infection. this study included 32 laboratory confirmed mers corona virus patients who were admitted into icu. consent was taken from the patients or their relatives. all studied cases were subjected to: (1) full medical history. (2) thorough clinical examination. (3) calculation of body mass index (bmi): a bmi of >30 is considered obese according to who [3] . (4) routine laboratory investigations (complete blood count, kidney and liver functions, and blood sugar testing). (5) radiological assessment: chest x-ray (posteroanterior and lateral views) and computed tomography (ct). (6) arterial blood gases, including; ph, pao 2 , sao 2 , paco 2 and hco 3 . (7) acute physiology and chronic health evaluation ii (apache ii) score, the sequential organ failure assessment score (sofa) and the clinical pulmonary infection score (cpis). (8) throat swab (eurotubo, deltalab, 08191 rubı´, barcelona, spain), sputum, tracheal aspirate or bronchoalveolar lavage specimens were taken and stored at 28°c, and transported within 72 h to the reference laboratories, where they were subjected to real-time reverse-tran scriptase-polymerase-chain-reaction (rrt-pcr) assays to test for mers-cov (altona diagnostics gmbh, 22767 hamburg, germany). for all patients, the results of rrt-pcr tests were confirmed by measuring cyclethreshold values for viral load [4] . the statistical analysis was performed with the statistical package for the social sciences, version 16 for windows (spss inc., chicago, il, usa). chi-square test was used to measure association. pearson's correlation test was used to measure correlation. values of p < 0.05 were considered statistically significant. in this study, there were 20 (62.50%) males and 12 (37.50%) females. the mean age was 43.99 ± 13.03 years. the main symptoms among the included patients were: fever (96.87%), cough (93.75%), dyspnea (90.62%), sore throat (75%), runny nose (75%), sputum (50%), headache (43.75%), myalgia (40.62%), chest pain (37.50%), hemoptysis (37.50%), nausea and vomiting (34.37%), abdominal pain (28.12%), abdominal pain (21.87%) and diarrhea (15.62%). the mean duration of symptoms before seeking medical advice was 5.30 ± 3.25 days (table 1) . the chest radiological findings among the included patients were as follows: 8 (25%) normal chest radiology, 5 (15.63%) increased bronchovascular markings, 7 (21.87%) unilateral infiltrates, and 12 (37.50%) diffuse bilateral infiltrates. pneumothorax occurred in 5 (15.63%) ventilated patients, for 3 of them it was unilateral and for other 2 patients bilateral ( table 1) . regarding the abgs findings in our study: mean ph: 7.26 ± 0.33, mean pao 2 : 64.04 ± 20.50 mmhg, mean o 2 saturation: 83.92 ± 17.45, mean paco 2 : 38.75 ± 4.17 mmhg, and mean hco 3 19.44 ± 8.5 meq/l ( table 1 ). the mean apache ii score was 22.46 ± 4.01, the mean sofa score was 7.15 ± 2.85 and mean cpis was 8.03 ± 1.35 (table 1) . regarding the presence of co-morbidity, 21 (65.62%) patients out of the included 32 patients were associated with one or more co-morbidities and 11 (34.38%) patients were without co-morbidity ( table 2) . all patients were admitted in icu and a mean duration of icu stay was 11.32 ± 4.50 days. regarding mechanical ventilation need, there were 23 (71.87%) ventilated patients and 9 (28.13%) patients without mechanical ventilation. for ventilated patients the mean duration of mechanical ventilation was 6.21 ± 3.76 days. the mean total hospital stay was 15 ± 3.6 days ( table 1) . according to the outcome of 32 patients included in this study, there were two groups: (1) the survivor group that included 18 patients (56.25%) with a mean age of 37.25 ± 15.50 years. (2) the expired group that included 14 patients (43.75%) with a mean age of 50.47 ± 13.80 years. there was a highly statistically significant (p < 0.01) difference between the expired group and the survivors regarding the duration of mechanical ventilation (expired: 9.64 ± 2.46, survivors: 3.55 ± 2.00), icu stay (expired: 13.98 ± 3.50, survivors: 8.66 ± 2.40) and total hospital stay (expired: 17.50 ± 2.92, survivors: 13.06 ± 2.81) ( table 2) . in this study, in spite of a statistically significant (p < 0.05) increase in occurrence of corona virus infection among males in comparison with females (62.50% males and 37.50% females), there was no statistically significant difference (p > 0.05) in the mortality among them (45% among males and 41.67% among females) (p > 0.05) ( table 2) . current smoking and smoking severity were statistically significantly (p < 0.01) higher in the expired group compared to the survivors. non smoking and ex smoking were statistically significantly (p < 0.01) higher among survivors in comparison with the expired group. also, there was a statistically (p < 0.05) significant positive correlation between mortality and smoking severity (r = 0.640) ( table 2) . in this study, there were no statistically significant differences between the expired and survivors groups as regards fever, cough, dyspnea, runny nose, sore throat, chest pain, hemoptysis, myalgia or headache (p > 0.05), but there was a statistically significant increase in frequency of nausea, vomiting and diarrhea among the expired group than that among the survivors (p < 0.05). also, there was a statistically significant increase in the duration of the symptoms before the hospitalization among the expired group than that among the survivors (p < 0.05) ( table 2 ). there was a statistically significant (p < 0.05) positive correlation (r = 0.681) between mortality and prolonged duration of illness before hospitalization. our study showed that most of the expired patients presented with bilateral pulmonary infiltrates or unilateral infiltrates, but most of the survivors presented with normal radiology or increased bronchovascular markings, and this difference in the results was statistically highly significant (p < 0.01) ( table 2) . there were statistically highly significant (p < 0.01) differences in the mean values of apache ii score (21.11 ± 3.70 vs 24.21 ± 3.82), sofa score (5.83 ± 2.64 vs 8.85 ± 2.17) and table 2) . this study found a statistically significant decrease in ph, pao 2 , o 2 saturation and hco 3 (p < 0.05) among the expired group in comparison with the survivors, but no statistical difference regarding paco 2 (p > 0.05) ( table 2) . as regards the correlation between mortality and specific co-morbidities, there was a statistically significant positive correlation between mortality and old age (r = 0.633, smoking (r = 0.640), obesity (r = 0.712), diabetes mellitus (r = 0.685), renal failure (r = 0.705), chronic heart diseases (0.591), copd (r = 0.523), malignancy (r = 0.692), kidney transplantation (r = 0.644) and liver cirrhosis (r = 0.525) (p < 0.05), (table 3) . there was a statistical (p < 0.05) positive correlation between the number of associated comorbidities and mortality (r = 0.735). in this study, there were 20 (62.50%) males and 12 (37.50%) females. this sex distribution showed a significant (p < 0.05) increase in the occurrence of corona virus infection among males, but there was no statistically significant difference (p > 0.05) in the mortality among them (45% among males and 41.67% among females). our results agree with other workers' results [6, 7] . the increased occurrence of corona virus infection among males can be explained by excess movement of the males through community with more exposure to the infected patients. in this study, there was high mortality (14 patients (43.75%) out of 32) among corona virus infected patients and there was a statistically significant (p < 0.05) increase in mortality in middle and old aged patients in comparison to young patients (mean ages: 50.47 ± 13.80 among the expired group vs 37.25 ± 15.50 among survivors). also, there was a statistically significant (p < 0.05) positive correlation (r = 0.633) between age and mortality. other researchers found similar results [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . this high mortality rate among corona virus infected old patients is explained by the high virulence of the new virus in the absence of previous immunity and already compromised immune system among old peoples. the most common symptoms among the included patients were: fever (96.87%), cough (93.75%), dyspnea (90.62%), sore throat (75%), runny nose (75%), and sputum (50%). there were statistically significant differences between the expired and survivors groups regards fever, cough, dyspnea, runny nose, sore throat, chest pain, hemoptysis, myalgia or headache (p > 0.05), but the frequency of nausea, vomiting and diarrhea were statistically significant higher among the expired group in comparison to the survivors (p < 0.05). these findings could be explained by the fact that the presence of nausea, vomiting and diarrhoea leads to dehydration (due to poor oral intake and fluid loss) with its detrimental effects. the duration of symptoms before hospitalization, icu stay, mechanical ventilation and hospital stay in our studied patients were statistically significantly prolonged in the expired group compared to the survivors. also, there was a statistically significant (p < 0.05) positive correlation (r = 0.681) between mortality and prolonged duration of illness before hospitalization. other investigators found similar results [5] [6] [7] [8] . these results can be explained by a delay in diagnosis and management of corona infected patients' result in deterioration in their health status with more need for icu admission, mechanical ventilation, increased total hospital stay and poor outcome. also, al-tawfiq et al. [9] found that corona patients in comparison with controls were more likely to be admitted to the intensive care unit (53% vs 20%; or, 4.65; p = .025) and to have a high mortality rate (76% vs 15%; or, 18.96; p < 0.001). the chest radiological findings among the included patients were as follows: 8 (25%) normal chest radiology, 5 (15.63%) increased bronchovascular markings, 7 (21.87%) unilateral infiltrates, and 12 (37.50%) diffuse bilateral infiltrates. pneumothorax occurred in 5 (15.63%) ventilated patients, for 3 of them it was unilateral and for other 2 patients bilateral. most of the expired patients presented with bilateral pulmonary infiltrates or unilateral infiltrates, but most of the survivors presented with normal radiology or increased bronchovascular markings, and this difference in the results was statistically highly significant (p < 0.01). our findings agree with other investigators who detected similar results [7] [8] [9] 15] . these extensive radiological shadows are denoting extensive lung pathology with its detrimental effects on pulmonary gas exchange and general condition of the patients with more need for mechanical ventilation and are associated with bad prognosis. this study found a statistically significant decrease in ph, pao 2 , o 2 saturation and hco 3 (p < 0.05) among the expired group in comparison to the survivors, but no statistical difference regarding paco 2 (p > 0.05). our results are in accordance with that of reyes et al. [16] who reported that one of the independent variables that were associated with high mortality was hypoxemia among sars patients caused by a corona virus. current smoking and smoking severity were statistically significantly (p < 0.01) higher in the expired group compared to the survivors. non smoking and ex smoking were statistically significantly (p < 0.01) higher among the survivors in comparison with the expired group. also, there was statistically (p < 0.05) significant positive correlation between mortality and smoking severity (r = 0.640). these results are in agreement with previous data that showed that smoking is a major determinant of morbidity and mortality in respiratory tract infection, especially in populations that smoke heavily [6] [7] [8] . cigarette smoking is associated with a variety of alterations in cellular and humoral immune system functions. these alterations include a decreased level of circulating immunoglobulins, a depression of antibody responses to certain antigens, a decrease in cd4+ lymphocyte counts, an increase in cd8+ lymphocyte counts, depressed phagocyte activity and a decreased release of pro inflammatory cytokines. [17] . smoking increases cd8+ lymphocytes in the cell mediated system and suppresses the host defense against infections [18] . there was a statistically significant (p < 0.05) positive correlation between mortality and obesity (r = 0.712). several studies indicate that morbid obesity may be an independent risk factor for complications and mortality from mers corona virus infection [6] [7] [8] [9] . there are some explanations for these results. obesity can impede pulmonary function (reduced functional residual capacity and expiratory volume). a subsequent ventilation-perfusion abnormality may decrease ventilatory reserve and predispose the obese to respiratory failure after even mild pulmonary challenges [19, 20] . obstructive sleep apnea is present in 40% of obese persons and is associated with systemic hypertension, pulmonary hypertension, and cor pulmonale [21, 22] . obese persons are at an increased risk of developing pulmonary emboli and aspiration pneumonia. morbid obesity is associated with complications in the intensive care unit including prolonged stay, prolonged ventilation and death [23, 24] . in addition to its effects on pulmonary function, obesity is frequently, but not always associated with diabetes, hypertension, hyperlipidemia, cardiovascular disease and high overall mortality [20] . our work found a statistically significant positive correlation between mortality and diabetes mellitus (r = 0.685), renal failure (r = 0.705), chronic heart diseases (0.591), copd (r = 0.523), malignancy (r = 0.692), kidney transplantation (r = 0.644) and liver cirrhosis (r = 0.525) (p < 0.05), (table 3) . there was a statistically (p < 0.05) positive correla-tion between the number of associated co-morbidities and mortality (r = 0.735). our results are in accordance with other workers' results [6] [7] [8] [9] [10] [11] [12] [13] [14] . also, arabi et al. [25] concluded that people with dm, renal failure, and chronic lung disease and immuno-compromised persons are considered to be at a high risk of severe disease from mers cov infection. also, our results are in agreement with previous data showing that patients who are immuno-compromised as a result of chronic diseases, malignancy, receiving treatment related to solid organ transplants and dm are at a high risk of respiratory tract infection-related complications including mortality [26, 27] . immuno-compromised patients have an increased attention because of the documentation of prolonged viral shedding in critically ill patients, with the subsequent emergence of resistance to neuraminidase inhibitor drugs [28] . renal failure population has many factors such as overwhelming uremia, neutrophil dysfunction, malnutrition, trace elements' deficiencies, iron overload, impaired glucose metabolism, and hyperparathyroidism, and the use of immunosuppressive drugs to treat and control underlying diseases leads to increased morbidity and mortality of infection [29] . casqueiro et al. [30] concluded that the infectious diseases are more frequent and/or serious in patients with dm, which potentially increases their morbidity and mortality due to many factors as a deficiency of the c4 component in dm, this reduction of c4 is probably associated with polymorphonuclear dysfunction and reduced cytokine response decrease secretion of inflammatory cytokines as interleukin-1 (il-1) and il-6 decreased mobilization of polymorphonuclear leukocytes, chemotaxis and phagocytic activity. cd4 t-lymphocytes and their response to antigens are impaired. most mers corona patients present with fever, cough, dyspnea, sore throat, runny nose and sputum. the presence of abdominal symptoms may indicate bad prognosis. prolonged duration of symptoms before patients' hospitalization, prolonged duration of mechanical ventilation and hospital stay, bilateral radiological pulmonary infiltrates, and hypoxemic respiratory failure were found to be strong predictors of mortality in such patients. also, old age, current smoking, smoking severity, presence of associated co-morbidities like obesity, diabetes mellitus, chronic heart diseases, copd, malignancy, renal failure, renal transplantation and liver cirrhosis associated with a poor outcome of icu admitted mers corona virus infected patients. middle east respiratory syndrome corona virus (mers-cov) summary and literature update isolation of a novel coronavirus from a man with pneumonia in saudi arabia global database on body mass index detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction estimation of mers-coronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data middle east novel corona mers-cov infection. epidemiology and outcome update epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study ksa mers-cov investigation team, hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients severe respiratory illness associated with a novel coronavirus -saudi arabia and qatar epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirus superspreading event involving 81 persons jordan mers-cov investigation team, hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description an update on middle east respiratory syndrome: 2 years later acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients risk factors of a/h1n1 etiology in pneumonia and its impact on mortality effects of cigarette smoking on the immune system: follow-up studies in normal subjects after cessation of smoking reversible alterations in immunoregulatory t cells in smoking: analysis by monoclonal antibodies and flow cytometry a prospective study of age and lifestyle factors in relation to communityacquired pneumonia influenza in australia and new zealand excess deaths associated with underweight, overweight and obesity sleep apnea and sleep disruption in obese patients cardiovascular evaluation and management of severely obese patients undergoing surgery. a science advisory from the obesity related excess mortality rate in an adult intensive care unit: a risk-adjusted matched cohort study morbid obesity as a risk factor for hospitalization and death due to 2009 pandemic influenza a(h1n1) disease clinical course and outcomes of critically ill patients with middle east respiratory syndrome corona virus infection outcome of influenza infections in outpatients after allogeneic hematopoietic stem cell transplantation mortality of 2009 pandemic influenza a (h1n1) in germany oseltamivirresistant novel influenza a (h1n1) virus infection in two immuno suppressed patients infectious complications in chronic kidney disease infections in patients with diabetes mellitus: a review of pathogenesis key: cord-344217-kci4uw7u authors: majid, sabhiya; farooq, rabia; khan, mosin s.; rashid, samia; bhat, showkat a.; wani, hilal a.; qureshi, waseem title: managing the covid-19 pandemic: research strategies based on the evolutionary and molecular characteristics of coronaviruses date: 2020-08-25 journal: sn compr clin med doi: 10.1007/s42399-020-00457-z sha: doc_id: 344217 cord_uid: kci4uw7u coronavirus disease 2019 (covid-19), an ongoing global health emergency, is a highly transmittable and pathogenic viral infection caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). emerging in wuhan, china, in december 2019, it spread widely across the world causing panic—worst ever economic depression is visibly predictable. coronaviruses (covs) have emerged as a major public health concern having caused three zoonotic outbreaks; severe acute respiratory syndrome-cov (sars-cov) in 2002–2003, middle east respiratory syndrome-cov (mers-cov) in 2012, and currently this devastating covid-19. research strategies focused on understanding the evolutionary origin, transmission, and molecular basis of sars-cov-2 and its pathogenesis need to be urgently formulated to manage the current and possible future coronaviral outbreaks. current response to the covid-19 outbreak has been largely limited to monitoring/containment. although frantic global efforts for developing safe and effective prophylactic and therapeutic agents are on, no licensed antiviral treatment or vaccine exists till date. in this review, research strategies for coping with covid-19 based on evolutionary and molecular aspects of coronaviruses have been proposed. coronaviruses (covs) are spherical rna viruses, deriving name from latin word "corona" or crown as they appear like a royal crown under the electron microscope due to characteristic spike projections on their spherical surface. covs belong to family coronaviridae within order nidovirales, which further has two subfamilies: orthocoronavirinae and torovirinae. subfamily orthocoronavirinae encompasses four genera: alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus [1] . beta coronaviruses are a subgroup of the coronavirus family, large enveloped positive-sense singlestranded rna (+ssrna) viruses able to infect a wide variety of mammals and avian species, causing mainly respiratory or enteric diseases [2] . discovered long back in 1960s, hcov-229e and hcov-oc43 were known to cause common cold in humans. to date, seven beta coronaviruses are known to cause human disease-the prevalent strains, hcov 229e, hku1, nl63, and oc43, typically cause mild infections of the upper respiratory tract in humans [3] [4] [5] . however, scenario has altered in the past two decades with covs becoming a major public health concern as three strains, sars-cov [6, 7] , mers-cov [8, 9] , and the newly identified sars-cov-2, are associated with serious respiratory diseases and have led to severe zoonotic outbreaks [10] [11] [12] . the sars-cov pandemic initiated in guangdong province, china, in 2002. the rabia farooq and mosin s. khan have contributed equally as second authors infected patients exhibited pneumonia symptoms with a diffused alveolar injury which lead to acute respiratory distress syndrome (ards); the disease quickly spread worldwide causing serious illnesses and nearly 800 deaths in 2002-2003 [13, 14] . mers-cov epidemic took place in 2012 [8, 9] . in both cases, the infected patients manifested severe acute pneumonia, but while sars-cov infected mainly the lower respiratory tract, mers-cov caused more pronounced gastrointestinal symptoms, often associated with kidney failure [15, 16] . the world health organization (who) has reported a total number of 2519 mers-cov cases with 866 associated deaths from 2012 to 31 january 2020 [17] . sars-cov-2 and the covid-19 pandemic currently, the novel coronavirus, sars-cov-2 alternatively termed 2019 ncov, has caused panic with who declaring it as a global health emergency on 30 january 2020 [18] . it was initially identified in the city of wuhan, china, in december 2019; patients presented with severe viral pneumonia and respiratory illness. the number of cases has been mounting since then to whopping figure of around six and half million globally [19] [20] [21] [22] . the disease caused by sars-cov-2 has been named covid-19, a highly transmittable and pathogenic respiratory infection, which has become a public health emergency of international concern as no clinically approved antiviral drug or vaccine is available-though few broad spectrum antiviral drugs and drug combinations in clinical trials have resulted in clinical recovery [23] [24] [25] [26] [27] . convalescent plasma (cp) therapy has recently been shown to be well tolerated, and severe covid-19 cases could potentially improve the clinical outcomes by neutralizing viremia [28] . transmission is via respiratory droplets or aerosols on exposure to coughing, sneezing, and close contact with an infected person [29] . social distancing, face masks, and frequent hand sanitization are recommended preventive measures. personal protective gear as recommended is to be used by healthcare workers for their safety. containing the outbreak before it can spread is the best way to prevent pandemics. border closures, screening at airports, and checkpoints-classical measures implemented in pandemics-can reduce spread of the virus but will not be a fool-proof strategy [30, 31] . millions of people travel every day from one country to another; a disease originating in one country can rapidly spread to other countries regardless of distances between them. this is particularly visible in the rapid spread of covid-19, which affected almost every country in the world within 3 months of the first reported case. information technology is playing its own role with numerous apps coming up, purportedly helping cope with awareness, management, and controlling spread of this disease-like "aarogya setu app" in india. this disease can remain asymptomatic during around 14 days incubation period of sars-cov-2. the sars and mers covs evade immune responses during longer incubation periods. the spectrum of covid-19 disease is broad-most commonly reported symptoms of are fever, myalgia or fatigue, dry cough, and dyspnea. around 80% of infections are mild, resolving within a week without any specific treatment or hospitalization. amongst hospitalized cases, pneumonia, sepsis, respiratory failure, and ards are frequently encountered complications, and in severe cases, progressive respiratory failure can be fatal [32] [33] [34] . overproduction of early response pro-inflammatory cytokines can result in cytokine storm leading to vascular hyperpermeability, reduced anticoagulant concentration, impaired anticoagulant-procoagulant balance predisposing to development of microthrombosis, disseminated vascular coagulation, and multi-organ failure [35] . this is evidenced in severe covid-19 pneumonia where raised d-dimer is a poor prognostic feature and disseminated intravascular coagulation is common in non-survivors [36] . as clinical manifestations of covid-19 range from mild to moderate, more systematic symptoms and severe radiological abnormalities are seen in older patients [37] . children and younger adults can remain as asymptomatic carriers [38] [39] [40] . the possibility for gastrointestinal involvement in sars-cov-2 infection and feco-oral transmission has been suggested [41] . who has estimated overall mortality rate of 3.7% in spite of the significant infectivity rate, majority deaths typically occurring amongst elderly patients, patients having multiple comorbidities, and immune compromised population [42] . the covid-19 epidemic has reached worldwide resonance; global efforts are being undertaken to characterize the molecular features and evolutionary origins of this virus. covs are typically harboured in mammals and birds-being common in camels, cattle, cats, bats, and other animals [43] [44] [45] . alpha and beta coronaviruses circulate in mammals, including bats. gamma coronaviruses mostly infect avian species and a few mammalian species, whereas delta coronaviruses infect birds and mammals [46, 47] . animal covs are known to cause important diseases in animals and are responsible for economic losses in domestic animals or bird. although rare, zoonotic events do occur wherein animal covs acquire the ability to infect humans and further spread through human-to-human transmission [48, 49] . studies have shown that bats harbour covs that are ancestral to sars-cov which have been circulating in bats for a long time before genetically changing and jumping to humans [50] . rhinolophus bats were found to have anti-sars-cov antibodies suggesting bats as a source of viral replication [51] . further, studies have revealed that palm civets and raccoon dogs, of chinese local food markets, harbour sars-related covs (sarsr-covs) suggesting that they could be key reservoir of infection and may be secondary hosts. this detection of sarsr-covs in bats and small animals in retail markets may indicate an interspecies transmission from bats to small animals and finally to humans [52] . in 2001, samples from the healthy persons of hong kong on molecular assessment showed 2.5% frequency rate of antibodies against sars-cov, indicating that sars-cov might have been circulating in humans before causing the outbreak in 2003 [53] . mers-cov reportedly has camels as a zoonotic source or primary host [44] . in a recent study, mers-cov was also detected in pipistrellus and perimyotisbats, favouring bats as key host and transmitting medium of this virus [54] . metagenomics analysis of phylogenetic relationships between sars-cov-2, sars-cov, bat sars r-covs, and bat cov revealed that sars-cov-2 genome (~29.9 kb) shares 79.5% sequence identity with sars-cov. again, close phylogenetic relationship between sars-cov-2 and a bat cov, 96% identical at whole genome level, points to bat origin of sars-cov-2 and bats as probable "key reservoirs" [55] . overall, these studies highlight bats as carriers of viruses with zoonotic and devastating potential. however, intermediate source of origin and transfer to humans is not known; further studies are needed to determine whether the virus was transmitted to humans by an intermediate host. nevertheless, rapid human-to-human transfer has been widely confirmed, already reported from more than 100 countries in the world [56, 57] . coronaviruses will continue to infect multiple species and cell types, including humans due to their ability to recombine and mutate. the source and spread of these covs necessitate to be evidently firmed; urgency finance for investigation on systematic basis of their spread and their pools is required to frame preventive plans to hold and regulate such outbreaks. in the phylogenetic connection between extremely pathogenic covs, their transitional zoonotic source requires to be vigorously studied. role of variations in human doings, biomes, and environment in leading to such pandemics requires to be studied as the changes in pathogen types, disease burden, and distribution have arisen largely due to human activity. in the last century, there was a reduced burden of infectious diseases due to improved nutrition, better hygiene, and use of vaccines and antimicrobials. however, in recent decades, there is an upsurge of disease emergence and propensity to pandemics much due to swelling global travel and trade, increasing human and livestock populations. viruses have broad range of hosts and are emerging pathogens [58] . in 2016, the united nations environment programme (unep) warned that zoonotic diseases are related to the health of ecosystems. zoonoses are opportunistic and increase with changes in animal or human hosts, environment, or pathogen itself. pathogens have passed between animals and humans in the last century due to reduction in ecosystems by human intervention and population growth. humans have encroached into animal habitats and disturbed natural buffer zones between animals and humans leading to emergence of zoonosis. livestock serves as a bridge between wildlife and human infections and forms a part of wildlife-livestock-human interface. consumer demand for livestock has increased due to economic growth leading to rigorous livestock farming near and around cities, thereby, expanding chances of zoonotic diseases. in addition, natural source of disease resistance is genetic diversity but "intensive livestock rearing" often produces genetic similarities within herds and flocks, making them susceptible to pathogen spillover from wild animals. consumption of wild animals in wet markets can also facilitate animal to human transmission. frequent climate change tells upon the survival of microbes in the environment, suggesting the more frequency of pandemics due to rapid change in climate [59] . thus, if we need to stop such pandemics in the future, we need to focus on the influence of human activities on ecosystems. experiments on monitoring of human and wildlife health will improve understanding and preparedness for potential pandemics. it invites for collaborative, trans-disciplinary, and international efforts as summarized by the "one health approach"-a concept of combining human, animal, and environmental components to address global health challenges having ecological interdependence. "one health approach" wherever adopted is seen to have significant impacts on control of infectious diseases [60] . ultimately, "if we have a robust plan for protecting nature, nature will protect humanity". last but not the least, strategies to curb the transmission of virus should be put in place in a more stringent way. the correct use of personal protective equipment (ppe) and regular and thorough hand hygiene are key measures in the prevention and control against infection through contact transmission, droplet transmission, and airborne virus particles. in addition, maintaining a constant and regular supply of drugs is indispensable during the pandemic. healthcare professionals are at three times higher risk than common individuals so their body temperature, nucleic acid, and specific antibodies for covid-19 should be monitored regularly. during pandemic, psychological illness is rampant so individuals especially healthcare workers should be evaluated for psychological illness, if any. our understanding of the virus deepens, and with the constant improvement of diagnosis, treatment, and strategies for prevention and control, healthcare professionals should continue to actively collect and improve their services based on the latest information [61] . a crucial need to counteract the covid-19 pandemic has arisen; no specific drug or vaccine is available as yet. for effective therapeutics and vaccine development elucidation of genetic features and molecular constituents of covs including sars-cov-2, unravelling the mechanisms of infection, molecular basis of virus-host interaction, its regulation, and host responses is needed. global efforts are being undertaken-biotechnological, molecular, and bioinformatic advances have been helpful in making available details related to the genomics and proteomics of these covs, especially sars-cov-2 in a very short time [62] . the covs are~65-125 nm in diameter, containing singlestranded rna ranging from 26 to 32 kbs in length as nucleic material, being enveloped in lipid bilayer derived from the host cell membrane. the structural together with few nonstructural proteins (nsps) are coded within the 3′ end of the viral genome, whereas the 5′ two-thirds of the genome codes for nsps that are important in viral replication, including the rna-dependent rna polymerase (rdrp) [63, 64] . rna of covs codes for four major structural proteins involved in its replication as well, termed as spike (s), membrane (m), envelope (e), and nucleocapsid (n) proteins. some beta coronaviruses also code for protein hemagglutinin esterase (he). the s, m, and e proteins lie in the viral envelope. the s protein is heavily glycosylated forming homotrimeric spikes on the surface of the viral particle required for binding and entry into host cells. amongst the four, m protein is the most abundant and important protein giving the virion primarily its shape; besides, it interacts with other structural proteins to perform various functions. interaction of s proteins with m proteins is needed for its incorporation into new virions [65] [66] [67] . in the same way, m protein along with the smallest one e protein which is found in small quantities is involved in virus assembly, forming of mature viral envelopes, and release of viral particles from host cells. e protein is mostly expressed in the infected cell; it is important for production and maturation of the virus, and its interaction with psd95/dlg/zo-1(pdz) proteins involved in host cell processes is important for viral infection. the n protein is located in the core of the viral particle forming the nucleocapsid of viral rna and is involved in replication [68, 69] . generally, the transcription and replication of covs take place in the host cell cytoplasm following viral entry. spike proteins of the virus bind with the host receptor, structural changes occur followed by endocytosis, which is ph dependent [70] , and the virus releases its rna inside the host cell cytoplasm. translation of the 5′ end of viral rna produces the rdrp, which uses viral rna as a template to generate series of virus-specific mrnas or sub-genomic mrnas from sub-genomic negative strand intermediates. translation of sub-genomic mrnas leads to production of structural and nonstructural viral proteins; they share the same 3′ ends and the same leader sequence of 70-90 nucleotides at their 5′ ends [71] . once sufficient, structural proteins and genomic viral rna are synthesized; viral assembly and budding occur in smooth-walled vesicles in the endoplasmic reticulum-golgi intermediate compartment (ergic) [72] . rna of coronavirus is polycistronic containing approximately seven genes: 5′ region mainly contains large replicase gene for replication and transcription process, and the 3′ region contains nonessential accessory proteins expressed from sub-genomic mrnas [73] . the large replicase gene at 5′ end encodes replication-transcription complexes (rtcs) amongst covs which comprise of two overlapping open reading frames (orfs), orf1a and orf1b. translation of these orfs results in two very large polyproteins, polyprotein 1a (pp1a) and polyprotein 1ab (pp1ab), further leading to formation of nonstructural proteins by co-and post-translational modifications by various proteinases. orf1b encodes enzymes which are needed in rna replication and its transcription. all covs contain specific genes in orf1 downstream regions that encode proteins for viral replication, nucleocapsid, and spike formation [74, 75] . covs contain doublemembrane vesicles (dmvs) which are attached with rtcs; these dmvs are derived from network of modified er membranes, also referred to as convoluted membranes (cms) [76] . the replication of covs is inhibited by addition of drugs in the early-secretory phase or by addition of rnai [77] . genome of this lethal virus has been isolated; a total of 120 whole genome sequences of sars-cov-2 could be downloaded from the gisaid database as depicted in fig. 1 (https://www.gisaid.org/cov2020/). sequences that likely had spurious mutations resulting from sequencing errors were indicated in the comment field of the gisaid data. its genome is a +ssrna virus having 29,903 bp length with 3′ poly a tail and 5′ cap. genbank: mn908947.3; locus mn908947 (https://www. ncbi.nlm.nih.gov/nuccore/mn908947), having 38 aa protein sequence; locus qhi42199 (https://www.ncbi. nlm.nih.gov/protein/qhi42199.1). the genome of the sars-cov-2 reportedly is over 80% identical to the previous sars-like bat cov, and according to the evolutionary tree, sars-cov-2 lies close to sars-cov. the orf1ab is the largest gene in sars-cov-2 which encodes the pp1ab protein and 15 nsps. the orf1a gene encodes for pp1a protein which also contains 10 nsps. further, the pp1a and pp1ab encoded by the orf1a/b give rise to viral proteases, papain-like protease (plpro), and 3c cleavage-like protease (3clpro/mpro), for creating non-structural proteins (rdrp, helicases) [78] . mpro is a crucial enzyme facilitating viral replication and transcription. recent studies have indicated notable variations in sars-cov and sars-cov-2 such as the absence of 8a protein and fluctuation in the number of amino acids in 8b and 3c protein in sars-cov-2 [79, 80] . spike glycoprotein of the sars-cov-2 has reportedly modified via homologous recombination; it is a mixture of bat sars-cov and a not known beta-cov [81] . attachment and entry of covs into host cells is mediated by "s" protein on outer surface of covs. s protein has two domains: s1 and s2 [82] . within s1 domain, receptor-binding domain (rbd), located at the c-terminus in sars-cov and mers-cov, mediates binding to the associated host cell receptor, while the s2 domain brings about the merger between viral and host cell membranes through endosomal pathway, leading to the entry of the viral genome into the cytoplasm of host cell. the virus may infect multiple hosts as the receptorbinding domain (rbd) is loosely attached to it [83, 84] . covs mostly identify carbohydrates or amino peptidases as a key receptor for entry to human cells [85] . the mechanism of entry of cov in host cell is governed by cellular proteases including human airway trypsin-like protease (hat), cathepsins, and transmembrane protease serine 2 (tmprss2) which cleave spike protein and start further infiltration [86] . varied covs bind to diverse receptors on host cells. mers-cov binds to dipeptidyl-peptidase 4 (dpp4, also known as cd26) receptor. human angiotensin-converting enzyme 2 (hace2), a zinc-dependent carboxypeptidase, responsible for regulating blood pressure, also acts as a receptor of entry for sars-cov, hcov-nl63, and sars-cov-2 [87] [88] [89] . the critical lysine 31 residue on the hace2 receptor recognizes glutamine 394 residues in the rbd region of sars-cov-2 [90] . comprehensive pathogenic mechanism of sars-cov-2 is depicted in fig. 2 [91] . current studies have indicated 74% homology between amino acid sequence of rbd of sars-cov-2 and sars-cov [92] and notable variations between sars-cov and sars-cov-2 such as the absence of 8a protein and variation in the number of amino acids in 8b and 3c protein in sars-cov-2 [93] . as per a recent study, s protein of sars-cov-2 has higher affinity to ace2 receptor as compared with s protein of sars-cov which might be attributed to the n501t mutation in spike protein of sars-cov-2. the spike protein of sars-cov-2 binds to hace2 which is highly expressed in the lungs and heart. studies suggest that there is an elevation in angiotensin ii in covid-19 patients which advocates that binding of covid-19 to ace2 leads to enhanced conversion of angiotensin ii from angiotensin i through the renin-angiotensin-aldosterone system (raas) thereby increasing cardio-myocyte hypertrophy and high blood pressure leading to myocardial injury, myocarditis, and cardiac arrhythmias [94] . in case of diabetics, there is an altered production of cytokine, impaired t cell-mediated immune response, inhibition of neutrophil chemotaxis, ineffective microbial clearance, and phagocytic cell dysfunction which together with the entry of coronavirus into host pneumocytes mediated by ace2 receptor adds to the severity of covid-19 in patients with diabetes mellitus. so, ace2 may play a key role in the severity of covid-19 infection in diabetic patients [94] . the coronavirus-human interactome x-ray crystallography has determined the crystal structures of cov-human interactomes to fully understand the initial step of infection at molecular level. for the first time, crystalline structure for hcov-nl63 s1-ctd complexed with human ace2 was discovered followed by sars-cov s1-ctd complexed with ace2, displaying common receptors [95, 96] which was followed by mers-cov s1-ctd complexed with human dpp4 [97] . overall binding mode of ace2 with sars-cov-2-rbd is nearly identical to that of the sars-cov-rbd as per the recent elucidation of their crystal structure. there are amino acid residues in rbd of sars-co-v, majority of which are highly conserved, or share alike side chain properties with rbd of sars-cov. crystal structure of sars-cov-2-rbd complexed with ace2 is available at protein data bank (pdb) [98] . presently, no registered antiviral drug for use in patients with covid-19 is there. on top of it, there is no efficient vaccine available for covid-19 in humans. the standard of care is "supportive" and includes drugs like protease inhibitors (lopinavir/ritonavir; darunavir + ritonavir; darunavir/ cobicistat); chloroquine or hydroxychloroquine; tocilizumab, monoclonal antibody against chimeric antigen receptor t cells; nucleotide inhibitor like remdesivir (a broad-spectrum antiviral); methylprednisolone 20 mg × 2/day; and antibiotic therapy using third-generation cephalosporin, clarithromycin, or azithromycin or alternatively fluoroquinolones in case of secondary bacterial infections [99] . a crucial need for a clinical research and therapeutic strategy to counteract this epidemiological outbreak remains. we have no empirical enveloped cure or vaccine for this potentially fatal disease; coordinated international efforts for developing therapeutics and vaccines for sars-cov-2 are needed. although several clinical trials are currently underway to test possible therapies, current response to the covid-19 outbreak has been largely limited to monitoring/containment. various biotechnological, molecular tools, and advances in bioinformatics have been instrumental in making available details related to the genomics and proteomics of this virus. some light has been shed on genetic features, molecular constituents, and mechanisms of infection. armed with this information research is progressing at a rapid pace to develop therapeutic strategies, neutralizing antibodies and vaccines to address this disease covid-19 which has spread as a pandemic of mammoth proportions. a wide variety of therapeutic options are being evaluated in an earnest attempt to find a cure. a drug, from time of its inception to qualifying all three phases of clinical trials, can take years to reach market; nevertheless, coordinated international efforts and adequate funding can make drugs against covid-19 available within a record time. bioinformatics and in silico drug modelling, a boon for speedy drug development, are being exploited. recent in silico master regulator analysis has shed light on sars-cov-2/human interactome detailing the host receptor recognition. features of the human interactome most affected by the infection including apoptotic and mitochondrial mechanisms and downregulation of the ace2 protein receptor have been propounded [100] . there is an upregulation of ace2 in diabetes and hypertension as they are being treated by ace inhibitors and angiotensin ii type-i receptor blockers (arbs). ace2 is also increased by drugs such as thiazolidinediones and ibuprofen. consequently, the increased expression of ace2 would facilitate infection with covid-19 and increase the risk of developing severe fatal covid-19 viral infection. a further aspect that should be investigated is the genetic predisposition for an increased risk of sars-cov-2 infection, which might be due to ace2 polymorphisms that have been linked to diabetes mellitus, cerebral stroke, and hypertension, specifically in asian populations. summarizing this information, the sensitivity of an individual might result from a combination of both therapy and ace2 polymorphism [101] . expectedly, development of neutralizing antibodies may take less time due to their speedy trials and high specificity, as a prompt option repurposing of broad spectrum antiviral agents and their combinations is being frantically evaluated. since its emergence, sars-cov-2 has drawn well-deserved attention. it led to a pandemic that has shocked and devastated the human world, shattering its economy-massive economic recession is predicted. it is likely that these coronaviruses will continue to emerge and evolve, causing both human and veterinary outbreaks due to their ability to recombine, mutate, and infect multiple species and cell types, including humans. the future of human cov outbreaks will depend not only on how the viruses will evolve but also on the development of efficient prevention, treatment strategies, and our preparedness to deal with them. priority funding for research on mechanistic basis of transmission of coronaviruses from one species to other, their reservoirs, zoonotic diseases, possible role of environmental changes, and pollution in leading to such epidemics is needed to face the imminent challenge of such repeated outbreaks. very rapid and efficient human-to-human transmission is confirmed which is of immense concern necessitating speedy development of therapeutic modalities. neutralizing antibodies and vaccines could play significant roles in controlling covid-19. studies on humancoronavirus interactome developing suitable therapeutic agents and vaccines are needed. research at accelerated pace is needed to translate into therapies and vaccines. hopefully, lessons from handling this outbreak will allow us to be better prepared in the future-the viruses can keep coming. coronaviruses of man discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus occurrence and frequency of coronavirus infections in humans as determined by enzyme-linked immunosorbent assay human coronaviruses: what do they cause? antivir ther viral infection of the lower respiratory tract identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus outbreak of global health concern management and prevention of sars in china 16. van den brand jma, smits sl, haagmans bl. pathogenesis of middle east respiratory syndrome coronavirus who. middle east respiratory syndrome coronavirus world-health-organization statement on the second meeting of the international health regulations centers-of-disease-control-and-prevention confirmed 2019-ncov cases globally european centre for disease prevention and control data china's cdc detects a large number of new coronaviruses in the south china seafood market in wuhan novel coronavirus: where we are and what we know return of the coronavirus clinical features of patients infected with 2019 novel coronavirus in wuhan the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan, china clinical features of patients infected with 2019 novel coronavirus in wuhan effectiveness of convalescent plasma therapy in severe covid-19 patients epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study pneumonia of unknown origin -china from sars-cov to wuhan 2019ncov outbreak: similarity of early epidemic and prediction of future trends a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection a novel coronavirus from patients with pneumonia in china covid-19: consider cytokine storm syndromes and immunosuppression d-dimer levels on admission to predict in-hospital mortality in patients with covid-19. j thrombosis hemostasis ct imaging features of 2019 novel coronavirus (2019-ncov) clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical course and risk factors for mortality of adult in patients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of 140 patients infected by sars-cov-2 in wuhan analysis of factors associated with disease outcomes in hospitalised patients with 2019 novel coronavirus disease an epidemiological study on covid-19: a rapidly spreading disease coronaviruses in avian speciesreview with focus on epidemiology and diagnosis in wild birds middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study severe acute respiratory syndrome coronavirus-like virus in sn compr chinese horseshoe bats molecular evolution of human coronavirus genomes epidemiology, genetic recombination, and pathogenesis of coronaviruses epidemiology and cause of severe acute respiratory syndrome (sars) in people's republic of china zoonotic origins of human coronaviruses the proximal origin of sars-cov-2 possible bat origin of severe acute respiratory syndrome coronavirus 2. emerg infect dis wet markets-a continuing source of severe acute respiratory syndrome and influenza? covid-19 infection: origin, transmission, and characteristics of human coronaviruses bat origin of human coronaviruses the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status molecular epidemiology, evolution and phylogeny of sars coronavirus world-health-organization coronavirus disease (covid-19) outbreak human coronaviruses: a review of virus-host interactions zoonosis emergence linked to agricultural intensification and environmental change zoonoses and one health: a review of the literature wuchang fangcang shelter hospital: practices, experiences, and lessons learned in controlling covid-19. sn compr clin med cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion the molecular biology of coronaviruses coronavirus: organization, replication and expression of genome mechanisms and enzymes involved in sars coronavirus genome expression pre-fusion structure of a human coronavirus spike protein the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles the role of flexibility and conformational selection in the binding promiscuity of pdz domains genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding role of endocytosis and low ph in murine hepatitis virus strain a59 cell entry subgenomic negative-strand rna function during mouse hepatitis virus infection a contemporary view of coronavirus transcription current insights into the genomics of novel coronavirus: sars-cov-2 an overview of their replication and pathogenesis intracellular processing of the n-terminal orf 1a proteins of the coronavirus mhv-a59 requires multiple proteolytic events sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum e2glycoprotein and broadly neutralizing antibodies that target them coronaviruses: drug discovery and therapeutic options structure-based design of antiviral drug candidates targeting the sars-cov-2 main protease emerging coronaviruses: genome structure, replication, and pathogenesis discovery of bat coronaviruses through surveillance and probe capture-based next-generation sequencing. msphere multibasic cleavage site in the spike protein of sars-cov-2 is essential for infection of human lung cells structure of sars coronavirus spike receptor binding domain complexed with the receptor structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 the bittersweet promise of glycobiology the novel coronavirus2019 (2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human coronavirus nl63employs the severe acute respiratory syndrome coronavirus receptor for cellular entry ace2 of the heart: from angiotensin i to angiotensin (1-7) identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza c virus and bovine coronavirus covid-19 infection: origin, transmission, and characteristics of human coronaviruses genome composition and divergence of the novel coronavirus (2019-ncov) originating in china the association of cardiovascular diseases and diabetes mellitus with covid-19 (sars-cov-2) and their possible mechanisms crystal structure of nl63 respiratory coronavirus receptor-binding complexed with its human receptor structural basis of receptor recognition by sars-cov-2 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 crystal structure of sars-cov-2 spike receptor-binding domain bound with ace clinical management of adult coronavirus infection disease 2019 (covid-19) positive in the setting of low and medium intensity of care: a short practical review master regulator analysis of the sars-cov-2 /human interactome covid-19 outbreak: an update on therapeutic options publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we acknowledge all the patients who have been key: cord-347374-mryazbnq authors: okba, nisreen m.a.; müller, marcel a.; li, wentao; wang, chunyan; geurtsvankessel, corine h.; corman, victor m.; lamers, mart m.; sikkema, reina s.; de bruin, erwin; chandler, felicity d.; yazdanpanah, yazdan; le hingrat, quentin; descamps, diane; houhou-fidouh, nadhira; reusken, chantal b.e.m.; bosch, berend-jan; drosten, christian; koopmans, marion p.g.; haagmans, bart l. title: severe acute respiratory syndrome coronavirus 2−specific antibody responses in coronavirus disease patients date: 2020-07-17 journal: emerg infect dis doi: 10.3201/eid2607.200841 sha: doc_id: 347374 cord_uid: mryazbnq a new coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has recently emerged to cause a human pandemic. although molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. validated serologic assays are needed for contact tracing, identifying the viral reservoir, and epidemiologic studies. we developed serologic assays for detection of sars-cov-2 neutralizing, spike protein–specific, and nucleocapsid-specific antibodies. using serum samples from patients with pcr-confirmed sars-cov-2 infections, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial elisas. we demonstrated that most pcr-confirmed sars-cov-2–infected persons seroconverted by 2 weeks after disease onset. we found that commercial s1 igg or iga elisas were of lower specificity, and sensitivity varied between the 2 assays; the iga elisa showed higher sensitivity. overall, the validated assays described can be instrumental for detection of sars-cov-2–specific antibodies for diagnostic, seroepidemiologic, and vaccine evaluation studies. dition, these epidemiologic studies can help identify the extent of virus spread in households, communities, and specific settings, which could help guide control measures. serologic assays are also needed for evaluation of results of vaccine trials and development of therapeutic antibodies. among the 4 coronavirus structural proteins, the spike (s) and nucleocapsid (n) proteins are the main immunogens (5) . we describe development of serologic assays for detection of virus neutralizing antibodies and antibodies to the n protein and various s protein domains, including the s1 subunit, and the receptor-binding domain (rbd) of sars-cov-2 in an elisa format. using a well-characterized cohort of serum samples from pcr-confirmed sars-cov-2 and patients pcr-confirmed to be infected with seasonal coronaviruses and other respiratory pathogens, we validated and tested various antigens in different platforms developed in-house, as well as a commercial platform. we used serum samples (n = 10) collected from 3 pcrconfirmed patients: 2 with mild covid-19 and 1 with severe covid-19 (table 1) from france in accordance with local ethics approvals (f.-x. lescure et al., unpub. data, https://doi.org/10.1101/2020.03.11.987958). for assay validation, we used samples obtained from persons who had pcr-diagnosed infections with human coronaviruses (hcov-229e, nl63, or oc43), sars-cov, mers-cov, or other respiratory viruses (table 1 ) as reported (6) . we also included samples from patients who had recent infections with cytomegalovirus, epstein-barr virus, or mycoplasma pneumoniae because these pathogens have a higher likelihood of causing false-positive results. as negative controls, we used serum samples from 45 healthy blood donors (sanquin blood bank, https://www. sanquin.nl) (cohort a). we also tested serum samples from sars patients (7) . all samples were stored at -20°c until use. the sanquin blood bank obtained written informed consent for research use of samples from blood donors. use of serum samples from the netherlands was approved by the local medical ethics committee (approval no. 2014-414). all serum samples (n = 31) from patients with pcrconfirmed cases of covid-19 cases were previously analyzed by a recombinant sars-cov-2 s protein-based immunofluorescence test and plaque reduction neutralization (r. wölfel et al., unpub. . we tested serum samples as part of an extended diagnostic regimen after we obtained informed written consent from patients. we obtained non-sars-cov-2-infected serum samples (n = 31) from the serum collection of the national consiliary laboratory for coronavirus detection at charité-universitätsmedizin berlin (berlin, germany). samples were collected after we obtained informed written consent. the collection contained followup antibody-positive serum samples from pcrconfirmed virus-infected cases: hcov-229e (n = 4), hcov-hku1 (n = 3), hcov-oc43 (n = 7), mers-cov (n = 3), hcov-nl63 (n = 6), sars-cov (n = 3), and common cold cov (n = 6). we expressed the s ectodomains of sars-cov-2 (residues 1-1,213, strain wuhan-hu-1, genbank accession no. qhd43416.1), sars-cov (residues 1-1,182, strain cuhk-w1, accession no. aap13567.1), and mers-cov (residues 1-1262, strain emc, accession no. yp_009047204.1) in hek-293t cells by using a c-terminal trimerization motif, strep-tag, and the pcaggs expression plasmid. likewise, we expressed the sars-cov-2 s1 subunit or its subdomains (s;s1, residues 1-682; s1 a , residues 1-294; rbd, residues 329-538; accession no. qhd43416.1) in 293t cells, as described (c. wang et al., unpub. data, https://doi. org/10.1101/2020.03.11.987958). we produced s1 proteins of other hcovs: hku1 (residues 1-750), oc43 (residues 1-760), nl63 (residues 1-717), 229e (residues 1-537), sars-cov (residues 1-676), and mers-cov as described (6, 8) . we affinity purified all recombinant proteins from culture supernatant by using protein-a sepharose beads (catalog no. 17-0780-01; ge healthcare, ge healthcare, https://www.gehealthcare.com) or strep-tactin beads (catalog no. 2-1201-010; iba lifesciences, https://www.iba-lifesciences.com). we checked purity and integrity of all purified recombinant proteins by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with coomassie blue. we used the plaque reduction neutralization test (prnt) as a reference for this study because neutralization assays are the standard for coronavirus serologic analysis. we tested serum samples for their neutralization capacity against sars-cov-2 (german isolate; gisaid id epi_isl 406862; european virus archive global #026v-03883) by using prnt as described with some modifications (9). we 2-fold serially diluted heat-inactivated samples in dulbecco modified eagle medium supplemented with nahco 3 , hepes buffer, penicillin, streptomycin, and 1% fetal bovine serum, starting at a dilution of 1:10 in 50 µl. we then added 50 µl of virus suspension (400 plaque-forming units) to each well and incubated at 37°c for 1 h before placing the mixtures on vero-e6 cells. after incubation for 1 h, we washed, cells supplemented with medium, and incubated for 8 h. after incubation, we fixed the cells with 4% formaldehyde/ phosphate-buffered saline (pbs) and stained the cells with polyclonal rabbit anti-sars-cov antibody (sino biological, https://www.sinobiological.com) and a secondary peroxidase-labeled goat anti-rabbit igg (dako, https://www.agilent.com). we developed signal by using a precipitate forming 3,3′,5,5′-tetramethylbenzidine substrate (true blue; kirkegaard and perry laboratories, https://www.seracare.com) and counted the number of infected cells per well by using an immunospot image analyzer (ctl europe gmbh, https://www.immunospot.eu). the serum neutralization titer is the reciprocal of the highest dilution resulting in an infection reduction of >50% (prnt 50 ). we considered a titer >20 to be positive. we performed the prnt for serum samples from germany by using vero e6 cells, as described (r. wölfel et al., unpub. data, https://doi.org/10.11 01/2020.03.05.20030502) (10) and 24-well plates. before the prnt, we heat-inactivated patient serum samples at 56°c for 30 min. for each dilution step (in duplicate), we diluted patient serum samples in 200 µl of optipro serum-free medium (https://www. thermofisher.com) and mixed 1:1 with 200 µl of virus solution containing 100 pfus. we vortexed the 400-µl serum-virus solution gently, incubated at 37°c for 1 h, and then incubated each 24-well plate with 200 µl serum-virus solution. after incubation for 1 h at 37°c, we discarded supernatants, washed cells once with pbs, and supplemented them with 1.2% microcrystalline cellulose solution in dulbecco modified eagle medium. after 3 days, we fixed and inactivated the plates by using a 6% formaldehyde/pbs solution and stained with crystal violet. we performed anti-sars-cov-2 s1 igg and iga elisas by using β-versions of 2 commercial kits (euroimmun medizinische labordiagnostika ag, https://www.euroimmun.com) and performed the assay according to the manufacturer's protocol. we detected optical density (od) at 450 nm and calculated a ratio of the reading of each sample to the reading of the calibrator, included in the kit, for each sample (od ratio). because the β-version of the kit awaits validation and marking, we determined an in-house cutoff value based on the mean background reactivity of all sars-cov-2-negative serum samples in the study multiplied by 3. the od ratio was 0.9 for iga and 0.3 for igg. we performed the in-house elisas by coating 96-well microtiter elisa plates with in-house-produced s antigens (s or s1 of sars-cov-2, sars-cov or mers-cov; sars-cov-2 s1 a ; or rbd proteins) or sars-cov n protein (sino biological) in pbs overnight at 4°c. after blocking, we added diluted serum (diluted 1:100 or 2-fold serially diluted for titers) and incubated at 37°c for 1 h. antigen-specific antibodies were detected by using peroxidase-labeled rabbit anti-human igg (dako) and 3,3′,5,5′-tetramethylbenzidine as a substrate. the absorbance of each sample was measured at 450 nm, and we set the cutoff value at 6 sd above the mean value for the negative cohort. serum samples were previously tested for antibodies against s1 of different coronaviruses. we used a protein microassay that has been described (6). we analyzed the correlations between antibody responses detected by different elisas and those detected by prnt, which is the standard for coronavirus serologic analysis. we used graphpad prism version 8 (https://www.graphpad.com) for this analysis. we evaluated sars-cov-2-specific antibody responses in severe and mild cases by using serum samples collected at different times postonset of disease from 3 pcr-confirmed covid-19 patients from france. we tested serum samples for sars-cov-2specific antibodies by using different elisas. after infection, all 3 patients seroconverted between days 13 and 21 after onset of disease (figure 1) , and antibodies were elicited against the sars-cov-2 s, s1 subunit, and rbd, but only 2/3 patients had detectable antibodies to the n-terminal (s1 a ) domain. because the n protein of sars-cov-2 is 90% similar to that of sars-cov (table 2) , we used sars-cov n protein as an antigen to test for sars-cov-2 n protein-directed antibodies in an elisa format. we found that antibodies were elicited against the n protein in all three patients. when tested in a prnt, serum samples from all three patients neutralized sars-cov-2 infection. antibody responses detected by different assays correlated strongly with neutralizing antibody responses ( figure 2 ). we observed cross-reactivity with the sars-cov s and s1 proteins, and to a lower extent with mers-cov s protein, but not with the mers-cov s1 protein ( figure 1 , panels g, h). this finding was evident from analyzing the degree of similarity of the different coronavirus s protein domains to their corresponding sars-cov-2 proteins ( table 2 ). this analysis showed that the s2 subunit is more conserved and thus plays a role in the cross-reactivity seen when the whole s was used as antigen. thus, s1 is more specific than s as an antigen for sars-cov-2 serologic diagnosis. we further assessed the specificity of the s1 assay by using cohorts a-e (table 1) , which were composed of serum samples from healthy blood donors (a), pcr-confirmed acute respiratory non-cov infections (b), acute-phase and convalescent-phase pcr-confirmed αand β-hcov infections (c), pcrconfirmed mers-cov infections (d), and pcr-confirmed sars-cov infections (e). none of the serum samples from specificity cohorts a-d were reactive in our in-house s1 elisa at the set cutoff value, indicating 100% specificity, whereas serum samples from sars-cov patients cross-reacted (figure 3, panel a) . the specificity of s1 as an antigen for sars-cov-2 serologic analysis was further supported by the fact that 87%-100% of serum samples in cohorts a-c included in this study were seropositive for endemic hcovs (hcov-hku1, hcov-oc43, hcov-nl63, and hcov-229e), as determined by the s1 protein microarray (figure 3, panel b) . nonetheless, all serum samples were seronegative for sars-cov and mers-cov. using the same cohort, we also validated the specificity of the n protein igg and rbd igg elisas for detecting sars-cov-2-specific antibodies. at the set cutoff, except for serum samples from sars-cov patients, none of the control serum samples was positive for rbd antibodies, and 1 mers-cov-positive serum sample was weakly positive for n protein antibodies ( figure 3 , panels c, d). we also detected seroconversion among the 3 patients with covid-19. because serum samples from the 3 patients were collected at a limited number of time points, it was difficult to accurately assess time for seroconversion. to accurately assess time of seroconversion, a larger number of longitudinal samples is needed. overall, these validated elisas for different antigens can be useful for epidemiologic studies and for evaluation of vaccine-induced immune responses. next, we validated the sensitivity and specificity of 2 commercial elisa kits for detecting s1-specific igg and iga by using the same cohort (table 1 ; (figure 4) . we also detected reactivity of serum samples from the validation cohorts a-d; 11/203 for iga and 8/203 for igg elisas. serum samples from 2 patients infected with hcov-oc43 (a betacoronavirus) were reactive in both igg and iga elisa kits. we have reported the cross-reactivity of these serum samples in a mers-cov s1 igg elisa kit (6) . we confirmed the cross-reactivity of the 2 serum samples by testing 12 serum samples from both patients that were collected at different time points were collected 4-56 days after onset of disease onset ( figure 5 ). all 9 covid-19 patients were previously confirmed to seroconvert at days 6-15 after onset of disease by use of a recombinant immunofluorescence test and prnt. a total of 8/9 seroconverted patients showed reactivity above the implemented cutoff values in the igg and iga elisa. a serum sample from 1 patient ( figure 5, panels a, b) had an antibody level slightly below the cutoff value, which might be explained by an overall reduced antibody response of this patient (prnt 90 = 10). overall, the iga-based elisa kit was more sensitive but less specific than the igg-based elisa kit. finally, we compared the performance of different elisas for detection of antibodies among pcrconfirmed covid-19 patients with that of prnt, which is the standard for coronavirus serologic analysis (tables 3, 4 ). the prnt 50 correlated strongly with different elisas; the commercial iga elisa showed the strongest correlation, followed by the s and n elisa, which indicated their capacity to detect sars-cov-2-specific antibodies. however, a larger patient cohort is needed to assess the sensitivities of these platforms. validated sars-cov-2 serologic assays are urgently needed for contact tracing, epidemiologic and vaccine evaluation studies. because the n and s proteins are the main immunogenic coronavirus proteins, we developed elisa-based assays that were able to detect antibodies to these 2 proteins, and to the 2 s domains, s1 a , and rbd. results for these assays correlated strongly with results of the prnt 50 . because most humans have antibodies against the 4 endemic human coronaviruses, it was crucial to verify the specificity of these assays to avoid false-positive results. in addition, the 2 zoonotic coronaviruses, sars-cov and mers-cov, are also betacoronaviruses, increasing the potential for cross-reactivity. among the s antigens tested, s1 was more specific than s in detecting sars-cov-2 antibodies, as mers-cov s cross-reactive antibodies were detected in serum of 1 of the covid-19 patients, which was not seen when mers-cov s1 was used for testing. this finding could be explained by the high degree of conservation in the coronavirus s2 subunit relative to s1 (table 2) . therefore, consistent with our earlier findings for serologic analysis of mers-cov (6), s1 is a specific antigen for sars-cov-2 diagnostics. when testing the specificity of s1 or its rbd for detecting sars-cov-2 antibodies, none of the serum samples from the validation cohorts (a-e) showed any reactivity, except for serum samples from patients with sars-cov. this finding is not unexpected because cross-reactivity resulted from the high degree of similarity between s1 and rbd of sars-cov and sars-cov-2 (table 2) . however, sars-cov has not circulated in the human population since 2003 (i.e., 17 years ago), and an earlier study reported waning of sars-cov-specific antibodies, which made them undetectable in 21 (91%) of 23 serum samples tested 6 years after infection (11) . it is therefore unlikely that antibodies to this virus are present in the population, and thus it is unlikely that false-positives results are caused by reactivity of sars-cov antibodies. we used the high degree of similarity between the sars-cov and sars-cov-2 proteins to develop a new in-house n protein elisa, in which we used sars-cov n protein (90% similar to sars-cov-2 n protein) as antigen. the n protein elisa could detect sars-cov-2-specific antibodies with high specificity and sensitivity. using the 3 different validated elisas, we found that antibody levels were higher after severe infection than after mild infections; similar findings have been reported earlier for mers-cov (12, 13) . however, this finding needs to be confirmed in a larger cohort of patients with various degrees of disease severity, and it highlights the potential need for a sensitive 1486 emerging infectious diseases • www.cdc.gov/eid • vol. 26, no. 7, july 2020 assay to avoid missing persons who have milder infections in epidemiologic studies. in addition, igg seroconversion can be reliably confirmed in the second week after disease onset. however, because of the limited number of longitudinal serum samples from covid-19 patients tested by the in-house assays, it was difficult to accurately assess time for seroconversion. for this assessment, a larger number of longitudinal samples is needed. in the 3 in-house elisas tested, the rbd and n protein elisas were more sensitive than s1 elisa in detecting antibodies in mildly infected patients and showed stronger correlations with prnt 50 titers. therefore, detecting antibodies against 2 different antigens might be needed to confirm the findings and avoid false-negative results in surveillance studies. however, the sensitivities of the assays need to be further validated with a larger cohort. we validated β-versions of iga and s1 igg commercial elisas in 2 different laboratories. the igabased elisa showed higher sensitivity than the igg-based elisa, whereas the igg elisa showed higher specificity than the iga elisa. the iga and igg assays can be used for serologic diagnosis, igg is longer lived (14) and thus is preferred for serosurveillance studies. we observed some cross-reactivity in both elisas with serum samples from the same 2 hcov-oc43 patients in which these samples showed cross-reactivity in a mers-cov s1 igg eli-sa (6) despite the different antigen used. this finding indicates a response to another protein that could be in the blocking or coating matrix, apart from the specific antigen coated, resulting in this consistent false-positive result. overall, the assays developed and validated in this study could be instrumental for patient contact tracing, serosurveillance studies, and vaccine evaluation studies. however, because various studies will be conducted in different laboratories, it is crucial to calibrate and standardize assays developed by different laboratories by using well-defined standard references as part of diagnostic assay validation. this standardization is not only needed to reduce interassay variability, but to also correlate results obtained from different laboratories that use various assays (15) . this correlation is crucial for better comparison and interpretation of results from different studies; evaluating vaccine trials; enabling uniform assessment of immunogenicity, efficacy; and better understanding of correlates of immune protection (16) . thus, setting up reference panels is a vital element in our preparedness approaches to emerging viruses. a pneumonia outbreak associated with a new coronavirus of probable bat origin coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. nat microbiol world health organization. coronavirus disease (covid-2019) situation reports detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr serological assays for emerging coronaviruses: challenges and pitfalls sensitive and specific detection of low-level antibody responses in mild middle east respiratory syndrome coronavirus infections newly discovered coronavirus as the primary cause of severe acute respiratory syndrome dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc blocking transmission of middle east respiratory syndrome coronavirus (mers-cov) in llamas by vaccination with a recombinant spike protein transmission of mers-coronavirus in household contacts lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study mers-cov antibody responses 1 year after symptom onset, south korea antibody response and disease severity in healthcare worker mers survivors chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus study participants. comparison of serologic assays for middle east respiratory syndrome coronavirus international biological reference preparations for epidemic infectious diseases we thank malik peiris for providing serum samples from sars patients. dr. okba is a postdoctoral researcher in the viroscience department, erasmus medical center, rotterdam, the netherlands. her primary research interest is development of diagnostic and intervention strategies for emerging viruses. key: cord-336150-l8w7xk0b authors: rathore, jitendra singh; ghosh, chaitali title: severe acute respiratory syndrome coronavirus-2 (sars-cov-2), a newly emerged pathogen: an overview date: 2020-08-25 journal: pathog dis doi: 10.1093/femspd/ftaa042 sha: doc_id: 336150 cord_uid: l8w7xk0b coronavirus disease 2019 (covid-19) is a viral pneumonia, responsible for the recent pandemic, and originated from wuhan, china, in december 2019. the causative agent of the outbreak was identified as coronavirus and designated as severe acute respiratory syndrome coronavirus 2 (sarscov-2). few years back, the severe acute respiratory syndrome coronavirus (sarscov) and the middle east respiratory syndrome coronavirus (mers-cov) were reported to be highly pathogenic and caused severe infections in humans. in the current situation sars-cov-2 has become the third highly pathogenic coronavirus that is responsible for the present outbreak in human population. at the time of this review, there were more than 14 007 791 confirmed covid-19 patients which associated with over 597 105 deaths in more then 216 countries across the globe (as reported by world health organization). in this review we have discussed about sars-cov, mers-cov and sarc-cov-2, their reservoirs, role of spike proteins and immunogenicity. we have also covered the diagnosis, therapeutics and vaccine status of sars-cov-2. on december 31, 2019, several cases of severe pneumonia were reported from wuhan, china. the causative agent of the outbreak was identified as betacoronavirus. genome sequencing revealed that it is closely related to the sars-cov (severe acute respiratory syndrome coronavirus) which had emerged in 2003, and is designated as sars-cov-2 (gorbalenya et al. 2020; zhou et al. 2020) . in a very short duration, more than 80 000 infectious cases including more than 3000 deaths were reported in china as on march 15, 2020. at the time of this review (18 may, 2020), the disease, termed as covid-19 (corona virus disease 2019), had already become pandemic and spread to more than 216 countries and territories, including community transmissions in countries like the united states, germany, france, spain, japan, singapore, south korea, iran, italy and india. as on july 19, more than 14 007 791 cases and 597 105 deaths had been reported globally, with the rapid growth of numbers in many countries. for the up-to-date information about covid-19, visit the world health organization (who) website (https://www.who.int/emer gencies/diseases/novel-coronavirus-2019). the bats are likely to be the origin of sars-cov-2, but the role of an intermediate host cannot be ruled out at this stage. initial studies showed that sars-cov-2, can use angiotensinconverting enzyme 2 (ace2) from bats, cats, civet cats, swine, ferrets, non-human primates (nhps) and humans as a receptor (letko, marzi and munster 2020; wan et al. 2020a; zhou et al. 2020) . a pet dog in hong kong and a tiger in bronx zoo in the united state of america tested positive with sars-cov-2 infection, indicating that canine ace2 can also be recognized by sars-cov-2. pangolins, which are endangered animals and are illegally imported into southern china (guangdong and guangxi provinces), have been considered as a potential intermediate host (lam et al. 2020; zhang et al. 2020b) . the initial reports showed that in most of the covid-19 cases there was mild to moderate infection. however, approximately 20% of the cases were reported severe (chen et al. 2020; wang et al. 2020a) . in this review, we will discuss about sars-cov, mers-cov and sarc-cov-2. we have also discussed about various reservoirs, associated with them. in the end, we have covered the role of spike proteins and their immunogenicity along with the diagnosis, therapeutics and vaccine status of sars-cov-2. zoonotic coronaviruses are becoming a global concern as there was emergence of earlier two coronaviruses, sars-cov and mers-cov (middle east respiratory syndrome coronavirus) which created a havoc and recently emergence of the third highly pathogenic sarc-cov-2. it has been observed that members of the family coronaviridae are known to infect a wide range of vertebrates and humans. before the outbreak of sars (severe acute respiratory syndrome), only two coronaviruses including hcov-229e and hcov-oc43 were known to infect humans. however, post-sars outbreak, the sars coronavirus (sars-cov), human coronavirus hcov-nl63, human coronavirus hcov-hku1 and mers-cov have been isolated from humans. similar to sars-cov and mers-cov, the newly isolated sarc-cov-2 is highly pathogenic in humans and causes severe acute respiratory distress (shi, guo and rottier 2016) . the genomes of coronaviruses consist of a positive and single-stranded rna genome of about 30 kb. the 5 terminus encodes the enzyme viral replicase/transcriptase, which is involved in virus replication, whereas the 3 terminus encodes viral structural proteins and virus group specific accessory proteins. functional studies of these viral proteins in detail are essential for antiviral drug screening and vaccine development. the earliest available genome sequencing data of sars-cov-2 made it possible to compare it with the genomes of sars-cov and other coronaviruses. results showed that sars-cov-2 belongs to the genus betacoronavirus and subgenus sarbecovirus, which also includes sars-cov. however, the mers-cov belongs to another subgenus, merbecovirus (lu et al. 2020; zhou et al. 2020) . the comparison study also showed that there is 79% nucleotide similarity between sars-cov-2 and sars-cov. the essential surface glycoprotein of sars-cov-2 known as spike (s) protein, essential for host cell receptor binding, showed only 72% similarity with sars-cov at the nucleotide level. the genomic organization of sars-cov-2 resembles those of other betacoronaviruses, including 5'-orf1ab-s (surface glycoprotein)-orf3a-e (envelope)-m (membrane glycoprotein)-n-3' as shown in fig. 1 . comparative genome analysis of ratg13, a virus from a rhinolophusaffinis (i.e. horseshoe) bat sampled from yunnan province in china in 2013, with sars-cov-2, showed that sars-cov-2 has 96% similarity at the nucleotide sequence level . although the sars-cov-2 and ratg13 have high similarity, yet they differ in some genomic features, such as sars-cov-2 contains a polybasic (furin) cleavage site insertion (residues prra) between the s1 and s2 subunits of the surface glycoprotein s protein (coutard et al. 2020) . polybasic insertion may increase the infectivity of the virus, as it is absent in other related betacoronaviruses. however, similar polybasic insertions are observed in different human coronaviruses, such as hcov-hku1 and highly virulent strains of avian influenza viruses. therefore, whether polybasic insertion between s1 and s2 subunits of s protein occurs due to the natural evolution in sars-cov-2 or by other means is going to be the topic of debate in the future. however, an independent insertion(s) of the amino acids paa at the s1/s2 cleavage site was also observed in armyn02 virus (having 72% similarity in spike protein and 97% similarity in replicase nucleotide) isolated from rhinolophus bat in yunnan province in mid-2019, indicating that these insertion events may be a natural part of ongoing coronavirus evolution . the receptor-binding domain (rbd) of s protein is essential to interact with the ace2 receptor present on the surface of the target cells of the host. therefore, comparative sequence analysis was performed and results showed that there is 85% similarity in rbd between sars-cov-2 and ratg13, but they share only one amino acid among the six key amino acid residues. further, due to the proteinaceous nature of spike, structural comparisons were also performed, suggesting that the rbd domain of the sars-cov-2 is well suited to interact with the human ace2 receptor. interestingly the same receptor was also utilized by sars-cov to cause infection (wrapp et al. 2020) . the sars-cov-2 is closely related to sars-cov and mers-cov having bat as reservoirs, but there are huge biological differences in the former as compared to the other two. the sars-cov-2 is markedly more infectious and has very different epidemiological dynamics. moreover, the mers-cov has never been able to fully adapt to human transmission (sabir et al. 2016) , whereas there is the remarkable local and global spread of sars-cov-2. as in the case of sars and mers, the intermediate host including civets and camels, respectively, played an important role and may be considered as a true reservoir host (sabir et al. 2016) . therefore, due to the ecological separation between a bat (reservoir) and humans, 'intermediate' or 'amplifying' mammalian host is a must to acquire mutations in sars-cov-2, and is essential for the efficient human transmission. to determine the intermediate host, it is essential to perform a wider sampling of animals that live close to human populations or available in wet markets for human consumption. surprisingly, there was discovery of viruses, closely related to sars-cov-2 from the malayan pangolins (manisjavanica) that are illegally imported into southern china (guangdong and guangxi provinces). it has been observed that rbd domain of guangdong pangolin viruses are particularly closely related to sars-cov-2. there is a 97% amino acid sequence similarity and contain all six critical key mutations that are essential for binding to the ace2 receptor in these viruses. however, the rest of the genome is highly divergent from sars-cov-2. hence, the evolution of coronaviruses in animal reservoirs as well as in intermediate hosts is required to explain the emergence of sars-cov-2 in humans. it might be possible that due to its asymptomatic infection, the virus could have acquired some of its essential mutations during a period the ''cryptic'' spread in humans before it was first detected in december 2019. recombination is another possibility, which cannot be ruled out as sarbeviruses, and coronaviruses experience widespread recombination. the genome of sarbeviruses experience recombination at multiple locations, including spike protein. there are studies, which showed that recombination does occur among sars-cov-2, ratg13 and the guangdong pangolin covs (lam et al. 2020) . the genome of rmyn02 too has been impacted by recombination . because of the small recombinant region, which may likely change as we increase the sample size of viruses related to sars-cov-2, it would be difficult to determine the pattern and genomic ancestry of recombination. a total of 8422 cases with 916 deaths in 29 countries including china were reported due to the human respiratory disease during 2002-2003, caused by the sars-cov. the studies showed that bats acted as a natural reservoir of sars-cov that caused the outbreaks (chan-yeung and xu 2003) . later, the sars-cov like similar antibody and genomic sequences were also discovered in rhinolophus bat, such as in r. ferrumequinum, r. pearsoni, r. sinicus, r. pusillus and r. macrotis (lau et al. 2005) . the comparative study of the genomes revealed that bat sars-like covs (sl-cov) have 78-92% nucleotide sequence identities with sars-cov and also among themselves, and hence display great genetic diversity. further, the phylogenetic analysis pointed out that rhinolophus bat might be the direct progenitor of human sars-cov (hon et al. 2008) . various sars-cov groups were isolated in different epidemic periods and hosts. several methods have been adopted to investigate the selective pressure. results have shown that the most functional proteins of sars-cov adopted the stepwise adaptive evolutionary pathway. for example, the spike protein showed strong positive selection in the early as well as middle phases, and not in the late phase. however, the replicase enzyme experienced positive selection only in humans, and assembly proteins experienced the same in the middle and late phases. interestingly, no such positive selection was observed in any proteins of bat sars-like-cov. however, specific amino acid sites that may be the targets of positive selection in each group were identified (tang et al. 2009 ). later in 2010, a study suggested the presence of two distinct genotypes of bt-slcov in r. sinicus (i.e. rp3/rs672 and hku3/rs806). the results also showed the evidence for the recombinant origin of rp3 and rs672. the phylogenetic study showed that their major parent has a relatively closer relationship with hu-scovs. therefore, there may be a possibility for the presence of a bt-scov lineage in r. sinicus, that may have hu-scovs as their direct ancestor, as reported earlier (hon et al. 2008) . however, these speculations are based on studies done on limited strains only, therefore an extensive analysis for the prevalence of such genotype is required for its credibility (yuan et al. 2010) . in 2012, globally, 64 human cases were confirmed resulting in 38 deaths by june 17, 2013, by a disease having symptoms similar to sars, that emerged in saudi arabia (who 2013). later, it was found that the disease was caused by a virus designated as a novel human coronavirus, mers-cov, phylogenetic data showed that it belonged to lineage c of the betacoronavirusgenus and was highly similar to bat coronaviruses hku4 (tylonycterispachypus) and hku5 (pipistrelluspipistrellus; lau et al. 2013) . comparative genomic results showed that mers-cov has a 50% nucleotide identity in the entire genome with hku4 and hku5. moreover, the rna dependent rna polymerase (rdrp) gene has 82% nucleotide identity. later, while studying the mode of entry into the cell it was confirmed that mers-cov uses dipeptidyl peptidase 4 (dppiv), also known as cd26. as dppiv is evolutionarily conserved among mammals, therefore mers-cov can infect a broad range of mammalian cells (humans, pigs, monkeys and bats) and may be efficient in cross-host transmission (raj et al. 2013) . similar to the case for sars-cov and mers-cov , the bat is still a probable species for origin of sars-cov-2, as it shares 96% whole-genome identity with a bat cov, batcov ratg13, from rhinolophusaffinis from yunnan province ). however, sars-cov and mers-cov before entering humans pass through intermediate hosts, such as civets or camels (cui, li and shi 2019) . this fact indicates that sars-cov-2 was probably transmitted to humans by other animals. by comparing the overall genome identity, it was concluded that pangolin-cov genome sequence is 90.55% identical to ratg13 and 91.02% identical to sars-cov-2. however, there was 96.2% identity between sars-cov-2 and ratg13 . other sar like covs (sl-covs) are also showed similarity with pangolin-cov, as its was 85.65% similar to zxc21 and 85.01% with zc45. in a comparative genome analysis between pangolin-cov and sars-cov-2 (genbank: mn908947), result showed 45.8-100% coverage range (average coverage 76.9%). moreover, pangolin-cov genes shared high average nucleotide (93.2%) and amino acid identity (94.1%) with sars-cov-2 (genbank mn908947). similar results were obtained when pangolin-cov genes were compared with ratg13 where 92.8% nucleotide and 93.5% amino acid identity was observed (zhang, wu and zhang 2020) . interestingly, some of the pangolin-cov genes showed higher amino acid sequence identity to sars-cov-2 genes than to ratg13genes. for example orf1b of pangolin-cov 73.4%, the spike (s) protein 97.5%, orf7a 96.9% and orf10 is 97.3% identical to sars-cov-2. similarly, orf1b 72.8%, the spike (s) protein 95.4%, orf7a 93.6% and orf10 is 94.6% identical to ratg13. the high s protein amino acid identity governs the functional similarity between sars-cov-2 and pangolin-cov. a comprehensive phylogenetic analysis was performed based on the nucleotide sequences of whole-genome sequence, rna-dependent rna polymerase gene (rdrp), non-structural protein genes orf1a and orf1b, and main structural proteins encoded by the s and m genes. results showed that in all phylogenetic trees, pangolin-cov, ratg13 and sars-cov-2 were clustered into a well-supported group designated as ''sars-cov-2 group'' which represents a novel betacoronavirus group. however, within this group, ratg13 and sars-cov-2 were grouped together, and pangolin-cov was their closest common ancestor (zhang, wu and zhang 2020) . recently, an extensive study including localized genomic analysis and the pattern of evolutionary recombination was done. the results showed that the strong purifying selection among coronaviruses from distinct host species as well as cross-species infections is responsible for the origin of sars cov-2 ). therefore, we may summarize the origin and intermediate hosts of sars-cov, mers-cov and sars-cov-2 as shown in fig. 2 . in the s1 domain of s protein, followed by fusion with cell membrane. sars-cov is responsible to cause severe acute respiratory syndrome. sars-cov utilizes angiotensin-converting enzyme 2 (ace2) receptor present on the surface of host cells, as shown in fig. 3 . sarc like-covs and sars-covs have identical genetic organizations with high sequence identities. the schematic representation of spike protein (s) from sars-cov-2 is shown in fig. 4 . however, there is some important exception at the n' terminus of spike protein (s), essential for receptor binding in covs. there is a study to investigate the receptor usage by full-length s of sl-cov, sars-cov and a series of s chimeras. different ace2 receptors from human, civet, or horseshoe bat were expressed in cell lines by using human immunodeficiency virus-based pseudovirus system. several important observations were made in the study. first, the sl-cov s was unable to use any of the three receptors. second, the sars-cov s was unable to enter the cells expressing bat ace2. third, the chimeric s enters the cells with different efficiencies for different constructs via human ace2. fourth, a minimal insert region (amino acids 310-518) was sufficient to convert the sl-cov s from non-ace2 binding to human ace2 binding, indicating that the sl-cov s is largely compatible with sars-cov s protein, both in structure and function (ren et al. 2008) . detailed structural study of human sars-cov rbd complexed with human ace2 receptors was performed. results revealed that it is the truncations in the receptor-binding motif (rbm) region of sl-cov spike protein, which abolished its human ace2-binding ability (li 2008) ). therefore, we may hypothesize that the sl-cov found in horseshoe bats is not the direct ancestor of human sars-cov. moreover, it has been observed that the human sars-cov, as well as its closely related civet sars-cov spike proteins, were not able to use a horseshoe bat (r. pearsoni) ace2 as a receptor for cell entry (ren et al. 2008) . these findings highlight a critical missing link (an intermediate host) in the bat-to-civet/human transmission chain of sars-cov (hou et al. 2010 ). an earlier study showed that ace2 from horseshoe bat could not function as a receptor for sars-cov. however, changing 3 amino acids (40, 41 and 42 amino acids) from she to fyq was found adequate to convert the nonfunctional bat ace2 into a fully active receptor for sars-cov. further, an ace2 molecule from a fruit bat, which naturally has the fyq motif, supports sars-cov entry into the cells thus causing infection. this result indicates that there must be a wide host range for sars-cov-related viruses among different bat populations (yuan et al. 2010) . in the case of sars-cov-2, the structural bioinformatics approaches accurately predicted that sars-cov-2 spikes bind human ace2 (wan et al. 2020b ). when cell lines over-expressed the transmembrane protein 'angiotensin-converting enzyme 2' (ace2) from humans, bats, pig or civet cats and were infected with sars-cov-2, results showed that they became hypersensitized to infection, thus indicating that ace2 is a sars-cov-2 receptor . the binding studies also revealed that receptor-binding domains on the sars-cov-2 s proteins have a high affinity to human ace2 (wrapp et al. 2020 ) which makes it more virulent. however, apart from ace2 interaction, the n-terminal domain (ntd) of the sars-cov-2 s proteins may show binding to alternative host-cell receptors . sars-cov-2 s proteins have also acquired a furin protease cleavage site, by acquiring several basic residues (rrar/s). the sars-cov-2 furin substrate site facilitates the prime cleavage step, which further sensitizes s proteins for subsequent activation of cleavages occurring on susceptible target cells, and finally facilitates virus to enter the cells and cause infection (qing and gallagher 2020) . human sars-cov and sars-like coronavirus (sl-cov) in bats have a similar genomic organization; therefore their corresponding gene products are highly conserved. as far as s protein is concerned, it has only a 63-64% sequence identity at the n-terminal region. it is the n-terminal region of coronavirus s protein that is responsible for receptor interaction. when the immunogenicity of the sl-cov s protein was analyzed and compared with that of sars-cov, results revealed that they shared only a limited number of immunogenic epitopes in their s proteins. moreover, major neutralization epitopes were also different ). in another study, a pseudovirus expressing full-length sl-cov s protein was used to raise mouse sera and monoclonal antibody. series of constructs expressing truncated s protein were prepared and analyzed with elisa, as well as western blot. results showed that amino acids 280-455 and amino acids 561-666 are two immunogenic determinants in mice. further, it was also shown that 280-455 amino acids are more immunogenic, as it was recognized by polyclonal as well as monoclonal antibodies. earlier studies also showed that amino acids 528-635 from sars-cov are immunodominant determinants (he et al. 2004 ). due to the high sequence similarity with sl-cov s protein in the same region, the amino acids 561-666 of s protein also demonstrated immune response in mouse (zhou et al. 2013 ). in a cross-reactivity test with antibodies against rbd domain sars-cov, some of the sl-cov strains (wivi) have shown positive results whereas some strains (shc014) failed too. this difference in reactivity is due to the low sequence identity in the rbd domain of shc014 and high sequence identity in the rbd domain of wivi with rbd domain of sars-cov (zeng et al. 2017 ). detection of novel coronavirus is done by different molecular biology techniques including real-time reverse transcription pcr (rrt-pcr), reverse transcription pcr (rt-pcr), reverse transcription loop-mediated isothermal amplification (rt-lamp), multiplex nucleic acid amplification, real-time rt-lamp and microarray-based assays . who also recommended a pan-coronavirus assay for characterization and confirmation.). viral culture and rt-pcr are among the most efficient and reliable methods for the diagnosis of sars-cov-2 infection. these methods are time consuming and generally takes hours to detect the nucleic acid and many days to isolate the virus from the samples. apart from that, specialized equipments and expertise are also required. to overcome these limitations, rapid diagnosis of sars-cov-2 infection can be done with rapid antigen detection (rad) tests. in rad tests, the immobilized sars-cov-2 antibody on the device can detect viral antigen in the sample. the results of rad tests are prompt and interpreted without specialized instrument. hence, rad tests could be beneficial reduce the workload in diagnostic laboratories and hospitals (mak et al. 2020) . however, as per the who, rad tests for sars â�� cov-2 antigen detection, further needs evaluation and is not recommended for clinical diagnosis (laboratory testing strategy recommendations for covid-19: interim guidance 2020). the immune response to sars-cov2 in the early weeks of the infection can be detected using enzyme-linked immunosorbant assay (elisa), automated chemiluminescence immunoassay (clia), and lateral flow immunoassay (lfia), plaque reduction neutralization tests (prnt), or a combination of these methods (espejo et al. 2020) . the most commonly antigens used in these assays were the spike glycoprotein s1 including the receptor binding domain (jin et al. 2020 ), the nucleocapsid protein or both (pang et al. 2020) . application of inhibitor to halt virus interactions with the host may be one of the prophylactic methods. in this direction, an engineered pan-cov fusion inhibitor has been designed and designated as ek1 peptide. it has shown promising results in mice by inhibiting the infection in five human coronaviruses, including sars-cov, mers-cov and three bat-sl-covs (xia et al. 2019) . it has also been reported that intranasal application of engineered ek1 peptide before or post viral infection showed protection in human dpp4-transgenic mice against mers-cov infection, indicating its potential prophylactic and therapeutic effect. another approach is designing of neutralizing antibody, which may block the interaction with the host cell. the s proteins of sars-cov and mers-cov are immunogenic. the rbd domains of sars-cov and mers-cov are known to have nonsequential epitopes that induce a more potent neutralizing antibody and give protection against sars-cov and mers-cov (du et al. 2009; zhou et al. 2019) . the modification on the structural basis for mers-cov s-rbd amino acid has improved the efficacy against mers-cov infection (zhou et al. 2019) . therefore, we may suggest that sars-cov-2 s-rbd or modified s-rbd of another related coronavirus could be used as target to develop a vaccine against sars-cov-2. recently, neutralizing monoclonal antibodies and nanobodies against the rbd domain of s protein showed protection against sars-cov and mers-cov (du et al. 2009; zhou et al. 2019) . although the ntd and s2 unit of s protein from sars-cov and/or mers-cov was also studied to develop neutralizing antibodies, but the efficacy was found to be very low (du et al. 2009 ). therefore, rbd of s protein sars-cov-2 would be a key target for developing neutralizing antibodies as shown in fig. 5 . cross protection by the antibodies developed against sars-cov, has been observed against bat-sl-cov-w1v1 and bat-sl-cov-shc014 (zeng et al. 2017) . therefore, the development of cross-neutralizing antibodies can be another possible way for urgent prevention and treatment of sars-cov-2 infection. currently, plasma therapy in which polyclonal antibodies from recovered sars-cov-2-infected patients have been used to treat sars-cov-2 infection is also being considered. researchers are working hard to develop monoclonal abs (mabs) and once such antibodies are produced, the next step will involve in vitro testing for neutralizing and/or crossneutralizing activity as well as in vivo evaluation for protective efficacy in available covid-19 animal models. preclinical and clinical trials testing the safety and and efficacy before they are approved for clinical applications are also necessary. recently, 1000 memory b cells specific to sars-cov-2 s1 or rbd (receptor binding domain) have been purified. among these, 178 antibodies showed positive results in antigen binding assays with the top 17 binders having ec 50 below 1 nm specific for rbd. further, among 11 neutralizing antibodies, eight of them have shown an ic 50 value within 10 nm, whereas 414-1 best among all have ic 50 of 1.75 nm. in epitope mapping, three main epitopes recognized by monoclonal antibodies have been identified in rbd domain. interestingly, 515-5 monoclonal antibody from same study, also showed cross-neutralizing property in the sars-cov pseudovirus assay (wan et al. 2020a) . in another study, 61 sars-cov-2-neutralizing monoclonal antibodies were isolated from five infected patients. 19 among them have shown positive result in in vitro neutralization assay and nine among them shown 50% virus-inhibition at the concentrations of 1-9 ng/ml. epitope mapping showed that receptor-binding domain (rbd) and the n-terminal domain (ntd), both are immunogenic in nature. further, structural studies of these monoloclonal antibodies have proven that one is targeting rbd, second one is targeting ntd and a third bridging rbd and ntd. therefore, several of these monoclonal antibodies are promising candidates for clinical development as potential therapeutic and/or prophylactic agents against sars-cov-2 (l et al. 2020). due to the high sequence identity of s protein between sars-cov-2 and its closely related sars-cov , sars-cov nabs have been tested for its cross-reactivity and/or cross-neutralizing activity against sars-cov-2 infection. interestingly, a sars-cov rbd-specific human neutralizing mab, cr3022, have shown the binding of sars-cov-2 rbd with high affinity and may recognize an epitope on the rbd that does not overlap with the ace2-binding site (tian et al. 2020) . further, sars-cov-2 entry and infection may be blocked by crossreacting the sera isolated by convalescent sars patients or from animals specific for sars-covs1 (hoffmann et al. 2020) . moreover, it has been observed that polyclonal antibodies against the rbd domain of sars-cov have been cross-reacted with the rbd protein of sars-cov-2. they cross-neutralized sars-cov-2 infection in hek293t cells expressing the human ace2 receptor. such findings may open new avenues for the potential development of sars-cov rbd-based vaccines that might eventually prevent sars-cov-2 and sars-cov infection (tai et al. 2020) . it may be possible that sars-cov rbd-targeting neutralizing antibodies could be applied for treatment/prophylaxis of sars-cov-2 infection in the current absence of a specific vaccine against sars-cov-2. remdesivir has been recently recognized as a promising antiviral drug in cultured cells, mice and nonhuman primate (nhp) models, against rna viruses including sars-cov and mers-cov (sheahan et al. 2017) . it is currently under clinical trials for the treatment of ebola virus infection (mulangu et al. 2019) . recently studies have shown that ec 90 value of remdesivir against 2019-ncov in vero e6 cells was 1.76 î¼m. these data suggest that its working concentration is likely to be achieved in nhps remdesivir have shown the efficient in vitro antiviral activity against sars-cov-2. however, the controversial evidence of clinical improvement in severe covid-19 patients has been reported recently in france. the five covid-19 patients admitted in icu and treated with remdesivir. treatment showed significant reduction of sars-cov-2 viral load from upper respiratory in most of the cases, however but two patients died with active sars-cov-2 replication in their lower respiratory tract. remdesivir treatment was interrupted for its side effects among four patients due the complexity in such critically ill patients (dubert et al. 2020 ). the first covid-19 case in the united states was intravenously treated with remdesivir (iv) (holshue et al. 2020) . within 24 h of remdesivir treatment, the patient showed recovery sign. as the viral loads was decreasing before remdesivir treatment, therefore it cannot be determined if further viral load reduction and clinical improvement were as the direct result of remdesivir treatment. in another study, the compassionate use of remdesivir (n = 53) reported 68% improved oxygenation, 47% discharge and 13% death. this study was not most significantly as it lacks of a paired control group (grein et al. 2020) . recent study at the national institutes of health (nih), showed preliminary results of the adaptive covid-19 treatment trial (actt, n = 1063). in this randomized controlled trial (rct), remdesivir treatment showed 31% faster time to recovery as comparative to the placebo group (p < 0.001). the mortality rate was also showed reduction in remdesivir group, however it was not statistically significant (8% vs. 11.6%, p = 0.059). so far, remdesivir has not shown any significant benefit in the reduction of mortality rate. currently, remdesivir is recommended by the nih for hospitalized severe covid-19 cases as defined by oxygenation needs (clinical management of covid-19). another drug, like chloroquine (c), has recently been reported as a potential broad-spectrum antiviral drug (savarino et al. 2006) . it inhibits the virus infection by increasing endosomal ph, which is essential for virus/cell fusion, as well as by interfering with the glycosylation of cellular receptors of sars-cov (vincent et al. 2005) . recent studies demonstrated that chloroquine is effective at both entry, as well as at postentry stages of the sars-cov-2 infection in vero e6 cells . apart from antiviral activity, chloroquine also has immune-modulating activity. therefore, it may synergistically enhance antiviral effect in vivo. chloroquine gets widely distributed in the whole body after oral administration, including lungs. the ec 90 value of chloroquinein vero e6 cells against the sars-cov-2 was 6.90î¼m, therefore it could be clinically achievable . the effect of hydroxychloroquine (hcq) and chloroquine (cq) in vitro was also tested by yao et al. in a systematic way, they had divided the experiment into two phases: treatment study and prophylaxis. in the treatment study, they determined the ec 50 values for chloroquine. results showed that it was 23.90 î¼m and 5.47 î¼m at 24 and 48 h, respectively. however, in the case ofhydroxychloroquine,the ec 50 values were 6.14 î¼m and 0.72 î¼m at 24 and 48 h, respectively. on the other hand in the prophylaxis study for chloroquine, the ec 50 values were more than100 î¼m and 18.01 î¼m at 24 and 48 h, respectively. similarly, for hydroxychloroquine, the ec 50 values were 6.25 î¼m and 5.85 î¼m at 24 and 48 h, respectively (yao et al. 2020) . hence, they found that hydroxychloroquine is more effective in vitro than chloroquine for both prophylaxis and treatment. a study in united states, where covid-19 patients hospitalized within 24 h of diagnosis was treated with hydroxychloroquine alone (hcq) or with hydroxychloroquine and azithromycin (hcq + azm) or no hcq as treatments. among patients, there was no significant reduction in mortality rate or in the need of ventilation with hydroxychloroquine alone or with hydroxychloroquine and azithromycin (magagnoli et al. 2020) . a new york hospital stated the qtc prolongation associated with hcq + azm (n = 84; chorin et al. 2020) . it was amplified from a baseline of 435 â± 24 ms to a maximal value of 463 â± 32 ms (p < 0.001) on day 3.6 â± 1.6 of the treatment. till date, researchers present conflicting data's related to the treatment with cq and hcq. therefore, significant randomized control tests (rcts) with improved study designs are required to examine the efficacy and the clinical benefits of hcq/cq treatment over its risks. currently, the nih recommendation are against cq/hcq and hcq + azm treatment for covid-19, except for clinical trials. due to the potential toxicity, nih recommendations are also against the high-dose of cq (600 mg) twice daily for 10 days in all settings (coronavirus disease 2019 (covid-19) treatment guidelines). nitazoxanide has demonstrated potent in vitro activity against sars cov-2, with an ec 50 at 48 h of 2.12 î¼m in vero e6 cells . this potent activity is consistent with ec 50 values for nitazoxanide and its active metabolite, tizoxanide, against mers-cov in llc-mk2 cells where ec 50 values of 0.92 î¼m and 0.83 î¼m respectively, have been demonstrated (rossignol 2016) . dexamethasone, a synthetic glucocorticoid, has antiinflammatory and immunosuppressive properties. there is a hyper inflammatory response involved in the clinical course of patients with pneumonia due to sars-cov-2. the elevated level of c-reactive protein (crp) in sars-cov-2 patients has significantly decreased from 129.52 to 40.73 mg/l at time of discharge. 71% of the patients were discharged home with a mean length of stay of 7.8 days. none of the patients had escalation of care, leading to mechanical ventilation (selvaraj et al. 2020) recently, a randomized, controlled clinical trial in the united kingdom save the lives of people seriously ill with covid-19 when treated with dexamethasone. results showed the reduction of number of death by one-third (ledford 2020) . dexamethasone may be useful for the short-term in severe sars-cov-2 patients as it inhibit the protective function of t cells and block b cells from making antibodies (theoharides and conti 2020). the development of successful vaccine for humans can take several years. as no coronavirus vaccines are available in the market as of now, therefore, the development of a vaccine for the first time can be difficult and time-consuming. however, a mrna-based vaccine has been co-developed by moderna and the vaccine research center at the national institutes of health. in this vaccine, the target antigen's mrna, encapsulated in lipid nanoparticles are injected into vaccinee and antigen expresses in vivo. the phase i clinical trial has been recently started (clini-caltrials.gov: nct04283461). curevac is also using the same platform but they are still in the pre-clinical phase. apart from this, there are various other approaches including dna vaccine, recombinant protein-based vaccine, recombinant vector vaccine, inactivated vaccine and attenuated vaccine. different research companies, universities and institutes are targeting s protein of sars-cov-2 to develop recombinant proteinbased vaccines including novavaxexpress2ion, ibio, sichuan clover biopharmaceutical, baylor college of medicine and the university of queensland. similarly, cansino biologics, geovax vaxart and the university of oxford are using viral-vector-based vaccine platform especially focused on the s protein. applied dna sciences and inovio are using dna vaccines platform again focused on the s protein (amanat fatima 2020). apart from the above-mentioned platforms, the whole microorganism based vaccine platform like inactivated and attenuated virus vaccine is also in consideration. codagenix with the serum institute of india is using live attenuated vaccine platforms. the recombinant vector-based platform (adenovirus vector) is adopted by johnson and johnson, and on the other hand, sanofi is also using the same platform (recombinant influenza vector) to develop a vaccine against sars-cov-2. at this stage, it is difficult to predict the best platform for a vaccine against sars-cov-2 as all the above mentioned platforms have some advantages as well as disadvantages (amanat fatima 2020). coronaviruses have shown the capability to jump species boundaries and adapt to new hosts. therefore, we may face more such kind of outbreaks in the future. role of the intermediate host is also of major importance, as they provide direct pathway for virus transmission in humans. the enormous diversity of viruses in animals and their ongoing evolution makes it important to limit our exposure to animal pathogens as much as possible. based on the metagenomic data it is predicted that the pangolin-cov is most closely related to sars-cov-2. pangolin-cov genome showed 91.02% nucleotide identity with the sars-cov-2 genome. due to a very limited knowledge of this novel virus, it is difficult to explain the significant number of amino acid substitutions that occurred between the sars-cov-2 and sars or sl-covs. for example, in sars-s-cov, six mutations occurred in the regions other than that of the rbd domain, but interestingly no amino acid substitutions were present in the receptor-binding motifs that directly interact with human receptor ace2 protein. therefore, such differences that could affect sars-cov-2 transmission property as compared to sars-cov are of importance for future investigation. sars-cov-2 continues to infect people globally; therefore it is imperative to develop new, safe, accurate, fast and simple new technologies for detecting sars-cov-2. apart from diagnosis, effective prophylactic and therapeutic agents are also required to control and prevent infection. various therapeutic agents including dexamethasone have shown promising results in the in vitro studies to control infection. however, there is an urgency to develop vaccine against coronaviruses. in this direction, studies on neutralizing antibodies from sars-cov and mers-cov against s protein and its many fragments including s1-ntd, rbd and s2 may provide important guidelines for development of vaccine against sars-cov-2. apart from neutralizing antibody against s protein, other approaches including dna vaccine, recombinant vector vaccine, inactivated vaccine and attenuated vaccine are also in pipeline to develop vaccines against sars-cov-2. so far, the traditional public health measures including detection of active cases, isolation of such cases, tracing of all contacts and their quarantine, maintaining social distancing, as well as community quarantine were found to be successful. only after this pandemic ends, we will be in a position to assess the social, health and economic impact of such a massive outbreak. therefore, we must learn lessons for our future from such outbreaks, as new viruses will keep coming. none declared. sars-cov-2 vaccines: status report epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the qt interval in patients with covid-19 treated with hydroxychloroquine and azithromycin clinical management of covid-19 covid-19) treatment guidelines the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade origin and evolution of pathogenic coronaviruses case reports study of the first five patients covid-19 treated with remdesivir in france the spike protein of sars-cov -a target for vaccine and therapeutic development review of current advances in serologic testing for covid-19 the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 compassionate use of remdesivir for patients with severe covid-19 identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor first case of 2019 novel coronavirus in the united states evidence of the recombinant origin of a bat severe acute respiratory syndrome (sars)-like coronavirus and its implications on the direct ancestor of sars coronavirus angiotensin-converting enzyme 2 (ace2) proteins of different bat species confer variable susceptibility to sars-cov entry diagnostic value and dynamic variance of serum antibody in coronavirus disease 2019 laboratory testing strategy recommendations for covid-19: interim guidance identification of 2019-ncov related coronaviruses in malayan pangolins in southern china genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus breakthrough: dexamethasone is first drug shown to save lives functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections potent neutralizing monoclonal antibodies directed to multiple epitopes on the sars-cov-2 spike bats are natural reservoirs of sars-like coronaviruses emergence of sars-cov-2 through recombination and strong purifying selection genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19 evaluation of rapid antigen test for detection of sars-cov-2 virus a randomized, controlled trial of ebola virus disease therapeutics potential rapid diagnostics, vaccine and therapeutics for 2019 novel coronavirus (2019-ncov): a systematic review dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like cronavirus of bat origin nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia new insights into the antiviral effects of chloroquine short-term corticosteroids in sars-cov2 patients: hospitalists' perspective broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronavirus: epidemiology, genome replication and the interactions with their hosts characterization of the receptorbinding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine differential stepwise evolution of sars coronavirus functional proteins in different host species dexamethasone for covid-19? not so fast potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro human igg neutralizing monoclonal antibodies block sars-cov-2 infection receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus who| novel coronavirus summary and literature update -as of 17 cryo-em structure of the 2019-ncov spike in the prefusion conformation a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans cross-neutralization of sars coronavirus-specific antibodies against bat sars-like coronaviruses recent advances in the detection of respiratory virus infection in humans probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak identification of immunogenic determinants of the spike protein of sars-like coronavirus immunogenicity difference between the sars coronavirus and the bat sars-like coronavirus spike (s) proteins a pneumonia outbreak associated with a new coronavirus of probable bat origin advances in mers-cov vaccines and therapeutics based on the receptor-binding domain key: cord-354474-hbl2ywix authors: temsah, m. h.; alhuzaimi, a. n.; alamro, n.; alrabiaah, a.; al-sohime, f.; alhasan, k.; kari, j. a.; almaghlouth, i.; aljamaan, f.; al-eyadhy, a.; jamal, a.; al amri, m.; barry, m.; al-subaie, s.; somily, a. m.; al-zamil, f. title: knowledge, attitudes and practices of healthcare workers during the early covid-19 pandemic in a main, academic tertiary care centre in saudi arabia date: 2020-08-28 journal: epidemiol infect doi: 10.1017/s0950268820001958 sha: doc_id: 354474 cord_uid: hbl2ywix as the middle east respiratory syndrome coronavirus (mers-cov) continues to occur in small outbreaks in saudi arabia, we aimed to assess the knowledge, attitudes and intended practices of healthcare workers (hcws) during the early stage of the covid-19 pandemic and compare worry levels with previous findings during the mers-cov outbreak in 2015. we sent an adapted version of our previously published mers-cov questionnaire to the same cohort of hcws at a tertiary hospital in saudi arabia. about 40% of our sample had previous experience with confirmed or suspected mers-cov patients, and those had a significantly higher knowledge score (13.16 ± 2.02 vs. 12.58 ± 2.27, p = 0.002) and higher adherence to protective hygienic practices (2.95 ± 0.80 vs. 2.74 ± 0.92, p = 0.003). the knowledge scores on covid-19 were higher in the current cohort than the previous mers-cov outbreak cohort (68% vs. 79.7%, p < 0.001). hcws from the current cohort who felt greater anxiety from covid-19 compared to mers-cov were less likely to have been exposed to mers-cov infected/suspected cases (odds ratio (or) = 0.646, p = 0.042) and were less likely to have attended the hospital awareness campaign on covid-19 (or = 0.654, p = 0.035). we concluded that previous experience with mers-cov was associated with increased knowledge and adherence to protective hygienic practices, and reduction of anxiety towards covid-19. since the world health organization (who) declared coronavirus disease 2019 (covid19) as a pandemic, it has become a major challenging public health problem worldwide [1, 2] . this pandemic has affected all aspects of people's life in almost all nations and among all socioeconomic groups. healthcare workers (hcws) of all types are facing an unprecedented crisis with the rapid spread of covid-19 and severity of the disease in many infected individuals. as such many healthcare systems have been overwhelmed and hcws presented with depression and anxiety [3, 4] . there is a potential shortage of physical resources, such as ventilators and intensive care unit beds, needed to care for surges of critically ill patients [5] ; however, additional medical supplies and beds will be of limited help unless there is an adequate medical workforce. the mental impact of the covid-19 epidemic on general population [6] , psychiatric patients [7] , workers [8] patients [9] , children [10] , older adults [11] and medical students [12] has been reported. however, little attention has been paid to the psychological wellbeing and fatigue levels among hcws [13, 14] . to further understand the knowledge, attitudes and intended practices of hcws during the early stage of the covid-19 pandemic, it is particularly beneficial to obtain their input, especially in an area of the world where other respiratory viral illnesses are either endemic, such as mers-cov, or seasonal, such as influenza. this study was the baseline for a serial, cross-sectional survey among hcws in a tertiary care hospital with a 1000-bed capacity in riyadh, saudi arabia. the hcws had multinational backgrounds; in addition to saudi nationals, they were mostly from the indian subcontinent and the philippines. data were collected between 5 and 16 february 2020, which was just before the presentation of the first case of covid-19 in saudi arabia. the survey was a pilot-validated, self-administered questionnaire that was sent to hcws online. the questionnaire was similar to that used in our previous mers-cov study [15] , with modification and additional questions related to the current covid-19 pandemic. the questions queried the demographic characteristics of the respondents ( job category, age, sex, work area and years of clinical experience) and previous exposure to mers-cov patients, either suspected or confirmed cases. we assessed the following domains for every participant: kap scores [16] , knowledge about covid-19 [17] , hcw attitudes toward infection control measures [18] and hygienic practice change scores. in addition, we assessed [19] the perceived adequacy of covid-19 information and [15] perceived high fear/stress from the covid-19 pandemic as compared to the previous mers-cov outbreak. the hcws knowledge of covid-19 disease was tested using eight true/false questions (supplementary table s1 ). the perceived adequacy of knowledge, hygienic practice changes and hcw attitudes toward infection control measures were assessed using a series of likert-based questions (supplementary tables s2-s4 ). the practice score is measuring the degree of improvement in protective practices after covid-19, from 1 to 4, with 1 representing no change, 2 little change, 3 moderate change and 4 significant change. the attitude score represents the level of agreement with protective hcws' attitudes toward infection control. the score ranges from 1 to 5, with 1 representing 'strongly disagreeing' and 5 representing 'strongly agreeing' with attitudes. additionally, the sources of information on the outbreak and attendance at the covid-19 awareness campaign (educational day conference) that was conducted at the hospital in the first week of february 2020 were evaluated. the participants were asked about their worry level regarding the current covid-19 compared to their worry level from the mers-cov outbreaks. we analysed the data using spss ibm v20 (spss, inc., chicago, il, usa). for all tests, statistical significance was set at p < 0.05. for the five domains we used a summative score to summarise the results from continuous likert's scale-based questions for each participant. an unpaired t test and analysis of variance (anova) analysis followed various post hoc tests to compare the means of different groups. the model was significant based on a model goodness-of-fit hosmer-lemeshow test [χ 2 (8) = 7.4, p = 0.490, model auc roc = 74%, χ 2 (16) = 103.8, p < 0.001]. the pearson's (r) test was used to assess the bivariate associations between the measured hcw scores. fisher's exact tests were used to establish the differences between hcw groups (physicians vs. non-physicians) for nominal variables. we compared this current analysis with data from our previous study conducted during the mers-cov outbreak in 2015 in the same institution [15] . the study was approved by the institutional review board at the college of medicine and king saud university medical city (approval no. 20/0065/irb). the questionnaire was sent to 800 hcws with a 72.8% response rate; 582 hcws completed the questionnaire. the majority of participating hcws were female (75%) and the most common age group was 31-39 years (38.3%, mean 38.6). nurses constituted 62% of our study population. the majority of respondents were hcws working in critical care units (44.8%) followed by outpatient clinics (28%), and inpatients wards (19.4%), see table 1 . hcws used multiple sources of information about covid-19, as shown in figure 1 . the mean knowledge score among the whole sample was 12.75 ± 2.2 of a total score of 16. there was no difference in the knowledge scores for clinical role groups, gender or hospital working areas (table 1 ). nearly 40% of the whole sample attended the hospital's educational day conference. a higher perceived adequacy of covid-19 information (mean: 3.97 ± 1.00 vs. 3.7 ± 0.92, p = 0.001) and better hygienic practices (3.10 ± 0.84 vs. 2.65 ± 0.87, p < 0.001) were observed among hcws who attended the educational day conference (fig. 2) . however, there was no difference in knowledge scores and attitudes toward selected preventive measures between those who attended and those who did not attend the educational day conference. the mean hygienic practice score was 2.76 ± 0.91 for the whole sample. there was no difference in knowledge scores between males and females. however, female hcws scored higher in terms of adherence to hygienic practices (2.98 ± 0.82 vs. 2.36 ± 0.90, p < 0.001), attitudes toward infection control measures (3.85 ± 0.93 vs. 4.15 ± 1.10, p = 0.003), and perception of adequacy of knowledge (3.88 ± 0.93 vs. 3.58 ± 1.03, p = 0.002; fig. 3 ). there was no significant difference in knowledge scores among different hcws across their different clinical roles. however, nurses had significantly higher hygienic practice scores (3.20 ± 0.68, p < 0.001) compared to all levels of physicians, in addition to significantly higher attitude scores toward infection control practices compared to resident physicians (p = 0.041). taking clinical area assignment into consideration, staff working in critical care units had significantly higher perceived information regarding covid-19, which was reflected by significant higher hygienic behaviour compliance. the younger hcws (⩽30 years) had significantly less adherence to hygienic practices compared to older hcws (31-39 years; p < 0.001) but both the young age group and the group working in critical care area did not show any significant difference in terms of attitude toward infection control practices. fig. 4 ). however, there was no significant difference in the protective attitude toward domestic hygiene among hcws who had previous experience with mers-cov cases compared with the hcws who did not. almost one-third (29.4%) of hcws did not adhere to flu vaccinations. kap scores analysed based on adherence to influenza vaccinations showed higher knowledge mean scores (13.00 ± 2.10 vs. 12.23 ± 2.25, p < 0.001) and hygienic practice scores (3.01 ± 0.81 vs. 2.38 ± 0.89, p < 0.001; fig. 5 ). there was no difference in attitude scores. there was a moderate positive correlation between hcw infection control attitudes and their perceived adequacy of information about covid-19 (r = 0.53, p value <0.01) ( table 2 ). in addition, there was a weak but significant correlation between hygienic practice scores and perceived adequacy of information (r = 0.32, p < 0.01) and with hcw knowledge scores (r = 0.22, p < 0.01). these findings suggested that education plays an important role in improving hcw attitudes and practices to prevent covid-19. hcw's perceived higher fear/stress from covid-19 than from previous mers-cov outbreaks the hcws were asked about their worry level from current covid-19 compared to their worry level from previous mers-cov outbreaks. multivariate logistic regression analysis of the predictors of the hcw's higher stress from covid-19 compared to mers-cov was performed and the results are displayed in table 3 . hcws previously exposed to mers-cov infected/suspected cases were significantly less likely to have higher stress from the covid-19 outbreak (odds ratio (or) = 0.646, p = 0.042). we found that hcws who had attended their hospital educational day on covid-19 were less likely to have stress related to covid-19 compared to stress related to mers-cov (or = 0.654, p = 0.035), accounting for other confounders. hcw worry levels based on contacting covid-19 themselves was associated with higher stress from covid-19 disease compared to mers-cov (or = 1.933 times higher, p < 0.001). hcws' hygienic practice scores converged significantly on higher odds of having high stress from covid-19 compared to mers-cov (or = 1.303, p = 0.046). nonetheless, the hcws' attitude scores correlated positively and significantly with higher odds of being highly stressed from covid-19 disease (or = 1.27, p = 0.032), accounting for the other predictors. we compared our current analysis with data from the previous study conducted in the same institution during the mers-cov outbreak in 2015 [15] . the resulting data analysis (table 4 ) suggested that the hcws' worry levels for contracting covid-19 and passing it on to their families and friends were significantly lower than those measured during the mers-cov (p < 0.001). also, the hcws' worry levels for contracting covid-19 was significantly lower than those measured during the previous mers-cov outbreak in 2015 (p < 0.001). this might be due to in part because this study was done before who declared covid-19 a global pandemic. interestingly, the proportion of hcws who underwent annual influenza vaccination at the current time significantly exceeded those measured during the 2015 mers-cov outbreak; the difference in percentages of hcws who immunised annually indicated an 18.86% rise in the proportion of hcws who immunised against seasonal influenza during this current point of time in 2020 compared to the same period in 2015 (p < 0.001). however, comparing the hcws' perceived hygienic changes and intents to be absent from their work did not differ significantly between the current study and the previous mers-cov (2015) study (p > 0.05 for each). the world has experienced several epidemics with novel coronaviruses; namely, sars-cov-1, which emerged in china in 2003 followed by middle east respiratory syndrome coronavirus (mers-cov) in the middle east in 2012, and the current severe acute respiratory syndrome corona virus-2 (sars-cov-2) pandemic [16] . mers-cov continues to be endemic in saudi arabia with weekly reported cases. with the ongoing circulation of mers-cov and continuing zoonotic spillover with 70% of the cases resulting from hospital outbreak, the emergence of covid-19 within the same setting will be overwhelming to healthcare facilities and workers [17] . therefore, it is of great importance to know the impact of such epidemics on hcws. this is an expected finding since there are established guidelines on the treatment of mers-cov and seasonal influenza and lack of comprehensive knowledge and experience with sars-cov-2. as the understanding of the epidemiology of sars-cov-2 evolved, human-to-human transmission was confirmed with the potential for asymptomatic transmission as well [18] . sars-cov-2 has also demonstrated a very rapid transmission rate with a reported r 0 = 2.5; i.e. each patient can spread the virus to two other patients [20] . hospital transmission was also reported and was estimated to account for 41.3% of cases [19] . this highlights the importance of strict infection control measures and continuous hcw education and competency, not only to decrease transmission but to limit hcw anxiety, which will result in better compliance, performance and patient care. these hcws reported that their main concern was the risk of transmitting the infection to their families (2.71/5) or acquiring it themselves (2.57/5) [21] . in response to the global crisis, as part as hospital preparedness [22] , king saud university medical city (ksumc) arranged an educational day for all hospital staff in an attempt to increase awareness and improve the preparedness of hcws. a survey was distributed afterward and completed by 582 hcws working at king khalid university hospital at ksumc to ensure the adequacy of education. the majority (62.3%) of the responding hcws were nurses, who were approximately half of the attendees (46.2%), and they reported adequacy of the information given regarding symptoms, treatment, prognosis and prevention of covid-19 disease. we observed a better hygienic practices and higher perceived adequacy of covid-19 information among those who attended the hospital's educational day conference. however, only 40% of the whole sample attended this activity. therefore, a different approach is required to improve the attendance of educational activities. suggested approaches include making the covid-19 educational activities periodic, making the attendance mandatory for all staff and utilising alternative or additional online modules. these results showed that a greater proportion (53.8%) still did not get a proper education and, hence, there was a need for more awareness activities and campaigns, including for example but not limited to lectures, departmental educational activity, concise educational leaflets, in addition to newly innovated methods including virtual lectures, smart phone applications (apps) and hotline access during the current pandemic to answer questions and queries. covid-19 pandemic has been accompanied by an overabundance of information that makes it difficult to obtain what is true from false, as most individuals get their information from social media [17] , and this might contribute to higher levels of misconception or anxiety. in this survey, the top source accessed by hcws was hospital announcements (77.8%), which were similar to the main sources used in the previous mers-cov outbreak [15] . this finding again highlights the importance of having a dedicated team to provide accurate information from trusted sources as most hcws rely on it. social network news remained a source of information among 61.7% of the hcws, which might elevate anxiety and should be discouraged in future awareness campaigns. the current study showed that hcws who had previous experience with mers had higher knowledge scores and more adherence to protective hygienic practices. these results could be explained by the fact that previous hospital educational campaigns and managing previous mers-cov cases may have enhanced their knowledge and intentions to be in compliance with infection control practices [23] . another speculation is that the occurrence of mers in saudi arabia is ongoing and there is more awareness about it among hcws [24, 25] . similarly, hcws who were more adherent to receiving the annual influenza vaccine had higher knowledge mean score and higher compliance with hygienic practices. the association between the effect of knowledge, among other multimodal interventions, and compliance with influenza vaccination, has been demonstrated in previous studies, including those conducted in saudi arabia [26] [27] [28] . influenza vaccine utilisation may indicate general awareness and initiative for self-healthcare. most hcws had strong perceptions of the importance of change in hygienic behaviours, including compliance to hand hygiene, infection control measures, avoiding contact with people who have flu-like symptoms, and decreasing handshaking and social visits. the level of knowledge of hcws toward viral infection outbreaks during the current covid-19 pandemic are much higher compared to the previous study conducted in the same institution during mers-cov a few years ago [15] . this finding is promising as it is expected that hcws would be more compliant with infection control measures. for different subgroups we observed higher scores in hygiene practices for hcw attending educational day conference, especially among females and nurses. this is directly reflective on the higher number of nurses attending such activities and the predominance of female nurses in our institution. hcw contribution to proper management of covid-19 infected patients is substantial, as the more knowledgeable they become, the more likely their management to be more appropriate. hcw not wearing masks have a 30% chance of developing infection [29] , meaning at least one in every three hcw not adherent to ppe will get infected. this highlights the importance of sending correct information to enhance their knowledge which would reflect on their attitudes and practices. as of 17 august 2020 within the eastern mediterranean region, the kingdom of saudi arabia ranks second only to the islamic republic of iran in total number of infected and active cases, follow by pakistan then iraq, yet the case fatality rate (cfr) is 1.1 in saudi arabia compare to 5.75, 2.14 and 3.31 in iran, pakistan and iraq, respectively [30] . this lower cfr in comparison to high attack rate can be attributed to the healthcare system in saudi arabia that can be anticipated in a country that dealt with several outbreaks of middle east respiratory syndrome coronavirus (mers-cov). moreover, the strict social distancing and lockdown strategies that were implemented in saudi arabia since the early stages of the pandemic contributed in slowing down the spread of covid-19 and decreased the cfr via preventing the healthcare system from overwhelming covid-19 patients, and thus allowing better and lifesaving care. evaluations of the related covid-19 cfr have suggested that cfr was dependent on the efficacy of local response efforts [31, 32] . a study from china, for example, found that the timely supply of adequate medical resources lowered the cfr from around 4.5-0.5% [33] . we suggest that high cfrs in the abovementioned countries were in part determined by hospital and healthcare surge capacity being exceeded and, as a result, patients potentially receiving suboptimal care during the crisis. recently, nguyen et al. reported that front-line hcws had at least a threefold increased risk of covid-19 infections [34] . given the above-mentioned initial high attack rates in saudi arabia and low cfr, future research that defines the associated risks and outcomes for hcws who contracted covid-19 in saudi arabia is advised. there are several limitations to this study. first, it was done in a single centre and therefore it cannot be generalised to the entire population and this also contributes to selection bias. second, a self-administered electronic questionnaire was used, which increases the chances of recall bias; however, this was balanced with previous data that was collected using the same method from the same cohort. third, although there were statically significant differences in knowledge, attitude and practice scores between different groups, but a small difference should be interpreted with caution. finally, the study was done early in the pandemic and not much information was available and thus it corresponded to a variable level of anxiety and stress. despite these limitations, the study has highlighted the importance of addressing hcw stress levels and ensuring providing information from trustable sources, which will all contribute to better compliance with infection control measures and limiting disease spread. the hcws' worry levels regarding contracting or transmitting mers-cov were higher than for covid-19 during the early stage of the covid-19 pandemic. the current study showed that previous experience with mers-cov and subsequent awareness campaigns that were conducted were associated with increased knowledge, adherence to protective hygienic practices and reduction of anxiety toward the covid-19 pandemic. supplementary material. the supplementary material for this article can be found at https://doi.org/10.1017/s0950268820001958. the novel coronavirus originating in wuhan, china: challenges for global health governance world health organization. situation report -51. who website psychological impact of the covid-19 pandemic on health care workers in singapore a multinational, multicentre study on the psychological outcomes and associated physical symptoms amongst healthcare workers during covid-19 outbreak ihme covid-19 health service utilization forecasting team, murray cjl (2020) forecasting covid-19 impact on hospital bed-days, icu-days, ventilator-days and deaths by us state in the next 4 months immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (covid-19) epidemic among the general population in china do psychiatric patients experience more psychiatric symptoms during covid-19 pandemic and lockdown? a case-control study with service and research implications for immunopsychiatry is returning to work during the covid-19 pandemic stressful? a study on immediate mental health status and psychoneuroimmunity prevention measures of chinese workforce mental health and the covid-19 pandemic covid-19 and its impact on child and adolescent psychiatry -a german and personal perspective mental health services for older adults in china during the covid-19 outbreak. the lancet the psychological impact of the covid-19 epidemic on college students in china how essential is to focus on physician's health and burnout in coronavirus (covid-19) pandemic? cureus 12 dentistry and coronavirus (covid-19) -moral decision-making middle east respiratory syndrome coronavirus epidemic impact on healthcare workers' risk perceptions, work and personal lives isolation of a novel coronavirus from a man with pneumonia in saudi arabia covid-19 in the shadows of mers-cov in the kingdom of saudi arabia severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the psychological impact of covid-19 pandemic on health care workers in a mers-cov endemic country coronavirus disease 2019 pandemic in the kingdom of saudi arabia: mitigation measures and hospital preparedness knowledge and attitudes towards middle east respiratory syndrome-coronavirus (mers-cov) among health care workers in south-western saudi arabia command and control centre, ministry of health, saudi arabia knowledge and attitude of healthcare workers about middle east respiratory syndrome in multispecialty hospitals of qassim, saudi arabia attitudes, believes, determinants and organisational barriers behind the low seasonal influenza vaccination uptake in healthcare workers -a cross-sectional survey healthcare professionals' knowledge, attitude and acceptance of influenza vaccination in saudi arabia: a multicenter cross-sectional study influenza vaccine uptake, determinants, motivators, and barriers of the vaccine receipt among healthcare workers in a tertiary care hospital in saudi arabia association between 2019-ncov transmission and n95 respirator use world health organization (2020) coronavirus disease (covid-19) pandemic how will country-based mitigation measures influence the course of the covid-19 epidemic? flattening-the-curve associated with reduced covid-19 case fatality rates -an ecological analysis of 65 countries wuhan and hubei covid-19 mortality analysis reveals the critical role of timely supply of medical resources risk of covid-19 among front-line health-care workers and the general community: a prospective cohort study acknowledgement. the authors are thankful to all the healthcare workers who contributed to this research, especially those in the frontline during any infectious disease outbreak. data availability. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. key: cord-328000-i9tzr13z authors: cockrell, adam s.; leist, sarah r.; douglas, madeline g.; baric, ralph s. title: modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice date: 2018-07-24 journal: mamm genome doi: 10.1007/s00335-018-9760-9 sha: doc_id: 328000 cord_uid: i9tzr13z the emergence of highly pathogenic human coronaviruses (hcovs) in the last two decades has illuminated their potential to cause high morbidity and mortality in human populations and disrupt global economies. global pandemic concerns stem from their high mortality rates, capacity for human-to-human spread by respiratory transmission, and complete lack of approved therapeutic countermeasures. limiting disease may require the development of virus-directed and host-directed therapeutic strategies due to the acute etiology of hcov infections. therefore, understanding how hcov–host interactions cause pathogenic outcomes relies upon mammalian models that closely recapitulate the pathogenesis of hcovs in humans. pragmatism has largely been the driving force underpinning mice as highly effective mammalian models for elucidating hcov–host interactions that govern pathogenesis. notably, tractable mouse genetics combined with hcov reverse genetic systems has afforded the concomitant manipulation of virus and host genetics to evaluate virus–host interaction networks in disease. in addition to assessing etiologies of known hcovs, mouse models have clinically predictive value as tools to appraise potential disease phenotypes associated with pre-emergent covs. knowledge of cov pathogenic potential before it crosses the species barrier into the human population provides a highly desirable preclinical platform for addressing global pathogen preparedness, an overarching directive of the world health organization. although we recognize that results obtained in robust mouse models require evaluation in non-human primates, we focus this review on the current state of hcov mouse models, their use as tractable complex genetic organisms for untangling complex hcov–host interactions, and as pathogenesis models for preclinical evaluation of novel therapeutic interventions. the etiologies of coronaviruses (covs) comprise an extensive host range that includes reptiles, birds, pigs, dogs, cats, cattle, rodents, bats, camels, and humans. the extensive nonhuman host range may serve as reservoirs for pre-emergent coronaviruses poised for zoonotic transmission into human and animal populations (forni et al. 2017; zhou et al. 2018) . there are six known human coronaviruses (oc43, 229e, nl-63, hku1, sars-cov, and mers-cov), four of which hku1, were identified in the last 15 years. considering that bats may be the pinnacle reservoir for the origin of most hcovs (sars-cov, mers-cov, 229e, and nl63), evidence for sars-cov and mers-cov indicates that intermediate zoonotic hosts (civet cat and camel, respectively) may be as important for the evolution of a pathogenic cov to emerge into the human population (forni et al. 2017) . the emergence of novel pathogenic covs into the human population can result in highly lethal respiratory hcovs with global pandemic potential through human-to-human spread, which is underscored by the global spread of sars-cov (~ 10% mortality) in 2002-2003 and the ongoing re-introduction of mers-cov (~ 35% mortality) into humans on the arabian peninsula. human-to-human transmission of mers-cov was most apparent in the south korean outbreak in 2015, established by a single individual returning from a visit to the arabian peninsula, and resulting in 186 individuals infected with an ~ 20% mortality rate (lee 2015) . the high mortality rate in south korea also established a society-wide panic that led to a severe economic toll, anticipated to cut 1 3 0.1% from the gdp rate in 2015 (lee 2015) . once an hcov emerges in humans new tools and robust mouse models are needed to decipher pathogenic mechanisms. ultimately, understanding the hcov-host molecular interaction networks that regulate pathogenesis can guide the engineering of therapeutic countermeasures. an ideal strategy for development of models that recapitulate human respiratory covs clearly includes the development of a genetically tractable mammalian model that effectively recapitulates human pathogenic phenotypes, and an efficient reverse genetics system for seamless manipulation of the hcov genome. genetic control of both host and hcov genomes facilitates the ability to draw causeand-effect relationships between virus/host genetics and pathogenic outcomes. reverse systems genetic tools were developed to manipulate the large hcov genomes (~ 30 kb) that encode 16 non-structural proteins; structural proteins including spike (s), envelope (e), membrane (m), and nucleocapsid (n); and a number of accessory proteins that vary in sequence and function ( fig. 1) [reviewed in (cockrell et al. 2017) ]. a number of in vitro experiments (biochemical and tissue culture based) demonstrated that hcov proteins interact with host cell proteins to ensure fidelity during virus replication or facilitate evasion of host cell immune responses. the most prominent hcov-host cell interaction is between the major determinant of viral tropism, the spike protein, and a host cell-specific receptor. the spike protein forms a trimer on the surface of the virus with each monomer harboring a receptor binding domain (rbd) that interacts with its cognate receptor on the host cell (ace2 for sars-cov and dpp4 for mers-cov) (fig. 2) . the spike protein, especially the rbd, is a major target for development of antibody and vaccine therapeutics. other highly conserved structural and non-structural proteins, chiefly enzymatic proteins, may serve as targets for broadly effective anti-hcov therapeutic targets. the accessory proteins are not conserved between hcovs, and numerous studies revealed that these gene sets encode critical determinants of species-specific pathogenesis by modulating host immune responses (fig. 1) . in vitro studies have shown a number of accessory proteins to be important for evasion of host innate immune responses through interactions with host proteins [reviewed in (de wit et al. 2016; totura and baric 2012) ]. although in vitro studies are imperative for understanding molecular and biochemical mechanisms, only in vivo studies can decipher how complex hcov-host interactions relate to pathogenic phenotypes following a respiratory infection. discerning the fine balance between pathogenesis and protection, that is governed by the relationship between host innate and adaptive immune responses, requires the development of mammalian models that effectively recapitulate the pathogenesis of respiratory hcovs. the challenges associated with modeling pathogenesis of emergent and pre-emergent hcovs are borne out of models developed for sars-cov and mers-cov over the last 15 years (table 1 ; fig. 2 ). mice, ferrets, and non-human primates (nhps) are traditionally used for the studies involving respiratory pathogens and are among the species initially investigated for sars-cov and mers-cov replication and pathogenesis in the lungs. taking into account a number of practical considerations when comparing models for both sars-cov and mers-cov, mouse models appear to be the most pragmatic (table 1) . although we emphasize the practicality of hcov mouse models for the purposes of this review and the absolute critical need for robust primate models for downstream drug and vaccine testing, it is important to note that research (e.g., efficacy of therapeutic countermeasures) established in a robust mouse model should effectively translate to large animal models, such as nhps, that more closely reflect human physiology. in general, mouse models for both sars-cov and mers-cov are affordable, readily available, require fig. 1 emergent and pre-emergent coronavirus genome organization. the orf1a and orf1b genes (green) encode 16 non-structural proteins (nsp1-nsp16) that are highly conserved throughout coronaviruses. the structural genes (red) encode the structural proteins spike (s), envelope (e), membrane (m), and nucleocapsid (n), which are common to all coronaviruses. the accessory genes (dark shade) are unique to different coronaviruses with regard to number, genomic organization, sequence, and function nominal training for handling, amenable to manipulation under bsl3 conditions, amenable to analysis with commercially available molecular reagents, amenable to large numbers for purposes of experimental reproducibility, and are genetically tractable (table 1) . despite these commonalities, establishing a mouse model for mers-cov presented challenges not encountered with sars-cov (table 1) . even now, 6 years after the first case of mers-cov, one of the biggest challenges remains a very limited understanding of the pathology of mers-cov in humans. only two studies have described the pathology of mers-cov in two fatal human cases (alsaad et al. 2017; ng et al. 2016 ). the two fatal human mers-cov cases were similar to those observed in cases of sars-cov, wherein patients exhibited severe acute respiratory distress with diffuse alveolar damage and formation of hyaline membranes, alveolar fibrin deposits, edema, hemorrhaging, cellular debris, alveolar and interstitial inflammation throughout lungs, and extensive pulmonary tissue necrosis (alsaad et al. 2017; ng et al. 2016) . fatal cases of sars-cov and mers-cov were commonly associated with a gradual loss of respiratory function with available intervention restricted to mechanical respiratory support and oxygen supplementation [reviewed in ]. therefore, effective mouse models for sars-cov and mers-cov should minimally be able to recapitulate fatal respiratory disease having pathology similar to that observed in humans. as described below, a number of mouse models exhibiting fatal respiratory disease were developed for sars-cov and mers-cov; however, a single impediment was realized early in model development for mers-cov that was not confronted for sars-cov. the mouse orthologue of the human receptor for mers-cov, dipeptidyl peptidase 4 (dpp4), did not support interaction with the mers-cov spike glycoprotein rbd (cockrell et al. 2014) . therefore, unlike sars-cov, commercially available mice were not susceptible to mers-cov (towler et al. 2004) ]. in order to establish lethal mouse models, the spike protein, and other genomic determinants, have been modified through adaptive evolution in mouse lungs (day et al. 2009; frieman et al. 2012; roberts et al. 2007a ). thereby, any mouse subspecies that exhibits an unaltered mouse ace2 can be infected with the mouse-adapted sars-cov. wild-type mers-cov column (green). mers-cov infects humans via an interaction of the spike protein [rcsb pdb id: 5x5f (yuan et al. 2017) ] with its cognate receptor, dpp4 [rcsb pdb id: 2onc (feng et al. 2007) ]. the mers-cov spike protein is not able to interact with the mouse orthologue of human dpp4 (cockrell et al. 2014; coleman et al. 2014) . therefore, the mouse dpp4 gene had to be genetically modified in order to allow for infection with mers-cov (cockrell et al. 2016; li et al. 2017; pascal et al. 2015) . pre-emergent covs column (grey). pre-emergent covs can use either known human receptors for covs (ace2 or dpp4) (ge et al. 2013; menachery et al. 2015; wang et al. 2014; yang et al. 2016 yang et al. , 2014 , or novel, unknown receptors to infect their host. the genetically highly diverse panel of mouse strains from the collaborative cross has the potential to provide mouse models for studies of newly emerging covs due to the high genetic variability present in this resource. for all virus images: yellow represents the envelope protein; light/dark blue represents the membrane protein; and red represents the nucleocapsid protein infection and replication (coleman et al. 2014 ). susceptibility and mers-cov pathogenesis was achieved using unique approaches to genetically engineer mice, as described in detail below. establishing mouse models of fatal disease for sars-cov and mers-cov has afforded the necessary knowledge to institute a preclinical in vivo platform that can be used to evaluate pre-emergent hcov respiratory pathogens with unknown disease outcomes (fig. 2) . the strategy is widely portable to other important human pathogens that replicate poorly or not at all in the mouse. the need for sars-cov mouse models was recognized shortly after the sars-cov outbreak that spread globally in 2002-2003. three different strategies were employed for development of sars-cov mouse models: (i) different mouse species (or subspecies) were challenged with wildtype human sars-cov isolates in order to find a model that allows for replication and reflects severe respiratory disease symptoms observed in infected human patients; (ii) mice were genetically engineered to modify the host receptor, which facilitated productive sars-cov replication and pathogenesis; and (iii) adaptive evolution of wild-type sars-cov to a chosen mouse species was done to enhance pathogenesis, and associated clinical phenotypes in vivo. initial studies to develop a mouse model for sars-cov evaluated the susceptibility of various mouse subspecies to clinical isolates of sars-cov. in early 2004, the first study using the mouse as a model organism for sars-cov was reported ). 4-6 weeks old female balb/c mice were intra-nasally infected with a clinical isolate of sars-cov (urbani) and efficient viral replication was observed from the upper and lower respiratory tract with peak titers on day 3 and clearance by day 7, whereas viral replication was not detected in other organs. however, mice continued to gain weight and did not show other signs of clinical disease. passive transfer of immune sera to naïve mice was sufficient to prevent viral replication in the respiratory tract, indicating that wild-type sars-cov could elicit an effective humoral immune response. nonetheless, in the absence of pathogenesis with no clinical signs of disease, it is difficult to properly evaluate the efficacy of therapeutic countermeasures. in a second study, glass et al. used c57bl/6 mice which were known to exhibit a th1-biased immune response in contrast to balb/c mice table 1 practical considerations for establishing an effective hcov animal model a yes-required virus adaptation b yes-after genetically engineering a mouse c affordability-more cost effective relative to ferret and nhp models d availability-developed models that can be readily acquired and studied by the broader scientific community e ease of handling-mice require least amount of specialized training compared to ferrets and nhps f amenable to absl3 conditions-mice are most amenable to absl3 conditions due to space limitations, specialized handling requirements, and personnel limitations g maybe-dependent on species and techniques h n/d-not determined that were considered to be th2-biased (glass et al. 2004) , which may account for differences in pathogenic outcomes. like the balb/c study, 5-6 weeks old c57bl/6 mice were infected with the clinical sars-cov urbani isolate. infected c57bl/6 mice exhibited slower weight gain than mock-infected mice and sars-cov was found not only in the upper and lower respiratory tract but also in the brain, which was not associated with clinical disease in humans. developing a sars-cov model in c57bl/6 mice had the added benefit of expanding the applicability of the model to genetically modified knock-out (ko) mice that are predominantly generated on a c57bl/6 background, thereby facilitating the investigation of host genetics on sars-cov pathogenesis (glass et al. 2004) . utilizing cd-1 −/− and rag −/− knock-out mice, glass et al. demonstrated that nk cells, nk-t cells, or t and b lymphocytes are not required for clearance of sars-cov in the mouse (glass et al. 2004 ). these initial studies to evaluate different murine subspecies as models for sars-cov resulted in replication in the respiratory tract with no indication of the clinical pathogenesis often observed in human cases experiencing severe respiratory distress. knock-out mice deficient in the innate immune response were the first to exhibit clinical alterations that included weight loss and progressively worsening pulmonary disease (hogan et al. 2004 ). 129svev and stat1 −/− mice on a 129svev background infected with a sars-cov clinical isolate (toronto-2) showed that stat1 was required for the resolution of sars-cov infection. clinical signs of disease were only observed in ko mice, whereas efficient viral replication in the lower respiratory tract caused no clinical symptoms in 129svev wild-type mice. in contrast, stat1 −/− mice supported viral replication with peak titers on day 3 and persistence throughout day 22 (hogan et al. 2004 ). most features of pathology that were found in stat1 −/− mice were different from those found in humans. however, some pathological features common to humans, such as bronchiolar injury with focal respiratory epithelial cell necrosis, supported the idea that the mouse species could be a good model for sars-cov-associated clinical findings observed in the respiratory tract of infected human patients. increased fatality as consequence of acute respiratory distress syndrome (ards) from sars-cov infection was often associated with advanced age (> 60 years old) in humans (booth et al. 2003; donnelly et al. 2003; tsui et al. 2003) , which was exploited to improve mouse models of sars-cov pathogenesis. to replicate advanced age in humans, 12-14 months old balb/c (roberts et al. 2005) , c57bl/6, and 129s6 (roberts et al. 2008 ) mice were intra-nasally infected with a clinical isolate of sars-cov (urbani). balb/c and c57bl/6 mice showed comparable levels and kinetics of viral replication while aged 129s6 mice cleared sars-cov by day 5 (roberts et al. 2008 ). the predominant clinical feature regarding mouse studies is weight loss, a consequence of appetite loss and dehydration. all three lines of aged mice showed transient signs of clinical disease including weight loss, ruffled fur, hunching, and dehydration that resolved by day 7. moreover, all three strains exhibited histopathological findings such as perivascular and peribronchiolar infiltrates, necrotic debris in bronchioles, and interstitial pneumonitis. importantly, extensive alveolar damage in aged balb/c mice persisted through day 9 post-infection (p.i.), findings that closely resembled pathology observed in humans. these are some of the first controlled studies to demonstrate that differences in host genetics can significantly influence sars-cov pathogenesis, a topic that is revisited below with regard to using a tractable population of genetically distinct mice referred to as the collaborative cross. concurrent with these studies, rockx et al. demonstrated that pathogenesis related to sars-cov clinical isolates was also dependent upon the genetics of the sars-cov strain evaluated in vivo (rockx et al. 2007 ). using reverse genetics rockx et al. modified the spike protein from the sars-cov urbani strain to encode the spike from middle (cuhk-w1) and early (gz02) phase clinical isolates or a civet strain spike gene from hc/ sz/61/03 (rockx et al. 2007 ). an infectious clone harboring the clinical isolate gz02 or civet hc/sz/61/03 spikes exhibited severe weight loss and death, as well as pathology consistent with acute respiratory distress syndrome in aged, but not young mice (rockx et al. 2007 ). despite these early achievements with aged mice for sars-cov mouse model development, it is impractical to maintain large colonies of aged mice; and immune senescence in aged mice may complicate studies involving pathogenesis and effective evaluation of immune responses to therapeutic countermeasures. in addition to these limitations, full-length clinical isolates of sars-cov were not shown to be lethal in aged mice, a clinical outcome that impacted nearly 10% of all human cases. lethality was not achievable using clinical sars-cov isolates in various young wild-type or immune-incompetent mouse strains. with this in mind, attention turned to genetic modification of mice to acquire a lethal model for sars-cov pathogenesis. in order to continue working with unaltered human clinical isolates of sars-cov, mouse strains constitutively expressing the human receptor for sars-cov, human angiotensin converting enzyme 2 (hace2), were generated. the logic followed that human clinical isolates of sars-cov are evolved to use the hace2 receptor more effectively than the innate mouse ace2 (mace2) receptor; therefore, may elicit pathology consistent with a lethal respiratory infection. different constitutive promotors were used to express hace2 in mice: cytokeratin promotor (mccray et al. 2007; netland et al. 2008) , chicken beta-actin promotor with a cytomegalovirus ie enhancer (tseng et al. 2007) , and the mouse ace2 promotor (yang et al. 2007 ). expression levels of human ace2 correlated with disease severity in all transgenic mouse models. human ace2 overexpression mice generated with the cytokeratin promoter and the mace2 promoter were attempts to limit expression to respiratory cells and cells that exhibited innate mace2 receptor expression, respectively (mccray et al. 2007; netland et al. 2008; yang et al. 2007 ). even though these transgenic mice showed infection of airway epithelium, all of them exhibited high levels of hace2 expression in the brains of transgenic mice which supported increased viral load in brain tissue (mccray et al. 2007; netland et al. 2008; tseng et al. 2007; yang et al. 2007 ). the increased viral loads in the brains of infected animals ultimately led to mortality caused by extensive dissemination and encephalitis in the brain (mccray et al. 2007; netland et al. 2008; tseng et al. 2007; yang et al. 2007) . accordingly, despite the successful generation of lethal hace2 overexpression mouse models for sars-cov pathogenesis, neurological-related mortality confounded their value as models that could effectively mimic lethal respiratory disease often observed in infected humans. adaptive experimental evolution has been a staple of virology for > 60 years (kalter 1949) , providing critical insights into pathogenic mechanisms while bestowing robust, lethal mouse models to interrogate the efficacy of vaccines and therapeutics (bolles et al. 2011; cockrell et al. 2016; rasmussen et al. 2014; roberts et al. 2007a ). adaptive experimental evolution is performed by serial in vivo passages in the tissue of interest to adapt the virus to the host innate and adaptive immune responses. this is similar to the ongoing battle between a virus and the host immune responses occurring in nature, but on an expedited time-scale and in a tissue-specific manner. tissue-specific adaptive evolution places the virus under selective pressure for mutations that allow for more efficient replication. to adapt sars-cov to cause severe acute respiratory disease in mouse lungs, 6-week-old female balb/c mice were intra-nasally infected with the clinical urbani isolate (roberts et al. 2007a ). this process was repeated (15 rounds) until signs of clinical disease were observed, predominantly weight loss that reached clinical endpoints for humane euthanasia (considered humane lethality) (roberts et al. 2007a ). the resulting mouse-adapted sars-cov was designated ma15. rapid weight loss was accompanied by high viral titer in the lungs, viremia and dissemination to extrapulmonary sites, lymphopenia, neutrophilia, and histopathological changes in the lung commonly associated with pneumonitis. accordingly, mortality was a consequence of extensive viral replication which led to virus-associated destruction of pneumocytes and epithelial cells. only six coding mutations were sufficient to render the sars-cov urbani human clinical isolate 100% lethal in 4 weeks old, 6-8 weeks old, and aged balb/c mice (roberts et al. 2007a ). the ma15 sars-cov has since been used in a multitude of studies and proven to not only cause lethal disease in balb/c mice but also in other mouse strains (deng et al. 2014; frieman et al. 2010; gralinski et al. 2015 gralinski et al. , 2017 totura et al. 2015; zhao et al. 2012) . additional passaging of ma15 resulted in the generation of other mouse-adapted sars-cov strains including ma20 (20 passages) (frieman et al. 2012) , while an independent research group acquired a lethal sars-cov model by passaging the human urbani isolate 25 rounds through the lungs of balb/c mice, resulting in v2163 (day et al. 2009 ). in addition to acquiring lethal sars-cov models of disease, comparing various passages of mouse-adapted sars-cov provides the unique advantage of being able to identify which sars-cov proteins acquire specific mutations that can elicit severe respiratory phenotypes. identification of adaptive mutations acquired in specific sars-cov proteins provided insight into virus-host interaction networks that may be used for the development of virusdirected therapeutic countermeasures. combining mouseadapted sars-cov strains with mouse models that support interrogation of the host genome for molecules that influence sars-cov pathogenesis can reveal import interaction nodes amenable to host-directed therapeutic intervention. as mentioned above, a number of early sars-cov mouse model studies showed that disease progression and outcome are mouse strain dependent, indicating that host genetics have a considerable influence on sars-cov pathogenesis. this is not surprising since human gene association studies have indicated that differences in an individual's genetics may govern susceptibility and clinical outcomes of respiratory viral pathogenesis (everitt et al. 2012; forton et al. 2009; kenney et al. 2017; mills et al. 2014; pasanen et al. 2017; patarcic et al. 2015; zhou et al. 2012 ), including retrospective studies of sars-cov-infected patients (chan et al. , 2006 ching et al. 2010; wang et al. 2008; zhu et al. 2011 ). however, human genetic associations do not indicate cause-and-effect, which require the capacity to model the impact of host genetics on respiratory viral pathogenesis, in this case sars-cov pathogenesis. a recently developed, innovative resource for genetic mapping, called the collaborative cross (cc), comprises a panel of recombinant inbred mouse strains containing tractable genetic diversity that approaches the genetic diversity in the human population (churchill et al. 2004; threadgill et al. 2002) . using an octo-parental breeding scheme that includes classical laboratory stains (a/j, c57bl/6j, and 129/svimj), mouse models for human diseases (nod/shiltj for diabetes; nzo/hlltj for obesity), and wild-derived mouse strains (cast/eij, pwk/phj, and wsb/eij), the cc captures 90% of the genetic variation present in the three major mouse subspecies (mus musculus musculus, mus musculus domesticus, mus musculus castaneus) (roberts et al. 2007b) . virus infection studies in cc mouse lines, including sars-cov, have led to mapping of high and low host response alleles as they relate to development of clinical signs of disease following viral pathogenesis (bottomly et al. 2012; brinkmeyer-langford et al. 2017; ferris et al. 2013; gralinski et al. 2015 gralinski et al. , 2017 green et al. 2017; rasmussen et al. 2014) . rapid genetic mapping is most successful through comparative analysis of cc strains that show extreme phenotypes such as high versus low weight loss, or high versus low lung titers. extreme phenotypes in select cc strains may reflect clinical outcomes observed in human disease, as observed in testing mouse-adapted ebola virus in different cc strains . applying the cc technology to sars-cov pathogenesis identified two cc strains (cc003/ unc and cc053/unc) with opposing susceptibility profiles . genetic mapping revealed an adaptor protein (ticam2) in the toll-like receptor pathway as a strong candidate driving severe respiratory disease phenotypes . based on these observations, the cc mouse platform can be used to identify novel mouse models that recapitulate human clinical outcomes resulting from pathogenic viruses. the cc mouse strains are an extraordinarily powerful platform to establish novel mouse models for emergent and pre-emergent isolates of respiratory coronaviruses. harnessing the power of cc host genetic mapping will support the identification of novel hcov-host molecular interaction networks that can be subsequently validated in genetically engineered mice. innovations in gene editing technologies such as crispr/cas9, talens, and synthetic zinc fingers have augmented the efficiency of genetic engineering in mice, making genomic modifications (i.e., allele-specific mutations, allele swaps, knock-outs, knock-ins) feasible in multiple mouse species, on a large-scale (doench 2018; kim and kim 2014) . coincidentally, advances in the crispr/ cas9 technology overlapped with the outbreak of mers-cov in 2012, which was fortuitous for the development of lethal mers-cov mouse models. building on expertise from the sars-cov outbreak, coronavirus researchers immediately recognized the overwhelming need for an effective mers-cov mouse model following the emergence of mers-cov in 2012. however, researchers were perplexed to find that mouse lines conventionally susceptible to sars-cov infection/replication were completely resistant to infection with clinical mers-cov isolates (coleman et al. 2014) . initial studies demonstrated that non-human primates were susceptible to clinical isolates of mers-cov (de wit et al. 2013b; falzarano et al. 2014; johnson et al. 2016 johnson et al. , 2015 munster et al. 2013) ; therefore, the inability to infect mice with mers-cov was likely not due to restriction by the host immune responses. this was validated in immune-incompetent mouse lines that also lacked the capacity for infection/replication of mers-cov (coleman et al. 2014) . concurrent with some of the initial mers-cov studies in mice, raj et al. identified a novel receptor for mers-cov, human dipeptidyl peptidase 4 (hdpp4) (raj et al. 2013) . unlike sars-cov which could infect mice through interaction of the rbd in the spike protein with the mace2 receptor, mouse dpp4 (mdpp4) did not support an interaction with the mers-cov spike protein (cockrell et al. 2014) . crystal structures of the mers-cov spike-hdpp4 interface revealed a number of specific amino acids necessary for an interaction between the rbd in spike and two specific domains, referred to as blades iv and v on the β-propeller structure, of the hdpp4 structure (lu et al. 2013; wang et al. 2013) . ectopic expression studies with the mouse dpp4 receptor revealed that altering a minimum of two amino acids on the mdpp4 (positions a288 and t330) conferred susceptibility of the human fibroblast cell line, 293t, to mers-cov infection (cockrell et al. 2014; peck et al. 2015) . importantly, in mice t330 is an n-linked glycosylation site that may sterically hinder mers-cov infection, and is not present in dpp4 orthologues from susceptible species including human, nhps, bats, and camels (peck et al. 2015) . apparently, ferret and hamster dpp4 may also have similar glycosylation's, which may explain the inability of mers-cov to also infect/replicate in these conventional small animal models (de wit et al. 2013a; peck et al. 2017; raj et al. 2014; van doremalen et al. 2014 ). species-specific challenges utilizing the dpp4 receptor not only limited development of mouse models, but stymied development of all small animal models required for evaluating therapeutic countermeasures. accordingly, the initial development of mouse models for mers-cov required employing a number of methods that focused on engineering mice to express/overexpress the human dpp4 receptor (agrawal et al. 2015; li et al. 2016; zhao et al. 2015 zhao et al. , 2014 (fig. 3) . the first mouse model, developed by zhao et al., established transient expression of human dpp4 in the airway of balb/c mice, c57bl/6 mice, and several knock-out mice using an adenoviral vector to transduce an hdpp4 overexpression cassette (ad-hdpp4) to the lungs of mice (zhao et al. 2014) . transient hdpp4 expression rendered mice susceptible to clinical isolates of mers-cov infection [1 × 10 5 plaque forming units (pfu) dose], supporting viral replication in the lungs and producing transient weight loss with mild pneumonia, particularly in older and immunodeficient mice (zhao et al. 2014) . no mortality or signs of advanced clinical respiratory disease were observed (zhao et al. 2014) . as a platform strategy that could be applied to novel emerging viruses, the ad-hdpp4 model provided a means to rapidly evaluate mers-cov-directed therapeutic countermeasures with regard to interfering with replication in the lungs of infected mice. nevertheless, the ad-hdpp4 model was not an effective means of understanding how therapeutic countermeasures could prevent development of severe respiratory disease that resulted in ~ 35% mortality in infected human cases. to develop more clinically relevant mouse models, a number of groups turned to more conventional hdpp4 knock-in approaches, resulting in ubiquitous, constitutive overexpression of hdpp4 throughout the mouse (fig. 3) . in 2015, agrawal et al. reported development of a robust mers-cov mouse model by placing expression of hdpp4 under the cag promoter (agrawal et al. 2015) . infection with clinical isolates of mers-cov resulted in high viral loads in the lungs that produced severe respiratory disease and was fatal by day 6 post-infection (agrawal et al. 2015) . however, disease was confounded by high viral loads in extrapulmonary tissues including the brain, heart, spleen, kidney, and intestines (agrawal et al. 2015) . in an independent study, zhao et al. demonstrated multi-organ damage in an hdpp4 overexpression mouse model employing the same promoter (zhao et al. 2015) . the mers-cov viral loads were nearly two logs higher in the brains of infected mice than in the lungs and corresponded to viral encephalitis observed in the brain by day 9, with mice exhibiting signs of paralysis (zhao et al. 2015) . li et al. went a step further by generating hdpp4 overexpression models with the goal of restricting expression to epithelial cells . two different models were generated using either the cytokeratin 18 (k18) or surfactant c protein (spc) promoter to drive expression of hdpp4. mouse lines derived from the spc-hdpp4 cassette did not exhibit mortality or signs of respiratory disease. in contrast, infection of k18-hdpp4 mice with 1 × 10 5 pfu of a mers-cov clinical isolate (emc_2012) produced weight loss, lung hemorrhaging and mortality by 6-7 days p.i., and lung pathology consistent with severe respiratory disease . unfortunately, the k18 promoter did not limit mers-cov infection to the lungs, instead high viral loads in the brains of infected mice caused neurological disease that was consistent with the observed mortality . in a novel genetic engineering approach, pascal et al. employed regeneron's velocigene technology to precisely replace the entire mdpp4 coding region (~ 79 kb) with the hdpp4 gene, which included ~ 82 kb of sequence encoding both introns and exons (pascal et al. 2015) (fig. 3) . despite removal of nearly the entire mdpp4 gene, the 5′ and 3′ untranslated regions of mdpp4 were maintained; thereby, retaining the endogenous mdpp4 promoter and rna termination regions conferred expression levels and tissue distribution for hdpp4 similar to that obtained for mdpp4. infection with 2 × 10 5 pfu of a mers-cov clinical isolate resulted in viral replication in the lungs with mild clinical disease and pathology indicative of inflammation, with no weight loss or mortality through day 4 p.i. (pascal et al. 2015) . subsequent studies with the velocigene mice comprised a more detailed evaluation of the model, wherein mice infected with 2.5 × 10 4 pfu achieved dramatic weight loss and mortality (using 20% cut-off) by 7 days p.i. . c dpp4 sequence used in velocigene hdpp4 knock-in mouse model (pascal et al. 2015) . mouse genomic sequence, from exon 2 through the stop codon in exon 26, was deleted and replaced with exon 2 through exon 26 and a portion of 3′ untranslated sequence of human genomic sequence. d dpp4 sequence used in hdpp4 knock-in model (li et al. 2017) . mouse genomic sequence from codon i264 in exon 10 to codon v340 in exon 12 was replaced with the human equivalent. e dpp4 sequence used in 288-330 +/+ mouse model (cockrell et al. 2016) . crispr/cas9 technology used to make a288l substitution in exon 10 and t330r substitution in exon 11 of mouse dpp4. f dpp4 construct used in hdpp4 overexpression mouse models. constructs included cdna from human dpp4 flanked by constitutive promoter, polyadenylation signals, and other regulatory elements (agrawal et al. 2015) ; or by 5′ and 3′ genomic regions of human cytokeratin 18 or human surfactant protein c ) (coleman et al. 2017) . lung pathology indicated that mice developed moderate signs of respiratory infection with little indication of severe respiratory disease. these studies were also the first indication that cd8 + t cells and macrophages play a significant role in mers-cov pathogenesis (coleman et al. 2017) . importantly, mers-cov infection/ replication was primarily in the lungs, with little involvement of extrapulmonary tissues (coleman et al. 2017; pascal et al. 2015) . the lack of brain lesions in this model was a significant advancement over transgenic models that relied on global/ubiquitous hdpp4 expression. nonetheless, this model is not readily available to the scientific community by regeneron, and if the model is obtained it comes with a number of commercial restrictions from regeneron that limit its utility when considering development of therapeutic countermeasures (declaration by the authors of this manuscript). another limitation often overlooked with hdpp4 expression models is the innate functions of dpp4 that have evolved in a species-specific manner to maintain various physiological processes inherent to the host. altered dpp4 activity and/or expression is associated with many pathological conditions that include psycho-neuroendocrine disorders, solid tumor cancers, hematological malignancies, infectious disease, and autoimmune/inflammatory diseases (klemann et al. 2016) . the enormous biological breadth of dpp4 is predominantly through its enzymatic activity wherein it cleaves off the amino terminal dipeptides of various biological substrates having l-alanine or l-proline at the penultimate position (klemann et al. 2016) . one of the more important physiological processes of dpp4, also known as cd26, is the modification of numerous cytokine and chemokines involved in the maintenance of immunological homeostasis (klemann et al. 2016; ohnuma et al. 2008) . as a cell surface molecule on cd4 + and cd8 + t cells, dpp4 influences numerous immunological processes including t cell activation and proliferation, cytokine production, differentiation to immunoglobulin producing plasma cells, and transendothelial migration (ohnuma et al. 2008) . therefore, overexpressing full-length hdpp4 or replacing mdpp4 with full-length hdpp4 in the knock-in mouse models may significantly alter the inherent biological and immunological processes that mdpp4 has evolved to execute in a species-specific manner, in the mouse. ultimately, if hdpp4 expression results in a modified immune response in mice, these could artificially influence the pathogenic outcomes of mers-cov infections and immunological responses that are central to evaluating the efficacy of therapeutic countermeasures. the goal of the most recent generation of mouse models was to genetically modify the mdpp4 receptor so that it would be amenable to interaction with the mers-cov spike rbd and subsequent infection, thereby avoiding the need to introduce the hdpp4 receptor. achieving a mers-cov susceptible mdpp4 with minimal modifications would be central to limiting any functional alteration to mdpp4 that could interfere with its broad influence over multiple host physiological processes. in 2016, cockrell et al. utilized the crispr/cas9 technology to make two amino acid substitutions in the mdpp4 gene (a288l and t330r) of c57bl/6j mice (cockrell et al. 2016) (fig. 3) . as discussed above, in vitro overexpression of the modified mdpp4 was previously found to alter the susceptibility of cell lines to mers-cov infection (cockrell et al. 2014; peck et al. 2015) . mice with the modified dpp4 (referred to as 288-330 +/+ mice) encode both amino acid substitutions on both chromosomes. characterization of this model demonstrated that innate mdpp4 expression profiles and physiological processes influenced by dpp4 (glucose metabolism and t cell activation) were not altered by the two amino acid modifications of mdpp4. the 288-330 +/+ mice infected with various isolates of mers-cov (clinical isolates, a camel isolate, and molecular clones) supported high viral replication in the lungs, but failed to exhibit clinical signs of disease (cockrell et al. 2016) . adaptive evolution through 15 rounds of serial passage in 288-330 +/− mice yielded a mouse-adapted mers-cov capable of achieving lethality, decreased respiratory function, and lung pathology indicative of severe acute respiratory distress syndrome at viral doses of 5 × 10 6 pfu (cockrell et al. 2016) . mers-cov infected 288-330 +/+ mice that exhibited severe respiratory disease were absent of any detectable virus in the brain. the development of severe respiratory disease and mortality could be prevented using a mers-cov spike-directed vaccine, and through prophylactic administration of an antibody therapeutic (cockrell et al. 2016) . although the 288-330 +/+ model is highly effective for studying mers-cov pathogenesis and evaluating therapeutic countermeasures, the high infectious dose (5 × 10 6 pfu) required to achieve severe respiratory disease and mortality left room for improvement. an additional 20 rounds of passaging resulted in a mouse-adapted virus capable of achieving similar severe respiratory disease and mortality, but at substantially lower doses (10 3 -10 5 pfu) (douglas et al. 2018) . overall, limiting changes in the mdpp4 receptor to two amino acids, researchers were able to minimize disruption of the natural expression levels, distribution, and biological functions of mdpp4. the 288-330 +/+ mers-cov mouse model continues to have a central role in studies investigating mers-cov pathogenesis and therapeutic countermeasures (menachery et al. 2017b, c) . the most recent mouse model consists of a genetically modified mdpp4 receptor through replacement of exons 10-12 (including introns) of the mdpp4 locus with the hdpp4 equivalent, resulting in a minimal knock-in mouse model referred to as hdpp4-ki (li et al. 2017) (fig. 3) . modification of mdpp4 in the hdpp4-ki model is much smaller than the regeneron full-length hdpp4 knock-in model, but larger than the two amino acid modifications to mdpp4 in the 288-330 +/+ model, placing it somewhere between the two types of models. regarding biological and immunological function, it is not clear if the modified mdpp4 in the hdpp4-ki model has altered expression, biodistribution, or functional profiles compared to wild-type mdpp4 receptor. nevertheless, the hdpp4-ki mice were susceptible to infection with clinical isolates of mers-cov, resulting in high levels of viral replication in the lungs but no signs of respiratory disease. a mouse-adapted mers-cov was achieved through 31 serial passages of mers-cov, inducing severe respiratory disease accompanied by high mortality at infectious doses of 10 4 -10 5 pfu (li et al. 2017) . similar to the 288-330 +/+ model, the hdpp4-ki model exhibited little involvement of extrapulmonary tissues in mers-cov pathogenesis (li et al. 2017 ). the mouse-adapted viruses acquired by li et al. revealed unique mutations in specific viral proteins that may be critical for achieving and evading host immune responses. comparison of these viruses to those obtained by douglas et al. demonstrated that changes at amino acid 222 in the mers-cov spike protein may be critical to achieve lethal infection at low infectious doses (douglas et al. 2018; li et al. 2017 ). importantly, this mutation was not present in earlier passages by cockrell et al. in the 288-330 +/+ model, which required high infectious doses to achieve severe respiratory disease and lethality (cockrell et al. 2016) . mouse-adapted viruses, such as those used by cockrell et al., douglas et al. and li et al. can help provide insight into adaptive evolution and identify specific mutations or regions of the virus that are important for enhancing virus fidelity in the host and/or evading the host immune responses. adaptations in mers-cov most notably occur in the spike region, which is an important determinant of host tropism. adapted mers-cov strains with mutations in this region may not provide an accurate representation of the observed viral pathogenesis in humans. currently, achieving lethal disease in mice with clinical isolates of mers-cov require expression of full-length hdpp4, as demonstrated for the regeneron hdpp4 knock-in mice. however, altering dpp4 significantly through overexpression or replacement of mdpp4 can disrupt the immunological homeostasis of the animal, making it difficult to analyze disease etiology and immune responses to therapeutic countermeasures. a mers-cov mouse model would ideally involve infection with clinical mers-cov isolates in an unmodified mouse. one possibility is utilizing cc mice, as described above for sars-cov, which offer a wider range of genetic variation than the genetically engineered c57bl/6 mice used in currently established mouse models. however, unlike sars-cov, the cc mice are not susceptible to mers-cov. crossing various cc lines with mice that have minimal alterations to mdpp4 (e.g., 288-330 +/+ mice), or direct genetic engineering of cc lines using crispr/cas9 technologies, can produce mouse lines that maintain native mouse dpp4 expression, distribution, and functional profiles, but can be screened for differential susceptibility to clinical isolates of mers-cov. the capacity to genetically engineer receptors to alter mouse susceptibility to hcov infection, combined with adaptive hcov evolution and genetically diverse cc mouse strains, establishes a platform that can be used to evaluate the pathogenesis of pre-emergent hcovs with unknown etiologies. the mouse tools founded from studies of sars-cov and mers-cov pathogenesis will aid in understanding if specific pre-emergent covs have pathogenic potential in mammalian models of severe respiratory disease. most hcovs (sars, mers, 229e, and nl63) are considered to have their origin in bats [reviewed in (forni et al. 2017; menachery et al. 2017a) ]. metagenomics analysis of various bat species has identified a diverse repertoire of sarslike (sl-bcovs) and mers-like (ml-bcovs) bat coronaviruses (ge et al. 2012 (ge et al. , 2013 he et al. 2014; lau et al. 2015 lau et al. , 2010 lau et al. , 2005 wu et al. 2016; yang et al. 2016) . those with pre-emergent potential include sl-covs wiv1 and wiv16, shown to utilize the ace2 receptor (ge et al. 2013; menachery et al. 2015; yang et al. 2016) , and an ml-cov hku4, demonstrated to utilize the dpp4 receptor for infection yang et al. 2014) . notably, wiv1 and wiv16 are the only pre-emergent covs directly isolated from bat feces on vero e6 cells and shown to replicate in various cell lines expressing the human, or nhp, ace2 receptor (ge et al. 2013; yang et al. 2016) . in contrast to wiv1 and wiv16, isolated hku4 was not demonstrated to infect cells, but rather lentiviral particles pseudotyped with hku4 spike protein supported efficient binding and infection of cells expressing hdpp4 yang et al. 2014) . the structure of cov spike proteins in the context of pseudotyped lentiviral particles may differ to that in cov particles, and may account for the difficulty of hku4 infecting and replicating on cells expressing hdpp4. the spike proteins of many pre-emergent bat covs (bcovs) have amino acid differences that preclude efficient interaction with known human host receptors, such as hace2 or mace2. transmission to intermediate hosts (civets or camels) and humans may require additional adaptations in the spike protein [reviewed in (graham and baric 2010; menachery et al. 2017a) ]. therefore, to evaluate pathogenesis of pre-emergent bcovs in mouse models, the spike proteins were replaced by generating infectious molecular clones with known spike proteins from clinical isolates of sars or mouse-adapted sars-cov becker et al. 2008; menachery et al. 2015 menachery et al. , 2016 . the first of these studies derived an infectious molecular clone sl-bcov from sequences of four previously identified sl-bcovs (hku3-1, hku3-2, hku3-3, and rp3) (becker et al. 2008 ). the chimeric virus was able to replicate in balb/c mouse lungs, but did not elicit signs of respiratory disease commonly associated with sars-cov (becker et al. 2008) . similarly, a chimeric wiv1 (wiv1-ma15) replicated in balb/c mouse lungs, but was attenuated with regard to causing respiratory disease (menachery et al. 2016) . attenuation of wiv1-ma15 was partially overcome by infecting hfh4 mice that overexpress the hace2 receptor (menachery et al. 2016) , indicating that mouse adaptation of the wiv1-ma15 to mace2 could enhance disease in wild-type balb/c mice. indeed, agnihothram et al. demonstrated that mouse adaptation of an infectious molecular clone of an hku5-se chimeric resulted in dramatic respiratory disease in aged balb/c mice . hku5 is closely related to mers-cov, but since there was no mouse model available for mers-cov at the time, it was more practical to replace the hku5 spike with the mouse-adapted sars-cov spike. these studies demonstrated that adaptive mutations outside of the spike are necessary to elicit respiratory disease in different mammalian species. however, this was not the case for the shc014 bcov chimeric virus (shc014-ma15), which caused significant weight loss in balb/c mice and replicated to titers comparable to sars-cov ma15 in the lungs . moreover, wild-type shc014 spike protein supported infection/replication in the lungs of balb/c mice, but did not result in disease. applying the classical approach of adapting shc014 to mice by serial passaging may be beneficial, but can be time-consuming. screening shc014, or other pre-emergent bcovs, in cc mouse strains has the potential to rapidly lead to mouse models capturing different, or even all, aspects of clinical disease in human patients. the ultimate goal for mouse model development is capturing disease etiologies that closely reflect what is observed during human cov infection, so the efficacy of various therapeutic countermeasures can be evaluated for effective resolution of pathogenic outcomes. a number of hcov-directed drug, antibody, and vaccine therapeutics were evaluated for efficacy against sars-cov until 2012 when the focus of therapeutic development shifted to mers-cov [reviewed in (dyall et al. 2017; zumla et al. 2016) ]. currently, there are no fda-approved therapeutics for the specific treatment of sars-cov or mers-cov. various combinations/types of broad spectrum antivirals (e.g., ribavirin) combined with interferon therapies (interferon α or β), and/ or immune modulators (e.g., corticosteroids) were ineffective for sars-cov and mers-cov, and in some cases appeared to worsen disease [reviewed in (dyall et al. 2017; zumla et al. 2016)] . a recent clinical trial seeks to interfere with mers-cov replication using a combination therapy that includes fda-approved protease inhibitors (lopinavir/ ritonavir) (https ://clini caltr ials.gov/ct2/show/study /nct02 84584 3). repurposing fda-approved drugs has been the interest of numerous therapeutic studies, but little is known regarding the efficacy of drug therapeutics against sars-cov or mers-cov outside of cell-based in vitro studies [reviewed in (dyall et al. 2017) ]. mouse models confer distinct advantages for therapeutic examination including cost savings through small scale production for testing, and experimental reproducibility. the recent development of a novel nucleotide prodrug, gs-5734, demonstrated efficacy against ebola virus in nhps (warren et al. 2016) , and has since moved into phase 2 clinical trials for ebola [https ://clini caltr ials.gov/ct2/show/ nct02 81858 2?cond=preva il+iv&rank=1; (dornemann et al. 2017)] . a recent study demonstrated broad spectrum efficacy of gs-5734 against multiple hcovs (sars-cov, mers-cov, and nl63) and pre-emergent bcovs (hku3, hku5, sch014, and wiv1) on primary human airway epithelial cells (sheahan et al. 2017) . importantly, gs-5734 exhibited in vivo efficacy against sars-cov-induced immunopathology if the drug was administered prophylactically, or a therapeutic dose 24-h post-infection (sheahan et al. 2017) . delaying treatment until 48-h post-infection did not ameliorate disease symptoms. although the kinetics of sars-cov pathogenesis will differ between mouse and human, these results indicate that the molecular programs leading to respiratory immunopathology are established shortly after infection, thereby limiting effective therapeutic treatment to a short window, post-infection. the study by sheahan et al. also demonstrates the importance of having mouse models that recapitulate severe respiratory disease often seen in humans (sheahan et al. 2017 ). an effective therapeutic should not only prevent viral replication, but more importantly should effectively restrict respiratory pathogenesis. achieving both may require therapeutic intervention with a combination of an hcov antiviral and a hostdirected therapy that curtails immunopathology associated with acute respiratory distress syndrome. only in the last 3 years have mers-cov mouse models, described above, become available to assess therapeutics; therefore, future studies will include evaluating in vivo efficacy of gs-5734 against mers-cov in the 288-330 +/+ model described above. as an ongoing threat to worldwide public health, a plethora of therapeutic research focused on development of mers-cov-specific antibodies and vaccines, both of which have been extensively reviewed [reviewed in dyall et al. 2017; okba et al. 2017)] . importantly, few studies have evaluated the therapeutic efficacy of mers-cov antibodies and vaccines in mammalian challenge models that elicit severe respiratory disease, often resulting in death (cockrell et al. 2016; munster et al. 2017; tai et al. 2016; van doremalen et al. 2017; wang et al. 2018 ). rather, many preclinical studies exhibited efficacy in the mouse or nhp challenge models with protection defined as reduced viral loads in the lungs by plaque assay or rt-pcr. preclinical results have expedited the process of moving some antibody and vaccine therapeutics into early clinical trials [reviewed in dyall et al. 2017; okba et al. 2017) ]. based on results obtained in studies with gs-5734, it will be critical to determine efficacy of antibody and vaccine therapeutics in lethal respiratory models of mers-cov infection. vaccine countermeasures are not only being considered as intervention strategies for staving off disease in humans, but have also exhibited efficacy in dromedary camels, the mers-cov zoonotic host (haagmans et al. 2016; muthumani et al. 2015) . targeting zoonotic hosts, such as dromedary camels or bat populations, may be an effective means of curtailing transmission into the human population. thinking beyond vaccine countermeasures, we now have the ability to harness genome editing technologies that could allow us to intervene with the process of zoonotic transmission. c57bl/6j mice were made susceptible to mers-cov by using crispr/cas9 technologies to modify two amino acids at positions 288 and 330 on mouse dpp4, to the orthologous amino acids encoded by human dpp4 (cockrell et al. 2016) . genetic modification of the human dpp4 orthologues in camel and bat to look like mouse dpp4 at these positions may result in camels and bats that are now resistant to mers-cov infection. although introduction of mers-cov-resistant bats into the environment may take a long time, camels are considered farm animals in many parts of northern africa and the middle east with controlled breeding practices; therefore, it may be feasible to establish resistant herds that are unable to transmit mers-cov to humans. genome editing of farm animals is clearly achievable as recently demonstrated in pigs modified with crispr/cas9 for genome-wide inactivation of porcine endogenous retroviruses (pervs), thereby eliminating the potential of cross-species transmission of pervs during xenotransplantation of pig organs into humans, with the intention of easing the shortage of available human organs (niu et al. 2017; yang et al. 2015) . the combined power of genetic engineering of mice, reverse genetic systems for covs, mouse-adaptation, and genetically diverse mouse populations afford the tools to develop models that reproducibly recapitulate severe respiratory disease that can be caused by hcovs in humans. establishing mouse models for sars-cov and mers-cov have largely depended on one or more of these tools. regardless of how effective models for sars-cov and mers-cov are for studying pathogenesis and therapeutic interventions, there are drawbacks related to mouse-adapted and transgenic mouse models that alter susceptibility and pathogenesis to hcovs. acquired mutations associated with severe respiratory disease in mouse-adapted hcovs may have no bearing on human pathogenesis or have altered pathogenic outcomes in humans. genetically engineering a specific host species to enhance susceptibility restricts applications of the mouse model, requiring cross-breeding to introduce novel host mutations from knock-out mouse lines and cc mouse strains. this process is labor intensive and requires an extensive timeline. alternatively, one could introduce mutations directly into genetically diverse lines; however, this is not cost effective. additionally, it is difficult to replicate and study co-morbidities in mice that are associated with lethal respiratory disease seen in humans. pre-existing conditions such as advanced age, diabetes, chronic lung diseases, heart disease, and kidney disease have been reported in many of the severe/lethal mers-cov human cases [reviewed in ]. one way to address many of these concerns could be to screen genetically diverse populations of mice (e.g., collaborative cross) with clinical isolates or infectious molecular clones of wild-type strains from emergent, or pre-emergent, hcovs. screening in genetically diverse mouse populations may yield mouse models with a range of clinically relevant phenotypes that more closely reflect the plethora of respiratory disease outcomes observed in the human population. experiences from sars-cov and mers-cov have taught researchers that each hcov may require unique approaches for mouse model development. the mouse tools instituted for sars-cov and mers-cov constitute a versatile preclinical platform for addressing global pathogen preparedness, a directive of the world health organization. open access this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. a mouse model for betacoronavirus subgroup 2c using a bat coronavirus strain hku5 variant generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study middle east respiratory syndrome synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge clinical features and short-term outcomes of 144 patients with sars in the greater toronto area expression quantitative trait loci for extreme host response to influenza a in pre-collaborative cross mice host genetic background influences diverse neurological responses to viral infection in mice homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection association of icam3 genetic variant with severe acute respiratory syndrome significance of the myxovirus resistance a (mxa) gene -123c> a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection a mouse model for mers coronavirus-induced acute respiratory distress syndrome efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses wildtype and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus cd8 + t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques sars and mers: recent insights into emerging coronaviruses a chimeric virus-mouse model system for evaluating the function and inhibition of papain-like proteases of emerging coronaviruses am i ready for crispr? a user's guide to genetic screens epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong first newborn baby to receive experimental therapies survives ebola virus disease adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies ifitm3 restricts the morbidity and mortality associated with influenza infection with mers-cov causes lethal pneumonia in the common marmoset discovery of alogliptin: a potent, selective, bioavailable, and efficacious inhibitor of dipeptidyl peptidase iv modeling host genetic regulation of influenza pathogenesis in the collaborative cross molecular evolution of human coronavirus genomes genetic association study for rsv bronchiolitis in infancy at the 5q31 cytokine cluster sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in china isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission genome wide identification of sars-cov susceptibility loci using the collaborative cross allelic variation in the toll-like receptor adaptor protein ticam2 contributes to sarscoronavirus pathogenesis in mice oas1b-dependent immune transcriptional profiles of west nile virus infection in the collaborative cross an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in china resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012. virology hemagglutinating behavior of mouse and egg-adapted type a (pr8) influenza virus human genetic determinants of viral diseases a guide to genome engineering with programmable nucleases cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events severe acute respiratory syndrome (sars) coronavirus orf8 protein is acquired from sars-related coronavirus from greater horseshoe bats through recombination costly lessons from the 2015 middle east respiratory syndrome coronavirus outbreak in korea middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence jumping species-a mechanism for coronavirus persistence and survival middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis mers-cov accessory orfs play key role for infection and pathogenesis ifitm3 and susceptibility to respiratory viral infections in the community pneumonia from human coronavirus in a macaque model protective efficacy of a novel simian adenovirus vaccine against lethal mers-cov challenge in a transgenic human dpp4 mouse model a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates inactivation of porcine endogenous retrovirus in pigs using crispr-cas9 revisiting an old acquaintance: cd26 and its molecular mechanisms in t cell function middle east respiratory syndrome coronavirus vaccines: current status and novel approaches genome-wide association study of polymorphisms predisposing to bronchiolitis pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection the role of host genetic factors in respiratory tract infectious diseases: systematic review, meta-analyses and field synopsis glycosylation of mouse dpp4 plays a role in inhibiting middle east respiratory syndrome coronavirus infection permissivity of dipeptidyl peptidase 4 orthologs to middle east respiratory syndrome coronavirus is governed by glycosylation and other complex determinants dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus host genetic diversity enables ebola hemorrhagic fever pathogenesis and resistance aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice the polymorphism architecture of mouse genetic resources elucidated using genome-wide resequencing data: implications for qtl discovery and systems genetics animal models and vaccines for sars-cov infection synthetic reconstruction of zoonotic and early human severe acute respiratory syndrome coronavirus isolates that produce fatal disease in aged mice broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection genetic dissection of complex and quantitative traits: from fantasy to reality via a community effort sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection ace2 x-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor severe acute respiratory syndrome: clinical outcome and prognostic correlates host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets roles of tnf-alpha gene polymorphisms in the occurrence and progress of sars-cov infection: a case-control study structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape orf8-related genetic evidence for chinese horseshoe bats as the source of human severe acute respiratory syndrome coronavirus mice transgenic for human angiotensin-converting enzyme 2 provide a model for sars coronavirus infection receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus genome-wide inactivation of porcine endogenous retroviruses (pervs) isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections rapid generation of a mouse model for middle east respiratory syndrome multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus a functional variation in cd55 increases the severity of 2009 pandemic h1n1 influenza a virus infection fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin genetic variation of the human alpha-2-heremans-schmid glycoprotein (ahsg) gene associated with the risk of sars-cov infection coronaviruses-drug discovery and therapeutic options key: cord-340163-ex03l0pc authors: hu, tingting; liu, ying; zhao, mingyi; zhuang, quan; xu, linyong; he, qingnan title: a comparison of covid-19, sars and mers date: 2020-08-19 journal: peerj doi: 10.7717/peerj.9725 sha: doc_id: 340163 cord_uid: ex03l0pc in mid-december 2019, a novel atypical pneumonia broke out in wuhan, hubei province, china and was caused by a newly identified coronavirus, initially termed 2019 novel coronavirus and subsequently severe acute respiratory syndrome coronavirus 2 (sars-cov-2). as of 19 may 2020, a total of 4,731,458 individuals were reported as infected with sars-cov-2 among 213 countries, areas or territories with recorded cases, and the overall case-fatality rate was 6.6% (316,169 deaths among 4,731,458 recorded cases), according to the world health organization. studies have shown that sars-cov-2 is notably similar to (severe acute respiratory syndrome coronavirus) sars-cov that emerged in 2002–2003 and middle east respiratory syndrome coronavirus (mers-cov) that spread during 2012, and these viruses all contributed to global pandemics. the ability of sars-cov-2 to rapidly spread a pneumonia-like disease from hubei province, china, throughout the world has provoked widespread concern. the main symptoms of coronavirus disease 2019 (covid-19) include fever, cough, myalgia, fatigue and lower respiratory signs. at present, nucleic acid tests are widely recommended as the optimal method for detecting sars-cov-2. however, obstacles remain, including the global shortage of testing kits and the presentation of false negatives. experts suggest that almost everyone in china is susceptible to sars-cov-2 infection, and to date, there are no effective treatments. in light of the references published, this review demonstrates the biological features, spread, diagnosis and treatment of sars-cov-2 as a whole and aims to analyse the similarities and differences among sars-cov-2, sars-cov and mers-cov to provide new ideas and suggestions for prevention, diagnosis and clinical treatment. for disease control and prevention (cdc, https://www.cdc.gov/), the who (https://www.who.int/), the national administration of traditional chinese medicine (http://www.satcm.gov.cn/) and the national health commission of the people's republic of china (http://www.nhc.gov.cn/). the following search parameters and mesh terms "sars-cov-2", "sars-cov", "mers-cov", "epidemiology", "clinical presentations". "laboratory diagnosis" and "radiological features" delivered 151 hits (see fig. 1 ). we focused on the similarities and differences of covid-19, sars and mers in all respects to be clinically relevant to a great extent. thus, inclusion criteria were the following: biological studies of sars-cov-2, sars-cov and mers-cov collection and analysis of at least one of the following biomaterials: faecal, urinary or blood samples of confirmed/suspected individuals analysis of isolated sars-cov-2, sars-cov or/and mers-cov by sequencing et al. the latest diagnostic criteria of covid-19, sars and mers including clinical presentations, labora tory diagnosis and radiological feature latest treatment and prevention methods of published in a peer-reviewed article availability of the full text publication availability of the paper in english most of the included papers were published between 2005 and 2020 and the evalutation of papers was conducted in the procedure above by two experienced researchers. among the included papers, six papers were excluded because the full texts were not available; besides, three non-english papers were excluded and five papers were also excluded for lacking of any relevance to the subject. totally, 137 texts were selected for this literature review. researchers determined the sequence of sars-cov-2 through high-throughput sequencing. in addition to the published references on sars-cov and mers-cov, we compared the biological features of sars-cov-2, sars-cov and mers-cov, which are summarized in table 1 . as shown in this table, sars-cov-2 can cause severe acute respiratory syndrome and has a 4.2% mortality rate; similarly, sars-cov and mers-cov can cause severe acute respiratory syndrome and have mortality rates of 11% and 34%, respectively . according to previous studies, sars-cov-2 has been confirmed to share 79.5% sequence identity with sars-cov and 94.6% sequence identity with sars-cov in orf1a/b; the sequencing results were used for cov species classification and revealed that both of these viruses are lineage b betacoronaviruses lu et al., 2020) . however, sars-cov-2 shares only 40% sequence identity with mers-cov, which is a lineage c betacoronavirus (chan et al., 2020) . civets are the intermediate hosts of sars-cov, whereas dromedary camels are the intermediate hosts of mers-cov lu et al., 2020) . however, the intermediate hosts of sars-cov-2 have not been determined. at present, the prevailing viewpoints suggest malayan pangolins and turtles . covs are rna viruses characterized by a single-stranded, 5′-capped, positive strand rna molecule ranging from 26 to 32 kb, and the rna includes at least six open reading frames (orfs). the first orf (orf1a/b) comprises approximately 2/3 of the genome and encodes replicase proteins, and the remaining orfs mainly encode four structural proteins: spike (s), membrane (m), envelope (e) and nucleocapsid (n) (perlman & netland, 2009) . the genome organization of sars-cov-2, sars-cov and mers-cov are described in fig. 2 , and the major distinctions between sars-cov-2 and sars-cov are in open reading frame-3b (orf3b), spike and open reading frame-8 (orf8), especially in spike s1 and orf8 (perlman & netland, 2009) . orf8 is an accessory protein. the sars-cov orf8b can activate nlrp3 inflammasomes and trigger pyroptotic cell death, whereas the new orf8 of sars-cov-2 does not contain a known functional domain or motif (shi et al., 2019) . the spike protein can mediate coronavirus entry into host cells. this protein has three segments: a short intracellular tail, a single-pass transmembrane anchor (tm) and a large ectodomain (ic), which consists of two subunits (s1 and s2). the s1 subunit can recognize and bind to receptors, and when s1 binds to a host receptor, the s2 subunit is exposed and cleaved by host proteases, mediating viral and host membrane fusion (li, 2016; belouzard, chu & whittaker, 2009) . the coronavirus structure is described in fig. 3 . the n protein is important for the virus capsid and modulates the initial innate immune response by inhibiting type i interferon (ifn) production (hui, 2013) . the m protein and the e protein are involved in virus morphogenesis, assembly and budding. the s protein mediates virus entry into cells. the receptor of sars-cov-2 is angiotensin converting enzyme ii (ace2), which is also the receptor of sars-cov. ace2 binds to the sars-cov-2 s ectodomain with 15 nm affinity, which is approximately 10-to 20-fold higher affinity than ace2 binding to sars-cov s. in contrast, the receptor of mers-cov is dpp4 (wrapp et al., 2020) . both ace2 and dpp4 are recognized by the c-terminal domain in s1 (s1-ctd) as a receptor-binding domain (rbd). as previously mentioned, s1 is one of the major distinctions between sars-cov-2 and sars-cov. these realizations lead one to wonder how these viruses can recognize the same receptor of the host cell despite their inherent differences. the rbd of sars-cov contains two subdomains: a core and an extended loop. the core is constructed of a five-stranded anti-parallel β sheet (β1 to β4 and β7) and three short connecting a helices (aa to ac). the extended loop subdomain is positioned to one side of the core, and a two-stranded β sheet (β5 and β6) forms a gently concave outer surface, the base of which cradles the n-terminal helix of ace2 (wang et al., 2013) . although only 9 out of 13 glycans in the table 1 a comparison of biological features among sars-cov-2, sars-cov and mers-cov. the data are from the following studies: zhou et al. (2020) , lu et al. (2020 ), chan et al. (2020 ), belouzard, chu & whittaker (2009 ), hui (2013 , wang et al. (2013) and . homology * -79.5% 40% bat lu et al., 2020) bat lu et al., 2020) bat lu et al., 2020) possible intermediate host malayan pangolins and turtles lu et al., 2020) palm civets lu et al., 2020) camel lu et al., 2020) the lineage of betacoronaviruses b lu et al., 2020) b lu et al., 2020) c lu et al., 2020) predominant cellulart receptor ace2 (belouzard, chu & whittaker, 2009; hui, 2013) ace2 (belouzard, chu & whittaker, 2009; hui, 2013) dipeptidyl peptidase 4 (dpp4, also known as cd26) (wang et al., 2013) symptoms severe acute respiratory syndrome, 4.2% mortality rate severe acute respiratory syndrome, 11% mortality rate severe acute respiratory syndrome, 34% mortality rate note: * homology with sars-cov-2. s1 subunit are conserved among sars-cov-2 s and sars-cov s, their overall structures are similar, and the most notable difference is the position of the rbds in their respective down conformations: tightly against the n-terminal domain (ntd) in sars-cov and angled closer to the central cavity of the trimer in sars-cov-2 (wang et al., 2013) . in contrast to sars-cov and other sarsr-covs, there is a four amino acid residue insertion at the s1/s2 boundary of sars-cov-2 s that results in the presence of a furin cleavage site (walls et al., 2020) . the s1-s2 site cleaved during biosynthesis is not necessary for s-mediated entry, but it may contribute to the high affinity of sars-cov-2 s for human ace2 (letko, marzi & munster, 2020) . angiotensin converting enzyme ii is the receptor of sars-cov-2 and sars-cov, and it is one of the enzymes in the renin angiotensin system (ras). it can convert angiotensin i (angi) to angiotensin 1-9 (ang 1-9) and angiotensin ii (ang ii) to angiotensin 1-7 (ang 1-7) to decrease angii, which can increase aldosterone and vasopressin secretion, cause vasoconstriction, and induce myocardial and renal fibrosis (flores-muñoz et al., 2011; serfozo et al., 2020) . as counterregulatory components of the ace-ang ii-at1 axis, ace2 and (ang 1-7) can control inflammation and fibrosis in cardiovascular and renal disease (simões e silva & teixeira, 2016). expression of the ace2 receptor is found in many tissues, including lung, heart, kidney, liver, endothelium, intestine, oral mucosa and even testis venkatakrishnan et al., 2020; . ace2 is reported to improve acute lung injury, suppress hypertension and cardiac dysfunction, reduce glomerular and biliary fibrosis, stimulate brown adipose tissue and induce browning in white adipose tissue (minato et al., 2020; rajapaksha et al., 2019; kawabe et al., 2019; chen et al., 2019) . all these factors could be targets for sars-cov-2 to damage human health. in addition, the level of ace2 was reported to be related to il-6, which plays an important role in cytokine storm syndrome (css), by long chen1 and li zhong on preprint.org. css is considered to increase the illness severity of sars-cov-2 . the lungs are the main target organ of sars-cov-2. according to recent research, ace2 is expressed in 0.64% of all human lung cells, and the majority of them are type ii alveolar cells (at2) (average 83%) . consistent with these findings, sars-cov-2 presents as lesions involving mainly destruction of the distal alveoli. other lung cells, such as type i alveolar cells (at1), endothelial cells, airway epithelial cells, fibroblasts and macrophages, were also reported to express ace2. though their ratio is low and variable among individuals, they may also be the target of sars-cov-2 (hamming et al., 2004) . severe acute respiratory syndrome coronavirus 2 also causes gastrointestinal symptoms, and approximately 3% of patients develop diarrhoeal symptoms (holshue et al., 2020) . accordingly, oesophageal upper and stratified epithelial cells as well as absorptive enterocytes from the ileum and colon were also reported to be ace2 positive . sars-cov-2 was also reported by the third people's hospital of shenzhen to be present in faecal samples. based on the above-mentioned studies, many researchers speculated thatthe faecal-oral route may be one route of sars-cov-2 transmission; however, it has been fairly settled by who that the fecal-oral route does not drive transmission and we should not read too much into this mode of transmission. according to several recent studies, acute kidney injury (aki) has been reported in over 20% of patients who suffered from covid-19 in china and the us richardson et al., 2020) . besides, a meta-analysis showed the prevalence of chronic kidney disease was higher in severe patients with covid-19 (3.3% vs. 0.4%; odds ratio 3.03, 95% ci [1.09-8.47]) (henry & lippi, 2020) . this outcome may be attributed to ace2 because ace2 is expressed in the kidney, especially in proximal tubular cells and bile duct cells (wu et al., 2018; macparland et al., 2018; soler et al., 2019) . notely, ace2 is also expressed by endothelial cells, and other major clinical events commonly observed in covid-19 patients including high blood pressure, thrombosis, pulmonary embolism, cerebrovascular and neurologic disorders (lovren et al., 2008; poissy et al., 2020; aggarwal, lippi & michael henry, 2020; mao et al., 2020; baumgartner-parzer & waldhausl, 2001) . the sars-cov-2 pneumonia outbreak in wuhan began in early december 2019. among the first 41 laboratory-confirmed patients, 27 (66%) had been exposed to the huanan seafood market, where wild animals are sold, suggesting that covid-19 may be passed to humans from wild animals . the median incubation period of covid-19 is 3.0 days (range, 0-24.0 days) (chen et al., 2020). the disease is highly contagious, and the speed of the spread and the infectivity of covid-19 dramatically exceeded those of mers-cov and sars-cov (goh et al., 2020) . the basic reproduction number (r0) reflects the rate of disease transmission. recent data revealed an r0 for covid-19 of 2.56 (95% ci [2.49-2.63]), indicating that one patient could transmit the disease to 2.56 other people . according to recent data from the who, centers for disease control and prevention (cdc), and reports to the who from various countries and their allied agencies, as of 22 july 2020, a total of 14,562,550 individuals were reported as infected with sars-cov-2 among 216 countries, areas or territories with recorded cases, and the overall case-fatality rate (cfr) was 4.2% (607,781 deaths among 14,562,550 recorded cases). in a report from the who (https://www.who.int/ emergencies/diseases/novel-coronavirus-2019/situation-reports), europe and the americas became the locations of the most serious epidemics, instead of china, which had previously been the location for the most serious outbreaks. in china, people with overseas exposures have become the high-risk population, as with wuhan-related exposures. male sex and older age are two significant risk factors . according to a study among 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china, the male-to-female ratio was 1.06:1, and the median age was 56 years (interquartile range, 42-68; range, 22-92 years) (wu & mcgoogan, 2020; wang et al., 2020) . of the 1,591 patients requiring treatment in an intensive care unit (icu) in the lombardy region of italy, the median (iqr) age was 63 (richardson et al., 2020; borghesi et al., 2020; zheng et al., 2020; de wit et al., 2016; gao et al., 2016) years, and 1,304 (82%) were male (grasselli et al., 2020) . according to the data from 5,700 hospitalized patients with confirmed covid-19 in new york city, the median age was 63 years (interquartile range, 52-75; range, 0-107 years), and 39.7% were female (richardson et al., 2020) . one study showed that males aged 50 years or older and females aged 80 years or older showed the highest risk (borghesi et al., 2020) . health workers are one of the high-risk groups. a total of 3.8% of confirmed covid-19 patients were health workers (aggarwal, lippi & michael henry, 2020) . diabetes could be one of the risk factors for progression to severe/critical outcomes . human-human transmission is considered a major transmission mode of covid-19 by the sixth version of the guidance for diagnosis and treatments for covid-19 issued by the national health commission of china. and according to the latest reports from the who, the driving transmission of covid-19 contains droplet transmission, contact transmission and aerosol transmission. from november 2002, when the first known case of sars occurred in foshan, china, to july 2003, when the who declared the sars pandemic over, a total of 8,096 cases were reported in 27 countries, including 774 deaths for a cfr of 9.6% (de wit et al., 2016) . among these 8,096 cases, 23.1% were health workers, the male-female ratio was 1:1.25, and the mean age was 39.9 years, ranging from 1 to 91 (rota et al., 2003) . the mean incubation period was 6.4 days (range 2-10) (park et al., 2019) . carrying an hlacw ã 0801 allele is a risk factor for sars (chen et al., 2006) . older age and male gender were predictive of poor prognosis (chan, tsui & wong, 2007) . human-human transmission is also the major transmission mode of sars-cov, and the primary route of transmission for sars is contact of the mucous membranes with respiratory droplets or fomites (https://www.who.int/zh). mers first emerged in september 2012 in saudi arabia and is still not contained. according to data from the who (https://www.who.int/csr/don/24-february-2020mers-saudi-arabia/zh/), as of 31 january 2020, 2,519 cases of mers had been laboratoryconfirmed, including 858 associated deaths (cfr: 34.4%) that were reported in the 27 countries globally that have had cases of mers-cov; the proportion of healthcare workers infected in saudi arabia from january 2013 to november 2019 was 19.1%. based on the analysis of who data from 23 september 2012 to 18 june 2018, ahmadzadeh et al. (2020) described the epidemiological characteristic of mers as follows: the cfr was 32% (95% ci [29.4-34.5]); the male-to-female ratio was 2.52; the mean age was 50.21 ± 18.73 years and ranged from 2 to 109 years of age; and the risk factors for mers included age >30 years old, saudi nationality, comorbidities, and the interval time from symptom onset and admission to the hospital >14 days. the median incubation period was seven days (range 2-17) (cho et al., 2016) . human-human transmission in mers-cov is limited and not sustained (bernard-stoecklin et al., 2019) . camels were thought to be involved in its spread to humans, but in what way remains unclear. according to a report from the who (https://www.who.int/news-room/fact-sheets/ detail/middle-east-respiratory-syndrome-coronavirus-(mers-cov)), people become infected through unprotected contact with infected dromedary camels or infected people. in conclusion, we summarized the epidemiological comparisons of covid-19 with sars and mers in table 2 . covid-19, sars, and mers show several similarities regarding their symptoms, which include fever, cough, myalgia, fatigue and lower respiratory signs. however, the symptoms vary with the state of the illness and in the process of disease progression. notably, 60% of patients suffering from sars have watery diarrhoea in addition to the abovementioned symptoms with the characteristics that there is a representative biphasic clinical course (hui & zumla, 2019; monaghan, 2016) . patients who suffer from mers have symptoms that include fever, cough (predominantly dry), malaise, myalgia, nausea, vomiting, diarrhoea, headache and even renal failure (arabi et al., 2014; gao et al., 2016) . not surprisingly, the symptoms of mers resemble sars, but the clinical course is unpredictable and changeable. more than half of the mers patients are reported to develop acute renal damage at an average time of approximately ten days after the onset of symptoms; additionally, the majority of the cases require renal replacement therapy (arabi et al., 2014; assiri et al., 2013; memish et al., 2014) . however, approximately 6.6% of patients who are infected with sars develop acute renal failure at a median time of 20 days after onset, and only 5% call for replacement therapy (chan, tsui & wong, 2007) . direct renal injury is considered to contribute to the common symptoms of acute renaldamage in mers patients (monaghan, 2016) ; namely, dpp4 is present in renal cells, and mers-cov can be detected in urine. the majority of patients with covid-19 infections present with fever (98%), cough (76%), and myalgia or fatigue (44%). it has been (2020), wang et al. (2020) , grasselli et al. (2020) , richardson et al. (2020) and borghesi et al. (2020) . other data were obtained the world health organization (who, https://www.who.int/emergencies/diseases/novel-coronavirus-2019), the centers for disease control and prevention (cdc, https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/index.html), and reports from various countries and their allied ministries to the who (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-theyhappen; https://www.worldometers.info/coronavirus/?utm_campaign=homeadvegas1; https://www.ecdc.europa.eu/en/covid-19-pandemic). reported that 55% of patients can present with dyspnoea, which develops a median of eight days after the onset of initial symptoms (kanne, 2020). in light of the studies from all over the country, the symptoms suggest that the target cell is likely present in the lower respiratory tract, as patients who are infected with covid-19 seldom have conspicuous upper respiratory symptoms such as sneezing or sore throat . the autopsy reports of new coronavirus pneumonia (ncp) patients indicate that the disease mainly causes distal airway inflammatory reactions and alveolar damage, which is coincidental with the abovementioned symptoms. the fibrosis and consolidation in the lungs of sars patients are more serious than the lesions caused by covid-19, which indicates that the chest lesions are not primarily serous inflammation; instead, the exudative reaction of sars is less than that of covid-19. specimens laboratory diagnosis plays a leading role in the early detection of infected individuals, which enables an earlier discovery of the source of infection and interruption of epidemic transmission (yan, chang & wang, 2020) . the first and crucial step for laboratory testing is the collection and processing of suitable specimens. in light of the published studies, viral rna, considered one of the gold standards of detection, can be found in the upper respiratory tract (urt), lower respiratory tract (lrt), stool, blood, and urine of patients who are infected with sars-cov, mers-cov, and sars-cov-2. the detection rates of the abovementioned specimens for sars-cov, mers-cov, and sars-cov-2 infection are summarized in table 3 young et al., 2020; peiris et al., 2003; poon et al., 2003; corman et al., 2016; ng et al., 2003; ling et al., 2020) . we can select the specimens showing higher sensitivity in different stages. currently, nucleic acid tests (nat) are widely considered the optimal method for diagnosis, since specific primers and standard operation procedures have been established during sequencing of the total genome of the coronavirus (monaghan, 2016) . in general, real-time polymerase chain reaction (rt-pcr) is thought to be the preferred and most widely used nat method (gootenberg et al., 2017) . the orf1a, orf1b, s gene, and n gene, in addition to the m gene and 3′utr, are all gene targets of rt-pcr assays, which can have high sensitivity. the rt-pcr methods for sars-cov, mers-cov and sars-cov-2 varied in genome target, sequence, assay use, etc. (lu et al., 2014; emery et al., 2004; yan, chang & wang, 2020) . as shown, most of the in-house assays, as well as commercial kits, can detect two or three regions of the virus genome. however, there are many knowledge gaps and limitations to overcome, including the difficulties of obtaining testing kits due to the global shortage, the requirements of having access to sophisticated equipment, and the management of false negatives that need to be retested (al-tawfiq & memish, 2020; loeffelholz & tang, 2020). the abovementioned limitations have resulted in the development in recent years of many kinds of nat testing kits used for routine detection of sars-cov and mers-cov, such as nucleic acid sequence-based amplification (nasba), loop-mediated isothermal amplification (lamp), rolling-circle amplification (rca), clustered regularly interspaced short palindromic repeats (crispr) system, etc., the details of which are summarized in table 4 . as shown, a real-time nasba assay was developed by chantratita et al. (2004) that can detect only one strand of sars-cov rna. in the same year, the rt-lamp assay for sars-cov can detect 0.01 pfu, which is 100-fold greater than rt-pcr sensitivity . several rt-lamp methods of mers-cov that can detect 3.4-15.8 copies of mers-cov rna have been developed (shirato et al., 2018; lee et al., 2017) . rt-pcr can detect sars-cov and mers-cov with sensitivities of five copies and 10 copies, respectively abd el wahed et al., 2013) . at present, many nat kits have been developed for sars-cov-2, especially rt-pcr. however, according to previous studies, the currently available rt-pcr kits are variable table 3 the detection rates of sars-cov-2, mers-cov, and sars-cov infection. data are converted directly from raw data or estimated from figures. data are all from the following studies: hui & zumla (2019) and offer sensitivities ranging between 45 and 60% (yi, 1990; al-tawfiq & memish, 2020) . in addition, these methods are not satisfactory, especially in resource-limited regions. there is no doubt that nat based on clinical manifestations and epidemiology can be an early and rapid screening diagnosis. if nat is not available, ct imaging also plays a vital role in the diagnosis. medical imaging technology that is commonly used to diagnose sars, mers and covid-19 includes chest x-ray (cxr), computed tomography (ct) and high resolution computed tomography (hrct). the spatial resolution of cxr is high, but the density resolution is not very good, which leads to a relatively high omission diagnostic rate. instead, ct has a higher density resolution because it can quantitatively evaluate images. not surprisingly, hrct has a high spatial and density resolution and can detect small parenchymal lesions early. the earliest radiographic abnormalities were described as ggos, and in the majority of patients, the radiographic abnormalities progressed rapidly to focal, multifocal or diffuse consolidation. airspace shadowing is the most common abnormality found on the cxr at presentation but it is nonspecific because, according to the survey, 10-40% of symptomatic patients in published reports, irrespective of age, have normal initial cxrs, and the findings may be indistinguishable from pneumonia of other causes (ooi & daqing, 2003) . in approximately two-thirds of patients, unilateral lung involvement is found at presentation, and as the condition progresses, it can become bilateral with maximal lung involvement. in addition, consolidation tends to be peripheral in distribution with lower zone predominance (ooi & daqing, 2003) . moreover, cxr obtained in most sars patients at initial presentation is abnormal, and the disease progression in these patients can be monitored with serial chest radiography (paul et al., 2004) . ct findings ground-glass opacification, consolidation, and inter-lobular interstitial and intralobular septal thickening are the most common findings in hrct scans, mainly with predominant involvement of the periphery and lower lobe. according to a previous study, high-resolution ct scans (hr-ct) may be useful in detecting opacities in patients with normal cxr (paul et al., 2004; groneberg et al., 2003) . in early stages, abnormalities described on ct include ground-glass opacification, consolidation, interlobular interstitial and intralobular septal thickening . when in the progressive stage, the disease findings by hrct, including lesion size, range and severity, progressed steadily. when in the late stages, consolidation can be indistinguishable from acute respiratory distress syndrome (ards). above all, ct is more sensitive in depicting sars than conventional cxr, and ct images obtained in patients with normal chest radiographs may show extensive disease and airspace consolidation . cxr findings a study by das et al. (2016) indicated that abnormalities can be detected on initial cxr in 83% of patients who suffered from mers-cov, and the main cxr findings of mers-cov include ground-glass opacity (66%) and consolidation (18%), both of which are distinct in 16% of infection cases. the consolidation can be patchy, coalesced or show opaque rounded nodules. the primary cxr of infected cases appears as lung parenchymal abnormalities with the dominating images of a peripheral mid-lung zone and peripheral lower lung zone. involvement of multifocal lung parenchymal abnormalities is less common than unifocal involvement. furthermore, serial cxr obtained during the progression of the disease can be used to evaluate the degree of radiographic deterioration. in general, the progression of mers can be divided into four types (i.e., types 1-4). type 1 is the disease development period that appears as initial radiographic deterioration with improvement. type 2 is the quiet period that shows no apparent changes in cxr. type 3 manifests as fluctuating radiographic changes with at least two radiographic peaks divided by a stage of soft remission. type 4 is the progression of radiographic deterioration (das et al., 2016) . ct findings on the basis of the statistics and the published references, mers ct imaging findings are more sensitive than cxr; unquestionably, ct is used to confirm or assess the progress of highly suspected mers (das et al., 2016) . the most common ct findings of mers are that of bilateral, predominantly subpleural and basilar airspace changes, with more extensive ggos than consolidation (ajlan, 2015) . ct scans were also assessed for the presence of consolidation, cavitation, centrilobular nodules, tree-in-bud pattern, septal thickening, perilobular opacities, reticulation, architectural distortion, subpleural bands, traction bronchiectasis, bronchial wall thickening, intrathoracic lymph node enlargement, interlobular thickening and pleural effusions (das et al., 2015) . generally speaking, ground-glass opacities, consolidation and a combination of the abovementioned image results are commonly found during the first week of infection; additionally, pleural effusion and interlobular thickening can also be seen on ct imaging findings of some infected cases (das et al., 2015) . other ct imaging findings can be seen in different stages with the development of the disease. interestingly, petruzzi, shanthly & thakur (2009) proposed that pet/ct, a new imaging technology, can be more sensitive and specific than traditional imaging technology such as ct and cxr. their research also confirmed that pet/ct can quantitatively describe pulmonary infections. in 2016, a study by das et al. (2016) proposed that distinctly increased β-2-(18f)-fluoro-2-deoxy-d-glucose (18f-fdg) might be seen in patients who are infected with mers . cxr findings in the early phase, cxr of covid-19 patients is not highly recommended for clinical diagnosis because of its low sensitivity in detecting sars-cov-2 pneumonia. nevertheless, in some areas with limited resources, cxr, as a radiological technology, might have some practical applicability (paul et al., 2004) . the earliest radiographic abnormalities can be negative or only a few patchy increased density shadows. as the disease progresses, cxr can detect double lung patterns and patchy increased density shadows, which are mainly in the lower bilateral lung fields. furthermore, severely ill patients can exhibit diffuse consolidation of both lungs, even showing a "white lung" pan et al., 2020; jakobsson et al., 2014) . the imaging findings of covid-19 are different regarding the age of the patients, the stages of disease, etc. (li & xia, 2020). the ct imaging findings of covid-19 are divided into early stage, progressive stage and severe stage considering the scope and evolution of lesions, and the characteristics of each stage are as follows: (1) early stage: single or multiple scattered patchy or conglomerate ggo, mainly in the middle and lower lungs and along the bronchovascular bundles; (2) progressive stage: increase of imaging fingdings in density; extent coexisting with the new areas of disease; consolidation growing with air bronchograms.; (3) severe stage: diffuse consolidation of the lungs of varying density; the fibrous exudating; air-bronchograms; bronchial dilation.; (4) dissipation stage: gradual resolution of the ggo; consolidation in the lungs with some residual curvilinear opacities compatible with fibrosis (kanne, 2020; . the ct imaging features of lesions are as follows: (1) the predominant distribution is in the subpleural region along the bronchial tract; (2) single and double lesions are rarely seen on ct scans, instead, multiple lesions are more common; (3) the lesions can be patchy, nodular, lumpy, honeycomb-like, etc.; (4) the density is commonly uneven, and ggos, interlobular thickening, consolidation, crazy-paving change, etc. can be seen on ct scans; and (5) pleural effusion is rarely seen, but concomitant signs, such as air bronchogram, in addition to mediastinal lymph node enlargement, are common. many published studies have indicated that chest ct should be considered as a major role in the diagnosis and management of covid-19. for instance, research by li. y found that chest ct has a low false negative rate in detecting covid-19 (3.9% 2/51 cases); thus, a negative chest ct might aid in the management of covid-19, that is, guide the doctors to make a decision whether the patient should be isolated during the incubation window (das et al., 2016) . however, some researchers still raise objections that a negative chest ct does not ensure that a person is not infected with sars-cov-2; equally, the abnormalities of the chest ct are not exclusive for covid-19 (adair & ledermann, 2020) . in general, we should use chest ct reasonably, and there is doubt that early diagnosis based on chest ct can contribute to overcoming the outbreak as soon as possible. a summary and a comparison of the three kinds of coronavirus pneumonia covid-19 pneumonia tends to show similarities with sars and mers pneumonia on cxr, with a predominance of bilateral ground-glass opacities and consolidative lesions in the peripheral lung. covid-19 seems radiologically milder than sars and mers despite the similarities in ct findings (yoon et al., 2020) . each disease undoubtedly has unique radiological features, and the summary data are in table 5 (kanne, 2020; das et al., 2015; wang et al., 2003) . in addition, changes in radiographic scores of sars in general tended to reflect temporal changes in clinical and laboratory parameters such as oxygen saturation (sao2). moreover, there were significant inverse relationships between radiographic scores and sao2. in a study involving a cohort of patients with serologically confirmed sars, all patients with diffuse consolidation at maximal radiographic change required oxygen supplementation, including mechanical ventilation . therefore, we can speculate that it may also be suitable for covid-19. the available pathology data for sars and mers infections mainly rely on limited numbers of autopsy and biopsy cases. according to previous studies, oedematous lungs with increased gross weights and multiple areas of congestion are the predominant visceral macroscopic changes in fatal sars cases. moreover, enlargement of the lymph nodes in the pulmonary hila and the abdominal cavity, as well as diminished spleen size and reduced spleen weights, are also the most common changes (nicholls et al., 2003) . morphological changes of sars include bronchial epithelial denudation, loss of cilia, and squamous metaplasia (nicholls et al., 2003; ding et al., 2003; . in the early phase, the histological features of pulmonary sars infections may be commonly connected with acute diffuse alveolar damage; on the contrary, a combination of diffuse alveolar damage and acute fibrinous and organizing pneumonia are demonstrated in the later phases of the disease (gu et al., 2005) . some studies suggest that the pathological features of mers infection are varied and include exudative diffuse alveolar damage with hyaline membranes, pulmonary oedema, type ii pneumocyte hyperplasia, interstitial pneumonia (which is predominantly lymphocytic), and multinucleate syncytial cells. moreover, bronchial submucosal gland necrosis was also observed, and it includes the pathologic basis for respiratory failure and radiologic abnormalities of mers infection (alsaad et al., 2018) . ultra-structurally, viral particles could be found in the pneumocytes, pulmonary macrophages, macrophages infiltrating the skeletal muscles, and renal proximal tubular epithelial cells (walker, 2016) . on 17 february 2020, the team of academician wang of pla general hospital performed a pathologic dissection on a patient who died of covid-19. tissue samples were taken from the patient's lung, liver and heart tissues, and histological examination of the lung revealed bilateral diffuse alveolar injury with fibrous mucinous exudation. in the right lung tissue, prominent alveolar epithelial exfoliation and clear membrane formation suggested ards, and in the left lung, tissue pulmonary oedema and clear membrane formation suggested the early stage of ards. mononuclear inflammatory infiltrations of lymphocytes in the stroma were manifested in both lungs, multinucleated giant cells and uncharacteristically enlarged alveolar cells were found in the alveolar exudate, and the latter contained larger nuclei, bi-tropic intracytoplasmic granules and prominent nucleoli, which showed viral cytopathic-like changes. additionally, no obvious intracytoplasmic or nuclear virus inclusion bodies were found. the severity of pulmonary lesions in sars, mers and covid-19 is different, especially the destruction of alveoli and the degree of necrotic lung. according to the comparison of the pathology, covid-19 seems to be not as severe as sars. indeed, they show some similarities in pathology in that they all have type ii pneumocyte hyperplasia. however, they also have some differentia. in general, the pathological features of sars are that a large proportion of type ii pneumocytes fall off into the alveolar cavity, while the pathological features of sars are that there are many cells proliferating and active in the alveolar walls, and some cells even fall into the lumen in clumps, especially in the late stage (qu et al., 2020; barrett et al., 2020) . indeed, the degree and progression of alveolar fibrosis are varied. in summary, these results suggest that pathology may also play a relevant role in the development of disease severity. during the sars-cov epidemic, there were no treatments available to reduce sars-related diseases and deaths. the earliest treated patients received intravenous injection (iv) ribavirin, which was based on its broad-spectrum antiviral activity, because there was insufficient time to perform efficacy studies (poutanen et al., 2003) . after confirming that sars-cov was the causative agent, many studies on treatments for sars were started. the most commonly used treatments for sars include ribavirin, lpv/r, corticosteroids, interferon (ifn) and convalescent plasma or immunoglobulins. unfortunately, the abovementioned treatments are associated with adverse effects, including avascular necrosis and osteoporosis (stockman et al., 2006) . therapeutically, mers therapies resemble sars therapies, among which antiviral therapy has been widely studied. broad spectrum antiviral drugs with low toxicity, including interferon, ribavirin and cyclophilin inhibitors, which were used effectively for sars, were also proven to be effective in mers patients (de wilde et al., 2013; omrani et al., 2014) . severe studies tested and compared the activity of sars-cov and mers-cov to antiviral drugs. for instance, de wilde et al. (2013) and de wit et al. (2016) indicated that sars-cov was fifty to one hundred times less sensitive to ifn-a treatment than mers-cov (gu et al., 2005) . the therapy of using antibodies targeting the virus was widely recognized during the epidemic of sars-cov (ter meulen et al., 2004) . to date, many antibodies targeting sars-cov and mers-cov have been identified. however, the difficulties of use worldwide relate to the emergence of possible escape mutants. at present, no approved antiviral therapy is available for sars or mers, nor for covid-19. the drugs available for covid-19 contain broad-spectrum antiviral drugs such as nucleoside analogues and hiv-protease inhibitors . in light of recent studies, broad-spectrum antivirals, including lopinavir, neuraminidase inhibitors, peptides (ek1), and rna synthesis inhibitors, are considered reasonable for treatment (rothan & byrareddy, 2020) . to date, a randomized, controlled clinical trial by ledford et al. found that dexamethasone is first drug shown to save lives and reduce deaths from the covid-19 (ledford, 2020). the other drug shown to benefit people who suffered from covid-19 is the antiviral drug remdesivir. surprisingly, remdesivir was testified to have the function of shortening the length of stay (los) of people with covid, but it did not have significant effects on deaths (oh et al., 2016) . in general, both remdesivir and dexamethasone are approved in the united states (usa) to treat covid-19. besides, many scientists are working diligently to study covid-19 to gain a better understand of virus-host interactions, in addition to creating novel therapeutics and testing potential vaccines. the novel atypical pneumonia that emerged in wuhan, hubei province has been proven to be caused by sars-cov-2, which has homology with sars-cov and mers-cov to some extent . the outbreaks of sars-cov and mers-cov provide us with significant lessons, including valuable experiences and clinical opinions, on how to better fight the sars-cov-2 epidemic. furthermore, the previous treatments can be used as the basis to begin to control the condition. given the above findings, we present our suggestions. to fully control the situation, we first need to confirm the transmission of sars-cov-2 by performing well-designed large-scale case-control studies; then, proper interventions can be taken to control the cause of disease. second, monitoring signs of viral genome mutations will play a major role in future research. third, assessing the effects of antiviral drugs by conducting a randomized controlled study (rct) is also important. finally, further research on animal vaccines is especially necessary. tingting hu conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. ying liu conceived and designed the experiments, performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft. mingyi zhao conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft. quan zhuang conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft. linyong xu conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft. qingnan he analyzed the data, authored or reviewed drafts of the paper, and approved the final draft. the following information was supplied regarding data availability: this is a review article; there is no raw data. from sars to mers: evidence and speculation rigidity of the outer shell predicted by a protein intrinsic disorder model sheds light on the covid-19 (wuhan-2019-ncov) infectivity nucleic acid detection with crispr-cas13a/c2c2 severe acute respiratory syndrome: global initiatives for disease diagnosis multiple organ infection and the pathogenesis of sars tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis chronic kidney disease is associated with severe coronavirus disease 2019 (covid-19) infection first case of 2019 novel coronavirus in the united states development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus clinical features of patients infected with 2019 novel coronavirus in wuhan genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding single cell rna sequencing of human liver reveals distinct intrahepatic macrophage populations neurologic manifestations of hospitalized patients with coronavirus disease screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study b38-cap is a bacteria-derived ace2-like enzyme that suppresses hypertension and cardiac dysfunction emerging infections-implications for dental care quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome viral load kinetics of mers coronavirus infection ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study sars: radiological features transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination initial ct findings and temporal changes in patients with the novel coronavirus pneumonia (2019-ncov): a study of 63 patients in wuhan, china emerging respiratory infections threatening public health in the asia-pacific region: a position paper of the asian pacific society of respirology radiologic pattern of disease in patients with severe acute respiratory syndrome: the toronto experience coronavirus infections-more than just the common cold clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study coronaviruses post-sars: update on replication and pathogenesis recent trends in soft-tissue infection imaging pulmonary embolism in patients with covid-19: awareness of an increased prevalence early diagnosis of sars coronavirus infection by real time rt-pcr identification of severe acute respiratory syndrome in canada cell-based therapy to reduce mortality from covid-19: systematic review and meta-analysis of human studies on acute respiratory distress syndrome liver-targeted angiotensin converting enzyme 2 therapy inhibits chronic biliary fibrosis in multiple drug-resistant gene 2-knockout mice presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area characterization of a novel coronavirus associated with severe acute respiratory syndrome the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak ang ii (angiotensin ii) conversion to angiotensin-(1-7) in the circulation is pop (prolyloligopeptidase)-dependent and ace2 (angiotensin-converting enzyme 2)-independent characteristics and diagnosis rate of 5630 subjects receiving sars-cov-2 nucleic acid tests from wuhan sars-coronavirus open reading frame-8b triggers intracellular stress pathways and activates nlrp3 inflammasomes development of fluorescent reverse transcription loop-mediated isothermal amplification (rt-lamp) using quenching probes for the detection of the middle east respiratory syndrome coronavirus ace inhibition, ace2 and angiotensin-(1-7) axis in kidney and cardiac inflammation and fibrosis ace2 and ace in acute and chronic rejection after human heart transplantation sars: systematic review of treatment effects human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets knowledge synthesis of 100 million biomedical documents augments the deep expression profiling of coronavirus receptors value of autopsy emphasized in the case report of a single patient with middle east respiratory syndrome structure, function, and antigenicity of the sars-cov-2 spike glycoprotein clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 plain radiograph and ct features of 112 patients with sars in acute stage world health organization (who). 2020. coronavirus disease (covid-19) pandemic cryo-em structure of the 2019-ncov spike in the prefusion conformation novel coronavirus pneumonia (covid-19) ct distribution and sign features characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention comparative analysis and refinement of human psc-derived kidney organoid differentiation with single-cell transcriptomics pathological findings of covid-19 associated with acute respiratory distress syndrome high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa laboratory testing of sars-cov, mers-cov, and sars-cov-2 (2019-ncov): current status, challenges, and countermeasures the deadly coronaviruses: the 2003 sars pandemic and the 2020 novel coronavirus epidemic in china the role of imaging in 2019 novel coronavirus pneumonia (covid-19). european radiology. epub ahead of print 15 clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study an ultrahistochemical study of the trabecular meshwork in normal and open-angle glaucomatous eyes chest radiographic and ct findings of the 2019 novel coronavirus disease (covid-19): analysis of nine patients treated in korea for the singapore 2019 novel coronavirus outbreak research team. 2020. epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore molecular and serological investigation of 2019-ncov infected patients: implication of multiple shedding routes estimating the unreported number of novel coronavirus (2019-ncov) cases in china in the first half of january 2020: a data-driven modelling analysis of the early outbreak epidemiological and clinical characteristics analysis of covid-19 in the surrounding areas of wuhan, hubei province in 2020 a pneumonia outbreak associated with a new coronavirus of probable bat origin we thank the language modification of this review by company aje. this study was supported by grants from the hunan innovative provincial construction project (2019sk2211) and the changsha science and technology plan project (kq2001044). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the following grant information was disclosed by the authors: hunan innovative provincial construction project: 2019sk2211. changsha science and technology plan project: kq2001044. the authors declare that they have no competing interests. key: cord-338436-0z828org authors: tzou, philip l.; tao, kaiming; nouhin, janin; rhee, soo-yon; hu, benjamin d.; pai, shruti; parkin, neil; shafer, robert w. title: coronavirus antiviral research database (cov-rdb): an online database designed to facilitate comparisons between candidate anti-coronavirus compounds date: 2020-09-09 journal: viruses doi: 10.3390/v12091006 sha: doc_id: 338436 cord_uid: 0z828org background: to prioritize the development of antiviral compounds, it is necessary to compare their relative preclinical activity and clinical efficacy. methods: we reviewed in vitro, animal model, and clinical studies of candidate anti-coronavirus compounds and placed extracted data in an online relational database. results: as of august 2020, the coronavirus antiviral research database (cov-rdb; covdb.stanford.edu) contained over 2800 cell culture, entry assay, and biochemical experiments, 259 animal model studies, and 73 clinical studies from over 400 published papers. sars-cov-2, sars-cov, and mers-cov account for 85% of the data. approximately 75% of experiments involved compounds with known or likely mechanisms of action, including monoclonal antibodies and receptor binding inhibitors (21%), viral protease inhibitors (17%), miscellaneous host-acting inhibitors (10%), polymerase inhibitors (9%), interferons (7%), fusion inhibitors (5%), and host protease inhibitors (5%). of 975 compounds with known or likely mechanism, 135 (14%) are licensed in the u.s. for other indications, 197 (20%) are licensed outside the u.s. or are in human trials, and 595 (61%) are pre-clinical investigational compounds. conclusion: cov-rdb facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies prioritize the most promising compounds and repurposed drugs for further development. cov-rdb contains four main types of antiviral experimental data, six main lookup/explanation tables, and a registry of ongoing or planned clinical trials. the four main types of antiviral experimental data include (i) cell culture and entry assay experiments; (ii) biochemical experiments; (iii) animal model studies; and (iv) clinical studies. the six main lookup/explanation tables provide information on viruses, virus strains/isolates, tested compounds, compound targets, cell types, and animal models. cov-rdb data are stored in a postgresql relational database, but there is not necessarily a oneto-one relationship for the tables displayed on the web and their underlying database structure. indeed, several of the website tables contain information from more than one underlying database table. as of 14 august 2020, the cov-rdb contains data from more than 1800 virus cell culture experiments, 465 entry assay experiments, 519 biochemical experiments, 259 animal model experiments, and 71 clinical studies from more than 310 peer-reviewed publications and 90 preprints. research articles are identified through incremental daily searches of pubmed and biorxiv using the search term "coronavirus" and by citations identified through reading these papers. publications containing experimental data are imported into a staging zotero database and then annotated to extract the data described in the sections below. preprints that are subsequently published in a peer-reviewed journal are identfied using a computer script that parses datasets downloaded from the stephen b. thacker cdc (centers for disease control and prevention) library the cell culture experiments table contains 13 fields, including four fields present in each of the experiment tables: reference, compound, virus category, and virus isolate/strain. the nine fields unique to the cell culture experiments table include six that describe experimental conditions and the three experiment results are the half-maximal effective concentration (ec 50 ), percent inhibition, and the 50% cytotoxic concentration (cc 50 ). the ec 50 can only be determined using a series of compound dilutions. while the ec 50 is usually reported as µm, inhibitory activity for interferons is also often reported as international units (iu)/ml and inhibitory activity for monoclonal antibodies is often reported as ng/ml. the ec 50 is available for the vast majority of in vitro cell culture experiments. however, for a few experiments, the experimental setup involved a single compound concentration (rather than a dilution series). for these experiments, the percent virus inhibition with the single compound concentration is reported. there are two tables for entry assay experiments-one for pseudovirus entry assays and another for cell-cell fusion assays. the pseudovirus assay table contains the following six unique fields: (i) pseudovirus vector, (ii) pseudovirus number, (iii) target cell type, (iv) time to addition of drug, (v) indicator of virus replication, and (vi) ec 50 . in the pseudovirus experiments, the virus strain is a virus construct composed of a virus that does not require a biosafety level 3 (bsl-3) laboratory, such as vesicular stomatitis virus (vsv) or human immunodeficiency virus type 1 (hiv-1), into which the coronavirus spike (s) gene has been cloned. this construct also has a reporter gene such as luciferase or gfp. the cell-cell fusion assay table contains the following seven unique fields: (i) effector cell type, (ii) effector cell number, (iii) target cell type, (iv) target cell number, (v) time to addition of drug, (vi) indicator of virus replication, and (vii) ec 50 . the biochemical experiments table contains two unique fields: the biochemical target and the half maximal inhibitory concentration (ic 50 ). the biochemical target is usually one of the virus enzymes including rna-dependent rna polymerase (rdrp), main protease (also called 3c-like protease; 3cl pro ), papain-like protease (pl pro ), and helicase. however, cell-free assays that test inhibitors of the spike (s) protein binding to angiotensin converting enzyme 2 (ace2) are also included. the animal model experiments are characterized by comparisons between a group of animals receiving a treatment intervention either shortly before or after virus infection and a group of untreated virus-infected control animals. the animal model experiments table has two parts-experimental conditions and experimental results. the experimental conditions include the (i) animal model, (ii) size and route of virus challenge, (iii) treatment intervention, (iv) treatment dosage, (v) treatment timing in relation to the addition of virus, (vi) number of treated subjects, and (vii) number of control subjects. the experimental results, which often depend on the study, include endpoints such as mortality, weight loss, fever, respiratory rate, lung pathology, and virus load measurements. the reduction of endpoint severity is reported on an ordinal scale ranging from 0 to 3. there are more than 59 references containing more than 259 animal model experiments, nearly all involving sars-cov-2, sars-cov, or mers-cov. approximately 70% of the studies involve mice; 10% involve non-human primates (rhesus macaques, marmosets, and cynomolgus macaques); and 20% involve hamsters, ferrets, cats, dogs, or rabbits. the most commonly studied interventions have included monoclonal antibodies, fusion inhibitors, interferons, and the nucleoside analog polymerase inhibitors. the clinical studies are represented using several enumerated and free-text fields. the enumerated fields include the reference, virus category, and type of study (e.g., observational, randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not randomized trial, randomized placebo-controlled trial). the free-text fields include descriptions of the interventions and regimen details, the study population and methods, and the study findings. cov-rdb does not provide an assessment of study quality such as validity and risk of bias as there are other research groups providing this type of assessment. antiviral data on coronaviruses other than sars-cov-2 provide insight into the robustness of an antiviral compound, in that, compounds that are active against multiple viral species will be more likely to inhibit future pandemic coronaviruses and will be less vulnerable to the development of drug-resistance mutations. indeed, for many drug targets, such as the virus rdrp and 3cl pro enzymes, and for host processes upon which coronaviruses depend, inhibitory compounds will likely have broad-spectrum activity. cov-rdb contains antiviral data for six categories of coronaviruses: sars-cov-2, sars-cov, mers-cov, endemic human coronaviruses, bat coronaviruses, and non-bat mammalian coronaviruses. sars-cov-2 (37%), sars-cov (31%), and mers-cov (17%) account for 85% of the data. however, the proportion of data associated with sars-cov-2 is rapidly increasing. figure 2 shows the distribution of study types for sars-cov-2, sars-cov, and mers-cov. sars-cov and sars-cov-2 belong to the same betacoronavirus 2b (sarbecovirus) clade, and their amino acids are approximately 97% identical in the rdrp and 3cl pro enzymes and 84% identical in the spike protein. in contrast, mers, a clade 2c betacoronavirus, is approximately 75% identical to sars-cov and sars-cov-2 in the rdrp gene, 60% identical in the 3cl pro gene, and 40% identical in the s gene. within each of these three viruses, there is little diversity, with median pairwise distances ranging between 0% and 0.2%. the four endemic human coronaviruses include two clade 2a betacoronaviruses and two alphacoronaviruses. bat coronaviruses are distributed widely among different clades [2, 3] . indeed, 4 of the 9 betacoronavirus clades and 7 of 11 coronavirus clades are found only in bats. the mammalian coronaviruses include murine hepatitis virus (mhv), which is a longstanding experimental model for coronavirus infection, and several other coronaviruses that have been studied because they are important livestock diseases [4] . although infectious bronchitis virus is an avian gammacoronavirus, we have included it in the non-bat mammalian coronavirus category. cov-rdb uses the terms isolate and strains to describe the different viruses used in antiviral studies, although "strains" is usually reserved for describing isolates that have distinct phenotypic properties [5] . where possible, isolates are named according to the recommendations from the international committee on taxonomy of viruses [6] , i.e., virus/host/location/isolate/date. most sars-cov-2 isolates are nearly identical to one another with the upper limit for the pairwise amino acid distance being about 0.1%, although this number varies depending upon the gene [7] . therefore, the biological significance of the isolate used for a particular study is not known. however, for some treatments such as monoclonal antibodies, changes in the sequence encoding the relevant epitope, specifically in the s protein receptor binding domain may prove to have biological and clinical significance [8, 9] . although sars-cov resulted from at least two zoonotic introductions from civet cats [4] , and although mers-cov resulted from multiple zoonotic introductions from dromedary camels, these viruses also demonstrate little genetic variability. several of the most commonly used isolates have been cloned, either as intact viruses (e.g., by plaque purification or limiting dilution) or by constructing a cdna copy representing a single sequence variant. modification of these clones, such as selection of a resistant variant in vitro [10] or introduction of a reporter gene like gfp or luciferase, presumably retains the characteristics of the original parental virus isolate or strain [11] . commonly used isolates that have been cloned and manipulated in the laboratory include mers-cov/human/amsterdam/emc/2012 [12] , sars-cov/human/hanoi/urbani/2003 [13] , sars-cov-2/human/usa/wa1/2020 [14] , and sars-cov-2/human/munich/929/2020 [15] . the cell lines table provides descriptions for the cell lines used in cell culture and entry assay experiments. it contains four fields: (i) the cell line's commonly used name, (ii) the source of the cell line, (iii) closely related cell lines, and (iv) a description of the cell line and one or more of the closely related cell lines. the most commonly used cell lines for sars-cov and sars-cov-2 include a variety of different vero cell clones [16] [17] [18] [19] [20] , huh7 [16, 21] , caco-2 [22] , calu-3 [23] , and 293t/ace2 cells [16] [17] [18] ( table 1) . while each of these cell lines expresses ace2, only calu-3 cells were originally derived from lung epithelial cells. the 293t cells are typically used for cell-cell fusion and pseudovirus entry experiments. several studies have also used human alveolar epithelial cells or a variety of different respiratory system or kidney organoids [24, 25] . the cell lines used for mers-cov are similar, with the main exception that 293t/dpp4 (dipeptidyl peptidase 4) cells are used instead of 293t/ace2 cells because dpp4 (aka cd26) is the mers-cov receptor [18] . vero cells support the replication of many viruses often producing a visual cytopathic effect [16] [17] [18] . they express ace2, the receptor for sars-cov and sars-cov2, and dpp4, the receptor for mers-cov. although vero cells are ifn-deficient, they express the ifn-α/β receptor and thus retain the ability to respond to exogenous ifn [19] . vero e6 cells engineered to express greater amounts of tmprss2 produce higher sars-cov-2 titers of sars-cov-2 [20] . drugs that target tmprss2 are often inactive in vero cells. human lung epithelial cell line calu-3 cells form differentiated pseudostratified columnar epithelia highly permissible to coronavirus infection. they are polarized with an apical domain facing the airway lumen and a basolateral domain facing internally. they produce a visual cytopathic effect. the 2b4 clone has high ace2 expression. they are often used for the preclinical development of respiratory drugs [23] . heterogeneous human epithelial colorectal adenocarcinoma caco-2 cells are considered to be more pharmacologically relevant than vero cells for some studies because of their human origin [22] . human hepatoma huh-7 cells express ace2 and tmprss2, yet do not support levels of replication as high as vero cells [16, 21] . the 293t cells are derived from the human embryonic kidney 293 cell line. 293t cells contain the sv40 large t-antigen, which facilitates replication of transfected plasmids containing the sv40 origin of replication. the 293t/ace2 cells are transfected to express ace2 and have been used for many sars-cov cell-cell fusion and pseudovirus entry inhibitor studies [16] [17] [18] . human airway epithelial cells differentiated human airway cells have occasionally been used to study antiviral agents, although they are more commonly used to study viral pathogenesis [24, 25] . tmprss2-transmembrane serine protease 2; ace2-angiotensin converting enzyme 2; ifn-interferon; dpp4-dipeptidyl peptidase 4; sv40-simian virus 40. the over 10 different animal models used in experiments described in the cov-rdb include three non-human primate models (rhesus macaques, cynomolgus macaque, and marmosets), multiple transgenic and non-transgenic mouse models, and several additional rodent models including hamsters and ferrets [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . the transgenic mice have been modified in multiple ways, including to express hdpp4 so that they can be infected with mers-cov, to knock out the interferon (ifn)-α/β receptor to compromise innate immunity [44] ; to knock out recombination activating gene 1 (rag1) to compromise adaptive immunity [45] ; to knock out carboxylesterase 1c, which causes poor plasma stability of remdesivir; and to express human rather than mouse ace2 [39] [40] [41] . table 2 describes the utility of the most common non-human primate and mammalian models for studies of the pandemic coronaviruses. pathological changes observed in the aged mouse model infected with sars-cov more closely resemble those observed in humans [29] . rag −/− mice lack t and b cells and lack adaptive immunity and experience prolonged coronavirus shedding [45] . ifnar −/− mice are vulnerable to greater coronavirus disease severity [44] . there are many hace2 transgenic mouse models. these mice are more likely to experience weight loss, detectable virus loads, and interstitial pneumonia following challenge with sars-cov-2 than those with the murine ace2 receptor [39] [40] [41] . rhesus macaque infection causes a self-limiting disease associated with virus replication. radiographic and pathologic examination of sars-cov-2-infected animals display evidence of pneumonia [26, 30, 32, 42] . cynomolgus macaque infection results in a productive infection in respiratory epithelial cells. symptoms are minimal but virus shedding can last up to 2 weeks. chest radiographs reveal unifocal or multifocal pneumonia. autopsy reveals variable amounts of foci of diffuse alveolar damage [31, 33] . rag-recombination activating gene; ifnar-interferon-α/β receptor; hace2-human angiotensin-converting enzyme 2. the target table has two main fields: name and description. the target classification organizes drugs, treatments, and compounds according to the virus or host process targeted by a compound including virus enzymes, virus entry into cells, host immunological responses, and other host processes. there are three virus enzyme inhibitor classes: rdrp, protease (including 3cl pro , pl pro ), and helicase inhibitors. there are four inhibitor classes targeting virus entry: convalescent plasma and polyclonal antibody preparations, monoclonal antibodies, miscellaneous other compounds that inhibit virus receptor binding, and fusion inhibitors. there are two classes that target host immunological responses: interferons and other potential immunostimulatory compounds. although there are potentially many mechanisms by which targeting a host processes may interfere with virus replication, we have divided these into two broad categories: host protease enzymes used by coronaviruses to cleave the spike protein, thus, priming it for fusion and other host proteins or pathways utilized by coronaviruses. the classification of host-acting compounds is likely to continue to evolve as mechanistic pathways become better defined. table 3 describes each of the targets and compound classes described above. figure 3 shows the distribution of experimental data types according to target. the database contains experiments involving approximately 1650 compounds. more than 1240 of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs-441524 (i.e., remdesivir) and β-nhydroxycytidine (i.e, eidd-28014), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of 975 compounds with a known or likely mechanism of action, 135 (14%) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid-19), 197 (20%) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and 595 (61%) are preclinical investigational compounds. the database contains experiments involving approximately 1650 compounds. more than 1240 of these compounds appear in the online compounds tables, which contain the following fields: (i) name, (ii) synonyms including abbreviations, (iii) closely related compounds, (iv) drug availability, (v) drug class, (vi) target, and (vii) description. for interferons, the drug class is the type of interferon (α, β, γ, or λ). for monoclonal antibodies, additional data are stored and displayed, including the antibody source and information on sequence and structure availability. the closely related compounds are subjectively defined as those that we intend to be returned by a query even if that compound was not entered by the user. there are five broad categories of closely related compounds: (i) monoclonal antibodies described in the same publication, (ii) interferons belonging to the same type (i.e., α, β, γ, or λ), (iii) a series of compounds derived from the same lead compound, (iv) prodrugs such as those for gs-441524 (i.e., remdesivir) and β-n-hydroxycytidine (i.e, eidd-28014), (v) drug combinations such as lopinavir and ritonavir-boosted lopinavir (lopinavir/r), and (vi) compounds presumed to act by a highly similar mechanism of action (e.g., hydroxychloroquine and chloroquine). the drug availability category indicates whether the compound has been licensed in the u.s. or another country or has been studied in humans. of 975 compounds with a known or likely mechanism of action, 135 (14%) are u.s. fda (u.s. food and drug administration) -approved drugs (for indications other than covid-19), 197 (20%) have been or are currently being evaluated in human clinical trials or are approved outside the u.s., and 595 (61%) are preclinical investigational compounds. table 3 . antiviral coronavirus therapy targets. inhibitors of the coronavirus rna-directed rna polymerase (rdrp) enzymes include nucleoside analogs that cause immediate chain termination, delayed chain termination, or viral mutagenesis. coronaviruses contain two protease enzymes: 3 chymotrypsin-like cysteine protease (3cl pro or main (m)-pro) and papain-like (pl pro ). coronavirus helicases catalyze the unwinding of duplex rna molecules into single strands. entry convalescent plasma and polyclonal sera. convalescent plasma is one of the most widely studied treatments for covid-19. polyclonal sera and immunoglobuline preparations have also entered clinical trials. recovery during sars-cov, mers-cov, and sars-cov-2 is usually associated with the development of neutralizing antibodies. most sars-cov-2 neutralizing mabs target the part of the receptor binding domain (rbd) that binds ace2 while most mers-cov neutralizing mabs target the part of the rbd that binds dpp4. many highly potent neutralizing mabs targeting each of the pandemic coronaviruses have shown protection in vitro and in animal models. structural studies have defined specific rbd epitopes recognized by individual mabs and identified amino acid residues that are critical for mab binding. other receptor binding inhibitors sars-cov and sars-cov-2 spike s1 binds to the cellular angiotensin converting enzyme 2 (ace2) receptor. mers-cov binds to dipeptidyl peptidase 4 (dpp4). a variety of compounds including non-antibody proteins, peptides, and small molecules have been shown to prevent the binding of the coronavirus spike protein to its cellular receptor. following receptor binding and spike s1/s2 cleavage and s2 priming, heptad region 1 (hr1), which is close to the fusion peptide sequence, and hr2, which is close to the virus membrane, collapse on to one another to bring virus and cell membranes together. nearly all fusion inhibitors are hr2-mimicking peptides less than 70 kda that bind hr1, thus preventing hr1−hr2 binding. interferons have been extensively studied for their ability to inhibit each of the pandemic coronaviruses in cell culture, animal models, and/or clinical studies [47] . sars-cov-2 may be more susceptible to interferons than sars-cov is [48] . there are several clinical trials using immunostimulatory cytokines and compounds purported to induce interferon. cleavage of coronavirus spike proteins is necessary for the virus to transition from receptor attachment to cell fusion. for sars-cov-2, there is a poly-basic furin cleavage site at the s1/s2 boundary and another cleavage site within s2 believed to be cleaved at the cell surface by host tmprss2 enzymes [49] . multiple intracellular processes essential to virus replication are vulnerable to pharmacologic inhibitors including endosomal acidification, membrane formation, various signaling pathways, nucleotide biosynthesis, and autophagy [50] . many compounds with uncertain mechanisms of action have been found to inhibit coronaviruses in vitro. several of these are also being studied in clinical trials. mabs-monoclonal antibodies. figure 4 displays ec 50 values for many of the directly acting antiviral compounds currently in clinical trials for the treatment of covid-19 including six polymerase inhibitors (remdesivir, eidd-2801, favipiravir, ribavirin, galidesivir, and sofosbuvir), three hiv-1 protease inhibitors (lopinavir, atazanavir, and darunavir), and three entry inhibitors (receptor binding monoclonal antibodies, soluble recombinant human ace2, and umifenovir). figure 5 displays ec 50 values for many of the repurposed compounds that target host processes required for virus replication including two host pis that target the transmembrane serine protease 2 (tmprss2) enzyme (camostat and nafamostat), three chloroquine analogs that interfere with endosomal acidification (chloroquine, hydroxychloroquine, and mefloquine), three other compounds believed to interfere with membrane trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). trafficking (niclosamide, imatinib, and chlorpromazine), and four compounds acting by a variety of different cellular mechanisms (ivermectin, nitazoxanide, ciclesonide, and cyclosporin). figures 4 and 5 show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec50s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above 100 μm. however, there is also marked heterogeneity in the ec50 values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss2 inhibitors camostat and nafamostat are practically inactive against sars-cov-2 in vero cells but have ec50s consistently below 1 μm in caco-2 and calu-3 cells, because these cells require tmprss2 for virus replication whereas vero cells do not [22, [51] [52] [53] [54] [55] [56] . 5 show that the potency of currently studied compounds extends over several orders of magnitude with monoclonal antibodies having ec 50 s in the high picomolar to low nanomolar range and some compounds displaying no activity at concentrations above 100 µm. however, there is also marked heterogeneity in the ec 50 values for the same compound in different experiments. for several drugs, the heterogeneity can be explained by the type of cells used, inoculum size, drug timing, and culture duration. for example, the host tmprss2 inhibitors camostat and nafamostat are practically inactive against sars-cov-2 in vero cells but have ec 50 s consistently below 1 µm in caco-2 and calu-3 cells, because these cells require tmprss2 for virus replication whereas vero cells do not [22, [51] [52] [53] [54] [55] [56] . viruses 2020, 12, x for peer review 11 of 22 table 4 describes a set of the most promising compounds for the treatment of sars-cov-2 based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display submicromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table 4 . promising sars-cov-2 antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a 1′cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov-2 particularly in cells other than vero cells [21, 22, [57] [58] [59] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [58, 60] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from 15 to 11 days (p < 0.001) and a non-statistically significant reduction in day 14 mortality of 11.9% vs. 7.1% (p = 0.06) [61] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid-19. ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n4hydroxycytidine-5′isopropyl ester (eidd-2801) eidd-2801 is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov-2 [62] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [62] . it is being evaluated in two phase ii clinical trials. table 4 describes a set of the most promising compounds for the treatment of sars-cov-2 based on the following criteria: (i) act by a validated direct or indirect antiviral mechanism, (ii) display sub-micromolar activity in vitro and/or inhibitory activity in an animal model, and (iii) have a record of safety and favorable pharmacokinetics in human subjects. the majority of these compounds are being studied in clinical trials, although the numbers of these trials are far fewer than those for less promising compounds. table 4 . promising sars-cov-2 antiviral compounds. remdesivir is a delayed chain terminator monophosphate prodrug of a 1 -cyano-substituted adenine c-nucleoside analog. it has high nanomolar inhibitory activity in vitro against sars-cov-2 particularly in cells other than vero cells [21, 22, [57] [58] [59] . it reduces viral replication and lung pathology in mice and rhesus macaques when administered shortly after infection [58, 60] . in a double-blind randomized clinical trial, its intravenous administration led to a significant reduction in time to recovery from 15 to 11 days (p < 0.001) and a non-statistically significant reduction in day 14 mortality of 11.9% vs. 7.1% (p = 0.06) [61] . based on this trial, the fda issued an emergency use authorization for remdesivir in patients with severe covid-19. ongoing trials are examining its safety and efficacy when administered subcutaneously or via inhalation. β-d-n4hydroxycytidine-5isopropyl ester (eidd-2801) eidd-2801 is a nucleoside analog, which like remdesivir has high nanomolar inhibitory activity in vitro against sars-cov-2 [62] . it reduces sars-cov and mers-cov replication and lung pathology in a mouse model [62] . it is being evaluated in two phase ii clinical trials. regn10933 and regn10987 are mabs with subnanomolar inhibitory activity that bind to non-overlapping ace2-competing sars-cov-2 spike receptor binding domain epitopes [8, 63] . this mab combination also reduces virus replication and lung pathology in syrian hamsters and rhesus macaques [64] . the combination is being evaluated in phase iii trials for preventing and treating sars-cov-2 infection. ly3819253 is a sars-cov-2 mab in phase iii trials for preventing and treating covid-19. as of august 2020, there are no associated published preclinical data. mabs (phase i/ii trials) azd7442, brii-196, js016, scta01, sti-1499, and ty027 are mabs in phase i/ii trials. as of august 2020, there are no associated published preclinical data linked to mabs with these names. many research groups that have published preclinical data on one or more mabs (or mab variants such as nanobodies) including their in vitro inhibitory activity, genetic sequence data, three-dimensional structural data, and/or animal model data [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] . interferons ifn-α, ifn-β, and ifn-λ ifn-α, ifn-β, and ifn-λ each inhibit sars-cov-2 by 90%-99% at low concentrations of about 100 international units (iu)/ml [48, [81] [82] [83] [84] . inhalational ifn-α and parenteral ifn-β were associated with modest reductions in disease severity and/or virus loads in two small open-label clinical trials [84, 85] . an inhaled formulation of ifn-β was reported in the news to significantly reduce the odds of developing severe disease or death in a blinded randomized control trial (sng016) of 220 patients that has not yet been published (https://www.synairgen.com/covid-19/). there are currently four planned or ongoing placebo-controlled trials of parenteral or inhaled ifn-β (~1800 patients) and of parenteral ifn-λ (~400 patients). camostat and nafamostat are tmprss2 inhibitors with nanomolar coronavirus inhibitory activity in biochemical and cell culture assays [51, [53] [54] [55] [56] [86] [87] [88] . both drugs are used in japan for the treatment of pancreatitis, while nafamostat is also used as an anticoagulant and for the treatment of disseminated intravascular coagulation. although nafamostat has approximately 10-fold greater inhibitory activity than camostat, it may be associated with greater toxicity. camostat is being studied in two blinded and two open-label randomized controlled studies totaling about 900 patients. nafamostat is being studied in three small randomized open-label studies totaling about 200 patients. apilimod was found to inhibit sars-cov-2 at two-digit nanomolar levels with high selectivity indexes in multiple drug screens [89] [90] [91] [92] . it inhibits the membrane protein pi (3, 5) p2 by inhibiting the enzyme pi-3p-5-kinase (pikfyve) thus interfering with endosomal trafficking of sars-cov-2 and additional viruses utilizing the same endosomal pathway [93, 94] . it has been studied in humans in multiple clinical trials and been found to be safe and well tolerated. it is being studied for the treatment of mild sars-cov-2 infections in one randomized placebo-controlled phase ii trial. ptc299 is an inhibitor of dihydroorotate dehydrogenase (dhodh), a rate limiting enzyme in the pyrimidine biosynthesis pathway [95] . dhodh inhibitors are therapeutic targets for autoimmune disases and viral infections [96, 97] . ptc299 has been found to be safe and have favorable pharmacokinetics in more than 300 human subjects and has low nanomolar sars-cov-2 inhibitory activity and a high selectivity index [98] . as both viral replication and cytokine overproduction depend on pyrmidine synthesis, dhodh inhibition may have a dual role in covid-19 treatment. dhodh inhibition is synergistic with viral polymerase inhibition [97] . there is one phase ii/iii trial of ptc299 for patients with severe but not critical covid-19. leflunomide, another repurposed dhodh inhibitor was found to reduce virus load in an open-label pilot study of 27 patients [99] . soluble recombinant human ace2 (rhace2) rhace2 protects mice for sars-cov-1 ards and has been studied as a treatment for ards in humans [100, 101] . it inhibits sars-cov-2 spike binding at nanomolar concentrations in a wide variety of cell lines [102, 103] . there are two ongoing phase ii trials of an intravenous commercial rhace2 preparation. notes: some compounds with sub-micromolar in vitro activity are not included in this table either because (i) they have not been used in humans, (ii) they have been identified only in high-throughput drug screens, or (iii) they have unfavorable pharmacokinetics. several compounds that inhibit sars-cov-2 less potently but are being studied as inhalational and/or intranasal therapies are also not shown. this last category includes (i) compounds that inhibit the interaction of sars-cov-2 spike and cell surface heparin sulfate proteoglycans such as lactoferrin and heparin; and (ii) ciclesonide, an inhaled corticosteroid that may interfere with membrane trafficking by binding directly to nsp-3 or nsp-4 or indirectly through a host protein. the clinical trials registry table is a regularly updated, annotated list of ongoing, planned, or completed clinical trials obtained from the clinicaltrials.gov, who ictrp, and chinese clinical trial websites. it contains trials of compounds with potential antiviral activity but not studies of non-antiviral interventions, such as those designed to optimize intensive-care management or reduce the inflammatory response associated with severe covid-19. the clinical trials registry classifies trials according to the compound target, the type of trial (e.g., observational or randomized controlled study), the status of the trial (pending, active, or completed), and the population studied. as of 6 august 2020, it contains more than 700 trials of which about 73% are listed on clinicaltrials.gov and 27% are listed only on the who international clinical trials platform. figure 6a displays the distribution of planned, ongoing, and published studies according to the compound targets of the drugs studied. figure 6b displays the same distribution for those drugs in three or more studies. it is notable that many of the most commonly studied compounds have either little or no activity against sars-cov-2, including several drugs used for non-coronavirus infections such as the hiv protease inhibitors lopinavir and darunavir and the influenza inhibitors favipiravir, oseltamivir, and umifenovir. the chloroquine analogs, chloroquine and hydroxychloroquine, have weak in vitro activity but have failed to show clinical efficacy in multiple clinical trials [104] [105] [106] [107] [108] [109] [110] . the search function allows users to specify one or more of the following options from four drop-down lists: (i) compound target, (ii) compound, (iii) virus category, and (iv) study type. if the user selects "any" for one of these and leaves the others in their default position, the search function returns the database's complete set of cell culture experiments, biochemical experiments, entry assay experiments, animal model studies, and published clinical studies. by selecting one or more of the above options, the search function restricts the data returned to those meeting the search criteria. by using the "copy to clipboard" link, users can import the results of any query into a spreadsheet where they can further sort and filter query results. the search function also provides a link to the trials in the clinical trials registry for selected compounds and compound targets. the compound drop-down list displays 60 of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional 363 closely related compounds (as described in the compound table section 2.2.6). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. the compound drop-down list displays 60 of the most well recognized compounds. selecting a compound returns the data for that compound as well as for an additional 363 closely related compounds (as described in the compound table section 2.2.6). if the user selects the compound target from the dropdown menu, then the compound menu will list all compounds designed to inhibit the selected target. the compounds entry on the compounds page also links to all the data on that compound in the database. to prioritize licensed drugs and investigational compounds for the treatment of covid-19, it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal to prioritize licensed drugs and investigational compounds for the treatment of covid-19, it is necessary to compare their relative antiviral activities. compounds that are not active in vitro will almost certainly not be useful clinically. therefore, pre-clinical data are necessary to prioritize animal model and clinical studies. compounds that are active in vitro, however, may also not be clinically useful if their associated in vitro data do not reflect physiologic conditions or if standard dosing with these compounds does not result in sufficient inhibitory concentrations at sites of infection. the creation of the cov-rdb was primarily motivated by the observation that many of the drugs being evaluated in covid-19 clinical trials demonstrate little or no in vitro anti-coronavirus activity. for example, as recently as july 2020, four of the most commonly studied drugs-chloroquine analogs, azithromycin, lopinavir/r, and favipiravir-demonstrated little if any in vitro activity. the creation of the cov-rdb was secondarily motivated by the observation that results for the same compound often vary across different laboratories as a result of experimental design such as cell line, inoculum size, drug-addition timing, duration of culture, and method for measuring virus replication. given sufficient data, a database makes it possible to eventually identify the experimental features responsible for the heterogeneity in published results, thus improving the ability to compare the antiviral activity of different compounds. indeed, we have already noted in this manuscript several compound classes for which viral inhibition is influenced by the cells used for virus culture. the cov-rdb is also designed to be educational as it provides multiple lookup tables for the viruses, drugs, cell lines, and animal models used in reported experiments. these tables contain descriptions of viruses, virus isolate/strains, cell lines, animal models, and more than 300 licensed and investigational compounds. work is underway to also add comprehensive, yet detailed, summaries of sars-cov-2 monoclonal antibodies and of pharmacokinetic data for those drugs with well-documented in vitro inhibitory activity. there are several additional web resources devoted to coronavirus drug development including sites devoted to high-throughput drug screening [111] , the genetics of monoclonal antibodies [112] , and meta-analyses of published clinical trials [113, 114] . the national institutes of health (nih) recognizes the importance of such resources and has recently announced a notice of special interest: national institute of allergy and infectious diseases (niaid) priorities for biomedical knowledgebases and repositories (not-ai-20-044). the cov-rdb database, user interface, and underlying computer code represent a framework for organizing a vast amount of data and for facilitating data curation. however, the value of this resource depends upon ongoing manual data curation and annotation. in conclusion, the cov-rdb provides a uniquely integrated interdisciplinary synthesis of in vitro, animal model, and clinical studies of compounds with proven or possible anti-coronavirus activity. it helps researchers place their findings in the context of previously published data and it facilitates comparisons between different candidate antiviral compounds, thereby helping scientists, clinical investigators, public health officials, and funding agencies to prioritize the most promising compounds and repurposed drugs for further development. the authors declare no conflict of interest. centers for disease control and prevention (cdc) the stepehn b. thacker cdc library research articles downloadable database origin and evolution of pathogenic coronaviruses viral determinants of interspecies transmission virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family filoviridae coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 the establishment of reference sequence for sars-cov-2 and variation analysis antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies escape from neutralizing antibodies by sars-cov-2 spike protein variants coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease coronavirus reverse genetic systems: infectious clones and replicons reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus an infectious cdna clone of sars-cov-2 rapid reconstruction of sars-cov-2 using a synthetic genomics platform sars-coronavirus-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology sars-associated coronavirus replication in cell lines differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation regulation of the interferon system: evidence that vero cells have a genetic defect in interferon production enhanced isolation of sars-cov-2 by tmprss2-expressing cells morphological cell profiling of sars-cov-2 infection identifies drug repurposing candidates for covid-19 identification of inhibitors of sars-cov-2 in-vitro cellular toxicity in human (caco-2) cells using a large scale drug repurposing collection comparative analysis of antiviral efficacy of fda-approved drugs against sars-cov-2 in human lung cells: nafamostat is the most potent antiviral drug candidate characterization and treatment of sars-cov-2 in nasal and bronchial human airway epithelia replication of sars-cov-2 in human respiratory epithelium replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys pneumonitis and multi-organ system disease in common marmosets (callithrix jacchus) infected with the severe acute respiratory syndrome-associated coronavirus animal models for sars animal models for sars and mers coronaviruses treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques cynomolgus macaque as an animal model for severe acute respiratory syndrome respiratory disease in rhesus macaques inoculated with sars-cov-2 comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model rapid isolation of potent sars-cov-2 neutralizing antibodies and protection in a small animal model susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 antiviral efficacies of fda-approved drugs against sars-cov-2 infection in ferrets infection and rapid transmission of sars-cov-2 in ferrets pathogenesis and transmission of sars-cov-2 in golden hamsters the pathogenicity of sars-cov-2 in hace2 transgenic mice pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2 covid-19 preclinical models: human angiotensin-converting enzyme 2 transgenic mice infection with novel coronavirus (sars-cov-2) causes pneumonia in rhesus macaques syrian hamsters as a small animal model for sars-cov-2 infection and countermeasure development passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset type i and type iii interferons-induction, signaling, evasion, and application to combat covid-19 sars-cov-2 sensitive to type i interferon pretreatment coronavirus membrane fusion mechanism offers a potential target for antiviral development coronaviruses -drug discovery and therapeutic options the anticoagulant nafamostat potently inhibits sars-cov-2 s protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner. viruses identification of antiviral drug candidates against sars-cov-2 from fda-approved drugs sars-cov-2 and sars-cov differ in their cell tropism and drug sensitivity profiles comparative analysis of antiviral efficacy of fda-approved drugs against sars-cov-2 in human lung cells sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor nafamostat mesylate blocks activation of sars-cov-2: new treatment option for covid-19 a large-scale drug repositioning survey for sars-cov-2 antivirals remdesivir inhibits sars-cov-2 in human lung cells and chimeric sars-cov expressing the sars-cov-2 rna polymerase in mice drug repurposing screens reveal fda approved drugs active against sars-cov-2 clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 remdesivir for the treatment of covid-19 -preliminary report an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice studies in humanized mice and convalescent humans yield a sars-cov-2 antibody cocktail regn-cov2 antibody cocktail prevents and treats sars-cov-2 infection in rhesus macaques and hamsters a human neutralizing antibody targets the receptor binding site of sars-cov-2 human neutralizing antibodies elicited by sars-cov-2 infection cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody convergent antibody responses to sars-cov-2 in convalescent individuals isolation of potent sars-cov-2 neutralizing antibodies and protection from disease in a small animal model potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells neutralization of sars-cov-2 by destruction of the prefusion spike neutralizing nanobodies bind sars-cov-2 spike rbd and block interaction with ace2 potently neutralizing and protective human antibodies against sars-cov-2 a cross-reactive human iga monoclonal antibody blocks sars-cov-2 spike-ace2 interaction a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2 broad neutralization of sars-related viruses by human monoclonal antibodies a potently neutralizing antibody protects mice against sars-cov-2 infection analysis of a sars-cov-2-infected individual reveals development of potent neutralizing antibodies with limited somatic mutation inhibition of sars-cov-2 by type i and type iii interferons antiviral activities of type i interferons to sars-cov-2 infection type i and type iii ifn restrict sars-cov-2 infection of human airway epithelial cultures sars-cov-2 clearance in covid-19 patients with novaferon treatment: a randomized, open-label, parallel group trial triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial alpha-1 antitrypsin inhibits sars-cov-2 infection molecular mechanism of sars-cov-2 cell entry inhibition via tmprss2 by camostat and nafamostat mesylate an enzymatic tmprss2 assay for assessment of clinical candidates and discovery of inhibitors as potential treatment of covid-19 discovery of sars-cov-2 antiviral drugs through large-scale compound repurposing the global phosphorylation landscape of sars-cov-2 infection drug repurposing screen for compounds inhibiting the cytopathic effect of sars-cov-2 oral drug repositioning candidates and synergistic remdesivir combinations for the prophylaxis and treatment of covid-19 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov inhibition of pikfyve kinase prevents infection by zaire ebolavirus and sars-cov-2 targeting of hematologic malignancies with ptc299, a novel potent inhibitor of dihydroorotate dehydrogenase with favorable pharmaceutical properties broad-spectrum inhibition of common respiratory rna viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response enhancing the antiviral efficacy of rna-dependent rna polymerase inhibition by combination with modulators of pyrimidine metabolism the dhodh inhibitor ptc299 arrests sars-cov-2 replication and suppresses induction of inflammatory cytokines efficacy and safety of leflunomide for refractory covid-19: an open-label controlled study angiotensin-converting enzyme 2 protects from severe acute lung failure a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig effect of hydroxychloroquine in hospitalized patients with covid-19: preliminary results from a multi-centre, randomized hydroxychloroquine in nonhospitalized adults with early covid-19: a randomized trial hydroxychloroquine with or without azithromycin in mild-to-moderate covid-19 hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial a cluster-randomized trial of hydroxychloroquine as prevention of covid-19 transmission and disease hydroxychloroquine for early treatment of adults with mild covid-19: a randomized-controlled trial a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 an opendata portal to share covid-19 drug repurposing data in real time the coronavirus antibody database. biorxiv 2020 characteristics of registered studies for coronavirus disease 2019 (covid-19): a systematic review ongoing clinical trials for the management of the covid-19 pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-326133-d46wbfrx authors: nakayasu, ernesto s.; nicora, carrie d.; sims, amy c.; burnum-johnson, kristin e.; kim, young-mo; kyle, jennifer e.; matzke, melissa m.; shukla, anil k.; chu, rosalie k.; schepmoes, athena a.; jacobs, jon m.; baric, ralph s.; webb-robertson, bobbie-jo; smith, richard d.; metz, thomas o. title: mplex: a robust and universal protocol for single-sample integrative proteomic, metabolomic, and lipidomic analyses date: 2016-05-10 journal: msystems doi: 10.1128/msystems.00043-16 sha: doc_id: 326133 cord_uid: d46wbfrx integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. the metabolite, protein, and lipid extraction (mplex) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. to illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with middle east respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. the mplex method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental, in vitro, and clinical). importance in systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. thus, the prospect of extracting different types of molecules (e.g., dnas, rnas, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample. author video: an author video summary of this article is available. given metabolite can be measured by metabolomics, which can result from the regulation of either its biosynthetic or degradation pathways. however, also measuring the levels of enzymes of each pathway using proteomics can reveal which mechanism is being regulated. further, measurements of the enzyme rna levels can also provide key information on whether the regulation occurs at the transcriptional or posttranscriptional level. for example, bordbar et al. built a metabolic network model based on available genomic sequences to study macrophage activation and subsequently used transcriptomics, proteomics, and metabolomics information to further refine the model, which led to a better understanding of the impact of metabolism during an inflammatory response (1) . in the context of multi-omics analyses, being able to perform multiple measurements on the same sample can also decrease experimental variation. additionally, this approach can be very useful when samples are difficult to obtain, i.e., for some environmental and patient samples (e.g., biopsy specimens) and for samples from high-biosafety-level laboratories, where working conditions are not optimal and are otherwise rigorously controlled. in addition, limited volumes or amounts of samples may preclude splitting them prior to performing parallel extractions and sample processing. recent studies have evaluated the use of variations of chloroform/methanol extraction methods to isolate proteins, metabolites, and lipids or to sequentially extract dna, rna, proteins, metabolites, and lipids, sometimes with the use of different commercial kits, and all from the same sample (5) (6) (7) (8) (9) . while the use of chloroform/ methanol mixtures is well established for metabolomics and lipidomics sample preparation (we routinely use such a protocol in our laboratory), the reproducibility of proteomics, transcriptomics, and genomics measurements and their applicability for a diverse range of samples require further investigation. indeed, we have found only a single report of an evaluation of the reproducibility of extraction of rna and protein and of the reproducibility of the resulting proteomics data from a single sample type; weckwerth et al. found that rna and protein that were extracted from arabidopsis thaliana had coefficients of variation (cvs) of 30% and 17%, respectively, when using a multi-omic extraction protocol based on the use of chloroform/methanol (7) . targeted quantification of peptides mapping to 22 proteins showed cvs of 17% on average. recent analysis of the material obtained using different kits for multiple extractions showed reduced yields and/or quality of the end products (10) . this could have been due to the fact that optimum buffers and solutions differ for extracting dna, rna, proteins, or metabolites and that longer extraction protocols may lead to material degradation. methods employing organic solvent extractions, such as the combination of chloroform, methanol, and water, have been widely used for extracting lipids and other metabolites (11, 12) . in this procedure, a chloroform and methanol solution is added to samples resuspended in water or aqueous buffer, or directly to samples that have sufficient water content, so as to induce the formation of two solvent layers-an upper aqueous phase, containing hydrophilic metabolites, and a lower organic phase, containing lipids and other hydrophobic metabolites-while proteins precipitate in the interphase. since organic solvent extraction is a simple and quick procedure, we reasoned, as others have (5) (6) (7) (8) , that it would prevent protein loss by degradation and make possible the simultaneous extraction of lipids, metabolites, and proteins for subsequent omics analyses. furthermore, organic solvents can be easily removed by evaporation, minimizing the introduction of artifacts during sample preparation. in this work, we sought to develop a robust protocol for simultaneous metabolite, protein, and lipid extraction (mplex) from the same samples for integrative multi-omic analyses. we based the protocol on a chloroform-methanol-water extraction method routinely used in our laboratory to simultaneously prepare metabolite and lipid extracts from the same sample. others have demonstrated the reproducibility of the resulting metabolomics and lipidomics data in using variations of this protocol for select sample types (5, 7, 9, 13) . to evaluate the broad applicability of expansion of this method for proteomics, we performed comprehensive proteomics analyses of the protein material extracted with the mplex procedure from a variety of samples, including a gramnegative bacterium, an archaeon, an environmental microbial community, a plant leaf, a murine tissue, a human body fluid, and a cell line. we found that the proteome coverage for this diverse set of samples was very similar to that seen with matched control samples prepared in parallel using a standard proteomics sample preparation method, suggesting the broad applicability of the protocol. we then applied this methodology and integrated proteomic, lipidomic, and metabolomic analyses in the study of middle east respiratory syndrome coronavirus (mers-cov) infections in a lung epithelial cell line, which showed the impact of viral infection on different host metabolic pathways. impact of different metabolite extraction methods on proteomic analysis. integrative multi-omics analysis is a powerful approach to study complex biological responses and has gained popularity in recent years (1) (2) (3) . in this context, the prospect of being able to perform multiple omics measurements on the same sample is very attractive but the method is still difficult to implement, likely due to the distinct optimal conditions for extracting different types of molecules. aiming to develop a protocol for global multi-omic analyses of the same sample, we modified an extraction approach based on a chloroform-methanol-water solution to simultaneously obtain metabolite, protein, and lipid fractions. since the protocol is well established and since we have applied it successfully for the analysis of lipids and other metabolites in several studies (14) (15) (16) (17) (18) (19) , we focused our efforts on determining if the method is applicable for global proteomic analysis and the associated quantification of relative amounts of proteins (i.e., the determination of fold increase or decrease in protein expression). we tested the mplex method with the gram-negative bacterium shewanella oneidensis by extracting its proteins, lipids, and metabolites (n ϭ 5). as a comparison, we also performed extractions using 100% methanol (meoh) or 100% acetonitrile (acn) (n ϭ 5 [each]), which are commonly used solvents for metabolomics extractions. we found that significantly reduced total protein fractions were recovered after extraction of metabolites and lipids by all three methods compared to control samples prepared using a standard protocol (control) (fig. 1a) . these results are in agreement with previous data from the literature showing that some protein mass is lost during precipitation procedures (20) . we then evaluated if these protein losses affected the ability to obtain useful proteomic data, since a method that can simultaneously extract multiple omics sources from the same sample would be extremely useful for systems biology experiments and subsequent integrated data analysis, as well as in cases where limited sample amounts are available (e.g., a survey of data from the national cancer institute showed that obtaining an adequate number of samples to conduct a study is a major difficulty facing researchers [21] ). thus, we investigated whether extraction with organic solvents would have a major impact on the coverage and the quantitative aspect of the associated proteomic analysis. to explore this issue, proteins extracted with mplex, acn, and meoh methods were digested in parallel with control samples, normalized by bicinchoninic acid (bca) assay, and analyzed by liquid chromatography mass spectrometry (lc-ms) using the accurate mass & time (amt) tag approach (22) . the results of the proteomic analysis of samples extracted with different methods showed that the numbers of peptides detected in the mplex samples were very similar (no significant difference) to the numbers seen with controls (fig. 1b) . a significant increase in the levels of peptides was identified in samples extracted with acn, but no significant differences between the control and meoh extractions in the numbers of peptides were observed (fig. 1b) . the overlap of the numbers of peptides identified in samples extracted with all protocols was very high, as shown by a similarity matrix (fig. 1c) . the similarities between samples were even higher at the protein level ( fig. 1d and e). the similarity of the proteome coverage results obtained by the different extraction methods is remarkable, considering that much larger (up to 3-fold to 4-fold) differences are observed just by digesting proteins using different buffers, surfactants, or denaturing agents, even without any previous extraction (23, 24) . our results show that despite some protein mass losses, the choice of extraction protocol did not significantly affect the proteome coverage. the selective loss of a few proteins during the extraction procedure is expected and has been shown in a study carried out with human plasma samples only (20) . another important feature for multi-omic analysis is that of being able to accurately identify differentially expressed or abundant molecules. in this context, if the extraction procedure affects the quality of the proteins, then it might increase the variance across different samples. thus, we examined the correlation of the proteomics data between samples extracted with different organic solvents, and the results showed remarkable similarity at both peptide and protein levels ( fig. 1f and g). we then calculated the variance of protein measurements by comparing different extraction protocols. indeed, no significant differences in the distributions of coefficients of variance (cv) were observed comparing mplex with controls, with the cvs of the great majority of the proteins smaller than 25%, with peaks of ͻ10% (fig. 1h ). meoh extraction led to cvs that were similar to but slightly smaller than those seen with the mplex and control samples (fig. 1h ). on the other hand, acn extraction had a bimodal distribution, with very low and very high cvs (fig. 1h) , suggesting that some proteins are not reproducibly precipitated with this solvent. this phenomenon might be due to the fact that acetonitrile does not fully precipitate small proteins (25) . taken together, these results showed that mplex did not affect the proteome coverage or the results of quantitative analysis of the s. oneidensis samples. to investigate whether the mplex protocol is robust and broadly applicable, we performed proteomic analyses of a very diverse set of samples that included the archaeon sulfolobus acidocaldarius, a unicyanobacterial consortium (26) , mouse brain cortex tissue, human urine, cells of the calu-3 human lung epithelial cell line, and leaves from arabidopsis thaliana. whereas we compared mplex results to control results for most of these samples, the a. thaliana sample results were compared to results of extractions performed with saturated phenol or trichloroacetic acid (tca), because plant leaves are rich in phenolic compounds that need to be removed and that otherwise would interfere with mass spectrometric analysis, and these alternative protocols have been shown to perform well in preparations of plant samples (27) . as observed for s. oneidensis, the proteome coverage was very high at both the peptide (see fig. s1 in the supplemental material) and protein ( fig. 2 ) levels across the diverse set of samples when using mplex and comparable to that obtained using the standard protein extraction protocol, although minor differences were detected for the unicyanobacterial consortium and human urine samples. in the case of a. thaliana, similar proteome coverage results were observed in samples extracted using either tca or mplex ( fig. 2e ; see also fig. s1e). however, despite repeating the experiment twice, we had very limited success in extracting leaf proteins using the phenol protocol. in terms of quantitative measurements, similar correlations were observed across different samples by comparing mplex results to control or tca extraction results at both the peptide and protein levels, although minor differences were observed in the results from the human urine samples ( fig. 2 ; see also fig. s1). overall, comparing mplex to control or tca extraction, the levels of proteome coverage and correlation between samples were very similar (see fig. s2 ), suggesting no qualitative losses. the fact that the proteome coverage, correlation, and variability results of comparisons of samples using mplex are not different from those seen with the standard protocol indicates that the relative quantification of proteins, which is the type of quantification employed in the vast majority of proteomics studies, is not compromised. nonetheless, we investigated any losses of specific proteins that could affect studies focusing on absolute quantification of protein copy numbers. only 1.1% and 1.9% of the proteins in shewanella oneidensis were shown to be significantly enriched and depleted by more than 2-fold, respectively ( table 1 ). the acn extraction showed a smaller number of significantly enriched or depleted proteins, which was likely a consequence of the higher variability in the replicates observed using this solvent (table 1 ). in contrast, the meoh extraction showed much higher losses than mplex (table 1) . with the exception of the human urine sample, all samples had losses corresponding to less than 5% of the proteins (table 1) . to investigate possible causes of protein enrichment or depletion using mplex, several physical-chemical properties of the significantly enriched or depleted proteins were examined, including the number of proteins with transmembrane domains, molecular weight, length, hydrophobicity calculated by grand average of hydropathy (gravy), and isolectric point (pi). no pattern was consistently observed across the different samples for any of the tested physical-chemical properties, indicating that the small amount of enrichment or depletion of proteins induced by mplex is not based on such properties. although these small differences in protein extraction results seen using mplex should be considered in proteomics studies employing absolute quantification, they likely do not introduce artifacts in the results, as these studies typically have very small (up to 15%) errors when stable isotope-labeled peptides are used as internal standards (28) and up to 2-fold to 3-fold variations in label-free analyses (29, 30) . although protein oxidation is an important physiological posttranslational modification, it is also an artifact introduced during sample processing for proteomic analysis. considering that there is more o 2 dissolved in organic solvents than in water (31), it is reasonable to suspect that extraction performed with such solvents could increase the oxidation of peptides. thus, the number of peptides containing oxidized methionine residues was counted in each sample, and an increase in methionine oxidation was observed only in the s. acidocaldarius sample extracted with the mplex protocol (see fig. s3 in the supplemental material). however, the opposite trend was observed in s. oneidensis, mouse brain cortex, and unicyanobacterial consortium samples, and no difference was observed in the other samples (see fig. s3 ). these results suggest that the oxidation of peptides is sample dependent and that it is not induced by mplex. taken together, our data show that mplex is a robust protocol and can be applied for a variety of sample types without compromising the proteome coverage or quantitative measurements or inducing oxidation artifacts. to illustrate an application for mplex and the value of multiple omics measurements obtained from the same sample, we applied the method to study mers-cov infection. we specifically chose mers-cov because it is a deadly emerging infectious agent with subsequent disease mortality rates of approximately 40% and because there are currently no effective drugs available for treatment (32) . since mers-cov is a newly emergent virus, information about the mechanism of virulence of the infection is very scarce in the literature and any new data would immensely contribute to a better understanding of the disease. in addition, experiments investigating mers-cov need to be performed in biosafety level 3 (bsl3) facilities, which require extensive safety and decontamination procedures. thus, being able to analyze multiple omics from the same sample would significantly reduce the time of exposure risk of the researcher inside the biosafety facility. for this experiment, we used human lung epithelial calu-3 cells, which we initially tested as described above and which showed good proteome coverage (fig. 2d) . nine replicates of cell cultures were infected for 18 h with mers-cov, while 3 replicates were left uninfected as mock controls. samples were subjected to mplex and submitted for global proteomic, metabolomic, and lipidomic analyses. in total, 2,670 proteins, 51 metabolites, and 236 lipid species were identified and quantified (see tables s1 to s4 in the supplemental material). data from all three global measurements were then integrated using the metscape plugin of cytoscape (fig. 3a) (33, 34) . we also performed a function-enrichment analysis based on the kegg database using the lrpath tool (35) and combined this information into metscape. the lrpath analysis showed that 25 pathways were significantly enriched in differentially abundant proteins (see table s5 ) and that 5 of the pathways were from the central metabolism of the cell (fig. 3a) . from these pathways, we chose the glycolysis and gluconeogenesis pathways due to their complexity and the fact that these two pathways share most of the metabolites and enzymes therein. being able to determine which of these pathways is affected more during infection would result in valuable information for better understanding the disease. in fig. 3a , the nodes highlighted in yellow represent the glycolysis/gluconeogenesis pathway, which was separated into a subnetwork in fig. 3b for a better visualization. this pathway showed several proteins that were downregulated during mers-cov infection, which are represented in metscape by the small nodes (fig. 3b ). this pathway was then manually curated and visualized using the vanted tool (36) (fig. 3c) , showing quantitatively that almost all proteins in the glycolysis/gluconeogenesis pathway were reduced in abundance during the infection with mers-cov (fig. 3c) . although limited numbers of metabolites from the glycolysis/gluconeogenesis pathways were detected, the reduced levels of glucose 6-phosphate (g6p), dihydroxyacetone phosphate (dhap), and 3-phospho-d-glycerate (3pg) further support the idea of a decrease in activity of this central pathway (fig. 3c) . since glycolysis and gluconeogenesis share the same enzymes, proteomics alone is insufficient to determine exactly which process is affected. however, results from the addition of metabolomics, specifically, the observation that the initial substrate, glucose (glc), had accumulated, indicated that glycolysis was more likely than gluconeogenesis to have been affected by the viral infection (fig. 3c) . to conclude, the proteomics analysis by itself would show differences only in the abundances of the enzymes from the glycolysis/ gluconeogenesis pathway, but the addition of metabolite measurements helps confirm that the pathway activity is reduced and which direction is the more affected, clearly illustrating the advantage of integrating multi-omic measurements for studying specific metabolic pathways. to further demonstrate the utility of multi-omic analyses facilitated by the mplex protocol, we investigated mers-cov-stimulated changes in the calu-3 lipidome by integrating the measurements of sphingolipids and glycerophospholipids from the lipidomics analysis, free fatty acids from the metabolomics analysis, and enzymes from the proteomic analysis using the vanted tool (fig. 4) . increases in the levels of all 5 detected fatty acid species were observed in mers-cov-infected cells compared to mock controls (fig. 4) . the increases in fatty acid levels appear unrelated to lipid synthesis itself, since almost all the enzymes of the synthesis pathway are downregulated with infection (fig. 4) . conversely, the decrease in levels of enzymes in the fatty acid degradation pathway might be contributing to the accumulation of fatty acids (fig. 4) . in addition, degradation of phosphatidylcholines (pc), lyso-pc, phosphatidylserines (ps), and lyso-ps by phospholipases might also have been contributing to the accumulation of fatty acids during infection (fig. 4) . although the responsible phospholipase was not detected in the proteomic analysis, it seems to be specific to pc and ps, since other classes of glycerophospholipids and glycerolipids remained mostly unchanged during infection (fig. 4) . more-extensive changes in abundance were observed in members of sphingolipid classes than in phospholipids. the abundance of hexosylceramide increased during mers-cov infection, seemingly due to a decrease in the levels of its degradation enzyme glucosylceramidase (gba) (fig. 4 ). an increase of ceramide levels was also detected during infection which did not appear to be related to synthesis, since the abundance of serine palmitoyltransferase (sptlc1), the enzyme that catalyzes the first step of ceramide synthesis by condensing serine and palmitate into 3-ketosphinganine, was decreased (fig. 4) . the accumulation of ceramides was most likely due to the degradation of sphingomyelin in combination with a decrease in levels of the ceramidase (asah1) (fig. 4) . sphingolipids have been reported to play an integral role in viral uptake, replication, maturation, and budding during viral infection. membrane domains enriched with ceramides have been proposed to facilitate the entry of enveloped viruses into host cells by changing the membrane fluidity and enhancing vesicular fusion (37) . ceramides are also known to trigger apoptosis and death of the host cells (38, 39) . indeed, apoptosis has already been reported in bronchial epithelial cells infected with mers-cov (40), but its relationship with the increased levels of ceramides still needs to be further investigated. overall, the lipid metabolic network built by integrating multi-omics measurements shows a much more complete and likely more accurate view of the lipid landscape compared to lipidomics alone and provides more insights concerning the mechanism of lipid regulation. integration of multi-omics measurements has been consolidated as a technique for studying complex biological systems (1-3) . thus, methods that enable multiple omics measurements on the same sample are not only attractive but the only choice in cases of samples with limited availability. in this context, the mplex method can be an excellent alternative since it has been shown to be robust and applicable for a variety of samples ranging from bacterial cells to environmental samples to animal tissue. it is worth noting that, in addition to metabolomics, proteomics, and lipidomics, it is very likely that mplex can be used for the analysis of posttranslational modifications. indeed, a preliminary unpublished phosphoproteomic analysis using mplex led to the identification of several thousand phosphopeptides, although more careful analysis is required to determine if there are losses in this process. to conclude, we demonstrate the utility of multi-omics integration using mplex to study a lung epithelial cell line infected with mers-cov, which showed major differences in central carbon and lipid metabolism during infection. samples. for this study, we chose a variety of sample types: plant leaves from arapdopsis thaliana, human urine as an example body fluid, the gram-negative bacterium shewanella oneidensis, the cultured tissue cell line calu-3, a unicyanobacterial consortium isolated from hot lake, wa, usa (26) , mouse brain cortex tissue, and the archaeon sulfolobus acidocaldarius strain dsm 639. calu-3 cell infection with mers-cov was performed as described in text s1 in the supplemental material. s. oneidensis, the unicyanobacterial consortium, and s. acidocaldarius cells were lysed by bead beating in a bullet blender (next advance, averill park, ny) with 0.1-mm-diameter zirconia beads at speed 8 for 3 min at 4°c, and the lysate was spun into a falcon tube at 2,000 ϫ g for 10 min at 4°c. additional lysis was done via pressure cycling technology (pct) using a barocycler (pressure biosciences inc., south easton, ma). the suspended cells were subjected to 20 s of high pressure at 35,000 lb/in 2 followed by 10 s of ambient pressure for 10 cycles. a. thaliana leaves were frozen with liquid nitrogen and mechanically disrupted on a mortar with a pestle. mouse brain cortex tissue was homogenized in ice-cold nanopure h 2 o at full speed with a hand-held omni tool and a disposable probe (omni, kennesaw, ga) for 30 s, allowed to cool, and homogenized again. extraction methods. each sample was processed in 5 replicates using the following protocols. (i) metabolite, protein, and lipid extraction (mplex). the extraction procedure was adapted from the method of folch et al. (41) by keeping the same final solvent proportions; however, the monophasic extraction step was not performed, as water was initially added to the sample along with the chloroform and methanol to simultaneously extract and partition molecules into the three different phases. cell pellets or lysates were resuspended in water, and 5 volumes of cold (ϫ20°c) chloroform-methanol (2:1 [vol/vol]) solution was added to the samples. samples were incubated for 5 min on ice, subjected to vortex mixing for 1 min, and centrifuged at 12,000 rpm for 10 min at 4°c. for the samples for which metabolomics and lipidomics analyses were performed, the upper aqueous phase and bottom organic phase, containing hydrophilic metabolites and lipids, respectively, were collected in glass autosampler vials. the interphases, containing proteins, were washed by adding 1 ml of cold (ϫ20°c) methanol, vortex mixed for 1 min, and centrifuged at 12,000 rpm for 10 min at 4°c. the supernatants were discarded, and the resulting pellets were dried in a vacuum centrifuge for 5 min. (ii) phenol extraction. powdered a. thaliana leaves were resuspended in 10 ml of phenol extraction buffer (0.5 m tris-hcl [ph 7.5], containing 0.7 m sucrose, 0.1 m kcl, 50 mm edta, 2% [vol/vol] ␤-mercaptoethanol, and 1 mm phenylmethanesulfonylfluoride), and then 10 ml of phenol solution saturated with 10 mm tris-hcl (ph 7.5) was added to each tube. samples were shaken for 30 min at 4°c and centrifuged at 5,000 ϫ g for 30 min at 4°c. the upper phenolic phase was collected into a fresh tube and washed twice by adding 10 ml of phenol extraction buffer, followed by centrifugation at 5,000 ϫ g for 30 min at 4°c, and discarding of the lower phase. the upper phenolic phase was collected in a fresh tube, and 5 volumes of 0.1 m ammonium acetate in methanol was added. samples were incubated overnight at ϫ20°c and centrifuged at 5,000 ϫ g for 30 min at 4°c. protein pellets were then washed twice with 10 ml ice-cold methanol and twice with 10 ml ice-cold acetone by adding the solvent, centrifuging at 5,000 ϫ g for 30 min at 4°c, and discarding of the supernatant. the resulting protein pellet was dried under a stream of n 2 . (iii) tca extraction. 10 ml of freshly prepared ice-cold tca-acetone extraction buffer (0.61 m trichloroacetic acid-90% acetone) was added to powdered a. thaliana leaves, and the mixture was incubated overnight at ϫ20°c. proteins were then precipitated by centrifuging for 30 min at 5,000 ϫ g at 4°c, and the supernatant was discarded. the protein pellet was washed three times by adding 10 ml of ice-cold acetone, followed by centrifugation for 10 min at 5,000 ϫ g at 4°c, and discarding of the supernatant. the resulting protein pellet was dried under a stream of n 2 . (iv) acetonitrile extraction. lysates were resuspended in 4 volumes of ice-cold (ϫ20°c) pure acetonitrile and incubated for 10 min at 4°c to precipitate the proteins. the samples were centrifuged for 10 min at 4°c at 12,000 ϫ g to pellet the protein. the supernatant was removed, and the protein pellets were dried by evaporation before digesting with trypsin. (v) methanol extraction. the methanol extraction was performed with the exact same procedure as the acetonitrile extraction, with the difference that the organic solvent was replaced by methanol. proteomic, lipidomic, and metabolomic analyses. the detailed methodology of proteomic, lipidomic, and metabolomic analyses are provided in text s1 in the supplemental material. for proteomic analysis, proteins were digested with trypsin into peptides and analyzed using the accurate mass & time (amt) tag approach (22) . peptides were separated by nano-capillary liquid chromatography (nano-lc), and eluting peptides were directly analyzed using ltq-orbitrap velos or exactive mass spectrometers (thermo fisher scientific). peptides were identified by matching to the appropriate mass tag database, and the peak areas were extracted using viper (42) . matching results were filtered with statistical tools for amt tag confidence and uniqueness probability scores (43) . lipids extracted from calu-3 cells infected with mers-coronavirus were analyzed by lc-tandem ms (lc-ms/ms) using an ltq-orbitrap velos mass spectrometer as previously described (14) . then, raw data files were analyzed using liquid (lipid informed quantitation and identification) software developed in-house for semiautomated identification of lipid molecular species followed by manual validation of identified species. polar metabolites extracted from calu-3 cells infected with mers-coronavirus were derivatized with n-methyl-n-(trimethylsilyl)trifluoroacetamide (mstfa) and analyzed by gas chromatography-mass spectrometry (gc-ms) as described previously (16) . the raw data files were processed using metabolitedetector (44) and manually validated. comparative analysis of different extractions. the proteomic analyses comparing the different extraction methods were performed by rolling up the intensity values of peptides into values corresponding to proteins using the r rollup function of inferno rdn (formerly dante) (45) . only proteins with two or more peptides that were unique were considered for further analysis. the intensity values were transformed to log 2 values and submitted to standard paired t tests and g tests (46) (considering only proteins present in 0 or 1 of 5 replicates). for analyses of proteomics, lipidomics, and metabolomics data from the calu-3 cells infected with mers-cov, the quantitative data profiles were evaluated for extreme outlier behavior (47) . no outlier samples were observed in the metabolomics and lipidomics data; however, one proteomics replicate from the infected group showed extremely poor coverage and correlation, indicating an issue with the protein extraction. that one sample was removed from subsequent analyses. further quality assessment of the proteomics data included evaluation of individual peptides to identify those with inadequate coverage for either statistical analyses or protein quantification (46) . metabolomic and lipidomic data were normalized via standard median centering, and proteomics data were normalized via median centering against a rank-invariant peptide subset identified to reduce bias (48) . to allow evaluation of the proteomic data at the protein level, a signature-based protein quantitation methodology was employed (49) . finally, the protein, metabolite, and lipid data were evaluated for quantitative differences between the results of mock infection and mers-cov infection via a standard two-sample t test. multi-omics data integration. accession numbers from proteomics data of the mers-cov-infected cells were converted into entrez gene identifiers (id) and uploaded to lrpath for function-enrichment analysis (35) . then, expression values of metabolomics, lipidomics (both converted to kegg compound ids), and proteomics data were integrated using metscape v. supplemental material for this article may be found at http://dx.doi.org/10.1128/ msystems.00043-16. text s1, docx file, 0.04 mb. figure s1 , tif file, 0.5 mb. figure s2 , tif file, 0.4 mb. figure s3 , tif file, 0.4 mb. model-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation personal omics profiling reveals dynamic molecular and medical phenotypes multi-omics of permafrost, active layer and thermokarst bog soil microbiomes a systems biology approach to infectious disease research: innovating the pathogen-host research paradigm a biomolecular isolation framework for eco-systems biology a universal protocol for the combined isolation of metabolites, dna, long rnas, small rnas, and proteins from plants and microorganisms process for the integrated extraction, identification and quantification of metabolites, proteins and rna to reveal their co-regulation in biochemical networks simultaneous extraction of proteins and metabolites from cells in culture a comparison of cell and tissue extraction techniques using high-resolution 1h-nmr spectroscopy simultaneously extracting dna, rna, and protein using kits: is sample quantity or quality prejudiced? a rapid method of total lipid extraction and purification preparation of lipide extracts from brain tissue two strings to the systems biology bow: co-extracting the metabolome and proteome of yeast a reversed-phase capillary ultra-performance liquid chromatography-mass spectrometry (uplc-ms) method for comprehensive top-down/ bottom-up lipid profiling comprehensive metabolomic, lipidomic and microscopic profiling of yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis salmonella modulates metabolism during growth under conditions that induce expression of virulence genes diel metabolomics analysis of a hot spring chlorophototrophic microbial mat leads to new hypotheses of community member metabolisms the fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation decreased abundance of type iii secretion system-inducing signals in arabidopsis mkp1 enhances resistance against pseudomonas syringae comparison of protein precipitation methods for sample preparation prior to proteomic analysis assessing the need for a standardized cancer human biobank (cahub): findings from a national survey with cancer researchers advances in proteomics data analysis and display using an accurate mass and time tag approach a quantitative study of the effects of chaotropic agents, surfactants, and solvents on the digestion efficiency of human plasma proteins by trypsin quantitative assessment of in-solution digestion efficiency identifies optimal protocols for unbiased protein analysis organic solvent extraction of proteins and peptides from serum as an effective sample preparation for detection and identification of biomarkers by mass spectrometry phototrophic biofilm assembly in microbial-mat-derived unicyanobacterial consortia: model systems for the study of autotrophheterotroph interactions sample extraction techniques for enhanced proteomic analysis of plant tissues advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics exponentially modified protein abundance index (empai) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein global quantification of mammalian gene expression control a new method to measure oxygen solubility in organic solvents through optical oxygen sensing middle east respiratory syndrome metscape 2 bioinformatics tool for the analysis and visualization of metabolomics and gene expression data cytoscape: a software environment for integrated models of biomolecular interaction networks lrpath: a logistic regression approach for identifying enriched biological groups in gene expression data integration of -omics data and networks for biomedical research with vaned sphingolipids in viral infection sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide anti-dengue virus nonstructural protein 1 antibodies cause no-mediated endothelial cell apoptosis via ceramideregulated glycogen synthase kinase-3beta and nf-kappab activation bilateral entry and release of middle east respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells a simple method for the isolation and purification of total lipides from animal tissues viper: an advanced software package to support high-throughput lc-ms peptide identification a statistical method for assessing peptide identification confidence in accurate mass and time tag proteomics metabolitedetector: comprehensive analysis tool for targeted and nontargeted gc/ms based metabolome analysis dante: a statistical tool for quantitative analysis of -omics data combined statistical analyses of peptide intensities and peptide occurrences improves identification of significant peptides from ms-based proteomics data improved quality control processing of peptide-centric lc-ms proteomics data a statistical selection strategy for normalization procedures in lc-ms proteomics experiments through dataset-dependent ranking of normalization scaling factors bayesian proteoform modeling improves protein quantification of global proteomic measurements key: cord-347587-auook38y authors: zhao, guangyu; he, lei; sun, shihui; qiu, hongjie; tai, wanbo; chen, jiawei; li, jiangfan; chen, yuehong; guo, yan; wang, yufei; shang, jian; ji, kaiyuan; fan, ruiwen; du, enqi; jiang, shibo; li, fang; du, lanying; zhou, yusen title: a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov date: 2018-08-29 journal: j virol doi: 10.1128/jvi.00837-18 sha: doc_id: 347587 cord_uid: auook38y the newly emerged middle east respiratory syndrome coronavirus (mers-cov) continues to infect humans and camels, calling for efficient, cost-effective, and broad-spectrum strategies to control its spread. nanobodies (nbs) are single-domain antibodies derived from camelids and sharks and are potentially cost-effective antivirals with small size and great expression yield. in this study, we developed a novel neutralizing nb (nbms10) and its human-fc-fused version (nbms10-fc), both of which target the mers-cov spike protein receptor-binding domain (rbd). we further tested their receptor-binding affinity, recognizing epitopes, cross-neutralizing activity, half-life, and efficacy against mers-cov infection. both nbs can be expressed in yeasts with high yield, bind to mers-cov rbd with high affinity, and block the binding of mers-cov rbd to the mers-cov receptor. the binding site of the nbs on the rbd was mapped to be around residue asp539, which is part of a conserved conformational epitope at the receptor-binding interface. nbms10 and nbms10-fc maintained strong cross-neutralizing activity against divergent mers-cov strains isolated from humans and camels. particularly, nbms10-fc had significantly extended half-life in vivo; a single-dose treatment of nbms10-fc exhibited high prophylactic and therapeutic efficacy by completely protecting humanized mice from lethal mers-cov challenge. overall, this study proves the feasibility of producing cost-effective, potent, and broad-spectrum nbs against mers-cov and has produced nbs with great potentials as anti-mers-cov therapeutics. importance therapeutic development is critical for preventing and treating continual mers-cov infections in humans and camels. because of their small size, nanobodies (nbs) have advantages as antiviral therapeutics (e.g., high expression yield and robustness for storage and transportation) and also potential limitations (e.g., low antigen-binding affinity and fast renal clearance). here, we have developed novel nbs that specifically target the receptor-binding domain (rbd) of mers-cov spike protein. they bind to a conserved site on mers-cov rbd with high affinity, blocking rbd's binding to mers-cov receptor. through engineering a c-terminal human fc tag, the in vivo half-life of the nbs is significantly extended. moreover, the nbs can potently cross-neutralize the infections of diverse mers-cov strains isolated from humans and camels. the fc-tagged nb also completely protects humanized mice from lethal mers-cov challenge. taken together, our study has discovered novel nbs that hold promise as potent, cost-effective, and broad-spectrum anti-mers-cov therapeutic agents. ities, neutralization mechanisms, cross-neutralizing activity against divergent mers-cov strains, half-life, and protective efficacy against lethal mers-cov infection in an established hdpp4-tg mouse model (38) . this study reveals that efficacious, robust, and broad-spectrum nbs can be produced to target mers-cov s protein rbd and that they hold great promise as potential anti-mers-cov therapeutics. identification and characterization of mers-cov-rbd-specific nbs. to construct the nb (i.e., vhh) library, we immunized llama with recombinant mers-cov rbd (residues 377 to 588, emc2012 strain) containing a c-terminal human igg1 fc tag (i.e., rbd-fc) and isolated peripheral blood mononuclear cells (pbmcs) from the immunized llama. after four rounds of bio-panning and screening using mers-cov rbd-fc, we isolated a positive clone with the highest binding affinity for the rbd. the gene encoding this rbd-specific nb was subcloned into yeast expression vector to construct nbms10 (which contains a c-terminal his 6 tag) and nbms10-fc (which contains a c-terminal human igg1 fc tag) nbs (fig. 1) . both nbms10 and nbms10-fc were nbms10-fc nbs. blood was collected from mers-cov rbd-fc protein-immunized alpaca after the last immunization to isolate pbmcs. rna was then extracted to synthesize cdna via rt-pcr. this was followed by pcr amplification of the n-terminal igg heavy-chain fragment (ϳ700 bp), including the vhh gene, while the latter was used as the template to amplify the vhh gene fragment (ϳ300 to 450 bp). the vhh dna sequence was further ligated into phagemid vector pcantab5e and transformed into e. coli tg1 competent cells to construct vhh library. vhh phage display was carried out to isolate rbd-specific clones. after four rounds of bio-panning, the rbd-specific vhh coding sequence was confirmed from the selected positive clones. the identified vhh coding gene containing a c-terminal his 6 or human igg1 fc was inserted into pichia pastoris yeast expression vector ppicz␣a to construct nbms10 and nbms10-fc, respectively, for further soluble expression and purification. expressed in yeast cells, secreted into the cell culture supernatants, and purified to homogeneity ( fig. 2a, left) . the estimated molecular weights were about 16 kda for nbms10 and 50 kda for nbms10-fc, since the latter formed a dimer. these mers-cov rbd-specific nbs from llama, but not severe acute respiratory syndrome coronavirus (sars-cov) rbd-specific mabs from mice, were recognized by anti-llama antibodies ( fig. 2a, right) . thus, the yeast-expressed nbs maintained their native conformation and antigenicity. to characterize their functions, we examined how the nbs interact with mers-cov rbds. first, we evaluated the binding between the nbs and mers-cov rbd using elisa. the result showed that both nbs bound strongly to recombinant mers-cov rbd containing a c-terminal folden tag (rbd-fd) and mers-cov s1 containing a c-terminal his 6 tag (s1-his) in a dose-dependent manner (fig. 2b) . second, we determined the binding affinity of the two nbs for mers-cov rbd using surface plasmon resonance (spr). the result showed that the k d between nbms10 and rbd-fc was 0.87 nm, the nbs were subjected to sds-page (left) or western blotting (right), followed by detection using anti-llama antibody. the molecular weight marker (in kda) is indicated on the left. (b) detection of binding between nbms10 or nbms10-fc and mers-cov s1 (mers-s1) or rbd (mers-rbd) protein by elisa. the plates were coated with mers-cov s1-his or rbd-fd protein (2 g/ml), followed by sequential incubation with respective nbs and goat anti-llama and hrp-conjugated anti-goat igg antibodies. the data are presented as mean a 450 values ϯ the standard deviation (sds) (n ϭ 2). significant differences (*; **, and ***) are shown in the binding of nbs to mers-s1 or mers-rbd at various concentrations. (c) the binding kinetics between nbms10 or nbms10-fc and mers-cov rbd or s1 protein were measured by spr. mers-cov rbd-fc protein was used for binding to nbms10 (containing a c-terminal his 6 ), and s1-his protein was used for binding to nbms10-fc (containing a c-terminal human fc). (d) detection of nbms10 and nbms10-fc neutralizing activity against mers-cov infection (emc2012 strain) by a microneutralization assay. the nb-mers-cov mixtures were incubated with vero e6 cells and observed for the presence or absence of cpe. neutralizing activity of nbs was recorded as the concentration of nbs in complete inhibition of mers-cov-induced cpe in at least 50% of the wells (nd 50 ). the data are expressed as mean nd 50 ϯ the sd (n ϭ 3). the experiments were repeated twice, and similar results were obtained. the "(ϫ) control" in panels a, b, and d refers to sars-cov 33g4 mouse mab. whereas the k d between nbms10-fc and s1-his was 0.35 nm (fig. 2c) . third, we carried out mers-cov neutralization assay. the result showed that the nbs efficiently neutralized the infection of live mers-cov (emc2012 strain) in vero cells. the measured 50% neutralization doses (nd 50 ) were 3.52 g/ml for nbms10 and 2.33 g/ml for nbms10-fc (fig. 2d ). taken together, the nbs strongly bound to mers-cov rbd and neutralized mers-cov infection. molecular mechanism underlying the neutralizing activities of nbs. to investigate the mechanism underlying the neutralizing activities of nbs, we evaluated the competition between the nbs and hdpp4 for the binding to mers-cov rbd. first, we carried out a flow cytometry assay where recombinant mers-cov rbd interacted with cell-surface-expressed dpp4 in the presence or absence of recombinant nbs. the result showed that both nbs significantly blocked the binding of rbd to cell-surface dpp4 in a dose-dependent manner ( fig. 3a and b) . as a negative control, sars-cov-rbdspecific 33g4 mab did not block the binding between mers-cov rbd and cell surface dpp4 ( fig. 3a and b) . second, we carried out an enzyme-linked immunosorbent assay (elisa) where recombinant mers-cov rbd and recombinant hdpp4 interacted in the presence or absence of recombinant nbs. the result showed that both nbs, but not were coated with mers-cov rbd-fc protein (2 g/ml), followed by sequential incubation with serial dilutions of nbs or hdpp4 protein (2 g/ml), goat anti-hdpp4, and hrp-conjugated anti-goat igg antibodies. the percent inhibition was calculated as the rbd-hdpp4 binding in the presence or absence of nbs according to the following formula: (1 ϫ rbd-hdpp4-nb/rbd-hdpp4) ϫ 100. a significant difference (***) occurred between nbms10 and nbms10-fc in inhibition of rbd-hdpp4 binding. the "(ϫ) control" in panels b and c refers to sars-cov 33g4 mab. the data are presented as the mean percent inhibition ϯ the sd (n ϭ 2). the experiments were repeated twice, and similar results were obtained. 33g4 mab, blocked the binding between mers-cov rbd and dpp4 in a dosedependent manner. moreover, compared to nbms10, nbms10-fc blocked the rbd-dpp4 binding more efficiently (fig. 3c) . these data reveal that the nbs can compete with hdpp4 for the binding to mers-cov rbd, suggesting that the nb-binding site and the dpp4-binding site overlap on the mers-cov rbd. to map the binding site of the nbs on mers-cov rbd, we performed alanine scanning on the surface of mers-cov rbd and detected the binding of nbs to the alanine-containing rbd mutants. the results showed that nbms10 demonstrated tight binding to mers-cov rbd containing the single mutations l506a, d510a, r511a, e513a, e536a, w553a, v555a, and e565a and slightly reduced binding to rbd containing triple mutations l506f-d509g-v534a, suggesting that these rbd residues do not play significant roles in nb binding. instead, single mutation d539a and double mutations e536a-d539a on mers-cov rbd both ablated the binding of nbms10 to the rbd (fig. 4a) , suggesting that rbd residue asp539 plays an important role in nb binding. we further investigated the role of asp539 in nb binding using the mers-cov pseudovirus entry assay. neither nbms10 nor nbms10-fc could neutralize the cell entry of mers-cov pseudovirus bearing the d539a mutation, again confirming that residue asp539 is critical for nb binding (fig. 4b ). to examine of the role of the d539a mutation in dpp4 binding, we carried out an elisa to detect the binding between dpp4 and the plates were coated with rbd-fd protein (2 g/ml) and treated with or without dtt, followed by sequential incubation with serial dilutions of nbms10 or nbms10-fc and goat anti-llama and hrp-conjugated anti-goat igg antibodies. the data are presented as mean a 450 values ϯ the sd (n ϭ 2). the "(ϫ) control" in panels b and d refers to sars-cov 33g4 mab. the above-described experiments were repeated twice, and similar results were obtained. mers-cov rbd bearing the d539a mutation. the result showed that the d539a mutation significantly reduced the binding of the rbd to dpp4 (fig. 4c ). overall, these results demonstrate that nbs recognize the asp539-containing epitope on mers-cov rbd and that this epitope also plays an important role in dpp4 binding. therefore, the nbs and dpp4 compete for the same region on mers-cov rbd, and mutations in this region can reduce the binding of both the nbs and dpp4. to investigate whether nb-recognized epitopes on mers-cov rbd are conformational or linear, we detected the binding of nbs to mers-cov rbd with its conformational structure disrupted. to this end, we treated mers-cov rbd with reducing agent dithiothreitol (dtt) to break the disulfide bonds in the protein, and performed an elisa on the binding between nbs and dtt-treated rbd. the result showed that neither nbms10 nor nbms10-fc bound to the dtt-treated rbd (fig. 4d) . as a control, both nbs bound to untreated rbd with high affinity. thus, the nbs recognize the conformational epitope on the rbd. to understand the structural mechanism underlying the neutralizing activities of the nbs, we examined the competitive interactions among the nbs, dpp4, and mers-cov rbd using structural modeling (fig. 5 ). in the absence of the nbs, mers-cov rbd binds tightly to the dpp4 receptor, with d539 of rbd serving as a key residue at the binding interface (fig. 5a) . here, rbd residue d539 forms a critical salt bridge with dpp4, and it interacts with the surrounding key rbd residues via van der waals contacts and hydrogen bonds (fig. 5b) , enabling rbd and dpp4 to maintain strong binding interactions. the nbs bind tightly to the rbd in the same d539-containing region, abolishing the binding between rbd and dpp4 (fig. 5c) . cross-neutralizing activity of nbs against divergent mers-cov strains. to investigate the cross-neutralizing activity of nbs against divergent mers-cov isolates, we performed mers-cov pseudovirus entry assay in the presence of the nbs where the pseudoviruses encode the s gene of various mers-cov isolates from different countries (saudi arabia, qatar, and south korea), hosts (human and camels), and time periods (2012 to 2015). these mers-cov strains all contain mutations in their rbds. the results showed that both nbs potently neutralized the cell entry of all of the mers-cov pseudoviruses, with the nd 50 values ranging from 0.003 to 0.979 g/ml (for nbms10) and from 0.003 to 0.067 g/ml (for nbms10-fc) ( table 1) . therefore, although the nbs were developed using the rbd from one mers-cov strain (emc2012), they have broad-spectrum cross-neutralizing activity against existing mers-cov strains, as well as potentially future emerging mers-cov strains. in vivo half-life of nbs. to evaluate the in vivo half-life of the nbs, we injected the nbs into mice, collected the sera from the mice after different time intervals, and measured the binding between the sera and recombinant mers-cov s1 using elisa. the results showed that the sera collected from nbms10-injected mice gradually lost their binding affinity for mers-cov s1, and completely lost their binding for mers-cov s1 10 days postinjection (fig. 6a ). in comparison, nbms10-fc demonstrated stable binding for recombinant mers-cov s1 at 10 days postinjection (fig. 6b) . as a control experiment, sera collected from pbs-injected mice showed no binding for recombinant mers-cov s1 (fig. 6c) . thus, compared to monomeric nb, fc-fused nb has a significantly extended in vivo half-life likely due to its dimeric structure, which increases the molecular weight of nb from 16 to 50 kda and hence may slow down its renal clearance. prophylactic and therapeutic efficacy of nb in transgenic mice. because mers-cov does not infect wild-type mice, we previously developed hdpp4-tg mice (38) as the susceptible animal model for mers-cov research. to evaluate the prophylactic efficacy of nbms10-fc, mice were injected with a single dose of nbms10-fc 3 days before they were infected with a lethal dose of mers-cov and were subsequently monitored for their weight and survival. trastuzumab, an antibody used for treating breast cancer, was used as a control. the result showed that after mers-cov infection, mice treated with nbms10-fc had a 100% survival rate (fig. 7a, above) and steady weight (fig. 7a, below) . in comparison, mice treated with trastuzumab all died on day 8 postinfection, and their weight also sharply decreased starting from day 4 postinfection (fig. 7a) . to evaluate the therapeutic efficacy of nbms10-fc, mice were first infected with mers-cov and then treated with single-dose nbms10-fc either 1 or 3 days postinfection. the result showed that mice treated with nbms10-fc on day 1 postinfection had a 100% survival rate and steady weight (fig. 7b ). in addition, mice treated with nbms10-fc on day 3 postinfection also had a 100% survival rate (fig. 7c , above); although their weights first decreased on day 5 postinfection, it rebounded on day 7 postinfection (fig. 7c, below) . in comparison, mice receiving trastuzumab all died on day 8 after infection, and their weights continuously decreased ( fig. 7b and c) . overall, nbms10-fc has potent prophylactic and therapeutic efficacy in protecting susceptible animal models against lethal mers-cov challenge. . virus-challenged mice were monitored for 14 days to evaluate survival rate (above) and body weight changes (below). the body weight data are presented as means ϯ the sd of mice in each group (n ϭ 6). significant differences (** and ***) are indicated between the nbms10-fc and control groups. mers-cov continues to infect humans with a high fatality rate. because camels likely serve as the transmission hosts for mers-cov and also because humans have contact with camels, the constant and continuing transmissions of mers-cov from camels to humans make it difficult to eradicate mers-cov from the human population. thus, efficacious, cost-effective, and broad-spectrum anti-mers-cov therapeutic agents are needed to prevent and treat mers-cov infections in both humans and camels. nbs have been gaining acceptance as antiviral agents because of their small size, good tissue permeability, and cost-effective production, storage, and transportation. however, their small size may also lead to relative low antigen-binding affinity and quick clearance from the host body. in this study, we have developed a novel mers-covtargeting nb, nbms10, and its fc-fused version, nbms10-fc, both of which demonstrate great promise as anti-mers-cov therapeutic agents. nbms10 and nbms10-fc present superior characteristics common to other nbs. they target the mers-cov rbd, which plays an essential role in cell entry of mers-cov by binding to its receptor hdpp4. both nbs can be expressed in yeast cells with high purity and yields and are soluble in solutions. all of these properties suggest costeffective production, easy storage, and convenient transportation of these nbs in potential commercial applications. the mers-cov rbd-targeting nbs developed also demonstrate good qualities comparable to previously reported mers-cov rbd-specific conventional iggs. first, the nbs bind to mers-cov rbd with high affinities. the k d values for nbms10 and nbms10-fc to bind mers-cov rbd were 8.71 ϫ 10 ϫ10 m and 3.46 ϫ 10 ϫ10 m, respectively. the k d values for rbd-targeting conventional iggs to bind mers-cov rbd range from 7.12 ϫ 10 ϫ8 m to 4.47 ϫ 10 ϫ11 m (29, 35, 36) . moreover, the nd 50 values for nbms10 and nbms10-fc to neutralize mers-cov (emc2012 strain) infection in cultured cells were 3.52 and 2.33 g/ml, respectively. the nd 50 values for rbd-specific conventional iggs to neutralize various mers-cov strains ranged from micrograms/ml to nanograms/ml (30, 32, 35, 39, 40) . thus, the nbs developed in this study and conventional iggs reported previously have comparable mers-cov rbd-binding affinities and mers-covneutralizing activities. structural comparisons of conventional iggs and nbs have shown that the antigen-binding site of iggs consists of paired heavy-chain and lightchain variable (vh-vl) domains, whereas nbs lack the light chain and hence cannot form the paired vh-vl domains (8, 41) . instead, nbs have an extended cdr3 region (ͼ16 amino acid residues), longer than that of the vhs of conventional iggs (average length 12 amino acid residues) (42) (43) (44) . moreover, the nbs developed here contain a 22-amino-acid cdr3; the extended cdr3 enables the nbs to bind to the antigens with higher affinity (37) . furthermore, although the single-domain nb (i.e., nbms10) is small and can be cleared from the serum relatively quickly, the fc-fused nb (i.e., nbms10-fc) with relatively increased size demonstrates extended in vivo half-life. therefore, the potential short half-life of nbs can be overcome by adding the appropriate tag to the nbs to increase their half-life. overall, the present study has shown the feasibility of overcoming the potential limitations of nbs. the mers-cov rbd-targeting nbs potently neutralize mers-cov entry into host cells. the k d values between the nbs and mers-cov rbd are significantly lower than that between mers-cov rbd and hdpp4 receptor. as a result, the nbs can outcompete hdpp4 for the binding of mers-cov rbd, thereby blocking the binding of mers-cov to dpp4, as well as mers-cov entry into host cells. it is worth noting that the rbd on the mers-cov s trimer frequently undergoes conformational changes, switching between a lying down, receptor-inaccessible conformation and a standing-up, receptoraccessible conformation. hence, in the context of the virus particles where the rbd is part of the s protein, the nbs would need to bind the rbd when the rbd is in the standing-up conformation (45) . importantly, the nbs demonstrate strong crossneutralizing activities against various mers-cov strains isolated from different hosts (humans and camels) and from different time points during mers-cov circulation in humans (from years 2012 to 2015). nbms10 had a relatively high nd 50 against the agv08584/2012 strain containing a v534a mutation, which is consistent with the slightly reduced binding affinity between nbms10 and mers-cov rbd containing the v534a mutation (fig. 4a) . the broad neutralizing spectrum of the nbs results from the binding site of the nbs on mers-cov rbd, which is located in the asp539containing region that plays a critical role in dpp4 binding. interestingly, several mers-cov rbd-specific conventional iggs also bind to the same epitope (39, 46) , suggesting that this region is a hot spot for immune recognition. although mutations in this region can eliminate the binding of the nbs to mers-cov rbd and hence lead to viral immune evasion, they also reduce the binding of mers-cov rbd to receptor dpp4 and hence decrease the efficiency of viral entry. thus, viral immune evasion from the inhibition of the nbs through mutations can be costly to mers-cov itself. indeed, residue asp539 in s protein rbd is highly conserved in almost all of the natural mers-cov strains published to date (fig. 8) . therefore, the mers-cov-specific nbs can potentially be developed into broad-spectrum anti-mers-cov therapeutic agents. despite the above analysis, this study did not examine all possible mutations in the nb-binding region (since the atomic structures of mers-cov rbd complexed with the nbs are still unknown), and thus it is possible that future escape mutations may occur to residues that this study did not cover. in that case, a combination of the current nbs and other antibodies targeting other s regions or various rbd epitopes may be helpful in battling the emergence of immune escape mers-cov strains. in sum, the mers-cov-specific nbs developed in the present study possess superior qualities common to all nbs such as their small size and cost-effective production. they also overcome potential limitations of other nbs by maintaining a high binding affinity for their target mers-cov rbd and an optimized half-life. moreover, they recognize a functionally important region on mers-cov rbd, rendering viral immune evasion costly and at the same time making themselves good candidates as broad-spectrum anti-mers-cov therapeutics. we have confirmed the effectiveness of the nbs by showing that the fc-fused nb completely protected animal models from lethal mers-cov challenge. thus, the nbs can potentially be used in both humans and camels to prevent and treat mers-cov infections in either of these hosts and also block the camel-tohuman transmission of mers-cov. overall, our study proves the feasibility of developing highly effective nbs as anti-mers-cov therapeutic agents and points out strategies to preserve the advantages of nbs, as well as to overcome the potential limitations of nbs. construction of vhh library and screening for mers-cov-rbd-specific nbs. construction of the nb (i.e., vhh) library and screening of mers-cov-rbd-specific nbs were performed as previously described (47) . briefly, male and female alpacas (llama pacos, 1 year) were subcutaneously immunized with recombinant rbd-fc (260 g/alpaca) (48) plus freund complete adjuvant, and boosted three times with the same immunogen plus freund incomplete adjuvant (invivogen). blood was collected 10 days after the last immunization, and then pbmcs were isolated using ficoll-paque gradient centrifugation (ge healthcare). total rna was extracted with trizol reagent (invitrogen). cdna was synthesized by reverse transcription-pcr (rt-pcr) using a transscript cdna synthesis supermix (transgen biotech, china), followed by pcr amplification of the n-terminal igg heavy-chain fragment (ϳ700 bp), using the forward primer vhh-l-f (5=-ggtggtcctggctgc-3=) and the reverse primer ch2-r (5=-ggtacgtgctgttgaact gttcc-3=). the vhh gene (ϳ300 to 450 bp) was further amplified using the above dna fragment as the template and the forward primer vhh-fr1-d-f (5=-tttctattactaggcccagccggccgagtctggaggrr gcttggtgca-3=) and the reverse primer vhh-fr4-d-r (5=-aaaccgttggccataatggcctgaggagacgr tgacstsggtc-3=) (the sfii restriction site is underlined). the sfii-digested vhh dna fragment was then inserted into phagemid vector pcantab5e (bio-view shine biotechnology, china) to construct the vhh phage display library (49) . phage particles were analyzed by elisa using recombinant mers-cov rbd-fc and fc of human igg1 proteins as the positive and negative target proteins, respectively, to screen for rbd-specific nbs. after four rounds of bio-panning, one of five positive clones, cab10, with the highest binding to mers-cov rbd, was selected for further analyses (fig. 1) . expression of mers-cov-rbd-specific nbs in yeast cells. nbms10 and nbms10-fc nbs containing a c-terminal his 6 and fc of human igg1, respectively, were constructed based on the aforementioned cab10 vhh. the dna sequences encoding nbab10 and nbab10-fc were synthesized (genscript) and inserted into the pichia pastoris secretory expression vector, ppicz␣a (invitrogen) (fig. 1) . the recombinant nbms10 and nbms-fc were expressed in pichia pastoris gs115 cells and purified using a ni-nta column (for nbms10; ge healthcare) and a protein a sepharose 4 fast flow column (for nbms10-fc; ge healthcare), respectively. sds-page and western blotting. the purified anti-mers-cov-rbd nbs were analyzed using sds-page and western blotting (23, 48) . briefly, nbs (3 g) were loaded onto 10% tris-glycine sds-page gels and stained using coomassie brilliant blue or transferred to nitrocellulose membranes. after being blocked overnight at 4°c with 5% nonfat milk/phosphate-buffered saline-tween 20 (5% pbst), the membranes were incubated sequentially with goat anti-llama igg (1:3,000; abcam) and horseradish peroxidase (hrp)-conjugated anti-goat igg (1:1,000; r&d systems) antibodies for 1 h at room temperature and then with ecl western blot substrate reagents. finally, the membranes were visualized using amersham hyperfilm (ge healthcare). a sars-cov-rbd-specific mab, 33g4 (50) , was used as a control. elisa. elisa was performed to detect the binding between nbs and mers-cov s1 or rbd proteins (23, 51) . briefly, elisa plates were coated overnight at 4°c, respectively, with recombinant mers-cov s1-his (48), rbd-fc (48), rbd-fd (51), or one of the mutant rbds containing a c-terminal human fc tag (28) . after being blocked with 2% pbst for 2 h at 37°c, the plates were further incubated sequentially with serially diluted nbs (containing a c-terminal his 6 or fc tag), either goat anti-llama (1:5,000) or mouse anti-his (1:3,000) antibody (sigma) and either hrp-conjugated anti-goat igg (1:3,000) or hrp-conjugated anti-mouse igg (1:5,000) antibody (ge healthcare) for 1 h at 37°c. elisa substrate (3,3=,5,5=tetramethylbenzidine [tmb]; invitrogen) was added to the plates, and the reactions were stopped with 1 n h 2 so 4 . the absorbance at 450 nm (a 450 ) was measured using a tecan infinite 200 pro microplate reader (tecan). to detect the binding between nbs and denatured mers-cov rbd protein, elisa plates were coated with rbd-fd protein (2 g/ml) overnight at 4°c and then sequentially incubated with dtt (10 mm) and iodoacetamide (50 mm) (sigma) for 1 h at 37°c (28) . after three washes using pbst, elisa was performed as described above. inhibition of the binding between mers-cov rbd and hdpp4 proteins by nbs was performed using elisa as described above, except that recombinant hdpp4 protein (2 g/ml; r&d systems), and serially diluted nbs were added simultaneously to the rbd-fc-coated plates. the binding between rbd and dpp4 was detected using goat anti-hdpp4 antibody (1:1,000; r&d systems) and hrp-conjugated anti-goat igg (1:3,000). the percent inhibition was calculated based on the a 450 values of rbd-hdpp4 binding in the presence or absence of nbs. sars-cov 33g4 mab was used as a negative control to nbs. surface plasmon resonance. the binding between nbs and mers-cov s1 or rbd protein was detected using a biacores200 instrument (ge healthcare) as previously described (29) . briefly, recombinant fc-fused mers-cov rbd-fc protein or nbms10-fc nb (5 g/ml) was captured using a sensor chip protein a (ge healthcare), and recombinant his 6 -tagged mers-cov s1-his protein or nbms10 nb at various concentrations was flown over the chip surface in a running buffer containing 10 mm hepes (ph 7.4), 150 mm nacl, 3 mm edta, and 0.05% surfactant p20. the sensorgram was analyzed using biacore s200 software, and the data were fitted to a 1:1 binding model. flow cytometry. this assay was performed to detect the inhibition of the binding between mers-cov rbd and cell surface hdpp4 by nbs (28) . briefly, huh-7 cells expressing hdpp4 were incubated with mers-cov rbd-fc protein (20 g/ml) for 30 min at room temperature in the absence or presence of nbs at various concentrations. cells were incubated with fluorescein isothiocyanate-labeled antihuman igg antibody (1:50, sigma) for 30 min and then analyzed by flow cytometry. the percent inhibition was calculated based on the fluorescence intensity of rbd-huh-7 cell binding in the presence or absence of nbs. mers pseudovirus neutralization assay. neutralization of mers pseudovirus entry by nbs was performed as previously described (23, 52) . briefly, 293t cells were cotransfected with a plasmid encoding env-defective, luciferase-expressing hiv-1 genome (pnl4-3.luc.re) and a plasmid encoding mers-cov s protein. the mers pseudoviruses were harvested from supernatants at 72 h posttransfection and then incubated with nbs at 37°c for 1 h before being added to huh-7 cells. after 72 h, the cells were lysed in cell lysis buffer (promega), incubated with luciferase substrate (promega), and assayed for relative luciferase activity using tecan infinite 200 pro luminator (tecan). the nd 50 of the nbs was calculated as previously described (53) . mers-cov microneutralization assay. neutralization of mers-cov infection by nbs was performed as previously described (28, 54) . briefly, mers-cov (emc2012 strain) at an amount equal to 100 median tissue culture infective doses (tcid 50 ) was incubated with nbs at different concentrations for 1 h at 37°c. the nb-virus mixture was then incubated with vero e6 cells for 72 h at 37°c in the presence of 5% co 2 . the cytopathic effect (cpe) was observed daily. the neutralizing activity of nbs was reported as the nd 50 . the reed-muench method was used to calculate the nd 50 value for each nb (55) . measurement of half-life of nbs. male and female c57bl/6 mice (6 to 8 weeks old) were intravenously injected with nbs (50 g in 200 l per mouse) into the tail vein. sera were collected at different time points (30 min, 2 h, 6 h, 1 day, 5 days, and 10 days postinjection). the concentrations of nbs in the sera were detected by elisa, as described above. briefly, mers-cov s1-his protein (2 g/ml) was used to coat elisa plates, and then sera, goat anti-llama antibodies (1:5,000), and hrp-conjugated anti-goat igg antibodies (1:3,000) were sequentially added for elisa reactions. evaluation of protective efficacy of nbms10-fc nb. the prophylactic and therapeutic efficacy of nbms10-fc was evaluated in hdpp4-tg mice as previously described (29) . briefly, male and female mice (8 to 10 weeks old) were intraperitoneally anesthetized with sodium pentobarbital (5 mg/kg of body weight) before being intranasally inoculated with lethal dose of mers-cov (emc2012 strain, 10 5.3 tcid 50 ) in 20 l of dulbecco modified eagle medium. either 3 days preinfection or 1 or 3 days postinfection, the mice were intraperitoneally injected with nbms10-fc (10 mg/kg). trastuzumab mab was used as a control to the nb. the infected mice were observed daily for 14 days, and their body weights and survivals were recorded. statistical analysis. statistical analysis was performed using graphpad prism version 5.01. to compare the binding of nbs to mers-cov s1 or rbd protein, as well as the rbds with or without d539a mutation to hdpp4 receptor, a two-tailed student t test was used. one-way analysis of variance was used to compare the inhibition of nbs to rbd-hdpp4 binding. statistical significance between survival curves was analyzed using kaplan-meier survival analysis with a log-rank test. p values lower than 0.05 were considered statistically significant. in the figures, "*," "**," and "***" indicate p ͻ 0.05, p ͻ 0.01, and p ͻ 0.001, respectively. data availability. all data needed to evaluate the conclusions presented here are included. additional data related to this study may be requested from the authors. camelid and shark single domain antibodies: structural features and therapeutic potential nanobody-based products as research and diagnostic tools global analysis of vhhs framework regions with a structural alphabet application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies® as inhaled biotherapeutics for lung diseases generation and characterization of alx-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection nanobodies as therapeutics: big opportunities for small antibodies nanobodies: natural single-domain antibodies caplacizumab for acquired thrombotic thrombocytopenic purpura phase i study of 68ga-her2-nanobody for pet/ct assessment of her2 expression in breast carcinoma the titan trial-assessing the efficacy and safety of an anti-von willebrand factor nanobody in patients with acquired thrombotic thrombocytopenic purpura isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction molecular evolution of mers coronavirus: dromedaries as a recent intermediate host or longtime animal reservoir? mers-cov spike protein: a key target for antivirals mers-cov spike protein: targets for vaccines and therapeutics receptor recognition mechanisms of coronaviruses: a decade of structural studies structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure, function, and evolution of coronavirus spike proteins recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce crossneutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus vaccines for the prevention against the threat of mers-cov introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies nanobodies and nanobodybased human heavy chain antibodies as antitumor therapeutics multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution single-domain antibodies as versatile affinity reagents for analytical and diagnostic applications single domain antibodies: promising experimental and therapeutic tools in infection and immunity single domain camel antibodies: current status engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 isolation of antigen specific llama vhh antibody fragments and their high level secretion by saccharomyces cerevisiae searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments: the importance of immunofocusing in subunit vaccine design single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection rapid human metapneumovirus microneutralization assay based on green fluorescent protein expression this study was supported by the national key plan for scientific research and summarized and analyzed the data. j.s. and f.l. performed the structural analysis. g.z., f.l., l.d., and y.z. wrote the manuscript. s.j., f.l., l.d., and y.z. revised the manuscript. key: cord-349262-gnqbyc6t authors: hemida, maged gomaa; ali, mohammed; alhammadi, mohammed; alnaeem, abdelmohsen title: the middle east respiratory syndrome coronavirus in the breath of some infected dromedary camels (camelus dromedarius) date: 2020-10-14 journal: epidemiol infect doi: 10.1017/s0950268820002459 sha: doc_id: 349262 cord_uid: gnqbyc6t dromedary camels remain the currently identified reservoir for the middle east respiratory syndrome coronavirus (mers-cov). the virus is released in the secretions of the infected camels, especially the nasal tract. the virus shedding curve through the nasal secretions was studied. although human transmission of the virus through the respiratory tract of close contact people with dromedary reported previously, the exact mechanism of transmission is still largely unknown. the main goal of this study was to check the possibility of mers-cov shedding in the exhaled air of the infected camels. to achieve this goal, we conducted a follow-up study in one of the dromedary camel herds, december 2018–april 2019. we tested nasal swabs, breath samples from animals within this herd by the real-time pcr. our results showed that some of the tested nasal swabs and breath were positive from 24 march 2019 until 7 april 2019. the phylogenetic analysis of the obtained s and n gene sequences revealed the detected viruses are clustering together with some human and camel samples from the eastern region, especially from al-hufuf city, as well as some samples from qatar and jordon. these results are clearly showing the possibility of shedding of the virus in the breath of the infected camels. this could explain, at least in part, the mechanism of transmission of mers-cov from animals to humans. this study is confirming the shedding of mers-cov in the exhaled air of the infected camels. further studies are needed for a better understanding of the mers-cov. it has been almost 8 years since the emergence of the middle east respiratory syndrome coronavirus (mers-cov) in 2012 in the arabian peninsula. the majority of human cases were reported in the arabian peninsula [1] [2] [3] . more recently, there are many reports about the potential risk of infection with mers-cov in africa [1] . the only known reservoir so far is the dromedary camels [4] . there are many reports about the animal-human transmission in the context of mers-cov [5, 6] . it is now well documented that mers-cov is released in the respiratory secretions detected in the nasal swabs by several research groups [5, [7] [8] [9] [10] [11] . although no report on the secretion of the virus in the milk, it is highly recommended to boil it before consumption to avoid any potential contamination of the raw milk during the process of milking and handling [12] [13] [14] . similar concept for eating the meats and raw organs, particularly the liver, it should be cooked well before consumption to avoid any potential hazards of infection as suggested by the world health organization (who) [12, 14] . the virus was also detected in the air around some positive camel herd. detection of the mers-cov-rnas of identical sequences from the owner and the herders of this particular herd was also reported [5, 6] . however, some other studies failed to detect the virus in the urine of some infected animals [15] . earlier studies succeeded in the detection of the mers-cov nucleic acids in the air circulated by an infected camel herd as well as in the people of close contact of this particular herd [5, 6] . meanwhile, several reports showed the possibility of detection of the mers-cov nucleic acids in the circulating air in some health care settings during the korean outbreak of mers-cov in 2015 [16, 17] . the main goal of the current study was to check the possibility of mers-cov shedding in the infected animal breath. we conducted this study according to the guidelines of the animal ethics protocols and the national committee of bio-ethics, king abdul-aziz city of science and technology, royal decree no. m/59 (http://www.kfsh.med.sa/kfsh_website/users uploadedfiles%5cncbe%20regulations%20english.pdf). the animal experiments were approved by the animal care committee of the deanship of scientific research, king faisal university, saudi arabia. we conducted molecular surveillance for the mers-cov in a dromedary camel herd during 2019. this herd consists of 52 animals held together in wire fenced yards. the male animals were kept in a separate individual partition while the female was kept together in multiple partitions. there is a shared open yard where all females may be allowed to be mixed together. the males only approach females during the mating season from november to march per each year. during the time from 10 march 2019 until 7 april 2019, we randomly selected nine animals out of the 52 (18%) from the camel herd to conduct our molecular surveillance. all animals were of variable ages ranging from 2 to 8 years old. the nine animals selected from different colour coat-based breeds, including magaheem, wodoah and sofr, were selected. these nine animals were mixed with the rest of the animals in the herd all the time. our molecular surveillance lasts from 10 march to 8 april 2019, with a biweekly sampling interval. we collected nasal swabs, breath samples from these nine animals, as previously described. a paired nasal and breath samples were collected per each animal at each time point, as described in detail below. nasal swabs were collected from at least 15% of the dromedary camel herd from november until march 2019. swabs were transferred into the tubes containing viral transport media including the dmem media containing 10% foetal bovine sera and antibiotic cocktail (penicillin and streptomycin). the processing of the collected swabs was done as previously described. briefly, the swabs were vortexed, then centrifuged at 5000 rpm for 10 min in a cooling centrifuge at 4°c. the supernatants were collected and stored at −80°c for further testing. the breath samples were collected in sterile tubes containing viral transport media as used for the collection of the nasal swabs. the breath collection technique was done in a simple way. briefly, the animals were secured in a specially designed camel crush. the animals were allowed to settle down for 5 min. with the help of an assistant, the nasal orifice is dilated, and the tube containing the media is slightly inserted inside (2-3 cm) the orifice without touching the skin or the mucous membranes of these animals during sampling. the collection tubes were held in a slope position facing the air stream coming from the animal during the exhalation. the tubes were kept in such position until the animal completes at least five cycles of breathing, including five exhalation streams. any container that touched the skin or the mucous membrane of the animals was discarded and not included in our analysis. the collection tubes were shaken immediately for 3 min and inverted upside down several times, then placed into ice tanks until transferred to the laboratory. the viral rnas were extracted from paired swabs and breath samples collected from dromedary camels using the qiagen viral rna (rneasy mini kit, qiagen, hilden, germany) extraction kit, according to the manufacturer's protocol. the concentration of the extracted viral rnas was measured by the nanodrop machine (thermo scientific nanodrop 2000, applied biosystems, 850 lincoln centre drive, foster city, ca 94404, usa). the eluted rnas were stored at −80°c for further testing. the rt-pcr targeting the upstream gene (up-e) of mers-cov was used for screening [13] . confirmation was done using the open reading frame (orf) 1a. five μl of the extracted rna was subjected to the real-time pcr testing using upe primers using lightmix molecular dx mers-cov-upe kits (roche, roche molecular systems, inc, pleasanton, ca 94588, usa) according to the manufacturer's protocol. all positive samples by the upe assay were confirmed by orf1a, as previously described [7] . positive samples were considered when there is an overlapping between the results from two targets. samples (nasal swabs and breath) that had ct values <39 were considered positive per each target. we used several sets of primers to amplify the partial mers-cov-s, mers-cov-n genes. the sequences of these primers are as follow: nf-5 ′ -cct tcg gta cag tgg agc ca-3 ′ and nr-5 ′ -gat ggg gtt gcc aaa cac aaa c-3 ′ , primers for the mers-cov-s gene sf-5 ′ -ccaattta-cgccaggatgat-3 ′ and sr-5 ′ -aatagaggcgg aaatagcac-3. we used the primers listed above to amplify the partial mers-cov-s and n genes using the qiagen one-step rt-pcr kit (cat no./id: 210210) to synthesis the cdna then doing the pcr in one reaction. the procedure of this one-step reaction was carried out as per the kit's instructions as well as previously described. we ran 5 μl per each amplified mers-n and s amplicons on a 1% agarose gel containing sybr® safe dna gel stain (invitrogen, thermo fisher scientific, waltham, ma, usa). the amplicons were visualised under ultraviolet light, then the gel pictures were photographed with the gel documentation system (bio-rad laboratories, inc., hercules, ca, usa). we purified the desired pcr products from the target bands by using the gel-based pcr extraction kits (qiagen, cat no/id: 28704), as per the instructions of the kits. we eluted the purified pcr products in 50 μl elution buffer as suggested by the kits, then stored at −20°c. we sequenced some positive samples from dromedary camels having low ct values (≤29) on the real-time pcr. the sequencing was done by the sanger method using the applied biosystems® we used the obtained sequences from dromedary camels to develop the phylogenetic tree based on the reported partial mser-cov-s and n genes sequences. the phylogenetic trees were built using the neighbour-joining method by the mega-7 software, as previously described. the scale bar represents the tree distance corresponding to 0.6 nucleotide substitution/nucleotides. a series of two by two tables, utilizing the fisher's exact test, were used to test the association between the results of the molecular surveillance, the sampling intervals and the sampling technique [18] . all the statistical data analyses were performed by spss version 21.0 (ibm corp, 2012). the values (<0.05) were considered significant (fig. 1) . our molecular testing for the selected animals at three time points starting 10 march 2019, until 7 april revealed that 1/9 (11%) of the tested nasal swabs were positive; all the nine animals were negative from the breath samples (table 1) . two weeks later, on 24 march, 6/9 (77%) nasal swabs were positive, and 5/9 (55%) breath samples were positive (table 1) . finally, on 7 april, 3/9 nasal swabs (33%) were positive, while 1/9 (11%) of the breath samples were positive ( table 1 ). the prevalence of positive nasal and breath samples was higher on the second sampling batch (24 march) compared to first (10 march) and third (7 april) batches. however, the difference was statistically significant (p < 0.05) only between the first vs. second sampling batches. similarly, consolidated results obtained from both nasal swaps and breath were significantly higher in the second batch vs. the first batch (p < 0.05). no significant statistical differences were observed between the prevalence of positive results obtained from nasal swabs and breath samples. the detection of the mers-cov-rnas in the breath of all three time intervals was statistically significant (p > 0.05) ( table 1) . the sequencing analysis of the partial mer-cov-s and n genes (figs 2 and 3 , respectively) from this positive mers-cov herd showing the detected viruses were closely related to the sequence from dromedary camels and humans from the arabian peninsula. the reported partial mers-cov-s gene clustered together with the other human sequences reported from al-hufuf city in the same regions in 2015 as well as sequences from qatar and jordon-2015 (fig. 2 ). the mers-cov continues to pose a significant risk for humans, especially some of those who may come in close contact with dromedary camels [12, 13] . despite the emergence of sars-cov-2 late 2019 [19] , mers-cov still has the high case fatality rates among the affected populations compared to sars-cov and sars-cov-2. the mers-cov case fatality rate started at 52% in 2012 then dropped down to 32% in 2019 [20] . the presence of mers-cov in the air of the proximity of patients and infected dromedary camels was previously documented independently by many research groups [6, 16, 17] . this result confirms that mers-cov may be transmitted through droplet infection. however, little is still known about the mechanism of transmission of mers-cov from the dromedary camels to humans. the roles of some camel secretions and excretions in the context of the mers-cov infection have been studied [5, 13, 15] . our results show that about 11% of the tested nasal swabs were positive during the first batch of the collection, while none of the breath samples was positive ( table 1 ). the reasons behind the inability to detect the viral rnas in the breath during the first round of sampling may be attributed to the dilution of the viral rna in the breath, and the rna may be present at the nondetectable limit by the real-time pcr. a high consistency was observed between the detection of the viral nucleic acids in both the nasal and breath samples in all three tested batches (p > 0.05), indicating similar opportunities to detect the virus nucleic acid in both methods (table 1 ). this may be attributed to several factors. first, during the early stage of the virus infection, the rna concentration in the breath may be at an undetectable level by the real-time pcr. second, the virus replication reached the peak of each animal; therefore, high virus concentration was achieved as previously reported [7, 8] . these results showed that mers-cov could be shed in the breath of infected animals for a while. however, the nasal swabs are still the sample of choice in the diagnosis of mers-cov among the infected dromedary camel population. spreading of the virus from animal to another in the same herd could potentially happen through the respiratory routes taking into consideration the very close contact between animals within the same herd. it may also occur through sharing the contaminated food and water from infected animals; however, all these aspects still need further investigations. detection of the virus in the air of positive camel's herd [5, 6] may suggest the virus is excreted in the breath of the infected animals in high concentration. however, this hypothesis was never tested before. detection of the foot and mouth diseases virus (fmdv) in the breath of some infected cattle was previously documented [21] . this study confirmed the potential spread of the fmdv between animals and among various herds in close proximity or within a distance. the aim of our study was to test the possibility of mers-cov shedding in the breath of the infected dromedary camels. our results are clearly showing the potential secretion of mers-cov in the breath of infected animals ( table 1) . the phylogenetic analysis based on the partial mers-cov-s and n genes in these infected animals revealed high identity to other mers-cov previously detected in the arabian peninsula (figs 2 and 3) [7, 8, 11] . a recent study showed that the sars-cov-2 could be transmitted through the droplet infection in the air [22] . although detection of the mers-cov-rnas in the breath does not conclude the viability of the detected virus particles. this may require virus isolation and the plaque assay to ensure the virus infectivity in the collected samples. these techniques should be done at a biosafety contaminant-3 laboratory. however, the overlapping between the viral detection in the nasal swabs and breath, as one of the gold standard techniques for the detection of the virus as well as the high degree of identity of the reported sequences in this study, may provide a piece of strong evidence about the potential shedding of the mers-cov in the breath of infected animals at various levels. further large-scale studies are highly recommended to document the curve of the mers-cov shedding in various body secretions and execrations as well as in the breath. this will lead to a better understanding of the dynamics of the virus spread among certain dromedary camel populations as well as in the environment. taken together all these evidence, we may conclude that mers-cov could be transmitted through the breath of infected animals. the virus spread from animal to animal and from animal to human come in their close contact (fig. 4) . however, large-scale controlled studies are still required to further enrich our understandings about the mechanism of mers-cov transmission between animals as well as from animals to humans. middle east respiratory syndrome coronavirus (mers-cov) neutralising antibodies in a high-risk human population comparative serological study for the prevalence of anti-mers coronavirus antibodies in high-and low-risk groups in qatar middle east respiratory syndrome coronavirus during pregnancy middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia evidence for camel-to-human transmission of mers coronavirus detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in africa and middle east genetic evidence of middle east respiratory syndrome coronavirus (mers-cov) and widespread seroprevalence among camels in kenya prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate mers-cov at the animal-human interface: inputs on exposure pathways from an expert-opinion elicitation middle east respiratory syndrome coronavirus and the one health concept dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) failure to detect mers-cov rna in urine of naturally infected dromedary camels risk of global spread of middle east respiratory syndrome coronavirus (mers-cov) via the air transport network airflow as a possible transmission route of middle east respiratory syndrome at an initial outbreak hospital in korea handbook of biological statistics a novel coronavirus from patients with pneumonia in china some one health based control strategies for the middle east respiratory syndrome coronavirus detection of foot-and-mouth disease virus in the breath of infected cattle using a hand-held device to collect aerosols airborne transmission of sars-cov-2: the world should face the reality acknowledgements. we wish to thank the king abdul-aziz city for science and technology (kacst), saudi arabia, for their generous funding through the mers-cov research grant programme (number 20-0004/24-1), which is a part of the targeted research program (trp). data availability statement. data are available upon request. key: cord-343789-6tq0kcfd authors: al-tawfiq, jaffar a.; momattin, hisham; dib, jean; memish, ziad a. title: ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study date: 2014-01-06 journal: int j infect dis doi: 10.1016/j.ijid.2013.12.003 sha: doc_id: 343789 cord_uid: 6tq0kcfd background: the middle east respiratory syndrome coronavirus (mers-cov) has been reported to have a high case-fatality rate. currently, there is no specific therapy or vaccine with proven effectiveness for mers-cov infections. methods: a combination of ribavirin and interferon therapy was used for the treatment of five mers-cov-positive patients. we reviewed the therapeutic schedule and the outcome of these patients. results: all patients were critically ill with acute respiratory distress syndrome treated with adjunctive corticosteroids and were on mechanical ventilation at the time of initiation of therapy. the median time from admission to therapy with ribavirin and interferon was 19 (range 10–22) days. none of the patients responded to the supportive or therapeutic interventions and all died of their illness. conclusions: while ribavirin and interferon may be effective in some patients, our practical experience suggests that critically ill patients with multiple comorbidities who are diagnosed late in the course of their illness may not benefit from combination antiviral therapy as preclinical data suggest. there is clearly an urgent need for a novel effective antiviral therapy for this emerging global threat. since the discovery of middle east respiratory syndrome coronavirus (mers-cov), the virus has caused 163 cases of disease, with a fatality rate of 43-50%. 1, 2 the disease was initially described in a patient from saudi arabia in june 2012. 3 mers-cov has caused sporadic cases and clusters in families and healthcare settings. [4] [5] [6] [7] the best treatment option for the virus is not known. in view of the high case-fatality rate and the potential global spread of the virus, there is an urgent need to develop effective therapies for this infection. in a recent review, based on therapies used for the related severe acute respiratory syndrome (sars) coronavirus, the possible use of interferon and ribavirin was considered as a therapeutic option. 8 the purpose of this study was to describe the outcome of the use of a combination of interferon-a2b and ribavirin in the management of five patients with mers-cov infections. a 62-year-old woman with diabetes, hypertension, and endstage renal disease (esrd) on hemodialysis was admitted with fever, cough, and respiratory failure of 3-day duration (table 1) . she tested positive for mers-cov by pcr. baseline laboratory data are shown in tables 2 and 3. on admission, the patient was started on oseltamivir 75 mg once daily for 5 days and levofloxacin 500 mg intravenous (iv) every 2 days for 15 days. imipenem was added on day 5 for 3 days. on day 21 after admission, she was started on ribavirin for 5 days with a loading dose of 2000 mg via nasogastric tube, followed by 400 mg per os (po) every 8 h, one dose of interferon-a2b 130 mg subcutaneously, and methylprednisolone 40 mg iv every 8 h for 1 day, then twice daily for 1 day, then daily for 2 days. she had a mild rise in amylase and lipase (from 61 to 126 u/l and from 274 to 348 u/l, respectively). the patient remained critically ill on mechanical ventilation requiring inotropic support. she died 34 days after admission from respiratory and multi-organ failure. a 58-year-old man with diabetes, hypertension, and esrd on hemodialysis was admitted with cough, fever, and hypoxemic respiratory failure of 2-day duration. he tested positive for mers-cov by pcr. baseline laboratory data are shown in tables 2 and 3. on admission, the patient was started on oseltamivir 75 mg once daily for 5 days, levofloxacin 500 mg iv every 2 days for 6 days, and imipenem 250 mg iv twice daily for 7 days. on day 6, he was prescribed methylprednisolone 60 mg iv every 6 h for 6 days, then every 8 h for 1 day, then 40 mg every 12 h for 1 day, followed by a prednisolone tapered dose for another 10 days. on day 13 after admission, he was started on ribavirin for 5 days with a loading dose of 2000 mg po, followed by 400 mg every 8 h and two doses of interferon-a2b 100 mg subcutaneously once per week. two weeks after the initiation of the therapy, platelets dropped from 408 to 43 â 10 9 /l ( table 2 ). the patient remained on the ventilator for more than 30 days. he died 35 days after admission from multiorgan failure. a 63-year-old woman with a history of severe bronchial asthma and obstructive sleep apnea (on continuous positive airway pressure (cpap)), with coronary artery disease, diabetes, hypertension, and chronic kidney disease (estimated glomerular filtration rate 15.7 ml/min), was admitted with fever, cough, and dyspnea of 3-day duration and a diagnosis of pneumonia. she tested positive for mers-cov by pcr. baseline laboratory data are shown in tables 2 and 3. on admission, the patient was also started on oseltamivir 75 mg once daily for 5 days, levofloxacin 750 mg iv every 2 days for 7 days, and imipenem 500 mg iv every 6 h for 8 days. on day 11, she was started on ribavirin for 5 days, with a loading dose of 2000 mg via nasogastric tube followed by 600 mg po every 8 h, one dose of interferon-a2b 144 mg subcutaneously, and prednisone 40 mg po daily, tapered to 10 mg po daily for 10 days. one week after initiation of the antiviral and steroid therapy, serum creatinine, aspartate aminotransferase (ast) tables 2 and 3, and amylase increased (from 0.6 to 2.9 mg/dl, from 6.3 to 20 â 10 9 / l, from 25 to 126 iu/l, and from 35 to 255 u/l, respectively). the patient was diagnosed with pancreatitis and thus further interferon and ribavirin was not given. the patient remained critically ill on mechanical ventilation. she died 45 days after admission from multi-organ failure. an 81-year-old man with a history of atrial fibrillation, diabetes mellitus, and hypertension was admitted with progressive respiratory distress and hypoxemia with fever for 2 days. he tested positive for mers-cov by pcr. baseline laboratory data are shown in tables 2 and 3. on the first day of hospitalization, the patient was started on oseltamivir 75 mg once daily for 6 days, levofloxacin 750 mg every 48 h for 5 days, and imipenem 250 mg iv every 6 h for 6 days. on day 20 after admission, he was started on ribavirin for 9 days, with a loading dose of 2000 mg via nasogastric tube followed by 800 mg via nasogastric tube every 8 h, two doses of interferon-a2b 100 mg subcutaneously once per week on hospital days 20 and 27, and methylprednisolone 40 mg iv every 8 h for 4 days, then 40 mg iv twice daily for 2 days, then 40 mg daily for 2 days (started on hospital day 22). three weeks after the initiation of therapy, his hemoglobin dropped (from 11.7 to 8.7 g/dl) ( table 2) , while bilirubin increased (from 0.7 to 7.4 mg/ dl), suggestive of hemolysis. the patient died 52 days after admission from multi-organ failure. a 24-year-old man with a history of esrd on hemodialysis was admitted with a febrile illness, a cough for 10 days, and respiratory failure requiring mechanical ventilation. he tested positive for mers-cov by pcr. baseline laboratory data are shown in tables 2 and 3. on the first day of hospitalization, the patient was started on oseltamivir 75 mg once daily for 2 days and imipenem 250 mg iv twice daily for 11 days. on day 19 of admission, he was started on ribavirin for 11 days, with a loading dose of 2000 mg via nasogastric tube, followed by 400 mg via nasogastric tube every 8 h, and two doses of interferon-a2b 100 mg subcutaneously once per week on hospital days 19 and 26. on the same day he started ribavirin, he was also started on methylprednisolone 40 mg iv every 8 h for 4 days, then 40 mg iv twice daily for 2 days, and then 40 mg daily for 2 days. two weeks after the initiation of therapy, lipase increased (from 561 to 612 u/l). the patient died 32 days after admission from multi-organ failure. we reviewed the cases of five patients with mers-cov (table 1) . their median age was 62 (range 24-81) years. the patients had chronic kidney disease and four were on maintenance hemodialysis. there were three men and two women. all patients tested negative for influenza. the median number of days from admission to therapy with ribavirin and interferon was 19 (range 10-22) ( table 4 ). all patients received adjunctive corticosteroid therapy for acute respiratory distress syndrome. two patients developed increased lipase after the initiation of therapy, but both of them were given corticosteroids at the same time as antiviral therapy. one patient (case 2) developed thrombocytopenia, with a platelet count that dropped from 408 to 49 â 10 9 /l (tables 2 and 3) and one patient had possible hemolysis (case 3). all patients had severe disease with progressive respiratory failure, developed multi-organ failure, and died a mean 39.6 (standard deviation 8.5) days after admission. none of the patients had bacteremia or fungemia during their hospital stay. figure 1 shows the median, minimum, and maximum values for amylase, lipase, and lactate dehydrogenase (ldh) for all the patients before initiation of therapy and at week1 and 2 after therapy initiation. since the emergence of mers-cov in 2012, the virus has caused a total of 163 cases of disease, with a high case-fatality rate of 43%. 2 there is an urgent need for effective therapeutic agents. to date, no data are available on the use of any agents for the therapy of mers-cov-positive patients. in this report we have described the therapy of five mers-cov patients who received interferon and ribavirin. the patients were treated during the initial phase of the al-hasa outbreak in april and may 2013, a time at which the disease epidemiology and clinical characteristics were not known. hence, there was an inevitable time lapse from admission to the initiation of therapy. all patients were already on mechanical ventilation by the time interferon and ribavirin therapy was instituted and all had a fatal outcome. the patients received the therapy late in the course of the disease, at a median of 19 (range 10-22) days after admission. late therapy may have contributed to the poor outcome, in addition to the severity of the disease and the multiple comorbidities of the patients. the timing of initiation of antiviral therapy is critical in the treatment of most viral infections. in the treatment of sars-cov, no effect of oseltamivir or ribavirin was observed when these agents were started 6-14 days after symptom onset. [11] [12] [13] early therapy with ribavirin, within 48 h of hospitalization or after the diagnosis of sars, has been shown to be associated with better outcomes, although the numbers of patients enrolled in these studies has been small. 7, [14] [15] [16] [17] in the treatment of influenza, oseltamivir therapy early in the disease resulted in reduced mortality when this was started not later than 8 days after the onset of symptoms. 18 early in vitro studies showed that ribavirin and interferon have anti-mers-cov activity. 19 the in vitro activity of interferon was augmented by the concomitant use of ribavirin. 20 in a rhesus macaque model of mers-cov infection, the combination of interferon-a2b and ribavirin therapy was effective in limiting the disease and resulted in very mild radiographic evidence of pneumonia. 21 the treatment was given within 8 h after inoculation of the rhesus macaques. treated animals had lower levels of systemic and local proinflammatory markers and fewer viral genome copies. 21 although these preclinical data are promising, our report illustrates some of the real world issues of dealing with a novel emerging viral infection. our patients were not diagnosed until a week into their illnesses, by which time all five were on mechanical ventilation. they had multiple comorbidities, which most likely adversely affected their clinical outcomes. in addition, we had no serial virologic determination of mers-cov levels in airway secretions to shed light on the possibility of virological clearance, virological failure, or clinical failure of therapy. in our small series, adverse effects from combination ribavirin and interferon therapy were observed in three cases. these side effects have been noted before. 22 two patients had pancreatic enzyme elevation and one had significant hemolysis. these findings were complicated by the presence of abnormal laboratory findings even before the initiation of combination therapy, so it is difficult to determine which side effects were due to disease progression and which were due to the therapeutic drugs. there is an urgent need for large-scale clinical trials to determine the safest and most effective regime for the treatment of this novel highly fatal emerging infection. while ribavirin and interferon may show promise, their use needs to be prompt and adverse effects monitored closely. this therapeutic approach should be tested in careful clinical studies. taking stock of the first 133 mers coronavirus cases globally-is the epidemic changing? middle east respiratory syndrome coronavirus (mers-cov)-update isolation of a novel coronavirus from a man with pneumonia in saudi arabia a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus family cluster of middle east respiratory syndrome coronavirus infections therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts severe acute respiratory syndrome: report of treatment and outcome after a major outbreak investigational use of ribavirin in the treatment of severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china effectiveness of antiviral treatment in human influenza a(h5n1) infections: analysis of a global patient registry broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus inhibition of novel b coronavirus replication by a combination of interferon-a2b and ribavirin treatment with interferon-a2b and ribavirin improves outcome in mers-covinfected rhesus macaques product information: rebetol (r) oral capsules solution, ribavirin oral capsules solution the authors (jat, hm, and jd) wish to acknowledge the use of the saudi aramco medical services organization (samso) facilities for the data and study, which resulted in this paper. opinions expressed in this article are those of the authors and not necessarily of samso. the authors thank dr paul anantharajah tambyah of the national university of singapore department of medicine for his critical review of the manuscript.financial support: all authors have no funding. conflict of interest: all authors have no conflict of interest to declare. key: cord-342144-awtiqxx5 authors: hufert, f.; spiegel, m. title: coronaviren: von der banalen erkältung zum schweren lungenversagen: chronologie einer pandemie date: 2020-04-01 journal: monatsschr kinderheilkd doi: 10.1007/s00112-020-00910-2 sha: doc_id: 342144 cord_uid: awtiqxx5 in december 2019 a new human coronavirus emerged in wuhan, china, which is known as sars-cov‑2. the clinical course of the disease known as coronavirus disease 2019 (covid-19) ranges from mild respiratory symptoms to severe lung failure. the virus is currently rapidly spreading around the world and pushing health systems to the limits of their capacity due to the exponential increase in the number of cases. the origin of sars-cov‑2 lies in the bat coronavirus pool and has now emerged in the human population due to interspecies transmission. molecular diagnostic methods have been established in a very short time and a number of clinical studies on the effectiveness of different antiviral drugs are ongoing. the development of a vaccine using different approaches is also under investigation. considering the high number of cases and mortality rates of up to 9% there is an urgent need for action. this article summarizes the current state of knowledge on human coronaviruses with a strong focus on the current data on sars-cov‑2. due to the daily changing level of knowledge, the article reflects the status up to 21 march 2020. humane coronaviren sind häufige vertreter der familie coronaviridae (subfamilie orthocoronavirinae) und verursachen hauptsächlich respiratorische infektionen ("respiratory tract infections", rti) unterschiedlicher schweregrade. die klassischen vertreter der humanen coronaviren sind: hcov-229e, hcov-nl63, hcov-oc43 und hcov-hku1. sie sind für ein drittel aller erkältungserkrankungen weltweit verantwortlich. in den meisten fällen sind infektionen mit diesen hcov unkompliziert, es kann aber auch zu schweren verläufen mit lebensbedrohlicher pneumonie und bronchiolitis kommen. letztere treten v. a. bei immunsupprimierten patienten und im senium, aber auch bei kindern auf. im kindesalter sind 9 % aller rti durch hcov verursacht. die hospitalisierungsraten von hcov-infizierten kindern mit infektionen des unteren respirationstrakts betragen 1,5 (<5 jahre) bis 2,8 (<1 jahr) pro 1000 [1] . neben der bekannten respiratorischen symptomatik können neuronale gewebe und zellen des gastrointestinaltrakts infiziert werden, sodass auch mit neurologischen symptomen und gastrointestinalen beschwerden zu rechnen ist. im jahr 2002 trat in der provinz guangdong (china) erstmalig ein neues coronavirus in der menschlichen population auf, das sars-cov (sars: "severe acute respiratory syndrome"). die infektion verlief influenzaartig mit fieber, kopfschmerzen, husten und myalgien. im weiteren kam es aber auch zu pneumonien, die zum lebensbedrohlichen lungenversagen führten. beim ausbruch des sars-cov im zeitraum von 2002-2004 wurden mehr als 8000 fälle in 29 ländern registriert. die mortalität betrug ca. 10%. molekularbiologische untersuchungen der genomsequenzen zeigten, dass sars-cov aus dem pool der fledermaus-coronaviren stammt und über andere säuger (z. b. zibetkatze) als zwischenwirt auf den menschen übertragen wurde. es handelte sich also um ein zoonotisches virus, das neu in die menschliche population eingebracht worden war. die epidemie konnte gut eingedämmt werden, da die übertragung von mensch zu mensch erst mit erkrankungsbeginn erfolgte, sodass schon einfaches temperaturmessen und definierte kontrollmaßnahmen den ausbruch stoppen konnten. im jahr 2012 traten klinisch ähnliche fälle in saudi-arabien auf, die ebenfalls mit schwerem lungenversagen assoziiert waren. hier zeigte sich, im gegensatz zu sars-cov, das auch über "superspreader"-ereignisse übertragen wurde und sich dadurch schnell weltweit verbreitete, ein mehr geografisch limitiertes auftreten der infektionen. ursache dieses ausbruchs war ein sars-verwandter erreger, das mers-cov (mers: "middle east respiratory syndrome"). bis heute sind weltweit 2243 fälle gemeldet worden, 85 % traten in saudi-arabien und den vereinigten arabischen emiraten auf. die restlichen fälle verteilen sich weltweit auf 25 länder und sind alle aus dem nahen osten importiert worden. im gegensatz zu sars-cov ist die mortalität von mers-cov mit 35 [3] , der eingriff in zelluläre apoptosemechanismen und die inhibition der antigenpräsentationsfunktion dendritischer zellen [4] . die diagnostik respiratorischer infektionen erfolgt heute mithilfe der echtzeit-polymerase-kettenreaktion ("realtime polymerase chain reaction", rt-pcr) im rahmen einer panel-diagnostik, die verschiedene erreger simultan nachweisen kann. dieses verfahren erlaubt den zeitnahen, sehr sensitiven und hochspezifischen nachweis viraler und bakterieller genome und ist den antigennachweisverfahren hinsichtlich sensitivität und spezifität deutlich überlegen. infektionen mit hcov werden weitgehend symptomatisch therapiert. chloroquin zeigt im mausmodell bei infektionen des zentralnervensystems (zns) einige erfolge [5] . ein klinischer nutzen konnte beim einsatz der eigentlich für die behandlung von humane-immundefizienz(hiv)-infektionen verwendeten proteaseinhibitoren lopinavir und ritonavir (kaletra ® ) zur therapie des sars-cov nachgewiesen werden [6] . im dezember 2019 trat in china erstmalig ein neues coronavirus auf, das zunächst als 2019-ncov bezeichnet wurde und nach aktueller nomenklatur des international committee on taxonomy of viruses (ictv) nun als sars-cov-2 bezeichnet wird [7] . die erkrankung, die das virus auslöst, erhielt von der world health organization (who) den namen "coronavirus disease 2019" (co-vid-19). betroffen war die provinz hubei und hier v. a. die 15 mio. einwohner zählende region der provinzhauptstadt wuhan. bis heute ist das virus bereits in zahlreiche länder der erde importiert worden. infolgedessen hat die who den internationalen gesundheitsnotstand ("public health emergency of international concern") ausgerufen. wissenschaftler arbeiten weltweit mit hochdruck an den zentralen biologischen, medizinischen und epidemiologischen fragestellungen zu covid-19 und dem erreger sars-cov-2; daher ändert sich die datenlage täglich. dieser beitrag erfasst den datenstand bis zum zeitpunkt mitte märz 2020. das sars-cov-2 ist das neueste der coronaviren, die in den letzten 20 coronavirus: from common cold to severe pulmonary failure. in december 2019 a new human coronavirus emerged in wuhan, china, which is known as sars-cov-2. the clinical course of the disease known as coronavirus disease 2019 (covid-19) ranges from mild respiratory symptoms to severe lung failure. the virus is currently rapidly spreading around the world and pushing health systems to the limits of their capacity due to the exponential increase in the number of cases. the origin of sars-cov-2 lies in the bat coronavirus pool and has now emerged in the human population due to interspecies transmission. molecular diagnostic methods have been established in a very short time and a number of clinical studies on the effectiveness of different antiviral drugs are ongoing. the development of a vaccine using different approaches is also under investigation. considering the high number of cases and mortality rates of up to 9% there is an urgent need for action. this article summarizes the current state of knowledge on human coronaviruses with a strong focus on the current data on sars-cov-2. due to the daily changing level of knowledge, the article reflects the status up to 21 march 2020. [23] . untersuchungenvonchenetal.konntenzeigen,dasseine schwangerschaftkeinen einfluss auf den verlauf der covid-19-pneumonie hat. es ist bisher keine intrauterine übertragung des virus auf das ungeborene in der spätschwangerschaft beschrieben worden [24] . aufgrund der noch geringen fallzahl von 9 infektionen in der schwangerschaft können diese ergebnisse noch nicht als gesichert gelten. die diagnostik von sars-cov, mers-cov und sars-cov-2 sowie auch von anderen coronaviren wird als molekularbiologischer direktnachweis mithilfe der rt-pcr durchgeführt [25] [26] [27] . als material sind ein nasen-und rachenabstrich sowie ein sekret der tiefen atemwege (sputum, probe einer bronchoalveolären lavage [bal] oder trachealsekret) zu gewinnen. hierzu sind spezielle abstrichtupfer zu verwenden, die entweder trocken oder besser mit einem virustransportmedium (evtl. auch 500 μl nacl-lösung) getränkt sind. gelhaltige abstrichtupfer sind für die pcr-diagnostik unbrauchbar. verschiedene pcr-testsysteme sind weltweit im einsatz. zurzeit wird in deutschland v. a. die pcr nach corman et al. durchgeführt [28] . die autoren des vorliegenden beitrags haben ebenfalls gute erfahrungen mit der vom amerikanischen cdc publizierten rt-pcr gemacht [29] . momentan wird an einem molekularen schnelltest auf der basis der isothermen rekombinase-polymerase-amplifikation gearbeitet, der einen vor-ort-nachweis in ca. 30 min erlaubt. die aktuellen testverfahren weisen un-terschiedliche genabschnitte des virus nach. aufgrund der hohen mutationsraten von rna-viren sollte, zum sicheren ausschluss von falsch-negativen ergebnissen, die pcr-diagnostik von zwei genabschnitten durchgeführt werden. zu beachten ist auch, dass mit diesem testverfahren kein infektiöses virus nachgewiesen wird, da virale gene oftmals länger nachgewiesen werden können, als tatsächlich ansteckungsgefahr besteht; ein beispiel hierfür ist die infektion mit noroviren. in diesem zusammenhang istdie tatsache erwähnenswert, dass bei mit sars-cov-2 infizierten kindern virusmaterial noch im stuhl nachgewiesen werden konnte, während zum selben zeitpunkt erfolgte rachenabstriche bereits negativ waren. auch hier allerdings ist unklar, ob es sich um ein infektiöses virus handelt [12] . zum nachweis der infektiosität muss eine virusisolierung in der zellkultur durchgeführt werden. erste untersuchungen am institut der bundeswehr in münchen haben gezeigt, dass das virus mindestens 4 tage nach erkrankungsbeginn noch isolierbar (pd dr. dobler, institut für mikrobiologie der bundeswehr; persönliche mitteilung) und damit der patient infektiös ist. daten zur infektionsdosis von sars-cov-2 in bezug auf die genomzahlkopien liegen noch nicht vor. infektiosität muss eine virusisolierung in zellkultur durchgeführt werden differenzialdiagnostisch muss die milde verlaufsform von covid-19 von anderen viralen infektionen des oberen rachentrakts abgegrenzt werden. die covid-19-pneumonie muss von einer pneumonie durch influenza-, adeno-, humanem respiratorischen synzytialvirus und anderen viral verursachten pneumonien sowie von einer durch mycoplasma pneumoniae verursachten pneumonie unterschieden werden. bei verdachtsfällen sind schnellstmöglich ein "point-of-care"-test (poct) und eine pcr-diagnostik auf krankheits-erreger der atemwege durchzuführen [14] . für die antivirale therapie von covid-19 stehen bislang weder zugelassene medikamente noch impfstoffe zur verfügung. in begrenztem umfang bestehen jedoch erfahrungen bei der therapie der sars-cov-infektion, die aufgrund der engen verwandtschaft der viren herangezogen werden können. so ist der kombinierte einsatz der proteaseinhibitoren lopinavir und ritonavir bei sars-cov-patienten von klinischem nutzen [6] . eine weitere studie mit mers-cov-infizierten wird in saudi-arabien durchgeführt, bei der mit lopinavir/ritonavir plus interferon-β behandelt wird [30] ; daten zu den ergebnissen liegen noch nicht vor. es zeigte sich aber im mausmodell, dass die kombination lopinavir/ritonavir plus interferon-β die viruslast bei der mers-cov-infektion nur geringgradig senken konnte und wenig einfluss auf die pathologischen veränderungen der lungen hat. erklären ließe sich dieses phänomen über die hohe plasmabindung der proteaseinhibitoren, sodass die freie wirkstoffkonzentration für einen antiviralen effekt gegen coronaviren evtl. nicht ausreicht. in einer aktuellen studie zeigte die behandlung von covid-19 patienten mit lopinavir/ ritonavir keinen therapeutischen nutzen [31] , sodass diese wirkstoffkombination wohl als therapiemöglichkeit ausfällt. therapie stehen bislang keine zugelassenen medikamente und impfstoffe zur verfügung bei der infektion mit mers-cov erzielte der polymeraseinhibitor remdesivir in verbindung mit interferon-β in vitro und im tiermodell eine sehr gute wirkung. im mausmodell konnten eine präventive und eine therapeutische wirkung nachgewiesen werden. die gabe von remdesivir plus interferon-β führte zur deutlichen reduktion der viruslast, einer verbesserte lungenfunktion und verminderter pathologischer lungenveränderungen [32] . auch im affenmodell erzielte remdesivirsehrgute erfolge [33] . hierbei ist zu beachten, dass die frühe gabe die wirkung deutlich verbessert. diese erklärt sich dadurch, dass die virusreplikation sehr schnell erfolgt und die antivirale wirkung, wenn der zell-, und gewebeschaden bereits gesetzt ist, deutlich geringer ist. antivirale therapeutische eingriffe müssen damit so früh wie möglich durchgeführt werden. remdesivir als rna-polymerase-inhibitor hat auch eine gute wirkung gegen andere rna-viren [34] . neben remdesivir besitzt chloroquin in vitro eine gute antivirale aktivität gegen coronaviren, einschließlich sars-cov-2 [5, 34] . der antivirale effekt beruht vermutlich auf den folgenden 2 mechanismen: (1) einer erhöhung des endosomalen ph-werts, die zur störung der viruszellfusion führt, (2) einer interferenz mit der glykosylierung des zellulären rezeptors ace2, die mit einer weniger effizienten bindung von sars-cov-2 einhergeht. die ec90 von chloroquin in der zellkultur, also die konzentration, bei der die sars-cov-2 replikation zu 90 % gehemmt wird, beträgt 6,9 μm. dies wäre (wie bei der therapie der rheumatoiden arthritis) durch die chloroquingabe von 3-4 mg/kgkgw und tag entsprechend der hydroxychloroquingabe von 6 mg/kgkgw und tag zu erreichen. hydroxychloroquin weist zusätzlich eine antiinflammatorische aktivität auf, die bei der behandlung von co-vid-19 von vorteil sein könnte [34] [35] [36] . erste klinische daten aus china legen den therapeutischen nutzen für chloroquin nahe [37] . entsprechend wurde die behandlung mit chloroquin in die aktuelle leitlinie der nationalen chinesischen gesundheitskommission zum umgang mit der covid-19-epidemie aufgenommen [14] . allerdings ist keines der angeführten medikamente aktuell für die co-vid-19-therapie zugelassen. sollte daher z. b. chloroquin eingesetzt werden, entspricht dies einer off-label-anwendung, und das einverständnis der sorgeberechtigten bzw. des patienten ist einzuholen. da remdesivir zurzeit nicht verfügbar ist und sich die wirksamkeit von lopinavir/ritonavir nicht bestätigt hat, bleibt, neben der symptomatischen therapie, im wesentlichen die gabe von chloroquin bzw. hydroxychloroquin übrig. diese scheint von den bisher damit behandelten covid-19 patienten gut vertragen zu werden [38] . schwere nebenwirkungen wie makuladegeneration und kardiotoxizität/verlängerung des qt-intervalls, die nach fortgesetzter einnahme auftreten können, sind aufgrund der kurzen behandlungsdauer von 7 tagen weniger wahrscheinlich. dennoch ist insbesondere bei patienten mit chronischen erkrankungen (z. b. mit eingeschränkter nierenfunktion oder lebererkrankungen) oder patienten, die medikamente erhalten, die zum auslösen von arrhythmien beitragen können, vorsicht geboten [38] . aus der behandlung von juvenilerrheumatoiderarthritis und systemischem lupus erythematodes ist bekannt, dass kinder die zur behandlung voncovid-19 vorgeschlagenenchloroquin-und hydroxychloroquindosen vertragen [39] . eine überdosierung ist insbesondere bei kleinkindern jedoch unbedingt zu vermeiden, da diese tödlich sein kann [40] . hierbei ist zu beachten, dass bei übergewicht das idealgewicht zur dosierung zugrunde gelegt werden muss. weitere behandlungsmöglichkeiten sind laut der aktuellen chinesischen leitlinie die eher unspezifische alleinige gabe von interferon-α oder in kombination mit ribavirin oder die gabe des fusionsinhibitors umifenovir, der in europa und usa allerdings nicht zugelassen ist. in einer aktuellen studie von hoffmann et al. konnte gezeigt werden, dass die zelluläre protease tmprss2 für die infektiosität von sars-cov-2 essenziell ist und eine hemmung dieser protease mithilfe von camostat-mesilat die vermehrung des virus u. a. in kultivierten lungenzellen hemmt [41] . camostat-mesilat ist in japan zur behandlung von chronischer pankreatitis und refluxöso-phagitis zugelassenund erzielte intierexperimenten bereits eine hemmende wirkung auf die vermehrung von sars-cov [42] . zudem haben pharmakokinetische untersuchungen ergeben, dass camostat-mesilat nach i.v.-gabe in größeren mengen in den lungen als im pankreas verfügbar ist [43] . mit einem impfstoff gegen covid-19 könnte der aktuelle globale ausbruch eingedämmt werden. deswegen ist die impfstoffentwicklung von zentraler bedeutung. es ist aber fraglich, ob dieses ziel unter den gegebenen zeitlichen umständen und wahrung aller rechtlichen zulassungsverfahren in wenigen monaten erreicht werden kann. wahrscheinlich kommt der einsatz eines impfstoffs für den derzeitigen ausbruch zu spät. zur impfstoffentwicklung werden unterschiedliche ansätze auf verschiedenen technologieplattformen von der coalition for epidemic preparedness innovations (cepi) gefördert. hierzu zählen der mers-dna-impfstoffansatz, "molecular-clamp"-technologien und die entwicklung einer mrnabasierten vakzine. alle diese ansätze benötigen jedoch noch viel forschungstätigkeiten, und diverse dna-impfstoffe waren bisher im primatenmodell nur wenig erfolgreich. ziel eines viralen impfstoffs sollte es sein, eine nachhaltige immunantwort zu erzeugen. die induktion einer t-zellbasierten und einer humoralen virusneutralisierenden antwort ist von zentraler bedeutung. human coronavirus in hospitalized children with respiratory tract infections: a 9-year population-based study from norway genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding human coronaviruses: a review of virus-host interactions interaction of severe acute respiratory syndrome-associated coronavirus with dendritic cells non-invasive bioluminescence imaging of hcov-oc43 infection and therapy in the central nervous system of live mice role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 the global spread of 2019-ncov: a molecular evolutionary analysis. pathog glob health transmission of 2019-ncov infection from an asymptomatic contact in germany epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding angiotensin-converting enzyme 2 protects from lethal avian influenza a h5n1 infections chinese clinical guidance for covid-19 pneumonia diagnosis and treatment substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov2) the effect of travel restrictions on the spread of the 2019 novel coronavirus (covid-19) outbreak on the origin and continuing evolution of sars-cov-2 comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations potential presymptomatic transmission of sars-cov-2 clinical characteristics of coronavirus disease 2019 in china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china clinical features of patients infected with 2019 novel coronavirus in clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus high-efficiency detection of severe acute respiratory syndrome virus genetic material a new approach for diagnosis of bovine coronavirus using a reverse transcription recombinase polymerase amplification assay detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr coronavirus disease 2019 (covid-19) -information for laboratories infection treated with a combination of lopinavir /ritonavir and interferon beta-1b (miracle a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov prophylacticandtherapeuticremdesivir(gs-5734) treatment in the rhesus macaque model of mers-cov infection remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro antimalarial drugs for rheumatoid arthritis hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibitingsars-cov-2infectioninvitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies coronavirus disease 2019 (covid-19)-informati-onforcliniciansontherapeuticoptionsforcovid-19 patients dosage of antimalarial drugs for children with juvenile rheumatoid arthritis and systemic lupus erythematosus: a clinical study with determination of serum concentrations of chloroquine and hydroxychloroquine are 1-2 dangerous? chloroquine and hydroxychloroquine exposure in toddlers sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor protease inhibitors targeting coronavirus and filovirus entry metabolic fate of 14c-camostat mesylate in man, rat and dog after intravenous administration safety and immunogenicity of a highly attenuated rvsvn4ct1-ebovgp1 ebola virus vaccine: a randomised, doubleblind, placebo-controlled, phase 1 clinical trial first fda-approved vaccine for the prevention of ebola virus disease, marking a critical milestone in public health preparedness and response a single dose of a vesicular stomatitis virus-based influenza vaccine confers rapid protection against h5 viruses from different clades a recombinant vsv-vectored mers-cov vaccine induces neutralizing antibody and t cell responses in rhesus monkeys after single dose immunization a prophylactic multivalent vaccine against different filovirus species is immunogenic and provides protection from lethal infections with ebolavirus and marburgvirus species in non-human primates a monovalent chimpanzee adenovirus ebola vaccine boosted with mva safety and immunogenicity of novel adenovirus type 26-and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels the ebola epidemic in west africa: proceedings of a workshop. 2, the outbreak incorporating individual health-protective decisions into disease transmission models: a mathematical framework rational use of face masks in the covid-19 pandemic werden [44] . der impfstoff ervebo, der auf diesem chimären vsv basiert, ist deshalb kürzlich von der food and drug administration (fda) zugelassen worden [45] . dieses konzept ist auch mit anderen viralen hüllproteinen, z. b. bei h5n1-influenzaviren, erfolgreich getestet worden [46] . ein s-protein-basierter impfstoff auf vsv-basis wurde ebenso für mers-cov entwickelt und konnte im primatenmodell neutralisierende antikörper und eine t-zellantwort induzieren [47] . nach anfänglichem zögern sind in deutschland und anderen europäischen ländern weitgehende reise-und mobilitätsbeschränkungen angeordnet worden. diese maßnahmen sind in einer globalisierten gesellschaft zwar unpopulär, seuchenhygienisch aber geboten und effizient, wie es die maßnahmen und erfahrungen während der ebola-epidemien in westafrika 2015 zeigten [52] . zudem hat eine studie anhand von modellrechnungen nachgewiesen, dass nur die kombination aus reise-und mobilitätsbeschränkungen sowie quarantänemaßnahmen die ausbreitung von sars-cov-2 wirkungsvoll eindämmen kann [16] . dementsprechend key: cord-352527-eeyqh9nc authors: zhou, yusen; yang, yang; huang, jingwei; jiang, shibo; du, lanying title: advances in mers-cov vaccines and therapeutics based on the receptor-binding domain date: 2019-01-14 journal: viruses doi: 10.3390/v11010060 sha: doc_id: 352527 cord_uid: eeyqh9nc middle east respiratory syndrome (mers) coronavirus (mers-cov) is an infectious virus that was first reported in 2012. the mers-cov genome encodes four major structural proteins, among which the spike (s) protein has a key role in viral infection and pathogenesis. the receptor-binding domain (rbd) of the s protein contains a critical neutralizing domain and is an important target for development of mers vaccines and therapeutics. in this review, we describe the relevant features of the mers-cov s-protein rbd, summarize recent advances in the development of mers-cov rbd-based vaccines and therapeutic antibodies, and illustrate potential challenges and strategies to further improve their efficacy. middle east respiratory syndrome (mers) coronavirus (cov) is an infectious virus that was first reported in june 2012 [1] . mers-cov may infect people of any age, but older age, underlying comorbidity (such as diabetes mellitus, renal disease, respiratory disease, heart disease, and hypertension), and delayed confirmation or late diagnosis are all factors that affect mers disease outcomes and mortality [2] [3] [4] [5] [6] [7] . sex could be a factor in mers epidemiology, as more males seem to be affected than females [8] [9] [10] . mers-cov infection of women during pregnancy has adverse outcomes, with fetal mortality of~27%; however, only a limited number of pediatric mers-cov infections occur [11] [12] [13] [14] . at the end of december 2018, 2,279 laboratory-confirmed mers infections were reported globally (in 27 countries), leading to 806 deaths, and a mortality of 35.3%. among these infections, 1,901 (83.4%) were reported in saudi arabia, with mortality in 732 individuals (38.5%) (http://www.emro.who.int/health-topics/mers-cov/mers-outbreaks.html). the largest mers outbreak outside saudi arabia occurred in south korea in 2015, with 186 cases and 38 deaths [9, 15, 16] . the most recent mers cases were reported in 2018 in south korea, the united kingdom, and malaysia, in addition to saudi arabia, the united arab emirates, and oman (http://www.who.int/emergencies/mers-cov/en/). mers-cov is thought to have originated in bats [17] [18] [19] [20] . mers-like viruses have been isolated from bats that use (at lower efficiency) the same receptor for cell entry as the mers-cov isolated from humans [21] [22] [23] . dromedary camels are potential intermediates for long-term evolution of mers-cov and seasonal zoonotic transfer of virus to humans [24] [25] [26] [27] . antibodies specific to host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [71, 78] . the dpp4 receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to speciesspecific variations [79] [80] [81] . in addition to dpp4, mers-cov can bind to sialic acid via the s1 subunit of s protein, or utilize the membrane-associated 78 kda glucose-regulated protein (grp78) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [82, 83] . the structures of mers-cov rbd alone and complexed with dpp4 have been determined ( figure 2 ) [77, 84, 85] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [77] . a number of rbd residues are located at the dpp4-binding interface, and they have a critical role in rbd-dpp4 binding [77, 84, 85] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp4. notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp4 [86] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd events [87] . in configurations with one, two, or three rbds rotated out, rbd determinants are exposed at the apex of the rbd-dpp4 complex, and they are accessible for interaction with dpp4 ( figure 3 ) [87] . mers-cov s protein has an important role in viral pathogenesis, determining host tropism and entry into host cells [58, 70, 71] . the s protein contains an s1 subunit at the n terminus and an s2 subunit at the c terminus. the s1 subunit is composed of the n-terminal domain (ntd) and rbd [58, 72, 73] . the rbd has a key role in the mediation of binding of mers-cov to cells expressing dipeptidyl peptidase 4 (dpp4) receptor, enabling the virus to enter into target cells by fusing with cell membranes through the formation of a fusion core ( figure 1c ) [74] [75] [76] [77] . the s protein requires host cellular proteases for its activity in viral entry, but although evidence initially indicated that cellular furin activates s protein, subsequent results have demonstrated no evidence for the involvement of furin during viral entry [71, 78] . the dpp4 receptor varies among different host species, and mers-cov is thought to use multiple pathways to enable rapid adaptation to species-specific variations [79] [80] [81] . in addition to dpp4, mers-cov can bind to sialic acid via the s1 subunit of s protein, or utilize the membrane-associated 78 kda glucose-regulated protein (grp78) to attach to target cells, suggesting that these proteins may also have roles in virion attachment [82, 83] . the structures of mers-cov rbd alone and complexed with dpp4 have been determined ( figure 2 ) [77, 84, 85] . the rbd has a fold-rich tertiary structure, which consists of a core and a receptor-binding motif (rbm), with stabilization provided by four disulfide bonds and two glycans [77] . a number of rbd residues are located at the dpp4-binding interface, and they have a critical role in rbd-dpp4 binding [77, 84, 85] . structural analysis of mers-cov trimeric s protein has identified specific features of the rbd and its complex with dpp4. notably, in the prefusion conformation of the s trimer, individual rbds are either buried (lying state) or exposed (standing state), and this flexibility presumably facilitates recognition by dpp4 [86] . other structural studies have revealed four s-trimer conformational states, in which each rbd is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation, suggesting fusion initiation through sequential rbd the mers-cov rbd core is colored in blue, the rbm is colored in red, and dpp4 is colored in green. the rbm residues directly involved in dpp4 binding are shown as sticks. dpp4, dipeptidyl peptidase 4; rbd, receptor-binding domain; rbm, receptor-binding motif; s, spike protein. the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus 5 (ad5)-directed expression of human dpp4 (ad5/hdpp4), hdpp4-transgenic (hdpp4-tg) mice, and non-human primates (nhps) [88] [89] [90] [91] [92] [93] [94] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table 1 . the function and structure of the s-protein rbd demonstrate that it is an important target for development of vaccines and therapeutic agents against mers-cov. a number of mers vaccines have been developed based on viral rbd, including nanoparticles, virus-like particles (vlps), and recombinant proteins, and their protective efficacy has been evaluated in animal models, including mice with adenovirus 5 (ad5)-directed expression of human dpp4 (ad5/hdpp4), hdpp4-transgenic (hdpp4-tg) mice, and non-human primates (nhps) [88] [89] [90] [91] [92] [93] [94] . features of these rbd-based vaccines, in terms of functionality, antigenicity, immunogenicity, and protective ability, are shown in table 1 . a soluble nanoparticle vaccine formed in escherichia coli by the rna-mediated folding of a rbd-ferritin (fr) hybrid elicits robust rbd-specific antibody and cellular immune responses in mice, producing antisera that effectively block the binding of rbd to hdpp4 in vitro [89] . the adjuvants alum and the squalene-based mf59 significantly augment the antibody titers and t-cell responses induced by rbd-fr nanoparticle vaccines engineered with or without a ssg linker [89] . similarly, a chimeric, spherical vlp (svlp) vaccine expressing mers-cov rbd induces specific antibody and cellular immune responses in mice, preventing pseudotyped mers-cov entry into susceptible cells [90] . the protective efficacy of these two types of mers vaccine does not yet seem to have been investigated in a viral-challenge animal model. recombinant vaccines involving rbd subunits have been extensively studied for protection against mers-cov infection in mers-cov-susceptible animal models [93, [95] [96] [97] 100, 101] . a recombinant rbd (rrbd) fragment (residues 367-606) expressed in insect cells elicits an antibody response and the production of neutralizing antibodies in mice and nhps [88, 91] . it gives incomplete protection in mers-cov-challenged nhps, with the alleviation of pneumonia and clinical manifestations, as well as the reduction of viral load in lung, trachea, and oropharyngeal swabs [91] . a mers-cov s-protein rbd fragment containing residues 377-588 has been identified as a critical neutralizing domain [95] . a treatment regimen involving two doses of a fusion of this fragment and the fc region of human igg (s377-588-fc) four weeks apart is able to induce strong, long-term antibody responses (including production of neutralizing antibodies) in mice [98] . these responses are significantly greater than those with a single dose or two doses at intervals of one, two, or three weeks [98] . rrbds with single or multiple mutations corresponding to s-protein sequences of mers-cov strains isolated from humans or camels from 2012 to 2015 have also been studied [100] . all these rrbds bind rbd-specific neutralizing monoclonal antibodies (mabs) and dpp4, and are highly immunogenic, eliciting the production of s1-specific antibodies in mice, which cross-neutralizes multiple mers pseudoviruses and live mers-cov [100] . a trimeric rbd-fd protein formed by fusing a mers-cov rbd fragment (residues 377-588) to the foldon trimerization motif, binds strongly to dpp4, and elicits robust and long-term responses with the production of mers-cov s1-specific antibodies and neutralizing antibodies in mice, and protects hdpp4-tg mice against mers-cov infection [94] . the protection provided by existing subunit vaccines based on wild-type mers-cov rbd is not complete, with survival rates in hdpp4-tg mice after a mers-cov challenge of~67% for s377-588-fc and 83% for rbd-fd [94, 98] . however, a variant rbd (t579n) vaccine produced by masking a non-neutralizing epitope at residue 579 with a glycan probe has both functionality in binding dpp4, and antigenicity in binding four potent mers-cov rbd-specific neutralizing mabs (hhs-1, m336, m337, and m338) [93] . the t579n vaccine has significantly greater efficacy than the wild-type rbd vaccine, and it fully protects against a lethal mers-cov challenge in immunized hdpp4-tg mice [93] , demonstrating the possibility of developing rbd-based mers-cov vaccines with high efficacy. [102] [103] [104] [105] [106] [107] [108] [109] . these antibodies generally have greater neutralizing activity against mers-cov infection than non-rbd s1-based or s2-based antibodies [58, 103, 110, 111] . the prophylactic and therapeutic efficacies of rbd-targeting antibodies have been tested in ad5/hdpp4 mice, hdpp4-tg mice, and nhps [102, 104, [112] [113] [114] . in an earlier review, we described the antiviral mechanisms, in vivo protection, and crystal structures of previously reported mers-cov rbd-specific mabs, including mouse mabs mersmab1, 2e6, 4c2, f11, and d12, and human mabs lca60, mers-4, mers-27, regn3048, regn3051, 1e9, 1f8, 3a1, 3b11, 3b12, 3b11-n, 3c12, m14d3, m336, m337, m338, hms-1, and 4c2h [58] . in this review, we focus on newly reported antibodies targeting mers-cov s-protein rbd, or on newly identified features of existing mabs that were not described previously (table 2 ) [102, [112] [113] [114] [115] . rbd-targeting human mabs have been extensively reported. most of these mabs can neutralize pseudotyped or live mers-cov in vitro, and some have shown protection against mers-cov infection in animal models in vivo [102, [112] [113] [114] [115] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure 4) [102, [112] [113] [114] [115] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp4 (as is the case for gd-27, mca1, and cdc2-c2), or with binding to the rbd residues outside of the dpp4-binding interface (as seen with mers-4) ( table 2) . infection in animal models in vivo [102, [112] [113] [114] [115] . the structures of several of these mabs with their antigen-binding fragments (fabs) or single-chain variable fragments (scfvs) complexed with rbd are known ( figure 4) [102, [112] [113] [114] [115] . binding of these mabs to rbd involves two major recognition modes, with binding to rbd residues contacted by or overlapping with dpp4 (as is the case for gd-27, mca1, and cdc2-c2), or with binding to the rbd residues outside of the dpp4-binding interface (as seen with mers-4) ( table 2) . the human mabs mers-gd27 and mers-gd33 each recognize distinct regions of the rbd [113] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic50) for their use in combination than separately [113] . an analysis of crystal structures has indicated that mers-gd27 binds rbd at the dpp4-binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m336 [106, 113] . the mers-gd27 mab protects hdpp4-tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day 5 postchallenge, and higher survival rates (60% for pre-challenge vaccination and 40% for post-challenge vaccination) relative to control mice treated with an irrelevant mab [112] . the human mab mca1 was isolated from a mers survivor via the construction of a phagedisplay antibody library from peripheral b cells [114] . crystal structure analysis indicates that mca1 binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp4 ( figure 4a ) [114] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced the human mabs mers-gd27 and mers-gd33 each recognize distinct regions of the rbd [113] . these mabs have a synergistic effect in the neutralization of pseudotyped mers-cov in vitro, with a much lower half-maximal inhibitory concentration (ic 50 ) for their use in combination than separately [113] . an analysis of crystal structures has indicated that mers-gd27 binds rbd at the dpp4-binding site, and that the neutralization and recognition epitopes almost completely overlap this site, as seen previously for mers-cov rbd-targeting neutralizing mabs, such as m336 [106, 113] . the mers-gd27 mab protects hdpp4-tg mice from mers-cov challenge, both preventively and therapeutically, with significantly lower lung virus titers and rna copy numbers at day 5 post-challenge, and higher survival rates (60% for pre-challenge vaccination and 40% for post-challenge vaccination) relative to control mice treated with an irrelevant mab [112] . the human mab mca1 was isolated from a mers survivor via the construction of a phage-display antibody library from peripheral b cells [114] . crystal structure analysis indicates that mca1 binds mers-cov s-protein rbd at residues involved in receptor binding, thus interfering with rbd binding to hdpp4 ( figure 4a ) [114] . this mab prophylactically and therapeutically inhibits mers-cov replication in common marmosets, resulting in significantly improved outcomes and reduced lung disease, compared with unvaccinated controls, and undetectable virus titers 3 days post-challenge [114] . a probe-based single-b-cell cloning strategy has been used for the isolation of cdc2-c2 and cdc2-c5 mabs from a patient convalescing from mers, as well as for the isolation of jc57-11 and jc57-14 mabs from nhps immunized with mers-cov full-length s dna and protein [102] . all these antibodies have neutralizing activities against both pseudotyped and live mers-cov. among them, cdc2-c2 is the most potent against 10 pseudotyped mers-cov strains, with neutralization ic 50 values ranging from 0.002 âµg/ml to 0.011 âµg/ml [102] . crystal-structure analysis of the cdc2-c2 and jc57-14 fab-rbd complexes indicates that both mabs bind rbd in the "out" (exposed) position, with the cdc2-c2 rbd binding overlapping with the dpp4-contacting residues ( figure 4b ,c) [102] . in addition, cdc2-c2 prophylactically protects hdpp4-tg mice from mers-cov infection, resulting in no detectable viral replication in the lungs three days post-challenge, and no fatalities over 28 days of observation [102] . the human mab mers-4 also neutralizes pseudotyped mers-cov and, notably, displays synergistic neutralization in combination with the mers-cov s-protein rbd-targeting mers-27 and m336 mabs [106, 118] , as well as the s-protein ntd-targeting 5f9 mab, in each case with dramatic reduction of the ic 50 compared with individual mabs [115] . structural analysis of a mers-4-fab-rbd complex revealed that mers-4 binds the rbd from outside the dpp4-binding interface, rather than competing with dpp4 ( figure 4d ). unlike mers-27, which binds rbd regardless of its conformational state within the s trimer, mers-4 binds rbd in the "standing" position where its epitopes are readily exposed and accessible [115] . thus, mers-4 displays unique epitope specificity, and an unusual mechanism of action involving indirect interference with dpp4 binding through conformational changes, which may explain the observation of synergistic neutralization in combination with other mabs [115] . single-domain antibody fragments (vhhs), or nanobodies, are the antigen-recognition regions of camelid heavy-chain-only antibodies (hcabs), which do not contain light chains. vhhs are easily expressed with high yield, and they have intrinsic stability, strong binding affinity, and specificity to target antigens, and they have therefore been developed as important therapeutic tools against viral infection, including that of mers-cov [116, 117, [119] [120] [121] [122] [123] . four vhhs (vhh-1, vhh-4, vhh-83, and vhh-101) have been identified from bone marrow cells of dromedary camels immunized with modified vaccinia virus (mva) expressing mers-cov s protein, and challenged with mers-cov [116] . these vhhs bind mers-cov s protein with low k d values (0.1-1 nm), recognize an epitope at residue d539 of rbd, and neutralize mers-cov (prnt 50 , 0.0014-0.012 âµg/ml) [116] . these four monomeric vhhs have each been fused with a c-terminal human igg2 tag to generate four hcabs (hcab-1, hcab-4, hcab-83, and hcab-101), with a higher binding affinity and a longer half-life than the free vhhs [116] . studies of protective efficacy show that hdpp4-tg mice (k18) injected with monomeric vhh-83 (20 or 200 âµg per mouse) lose weight, and die within seven days post-infection, possibly because of the short half-life of the vhh. however, when the mice are injected with hcab-83 (200 âµg per mouse), which has an extended half-life (~4.5 days), protection against mers-cov is complete, with no viral titers or pathological changes in the lungs of virally challenged mice [116] . by immunizing llamas with a recombinant rbd fragment (residues 377-588) fused to a c-terminal human igg fc tag (s377-588-fc), we constructed a vhh library, and we used it to generate a monomeric vhh, nbms10, and a human fc-fused vhh, nbms10-fc [117] . both vhhs can be expressed in a yeast expression system to high purity, and bind rbd with high affinity, recognizing a conformational epitope (residue 539) at the rbd-dpp4 interface, and blocking the binding of rbd to dpp4. these vhhs, particularly nbms10-fc, potently cross-neutralize pseudotyped mers-cov strains isolated from different countries, hosts, and time periods [117] . importantly, the fc-fused nbms10-fc significantly improves the serum half-life of nbms10, and a single-dose treatment of hdpp4-tg mice with this agent completely protects them against lethal mers-cov challenge [117] . these single-domain vhhs demonstrate the feasibility of developing cost-effective, potent, and broad-spectrum therapeutic antibodies against mers-cov infection. compared with vaccines based on mers-cov full-length s protein, which have the potential to attenuate neutralizing activity or enhance immune pathology, vaccines developed from mers-cov s-protein rbd are safer, and they do not cause immunological toxicity or eosinophilic immune enhancement [55, 99, 110, 124] . moreover, rbd-based therapeutic antibodies are generally more potent than non-rbd s1-based or s2-based antibodies [58, 104, 111] . hence, rbd-based vaccines and therapeutic antibodies have the potential for further development as effective tools to prevent and treat mers-cov infection. despite their acknowledged advantages, there are some issues associated with rbd-based interventions that need to be addressed. for example, rbd is under a high level of pressure of positive selection, and mutations occur in the rbd-dpp4 binding interface that might reduce the efficacy of these treatments [100, [125] [126] [127] . one possible way to avoid this effect, and to delay the emergence of escape mutants is to combine rbd-targeting therapeutics with those targeting other regions of the s protein, or to combine antibodies recognizing distinct epitopes within the rbd [102, 128] . such combinatorial strategies could also dramatically reduce antibody neutralization doses, providing feasible means to combat the continual threat of mers-cov. some recent advances have been made in the structure-guided design of anti-mers-cov interventions. structurally designed inhibitors of the 3cl protease have demonstrated potency against mers-cov [129] . also, a structurally designed s-protein trimer in the optimal prefusion conformation is shown to elicit production of high titers of anti-mers-cov neutralizing antibodies [87] . indeed, based on the previous studies on the structural design of mers-cov rbd, non-neutralizing epitopes in the rbd can be masked, to refocus the immunogenicity of the rbd on the neutralizing epitopes, and thus to enhance its ability to confer immune protection [93] . results from these structure-based studies will help to inform the design of innovative rbd-based anti-mers-cov vaccines and therapeutics with improved efficacy. isolation of a novel coronavirus from a man with pneumonia in saudi arabia fatality risks for nosocomial outbreaks of middle east respiratory syndrome coronavirus in the middle east and south korea risks of death and severe disease in patients with middle east respiratory syndrome coronavirus impact of comorbidity on fatality rate of patients with middle east respiratory syndrome clinical determinants of the severity of middle east respiratory syndrome (mers): a systematic review and meta-analysis prevalence of comorbidities in the middle east respiratory syndrome coronavirus (mers-cov): a systematic review and meta-analysis epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study sex matters -a preliminary analysis of middle east respiratory syndrome in the republic of korea middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature mers-cov infection in a pregnant woman in korea middle east respiratory syndrome coronavirus in pediatrics: a report of seven cases from saudi arabia middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia an outbreak of middle east respiratory syndrome coronavirus infection in south korea probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus rapid detection of mers coronavirus-like viruses in bats: pote1ntial for tracking mers coronavirus transmission and animal origin receptor usage of a novel bat lineage c betacoronavirus reveals evolution of middle east respiratory syndrome-related coronavirus spike proteins for human dipeptidyl peptidase 4 binding mers-cov spillover at the camel-human interface prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation high prevalence of mers-cov infection in camel workers in saudi arabia the prevalence of middle east respiratory syndrome coronavirus (mers-cov) antibodies in dromedary camels in israel serologic evidence for mers-cov infection in dromedary camels sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia dromedary camels in northern mali have high seropositivity to mers-cov cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission unusual presentation of middle east respiratory syndrome coronavirus leading to a large outbreak in riyadh during 2017 outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study outbreak of middle east respiratory syndrome at tertiary care hospital transmission of middle east respiratory syndrome coronavirus infections in healthcare settings hospital-associated middle east respiratory syndrome coronavirus infections hospital-associated middle east respiratory syndrome coronavirus infections family cluster of middle east respiratory syndrome coronavirus infections healthcare-associated infections: the hallmark of middle east respiratory syndrome coronavirus with review of the literature clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus persistence of antibodies against middle east respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study prospects for a mers-cov spike vaccine current advancements and potential strategies in the development of mers-cov vaccines is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? cov spike protein: a key target for antivirals genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus the endonucleolytic rna cleavage function of nsp1 of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines mers coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral rna middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis structural and biochemical characterization of endoribonuclease nsp15 encoded by middle east respiratory syndrome coronavirus structural insights into the middle east respiratory syndrome coronavirus 4a protein and its dsrna binding mechanism middle east respiratory coronavirus accessory protein 4a inhibits pkr-mediated antiviral stress responses inhibition of stress granule formation by middle east respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication sola, i. mers-cov 4b protein interferes with the nf-kappab-dependent innate immune response during infection proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein structure, function, and evolution of coronavirus spike proteins mers-cov spike protein: targets for vaccines and therapeutics dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus spike protein is not activated directly by cellular furin during viral entry into target cells receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 adaptive evolution of mers-cov to species variation in dpp4 middle east respiratory syndrome coronavirus and bat coronavirus hku9 both can utilize grp78 for attachment onto host cells identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles novel chimeric virus-like particles vaccine displaying mers-cov receptor-binding domain induce specific humoral and cellular immune response in mice recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus introduction of neutralizing immunogenicity index to the rational design of mers coronavirus subunit vaccines a recombinant receptor-binding domain of mers-cov in trimeric form protects human dipeptidyl peptidase 4 (hdpp4) transgenic mice from mers-cov infection searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design receptor-binding domain-based subunit vaccines against mers-cov optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus receptor-binding domain of mers-cov with optimal immunogen dosage and immunization interval protects human transgenic mice from mers-cov infection engineering a stable cho cell line for the expression of a mers-coronavirus vaccine antigen recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies middle east respiratory syndrome: current status and future prospects for vaccine development evaluation of candidate vaccine approaches for mers-cov a novel human mab (mers-gd27) provides prophylactic and postexposure efficacy in mers-cov susceptible mice ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein chimeric camel/human heavy-chain antibodies protect against mers-cov infection a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov structural basis for the neutralization of mers-cov by a human monoclonal antibody mers-27 application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies(r) as inhaled biotherapeutics for lung diseases generation and characterization of alx-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection nanobodies as therapeutics: big opportunities for small antibodies nanobodies: natural single-domain antibodies vaccines for the prevention against the threat of mers-cov evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak mutations in the spike protein of middle east respiratory syndrome coronavirus transmitted in korea increase resistance to antibody-mediated neutralization combining a fusion inhibitory peptide targeting the mers-cov s2 protein hr1 domain and a neutralizing antibody specific for the s1 protein receptor-binding domain (rbd) showed potent synergism against pseudotyped mers-cov with or without mutations in rbd structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this study was supported by the nsfc grant 81571983, and the nih grants r21ai128311, r01ai137472, and r01ai139092. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. the authors declare no competing interests. key: cord-351760-698voi9y authors: han, hui-ju; liu, jian-wei; yu, hao; yu, xue-jie title: neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection date: 2018-11-30 journal: viruses doi: 10.3390/v10120680 sha: doc_id: 351760 cord_uid: 698voi9y since emerging in 2012, middle east respiratory syndrome coronavirus (mers-cov) has been a global public health threat with a high fatality rate and worldwide distribution. there are no approved vaccines or therapies for mers until now. passive immunotherapy with neutralizing monoclonal antibodies (mabs) is an effective prophylactic and therapeutic reagent against emerging viruses. in this article, we review current advances in neutralizing mabs against mers-cov. the receptor-binding domain (rbd) in the spike protein of mers-cov is a major target, and mouse, camel, or human-derived neutralizing mabs targeting rbd have been developed. a major problem with neutralizing mab therapy is mutant escape under selective pressure, which can be solved by combination of neutralizing mabs targeting different epitopes. neutralizing mabs are currently under preclinical evaluation, and they are promising candidate therapeutic agents against mers-cov infection. middle east respiratory syndrome (mers) emerged in 2012 in saudi arabia with the death of a man with pneumonia; the causative agent was subsequently identified as mers-cov, which belonged to lineage c betacoronaviruses [1] . with dromedary camels (camelus dromedarius, also known as arabian camel) as direct sources and bats as potential reservoirs [2] , mers-cov has been frequently introduced into human populations. once mers-cov is introduced into a person, person-to-person transmission might occur, and is responsible for approximately 40% of mers cases globally [3] . mers-cov has been a consistent threat to humans. as of october 2018, mers-cov has caused 2254 laboratory-confirmed human cases, including 800 deaths in 27 countries, with the fatality rate as high as 35% (http://www.who.int/emergencies/mers-cov/en/). although mers cases are primarily reported in the middle east, facilitated by international travelling, mers-cov can also be a worldwide threat, which is well illustrated by the mers outbreak in south korea in 2015 [4] . given the potential risk of causing worldwide public health emergencies and the absence of licensed vaccines and antiviral therapeutics, the world health organization has listed mers-cov in the "list of blueprint priority diseases" (http://www.who.int/blueprint/priority-diseases/en/). vaccines are the most important approach against viral infections, but usually take a long time to develop. they are also unable to provide either immediate prophylactic protection or treat ongoing viral infections. neutralizing monoclonal antibodies (mabs) have recently emerged as a powerful tool to provide prophylactic and therapeutic protection against emerging viruses [5] . potent neutralizing mabs can be achieved by various technologies, such as hybridoma technology, humanized mouse, phage or yeast display, and single b cell isolation [5] . mers-cov is a single, positive-stranded rna virus of about 30 kb, which encodes four major viral structural proteins-including spike (s), envelope (e), membrane (m) and nucleocapsid (n)-as well as several accessory proteins [6] . the s protein (1353 aa) plays an important role in virus infection and consists of a receptor-binding subunit s1 (aa 18-751) and a membrane-fusion subunit s2 (aa 752-1353). s1 mediates viral attachment to host cells and s2 mediates virus-cell membrane fusion [7] . the s1 subunit contains a receptor-binding domain (rbd) (aa 367-606) [8] that can bind to cell receptor dipeptidyl peptidase 4 (dpp4, also known as cd26), and mediates viral attachment target cells [9] . the rbd consists of a core subdomain and a receptor-binding motif (rbm) (aa 484-567). the schematic representation of mers-cov s protein is shown in figure 1a . neutralizing mabs binding to the s protein of mers-cov can prevent viral attachment to the cell receptor and inhibit viral entry [7] . the s protein of mers-cov is a key target for antivirals, and rbd is the most popular focus. in this study, we review the current knowledge on neutralizing mabs targeting the rbd of mers-cov. stable hybridoma cell lines were generated by fusing myeloma cells with splenocytes of mice that were immunized with mers-rbd protein. two neutralizing mabs, 4c2 and 2e6, had high affinity for the rbd of mers-cov and blocked both pseudovirus and live mers-cov entry into cells with high efficacy [10] . humanized 4c2 showed similar neutralizing activity in cell entry tests. in vivo tests indicated that 4c2 could significantly reduce the virus titers in the lungs of ad5-hcd26-transduced mice which were infected with mers-cov, highlighting its potential application in humans not only for preventing but also treating mers-cov infection. crystallization of the 4c2 fab/mers-rbd complex showed that the 4c2 recognized conformational epitopes (y397-n398, k400, l495-k496, p525, v527-s532, w535-e536, and d539-q544), which were partially overlapped the receptor-binding footprint in the rbd of mers-cov. the 4c2 complex interfered with mers-cov binding to dpp4 by both steric hindrance and interface-residue competition. 2e6 competed with 4c2 to bind to mers-rbd, indicating that they recognized proximate or overlapping epitopes [10] . neutralizing mab mersmab1 was obtained by fusing myeloma cells with splenocytes of a mouse that was immunized with recombinant mers-cov s1 [11] . mersmab1 effectively blocked the entry of pseudovirus and live mers-cov into cells. structural analysis showed that mersmab1 bound to the rbd of mers-cov through recognizing conformational epitopes, and all of the residues critical for mersmab1 binding were located on the left ridge of rbm. mersmab1 neutralized mers-cov by competitively blocking the binding of mers-cov rbd to dpp4. based on escape mutant analysis of the key residues on the rbd, it was found that residue l506, d510, r511, e513, and w553 were critical for mersmab1 binding to the rbd, while mutation of e536, d539, or e565 did not affect the interaction of mersmab1 and the rbd at all [11] . an ultra-large nonimmune human antibody-phage display library was constructed with b cells of unimmunized donors. with a unique spanning strategy, seven human neutralizing mabs with varying neutralization efficacy to mers-cov were identified [12] . binding detection demonstrated that the epitopes of these mabs lay within aa 349-590 of the s protein, which overlapped a large part of the rbd of mers-cov. binding competition assays showed that these mabs recognized at least three distinct epitope groups, which was further confirmed by escape studies. with no cross-epitope resistance, these mabs neutralized mers-cov by competitively blocking the binding of the rbd of mers-cov to dpp4. escape mutant assays showed that five residues were critical for neutralization of these mabs, namely l506, t512, y540, r542, and p547. of the seven mabs, 3b11 exhibited the best neutralization activity against both pseudovirus and live mers-cov infectivity in cells. moreover, under the selective pressure of these mabs, the igg form of 3b11 was superior, since it did not induce neutralization escape [12] . in vivo tests demonstrated that 3b11 reduced lung pathology in rhesus monkeys infected with mers-cov [13] . with its high neutralizing activity and suppression of mutant escape, 3b11 in the igg form is a promising therapeutic mab against mers-cov. three human mabs-m336, m337, and m338-were identified from a large naïve human phage display antibody library, which was constructed with peripheral blood mononuclear cells from healthy volunteers [14] . the binding sties of the three mabs were within the rbd of mers-cov (aa 377-588), therefore they neutralized mers-cov by competing with dpp4 binding to the rbd. the three mabs also competed with each other to bind to the rbd of mers-cov, and mutant analysis showed that the three mabs possessed overlapping but distinct epitopes. of the three mabs, m336 neutralized both pseudovirus and live mers-cov infectivity in cells with exceptional potency (m336 inhibited 90% mers-cov pseudovirus infection at a concentration of 0.039 g/ml, and neutralized live mers-cov with ic 95 of 1 g/ml and ic 50 of 0.07 g/ml). residues in the rbd crucial for m336 binding were l506, d510, e536, d539, w553, and v555 [14] . in vivo study demonstrated that prophylaxis with m336 reduced virus titers in the lung of rabbits infected with mers-cov [15] , and m336 also provided transgenic mice expressing human dpp4 with full prophylactic and therapeutic protection from mers-cov [16] . however, another study with a non-human primate, the common marmoset showed that m336 could only alleviate the severity of the disease, and did not provide complete protection against mers-cov [17] . igg+ memory b cells were isolated from a mers patient, and were subsequently immortalized with epstein-barr virus. a neutralizing mabs, lca60, was identified, and was the first fully human neutralizing mab with naïve heavy and light chain pairs [18] . lca60 efficiently neutralize mers-cov infectivity in cells. in vivo study showed that lca60 provided balb/c mice transduced with adenoviral vectors expressing human dpp4 (hdpp4) with both prophylactic and postexposure protection against mers-cov. furthermore, the neutralizing efficacy of lca60 was evaluated in ifn-α/β receptor-knockout mice that were more stringent models of mers-cov infection. after transducing with hdpp4, these mice showed more profound clinical symptoms when challenged with mers-cov [19] ; administration of lca60 reduced mers-cov titer in the lungs of these mice more effectively (lung viral titer reduced by three logs in one day for ifn-α/β receptor-knockout mice vs. three days for balb/c mice) [18] . with naïve heavy and light chain pairs, lca60 was more potent than 3b11 and comparable to m336. cross-competition experiment demonstrated that lca60 competed with 3b11 to bind to the rbd. lca60 interacted with rbd residues around k493, and the lca60 footprint on the rbd was partially overlapped with that of dpp4. four residues in the rbd affected the binding of lca60-namely t489, k493, e536, and e565-which were conserved in all mers-cov isolates. moreover, compared with dpp4, the binding affinity of lca60 to rbd was significantly higher (~500-fold). therefore, one major neutralization mechanism of lca60 was to competitively inhibit the interaction of the rbd with dpp4. interestingly, virus escape studies demonstrated that under the selective pressure of lca60, a mutant variant (v33a) in the n-terminal domain (ntd) of mers-cov s1 subunit was also generated [18] . a gmp-approved cell line (lca60.273.1) that expresses lca60 in high concentrations has been established, highlighting its application as promising therapeutics against mers-cov infection [20] . hybridoma b cells producing neutralizing mabs against the s protein of mers-cov were generated by immunizing humanized transgenic mice (velocimmune mice) with dna encoding the mers-cov s protein. two fully human neutralizing mabs, regn3051 and regn3048, were obtained [21] . the two mabs bound with high affinity to distinct epitopes on the rbd of mers-cov, which were conserved during the natural evolution of mers-cov. mutation as a result of selective pressure by one mab should not affect the binding of the other mab. regn3051 neutralized a broad range of mers-cov isolates, the prototype emc/2012 strain and all clinical mutants including a431p, s457g, s460f, a482v, l506f, d509g, and v534a. with the exception of v534a variant, regn3048 achieved similar neutralizing activity. in vivo study demonstrated that regn3051 and regn3048 reduced mers-cov replication in humanized dpp4 mice in both prophylactic and therapeutic settings [21] . when evaluated in the common marmoset, both mabs seemed to be more effective for prophylaxis rather than for treatment of mers-cov infection [22] . an anti-mers-cov phage display antibody library was constructed with the peripheral b cells of a mers survivor, and a human neutralizing mab against mers-cov, mca1, was identified [23] . mca1 showed potent neutralizing activity against mers-cov in cell entry tests. in vivo, mca1 completely inhibited the replication of mers-cov in common marmosets when administrated prophylactically or therapeutically. structure analysis of the mca1 fab-rbd complex showed that mca1 formed direct contacts with the receptor-binding site (rbs) subdomain on the rbd. epitopes on the rbs critical for mca1 binding were d510, w535, e536, d539, y540, r542, and q544. superimposed structure analysis of mca1-rbd and hdpp4-rbd complexes showed that the binding interface of mca1 was largely overlapped with that of hdpp4. therefore, the neutralizing mechanism of mca1 was achieved by competing with dpp4 for binding to the rbd [23] . two potent human neutralizing mabs, mers-4 and mers-27, were derived from a nonimmune human yeast display antibody library, which was constructed with spleen and lymph node polyadenylated rna from normal humans [24] . [24] . further structural analysis showed that mers-4 bound to unique epitopes and caused conformational changes in the rbd interface critical for accommodating dpp4, therefore indirectly disrupting the interaction between the two. moreover, mers-4 also demonstrated synergistic effects with m336 and 5f9 (a ntd-specific mab). the special neutralizing mechanism made mers-4 a valuable addition for the combined use of mabs against mers-cov infection [25] . thirteen ultrapotent neutralizing mabs, which all targeted the rbd of mers-cov were generated following a protocol for the rapid production of antigen-specific human mabs [26] . briefly, antibody-secreting b cells were isolated from the whole blood of a mers patient, and the antibody genes were amplified and cloned into vectors to transfect human cell lines for mab production. of the 13 mabs, mers-gd27 and mers-gd33 exhibited the strongest neutralizing activity against both pseudovirus and live mers-cov in cell infection tests. mers-gd27 directly competed with dpp4 to bind to the rbd to dpp4, and the crystal structure of mers-gd27 showed that its epitopes were almost completely overlapped with dpp4-binding sites. mers-gd27 and mers-gd33 recognized distinct epitopes on the rbd, and had a low level of competing activity. the combined use of the two mabs demonstrated synergistic effects in neutralization against pseudotyped mers-cov. mutant analysis demonstrated that residues l506, d509, v534, e536, and a556 on rbd were important for the neutralizing activity of mers-gd27, and residue r511was critical for mers-gd33 [26] . moreover, in vivo study found that mers-gd27 could provide both prophylactic and therapeutic protection for hdpp4-trangenic mice against mers-cov infection [27] . dromedary camels exposed to mers-cov showed mild clinical signs but developed exceptionally potent neutralizing antibodies. camelid species naturally produced heavy chain-only antibodies (hcabs) [28] , which are dimeric and devoid of light chains, and their antigen recognition region is solely formed by the variable heavy chains (vhhs) (also called nanobodies, nbs). vhhs or nbs have long complementarity-determining region 3 (cdr3) loops and are capable of binding to unique epitopes not accessible to conventional antibodies [29] . notably, camelid vhhs are relatively stable and can be produced with high yields in prokaryotic systems [30] . because of their small size; good tissue permeability; and cost-effective production, storage, and transportation [31] [32] [33] , vhhs or nbs have been gaining acceptance as antiviral agents. a vhh complementary dna library was constructed with the bone marrow of dromedary camels infected with mers-cov. four vhhs (vhh-1, vhh-4, vhh-83, and vhh-101) with high neutralizing activity were identified by direct cloning and screening of the phage display antibody library [34] . the four vhhs competed for a single epitope that partially overlapped with the rbd-dpp4 interface. mutant analysis showed that the four vhhs did not bind to the d539n variant, which was a critical residue on the rbd for dpp4 binding [8, 35] . therefore, these vhhs most likely neutralized mers-cov by blocking its binding to dpp4. of the 4 vhhs, vhh-83 showed the best neutralizing activity and epitope recognition. vhh-83 efficiently blocked the entry of mers-cov into cells, and it also prophylactically protected k18 transgenic mouse expressing hdpp4 from mers-cov infection. to extend the half-life of vhh-83 in serum, it was linked to a human fc domain lacking the ch1 exon to construct the chimeric camel/human hcab-83, which showed similar neutralizing activity as vhh-83. the chimeric camel/human hcab-83 was highly stable in mice and provided k18 mice with fully prophylactic protection against mers-cov infection [34] . alpacas were immunized with recombinant mers-cov rbd-containing a c-terminal human igg1 fc tag, and vhhs were amplified from their peripheral blood mononuclear cells to construct a vhh phage display library. a neutralizing nb, nbms10, which bound with high affinity to the rbd of mers-cov and blocked the binding of rbd to dpp4, was identified [36] . to extend its in vivo half-life, the human-fc-fused version, nbms10-fc, was constructed. nbms10 competed with dpp4 to bind to rbd, indicating that the binding site of nbms10 on rbd overlapped with that of dpp4. the binding site of the nbms10 on the rbd was mapped to be around residue d539, which is part of a highly conserved conformational epitope at the receptor-binding interface in almost all the natural mers-cov published to date. nbms10 did not neutralize psuedotyped mers-cov bearing a mutation in d539, confirming that residue d539 was critical for nbms10 binding. nbms10 efficiently neutralized the cell entry of live mers-cov. moreover, nbms10 showed potent prophylactic and therapeutic efficacy in protecting hdpp4-transgenic mice against mers-cov infection [36] . for their exceptionally high neutralization activity in vitro and in vivo, these newly identified neutralizing mabs are promising candidate therapeutics against the infection of mers-cov. however, the use of a single neutralizing antibody bears the risk of selecting escape mutants, a fact that has been observed for lca60 and other described antibodies [12, 18, 37] . notably, the majority of these escape mutations had little impact on viral fitness and the interaction of dpp4 with the rbd [12] . moreover, mutants of mers-cov during natural infection have also been reported [38] . escape from neutralization is a major concern with therapeutic neutralizing mabs, however, this potential problem can be solved by combining mabs that target distinct epitopes and show different neutralizing mechanisms [37] . this strategy can take advantage of the synergistic effects while decreasing the possibility of viral escape. currently, most of the mers-cov neutralizing mabs compete with dpp4 binding to the rbd, and residues on the rbd critical for mab neutralization are identified by mutant analysis. almost all of the residues identified critical for mab neutralization are located in rbm, and overlap with those critical for dpp4 binding ( figure 1b) . with the availability of crystal structure of mab fab-rbd complex, the neutralization mechanism of these mabs will be better illustrated. based on the crystal structure of rbd-dpp4, it was found that several conserved residues in the rbd are critical for the interaction of the rbd with dpp4 (y499, l506, d510, e513, e536, d537, d539, y540, r542, w553, and v555) [35, 39] . development of therapeutic neutralizing mabs targeting those critically conserved residues might be important for combating mers-cov. moreover, a study found a mouse-derived neutralizing mab, 5f9, which bound to a possible linear epitope in the ntd of the mers-cov s1 subunit, exhibited efficient neutralizing activity against pseudovirus and live mers-cov in cell entry tests. this study highlighted the important role of ntd during the infection process of mers-cov. ntd might have significant implications for the development of prophylactic and therapeutic mabs against mers-cov infection [40] . although the in vitro neutralizing potency of 5f9 was approximately 10-fold lower than that of the rbd-targeting neutralizing mabs [40] , it may provide an alternative for the immunotherapy against mers-cov, once the virus mutates and is no longer susceptible to rbd-specific mabs. so far, there is a lack of appropriate animal models to mimic the pathology of merd-cov in humans. commonly-used laboratory animals-such as wild-type mouse, ferret, hamster, and guinea pig-are not susceptible to mers-cov infection due to differences in critical amino acids in the s-binding domain of their dpp4 [41] [42] [43] . new zealand rabbits, hdpp4-transduced/transgenic mice, camelids and non-human primates (rhesus macaque and common marmoset) are susceptible to mers-cov infection, however, rabbits showed asymptomatic infection [44] ; dromedary camels displayed different clinical manifestations to that of humans [45] ; rhesus macaque only showed transient lower respiratory infection [46] , while common marmoset developed progressive pneumonia [47] ; hdpp4-trangenic mouse expressed hdpp4 extensively, and resulted in multiple organ damage [48] ; hdpp4-transduced mouse only exhibited mild transient clinical diseases [19] . with robust animal models, the protective effects of these neutralizing mabs will be better evaluated. furthermore, ongoing efforts on developing therapeutic neutralizing mabs against mers-cov should also consider the different target populations (dromedary camels and humans) and their protective efficacy. isolation of a novel coronavirus from a man with pneumonia in saudi arabia evidence for zoonotic origins of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea human monoclonal antibodies as candidate therapeutics against emerging viruses. front genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans mers-cov spike protein: a key target for antivirals structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012. virology exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection efficacy of antibody-based therapies against middle east respiratory syndrome coronavirus (mers-cov) in common marmosets prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus rapid generation of a mouse model for middle east respiratory syndrome rapid generation of a human monoclonal antibody to combat middle east respiratory syndrome pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylactic and therapeutic efficacy of mab treatment against mers-cov in common marmosets human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein structural definition of a unique neutralization epitope on the receptor-binding domain of mers-cov spike glycoprotein ultrapotent human neutralizing antibody repertoires against middle east respiratory syndrome coronavirus from a recovered patient a novel human mab (mers-gd27) provides prophylactic and postexposure efficacy in mers-cov susceptible mice naturally-occurring antibodies devoid of light-chains molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies nanobodies: natural single-domain antibodies application of camelid heavy-chain variable domains (vhhs) in prevention and treatment of bacterial and viral infections nanobodies(r) as inhaled biotherapeutics for lung diseases generation and characterization of alx-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection chimeric camel/human heavy-chain antibodies protect against mers-cov infection molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 a novel nanobody targeting middle east respiratory syndrome coronavirus (mers-cov) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against mers-cov importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike to avoid neutralization escape severe respiratory illness caused by a novel coronavirus identification of residues on human receptor dpp4 critical for mers-cov binding and entry a novel neutralizing monoclonal antibody targeting the n-terminal domain of the mers-cov spike protein wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus asymptomatic middle east respiratory syndrome coronavirus infection in rabbits experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4 an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus funding: this study was supported by a grant from national natural science funds of china (nos. 31570167). the authors declare that they have no conflict of interest. key: cord-343528-5283jsnu authors: zhang, zhao; shen, libing; gu, xun title: evolutionary dynamics of mers-cov: potential recombination, positive selection and transmission date: 2016-05-04 journal: sci rep doi: 10.1038/srep25049 sha: doc_id: 343528 cord_uid: 5283jsnu middle east respiratory syndrome coronavirus (mers-cov) belongs to beta group of coronavirus and was first discovered in 2012. mers-cov can infect multiple host species and cause severe diseases in human. we conducted a series of phylogenetic and bioinformatic analyses to study the evolution dynamics of mers-cov among different host species with genomic data. our analyses show: 1) 28 potential recombinant sequences were detected and they can be classified into seven potential recombinant types; 2) the spike (s) protein of mers-cov was under strong positive selection when mers-cov transmitted from their natural host to human; 3) six out of nine positive selection sites detected in spike (s) protein are located in its receptor-binding domain which is in direct contact with host cells; 4) mers-cov frequently transmitted back and forth between human and camel after it had acquired the human-camel infection capability. together, these results suggest that potential recombination events might have happened frequently during mers-cov’s evolutionary history and the positive selection sites in mers-cov’s s protein might enable it to infect human. acid substitutions in its s protein receptor binding domain along with its switching host from civet to human 20 . moreover, two amino acid substitutions in the s protein's c-terminal of hku4, a bat beta-coronavirus, enable its entry to human cells and the same amino acid substitutions are also found in mars-cov 21 . furthermore, heptad repeat regions in c-terminal of mers-cov and related coronaviruses also play important roles in viral adaptive evolution 22 . in summary, those studies introduced above suggested that s protein plays a vital role in mers-cov's cross-species transmissibility. however, the evolutionary mechanism of how mers-cov's s and other proteins facilitated the cross-species transmission of mers-cov remains to be investigated. here, we performed a series of phylogenetic and bioinformatic analyses for mers-covs. we systematically investigated the recombination events in mers-covs, the potential transmission route of mers-covs in five different host species and the evolutionary pressure of each mers-cov's protein during cross-species transmission. our study might offer some insight in explaining the possible mechanism in mers-cov's adaptive evolution. epidemic description and phylogenetic analysis of mers-cov. by far, the largest mers-cov outbreak is in saudi arabia and almost all human cases have a direct or indirect link to arabian peninsula. in this study, we collected 74 human mers-cov whole genome sequences from 9 countries (supplementary table 1 ). the geographic distribution of these samples is shown in supplementary fig. 1a . the majority of them are from the countries in arabian peninsula (78.4%, 58/74) and more than half of them are from saudi arabia (64.9%, 48/74) (supplementary fig. 1a) . the peak season for mers is between april (26.5%) and may (25.0%) (supplementary fig. 1b) . based on the whole-genome alignment of our collected sequences (supplementary table 1 ), we performed the phylogenetic analysis for these sequences with two sras-covs serving as the outgroup. our phylogenetic tree shows that all camel and human mers-covs are clustered together. the bat and hedgehog mers-covs formed a basal paraphyletic group to all camel and human mers-cov clade (fig. 1a) . a single camel mers-cov isolated in egypt (gi: 589588051) forms a single basal clade to human and the other camel mers-covs (fig. 1b) . the human-camel mers-cov cluster can be further divided into two clades-clade a and clade b, as previously reported 16 . clade a contains four human strains isolated in jordan and saudi arabia while 70 human and 17 camel mers-covs are mixed in clade b. there are five groups in clade b and we named them as group i to group v as the previous study 15 . there are 25, 17, 14, 2 and 29 mers-cov sequences in group i to group v, respectively (fig. 1b) . we performed the recombination analysis on the collected full-length mers-cov sequences. we find that there are 28 of them experienced potential recombination events (30.4%, 28/92), including three camel mers-covs and 25 human mers-covs (supplementary table 1 ). we divided 28 potential recombinant sequences into seven different types and named them as type 1 to type 7 ( fig. 1btable 1 ). type 1 means the recombination happened between group ii and group v, which includes 3 sequences and is about 11% of total recombinant sequences. type 2 means the recombination happened between group iii and group v, which includes 6 sequences (22%). interestingly, the mers-covs newly found in 2015 in south korea and china are type 2 recombinants 15, 23 . type 3 means the recombination happened between group i and group iii, which includes 2 sequences (7%). type 4, 5 and 6 are the recombination happened between different genomic regions of group iv and group v, which include 7, 4 and 4 sequences (25%, 14% and 14%), respectively. type 7 is the recombination happened among three groups (group i, iv and v), which includes 2 sequences (7%). our phylogenetic analysis showed type 1 belongs to phylogenetic group ii while type 2 and 3 belong to phylogenetic group iii, and type 4 to 7 belong to phylogenetic group v. there is no recombination found in phylogenetic group i and group iv (fig. 1b) . we also reconstructed the phylogenetic tree using non-recombinant sequences only and found that its topology is consistent with the tree based on all sequences (supplementary fig. 2) . we also performed the snp (single-nucleotide polymorphisms) analyses for each recombinant types and found the large recombination segments in type 2, 4, 6, 7 are conspicuous but in type 1, 3, 5 are obscure (supplementary fig. 3 ). in order to explore the selection pressure on the mers-cov proteins when it transmitted from animal host to human, we performed the adaptive evolution analyses for all mers-cov protein in absence of recombinant strains. firstly, we set camel and human mers-covs as the foreground branch and bat and hedgehog mers-covs as the background branch to preform branch-site test in codeml of paml program (see fig. 1a ). the strong positive selection is detected in spike (s) glycoprotein between these two branches (p < 0.001), while there is no significant positive selection in the other mers-cov genes ( table 1) . we find nine positive selection sites in mers-cov spike (s) glycoprotein and eight of them are statistically significant (table 1) . six significant positive selection sites are located in the receptor binding domain of s protein (fig. 2a) . we utilized a published crystal structure (pdb id 4l72 in rcsb protein data bank), the receptor binding domain (rbd, aa 367-606, fig. 2b ) of mers-cov spike glycoprotein complexed with the human receptor dipeptidyl peptidase 4 (ddp4), to demonstrate their locations in a 3d environment (fig. 2b) . the receptor binding domain of mers-cov s protein can be further divided into a receptor-binding sub-domain and a core sub-domain. two significant positive selection sites, k511r and g521n, are in the receptor-binding sub-domain and k511r is in direct contact with human receptor ddp4. q419s, g436n, d472s and r479l are in the core sub-domain. moreover, we also detected a positive selection site in s protein's c-terminal, l775s. secondly, we screened the positive selection sites among human-camel mers-covs (table 2 ). five significant (table 3 ). the genome-wide average nucleotide substitution rate of camel and human mers-covs was 4.81 × 10 −4 substitutions per site per year. open reading frame 3 (orf3) has the fastest substitution rate while orf5 has the slowest substitution rate. the orf4b, nucleocapsid (n) glycoprotein, and spike (s) glycoprotein, have a similar substitution rate which is faster than the whole-genome substitution rate. in order to study the temporal and spatial pattern of mers-cov transmission, a maximum clade credibility (mcc) tree was constructed using mers-cov whole genome sequences without recombinant strains (fig. 3c) . the ancestral host state with time reference was estimated for each tree node and marked with different colors. we named six important nodes in mers-cov divergence on mcc tree for node a to f (fig. 3c ). the possible transmission time for each node and its 95% highest posterior density (hpd) are shown in fig. 3b . we found that the origin time of human-camel mers-cov is relatively late (node d). furthermore, the tmrca for clade b is in ~2012 (fig. 3b , node f) and clade a and clade b are divergent in ~2011 (fig. 3b , node e). interestingly, the mcc tree shows that there are six cross-species transmission events with high posterior probabilities in clade b. five of them are human-to-camel transmission events and one of them are camel-to-human transmission events (fig. 3c ). additionally, with the mers-cov of human/camel and bat/hedgehog mers-cov together, we inferred the ancient mers-cov exists for decades of years (fig. 3b,c) . the tmrca of the mers-covs for vespertilio superans, neoromicia capensis or erinaceus europaeus can be traced back to 2006 (node c), 2003 (node b) and 1996 (node a), respectively. before the emergence of human-camel mers-cov, the estimated tmrca for all mers-covs appeared in ~1996 (fig. 2c , node a). we also preformed root-to-tip analysis using the consistent dataset (fig. 3a) , and the result shows that the origin time of tmrca is in ~1995 with high statistical supports (r 2 = 0.874, p value < 0.001). together these results suggest that the ancient mers-cov should have existed for decades in animal host and got the ability to infect human or camel recently. mers-cov belongs to coronavirus, beta-coronavirus, lineage c. since it was discovered in 2012, mers-cov has attracted extensive attention due to its human-to-human infection capability and high mortality rate. recombination events have been confirmed in human mers-cov 23 . the fact that mers-cov can be found in multiple species proposes its cross-species transmissibility [4] [5] [6] [7] 11 . by far, the evolutionary details of how mers-cov transmitted to human are still unknown. based on the most comprehensive collection of mers-cov genome sequences so far, we tried to elucidate the evolution and transmission of mers-cov among different species. mers-cov has been reported in five species including european hedgehog, two species of bats, dromedary camel, and human. we used the ml method to reconstruct the whole-genome phylogenetic tree of mers-covs isolated from these species. the ml tree shows that the hedgehog mers-covs are basal to all the other mers-covs and two bat mers-covs are basal to camel and human mers-covs. this result suggests that the ancestor of camel and human mers-covs may be from other animal host, such as the hedgehog or bat. we also reconstructed the phylogenetic tree of mers-cov using nj method or based on each mers-cov protein. these trees show a consistent topology, which proposes that the phylogenetic relationship estimated in our study is credible (supplementary fig. 5) . we divided clade b into five groups as pervious study to detect the recombination of mers-cov 15 . because the evolutionary distances among mers-covs are close (table 3) , no large segment recombination could be detected among them. thus, according to discontinuous recombination segments, we defined potential recombination events in mers-covs. this method has been used in the previous study to label potential recombination 24 . in our study, we found 28 strains form seven recombinant types, which took more than 30% of all isolated mers-covs in human and camel. among them, we found 26 strains in six recombinant types (type 1 to type 6) between two phylogenetic groups and two strains in one type (type 7) among three phylogenetic groups. for now, the recombination of mers-cov was confirmed in previous study, but no report about the recombination among more than two groups of mers-cov. interestingly, most recombinant types (type 1, 2, 4, 5, 6 and 7) are related to group v and they make up 92.9% of total recombinant strains (26/28). the result suggests that recombination events might happen frequently and the recombinant types involving group v might happen broadly. additionally, multiple recombination events indicate that double infection and super infection likely existed during the transmission history of mers-cov. we failed to detect possible large recombination segments in type table 3 . evolutionary distance, nucleotide substitution rate and coefficient of variation of substitution rate for human and camel mers-cov proteins. 1, 3 and 5. by comparing the snps (single-nucleotide polymorphisms) of reference sequences with recombinant sequences, we reckoned that specific nucleotide mutation might influence the results of recombination analysis. this problem can be solved by discovering more mers-cov sequences or developing more detailed genotype classification for mers-covs in the future. we also performed phylogenetic analyses for potential recombinant region and got similar results (supplementary fig. 3) . interestingly, the east asian mers-cov strains (china and south korea) belong to type 2 recombinant and the previous study show that their tmrca might be a result of potential recombination event 23 , which indicates the recombinant strains have transmitted broadly. moreover, one recombinant mers-cov lineage has led to the large-scale outbreak in both camel and human 26 . it proposes that recombinant mers-covs have experienced cross-species infection. additionally, our study reveals that the number of recombinant strains is large and the potential recombinant types are abundant. together these findings highlight that we should take more attention to recombinant mers-cov transmission. although how the mers-cov transmitted from its natural host to human is still unknown, it is confirmed the mers-cov have been found in many animal hosts, such as bats and hedgehog. to study the evolutionary pressure on each mers-cov's protein during its potential cross-species transmission, we conducted a comprehensive scan for positive selection sites in mers-cov's proteins. recombinant strains were excluded in this analysis. we set camel-human mers-covs as the foreground branch and hedgehog-bat mers-cov as the background branch and estimated the relative evolutionary pressure on the foreground branch compared to the background branch. we only found that mers-cov's s protein underwent strong positive selection and there are nine significant positive selection sites in s protein. it suggests that s protein was under strong evolutionary pressure during the transmission from its natural host to human. among significant positive selection sites, six of them are located in s protein's receptor binding domain (rbd). rbd is crucial for virus to enter host cells and it comprises of one binding region and one core region. based on the rbd's 3d model, we find that two sites are located in the binding region of rbd, which suggests that these amino acid substitutions might change mers-cov's binding capability to host cells and thus facilitate its cross-species transmission. the other four sites are located in the core region of rbd. these amino acid substitutions might change the structure of core region and indirectly influence mers-cov's cross-species capability. in order to eliminate the sample bias between human/camel group and bat/hedgehog group, we also did random sampling for the 68 non-recombinant human and camel sequences. we tried 10, 20 and 50 random sampling, respectively. using random sampled sequences together with bat and hedgehog mers-cov sequences, we performed branch-site analyses and got the same results as our aforementioned (data not show). we also estimated the nucleotide substitution rates of camel-human mers-covs to investigate its evolutionary dynamics after it infected camel and human. our estimated nucleotide substitution rate for mers-cov's whole genome is 4.81 × 10 −4 , which is slower than the previous estimation 9 . one explanation for the phenomenon is that we used a larger dataset than the previous study, which includes all available mers-cov whole genome sequences. our estimated confidence interval of the substitution rate of mers-cov genome is largely overlapped with the result from another study 16 . the estimated nucleotide substitution rates show that four proteins experienced the accelerated evolution. through evolutionary pressure analysis, we found camel-human mers-cov's four proteins underwent positive selection and detected five significant positive selection sites. one of them is located in m proteins. there is evidence that m proteins are powerful interferon antagonist 26 , which proposes that the evolutionary pressure on m proteins are from host's immune system. two out of five significant sites are found in n protein which is fundamental for mers-cov self-assembly. coronavirus n protein is able to bind to different host cell proteins and demonstrated to have various functions, one of which is also counteracting host interferon as shown in sars-cov [27] [28] [29] [30] . it is reasonable to speculate that mers-cov n protein under intensive selection because its functions were similar to those of sars-cov n protein. the results above suggest that the arm race between mers-cov proteins and host's immune system might be the main evolutionary driving force behind mers-cov's adaptive evolution after it began to infect camel and human. mers-cov spike (s) glycoprotein evolves slightly faster than the genome-wide average rate, which indicates that the nucleotide substitution rate of mers-cov s protein still maintains a fast speed even after it crossed the species boundary. the positive selection site we found in mers-cov s protein with site-specific test is identical to the previous study's result 3 . this site is located in heptad repeats 1 which is a key component in membrane fusion architecture and required for mers-cov entering host cells 31 . in absence of recombinant strains, we performed the mcc analysis using mers-cov whole-genome sequences in order to infer the time and source species when mers-cov crossed the species boundary. the topology of the mcc tree is highly congruent with that of the whole-genome phylogenetic tree. we defined six nodes (a-f) to explain transmission. the posterior probability for the ancestral sate of node a, b or c is not very high in our mcc analysis and the 95% highest posterior density (hpd) of the divergence time for these three nodes is quite long. so these results are weak for demonstrating the exact origin time or ancestor state of mers-cov. however, these estimations still provided the evidence that the ancestor mers-cov should have been infected a number of animal hosts, such as bat or hedgehog, for decades of years (supplementary fig. 4) . the x-intercept (tmrca) in root-to-tip is ~1995 with high statistical supports, which is close to the estimated time of tmrca in mcc analysis. this hypothesis is in agreement with the result of serological studies 2,3 . the appearance of the common ancestor of human-camel mers-cov is in 2010 and the appearance of the tmrca of clade a and b is in 2011, which are exactly the same as the previous report 16 . in clade b, we detected five possible human-to-camel transmission events and one camel-to-human transmission event. it suggests that mers-cov frequently transmitted back and forth between human and camel after it acquired the capability of infecting both hosts. actually, there is at least one confirmed case of camel-to-human mers-cov transmission 32 . scientific reports | 6:25049 | doi: 10.1038/srep25049 in conclusion, we found that potential recombination events are common in mers-cov's evolutionary history and potential recombinant mers-covs can be divided into seven types. the amino acid sites under positive selection in mers-cov s protein, especially those in its receptor binding domain, might have facilitated its cross-species transmission from animal host to human. we detected the strong positive selection in four proteins of camel-human mers-covs, which indicates that they probably experienced strong adaptive evolutionary pressure from host's immune system. additionally, we also found six possible cross-species transmission cases between human and camel. our study investigated the evolutionary dynamics of mers-cov, which shall provide a basis for mers-cov control and treatment. sequence data. the complete genomic nucleotide sequences of 91 mers-covs and two sars-covs were downloaded from ncbi nucleotide database. among 91 mers-cov genomic sequences, 68 of them are from human, 18 of them are from dromedary came, two of them from two bat species neoromicia capensis and vespertilio superans, and 3 of them are from european hedgehog erinaceus europaeus. two sars-cov genomic sequences are from human and bat rhinolophus ferrumequinum, respectively. we used sequences 453061240 as reference to extract open reading frames from each mers-cov genome in this study. genomic sequence alignment and phylogenetic analysis. total 93 collected genomic sequences were aligned using the muscle software with default parameters 33 . clustalw and mafft used to validate the muscle result 34, 35 . alignments were refined manually in bioedit (http://www.mbio.ncsu.edu/bioedit/ bioedit.html). only unambiguously aligned positions were used for subsequent phylogenetic analyses (supplementary files). we used the jmodeltest 3.1 to estimate the best nucleotide substitution model for our alignment 36 , which is gtr+ i+ g. we used the phyml 3.1 to perform the phylogenetic analysis for 93 collected genomic sequences based on their genomic sequence alignment 37 . the branch support values were calculated with shimodaira-hasegawa test integrated in phyml. in clade b, we estimated the consensus sequences for every phylogenetic group using cons tool in emboss explorer (http://bioinfo.nhri.org.tw/cgi-bin/emboss/cons). five consensus sequences were set as reference and every sequence in clade b was used as the query to detect the possible recombination using simplot software 25 . the window is set to 200 bp and the step is set to 20 bp. positive selection analysis. we extracted the coding region of each mers-cov protein using mers-cov 453061240 strain as a reference template. the codeml program implemented in paml 4.7 package was used to detect the positive selection in the codon alignment of each mers-cov protein set 38 . in the branch-site model, the group of human-camel mers-covs was set to be foreground, the group of bat-hedgehog mers-covs was set to be background, and model a with estimated ω value was compared with the null model (model a') with fixed ω value. to reduce the bias from sample size, we performed random sampling on 68 human and camel mers-covs (clade a and b) which are all non-recombinant sequences. we used 10, 20 and 50 as the random sample size with and without replacement. the random sampled sequences together with bat and hedgehog mers-covs were used to generate the datasets for branch-site model analysis as aforementioned method (script see supplementary files). moreover, for each random sample size, we repeatedly drew five times in order to make results robust. we also used the site-specific model to detect positive selection in the human-camel clade. the site-specific model was performed by comparing the models m2a (positive selection) and m8 (beta & ω ) vs. the null models the crystal structure of the receptor binding domain (rbd) of mers-cov spike (s) glycoprotein in complex with the human receptor dipeptidyl peptidase 4 (ddp4) was displayed using jmol (jmol: an open-source java viewer for chemical structures in 3d we estimated the transmission of mers-cov among different hosts or geographic areas. the sampling time and host/geographic location of each sequence were also used in analysis. the nucleotide substitution rates and the origin time of most recent common ancestor (mrca) on various nodes of mcc tree were also estimated using the beast package. a relaxed molecular clock with an uncorrelated log-normal distribution, and a constant population size model were used in bayesian coalescence analysis. according to the outcome of jmodeltest3.1, the gtr+ gamma+ i model of nucleotide substitution was employed in mcc analysis. statistical uncertainty in parameter estimations was reflected by the 95% highest posterior density (hpd) values. mcmc analysis was run for 500/100 million generations for hosts/geographic transmission with sampling every 50,000/10,000 generations to achieve parameter convergence and adequate effective sample sizes (ess > 200). we summarized the trees using treeannotator implemented in the beast v1.8.2 package. the initial 25% samples were discarded as burn-in, leaving 75% trees per run to produce the consensus tree middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group mers coronavirus neutralizing antibodies in camels antibodies against mers coronavirus in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat mers-related betacoronavirus in vespertilio superans bats characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs isolation of a novel coronavirus from a man with pneumonia in saudi arabia spread, circulation, and evolution of the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description evidence for camel-to-human transmission of mers coronavirus molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku14, from domestic rabbits origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus the heptad repeat region is a major selection target in mers-cov and related coronaviruses the recent ancestry of middle east respiratory syndrome coronavirus in korea has been shaped by recombination full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists nucleocapsid protein of sars coronavirus tightly binds to human cyclophilin a the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 sars-cov nucleocapsid protein binds to hubc9, a ubiquitin conjugating enzyme of the sumoylation system sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus evidence for camel-to-human transmission of mers coronavirus muscle: multiple sequence alignment with high accuracy and high throughput clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform jmodeltest: phylogenetic model averaging new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 paml 4: phylogenetic analysis by maximum likelihood mega6: molecular evolutionary genetics analysis version 6.0 bayesian phylogenetics with beauti and the beast 1.7 this work was supported by the grant from the national science foundation of china (31571355). we thank the anonymous reviewers. z.z., l.s. and x.g. designed the experiments; z.z. and l.s. collected viral sequences, performed phylogenetic analyses, adaptive evolutionary analyses and transmission analyses. z.z., l.s. and x.g. wrote the manuscript. all authors reviewed the manuscript. supplementary information accompanies this paper at http://www.nature.com/srep key: cord-352741-0pdeehai authors: geramizadeh, bita; marzban, mahsa title: histopathologic findings of coronavirus in lung: a mini-review date: 2020-10-12 journal: clin pathol doi: 10.1177/2632010x20951823 sha: doc_id: 352741 cord_uid: 0pdeehai coronaviruses (covs) are important human and animal pathogens. there have been several outbreaks of lung involvement by this category of viruses in the world, ie, severe acute respiratory syndrome (sars-cov-1) in 2002 and 2003, the middle east respiratory syndrome (mers-cov) in 2012, and the new coronavirus (2019-ncov) outbreak of pneumonia from wuhan, china, since december 2019. there have been several studies about the clinical features and imaging features, but very few reports have been published about pathologic findings in lung tissue, which was partly because of the lack of tissue diagnosis secondary to suddenness of the outbreak. overall, less than 30 reports have been published in the literature about histologic findings of lung in these viruses, so far. in this report, we will review the published reports about the histopathologic findings of lung tissue in the patients infected with sars-cov-2 in comparison with 2 other coronaviruses that have caused outbreaks, ie, sars-cov-1 and mers-cov. illness, but less than 20% eventually fall in the severe group, needing assisted ventilation. this group of patients has a high mortality rate, mostly in older age, and patients with underlying diseases such as diabetes or on treatment with immunosuppressive like transplant patients. there have been several studies about the clinical features and imaging features, but very few reports have been published about pathologic findings in lung tissue. this has been due to the outbreak that did not let the researchers to perform enough numbers of autopsies. 4 in this report, we will review the published reports about the histopathologic findings of lung tissue in the patients infected with sars-cov-2 in comparison with 2 other covs that have caused outbreaks, ie, sars-cov-1 and mers-cov. case reports and case series that have been published in the medical literature have been gathered by searching in pubmed and google scholar. the keywords for searching were "lung," " pulmonary," and covs, ie, "severe acute respiratory syndrome coronavirus 2 [sars-cov-2])," "coronavirus disease (covid-19)," "pathology," "biopsy," "autopsy," "histopathology," "severe acute respiratory syndrome (sars)," and "middle east respiratory syndrome (mers)." each keyword was used alone and together with other key words to find the most relevant papers regarding the histopathologic findings to be selected and included in this review. during the last years since 2002, from the beginning of sars-cov-1 epidemics, then mers-cov and now for sars-cov-2, less than 20 papers in english have been published with specific consideration of the histopathologic findings of lung tissue in human, most of which have been autopsy findings after the patients' death; therefore, most of the findings demonstrate the fatal forms of the diseases. table 1 shows the characteristic findings of 3 covs in the lung. the macroscopic picture of the lung shows edema with congestion. cut sections of the lung show irregular and patchy areas of consolidation (pneumonia). frequent bronchopneumonia has also been reported. mucopurulent material was seen in the upper respiratory tract. lung histomorphology of early phase of the disease (<10 days) mostly shows acute diffuse alveolar damage. later phase of the disease also shows "diffuse alveolar damage" with "acute fibrinous and organizing pneumonia." other findings were "pulmonary infarction, squamous metaplasia, giant cell transformation, and microthrombi. intracytoplasmic viral inclusions have also been reported in alveolar epithelial cells." 5, 6 in other studies, "desquamative alveolitis and bronchitis, with proliferation of alveolar epithelial cells, exudation of mononuclear cells, lymphocytes and plasma cells" were reported. the epithelial cells are enlarged and fuse to create syncytia. 7 there is also a case report about the histologic findings of a lung biopsy taken during surgery approximately a week after the beginning of symptoms in a patient finally diagnosed as sars-cov-1. histopathologic findings of this biopsy revealed nonspecific change such as mild increase in interstitial lymphocytes with moderate increase of intra-alveolar macrophages as well as formation of "hyaline membrane." pneumocytes have been reported to show cytomegaly and karyomegaly with prominent nucleoli. 8 large syncytial cells with multiple nuclei were also reported. small vessel vasculitis was also seen. 9 another finding in lung tissue has been severe neutrophilic capillaritis and small vessel injury that has been described to be correlated with the severity of lung injury. 10 in addition to lung, systemic organ involvement has been reported, as "degeneration and necrosis" of the hepatocytes, parenchymal cells of kidney and adrenal as well as heart myocardial cells. 7 in the reported cases of sars, patients' outcome has not been directly correlated with the pathology of injury of the lung and other organs. 7,11 -14 none of the histopathologic findings were specific and characteristic for the diagnosis of sars disease. 14,15 there has been only a single autopsy case reported by ng et al on a patient who died after infection with this mers-cov. histopathologic features include diffuse alveolar damage, pulmonary consolidation, edema, and exudative pneumonia. hyaline membrane has also been reported. pneumocytes, multinucleated epithelial cells, and submucosal glands of bronchi were the main infected cells. 16 another reported patient was a known case of lymphoma on chemotherapy who has been infected by mers, also showed diffuse alveolar damage with necrotizing pneumonia as well as congestion. ultrastructural studies showed viral inclusions in the respiratory epithelium. there is also the possibility of necrosis to be secondary to prior chemotherapy. this case was reported based on the findings on a needle biopsy. 16, 17 the histopathologic changes in this patient showed heterogeneous severity. alveolar spaces and interstitium showed fibrin deposits as well as pigmented pulmonary macrophages and lymphocytes. in the mostly injured parts of the lung, the alveolar spaces showed a lot of blood and fibrin, mixed inflammatory cell infiltrate, and cellular debris. pneumocyte hyperplasia and reactive changes, denudation and sloughing of alveolar cells, rare multinucleated syncytial cells, congestion of the alveolar walls, and hyaline membrane formation were also reported. peribronchiolar spaces showed focal infiltration of polymorphonuclear (pmn) leukocytes and lymphocytes. the infiltration of cd4-positive t-helper lymphocytes in the wall of arteries ("intimal arteritis") is consistent with an active immune response to the lung injury secondary to viral infection. 17 it is worthy to note that there have been very few autopsy cases of mers, but pulmonary pathology reported from animal studies was compatible with human cases. 18 in mers cases, similar histopathologic changes such as multinucleated cells and viral inclusion have been reported in many other organs such as brain, kidney, and liver. 18 histological examination of lung in rare cases reported from sars-cov-2 showed "edema, bilateral diffuse alveolar damage with cellular fibromyxoid exudates, desquamation of pneumocytes and hyaline membrane formation," indicating acute respiratory distress syndrome. interstitial lymphocytic infiltrates were also reported. "syncytial cells with multiple nuclei and large and atypical pneumocytes have been reported with large nuclei, amphophilic granular cytoplasm, and prominent nucleoli in the intra-alveolar spaces, showing viral cytopathic-like changes." 19 two other cases have reported resected lung tissue for adenocarcinoma, which turned out to be infected with sars-cov-2 at the time of surgery. sections away from the tumor showed diffuse alveolar damage, with edema and proteinaceous exudates. prominent inspissated globules of secretions were also noticed. vascular congestion and mild inflammatory infiltration were also present. multinucleated giant cells were reported within the alveolar spaces. there were also histopathologic findings of ongoing "reparative process," such as marked and patchy hyperplasia of pneumocytes and abundant alveolar macrophages as well as interstitial thickening, proliferation of fibroblasts, and fibroblast plugs. histopathologic findings of viral cytopathy such as inclusions were also noted in some of the pneumocytes. 4 in another study, the blood vessels of alveolar septum showed congestion, edema, and moderate infiltration of monocytes and lymphocytes as well as exfoliation of bronchial epithelial cells. hyaline thrombi were seen in a few of small vessels. focal hemorrhage in lung tissue, organization of exudates in alveolar spaces, and pulmonary interstitial fibrosis were also noted. 20 another finding that has just reported is the consolidation created by fibroblastic proliferation with extracellular matrix and fibrin forming clusters in airspaces. 21 the pathological features of sars-cov-2 are very similar to those seen in sars-cov-2 and mers-cov infections. there is no specific finding in these pulmonary infections. 18, 21 even the autopsy findings of h1n1 pandemic of influenza in the lung of victims in 2009 were very similar, and small vessel thrombosis and diffuse alveolar damage have been the main features that are very similar to cov-infected lungs. 15 table 1 summarizes the histopathologic findings of the lung in these 3 covs reported during their epidemics and pandemics. the most important finding in all of pulmonary involvement in all 3 of them is diffuse alveolar damage that clinically is presented as acute respiratory tract disease. 4,15 microvascular involvement and alveolar capillary microthrombi, as well as new vessel formation, were reported to be significantly more common in patients with covid-19 compared with influenza. 19 one histopathologic finding in the new sars-cov-2infected lung disease that has not been reported in the previous epidemics of coronaviruses is the presence of pulmonary fibrosis that can be indicative of future pulmonary dysfunction if the patient recovers. 4, 20 progressive pulmonary fibrosis and marked thickening of alveolar wall were also reported, which is the main factor leading to pulmonary dysfunction. in the early stages, there is no mature fibrosis, but survived patients for longer periods have reported to show advanced stages with extracellular matrix deposition and interstitial fibrosis. 19, [21] [22] [23] [24] another important finding in the victims of these 3 viruses is the involvement of vital organs such as kidneys and livers, especially in the fatal cases. 4, 12, 15, 16 ultrastructural findings in 3 viruses showed evidences of viral entry via different organs, and viral inclusions have been reported especially in the respiratory epithelium, kidney, and heart. 16, 17, 25 there are some limitations in these studies: 1. most of these histopathologic findings have been based on autopsy findings that are related to the fatal cases. there is very limited data related to mild or moderate infections with recovery. 2. in the cases of mers-cov, the number of human cases is very limited and cannot be accurately distributed. for sars-cov-2, it is still soon to decide about the details of pulmonary histopathology, although the main findings have already been reported and analyzed. coronaviruses: an overview of their replication and pathogenesis emerging coronaviruses: genome structure, replication, and pathogenesis coronavirus disease 2019 (covid-19): a systematic review of imaging findings in 919 patients pulmonary pathology of earlyphase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore. challenges in determining a sars diagnosis the clinical pathology of severe acute respiratory syndrome (sars): a report from china lung pathology of fatal severe acute respiratory syndrome multiple organ infection and the pathogenesis of sars pulmonary pathology and covid-19: lessons from autopsy. the experience of european pulmonary pathologists pulmonary pathology of severe acute respiratory syndrome in toronto the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h5n1-a review fatal aspergillosis in a patient with sars who was treated with corticosteroids immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan emerging respiratory infections: the infectious disease pathology of sars, mers, pandemic influenza, and legionella clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection − clinicopathological and ultrastructural study value of autopsy emphasized in the case report of a single patient with middle east respiratory syndrome pathological findings of covid-19 associated with acute respiratory distress syndrome a pathological report of three covid-19 cases by minimally invasive autopsies pathological study of the 2019 novel coronavirus disease (covid-19) through post-mortem core biopsies pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 postmortem examination of covid-19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction covid-19 in a hispanic woman autopsy report with clinical pathological correlation sars-cov-2 identification in lungs, heart and kidney specimens by transmission and scanning electron microscopy the pathological features of sars-cov-2 are similar to those seen in sars-cov and mers-cov infections, the most important of which are diffuse alveolar damage and hyaline membrane disease. however, pulmonary vascular damage, fibrosis and alveolar wall thickening, and extracellular matrix deposition are mostly seen in sars-cov-2. bita geramizadeh: idea of the research, literature search, writing the paper. mahsa marzban: literature search, editing the paper. bita geramizadeh https://orcid.org/0000-0003-1009-0049 key: cord-324926-3c5ab73l authors: xia, shuai; yan, lei; xu, wei; agrawal, anurodh shankar; algaissi, abdullah; tseng, chien-te k.; wang, qian; du, lanying; tan, wenjie; wilson, ian a.; jiang, shibo; yang, bei; lu, lu title: a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike date: 2019-04-10 journal: sci adv doi: 10.1126/sciadv.aav4580 sha: doc_id: 324926 cord_uid: 3c5ab73l continuously emerging highly pathogenic human coronaviruses (hcovs) remain a major threat to human health, as illustrated in past sars-cov and mers-cov outbreaks. the development of a drug with broad-spectrum hcov inhibitory activity would address this urgent unmet medical need. although previous studies have suggested that the hr1 of hcov spike (s) protein is an important target site for inhibition against specific hcovs, whether this conserved region could serve as a target for the development of broad-spectrum pan-cov inhibitor remains controversial. here, we found that peptide oc43-hr2p, derived from the hr2 domain of hcov-oc43, exhibited broad fusion inhibitory activity against multiple hcovs. ek1, the optimized form of oc43-hr2p, showed substantially improved pan-cov fusion inhibitory activity and pharmaceutical properties. crystal structures indicated that ek1 can form a stable six-helix bundle structure with both short α-hcov and long β-hcov hr1s, further supporting the role of hr1 region as a viable pan-cov target site. coronaviruses (covs) are enveloped viruses with a positive-sense, single-stranded rna and are associated with various natural hosts. covs are divided into alpha, beta, gamma, and delta groups, and the beta group is further composed of a, b, c, and d subgroups (fig. 1a) (1). among them, six covs can infect humans (hcovs), including hcov-229e (229e) and hcov-nl63 (nl63) in the alpha group, hcov-oc43 (oc43) and hcov-hku1 (hku1) in beta subgroup a, severe acute respiratory syndrome cov (sars-cov) in beta subgroup b, and middle east respiratory syndrome cov (mers-cov) in beta subgroup c (2) . in this century, sars-cov and mers-cov have emerged in the human population and caused severe pulmonary disease with alarmingly high case-fatality rates. in 2002, sars-cov infections first appeared in china and then quickly spread as a global epidemic in more than 30 countries with 8273 infections and 775 deaths (nearly 10% mortality) (2) . in 2012, mers-cov emerged in saudi arabia and spread throughout the middle east. in 2015, the second pandemic of mers-cov occurred in south korea, causing super-spreading events with third-and fourth-generation cases of infection. the world health organization has reported 2229 laboratory-confirmed cases of mers-cov infection, including 791 deaths (about 35% case fatality) in 27 countries as of august 2018 (www.who.int/emergencies/ mers-cov/en/). meanwhile, the remaining common hcovs, such as 229e, oc43, and nl63, usually infect the human upper respiratory tract and cause the common cold, but they also are responsible for severe and even fatal diseases in children, elderly, and immunocompromised patients (3) (4) (5) . these scenarios suggest that those common hcovs might also pose a lethal threat to humans. note that hcovs are rapidly evolving. oc43 isolates with novel genomes are being continuously identified (6) (7) (8) . the existence of sars-like cov (sl-cov) and mers-like cov (ml-cov) also pose great threats to public health worldwide. recent studies identified some types of sl-cov, such as sl-wiv1-cov and sl-shc014-cov, whose spike (s) proteins highly resemble those of sars-cov. these sl-covs could use the same sars-cov receptor, i.e., angiotensin-converting enzyme-2 (ace2), to directly enter permissive human cells without need for adaptation (9) (10) (11) . in addition, the ml-cov, tylonycteris bat cov hku4, was shown to recognize the mers-cov receptor cd26 and infect human cells either after introduction of two single mutations (s746r and n762a) into its s protein or with the help of exogenous protease (12, 13) . from a historical perspective, zoonotic covs harbor a strong ability to cross species barriers to infect humans rapidly and unpredictably, as illustrated by newly emerging hcovs (2, 14) . thus, developing a specific drug that only targets a single hcov would be ineffective against newly emerging hcovs (9) (10) (11) . since no broad-spectrum anti-hcov drug is currently available for clinical use, it is incumbent to search for a common or conserved target site based on existing hcovs. the s glycoprotein is a type i transmembrane glycoprotein that plays an important role in mediating viral infection and is common to all hcovs. the s proteins consist of two subunits, s1 and s2 (fig. 1b) . the s1 subunit binds the cellular receptor through its receptor-binding domain (rbd), followed by conformational changes in the s2 subunit, which allows the fusion peptide to insert into the host target cell membrane. the heptad repeat 1 (hr1) region in the s2 subunit forms a homotrimeric assembly, which exposes three highly conserved hydrophobic grooves on the surface that bind heptad repeat 2 (hr2). this six-helix bundle (6-hb) core structure is formed during the fusion process and helps bring the viral and cellular membranes into close proximity for viral fusion and entry ( fig. 1b) (15) . thus, the s protein is an important target protein for the development of specific drugs. in particular, the s1 rbd is a very good target site, and both rbd-specific antibodies and rbd-based vaccines have previously exhibited effective antiviral activity or protective effect in blocking binding of virus to host receptors (16) . however, from an evolutionary perspective, the rbd of cov is part of a highly mutable region and, thus, is not an ideal target site for broadspectrum antiviral inhibitor development (14) . the sars-cov rbdspecific antibody fm6 failed to block infection mediated by the s protein of sl-cov-shc014 (9) (10) (11) 17) . in contrast, the hr region in the s2 subunit is conserved among various hcovs and plays a pivotal role in hcov infections by forming the 6-hb that mediates viral fusion (fig. s1a). furthermore, the mode of interaction between hr1 and hr2 is conserved among covs such that residues located at the "e" and "g" positions in the hr1 helices interact with residues at the "a" and "d" positions in the hr2 helices (fig. s1b) (18) . previous studies have reported that peptides derived from the hr2 (or c-terminal hr) region of class i viral fusion proteins from some enveloped viruses, including hiv-1 (19) (20) (21) , respiratory syncytial virus (rsv) (22) , ebola virus (23) , paramyxoviruses sv5 (24) , nipah virus (25) , and murine hepatitis virus (mhv) (26) , could competitively bind the viral hr1 (or n-terminal hr) and effectively inhibit viral infection. therefore, it is reasonable to speculate that hr1 could also be a good target for the development of fusion inhibitors against highly pathogenic hcovs. we and others have reported that peptides derived from the hr2 regions of sars-cov and mers-cov s proteins can competitively inhibit viral 6-hb formation, thereby preventing viral fusion and entry into host cells (18, 27) . for example, the peptide cp-1, derived from the sars-cov spike protein hr2 region, was able to inhibit sars-cov entry in a manner similar to that of mers-hr2p against mers-cov infection (18, 27) . however, we note that those peptides lack broad-spectrum antiviral activity against heterologous hcovs. for example, cp-1 and mers-hr2p peptides failed to cross-inhibit mers-cov and sars-cov infection, respectively. furthermore, the cov fusion core hr regions can be divided into two groups (28, 29) : short hrs, such as mers-cov, sars-cov, and oc43 hrs; and long hrs, such as 229e and nl63 hrs. the difference between short and long hrs arises from an insertion of 14 amino acids in the long hrs, further increasing the difficulty in designing broad-spectrum peptide fusion inhibitors. to address this challenge, we herein report the successful screening of a peptide oc43-hr2p with broad-spectrum fusion inhibitory activity. furthermore, a modified oc43-hr2p peptide, ek1, shows promising potency and breadth in inhibiting infection by multiple hcovs. in vivo studies demonstrate that administration of ek1 via the nasal route exhibits highly protective effects and safety profiles, highlighting its clinical potential. moreover, structural studies of ek1 in complex with hr1s from different hcovs explain the conserved basis for the hr1-ek1 interaction, further indicating that hr1 region could serve as a promising target site for the development of broad-spectrum pan-cov fusion inhibitors. on the basis of the 6-hb fusion core structure, we previously reported on peptides mers-hr1p and mers-hr2p that were derived from mers-cov hr1 and hr2, respectively (fig. s1c), and these two peptides displayed good interaction with each other (27) . here, we located the conserved hr region of multiple hcovs by sequence comparisons and then synthesized hr1-and hr2-derived peptides, termed hr1p and hr2p (fig. 1c) . notably, we observed 14-amino acid insertions in both hr1p and hr2p in the hrs of two -hcovs, i.e., 229e and nl63. to systematically assess the inhibitory activities of these peptides against different hcovs, we developed multiple cell-cell fusion assays that were mediated by the s protein of various hcovs (fig. s1d). consistent with previous results, mers-hr2p had inhibitory activity against cell-cell fusion mediated by mers-cov s protein with a concentration for 50% inhibition (ic 50 ) of 1.01 m, whereas it showed little inhibitory activity in other hcov s-mediated cellcell fusion assays even with concentrations up to 5 m ( fig. 1d and table s1). similarly, sars-hr2p specifically inhibited sars-cov s-mediated cell-cell fusion with an ic 50 of 0.52 m. on the other hand, hr2p peptides derived from the two -hcovs, i.e., 229e and nl63, showed potent and broad inhibitory activity against -hcov s-mediated cell-cell fusion with ic 50 values ranging from 0.13 to 0.51 m or from 0.21 to 0.56 m, respectively, but no effective inhibitory activity against -hcovs (including mers-cov, sars-cov, and oc43) s-mediated fusion ( fig. 1d and table s1 ). oc43-hr2p exhibited broad and potent fusion inhibitory activity with ic 50 values of 0.39, 0.54, and 0.66 m against mers-cov, sars-cov, and oc43, respectively ( fig. 1d and table s1 ). unexpectedly, oc43-hr2p, a -hcov hr2-derived peptide, and thus 14 residues shorter than 229e-hr2p and nl63-hr2p, exhibited effective activity against -hcovs with an ic 50 of 0.84 m on 229e-s-mediated cell-cell fusion and an ic 50 of 0.94 m on nl63-s-mediated cell-cell fusion. among all hr1ps, only 229e-hr1p exhibited moderate inhibitory effects on 229e-s-and nl63-s-mediated cell-cell fusion (fig. 1d) . thus, compared to hr1ps, all hcov hr2-derived peptides exhibited excellent self-specific fusion inhibitory activity, whereas oc43-hr2p showed broad-spectrum and potent fusion inhibitory activity against both -hcov and -hcov s-mediated cell-cell fusion ( fig. 1d and table s1 ). we also measured the -helicity of oc43-hr2p and hcov-hr1ps by circular dichroism and found that the peptides alone exhibited limited -helicity ranging from 11.3 to 42.3% ( fig. s1e ). in contrast, the mixtures of oc43-hr2p and each of these hr1 peptides, respectively, exhibited high -helicity (70.9 to 86.7%) with melting transition temperature (t m ) values that ranged from 48.5° to 91.5°c ( fig. s1e ), further implying that oc43-hr2p can bind hr1s of different hcovs and form stable complexes, thereby blocking s protein-mediated fusion. it was previously reported that introduction of negatively and positively charged amino acids glu (e) and lys (k), at the i to i + 3 or i to i + 4 positions in a helix, into an hiv-1 fusion inhibitory peptide can form intramolecular e-k or k-e salt bridges that result in substantial enhancement of the peptide's stability, solubility, and antiviral activity (30, 31) . using a similar design, we further optimized the sequence of oc43-hr2p by introducing glu or lys at appropriate sites in the peptide to increase the solubility and thereby the antiviral activity of the peptide. moreover, on the basis of the structure of mers-cov s 6-hb, we also introduced mutations at some sites that are not expected to be involved in hr1 binding, such as 4q, 14y, 32d, and 36l, to further enhance the fusion inhibitory activity of the peptide (table s2) . among the series of optimized peptides, peptides ek0-1, ek0-2, ek0-3, and ek1 showed gradually increased solubility and excellent inhibitory activity in cell-cell fusion assays. the final peptide, ek1, exhibited the most potent pan-cov antiviral fusion activity with ic 50 values in the range of 0.19 to 0.62 m (table s2) . in some hcov cell-cell fusion assays, ek1 exhibited even more effective inhibitory activity than the autologous peptide, such as mers-hr2p and sars-hr2p. akin to its ancestor oc43-hr2p, ek1, which is derived from the short 6-hb fusion core of oc43, was also able to potently inhibit the cell-cell fusion mediated by 229e and nl63 s proteins, both of which harbor the long fusion core. these results further underscore the broad-spectrum anti-hcov potential of ek1 (fig. 2 , a to e, and table s2). furthermore, ek1 also had superior pharmaceutical properties and solubility in phosphatebuffered saline (pbs) and water, which increased by 478-fold and 3.5-fold, respectively, as compared to the original oc43-hr2p. together, these results establish an important framework and platform for subsequent development of ek1 as a potential therapeutic (table s2) . sl-covs, including wiv1, rs3367, and shc014 covs, all manifest potential for human infection (9) (10) (11) . to further assess the breadth of fusion inhibitory activity, as demonstrated by ek1, we established cell-cell fusion assays mediated by the s protein of these sl-covs ( fig. s2) . notably, many studies have suggested diversity in the receptor-binding motif (rbm) of sl-cov's rbd (9-11), which plays an important role in binding the cellular receptor and serves as an effective target site for the development of cov-specific antibodies or vaccines (32) . specifically, the rbm of shc014 cov has only 80% similarity and 64% identity to that of sars-cov, and an antibody targeting the sars-cov rbd could not prevent infection mediated by the shc014 s protein (9) (10) (11) 17) . in contrast, the hr1 and hr2 sequences of sl-cov are 100% identical to those of sars-cov ( fig. s2b ). correspondingly, ek1 exhibited greater inhibitory activity than the autologous peptide sars-hr2p against cell-cell fusion mediated by the s protein of the three sl-covs tested (fig. 2 , f to h), while an ek1-scrambled peptide showed no inhibitory activity on cell-cell fusion mediated by any of these cov s proteins (fig. 2, a to h) . the pseudovirus assay is a good model to mimic the process of virus entry into the target cell and has been widely used in previous studies to assess the inhibitory activity of antiviral agents against related cov infection (33) . we also used here pseudovirus assays to assess the inhibitory activity of ek1 against different pseudotyped hcovs. in a mers-cov pseudovirus infection assay, both ek1 and mers-hr2p showed inhibitory activity with ic 50 values of 0.26 and 1.06 m, respectively (fig. 2i) (fig. 2, n and o) . in contrast, ek1 exhibited no inhibitory activity against vsv-g-mediated viral infection (fig. 2p) . the ek1-scrambled peptide showed no inhibitory activity on infection by any of these pseudoviruses (fig. 2 , i to p). as previously reported (10), we failed to assemble a pseudotyped shc014 and thus did not perform pseudovirus infection assays on this virus. collectively, pseudovirus infection by all hcovs and sl-covs could be efficiently blocked by ek1, further indicating that ek1 has broad-spectrum antiviral activity against infection by pan-hcovs, including -hcovs, -hcovs, and sl-covs. blam-vpr assay is a sensitive model to characterize the inhibitory mechanism and activity of antiviral agents on virus-cell membrane fusion. this assay has been widely used for assessing the membrane fusion activity of various viruses, including hiv-1, ebola virus, and henipavirus (34) (35) (36) (37) . here, we developed the blam-vpr assays for mers-cov and sars-cov to further clarify the fusion inhibitory mechanism of ek1. we found that ek1 effectively inhibited the entry of mers-cov blam-vpr virions and sars-cov blam-vpr virions into huh-7 cells and ace2-293 t cells, respectively, in a dose-dependent manner ( fig. s2d ), thus confirming that ek1 does act as an hcov fusion inhibitor. subsequently, we further evaluated the inhibitory activity of ek1 against live hcov infection, including mers-cov, oc43, 229e, and nl63. we found that ek1 could effectively inhibit the infection and replication of these hcovs at cellular level in a dose-dependent manner ( the respiratory tract is the primary target tissue of hcov infection. to investigate the preclinical potential of the ek1 peptide, we administered it via an intranasal route and assessed its distribution within the respiratory tract. we observed the fluorescent signals of cy5-ek1 mainly in the upper and lower respiratory tract of mice (fig. 3 , a and b). compared with those from pbs-treated mice, the lungs obtained from cy5-ek1-treated mice exhibited significantly higher fluorescence signals (p < 0.0001, n = 3) (fig. 3 , c and d). together, these results suggest that ek1 (intranasal) can widely be distributed in the whole respiratory tract and be enriched in the lung. we also noted that cy5-ek1 administered intranasally could be detected in significant amounts in several extrapulmonary organs, including the liver, kidney, and spleen, in some animals, suggesting that ek1 could enter into the blood circulation and other organs (fig. s3, a and b). this observation suggests that intranasal administration of ek1 could also be beneficial for multiorgan infection or systemic infection of hcovs, both of which are common in mers-cov infection. next, we assessed the protective effect of ek1 in vivo on oc43 and mers-cov infection mouse models. in the oc43-infected mouse model, we treated newborn mice with ek1 at a dose of 5 mg/kg or with pbs 30 min before or after challenge with hcov-oc43 at 10 2 tcid 50 (50% tissue culture infectious dose). body weight of mice in the viral control group decreased, starting from 5 days postinfection (dpi), and mice succumbed to infection by 10 dpi with 100% mortality (fig. 3 , e to g). in contrast, the final survival rate of mice in the ek1 prophylactic and therapeutic groups was 100 and 66.7%, respectively (fig. 3e) , and their body weight either appeared normal (prophylactic) or rapidly recovered at 16 dpi (therapeutic) (fig. 3f ). meanwhile, we tested the viral titers in brains of mice of all groups at 5 dpi. infectious virus was readily detectable in the viral control group, whereas infectious virus titers were below the limit of detection (2 log tcid 50 /g) in the ek1 prophylactic mice or very low in the ek1 therapeutic mice (fig. 3g) . however, the infectious virus titers in the brains of mice that died during ek1 therapeutic treatment were as high as those in the brains of viral control mice without ek1 treatment, while the viral titers in the brains of survival mice with ek1 therapeutic treatment and in those of normal control mice were both undetectable ( fig. s2e ). consistently, the brains of mice that died during ek1 therapeutic treatment exhibited similar histopathological changes as those of the viral control mice, i.e., similar amount of vacuolation, degeneration, and infiltration ( fig. s2f ). in contrast, the brains of the survival mice with ek1 therapeutic treatment and those of the normal control mice showed no apparent histopathological changes ( fig. s2f ). to evaluate the prophylactic and therapeutic potentials of ek1 against mers-cov infection, we took advantage of the well-characterized transgenic (tg) mice globally expressing human dipeptidyl peptidase iv (dpp4) viral receptor. we treated mice with 200 g of ek1 or with pbs 30 min before or after challenge with mers-cov at 10 4 tcid 50 . likewise, the weight loss of tg mice treated with ek1 before or after mers-cov challenge was insignificant and rapidly recovered at 16 dpi, while untreated mice progressively lost significant weight before succumbing to infection within 9 dpi with 100% mortality (fig. 3 , h to j). in contrast, the survival rates in the ek1 prophylactic and therapeutic groups were 100 and 75%, respectively ( fig. 3 , h to j). when the yield of infectious viruses in lungs was used as the end point for assessing the efficacy of ek-1 on mers-cov infection, we were unable to recover any infectious virus from both ek1 prophylactic and therapeutic groups, in sharp contrast to the untreated controls (fig. 3j ). together, these results indicated that the peptide ek1 has broad and potent prophylactic and therapeutic efficacy against hcov infections. safety is obviously very important for the development of ek1 in clinical applications, and therefore, we first tested its in vitro cytotoxicity on various target cells. ek1 is not cytotoxic at concentrations up to 1 mm, which is more than 200 times higher than its ic 50 for inhibiting any hcov s-mediated cell-cell fusion and pseudovirus entry ( fig. s3c ). we then further investigated its cytotoxicity in vivo. we continuously administered mice with pbs, low-dose ek1 (20 mg/kg), or high-dose ek1 (100 mg/kg) by intranasal route every day for 1 week, and we recorded the body weight changes for the following 2 weeks ( fig. s3d ). the ek1-treated mice in both high-and low-dose groups lived normally, with no apparent difference in weight gain/loss observed as compared to pbs-treated mice. meanwhile, we used enzymelinked immunosorbent assay (elisa) to measure ek1-specific antibodies in sera of mice 2 weeks after intranasal administration. no ek1-specific antibodies were detected in either the ek1treated group or in the pbs-treated group ( fig. s3e ). on the basis of the distribution of ek1 by intranasal administration ( fig. s3, a and b) , we postulated that ek1 could penetrate the air-blood barrier to enter blood circulation and become enriched in some important organs, such as the liver and kidney. therefore, we further examined levels of alanine aminotransferase (alt; fig. s3f ) and creatinine ( fig. s3g ) in the sera of mice. alt and creatinine showed no significant difference (p > 0.05) at all time points between ek1-and pbs-treated groups, suggesting that nasal application of ek1 at high or low doses did not affect mouse hepatic and renal function. we further compared the potential histopathological changes of ek1-and pbs-treated groups 4 weeks after administration. the hematoxylin and eosin staining of lung, liver, kidney, and spleen sections from mice treated with ek1 at different doses exhibited no pathological abnormality, when compared to control mice treated with pbs ( fig. s3h ). none of these organs showed evidence of cell degeneration, necrosis, or infiltration of inflammatory factors. hence, ek1 appears to be generally safe via nasal application. to investigate the structural basis for the pan-cov inhibitory effect of ek1 peptide, we crystallized ek1 in complex with hr1 peptides from three representative hcovs, including the most pathogenic sars-cov and mers-cov in -hcovs and 229e in -hcovs, the hr1 of which is 14 amino acids longer than those of -hcovs. (table s3) . in all three structures, ek1 snugly fits into the hydrophobic groove formed between two adjacent hr1 helices in an oblique and antiparallel manner (fig. 4 , a to c), producing a 6-hb structure resembling the 3hr1-3hr2 postfusion state of corresponding hcovs ( fig. s4) . notably, the binding sites of ek1 coincide with those of native hr2s (fig. s4 ). these results indicate that the presence of ek1 would preclude binding of hr2 onto their corresponding 3hr1 core, thereby blocking formation of the 3hr1-3hr2 6-hb, which is an indispensable step during host-viral membrane fusion. hence, administration of the ek1 peptide would block cellular entry of those hcovs (fig. 2) . in all three structures, the ek1 peptide adopts a mixed secondary structure conformation (fig. 4 , a to c). the central region of ek1 folds into a five-turn helix, which packs against two neighboring hr1 helices via extensive hydrophobic interactions (3hr1 cores of different hcovs are illustrated as electrostatic surfaces, while residues on ek1 that are involved in hydrophobic packing are shown as stick models; residues l12, e15, m16, l19, a22, i23, l26, s29, and y30 on ek1 locate within the five-turn helix of ek1 and form strong hydrophobic interactions with the 3hr1 cores; fig. 5a ) and a few electrostatic or polar interactions (residues e13, e15, k18, e20, e27, e28, and y30 locate within the five-turn helix region of ek1 and interact with hr1 residues through side chain-to-side chain hydrophilic interactions; fig. 6a ). the rest of ek1 adopts an extended conformation, except that one extra turn is formed at the c-terminal end of ek1 (around e35) in the hr1(229e)-l6-ek1 structure (fig. 6 , right panel). in the extended region of ek1, polar interactions between side chain (hr1 residues) and main chain (ek1 residues) dominate (compare fig. 6, b and a) . most of these side chain-to-main chain polar interactions cluster at either end of the ek1 helical region, which interweave into extensive h-bond networks and likely help secure the ek1 helical region in the correct register (fig. 6b) . hydrophobic residues in the extended region of ek1 also insert their bulky side chains into hydrophobic pockets on the surface of the 3hr1 cores (residues l2, i5, v7, l10, i31, and l36; fig. 5a ), further strengthening the adhesion of the ek1 extended region onto the 3hr1 cores. both hydrophobic pockets and ridges exist on the surface of 3hr1 cores (fig. 5a) . correspondingly, we observed two kinds of hydrophobic interactions between the ek1 and 3hr1 cores. in particular, certain ek1 residues insert their hydrophobic side chains into pockets on the 3hr1 cores (fig. 5a , shown as orange stick models and hereafter named as "burying residues"), and other ek1 residues pack their side chains against hydrophobic ridges on the 3hr1 cores (fig. 5a , shown as yellow stick models and hereafter named as "ridge-packing residues"). note that the residues on hr1 that medi. s5 ). together, extensive and highly conserved hydrophobic and hydrophilic interactions between ek1 and 3hr1 cores endow ek1 with the ability to bind the 3hr1 cores from different hcovs and, hence, the capability of blocking the association of different hr2s onto their corresponding 3hr1 cores (fig. 2 ). the ek1 peptide outcompetes self-derived hr2 of mers-cov in both cell-cell fusion and pseudovirus infection assays. we therefore also compared the interactions between ek1 and mers-hr1 ] than did mers-hr2, which likely accounts for its better ic 50 in cell-cell fusion and pseudovirus infection assays (fig. 2) . similar to hr1 from -hcovs, sequence alignment across hcovs revealed that a 14-amino acid insertion also exists in the hr2 region of -hcovs (fig. 1c) . such an insertion renders the hr2 helices of -hcovs four turns longer than those of -hcovs. the helical region of ek1 is only five turns long, much shorter than the nine-turn helix in 229e-hr2 (38) . nevertheless, the burying residues in the extended region of ek1 and their equivalents in 229e-hr2 all neatly insert their side chains into corresponding . furthermore, a total of 12 polar and electrostatic interactions occur between ek1 and 229e-hr1, including five side chain-to-side chain and seven side chain-to-main chain interactions [fig. 6, a and b (right panel) ]. together, these extensive hydrophobic and hydrophilic interactions preserve the high affinity of ek1 toward long hr1 hcovs. that ek1 can form 6-hb structures with both short (sars-cov and mers-cov) and long (229e) hr1s highlights its broad structural compatibility in accommodating hr1s from different hcovs, thus consolidating its broad-spectrum inhibitory effect against pan-covs. no effective and broad-spectrum anti-hcovs drugs or vaccines are currently available in the clinic. notwithstanding, the rbd of the s protein has been proposed as a promising target for the develop-ment of specific antibodies and vaccines (39, 40) . for example, the rbd-specific antibody cdc2-c2 exhibits inhibitory activity against mers-cov infection. nevertheless, administration of the single-antibody cdc2-c2 could result in the emergence of escape mutations in mers-cov rbd (41) . unfortunately, the cov s rbd is hypervariable throughout evolution, which has led to marked difference in host receptor usage in different hcovs. even when the same host receptor is used by different hcovs, they frequently target different binding sites on the host receptor (42) . therefore, specific rbd-targeting antibodies or vaccines inevitably lack broad-spectrum activity against hcov infection. for example, menachery et al. (9) found that a sars-cov rbd-specific antibody could not protect mice from infection by the chimeric virus with the shc014 s proteins, although shc014 has high homology with sars-cov and can bind the same host receptor ace2. the lag time between emerging human cov outbreaks and development of new prophylactic treatments or vaccines is of concern. thus, there is an urgent need for the development of new, broad-spectrum drugs that target conserved sites in currently circulating and future emerging hcovs so as to prepare for future outbreaks of yet unknown hcovs. the 3hr1 cores are shown as electrostatic surfaces. at the positions where the hydrophobic surface on the 3hr1 core is deeply concave (pockets), ek1 residues that bury over 70% of their side-chain solvent accessible surface (sas) into these pockets are shown as orange stick models. at the locations where the hydrophobic surface on the 3hr1 core is relatively flat (ridges), ek1 residues that pack 50 to 70% of their side-chain sas against these ridges are shown as light yellow stick models. ek1 residues are labeled, where those labeled in red form both hydrophobic and hydrophilic interactions with 3hr1 cores. (b) hr1 residues involved in interactions with ek1 are conserved across different hcovs. ek1 and hr1 residues linked with dashed lines locate to the same layers on the 3hr1 triple helix. burying ek1 residues are shaded orange, and ridge-packing ek1 residues are shaded light yellow. hr1 residues that mediate assembly of the 3hr1 cores are shaded orange, while those involved in ridge packing are shaded yellow. hr1 residues that mediate conserved side chain-to-side chain and side chain-to-main chain hydrophilic interactions with ek1 residues are highlighted with cyan and red boxes, respectively. in the early 1990s, a series of peptides derived from the hiv-1 gp41 hr2 (or chr) domain, such as sj-2176 (19) , dp-178 (later t20) (20) , and c34 (21) , were reported to have highly potent inhibitory activity against hiv-1 gp41-mediated membrane fusion and hiv-1 infection with ic 50 values at low nanomolar levels. subsequently, numerous virus fusion inhibitory peptides overlapping the hr2 sequences of class i membrane fusion proteins from other enveloped viruses, including rsv (22) , ebola virus (23), paramyxoviruses sv5 (24), and nipah virus (25) , were also reported. in 2003, bosch et al. (26) discovered that a soluble hr2 peptide derived from the mhv s protein hr2 region inhibited virus cell entry, suggesting that the s glycoprotein of cov is a class i fusogen with the ability to form 6-hb during the s protein-mediated membrane fusion process. in 2004, the bosch (43) and jiang (18) groups independently reported that peptides derived from the hr2 region of sars-cov s protein could interact with the peptides from its hr1 region to form 6-hb and inhibited sars-cov s protein-mediated membrane fusion and sars-cov infection with moderate potency. in 2014, lu et al. (27) reported that peptide derived from the mers-cov s-hr2 could competitively inhibit 6-hb formation, thereby preventing fusion of the virus with host cells. in our previous studies, we found that the mers-cov-specific fusion inhibitor mers-hr2p could not block sars-cov pseudovirus infection and did not display any broad-spectrum inhibitory activity. thus, it was unclear whether 6-hb was a good target site for the development of a broad-spectrum anti-hcov inhibitor. to identify a pan-hcov fusion inhibition target site and preferably also to develop a potential pan-hcov inhibitor against infection of multiple hcovs in the human respiratory tract, we successfully established multiple hcov s-mediated cell-cell fusion assays to determine the cross-inhibitory spectrum between hr1ps and hr2ps, which are derived from the hr1 and hr2, respectively, of hcovs. unexpectedly, we found that oc43-hr2p harbored broad-spectrum binding activity to all hr1ps and manifested fusion inhibitory activity against all hcov s-mediated cell-cell fusion. we then further optimized the oc43-hr2p to improve its antiviral activity and solubility. the optimized peptide ek1 exhibited broad and potent fusion inhibitory activity against infection by hcovs and sl-covs on various in vitro models, i.e., cell-cell fusion assays and pseudotyped or live viral infection assays. we assessed the prophylactic and protective effects of ek1 through intranasal administration on oc43 and mers-cov mouse models. ek1 exhibited effective preventive and protective effects in these mouse models with an acceptable in vivo safety profile. furthermore, the in vivo pharmacokinetic profiling and safety studies helped us acquire useful data for potential human clinical trials in the future. the potency of hr2-derived peptides in inhibiting corresponding virus-cell fusion varies significantly. for example, anti-hiv-1 peptide t20 is about 900-fold more potent than the anti-sars-cov peptide sc-1, yet only 30-fold more potent than the anti-mers-cov peptide hr2p (18, 27, 44) . it is known that hiv-1 and sars-cov each enter their target cells mainly through plasma and endosomal membrane fusion, respectively, while mers-cov could infect its target cells via both plasma and endosomal membrane fusion (18, 27, 44) . the vast differences seen in potency thus suggest that only a limited number of the ek1 peptides can get into the endosome to inhibit virus-cell fusion. therefore, increasing the cell permeability of the fusion inhibitory peptides is expected to enhance the potency of these peptides. we have recently reported that addition of hydrocarbon stapling or a palmitic acid group (c16) to the mers-cov fusion inhibitory peptides significantly improves their antiviral potency and pharmacokinetic properties (45, 46) . in the near future, we will use the similar approaches to improving the antiviral activity of ek1. recent studies have reported that several emerging sl-covs, including wiv1, shc014, and rs3367, have the potential to infect humans with high virulence by further evolution or direct gene recombination with sars-cov (9, 11) . we found that the hr sequences of these sl-covs are the same as those of sars-cov, and we predicted that ek1 could prevent infection by these sl-covs. as (a) side chain-to-side chain hydrophilic interactions between ek1 and hr1. side chains of ek1 and hr1 residues involved in these interactions are shown as cyan and gray stick models, respectively, and are similarly color-coded. at least four pairs of this hydrophilic interaction are conserved across different ek1-hr1 complexes: y30 ek1 forms similar polar interactions with q1009 mers (left), q917 sars (middle), or t817 229e (right); e27 ek1 forms similar polar interactions with q1009 mers (left), q917 sars (middle), or s820 229e (right); e15 ek1 forms salt bridges with k1021 mers (left), k929 sars (middle), or k832 229e (right); and t8 ek1 makes similar interactions with n1028 mers (left), q936 sars (middle), or q839 229e (right). the conserved hr1 residues mentioned above are highlighted with cyan boxes. (b) main chain-to-side chain hydrophilic interactions between ek1 and hr1. ek1 and hr1 residues involved in these interactions are shown as green and gray stick models, respectively. the main-chain atoms of hr1 residues are not shown for clarity. similar to the side chain-to-side chain interactions, main chain-to-side chain interactions are also highly conserved across different ek1-hr1 complexes. the hr1 residues involved in conserved interactions are highlighted with red boxes. expected, ek1 efficiently inhibited sl-cov s protein-mediated cellcell fusion. consistent with previous studies (9, 11) , we herein also failed to establish a shc014 pseudovirus using an hiv-1 backbone vector; therefore, we assessed the antiviral effect of ek1 only on wiv1 and rs3367 pseudovirus infection and found that ek1 exhibited effective inhibitory activity against these two sl-covs. in addition, some animal covs within the cov family are very close to hcov, such as bovine coronavirus (bcov) and mhv. the hr sequences of these covs are very similar to those of oc43, indicating that peptide ek1 will likely also be effective on these viruses. one intriguing question is why only ek1 and the hr2 from oc43 manifested "pan-cov" inhibitory activity, while hr2 from other hcovs did not. hr2s from -hcovs are substantially longer than those from -hcovs (fig. 1c) , and docking of hr2s from 229e and nl63 onto the 3hr1 cores of -hcovs resulted in severe steric clashes (figure not shown). it is thus understandable why the hr2s from 229e and nl63 would not have broad-spectrum activity toward -hcovs (fig. 1d) . at the same time, sequence alignment between ek1 and hr2s from oc43, sars-cov, and mers-cov provided clues on the very limited inhibition breadth of sars-hr2 and mers-hr2. although most residues that mediated hydrophobic (shaded orange and yellow) and side chain-to-side chain hydrophilic interactions (boxed in cyan) are conserved between ek1 and different hr2s, sequence variability does exist at positions v7, y30, and some other ridge-packing residues ( fig. s7a ). we thus focused our analysis on these residues and their surrounding environments in different hcovs' 3hr1 cores. in both solved structures of mers(hr1)-ek1 and sars(hr1)-ek1, v7 ek1 fits its side chain neatly into the hydrophobic pockets on these two 3hr1 cores ( fig. s7b, second and fourth panels) . nevertheless, in the previously reported structure of the sars-cov fusion core, a1156 sars , the equivalent of v7 ek1 in sars-cov, would occupy the same cavity loosely ( fig. s7b, third panel) , while t1257 mers , the equivalent of v7 ek1 in mers-cov, could not fully bury its side chain owing to the polar nature of the thr residue ( fig. s7b, first panel) . thus, val, in ek1 and oc43-hr2, is more effective in fitting the hydrophobic cavities at this position than ala (seen in sars-hr2) or thr (seen in mers-hr2) ( fig. s7b ). in total, we observed four ridge-packing interactions between the ek1 and 3hr1 cores. the ridge to which s29y30 ek1 binds is surrounded by polar and positively charged residues in mers-cov and sars-cov but surrounded by polar and negatively charged residues in 229e [ fig. s7c (top panel) , side chains of surrounding residues are shown as stick models]. at this position, the polar pair "ser-tyr" in ek1 and oc43-hr2 appears to be more suitable than the "ser-leu" pair from sars-hr2 in accommodating the opposite electrostatic environments in different hcovs. moreover, the aromatic ring of tyr could also form additional polar interactions with hr1 side chains in different hcovs (fig. 6a) . likewise, the ridges to which e15m16 ek1 binds also exhibited better shape complementarity to met (in ek1 and oc43-hr2) than to ile (in sars-hr2). collectively, it seems that ek1 and oc43-hr2 have more optimal amino acid choices at all the abovementioned critical positions, which likely accounts for their unique pan-cov inhibitory activity. in its natural state, the s protein that is present on the cov surface is inactive. after receptor binding target cell proteases activate the s protein by cleaving the exposed enzyme target sites, leaving the s2 subunit free to mediate viral fusion and entry. cov can enter the target cell via two pathways: one is the endocytosis pathway and the other is direct fusion on the cellular surface. for example, when sars-cov enters the target cell through the endocytosis pathway, its s protein can be cleaved and activated by the ph-dependent cysteine protease cathepsin l in the endosome. on the other hand, recent studies have consistently reported that the sars-cov s protein can also be cleaved and activated by transmembrane protease serine 2 (tmprss2) and human airway trypsin-like protease, which are located on the cell surface, thus activating and allowing the s protein to mediate sars-cov infection at the plasma membrane (9) (10) (11) . in accord with such a finding, matsuyama et al. (47) reported that the hr2 peptide efficiently inhibited sars-cov entry into cells, while lysosome-tropic reagents failed to inhibit at all. similarly, tmprss2 also has the capacity to promote the entry of mers-cov through bypassing the endocytosis pathway and directly activating its s protein on the cellular surface (48) . recently, several studies have reported that tmprss2 is highly expressed on human respiratory epithelial cells surface and was even associated with several cov receptors, such as ace2 and dpp4 (48) (49) (50) . hence, the plasma membrane fusion pathway seems a preferred choice for hcov infection in the human respiratory tract. consistently, our previous study found that mers-cov-specific fusion inhibitor hr2pm2 effectively inhibited mers-cov infection in vivo by intranasal administration (33) . other studies have also reported that the current clinical isolates of 229e and oc43 are very sensitive to cell surface tmrrss2 but not to endosomal cathepsins (51, 52) . overall, for current circulating hcovs or emerging hcovs, the cell surface fusion pathway in human respiratory tract would appear to be very important. therefore, peptide fusion inhibitors and the strategy of intranasal administration are excellent choices for preventing hcov infection via the airway, which is a key site for hcov to rapidly establish infection and widely spread to other organs. currently, circulating hcovs pose a potential threat to humans; moreover, it is almost certain that other zoonotic covs will be transmitted to humans in the future. the availability of hcov-specific drugs with broad-spectrum inhibitory activity is therefore important for the prevention and control of a future hcov epidemic. the ek1 fusion inhibitor peptide targeting the conserved site in the spike hr1, but not the hypervariable rbd region, has potent and broad inhibitory activity against multiple hcov infections. ek1 through nasal administration exhibited effective and broadly anti-hcov activity with a satisfactory safety profile in vivo. hence, ek1 is a promising candidate for further development as an antiviral agent against infection of multiple hcovs, especially for use in infants and the elderly, as well as immunocompromised patients, who would be more vulnerable to hcov infections (53) (54) (55) (56) . meanwhile, this study provides clues and methods for the development of peptide fusion inhibitors with potency and breadth in inhibiting infections by other highly pathogenic enveloped viruses with class i membrane fusion proteins, such as ebola and marburg viruses, hendra and nipah viruses, and influenza viruses. were preserved in our laboratory. the dna sequence of the s protein of oc43 with a deletion of 17 amino acids in the c terminus was synthesized. dna encoding 229e-s or nl63-s-18 (containing a c-terminal 18-amino acid deletion) was provided by f. li. all peptides were synthesized by karebay biochem with a purity of >95% (tested by high-performance liquid chromatography). the ek1-scrambled sequence was "lkvllyeefklleslimeileyqkdsdikenaedtk." the coding sequence of ek1 peptide was individually fused to the 3′ end of the hr1 domain from sars-cov, mers-cov, and hcov-229e (residues 892 to 970, 984 to 1062, and 785 to 873, respectively) through a six-amino acid linker (l6: sggrgg). the resulting sequences encoding different hr1-l6-ek1 fusion proteins were then subcloned into a modified pet-28a vector, which contains a his 6 -sumo tag upstream of the multiple cloning site. the resulting constructs, pet-28a-his 6 -sumo-hr1-l6-ek1, were then expressed in escherichia coli bl21 (de3) at 16°c overnight in lb medium. we initially purified these fusion proteins using his-talon resin (clontech). eluted fractions from the his-talon column were then mixed with ulp1 enzyme [1:100 (w/w)] and dialyzed against buffer a [20 mm tris-hcl (ph 8.0), 150 mm nacl, and 1 mm dithiothreitol] at 4°c overnight. after sumo tag cleavage, the samples were reloaded onto the his-talon column, and flow-through fractions containing untagged hr1-l6-ek1 were pooled, concentrated, and gel-filtered in buffer b [20 mm tris (ph 8.0) and 150 mm nacl] on a hiload 16/60 superdex 75 (ge healthcare) column. peak fractions that contain hr1-l6-ek1 trimer were pooled and concentrated to 10 mg/ml through centrifugation (emd millipore). and 30% peg550mme, respectively. diffraction data were collected at beamline bl19u1 of shanghai synchrotron radiation facility (ssrf), china, and processed with the hkl3000 program (57) . a summary of the data collection statistics is provided in table s3. the structure of hr1(sars)-l6-ek1 was solved by molecular replacement, as implemented in the phaser program of phenix (58) . the programs used a sars-cov fusion core structure [protein data bank (pdb): 2bez] as the search model. for structure determination of hr1 (mers)-l6-ek1 and hr1(229e)-l6-ek1, the mers-cov fusion core [pdb: 4mod] and sars-cov fusion core [pdb: 2bez] were used as search models. the structural models were further improved by cycles of manual building and refinement using the coot (59) and phenix (58) programs. the quality of these models were analyzed with molprobity (60) . a summary of the structure refinement statistics is also given in table s3. the figures were all prepared using the pymol program (the pymol molecular graphics system, version 2.1, schrödinger llc). the electrostatic calculations were performed with pdb2pqr (61). models for hr1(oc43)-l6-ek1, hr1(hku1)-l6-ek1, and hr1(nl63)-l6-ek1 were derived by homology modeling using swiss model website (62) . the template for hr1(oc43)-l6-ek1 and hr1(hku1)-l6-ek1 were obtained from the crystal structures of hr1(sars)-l6-ek1 and hr1(mers)-l6-ek1, and the template for hr1(nl63)-l6-ek1 proteins was obtained from the crystal structure of hr1(229e)-l6-ek1. to relax and stabilize the interaction between ek1 and corresponding hr1, initial models were optimized by performing energy minimization, followed by a 5-ns molecular dynamics simulation using schrödinger suite 2017-4 (www.schrodinger.com). the simulation systems were solvated with full-atom tip3p water, containing cl − and na + ions at a concentration of 0.15 m to mimic physiological ionic strength. during the simulation, temperature t and pressure p were kept constant, at 310 k and 1 atm, respectively. circular dichroism spectra (195 to 260 nm) were collected on a j-815 spectropolarimeter (jasco inc., japan) to evaluate the secondary structure of the peptides and their complexes (63, 64) , individual peptides or complexes dissolved in pbs with the final concentration at 10 m. thermal denaturation of peptide complexes was monitored from 20° to 100°c at 222 nm with a thermal gradient of 5°c/min. the midpoint of the t m values was acquired by jasco software utilities. a pseudovirus bearing cov s protein or vsv-g protein and a defective hiv-1 genome that expresses luciferase as reporter was produced in 293 t cells, as previously described (27) , and its titer was quantitated by using hiv-1 p24 elisa. the pseudovirus was then used to infect target huh-7 cells (or ace2/293 t cells for pseudotyped sars-cov) (10 4 per well in 96-well plates) in the presence or absence of the test peptide at the indicated concentration. twelve hours after infection, culture medium was refreshed and then incubated for an additional 48 hours, followed by washing cells with pbs, lysing cells with lysis reagent (promega), and transferring the cell lysates to 96-well costar flat-bottom luminometer plates (corning costar) for the detection of relative light units using the firefly luciferase assay kit (promega) and an ultra 384 luminometer (tecan). as previously described (27) , 293 t effector cells were transfected with plasmid paav-ires-egfp encoding the egfp (293 t/egfp cells) or plasmid paav-ires-s-egfp encoding the corresponding hcov s protein (293 t/hcov s/gfp cells) as the effector cells. huh-7 cells, expressing various hcov receptors on the membrane surface, were used as target cells, as described below. the inhibitory activity of a test peptide on hcov s-mediated cellcell fusion was determined, as previously described (27) . briefly, effector cells (293 t/s/gfp) and target cells (huh-7 cells) were cocultured in the presence or absence of a test peptide at the indicated concentrations for fusion. after counting the fused and unfused cells, the percentage of cell-cell fusion was calculated, as described above. the percent inhibition of cell-cell fusion was calculated using the following formula as described elsewhere (27): . "e" represents the percentage of cell-cell fusion in the experimental group. "p" represents the percentage of cell-cell fusion in the positive control group, where 293 t/hcov s/egfp cells were used as effector cells, to which only pbs was added. "n" is the percentage of cell-cell fusion in negative control group, where 293 t/egfp cells were used as effector cells. the ic 50 was calculated using the calcusyn software provided by t. c. chou (65) . samples were tested in triplicate, and all those experiments were repeated twice. an hcov virion-based fusion assay was performed, as described elsewhere (34, 35) . briefly, huh-7 cells and ace2-293 t cells were used as target cells for the entry of mers-cov blam-vpr virions and sars-cov blam-vpr virions, respectively. these target cells were cultured at 37°c for 5 hours in six-well plates (with virions containing blam-vpr equivalent to 80 ng of p24-gag per well) in the presence or absence of ek1 at the indicated concentrations. the cells were washed with pbs, resuspended in 500 l of dmem, and then incubated with ccf4-am substrate at room temperature for 2 hours, as described by the manufacturer (invitrogen, germany). last, the cells were monitored via flow cytometry. after the virion fusion with the target cell, the ccf4-am (emission at 520 nm) substrate could be cleaved by blam-vpr into ccf4 (emission at 447 nm). flow cytometric data were collected with a bd facsdiva and analyzed with flowjo. the inhibitory activity of peptides against oc43 replication in hct-8 cells was assessed, as described elsewhere (66) . briefly, 100 tcid 50 of oc43 was mixed with a test peptide at graded concentration and incubated at 37°c for 30 min. the mixture was then applied in triplicate onto the monolayer of hct-8 cells grown in a 96-well microtiter plate. on day 5 after infection, viral titer in the culture medium was tested, and tcid 50 was calculated on the basis of the cytopathic effect (cpe) (67) . the inhibitory activity of the tested peptide against 229e replication in a549 cells and nl63 replication in llc-mk2 cells was evaluated in a similar way, as described above. the inhibitory activity of peptide against mers-cov replication was tested in calu-3 cells using a modified standard microneutralization assay, as previously described (68) . briefly, 60 l of a serially twofold diluted peptide was incubated with 60 l (120 tcid 50 ) of mers-cov in mem medium supplemented with 2% fbs (m-2 medium) in duplicate wells of 96-well plates for ~60 min at room temperature. one hundred microliters of the peptide/mers-cov mixtures was then transferred into confluent calu-3 cells grown in 96-well plates. wells of calu-3 cells cultured with m-2 medium with and without virus were included in these assays as positive and negative controls, respectively. while the advanced cpe of calu-3 cells could develop within 24 to 36 hours in response to mers-cov infection at higher multiplicities of infection (mois) of 1 or 0.1 (69), we did not observe any prominent formation of cpe until ~ 60 to 72 hours after infection at an estimated moi of ~ 0.001. hence, to more accurately measure the efficacy of peptide inhibitors against mers-cov infection, we harvested the supernatants at 72 hours and quantified infectious virus titers by the standard vero e6-based infectivity assays and expressed the titers as log 10 tcid 50 /ml. pregnant balb/c mice (18 days) purchased from the department of laboratory animal science, fudan university were separated into four groups after delivery of their offspring. eleven newborn mice were chosen for each group. mice in the prevention and treatment groups were intranasally administered peptide (5 mg/kg in 2 l of pbs) 30 min before or after intranasal challenge with a viral dose of 10 2 tcid 50 (in 2 l dmem). mice in the viral control group and the normal control group were intranasally administered with 2 l of pbs 30 min before viral challenge or without viral challenge. mouse survival rate and body weight variations were recorded up to 2 weeks after infection (70) . on day 5 after infection, five mice in each group were randomly selected for euthanasia to collect and assess the viral titer in mouse brain. briefly, mice expressing human dpp4 were randomly separated into three groups (n = 6): viral control, prophylactic group, and therapeutic group. in the viral control and therapeutic groups, each mouse was challenged intranasally with 10 4 tcid 50 [i.e., 1000 ld 50 (median lethal dose)] of mers-cov in a volume of 60 l, 30 min after viral challenge, and then treated via the intranasal route with 200 g of ek1 (in 50 l of pbs) or 50 l of pbs, respectively. prophylactic group mice were pretreated via the intranasal route with 200 g of ek1 per mouse 30 min before viral challenge. challenged mice were monitored daily for weight loss and mortality. in the interim, two mice were randomly euthanized (two of six) in each group at day 2 after challenge to determine the lung virus titers by cpe on vero e6 and expressed as tcid 50 /ml. six balb/c female mice (8 weeks old) were randomly assigned to two groups and nasally administered with 20 g of cy5-ek1 in 50 l of pbs (n = 3) or 50 l of pbs without peptide (n = 3) as control for background fluorescence measurement. one hour later, mice were imaged for distribution of cy5-ek1 by the ivis lumina k series iii in vivo imaging system (perkinelmer, waltham, ma, usa). mice were euthanized by intraperitoneal injection of sodium pentobarbital to obtain the lungs, livers, kidneys, spleens, and hearts for imaging and measuring their fluorescence value. the relevant radiant efficiency (p s −1 cm −2 sr −1 ) (w −1 cm 2 ) was then calculated by living image 4.4 software (56, 71, 72) . cytotoxicity of the peptides to the cells (293 t, 293 t/ace2, huh-7, a549, llc-mk2, and calu-3 cells) was tested by using the cell counting kit-8 (cck-8; dojindo, kumamoto, japan). briefly, each cell type was seeded into the wells of a 96-well microtiter plate (10 4 per well) and incubated at 37°c for 12 hours, replacing medium with dmed containing ek1 at graded concentrations. after incubation at 37°c for 2 days, cck-8 solution (10 l per well) was added, followed by an additional incubation for 4 hours. the absorbance was measured at 450 nm. in vivo safety of ek1 through intranasal administration as previously described (27) , female balb/c mice (8 weeks old) were assigned randomly to three groups (n = 5) and continuously administered with pbs, low-dose ek1 (20 mg/kg), or high-dose ek1 (100 mg/kg) by intranasal route every day for 1 week. the body weight changes were monitored for the following 2 weeks. alt and creatinine in the sera of each group of mice were measured using the alt and creatinine assay kits (njjcbio, nanjing, china) before the first treatment and 1, 3, and 5 days after the final treatment. the titer of immunoglobulin g sera to ek1 in each group of mice was evaluated by elisa 2 weeks after the final treatment. four weeks after administration, mice in each group mice were euthanized to harvest the lungs, livers, kidneys, and spleens for hematoxylin and eosin staining. the animal studies were approved by the institutional laboratory animal care and use committee at fudan university (20180302-019). analyses of independent data were performed by student's unpaired two-tailed t test. statistical analyses were carried out using graphpad prism 6.0. p values less than 0.05 were considered significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. the ic 50 was calculated using the calcusyn software (65) . supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/5/4/eaav4580/dc1 fig. s1 . interaction between hcov hr1 and hr2 regions in hcov s proteins. fig. s2 . identity and similarity in spike protein sequences of sars-cov and sl-covs and the effect of ek1 on hcov s protein-mediated cell fusion and virus-cell fusion, as well as the viral loads and histopathological changes in brains of hcov-infected mice. fig. s3 . in vivo safety of ek1 through intranasal administration. fig. s4 . structural comparison between the hr1-ek1 6-hb bundles and cognate hr1-hr2 6-hb bundles reveals that the ek1 peptide binds to the triple-helix hr1 core of different hcovs in a similar manner to that of the native hr2 of the corresponding hcov. fig. s5 . the ek1 peptide forms 6-helical bundle structures with the hr1 motifs from oc43, hku1, and nl63. fig. s6 . interactions between the hr1 and hr2 motifs of mers and 229e. fig. s7 . key residues at critical positions endow ek1 and oc43-hr2 with pan-cov activity. table s1 . inhibitory activity of peptides on multiple cell-cell fusion assays. table s2 . solubility and fusion inhibitory activities of different peptides. table s3 . data collection and structural refinement statistics. discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus epidemiology, genetic recombination, and pathogenesis of coronaviruses acute life threatening event (alte) in an infant with human coronavirus hcov-229e infection human coronavirus oc43 associated with fatal encephalitis fatal outcome of human coronavirus nl63 infection despite successful viral elimination by ifn-alpha in a patient with newly diagnosed all molecular epidemiology and evolutionary histories of human coronavirus oc43 and hku1 among patients with upper respiratory tract infections in kuala lumpur identification and evolutionary dynamics of two novel human coronavirus oc43 genotypes associated with acute respiratory infections: phylogenetic, spatiotemporal and transmission network analyses molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination a sars-like cluster of circulating bat coronaviruses shows potential for human emergence isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor sars-like wiv1-cov poised for human emergence two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines effects of human anti-spike protein receptor binding domain antibodies on severe acute respiratory syndrome coronavirus neutralization escape and fitness interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors hiv-1 inhibition by a peptide peptides corresponding to a predictive alpha-helical domain of human-immunodeficiency-virus type 1 gp41 are potent inhibitors of virus-infection a trimeric structural subdomain of the hiv-1 transmembrane glycoprotein heptad-repeat regions of respiratory syncytial virus f1 protein form a six-membered coiled-coil complex functional importance of the coiled-coil of the ebola virus glycoprotein membrane fusion machines of paramyxoviruses: capture of intermediates of fusion inhibition of henipavirus fusion and infection by heptad-derived peptides of the nipah virus fusion glycoprotein the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor influence of hydrophobic and electrostatic residues on sars-coronavirus s2 protein stability: insights into mechanisms of general viral fusion and inhibitor design core structure of s2 from the human coronavirus nl63 spike glycoprotein remodeling of gp41-c34 peptide leads to highly effective inhibitors of the fusion of hiv-1 with target cells helix stabilization by glu-…lys+ salt bridges in short peptides of de novo design structure of sars coronavirus spike receptor-binding domain complexed with receptor protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection a sensitive and specific enzyme-based assay detecting hiv-1 virion fusion in primary t lymphocytes hiv-1 virion fusion assay: uncoating not required and no effect of nef on fusion studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha a catalytically and genetically optimized -lactamase-matrix based assay for sensitive, specific, and higher throughput analysis of native henipavirus entry characteristics crystal structure of the post-fusion core of the human coronavirus 229e spike protein at 1.86 å resolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies recombinant receptor-binding domains of multiple middle east respiratory syndrome coronaviruses (mers-covs) induce cross-neutralizing antibodies against divergent human and camel mers-covs and antibody escape mutants importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on mers-cov spike glycoprotein to avoid neutralization escape receptor recognition mechanisms of coronaviruses: a decade of structural studies severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides hiv gp41 c-terminal heptad repeat contains multifunctional domains. relation to mechanisms of action of anti-hiv peptides de novo design of -helical lipopeptides targeting viral fusion proteins: a promising strategy for relatively broad-spectrum antiviral drug discovery discovery of hydrocarbon-stapled short -helical peptides as promising middle east respiratory syndrome coronavirus (mers-cov) fusion inhibitors efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss2 the tetraspanin cd9 facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease type ii transmembrane serine proteases in cancer and viral infections clinical isolates of human coronavirus 229e bypass the endosome for cell entry wild-type human coronaviruses prefer cell-surface tmprss2 to endosomal cathepsins for cell entry human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease severity and outcome associated with human coronavirus oc43 infections among children a peptide-based viral inactivator inhibits zika virus infection in pregnant mice and fetuses processing of x-ray diffraction data collected in oscillation mode phenix: a comprehensive python-based system for macromolecular structure solution coot: model-building tools for molecular graphics molprobity: all-atom structure validation for macromolecular crystallography pdb2pqr: an automated pipeline for the setup of poisson-boltzmann electrostatics calculations swiss-model: modelling protein tertiary and quaternary structure using evolutionary information determination of the helix and beta form of proteins in aqueous solution by circular dichroism determination of the hiv-1 gp41 fusogenic core conformation modeled by synthetic peptides: applicable for identification of hiv-1 fusion inhibitors theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies novel treatment with neuroprotective and antiviral properties against a neuroinvasive human respiratory virus biopolymeric nano/ microspheres for selective and reversible adsorption of coronaviruses blocking of exchange proteins directly activated by camp leads to reduced replication of middle east respiratory syndrome coronavirus bilateral entry and release of middle east respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells vacuolating encephalitis in mice infected by human coronavirus oc43 pharmacokinetics on a microscale: visualizing cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells mr molecular imaging of prostate cancer with a peptide-targeted contrast agent in a mouse orthotopic prostate cancer model designed the study. s.x. established the cell-cell fusion assays, performed the inhibition of hcov-oc43 infection in vivo and in vitro assays and ek1 safety study, and conducted circular dichroism spectroscopy and cytotoxicity assay. b.y. and l.y. performed the expression and purification of fusion protein hr1-l6-ek1 and structural study are inventors on a patent application related to this work assigned to fudan university (application no. cn201610070216.4, filed on a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike we thank f. li at the university of minnesota medical school for providing the hcov-229e and hcov-nl63 spike gene. we thank the staff from bl19u1 beamline of the national facility for protein science shanghai (nfps) at the shanghai synchrotron radiation facility for assistance during data collection. funding: this work was supported by the national megaprojects of china for major infectious diseases (2018zx10301403 to l.l.), the ministry of science and technology of the people's republic of china (2016yfc1201000 and 2016yfc1200405 to s.j. and 2016yfc1202901 to l.l.), the national natural science foundation of china (81661128041, 81672019, and 81822045 to l.l. and 31600619 to b.y.), the shanghai rising-star program (16qa1400300 to l.l.), and key: cord-339386-sxyeuiw1 authors: mcintosh, kenneth; perlman, stanley title: 157 coronaviruses, including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) date: 2015-12-31 journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: 10.1016/b978-1-4557-4801-3.00157-0 sha: doc_id: 339386 cord_uid: sxyeuiw1 nan the family coronaviridae, within the order nidovirales, contains two subfamilies, the coronavirinae and the torovirinae. coronaviruses (covs) are a large group of viruses infecting mammals and birds and producing a wide variety of diseases. they have been divided into four genera, two of which contain viruses infecting humans (see later). all human coronaviruses (hcovs) are primarily respiratory pathogens. during the winter of 2002 to 2003, an alarming new disease appeared, severe acute respiratory syndrome (sars), which was quickly attributed to a new cov, the sars-cov. the outbreak originated in southern people's republic of china, with evidence that the virus was first derived from bats and was transmitted to man through an intermediate host, probably the palm civet (paguma larvata) or raccoon dog (nyctereutes procyonoides). [1] [2] [3] the sars epidemic was controlled through a massive effort at case identification and containment, and the last known case occurred in mid-2004. in retrospect, the emergence of sars is consistent with what is known about covs as a group: they are important pathogens in animals causing a wide variety of diseases through a wide variety of pathogenic mechanisms, and they have been noted to mutate frequently and infect new species. 4, 5 more recently, a related but different cov producing severe respiratory disease has emerged, the middle east respiratory syndrome coronavirus (mers-cov). mers-cov was grown in june 2012, from a sputum sample obtained from a man in saudi arabia who died of overwhelming pneumonia. the virus was quickly identified as a new cov most closely related to several bat covs. 6 this report was followed by a number of other reports identifying a total of 537 infected individuals, all of whom had acute respiratory symptoms, severe in most, and fatal in 145 (as of may 11, 2014) . 7 human-to-human transmission has been documented, especially in hospital settings, 7a but appears to be inefficient. in 1965, tyrrell and bynoe 8 cultured a virus obtained from the respiratory tract of a boy with a common cold by passage in human embryonic tracheal organ cultures. the media from these cultures consistently produced colds in volunteers. the agent was ether sensitive but not related to any known human virus. subsequently, electron microscopy of fluids from infected organ cultures revealed particles that resembled infectious bronchitis virus of chickens. 9 at about the same time, hamre and procknow 10 recovered a cytopathic agent in tissue culture from medical students with colds. the prototype virus was named 229e and was found on electron microscopy to have a similar or identical morphology ( fig. 157-1) . 9 using techniques similar to those used by tyrrell and bynoe, mcintosh and colleagues 11 reported the recovery of several infectious bronchitis-like agents from the human respiratory tract, the prototype of which was named oc43 (oc for organ culture). at much the same time, mouse hepatitis virus and transmissible gastroenteritis virus of swine were shown to have the same morphology on electron microscopy. 12, 13 shortly thereafter, the name coronavirus (the prefix corona denoting the crownlike appearance of the surface projections) was chosen to signify this new genus. 14 the number of animal covs quickly grew, including viruses causing diseases in rats, mice, chickens, turkeys, various other bird species, cattle, several wild ruminants, beluga whales, dogs, cats, rabbits, and pigs, with manifestations in the respiratory and gastrointestinal tracts, central nervous system (cns), liver, reproductive tract, and others. through sequencing and antigenicity studies, the animal and human covs (hcovs) initially were divided into three groups: group 1, which • covs are members of the nidovirales order, single-stranded, positive-sense rna viruses with a large genome. they mutate and also recombine frequently. • laboratory diagnosis is best accomplished by finding viral rna through polymerase chain reaction. • there are no accepted effective antiviral drugs for covs. • prevention is through epidemiologic methods. the sars epidemic was halted through careful case finding, quarantine, and use of barrier precautions. 15 a cov was independently and almost simultaneously isolated from sars patients by several laboratories and found by sequencing to be only distantly related to previously characterized covs. [16] [17] [18] [19] [20] the sars outbreak stimulated a rapid and intense public health response coordinated by the world health organization, and by july 2003, transmission had ceased throughout the world. despite this effort, however, 8096 probable cases had occurred in 29 countries, with 774 deaths. 15 with the identification of the sars-cov, the hcov field became much more active. sensitive molecular methods were developed to detect rna from viruses identical or closely related to hcov-229e and hcov-oc43 in the respiratory tract, and two new species were discovered: nl63, an alphacoronavirus, and hku1, a betacoronavirus. [21] [22] [23] hcov-nl63 was found independently by three groups, two in the netherlands and, somewhat later, the third in new haven, figure 157-2 phylogenetic relationships among members of the subfamily coronavirinae. a rooted neighbor-joining tree was generated from amino-acid sequence alignments of coronaviridae-wide conserved domains in replicase polyprotein 1 (adrp, nsp3; mpro, nsp5; rdrp, nsp12; hel, nsp13; exon, nsp14; nendou, nsp15; o-mt, nsp16) for 21 coronaviruses, each a representative of a currently recognized coronavirus species. five of the six known human coronaviruses (hcov-229e, hcov-nl63, hcov-hku1, sars-cov, and mers-cov) are indicated. hcov-oc43 is closely related to bovine coronavirus, which is shown in the figure. equine torovirus berne served as the outgroup. virus names are given with strain specifications; species and genus names are in italics as per convention. the tree shows the four main monophyletic clusters, corresponding to genera alpha-, beta-, gamma to be established numerous reports of cov-like particles (covlps) found by electron microscopy in human fecal matter, but these particles have been difficult to characterize further. more recent efforts to detect cov rna in feces using polymerase chain reaction (pcr) and primers for respiratory hcovs have had limited success and have failed to associate covs with gastrointestinal disease. 28, 29 toroviruses were, like covs, first described in animals. they were first detected in the feces of cattle (breda virus) and horses (berne virus). 30, 31 shortly thereafter, beards and colleagues 32 examined human fecal material and reported finding particles with a similar appearance that aggregated in the presence of antiserum to the bovine and equine viruses. the human toroviruses, like the bovine toroviruses, do not grow in tissue culture, and thus almost all existing information about them is based on electron micrographic data. unlike animal toroviruses, 33,34 pcr-amplified torovirus rna sequences have not been found in human stool samples. a report of genome sequences amplified from particles purified from human stool 35 was subsequently retracted and considered to reflect laboratory contamination from porcine strains. 36 at this time, there are no reports definitively showing the existence of human toroviruses. the cov nucleic acid is rna, approximately 30 kilobases in length, of positive sense, single stranded, polyadenylated, and infectious. the rna, the largest known viral rna ( fig. 157-3) , codes for (in order from the 5′ end) a large polyprotein that is cleaved by virus-encoded connecticut. 24 in all three cases, positive samples were from infants and children with respiratory disease. notably, hcov-nl63 and hcov-229e were estimated to originate from a common precursor and diverge approximately 1000 years ago. 25 hcov-hku1 was found in hong kong in an adult with respiratory disease. these two new hcov strains subsequently have been found worldwide and appear to have pathogenicity similar to that of hcov-229e and hcov-oc43, with the possible exception that nl63 is more frequently found in children with croup. 26, 27 the mers-cov was found when a man was admitted in june 2012 to a hospital in jeddah, saudi arabia with overwhelming acute pneumonia with renal failure, and a sample of sputum grew a cytopathic virus that, on sequencing, proved to be a cov, classified as a betacoronavirus, and most closely related to two bat covs, hku-4 and hku-5. 6 over the next 23 months 536 additional cases (145 fatal) were found, all but a few of them sporadic or hospital-based and in individuals living or traveling in the middle east. in the remainder of this chapter, the group of respiratory hcovs first discovered in the 1960s and containing hcovs 229e, oc43, nl63, and hku-1 are referred to as community-acquired hcovs to distinguish them from the sars-cov and the mers-cov. in view of the prominence of covs in animal enteric diseases, there have been extensive efforts to identify enteric hcovs. there are figure 157-3 genome organization of representative human coronaviruses. all coronavirus genomes have the same basic structure and mechanism of replication. 4 the 5′ end of each genome encodes a leader sequence, which is attached to each virus-specific messenger rna transcript by a novel mechanism of discontinuous replication. the first two thirds of each genome encode replicase-associated genes. gene 1 is translated as two large polyproteins, with the first expressed from orf1a and the second from orf1a/b following a −1 frameshift event. these polyproteins are then cleaved into individual proteins by two virus-encoded proteases. the major structural genes, the hemagglutinin-esterase (he), surface (s), envelope (e), transmembrane (m), and nucleocapsid (n) proteins, are indicated in green. the nonreplicase, accessory genes located at the 3′ end of the genome are indicated with open boxes. the functions of these proteins are largely not known, and there is no sequence homology between accessory proteins of different coronaviruses. some of these proteins are virion associated, but none are required for virus replication. the open reading frames (orfs) encoding these proteins are numbered in order of appearance from the 5′ end of the genome, with the exception that ns12.9 of hcov-oc43. i is an internal protein expressed from an alternative reading frame located within the n gene. it is equivalent to sars-cov-specific protein 9b and the mers-cov-specific protein 8b. (figure prepared by 4 (dpp-4), which, like ace2 and apn, is an ectopeptidase that is abundantly expressed in the respiratory tract. 46 all the community-acquired respiratory hcovs grow only with difficulty in tissue culture. despite this, both 229e and nl63 were discovered because they produced a detectable cytopathic effect, the first in human embryonic kidney 10 and the second in llc-mk2 cells. 21 both the sars-cov and the mers-cov were initially isolated and grew readily in vero cells. 6, 17 hcovs oc43 and hku-1 have been grown in tissue culture after laboratory adaptation or in primary human airway epithelial cells. [47] [48] [49] detection of all these viruses in clinical specimens is most conveniently and sensitively achieved using pcr. likewise, the enteric covs have been difficult to cultivate in vitro. all but a few strains have been detected only by electron microscopy of human fecal material. [50] [51] [52] 53, 54 some strains have been characterized by immune electron microscopy and found to be related to hcov-oc43. 55 two strains obtained from an outbreak of necrotizing enterocolitis in texas and passaged in intestinal organ cultures were reported to contain four or five proteins with apparent molecular weights similar to those of other covs but not related antigenically to known human strains or mouse hepatitis virus a59. 56 the evidence favors the view that these isolates, as well as particles antigenically related to hcov-oc43, are members of the family coronaviridae, although their association with human disease is not yet proven. surveys of children with diarrhea using pcr imply that diarrhea associated with the four well-described hcov strains is unusual. 28, 29 epidemiology evidence of community-acquired respiratory cov infections has been found wherever in the world it has been sought. in temperate climates, respiratory cov infections occur more often in the winter and spring than in the summer and fall. the contribution of cov infections to the total number of upper respiratory illnesses may be as high as 35% during times of peak viral activity. overall, the proportion of adult colds produced by covs may be reasonably estimated at 15%. early studies of hcov-oc43 and 229e in the united states demonstrated periodicity, with large epidemics occurring at 2-to 3-year intervals. 57 strain hcov-229e tended to be epidemic throughout the united states, whereas strain hcov-oc43 appeared in localized outbreaks. similar studies of nl63 and hku1 have not been done, but it seems from the available data that they also vary widely in incidence from year to year and place to place. reinfection is common and may be due to the rapid diminution of antibody levels after infection. 58 infection occurs at all ages but is most common in children. the ratio of symptomatic to total infections varies between 50% and 90%, depending on the age of the population studied, the method of virus detection, and the definition of "infection. " 59, 60 among adult volunteers 72% of those infected with hcov-229e developed colds. 61 the first report of a new cov causing severe pneumonia appeared in promed mail on september 20, 2012. a man from jeddah, saudi arabia had developed pneumonia in june and died of respiratory and renal failure, and a virus was grown from a sputum sample that was subsequently sequenced and found to be a betacoronavirus thought at the time to be most closely related to bat covs hku4 and hku5. 6 between then and may 2014, a total of 537 cases occurred, all infected by this virus, now termed the middle east respiratory syndrome coronavirus (mers-cov). one hundred forty-five of the cases were fatal. 7 more than 400 of these have been acquired and diagnosed in the kingdom of saudi arabia, with most of the remainder in the united arab emirates, qatar, jordan, and kuwait. cases originating in the arabian peninsula have also occurred in travelers to egypt, tunisia, germany, italy, great britain, malaysia, the philippines, and the united states, with a few secondary cases sometimes occurring in those locations through close family or hospital spread. in the united states,these include two unrelated mers cases, and a third case related to one of these. 61a while infections with severe respiratory involvement have proteases to form several nonstructural proteins, including an rnadependent rna polymerase, methyltransferases, and a helicase, followed by either four or five structural proteins intermingled with a variable number of nonstructural and minor structural proteins. 37 the first of the major structural proteins is a surface hemagglutinin-esterase protein, present on hcovs oc43 and hku1 and some animal betacoronaviruses, that may play some role in the attachment or release of the particle, or both, at the cell surface. this gene contains sequences similar to the hemagglutinin of influenza c virus, likely evidence of an interfamily recombinational event that occurred many years ago. the next gene encodes the surface glycoprotein that forms the petal-shaped surface projections and is responsible for attachment and the stimulation of neutralizing antibody. this is followed by a small envelope protein, a membrane glycoprotein, and a nucleocapsid protein that is complexed with the rna. there are several other open reading frames whose coding functions are not clear. the strategy of replication of covs is similar to that of other nidoviruses, in that all messenger rnas form a nested set with common polyadenylated 3′ ends, with only the unique portion of the 5′ end being translated. 4 as in other rna viruses, mutations are common in nature, although the mutation rate is much lower, approximately 2 × 10 −6 per site per replication cycle. 38 unlike other rna viruses, covs encode a 3′-5′ exonuclease that has proofreading activities, playing a critical role in maintaining replication fidelity in cell cultures and in animals. 39 covs are also capable of genetic recombination if two viruses infect the same cell at the same time. all covs develop exclusively in the cytoplasm of infected cells (fig. 157-4) . they bud into cytoplasmic vesicles from membranes of the pre-golgi endoplasmic reticulum. these virus-filled vesicles are then extruded by the exocytic secretory pathway. 40 the resultant virus particles have a diameter of 70 to 80 nm on thin-section electron microscopy and 60 to 220 nm on negative staining. they are pleomorphic, with widely spaced, petal-shaped projections 20 nm long (see fig. 157-1) . the cellular receptor for 229e and most other alphacoronaviruses is aminopeptidase n (apn). 41 interestingly, nl63, the other known human alphacoronavirus, uses as its cellular receptor angiotensinconverting enzyme ii (ace2), 42 the same receptor as is used by the sars-cov. 43 mouse hepatitis virus, a betacoronavirus related to strain oc43, uses as its receptor a member of the carcinoembryonic antigen family. 44 hcov-oc43 and bcov, which is closely related to hcov-oc43, bind to 9-o-acetylated neuraminic acid as part of the entry process. 45 the host cell receptor for mers-cov is dipeptidyl peptidase as 50%. there was no mortality in children or young adults younger than the age of 24 years. in response to the global spread and associated severe disease, the world health organization coordinated a rapid and effective control program that included isolation of cases, careful attention to contact, droplet and airborne infection control procedures, quarantine of exposed persons in some settings, and efforts to control spread between countries through travel advisories and travel alerts. presumably as a result of these efforts, global transmission ceased by july 2003. a few subsequent cases of sars were detected, but all were either a result of laboratory spread or individual cases related to presumed contact with civet cats or other intermediate hosts. the last known case occurred in mid-2004. spread of sars to humans is thought to have occurred primarily through droplet or contact transmission, with a possible role for fomites. in most instances, an individual case transmitted to very few others, although several well-documented instances of small-particle airborne transmission occurred, resulting in super-spreading events. 67 spread in hospital settings appeared to be surprisingly efficient, but it could be effectively suppressed with the enforcement of droplet and contact precautions. 68 containment measures were efficacious, in part, because patients were most contagious only after lower respiratory disease developed. 69 the chain of spread was finally broken in the people's republic of china, the last country to experience endemic spread, in june 2003. it now seems almost certain that the human epidemic began with the spread of a closely related bat virus first to palm civets or other animals sold in live wild game markets and then to humans in guangdong province in the people's republic of china, and that the virus adapted itself through mutation and possibly recombination, until it transmitted readily among humans. [70] [71] [72] the virus that spread worldwide came largely from a single infected individual who traveled from occurred at all ages, the elderly and those with underlying conditions (diabetes, renal disease, immunosuppression) have been most often severely or fatally affected. the who and the cdc have published a case definition, as well as surveillance instructions to aid in epidemiologic control of the mers-cov. 62, 63 the putative bat origin of this virus was strengthened by the finding that the virus grew readily in primary bat tissue culture. 64 nevertheless, and while bats sampled in the middle east, africa, and europe were found to carry viruses closely related to the mers-cov, 65 epidemiologic studies suggested there was likely to be at least one intermediate host. serologic and virologic studies indicated that camels in the middle east and africa were frequently infected by viruses very similar to some of those found in human mers cases. 65a acquisition of mers from camels appears likely, although the proportion of camel-acquired cases (versus those acquired from person-to-person contact or through another animal intermediate) is not clear. case clusters indicate that person-to-person hospital spread is more common than spread within families, and that casual-contact spread is unusual. 7a the sars epidemic began in guangdong province in the people's republic of china in mid-november 2002. 66 it came to worldwide attention in march 2003 when cases of severe, acute pneumonia were reported to the world health organization from hong kong, hanoi, and singapore. disease spread in hospitals to health care workers, visitors, and patients, among family members, and, on occasion, in hotels, apartment complexes, markets, and airplanes. worldwide spread was rapid but focal ( fig. 157-5) . the largest numbers of cases were reported from the people's republic of china, hong kong, taiwan, singapore, and toronto, canada. the overall case-fatality rates in these locations ranged from 7% to 17%, but persons with underlying medical conditions and those older than 65 years of age had mortality rates as high involvement in other organ systems, including diarrhea, leukopenia, thrombocytopenia, and, most notably, pan-lymphopenia. 83 virus has been detected in respiratory secretions, blood, stool, and urine specimens and tissue from the lung and kidney. on the basis of pcr testing, virus titer is highest during the second week of illness 84 and can often be detected into the third week of illness and sometimes for as long as several months. 18, 85 pulmonary symptoms may worsen late in the course of the illness, with the development of adult respiratory distress syndrome. 84 there may also be late evidence of liver and kidney involvement. the pulmonary pathology of infection by the sars-cov has been described extensively, 17,86,87 but little has been published about the pathology in other organ systems. [87] [88] [89] the extrapulmonary pathologic changes found most consistently at autopsy are extensive necrosis of the white pulp of the spleen and a generalized small vessel arteritis. 87, 88 in the lung, there is hyaline membrane formation, interstitial infiltration with lymphocytes and mononuclear cells, and desquamation of pneumocytes in the alveolar spaces. giant cells are a constant finding and usually have macrophage markers. in bronchoalveolar lavage, biopsy, and autopsy specimens viral particles have been noted in type i and ii pneumocytes. 90 at this point, little is known about the pathogenesis of mers-cov because the infection was only recently described and no pathological specimens are yet available. it is anticipated that the pathologic changes in the lungs of patients with severe disease will be similar to those observed in patients with sars or other patients with acute respiratory distress syndrome (ards). almost all the antigenically distinct respiratory cov strains that were isolated in the 1960s have been administered to volunteers, and all these produce illness with similar characteristics. 8,91 a summary of these characteristics is given in table 157 -1, in which a comparison is made with colds produced by rhinoviruses in similarly inoculated volunteers. the incubation period of cov colds was longer and their duration somewhat shorter, but the symptoms were similar. asymptomatic infection was sometimes seen and, indeed, has been a feature guangdong province to hong kong and infected a large number of individuals before himself succumbing to the disease. in contrast, the virus that was epidemic in the people's republic of china was more variable. although an etiologic role is not proven, enteric covs (or covlps) have been most frequently associated with gastrointestinal disease in neonates and infants younger than 12 months. particles have been found in the stools of adults with the acquired immunodeficiency syndrome. 73, 74 asymptomatic shedding is common, particularly in tropical climates 75 and in populations living in poor hygienic conditions. 76 the viruses can be detected for prolonged periods 51,53,77 and without any apparent seasonal pattern. 78 community-acquired respiratory covs (hcov-229e, oc43, nl63, hku-7) generally replicate in ciliated and nonciliated (hcov-229e) epithelial cells of the nasopharynx, 79 probably producing both direct cell degeneration 80 and an outpouring of chemokines and interleukins, with a resultant common-cold symptom complex similar to that produced by rhinovirus infection. 81 the incubation period is, on average, 2 days, and the peak of respiratory symptoms, as well as viral shedding, is reached at approximately 3 or 4 days after inoculation. 61 the pattern of virus replication of covs must be at least in part determined by virus-receptor interaction. the two best-defined receptors for the respiratory covs are aminopeptidase n for strain hcov-229e and angiotensin-converting enzyme ii for nl63. the pathogenicity of sars is more complex and involves systemic spread. the route of infection of the sars-cov is probably through the respiratory tract. after an incubation period that is usually 4 to 7 days, but can be as long as 10 to 14 days, the disease begins, starting usually with fever and other systemic (influenza-like) symptoms, with cough and dyspnea developing a few days to a week later. 82 although the lung is the focus of the disease process, there are often signs of levels decreased, suggesting that disease was partly immune mediated. 84 clinical improvement was associated with the onset of a virusspecific antibody response. 119 pediatric disease was, interestingly, significantly less severe than adult disease, although the features were similar. 121 disease during pregnancy was severe, with high mortality in both the mother and fetus. 122 congenital transmission was not described. the nature of the illness associated with enteric cov infection is much less clear. one study found a significant association of gastroenteritis in infants 2 to 12 months of age with the presence of covlps in the stool. 55 another study, confined to infants in a neonatal intensive care unit, found highly significant associations between the presence of covlps in the stool and the presence of water-loss stools, bloody stools, abdominal distention, and bilious gastric aspirates. 53 a further study of symptomatic infants shedding covlps pointed to possible differences between covlp-associated diarrhea and rotavirus diarrhea: although fever and vomiting were of similar incidence, stools were more often occult blood positive (18% in covlp-associated vs. 0% in rotavirus-associated disease), less often watery (66% vs. 92%), and more often mucoid (32% vs. 8%). 77 finally, covs have been associated with at least three outbreaks of necrotizing enterocolitis in newborns, 52, 53, 56 and the best characterized strains 56 were isolated in infants with this illness. surveys seeking hcov rna by pcr using primers that would detect the known community-acquired respiratory hcovs in stool have been quite disappointing. in one study of 878 fecal samples from children with gastrointestinal complaints tested over 2 years, all four hcov species were found, but in all but 4 of 22 hcov-positive cases, either rotavirus or norovirus was also present. 28 in addition, about half of the children in this survey had respiratory and gastrointestinal symptoms. in the same study, 112 asymptomatic children were sampled and 2 were positive. another study sampled 151 symptomatic children, and 2 were found to have hku1 rna. 29 molecular studies using primers that would broadly detect new covs should be considered to resolve questions about the role of covs in human gastrointestinal disease. 123 like many other viruses, covs have been sought as possible etiologic agents in multiple sclerosis. the search has been stimulated by the capacity of jhm, a well-studied strain of mouse hepatitis virus, to produce in mice and rats an immune-mediated chronic demyelinating encephalitis histologically similar to multiple sclerosis. 124 hcov-oc43 125, 126 and hcov-229e 127 have been detected in brain tissue from multiple sclerosis patients using virus isolation, 125 in situ hybridization, immunohistology, 126 and pcr. 127 moreover, t-cell lines established from patients with multiple sclerosis by stimulation with myelin basic protein or hcov-229e were found to be cross-reactive with the opposite antigen, suggesting that molecular mimicry might be a possible pathogenic mechanism for the disease association. 128 an adolescent boy with acute demyelinating encephalitis was found to have hcov-oc43 rna in both the respiratory tract and the cerebrospinal fluid. 129 despite these intriguing reports, compelling evidence is lacking to establish an etiologic or pathogenetic association of covs with cns disease in humans. although some human respiratory covs grow in tissue culture directly from clinical samples and although antigen detection systems have been developed for both hcov-oc43 and hcov-229e, 130, 131 laboratory diagnosis of cov respiratory infections is best accomplished by molecular methods. reverse-transcriptase pcr (rt-pcr) systems have been developed using many different primers and detectors. from a clinical point of view, a single generic test for respiratory covs would be desirable, and such tests have been developed. however, when tested side by side with specific systems, the generic systems have a somewhat lower sensitivity. 95 systems that combine primers and probes specific for several covs have also had considerable success. 132 of both serologic surveys and pcr-based studies of natural infection of infants, children, and adults. 92, 93 more serious respiratory tract illness is probably also caused by all four strains of community-acquired hcov. the evidence for this is not conclusive, but it seems likely that all strains can produce pneumonia and bronchiolitis in infants, 24, 27, 94, 95 otitis and exacerbations of asthma in children and young adults, [96] [97] [98] pneumonia in healthy adults, 99 exacerbations of asthma and chronic bronchitis in adults, 100, 101 both serious bronchitis and pneumonia in the elderly, 102, 103 and pneumonia in the immunocompromised host. 104, 105 hcovs are found in asymptomatic individuals of all ages, and, when accompanied by illness, are also sometimes accompanied by infections with other potential respiratory pathogens. these characteristics (infection without disease, coinfection during disease) are features of many respiratory pathogens, including particularly rhinoviruses, adenoviruses, human metapneumovirus, human bocavirus, and parainfluenza viruses, but also (although less frequently) respiratory syncytial virus and influenza virus. because infections with respiratory hcovs are so common, however, it is possible that they are responsible for a significant portion of these serious lower respiratory tract diseases, even though the basic pathogenicity of hcovs (judging from volunteer studies) is similar to that of rhinoviruses, and clearly less than that of respiratory syncytial virus, influenza viruses, and certain adenovirus types. there is some evidence that hcov-oc43 is more pathogenic in the elderly than hcov-229e 106 and also some evidence that infection with nl63 in children is different from the other respiratory hcovs in that several series have found an excess of children with croup. 26, 27 information about the clinical presentation of patients infected with the mers-cov is limited. it is clear that there is a spectrum of illness with some infections consisting of mild upper respiratory symptoms only, and others characterized by cough and fever with progression to respiratory failure over about a week. 6, 7, 62, 107 renal failure, as well as pericarditis and adult respiratory distress syndrome has been part of the reported clinical picture. the mers-cov host cell receptor, dpp-4, is expressed at high levels in the kidney, 108 raising the possibility that direct infection of this organ contributes to renal disease. a case definition that will lead to further epidemiologic studies has been published by the world health organization. 109 severe acute respiratory syndrome coronavirus the first symptom in most cases of sars was fever, usually accompanied by headache, malaise, or myalgia. this was followed, usually in a few days, but as long as a week later, by a nonproductive cough and, in more severe cases, dyspnea. approximately 25% of patients had diarrhea. interestingly, upper respiratory symptoms such as rhinorrhea and sore throat usually did not occur. 82, 84, [110] [111] [112] [113] the chest radiograph was frequently abnormal, showing scattered air-space opacification, usually in the periphery and lower zones of the lung. 114 spiral computed tomography demonstrated both ground-glass opacification and consolidation, often in a subpleural distribution. [115] [116] [117] lymphopenia was common, 84, 86, 110 with normal or somewhat depressed neutrophils. paradoxically, neutrophilia was associated with poor outcomes. 83 the decrease in lymphocytes in the blood was most marked for cd4 cells but was seen in all t-cell phenotypes, including cd3 and cd8, as well as natural killer cells. creatine kinase was often abnormal, as were lactic dehydrogenase and aspartate aminotransferase. levels of proinflammatory cytokines were elevated at early times during infection in patients with severe clinical disease 118 and decreased in those patients who resolved the infection. 119 approximately 25% of patients developed severe pulmonary disease that progressed to adult respiratory distress syndrome. adult respiratory distress syndrome with sars-cov infection was most likely to develop in patients older than 50 years or with underlying disease such as diabetes, cardiac disease, and chronic hepatitis. 84, 110, 111, 120 the overall mortality rate was between 9% and 12%, with the highest rates in the elderly and adults with underlying liver disease. in some patients, clinical deterioration occurred during the second week of illness, as virus ribavirin. 134 it is now known that ribavirin has little activity against sars-cov in vitro, and there is no evidence that either intervention improved outcomes. 135, 136 lopinavir/ritonavir and intravenous immune globulin were also used in some patients, again without conclusive evidence that they were helpful or harmful. there is anecdotal and at least partially controlled evidence of the benefit of either interferon-α or interferon-β treatment. further, treatment of sars-cov-infected cynomolgus monkeys with pegylated interferon-α resulted in improved outcomes, 137 lending credence to the use of this therapy if sars recurred. treatment of mers-cov infection depends entirely on supportive measures. no antiviral drugs are recommended, although several studies have indicated that mers-cov is more sensitive to interferonα or interferon-β than sars-cov. 138, 139 standard droplet precautions should be used, with aerosol precautions during certain high-risk procedures. 109 rigorous application of hospital infection control procedures, particularly those directed at contact and droplet spread, was shown to have a major beneficial effect on the spread of the sars-cov. 68 the containment of the global sars outbreak is a testament to the power of the cooperation and collaboration engendered by the world health organization to address a major public health threat. similar precautions are recommended for patients with suspected or confirmed mers-cov infections. 109 vaccines for animal covs have been developed and widely used with variable efficacy. in one instance, a vaccine for feline infectious peritonitis appeared to lead to enhanced disease with subsequent natural infection. if sars does return or mers reaches epidemic proportions, an effective vaccine would be extremely helpful in control efforts, and a variety of vaccination strategies, including inactivated, subunit, and live-attenuated vaccines, are being pursued. 135, 140 in addition, hospitals have been advised on improvement of infection control procedures so that in future epidemics of respiratory viruses, they will not be a major source of spread of infection, as occurred in the 2002 epidemic of sars. the mers-cov was originally isolated in vero and llc-mk2 cells, and there are several published methodologies for detection and identification by pcr. 133 current recommendations from who are that definition of a possible case should be immediately reported to national authorities, and clinical, epidemiologic, and microbiologic investigations should be carried out. 63 although sars-cov was grown from respiratory tract specimens in vero e6 and fetal rhesus monkey kidney cells, the more sensitive and rapid rt-pcr assays were most widely used to detect infection. virus was detected by rt-pcr in upper and lower respiratory tract, blood, stool, and urine specimens. early in the illness, specimens were found positive only in approximately one third of patients. 84 use of samples from multiple sources increased the yield. virus was detected most frequently during the second week of illness. 18, 84, 85 antibody tests have been developed using tissue culture-grown virus and indirect immunofluorescence or enzyme-linked immunosorbent assay. immunoglobulin m antibody can be detected in most patients for a limited period of time, and immunoglobulin g antibody appears first approximately 10 days after onset of fever in patients with good outcomes and becomes essentially universal after 4 weeks. 84 laboratory diagnosis of the gastrointestinal covs depends now entirely on electron microscopy of stool specimens and detection of characteristic particles in negatively stained specimens. such testing is best performed in laboratories with extensive previous experience. given the severity of sars, clinicians throughout the world empirically treated most patients with corticosteroids and intravenous or oral the complete reference list is available online at expert consult. severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of viruses related to the sars coronavirus from animals in southern china coronaviruses post-sars: update on replication and pathogenesis isolation of a novel coronavirus from a man with pneumonia in saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus cultivation of a novel type of common-cold virus in organ cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia mosaic structure of human coronavirus nl63, one thousand years of evolution croup is associated with the novel coronavirus nl63 detection of human coronaviruses in children with acute gastroenteritis unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) human aminopeptidase n is a receptor for human coronavirus 229e human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc association of coronavirus infection with neonatal necrotizing enterocolitis medical reviews: coronaviruses a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection effects of a "new" human respiratory virus in volunteers available at www.cdc .gov.coronavirus/mers/index middle east respiratory syndrome (mers): case definitions interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) a review of studies on animal reservoirs of the sars coronavirus molecular evolution of the sars coronavirus during the course of the sars epidemic in china clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study multiple organ infection and the pathogenesis of sars time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction respiratory viruses and exacerbations of asthma in adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome clinical disease in children associated with newly described coronavirus subtypes sars: systematic review of treatment effects the spike protein of sars-cova target for vaccine and therapeutic development severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats evolutionary insights into the ecology of coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china coronaviruses post-sars: update on replication and pathogenesis isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 9 hospital outbreak of middle east respiratory syndrome coronavirus cultivation of a novel type of common-cold virus in organ cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease morphologic characteristics and nucleic acid type of transmissible gastroenteritis virus of pigs severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children mosaic structure of human coronavirus nl63, one thousand years of evolution croup is associated with the novel coronavirus nl63 the association of newly identified respiratory viruses with lower respiratory tract infections in korean children detection of human coronaviruses in children with acute gastroenteritis human coronaviruses are uncommon in patients with gastrointestinal illness studies with an unclassified virus isolated from diarrheic calves purification and partial characterization of a new enveloped rna virus (berne virus) an enveloped virus in stools of children and adults with gastroenteritis that resembles the breda virus of calves identification and characterization of a porcine torovirus phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events characterization of torovirus from human fecal specimens notice of retraction to "the novel hemagglutinin-esterase genes of human torovirus and breda virus unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) human aminopeptidase n is a receptor for human coronavirus 229e human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the adaptation of two human coronavirus strains (oc38 and oc43) to growth in cell monolayers detection of the new human coronavirus hku1: a report of 6 cases culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures pleomorphic virus-like particles in human faeces chronic enterocyte infection with coronavirus: one possible cause of the syndrome of tropical sprue? association of coronavirus infection with neonatal necrotizing enterocolitis pleomorphic, enveloped, virus-like particles associated with gastrointestinal illness in neonates human enteric coronaviruses: antigenic relatedness to human coronavirus oc43 and possible etiologic role in viral gastroenteritis isolation and propagation of a human enteric coronavirus medical reviews: coronaviruses the time course of the immune response to experimental coronavirus infection of man a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls effects of a "new" human respiratory virus in volunteers available at www.cdc.gov. coronavirus/mers/index middle east respiratory syndrome (mers): case definitions interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china severe acute respiratory syndrome-singapore effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) viral loads in clinical specimens and sars manifestations a review of studies on animal reservoirs of the sars coronavirus cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human molecular evolution of the sars coronavirus during the course of the sars epidemic in china detection of coronavirus-like particles in homosexual men with acquired immunodeficiency and related lymphadenopathy syndrome stool viruses, coinfections, and diarrhea in hiv-infected patients. berlin diarrhea/wasting syndrome study group coronaviruslike particles and other agents in the faeces of children in efate, vanuatu coronavirus-like particles in adults in melbourne, australia coronaviruslike particles in human gastrointestinal disease: epidemiologic, clinical, and laboratory observations an eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles isolation and characterization of current human coronavirus strains in primary human epithelia cultures reveals differences in target cell tropism ultrastructure of human nasal epithelium during an episode of coronavirus infection signs and symptoms in common colds epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study detection of sars-cov rna in stool samples of sars patients by nest rt-pcr and its clinical value a major outbreak of severe acute respiratory syndrome in hong kong lung pathology of fatal severe acute respiratory syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china multiple organ infection and the pathogenesis of sars time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars antigenic relationships amongst coronaviruses a case-control study of acute respiratory tract infection in general practice patients in the netherlands role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study coronavirus infection in acute lower respiratory tract disease of infants genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children recurrent wheezy bronchitis and viral respiratory infections detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction coronavirus infections in military recruits: three-year study with coronavirus strains oc43 and 229e respiratory viruses and exacerbations of asthma in adults pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity rhinovirus and coronavirus infection-associated hospitalizations among older adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection coronavirus 229e-related pneumonia in immunocompromised patients clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations the united kingdom public health response to an dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv world health organization. global alert and response (gar): infection prevention and control clinical features and short-term outcomes of 144 patients with sars in the greater toronto area acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome severe acute respiratory syndrome in children: experience in a regional hospital in hong kong severe acute respiratory syndrome among children severe acute respiratory syndrome: radiographic appearances and pattern of progression in 138 patients imaging of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome: radiographic and ct findings thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease early enhanced expression of interferon-inducible protein-10 (cxcl-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome critically ill patients with severe acute respiratory syndrome clinical presentations and outcome of severe acute respiratory syndrome in children severe acute respiratory syndrome and pregnancy viral discovery and sequence recovery using dna microarrays corona virus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients detection of coronavirus rna and antigen in multiple sclerosis brain human coronavirus gene expression in the brains of multiple sclerosis patients long-term human coronavirus-myelin cross-reactive t-cell clones derived from multiple sclerosis patients detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis diagnosis of human coronavirus infection by immunofluorescence: method and application to respiratory disease in hospitalized children surveillance of community-acquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995 clinical disease in children associated with newly described coronavirus subtypes detection of a novel human coronavirus by real-time reversetranscription polymerase chain reaction development of a standard treatment protocol for severe acute respiratory syndrome treatment and vaccines for severe acute respiratory syndrome sars: systematic review of treatment effects pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus the spike protein of sars-cova target for vaccine and therapeutic development key: cord-348278-is20odaq authors: al-tawfiq, jaffar a.; memish, ziad a. title: drivers of mers-cov transmission: what do we know? date: 2016-02-29 journal: expert rev respir med doi: 10.1586/17476348.2016.1150784 sha: doc_id: 348278 cord_uid: is20odaq middle east respiratory syndrome coronavirus (mers-cov) emerged in 2012 has since resulted in sporadic cases, intra-familial transmission and major outbreaks in healthcare settings. the clinical picture of mers-cov includes asymptomatic infections, mild or moderately symptomatic cases and fatal disease. transmissions of mers-cov within healthcare settings are facilitated by overcrowding, poor compliance with basic infection control measures, unrecognized infections, the superspreaders phenomenon and poor triage systems. the actual contributing factors to the spread of mers-cov are yet to be systematically studied, but data to date suggest viral, host and environmental factors play a major role. here, we summarize the known factors for the diverse transmission of mers-cov. the middle east respiratory syndrome (mers) was initially recognized in 2012 and since then a total of 1621 cases have been reported from 26 countries, with a case fatality rate of 36% [1] . the disease has a wide range of clinical presentation and epidemiology [2] [3] [4] [5] [6] . three main factors contribute to the transmission of mers coronavirus (mers-cov): the virus, the host, and the environment. with regard to the patterns of transmission, three patterns have been recognized: sporadic transmission, limited intrafamilial transmission, and healthcare-associated transmissions. here, we review the available data regarding the diversity of transmission and summarize known risk factors for such transmission. one of the reasons for the delayed diagnosis of mers cases is the wide clinical spectrum of mers, which ranges from asymptomatic to mild upper respiratory tract symptoms to acute fulminant pneumonia associated with multisystem failure and death [2] [3] [4] [5] [6] . the most common presentations among hospitalized mers patients were fever, cough, shortness of breath, and clinical and radiological evidence of pneumonia [7] [8] [9] [10] . patients may also present with nonspecific symptoms such as fatigue, myalgia, fever, cough, headache, vomiting, and diarrhea [7] [8] [9] [10] . patients with severe disease develop respiratory failure secondary to acute lung injury, and may develop renal failure and coagulopathy [11] [12] [13] [14] [15] . the case fatality rate is related to the viral load, severity of the disease, and the presence of comorbidities [11] [12] [13] [14] [15] . in a recent study, multivariate analysis revealed predictors of mortality as: age >60 years (odds ratio [or] 11.7), underlying illness (or 5.19) , lower c t values where the odds of death increased 17% for each 1 point drop in c t (or 1.17) [16] . the risk of death in patients with mers was significantly associated with age >65 years (9.3 times that of younger patients) and treatment for underlying diseases (7.8 times that of other patients) [17] . the majority (>95%) of the cases of mers infection occur in adults and older adults with one or more comorbidities, and few cases occurred among children [18, 19] . comorbidities associated with mers-cov infection include: diabetes mellitus, renal failure, and hypertension, as shown in table 1 [7, 9, 12, 13, 20] . the diagnosis of mers-cov infection is based on the detection of viral rna in respiratory samples such as nasopharyngeal swabs, bronchoalveolar wash, or sputum using real-time reverse transcriptase polymerase chain reaction (rt-pcr) assays targeting upe and orf1b regions of the mers-cov genome [7, 21] . currently, the detection of mers-cov in respiratory samples is considered the gold standard for the diagnosis of mers in a clinical setting. lower or deeper respiratory samples were more likely to be positive for mers-cov than upper respiratory samples and thus these samples are the preferred method of testing using real-time pcr [16, 21] . viral loads in lower respiratory samples were at least 100 times more compared to upper respiratory tract samples (5 × 10 6 vs. 1.9 × 10 4 cop/ml, respectively) [22] . serology using immunofluorescence, serum neutralization, or protein microarray assays adds additional diagnostic methods for the detection of mers-cov antibodies, but most of these tests require a minimum of 3 weeks postinfection to reveal positive results [21, [23] [24] [25] [26] . different serologic tests were used for the diagnosis of mers-cov infection, which included: plaque reduction neutralization tests (prnts), microneutralization (mn) test, mers-cov-spike pseudoparticle neutralization test (ppnt), and mers-cov s1-enzyme-linked immunosorbent assay (elisa) antibody test [27] . these antibody detection tests (prnt, mn, ppnt, and elisa test) have an excellent sensitivity of 94-100% [27] . the ppnt does not require biosafety level (bsl)-3 containment [27] [28] [29] [30] . in one study, serum dilutions causing plaque reductions of 90% (prnt90) and 50% (prnt50) showed that the use of prnt50 detects few infections that were not detected by prnt90 tests [25] . however, these tests were not used for diagnostic purposes, and the exact sensitivity and specificity of mers-cov antibody tests in clinical settings are not known. three main patterns of the transmission of mers-cov are well characterized. these include: sporadic transmission, intrafamilial transmission, and health-care-associated transmissions [31] [32] [33] [34] [35] [36] [37] [38] . of all the cases in saudi arabia, 45% were health-care-associated infections, 38% were primary cases, and 13% were among household contacts [39] . the source of the infection in the remaining 4% was not reported [39] . the dynamics of the transmission of mers-cov possibly involves small animals, such as bats, and dromedary camels of africa, with the importation of camels into the kingdom of saudi arabia resulting in subsequent zoonotic transmission. these sporadic cases then lead to intrafamilial transmission and outbreaks in the health-care settings ( figure 1 ). primary cases occurring in the community are thought to be linked to dromedary camels [40, 41] . the evidence for dromedary camels as a potential source of infections relies on al-tawfiq et al. [9] arabi et al. [12] asiri et al. [7] shalhoub et al. [15] assiri et al. [13] korean cdc [20] grand total % of the total four different lines of evidence. these lines of evidence are: the high seroprevalence of mers-cov antibodies in dromedary camels, shepherds and abattoir, [42] [43] [44] [45] as well as similarity in the genome sequences of mers-cov from dromedary and human specimens, [40, [44] [45] [46] [47] the isolation of mers-cov from camels, [48] [49] [50] and serological evidence that animal infection preceded the human infection in the jeddah case [41, 51] . the exact mode of transmission of mers-cov from dromedary camels to humans remains to be elucidated. in a recent case-control study, 30 primary cases were matched to 116 controls [52] . the answers of the questions for the primary cases were provided by family members in 57% of instances compared to 1% in the control group [52] . in a univariate analysis, the following risks were significant: direct physical contact with a dromedary camel during the last 6 months, household members frequently visited farms with dromedaries, household members had direct contact with dromedaries during the exposure period, dromedaries were present on farm, milked dromedaries while on farm, household members visited a farm with dromedaries during the exposure period, and any direct contact with a dromedary camel during the exposure period [52] . in a multivariate analysis, risks were associated with direct dromedary exposure in the preceding two weeks, diabetes mellitus, heart disease, and current tobacco smoking [52] . in june 2012, the investigation of the source of mers-cov around the house of the first patient in bisha showed that a sample from a taphozous perforatus bat (egyptian tomb bat) had 100% identity to the human mers-cov cloned from the index case-patient [53] . there are no studies of bats in saudi arabia to further confirm this finding. mers-cov seems to have two seasons for the transmission: march-may and september-november [54] [55] [56] . the reason for the increased cases in april-may 2014 was the occurrence of the jeddah outbreak, the associated increase in the number of tests performed, [57] and increased health-care-associated transmission [5, 57] . so increased surveillance identified more cases, although the overall percentage of patients identified did not differ significantly between jeddah (4.6%) and other cities (5.2%) in the same time period [57] . in addition, data showed that the seroepidemiology is low in saudi arabia with an overall prevalence of 0.15% of 10,007 screened individuals [24] . any seasonal variation may reflect the risk of transmission of mers-cov between animals and humans, seasonal variation in the circulation of the virus in animals, and the natural reservoirs of mers-cov [56] . a study evaluating the seasonality of mers-cov transmission showed that mers-cov infections followed influenza a epidemics, and most of the influenza waves did not co-occur with the mers-cov waves [58] . it is important to study the factors leading to this seasonality of mers-cov, such as parturition of camels. the majority of reported mers cases were secondary cases due to human-to-human transmission. the transmission occurred within family clusters, [2] [3] [4] 25] community settings, [32, 59] travel-associated transmission, [60] [61] [62] [63] [64] and health-care settings [4, 9, 20, 57, [65] [66] [67] [68] [69] [70] [71] . the transmission within any particular family is also variable and the factors leading to heterogeneity in transmission had not been elucidated. in an outbreak in al-madinah al-munawarah, 61% were health-care-associated, 28% were primary cases, and 11% were among family members [59] . genomic testing of mers-cov is needed to accompany any cluster investigation to examine the relatedness of the cases to each other. an investigation of a family cluster in hafr al-bain, saudi arabia, indicated that certain cases in the same family were not related to the cluster, but these cases were caused by community transmissions [72] . the evaluation of contacts of the first mers case was extended to the patient's family contacts, which included three wives, 10 sons, 11 daughters, 12 grandchildren, and one house maid; two shepherds and 14 health-care workers [33] . none of the 53 contacts was mers-cov-positive by pcr [33] . the largest data on contact investigation revealed that the percentage of positive cases was 2.5%, 1.12%, and 3.03% among hospital patients, health-care worker contacts, and family contacts, respectively [73] . thus, the transmission of mers-cov among family members seems to be variable. possible factors leading to heterogeneity in transmissions may include: genetic predisposition, severity of the disease, and other environmental factors. it is clear that health-care-associated infections of mers-cov contribute significantly to the increased number of cases. since 2012, there had been many large outbreaks in the health-care setting involving many facilities. examples of these outbreaks include: al-hasa outbreak, zarqa outbreak in jordan, jeddah, taif, and the republic of korea [13, 20, 57, 58, 65, 67, 71] . in al-hasa, saudi arabia, 23 confirmed cases and 11 probable cases constituted the outbreak [13] . further genotyping studies revealed multiple introduction of mers-cov into the hospital outbreak [74] . between 17 february and 26 april 2014, a multiple health-care-associated mers outbreak took place in jeddah and involved 14 hospitals [5, 57] . of all the cases in that outbreak, 60% of the cases resulted from health-care-associated transmission [75] . factors contributing to the transmissions and increased number of cases included: sensitive case definition, active search for cases, and contact tracing [57] . in 2015, a large mers outbreak occurred in the republic of korea [76] . the outbreak was epidemiologically linked to a single patient who visited multiple countries in the arabian peninsula [76] . the outbreak resulted the quarantine of 17,000 contacts as of 20 july 2015 [77] [78] [79] [80] . and the outbreak resulted in a total of 186 cases and 36 deaths in a short time [81] . variable intrahospital transmissions occurred among contacts of mers patients. among the contacts of the first case, none of the 48 health-care workers who had significant contact during the patient's stay of 10 days in the private hospital in jeddah were positive [12] . the described health-care-associated transmissions are driven by multiple factors. these factors include: late recognition, overcrowding, inadequate infection control precautions, prolonged viral shedding, and the occurrence of superspreading events [32] . the exact definition of a superspreader is not well defined. in one study of sars, a 'superspreading event' was defined as transmission of sars to at least eight contacts, [82] and others used the term to refer to individuals infecting unusually large numbers of secondary cases [83, 84] . a superspreading event was recognized during the sars outbreak when a flight attendant infected more than 100 patients in singapore [85] [86] [87] . in the al-hasa outbreak, a single patient caused seven secondary cases; [13] thus, a superspreading event may have contributed to the outbreak. the index case in the republic of korea caused 27 secondary cases; one of these cases caused 24 tertiary cases while another patient caused 73 tertiary cases [80] . in the republic of korea outbreak, superspreaders contributed to the infection of 85, 28, 23, 11, and 6 from patients' number 14, 1, 16, 76 and 15, respectively [20] . another secondary patient resulted in 91 tertiary mers cases, of which 39% were other patients in the emergency room, and 13% were health-care workers [88] . table 2 provides a summary of possible superspreaders in different sars and mers outbreaks [8, 13, 20, 59, 72, 88] . many theories have been given to explain the occurrence of superspreading events and cited factors include the virus characteristics, the host, the environment, and cultural and travel-related behaviors. other contributing factors include: prolonged duration of exposure, the practice of seeking care at multiple health-care facilities, frequent interhospital transfer, and large numbers of contacts [20] . additional factors include: multi-bedded hospital rooms, crowded hospital rooms, and aerosol-generating procedures [20] . additional influencing factors include: viral mutation, duration of contact with an infectious host and routes of transmission of infections, genetic susceptibility, and underlying comorbidities [89] . a higher viral load, more environmental contacts, more interpersonal contacts, and complex network of interactions made by individuals may also play further roles in superspreading events. although initial studies showed that no significant mutation was detected among mers-cov isolates, [57, 90, 91] complete genome analysis of mers-cov showed genetic recombination events between group 3 and group 5 of clade b [92] . the significance of this recombination in the transmissibility is not known [92] . many patients with mers have underlying comorbidities [7] . in one study, 96% had underlying comorbid medical disorders, including diabetes (68%), hypertension (34%), chronic cardiac disease (28%), and chronic renal disease (49%) [7] . the viral shedding characteristics in those with comorbid diseases might be different from healthy individuals. after 12 days from the initial positive samples, 30% of contacts and 76% of cases were still positive for mers-cov by pcr [89] . a health-care worker shed mers-cov for about 42 days after initial sample [93] . environmental persistence of the mers-cov virus was recently investigated. in one study, it was found that mers-cov survives well on surfaces and in the air and that the virus is more stable at low temperature and low humidity conditions [94] . most of the touchable environments in rooms where mers-cov patients were cared for were contaminated, and viable virus could be isolated in three of the four enrolled patients on days 18-27 after symptom onset [95] . specimens from bed controller and thermometer were pcrpositive until the fifth day from the last positive pcr of the patient's respiratory specimen [95] . environmental factors contributing to a superspreading event include air recirculation, as occurred in the sars outbreak at the hotel metropole, amoy gardens housing complex, and a flight between china and canada [96] . the contribution of air recirculation to the spread of mers-cov is yet to be investigated. the custom and behavior of the affected population may also contribute to a superspreading event, and these behaviors include: 'doctor shopping', traditional ways of greetings, hugging and kissing. in the republic of korea outbreak, the presence of many visitors and family members staying with patients contributed to the superspreading event [85] . hospital environment may also contribute 11 [88] to the spread of the virus, and these include overcrowding, multi-bedded rooms, and inadequate environmental cleaning. it is estimated that between one-fourth and one-fifth of laboratory-confirmed mers cases are asymptomatic. the exact contribution of asymptomatic individuals and those with prolonged viral shedding to the epidemiology and transmission of mers-cov are not well characterized. a recent survey of 225 patients with laboratory-confirmed mers found that 64 (25.1%) were reported as asymptomatic at time of specimen collection; however, when 33 (52%) of those patients were interviewed, 26 (79%) reported at least one symptom that was consistent with a viral respiratory illness [5, 97] . it is estimated that between 20% and 25% of laboratory-confirmed mers cases are asymptomatic. prolonged viral shedding of patients and asymptomatic contacts pose potential, important challenges for infection control [25] . mers-cov was detected by rt-pcr for 18-24 days from the respiratory tract secretions [98] . asymptomatic individuals were also described to harbor mers-cov by rt-pcr [21] . asymptomatic health-care workers shed mers-cov for 42 days from 24 april to 12 june 2014 [93] . since the emergence of mers-cov in 2012, the virus caused sporadic cases, intrafamilial transmission, and major outbreaks in health-care settings. in particular, the transmission within health-care settings is of major concern due to the potential to cause large outbreaks even outside the arabian peninsula. this is exemplified by the large outbreak in the republic of korea. transmissions of mers-cov within health-care settings are facilitated by overcrowding, poor compliance with basic infection control measures, unrecognized infections, the superspreader phenomenon, and poor triage systems. the actual contributing factors to the spread of mers-cov are yet to be systematically studied, but data to date suggest that viral, host, and environmental factors play a major role. understanding these factors and the contribution of each factor to the superspreading events would further enhance the control measures of mers-cov transmission. the epidemiology of the disease was studied in many aspects and showed propensity for the older and those with underlying medical conditions. there is yet an unexplained low rate of involvement of children. the clinical picture of mers had been further elucidated to include asymptomatic infections, mild or moderately symptomatic cases, and fatal disease. this heterogeneity in the clinical presentation is similar to other known infectious diseases and might be related to the immune response. mers-cov is present in the lower respiratory tract system for a prolonged period of time and at higher concentrations than those of the upper respiratory tract, the urine, and the stool. this finding puts the mers-cov dynamics in the same clinical presentation as sars. the finding also has an important role for the prospect of control in health-care settings. in the following years and months to come, it is important to fill the knowledge gap in our understanding of the epide the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. • mers-cov causes sporadic cases, intrafamilial transmission, and nosocomial infections. • mers clinical presentation ranges from mild or asymptomatic cases to severe and fatal disease. • transmission of mers-cov within health-care settings is facilitated by overcrowding, poor compliance with basic infection control measures, unrecognized infections, the superspreader phenomenon, and poor triage systems. • contributing factors for superspreading phenomena include viral, host, and environmental factors. • the virus is present in the lower respiratory tract system for prolonged period of time and at higher concentrations than the upper respiratory tract, the urine, and the stool. family cluster of middle east respiratory syndrome coronavirus infections a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case family cluster of middle east respiratory syndrome coronavirus infections mers-cov outbreak in jeddah-a link to health care facilities • an interesting study of one of the largest outbreak in jeddah middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study •• first study describing the clinical features of mers cases clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome-coronavirus (mers-cov): a case-control study of hospitalized patients case control study of hospitalized patients with severe acute respiratory illness (sari) molecular epidemiology of hospital outbreak of middle east respiratory syndrome characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection outcome of critical mers cases admitted to the intensive care unit hospital outbreak of middle east respiratory syndrome coronavirus • first description of hospital outbreak of mers cases ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ifn-alpha2a or ifn-beta1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study association of higher mers-cov virus load with severe disease and death, saudi arabia real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in middle east respiratory syndrome coronavirus outbreak in the republic of korea respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome viral shedding and antibody response in 37 patients with mers-coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study transmission of mers-coronavirus in household contacts occupational exposure to dromedaries and risk for mers-cov infection comparison of serological assays in human middle east respiratory syndrome (mers)-coronavirus infection seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralization assays reveal a high prevalence of antibody in dromedary camels in egypt seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity lack of middle east respiratory syndrome coronavirus transmission from infected camels managing mers-cov in the healthcare setting middle east respiratory syndrome coronavirus in healthcare settings an update on middle east respiratory syndrome: 2 years later middle east respiratory syndrome coronavirus infection control: the missing piece? middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution coronaviruses: severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus in travelers emerging respiratory viral infections: mers-cov and influenza middle east respiratory syndrome-coronavirus infection: an overview saudi ministry of health middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation human infection with mers coronavirus after exposure to infected camels, saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan antibodies against mers coronavirus in dromedary camels dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels mers coronaviruses in dromedary camels mers coronavirus in dromedary camel herd, saudi arabia isolation of mers coronavirus from a dromedary camel evidence for camel-tohuman transmission of mers coronavirus risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome coronavirus in bats, saudi arabia molecular epidemiology and genetic analysis -origin and evolution an observational, laboratorybased study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands travelrelated mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia saudi arabia s arabia probable transmission chains of middle east respiratory syndrome coronavirus and the multiple generations of secondary infection in south korea mers outbreak in korea: hospital-to-hospital transmission epidemiological investigation of mers-cov spread in a single hospital in south korea health-care associate transmission of middle east respiratory syndrome corona virus, mers-cov, in the kingdom of saudi arabia health care worker contact with mers patient, saudi arabia hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 9 middle east respiratory syndrome coronavirus (mers-cov) -republic of korea south korea coronavirus mers case list -including imported and exported cases middle east respiratory syndrome coronavirus (mers-cov): summary and risk assessment of current situation in the republic of korea and china -as of 19 middle east respiratory syndrome coronavirus (mers-cov). mers-cov in republic of korea at a glance preliminary epidemiological assessment of mers-cov outbreak in south korea middle east respiratory syndrome coronavirus (mers-cov) nosocomial outbreak in south korea: insights from modeling superspreading sars events superspreading and the effect of individual variation on disease emergence an event-based model of superspreading in epidemics role of disease-associated tolerance in infectious superspreaders infectious disease: the tolerance of superspreaders severe acute respiratory syndrome (sars): status of the outbreak and lessons for the immediate future middle east respiratory syndrome coronavirus superspreading event involving 81 persons, korea middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications complete genome sequence of middle east respiratory syndrome coronavirus kor/knih/ 002_05_2015, isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea the role of super-spreaders in infectious disease middle east respiratory syndrome: sars redux? virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen key: cord-349010-n4s8dzgp authors: al-tawfiq, jaffar a.; memish, ziad a. title: update on therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) date: 2016-12-24 journal: expert rev anti infect ther doi: 10.1080/14787210.2017.1271712 sha: doc_id: 349010 cord_uid: n4s8dzgp introduction: the middle east respiratory syndrome coronavirus (mers-cov) is an important emerging respiratory pathogen. mers-cov resulted in multiple hospital outbreaks within and outside the arabian peninsula. the disease has a high case fatality rate, with the need for a therapeutic option. areas covered: in this review, we provide an overview of the progress in the development of therapeutic strategies for mers. we searched pubmed, embase, cochrane, scopus, and google scholar, using the following terms: ‘mers’, ‘mers-cov’, ‘middle east respiratory syndrome’ in combination with ‘treatment’ or ‘therapy’. expert commentary: there are multiple agents tried in vitro and in vivo. none of these agents were used in large clinical studies. available clinical studies are limited to the use of the combination of interferon and other agents. these clinical studies are based solely on case reports and case series. there are no prospective or randomized trials. there is a need to have prospective and randomized clinical trials for the therapy of mers-cov. however, this strategy might be hampered by the sporadic cases outside the large hospital outbreaks. the middle east respiratory syndrome coronavirus (mers-cov) emerged as an important virus in 2012 and since then has caused multiple outbreaks in hospitals especially in the kingdom of saudi arabia and outside the arabian peninsula [1] [2] [3] . since the emergence of mers-cov, a total of 1800 cases including 640 deaths were reported by the world health organization (who) [4] . due to the increased morbidity and mortality of mers-cov infection, the attention was directed toward the development of prevention strategies and the establishment of therapeutic modalities. an earlier review was based on the severe acute respiratory syndrome (sars) experience and had suggested few possible options for the treatment of mers-cov infection [5] . in this review, we provide an overview of the progress in the development of therapeutic strategies for mers. we searched pubmed, embase, cochrane, scopus, and google scholar using the following terms: 'mers,' 'mers-cov,' 'middle east respiratory syndrome' in combination with 'treatment' or 'therapy. ' we also reviewed the references of each article to further include other studies or reports not identified by the search. we classified the studies into the following categories: in vivo and in vitro studies, animal studies, and human case reports or case series. for clinical studies, we graded the level of the evidence based on the 'oxford centre for evidence-based medicine' [6] . in vitro studies showed variable activity of various agents against mers-cov (table 1) . these agents include: interferon, ribavirin, hiv protease inhibitors (nelfinavir, ritonavir, and lopinavir). interferon is antiviral type i ifn system, a major part of the innate immune response [7, 8] . in vitro studies showed that ifn-β has an ic 50 of 1.37 u/ml and that ifn-β has anti-mers-cov activity of 16-, 41-, 83-, and 117-fold higher than ifn-α2b, ifn-γ, ifn-universal type 1 and ifn-α2a, respectively [9] . in vitro studies showed that ifn-β has a lower ic50 for mers-cov compared to ifn-a2b [9] . ribavirin is a nucleoside analog that is activated by host kinases to a nucleotide [7, 10, 11] . it was shown that in vitro doses of ribavirin required to inhibit mers-cov replications are too high to be achieved in vivo [7, 10] . nelfinavir and lopinavir inhibit mers-cov in vitro [7, 12] . the mean 50% effective concentration (ec50) of lopinavir using vero e6 and huh7 cells was 8.0 μm [13] . camostat and the heptad repeat 2 peptide (hr2p) are two mers-cov fusion inhibitors that were tested in vitro [14, 15] . the fusion inhibitor, camostat, inhibited viral entry into human bronchial submucosal gland-derived calu-3 cells but not the immature lung tissue [14] . the second fusion inhibitor, hr2p, inhibits mers-cov replication and the spike protein-mediated cell-cell fusion [15] . cyclosporin affects the function of many cyclophilins that act as chaperones and facilitate protein folding [16, 17] . in vitro, cyclosporine inhibited mers-cov replication [16, 17] . nitazoxanide, a broad-spectrum antiviral agent, and teicoplanin, an inhibitor of cathepsin l in the late endosome/lysosome and blocker of the entry of mers-cov, also showed inhibitory effect of mers-cov in vitro [18, 19] . there are few studies evaluating various agents as therapy for mers-cov in animal models (table 2) [12, [21] [22] [23] [24] . in the rhesus macaques model, interferon-α2b-ribavirin combination decreased viral replication within 8 h of mers-cov infection [25] . in a primate model, the mortality rate at 36 h post-inoculation was reduced from 67% in untreated to 0-33% in animals treated with a combination of interferon-β1b and either lopinavir or ritonavir [12] . intranasal use of an hr2p analog with improved pharmaceutical property, hr2p-m2, was protective in mice model [21] . in an animal model using mers-cov infected mice, the use of high titer mers immune camel serum was effective in reducing lung injury and acceleration of virus clearance [22] . mycophenolate has a direct and indirect antiviral activity by modulation of ifn response [20] . the use of mycophenolate in the common marmoset animal model resulted in higher mortality than untreated animals [12] . a monoclonal antibody designated as m336 is an antibody derived from a large phage-displayed antibody library from b cells of healthy donors [26] . the use of this m336 in mice showed promising results as a therapeutic and a prophylactic agent [23] . currently, there is no animal model that completely reflects the course of mers-cov disease in humans and thus the data obtained from these animal models are to be interpreted cautiously. and animal models utilize therapy shortly after infection. based on analysis of sars data, interferon-ribavirin combination was suggested as a possible therapeutic option for the treatment of mers-cov infections [5] . limited data are available regarding the clinical efficacy of antiviral agents [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] ( table 3 ). the first use of the combination of ribavirin-interferon therapy was in five patients with mers infection [27] . the therapy was started late in the course of the disease with a median time from admission to therapy of 19 days [27] . of the included 5 patients, none responded to therapy [27] . in a subsequent retrospective cohort study, 20 mers patients received ribavirin-interferon compared to 24 patients who did not [28] . the 14-day survival rate was better in those who received the combination therapy (70% vs. 29%, p = 0.004); however, the 28-day survival rate was not statistically different (30% vs. 17%) ( table 1 ) [28] . in another case series, 11 mers patients had ribavirin and peg-interferon α-2a [29] . ribavirin-ifn-α2a was compared to ribavirin-ifn-β1a in a study of 13 and 11 patients, respectively [30] . the mortality rate was not statistically different between the two groups (85% vs. 64%) [30] . in a large cohort study of 51 patients, various combinations of interferon and ribavirin were used with different outcomes (table 3 ) [31] . in a case series of 6 patients, 3 patients received ribavirin and interferon-alfa 2b within 1-2 days of admission and they survived compared to the other 3 patients who died as they received the therapy 12-19 days after admission [36] . another study evaluated the use of interferon beta, interferon alpha, or ribavirin and showed survival rates of 18/23 (78.3%), 6/8 (75%), and 13/19 (68.4%), respectively (table 3 ) [31] . the combination therapy was also used in other case reports (table 3 ) [33, 34] . the role of the combination of ribavirin and ifn was also tried as a treatment and a prophylaxis [34] . the current studies of the use of ribavirin and ifn combination therapy for mers-cov infection rely on small number of patients but there is a trend for improvement. thus, it was suggested that the combination of type 1 interferon and ribavirin could be used [37] . due to the inhomogeneous nature of available studies and the limited data that are available, a precise recommendation on therapy of mers could not be established. the combination of lopinavir/ritonavir, ribavirin and interferonalpha was used in one case [32] . one patient received pegylated interferon, ribavirin, and lopinavir/ritonavir from day 13 of illness and the patient had continued mers-cov in the respiratory tract secretions until the fourth week of illness [33] . however, viremia was detected for only 2 days after initiation of triple therapy [33] . in a case series, eight patients received mycophenolate mofetil and all survived [31] . in the sars epidemic, passive immunotherapy with neutralizing antibodies was considered as a therapeutic approach. there are multiple antibodies against mers-cov [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] (table 4 ). in the mers-cov infection, the production of large quantities of mers-cov neutralizing human polyclonal antibodies was possible using gamma-irradiated whole-killed virion vaccine or a spike protein nanoparticle vaccine in a bovine model [49] . utilizing one dose of these antibodies prevented infection in mice [49] . these antibodies were effective when given 12 h before or 24 and 48 h after mers-cov infection [49] . corti et al. isolated a potent mers-cov-neutralizing antibody (lca60) from memory b cells of an infected individual. the lca60 antibodies bind to a site on the spike protein and neutralize mers-cov infection [48] . these lca60 antibodies were used successfully in mice model [48] . similarly, utilizing a humanized mouse model of mers-cov infection, antibodies against the spike protein were efficacious as prophylaxis [39] . antibodies obtained from the sera of mers immune camels were supportive of the clearance of the virus, and reduction of the severity of the disease in mers-covinfected mice [22] . however, the purification and safety of these antibodies in humans has not been established yet. the cellular dipeptidyl peptidase iv (ddp iv; known as cd26 or adenosine deaminase (ada)-complexing protein-2) [48] is an important receptor that mediates mers-cov infection through the viral spike (s) protein [46, 50] . the mers-cov receptor binding domain (rbd), present on the surface spike protein (s), binds to the host cells receptor dpp iv [46, 50, 51] . in humans, dpp iv is present mainly on the lower respiratory tract area such as the bronchial epithelial and alveolar cells [52, 53] . although ddp iv is important for the viral entry into host cells, the use of dpp iv inhibitors, sitagliptin, vildagliptin, and saxagliptin, does not block the infection of mers-cov [50] . in vitro use of monoclonal antibodies (mers-4) exhibited ic50 of 0.056 µg/ml [43] . other possible human mab (m336, m337, and m338) neutralize pseudovirus and live virus [45] . in rhesus model of mers-cov infection, a human monoclonal antibody, 3b11-n, against mers-cov was effective in reducing the pathology of mers-cov [47] . the use of polyclonal antibody (pab) against cd26 inhibits mers-cov infection in vitro [50] . humanized anti-cd26 monoclonal antibodies (mab) such as mab ys110 and 2f9 significantly inhibit mers-cov infection in vitro [38] . polyclonal antibodies against the mers-cov s1 domain neutralize the virus infection [46] . many other mers-cov antibodies are being developed and tested [38] . in the sars epidemic, convalescent plasma was thought to improve the outcome of sars patients [5] . previous studies suggest that convalescent plasma may be used for patients with sars and severe influenza and may result in decreased viral load and a lower mortality rate [54] [55] [56] [57] . however, most of the studies were of low or very low quality, lacked control groups, and had risk of bias [58] . two patients with mers-cov infection received intravenous immunoglobulin in an attempt to treat the infection, one patient was in saudi arabia [59] and the other was in the usa [60] . a protocol for the use of convalescent plasma as a therapeutic option for mers was suggested [61] . plasma donors were identified as those with anti-mers-cov indirect immunofluorescence assay (ifa) antibodies (titer of ≥1:160) with no evidence of active mers-cov infection [61] . in nine confirmed survivors of mers-cov infection, 55%, 33%, and 22% of them had positive mers antibodies by ifa at 3, 10, and 18 months, respectively [62] . the two patients who had long lasting antibodies had severe disease; however, the titer of the ifa antibodies was not measured in the study [62] . in a larger study, mers-cov neutralizing antibodies were produced at low levels and were short-lived [63] . further studies of the kinetics of the mers-cov antibodies showed that all surviving patients and 50% of fatal cases produced igg and neutralizing antibodies [64] . the presence of antibodies did not lead to the elimination of virus from the lower respiratory tract [64] . in a study of 12 patients from south korea, nine patients had prnt50 titers >1:320 by day 21 and two had titers >1:320 by day 28 [65] . in a study of 443 samples, 12 (2.7%) had reactive elisa results, and 9 of those had reactive indirect fluorescent antibody and microneutralization assay titers [66] . thus, the use of convalescent plasma for the treatment of mers-cov in a clinical trial may be challenging due to a small pool of potential donors with sufficient antibody titers [66] . based on sars experience, some authors suggested that steroid therapy might be beneficial for severe mers-cov infection [67] . corticosteroid use for patients with sars showed that early use of corticosteroids significantly increased viral load, and 20.7-fold increase in risk of icu admission and mortality [68] . in another study of 16 non-icu patients, the median time for sars-cov to become undetectable in plasma was 12 days compared to 8 days in patients who did and did not receive early corticosteroid therapy [69] . corticosteroids were used as adjunct therapy for many patients with mers-cov [27, 70] . in a study of 13 patients with mers-cov infection, one patient received steroid and intravenous immunoglobulin for thrombocytopenia [59] . initial study of five patients, three patients received a combination of interferon and ribavirin in addition to adjunct steroid on day 0-21 and all died during hospitalization [27] . however, steroids were not evaluated systematically as therapy for mers patients. the host protease, furin, is an important factor to break down the s1-s2 region of the mers-cov [71] . in vivo studies showed activity of multiple agents against mers-cov and include: chloroquine, chlorpromazine, cyclosporine, and mycophenolic acid [7, 72] . in addition, the us fda approved repurposed agents with broad antiviral activity including in vitro activity against mers-cov [73] . these agents include the polymerase inhibitor bcx4430 and the helicase inhibitor ssya10-001, spike binding (immunoadhesin (dpp4-fc)) [73] . there are many other agents currently in preclinical investigation such as: fluspirilene, thiothixene, fluphenazine hydrochloride, promethazine hydrochloride, astemizole and chlorphenoxamine hydrochloride [72] . the emergence and continued cases of mers-cov infection require the availability of mers therapy. there is an urgent need for the development of standardized animal models and the establishment of standardized clinical therapeutic protocols. the current clinical studies are limited to case reports and case series with no control arm. the quality of evidence these studies offer is too low to make a conclusion. it is difficult to draw conclusion in the face of these limited studies. the most used combination of therapy was interferon and ribavirin as developed initially in 2013. the development of novel therapeutic agents or the repurposing old therapeutic agents against mers-cov are needed as alternative pathways for testing and clinical trials. it was thought that convalescent sera may provide an exciting alternative as a therapeutic agent; the present data does not support the wide adaptation of this therapy. the current mers therapy relies on supportive care and providing circulatory and ventilation support. it is expected that the development and the use of repurposed drugs would allow the development of therapeutic agents for mers-cov. the best location to provide a randomized controlled trail was thought to be the intensive care units for the use of convalescent sera; the presence of milder disease may necessitate the development of therapeutic protocols for patients with severe and those with milder disease. monoclonal and polyclonal antibodies may offer further therapeutic options for the disease in humans. since the prospect for a randomized clinical trial is low due to the sporadic nature of the disease outside hospital outbreaks, it is prudent to have well-conducted prospective clinical studies. the proteins involved in mers-cov entry and replication are attractive targets for the development of antiviral therapeutics. clinical studies utilizing anti-mers-cov antibodies as therapeutic options would add to the prospect to develop therapeutic agents for this syndrome. although many drugs appear to be effective in vitro, a consideration of their availability, pharmacokinetic/pharmacodynamic properties, and side effects should be taken into consideration. of medications with an attractive use, lopinavir, interferon, and mycophenolate are among these agents. accelerated and preferably randomized controlled trails should be conducted. neutralizing antibodies are also promising and the use of these agents in humans. targeting the ddp4 receptor may be of particular importance; however, it is important to keep in mind that the development of any mutation in the binding sites may limit the use of these agents [74] . few monoclonal antibodies showed protective efficacy as a prophylaxis in animal models [39, 48] . the development and testing of monoclonal antibodies are associated with high costs and lack of an undefined population for their use [75] . these monoclonal bodies had not been used in phase 1 clinical trials and that further development of these agents require time and cost. • mers-cov emerged in 2012 and has caused multiple hospital outbreaks. this paper was not funded. managing mers-cov in the healthcare setting • a review of mers-cov in healthcare setting hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus in healthcare settings middle east respiratory syndrome coronavirus (mers-cov) n d therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy oxford centre for evidence-based medicine -levels of evidence broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays • a study of the inhibitors of mers-cov inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin future treatment strategies for novel middle east respiratory syndrome coronavirus infection treatment with lopinavir/ ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset • a primate model of mers-cov infection screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture middle east respiratory syndrome coronavirus (mers-cov) infection mediated by the transmembrane serine protease tmprss2 structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment cyclosporin a inhibits the replication of diverse coronaviruses nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis c virus infection in vitro and in vivo protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection passive transfer of a germline-like neutralizing human monoclonal antibody protects transgenic mice against lethal middle east respiratory syndrome coronavirus infection single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal middle east respiratory syndrome (mers)-coronavirus infection treatment with interferon-alpha 2b and ribavirin improves outcome in mers-covinfected rhesus macaques junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study the first study of interferon and ribavirin in mers-cov ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study •• a large cohort study of ribavirin-interferon in mers-cov patients acute management and longterm survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia combination therapy with lopinavir/ ritonavir, ribavirin and interferon-alpha for middle east respiratory syndrome: a case report virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen ribavirin and interferon-α2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases middle east respiratory syndrome coronavirus during pregnancy middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia guidelines for the laboratory diagnosis of middle east respiratory syndrome coronavirus in korea inhibition of middle east respiratory syndrome coronavirus (mers-cov) infection by anti-cd26 monoclonal antibody pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines a conformation-dependent neutralizing monoclonal antibody specifically targeting receptor-binding domain in middle east respiratory syndrome coronavirus spike protein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv the multifunctional or moonlighting protein cd26/dppiv convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? successful treatment of avianorigin influenza a (h7n9) infection using convalescent plasma successful treatment of avian influenza with convalescent plasma the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus (mers-cov) into the united states feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol antibody response and disease severity in healthcare worker mers survivors transmission of merscoronavirus in household contacts viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection •• a study of viral shedding of mers-cov kinetics of serologic responses to mers coronavirus infection in humans feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia clinical recommendations from an observational study on mers: glucocorticoids was benefit in treating sars patients the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans mechanisms of coronavirus cell entry mediated by the viral spike protein repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection development of medical countermeasures to middle east respiratory syndrome coronavirus towards the prophylactic and therapeutic use of human neutralizing monoclonal antibodies for middle east respiratory syndrome coronavirus (mers-cov) • a study of use of human neutralizing monoclonal antibodies for mers-cov research and development activities for middle east respiratory syndrome: the current landscape. n d the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. key: cord-343196-vlwzzrgc authors: kiambi, stella; corman, victor m.; sitawa, rina; githinji, jane; ngoci, james; ozomata, abdullahi s.; gardner, emma; von dobschuetz, sophie; morzaria, subhash; kimutai, joshua; schroeder, simon; njagi, obadiah; simpkin, piers; rugalema, gabriel; tadesse, zelalem; lubroth, juan; makonnen, yilma; drosten, christian; müller, marcel a.; fasina, folorunso o. title: detection of distinct mers-coronavirus strains in dromedary camels from kenya, 2017 date: 2018-11-28 journal: emerg microbes infect doi: 10.1038/s41426-018-0193-z sha: doc_id: 343196 cord_uid: vlwzzrgc nan between july 2016 and october 2017, nasal swabs were randomly taken from n = 1421 dromedaries in five counties, namely, turkana (n = 417), marsabit (n = 370), isiolo (n = 403), laikipia (n = 181), and nakuru (n = 50). in addition, monthly repeated sampling was performed on 430 dromedaries from four herds in two different countries (isiolo and nakuru) for a period of 7 months (from april to october 2017). in total, n = 2175 nasal swab samples were collected. all samples were stored frozen in trizol buffer at −80°c. rna extraction (direct-zol™ rna kit, zymo research) and mers-cov nucleic acid detection were performed following the manufacturer's instructions and according to previously established protocols 8 . in seven of 2175 (0.23%) tested nasal swabs, mers-cov rnas were detected by the upe mers-cov rt-pcr screening assay (supplementary table) . for 2/7 samples, which had very low mers-cov rna concentrations (<2 x 10 4 copies/ml), confirmatory rt-pcr testing and sequencing were unsuccessful. the mean viral load for 5/7 samples was 1.1 x 10 7 (range 1.2 x 10 5 -5.0 x 10 7 ) rna copies per ml buffer. four of the five mers-cov rna-positive animals were female and <1 year old, consistent with previous observations that juvenile dromedaries and possibly females may be the main sources for mers-cov excretion 9 . the mers-cov rna-positive animals belonged to two different dromedary camel herds in dabel and lombolio, which are both located within isiolo country. however, the herds neighbor each other and share common pastures and water sources. introduced into the two herds. the dromedaries were sampled on the same day, suggesting simultaneous cocirculation of two different mers-cov strains and perhaps an unexplored infection dynamic in isiolo, which is a camel congregation location. to experimentally confirm the presence of two independently circulating mers-cov strains and to rule out sample cross-contamination, we generated complete mers-cov genome sequences using a previously established protocol 10 . full genome sequences were generated for one specimen of each of the two positive herds using the samples with the highest mers-cov rna concentrations (5.0 x 10 7 and 3.7 x 10 6 copies/ml). other confirmed mers-covpositive samples were assigned to two different mers-cov isolates ("dabel" or "lombolio") by amplifying and sequencing single-nucleotide polymorphisms in the spike gene and the open reading frame 3 (supplementary table) . all three viruses from dabel and both viruses from lombolio shared the same polymorphism patterns. for phylogenetic analysis, we included two representatives of mers-cov lineages representing mers-cov clades a and b, as defined earlier 11 , along with all published clade c (non-a, non-b) mers-cov complete genomes (genbank accessed 2 april 2018). a phylogenetic tree was constructed using the maximum-likelihood method based on the general time reversible model and 500 bootstrap replicates using the phyml plugin in geneious r11 (www.geneious.com, biomatters ltd, new zealand). as shown in fig. 1 , both kenyan dromedary mers-cov isolates clustered with the proposed clade c viruses from ethiopia and egypt in a sister relationship to all arabian mers-covs (clades a and b). the two kenyan mers-cov isolates diverged by 0.02% at the nucleotide level, confirming the circulation of at least two different mers-cov strains in kenya. the next closest mers-cov relative was obtained from a dromedary sampled in egypt in 2014 (nrce-nc163/2014; acc no. ku74020, clade c) and showed 0.23-0.24% nucleotide distance. recombination analysis by rdp v4.95 indicated that none of the two kenyan mers-cov strains had recombined with any of the known clade a, b, or c strains. the previously described clade c african mers-cov strains 4,5 had several mutations in the spike protein, which is responsible for cellular receptor interaction, virus entry, and antibody-directed virus neutralization 12 . an alignment of the amino-acid sequences of all known mers-cov spikes showed that the kenyan mers-cov strains had one unique amino-acid change (s528p) within the core part of the receptor-binding domain (supplementary figure) . as the mutation was not among the 14 amino-acid residues that directly interact with the dipeptidyl peptidase-4 receptor 12 , phenotypic traits of these new clade c mers-cov strains may be comparable to epidemic mers-cov strains as described previously 4 . however, without further extensive experimental assessment, we cannot rule out the possibility that the observed mutation in the spike protein causes differences in the receptor interaction or receptor binding affinity, which may influence virus transmission or host tropism. recently described mers-cov strains from western africa had genomic deletions within open reading frame (orf) 4 a/b that were not seen in eastern african mers-cov strains 4 . both of the encoded proteins, proteins 4a and 4b, have anti-immune functions 13, 14 and may represent important virulence factors in vivo. we provide independent evidence that mers-cov from eastern african dromedaries encode a complete orf4a/b. the observation that mers-cov strains in different parts of eastern africa have a complete orf4a/b suggests the predominance of these strains on the african continent and emphasizes that the orf4a/b deletion is most likely geographically restricted to western africa. taken together, differential spike-receptor interactions and anti-immune activity may influence virus replication and transmission. the limited number of human mers cases in africa would certainly favor the idea that mers-cov strains differ in virulence and transmissibility. further experimental confirmation, preferably by animal transmission experiments in combination with coronavirus reverse genetics 15 , are warranted. the phylogenetic relationship of mers-cov strains from the african continent (clade c) with the strains circulating on the arabian peninsula (clades a and b) hints at the divergence of these clades some time ago. the putative absence of clades a and b mers-covs on the african continent may be explained by a lack of surveillance and testing and/or by the genetic drift of mers-cov on the arabian peninsula. the unidirectional export routes from africa to the arabian peninsula may prevent the reintroduction opportunities of clades a and b mers-covs into african dromedary herds. interestingly, to date, no clade c mers-cov strains from africa have been detected on the arabian peninsula, which is rather surprising, given the continuous and extensive export of african dromedaries to the arabian peninsula. an explanation for this observation may again be a lack of testing of imported animals and/or the fact that previous clade a/b mers-cov infections may have established herd immunity in the arabian dromedary populations. as cov infections do not elicit long-lasting (mucosal) immunity, the introduction of clade cmers-cov strains on the arabian peninsula may be possible in the future and should therefore be monitored. to shed light on possible reasons for the restricted geographic circulation of different mers-cov strains, enhanced virological surveillance of mers-cov is urgently needed in dromedary populations of the affected regions. putative underlying evolutionary and molecular mechanisms that influence the geographic distribution of differentially virulent mers-cov strains should be assessed through phenotypic characterizations of different mers-cov strains. the early detection and characterization of emerging mers-cov strains with new phenotypic features will be highly relevant for future vaccination strategies and the prediction of epidemics in humans. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers and the dromedary camel trade between africa and the middle east mers coronaviruses from camels in africa exhibit regiondependent genetic diversity cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt antibodies against mers coronavirus in dromedary camels mers-cov antibodies in humans detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction time course of mers-cov infection and immunity in dromedary camels rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 mers-cov 4b protein interferes with the nf-kappabdependent innate immune response during infection middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist transgene expression in the genome of middle east respiratory syndrome coronavirus based on a novel reverse genetics system utilizing redmediated recombination cloning key: cord-354302-l2kywzro authors: adney, danielle r.; van doremalen, neeltje; brown, vienna r.; bushmaker, trenton; scott, dana; de wit, emmie; bowen, richard a.; munster, vincent j. title: replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels date: 2014-12-17 journal: emerg infect dis doi: 10.3201/eid2012.141280 sha: doc_id: 354302 cord_uid: l2kywzro in 2012, a novel coronavirus associated with severe respiratory disease in humans emerged in the middle east. epidemiologic investigations identified dromedary camels as the likely source of zoonotic transmission of middle east respiratory syndrome coronavirus (mers-cov). here we provide experimental support for camels as a reservoir for mers-cov. we inoculated 3 adult camels with a human isolate of mers-cov and a transient, primarily upper respiratory tract infection developed in each of the 3 animals. clinical signs of the mers-cov infection were benign, but each of the camels shed large quantities of virus from the upper respiratory tract. we detected infectious virus in nasal secretions through 7 days postinoculation, and viral rna up to 35 days postinoculation. the pattern of shedding and propensity for the upper respiratory tract infection in dromedary camels may help explain the lack of systemic illness among naturally infected camels and the means of efficient camel-to-camel and camel-to-human transmission. in 2012, a novel coronavirus associated with severe respiratory disease in humans emerged in the middle east. epidemiologic investigations identified dromedary camels as the likely source of zoonotic transmission of middle east respiratory syndrome coronavirus (mers-cov). here we provide experimental support for camels as a reservoir for mers-cov. we inoculated 3 adult camels with a human isolate of mers-cov and a transient, primarily upper respiratory tract infection developed in each of the 3 animals. clinical signs of the mers-cov infection were benign, but each of the camels shed large quantities of virus from the upper respiratory tract. we detected infectious virus in nasal secretions through 7 days postinoculation, and viral rna up to 35 days postinoculation. the pattern of shedding and propensity for the upper respiratory tract infection in dromedary camels may help explain the lack of systemic illness among naturally infected camels and the means of efficient camel-to-camel and camel-tohuman transmission. t he middle east respiratory syndrome coronavirus (mers-cov) was first recognized in 2012 related to a fatal human case of pneumonia in saudi arabia (1) . currently, >800 cases of mers have been identified, and the estimated case-fatality rate is ≈35% (2) . most cases have been identified on the arabian peninsula, but several travel-associated cases have been reported (2) (3) (4) . human-to-human transmission has been reported, predominantly among persons in health care facilities and households; the rate of human infection by zoonotic transmission from a reservoir source is currently not known (4) (5) (6) . the close phylogenetic relationship of human mers-cov isolates with those obtained from bats initially suggested a direct link between the emergence of mers-cov and a putative natural reservoir (7) (8) (9) . anecdotal reports mentioned contact of mers-cov-infected patients with camels and goats, suggesting that livestock might be the intermediate reservoir host for mers-cov (4,10-12). serologic studies revealed widespread prevalence of mers-cov-specific antibodies in dromedary camels from several countries that reported mers cases (4, (13) (14) (15) (16) (17) (18) (19) . further, mers-cov rna was detected in nasal swab samples obtained from 3 camels on a farm linked to 2 human mers-cov cases, and the virus was isolated from nasal swab samples from dromedary camels in qatar (14) . mers-cov isolation and subsequent full genome sequencing directly linked a dromedary camel and a fatal mers-cov case in a person in saudi arabia (20, 21) . despite these associations, the role of camels as a primary reservoir for mers-cov is still debated (22, 23) . here we report on the experimental inoculation of 3 camels with a human isolate of mers-cov. mers-cov (strain hcov-emc/2012) was provided by the department of viroscience, erasmus medical center, rotterdam, the netherlands. the virus was propagated in vero e6 cells cultured in dulbecco modified eagle medium (invitrogen, carlsbad, ca, usa) supplemented with 2% fetal bovine serum, 2 mmol/l glutamine, 50 u/ml penicillin, and 50 µg/ml streptomycin. three native-born adult male dromedary camels (camelus dromedarius) were obtained through private sale; the animals tested negative by neutralization assay for mers-cov and for bovine coronavirus by elisa. camels 1, 2, and 3 were 2, 3, and 5 years old, respectively. camels 1 and 2 were intact males, and camel 3 had been castrated. animals were housed in an animal biosafety level 3 facility for the duration of the experiment and fed ad libitum. camels were acclimated to the facility for 2 weeks before virus inoculation. we sedated the camels with xylazine, then inoculated them with a total dose of 10 7 50% tissue culture infective dose (tcid 50 ) of mers-cov (strain hcov-emc/2012) in a total volume of 15 ml, by way of intratracheal (8 ml using transcutaneous catheter), intranasal (3.3 ml in each nostril by expulsion from a syringe), and conjunctival (0.2 ml in each conjunctival sac) routes. the routes of inoculation and infectious dose were chosen to reflect a combination of most likely routes of exposure and to increase the potential of infection. the animals were observed at least 1× daily for the duration of the experiment for behavior, food consumption, activity level, and nasal discharge. rectal temperature was taken daily from 2 to 7 days postinoculation, then 3× weekly until the animals were euthanized. nasal and oral swab samples and fecal samples were collected into virus transport medium or virus lysis buffer daily from 0 to 7 days postinoculation (dpi), then 3× weekly until the animal was euthanized. blood was collected into evacuated edta and serum-separating tubes daily at 0-7 dpi and 3× weekly thereafter. urine was collected by convenience and at necropsy. to evaluate whether virus is exhaled from infected camels, a funnel was placed over the muzzle of each camel and connected to a vacuum pump to capture exhaled air in tissue culture media (10 ml dulbecco modified eagle medium, 1% fetal bovine serum, 0.013% se-15 (anti-foam) with an all glass impinger (ace glass inc., vineland, nj, usa). exhaled breath was collected for ≈2 minutes and analyzed by quantitative real-time pcr (qpcr) and virus titration. on days 5, 28, and 42, camels 1, 2, and 3, respectively, were euthanized, and samples were collected from nasal turbinates, lungs, trachea, larynx, pharynx, liver, spleen, kidney, bladder, urine, duodenum, jejunum, colon, rectum, abomasum, forestomachs, prescapular lymph node, retropharyngeal lymph node, tracheobronchial lymph node, mediastinal lymph node, mesenteric lymph node, medulla, and olfactory cortex. we extracted rna from swab samples, fecal samples, and serum samples using the qiaamp viral rna kit (qia-gen, valencia, ca, usa) according to the manufacturer's instructions. for detection of viral rna, we used 5 ml of rna in a one-step real-time reverse transcription pcr upe assay (24) using the rotor-genetm probe kit (qiagen) according to manufacturer's instructions. standard dilutions of a titered virus stock were run in parallel, to calculate tcid 50 equivalents in the samples (25) . we titrated swab samples in viral transport medium, whole blood, and homogenized tissues (≈10% wt/vol) for mers-cov virus by plaque assay. briefly, 10-fold serial dilutions of samples were prepared in ba-1 medium (mem, 1% bovine serum albumin, 350 mg/l sodium bicarbonate, 50 mm tris, ph 7.6, 5 mg/l phenol red) containing 100 mg gentamicin, 200,000 u penicillin g, 100 mg streptomycin, and 5 mg amphotericin/l; plaque assay was conducted as horizontal lines indicate the normal temperature range observed among these dromedary camels as calculated by mean ± 3×, the sd before inoculation. described for west nile virus (26) . plaques were counted on days 1 and 3 after the second overlay and virus titers were expressed as pfus per ml or gram. we determined neutralizing antibody titers by plaque reduction neutralization test as described (26), using a 90% neutralization cutoff. tissues were fixed for >7 days in 10% neutral-buffered formalin and embedded in paraffin. tissue sections were stained with hematoxylin and eosin. to detect mers-cov antigen, we completed immunohistochemical testing using a rabbit polyclonal antiserum against hcov-emc/2012 (1:1,000) as a primary antibody. each camel showed minor clinical signs of disease, consisting of rhinorrhea (figure 1, panel a) and a mild elevation in body temperature at 2 dpi and 5-6 dpi ( figure 1, panel b) ; no other clinical signs were observed. rhinorrhea developed in all 3 camels beginning at 2 (camels 1 and 3) and 5 (camel 2) dpi, and persisted <2 weeks. the nasal discharge drained from both nares and varied in character from serous to purulent; minor hemorrhage was observed on some occasions, but may have been caused by trauma that occurred during collection of samples. mers-cov shedding started during 1-2 dpi, as detected by the presence of infectious virus and viral rna by qpcr in nasal swab samples. infectious virus shedding was detected <7 dpi, and shedding of viral rna was detected <35 dpi in nasal swab samples (figure 2 ). low concentrations of infectious virus and viral rna were detected in oral samples, likely originating in drainage from the nasal cavity ( figure 3) . no viral rna was detected in fecal samples or in urine samples collected by convenience or at necropsy at 0, 1, 5, 14, 21, 28, and 42 dpi from the 3 camels. no infectious virus or viral rna was detected in any of the serum or whole blood samples. small quantities of mers-cov rna were detected in exhaled breath by qpcr (10 1.2 and 10 1.4 tcid 50 equivalent/ml) at 3 and 5 dpi, but infectious virus was not detected. infectious virus was detected in tissues from camel 1, which was euthanized on 5 dpi, but not in tissues obtained from camels 2 and 3, which were euthanized at28 and 42 dpi, respectively. infectious virus was detected in tissues of the upper respiratory tract (urt), including nasal turbinates, olfactory epithelium, pharynx, and larynx. in the lower respiratory tract, infectious virus was detected in the trachea and in 1 of 4 lung lobes tested. infectious virus was also detected in the retropharyngeal, mediastinal, mesenteric, and tracheobronchial lymph nodes (figure 4 ). on necropsy of camel 1 at 5 dpi, histologic lesions were found in the pseudostratified epithelial cells in the urt and the lower respiratory tract (trachea, bronchi, and bronchioles) but not in the alveoli ( figure 5) . the lesions were characterized as mild to moderate acute intraepithelial and submucosal inflammation with multifocal necrosis and loss of pseudostratified epithelial cells, comparable to the common cold among humans. multifocal loss of epithelial polarity and cilia with squamous metaplasia were observed. the epithelium was infiltrated by small-to-moderate numbers of neutrophils with fewer macrophages; similar inflammatory cells also permeated the submucosa. the submucosal glands of the trachea were multifocally necrotic and infiltrated by small numbers of neutrophils. viral antigen was detected within the epithelial cells of the nasal turbinates, larynx, trachea, bronchi, and bronchioles, but not the alveoli. in addition, viral antigen was present at the follicular mantle zone of the tonsils and mediastinal and retropharyncheal lymph nodes ( figure 5 ). the nasal turbinates, larynx, and trachea of camel 2 (necropsied at 28 dpi) had similar but milder lesions when compared with those of camel 1. the nasal turbinate, larynx, and bronchus showed small numbers of infiltrating neutrophils; however, in contrast with the condition of camel 1, the cilia and goblet cells were intact. the remainder of the respiratory tract of camel 2 was unaffected. immunohistochemical testing revealed the presence of limited viral antigen in the nasal turbinate but not in any of the other tissues at that time. no lesions or viral antigens were detected in camel 3 at 42 dpi. serum samples were collected weekly from the camels to monitor the generation of neutralizing antibodies specific to mers cov. each of the 3 camels was seronegative before inoculation. robust mers-cov specific antibody responses developed in camels 2 and 3 (euthanized on 28 and 42 dpi, respectively), detected first on 14 dpi with a plaque-reduction neutralization test titer from 20 to 40 that increased to 640 at 35 dpi (table) . camel 1 was euthanized at 5 dpi and was not tested for development of antibodies against the virus. tissues were collected at 5 days postinoculation (dpi) for camel 1, 28 dpi for vamel 2 and 42 dpi for camel 3. detectable infectious virus in the collected tissues was found only in camel 1. nasal turbinates were sampled in 3 different sections: anterior, medial, and posterior. infectious titers were determined by plaque assay. ln, lymph node. epidemiologic and surveillance data on the emergence of mers-cov strongly point toward a role for dromedary camels as a reservoir for zoonotic transmission (13) (14) (15) (16) (17) (18) (19) (20) (21) 27, 28) . to understand the ecology of mers-cov in the most likely reservoir host, we experimentally inoculated 3 young adult dromedary camels with mers cov. the disease observed was clinically benign, in agreement with the absence of overt illness reported from field surveillance studies (14, 17, 19, 21) . a large quantity of mers-cov and viral rna was detected in nasal swab specimens from each of the 3 camels. infectious virus was detected through 7 dpi, and rna was detected through 35 dpi in camel 3, which was euthanized on day 42. this route of shedding is consistent with data on naturally infected camels (14, 18, 21, 28, 29) , and the pattern of shedding suggests that the infectious period of camels may be short. mers-cov was not detected in either urine or feces, again consistent with field observations (21, 28) . the large quantities of mers-cov shed in nasal secretions by each of the 3 camels suggest that camel-tocamel and camel-to-human transmission may occur readily through direct contact and large droplet, or possibly fomite transmission. histopathologic examination revealed that the urt, specifically the respiratory epithelium in the nasal turbinates, is the predominant site of mers-cov replication in camels. neutralizing antibodies were detected from 14 dpi onward, reaching a maximum neutralizing titer of 640 after 35 days. serologic studies in camels in the field have reported mers-cov neutralizing titers as high as 5,120 (14, 16) , potentially indicative of repeated exposure and re-infection. the study reported here was done on the basis of inoculation of 3 male animals with a human isolate of mers-cov, and the study design we used imposed several limitations on how these data inform what occurs in natural infections. the camels we inoculated were exposed to a high dose of virus by 3 simultaneous routes of inoculation. in retrospect, the inoculation dose does not seem excessive, based on the large quantity of virus shed nasally in all 3 animals ( figure 2 ). the total dose inoculated was relatively equivalent to the amount of virus present in a single nasal swab sample taken during the first days postinoculation, and it seems probable that a camel shedding this quantity of virus would readily infect other camels or humans with which it had direct contact. the fact that we inoculated the camels with the virus by 3 routes precludes drawing conclusions regarding efficiency of transmission by a particular route, which is a topic that should be addressed in future studies. the influence of camel age on susceptibility and dynamics of virus shedding is another notable parameter that requires further study. it seems likely that productive infection and shedding of virus in natural settings occurs predominantly in juvenile camels (28) . this could be the result of an intrinsic difference in age-related susceptibility, but is more likely related to the immunologically naïve status of the animals in the context of a high force of infection after decay of passively acquired antibodies. the animals we infected were young adults, but were seronegative and therefore probably as susceptible as juveniles from mers-cov-endemic regions. another aspect of pathogenesis not addressed here is whether virus is present in milk or meat from infected camels and thereby poses another potential route of exposure to humans who consume such products. despite these limitations, the magnitude and pattern of virus shedding was essentially identical in all 3 animals and supports the available epidemiologic data indicating that camels are likely a major reservoir host for mers-cov. additional experimental and field studies are clearly required to address the duration of shedding of infectious mers-cov from infected camels, to determine whether infection results in protective immunity, and to clarify the burden of illness among humans resulting from transmission from camels. emerging infectious diseases • www.cdc.gov/eid • vol. 20, no. 12, december 2014 isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. global alert and response. middle east respiratory syndrome coronavirus (mers-cov)-update investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in florence the emergence of the middle east respiratory syndrome coronavirus hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans middle east respiratory syndrome coronavirus in bats, saudi arabia a decade after sars: strategies for controlling emerging coronaviruses recovery from severe novel coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia evidence for camel-to-human transmission of mers coronavirus concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques dynamics of passive immunity to west nile virus in domestic chickens (gallus gallus domesticus) middle east respiratory syndrome coronavirus antibody reactors among camels in dubai mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through wholegenome analysis and virus cultured from dromedary camels in saudi arabia we thank bart haagmans and ron fouchier, for providing mers-cov (isolate hcov-emc/2012); tina thomas, dan long, and rebecca rosenke for histopathologic examination; and anita mora and ryan kissinger for figure preparation. all animal work in this study was approved by the institutional animal care and use committee of colorado state university and was performed in compliance with recommendations in the guide for the care and use of laboratory animals of the national institute of health.ms adney is a graduate student at colorado state university in fort collins, colorado. her research focus is on the pathogenesis of emerging infectious diseases. key: cord-320921-eumuid3r authors: widagdo, w.; okba, nisreen m. a.; richard, mathilde; de meulder, dennis; bestebroer, theo m.; lexmond, pascal; farag, elmoubasher a. b. a.; al-hajri, mohammed; stittelaar, koert j.; de waal, leon; van amerongen, geert; van den brand, judith m. a.; haagmans, bart l.; herfst, sander title: lack of middle east respiratory syndrome coronavirus transmission in rabbits date: 2019-04-24 journal: viruses doi: 10.3390/v11040381 sha: doc_id: 320921 cord_uid: eumuid3r middle east respiratory syndrome coronavirus (mers-cov) transmission from dromedaries to humans has resulted in major outbreaks in the middle east. although some other livestock animal species have been shown to be susceptible to mers-cov, it is not fully understood why the spread of the virus in these animal species has not been observed in the field. in this study, we used rabbits to further characterize the transmission potential of mers-cov. in line with the presence of mers-cov receptor in the rabbit nasal epithelium, high levels of viral rna were shed from the nose following virus inoculation. however, unlike mers-cov-infected dromedaries, these rabbits did not develop clinical manifestations including nasal discharge and did shed only limited amounts of infectious virus from the nose. consistently, no transmission by contact or airborne routes was observed in rabbits. our data indicate that despite relatively high viral rna levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission. middle east respiratory syndrome coronavirus (mers-cov) is a novel pathogen that is known to infect dromedary camels and humans [1, 2] . seroepidemiological studies indicate that this virus has been circulating in dromedary camels in the arabian peninsula and africa for decades [3] [4] [5] . mers-cov sequences obtained from these camels are largely similar to those obtained from human mers cases in corresponding regions, thus providing evidence that dromedary camels act as the zoonotic source of this virus [6, 7] . however, many primary human mers cases do not have a history of direct contact with these animals [8] . this suggests the presence of unidentified routes of human-to-human transmission or the involvement of other animal species in spreading the virus to humans. besides dromedary camels, other animal species, i.e. llamas, alpacas, and pigs have been shown to be susceptible and develop upper respiratory tract infection upon experimental intranasal mers-cov inoculation [9] [10] [11] . this is in line with the expression of the mers-cov receptor, dipeptidyl peptidase-4 (dpp4), in their nasal epithelium [9] . mers-cov-seropositive llamas and alpacas have been reported in the field, and mers-cov-experimentally-inoculated alpacas have also been shown to transmit the virus via contact [12] [13] [14] . to further understand the zoonotic potential of mers-cov, it is crucial to delineate the factors involved in the spread of the virus among dromedaries as well as other animal species. in order to gain insight into these factors, we performed mers-cov transmission experiments in rabbits. we have previously shown that rabbits are susceptible to mers-cov and develop both upper and lower respiratory tract infection upon virus inoculation [15] . naïve recipient rabbits were housed with mers-cov-inoculated donor rabbits either in the same or in adjacent cages to determine whether mers-cov can be transmitted via contact or airborne routes, respectively [16] . donor rabbits were found to shed high levels of viral rna but a limited amount of infectious virus thus potentially restricting mers-cov transmission in these animals. in vivo experiments in this study were performed using passage 7 human isolate mers-cov emc strain (hcov-emc/2012) and passage 3 isolate mers-cov (qatar15/2015; genbank acc. no. mk280984) that were propagated in vero cells as described earlier [9] . qatar15 was isolated from a 69 years old qatari man that developed severe pneumonia and was pcr confirmed to have a mers-cov infection [17] . animal experiments were approved and performed according to the guidelines from the institutional animal welfare committee (no. 201300121 approved on 17 july 2013, 122-17-01 approved on 28 september 2017, and avd277002015283-wp01 approved on 2 november 2016). the studies were performed under biosafety level 3 (bsl3) conditions. to compare whether different routes of mers-cov inoculation generate similar clinical outcomes, twelve 6-month-old new zealand rabbits (oryctolagus cuniculus), specific pathogen free, and seronegative for mers-cov were divided into four groups. animals were inoculated under ketamine-medetomidine anesthesia either (a) intranasally with 200 µl of 1 × 10 6 tcid 50 /ml mers-cov; (b) intranasally with 1 ml 1 × 10 6 tcid 50 /ml mers-cov; (c) intranasally with 200 µl of 1 × 10 6 tcid 50 /ml mers-cov and intratracheally with 3 ml 4 × 10 6 tcid 50 /ml mers-cov; or (d) intranasally with 1 ml pbs. the intranasal inoculums were divided equally over both nostrils. nasal and throat swabs were obtained daily from day 1 up to day 4 post inoculation. these animals were then sacrificed on day 4 and respiratory tract tissues were collected. to compare the clinical outcomes of the mers-cov emc strain and the qatar15 strain, ten new zealand rabbits were divided into two groups and inoculated with 1 ml of 1 × 10 6 tcid 50 /ml of each mers-cov strain intranasally. nasal and throat swabs were obtained from day 1 up to day 4 post inoculation and these animals were sacrificed on day 4. to study mers-cov transmission, a modified version of the previously described influenza a virus ferret transmission set-up was used. this set-up consists of two clear polymethyl methacrylate cages of different sizes. donor rabbits and direct contact recipients were housed in a cage of 35 cm × 30 cm × 65 cm (w × h × l), whereas airborne recipients were housed in a cage of 30 cm × 30 cm × 55 cm (w × h × l). these cages are separated by two stainless steel grids 10 cm apart to prevent direct contact but still allow airflow from the donor rabbit to the airborne recipient rabbit. these transmission cages allow the experiment to be conducted in negatively pressured isolators in the bsl3 facility, with hepa-filtered airflow <0.1 m/s [18] . since these cages were too small for new zealand rabbits, we chose a smaller-sized breed, the netherland dwarf rabbits (oryctolagus cuniculus), for the mers-cov transmission experiment. we used both male and female rabbits with an age viruses 2019, 11, 381 3 of 13 range of 6 months-3 years in these experiments. first, three mers-cov seronegative netherland dwarf rabbits were inoculated intranasally with 1 ml of 1 × 10 6 tcid 50 /ml mers-cov qatar15 strain (500 µl per nostril) to show that they are equally susceptible to mers-cov as the new zealand rabbits. nasal and throat swabs were obtained daily up to 4 days post inoculation. these animals were then sacrificed on day 4 and their respiratory tract tissues were collected. for the virus transmission experiment, twelve netherland dwarf rabbits were randomly distributed into four individually housed groups. one naïve rabbit from each group was inoculated intranasally with 1 ml of 1 × 10 6 tcid 50 /ml mers-cov (500 µl per nostril), thus acting as donor rabbits. the other two rabbits were used as direct contact and airborne recipients, respectively. donor and direct contact animals were of the same sex. all animals were sacrificed at 14 days post exposure and blood was collected to assess seroconversion. nasal swabs, throat swabs, and respiratory tract tissue samples were evaluated for the presence of infectious virus by virus titration, and for viral rna by rt-qpcr against the upe gene as previously described [9] . samples with a cycle threshold less than forty were considered as positive for mers-cov rna. viral rna was quantified as genome equivalents (ge) using mers-cov strain emc (containing 10 6 tcid 50 /ml) as a calibrator. virus titration was performed in serial 10-fold dilutions on vero cells. cells were monitored under a light microscope at day 6 for cytopathic effect. the amount of infectious virus in swab samples was calculated by determining the tcid 50 . statistical analysis was performed using the graphpad prism program (la jolla, ca, usa). kruskal-wallis test was applied due to the non-normal distribution of our data as priorly determined by shapiro-wilk test. the significant difference between groups was determined at a p-value < 0.05. respiratory tract tissue samples were collected in formalin and embedded in paraffin for pathological analysis. hematoxylin-eosin staining was performed for histopathological analysis. the presence of mers-cov nucleoprotein and mers-cov rna was detected by immunohistochemistry and in-situ hybridization, respectively, using previously published protocols [15] . the localization of dpp4 in the respiratory tract of non-infected new zealand rabbits was analyzed using an optimized immunohistochemical assay [15, 19] . collected serum samples were tested for mers-cov neutralizing antibodies using a virus neutralization assay and for mers-cov s1-specific antibodies using mers-cov s1 elisa according to the previously published protocols [9] . goat anti-rabbit igg conjugated with hrp (1:2000, dako, glostrup, denmark) was used as a secondary antibody in the elisa. rabbits are the smallest animal species that can be naturally infected by mers-cov. we previously reported that they develop both upper and lower respiratory tract infection upon mers-cov inoculation [15] , suggesting the expression of the viral receptor at these locations. using immunohistochemistry, we analyzed the dpp4 expression in rabbit respiratory tract tissues. in the upper respiratory tract, dpp4 is strongly expressed at the apical surface of both nasal respiratory and olfactory epithelium ( figure 1 ). in the lower respiratory tract, dpp4 is present in both bronchiolar and alveolar epithelial cells, although some variation in dpp4 expression was observed throughout the lungs. dpp4 is either absent, limitedly expressed on alveolar type ii cells, or expressed on both alveolar type i and ii cells ( figure 1 ). thus, these data highlight a broad dpp4 expression in the respiratory tract different human mers-cov strains have been isolated since the emc/2012 strain was first characterized [22, 23] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar15/2015) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar15 strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs ( figure 3 ). following this result, the mers-cov transmission experiment was performed using the qatar15 strain, the more recent strain of these two. type ii cells are indicated with arrows, and type i cells with an arrowhead. nasal epithelium and bronchiolar epithelium pictures were taken at a 400× magnification, and alveolar epithelium at 1000×. the isotype control showed no background signal in our assay. in our previous study, we inoculated rabbits both intranasally and intratracheally [15] . intratracheal inoculation is quite invasive, and thus requires a skillful operator to minimize procedure-related damage in the respiratory tract. in contrast, intranasal inoculation is less invasive and had been used in other studies to infect rabbits with mers-cov [20, 21] . here we investigated whether intranasal mers-cov inoculation is sufficient to induce both upper and lower respiratory tract infection in rabbits, in comparison to combined intranasal and intratracheal inoculation. three new zealand rabbits (oryctolagus cuniculus) were inoculated with mers-cov emc strain either intranasally with 200 µl of 1 × 10 6 tcid 50 /ml (group a); intranasally with 1 ml of 1 × 10 6 tcid 50 /ml (group b); intranasally with 200 µl of 1 × 10 6 tcid 50 /ml and intratracheally with 3 ml of 4 × 10 6 tcid 50 /ml (group c); or intranasally with 1ml of pbs as a negative control (group d). all three groups of mers-cov inoculated rabbits developed minimal clinical manifestations and histopathological lesions. the amount of viral rna shed in the nasal and throat swabs did not vary among the inoculated groups (figure 2a,b) . however, in the lungs of the rabbits, the amount of viral rna was significantly lower in group a than in groups b and c ( figure 2c ). in line with these observations, fewer mers-cov-infected cells were observed in the lungs of group a animals compared to groups b and c ( figure 2d ). based on these results, we decided to use intranasal inoculation with 1 ml of 1 × 10 6 tcid 50 /ml mers-cov for our subsequent experiments. pictures were taken at a 400× magnification, and alveolar epithelium at 1000×. the isotype control showed no background signal in our assay. different human mers-cov strains have been isolated since the emc/2012 strain was first characterized [22, 23] . however, studies that evaluate phenotypic differences between these strains in animals are currently lacking. we investigated whether a more recent mers-cov strain (qatar15/2015) replicates differently in rabbits in comparison to the emc strain. we found that rabbits inoculated with the mers-cov emc strain and those with the qatar15 strain developed an equally mild infection and shed similar levels of viral rna in their nasal and throat swabs (figure 3 ). following this result, to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, 10 cm apart [16] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar 15 strain to determine their susceptibility to the virus. similar to the new zealand rabbits [15] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure 4a,b) . identical to the new zealand rabbits [15] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and 24 h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day 7 or 9 post inoculation/exposure for the donor and direct contact rabbits, respectively, and day 9 post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~10 5 -10 6 tcid50 ge/ml) and the throat swabs (~10 3 -10 4 tcid50 ge/ml). the nasal and throat swabs were obtained from day 0 (before inoculation) until day 4 post inoculation. the amount of viral rna is displayed in genome equivalents per ml (ge/ml). dashed lines depict the detection limit of the assay. all error bars represent standard deviations. to study mers-cov transmission, an experimental set up previously used to investigate influenza a virus transmission between ferrets was used. this set up consists of two polymethyl methacrylate cages separated with two steel grids, 10 cm apart [16] . because this set-up was too small to house new zealand rabbits, we used a smaller-sized breed, the netherland dwarf rabbits. prior to the virus transmission experiment, netherland dwarf rabbits were inoculated with mers-cov qatar 15 strain to determine their susceptibility to the virus. similar to the new zealand rabbits [15] , netherland dwarf rabbits shed viral rna in the nasal and throat swabs as well as in the respiratory tract tissues upon intranasal inoculation ( figure 4a,b) . identical to the new zealand rabbits [15] , the mers-cov-inoculated netherland dwarf rabbits did not develop any clinical signs, including nasal discharge, and showed minimal histopathological lesions and immune cell infiltration in the respiratory tract. donor rabbits at day 1 post inoculation, and in one of the donors up to day 7. in the throat swabs, infectious virus was only detected in two donors on day 1, up to day 5 in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration (figure 5c,d) . serological analysis of samples collected 14 days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure 6a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [15, 21] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. to study mers-cov transmission, four netherland dwarf rabbits were intranasally inoculated with mers-cov. six-hours later, each of them was co-housed with one naïve rabbit in the same cage, and 24 h later another one was co-housed in an adjacent cage to determine whether mers-cov could be transmitted through contact and/or airborne routes. nasal and throat swabs were collected every other day up to day 7 or 9 post inoculation/exposure for the donor and direct contact rabbits, respectively, and day 9 post exposure for the airborne recipient ones. both viral rna and infectious virus were quantified in these samples. we found that all donor rabbits shed high loads of viral rna in both the nasal swabs (~10 5 -10 6 tcid 50 ge/ml) and the throat swabs (~10 3 -10 4 tcid 50 ge/ml). the amount of viral rna shed by the inoculated rabbits remained high until day 7 post inoculation. on the other hand, recipient rabbits housed in the same cage (direct contact recipients), or adjacent cage (airborne recipients), shed limited amounts of viral rna (~10 tcid 50 ge/ml) in both nasal and throat swabs. among four animals in each group, only two direct contact recipient and two airborne recipient rabbits had detectable viral rna up to day 5 post inoculation in the nasal swabs, while in the throat swabs, viral rna was only detected in one direct contact recipient and one airborne recipient rabbit at day 1 post inoculation ( figure 5a,b) . infectious virus was detected at low level (~10 2 tcid 50 /ml) both in the nasal and throat swabs of the donor rabbits; in the nasal swabs of all donor rabbits at day 1 post inoculation, and in one of the donors up to day 7. in the throat swabs, infectious virus was only detected in two donors on day 1, up to day 5 in one of them. in contrast, none of the swabs from recipient rabbits was positive for virus titration ( figure 5c,d) . serological analysis of samples collected 14 days after exposure showed that only the donor rabbits seroconverted and developed neutralizing antibodies ( figure 6a,b) . the antibody response of these directly inoculated rabbits was relatively low, confirming the results of previous studies [15, 21] . this indicates that these rabbits developed mers-cov infection while the contact and airborne-exposed rabbits did not, supporting the results of the virus titration. current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [3] [4] [5] 24] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of current data indicate that mers-cov is highly endemic in dromedary camels in the arabian peninsula and africa and has been circulating in these animals for decades [3] [4] [5] 24] . this suggests that this virus is easily transmitted between dromedary camels. from an epidemiological point of view, it is important to know whether other animal species in the region may also spread the virus to humans or other animal species. in vitro, mers-cov has been found to infect cells from a broad range of animal species including old and new world camelids as well as primates, bats, cows, sheep, goats, pigs, horses, and rabbits [15, 25, 26] . the dpp4 viral receptor of these species, especially rabbits, has high similarity to that of humans and dromedary camels, especially in the region that interacts with the spike protein, and thus can facilitate mers-cov infection [25] [26] [27] . the new world camelids, i.e. llamas and alpacas, have been shown to seroconvert to mers-cov when present in regions where mers-cov is circulating and may transmit the virus [12] [13] [14] . it is currently unclear why, besides camelids, other livestock animals do not seem to transmit the virus to humans [24, [28] [29] [30] . to further understand the transmission potential of mers-cov, we performed virus transmission experiments using rabbits as animal model. to perform the virus transmission experiments, we housed mers-cov-inoculated rabbits together with naïve contact rabbits either in the same or adjacent cages. rabbits developed both upper and lower respiratory tract infection upon mers-cov inoculation [15] , either via intranasal or combined intranasal and intratracheal routes, in line with the localization of dpp4 in their respiratory tract epithelium. the amount of viral rna being shed by the inoculated rabbits during the first three days post inoculation is almost as high as that of the mers-cov-inoculated dromedary camels [31, 32] . however, none of the direct contact and airborne-exposed rabbits developed any clinical signs, shed significant levels of viral rna, shed infectious virus, nor did they seroconvert. one possible reason for this lack of transmission is the limited amount of infectious virus being shed by the inoculated rabbits [21] . previous studies have shown that in the nasal and lung tissues of experimentally infected rabbits, infectious virus was generally detected in a limited amount despite the abundant presence of viral rna and virus nucleoprotein [15, 21] . alternatively, low levels of infectious virus transmitted to recipient animals may be unable to initiate a productive infection due to the presence of host proteins that restrict replication. comparable to rabbits, mers-cov-infected pigs and goats develop minimal clinical signs, hardly shed infectious virus, and barely spread the virus to naïve animals [28, 33] . in contrast, mers-cov-infected dromedary camels develop nasal discharge and shed a high amount infectious virus (10 4 -10 5 tcid 50 /ml), almost equal to the amount of viral rna being shed (10 5 -10 6 ge/ml), during the first 4 days post inoculation [31, 32] . these interspecies differences may indicate presence of nasal discharge and infectious virus shedding as critical factors in mers-cov transmission. in humans, levels of infectious virus shed by mers-cov patients have rarely been reported. however, mers-cov patients that transmit the virus have been shown to shed a higher amount of viral rna in their swabs compared to those that do not, supporting the quantity of virus shed as an important factor in the transmission of mers-cov between humans [34] . for influenza a viruses, infectious virus shedding has been documented as one of the main determinants of airborne virus transmission. using ferrets as an animal model, it has been reported that a reduction in infectious virus shedding in the nasal swabs can subsequently limit virus transmission [35] [36] [37] . our results show that despite the presence of dpp4 in the upper respiratory tract, accompanied by mers-cov replication at this site, a limited amount of infectious virus was shed. similarly, titration of rabbit lung homogenates that show high levels of viral rna and presence of nucleoprotein ( figure 2c ,d) resulted in only low levels of infectious virus, in line with earlier observations [15, 21] . at this stage, it is not clear which host mediated mechanisms limit the production of infectious virus while allowing viral rna to still be shed at high levels. since restriction of infectious virus shedding in the rabbits already occurred one day post inoculation, activation of host innate immune responses, including type i interferon induction, may be relevant. in vitro studies have shown that mers-cov is relatively sensitive to type i interferon-mediated antiviral activities [38, 39] . in human plasmacytoid dendritic cells, mers-cov inoculation leads to secretion of large amount of type i and iii interferons and production of viral rna, but hardly any infectious virus is being produced [40] . it is also possible that most infectious virus shed by these rabbits is defective, lacking the capacity to efficiently infect target cells. further studies are needed to elucidate the mechanisms that restrict mers-cov replication in rabbits compared to dromedary camels. potentially, some of the mers-cov accessory proteins shown to antagonize immune responses including production of interferon, may not work efficiently in some mers-cov susceptible species, including rabbits. it is intriguing to investigate whether a similar phenomenon occurs in some human mers-cov infections and whether this is linked to the development of asymptomatic to mild clinical manifestations [41] . this might partly explain why mers-cov transmission in humans is rather inefficient in comparison to dromedary camels [42, 43] , and why camelids that secrete high levels of infectious virus are the only known zoonotic source of mers-cov [2, 7, 14, 31] . deciphering these mechanisms could potentially offer insight into understanding mers-cov transmission as well as developing novel treatments to tackle the ongoing outbreaks. isolation of a novel coronavirus from a man with pneumonia in saudi arabia isolation of mers coronavirus from a dromedary camel middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia geographic distribution of mers coronavirus among dromedary camels zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels infection, replication, and transmission of middle east respiratory syndrome coronavirus in alpacas asymptomatic middle east respiratory syndrome coronavirus infection in rabbits airborne transmission of influenza a/h5n1 virus between ferrets new case-qatari supreme council of health pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody deletion variants of middle east respiratory syndrome coronavirus from humans epidemiology of a novel recombinant middle east respiratory syndrome coronavirus in humans in saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 spiking the mers-coronavirus receptor inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus experimental transmission using a pig model risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea impact of prior seasonal h3n2 influenza vaccination or infection on protection and transmission of emerging variants of influenza a(h3n2)v virus in ferrets efficacy of seasonal live attenuated influenza vaccine against virus replication and transmission of a pandemic 2009 h1n1 virus in ferrets predicting "airborne" influenza viruses: (trans-) mission impossible? mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-alpha treatment inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus world health organization mers situation update transmission of mers-coronavirus in household contacts mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-331228-wbd0s4fo authors: shehata, mahmoud m.; gomaa, mokhtar r.; ali, mohamed a.; kayali, ghazi title: middle east respiratory syndrome coronavirus: a comprehensive review date: 2016-01-20 journal: front med doi: 10.1007/s11684-016-0430-6 sha: doc_id: 331228 cord_uid: wbd0s4fo the middle east respiratory syndrome coronavirus was first identified in 2012 and has since then remained uncontrolled. cases have been mostly reported in the middle east, however travel-associated cases and outbreaks have also occurred. nosocomial and zoonotic transmission of the virus appear to be the most important routes. the infection is severe and highly fatal thus necessitating rapid and efficacious interventions. here, we performed a comprehensive review of published literature and summarized the epidemiology of the virus. in addition, we summarized the virological aspects of the infection and reviewed the animal models used as well as vaccination and antiviral tested against it. coronaviruses (cov) became known to cause human disease in the twentieth century. hcov-229e and hcov-oc43 were discovered in the 1960s and shown to cause respiratory infections in humans [1, 2] . with the emergence of sars-cov in 2003 [3] , two other human coronaviruses were discovered, hcov nl63, hcov hku1 [4] . in 2012, a new type of coronavirus was detected as the cause of severe respiratory illness in humans. the first case was a 60-year-old male from saudi arabia admitted to hospital with acute respiratory illness leading to pneumonia and acute renal failure. the virus initially named as human corona virus-emc [5] , is currently known as the middle east respiratory syndrome coronavirus (mers-cov) [6] . classification and nomenclature of mers-cov phylogenetically, mers-cov is a lineage c β coronavirus (β-cov) and is closely related to bat coronaviruses hku4 and hku5. the rooted phylogenetic analysis showed that mers-cov had an amino acid sequence identity less than 90% to all other known covs [7] . the virus initially named by many different working groups as novel coronavirus, human coronavirus emc, human b coronavirus 2c emc, human b coronavirus 2c england-qatar, human b coronavirus 2c jordan-n3, and b coronavirus england 1, which represented the places where the first complete viral genome was sequenced (erasmus medical center, rotterdam, the netherlands) or where the first laboratory-confirmed cases were identified or managed (jordan, qatar, and england) was later named as mers-cov by the coronaviruses study groups of ictv [5, 6, 8] . mers-cov is an enveloped virus with a positive sense rna genome. coronavirus genomes range between 25 to 32 kb in size. the complete sequence of hcov-emc-2012 resulted in 30 119 nucleotides sequence [7] . coronavirus genomes are polycistronic with large replicase open reading frames orf1a and orf1b which are subsequently cleaved into 15 or 16 nonstructural proteins (nsps). the region downstream of orf1b encode smaller genes including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural protein [9] [10] [11] . the functional receptor for mers-cov is the dipeptidyl peptidase 4 (dpp4) which is present on human nonciliated bronchial epithelial cells surfaces [12] . the dpp4 protein displays high amino acid sequence conservation across different species, including the sequence that was obtained from bat cells. cell lines susceptibility studies showed that mers-cov infected several human cell lines, including histiocytes as well as respiratory, kidney, intestinal, and liver cells [13] . the range of tissue tropism in vitro was broader than that for any other known human coronavirus [14] . mers-cov can also infect nonhuman primate, porcine, bat, civet, rabbit, and horse cell lines all possessing the dpp4 receptor [15] . the replication cycle of mers-cov consists of numerous steps as illustrated by lu et al. [30] . the mers-cov s protein is a class i fusion protein composed of two subunits: the amino n-terminal receptor binding s1 and carboxyl c-terminal membrane fusion s2 subunits. the s1/s2 junction is a protease cleavage site which is responsible for membrane fusion activation, virus entry, and syncytium formation. the s1 c domain contains the receptor binding domain (rbd), and an n domain [13] . neutralizing monoclonal antibodies against the rbd may inhibit virus entry into cells and receptor-dependent syncytium formation in cell culture, hence vaccines containing the rbd induced high levels of neutralizing antibodies in mice and rabbits [16] [17] [18] . dpp4 is the cell key functional receptor for the mers-cov s protein [19] . mers-cov is the first cov that has been identified to use dpp4 as a receptor [19, 20] . dpp4 has important roles in glucose metabolism, t cell activation, chemotaxis modulation, cell adhesion, and apoptosis [19, 21] . the s2 subunit contains five domains: a fusion peptide, the heptad repeat 1 (hr1) and hr2 domains, a transmembrane domain, and a cytoplasmic domain, which form the stalk region of s protein that facilitates fusion of the viral and cell membranes [22, 23] . the binding of the s1 subunit to the cellular receptor triggers conformational changes in the s2 subunit, which inserts its fusion peptide into the target cell membrane to form a six-helix bundle fusion core between the hr1 and hr2 domains that approximates the viral and cell membranes for fusion. mers-cov utilizes many pathways for membrane fusion depending on available host proteases, such as transmembrane protease serine protease 2 (tmprss2), trypsin, chymotrypsin, elastase, thermolysin, endoproteinase lys-c, and human airway trypsin-like protease. proteases cleave the s protein into the s1 and s2 subunits to activate the mers-cov s protein for endosome-independent host cell entry at the plasma membrane [24] [25] [26] . in addition to the pervious fusion proteases furin has been identified recently to play an essential role in the mers-cov s protein cleavage activation into their biologically active forms [27, 28] . after cell entry, the virion particle disassembles to release the nucleocapsid and viral rna into the cytoplasm for expression of viral polyproteins pp1a and pp1ab. doublemembrane vesicles and convoluted membranes are formed by the attachment of the hydrophobic domains of the mers-cov replication machinery to the limiting membrane of auto-phagosomes [29] . the viral polyproteins pp1a and pp1ab are cleaved by papain-like protease and 3c-like protease into nsp1 to nsp16 [7, 30, 31] . these nonstructural proteins form the replication-transcription complex, which regulates transcription and viral protein expression [29] . after the production of abundant viral rna and structural and accessory proteins, the n protein binds to the genomic rna in the cytoplasm to form the helical nucleocapsid (viral core). the viral core is enveloped by budding through intracellular membranes between the endoplasmic reticulum and golgi apparatus [32] . the s, e, and m proteins are transported to the budding virion, where the nucleocapsid probably interacts with m protein to generate the basic structure and complexes with the s and e proteins to induce viral budding and release from the golgi apparatus [33] . mers-cov replication cycle is completed by releasing the progeny virions through the cell membrane via exocytosis pathway. mice mers-cov strain hcov-emc/2012 was inoculated to three different mouse strains (immunocompetent balb/c mice, 129s6/svev and innate immune-deficient 129/ stat1 -/mice) intranasally. no significant weight loss was observed and infectious virus could not be detected in the lungs. only moderate pathological lesions were observed in the lungs. hence no viral replication was observed in these strains of mice [34] . zhao et al. developed a mouse model transduced with a recombinant adenovirus vector expressing hdpp4 (ad5-hdpp4) in lung tissue. inoculation of mers-cov in these mice resulted in mers-cov replication but without mortality. young mice cleared from mers-cov in 6-8 days and old mice in 10-14 days. perivascular and peribronchial lymphoid infiltration was observed, with progression to an interstitial pneumonia postinfection [35] . in another study, transgenic mice expressing hdpp4 were susceptible to mers-cov infection. infectious virus was isolated from lung and brain tissue and weight loss was observed [36] . pascal et al. developed humanized transgenic mouse. no mortality or clinical signs was observed but interstitial pneumonia and significant lung disease were observed histopathologically, suggesting that humanized dpp4 mouse is a model for mers-cov infection in which pathological changes resembles mers-cov infection in humans [37, 38] . the rhesus macaque was the first animal model used for mers-cov infection as it possessed dpp4 receptor [38, 39] . in infected animals, an increase in respiratory rates, body temperature, cough and reduced appetite was observed with mild to moderate severity. infectious virus isolated only from the lower respiratory tract. viral rna was detected in the conjunctiva, nasal mucosa, tonsils, pharynx, trachea, bronchus and lungs. mild to marked interstitial pneumonia with dark red lesions appeared in lungs. seroconversion of neutralizing antibodies began at 7 dpi and increased in titer with time. the development of a transient pneumonia, rapid replication, and tropism of mers-cov for the lower respiratory tract resembled the severity of the disease observed in humans [38, 40, 41] . similarly, the common marmoset was shown to possess the dpp4 receptor [42] . radiographic imaging showed mild to severe bilateral interstitial infiltration and extensive bronchointertitial pneumonia in infected animals. infectious virus was detected in lower and upper respiratory tract tissue and viral rna was detected in nasal mucosa, oropharyngeal swabs, blood, conjunctiva, lymph nodes, gastrointestinal tract, kidney, heart, adrenal gland, liver, spleen, brain and lungs [42] . inoculation of syrian hamsters and ferrets with mers-cov did not result in infection [12, 43] . rabbits may be used as a model to study pathogenesis, transmission, and disease control strategies of mers-cov in vivo as they seroconvert and shed virus after inoculation [44] . in september 2012, a novel coronavirus infection was noted in promed mail [45] . the virus was isolated from the sputum of a 60-year-old saudi male, who was admitted to a hospital with pneumonia and acute kidney injury in june 2012. a few days later, another report appeared describing an almost identical virus detected in a patient in qatar with acute respiratory syndrome and acute kidney injury. the patient had a recent travel history to saudi arabia and then traveled to uk for further medical care [5, 46, 47] . two cases from jordan (april 2012) were retrospectively diagnosed as mers patients. since that time, more than 1542 cases of mers-cov infection have been reported including 544 deaths [48] . the actual number of cases could be higher than those reported [49] . an outbreak of more than 180 confirmed cases including 36 deaths occurred in south korea in may and june 2015. the median age of korean cases were 55 years (range: 16 to 87 years), 60% were men, and 14% were health care professionals. the index case was a 68-year-old male who had recently traveled to several countries in the arabian peninsula [50] . disease control and prevention (cdc), and the ministry of health of saudi arabia (mohsa) as asymptomatic, mild, severely symptomatic, or mortal. cases may be classified into suspected, probable, and confirmed [52, 53] . any person with laboratory confirmation of infection with mers-cov irrespective of clinical signs and symptoms is considered as a confirmed case. who criteria for laboratory confirmation require detection of viral rna or acute and convalescent serology. the presence of nucleic acid can be confirmed by positive results from at least two sequence-specific rrt-pcrs or a single sequence-specific rrt-pcr test and direct sequencing from a separate genomic target [54] . a case confirmation by serological methods requires demonstration of seroconversion in two samples collected at least 14 days apart using at least one screening assay (enzyme-linked immunoassay, immunofluorescence assay) and a neutralization assay. a probable case is defined by the following criteria, a febrile acute respiratory illness as pneumonia or acute respiratory distress syndrome, direct contact with a confirmed mers-cov case and unavailability of mers-cov testing or results being inconclusive for a single inadequate specimen. any person who developed a fever and pneumonia or acute respiratory distress syndrome with a history of travel to countries in or near the arabian peninsula within 14 days before symptom onset or was in contact with a traveler from this region who developed a febrile respiratory illness is considered as a mers-cov suspected case. the who, cdc, and mohsa recommended laboratory diagnostics for mers-cov infection [6, 9, 51, 55, 56] . mers-cov cases must be confirmed by at least two positive qrt-pcr tests on two different specific genomic regions or single positive qrt-pcr with a sequence of another positive genome fragment [57] . the who algorithm for testing mers-cov relies on qrt-pcr and sequencing [58] . available real-time tests include an assay targeting the rna upstream of the e gene (upe) as a highly sensitive screening assay and three confirmatory assays targeting open reading frames (orf 1a and 1b) and/ or n gene. the orf 1a assay is of equal sensitivity to the upe assay. the orf 1b assay is less sensitive but is useful for confirmation. these assays are specific for mers-cov and have not shown cross-reactivity with other respiratory human coronaviruses. for sequencing, two target genes, the rna-dependent rna polymerase (rdrp, present in orf 1b) and n genes are enough to confirm the existence of mers-cov rna in the samples of a patient [57] . several serologic assays including immunofluorescence assays, protein microarray assay, enzyme-linked immunosorbent assay (elisa) have been developed for the detection of mers-cov antibodies [57, [59] [60] [61] . any positive test by one of these assays should be confirmed with a neutralization assay. single serological result may be valuable for definition of probable case and should be followed by further testing for confirmation of mers-cov infection [62] [63] [64] . incubation period of mers-cov infections was studied by assiri et al. in 2013. the median incubation period was 5.2 days (95% ci 1.9-14.7 days) [65] . in another report from france of a secondary case, a patient who shared a room with an infected patient, the incubation period was estimated at 9 to 12 days [66] . in the recent outbreak in south korea during may/june 2015, the median incubation period was 6.3 days [67] . who and cdc recommended that individuals that returned from the arabian peninsula and other affected countries must be evaluated for mers-cov infections up to at least 14 days [68] . clinical features of mers-cov infections range from asymptomatic cases to mildly ill, severe pneumonia, acute respiratory distress syndrome, septic shock and mortal with multi-organ failure (table 1) [64, 65] . many other clinical features such as gastrointestinal symptoms (anorexia, nausea, vomiting, abdominal pain, diarrhea), pericarditis, and disseminated intravascular coagulation were reported [65, 69, 70] . specific clinical conditions (comorbidities) were apparently proportionate with high severity of mers-cov infections. a study by assiri et al. in saudi arabia showed that of a total of 47 patients with mers-cov infection in 2013, 45 (96%) had underlying clinical conditions, including diabetes mellitus (68%), hypertension (34%), chronic cardiac disease (28%), and chronic kidney disease (49%) [65] . this high rate of comorbidities must be interpreted with some caution, since diabetes mellitus is common in saudi arabia, and because approximately half of those 47 were part of an outbreak in a hemodialysis unit, where rates of comorbidities might be high due to chronic kidney disease [65, 71] . in another study, being on dialysis, diabetes mellitus, and age > 50 years was associated with mortality [72] . in this study, testing positive for mers-cov in a plasma sample was a predictor of severe outcome [72] . younger adults and children appeared to be less susceptible to mers-cov infection. only one study described mers-cov infection in children [73] . all of those children were discovered during contact investigations of older patients. only 2 of 11 children developed symptoms of mers-cov infection. these two children had underlying conditions (cystic fibrosis and down syndrome). the other 9 children were asymptomatic. there are few reports of mers-cov infections in pregnant women. a five-month pregnant female developed vaginal bleeding and abdominal pain after one week, then delivered a stillborn infant [74] . another case in the united arab emirates was near term phase, she gave birth to an apparently healthy baby, and died after delivery [52] . mild and asymptomatic mers-cov infections have been reported, a majority of whom were identified among the contacts of patients [62, 75, 76] . in a report from mohsa, more than 3000 contacts of patients were screened using qrt-pcr and seven healthcare workers with mers-cov infection were identified, two of whom were asymptomatic and five of whom had mild upper respiratory tract symptoms [75] . epidemiological and virological studies were conducted in attempts to determine person to person transmission of mers-cov. they studied case clustering in household and hospital outbreaks in the uk, tunisia, italy, and in healthcare facilities in saudi arabia, france, iran, and lately in south korea. those studies provided strong evidence that human-to-human transmission occurs [70, [77] [78] [79] [80] . the number of contacts infected by individuals with confirmed infections, however, appears to be limited [62] , except the outbreak of south korea in may/ june 2015, where most cases were secondary and some cases were tertiary infections [67, 81] . secondary cases often were milder or symptomless [62] . possible modes of transmission include droplet and close contact transmission, air borne transmission, and fomite transmission [82] . the majority of all laboratory-confirmed secondary cases have been associated with healthcare settings [82] . the majority of cases of jeddah, saudi arabia hospital outbreak during the spring of 2014 were acquired through human-to-human transmission due to systematic weaknesses in infection control [76] . secondary transmission rates were assessed within households and the transmission rate was around 4%, suggesting that the actual number of infection is greater than reported [62] . during the outbreak in south korea during may/june 2015, 25 secondary infections were associated with the index case, who was hospitalized from may 15 to may 17 and 11 were tertiary [83] . the median incubation period was six days for secondary cases and six days for tertiary cases. this outbreak also clearly demonstrated roles of "superspreaders," who may be responsible for a high proportion of cases [83] . for instance, a single patient infected more than 70 other people while being treated in the emergency room of a hospital in south korea for three days, 27-29 may 2015. transmissibility and epidemic potential studies of mers-cov revealed that the reproduction number (r 0 ) of patients infected with mers-cov ranged between 0.6 to 0.69 [84, 85] . the finding of an r 0 < 1 suggests that mers-cov does not yet have pandemic potential. other study suggested that r 0 values might reach to 0.8 to 1.3 in the absence of infection control [49] . shedding periods of mers-cov in humans was reported to be long as viruses were detected in lower respiratory samples of symptomatic patients for more than two weeks [86] . at instances, prolonged shedding for 6 weeks was detected in an asymptomatic healthcare worker. these findings raise concerns that asymptomatic persons could transmit infection to others in a silent manner [87] . the majority of cases have occurred in saudi arabia and united arab emirates [88] [89] [90] . many cases have also been reported outside the arabian peninsula in north africa, europe, asia, and north america as shown in table 2 . almost all cases reported outside the arabian peninsula had a travel history to it. the first cluster was in october/november 2012 in four men of the same family in riyadh, saudi arabia, two of whom died [75] . the second cluster was reported in jordan in april 2012 involving 10 healthcare workers exposed to fatal patients. in addition, seven surviving hospital contacts seroconverted suggesting that they had mers-cov infection [91] . the third cluster was reported in uk during january/ february 2013. an english resident had a travel history to saudi arabia and pakistan in january, developed a severe respiratory illness, and tested positive for both mers-cov and h1n1 influenza a, and died in march 2013 after infecting several contacts [92] . a cluster of 43 cases of mers-cov was reported in al-hasa in saudi arabia during april 2013. all those cases were directly linked to human to human contact in the same hospital. there were only two confirmed cases of healthcare workers, and three family members were detected by a survey of over 200 household contacts that visited this hospital [77] . in france, may 2013, an infection of mers-cov was reported in a patient who recently traveled to the united arab emirates. a second case who shared the hospital room with the first case tested positive. the first patient died and the second patient was critically ill. a survey of 100 healthcare workers found no other infections with mers-cov, despite the lack of use of personal protective equipment [70] . a surge in mers-cov cases was reported in saudi arabia and the united arab emirates during march and april 2014 [55, 76] . the majority of cases were associated with hospital-based outbreaks jeddah, riyadh, tabuk, and madinah in saudi arabia as well as in al ain, and abu dhabi in united arab emirates. cases included several healthcare workers, visitors, patients, and ambulance staff. person to person transmission was confirmed in > 75% of cases. the majority of infected health care workers developed mild symptomatic or asymptomatic infection, but about 15% had severe illness or died [93] . the recent outbreak of south korea occurred in may 2015. the index case was a man who had recently traveled to bahrain, the united arab emirates, saudi arabia, and qatar [55] . as of late july 2015, > 180 secondary cases were reported including 36 death and many cases had been reported among household and hospital contacts [55, 67] . in china, one case occurred in a man who traveled to china from korea following exposure to two relatives with mers-cov infection [55] . in spite of reporting of mers-cov infections throughout the year, some evidence on disease seasonality occurred. the first identified cases of mers-cov infection were reported in april and june 2012 [5, 46, 47] . a high increase in cases was reported in april and may 2013 followed by a surge in case reporting in april and may 2014. increase in case reporting in march to may 2013 were attributed to infection from infected young camels [94, 95] sources and modes of transmission of mers-cov are still unclear. initially, a bat origin of mers-cov was suggested based on the relation of genome sequences between mers-cov and bat coronaviruses [96] . cell tropism studies showed that both bat coronavirus hku4 and mers-cov shared the same cell type receptors, dpp4 [4, 7, 75] . mers-cov grows readily in several bat-derived cell lines [14] . there is no evidence for direct or indirect transmission of mers-cov from bats to humans. virological studies performed in europe, africa, and asia, including the middle east, have shown that coronavirus rna sequences are found frequently in bat feces. some of the sequences were closely related to mers-cov sequences [97] [98] [99] . in a survey from saudi arabia, 823 fecal and rectal samples were tested by pcr for mers-cov, many coronaviruses sequences were detected [97] . most of the detected sequences were unrelated to mers-cov, but one sequence of 190 nucleotide in the rna-dependent rna polymerase (rdrp) gene had a 100% identity with a mers-cov. this sequence was detected from feces of a taphozous perforatus bat captured near the home of the index saudi patient. uncommon contact of humans with bats indicates that bats are not the intermediate host of mers-cov transmission but may be the reservoir of the virus [100] . dromedary camels (camelus dromedarus) appear to be the source of mers-cov. other animals like sheep, goats, and cows tested negative to anti-mers-cov antibodies. camel sera from oman, canary islands, and egypt were positive for anti-mers-cov antibodies in about 100%, 14%, and > 90% of the samples respectively [63, 101, 102] . retrospective studies on archived human sera showed no evidence of infection with mers-cov before 2012 [103] , but anti-mers-cov antibodies were detected in archived camel sera in saudi arabia in 1993 [95] , and united arab emirates in 2003 [104] , indicating circulation of mers-cov in camels for many years. bactrian camels in mongolia tested negative for mers-cov antibodies [105] . serologic studies from around the middle east suggested that camels are one of the sources of mers-cov as > 90% of adult camels tested positive and had high titers of antibodies. seropositivity was different in juvenile camels and was usually lower than in adults. these results suggested that mers-cov infections in camels occurred in young ages followed by frequent boosting [94, 95, 102, 104] . camels in other parts of the world, far from the middle east like in europe, australia, and the americas do not have mers-cov antibodies and have no evidence of infection [106] . table 3 summarizes camel serologic studies. in a study aimed to evaluate virus infectivity and shedding in camels, three adult dromedary camels were inoculated with mers-cov intratracheally, intranasally, and conjunctivally. those camels shed large quantities of virus from the upper respiratory tract and infectious virus was detected in nasal secretions for 7 days post-inoculation and viral rna for up to 35 days post-inoculation [113] . human infections with mers-cov were linked to camels. the first evidence was a study in saudi arabia in which the mers-cov full genome sequences of isolates from a man with fatal infection and from one of his camels were identical. this patient had a direct contact with his deceased camels some days before the onset of symptoms. these results suggested that mers-cov can infect dromedary camels and can be transmitted from them to humans by direct close contact [86] . in other studies, phylogenetic analyses of camel and human isolates of the mers-cov genome demonstrated that the viruses were highly identical or in some cases were similar to each other [107, 108, 114] . seroepidemiological studies shown low prevalence of mers-cov antibodies in humans in saudi arabia [103, 115] . a survey of 10 009 individuals representative of the general population of saudi arabia resulted in 15 seropositive subjects (0.15%), however, seropositivity increased 15-23 folds in camel-exposed individuals [78] . in a separate report, 7 of 87 camel shepherds and 140 slaughterhouse workers (3.1%) tested positive for mers-cov antibodies [103] . an overview of mers-cov transmission routes is illustrated in fig.2 . the development of an effective vaccine is critical for prevention of a mers-cov pandemic. some investigators have indicated that the rbd protein of mers-cov s protein is a good candidate antigen as a subunit vaccine. various rbd fragments showed the highest dpp4 binding affinity and induced the highest-titer of igg ab and neutralizing ab in mice and rabbits [17, 18, [116] [117] [118] [119] [120] . a robust neutralizing antibody response was elicited in balb/c mice against mers-cov after immunization with purified full s protein nanoparticles produced in sf9 cells infected with specific recombinant baculovirus containing the s gene [121] or a recombinant human adenoviral vectors (rad5 or rad41) containing the s or s1 genes [122, 123] . vaccinia ankara was encoded with full s protein and inoculated to balb/c mice that developed high levels of neutralizing antibodies and had induction of humoral and cell-mediated immunity [124, 125] . another study using ad5-hdpp4-transduced balb/c mice immunized with venezuelan equine encephalitis virus replicon particles containing s protein elucidated a reduction of viral titers to nearly undetectable levels and increased neutralizing antibodies [35] . recently, wang et al. developed two candidate vaccines, a subunit (full s and s1 protein fraction) and a dna vaccine (full s and s1 gene in a mammalian vrc8400 vector). the vaccine containing the full s dna and s1 protein was the most efficacious in mice and rhesus macaques [126] . using antibodies to deter mers-cov infection appears to have some promise. transfer of sera containing anti-mers-cov-s protein to or seropositive camel sera to ad5-hdpp4-transduced mice accelerated virus clearance, inhibited virus attachment, and reduced weight loss [35, 37, 127] . recently, corti et al. successfully isolated monoclonal antibodies from serum obtained from a mers-cov survivor after 200 days of infection [128] . transduced ad5-hdpp4 balb/c mice were immunized with 15 mg/kg of the mab and showed decreased lung [105] viral titers, no weight loss, and decreased peribronchial lymphoid infiltration [128] . no approved antivirals for use against mers-cov infection are yet available. the first approach performed when a new unknown virus like mers-cov emerges is testing drugs used as antiviral for similar viruses [29, 129, 130] . type i interferons and ribavirine combination exhibited acceptable results in cell culture and rhesus macaques by decreasing the host inflammatory response, replication of virus, and improved clinical outcome [129, 131, 132] . a human cohort study in saudi arabia showed that treatment with combination of ribavirin and interferon-α2b to 5 did not improve clinical outcomes but this may have been due to late treatment or due to the immunocompromised state of the patients [133] . in a retrospective study of 20 mers-cov infected patients treated with ribavirin and interferon α-2a, results showed 14-day and 28-day survival was improved by 70% and 28% in the treated group as compared to an untreated group [134] . the second approach is screening of approved drugs with known safety profiles and transcriptional signatures in different cell lines. several drugs, including antiparasitics, neurotransmitters, antibacterials, inhibitors of clathrinmediated endocytosis estrogen receptor, lipid or sterol metabolism, protein processing, and dna synthesis or repair were tested on culture cells [119, [135] [136] [137] [138] [139] . lopinavir-ritonavir combined with pegylated interferon and ribavirin therapy showed improved outcomes in infected marmosets [140] . the third approach involves in vitro inhibition of s protein to block virus entry into host cells using designed antiviral peptides targeting the hr2 domain of the s2 subunit of the mers-cov and preventing the interaction between the hr1 and hr2 domains required for the formation of the heterologous six-helix bundle in viral fusion core formation [22, 23] . other drugs that act as inhibitors for viral proteases and helicase to suppress mers-cov infection were tested [141] [142] [143] [144] [145] . other investigators studied inhibition of mers-cov infection by competitive inhibition of dpp4 cell receptor using compounds such as sitagliptin, vildagliptin, and saxagliptin [19, 146] . more than three years have passed since the first detection of mers-cov human infection and the virus, uncontrolled, continues to cause major outbreaks in the middle east. the recent outbreak in korea demonstrated that a single index case can lead to 185 more infections in a short period of time, hence raising questions about the accuracy of the number of cases being reported in the middle east. furthermore, the korean outbreak confirmed the high fatality rate of mers-cov infection as being true rather than overestimated in case only the more severe cases are detected. in all, public health, veterinary health, and research efforts need to be consolidated in order to answer the following high priority questions: -what is the true extent of human infection with mers-cov? -what antivirals and vaccines are to be used in humans? -what infection control measures are needed in healthcare settings to prevent nosocomial outbreaks? -what measures need to be in place in order to prevent zoonotic infections from camels? -is it possible to control the virus in the camel population and if so, how? -are there other animal species involved in the mers-cov transmission cycle? a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease coronavirus as a possible cause of severe acute respiratory syndrome discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public nidovirales: evolving the largest rna virus genome virus-encoded proteinases and proteolytic processing in the nidovirales adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines serologic assessment of possibility for mers-cov infection in equids identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc interspecies transmission and emergence of novel viruses: lessons from bats and birds dipeptidylpeptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein furin at the cutting edge: from protein traffic to embryogenesis and disease mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features characterization of the coronavirus m protein and nucleocapsid interaction in infected cells sars-beginning to understand a new virus wildtype and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection pneumonia from human coronavirus in a macaque model host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters asymptomatic middle east respiratory syndrome coronavirus infection in rabbits novel coronavirus-saudi arabia: human isolate. archive number: 20120920.1302733. published date patient with new strain of coronavirus is treated in intensive care at london hospital middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility mers-cov in the republic of korea at a glance. 2015 51. who. laboratory testing for middle east respiratory syndrome coronavirus -interim recommendations (revised) revised case definition for reporting to who-middle east respiratory syndrome coronavirus-interim case definition as of 14 middle east respiratory syndrome (mers), case definition middle east respiratory syndrome coronavirus, case definition for reporting to who, interim case definition as of 14 middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (puis) for middle east respiratory syndrome coronavirus (mers-cov) assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections laboratory testing for middle east respiratory syndrome coronavirus cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests contact investigation of a case of human novel coronavirus infection treated in a german hospital specific serology for emerging human coronaviruses by protein microarray transmission of mers-coronavirus in household contacts seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt laboratory testing for middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study che d; investigation team. first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission preliminary epidemiological assessment of mers-cov outbreak in south korea update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide family cluster of middle east respiratory syndrome coronavirus infections clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission is mers another sars? ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study middle east respiratory syndrome coronavirus disease in children cov investigation team stillbirth during infection with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infections in health care workers mers-cov outbreak in jeddah-a link to health care facilities hospital outbreak of middle east respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study world health organization global outbreak alert and response network middle east respiratory syndrome coronavirus international investigation team. family cluster of middle east respiratory syndrome coronavirus infections spread of mers to south korea and china summary and risk assessment of current situation in republic of korea and china notice to health care providers: updated guidelines for evaluation of severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov) epidemiological investigation of mers-cov spread in a single hospital in south korea interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk assessing the pandemic potential of mers-cov evidence for camel-to-human transmission of mers coronavirus a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment-as of 5 hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) mers coronavirus in dromedary camel herd, saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia ) and characterisation of assay specificity rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus in bats, saudi arabia human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe close relative of human middle east respiratory syndrome coronavirus in bat the lancet infectious diseases. need for global cooperation in control of mers-cov middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan koopmans mp. middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 antibodies against mers coronavirus in dromedary camels absence of mers-coronavirus in bactrian camels, southern mongolia mers coronavirus neutralizing antibodies in camels mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus antibody reactors among camels in dubai geographic distribution of mers coronavirus among dromedary camels antibodies against mers coronavirus in dromedary camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vectorbased vaccines carrying the spike protein of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara (mva) delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin cyclophilins as modulators of viral replication broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon α-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis testing of middle east respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus the newly emerged sars-like coronavirus hcov-emc also has an "achilles' heel": current effective inhibitor targeting a 3c-like protease inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody mahmoud m. shehata, mokhtar r. gomaa, mohamed a. ali, and ghazi kayali declare that they have no conflict of interest. this manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. key: cord-347889-lpd1olqq authors: weston, stuart; matthews, krystal l.; lent, rachel; vlk, alexandra; haupt, rob; kingsbury, tami; frieman, matthew b. title: a yeast suppressor screen used to identify mammalian sirt1 as a proviral factor for middle east respiratory syndrome coronavirus replication date: 2019-05-29 journal: journal of virology doi: 10.1128/jvi.00197-19 sha: doc_id: 347889 cord_uid: lpd1olqq viral proteins must intimately interact with the host cell machinery during virus replication. here, we used the yeast saccharomyces cerevisiae as a system to identify novel functional interactions between viral proteins and eukaryotic cells. our work demonstrates that when the middle east respiratory syndrome coronavirus (mers-cov) orf4a accessory gene is expressed in yeast it causes a slow-growth phenotype. orf4a has been characterized as an interferon antagonist in mammalian cells, and yet yeast lack an interferon system, suggesting further interactions between orf4a and eukaryotic cells. using the slow-growth phenotype as a reporter of orf4a function, we utilized the yeast knockout library collection to perform a suppressor screen where we identified the ydl042c/sir2 yeast gene as a suppressor of orf4a function. the mammalian homologue of sir2 is sirt1, an nad-dependent histone deacetylase. we found that when sirt1 was inhibited by either chemical or genetic manipulation, there was reduced mers-cov replication, suggesting that sirt1 is a proviral factor for mers-cov. moreover, orf4a inhibited sirt1-mediated modulation of nf-κb signaling, demonstrating a functional link between orf4a and sirt1 in mammalian cells. overall, the data presented here demonstrate the utility of yeast studies for identifying genetic interactions between viral proteins and eukaryotic cells. we also demonstrate for the first time that sirt1 is a proviral factor for mers-cov replication and that orf4a has a role in modulating its activity in cells. importance middle east respiratory syndrome coronavirus (mers-cov) initially emerged in 2012 and has since been responsible for over 2,300 infections, with a case fatality ratio of approximately 35%. we have used the highly characterized model system of saccharomyces cerevisiae to investigate novel functional interactions between viral proteins and eukaryotic cells that may provide new avenues for antiviral intervention. we identify a functional link between the mers-cov orf4a proteins and the ydl042c/sir2 yeast gene. the mammalian homologue of sir2 is sirt1, an nad-dependent histone deacetylase. we demonstrate for the first time that sirt1 is a proviral factor for mers-cov replication and that orf4a has a role in modulating its activity in mammalian cells. camels (2) (3) (4) (5) . the virus has yet to show sustained transmission between humans, but, with no currently approved antiviral therapeutics or vaccines, mers-cov is considered a "threat to global health" by the who. identifying novel virus-host interactions is essential to guide future intervention strategies. coronaviruses have large (ϳ30-kb) positive-sense, single-stranded rna genomes. approximately two-thirds of this rna is dedicated to the production of 16 nonstructural proteins which regulate replication of the viral genome. the remaining third encodes the structural and accessory proteins (6) .the accessory proteins largely function to promote viral replication through modulating cellular responses to infection. one such protein, produced by mers-cov, is orf4a. this accessory protein has been shown in vitro to bind double-stranded rna and to disrupt the innate immune sensing pathways of rig-i and mda5 to suppress the interferon response (7) (8) (9) . additionally, orf4a has been found to inhibit the pkr and stress granule response in cells (10) (11) (12) , further interfering with cellular function to promote viral infection. the yeast saccharomyces cerevisiae have been used extensively throughout the history of eukaryotic cell biology, including its being the first eukaryotic genome to be fully sequenced (13) . the genome is now highly annotated, due in part to experiments performed with the yeast knockout library collection (14, 15) . the yeast knockout collection systematically knocked out each gene in s. cerevisiae with a known dna cassette containing a unique "barcode" region. therefore, by taking any yeast cell from within this library and pcr amplifying and sequencing the barcode region, it is possible to determine which gene has been deleted from that cell. the project managed to knock out ϳ4,600 of the ϳ6,000 yeast genes; deletion of the others resulted in an inability to produce viable yeast, and they were deemed essential for growth in glucose (glu) media. therefore, the yeast knockout collection represents a widely available, semi-genome-wide knockout library of yeast. while extensively used to study eukaryotic cell biology, yeast species have also been used to look for factors involved in replication of rna viruses (16) (17) (18) (19) (20) (21) (22) . we and others have previously demonstrated that expression of certain proteins from viruses and bacteria in the yeast s. cerevisiae can inhibit growth (23) (24) (25) (26) (27) (28) (29) (30) . this growth phenotype can be leveraged to identify compounds with antiviral properties or functionally important residues of the viral proteins. in this study, we utilized the slow-growth phenotype to investigate novel genetic interactions between the viral protein and eukaryotic cells. through screening the mers-cov proteome, we identified various proteins that, when expressed in yeast, caused a slow-growth phenotype, including the protein encoded by the orf4a accessory gene. yeast lack an interferon response, and since orf4a has largely been studied in experiments designed to disrupt these pathways, we hypothesized that orf4a must have further functions in a eukaryotic cell that have yet to be analyzed. to investigate the cellular pathways that orf4a interacts with in yeast, we took advantage of the yeast knockout library. we hypothesized that removal of genes involved in the orf4a-mediated slow growth would cause a reversion of the slow-growth phenotype, allowing the identification of genetic suppressors. this screening approach identified the ydl042c/sir2 yeast gene as a suppressor of orf4a-mediated slow growth, and that gene became the focus of our study. ydl042c/sir2 is a sirtuin protein, first identified through its role in silencing the cryptic mating-type loci hml and hmr (31) . since that initial identification, sirtuins have been defined as nad-dependent histone deacetylases (32) involved in regulating a multitude of cellular functions, including metabolism, apoptosis, stress responses, dna repair, and gene expression (33) (34) (35) (36) . the mammalian homologue of sir2, sirt1, has been shown to have both proviral and antiviral roles, depending on the virus (37) (38) (39) (40) (41) (42) (43) , making this an attractive candidate hit from our yeast-based screening for study in mammalian cells. moreover, it has previously been suggested that resveratrol, a compound that activates sirtuins, inhibits mers-cov replication, and yet no mechanism was investigated in that work (44) . given the combination of sirt1 being shown to have roles in replication of mammalian viruses and the fact that sir2 was the most com-monly identified gene in our suppressor screen, this sirtuin became the focus of the study. other genes identified in yeast are yet to be examined in the mammalian context. we found that chemical inhibition of sirt1 or knockdown with either small interfering rna (sirna) or clustered regularly interspaced short palindromic repeat interference (crispri) inhibited replication of mers-cov, demonstrating that sirt1 is a proviral factor for viral replication. the sirtuin activator resveratrol was found to inhibit mers-cov replication, but this effect was not dependent on the presence of sirt1, suggesting that other sirtuin proteins may have antiviral roles, while sirt1 appears to have a proviral role. the yeast-based screening system demonstrated a functional link between orf4a and sir2. we did not observe a direct interaction between orf4a and sirt1 in mammalian cells. however, we do show that orf4a can modulate sirt1mediated enhancement of nf-b signaling, suggesting a functional link between the two proteins in mammalian cells. our work demonstrates the utility of yeast for identifying novel interactions between viral proteins and eukaryotic cells and defines sirt1 as a proviral factor for mers-cov replication. mers-cov orf4a causes a slow-growth phenotype in yeast, and ydl042c/sir2 is a genetic suppressor. work performed in our laboratory and others previously found that certain proteins from various pathogens can cause a slow-growth phenotype in s. cerevisiae (23) (24) (25) (26) (27) (28) (29) (30) . this phenotype can be used to investigate functional aspects of these proteins in eukaryotic cells by the use of suppressor screens. in these screens, yeast genes are knocked out (as in this study) or overexpressed to identify genes that suppress the growth phenotype caused by the expression of the pathogen protein. we took advantage of the yeast model system to investigate cellular interactions of proteins from mers-cov. genes from the mers-cov genome were cloned into a galactose (gal)-inducible (gal1) expression vector, based on pbr416 (45) , with a c-terminal green fluorescent protein (gfp) tag, and were transformed into yeast. grown in the presence of 2% glucose (glu), the expression of viral genes was inhibited and the yeast strain containing this plasmid was able to grow similarly to yeast transformed with a vector control ( fig. 1b and data not shown). however, grown in the presence of 2% galactose (gal), viral genes were expressed. we analyzed whether any of the mers-cov-encoded proteins could inhibit yeast growth by performing 48 h growth curves, measuring the optical density at 600 nm (od 600 ) on an automated plate reader as the readout for growth (fig. 1a) . we found that the following four mers-cov proteins inhibited growth of yeast: nsp5 (green dotted/dashed line in fig. 1a ), nsp10 (maroon line), nsp15 (orange line), and orf4a (gray line). the other mers-cov genes examined had only a modest impact or no impact on growth, suggesting that expression of gfp-tagged proteins alone was not responsible for the slow growth observed. we decided to focus our further study on orf4a as this accessory protein has been best described for roles in inhibiting the mammalian interferon (ifn) response, which is not conserved in yeast, therefore suggesting the possibility of hitherto-undescribed functions of orf4a in eukaryotic cells. the growth inhibition caused by orf4a was further assessed on solid media, where the levels of growth on glu plates (no protein expression) were indistinguishable between vector control and orf4a-expressing yeast whereas growth on gal plates displayed growth inhibition of yeast expressing orf4a (fig. 1b) . the results from an initial liquid-culture screening were also further confirmed (fig. 1c) . we reasoned that the slow-growth phenotype induced by orf4a was a result of the viral protein disrupting normal cellular function, allowing us to perform a suppressor screen. we hypothesized that knockout of yeast genes involved in the orf4a-mediated slow growth might reduce the level of inhibition and therefore increase the growth rate. to test this hypothesis, the inducible orf4a plasmid was transformed into a pooled collection of the yeast knockout library. this library consists of ϳ4,600 nonessential gene knockouts. when this transformed library was plated onto glu plates, the yeast colonies that formed were of similar sizes (fig. 1d, glu) . however, plated to gal plates to induce orf4a expression, a broad range of colony sizes were observed (fig. 1d , gal). of most interest were the large colonies, as these were thought to have been formed by yeast cells that contain deletions of genes that confer a suppressor phenotype on orf4a-mediated slow growth. large colonies were picked from gal plates and were tested for growth in liquid culture and for continued expression of orf4a (data not show). suppressors were determined as knockout yeast that expressed orf4a and showed enhanced growth compared to orf4a-expressing wild-type (wt) yeast. the identity of the knockout was determined by pcr amplification and sequencing of the unique barcode regions that are used in the yeast knockout library. for the screening process, ϳ150 colonies were assessed for growth phenotypes and 56 were found to have suppressor phenotypes to various degrees (data not shown). these 56 colonies determined as hits were sequenced and found to represent 42 different genes ( table 1 ). the most commonly detected gene knockout was ydl042c/ sir2 (no other sirtuin genes were detected). tested in liquid culture, δydl042c/sir2 colonies picked from gal plates showed a suppressor phenotype. panel e of fig. 1 shows an example of a single colony that was picked from a galactose screening plate that had a suppressor phenotype. when the growth rate of the initial fast-growing colony was assessed, the identity of the gene knockout was unknown. subsequent sequencing determined it to be δydl042c. this colony represented one of the five δydl042c colonies that were identified as suppressors in the screen. the colony displayed in fig. 1e still maintained expression of orf4a, such that the enhanced growth was not a consequence of a lack of expression of the viral protein (fig. 1f) . to validate the hypothesis that ydl042c/sir2 is indeed a suppressor of orf4a, a confirmed ydl042c/sir2 deletion strain was transformed with the orf4a expression plasmid. when the known transformed deletion strain was tested for growth ( fig. 1g ) and protein expression (fig. 1h) , the suppressor phenotype was maintained. to further validate identification of ydl042c as a specific suppressor of orf4a, δydl042c yeast cells were also transformed with an empty vector or a vector expressing sars-cov orf3b with a gfp tag (which does not cause a slow-growth phenotype). empty vector and orf3b-gfp δydl042c yeast grew at rates similar to those seen with the vector control yeast, suggesting that the δydl042c phenotype did not stimulate enhanced growth and therefore that the suppressor phenotype seen when orf4a was present was specific (fig. 1i ). combined, these data argue that ydl042c/sir2 is a bona fide genetic suppressor of orf4a-mediated slow growth. orf4a and sirt1 do not directly interact in mammalian cells. the mammalian homologue of yeast ydl042c/sir2 is the histone deacetylase sirtuin protein sirt1 (67% similarity at the protein level). we therefore hypothesized that orf4a may interact with raffinose to reach saturation. yeast cells were diluted and cultured for 48 h in 2% gal media at 30°c on a plate reader. every 30 min, the plate was shaken for 30 s and the od 600 was read. the growth curve has od 600 plotted against time. data are from one representative experiment performed using two separate colonies of yeast. error bars represent deviations between results from the two colonies. (b) yeast cells transformed with a gal-inducible plasmid to express mers-cov orf4a or a vector control were cultured for 2 days in media containing 2% raffinose to reach saturation. the od 600 was measured, yeast cells were diluted to an od 600 of 0.1 in 2% raffinose media, and then a 1:5 dilution series was made. this dilution series of the yeast was then replica plated on agar plates containing 2% glucose (glu) or 2% gal. (c) as described for panels a and b, vector control or orf4a plasmid yeast cells were grown for 2 days in 2% raffinose to reach saturation. yeast cells were diluted and cultured for 48 h in 2% gal media at 30°c on a plate reader. every 30 min, the plate was shaken for 30 s and the od 600 was read. the growth curve has od 600 plotted against time. data are from three independent experiments, with error bars showing standard deviations. (d) the gal-inducible orf4a plasmid was transformed into a pool of the yeast knockout (yko) library collection and plated onto agar plates containing glu or gal. a suppressor screen was performed by picking large colonies from the orf4a yko library on gal plates and analyzing growth rates for colonies that no longer had the slow-growth phenotype caused by expression of orf4a. the most commonly detected gene deletion was ydl042c/sir2. (e) a representative graph for one of the large colonies picked from a gal plate that was later determined to be δydl042c/sir2. (f) western blot to demonstrate that the suppressor phenotype observed in δydl042c/sir2 colonies was not a result of a lack of orf4a expression. (g) a known δydl042c/sir2 strain was picked from an arrayed collection of the yko library and transformed with gal-inducible orf4a plasmid. data show growth curves of these transformed yeast cells from 3 independent experiments (error bars represent standard deviations). (h) western blot to confirm that orf4a was still expressed in the transformed δydl042c/sir2 cells. (i) control experiments testing whether δydl042c/sir2 yeast cells do or do not grow faster than wild-type yeast. sars-cov-orf3b and an empty vector were transformed into yeast that were subsequently grown for 48 h in 2% raffinose media prior to growth to produce growth curves as described above. sirt1 in mammalian cells to regulate mers-cov replication. endogenous sirt1 is localized to the nucleus in huh7 cells, with areas that appear to have lower-intensity labeling ( fig. 2a) . transfected into huh7 cells, a plasmid encoding orf4a-gfp showed gfp signal in the cytosol, with additional puncta in the nucleus (fig. 2b ), in agreement with previously published data (7) . the colabeling of these transfected cells with antibodies against sirt1 showed both proteins to be in the nucleus. however, the nuclear puncta of orf4a-gfp appeared to be in regions with lower levels of sirt1 labeling (fig. 2b) . in cells infected with mers-cov, labeled with antisera to orf4a, and colabeled with anti-sirt1 antibody, orf4a was founded in the nucleus and in cytosolic puncta, while sirt1 was still localized to the nucleus (fig. 2c) . the more punctate appearance of cytosolic orf4a in infected cells was possibly a result of lower expression levels compared to the overexpression construct. overall, it appears that endogenous sirt1 was localized to the nucleus, while gfp-tagged orf4a and orf4a expressed during infection were both localized in the nucleus and cytosol. to thoroughly test whether orf4a and sirt1 could interact in huh7 cells, we acquired both wt and catalytically dead (h363y) flag-tagged expression plasmids of sirt1 (fig. 3a) , the data in the column labeled "no. of hits" indicate numbers of individual colonies that were found to have the same gene deletion. yko, yeast knockout; orf, open reading frame. reasoning that a higher level of expression of sirt1 would allow us to detect even low levels of interaction. the overexpression constructs appeared to show a localization pattern similar to that seen with endogenous sirt1 when expressed individually in cells (data not shown) and when cotransfected with orf4a-gfp (fig. 3b) , suggesting that these are useful for investigating interaction of orf4a and sirt1 proteins. once again, both sirt1-flag and orf4a-gfp were seen in the nucleus, suggesting that they are in the same compartment of cells and therefore could potentially interact. to investigate this more directly, we used coimmunoprecipitation (co-ip) experiments. cells were transfected with orf4a-gfp alone or in combination with wt or h363y sirt1-flagexpressing plasmids. anti-gfp antibody was used to immunoprecipitate orf4a-gfp, which could be detected in the ip fraction in blotting with anti-gfp antibody (fig. 3c) . in cells cotransfected with orf4a-gfp and either of the sirt1 constructs, while orf4a-gfp could be precipitated, sirt1 was detected only in the nonspecific and flowthrough fractions and never in the ip fraction (fig. 3c) . these data suggest that orf4a and sirt1 do not directly interact in huh7 cells. sirt1 is a proviral factor for mers-cov replication. yeast sir2 was detected as a genetic suppressor of orf4a-mediated slow growth, and yet orf4a and sirt1 were huh7 cells grown on coverslips were transfected with a plasmid expressing orf4a-gfp and were subsequently subjected to immunofluorescence labeling for sirt1 (visualized with anti-mouse texas red). arrows point toward nuclear and arrowheads point toward cytosolic orf4a localizations. (c) huh7 cells were plated to coverslips 1 day prior to infection with mers-cov at an moi of 1 (uninfected cells were grown as a control). infection was allowed to proceed for 18 h prior to fixation and immunofluorescence labeling for sirt1 (visualized with anti-mouse texas red) and orf4a (visualized with anti-rabbit fitc). arrows point toward nuclear and arrowheads point toward cytosolic orf4a localizations. in all experiments, nuclei were labeled with dapi. scale bars ϭ 50 m. found to not directly interact in mammalian cells. the genetic suppression observed in yeast does not necessitate a direct interaction between the two proteins. we therefore hypothesized that orf4a may modulate sirt1 activity indirectly to regulate mers-cov replication. if this were the case, modulation of sirt1 activity should alter the replication of mers-cov. it has previously been suggested that resveratrol, a chemical compound that broadly activates sirtuin proteins, can inhibit mers-cov replication (44) . we looked to reproduce these results and indeed found that at higher concentrations, resveratrol was capable of inhibiting mers-cov replication as judged by assessing viral titer released into the supernatant (fig. 4a ). this inhibition was seen when the drug was added 2 h before, at the same time as, or 2 h after virus was added to the cells, suggesting that resveratrol was not inhibiting entry stages of infection. we additionally tested the impact of ex527, a chemical that inhibits sirt1. these data showed that sirt1 inhibition also resulted in inhibition of mers-cov replication (fig. 4b) . this result was again seen regardless of time of addition. neither of these results appeared to be fully explainable by cell death, as only small differences were observed at the higher concentrations of drug in assessments of cell viability by celltitre-glo assay (fig. 4c) . in order to investigate the role of sirt1 more directly, sirna was used to knock down expression of the protein. we used two sirna sequences targeting sirt1 (42) . both sirna sequences were capable of knocking down sirt1 expression (fig. 5a) , and both resulted in minor drops in cell viability as assessed by celltitre-glo assay (fig. 5b ). when these samples were infected with mers-cov, significant inhibition of infection was detected (fig. 5c ). while knockdown of sirt1 was clearly capable of inhibiting mers-cov replication, overexpression of wt or h363y sirt1-flag did not have any impact on virus production (fig. 5d) , suggesting that the endogenous levels of sirt1 may be sufficient for replication. these data suggest that sirt1 is a proviral factor for mers-cov infection. however, this result is at odds with the data showing that resveratrol, a sirutin activator, inhibited infection. resveratrol is a broad activator of sirtuin proteins, of which there are 7 in the human genome. we hypothesized that different sirtuin proteins may have different impacts on mers-cov infection. this was tested by transfecting cells with sirt1 sirna, treating with resveratrol, and infecting with mers-cov. resveratrol was capable of inhibiting mers-cov infection even in the absence of sirt1 (fig. 5e) . these data suggest that resveratrol inhibits mers-cov infection independently of sirt1. crispri further demonstrates sirt1 is a proviral factor for mers-cov replication. our sirna-mediated knockdown of sirt1 in fig. 5 suggests that sirt1 is a proviral factor for mers-cov replication. in order to further assess the effect of sirt1 on mers-cov replication, a crispri approach was used (46) . for this, huh7 cells that stably express a catalytically dead version of cas9 (dcas9) were produced through lentiviral transduction. the dcas9 can be inducibly expressed and binds to dna as directed by the guide rna but does not cleave, instead acting as a steric block to transcription. moreover, a krab element on the dcas9 further enhances transcriptional repression. these stable huh7-dcas9 cells were further transfected to stably express a short guide rna (sgrna) sequence to target the sirt1 gene. two lentiviral preparations were used to independently transfect two separate populations of huh7 cells stably expressing the dcas9 construct (huh7-dcas9 cells) . the sgrna is constitutively expressed, while the dcas9 is doxycycline (dox) inducible; therefore, the stable cell populations can be divided into dox-treated and untreated populations for internal controls of target gene knockdown. both huh7-dcas9-sgrna cell populations demonstrated knockdown of sirt1 under conditions of treatment with dox for 7 days (fig. 6a ). when these doxycycline-treated cells were infected with mers-cov, there was a significant inhibition of replication in both populations (fig. 6b ). knockdown of sirt1 by both sirna and crispri inhibited mers-cov replication, showing this protein to be a proviral factor for replication. using these crispri cells, we also confirmed that resveratrol inhibited mers-cov replication even in the absence of sirt1 (fig. 6c) , validating the sirna results (fig. 5e ). the level of inhibition of mers-cov replication in control cells appears to have been slightly lower in this experiment; however, under conditions of testing over multiple infections (fig. 6b) , the inhibition results were statistically significant. overall, it appears that even though the sirtuin activator resveratrol has antiviral activity with respect to mers-cov replication, sirt1 is dispensable for this effect. orf4a modulates sirt1 activity as assessed by an nf-b pathway readout. sirt1 appears to function as a proviral factor for mers-cov replication ( fig. 5 ; see also fig. 6 ). our suppressor screen in yeast detected a genetic link between mers-cov orf4a and sir2 (fig. 1 ), but we were unable to detect a direct interaction between orf4a and mammalian sirt1 (fig. 3) . we therefore speculated that orf4a may have an indirect influence on sirt1 activity in promoting mers-cov replication. sirt1 has previously been shown to modulate nf-b signaling through deacetylation of rela/p65 (47) . we therefore decided to use nf-b signaling as a readout of sirt1 activity to determine whether orf4a alters this. hek293t cells were transfected with combinations of orf4a, sirt1 (wt or h363y mutant), and a b-luciferase plasmid (fig. 7a) . the following day, cells were treated with tumor necrosis factor alpha (tnf-␣) for 6 h and lysed, and the amount of luciferase produced was determined. (results of controls performed for analysis of background luminescence can be seen in fig. 7d .) at odds with previously published data, we found that transient transfection of sirt1 in our system significantly enhanced luciferase production following tnf-␣ treatment compared to the results seen with control transfected cells ( fig. 6 ; see also fig. 7b ). cells transfected with h363y sirt1 also showed an enhanced level of luciferase production (fig. 7b) . when mers-cov orf4a was transfected into cells, inhibition of luciferase production was observed (fig. 7b) . importantly, when orf4a was transfected into cells alongside sirt1 (wt or h363y), the luciferase levels were similar to those seen with orf4a alone. these results suggest that sirt1 no longer enhanced luciferase production in response to tnf-␣ when orf4a was present, suggesting that orf4a was altering this readout of sirt1 activity (fig. 7b) . treatment of cells with resveratrol also gave an enhanced level of nf-b signaling in response to tnf-␣, further suggesting a role for sirtuins in regulating this pathway in hek293t cells (fig. 7c ). however, ex527 had no effect in our tests (fig. 7c) . a small but significant increase in nf-b signaling was seen when orf4a-transfected cells were treated with resveratrol, but luciferase production was largely blocked compared to the levels seen with cells without transfected orf4a (fig. 7b) . these data further suggest that orf4a is capable of modulating sirt1-mediated enhancement of nf-b signaling stimulated by tnf-␣ treatment. overall, the results from the sirt1 transfection and the resveratrol treatment suggest that orf4a alters a sirt1-mediated response in cells, suggesting a functional link between the two. we have demonstrated that sirt1 is a proviral cellular protein for mers-cov replication and that yeast cells represent a powerful tool to identify previously unknown virus-host interactions. our initial work started with a suppressor screen in the yeast s. cerevisiae. we found that expression of mers-cov orf4a in yeast caused slow growth. orf4a functions as a double-stranded rna binding protein that disrupts rig-i, mda5, pkr, and stress granule responses in infected cells to promote viral replication (7) (8) (9) (10) (11) (12) . orf4a is well characterized as an inhibitor of the interferon response. yeast species do not have an interferon response and yet show growth inhibition by orf4a expression, suggesting further functional cellular interactions for the protein. to investigate the ways in which orf4a might inhibit yeast growth, a suppressor screen was utilized. by screening the yeast knockout library for suppressors of orf4a-induced growth attenuation, the ydl042c yeast gene was identified. the ydl042c gene encodes the sir2 protein, and our work validated this as a bona fide suppressor of the the orf4a-mediated slow-growth phenotype. the suppressor screen results suggest that there is a functional link between the two proteins but that the link does not rely on a direct physical interaction. while yeast is a powerful starting point for investigating eukaryotic cell biology, in the context of mers-cov infection, it is important to establish relevance in a mammalian system. we did not find a direct interaction between orf4a and sirt1 in huh7 cells by coimmunoprecipitation. however, when sirt1 was inhibited either by ex527 treatment or by knockdown by sirna or crispri, mers-cov replication was inhibited. this establishes sirt1 is a proviral factor for mers-cov replication. interestingly, our data also suggest that other sirtuin proteins may have antiviral roles in the context of mers-cov replication. resveratrol, a compound derived from various plants, has been shown to activate sirtuin proteins (48) and was previously shown to inhibit mers-cov replication (44) , a result that we replicated here. however, we demonstrated that knockdown of sirt1 by sirna or cripsri had no impact on the resveratrol-mediated inhibition of mers-cov replication, suggesting that other members of the sirtuin family that are activated by the compound may have antiviral properties. alternatively, resveratrol may have other effects on cell biology that inhibit mers-cov replication. since sirt1 was shown to be a proviral factor by our data and since resveratrol can activate sirt1 function, it appears that the antiviral properties of resveratrol may outweigh the potential proviral function of sirt1 or that the proviral role of sirt1 is not based on its catalytic activity. indeed, our experiments looking at the nf-b pathway (discussed further below) suggest that sirt1 may have activity in a catalytically dead form, arguing for a role in mers-cov replication that does not require enzymatic function. the role of other sirtuins in mers-cov replication and in how mers-cov modulates these to promote infection is the focus of future studies. the yeast-based screening system identified a genetic interaction between orf4a and sir2, which led us to hypothesize that orf4a may have a functional link with mammalian sirt1 to regulate its function and promote mers-cov replication. sirt1 has previously been shown to modulate nf-b signaling in response to tnf-␣ (47) . this work demonstrated a direct interaction between sirt1 and p65/rela and that sirt1 inhibited the pathway by deacetylation of this nf-b protein. we therefore reasoned that this modulation of the nf-b pathway could be used as a readout to determine whether orf4a expression alters sirt1 function. interestingly, we found that sirt1 overexpression in hek293t cells actually resulted in an enhanced nf-b response following tnf-␣ treatment and that this enhancement occurred regardless of the level of catalytic activity of sirt1. this discrepancy with previously published work could represent a result of the use of different cell lines. our data suggest that sirt1 promotes nf-b signaling in hek293t cells, perhaps by directly binding nf-b and acting as a scaffold protein or by altering steps downstream in the signaling pathway, but that it does so independently of catalytic activity. while we found the effects of sirt1 on nf-b to be different from those reported from previous studies in different cell lines, sirt1 still altered the nf-b response, allowing us to investigate the role of orf4a in this pathway. our experiments demonstrated that orf4a was a potent suppressor of nf-b signaling in hek293t cells. when orf4a and sirt1 were coexpressed in cells, there was no longer an enhancement in luciferase production from the b promoter. we cannot rule out the possibility that orf4a may influence nf-b signaling independently of sirt1, but sirt1 overexpression was not able to overcome orf4a-mediated inhibition. these data, along with the genetic link observed in yeast, suggest that orf4a and sirt1 may have a functional connection without direct binding and that there is an additional node of regulation between the two proteins, which is under investigation. this work adds sirt1 to the list of host factors that mediate mers-cov replication such as stress granule formation and host innate immune machinery (9) (10) (11) (12) . the magnitude of inhibition when those factors are affected is ϳ1 log, demonstrating that, as seen in sirt1, host machinery interacts with various virus replication steps to potentially hinder replication. all of these factors demonstrate novel therapeutic targets for future development. overall, we have demonstrated the utility of yeast for investigating the function of viral proteins in a highly tractable heterologous expression platform. we found that studies in yeast are relevant in a mammalian system, as the identification of yeast sir2 as a suppressor was used to determine that the mammalian deacetylase, sirt1, is a proviral factor for mers-cov replication. we currently do not understand how sirt1 enhances mers-cov replication or the role that orf4a plays in this. we hypothesize that acetylation of additional cellular or viral proteins during mers-cov replication is required for efficient replication and that orf4a has evolved to modulate this activity. the multifunctional property of orf4a has revealed novel sirt1 functions and pathways that mers-cov and potentially other viruses utilize to impact their replication. was used for all subsequent steps) with 4% (vol/vol) formaldehyde (thermo) in phosphate-buffered saline (pbs). cells were washed with pbs and excess formaldehyde quenched by 15 min of incubation with 50 mm nh 4 cl-pbs. cells were again washed and lysed in 0.1% (vol/vol) triton x-100 (sigma) and then incubated for 10 min. samples were blocked for 10 min with 0.2% bovine serum albumin (bsa)-pbs. primary antibodies were then incubated with the sample diluted in 0.2% bsa for 1 h. samples were washed in 0.2% bsa three times for 5 min each time and were then incubated with secondary antibodies in 0.2% bsa for 45 min. finally, coverslips were washed with pbs four times for 5 min each time and mounted to glass slides using prolong gold antifade reagent with dapi (4=,6-diamidino-2-phenylindole; invitrogen). for the experiment looking at orf4a localization with mers-cov infection, huh7 cells were plated on coverslips 1 day prior to infection with mers-cov. cells were infected for 18 h prior to being fixed with 4% pfa for 24 h per the standard operating practice (sop) for the biosafety level 3 (bsl3) containment facility. cells were then labeled for immunofluorescence microscopy as described above. the antibodies used were as follows: mouse anti-sirt1 (clone 1f3; abcam) (1 mg/ml) and anti-mouse texas red and anti-rabbit fluorescein isothiocyanate (fitc; vector labs) (1.5 mg/ml). all antibodies were diluted 1:100 for use. rabbit anti-orf4a serum (a kind gift from stanley perlman; used as described previously [55] ) was diluted 1:100 for use. imaging was performed using an echo revolve microscope. sirna knockdown. a double-sirna-transfection approach was used to achieve knockdown. cells were plated in a 6-well dish the morning of day 1 and allowed to settle on the plates for 3 to 4 h. cells were then transfected with one of two sirt1-targeting sirnas (the sequences are described above in the "plasmids and compounds" section and were the same as those reported in reference 42) or with scrambled sirna (mission sirna universal negative-control no. 1 [sigma]) using oligofectamine reagent (thermo) and opti-mem (gibco). for a 6-well dish, 10 l oligofectamine and 20 l opti-mem were mixed and the mixture was incubated for 5 min at rt and mixed with 160 l opti-mem and 4 l 50 m sirna. this transfection mixture was incubated for 20 min at rt. media were removed from cells and replaced with 800 l opti-mem, the transfection mixture was added, and the resulting mixture was incubated at 37°c/5% co 2 for 4 h. after this incubation period, a 1:1 volume of 20% fbs-dmem was added to the cells. the following day, media were changed for 10% fbs-dmem. cells were collected on day 3 and replated for a second round of transfection using the same approach. cells were then collected on day 5 and prepared for experimental use. crispri. huh7 cells were transduced with phage tre dcas9-krab lentiviruses, and the transduced cells were selected with geneticin (invitrogen) (0.5 mg/ml). transduced cells were screened for inducible dcas9 expression to generate a doxycycline-inducible (1 g/ml dox; sigma) gene repression cell line. this cell line was then further transduced with sirt1 crispri sgrna lentiviruses (sequences for the sgrna are provided above in the "plasmids and compounds" section). sirt1 crispri sgrnas were designed using the broad institute sgrna design tool. of three sgrnas initially tested, only one resulted in clear and reproducible sirt1 repression. in each of the tested sgrna sequences, two independent lentivirus preparations (see below) were used for transduction on separate bulk populations of the huh7-dcas9 inducible cell lines. transduction of sirt1 sgrnas was selected for by the use of puromycin (sigma) (1 g/ml). the huh-dcas9-sgrna cells were continuously cultured in dmem supplemented with 10% fbs, 1% pen/strep, 0.5 mg/ml neomycin, and 1 g/ml puromycin. to achieve knockdown of sirt1, these cells were cultured in growth medium containing 1 g/ml dox, with this medium being changed every 2 days over a 7-day period prior to use of the cells for experiments. lentivirus production and transduction. all lentiviruses were produced in hek293t cells. lentivectors were cotransfected with vesicular stomatitis virus envelope glycoprotein (vsv-g)-expressing plasmid pmd2.g (a gift from didier trono; addgene plasmid no. 12259) and packaging plasmid pspax2 (a gift from didier trono; addgene plasmid no. 12260) and were concentrated using peg-it (system biosciences). for transduction, cells were plated to achieve approximately 70% confluence in a 24-well dish. lentivirus preparations were added to cells in media containing 4 g/ml polybrene (americanbio). cells were then subjected to spinoculation at 1,000 rpm for 45 min and incubated overnight prior to addition of puromycin selection media. mammalian cell western blot sample preparation. cells for western blotting were plated 1 day prior to sample collection. plates were placed on ice and washed twice with 4°c pbs. cells were lysed on ice for 15 min by incubation with radioimmunoprecipitation assay (ripa) buffer (sigma) containing 1ϫ complete protease inhibitor cocktail (complete mini; roche). lysate was collected and centrifuged at 21,000 ϫ g for 10 min. supernatant was collected and used for western blotting or stored at ϫ80°c prior to use. the protein content of the sample was quantified using a bicinchoninic acid (bca) system (thermo scientific). a 40-g volume of whole-cell lysate (wcl) was used in all western blotting experiments. western blotting. samples were separated using 4% to 20% mini-protean tgx gels (bio-rad) and transferred to polyvinylidene difluoride (pvdf) membranes (immoblion-fl; millipore) using a trans-blot turbo system (bio-rad) or a wet transfer approach (1 h of transfer at 350 ma). membranes were blocked using aquablock (east coast bio) for 1 h at rt and were then incubated with primary antibody (diluted in aquablock) overnight at 4°c. the following day, membranes were washed with tris-buffered saline with tween 20 (tbst; 50 mm tris, 150 mm nacl, 0.05% tween 20) and incubated with secondary antibodies diluted in aquablock for 1 h at rt. membranes were further washed with tbst prior to imaging using a chemidoc imaging system (bio-rad). blot visualization was achieved using either fluorescence or horseradish peroxidase (hrp)-conjugated secondary antibodies, detected by the use of amersham ecl prime reagent (ge healthcare life sciences). the primary antibodies used were as follows: mouse anti-sirt1 (as described in the "immunofluorescence microscopy" section), mouse anti-flag (clone m2; sigma) (1 mg/ml), mouse anti-tubulin (clone dma1a; sigma), rabbit anti-gfp (sigma) (1 mg/ml), and mouse anti-actin (clone 1a4; sigma). all primary antibodies were diluted 1:1,000 for western blotting. the secondary antibodies used were as follows: goat anti-mouse hrp (thermo scientific) (0.8 mg/ml), goat anti-rabbit hrp (thermo scientific) (0.8 mg/ ml), goat anti-mouse alexa fluor 546 (life technologies) (2 mg/ml), and goat anti-rabbit alexa fluor 633 (life technologies) (2 mg/ml). hrp-conjugated secondary antibodies were diluted 1:10,000, and fluorescent secondary antibodies were diluted 1:2,000. immunoprecipitation. cells were plated 1 day prior to use and lysed in ripa buffer containing 1ϫ complete protease inhibitor cocktail as described above (similarly to the co-ip protocols previously described in references 56 and 57, for example). protein content in the whole-cell lysate was determined by bca assay, following the instructions of the manufacturer (thermo scientific). wcl (450 g) was used in the ip. nonspecific binding to beads was cleared by incubating the 450 g of wcl for 1 h at 4°c with protein a magnetic beads (cst). beads were separated from lysate with a magnetic rack, and the supernatant was collected. the nonspecific fraction was prepared by adding 2ϫ lsb with ␤-mercaptoethanol and heating at 95°c for 5 min. the collected supernatant was added to fresh magnetic beads and incubated with anti-gfp antibody (5 g) overnight at 4°c. the following day, the beads were separated with a magnetic rack and the supernatant was collected as the flowthrough fraction. beads were washed five times with ripa buffer before addition of 2ϫ lsb with ␤-mercaptoethanol and heating at 95°c for 5 min to prepare the immunoprecipitation fraction. viral infection. mers-cov (jordan strain-genbank accession no. kc776174.1; mers-cov-hu/jordan-n3/2012) stocks were prepared by infection of vero cells, and titers were determined by plaque assay using these cells (as described previously [58] ). all experimental infections were performed with huh7 cells at a multiplicity of infection (moi) of 0.1 unless indicated otherwise. for quantification of virus production, medium samples were collected 24 h after infection, centrifuged in a table top centrifuge for 3 min at full speed, and stored at ϫ80°c prior to use. tcid 50 (50% tissue culture infective dose) assays of vero cells were used to quantify the amounts of virus in the collected samples (58) . luciferase assays. for celltiter-glo (ctg) assays, cells were plated in opaque 96-well plates 1 day prior to use. for drug treatments, resveratrol or ex527 was added at the indicated concentrations and incubated for 24 h before ctg assays were performed. for sirna samples, cells were plated and grown overnight and then used for ctg assays. a celltiter-glo luminescent cell viability assay (promega) kit was used per the manufacturer's instructions. luminescence was read using a synergy htx multimode plate reader. reporter assays were used with b luciferase plasmids as previously described (49) . briefly, hek293t cells were plated 1 day prior to transient transfection as described above. these transfections were performed in 48-well plates using a total of 400 ng of dna per well. at most, three plasmids were required for transfection (sirt1/orf4a/luciferase for example); in instances of fewer experimental proteins being needed, preceive-gfp was used to balance the amounts of dna. cells were transfected for 18 h before further manipulation. in the cases of drug treatment, cells were pretreated with resveratrol or ex527 for 2 h and tnf-␣ stimulation was performed for 6 h using 10 ng per well (recombinant tnf-␣ was purchased from biolegend). the luciferase assay was performed using a pierce firefly luciferase glow assay (thermo) kit, following the manufacturer's instructions. luminescence was read on the synergy htx reader as described above. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia mers coronaviruses in dromedary camels genetic relatedness of the novel human group c betacoronavirus to tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 genomic characterization of newly discovered coronavirus associated with acute respiratory distress syndrome in humans sars and mers: recent insights into emerging coronaviruses middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists structural insights into the middle east respiratory syndrome coronavirus 4a protein and its dsrna binding mechanism middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pactinduced activation of rig-i and mda5 in the innate antiviral response middle east respiratory coronavirus accessory protein 4a inhibits pkr-mediated antiviral stress responses inhibition of stress granule formation by middle east respiratory syndrome coronavirus 4a accessory protein facilitates viral translation, leading to efficient virus replication life with 6000 genes the yeast deletion collection: a decade of functional genomics functional profiling of the saccharomyces cerevisiae genome systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus genome-wide analysis of host factors in nodavirus rna replication yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of rna viruses an influenza virus replicon system in yeast identified tat-sf1 as a stimulatory host factor for viral rna synthesis expression of the rna genome of an animal virus in saccharomyces cerevisiae genome-wide screen identifies host genes affecting viral rna recombination how yeast can be used as a genetic platform to explore virus-host interactions: from "omics" to functional studies novel influenza virus ns1 antagonists block replication and restore innate immune function yeast based small molecule screen for inhibitors of sars-cov identification of novel amino acid residues of influenza virus pa-x that are important for pa-x shutoff activity by using yeast poliovirus 2apro expression inhibits growth of yeast cells n-terminal acetylation by natb is required for the shutoff activity of influenza a virus pa-x multifunctional analysis of chlamydia-specific genes in a yeast expression system a functional genomic yeast screen to identify pathogenic bacterial proteins large-scale identification of legionella pneumophila dot/icm substrates that modulate host cell vesicle trafficking pathways four genes responsible for a position effect on expression from hml and hmr in saccharomyces cerevisiae transcriptional silencing and longevity protein sir2 is an nad-dependent histone deacetylase sirtuins in mammals: insights into their biological function sirtuins, metabolism, and dna repair mammalian sirtuins: biological insights and disease relevance the substrate specificity of sirtuins interplay between sirt1 and hepatitis b virus x protein in the activation of viral transcription resveratrol treatment reveals a novel role for hmgb1 in regulation of the type 1 interferon response in dengue virus infection sirt1 inhibits ev71 genome replication and rna translation by interfering with the viral polymerase and 5=utr rna resveratrol enhances hbv replication through activating sirt1-pgc-1␣-ppar␣ pathway the sirt1 inhibitor, nicotinamide, inhibits hepatitis b virus replication in vitro and in vivo sirtuins are evolutionarily conserved viral restriction factors sirt1 suppresses human t-cell leukemia virus type 1 transcription effective inhibition of mers-cov infection by resveratrol regulatable promoters of saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression crispr interference (crispri) for sequence-specific control of gene expression modulation of nf-b-dependent transcription and cell survival by the sirt1 deacetylase small-molecule allosteric activators of sirtuins the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling stress-dependent regulation of foxo transcription factors by the sirt1 deacetylase cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells transcriptional regulation with crispr/cas9 effectors in mammalian cells dynamic imaging of genomic loci in living human cells by an optimized crispr/cas system transformation of yeast by lithium acetate/ single-stranded carrier dna/polyethylene glycol method antagonism of dsrna-induced innate immune pathways by ns4a and ns4b accessory proteins during mers coronavirus infection posting date. jnk1 phosphorylates sirt1 and promotes its enzymatic activity accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitindependent rapid degradation of interferon regulatory factor 3 growth and quantification of mers-cov infection, p 15e.2.1-15e.2.9 we thank kirsten kulcsar and william jackson for valuable comments and critical readings of the manuscript, brendan cormack for the yeast knockout library collection, and stanley perlman for the orf4a antisera. we also thank carolyn frenkil for her generous donation that initiated this work. key: cord-346502-x2b0ao3q authors: arabi, yaseen m; shalhoub, sarah; mandourah, yasser; al-hameed, fahad; al-omari, awad; al qasim, eman; jose, jesna; alraddadi, basem; almotairi, abdullah; al khatib, kasim; abdulmomen, ahmed; qushmaq, ismael; sindi, anees a; mady, ahmed; solaiman, othman; al-raddadi, rajaa; maghrabi, khalid; ragab, ahmed; al mekhlafi, ghaleb a; balkhy, hanan h; al harthy, abdulrahman; kharaba, ayman; gramish, jawaher a; al-aithan, abdulsalam m; al-dawood, abdulaziz; merson, laura; hayden, frederick g; fowler, robert title: ribavirin and interferon therapy for critically ill patients with middle east respiratory syndrome: a multicenter observational study date: 2019-06-25 journal: clin infect dis doi: 10.1093/cid/ciz544 sha: doc_id: 346502 cord_uid: x2b0ao3q background: the objective of this study was to evaluate the effect of ribavirin and recombinant interferon (rbv/rifn) therapy on the outcomes of critically ill patients with middle east respiratory syndrome (mers), accounting for time-varying confounders. methods: this is a retrospective cohort study of critically ill patients with laboratory-confirmed mers from 14 hospitals in saudi arabia diagnosed between september 2012 and january 2018. we evaluated the association of rbv/rifn with 90-day mortality and mers coronavirus (mers-cov) rna clearance using marginal structural modeling to account for baseline and time-varying confounders. results: of 349 mers patients, 144 (41.3%) patients received rbv/rifn (rbv and/or rifn-α2a, rifn-α2b, or rifn-β1a; none received rifn-β1b). rbv/rifn was initiated at a median of 2 days (q1, q3: 1, 3 days) from intensive care unit admission. crude 90-day mortality was higher in patients with rbv/rifn compared to no rbv/rifn (106/144 [73.6%] vs 126/205 [61.5%]; p = .02]. after adjusting for baseline and time-varying confounders using a marginal structural model, rbv/rifn was not associated with changes in 90-day mortality (adjusted odds ratio, 1.03 [95% confidence interval {ci}, .73–1.44]; p = .87) or with more rapid mers-cov rna clearance (adjusted hazard ratio, 0.65 [95% ci, .30–1.44]; p = .29). conclusions: in this observational study, rbv/rifn (rbv and/or rifn-α2a, rifn-α2b, or rifn-β1a) therapy was commonly used in critically ill mers patients but was not associated with reduction in 90-day mortality or in faster mers-cov rna clearance. the middle east respiratory syndrome (mers) is a severe respiratory infection caused by a novel coronavirus (mers-cov) and is associated with high mortality [1] . to date, there is no specific therapy of proven effectiveness for mers. based on prior experience with severe acute respiratory syndrome and on preclinical data, ribavirin and recombinant interferon (rbv/rifn) therapy has been used in managing patients with mers [2] [3] [4] . rbv is a guanosine analog that has antiviral activity against multiple rna viruses [4] . different preparations of recombinant rifns (rifn-α2a, rifn-α2b, rifn-β1a, and rifn-β1b) are active against mers-cov in vitro [5] . rbv and rifn at relatively high concentrations inhibited mers-cov replication in vero and llc-mk2 cells, but when used in combination, lower concentrations achieved comparable endpoints [2] . high doses of rbv/rifn-α2b administered 8 hours after inoculation of rhesus macaques with mers-cov resulted in reduced viral loads and was partially effective in preventing progression to pneumonia compared to animals that were untreated [3] . however, clinical data on rbv/rifn in mers have been limited to small single-center studies [6] [7] [8] [9] . in one report, all 5 patients who received rbv/rifn-α2b at a median of 19 days from admission died [7] . in a retrospective study (n = 32), patients who received rbv/ifn-α2a had a mortality rate of 85% compared with 64% in those who received rbv/ifn-β1a (p = .24) [8] . another study (n = 44) showed that rbv/rifn-α2a therapy compared to control was associated with a significant reduction in 14-day mortality (30% vs 71%; p = .004) but not in 28-day mortality (70% vs 83%; p = .054). recipients of rbv/rifn-α2a had a significant reduction (>2 g) in hemoglobin level, raising concerns of a previously described complication of hemolysis [9] . another study (n = 51) found that mers therapy using different regimens of rifn-β, rifn-α, rbv, and mycophenolate mofetil was associated with lower mortality on univariable analysis but not on multivariable analysis [10] . these studies have not provided clear evidence upon which to base treatment recommendations because of their nonrandomized design, inconsistent results, small sample sizes with limited power, and the lack of adjustment for unbalanced covariates. in addition, because of therapy initiation was not standardized, such studies are prone to 2 sources of bias: immortal time bias and indication bias. immortal time bias may occur because patients in the therapy group have survived for a period of time before receiving the therapy. because the outcome (death) could not possibly have occurred during this period in this group, this type of bias systematically underestimates adverse outcomes (eg, death) [11, 12] . indication bias occurs when the association of the therapy and outcome is caused by the indication for which the therapy was used and not to the therapy itself. if physicians typically prescribe certain therapy, such as rbv/rifn, only when the clinical condition is not improving or is worsening, standard multivariable analyses may overestimate the association with poor outcome. addressing both immortal time and indication bias in observational studies requires accounting for baseline and time-varying confounding associated with the decision to initiate treatment. the objective of this study was to examine the effect of rbv/ rifn therapy in a large cohort of critically ill patients with mers on the 90-day mortality and mers-cov rna clearance by accounting for baseline and time-varying confounders. some of these findings have been previously presented in an abstract form [13] . this retrospective analysis was conducted on a multicenter database of all critically ill patients with mers admitted to the intensive care units (icus) of 14 hospitals in 5 cities in saudi arabia between september 2012 and january 2018 [14] . details of the cohort have been reported previously [14] . some of the patients in the current analysis may have been included in previous single-center studies [8] [9] [10] . patient-level informed consent was not required. the institutional review boards (irbs) of all participating centers approved the study. participating centers followed the saudi arabian ministry of health guidelines for mers diagnostic testing. patients were defined to have mers if they had a positive mers-cov real-time reverse transcription polymerase chain reaction result (rrt-pcr) targeting amplifications of the upstream e protein (upe gene) and open reading frame 1a. specimens were obtained from nasopharyngeal swabs or sputum in nonintubated patients and from tracheal aspirates or bronchoalveolar lavage in intubated patients [15] . for patients who tested positive for mers-cov, follow-up respiratory samples were collected at the discretion of the treating teams approximately 1-2 times per week for infection control purposes. in the current analysis, we excluded patients who were enrolled in a randomized controlled trial (rct) for mers antiviral therapy that started enrolling patients in november 2017 (mers-cov infection treated with a combination of lopinavir/ritonavir and interferon β-1b [miracle] trial) [16] . the main exposure was rbv/rifn therapy, defined as the use of rbv/rifn combination, rbv alone, or rifn alone. the comparator group was the use of neither rbv nor rifn. three different types of rifns were used in the current cohort: rifn-α2a (pegasys, hoffmann-la roche, c/o genentech, south san francisco, california); rifn-α2b (peg-intron, merck sharp & dohme, whitehouse station, new jersey); and rifn-β1a (rebif, serono, rockland, massachusetts). commonly used dosing protocols for rbv/rifn for patients with mers in the participating hospitals are shown in supplementary table 1 . using standardized case report forms, we documented demographics, clinical presentation, underlying comorbidities, and final outcomes [17] . we collected sequential organ failure assessment (sofa) score and physiologic and laboratory parameters on icu days 1, 3, 7, 14, and 28 [17, 18] . we documented therapeutic interventions, including corticosteroids, mechanical ventilation, oxygen rescue therapies (nitric oxide, prone ventilation, high frequency oscillatory ventilation or extracorporeal membrane oxygenation), packed red blood cell transfusion, vasopressor therapy, and renal replacement therapy. the primary outcome was 90-day mortality. we also assessed time to mers-cov rna clearance in respiratory samples in patients who had at least 1 follow-up rrt-pcr performed after the diagnostic test. clearance was defined as the time from icu admission until the test was negative on 2 occasions, without a positive test afterward. to assess rbv/rifn safety profile, we evaluated serial levels of hemoglobin, white blood cell (wbc) count, platelet count, aspartate aminotransferase (ast), alanine aminotransferase (alt), bilirubin, international normalized ratio (inr), lactic acid, and creatinine. other secondary outcomes were icu and hospital mortality, and icu and hospital length of stay. we compared baseline characteristics, interventions, and outcomes of patients who received rbv/rifn to those who did not, using χ 2 test or fisher exact test for categorical variables and student t test or mann-whitney u test for continuous variables. for serial measurements, we tested differences between the 2 groups over time using repeated-measures analysis of variance with bonferroni correction for multiple comparisons and with no imputation for missing values. we constructed 3 models to assess the association of rbv/ rifn therapy with 90-day mortality adjusting for baseline characteristics and time-varying confounders. these analytical models were detailed in a previous study [19] . first, we created a logistic regression model adjusting for the following a priori baseline variables of clinical interest and all significant variables at the univariable level (p ≤ .2). these variables included diabetes, liver disease, renal disease, malignancy, sofa score on the first day of icu admission, source of infection, and year (before july 2014 and after) by applying the proc genmod procedure (sas software). second, we created a cox proportional hazards model adjusting for the same covariates and accounting for the rbv/ rifn therapy as a time-varying covariate. third, we created a marginal structural model analysis with inverse probability of treatment weighting to account for timevarying confounders that are likely to influence the decision to initiate rbv/rifn therapy and at the same time may be associated with mortality [20] [21] [22] [23] . we calculated stabilized weights for the probability that each subject received the treatment, censored on the day of therapy, icu discharge, or 28-day mortality, whichever came first. we included in this model selected baseline characteristics as well as time-dependent variables (sofa on the index day of rbv/rifn initiation and the previous day, ventilation status on the index day and on the previous day [0: not ventilated, 1: noninvasive ventilation, 2: invasive mechanical ventilation, 3: oxygen rescue therapy], hemoglobin, wbc count, ast and creatinine on the index day, and corticosteroid therapy on the index day). we included corticosteroid therapy as a time-varying covariate, as we have shown previously that corticosteroid therapy in critically ill mers patients might prolong mers-cov rna clearance although it was not associated with difference in mortality [19] . because these values were recorded on days 1, 3, 7, 14, and 28, we imputed missing values for the remaining days (supplementary methods). we used a weight-trimming approach to deal with extreme weights; weights <5th percentile value were fixed at the 5th percentile value and weights >95th percentile value were fixed at the 95th percentile value. this process continued until the average weight reached approximately 1. then, we used a weighted regression model using these weights taking in consideration the repeated-measures nature of the data to estimate the association of rbv/rifn with 90-day mortality. we carried out sensitivity analyses examining the association of rbv therapy alone compared to no rbv and rifn therapy alone compared to no rifn. to account for the possible variation by site, we carried out a sensitivity analysis using a logistic regression model adjusting for clustering by centers in addition to the previously mentioned baseline variables. we further evaluated the association of different types of rifn (rifn-α2a, rifn-α2b, and rifn-β1a) on 90-day mortality using a similar logistic regression model and adjusting for clustering by center. we also created 2 models to assess the association of rbv/ rifn therapy with mers-cov rna clearance adjusting for baseline characteristics and time-varying confounders. first, we carried a cox proportional hazards model adjusting for the same baseline covariates mentioned earlier and accounting for the rbv/rifn therapy as a time-varying covariate. we censored patients if they never cleared mers-cov rna or at the time of last rrt-pcr test. second, we created a marginal structural cox proportional hazards model incorporating the stabilized weights to estimate the effect of rbv/rifn therapy on mers-cov rna clearance in a similar approach to the marginal structural model used for 90-day mortality. analyses were conducted using sas version 9.4 software (sas institute, cary, north carolina). the study was approved by the national guard health affairs irb and by the irbs of all participating sites. informed consent was waived by the irb because of the retrospective nature of the study. of the 355 patients with mers in the cohort, 6 patients were excluded because they were enrolled in the miracle trial. the remaining cohort included 349 patients, of whom 144 (41.3%) received rbv/rifn therapy (table 1) . patients in the 2 groups were similar in age, sex, body mass index, and source of admission (table 1) . comorbidities were common in both those who received rbv/rifn and those who did not (84.0% vs 78.0%; p = .17). patients who received rbv/rifn were more likely to have diabetes (58.3% vs 42.0%; p = .003) and chronic renal disease (36.8% vs 27.3%; p = .06), but less likely to have chronic liver disease (2.1% vs 7.8%; p = .02). of note, the rbv/rifn recipients had lower sofa scores than those not treated (median, 8 [q1, q3: 5, 11] vs 10 [q1, q3: 6, 13]; p = .01; table 1 ). rbv/rifn therapy was initiated at a median of 2 days (q1, q3: 1, 3) from icu admission, which corresponded to 5.0 days (q1, q3: 2.0, 9.0) from hospital admission and 9.0 (q1, q3: 6.0, 12.0) from onset of symptoms (table 2 and figure 1 ). of these patients, 117 (81.3%) patients received rbv/rifn combination, 18 (12.5%) rbv alone, and 9 (6.3%) rifn alone. a total of 73 (57.9%) received rifn-α2a, 22 (17.5%) received rifn α-2b, 31 (24.6%) received rifn-β1a, and none received rifn-β1b ( table 2 ). the use of rbv/rifn therapy and the type of rifn varied by site ( supplementary figures 1 and 2) . during icu stay, the provision of mechanical ventilation, oxygen rescue therapies, renal replacement therapy, and vasopressor therapy was similar in the 2 groups (table 3) . during the icu stay, patients who received rbv/rifn therapy were more likely to receive corticosteroid therapy compared with those who did not receive rbv/rifn (59.7% vs 44.9%; p = .006; table 3 ). crude 90-day mortality was higher in patients who received rbv/rifn therapy compared to those who did not analyses of rbv therapy vs no rbv and rifn vs no rifn were consistent with results to the primary analysis, with no significant association with 90-day mortality or mers-cov rna clearance using marginal structural modeling (table 4 ). when the logistic regression model was adjusted for clustering by centers in addition to the previously mentioned baseline variables, there remained no association of rbv/ rifn with 90-day mortality. examining different types of rifn on 90-day mortality using a similar logistic regression model and adjusting for clustering by center showed similar results (supplementary table 2 ). there were no differences between the 2 groups group over time in hemoglobin, wbc count, platelet count, ast, alt, bilirubin, inr, lactic acid, or creatinine (supplementary figure 3) . however, patients treated with rbv/rifn received more blood transfusions compared with those who were not treated with rbv/rifn (58/144 [40.3%] vs 58/205 [28.3%]; p = .02; table 3 ). while benefit of rbv/rifn was suggested by preclinical studies, our observational study that accounted for baseline and time-varying differences among 349 critically ill patients with mers treated with rbv/rifn, or not, demonstrates that rbv/ rifn was not associated with decreased mortality or with faster mers-cov rna clearance. what are the possible explanations for lack of clinical and virological benefit? first, it has been shown that the rbv concentrations required to inhibit mers-cov replication are much higher than clinically achievable concentrations with oral dosing [5] . second, the lack of rifn effectiveness may be related to the type used. one study examined the in vitro mers-cov susceptibility to different rifn preparations (rifn-α2b, rifn-γ, rifn-universal, rifn-α2a, rifn-β) and found that rifn-β had the strongest mers-cov inhibition, at 41 times lower than the previously reported 50% inhibitory concentration (56.08 u/ml) of rifn-α2b [5] . another in vitro study found that serum concentrations achievable at therapeutic doses of rifn-β-1b were 3-4 times higher than the in vitro inhibitory concentrations of mers-cov, whereas those of other rifn preparations and rbv were lower than inhibitory levels [24] . of note, none of the patients in the current cohort received rifn-β-1b. an rct (miracle) is currently recruiting patients examining the effect of a combination of lopinavir/ritonavir and rifn-β-1b on mortality of hospitalized patients with mers [16] . third, the positive, but modest, effect observed in previous rhesus macaque experiments occurred after very early treatment (8 hours after inoculation of with mers-cov) and with the administration of high doses of rbv/rifn-α2b. in contrast, it took a median of 5 days for patients in our cohort to present to the hospital and another 4 days to start therapy. to assess safety profile of rbv/rifn, we compared the levels of hemoglobin, wbc count, platelet count, ast, alt, bilirubin, inr, lactate, and creatinine and we found no difference between the 2 groups over the icu stay. however, these data should be interpreted in the context of the potential for time-varying confounding. of note, we found that patients who were treated with rbv/rifn received more blood transfusions than patients who were not treated with rbv/rifn, which is consistent with the findings of a previous study [9] . these changes may be related to hemolysis induced by rbv therapy. our study demonstrates that not accounting for time-varying confounding can substantially influence the results of observational studies. our study found an association of rbv/rifn with higher crude mortality on crude analysis, with adjustment for baseline characteristics alone (by logistic regression) and with adjustment for baseline characteristics including the time to initiation of rbv/rifn (by cox proportional hazards analysis). ultimately, we did not find evidence that rbv/rifn therapy was associated with reduced mers mortality when adjusting for baseline and time-dependent covariates using a marginal structural model. this finding suggests that much of the observed increased mortality may have been related to confounding due to indication bias, and calls for caution when interpreting observational studies that do not account for timevarying confounders. our study examined rbv/rifn therapy in a large multicenter cohort of critically ill patients with mers. limitations include its retrospective nature and lack of randomization (and therefore inevitable initial imbalance in potential confounders). although marginal structural models adjust for time-varying confounding, unmeasured confounders cannot be entirely excluded. quantitative data on viral loads were not available. because practices for repeating mers-cov rrt-pcr varied among centers and because of competing risk of mortality, sufficient data to assess mers-cov rna clearance were available for only about 50% of the patients. rcts remain the best approach to derive the most unbiased estimates of treatment effect. most patients included in this study were diagnosed with mers prior to the launch of the first therapeutic rct mentioned earlier (miracle). since then, efforts have focused on considering eligible patients in this trial. .71 logistic regression and cox proportional hazards regression models were adjusted for the following variables: diabetes with chronic complications, liver disease, renal disease, any malignancy including leukemia or lymphoma, sequential organ failure assessment (sofa) score on day 1, source of infection (community-acquired/healthcare worker, hospital-acquired/non-healthcare worker, hospital-acquired), and year (before 1 july 2014 and after). for logistic regression, hosmer-lemeshow goodness-of-fit test was used to assess the model fitness, and p values were not significant for all analyses. for marginal structural model, adjustment was made for the same afore mentioned baseline characteristics and for the following time-varying covariates: sofa on the index day of rbv/rifn initiation and the previous day, ventilation status on the index day and on the previous day (0: not ventilated, 1: noninvasive ventilation, 2: invasive mechanical ventilation, 3: oxygen rescue therapy), hemoglobin, white cell count, aspartate aminotransferase and creatinine on the index day, and corticosteroid therapy on the index day. for cox proportional hazards regression model and marginal structural cox proportional hazards model, we censored patients if they never cleared mers-cov rna or at the time of last real-time reverse-transcription polymerase chain reaction test. abbreviations: ahr, adjusted hazard ratio; aor, adjusted odds ratio; ci, confidence interval; mers-cov, middle east respiratory syndrome coronavirus; rbv, ribavirin; rifn, recombinant interferon. in conclusion, rbv/rifn therapy (rbv and/or rifn-α2a, rifn-α2b, or rifn-β1a) is not associated with reduction in 90-day mortality or with faster mers-cov rna clearance. future studies should test the antiviral and clinical effectiveness of newer antiviral interventions that show more promising results in relevant animal models [25, 26] . middle east respiratory syndrome inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques therapeutic options for middle east respiratory syndrome coronavirus (mers-cov)-possible lessons from a systematic review of sars-cov therapy interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays ribavirin and interferon-α2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia immortal time bias in pharmaco-epidemiology effectiveness of inhaled corticosteroids in chronic obstructive pulmonary disease: immortal time bias in observational studies effect of ribavirin and interferon on the outcome of critically ill patients with mers saudi critical care trial group. critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections miracle trial group. treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial international severe acute respiratory and emerging infection consortium (isaric) use of the sofa score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. working group on "sepsis-related problems" of the european society of intensive care medicine saudi critical care trial group. corticosteroid therapy for critically ill patients with middle east respiratory syndrome canadian critical care trials group h1n1 collaborative. the influence of corticosteroid treatment on the outcome of influenza a(h1n1pdm09)-related critical illness marginal structural models to estimate the causal effect of zidovudine on the survival of hiv-positive men analysis of longitudinal observational data using marginal structural models marginal structural models and causal inference in epidemiology broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses safety and tolerability of a novel, polyclonal human anti-mers coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study supplementary materials are available at clinical infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-351186-llnlto7p authors: park, yong-shik; lee, changhwan; kim, kyung min; kim, seung woo; lee, keon-joo; ahn, jungmo; ki, moran title: the first case of the 2015 korean middle east respiratory syndrome outbreak date: 2015-11-14 journal: epidemiol health doi: 10.4178/epih/e2015049 sha: doc_id: 351186 cord_uid: llnlto7p this study reviewed problems in the prevention of outbreak and spread of middle east respiratory syndrome (mers) and aimed to provide assistance in establishing policies to prevent and manage future outbreaks of novel infectious diseases of foreign origin via in-depth epidemiological investigation of the patient who initiated the mers outbreak in korea, 2015. personal and phone interviews were conducted with the patient and his guardians, and his activities in saudi arabia were investigated with the help of the saudi arabian ministry of health. clinical courses and test results were confirmed from the medical records. the patient visited 4 medical facilities and contacted 742 people between may 11, 2015, at symptom onset, and may 20, at admission to the national medical center; 28 people were infected and diagnosed with mers thereafter. valuable lessons learned included: (1) epidemiological knowledge on the mers transmission pattern and medical knowledge on its clinical course; (2) improvement of epidemiological investigative methods via closed-circuit television, global positioning system tracking, and review of health insurance review and assessment service records; (3) problems revealed in the existing preventive techniques, including early determination of the various people contacted; (4) experiences with preventive methods used for the first time in korea, including cohort quarantine; (5) reconsideration of the management systems for infectious disease outbreaks across the country, such as this case, at the levels of central government, local government, and the public; (6) reconsideration of hospital infectious disease management systems, culture involving patient visitation, and emergency room environments. after landing in korea in may of 2015, middle east respiratory syndrome (mers) resulted in a total of 186 confirmed cases and 36 deaths from may 11, when the symptoms occurred in the first patient, to july 4, when the last patient developed the symptoms [1] . a novel coronavirus confirmed for the first time in 2012 is suspected to be the cause of mers and camels may be the host. human-to-human transmission is known to occur after transmission from a camel to a human, but the exact route has not been sufficiently investigated [2] . the outbreak in korea started with a patient who arrived in may 2015 from saudi arabia, a country that had an outbreak of a large number of mers cases, and subsequently spread. a majority of infected patients were revealed to be cases of hospital-acquired infection [2, 3] . this report is an epidemiological study of the first patient (patient #1) who initially brought mers into korea in 2015. from may 20, when a diagnosis of mers was confirmed, the division of epidemic intelligence service, korea centers for disease control and prevention (kcdc), in cooperation with a group of civilian volunteers in epidemiology, traced the infection route and performed preventive measures for the spread of additional infections. through personal and phone interviews we contacted employees at business facility in saudi arabia who may have had contact with patient #1 during the incubation period; we investigated the places he visited, presence or absence of mers symptoms in the individuals he contacted, history of visiting medical facilities in the middle east, and history of consuming camel milk or meat, among other things. the patient's specific activities in saudi arabia were verified with the help of the saudi arabian ministry of health. additionally, the timing of symptom occurrence and the initial symptoms were reevaluated by confirming the history of visiting domestic medical facilities after arrival in korea via personal interviews with the patient and his guardians and examining his medical records. the kcdc performed a diagnostic serum antibody test for mers for individuals whose contact with patient #1 was confirmed by personal interviews or closed-circuit television (cctv) reviews. this study was conducted as an epidemiological investigation of the mers outbreak, and thus, additional processes for review and approval by institutional review board were not required on the basis of the life ethics and safety law enforcement rule item 2 (human subject studies). patient #1 was a 68-year-old man and had underlying diseases including asthma, hypertension, dyslipidemia, and benign prostate hypertrophy. at the time of the study, he was a current smoker. he was in the greenhouse building business with a business facility in the middle east, specifically bahrain, as well as at a domestic business facility in asan, chungnam, korea. he visited the middle east region about once every 2 months and stayed for approximately 3 weeks during each visit. the most recent business trip was between april 24, and may 4, 2015, for 11 days. he had business visits to saudi arabia (may 1 to 2) and the united arab emirates (april 29 to 30) with bahrain as the base; both departure from and arrival at korea was via qatar. during his stay in the middle east, he had no history of direct contact with camels, consuming camel by products such as milk or meat, or visiting the medical facilities there. in saudi arabia, he stayed at his business facility and the hotel in the area of al muzahimiyah outside of the capital, riyadh. during his trips within the middle east, he had no contact with animals and did not eat or drink outside the hotels where he stayed. while staying in riyadh, he travelled with a driver, a guide, and the bahrain business facility manager, none of whom showed the symptoms suspected of mers. patient #1 returned to korea without any abnormal symptoms on may 4 via flight oz6888 and went to his domestic business place in asan on may 11. on may 11, fever broke for the first time, and he visited asan seoul clinic the next day (may 12). at the time of the visit, his body temperature was 37.0°c, and he complained of febrile sense and myalgia. on may 14, he visited the same place with a persistent high temperature (38.9°c) and myalgia, and on may 15, he was transferred to pyeongtaek st. mary's hospital for inpatient treatment, because respiratory symptoms such as cough and sputum had developed, his body temperature was 38.3°c, and the myalgia had worsened. after the transfer, he was in an outpatient exam room, a laboratory room, and a chest radiography room, and was put in a double-occupancy room at approximately 2 pm (room 8104, 8th floor). around 7:15 pm, a chest computed tomography (ct) scan was performed on the first underground floor. on the following day (may 16) at approximately 7:15 am, he visited the radiography room on the first underground floor to undergo a chest radiogram. pneumonia in the right upper lobe was found on the chest ct scan, and infections with haemophilus influenzae and streptococcus pneu monia were confirmed on bacterial culture testing. from the evening of may 15, breathing difficulty and chest pain developed. symptoms did not improve, and the patient was discharged from the hospital at 10 am on sunday, may 17; thereafter, his wife drove him for treatment to the 365 clinic, where he usually went for examination. the 365 clinic referred him to a high-level medical center. he went to the samsung medical center in seoul emergency department, but returned home because a patient room was not available. on the following day (may 18) at approximately 10 am, his wife drove him again to the samsung medical center in seoul emergency department, and he was admitted. from 2 pm, he wore a facial mask. on may 19, he was reported to the kcdc as a case suspected of mers in consideration of the clinical courses that deteriorated despite the antibiotic therapy and his history of travel to the middle east within the previous two weeks, which was revealed during a consultation with physician. on may 20, his sputum polymerase chain reaction (pcr) test was determined positive, following which he was transferred to the national medical center. patient #1 received antiviral, interferon, and antibiotic treatments, and me chanical ventilation therapy after an endo-tracheal intubation was performed due to worsening respiratory symptoms. on june 30, a sputum pcr test was confirmed negative for mers virus, and it was decided that he was cured. on september 25, he was discharged after completing treatment for a sacral sore (figure1). individuals who made contact with patient #1 and preventive efforts for them individuals who were in direct contact with patient #1 without appropriate personal protective equipment or who stayed with him in an enclosed space (patient room, office, etc.) were considered cases of close contact and were put under surveillance. the scope of close contact surveillance gradually expanded with the spread of the mers outbreak, and the final count of individuals under close contact surveillance was 742: 4 employees at patient #1's domestic business facility in asan, 4 medical staff members at the asan seoul clinic, 672 medical staff members and patients (including patient #1's wife) at pyeongtaek st. mary's hospital, 39 medical staff members and patients at the 365 clinic, and 23 medical staff members and patients at the samsung medical center in seoul. depending on the extent of contact, they were classified into either 1) quarantine of a person or a place or 2) active observational surveillance and were closely tracked and observed ( figure 1 ). during surveillance, when symptoms suspected as mers occurred, respiratory samples were collected for testing, the suspected cases were transferred to a quarantined hospital, and epidemiological investigations were conducted. a total of 742 people had contact with patient #1 from may 11, when the first symptoms occurred, to may 20, when he was transferred to the national medical center. of those, 28 became infected, with an infection rate of 3.8%. according to the characteristics of the contacted individuals, 4 out of 235 medical staff members (1.7%); 11 out of 206 patients (5.3%); and 13 out of 301 guardians, caregivers, and visitors (4.3%) were infected. however, trying to correlate patient #1's transmission capacity or epidemiological characteristics to the infection rate had limitations because with the spread of mers, the scope of surveillance of contacted individuals gradually expanded, and the standards were not consistent across different institutions. nonetheless, based on a study analyzing epidemiological characteristics of the mers outbreak around pyeongtaek st. mary's hospital, the rate of infection was 3.9% overall and was 1.1% among medical staff members; 7.6% among patients; and 3.3% among guardians, caregivers, and visitors [4] . aside from patient #1, 3 of 28 individuals, in particular, with a confirmed diagnosis (#14, #15, and #16) individually infected more than 5 individuals whose diagnosis was additionally confirmed, and they were thus labeled as "super spreaders." it is thought that, after contact with patient #1, they visited different medical facilities and became epidemiologically associated with additional patients with a confirmed diagnosis [5] . the 2015 mers outbreak in korea started with the infection of a businessman in his 60s who often traveled to the middle east for business. he did not show symptoms at his arrival in korea. a week later, he started experiencing symptoms and visited medical facilities. however, it took 9 days for the mers infection to be confirmed at the 4th medical facility he visited, and, by then, the infection transmission had already begun at the previous 3 medical facilities. thereafter, a "mers crisis" transpired for the first time wherein a total of 186 patients were confirmed with mers infection; 36 deaths occurred among these patients, and 16,693 individuals were quarantined for prevention. the clinical symptoms in the first patient deteriorated because of a delay in receiving appropriate treatment, and he was assisted with mechanical ventilation. on day 42 after admission to the 4th hospital, tests for mers virus were negative, and the patient's condition recovered. from the epidemiological investigation, the course of transmission to this patient in the middle east was not clear. it can only be inferred that the first patient became infected while visiting saudi arabia, on the basis of a recent study showing that the mers virus that broke out in korea is genetically closest to the qatar strain [5, 6] . however, to uncover the specific course of transmission, we must perform an epidemiological investigation in coordination with saudi arabia. various factors are considered as causes of the widespread outbreak. the first is health care workers' lack of knowledge regarding mers. since june 2013, the kcdc had organized a national mers management team in preparation of a mers outbreak in korea, and mockup training had been performed to enhance the capacity to manage outbreaks of novel infectious diseases at quarantine stations and at the level of local government of cities and provinces. however, in cases such as the present case, wherein the symptoms do not manifest themselves when patients enter korea, preventing an outbreak of a novel infectious disease by imposing quarantine is not practical. in particular, health care workers did not receive sufficient education or communication against mers in anticipation of a case wherein a mers-infected individual, on whom quarantine was not imposed, would go to a regional medical facility. until the diagnosis was confirmed for patient #1, the first 3 medical facilities that he visited were unable to suspect a mers case. the doctor at the 3rd medical facility, who examined patient #1 and subsequently became infected, in an interview with the media after his discharge stated that he had not even heard of mers. the second factor communication about mers among travelers to the middle east was insufficient. since 2013, when the likelihood of spread of mers would spread within the middle east region was indicated, the kcdc persistently promoted mers awareness among the travelers to that region. however, patient #1, who frequently visited the region and his family members, had no idea about mers. thus, when he visited the general medical facilities with respiratory symptoms, he did not mention about his travel to the middle east, and only mentioned about his visit to bahrain when inquired about a travel history involving the middle east at the medical center where his diagnosis was confirmed. therefore, mers could not be diagnosed sooner. a third factor contributing to the rationale behind the outbreak was not kept in check earlier was that quarantine was not thoroughly imposed. after patient #1's diagnosis was confirmed, thorough quarantine was neither imposed on individuals who had close contact with him nor on those with casual contact (such as those who had a possibly contacted the doctors or the patients or those who were exposed to a space infected by him). lastly, in this outbreak, most patients were infected via hospital-acquired infection because korean medical institutions' patient rooms and emergency rooms were very crowded, and infected patients had to visit several hospitals because of the shortcomings of the medical care delivery system, which played a critical role in the spread of mers from hospital to hospital. through an investigation on the mers outbreak within korea, valuable lessons were learned. they include (1) accumulation of epidemiological knowledge of the pattern of mers transmission as well as medical knowledge on its clinical courses; (2) improvement of epidemiological investigative techniques using cctv, global positioning system tracking, and review of health insurance review and assessment service records, among other things; (3) problems revealed in the existing preventive techniques, including the early determination of the scope of contacted people; (4) accumulation of experiences with the preventive techniques used for the first time in korea, such as cohort quarantine; (5) reconsideration of the management systems that deal with an outbreak of infectious diseases across the country, such as in the present case, at the levels of central government, local government, and the public; and (6) reconsideration of infectious disease management system at hospitals, culture involving patient visitation, and emergency room environments. the 2015 mers outbreak in korea began with an infected businessman in his 60s who visited the middle east region. however, the spread of outbreak is attributed to insufficient preparation and management by the ministry of health and welfare. to prepare for an outbreak of novel infectious diseases in future, government organizations responsible for infectious diseases need to train personnel specialized in the area, and also promote awareness among medical staff at the primary and secondary medical facilities as well as among the public. the mers outbreak should serve as an opportunity to improve the level of managing hospital-acquired infection in korea. mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome current epidemiological situation of middle east respiratory syndrome coronavirus clusters and implications for public health response in south korea epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control the authors have no conflicts of interest to declare for this study. supplementary material (korean version) is available at http: //www.e-epih.org/. key: cord-333144-gyuh2fvl authors: siddiqui, arif jamal; jahan, sadaf; ashraf, syed amir; alreshidi, mousa; ashraf, mohammad saquib; patel, mitesh; snoussi, mejdi; singh, ritu; adnan, mohd title: current status and strategic possibilities on potential use of combinational drug therapy against covid-19 caused by sars-cov-2 date: 2020-08-05 journal: journal of biomolecular structure & dynamics doi: 10.1080/07391102.2020.1802345 sha: doc_id: 333144 cord_uid: gyuh2fvl the spread of new coronavirus infection starting december 2019 as novel sars-cov-2, identified as the causing agent of covid-19, has affected all over the world and been declared as pandemic. approximately, more than 8,807,398 confirmed cases of covid-19 infection and 464,483 deaths have been reported globally till the end of 21 june 2020. until now, there is no specific drug therapy or vaccine available for the treatment of covid-19. however, some potential antimalarial drugs like hydroxychloroquine and azithromycin, antifilarial drug ivermectin and antiviral drugs have been tested by many research groups worldwide for their possible effect against the covid-19. hydroxychloroquine and ivermectin have been identified to act by creating the acidic condition in cells and inhibiting the importin (impα/β1) mediated viral import. there is a possibility that some other antimalarial drugs/antibiotics in combination with immunomodulators may help in combatting this pandemic disease. therefore, this review focuses on the current use of various drugs as single agents (hydroxychloroquine, ivermectin, azithromycin, favipiravir, remdesivir, umifenovir, teicoplanin, nitazoxanide, doxycycline, and dexamethasone) or in combinations with immunomodulators additionally. furthermore, possible mode of action, efficacy and current stage of clinical trials of various drug combinations against covid-19 disease has also been discussed in detail. communicated by ramaswamy h. sarma human coronaviruses are a group of spherical or pleomorphics medium size (100-150 nm), enveloped rna viruses containing petal or club shaped peplomers on the surface. they belong to the family coronaviridae. these viruses infect mammals and birds causing diseases of the respiratory tract, gastrointestinal tract, liver, kidney and nervous system. human coronavirus is responsible for human respiratory disease and a causative agent of common cold. the us national institute of allergy and infectious diseases (niaid) research efforts build on earlier research on severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), which also are caused by coronaviruses (acter et al., 2020) . mers was firstly erupted in saudi arabia in september 2012. according to who, this disease has since spread to 27 countries (chafekar & fielding 2018) . patients infected with mers coronavirus (mers-cov) develop severe acute respiratory illness, consisted with shortness of breath, cough and fever. infection with sars coronavirus (sars-cov) can also cause a severe viral respiratory illness. it was first reported in china in february 2003 though cases subsequently were noticed from november 2002 (rabaan et al., 2020) . by the time it was being contained, sars spreaded to 26 countries in the span of four months. the evidence of research suggested that mers-cov and sars-cov are originated from bats and in the same manner as covid-19 was spread as well (rabaan et al., 2020) . furthermore, sars-cov spreads from infected civets to the human, while mers-cov spreads from infected dromedary camels to human. scientists are still trying to find out how sars-cov-2 spread from an animal reservoir to human (mahanta et al., 2020; rabaan et al., 2020) . currently, the world is suffering from a pandemic disease covid-19 caused by a novel strain of coronavirus, called as sars-cov-2 (aanouz et al., 2020) . availability of number of covid-19 diagnostic test/kits or poor health services and sectors in lower income countries (mahanta et al., 2020) . currently till date (21 june 2020), countries with the most number of recorded infected cases are united states (2,255,119 cases, 119,719 deaths and 975,038 recovered), followed by brazil (1, 067, 579 cases, 49, 976 deaths and 543, 186 recovered) , russia (576, 952 cases, 8002 deaths and 339, 711 recovered) , india (410, 461 cases, 13, 254 deaths and 235, 328 recovered), united kingdom (303, 110 cases, 42, 589 deaths) , spain (245, 938 cases, 28, 322 deaths) , peru (251,338 cases, 7861 deaths and 138,763 recovered), italy (238,275 cases, 34,610 deaths and 182,893 recovered) , chile (236, 748 cases, 4295 deaths and 196, 609 recovered) , iran (202,584 cases, 9507 deaths and 163,591 recovered) and germany (189,822 cases, 8882 deaths and 174,900 recovered) (ecdc 2020; who 2020; worldometer 2020) (figure 1 ). according to this data, it can be concluded that infection rate due to covid-19 is spreading exponentially in new hotspots like united states and europe, when compared to first epicenter china, where the number of new cases is rapidly declining. therefore, at this stage, the urge and requirement of effective drug or vaccine to control the covid-19 disease is at its peak (islam et al., 2020; k et al., 2020) . discovery of new drug or vaccine development after all trials for human use will take approximately further time of 1-2 years or more. however, some countries have already started efforts in making vaccine or chemoprophylaxis against covid-19 disease, even initiation of human trials are on track (choudhary & sharma 2020; khan et al., 2020; muralidharan et al., 2020) . while some drugs have shown therapeutic effect against covid-19 infection such as hydroxychloroquine (al-kofahi et al., 2020; choudhary & sharma 2020; liu et al., 2020; sinha & balayla 2020) , azithromycin, (andreani et al., 2020a; choudhary & sharma 2020) ivermectin (caly et al., 2020; chaccour et al., 2020; choudhary & sharma 2020) and some other antivirals (asai et al., 2020; boopathi et al., 2020; lian et al., 2020) . however, it is still not clear that whether these drugs have a better therapeutic effect, when compared to other drugs or combination therapy of multiple drugs with immunomodulators will prove to provide us with better results. consequently, this review will provide an insight and comprehensive view on different therapeutic approaches including combining of different known anti-parasitic drugs, as well as proposing novel suggestions of chemoprophylaxis drug therapy, which can be used in the current treatment and vaccine development strategies against covid-19 disease. all peer reviewed scientific papers were used for this review article. extensive literature searches have been performed using various literature search engines, including science direct, pubmed, web of science, google scholar, scopus and researchgate with several terms: (1) mers-cov, sars-cov-1 and sars-cov-2 related articles; (2) antimalarial drugs usages in covid-19 disease; (3) antiviral drugs; and (4) immunomodulators and vaccine related compounds. all relevant studies meeting search criteria were included in this review. lacking specific antiviral treatment, both sars-cov and mers-cov pose major clinical management challenges (lu et al., 2015) . many drugs and therapies are still under clinical trials and substantial efforts are underway to discover new therapeutic agents for coronavirus infections (lin et al., 2018; zumla et al., 2016) . the isaric (international severe acute respiratory & emerging infection consortium), collected drugs list in july 2013 which are easily available for the treatment of pandemic influenza, mers-cov, sars-cov-1. sars-cov-1 came into picture first time in southern china and quickly spreaded around the globe in 2002 -2003 (rabaan et al., 2020 . with high rate of nosocomial transmission having symptoms of high fever and unusual epidemic of a typical pneumonia to health care workers was happened in foshan, guangdong, china on november 2002 (sims et al., 2008) . the most promising and clinically available drugs were ribavirin and interferon (ifn), or a combination of the two. the combination of drugs have shown the efficacy in an in vivo model for mers-cov infection ( figure 2 ) (falzarano et al., 2013) . although, the combination could not fulfill the recovery criteria in the small number of severely ill mers-cov patients (dyall et al., 2017) . in an in vitro study, mycophenolic acid (mpa) and ifn-b were also found to be highly effective against mers-cov infection . mpa was found to be effective and specific to mers-cov, and also with some activity were detected against sars-cov infection (chan et al., 2015; hart et al., 2014) . clinical trials have demonstrated the expression of ip-10, ifn-c, il-8 and il-6 and these cytokines are denoted the severity of the disease (russell et al., 2020) . three fda-approved broad-spectrum inhibitors (chlorpromazine, chloroquine, toremifene) that were shown to be effective against mers-cov infection in immortalized cell lines and evaluated their antiviral activities (cong et al., 2018) . among all three drugs, toremifene is known as an estrogen receptor modulator which has been found to restrict filoviruses and thus inhibit both mers-cov and sars-cov (cong et al., 2018) . previously, chloroquine (cq), a well-established anti-parasitic agent, showed strong inhibition on mers-cov and sars-cov with low toxicity (cong et al., 2018) . cq likely accumulates in lysosomes, where it sequesters protons and increases the ph. the drug interacts with a variety of host proteins and cellular processes, resulting in modulation of immune response (cong et al., 2018) . cq has also been reported to inhibit replication of multiple viruses such as flaviviruses, influenza viruses, human immunodeficiency virus (hiv), ebola and nipah viruses in vitro . however, in other study cq had showed the least anti-mers activity with very low toxicity during the treatment of coronavirus (cong et al., 2018) . although, another drug chlorpromazine, belongs to neurotransmitter inhibitor, considered as first established antipsychotic drug and was used for schizophrenia treatment (de wilde et al., 2014). in addition, the mode of action of chlorpromazine is by inhibiting the clathrin-mediated endocytosis via blocking the formulation of clathrin-coated pits at the plasma membrane (de wilde et al., 2014). according to earlier work, which has stated that chlorpromazine have potential to inhibit mers-cov infection of huh 7 cells with an ec 50 of 4.9, vs cc 50 of 21.3 lm (cong et al., 2018) . however, all these studies determined the tested compounds, each of which were shown to be efficacious in continuous cell lines, and could be a promising therapeutic drugs to treat the coronaviruses related diseases. current status of various drug therapy in use for the treatment of covid-19 with their possible antiviral effects and mechanism of action there are many drugs which are currently in use for the treatment of covid-19 disease and most of the drugs are under clinical trials (table 1) . the hcq is the derivative of cq, and is the first category drug which is working as a therapeutic agent against covid-19 infection (beura & prabhakar 2020; choudhary & sharma 2020; sinha & balayla 2020) . this drug has also been previously used for the treatment of rheumatoid arthritis and systemic lupus erythematosus (adeoye et al., 2020; sinha & balayla 2020) and is a first line drug for malaria treatment (azad et al., 2017; bhardwaj et al., 2016; roesch et al., 2020; siddiqui et al., 2015) . effect of hcq on viral replication goes beyond cytokines inhibition (asai et al., 2020; bhardwaj et al., 2015; siddiqui, bhardwaj, et al., 2020; sinha & balayla 2020) . it acts as weak base and tend to increase the ph within the intracellular vacuole (choudhary & sharma 2020; hasan et al., 2020; sinha & balayla 2020) . due to the weak base of this medication, it may affect acid balance and can inhibit many enzymes. this specific feature of hcq possibly inhibits the viral entry to the cell (asai et al., 2020; sinha & balayla 2020) . it is also known to obstruct the viral post-translational modifications and glycosyl-transferases (choudhary & sharma 2020) . furthermore, it has been known to modify the processes such as degradation of protein through acidic hydrolases in the lysosome ( figure 3 ) (choudhary & sharma 2020) . the impact of antiretroviral has been considered due to inhibition of viral glycosylation; a significant antiviral mechanism of hcq (asai et al., 2020) . additionally, it also inhibits the protein replication of viruses by blocking/distracting the pathway of endosome/lysosome or viral protein maturation. some studies have revealed the possible mechanism of hcq and its activity against covid-19 sinha & balayla 2020) . most of the countries are currently using hcq for the treatment and management of covid-19. however, many recent studies showed that the use of hcq in hospitalized covid-19 patients had no impact on the risk of the most severe outcomes from the disease (ferner & aronson 2020a; geleris et al., 2020) . furthermore, latest published data showed that, treatment with hcq in 97 hospitalized patients of covid-19, reduced the risk of mechanical ventilation. although, mortality rate was higher in patients who got the treatment with hcq alone (magagnoli et al., 2020) . another study also showed similar data that high dose of hcq drug is not of any apparent benefit and also the mortality rate is still high (borba et al., 2020) . likewise, joshua geleris et al., also treated 811 covid-19 patients with hcq (600 mg twice on day 1, then 400 mg daily for a median of 5 days), and the authors said the results should not be rule out either benefit or harm from hcq use, however, the finding of this study do not support continued use of hcq drug in covid-19 patients (geleris et al., 2020) . furthermore, gautret et al., study showed that hcq treatment is significantly associated with viral load reduction/disappearance in covid-19 patients (gautret et al., 2020a) . similarly, zhaowei chen et. al, study showed that the use of hcq drug in patients with covid-19 was just providing a significant recovery in the short period . data collected by who, regarding safety and efficacy of hcq as the treatment of covid-19, the team of executive group of the solidarity trial decided to implement a temporary pause on hcq trials as a precautionary measure. however, hcq has been registered for clinical trial in more than 13 different countries and approximately 40 randomized clinical trials started giving answer about hcq's efficacy against covid-19 (bienvenu et al., 2020) . we need to wait to shed more light, based on clinical evidences for using hcq against covid-19 patients. azithromycin is the second choice of antimalarial drug to use against covid-19 and it belongs to the class of antibiotics (andreani et al., 2020a; bakheit et al., 2014; soni et al., 2015) . many studies have recently revealed the therapeutic effect of azithromycin against the covid-19 infection (andreani et al., 2020a; asai et al., 2020; choudhary & sharma 2020) . however, the exact mechanism is still unknown, but multiple mechanisms have been proposed for the putative antiviral properties determined with azithromycin drug. some researches states that azithromycin acts as acidotropic lipophilic weak base, which changes (increase) the ph level of endosome maturation and trans-golgi network (asai et al., 2020; choudhary & sharma 2020) . moreover, it can potentially inhibit endocytosis and viral genetic shedding from lysosomes, ultimately restricting viral replication (gravesen & judy 2020) . similarly, influenza and hiv also needs an acidic environment for the uncoating of the envelop. however, coronavirus also belongs to enveloped virus and it might have similar mechanism (choudhary & sharma 2020; thanh le et al., 2020) . likewise, these kinds of mechanisms have also been found inhcq. furthermore, azithromycin directly acts on bronchial epithelial cells by decreasing mucus secretion to enable and enhances the lung function (choudhary & sharma 2020) . recently, quantum mechanical modeling proposed a possible role of azithromycin drug in interfering with viral entry through binding collaboration between the covid-19 spike protein and host receptor ace2 protein (angiotensin converting enzyme 2) ( figure 3 ) (basit et al., 2020; choudhary & sharma 2020; lobo-galo et al., 2020) . however, clinical trials are still necessary to confirm the role of the drug and its work as prophylaxis in reducing the infection rate. azithromycin is currently table 1 . list of drugs used in the treatment of mers-cov, sars-cov-1 and sars-cov-2 with their chemical structure, biological activity and possible mechanism. chemical structure biological activity/therapeutic effect mode of action references hydroxychloroquine used in the treatment of malarial and inflammatory activities. it is also now often used as an antirheumatologic agent in systemic lupus erythematosis and rheumatoid arthritis. currently, this drug is used against sars-cov-2. inhibits terminal glycosylation of ace2, the receptor that sars-cov-2 target for cell entry. ace2 that is not in the glycosylated state may less efficiently interact with the sars-cov-2 spike protein, further inhibiting viral entry. (gautret et al. 2020a; liu et al. 2020) azithromycin it is used orally in children for the treatment of acute otitis media, malaria and also against sars-cov-2 interferes with viral entry through binding collaboration between the covid-19 spike protein and host receptor ace2 and block the viral entry. (andreani et al. 2020b; asai et al. 2020) ivermectin it is a macrocyclic lactone derived from streptomyces avermitilis with antiparasitic activity such as nematodes, scabies and onchocerciasis (river blindness). now this drug is also used against sars-cov-2. acts and prevents the import (integrase protein and importin (imp) a/b1 heterodimer) through the rise in antiviral response, inhibits the nuclear import of viral and host protein under clinical trial and has reached phase iv with promising results (nct02048007). ivermectin is generally used for the treatment and control of filarial diseases (murthy 2019). recently, its usage against covid-19 infection showed some positive results and patients response. in vitro study on ivermectin have also been found very effective against positive-sense single-stranded rna viruses, such as dengue, zika and yellow fever, via inhibiting the viral replication (azeem et al., 2015; choudhary & sharma 2020; gotz et al., 2016; mastrangelo et al., 2012) . similarly, recent report suggested that ivermectin have a potential to inhibit covid-19 replication in vitro (caly et al., 2020) . moreover, the treatment of it is a synthetic, broadspectrum tetracycline antibiotic exhibiting antimicrobial activity. currently, it is also in use for treatment against covid-19 disease. chelate zinc from mmps and on the basis of chelating activity of this antibiotic, it might help in inhibiting sars-cov-2. (conforti et al. 2020; malek et al. 2020; szolnoky 2020) ribavirin it is a nucleoside analogue and antiviral agent used in therapy of chronic hepatitis c, other flavivirus and coronavirus infections. ribavirin is incorporated into viral rna, thereby inhibiting viral rna synthesis, and inhibiting normal viral replication. (dyall et al. 2017; falzarano et al. 2013 ) it is an antineoplastic antibiotic derived from various penicillium fungal species. it is an active metabolite of the prodrug mycophenolate mofetil and has antibacterial, antifungal, and antiviral activities. the mechanism of action of this drug is still unknown for sars-cov-2. however, in case of mers-cov, they synergistically inhibit the papain-like protease (plpr). (chan et al. 2015; hart et al. 2014 ) it is a phenothiazine that was once the most commonly prescribed antipsychotic agent, but it is now rarely used. coronaviruses has been using clathrin-mediated endocytosis pathway to enter human cells. this drug has potential to inhibit clathrin-mediated endocytosis pathway for entry of viruses. (cong et al. 2018; de wilde et al. 2014 ) it is an aminoquinoline used for the prevention and therapy of malaria. it is also effective in extraintestinal amebiasis and act as an antiinflammatory agent for therapy of rheumatoid arthritis and lupus erythematosus does not affect the level of ace2 expression on cell surfaces, but inhibits terminal glycosylation of ace2, the receptor that sars-cov and sars-cov-2 target for cell entry (cong et al. 2018; dyall et al. 2014 ) it is a non-steroidal antiestrogen that is used in the treatment of estrogen receptor positive breast cancer. long term toremifene therapy has been associated with development of fatty liver, steatohepatitis, cirrhosis, and rare instances of clinically apparent acute liver injury inhibits the spike protein and nsp14 (methyltransferase non-structural protein) of sars-cov-2. zhou et al. 2020 ) it is a synthetic corticosteroid with anti-inflammatory and immunomodulating properties. methylprednisolone binds to and activates specific nuclear receptors, resulting in altered gene expression and inhibition of proinflammatory cytokine production. zhu et al. 2020) covid-19 by the single dose of ivermectin was found to reduce the viral load up to 5000 fold in vitro culture within 48 h (caly et al., 2020) . however, the best part of this drug is that no toxicity was observed during in vitro culture. mechanism of action of this drug against covid-19 is still unknown, though researchers believed that this drug is working in a similar way it acts on other viruses. single stranded rna viruses are mostly dependent on integrase protein and importin (imp) a/b1 heterodimer during infection, and this drug acts and prevents the import through the rise in antiviral response that ivermectin inhibits the nuclear import of viral and host protein (caly et al., 2020; choudhary & sharma 2020) (figure 3 ). ivermectin has passed the phase i of clinical trial and now 200 mcg/kg dose of ivermectin is under phase ii clinical trial (nct04407130). favipiravir is generally used for the treatment and control of influenza virus (delang et al., 2018; elfiky 2020; furuta et al., 2017) . this medicine was first licensed by japan and largely used as an anti-influenza drug. it's efficacy in inhibiting other viruses such as ebola, lassa and rabies have been seen effectively (asai et al., 2020) . this drug is a derivative of pyrazinecarboxamide and it is being developed and manufactured by fujifilm group. it targets rdrp enzymes, that is important for the transcription and replication of viral genomes (coomes & haghbayan 2020) . furthermore, favipiravir also has a potential to use for the treatment of avian influenza and other influenza strains that are resistant to neuramidase inhibitors (coomes & haghbayan 2020) . the mechanism of action of favipiravir is described previously; it acts on viral rna synthesis as a chain terminator at the specific site, where the rna is incorporated into the host cell ( figure 3 ). furthermore, this drug is not effective against dna based viruses. conversely, this specific property of favipiravir proves to have a positive outcome in the treatment of covid-19 disease (asai et al., 2020) . currently, favipiravir is under clinical trial and it has passed phase i and ii with promising results (nct04359615). significantly, some study showed that the treatment of covid-19 patients with favipiravir showed reduction in the load of sars-cov-2 when compared with control groups (cai et al., 2020; coomes & haghbayan 2020; lu et al., 2020) . the pharmaceutical company glenmark, india launched antiviral drug favipiravir under the brand name fabiflu on 20 th june 2020 for the treatment of mild to moderate covid-19 patients. however, fabiflu is the first oral favipiravir drug approved medication in india for the treatment of covid-19 infected patients. dose of favipiravir is administrated orally, 1800 mg twice daily on day 1 than 800 mg twice daily up to 14 days. ã sars-cov-2 enters the human cell and initiate its replication cycle. first stage starts with the binding of sars-cov-2 virus with ace2 receptor, followed by transfer of viral rna in to the human cell. rna-dependent rna polymerase (rdrp) enzyme initiates the production of viral rnas. during rna methylation, rna cap is formed, which protects against the innate immune responses. furthermore, during the process of viral rna synthesis, translation of proteins is associated with ph-dependent membrane stress, which possibly elicits adverse effects against immune cells and cytokines. during this stage, if the viral replication cycle is not inhibited or infected cells are not eradicated; packed/assembled viruses will get disseminated and transfect other healthy host cells. triphosphate (atp) analog. furthermore, after administration it gets metabolized into its active form gs-441,524 . its activity against many other viruses such as lassa fever, nipah, hendra, respiratory syncytial, junin, mers and sars coronaviruses has also be seen (asai et al., 2020; hendaus 2020) . however, the mechanism of action of this drug is shown by the inhibition of rdrp enzymes, conceivably through the interruption of rna chain termination in the host cells (figure 3 ) (augustin et al., 2020) . consequently, it has been suggested that, remdesivir may be one of most useful and effective drug for the treatment of covid-19 disease (asai et al., 2020; ferner & aronson 2020b) . in vitro testing with remdesivir has showed good results with significant reduction in sars-cov-2 load (grein et al., 2020) . moreover, remdesivir has already reached phase iii clinical trials against sars-cov-2 (nct04365725) after encouraging pre-clinical results against sars-cov (agostini et al., 2018; sheahan et al., 2017) and mers-cov (de wit et al., 2020) . umifenovir is commonly used for the treatment of influenza a and b viruses and hepatitis c virus (pshenichnaya et al., 2019) . umifenovir is a derivative of indole carboxylic acid. this drug was firstly developed by russia in 1988 (pshenichnaya et al., 2019) . however, after in vitro demonstration, this drug showed some potential to treat ebola virus, human herpes virus, tacaribe arenavirus (pshenichnaya et al., 2019) . the mechanism of action of umifenovir is known, it obstructs viral cell membrane fusion as well as virus endosome fusion with the host cell membrane. it also interferes with hydrogen bonding network of phospholipid (costanzo et al., 2020) . however, in other diseases caused by influenza virus, umifenovir directly interacts with virus particles to stabilize hemagglutinin. blaising et.al 2020 first showed the in vitro activity of umifenovir with good results against sars-cov-1 and sars-cov-2 . currently, umifenovir is undergoing clinical trials as a single agent (nct04260594, nct04255017) and also in randomized human clinical trial for comparison with favipiravir (chictr2000030254). teicoplanin is a glycopeptide antibiotic and basically used in the prophylaxis and treatment of bacterial infections caused by gram positive bacteria such as staphylococcus aureus and enterococcus faecalis (shea & cunha 1995) . however, teicoplanin have also showed good efficacy against many viruses such as hiv, ebola virus, hepatitis c virus (hcv), flavivirus, influenza virus, as well as mers-cov and sars-cov infection (baron et al., 2020) . recently, in vitro study suggests that, teicoplanin has potential efficacy to inhibit the virus activity and reduces the load of sars-cov-2 (pandey et al., 2020; zhang, ma, et al., 2020) . furthermore, suggested that the concentration of teicoplanin antibiotic requires to inhibit the viral activity that is ic 50 value of 1.66 um (zhang, ma, et al., 2020) . it acts on early stage of the viral life cycle by blocking or inhibiting the low-ph cleavage of the viral spike protein through cathepsin l in the late endosome. thus, this way it stops the releasing of genomic viral rna and preventing the viral replication cycle inside the host cell (pandey et al., 2020) . therefore, teicoplanin needs to be further investigated for its potential effect against sars-cov-2 by randomized clinical trials, and hopefully this antibiotic will come out with some promising results. nitazoxanide is used for the treatment of parasitic diseases (cryptosporidiosis and giardiasis) that cause diarrhea, and viral diseases (hiv, hcv, hepatitis b virus (hbv), rotavirus, influenza virus, mers-cov) (pepperrell et al., 2020; simsek yavuz & unal 2020) . previous in vitro study suggested that, tizoxanide; the active circulating metabolite of nitazoxanide, inhibits the replication of the viruses (calderon et al., 2020) . the amount of drug required to inhibit viral replication by 50% (ic50s) are between 0.2 and 1.5 mg/ml in human and canine cell lines (pepperrell et al., 2020) . furthermore, nitazoxanide has a potential capacity to enhance the production of interferon-a and interferon-b, which has been previously shown in vitro to exhibit activity against mers-cov, rotavirus, hbv, hcv, influenza virus and sars-cov-1 (calderon et al., 2020) . this drug has been shown to selectively inhibit the maturation of the hemagglutinin glycoprotein at the posttranslation stage (calderon et al., 2020) . nitazoxanide is currently under clinical trial to confirm its effectiveness with a dose of 500 mg alone or in combination with other drugs against covid-19 (nct04406246). nitazoxanide is known for its safety in humans. research showed the tolerability of single doses up to 4 g with minimal gastrointestinal side effects (rajoli et al., 2020) . doxycycline belongs to tetracycline antibiotic, that work against and inhibit various bacterial infections such as urinary tract infection, intestinal infection, gonorrhea, chlamydia and others (ali et al., 2017) . currently, doxycycline is also under use for the treatment against covid-19, because coronavirus is well-known to bind with metalloproteases (mmps) of the host cells, in particular to ensure viral survival (conforti et al., 2020) . furthermore, doxycycline is known to chelate zinc from mmps, and on the basis of chelating activity of this antibiotic might help in inhibiting sars-cov-2 (conforti et al., 2020; szolnoky 2020) . on the other hand, it is also known that these class of antibiotics have the ability to inhibit the replication of positive polarity single stranded rna viruses (szolnoky 2020) . it is also used for the treatment of inflammatory skin diseases for long time, due to modulatory activity of innate immune response generation by this antibiotic (malek et al., 2020) . due to modulating effect, it can decrease the expression of nf-jb and releases the inflammatory cytokines such as tumor necrosis factor-a, interleukin-1b and interleukin-6, which can inhibit granulomas inflammatory response and release free radicals (malek et al., 2020) . therefore, due to these properties, possibility to inhibit the viral replication of sars-cov-2 inside the human cells is high (malek et al., 2020) . currently, doxycycline has already reached phase iii clinical trials with 200 mg of daily dose given to patients that are infected with sars-cov-2 (nct04371952). the drug dexamethasone is used since long time for the treatment of arthritis and asthma. currently, it is used in treatment of covid-19 infected patients in united kingdom and under clinical trials. according to one research team, they got effectual and promising results (al saleh et al., 2020) . however, it is known that patients with advance stage of covid-19 infection have severe lung inflammation, but clinicians are checking for the dexamethasone effectiveness results in last stage of infection (theoharides & conti 2020) . 2104 covid-19 infected patients were randomly selected for clinical trial and all patients were administrated with 6 mg dexamethasone once a day for 10 days (theoharides & conti 2020) . in addition, it has reduced the death rate by one third in ventilated patients, according to press release from the recovery trial organizers (nct04381936). more than 80 countries have started the development strategies to make a vaccine against covid-19. currently, over 90 different vaccines are being developed by these countries using various approaches and novel technologies (mandal 2020; thanh le et al., 2020) . however, some groups are already in a stage of human trials, while others are still testing on animals (thanh le et al., 2020) . presently, formulation of vaccines against covid-19 are divided into eight categories i.e. virus-weakened form, virus-inactivated form, replicating viral vector vaccine, non-replicating viral vector vaccine, nucleic acid based vaccine (dna and rna), protein based vaccine (protein sub-unit vaccine and virus like particles vaccine) (thanh le et al., 2020) . currently, there are many drugs which are in use and trials for curing this pandemic disease covid-19. many studies have showed and determined that covid-19 infection suppresses the immune system yaqinuddin & kashir 2020) . therefore, we need to ponder over other options which might have therapeutic potential: (1) use of combinational therapy with effective immunomodulators with known mechanism of action, which can enhance the functioning and response of immune system. some important immunomodulators with their pharmacological effects have been listed in table 2; (2) use of some other anti-malarial drugs such as artemisinin derivative and amodiaquine, because artemisinin has also shown antiviral activity. mode of action of artemisinin is still not clear, but there is a possibility that it interferes with the viral replication, because of its usage for the treatment of severe malaria, helminths parasites and cancer. however, amodiaquine's mode of action is similar to hcq, (3) similarly, we must think of new drug approaches which can act on ace 2 receptor, because covid-19 virus uses its surface of spike protein to block onto ace 2 receptor on the surface of host receptor, (4) another possible suggestion is to use two or three different combination of drugs i.e. ivermectin, hcq and azithromycin antimalarial drug in combination with favipiravir or remdesivir antiviral drug with immunomodulators or bcg vaccine for the treatment of covid-19 infection. however, researchers have already started using combination therapy for treating the covid-19 patients, but new approaches and combination of drugs are still needed, which can work in multiple way to cure the patients infected with sars-cov-2. clinicians all over the world using combination drug therapy for the treatment of covid-19, which includes varieties of drugs in combination with different drugs and immunomodulators or natural remedies. some drugs work well, while others not. below are some drug combinations which are currently in use to treat covid-19 infected patients. hcq þ az based treatment showed an apparent accelerated virus load clearance in the patients. this combination is already in use with phase iii clinical trials underway (andreani et al., 2020b; gautret et al., 2020b) . administration of hcq þ az in the covid-19 patients are given for 5 days with loading dose of 400 mg (hcq) þ 500 mg (az) for first day and 200 mg (hcq) þ 250 mg (az) for the next four days (nct04347512). fruitful results have been observed. another combination for the treatment of covid-19 patients has reached phase ii clinical trials. in this combination (hcq þ nz), both drugs have different mode of action and both works in two different sites of virus. however, the administration of hcq þ nz drugs for covid-19 patients are given for 10 days, three times daily with loading dose of 200 mg (hcq) and 500 mg (nz) given orally twice daily for 6 days (nct04361318). still, we need to wait further for the evaluation and efficacy of clinical results of this combination. this combination of drugs (nz þ iv) have also been in use for the treatment of covid-19 infected patients (pepperrell et al., 2020; simsek yavuz & unal 2020) , and is in phase ii of clinical trial with 100 participants. both drugs have the potential to inhibit sars-cov-2 infection. in addition, mode of action of both drugs is different, they inhibit viral protein synthesis and viral replication respectively. however, the administration of these two drugs are orally with 200 mcg/kg ivermectin on empty stomach plus 500 mg nitazoxanide twice daily with food for 6 days (nct04360356). another important combination will soon be going for clinical trials. these two drugs (az and nz) have shown good efficacy against sars-cov-2 ( kelleni 2020) , while acting on different sites of virus life cycle as described in table 1 . administered dose is 600 mg of nz drug alone is currently in use for the treatment of covid-19 (kelleni 2020) . this combination needs to be considered and tested for positive and effective results. iv and dx showed good results in using alone administered treatment in covid-19 patients. due to the effective response, this combination is under phase ii clinical trial. here, the mode of action of these two drugs is different and doxycycline also increases the pro-inflammatory response after inhibiting nf-jb pathway (malek et al., 2020) . administration dose of these two drugs is 200 mcg/kg ivermectin as single dose with 200 mg doxycycline on day 1, followed by 100 mg doxycycline at every12 hour for 4 days (nct04407130). however, this is still in very initial phase of clinical trials, thus we need to wait more for the outcome of this combination clinically. the new way of combination drug therapy in use for the treatment of covid-19 such as antiviral and antimalarial drugs fv þ hcq and fv þ az combinations. the mode of action of fv is different when compared with hcq and az, which is a good sign where combination will prove to provide with good results (jean et al., 2020) . furthermore, these combinations has already reached the phase iii clinical trials. administration of these two drugs are 800 mg hcq on day 1, followed by 400 mg doses for 2-5 days with 3200 mg fv on day 1, followed by 1200 mg on 2-5 days. on the other hand, the current drug combination of fv and az dose used against covid-19 infected patients are 500 mg az on day 1, followed by 250 mg doses for 2-5 days with 3200 mg fv on day 1, followed by 1200 mg on 2-5 days (nct04411433). another approach to create new drug combination for the treatment of covid-19 infected patients is by using antiviral drug plus cytokine (il-6) blocking agent such as tz. tz is currently used to treat cytokines release syndrome (crs) and arthritis . during sars-cov-2 infection, the researcher observed that many cytokines increases and one of them is interleukin-6. the mechanism of action of tz is still not clear. however, this drug has been started using in clinical trials with combination of fv and rd and have reached phase iii of clinical trials. administration of these two drugs are 1600 mg of fv twice on day 1, followed by 600 mg doses twice for 2-7 days with 400 mg tz on day 1 (nct04310228). moreover, drug combination with tz þ rd has also reached the clinical trial level (nct04409262). recently, antiviral drugs such as uf, lp/rt, hcq (anti-malarial) and immunomodulator interferon-b 1a have been used as potential effective agents against covid-19. furthermore, these combinations have already reached phase iv clinical trials for the treatment of covid-19 patients (nct04350684). however, there is another drug combination ia þ lp/rt þ ribavirin, which was also used to treat covid-19 patients and shown good results of viral load elimination from nasopharyngeal swabs in phase ii clinical trials (jalkanen et al., 2020) . till date, there is no specific drug or vaccine has been developed against covid-19 disease. it is important to develop a specific and novel inhibitor for blocking the viral entry and echinacea species extract uses in respiratory tract infections and inflammatory conditions, including common cold, coughs, bronchitis, and inflammation of mouth and pharynx its replication on the host cells, which will essentially control this pandemic disease. as we know that, there are many clinical trials related to new drugs, drug repositioning and vaccine development studies against novel coronavirus are on track. however, novel strategies like combinational therapeutic approaches with different drugs and immunomodulators will possibly show a better path in controlling covid-19 infection. furthermore, at this stage, computational approaches could help and lead us in developing or designing new therapeutic drugs at rapid level. on the other hand, successful vaccine development can only be achieved by using variety of therapeutic approaches and correctly shared information. this is significant and a must requirement to develop an important vaccine against covid-19 and eradicate this pandemic from the world. the authors have declared no conflict of interest. arif jamal siddiqui http://orcid.org/0000-0002-6236-0920 mitesh patel http://orcid.org/0000-0002-9283-2124 mohd adnan http://orcid.org/0000-0002-7080-6822 moroccan medicinal plants as inhibitors against sars-cov-2 main protease: computational investigations evolution of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as coronavirus disease 2019 (covid-19) pandemic: a global health emergency. the science of the total environment repurposing of chloroquine and some clinically approved antiviral drugs as effective therapeutics to prevent cellular entry and replication of coronavirus coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease the desperate search for effectiveness multiple myeloma in the time of covid-19 doxycycline as potential anti-cancer agent. anti-cancer agents in medicinal chemistry in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov-2 shows synergistic effect in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov-2 shows synergistic effect covid-19 drug discovery using intensive approaches synthesis of primaquine glyco-conjugates as potential tissue schizontocidal antimalarial agents evaluation of cytotoxicity and antiviral activity of ivermectin against newcastle disease virus elevated interleukin-6 and severe covid-19: a meta-analysis development of remdesivir repositioning as a nucleotide analog against covid-19 rna dependent rna polymerase azithromycin. profiles of drug substances, excipients, and related methodology teicoplanin: an alternative drug for the treatment of covid-19? truncated human angiotensin converting enzyme 2; a potential inhibitor of sars-cov-2 spike glycoprotein and potent covid-19 therapeutic agent in-silico strategies for probing chloroquine based inhibitors against sars-cov-2 repetitive live sporozoites inoculation under arteether chemoprophylaxis confers protection against subsequent sporozoite challenge in rodent malaria model host immune response is severely compromised during lethal plasmodium vinckei infection systematic review of registered trials of hydroxychloroquine prophylaxis for covid-19 health-care workers at the first third of 2020. one health novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial experimental treatment with favipiravir for covid-19: an open-label control study treatment with hydroxychloroquine vs hydroxychloroquine þ nitazoxanide in covid-19 patients with risk factors for poor prognosis: a structured summary of a study protocol for a randomised controlled trial the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro remdesivir for severe acute respiratory syndrome coronavirus 2 causing covid-19: an evaluation of the evidence ivermectin and novel coronavirus disease (covid-19): keeping rigor in times of urgency mers-cov: understanding the latest human coronavirus threat. viruses, 10, e93 middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease efficacy of hydroxychloroquine in patients with covid-19: results of a randomized clinical trial potential use of hydroxychloroquine, ivermectin and azithromycin drugs in fighting covid-19: trends, scope and relevance doxycycline, a widely used antibiotic in dermatology with a possible anti-inflammatory action against il-6 in covid-19 outbreak mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells favipiravir, an antiviral for covid-19? sars-cov-2: recent reports on antiviral therapies based on lopinavir/ritonavir, darunavir/umifenovir, hydroxychloroquine, remdesivir, favipiravir and other drugs for the treatment of the new coronavirus screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection favipiravir as a potential countermeasure against neglected and emerging rna viruses repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection middle east respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies european centre for disease prevention and control sars-cov-2 rna dependent rna polymerase (rdrp) targeting: an in silico perspective treatment with interferon-a2b and ribavirin improves outcome in mers-cov-infected rhesus macaques chloroquine and hydroxychloroquine in covid-19 remdesivir in covid-19 favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial observational study of hydroxychloroquine in hospitalized patients with covid-19 corrigendum: influenza a viruses escape from mxa restriction at the expense of efficient nuclear vrnp import effect of biosolids characteristics on retention and release behavior of azithromycin and ciprofloxacin compassionate use of remdesivir for patients with severe covid-19 interferon-b and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cellbased assays a review on the cleavage priming of the spike protein on coronavirus by angiotensin-converting enzyme-2 and furin comparative effectiveness of tamoxifen, toremifene, letrozole, anastrozole, and exemestane on lipid profiles in breast cancer patients: a network meta-analysis remdesivir in the treatment of coronavirus disease 2019 (covid-19): a simplified summary a molecular modeling approach to identify effective antiviral phytochemicals against the main protease of sars-cov-2 interferon beta-1a for covid-19: critical importance of the administration route treatment options for covid-19: the reality and challenges design of multi-epitope vaccine candidate against sars-cov-2: a in-silico study nitazoxanide/azithromycin combination for covid-19: a suggested new protocol for early management targeting sars-cov-2: a systematic drug repurposing approach to identify promising inhibitors against 3c-like proteinase and 2 0 -o-ribose methyltransferase umifenovir treatment is not associated with improved outcomes in patients with coronavirus disease 2019: a retrospective study disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro fda-approved thiol-reacting drugs that potentially bind into the sars-cov-2 main protease, essential for viral replication potential therapeutic agents against covid-19: what we know so far bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19. medrxiv potential anti-viral activity of approved repurposed drug against main protease of sars-cov-2: an in silico based approach doxycycline as a potential partner of covid-19 therapies. idcases, 21, e00864 mobilizing the research ecosystem for scientific advances towards positive impact in the context of the covid-19 ivermectin is a potent inhibitor of flavivirus replication specifically targeting ns3 helicase activity: new prospects for an old drug computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 strategies to control human lymphatic filarial infection: tweaking host's immune system potential therapeutic targets for combating sars-cov-2: drug repurposing, clinical trials and recent advancements review of safety and minimum pricing of nitazoxanide for potential treatment of covid-19 clinical efficacy of umifenovir in influenza and arvi (study arbitr) sars-cov-2, sars-cov, and mers-cov: a comparative overview dose prediction for repurposing nitazoxanide in sars-cov-2 treatment or chemoprophylaxis impact of the first-line treatment shift from dihydroartemisinin/piperaquine to artesunate/mefloquine on plasmodium vivax drug susceptibility in cambodia associations between immune-suppressive and stimulating drugs and novel covid-19-a systematic review of current evidence broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses immune responses in liver and spleen against plasmodium yoelii pre-erythrocytic stages in swiss mice model assessment of real-time method to detect liver parasite burden under different experimental conditions in mice infected with plasmodium yoelii sporozoites neurological disorder and psychosocial aspects of cerebral malaria: what is new on its pathogenesis and complications? a minireview sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium antiviral treatment of covid-19 hydroxychloroquine and covid-19 cloning, expression and functional characterization of heme detoxification protein (hdp) from the rodent malaria parasite plasmodium vinckei further aspects of doxycycline therapy in covid-19. dermatology and therapy, e13810 mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir the covid-19 vaccine development landscape dexamethasone for covid-19? not so fast a retrospective cohort study of methylprednisolone therapy in severe patients with covid-19 pneumonia world health organization coronavirus worldometer an update on current therapeutic drugs treating covid-19 innate immunity in covid-19 patients mediated by nkg2a receptors, and potential treatment using monalizumab, cholroquine, and antiviral agents teicoplanin potently blocks the cell entry of 2019-ncov rational use of tocilizumab in the treatment of novel coronavirus pneumonia network-based drug repurposing for novel coronavirus 2019-ncov/ sars-cov-2 successful recovery of covid-19 pneumonia in a renal transplant recipient with long-term immunosuppression coronaviruses -drug discovery and therapeutic options key: cord-354738-4rxradwz authors: kohl, claudia; kurth, andreas title: european bats as carriers of viruses with zoonotic potential date: 2014-08-13 journal: viruses doi: 10.3390/v6083110 sha: doc_id: 354738 cord_uid: 4rxradwz bats are being increasingly recognized as reservoir hosts of highly pathogenic and zoonotic emerging viruses (marburg virus, nipah virus, hendra virus, rabies virus, and coronaviruses). while numerous studies have focused on the mentioned highly human-pathogenic bat viruses in tropical regions, little is known on similar human-pathogenic viruses that may be present in european bats. although novel viruses are being detected, their zoonotic potential remains unclear unless further studies are conducted. at present, it is assumed that the risk posed by bats to the general public is rather low. in this review, selected viruses detected and isolated in europe are discussed from our point of view in regard to their human-pathogenic potential. all european bat species and their roosts are legally protected and some european species are even endangered. nevertheless, the increasing public fear of bats and their viruses is an obstacle to their protection. educating the public regarding bat lyssaviruses might result in reduced threats to both the public and the bats. the european continent is inhabited by 52 hibernation. many bat species migrate over vast distances while others are rather territorial. all bats in europe utilize echolocation to navigate. contrary to the worldwide efforts in protecting bats, they have been increasingly gaining attention as potential reservoir hosts of some of the most virulent viruses we know. various publications reviewed bats globally as carriers and potential reservoir hosts of human-pathogenic and zoonotic viruses [3] [4] [5] [6] [7] [8] [9] [10] , while hardly anything is known about human-pathogenicity of european bat viruses apart from lyssaviruses. in this review, we discuss a selection of viruses as possible threats posed by european bats to the public from our point of view. a summary of viruses that have been detected in european bats is given in table 1 at the end of the manuscript. a more comprehensive and up-to-date list of bat-associated viruses can be found online at the database of bat-associated viruses (dbatvir) [11] . european bat lyssaviruses (family rhabdoviridae) are the most important zoonotic bat-borne viruses in europe and have been comprehensively reviewed by banyard et al. in this special issue on bat viruses (title: lyssavirus infections of bats: emergence and zoonotic threat) [12] . therefore, we will provide a short overview. [13, 14] nyctalus noctula rhinolophus ferrumequinum hungary pcr [15] myotis myotis germany pcr [16] astroviridae myotis myotis germany pcr [16] mamastrovirus italy pcr [22, 28] the postulates drafted by jacob henle and robert koch in the late 19th century constitute a framework regarding the principles of cause-and-effect in microbiology [55] . back then, it was comparatively straightforward to limit cause-and-effect to four postulates, although viruses had not yet been discovered nor was molecular biology developed ( table 1 ). all of the postulates are hard to fulfill for viruses, as they do not grow on nutrient media, but require living cells for replication. when looking for viruses on a molecular level, it is necessary to consider that only the first postulate can be accomplished. studies identifying a host-pathogen relationship solely at the molecular level do not take into consideration that detection does not equal etiology. even though polymerase chain reaction (pcr) screening and metagenomic studies are indispensable and valuable tools, virologists should stay close to the henle-koch postulates when assuming a possible virulence of viruses detected in bat hosts. a plethora of coronaviruses has been detected in bats, mostly belonging to the alphaand betacoronaviruses [11, 56] . the genus alphacoronavirus hosts human-pathogenic strains (i.e., human cov 229e and nl63); however, in this review, we will focus on selected highly human-pathogenic betacoronaviruses and their european bat virus relatives [56] . from november 2002 until july 2003 the world was confronted with the first pandemic of the new millennium, caused by a novel coronavirus (cov) inducing the severe acute respiratory syndrome in humans (sars) [57] [58] [59] . the pandemic spread from its origin, a wet-market in the guangdong province in china, through 33 countries on five continents resulted in more than 8000 infected humans of whom more than 700 eventually died [60, 61] . the search for the animal reservoir began, identifying masked palm civets and bats as possible sources. subsequently, a plethora of diverse coronaviruses of distinct groups have been detected in various bat species around the world via molecular-biological techniques. in 2012, another human-pathogenic coronavirus, called middle east respiratory syndrome coronavirus (mers-cov), began spreading from the arabian peninsula, so far resulting in globally 707 laboratory-confirmed cases of infection with mers-cov, including at least 252 deaths [62] . dromedaries and bats are suspected as reservoirs for mers-cov [63] . recent findings support the plausibility of dromedaries as reservoir species [64] . although numerous studies in european bats report the presence of sars-like-cov and mers-like-cov sequences [21, [24] [25] [26] 65] , no final conclusion can be drawn regarding their zoonotic potential. a related virus detected in bats cannot necessarily be considered as zoonotic. a few alterations in the sars-cov spike protein enabled its binding to the host receptor ace-2, thus sars-cov became capable of infecting humans [66] . so far, the sars-like cov detected in european bats lack these alterations and thus are not predicted to be capable of infecting humans. although virus strains might be similar or related on a nucleic acid level, the distinct function of proteins is crucial when determining the host range. therefore, mere similarity is not sufficient to examine the potential of viruses to infect humans or even predict their virulence. it took ten years from the emergence of sars-cov for the first bat cov to be isolated from rhinolophus bats in china, that displayed the human ace-2 receptor, which enabled the virus to infect human cells [67] . these findings provide evidence for the reservoir theory. from the european perspective, nevertheless, no sars-like cov or mers-like cov has been isolated from any european bat, nor has any transmission of sars-like cov or mers-like cov to humans been reported. the case of mers-cov is slightly different, as a sequence of 190 base pairs with 100% identity to mers-cov was detected in a bat (taphozous perforates-the species identification performed was not beyond doubt, as it was based on exclusion criteria (no cytochrome b sequence of taphozous perforates is available in genbank [68] )) in saudi arabia [8] . this finding initiated a controversy among leading cov experts, as the journal nature recently reported [69] . they discussed that the complete genome sequence of mers-cov obtained from the bat should confirm that the virus was indeed identical and not coincidentally just a short conserved region of the virus genome. furthermore, a prevalence study might provide insights into the distribution of mers-cov in bat populations. although taphozous perforates are not abundant in europe, climate change and environmental factors may have an effect on the future distribution of this bat species (figure 1 ) [70] . the case of mers-cov emergence impressively demonstrates the necessity of virus discovery and prevalence studies. with the first sequence of mers-cov that became available, bats were suspected as reservoir hosts, not only because mers-cov is a sars-cov relative, but also because previous bat virus discovery studies had provided eligible sequences of bat cov to genbank, allowing for correlations with the novel mers-cov. recently, a quasi-species of mers covs was recovered from nasal swabs of dromedaries of the kingdom of saudi arabia [64] . the mers cov consensus genome variants from dromedaries and humans are indistinguishable, supporting the plausibility of dromedaries in the role of transmission [64] . in 2002, the first reported outbreak of filovirus, named lloviu virus (llov), in a european bat population occurred in france, spain, and portugal [29] . several colonies of schreiber's bats (miniopterus schreibersii) suddenly declined due to an unknown disease. llov was found in animals that showed signs of viral infection, but not in healthy bats co-roosting in the caves (myotis myotis). llov is distinctly related to filoviruses found in african bats and was classified in 2013 as type species of the novel genus cuevavirus [56] . unfortunately, the lack of successful isolation of llov prohibits the experimental infection of schreiber's bats to clarify whether llov is the first filovirus capable of inducing disease in bats. this would challenge the hypothesis of bats as potential reservoir hosts for other filoviruses like ebola and marburg virus. schreiber's bats are distributed in distinct lineages throughout oceania, africa, southern europe, and south-east asia (figure 2 ) [72] . they are thought to transmit and maintain llov across different lineages throughout their habitats, although no studies are available to prove this hypothesis. consequently, the sole demonstration of a novel filovirus sequence does not provide evidence of a possible public health threat. following the henle-koch postulates, the virus should be isolated and further characterized to draw conclusions on the evolution of filoviruses in their respective bat host. as most filoviruses are described as highly pathogenic for humans, the occurrence of llov should be carefully monitored by prevalence studies in the highly abundant miniopterus schreibersii (figure 2 ). in 2012, three distinct paramyxoviruses were detected in german bats, two of which were related to the proposed genus jeilongvirus (myotis mystacinus, pipistrellus pipistrellus) and one was related to the genus rubulavirus (nyctalus noctula) [34] . another study published in the same year described another 12 different paramyxoviruses in bats from germany (myotis bechsteinii, m. daubentonii, m. myotis, and m. mystacinus) and bulgaria (myotis alcathoe and m. capaccinii), all of which belong to the genus morbillivirus [35] . none of the novel bat paramyxoviruses are closely related to viruses of the highly pathogenic genus henipavirus or other human-pathogenic paramyxoviruses [34, 35] . there is no evidence to suggest that any of these novel paramyxoviruses are capable of infecting humans. similar to the case of the llov filovirus, virus isolates and prevalence studies in both humans and bats could improve knowledge and clarify their zoonotic potential. few studies have documented the negative results from pcr testing of european bats for other human-pathogenic viruses. for instance following generic pcr screening for flavi-, hanta-and influenza-a viruses in 210 european bats in 2011 [73] , testing of another 1369 central european bats for influenza-a viruses [74] and testing 42 european bats for hepadnaviruses in 2013 did not lead to the detection of any viral nucleic acids [75] . pcr screening of 468 european bats for orthopoxviruses has not revealed any known or novel virus sequences [76] . so far, the only virus isolates (beside lyssaviruses) obtained from european bats are one bunyavirus, one adenovirus and 22 orthoreoviruses [13, 19, 36, 37] . these represent the only isolates that would allow for further characterization and potential clarification of their zoonotic potential. nevertheless, recombinant viruses, constructed on sequence information, are also valuable tools to study prevalence and pathogenicity in vitro. toscana virus (tosv) was isolated from a bat's brain in 1988, while simultaneously tosv was isolated from sandflies in the laboratory [19] . as tosv has never been reported in bats afterwards and no hemagglutination-inhibiting antibodies has been initially found in the bat, there is a reasonable chance that this tosv isolation may have been a cross-contamination [77] . bat adenovirus 2 (bat adv-2) was isolated from a bat's intestine in 2009 [13] , and the whole genome was obtained and circumstantially analyzed [14, 78] . bat adv-2 displays a monophyletic relationship to the adenoviruses of canids (cadv). moreover, open trading frames (orf) in the bat adv-2 genome and the cadv are identical and not present in other members of the mastadenoviruses. the closely related canine adv contribute to the severe kennel cough syndrome in canids and show an unusually broad host range [79] . this provides evidence suggesting an ancestral inter-species transmission of mastadenoviruses between bats and canids. like in the case of rabies virus, which is prevalent in both bats and terrestrial mammals (e.g., dogs, raccoons, skunks, and foxes) of the americas, a continuing exchange and transmission between bats and canids or other terrestrial animals might be possible [80] . there is no evidence of a zoonotic potential of bat adv-2. in 2012, three novel orthoreoviruses were isolated from plecotus auritus and myotis mystacinus in germany [36] . a subsequent pcr screening obtained identical viral sequences also in other bat species: pipistrellus pipistrellus, pipistrellus nathusii, pipistrellus kuhlii, and nyctalus noctula. at the same time, a group in italy detected further 19 orthoreoviruses in myotis kuhlii, rhinolophus hyposideros, tadarida teniotis, and vespertilio murinus [37] . summing up the data for the reovirus isolates from germany and italy, a close relationship was revealed to the genus mammalian orthoreovirus (mrv), in particular to an orthoreovirus obtained from a dog (strain t3/d04) with hemorrhagic enteritis in italy [36, 37, 81] . no ancestral relationship was assumed here, but rather an opportunistic -behavior‖ of the novel closely related mrvs, as they were detected in various different bat species. moreover, the newly isolated mrvs are phylogenetically related to viruses capable of inducing severe meningitis in humans [82] . recently, a study published by steyer et al. described the detection of an mrv from a child hospitalized with acute gastroenteritis in slovenia [83] . the causative agent was determined to be an mrv with the highest similarity of 98.4%-99.0% in the respective segments to a bat mrv (t3/bat/germany/342/08) [83] . this might indicate a human-pathogenic potential of strain t3/bat/germany/342/08. as the case of sars-cov has shown that even small changes in the genome are important for determining the host range, this has to be determined for the bat mrvs in further studies. interestingly, no contact was reported between the infected child and bats, but contact to a domestic dog was assumed [83] . the isolated viruses will allow for a seroprevalence study (cross-reactivity and cross-neutralization with other strains) in humans, which shall be initiated to examine the prevalence of specific antibodies to bat mrvs in germany and italy (where these viruses have been found) to clarify their zoonotic potential. this is especially interesting as asian bat orthoreoviruses of the genus pteropine orthoreovirus have already been linked to potentially zoonotic respiratory diseases in humans [84, 85] . rhabdoviruses of the genus lyssavirus that have been detected in europe are considerably harmful and truly zoonotic agents, inevitably causing the death of unvaccinated humans if not treated in time before onset of the rabies disease [86] . even though bat-transmitted lyssaviruses have a fatality rate of virtually 100% and are suspected to be transmissible by bat biting and scratching, the reported total number of human fatalities in europe is low (n = 2-5 since 1963) [86] [87] [88] . all described hosts of european bat lyssaviruses (eblv-1 and eblv-2) are synanthropic, hence sharing their habitats with humans [87] . eblv-1 has been predominantly detected in eptesicus serotinus and e. isabellinus in europe, both living in buildings, roofs, and attics usually in the southern regions of europe (e. serotinus until 55° north, e. isabellinus in southern portugal-e. isabellinus is a north african population of e. serotinus that is controversially but not concludingly discussed as a novel species [1]), and male bats are reported to co-roost with multiple bat species [90] . eblv-1 was also detected in v. murinus, m. schreibersii, m. myotis, m. nattereri, r. ferrumequinum, and t. teniotis. whether these bat species constitute accidental hosts infected by spillover from co-roosting e. serotinus species, or whether they are additional reservoirs, has not yet been determined [38] [39] [40] [41] 91] . two human cases described by johnson et al. were confirmed as infected with eblv-2, which is prevalent in european m. daubentonii and m. dasycneme [40, 86] . m. daubentonii is prevalent in north-eastern europe and is frequently found co-roosting with p. pipistrellus and m. nattereri, whereas m. dasycneme is found throughout europe and in the mediterranean, co-roosting with m. capaccinii. so far, none of the co-roosting bats have been reported to carry eblv-2 [90] . however, spillover transmission to other animals (stone-marten, sheep, and cat) was described for eblv-1 [92] [93] [94] . overall, lyssaviruses prevalent in european bats pose a risk to public health, and preventive measures have already been implemented by many european countries for decades (e.g., surveillance, vaccination plans, and post exposure prophylaxis) [87] . especially the high-risk occupational groups (i.e., bat workers, bat carers in bat bat hospitals) are at increased risk. however, lyssavirus prevalence in european bats is very difficult to determine and results are very heterogenic [40] . the lyssavirus prevalences are considerably low, but changes of behavior as a result of a lyssavirus infection may be more likely to bring bats into contact with humans. however, it is necessary to balance the risk with the total number of fatal human cases during the last 35 years (five cases in 590 million people living in greater europe) [87] . accordingly, the risk is relatively low and would probably fall to zero if people were educated appropriately. direct contact (bites and scratches) with certain bat species might be risky and require post exposure prophylaxis. only few of the european bat species are known to be reservoirs of eblv-1 and eblv-2, but all of the european species are endangered or close to extinction. relocation or culling of bat colonies, in spite of being an obvious solution from the viewpoint of the general public, increases the risk of lyssavirus exposure and transmission and should not be considered [95] . only education can channel public fear to avoid further threats to the bats and the general public. alexander von humboldt discovered the latitudinal gradient in species diversity as early as 1799 [96] : the richness of species is subject to a global diversity gradient, abating from the species-rich tropics toward the higher latitudes [97] . bats influence this gradient significantly. more than 1100 bat species have been described worldwide. although they are abundant worldwide except for the polar regions, a steep diversity gradient is present from the tropics towards the poles [97] [98] [99] [100] . are fewer viruses prevalent in european bats because of the lower abundance of species in the more temperate europe? and is the zoonotic risk posed by bats decreased accordingly? only few studies on the biogeography of microorganisms are available. these studies indicate that the latitudinal diversity gradient has either no or a top-down effect on microbial diversity [101] [102] [103] [104] [105] . two studies hypothesized that the local diversity and dispersal of viruses is very high, though overall, the viral diversity is limited on the global scale [106, 107] . therefore, no assumptions can be made regarding the viral diversity in species abundant in temperate climates. as the total number of abundant species might not be essential, the change in biodiversity might play a role. the effect of decline in biodiversity on the emergence of diseases is subject of numerous publications [108] [109] [110] [111] [112] [113] [114] . basically, there are arguments in favor of two controversial theories; reduced biodiversity could either increase (dilution effect) or decrease the risk of disease transmission. for almost half of the zoonotic diseases that have newly emerged by spillover since 1940, a preceding change in land-use, agriculture, and wildlife hunting was reported [108] . all of the above-mentioned effects contribute to changes in biodiversity and increased contact situations between human and animal hosts, also in europe. once spillover in novel hosts has occurred, a high density of the novel host population eventually facilitates the establishment in the novel niche. thus, human overpopulation and a decreased biodiversity might be mutual factors promoting the establishment of emerging infectious diseases. in conclusion, the baas becking hypothesis from 1932 might still be appropriate: -everything is everywhere, but the environment selects‖ [115] . until now, lyssaviruses have been the only proven zoonotic viruses in european bats and may cause rabies in humans. however, only few bat species are known to transmit lyssaviruses in europe, and the number of human cases is rather low. nevertheless, education of the general public should be intensified to avoid easily preventable infections. although viruses with zoonotic potential have been detected in european bats, no clear assumption can be made without further studies. sero-prevalence studies should be conducted on the orthoreoviruses isolated from european bats, especially as a closely related virus was detected in a diseased child in slovenia [83] . other bat viruses detected by using molecular techniques should be isolated (e.g., mers-like cov or bat bunyavirus) to allow for characterization and follow-up sero-prevalence studies. in general, bats are special reservoir hosts because of their biological features, long-time co-evolution and high diversity of viruses that can be found. furthermore, there is neither a clearly decreased risk in the emergence of zoonotic viruses in temperate climates compared to the tropics nor a decreased risk in regions of lower biodiversity. in conclusion, many drivers of emergence in the tropics also have validity in europe. however, european bats are endangered species, and some of them are threatened by extinction. although lyssaviruses are prevalent in european bats, and some viruses might have a zoonotic potential, the overall hazard for humans is comparably low. moreover, the protection of bats (and any wildlife) will consecutively also protect the general public. international union for conservation of nature and natural resources (iucn) convention on the conservation of migratory species of wild animals bats: important reservoir hosts of emerging viruses bats prove to be rich reservoirs for emerging viruses bats, emerging infectious diseases, and the rabies paradigm revisited bats and their virome: an important source of emerging viruses capable of infecting humans bat zoonoses: the realities. in food security in a global economy veterinary medicine and public health bats and viruses: a brief review mass extinctions, biodiversity and mitochondrial function: are bats -special‖ as reservoirs for emerging viruses? bats as a continuing source of emerging infections in humans dbatvir: the database of bat-associated viruses lyssaviruses and bats: emergence and zoonotic threat new adenovirus in bats genome analysis of bat adenovirus 2: indications of interspecies transmission novel adenoviruses and herpesviruses detected in bats amplification of emerging viruses in a bat colony novel european lineages of bat astroviruses identified in hungary a preliminary study of viral metagenomics of french bat species in contact with humans: identification of new mammalian viruses ecology of viruses isolated from sand flies in italy and characterized of a new phlebovirus (arabia virus) detection and prevalence patterns of group i coronaviruses in bats ibá ñez, c.; garin, i.; et al. detection of alpha and betacoronaviruses in multiple iberian bat species detection of coronaviruses in bats of various species in italy alphacoronavirus detected in bats in the united kingdom. vector borne zoonotic dis genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences identification of sars-like coronaviruses in horseshoe bats (rhinolophus hipposideros) in slovenia human betacoronavirus 2c emc/2012-related viruses in bats circulation of group 2 coronaviruses in a bat species common to urban areas in western europe. vector borne zoonotic dis alpha and lineage c betacov infections in italian bats discovery of an ebolavirus-like filovirus in europe bats worldwide carry hepatitis e-related viruses that form a putative novel genus within the family hepeviridae discovery of herpesviruses in bats detection of a novel bat gammaherpesvirus in hungary novel papillomaviruses in free-ranging iberian bats: no virus-host co-evolution, no strict host specificity, and hints for recombination novel paramyxoviruses in free-ranging european bats bats host major mammalian paramyxoviruses isolation and characterization of three mammalian orthoreoviruses from european bats identification of mammalian orthoreovirus type 3 in italian bats antigenic and molecular characterization of bat rabies virus in europe european bat lyssaviruses: an emerging zoonosis bat rabies surveillance in europe european bat lyssaviruses genomic diversity and evolution of the lyssaviruses application of broad-spectrum resequencing microarray for genotyping rhabdoviruses evidence of two lyssavirus phylogroups with distinct pathogenicity and immunogenicity epidemiology of bat rabies in germany genetic analysis of european bat lyssavirus type 1 isolates from france evolution of european bat lyssaviruses novel lyssavirus in natterer's bat first encounter of european bat lyssavirus type 2 (eblv-2) in a bat in finland phylogeny of european bat lyssavirus 1 in eptesicus isabellinus bats isolation of a european bat lyssavirus type 2 from a daubenton's bat in the united kingdom isolation of bokeloh bat lyssavirus in myotis nattereri in france molecular diagnostics for the detection of bokeloh bat lyssavirus in a bat from bavaria detection of rhabdovirus viral rna in oropharyngeal swabs and ectoparasites of spanish bats die aetiologie der tuberkulose international committee on taxonomy of viruses (ictv) online 2013 identification of a novel coronavirus in patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome severe acute respiratory syndrome state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia close relative of human middle east respiratory syndrome coronavirus in bat animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor middle east respiratory syndrome coronavirus in bats, saudi arabia deadly coronavirus found in bats predicting species distribution and abundance responses to climate change: why it is essential to include biotic interactions across trophic levels molecular systematics and biogeography of the bent-wing bat complex miniopterus schreibersii (kuhl, 1817) (chiroptera: vespertilionidae) diseases and causes of death in european bats: dynamics in disease susceptibility and infection rates no virological evidence for an influenza a-like virus in european bats bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human hepatocytes genomic characterization of m and s rna segments of hantaviruses isolated from bats create two species, bat adenovirus b and murine adenovirus b, in the genus mastadenovirus, family adenoviridae ictv: 2011, ictv 2011.024av. available online canine respiratory viruses the evolutionary history and dynamics of bat rabies virus virological and molecular characterization of a mammalian orthoreovirus type 3 strain isolated from a dog in italy isolation and molecular characterization of a novel type 3 reovirus from a child with meningitis high similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in europe a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans identification and characterization of a new orthoreovirus from patients with acute respiratory infections human rabies due to lyssavirus infection of bat origin bat rabies, public health and european bat conservation human rabies of bat origin in europe case report: isolation of a european bat lyssavirus type 2a from a fatal human case of rabies encephalitis handbuch der fledermäuse europas und nordwestafrikas european bat lyssavirus infection in spanish bat populations spill-over of european bat lyssavirus type 1 into a stone marten (martes foina) in germany natural and experimental infection of sheep with european bat lyssavirus type-1 of danish bat origin ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control ansichten der natur, mit wissenschaftlichen erläuterungen phylogeny, niche conservatism and the latitudinal diversity gradient in mammals taxonomy of chiroptera a molecular phylogeny for bats illuminates biogeography and the fossil record bat species density gradients in the new world: a statistical assessment reverse latitudinal trends in species richness of pitcher-plant food webs the diversity and biogeography of soil bacterial communities body size determines the strength of the latitudinal diversity gradient microbial biogeography: putting microorganisms on the map biogeography of diseases: a framework for analysis here a virus, there a virus, everywhere the same virus? microbial biogeography? impacts of biodiversity on the emergence and transmission of infectious diseases biodiversity loss and emerging infectious disease: an example from the rodent-borne hemorrhagic fevers biodiversity loss and the rise of zoonotic pathogens pangloss revisited: a critique of the dilution effect and the biodiversity-buffers-disease paradigm a meta-analysis suggesting that the relationship between biodiversity and risk of zoonotic pathogen transmission is idiosyncratic host range and emerging and reemerging pathogens population biology of multihost pathogens geobiologie of inleiding tot de milieukunde the authors are grateful to ursula erikli for copy-editing and the two anonymous reviewers for their valuable comments. both authors reviewed the literature and wrote the manuscript. the authors declare no conflict of interest. key: cord-349287-mwj2qby4 authors: mackay, ian m.; arden, katherine e. title: mers coronavirus: diagnostics, epidemiology and transmission date: 2015-12-22 journal: virol j doi: 10.1186/s12985-015-0439-5 sha: doc_id: 349287 cord_uid: mwj2qby4 the first known cases of middle east respiratory syndrome (mers), associated with infection by a novel coronavirus (cov), occurred in 2012 in jordan but were reported retrospectively. the case first to be publicly reported was from jeddah, in the kingdom of saudi arabia (ksa). since then, mers-cov sequences have been found in a bat and in many dromedary camels (dc). mers-cov is enzootic in dc across the arabian peninsula and in parts of africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. the ksa is the focal point of mers, with the majority of human cases. in humans, mers is mostly known as a lower respiratory tract (lrt) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. however, mers-cov has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. older males most obviously suffer severe disease and mers patients often have comorbidities. compared to severe acute respiratory syndrome (sars), another sometimesfatal zoonotic coronavirus disease that has since disappeared, mers progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. most human cases of mers have been linked to lapses in infection prevention and control (ipc) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (hcws) and higher exposures in those with occupations that bring them into close contact with camels. sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics have been available almost from the start of the emergence of mers. while the basic virology of mers-cov has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. electronic supplementary material: the online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. an email from dr ali mohamed zaki, an egyptian virologist working at the dr soliman fakeeh hospital in jeddah in the kingdom of saudi arabia (ksa) announced the first culture of a new coronavirus to the world. the email was published on the website of the professional emerging diseases (promed) network on 20 th september 2012 [1] (fig. 1) and described the first reported case, a 60 year old man from bisha in the ksa. this information led to the rapid discovery of a second case of the virus, this time in an ill patient in the united kingdom, who had been transferred from qatar for care [2] . the new virus was initially called novel coronavirus (ncov) and subsequentlty entitled the middle east respiratoy syndrome coronavirus (mers-cov). as of 2 nd of september 2015, there have been 1,493 detections of viral rna or virus-specific antibodies across 26 countries (additional file 1: figure s1 ) confirmed by the world health organization (who), with over a third of the positive people dying (at least 527, 35 %) [3] . since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (dc; camelus dromedarius) in the ksa [4] , also dcs across the arabian peninsula and parts of africa that are a source of dc imports for the ksa [5] . to date, mers-cov has not been detected in dcs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . occasionally, virus is transmitted from infected dcs to exposed humans. subsequent transmission to other humans requires relatively close and prolonged exposure [10] . the first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . however, sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (dpp4; also called cd26); that mers-cov has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than sars-cov; is widespread in dcs; has the potential to infect other animals and that mers kills its human host more often than sars did (20-40 % versus 9 % for sars [14] ) [15] [16] [17] [18] [19] . in humans, overt disease was given the name middle east respiratory syndrome, with the acronym mers. from intermittent animal-to-human spill-over events, the mers-cov spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. the spread of mers-cov among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (hcws). although dcs appear to suffer the equivalent of a 'common cold' from mers-cov infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. it has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . studies have established that the mean incubation period for mers is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . the first who case definition [27] defined probable cases of mers based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (lrt) involvement. it also included roles for contact with a probable or confirmed case or for travel or residence within the arabian peninsula. if strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . from july 2013, the revised who case definition included the importance of seeking out and understanding the role of asymptomatic cases and from june 2014, the who definition more clearly stated that a confirmed case included any person whose sample was rt-pcr positive for mers-cov, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] apart from the who and the ksa ministry of health reports, asymptomatic or subclinical cases of mers-cov infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . the ksa definition of a case became more strict on 13 th may 2014, relying on the presence of both clinical features and laboratory confirmation [33] . testing of asymptomatic people was recommended against from december 2014 [34] , reinforced by a case definition released by the ksa ministry of health in june 2015 [35] . the ksa has been the source of 79 % of human cases. severe mers is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . interestingly in june 2015, an outbreak in south korea followed a similar distribution [39, 40] . among laboratory confirmed cases, fever, cough and upper respiratory tract (urt) signs and symptoms usually occur first, followed within a week by progressive lrt distress and lymphopaenia [37] . patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . mers has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the ksa, yet only 19 % of cases in south korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . general supportive care is key to managing severe cases [43] . children under the age of 14 years are rarely reported to be positive for mers-cov, comprising only 1.1 % (n = 16) of total reported cases. between 1 st september 2012 and 2 nd december 2013, a study described the then tally of paediatric cases in the ksa, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . in amman, jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for mers-cov rna, despite being collected at a similar time to the first known outbreak of mers-cov in the neighbouring town of al-zarqa [45] . a second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not rt-rtpcr positive, the mother did subsequently develop antibodies to mers-cov, suggestive of recent infection [46] . her exposure history to a mers-cov rt-rtpcr positive relative and an antibody-reactive husband, her incubation period and her symptom history met the who criteria for being a probable mers-cov case [46] . diagnostic methods were published within days of the promed email announcing the first mers case [47] , including several now gold standard in-house rt-rtpcr assays (fig. 2 ) as well as virus culture in vero and llc-mk2 cells [18, 47, 48] . a colorectal adenocarcinoma (caco-2) epithelial cell line has since been recommended for isolation of infections mers-cov [49] . we previously [18] .). open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (utr; grey rectangles). fs-frame-shift. predicted regions encompassing recombination break-points are indicated by orange pills. created using geneious v8.1 [211] and annotated using adobe illustrator. beneath this is a schematic depicting the location of rt-pcr primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest rt-rtpcr screening assays and conventional, semi-nested (three primers) rt-pcr confirmatory sequencing assays [47, 48] . publication order is noted by first [27 th september 2012; red] and second [6 th december 2012; orange] coloured rectangles; both from corman et al. [47, 48] those assays recommended by the who are highlighted underneath by yellow dots [53] . the nseq reverse primer has consistently contained one sequence mismatch with some mers-cov variants. an altered version of that from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res 2015 vol 202:60-88 with permission from elsevier [5] reviewed the broad tropism of mers-cov [5] . however, as is well described, cell culture is a slow, specialised and insensitive method [50] while pcr-based techniques are the preferred method for mers-cov detection. the first open reading frames (orf 1a and 1b; fig. 2 ) have become a key diagnostic and taxonomic target for cov species identification. with less than 80 % identity between the amino acid sequence of mers orf 1ab and betacoronavirus relatives, tylonycteris bat hku4 and pipistrellus bat hku5, it can be concluded that it is a novel and distinct virus. mers-cov is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . the structural proteins include the spike (s), envelope (e), membrane (m) and nucleocapsid (n) [52] . the products of orf1a and orf1b are predicted to encode nonstructural proteins. the majority of specimen testing to date has employed validated rt-rtpcr assays shown to be sensitive and specific [47, 48, 53] . the realstar® kit uses these whorecommended assays [54] . the target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (imm observation). other rt-rtpcr assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. the detection of mers-cov antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. detection of viral proteins rather than viral rna indicates the likely presence of infectious virus. the first rapid immunochromatographic tool described could detect recombinant mers-cov nucleocapsid protein from dc nasal swabs with 94 % sensitivity and 100 % specificity compared to rt-rtpcr [61] . a different approach used a monoclonal antibody-based capture elisa targeting the mers-cov nucleocapsid protein with a sensitivity of 10 3 tcid 50 and 100 % specificity [62] . demonstration of a seroconversion to a mers-cov infection meets the current who definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior mers-cov infection and help support transmission studies. because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral rna [63] . strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. much of the arabian peninsula and all of the horn of africa lack baseline data describing the proportion of the community who may have been infected by a mers-cov. however, sero-surveys have had widespread use in elucidating the role of dcs as a transmission source for mers-cov. because of the identity shared between dc and human mers-cov (see molecular epidemiology: using genomes to understand outbreaks), serological assays for dc sero-surveys should be transferrable to human screening with minimal re-configuration. also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested mers-cov isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single mers-cov serotype [49] . the development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to mers-cov, as well as to likely sources of cross-reaction [64] . obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . a number of commercial elisa kits, immunofluorescent assays (ifa) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . initially, conventional ifas were used for human sero-surveys. these relied on mers-cov-infected cell culture as an antigen source, detecting the presence of human anti-mers-cov igg, igm or neutralizing antibodies in human samples [18, 48, 69] . no sign of mers-cov antibodies was found among 2,400 sera from patients visiting hospital in jeddah, from 2010 through 2012, prior to the description of mers-cov [18] . nor did ifa methods detect any sign of prior mers-cov infection among a small sample of 130 healthy blood donors from another hospital in jeddah (collected between jan and dec 2012) [70] . of 226 slaughterhouse workers, only eight (3.5 %) were positive by ifa, and those sera could not be confirmed by virus neutralization (nt) test. the study indicated that hcov-hku1 was a likely source of crossreactive antigen in the whole virus ifa [70] . whole virus mers-cov ifa also suffered from some cross-reactivity with convalescent sars patient sera and this could not be resolved by an nt test which was also cross-reactive [71] . ifa using recombinant proteins instead of whole-virus ifa, has been shown to be a more specific tool [31] . since asymptomatic zoonoses have been posited [72] , an absence of antibodies to mers-cov among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering dcs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. ifa using recombinant proteins instead. some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the mers-cov nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing mers-cov spike protein and luciferase [74, 75] . a pseudo particle neutralization (ppnt) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (mnt) test. [10, 74, [76] [77] [78] ] studies using small sample numbers and ppnt found no evidence of mers-cov neutralizing antibody in sera from 158 children with lrt infections between may 2010 and may 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the jazan region of the ksa during 2012 [79, 80] . similarly, a study of four herdsmen in contact with an infected dc herd in al-ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in al-ahsa and 146 controls who were not exposed to dcs in any professional role, found none with serological evidence of past mers-cov infection using the ppnt assay [10] . a delay in the neutralizing antibody response to mers-cov infection was associated with increased disease severity in south korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . the implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. a jordanian outbreak of acute lrt disease in a hospital in 2012 was retrospectively found to be associated with mers-cov infection, initially using rt-rtpcr, but subsequently, and on a larger scale, through positivity by elisa and ifa or mnt test. [46, 82, 83] this outbreak predated the first case of mers in the ksa. the elisa used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-cov hku5 to identify antibodies against the equivalent crossreactive mers-cov protein [71] . it was validated using 545 sera collected from people with prior hcov-oc43, hcov-229e, sars-cov, hcov-nl63, hrv, hmpv or influenza a(h1n1) infections but was reportedly less specific than the recombinant ifa discussed above. it was still considered an applicable tool for screening large sample numbers [82] . a protein microarray expressing the s1 protein subunit has also been validated and widely used for dc testing [5, 84] . detection of mers-cov infection using elisa or s1 subunit protein microarray [84] is usually followed by confirmatory ifa and/ or a plaque-reduction neutralization (prnt) [69, 70, 85] or mnt test. [74, 85, 86] this confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in dcs (bovine cov, bcov) or humans (hcov-oc43, hcov-229e, hcov-nl63, hcov-hku1, sars-cov). in the largest study of human sera, a tiered diagnostic process assigned both recombinant ifa and recombinant elisa positive sera to 'stage 1' seropositivity. a stage 2 seropositive result additionally required a suitably titred prnt result [87] . the study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 ksa provinces contained mers-cov antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . contemporary surveys are needed. mers-cov does not appear to be easily transmitted from dcs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. this was reinforced when a false positive us case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, nt assay and was subsequently retracted [88, 89] . the who recommends sampling from the lrt for mers-cov rt-rtpcr testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] lrt samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . recommended sample types include bronchoalveolar lavage (bal), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°c [90] . if available, lung biopsy or autopsy tissues can also be tested [53] . the urt is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when urt sampling is to be conducted [90] . paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting rt-rtpcr [53, 90] . human urine and stool have been found to contain mers-cov rna 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . in two cases that arrived in the netherlands, urine was rt-rtpcr negative but faeces was weakly positive and sera were rt-rtpcr positive for five days or more [25] . the finding of mers-cov viral rna in serum provides an avenue for retrospective pcr-based studies if respiratory samples are unavailable [83] . rnaaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . clinically suspected mers cases may return negative results by rt-rtpcr. data have shown one or more negative urt samples may be contradicted by further urt sampling or the use of lrt samples, which is preferred [2, 43, 93] . higher viral loads occur in the lrt compared to the urt. [22, 69, 88, 94] this fits with the observation that the majority of disease symptoms are reported to manifest as systemic and lrt disease [21] . however, on occasion, even lrt specimens from mers cases may initially be negative, only to later become positive by rt-pcr [95] . this may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . despite this both the largest human mers-cov studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the urt. it is then noteworthy that one study reported an association between higher loads in the urt and worse clinical outcome including intensive care and death [94] . at writing, no human data exist to define whether the virus replicates solely or preferentially in the lrt or urt, or replicates in other human tissues in vivo although mers-cov rna has been detected from both the urt and lrt in a macaque monkey model [100] .the distribution of dpp4 in the human upper airways is also not well described. individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] in one instance, a hcw shed viral rna for 42 days in the absence of disease [99] . it is an area of high priority to better understand whether such cases are able to infect others. over three quarters of mers cases shed viral rna in their lrt specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding rna in their urt specimens [91, 102] . in the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (npa; an urt sample), 30 tracheal aspirates, 13 sputa and three bal were examined. the tracheal aspirates and bal returned the highest viral load values followed by npa and sputum. unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in npa testing, were significantly correlated with severe disease and death [49, 94, 103] . this study demonstrated the importance of lrt sampling for whole genome sequencing. when tested, samples positive for mers-cov are often negative for other pathogens [2, 25, 93, 104] . however, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . when viruses are sought, they have included human herpesvirus (hhv), rhinoviruses (hrv), enteroviruses (ev), respiratory syncytial virus (rsv), parainfluenzavirus types 1, 2 and 3 (pivs),influenzaviruses (ifvs), endemic hcovs, adenoviruses (advs) metapneumovirus (mpv) and influenza a\h1n1 virus; co-detections with mers-cov have been found on occasion [2, 22, 37, 69, 97] . bacterial testing is sometimes included (for example, for legionella and pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . further testing of the lrt sample from the first mers case used ifa to screen for some viruses (negative for ifv, pivs, rsv and advs) and rt-pcr for others (negative for adv, evs, mpv and hhvs) [18] . rt-pcr also detected mers-cov. the who strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both mers cases and their contacts. little is known of other causes of mers-like pneumonia in the ksa or of the general burden of disease due to the known classical respiratory viruses. testing of adult pilgrims performing the hajj in 2012 to 2014 has not detected any mers-cov. in 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the ksa were tested [47] . in 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . it should be noted that most pilgrims arrived from mers-free countries. a further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . in earlier hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among hajj pilgrims. [110] a lrt sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of mers-cov infection and replication; it may have been assumed it was the lrt because disease was first noticed there but the urt may be the site of the earliest replication. in jeddah between march and july 2014 (hereafter called the jeddah-2014 outbreak; fig. 3 ), there was a rapid increase in mers cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 mers-cov detections (~3 % prevalence) [111] . among 5,065 individuals sampled and tested across the ksa between october 2012 and september 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated hcws (n = 1,695; 33.5 %) [32] . among the detections, 19 (17.8 %) were hcws and 10 (9.3 %) were family contacts [32] . the 2-3 % prevalence of active mers-cov infections is not dissimilar to the hospital-based prevalence of other human covs. [112] however, the proportion of deaths among those infected with mers-cov is much higher than that known for the hcovs nl63, hku1, 229e or oc43 in other countries, and even above that for sars-cov; it is not a virus that could reasonably be described as a "storm in a teacup". it is the low transmission rate that has prevented worldwide spread, despite many "opportunities". very early in the mers outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of mers-cov with three of the first five cases having contact with dcs [73, 113, 114] . today, animal mers-cov infections must be reported to the world organization for animal health as an emerging disease [115] . a summary of the first mers cases reported by the who defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . this definition made no specific allowance for acquisition from dcs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . camels are known to produce high levels of mers-cov rna in their urt and lungs [116] . providing support for a droplet transmission route and perhaps indicating the presence of rna in smaller, drier droplet nuclei, mers-cov rna was identified in a high volume air sample collected from a barn housing an infected dc [117] . the precise source from which humans acquire mers-cov remains poorly studied but it seems likely that animal and human behavioural factors may play roles (fig. 3) [118] . these factors may prove important for human cases who do not describe any dc contact [119] nor any contact with a confirmed case. whether the who definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. wording focuses on consumption of dc products but does not specifically ascribe risk to a droplet route for acquisition of mers-cov from dc [120] . some mers patients are listed in who disease notices as being in proximity to dcs or farms, but the individuals have not described coming into contact with the animals. no alternative path for acquiring infection is reported in many of these instances. what constitutes a definition of "contact" during these interviews has been defined for one study [72] . despite this lack of clarity, the who consider that evidence linking mers-cov transmission between dcs to humans is irrefutable (fig. 4) [120] . the possibility that bats were an animal host of mers-cov was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . conclusive evidence supporting bats as a source for human infections by mers-cov has yet to be found, but bats do appear to host ancestral representatives [53, 125] . however, these are not variants of the same virus nor always within the same phylogenetic lineage as mers-cov; they are each a genetically distinct virus. bat-to-human infection by mers-cov is a purely speculative event. the only piece of mers-cov-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the rnadependent rna polymerase gene of the mers-cov genome, identified in a faecal pellet from an insectivorous emballonuridae bat, taphozous perforatus found in bisha, the ksa [121] . while very short, the sequence of the fragment defined it as a diagnostic discovery. subsequently a link to dcs was reported [85] and that link has matured into a verified association [38, 126] (fig. 4) . (see figure on previous page.) fig. 3 monthly detections of mers-cov (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th september 2015. an approximation of when dc calving season [128] and when recently born dcs are weaned is indicated. spring (green) and summer (orange) in the arabian peninsula are also shaded. note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. sources of these public data include the who, ministries of health and flutrackers [207] [208] [209] . earlier and subsequent versions of this chart are maintained on a personal blog [210] . modified and reprinted from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res 2015 vol 202:60-88 with permission from elsevier [5] dcs, which make up 95 % of all camels, have a central presence in the arabian peninsula where human-dc contact ranges from little to close [119] . contact may be commonplace and could occur in variety of ways (fig. 4a) . there are several large well-attended festivals, races, sales and parades which feature dcs and dcs are also kept and bred close to populated areas in the ksa [127, 128] . dc milk and meat are widely consumed and the older dc is an animal of ritual significance after the hajj pilgrimage [129] . however, mers-cov infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing dc products. daily ingestion of fresh unpasteurized dc milk is common among the desert bedouin and many others in the ksa. dc urine is also consumed or used for supposed health benefits. despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among mers cases; this may simply be a reporting issue rather than an unexplainable absence of mers. a small case-control study published in 2015 identified direct dc contact, and not ingestion of products, to be associated with onset of mers [38] . the first sero-survey of livestock living in the middle east region was conducted during 2012-2013 [85] . dcs were sampled from a mostly canary island-born herd and from omani dcs (originally imported from the horn of africa) [85] . a neutralising antibody assay found only 10 % of strongly seropositive canary island [5] . b camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor ipc in healthcare settings where transmission is amplified, accounting for the bulk of cases. there are human mers cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) mers-cov infection may be implicated in transmission dc sera could neutralise mers-cov while all omani dc sera had high levels of specific mers-cov neutralizing antibody [85] . this indicated that dcs had in the past been infected by mers-cov, or a very similar virus. since this study, a host of peer-reviewed reports have looked at both dcs and other animals, and the possibility that they may host mers-cov infection. seropositive dcs have been found throughout the arabian peninsula including oman, the ksa, qatar, jordan, the united arab emirates (uae), kuwait as well as sudan, somalia, egypt, tunisia, nigeria, kenya and ethiopia in africa and the canary islands [85, [130] [131] [132] [133] [134] . other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, bactrian camels, llamas and guanaco (south american camelids) but none had detectable neutralising antibody against mers-cov [4, 74, 78, 85, 86, 135, 136] . no virology or serology studies of human samples from areas in africa where there are camels with a history of mers-cov have been reported to date. however,an absence of unexplained pneumonia that may be attributable to mers-cov infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. it is thus unclear whether mers-cov, or an antigenically related cov, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the arabian peninsula [133] . mers-cov rna has also been detected in dc samples, and recovery of infectious virus has also been achieved from dc samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . from some of these, full or majority length genomes of mers-cov have been sequenced [77, 137, 138] . dc versions of mers-cov were found to be as similar to each other, as were variants detected from different humans over time and across distance. antibody screening assays have also detected crossreactive antibodies in sera. these were identified as such by screening sera against similar viruses, for example bcov or hcov-oc43 (as an antigenic facsimile for bcov). it is possible that other mers-cov-like viruses also reside within dcs, but this does not detract from the definitive finding of mers-cov genetic sequences in both dcs and humans [117, 142, 143] . screening studies have shown that juvenile dcs are more often positive for virus or viral rna while older dcs are more likely to be seropositive and rna or virus negative [76, 77, 144] . in adult dcs, mers-cov rna has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . viral loads among positive dcs can be very high [4, 76, 77, 139, 144] and dcs have been found positive both when ill with urt respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . these findings indicate dcs host natural mers-cov infections. furthermore, stored dc sera have revealed signs of mers-cov in dcs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . older sera have not been tested and so precisely how long dcs have been afflicted by mers-cov, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in africa or the arabian peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. researchers sought to determine a direction for infection; were dcs transmitting virus to humans or were humans infecting dcs? at a qatari site, a farm owner and his employee became ill in mid-october 2013 and tested positive for mers-cov rna in a sputum and throat swab sample, respectively. rt-rtpcrs found mers-cov rna in 11 of 14 positive dc nasal swabs at the farm; six (43 %) positive by two or more assays [138] . the results indicated a recent outbreak had occurred in this herd; the first indication of mers-cov rna found within dcs with a temporal association to human infections. three positive dc samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and dc mers-cov sequences [138] . the dcs and human contacts yielded orf1a and orf4b sequences differing by only a single nucleotide each, clustering closely with the hafr-al-batin_1_2013 variant [138] . subsequent case studies found evidence of a concurrent human and dc infection and the direction of that infection was inferred to be from the ill dcs and to their human owners [117, 142, 146] . partial genome sequences indicated that a human and a mers-cov rt-rtpcr positive dc had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] all nine dc in the owner's herd, serially sampled, reacted in a recombinant s1 antigen elisa, with the two animals that had been rt-rtpcr positive showing a small, verifiable rise in antibody titre [142] . a rise in titre theoretically begins 10 to 21 days after dc infection [142] . the authors suggested that the rise in titre in dc sera which occurred alongside a declining rna load, while the patient was actively ill and hospitalized, indicated that the dcs were infected first followed by the owner [117, 142] . bcov antibodies were also present, and rising in one of the two rt-rtpcr positive animals but no animal's antibodies could neutralise bcov infection [142] . camel calving season occurs in the winter months (between late october and late february; fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve dc populations [128] . what role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . juvenile dcs appear to host active infection more often than adult dcs and thus the sacrificial slaughter of dcs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. in contrast to earlier results, slaughterhouse workers who kill both younger and older dcs, may be an occupational group with significantly higher incidence of seropositivity to mers-cov when animals have active mers-cov infections [129, 139, [147] [148] [149] . expanded virological investigations of african dcs may lead to more seropositive animals and geographic areas in which humans may be at risk. it is possible that there are areas where humans already harbour mers-cov infections that have not been identified because of an absence of laboratory surveillance. virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . infectious mers-cov added to dc, goat or cow milk and stored at 4°c could be recovered at least 72 h later and, if stored at 22°c, recovery was possible for up to 48 h [150] . mers-cov titre decreased somewhat when recovered from milk at 22°c but pasteurization completely ablated mers-cov infectivity [150] . in a subsequent study, mers-cov rna was identified in the milk, nasal secretion and faeces of dcs from qatar [151] . a single study has examined the ability of mers-cov to survive in the environment [150] . plastic or steel surfaces were inoculated with 10 6 tcid 50 of mers-cov at different temperature and relative humidity (rh) and virus recovery was attempted in cell culture. at high ambient temperature (30°c) and low rh (30 %) mers-cov remained viable for 24 h [150] . by comparison, a well known and efficently transmitted respiratory virus, influenza a virus, could not be recovered in culture beyond four hours under any conditions [150] . aerosol experiments found mers-cov viability only decreased 7 % at low rh at 20°c. in comparison, influenza a virus decreased by 95 % [150] . mers-cov survival is inferior to that previously demonstrated for sars-cov [152] . for context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. mers-cov's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . whether mers-cov can remain adrift and infectious for extended periods (truly airborne) remains unknown. such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. the nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the al-ahsa hospital, the ksa and the south korean outbreaks [21, 23, 154, 155] . by extrapolation, aerosol-generating events involving dcs (urination, defecation, and preparation and consumption of dc products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. the provision of evidence supporting the best formulation of personal protective equipment to be worn by hcws who receive, manage or conduct procedures on infectious cases remains a priority. mers-cov was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to mers-cov transmission chains. however, transmission of mers-cov is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] in a household study, 14 of 280 (5 %) contacts of 26 mers-cov positive index patients were rna or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . it seems that the majority of human cases of mers-cov, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of mers-cov has not been self-sustaining [157] [158] [159] [160] [161] . that is to say, the basic reproduction number (r 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. if r 0 was greater than 1, a sustained increase in case numbers would be expected. some r o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. that mers has had a constant presence in the arabian peninsula since 2012 is due to ongoing, sporadic spill-over events from dcs amplified by poorly controlled hospital outbreaks. the first known mers human-to-human transmission event was one characterized by acute lrt disease in a healthcare setting in jordan. in stark contrast, a sero-survey of hcw who were sometimes in close and prolonged contact with the first, fatal mers-cov case in 2012 [162] , found none of the hcw had seroconverted four months later, despite an absence of eye protection and variable compliance with required ppe standards [162] . early on in the mers story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. contacts of confirmed mers cases were often observed for clinical illness, but not tested. these omissions may have confounded our understanding of mers-cov transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. case-control studies were not a focus. as testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . a rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the jeddah-2014 outbreak. historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. upon follow-up, over three-quarters of such mers-cov rna positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. the proportion of fatal mers cases within the ksa compared to outside the ksa, as well as the age, and sex distribution change in different ways when comparing mers outbreaks. approximately 43 % of mers cases (549 of 1277) in the ksa were fatal betwen 2012 and december 2015 while 21 % (72 of 330) died among those occurring outside of the ksa. the total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. however the proportion of male deaths from total males with mers is a similar figure to that for females. in the ksa, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 south korean or the jeddah-2014 outbreaks (additional file 2: figure s2 ). why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . as a group, hcws comprised 16 % of mers cases in the ksa and south korea. it is apparent that the weekly proportion of infected hcws increases alongside each steep rise in overall detections (fig. 5) . in may 2013, the who published guidelines for ipc during care of probable or confirmed cases of mers-cov infection in a healthcare setting [166] . this is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . these rises in mers-cov detections can decrease the average age during each event because hcws are usually younger than inpatients with mers. healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (ipc) procedures [115, 118] . most of the analysis of mers-cov genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . mers-cov was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. the technique can produce genomic [207] [208] [209] . earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during mers-cov characterization [48] . as more genomes from both humans and dcs have been characterized, two clades have become apparent; a and b (fig. 6) . clade a contains only human-derived mers-cov genomes from jordan, while clade b comprises the majority of human and camel genomes deduced thus far [168] . two studies during 2015, one looking at jeddah-2014 mers-cov variants and another looking at a variant exported from south korea to china, have now identified signs of genetic recombination among mers-cov variants. while human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . shared identity implies that the major source for human acquisition is the dc, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. over a month, a dc virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that dcs are the natural, rather than intermediate, host for the mers-cov we know today [77] . to date, recombination has been localised to breakpoints near the boundary between orf1a and orf1b regions, within the spike gene [170] and in the orf1b region (fig. 2) [172] . it is not unexpected that recombination should occur since it is well known among other covs [124] and because the majority of mers-cov whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. if we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely fig. 6 the genetic relationship between mers-cov nucleotide sequences (downloaded from genbank using the listed accession numbers and from virological.org [212] ). this neighbour joining tree was created in mega v6 using an alignment of human and dcderived mers-cov sequences (geneious v8.1 [211] ). clades are indicated next to dark (clade a) or pale (clade b) blue vertical bars. camel icons denote genomes from dcs. healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. genetic mutations noted during the largest of human outbreaks, jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . however, we understand very little of the phenotypic outcomes that result from subtle genetic change in mers-cov genomes. to date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . but vigilance and larger, more contemporary and in vivo studies are needed. genome sequence located to a distinct clade were identified from an egyptian dc that was probably imported from sudan. this does not fit into either of the current clades [125, 168, 177] . a virus sequenced from a neoromicia capensis bat was more closely related to mers-cov than other large bat-derived sequences had been to that point, but the genome of a variant of a mers-cov has yet to be discovered and deduced from any bat [125] . analyses of mers-cov genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (fig. 2) , which encodes the spike protein and accessory proteins [168] . at least nine mers-cov genomes contained amino acid substitutions in the receptor binding domain (rbd) of the spike protein and codons 158 (n-terminal region), 460 (rbd), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . the spike protein had not changed in the recombinant mers-cov genome identified in china in 2015 but was reported to have varied at a higher rate than that for complete mers-cov genomes, among south korean variants [172, 178] . this highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. despite this, one assay amplifying a 615 nucleotide fragment of the spike s2 domain gene for sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (fig. 6 ) [169] . this approach was employed when defining the geographically constrained mers hospital outbreak in al-ahsa, which occurred between 1 st april and 23 rd may 2013, as well as clusters in buraidah and a community outbreak in hafr al-batin, the ksa. genomic sequencing identified that approximately 12 mers-cov detections from a community outbreak in hafr al-batin between june and august 2013 may have been triggered by an index case becoming infected through dc contact [175] . sequencing mers-cov genomes from the 2013 al-ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . however, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . in riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . riyadh is a nexus for camel and human travel and has had more mers cases than any other region of the ksa to date but also harbours a wide range of mers-cov variants [128, 167, 179] . however the south korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . for many mers cases detected outside the arabian peninsula, extensive contact tracing has been performed and the results described in detail. contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. results of contact tracing to date have found that onward transmission among humans is an infrequent event. for example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in germany who travelled from the uae but no sign of virus or antibody were found in any of them [73] . the very first mers case had made contact with 56 hcws and 48 others, but none developed any indication of infection [162] . in a study of 123 contacts of a case treated in france, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . none of the contacts of the first two mers cases imported into the usa in 2014 contained any mers-cov footprint [182] and none of the 131 contacts of two travellers returning to the netherlands developed mers-cov antibodies or tested rna positive [25, 183] . analyses of public data reveal many likely instances of nosocomial acquisition of infection in the arabian peninsula and these data may be accompanied by some details noting contact with a known case or facility. one example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple south korean hospitals [184] . such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (fig. 4c) . the hospital-associated outbreak in jeddah in 2014 was the largest and most rapid accumulation of mers-cov detections to date. the greatest number of mers-cov detections of any month on record occurred in jeddah in april. the outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . a rise in fatalities followed the rapid increase in case numbers. in 2015 two large outbreaks occurred. south korea was the site of the first large scale outbreak outside the arabian peninsula and produced the first cases in both south korea and china, occurring between may and july 2015. this was closely followed by a distinct outbreak in ar riyad province in the ksa which appeared to come under control in early november. after staying in bahrain for two weeks, a 68 year old male (68 m) travelled home to south korea via qatar, arriving free of symptoms on the 4 th may 2015 [187] . he developed fever, myalgia and a cough nearly a week later (11 th ). he visited a clinic as an outpatient between the 12 th and 15 th of may and was admitted to hospital a on the 15 th [188] . he was discharged from hospital a on the 17 th then visited and was admitted to the emergency department of hospital b on the 18 th . during this second stay, a sputum sample was taken and tested positive for mers-cov on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the south korean outbreak. approximately 34 people were infected during this time [187] . in total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 m; 37 cases died [189] . in south korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with mers, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of korean society, crowded emergency rooms, poor ipc measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . all of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. the hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . early in the outbreak, a infected traveller, the son of an identified case in south korea, passed through hong kong on his way to china where he was located, isolated and cared for in china [91, 199, 200] . no contacts became ill. the outbreak was brought under control in late july/ early august [201] after improved ipc measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . a review of public data showed that, as for mers in the ksa, older age and the presence of underlying disease were significantly associated with a fatal outcome in south korea. [40] even though r 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from mers-cov infection [204] . the dynamic of an outbreak depends on the r 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . in the region of ar riyad, including the capital city of riyadh, a hospital based cluster began, within a single hospital, from late june 2015 [205] . by mid-september there had been approximately170 cases reported but the outbreak appeared to been brought under control in november. it became apparent early on that mers-cov spread relatively ineffectively from human-to-human. despite ongoing and possibly seasonal introduction of virus to the human population via infected dcs and perhaps other animals yet to be identified, the vast majority of mers-cov transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. this opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. it remains unclear if this group are uniquely affected by mers-cov or if other respiratory virus infections, including those from hcovs, produce a similarly serious impact. in south korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. household human-to human transmission occurs but is also limited. educational programs will be essential tools for combatting the spread of mers-cov both within urban and regional communities and for the health care setting. vigilance remains important for containment since mers-cov is a virus with a genetic makeup that has been observed for only three years and is not stable. among all humans reported to be infected, nearly 40 % have died. continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. whole genome sequencing has been used extensively to study mers-cov travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. mers and sars have some clinical similarities but they also diverge significantly [206] . defining characteristics include the higher pfc among mers cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of sars) and the higher association between fatal mers and older males with underlying comorbidities. for the viruses, mers-cov has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to sars-cov. there appears to be a 2-3 % prevalence of mers-cov in the ksa with a 5 % chance of secondary transmission within the household. there is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any r 0 would predict on face value. nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of mers or mers-cov during or immediately after these events. there is no evidence that mers-cov is a virus of pandemic concern. nonetheless, hospital settings continue to describe mers cases and outbreaks in the arabian peninsula. as long as we facilitate the spread of mers-cov among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. the mers-cov appears to be an enzootic virus infecting the dc urt with evidence of recent genetic recombination. it may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. rt-pcr testing of lrt samples remains the gold standard for mers-cov confirmation. serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. similarly, the important question of whether those who do shed mers-cov rna for extended periods are infectious while appearing well, continues to go unanswered. it is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. while the basic virology of mers-cov has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. additional file 1: figure s1 . the severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd absence of mers-coronavirus in bactrian camels, southern mongolia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity middle east respiratory syndrome coronavirus infection not found in camels in japan absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread lack of middle east respiratory syndrome coronavirus transmission from infected camels tensions linger over discovery of coronavirus as outbreak continues middle east respiratory syndrome coronavirus (mers-cov): evidence and speculations cumulative number of reported probable cases of sars in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection replicative capacity of mers coronavirus in livestock cell lines human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-tohuman transmission case definition for case finding severe respiratory disease associated with novel coronavirus middle east respiratory syndrome coronavirus case definition for reporting to who. interim case definition as of 14 middle east respiratory syndrome coronavirus | case definition for reporting to who 14 revised interim case definition for reporting to who -middle east respiratory syndrome coronavirus (mers-cov): interim case definition as of transmission of mers-coronavirus in household contacts screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study case definition and surveillance guidance for mers-cov testing in saudi arabia infection prevention/control and management guidelines for patients with middle east respitaory syndrome coronavius (mers-cov) infection infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection middle east respiratory syndrome coronavirus in children characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia preliminary epidemiological assessment of mers-cov outbreak in south korea mortality risk factors for middle east respiratory syndrome outbreak, south korea clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea invited editorial: mers-cov an emerging viral zoonotic disease: three years after and counting middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in stillbirth during infection with middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia development and evaluation of a 'real-time' rt-pcr for the detection of enterovirus and parechovirus rna in csf and throat swab samples mers: emergence of a novel human coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans laboratory testing for middle east respiratory syndrome coronavirus | interim guidance performance and clinical validation of the realstar mers-cov kit for detection of middle east respiratory syndrome coronavirus rna real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus laboratory testing for middle east respiratory syndrome coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses realtime sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) a sweet spot for molecular diagnostics: coupling isothermal amplification and strand exchange circuits to reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in serological assays for emerging coronaviruses: challenges and pitfalls middle east respiratory syndrome coronavirus immunoassays test characteristics: anti-mers-cov elisa camel (igg) recombivirus anti-middle east respiratory syndrome coronavirus (mers-cov) elisa kits ncov / novel coronavirus) nucleocapsid antibody, mouse mab is mers another sars? investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates contact investigation for imported case of middle east respiratory syndrome seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012. influenza other respir viruses kinetics of serologic responses to mers coronavirus infection in humans hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological findings from a retrospective investigation specific serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study cdc concludes indiana mers patient did not spread virus to illinois business associate middle east respiratory syndrome: what clinicians need to know interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (puis) for middle east respiratory syndrome coronavirus (mers-cov characteristics of traveler with middle east respiratory syndrome distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case association of higher mers-cov virus load with severe disease and death, saudi arabia an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms health protection agency ukncit. evidence of person-to-person transmission within a family cluster of novel coronavirus infections prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications respiratory tract samples, viral load and genome fraction yield in patients with middle east respiratory syndrome ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response a case of imported middle east respiratory syndrome coronavirus infection and public health response viral respiratory infections among hajj pilgrims in 2013 influenza a and b viruses but not mers-cov in hajj pilgrims acute respiratory infections in travelers returning from mers-cov-affected areas changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a 10-year retrospective analysis of data soaring mers cases cause pandemic jitters, but causes are unclear co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 recovery from severe novel coronavirus infection human isolate 115. who statement on the fifth meeting of the ihr emergency committee concerning mers-cov | who statement mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia evidence for camel-to-human transmission of mers coronavirus taking stock of the first 133 mers coronavirus cases globally-is the epidemic changing? human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection. zoonoses public health summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus in bats, saudi arabia emerging infectious diseases associated with bat viruses bats and their virome: an important source of emerging viruses capable of infecting humans coronavirus diversity, phylogeny and interspecies jumping rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease link between mers virus and camels worries breeders dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) fiqh us-sunnah antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries mers coronavirus neutralizing antibodies in camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus antibody reactors among camels in dubai serologic assessment of possibility for mers-cov infection in equids mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate human infection with mers coronavirus after exposure to infected camels, saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels evidence for camel-to-human transmission of mers coronavirus the holy qur'an sacrificing an animal at mina occupational exposure to dromedaries and risk for mers-cov infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels the effects of temperature and relative humidity on the viability of the sars coronavirus viability of pseudomonas aeruginosa in cough aerosols generated by persons with cystic fibrosis middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? an observational, laboratory-based study of outbreaks of mers-coronavirus in jeddah and riyadh, kingdom of saudi arabia assessment of the mers-cov epidemic situation in the middle east region assessing the pandemic potential of mers-cov interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility transmission dynamics and control of ebola virus disease (evd): a review health care worker contact with mers patient, saudi arabia middle east respiratory syndrome coronavirus: epidemiology and disease control measures age-specific and sex-specific morbidity and mortality from avian influenza a(h7n9) mers-cov outbreak in jeddah-a link to health care facilities infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers-cov recombination: implications about the reservoir and potential for adaptation middle east respiratory syndrome coronavirus recombination and the evolution of science and public health in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 9 reliable typing of mers-cov variants with a small genome fragment variations in spike glycoprotein gene of mers-cov molecular epidemiology of hospital outbreak of middle east. respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) in the republic of korea microevolution of outbreak-associated middle east respiratory syndrome coronavirus, south korea first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities followup of contacts of middle east respiratory syndrome coronavirus-infected returning travelers, the netherlands press release -who, korea-who joint mission on mers cov call for infection control to stem mers infection control and mers-cov in health workers an outbreak of middle east respiratory syndrome coronavirus infection in south korea middle east respiratory syndrome coronavirus (mers-cov) -republic of korea samsung hospital to invest w100b in post-mers improvements why the panic? south korea's mers response questioned intensified public health measures help control mers-cov outbreak in the republic of korea south korean mers outbreak spotlights lack of research complete genome sequence of middle east respiratory syndrome coronavirus isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china urgent call for research on middle east respiratory syndrome (mers) in korea middle east respiratory syndrome (mers) in asia: lessons gleaned from the south korean outbreak no respite for korean economy even as mers patients recover middle east respiratory syndrome coronavirus (mers-cov) -china mers in south korea and china: a potential outbreak threat? objective determination of end of mers outbreak, south korea updates on mers outbreak (as of 6:00 on surveillance operation for the 141st confirmed case of middle east respiratory syndrome coronavirus in response to the patient's prior travel to jeju island the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission moh holds the first press conference to update community and media on mers severe acute respiratory syndrome vs. the middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) 2012-2015 case list of moh/who novel coronavirus mers ncov announced cases geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data recent evolution patterns of the middle east respiratory syndrome coronavirus (mers-cov) preliminary analysis of middle east respiratory syndrome coronavirus (mers-cov) sequences from korea and china any unreferenced opinions expressed herein are those of the authors and do not necessarily represent the views of any employer or institution. our thanks go to andrew rambaut, maia majumder, marion koopmans and hale abdali for helpful discussions on social media and cmam and riam for patience. the authors declare that they have no competing interests.author's contributions imm and kea contributed equally to this work and read an approved the final manuscript. key: cord-338776-2wa30218 authors: zhao, xiaoyu; chu, hin; wong, bosco ho-yin; chiu, man chun; wang, dong; li, cun; liu, xiaojuan; yang, dong; poon, vincent kwok-man; cai, jianpiao; chan, jasper fuk-woo; to, kelvin kai-wang; zhou, jie; yuen, kwok-yung title: activation of c-type lectin receptor and (rig)-i-like receptors contributes to proinflammatory response in middle east respiratory syndrome coronavirus-infected macrophages date: 2020-02-15 journal: j infect dis doi: 10.1093/infdis/jiz483 sha: doc_id: 338776 cord_uid: 2wa30218 background: human infection with middle east respiratory syndrome coronavirus (mers-cov) poses an ongoing threat to public health worldwide. the studies of mers patients with severe disease and experimentally infected animals showed that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of mers-cov. we sought to identify pattern recognition receptor (prr) signaling pathway(s) that mediates the inflammatory cascade in human macrophages upon mers-cov infection. methods: the potential signaling pathways were manipulated individually by pharmacological inhibition, small interfering ribonucleic acid (sirna) depletion, and antibody blocking. the mers-cov-induced proinflammatory response was evaluated by measuring the expression levels of key cytokines and/or chemokines. reverse transcription-quantitative polymerase chain reaction assay, flow cytometry analysis, and western blotting were applied to evaluate the activation of related prrs and engagement of adaptors. results: mers-cov replication significantly upregulated c-type lectin receptor (clr) macrophage-inducible ca(2+)-dependent lectin receptor (mincle). the role of mincle for mers-cov-triggered cytokine/chemokine induction was established based on the results of antibody blockage, sirna depletion of mincle and its adaptor spleen tyrosine kinase (syk), and syk pharmacological inhibition. the cytokine and/or chemokine induction was significantly attenuated by sirna depletion of retinoic acid-inducible-i-like receptors (rlr) or adaptor, indicating that rlr signaling also contributed to mers-cov-induced proinflammatory response. conclusions: the clr and rlr pathways are activated and contribute to the proinflammatory response in mers-cov-infected macrophages. middle east respiratory syndrome coronavirus (mers-cov) has been identified as a novel pathogen causing human respiratory infections since 2012 [1] . most mers patients presented with viral pneumonia, and some of them developed acute respiratory distress syndrome and multiorgan failure with a case-fatality rate over 30% [2] . cytological examination of bronchoalveolar lavage fluids from mers patients exhibited large numbers of neutrophils and macrophages, indicating a massive pulmonary inflammation [3] . the postmortem study of a mers patient revealed compatible pathological changes, including edematous alveolar septa with infiltration of lymphocytes, neutrophils, and macrophages, as well as diffuse alveolar damage [4] . a detailed examination of lung tissues of mers-cov-infected nonhuman primates including common marmosets and rhesus macaques revealed similar pathological changes, reminiscent of prominent lung disease in severe mers patients. thus, the authors reached a central conclusion that robust viral replication, together with an intense local immune response to mers-cov infection, may result in severe respiratory disease in these experimental animals [5] . macrophages are important sentinel cells of the innate immune system. macrophages sense and recognize invading pathogens or endogenous ligands through a broad range of sensors, and they generally elicit effective clearance via phagocytosis. on the other hand, macrophages may fail to eliminate invading microbes. instead, they become inappropriately activated and initiate dysregulated inflammatory responses in some cases [6, 7] . we have previously reported that mers-cov productively infected human monocyte-derived macrophages (mdms) and triggered aberrant proinflammatory response [8] , providing direct evidence to explain the massive inflammation observed in severe mers patients and experimentally infected nonhuman primates. upon viral infection, host germline-encoded pattern recognition receptors (prrs) including retinoic acid-inducible gene (rig)-i-like receptors (rlrs), toll-like receptors (tlrs), nucleotide-binding oligomerization domains-like receptors (nlrs), and c-type lectin receptors (clrs) detect the presence of foreign motifs or ligands known as pathogen-associated molecular patterns [9] [10] [11] [12] . the intracellular signaling cascades triggered by these prrs lead to transcriptional activation of type i interferons (ifns) and inflammatory mediators that coordinate the elimination of pathogens and infected cells and, meanwhile, contribute to the inflammation and clinical symptoms of viral infections. the rlrs, such as rig-i and melanoma differentiationassociated gene 5 (mda5), have been extensively characterized as essential cellular sensors to recognize rna viruses [11, 13] . ding et al [14] demonstrated the rlr-mediated signaling for nuclear factor (nf)-κb activation in transmissible gastroenteritis virus infection. zalinger et al [15] elucidated the importance of mda5 to host defense during murine cov infection. meanwhile, clr signaling has shown the increasing importance for triggering inflammatory response upon viral infections [16, 17] . the transcription factor nf-κb is an essential mediator of inducible gene expression of cytokines and chemokines. clr induced signal transduction seems to mainly activate and modulate nf-κb functions [17, 18] . the mechanisms by which covs evade host innate antiviral response, including mers-cov and severe acute respiratory syndrome (sars)-cov, have been extensively investigated [19] [20] [21] . however, it is poorly understood how the highly pathogenic mers-cov triggers the aberrant proinflammatory response, one of the pathological bases of severe respiratory diseases in mers patients and experimentally infected animals. in this study, we sought to elucidate the contribution of rlr and clr signaling pathway for mediating the exuberant inflammatory responses in macrophages upon mers-cov infection. the mers-cov of strain emc/2012 [1] was propagated in vero e6 cells. three days after virus inoculation, the cell-free media were collected and stored at −80°c in aliquots. ultraviolet (uv) inactivation of mers-cov was performed by exposing the virus to uv cross-linker for 10 minutes as described previously [22] . peripheral blood was obtained from healthy blood donors at the hong kong red cross blood transfusion center according to a protocol approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster. monocyte preparation and differentiation were performed according to a well established protocol described previously [23] . for viral infection, treated or mock-treated mdms were inoculated with mers-cov at a multiplicity of infection (moi) of 2 or were mock inoculated for 1 hour at 37°c. cell lysates were harvested at 24 hours postinfection (hpi) for quantification of messenger rna (mrna) expression levels of cellular genes and detection of viral load. tank-binding kinase 1 (tbk1) and inhibitor of nf-κb kinase epsilon (ikkε) inhibitor amlexanox, spleen tyrosine kinase (syk) inhibitor r406, and caspase-1 inhibitor vx-765 were purchased from invivogen. the individual inhibitors or a neutralization antibody against human mincle (invivogen, mabg-hmcl) or mouse monoclonal immunoglobulin (ig)g2b (mab004; r&d) were administered to mdms 1 hour before virus inoculation at the indicated concentrations and supplemented in the culture media throughout after infection. silencer select small interfering rna (sirna) targeting human mitochondrial antiviral-signaling proteins (mavs) (s33179; thermo fisher scientific), syk (s13681), rig-i (s223615), mda5 (s34499), mincle (s25297), and scrambled sirna were transfected to mdms using lipofectamine 3000 (thermo fisher scientific) according to the manufacturer's instruction. in brief, mdms were transfected with 200 nm sirna for 2 consecutive days. at day 3 after sirna transfection, the cells were inoculated with mers-cov. detection of cellular mrna expression and viral load was performed as described previously [24] . in brief, cell lysates were applied to rna extraction, followed by reverse transcription (rt) using oligo(dt) or the virus-specific primer. the resultant complementary deoxyribonucleic acids (cdnas) were used for quantitative polymerase chain reaction (qpcr) assay to measure mrna expression level of cellular gene and viral load. the primer sequences used in qpcr assay are shown in the supplementary table. flow cytometry analysis, immunofluorescence staining, and western blot immunostaining and flow cytometry analysis were performed according to the standard protocol as described elsewhere [25, 26] . in brief, after detachment, fixation, and permeabilization, mdms were labeled with a mouse antibody against human mincle (ab100846; abcam) and in-house made antibody against mers-cov np or antibody of isotype control, followed by the corresponding secondary antibodies. flow cytometry analysis was performed using a bd facscanto ii flow cytometer (bd biosciences), and the data were analyzed using flowjo vx (tree star). for immunofluorescence staining, mdms seeded on glass coverslips were inoculated with mers-cov at 5 moi. at 24 hpi, the cells were fixed with 4% paraformaldehyde and labeled with anti-np and anti-mincle, followed by corresponding secondary antibodies. slides were mounted with prolong gold antifade reagent with 4' ,6-diamidino-2-phenylindole (themo fisher scientific) and imaged with a carl zeiss lsm 800 confocal microscope. the whole-cell extracts of infected and mock-infected macrophages were separated in sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. after blocking, the membranes were probed with antibodies against mavs (ab31334; abcam), syk (ab40781), rig-i (ab45428), mda5 (ab126630), mincle (ab100846), and card9 (ab124922), np antibody or mouse β-actin antibody (a5441; sigma), followed by secondary staining. the blots were visualized by luminata classico western hrp substrate (wbluc0500; millipore). quantification was performed using imagej software. a human ip-10 elisa kit (ab173194; abcam) was used for quantification of ip-10 secretion in the culture media. an unpaired t test was performed for data analysis using graphpad prism 6. p < .05 was considered to be statistically significant. data are presented as mean and standard deviation of representative experiments. we first used the inhibitors of rlr and clr signaling pathway to evaluate their potential contribution for mediating the proinflammatory response in mers-cov-infected macrophages. amlexanox, an inhibitor of rlr signaling pathway, specifically supresses the noncanonical ikb kinases ikkε and tbk1, and both are essential players for the coordination of ifn regulatory factor 3 (irf3)-and nf-κb-mediated innate immune response [27, 28] . r406 is a specific, atp-competitive inhibitor of syk, the essential adaptor of clr pathway [29] . caspase-1 inhibitor vx-765, a commonly used inhibitor for nlr signaling, is used for comparison [30] . mtt assay was used to detect the 50% cytotoxic concentration (cc 50 ) of each inhibitor in mdms (supplementary figure 1) , to ensure that the concentrations used for inhibition of prrs have no adverse effect on macrophage viability. based on the effective concentration of each inhibitor and its cc 50 , the indicated working concentrations ( figure 1a ) were used throughout the study. to assess the contribution of rlr or clr pathway to induce proinflammatory response, we measured the expression levels of a series of key proinflammatory cytokines and/or chemokines in mers-cov-infected mdms in the presence of these inhibitors ( figure 1b ). consistent with our previous observation, mers-cov infection globally stimulated an array of proinflammatory cytokines and chemokines including interleukin (il)-6, tumor necrosis factor (tnf)-α, macrophage-inflammatory protein (mip)-1α, rantes, ifn-γ, and ip-10 [8] . the induction of cytokine and/or chemokine was dependent on viral replication because the inoculation of the uv-inactivated viruses was unable to trigger these inflammatory mediators, except rantes (supplementary figure 2) . it is notable that mers-cov-mediated induction of tnf-α, mip-1α, ifn-γ, and ip-10 was generally diminished in the presence of amlexanox and r406, although amlexanox treatment only marginally reduced tnf-α production. in addition, the treatment of amlexanox, but not r406, significantly attenuated the induction of il-6 and rantes. however, nlr signaling inhibitor vx-765 seemed to have minimal effect on these inflammatory mediators. because the replication of mers-cov is the driving force for the induction of most cytokines and/or chemokines, we evaluated the viral replication in the presence of the inhibitors. it was shown that the addition of these inhibitors did not affect viral replication (supplementary figure 3) . accumulating evidence suggested that rlrs and clrs are inducible upon stimulation or microbial infection [14, [31] [32] [33] . among clrs, dectin-1, dectin-2, and mincle are highly expressed in myeloid cells such as monocytes, macrophages, and dendritic cells [17] . thus, we measured the mrna profiles of rlrs and myeloid clrs in mers-cov-infected macrophages. as shown in figure 2a , the mrna expression levels of rlrs including rig-i, mda5, and lpg2, as well as clrs mincle and dectin-2 were significantly upregulated at 24 hpi. the positive modulation pattern of rlr and clr, especially the increased expression of rig-i and mincle, was manifested as early as 6 hpi ( figure 2b ), suggesting an accelerating activation of these prrs during mers-cov infection. overall, the above results suggested that rlr and clr signaling might be involved in viral recognition and trigger the proinflammatory response upon mers-cov infection in macrophages. to dissect the role of rlr and clr signaling in inducing proinflammatory response, we depleted the rlr adaptor, mavs, or the clr adaptor, syk, by sirna knockdown and examined mers-cov-elicited cytokine and/or chemokine response. the effective depletion of both adaptors was shown by rt-qpcr assay at 48 hours post-sirna transfection, to approximately 20% for syk and 40% for mavs relative to the control cells ( figure 3a ). the reduced expression of the adaptors was also verified by western blot. subsequently, we infected the transfected cells with mers-cov and measured the expression profiles of the cytokines and/or chemokines. as shown in figure 3b , sirna depletion of either mavs or syk significantly diminished the induction of all tested inflammatory molecules including il-6, tnf-α, mip-1α, rantes, ifn-γ, and ip-10. rig-i, mda5, and mincle were upregulated the most among rlr and clr receptors ( figure 2 ). we then tested whether sirna depletion of rig-i, mda5, and mincle have any effect on mers-cov-triggered immune activation. as shown in figure 4a , the receptors were depleted to approximately 30% at transcriptional level, which were verified by western blot analysis. in rig-i-, mda5-, and mincledepleted cells, the induction of ifn-γ and ip-10 was generally abolished; and mip-1α and rantes expression were significantly reduced ( figure 4b) . a significantly attenuated il-6 induction was observed in rig-i-depleted cells, but not in mda5-or mincle-depleted cells; whereas tnf-α production was basically unaffected after depletion of these receptors individually. the induction of ifn-γ and ip-10 was constantly and significantly reduced in various assays, including the inhibitor treatment and sirna depletion. in addition, these 2 proinflammatory mediators are highly stimulated in severe sars and mers patients [34] [35] [36] . thus, we examined mers-cov-induced secretion of ifn-γ and ip-10 after genetic depletion of the above-mentioned rlr and clr receptors and adaptors. ip-10 secretion was readily detectable from both mers-cov-infected and mock-infected mdms; whereas ifn-γ secretion was below the detection limit, even in the virus-infected cells. thus, we examined mers-cov-induced ip-10 secretion in mdms upon sirna transfection. as shown in figure 4c , genetic depletion of rig-i, or syk and mincle resulted in a signification reduction of ip-10 secretion. knockdown of rig-i adaptor mavs also marginally attenuated ip-10 production. taken together, we found that disrupting rlr or clr signaling, especially at the adaptor level, largely dampened the production of mers-cov-induced proinflammatory cytokines and chemokines. the interplay of clr signaling and human covs has not been appreciated yet. to further characterize the role of mincle for immune activation in mers-cov-infected macrophages, we measured the mers-cov-related ifn-γ induction in the presence of increasing concentrations of a neutralization antibody against mincle. the results showed that the addition of α-mincle significantly suppressed mers-cov-triggered ifn-γ induction in a dose-dependent manner ( figure 5a ). in addition, treatment of α-mincle significantly diminished the induction of il-6, rantes, ifn-γ, and ip-10 ( figure 5b ). however, the inhibitory effect was negligible for tnf-α and mip-1α. taken together, in line with the observations from mincle sirna depletion, clr receptor mincle contributed to the immune activation in mers-cov-infected macrophages. to characterize the engagement of mincle in mers-cov-infected cells, we assessed mincle protein expression by flow cytometry analysis and immunofluorescence staining. as shown in figure 6a , in the mock-infected cells, approximately 50% of macrophages were mincle + , indicating that mincle is indeed highly expressed in macrophages as reported previously [17] . the percentage of np + (infected) cells increased from 2.9% at 3 hpi to 37.8% at 24 hpi, indicating a productive viral replication. we then compared mincle expression in np + cells versus that in np − cells at 24 hpi. in np − cells, mincle protein expression, ie, the percentage of mincle-expressing cells and the abundance of mincle protein (mean fluorescence intensity), remained similar in the np − cells and the mock-infected cells. it is notable that mincle protein expression was significantly elevated in the np + cells ( figure 6b ). mincle upregulation in the virus-infected cells was verified by costaining of mincle and np. the np + macrophages displayed the substantially elevated mincle expression ( figure 6c ). therefore, the results suggested that mers-cov replication significantly enhanced mincle expression in macrophages. engagement of adaptor syk with clrs, via card9, activates mitogen-activated protein kinases and transcription factor nf-kb and results in the induction of proinflammatory cytokines [9] . card9 activation is not only mediated by phosphorylation and ubiquitination processes, but it is also enabled by upregulation [37] . r406 had a negligible effect on card9 expression in the naive macrophages. however, card9 was significantly upregulated in mers-cov-infected macrophages. in contrast to the mockinfected cells, inhibition of clr signaling by r406 significantly suppressed the degree of card9 activation. thus, the results revealed the importance of card9 as an essential adaptor to relay clr signaling in mers-cov-infected macrophages. mers-cov, the most virulent human cov identified so far, has posed an ongoing threat to public health worldwide. it has been recognized that the rapid viral replication in lung tissues, massive inflammation, and elevated proinflammatory cytokine and chemokine response collectively contribute to acute lung injury and underlie the high pathogenicity of this life-threatening human cov [2, 5, 34, 38] . the postmortem examination of mers-cov patient's lung tissues, which were obtained by needle sampling shortly after death, revealed severe pneumonia with diffuse alveolar damage and mixed inflammatory cell infiltration. immunostaining revealed numerous intra-alveolar macrophages and infiltrating t lymphocytes in the parenchyma [4] , which are consistent with the histopathological changes in lung tissues of mers-covinfected marmosets. it is notable that profound macrophage proliferation was the most prominent pathological change in the postmortem examination of lung tissues of fatal sars patients [39] . it was postulated that the proinflammatory cytokines and chemokines released from the proliferative alveolar macrophages play a prominent role in the pathogenesis of sars [40] [41] [42] . we have reported that mers-cov can productively infect human macrophages and trigger exuberant proinflammatory response [8] . it is conceivable that the proliferating macrophages in lung tissue of a mers patient and the dysregulated immune response in these cells may contribute significantly to the high pathogenicity of mers-cov. in this study, we sought to identify the cellular signaling pathway(s) that mediate the proinflammatory response in mers-cov-infected human macrophages. the initial observations from the effect of the pathway inhibitors and profiling of receptor activation hinted at the possible involvement of rlr and clr signaling in mers-cov-induced immune activation in macrophages. we then performed a series of experiments to characterize the role of rlr and clr, especially clr, in mers-cov-infected macrophages, with the intention to better understand the pathogenesis of human mers. the role of rlr and clr for mers-cov for eliciting immune activation was noted upon genetic depletion of the rlr adaptor mavs or the clr adaptor syk. the genetic depletion generally compromised or abolished the induction of cytokines and/or chemokines. the attenuated production of most proinflammatory mediators including mip-1α, rantes, ifn-γ, and ip-10 was replicated when rlr receptors (rig-i or mda5) or clr receptor (mincle) were depleted individually. however, the dampened inflammatory response was more prominent in adaptor depletion than receptor depletion, which could be ascribed to the fact that the activation signals from multiple receptors (eg, rig-i and mda5) converge at 1 adaptor (eg, mavs). upon mers-cov infection, multiple prrs were activated in the macrophages ( figure 2 ). therefore, depleting individual receptors was unable to entirely block the activation signals. the rlrs are ubiquitously expressed in many cell types and are among the first sensors to detect many viral infections. they are localized in the cytoplasm and recognize the genomic rna of double-stranded rna (dsrna) viruses and dsrna generated as the replication intermediate of single-stranded rna viruses. the rig-i and mda5 are inducible upon ifn treatment or virus infection. the activated rig-i or mda5 interacts with an essential rlr adaptor, mavs, initiating a signaling cascade that includes the activation of tbk1 and ikkε kinases, which subsequently phosphorylate and activate the transcription factor irf3 and nf-κb. in this study, we demonstrate that rig-i or mda5 were both induced in macrophages upon mers-cov infection. the engagement of the receptors and subsequent signaling transduction were manifested in sirna depletion of both receptors and their adaptor mavs, as well as tbk1/ikkε inhibition. thus, rlrs indeed recognize replicating mers-cov and mediate the proinflammation response in macrophages. recent studies have identified clrs as an important family of prrs that are involved in the induction of inflammation response to specific pathogens. dectin-1, dectin-2, and mincle are members of a family of c-type lectins that are typically expressed by dendritic cells and macrophages [17] . mincle is known to associate noncovalently with the adaptor molecule fcrγ. the phosphorylation of the adaptor serves to recruit syk and induces downstream nf-κb activation through the syk-card9-bcl10-malt1 complex. the in vivo genetic studies have revealed an essential role of clr-triggered card9 signaling in host protection. card9-deficient mice exhibited deregulated innate responses and failed to control infection of mycobacterium tuberculosis [43] . in addition, mincle was identified as a lipopolysaccharide-inducible protein in mouse macrophages and was transcriptionally upregulated after exposed to various stimuli and cellular stress [44] . in human monocytes, fungus infection elevated mincle expression [31] . in this study, we demonstrated that mers-cov replication upregulated mincle expression. the antibody blockage of mincle, sirna depletion of mincle, syk pharmacological inhibition, and card9 activation invariably highlighted the role of mincle for mediating the proinflammatory cascade in mers-cov-infected macrophages. in terms of cellular inflammatory response, 4 major family of prrs, including tlrs, rlrs, clrs, and nlrs, share overlapping ligand specificities and converge on common downstream pathways. the cooperative detection by different prrs has been reported in other dna and rna viruses [33, 45] and bacteria [46] . in rhinovirus-infected bronchial epithelial cells, the innate immune response requires a coordinated recognition, initially via tlr3 or trif and followed by rna helicases rig-i and mda5 [33] . our results indicate that clr and rlr signaling may be involved in mediating the immune activation in mers-cov-infected human macrophages. however, the possible crosstalk or coordination of different pathways has yet to be investigated. taken together, our study has advanced current knowledge of the recognition of human covs by cellular prrs. in agreement with previous literature on other human covs, our data revealed the important role of rlrs in mediating the proinflammatory response upon mers-cov infection. more important, our study further investigated the involvement of clrs in sensing mers-cov and provided the first evidence that clrs contributed to the recognition of mers-cov. in addition, we demonstrated that a significant proportion of mers-cov-triggered proinflammatory response was governed by mincle. in this regard, clrs may play an important role in modulating the pathogenesis of mers-cov. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. supplementary figure 1 . determination of 50% cytotoxic concentration (cc 50 ) of 3 pattern recognition receptor (prr) pathway inhibitors. monocyte-derived macrophages (mdms) were treated with the indicated concentrations of inhibitors and incubated at 37°c for 24 hours. the cells were then applied to mtt assay. the results are used for calculation of the cc 50 of each inhibitor. data are presented as mean ± standard deviation (sd) of triplicate wells of mdms from 3 different donors. isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study an acute immune response to middle east respiratory syndrome coronavirus replication contributes to viral pathogenicity protective and pathogenic functions of macrophage subsets disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in crohn's disease active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis signaling in innate immunity and inflammation pattern recognition receptors and inflammation differential roles of mda5 and rig-i helicases in the recognition of rna viruses rna viruses promote activation of the nlrp3 inflammasome through a rip1-rip3-drp1 signaling pathway the zinc-finger protein zcchc3 binds rna and facilitates viral rna sensing and activation of the rig-i-like receptors transmissible gastroenteritis virus infection induces nf-κb activation through rlrmediated signaling mda5 is critical to host defense during infection with murine coronavirus clec5a is critical for dengue-virus-induced lethal disease myeloid c-type lectin receptors in viral recognition and antiviral immunity c-type lectin mincle is an activating receptor for pathogenic fungus severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3. tank.tbk1/ikkepsilon complex middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response antagonism of the interferoninduced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways tetherin/bst-2 is essential for the formation of the intracellular virus-containing compartment in hiv-infected macrophages differentiated human airway organoids to assess infectivity of emerging influenza virus a functional variation in cd55 increases the severity of 2009 pandemic h1n1 influenza a virus infection identification and characterization of gldc as host susceptibility gene to severe influenza an inhibitor of the protein kinases tbk1 and ikk-ɛ improves obesity-related metabolic dysfunctions in mice the non-canonical iκb kinases ikkε and tbk1 as potential targets for the development of novel therapeutic drugs syk kinase signalling couples to the nlrp3 inflammasome for anti-fungal host defence cell death by pyroptosis drives cd4 t-cell depletion in hiv-1 infection mincle polarizes human monocyte and neutrophil responses to candida albicans murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 co-ordinated role of tlr3, rig-i and mda5 in the innate response to rhinovirus in bronchial epithelium pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology an interferon-gammarelated cytokine storm in sars patients comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity regulation of dectin-1-mediated dendritic cell activation by peroxisome proliferator-activated receptor-gamma ligand troglitazone middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis lung pathology of fatal severe acute respiratory syndrome immunopathogenesis of coronavirus infections: implications for sars multiple organ infection and the pathogenesis of sars dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice the adaptor molecule card9 is essential for tuberculosis control a novel lpsinducible c-type lectin is a transcriptional target of nf-il6 in macrophages innate immune sensing of modified vaccinia virus ankara (mva) is mediated by tlr2-tlr6, mda-5 and the nalp3 inflammasome mycobacterium paratuberculosis is recognized by toll-like receptors and nod2 supplementary figure 2 . inactivated middle east respiratory syndrome coronavirus (mers-cov) was unable to induce proinflammatory cytokines and chemokines. ultraviolet (uv) inactivation was performed by exposing the viruses to uv crosslinker for 10 minutes at 37°c. the inactivation was confirmed after inoculation in vero e6 cells. monocyte-derived macrophages (mdms) were infected with mers-cov or the uv-inactivated mers-cov at a multiplicity of infection (moi) of 2 or were mock infected. at 24 hours postinoculation, cells were harvested for detecting the expression levels of cytokines and chemokines by reverse transcription-quantitative polymerase chain reaction (rt-qpcr) assay. data are presented as mean ± standard deviation (sd) of mdms from 3 different donors.supplementary key: cord-321918-9jwma2y6 authors: xiu, siyu; dick, alexej; ju, han; mirzaie, sako; abdi, fatemeh; cocklin, simon; zhan, peng; liu, xinyong title: inhibitors of sars-cov-2 entry: current and future opportunities date: 2020-06-15 journal: j med chem doi: 10.1021/acs.jmedchem.0c00502 sha: doc_id: 321918 cord_uid: 9jwma2y6 [image: see text] recently, a novel coronavirus initially designated 2019-ncov but now termed sars-cov-2 has emerged and raised global concerns due to its virulence. sars-cov-2 is the etiological agent of “coronavirus disease 2019”, abbreviated to covid-19, which despite only being identified at the very end of 2019, has now been classified as a pandemic by the world health organization (who). at this time, no specific prophylactic or postexposure therapy for covid-19 are currently available. viral entry is the first step in the sars-cov-2 lifecycle and is mediated by the trimeric spike protein. being the first stage in infection, entry of sars-cov-2 into host cells is an extremely attractive therapeutic intervention point. within this review, we highlight therapeutic intervention strategies for anti-sars-cov, mers-cov, and other coronaviruses and speculate upon future directions for sars-cov-2 entry inhibitor designs. coronaviruses (covs) are enveloped positive-stranded rna viruses. they belong to the order of nidovirales and are classified into four genera: α, β, γ, and δ. 1 coronaviruses are animal viruses with circulating reservoirs in mammals and birds. for most coronaviruses, the lifecycle can be dissected into four steps, including viral entry, replication, assembly, and release. 2 until last year, six strains of coronaviruses have been identified that are pathogenic to humans. among them are cov-nl63, cov-oc43, cov-hku1, and cov-229e that could cause mild respiratory tract diseases. 3 however, two of the β-covs, the severe acute respiratory syndrome coronavirus (sars-cov), and the middle east respiratory syndrome coronavirus (mers-cov) have caused severe epidemics in the past. 4, 5 in april 2003, sars-cov was responsible for 8098 infections, with a fatality rate of ∼10% by the end of september 2003. 6 mers-cov emerged from its zoonotic reservoir in 2012 and infected 2494 people with a fatality rate of ∼34% by the end of 2019. 7 both outbreaks having such high fatality rates, highlight the need for surveillance of coronavirus emergence. while efforts for the development of antivirals against sars-cov or mers-cov are still in process, a new coronavirus (sars-cov-2) has emerged from an epicenter located in wuhan, china, in december 2019. 8 sars-cov-2 is highly contagious and has quickly spread in and beyond china. as of may 28, 2020, there have been more than 5 596 550 diagnosed cases around the world, with 353 373 confirmed deaths (figure 1 ). 9 the united states of america and brazil reporting the majority of the confirmed cases in the americas, with 1 658 896 and 391 222 cases, respectively. recently the genome of sars-cov-2 was determined, which revealed 80% identity with that of some sars-cov strains (gz02, bj01, tor2, sz3, pc4-227) and interestingly 96% identity to the bat coronavirus batcov ratg13. 11 the receptor-binding spike (s) protein is highly divergent from other covs and displays nucleotide sequence identities of 75% or less to all other previously described sars-covs. however, again, the new sars-cov-2 s protein shares 93.1% identity to the ratg13 s protein. 11 the glycoprotein or s protein is responsible for receptor recognition and viral entry into host cells. the spike protein can be divided into two domains; s1 is responsible for angiotensin-converting enzyme ii(ace2) recognition, the recently identified host cell receptor, and s2 mediates membrane fusion (figure 2 ). 12 structural alignment of sars-cov-2 s protein with sars-cov s protein shows that both s proteins are similarly with a root-mean-square deviation (rmsd) of 3.8 å over 959 cα atoms, while the s2 domain, responsible for membrane fusion, display the most substantial similarities with an rmsd of 2.0 å ( figure 2c ). engagement of the host cell receptor ace2 is important for viral entry; however, subsequent entry steps can vary and are cell-type specific. sars-cov can enter the host cell via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon ace2 binding. 13, 14 the clathrin-mediated pathway includes the s protein binding to ace2 and subsequent dynamin/clathrinmediated internalization of endosomal vesicles that maturate to late endosomes. within the late endosomes and lysosomes, acidification of the internalized endosomes and h + -dependent activation of the cellular cathepsin l proteinase takes place that cleaves and activates the s protein, therefore initiating viral fusion with the endosomal/lysosomal membrane (figure 3 ). in the case of sars-cov, cell culture studies revealed that the entry process is delayed with a lag phase of around 30 min, suggesting substantial maturation requirements. 15 in accordance with findings that mouse hepatitis coronavirus (mhv) and feline coronavirus (fcv) infections of hela cells are also heavily dependent on endosomal maturation, the clathrindependent entry and endosomal maturation are key to entry across coronaviridae. 16 for sars-cov-2, a recent study also confirms that virus can use host cell receptor cd147 to gain entry into the host cells besides ace2. 17 in addition to the endosome-mediated entry pathway, host proteases also play critical roles in the nonendosomal entry of coronaviruses. 5 host proteases such as the transmembrane protease serine 2 (tmprss2) and tmprss11d can cleave the s protein at the s1/s2 cleavage site (figure 2 ) to prime and activate the s protein for membrane fusion during the nonendosomal pathway. 18 a recent study also confirms that tmprss2 expressing veroe6 cells are highly susceptible to sars-cov-2 infection, highlighting the importance of tmprss2 in the replication cycle. 19 mers-cov can also be activated by furin (serine endoprotease) to initiate the nonclathrin mediated membrane fusion event. 20 interestingly, in the new sars-cov-2 s figure 3 . entry model of sars-cov-2 into the host cell. binding of the s1 domain within the spike (s) protein to the cellular ace2 receptor triggers conformational changes in the s2 domain that results in internalization and subsequent membrane fusion ((a) endosomal/clathrindependent pathway). the endosomal pathway is facilitated by a low ph and the ph-dependent cysteine protease cathepsin l. alternatively, sars-cov-2 can enter the cell via the nonendosomal/clathrin-independent pathway (b). during this route, ace2 recognition by the sars-cov-2 s protein (comparable to route a) is followed by additional activation/cleavage of the s protein into s1 and s2 domains by cell membrane-associated serine proteases such as tmprss2 and tmprss11d. the figure was prepared with https://biorender.com/. . genome organization of sars-cov-2. genome organization of the sars-cov-2 and location the central genes within the genome (numbers in brackets). 29 the figure was prepared with https://biorender.com. protein, additional amino acid insertions at the s1/s2 cleavage site results in an "rrar" furin recognition site absent in sars-cov s protein. 21 this polybasic insertion sequence has possible implications for the sars-cov-2 replication cycle and its increased pathogenicity. indeed, polybasic furin sites have been observed in hemagglutinin (ha) proteins of highly virulent avian and human influenza viruses, and similar furinlike processing events are also observed for other rna viruses such as ebola virus and marburg virus, human immune deficiency virus (hiv), and flaviviruses. 22 to activate the s protein for membrane fusion with the cellular membrane, structural rearrangements within the s2 domain are required. two heptad repeats, hr1 (dark blue in figure 2 ) and hr2 can interact to form a six-helix bundle (6-hb), a common postfusion structure shared by all type i viral glycoproteins, to bring viral and cellular membranes in close proximity. additionally, the s2 domain contains a membrane interacting domain or fusion peptide that is exposed upon specific triggers such as receptor binding or low endosomal ph. to date, three membrane interacting regions with hostmembrane destabilizing effects have been identified in the sars-cov s protein: two conserved sequences across coronaviridae, with residues 798−815 23 and residues 864− 886, 24 both c-terminal positioned at the second cleavage site in the s protein termed s2′ at arg 797 and a less conserved third region with membrane disordering properties residues 770−788. 25 once in the host cell, the viral particle uncoats and is ready for transcription and translation. 26 the first orf codes for approximately 67% of the genome and is separated into open reading frames (orf) 1a and 1b (figure 4 ). orf1a and orf1b are translated into polyproteins pp1a (4382 amino acids) and pp1ab (7073 amino acids) that are processed by 3-c-like protease (3clpro) and papain-like protease (plpro). the processing of these polyproteins produces a variety of nonstructural proteins (nsps), including rna-dependent rna polymerase (rdrp) and helicase, to catalyze viral genome replication and protein synthesis. 27 the remaining orfs in the sars-cov-2 genome code for accessory and structural proteins. following further assembly, the mature virions are transported to the cell surface in vesicles and released by exocytosis. 28 any protein involved in the replication process could be a potential target for the development of antiviral agents. as mentioned previously, zhang et al. determined the fulllength genome sequence of sars-cov-2 and revealed that the virus was very similar (89.1% nucleotide similarity) to a group of sars-like coronaviruses. 30 simultaneously, shi et al. found that sars-cov-2 shares 96% sequence identity at a wholegenome level to a bat coronavirus, and importantly, they confirmed that sars-cov-2 utilizes the same cell entry receptor, ace2, as sars-cov. 11 recently, the cryo-em structure of full-length human ace2 bound to the rbd of the sars-cov-2 was solved, providing an important structural foundation for intervention strategies. 31 conservation analysis also revealed that the rdrp and the 3clpro are highly conserved between sars-cov-2 and sars-cov. 32 therefore, it is widely accepted that sars-cov-2 would behave similarly to sars-cov with regards to viral entry and replication. being the first step in the infection process, the entry of pathogenic viruses into susceptible cells is an extremely attractive intervention point. as with other well-known viruses, such as hiv-1 and ebola, viral entry of coronaviruses is a complex multiple-step process with numerous interactions and processing points that, in theory, could be targeted. 33 in this review, we summarize case studies and highlight efforts in designing entry inhibitors against sars-cov, mers-cov, and other coronaviruses that can provide important information to combat the current sars-cov-2 outbreak. ■ host cell ace2 receptor recognition by the sars-cov-2 spike (s) as a promising antiviral target binding of the sars-cov-2 spike (s) protein to the cellular ace2 receptor represents the first encounter (in both the endosomal and nonendosomal pathway) in the viral replication cycle and provides prophylactic intervention opportunities. 34 sars-cov-2 spike (s) recognizes with its rbd the cellular ace2 receptor with high affinity (k d = 14.7 nm) 12 as judged by surface plasmon resonance (spr) interaction analysis, and intervention at the rbd-ace2 interface can potentially disrupt infection efficiency. recently the cryo-em and crystal structures of sars-cov-2's rbd in complex with ace2 were solved and provide important structural guidance for inhibitor design ( figure 5 ). 31 the interface can be divided into three contact sides, mainly polar in nature, and is similar to the sars-cov-ace2 complex. 35, 36 in this structure, an extended loop of the rbd contacts an arch-like helix α1 of the proteolytic domain (pd) of ace2 via an n-(cluster 1), central (cluster 2), and cterminal (cluster 3) portion ( figure 5 purple box). additionally, helix α2 and loop 3−4 (connecting β3 and β4) of ace2 provide limited contacts. at the n terminus of α1 (cluster 1), gln498, thr500, and asn501 of the rbd interact via hydrogen bonds with tyr41, gln42, lys353, and arg357 from ace2. the middle portion (cluster 2) of the rbd loop contacts via tyr453, the ace2 pd at residue his34. at the c terminus of α1 (cluster 3), gln474 of rbd contacts gln24 of ace2, and phe486 of rbd interacts with met82 of ace2 through van der waals interactions ( figure 5 ). the structures of the rbds from the sars-cov-2-ace2 complex and the sars-cov-ace2 complex are quite similar, with an rmsd of 0.68 å over 139 cα atoms ( figure 6 ). 31 a comparison of both structures, however, also highlights some deviations at all three clusters summarized in table 1 . these deviations need to be considered carefully during the inhibitor design process. in addition, helix α2 and the loop 3−4 connecting β3 and β4 are also contributing to the interface. sars-cov-2 s protein monomer was obtained from pdb 6vsb and rbd-ace2 complex from pdb 6vw1. boxes 1, 2, and 3 highlight polar clusters 1, 2, and 3, respectively. ■ targeting the rbd peptide analogues, monoclonal antibodies, and protein chimeras as rbd inhibitors. both sars-cov and sars-cov-2 use ace2 to gain entry into the host cells. as such, this critical interaction can be blocked to stop viral entry. 19 this strategy was first demonstrated by hsiang et al. using a biotinylated enzyme-linked immunosorbent assay (elisa), hsiang et al. reported the disruption of the sars-cov s protein-ace2 interaction by small peptides. from a total of 14 designed peptides, peptides sp-4, sp-8, and sp-10 ( figure 7 and table 2 ) significantly blocked the interaction of the sars-cov s protein with ace2 with ic 50 values of 4.30, 6.99, and 1.88 nm, respectively. additional immunofluorescence assay (ifa) studies with s-protein-pseudotyped retroviruses, revealed a novel mechanism of infection inhibition of vero e6 cells by sp-10. 37 38 in light of the successful inhibition of sars-cov with this linked peptide, a similar strategy could potentially be effective against the new sars-cov-2. the recently solved cryoem structure of sars-cov-2 in complex with the human ace2 receptor can provide a structural rationale for the peptide design. 31 monoclonal antibodies (mab) have potential applications for diagnosis, prophylaxis, and treatment of established and evolving viral infections. 39−41 prabhakar et al. isolated specific antibodies from b cells in xenomouse immunized with sars-cov. further investigation revealed that several abs directly react with the rdb domain, and a combination of two abs (4d4 and 3c7) displayed near-complete neutralization efficiency as compared to a single ab application. 42 two additional potent monoclonal antibodies, mab201 and mab68, could be isolated from transgenic mice immunized with the soluble ectodomain of sars-cov s protein. 43 this mab could bind sars-cov s protein directly with affinities of 34 nm (mab 201) and 83 nm (mab 68) as judged by spr analysis. mice that received 40 mg/kg of mab 201 or mab 68 before sars-cov infection showed complete protection from reinfection of lung tissues. 43, 44 cross-reactivity of mabs is highly desirable, and dimitrov et al. identified the human mab m396 that binds sars-cov with high affinity (k d = 20 nm). 45 mice that received 200 μg of m396 were nearly completely protected from infection by urbani and gd03 virus strains. 46 m396 did compete with the sars-cov receptor, ace2, for binding to the rbd, suggesting that m396 inhibits sars-cov-ace2 binding as the predominant mechanism of action. 45 however, sars-cov-2 showed some complexities for rbd directed antibodies. for instance, wrapp et al. tested crossreactivity of three antibodies, including s230, m396, and 80r, against sars-cov-2 rbd. despite the partly high degree of structural homology between the sars-cov-2 and sars-cov, no binding to the sars-cov-2 rbd was detected for any of the three antibodies at the concentration of 1 μm. it can be concluded that sars-cov antibodies will not necessarily be cross-reactive for sars-cov-2. 12 in a different approach, hu et al. generated a novel chimeric recombinant protein recently by connecting the extracellular domain of human ace2 to the fc region of human immunoglobulin igg1. these chimeric constructs displayed high-affinity for the sars-cov-2 and sars-cov rbd binding and potently neutralized sars-cov and sars-cov-2 in vitro, with ic 50 values between 0.8 and 0.1 μm, respectively. these journal of medicinal chemistry pubs.acs.org/jmc perspective recombinant chimeras also showed cross-reactivity and could have, therefore, useful applications for diagnosis, prophylaxis, and treatment of sars-cov-2. 47 using the velocimmune platform, pascal et al. generated several human, noncompeting monoclonal antibodies that target mers-cov s protein and block viral entry into host cells. among them, two antibodies, regn3051 and regn3048, can significantly inhibit mers-cov pseudoparticles, with ic 50 values of 460 and 180 pm, respectively. 48 in addition, regn3051 and regn3048 showed a good performance in a novel transgenic mouse model, which was developed by replacing the mouse dpp4 coding sequence with that encoding human dpp4. results suggested that both regn3051 and regn3048 were able to potently reduce mers-cov specific rna levels in the lungs at a 200 μg per mouse dose compared with the isotype control antibody. at the 20 μg dose, regn3051 was more effective at decreasing mers-cov rna levels compared with regn3048 at the same dose. 48 recently, in the common marmoset model of mers-cov infection, de wit et al. tested the prophylactic and therapeutic efficacy of regn3051 and regn3048. data demonstrated that their protection might be more effective in a prophylactic treatment process rather than treatment of mers-cov. 49 in the latest attempt, chen et al. identified sars-cov-2 rbd specific antibodies from samples of 26 recovered covid-19 patients using an rbd-specific elisa binding study. among them, 311mab-31b5 and 311mab-32d4 effectively neutralized pseudovirus entry, with ic 50 values of 0.0338 and 0.0698 μm, respectively. 50 recently, in an elisa based (cross)reactivity assay, assessing antibody-containing supernatants of a collection of 51 sars-s hybridoma's derived from immunized transgenic h2l2 mice that encode chimeric immunoglobulins, wang et al. identified a chimeric mab 47d11 that targets rbd. 47d11 exhibited cross-neutralizing activity of sars-cov-s protein and sars-cov-2-s protein pseudotyped vsv infection with ic 50 values of 0.19 and 0.57 μm, respectively. 51 brouwer et al. used cross-sectional blood samples from three pcr-confirmed sars-cov-2-infected individuals to screen for binders to a soluble prefusion-stabilized s protein of sars-cov-2 using an elisa-based approach. all three blood samples did bind to the prefusion-stabilized s protein and prompted subsequent sorting of sars-cov-2 s proteinspecific b cells for mab isolation. nineteen nabs could be identified that target a diverse range of antigenic sites on the s protein and showed remarkable picomolar inhibiting activities with the two most potent ic 50 values of 0.010 and 0.007 μg/ ml (cova1-18 and cova2-15, respectively) against live sars-cov-2 virus. 52 large antibody libraries are crucial in response to rapidly emerging pathogens. using eight large phage-displayed vh, scfv, and fab libraries and panning against the rbd of the sars-cov-2, li et al. identified an exceptional potent (k d to rbd of 160 pm as judged by biolayer interferometry) mab igg1 ab1 that competes with ace2 in vitro and protected transgenic mice expressing hace2 from high-titer intranasal sars-cov-2 challenge. 53 in two different assays using replication-competent sars-cov-2 in a microneutralizationbased assay, 100% neutralization at <400 nm, and in a luciferase reporter gene assay, an ic 50 of 200 nm was reported. moreover, transgenic mice expressing human ace2 administrated with 0.3 mg of ig1 ab1 prior intranasal infection with sars-cov-2 did not show any detectable replicationcompetent virus, demonstrating the preventive effect of igg1 ab1. 53 small molecules targeting the rbd. besides peptides, mab, and protein chimeras, small molecules are still the preferred modality for a drug. this is due to improved pharmacokinetics, stability, and dosage logistics compared to proteins or peptides. 54, 55 in addition, small molecules have advantages compared to peptides/proteins regarding dissemination logistics in remote areas and the high expenses of peptide/protein production. 54, 55 to identify small molecule entry inhibitors against the sars-cov s protein, sarafianos et al. screened a chemical library composed of 3000 compounds according to lipinski's rule of five 56 and identified an oxazole-carboxamide derivative, ssaa09e2 (1, table 3), that blocks the binding of the rbd of 57 lundin et al. screened a library of 16 671 diverse compounds and found a small molecule inhibitor, k22 (2), which was able to inhibit hcov-229e with an ic 50 value of 0.7 μm and cc 50 value of 110 μm. studies for mechanism showed that k22 targeted a very early step in the hcov-229e life cycle and may interact with viral particles, thus inactivating their binding. 58 ■ targeting the cellular receptor peptide analogues as ace2 inhibitors. human angiotensin-converting enzyme (ace) is a highly glycosylated type i integral membrane protein and has been identified as a fundamental regulator of the renin−angiotensin system (ras) in humans and is an important target in regulation of blood pressure homeostasis. ace2 is a human homologue of ace. 59 it contains a single zinc-binding catalytic domain, which is 42% similar to the human ace active region. 60 ace2 can catalyze the cleavage of angiotensin i into angiotensin 1-9, and angiotensin ii into the vasodilator angiotensin 1-7 and its organ-and cell-specific expression also suggests a role in the regulation of cardiovascular and renal function and fertility. 60 ace2 is a functional receptor to the sars-cov during viral entry, and recent research demonstrated that sars-cov-2 also utilizes ace2 for infection. 61 however, ace2 cannot be inhibited by ace inhibitors, so there is an urgent need to develop specific ace2 inhibitors that would prevent infection by both sars-cov and sars-cov-2. one of the first efforts to target the ace2 receptors was documented by liu et al. using a novel epitope assembling assay, liu et al. identified linear b-cell immuno-cross-reactive epitopes of sars-cov s protein by synthesizing 22 longer peptides. five of these peptides showed serologically highly cross-reactivity in all tested sars patients sera. among them, peptide s 471-503 could significantly block the binding of rbd to ace2. s 471-503 , derived from the s1 fragment ( figure 7 and table 2 ) could target ace2, and showed antiviral activity against sars-cov infection in vitro, with an ec 50 value of 41.6 μm, providing an important basis to explore the antiviral potential of s 471-503 against sars-cov-2. 62 another peptide derived from the rbd, rbd-11b, located in s1 of the sars-cov s protein, is crucial for binding to the host cells ace2 receptor 62 (figure 7 and table 2 ). given the vital role of this motif, meyer et al. confirmed the binding to ace2 of a synthesized peptide mimicking this region ( 438 ykyryl 443 ) with a k d of around 46 μm. moreover, rbd-11b displays no toxicity, as judged by an mtt (3-(4,5)dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumbromide) cell proliferation assay, on veroe6 cells. in addition, rbd-11b showed antiviral activity to hcov-nl63 at a peptide concentration of 7 mm in caco2 cells, which also used ace2 as a functional receptor. 63 constrained peptides are receiving more attention in the drug development field, combining the best attributes of antibodies and small molecules. linear peptides are often highly flexible and unstructured in solution, only forming structures upon target binding. this can sometimes reduce the affinity of such peptides for their target by an entropic penalty mechanism. however, stabilization methods such as cyclization or hydrocarbon stapling can increase the physicochemical characteristics and drug-like properties while negating the entropic penalty of binding and having a positive impact on affinity. 64 using a constrained peptide library displayed on filamentous phages, ladner et al. identified several peptides inhibiting ace2 function with the most potent being dx600 (table 2) . dx600, an n-terminal acetylated and c terminal amidated peptide, was a potent ace2 peptide inhibitor with an ic 50 value of 10 nm and a k i value of 2.8 nm. dx600 did not inhibit ace activity and thus is specific to ace2. in addition, dx600 was chemically stable and not hydrolyzable by ace2. 65 although it is not clear whether dx600 can inhibit coronavirus, as an effective ace2 inhibitor, anticoronavirus tests should be conducted in the future. small molecule as ace2 inhibitors. as discussed previously, peptide and constrained peptide inhibitors have inherent caveats concerning their use as drugs. 64 therefore, screening for small molecule inhibitors, guided by information gleaned from the previous studies is the next logical step. a virtual screen targeting the ace2 catalytic site with around 140 000 compounds combined with a molecular docking approach led to the identification of naae (n-(2-aminoethyl)-1 aziridine-ethanamine) (3, table 3 ). 3 showed a dosedependent inhibition of ace2 catalytic activity with an ic 50 value of 57 μm and a k i of 459 μm. despite its micromolar potency in inhibiting a sars-cov pseudotyped virus, cytotoxicity data is not available to date. 66 chloroquine (4) currently has applications for malaria and amoebiasis treatment. interestingly, nichol et al. showed that chloroquine could also block the interaction of rbd of sars-cov to ace2 under cell culture conditions with an ed 50 value of 4.4 μm. 67 recently, wang et al. found that 4 blocked sars-cov-2 virus infection, with an ic 50 value of 1.13 μm and a cc 50 > 100 μm in vero e6 cells. 68 chloroquine possibly increases endosomal ph required for virus/cell fusion as well as impairs with the terminal glycosylation of the cellular ace2 receptor, thereby reducing the affinity of sars-cov/sars-cov-2 to ace2. besides its antiviral activity, chloroquine may synergistically enhance its antiviral effect with immunemodulating activity in vivo. 68 at present, chloroquine is carried out in clinical research in china for the treatment of sars-cov-2 (chictr2000029609). 69 hydroxychloroquine (5) is an analogue of chloroquine, which shares the same mechanism of action as chloroquine but displays a more tolerable safety profile. 70 71 recent studies suggest that 4 and 5 could cause ventricular arrhythmias, 72 qt prolongation, 72,73 retinopathy, 74 and other cardiac-related toxicity, which may pose a particular risk to critically ill patients. although both show antiviral activity, safety, and effectiveness, they require further clinical research. turner et al. identified that the sars-cov receptor, ace2, undergoes proteolytic shedding, releasing an enzymatically active ectodomain during viral entry. 75 further research identified that a disintegrin and metalloproteinase (adam17) is responsible for shedding regulation of ace2. inhibiting adam activity with the adam-specific inhibitor gw280264x (6) reduced shedding of ace2 at 1 nm against sars-cov. 76 another enzyme involved in ace2 shedding is tace (tnf-α converting enzyme, a member of the adam family). two tace inhibitors, tapi-0 (7) and tapi-2 (8), reduced ace2 shedding against sars-cov, with ic 50 values of 100 and 200 nm, respectively. 75 perhaps the most promising small molecule described to date is the very potent ace2 inhibitor mln-4760 (9). 9 can inhibit the catalytic activity of ace2 with an ic 50 of around 440 pm. 77 the crystal structure of the apo and 9 bound ace2 complex revealed a significant subdomain movement of the nterminal and c-terminal subdomains of ace2 upon 9 binding. this movement is important to position critical residues to stabilize the bound inhibitor. its high potency makes 9 a very attractive candidate for sars-cov-2 interference; however, no antiviral coronavirus data is available at this time. milewska et al. synthesized several polymer-based compounds showing prominent anticoronaviral activity. among them, a cationically modified chitosan derivative, n-(2hydroxypropyl)-3-trimethylammonium chitosan chloride (htcc, 10), and hydrophobically modified htcc (hm-htcc, 11) were found that could inhibit hcov-nl63 replication. for both tested polymers, their ic 50 values were relatively low in llc-mk2 cells, amounting to ∼50 nm for 10 and ∼230 nm for 11. cc 50 values were ∼0.8 and ∼1 μm for 10 and 11, respectively. 78 recent research showed that 10 and 11 blocked the interaction of hcov-nl63 with its ace2 receptor and thus interfered with the process of viral entry. 79 despite the availability of many compounds with inhibitory effects on ace2, the corresponding admet data in a preclinical model is not available. regardless, direct inhibition of ace2 is probably not a viable therapeutic modality, however. this is due to its important normal physiological roles, in addition to its lung injury protective role in acute respiratory distress syndrome from a variety of causes, including sars-cov infection. 80, 81 as such, directly inhibiting ace2 as an antiviral strategy appears to be physiologically unsound, and virally targetted blockers of its interaction with the sars-cov/sars-cov-2 s protein hold greater promise. membrane fusion is a crucial step in the mers/sars infection cycle in both described pathways (see section 1). within the endosomal/clathrin-dependent route, internalized viral particles need to fuse with the endosomal membrane to escape the endosomal/lysosomal environment. this is achieved via a conformational change of the s protein (s2 domain) within the acidic milieu followed by membrane fusion activation by the host protease cathepsin l. membrane fusion is also essential during the nonendosomal/clathrin-independent route to fuse with cellular membranes facilitated by host protease cleavage of the s protein by cell membrane-associated proteases such as tmprss2. 19 in conclusion, the s2 domain of the sars-cov s protein and host proteases such as 84, 85 hr1 and hr2 can interact with each other to form a 6-hb to bring viral and cellular membranes close (for exact location, see figure 2 ). on the basis of this requirement, bosch et al. obtained peptides corresponding to region hr2 within the hr. hr2-8 displayed in an infection inhibition assay with pseudotyped sars-cov s protein in vero cells an ec 50 value of 17 μm (figure 7 and table 2 ). 84 moreover, hr2-8 demonstrated concentration-dependent inhibition of hcov-nl63 infection with an ic 50 value of 0.5 μm and a cc 50 value of 20 μm. 86 on the basis of these initial results, further development of the hr2-8 peptide is necessary to develop a more potent human coronaviruse (hcov) peptide inhibitor. similarly, ngai et al. obtained three hr derived peptides, including hr1-a, gst-removed-hr2, and hr2 peptide, with remarkable inhibitory activity against sars-cov ( figure 7 and table 2 ). virus entry inhibition studies suggested that hr1-a, derived from the hr1 region, had an ec 50 value of 1.61 μm. gst-removed-hr2 peptide and hr2 peptide, derived from the hr2 region, had ec 50 values of 2.15 and 0.34 μm, respectively. 87 hr2p, spanning residues 1251−1286 in hr2 domains, could effectively inhibit mers-cov infection and s protein-mediated membrane fusion (figure 7 and table 2 ). this study indicates that hr2p could specifically inhibit mers-cov in vero cells, with an ic 50 value of ∼0.6 μm and a cc 50 value of >1000 μm. hr2p also demonstrated high selectivity, as indicated by its high selectivity index (si > 1667). importantly, the introduction of arg, lys, or glu residues into the hr2p peptide increased stability, solubility, and anti-mers-cov activity. 88 to improve the stability, solubility, and antiviral activity of hr2p, channappanavar et al. designed and synthesized an hr2p analogue named hr2p-m2. hr2p-m2 strongly blocked s protein-mediated cell−cell fusion in a dose-dependent manner at ic 50 values of 0.55 μm in vitro. in vivo, hr2p-m2 intranasal administration to ad5/ hdpp4 transgenic mice protected them from mers-cov infection and reduced the lung viral titers by more than 1000fold. moreover, combination treatment with ifn-β was demonstrated to enhance the protective effect. 89 the development of a drug with broad-spectrum hcov inhibitory activity is increasingly becoming an attractive approach. xia et al. found that the ek1 peptide showed pan-cov fusion inhibitory activity against multiple hcovs ( figure 7 and table 2 ). 90 further investigation revealed that ek1 directly reacts with the hr1 region and can competitively inhibit viral 6-hb formation. the pseudovirus assay suggested that the antiviral activity of ek1 against hcov-oc43, hcov-nl63, and hcov-229e infection with ic 50 values of 1.81, 6.02, and 3.35 μm, respectively. in vitro cytotoxicity assay determined that ek1 is not cytotoxic at concentrations up to 1 mm. mice that received 5 mg/kg of ek1 were nearly completely protected from infection by hcov-oc43 and 200 μg of ek1 against mers-cov infection. recently, this team found that ek1 could also potentially inhibit sars-cov-2 with an ic 50 value of 2.38 μm in pseudovirus assay and an ic 50 value of 0.19 μm in fusion inhibitory assay. 91 to improve the inhibitory activity of ek1 against sars-cov-2, they conjugate the cholesterol molecule to the ek1 peptide and found that a new peptide, ek1c4, exhibited highly potent inhibitory activity inhibit sars-cov-2 s-mediated membrane fusion and pseudovirus infection with ic 50 values of 1.3 and 15.8 nm, the cc 50 of ek1c4 was 5 μm, and the selectivity index was >136. in the oc43-infected mouse model, mice that received 0.5 mg/kg of ek1c4 were nearly completely protected from infection by hcov-oc43. these data suggested that ek1c4 could be used for inhibition and treatment of infection by currently circulating sars-cov-2. 92 mers-5hb, a polypeptide derived from the hr1 and hr2 region, was synthesized by gong et al., and affinity analysis demonstrated a low k d value of 0.24 nm, and an ic 50 value of 1 μm against mers-cov and cc 50 > 50 μm. hr derived peptides is a highly promising strategy for viral fusion inhibition. successful hr peptides have been used in the past to block entry of other virus families such as the hiv with the gp41 derived peptide fuzeon (t20), the only approved fusion inhibitor for hiv-1 treatment to date. 93 therefore, hr derived peptides highlight a promising strategy for inhibitor development combating the new sars-cov-2. xia et al. reported that two peptide-based membrane fusion inhibitors, 229e-hr1p and 229e-hr2p (figure 7 and table 2 ), targeting the hcov-229e s protein hr1 and hr2 domains, could competitively inhibit the viral autologous 6-hb formation and inhibit hcov-229e s protein-mediated viruscell membrane fusion with ic 50 values of 5.7 and 0.3 μm, respectively. moreover, neither 229e-hr1p nor 229e-hr2p had significant cytotoxicity to huh-7 and a549 cells at concentrations up to 1000 μm. in addition, 229e-hr2p potentially inhibited pseudotyped and live hcov-229e infection with ic 50 values of 0.5 and 1.7 μm, respectively. 94 the s2 domain is the most conserved motif between the sars-cov and the new sars-cov-2 s protein. 92 it represents an ideal immunogen for the generation of a novel or repurposing sars-cov s2 domain targeting mabs with cross-reactive potential. 95 sasazuki et al., for example, could successfully isolate the human mab 5h10 from immunized kunming (km) mice. 5h10 displayed an anti-sars-cov neutralizing activity of around 5 μg/ml. cell fusion assays indicate that 5h10 can inhibit viral fusion and entry rather than viral attachment to the surface of host cells or cleavage of the s protein. consequently, the s protein of sars-cov might be the direct target of 5h10; however, further studies are required to confirm this hypothesis. 96 tan et al. identified mab 1a9 (ic 50 value between 25 and 50 μg/ml), an anti-sars-cov s2 domain mab, that binds to a conserved loop region between the hr1 and hr2 domains of the s2 domain. 97 tsunetsugu-yokota et al. found that antibody skot20 can inhibit sars-cov with an ec 50 value of 5 μg/ml in vero e6 cells sars-cov. mutational studies indicate that skot20 restrict conformational changes within the s2 domain, essential for viral entry. 98 99 on the basis of this approach, they identified two small molecules, tgg (12, table 4 ) and luteolin (13) , that can bind avidly to the sars-cov s2 protein and inhibit viral entry of sars-cov into vero e6 cells with ic 50 values of 4.5 and 10.6 μm, respectively. cytotoxicity assay showed that the cc 50 of 12 and 13 were 1.08 and 0.155 mm, respectively. therefore, the selectivity index of 12 and 13 were 240.0 and 14.62, respectively. further acute toxicity suggested that the 50% lethal doses of 12 and 13 were ∼456 and 232.2 mg/kg, respectively. these indicated that these small molecules could be used at relatively high concentrations in mice. 98 quercetin (14), an analogue of 13, also showed antiviral activity against sars-cov, with an ic 50 arbidol (16), a broad-spectrum drug, has been licensed for decades in russia and china against influenza by binding to the ha protein to block the viruses−cell fusion. 103 recently, wang et al. identified that 16 efficiently inhibited sars-cov-2 virus infection in vitro with an ic 50 value of 4.11 μm, a cc 50 value of 31.79 μm, and an si of 7.73. 104 vankadari compared protein sequence analysis and found that a small region of the s2 domain (aa947−aa1027) of the sars-cov-2 spike glycoprotein resembles that of the influenza virus h3n2 ha. so the mechanism of 16 was to target the sars-cov-2 spike glycoprotein and blocked its trimerization, which may inhibit host cell adhesion and hijacking. 105 in january 2020, in wuhan, china, a clinical pilot trial conducted with 36 patients with sars-cov-2 virus infection received 400 mg 16 three times a day for 9 days; 31 untreated sars-cov-2 patients served as a control group. in this trial, patients with 16 showed a tendency to decrease viral load as determined by rt-pcr and reduced mortality (0% vs 16%), as compared to the control group. 106 the hr regions of sars-cov and sars-cov-2 s protein share a high degree of conservation, and the described small molecules as fusion inhibitors can have potential applications in inhibiting sars-cov-2 fusion. indeed, targeting virus surface protein is a promising antiviral strategy, whether inhibiting rbd or s2 domain. during clathrin-dependent viral entry, the host cellular cathepsin l protease plays a key role in infection efficiency by activation of the s protein into a fusogenic state to escape the late endosomes, and cathepsin l (lysosomal endopeptidase) cleavage is believed to expose a hydrophobic fusion peptide essential to initiate membrane fusion. 107 in light of its vital role in the sars cov infection cycle, cathepsin l is a desirable target to interfere with virus−cell entry. 83 cathepsin l consists of a pro-and a mature-domain. in a low ph milieu, the pro-domain is autocatalytically cleaved to obtain the papain-like folded mature-domain consisting of an n-terminal helical domain and a c-terminal β-sheet domain (figure 8 ). 108 a well conserved cys-his-asn triad in the active site is crucial for substrate binding and catalysis. in light of its importance in the sars-cov-2 replication cycle, cathepsin l is a highly desirable target that will be described in the following section. 109 teicoplanin is a glycopeptide antibiotic, with applications in the treatment of serious infections caused by gram-positive bacteria such as streptococcus and staphylococcus aureus. 110 interestingly, teicoplanin was shown to block the entry of sars, mers, and ebola virus by specifically inhibiting the cathepsin l activity. 111 more recently, zhang et al. showed that teicoplanin could also block the entry of the new sars-cov-2 pseudoviruses with an ic 50 value of 1.66 μm. as a routinely used clinical antibiotic, teicoplanin could be potentially used immediately to combat the current sars-cov-2 outbreak. 112 small molecules as cathepsin l inhibitors. human cathepsin l plays numerous critical roles in diverse cellular settings associated with human diseases. 113 previous studies also highlighted the feasibility of targeting this cysteine endopeptidase with small molecules with implications for possible intervention strategies of sars-cov-2 infection. 113 a high-throughput screen (hts) of a 1000-compound library that resulted in the identification of mdl28170 (17 , table 4 ) by bates et al., and in an antiviral activity assay, 17 specifically inhibited cathepsin l-mediated substrate cleavage and blocked sars-cov viral entry, with an ic 50 value of 2.5 nm and ec 50 value in the range of 100 nm. however, despite its potent inhibitory activity, no cytotoxicity data for 17 is currently available. 83 two small molecules, cid 16725315 (18) and cid 23631927 (19) , were reported by diamond et al. as viral entry inhibitors of the sars-cov. in a cathepsin l inhibition assay, 19 could block cathepsin l with an ic 50 value of 6.9 nm, while 18 showed slightly weaker potency with an ic 50 value of 56 nm. interestingly, besides inhibiting sars-cov, compound 19 (ec 50 value of 273 nm) showed some inhibition activity for ebola virus infection (ec 50 value of 193 nm) of human embryonic kidney 293t cells. importantly, 19 did not show any sign of toxicity to human aortic endothelial cells at 100 μm. this data offers a new promising point for the treatment of sars and ebola virus infections. 114 recently, in a cell-based assay screen of ∼14 000 compounds, ssaa09e1 (20) was identified that could specifically bind to the cathepsin l proteinase and interference sars-s protein during viral entry, with an ic 50 value of 5.33 μm. in a pseudotype-based assay in 293t cells, the ec 50 value of 20 was around 6.4 μm, and no cytotoxicity was detected below 100 μm. 57 using sars-cov entry assays, zhou et al. screened 2100 cysteine protease inhibitors with confirmed activity to inhibit human cathepsins. among them, k11777 (21) demonstrated the most robust activity. results demonstrated that 21 blocked sars-cov pseudovirus entry at an ic 50 value of 0.68 nm while no toxicity was observed, cc 50 value >10 μm. interestingly, for other coronaviruses, 21 showed broadspectrum antiviral activity with ic 50 values of 1.48, 6.78, and 46.12 nm against hcov-229e, hcov-nl63, and mers-cov, respectively. 115 inhibitors of cell membrane-associated tmprss2. either the endosomal cysteine proteases cathepsin l or the cell membrane-associated serine protease tmprss2 can facilitate sars-cov virus entry into host cells by cleavage of the viral s protein. 19 this cleavage exposes fusion-competent motifs known as fusion peptides, and importantly, for sars-cov, the interference of both proteases is required for efficient inhibition of virus replication. 19 matsuyama et al. identified camostat (22 , table 4 ), a commercially available serine protease inhibitor that can efficiently prevent sars-cov infections at 10 μm by inhibiting tmprss2 activity. however, even at high concentrations (100 μm) of 22, the inhibition of viral entry via sars s protein-mediated cell fusion never exceeded 65% (inhibition efficiency), indicating that despite the inhibition of tmprss2, 35% of virus entry takes place via the endosomal cathepsin pathway. therefore, they examined the activity of pseudotyped viruses when treated with a combination of (23,25)trans-epoxysuccinyl-l-leucylamindo-3methylbutane ethyl ester (est, a cathepsin inhibitor) and 22. the results suggested that simultaneous treatment with est and 22 remarkably blocked infection (>95%). 116 similarly, poḧlmann et al. reported that 22 could prevent the viral entry of sars-cov-2. importantly, full inhibition efficiency was attained when treated with both 22 and e-64d (a cathepsin inhibitor). both studies indicate that sars-cov and sars-cov-2 enter cells via a similar mechanism, showing the potential of 22 as a promising candidate for further development as a sars-cov-2 treatment. 19 inhibitors of the furin cleavage site in the coronavirus spike proteins. elevated levels of furin expression were able to facilitate mers-cov pseudovirion infection, and viral entry could be reduced by furin sirna silencing. 20 decanoyl-rvkr-chloromethylketone (23, dec-rvkr-cmk), a furin inhibitor, was shown to block mers-cov s protein-mediated entry as well as virus infection, with journal of medicinal chemistry pubs.acs.org/jmc perspective an ic 50 value of 75 μm in hek-293t cells. furthermore, when cathepsin inhibitor camostat was used in combination with 23, a significant inhibition in infectivity was characterized compared to camostat alone. 20 recently, bestle et al., showed that the potent peptidomimetic inhibitor mi-1851 (24) could prevent proteolytic processing of the s protein from sars-cov-2 by endogenous furin in hek293 cells. however, no antiviral data is available for 24 yet. 117 the peculiar furin-like cleavage site (s1/s2-site in figure 2 ) in sars-cov-2 that is absent in the sars-cov and other sars-like covs indicates that furin inhibitors could play a significant role in blocking the viral entry process. 117, 118 ■ host factor inhibitors sars-cov-2 cell entry also relies on host cell factors. therefore, these host cell factors can play an essential role as targets for sars-cov-2 inhibition. 119 chlorpromazine (25 table 5 ) is an antipsychotic drug developed for the treatment of schizophrenia. it has also been reported to inhibit the infection of hepatic c virus (hcv), 120 mouse hepatitis virus (mhv-2), 27 and alphavirus. 121 (28), an abelson kinase signaling pathway inhibitor that could inhibit abelson tyrosine−protein kinase 2 (abl2) to block mers-cov virion fusion with endosomal membranes with an ic 50 value of 10 μm. 28 showed no cytotoxic effects in vero cells at 100 μm. 123, 124 another abl inhibitor, dasatinib (29) , was active against both mers-cov and sars-cov, with ic 50 values of 5.4 and 2.1 μm, respectively. 125 on the basis of an hts assay using cytopathic-effect ( phenotypic screening methods are usually used to identify firstin-class drugs without knowing the actual target and mechanism of action of the drug, while target-based screening identifies best-in-class drugs. 127−129 although the phenotypic screening approach often is limited in terms of capacity compared to in silico target-based screening, it can have advantages in identifying cell-active compounds providing information on drug solubility or cell uptake. 127−129 many drugs, especially natural products, have an unknown mechanism of action but were shown to inhibit coronavirus entry. 130 hsiang et al. screened a library of 121 chinese herbs using a biotinylated enzyme-linked immunosorbent assay to search for active compounds that could potentially inhibit sars-cov s protein binding to ace2. further studies identified emodin (31 , table 5 ), the active component from polygonum multiflorum and rheum officinale, could block the interaction of sars-cov s protein to ace2, with an ic 50 value of 200 μm in an s protein-pseudotyped retrovirus assay using vero e6 cells. however, the mechanism of action of 31 still needs to be determined. 131 sarafianos et al. found that ssaa09e3 (32), a benzamide derivative, could prevent sars-cov virus−cell membrane fusion in pseudotyped-based and antiviral-based assays, with an ic 50 value of 9.7 μm, but a cc 50 value of 20 μm indicates additional unknown cellular targets. 57 out of an hts, ve607 (33) was identified using a phenotype-based screen from a 50 240 structurally diverse small-molecule compound library. pseudotype virus entry assay suggested ve607 can specifically inhibit sars-cov virus entry into cells with an ec 50 value of 3 μm and inhibited sars-cov plaque formation with an ic 50 of 1.6 μm. 132 a similar hts approach was employed by zhang et al. for screening a compound library consisting of 727 structurally diverse small molecules. eighty-four compounds were identified with significant anticoronavirus potential. further studies revealed that 51 compounds inhibited virus entry, while 19 others interfered with viral replication. 133 natural products should, however, be considered with caution due to their unknown mechanism of action and possible toxic side effects. the recent sars-cov-2 outbreak, with its high fatality rate, has raised global concerns and was declared as a global pandemic by the who. the number of infections continues to rise, and numerous research groups around the globe have prioritized the identification and development of new covid-19 treatments. still, there are no effective treatments to date. viral entry is the first step in the viral life cycle and represents an attractive intervention point by blocking the coreceptor interaction or the virus−cell membrane fusion event. sars-cov-2 and other coronaviruses have similar infection mechanisms. this is especially true for sars-cov and cov-nl63, which share the same human ace2 receptor crucial for viral entry. therefore, already developed inhibitors against known hcovs could potentially be used to combat sars-cov-2. these efforts identified a large number of inhibitors, including peptides, antibodies, small-molecule compounds, and natural products with anticoronavirus activity. although many inhibitors demonstrated efficacy in inhibiting coronavirus virus infection, no specific prophylactic or postexposure therapy is currently available for hcovs. one of the main reasons causing this is that most of the potenial agents were not adequately evaluated for in vitro and in vivo studies. most drugs are in the preclinical stage and stopped in animal models due to poor bioavailability, safety, and pharmacokinetics so that few entered human trials. in light of the urgency of the current outbreak, repositioning of already approved drugs is increasingly becoming a promising approach, especially with toxicity and safety data in hand. the most effective measure to prevent viral diseases is vaccination. coronavirus vaccine development mainly focused on s protein, and some of them reported can inhibit sars, 134−136 and mers. 137 although vaccination strategies were developed in the context of previous epidemics, no vaccine for sars-cov-2 infections is yet available. since the recent sars-cov-2 outbreak, research groups around the world are now stepping up to develop vaccines targeting sars-cov-2, and vaccine research routes include nucleic acid vaccines, viral vector vaccines, inactivated vaccines, and recombinant protein vaccines. typical vaccine development is time, resource, and financially consuming, although this pandemic has created initiatives that hope to speed the development of a sars-cov-2 vaccine. even the most optimiztic views regarding an effective sars-cov-2 vaccine being created are at least one year away. even after creation, other hurdles for the sars-cov-2 include global implementation and distribution, and different strategies for containing this contagion should be explored simultaneously as the vaccine efforts. in addition to small-molecule inhibitors, monoclonal antibodies, and vaccine development, convalescent sera from sars-cov-2 survivors (convalescent-phase sera) is an additional option for covid-19 treatment. passive immunization was well established for viral infection prophylaxis. 138 by metaanalysis of studies about the 1918 influenza, h1n1 influenza epidemic demonstrated that early treatment of convalescent blood products decreased the risk ratio caused by pneumonia from 37% to 16%. 139 nevertheless, the appropriate titer of the convalescent-phase sera antibody remains to be determined, which was required for therapeutic efficacy to inhibit sars-cov-2. research carried out with mers-cov suggested that sera from patients recovering from infections did not contain sufficient antibody titers for therapeutic use. 140 recent initiatives such as the governmental (usa) operation warp speed (ows) to support the development, manufacturing, and distribution of covid-19 vaccines, therapeutics, and diagnostics or the accelerating covid-19 therapeutic interventions and vaccines (activ) public− private partnership coordinated by the national institutes of health (nih) are crucial milestones in a coordinated effort to accelerate and prioritize the development of the most promising vaccines and treatments. initiatives like these that bridge government, academia, and industry should also be continued past the current covid-19 crisis so that we can respond to future novel outbreaks rapidly and adequately. severe acute respiratory syndrome (sars): a year in review middle east respiratory syndrome coronavirus (mers-cov) in silico design of antiviral peptides targeting the spike protein of sars-cov-2 covid-2019) situation reports; world health organization covid-19) cryo-em structure of the 2019-ncov spike in the prefusion conformation clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to npc1+ endolysosomes is a rate-defining step coronavirus cell entry occurs through the endo-/ lysosomal pathway in a proteolysis-dependent manner middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein s2 domain with characteristics of a viral fusion peptide genetic analysis of the sars-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion identification and characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated coronavirus spike protein drug targets for corona virus: a systematic review mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of eps15 a decade after sars: strategies for controlling emerging coronaviruses severe acute respiratory syndrome coronavirus 2 isolate wuhan-hu-1 a new coronavirus associated with human respiratory disease in china structural basis for the recognition of the sars-cov-2 by full-length human ace2 learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov pathology and pathogenesis of severe acute respiratory syndrome role of changes in sars-cov-2 spike protein in the interaction with the human ace2 receptor: an in silico analysis cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 structure of sars coronavirus spike receptor-binding domain complexed with receptor design and biological activities of novel inhibitory peptides for sars-cov spike protein and angiotensin-converting enzyme 2 interaction identification of critical determinants on ace2 for sars-cov entry and development of a potent entry inhibitor faccin-galhardi, l. c. antibody therapy for the control of viral diseases: an update monoclonal antibodies for prophylaxis and therapy of infectious diseases. expert opin. emerging drugs monoclonal antibodies against viruses and bacteria: a survey of patents human monoclonal antibodies to sars-coronavirus inhibit infection by different mechanisms development and characterization of a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody that provides effective immunoprophylaxis in mice generation and characterization of human monoclonal neutralizing antibodies with distinct binding and sequence features against sars coronavirus using xenomouse structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies potent neutralization of 2019 novel coronavirus by recombinant ace2-ig. biorxiv. 2020 pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylactic and yherapeutic efficacy of mab treatment against mers-cov in common marmosets human monoclonal antibodies block the binding of sars-cov-2 spike protein to angiotensin converting enzyme 2 receptor a human monoclonal antibody blocking sars-cov-2 infection rapid selection of a human monoclonal antibody that potently neutralizes sars-cov-2 in two animal models therapeutic potential of small molecules and engineered proteins what are the drugs of the future? experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings novel inhibitors of severe acute respiratory syndrome coronavirus entry that act by three distinct mechanisms targeting membrane-bound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus angiotensin-converting enzyme 2: cardioprotective player in the renin-angiotensin system? hypertension a novel angiotensin-converting enzyme−related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1−9 angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target screening and identification of linear b-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (sars)-associated coronavirus using synthetic overlapping peptide library a hexapeptide of the receptor-binding domain of sars corona virus spike protein blocks viral entry into host cells via the human receptor ace2 using peptidomimetics and constrained peptides as valuable tools for inhibiting protein−protein interactions novel peptide inhibitors of angiotensin-converting enzyme 2 structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor chloroquine is a potent inhibitor of sars coronavirus infection and spread remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia conduction disorder and qt prolongation secondary to long-term treatment with chloroquine chloroquine and hydroxychloroquine retinopathy-related risk factors in a turkish cohort tumor necrosis factor-alpha convertase (adam17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (sars-cov) receptor, angiotensin-converting enzyme-2 (ace2) tace antagonists blocking ace2 shedding caused by the spike protein of sars-cov are candidate antiviral compounds ace2 x-ray structures reveal a large hingebending motion important for inhibitor binding and catalysis szczubialka, k. novel polymeric inhibitors of hcov-nl63 htcc: broad range inhibitor of coronavirus entry angiotensin-converting enzyme 2 protects from severe acute lung failure the discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice cell-based antiviral screening against coronaviruses: developing virus-specific and broad-spectrum inhibitors inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry severe acute respiratory syndrome coronavirus (sars-cov) infection inhibition using spike protein heptad repeat-derived peptides structures and polymorphic interactions of two heptad-repeat regions of the sars virus s2 protein inhibition of human coronavirus nl63 infection at early stages of the replication cycle fusion core structure of the severe acute respiratory syndrome coronavirus (sars-cov): in search of potent sars-cov entry inhibitors protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection a pan-coronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein inhibition of sars-cov-2 infection (previously 2019-ncov) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion identification of a novel inhibitor against middle east respiratory syndrome coronavirus peptide-based membrane fusion inhibitors targeting hcov-229e spike protein hr1 and hr2 domains sars-cov-2) based on sars-cov immunological studies fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (sars) coronavirus s protein neutralizes the virus in a rhesus macaque sars model substitution at aspartic acid 1128 in the sars coronavirus spike glycoprotein mediates escape from a s2 domain-targeting neutralizing monoclonal antibody a single amino acid substitution in the s1 and s2 spike protein domains determines the neutralization escape phenotype of sars-cov a small-molecule fusion inhibitor of influenza virus is orally active in mice small molecules that bind the inner core of gp41 and inhibit hiv envelope-mediated fusion small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov structural basis of influenza virus fusion inhibition by the antiviral drug arbidol the anti-influenza virus drug, arbidol is an efficient inhibitor of sars-cov-2 in vitro arbidol: a potential antiviral drug for the treatment of sars-cov-2 by blocking trimerization of the spike glycoprotein clinical features of 69 cases with coronavirus disease 2019 in wuhan, china cathepsin l functionally cleaves the severe acute respiratory syndrome coronavirus class i fusion protein upstream of rather than adjacent to the fusion peptide cysteine proteases: modes of activation and future prospects as pharmacological targets caught in the act: the crystal structure of cleaved cathepsin l bound to the active site of cathepsin l serruys-schoutens, e. clinical evaluation of teicoplanin for therapy of severe infections caused by gram-positive bacteria glycopeptide antibiotics potently inhibit cathepsin l in the late endosome/lysosome and block the entry of ebola virus, middle east respiratory syndrome coronavirus (mers-cov), and severe acute respiratory syndrome coronavirus (sars-cov) teicoplanin potently blocks the cell entry of 2019-ncov a review of small molecule inhibitors and functional probes of human cathepsin l a small-molecule oxocarbazate inhibitor of human cathepsin l blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney 293t cells protease inhibitors targeting coronavirus and filovirus entry simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry boẗtcher-friebertshaüser, e. tmprss2 and furin are both essential for proteolytic activation and spread of sars-cov-2 in human airway epithelial cells and provide promising drug targets a multibasic cleavage site in the spike protein of sars-cov-2 is essential for infection of human lung cells perspectives for repurposing drugs for the coronavirus disease 2019 hepatitis c virus entry depends on clathrin-mediated endocytosis inhibitors of alphavirus entry and replication identified with a stable chikungunya replicon cell line and virus-based assays testing of middle east respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry coronavirus s protein-induced fusion is blocked prior to hemifusion by abl kinase inhibitors abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection saracatinib inhibits middle east respiratory syndrome-coronavirus replication in vitro phenotypic vs. target-based drug discovery for first-in-class medicines overview of recent strategic advances in medicinal chemistry new techniques and strategies in drug discovery natural products and their derivatives against coronavirus: a review of the non-clinical and preclinical data emodin blocks the sars coronavirus spike protein and angiotensinconverting enzyme 2 interaction identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics a screen of the nih clinical collection small molecule library identifies potential anti-coronavirus drugs severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region sars coronavirus spike polypeptide dna vaccine priming with recombinant spike polypeptide from escherichia coli as booster induces high titer of neutralizing antibody against sars coronavirus prospects for a mers-cov spike vaccine passive immunization. prim care meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol key: cord-345827-yo3uq03v authors: antiochia, riccarda title: developments in biosensors for cov detection and future trends date: 2020-10-28 journal: biosens bioelectron doi: 10.1016/j.bios.2020.112777 sha: doc_id: 345827 cord_uid: yo3uq03v this review summarizes the state of art of biosensor technology for coronavirus (cov) detection, the current challenges and the future perspectives. three categories of affinity-based biosensors (abbs) have been developed, depending on their transduction mechanism, namely electrochemical, optical and piezoelectric biosensors. the biorecognition elements include antibodies and dna, which undergo important non-covalent binding interactions, with the formation of antigen-antibody and ssdna/oligonucleotide-complementary strand complexes in immunoand dna-sensors, respectively. the analytical performances, the advantages and drawbacks of each type of biosensor are highlighted, discussed, and compared to traditional methods. it is hoped that this review will encourage scientists and academics to design and develop new biosensing platforms for point-of-care (poc) diagnostics to manage the coronavirus disease 2019 (covid-19) pandemic, providing interesting reference for future studies. coronaviruses are a large family of viruses that usually cause mild to moderate upper-respiratory tract illnesses, like the common cold (peiries, 2012) . there are hundreds of coronaviruses, most of which circulate among animals, such as pigs, camels, bats and cats. sometimes those viruses jump to humans, a process called "spillover", and cause a disease. to date, seven known human coronaviruses (hcov) have been identified that affect the human population (table 1) . four of them (hcov-229e, hcov-nl63, hcov-oc43 and hcov-hku1) cause only mild disease, being responsible of approximately one-third of common flu infections in humans (lim, 2016; fung, 2019; pene, 2003; vijgen, 2005; van der hoeck, 2007; walsh, 2007) . three of them can cause more serious, even fatal, disease: sars coronavirus (sars-cov), mers coronavirus (mers-cov) and the novel sars coronavirus (sars-cov-2). mers-cov causes the middle east respiratory syndrome (mers). it is transmitted from an animal reservoir in camels. it was identified in september 2012 and continues to cause sporadic and localized outbreaks, mostly in the middle east region (assiri, 2013) . the third novel coronavirus, which emerged recently in this century, is called sars-cov-2, for its genetic similarities with sars-cov (paudel et al., 2020; wu et al., 2020) . it causes coronavirus disease 2019 , which emerged in china in december 2019 and was declared a global pandemic by the world health organization (who) on march 11, 2020 (udugama et al., 2020; wang et al., 2020; who, 2020; zhu et al., 2020b) . the covid-19 clinical manifestations can range from very mild symptoms to life-threatening conditions huang et al., 2019) . some people may be asymptomatic or have only flu-like symptoms, while others may experience worsened symptoms, such as interstitial pneumonia, possibly caused or accompanied by a thrombophilic vasculitis in the lung (boraschi, 2020) . all coronaviruses are enveloped positive single-stranded rna viruses with a diameter of about 100 nm and a "crown-like" characteristic surface, responsible for their name ("corona" in latin means "crown"). there are four types, depending on the sequence of entire viral genomes: α-, β-, γand δcoronavirus. sars-cov, mers-cov and sars-cov-2 belong to the β-coronavirus family . genoma analysis of sars-cov-2 sequences showed that the complete genome sequence recognition rates of sars-cov and bat sars coronavirus were 79.5% and 96%, respectively (wang j o u r n a l p r e -p r o o f et al., 2020) . therefore, the bat origin of sars-cov-2 seems to be the most probable hypothesis (chan et al., 2013; hu et al., 2015) . compared to sars-cov and mers-cov, with a mortality rate of 10% and 35% respectively, the novel sars-cov-2 has higher transmissibility and infectivity but lower mortality rate (huang, 2019; , although it led to over 1,150,000 deaths globally, as of 24 october 2020. since mers and covid-19 are highly contagious diseases with the potential to cause a pandemic, in absence of a specific vaccine or effective therapeutic drugs, it is of extreme importance to find rapid and accurate detection methods for control and prevention of virus spread. the key-point to control a virus outbreak is usually the isolation of individuals presenting mild infection symptoms through a strict quarantine, thus blocking the infection transmission process. during the current covid-19 pandemic, the centers for disease control and prevention (cdc) established the requirement of at least two negative test results in a row, at least 24 hours apart, during the quarantine period before a person is declared recovered. people with more severe symptoms are normally sent to hospitals, where they receive inappropriate treatments, as no specific antiviral drugs have been developed, with the consequent overloading of the hospitals which become the main places of spread of the virus (signorelli et al., 2020) . another important issue are the asyntomatic individuals who were diagnosed with sars-cov-2 infections but without any relevant clinical symptoms. increasing evidence has shown that asymptomatic individuals can efficiently spread the virus, causing serious difficulties in the control of the epidemic . therefore, early diagnostic tests, based on sensitive, specific, accurate and rapid methods, are crucial for successful outbreak containment. an adequate detection system is essential in helping to stop or decrease the spread of a virus outbreak before human and economic consequences become devastating. there are two main biorecognition strategies for detection of virus diseases: i) detection of viral nucleic acid (dna and rna); ii) detection of specific viral biomarkers, such as antigens or antibodies generated against the virus by the patient's immune system response (boonham et al. 2014; ozer, 2020) . recent traditional methods of detection of these analytes include polymerase chain reaction (pcr) (shen et al., 2020) and enzyme-linked immunosorbent assay (elisa), which can directly detect dna/rna and antibodies/antigens, respectively. both methods show high sensitivities but present some disadvantages, as they require virus isolation and the use of sophisticated laboratory equipment. they are also difficult to use at the point-of-care (poc), requiring well-trained staff, expensive instruments and time-consuming processes. moreover, some viruses are hard or impossible to cultivate (krejcova et al., 2015) . biosensors may represent a valid alternative, as they offer a rapid detection of viral diseases with high sensitivity and selectivity. a typical biosensor consists of three components: a biological element, a transducer for converting the recognition process into a quantitative signal, and an electronic system for amplification and signal processing, as schematized in figure 1 . on the basis of the biological element being used, biosensors can be divided into four classes: enzymatic, antibody/antigen-based, nucleic acid/dna-based and whole cells-based (kawamura and miyata, 2016) . depending on the transducer type being used, biosensors can be classified as: i) electrochemical biosensors (ronkainen and halsall, 2010; thèvenot et al., 2001) , in turn divided into amperometric, potentiometric, fet (field effect transistor)-based (mazarine de moares and kubota, 2016; vu and chen, 2019) and impedimetric biosensors; ii) optical biosensors (long and zhu, 2013) ; iii) thermal biosensors; iv) piezoelectric biosensors (skladal, 2016) . the classification of these biosensors is represented in figure 1 . additionally, another classification can be made on the basis of the biorecognition principle: i) catalytic biosensors (typically with enzymes and cells); ii) affinity-based biosensors (abbs) (typically with dna, antibodies and aptamers) (kawamura and miyata, 2016) . as schematized in figure 2 , the principle of the biocatalytic role is the conversion of the analyte (a), during the chemical reaction by the biological element (b), to form a product (p), able to generate a signal measurable by a transducer. in case of the bioaffinity role, the analyte (a) is bound specifically and selectively by the biological element (b) to form a complex (ab), detectable by a transducer. abbs are considered the most suitable biosensors for virus detection, especially for the reversible interaction between analyte and biological element, allowing the biodevice reuse (antiochia et al., 2015; antiochia et al., 2016; pejcic et al., 2006) . among the abbs, biosensors employing antibodies or antigens as biological elements are referred as "immunosensors". on the basis of the detection principle, abbs can be divided into two groups: "label-free", based on the direct measurement of the signal produced during the biochemical reaction, and "label-based or labelled" biosensors, based on the indirect measurement of the signal generating from a specific label, which makes them highly sensitive. label-based biosensors can be, in turn, subdivided into two main formats, competitive and non-competitive ("sandwich" format), depending on their mechanism, as shown in figure 3 . the peculiar characteristics of the abbs biosensors allow them to complement current methods of screening and monitoring for an early warning of a viral disease outbreak, especially when in situ and real-time analysis is required. thanks to the recent progress in electronics, the abbs biosensors can be miniaturized as lab-on-chip or handheld devices for on-site monitoring (lafleur, 2016; zhu et al., 2020a) . moreover, the recent development of nanotechnology provides a powerful tool to improve the performances of the abbs. nanomaterials have been largely used as signal amplifiers to improve the sensitivity of the biosensors, thanks to their excellent conductivity and extraordinary photoelectrochemical properties (holzinger et al., 2014; mokhtarzadeh et al., 2017; zhang et al, 2009; mujawar et al., 2020) . the objective of this review is to address the developments of abbs for coronavirus detection. the review covers papers that have been published in the last 15 years and is structured into three main sections, depending on the type of the biosensor transduction mode. another section has been specifically dedicated to the current methods of detection of sars-cov-2, with particular attention to the biosensing devices, as most of the cov research today is focused on covid-19 management. the electrochemical transduction shows several advantages compared to other transduction methods, such as low cost, high sensitivity, ease of miniaturization for poc use and relatively simple instrumentation. biosensors involving amperometric detection usually employ an electroactive label, as both antibody/antigen and dna hybridization reactions do not generate a significant signal on their own. many of the studies reported in literature employ ferro/ferricyanide, as redox probe. the current signals arising from non-specific adsorption of proteins or other biomaterial and the biofouling of the electrode surface represent the main limitations of this type of biosensor. for this reason, a great deal of effort has to be devoted to control the surface structure, especially for measurements in complex matrices, such as blood (thevenot et al., 2001) . one common strategy to prevent non-specific binding (nsb) is the use of blocking reagents, such as bovin serum albumin (bsa), gelatin and casein, which occupy all the remaining nsb sites after the adsorption of the coated protein (balcer et al., 2003) . however, when using a complex biological sample such as serum, these blocking solutions might not be enough. chemical modification and functionalization of the electrode surface is generally performed to suppress the nsb and, at the same time, to enhance the biocompatibility of the electrode surface towards antibodies or proteins and the biosensor sensitivity. thiol terminated polyethylene glycol (peg) has become quite popular for reducing nsb, by forming self-assembled monolayers j o u r n a l p r e -p r o o f (sam) on the metal coated sensor electrode, providing also functional groups for surface immobilization (contreras-naranjo et al., 2019). the first electrochemical biosensor for sars-cov detection was developed by ishikawa and coworkers (2009) . it is a fet-based immunosensor, where the change in conductance generated by the antigen-antibody binding can be measured and correlated to the analyte concentration. the virus antigen nucleocapsid n protein has been used as sars biomarker. instead of conventional antibodies, antibody mimic proteins (amps) have been utilized as affinity binding agents. these amps can be easily produced in vivo and are smaller and more stable than normal antibodies. the fet sensor has been opportunely modified with a fibronection-based protein (fn) as amp capture agent to selectively bind the antigen n protein. the exposed gate region of fet-based immunosensor was modified with in 2 o 3 nanowires on a si/sio 2 substrate in order to improve the immobilization of the amps and the signal transducing. at the working ph=7.4, the n proteins are positively charged and therefore their binding on a p-type channel causes depletion of charge carriers (holes) and a consequent decrease in conductance. the so-developed platform was able to detect the n protein at sub-nanomolar concentrations, with a sensitivity comparable to current immunological detection methods, but with a shorter time and without the need of labelled reagents. in 2019, layqah and eissa (2019) described the first amperometric immunosensor for mers-cov virus detection. in particular, the spike protein s1 was utilized as mers biomarker. the biosensor's working principle is an indirect competition between the free virus in the sample and immobilized mers-cov recombinant spike protein s1 for a fixed antibodies concentration added to the sample. the immunosensor was realized on an array electrodes system to allow the simultaneous detection of different types of human coronavirus. the surface of the carbon electrodes was modified with gold nanoparticles (aunps) in order to enhance the electrochemical properties of the electrode, providing a higher surface area and a faster electron transfer rate. successively, mers-cov and hcov antigens were immobilized onto the aunps/carbon electrode. the non-specific adsorptions were minimized by incubating the electrode in a bsa solution, in order to block the unreacted aldehyde groups and the free gold surface. the antibody concentration to be used for incubation of the antigen-modified electrode and the binding time were carefully optimized. the optimum conditions resulted to be 10 µg/ml and 20 minutes for antibody concentration and binding time, respectively. the current signal was obtained with square wave voltammetry (swv), by measuring the peak current of the ferro/ferricyanide redox probe (label), properly added to the solution. when the antibodies bind to the immobilized antigens, a decrease of the swv peak current is clearly observed, because of the "coverage" of the electrode surface by the antibody molecules. as a direct consequence, a decrease of both electron transfer efficiency and current is registered. the immunosensor showed a good linear 7 response from 0.001 to 100 ng/ml and from 0.01 to 10.000 ng/ml for mers-cov and hcov, respectively, and a very high sensitivity, with a detection limit of 0.4 and 1.0 pg/ml for mers-cov and hcov, respectively, definitely lower values than those obtained with elisa method (1 ng/ml) (chen et al., 2015) . the selectivity against virus proteins such as flua and flub resulted very good, attesting no cross-reactivity of the proposed biosensor. moreover, the possibility of use of the biosensor for simultaneous multiplex detection of different types of cov was confirmed, by mixing the two proteins mers-cov and hcov on the same electrode surface. as for the sensor stability, the biosensor resulted to be stable for about 2 weeks, showing only 2% current decrease during this period. finally, the proposed immunosensor was successfully tested in artificial nasal samples spiked with mers-cov and hcov antigens, showing good recovery percentages and a good reproducibility (rsd 3-6%). although several types of optical transducers are used in affinity-based biosensing, fluorescence and surface plasmon resonance (spr) are undoubtedly the most validated and assessed transduction techniques. in particular, fluorescence measurements are of great interest thanks to their high sensitivity (stefan et al., 2000) . in most fluorescent-based immunosensors, molecules called fluorochromes are used to label the biomolecules and generate the fluorescent signal, as no fluorescence properties are usually exhibited by antigens and antibodies. spr technique can be considered the most advanced and developed optical label-free technology in recent biosensing. spr transducers present interesting features compared to other physicochemical transducers, allowing real-time monitoring of bioanalytes, without the need of labelling. the biotransducer immobilized onto the sensor disk surface interacts with the analyte producing a local increase in the refractive index at the metal surface which promotes an spr signal shift (lu et al., 2000; abdulhalim et al., 2008) . unfortunately, also spr based affinity biosensors present the drawback of the nsb phenomena onto the spr disk, which can affect the accuracy of the measurements in biological fluids. another problem is the difficulty of immobilization of large bioreceptor molecules because of their steric hindrance. the key issue to overcome these drawbacks is a proper sensor chip modification (sarano et al., 2015) . more importantly, spr based biosensors show very high sensitivities, comparable to elisa immunoassay (gomara et al., 2000) . the first use of a spr biosensor was carried out by chen and coworkers for sars-cov morphological study (chen et al., 2005) . it was shown that spr was able to verify that the n-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer. the first spr-based biosensor for the diagnosis of sars was developed by park et al. (2009) . the biorecognition element was the antigen sars-cov membrane-envelope (scvme) protein, genetically fused to a gold binding polypeptide (gbp), able to bind a gold surface. the fusion proteins were directly self-assembled onto the spr gold chip, thus realizing a specific sensing platform for anti-scvme antibodies. the fusion protein-coated spr chip showed a low detection limit of 200 ng/ml, high selectivity and a fast response time of 10 minutes. selectivity studies were also carried out, using mouse igg as negative controls, showing very low spr responses by the nsb of mouse igg, thus attesting no significant cross-reactivity of the proposed spr immunosensor. in the same year, huang et al. (2009) described the first spr fluorescence fiber-optic immunosensor for detection of sars-cov nucleocapsid protein n in human serum. nucleocapsid protein n is one of the virus early expressed protein which can be detected one day after infection. therefore, it is an important biomarker for an early diagnosis and for an accurate prevention of the virus spread. the biosensor combines the properties of a "sandwich immunoassay" with the localized surface plasmon (lsp) technique. monoclonal antibodies against n (anti-n1) protein were immobilized on elisa plates as capture antibodies for n protein and polyclonal anti-n antibodies were used as detection antibodies. at the beginning of the assay, a rapid increase in fluorescence signal is registered as a large number of lsp coupled fluorescence (lspcf) probes migrate into the evanescent field interaction region, close to the fiber core surface. however, some n proteins (target antigens) are captured by the immobilized anti-n1 protein antibodies (capture antibodies) on the fiber core surface and therefore not all lspcf probes in the interaction region bind to n proteins, diffusing out of the interaction region, thus causing a decrease of the fluorescence intensity. a linear dependence between the fluorescence signal and n protein concentration was obtained over the range from 1 pg/ml to 10 ng/ml. the detection limit of the biosensor was found to be 1 pg/ml, much lower that the value reported for conventional elisa assay (37.5 pg/ml) (he et al., 2005) . a similar lod value of 1.56 pg/ml was only obtained with a chemiluminescence enzyme immunoassay (cleia) based system (fujimoto et al., 2008) . the presented lspcf fiber-optic biosensor presents many interesting and useful features, as it measures the fluorescence signal close to the reaction region resulting in a significant increase of the fluorescence efficiency (chang et al 1996) , showing also a high specificity, thanks to the sandwich immunoassay configuration. a fluorescence immunosensor has been realized by weng and neethirajan (2018) piezoelectric detection is based on the principle that frequency variations of a piezoelectric quartz crystal (pqc) correspond to mass changes, as a result of an affinity interaction event, such as antibody-antigen interaction or dna hybridization (holford et val., 2012) . in a general way, a piezoelectric biosensor can be constructed by immobilising a receptor (antibody, nucleic acid, etc.) onto the surface of a pqc and monitoring the frequency changes, due to the binding of the specific ligand (antigen, nucleic acid, etc.). the increased mass, associated with the biorecognition reaction, results in a decrease of the oscillating frequency. although initially in 1990's some researchers have evaluated that the detection limit with the piezoelectric method is inferior compared to electrochemical and optical detectors (ivnitski et al., 1999) , more recently the large number of articles appeared in literature clearly demonstrated that piezoelectric immunosensors are one of the most sensitive analytical instruments developed to date, especially for detection of a wide range of viruses, being capable of detecting antigens in the picogram range. moreover, this type of device has the potential to detect antigens in the gas phase as well as in the liquid phase without the need of a label. the first piezoelectric immunosensor reported in literature for coronavirus detection was published by zuo et al. (2004) , regarding an immunosensor for sars-cov detection in sputum. the horse policlonal antibodies against sars-cov were immobilized onto a pqc surface. the detection of the antigen was achieved by spraying it in the form of an aerosol via ultrasonic oscillation. in particular, the antigen powder was dissolved into the sputum of a healthy person and successively the solution was sprayed into the aerosol. the frequency shift obtained was proportional to the antigen concentration in the range 0.6-4 μg/ml. the biosensor showed very good reproducibility and stability, as it can be reutilized 100 times without a significant lack of activity, remaining stable for more than 2 months, if stored at 4-6°c. a second piezoelectric immunosensor, based on microcantilever technology, was realized by velanki and ji (2006) for feline coronavirus detection. feline coronavirus is prevalent in the cat population causing a deadly disease, called feline infectious peritonitis. the fip (feline infectious protein) type i virus antigen was used as biomarker. the silicon microcantilever surface was coated with a thick sio 2 layer and then modified by immobilization of fip virus type i polyclonal antibodies. the deflection amplitudes of the microcantilever resulted in proportion to the fip i antigen injected into the fluid cell. the proposed biosensor allowed to detect fip i with a detection limit of 0.1 µg/ml. current methods used for screening and diagnosis of novel covid-19 are based on three different approaches: sars-cov-2 antigen detection in nasopharyngeal secretions through molecular biology techniques, computed tomography, and sars-cov-2 antibody analysis in serum using immunoassay methods (carter et al., 2020) . among the molecular techniques, reverse transcription polymerase chain reaction (rt-pcr), a well documented technique used in medicine for around 20 year to detect genetic information, has been endorsed for clinical diagnosis of sars-cov-2 by both the who and the us cdc. many laboratories have developed real time rt-pcr assays, which have several advantages over traditional rt-pcr, such as high specificity for sars-cov rna, as they use internal probes as well as amplification primers, high sensitivity, with consistent detection limits of between 1 and 10 sars-cov rna copies per reaction, fast reaction time and reduced risk of contamination in the laboratory, as real-time pcr assays operate as closed systems. however, a number of false-negative and false-positive results have been reported . false-negative results can arise from poor sample collection or degradation of the viral rna during shipping or storage. application of appropriate assay controls that identify poor-quality samples can help avoid most false-negative results. the most common cause of false-positive results is contamination with previously amplified dna. the use of real-time rt-pcr helps mitigate this problem by operating as a contained system. a more difficult problem is the cross-contamination that can occur between specimens during collection, shipping, and aliquoting in the laboratory. liberal use of negative control samples in each assay and a well-designed plan for confirmatory testing can help ensure that laboratory contamination is detected and that specimens are not inappropriately labeled as sars-cov positive. in addition, any positive specimen should be retested in a reference laboratory to confirm that the specimen is positive. technique where the amplification is conducted at a single temperature and does not need specialized laboratory equipment. tests for covid-19 with rt-lamp are still being assessed in clinical settings. both molecular methods have the known drawback to give information only if the patient is currently infected and are not suitable for poc testing (carter et al., 2020; udugama et al., 2020) . due to false negative and positive results of rt-pcr, computed tomography (ct) scans started to be used in several hospitals as a clinical diagnostic tool for covid-19 (udugama et al., 2020) . chest ct scans are a non-invasive procedure consisting in taking many x-ray measurements at different angles to obtain cross-sectional images. this method shows higher sensitivity but a lower specificity compared to rt-pcr, as the imaging features overlap with other viral pneumonia (ai et al., 2020) . among the immunoassay methods, elisa for detecting immunoglobulin g, m and a (igg, igm, iga) from human serum of covid-19 patients is in development. it has the advantage to be a simple, cheap and quick method to be done in a normal laboratory. it must be stressed that antibody assays are the most reliable indicators of sars-cov infection when applied to convalescent-phase serum specimens. in some patients, antibody becomes detectable within 8 to 10 days, and most have detectable antibody by 2 or 3 weeks. however, some persons do not develop detectable antibodies until 28 days after onset of illness. overall, the medium seroconversion time for iga, igm, and igg are 4-6, 4-6, and 5-10 days post symptom onset, respectively. iga and igm detection show the highest sensitivity during about 4-25 days after illness onset, and therefore they can provide a better diagnosis outcome in early stages compared to igg, which reachs its peak during 21-25 days after illness onset, and stays at a relatively high reading until 31-41 days, thus being powerful for diagnostics at later stages . for these reasons, antibody tests have limited diagnostic use. they do not serve for an early diagnosis and can be used as a complement to the virus detection tests for patients presenting late, after symptoms onset, to healthcare facilities or when virus detection tests are negative despite strong indications of infection. moreover, more importantly, antibody tests are utilized for sero-epidemiological surveys and studies. according to the food and drug administration (fda) emergency use authorizations (euas) for covid-19 diagnostics, in addition to the most common elisa method, there are two serological assays used for the detection of antibodies generated against sars-cov-2, the chemiluminescence immunoassays (clia) and the lateral flow immunoassays (lfia). lfia is an j o u r n a l p r e -p r o o f immunochromatography test commonly used for pregnancy tests, utilizing the same principle as elisa and usable at the poc. these tests are rapid (10-30 minutes), can be utilized directly by the patients but are more expensive for a large screening than normal elisa tests and suffer from poor analytical sensitivity (carter et al., 2020) . chen and co-workers have recently presented a simple and rapid lfia that uses lanthanide doped nanoparticles for detecting anti-sars-cov-2 igg in human serum with high diagnostic accuracy . there are important issues about serological antibody assays that still need to be clarified, such as to establish whether the anti-sars-cov-2 antibodies can be considered neutralizing, their persistence in blood and the possible cross-reactivity with other coronaviruses, with the risk of false-positive results. the duration of immunity after infection is a key issue for the "immunity shield", which gives protection against short-term or long-term reinfection and for taking important decisions on physical distancing and social restrictions. despite more than 300 serological assays for covid-19 have been developed, the fda has approved only 12 serological tests intended for use in clinical laboratories under the emergency use authorization (eua), among which the most commonly used in italy are clia tests, produced by abbott laboratories and diasorin (carter et al., 2020) . biosensors based on specific biomolecular interactions offer an alternative and reliable solution to current methods for clinical diagnosis of covid-19, due to their high sensitivity, low-cost, easy to use and possibility of poc utilization. up to date, there are only two papers describing affinity-based biosensors for covid-19 detection. seo and co-workers proposed a new fet based biosensor for sars-cov-2 virus detection. the sensing area of the device is a graphene sheet, modified with sars-cov-2 spike antibody, properly immobilized onto the graphene sheet surface, as schematized in figure 4 . the device was able to detect the sars-cov-2 antigen spike protein at concentrations as low as 1 fg/ml in phosphate buffer, value much lower than that reported with elisa platform. moreover, the biosensor was tested in the universal transport medium (utm), used for suspending the nasopharyngeal swabs for real clinical analysis. no reagent contained in utm affected the measurements and therefore the covid-19 fet biosensor can be successfully utilized to detect antigens in clinical samples without any preparation or pre-processing. furthermore, the device exhibited no measurable cross-reactivity with mers-cov antigen (seo et al., 2020) . most recently, pathsensors inc. announced the development of a "canary" fast biosensor to detect the novel sars coronavirus. the proposed platform utilizes a cell-based immunosensor that couples capture of the virus with signal amplification and provides a result in 3-5 min. the initial application of the pathsensors device will be for testing of environmental swabs and air monitoring in sensitive spaces, such as hospitals, offices, food services, etc. validation data of the new biosensors will be available in july 2020 (pathsensors, 2020). when assessing a patient with covid-19 infection, the identification of effective biomarkers, different than immunoglobulins, can be useful to clinicians in starting treatment, monitoring the progression of the disease and closing monitoring. moreover, on the basis of biomarkers values, patients can be classified into different risk groups for a better clinical management and prevention of serious complications. of course, the identification of novel biomarkers is related to the understanding of the viral pathogenetic mechanisms, as well as cellular and organ damage. the analysis of recently published studies highlights the role of systemic vasculitis and cytokine mediated coagulation disorders as the principal actors of multi-organ failure in patients with severe covid-19 complications (ponti et al., 2020) . it seems that hematological (lymphocyte count, neutrophil count), inflammatory (c-reactive protein, erythrocyte sedimentation rate, interleukin-6), and especially biochemical (d-dimer, troponins, creatine kinase) biomarkers are strongly correlated with severe prognosis or exitus in covid-19 patients and can therefore be used in clinical practice as predictive biomarkers (kermali et al., 2020; morales-narvaez et al., 2020) . in addition to the above discussed laboratory parameters, novel biomarkers are under investigation, among which homocystein, angiotensin ii, -(1-7), -(1-9) and alamandine, in order to clearly determine j o u r n a l p r e -p r o o f their predictive clinical value as indicators of severe prognosis in covid-19 patients (ponti et al., 2020) . in this context, the development of novel biosensing devices or the modification of existing ones for the multiplexing and simultaneous detection of the above mentioned biomarkers is another challenging approach to perform effective assessment of clinical progresses or critical trends of covid-19 infection. biosensors represent an attractive tool in diagnostics, as they have the potential to detect the outbreak of a virus, crucial for the control and prevention of the disease. this review describes the recent developments in fabrication of abbs for cov detection. the reported papers prove that electrochemical, optical and piezoelectric biosensors offer advantages over conventional methods, such as rt-pcr and elisa tests, due to their characteristics, such as fast response time, low cost, easy-to-use, portability, real-time and in situ analysis. the main characteristics of the biosensors reported in this review are summarized in table 2 . it is interesting to note that the amperometric (layqah and eissa, 2019) and fet-based (seo, et al., 2020) biosensors achieved detection limits lower than picomolar levels, thanks to the nanomaterials employed in their fabrication, aunps and graphene, respectively. further improvements in terms of abbs sensitivity and selectivity will certainly be obtained by developing novel nanostructured biosensing platforms. another interesting feature of the biosensors is the possibility to use microarrays integrated within the device, thus allowing multiplexing simultaneous virus detections. although the reported biosensors demonstrated surprising characteristics, some of them still need to be validated in real samples. there is an urgent and growing need for reliable diagnostic solutions for early detection of viral diseases, especially covid-19. early detection can support important decisions in efficiently managing epidemiological and infection control measures, allowing to isolate patients in a timely manner, in order to cut off the route of transmission and take the necessary safety measures, thus facilitating the return to normal human, social and working activities. currently, the conventional diagnostic systems for covid-19 are expensive and located in hospitals or specialized laboratories (carter et al., 2020) . rapid serological tests are in development. they have been designed to give a fast result (10-30 minutes compared to 4-5 hours for conventional methods) and for use in hospitals or near to the poc. however, they are still available for healthcare professionals, and not for patients directly. the need of a rapid home test kit, easily usable by patients is expected to be in high demand. the fundamental concept of the poc is to carry out the test in the most comfortable and immediate way for the patient, who can take the test, obtain immediate medical reports and receive the first treatment directly at home, without having the discomfort to go to the reference hospital, where the risk of covid-19 infection is very high. nano-enabled abbs possess the ideal requirements for miniaturization and therefore for poc applications. another important strength of abbs is their wireless link capability, which allows the transmission of the measured data to a remote medical database or to a health care provider. the measured data could be automatically uploaded via bluetooth to the patient's smartphone or tablet and then directly transferred to health centers, thus monitoring the disease outbreak . moreover, a big data "internet of things" (iot) system for healthcare is emerging, so that machine learning and artificial intelligence (ai) approaches can be used to extract the maximum amount of information from the analytical responses of the developed biosensors and to allow the results to rapidly inform health authorities to tackle infection disease outbreak, to make epidemiological models and to prevent novel pandemic outbreaks. unfortunately, at present, a wireless iot abb for covid-19 is not available. in summary, as future research, it is highly recommended to scientists to invest a lot of effort in developing ai and iot supported nano-based biosensing devices as diagnostics tools to manage covid-19 pandemic and to prevent other possible disease outbreaks. enzyme cells in the "competitive" format (steps a and b), an analyte displaces bound labelled analyte, which is then detected or measured. in the "sandwich" format an unlabeled analyte is "sandwiched" between two antibodies, the unlabelled capture antibody and the labelled detection antibody. effects of blocking buffers and plasma proteins on the protein c biosensor performance biomaterials nanoarchitectonics announced the development of a sarscov-2 biosensor coronaviruses, in: medical microbiology who, world health organization list of abbreviations: ab=antibody; amp=antibody mimics proteins; scvme=sars-cov membrane-envelope protein; gbp=gold binding polypeptide; pqc=piezoelectric quartz cristal; ibv=infectious bronchitis virus; mc=microcantilever; fip=felin infectious protein; ppt=plasmonic photothermal localized spr the author thanks sapienza university of rome for financial support. host symptoms references key: cord-329959-4yecwdlo authors: lin, min-han; moses, david c.; hsieh, chih-hua; cheng, shu-chun; chen, yau-hung; sun, chiao-yin; chou, chi-yuan title: disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes date: 2017-12-28 journal: antiviral res doi: 10.1016/j.antiviral.2017.12.015 sha: doc_id: 329959 cord_uid: 4yecwdlo severe acute respiratory syndrome coronavirus (sars-cov) emerged in southern china in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. ten years later, a second highly pathogenic human cov, mers-cov, emerged in the middle east and has spread to other countries in europe, north africa, north america and asia. as of november 2017, mers-cov had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against mers-cov. here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (pl(pro)s) of mers-cov and sars-cov. our findings suggest that disulfiram acts as an allosteric inhibitor of mers-cov pl(pro) but as a competitive (or mixed) inhibitor of sars-cov pl(pro). the phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of sars-cov pl(pro) by disulfiram, while synergistic inhibition of mers-cov pl(pro) by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs. before 2002, human coronaviruses (covs) had the reputation of occasionally emerging from zoonotic sources and causing mild respiratory tract infections. in late 2002, however, without any warning, severe acute respiratory syndrome (sars) emerged and spread by coronaviral infection to become a pandemic, mainly in asia but also in other regions, with a fatality rate of 10% (hilgenfeld and peiris, 2013) . ten years later, when sars had almost been forgotten, a second highly pathogenic human cov, mers, caused the severe respiratory syndrome in the middle east and then spreading to other countries due to human activity (zaki et al., 2012) . mers-cov has infected at least 2100 people with a high mortality rate of 35% since 2012 (http://www.who.int/csr/ don/7-november-2017-mers-saudi-arabia/en/). because of international travel and climate change, we cannot rule out the possibility of the emergence of additional highly pathogenic covs in the near future (menachery et al., 2015 (menachery et al., , 2016 . thus, the development of antiviral drugs effective against covs is urgently needed. covs are positive-sense single-stranded rna viruses. after the virion has entered the host cell, two polyproteins, pp1a and pp1ab, are directly translated and then cleaved by two viral proteases, main protease (m pro ) and papain-like protease (pl pro ) (perlman and netland, 2009 ). pl pro is responsible for the cleavage of non-structural proteins (nsp) 1, 2 and 3 while m pro cleaves all junctions downstream of nsp4 (perlman and netland, 2009 ). in addition, pl pro can deubiquitinate or deisgylate host cell proteins, including interferon factor 3 (irf3), and inactivate the pathway of nuclear factor κ-light-chain-enhancer of activated b cells (nf-κb), resulting in the immune suppression of host cells (clementz et al., 2010; frieman et al., 2009; yang et al., 2014; zheng et al., 2008) . due to its multiple roles in viral replication and host cell control, pl pro is considered a potential antiviral target. disulfiram is a drug which has been approved by the united states food and drug administration (fda) for use in alcohol aversion therapy since 1951 (bell and smith, 1949; krampe and ehrenreich, 2010; moore et al., 1998) . it is known to irreversibly inhibit hepatic aldehyde dehydrogenase (lipsky et al., 2001) . recent studies indicate t that disulfiram is able to inhibit other enzymes, such as methyltransferase, urease and kinase, all by reacting with important cysteine residues, suggesting broad-spectrum characteristics (diaz-sanchez et al., 2016; galkin et al., 2014; paranjpe et al., 2014) . in addition, there has been a clinical trial investigating the usage of disulfiram for reactivating latent hiv in order to make it accessible to highly active anti-retroviral therapy (elliott et al., 2015) , and the drug has also been shown to act as a "zinc ejector" with respect to hepatitis c virus ns5a protein (lee et al., 2016) . however, the effect of disulfiram on viral cysteine proteases is still unknown. in the present study, we demonstrate that disulfiram is an inhibitor of mers-cov and sars-cov pl pro s, and furthermore that disulfiram acts on mers-cov and sars-cov pl pro via different inhibition mechanisms. moreover, we investigated the synergies between a number of known pl pro inhibitors and disulfiram, and our results point to the possibility of using combination treatments involving disulfiram and other clinically available drugs against covs. the sars-cov pl pro c271a mutation was introduced using the quikchange mutagenesis kit (stratagene) and was verified by dna sequencing. the forward primer was 5′-gtacactggtaactatcaggcgggtcatt acactcatata and the reverse primer was 5′-tatatgagtgtaatgacccgcctgatagttaccagtgtac. the mers-cov and sars-cov pl pro s and the sars-cov pl pro c271a mutant protein were produced and purified as previously described (chou et al., 2012 lin et al., 2014) . briefly, the cultures were grown at 37°c for 4 h, then induced with 0.4 mm isopropyl β-d-1-thiogalactopyranoside and grown at 20°c for 20 h. the cell pellet was resuspended in lysis buffer (20 mm tris, ph 8.5, 250 mm nacl, 5% glycerol, 0.2% triton x-100, 2 mm β-mercaptoethanol (βme)), lysed by sonication and then centrifuged to remove the insoluble pellet. the target protein was purified from the fraction of soluble proteins via nickel affinity chromatography, then loaded onto an s-100 gel-filtration column (ge healthcare) equilibrated with running buffer (20 mm tris, ph 8.5, 100 mm nacl, 2 mm dithiothreitol). for the crystallization of sars-cov pl pro in complex with glycerol, the reductant was removed and 50 μm disulfiram was added to each buffer during the purification process. the purity of the fractions collected was analyzed by sds-page and the protein was concentrated to 30 mg/ml using an amicon ultra-4 30-kda centrifugal filter (millipore). the dub assay was carried out as previously described (cheng et al., 2015; chou et al., 2008; lin et al., 2014) . the fluorogenic substrate ub-7-amino-4-trifluoro-methylcoumarin (ub-afc) (boston biochem) was added at a concentration of 0.25 μm along with various concentrations of inhibitors into 20 mm phosphate (ph 6.5) and each mixture was incubated at 30°c for 3 min. after adding 0.2 μm coronaviral pl pro , enzymatic activity was determined by continuously monitoring fluorescence intensity at excitation and emission wavelengths of 350 and 485 nm, respectively. the data was fitted to obtain ic 50 according to eq. (1): (1) in which v is the initial velocity in the presence of inhibitor at concentration [i] and v 0 is the initial velocity in the absence of inhibitor, while n is the hill constant. in addition, to test for the recoverability of activity, coronaviral pl pro was incubated with or without 200 μm disulfiram for 1 h and then desalted using a sephadex g-25 column. the dub activity of 0.2 μm treated enzyme was then determined in the presence or absence of 5 mm βme. the peptidyl substrate dabcyl-frlkggapikgv-edans was used to measure the proteolytic activity of pl pro . fluorescence intensity was monitored at 329 nm (excitation) and 520 nm (emission) and converted to the amount of hydrolyzed substrate based on previous studies (cheng et al., 2015; chou et al., 2008) . for inhibition studies, the reaction mixture contained 9-80 μm peptide substrate with 0-200 μm disulfiram in 20 mm phosphate (ph 6.5). mers-cov pl pro at 0.6 μm and wild-type sars-cov pl pro and c271a mutant at 0.05 μm was used, respectively. after adding the enzyme to the reaction mixture, fluorescence intensity was continuously monitored at 30°c. the increase in fluorescence was linear for at least 1 min, and thus the slope of the line represented the initial reaction velocity (v). the data obtained for the inhibition of mers-cov pl pro by disulfiram was found to best fit a noncompetitive inhibition pattern in accordance with eq. (2): while the data obtained for the inhibition of sars-cov pl pro by disulfiram was found to best fit a competitive inhibition pattern in accordance with eq. (3) or a mixed inhibition pattern in accordance with eq. (4): in which k cat is the rate constant, [e], [s] and [i] denote the enzyme, substrate and inhibitor concentrations, and k m is the michaelis-menten constant for the interaction between the peptide substrate and the enzyme. k is is the slope inhibition constant for the enzyme-inhibitor complex and αk is is the slope inhibition constant for the enzyme-substrate-inhibitor complex. the program sigmaplot 12.5 (systat software inc., usa) was used for data analysis. to characterize the mutual effects of disulfiram and other known pl pro inhibitors, the activity of mers-cov pl pro was measured with and without either 6-thioguanine (6tg) (0 and 15 μm) or mycophenolic acid (mpa) (0 and 150 μm) in the presence of various concentrations of disulfiram (0-30 μm), and that of sars-cov pl pro was measured with and without either 6tg or n-ethylmaleimide (nem) in the presence of various concentrations of disulfiram (0-24 μm). the concentrations of the peptidyl substrate and mers-cov pl pro were 20 and 0.6 μm, respectively, while those of the substrate and sars-cov pl pro were 15 and 0.05 μm, respectively. data obtained from the reactions were fitted to eq. (5): where v is the initial velocity in the presence of both inhibitors, [i] and [j] are the concentrations of the two inhibitors, v 0 is the velocity in the absence of inhibitors, k i and k j are the apparent dissociation constants for the two inhibitors, and α is a measurement of the degree of interaction between the two inhibitors (copeland, 2000; yonetani and theorell, 1964) . release of zinc ions from coronaviral pl pro s was monitored as the increase in fluorescence emission from the zinc-specific fluorophore fluozin-3 (thermo fisher scientific) (lee et al., 2016) . briefly, the protein and fluozin-3 were mixed in 20 mm phosphate buffer (ph 6.5) to concentrations of 5 μm and 1 μm, respectively, in the presence or absence of 5 μm disulfiram. fluorescence emission was continuously measured at 25°c using emission and excitation wavelengths of 494 nm and 516 nm, respectively, in a perkinelmer ls50b luminescence spectrometer. the change in secondary structure of coronaviral pl pro s in the absence and presence of 5 μm disulfiram was continuously measured using ellipticity at 222 nm as the temperature was ramped from 30 to 85°c in a jasco j-810 spectropolarimeter. the protein at 5 μm was dissolved into 20 mm phosphate buffer, ph 6.5. the width of the cuvette was 1 mm. for the inactivation studies, sars-cov pl pro (0.05 μm in 20 mm phosphate buffer, ph 6.5) was incubated with different concentrations of disulfiram and peptide substrate, and enzymatic activity was traced for 5 min. all progress curves recorded showed an exponential course and were analyzed according to the following integrated rate equation (eq. (6)) (copeland, 2000) : in which v i is the initial velocity, v s is the steady-state velocity, and d is the displacement on the y-axis. the replot of k inact versus the concentration of disulfiram was fitted to a saturation curve according to eq. (7) (copeland, 2000) : in which k inact is the dissociation constant of the enzyme-disulfiram complex and k max is the maximum inactivation rate constant. crystals of sars-cov pl pro in complex with βme or glycerol were obtained at 22°c by the sitting-drop vapor-diffusion method. for the pl pro -βme complex, the protein at 15 mg/ml was incubated with 0.4 mm disulfiram for 1 h and then crystallized. single crystals were grown in reservoir solution containing 16% (w/v) peg 3350 and 0.1 m bis-tris propane (ph 8.0). for the pl pro -glycerol complex, protein purified with the addition of 50 μm disulfiram into each buffer during the purification process was crystallized at 12.5 mg/ml. single crystals were grown in reservoir solution containing 6% (w/v) peg 8000 and 0.1 m hepes (ph 8.0). all crystals were cryoprotected in reservoir solution supplemented with 15% and 25% (v/v) glycerol for pl pro -βme and pl pro -glycerol, respectively, and then flash-cooled in liquid nitrogen. 2.9. data collection and structure determination x-ray diffraction data was collected at 100 k on the spxf beamline 15a1 at the national synchrotron radiation research center, taiwan, roc using a rayonix mx300he ccd detector at a wavelength of 1 å. the diffraction images were processed and then scaled with the hkl-2000 package (otwinowski and minor, 1997) . the structure was solved by the molecular-replacement method with phaser (mccoy et al., 2007) using the structure of wild-type sars-cov pl pro (pdb entry 2fe8; (ratia et al., 2006) ) as the search model. manual rebuilding of the structure model was performed with coot (emsley and cowtan, 2004) . structure refinement was carried out with refmac (murshudov et al., 2011) . data-processing and refinement statistics are summarized in table 3 . the crystal structures of the sars-cov pl pro -βme complex and sars-cov pl pro -glycerol complex have been deposited in the protein data bank (pdb entries 5y3q and 5y3e for pl pro -βme and pl pro -glycerol, respectively). 3.1. the inhibition of mers-cov and sars-cov pl pro s by disulfiram pl pro s are cysteine proteases that use the thiol group of cysteine as a nucleophile to attack the carbonyl group of the scissile peptide bond han et al., 2005; verma et al., 2016) . inhibition can be expected if the catalytic cysteine of a pl pro is interfered with or modified (cheng et al., 2015; chou et al., 2008) . disulfiram is known to be a thiol-reactive compound that can covalently modify cysteine residues (diaz-sanchez et al., 2016; galkin et al., 2014; lipsky et al., 2001; paranjpe et al., 2014) . to determine whether disulfiram can inhibit coronaviral pl pro s, the dub activity of mers-cov and sars-cov pl pro was measured in the presence of various concentrations of disulfiram. interestingly, disulfiram showed a dose-dependent inhibitory effect on both proteases with ic 50 values in the micromolar range ( fig. 1) . next, to elucidate the kinetic mechanisms of the interactions between disulfiram and the two pl pro s, the proteolytic activity of each enzyme was measured in the presence of various concentrations of a peptidyl substrate and disulfiram. the results were then fitted to different kinetic models (competitive, noncompetitive, uncompetitive and mixed inhibition). surprisingly, disulfiram showed a noncompetitive inhibition pattern against mers-cov pl pro ( fig. 2a ) but a competitive inhibition pattern against sars-cov pl pro (fig. 2b ). this inconsistency is quite intriguing since the two enzymes share a similar overall structure and an identical catalytic triad (bailey-elkin et al., 2014; chou et al., 2014; lei et al., 2014; ratia et al., 2006) , albeit the inhibition constant (k is ) of disulfiram for mers-cov pl pro is 4.4-fold higher than that for sars-cov pl pro (table 1 ). perhaps this discovery should not be surprising given that disulfiram is also a noncompetitive inhibitor for citrullus vulgaris urease with a k is of 67.6 μm (diaz-sanchez et al., 2016), while its ic 50 for giardia lamblia carbamate kinase is 0.6-1.4 μm (chen et al., 2012) . similarly, a previous study mentions that their compound 4 also has different recognition specificity for the two pl pro s . our study once again suggests broad-spectrum potency for disulfiram, given the versatility it shows even against two coronaviral pl pro s. the inconsistent inhibitory effect of disulfiram against the two pl pro s suggests that the binding modes of disulfiram on the two enzymes may be different. to verify this, multiple inhibition assays using disulfiram and other known pl pro inhibitors, including 6tg, mpa and nem, were performed (fig. 3) (chen et al., 2009; cheng et al., 2015; yonetani and theorell, 1964) . interestingly but not surprisingly, we found that disulfiram displays a synergistically inhibitory effect with either 6tg or mpa on mers-cov pl pro , with the lines in the yonetani-theorell plots intersecting above the x-axis and α values below 1 in both cases (fig. 3a and b) (copeland, 2000) . in contrast, in the case of sars-cov pl pro , each of the plots displays two parallel lines and both α values are significantly higher than 1 (fig. 3c and d) , suggesting that binding of disulfiram and of 6tg or nem are mutually exclusive on sars-cov pl pro (copeland, 2000) . since 6tg is a competitive inhibitor of both pl pro s (cheng et al., 2015; chou et al., 2008) , the contrasting synergy of disulfiram and 6tg on the two pl pro s confirms the inconsistent inhibitory pattern of disulfiram (figs. 2 and 3) . furthermore, mpa has previously been shown to be a noncompetitive inhibitor of mers-cov pl pro and to work synergistically with 6tg to inhibit mers-cov pl pro (cheng et al., 2015) . combining those results with our results regarding the binding synergy of disulfiram and 6tg or mpa (fig. 3a and b), we propose that disulfiram may occupy a third binding site on mers-cov pl pro , neither a site at the active center nor the mpa binding site. next, we evaluated pl pro inhibition in the presence of disulfiram combined with 6tg and/or mpa by proteolytic assays using a peptidyl substrate. we found that the ic 50 of disulfiram against mers-cov pl pro showed a 1.6-fold decrease in the presence of 15 μm 6tg and a 5.2-fold decrease at 15 μm 6tg when it was tested in combination with 150 μm mpa (table 2) . for comparison, in the case of disulfiram against sars-cov pl pro , there is no enhanced inhibitory effect in the presence of 6tg or nem. our results suggest a potential for using the above three fdaapproved drugs in combination treatments against mers-cov. incidentally, previous studies have suggested that mpa may be used in combination treatments with interferon against mers-cov (chan et al., 2013) . the experiments were repeated to ensure reproducibility. kinetic parameters such as k m , k cat and k is from the best-fit results are shown in table 1 . in the presence of disulfiram, the best-fitted kinetic parameters and k is were determined in accordance with competitive (eq. (3)) or mixed inhibition (eq. (4)) and noncompetitive (eq. (2)) inhibition models for sars-cov and mers-cov pl pro , respectively. c the value is αk is , the inhibition constant for the enzyme-substrate-inhibitor complex. d k inact and k max values are from the best fit to the saturation equation (eq. (7)). previous studies suggested that disulfiram can bind to the zincbound cysteines in hepatitis c virus ns5a protein (lee et al., 2016) . as there are four cysteines bound to a zinc ion in pl pro s ( fig. s2c and s2d ) (bailey-elkin et al., 2014; chou et al., 2014) , we performed zinc ejection assays to test whether these zinc-bound cysteines may be a candidate for the aforementioned "third binding site" occupied by disulfiram on mers-cov pl pro . in the present study, the zinc-specific fluorophore, fluozin-3, was used to identify the release of zinc ion due to the binding of disulfiram to the enzyme (fig. 4a) . unexpectedly, we observed significant zinc release in the presence of disulfiram not only from mers-cov pl pro but also from sars-cov pl pro . this result indicates that disulfiram may bind not only to the active site but also to the zinc-binding sites in sars-cov pl pro . following this finding, we tried to fit our inhibitory results to a mixed inhibition model (fig. s1 ). the two k is for the enzyme-substrate and enzyme-substrate-inhibitor complexes were 6.0 and 43.8 μm, respectively, showing a 7.3-fold difference in the binding affinity for the two putative binding sites (table 1 ). this significant difference may explain why the inhibitory pattern of disulfiram against sars-cov pl pro looks more like competitive inhibition. next, the thermostability of the two pl pro s in the absence and presence of disulfiram was evaluated (fig. 4b) . not surprisingly, the melting temperature of both pl pro s decreased 10-15°c in the presence of disulfiram. these results conform to our earlier finding that the release of zinc ion can destabilize pl pro (chou et al., 2012) . 6)). (c) the observed inactivation rate constants (k inact ) from panel b were replotted against disulfiram concentration. the solid line is the best-fit result in accordance with the saturation equation (eq. (7)). kinetic parameters k inact and k max corresponding to the best-fit curve are shown in table 1 . disulfiram is known to covalently modify cysteine residues and leave a diethyldithiolcarbamate (ddc) moiety to inactivate the carbamate kinase of giardia lamblia (galkin et al., 2014) . in the presence of 5 mm βme, however, the inhibitory effect of disulfiram against pl pro s is minor and the ic 50 is larger than 300 μm (table 2 ). this suggests that the reductant can protect the enzyme and, therefore, that disulfiram may inhibit the enzyme by modifying the cysteine in the catalytic triad (cys112-his273-asp287). to further investigate this possibility, the dub activity of the enzyme was measured after incubation with 200 μm disulfiram for 1 h followed by removal of the small molecules using a sephadex g-25 column. this treatment resulted in an 84% loss of activity, suggesting irreversible inhibition of sars-cov pl pro by disulfiram (fig. 5a, right panel) . similarly, in a previous in vivo study, disulfiram-treated aldehyde dehydrogenase showed 77% enzyme inhibition as compared to the activity of the control (lipsky et al., 2001) . next, the disulfiram-treated sars-cov pl pro was incubated with 5 mm βme for 10 min, after which activity was measured to test for re-activation. we found that 30% of the enzyme's activity was restored after treatment with βme (fig. 5a, right panel) . the rescuing effect of the reductant suggests that the modification was due to the disulfide bonding interaction between the enzyme and the inhibitor. however, in the case of mers-cov pl pro , we found that treatment with disulfiram resulted in an irreversible loss of activity which was not rescued by the addition of the reductant (fig. 5a, left panel) . previous studies have suggested that the release of zinc ion following treatment with edta will lead to a 62% loss of pl pro activity (chou et al., 2012) . this result is consistent with the effect of disulfiram on pl pro s. also, the inability of the reductant to rescue the dub activity of mers-cov pl pro , suggesting that disulfiram cannot influence its active site, is compatible with disulfiram's noncompetitive mode of inhibition of the enzyme. on the other hand, proteolytic assays of sars-cov pl pro at various concentrations of disulfiram showed dose-and time-dependent decay when enzyme activity was measured for 5 min (fig. 5b) . by fitting the data to eq. (6), different k inact values at various concentrations of disulfiram were determined and then plotted versus those disulfiram concentrations (fig. 5c ). the saturated curvature suggests a slowbinding phenomenon due to covalent inactivation (copeland, 2000) , a conclusion supported by the irrecoverability of enzyme activity after disulfiram removed (fig. 5a) . best-fit analysis determined a k inact of 5.4 μm and a k max of 0.011 s −1 (fig. 5c and table 1 ). interestingly, the k inact value is close to k is , indicating that disulfiram may inactivate the enzyme very soon after binding. for comparison, previous studies have indicated that 6-mercaptopurine and 6tg are also slow-binding inhibitors against the same enzyme, albeit enzyme activity was recovered after removing the inhibitors (chou et al., 2008) . the structure of sars-cov pl pro in complex with disulfiram should allow us to understand the binding mechanism more clearly. accordingly, we attempted to crystallize sars-cov pl pro in the presence of disulfiram. unfortunately, although crystals of the protein were formed in the presence of 0.4 mm disulfiram, the crystal structure showed only βme-like electron density near the active-site cysteine with no omit electron density shown near the zinc-binding site ( fig. s2a and s2c ). βme is a reducing agent that is added into the purification buffer to stabilize the protein, and which is also known to reverse the effect of disulfiram (table 2, fig. 5a and kitson, 1975) . to avoid this effect, we eliminated all reducing agents from the purification process, added 50 μm disulfiram into all purification buffers, and then attempted to crystallize the protein purified under these conditions. although we were able to grow crystals under different crystallization conditions, we again obtained an unexpected result, as the only omit electron density near the catalytic site was fitted as a glycerol molecule ( fig. s2b and s2d ). this result might be due to the crystals having been cryoprotected in reservoir solution supplemented with 25% (v/v) glycerol. nevertheless, the binding of βme and glycerol near the active site suggests that the active site may be accessible to disulfiram. next, using the aforementioned two complex structures, a disulfiram and a ddc molecule were docked into the glycerol and βme binding sites, respectively (fig. 6) . ddc may be able to covalently bind to residue cys112 in a manner similar to that of βme (fig. 6a ), while disulfiram may be able to occupy the glycerol site (fig. 6b) . interestingly, in the docking structure of the pl pro -disulfiram complex, we can see that one sulfur atom of the disulfide bond of disulfiram is within 4 å of residue cys271 at blocking loop 2 (bl2), which is very important for substrate and inhibitor binding ratia et al., 2008) . for comparison, there is a valine at the same site in mers-cov pl pro (bailey-elkin et al., 2014; chou et al., 2014) . to verify the possible inhibitory effect of disulfiram due to binding to residue cys271, inhibition of the sars-cov pl pro c271a mutant by disulfiram was measured (fig. s3) . interestingly, we can see a 4.4-fold increase in ic 50 ( table 2 ) compared with that for inhibition of wild-type sars-cov pl pro by disulfiram. in addition, the decrease of the melting temperature of the c271a mutant following treatment with disulfiram is 6°c, lower than that of wild-type sars-cov pl pro treatment with the same inhibitor ( fig. 4b and c) . these findings suggest that disulfiram may inhibit sars-cov pl pro partly via the residue cys271 and support the reliability of the docking of disulfiram on the glycerol binding site. based on our kinetic and structural results, we propose kinetic mechanism schemes for the inhibition of the two pl pro s by disulfiram (fig. 7) . similar to the mechanism in the case of disulfiram-treated urease (diaz-sanchez et al., 2016) , disulfiram may form a covalent adduct with sars-cov pl pro and then leave a ddc on the active-site cys112, preventing downstream acylation and thereby inactivating the fig. 6 . binding of disulfiram to sars-cov pl pro . overlay of model structure of sars-cov pl pro in complex with ddc (magenta) (a) or disulfiram (orange) (b) with the crystal structure of sars-cov pl pro in complex with ubiquitin (gray, pdb code: 4m0w). ddc and disulfiram are modeled based on the binding sites of βme and glycerol, respectively. the red dashed lines show putative polar interactions while the black dashed line shows the distance between residue cys271 and disulfiram as 4.0 å. (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) enzyme. in contrast, disulfiram shows a noncompetitive inhibitory effect against mers-cov pl pro and can synergistically inhibit that enzyme with 6tg and mpa. in this study, we found that disulfiram is, respectively, a noncompetitive and competitive (or mixed) inhibitor of mers-cov and sars-cov pl pro s. multiple inhibition assays also support a kinetic mechanism by which disulfiram together with 6tg and/or mpa can synergistically inhibit mers-cov pl pro , but not, due to its competitive mode of inhibition, sars-cov pl pro . on the other hand, the results of kinetic assays, continued inactivation after the removal of disulfiram, reactivation by reductant, and the phenomenon of slow-binding inhibition suggest that disulfiram may act at the active site of sars-cov pl pro , forming a covalent adduct with residue cys112. crystal structures of the enzyme in complex with glycerol and βme imply that the active site is solvent-exposed and accessible for disulfiram or ddc binding. fig. 7 . schemes of proposed kinetic mechanisms for the inhibition of sars-cov and mers-cov pl pro by disulfiram. the upper diagram denotes enzyme catalysis, mixed inhibition and inactivation of sars-cov pl pro by disulfiram. the lower diagram shows noncompetitive inhibition of mers-cov pl pro by disulfiram and triple inhibition with two other fda-approved drugs, 6tg and mpa. sh symbolizes the thiolate of catalytic triad residue cys. crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papainlike protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression preliminary report on clinical trials of antabuse broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus a homogenous luminescence assay reveals novel inhibitors for giardia lamblia carbamate kinase thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases enzymes: a practical introduction to structure, mechanism, and data analysis inhibition of urease by disulfiram, an fda-approved thiol reagent used in humans short-term administration of disulfiram for reversal of latent hiv infection: a phase 2 dose-escalation study coot: model-building tools for molecular graphics severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling structural basis for inactivation of giardia lamblia carbamate kinase by disulfiram papainlike protease 2 (plp2) from severe acute respiratory syndrome coronavirus (sars-cov): expression, purification, characterization, and inhibition from sars to mers: 10 years of research on highly pathogenic human coronaviruses the effect of disulfiram on the aldehyde dehydrogenases of sheep liver supervised disulfiram as adjunct to psychotherapy in alcoholism treatment inhibitor recognition specificity of mers-cov papain-like protease may differ from that of sars-cov using an old drug to target a new drug site: application of disulfiram to target the zn-site in hcv ns5a protein crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features structural and functional characterization of mers coronavirus papainlike protease in vivo inhibition of aldehyde dehydrogenase by disulfiram phaser crystallographic software a sars-like cluster of circulating bat coronaviruses shows potential for human emergence sars-like wiv1-cov poised for human emergence sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases refmac5 for the refinement of macromolecular crystal structures processing of x-ray diffraction data collected in oscillation mode disulfiram is a direct and potent inhibitor of human o6-methylguanine-dna methyltransferase (mgmt) in brain tumor cells and mouse brain and markedly increases the alkylating dna damage coronaviruses post-sars: update on replication and pathogenesis a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme cysteine proteases: modes of activation and future prospects as pharmacological targets proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease studies on liver alcohol hydrogenase complexes. 3. multiple inhibition kinetics in the presence of two competitive inhibitors isolation of a novel coronavirus from a man with pneumonia in saudi arabia plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production we would like to thank ziad omran for helpful suggestions. this research was supported by grants from the ministry of science and technology, taiwan, roc (104-2320-b-010-034, 105-2320-b-010-012 and 106-2320-b-010-013) to cyc and a cgmh-nymu joint research grant (cmrpg2f0431) to cys and cyc. we are grateful for the experimental facilities and the technical services provided by the synchrotron radiation protein crystallography facility, which is supported by the national core facility program for biotechnology, ministry of science and technology, taiwan, roc, and the national synchrotron radiation research center, a national user facility supported by the ministry of science and technology, taiwan, roc. supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.antiviral.2017.12.015. key: cord-330343-p7a8chn4 authors: kelly-cirino, cassandra; mazzola, laura t; chua, arlene; oxenford, christopher j; van kerkhove, maria d title: an updated roadmap for mers-cov research and product development: focus on diagnostics date: 2019-02-01 journal: bmj glob health doi: 10.1136/bmjgh-2018-001105 sha: doc_id: 330343 cord_uid: p7a8chn4 diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. ► the middle east respiratory syndrome-coronavirus is a high-priority pathogen identified by the who r&d blueprint because of its high fatality rate, large geographical range of the dromedary camel reservoir and lack of medical interventions. ► accurate and accessible diagnostic tests are essential to outbreak containment and case management, as well as surveillance in both humans and animals, but available diagnostic tests are limited by inconsistent quality assessment, specimen acquisition issues and infrastructure requirements. ► diagnostic research and development (r&d) needs to include point-of-care testing options, syndromic panels for differential diagnosis, a greater understanding of viral and antibody kinetics, improved access to clinical specimens, and establishment of international reference standards. diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. the middle east respiratory syndrome-coronavirus (mers-cov) is an emerging virus associated with severe respiratory illness, first detected in 2012 in saudi arabia. 1 5 provides an overview to the current status of mers-cov diagnostics, including feedback from subject matter expert and developer interviews on the common challenges with test development and implementation, and identifies gaps for further research and development (r&d). mers-cov is a zoonotic virus, and dromedary camels (camelus dromedarius) are the reservoir host and the source of zoonotic transmission to humans. [6] [7] [8] dromedaries appear to be only mildly symptomatic following infection and present a significant reservoir risk for spillover events. 2 6 9 mers-cov rna has been detected in dromedary camels in a number of countries, including egypt, oman, qatar and saudi arabia, with evidence suggesting that mers-cov is also widespread in the middle east, africa and south asia. 5 8 10-35 infection in camels is notifiable to the oie. 36 individuals with close and frequent contact with dromedaries are at a higher risk for mers-cov infection than the general population. 37 38 clinical indications and management coronaviruses are a family of viruses that can cause diseases in humans, ranging from the common cold to severe acute respiratory syndrome (sars). the clinical spectrum of mers ranges from no symptoms (or asymptomatic infection), mild symptoms including fever, cough, gastrointestinal illness and shortness of breath, to severe disease including pneumonia, acute respiratory distress syndrome and death. 2 39 severe cases of mers can result in respiratory failure, requiring mechanical ventilation and support in intensive care. risk factors for severe disease include a weakened immune system, older age (>60 years), and comorbidities such as diabetes, cancer, renal disease and chronic lung disease. 40 41 human-tohuman transmission spreads through close and unprotected human contact, and more than half of reported mers cases have occurred through nosocomial transmission. [42] [43] [44] [45] to prevent nosocomial infections, who and others recommend using standard infection and prevention control measures when caring for patients. [46] [47] [48] who also recommends that contact tracing of all symptomatic and asymptomatic close contacts of the primary patient should be conducted routinely. 49 the molecular epidemiology for mers-cov has not changed significantly since the initial human cases were detected in 2012. the current virus remains 99% identical to the sequences seen in the first human cases from 2012 as well as archived camel sera from 1983, with no increase in pathogenicity observed in the animal host. [50] [51] [52] as genetic mutations could impact detection, bmj global health immunotherapy and vaccine development efforts, 53 sequencing of mers-cov strains from camels and humans (after a zoonotic spillover) is important and is regularly being conducted in affected member states (who, personal communication, 2018). there are currently no prophylactic or therapeutic interventions of proven efficacy for any coronavirus infections. without a specific therapy for mers, treatment is supportive. 5 54 55 effective mers therapeutics are still in the early stages of research and evaluation. several broad-spectrum antiviral agents including nitazoxanide, 56 viral methyltransferase inhibition 57 and nucleotide prodrugs 58 have shown in vitro activity against mers-cov. early results for novel mers-specific therapeutics that inhibit viral replication or have specific neutralising activity are promising. 47 59 60 the who r&d blueprint for mers has called for three types of vaccines: (1) dromedary camel vaccine to prevent zoonotic transmission, (2) human vaccine for long-term protection of persons at high exposure risk and (3) human vaccine for reactive use in outbreak settings. 55 61 mers-cov vaccines are in the early stages of development, 55 62 63 with one candidate vaccine in phase i clinical trials (nct02670187). 64 neutralising monoclonal antibodies have been designed to target the mers-cov spike protein, 53 65 with chadox1 and modified vaccinia ankara vectors also strong vaccine candidates, 60 66 but none have yet advanced to clinical trials. to accelerate the process, the coalition for epidemic preparedness innovation has recently launched a call for proposals for the development of a human mers-cov vaccine in order to engage with developers interested in supporting these efforts. 67 the who laboratory guidelines recommend nucleic acid amplification tests (naat) for diagnosis, using serology for diagnosis only when naat is not available. 68 in suspected patients, a single negative test result does not exclude diagnosis. repeat sequential sampling and testing is strongly recommended. the kinetics of mers-cov infection has been shown to vary widely across cases, 40 69-72 prompting a more detailed investigation of viral and antibody dynamics across the broad range of sample types, disease states and host factors. 73 74 the best naat test sensitivity is achieved using specimens from the lower respiratory tract (sputum, tracheal aspirates or bronchoalveolar lavage), where mers-cov replication occurs at higher and more prolonged levels of mers-cov rna, typically between 10 6 and 10 10 copies/ml. 72 75 mers-cov viral load is generally higher for severe cases, with more prolonged viral shedding than mild cases. viral load concentrations, which may be undetectable at early-stage infection, generally peak in the second week after symptom onset, and then drop to undetectable in survivors by the fourth week from onset. upper respiratory tract specimens (nasopharyngeal or oropharyngeal swabs) may also be used, but demonstrate 100×-1000× lower viral load and can test negative for mild cases. 76 77 if possible, both upper and lower respiratory tract sampling are advised. specimens outside the respiratory tract are not recommended for diagnosis, as they can test negative in both severe and mild presentation. viral rna has been detected in stool samples (10 4 copies/ml), serum samples (10 3 copies/ml) and urine (10 2 copies/ml), more likely an indicator of severity as it typically precedes a poor clinical outcome. 71 76 78 serological diagnosis can be made using paired samples, more often used for research rather than diagnostic purposes, preferably with the initial sample collected in the first week of illness and the second collected 3-4 weeks later. if only a single serum sample can be collected, this should occur at least 3-4 weeks after onset of symptoms for determination of a probable case. table 1 presents an overview of the implementation requirements for mers-cov diagnostics (detailed commercial product information is presented in online supplementary tables s1 and s2). molecular diagnostics such as naat (eg, pcr) typically require sophisticated laboratory infrastructure including biosafety cabinets, 79 while most serological tests (elisa, indirect immunofluorescence test (iift)) can be run on the benchtop in a more modest laboratory environment, depending on sample preparation precautions. 80 81 point-of-care (poc) tests are designed to be used outside of a traditional laboratory; near-poc tests are defined for rapid use in a laboratory near the patient, but are more automated and easy to use than the traditional laboratory test. 72 75 poc tests such as low-complexity rapid diagnostic tests (rdts) can be used at the bedside, typically with non-invasive samples after minimal training. inhouse tests are described in sections below; commercial sources are listed in online supplementary tables s1 and s2. naats are currently the standard for mers-cov diagnosis, as these tests (typically reverse transcriptase pcr (rt-pcr)) have the highest sensitivity at the earliest time point during the acute phase of infection. following the who guidelines, two different targets on the mers-cov need to be detected by rt-pcr to confirm a case. mers-cov assays to detect the upstream envelope gene (upe) followed by confirmation of open reading frame 1a (orf1a), 1b (orf1b) genes or nucleocapsid (n) genes for confirmation have been developed. 55 82 most commercial pcr tests perform parallel screening for the upe gene with confirmation by the orf1a, orf1b or n genes (most commonly upe + orf1a). initial naat tests for mers-cov were developed as inhouse tests, following the first detection of mers-cov in the middle east. [83] [84] [85] [86] inhouse tests are not necessarily subject to quality control or regulation, and may not be rigorously validated; in some cases, inhouse tests are eventually developed into commercial products through collaboration and licensing efforts. 50 83 84 87-89 commercial assays may undergo an international and/or incountry regulatory process; once on the market they can be independently evaluated for sensitivity, specificity and limit of detection. 78 90 as of 2018, there are several commercial naat tests available for mers-cov, including duplex and multiplex panels (see online supplementary table s1). serology is not widely performed for diagnosing acute mers-cov infection; however, it has been a useful tool bmj global health to determine the extent of infection around clusters and in seroepidemiological studies in animals and humans. seroconversion typically occurs during the second and third week after symptom onset; data suggest that low antibody titre in the second week or delayed seroconversion is more closely associated with mortality than high viral load. 71 74 mers-cov seroconversion may not be observed for some patients, notably with mild or asymptomatic infection, and can show cross-reactivity with antibodies to other coronaviruses. 42 69 serological methods for the detection of antibodies against mers-cov include elisa, iift and neutralisation tests. mers-cov serological assays can employ commercial reagents or proprietary monoclonal antibodies as capture agents. 87 91 92 many mers-cov elisa tests are labelled for research use only, with little or no clinical validation data available. similar to the elisa, iift is used when it is difficult to evaluate specific antigens individually by enzyme immunoassays or there is a preference for broader analysis of an immobilised specimen. iift microscopy assay can probe the entire antigen spectrum of the specimen, and is often designed for simultaneous detection of antibodies against biochemically distinct antigens. neutralisation is a method for detecting anti-mers-cov antibody activity via inhibition of infection or replication, 69 93 performed as plaque reduction neutralisation, microneutralisation (mn) and pseudoparticle neutralisation (ppnt). mn is labour-intensive and slow, requiring at least 3-5 days for results; neutralisation techniques other than ppnt require biosafety level 3 containment as they involve live virus cultures. 94 rdts can leverage the same antibody/antigen capture agents as elisa but in a lateral flow strip cartridge. 95 this enables a fast 10-30 min time to result, but with a 100-fold lower detection sensitivity than elisa. 91 92 follow-up confirmatory testing is therefore required. rdts are typically paired with minimally invasive specimen collection (blood, oral fluid, nasal swabs) so that they can be used with minimal training outside of laboratory settings. early prototypes for mers-cov rdts have been developed, 87 92 96 with commercial rdts for detection of mers-cov in camels and humans available (online supplementary table s2). the human mers-cov rdt does not appear to be widely used, perhaps due to the more invasive processing required for lower respiratory specimens, as well as sensitivity issues for upper respiratory specimens. the camel mers-cov rdt is used with upper respiratory specimens; however, test sensitivity varies depending on specimen sampling and infection kinetics. 97 multiplex panels at the early stages, the symptoms of mers-cov infection can mimic diseases such as influenza, pneumonia, sars and other respiratory infections. a syndromic approach involves testing for pathogens based on a syndrome such as fever or acute respiratory distress; a shift from individual tests to multiplex panels can quickly identify or eliminate likely pathogens from a single specimen. for analysis of circulating reservoirs, multiplex microbead-based immunoassays have been used to detect igg antibodies for multiple pathogens. 98 99 multiplex, syndromic panels that include mers-cov have been demonstrated using pcr-based panels including mers-cov, showing similar limits of detection to single assays. 89 100 101 commercial respiratory panel tests including mers-cov have also recently been developed (see online supplementary table s1). there is a need for international consensus and adoption of minimum standards for tests used in diagnosis, surveillance and research, following who's recommended algorithm for human cases 82 and oie recommendation for animal health. 36 harmonisation of the testing process can be achieved by building consensus and capacity across international and incountry laboratories. in order to enable and sustain the capacity for a rapid outbreak response, laboratories must have access to high-quality reagents and instrumentation, along with technical support and cold-chain transport when necessary. in addition, international reference panels would achieve a more standardised training for external quality assessment (eqa) and quality control. building on mandatory case reporting, 102 an international mers-cov data sharing platform that includes case exposure history and sequence data would greatly facilitate the knowledge base across the mers-cov community. [103] [104] [105] [106] clinical validation understanding mers-cov viral dynamics across a broad range of specimen types is critical to establishing the limits of detection and timing of diagnostics in order to make the greatest impact for diagnosis, case management and surveillance. ensuring a test has appropriate sensitivity and specificity is a major challenge in the development of diagnostics for novel and rare pathogens, as there is often a very limited supply of well-characterised clinical material. especially during the early stage of an outbreak, clinical evaluation must often be performed in the affected countries by laboratories working closely with the ministries of health. typically only a small number of patient specimens are shared outside of the affected countries due to strict import and export regulations, particularly for 'dual-use' pathogens. 107 108 specifically, the provisions of the nagoya protocol have significant impact on the access to genetic materials for both commercial and non-commercial applications. 109 110 in particular, the development and validation process for new diagnostics could be accelerated if well-characterised specimens and reference standards could be more easily obtained. eqa can be useful for evaluation of test performance, as shown with evaluations of both inhouse and commercial assays for mers-cov, [111] [112] [113] and bmj global health more recently a global proficiency testing programme used to assess laboratory detection of mers-cov. 114 even after validation, a substantial amount of reference material is required for quality control; often manufacturers must develop their own calibration standards to maintain supply and to control lot-to-lot variability. international reference standards and qualified specimen panels can accelerate the development and validation of diagnostic tests. in particular, the who international biological reference preparations (as provided by member states) serve as reference sources for ensuring the reliability of in vitro biological diagnostic procedures used for diagnosis of diseases and treatment monitoring, including mers-cov. several international institutes also provide specimens for validation; these groups typically have a defined pathogen/disease focus with a corresponding archive of biological reference materials; however, the supplies may be limited (see online supplementary material 1). currently, mers-cov diagnosis by pcr requires a laboratory with sophisticated facilities and biosafety cabinets. the turnaround time to receive a test result can take days to weeks, depending on laboratory proximity, sample transport options and laboratory processing capacity, 72 75 and infrastructure requirements place most pcr systems in reference laboratories, which may not be ideal for diseases like mers-cov that recommend immediate isolation for infections detected across a variety of settings. 81 115 116 a more nimble approach is needed for mers-cov case detection and triage, 92 117 and at border crossings for animal surveillance, quarantine and targeted vaccination. 11 21 87 118 the fao-oie-who mers technical working group has given a clear call for the development of an rdt to improve identification and isolation of primary human cases in healthcare facilities. 5 serological rdts are ideal for low infrastructure settings such as a primary health clinic, home or field testing. however, specimen collection remains a key challenge for mers-cov, as the recommended lower respiratory specimens are difficult to obtain outside of a hospital setting. upper respiratory specimens such as nasal swabs are easy to obtain and work well in conjunction with rdts for camels, but these specimens generally have low virus titre in humans, thus limiting current use of rdts to animal testing. 87 92 96 improvement of the current rdt detection chemistry, if feasible, may support the future use of these tests in humans, at least for rapid triage in highly infectious cases. poc and near-poc microfluidic platforms enable a more flexible, but still highly sensitive approach for near-patient naat testing in decentralised settings. near-poc naat platforms are compact and self-contained, with automated sample preparation for processing in minimal laboratory settings, which most healthcare workers can be trained to operate within a day. [119] [120] [121] recent publications describe mers-cov assays designed for poc pcr, 89 loop-mediated isothermal amplification assay 122 and paper-based sensor detection 123 ; however, no mers-cov assays are currently available for the existing near-poc platforms. given that pcr is now the standard for mers-cov diagnosis, it would be highly desirable to have an automated, self-contained naat assay that can be readily deployed in a field or clinic setting. syndromic testing can be valuable during the early stages of an outbreak, in order to distinguish mers-cov from other respiratory infections or identify cases of coinfection. 100 124 a syndromic panel could be effective in expediting pathogen and outbreak identification, especially with technologies that can screen for multiple pathogens simultaneously. 125 using the panel approach, a definitive diagnosis could enable timely decisions about triage, treatment, infection control and contact tracing. 126 while the per-test cost rises with test complexity, including additional reagents and more sophisticated instrumentation, a rapid and efficient diagnosis scheme can impact intervention and infection control and can be cost-saving overall. 127 128 as respiratory diseases are both regional and seasonal, 129-131 region-specific panels may be more cost-effective. 132 multiplex panels offer the alternative for a 'bundled' testing paradigm; however, if not routinely used (if the market is small), then developers may be reluctant to support the test for diagnostic use, which requires additional investment for validation and regulation. surveillance can be an effective method to identify the initial stages of outbreak, but it requires routine and effective sampling. the impact of surveillance testing depends on the test sensitivity and specificity, sampling rates, kinetics of the disease, and whether the target is animal or human populations. most surveillance sampling is performed in the field, either through population-based or 'hot spot' sampling. for mers-cov, it may be difficult and expensive to implement routine surveillance in dromedary camel stock, as they represent a significantly large reservoir but may suffer only mild effects from mers-cov infection, if any. the ideal surveillance tool would be a highly sensitive and field-appropriate screening test. per-test cost is also an important factor along with ease of implementation. this review has identified diagnostics currently available for mers-cov and highlighted ongoing challenges caused by critical gaps in diagnostics to support outbreak management. rdts offer the potential for rapid poc screening for mers-cov; however, there are practical limits to implementing lower respiratory sample acquisition outside of a hospital setting, limiting feasibility. poc or near-poc naat platforms provide an opportunity for implementation of automated, self-contained bmj global health testing in hospitals and clinics with limited training in endemic-prone areas. expansion of test menu options for existing poc or near-poc naat platforms will strengthen incountry response capacity to endemic diseases and simultaneously ensure countries are prepared for future pandemics. syndromic multiplex panels may expedite differential diagnosis of mers-cov from other endemic respiratory diseases, but further analysis is needed to inform implementation and cost-effectiveness in the context of regional and seasonal detection. there is also a need for more sensitive serological assays with lower cost and minimum cross-reactivity that can be used as surveillance tools. a more detailed understanding of mers-cov viral and antibody kinetics is needed across the broad range of sample types in order to optimise the use of existing assays and to address ongoing technical challenges in the detection of mild and asymptomatic infections. surveillance continues to be important for the detection of mers-cov spillover events; however, questions remain on the cost-effectiveness of routine screening of the large reservoir camel population. in addition, support towards sample biobanks with well-characterised specimens and reference standards will facilitate diagnostic development and quality assurance for mers-cov diagnostics worldwide. in order to achieve the goals of the r&d blueprint efforts, who is identifying key target product profiles for diagnostics in order to mobilise funding and resources to support the development and implementation of the most critically needed tests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who | middle east respiratory syndrome coronavirus (mers-cov). who mers-cov r&d blueprint plan of action r&d blueprint for action to prevent epidemics progress on the global response, remaining challenges and the way forward evidence for camel-tohuman transmission of mers coronavirus human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir risk factors for mers coronavirus infection in dromedary camels in burkina faso cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt mers coronaviruses in dromedary camels geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan absence of middle east respiratory syndrome coronavirus in camelids antibodies against mers coronavirus in dromedary camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia lack of middle east respiratory syndrome coronavirus transmission from infected camels mers coronavirus in dromedary camel herd, saudi arabia cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia dromedary camels in northern mali have high seropositivity to mers-cov middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels serologic evidence for mers-cov infection in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation mers-cov situation update, map 1. mers-cov livestock field surveys by country epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate middle east respiratory syndrome coronavirus antibody reactors among camels in dubai identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates mers cov: oie -world organisation for animal health occupational exposure to dromedaries and risk for mers-cov infection risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during 2013-2014: a case-control study middle east respiratory syndrome (mers) | symptoms & complications | cdc middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia transmission of merscoronavirus in household contacts hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description the role of super-spreaders in infectious disease super-spreading events of mers-cov infection mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study development of medical countermeasures to middle east respiratory syndrome coronavirus who | infection prevention and control (ipc) guidance summary. who who recommended surveillance standards an observational, laboratorybased study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia microevolution of outbreakassociated middle east respiratory syndrome coronavirus, south korea middle east respiratory syndrome coronavirus: virology, pathogenesis, and epidemiology middle east respiratory syndrome coronavirus vaccines: current status and novel approaches challenges presented by mers corona virus, and sars corona virus to global health a roadmap for mers-cov research and product development: report from a world health organization consultation nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronaviruses -drug discovery and therapeutic options a novel neutralizing monoclonal antibody targeting the n-terminal domain of the mers-cov spike protein vaccine development for emerging virulent infectious diseases rapid development of vaccines against emerging pathogens: the replication-deficient simian adenovirus platform technology report from the world health organization's third product development for vaccines advisory committee (pdvac) meeting middle east respiratory syndrome vaccines chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice emerging infectious diseases: a proactive approach laboratory testing for middle east respiratory syndrome coronavirus kinetics of serologic responses to mers coronavirus infection in humans viral load kinetics of mers coronavirus infection viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection the role of laboratory diagnostics in emerging viral infections: the example of the middle east respiratory syndrome epidemic mers coronavirus: data gaps for laboratory preparedness predictors of mortality in middle east respiratory syndrome (mers) mers-cov diagnosis: an update clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak who | laboratory biosafety manual -third edition challenges and opportunities for the implementation of virological testing in resource-limited settings advances in addressing technical challenges of point-of-care diagnostics in resource-limited settings who | laboratory testing for middle east respiratory syndrome coronavirus. who mers-cov lab detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction performance and clinical validation of the realstar mers-cov kit for detection of middle east respiratory syndrome coronavirus rna real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus development of dual taqman based one-step rrt-pcr assay panel for rapid and accurate diagnostic test of mers-cov: a novel human coronavirus, ahead of hajj pilgrimage development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels an isothermal, label-free, and rapid one-step rna amplification/detection assay for diagnosis of respiratory viral infections comparison of eplex respiratory pathogen panel with laboratory-developed real-time pcr assays for detection of respiratory pathogens clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of middle east respiratory syndrome coronavirus from upper respiratory tract specimens a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus a highly specific rapid antigen detection assay for on-site diagnosis of mers seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity lateral flow assays development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen middle east respiratory syndrome (mers) coronavirus and dromedaries identification of mycoplasma suis antigens and development of a multiplex microbead immunoassay serosurveillance of viral pathogens circulating in west africa two-tube multiplex real-time reverse transcription pcr to detect six human coronaviruses a multiplex liquid-chip assay based on luminex xmap technology for simultaneous detection of six common respiratory viruses surveillance and testing for middle east respiratory syndrome coronavirus using healthmap to analyse middle east respiratory syndrome (mers) data progress in promoting data sharing in public health emergencies who | influenza surveillance outputs data sharing: make outbreak research open access the weapon potential of a microbe biological agents: weapons of warfare and bioterrorism explanation of the nagoya protocol on access and benefit sharing and its implication for microbiology global scientific research commons under the nagoya protocol: towards a collaborative economy model for the sharing of basic research assets first international external quality assessment of molecular diagnostics for mers-cov external quality assessment of mers-cov molecular diagnostics during the 2015 korean outbreak external quality assessment for the molecular detection of mers-cov in china proficiency testing for the detection of middle east respiratory syndrome coronavirus demonstrates global capacity to detect middle east respiratory syndrome coronavirus point-of-care testing for infectious diseases: diversity, complexity, and barriers in low-and middle-income countries diagnostic point-of-care tests in resource-limited settings response to emergence of middle east respiratory syndrome coronavirus an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels evaluation of the whole-blood alere q nat point-of-care rna assay for hiv-1 viral load monitoring in a primary health care setting in mozambique point-of-care cepheid xpert hiv-1 viral load test in rural african communities is feasible and reliable performance of the samba i and ii hiv-1 semi-q tests for viral load monitoring at the point-of-care one-pot reverse transcriptional loop-mediated isothermal amplification (rt-lamp) for detecting mers-cov multiplex paperbased colorimetric dna sensor using pyrrolidinyl peptide nucleic acid-induced agnps aggregation for detecting mers-cov, mtb, and hpv oligonucleotides the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus wpro | second meeting on laboratory strengthening for emerging infectious diseases in the asia pacific region point-counterpoint: large multiplex pcr panels should be first-line tests for detection of respiratory and intestinal pathogens cost analysis of multiplex pcr testing for diagnosing respiratory virus infections impact of a rapid respiratory panel test on patient outcomes pcr for detection of respiratory viruses: seasonal variations of virus infections global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study prevalence and seasonal distribution of respiratory viruses during the 2014 -2015 season in istanbul development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay acknowledgements we gratefully acknowledge input to the roadmap from all those who attended the fao-oie-who global technical meeting on mers-cov in september 2017. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organisations with which they are affiliated. editorial assistance for later drafts was provided by rachel key: cord-353495-c3s5n5vo authors: yao, yanfeng; bao, linlin; deng, wei; xu, lili; li, fengdi; lv, qi; yu, pin; chen, ting; xu, yanfeng; zhu, hua; yuan, jing; gu, songzhi; wei, qiang; chen, honglin; yuen, kwok-yung; qin, chuan title: an animal model of mers produced by infection of rhesus macaques with mers coronavirus date: 2014-01-15 journal: j infect dis doi: 10.1093/infdis/jit590 sha: doc_id: 353495 cord_uid: c3s5n5vo in 2012, a novel coronavirus (cov) associated with severe respiratory disease, middle east respiratory syndrome (mers-cov; previously known as human coronavirus–erasmus medical center or hcov-emc), emerged in the arabian peninsula. to date, 114 human cases of mers-cov have been reported, with 54 fatalities. animal models for mers-cov infection of humans are needed to elucidate mers pathogenesis and to develop vaccines and antivirals. in this study, we developed rhesus macaques as a model for mers-cov using intratracheal inoculation. the infected monkeys showed clinical signs of disease, virus replication, histological lesions, and neutralizing antibody production, indicating that this monkey model is suitable for studies of mers-cov infection. coronaviruses (covs) can infect humans and a wide variety of animals, causing respiratory, enteric, hepatic, and neurological diseases of varying clinical severity. based on their genotypic and serological characteristics, they have been classified into 3 genera: alphacoronavirus, betacoronavirus, and gammacoronavirus [1] . coronaviruses are well known for their high frequency of recombination and high mutation rates, which may allow them to adapt to new hosts and ecological niches. this is best exemplified by the severe acute respiratory syndrome (sars) epidemic, which was caused by sars-cov [2] . sars-cov was shown to have originated from animals, with horseshoe bats (rhinolophus sinicus) as the natural reservoir and the palm civet (paguma larvata) as the intermediate host, allowing animal-to-human transmission [3] . since the sars epidemic, many other novel covs have been discovered in both humans and animals. a novel betacoronavirus lineage c, including the tylonycteris bat cov hku4 (ty-batcov hku4) and pipistrellus bat cov hku5 (pi-batcov hku5), was discovered in the lesser bamboo bat (t. pachypus) and the japanese pipistrelle (p. abramus) in 2007 in hong kong, china. these viruses were found to be closely related to a novel strain of human cov, referred to as middle east respiratory syndrome cov (mers-cov), which was identified in saudi arabia [4] [5] [6] . this novel human virus is now classified as a lineage c betacoronavirus that differs from other covs previously found in humans, including sars [7] . to date, 114 human cases of mers-cov have been reported, with 54 fatalities [8] . mers-cov is associated with severe respiratorytract infection, renal failure, and fatalities. similar to sars-cov, mers-cov is closely related to bat covs, suggesting that bats may be the natural reservoir of this family of viruses [9, 10] . specifically, a virus from egyptian tomb bats showed 100% nucleotide identity to virus from the human index case-patient [11] . it is currently unclear whether the human cases were a result of direct zoonotic transmission from bats to humans or whether an intermediate host was involved. however, a recent study showed mers-cov reactive antibodies in retired racing camels in oman, which neighbors saudi arabia [12] . in middle eastern nations, large amounts of camel meat were consumed every year, much of which was imported from african countries like egypt. this epidemiological evidence may suggest a bat-camel-human viral linkage, while more investigations are needed to define the source of mers-cov. currently, there is no evidence of efficient transmission between humans by mers-cov. however, the occurrence of some clusters of cases suggests that human-to-human transmission is possible and has raised concerns regarding the potential of this virus to cause a pandemic similar to that caused by sars-cov in 2002/ 2003 [13, 14] . the pathogenic mechanism of mers-cov is not clear, and neither an effective vaccine nor therapeutic drugs are available for prevention and treatment. the development of animal models for mers-cov infection of humans is of utmost importance to study the pathogenesis of this virus and to test the efficacy of potential therapeutic or prophylactic intervention strategies. at present, there are few reports of animal model for mers-cov infection, thus limiting further study [15, 16] . nonhuman primates have played an essential role in our understanding of the various forms of the pathogen, which could reflect variable clinical symptoms and pathology in humans. previous studies have reported nonhuman primate disease models for influenza, sars, and other viruses [17] [18] [19] . therefore, in the present study, we explored the suitability of the rhesus monkey as an animal model for mers-cov isolate human coronavirus-erasmus medical center (hcov-emc) infection or disease. the research on mers-cov virus was discussed among the staff members of the department of pathogen biology at the institute of laboratory animal science (ilas) of the chinese academy of medical sciences and peking union medical college (pumc). the mers-cov animal model experiments and protocols were discussed explicitly and extensively among the staff members of the department of pathogen biology. these discussions were followed by additional discussions with biosafety officers and facility managers at the ilas of pumc, as well as with numerous specialists from the sars-cov and general infectious disease fields throughout china. all research procedures were approved by the ilas institutional animal care and use committee and laboratory safety committee (lsc). the committee recommended that the number of animals be reduced to comply with the 3r (reduction, replacement, refinement) principles; we therefore designed the experiments to include 6 animals to test the animal model of mers-cov, 4 monkeys infected with virus and 2 uninfected monkeys as controls. the approved registration number is ilas-pc-2013-004. all experiments were conducted within the animal biosafety level 3 (absl-3) facility, which was constructed and accredited based on national standard gb19489 at the ilas of pumc, beijing, china. mers-cov strain hcov-emc was a kind gift from professor fouchier [4] . seed stocks of hcov-emc were propagated in vero cells. the seed stocks were diluted to the designated titer and used for determining the hcov-emc 50% tissue culture infection dose (tcid 50 ) and performing the neutralizing antibody assays. vero cells were maintained in dulbecco's modified eagle medium (dmem; invitrogen) supplemented with 10% fetal bovine serum (fbs), 100 international units (iu)/ml penicillin, and 100 µg/ml streptomycin and cultured at 37°c in 5% co 2 . four monkeys, aged 2-3 years, were inoculated intratracheally with hcov-emc (6.5 × 10 7 tcid 50 /1 ml) diluted in dmem. the monkeys were anesthetized, and 1 ml of the inoculum was administered intratracheally. mock-infected monkeys (2 monkeys) intratracheally inoculated with dmem were included as controls. the monkeys were observed twice daily, with detailed recording of clinical signs, symptoms, morbidity, and mortality, including the nature, onset, severity, and duration of all gross or visible changes. chest x-rays were performed before inoculation and 3 and 5 days postinoculation (d.p.i.) with mers-cov. two infected monkeys and a control monkey were sacrificed at 3 d.p.i. tissue specimens, including lung, trachea, heart, spleen, kidney, brain, liver, and colon tissue, were collected for various pathological, virological, and immunological tests. for the virological and immunological tests, swab samples of the oropharyngeal, nasal turbinates, and cloacal regions were collected at 1, 3, 5, 7, 9, 11, 14, 21, and 28 d.p.i., and blood was collected at 7, 14, 21, and 28 d.p.i. total rna was isolated from individual samples using the rneasy mini kit (qiagen) according to the manufacturer's instructions. reverse transcription (rt) reactions were performed using the superscript iii first strand synthesis kit (invitrogen) according to the manufacturer's instructions. all complementary dna (cdna) samples were stored at −20°c until use in the polymerase chain reaction (pcr). for most experimental samples and the positive control (hcov-emc), duplicate cdna samples were obtained from each total rna sample. the primers used for hcov-emc were described previously [20] and are specific for the rna-dependent rna polymerase (rdrp) gene. the sequences of the primers used for pcr detection were as follows: rdrpseq-fwd (tgctat wagtgctaagaatagrgc; r = a/g, w = a/t) and rdrpseq-rev (gcatwgcncwgtcacacttagg; w = a/ t, n = a/c/t/g). the amplification protocol was as follows: 95°c for 3 minutes, followed by 45 cycles of 95°c for 15 seconds, 56°c for 15 seconds, and 72°c for 30 seconds, with a terminal elongation step of 72°c for 2 minutes. for cases in which no amplification products were obtained with the pcr assay, a 50-µl second-round reaction was set up containing 2 µl of the reaction mixture from the first round, the primer rdrpseq-fwd (the same as that used in the first round), and rdrpseq-rnest (cacttaggrtartcccawccca). thermal cycling was performed as follows: 95°c for 3 minutes, followed by 45 cycles of 95°c for 15 seconds, 56°c for 15 seconds, and 72°c for 30 seconds, followed by a 2-minute extension step at 72°c. tissue samples were homogenized to a final 10% (weight per volume) suspension in dmem and clarified by low-speed centrifugation at 4500 g for 30 minutes at 4°c. swab samples were immersed in 1 ml dmem, vortexed, and clarified by low-speed centrifugation at 5000 g for 10 minutes at 4°c. virus titers were determined in vero cells monolayers grown in 96-well plates. vero cells were seeded (1.5 × 10 4 /well) in a 96-well plate and incubated overnight at 37°c in a co 2 incubator. then, 100 µl of 10-fold serially diluted suspension was added to each well in quadruplicate. the virus was allowed to adsorb to the cells at 37°c for 1 hour. after adsorption, the viral inocula were removed, and 0.1 ml medium (dmem, 2% fbs) was added to each well. the plates were incubated in a co 2 incubator at 37°c for 3 days, after which the cytopathic effects (cpes) were observed microscopically at 40× magnification. the virus titer of each specimen, expressed as the tcid 50 , was calculated by the reed-muench method. for the neutralization test, serum samples were diluted 2-fold from an initial 1:10 dilution to a final dilution of 1:1280, mixed with 50 µl of 2000 tcid 50 /ml virus, and incubated at 37°c for 1 hour; this step was performed in quadruplicate. thereafter, 100 µl virus-serum mixture was added to vero cells previously seeded at 1.5 × 10 4 /well. the inoculated plates were incubated in a co 2 incubator at 37°c for 3 days, after which the cpes of the virus were observed microscopically at 40× magnification. animal necropsies were performed according to a standard protocol. samples for histological examination were stored in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin prior to examination by light microscopy. alternatively, the sections were immunohistochemically stained using a polyclonal antibody against the nucleoprotein of hcov-emc. the monkeys were observed daily for clinical signs of disease for 28 d.p.i., and body weight and temperature were recorded. the rectal temperature was obtained daily in the first week after challenge, after which the temperature was measured at specific time points; graphs of the temperature profiles (figure 1 ) depict the mean temperatures for the mock-infected and mers-cov-infected monkeys. the temperature of the infected monkeys increased at 1-2 d.p.i, and returned to normal thereafter. moreover, within the first few days, the infected monkeys drastically decreased their water intake. none of the infected monkeys showed overt weight loss when compared with the mock-infected monkeys. radiographic imaging revealed varying degrees of localized infiltration and interstitial markings ( figure 2 ). none of the infected monkeys died during the experiment. total rna was isolated from tissue homogenates and oropharyngeal, nasal turbinates, and cloacal swab samples and analyzed using primers specific for the hcov-emc rdrp gene. viral rna was detected in lung homogenates obtained from the infected monkey at 3 d.p.i.; however, the other organs remained rt-pcr negative (table 1) . no viral rna was detected in any of the swab samples (table 2) . nasal turbinates, oropharyngeal, and cloacal swabs were collected from inoculated animals at 1, 3, 5, 7, 11, 14, 21, and 28 d. p.i., and virus titers were determined by end-point titration in vero cells. the oropharyngeal, nasal turbinates, and cloacal swabs contained no detectable virus titers during the experiment, which was in agreement with the rt-pcr results. samples of the tissues described above were homogenized, and virus titers were determined. for the animals inoculated with mers-cov, virus could be detected in the lung (10 1.67 tcid 50 / ml); however, samples from the kidney, trachea, brain, heart, liver, spleen, and intestine had no detectable virus titers. the levels of hcov-emc-neutralizing antibody were evaluated. no neutralizing antibodies were detected in mock-infected monkeys. by contrast, in the infected monkeys, neutralizing antibodies were detected beginning at 7 d.p.i., reaching a peak titer of 1:320 at 14 d.p.i., and decreasing slightly to 1:160 at 28 d.p.i. (figure 3 ). two mers-cov-infected animals were euthanized at 3 d.p.i., and the trachea, lung, brain, heart, liver, spleen, kidney, and intestine were histologically and virologically examined. lung lesions were present in the inoculated monkeys. grossly, there was congestion, and palpable nodules, which showed as being scattered in distribution, were particularly evident in the posterior regions of the lobes (figure 4) . microscopically, lung tissue showed multifocal mild-to-moderate interstitial pneumonia and exudative pathological changes ( figure 5) . immunohistochemistry was also performed to assess the presence of mers-cov-infected cells in tissues collected from the infected animals and control animals ( figure 5 ). in monkeys inoculated with the mers-cov, infected cells were identified in the lung and in the same regional area as the pathological changes. types i and ii pneumocytes and alveolar macrophages were found to contain viral antigen. no vascular endothelium was observed ( figure 5 ). in addition, infected cells were not observed in other organs. in the control group, no positive areas were identified in any organs. currently, humans infected with mers-cov have been reported to develop a severe acute respiratory illness with symptoms of fever, cough, shortness of breath, and often, renal failure. more than half of the reported patients have died. some laboratory-confirmed cases reportedly experience a mild respiratory illness. the development of animal models that reflect these variations in the human population is challenging. in this study, we sought to develop an animal model that displays these symptoms. our studies have tested mouse, ferret, and guinea pig models and found these animals are not susceptible to mers-cov (unpublished data). nonhuman primates have been useful for evaluating vaccines and studying disease pathogenesis for several respiratory viruses, including influenza, sars-cov, respiratory syncytial virus, and human parainfluenza viruses. in this study, the rhesus monkey was explored as a model for mers-cov. not only did this monkey model support viral growth, it also manifested respiratory and generalized illness along with tissue pathology. the mers-cov dose used in this study was 6.5 × 10 7 tcid 50 by intratracheal infection. after infection, we observed a temperature increase in the infected animals. fever is one of the markers of mers-cov infection in humans [21] , and in the included monkeys, the temperature increase was significant from 1-2 d.p.i. the body weight of each monkey was also recorded prior to viral infection, but no significant change in mean weight was observed. virus replication was detected in the lungs of the infected animals. however, we did not detect virus rna or measurable titers in the nasal turbinate or oropharyngeal samples, suggesting that the monkeys did not shed virus in the upper-respiratory tract. in humans, mers-cov was detected in lower-respiratory-tract specimens with a high viral load, whereas nasopharyngeal samples were weakly positive or inconclusive [22] . this monkey model showed similar results. the primary mers-cov infection-related pathology in humans was acute pneumonia accompanied by multiorgan dysfunction and acute renal failure, which is compatible with a broad range of tissue tropisms in cell culture susceptibility testing [23] . the pathological presentation of pneumonia was also observed in the infected monkeys ( figure 3) ; however, no extrapulmonary lesions were found. these findings are consistent with the report by vincent et al, who reported similar findings of acute localized-to-widespread pneumonia that resulted in mild-to-moderate clinical disease [15] . in humans, renal failure is a major symptom of mers; however, we observed no pathological changes in the kidneys. the reasons for this discrepancy are not clear and deserve further study. it is possible that dissemination of the virus may not yet have occurred and the virus was limited to the lung, as we did not detect the virus in the blood. to determine whether the animals were infected, we developed a neutralizing antibody assay against mers-cov. there was evidence of seroconversion in all the inoculated animals. mers-cov-specific antibody was detected in the monkey serum samples beginning at 7 d.p.i., and the titer increased gradually. neutralizing antibody protection assays showed that the infected monkeys produced neutralizing antibodies that could prevent infection due to rechallenge by mers-cov. the results also indicate the potent immunogenicity of mers-cov, suggesting the possibility of using the virus as an inactivated or attenuated vaccine. in summary, the viral detection assays, immunological response, and pathological changes in the lungs of the infected animals suggest that mers-cov was able to establish an infection in the inoculated monkeys. additionally, the rhesus macaque appears to be a suitable model for studies of mers-cov pathogenesis or potential prophylactic or therapeutic intervention strategies. up to this stage, pathogenesis of mers-cov in human is still not clear. rhesus macaques can be infected by mers-cov in the laboratory, but we recognize that it is necessary to search for more suitable animal model that will have similar disease presentations as that observed among those laboratoryconfirmed human cases. however, before another animal model is defined, rhesus macaques may be used as an infection model for the evaluation of vaccine and antiviral drugs as stated in a recent paper [24] . is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation of a novel coronavirus from a man with pneumonia in saudi arabia comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features genetic characterization of vetacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans world health organization. global alert and response: novel coronavirus infection-update human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe coronaviruses in bats from mexico middle east respiratory syndrome coronavirus in bats, saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence of person-to-person transmission within a family cluster of novel coronavirus infections family cluster of middle east respiratory syndrome coronavirus infections pneumonia from human coronavirus in a macaque model the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters cross-reactive t cells are involved in rapid clearance of 2009 pandemic h1n1 influenza virus in nonhuman primates an animal model of sars produced by infection of macaca mulatta with sars coronavirus persistent pneumocystis colonization leads to the development of chronic obstructive pulmonary disease in a nonhuman primate model of aids assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques key: cord-348401-x2q9vyf2 authors: millet, jean k.; séron, karin; labitt, rachael n.; danneels, adeline; palmer, kenneth e.; whittaker, gary r.; dubuisson, jean; belouzard, sandrine title: middle east respiratory syndrome coronavirus infection is inhibited by griffithsin date: 2016-07-15 journal: antiviral res doi: 10.1016/j.antiviral.2016.07.011 sha: doc_id: 348401 cord_uid: x2q9vyf2 highly pathogenic human coronaviruses associated with a severe respiratory syndrome, including middle east respiratory syndrome coronavirus (mers-cov), have recently emerged. the mers-cov epidemic started in 2012 and is still ongoing, with a mortality rate of approximately 35%. no vaccine is available against mers-cov and therapeutic options for mers-cov infections are limited to palliative and supportive care. a search for specific antiviral treatments is urgently needed. coronaviruses are enveloped viruses, with the spike proteins present on their surface responsible for virus entry into the target cell. lectins are attractive anti-coronavirus candidates because of the highly glycosylated nature of the spike protein. we tested the antiviral effect of griffithsin (grft), a lectin isolated from the red marine alga griffithsia sp. against mers-cov infection. our results demonstrate that while displaying no significant cytotoxicity, griffithsin is a potent inhibitor of mers-cov infection. griffithsin also inhibits entry into host cells of particles pseudotyped with the mers-cov spike protein, suggesting that griffithsin inhibits spike protein function during entry. spike proteins have a dual function during entry, they mediate binding to the host cell surface and also the fusion of the viral envelope with host cell membrane. time course experiments show that griffithsin inhibits mers-cov infection at the binding step. in conclusion, we identify griffithsin as a potent inhibitor of mers-cov infection at the entry step. coronaviruses belong to the coronaviridae family. they are enveloped viruses with a large single stranded, positive-sense rna genome and infect both humans and animals. until 2003, human coronaviruses were responsible for only mild respiratory diseases, mainly common colds. this changed with the emergence of a highly pathogenic coronavirus associated with a severe respiratory syndrome, namely the severe acute respiratory syndrome coronavirus (sars-cov). in 2012, a new deadly human coronavirus emerged in the arabian peninsula, middle east respiratory syndrome coronavirus (mers-cov) responsible for severe pneumonia that can be associated with additional clinical symptoms such as vomiting, diarrhea or renal failure (alhogbani, 2016; zaki et al., 2012) . the mers-cov epidemic is still ongoing, and so far more than 1700 laboratory-confirmed cases have been diagnosed with a mortality rate of approximately 35%. there are neither clinically approved antivirals nor vaccines available against mers-cov and therapeutic options for mers-cov infections are limited to palliative and supportive care. the mers-cov spike protein is a main determinant of virus entry into host cells as it mediates both binding to the dpp4 (dipeptidyl peptidase 4) receptor and fusion of the viral envelope with host cell membrane (millet and whittaker, 2014; raj et al., 2013) . it is a type i fusion protein and is highly glycosylated with 19 predicted n-glycosylation sites. one option to block mers-cov infection is to take advantage of the highly glycosylated nature of its spike protein by using lectins in order to inhibit mers-cov entry and thus virus propagation. griffithsin (grft) is a 121 amino acid long lectin that was isolated from the red marine alga griffithsia sp. griffithsin has been shown to have antiviral activity against hiv-1 within the picomolar range (mori et al., vero-81 cells were treated with increasing concentrations of griffithsin (a) or with 0 mg/ml (à) or 1 mg/ml (þ) of the lectin (b). for (a), after 2 h of incubation, supernatants were replaced with medium without griffithsin and cells were incubated at 37 c for 7 h. in (b), cells were left treated with griffithsin for 24 h or 48 h. cell viability was measured using a luminescence-based atp quantitation assay. results are expressed as average of cell viability (% of 0 mg/ml control), with error bars representing sd from the average of three independent experiments. data were statistically analyzed using one-way anova test (a) or using a two-tailed student t-test (b), with the following convention for p-value significance: not significant (n.s.), p > 0.05. (c) dose-response analysis of griffithsin on mers-cov infection. immunofluorescence assay of mers-cov-infected huh-7, mrc-5, and vero-81 cells in presence of increasing concentrations of griffithsin. cells were infected with mers-cov strain emc/2012 at an m.o.i. of 10, in presence of increasing amounts of griffithsin for 2 h at 37 c. pbs was used for the non-treated control condition. cells were washed with pbs and the inoculum replaced with cell growth medium. the cells were incubated at 37 c 5% co 2 for an additional 7 h. cells were then fixed, immunolabeled for mers-cov s, and stained for nuclei (dapi) . n.i.: non infected control. (d) quantification of mers-cov-positive cells in presence of increasing concentration of griffithsin. for each condition, five 10 â objective fields were randomly acquired and analyzed for total number of 2005). characterization of griffithsin structure has demonstrated that it is a domain-swapped homodimer with three carbohydrate binding domains on each monomer that bind to terminal mannose residues linked on hiv envelope protein gp120 n-glycans (zi ołkowska et al., 2006) . griffithsin has also been identified as a potent inhibitor of hepatitis c virus (hcv) and sars-cov infection in vitro and in vivo with minimal toxicity (meuleman et al., 2011; o'keefe et al., 2010) . therefore, we evaluated the anti-mers-cov activity of griffithsin. hek-293t (atcc), huh-7 (nakabayashi et al., 1982) , mrc-5 (atcc), and vero-81 (atcc) cells were grown at 37 c 5% co 2 in dmem (corning) supplemented with 10% fetal bovine serum (thermofisher), 10 mm hepes (corning), 100 iu/ml penicillin and 100 mg/ml streptomycin (corning). baric (unc chapel hill) and bart haagmans (utrecht university). the virus was propagated in vero-81 cells, with viral titrations performed using tcid 50 assays. all experiments involving authentic mers-cov were performed in a biosafety level 3 facility (animal health diagnostic center, cornell university). mannonanose-di-(n-acetyl-d-glucosamine) (man-9 glycan), was purchased from sigma aldrich. celltiter-glo luminescent cell viability assay reagents and luciferase assay kit were purchased from promega. 2.5 â 10 4 huh-7, mrc-5, and vero-81 cells were seeded in 96well plates and incubated for 16 h at 37 c 5% co 2 incubator. the cells were then treated with increasing concentrations of griffithsin, from 0 (pbs) up to 2 mg/ml, a range used in subsequent experiments. the treated cells were incubated at 37 c 5% co 2 for 2 h. the medium was replaced with fresh growth medium without griffithsin and cells were incubated for another 7 h at 37 c 5% co 2 . to test the effect of griffithsin for longer incubation times, the cells were left treated with either 0 or 1 mg/ml griffithsin for 24 h or 48 h. the cells were then lysed and viability assayed using the celltiter-glo kit according to manufacturer's guidelines. luminescence (relative luminescence units -rlu) readings were performed using a glomax 20/20 luminometer (promega). the percent cell viability was calculated by the following equation: the assays were carried out using triplicate wells, and data corresponds to the average of three independent experiments. 1.25e1.5 â 10 5 huh-7, mrc-5, and vero-81 cells were seeded in wells of 8-well chamber slides (ibidi) and incubated at 37 c 5% co 2 for 16 h. increasing concentrations of griffithsin (0e2 mg/ml) were added to mers-cov strain emc/2012 viral inoculums that were used to infect cells at a multiplicity of infection (m.o.i.) of 10. noninfected cells and pbs-treated virus inoculum were used as negative controls. the infected cells were incubated at 37 c 5% co 2 for 2 h, after which the inoculum was removed, followed by pbs washing to remove unbound virions and addition of cell culture medium. the infection was left to proceed at 37 c 5% co 2 for an additional 7 h. the wells of chamber slides were then processed for immunofluorescence assay as described in 2.8. immunofluorescence microscopy assays. 3 â 10 5 huh-7 and mrc-5 cells were seeded in 24-well plate wells and incubated at 37 c 5% co 2 for 16 h. cells were infected with virus inoculum that was untreated or treated with 1 mg/ml griffithsin. the infected cells were incubated at 37 c 5% co 2 for 2 h, after which the inoculum was removed, followed by pbs washing to remove unbound virions and addition of cell culture medium containing or not 1 mg/ml griffithsin. cells were left to incubate at 37 c 5% co 2 . at 24 h and 48 h post infection time points, cell culture supernatants were harvested and stored at à80 c. cell titrations were performed using a tcid 50 assay on vero-81 cells. results are expressed as log 10 tcid 50 /ml and represent averages of two independent experiments. particles pseudotyped with either mers-cov-s envelope proteins (merspp), genotype 2a hcv envelope proteins (hcvpp), or the g envelope glycoprotein of vesicular stomatitis virus (vsv-gpp) were produced as previously described (belouzard et al., 2009; op de beeck et al., 2004) . pseudotyped virions were used to inoculate huh-7 cells in 48-well plates for 3 h in the presence of griffithsin. the inoculum was removed and cells were further incubated with culture medium for 45 h. cells were lysed and luciferase activity was detected by using a luciferase assay kit (promega) and light emission measured by using a tristar lb 941 luminometer (berthold technologies). for competition experiments with man-9, griffithsin was preincubated with man-9 at different concentrations for 3 h at 37 c before being added to merspp during the 3 h inoculation step of huh-7 cells. the inoculum was removed and luciferase activity quantified 45 h post inoculation as described above. 1.5 â 10 5 huh-7 cells were seeded in wells of 8-well chamber slides (ibidi) and incubated at 37 c 5% co 2 for 16 h. virus infection was divided into four steps: 1) inoculation and attachment of virus for 1 h at 4 c, 2) pbs wash to remove unbound virus, followed by further binding of bound virus for 1 h at 4 c, 3) internalization for 1 h at 37 c, and 4) infection for 8 h at 37 c. in all conditions, cells were infected with mers-cov strain emc/2012 at an m.o.i. of 10, with griffithsin (1 mg/ml) added or not at different steps during virus entry. in experimental condition a, griffithsin was not present at any step. in condition b, griffithsin was only present during initial attachment of virus on cells. in condition c, griffithsin was added cells (dapi nuclei stain) and s-positive cells (infected cells). results are expressed as percentage infected cells, with error bars representing sd from the average of three independent experiments. data were statistically analyzed using one-way anova test with the following convention for p-value significance: very highly significant (***), p 0.001. only during the further binding step. in condition d, griffithsin was present only during internalization. finally, in condition e, griffithsin was present during initial attachment, further binding and internalization steps (1e3), but not during infection. after mers-cov infection was left to proceed for 8 h at 37 c, the wells of the chamber slides were processed for immunofluorescence assay as described in 2.8. immunofluorescence microscopy assays. immunofluorescence were performed as previously described (millet and whittaker, 2014) . for quantifications of each condition, 5 fields were randomly acquired. for each field, the total number of cells (dapi stain) and number of infected cells (spike labeling) were counted using automatic cell counting software (imaris 7.6.5). the results of quantification are expressed in percent-infected cells. the experiment was performed using duplicate wells, and data corresponds to the average of three independent experiments. purified v5-tagged soluble mers-cov spike proteins were incubated in 1% triton x-100 in pbs in presence or absence of 2 mg/ ml of griffithsin for 2 h at 4 c followed by addition of polyclonal anti-griffithsin antibodies and a 2 h incubation. the proteinantibodies complexes were precipitated with protein a sepharose beads pre-incubated with 5% bsa to avoid nonspecific binding. proteins were separated by 8% sds-page and were transferred to nitrocellulose membrane (amersham). spike proteins were immunodetected with an anti-v5 antibody (thermofisher). analysis of experimental data, calculations of standard deviations (s.d.) and standard error of the mean (s.e.m.), and graph plotting were performed using graphpad prism 6.0. statistical analyses were performed using one-way anova test (dose-responses for cell viability and viral infectivity) and two-tailed student's t-test (viral titer assays, competition with man9, and binding assay). for p-value significance, the following convention was used: not significant (n.s.) p > 0.05, significant (*) p 0.05, highly significant (**) p 0.01, very highly significant (***) p 0.001. we assessed the effect of griffithsin on cell viability and tested its potential cytotoxicity by treating huh-7, mrc-5 and vero-81 cells with a range of griffithsin concentrations that was used in subsequent experiments, 0e2 mg/ml (fig. 1a) , or with 1 mg/ml for longer treatment exposure times (fig. 1b) . griffithsin does not decrease cell viability at the doses and times tested, results that are in agreement with a previous investigation (nixon et al., 2013) . to determine the inhibitory activity of griffithsin on mers-cov infectivity, we have carried out dose-response infection experiments in three different cell lines (fig. 1c and d) . huh-7, mrc-5, and vero-81 cells were infected with strain emc/2012 of mers-cov at an m.o.i. of 10 in presence of increasing concentrations of griffithsin (0e2 mg/ml). the infected cells were fixed at an early infection time point (9 h) and assayed by immunofluorescence microscopy analysis (fig. 1c ) followed by quantification of the percentage of infected cells for each concentration (fig. 1d) . griffithsin significantly decreased infectivity by mers-cov, with a clear, dosedependent inhibitory effect in all cell lines tested ( fig. 1c and d) . even at the most diluted concentration of 0.125 mg/ml tested, the inhibitory activity of griffithsin on mers-cov infection was significant, with a minimal drop of 44.7% in infectivity for huh-7 cells and a maximal drop of 63.2% for vero-81 cells compared to the condition without the lectin. at 2 mg/ml, griffithsin inhibited viral infectivity by more than 90% in all cell lines tested. because we have performed our assay using high m.o.i. and a short infection time, these results show the strong inhibitory activity of griffithsin on early steps of the mers-cov viral cycle. we investigated the effect of griffithsin on mers-cov virus titers (fig. 2) . huh-7 and mrc-5 cells were infected with mers-cov in presence or not of 1 mg/ml griffithsin. at 24 h and 48 h post infection, cell supernatants were harvested and analyzed for virus titers by performing a tcid 50 assay (fig. 2) . for both cell lines, griffithsin significantly decreased virus titers at both 24 and 48 h fig. 2 . effect of griffithsin on mers-cov virus titers. huh-7 and mrc-5 cells were seeded in wells of 24-well plates and infected with mers-cov in presence or not of 1 mg/ml griffithsin. after 2 h of incubation, the inoculum was removed, and cells were incubated in presence or absence of 1 mg/ml of griffithsin. at 24 h and 48 h post infection, supernatants were harvested. tcid 50 assays were performed on the collected supernatants using vero-81 cells. results are expressed as average of log 10 tcid 50 /ml, with error bars representing sd from the average of two independent experiments. data were statistically analyzed using two-tailed student t-test with the following convention for p-value significance: significant (*), p 0.05. time points, confirming the inhibitory role of the lectin on mers-cov infection. griffithsin has been shown to inhibit cellular entry of numerous viruses including sars-cov. to confirm the results obtained with mers-cov and determine the activity of this molecule on entry mediated by mers-cov spike protein, mers-cov pseudotyped virions (merspp) were produced and used to infect huh-7 cells. a dose-dependent inhibition of merspp infection was observed in the presence of griffithsin (fig. 3a ) suggesting that this compound is able to inhibit mers-cov entry. as expected, griffithsin was also able to inhibit hcvpp infection and had no effect on vsv-gpp infection as previously described (meuleman et al., 2011) . griffithsin is known to interact with n-linked high mannose oligosaccharides. the antiviral effect of this compound was shown to be due to the interactions of griffithsin with the mannoses linked to the viral envelope protein (moulaei et al., 2010) . to confirm that the inhibition observed is specific of griffithsin activity, a competition assay using n-linked high mannose oligosaccharides mannonosedi-(n-acetyl-d-glucosamine) (man-9 glycan) was performed. as shown in fig. 3b , incubation of merspp with griffithsin preincubated first with man-9 glycan significantly restores the infectivity of pseudotyped particles which suggests that griffithsin interacts with mannoses of mers-cov s envelope and impairs its functions during entry. to further confirm that griffithsin interacts with mers-cov spike protein, we performed a coimmunoprecipitation assay. soluble mers-cov spike proteins were incubated in presence or absence of griffithsin. the presence of spike proteins complexed with griffithsin was analyzed by western blot after immunoprecipitation with a polyclonal antigriffithsin antibody (fig. 3c) . western blot analysis revealed that spike protein could be coimmunoprecipitated only in presence of griffithsin. to better define which stage in the virus life cycle griffithsin acts on, we performed an infection assay using authentic mers-cov with griffithsin present at different times during viral entry steps (fig. 4a) . in this experiment, viral entry steps were divided into attachment at 4 c, further binding at 4 c, internalization at 37 c. in the control conditions, griffithsin was either not present during entry steps (a) or present during all entry steps (e). in the latter condition, griffithsin had a significant inhibitory effect on infectivity compared to control condition (a), a result which recapitulates well the dose-response results ( fig. 4b and c) . we do note however that the infectivity in condition (a) (31.7%) is lower for 3 h at 37 c before being added to the merspp for infection of huh-7. intracellular firefly luciferase signal was measured as described above. experiment was performed three times in duplicate or triplicate. error bars represent sem. data were statistically analyzed using two-tailed student's t-test comparing results with non-treated condition with the following convention for p-value significance: not significant (n.s.), significant (*) p > 0.05; highly significant (**) p 0.01, very highly significant (***), p 0.001. (c) soluble mers-cov spike protein was incubated in presence or absence of 2 mg/ml griffithsin. then griffithsin was immunoprecipitated and mers-cov spike protein was detected in western blot. than observed in the non-treated control of the dose response experiments (84.3%). we attribute this drop to differences in the infection assays used, such as shorter inoculum contact time and multiple pbs washes. in condition (b), where griffithsin is only present during the first attachment step, a significant decrease in infectivity was measured (fig. 4c) . such significant inhibition was not observed for conditions (c), and (d), where griffithsin was present only at later steps in entry (further binding without inoculum, and internalization, respectively). taken together, these data reveal that griffithsin acts on very early stages of virus entry, probably by directly interacting with the glycosylated part of mers-cov spike protein and inhibiting its binding function. mers-cov emerged in 2012 in saudi arabia causing severe pneumonia less than 10 years after the sars-cov emergence in china. in the case of mers-cov, person-to-person transmission has during the second step, the viral inoculum was removed and cells were washed three times with cold pbs and kept at 4 c for 1 h, enabling further binding of virus to cell surfaces, in presence (c and e) or absence (a, b, and d) of 1 mg/ml griffithsin. for the third step, the cells were placed at 37 c for 1 h allowing for internalization of virions to occur, in presence (d and e) or absence (a, b, and c) of 1 mg/ml griffithsin. the infection was left to proceed in a last 8 h step at 37 c in absence griffithsin. (b) immunofluorescence assay of mers-covinfected huh-7 cells with griffithsin present at different stages of infection. cells were infected and treated as described in (a) then fixed, immunolabeled and analyzed as described previously in fig. 2. (c) quantification of mers-cov-positive huh-7 cells for each condition tested. cells were analyzed as described in fig. 2 . results are expressed as percentage infected cells, with error bars representing sd from the average of three independent experiments. data were statistically analyzed using two-tailed student's t-test comparing results with non-treated condition (a) with the following convention for p-value significance: not significant (n.s.), p > 0.05; very highly significant (***), p 0.001. occurred, however transmission is not sustained, with a basic reproduction number below 1. mers-cov is a zoonotic virus, and camels and bats are its most likely reservoir. sars-cov-like viruses have been isolated in bats and it is likely that bats are the reservoir of a sars-cov ancestor. considering the increasing number and diversity of coronaviruses identified/isolated in bats and the proven propensity of coronaviruses to cross the species barrier and infect new hosts, it is likely that new coronaviruses causing severe diseases in humans may emerge in the future. currently, there are no drugs with proven efficacy to treat mers-cov or any other coronavirus infection. treatments are limited to supportive care for respiratory and circulatory functions and preventive care to protect renal, hepatic and neurological functions and to prevent secondary infection. as a consequence, new therapies are urgently needed. virus features conserved among the coronavirus family are attractive targets for the development of antiviral therapy to fight current and future emerging coronavirus infections. coronavirus spike proteins are the main determinant of virus entry and tropism (belouzard et al., 2012) . these glycoproteins are variable in sequence and length but share the same organization with two domains s1 and s2. the s1 domain is responsible for receptor binding and attachment of the virus to the host cell surface whereas s2 contains the fusion machinery. a common feature of coronavirus spike proteins is their high glycosylation with 19e39 potential glycosylation sites, coronavirus spike proteins being exclusively n-glycosylated. therefore, the carbohydrate-binding proteins lectins are attractive anti-coronavirus candidates. griffithsin is a lectin that was isolated from the red marine alga griffithsia sp. and has been shown to inhibit hiv, hcv, and sars-cov within the nanomolar range. we demonstrated that while griffithsin has minimal to no effect on cell viability, it is a potent inhibitor of mers-cov infectivity and production in vitro. by using particles pseudo-typed with the mers-cov spike protein, we have shown that griffithsin acts during mers-cov entry. among the 3 viral proteins anchored in the mers-cov envelope, in addition to the spike protein, only the m protein harbors a potential n-glycosylation site in its short n-terminal domain. for mhv, it has been shown that the m protein may be an additional target for lectins and enhance its antiviral effects. griffithsin inhibits mers-cov pseudotyped-particles to the same extent than mers-cov infection, suggesting that the spike protein is the main target of griffithsin for its antiviral effect. indeed, our results indicate a direct interaction of griffithsin with mers-cov spike protein. for hiv, griffithsin binds to the terminal mannoses of the n-glycans of gp120, this binding slightly interferes with cd4 binding and likely prevents conformational rearrangement required during entry (mori et al., 2005) . for sars-cov, it has been shown that griffithsin does not affect ace2 binding and it has been suggested that griffithsin acts at a post-binding step during entry . other plant lectins, galanthus nivalis agglutinin (gna), hippeasttrum hybrid lectin (hha) and urtica dioica agglutinin (uda) have also been shown to prevent mhv entry at a post-binding step probably by interfering with the conformational rearrangements of the spike protein that drive the fusion of the viral envelope with the host cell membrane (van der meer et al., 2007) . for mers-cov, our results show that griffithsin inhibits infection with a different mechanism. in fact, we found that griffithsin interferes with infection as early as during the attachment step, likely by preventing interaction between spike and dpp4, but is not able to prevent mers-cov infection when added at a postbinding step. indeed, a similar mechanism of receptor binding interference has already been observed for hcv (meuleman et al., 2011) . crystal structures of mers-cov receptor binding domain (rbd, residues 367 to 606) have been resolved in combination with dpp4 or in its absence. the mers-cov rbd contains a core domain with an accessory subdomain lying on the edge of the core structure (chen et al., 2013; lu et al., 2013) . both subdomains contain nlinked glycans on n210 in the core domain and on n487 in the accessory domain. the contributions of these sugar moieties to the virus-receptor interaction are unknown, but one can imagine that any involvement of one of these n-glycans in receptor binding could be impaired by griffithsin. in conclusion, griffithsin has a low cytotoxicity, likely interacts with any coronavirus spike proteins because of their highly glycosylated nature and is able to hamper coronavirus spike protein functions. griffithsin should be considered as an interesting drug candidate to develop for the treatment and/or prevention of current but also future emerging coronavirus infections. acute myocarditis associated with novel middle east respiratory syndrome coronavirus activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites mechanisms of coronavirus cell entry mediated by the viral spike protein crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 griffithsin has antiviral activity against hepatitis c virus host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity growth of human hepatoma cells lines with differentiated functions in chemically defined medium griffithsin protects mice from genital herpes by preventing cell-to-cell spread broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae characterization of functional hepatitis c virus envelope glycoproteins the carbohydratebinding plant lectins and the non-peptidic antibiotic pradimicin a target the glycans of the coronavirus envelope glycoproteins domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding this work was supported by the french national agency for research (anr, anr-14-ce15-0008) (ks, jd, and sb). work in the whittaker lab is supported by the national institutes of health grant r21 ai111085. we are grateful to sophana ung for his assistance in the illustrations. we also thank susan daniel and the whittaker lab for helpful discussions. key: cord-352256-qxdakdk0 authors: yousefi, bahman; valizadeh, saeid; ghaffari, hadi; vahedi, azadeh; karbalaei, mohsen; eslami, majid title: a global treatments for coronaviruses including covid‐19 date: 2020-05-11 journal: j cell physiol doi: 10.1002/jcp.29785 sha: doc_id: 352256 cord_uid: qxdakdk0 in late december 2019 in wuhan, china, several patients with viral pneumonia were identified as 2019 novel coronavirus (2019‐ncov). so far, there are no specific treatments for patients with coronavirus disease‐19 (covid‐19), and the treatments available today are based on previous experience with similar viruses such as severe acute respiratory syndrome‐related coronavirus (sars‐cov), middle east respiratory syndrome coronavirus (mers‐cov), and influenza virus. in this article, we have tried to reach a therapeutic window of drugs available to patients with covid‐19. cathepsin l is required for entry of the 2019‐ncov virus into the cell as target teicoplanin inhibits virus replication. angiotensin‐converting‐enzyme 2 (ace2) in soluble form as a recombinant protein can prevent the spread of coronavirus by restricting binding and entry. in patients with covid‐19, hydroxychloroquine decreases the inflammatory response and cytokine storm, but overdose causes toxicity and mortality. neuraminidase inhibitors such as oseltamivir, peramivir, and zanamivir are invalid for 2019‐ncov and are not recommended for treatment but protease inhibitors such as lopinavir/ritonavir (lpv/r) inhibit the progression of mers‐cov disease and can be useful for patients of covid‐19 and, in combination with arbidol, has a direct antiviral effect on early replication of sars‐cov. ribavirin reduces hemoglobin concentrations in respiratory patients, and remdesivir improves respiratory symptoms. use of ribavirin in combination with lpv/r in patients with sars‐cov reduces acute respiratory distress syndrome and mortality, which has a significant protective effect with the addition of corticosteroids. favipiravir increases clinical recovery and reduces respiratory problems and has a stronger antiviral effect than lpv/r. currently, appropriate treatment for patients with covid‐19 is an ace2 inhibitor and a clinical problem reducing agent such as favipiravir in addition to hydroxychloroquine and corticosteroids. healthcare workers are advised to be vaccinated every year. but sometimes more dangerous and even deadly forms occur that are likely to lead to epidemics and pandemics (andrews, 2006) . in late december 2019 in wuhan, china, several patients were diagnosed with viral pneumonia, now known as the 2019 novel coronavirus (2019-ncov). subsequently, a large number of cases of the disease were found to either reside in wuhan or travel to the city. the researchers were able to obtain the 2019-ncov genome sequence by performing bronchoalveolar lavage and culture of patients' lungs and by conducting phylogenetic analyzes of the genome and investigating the evolutionary relationship between 2019-ncov and other coronaviruses, they found the origin of 2019-ncov and its evolutionary history. the 2019-ncov genomic sequence obtained from the patients showed that 98.99% of their gene sequences matched. the genome also has an 88% similarity to the genome of the bat-sl-covzc45 and bat-sl-covzxc21 bat coronavirus genomes that appeared in zhoushan, china, but with the severe acute respiratory syndrome-related coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) coronaviruses, 79% and 50% were similar, respectively. also by homology modeling, a model for the virus receptor was obtained, which, by comparing homology models, concluded that despite differences in some amino acids, the structure of the domains attached to the receptor at 2019-ncov and sars-cov was similar. sars-cov spike (s) protein is essential for entry into the host cell. the spike s1 subunit contains the receptor-binding domain (rbd). the sars-cov virus uses the angiotensin-converting-enzyme 2 (ace2) receptor to bind the host cell, that contains a diversity of respiratory epithelial cells, alveolar macrophages, and monocytes. considering the relatively high identity of the rbd in 2019-ncov and sars-cov, it is crucial to evaluate the cross-reactivity of anti-sars-cov antibodies with 2019-ncov spike protein, which could have significant implications for rapid development of vaccines and therapeutic antibodies against 2019-ncov. these structures displayed that sars-cov rbd contains a core structure and a receptor-binding motif (rbm), and that the rbm binds to the outer surface of ace2. the ace2 is the only known human homolog of the key regulator of blood pressure ace. sars-cov-2 rbd showed significantly higher binding affinity to ace2 receptor than sars-cov rbd and could block the connection and, therefore, binding of sars-cov-2 and sars-cov rbd to ace2expressing cells, so inhibiting their infection to host cells. therefore, ace2 also serves as the cellular entry site for the sars-cov, a main target for pharmacological intervention and improved new monoclonal antibodies that could attach specifically to 2019-ncov rbd. sars-cov rbd-specific antibodies possibly will cross-react with sars-cov-2 rbd protein, and sars-cov rbd-induced antisera could neutralize sars-cov-2, suggesting the potential to improve sars-cov rbd-based vaccines for inhibition of sars-cov-2 and sars-cov infection (tai et al., 2020; wan, shang, graham, baric, & li, 2020) . bats host a reservoir of various zoonotic viruses, including coronaviruses. regulatory studies and phylogenetic analyzes have shown that there is a high genetic diversity among sars-like viruses in bats, allowing for the recombination and evolution of new species. it has been shown that the bat virus with 96% nucleotide sequence similarity to human sars-cov can use human ace2 as a receptor. this represents the same state of entry into the cell as human sars-cov. for example, bat sl-cov-wiv1 can grow on human epithelial cells and vero e6 cells, which is neutralized by human sars-cov convalescent sera (ge et al., 2013; yang et al., 2016) . the recent crisis was the outbreak of novel coronavirus from emerged wuhan in central china, referred to as 2019-ncov, has recently caused a pandemic scale of pneumonia in humans and resulted in a huge threat to the global public and a high number of hospitalizations. the damage to the lungs, which leads to fluid leaking from small blood vessels in the lungs. the fluid collects in the lungs' air sacs or alveoli. this makes it difficult for the lungs to transfer oxygen from the air to the blood. while there's a shortage of information on the type of damage that occurs in the lungs during 2019-ncov wu, leung, & leung, 2020 a glycopeptide antibiotic commonly used to treat a bacterial infection is active against sars-cov and is on the list of drugs that are also used to treat covid-19 (j. . this antibiotic is chloroquine has recently been shown to be an inhibitor of sars-cov-2 in vitro and the hydroxylated form has been suggested as a potential treatment for patients with sars-cov-2 infection. importantly, the overdose of this drug is toxic and can cause death (devaux, rolain, colson, & raoult, 2020; weniger & world health organization, 1979) . m. wang et al. (2020) reported that the ec50 of chloroquine against covid-19 tested on vero e6 was 1.13 μm and the ec90 was 6.90 μm. chloroquine inhibits sars-cov entry, which exerts its inhibitory effect by altering glycosylation of the ace2 receptor and spike protein. in patients with covid-19, hydroxychloroquine has been shown to decrease inflammatory responses and decrease cytokine storm in vitro. chloroquine also interferes with proteolytic processing of m protein and modifies virion assembly and budding. also, chloroquine indirectly act against covid-19 by reducing the production of proinflammatory cytokines and by activating anti-sars-cov-2 cd8 + t-cells (devaux et al., 2020) . it has also been used in the treatment of intracellular bacterium coxiella burnetii both in vitro and in vivo and has resulted f i g u r e 1 molecular mechanisms and therapeutic targets of drugs that have been used to treat covid-19. ace2, angiotensin-converting enzyme 2 receptor; covid-19, coronavirus disease-19; er, endoplasmic reticulum; impdh, inosine monophosphate dehydrogenase; rdrp, rna-dependent rna polymerase in its elimination (savarino, lopinavir is an antiretroviral protease inhibitor used in combination with ritonavir to treat patients with aids and hiv infection. lopinavir is a potent and highly specific inhibitor of hiv-1 protease. ritonavir inhibits the metabolism of lopinavir, therefore coadministration of lopinavir and ritonavir in healthy volunteers increases the area under the lopinavir plasma concentration-time curve> 100-fold (hurst & faulds, 2000) . in a study by cao et al. (2020) , can be obtained (lim et al., 2020) . but in a study by jun zhang at xixi hospital in hangzhou, china. a 2020 study showed that 400 mg daily lopinavir with ifn-α2b atomization inhalation, 5 million u twice daily, as well as the use of lopinavir alone could be useful for patients with covid-19 and increased eosinophils have been suggested as an indicator of improvement in patients, but after starting treatment with this drug, complications such as digestive adverse effect and hypokalemia should be considered by physicians . jinyu xia's study also showed that the combined effect of arbidol with lpv/r was greater than that of lpv/r (16 patients received oral arbidol with lpv/r and 17 patients received lpv/r alone). arbidol has been shown to have a direct antiviral effect on the initial in vitro replication of sars-cov (deng et al., 2020; khamitov et al., 2008) . arbidol (umifenovir) is a broad-spectrum antivirus that works on both enveloped and nonenveloped viruses. it is active on viruses such as influenza a and b and is also known to inhibit hepatitis c. this drug can prevent the fusion of the virus with the target membrane and block the entry of the virus into the target cell (boriskin, leneva, pecheur, & polyak, 2008 ). oseltamivir is a neuraminidase inhibitor (nais) used to treat influof recent concern about the use of oseltamivir was the emergence of resistance in 23% of patients with h1n1, which was asso ribavirin is a guanosine nucleoside analog and antiviral compound (graci & cameron, 2006; martinez, 2020) . in animal studies of coronaviruses, ribavirin, although weakly inhibitory, can reduce the release of macrophage proinflammatory cytokines in mice and alter the th-2 response to th-1 response and act as an immunomodulator (ning et al., 1998) . in most cases, ribavirin is combined with ifn, and although the results of ribavirin and ifnα-2b yousefi et al. | 5 in a mers-cov rhesus macaque model were promising, with the results of the trial and the effect of ribavirin and ifn (either α2a or β1) on mers-cov infected patients it was different, however, ribavirin lowers hemoglobin concentrations in respiratory patients and therefore reduces its potential as an antiviral against sars-cov-2 (arabi et al., 2017; falzarano et al., 2013) . ifnα-2b and ribavirin, when used for mers-cov treatment, showed that they were 8-fold and 16-fold reduced in their single-dose regimen, respectively and they further stimulate cytokine release and immune responses, reducing viral replication, modulating host response, and improving clinical outcomes. studies using ribavirin plus lopinavir/ritonavir on patients with sars have reduced ards and mortality. there was also a significant decrease in ards and mortality in patients with sars-cov who received corticosteroids with ribavirin, lopinavir/ ritonavir (chu et al., 2004; zheng & wang, 2016 ). favipiravir (fpv) a guanosine analog and an oral anti-influenza drug that targets rna-dependent rna polymerase (rdrp), which converts to active phosphoribosylated form in cells and acts as an rna polymerase inhibitor. also, favipiravir is a broad-spectrum drug that blocks the replication of flavivirus, poliovirus, rhinovirus, filovirus, and arenaviruses. the effective dose of favipiravir used is day 1: 1,600 mg twice daily, days 2-5: 600 mg twice daily but its effective dose for treatment of covid-19, and 600 mg tid with 1,600 mg first loading dosage for no more than 14 days. however, favipiravir is contraindicated in pregnant women due to teratogenicity and embryotoxicity in animals and cannot be used in this group of patients (delang, abdelnabi, & neyts, 2018; shiraki & daikoku, 2020) . a study by fang et al. (2020) found that favipiravir could play an important role in protecting mice from the fatal infection of wild-type and oseltamivir-resistant-influenza b viruses. favipiravir has also been used in combination with zanamivir to successfully clear the type b influenza virus in immunocompromised children (lumby et al., 2020) . in a randomized clinical trial, chen et al. (2020) showed that in patients with moderate covid-19 who were untreated with antiviral drugs, favipiravir increased clinical recovery over 7 days and reduced fever, cough, and respiratory problems, it was therefore used as an effective drug in these patients. another study by jianjun gao on 80 patients with covid-19 at the hospital of shenzhen, china, showed that favipiravir had a stronger antiviral effect than the lopinavir/ritonavir drug combination and reported no adverse effects (furuta, komeno, & nakamura, 2017) . a cellular inosine monophosphate dehydrogenase inhibitor and an immunomodulator that has antiviral effects on a range of viruses, including influenza a, reovirus, flavivirus, and coxsackie b3 virus, it is also used as an inhibitor of nucleotide synthesis in lymphocytes and an immunosuppressant in transplantation and treatment of autoimmune diseases (diamond, zachariah, & harris, 2002; padalko et al., 2003) . it has also been shown to inhibit hepatitis b and hepatitis c virus replication in combination with ifn-α and cyclosporine a. mycophenolic acid (mpa) in use with cyclosporine, it is also a potent inhibitor of mers-cov but has a less inhibitory effect on sars-cov. in studies of coronaviruses in the mouse model, it did not affect sars-cov, but at standard concentrations at oral doses, it was able to inhibit mers-cov (chan et al., 2013; henry et al., 2006; markland, mcquaid, jain, & kwong, 2000) . mpa acts as a synergism with ifn-β1b and thiopurine analogs but it has less protective effect than the combined effect of 6 | lopinavir/ritonavir with ifn-β1b. the use of this compound in bedside coronaviruses was that with the ifn-β1b used in saudi arabia to treat mers-cov all patients using this drug combination could survive but patients had lower acute physiology and chronic health (allison & eugui, 1993) . mpa also had a significant inhibitory effect on the proliferation of hcov-oc43 and hcov-nl63 at ec50 concentrations of 1.95 μm and 0.19 μm, respectively (shen et al., 2019) . lin et al. (2018) also showed that mpa along with disulfiram and 6-thioguanine was synergistically inhibitors of the mers-cov papain-like protease (mers-cov pl pro ). angiotensin-converting enzyme 2 gene (ace2) is a metalloproteinase with 805 amino acids which is an important receptor for entry of sars-cov into the host cell. domain s1 in the sars-cov spike protein binds the virus to the ace2 cell receptor present in the host cells. therefore understanding the association between sars-cov, sars-cov-2, and ace2 can be an effective therapeutic approach (lin et al., 2018) . ace2 is a homolog of carboxy peptidase that is best known for cleaving different peptides in the renin-angiotensin system (ras) and other substrates, such as apelin (batlle, wysocki, & satchell, 2020) . this enzyme is mostly present in organs such as the kidneys, gastrointestinal tract, and relatively low level in the lungs and its expression has been reported in type 2 pneumocytes (hamming et al., 2004) . the extracellular domain of ace2 acts as the sars-cov and sars-cov-2 spike protein receptor. the new coronavirus also uses membrane-bound ace2 as the receptor. ace2 neutralizes sars-cov-2 in vitro by combining with the fc protein of immunoglobulin (lei et al., 2020) . in addition, sars-cov-2 binds to ace2 with a higher affinity than sars-cov. in this context, the preparation of a soluble and recombinant form of ace2 protein in humans could be useful as a novel biological treatment to counter or limit the progression of infection caused by coronaviruses that use ace2 as a receptor (wrapp et al., 2020 to treat covid-19, it is clear that the use of these drugs alone will not produce the desired results, and some drugs will cause side effects in certain groups of people. for example, excessive use of chloroquine is toxic and can even be fatal, or the use of favipiravir in pregnant women is not recommended, as it is associated with teratogenicity and embryotoxicity in animals or corticosteroids that are immunosuppressive and are not recommended for mild or early ards, because early use of corticosteroids can delay the clearance of the virus and increase the risk of death, long-term administration of high doses of corticosteroids in the early stages of treatment has negative effects. generally, corticosteroids should be avoided unless the patient has symptoms such as moderate or severe ards, sepsis or septic shock. mpa also has no effective inhibitory effect on sars-cov, so they are not recommended for treatment. lopinavir is associated with side effects such as digestive adverse effects and hypokalemia that should be considered by physicians. the use of neuraminidase inhibitors is also invalid due to resistance to ncov-2019 and is not recommended for the treatment of patients. however, the use of hydroxychloroquine as prophylaxis is appropriate because it blocks the entry of the virus and prevents virus replication, and remdesivir has extensive antiviral activity against coronaviruses. therefore, the appropriate recommendation for the treatment of patients with covid-19 is a combination of existing drugs. ifnα-2b together with ribavirin, when used for the treatment of mers-cov, showed that their effective dose was decreased compared to the single-dose regimen and better-stimulated cytokine release and inflammatory responses, and decreased viral replication, modulated host response, and improved clinical outcomes. ribavirin plus lopinavir/ritonavir in patients with sars-cov reduces ards and mortality, and in patients with sars-cov who receive corticosteroids with ribavirin, lopinavir/ritonavir, their ards and mortality significantly decreased. lopinavir also has a stronger antiviral effect than the lpv/r, and the combination of arbidol and lpv/r is more effective than lpv/r. in addition to these drugs, if ace2 is presented in its soluble form as a suitable recombinant protein, it may be a new factor in counteracting the spread of coronavirus as well as its proliferation in infected individuals. another possible treatment option could be the use of serums that are obtained from the blood of patients infected with the new coronavirus during their recovery and also targeting the cellular components involved in the host's inflammatory response to the virus is also a good way of targeting viral damage by targeting a cellular protein. yousefi et al. effectiveness of antiviral treatment in human influenza a (h5n1) infections: analysis of a global patient registry immunosuppressive and other effects of mycophenolic acid and an ester prodrug, mycophenolate mofetil the flu…and you corticosteroid therapy for critically ill patients with middle east respiratory syndrome effect of ribavirin and interferon on the outcome of critically ill patients with mers. viral respiratory infections soluble angiotensinconverting enzyme 2: a potential approach for coronavirus infection therapy oseltamivir resistance in severe influenza a (h1n1) pdm09 pneumonia and acute respiratory distress syndrome: a french multicenter observational cohort study arbidol: a broadspectrum antiviral compound that blocks viral fusion broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus favipiravir versus arbidol for covid-19: a randomized clinical trial. medrxiv role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings favipiravir as a potential countermeasure against neglected and emerging rna viruses arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? mycophenolic acid inhibits dengue virus infection by preventing replication of viral rna treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques effectiveness of favipiravir (t-705) against wildtype and oseltamivir-resistant influenza b virus in mice favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor mechanisms of action of ribavirin against distinct viruses tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis mycophenolic acid inhibits hepatitis c virus replication and acts in synergy with cyclosporin a and interferon-α what can we predict about viral evolution and emergence? current opinion in lb16. phase 3 trial of baloxavir marboxil in high-risk influenza patients (capstone-2 study) efficacy and safety of glucocorticoids in the treatment of severe communityacquired pneumonia: a meta-analysis antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in wuhan, china potent neutralization of 2019 novel coronavirus by recombinant ace2-ig therapeutic options for the 2019 novel coronavirus (2019-ncov) case of the index patient who caused tertiary transmission of coronavirus disease 2019 in korea: the application of lopinavir/ ritonavir for the treatment of covid-19 pneumonia monitored by quantitative rt-pcr disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes patients of covid-19 may benefit from sustained lopinavir-combined regimen and the increase of eosinophil may predict the outcome of covid-19 progression gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses developing therapeutics for ebola virus disease: a multifaceted approach, neglected tropical diseases: drug discovery and development favipiravir and zanamivir cleared infection with influenza b in a severely immunocompromised child broad-spectrum antiviral activity of the imp dehydrogenase inhibitor vx-497: a comparison with ribavirin and demonstration of antiviral additivity with alpha interferon compounds with therapeutic potential against novel respiratory 2019 coronavirus a randomized, controlled trial of ebola virus disease therapeutics ribavirin inhibits viral-induced macrophage production of tnf, il-1, the procoagulant fgl2 prothrombinase and preserves th1 cytokine production but inhibits th2 cytokine response mycophenolate mofetil inhibits the development of coxsackie b3-virus-induced myocarditis in mice efficacy and safety of the nucleoside analog gs-441524 for treatment of cats with naturally occurring feline infectious peritonitis surviving sepsis campaign: international guidelines for management of sepsis and septic shock new insights into the antiviral effects of chloroquine broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov high-throughput screening and identification of potent broadspectrum inhibitors of coronaviruses favipiravir, an anti-influenza drug against life-threatening rna virus infections human coronavirus-receptor interactions characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine an evaluation of chloroquine as a broad-acting antiviral against hand, foot and mouth disease pulmonary pathology of early phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer clinical practice guidelines by the infectious diseases society of america: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenza receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus prompt oseltamivir therapy reduces medical care and mortality for patients with influenza infection: an asian population cohort study remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys review of side effects and toxicity of chloroquine prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection. roceedings of the national academy of sciences of the united states of america cryo-em structure of the 2019-ncov spike in the prefusion conformation nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study antimalaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus influenza-like illness caused by the 2019 novel coronavirus (2019-ncov) via the person-to-person transmission. preprints teicoplanin potently blocks the cell entry of 2019-ncov bioinformatics analysis on molecular mechanism of ribavirin and interferon-α in treating mers-cov potential benefits of precise corticosteroids therapy for severe 2019-ncov pneumonia a global treatments for coronaviruses including covid-19 key: cord-339146-ifdgl2bj authors: lu, xiaoyan; rowe, lori a.; frace, michael; stevens, james; abedi, glen r.; elnile, osman; banassir, taleb; al‐masri, malak; watson, john t.; assiri, abdullah; erdman, dean d. title: spike gene deletion quasispecies in serum of patient with acute mers‐cov infection date: 2016-08-22 journal: j med virol doi: 10.1002/jmv.24652 sha: doc_id: 339146 cord_uid: ifdgl2bj the spike glycoprotein of the middle east respiratory coronavirus (mers‐cov) facilitates receptor binding and cell entry. during investigation of a multi‐facility outbreak of mers‐cov in taif, saudi arabia, we identified a mixed population of wild‐type and variant sequences with a large 530 nucleotide deletion in the spike gene from the serum of one patient. the out of frame deletion predicted loss of most of the s2 subunit of the spike protein leaving the s1 subunit with an intact receptor binding domain. this finding documents human infection with a novel genetic variant of mers‐cov present as a quasispecies. j. med. virol. 89:542–545, 2017. © 2016 wiley periodicals, inc. since first being recognized in 2012, middle east respiratory syndrome coronavirus (mers-cov) cases have continued to be reported from saudi arabia and neighboring countries. although considerable progress has been made in our understanding of the epidemiology and clinical features of mers-cov infection, less is known about this virus's capacity for genetic variation. beginning in september 2014, an outbreak of mers-cov was reported from multiple healthcare facilities located in taif, in the makkah region of saudi arabia. an investigation of this outbreak by the saudi ministry of health and u.s. centers for disease control and prevention (cdc) was recently reported [assiri et al., 2016a] . among 38 laboratory-confirmed mers-cov cases, serum samples from 17 were sent to cdc for serologic and molecular evaluation. spike gene sequences were obtained from 10 patients, including two patients with identical sequences with overlapping stays at a private hospital (hospital d). the first patient (#27) was a 75-year-old woman who was evaluated for severe acute respiratory symptoms at another hospital and transferred to the intensive care unit of hospital d on november 1 where she was confirmed positive for mers-cov. on november 11, an 81-year-old inpatient (#30) staying on the same floor as patient #27, developed respiratory symptoms and also tested positive for mers-cov. both patients subsequently died. further analysis of the serum sample from patient #27 identified a second sequence with a large deletion in the spike gene. in this report, we describe this variant and speculate on its possible origins and implications for predicted spike protein function. serum specimens from taif patients were screened for mers-cov by real-time rt-pcr and positive samples were further subjected to rt-pcr and sanger sequencing of the spike gene as previously reported [assiri et al., 2016a] . to confirm our finding of a deleted spike gene sequence amplified by primer set sf6/sr6 [assiri et al., 2016b] from patient #27, sample extracts were reamplified using a different primer set sf6/ssr6 [assiri et al., 2016b] using superscript iii one-step rt-pcr system with platinum taq dna polymerase (thermo fisher scientific, carlsbad, ca). the amplicons were sequenced on both sanger (3130xl genetic analyzer, fischer scientific) and next generation (pacbio rs ii, pacific biosciences, san francisco, ca) platforms. sequencher 4.8 software (gene codes, ann arbor, mi) was used for sanger sequence assembly and editing. pacbio data analysis was performed using cdc disclaimer: the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. clc genomics workbench v6 (waltham, ma). wildtype and deleted genome sequences of the quasispecies were prepared from the serum of patient #27 using 20 overlapping primer sets [assiri et al., 2016b] and deposited in genbank (ku710264; ku710265). as previously reported [assiri et al., 2016a] , realtime rt-pcr testing of serum specimens from a mers-cov outbreak in taif identified two epidemiologically linked case-patients (#27 and #30) with identical spike gene sequences. on further analysis, amplicons generated from patient #27 revealed a second smaller amplicon that was not present in the samples from patient #30 or other case-patients ( fig. 1 ). repeated rt-pcr confirmed presence of the smaller product and sanger sequencing identified a 530 nucleotide deletion which mapped to the region encoding subunit two of the spike protein gene (referred to from here forward as s530d). to confirm that this finding was not an artifact of our sequencing method, new amplicons generated from the serum sample were subjected to deep sequencing. approximately, 22,000 reads were obtained providing a minimum coverage of a few hundred bases throughout, reaching a maximum of over 11,000â coverage at nondeletion loci. a large population of reads with a relative coverage gap of 530 bases was obtained confirming the size and position of the predicted deletion. s530d was abundant in the cell-free serum sample, with an approximate ratio of 4-to-1 deleted to intact sequence reads. no other sequence variants were detected. mers-cov genome sequences were subsequently obtained from patient #27; genome sequencing was not attempted on the sample from patient #30 due to limited sample volume and low virus load. the wild-type mers-cov spike precursor protein is comprised of 1353 residues that are organized into two subunits: an amino-terminal subunit (s1, aa 1-751) that contains the receptor binding domain (rbd), and a carboxy-terminal subunit (s2, aa 752-1353) that contains the putative fusion peptide, two heptad repeat domains and the transmembrane and intracellular domains (fig. 1) . determinants of cellular tropism and interaction with the target cell receptor reside within the s1 domain, while mediators of membrane fusion are located within the s2 domain [gao et al., 2013] . in the endoplasmic reticulum (er)-golgi compartments of the infected cell, the precursor spike protein is first cleaved by a host protease such as furin at position 751r/752s into the s1 and s2 subunits that remain non-covalently linked [gierer et al., 2013; millet and whittaker, 2014] . trimers of these proteins form with the s2 subunit embedded in the er membrane and the s1 projecting outward. after release from the cell, the mature virus particle binds by the s1 rbd to the dipeptidyl peptidase-4 (dpp4) receptor on the surface of a new cell [raj et al., 2013] . a proteolytic cleavage at a second site in s2 located at position r887/s888 upstream from the putative fusion peptide then occurs that facilitates membrane fusion and virus entry into the host cell [gao et al., 2013; millet and whittaker, 2014] . the deleted gene would predict an 801 amino acid truncated protein prematurely terminating at an outof-frame stop codon (fig. 1) . this protein would contain the entire n-terminal s1 subunit, including the virus rbd, 20 in-frame residues immediately c-terminal to the r751/s752 protease cleavage site and 30 outof-frame non-spike residues. all key components of the membrane fusion architecture of the s2 subunit located anterior to the premature stop codon, including the proposed fusion peptide (aa 949-970) [ou et al., 2016] , would be predicted to be lost. the 30 non-spike residues (hifawqhsrcwldcwlillccysic-teyfl) at the c-terminus of the truncated protein include 14 hydrophobic residues (ala, phe, gly, ile, leu, met, val, or trp) and five cysteines. rna viruses are prone to rapid expansion of genomic variants or quasispecies that may aid virus escape from immunesurveillance and expand tissue tropism and host range [duarte et al., 1994] . well documented among coronaviruses generally, mers-cov quasispecies have been identified in naturally [briese et al., 2014] and experimentally [borucki et al., 2016] infected dromedary camels and sars-cov in humans [tang et al., 2006 ], but no similar instances have been reported for human mers-cov infections. moreover, although naturally occurring deletion mutations of varying size and location have been previously identified among human coronaviruses, including sars-cov [chiu et al., 2005] , hcov-oc43 [vijgen et al., 2005] , and mers-cov [lamers et al., 2016] , most have been restricted to the nucleocapsid or non-structural accessory protein genes located near the 3 0 -end of the viral genome. with the exception of a single codon deletion (residue 1293) in the spike transmembrane domain that was reported for a mers-cov derived from a dromedary camel [chu et al., 2014] , no naturally occurring deletions in the spike gene have been previously reported from human derived virus. modification of the coronavirus spike protein through natural and experimentally induced mutations has been shown to change cell and organ tropism leading in some cases to changes in virus pathogenicity and host range [rasschaert et al., 1990; wesley et al., 1991; vijgen et al., 2005; brandão et al., 2006; terada et al., 2012] . although most spike gene deletion mutations have been found in the s1 region, some studies have also documented mutations in the s2 region with similar effects. for example, specific mutations introduced into the s2 region of feline coronavirus have been shown to change virus tropism from the gut epithelium to macrophages with associated changes in pathogenicity from a mild enteric infection to fatal immune-mediated disease, respectively [rottier et al., 2005] . the spike gene deletion described here would most likely to render the virus defective, either non-infectious or with substantially reduced infectivity. loss of the s2 subunit would likely disrupt membrane anchoring of the spike protein and prevent fusion of the virus and host cell. propagation of defective viruses requires a helper virus to compensate for lost function. in the case of s530d, it is interesting to speculate how this mutation might conversely help sustain wild-type mers-cov infection. one hypothetical outcome of loss of the s2 transmembrane domain would be to yield a free s1 subunit with a "sticky" hydrophobic tail that might lead to aggregated/misfolded protein due to the additional disulfide bounds that might form. assuming that s530d still forms stable trimer complexes that retain biding affinity for dpp4, this form of the spike protein might act as a "decoy," blocking spike-specific mers-cov neutralizing antibodies. a similar concept has been hypothesized as an immune escape strategy used by ebola virus, termed "antigenic subversion" [mohan et al., 2012] . it has been shown that infection of susceptible cells with mers-cov can be inhibited with a soluble form of the dpp4 receptor [raj et al., 2013] . conversely, free spike protein fragments could theoretically bind and block anti-rbd neutralizing antibodies. arguing against this hypothesis is that anti-mers-cov antibodies were not detected (titer <400) in this patient by a sensitive enzyme immunoassay [assiri et al., 2016a] and the paucity of other reports of mers-cov spike gene deletions suggest that this event is rare and not of deliberate design. this study had several limitations. different specimen types from different time-points were not available from this patient or other patients in the predicted transmission chain preventing determination s530d persistence in the patient or capacity for transmission. limited available serum also prevented culture attempts, which would have allowed assessment of virus viability and direct assessment of protein form and function. nevertheless, our finding provides new insights into the capacity of mers-cov for genetic variation that may have unforeseen public health implications. multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia epidemiology of a novel recombinant mers-cov in humans in saudi arabia middle east respiratory syndrome coronavirus intra-host populations are characterized by numerous high frequency variants molecular analysis of brazilian strains of bovine coronavirus (bcov) reveals a deletion within the hypervariable region of the s1 subunit of the spike glycoprotein also found in human coronavirus oc43 middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia tracing sars-coronavirus variant with large genomic deletion mers coronaviruses in dromedary camels rna virus quasispecies: significance for viral disease and epidemiology structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies circulation of mers-cov deletions variants in humans host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein antigenic subversion: a novel mechanism of host immune evasion by ebola virus identification of the fusion peptide-containing region in betacoronavirus spike glycoproteins dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein the large 386-nt deletion in sars-associated coronavirus: evidence for quasispecies? feline infectious peritonitis virus with a large deletion in the 5'-terminal region of the spike gene retains its virulence for cats complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus key: cord-320548-oigyut2k authors: zumla, alimuddin; memish, ziad a; maeurer, markus; bates, matthew; mwaba, peter; al-tawfiq, jaffar a; denning, david w; hayden, frederick g; hui, david s title: emerging novel and antimicrobial-resistant respiratory tract infections: new drug development and therapeutic options date: 2014-09-01 journal: lancet infect dis doi: 10.1016/s1473-3099(14)70828-x sha: doc_id: 320548 cord_uid: oigyut2k the emergence and spread of antimicrobial-resistant bacterial, viral, and fungal pathogens for which diminishing treatment options are available is of major global concern. new viral respiratory tract infections with epidemic potential, such as severe acute respiratory syndrome, swine-origin influenza a h1n1, and middle east respiratory syndrome coronavirus infection, require development of new antiviral agents. the substantial rise in the global numbers of patients with respiratory tract infections caused by pan-antibiotic-resistant gram-positive and gram-negative bacteria, multidrug-resistant mycobacterium tuberculosis, and multiazole-resistant fungi has focused attention on investments into development of new drugs and treatment regimens. successful treatment outcomes for patients with respiratory tract infections across all health-care settings will necessitate rapid, precise diagnosis and more effective and pathogen-specific therapies. this series paper describes the development and use of new antimicrobial agents and immune-based and host-directed therapies for a range of conventional and emerging viral, bacterial, and fungal causes of respiratory tract infections. the emergence of diffi cult-to-treat known and novel bacterial, viral, and fungal respiratory tract pathogens with epidemic potential is of major global concern. treatment options are limited by increasing antimicrobialdrug resistance. however, new viral infections causing severe respiratory tract disease with pandemic potential have focused global attention. 1 a substantial rise in the number of patients with multidrug-resistant pulmonary tuberculosis 2 and pan-drug-resistant bacteria 3 has been noted. increasing use of immunosuppressive agents, broad-spectrum antibiotics, and anticancer agents, coupled with resistance to azoles, has led to an increase in the number of invasive pulmonary fungal infections 4 with resultant high morbidity and mortality. successful treatment outcomes for patients with respiratory tract infections across all health-care settings require appropriate, eff ective, and pathogen-specifi c drug or alternative treatments. we describe a range of conventional and emerging viral, bacterial, and fungal causes of respiratory tract infections for which new antimicrobial drugs and immune-based and host-directed therapies are being developed and studied. the outbreak of severe acute respiratory syndrome coronavirus (sars-cov), 5 re-emergence of avian infl uenza a h5n1, 6 global circulation of oseltamivirresistant seasonal infl uenza a h1n1, 7 and subsequent emergence of the pandemic infl uenza a h1n1 strain pdm09 virus (which continues to circulate), 8 have shown the potential limitations of current antiviral treatments for severe respiratory viral infections. epidemic waves of avian infl uenza a h7n9, 9 sporadic cases of avian infl uenza a h10n8, 10 the ongoing outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection, and the burden of common respiratory viruses 11 -such as seasonal infl uenza, respiratory syncytial virus, rhinoviruses, and adenoviruses-show that the development of more eff ective therapies to reduce morbidity and mortality is urgently needed. research is focused on the repurposing of available antiviral drugs for generic or specifi c use and for two classes of antiviral drugs are approved for the prevention and treatment of infl uenza in most countries: m2 inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir, peramivir, zanamivir, and laninamivir; table 1). in general, antiviral treatment is indicated as early as possible for any patient with confi rmed or suspected infl uenza who has severe, complicated, or progressive illness or is admitted to hospital, and in outpatients at higher risk of infl uenza complications. 12, 13 time to treatment after onset of symptoms, illness severity, and extent of viral replication are key variables with respect to response. starting of treatment should not be delayed for diagnostic testing. m2 inhibitors-also known as adamantanes-are ineff ective against infl uenza b viruses and recently circulating infl uenza a h3n2 and 2009 pandemic infl uenza a h1n1 viruses, which are resistant because of an s31n mutation in the m2 ion channel. 12 however, a proportion of avian infl uenza a h5n1 strains will be susceptible, 14 and the combined use of an adamantane and a neuraminidase inhibitor improves antiviral activity for susceptible isolates. 15 two neuraminidase inhibitors are approved for use in most countries: oseltamivir and zanamivir. laninamivir is approved for use in japan only, and peramivir in china, japan, and south korea. several observational studies have shown that when adults admitted to hospital with severe infl uenza are given oseltamivir, mortality falls and clinical outcomes improve, especially when treatment is initiated within 2 days of the onset of symptoms (but positive eff ects are noted when it is begun as late as 4-5 days after onset). 12, 13, 16, 17 oseltamivir reduces mortality in infl uenza a h5n1 infection when given before the onset of respiratory failure, 18 and might be benefi cial when started as late as 6-8 days after symptom onset. 19 in patients admitted to hospital with severe infl uenza a h7n9 infection, reduction of viral load after treatment with oseltamivir correlated with improved outcome, whereas the emergence of virus resistant to neuraminidase inhibitors that harbours an arg292lys substitution is associated with poor outcomes and poor response to oseltamivir and peramivir. 20 the standard duration of oseltamivir treatment is 5 days; longer treatment is recommended for critically ill patients with respiratory failure, who often have prolonged viral replication in the lower respiratory tract despite treatment. 13 whether increased doses provide greater antiviral eff ects in such patients is under investigation. a randomised controlled trial 21 of patients in hospital (76% of whom were children) showed no virological or clinical advantages when a double dose of oseltamivir was given rather than a standard dose. no additional benefi t was noted with high-dose oseltamivir in adults admitted with infl uenza a, although a faster virological response was noted in those with infl uenza b. 22 however, in a randomised controlled trial 23 of 18 critically ill patients with 2009 pandemic infl uenza a h1n1, a triple-dose oseltamivir regimen was associated with signifi cantly higher proportions of viral clearance at 5 days than was standard therapy (78% vs 11%; p=0·015). 23 studies of intravenous neuraminidase inhibitors that are underway should provide further data on the value of high-dose therapy. zanamivir and laninamivir have generally similar profi les of susceptibility. for example, the his275tyr mutation confers high-level resistance to oseltamivir carboxylate and reduced susceptibility to peramivir in n1containing viruses but does not substantially diminish susceptibility to zanamivir and laninamivir. 30 inhaled zanamivir has not been studied in detail in severely ill patients or those admitted to hospital, in whom eff ective delivery to sites of viral replication and tolerability could be an issue. by contrast, intravenous zanamivir has been used widely on a compassionate basis since the 2009 h1n1 pandemic, particularly for late treatment of critically ill adults with 2009 pandemic infl uenza a h1n1 virus infection and those with suspected or proven oseltamivir resistance. 31 one trial 32 has shown no drug-related trends in safety measures, and a subset of 93 patients positive at baseline for infl uenza showed a median decrease in nasopharyngeal viral rna load of 1·42 log 10 copies per ml after 2 days of treatment. a phase 3 trial in patients who have been admitted to hospital is underway (nct01014988). a phase 2 randomised controlled trial of inhaled laninamivir in uncomplicated infl uenza failed to show superiority in illness alleviation (primary endpoint) compared with placebo. the trial, involving 639 patients, tested 40 mg and 80 mg doses of the inhaled drug. the median time to alleviate fl u symptoms was 102·3 h for the 40 mg dose and 103·2 h for the 80 mg dose, compared with 104·1 h for the placebo (nct01793883). das181 has host-directed receptor-destroying action, which is inhibitory for parainfl uenza and infl uenza viruses, including those resistant to amino adamantanes and neuraminidase inhibitors. 15 when delivered topically, it is eff ective in animal models of lethal infl uenza caused by the h5n1 and h7n9 viruses, including the neuraminidase-inhibitor-resistant arg292lys -containing variant. 35 in a phase 2 randomised controlled trial, 36 inhaled das181 reduced pharyngeal viral replication in uncomplicated infl uenza but did not reduce nasal viral loads or improve clinical outcomes. case reports 37 suggest that inhaled or nebulised das181 might be eff ective in immunocompromised hosts with severe parainfl uenza lung disease. favipiravir (t-705; 6-fl uoro-3-hydroxy-2-pyrazinecarboxamide) is active against infl uenza a, b, and c viruses, including strains resistant to approved antivirals, and a broad range of other rna viruses when given at series somewhat higher concentrations. 38 combinations of favipiravir and neuraminidase inhibitors have additive and synergistic eff ects in preclinical models, 39 but clinical trials have been restricted to uncomplicated infl uenza so far. these clinical trials (combination amantadine, ribavirin, and oseltamivir vs oseltamivir mono therapy [nct01227969], nitazoxanide vs oseltamivir vs combination vs placebo [nct01610245], favipiravir vs placebo randomised controlled trial in outpatients [nct02008344, nct2026349]), which have not been published, suggest that favipiravir has antiviral eff ects similar to those of oseltamivir. 40 a randomised controlled trial 41 showed that favipiravir shortened the time to alleviation of infl uenza symptoms by about 15 hours compared with placebo, and further studies are underway. nitazoxanide is an oral antiparasitic drug with immunomodulatory eff ects, including upregulation of interferon and various interferon-inducible genes and a specifi c infl uenza-inhibitory eff ect related to blockade of haemagglutinin maturation. 42 nitazoxanide inhibits infl uenza replication in vitro 43 and in a phase 2 randomised controlled trial 44 had signifi cant antiviral eff ects (1·0 log 10 reduction in nasal viral loads) and resulted in a signifi cantly faster time to alleviation of illness (roughly 20 h diff erence in medians from placebo) in uncomplicated infl uenza. 44 a placebocontrolled randomised trial of nitazoxanide versus oseltamivir-and the com bination thereof-in uncomplicated infl uenza and a hospital-based study of its use in severe respiratory illness are in progress (nct01610245). non-randomly assigned studies and case reports suggest that convalescent plasma with neutralising antibodies is a useful add-on therapy for patients with sars and severe infl uenza pneumonia, including that caused by infl uenza a h5n1. 45 a recently published systematic review of available sars and infl uenza treatment studies employing convalescent plasma or serum found a signifi cant overall mortality benefi t. 46 a prospective observational study 47 showed lower crude mortality and faster nasopharyngeal viral clearance in plasma-treated patients who were admitted with severe 2009 pandemic infl uenza a h1n1 infection, whereas in a randomised controlled trial 48 a reduction in mortality was reported in severe illness when hyperimmune globulin was given within 5 days of the onset of symptoms (table 2) . heterosubtypic haemagglutinin stem-neutralising antibodies, which are highly eff ective in animals, 49 are entering clinical evaluation in human beings. the combination of antivirals with diff erent mechanisms of actions (eg, a neuraminidase inhibitor with a polymerase inhibitor such as favipiravir, 38 a broad-spectrum antihaemagglutinin-neutralising antibody, 49 (20) oral rimantadine and nebulised saline (21) post-hoc analysis showed faster cough resolution but no signifi cant diff erences in the proportion of patients shedding virus by treatment day 3 (57% zanamivir plus rimantadine, 67% placebo plus rimantadine), or in the durations of hospitalisation and supplemental oxygen use underpowered because of low enrolment oseltamivir and corticosteroids (19) more rapid improvement in partial pressure of oxygen, fraction of inspired oxygen, and sequential organ failure assessment scores; shorter ventilator use (median 7 days vs 15 days, p=0·03); and faster viral clearance in the sirolimus than in the control group rct=randomised controlled trial. 51 in a retrospective study 53 of critically ill adults, mortality rates did not diff er between those who received a triple combination of antiviral drugs and those receiving oseltamivir only, and a randomised controlled trial sponsored by the national institute of allergy and infectious diseases in higher-risk outpatients is underway (nct01227969). host-directed therapies aim to reduce the damaging consequences of the host immune response to the pathogen. combinations of antivirals with host-directed therapies such as the immunomodulator sirolimus, an mtor inhibitor that blocks host pathways needed for viral replication (table 2) , 54 might also enhance antiviral activity. other host-directed therapies inhibiting cellular targets needed for effi cient viral replication (eg, the raf-mek-erk mitogenic kinase cascade and the ikk-nf-κb module) might provide future options for clinical testing. 15 the role of adjunctive immunomodulatory therapies in severe infl uenza and other respiratory viral infections remains uncertain. several observational studies show that systemic corticosteroids given for 2009 pandemic infl uenza a h1n1-associated viral pneumonia increased the risk of mortality and morbidity (eg, secondary infections), especially when there was a delay in initiation, or absence of, eff ective antiviral therapy. 45 their use might delay viral clearance and increase the risk of the emergence of resistance 20 and fungal infections. 45 other potential adjunctive therapies for infl uenza include intravenous immunoglobulin, n-acetylcysteine, statins, macrolides, peroxisome proliferator-activated receptor agonists, celecoxib, mesalazine, plasmapheresis, and haemo perfusion. 45 chloroquine was eff ective against infl uenza a h5n1 infection in one animal model 55 but was ineff ective in other animal models and one human randomised controlled trial. 56, 57 interferons mers-cov infection can cause severe respiratory disease, and has higher mortality in those with medical comorbidities. although empirical treatment with a range of antivirals has been tried for severe respiratory tract infections caused by mers-cov and sars-cov, no regimens have been rigorously assessed in clinical trials (panel). 58,59 mers-cov elicits attenuated innate immune responses with delayed proinfl ammatory cytokine induction in cell culture and in vivo. 60, 61 it is also readily inhibited by type 1 interferons (interferon alfa and especially interferon beta), suggesting a potential therapeutic use for interferons. early pegylated interferon alfa therapy was eff ective in a sars primate model, and treatment with interferon-alfa-consensus-1 plus systemic corticosteroids was associated with improved oxygen saturation and more rapid resolution of radiographic lung opacities than were systemic corticosteroids alone in an uncontrolled study of patients with sars patients. 62 further studies of interferons in mers-cov seem warranted. ribavirin was used extensively in patients with sars without any benefi cial eff ects and was complicated by haemolytic anaemia and metabolic disturbances in many cases. 58, 59 a combination of interferon alfa 2b and ribavirin reduced lung injury and moderately decreased viral replication (<1·0 log 10 reduction in lung titres) when given to rhesus macaques within 8 h of inoculation with mers-cov. 63 the treatment combination was given to several severely ill patients with mers, but the infections proved fatal, probably because of late administration in the advanced stage of the disease. 64, 65 ribavirin has in-vitro inhibitory eff ects against mers-cov. 66 the use of protease inhibitors with lopinavir and ritonavir as initial therapy in sars was associated with signifi cantly less death (2·3% vs 15·6%, p<0·05) and intubation (0% vs 11·0%, p<0·05) than was use of ribavirin alone in a matched historical cohort (n=44 for lopinavir and ritonavir as intial treatment vs n=634 for the matched historical cohort). 68 however, one study reported that nelfi navir and lopinavir have high 50% eff ective inhibitory concentrations (ec 50 ) against mers-cov in vitro, 66 whereas another found inhibition with lopinavir at clinically achievable concentrations. 69 several drugs have shown inhibitory eff ects against mers-cov in cell cultures, including interferons, ciclosporin, and mycophenolic acid. 66, 67, 69 mycophenolic acid was inhibitory at clinically achievable concentrations, and the combination of mycophenolic acid and interferon β1b lowered the ec 50 of each drug by one-to-three times. 66 dipeptidyl peptidase 4 (dpp4), also known as cd26, is a functional receptor for mers-cov, and an anti-cd26 polyclonal antibody showed in-vitro inhibitory eff ects on mers-cov. 70 by contrast, inhibitors of the enzymatic action of dpp4 (eg, gliptins) did not inhibit viral replication. timely administration of neutralising antibodies could have a high likelihood of therapeutic success. 46 treatment with convalescent plasma (from patients who have recovered from sars-cov infection) containing high levels of neutralising antibody within 2 weeks of illness onset resulted in a higher proportion of discharges at day 22 than did treatment more than 14 days after onset (58% vs 16%, p<0.001). 71 75 showed worse outcomes when systemic corticosteroids were given in sars. consequently, their use should be avoided unless a carefully controlled prospective study is done to test their eff ectiveness when combined with an antiviral. several observational studies have shown that systemic corticosteroids given for 2009 pandemic infl uenza a h1n1-asssociated viral pneumonia or acute respiratory distress syndrome increased the risk of mortality and morbidity (eg, secondary bacterial or fungal infections), especially if there is delay or lack of eff ective antiviral therapy. 45 use of systemic corticosteroids has probably contributed to delayed viral clearance and emergence of antiviral resistance in patients with severe infl uenza a h7n9 infection requiring extracorporeal membrane oxy genation. 20 infl uenza increases the risk of invasive aspergillosis, especially among immunocompromised patients, and this is often a silent infection in the early stages, 76 so direct surveillance with aspergillus antigen and pcr testing on respiratory secretions is advisable. patients treated for fungal infections will have to undergo antifungal therapeutic drug monitoring. 77 data are insuffi cient to support routine use of any of the immune therapies. better animal data and careful systematic clinical studies, including serial virological measurements of priority treatments such as convalescent plasma and interferons (and randomised controlled trials if case numbers are suffi cient), are needed. currently, clinical management of patients with severe respiratory tract infections due to mers-cov largely relies on meticulous intensive care supportive treatment and prevention of complications. research done in patients with haemopoietic stem-cell transplants shows that adoptive transfer of antigenspecifi c t cells can restore protective immunity and prevent or reverse disease due to opportunist viral infections such as cytomegalovirus. 78 in transplant recipients, transfer of donor-derived t cells can result in resolution of infection through expansion of virusspecifi c t cells, with associated clinical improvement. 79 transfer of donor t cells is associated with the risk of severe acute graft-versus-host disease, and thus most t-cell therapies have been done in patients who have low lymphocyte counts. lymphopenia enables only a very low number of t cells to be transferred, which then proliferate in lymphopenic hosts, most likely as a result of the interleukins 7 and 15 if the patient does not receive immunosuppressive treatment during t-cell therapy. 80 t-cell therapy targeting cytomegalovirus strains resistant to drug treatment is clinically relevant in lung transplant recipients. 81 t-cell expansion requires time to induce clinical regression of viral infection. several other approaches might be applicable in situations that necessitate fast clinical action-eg, use of synthetic mhc antigens loaded with the relevant peptide from the pathogen of interest (so-called tetramer or multimer mhc-peptide complexes), which engage pathogenspecifi c lymphocytes expressing the pathogen-specifi c t-cell receptors. pathogen-specifi c t cells can be isolated through use of soluble mhc-peptide complexes, and can immediately be transferred into patients for salvage treatments for viral infections. 82 t-cell expansion can also be achieved with several stimuli targeting several infectious pathogens. 83 expansion of t cells targeting several antigens of cytomegalovirus, epstein-barr virus, and adenovirus provides broad antiviral specifi city after stem-cell transplantation. 84 an alternative approach to series become independent of ex-vivo expansion of t cells is the identifi cation of t-cell receptors that would recognise viral infected cells that could be transferred into recipient eff ector cells. 85 t cells can also be engineered to produce an antiviral rna that would block viral infection. 86 synthetic antisense molecules, such as phosphorodiamidate morpholino oligomers, are structurally similar to rna but the phosphorodiester linkage is replaced with a neutral phosphorodiamidate linkage and the ribose ring with a six-membered morpho lino ring. 87 they change gene expression by inhibiting translation, disrupting rna secondary structure, and interfering with pre-mrna splicing. 88 the usefulness of phosphorodiamidate morpho lino oligomers coupled to argininerich cell-penetrating peptides has been repeatedly demonstrated against bacterial pathogens 89 and could be a viable option for any microbial gene of interest. specifi c biological therapy for infectious pathogens targets not only drug-resistant pathogens but also their immune evasion mechanisms. 90 an antibody directed against cd19 (a b-cell marker) fused to a t-cell signalling molecule can be expressed in t cells and could kill target cells once they encounter their nominal target antigen. such cd19 chimeric-antigen-receptor cells are used to remove epstein-barr-virus-positive lymphoma cells in the case of post-transplantation proliferative diseases. 91 similar approaches can be used for the eff ective removal of pathogen-infected cells when very specifi c antibodies exist and if target molecules are expressed on infected cells only. 92 the frequency and spectrum of resistance to antibiotics in specifi c bacterial pathogens that cause respiratory tract infections continues to increase worryingly. multidrugresistant streptococcus pneumoniae-with resistance to three or more antibiotics-was initially noted in 1977 in south africa 93 and subsequently in many other countries, with alarming rates of 30-50% of s pneumoniae that are multidrug resistant in the usa and spain. [94] [95] [96] the european antimicrobial resistance surveillance system showed that 22·2% of s pneumoniae were intermediate penicillin susceptible, 10·9% were penicillin resistant, and 21·1% were resistant to erythromycin. 97 concerns about multidrug-resistant and pan-antibioticresistant gram-negative bacteria 98, 99 are focused on klebsiella pneumoniae, enterobacter spp (production of extended spectrum β lactamase, klebsiella pneumoniae carba penemase, ndm1, and ampc), acinetobacter baumannii, and pseudomonas aeruginosa. in one survey of us health centres, 78% of gram-negative bacteria were resistant to all antibiotics except colistin (to which 62% of acinetobacter spp, 59% of pseudomonas spp, and 52% of enterobacter spp were resistant). 98 therapeutic options to treat these infections are limited. 100, 101 carbapenems are recommended for organisms that produce extended-spectrum β lactamases. 101 in a metaanalysis, [102] [103] [104] [105] [106] doripenem was more eff ective for p aeruginosa infections than were comparators in a modifi ed intention-to-treat analyses. polymyxin b and colistin are concentration-dependent bactericidal agents that bind to bacterial cell membranes and have reliable activity against acinetobacter spp. novel β-lactamase inhibitors 107 and antibiotic com bination therapies 108 might provide stopgap measures for fulfi lling clinical need. antibiotic development pipelines remain thin, 109, 110 and global attention is focused on increasing awareness for investments into the development of new antibacterial agents 111 and other antibacterial innovations, coupled to raising global awareness for more prudent use of available drugs. 112 in 2012, an estimated 1·3 million people died worldwide from tuberculosis, 170 000 of whom had multidrugresistant disease. 113 multidrug-resistant tuberculosis, which is caused by mycobacterium tuberculosis bacilli resistant to at least isoniazid and rifampicin, is now widespread globally, with an estimated half a million cases in 2012. 2 extensively drug-resistant tuberculosisresistance to rifampicin, isoniazid, any fl uoroquinolone, and at least one of the three injectable second-line drugs, amikacin, kanamycin, and capreomycin-has been reported in 92 countries. 113 who recommends use of second-line drugs for 18-24 months or longer for extensively drug-resistant or multidrug-resistant disease. 114, 115 treatment success rates are low in both individualised and standard regimens and new drugs and regimens are needed. in the past 5 years, a promising pipeline of new drugs for the treatment of multidrug-resistant and extensively drug-resistant tuberculosis has emerged. 115 progress has been made by repurposing drugs that are already available, including re-engineering existing antibacterial compounds and redesigning scaff olds, leading to discovery of new compounds. 116, 117 two new drugs, delamanid (opc-67683) and bedaquiline (tmc207 or r207910), have been approved by regulatory authorities. these new drugs are combined with older drugs to treat multidrug-resistant disease. 118, 119 host-directed adjunct therapies several approaches to rational development of adjunct immune-based therapies for multidrug-resistant tuberculosis have been developed. 120, 121 non-steroidal antiinfl ammatory drugs can reduce m tuberculosis load and series alleviate lung disease 122 in mice. 123 effl ux pump inhibitors such as verapamil and reserpine reduce macrophageinduced drug tolerance, and thus could be used as adjunct host-directed therapies. 124, 125 phosphodiesterase inhibitors such as cilostazol and sildenafi l improve mycobacterial clearance and decrease time to sterilisation by reducing tissue infl ammation. 126 a range of adjunct immunotherapy approaches implicating cytokines or their inhibitors and other biological immunomodulatory compounds are being assessed as means to limit damage from infl ammatory responses against m tuberculosis. various cytokine regimens, including interferon c or interleukin 2, have been assessed, with variable eff ect. 127, 128 the antiinfl ammatory eff ects of macrolide antibiotics need to be further studied. 129 whole genome sequencing might allow for rapid determination of resistance patterns of m tuberculosis strains, enabling tailored treatment regimens. other immunomodulatory strategies include restoration of eff ective antipathogen-directed immunoresponses-and consequent decreasing of damaging host responses in lung tissues-in multidrug-resistant tuberculosis with infusions of the patient's own bonemarrow-derived stromal cells. a phase 1 trial showed that the procedure is safe, 130 and phase 2 trials are planned to assess the eff ects of mesenchymal stromal cell adjunct therapy on clinical and microbiological outcomes. invasive fungal respiratory tract infections are increasingly reported worldwide (table 3) . 131, 132 the two most common pulmonary fungal pathogens are aspergillus fumigatus and pneumocystis jirovecii. they increasingly represent primary causes of morbidity and mortality in critically ill patients across europe, africa, and asia as a result of more people living with hiv, increased use of immunomodulatory drugs in patients with cancer, transplantations, and use of broad-spectrum antibiotics. some patients with relapsed or micro biologically unconfi rmed multidrug-resistant tuber culosis have alternative diagnoses, including chronic pulmonary aspergillosis, and more com prehensive searches for alternative fungal diagnoses in smear and culture negative cases should be done in patients with multidrug-resistant disease. 133 aspergillus is the most important fungal cause of invasive pulmonary disease, and a fumigatus is the cause in more than 75% of cases. voriconazole is the most eff ective treatment for invasive aspergillosis but resistance has been noted on all continents except south america. 134, 135 widespread use of the azoles as fungicides in agriculture has led to the environmental development of pan-azole resistance. 136 resistance can also emerge during treatment, typically to itraconazole, and is possibly linked to a combination of low blood concentrations of the drug and high fungal loads. [137] [138] [139] modelling suggests that more than 6·5 million people have severe asthma with fungal sensitisations, as much as 50% of adults with asthma who attend secondary care have fungal sensitisation, and an estimated 4·8 million adults have allergic bronchopulmonary aspergillosis. 140, 141 people with asthma who are sensitised to a fumigatus have a much higher rate of bronchiectasis than do those who are unsensitised. reclassifi cation of aspergillosis in adults with cystic fi brosis by aspergillus serology (ige and igg) and both pcr and antigen on sputum showed three distinct classes of aspergillosis. 18% had allergic bronchopulmonary disease, 15% had aspergillus sensitisation, and 30% had aspergillus bronchitis; the remaining patients had no disease. long-term oral antifungal therapy is benefi cial for 60-80% of patients with asthma, but is of unproven benefi t in cystic fi brosis. 142 resistance in a fumigatus has been reported throughout europe in roughly 4% of samples from patients with cystic fi brosis. 143, 144 a new fungus causing disseminated infections in patients with aids was identifi ed in 2009. 145 molecular identifi cation on the basis of its1 and its2 sequencing showed that all isolates of this new species were tightly clustered and were most similar to emmonsia pasteuriana and emmonsia parva, and slightly more distantly related to histoplasma capsulatum. clinical features of infection included fever, loss of weight, anaemia, skin lesions akin to those in disseminated histoplasmosis, and a chest radiograph similar to that noted in pulmonary tuberculosis. the fungus was cultured from skin and blood, but not sputum or csf. signifi cant clinical responses were noted when patients were given intravenous amphotericin b followed by itraconazole. 145 a large combination study 146 series did not reach its primary endpoint of reduced mortality, although patients with positive galactomannan seemed to benefi t most. guidelines for management of invasive aspergillosis still favour voriconazole over all other treatments and combination therapy is not usually recommended. a tablet formulation of posaconazole, which is more bioavailable than the oral suspension, is available and can be given once a day, 147 and the us food and drug administration has approved an intravenous suspension of the drug. the only new drug to be approved is isavuconazole, a broad-spectrum azole, which will be available in intravenous and oral forms (application for approval was submitted in july, 2014). itraconazole seems safe in the fi rst trimester of pregnancy, whereas fl uconazole increases the risk of fallot's tetralogy by a factor of three to one in 1000. 148 drivers for the development of new antifungal drugs include inadequate response rates, the absence of oral preparations of echinocandins, drug interactions, important drug toxic eff ects (especially amphotericin b and voriconazole), and triazole and echinocandin resistance. several drugs are being repurposed for use as antifungals, and new drugs are under development (table 4) . [149] [150] [151] [152] [153] [154] [155] sertraline, which is used for depression, has synergistic activity with fl uconazole in a murine model of cryptococcal infection. 156 calcineurin and targets of rapamycin inhibitors have antifungal activity, which is synergsitic with that of azoles. 157 hsp90 inhibitors initially developed for cancer treatment can improve fl uconazole activity in vitro and in animals. 158 enoxacin, a fl uoroquinolone antibiotic, shows activity in a murine candidiasis model. 159 although azoles are important for the treatment of invasive pulmonary aspergillosis, the degree of immunosuppression and other immunological factors have a role in treatment outcomes. antifungal immune responses could be improved by adaptive transfer of pathogen-specifi c t cells directed against invasive and pulmonary fungal infections, particularly infections with candida, aspergillus, and mucormycetes, especially after allogeneic stem-cell transplantation. t-cell responses are mhc class i restricted (for cd8positive t cells) or mhc class ii restricted (for cd4positive t cells), and thus an eff ective t-cell response needs to match the genetic background of the patient. t-cell transfer was developed on the basis of the promising fi nding that transfer of pathogen-specifi c t-cell clones induces clinically signifi cant responses. 160, 161 several approaches have been used to obtain these pathogen-specifi c t cells. anti-pathogenspecifi c t cells can be expanded ex vivo under appropriate conditions (usually with the help of recombinant cytokines, synthetic peptides, or cellular components representing the pathogen). responder t cells are identifi ed by interferon-γ production, removed via an interferon-capture assay, and transferred into the patient. this approach requires time for expansion of t cells (either the patient's own or those of an mhc-matched donor). this protocol enabled the expansion of aspergillus spp, candida spp, with the terms "respiratory tract", "pneumonia", "infections", "bacteria", "virus", "fungus", and "mycobacteria". we also combined these terms with the words "antibiotic", "antibiotic resistance", "treatment", "drugs", "drug development", "drug pipeline", "antibiotic development", "host-directed", "therapy", "adjunct therapy", "steroids", and "immunotherapy". we complemented the search with publications from who, the us centers for disease control and prevention, http://clinicaltrials. gov, and google scholar. we also reviewed studies cited by articles identifi ed by this search. and mucor spp-reactive t cells defi ned by interferon-γ production. upon re-encounter with the nominal target antigen, the t cells proliferated and increased the antifungal reactivity of phagocytes. 162 new and antimicrobial-resistant species of bacteria, viruses, and fungi continue to emerge because of the remarkable genetic and adaptable plasticity of the microbiota. 163 respiratory tract infections are among the top two causes of death globally. 164, 165 microorganisms do not respect international boundaries, and ease of travel and airborne spread make them a threat to global health security. the increasing frequency of antibiotic resistance and limited therapeutic options emphasise the urgent need for more international cooperation to tackle new emerging microbial threats and multidrug-resistant microbes. development of new therapeutic options needs to be coupled to international regulations on the use and prescription of antimicrobial drugs. dsh and az coordinated the writing of this series paper and wrote the draft outline, and subsequent and fi nal drafts. all authors contributed relevant text and tables on their expert sections or sections and contributed to fi nalising the paper. fgh has served as non-paid consultant for multiple companies engaged in marketing and/or clinical development of antivirals for respiratory viral infections including several whose therapeutics are discussed in this review (adamas, biocryst, gsk, genentech, janssen, roche, romark, toyama/medivector, visterra). dwd holds founder shares in f2g, a university of manchester spin-out company. he acts as a consultant to trinity group, t2 biosystems, glaxosmithkline, sigma tau, oxon epidemiology, and has consulted for merck and astellas and he has been paid to give talks on behalf of astellas, gilead, and pfi zer. all other authors declare no confl icts of interest. interspecies transmission and emergence of novel viruses: lessons from bats and birds clinical features and rapid viral diagnosis of human disease associated with avian infl uenza a h5n1 virus history of swine infl uenza viruses in asia writing committee of the world health organization consultation on northern hemisphere infl uenza vaccine composition for 2013-2014. who recommendations for the viruses used in the 2013-2014 northern hemisphere infl uenza vaccine: epidemiology, antigenic and genetic characteristics of infl uenza a(h1n1)pdm09, a(h3n2) and b infl uenza viruses collected from who. avian infl uenza a(h7n9) virus clinical and epidemiological characteristics of a fatal case of avian infl uenza a h10n8 virus infection: a descriptive study addressing the public health burden of respiratory viruses: the battle against respiratory viruses (brave) initiative for the niaid collaborative antiviral study group. safety and effi cacy of nebulized zanamivir in hospitalized patients with serious infl uenza antiviral agents for the treatment and chemoprophylaxis of infl uenza. recommendations of the advisory committee on immunization practices (acip) antiviral resistance among highly pathogenic infl uenza a (h5n1) viruses isolated worldwide in 2002-2012 shows need for continued monitoring newer infl uenza antivirals, biotherapeutics and combinations complications and outcomes of pandemic 2009 infl uenza a (h1n1) virus infection in hospitalized adults: how do they diff er from those in seasonal infl uenza? eff ectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with infl uenza a h1n1pdm09 virus infection: a metaanalysis of individual participant data determinants of antiviral eff ectiveness in infl uenza virus a subtype h5n1 eff ectiveness of antiviral treatment in human infl uenza a(h5n1) infections: analysis of a global patient registry association between adverse clinical outcome in human disease caused by novel infl uenza a h7n9 virus and sustained viral shedding and emergence of antiviral resistance eff ect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe infl uenza: double blind randomised controlled trial a prospective intervention study on higher-dose oseltamivir treatment in adults hospitalized with infl uenza a and b infections viral clearance with standard or triple dose oseltamivir therapy in critically ill patients with pandemic (h1n1) 2009 infl uenza intravenous peramivir for treatment of infl uenza a and b virus infection in high-risk patients a clinical trial of intravenous peramivir compared with oral oseltamivir for the treatment of seasonal infl uenza in hospitalized adults intravenous peramivir for treatment of infl uenza in hospitalized patients business wire. biocryst announces outcome from the peramivir phase 3 interim analysis early emergence of an h275y mutation in a hematopoietic cell transplant recipient treated with intravenous peramivir rapid selection of oseltamivir-and peramivirresistant pandemic h1n1 virus during therapy in 2 immunocompromised hosts cs-8958, a prodrug of the new neuraminidase inhibitor r-125489, shows long-acting anti-infl uenza virus activity clinical experience with intravenous zanamivir under an emergency investigational new drug program in the united states safety and pharmacokinetics of intravenous zanamivir treatment in hospitalized adults with infl uenza: an open-label, multicenter, single-arm, phase ii study long-acting neuraminidase inhibitor laninamivir octanoate versus oseltamivir for treatment of infl uenza: a doubleblind, randomized, noninferiority clinical trial a randomized double-blind controlled study of laninamivir compared with oseltamivir for the treatment of infl uenza in patients with chronic respiratory diseases an investigational antiviral drug, das181, eff ectively inhibits replication of zoonotic infl uenza a virus subtype h7n9 and protects mice from lethality a phase ii study of das181, a novel host directed antiviral for the treatment of infl uenza infection das181 treatment of hematopoietic stem cell transplant patients with parainfl uenza virus lung disease requiring mechanical ventilation favipiravir (t-705), a novel viral rna polymerase inhibitor combinations of favipiravir and peramivir for the treatment of pandemic infl uenza a/california/04/2009 (h1n1) virus infections in mice clinical eff ectiveness and safety of favipiravir, a novel anti-infl uenza drug with a selective inhibition activity against viral rna polymerase. 51st interscience conference on a phase 2, randomized, double blind, placebocontrolled, multicenter study evaluating the safety and pharmacokinetics of diff erent dosing regimens of favipiravir (t-705) in adult subjects with uncomplicated infl uenza, viii options for control of infl uenza a new class of anti-infl uenza molecules targeting viral hemagglutinin at the post-translational level romark announces clinical trial results for new infl uenza drug presented at idsa meeting eff ect of nitazoxanide in adults and adolescents with acute uncomplicated infl uenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial adjunctive therapies and immunomodulatory agents in the management of severe infl uenza the eff ectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral aetiology: a systematic review and exploratory meta-analysis convalescent plasma treatment reduced mortality in patients with severe pandemic infl uenza a (h1n1) 2009 virus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 infl uenza a(h1n1) infection an in vivo human-plasmablast enrichment technique allows rapid identifi cation of therapeutic infl uenza a antibodies antiviral combinations for severe infl uenza infections: a review triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant infl uenza virus strains in vitro effi cacy of oseltamivirzanamivir combination compared to each monotherapy for seasonal infl uenza: a randomized placebo-controlled trial triple-combination antiviral drug for pandemic h1n1 infl uenza virus infection in critically ill patients on mechanical ventilation adjuvant treatment with a mammalian target of rapamycin inhibitor, sirolimus, and steroids improves outcomes in patients with severe h1n1 pneumonia and acute respiratory failure anti-malaria drug chloroquine is highly eff ective in treating avian infl uenza a h5n1 virus infection in an animal model eff ectiveness of chloroquine against infl uenza chloroquine for infl uenza prevention: a randomised, double-blind, placebo controlled trial international severe acute respiratory and emerging infections consortium treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients severe acute respiratory syndrome and coronavirus tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicenter retrospective matched cohort study mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc use of convalescent plasma therapy in sars patients in hong kong contact investigation of a case of human novel coronavirus infection treated in a german hospital clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection eff ects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study invasive aspergillosis after pandemic (h1n1) pharmacokinetics and pharmacodynamics of a novel triazole, isavuconazole: mathematical modeling, importance of tissue concentrations, and impact of immune status on antifungal eff ect immune reconstitution after allogeneic transplantation and expanding options for immunomodulation: an update monoculture-derived t lymphocytes specifi c for multiple viruses expand and produce clinically relevant eff ects in immunocompromised individuals infusion of cytomegalovirus (cmv)-specifi c t cells for the treatment of cmv infection not responding to antiviral chemotherapy transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation clinical grade generation of hexon-specifi c t cells for adoptive t-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation rapid salvage treatment with virus-specifi c t cells for therapy-resistant disease expansion of t cells targeting multiple antigens of cytomegalovirus, epstein-barr virus and adenovirus to provide broad antiviral specifi city after stem cell transplantation morpholino antisense oligomers: design, preparation, and properties antisense-mediated modulation of splicing: therapeutic implications for duchenne muscular dystrophy viral strategies of immune evasion viral interference with antigen presentation to cd8+ t cells: lessons from cytomegalovirus stimulation of human ebv-and cmv-specifi c cytolytic eff ector function using allogeneic hla molecules t cell allorecognition via molecular mimicry treating b-cell cancer with t cells expressing anti-cd19 chimeric antigen receptors cytotoxic therapy for severe swine fl u a/h1n1 emergence of multiply resistant pneumococci molecular characterization of streptococcus pneumoniae invasive serotype 19a isolates from adults in two spanish regions trends in antibacterial resistance among streptococcus pneumoniae isolated in the usa: update from protekt us years 1-4 streptococcus pneumoniae: does antimicrobial resistance matter? antimicrobial activity of ceftaroline tested against drug-resistant subsets of streptococcus pneumoniae from us medical centers variation in defi nitions and isolation procedures for multidrug-resistant gram-negative bacteria: a survey of the society for healthcare epidemiology of america research network who. antimicrobial resistance-global report and surveillance variation in potency and spectrum of tigecycline activity against bacterial strains from u.s. medical centers since its approval for clinical use new developments in carbapenems meta-analysis of doripenem vs comparators in patients with pseudomonas infections enrolled in four phase iii effi cacy and safety clinical trials current control and treatment of multidrug-resistant acinetobacter baumannii infections polymyxins revisited global assessment of the antimicrobial activity of polymyxin b against 54 731 clinical isolates of gram-negative bacilli: report from the sentry antimicrobial surveillance programme in vitro activity of colistin (polymyxin e) against 3,480 isolates of gram-negative bacilli obtained from patients in canadian hospitals in the canward study determination of in vitro activities of polymyxin b and rifampin in combination with ampicillin/sulbactam or cefoperazone/ sulbactam against multidrug-resistant acinetobacter baumannii by the e-test and checkerboard methods antibiotics in phase ii and iii clinical trials antibiotics currently in clinical development who. who global strategy for containment of antimicrobial resistance discovery and preclinical development of new antibiotics who guidelines for the programmatic management of drug-resistant tuberculosis: 2011 update new antituberculosis drugs, regimens, and adjunct therapies: needs, advances, and future prospects advances in the development of new tuberculosis drugs and treatment regimens tuberculosis drug discovery in the post-post-genomic era provisional cdc guidelines for the use and safety monitoring of bedaquiline fumarate (sirturo) for the treatment of multidrugresistant tuberculosis rational development of adjunct immunebased therapies for drug-resistant tuberculosis: hypotheses and experimental designs host genotype-specifi c therapies can optimize the infl ammatory response to mycobacterial infections ibuprofen therapy resulted in signifi cantly decreased tissue bacillary loads and increased survival in a new murine experimental model of active tuberculosis antitubercular specifi c activity of ibuprofen and the other 2-arylpropanoic acids using the ht-spoti whole-cell phenotypic assay effl ux inhibition with verapamil potentiates bedaquiline in mycobacterium tuberculosis acceleration of tuberculosis treatment by adjunctive therapy with verapamil as an effl ux inhibitor adjuvant host-directed therapy with types 3 and 5 but not type 4 phosphodiesterase inhibitors shortens the duration of tuberculosis treatment diff erential gene expression in response to adjunctive recombinant human interleukin-2 immunotherapy in multidrug-resistant tuberculosis patients totally drugresistant tuberculosis and adjunct therapies anti-infl ammatory activity of macrolide antibiotics autologous mesenchymal stromal cell infusion as adjunct treatment in patients with multidrug and extensively drug-resistant tuberculosis: an openlabel phase 1 safety trial human fungal infections: the hidden killers the frequency of pneumocystis jirovecii in sputum samples of hiv and tb patients received at the central reference laboratory in windhoek, namibia global burden of chronic pulmonary aspergillosis as a sequel to pulmonary tuberculosis voriconazole resistance in aspergillus fumigatus-should we be concerned? risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in aspergillus species clinical implications of azole resistance in aspergillus fumigatus, the netherlands frequency and evolution of azole resistance in aspergillus fumigatus associated with treatment failure azole antifungal resistance in aspergillus fumigatus high frequency triazole resistance found in non-culturable aspergillus fumigatus from lungs of patients with chronic fungal disease global burden of abpa in adults with asthma and its complication chronic pulmonary aspergillosis allergic bronchopulmonary aspergillosis: review of literature and proposal of new diagnostic and classifi cation criteria allergic bronchopulmonary aspergillosis in cystic fi brosis. a european epidemiological study novel immunologic classifi cation of aspergillosis in adult cystic fi brosis high prevalence of azole-resistant aspergillus fumigatus in adults with cystic fi brosis exposed to itraconazole a dimorphic fungus causing disseminated infection in south africa a randomised, double-blind study of combination antifungal therapy with voriconazole and anidulafungin versus voriconazole monotherapy for primary treatment of invasive aspergillosis. 22nd european congress of clinical microbiology and infectious diseases a new solid oral tablet formulation of posaconazole: a randomized clinical trial to investigate rising single-and multiple-dose pharmacokinetics and safety in healthy volunteers oral fl uconazole during pregnancy and risk of birth defects therapy for fungal diseases: opportunities and priorities a phase 1, randomized, open-label crossover study to evaluate the safety and pharmacokinetics of 400 mg albaconazole administered to healthy participants as a tablet formulation versus a capsule formulation enfumafungin derivative mk-3118 shows increased in vitro potency against clinical echinocandin-resistant candida species and aspergillus species isolates activity of mgcd290, a hos2 histone deacetylase inhibitor, in combination with azole antifungals against opportunistic fungal pathogens modeling nikkomycin z dosing and pharmacology in murine pulmonary coccidioidomycosis preparatory to phase 2 clinical trials in vitro and in vivo antifungal activities of t-2307, a novel arylamidine advancing antifungal r&d. www.f2g.com/f2g-ltd-completes-30-million-fi nancing-round-to-fund-pre-clinical-and-clinical-developmentof-novel-anti-fungal-compounds/ (accessed may the antidepressant sertraline provides a promising therapeutic option for neurotropic cryptococcal infections signaling cascades as drug targets in model and pathogenic fungi progress and prospects for targeting hsp90 to treat fungal infections inhibition of candida albicans virulence factors by novel levofl oxacin derivatives restoration of viral immunity in immunodefi cient humans by the adoptive transfer of t cell clones adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specifi c t-cell lines clinical-scale generation of multi-specifi c anti-fungal t cells targeting candida, aspergillus and mucormycetes towards an ecology of the lung: new conceptual models of pulmonary microbiology and pneumonia pathogenesis deaths due to respiratory tract infections in africa: a review of autopsy studies global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study key: cord-339724-roj8ksvc authors: lan, jiaming; deng, yao; chen, hong; lu, guangwen; wang, wen; guo, xiaojuan; lu, zhuozhuang; gao, george f.; tan, wenjie title: tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen date: 2014-11-18 journal: plos one doi: 10.1371/journal.pone.0112602 sha: doc_id: 339724 cord_uid: roj8ksvc the development of an effective vaccine is critical for prevention of a middle east respiratory syndrome coronavirus (mers-cov) pandemic. some studies have indicated the receptor-binding domain (rbd) protein of mers-cov spike (s) is a good candidate antigen for a mers-cov subunit vaccine. however, highly purified proteins are typically not inherently immunogenic. we hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. therefore, the immunogenicity of a novel mers-cov rbd-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. different vaccination regimens were compared in balb/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant mers-cov rbd in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly i:c) or alum and cysteine-phosphate-guanine (cpg) oligodeoxynucleotides (odn). the immune responses of mice vaccinated with rbd, incomplete freund’s adjuvant (ifa) and cpg odn by a subcutaneous (s.c.) route were also investigated. we evaluated the induction of rbd-specific humoral immunity (total igg and neutralizing antibodies) and cellular immunity (elispot assay for ifn-γ spot-forming cells and splenocyte cytokine production). our findings indicated that the combination of alum and cpg odn optimized the development of rbd-specific humoral and cellular immunity following subunit vaccination. interestingly, robust rbd-specific antibody and t-cell responses were induced in mice immunized with the rrbd protein in combination with ifa and cpg odn, but low level of neutralizing antibodies were elicited. our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. the vaccination regimen used in this study is promising and could improve the protection offered by the mers-cov subunit vaccine by eliciting effective humoral and cellular immune responses. in 2012 a novel human coronavirus, middle east respiratory syndrome coronavirus (mers-cov), caused outbreaks of a sars-like illness in the middle east, and is now considered a threat to global public health [1, 2] . as of july 23, 2014, the world health organization (who) reported 837 confirmed cases of mers-cov infection, including 291 deaths (a case fatality rate of 34.8%) [3] . now, studies show that camels are a likely primary source of the mers-cov that is infecting humans [4, 5, 6] . but the routes of transmission between camels and people which is the key point to stop transmission of the virus, is far from clearly understood. the continued threat of mers-cov necessitates the development of an effective vaccine. some studies have indicated that recombinant receptor-binding domain (rrbd) protein of mers-cov spike (s) is a good candidate antigen for a mers-cov subunit vaccine [7, 8, 9, 10] . however, highly purified proteins are typically not inherently immunogenic, as they usually lack the means to directly stimulate the innate immune system [11] . besides, they are often prone to degradation. hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvant(s) to evoke the desired antigen-specific immune response phenotype enabling successful vaccination [12] . aluminium is one of the most common adjuvant in non-living vaccines, has a record of successful use in human vaccination where it promotes antibody-mediated protective immunity [13] . another classic adjuvant is that based on a water-in-oil-emulsion formulation, such as incomplete freund's adjuvant (ifa). recently, researches have focused on adjuvants that signal through pattern recognition receptors (prrs), such as toll-like receptors (tlrs) [14] . cysteine-phosphate-guanine (cpg) oligodeoxynucleotides (odns), which activate b cells and plasmacytoid dendritic cells via tlr9 and induce both innate and adaptive immunity, are currently being developed as a vaccine adjuvant [15] . another frequently used adjuvant is polyriboinosinic acid (poly(i:c)), a synthetic dsrna that mimics the effects of naturally occurring dsrna, a tlr3 agonist [16, 17] . beside of enhancing the immune response, adjuvant(s) can tailor-make the polarization immune response. for example, ppolarized th1-type immunity can be achieved by the addition of freund's adjuvant or cpg dna to an antigen. on the other hand, th2 antibody responses can be induced by the alum, as indicated by increased igg1 relative to igg2a [18, 19] . however, in situations where both th1 and th2 responses are required for protection, the choice of one regimen over another might be counter effective. this has led to additional research for alternative adjuvants or adjuvant combinations that promote balanced mixed th1/th2 responses [18] . in recent years, the combination of antigens with more than one adjuvant, called the adjuvant system approach has produced vaccines with the ability to generate effective immune responses adapted to both the pathogen and the target population [20] . by using multiple adjuvants in combination, antigen presenting cell (apc) activation is influenced at more than one level, guiding the subsequent adaptive pathways and ultimately inducing a more robust immune response [20] . the induction of a robust humoral, including potent neutralizing antibodies, and cellular immune response is likely essential for immediate and sustained protective immunity in a mers-cov vaccine design. in this study, different adjuvants combination regimens including alum, ifa, cpg and poly(i:c) were compared in an effort to promote balance between th1 and th2 immune response to bystander rrbd antigen spanning residues 367-606 of mers-cov s in a murine model to develop an effective vaccine against mers-cov infection. animal studies were carried out in strict compliance with the guide for the care and use of laboratory animals of the people's republic of china. the study protocol was approved by the committee on the ethics of animal experiments of the chinese centre for diseases control and prevention. all procedures were performed under ethylether anesthesia and all efforts were made to minimize suffering. mers-cov rrbd protein, containing a 240-amino-acid fragment spanning residues 367-606 ( figure 1a ) of genbank number jx869059, was prepared using a bac-to-bac baculovirus expression system as described in detail previously [21] . the required rrbd was measured by sds-page ( figure 1b) and western blot ( figure 1c ) with a mice polyclonal antibody against spike of mers-cov ( figure 1c ). before vaccination, the rrbd protein was quantified by bradford method. the rrbd protein was combined with different adjuvants immediately prior to immunisation. aluminium hydroxide was kindly provided by the north china pharmaceutical group corporation genetech biotechnology development company. the odn motif containing unmethylated cpg (59-tccat-gacgttcctgacgtt-39) was synthesised by takara bio inc. poly(i:c) and ifa were purchased from sigma (st. louis, mo). a single dose (10 mg) of rrbd protein (100 ml) was combined with either 100 mg of alum alone (rbd/a), alum plus 10 mg of cpg (rbd/a+c), alum plus 50 mg of poly(i:c) (rbd/ a+p) or 10 mg of cpg and 100 ml of ifa (rbd/i+c). six-to-eight-week-old female balb/c mice (animal care centre, chinese academy of medical science, beijing, china) were randomly distributed into eight groups. eight mice of each group were vaccinated three times with rrbd proteins at 3-week intervals by either an intramuscular (i.m.) or a subcutaneous (s.c.) route (table 1 ). sera were collected 2 weeks after each vaccination and heat-inactivated at 56uc for 30 min before detection of rbdspecific and neutralizing antibodies. mice were scarified 2 weeks after the last immunisation, and their lungs and spleens were harvested for detection. a schematic of the vaccination and analysis timeline is shown in figure 2 . elisa was used to detect the mers-cov rbd-specific antibody response in immunised mice. briefly, 96-well elisa plates were pre-coated with rrbd protein (100 ng/well) overnight at 4uc and blocked with 2% non-fat milk for 2 h at 37uc. serially diluted sera of eight mice in each group were added to the plates and incubated at 37uc for 1 h, followed by four washes with phosphate-buffered saline (pbs) containing 0.1% tween 80 (pbst). bound antibodies were incubated with hrp-conjugated anti-mouse igg, igg1, igg2a or igg2b (1:5,000, sigma) for 1 h at 37uc. the reaction was visualised by using 3, 39, 5, 59tetramethylbenzidine (tmb) peroxidase substrate solution (invitrogen) and stopped by addition of 2 m h 2 so 4 . absorbance at 450 nm was measured using an elisa plate reader (wellscan mk 3). the cut-off value was set 2.1-fold above that of the negative control. antibody avidity was determined using the elisa method described by vermont et al [22] . briefly, sera were diluted to a titre of 1:100, and an ascending concentration of the chaotropic agent nascn (0-7 m) was added to the plate. plates were incubated for 15 min at room temperature (rt) before washing and development to determine total igg. as a control for antibody specificity, elisa was used to measure the total anti-mers-cov igg titres of pre-and post-vaccination sera samples. the conventional neutralization assay using live mers-cov is cumbersome and has to be performed in biosafety level-3 facilities. therefore, we adapted a mers-cov pseudovirus system which is sensitive and quantitative, and can be conducted in biosafety level-2 facilities as reported by zhao et al [23] . in brief, 293t cells were co-transfected with a plasmid encoding codon-optimized mers-cov s protein and a plasmid encoding env-defective, luciferaseexpressing, hiv-1 genome (pnl4-3r-e-luc) using fugene hd reagents (roche, basel, switzerland). supernatants containing mers-cov pseudovirus were harvested 48 h post-transfection and used for single-cycle infection. huh7.5 cells were plated at 10 4 cells/well in 96-well tissue-culture plates and grown overnight. the supernatants containing pseudovirus were pre-incubated with 2-fold serially diluted mouse sera at 37uc for 1 h before addition to cells. the culture was refed with fresh medium 24 h later and incubated for an additional 48 h. cells were washed with pbs and lysed using lysis reagent included in a luciferase kit (promega). aliquots of cell lysates were transferred to 96-well costar flatbottom luminometer plates (corning costar), followed by addition of luciferase substrate (promega). relative light units were determined immediately in the gaomax luminometer (promega). all experiments were carried out in triplicate. pseudovirus inhibition (pi) rate was calculated as: (relative luciferase units of mock sera -relative luciferase units of immune serum for a given dilution)/relative luciferase units of mock sera. to evaluate the antigen-specific t-cell response induced by the vaccination regimes, an ifn-c elispot assay was performed as described previously [17] . briefly, 96-well plates were coated with 100 ml per well of 5 mg/ml anti-mouse ifn-c antibody (bd pharmingen) overnight at 4uc and then blocked for 2 h at rt. freshly harvested splenocytes (5610 5 per well) or lung lymphocytes of eight mice in each group were isolated as described previously [22] . then, 4 mg/ml of a synthesised 18-mer peptide library, which overlapped the mers-cov s rbd by 10 amino acids, was added to the wells in triplicate. next, a biotinylated detection antibody (bd pharmingen) and streptavidin-horseradish peroxidase were added. blots were developed by the addition of an aec (3-amino-9-ethylcarbazole) substrate solution, which produced a coloured spot after 5-min rt incubation in the dark. finally, ifn-c spot-forming cells (sfcs) were counted. phorbol 12-myristate 13-acetate (pma) and ionomycin were added to the positive-control group, whereas the negative-control group received no stimuli. the number of peptide-specific ifn-c secreting t cells was calculated by subtracting the negativecontrol value from the sfc count. a cba analysis was conducted to investigate the levels of th1and th2-type cytokine secretion [18] in mice after three times' immunization. in brief, splenocytes (5610 5 per well) of eight mice in each group were distributed in 96-well plates and stimulated with 4 mg/ml of pooled rbd peptide. plates were incubated for 24 h at 37uc and supernatants were harvested. the concentrations of cytokines, including il-2, il-4, il-6, il-10, tnf-a, il-17a and ifn-c, were measured using a mouse th1/th2/th17 cytokine kit (bd biosciences) and a facs calibur flow cytometer (becton dickinson). data were analysed using the fcap array software (becton dickinson). statistical analysees were conducted using the one-way anova function in the spss 17.0 software package. a p-value less than 0.05 were considered to indicate statistical significance. the vaccination regime affects the rbd-specific igg response in mice to assess the humoral immune response to different immunisation regimens, mice were immunised with rrbd protein combined with different adjuvants three times at 3-week intervals. serum samples were collected 2 weeks after each vaccination and total anti-mers-cov rbd igg antibody titres were determined by elisa. the results indicated that rrbd protein combined with any adjuvant, including alum, ifa, cpg or poly(i:c), could induce a rbd-specific igg antibody response in the majority of mice after the second immunisation. in a few vaccinated mice, rbd-specific igg antibodies could be detected even after the first immunisation. the seroconversion rates of the different groups following the first and the second immunisations are shown in table 1 . as shown in figure 3a , there was no discernible increase in igg titres after the third immunisation compared to the second immunisation. among the vaccination regimes, rbd/a+c and rbd/i+c elicited the highest total igg titres (p,0.05, figure 3a) . besides, the difference of igg titer in these two groups was not significant (p$0.05, figure 3a ). similarly shown in figure 3a , the difference of igg titer in rbd/a and rbd/a+p groups had no significance. the rbd-specific antibodies were lower than 1:10 in the adjuvant control groups at each of the three vaccinations. the responses to the various vaccination regimes were investigated using nascn antibody-displacement elisa to measure antibody avidity ( figure 3b ). mice received the rbd/ a+c or rbd/i+c regimes had higher antibody avidity 2 weeks after the final vaccination than those received the rbd/a or rbd/a+p regimes. it was also noteworthy that high antibody avidity correlated with a high igg titre in mice. to further characterise the immune response to the different vaccination regimes, igg isotype analyses were performed 2 weeks after the final vaccination using secondary antibodies against igg1, igg2a and igg2b. as shown in figures 3c, d , e and f, mice immunised with rbd/a+c or rbd/i+c produced higher igg1 and igg2a titres than mice immunised with rbd/a or rbd/a+p. also, the igg1 to igg2a ratio revealed a th1 skewed response in mice that received the rbd/a+c or rbd/i+c regimes. in contrast, the rbd/a and rbd/a+p regimes produced a higher igg1/igg2 ratio, indicating a th2 response. the titres of rbd-specific igg2b antibodies, however, were not significantly different among the vaccination groups (p$0.05). nneutralizing antibodies in the sera of mice immunised with different vaccination regimes were evaluated with a pseudovirusbased neutralization assay. a low level of neutralizing antibodies were detected 2 weeks after the first or second vaccination in aall of the sera tested, although the total igg antibody levels had almost peaked after the second vaccination. the highest level of neutralizing antibodies was induced after the last vaccination ( figure 4 ). the pseudovirus inhibition (pi) rates are shown in figure 4 . as shown, the rbd/a+c regime had the highest neutralizing antibody activity (p,0.01). surprisingly, there was low level of detectable neutralizing antibody in the sera of mice immunised s.c. with the rbd/i+c regime, although sera from this to characterise the cellular immune responses elicited by the vaccination regimes, single ifn-c-producing cells were quantified by elispot. both systemic and local cellular immune responses were assessed using lymphocytes from the spleen and lungs of immunised mice. the peptide library used to stimulate the lymphocytes was described in the materials and methods section. results are expressed as the number of sfcs per 10 6 input cells. adjuvants without rrbd did not elicit a clear cellular response in the spleen 2 weeks after the third immunisation ( figure 5a ). neither rbd/a nor rbd/a+p induced a significant cellular immune response. in contrast, rbd/a+c and rbd/i+c regimen enhanced a detectable systemic cellular immune response. furthermore, the rbd/i+c regimen induced the greatest cellular immune response with the greatest number of ifn-c-producing cells in the spleen (p,0.05). a significant cellular immune response in the lung was induced only by the rbd/i+c regime, although a few ifn-c producing cells were detected in all immunised mice ( figure 5b ). we therefore concluded that while both the rbd/a+ c and rbd/i+c regimes could induce a systematic cellular immune response in mice, only the rbd/i+c regime could elicit a significant local cellular immune response in the lung. a higher frequency of rbd-specific, tnf-a-and il-4producing t cells were induced with alum and cpg via an i.m. route the cytokine profiles of spleen cells from immunised mice were analysed after stimulation with rbd-specific peptides. during cba, splenocytes from mice immunised with rbd/a+c or rbd/i+c produced ifn-c ( figure 6a ). in contrast, il-2 was produced by splenocytes following immunisation with rrbd combination of any adjuvants ( figure 6b ). but the differences in ifn-c and il-2 production among the groups were not significant (p$0.05). compared with other groups, splenocytes from mice immunised with rbd/a+c induced significantly higher levels of tnf-a ( figure 6c ) and il-4 ( figure 6d ) (p,0.01). all these indicated that the adjuvants of alum and cpg combination could induce a th1 and th2 mixed immune responses in the rrbd antigen model of mers-cov, though the responses revealed a th1 polarization in the isotype elisa and elispot detection. similarly, the high levels of ifn-c, il-2 and il-6 also indicated a th1 and th2 mixed immune responses could be induced by the rbd/i+c regimes. (figure 6a, 6b, 6e) . different from all of these, as shown in figure 6f , the rbd/a+p regimes induce the highest level of il-10 (p,0.01), which indicated a th2 response inclination consistent with the results of igg isotype. however, il-17a was not detected in any of the vaccination groups (data not shown). coronaviruses can adapt rapidly to new hosts, and an adaptation of mers-cov that allowed the virus to efficiently replicate in humans would be a major public health concern, since such an adaptation could trigger a pandemic [24] . the development of an effective vaccine is critical to prevent a potential mers-cov pandemic. previous studies have shown that vaccination with the sars-cov rbd induces highly potent neutralizing antibodies and significantly inhibits sars-cov infection [25, 26] . therefore it was proposed that vaccination with the rbd of mers-cov, which belongs to the same betacoronavirus genus as sars-cov [27, 28] , might also inhibit mers-cov infection and induce a neutralizing antibody response against mers-cov. du et al [8] identified a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated rbd of mers-cov spike protein fused with human igg fc fragment (s377-588-fc) was able to induce in the vaccinated mice strong mers-cov sspecific antibodies, which blocks the binding of rbd to dipeptidyl peptidase 4 (dpp4), the human mers-cov receptor [29] and effectively neutralizes mers-cov infection. besides, they [9] showed that residues 377 to 662 in the s protein of mers-cov induced significant neutralizing antibody responses, suggesting that this region had a potential to be developed as a mers-cov vaccine. mou et al [10] showed the polyclonal antibodies in rabbits against the rbd in the s protein to a 231-amino-acid fragment (residues 358 to 588) efficiently neutralized virus infectivity. however, none of the studies evaluated the immunogenicity of rrbd protein systematically in an animal model. recently, ma et al [7] ssuggested the possibility of developing a recombinant rbd protein containing residues 377-662 into an effective and safe mucosal mers vaccine through the intranasal route in the presence of the only poly(i:c) adjuvant in a mouse model. while the need for vaccines with the ability to generate an effective immune response has led to the combination of antigens with more than one adjuvant, the 'adjuvant system' approaches. the adjuvant system approach aids in the development of vaccines that generate effective immune responses [11, 30] . in this study, the roles of three adjuvants-alum, ifa, cpg and poly (i:c)-in rrbd subunit vaccination were investigated aimed at inducing an effective immune response through use of tailored adjuvant combinations and delivery routes. consistent with above studies, all vaccination regimes containing rrbd induced an rbd-specific cellular and humoral immune response. however, a more robust immune response was elicited when mice were immunised with the rbd/a+c and rbd/i+c regimes. an unexpected result was the absence of neutralizing antibodies in the sera of rbd/i+c immunised mice, despite anti-rbd specific igg titres being similar for the rbd/i+c and rbd/ a+c regimes. to further understand the riddle, we detected the aantibody avidity of different vaccination regimes by aavidity elisa. however, the results showed the high antibody avidity correlated with a high igg titre in mice of rbd/i+c and rbd/ a+c groups. so, we speculated maybe the adjuvants of destroyed the conformation of rrbd and covered the antigen binding sites. another probable cause of the low titer of neutralizing antibodies in the sera of rbd/i+c immunised mice was the delivery route of subcutaneous. as known, the subcutaneous may be associated with degradation at injection site, which leads to decreased bioavailability [31] . whatever, further studies are in process. compared with other studies, the regimes in this study induced lower titres of neutralization antibodies. for example, the pi 50 of alum plus cpg, the group showing the highest titer of neutralizing antibody in all immunization groups was 1:500. while the rrbd protein in the above studies acquired a 1:1000 in mice neutralization antibody titre. the differences may be caused by the detection methods of neutralization antibody. as showed in the materials and methods parts, the neutralization antibodies in this study were detected by a pseudovirus system which can be conducted in biosafety level-2 facilities. while the differences of induced neutralization antibodies among different groups can be shown clearly. the subclass of immunoglobulin induced after immunization is an indirect measure of the relative contribution of th1-type cytokines vs. th2-type cytokines [18] . to characterise the immune response of the different vaccination regimes, igg isotype including igg1, igg2a and igg2b analyses were performed. as expected, the rbd/a regimes produced a th2 response with high igg1/igg2 ratio. in contrast, mice received the rbd/a+c or rbd/i+c regimes revealed a th1 skewed response. consistently, the rbd/a+c or rbd/i+c regimes induced a systematic cellular immune response in mice by elispot analysis. the high level of ifn-c and il-2 in the cba was also a proof of the cellular immune response in mice. besides, the mice in the rbd/a+c group had a high level of il-4 and il-10, which were an index of th2 skewed response. taken together, the rbd/a+c induced a th1 and th2 mixed immune responses, though the responses had a th1 inclination. it was our original intention of mixed th1/th2 responses for better protection. similarly, the rbd/i+c regimes induced a mixed th1 and th2 responses. however, it was a pity that the rbd/i+c regimes could not induce an effective neutralization antibody, which was the most important factor of a prophylactic vaccine. above all, in this study, mers-cov s rrbd combined with the adjuvants alum and cpg produced the most robust immune response. it indicates that the combination of alum and cpg was the optimal strategy for i.m. rrbd antigen delivery in a murine model. this result will facilitate future mers-cov vaccine design. the results of the present study also support the importance of the adjuvant system approach, although adjuvant combinations do not always produce the desired response, as seen with rbd/i+c. consistent with the results of the present study, cpg plus alum was found to induce protective humoral, as well as cellular immunity, in mice immunised with a recombinant haemagglutinin vaccine that protected against influenza virus challenge [32] . the ideal immunity of the cpg and alum combination may be the result of mutual complementation of these two adjuvants. it is well known that alum can promote antibody-mediated protective immunity. however, alum is a poor inducer of cellular immune responses [12] . recently, adjuvants including oil-in-water emulsions have shown improved efficacy for avian influenza protection suggesting that even for diseases where humoral immunity can confer protection, cellular immune responses may be necessary in vaccine design [33] . the key features of cpg-odn used as a vaccine adjuvant, include the ability to elicit th1 cell, but only under certain conditions, cd8+ cytotoxic t cell responses and an additional ability to divert the pre-existing th2 response in neonates and elderly mice toward a th1 phenotype [34] . thus, we expect that the combination of alum and cpg will prove applicable in a range of infectious diseases that have defeated current immunisation strategies. except for a choice of adjuvants in combination with optimal protective antigen, practical items such as the antigen: adjuvant ratio, dose, vaccination regimen and often route of administration will strongly impact on both the effectiveness and safety of the vaccine formulation. in most cases, an experimental vaccine will be initially tested in an animal model [29] . to evaluate the immunogenicity of rrbd protein thoroughly, it is necessary to test the protective effects of rrbd subunit immunisation in an animal model of mers-cov infection. to date, rhesus macaques have been reported to generate pneumonia-like symptoms within 24 h of mers-cov infection [35] , and we are testing the effects of rrbd immunisation in rhesus macaques. considerable efforts are being made to establish a small animal model of mers-cov infection. though the lung cells of the syrian hamster express the receptor for mers-cov, they are not susceptible to mers-cov infection [35, 36] . recently, a mouse model of mers-cov infection was reportedly generated by transduction of mice with adenoviral vectors expressing dpp4 [24] . in the future, we expect the protective effect of the rbd/a+c vaccination should be investigated in this murine model of mers-cov infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread middle east respiratory syndrome coronavirus (mers-cov) -update evidence for camel-to-human transmission of mers coronavirus concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines identification of a receptorbinding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies vaccine adjuvants: mode of action ) ph-triggered microparticles for peptide vaccination immunomodulatory properties of the vaccine adjuvant alum tlr-based immune adjuvants cpg motif-based adjuvant as a replacement for freund's complete adjuvant in a recombinant lhrh vaccine synthetic double-stranded rnas are adjuvants for the induction of t helper 1 and humoral immune responses to human papillomavirus in rhesus macaques poly(i:c)/alum mixed adjuvant priming enhances hbv subunit vaccine-induced immunity in mice when combined with recombinant adenoviral-based hbv vaccine boosting the adjuvanticity of an o. volvulus-derived rov-asp-1 protein in mice using sequential vaccinations and in non-human primates adjuvant-dependent modulation of th1 and th2 responses to immunization with beta-amyloid unmet needs in modern vaccinology: adjuvants to improve the immune response molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 antibody avidity and immunoglobulin g isotype distribution following immunization with a monovalent meningococcal b outer membrane vesicle vaccine a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov rapid generation of a mouse model for middle east respiratory syndrome receptor-binding domain as a target for developing sars vaccines roadmap to developing a recombinant coronavirus s protein receptor-binding domain vaccine for severe acute respiratory syndrome is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? genetic characterization of betacoronavirus lineage c viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus hku5 in japanese pipistrelle: implications for the origin of the novel middle east respiratory syndrome coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc randomized, double-blind, phase 2a trial of falciparum malaria vaccines rts,s/as01b and rts,s/as02a in malaria-naive adults: safety, efficacy, and immunologic associates of protection subcutaneous drug delivery: a route to increased safety, patient satisfaction, and reduced costs insect cell-expressed hemagglutinin with cpg oligodeoxynucleotides plus alum as an adjuvant is a potential pandemic influenza vaccine candidate trends in vaccine adjuvants adjuvant activity of cpg-odn formulated as a liquid crystal pneumonia from human coronavirus in a macaque model the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters key: cord-346320-ysgz6adr authors: arabi, yaseen m.; hajeer, ali h.; luke, thomas; raviprakash, kanakatte; balkhy, hanan; johani, sameera; al-dawood, abdulaziz; al-qahtani, saad; al-omari, awad; al-hameed, fahad; hayden, frederick g.; fowler, robert; bouchama, abderrezak; shindo, nahoko; al-khairy, khalid; carson, gail; taha, yusri; sadat, musharaf; alahmadi, mashail title: feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia date: 2016-09-17 journal: emerg infect dis doi: 10.3201/eid2209.151164 sha: doc_id: 346320 cord_uid: ysgz6adr we explored the feasibility of collecting convalescent plasma for passive immunotherapy of middle east respiratory syndrome coronavirus (mers-cov) infection by using elisa to screen serum samples from 443 potential plasma donors: 196 patients with suspected or laboratory-confirmed mers-cov infection, 230 healthcare workers, and 17 household contacts exposed to mers-cov. elisa-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. of the 443 tested samples, 12 (2.7%) had a reactive elisa result, and 9 of the 12 had reactive indirect fluorescent antibody and microneutralization assay titers. undertaking clinical trials of convalescent plasma for passive immunotherapy of mers-cov infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness. we explored the feasibility of collecting convalescent plasma for passive immunotherapy of middle east respiratory syndrome coronavirus (mers-cov) infection by using eli-sa to screen serum samples from 443 potential plasma donors: 196 patients with suspected or laboratory-confirmed mers-cov infection, 230 healthcare workers, and 17 household contacts exposed to mers-cov. elisa-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. of the 443 tested samples, 12 (2.7%) had a reactive elisa result, and 9 of the 12 had reactive indirect fluorescent antibody and microneutralization assay titers. undertaking clinical trials of convalescent plasma for passive immunotherapy of mers-cov infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness. m iddle east respiratory syndrome coronavirus (mers-cov) was initially identified in september 2012 when a patient in saudi arabia with a severe, acute respiratory infection and acute renal failure died (1) . as of june 19, 2016, more than 1,733 mers-cov cases and at least 628 associated deaths had been identified; >80% of the cases occurred in saudi arabia (2) . more than 20 countries outside of the arabian peninsula have reported mers-cov cases, and the 2015 outbreak in south korea with attendant mortality has reinforced concerns about international outbreaks (3) . no specific treatment has been proven effective for mers-cov infection. convalescent plasma containing mers-cov-specific antibodies from recovered patients has been suggested as a potential therapy for infected persons (4) . convalescent plasma has been used to treat several other viral infections, including those caused by the severe acute respiratory syndrome coronavirus (sars-cov), avian influenza a(h5n1) virus, and influenza a(h1n1)pdm09 virus (5) (6) (7) (8) (9) (10) . a recent metaanalysis of studies using passive immunotherapy for treatment of severe acute respiratory infections of viral etiology suggests that the timely use of convalescent blood products, particularly those with neutralizing antibodies, results in a reduced death rate (11) . public health england and isaric (the international severe acute respiratory and emerging infection consortium) published a decision-making support tool on potential therapies for mers-cov that highlights convalescent plasma and other neutralizing antibody-containing immunotherapeutics (e.g., hyperimmune immunoglobulins and monoclonal antibodies) as the most promising potential treatments for serious mers-cov illness and deserving of evaluation in human clinical trial(s) (4) . however, no data support the feasibility of obtaining convalescent plasma from patients who have been exposed to mers-cov or recovered from infection with the virus. camels are the likely source for most animalto-human transmission and appear to have long-lasting antibody responses; in preclinical models, such antibodies appear effective in reducing the severity of pathologic plasma immunotherapy for mers-cov infection, saudi arabia changes in infected lungs (12) . however, the antibody response to mers-cov infection in humans is poorly defined. thus, we planned a 2-phase study to 1) determine the feasibility of collecting high-titer convalescent plasma from mers-cov patients and contacts and, if successful, to 2) conduct a pilot therapeutic study using convalescent plasma in symptomatic mers-cov patients with moderate to severe illness. herein, we report on the feasibility study. in collaboration with the king abdullah international medical research center, the gulf cooperation council infection control center, and the world health organization (who)-international severe acute respiratory and emerging infection consortium mers-cov working group, we developed a study protocol to screen potential donors, collect high-titer convalescent plasma, and administer the plasma in a clinical trial (13) . the study was approved by the ministry of the national guard health affairs institutional review board (approval no. irbc/233/14, june 9, 2014) and registered in clinicaltrials.gov (nct02190799). we conducted the study at king abdulaziz medical city, a 1,100-bed tertiary care center in riyadh, saudi arabia. the hospital is accredited by the joint commission international, and the hospital's department of pathology and laboratory medicine is accredited by the college of american pathologists and the american association of blood banks. we screened potential convalescent plasma donors from 3 cohorts: 1) patients with acute respiratory illness who were suspected of having mers-cov or who were confirmed mers-cov-positive by real-time reverse transcription pcr (rrt-pcr) of upper or lower respiratory secretions; 2) healthcare workers exposed to a laboratoryconfirmed mers-cov patient, as identified by ongoing active surveillance of the hospital infection prevention and control department; and 3) household contacts of patients with laboratory-confirmed mers-cov infection. we obtained written informed consent for mers-cov serologic testing from all healthcare workers and household contacts. medical teams ordered serologic testing as part of the clinical care for patients with suspected or confirmed mers-cov infection; no additional informed consent was required. healthcare workers completed a self-administered survey that asked questions about the nature, duration, and degree of exposure to patients with laboratory-confirmed mers-cov infection. for all study participants, we documented the time that had elapsed from symptom onset or exposure to the collection of samples for testing. during july-october 2015, we screened serum samples from study participants by using a spike protein subunit 1 (s1)-based elisa. to confirm results of elisa-reactive samples, we used indirect immunofluorescent antibody (ifa) and microneutralization (mn) assays (14) (15) (16) . for mers-cov patients with a nonreactive elisa result, we collected a follow-up sample 14-21 days later for repeat elisa. study participants were considered candidates for plasma donation if they 1) had a reactive elisa result; 2) had a mn assay titer of >100; 3) had no clinical or laboratory evidence of ongoing mers-cov infection; and 4) met the eligibility for plasma donation according to the institutional criteria, which were in accordance with who guidelines (17) . persons who met all criteria were eligible for plasma donation according to the position paper of the who blood regulators network (18) . the primary outcome of this first phase of the study was the feasibility of conducting the second phase. feasibility was measured by our ability to screen and identify a sufficient number of potential plasma donors to provide enough high-titer, fresh-frozen plasma to enroll and provide transfusions to 20 patients over 12 months. each phase 2-enrolled patient would require 2 fresh-frozen plasma units (250-350 ml/unit). we first conducted testing for mers-cov by rrt-pcr. we extracted rna from respiratory specimens (nasopharyngeal swab, tracheal aspirate, bronchoalveolar lavage) using the magna pure 96 viral na kit (roche applied science, indianapolis, in, usa). we tested the extracted nucleic acids by rrt-pcr targeting the upstream envelope protein gene (upe) and open-reading frame 1a (orf1a) regions of the mers-cov genome on a lightcycler 480 system (roche diagnostics, mannheim, germany) realtime pcr (19) . a positive control for orf1a and upe rrt-pcr was performed according to the manufacturer's instructions. to be consistent with the cutoff used by the saudi arabia ministry of health reference laboratory, we considered a cycle threshold (c t ) of <35 the cutoff for upe and orf1a. for c t s >35, we repeated the testing using different samples, preferably from the lower respiratory tract, to avoid false-positive results. we detected mers-cov antibodies by elisa (euroimmun ag, lubeck, germany), using wells coated with mers-cov s1 antigen (20, 21) . serum samples were diluted (1:100) and incubated with antigens according to the elisa manufacturer's instructions. positive and negative control serum and calibration samples were included. antibodies were detected by adding peroxidase-labeled rabbit anti-human igg (euroimmun ag, lubeck). results were reported as the optical density (od) ratio, which was calculated as the od value of the patient's sample divided by the calibrator od value. we used cutoff values recommended by the elisa kit manufacturer: a ratio of <0.8 was considered negative, >0.8 to <1.1 was considered borderline, and >1.1 was considered positive. we used an ifa (euroimmun ag) according to the manufacturer's instructions to detect mers-cov antibodies. serum samples were diluted in doubling dilutions, starting with 1:10 and ending with 1:1,280, in sample buffer and then incubated with vero b4 cells infected with hcov-emc (euroimmun ag). mers-cov igg was detected by adding fitc-labeled goat anti-human igg (euroimmun ag); positive and negative controls were included. samples with an ifa titer of >1:10 were considered reactive according to the ifa manufacturer's instructions. our original protocol used an ifa cutoff of >160 to define suitable donors for plasma (13) . in the course of the study, mn became available, and we revised the criteria for plasma donation to be based on mn assay results. the presence of neutralizing mers-cov antibodies was also assessed using a mn assay (16) . in brief, 2 × 10 4 vero cells/well were plated onto a 96-well microtiter plate. after 24 h, 2-fold serial dilutions of serum samples (heat-inactivated at 56°c for 30 min) were incubated with an equal volume of the mers-cov strain jordan-n3/2012 (200 tcid 50 [50% tissue culture infectious doses]) for 1 h at 37°c (16) . medium was aspirated from the microtiter plate, and 200 ml of the serum-virus mixture was added to the wells in triplicate. the plate was incubated for 48 h at 37°c in a humidified chamber with 5% carbon dioxide, after which the serum-virus mixture was aspirated and the cells were fixed by adding 100 ml of a 1:1 mixture of cold ethanol and methanol. the plate was then incubated at -80°c for 30 min, washed 5 times with pbs, and processed as described above for elisa, using rabbit anti-coronavirus spike protein antibody and horseradish peroxidase-conjugated goat anti-rabbit igg secondary antibody. plates were developed using abts substrate (kpl inc., gaithersburg, md, usa); od was measured at 405 nm. controls consisted of uninfected cells and cells infected with 200 tcid 50 of mers-cov. the highest dilution of serum sample that resulted in a >50% reduction in od, compared with the control containing no antibody, was reported as the 50% virus neutralization titer. rrt-pcr, elisa, and ifa testing for mers-cov were performed at the king abdulaziz medical city laboratory. mn was performed at the naval medical research center (silver spring, md, usa). we used descriptive statistics (i.e., numbers and proportions, means ± sd, and medians with quartile 1 [q1] and q3 values) for measurements for eligible donors and participants with seroreactive test results. we used the pearson correlation to test for correlations between elisa od and ifa and mn titers. the identity of study participants with mers-cov was known only to investigators listed on the approved king abdullah international medical research center protocol. all samples were delinked from any identifiable personal information when provided to nonlisted investigators. we contacted 692 healthcare workers who had a history of exposure to or a diagnosis of mers-cov infection. of those 692 healthcare workers, 230 (33%) consented to serum sampling and were tested (table 1) ; 11 had a history of laboratory-confirmed mers-cov infection, and 219 had a history of exposure but were mers-cov rrt-pcr negative during their asymptomatic or potential immediate postincubation period. only 4 (36.7%) of 11 healthcare workers who had a history of laboratory-confirmed mers-cov infection had elisa-reactive serum samples after a median of 381 days (q1 246 days, q3 485 days) after infection ( figure 1 ). the confirmatory ifa was reactive for all 4 of those healthcare workers, and mn was reactive for 3 (table 2) . however, only 1 healthcare worker (participant no. 9) had a high mn titer (800) ( table 2 ), but she was not considered a candidate for plasma donation because of a previous pregnancy. exposed healthcare workers who had negative mers-cov rrt-pcr results also had nonreactive elisa results. a total of 196 patients with suspected or laboratoryconfirmed mers-cov infection were tested; 183 (88.8%) were hospitalized in the emergency department, 11 (5.8%) in the intensive care unit, and 2 (0.97%) in the medical wards (table 1) . two (40%) of 5 patients with laboratoryconfirmed mers-cov and 6 (3%) of 191 who were mers-cov rrt-pcr negative had elisa-reactive serum samples. ifa and mn assay results were positive for 6 (75%) of 8 patients who had elisa-reactive serum samples; the 2 patients who had nonreactive ifa results also had nonreactive mn results. one of the 6 patients (no. 7) had high ifa (1:1,280) and mn (400) titers (table 2) . this patient, a 69-year-old man, was admitted to the intensive care unit with mers-cov infection resulting in acute respiratory distress syndrome, acute kidney injury, and shock. he required mechanical ventilation, renal replacement therapy, and vasopressors ( figure 2 ). the high titer occurred while he was in intensive care, 32 days after symptom onset. his serologic titers by elisa, ifa, and mn declined progressively as he recovered clinically; elisa and ifa were nonreactive by 8 months after hospital admission ( figure 2 ). of the 6 patients, 5 (nos. 1-5) had mn titers >100 (table 2), but these patients did not meet clinical criteria for plasma donation because of age, concurrent conditions, or previous pregnancy. of note, 3 patients with laboratory-confirmed mers-cov infection had a nonreactive elisa; these 3 samples were collected 3, 6, and 36 days after symptom onset. two of the patients died before the test was repeated. for the third patient, repeat elisas at 2 and 4 weeks after the first nonreactive elisa were negative. a median of 34 days (q1 34 days, q3 34 days) after 2 patients received a laboratory diagnosis of mers-cov infection, we tested 3 household contacts for 1 of the patients and 14 for the other (table 1) . serum samples for all 17 contacts were nonreactive by elisa (figure 1 ; table 2 ). elisa and mn results were highly correlated (pearson correlation coefficient 0.70, p = 0.001) (figure 3) . however, elisa and ifa results showed only a modest correlation (pearson correlation coefficient 0.55, p = 0.015), and ifa and mn results were not statistically table 1 . characteristics of participants in a study for the feasibility of collecting convalescent plasma from persons who had been infected with or exposed to mers-cov, saudi arabia, july-october 2015* characteristic value healthcare workers exposed to laboratory-confirmed mers-cov patients, n significantly correlated (pearson correlation coefficient 0.38, p = 0.12). our results indicate that it would be possible to obtain quantities of convalescent plasma large enough to use in therapeutic studies or in a large number of mers-cov patients; however, large-scale screening would be required because of the limited availability of eligible potential donors with sufficient levels of antibody. our findings suggest that recently recovered mers patients may be suitable potential donors, provided they meet other plasma donation criteria. of note, none of the seropositive persons in our study met our clinical and laboratory criteria for plasma donation. our findings show that serum antibody to mers-cov was infrequently reactive by elisa; however, reactivity may have been affected by the timing of sample collection or severity of the illness. most of the small subset of participants with elisa-reactive serum samples had mers-cov antibodies as assessed by ifa and mn. elisa results and mn titers were highly correlated; ifa and mn were not. one healthcare worker had high mn titers, but she did not meet the clinical criteria for plasma donation. another critically ill patient had high antibody titers by the 3 assays, but antibody titers declined quickly as the patient recovered clinically, and he was not eligible to donate plasma. in accordance with who and us centers for diseases control and prevention guidelines, we used elisa (figure 2 ). values shown are the highest values for the patient. to screen for mers-cov igg and ifa and mn assays to confirm positive results (14, 15) . the elisa is based on the virus s1 protein as antigen, and the ifa is based on detection of virus-specific antibodies, using cell cultures infected with the virus. the mers-cov spike protein is a glycoprotein that forms the spikes of the virus, and the n terminal component (s1) is believed responsible for the first step of virus entry into the host cell (22) . our original protocol used ifa as a confirmatory test; however, we switched to mn when that assay became available. our findings showed a high correlation between elisa and mn results but not between ifa and mn results, which may indicate that mn is a better confirmatory test. however, for 1 patient in our study, the 3 tests showed similar results (figure 2 ), and other studies have shown good correlation between the tests (20) , which may indicate that the lack of correlation shown between ifa and mn in our study was associated with sample size. our findings suggest that the low prevalence of seroreactivity for mers-cov, even among persons with confirmed or suspected infection, may be a reflection of a short-lasting antibody response. it is possible that some of the study participants were seronegative at the time of testing because the window for positive serologic results had passed. a short-lasting immune response may also partly explain why negligible or low levels of mers-cov seroreactivity have been detected in persons at risk for the disease (i.e., camel and abattoir workers) in saudi arabia and elsewhere (23) (24) (25) (26) . a seroprevalence study conducted during december 2012-december 2013 showed mers-cov antibodies in only 0.15% of the general population (n = 10,009) in all 13 provinces in saudi arabia (21) . seroprevalence was also low among camel shepherds (2.3%, n = 87) and slaughterhouse workers (3.6%, n = 140), albeit higher than in the general population (21) . the clinical relevance of antibody titers in protecting against subsequent mers-cov infection is uncertain. similar findings have been described with other coronaviruses. cao et al. (27) studied specific and neutralizing antibody titers in 56 patients who recovered from sars-cov infection. their findings showed that sars-cov igg and neutralizing antibodies peaked at 4 months and then began diminishing, reaching undetectable levels in 25.6% (igg) and 16.1% (neutralizing antibodies) of patients at 36 months. xie et al. (28) showed that sars-cov igg decreased over 1 year in recovering sars patients. in an experiment of intranasal inoculation of cov 229e in human volunteers, callow et al. (29) studied the time course of specific antibody response and found that those antibodies peaked 1 week after the inoculation and then began declining. furthermore, it appears that the antibody immune response to mers-cov in humans differs from that in camels. alagaili et al. (30) showed that 74% of 150 camels from different parts of saudi arabia have antibodies to mers-cov by elisa, and the prevalence of antibodies is higher in older camels (95%). two patients in the 2015 mers-cov outbreak in south korea were reported to have received convalescent plasma collected from recovered patients (31) . it is unclear whether the plasma was tested for the presence of mers-cov antibodies. our study demonstrates that such testing should be mandatory for donated convalescent plasma because of the low prevalence of mers-cov antibodies, even in patients with past laboratory-confirmed mers-cov infection. without such testing, the presence of antibodies to mers-cov cannot be confirmed, and the convalescent plasma may not be associated with a protective effect. our study also highlights the need for prospective serology studies to better understand the humoral response to mers-cov infection. the strengths of our study are that we screened a large number of persons, including patients with laboratoryconfirmed mers-cov infection, and used screening and confirmatory antibody assays. a study limitation was the small number of household contacts who were screened, although, based on our findings and those of others (20) , a small proportion of household contacts are likely to show seroreactivity. only one third of invited healthcare workers participated in the study. we used an s1-based elisa for screening, and our study did not address the magnitude and duration of other antibody isotypes in the immune response. the interval between illness and recovery was prolonged in most of the exposed healthcare workers (median interval >1 year). it is possible that earlier sampling would have resulted in the detection of more reactivity and higher antibody titers. our study, which was designed to screen for antibodies in convalescent plasma, was not designed to characterize the immune response to mers-cov infection or to identify clinical correlates of the presence or absence of mers-cov antibodies. further testing is needed to determine whether the antibodies in convalescent plasma are clinically effective against mers-cov infection. our findings suggest the need to explore other passive immunotherapeutic approaches, such as monoclonal or polyclonal human antibodies from transchromosomic bovines (16, 32) and, possibly, polyclonal antibodies from camels (12) . our findings also raise questions about whether naturally occurring infections and potential mers-cov vaccines will offer longlasting immunity. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus outbreak in the republic of korea treatment of mers-cov: information for clinicians. clinical decision-making support for treatment of mers-cov patients hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a(h1n1) infection sars: systematic review of treatment effects convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? use of convalescent plasma therapy in sars patients in hong kong successful treatment of avian influenza with convalescent plasma the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection feasibility, safety, clinical, and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol centers for disease control and prevention. cdc laboratory testing for middle east respiratory syndrome coronavirus world health organization. laboratory testing for middle east respiratory syndrome coronavirus: interim recommendations (revised) human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo world health organization. guidelines on assessing donor suitability for blood donation position paper on collection and use of convalescent plasma or serum as an element in middle east respiratory syndrome coronavirus response detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction transmission of mers-coronavirus in household contacts presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies mers coronaviruses in dromedary camels lack of mers coronavirus neutralizing antibodies in humans investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012. influenza other respir viruses disappearance of antibodies to sars-associated coronavirus after recovery dynamic changes of serum sars-coronavirus igg, pulmonary function and radiography in patients recovering from sars after hospital discharge the time course of the immune response to experimental coronavirus infection of man middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia south korea tries old weapon to fight mers pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection key: cord-349781-l93978vq authors: cong, yu; hart, brit j.; gross, robin; zhou, huanying; frieman, matthew; bollinger, laura; wada, jiro; hensley, lisa e.; jahrling, peter b.; dyall, julie; holbrook, michael r. title: mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells date: 2018-03-22 journal: plos one doi: 10.1371/journal.pone.0194868 sha: doc_id: 349781 cord_uid: l93978vq middle east respiratory syndrome coronavirus (mers-cov) presents an emerging threat to public health worldwide by causing severe respiratory disease in humans with high virulence and case fatality rate (about 35%) since 2012. little is known about the pathogenesis and innate antiviral response in primary human monocyte-derived macrophages (mdms) and dendritic cells (mddcs) upon mers-cov infection. in this study, we assessed mers-cov replication as well as induction of inflammatory cytokines and chemokines in mdms and immature and mature mddcs. immature mddcs and mdms were permissive for mers-cov infection, while mature mddcs were not, with stimulation of proinflammatory cytokine and chemokine upregulation in mdms, but not in mddcs. to further evaluate the antiviral activity of well-defined drugs in primary antigen presenting cells (apcs), three compounds (chloroquine, chlorpromazine and toremifine), each with broad-spectrum antiviral activity in immortalized cell lines, were evaluated in mdms and mddcs to determine their antiviral effect on mers-cov infection. while chloroquine was not active in these primary cells, chlorpromazine showed strong anti-mers-cov activity, but it was associated with high cytotoxicity narrowing the potential window for drug utilization. unlike in established cells, toremifene had marginal activity when tested in antigen presenting cells, with high apparent cytotoxicity, also limiting its potential as a therapeutic option. these results demonstrate the value of testing drugs in primary cells, in addition to established cell lines, before initiating preclinical or clinical studies for mers treatment and the importance of carefully assessing cytotoxicity in drug screen assays. furthermore, these studies also highlight the role of apcs in stimulating a robust protective immune response to mers-cov infection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 middle east respiratory syndrome coronavirus (mers-cov) was first isolated in saudi arabia in 2012 from a patient with severe acute respiratory disease complicated by renal failure [1, 2] . since that time, the virus has caused sporadic outbreaks of mild-to-severe respiratory disease. approximately 80% of human cases have been reported in saudi arabia with 211 cases occurring in the first 9 months of 2017 [3] . beginning in may 2015, a large hospital-associated outbreak of mers occurred in the republic of korea. the outbreak in korea resulted in a total of 186 mers-cov cases, including 36 deaths, and was the largest outbreak of mers occurring outside of the arabian peninsula [4] . this outbreak highlighted the risk of international dissemination of mers-cov and the continued risk of nosocomial infection. as of september 6, 2017, the number of confirmed global cases of mers-cov infection reported to world health organization was 2079 cases in 27 countries with 722 fatalities, resulting in a case fatality rate around 35% [3] . mers-cov is a zoonotic virus that is transmitted from animals to humans with camels likely serving as the principal host for mers-cov [5] . while nosocomial infections are common, barrier nursing practices can limit spread of the virus as the virus does not seem to pass easily from person-to-person unless close contact occurs [6] . in humans, mers-cov infection typically causes a lower respiratory tract disease such as pneumonia, and common symptoms include fever, cough, sore throat, myalgia, and shortness of breath [7] . symptoms such as gastrointestinal complications and renal failure have also been reported in patients, especially those with severe chronic illness such as diabetes [6, 8] . systemic dissemination has been documented in locations such as the circulatory system and respiratory tract [9] . in the studies presented here, we had two principal objectives. the first was to determine whether human antigen presenting cells (apcs) were permissive to mers-cov infection. the second objective was to determine if these cells were suitable or appropriate for secondary screens for drugs that have been identified as effective in continuous culture cell lines. macrophages and dendritic cells (dcs) are professional apcs linking innate and adaptive immunity. these and other apcs act as a first defense against viral infection by stimulating immune surveillance, priming, and tolerance [10, 11] . appropriately functioning apcs are critical for the ability to mitigate infection and limit the development of disease. apcs are abundant in the respiratory tract where they provide immune surveillance and respond to local tissue inflammation in the airways and the distal lung. an important role of apcs is mitigating infection by producing cytokines that stimulate an inflammatory response and recruiting memory and effector cells to the site of infection [12] . professional apcs are also an important source of type i interferons (ifn-α/β). type i ifns have a significant bystander effect on uninfected neighboring cells by inducing an antiviral state, activating innate immune cells, and priming adaptive immunity. currently, no prophylactic or therapeutic options are proven as effective interventions for infection with mers-cov, severe acute respiratory syndrome coronavirus (sars-cov), or any other coronaviruses. to rapidly identify potential therapeutic options against emerging viral infections, investigators have adopted the approach of screening existing licensed drugs for efficacy against novel viral pathogens. screening licensed drugs could expedite the implementation of new medical countermeasures by providing an avenue for off-label use of compounds shown to be useful for the treatment of specific viral diseases. a number of pharmaceutical agents have potential for the treatment of coronaviruses, including neurotransmitter inhibitors, estrogen receptor antagonists, kinase signaling inhibitors, protein-processing inhibitors, and antiparasitic agents [13, 14] . results from previous studies found toremifene citrate (tomf), chlorpromazine (cpz) and chloroquine (cq) to be [13, 14] . we established a cell differentiation method to generate large amounts of monocyte-derived macrophages (mdms) and dendritic cells (mddcs) of high quality (>95% purity) for evaluating compounds against other viruses such as yellow fever and ebola viruses [15] . to address pharmaceutical efficacy against mers-cov infection in primary cells critical for blocking infection, we tested several candidate mers-antivirals in human mdms and immature dendritic cells. the tested compounds included tomf, an estrogen receptor antagonist, cpz, a neurotransmitter inhibitor, and cq, an antimalarial agent. all viral infection-related work was performed in the biocontainment laboratory at the integrated research facility (irf) of national institute of allergy and infectious disease (niaid)/ national institute of health (nih) using procedures and processes appropriate for the facility and the agent under study. all staff performing this work were trained to work in the biocontainment space and with mers-cov. human mdms and mddcs were differentiated at the irf as described previously [15] . briefly, cd14 + monocytes were isolated from human peripheral blood mononuclear cells for differentiation into mdms and mddcs. mdms were differentiated by culturing the purified cd14+ monocytes with human macrophage-colony stimulating factor (m-csf) (r&d systems, minneapolis, mn) and kpb-m15 conditioned medium (scgf research laboratory, ukyo-ku, jp) for 7 days. to generate mddcs, cd14+ monocytes were supplied with 20 ng/ml of human granulocyte macrophage-colony stimulating factor (gm-csf) and 10 ng/ml of interleukin (il)-4 in r-10 medium [rpmi-1640 (lonza, allendale, nj)] containing 10% heat inactivated fbs (millipore sigma). the cells were then incubated for 6 days at 37˚c and 5% co 2 with a change of medium every second day. immature mddcs were harvested at this point or stimulated to induce maturation. to induce maturation, the medium was replaced with a mddc maturation cocktail (r-10 medium containing 10 ng/ml of tumor necrosis factor (tnf)-α, 10 ng/ml of il-1β, 15 ng/ml of il-6, 1 μg/ml of prostaglandin e2 (peg2), 20 ng/ ml of gm-csf, 10 ng/ml of il-4) on day 6 post-plating and incubated overnight at 37˚c and 5% co 2 . the cells were then collected for phenotyping and further assays. mdms and mddcs were stained and analyzed as mentioned previously [15] . mdms and mddcs (immature and mature) were seeded at 5 × 10 5 cells per well in 48-well plates and incubated overnight at 37˚c and 5% co 2 . the next day, the cells were infected at a moi of 2 for 1 (hour) h at 37˚c and 5% co 2 . after the inoculum was removed, cells were washed twice with phosphate-buffered saline and cultured with r-10 medium. cell-free supernatants were collected, aliquoted to multiple microtubes at the indicated time-points, and frozen at -80˚c until used. the titer of virus stocks and cell-free supernatants collected from kinetics studies were determined by plaque assay on vero e6 cells. test samples were diluted serially 1:10, and 100 μl of the sample was added to each well of 6-well plates containing an 80-90% confluent monolayer of vero e6 cells. the plates were then incubated for 1 h at 37˚c and 5% co 2 with rocking every 15 mins for virus adsorption and infection. following incubation, a pre-warmed semisolid overlay (0.8% tragacanth [f/c] (millipore sigma) mixed in 1x eagle's minimum essential medium containing 2% fbs) was gently added to the cells. the plates were then incubated for 5 days at 37˚c and 5% co 2 without being disturbed. to fix the plates, the overlay was aspirated, and the cells were fixed and stained with neutral buffered formalin (nbf) (thermo fisher scientific) containing 0.2% crystal violet (f/c) (millipore sigma) for 30 min at room temperature. the plates were then washed with running water and air-dried. the plaques were then enumerated, and the virus titer was calculated. inflammatory mediator expression was measured using a custom human 10-plex cytokine bead arrays (cat# hcytomag-60k, millipore sigma, billerica, ma) on a luminex flex-map 3d tm system equipped with xponent 4.2 software. (luminex, austin, tx). cytokines and chemokines included in the panel were: ifn-α2, ifn-γ, tnf-α, il-6, il-8, il-12p40, monocyte chemotactic protein (mcp)-1, macrophage inflammatory protein (mip)-1α, interferon-inducible protein (ip)-10, and regulated upon activation normal t-cell expressed and secreted (rantes). the assays were performed according to the manufacturers' instructions in 96-well mylar plates (millipore) as described previously [15] . cell-free supernatants harvested from kinetics studies were mixed with beads coated with capture antibodies to various cytokines and chemokines. the mixtures were incubated with biotinylated detection antibodies. finally, pe-conjugated streptavidin was added, and the fluorescent signals were measured using a flexmap equipped with xponent 4.2 software (luminex corp). the raw data were measured as mean fluorescence intensity (mfi), and the concentration of each analyte was calculated using a 4-or 5-parameter logistic fit curve generated for each analyte from the 6-point concentration standard curve. the concentrations of all the analytes in the quality control reagents were found to be within the expected ranges. the lower limit of quantification (lloq) was determined using the lowest point on the standard fit curve that was at least 3 times above background. the calculation of the lloq was performed by subtracting the mfi of the background (diluent) from the mfi of the lowest standard concentration and back-calculating the concentration from the standard curve. data were further analyzed using prism 6.0 software (graphpad software, la jolla, ca). mdms or mddcs (100 μl) were plated in black opaque (cytotoxicity assay) or clear bottom (testing viral inhibition) 96-well greiner microplates (greiner bio-one, monroe, nc, 655948) at 1 × 10 5 cells/well and incubated at 37˚c and 5% co 2 overnight. drugs in powder formulations were dissolved in (dmso) (millipore sigma). final dmso concentrations were lower than 0.05%. drug solutions diluted in rpmi-1640 with 10% fbs (r-10 medium) to a 4x concentration were added into a 96-well, cell-free, drug dilution plate. drugs (50 μl/well) were transferred from the drug dilution plates to cell plates using the 96-well liquidator (rainin instrument, oakland, ca) 1 h prior to infection. duplicate cell plates for each cell type were infected with mers-cov at a moi of 0.1 by adding 50 μl of virus diluted in r-10 medium directly to the drug mixture in the cell plate and the plates incubated at 37˚c and 5% co 2 . after 48 h, cell culture supernatants were collected, and the cells were fixed with 10% nbf. the collected supernatants were clarified by centrifugation and stored at -80˚c for future use. to quantify the antiviral activity of the candidate compounds, a cell based immunofluorescence enzyme-linked immunosorbent assay was used as described previously [13] . the fixed plates, as described above, were stained with a rabbit polyclonal antibody to the mers-cov--emc/2012 s protein (sino biological) at 1:1000 followed by staining with alexa fluor 594-conjugated goat anti-rabbit igg (h+l) antibody (thermo fisher scientific) at 1:2500. fluorescence was quantified on a plate reader (infinite m1000 pro; tecan us, morrisville, nc) with an excitation wavelength of 590 nm and an emission wavelength of 617 nm. cytotoxicity assays were performed in parallel to measure drug toxicity. one black opaque cell plate for each cell type was mock infected using r-10 medium under the same conditions cd14 + monocytes were isolated from human peripheral blood mononuclear cells (pbmcs). cells were then differentiated to monocyte-derived macrophages (mdms) by adding macrophage-colony stimulating factor (m-csf) or to immature monocyte-derived dendritic cells (mddcs) by adding granulocyte macrophage-colony stimulating factor (gm-csf) and interleukin-4 (il-4). immature mddcs were exposed to mddc maturation cocktail (r-10 medium containing 10 ng/ml of tumor necrosis factor (tnf)-α, 10 ng/ml of il-1β, 15 ng/ml of il-6, 1 μg/ml of prostaglandin e2 (peg2), 20 ng/ml of gm-csf, 10 ng/ml of il-4) overnight for maturation. mdms and mddcs were characterized by flow cytometry for expression of major cell-surface markers. using these protocols, the mdms are predominantly cd14+, cd11b+, hla-dr+, cd169+, cd163+, and cd86+. the mddcs are predominantly cd14-, cd11c+, hla-dr+, cd80+, and cd86+. mature mddcs are cd83+, while immature mddcs are cd83-. https://doi.org/10.1371/journal.pone.0194868.g001 as the infected cells, and cell viability was measured using the celltiter glo luminescent cell viability assay kit (promega, madison, wi) according to the manufacturer's instructions. luminescence was read on the infinite1 m1000 pro plate reader (tecan). antiviral activity was evaluated by means of a 50% tissue culture infectious dose (tcid 50 ) assay using the spearman/kaerber method [16, 17] . briefly, a 10-fold serial dilution, 10 −1 to 10 −7 , was made from cell-free supernatant samples collected from the drug efficacy evaluation studies for cell-based enzyme-linked immunosorbent assay. these dilutions were used to infect vero e6 cells on 96-well plates with seven infected wells for each dilution. samples containing only cell culture media were used as a negative control, and virus stock was used as a positive control. test samples were added to each well at a volume of 100 μl. the plates were then incubated for 6 days at 37˚c and 5% co 2 to observe the mers-cov-induced cytopathology. following the incubation, the media were removed, and the cells were fixed and stained with 10% nbf containing 0.2% crystal violet. the plates were incubated for 30 min at room temperature and then washed with running water. the tcid 50 titer was determined by identifying the 50% endpoint in cytopathic effects in the cell monolayer. the results were calculated by applying the spearman/kaerber method and presented as log 10 tcid 50 /ml. non-linear regression analysis was performed to calculate 50% effective concentration (ec 50 ) of compounds and their specific toxicity (50% cytotoxicity concentration, cc 50 s) using prism 6.0 software (graphpad software) from the dose-response curves of the drugs. selectivity index (si) is defined as the ratio of the concentration of 50% cellular cytotoxicity to 50% effective concentration (si = = cc 50 äec 50 ), indicating the relative efficacy of a compound in viral replication inhibition as opposed to causing cytopathic effect. error bars represent the standard deviation of three replicates unless indicated otherwise. statistical significance determinations were performed using the results of at least two individual experiments or duplicate or triplicate replicates from a single experiment. for these studies, mdms and mddcs were carefully characterized to ensure uniformity and reproducibility between experiments. differentiated mdms and mddcs were characterized by flow cytometry as performed previously [15] . the mdms generated were predominantly cd14 + , cd11b + , hla-dr + , cd169 + , cd163 + , and cd86 + , confirming that the mdm population was highly purified, and over 95% of the cells generated were positive for macrophages (fig 1) . phenotyping of human mddcs showed they were cd14 -, cd11c + , hla-dr + , cd40 + , cd80 + , and cd86 + . the dc maturation marker, cd83, was present on 95.1% of mature mddcs (fig 1) compared to 8.13% of the immature mddcs and 0.98% of the unstained control cells (fig 1) . the isolation protocol used in these studies consistently generated highly purified phenotypically homogeneous apcs with minimal inter-experimental or inter-operator variability. to determine if primary apcs are permissive to mers-cov infection, kinetics studies were performed to measure virus titer following infection using cells isolated from four different donors. a 2-to 4-log 10 increase in viral titers was observed in mdms and immature mddcs despite variation between donors (fig 2a and 2b) . no significant increase in viral titer was observed in mature mddcs up to 8 days pi (fig 2c) indicating a restriction in virus infection or virus replication. virus clearance in mdms began at day 3 pi, and no virus could be detected by 6 to 8 days pi. however, mers-cov continued to propagate in immature mddcs up to 8 days pi, demonstrating differential infection and replication capabilities in mdms and immature mddcs. to compare the ability of mers-cov to induce innate immune responses in three types of apcs, the release of cytokines and chemokines was measured from virus-or mock-infected cells. the 10-plex human panel included the antiviral cytokines (ifn-α2 and ifn-γ), proinflammatory cytokines (tnf-α, il-6, and il-12p40), and chemokines (ip-10, mcp-1, mip-1α, rantes and il-8). in mdms, mers-cov infection resulted in increased concentrations of almost all antiviral or proinflammatory cytokines and most chemokines (except mcp-1), many of these cytokines or chemokines were released as soon as 1 h pi ( fig 3a) and may have been the result of direct cell stimulation rather than active infection. the high induction of ifn-γ, and ip-10 over the course of the infection in mdms was dramatic, indicating a very strong antiviral response was induced upon mers-cov infection. mers-cov infection repressed il-6 expression from mdm and this repression decreased over the course of several days ( fig 3a) with a similar response profile seen with chemokines mip-1α and mcp-1. increased mip-1α and ip-10 expression was observed in immature mddcs (fig 3b) . however, other than a bolus of ifn-γ released immediately after the infection of mddc, the release of most of the cytokines or chemokines from immature and mature mddcs were negligible over the course of the mers-cov infection relative to mock-infected cells (fig 3b and 3c) . as expected in a human population, considerable donor-to-donor variation in the cytokine response was observed following infection with mers-cov. a cell-based immunofluorescence assay was applied to evaluate the antiviral effectiveness of cq, cpz, and tomf in mdms, which adhere to 96-well plates and could be fixed and stained. cytotoxicity assays were performed in parallel using mock-infected cells after drug addition. tomf performs well inhibiting mers-cov replication in vero e6 cells (ec 50 = 12. 9 μm) [13] . in mdms, the activity of tomf at the dose range tested was too low to determine an ec 50 and the cytotoxicity was relative high (fig 4a) . unlike in vero e6 cells (ec 50 = 6.3 μm) [13] , cq did not show any detectable antiviral activity in mdms, although no cytotoxicity was associated with this compound (fig 4b) . cpz inhibited mers-cov in vero e6 cells (ec 50 = 9.5 μm) with low cytotoxicity [13] . however, in mdms, the cytotoxicity of cpz was relatively high (fig 4c, cc 50 = 25.64 μm. antiviral activity of cpz (ec 50 = 13.58 μm) did not separate well from cytotoxicity and led to a low selectivity index (si = 1.9). the cell-based immunofluorescence assay could not be applied to immature mddcs due to their poor adherence to the 96-well plate; cells could not be fixed for further antibody staining. therefore, the efficacy of the candidate compounds on mers-cov-infected apcs was evaluated by a median tcid 50 assay. mature mddcs did not produce detectable infectious virus after infection and were therefore excluded (fig 2c) in these drug activity studies. cell culture supernatants saved from the cell-based assay were titrated by tcid 50 to detect mers--cov to evaluate the efficacy of the compounds. there was no virus yield reduction in cqtreated mdms or immature mddcs, though the cytotoxicity was relativity low (fig 5a and 5b ). in cpz treated cells, around a 2 log 10 virus reduction was observed in both cell types. however, high cytotoxicity narrows the therapeutic window in both mdms and mddcs ( fig 5a and 5b) . a 1-1.5 log 10 virus reduction was measured in tomf-treated cells, but only when the dose is higher than 20 μm, with a corresponding increase in cytotoxicity (fig 5a and 5b) . these observations were similar when compared to the cell-based assay mentioned above. virus yield reduction data for tomf, cq and cpz in vero e6 cells showed that cpz was active against mers-cov infection with a 3.1 log 10 drop at the drug dosage of 15 μm, but tomf and cq were not active (fig 5c) . these data suggest a strong correlation between veroe6 cells and mdms when virus titers are determined by tcid 50 . as sporadic outbreaks of mers continue to occur, our current options for treatment of lifethreatening zoonotic coronavirus infections in humans are still lacking. the lack of treatment is due to the poorly understood pathogenesis of mers and other coronavirus infections, despite the extensive research efforts triggered by the 2003 sars outbreak [18] . human apc cells, mdms and mddcs, play important roles in bridging innate and adaptive immunity during viral infections by presenting viral antigens to t cells and by secreting appropriate cytokines and chemokines [19] . mdms and immature mddcs reside at the sites where most infections occur, such as the mucosal surfaces within the respiratory tract. in their role in the immune response, apcs are also a potential initial site for viral replication. additionally, apcs can act as viral reservoirs for further dissemination to organs as already shown for other human infectious diseases like measles and human immunodeficiency virus (hiv) infection [20, 21] . in this study, we assessed the characteristics of mers-cov growth kinetics in mdms and mddcs. we found that mers-cov can readily infect and robustly replicate in human mdms and immature mddcs, while the virus does not establish a notable infection in mature mddcs. high viral infectivity in mdms and immature mddcs indicates that the virus can overcome the initial antiviral response, but that the maturation of mddc imparts a restriction that prevents virus replication. the capacity of mddc to stimulate t cells is closely related to their maturation stage [22] . immature dcs are highly specialized at antigen uptake and processing, but are poor stimulators of primary immune responses [23] [24] [25] . productive infection of immature dcs may delay the activation of t cells, therefore allowing more time for mers-cov replication or dissemination. an additional reason that immature mddcs may be more susceptible to mers-cov infection than mature mddcs to mers-cov could be their ability to internalize and process viral antigens. after uptake of antigens or pathogens, immature mddcs may undergo maturation, migrate to lymphatic tissues, and trigger adaptive immune responses. in theory, the ability of sampling and presenting antigen should be enhanced through mddcs maturation. for example, immature mddcs may promote viral dissemination of sars-cov within the host by recruiting the virus to enter and damage the lymphatic tissues [26]. a recent study has shown that abortive herpes herpesvirus (hhv)-1 (herpes simplex virus-1) infection of mature mddc is able to modulate their inhibition of t-cell stimulatory function [27] . endocytosis in mature dcs is slowed somewhat relative to immature dcs, potentially the result of changes to their endocytic mechanism or receptor expression [28] . similarly, virus was disseminated systemically in a patient with mers [29]. following mers-cov infection, the induced immune response in apcs was consistent with their susceptibility to infection and the kinetics of virus propagation. the production of proinflammatory cytokines is essential for host resistance against mers-cov infection. infection of mdms with mers-cov appreciably induced the secretion of antiviral cytokines (ifn-α2, ifn-γ, and il-12p40), moderately or appreciably up-regulated proinflammatory cytokines (tnf-α, il-6), and variably upregulated inflammatory chemokines (mip-1α, ip-10, rantes, il-8). clinical studies have shown that expression of proinflammatory cytokines/chemokines, such as il-6, il-8, ifn-γ, or ip-10, is highly correlated with disease severity and mortality of sars patients [30] . nicholls et al reported that macrophages in lung tissues of sars patients were identified as the location where the "cytokine storm" and virus pathogenesis originated [31] . mdms secrete proinflammatory cytokines and chemokines to activate antiviral mechanisms, which may cause the dysregulated production of these inflammatory mediators leading to damage to neighboring tissues [32] . expression of il-6 and tnf-α are a potential indicators of severe respiratory viral infection, such as sars and human infection by avian influenza viruses [33] . our data indicate the possibility of an ifnγ th1-mediated inflammatory response that may cause severe mers disease before an adaptive host immune response is mounted. however, restriction of virus growth in mature mddcs results in no antiviral activation by ifn-α2. nevertheless, only moderate expression of ip-10 and minimal induction of mip-1α, mcp-1, and rantes were observed in immature mddcs. significant upregulation of most cytokines and chemokines was not observed in either mature or immature mddcs. overall, the limited antiviral cytokine response in mddcs suggests a possible mechanism of immune escape during mers-cov infection as seen in sars-cov infection [26] . a review of the literature suggests that there is a significant gap in knowledge regarding the role on intracellular regulatory mechanisms such as pathogen recognition receptors (prr) in the control of coronavirus infection. in this study there was essentially no change in the concentration of cytokines/chemokines measured in mature (and non-permissive) mddcs. in immature mddcs, while the measured concentration was less than the control cells in many cases, the concentrations of a number of cytokines/chemokines (il-6, ip-10, mip-1α, mcp-1) increased over the course of the infection, specifically on days 6 and 8 when titers were highest. these data suggest that prr may have been activated, as would be anticipated in a productive viral infection, but the scale and scope was not evaluated. in addition, data obtained for cytokine release very early after infection cannot be properly evaluated due to the potential presence of cellular components in the virus preparations. in several reports of immune responses triggered by apc, findings are similar, or conflicting compared to our results. [19, 32] . these discrepancies may relate to differences in experimental conditions, the protocols for generating and phenotyping mdms and mddcs in different laboratories, or variability of individual blood donors [36] . appreciable efforts have been made to identify novel antiviral therapeutics for mers-cov since the initial outbreak occurred. compounds that target host effectors could be beneficial during the course of infection [37] . mers-cov drug screens have been performed mostly in established cell lines such as vero and huh 7 cells. very little drug evaluation has been performed in primary cell lines. here we picked three fda-approved broad-spectrum inhibitors (cpz, cq, tomf) that were shown to be effective against mers-cov infection in immortalized cell lines [13, 14] and evaluated their antiviral activities in mdms and mddcs. we excluded mature mddcs for drug screening due to the lack of productive mers-cov infection. surprisingly, our findings for tomf activity in mers-cov-infected mdms and immature mddcs differed from results of previous studies in vero e6 cells. tomf showed minimal activity against mers-cov infection in mdms. tomf also had little antiviral activity in immature mddcs until reaching a high concentration (>20 μm) that was associated with high cytotoxicity. tomf is an estrogen receptor modulator that has been shown to inhibit filoviruses [38] and block both mers-cov and sars-cov with ec 50 s12.9 and 11.97μm, respectively, in vero e6 cells [13] . this drug inhibits late phrase of virus entry by interfering the fusion of filoviruses [38] . however, in primary cells, the ec 50 increased to 38 μm though lower cytotoxicity was seen (cc 5 0 = 22.54 μm). thus, the therapeutic window for effective treatment with tomf in primary apcs is narrow. previously, cq, a well-established antiparasitic agent, showed strong inhibition on mers-cov and sars-cov with low ec 50 s (range from 5.76 to 12.9 μm) and low toxicity [13] . cq likely accumulates in lysosomes where it sequesters protons and increases the ph. the drug interacts with a variety of host proteins and cellular processes, resulting in modulation of immune responses [39] . cq has also been reported to inhibit replication of multiple viruses such as flaviviruses [40] , influenza viruses [41, 42] , hiv [40] , ebola [43] and nipah viruses [44] in vitro. in our study, cq had minimal anti-mers activity in either mers-cov-infected mdms or immature mddcs, though appreciable cytotoxicity was not observed. cpz is a neurotransmitter inhibitor, which was the first antipsychotic drug developed for treatment of schizophrenia treatment [45] . cpz inhibits clathrin-mediated endocytosis through preventing the formulation of clathrin-coated pits at the plasma membrane [46] . previous work has reported that cpz inhibits mers-cov infection of huh 7 cells with an ec 50 of 4.9, vs cc 50 of 21.3 μm [14] . in our primary cells, some activity against mers-cov was observed in cpz-pretreated mdms and immature mddcs (ec 50 = 13.58 μm). however, the high cytotoxicity (25.64 μm) observed with cpz on both types of primary cells precludes further development. these studies demonstrate the tested compounds, each of which were shown to be efficacious in continuous cell lines, were not effective at inhibiting mers-cov infection in primary apcs. in conclusion, mers-cov infection in primary mdms induces overexpression of inflammatory cytokines/chemokines potentially leading to tissue damage and systemic virus dissemination. on the other hand, the mechanism of immune escape through mddc results in inefficient viral clearance, which may explain the high pathogenicity and clinical manifestations seen in mers. this study also provides the first demonstration of medical countermeasure evaluation for mers-cov in primary apcs and illustrates the importance of using nonimmortalized primary cells for the evaluation of drug efficacy. these data also suggest that, given the established mechanisms of action of tomf, cq and cpz, the mechanisms of virus internalization and exocytosis may be different from those used in established cell lines. before performing in vivo studies, potential compounds should be evaluated in primary cells as major differences may occur between immortalized cell lines and primary cells. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia. disease outbreak news. geneva, ch: world health organization computer-generated vs. physiciandocumented history of present illness (hpi): results of a blinded comparison middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) severe acute respiratory syndrome vs. the middle east respiratory syndrome. current opinion in pulmonary medicine clinical spectrum of the middle east respiratory syndrome coronavirus (mers-cov) journal of clinical virology: the official publication of the pan american society for clinical virology gammadelta t cells present antigen to cd4+ alphabeta t cells infection of human alveolar macrophages by human coronavirus strain 229e. the journal of general virology the development and function of lung-resident macrophages and dendritic cells repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection. antimicrobial agents and chemotherapy screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture. antimicrobial agents and chemotherapy characterization of yellow fever virus infection of human and non-human primate antigen presenting cells and their interaction with cd4+ t cells monte carlo simulation of the spearman-kaerber tcid50 a decade after sars: strategies for controlling emerging coronaviruses productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response. virology measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and t cells effects of chloroquine on viral infections: an old drug against today's diseases? chloroquine could be used for the treatment of filoviral infections and other viral infections that emerge or emerged from viruses requiring an acidic ph for infectivity antiviral therapies against ebola and other emerging viral diseases using existing medicines that block virus entry a systematic screen of fda-approved drugs for inhibitors of biological threat agents simulating henipavirus multicycle replication in a screening assay leads to identification of a promising candidate for therapy pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents infectious entry of west nile virus occurs through a clathrin-mediated endocytic pathway we thank irf cell culture staff in preparing the established cells used in this study. we thank irf immunology staff in characterizing the primary cells used in this study. we thank dr. key: cord-356219-wl9htpp2 authors: farag, elmoubasher a. b. a.; reusken, chantal b. e. m.; haagmans, bart l.; mohran, khaled a.; raj, v. stalin; pas, suzan d.; voermans, jolanda; smits, saskia l.; godeke, gert-jan; al-hajri, mohd. m.; alhajri, farhoud h.; al-romaihi, hamad e.; ghobashy, hazem; el-maghraby, mamdouh m.; el-sayed, ahmed m.; al thani, mohamed h. j.; al-marri, salih; koopmans, marion p. g. title: high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases, qatar 2014 date: 2015-07-15 journal: infect ecol epidemiol doi: 10.3402/iee.v5.28305 sha: doc_id: 356219 cord_uid: wl9htpp2 two of the earliest middle east respiratory syndrome (mers) cases were men who had visited the doha central animal market and adjoining slaughterhouse in qatar. we show that a high proportion of camels presenting for slaughter in qatar show evidence for nasal mers-cov shedding (62/105). sequence analysis showed the circulation of at least five different virus strains at these premises, suggesting that this location is a driver of mers-cov circulation and a high-risk area for human exposure. no correlation between rna loads and levels of neutralizing antibodies was observed, suggesting limited immune protection and potential for reinfection despite previous exposure. d romedary camels are likely the primary source of middle east respiratory syndrome virus (mers-cov) infection in humans, but further evidence is needed to support their role in zoonotic transmission. two of the earliest diagnosed cases in qatar were men who had visited the doha central animal market and the adjoining central slaughterhouse (farag, pers. comm.). therefore, pre-and postmortem sampling was conducted on dromedary camels (n0105) at the central slaughterhouse in doha, qatar. nasal, oral, and rectal swabs collected prior to slaughter were tested for the presence of mers-cov rna. most of the camels that were sampled showed evidence for mers-cov shedding at the time of slaughter (59%). sequence analysis showed the circulation of at least five different virus strains at the slaughterhouse premises. an understanding of the extent and pattern of mers-cov shedding by dromedaries presenting for slaughter provides insight into the risks for mers-cov exposure of persons with occupational contact with live camels and their carcasses. illness associated with infection with mers-cov is characterized primarily by mild-to-severe respiratory $ these authors contributed equally to the manuscript. complaints, most requiring hospital admission for pneumonitis or acute respiratory distress syndrome. as of june 11, 2015, ecdc has reported 1,288 laboratoryconfirmed cases, including 498 deaths (1) . human-to-human transmission seems limited to family and health care settings. overall, a large proportion of mers cases is suspected to be a result of zoonotic transmission (1) with growing evidence for dromedary camels (camelus dromedarius) as a reservoir. mers-cov-specific antibodies have been detected in camels across the middle east and the african continent, suggesting a geographically widespread distribution (2) . analysis of an outbreak associated with a barn in qatar found dromedaries and humans to be infected with nearly identical strains of mers-cov (3) and further support for camels as reservoir came from a study in saudi arabia (ksa) that found widespread circulation of different genetic variants of mers-cov in camels, with geographic clustering of human and camel mers-cov sequences (4) . however, few other studies provided evidence for zoonotic transmission of mers-cov from camels (5) . the routes of direct or indirect zoonotic transmission are yet unknown. we investigated the rate of mers-cov circulation in dromedaries at the slaughterhouse in qatar, previously linked to two mers cases in qatar. a random group of 105 camels that presented for slaughter in february (n053) and march (n052) 2014 were sampled for mers-cov analysis (table 1) . animals either had come directly from within qatar or ksa, or had been sold through the central animal market (cm). swabs and lymph nodes were tested for mers-cov rna by internally controlled rt-pcr targeting upe and n genes, as described (3, 6) . the first camel isolate of mers-cov as described by raj et al. (7) was obtained from the first group of 53 samples and among others sequences generated from this group have been used to define a general mers-cov typing fragment (8) . in total, 59% of the camels showed evidence for virus shedding in at least one type of swab at the time of slaughter ( table 1 ). the percentage positive samples was the highest for nasal samples, followed by oral swabs, fecal swabs, and bron-chial swabs. all but one animals with virus shedding from any sample had a positive nasal swab. for saliva (oral), the percentage of positive samples was the highest for animals between 7 and 12 months of age. lymph nodes from 53 animals were tested, yielding five positives. approximation of the viral loads in the samples using the ct values obtained with the upe target showed no significant differences between types of samples and age groups (fig. 1 ) it should be noted that viral loads with dct !20 were observed only in the nasal swabs and the nasal swab sample with the highest viral load was found to contain infectious virus (7). to obtain further insight in the diversity of the viruses that circulated in dromedary camels at the slaughterhouse, mers-cov strains were sequenced according to a recently developed technique that enables the identification of divergent mers-cov types [sequences and technique in (8)]. in total, five different sequence types were identified with three different types found at both sampling moments (table 2) . camels either came from the large al-shahaniya international racing complex (ash) or from different sources elsewhere in qatar (indicated by the initial arrow for animals 6á8 and 10á12 in (table 2) . subsequently, they were either brought to a showing area (al mazad, am), to the barns at the cm for a holding period, or immediately sent to the slaughterhouse (sh). therefore, the sampling for animals 1á5 and 9á13 reflects mers-cov sequence diversity as a result of import from other regions in qatar, whereas virus circulation at the cm more likely explains the virus diversity for animals 6á8. antibodies to mers-cov s1 were found in 100 out of 103 animals tested by micro-array technology (9) . for 53 animals, antibody levels were also determined by virus neutralization assay as described earlier (9) . almost all animals had detectable neutralizing antibodies with no obvious age pattern and no significant difference in proportion of animals with low antibody levels ( b20) (fig. 2a) . there was no correlation between antibody levels and the viral load as reflected by ct values (fig. 2b) . (10) a study at four slaughterhouses in egypt showed an overall rna prevalence in nasal swabs of 3.6% among 110 camels (11) , which is significantly lower than in our study. a comparison of the organization of the meat markets between egypt and qatar could provide insight in the observed differences. the camels that are put together for a holding period of weeks prior to slaughter in doha have a wide variety of origins with varying initial immune status, which might provide a platform for extensive virus circulation. these include naïve camels from australia (12) and camels from areas in the horn of africa and the gulf region with known differences in immune status (2, 13, 14) . we observed a positivity rate in rectal swabs of 15 out of 103 animals that were analyzed (of which 61 were positive in nasal swabs). other studies observed none to very low numbers of camels shedding mers-cov rna in feces (3, 15) . however, the total numbers of animals in these studies were too low to make a significant comparison with the data presented here. in the current views on mers-cov epidemiology, young camels ( 51year) with primary infections are thought to play a bigger role in mers-cov transmission than older animals for which less frequent shedding is observed (4, 15) and who demonstrate higher rates of seroconversion reviewed in (ref. 2) . however, we observed no significant differences in mers-cov rna shedding between different age groups. moreover, the lack of correlation between viral rna loads and levels of neutralizing antibodies in the animals suggests limited protection and potential for reinfection despite previous exposure, similar to the situation in humans with the four common human covs and as observed in a camel herd in ksa (15) . a problem is that the time since onset of infection could not be determined as the animals did not show overt symptoms. therefore, it remains to be determined how the kinetics of infection are. in theory, the observed shedding of virus in the presence of neutralizing antibodies could represent sampling toward the end of an infection cycle. alternatively, the data may reflect limited mucosal immunity as has been shown for other animal coronaviruses (16) . the possibility of camel vaccination has been suggested as a possible approach to controlling mers-cov transmission to humans. however, this may prove to be a challenging task in light of the above observations. given the high numbers of animals shedding these viruses in dynamic environments like the doha market and abattoir, potential human health risks need to be considered and the implementation of management alternatives (e.g. separation of naïve animals from previously exposed animals and personal protective equipment for employees) might reduce the burden of mers-cov exposure to humans. severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) á seventeenth update middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction isolation of mers coronavirus from a dromedary camel reliable typing of mers-cov variants with a small genome fragment middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study occupational exposure to dromedaries and risk for mers-cov infection mers coronaviruses in dromedary camels seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia animal coronavirus vaccines: lessons for sars we are grateful to berend-jan bosch for supply of antigens for micro-array testing. samples were collected according to national regulations with regard to animal health and welfare under the institutional animal care and use committee (iacuc), permit number 2014-01-001. all animal samples were transported in agreement with dutch import regulations with regard to animal disease legislation. the authors have not received any funding or benefits from industry or elsewhere to conduct this study. key: cord-352899-bt2xg0ha authors: van kerkhove, maria d.; peiris, malik j. s.; malik, mamunur rahman; ben embarek, peter title: interpreting results from environmental contamination studies of middle east respiratory syndrome coronavirus date: 2016-10-15 journal: clin infect dis doi: 10.1093/cid/ciw478 sha: doc_id: 352899 cord_uid: bt2xg0ha nan to the editor-middle east respiratory syndrome coronavirus (mers-cov) continues to pose a threat to human health among populations in contact with infected dromedary camels and globally through the travel of persons who acquired infection from areas with enzootic dromedary infection. transmission of mers-cov is categorized as zoonotic, with repeated introductions into the human population after contact with infected dromedaries, resulting in limited human-to-human transmission, notably in healthcare settings [1] the mers-cov outbreak in south korea in 2015, resulting in 186 cases and 38 deaths [2] , reminds us that the introduction of a single case into an unsuspecting healthcare system can trigger a very large outbreak with serious public health and socioeconomic consequences. although we have known for quite some time that nosocomial transmission is responsible for more than half of reported mers-cov cases worldwide [1] , we know little about how transmission occurs in healthcare settings. we have suspected, and our involvement in several missions to affected countries in the kingdom of saudi arabia, jordan, and south korea has confirmed, that delayed recognition and slow isolation of infected patients, extended stays in overcrowded emergency rooms, and suboptimal adherence to infection prevention and control procedures have been responsible for secondary cases in these settings [3] , but the role of environmental contamination has received relatively little attention. researchers in korea have recently published studies [4, 5] evaluating environmental and/or air contamination by mers-cov and should be commended for their efforts to evaluate the hospital outbreaks in their country. taken together, these results from kim et al [4] and others [5] [6] suggest that mers-cov can persist on the surfaces in contaminated environments, such as patient rooms and equipment, and underscore the critical importance of adequate and thorough disinfection of patient rooms during and after their treatment in healthcare settings. although the findings from kim et al [4] are very interesting and raise questions about the potential presence of mers-cov in the air, they provide no evidence of airborne transmission. some concerns have been expressed that the claimed "virus isolation" did not yield virus isolates but rather was based on reverse-transcription polymerase chain reaction detection of infected cells [7] . kim et al [4] did go further to demonstrate passage of virusinfected cells leading to detectable viral antigen detection, which is better evidence of replicating virus. we are aware of a number of other environmental studies conducted in hospitals that have found surface contamination but have not been able to isolate virus from air samples. these studies have not yet been published in the peer-reviewed literature. it is important that these and any negative results are also published, or else we will be left with a skewed selection of positive findings. the partial inconsistencies in results between studies will need further examination. in particular, it is important that future studies include "negative controls" with comparable sampling and testing strategies applied in settings where patients with mers were not housed. to fully understand the possible role of environmental contamination, including the possible detection of mers-cov in the air, additional studies must be conducted to see whether these results can be replicatedfor example, in hospitals in the middle east where patients with mers-cov are treated, to evaluate virus persistence in hospital environments. a more complete understanding of the results of environmental and air contamination studies will have important implications for the application of infection prevention and control measures [8] currently used in hospitals treating patients with mers-cov, possibly leading to more detailed recommendations. potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. maria d. van kerkhove, 1,2 malik j. s. peiris, 3 mamunur rahman malik, 4 and peter ben embarek 2 middle east respiratory syndrome coronavirus (mers-cov): current situation 3 years after the virus was first identified preliminary epidemiological assessment of mers-cov outbreak in south korea notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers outbreak units environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions environmental contamination and viral shedding in mers patients middle east respiratory syndrome coronavirus (mers-cov) key: cord-350733-0zghspb8 authors: aronson, jeffrey k.; auker‐howlett, daniel; ghiara, virginia; kelly, michael p.; williamson, jon title: the use of mechanistic reasoning in assessing coronavirus interventions date: 2020-07-15 journal: j eval clin pract doi: 10.1111/jep.13438 sha: doc_id: 350733 cord_uid: 0zghspb8 rationale: evidence‐based medicine (ebm), the dominant approach to assessing the effectiveness of clinical and public health interventions, focuses on the results of association studies. ebm+ is a development of ebm that systematically considers mechanistic studies alongside association studies. aims and objectives: to explore examples of the importance of mechanistic evidence to coronavirus research. methods: we have reviewed the mechanistic evidence in four major areas that are relevant to the management of covid‐19. results and conclusions: (a) assessment of combination therapy for mers highlights the need for systematic assessment of mechanistic evidence. (b) that hypertension is a risk factor for severe disease in the case of sars‐cov‐2 suggests that altering hypertension treatment might alleviate disease, but the mechanisms are complex, and it is essential to consider and evaluate multiple mechanistic hypotheses. (c) confidence that public health interventions will be effective requires a detailed assessment of social and psychological components of the mechanisms of their action, in addition to mechanisms of disease. (d) in particular, if vaccination programmes are to be effective, they must be carefully tailored to the social context; again, mechanistic evidence is crucial. we conclude that coronavirus research is best situated within the ebm+ evaluation framework. often measured at the same time. not all association studies are equal, for example, experimental studies such as randomized controlled trials (rcts) are favoured over observational studies, ceteris paribus. studies other than association studies tend to be regarded as less useful by ebm. for example, mechanistic reasoning, which appeals to features of the mechanisms by which the intervention is hypothesized to lead to the outcome and to the mechanistic studies that investigate these features, is viewed as inferior to association studies by presentday ebm. 1 ebm+ is a development of ebm that treats mechanistic studies on a par with association studies. 2 figure 1 portrays the ebm+ view of the assessment of a causal claim. association studies provide direct evidence that the putative cause a and the putative effect b are correlated (pathway c 1 in figure 1 ). but correlation is insufficient for causation: a correlation may be attributable to chance, bias, uncontrolled confounders, inappropriately controlled colliders, or relationships other than causation. what distinguishes correlations that are causal from those that are spurious is the existence of a mechanism complex, by which instances of a explain instances of b. so, in order to establish causation, one needs to establish the existence of a mechanism (or mechanisms) of action as well as the existence of a correlation. experimental studies such as rcts are valuable precisely because they can indirectly support the existence of a mechanism (channel c 2 ), by making confounding and bias less likely to corrupt the relationship between the dependent and independent variables. but mechanistic studies also provide evidence of the existence of a mechanism, by confirming or disconfirming specific hypotheses about features of the mechanism complex linking a and b (channels m 1 and m 2 ). (in some cases, these features can also support or undermine the claim that a and b are correlated-channel m 3 .) reasoning that proceeds along channels m 1 , m 2 , and/or m 3 is mechanistic reasoning. mechanistic reasoning is particularly pertinent when specific hypotheses about key features of the mechanism complex are established (or ruled out) by mechanistic studies (see section 3) . given this more nuanced picture of causal assessment, it can be important to explicitly and systematically scrutinize mechanistic studies when assessing causal claims. this need is now often recognized when assessing the effects of environmental exposures 4 but less so when assessing the effects of interventions 5, 6 and of infectious diseases. 7 in this paper, we aim to redress the balance by showing that there is a need to assess mechanistic studies explicitly and systematically when interrogating the effects of interventions in infections with coronaviruses. indeed, this need is particularly urgent in diseases such as sars, mers, and covid-19 (due respectively to sars-cov-1, mers-cov, and sars-cov-2), because outbreaks are rapid, limiting the opportunity to conduct high-quality rcts. thus, association studies on their own tend to provide evidence that is too weak to establish causation. when rapid outbreaks are coupled with severe disease, effective interventions need to be identified very quickly. in these circumstances, only by considering mechanistic studies alongside association studies is it possible to build an evidence base that distinguishes those interventions that are effective from those that are ineffective. in the following sections, we provide several examples of the importance of mechanistic evidence to coronavirus research. the assessment of combination therapy for mers highlights the difficulties that can be encountered when suggesting treatments on the basis of poor mechanistic reasoning (section 2). that treatment for hypertension is a risk factor for severe disease in the case of sars-cov-2 suggests that altering hypertension treatment might alleviate disease, but the mechanisms are complex, and it is essential to consider and evaluate more than one mechanistic hypothesis (section 3). to successfully limit the spread of an infection, public health interventions need to take account of all relevant social mechanisms, rather than just targeting risk factors. moreover, interventions shown to be effective in one country cannot be successfully extrapolated to other countries unless the relevant social mechanisms are sufficiently similar, and this also calls for scrutiny of mechanistic evidence (section 4). finally, if vaccination programmes are to be effective, they must be carefully tailored to the social context; again, mechanistic evidence is crucial (section 5). we conclude that coronavirus research is best situated within the ebm+ evaluation framework (section 6). one strategy in seeking interventions for diseases caused by novel viruses is to repurpose existing drugs. this strategy makes much use of evidence from mechanistic studies: evidence that the drug has some action against the novel virus in the laboratory, both in vitro and in vivo in experimental animals, is used to justify the decision to use the treatment clinically. covid-19 is no different. for example, the antimalarial drug hydroxychloroquine inhibits sars-cov-2 replication in vitro, 8 leading to the suggestion that it would be a good intervention for covid-19. the motivation for this strategy is twofold. f i g u r e 1 evidential relationships for establishing a causal claim. 3 the existence of an appropriate correlation and an appropriate mechanism together confirm causation-it is not enough to have one without the other. arrows signify potential positive or negative evidential relationships because of the novelty of the virus, there are no disease-specific drugs ready for testing. and as a result of the intensity of the disease, there is a pragmatic motivation for using compounds that may not yet have been rigorously tested. the end goal in the standard approach is still testing of compounds in randomized trials. however, high-quality trials are time-consuming. responses to covid-19 thus rely heavily on the use of evidence from mechanistic studies. can this strategy be improved by taking an ebm+ approach? standard ebm says little about the role mechanistic reasoning can play in attempts to identify effective treatments. as a result, mistakes may be made in how such evidence is handled. on the other hand, ebm+ takes mechanistic studies into account and offers explicit guidance regarding their evaluation. the following analysis of the way repurposing was carried out in response to mers shows how the ebm+ approach uses mechanistic reasoning to identify effectiveness. as a repurposing strategy, a combination of an interferon (ifn) and ribavirin has been suggested for treating mers. the mechanism of action of ifn is fairly well known, 9 and there was some evidence that replication of mers-cov is inhibited by ifn in vitro and in vivo 10, 11 . a major problem for the use of ifn was that it only inhibited the virus at clinically unobtainable concentrations. however, a synergistic effect was observed by using it in combination with ribavirin, in vitro inhibition of mers-cov being observed at clinically obtainable concentrations of both compounds. 12, 13 moreover, rhesus macaques infected with mers-cov displayed signs of recovery when given combination therapy. 14 at no point did these studies establish that this treatment would be effective, but this evidence nevertheless provided the motivation for clinical use of combination therapy. the problem here is that merely having some evidence from mechanistic studies is not enough to conclude that a mechanism of action exists. arguably, for a treatment to be put forward for clinical use, the existence of a mechanism linking the treatment and recovery from the disease must be very plausible. however, evidence that a drug inhibits viral replication in vitro is only weak evidence that it will inhibit viral replication in vivo. evaluating the extent to which mechanistic studies confirm the plausibility of mechanistic hypotheses is something that ebm+ adds to standard ebm. in animals is not enough to conclude that the mechanism is relevant to the clinical outcome in humans for the following reasons. in addition to viral replication, the pathology of mers may include a mechanism by which the immune system contributes to lung damage, even after viral load is reduced. 15 reductions in lung pathology in rhesus macaques supported combination therapy; hence, one might be tempted to say that there is evidence for both important features of the mechanism by which combination therapy affects outcomes in mers. the problem with this conclusion is that rhesus macaques are inappropriate animals for investigating whether an intervention can reduce respiratory pathology. this is because they only develop a mild form of mers that lacks the kind of severe lung damage observed clinically. 16 full evaluation of this evidence thus shows that it is only moderately plausible that a mechanism exists linking combination therapy and recovery from mers in humans. of course, it will be no surprise to proponents of ebm that evidence from mechanistic studies may struggle to prove the existence of a relevant mechanism. however, it is not enough merely to note that results are not easily extrapolated from cell cultures or animals to humans, and to apply this scepticism rigidly to all experimental systems. common marmosets, for example, develop a form of mers very similar to that in humans, and evidence obtained using these animals is much more relevant to humans than evidence from rhesus macaques. 16, 17 without explicitly evaluating mechanistic evidence, it is not possible to distinguish a plausible (strong) mechanistic hypothesis from an implausible (weak) hypothesis. moreover, when repurposing drugs, compounds that are more likely to be effective should be prioritized. these considerations favour the ebm+ approach, as does an analysis of the evidence for another putative treatment for mers. mycophenolic acid was proposed as an intervention for mers, based on evidence that replication of mers-cov was inhibited in vitro. 18 moreover, it was more efficacious than combination therapy. [19] [20] [21] accordingly, mycophenolic acid was suggested as a potential treatment for mers and saw some clinical use. 22 however, mycophenolic acid is an immunosuppressant, and its mechanism of action involves selective depletion of the dna and rna precursor guanosine in t cells. 23 indeed, it was subsequently found to be associated with greater mortality in common marmosets. 17 testing of mycophenolic acid should not have proceeded to clinical testing; the immunosuppression mechanism should have been considered. ebm+ requires that the whole complex of mechanisms by which mycophenolic acid operates should be scrutinized, rather than isolated mechanistic hypotheses. this practice is not always followed, and in this case, a compound made its way into clinical testing that should not have. this is thus another case in which a full evaluation of evidence from mechanistic studies would improve on the current repurposing strategy. pharmacological risk surveillance is another area in which an evidentially diverse approach to causal evaluation is desirable. 24, 25 knowing a mechanism that links a treatment and an adverse event can help explain their observed correlation. take, for example, antihypertensive drugs that act on components of the renin-angiotensin system (ras) and might be either beneficial or harmful, as competing mechanistic hypotheses suggest. [26] [27] [28] angiotensin converting enzyme 2 (ace-2) is the receptor to which sars-cov-2 binds to enter host cells. 29, 30 some antihypertensive drugs increase ace-2 expression. it has therefore been suggested that those drugs may worsen the intensity of covid-19 by providing a greater opportunity for sars-cov-2 to enter host cells. 27 on the other hand, ace-2 protects against lung injury by regulating concentrations of angiotensin ii, which is vasoconstrictive, pro-inflammatory, and pro-oxidative. 31 hence, it has been suggested that increased expression of ace-2 from using antihypertensive drugs might reduce the intensity of covid-19. 28 this evidence suggests two mechanism hypotheses: h1, by which the drugs increase the intensity of the disease, and h2, by which they reduce it ( figure 2 ). the status of h1 and h2 is problematic. there are two separate issues. first, the mechanistic studies from which the evidence is obtained have not been properly evaluated, and neither has the plausibility of either mechanism. the mechanism by which ace2 protects against lung injury (h2) is fairly well established, 32,33 but the evidence of a protective effect against sars-cov-2 induced lung injury 34 needs evaluation, since it may be insufficient to support the claim of a protective mechanism. for example, an error may have been committed during the study, or the experimental system and the target system may be dissimilar. 2 only by evaluating the evidence that supports the proposed mechanisms can this question be settled. secondly, these mechanisms are plausibly not mutually exclusive. that they both operate is a distinct possibility, although if so, they probably operate in sequence rather than in parallel: by h1, more virions enter the host cell, and by h2, the damage caused by the virus is reduced. ebm+ recommends evaluating the whole complex of mechanisms at work. focusing on isolated mechanisms risks missing interactions in the way they influence the intensity of the disease. despite the absence of high-quality clinical trials, we nevertheless have some evidence of possible causal relationships. in this sort of scenario, the ebm+ approach enables evaluation of the plausibility of causality. we might obtain evidence of a correlation between antihypertensive drug therapy and the intensity of covid-19 through association studies with lower quality designs, in advance of better ones. such studies cannot prove causation, because they are subject to biases. however, the ebm+ approach to causal evaluation combines this kind of evidence with (properly evaluated) evidence of mechanisms to obtain a better assessment of causality than either strand of evidence provides alone. 35 the importance of mechanistic reasoning to conclusions about the effects of antihypertensive drugs depends on the extent to which mechanistic studies shed light on the relevant mechanisms. for mechanistic reasoning to be strong, key features of the mechanism complex should be well established, and this can be achieved by meeting the following conditions: • ideally, the operation of each feature should have been demonstrated in vitro in different types of relevant cells/tissues/organ, and in vivo in a range of species. when the experimental system differs from the target system (eg, a macaque model investigating a treatment in humans), a mechanism that exists in one experimental species and not another is unlikely to be helpful. • such demonstrations should include evidence that the mechanism exists, that enhancing it leads to measurable outcomes, and likewise for inhibiting it or abolishing it. other criteria that, if fulfilled, would strengthen the mechanistic reasoning further include: • demonstrating the anatomical location of the mechanism and its relevance. do strategic lesions cause predictable changes? • demonstrating the time course of the mechanism. does it change in response to interventions such as upregulation or downregulation? • do genetic polymorphisms, 37-39 physiological variables, diseases, and drug interactions alter it predictably? • when any changes occur, is dose-responsiveness maintained? establishing key features of the whole complex of relevant mechanisms through a variety of experimental sources thus makes mechanistic reasoning strong. many public health interventions attempt to change or influence human behaviour. for decades, most of this effort has gone into preventing non-communicable diseases-for example, by preventing smoking, obesity, and alcohol misuse and encouraging physical activity. 40 although human behaviour is also central to the transmission of infectious disease, efforts to change behaviour have mostly been applied to hiv, other sexually transmitted infections, and tuberculosis. one important finding from work in non-communicable diseases, as well as hiv, has been the realization that social and psychological mechanisms are relevant both to aetiology and to the effectiveness of preventive interventions. 41 thirdly, one needs to consider mechanisms that affect take-up of interventions. the use of testing, for example, is based on the assumption that people will avail themselves of the test and understand what the result means. not everyone will, and different individuals and groups will respond differently to the offer. this response is governed not only by availability, but also by the social and psychological mechanisms in play. the same will be true inter alia of other offers, such as tracing apps and any vaccine, should one become available. these mechanisms are not as well understood as we need them to be, and there is an urgent need for programmes of research and evidence synthesis. these mechanisms will also have to be integrated into decision-making processes in future coronavirus pandemics. the knowledge base, such as it is, is highly anglo-american-centric, and its transferability to other settings is not known. the idea that strategies that have been used in north america and europe could or should be applied elsewhere is not sound. likewise, applying strategies that appear to have worked in south korea, singapore, or china to the united kingdom are subject to the same uncertainty. the uk strategy of closing schools assumed that this protects children and families, because schools are potentially infection rich. however, this was premised on the biology of influenza, which thrives in schools, making children vectors of infection. 48 but covid-19 is different. children do not appear to be at great risk from sars-cov-2, though their infectivity towards adults is not clear at present. as a preventive mechanism, school closure is a blunt instrument. when schools reopen, various social and behavioural mechanisms come into play, such as parents' reluctance to return their children to school, teachers' fears of returning to work, and the need for transport to and from schools. applied in settings outside the united kingdom, this strategy is blunter still. school closures have many potential consequences. these include, but are not limited to, increased risks from accidents and injury outside the relative safety of the school, sexual violence to young girls, pressures and demands on parents, including economic costs of staying away from work, living conditions in multiple-generation households (which, unlike schools, are likely to be important cockpits of infection in covid-19), long-term educational disadvantages through missing schooling (more likely to damage girls), and pressures on the social fabric to which schools contribute. 49 all these will interact with local socio-economic and cultural settings. there are thus two strategies for jurisdictions that seek to establish public health measures to deal with coronavirus pandemics. one is to develop local solutions, building from the ground up, rather than naively importing solutions that may never have been understood properly in the countries that implemented them. the other is to extrapolate interventions that have worked well elsewhere. however, successful extrapolation requires a detailed evaluation of the similarities and differences between the relevant mechanisms in the source and target jurisdictions. 2, 50 these two strategies are not mutually exclusive and can be combined. by june 2020, researchers worldwide haddeveloped 149 vaccine candidates against covid-19, of which 17 are in clinicalevaluation and 132 in preclinical evaluation. although the development of an effective vaccine will mark a major step forward, the mere existence of a vaccine will not ensure high uptake. as noted in the "tailoring immunization programmes" guide published by the who's regional office for europe, mechanistic studies are crucial in order to understand the psychological, contextual, and social mechanisms that influence vaccination behaviours. 51 in recent years, several vaccination studies have investigated barriers to vaccination, and have identified evidence, which confirms general mechanisms that appear to work in diverse situations, as well as more specific mechanisms that work only in particular contexts. 52 most of these mechanistic studies can provide useful insights for coronavirus vaccination and the assessment of its effects. some evidence, for instance, supports a behavioural mechanism known as the "intention-behaviour gap," 53 in sum, the explicit and systematic assessment of mechanistic studies is essential for the development of effective vaccination programs in a given context. we have presented a range of contexts in coronavirus research in which it is beneficial to systematically assess mechanistic studies alongside association studies. the cases we have discussed are illustrative rather than exhaustive. indeed, we have not touched on assessment of medical devices, for which mechanistic reasoning is clearly essential; for example, the use of less invasive respiratory devices 64 moreover, social behaviours can change radically during an epidemic, partly in response to perceptions about the organism and its effects and partly in response to public health interventions. only by explicitly articulating and systematically assessing these potential mechanisms can one ensure that coronavirus interventions are effective. evidence-based medicine. a new approach to teaching the practice of medicine evaluating evidence of mechanisms in medicine: principles and procedures the feasibility and malleability of ebm+ evidential proximity, independence, and the evaluation of carcinogenicity the use of mechanistic evidence in drug approval reinforced reasoning in medicine establishing the teratogenicity of zika and evaluating causal criteria. synthese, published online remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro mechanism of action of interferon and ribavirin in treatment of hepatitis c pegylated interferon-α protects type 1 pneumocytes against sars coronavirus infection in macaques mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporine a or interferon-a treatment ribavirin and interferon-β synergistically inhibit sars-associated coronavirus replication in animal and human cell lines inhibition of novel β-coronavirus replication by a combination of interferon-α2b and ribavirin treatment with interferon-α2b and ribavirin improves outcome in mers-cov infected rhesus macaques middle east respiratory syndrome a comparative review of animal models of middle east respiratory syndrome coronavirus infection treatment with lopinavir/ritonavir or interferon-α1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset the emergence of the middle east respiratory syndrome coronavirus. pathogens and disease broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia pharmacology and toxicology of mycophenolate in organ transplant recipients: an update reviewing the mechanistic evidence assessors e-synthesis and ebm +: a case study of amoxicillin and drug reaction with eosinophilia and systemic symptoms (dress) e-synthesis: a bayesian framework for causal assessment in pharmacosurveillance drugs and the renin-angiotensin system in covid-19 are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor structural and functional basis of sars-cov-2 entry by using human ace2 angiotensin-converting enzyme 2 and antihypertensives (angiotensin receptor blockers and angiotensin-converting enzyme inhibitors) in coronavirus disease 2019 a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin converting enzyme 2: sars-cov-2 receptor and regulator of the renin-angiotensin system inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 mechanisms and the evidence hierarchy inhibitors of the renin-angiotensin-aldosterone system and covid-19 the combination of ace i/d and ace2 g8790a polymorphisms revels susceptibility to hypertension: a genetic association study in brazilian patients antihypertensive treatment with acei/arb of patients with covid-19 complicated by hypertension is hypertension in african-descent populations contributed to by an imbalance in the activities of the ace2/ang-(1-7)/mas and the ace/ang ii/at1 axes? changing population behavior and reducing health disparities: exploring the potential of "choice architecture" interventions causal narratives in public health: the difference between mechanisms of aetiology and mechanisms of prevention in non-communicable diseases the integration of social, behavioural and biological mechanisms in models of pathogenesis prisoners of the proximate: loosening the constraints on epidemiology in an age of change the human behaviour-change project: harnessing the power of artificial intelligence and machine learning for evidence synthesis and interpretation from theory-inspired to theory-based interventions: a protocol for developing and testing a methodology for linking behaviour change techniques to theoretical mechanisms of action aids: don't die of prejudice harnessing behavioural science in public health campaigns to maintain 'social distancing' in response to the covid-19 pandemic: key principles effectiveness of school closure for the control of influenza. national collaborating centre for infectious diseases children's ebola recovery assessment: sierra leone across the boundaries: extrapolation in biology and social science the guide to tailoring immunization programmes (tip): increasing coverage of infant and child vaccination in the who european region increasing vaccine uptake in low-and middle-income countries. opportunities for behavioural science research. the behavioural insights team the intention-behavior gap psychological factors associated with uptake of the childhood influenza vaccine and perception of post-vaccination side-effects: a cross-sectional survey in england the state of vaccine confidence 2016: global insights through a 67-country survey parental reminder, recall and educational interventions to improve early childhood immunisation uptake: a systematic review and meta-analysis beliefs underlying uk parents' views towards mmr promotion interventions: a qualitative study a systematic review of interventions for reducing parental vaccine refusal and vaccine hesitancy on the benefits of explaining herd immunity in vaccine advocacy vaccinating to help ourselves and others inviting free-riders or appealing to prosocial behavior? game-theoretical reflections on communicating herd immunity in vaccine advocacy the roles of altruism, free riding, and bandwagoning in vaccination decisions increasing vaccination: putting psychological science into action the use of mechanistic reasoning in assessing coronavirus interventions the authors declare no conflict of interest. key: cord-355290-m8875kdy authors: meyer, benjamin; garcía-bocanegra, ignacio; wernery, ulrich; wernery, renate; sieberg, andrea; müller, marcel a.; drexler, jan felix; drosten, christian; eckerle, isabella title: serologic assessment of possibility for mers-cov infection in equids date: 2015-01-17 journal: emerg infect dis doi: 10.3201/eid2101.141342 sha: doc_id: 355290 cord_uid: m8875kdy nan bats, pigs, and goats (6) . mers-cov uses the receptor dipeptidyl-peptidase-4 (dpp-4) to enter its host cell (7) . sequence comparison between the receptor-binding domain of the mers-cov spike protein and several mammalian dpp-4 sequences showed a higher percentage identity in the amino acid residues critical for virus entry between human and horse dpp-4 than between human and dromedary dpp-4 (8) . it has been shown that mers-cov can use horse dpp-4 expressed on nonsusceptible cells (9) , but no data are available on susceptibility of primary horse cells. therefore, members of the family equidae, which include domestic horses, donkeys, and mules, might be susceptible to mers-cov infection. according to the food and agricultural organization of the united nations (http://faostat. fao.org), >800,000 equids (horses, mules, and donkeys) are present on the arabian peninsula, but their role as putative mers-cov animal reservoirs has not been investigated. therefore, we assessed in vitro susceptibility of primary horse cells to mers-cov infection and searched for serologic evidence of infection with mers-cov in equids originating from spain and the united arab emirates. primary cells derived from the kidney of 2 horses (termed pn-r and pfn-r) and an interferon-deficient primate cell line (verob4) were infected with mers-cov at a multiplicity of infection of 0.5 pfus. virus replication was quantified by real-time reverse transcription pcr (mers-cov upe assay) (10) and by plaque assay in vero cells to confirm the production of infectious virus particles. endurance horses from the united arab emirates that were collected for routine veterinary purposes; and 861 samples from 697 horses, 82 donkeys, and 82 mules in spain. because the reactivity of equid serum against mers-cov has not been investigated, we established a 2-stage algorithm for serologic testing that did not involve the determination of reactivity cutoff values. the screening stage involved testing of all serum samples by using a previously described elisa with the spike protein s1-domain of mers-cov as the test antigen (4). the elisa was adapted for use with horse serum by exchange of the secondary antibody. all serum samples reacted with low to medium od values (range 0.0-0.55) (figure, panel c) . we then tested the 50 most reactive serum samples (optical density range 0.22-0.55) by using recombinant immunofluorescent and microneutralization assays (1). these assays are more specific than the elisa assay and therefore can be used for confirmation. none of the tested serum samples showed reactivity in the recombinant immunofluorescent or microneutralization assays; this finding suggests that no previous exposure of equids to mers-cov has occurred in the united arab emirates and spain. identifying all potential animal reservoirs is a critical step in controlling zoonotic diseases. molecular data suggest that horses may be highly susceptible to mers-cov because of their high similarity in dpp-4 amino acids at positions critical for binding of the mers-cov spike protein (8) . our in vitro data confirm the susceptibility of primary horse cells, showing production not only of viral rna but also of infectious virus progeny, which is a prerequisite for transmission. the lower replication observed in horse cells than in verob4 cells may be the result of a difference in the interferon competence of the cells; replication levels in horse cells are comparable to those in bat cells (6) . although we did not find evidence for equid infections with mers-cov in this study, the general susceptibility on the cell culture level suggests that equids from mers-cov-endemic areas, such as africa and the arabian peninsula, should be further investigated for possible infection with mers-cov. middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia middle east respiratory syndrome coronavirus antibody reactors among camels in dubai replicative capacity of mers coronavirus in livestock cell lines dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc spiking the mers-coronavirus receptor receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction we thank dagmar hensel for excellent technical help and matthias lenk for horse cell lines. we also thank victor corman, monika eschbach-bludau, tobias bleicker, and sebastian brünink for help with horse serum samples.this study was supported by the european commission under project antigone (contract no. 278976) and the german centre for infection research. key: cord-345046-str19r9a authors: al ghamdi, mohammed; alghamdi, khalid m.; ghandoora, yasmeen; alzahrani, ameera; salah, fatmah; alsulami, abdulmoatani; bawayan, mayada f.; vaidya, dhananjay; perl, trish m.; sood, geeta title: treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia date: 2016-04-21 journal: bmc infect dis doi: 10.1186/s12879-016-1492-4 sha: doc_id: 345046 cord_uid: str19r9a background: middle eastern respiratory syndrome coronavirus (mers-cov) is a poorly understood disease with no known treatments. we describe the clinical features and treatment outcomes of patients with laboratory confirmed mers-cov at a regional referral center in the kingdom of saudi arabia. methods: in 2014, a retrospective chart review was performed on patients with a laboratory confirmed diagnosis of mers-cov to determine clinical and treatment characteristics associated with death. confounding was evaluated and a multivariate logistic regression was performed to assess the independent effect of treatments administered. results: fifty-one patients had an overall mortality of 37 %. most patients were male (78 %) with a mean age of 54 years. almost a quarter of the patients were healthcare workers (23.5 %) and 41 % had a known exposure to another person with mers-cov. survival was associated with male gender, working as a healthcare worker, history of hypertension, vomiting on admission, elevated respiratory rate, abnormal lung exam, elevated alanine transaminase (alt), clearance of mers-cov on repeat pcr polymerase chain reaction (pcr) testing, and mycophenolate mofetil treatment. survival was reduced in the presence of coronary artery disease, hypotension, hypoxemia, cxr (chest x-ray) abnormalities, leukocytosis, creatinine >1 · 5 mg/dl, thrombocytopenia, anemia, and renal failure. in a multivariate analysis of treatments administered, severity of illness was the greatest predictor of reduced survival. conclusions: care for patients with mers-cov remains a challenge. in this retrospective cohort, interferon beta and mycophenolate mofetil treatment were predictors of increased survival in the univariate analysis. severity of illness was the greatest predictor of reduced survival in the multivariate analysis. larger randomized trials are needed to better evaluate the efficacy of these treatment regimens for mers-cov. coronaviruses cause a spectrum of illness from asymptomatic disease to respiratory failure. early reports of coronavirus infections suggested that most infections were mild until the 2003 sars epidemic that was associated with significant morbidity and mortality [1] . in september 2012, a novel coronavirus was identified in a 60-year old man in saudi arabia [2] . a second case was identified in a qatari patient hospitalized in the united kingdom [3] . the two coronaviruses were genetically identical and similar to isolates obtained from bats [4] . in july 2013, the coronavirus study group named this new virus middle east respiratory syndrome coronavirus (mers-cov) [5] . as of december 21, 2015, there have been 1625 cases worldwide with 586 deaths [6] . the epidemiology and clinical manifestations of this disease have described a spectrum of illness from asymptomatic infection to severe respiratory failure and death. the overall mortality rate remains at 37 % [7] [8] [9] [10] [11] [12] [13] [14] [15] . importantly, there are no known effective treatments. in 2014 there was an increase in mers-cov cases reported from the jeddah region of saudi arabia. to describe the changing epidemiology and outcomes, we report the clinical features and treatment outcomes of patients admitted to a regional referral hospital in jeddah, saudi arabia. king fahd general hospital is an 800-bed hospital in jeddah, kingdom of saudi arabia and is a regional coronavirus referral center. there are 36 icu beds and one infectious disease physician that serves the hospital. between january through december 2014, all patients admitted or transferred to king fahd hospital with a positive mers coronavirus pcr from clinical nasal swabs or nasopharyngeal aspirates were included. all pcr testing was performed at the ministry of health regional lab in jeddah. the magna pure compact/ magna pure 96 (roche) automated system was used to extract rna from samples. primers and probes for upe and orf 1a targets of mers-cov were used from tib molbiol (germany) along with master mix from roche for the light cycler 480 ii (roche) were used to amplify upe and orf 1a gene targets. samples that tested positive for both upe and orf 1a gene targets with a cycle threshold time of less than 37 were considered confirmed cases. positive and negative controls were used to monitor the amplification process & to check for any inhibition of amplification. medical charts for all patients were reviewed and data abstracted on standardized data collection forms by an infectious disease trained physician. demographic, clinical and laboratory data were entered into a database. to understand the epidemiology, age was categorized as <30, 30-60 and >60. hypotension was defined as blood pressure <90/60 mm hg, tachypnea as a respiratory rate greater than 16, hypoxia as an oxygen saturation <90 %, thrombocytopenia as platelets <150,000/ cubic millimeter, leukopenia was defined as a white blood cell count <5000 cells/cubic millimeter and leukocytosis as a white blood cell count >10,000 cells/ cubic millimeter. renal insufficiency was defined as a creatinine >1.5 mg/dl. liver function abnormalities were defined as a lactate dehydrogenase (ldh) >300 u/liter, alanine transaminase (alt) > 50 u/liter and aspartate aminotransferase (ast) >40 u/liter. immunosuppression was defined as aids, history of organ transplant, neutropenia, known malignancy, taking immunosuppressive medication and congenital immunodeficiency. pregnancy was considered an immunosuppressed state. a modified acute physiologic and chronic health evaluation (apache 2) score was calculated using age, temperature, mean arterial blood pressure, respiratory rate, potassium, creatinine, acute renal failure, and comorbid conditions to estimate severity of illness [16] . pao2 was estimated using pulse oximetry oxygen saturation results and hematocrit was calculated by multiplying the hemoglobin times three. all statistical analyses were performed using stata software (version 13.1, college station, tx). the percent distribution of clinical variables among patients who survived and those who died were compared using the fisher exact test. a multivariate logistic regression was done on treatments administered and severity of illness to determine which treatments were associated with survival. mycophenolate mofetil was not included in this logistic regression analysis because 100 % of patients receiving mycophenolate mofetil survived. the association between severity of illness and treatments administered was assessed by performing a linear regression of treatments administered onto the modified apache 2 score. there were a total of 51 cases, thirty patients (58.8 %) of whom were saudi nationals, and 21 (41.2 %) were foreign nationals. the median age was 54 years old (iqr 36.5-58). most were male (n = 40, 78.4 %). twenty-one patients (41.2 %) had exposure to a known patient with mers coronavirus and 12 (23.5 %) were healthcare workers. none of the patients had animal exposure. two patients (3.9 %) were on pilgrimage to mecca. overall, 71 % of patient had at least one co-morbid condition. seventeen patients had diabetes (33.3 %), 25 had hypertension (49 %), 14 (27.5 %) had end stage renal disease, eight (15.7 %) had coronary artery disease and six (11.8 %) patients were immunosuppressed, two of whom were pregnant. patients received a variety of novel treatments including immunosuppressants and antivirals. forty-two (82.4 %) patients received broad-spectrum antibiotics and five (9.8 %) received hydrocortisone. thirty one patients received antiviral treatment. twenty-three patients (45.1 %) were treated with interferon beta, eight (15.7 %) were treated with interferon alpha. a variety of anti-viral combinations were used. eight patients (15.7 %) received mycophenolate mofetil, seven of these patients received it in combination with interferon beta. nineteen (37.3 %) patients required intensive care unit (icu) care, and 10 patients received extracorporeal membrane oxygenation (ecmo). all patients treated in the icu and all patients receiving ecmo died. in this recent cohort, when comparing survivors to nonsurvivors, survival was associated with male gender, vomiting on admission, elevated respiratory rate, abnormal lung exam on physical exam, working as a healthcare worker, history of hypertension, elevated alt, clearance of mers cov on repeat pcr testing, and receiving mycophenolate mofetil or beta interferon (table 1 ). in contrast, markers of severe disease like hypotension, hypoxemia, chest radiographic abnormalities, leukocytosis, elevated creatinine, thrombocytopenia, anemia, renal failure were associated with death. treatments given were based as indicated based on the clinical assessment of the infectious disease consult team. thirty-one patients received antivirals, ribavirin or alpha or beta interferon, and 13 patients received immunosuppressive medication. most patients received a combination of alpha interferon and ribavirin (5, 9.8 %), beta interferon and ribavirin (10, 19.5 %) or beta interferon alone (11, 21.6 %). two patients received alpha interferon alone (3.9 %). eight patients received mycophenolate mofetil (15.7 %) and seven of them received this in combination with beta-interferon. five patients received hydrocortisone; two in combination with beta interferon and ribavirin and 3 in combination with alpha interferon and ribavirin. all eight patients given mycophenolate mofetil survived therefore mycophenolate mofetil could not be evaluated in this model. while the results of the univariable analysis demonstrated improved survival in patients treated with betainterferon and mycophenolate mofetil, the multivariable analysis which included a marker of severity of illness, demonstrated a strong association between severity of illness and reduced survival, and no association between treatment with beta interferon and survival. mycophenolate mofetil was not evaluable in this model ( table 2 ). in analyzing the relationship between severity of illness and treatments administered, beta interferon and mycophenolate mofetil were given to less severely ill patients (table 3) discussion mers-cov is an emerging disease for which the initial epidemiology has been described, but in-depth clinical studies and the role of therapy in incompletely understood. while the clinical features for mers-cov have been described in several large case series [6] [7] [8] [9] [10] [11] [12] [13] [14] , there is a paucity of literature on therapy. our results from a relatively large number of patients demonstrate similar clinical features and mortality to previous studies [6] [7] [8] [9] [10] [11] [12] [13] [14] . in our cohort, treatment with beta interferon and mycophenolate mofetil may be predictive of survival, but the greatest predictor of survival is the severity of illness on presentation. improved diagnostics have demonstrated an expanded spectrum of disease that includes less severe cases than previously reported. we now understand that mers-cov causes an acute respiratory disease syndrome and [7] [8] [9] [10] [11] [12] [13] [14] [15] . this may be partially related to the epidemiology of increased disease transmission in healthcare settings rather than a true host risk factor. laboratory findings have been nonspecific and consistent with other viral infections. thrombocytopenia (75 %) and lymphopenia (58 %) have been commonly described in these patients [7, [9] [10] [11] [12] [13] 15] . forty three percent had acute kidney injury [7, [11] [12] [13] 17] and 76-100 % had cxr abnormalities with bibasilar infiltrates as the most common finding [8-13, 15, 18] . the outcomes in these more severely ill patients remain poor. between 50-90 % required icu care [10, 11, 13, 15] and 67-100 % in the icu setting required invasive ventilation for a median of 7-16 days [8, 10, 12] . in addition to mechanical ventilation, several patients have received extracorpeal membrane oxygenation (ecmo) to support ventilation. from non-randomized data from the world health organization, five out of six patients receiving ecmo died [9] . fifty-eight to 75 % required renal replacement therapy [11, 12, 17] and 30-60 % of hospitalized patients died [7] [8] [9] [10] [11] [12] [13] [14] [15] . the severity of illness can be partially explained by the widespread lung disease caused by mers-cov and it appears that mortality in those patients requiring intensive care is extremely high. although no autopsy data is available, in explanted lung, infection with mers-cov causes widespread infection and alveolar disease [19, 20] . the clinical features in our cohort similarly also show a high proportion of patients with fever (96 %) and cough (80.4 %) shortness of breath (90 %), and almost one third of patients (29.4 %) with gastrointestinal symptoms. our cohort consisted of ill patients with hypotension (15.7 %), tachypnea (76.9 %) and hypoxia (33 %). thirty seven percent required icu care and 10 patients received ecmo. similar to previous results, all of the patients who received ecmo died [9] . there is no known effective treatment for mers cov. many compounds have been screened in vitro for possible activity against this coronavirus [21] [22] [23] [24] , however, the in vivo efficacy has not been subjected to clinical investigation. in vitro data suggests that mers-cov inhibits host interferon production through various molecular pathways [25] [26] [27] [28] [29] [30] mycophenic acid, the active agent of prodrug mycophenolate mofetil, and cyclosporine strongly inhibit mers coronavirus in human and monkey cell lines even more so than they inhibit sars coronavirus [24, [31] [32] [33] . interferon alpha and interferon beta reduce mers coronavirus replication in explanted lung tissue [19] . in vivo, comparing host response in two patients with mers coronavirus and differing outcomes, the patient who was able to clear mers cov infection was able to mount an interferon response and the patient who died had low levels of interferon alpha suggesting a therapeutic role for interferon [34] . the combination of interferon alpha and ribavirin has been used successfully in rhesus monkeys infected with mers coronavirus [35] , and in a few small case series [36] [37] [38] . beta-interferon seems to be an even more potent inhibitor of mers coronavirus in vitro [19] [24, [31] [32] [33] . one small study with exceptionally high mortality rates using interferon beta for treatment found no difference in mortality between interferon beta use and interferon alpha use [39] . our data, albeit from a retrospective cohort support the findings that interferon beta is associated with a decrease in mortality. there are limited data on the efficacy of treatment regiments for this virulent disease. we present data from a retrospective cohort of ill patients with mers-cov and the results of the evaluation of the clinical efficacy of beta interferon beta, alpha interferon, ribavirin and mycophenolate mofetil in addition to routine supportive care. forty five percent of patents (23 patients) received interferon beta and in this cohort, sixteen percent of patients received interferon alpha (8 patients) and 37 % of patients (9 patients) received ribavirin, either in conjunction with interferon alpha or interferon beta, and 8 patient received mycophenolate mofetil. patients receiving beta interferon and mofetil had improved survival, however this was confounded by the severity of illness on presentation for beta interferon. all of the patients who received mycophenolate mofetil survived however because of the small number, we could not analyze the independent efficacy of mycophenolate mofetil. while this is a relatively large series of mers-cov cases, the primary limitation of our study is that it is a retrospective review of cases and not a randomized trial and thus subject to confounding as seen in our cohort. we used a modified apache 2 score without all of the clinical variables, which may have underestimated the association of severity of illness with reduced survival. importantly, the mortality in patients receiving additional therapies that modulate the immune response was low. all of the eight patients who received mycophenolate mofetil in our study survived. hence, it may be reasonable to further study this agent in controlled trials. this observational study investigates novel treatment options like beta interferon and mycophenolate mofetil for mers-cov in humans which have in vitro activity. our cohort demonstrated severity of illness is an important effect modifier and needs to be considered in evaluating novel agents. to better assess the efficacy of these therapies, international prospective randomized trials with adequate numbers of patients are needed to further evaluate the impact of these treatments in addition to routine supportive care when compared to other treatment options. this study was reviewed and approved by johns hopkins university institutional review board and the directorate of health affairs. data supporting the findings are in the manuscript, additional data available upon request. abbreviations aids: acquired immune deficiency syndrome; alt: alanine transaminase; apache 2: acute physiologic and chronic health evaluation; ast: aspartate aminotransferase; cxr: chest x ray; ecmo: extracorporeal membrane oxygenation; icu: intensive care unit; ldh: lactate dehydrogenase; mers co-v: middle eastern respiratory syndrome coronavirus; pcr: polymerase chain reaction. the authors declare that they have no competing interests. authors' contributions ma conceived of the study, participated in its design and helped draft the manuscript. ka participated in data collection and analysis, and reviewed the manuscript. yg participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. fs participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. mb participated in data collection and analysis, and reviewed the manuscript. tmp participated in the design and analysis as well as the writing of the manuscript. dv participated in the statistical analysis of the study. gs helped analyze the data and write the manuscript. all authors read and approved the final manuscript. the severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia patient with new strain of coronavirus is treated in intensive care at london hospital latest outbreak news from promed-mail: novel coronavirus -middle east middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group who summary family cluster of middle east respiratory syndrome coronavirus infections ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection jordan mers-cov investigation team. hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description middle east respiratory syndrome coronavirus infections in health care workers middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients apache ii: a severity of disease classification system in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures emerging human middle east respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses screening of an fda-approved compound library identifies four smallmolecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus molecular pathology of emerging coronavirus infections middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon (ifn)-alpha-2b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study there are no acknowledgements. there was no external funds provided for this project. key: cord-356192-8b96rgqa authors: xie, qian; cao, yujuan; su, juan; wu, jie; wu, xianbo; wan, chengsong; he, mingliang; ke, changwen; zhang, bao; zhao, wei title: two deletion variants of middle east respiratory syndrome coronavirus found in a patient with characteristic symptoms date: 2017-04-18 journal: arch virol doi: 10.1007/s00705-017-3361-x sha: doc_id: 356192 cord_uid: 8b96rgqa significant sequence variation of middle east respiratory syndrome coronavirus (mers cov) has never been detected since it was first reported in 2012. a mers patient came from korea to china in late may 2015. the patient was 44 years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with mers, and had had close contact with individuals with confirmed cases of mers.after one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. a nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of mers cov. deletions of 414 and 419 nt occurred between orf5 and the e protein, resulting in a partial protein fusion or truncation of orf5 and the e protein. functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package mers cov. however, the mechanism of how these deletions occurred and what effects they have need to be further investigated. electronic supplementary material: the online version of this article (doi:10.1007/s00705-017-3361-x) contains supplementary material, which is available to authorized users. with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. a nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of mers cov. deletions of 414 and 419 nt occurred between orf5 and the e protein, resulting in a partial protein fusion or truncation of orf5 and the e protein. functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package mers cov. however, the mechanism of how these deletions occurred and what effects they have need to be further investigated. qian middle east respiratory syndrome coronavirus (mers cov) has been reported in more than 23 countries [1] since the first case was identified in 2012 [2] . infection with this virus leads to a mortality rate of about 40%, but its origin is still not known [3] [4] [5] [6] [7] . mers cov belongs to lineage c of the betacoronaviruses and has a single-stranded, positivesense, 30.1-kb rna genome. the viral genomic rna encodes four structural proteins, i.e., spike glycoprotein (s), envelope (e), matrix (m) and nucleocapsid (n), as well as several nonstructural proteins, including orf3-5 and orf8b [8] . recently, 186 individuals were confirmed to be infected with mers cov in korea. during the epidemic, one person who was in close contact with a mers cov patient started to show mers symptoms shortly after he traveled to guangdong province of china and was confirmatively diagnosed with mers cov by lab tests. the patient was cured after 31 days of treatment with antiviral, anti-infection, and immune-enhancing agents. in order to better understand the transmission and evolution of this virus [9] , viral rna was isolated from a nasopharyngeal swab sample of the korean patient and sequenced. in addition to the wild-type (wt) virus, two deletion variants of mers cov were detected in this patient. the cdna was amplified using 24 pairs of primers (supplemental table 1 ). each fragment amplified by rt-pcr was about 1500 bp in length. after electrophoresis, pcr products were recovered using a pcr purification kit and sequenced on an ab3730 sequencer (life technologies, guangzhou, china). the sequences obtained from pcr products were assembled into a fulllength genome sequence using dnastar (version 7.0, dnastar inc., madison, wi, usa). [10] . rna was extracted from nasopharyngeal swab specimens collected on days 4, 5, 10, and 13 after onset of fever. reverse transcription of rna into cdna was performed as described previously. the cdna was used as the template for pcr amplification with la-taq mix (takara) and primer pair no. 22. pcr products were analyzed by 1% agarose gel electrophoresis. protein sequences were aligned using mega (version 6.0) [11] . transmembrane software was used to predict the transmembrane domain of the orf5 protein (http://www.cbs.dtu.dk/services/ tmhmm/) [12] . rna secondary structure was predicted using rnafold software, available at http://rna.tbi.uni vie.ac.at/cgi-bin/rnafold.cgi [13] . all products yielded usable sequences except those produced using primer pair no. 22 . two specific products obtained by nested pcr (fig. 1a) were purified, cloned and sequenced. the lower-molecular-weight band was composed of two variants that differed by 5 bp. variant 2 was longer than variant 1, with the sequence tatgg adjacent to the sequence ctcatgg). the upper band (wt) was 414 bp longer than variant 2 after the sequence ctcatggtatgg. all fragments of the sequences were assembled into three contigs of wt, variant 1 and variant 2. the genomic sequences have been uploaded to genbank as kt036372 [14] , kt036373 and kt036374, and the main differences in their nucleotide sequences are shown in fig. 1b . the predicted changes in the primary structures of the orf5 and e proteins are shown in (fig. 1c) . variant 2 encodes a fusion protein of the orf5 and e proteins (orf5-e) with an 81-amino-acid (aa) deletion at the c-terminus of orf5 and a 31-aa deletion at the n-terminus of the e protein. variant 1 encodes two truncated proteins: a 143-aa fragment of the n-terminus of orf5 with an additional 5 aa (fpygy), and a 52-aa fragment of the c-terminus of the e protein. until now, no such variant has been found in the ncbi database. although the function of the s protein has been examined previously [15] [16] [17] [18] [19] , our knowledge of orf5 and e protein functions in mers cov is limited [20] . moreover, the effects of orf5 and e protein mutations on viral packaging, infection and disease development have not been evaluated. based on studies of other coronaviruses, it is believed that the e protein is important for virus packaging and replication [20] [21] [22] . the conserved hydrophobic transmembrane n-terminal domain of the e protein is necessary for cov to be implanted in the membrane. even single point mutations in the transmembrane protein of the infectious bronchitis virus (ibv) e protein [23] , or amino acid changes in the n-terminus of the sars-cov e protein can result in attenuation of virulence [24] . to predict the function of the e protein of mers cov, we aligned the e and orf5-e protein sequences of mers cov with those of two other coronaviruses, sars-cov and china rattus coronavirus hku24, using mega software (version 6.0) [11] . the results showed that the e protein of mers cov shares high similarity with the other two coronavirus (45% for sars cov; 60% for hku24 cov) in the n-terminal, c-terminal and transmembrane domains (fig. 2a) . the truncated e protein with a deletion of aa 1-30 lacks the n-terminus and a major part of the hydrophobic transmembrane domain in mers cov variant 1, which might directly impair virus packaging and replication [24] . the putative fusion orff5-e protein (fig. 2b ) encoded by variant 2 is predicted to have three transmembrane regions (transmembrane hidden markov models [12] ), and it remains unclear whether it is able to function like the wildtype e protein. almazán et al. reported that mers cov with a deletion in the e gene produced replication-competent but propagation-defective virus particles and proposed that this defective virus should be a potential vaccine candidate for preventing mers cov infection [25] . the two variants identified in this study carried mutations in the n-terminal domain, which is dispensable for the function of the e protein. however, variations in this region lead to changes in the location of this protein, and therefore, the virulence of these two variants might be impaired to some extent. this needs to be investigated using a recombinant virus. the orf5 gene of both variants of mers cov in this study was truncated and fused with the e protein. the effect of these variations on the virus could not be predicted because the function of the orf5 gene is not well understood. however, scobey et al. found that the effect of orf5 deletions on the viral replication is minimal, but deletion of the whole orf5 gene significantly enhances s protein expression [26] . more investigations are required to determine the effects of the orf5 mutant in these two variants. intragenomic sequence deletions have been found in some coronavirus [27, 28] . it has been proposed that this occurs by a copy-choice or template-strand-switching mechanism [29] . one important condition is for there to be a specific leader sequence flanked by the deletion region and a stem-loop structure [30] . leader sequences corresponding to the ucuaaac sequence of murine hepatitis virus (mhv) or the cuuaaca sequence of infectious bronchitis virus (ibv) were not found in mers cov in this study. maori et al. have found that inverted repeats facilitate looping out of the middle genomic sequences during rna replication, resulted in a defective rna genome [31] . an rna secondary structure predicted using the rnafold webserver [13] suggested that the inverted repeat sequence contains long complementary sequences at each end and forms a strong stem-loop structure in the deletion region (fig. 2c) . the deleted sequence was closely adjoined, characterized by a 14-bp nearly complete inverted repeat sequence consisting of 27131-gtcata-cacaccaa-27144 and 27527-ttggtgtgtatggc-27540, which would result in rna replicase jumping from one segment to another distant segment. whether this feature is linked to rna intramolecular recombination remains to be investigated. wild-type mers cov and two variants were isolated for the first time from a patient who had traveled from korea to china. genomic sequencing revealed 414-bp and 419-bp deletions between orf5 and the e protein that would result in partial fusion or truncation of these proteins. whether this finding is a special case or not needs to be investigated by sequencing more samples. based on previous studies of e protein localization [23-25, 32, 33] , we conclude that the two variants might affect virus packaging, which could result in the attenuation of virulence and therefore be relevant for studies related to vaccine development, pathogenesis and viral evolution. 2012zx10004-213. conflict of interest the author of this study has no conflict of interest. ethical approval the mers patient was informed of an urgent clinical test. this study was approved by the medical ethics committee of southern medical university on may 28 and is in accordance with the 1964 helsinki declaration and its later amendments or comparable ethical standards. open access this article is distributed under the terms of the creative commons attribution 4.0 international license (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. an update on middle east respiratory syndrome: 2 years later severe respiratory illness caused by a novel coronavirus mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in bats, saudi arabia close relative of human middle east respiratory syndrome coronavirus in bat evidence for camel-tohuman transmission of mers coronavirus lack of middle east respiratory syndrome coronavirus transmission from infected camels the input sequence was based on kt036372, nt 26963-27610. the solid and broken lines indicate the inverted repeat sequence of nt 27131-gtcatacacaccaa-27144 and nt 27527-ttggtgtgtatggc-27540, respectively 8. zaki am transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study dnastar's lasergene sequence analysis software mega6: molecular evolutionary genetics analysis version 6.0 evaluation of methods for the prediction of membrane spanning regions memory efficient folding algorithms for circular rna secondary structures genomic sequencing and analysis of the first imported middle east respiratory syndrome coronavirus (mers cov) in china identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein mers coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis a single polar residue and distinct membrane topologies impact the function of the infectious bronchitis coronavirus e protein severe acute respiratory syndrome coronaviruses with mutations in the e protein are attenuated and promising vaccine candidates engineering a replication-competent, propagation-defective middle east respiratory syndrome coronavirus as a vaccine candidate reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus leader switching occurs during the rescue of defective rnas by heterologous strains of the coronavirus infectious bronchitis virus the ucuaaac promoter motif is not required for high-frequency leader recombination in bovine coronavirus defective interfering rna primary structure of poliovirus defective-interfering particle genomes and possible generation mechanisms of the particles regulation of coronavirus mrna transcription isolation and characterization of israeli acute paralysis virus, a dicistrovirus affecting honeybees in israel: evidence for diversity due to intra-and inter-species recombination subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein a coronavirus e protein is present in two distinct pools with different effects on assembly and the secretory pathway acknowledgements we thank dr. zhu hong (zhejiang university) for scholarly advice, and dr. xu jin (southern medical university) for language editing. this study was partially supported by grants from guangdong provincial science and technology (nos. 2010a040302003 and 2011b031800163), national natural science foundation of china (no. 31670168) and the 12th five-year-majorprojects of china's ministry of public health, grant no. key: cord-354272-99vw735a authors: darling, n. d.; poss, d. e.; schoelen, m. p.; metcalf-kelly, m.; hill, s. e.; harris, s. title: retrospective, epidemiological cluster analysis of the middle east respiratory syndrome coronavirus (mers-cov) epidemic using open source data date: 2017-10-24 journal: epidemiol infect doi: 10.1017/s0950268817002345 sha: doc_id: 354272 cord_uid: 99vw735a the middle east respiratory syndrome coronavirus (mers-cov) is caused by a novel coronavirus discovered in 2012. since then, 1806 cases, including 564 deaths, have been reported by the kingdom of saudi arabia (ksa) and affected countries as of 1 june 2016. previous literature attributed increases in mers-cov transmission to camel breeding season as camels are likely the reservoir for the virus. however, this literature review and subsequent analysis indicate a lack of seasonality. a retrospective, epidemiological cluster analysis was conducted to investigate increases in mers-cov transmission and reports of household and nosocomial clusters. cases were verified and associations between cases were substantiated through an extensive literature review and the armed forces health surveillance branch's tiered source classification system. a total of 51 clusters were identified, primarily nosocomial (80·4%) and most occurred in ksa (45·1%). clusters corresponded temporally with the majority of periods of greatest incidence, suggesting a strong correlation between nosocomial transmission and notable increases in cases. middle east respiratory syndrome coronavirus (mers-cov) is a respiratory illness caused by a novel coronavirus originally discovered in 2012. mers-cov can cause severe acute respiratory symptoms, including fever, cough, and shortness of breath, and is fatal in approximately one-third of reported cases. presently, there is no vaccine to prevent infection and no specific antiviral treatment for those infected with the virus [1] . mers-cov is the sixth strain of human coronavirus identified. although mers-cov cases were first reported from the kingdom of saudi arabia (ksa) in september 2012, the first two known cases were retrospectively discovered in jordan from april 2012 [2] . since its discovery in 2012, mers-cov cases have predominately been reported from ksa; however, cases have also been reported from algeria, austria, bahrain, china, egypt, france, germany, greece, iran, italy, jordan, kuwait, lebanon, malaysia, the netherlands, oman, philippines, qatar, republic of korea (rok), thailand, tunisia, turkey, united arab emirates (uae), united kingdom (uk), united states (usa), and yemen [2] . according to the armed forces health surveillance branch (afhsb), which is an organization under the defense health agency that utilizes biosurveillance to protect and promote the health of the us armed forces, as of 1 june 2016, 1806 cases of mers-cov have been reported, including at least 564 deaths [3] . afhsb's death count (case fatality proportion -31%) includes only those deaths which have been publicly reported and verified. the dynamics of the transmission of mers-cov is essential to understanding the risk posed by the virus as well as instituting effective infection control and prevention practices in areas where humans are at a greater risk of exposure. despite beliefs that camels are the most likely reservoir of the virus, the limited camel-to-human transmission has been reported by cdc and who [4, 5] . many published findings suggest that camel calves play a potential role in mers-cov transmission [6] [7] [8] . they found the 'rate of virus isolation [was] significantly higher in calves', and calves are often more acutely infected with mers-cov than adult camels, suggesting increased infectivity among calves [6] [7] [8] . although camel breeding season has been a proposed contributor to mers-cov transmission among camels, there is insufficient literature to support that human infections are more common during this time of year. human-to-human transmission of mers-cov has been investigated as a potentially significant route of spread. researchers have found that close contact with infected individuals is required to transmit the virus from one human to another, supporting the role of limited human-to-human transmission in the mers-cov epidemic and, more specifically, its role in nosocomial and household clusters [9] . while it is known that the virus can be transmitted through respiratory secretions, the exact routes through which the virus spreads are not well understood [9] . in an effort to better understand the patterns of transmission, a retrospective analysis of epidemiological clusters identified throughout the ongoing mers-cov epidemic was conducted using open-source data. epidemiological literature, classified by the tiered-source classification system (tscs), addressing mers-cov cluster analyses was collected and reviewed. several key search terms were utilized to capture all cluster-related literature, including 'mers-cov', 'nosocomial', 'cluster', 'transmission', 'superspreader', 'contact tracing', and 'healthcare worker'. see supplemental 1 for a comprehensive list of key search terms. publications selected for inclusion in the literature review were classified using tiers. this system was developed by afhsb to categorize the sources used in the literature review by their credibility. literature published by official sources, including the who and cdc, was considered a tier 1 source. literature published by reputable sources other than the who and cdc (e.g. all peer-reviewed journals regardless of perceived impact factor, including the lancet or nature) was considered a tier 2 source. literature from a foreign source, such as the ksa ministry of health (moh) or a media source, was classified as a tier 3 source. inclusion of a source required two or more of the key terms in supplemental 1. the literature included in the analysis encompassed translatable studies, situation reports from public health agencies, and publications updated to include more recent cluster information. studies related to mers-cov that were identified as having one or more of the following characteristics were excluded from the analysis: an investigational period prior to 2012; published in a non-translatable language; molecular-based; a focus on viral reservoirs, genealogy, or genetics, preventive measures, or other coronaviruses. in total, 80 studies were selected for inclusion. of these, 20 were classified as tier 1 sources, 22 as tier 2, and 38 as tier 3 (table 1 ). these sources were used to identify clusters as well as verify and characterize associations between cases. a mers-cov cluster was defined as two or more persons with onset of symptoms within a 14-day incubation period who are associated with a specific setting [10] . clusters were further categorized as exported, nosocomial, and/or household clusters. an exported cluster was defined as any cluster that resulted from verified travel of an index case (from an area of known mers-cov transmission) within one incubation period (14 days) of symptom onset. if the index case was asymptomatic, verified travel from an area of known mers-cov transmission within 14 days prior to the date of the index case was laboratory confirmed for mers-cov. a nosocomial cluster was defined as a cluster associated with a healthcare or hospital setting. a household cluster was defined as a cluster associated with the same family and/or physical household. case identification and data collection were performed on an ongoing basis by epidemiologists at afhsb beginning with the emergence of the mers-cov outbreak in 2012. case demographics, including city and country of origin, age, gender, date of symptom onset (if any), asymptomatic status, mortality, comorbidities, healthcare worker (hcw) status, and date reported, were collected on a daily basis. each case was verified using tiers 1 and 2 sources. if a tier 1 or 2 source failed to verify a case reported by a tier 3 source, it was not included in the afhsb case line list. if an epidemiological link between cases was identified through a tier 3 source, tiers 1 and 2 sources were used to verify the link. if a tier 1 or 2 source was not available, supporting data from at least three separate tier 3 sources were used as verification. due to the lack of data available at the local level in the arabian peninsula, in the event that multiple ongoing nosocomial outbreaks were known to have been occurring in one area, all cases reported to have been associated with that area and possibly epidemiologically linked to one of the ongoing clusters were categorized under one cluster (e.g. riyadh, jeddah). for all exported mers-cov clusters, the city and country of origin were determined by the reported travel history of the index case. if travel history was only available in the country level, the capital city of the country of travel was used as the point of origin for the index case. for two identified clusters, the index case had to travel to multiple countries with a history of confirmed autochthonous mers-cov transmission. as a result, the country of most probable exposure, determined by the duration of stay in the country as well as active transmission reported in that country at the time of travel, was used as the point of origin. in order to visually display the overlap of clusters on the epidemiological curve, the start and end dates of each identified cluster were defined. the start date of each cluster was the date of symptom onset of the index case. in the absence of symptom-onset data, the 'report date', or the date a case was publically reported, was used instead. the end date of each cluster was determined by adding 14 days to the date of symptom onset or date of death of the last case identified in the cluster. the 14-day period is representative of the maximum incubation period of a mers-cov case, ensuring no additional cases could have been associated with a given cluster [10] . for asymptomatic cases, date of diagnosis was used in place of date of symptom onset. if the date of diagnosis was not publically available, date reported was used. april 2012 and june 2016, 817 (45·2%) cases were determined to have been associated with at least one of the 51 clusters identified in this analysis [2, 11] . a small portion of cases associated with one or more clusters were hcws (n = 159), and 106 clusterassociated cases were asymptomatic ( from another mers-cov case, including 41 nosocomial infections and 13 household infections. as the ksa moh did not specify which mers-cov case these were acquired from, it is unclear if any of these batched cases were associated with the 51 identified mers-cov clusters or were part of separate clusters. of the 51 identified clusters, 41 (80·4%) were classified as nosocomial clusters; 12 (23·5%) were household clusters; and eight (15·7%) were exported (table 3 ). ten clusters were classified as more than one type of cluster, including four exported nosocomial clusters, three exported household clusters, and three clusters with both nosocomial and household characteristics. three clusters displayed both nosocomial and household transmission characteristics, four clusters were classified as both exported and nosocomial, and three clusters were classified as both exported and household. the two countries reporting the greatest number of nosocomial clusters were ksa and rok, with 18 and 15 nosocomial clusters, respectively. of the eight exported clusters, two (rok1, tunisia1) had index cases with travel to multiple countries with a history of confirmed autochthonous mers-cov transmission (see table 4 , technical appendix). the average duration of each cluster was 44·4 days, with a range of 14-119 days. cluster size ranged from two cases to 182 cases, with an average of 16 individuals affected. over 90% of the cluster-associated cases were acquired in ksa (n = 558, 68·3%) and rok (n = 186, 22·8%), supporting the notion that nosocomial transmission, which accounted for 100% of the clusters identified in rok and 78% of the clusters identified in ksa, was a prominent driver of clusters in this epidemic. figure 1 depicts the epidemiological curve of the mers-cov outbreak using the estimated epidemiological week of illness onset for each case. symptom-onset date was available for 1267 cases (70·2%), including 623 (72·6%) of the clusterassociated cases. for asymptomatic cluster-associated cases, date reported was used for 98 of the cases, and date of diagnosis was used for eight cases. the cluster bands seen above the epidemiological curve in figure 1 illustrate the duration of transmission within each cluster and the corresponding overlap of identified clusters with peak mers-cov incidence over the epidemic period of this study. most temporal peaks correspond with at least two ongoing clusters (see fig. 1 ). the time periods of greatest incidence (>35 incident cases at the period's peak) in 2014 and 2015 correspond with at least four ongoing mers-cov clusters, of which at least two were classified nosocomial in each of these periods. see table 4 (technical appendix) for individual cluster data, such as cluster duration and a number of cases affected. the 51 clusters identified in this analysis spanned the duration of the epidemiological curve, demonstrating the consistency of cluster-driven transmission throughout the epidemic. clusters also corresponded with each period of increased mers-cov incidence and accounted for over 45% of the total confirmed cases reported, supporting the notion that human-tohuman transmission is a prominent driver of the mers-cov epidemic. the alignment of nosocomial clusters with the time periods of greatest incidence (>35 incident cases at the period's peak) suggests nosocomial transmission is a key driver of transmission in the mers-cov epidemic as opposed to seasonal events, such as the hajj or camel-breeding season that occur in the fall and spring, respectively. the absence of apparent seasonality in the identified clusters further supports the significant role humanto-human transmission in healthcare settings plays in the propagation of mers-cov. classification of each cluster into one or more of the three disease transmission categories (nosocomial, household, or exported) elucidated common characteristics among the clusters, including the overall concentration of case clusters in cities and healthcare institutions. with nosocomial transmission accounting for 41 of the identified clusters (80·4%), the role of healthcare facilities, transportation protocols during inter-and intra-hospital transfers, and the contribution of cultural norms such as 'doctor shopping' became apparent themes observed throughout this analysis. in rok, practices such as seeking healthcare at multiple facilities, or 'doctor shopping', and being cared for by relatives while hospitalized are believed to have contributed to the emergence of the largest outbreak outside of ksa during the mers-cov epidemic [12, 13] . additionally, four superspreaders, defined as a confirmed mers-cov case responsible for infecting 10 or more secondary cases, played a critical role in the perpetuation of transmission in rok. the mers-cov outbreak in rok included 15 clusters at separate healthcare institutions, each with an index case related to an ongoing mers-cov cluster at another facility. between may 2015 and july 2015, 186 laboratory-confirmed mers-cov cases were reported. the geographic distribution and rapid pace at which mers-cov spread in rok demonstrates the susceptibility of healthcare environments to human-to-human transmission of the virus and their catalytic role in mers-cov transmission. in ksa, clusters were primarily concentrated in cities, specifically active transmission at healthcare facilities within those cities, as illustrated by the largest identified clusters in the country (ksa8, ksa9, ksa17, ksa20, and ksa23; see table 4 , technical appendix). the most common theme in the perpetuation of transmission among nosocomial clusters was the role of more transient departments in the healthcare setting, such as dialysis units and emergency departments, which were implicated in a majority of the nosocomial clusters identified in this analysis. at least seven of the 23 clusters identified in ksa occurred in designated mers-cov treatment centers, which were presumably best equipped to handle these infectious cases. however, the nosocomial transmission that persisted in these clusters suggests inconsistent infection control practices across different departments of those designated hospitals [14] . hcws likely contributed to the continued transmission among patients and between wards within a hospital, as hcws represent 159 (19·5%) of the clusterassociated cases. of the 41 clusters classified as nosocomial, 30 clusters (73·2%) involved hcws. the use of tier 1 data collected as the mers-cov outbreak progressed is one of the greatest strengths of this comprehensive cluster analysis. collection of the data and emerging literature in real time allowed for an inclusive literature review from which cluster identification and evaluation of key components of the outbreak could be analyzed. the availability of opensource data and information provided the opportunity to precisely map each confirmed case temporally and geographically. utilization of the date of symptom onset for the epidemiological curve created an accurate representation of the progression of the epidemic and corresponding cluster durations (fig. 1) . the availability of open-source data from certain regions also served as a limitation in this analysis. due to limited demographic information, it was occasionally necessary to broaden the case criteria for a particular cluster, specifically clusters ksa8, ksa9, and ksa20 (see table 4 , technical appendix). if a case was reported from the city during the estimated time in which there was ongoing nosocomial transmission, had no travel or camel exposure in the 14 days prior to illness onset, and had no known household contact with a confirmed mers-cov case, the case was included in the case count for that particular nosocomial cluster. although the particular epidemiological details regarding the exact location of exposure were generally not provided for cases during the three aforementioned clusters, tier 1 sources often denoted if a case was under investigation for a possible link to a hospital with the known ongoing transmission of mers-cov, which reinforced the inclusion of these cases in their respective clusters. in addition to limitations on precise case inclusion in the larger clusters denoted above, availability of date of symptom onset may have created an artifact in the cluster date and duration, potentially altering its appearance on temporal scales. of the known symptomatic cases, illness onset data were missing for 334 cases, including 96 cluster-associated cases, and date reported was used as an approximation. on average, afhsb observed an 8-9-day lag from the date of symptom onset to the date a case was reported. considering our analysis aggregated these cases by estimated epidemiological week of illness onset, the impact of this possible artifact is likely to be relatively insignificant to the overall trends observed in this analysis. additionally, ksa retrospectively released information on 113 confirmed mers-cov cases on 3 june 2014 and on 16 cases on 19 september 2014 with minimal geographic and demographic data. all cases released on 3 june 2014 occurred between 5 may 2013 and 6 may 2014, with a majority of the cases (n = 84) occurring after 1 march 2014. the rest of the cases (n = 29) occurred between 5 may 2013 and 28 february 2014. as no other date was available, the date reported was used to represent the cases released in these two batches on the epidemiological curve. asymptomatic cases accounted for 11·4% (n = 205) of the total cases in this study population. over 50% (n = 106) of these asymptomatic cases were related to a cluster. this finding may support who's assessment that contact tracing efforts intensified as the epidemic progressed and are responsible for the detection of asymptomatic cases [15, 16] . a study performed by cdc analyzing case data between september 2012 and january 2016 found that there was likely an underrepresentation of asymptomatic cases reported from the countries of the arabian peninsula. estimations suggest that the total number of mers-cov cases from the region may be 2·3 times greater than the total number of cases recorded to date [17] . inconsistencies in reporting of asymptomatic cases from entities such as the ksa moh may have contributed to an underrepresentation of not only the total number of mers-cov cases in the outbreak but also the number of clusters, as well as the breadth and duration of the identified mers-cov clusters. the supplementary material for this article can be found at https://doi.org/10.1017/s0950268817002345. middle east respiratory syndrome (mers): prevention & treatment centers for disease control and prevention military-health-topics/ health-readiness/armed-forces-health-surveillance-branch/integrated-biosurveillance/surveillance-summaries) world health organization. a roadmap for research and product development against middle east respiratory syndrome-coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir dromedary camels and middle east respiratory syndrome: mers coronavirus in the 'ship of the desert' co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia acute middle east respiratory syndrome coronavirus infection in livestock dromedaries guidance for monitoring and movement of persons with potential middle east respiratory syndrome coronavirus (mers-cov) exposure surveillance for human infection with middle east respiratory syndrome coronavirus (mers-cov): interim guidance novel coronavirus infection update middle east respiratory syndrome (mers) in the republic of korea middle east respiratory syndrome coronavirus outbreak in the republic of korea infection prevention and control guidelines for the middle east respiratory syndrome coronavirus (mers-cov) infection moh's command and control center forms rapid response teams including 120 health specialists middle east respiratory syndrome coronavirus (mers-cov) global summary and risk assessment accessed 23 estimation of severe middle east respiratory syndrome cases in the middle east the authors acknowledge the support provided by afhsb's us government partners, including the centers for disease control and prevention and the united states forces korea. this study received no specific grant from any funding agency, commercial, or not-for-profit sectors. none. key: cord-356007-6b0w36l9 authors: alanazi, khalid h.; killerby, marie e.; biggs, holly m.; abedi, glen r.; jokhdar, hani; alsharef, ali a.; mohammed, mutaz; abdalla, osman; almari, aref; bereagesh, samar; tawfik, sameh; alresheedi, husain; alhakeem, raafat f.; hakawi, ahmed; alfalah, haitham; amer, hala; thornburg, natalie j.; tamin, azaibi; trivedi, suvang; tong, suxiang; lu, xiaoyan; queen, krista; li, yan; sakthivel, senthilkumar k.; tao, ying; zhang, jing; paden, clinton r.; al-abdely, hail m.; assiri, abdullah m.; gerber, susan i.; watson, john t. title: scope and extent of healthcare-associated middle east respiratory syndrome coronavirus transmission during two contemporaneous outbreaks in riyadh, saudi arabia, 2017 date: 2018-12-31 journal: infect control hosp epidemiol doi: 10.1017/ice.2018.290 sha: doc_id: 356007 cord_uid: 6b0w36l9 objective: to investigate a middle east respiratory syndrome coronavirus (mers-cov) outbreak event involving multiple healthcare facilities in riyadh, saudi arabia; to characterize transmission; and to explore infection control implications. design: outbreak investigation. setting: cases presented in 4 healthcare facilities in riyadh, saudi arabia: a tertiary-care hospital, a specialty pulmonary hospital, an outpatient clinic, and an outpatient dialysis unit. methods: contact tracing and testing were performed following reports of cases at 2 hospitals. laboratory results were confirmed by real-time reverse transcription polymerase chain reaction (rrt-pcr) and/or genome sequencing. we assessed exposures and determined seropositivity among available healthcare personnel (hcp) cases and hcp contacts of cases. results: in total, 48 cases were identified, involving patients, hcp, and family members across 2 hospitals, an outpatient clinic, and a dialysis clinic. at each hospital, transmission was linked to a unique index case. moreover, 4 cases were associated with superspreading events (any interaction where a case patient transmitted to ≥5 subsequent case patients). all 4 of these patients were severely ill, were initially not recognized as mers-cov cases, and subsequently died. genomic sequences clustered separately, suggesting 2 distinct outbreaks. overall, 4 (24%) of 17 hcp cases and 3 (3%) of 114 hcp contacts of cases were seropositive. conclusions: we describe 2 distinct healthcare-associated outbreaks, each initiated by a unique index case and characterized by multiple superspreading events. delays in recognition and in subsequent implementation of control measures contributed to secondary transmission. prompt contact tracing, repeated testing, hcp furloughing, and implementation of recommended transmission-based precautions for suspected cases ultimately halted transmission. facilitate early recognition of suspect cases, followed by implementation of appropriate infection prevention and control (ipc) measures. 4 mers-cov may be transmitted by close contact that likely includes respiratory droplets, but transmission routes are still not fully understood. 7 both the saudi arabia ministry of health (moh) and the world health organization (who) recommend mers-cov-specific precautions for healthcare settings to reduce the risk of healthcare-associated transmission. 8, 9 from may 28 through june 19, 2017, 2 hospitals in riyadh, saudi arabia, reported mers cov outbreaks; epidemiologic links between the 2 hospitals were not apparent, and the extent of circulation was unknown. the outbreaks were initially reported by who in july 2017. 10 the moh and us centers for disease control and prevention (cdc) conducted an investigation to describe the scope of healthcare-associated transmission using epidemiologic, molecular, and serologic methods. the investigation was conducted at 4 healthcare facilities in riyadh, saudi arabia, where cases presented: (1) hospital a, a 1,200-bed tertiary-care moh hospital with 140 intensive-care unit (icu) beds and a busy emergency department (ed); (2) hospital b, a 200-bed moh specialty pulmonary hospital; (3) clinic c, an outpatient clinic; and (4) an outpatient dialysis unit. we defined a case as any patient with laboratory-confirmed mers-cov infection and an epidemiologic connection to the affected healthcare facilities as a patient, hcp, visitor or family member of a patient from may 28 through june 19, 2017. laboratory confirmation was performed either at the moh using a rrt-pcr assay targeting both the region upstream of the e gene (upe) and open reading frame (orf) 1a 11, 12 or at the cdc by genome sequencing. an indeterminate rrt-pcr result was defined as positive result on only 1 of the 2 gene targets required for confirmation. we defined a superspreading event as any interaction in which a mers case transmitted to ≥5 subsequent cases. patients with symptoms consistent with mers and contacts exposed to identified cases were tested. contact investigations were performed by hospital infection control personnel, local public health authorities, and moh personnel. moh recommends mers-cov testing of hcp identified with prolonged, close contact with a mers case (ie, >10 minutes within 1.5 m) if not properly wearing personal protective equipment (ppe). 13 in addition to recommended testing, ad hoc testing of hcp contacts with various levels of exposure and ppe use occurred. we reviewed available medical and public health records for all cases and conducted key-informant interviews with hcp. we collected sera and interviewed available hcp cases and hcp identified as rrt-pcr-negative contacts. interview forms included questions related to demographics, occupation, exposures, ppe use, symptoms, and underlying medical conditions. available mers-cov rrt-pcr-positive samples from confirmed cases collected from may 28 through june 19, 2017, were stored at −80°c and shipped to the cdc for further molecular analysis. sample aliquots (200 µl) were extracted on a nuclisens easy-mag (biomerieux, marcy-l'étoile, france), and 100 µl of total nucleic acid was recovered. the specimen extracts were retested by mers-cov n2 and/or n3 real-time rrt-pcr assays, 14 and genome sequencing was performed on positive samples with sufficient viral load using the previously described primer sets and protocol. 15, 16 the nucleotide sequences were first aligned in mafft version 7.013 multiple-sequence alignment software. phylogenetic trees were inferred using the maximum likelihood (ml) method with phyml version 3.0 software, 17 assuming a general time-reversible (gtr) model with a discrete γ-distributed rate variation among sites (γ 4 ) and an spr tree-swapping algorithm and visualized using mega version 6 software. 18 serology serum samples were tested at the cdc for anti-mers-cov antibodies using indirect elisas for nucleocapsid (n) and spike (s) proteins followed by a confirmatory microneutralization test (mnt) as previously described. 19 at the optical density cutoffs used by our laboratory, the n elisa has a sensitivity of 88.9% and a specificity of 92.2%, and the s elisa has a sensitivity of 90.8% and a specificity of 90.8% (unpublished data). mers-cov seropositivity was defined as having 2 of 3 positive assays, including n-elisa, s-elisa, and mnt, or positive by mnt alone. indeterminate seropositivity was defined as s elisa positive, but n elisa and mnt-negative. this investigation was determined by moh and cdc to be public health response, not research, and therefore was not subject to institutional review board (irb) review. signed consent was obtained from seroepidemiologic investigation participants. interviews were conducted in arabic, english, filipino, or malayalam. we identified 48 mers cases, including 38 linked to hospital a and 10 linked to hospital b ( fig. 1 and table 1 ). at both hospitals, transmission was traced to a single introduction by the respective index cases (fig. 2) . index patient a presented at hospital a on may 28, and index patient b presented at hospital b on june 2. no epidemiologic link was established between these cases. respiratory specimens from 36 mers-cov cases were received by the cdc: 35 were confirmed positive by rrt-pcr and 1 positive specimen could not be confirmed by mers-cov n2 and/or n3 rrt-pcr assays but was confirmed by sequencing the spike gene. phylogenetic analysis of 95 mers-cov genomes, including 21 complete or nearly complete genomes in this study, showed clustering of the outbreak sequences in lineage 5 within clade b 15, 20 (fig. 4) . the outbreak sequences from each hospital formed a monophyletic group and separated into 2 distinct clusters, suggesting 2 distinct outbreaks. the hospital a cluster appears to have been closely related to camel mers-cov (kt368879) and human mers-cov (mg011358) sampled at riyadh in 2015 and 2016 respectively. the hospital b cluster appears to have been more age, y, median (range) 58 ( among 38 cases linked to hospital a, 17 were patient cases, 17 were hcp cases, and 4 were family members (table 1) . index patient a was a 46-year-old factory worker with no history of contact with camels or camel products. he presented to the ed on may 28 with cough, shortness of breath, and chest pain. although er triage was in place, the patient was not initially considered a suspected mers case, and he remained in the ed for >14 hours prior to transfer to a medical ward (ward a). index patient a was directly linked to 19 subsequent cases: 1 ambulance driver exposed in the ambulance, 13 likely exposed in the ed, and 5 on ward a, where index a was intubated without airborne precautions in place. on hospital day 3, index patient a was suspected of mers and was transferred from ward a to a negative-pressure room with recommended isolation precautions. index patient a was confirmed rrt-pcr positive for mers-cov on may 31, and contact tracing began the same day. all secondary transmission at hospital a likely occurred before suspicion of mers in individual cases and the subsequent implementation of recommended transmission-based precautions. may june 26 27 28 29 30 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 in addition, 2 secondary cases, cases 5 and 6, overlapped with index patient a in the ed and were themselves associated with subsequent superspreading events (table 2 ). initially, case 6 was not identified as a contact of index patient a and was suspected to have had community exposure. however, later medical record review demonstrated overlap with index patient a during his initial ed visit for non-mers illness on may 28. he subsequently visited an outpatient dialysis unit, followed by a second ed presentation with admission to hospital a on june 1. case 6 was directly linked to 6 secondary cases: 1 patient at hospital a, 2 patients and 1 cleaner at the outpatient dialysis unit, and 2 household contacts. molecular evidence showed that case 6 clustered with index a and other subsequent cases at hospital a. case 5 was a known contact of index patient a in the ed, where he stayed for 2 days before transfer to a medical ward (ward b). he remained on ward b for 3 days, where he developed respiratory distress and was intubated on june 1. on june 2, mers was suspected, airborne precautions were implemented, and a sample was obtained for testing. mers-cov was confirmed on june 3, and the patient died the same day. case 5 was linked to 10 subsequent cases on ward b, including 6 hcp (fig. 2) , 4 of whom were present during the intubation procedure on case 5. of 17 hcp cases linked to hospital a, 10 were available for interview and serum collection. all 10 interviewed hcp cases reported ≥1 symptom when tested for mers-cov, with most hcp cases reporting mild upper-respiratory symptoms and/or diarrhea; none developed severe illness, and all survived. of these 10 available hcp, 9 reported prolonged, close contact with an unrecognized patient case before implementation of mers-cov ipc measures and with limited ppe use ( table 3 ). the remaining hcp case cared for a non-mers patient in the same room as a mers patient case. of these 10 hcp cases, 4 reported having been in the same room as a patient case during intubation, and none reported wearing an n95 mask or a powered air purifying respirator (papr). among the 10 interviewed hcp cases, the time from first positive mers-cov result to serum collection was 55-61 days, and 1 was seropositive: a 32-year-old female who had reported headache, muscle aches, and productive cough. additionally, we interviewed and collected serum from 66 hcp contacts of cases; none were seropositive. among all 15 hcp cases identified at hospital a and the ambulance driver, 8 tested positive on their first rrt-pcr test, and among these 8, the median time from likely exposure to positive sample collection was 5.5 days (range, 3-11 days). the 8 hcp cases who did not test positive on their initial test, tested positive on a second or later test, with a median time from likely exposure to first positive sample collection of 8 days (range, 5-12) (fig. 3) . ten cases were identified at hospital b; index patient b and 9 hcp cases who reported direct contact with him. index patient b was a 23-year-old butcher who slaughtered camels and contacted camel products. on may 28, he developed fever, cough, and rhinorrhea and presented to clinic c. he was discharged home but returned to clinic c 3 times over 4 days with worsening respiratory symptoms. on june 1, he was diagnosed with pneumonia and cardiomegaly and was referred to hospital b, where he presented to the ed on june 2. he was not initially suspected to have mers; however, a chest radiograph revealed bilateral infiltrates and additional history indicated camel contact. he was then placed on isolation precautions, and specimens were collected for mers-cov testing. he was intubated later that day after ipc measures for mers-cov had been implemented, including transfer to a negative pressure room. he died on june 3. at clinic c, index patient b had 15 hcp contacts, including 2 with close, prolonged contact; no rrt-pcr confirmed hcp-cases were documented at clinic c. of 15 hcp contacts of index b, 14 (93%) were interviewed and had serum collected. among these, 2 hcp were seropositive; both were physicians with initial indeterminate rrt-pcr test results. subsequent rrt-pcr testing was negative, and neither was recorded as a mers case. both cared for index b during multiple clinic visits and reported being within 1.5 m of index patient b for <10 minutes. one reported no ppe use, and the other reported wearing gloves and a surgical mask. both had diabetes, and 1 reported hypertension and smoking. both developed symptoms within 4-10 days of caring for index b, including fever. at hospital b, 27 healthcare contacts of index patient b were identified. among them, 9 hcp (33%) tested rrt-pcr positive for mers-cov; 5 reported contact with index patient b before isolation, and 4 reported contact following implementation of ipc measures for mers-cov in a negative-pressure room. of these latter 4, 3 participated in aerosolizing procedures, including intubation, open suctioning of airways, and/or cardiopulmonary resuscitation. all 3 reported wearing full ppe, including gloves, gown, n95 mask, and face shield. also, of these 4 hcp, 2 reported visible contamination of gloves or gown by bodily fluids during care of index patient b, who was reported to have had copious respiratory secretions. no transmission to patients or visitors at hospital b was identified. of the 9 hcp cases, 7 were interviewed and had serum collected; all 7 reported close, prolonged contact with index patient b. time from symptom onset to serum collection was 39-47 days. among these 7 hcp, 3 were seropositive, and 2 had an indeterminate result. among the 3 seropositive hcps, 2 had been diagnosed with pneumonia, 1 of whom also had diabetes mellitus. the third reported productive cough, dyspnea, and diabetes mellitus. among the 2 with an indeterminate result, 1 reported rhinorrhea and nonproductive cough, and the other had fever and upper respiratory tract and gastrointestinal symptoms; neither had comorbidities. the 2 seronegative hcp-cases reported mild upper-respiratory-tract symptoms; 1 also had fever and gastrointestinal symptoms. all 9 survived, and none were critically ill. at hospital b, 34 of 50 mers-cov rrt-pcr-negative hcp contacts of cases (68%) were interviewed and provided serum. one was seropositive, a physician who had close, prolonged contact with index b after isolation and while wearing recommended ppe; however, he had previously tested rrt-pcr positive for mers-cov in 2013. of 9 hcp cases identified at hospital b, 2 tested positive by rrt-pcr on their first test, 5 tested negative then subsequently tested positive, and 2 had an initial indeterminate rrt-pcr test result (fig. 3) . one hcp case with an initial indeterminate result was subsequently confirmed by rrt-pcr, the other was confirmed by genome sequencing. for the 8 hcp cases with a positive rrt-pcr test, the median time from known exposure to positive sample collection was 6.5 days (range, 2-10 days). a large mers-cov transmission event occurred in riyadh during may-june 2017, with cases initially reported from 2 hospitals. our molecular and epidemiologic investigation demonstrated separate virus introductions at the 2 facilities, each by a single index case. similar to previous outbreaks, 3, 21, 22 transmission was characterized by early superspreading events, which led to a rapidly escalating number of cases. during these 2 outbreaks, delays in the recognition and isolation of early cases, along with emergency intubation (sometimes precluding recommended airborne precautions), were associated with superspreading events. cases linked to superspreading events included 2 index cases and 2 secondarily infected hospitalized cases; all had severe illness, low cycle threshold values suggesting high viral loads, and all 4 died. these results are consistent with prior evidence that length of patient hospitalization before isolation and high viral loads have been linked to transmission. 23 although 2 of the cases associated with superspreading events were contacts of index a, they were not detected via contract tracing before developing symptoms and were associated with additional healthcareassociated transmission. the delay in recognition of index patient a due to the patient's comorbidities and complex presentation has been previously described. 24 this case patient was admitted to the ed without respiratory precautions despite initial triage, highlighting the need for strengthening triage practices. the presentation of index patient b before hospitalization is notable; this 23-year-old male had no known comorbidities and initially presented to clinic c with a mild illness, followed by further visits with worsening respiratory symptoms. furthermore, 2 physicians at clinic c tested seropositive after an indeterminate rrt-pcr test, suggesting transmission at clinic c. thus, increased testing for mers-cov in an outpatient setting for individuals with known risk factors and worsening respiratory symptoms might facilitate early recognition of mers cases. transmission from patient cases to hcp participating in aerosolizing procedures prior to airborne precautions was likely, despite taking recommended airborne precautions and wearing appropriate ppe. mers-cov has been detected in large quantities in respiratory secretions 25 and live virus isolated from environmental surfaces. 7 it is possible that inappropriate use of ppe (eg, insufficient fit testing) or contamination of ppe and inappropriate doffing resulted in transmission. transmission to hcp wearing isolation gowns and n95 respirators during intubation has been observed previously. 26, 27 hcp should ensure appropriate fit testing and donning and doffing of ppe to prevent mers-cov transmission. among the 17 hcp cases tested by serology, 11 (65%) had no detectable antibodies. the 4 seropositive hcp cases (24%) each had either evidence of pneumonia or symptoms suggestive of lower respiratory tract infection, consistent with previous evidence that hcp cases with lower-respiratory-tract symptoms are more likely to have detectable antibodies. 28 the use of serologic testing to detect unrecognized infections in asymptomatic or mildly symptomatic individuals may be limited. 29 in both outbreaks, rapid identification of contacts, symptom monitoring, and repeated testing allowed for efficient detection of secondary hcp cases and provided information to guide outbreak management. of the 25 hcp-cases, 10 were detected on initial rrt-pcr testing and 15 by repeated rrt-pcr testing, including multiple hcp cases who initially tested rrt-pcrnegative up to 7 days after known case exposure, indicating that asymptomatic or mildly symptomatic hcp may require repeated screening to rule out infection. although we found no evidence of transmission from hcp to hcp, rapid furlough of mers-cov positive hcp is important to avoid exposing susceptible individuals, particularly patients, to mers-cov positive hcp. our investigation had several limitations. complete medical records were not available for all patients. seropositivity may have been a result of unknown exposures outside of this outbreak. although hospitalized patients have been shown to develop mers-cov antibody responses after 3 weeks, 30 mers-cov antibody kinetics over time are not fully understood, particularly in asymptomatic or mildly ill individuals. genome sequencing was limited by sample quality, and full-genome sequences were not available from all patient samples. hcp ppe use was assessed via interview, so errors in recollection may have been incorporated into our data. due to the retrospective nature of this investigation, ipc practices during potential transmission events could not be confirmed by observation. the introduction of mers-cov into healthcare facilities continues to occur, resulting in substantial morbidity and mortality. in these 2 contemporaneous but epidemiologically unrelated outbreaks, superspreading events were associated with extensive transmission and disruptions to hospital operations, including large-scale furloughing of exposed hcp. early recognition of cases, rapid implementation of recommended ipc measures, and aggressive contact tracing and repeated testing are necessary to effectively prevent and interrupt transmission of mers-cov. (fig. 4) kt368879 camel/riyadh/ry86/2015 saudi arabia kt368835 camel/jeddah/d42/2014 saudi arabia kx108946 d1189 camel united arab emirates isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome hospital outbreak of middle east respiratory syndrome coronavirus description of a hospital outbreak of middle east respiratory syndrome in a large tertiary-care hospital in saudi arabia mers-cov outbreak in jeddah-a link to health care facilities multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea infection prevention and control guidelines for the middle east respiratory syndrome coronavirus (mers-cov) infection infection prevention and control during health care for probable or confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection. geneva: who world health organization. who mers-cov global summary and assessment of risk world health organization. laboratory testing for middle east respiratory syndrome coronavirus (mers-cov), interim guidance assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections saudi arabia ministry of health website real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus epidemiology of a novel recombinant mers-cov in humans in saudi arabia diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 mega6: molecular evolutionary genetics analysis version 6.0 inclusion of mers-spike protein elisa in algorithm to determine serologic evidence of mers-cov infection origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china: phylogenetics and coalescence analysis preliminary epidemiological assessment of mers-cov outbreak in south korea identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department risk factors for transmission of middle east respiratory syndrome coronavirus infection during the 2015 outbreak in south korea unusual presentation of middle east respiratory syndrome coronavirus leading to a large outbreak in riyadh during 2017 viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea antibody response and disease severity in healthcare worker mers survivors multihospital outbreak of a middle east respiratory syndrome coronavirus deletion variant kinetics of serologic responses to mers coronavirus infection in humans acknowledgments. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention.financial support. no financial support was provided relevant to this article. supplementary material. to view supplementary material for this article, please visit https://doi.org /10.1017/ice.2018.290 key: cord-354536-c9v9kbw8 authors: han, yan-jie; ren, zhi-guang; li, xin-xin; yan, ji-liang; ma, chun-yan; wu, dong-dong; ji, xin-ying title: advances and challenges in the prevention and treatment of covid-19 date: 2020-07-09 journal: int j med sci doi: 10.7150/ijms.47836 sha: doc_id: 354536 cord_uid: c9v9kbw8 since the end of 2019, a new type of coronavirus pneumonia (covid-19) caused by the severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has been spreading rapidly throughout the world. previously, there were two outbreaks of severe coronavirus caused by different coronaviruses worldwide, namely severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). this article introduced the origin, virological characteristics and epidemiological overview of sars-cov-2, reviewed the currently known drugs that may prevent and treat coronavirus, explained the characteristics of the new coronavirus and provided novel information for the prevention and treatment of covid-19. the spread of covid-19, which first appeared in december 2019, is still increasing rapidly. 1, 2 at present, cases of covid-19 caused by sars-cov-2 have been found in many countries around the world. 3, 4 according to the latest data of beijing time, may 6, 2020, there were 3,729,072 confirmed cases and 258,622 deaths worldwide. on january 31, 2020, the covid-19 epidemic requires a coordinated international response, indicating that it may pose risks to multiple countries. 5 sars-cov-2 was isolated from the airway epithelial cells of patients with viral pneumonia in wuhan. 6 compared with known coronaviruses that can infect humans, the sars-cov-2 structure has certain differences, so it is defined as the seventh coronavirus. 7 sars-cov-2 belongs to the order nidovirales, coronaviridae. 8 it has an envelope and contains a very large rna virus genome. the 3d structure of the coronavirus, obtained by cryo-electron tomography, reveals that it is spherical and has an envelope. 9 coronavirus is characterized by spikes sticking out from the surface, which in part have the same characteristics as sars-cov, and other four human coronaviruses hcov-nl63 (human coronavirus nl63), hcov-229e (human coronavirus 229e), hcov-oc43 (human coronavirus oc43), and hcov-hku1 (human coronavirus hku1), which only cause mild respiratory diseases. 6 virus-cell receptor binding and the production of structurally complete virus particles. 11, 12 importantly, homology modeling shows that the sars-cov-2 binding domain to the ace2 receptor is structurally similar to sars-cov. 13, 14 despite the presence of amino acid mutations in its receptor binding domain, multiple key amino acids are changed. 13, 14 however, the amino acid perfectly maintains the stability of the mutual structural conformation of the virus s-protein and the ace2 receptor in a holistic manner. preliminary revealed virus uses ace2 receptor to enter the cell. 13, 14 so far, there are no specific drugs specifically used to prevent and treat coronavirus. if there are no vaccine and/or specific antiviral drugs during a virus outbreak, non-specific therapeutic interventions are often used to prevent acute complications and reduce severe morbidity and mortality, such as providing supportive care, including adequate rest, rehydration and analgesics. 15 at the same time, traditional chinese medicine, hormonal drugs, broad-spectrum antibiotics, antivirals, antifungals and interferon-α2b can be utilized to minimize the risk of co-infection. 16 in addition, there is no direct clinical evidence which has shown specific anti-sars-cov-2 efficacy, and further substantial research is needed to pay attention to related adverse reactions. ribavirin (ribavirin) is a synthetic nucleoside antiviral agent with broad-spectrum antiviral activity and inhibits both dna and rna viruses. 17 it is usually used in aerosolized form for adults and children in treatment of respiratory syncytial virus pneumonia. 18 early in vitro studies have shown that administration of ribavirin can enhance the in vitro activity of interferon. 16 ribavirin has anti-mers-cov activity in vitro when used alone or in combination with interferon alpha. 19 clinical studies have shown that ribavirin and regulated interferon alpha-2a treatment can significantly improve the 14-day survival rate and slightly improve the 28-day survival rate in patients with mers-cov infection. 16 the timing of initiation of antiviral therapy is critical to the treatment of most patients with viral infections. adults: 500 mg/time, intravenous infusion, 2 to 3 times a day, the course of treatment does not exceed 10 days. 20 however, the side effects of ribavirin limit its use to some extent. the use of high-dose ribavirin may be related to hemolytic anemia, neutropenia, teratogenicity, and cardiopulmonary distress. 18 in view of the curative effect of ribavirin in the treatment of diseases caused by sars-cov and mers-cov, 21 it is expected to become one of the effective drugs to treat coronavirus. redesivir (rdv, gs-5734), a nucleoside analogue, is a drug under investigation, it has not been approved for marketing in any country yet. 22 it can exert therapeutic effects by inhibiting the synthesis of viral nucleic acids and has antiviral activity. 23 gilead sciences, inc. believes that antiviral nucleic acid analogs, such as ribavirin, will be cut out by the coronavirus exonuclease exon when integrated into viral rna during the treatment of coronavirus infection, but rdv is resistant to exon. the resistance results in rdv treatment of coronavirus are more effective than other nucleic acid drugs. previously, rdv was mainly used as a test drug against ebola virus, and it has a strong anti-filovirus efficacy in vitro. 24 in vitro tests, rdv can effectively inhibit the activity of sars-cov and mers-cov. 23 for both mers-cov and sars-cov, its half effective concentration (ec50) is 0.07 μmol/l. in contrast, lopinavir-ritonavir ec50 values are respectively 8 μmol/l and 17 μmol/l. 25 however, as an effective potential drug for sars-cov-2, rdv takes an emergency approach after weighing the risks and benefits. on february 3, 2020, beijing china-japan friendship hospital led two independent random, double-blind, controlled clinical trials, one for patients with new-type coronavirus mild-to-moderate pneumonia in hospitalized adults (308 cases), and one for patients with severe coronavirus-infected adults (453 cases), to verify the efficacy and safety of ribavirin. the experiments are currently undergoing. lopinavir and ritonavir (kaletra/aluvia) is the first-line drug for the clinical treatment of aids. 26, 27 developed by abbott, marketed in 2005, mainly combined with viral protease to inhibit protease function. lopinavir-ritonavir is a compound tablet consisting of lopinavir and ritonavir. lopinavir is a sensitive substrate for cytochromes cyp3a4 and p-glycoprotein. 26 it can block the division of gag-pol polyprotein and has a high protein binding rate in plasma. ritonavir is a substrate of cyp3a4, p-glycoprotein and cyp2d6, which inhibit hiv protease: enzymes cannot break down the precursor of gag-pol polyprotein. ritonavir can inhibit cyp3a-mediated lopinavir metabolism, resulting in higher lopinavir concentrations. 26 in vitro studies showed that lopinavir and ribavirin can inhibit the replication of mers-cov and sars-cov. 28 adults: 400 mg/100 mg each time, orally, bid, and the course of treatment does not exceed 10 days. 20 darunavir (prezista) is a second-generation hiv-1 protease inhibitor. it was first marketed in the united states in july 2006. it was developed by tibotec, a subsidiary of johnson & johnson. darunavir, ritonavir, ritonavir and the combination of other retroviral drugs can be used to treat hiv infection. 29 it can selectively inhibit the cleavage of hiv-encoded gag-pol polyprotein in virally infected cells, thereby inhibiting viral replication. 30 darunavir in particular patient population (including pregnant women, pediatrics, patients with hiv-2 infection and co-infection with viral hepatitis) is also safe and effective. 29 transmembrane protease serine 2 (tmprss2) inhibitors may be used to block sars-cov-2 infection and then used to treat covid-19. 31 ace2 is a metal peptidase, expressed on major viral target cells such as lung cells and intestinal epithelial cells, and its catalytic domain binds to the s protein of sars-cov with high affinity. 32 for viral infectivity, host cell proteases affect the s protein cleavage is crucial. tmprss2 can activate the spike protein of sars by lysing the spike protein on the cell surface, which in turn binds to ace2 and enters the host cell. 33 tmprss2 is expressed in ace2-positive cells in the human lung. 34 it is shown that tmprss2 may play an important role in the transmission of sars-cov in the human respiratory tract. 33 so far, multiple studies showed that sars-cov-2 is likely to bind to human ace2 receptors and thus invades the human body. 13, 14, 31 many sars-cov-2 prevention and treatment drugs are screened with the ace2 receptor as a target. in view of the important role of tmprss2 in influenza virus and coronavirus infections, tmprss2 inhibitors might be useful for clinical treatment. 35 favipiravir (avigan) is a broad-spectrum antiviral drug that selectively inhibits the rna polymerase of influenza viruses. 36 it was approved for marketing in japan in march 2014 and it is used for antiviral treatment of influenza a and b. 37 it is converted into an active phosphoribosylated form in the cell and recognized as a substrate by viral rna polymerase. 38 favipiravir is active against a variety of influenza viruses including a (h1n1) pdm09, a (h5n1), and a (h7n9) avian influenza viruses, and has a synergistic effect with oseltamivir. 37 in addition to its anti-influenza activity, favipiravir blocks the replication of many other rna viruses, including alphaviruses, flaviviruses (yellow fever and west nile virus), enteroviruses (poliomyelitis virus and rhinovirus), influenza a virus, respiratory syncytial virus and norovirus. 37, 39 a number of marketed drugs with antiviral activity, such as favipiravir, were discovered, and follow-up studies need further verification by animal experiments and clinical trials. (umifenovir) is a synthetic broad-spectrum antiviral compound developed by the ussr research center for medicinal chemistry, used to prevent and treat human influenza a and b influenza infections and post-influenza complications. 40 abidol was used as an anti-influenza virus drug for decades. 41 abidol is active against many dna/rna and enveloped/non-enveloped viruses and inhibits membrane fusion between virus particles and plasma membrane and between virus particles and endosome membrane by embedding in membrane lipids. 42 at present, darunavir and abidol are used in patients with pneumonia infected by a novel coronavirus in zhejiang province. 20 the follow-up treatment effect needs further verification. resveratrol is a natural compound found in grape seeds, peels and red wine. 43 44, 45 resveratrol has antiviral activity against respiratory viruses and certain anti-inflammatory effects, so it can be used as an adjuvant treatment for respiratory infections. 44 resveratrol has a wide range of antiviral effects by down-regulating inflammatory signal transduction, which is mainly associated with the inhibition of viral replication, viral protein synthesis, gene expression, and nucleic acid synthesis. [44] [45] [46] in vitro model evaluations show that resveratrol significantly inhibits mers-cov virus infection in a dose-dependent manner and prolongs the survival time of infected cells, which may be related to resveratrol's reduction of nucleocapsid (n) protein expression. 46 nucleocapsid (n) protein is a multifunctional protein essential for cov replication. 47 in addition, resveratrol down-regulates mers-cov-induced apoptosis in vitro. in the case of continuous administration, resveratrol can inhibit mers-cov at lower doses. 46 another study shows that resveratrol also has a significant inhibitory effect on the emerging positive sense rna chikungunya virus, indicating that resveratrol may become a candidate drug for the treatment of new rna viruses. 46 after a cell is infected by a virus, it can synthesize and secrete a substance that can interfere virus replication and enhance antiviral ability of nearby cells. 48 this substance is called interferon. human interferon is subdivided into type i and type ii. 49 the host's inherent interferon response is critical to control viral replication after infection. 50 interferon response can be enhanced by recombinant interferon or interferon inducer. 51 recombinant interferon α and interferon β can inhibit the replication of sars-cov and mers coronavirus in vitro and in animal models. 52 among all subtypes, interferon β1b has the best antiviral effect on mers-cov. 16, 53 various combinations of interferon alpha or interferon beta with other antivirals, such as ribavirin, lopinavir, and ritonavir were used to treat patients with sars and mers. in vivo and in vitro studies showed that the combination of ribavirin with ifnα may have a synergistic effect. 16 the "pneumonitis diagnosis and treatment scheme for new coronavirus infection (trial version 7)" states that aerosolized interferon alpha can be used as a trial treatment against sars-cov-2 virus to improve the virus clearance effect of respiratory mucosa in patients. 20 adults: 5 million iu or equivalent dose of interferon alpha each time, add 2 ml of sterile water for injection or normal saline, inhale by atomization, bid. 20 polyinosinic acid-polycytidylic acid is a synthetic analog of dsrna, which can strongly induce type i interferon. however, a recent study showed that the 4a protein of double-stranded rna-binding domain interacts with polyinosinic acid-polycytidylic acid, thereby inhibiting polyinosinic acid-polycytidylic acid or sendai virus-induced interferon production, 54 but experiments show that polycytidylic acid can still significantly reduce mers-cov load in balb/c mice. 51 nitroxanide (ntz) is another effective type i interferon inducer. it is currently used clinically to treat parasitic infections. it was originally marketed in mexico under the trade name daxon/colufas in 1996, then listed in the united states under the trade name alinia in 2002. it is a synthetic thiazolyl-salicylic acid derivative. in cell culture assays, nitrazine can inhibit the replication of a variety of rna and dna viruses, including respiratory syncytial virus, parainfluenza virus, and coronavirus. 51 nitroxanide has a broad-spectrum antiviral activity: rotavirus, norovirus, hbv, hcv, dengue virus, yellow fever, japanese encephalitis virus. 55 it has been evaluated in phase ii and phase iii clinical trials for the treatment of hcv infection and the treatment of influenza. 55, 56 cyclophilins (cyclophilins, cyps) belong to the peptidyl-prolyl isomerases (ppiase) family, which are involved in the replication of rna viruses, including human immunodeficiency virus 1, hepatitis c virus and influenza virus. 57 cyclophilin a is the main cellular target of the immunosuppressive drug cyclosporin a (csa). 57 csa is a classic immunosuppressive drug. it binds to the cell cyclophilin to inhibit calcineurin, thereby preventing the translocation of the nuclear factor of activated t cells from to the nucleus, interleukin2 and the transcription of genes. 58 csa can inhibit the replication of almost all species of coronavirus in a dose-dependent manner in vitro. 59 cell culture experiments indicated that csa strongly inhibited the replication of sars-cov, mers-cov, human coronavirus 229e, feline coronavirus, avian infectious bronchitis virus, and mouse hepatitis virus, but they showed a significant blocking effect only in the early stages of replication. 59 compared to other rna viruses (0.5-3 µm), a higher csa concentration (16 µm) is required to block coronavirus replication, which indicates that the coronavirus is less sensitive to csa treatment. 60 as an effective and broad-spectrum coronavirus inhibitor, csa and its analogs have good research and development prospects. 59 chloroquine phosphate is a widely used antimalarial and autoimmune disease drug and has recently been reported as a potential broad-spectrum antiviral drug. 61, 62 chloroquine phosphate can block virus infection by up-regulating the ph of endosomes, required low for virus-cell fusion, and inhibiting glycosylation of cellular receptors. it specifically interacts with sugar-modifying enzymes or glycosyltransferases in human cells. 61 it has been demonstrated that chloroquine phosphate may have an inhibitory effect on quinone reductase 2, which is structurally adjacent to udp-n-acetylglucosamine 2-epi-isomerase, thus affecting the biosynthesis of sialic acid. 61 the presence of a sialic acid moiety in the coronavirus receptor ace2 may explain the inhibitory effect of chloroquine phosphate on sars-cov replication and other functions. 61, 62 recent cellular experiments showed that chloroquine phosphate is very effective in controlling sars-cov-2 infection in vitro, and it plays a role in the entry phase and post-entry phase of sars-cov-2 infection in vero e6 cells. 56 in addition to its antiviral activity, chloroquine phosphate has immunomodulatory activity and can synergistically enhance its antiviral effect in vivo. 63 given the above characteristics of chloroquine phosphate, it is likely to be clinically applicable to sars-cov-2. chlorpromazine is an antipsychotic used to treat schizophrenia. 51 in 1952, chlorpromazine was marketed in france under the trade name largactil; in 1957, it was approved by the fda to be marketed under the trade name thorazine. after most viruses attach to host surface receptors, they enter cells using endocytosis mechanisms (catenin-dependent and independent pathways). sars uses clathrin-dependent mechanisms to enter host cells. 64 it has been revealed that chlorpromazine is a broad-spectrum virus inhibitor that can inhibit hcv, alpha virus, and various coronaviruses including human coronavirus 229e, sars-cov and mers-cov in vitro. 25 imatinib (gleevec) is a small molecule inhibitor. nobartis was first marketed in the united states in 2001. it is clinically used to treat chronic myeloid leukemia and malignant gastrointestinal stromal tumors. imatinib is specifically designed to target abelson tyrosine-protein kinase 2. abl2 kinase is a non-receptor tyrosine kinase that exists in the nucleus and mitochondria, and mediates a wide range from embryonic morphogenesis to viral infection cellular processes. 65 abl2 kinase is required for sars-cov and mers-cov to replicate in vitro. 66 as an in vitro inhibitor of sars-cov and mers-cov, imatinib can significantly reduce the titers and inhibit their development process. 66 imatinib's anti-coronavirus activity was mainly manifested in the early stages of infection and worked by inhibiting the fusion of viral particles on the endosomal membrane. 66 monoclonal antibodies (mab) were successfully used to treat various diseases. 67 passive immunotherapy using neutralizing monoclonal antibodies (mab) is an effective preventive and therapeutic agent for emerging viruses. 68 sars-cov and mers-cov have always been a global public health threat. to date, no vaccines or specific therapies have been approved for diseases caused by these two viruses. 68 neutralizing antibodies are a major component of protective immunity against human viral infections. in vivo antibody responses mobilize a mixture of dynamic mabs that work in concert to target various antigens on viral envelope glycoproteins. 69 neutralizing mabs can be achieved by a variety of techniques, such as hybridoma technology, humanized mice, phage or yeast, and single b cell isolation. 67 more and more mabs were developed, with high efficacy against emerging viruses in vitro and in infected animal models. 67 spike glycoproteins play a key role in mediating virus entry and have the capacity to induce protective antibody responses in infected individuals. consistently with it, spike proteins are the main targets of neutralizing antibodies. 70 regeneron et al. used velocimmune mice to generate fully human non-competitive monoclonal antibodies that bind to mers-cov spike protein, and the characterization of two lead monoclonal antibody candidates (regn 3051 and regn 3048) were performed in vitro and in vivo. 70 both mabs effectively neutralize mers-cov in vitro. moreover, in vivo studies in infected mouse models showed that treatment of infected mice can decrease lung virus titers. 70 currently, multiple mabs for sars-cov and mers-cov are undergoing the phase ⅰ clinical trial, which has promising prospects for the treatment of new viral diseases. based on the above studies, neutralizing antibodies to spike proteins on the surface of sars-cov-2 may be the first therapy considered by researchers. 71 recently, genbank has released the sars-cov-2 genomic sequence (mn908947.3). spike protein can be used as an immunogen to screen mice or rabbits for neutralizing antibodies. however, the development of this traditional method is slow. many time-saving methods have been developed, such as the rapid identification of viral neutralization lead candidate phages or yeast display libraries by expressing fragments of antibodies, and their application to the screening of neutralizing antibodies. 72 on march 4, 2020, a new virus fusion research team (cevi fusion research team) led by the korea chemical research institute found antibodies that can fight against sars-cov-2. the structure of the spike protein of the sars-cov-2 is very similar to that of middle respiratory syndrome (mers) and sars, and it was confirmed that the latter can be combined with its neutralizing antibody. 73 blood-derived immunotherapy based on recovered patients can be used to treat infections including measles virus, lhasa virus, sars coronavirus and influenza a h5n1 virus. 74 this method is also applicable to the treatment of sars-cov-2. rehabilitated patients with new coronavirus pneumonia will produce high-titer polyclonal antibody immune responses to different antigens of sars-cov-2. some of these polyclonal antibodies may neutralize the virus and prevent a new round of infection. 75 this can be achieved by donating plasma and whole blood into infected patients. 76 as of today there are more patients with new coronavirus pneumonia in rehabilitation, and plasma can be provided for the treatment of infections if necessary. on january 27, 2020, the state administration of traditional chinese medicine conducted clinical observations of the "qingfei paidu decoction" in shanxi, hebei, heilongjiang, and shaanxi provinces. by february 5, 214 confirmed cases have been treated. the total effective rate has reached more than 90%. on february 6, it began to be promoted and used in china. 77 the main drug composition of the prescription is "ephedra 9 g, roasted licorice 6 g, almond 9 g, raw gypsum 15 ~ 30 g (first fried), guizhi 9 g, alisma 9 g, polyporus 9 g, atractylodes 9 g, poria 15 g, bupleurum 16 g, scutellaria baicalensis 6 g, ginger pinellia 9 g, ginger 9 g, aster 9 g, winter flower 9 g, shegan 9 g, asarum 6 g, yam 12 g, citrus aurantium 6 g, tangerine peel 6 g, patchouli 9 g". 20 the prescription can be used to treat scov-infected pneumonia patients with light, normal, and severe types, and can also be used according to actual conditions in the treatment of critically ill patients. with the spread of the covid-19 epidemic, scientists around the world are actively exploring potentially effective drugs to fight against new coronaviruses, especially rdv for injection. after evaluating the risks and benefits, on february 6, 2020, active treatment was taken in the wuhan epidemic area, and a phase iii clinical trial was conducted, which brought dawn to the clinical treatment of covid-19. however, there are many difficulties in the development of coronavirus drugs, which restrict the development and application of drugs. firstly, coronaviruses are rna viruses with variability, and this is the reason why new types of coronaviruses with novel structures easily appear. the drugs used in the past may not be effective for this new type of coronavirus or have only weak effects. secondly, many drugs have high ec50/cmax ratios in the clinic and are prone to severe side effects. for example, the use of high-dose ribavirin may be related to hemolytic anemia, neutropenia, and cardiopulmonary distress. 78 in addition, virus research is highly risky, and general experimental conditions are difficult to meet biosafety requirements, so screening techniques, animal models, and suitable animal experimental platforms are limited. moreover, the severe epidemic caused by coronavirus has the characteristics of timeliness. there are also some difficulties in clinical trial resources and recruitment of patients with related diseases into clinical trials. although research of these coronaviruses are based on the results of genome sequencing and bioinformatics, some screening models such as sars and mers virus-infected cells, some small mechanisms based on different mechanisms of action such as nucleic acid synthesis inhibitors, protease inhibitors, and polymerase inhibitors have been established. since low specificity of research and exploration, there is no clinical evidence to show the efficacy of broad-spectrum antiviral drugs against coronavirus. further substantial research is needed and attention should be given to certain toxic side effects. 18 the most effective way to fight against the virus remains the vaccine. compared with the long cycle required for drug development, the vaccine takes a relatively short time and has a strong protective effect. monoclonal antibodies have better effects, stronger specificity, and higher safety, but they also require more time and cost to develop. compared to sars and mers, humans have made great progress in understanding the new coronavirus, the mechanism of drug action, research and development strategies, both in terms of knowledge and technology. in the waiting period for the development of new coronaviruses, china is now using multiple drugs to treat new coronaviruses. although there are many risks, it is undeniably the most effective approach at present. according to chinese anti-sars-cov-2 diagnosis and treatment program and treatment experience, a combination of multiple drugs is adopted, but it is not recommended to use 3 or more antiviral drugs (table 1) 20 . this will alleviate the symptoms of the patient and enhance the patient's immunity, thereby achieving the purpose of treating the disease. covid-19) outbreak a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster emerging understandings of 2019-ncov nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china_ a modelling study advances in covid-19: the virus, the pathogenesis, and evidence-based control and therapeutic strategies clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study a novel coronavirus from patients with pneumonia in china overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov stabilized coronavirus spikes are resistant to conformational changes induced by receptor recognition or proteolysis sars and mers: recent insights into emerging coronaviruses coronavirus envelope protein: current knowledge receptor recognition mechanisms of coronaviruses: a decade of structural studies genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin therapeutic options for middle east respiratory syndrome coronavirus (mers-cov) -possible lessons from a systematic review of sars-cov therapy ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study in vitro antiviral activity of ribavirin against severe fever with thrombocytopenia syndrome virus common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada diagnostic accuracy of the recognition of stroke in the emergency room (rosier) score and ct brain in an hiv population notice on the new coronavirus pneumonia diagnosis and treatment program observation on the curative effect of ribavirin and vidarabine monophosphate in the treatment on hand foot and mouth disease intravenous palivizumab and ribavirin combination for respiratory syncytial virus disease in high-risk pediatric patients coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease late ebola virus relapse causing meningoencephalitis: a case report screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture pharmacokinetics and drug-drug interactions of lopinavir-ritonavir administered with first-and second-line antituberculosis drugs in hiv-infected children treated for multidrug-resistant tuberculosis week 96 efficacy of lopinavir/ritonavir monotherapy in virologically suppressed patients with hiv: a randomized non-inferiority trial (anrs 140 dream) role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings darunavir stands up as preferred hiv protease inhibitor kinetics analysis of consecutive hiv proteolytic cleavage of the gag-pol polyprotein sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein efficient multiplication of human metapneumovirus in vero cells expressing the transmembrane serine protease tmprss2 tmprss2: a potential target for treatment of influenza virus and coronavirus infections a new antiviral drug-favipiravir favipiravir as a potential countermeasure against neglected and emerging rna viruses favipiravir (t-705), a novel viral rna polymerase inhibitor favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (rsv) the synthetic antiviral drug arbidol inhibits globally prevalent pathogenic viruses an antioxidant resveratrol significantly enhanced replication of hepatitis c virus in vitro antiviral activity of resveratrol against respiratory viruses antiviral activity of resveratrol effective inhibition of mers-cov infection by resveratrol the coronavirus nucleocapsid is a multifunctional protein fluvastatin interferes with hepatitis c virus replication via microtubule bundling and a doublecortin-like kinase-mediated mechanism antiviral actions of interferons pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses coronaviruses -drug discovery and therapeutic options ribavirin and interferon-β synergistically inhibit sars-associated coronavirus replication in animal and human cell lines severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response nitazoxanide: a first-in-class broad-spectrum antiviral agent effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial cyclophilins and cyclophilin inhibitors in nidovirus replication suppression of coronavirus replication by cyclophilin inhibitors the sars-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors cyclosporin a inhibits the replication of diverse coronaviruses new insights into the antiviral effects of chloroquine in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted coronavirus s protein-induced fusion is blocked prior to hemifusion by abl kinase inhibitors abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion human monoclonal antibodies as candidate therapeutics against emerging viruses neutralizing monoclonal antibodies as promising therapeutics against middle east respiratory syndrome coronavirus infection assessment of immunoreactive synthetic peptides from the structural proteins of severe acute respiratory syndrome coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection the ebola epidemic crystallizes the potential of passive antibody therapy for infectious diseases selection of vaccinia virus-neutralizing antibody from a phage-display human-antibody library screening of fda-approved drugs using a mers-cov clinical isolate from south korea identifies potential therapeutic options for passive immunotherapy: assessment of convalescent serum against ebola virus makona infection in nonhuman primates enhancement of infectivity of a non-syncytium inducing hiv-1 by scd4 and by human antibodies that neutralize syncytium inducing hiv-1 convalescent plasma: new evidence for an old therapeutic tool? progress in screening research on effective chinese medicine prescriptions adverse events associated with high-dose ribavirin: evidence from the toronto outbreak of severe acute respiratory syndrome this work was supported by the national natural science foundation of china (nos. 81670088, 81602708) and the henan provincial science foundation program (no.172102410019). the study was conceived by x. the authors have declared that no competing interest exists. key: cord-337825-ujq9mxk7 authors: chen, bin; tian, er-kang; he, bin; tian, lejin; han, ruiying; wang, shuangwen; xiang, qianrong; zhang, shu; el arnaout, toufic; cheng, wei title: overview of lethal human coronaviruses date: 2020-06-10 journal: signal transduct target ther doi: 10.1038/s41392-020-0190-2 sha: doc_id: 337825 cord_uid: ujq9mxk7 coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. seven coronaviruses that infect humans have been identified: hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1, sars-cov, mers-cov, and sars-cov-2. among them, sars-cov and mers-cov caused outbreaks in 2002 and 2012, respectively. sars-cov-2 (covid-19) is the most recently discovered. it has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. viruses are genetically simple, yet highly diverse. however, the recent outbreaks of sars-cov and mers-cov, and the ongoing outbreak of sars-cov-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. this paper mainly focuses on sars-cov, mers-cov, and sars-cov-2. here, recent studies are summarized and reviewed, with a focus on virus–host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment. coronaviruses are crown-like particles with spikes protruding from their surface. they are enveloped viruses with single-stranded, positive-sense rna (+ssrna) genomes of approximately 26-32 kb, which is currently the largest known genome size for an rna virus. 1 rna viruses are divided into five branches. 2, 3 certain groups in a particular branch might be related to and/or share features with viruses classified in another branch. in branch 1 are the leviviruses and eukaryotic relatives (e.g., mitoviruses, narnaviruses, ourmiaviruses); branch 2 represents several +rna virus families (of eukaryotes) (e.g., orders picornavirales and nidovirales, and the families caliciviridae, potyviridae, astroviridae, and solemoviridae) and several dsrna viruses (e.g., partitiviruses and picobirnaviruses); in branch 3 are certain +rna viruses such as the supergroups of alphaviruses and offlaviviruses, the nodaviruses, tombusviruses, and many small and novel groups; in branch 4 are dsrna viruses such as the cystoviruses, reoviruses, and totiviruses; branch 5 consists of −rna viruses. 2 coronaviruses were classified as most likely related to branch 2, and belong to the order nidovirales, and the family coronaviridae. in 2018, the international committee on taxonomy of viruses divided the coronaviridae into the orthocoronavirinae and letovirinae subfamilies. according to their host targets, coronaviruses can also be divided into animal and human coronaviruses. many diseases in domestic animals are related to animal coronaviruses, such as canine respiratory coronavirus, which causes respiratory disease in dogs. 4 the highly pathogenic human coronaviruses belong to the subfamily coronavirinae from the family coronaviridae. the viruses in this subfamily group into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. the classic subgroup clusters are labeled 1a-1b for the alphacoronaviruses and 2a-2d for the betacoronaviruses. to date, including the recently discovered sars-cov-2, there are seven coronaviruses that infect humans. human coronavirus (hcov)-229e, hcov-nl63, hcov-oc43, or hcov-hku1 cause only the common cold, 5 whereas the severe acute respiratory syndrome coronavirus (sars-cov) or the middle east respiratory syndrome coronavirus (mers-cov) cause relatively high mortality and emerged in 2002 6 and 2012, 7 respectively. notably, sars-cov-2 is currently causing a worldwide epidemic. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, 1, 8 whereas sars-cov-2 is a new member of the betacoronaviruses distinct from sars-cov and mers-cov. 9 figure 1 shows the phylogenetic tree of rna viruses. the estimated mutation rates of coronaviruses (covs) are moderate to high compared to those of other ssrna viruses. 1 there are two gene loci that are sites of variation in sars-cov. one of these sites is located in the spike (s) protein gene. the second major site of variation is the accessory gene open reading frame orf8. in mers-cov, the major sites of variation are located in the s, orf4b, and orf3 genes. 2 the major differences in the sequence of the s gene of sars-cov-2 are three short insertions in the n-terminal domain and changes in four out of five of the key residues in the receptor-binding motif. 9 the organizations of the genome and gene expression are similar for all coronaviruses. orf1a/b, located at the 5′ end, encodes 16 nonstructural proteins (nsps) (named nsp1-nsp16); other orfs at the 3′ end encode structural proteins, including the s, envelope (e), membrane (m), and nucleocapsid (n) proteins. the mutations in sars-cov-2 have become a hot research topic. the surface proteins of sars-cov and batcov-ratg13, the nearest potential bat precursors of sars-cov-2, have 76 and 97% sequence identity, respectively, to that of sars-cov-2. 10 compared to that of sars-cov, the antigenic surface of sars-cov-2 is highly divergent compared to other covs. since its outbreak began at the end of 2019, sars-cov-2 has acquired mutations throughout its genome, and there are already hundreds of virus strains distributed worldwide. according to gisaid.org, as of may 8, there were 16,004 full genome sequences available, and the s clade of the full genome tree contains the largest group of viruses, indicating that mutations in orf8-l84s are most frequent. the temporal resolution assumes an average nucleotide substitution rate of 5 × 10 −4 substitutions per site per year. based on these mutations, researchers found that sars-cov-2 can be divided into two subtypes. they reported that the snps at locations 8782 and 28,144 show strong linkage; the "ct" haplotype was defined as the "l" type because t28,144 encodes leucine, while the "tc" haplotype was defined as the "s" type because c28,144 encodes serine, 11 which is in accordance with another study that divided sars-cov-2 into two types. 12 the "l" type seems to be more prevalent and aggressive, but the "s" type may be ancestral, as sites 8782 and 28,144 were identical to the orthologous sites in the most closely related viruses. 13 patients may be infected by either or both types. 11 from the gisaid update, compared with ratg13, there are differences in the receptor-binding interface, which may have favored host switching. data related to the sequence and mutations of sars-cov-2 are also available and continuously updated online through the china national center for bioinformation (cncb) (https://bigd.big.ac.cn/ncov). the history of sars-cov, mers-cov, and sars-cov-2 the first case of sars was discovered in foshan, china, in november 2002. 6 infections occurred through either direct or indirect contact with patients. by july 2003, sars-cov had spread to over 30 countries, 12 causing 8096 reported cases and 774 deaths. after that, no additional infections were detected, and the sars pandemic was declared over. sars-cov was first isolated from himalayan palm civets (hpcs) from a live-animal market in guangdong, china. 12, 14 other animals, such as raccoon dogs (nyctereutes procyonoides), along with human workers from the same market, also showed evidence of viral infection. 12 multiple studies have demonstrated that the reservoirs of several coronaviruses, including sars-cov-like and mers-cov-like viruses, were bats. 8, 15 although palm civets might have been intermediary hosts of sars-cov, 16 researchers often concluded there was no evidence that these animals were the ultimate sources of sars-cov, and the viruses cannot circulate directly in palm civets in the wild. 17 in 2003, guan and zheng's team investigated a live-animal retail market in guangdong, focusing on recently captured wild animals and human consumption. the animals sampled included seven wild and one domestic animal species. they collected nasal fig. 1 phylogenetic tree of coronaviruses based on full-length genome sequences. all complete genome sequences of coronavirus were downloaded from the ncbi reference sequence database, refseq. the tree was constructed using maximum likelihood estimation (mle) by mega x, with clustal omega as the multiple sequence alignment method, and 1000 bootstrap replicates. only bootstraps ≥50% values are shown. the seven known human-infecting coronaviruses are indicated with a red star and fecal samples with swabs and then used reverse transcriptionpolymerase chain reaction (rt-pcr) to test for viral nucleic acid from the n gene of the human sars-cov. the rt-pcr assay results showed that samples from four of six hpcs were positive; the other seven species (beaver, chinese ferret-badger, chinese hare, chinese muntjac, domestic cat, hog-badger, and raccoon dog) sampled were negative. 12 mers-cov was first found in 2012 in a lung sample from a 60year-old patient who died of respiratory failure in jeddah, saudi arabia. on 15 september 2012, a similar type of virus named human coronavirus was isolated from a patient with severe respiratory infection. cases have also been reported in other countries. 18 mers has a 35% mortality rate, and since it emerged in the human population in june 2012, it has caused substantial morbidity and mortality. 19, 20 from 2012 until january 15, 2020, the total number of laboratory-confirmed mers-cov infection cases reported globally to the world health organization (who) was 2506, with 862 associated deaths, covering 27 countries (www. who.int/csr/don/31-january-2020-mers-united-arab-emirates/en). cases of mers from other countries were linked to travel to the middle east. 20,21 mers-cov was identified from the saliva of a patient with acute pneumonia and renal failure in jeddah (ksa). mers-cov can be detected in respiratory tract secretions, as well as in feces, serum, and urine. [22] [23] [24] in addition, it has been isolated from environmental objects such as bedsheets, bedrails, intravenous fluid hangers, and x-ray devices. 25 in the case of known camel infection, mers-cov was transmitted from a camel to a human, which was confirmed by rna sequencing. 26 at the end of december 2019, the first clusters of patients with pneumonia caused by sars-cov-2 were reported in wuhan, china. 27 the basic reproduction number of sars-cov-2 is approximately 2.2, indicating that each patient would on average spread the infection to 2.2 people. 28 human-to-human transmission of sars-cov-2 occurred rapidly, and the atypical symptoms during the early stage may be a further disadvantage. 29 moreover, human infection might have begun months prior to the official announcement of the outbreak. 30 to prevent further spread, stricter screening and surveillance are needed at travel hubs. 31 early detection, diagnosis, and treatment are also effective measures to contain the epidemic. 32 nonetheless, the fatality rate of sars-cov-2 is lower than that of sars, 33 with an estimated mortality risk of 2%. 34 sars-cov-2 was first isolated from clinical specimens using human airway epithelial cells and the vero e6 and huh-7 cell lines. 27 approaches to assess virions include isolation from lower respiratory tract specimens 35 and artificially infected specific pathogen-free human airway epithelial cells. 36 fluorescence quantitative pcr with primers designed according to specific sequences and serological testing aimed at igg and igm can be used to detect the virus. 9 sars-cov, mers-cov, and sars-cov-2 pose major challenges to global health, causing infections in large parts of the world. sars-cov was found in 30 countries and mers-cov in 27 countries, while sars-cov-2 was found in 213 countries by 8th may, 2020. details on the pandemics caused by sars-cov-2 are shown in box 1, highlighting the status of the coronavirus-caused diseases worldwide. epidemiological analysis and symptoms of sars, mers, and coronavirus disease 2019 (covid19) the clinical manifestations of sars include hypoxia, cyanosis, high fever (above 38°c), accelerated breathing or respiratory distress syndrome, and shortness of breath. x-rays show changes to the lungs to varying degrees. the who case definition (2003) includes the following: (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive test for sars-cov by one or more assays. 37 similar to sars-cov infection, mers-cov infection manifests as a severe lower respiratory tract infection with extrapulmonary involvement and high fatality rates. 19 symptomatic patients may present fever, chills, stiffness, myalgia, discomfort, cough, shortness of breath, and gastrointestinal symptoms of diarrhea, vomiting, and abdominal pain. pneumonia is common, and severe infection with acute respiratory failure, renal failure, and shock is particularly frequent among older patients. 20 previous studies have estimated that approximately 12.5-25% of mers-cov infections may be asymptomatic. 20, 38 it must be noted that immunocompromised people are at high risk of mers-cov infection. 21 for covid-19, fever, cough, myalgia, and fatigue are most commonly observed at illness onset; less commonly observed are sputum production, hemoptysis, and headache, among others. 29, 35 similar to sars and mers, some patients develop acute respiratory distress syndrome (ards). 39 around 81% of infected patients only develop mild symptoms, with mild pneumonia or no pneumonia. 14 and 5% are severe and critical status, respectively. 40 patients requiring icu care tend to be much older and are more likely to have underlying comorbidities, such as hypertension and diabetes. 29 additionally, symptoms such as pharyngeal pain and anorexia are reported at higher rates among those admitted to the icu than among non-icu patients. 29 histopathologically, symptoms such as diffuse alveolar damage (dad), loss of cilia, squamous metaplasia, denudation of bronchial epithelia, and giant-cell infiltrate often occur in the lungs of patients with sars. the number of macrophages increases prominently in the alveoli and the interstitium. 41 according to the morphological changes, observed, most authors have subclassified lung lesions in sars into two or three consecutive phases: an acute exudative inflammatory phase, a fibrous proliferative phase, and a final fibrotic stage, although there is considerable overlap in histological findings among these phases. 42 the main features of the first phase include extensive hyaline membrane formation, edema, alveolar hemorrhage, and fibrin exudation in alveolar spaces. the second phase includes widening of septae, pneumocyte hyperplasia, and organizing fibromyxoid and cellular exudates. 42, 43 the third phase includes septal and alveolar fibrosis. 42 several autopsies of sars patients have demonstrated secondary bronchopneumonia, and the pathogens identified mainly consist of staphylococcus aureus, mucor sp., aspergillus sp., pseudomonas aeruginosa, and cytomegalovirus. 43 sars-cov also greatly affects the immune system. for example, hemorrhagic necrosis is usually obvious in the lymph nodes and spleen. 44 as described above, sars-cov attacks immune cells, especially t cells and macrophages, with approximately 50% of lymphocytes and 30% of monocytes in the circulation becoming infected. 42, 45 under the influence of the virus, the patient's germinal centers disappear, and both t and b lymphocytes are depleted. in the spleen, the white pulp shrinks, hemorrhage and necrosis occur in the red pulp, and the periarterial lymphatic sheaths decrease. 42, 45, 46 with regard to the immune system, sars-cov can infect the lymphoid component of the intestine. for this reason, the patient's submucosal lymphoid tissue atrophies. 47 sars-cov can also infect epithelial cells in the mucosa of the small and large intestines, and the digestive tract may undergo mild diffuse inflammation and atrophy of the submucosal lymphoid tissues. 47 according to previous reports, symptoms in the liver include extensive hepatocyte mitosis, balloon degeneration of hepatocytes, mild to moderate lymphocytic infiltrates, fatty degeneration, and central lobular necrosis. 42 additionally, apoptosis of liver cells has been confirmed in some cases. 48 on autopsy, the kidneys of some sars patients showed focal bleeding and acute tubular necrosis of different degrees. one of the major complications of ards is acute kidney injury. 42, 49, 50 in many other cases, the features are nonspecific, including benign hypertensive nephrosclerosis and autolysis. 42 moreover, researchers have detected viral particles and genome sequences in the cytoplasm of neurons of the hypothalamus and cortex in the brain, 51 which suggests that the virus can cross the blood-brain barrier and cause degeneration and necrosis of neurons, edema, extensive glial cell hyperplasia, and cellular infiltration. 42 pneumocytes and epithelial syncytial cells are important targets of mers-cov. the lung tissue presents dad. severe peripheral vascular diseases, patchy cardiac fibrosis, and hepatic steatosis have also been found in other organs. 52 another symptom is bronchial submucosal gland necrosis. 52 renal biopsy may show acute tubulointerstitial nephritis and acute tubulosclerosis with protein casts. 22 in covid-19 patients, a bilateral (sometimes unilateral) distribution of patchy shadows and ground glass opacity in the lungs is typical, based on ct scans. 29 plasma concentrations of interleukins (ils) 2, 7, and 10, granulocyte-colony stimulating factor, interferon (ifn)-γ-inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and tumor necrosis factor α are increased in most dying patients. 39, 53 in addition, neutrophilia related to cytokine storms induced by virus invasion, coagulation activation related to a sustained inflammatory response, and acute kidney injury related to the effects of the virus, hypoxia, and shock, may be involved in mortality. 29 those who survive intensive care may experience long-term lung damage and fibrosis due to aberrant and excessive immune responses. 39 moreover, researchers found that as sars-cov-2 spread and changed across generations, clinical symptoms started to differentiate those infected previously from those infected more recently. the initial symptoms are becoming more insidious, which indicates that the virus may gradually evolve into an influenza-like virus and lie dormant for longer in asymptomatic patients. 54 in conclusion, in terms of outbreak times, sars-cov was the earliest, followed by mers-cov, then sars-cov-2. to date, mers-cov has the longest duration of infection, and sars-cov-2 appears to be the most infectious. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. the common transmission methods and symptoms of the three coronavirus infection are shown in fig. 2 . structural studies of sars-cov, mers-cov, and sars-cov-2 sars-cov is a coronavirus with a diameter of approximately 100 nm. coronaviruses are the largest +ssrna viruses and contain at least 14 orfs, 16 protein combines with viral rna to form a nucleocapsid, which is involved in the replication of sars-cov and is the most abundant protein in virus-infected cells. 56, 57 the m protein is a membrane glycoprotein that is involved in budding and envelope formation. the s protein of sars-cov is a type i transmembrane (tm) glycoprotein that is responsible for viral binding, fusion and entry into cells, as well as antibody induction. 16, 58 the s protein contains a signal peptide (sp: amino acids 1-12) and three domains called the extracellular domain (amino acids 13-1193), the tm domain (amino acids 1194-1215) and the intracellular region (amino acids 1216-1255). 16 the extracellular domain consists of two subunits: s1 and s2. the fragment located in the middle region of the s1 subunit (amino acids 318-510) is the angiotensin-converting enzyme 2 (ace2) receptor-binding region. the s2 subunit comprises a fusion peptide (fp) and two x 7-valent element repeats (hr1 and hr2) that are responsible for fusion between the virus and the target cell membrane. 58 thus, the s protein may be key to develop a vaccine to generate antibodies and block virus binding and fusion in the coronaviruses (fig. 3) . the 3′ end of the sars virus genome encodes 12 structural proteins and helper proteins, of which orf3a, orf6, orf7a, and orf7b have been proven to be viral structural proteins involved in the formation of viral particles. the 5′ end of the genome encodes 16 nonstructural proteins (nsps), which are important for virus assembly, and may enable the design of small molecule drugs and/or vaccines. mers-cov belongs to lineage c of the genus betacoronavirus (βcov) in the family coronaviridae, order nidovirales, and it is the first lineage c cov and the sixth cov known to cause human infection. mers-cov is an enveloped +ssrna virus similar to other covs, but the amino acid sequence homology between mers-cov and other known covs is less than 90%. 19 orf1a and orf1b located in the first two-thirds of the mers-cov genome are involved in virulence and encode nsps (nsp1-16). the remaining third of the genome encodes four structural proteins, the s, e, m, and n proteins, as well as five accessory proteins (orf3, orf4a, orf4b, orf5, and orf8b). [59] [60] [61] the flanking regions of the genome contain the 5′ and 3′ untranslated regions (utrs). 62 most mers-cov proteins are found in the cytoplasm. 63 to date, the ns4b protein and the orf3a-encoded protein are the only known coronavirus proteins to be detected in the nucleus. 64 nsp1 suppresses host gene expression in infected cells, and its rna cleavage-inducing function promotes virus assembly/budding. 65 nsp16 is a viral 2′-o-methyltransferase (2′-o-mtase) that encodes critical functions in immune modulation and infection, suggesting that nsp16 is a conserved universal candidate for rapid design of a live attenuated vaccine. 66 orf4a's protein can mask viral dsrna to play a role in immune evasion, including type 1 ifn and protein kinase r-mediated antiviral stress responses. 67 mers-cov nsp13 is a full-length coronavirus helicase that contains multiple domains, including an n-terminal cys/his rich domain with three zinc atoms, a beta-barrel domain and a c-terminal superfamily (sf) 1 helicase (sf1) core with two reca-like subdomains. this protein might interfere with the nonsensemediated mrna decay pathway to avoid degradation of viral rna. 68 the mers-cov s protein can recognize dipeptidyl peptidase 4 (dpp4) on the cell surface, promoting the fusion of the viral envelope and the cell membrane. the mers-cov s protein, a 1353-amino acid type i tm glycoprotein located on the viral envelope surface, is composed of s1 and s2 subunits. 69 the structure of the s protein consists of four parts: an sp located at the n terminus (amino acids 1-12), an extracellular domain (amino acids 13-1195), a tm domain (amino acids 1196-1215), and an intracellular domain (amino acids 1216-1255). 16 the s1 subunit contains two independent domains, an n-terminal domain (ntd, amino acids 18-353) 70 and a c-terminal domain (amino acids 367-588), both of which may function as receptor-binding domains (rbds); 71 these domains serve as critical targets for the development of vaccines and therapeutics 72, 73 and facilitate the involvement of the s1 subunit in cell receptor (dpp4) binding. the core structure of the mers-cov s protein rbd is a five-stranded antiparallel sheet with several short helices, in which three disulfide bonds stabilize the core by connecting c383 to c407, c425 to c478, and c437 to c585. 16, 73 the accessory subdomain of the c-domain is a hypervariable region for receptor recognition fig. 3 clustering of the spike protein of each coronavirus. fifty-four spike protein sequences filtered from each coronavirus coding sequences were clustered using the clans (cluster analysis of sequences) program on the website of mpi bioinformatics toolkit. each colored dot represents the spike protein sequence of each coronavirus. dots in the same color mean they are of the same genus, and each line shows the similarity of two sequences, with darker lines indicating higher similarity (lower e values). the coronaviridae family includes the following genera: alphacoronavirus (colored in green); betacoronavirus (red); gammacoronavirus (orange), and deltacoronavirus (blue). the indicated sars-cov, mers-cov, and sars-cov-2 belong to the genus of betacoronavirus and is called the receptor-binding motif (rbm). the accessory subdomains in sars-cov and mers-cov differ, resulting from divergent evolution and leading to the use of different receptors. 71 dpp4 consists of an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets 74 and the rbd. the s2 subunit of mers-cov contains an fp (residues 943-982), an hr1 domain (residues 984-1104), an hr2 domain (residues 1246-1295), a tm domain (residues 1296-1317), and an intracellular domain (residues 1318-1353). 69 the s2 subunit is responsible for fusion between the virus and the target cell membrane. after the s1 subunit binds to dpp4, the s2 subunit inserts its fp into the target cell membrane to change its conformation. its hr1 helix then forms a homotrimer with an exposed surface that has three highly conserved hydrophobic grooves, and binds with hr2 to form a six-helix bundle (6-hb) core structure, which facilitates fusion by bringing the viral and cell membranes into close proximity. 69 the genetic material of mers-cov then enters the host cell via the fusion pore. 69 sars-cov-2, which causes covid-19, is a spherical betacoronavirus with a diameter of 60-140 nm and unique spikes ranging from 9 to 12 nm. 27 its rna has the typical betacoronavirus organization, containing a 5′ utr, a gene encoding the replicase complex (orf1a/b), the s gene, e gene, m gene, and n gene, a 3′ utr, and several unidentified nonstructural orfs. 27, 75 the length of the sequences is 265 nt for the 5′ terminal region, 229 nt for the 3′ terminal region, 3822 nt for the s gene, 228 nt for the e gene, 669 nt for the m gene, and 1260 nt for the n gene. 75 the 21,291nt-long orf1a/b gene contains 16 predicted nsps. 75 similar to sars-cov, there is a predicted orf8 gene that is 366 nt in length and located between the m and n orf genes. 75 nucleotides 1, 1029, and 1652 may be the recombination breakpoints because based on the fragments from nt 1 to nt 1029 and nt 1652 to the end, sars-cov-2 seems to be related to bat-sl-covzc45 and bat-sl-covzxc21; however, when considering the region from nt 1030 to nt 1651, sars-cov-2 is mostly likely to be grouped with sars-cov and bat sars-like covs. more research is required to analyze the recombination of the genome as a whole. 75 with over 10% of its conserved replicase complex being different from those of other betacoronaviruses, sars-cov-2 solely occupies a newly created subclade within the sarbecovirus subgenus. 27, 75 bats may be its natural host, but owing to several arguments including the complicated environment in the wet markets in wuhan, this remains unclear, and further research is needed. 36, [75] [76] [77] the free energy of the spike protein of sars-cov-2 is much lower than that of sars-cov, indicating that sars-cov-2 is more stable than sars-cov. 78 the high similarity of the rbd sequences and of the spike protein structures between sars-cov-2 and sars-cov, 36, 75, 79 also the simulation of the spike protein of sars-cov-2 binding to ace2, 80 the receptor of sars-cov, and results shown that ace2 plays a vital role in sars-cov-2 entry into hela cells, 76 indicating that sars-cov-2 also uses ace2 for cell entry. structural modeling of the ace2-b0at1 complex (b0at1 is used to obtain stable ace2) suggests that the complex can bind two s proteins simultaneously, providing important clues to the molecular basis of coronavirus recognition and infection. 81 the rbd-ace2 binding free energy for sars-cov-2 is significantly lower than that for sars-cov, in accordance with the fact that sars-cov-2 is more infectious than sars-cov. 78 a recent study found that cd147, a kind of tm glycoprotein, facilitates cell entry of sars-cov-2 functionally, and its affinity constant with the s protein is 1.85 × 10 −7 m. 82 table 1 shows a comparison of the structures of sars-cov, mers-cov, and sars-cov-2. the specific function of the sars-cov-2 proteins, including s, e, m, and n proteins, need further study. in conclusion, sars-cov, mers-cov, and sars-cov-2 are similar in structure. their major proteins are structural proteins, including the s, e, m, and n proteins, accessory protein, and nsps (nsp1-16). all these viruses rely on the s protein to identify cell targets and fuse with membranes. their s proteins are composed of s1 and s2 subunits. the s1 subunit contains an ntd and a c-domain, in which there is an rbd. the accessory subdomain of the c-domain is a hypervariable region for receptor recognition and is called the rbm. the difference is that sars-cov recognizes ace2, sars-cov-2 recognizes ace2 and cd147, but mers recognizes dpp4, which results from the difference between the accessory subdomains of sars-cov, sars-cov-2, and mers-cov. the function of the accessory and nonstructural proteins of these three viruses is related to viral replication and assembly, hence linked to virulence. pathogenesis of sars-cov, mers-cov, and sars-cov-2 sars-cov and sars-cov-2 are nearly identical in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4. the human ace2 protein is a typical zinc metallopeptidase that comprises 805 amino acids and contains a single catalytic domain. it is a type i integral membrane glycoprotein that is oriented with an extracellular n terminus and a catalytic site that functions in metabolizing circulating peptides. 83 it is believed that there is a virus-binding hot spot on ace2 that is not pre-existent or preorganized but is induced to form upon virus binding. 84 the hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. the general structural features of the hot spot favor virus binding: it is located in a region on ace2 that is furthest from the membrane, relatively flat, and free of glycosylation and is thereby easily accessible to viruses. the hot spot is mainly an intrinsic property of ace2, but it is also a dynamic structure and receives structural contributions from both ace2 and the viruses, with the contributions of ace2 being more pronounced. dpp4 is a type ii transmembrane glycoprotein that consists of approximately 766 amino acids. dpp4 contains an n-terminal hydrolase and a cterminal β-propeller domain composed of eight lancets. 74 the s protein rbd binds to the side surface of the dpp4 propeller and the amino acid residues in lancets 4 and 5, including k267, q286, t288, r317, r336, q344, a291, l294, and i295. 74 the pathogenic mechanism of sars-cov is believed to include two parts: the virus injures target cells directly, and subsequent immune system dysfunction leads to indirect injury. 42 sars-cov spreads via droplet and contact transmission and via the fecal-oral route. through droplet inhalation, the virus enters the respiratory tract and invades epithelial cells. infection and replication of the virus and local inflammatory changes contribute to lung tissue damage. subsequently, sars-cov infects circulating and resident immune cells. the key cells in this step consist mainly of t cells and macrophages. the viruses are then carried to other organs, including secondary lymphoid organs, by these circulating immune cells. 42 sars-cov resembles hiv because both viruses attack immune cells and cause immunodeficiency. 42 sars-cov causes lung injury mainly by inhibiting the function of ace2. the virus binds to ace2 via the spike protein, downregulating its function and contributing to lung injury. ace2 is an important component of the renin-angiotensin system (ras). in this system, angiotensinogen is first converted to angiotensini (angi) by renin, and then angiis converted to ang ii under the influence of ace2. ace2 downregulates the levels of angi and ang ii, which can bind to the ang ii type i (at1) receptor and cause certain types of lung injury, mainly pulmonary hypertension, pulmonary fibrosis, and acute lung injury. 85 ang ii can directly induce pulmonary artery smooth muscle cells to grow or proliferate rapidly through at1, thus causing pulmonary hypertension. 86 ang ii contributes to pulmonary fibrosis by promoting the expression of the profibrotic cytokine transforming growth factor-b1, causing fibroblasts to convert to myofibroblasts and accumulate collagen. 70 several strategies can be used by the virus to escape the innate immune response. normally, viral pathogen-associated molecular patterns (pamps) are detected by host pattern recognition receptors (prrs). pamps include viral dsrna and mrna, and prrs include retinoic acid-inducible gene i protein (rig-i) and melanoma differentiation-associated protein 5 (mda5). production of type i ifn is induced via the nuclear factor-κb (nf-κb) pathway. when ifn binds to the ifnα/β receptor, signal transducer and activator of transcription (stat) proteins are activated, which increases the production of other antiviral proteins and thus blocks sars-cov replication for further infection. 11 additionally, sars-cov could encode several proteins that hinder the signaling cascades downstream of prrs. the cell receptor of mers-cov is dpp4, which is widely expressed on epithelial cells in the prostate, alveoli, kidneys, liver, and small intestine and on activated leukocytes; thus, the range of mers-cov tissue tropism is broader than that of any other similar coronavirus. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of proinflammatory cytokines and chemokines (such as tnf-α, il-6, cxcl-10, ccl-2, ccl-3, ccl-5, and il-8). 87 compared to sars-cov infection, mers-cov infection can cause higher expression levels of il-12, ifn-γ, ip-10/cxcl-10, mcp-1/ccl-2, mip-1α/ccl-3, rantes/ccl-5, and il-8. 87 induction of immune cell-recruiting cytokines/chemokines might lead to the infiltration of a large number of immune cells into the lower respiratory tract, causing severe inflammation and tissue damage. 87 mers-cov can also infect t cells and lead to apoptosis, avoiding the host immune response and facilitating faster spread. 88 mers-cov has also evolved a mechanism to escape the host cell immune system. when host sensors initiate signaling pathways, transcription of type i ifn genes begins, and the type i ifn response, which is an essential component of antiviral innate immunity, is initiated. [89] [90] [91] ifn activates the transcription of numerous ifn-stimulated genes (isgs) and synthesizes 2′,5′oligoadenylate (2-5a), which causes rnase l activation. [91] [92] [93] activated rnase l can cleave both viral and host ssrna, leading to translation stagnation and apoptosis and limiting virus replication and transmission in vitro and in vivo. 92, 94 however, the mers-cov protein ns4b has phosphodiesterase activity and antagonizes rnase l via enzymatic degradation, leading to inference with ifn signaling. although ns4b is mainly expressed in the nucleus, the expression level of the ns4b protein in the cytoplasm is sufficient to prevent activation of rnase l. 88 in addition, the mers-cov m, orf4a, orf4b, and orf5 proteins are reported to be strong ifn antagonists, among which the orf4a, orf4b, and orf5 proteins inhibit both type i ifn induction 61, 95, 96 and nf-κb signaling pathways, which are similar to the type i ifn signaling pathway, 96 providing a possible mechanism for mers-cov evasion of innate immunity. 88, 96 for sars-cov-2, the first cluster of patients was believed to be associated with a seafood market. 27 however, due to the identification of patients without direct exposure to the market, transmission between humans was proven. 97 hospital-related transmission has also been detected via infected medical workers and hospitalized patients. main routes of transmission include respiratory droplets, close contact, and extended exposure to aerosol environments loaded with high concentrations of virions. in addition to transmission through the respiratory tract, exposure of unprotected eyes to sars-cov-2 might cause acute respiratory infection. 98 notably, the virus can be transmitted during the incubation period. 99 the tm protease serine type 2 (tmprss2) primes the s protein of sars-cov-2 for cell entry. 100 based on analysis of the ace2 rna expression profile in normal human lungs, sars-cov-2 mainly infects type ii alveolar (at2) cells (only 0.64% of human lung cells can express ace2, 83% of which are at2 cells), and they express many other genes that facilitate sars-cov-2 infection; 101 moreover, cd147 was also reported that it probably functionally facilitates cell entry of sars-cov-2. 82 high expression of ace2 has also been detected in cells of the digestive system, such as upper esophageal cells; accordingly, this system is a potential route of infection. 102 in addition to the lungs and intestines, other organs, such as the heart, esophagus, kidney, bladder, testis, ileum, and adipose tissue, also express ace2, and the expression level is higher than that in the lungs. 103, 104 certain tumor tissues have higher expression of ace2, making cancer patients more vulnerable than other people. 104 in conclusion, sars-cov and sars-cov-2 bind cells expressing ace2. they affect the ras system due to the affinity for ace2 (much stronger with sars-cov-2 than with sars-cov), leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. the specific pathogenic mechanisms of these three viruses are illustrated in fig. 4 . diagnostic methods in addition to clinical manifestations, definitive diagnosis and confirmation of a coronavirus infection requires specific testing. the criteria for the diagnosis of sars from the who include (1) fever higher than 38°c or history of such in the past 2 days, (2) radiological evidence of new infiltrates consistent with pneumonia, (3) chills, cough, malaise, myalgia, or known history of exposure, and (4) positive tests for by at least one assay. as for mers, the criteria for the diagnosis according to the who include (1) fever, cough, and hospitalization with suspicion of lower respiratory tract lesion, (2) history of contact with probable or confirmed cases, (3) history of travel or residence within the arabian peninsula, and (4) positive tests by a pcr-based detection method using respiratory samples, such as nasopharyngeal swabs, nasopharyngeal or tracheal aspirates, bronchoalveolar lavage (bal) and induced sputum, or by serological tests such as the rapid immunochromatographic assay using highly selective monoclonal antibodies at room temperature. the initial criteria for the diagnosis of sars included (1) a history of travel or residence in wuhan within 2 weeks before the onset of illness, (2) exposure to patients from wuhan with fever and respiratory symptoms within 2 weeks before the onset of illness or the existence of clusters of diseases, (3) fever accompanied by radiographic features of pneumonia, a normal or decreased total number of white blood cells, or a decreased lymphocyte count, and (4) fluorescence pcr testing positive for nucleic acids of sars-cov-2. 37, 59, [105] [106] [107] early diagnosis of mers is difficult, but several highly specific and sensitive molecular and serologic assays exist for diagnosis in both animals and humans. 108 among them, a rapid immunochromatographic assay can detect mers-cov antigens based on the detection of the mers-cov nucleocapsid protein in a short time frame; the relative sensitivity and specificity of this test are 93.9 and 100%, respectively. 105 nucleic acid tests, such as real-time pcr tests, were mostly used to detect virus in the early stage of the outbreak of sars-cov-2, although this approach requires a relatively long time and is not conducive to accelerating diagnosis or large-scale application. therefore, alternative, rapid diagnostic kits for sars-cov-2 may be used. for example, the colloidal gold detection kit uses serum, plasma, or whole blood samples to detect igm/igg antibodies for diagnosis on the 7th day of infection or the 3rd day after onset and requires only 15 min. the nucleic acid detection kit for six respiratory viruses, including sars-cov-2 and influenza a virus, uses thermostatic amplifier chips for diagnosis. by detecting different infections, it may help distinguish people with influenza from those with other viral infections. a newly developed fig. 4 pathogenesis of sars-cov, mers-cov, and sars-cov-2. sars-cov and sars-cov-2 play a pathogenic role by inhibiting ace2. under the influence of renin and ace, angiotensinogen is converted into ang ii. through the at1 receptor, ang ii acts as a lung injury-promoting factor, and in some cases, may cause vascular constriction, an inflammatory response, cell proliferation, fibrosis, and apoptosis of alveolar epithelial cells, resulting in diseases such as pulmonary hypertension, pulmonary fibrosis, and acute lung injury. ace2 converts ang ii into ang (1-7), and through the mas receptor, ang (1-7) play roles in vasodilation, antiproliferative activity, and antioxidant activity. sars-cov downregulates the activity of ace2 and causes an increase in the amount of ang ii and lung injury. mers-cov infection of dendritic cells and macrophages can lead to the continuous production of pro-inflammatory cytokines and chemokines, leading to a large number of immune cells infiltrating a patient's lower respiratory tract, causing severe inflammation and tissue damage. mers-cov can infect t-cells from human lymphoid organs and causes the peripheral blood inducing apoptosis by intrinsic and extrinsic pathways, thus avoiding host immune response detection method, nanopore targeted sequencing, also has the potential for efficiently detecting viruses in a reasonable time. moreover, it could identify 40 other respiratory viruses and monitor changes in virulence and transmissibility caused by viral mutations. 109 therapeutic agents all patients should be treated in isolation. 53 based on previous studies, drugs may be developed to inhibit cell entry. several strategies have been investigated to identify specific antivirals targeting the s protein, including mers-rbd-targeted nabs that block viral attachment, peptide inhibitors targeting s2 to prevent the formation of the fusion core, and small molecules without defined mechanisms. 110 sdpp4 has been found to bind mers-cov rbd with high affinity, suggesting that sdpp4 could serve as a blocker for mers-cov attachment to and entry into cells. 111, 112 one study reported a monoclonal antibody, 7d10, that binds to the ntd to inhibit cellular entry of mers-cov. 113 experiments have shown that 7d10 not only inhibits the binding of mers-cov to cells but also has effects after viral-cell attachment. 7d10 inhibits virus invasion by blocking the binding of the s1 subunit to the receptor and interfering with the conformational transformation of the s2 subunit when the virus fuses with the cell membrane. 113 a peptide fragment spanning residues 736-761 of the s protein exerts neutralization activity by inhibiting membrane fusion and the entry of mers-cov, suggesting that it is possible to develop vaccines targeting this neutralizing epitope. 114 griffithsin, a lectin derived from red algae, binds to oligosaccharides on the surface of sars-cov s protein and may also prove effective against sars-cov-2. 34, 115 researchers found that antibodies raised against sars-cov, such as css-2, -3, -4, and -5, could cross-neutralize sars-cov-2 by reducing s-driven cell entry, although the efficiency was lower than that observed for sars-cov. 100 meplazumab, an anti-cd147 humanized antibody, binds with the cd147 in competition with the s protein, 116 thus significantly inhibiting virus from invading cells and reducing the time of negative conversion. 82, 106 chloroquine could effectively inhibit sars-cov-2 in vitro, and hydroxychloroquine was even more potent than chloroquine. 117, 118 hydroxychloroquine could significantly help reduce the virus load, and azithromycin could reinforce its effect. 119 arbidol could also contribute to condition improvement. 120 note that these studies were generally limited and further trials and development are necessary. protease inhibitors also have the potential for coronavirus therapeutics. protease inhibitors, including disulfiram, lopinavir, and ritonavir, have been reported to be effective against sars and mers, and disulfiram, a drug for alcohol dependence, was reported to inhibit the papain-like protease (plpro) of mers-cov and sars-cov in cell cultures. 34, 108 in vitro, sars-cov can be inhibited by both 6-mercaptopurine and 6-thioguanine targeting plpro. 121 when used together with ribavirin, the protease inhibitors lopinavir and ritonavir have improved the outcomes of patients with sars compared with those achieved with the use of ribavirin alone. 122 such inhibitors are also being tested in patients infected with sars-cov-2, but whether these inhibitors effectively suppress 3clpro and plpro of sars-cov-2 remains controversial. 34 drugs such as colistin, valrubicin, icatibant, bepotastine, epirubicin, epoprostenol, vapreotide, aprepitant, caspofungin, perphenazine, prulifloxacin, bictegravir, nelfinavir, and tegobuvir bind to the main protease of sars-cov-2, and thus, are potential candidates. 123 lianhua-qingwen formula, a kind of traditional chinese medicine, has been also recently described theoretically as a potential treatment candidate against the main virus protease. 124 the serine protease inhibitor camostat mesylate could partly block sars-cov-2 infection of lung cells by inhibiting tmprss2. 100 viral rna synthesis blockers also have potential. remdesivir has broad-spectrum activity against mers-cov and sars-cov in cell cultures and animal models and is currently in clinical development for the treatment of ebola virus disease. 34, 125 it was also reported positively based on a patient with sars-cov-2 infection in the us, 126 and another study showed that it was able to inhibit the virus. 117 ribavirin is often used to treat sars and mers, but it is uncertain whether it is potent enough against covid-19. 34, 108 regardless, when used alone, ribavirin has minimal activity against sars-cov and may cause strong side effects. it is often used together with corticosteroids. 14,127 a preclinical study of galidesivir (bcx4430), an adenosine analog, revealed antiviral activity against sars-cov and mers-covcov. 115 a study of favipiravir (t-705), a guanine analog, proved its activity against sars-cov-2, and randomized trials of favipiravir plus interferon-α (chictr2000029600), and favipiravir plus baloxavir marboxil (chictr2000029544) are in progress. 34 corticosteroids may be used to treat sars patients to suppress lung inflammation; although this therapy is not associated with mortality in patients with mers, it is associated with delayed clearance of mers-cov rna. 14,128 moreover, clinical evidence does not support the use of corticosteroids for covid-19 treatment. 129 regarding antibodies, plasma therapy using convalescent plasma from fully recovered patients is effective against these coronaviruses, including sars-cov-2. 108, 130 tocilizumab (antihuman il-6 receptor) may curb immunopathology and trials are ongoing now. 131 a study of novel monoclonal antibodies against the mers-cov spike protein suggests that mabs can be utilized for the identification of specific mutations of mers-cov. 132 wang et al. provided an all-hydrocarbon-stapled peptide that likely mimics the native conformation of the c-terminal short α-helical region of the mers-cov s protein, which can block the formation of the hexameric coiled-coil fusion complex to inhibit viral-cell membrane fusion. 133, 134 current treatments for sars mainly include ribavirin, ifn α, plasma therapy, host-directed therapies, and systemic corticosteroids. 14, 41 due to the lack of clinical trial data, adequate supportive care supplemented with different combinations of drugs remains the main treatment for patients. 14, 41 for mers, despite many studies in humans, there is no consensus on the best treatment; thus, randomized clinical trials are needed to assess potential treatment options. 134 several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn and novel therapies, including polyclonal antibodies and broad-spectrum antivirals. 108 antimicrobial peptides (amps) may be used as alternative therapeutic agents, 135 and many have been effective even against bacterial proteases agents. 136 antiviral drugs with effective activity in vitro include neutralizing monoclonal antibodies, antiviral peptides, mycophenolic acid, lopinavir, ifn, ribavirin, nitazoxamide, mycophenolate mofetil (mmf), alisporivir, silvestro, and corticosteroids. 19, 21, 59 although there is no agreement on the most ideal treatment option to cure covid-19, much research activities and pursued routes are underway. potential host-targeted agents the host-directed strategy is another approach for limiting viral replication. host proteases such as cathepsin b and cathepsin l can cleave the spike protein of sars-cov. drugs such as camostat inhibit host serine proteases and then interfere with the entry of sars-cov, but they may cause many significant side effects. 14, 135 pegylated ifn α-2a and -2b may be used to stimulate innate antiviral responses in patients, and chloroquine, an approved immune modulator, was reported to have inhibitory effects against sars-cov-2 under certain conditions. 34 researchers have also proposed that some commercially available drugs with suitable safety profiles, such as metformin, glitazones, fibrates, sartans, and atorvastin, as well as nutrient supplements and biologics, might reduce immunopathology, boost immune responses, and prevent or curb ards. in addition, ongoing cellular therapies using mesenchymal stromal cells from allogeneic donors have been shown to reduce nonproductive inflammation and affect tissue regeneration and are being evaluated in phase 1/2 trials in patients with ards; these therapies may be assayed in sars-cov-2-infected patients. 40, 137, 138 expansion of antiviral t cells as cellular drugs could aid in preparing t cell products for adjunct treatment of patients with severe infection. 40 overall, there are similarities and differences among the treatments for these three coronaviruses. notably, we summarize the potential drugs and vaccines in table 2 . vaccination could be used to prevent infection or to reduce disease severity. different kinds of vaccines, such as dna vaccines, recombinant proteins, subunit vaccines, and inactivated viruses, were described. 14 however, the highly sophisticated immune evasion mechanisms of viral pathogens and their high mutation rates make human vaccine development a major challenge. 136 the four nsps (3clpro, plpro, helicase, and rdrp), which are key enzymes in the viral life cycle, and the s protein, which is responsible for receptor binding during cell entry, are attractive targets for developing vaccines against coronaviruses. 115 among them, the s protein is most commonly targeted. 61 based on previous studies, it is believed that the s protein receptor-binding domain (s-rbd) located in the s1 subunit is an important target for the development of a sars vaccine, especially the key neutralizing region, cnd. indeed, cnd can induce a strong neutralizing antibody response and crossprotection against sars-cov mutants. one study showed that a recombinant fusion protein (designated rbd-fc) containing the 193-amino acid rbd and a human igg1 fc fragment can induce highly potent antibody responses in immunized rabbits. the antibodies inhibited sars-cov infection (serum dilution of 1:10,240), and they are believed to be safer than other types of vaccines. 139 additionally, with chloroplast transgenic technology, it is possible to combine the fusion gene s-rbd and the carrier molecule cholera toxin b subunit into the tobacco chloroplast genome to obtain a chloroplast transgenic tobacco plant that stably expresses the oral sars-cov subunit vaccine. 140 for mers, vaccines based on the viral s protein include full-length s or strimer protein-based vaccines such as a full-length s-based simian adenovirus vector vaccine (chadox1) and dna vaccine (gls-5300), 61 s1-based vaccines such as an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1) and an rabv vector encoding an s1-elicited antibody, 141, 142 rbd-based vaccines, and vaccines based on other regions. it has been confirmed that the s proteins of sars-cov and sars-cov-2 are quite similar, but researchers recently found that the three monoclonal antibodies developed to bind to the s protein of sars-cov, s230, m396, and 80r do not cross-react with the s protein rbd of sars-cov-2, simian adenovirus vector vaccine (chadox1);an ad vector encoding mers-cov s1 extracellular domain (ad5.mers-s1); an rabv vector encoding s1 elicit antibody; rbd-based vaccines 61, 138, 139, 140 overview of lethal human coronaviruses chen et al. suggesting that tailored vaccines and antibodies against sars-cov-2 must be designed. 143 dna vaccines are also quite promising. specific igg antibodies against sars-cov can be promoted by the s gene dna vaccine. as mentioned above, the s protein of sars-cov plays an important role during the pathogenic process, and synthetic peptides induced by dna vaccine in escherichia coli elicit specific antibodies against the sars-cov s protein which might provide another approach for further developing sars-cov vaccines. 42, 144 to evaluate the safety and immunogenicity of a plasmid dna vaccine (gls-5300) that expresses the s protein of mers-cov, a phase i clinical trial on healthy volunteers was conducted in 2016, but the results were not reported. another phase i trial utilizing the viral vector, chimpanzee adenovirus, oxford university #1 (chadox1), containing the mers-cov s protein expression gene was started by oxford university in january 2018. 145 in addition, camel vaccines against mers-cov are a consideration. at present, at least two promising candidate camel vaccines are undergoing development, and field trial evaluation is in progress. 108, 146 one study found that the rbd fragment covering spike residues 377-588 is a key neutralizing receptor-binding fragment and an ideal candidate for mers vaccines. 147 another potential neutralizing epitope is a peptide fragment covering 736-761 residues of the s protein which blocks the membrane fusion and cellular entry of mers-cov 114 (fig. 5) . other approaches recombinant viruses may be employed to generate an immune response against the viruses. there are two kinds of recombinant viruses which are promising for designing a protective vaccine. the first is a defective or nonpathogenic vector capable of expressing viral proteins, and the second is a vector that can be assembled in a test tube to stimulate the assembly of virus-like particles. adenosine deaminase (ada), a kind of human enzyme, could interact with dpp4, therefore, ada could compete with mers-cov as a natural antagonist when the virus attempts to bind to cells. 148 in addition, anti-dpp4 can play a similar role, 149 however, it is not practical to use this method in vivo because dpp4 has important functions in the regulation of several different signaling pathways. 150 questions concerning the coronavirus-host interactions this topic has intrigued the community. understanding certain mechanisms about virus interactions will support drug research activities. for instance, it was found that sars-cov-2 has a stronger, more rapid spreading ability than many known viruses. is it possible that it invades host cells via additional novel routes, not limited to ace2? for example, cd147 is probably involved in virus invasion. 82 is there an s-like protein involved in invasion? the affinity of the human ace2 protein for the rbd of the new coronavirus is 10-20 times higher than it is for that of the sars virus, which likely explains why sars-cov-2 spreads more swiftly. there is a distinct insert present in the peptide fragment spanning of s1/s2 loop of the sars-cov-2 s protein, which is not shared the similarity with sars-cov or any sars-related viruses. does this peptide loop impact on the entry pathway type of sars-cov-2, compared to other known betacoronavirus lineage b? furthermore, the s protein is likely cleaved during virus assembly and delivery to the cell surface by golgi-resident proprotein convertases such as furin, 151 which is different from the behavior of its close relatives. furin is found in a variety of human tissues, including the lungs, liver, and small intestine. therefore, which are exactly the cell types that sars-cov-2 may attack? yet, in relation to the last two questions, it should be noted that studies are also necessary to investigate any possibility of receptor-independent virus entry. the sars-cov-2 genome encodes nonstructural proteins, structural proteins, and accessory proteins. 3clpro, plpro, helicase, and rdrp, which belong to the first type, and the s protein, which belongs to the second, have been recognized as promising targets for developing antiviral agents against sars-cov and mers-cov, which therefore may be applied to sars-cov-2. nonetheless, the rapid identification of effective interventions against sars-cov-2 is a major challenge. however, the subject of using currently known antiviral drugs has been frequently brought up by the community as a potential short-term strategy to combat sars-cov-2. drugs affecting such targets and their status for sars-cov-2 are listed below. the main candidates as inhibitors of 3clpro include other agents for sars-cov-2 fig. 5 the targets of the different drug candidates against the three coronaviruses. common targets against the three coronaviruses are mainly the s protein and the s1/s2 subunits, pl protein, rdrp, 3cl protein, and helicase. the figure shows drug candidates (in black) and vaccines (in red). among them, remdesivir has been trending in the news recently. it inhibits the rdrp, is in phase iii for sars-cov-2, and may have an effect on the three viruses. ribavirin in combination with a pegylated interferon may also have an effect against the three viruses. ritonavir and lopinavir, which inhibit the 3clpro and are in phase iii for sars-cov-2, have an effect on both sars-cov-2 and mers-cov. dna vaccines and vaccines based on the s protein or subunits of the s protein are in development lopinavir, ritonavir, darunavir, cobicistat, and asc09f (phase iii, in combination with oseltamivir, for sars-cov-2). 152 candidates as inhibitors of plpro include thiopurine analogs, compound 6 (preclinical), and remdesivir (phase iii for mars-cov). 121, 153 favipiravir, ribavirin (randomized trial for sars-cov-2), and remdesivir are among the candidate inhibitors of rdrp. 118 previously, compounds that may interfere with atpase and helicase activities were reported (before covid-19), such as bananins, 5-hydroxychromone derivatives, and triazole derivatives (preclinical). 34, 78 candidates that may suppress viral entry by targeting the s protein include peptide p9 and α-helical lipopeptides (preclinical), 154 and those targeting the s2 subunit of the s protein mainly include hr1p, hr1m, hr1l, hr2l, hr2p, and hr2l (preclinical). 155 due to the strong affinity between ace2 receptor and the rbd of sars-cov-2, another opportunity might be through the development of antibodies to block ace2. the second method is to use a large amount of soluble ace2 to directly block the spike protein. the third method is to find a drug that directly inhibits the spike membrane fusion process, such as the aids drug enfuvirtide. the fourth approach is to identify an agent that inhibits the activity of disintegrin and metalloproteinase 17 (adam17), which may also prevent viral release and proliferation in cells and protect ace2 and the lungs. 87 mrna vaccine technology sars-cov-2 vaccines are already under development. the mrna and dna vaccines are promising approaches to prevent cellular invasion by similar coronaviruses in the future. in january 2020, the coalition for epidemic preparedness innovations (cepi) announced three partnerships to develop a new coronavirus vaccine. among them, moderna and inovio presented mrna and dna vaccine technologies, respectively. the goal of the three teams is to have at least one candidate vaccine capable of preventing coronavirus infection in 16 weeks, to be tested in a phase i clinical trial. moderna's vaccine model is designed to use mrnas to safely pre-expose the immune system to a small amount of encoded proteins usually generated by the pathogen, so that the immune system becomes prepared to fight future pathogens and prevent infection. the candidate genes developed by moderna contain mrnas, and combining multiple mrnas into a single vaccine might be useful to rapidly induce responses, which is an approach that may be applied for future, emerging pandemic threats too. at the same time, because the mrna in the cell will be degraded over time, the mrna vaccine will not continue to produce antigen components for a long time, which can avoid the risk of continued stimulation of the immune system. generally, mrna vaccines are also described as simple to prepare and to yield remarkable results. additional advantages, as well as disadvantages, of nucleic acid-based vaccines were described in detail. furthermore, the basis of traditional vaccines, proteins or polysaccharides, cannot be straight forward applied at present against many pathogens. therefore, mrna vaccines would be suitable for achieving rapid, mass production of emergency vaccines in the event of a major epidemic. however, because rna is easily degraded, inducing cells to absorb mrna quickly is a challenge. in recent years, the method for delivery of mrna into cells has been greatly improved, and stemirna and moderna have adopted nanolipid (lpp or lnp (lipid nanoparticle)) drug-loading technology. however, this is still to be developed for mrnas. furthermore, ensuring safety and effectiveness is another challenge for viral mrna vaccines, and clinical validation will have to be carried out carefully. moderna is a worldwide leader in mrna therapy, with currently nine mrna-type vaccine candidates (e.g., mrna-1273 against sars-cov-2). the national institutes of allergy and infectious diseases (niaid) of the national institutes of health (nih) and the mrna vaccine giant moderna have teamed up to bring the new coronavirus vaccine possibly to phase ii within a few months, and phase iii late this year. simultaneously, in january 2020, the translational medicine platform of dongfang hospital affiliated with tongji university cooperated with stemirna (shanghai) biotechnology co., ltd. to rapidly promote the development of a new coronavirus mrna vaccine. in february 2020, the chinese scientific research team announced that animal testing of the newly developed coronavirus vaccine has begun. if animal testing goes well, this new vaccine will enter human clinical trials within a few months. also in february 2020, zhuhai livanda biotechnology co., ltd. announced that the first batch of new coronavirus mrna vaccine standard samples completed during the spring festival had been delivered to relevant national authorities on for animal testing and efficacy verification. the researchers detected the production of target antibodies in mouse serum at day 12 following immunization. the company claimed that this was also the first time worldwide that new coronavirus vaccines developed based on mrna technology have resulted in antibodies in animals. livanda is currently pushing ahead with the project through extensive cooperation with the academy of military medical sciences, the guangdong provincial institute of supervision, and the macau university of science and technology. the antigen used in its development is the same as that used by moderna. dna vaccine technology dna vaccine technology may have many advantages (e.g., safe, fast, less technical barriers), along with potential limitations too. 156 a phase i human clinical trial of the mers-cov vaccine has proven its safety and effectiveness. 157 the dna vaccine based on the sars-cov s protein developed by the team of barney graham and gary nabel of the us-nih vaccine research center (vrc) has achieved positive results in animal experiments and a clinical phase i trial. 158, 159 inovio pharmaceuticals has accumulated extensive safety and immunogenicity data for mers-cov vaccine studies. because of the high degree of similarity between mers-cov and sars-cov-2, lessons from such a vaccine development process may be beneficial for the development of a dna vaccine against sars-cov-2. since january 2020, inovio has been collaborating with several experts and companies (some in china), and has currently already begun phase i clinical trials of the dna vaccine ino-4800. china's cansino biologics might be the leader as it was announced it has moved to phase ii testing of a vaccine called ad5-ncov. the latter is currently often being reported as a dna vaccine, but to be precise, it uses viral vectors (adenovirus) to deliver dna related to sars-cov-2. part of the project is carried out currently with the chinese military medical research institute. the vaccine candidate is constructed using genetic engineering methods and defective human type 5 adenovirus as a vector that can express the sars-cov-2 s antigen, to stimulate the body to produce strong humoral or cellular immunity. sars-cov and mers-cov belong to subgroups 2b and 2c of the betacoronaviruses, respectively, and sars-cov-2 is a new member of betacoronaviruses, distinct from sars-cov and mers-cov. sars-cov and sars-cov-2 are similar in terms of invasion and self-replication, whereas mers-cov has different targets. the cellular receptor of sars-cov and sars-cov-2 is ace2, plus cd147 for sars-cov-2, while that of mers-cov is dpp4 (cd26). all of them evolved a mechanism to escape the host cell immune system. sars-cov and sars-cov-2 affect the ras system by suppressing ace2, leading to the onset of symptoms. mers-cov causes symptoms by producing inflammatory cytokines and invading t cells. sars-cov, mers-cov, and sars-cov-2 infections have similar symptoms, including fever, cough, myalgia, and shortness of breath, among others. current treatments for sars mainly include ifn-α, antiviral treatments (e.g., ribavirin), plasma therapy, host-directed therapies, and systemic corticosteroids. for mers, several therapeutics are in development, including convalescent plasma, lopinavir/ritonavir, ribavirin, ifn, and novel therapies, including polyclonal antibodies, broad-spectrum antivirals, and amps. for covid-19, candidates mainly include drugs targeting the s protein, nonstructural proteins (3clpro, plpro, helicase, and rdrp), and viral rna synthesis blockers such as remdesivir and ribavirin. vaccines such as dna vaccine, mrna vaccine, and recombinant vaccines are being rapidly developed. so far this century, human beings have experienced several epidemic outbreaks, and each outbreak had a negative impact at different levels, including health, economy, and even psychology and human behavior. in the future, more precautious measures should be available to guide individuals and groups to take effective emergency measures and to support social stability, and physical and mental health. furthermore, additional studies of coronaviruses and disease epidemics should continue, to support the preparation for future responses, medical therapies, vaccines, and methods of relieving personal anxiety. this review has highlighted several approaches, and the names and progress related to several compounds and biologics currently under research and development, as well as the companies and researchers involved in these efforts. for future directions, it has also described the differences and similarities, as well as the potential routes and possibilities for targeting each of the main three viruses investigated here. epidemiology, genetic recombination, and pathogenesis of coronaviruses origins and evolution of the global rna virome classify viruses-the gain is worth the pain detection of a group 2 coronavirus in dogs with canine infectious respiratory disease human coronaviruses: what do they cause? epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china isolation of a novel coronavirus from a man with pneumonia in saudi arabia ecology, evolution and classification of bat coronaviruses in the aftermath of sars a pneumonia outbreak associated with a new coronavirus of probable bat origin characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov on the origin and continuing evolution of sars-cov-2 isolation and characterization of viruses related to the sars coronavirus from animals in southern china mutations, recombination and insertion in the evolution of 2019-ncov sars and mers: recent insights into emerging coronaviruses global epidemiology of bat coronaviruses from sars to mers, thrusting coronaviruses into the spotlight challenges presented by mers corona virus, and sars corona virus to global health middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease mers-cov as an emerging respiratory illness: a review of prevention methods the middle east respiratory syndrome (mers) viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection a case report of a middle east respiratory syndrome survivor with kidney biopsy results kinetics and pattern of viral excretion in biological specimens of two mers-cov cases environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea human intestinal tract serves as an alternative infection route for middle east respiratory syndrome coronavirus a novel coronavirus from patients with pneumonia in china early transmission dynamics in wuhan, china, of novel coronavirusinfected pneumonia clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china evolution and variation of 2019-novel coronavirus pattern of early human-to-human transmission of wuhan transmission dynamics of 2019 novel coronavirus (2019-ncov) modelling the epidemic trend of the 2019 novel coronavirus outbreak in china therapeutic options for the 2019 novel coronavirus (2019-ncov) clinical features of patients infected with 2019 novel coronavirus in wuhan genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding severe acute respiratory syndrome: clinical and laboratory manifestations mers-cov outbreak in jeddah-a link to health care facilities reducing mortality from 2019-ncov: host-directed therapies should be an option characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention severe acute respiratory syndrome: historical, epidemiologic, and clinical features pathogenetic mechanisms of severe acute respiratory syndrome studies of severe acute respiratory syndrome coronavirus pathology in human cases and animal models the clinical pathology of severe acute respiratory syndrome (sars): a report from spatiotemporal interplay of severe acute respiratory syndrome coronavirus and respiratory mucosal cells drives viral dissemination in rhesus macaques organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways severe acute respiratory syndrome associated coronavirus is detected in intestinal tissues of fatal cases sars-associated viral hepatitis caused by a novel coronavirus: report of three cases impact of acute kidney injury on outcome in patients with severe acute respiratory failure receiving extracorporeal membrane oxygenation acute renal impairment in coronavirus-associated severe acute respiratory syndrome multiple organ infection and the pathogenesis of sars clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study caution: clinical characteristics of covid-19 patients are changing at admission yeast expression and characterization of sars-cov n protein the sars coronavirus nucleocapsid protein-forms and functions nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle sars-associated coronavirus focus on middle east respiratory syndrome coronavirus (mers-cov) molecular characteristics, functions, and related pathogenicity of mers-cov prospects for a mers-cov spike vaccine mers-cov: understanding the latest human coronavirus threat the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists middle east respiratory syndrome coronavirus ns4b protein inhibits host rnase l activation the endonucleolytic rna cleavage function of nsp1 of middle east respiratory syndrome coronavirus promotes the production of infectious virus particles in specific human cell lines middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis structural insights into the middle east respiratory syndrome coronavirus 4a protein and its dsrna binding mechanism crystal structure of middle east respiratory syndrome coronavirus helicase middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein interaction of n-acetyl-seryl-aspartyl-lysyl-proline with the angiotensin-converting enzyme 2-angiotensin-(1-7)-mas axis attenuates pulmonary fibrosis in silicotic rats crystal structure of the receptor-binding domain from newly emerged middle east respiratory syndrome coronavirus potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 association of host tropism of middle east syndrome coronavirus with the amino acid structure of host cell receptor dipeptidyl peptidase 4 complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin host and infectivity prediction of wuhan 2019 novel coronavirus using deep learning algorithm molecular mechanism of evolution and human infection a new coronavirus associated with human respiratory disease in china evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission structure of dimeric full-length human ace2 in complex with b0at1 sars-cov-2 invades host cells via a novel route: cd147-spike protein the emerging role of ace2 in physiology and disease a virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses angiotensin-converting enzyme 2 in lung diseases a potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways activation of the chicken type i interferon response by infectious bronchitis coronavirus murine coronavirus mouse hepatitis virus is recognized by mda5 and induces type i interferon in brain macrophages/microglia ribonuclease l and metal-ion-independent endoribonuclease cleavage sites in host and viral rnas viral encounters with 2',5'-oligoadenylate synthetase and rnase l during the interferon antiviral response dimeric structure of pseudokinase rnase l bound to 2-5a reveals a basis for interferon-induced antiviral activity homologous 2',5'-phosphodiesterases from disparate rna viruses antagonize antiviral innate immunity middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist the orf4b-encoded accessory proteins of middle east respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster 2019-ncov transmission through the ocular surface must not be ignored transmission of 2019-ncov infection from an asymptomatic contact in germany sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes the single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to wuhan 2019-ncov infection. front two things about covid-19 might need attention development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? middle east respiratory syndrome coronavirus (mers-cov) infection: epidemiology, pathogenesis and clinical characteristics mers: progress on the global response, remaining challenges and the way forward nanopore target sequencing for accurate and comprehensive detection of sars-cov-2 and other respiratory viruses mers-cov spike protein: targets for vaccines and therapeutics identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection structural definition of a neutralization epitope on the n-terminal domain of mers-cov spike glycoprotein the amino acids 736-761 of the mers-cov spike protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents coronaviruses-drug discovery and therapeutic options meplazumab treats covid-19 pneumonia: an open-labelled, concurrent controlled add-on clinical trial remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study potential inhibitors against 2019-ncov coronavirus m protease from clinically approved medicines theoretical study of the anti-ncp molecular mechanism of traditional chinese medicine lianhua-qingwen formula (lqf) broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses first case of 2019 novel coronavirus in the united states development of a standard treatment protocol for severe acute respiratory syndrome corticosteroid therapy for critically ill patients with middle east respiratory syndrome clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients aberrant pathogenic gm-csf+ t cells and inflammatory cd14 +cd16+ monocytes in severe pulmonary syndrome patients of a new coronavirus characterization of novel monoclonal antibodies against merscoronavirus spike protein discovery of hydrocarbon-stapled short α-helical peptides as promising middle east respiratory syndrome coronavirus (mers-cov) fusion inhibitors a systematic review of therapeutic agents for the treatment of the middle east respiratory syndrome coronavirus (mers-cov) identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review cell therapy in acute respiratory distress syndrome challenges and responses in human vaccine development receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine expression of b subunit of e. coli heat-labile enterotoxin in the progenies of transgenic tobacco bred by crossing nuclear-and chloroplast-transgenic lines immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice one-health: a safe, efficient, dual-use vaccine for humans and animals against middle east respiratory syndrome coronavirus and rabies virus cryo-em structure of the 2019-ncov spike in the prefusion conformation dna vaccine of sars-cov s gene induces antibody response in mice chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody an emerging role of dipeptidyl peptidase 4 (dpp4) beyond glucose control: potential implications in cardiovascular disease structural modeling of 2019-novel coronavirus (ncov) spike protein reveals a proteolytically-sensitive activation loop as a distinguishing feature compared to sars-cov and related sars-like coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov disulfiram can inhibit mers and sars coronaviruspapain-like proteases via different modes de novo design of α-helical lipopeptides targeting viral fusion proteins: a promising strategy for relatively broad-spectrum antiviral drug discovery structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor new vaccine technologies to combat outbreak situations safety and immunogenicity of an anti-middle east respiratory syndrome coronavirus dna vaccine: a phase 1, open-label, single-arm, dose-escalation trial a dna vaccine induces sars coronavirus neutralization and protective immunity in mice a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial potent binding of 2019 novelcoronavirus spike protein by a sars coronavirus-specific human monoclonalantibody potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies exceptionally potent neutralizationof middle east respiratory syndrome coronavirus by human monoclonalantibodies interaction between heptad repeat 1 and 2 regions inspike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors fusion mechanism of 2019-ncov andfusion inhibitors targeting hr1 domain in spike protein the authors gratefully acknowledge the financial support from the national natural science foundation of china (grant no. 31870836), and the 1.3.5 project for disciplines excellence, west china hospital, sichuan university (zyyc20005 to w.c.). competing interests: the authors declare no competing interests.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. key: cord-346777-zmmnn9b2 authors: lester, sandra; harcourt, jennifer; whitt, michael; al-abdely, hail m.; midgley, claire m.; alkhamis, abdulrahim m.; aziz jokhdar, hani a.; assiri, abdullah m.; tamin, azaibi; thornburg, natalie title: middle east respiratory coronavirus (mers-cov) spike (s) protein vesicular stomatitis virus pseudoparticle neutralization assays offer a reliable alternative to the conventional neutralization assay in human seroepidemiological studies date: 2019-09-11 journal: access microbiol doi: 10.1099/acmi.0.000057 sha: doc_id: 346777 cord_uid: zmmnn9b2 middle east respiratory syndrome coronavirus (mers-cov) is a novel zoonotic coronavirus that was identified in 2012. mers-cov infection in humans can result in an acute, severe respiratory disease and in some cases multi-organ failure; the global mortality rate is approximately 35 %. the mers-cov spike (s) protein is a major target for neutralizing antibodies in infected patients. the mers-cov microneutralization test (mnt) is the gold standard method for demonstrating prior infection. however, this method requires the use of live mers-cov in biosafety level 3 (bsl-3) containment. the present work describes the generation and validation of s protein-bearing vesicular stomatitis virus (vsv) pseudotype particles (vsv-mers-cov-s) in which the vsv glycoprotein g gene has been replaced by the luciferase reporter gene, followed by the establishment of a pseudoparticle-based neutralization test to detect mers-cov neutralizing antibodies under bsl-2 conditions. using a panel of human sera from confirmed mers-cov patients, the vsv-mers-cov particle neutralization assay produced results that were highly comparable to those of the microneutralization test using live mers-cov. the results suggest that the vsv-mers-cov-s pseudotype neutralization assay offers a highly specific, sensitive and safer alternative method to detect mers-cov neutralizing antibodies in human sera. middle east respiratory syndrome coronavirus (mers-cov) is a zoonotic pathogen that is associated with respiratory virus infections ranging in severity from asymptomatic to severe respiratory illnesses and death. mers-cov was first isolated from a 60-year-old patient who died with viral pneumonia and acute renal failure in 2012 in saudi arabia [1, 2] . mers-cov cases have been identified in 27 countries, although all cases outside of the arabian peninsula have been associated with exportations from the arabian peninsula [3] [4] [5] [6] [7] . as of 28 february 2019, the world health organization (who) has reported 2374 confirmed cases of mers-cov infection globally, resulting in 823 deaths (https://www. who. int/ emergencies/ mers-cov/ en/). genetically, mers-cov most closely resembles severe acute respiratory syndrome coronavirus (sars-cov), a betacoronavirus known as the first coronavirus to cause severe respiratory infections in humans. sars-cov emerged from an animal host in 2002 and caused a worldwide outbreak comprising an estimated 8000 cases with 800 deaths [7] [8] [9] [10] . similar to sars-cov, mers-cov infections represent zoonotic transmission events [11] [12] [13] . several sero-epidemiology studies have demonstrated that dromedary camels from the arabian peninsula and north africa have high titres of neutralizing antibodies against mers-cov, which can be detected in camel sera collected over 30 years [14] [15] [16] [17] . in contrast to the sars outbreak, which was brief, mers-cov cases continue to occur 7 years after its identification. the continuous spread of mers-cov serves as a constant reminder of the propensity of novel human coronaviruses to emerge from zoonotic reservoirs and cause severe disease in humans, and so continuous serological surveillance efforts remain essential for these viruses. the risk factors for camel-human transmission, humanhuman transmission and hospital outbreaks, and the roles of asymptomatic cases in human-human transmission are not fully understood. the clinical symptoms for mers patients overlap with those for other lower respiratory tract infections, further demonstrating the need to develop low biosafety-level laboratory-based diagnostic assays with high specificity and sensitivity. furthermore, countermeasures for the treatment or prevention of mers-cov infection, including vaccines, are unavailable. therefore, methods for measuring the potency and breadth of neutralization antibodies against mers-cov are essential to address these gaps in our knowledge, as well as to strengthen disease surveillance and disease preparedness platforms. the spike (s) protein of mers-cov is a surface glycoprotein that facilitates receptor binding, membrane fusion and viral entry into host cells via attachment to the cellular receptor dipeptidyl peptidase iv (dpp4), thus playing a pivotal role in mers-cov infection of the host cell [18] . the s protein is a major immunogenic component of covs and is the major target for neutralizing antibodies [19] [20] [21] [22] . laboratory tests, such as immunofluorescence assays (ifas) and conventional enzyme-linked immunosorbent assays (elisas), are commonly used screening tools for detecting anti-mers-cov serum antibodies. however, these assays often yield false-positive reactions due to cross-reactivity with other common human coronaviruses, and so confirmatory tests such as neutralization tests are utilized [23] . the most widely used confirmatory serological assays to detect and measure neutralizing antibody responses to mers-cov utilize neutralization of live virus, through either the plaque reduction neutralization test (prnt) or the microneutralization test (mnt) methods. however, due to the highly pathogenic nature of mers-cov and the absence of treatments and vaccines for mers-cov infection, prnt and mnt tests must be conducted under strict bio-containment procedures in a biosafety level 3 (bsl-3) laboratory. this limits epidemiological and pathogenesis studies globally. the development of an alternative method that can be reliably and conveniently used to determine the prevalence of mers-cov antibodies at a population level is crucial to prevent the spread of mers-cov. a safe and convenient way to detect neutralizing antibodies against mers-cov in serum is the use of viral pseudotypes. viral pseudotype particles have proven to be very valuable tools for studying virus entry pathways, evaluating the efficacy of vaccines, serological surveillance and gene therapy studies [24] [25] [26] [27] [28] [29] [30] [31] . several reports have utilized mers-lentivirus pseudotyped viruses in sero-epidemiological studies and basic research investigations [21, [32] [33] [34] [35] [36] . additionally, vesicular stomatitis virus (vsv) pseudotypes with mers-cov spike glycoproteins have been utilized to investigate the role of the mers s protein in virus-receptor-mediated entry, virus/host tropism and drug screening [37] [38] [39] . however, systematic comparative equivalency analysis between vsv pseudotypes bearing mers-cov spike glycoproteins and the conventional mnt using human sera from confirmed mers-cov patients have not been performed. the purpose of this study was to develop a vsv pseudotypebased neutralization assay to detect mers-cov s protein neutralizing antibodies and calculate its equivalence to traditional neutralization assays, therefore circumventing the use of live virus and the need for widely unavailable high-level containment biosafety facilities. here, we validate a mers-cov neutralization assay using a membraned vsv pseudotype system, where its glycoprotein is replaced with a luciferase reporter gene and its membrane is decorated with mers-cov s proteins. additionally, we perform equivalence testing in comparison with the conventional mnt. we demonstrate that these pseudoparticles are neutralized by sera from mers-cov-infected patients. furthermore, most of the patient sera samples have neutralization activities, and these results were consistent with the neutralization activity that was measured by the conventional microneutralization assay using live mers-cov. these results demonstrate that the mers-cov-s protein pseudotyped vsv particle-based neutralization assay would serve as a safe, reliable and highly specific alternative method to detect mers-cov neutralizing antibodies to be used for future sero-epidemiological studies. baby hamster kidney-21 (bhk-21) cells were cultured and maintained in dulbecco's modified essential medium (dmem) containing 5 % fetal bovine serum (fbs) (life technologies/gibco), and 1× penicillin/streptomycin (p/s) (sigma) at 37 °c under a 5 % co 2 atmosphere. vero (african green monkey kidney epithelial) cells were cultured and maintained in dmem containing 10 % fbs and 1× p/s 37 °c under a 5 % co 2 atmosphere. different vsv-based pseudotypes bearing either mers-cov-s or vsv-g glycoproteins were produced in bhk-21 cells. the pseudoparticle titrations and neutralization assays were carried out in vero cells. pooled normal human serum (pnhs) was purchased from lee biosolutions and used as a negative control serum. human serum from a single patient with laboratory-confirmed mers-cov infection was used as the positive control serum. the positive control serum was collected from the first imported case of mers-cov in the usa during the case investigation, with a neutralizing titre of 320. a laboratory-confirmed sars-cov patient serum sample and a panel of human sera with confirmed high neutralizing antibody titres to human coronaviruses 229e, hku1, oc43 and nl63 were used in this study to evaluate the vsv-mers-cov-s particle-based neutralization assay for potential cross-neutralization. a total of 52 human sera samples from mers-cov-infected patients in saudi arabia were used to examine equivalences. anti-vsv-g monoclonal antibody was purchased from kerafast, inc. a codon-optimized s gene from the mers-cov florida isolate (genbank accession number: kj829365.1) was synthesized by genescript and sub-cloned into pcaggs 2.0 eukaryotic expression vector. vsv-mers-cov-s pseudoparticles were generated as previously described [24] . the pseudoparticle titres were determined in vero cells. vero cells were seeded in 96-well black and white tissue culture-treated plates (perkin-elmer) and were inoculated with 50 µl of pseudoparticles five fold serially diluted in serum-free dmem (1× p/s in triplicate in . after 1 h adsorption, the inocula were removed, replaced with fresh dmem containing 10 % fbs and 1× p/s, and incubated at 37 °c. at 24 h post-inoculation, luciferase activity was determined using the luciferase assay kit (promega, inc.) according to the manufacturer's instructions. the titre of the vsv-mers-cov-s pseudoparticles was defined as the highest dilution yielding 10 5 relative luciferase units (rlu). for the titration of vsv-mers-cov-s pseudoparticles, the pcaggs plasmids encoding vsv recombinant g and the empty vector (ev) pcaggs-ev were used to generate vsv-g pseudoparticles and pseudoparticles without heterologous protein (ev) as positive and negative controls, respectively. ten fold serial dilutions of vsv pseudotyped with mers-s (vsv-mers-s) or negative control pcaggs empty vector containing no glycoprotein (vsv-ev) were used to infect vero cells in 96-well plates. rlu were measured 24 h post-infection. the results are expressed as average rlu±standard deviation (sd) of triplicate wells from an experiment repeated three times with similar results. the error bars indicate the sd. briefly, vero cells were seeded in 96-well black and white tissue culture-treated plates 1 day prior to infection. human sera were heat-inactivated at 56 °c for 30 min, and diluted as two fold serial dilutions with an initial dilution of 1 : 40 in serum-free dmem containing 1× p/s. two hundred microlitres of each serum dilution were mixed thoroughly with 10 5 rlu-equivalent vsv-mers-cov-s pseudoparticles diluted to 200 µl and incubated at 37 °c under a 5 % co 2 atmosphere for 1 h. positive and negative control sera were included with each assay run. in triplicates, 100 μl of the pseudoparticleserum mixtures were transferred onto vero cell monolayers and incubated at 37 °c under a 5 % co 2 atmosphere for 1 h. after adsorption, 100 µl of dmem containing 10 % fbs p/s was added and incubated at 37 °c and 5 % co 2 for 23 h. at 24 h post-inoculation, luciferase activity was determined using the luciferase assay kit according to the manufacturer's instructions. the results are expressed as the percentage neutralization ±sd of three parallel wells from three independent experiments. to calculate neutralization, the luciferase activity from each serial diluted human sera sample was normalized to that from the negative control, pnhs. neutralization was calculated as: (average rlu value of pnhs−average rlu value with test serum/average rlu value of pnhs)×100. reciprocal endpoint titres were defined as the highest dilution of the test serum sample that decreased rlu values by 80 % or more. serum samples that failed to show at least an 80 % reduction at the 1 : 40 dilution were considered to be below the limit of detection. the slides were first coated with poly-l-lys and seeded with hek-293t cells 24 h prior to transfection. the cells were transfected with 1 µg of pcaggs-mers-cov-s plasmid dna or mock transfection. at 24 h post-transfection, the cells' expression of mers-cov-s was detected by convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera; 1 : 400), followed by fitc-labelled anti-human iga/igg/igm (1 : 500) (abcam). dapi stain was used and cells were imaged using a zeiss axioimager microscope at 20× magnification. all graphs were generated with the graphpad prism 7 software. all results were calculated and presented as the means±sd obtained from duplicate or triplicate independent experiments. pearson's correlation analysis and the bland-altman method were used to assess the comparative analyses between the vsv-mers-cov-s pseudoparticle neutralization assay and the mnt assay. statistical analyses were performed using graphpad prism 7 software. the specimens used in this study were determined to be nonhuman subject research by the centers for disease control and prevention (cdc) institutional review board (irb). to establish vsv-mers-cov-s pseudotype neutralization assays, we generated vsv-mers-cov pseudoparticles with surface s protein and a firefly luciferase reporter gene as previously described [24] . the expression of mers-cov s proteins was confirmed by immunofluorescence at 24 h post-transfection in hek293t cells (fig. 1a) . it is known that bhk-21 cells do not have the s-binding mers-cov receptor, dpp4, and are therefore not permissible to mers-cov infection, but vero cells do [18, 39] . efficient packaging of mers-cov s on the surface of the vsv pseudoparticles and the ability of the pseudoparticles to enter target cells through receptor binding were confirmed by comparing the titrated inoculation of vero and bhk-21 cells of negative control vsv pseudoparticles containing no viral glycoproteins on their surface (vsv-ev) with that for vsv pseudotyped with mers-cov-s proteins, designated vsv-mers-s (fig. 1b and c) . as expected, in the vero cells the luciferase activity was proportional to the dilution of the mers-s pseudoparticle inoculum (fig. 1) . in comparison to the control pseudoparticles, the mers-s pseudoparticles demonstrated an approximately six fold increase in luciferase activity (fig. 1) . neither the mers-s pseudoparticles nor the control pseudoparticles induced high levels of luciferase activity in bhk-21 cells (fig. 1) , further supporting the notion that bhk-21 cells are resistant to mers-cov infection [39] . based on the rlu values observed at each serial dilution, it was decided to use a 1 : 200 dilution as the input pseudoparticle dose for further experiments for the mers-cov s protein pseudotyped vsv particle-based neutralization assay. to establish optimal parameters for the mers-cov s protein pseudotyped vsv particle-based neutralization assay, the cell density was optimized for maximum luciferase activity. no significant differences in luciferase activities were observed at 10 3 -10 5 cellsper well, although maximum luciferase activity was reached at 10 4 cells per well, and so 10 4 cells per well was selected as the standard assay cell density (fig. s1a , available in the online version of this article). next, we investigated the incubation time for optimal luciferase activity. the maximum rlu values peaked at 24 h post-infection and the rlu values decreased with prolonged incubation times (fig. s1b ). therefore, 24 h post-infection was determined to be the most suitable infection time for mers-cov-s pseudoparticles. to investigate whether anti-mers-cov human sera neutralized vsv-mers-cov-s pseudoparticles, vsv-mers-cov-s pseudoparticles were preincubated with two fold serial dilutions of pnhs serum (negative control), convalescent sera from a mers-cov patient with a known neutralizing titre against live virus as a positive control (mers immune sera) or anti-vsv-g monoclonal antibody. luciferase activity was reduced when vsv-mers-cov-s was preincubated with positive control serum in a dose-dependent manner (fig. 2a) . in contrast, preincubation with the negative control pnhs or anti-vsv-g monoclonal antibody did not significantly reduce luciferase activity at any dilution factor, thus demonstrating that vsv-mers-cov-s pseudoparticles were specifically neutralized by serum from an individual with a confirmed prior mers-cov infection (fig. 2a) . as expected, at any dilution factor, preincubation with anti-vsv-g monoclonal antibody efficiently neutralized vsv-δg pseudoparticles packaged with vsv-g, although neither the mers-cov positive control serum or the negative control pnhs neutralized vsv-δg pseudoparticles packaged with vsv-g (fig. 2b) . neutralization may be assessed as the percentage reduction in infectivity at a single dilution. since our goal is to measure the endpoint neutralization titres of human sera that possibly contain mers-cov neutralizing antibodies, we evaluated the percentage neutralization reduction curve of vsv-mers-cov-s pseudoparticles when preincubated with a mers-cov immune serum with a known live-virus endpoint neutralization titre. the percentage reduction in rlu (% neutralization) was determined by calculating the difference between the average rlu number in triplicate wells with the anti-mers-cov serum and the average rlu number in triplicate wells with the negative control pnhs sample, dividing it by the average rlu number in triplicate wells with the negative control pnhs sample and then multiplying the result by 100. neutralization of vsv-mers-cov-s pseudoparticles was dependent on the serum dilution, reducing rlu between 99 and 53 % at the dilutions tested (fig. 3) . using a target value of 80 % reduction in rlu in comparison to the pnhs control was within the linear range of reduction. our standard positive control mers-cov immune serum crossed the 80 % reduction line between 320 and 640 dilutions. this same serum has a known endpoint titre of approximately 320 in neutralization assays using live virus (unpublished data), demonstrating relative equivalence in the two assays for this serum. we previously measured the endpoint neutralizing antibody titres of 52 human sera collected from mers-cov patients using a live virus mnt. to determine if the vsv-mers-cov-s pseudoparticle neutralization test results in equivalent endpoint titres in comparison to the live virus mnt, we compared the endpoint titres from the live virus mnt to the endpoint titre necessary to achieve an 80 % reduction in rlu with vsv-mers-cov-s pseudoparticles. neutralizing antibody titres ranging from 40 to 1280 from 52 human serum samples against mers-cov were successfully detected by both neutralization assays (fig. 4) . sixty-seven per cent (n=35) of the serum samples had the same endpoint neutralization titres as those measured by either technique. we observed that approximately 29 % (n=15) of the serum samples gave higher neutralizing titres in the vsv-mers-cov-s pseudoparticle neutralization assay than the conventional mnt. furthermore, five sera with neutralization titres below the limit of detection by mnt had neutralizing titres that were detectable by the vsv-mers-cov-s pseudoparticle neutralization assay, thus demonstrating that the vsv-mers-cov-s pseudoparticle neutralization assay may be more sensitive (fig. 4) . the titres from both assays were then analysed statistically. pearson's correlation analysis of all data showed a strong correlation between the two neutralization assays, with r=0.9348, p<0.0001, 95 % confidence interval (ci)=0.8886 to 0.9622 (fig. 4a) . bland-altman method comparison analysis further demonstrated that these two methods are highly comparable. the average of the logarithmic difference of the two methods was 0.3269 (fig. 4b) . collectively, these data indicate that the vsv-mers-cov-s pseudoparticle neutralization assay can be a reliable alternative to the live virus-based mnt. there are six known human coronaviruses that cause respiratory disease in humans. in order to test the specificity of our vsv-mers-cov-s pseudoparticles and their potential cross-reactivity with other common human coronaviruses, we tested 40 serum samples from humans with no known anti-mers-cov exposure. when using the 80 % reduction in rlu as the cut-off value, none of the serum samples neutralized vsv-mers-cov-s pseudoparticles at any dilution factor (fig. s2) . we also tested potential cross-neutralization of vsv-mers-cov pseudoparticles with sera from other humans with confirmed infections with the human coronaviruses 229e, hku1, oc43, nl63 and sars-cov. using the neutralization parameters of 80 % or greater reduction in rlu, we found no cross-neutralization with 229e, hku1, oc43, nl63 or sars-cov anti-sera (fig. 5) . these data confirm the specificity of the vsv-mers-cov-s pseudoparticles. mers-cov continues to circulate on the arabian peninsula and can cause severe infections, including fatalities. at present, there are no antiviral therapeutics or approved vaccines to combat mers-cov infections. the presence of serum neutralizing antibodies ≥20 is a reliable indicator of prior mers-cov exposure. neutralization assays using live viruses are recognized as the gold standard serological assays to confirm the antibody responses to mers-cov infection. however, one major obstacle to performing the mnt assay is the requirement for access to high-level biocontainment laboratories, due to the use of live mers-cov. thus, only a limited number of facilities and researchers are able to perform such neutralization assays. additionally, determining the neutralization of infectious virus in this assay requires the assessment of cytopathic effect in 96-well plates, which is labour-intensive and time-consuming. furthermore, because of the large viral genome, reverse genetics for mers-cov is challenging, and still needs to be used under bsl-3 containment, thereby limiting molecular manipulation and studies. these limitations highlight the need to develop alternative methods for detecting mers-cov neutralizing antibodies. an alternative approach to overcome these shortcomings is the use of viral pseudotypes. generally, lentiviral and vsvbased pseudotypes are used for the study of enveloped viruses. several studies have utilized lentiviral and vsv pseudotype systems harbouring mers-cov spike glycoproteins to study viral entry, viral tropism and other basic science questions [27, 29, [37] [38] [39] [40] . in addition to being utilized to probe basic science questions, pseudoparticle-based neutralization assays have been used for serological surveillance of reservoir species for several other zoonotic viruses in addition to mers-cov [17, 33, [41] [42] [43] [44] [45] [46] . studies have employed lentiviral and env-deficient hiv-1 backbone vector pseudotype systems for mers-cov seroepidemiological surveillance in humans [32] [33] [34] [35] [36] . using dromedary camel sera, hemida et al. demonstrated that lentiviral-based mers-cov s pseudoparticles detected antibodies specific to mers-cov. in our hands, lentiviral particles grew to lower titres than vsv particles, making it challenging to prepare high volumes of stock for use in multiple studies. standardizing many particles for use in multiple studies is important for making comparisons between studies in this context. barlan et al. utilized vsv pseudotyped with mers-cov-s and demonstrated that susceptibility to mers-cov infection is enhanced by cellular proteases that cleave the s protein [37] . fukuma et al. demonstrated that infection with vsv-based pseudoparticles bearing truncated c-terminal mers-cov-s protein and non-truncated mers-cov-s protein was inhibited by rabbit anti-mers-cov s serum and that infection was dependent on the expression of dpp4 [40] . this demonstrated that vsv-based mers-s pseudoparticles can be utilized for neutralization assays. nonetheless, studies featuring seroepidemiological surveillance in humans utilizing a vsv pseudotype system bearing mers-cov spike glycoproteins had not previously been performed. this report describes the first comparison between the neutralization assay using vsv-mers-cov-s pseudoparticles and the mnt using a panel of human sera. this study is limited by only utilizing 52 specimens, which limits its statistical power. although analysing more specimens might yield a more powerful comparative analysis, the complexities of limited availability, diplomatic issues and biosafety concerns make acquiring mers-cov human specimens less feasible. despite this limitation, statistical analyses comparing mnt and vsv-mer-cov pseudoparticle neutralization indicate there was a positive correlation between the two methods. although there was a strong correlation between the two methods, the vsv-mers-cov-s pseudoparticle neutralization assay is slightly more sensitive than the conventional mnt. one potential explanation for this is the surface density of s protein. the generation of pseudoparticles by transient transfection in continuous cell lines, rather than producer cells that are more physiologically relevant, may lead to the production of large amounts of virus with less s protein on its surface. thus, the pseudoparticles may be more susceptible to neutralization because fewer neutralizing antibodies are required to block the entry of mers-cov. however, in corroboration with our data, fukushi et al. previously demonstrated that a neutralization test based on a vsv-based sars-cov-s protein bearing pseudoparticles was also more sensitive than the conventional virus neutralization test using live sars-cov [47] . in comparison with the mnt method, the pseudoparticle neutralization assay required half as much time to test for mers-cov neutralizing antibodies. more importantly, this assay can be performed in a low-biosafety containment laboratory. in addition to being faster and more sensitive than conventional neutralization assays while maintaining specificity, our vsv-mers-cov-s pseudoparticle system offers a convenient approach for examining the antigenic and neutralizing epitope differences among different s proteins from divergent strains of mers-cov. the data we have presented here use one s plasmid, but the pseudoparticles can be packaged using the spike sequence from other virus strains. because s protein is provided in trans in a pcaggs vector, it can be easily mutated or swapped for any spike protein an investigator is interested in studying. there are existing reverse genetics systems for human coronaviruses, including mers-cov, but those systems are more difficult to generate and require bsl-3 facilities [48] [49] [50] . using those full-virus reverse genetics systems are essential for viral replication and pathogenesis studies, but for neutralization studies, the presence of spike alone is sufficient. mers convalescent sera did not neutralize vsv particles packaged with vsv-g, demonstrating that neutralization was due to serum antibodies binding specifically to mers s. one of the main concerns in designing a mers-cov neutralization test is the presence of five other known human coronaviruses, four of which cause common infections. it had previously been shown that anti-coronavirus sera crossneutralize amongst similar coronaviruses [51] . in this study we determined that vsv-mers-cov-s pseudoparticles were not neutralized by antisera from alpha-or betacoronaviruses, further demonstrating the assay's high specificity. in conclusion, we established a neutralization test using a vsv-based pseudotyped virus possessing mers-cov s protein and expressing a luciferase reporter gene. we used the vsv-mers-cov-s pseudoparticle neutralization assay at bsl-2 containment, improving the safety of mers-cov neutralization assays. additionally, we found the system to be more rapid and sensitive and easier to use than a conventional microneutralization assay. these characteristics make the vsv-mers-cov-s pseudoparticle neutralization assay a more attractive, convenient and suitable assay for mers-cov screening and confirmatory serology testing. it offers a safe, rapid alternative method to monitor the potency and breadth of neutralization antibodies against mers-cov exposure in future seroepidemiological studies. isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome emerging diseases. soaring mers cases in saudi arabia raise alarms the global spread of middle east respiratory syndrome: an analysis fusing traditional epidemiological tracing and molecular phylodynamics an outbreak of middle east respiratory syndrome coronavirus infection in south korea middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome vs. the middle east respiratory syndrome cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) human infection with mers coronavirus after exposure to infected camels, saudi arabia antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study mers coronavirus neutralizing antibodies in camels, eastern africa seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the amino acids 736-761 of the mers-cov spike protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents mers-cov spike protein: a key target for antivirals the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies the amino acids 736-761 of the mers-cov spike protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents serological assays for emerging coronaviruses: challenges and pitfalls generation of vsv pseudotypes using recombinant δg-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein novel functional hepatitis c virus glycoprotein isolates identified using an optimized viral pseudotype entry assay chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies pseudotyped lentiviral vectors: one vector, many guises comparison of serological assays in human middle east respiratory syndrome (mers)-coronavirus infection seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia evaluation of candidate vaccine approaches for mers-cov identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism adaptive evolution of mers-cov to species variation in dpp4 inability of rat dpp4 to allow mers-cov infection revealed by using a vsv pseudotype bearing truncated mers-cov spike protein development of a neutralization assay for nipah virus using pseudotype particles the production and development of h7 influenza virus pseudotypes for the study of humoral responses against avian viruses comparative serological assays for the study of h5 and h7 avian influenza viruses safe pseudovirusbased assay for neutralization antibodies against influenza a(h7n9) virus bat origins of mers-cov supported by bat coronavirus hku4 usage of human receptor cd26 receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to sars-cov reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus transgene expression in the genome of middle east respiratory syndrome coronavirus based on a novel reverse genetics system utilizing red-mediated recombination cloning cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests the authors wish to acknowledge financial support from cdc lassi 2016. the authors declare that there are no conflicts of interest. the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. names of specific vendors, manufacturers, or products are included for public health and informational purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by the centers for disease control and prevention or the us department of health and human services. the specimens used in this study were determined to be non-human subject research by the centers for disease control and prevention (cdc) institutional review board (irb). key: cord-344246-sf9cymhc authors: diriba, kuma; awulachew, ephrem; getu, eyob title: the effect of coronavirus infection (sars-cov-2, mers-cov, and sars-cov) during pregnancy and the possibility of vertical maternal–fetal transmission: a systematic review and meta-analysis date: 2020-09-04 journal: eur j med res doi: 10.1186/s40001-020-00439-w sha: doc_id: 344246 cord_uid: sf9cymhc background: coronavirus is challenging the global health care system from time to time. the pregnant state, with alterations in hormone levels and decreased lung volumes due to a gravid uterus and slightly immunocompromised state may predispose patients to a more rapidly deteriorating clinical course and can get a greater risk of harm for both the mother and fetus. therefore, this systematic review was aimed to assess the effect of coronavirus infection (sars-cov-2, mers-cov, and sars-cov) during pregnancy and its possibility of vertical maternal–fetal transmission. methods: a systematic search was conducted on pubmed, web of science, embase, google scholar and the cochrane library until the end of april. all authors independently extracted all necessary data using excel spreadsheet form. only published articles with fully accessible data on pregnant women infected with sars-cov, mars-cov, and sars-cov-2 were included. data on clinical manifestations, maternal and perinatal outcomes were extracted and analyzed. result: out of 879 articles reviewed, 39 studies involving 1316 pregnant women were included. the most common clinical features were fever, cough, and myalgia with prevalence ranging from 30 to 97%, while lymphocytopenia and c-reactive protein were the most common abnormal laboratory findings (55–100%). pneumonia was the most diagnosed clinical symptom of covid-19 and non-covid-19 infection with prevalence ranged from 71 to 89%. bilateral pneumonia (57.9%) and ground-glass opacity (65.8%) were the most common ct imaging reported. the most common treatment options used were hydroxychloroquine (79.7%), ribavirin (65.2%), and oxygen therapy (78.8%). regarding maternal outcome, the rate of preterm birth < 37 weeks of gestation was 14.3%, preeclampsia (5.9%), miscarriage (14.5%, preterm premature rupture of membranes (9.2%) and fetal growth restriction (2.8%). from the total coronavirus infected pregnant women, 56.9% delivered by cesarean, 31.3% admitted to icu, while 2.7% were died. among the perinatal outcomes, fetal distress rated (26.5%), neonatal asphyxia rated (1.4%). only, 1.2% of neonates had apgar score < 7 at 5 min. neonate admitted to icu was rated 11.3%, while the rate of perinatal death was 2.2%. in the current review, none of the studies reported transmission of cov from the mother to the fetus in utero during the study period. conclusion: coronavirus infection is more likely to affect pregnant women. respiratory infectious diseases have demonstrated an increased risk of adverse maternal obstetrical complications than the general population due to physiological changes occurred during pregnancy. none of the studies reported transmission of cov from the mother to the fetus in utero, which may be due to a very low expression of angiotensin-converting enzyme-2 in early maternal–fetal interface cells. coronaviruses (covs) are one of the major pathogens that are grouped in the family of coronaviridae, which primarily target the human respiratory system [1] . it is one of the emerging and reemerging viral outbreaks throughout the world. previous outbreaks of coronaviruses include the severe acute respiratory syndrome (sars)-cov epidemic in 2003 [2] and the middle east respiratory syndrome (mers)-cov in 2012 [3] , while the newly emergent coronavirus, initially referred to as 2019-ncov and subsequently termed sars-cov-2, the disease it produces has been termed covid-19, which causes respiratory infection and can progress to severe pneumonia and, in a small number of cases, death [4] . although these coronaviruses were isolated from different human and animal hosts at different times and locations, they all belong to the species severe acute respiratory syndromerelated coronavirus [5, 6] . the increasing mortality rate warrants that vulnerable populations in the society be identified and protected. when covid-19 and other cov infect women who are pregnant, it increases the risk of adverse obstetrical and neonatal outcomes and results in severe respiratory disease [5] . previous data from multiple studies of influenza and other respiratory infectious diseases have demonstrated an increased risk of maternal obstetrical complications when compared with nonpregnant women due to physiological changes occurring during pregnancy [7] . this association has also been previously demonstrated to occur when pregnant women became infected with either of the two pathogenic coronavirus infections (sars-cov 2 and mers-cov) [8] . coronavirus infection in pregnant women makes clinical management more difficult by prolonging and complicating the illness and compromises the treatment [9] . researchers are still in question regarding the transmission of the novel and previous coronavirus infection from a pregnant woman to her fetus, a process termed vertical transmission [10] [11] [12] . there are few published cases of coronavirus disease occurring during pregnancy and due to the possibility of mother-fetal vertical transmission, there is a concern that the fetuses may be at risk of congenital covid-19 and other cov outbreaks. due to the alarming spread of cov outbreaks throughout the world, a comprehensive understanding of the transmission of the virus from mother to fetus in utero like other emerging viral infections as zika virus and ebola virus [13, 14] , that can threaten the health and survival of an infected mother and fetus is essential for effective management of the infection and treatment. therefore, this systematic review and meta-analysis was aimed to assess the effect of coronavirus infection (sars-cov-2, mers-cov, and sars-cov) during pregnancy and its possibility of vertical maternal-fetal transmission. a systematic review and meta-analysis was aimed to assess the effect of coronavirus infection (sars-cov-2, mers-cov, and sars-cov) during pregnancy and its possibility of vertical maternal-fetal transmission following the methodological framework suggested by arksey and o'malley [15] . all relevant articles were searched without date limits using the following databases: pubmed, web science, embase, google scholar, cochrane library, and science direct according to the preferred reporting items for systematic reviews and meta-analysis (prisma) [16] . all searches were limited to article written in english given that such language restriction does not alter the outcome of the systematic reviews and meta-analysis [17] . the gray literature of observational studies was searched through the review of reference lists and input of content experts. we searched scientific publications from 2003 to 2020. all papers published until the end of april 30, 2020, and fulfill the inclusion criteria were considered. the search used the following keywords: coronavirus, novel coronavirus-2019 infection, pregnancy, middle east respiratory syndrome, severe acute respiratory syndrome, severe acute respiratory syndrome coronavirus-2, and vertical transmission. we searched all terms with the help of boolean operators like "and" or "or". coronavirus infection is more likely to affect pregnant women. respiratory infectious diseases have demonstrated an increased risk of adverse maternal obstetrical complications than the general population due to physiological changes occurred during pregnancy. none of the studies reported transmission of cov from the mother to the fetus in utero, which may be due to a very low expression of angiotensin-converting enzyme-2 in early maternal-fetal interface cells. studies selected for inclusion were assessed for methodological quality by all authors' independently using the standard critical appraisal instruments of the joanna briggs institute meta-analysis of statistics assessment review instrument (jbi-mastari) [18] . the disagreements were resolved by consensus. the primary outcome variable of this study was the pregnancy outcomes observed, listed as follows: preterm birth (ptb; either before 37 or 34 weeks of gestation), preeclampsia, preterm prelabor rupture of membranes, (pprom), fetal growth restriction (fgr), miscarriage, maternal death, mode of delivery and other clinical feature, laboratory findings and coexisting disease. the secondary outcomes were the perinatal outcomes observed as listed: fetal distress, apgar score < 7 at 5 min, neonatal asphyxia, admission to the neonatal intensive care unit (nicu), perinatal death, including both stillbirth, and neonatal death, evidence in utero transmission. the data were extracted using a standardized data extraction format, adapted from the joanna briggs institute (jbi), by three authors (kd, ea, and ag), independently extracting all necessary data using an excel spreadsheet form. then the extracted data were merged for systematic analysis. any disagreements during the data extraction were resolved through discussion and consensus. the main outcomes extracted from the study were: primary author, study design, publication year, country, patient demographics, maternal and perinatal outcome, and evidence of in utero transmission of coronavirus. all clinical characteristics of pregnant women infected with coronavirus, laboratory and radiological findings, treatment options, and data on associated risk factors were extracted by the authors. following data extraction, systematic review and metaanalysis were carried out using r software version 3.6.1 and stata statistical software (version 13) with usercontributed commands for meta-analyses: metaprop, metan, metainf, metabias, and metareg [19] . the effect sizes and ses of the studies were pooled using a random-effects model to calculate the pooled estimates of laboratory findings, other clinical features and coexisting diseases among patients with covid-19 infection. a meta-analysis was also planned to assess the association of various comorbidities and laboratory findings with the severity of disease. the standard error for each original study was calculated using the binomial distribution formula. evidence for statistical heterogeneity among reported prevalence was using the cochrane q-test and i 2 statics [20] . the pooled proportion was estimated by using the back-transform of the weighted mean of the transformed proportions for both the fixed-effects model and the random-effects model [21] . a significance level of p < 0.10 and i 2 > 50% was interpreted as evidence of heterogeneity [22, 23] . a potential source of heterogeneity was investigated by subgroup analysis and meta-regression analysis [24] . where statistical pooling was not possible, the findings were presented in a narrative form including tables and figures to aid in data presentation where appropriate. sensitivity analyses were conducted to weigh up the relative influence of each individual study on the pooled effect size using a user-written function, metainf. the presence of publication bias was assessed informally by visual inspection of funnel plots [25] . point prevalence, as well as 95% confidence intervals, was presented in the forest plot format. database searches identified a total of 879 articles. from these initial articles, 29 articles were excluded due to duplication. from the remaining 850 articles, 786 articles were excluded after review of their titles and abstracts confirmed nonrelevance to this review. therefore, 64 full-text articles were assessed for eligibility based on the preset criteria, which resulted in the further exclusion of 25 articles primarily due to the outcome of the interest being not reported in the study. ultimately, 39 studies met the eligibility criteria and were included in the final review ( fig. 1 and table 1 ). in the current review, 39 articles with a total of 1316 pregnant women with laboratory-confirmed cov [12, (table 1) . from commonly reported laboratory findings, lymphocytopenia was the most frequently reported in the three coronavirus infections with prevalence ranged from 63 to 100%, followed by elevated c-reactive protein and leukopenia with prevalence ranged from 45 to 100% (table 2) . the most common computed tomography imaging features in pregnant women infected with coronaviruses were ground-glass opacity followed by bilateral pneumonia with a prevalence of 65.8% and 57.9%, respectively (table 3) . in this study, ribavirin and oseltamivir were the most commonly used antiviral therapy used for the treatment of viral pathogens among pregnant women infected with coronaviruses with a prevalence of 65.2% and 56.5%, while the most common antibiotic therapy used for the treatment of common bacterial coinfection was azithromycin with prevalence of 35%. in this study, hydroxychloroquine was the leading drug used by people infected with coronaviruses with a prevalence of 79.7%. from the total coronavirus-infected pregnant women, around 78.8% were treated with oxygen therapy while 18.1% were supported by mechanical ventilation ( table 3 ). out of 1316 pregnant women infected with cov, 46.5% give birth at > 37 weeks of gestation, while the rates of ptb < 34 and < 37 weeks of gestation were 9.5% and 14.3%, respectively. preeclampsia was reported among 5.9% of pregnant women, while the rate of miscarriage for cov infection was 14.5%. pprom and fgr were rated 9.2% and 2.8%, respectively. from the total covinfected pregnant women, 31.3% were admitted to icu from which 2.7% were died. the prevalence of cesarean delivery was 56.9%, while 28.6% had undergone normal delivery. fetal distress was reported in 26.5%, while neonatal asphyxia was reported in only 1.4% of neonates. only, 1.2% of neonates had apgar score < 7 at 5 min. neonate admitted to icu was rated 11.3%, while the rate of perinatal death was 2.2%. in the current review, none of the studies reported transmission of cov from the mother to the fetus in utero during the follow-up period ( table 4 ). in our systematic review, different comorbidities that aggravate the infection were found among pregnant women infected with cov. from the total cov-infected pregnant women, the rate of gestational diabetes was 9.6%, while hypertension was reported in 8.5% of pregnant women infected with cov. the rate of asthma in pregnant women infected with cov was 5.5%, while cardiovascular disease and digestive disease rated 5.7% and 3.6%, respectively ( table 4 ). in this meta-analysis, mers-cov was the most predominant causative agent of severe cases among infected pregnant women with a prevalence of 77% with 95% ci , followed by sars-cov rated 48% with 95% ci . according to our findings, sars-cov-2 was the least causative agent of severe cases among the infected radiological finding pregnant women, which rated 25% with 95% ci [7-59] (fig. 2) . out of 39 eligible articles, 25 studies [12, reported information on infections caused by sars-cov-2 among a total of 1271 pregnancies. the prevalence of sars-cov-2 among preterm birth at < 37 and 34 weeks of gestation was 14.3% and 8.9%, respectively, while 46.2% of pregnant women give birth at > 37 weeks of gestation. preeclampsia was reported among 5.7% of pregnant women with covid-19. pprom was reported in 8.9%, while the rate of fetal growth restriction was reported in 1.2%. in this study, miscarriage was rated 2.4%. icuadmitted pregnant women were accounted for 28.5%, while the rate of maternal death was reported in 1.5%. the prevalence of cesarean delivery was 57% (table 5) . fetal distress was reported among 25%, while the rate of neonatal asphyxia was 1.6%. the prevalence of apgar score < 7 at 5 min was 1.4%. the rate of newborns admitted to nicu was 11.6% in which perinatal death was reported among 2.9%. none of the studies reported transmission of sars-cov-2 from the mother to the fetus in utero during the follow-up period (table 5) . out of 35 eligible articles, seven studies [50] [51] [52] [53] [54] [55] [56] reported information on 12 pregnant women infected with mers-cov. the prevalence of preterm birth among pregnant women of < 34 weeks of gestation was 33.3%, while 80% of pregnant women give birth at > 37 weeks of gestation. preeclampsia was observed among 5.7% of pregnant women infected with mers-cov. icu-admitted pregnant women accounted for 33.3%, while the rate of maternal death was reported to be 40%. the prevalence of pregnant women given birth by cesarean was 66.7%, while 16.7% of pregnant women underwent normal delivery. perinatal death was reported among 33.3% of the newborns, while none of the studies reported fetal distress, apgar score < 7 at 5 min, neonatal asphyxia, and admission to the neonatal intensive care unit. none of the studies reported transmission of mers-cov from the mother to the fetus in utero during the follow-up period (table 5) . out of 35 eligible articles, seven studies [57] [58] [59] [60] [61] [62] [63] reported information on 33 pregnant women infected with sars-cov. the prevalence of sars-cov among preterm birth < 37 and 34 weeks of gestation was 21.7% and 12%, respectively, while 38.5% of pregnant women give birth at > 37 weeks of gestation. pprom was reported in 12.5%, while the rate of fetal growth restriction was reported in 12.5%. miscarriage was reported among 38.1% of pregnant women. icu admitted pregnant women were accounted for 54.5%, while the rate of maternal death was reported in 12.5%. the prevalence of pregnant women giving birth by cesarean was 50%, while 22.2% of pregnant women underwent normal delivery. the prevalence of fetal distress and perinatal death was 33.3% and 10%, respectively, while none of the studies reported apgar score < 7 at 5 min, neonatal asphyxia and admission to the neonatal intensive care unit. none of the studies reported transmission of sars-cov from the mother to the fetus in utero during the follow-up period (table 5) . subgroup analysis was conducted to justify the cause of heterogeneity. subgroup analysis of the included studies showed that the possible cause of heterogeneity was a sample size difference, especially in sars-cov-2 (i 2 = 94%). the funnel plots suggest a publication bias for some of the study of the parameters (p < 0.02). we conducted a systematic review and meta-analysis to provide an overview of the effect of cov (sars-cov-2, mers-cov, and sars-cov) infection on maternal and perinatal, and the possibility of vertical transmission of the virus from pregnant women to the fetus. the rapidly spreading nature of covid-19 and previous cov infections could have a significant effect on human health. thus, attention should be given to pregnant women, and they should be included in preparedness and response plans. in previous outbreaks, clinicians did not volunteer to treat or vaccinate pregnant women because of concerns for fetal safety [64] . the pregnant state, with alterations in hormone levels and decreased lung volumes due to a gravid uterus and slightly immunocompromised state may predispose patients to a more rapidly deteriorating clinical course and face a greater risk of harm if they get respiratory infections. in the current study, the predominant signs and symptoms of hospitalized pregnant women suffering from covid-19 and other coronavirus infections were viral pneumonia, fever, cough, fatigue, and myalgia. the centre for disease control listed these symptoms to be the leading clinical feature of patients infected with covid-19 and other coronaviruses [65] . the high fever after delivery may have resulted from immunity reduction due to fatigue and blood loss in childbirth, anatomy of female genitalia, sweating during puerperium, and postpartum lactation. once postpartum fever occurred, obstetricians and gynecologists should follow and perform a differential diagnosis to exclude breast swelling, mastitis, urinary tract infections, common colds, and reproductive tract infections must be focused on. gastrointestinal symptoms like diarrhea and abdominal pain were also observed in pregnant women with covid-19 and other coronavirus infections. gestational diabetes and hypertension were the most common coexisting disorders with a prevalence of 9.6% and 8.5%. more than half of the pregnant women infected with coronaviruses were treated by antiviral and antibiotic therapy. in our study, the most frequently reported laboratory findings was lymphocytopenia (66.1%), followed by elevated c-reactive protein marker (56.6%), leukopenia (48.3%) and elevated lactate dehydrogenase (34.8%). this is similar to previous studies reporting on covid-19 and other cov infections [66] [67] [68] [69] . laboratory findings like leukopenia and lymphocytopenia were helpful to distinguish viral infections from common bacterial infections [70] . the most common computed tomography imaging features in pregnant women with covid-19 and non-covid-19 pneumonia were ground-glass opacity and bilateral pneumonia with a prevalence of 57.9% and 65.8%, respectively. the pathological basis of these changes could be due to inflammatory cell infiltration and interstitial thickening, cell exudation, and hyaline membrane formation. the most common treatment options used were hydroxychloroquine (79.7%), ribavirin (65.2%), oseltamivir (56.5%) and azithromycin (35%) to treat common bacterial pathogens when secondary infections occurred and treat viral pathogens. among covinfected pregnant women, oxygen therapy was 78.8%. based on the findings of the present study, higher prevalence of covid-19 and other cov was reported among preterm birth < 37 weeks of gestation. according to some studies report, infection with covid-19 during pregnancy can cause complications for both the mother and the fetus [59, 71] . this includes preeclampsia, preterm premature rupture of membranes, fetal growth restriction, and miscarriage. large numbers of cov-infected pregnant women with severe cases were admitted to the intensive care unit which even there is much controversy relating to the possibility of in utero transmission of sars-cov-2, mers-cov, and sars-cov. multiple samples were obtained at the time of labor and delivery to test for the presence of coronavirus by qrt-pcr, including amniotic fluid aspirated from the vagina during labor, cord blood, and segments of the umbilical cord, fetal membranes, and placenta, neonatal nasopharyngeal and throat swabs, gastric aspirate and meconium samples tested negative for the coronaviruses, suggesting there was no evidence of vertical transmission in women who developed covid-19 and non-covid-19 coronavirus pneumonia in late pregnancy [12, 63, 72] . studies conducted in london [73] reported a neonate born to a pregnant woman with covid-19 that tested positive for sars-cov-2 in the pharyngeal swab sample 36 h after birth, it was subsequently confirmed that qrt-pcr testing of the placenta and cord blood was negative for sars-cov-2, it is believed that the mother or a family member transmitted the infection to the infant through close contact after delivery, not in utero through placenta [39, 74] . in the current study, the systematic testing procedures for coronavirus infection, including chest radiograph and serial rt-pcr assays with multiple clinical samples did not demonstrate the presence of sars-cov-2, mers-cov and sars-cov in the newborn. cov antibody tests were performed with mother and newborn sera. in some mothers' , sera immunoglobulin g was detected by using serological tests. however, cov antibodies for igg, igm, and iga were detected in none of the newborn's blood samples [55, 75] . therefore, there is no evidence of intrauterine transmission of sars-cov-2 and other cov from mother to newborn infants. this may be due to a very low expression of angiotensin-converting enzyme 2 (ace2) in early maternal to fetal interface cells [76] . the virus can be transmitted through close contact or droplets to a new born after birth [73] . thus, mothers and their neonates should be taken care of in isolated rooms to prevent neonatal transmission and effective protection measures should be implemented during delivery and postdelivery care to prevent transmission of the virus from mothers to the newborn. only english articles or reports were considered for this review. the small number of cases in some of the included studies, the study design, and the lack of standardized criteria were the major limitations of this systematic review. additionally, there is a possibility that some patients were included in more than 1 report, although all authors independently reviewed all the included studies, carefully focusing on the different institutions reporting outcomes. we included case reports and case series, thus facing a higher risk of publication bias, which could affect the estimated outcome. furthermore, lack of denominator in case series used in this review is the other major factor that affects the estimated outcome. moreover, when focusing on the outcomes of covid-19 infection, and particularly perinatal outcomes, reported data are intuitively limited to a very short-term followup period and thus infections that occurred proximate to the delivery. this has the potential to overestimate the magnitude of risks such as ptb and underestimate more longitudinal risks such as fgr. in some of the studies, we did not find standardized criteria and timing of delivery of pregnancies affected by cov infection. in general, based on the published data collected, fever, cough, and myalgia were the most common clinical features, while the predominant abnormal laboratory findings reported were lymphocytopenia and c-reactive protein. bilateral pneumonia and ground-glass opacity were the most common radiological abnormal findings. oxygen therapy was the most common treatment option used while bacterial coinfection was treated by antibiotics therapy, and viral pathogen was treated by antiviral therapy. among the coronavirus species, mers-cov was the leading cause of severe cases in infected pregnant women. pregnant women infected with coronaviruses are at increased risk of adverse obstetrical outcomes, compared with the general population. the infection outcome was mainly associated with a relatively higher rate of cesarean delivery, preterm birth, intensive care unit admission, preeclampsia, miscarriage, fetal distress, and perinatal death. none of the studies reported transmission of cov from the mother to the fetus in utero. this may be due to a very low expression of angiotensin-converting enzyme 2 (ace2) in early maternal-fetal interface cells as suggested by different experts. isuog interim guidance on 2019 novel coronavirus infection during pregnancy and puerperium: information for healthcare professionals severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation of a novel coronavirus from a man with pneumonia in saudi arabia who director-general's opening remarks at the media briefing on covid-19-11 another decade, another coronavirus severe acute respiratory syndrome: historical, epidemiologic, and clinical features viral infections during pregnancy potential maternal and infant outcomes from (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections 225-management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) what are the risks of covid-19 infection in pregnant women? an analysis of 38 pregnant women with covid-19, their newborn infants, and maternal-fetal transmission of sars-cov-2: maternal coronavirus infections and pregnancy outcomes clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records maternal and infant death and the rvsv-zebov vaccine through three recent ebola virus epidemics-west africa, drc équateur and drc kivu: 4 years of excluding pregnant and lactating women and their infants from immunization pregnant in the time of ebola: women and their children in the 2013-2015 west african epidemic scoping studies: towards a methodological framework the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration the inclusion of reports of randomised trials published in languages other than english in systematic reviews systematic reviews of health promotion and public health interventions. melbourne: university of melbourne clinical features and chest ct manifestations of coronavirus disease 2019 (covid-19) in a single undue reliance on i 2 in assessing heterogeneity may mislead metaprop: a stata command to perform meta-analysis of binomial data explaining heterogeneity in meta-analysis: a comparison of methods the comparison of percentages in matched samples bias in meta-analysis detected by a simple, graphical test clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia detection of sars-cov-2 in placental and fetal membrane samples coronavirus disease 2019 (covid-19) during pregnancy: a case series coronavirus disease 2019 (covid-19) in pregnant women: a report based on 116 cases analysis of pregnancy outcomes of pregnant women during the epidemic of new coronavirus pneumonia in hubei clinical course of severe and critical covid-19 in hospitalized pregnancies: a us cohort study clinical analysis of pregnant women with 2019 novel coronavirus pneumonia possible vertical transmission of sars-cov-2 from an infected mother to her newborn perinatal transmission of covid-19 associated sars-cov-2: should we worry? clin infect dis clinical characteristics and risk assessment of newborns born to mothers with covid-19 early release-lack of vertical transmission of severe acute respiratory syndrome coronavirus 2, china a case of 2019 novel coronavirus in a pregnant woman with preterm delivery a 55-day-old female infant infected with 2019 novel coronavirus disease: presenting with pneumonia, liver injury, and heart damage clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan coronavirus disease 2019 infection among asymptomatic and symptomatic pregnant women: two weeks of confirmed presentations to an affiliated pair of new york city hospitals clinical manifestations and outcome of sars-cov-2 infection during pregnancy pregnancy and perinatal outcomes of women with coronavirus disease (covid-19) pneumonia: a preliminary analysis clinical and ct imaging features of the covid-19 pneumonia: focus on pregnant women and children clinical characteristics of 19 neonates born to mothers with covid-19 characteristics and outcomes of pregnant women hospitalised with confirmed sars-cov-2 infection in the uk: a national cohort study using the uk obstetric surveillance system (ukoss). medrxiv clinical characteristics of 46 pregnant women with a sars-cov-2 infection in washington state maternal mortality from covid-19 in mexico symptoms and critical illness among obstetric patients with coronavirus disease 2019 (covid-19) infection middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature mers-cov infection in a pregnant woman in korea emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report middle east respiratory syndrome coronavirus during pregnancy stillbirth during infection with middle east respiratory syndrome coronavirus impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome sars and pregnancy: a case report sars during pregnancy a casecontrolled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome pregnancy and perinatal outcomes of women with severe acute respiratory syndrome sars in pregnancy: this case study explores the first documented infection in the us the placentas of patients with severe acute respiratory syndrome: a pathophysiological evaluation severe acute respiratory syndrome in pregnancy pregnant women and the ebola crisis ct imaging features of 2019 novel coronavirus (2019-ncov) epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study a case series of children with 2019 novel coronavirus infection: clinical and epidemiological features hospital outbreak of middle east respiratory syndrome coronavirus multiple organ infection and the pathogenesis of sars analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia. jie he he hu xi za zhi = zhonghua jiehe he huxi zazhi = chin j tuberc respir dis pregnancy and perinatal outcomes of women with covid-19 pneumonia: a preliminary analysis clinical characteristics of covid-19 in pregnancy: analysis of nine cases newborn baby tests positive for coronavirus in london. the guardian a case report of neonatal covid-19 infection in china serologic evaluation of mers screening strategy for healthcare personnel during a hospitalassociated outbreak single-cell rna expression profiling of ace2 and axl in the human maternal-fetal interface the sars-cov-2 receptor ace2 expression of the maternal-fetal interface and fetal organs by singlecell transcriptome study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to acknowledge authors of primary study and dilla university, collage of health science and medicine and department of medical laboratory science. key: cord-353342-2n6kqyeo authors: corman, victor m.; albarrak, ali m.; omrani, ali senosi; albarrak, mohammed m.; farah, mohamed elamin; almasri, malak; muth, doreen; sieberg, andrea; meyer, benjamin; assiri, abdullah m.; binger, tabea; steinhagen, katja; lattwein, erik; al-tawfiq, jaffar; müller, marcel a.; drosten, christian; memish, ziad a. title: viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection date: 2016-02-15 journal: clin infect dis doi: 10.1093/cid/civ951 sha: doc_id: 353342 cord_uid: 2n6kqyeo background. the middle east respiratory syndrome (mers) coronavirus causes isolated cases and outbreaks of severe respiratory disease. essential features of the natural history of disease are poorly understood. methods. we studied 37 adult patients infected with mers coronavirus for viral load in the lower and upper respiratory tracts (lrt and urt, respectively), blood, stool, and urine. antibodies and serum neutralizing activities were determined over the course of disease. results. one hundred ninety-nine lrt samples collected during the 3 weeks following diagnosis yielded virus rna in 93% of tests. average (maximum) viral loads were 5 × 10(6) (6 × 10(10)) copies/ml. viral loads (positive detection frequencies) in 84 urt samples were 1.9 × 10(4) copies/ml (47.6%). thirty-three percent of all 108 serum samples tested yielded viral rna. only 14.6% of stool and 2.4% of urine samples yielded viral rna. all seroconversions occurred during the first 2 weeks after diagnosis, which corresponds to the second and third week after symptom onset. immunoglobulin m detection provided no advantage in sensitivity over immunoglobulin g (igg) detection. all surviving patients, but only slightly more than half of all fatal cases, produced igg and neutralizing antibodies. the levels of igg and neutralizing antibodies were weakly and inversely correlated with lrt viral loads. presence of antibodies did not lead to the elimination of virus from lrt. conclusions. the timing and intensity of respiratory viral shedding in patients with mers closely matches that of those with severe acute respiratory syndrome. blood viral rna does not seem to be infectious. extrapulmonary loci of virus replication seem possible. neutralizing antibodies do not suffice to clear the infection. the middle east respiratory syndrome coronavirus (mers-cov) was first isolated in 2012 in saudi arabia [1] . since 2012, at least 1595 laboratory-confirmed cases of mers-cov infection, mostly with respiratory tract illness, have been reported; 571 of these were fatal [2] . known cases and outbreaks have been linked to countries in the arabian peninsula [3] . large nosocomial outbreaks, such as in jeddah, kingdom of saudi arabia in 2014 and the republic of korea in 2015, have demonstrated the potential of the virus to spread in healthcare settings [4] [5] [6] . due to the sporadic nature of the disease, with cases and small outbreaks distributed over a wide geographic area, investigation of the natural history of infection has been limited. except for individual case descriptions, chronological data summarizing the main viral diagnostic parameters, such as viral load or antibodies, are lacking. better knowledge of the kinetics of viral shedding from different body regions could help prevent nosocomial transmission and inform clinical management. knowledge of serological features, such as the kinetics of antibody production, could guide decisions regarding diagnostic protocols and provide essential information regarding immunity and virus elimination. quantitative data, such as viral loads and antibody titers, could enable comparisons with related diseases, in particular, severe acute respiratory syndrome (sars), for which studies of natural history were conducted in the aftermath of the 2002-2003 epidemic [7] . patients were seen during a hospital-associated outbreak between 5 march and 1 may 2014. there was no prospective planning of statistical power. patients were selected for mers-cov testing by reverse transcription polymerase chain reaction (rt-pcr) based on general clinical condition, oxygen saturation, and their need for invasive or noninvasive ventilation. samples of patients who tested positive were taken at least daily, starting from 0-7 days after initial submission of samples for mers-cov diagnosis. the day of the first sample testing positive by rt-pcr was defined as the day of diagnosis. the mean delay between first positive sampling and return of laboratory results was 3 days. stored samples, for retrospective analysis, were not available. specimens were taken from tracheal secretions via suction catheters, from the throat and the eyes using sterile swabs, and from urine and stool via sterile containers. baseline information on enrolled patients is provided in supplementary table 1 . institutional review board approval was obtained from the research ethics committee at prince sultan military medical city. real-time rt-pcr was performed on rna extracts using the upe and orf1a target genes as described in [8, 9] . raw rna concentrations were transformed to absolute viral loads by conversion factors, according to sample type (supplementary table 2 ). virus isolation, with increased sensitivity via the use of caco2 human colon carcinoma cells, was performed as described in [10] . a recombinant enzyme-linked immunosorbent assay (elisa; anti-mers-cov elisa igg, euroimmun, lübeck, germany) was based on soluble mers-cov spike protein s1 domain expressed in hek-293t cells [11] . the test was conducted as described previously [12, 13] . sera were tested twice and the arithmetic mean of the 2 measurements was used. detection of immunoglobulin m (igm) antibodies was done using immunofluorescence slides carrying vero cells infected with full mers-cov, as described in corman et al [9] . these were converted into a homogenous reagent format by an in vitro diagnostics manufacturer (anti-mers-cov-iift; euroimmun). all sera were depleted of immunoglobulin g (igg) antibodies using eurosorb (euroimmun) reagent according to manufacturer instructions. a mers-cov microneutralization test (nt) was performed as described in [13] [14] [15] . predilution before setting up log 2 -dilution series was 1:10, defining 1:20 as the lowest possible significant titer for categorizing a sample as positive. statistical analyses were done using spss software (version 22). in all cases, correlation analyses and preliminary multiple regression analyses were conducted to exclude confounding due to patient age or disease duration. to determine kinetic virological parameters in mers-cov infection, we followed 37 hospitalized patients. mean age was 63 years (range, 24-90 years), and 73% of patients were male. mers-cov infection had been established in all cases by rt-pcr. sixty-five percent of all patients died during the course of study. sequencing of full or partial genomes from 35 of the study patients revealed the existence of at least 6 closely related virus lineages (supplementary figure 1 and table 1 ). some sequences had already been seen in an earlier study [5] . patients belonged to at least 3 nosocomial transmission clusters. three cases could not be associated with clusters. at time of positive diagnosis, patients had spent 11 days in hospital on average, with a maximum of 108 days. only 20 of the 37 patients had been hospitalized for less than a week. because of the unresolved timing of transmission events in nosocomial clusters and the existence of comorbidities in most patients, it was impossible to determine the day of onset of symptoms in the majority of patients. unambiguous knowledge of the day of onset of symptoms was available for only 9 patients. mean and median duration between symptom onset and admission was 3 days (range, 0-8 days). in these 9 cases, mean and median duration between onset and diagnosis was 8 days (range, 1-16 days). the mean age of the 9 cases was not significantly different from the mean age of all patients under study. to provide a common point of reference in the clinical course of all patients, the day of diagnosis (day of first rt-pcr-positive sample) was defined as day 0 in the subsequent analyses. eight hundred twenty-three specimens from the 37 patients were tested, including 661 tests for viral load in 6 different sample categories (supplementary table 2 ). because of the variable latency between diagnosis and enrollment, clinical samples were not evenly distributed over patients' courses of disease (supplementary figure 2 ). absolute viral rna concentrations and positive proportion of samples were determined in 661 samples. data are illustrated in figure 1 and supplementary table 2 . lower respiratory tract (lrt) samples had the highest viral loads, up to 6.3 × 10 10 copies/ml (mean, 5.01 × 10 6 copies/ml). average viral loads in all other sample types were significantly lower (2-tailed t test, p < .0001 for all comparisons). virus isolation trials using the 6 stool samples with the highest rna concentration had negative outcomes. almost half of all sera showed detectable viral loads during the first week after diagnosis (25 of 51 sera tested). virus isolation from 20 viremic serum samples (10 with and 10 without neutralizing antibodies) failed, despite a highly optimized protocol [10] . there was an inverse correlation between in vitro serum neutralization activity and viremia in 45 sera (pearson r = −0.31, p < .03). however, viral rna and neutralizing antibodies were codetected in several cases, suggesting that the detected viral rna may only in part represent infectious virions ( figure 2a ). concentrations of rna in serum did not correlate significantly with those in lrt samples collected on the same day (n = 31 pairs of serum and lrt samples; spearman correlation, p = .08; figure 2b ). distributions of average lrt viral load per patient were summarized in 3 subsequent time windows (figure 3 ). in particular, during the first 5 days after diagnosis, average viral loads were not normally distributed but had a skewed distribution with a preponderance of patients with high viral load. of note, the 17 patients in the 2 highest viral load categories (right-most columns in the top panel in figure 3 ) did not show a significantly increased proportion of fatal outcomes (χ 2 test, p = .12). the average viral load during the first week after diagnosis was 5 × 10 7 copies/ml in fatal cases and 3.9 × 10 6 copies/ml in survivors (2-tailed t test, p < .007). divergence of viral loads between survivors and fatal cases was more pronounced in the second week (1.6 × 10 5 and 7.8 × 10 6 copies/ml, respectively; p < .0006). serological courses could be followed for 35 patients. almost half of these (n = 17) were already reactive (via elisa) on the day of diagnosis. among 27 patients with complete serological follow-up during the first week after diagnosis, 89% (n = 24) had antibodies by end of the week in both elisa and neutralization tests. eighteen of these patients tested positive for igm by immunofluorescence assay (ifa) (titers >1:10) by end of the week. only 1 of the igm-positive patients did not have a concomitant positive elisa result by end of the week. all of the 12 patients with 2 weeks of serological follow-up seroconverted (elisa and neutralization tests). eleven of these 12 patients developed igm detectable by ifa. antibody kinetics averaged over all samples and tests are summarized in figure 4 . information on outcome was available for 34 patients with serological follow-up. all 12 patients who survived their infection showed seroconversion (elisa and nt) during the first week. all developed igm antibodies concomitantly. among 22 fatal cases, 14 showed seroconversion by elisa prior to death, the latest seroconversion occurring by day 11 postdiagnosis. twelve of 22 fatal cases developed neutralizing antibodies, and 11 developed detectable igm. antibody levels (elisa optical density [od] and log 2 nt titers) during the first week after diagnosis were not significantly different between surviving and fatal cases (2-tailed t test, p = .8). during the second week after diagnosis, the average elisa od values in survivors were significantly higher than in fatal cases (2.9 vs. 2.1, 2-tailed t test, p < .02). also, average nt titers were higher in survivors during week 2, but with less significant discrimination (2 7.5 vs 2 5.4 in survivors and fatal cases, respectively; 2-tailed t test, p < .06). elisa od values and log 2 nt titers were compared against log 10 viral loads in lrt samples. from 30 patients, elisa and viral load data were available based on matched serum and respiratory tract samples taken on the same days (198 matched data pairs, covering days 0-17 postdiagnosis). because of workload and biosafety, the number of nt assays had to be restricted. however, combined elisa, nt, and viral load data were available from 26 patients, with 91 matched datasets that covered days 1-17 after diagnosis. supplementary figure 3 summarizes the distribution of matched samples over time. pearson test identified significant linear correlation between antibodies and log 10 viral loads (elisa, r = −0.6; nt, r = −0.51; p < .001 in both analyses). however, plots of elisa and nt antibody results in matched sample pairs yielded no evidence of mutually exclusive occurrence of virus and antibodies ( figure 5a and 5b) . discussion we studied quantitative viral excretion and serum antibody kinetics of a substantial group of hospitalized patients infected with mers-cov. the time of diagnosis chosen as a chronological reference point represents the time when an infection is suspected in hospitalized patients, or when outpatients report to hospitals due to worsening symptoms. even though the day of onset was unknown in many of the studied cases, the presented virological courses represent the typical situation encountered during mers treatment in hospital settings. by providing absolute quantitative measures of virus excretion, we can for the first time compare results between mers and sars, a disease that is now thought to have involved higher pandemic potential than mers [16] . in our patients and elsewhere, the lrt was found to be the main source of mers-cov excretion [17] . unfortunately, there are few data on lrt virus excretion for sars-cov, because endotracheal sampling was widely discouraged during sars outbreaks to avoid nosocomial risks. we have shown, in one of the few available studies, that sars-cov was excreted from the lrt at mean concentrations of 1.2-2.8 × 10 6 copies/ml, reaching a maximum of 10 10 copies/ml [18] . that study was conducted in a similar clinical context (a treatment center in singapore) with similar timing of samples and clinical courses. determination of viral load was performed by the same laboratory, using equivalent calibrators and conversion factors. from this comparison, we can conclude that average and peak lrt viral loads in mers are equal to those in sars [18] . more data are available for upper respiratory tract (urt) viral loads in sars patients. peak urt rna concentrations can reach up to 5 × 10 5 copies/sample between days 7 and 10 after onset [18, 19] . the corresponding number for mers ( peaking at 4.1 × 10 6 copies/sample), is equivalent or higher. also, the 47.6% proportion positive for mers in urt exceeded the 38% proportion positive in 98 urt samples for sars patients in hong kong [19] . the timing of excretion is more difficult to compare, as many patients in studies on sars were outpatients who entered hospital because of their sars infection, whereas our mers patients were mostly inpatients [20] . in sars cases that occurred before hong kong authorities started active community contact tracing, the average time from symptom onset to admission was 4-7 days [20] . because case definitions and diagnostics were well-established during sars in hong kong, diagnostic samples would have been taken immediately upon admission. this can be aligned with the timing in our study based on a subcohort of patients for whom an onset of symptoms could be reliably determined. in these patients, the time from onset of symptoms to the initiation of laboratory diagnostics (8 days) was similar to that of early hong kong sars cases. the shedding peak in sars patients occurred after approximately 10-12 days from symptom onset, which is very similar to the shedding maximum observed in our study, under the assumption that the day of first diagnosis in our mers cases plausibly falls around the eighth day of symptoms (ie, directly after admission) [21, 22] . in summary, neither the virus concentration nor the timing of respiratory shedding provides an explanation for differences in transmissibility between mers-cov and sars-cov. experimental data suggest a higher sensitivity of mers-cov to respiratory epithelium-associated type i interferon, which might provide a plausible explanation for its lower transmissibility in comparison with sars-cov [23] . many other explanations are possible, however. the detection of mers-cov in serum is another similarity with sars. up to 79% of serum samples were found to contain sars-cov rna during the first week of illness, and around 50% during the second week [24] [25] [26] . these numbers match our observations for mers. free viremia seems unlikely as a cause of nosocomial transmission of mers, as no infectious virus was isolated from serum. there is evidence of sars-cov replicating in peripheral blood mononuclear cells, macrophages, and dendritic cells, albeit at low levels [27] [28] [29] [30] . in the present study, the absence of correlation of serum viral load with lrt viral load points to potential extrapulmonary replication. viremia despite the presence of neutralizing antibodies indicates a body region that is not accessible to neutralizing antibodies but which releases virus into the blood. sars-cov has been shown to replicate in several extrapulmonary organs without evidence of tissue damage [27] . one organ implicated in mers is the kidney. kidney failure has been reported in many cases, and mers-cov was originally isolated in kidney cells that express dpp4, the mers-cov entry receptor [11] . however, earlier healthcare-associated outbreaks have been centered near dialysis centers and nephrology departments, and have affected metabolically compromised patients who are predisposed to kidney failure when suffering from systemic disease affecting blood pressure and circulation. although patients with sars-cov showed viral rna detection rates up to 50% in urine [7, 22] , we rarely found urine-associated mers-cov rna in this study. as in sars, kidney failure in mers patients might well be explained by severe inflammatory reaction combined with the administration of potentially nephrotoxic drugs during intensive care [31] . nevertheless, it will be highly important to conduct postmortem examinations, particularly of the kidney, of patients who die during acute mers-cov infection. a clear difference from sars was the detection of viral rna in stool. in sars, the rna prevalence in stool samples was so high that testing of stool was proposed as a reliable and sensitive way to routinely diagnose the disease [7, 22] . active replication in the gut with live virus isolation has been demonstrated [32] . for mers, we found stool-associated rna in only 14.6% of samples, with rather low rna concentration, and had no success in isolating infectious virus. based on these data, fecal excretion may not have played a relevant role for nosocomial spread of mers-cov among the patients under study. as in sars, mers-cov nosocomial transmission was repeatedly ascribed to the potential of some patients to act as super-shedders or super-spreaders [6, 20] . our analysis of viral loads, particularly in the early acute phase of disease, supports the existence of a limited number of patients with extraordinarily high viral loads. as these patients were not more likely to die of the infection, they might not have had more severe symptoms, and thus might have been able to engage in social contact despite their disease. the course of mers antibody development resembles that of sars. patients infected with sars seroconverted during weeks 2 and 3 after onset [7] . most of the patients studied here had already seroconverted during the first week after diagnosis, which putatively represents the second week after onset. as in sars, igm was not detected earlier than igg, which limits its diagnostic utility, in particular when considering that igm against more prevalent human coronaviruses may cross-react with mers-cov [12, 33] . with current methodology, igm testing should be restricted to cases that require proof of recent and overcome mers-cov infection. information on the prognostic value of antibody response in sars is less clear. in the present study on mers, 36% (elisa) and 45% (nt) of fatal cases failed to mount an antibody response prior to death. however, differences became apparent only in the second week after diagnosis, pointing to only a weak protective effect against lung disease. the development of antibodies in serum was not followed by a rapid elimination of viral rna from the lung. neutralizing antibodies normally include immunoglobulin a (iga) secreted in respiratory fluids and saliva. we have recently shown that anti-mers-cov iga is indeed secreted in respiratory fluids [10] , suggesting that the development of iga comes too late to confer timely reduction of viral replication in infected mucosa. based on these data, vaccines against mers-cov should be designed so as to include and enhance cellular immune responses. supplementary materials are available at http://cid.oxfordjournals.org. consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. isolation of a novel coronavirus from a man with pneumonia in saudi arabia disease outbreak news: middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment. who/mers/ra/15 mers-cov outbreak in jeddah-a link to health care facilities an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia preliminary epidemiological assessment of mers-cov outbreak in south korea the aetiology, origins, and diagnosis of severe acute respiratory syndrome detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc transmission of mers-coronavirus in household contacts presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus detection of sars coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-pcr assays the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients detection of sars coronavirus in patients with suspected sars clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential detection of sars coronavirus in plasma by real-time rt-pcr quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome detection of severe acute respiratory syndrome coronavirus rna in plasma during the course of infection multiple organ infection and the pathogenesis of sars chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients sars-coronavirus replication in human peripheral monocytes/macrophages acute renal impairment in coronavirusassociated severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection contact investigation of a case of human novel coronavirus infection treated in a german hospital acknowledgments. we thank artem siemens, heike kirberg, monika eschbach-bludau, sebastian brünink, stephan kallies, and tobias bleicker (institute of virology, bonn, germany) for excellent technical assistance. we also thank dr terry c. jones, department of zoology, university of cambridge, united kingdom, for editing the final manuscript.financial support. this work was supported by european commission grant prepare (contract number 602525), as well as the deutsche forschungsgemeinschaft (dfg dr772/7-1).potential conflicts of interest. all authors: no potential conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-351413-3nfukrfl authors: al-ahmadi, khalid; alahmadi, sabah; al-zahrani, ali title: spatiotemporal clustering of middle east respiratory syndrome coronavirus (mers-cov) incidence in saudi arabia, 2012–2019 date: 2019-07-15 journal: int j environ res public health doi: 10.3390/ijerph16142520 sha: doc_id: 351413 cord_uid: 3nfukrfl middle east respiratory syndrome coronavirus (mers-cov) is a great public health concern globally. although 83% of the globally confirmed cases have emerged in saudi arabia, the spatiotemporal clustering of mers-cov incidence has not been investigated. this study analysed the spatiotemporal patterns and clusters of laboratory-confirmed mers-cov cases reported in saudi arabia between june 2012 and march 2019. temporal, seasonal, spatial and spatiotemporal cluster analyses were performed using kulldorff’s spatial scan statistics to determine the time period and geographical areas with the highest mers-cov infection risk. a strongly significant temporal cluster for mers-cov infection risk was identified between april 5 and may 24, 2014. most mers-cov infections occurred during the spring season (41.88%), with april and may showing significant seasonal clusters. wadi addawasir showed a high-risk spatial cluster for mers-cov infection. the most likely high-risk mers-cov annual spatiotemporal clusters were identified for a group of cities (n = 10) in riyadh province between 2014 and 2016. a monthly spatiotemporal cluster included jeddah, makkah and taif cities, with the most likely high-risk mers-cov infection cluster occurring between april and may 2014. significant spatiotemporal clusters of mers-cov incidence were identified in saudi arabia. the findings are relevant to control the spread of the disease. this study provides preliminary risk assessments for the further investigation of the environmental risk factors associated with mers-cov clusters. middle east respiratory syndrome coronavirus (mers-cov) is an emerging human viral respiratory infectious disease caused by a novel coronavirus. it was first reported in saudi arabia in 2012 [1] , and since then, it has spread to several other countries, resulting in global public health implications. from april 2012 through february 2019, a total of 2374 laboratory-confirmed mers-cov cases (with 823 deaths, 34.66%) were reported to the world health organization (who) by 27 countries, with the majority (1983 cases, 83.52%) being reported by saudi arabia (with 745 deaths, 37.56%) [2] . the risk assessment of mers-cov infection, transmission and severity is crucial in predicting and preventing further outbreaks of human infections and in enhancing control measures. recent studies have advised that dromedary camels (camelus dromedarius) serve as a reservoir host for mers-cov, and camel-to-human transmission can occur through sporadic zoonotic infections associated with exposure to infected dromedary camels and their products [3] [4] [5] . the risk factors were identified for primary mers-cov infection in persons with either direct or indirect exposure to camels. in particular, higher heterogeneity was more prominent in zoonotic than in human-to-human transmission in the middle east region; this result emphasizes the importance of the environmental component of the epidemic [32] . although approximately 83% of the globally reported mers-cov cases are found in saudi arabia, spatial patterns and clusters of the occurrence of this disease have not been addressed, leaving a wide gap in knowledge on this important issue. this study aimed to examine the spatiotemporal clustering of the mers-cov incidence in saudi arabia between 2012 and 2019 using spatial scan statistics and gis. all laboratory-confirmed mers-cov cases reported between june 13, 2012 and march 31, 2019 were compiled from the official websites of the saudi ministry of health (smoh) [33] and the who [34] . we undertook a detailed review of the mers-cov data and performed a range of checks for data consistency, completeness and fitness for the study purpose. we then developed a dataset with variables of interest for each individual with mers-cov. the dataset included the following: diagnosis date, gender, age, nationality, healthcare, employment status and source of infection, as well as the city, governorate and province of residence. a confirmed case is defined as a suspected case that has a laboratory confirmation of mers-cov infection. a suspected case is defined as either (i) an adult patient presenting with severe pneumonia or acute respiratory distress syndrome, based on clinical or radiological evidence, or (ii) an adult patient presenting with an unexplained deterioration of a chronic condition, such as congestive heart failure or chronic kidney disease being treated with hemodialysis, or (iii) a child or an adult patient exposed to a confirmed case of mers-cov or who has visited a healthcare facility where a mers-cov patient was recently identified, or has had a history of contact with dromedary camels or consumption of camel products within 14 days before symptoms and who presents with either (a) acute febrile illness (temperature ≥ 38 • c) with or without respiratory symptoms, or (b) gastrointestinal symptoms and leukopenia or thrombocytopenia. laboratory testing for mers-cov is performed at approved regional smoh and selected non-smoh governmental laboratories to confirm a clinically suspected case and to screen contacts by using validated, commercial, real-time, reverse-transcription polymerase chain reaction (rrt-pcr) assays. the laboratory confirmation of mers-cov infection requires either a positive rrt-pcr result for at least two specific genomic targets, or a region upstream and open reading frame1a (upe and orf1a) [35] . a primary case is defined as a person with a laboratory-confirmed mers-cov infection with no evidence of contact with infected individuals but is known or believed to have had direct or indirect exposure to camels or camel habitats. exposure to camels includes direct physical contact with camels or their surroundings (milking and handling excreta), drinking raw camel milk or other unpasteurized products derived from camel milk and handling raw camel meat. indirect contact includes casual contact with sites where camels have been (e.g., camel markets or farms) but without direct physical contact with camels, or living with a household member who has had direct contact with camels. by contrast, a secondary case is defined as a person who has shared the same enclosed space (e.g., a room or office) for frequent or extended periods with an individual with a symptomatic mers-cov infection. mers-cov is believed to spread between humans mainly through contact and respiratory droplets. however, transmission through small particle droplet nuclei (aerosols) may occur. environmental contamination during outbreaks in healthcare facilities can be extensive and might contribute to outbreak amplification, if adequate disinfection procedures are not followed [35] . the spatial database of the mers-cov incidence in saudi arabia was created in the format of an esri file geodatabase on the three spatial levels of city, governorate and province. saudi arabia consists of 13 administrative provinces, 136 governorates and more than 300 cities. mers-cov cases were grouped and aggregated to be represented by cities, governorates and provinces. the prevalence of intrinsic variance instability in estimating incidence rates as a result of the variation in populations across spatial units, which can possibly identify outliers, has received broad attention in the disease mapping field [36] . to address this issue, we used geoda [37] software for generating eb smoothed rate maps for mers-cov incidence. the number of mers-cov incidence cases for the governorates was used as an event variable, and the populations of governorates were estimated from the 2010 census [38] and used as base variables. we analyzed the spatiotemporal clustering of the mers-cov incidence in saudi arabia between 2012 and 2019 at the city level by using kulldorff's spatial scan statistics via satscan 9.6 [39] . we used purely temporal, seasonal, purely spatial and spatiotemporal retrospective analyses to scan, detect and evaluate the periods and geographical areas with the highest mers-cov risk incidence clusters. the purely spatial scan statistic is defined by a circular window on the map. the window is sequentially centered on each of several possible cities that are positioned throughout the study area. the spatiotemporal scan statistic imposes a cylindrical window with a circular geographic base and height corresponding to time. the temporal scan statistic uses a window that moves in one dimension, time, defined in the same way as the height of the cylinder used by the spatiotemporal scan statistic. the key feature that distinguishes the seasonal scan statistic from the purely temporal scan statistic is that the former ignores the year of the observation and retains only the day and month [40] . the number of mers-cov cases by city was used as the case file, the city population estimated from the 2010 census [38] was used as the population file and the latitude and longitude of each city were used in the coordinates file. in satscan, an analysis was conducted by progressively scanning a window across time and/or space through a comparison of the number of observed and expected cases of mers-cov incidence, assuming random distribution, inside the window at each city. the null hypothesis is that the incidence of mers-cov is randomly distributed, and the alternative hypothesis is that the incidence increases more inside the window than in areas outside it. the log likelihood ratio (llr) is the hypothesis-testing statistic estimated based on monte carlo randomization. the window with the maximum likelihood ratio is the most likely cluster; that is, it identifies the cluster that is least likely to occur by chance. in addition to the most likely cluster, satscan also designates secondary clusters for purely spatial and spatiotemporal analyses and ranks them in relation to their estimated llr statistic. satscan scans for clusters by using different criteria; the criterion recommended by satscan is the percentage of the population at risk, with a value of 50% [40] . we tested the percentage of the population at risk from 10% to 50%, and from the result, 30% performed best; that is, the value of 30% did not include neighboring cities that have a non-elevated risk. the four types of analyses (purely temporal, seasonal, purely spatial and spatiotemporal) were conducted using the poisson discrete-based model with 999 monte carlo permutations to test for statistical significance. only clusters with significance levels of 0.05 and only scans of cities with high rates were reported. for temporal analysis, values of 1 day, 1 month and 1 year were set as the time aggregation units for the daily, monthly and annual clusters, respectively, whereas for the seasonal cluster, 1 month was set. for spatiotemporal analysis, 1 month and 1 year were set for the monthly and annual spatiotemporal analyses, respectively. a total of 2008 laboratory-confirmed human mers-cov cases reported in saudi arabia during the period between june 2, 2012 and march 31, 2019 were included in this study. the primary cases accounted for 24.05% (n = 483) of the total number of confirmed cases; of these, 48.24% (n = 233) involved (direct and indirect) exposure to camels. secondary cases accounted for 40.90% (n = 821) of the total number of confirmed cases, whereas missing and unknown cases accounted for 18.02% (n = 362) and 17.03% (n = 342) of the total number of confirmed cases, respectively. on the incidence of mers-cov infection was mostly reported from riyadh (n = 722, 35.95%), jeddah (n = 276, 13.74%) and alahsa governorates (n = 129, 6.42%), figure 2 . the incidence in wadi addawasir, buraydah, taif, alkharj, najran, madinah and makkah governorates ranged from 51 to 81 cases. these were followed by five governorates in northern saudi arabia (hafr albatin, sakaka, hail, dumat aljundal and tabuk) and two governorates in eastern saudi arabia (alkhubar and dammam) with 20-26 cases. for the eb smoothed incidence rate of mers-cov infection, the wadi addawasir governorate showed the highest rate across the country, with 66.87 cases per 100,000 people ( figure 3 ). dumat aljundal and najran governorates followed with 33.46 and 20.02 cases per 100,000 people, respectively. alkharj, alhinakiyah and afif governorates exhibited an incidence rate in the range of 15.03-18.12 cases per 100,000 people. in riyadh, the capital of saudi arabia, the the incidence of mers-cov infection was mostly reported from riyadh (n = 722, 35.95%), jeddah (n = 276, 13.74%) and alahsa governorates (n = 129, 6.42%), figure 2 . the incidence in wadi addawasir, buraydah, taif, alkharj, najran, madinah and makkah governorates ranged from 51 to 81 cases. these were followed by five governorates in northern saudi arabia (hafr albatin, sakaka, hail, dumat aljundal and tabuk) and two governorates in eastern saudi arabia (alkhubar and dammam) with 20-26 cases. for the eb smoothed incidence rate of mers-cov infection, the wadi addawasir governorate showed the highest rate across the country, with 66.87 cases per 100,000 people ( figure 3 ). dumat aljundal and najran governorates followed with 33.46 and 20.02 cases per 100,000 people, respectively. alkharj, alhinakiyah and afif governorates exhibited an incidence rate in the range of 15.03-18.12 cases per 100,000 people. in riyadh, the capital of saudi arabia, the incidence rate of mers-cov infection was 13.92 cases per 100,000 people. in buraydah, alahsa and jeddah governorates, the incidence rates were 13.04, 12.06 and 7.98 cases per 100,000 people, respectively. temporal cluster analysis generated from the spatial scan test identified the years 2014, 2015 and 2016, the months of april and may of 2014, and the period from april 5 to may 24, 2014 as the strongly significant clusters of annual, monthly and daily mers-cov incidence, respectively (table 1) . seasonal cluster analysis revealed that april and may show strongly significant seasonal clusters of mers-cov incidence (table 1) . temporal cluster analysis generated from the spatial scan test identified the years 2014, 2015 and 2016, the months of april and may of 2014, and the period from april 5 to may 24, 2014 as the strongly significant clusters of annual, monthly and daily mers-cov incidence, respectively (table 1) . seasonal cluster analysis revealed that april and may show strongly significant seasonal clusters of mers-cov incidence (table 1) . the results of the purely spatial cluster analysis of mers-cov incidence from 2012 to 2019 revealed the most significant and secondary clusters at the city level (table 2 and figure 4 ). wadi addawasir in riyadh province had the most likely high-risk cluster, followed by a secondary significant cluster in alkharj and aldilm cities in the same province. spatial clusters in single cities were identified across the country; alhofuf (east), dumat aljundal (north), najran (south), alqunfidhah (southwest), alhinakiyah (west) and buraydah (center) represented the third, fourth, fifth, sixth, seventh and eighth secondary clusters, respectively. the results of the spatiotemporal cluster analysis of mers-cov infection, using years and months as the time aggregates from 2012 to 2019, showed significant most likely and secondary clusters in saudi arabia (table 3; table 4 and figure 5 ; figure 6 ). spatial variations existed between the annual and monthly spatiotemporal clusters. for the annual spatiotemporal clusters, a group of cities (n = 10) located in riyadh province was identified as the most likely high-risk cluster for mers-cov incidence between 2014 and 2016. this was followed by a secondary cluster that was found for three cities (jeddah, makkah and taif) in makkah province between 2014 and 2015. spatiotemporal clusters in single cities were also observed and varied in space and time across the country. wadi the results of the spatiotemporal cluster analysis of mers-cov infection, using years and months as the time aggregates from 2012 to 2019, showed significant most likely and secondary clusters in saudi arabia (table 3; table 4 and figure 5 ; figure 6 ). spatial variations existed between the annual and monthly spatiotemporal clusters. for the annual spatiotemporal clusters, a group of cities (n = 10) located in riyadh province was identified as the most likely high-risk cluster for mers-cov incidence between 2014 and 2016. this was followed by a secondary cluster that was found for three cities in this study, we examined the spatial pattern of mers-cov risk at the governorate level and the temporal, seasonal, spatial and spatiotemporal clustering of mers-cov incidence at the city level over a seven-year period. to the authors' knowledge, this is the first study that aims to analyze the spatiotemporal pattern and clustering of mers-cov in saudi arabia. a total of 2008 laboratory-confirmed mers-cov cases were reported in saudi arabia, representing approximately 83% of the global cases. overall, the majority of mers-cov cases were secondary infections (40.90%). this result indicates that secondary infections, either hospital or community acquired, remain a major challenge for the saudi healthcare system in the prevention and control of mers-cov outbreaks, despite the significant improvement in mers-cov surveillance. on the other hand, the primary cases accounted for only 24.05% of the total confirmed cases. although compared with the general population, people in close contact with dromedary camels have a higher risk of developing mers-cov infection via a primary source [3] [4] [5] , our results indicated that only 48.24% of the total primarily infected cases were associated with direct or indirect contact with camels. this result is consistent with previous findings [4] regarding the ambiguity of primary mers-cov infection transmission. in saudi arabia, camel milk and meat production has increased by 5.4% and 6.4% per year, respectively [41] . however, intensified animal production has epidemiological consequences, including increased risk of disease. recent trends in saudi arabia have indicated a tendency towards dromedary camel husbandry intensification since the 1960s, as evident in increased production in nearby cities by providing enhanced supplemental diets for the animals and improving camel health management [11, 42, 43] . these areas of intensified camel production are probable hotspots for the transmission and spread of mers-cov. in addition, dealing with camel products, consuming raw unpasteurized milk and conducting slaughter processes have been documented as risk factors for primary mers-cov transmission to humans [44] . the epidemic curve of mers-cov has varied significantly each year from 2012 to 2019, and it has exhibited variance in monthly peaks. a combination of sporadic and epidemic patterns as a result of animal-to-human, human-to-human and unknown exposure was observed. by contrast, the epidemic curve in south korea, where the largest outbreak outside of the arabian peninsula occurred, had a clear nosocomial epidemiological pattern [45] . purely temporal cluster analyses of mers-cov infection illustrated significant clusters in april and may of 2014. this finding is consistent with previous results [46] , which showed significant peaks in mers-cov incidence between march and may during a similar period. seasonal cluster analysis identified april and may as a strongly significant seasonal cluster of mers-cov infection. in accordance with our findings, it was reported in [47] that mers-cov infection occurred markedly in june, followed by may and april, and the lowest rates were seen in january. one possible reason for this trend is the seasonal variations in zoonotic infections in camels during the breeding season [48, 49] , when camel farms are considered an important potential source of mers-cov transmission [47] . moreover, a recent serological study in saudi arabia found a higher risk of mers-cov infection in camels in winter than in summer [50] . however, the low daily frequency and sporadic cases of mers-cov indicate a reduced likelihood of zoonotic-to-human transmission and increased possibility of human-to-human transmission, which is consistent with other findings [51] . the main mers-cov outbreaks in 2014 and 2015 were closely followed by human influenza a epidemics [52] . no coincidence was found between the peak of influenza occurrence and mers-cov occurrence, which suggests that the seasonal characteristics of mers-cov infections may vary from those of human influenza viral infections. in this study, no epidemics of mers-cov were observed during mass gatherings of pilgrims in the hajj season, which is consistent with previous findings [53] . this indicates another knowledge gap regarding the mode of transmission that needs further investigation. spatial clusters of mers-cov cases were mainly found in a group of cities in the provinces of riyadh, qassim, hail and najran located on the outskirts of larger deserts, which is the natural habitat for camels. however, significant spatial clusters of mers-cov cases were also reported from small general hospitals in single cities, such as wadi addawasir, dumat aljundal, alhinakiyah and alqunfidhah; in these settings, delays in the isolation of suspected patients, inadequate infection and control measures, and late case diagnosis and management are expected. the annual spatiotemporal high-risk mers-cov clusters occurred mainly in the early periods of the mers-cov epidemic (between 2014 and 2016) in major cities, such as riyadh, jeddah, taif, alhofuf and buraydah, whereas recent clusters (between 2017 and 2019) were observed in relatively small cities, such as dumat aljundal and wadi addawasir. this result can be explained by infection prevention and control practices, which were, to some extent, more effective in major cities than in small cities and remote areas. for the monthly spatiotemporal high-risk mers-cov clusters, the cities of jeddah, makkah and taif were identified as a part of the most likely high-risk mers-cov cluster for april and may of 2014, when the number of cases represented the largest accumulation of cases reported since the beginning of the mers-cov outbreak. the probable source of infection in the majority of the cases in this outbreak was secondary human-to-human transmission in jeddah that took place in healthcare facilities as a result of overcrowding and inadequate infection control measures, rather than a sudden increase in primary cases in the community [49] . the spatiotemporal cluster detected in riyadh between march 2014 and october 2015 could be attributed to several outbreaks, with the most prominent one occurring in a single healthcare setting in riyadh in august 2015 [54] . in addition, the most recent clusters of mers-cov incidence were identified in wadi addawasir in february 2019, in dumat aljundal in august 2017 and in buraydah in march 2016; according to [55] [56] [57] , the majority of the cases were associated with healthcare-acquired infections. this indicates persistent challenges related to nosocomial transmission, which require a thorough investigation of compliance to infection control measures by healthcare workers. mers-cov infection has global public health implications and has been labelled as an epidemic in saudi arabia. this study examined the spatial pattern and spatiotemporal clusters of mers-cov incidence in saudi arabia for the first time by using the latest publicly available mers-cov data. the results of this study provide initial risk assessments that can be used as the basis for the further investigation of the potential environmental risk factors that can explain the spatiotemporal clusters of mers-cov infection. the immediate isolation of suspected patients, adequate infection control measures and early case diagnosis and management remain the principal elements in controlling the spread of the disease, especially in small hospitals and in remote areas. future research investigating the effects of age, gender and occupation on mers-cov infection and mortality is needed. isolation of a novel coronavirus from a man with pneumonia in saudi arabia emergencies: middle east respiratory syndrome coronavirus (mers-cov) evidence for camel-to-human transmission of mers coronavirus community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study global status of middle east respiratory syndrome coronavirus in dromedary camels: a systematic review presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study high prevalence of mers-cov infection in camel workers in saudi arabia risk factors for mers-cov seropositivity among animal market and slaughterhouse workers a more detailed picture of the epidemiology of middle east respiratory syndrome coronavirus camel meat in the world peri-urban camel (camelus dromendarius) production system in saudi arabia: a note water, and agriculture in saudi arabia numbering and tracking camels electronically transmission of mers-coronavirus in household contacts middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission hospital outbreak of middle east respiratory syndrome coronavirus hospital-associated middle east respiratory syndrome coronavirus infections identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department outbreak of middle east respiratory syndrome at tertiary care hospital middle east respiratory syndrome coronavirus transmission in extended family occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update risk factors for primary middle east respiratory syndrome coronavirus illness in humans prevalence of comorbidities in cases of middle east respiratory syndrome coronavirus: a retrospective study spatiotemporal frameworks for infectious disease diffusion and epidemiology spatial methods for infectious disease outbreak investigations: systematic literature review mers-cov geography and ecology in the middle east: analyses of reported camel exposures and a preliminary risk map spatial modelling of contribution of individual level risk factors for mortality from middle east respiratory syndrome coronavirus in the arabian peninsula mapping potential amplification and transmission hotspots for mers-cov quantifying spatiotemporal heterogeneity of mers-cov transmission in the middle east region: a combined modelling approach ministry of health in saudi arabia. command and control center world health organization. emergencies preparedness, response. disease outbreaks by year middle east respiratory syndrome coronavirus; guidelines for healthcare professionals. version 5 bayesian disease mapping: hierarchical modeling in spatial epidemiology software for exploratory spatial data analysis (esda) general authority for statistics saudi arabia. the general population and housing census satscan tm v9.6. software for the spatial and space-time scan statistics guide for version 9 camel sciences and economy in the world: current situation and perspectives systems of camel management in saudi arabia awad-acharari, f. genetic and nongenetic effects for milk yield and growth traits in saudi camels mers-cov in upper respiratory tract and lungs of dromedary camels comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea a pandemic risk assessment of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia global seasonal occurrence of middle east respiratory syndrome coronavirus (mers-cov) infection emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives infection control and mers-cov in health-care workers the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection in livestock and temporal relation to locations and seasons interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk differences in the seasonality of middle east respiratory syndrome coronavirus and influenza in the middle east a systematic review of emerging respiratory viruses at the hajj and possible coinfection with streptococcus pneumoniae increase in middle east respiratory syndrome-coronavirus cases in saudi arabia linked to hospital outbreak with continued circulation of recombinant virus response: disease outbreak news: middle east respiratory syndrome coronavirus (mers-cov)-the kingdom of saudi arabia ministry of health saudi arabia world health organization. who mers-cov global summary and assessment of risk this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors wish to express their gratitude to king abdulaziz city for science and technology and king faisal specialist hospital and research centre for supporting this study, as well as the smoh and the who for providing the data. the authors declare no conflict of interest. key: cord-341795-zbqfs77n authors: sikkema, r. s.; farag, e. a. b. a.; islam, mazharul; atta, muzzamil; reusken, c. b. e. m.; al-hajri, mohd m.; koopmans, m. p. g. title: global status of middle east respiratory syndrome coronavirus in dromedary camels: a systematic review date: 2019-02-21 journal: epidemiol infect doi: 10.1017/s095026881800345x sha: doc_id: 341795 cord_uid: zbqfs77n dromedary camels have been shown to be the main reservoir for human middle east respiratory syndrome (mers) infections. this systematic review aims to compile and analyse all published data on mers-coronavirus (cov) in the global camel population to provide an overview of current knowledge on the distribution, spread and risk factors of infections in dromedary camels. we included original research articles containing laboratory evidence of mers-cov infections in dromedary camels in the field from 2013 to april 2018. in general, camels only show minor clinical signs of disease after being infected with mers-cov. serological evidence of mers-cov in camels has been found in 20 countries, with molecular evidence for virus circulation in 13 countries. the seroprevalence of mers-cov antibodies increases with age in camels, while the prevalence of viral shedding as determined by mers-cov rna detection in nasal swabs decreases. in several studies, camels that were sampled at animal markets or quarantine facilities were seropositive more often than camels at farms as well as imported camels vs. locally bred camels. some studies show a relatively higher seroprevalence and viral detection during the cooler winter months. knowledge of the animal reservoir of mers-cov is essential to develop intervention and control measures to prevent human infections. middle east respiratory syndrome (mers) is a highly fatal respiratory tract disease in humans that was first detected in 2012 in the kingdom of saudi arabia (ksa) [1] . after its first detection, mers-coronavirus (mers-cov) was being reported in human patients across the arabian peninsula, with occasional travel-related cases in other continents. as of the end of march 2018, a total of 2189 human laboratory-confirmed cases from 27 countries have been reported to the world health organisation (who), including 782 associated deaths [2] . dromedary camels (camelus dromedaries) have been shown to be the natural reservoir from where spill-over to humans can occur [3, 4] . human-to-human infection is also reported frequently, especially in healthcare settings [5] . sustained human-to-human transmission outside of hospital settings has not been shown yet [6] . direct or indirect human contact with camels has resulted in repeated introductions of mers-cov into the human population [7] . it has been suggested that camels may have acquired mers-cov from a spill-over event from a bat reservoir, but evidence for that remains inconclusive [8] . infections with mers-cov generally are thought to be mild or inapparent in camels [9] , and are therefore of low economical or animal welfare significance. this systematic review was done to compile and analyse all published data on mers-cov in the global camel population to provide an overview of current knowledge on the distribution, spread and risk factors of mers-cov infections in dromedary camels as a basis for the design of intervention and control measures to prevent human infections. on 2 may 2018, a literature search on pubmed was performed, using the terms 'middle east respiratory syndrome coronavirus' and 'mers-cov'. using the term 'mers' did not result in any additional articles that fit the scope of this review. only articles published in english were included. two reviewers individually selected all original research articles containing laboratory evidence of mers-cov infections in dromedary camels in the field. articles that were mentioned in food and agriculture organization (fao) updates [10] or in the references of included publications, but did not appear in the pubmed search were added. subsequently, abstracts, follow-up studies of mers-cov-positive camels and genome studies without prevalence data were excluded from the analysis. data on variables such as year of sampling, country, region, age, sex and animal origin were extracted and analysed. for each variable, the number of positive camels, total number of camels tested and the median percentage positivity was calculated. data from experimental infection studies were not included in this analysis, but they were included in the review to provide additional information and context to the field studies. additional information on the distribution and trade of dromedary camels was collected from references in the publications on mers-cov in camels and extracted from official fao and world organisation for animal health (oie) databases [11, 12] . the additional literature on camel trade was collected in a less systematic way from pubmed. the literature search resulted in a total of 53 papers (fig. 1 ). forty-three research papers described the results of crosssectional studies in dromedary camel populations, six papers described outbreak investigations, including an analysis of camel samples, and four papers described longitudinal studies. in total, 33 papers describe camel studies in the middle east, 13 studies investigated camels from africa and the remaining seven surveys were from spain, australia, japan, bangladesh and pakistan (table 1) . most recent fao statistics estimate the world population of camel to be around 29 million [11] , of which approximately 95% are dromedary camels [13] . however, it is believed that the true population size is even larger due to inaccurate statistics and feral camels, such as the feral dromedary camel population in australia that is estimated to be around 1 million [14] . over 80% of the camel population lives in africa. the main camel countries are chad (6 400 000), ethiopia (1 200 000), kenya (2 986 057), mali (1 028 700), mauritania (1 379 417), niger (1 698 110), sudan (4 830 000), somalia (7 100 000) and pakistan (1 000 000) [12] (table 2) . a large number of camels are being transported from the horn of africa to the middle east each year. these are mainly meat camels coming from the east of africa going to egypt, libya and the gulf states, and sudanese camels that are being imported into the middle east to participate in camel racing competitions [15] . for example, the fao reported that somalia exported 77 000 camels in 2014 [16] . the largest camel market in africa is the birqash market near cairo (egypt), where camels from sudan and ethiopia are most common, but trade routes include animals from chad, somalia, eritrea and kenya [17] . imported camels are usually quarantined for 2-3 days at the border before they are allowed to enter egypt [17] . most somali and sudanese camels that are exported to the ksa are shipped from the ports of berbera and bosaso in north somalia to the ksa ports of jizan and jeddah [15] . in general, only minor clinical signs of disease have been observed in animals infected with mers-cov and most mers-cov infections do not appear to cause any symptoms [9] . disease symptoms that have been described after experimental and field infections are coughing and sneezing, respiratory discharge, fever and loss of appetite [18] [19] [20] . although mers-cov rna can be detected in several organs after experimental infection, in studies of natural infectious virus it has only been detected in the tissues of the upper and lower respiratory tract and regional lymph nodes of the respiratory system in part of the infected camels. histologically, a mild-to-moderate inflammation and necrosis could also be seen on the upper and lower respiratory tract. no viral antigen or lesions were detected in the alveoli. histopathological examination showed that the nasal respiratory epithelium is the principal site of mers-cov replication in camels [18, 21] . in one study investigating experimental infection of camels, mers-cov shedding started 1-2 days post-infection (dpi). in that study, infectious virus could be detected until 7 dpi, and viral rna until 35 dpi in nasal swab samples and, in lower amounts, in oral swab samples [18] . no infectious virus or viral rna was detected in faecal or urine samples [18] . viral rna detection in nasal, but also rectal swabs of camels after experimental infection until day 14, has been confirmed in a recent vaccine study [21] . in the field surveys included in this review, mers-cov rna has been described in rectal swab samples, although other field studies report negative results [3, [22] [23] [24] and when viral rna can be detected, the positivity rate of rectal swabs is lower compared with nasal swab samples [19, [25] [26] [27] . oral swabs are usually negative or show a lower positivity rate even when nasal swabs test positive for mers-cov rna [3, 19, 26] . some studies have reported mers-cov rna in milk samples [27, 28] . longitudinal studies of camel herds show that pcr results of nasal swabs can remain positive after 2 weeks [27, 29] . when an interval of sampling of 1 or 2 months was maintained, nasal swabs become negative for viral rna in the next sampling round [24, 30] . mers-cov infections have also been detected in camels with mers-cov antibodies, both in calves with maternal antibodies as well as older camels that had already acquired antibodies from a previous infection. however, virus replication and thus the virus load is generally lower in infected seropositive animals compared with seronegative camels [19, 21, 23, 24, 30, 31] . little is known about the longevity of antibody titres after infection from longitudinal studies. a study following camels on a closed farm found that neutralizing antibodies remained consistent during a year [30] , while other studies found that antibody titres rapidly drop by 1-4-fold within a period often as short as 2 weeks [24, 27] . the first evidence of mers-cov in camels described so far is the detection of antibodies to mers-cov in camel sera from somalia and sudan from 1983 of which 81% tested positive [32] . additional serological evidence of the widespread presence of mers-cov infection in camels, included in this review, has been found in 18 additional countries: bangladesh, burkina faso, egypt, ethiopia, iraq, israel, jordan, kenya, ksa, mali, morocco, nigeria, oman, pakistan, qatar, spain, tunisia and the uae (fig. 2 ). in addition, promed mail reported that virus-positive camels had been found in kuwait and iran, the latter reportedly in imported animals (archive number 20140612.2534919 and 20141029.2912385). in 11 countries, serological findings were complemented with the finding of viral rna in dromedary camels: burkina faso, egypt, ethiopia, iraq, jordan, ksa, morocco, nigeria, oman, qatar and the uae. investigations of mers-cov circulation amongst dromedary camels in australia, japan, kazakhstan, usa and canada did not find any proof of mers-cov circulation. all countries where mers-cov circulates in the camel population, with the exception of spain (canary islands), pakistan and bangladesh, are located in the middle east or africa [4, 33] . one out of 17 camels that had mers-cov antibodies in bangladesh was born in bangladesh, 16 others were imported from india [34] . however, there have not been any additional reports of mers-cov in camels in india. there is no record of foreign origin of the seropositive camels from pakistan [35] . moreover, in previous studies there had already been evidence of seropositive camels that originate from pakistan [37, 58] . when combining serology data from all papers included in this review, the overall median seroprevalence of camels in africa is 81% (6106/8526; range 28-98%), compared with a median seroprevalence of 93% (3230/3846; range 53-100%) in camels from the middle east. based on viral shedding studies from african countries, the median rate of viral shedding was 5% (1108/6318; range 1-15%), compared with 12% in camels from the middle east (1191/14902; range 0-100%). the seroprevalence of mers-cov antibodies increases with age in camels, while the fraction of camels that test positive for mers-cov rna in their nasal swabs decreases with age [17, 31, 36, 38, 39] . when all serological results of papers that included sufficient age information is combined, the median seroprevalence of camels aged under 2 years is 52% (992/1972; range 0-100%), while the age groups 2-5 years (702/924; range 30-100%) and over 5 years old (1226/1370; range 0-100%) had a combined median seroprevalence of 97%. in the virological studies reporting age breakdown, the median rate of nasal shedding in 0-2 years old camels was 34% (718/2612; range 0-100%) of cases, compared with 2% (91/1142; range 0-100%) in camels older than 2 years. some individual studies show a significantly higher seroprevalence in female camels compared with males [27, 39] , while others show the opposite [38] or do not find any significant difference [17, 35] . similar disagreeing results are published for the presence of mers-cov rna in male vs. female camels [17, 27, 38, 39] . in the studies in this review where sex of camels was recorded, a total of 4810 serum samples from female camels and 3458 samples from male camels were collected and analysed for mers-cov antibodies, compared with 2007 vs. 2505 nasal swabs for viral rna testing. approximately three times more female camels were sampled at farms, while male camels were in the majority in studies that looked at mers-cov prevalence of camels at slaughterhouses, live animal markets and quarantine areas. the overall median seroprevalence of male and female camels in our review is 50% and 67%, respectively (range 0-100%; excluding results from israel and kazakhstan). the median percentage of presence of viral rna is 18% in nasal swabs of male camels (range 0-21%) compared with 9% in female camels (range 0-100%), in our review. in several studies, camels that were sampled at animal markets or quarantine facilities were seropositive more often than camels at farms [17, 22, 27, 34] . combining serological laboratory results of camels in our review with sufficient background information with regard to the sampling location does not result in the same pattern, with a median seroprevalence of 84% (5632/8115; range 0-100%; excluding australia and spain) in camels from farms and 80% (943/1005; range 28-98%) in the camel population sampled at markets and quarantine facilities. studies in egypt found a significantly higher pcr positivity rate in camels sampled in abattoirs or quarantine facilities, but these results could not be confirmed by other papers in this review [17, 27] . when comparing differences in seroprevalence or virus rnapositive rate in nomadic vs. sedentary camel herds, some authors did not find a statistical difference between the two herd management types [39, 40] , while others found some evidence of higher seroprevalences in nomadic herds [27, 36] . one study in kenya looked at the differences between herds with different levels of isolation, and did not find significant differences in mers-cov antibody levels [40] . most studies that compared local camels with imported camels suggested that imported camels are seropositive for mers-cov more often [9, 17, 27, 34, 41] , although not all differences were significant. two studies in egypt found a significantly higher rna positivity rate in imported camels from east africa compared with domestically bred camels [17, 27] , while another study executed in the ksa found a significantly higher number of mers-cov rna-positive results amongst local camels vs. camels from sudan and somalia [22] . although mers-cov was detected almost year-round in camels, some studies show a relatively higher seroprevalence and viral detection during the cooler winter months [17, 20, 27, 38] . mers-cov antibodies have been detected in llamas and alpacas in israel and in alpacas in qatar [42, 43] . to date, no mers-cov antibodies or viral rna have been detected in bactrian camels [4, 37, [44] [45] [46] [47] (table 1 and table 3 ). swine, goats and horses that were included in the field surveys in our review all tested negative for mers-cov rna and antibodies [4, 17, 31, [48] [49] [50] [51] [52] . mers-cov antibodies were detected in two studies in sheep in egypt and qatar, although in very low numbers [17, 51] . however, most surveys that investigated sheep did not find evidence of mers-cov infection or exposure [4, 23, 29, 31, 34, [48] [49] [50] [51] 53] . the publications in this review show that the mers-cov mainly circulates in dromedary camel populations in the middle east and part of africa, and has been infecting dromedary camels in africa for more than three decades. antibodies have also been found in arabic camel sera from the early 90s [31, 32] . however, mers-cov was discovered until 2012, after the first human cases appeared [1] , which is probably due to the minor clinical symptoms of mers-cov infections in camels [18] . most camel surveys were conducted in the middle east and some northern and eastern african countries, but significant data gaps currently still exist in the north and west of africa, in countries that have camel populations of 100 000 to more than a million animals, such as algeria, libya, mauritania and niger. even less is known about the central asian region. some evidence of mers-cov circulation in camels of pakistan and bangladesh was recently published, but data is lacking from afghanistan and india. knowledge on the presence of mers-cov in the animal reservoir is a crucial first step to assess whether mers-cov could be a relevant public health threat in these regions. mers-cov infections are mainly detected in calves and young camels [30, 31] . the research included in this review shows that the igg positivity rate increases gradually in dromedary camels of increasing age while the mers-cov rna detection rate decreases. maternal igg antibodies in camels are acquired through the intake of colostrum during the first 24 h post-parturition. after 24 h, antibody levels in the dam's milk decrease rapidly [54] . one study showed that maternal antibodies in calves peak at 7 days postparturition and decline in the following 6 months. after 5-6 months, over half of the calves did not have maternal neutralizing antibodies in their serum any longer [30] . however, in other field studies, the titre of mers-cov-specific antibodies is still low at 1 month of age and increases with age in dromedary calves [27, 55] . a lower or undetectable antibody levels in young camels is likely to explain the higher mers-cov rna detection rate. in adult camels, a much higher mers-cov seroprevalence can be found, which is probably due to a long-lasting immune response against a mers-cov infection or multiple re-infections with mers-cov. immunity is not sterilizing, as mers-cov infection and shedding have also been shown in adult camels that have mers-cov antibodies [19, 21, 23, 24, 30, 31] . several articles have analysed seroprevalence and virus shedding data in relation to factors, other than age, that may explain differences in seroprevalence and mers-cov rna-positive rate in camels, such as sex, sampling location, herd characteristics and animal origin. our review shows that there is considerable heterogeneity in results. in addition, comparison between studies is difficult given the lack of standardisation of study designs. a key factor to consider when comparing studies is the difference in distribution of male and female camels amongst different disciplines of camel husbandry. females are mainly used for milking and reproduction. as a result, they often stay at farms. male camels, especially of young age (<1 year old), are the predominant sex in slaughterhouses and amongst camels used for transport [39, 56] . this also influences the risk profile of acquiring a mers-cov infection. female camels are in closer contact with calves, who are more susceptible to infection and shed virus in higher quantities compared with older camels [30] . on the other hand, meat and transport camels (predominantly male) travel more, leading to increased contact with other camels and camel herds, and therefore a higher chance of exposure to mers-cov. some papers in this review suggest that there is a generally lower infection rate of domestically bred camels and camels on farms compared with imported camels and camels on animal markets or in quarantine facilities. this may be explained by the same increased contact rate and mixing of camel herds, leading to an increased chance of mers-cov exposure and spread. the increase in mers-cov circulation in winter and spring can have multiple explanations. firstly, the winter is the calving season [10] , which leads to a larger proportion of young animals that usually have a higher number of mers-cov infections and virus excretion. moreover, in winter season, there is a major increase of camel and human movements due to camel racing competitions, camel breeding, trading and movements to grazing grounds, which increases the chance of virus spread. additionally, cooler temperatures may facilitate coronavirus survival in the environment [57] . in experimental studies, llama's and alpaca's are shown to be susceptible to infection with mers-cov [58, 59] , which was confirmed by two papers in our review, describing serologically positive llamas and alpacas in israel and alpacas with mers-cov neutralizing antibodies in qatar [42, 43] . in experimental settings, animal-to-animal transmission has been shown for alpacas, making them a possible risk population for human infections [58] . two studies in our review also found anti-mers-cov antibodies in sheep [17, 51] but experimental inoculation of sheep did not result in mers-cov replication or antibody development [59, 60] . however, the dpp4 receptor, the entry receptor for mers-cov, is present in sheep tissues, making it possible for the virus to bind to the sheep respiratory tract which may explain the finding of mers-cov antibodies [61] . pigs also express the dpp4 receptor in their respiratory tract, and viral replication in experimental settings has been shown for pigs, but no antibodies or mers-cov rna have been found in pigs during field surveys [48, 59] . this may be explained by the limited viral shedding in pigs and the absence of animal-to-animal transmission [62, 63] . we show that dromedary camels are present in large parts of the african and asian continent, and that mers infections in dromedary camels are widespread. however, human infections due to spill-over from the dromedary camel reservoir have not been reported in africa [10] . several explanations for the difference in human cases between the arabian peninsula and africa have been suggested, such as differences in cultural habits, camel husbandry, prevalence of comorbidities, under detection or genetic factors in the local population [64] . moreover, west african viruses were found to be phylogenetically and phenotypically distinct from the mers-cov viruses that caused human disease in the middle east [65] . increased knowledge on the animal reservoir of mers-cov needs to be combined with research on mers prevalence and risk factors in humans to assess the true public health risk. moreover, the absence of human disease, combined with the mild symptoms in camels, caused by mers, will likely have a negative effect on the willingness to implement interventions and the cost-effectiveness of possible interventions in some areas. since the discovery of mers-cov in 2012, the dromedary camel has been identified as the animal reservoir of human infections with the mers-cov. however, the exact route of human primary infections is still unknown. moreover, the scale of the spread and prevalence of mers-cov in the camel reservoir is not fully known yet since there is still a lack of mers-cov prevalence data in some countries that harbour a very significant proportion of the world camel population. however, knowledge of the animal reservoir of mers-cov is essential to develop intervention and control measures to prevent human infections. prospective studies that include representative sampling of camels of different age groups and sex, within the different husbandry practices, are needed to fully understand the patterns of mers-cov circulation. such studies are important as they may give more information on critical control points for interventions to reduce the circulation of mers-cov and/or exposure of humans. author orcids. r. s. sikkema, 0000-0001-7331-6274 financial support. this study was financially supported by the european commission's h2020 programme under contract number 643476 (http:// www.compare-europe.eu/). none. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers situation update march middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission global summary and assessment of risk mers-cov spillover at the camel-human interface further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus mers coronaviruses in dromedary camels cov situation updates available at la production de viande de chameau: ã©tat des connaissances, situation actuelle et perspectives distribution and abundance of the feral camel (camelus dromedarius) in australia mers and the dromedary camel trade between africa and the middle east somalia registers record exports of 5 million livestock in 2014 cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels mers coronavirus in dromedary camel herd, saudi arabia mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector-borne and zoonotic diseases longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia novel betacoronavirus in dromedaries of the middle east high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate time course of mers-cov infection and immunity in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia mers coronavirus neutralizing antibodies in camels presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands middle east respiratory syndrome coronavirus antibodies in dromedary camels serologic evidence for mers-cov infection in dromedary camels antibodies against mers coronavirus in dromedary camels antibodies against mers coronavirus in dromedary camels cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia risk factors for mers coronavirus infection in dromedary camels in burkina faso, ethiopia, and morocco serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels absence of mers-coronavirus in bactrian camels absence of middle east respiratory syndrome coronavirus in bactrian camels in the west inner mongolia autonomous region of china: surveillance study results from absence of middle east respiratory syndrome coronavirus in camelids middle east respiratory syndrome coronavirus infection not found in camels in japan seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt middle east respiratory syndrome coronavirus antibody reactors among camels in dubai middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan serologic assessment of possibility for mers-cov infection in equids dromedary camels in northern mali have high seropositivity to mers-cov. one health studies on the supply of immunoglobulin g to newborn camel calves (camelus dromedarius) acute middle east respiratory syndrome coronavirus infection in livestock dromedaries the camel today: assets and potentials stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions experimental infection and response to rechallenge of alpacas with middle east respiratory syndrome coronavirus livestock susceptibility to infection with middle east respiratory syndrome coronavirus inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 middle east respiratory syndrome coronavirus experimental transmission using a pig model domestic pig unlikely reservoir for mers-cov mers-cov antibodies in humans mers coronaviruses from camels in africa exhibit region-dependent genetic diversity genetic diversity and phylogeographic structure of bactrian camels shown by mitochondrial sequence variations evidence for camel-to-human transmission of mers coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate phylogenetic analysis of merscov in human and camels in iraq no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection in livestock and temporal relation to locations and seasons key: cord-346389-gbmnoo84 authors: callender, lauren a.; curran, michelle; bates, stephanie m.; mairesse, maelle; weigandt, julia; betts, catherine j. title: the impact of pre-existing comorbidities and therapeutic interventions on covid-19 date: 2020-08-11 journal: front immunol doi: 10.3389/fimmu.2020.01991 sha: doc_id: 346389 cord_uid: gbmnoo84 evidence from the global outbreak of sars-cov-2 has clearly demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from covid-19. this is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. not all comorbidities confer the same risk, however, many affect the function of the immune system, which in turn directly impacts the response to covid-19. furthermore, the myriad of drugs prescribed for these comorbidities can also influence the progression of covid-19 and limit additional treatment options available for covid-19. here, we review immune dysfunction in response to sars-cov-2 infection and the impact of pre-existing comorbidities on the development of covid-19. we explore how underlying disease etiologies and common therapies used to treat these conditions exacerbate covid-19 progression. moreover, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of covid-19 in patients with pre-existing comorbidities. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and subsequent sars-cov-2 induced coronavirus disease 2019 has spread on an unprecedented scale. according to the world health organization (who), as of the 8th july 2020, there have been 11,669,259 cases and 539,906 covid-19 related deaths worldwide. evidence from the global outbreak has clearly demonstrated that individuals with pre-existing comorbidities such as hypertension, cardiovascular disease, and diabetes are at a much greater risk of dying from covid-19 (1, 2) . this is of great concern for individuals living with these conditions, and a major challenge for global healthcare systems and biomedical research. given that comorbidities are associated with high mortality among covid-19 patients, a better understanding of the biological mechanisms that underpin this risk are needed to enable development of appropriate preventative and therapeutic strategies. the immune system plays a vital role during covid-19, and the degree of immune dysfunction correlates with disease severity (3, 4) . severe covid-19 cases are associated with significant lymphopenia and an overactivated innate immune response resulting in hyperinflammation (5) . many covid-19 associated comorbidities affect the function of the immune system, which in turn directly impacts the response to covid-19. furthermore, the myriad of drugs prescribed for these comorbidities will also influence the progression of covid-19 and limit additional treatment options available for covid-19. here, we review the current sars-cov-2 literature and explore how preexisting comorbidities adversely affect covid-19 outcome. furthermore, we discuss the long-term challenges associated with the use of both novel and repurposed therapies for the treatment of covid-19 in patients with pre-existing comorbidities. in december 2019, several cases of an infectious pneumonia with an unknown etiology emerged in wuhan province, china. by january 2020, a novel coronavirus termed sars-cov-2 was identified as the cause. the virus spread rapidly and was classified as a pandemic by the who on the 11th march 2020. however, this is not the first pathogenic human coronavirus to emerge in the last decade. in 2002, a severe acute respiratory syndrome (sars) coronavirus (sars-cov) with animal to human transmission was reported in guangdong province, china (6, 7) . prior to sars-cov, four human coronaviruses belonging to the alpha and beta genera of the coronaviridae family had been identified: hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku (8) . however, unlike the previously identified coronaviruses, sars-cov was phylogenetically distinct (9) . furthermore, rather than causing upper respiratory tract infections with mild common cold symptoms, sars-cov caused severe lower respiratory tract infections, resulting in viral pneumonia and risk of developing acute respiratory distress syndrome (ards). the sars-cov outbreak lasted 8 months, and infected 8,098 individuals across 26 different countries, with a mortality rate of ∼10% (10) . in 2012, a second novel human coronavirus emerged in saudi-arabia and was termed middle east respiratory syndrome (mers) coronavirus (mers-cov). similar to sars-cov, infection with mers-cov can cause fatal pneumonia. to date, the who has reported 2,519 mers-cov cases, with a mortality rate of ∼30% (10) . while the previous two coronavirus outbreaks were relatively well-contained, the unprecedented spread of the current pandemic has demonstrated increased infectivity of sars-cov-2. early genome sequencing from china revealed that the 30 k base-pair viral genome of sars-cov-2 shared 79.6% sequence identity with sars-cov, whereas a bat coronavirus previously detected in rhinolophus affinis shared 96% sequence identity (11, 12) . whilst mers-cov utilizes dipeptidyl peptidase-4 (ddp4) for cell entry, sars-cov, and sars-cov-2 share the same cell entry receptor; angiotensin converting enzyme ii (ace2) (11, 12) . ace2 is recognized by the s1-subunit of the spike protein and is ubiquitously expressed in the epithelia of the nasal cavity, airway tract and the alveolar space. notably, reports have shown that the receptor binding domain of sars-cov-2 s1 has a higher affinity for ace2, which may be one contributing factor to the increased viral pathogenesis of sars-cov-2 (13, 14) . the scale of the ongoing pandemic demonstrates the need for a more comprehensive understanding of the disease, and of the contributing factors such as pre-existing comorbidities, which are proving detrimental for disease severity and outcome. clinical presentation of covid-19 varies greatly. a metaanalysis of 61 studies from 11 countries (59,254 patients) reported 81.4% of cases as mild, 13.9% as severe and 4.7% as critical (15) . most healthy individuals are asymptomatic or present with mild/moderate respiratory illness (16) . the majority of critical cases occur in older (≥60 years) or comorbid individuals (15, 17, 18) . covid-19 symptoms are typical of pathogenic human coronaviruses (figure 1 ) (22) , with fever and cough reported most commonly, however other common symptoms include dyspnoea, sore throat, sputum production, fatiguem and headache (17, 18, 23) . more recently, olfactory dysfunction such as anosmia has been described in covid-19 patients (20, (24) (25) (26) (27) . furthermore, rare gastrointestinal symptoms such as nausea, diarrhea and vomiting have also been described (17, 28, 29) . patients with severe covid-19 can develop serious and potentially fatal complications such as ards, thromboembolic events, septic shock and multiple organ failure (figure 1) . due to the severity of these complications, many are associated with critical covid-19 patients who require intensive care (19, 30) . due to underreporting and large inter-country variation, the fatality rate remains unclear. for instance, in italy the overall case-fatality rate has been reported as 7.2% compared to 2.3% in china (31) . however, despite the variation, the evidence clearly demonstrates a higher fatality rate among those who develop severe covid-19 (32) . the immune system plays a vital role during covid-19, and the degree of immune dysfunction correlates with disease severity (figure 2 ) (3, 4) . during sars-cov-2 infection the immune system becomes activated, resulting in local inflammation, the recruitment of monocytes, dendritic cells (dcs), natural killer (nk), t and b cells. this response may manifest as mild/moderate disease resulting in a fever, cough and fatigue, however this will be followed by resolution of both the infection and inflammation. in severe covid-19 cases, severe lymphopenia and the accumulation of functionally exhausted t and nk cells result in an inability to mount an effective antiviral immune response to clear sars-cov-2 (34, 35) . furthermore, interleukin 6 (il-6) levels remain elevated over time, and are accompanied by high levels of il-2, il-7, il-10, tumor necrosis factor-α (tnf-α), c-x-c motif chemokine 10 (cxcl-10), monocyte chemoattractant protein-1 (mcp-1), and macrophage inflammatory protein-1α (mip-1α) resulting in systemic cytokine storm (30) . this uncontrolled systemic hyperinflammation can cause the development of critical and potentially life-threatening complications such as severe pneumonia, ards, septic shock and multiple organ failure (17, 30, 33) . both lymphopenia and hyperinflammation figure 1 | prevalence of covid-19 symptoms and complications. data obtained from a quantitative meta-analysis of 19 studies (19) . it's important to note that 87% of all cases analyzed in the meta-analysis were hospitalized cases. therefore, the proportion of symptoms and complications are representative of this and the overall proportions with regards to all covid-19 cases will be much lower. the prevalence of anosmia (20) and thromboembolic events (21) were obtained independently from smaller cohorts and may therefore change as more data is published. figure 2 | immune response in mild/moderate and severe covid-19 cases. mild/moderate covid-19 is characterized by local inflammation, the recruitment of monocytes, dcs, nk cells, t and b cells, followed by resolution of the infection and inflammation. severe covid-19 is characterized by severe lymphopenia, t cell exhaustion, and systemic hyperinflammation that can cause the development of critical and potentially life-threatening complication such as severe pneumonia, ards, septic shock, and multiple organ failure (17, 30, 33) . are being reported in the majority of covid-19 cases admitted to hospital and is associated with a poor prognosis (4, 32, 36) . more detailed examination has demonstrated negative effects on all lymphocyte subpopulations including cd4 + and cd8 + t cells, b cells, and nk cells (3) . high-dimensional analysis of circulatory immune profiles in mild, moderate and severe covid-19 patients by mass cytometry revealed that proportions of naïve cd4 + t cells, tgfβ + cd28 − naïve cd4 + t cells, dcs, and macrophages are associated with mild cases, whereas a sharp decline in the proportion of cd8 + t cells and nk cells was observed in severe cases (3) . interestingly, single-cell rna sequencing of pbmcs isolated from hospitalized covid-19 patients revealed a novel population of developing neutrophils, which appeared to be closely related to plasmablasts, in patients that had developed ards (37) . as this was a small cohort of patients, further studies are needed to assess whether this novel subset of neutrophils plays a role in the development of ards and other covid-19 complications. functionally, cd8 + t cells and nk cells in severe covid-19 patients exhibited more signs of exhaustion than mild/moderate patients (34, 35) . for example, elevated programmed cell death protein-1 (pd-1), cytotoxic t-lymphocyte-associated protein-4 (ctla-4) and t cell ig and itm domain (tigit) on cd8 + t cells and increased nkg2a on nk cells (34, 35) . as exhausted t and nk cells are less able to mount an effective antiviral immune response, it is unsurprising that these subsets are unable to eradicate sars-cov-2 and correlate with severe covid-19 cases. in addition to t cell changes, humoral immunity against sars-cov-2 is starting to come to light. evidence of sars-cov-2 specific antibodies was demonstrated in a study of 173 hospitalized patients. igg and igm sars-cov2 antibodies were present in 40% of patients within 1 week of onset and 100% by day 15 (38) . in another study, most patients developed robust antibody responses between 17 and 23 days, and although delayed, a stronger antibody response was observed in critical patients (39) . these findings were also mirrored in a study of 285 covid-19 patients, who all showed a positive igg response by day 19, followed by seroconversion to igm (40) . interestingly, antibody titers were found to be higher among severe covid-19 patients (40) , however the authors acknowledge that interpreting an association between antibody response and disease severity is difficult due to the small sample size of severe and critical patients in their study. a recent study reported low variable plasma antibody titers in convalescent individuals, however they found binding domain specific antibodies with potent anti-viral activity in all individuals (41) . older individuals (≥60 years) are more prone to severe covid-19 and have a higher mortality rate (15, 18, 36, 42) . clinically, older patients have more pronounced immune dysfunction compared to younger patients, as lymphocyte counts are lower and pro-inflammatory cytokine levels higher (43) . this is not surprising as aged immune systems are associated with immunosenescence and chronic low-grade inflammation, termed inflammaging (44) . although immunosenescence affects all aspects of the immune system, much of the deterioration in protective viral immunity can be attributed to defective t cell immunity (45) . the decline of naïve t cell output due to thymic involution (46) and the accumulation of senescent t cells leads to reduced viral host immunity (47) . in mice, cd4 + t cells were shown to be crucial against sars due to their important role in sars-cov clearance. this protection was lost in aged mice as senescent cd4 + t cells responded poorly to antigen (48, 49) . moreover, in addition to inflammaging, the accumulation of senescent cd8 + t cells and b cells with distinct senescenceassociated secretory phenotypes (50, 51) in older individuals results in elevated baseline inflammation, further increasing susceptibility to hyperinflammation and cytokine storm upon sars-cov-2 infection. in addition to age, biological sex and ethnicity have also been implicated in covid-19 outcomes. although no major sex differences exist when examining absolute number of covid-19 cases, disease incidence is higher in males when comparing older individuals (≥60 years). furthermore, initial reports from china suggested a male bias in mortality (23, 36) , which has now been reported in 37 out of 38 countries that have reported sex-disaggregated data, revealing a global male case fatality rate of 7.3% compared to 4.4% in females (52, 53) . this finding is consistent with data obtained from the previous sars and mers epidemic (54) (55) (56) . the predominant hypothesis to explain these biological sex differences is that estrogen plays a protective role against covid-19. following the sars epidemic, studies in mice demonstrated that ovariectomy or pharmaceutical blocking of estrogen in female mice resulted in elevated immune cell infiltration in the lung and consequently a more severe disease outcome (57) . in support of this, researchers in china reported that lower levels of estrogen were associated with more severe covid-19 cases in women (58) . although the exact molecular mechanisms underpinning how estrogen protects against covid-19 are yet to be confirmed, the influence of estrogen on aging and immunity, ace2 levels, and sex-related risk factors for comorbidities have all been suggested (52, 53, 59) . due to these benefits, researchers in the uk have commenced investigations into the effects of hormonal therapies such as the contraceptive pill and hormone replacement therapy, however no data has been published yet. more recently, data has emerged suggesting that black, asian and minority ethnic (bame) individuals are at a greater risk of acquiring sars-cov-2 and have worse clinical outcomes (60) . for instance, in the uk two thirds of covid-19 fatalities among healthcare workers were bame individuals (61, 62) . underlying comorbidities, which are more prevalent in bame individuals, in addition to cultural, behavioral and socio-economic differences have been proposed as possible causes (60) . however, more data is needed in order to truly establish whether a relationship between covid-19 and ethnicity exists. evidence from the global outbreak has demonstrated that individuals with pre-existing comorbidities are at a much greater risk of dying from covid-19 (1, 2) . however, a greater understanding of the biological mechanisms that underpin this risk is needed to develop appropriate preventative and therapeutic strategies. here we review the major comorbidities identified in a number of meta-analyses ( figure 3 ) (1, 2). after identification, independent searches were conducted in order to comprehensively assess the impact of each comorbidity and its associated therapies on sars-cov-2 risk, covid-19 progression and outcome, and future covid-19 therapy options. hypertension has been repeatedly reported as the highest pre-existing comorbidity in covid-19 patients (1, 2, 17, 63-65). retrospective analysis revealed that patients with hypertension have an increased risk for severe infection and mortality (65, 66) . however, whether hypertension itself or the use of hypertensive therapies are responsible for these statistics is currently unknown. hypertensive patients are commonly treated with renin angiotensin system inhibitors, such as ace inhibitors (acei) and angiotensin-receptor blockers (arb). as acei and arb can significantly increase ace2 expression (67), many speculate they are responsible for the increased risk to hypertensive patients (68) . conversely, a retrospective review of 417 hospitalized covid-19 patients, of which 12.23% had underlying hypertensions, indicated that acei and arb may be protective effect against covid-19, as the percentage of severe cases were lower in patients treated with acei/arb (23.5%) when compared to those treated with other anti-hypertensive treatments such as calcium channel blockers, β-blockers, and diuretics (48%) (69) . however, patients treated with non-aci/arb were also found to have a higher incidence of additional comorbidities (69), which may have been responsible for the development of severe disease. due to the conflicting evidence and opinions among the scientific community, it remains unclear whether treatment with acei/arb has a positive or negative impact on covid-19 progression, however many are continuing to examine this. (1). hypertension (16%), cardiovascular disease (12.11%), and diabetes (7.87%) were the most prevalent pre-existing co-morbidities. as this data is representative of hospitalized patients, the prevalence of these among mild/moderate cases may be different, and as more data is analyzed these may change. frontiers in immunology | www.frontiersin.org cardiovascular disease has also been highly reported among covid-19 patients and is associated with an increased mortality rate (17, 63, 70) . furthermore, cardiovascular complications such as thromboembolic events, myocarditis, acute coronary syndrome, arrythmia, cardiogenic shock and heat failure, have been documented in covid-19 patients without prior cardiovascular disease (71), demonstrating a significant impact of sars-cov-2 infection on the heart. in a case series of 187 covid-19 patients, those with underlying cardiovascular disease had a mortality rate of 37.5%, which was further increased to 69.44% in a subset of patients who had both underlying cardiovascular disease and elevated troponin t levels, indicative of myocardial injury (70) . two possible explanations for the increased prevalence and mortality among patients with comorbid cardiovascular disease have been proposed. firstly, cardiovascular disease is commonly treated with renin angiotensin system inhibitors as described above (72, 73) , and secondly, ace2 is highly expressed in the heart (74) . a recent study analyzing the cellular distribution of ace2 in human heart tissue obtained from covid-19 patients identified that ace2 was highly expressed in pericytes, cardiomyocytes and fibroblasts (75) . furthermore, cd209, an additional binding receptor for sars-cov, was specifically expressed in macrophages (75) . due to the increased presence of macrophages in cardiovascular disease, the authors speculate that cd209 + macrophages may enhance viral entry into the human heart. collectively, this study indicated an intrinsic susceptibility to sars-cov-2 infection in the heart, which could explain the high susceptibility to covid-19 among cardiovascular disease patients and the higher incidence of acute cardiac injury in non-cardiovascular disease patients. according to numerous reports severe covid-19 patients are at heightened risk of thromboembolic events, with 20-30% of critically ill covid-19 patients reported to have developed thromboembolic complications (21, (76) (77) (78) (79) . systemic inflammation and subsequent activation of coagulation are both contributing factors to this increased risk (79, 80) . coagulation abnormalities have been reported throughout the covid-19 pandemic and the term covid-19-associated coagulopathy (cac) has been used to describe patients displaying coagulation changes (78) . elevated levels of prothrombin, fibrinogen and ddimer, in addition to elevated inflammatory markers such as c-reactive protein (crp) and il-6, are markers of cac (78) . in particular, increased d-dimer levels highly correlate with disease severity, as elevated d-dimer presenting at admission or over time are associated with increase mortality in covid-19 patients (81) . due to the high incidence of thromboembolic events, the use of thromboprophylaxis for patients admitted to hospital with severe covid-19 has been suggested (76, 78) , and cac should be monitored carefully in all hospitalized covid-19 patient, particularly those with pre-existing risk of thromboembolic events. diabetes is the third most prevalent underlying comorbidity in covid-19 patients (2, 17, 63, 82, 83) . type 2 diabetes is a multifactorial disease characterized by chronic inflammation and impaired metabolism and has become an increasing risk to human health. diabetic individuals have an increased susceptibility to infection (84) , and are at a high risk of developing multiple comorbidities such as cardiovascular disease (83) . one study on covid-19 found that diabetic patients were more likely to develop pneumonia and were responsible for 11.7% of severe cases, but only 4% of mild/moderate cases (85) . there are a number of reasons as to why people with diabetes are more likely to develop severe covid-19. firstly, chronic inflammation in diabetic patients increases their susceptibility to hyperinflammation and the development of cytokine storm. this has been already reported in covid-19 patients, as il-6 and crp levels were found to be significantly higher in diabetic patients (83) . secondly, it is well-documented that hyperglycaemia can impair the immune response, increase oxidative stress and is associated with the onset of premature senescence (86, 87) . consequently, diabetic patients that are unable to control their blood glucose levels may have an even greater vulnerability to severe disease. finally, in addition to disease etiology, the treatment of diabetes may also impact covid-19 development. as previously discussed for hypertension and cardiovascular disease, the use of renin angiotensin system inhibitors may increase susceptibility to sars-cov-2 infection (88, 89) . furthermore, dpp4 inhibitors commonly used to treat diabetes have an anti-inflammatory effect, resulting in reduced macrophage infiltration, which could impair the innate immune response during covid-19 (90) . obesity is associated with most of the common covid-19 comorbidities such as hypertension, cardiovascular disease and diabetes (91, 92) . the global prevalence of obesity varies greatly, for example obesity is more common in the united states and europe than it is in asian countries (93) . consequently, covid-19 severity and mortality rates may also vary as a result of this. one study reported a higher bmi in patients with severe infection, and when comparing survivors vs. nonsurvivors, it was reported that 88.2% of non-survivors had a bmi above 25 kg/m 2 , which was a significantly higher proportion than survivors (94) . however, the cohort for this study was small (n = 30) and further retrospective analysis of existing studies is needed to clarify the impact of obesity on covid-19. following the previous h1n1 pandemic, retrospective analyses reported that obesity was associated with increased risk of severe infection and mortality (95) , which is in line with the increased risk of infection in obese individuals (96) . in addition to increased risk of comorbidities, obesity is also linked to an impaired immune response (97) , with evidence of impaired antibody (98) and t cell (99) responses. furthermore, expression of ace2 is upregulated in adipocytes of obese individuals and therefore may act as a potential target for sars-cov-2 (100). the incidence of cancer as a covid-19 comorbidity has been low. in an extensive meta-analysis of 76,993 patients, malignancy accounted for just 0.92% of comorbidities reported (1). however, a nationwide analysis in china reported that 1.1% of covid-19 cases had active cancer, and these patients had a higher proportion of serious events in comparison to individuals without cancer (101) . at this current time, it is unclear whether cancer patients are at high risk of covid-19 due to an immunocompromised state linked to certain cancer therapies. in one particular study, patients that underwent recent chemotherapy or surgery had a higher risk of clinically severe events than those who did not. however, a limited number of just 19 patients were included in this analysis (102) , demonstrating the need for more thorough research. furthermore, not all cancer patients should be considered equally immunocompromised. cancer patients treated with immuno-oncology therapies such as immune checkpoint inhibitors (ici) could be more immunocompetent than patients undergoing chemotherapy (103) . nonetheless, there are two major concern associated with the use of immuno-oncology therapies during covid-19. firstly, there is potential overlap between covid-19 interstitial pneumonia and possible pneumological toxicity from anti-pd-1/pdl-1 agents, which can be fatal. although this is a rare immune-related adverse event, it has been reported in 2.5-5% of patients treated with anti-pd-1/pdl-1 monotherapy, and 7-10% of patients treated with anti-pd-1/anti-ctla-4 combination therapy (104, 105) . secondly, there is a risk of cytokine release syndrome associated with t cell-engaging immunotherapy, such as chimeric antigen receptor (car) t cells. given that cytokine storm has been linked to a negative outcome in covid-19 due to the development of ards and multiple organ failure, ici and car-t cell therapies may exacerbate this hyperinflammatory state and increase mortality in these patients (103) . overall, despite the lower incidence of cancer among covid-19 patients, those being treated with immunocompromising therapies such as chemotherapy, and those susceptible to immune-related adverse events in response to immuno-oncology therapies should be monitored carefully. as infection with sars-cov-2 results in an acute respiratory disease that can progress to ards, respiratory failure and potentially even death, it is reasonable to speculate that patients with pre-existing respiratory disease would be at increased risk of severe covid-19. surprisingly, this risk is not as striking as one might anticipate, as the prevalence of asthma was just 0.90% in a study of 548 covid-19 patients in china (106) . furthermore, the incidence of chronic obstructive pulmonary disease (copd) among hospitalized covid-19 patients was reported to be just 0.95% in a meta-analysis of 76,993 patients (1). conversely, the center for disease control and prevention (cdc) recently released data of us hospitalizations indicating that copd is present in 9.2% of patients (107) . a possible explanation for this global variation could be due to differences in therapeutics agents and disease management. for instance, a global survey assessing the severity and control of 7,786 asthmatic adults worldwide published that individuals in japan and asia-pacific regions reported less severe disease than those in europe and the united states (108). the prevalence of chronic liver disease in covid-19 patients is estimated to be 3% (19, 109) . however, liver damage has been repeatedly reported as a common complication in response to covid-19 (30, 36, 109) . in a study of 1,100 patients in china, elevated levels of the liver enzyme aspartate aminotransferase (ast) were reported in 18% of mild/moderate covid-19 patients, and 56% of severe covid-19 patients (36) . the same study also reported elevated levels of alanine aminotransferase (alt) in 20% of mild/moderate covid-19 patients and 28% of severe covid-19 patients (36) . consequently, it has been proposed that liver damage associated with severe covid-19 patients is due to dysregulated innate immunity against sars-cov-2, or hepatoxicity in response to treatments, rather than pre-existing liver disease. chronic kidney disease is associated with an increased risk of pneumonia, and elevated mortality from infectious diseases has been reported in patients with end stage renal disease (110) . in an extensive meta-analysis, chronic kidney disease accounted for 0.83% of comorbidities in covid-19 patients (1). while the prevalence of chronic kidney disease among covid-19 patients is low, those with pre-existing kidney disease have been associated with severe disease and increased mortalities (111) . ace2 expression in the kidney is elevated in chronic kidney disease (112) . however, elevated ace2 expression in the kidney does not appear to correlate with increased susceptibility to sars-cov-2 in the same way as it does in the heart. nonetheless, chronic kidney disease is associated with persistent, low-grade inflammation which could exacerbate covid-19 symptoms. several factors contribute to this inflammation such as elevated cytokines including il-6 and crp, oxidative stress and impaired metabolism (113) . therefore, the underlying pathogenesis of chronic kidney disease may increase vulnerability to hyperinflammation and cytokine storm upon sars-cov-2 infection, resulting in severe covid-19. autoimmune diseases are conditions characterized by inappropriate immune activation and destruction of healthy cells. lymphopenia is common among autoimmune diseases such as type 1 diabetes, rheumatoid arthritis and systemic erythematosus lupus (114) , and since lymphopenia is regarded as a major risk factor for developing severe covid-19, individuals living with an autoimmune condition may be perceived as high risk. however, unlike other comorbidities mentioned above, autoimmune diseases have not been reported as a risk factor in the current meta-analyses. one possible explanation could be that autoimmune diseases are often treated with drugs designed to restrict immune activation, many of which are now being repurposed for covid-19 and will be discussed below. furthermore, potential alterations in patient behaviors in order to shield themselves from infection could influence reporting of autoimmunity as a risk factor. as the pandemic continues to accelerate globally, so does the race to develop an effective therapy to protect against and treat covid-19 (figure 4) . however, robust efficacy and safety assessments are needed, particularly with regards to their use in comorbid patients, as reviewed below. the sars-cov-2 genome and spike protein structure was discovered very rapidly (115) , enabling focussed development of both rna-and protein-based vaccines. however, vaccine development is a challenging and time-consuming process. following the sars-cov epidemic, several vaccines were developed and assessed using animal models. vaccination with live virus was shown to cause complications in mice such as lung damage (116, 117) , and although recombinant spike protein based vaccines were able to protect animals from sars-cov challenge, they were ineffective at inducing sterilizing immunity (118) . other vaccines with inactivated sars-cov and mers-cov have demonstrated reduced viral titers, reduced morbidity and greater survival in animal models (119, 120) . however, the rapid eradication of sars-cov reduced demand for development. some mers-cov vaccines are currently in pre-clinical and clinical development (121) , however as mers-cov is less closely related, it is unlikely that these will cross-protect against sars-cov-2. learnings from previous coronavirus outbreaks indicate that antibody responses are not particularly long-lived, with sars-cov antibodies lasting on average just 2 years (122) . this apparent lack of long-term protection exacerbates the need for an effective vaccine or vaccine programme to protect against potential recurrent seasonal sars-cov-2 infections. on the 6th july 2020, the who reported 160 candidate sars-cov-2 vaccines under development worldwide (123) . some of the most promising include an mrna vaccine; mrna-1273 (124), a dna plasmid vaccine (125) , and an adenovirus vaccine; chadox1 ncov-19 (126) . although clinical evaluation is underway, outcomes are not available yet. furthermore, frontiers in immunology | www.frontiersin.org vaccine efficacy depends upon an individual's ability to mount a strong immune response against it. consequently, vaccine regimens that grant protection in healthy individuals may not be adequate for older individuals and those with comorbid conditions who have increased immunosenescence (127) . for influenza, the use of adjuvant (128) and high dose vaccines (129, 130) have been developed and implemented to increase vaccine immunogenicity for older and high-risk comorbid adults. therefore, similar measures are likely to be needed to fine tune a sars-cov-2 vaccine once it becomes available. in the interim, rolling out a sars-cov-2 vaccine with proven efficacy in healthy individuals could result in herd immunity, preventing transmission to those more vulnerable and offer indirect protection. another therapeutic approach intended to prevent covid-19 is convalescent plasma therapy. previous use during the sars (131) and mers (132) epidemics, and h1n1 pandemic (133), demonstrated reasonable efficacy and safety. evidence from those outbreaks revealed that convalescent plasma contained neutralizing antibodies (134) , and a meta-analysis from 32 sars-cov and influenza studies, reported a significant reduction in mortality following convalescent plasma therapy (135) . several convalescent plasma studies for sars-cov-2 have been reported. in china, a small pilot study was conducted to test convalescent plasma collected from 40 recently recovered patients on 10 severe covid-19 patients, four of which had underlying conditions including hypertension and cardiovascular disease (136) . from the 40 patients, 39 had high neutralizing antibody titers of ≥1:640. no serious adverse events were reported following transfusion, and all patients experienced improved symptoms within 1 to 3 days post-transfusion. however, there are many caveats to this study, the very small sample size and the use of concomitant treatments mean it is not possible to ascertain if clinical improvements were due to convalescent plasma. consequently, additional large scale, controlled, randomized trials are needed and are currently underway. despite no adverse events being reported in the small sample sizes currently being tested for covid-19, plasma transfusions are not without risk. as with any transfusion, there is a risk of transfusion transmitted infections, albeit small (137) . perhaps of more concern are the non-infectious risks such as allergic reactions, transfusion related acute lung injury characterized by acute hypoxemia and pulmonary oedema, and transfusion associated circulatory overload, characterized by hypertension, tachycardia, tachypnoea and dyspnea (138). these are of particular concern for severe covid-19 patients with extensive lung damage, and for those with pre-existing hypertension, cardiovascular disease or renal failure (138, 139). due to the increased prevalence of comorbidities among covid-19 patients, those predisposed to transfusion-related adverse events will need to be carefully evaluated. as transfusion volume and rate have both been identified as risk factors for transfusion associated circulatory overload (140) , covid-19 trials should examine these parameters to ensure safety in comorbid patients. while many scientists attempt to develop novel therapies to prevent covid-19, others are focusing their attention on repurposing drugs that are already on the market (figure 4) . at present, the most contested repurposed drug is chloroquine and its analog hydroxychloroquine. following the 2002 sars-cov epidemic, researchers found that in vitro treatment of chloroquine could increase endosomal ph and impair terminal glycosylation of ace2 receptors on the cell surface, therefore inhibiting sars-cov-ace2 interactions and preventing virus entry (141, 142) . similarly, after the 2012 mers-cov epidemic, researchers demonstrated that chloroquine could inhibit in vitro replication of mers-cov in well-established cell lines (143) and primary mature antigen presenting cells (144) . however, as sars-cov and mers-cov resolved relatively quickly, continued assessment of chloroquine remained limited. furthermore, although in vitro treatment can inhibit sars-cov-2 (145), the use of chloroquine and hydroxychloroquine for covid-19 in the clinic is highly controversial. a series of early clinical trials conducted in china reported apparent efficacy of chloroquine phosphate in treating covid-19 (146) . this was followed by a small, non-randomized study of just 36 patients from france, which demonstrated a clinical benefit from the use of hydroxychloroquine, but lacked an appropriate control group and a long-term follow-up (147) . however, an observational study of 1,376 patients treated with hydroxychloroquine report no sign of efficacy for covid-19 (148) . this was further confirmed by results from the large randomized evaluation of covid-19 therapy (recovery) trial that examined 1,542 hospitalized covid-19 patients treated with hydroxychloroquine and reported no significant difference in mortality when compared to 3,132 standard of care patients (149, 150) . lopinavir is a protease inhibitor often formulated with lowdose ritonavir and traditionally used to treat hiv patients (151) . lopinavir/ritonavir was previously assessed in a small, nonrandomized study conducted in hong kong during the sars-cov epidemic. this study demonstrated that lopinavir/ritonavir treatment resulted in reduced viral load and milder disease, with less recurrence of fever, diarrhea, and improved chest radiographs when compared with those treated with ribavirin, an alternative antiviral (152) . during the current outbreak, two independent case studies of covid-19 patients hospitalized in south korea, reported a reduction in viral load and subsequent recovery of patients following the administration of lopinavir/ritonavir (153, 154) . however, a randomized control study in china of 199 severe covid-19 patients demonstrated no difference in mortality, or in the amount of viral rna detected (155) . furthermore, recent findings from the recovery trial comparing 1,596 patients randomized to lopinavir/ritonavir to 3,376 standard of care patients concluded no clinical benefit from the use of lopinavir/ritonavir (149, 156) . moreover, protease inhibitors such as lopinavir/ritonavir have been associated with hepatotoxicity during hiv treatment (157, 158) , and drugto-drug interaction (ddi) with certain statins, for example rosuvastatin, can increase the risk of myopathy (159, 160) . therefore, administration to covid-19 patients with preexisting liver disease and those being treated with statins could prove detrimental. like other members of the protease inhibitor class, lopinavir/ritonavir has also been associated with metabolic changes that can result in hyperglycaemia (161) , hyperlipidaemia (162) , and insulin resistance (163) . due to the lack of efficacy and increased risk of toxicity to certain covid-19 patients lopinavir/ritonavir could be prove more harmful to patients and should therefore be avoided. oseltamivir and favipiravir, two antiviral treatments traditionally used to treat influenza, have also been examined for use in covid-19. oseltamivir is a neuraminidase inhibitor, which has been extensively used as a prophylactic against influenza. in a small, single-center study of 138 patients with sars-cov-2 pneumonia in china, 89.9% of patients received oseltamivir alongside anti-bacterial drugs such as moxifloxacin, ceftriaxone, azithromycin, and glucocorticoid therapy (18) . the study concluded no effective outcomes based on oseltamivir (18) . however, the small study size, lack of appropriate control and the fact that many patients remained hospitalized at the time of publications limits full interpretations. favipiravir is an antiviral with potent inhibitory activity against viral rna-dependent rna polymerase. experimentally, favipiravir demonstrated effective sars-cov-2 inhibition in vero e6 cells (145) . furthermore, a small, open-label, non-randomized comparative study of 80 patients in china, compared clinical outcomes of patients treated with favipiravir and lopinavir/ritonavir (164) . the median time until viral clearance was 4 days with favipiravir compared to 11 days with lopinavir/ritonavir. at day 14, ct scans of the chest from patients treated with favipiravir demonstrated significant improvements. adverse events occurred in 11% of favipiravir treated patients compared to 55% of lopinavir/ritonavir treated patients (164) . however, despite initial promise more robust clinical data is needed in order to establish the efficacy and safety of favipiravir as a treatment of covid-19. another antiviral agent being examined for use in covid-19 is remdesivir. when metabolized into its active form, remdesivir inhibits viral rna polymerases, causing a decrease in viral rna production. remdesivir has been shown to inhibit sars-cov and mers-cov in human airway epithelial in vitro models (165, 166) , and in combination with interferon beta, remdesivir has been shown to be superior to lopinavir/ritonavir in a mers-cov mouse model (167) . despite some initial controversial findings (168) , results from more robust clinical trials have demonstrated reasonable efficacy. for instance, preliminary results from the national institute of allergy and infectious diseases adaptive covid-19 treatment trial involving 1,063 patients, demonstrated that remdesivir accelerated recovered by 31% compared to placebo (169) . furthermore, recent results from the phase 3 simple trial investigating the use of remdesivir in patients with moderate covid-19 showed that patients receiving remdesivir treatment were 65% more likely to have improved clinically by day 11 than standard of care patients (170, 171 ). due to the success of these two trials, the use of remdesivir has been approved by the fda, ema, uk, and japan as a treatment for covid-19. despite proven efficacy, adverse events in response to remdesivir have been reported in 60% of patients of which 12% were severe (168) . these included septic shock, multiple organ dysfunction syndrome, acute kidney injury and hypotension. therefore, the use of remdesivir in comorbid patients with increased susceptible to these adverse events requires further evaluation. as severe covid-19 cases are characterized by hyperinflammation, the use of immune modulating and anti-inflammatory treatments to prevent severe lung injury and disease progression are being explored. due to their immunomodulatory properties, mscs are being clinically assessed to treat inflammatory conditions such as systemic lupus erythematosus (172) and graft vs. host disease following allogeneic haemopoietic stem-cell transplantation (173) . consequently, a pilot study was initiated to investigate the potential therapeutic benefit of mscs for covid-19 infected patients in china. the study involved just 10 covid-19 patients who were monitored for 14 days post-msc injection (174) . msc treatment was well-tolerated and no adverse events occurred during treatment. furthermore, virtually all clinical symptoms subsided, with 3 patients being discharged 10 days post-msc injection (174) . mass cytometry of pbmcs revealed that peripheral lymphocytes, regulatory cd14 + cd11c + cd11b mid dcs and il-10 increased. whereas, crp, tnf-α and overactivated cxcr3 + cd4 + /cd8 + /nk cells decreased 3 to 6 days following injection compared to placebo group. mscs were shown to be ace2 negative, meaning they were immune to sars-cov-2 infection (174) . although this pilot study shows promise, more robust clinical data is required to validate therapeutic benefit and safety. moreover, the use of mscs would require clinical grade msc production and may not be a plausible solution for many healthcare systems. il-6 is considered the key cytokine responsible for the induction of cytokine storm during sars-cov, mers-cov and sars-cov-2 infections (30, 175, 176) . consequently, the recombinant humanized anti-human il-6 receptor monoclonal antibody tocilizumab, currently used to treat rheumatoid arthritis (ra), has been examined for use during covid-19. tocilizumab first demonstrated effectiveness in a small retrospective study of 20 patients with severe covid-19 pneumonia in china (177) . oxygen intake was reduced, and improved symptoms occurred in 75% of patients. lymphocyte levels returned to normal in 56% of patients and crp levels reduced significantly in 84.2% of patients (177) . several case studies have also demonstrated rapid clinical improvements following tocilizumab (178) (179) (180) . although these initial studies are promising, the full clinical trial data is still unavailable. furthermore, although safety profiles for tocilizumab are well-established for intermittent use in ra, the safety of tocilizumab when used in combination with antiviral agents and other comorbid therapies has not been established. other anti-inflammatory treatments now being tested clinically for their use against covid-19 are janus kinase (jak) and bruton's tyrosine kinase (btk) inhibitors. the jak-signal transducer and activator of transcription (jak/stat) pathway mediates the signal transduction of numerous cytokines in a number of immune cells such as t cell, nk cells, and dcs (181) . consequently, jak inhibitors have emerged as effective treatments for many autoimmune and immunemediated disease. at present, a number of jak inhibitors such as ruxolitinib (182), baricitinib (183) , and fedratinib (184), are being assessed as a potential treatment for covid-19. preliminary results from a pilot study evaluating 88 hospitalized patients, 20 of which were treated with baricitinib, demonstrated significant reductions in serum il-6, il-1β, and tnf-α, as well as a rapid recovery of circulatory t and b cell frequencies following baricitinib treatment (185) . consequently, a phase 3 adaptive covid-19 treatment trial (attc-2) has now been established in order to evaluation the use of baricitinib in combination with remdesivir compared to remdesivir alone (186) . however, despite promising initial findings, as jak inhibitors block a wide range of cytokines including ifn-α, which is crucial during early innate immunity in response to viral infections, the impact of this on viral clearance needs to be evaluated. furthermore, ruxolitinib and baricitinib have both been associated with increased weight gain, cholesterol and albumin levels (187, 188) . although no causal association has been reported yet, the issue should not be dismissed and covid-19 patients with metabolic and cardiovascular comorbidities should be carefully considered before use. bruton's tyrosine kinase (btk) is a key regulator of cell surface receptors expressed primarily in b cells, but also in monocytes/macrophages and neutrophils (189) . currently, btk inhibitors are used to treat various b cell malignancies and chronic graft vs. host diseases (190) . as btk can regulate il-6, tnf-α, and mcp-1, btk inhibitors are being tested in combination with car-t cells (191) , to alleviate cytokine release syndrome. furthermore, in chronic lymphocytic leukemia, btk inhibitors have been shown to increase cd4 + and cd8 + t cell, and significantly downregulate pd-1 and ctla-4 (192) , highlighting a potential reversal of t cell exhaustion, which could be beneficial in covid-19. consequently, clinical trials to assess the use of btk inhibitors against covid-19 are underway. the use of corticosteroids to treat covid-19 remained largely uncertain until recently. although individuals being treated with long term corticosteroid were instructed to continue with their medication, the use of corticosteroids specifically to treat covid-19 was not recommended (193) . this was largely due to the unknown impact of immune suppression on viral clearance and potential adverse outcomes. however, preliminary data from the recovery trial evaluating 2,104 patients randomly allocated to receive dexamethasone has proven significant clinical improvements (194) . dexamethasone is a steroid used to reduce inflammation in a myriad of inflammatory conditions and now reported to reduce covid-19 related deaths among patients receiving respiratory support by one-third (194) . in response to these findings the demand for dexamethasone to treat the most critical covid-19 patients has surged globally (195) . whilst these findings are exciting, safety data detailing potential adverse events and the impact of co-medications, such as nonsteroidal anti-inflammatory drugs, has not yet been reported and thus dexamethasone should still be considered carefully prior to administration. patients with pre-existing comorbidities are at a greater risk of dying from covid-19. however, not all comorbidities confer the same risk. by exploring the underlying disease etiologies and common therapies used to treat these conditions, we have discussed their impact on covid-19. comorbidities closely associated with age, chronic inflammation and dysregulated metabolism such as hypertension, cardiovascular disease, and diabetes are the most prevalent comorbidities. however, many of these comorbidities are strongly associated with each other. consequently, many patients will have multiple comorbidities and therefore while we have discussed these individually, the reality is that a combination of factors will be at play. furthermore, as multiple drug use is inevitable for patients with pre-existing comorbidities, the impact of overlaying drugs on an already compromised state and the possibility of ddi leading to adverse events needs to be carefully considered. as the scale of this pandemic continues to accelerate globally, we hope this review provides healthcare professionals and biomedical researchers with a more comprehensive understanding of the impact of pre-existing comorbidities on covid-19 development and treatment. the authors lc and mc are fellows of the astrazeneca postdoc programme. figures were created with biorender.com. lc and mc wrote the first draft of the article. sb, mm, jw, and cb reviewed and edited the article before submission. all authors contributed extensively to the discussion of the content and researched data for the article. prevalence of underlying diseases in hospitalized patients with covid-19: a systematic review and meta-analysis prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and meta-analysis high-dimensional immune profiling by mass cytometry revealed immunosuppression and dysfunction of immunity in covid-19 patients lymphopenia predicts disease severity of covid-19: a descriptive and predictive study clinical and immunologic features in severe and moderate coronavirus disease 2019 epidemiology and cause of severe acute respiratory syndrome (sars people's republic of china identification of a novel coronavirus in patients with severe acute respiratory syndrome human coronavirus infections the genome sequence of the sars-associated coronavirus severe acute respiratory syndrome a pneumonia outbreak associated with a new coronavirus of probable bat origin a genomic perspective on the origin and emergence of sars-cov-2 structural basis of receptor recognition by sars-cov-2 structural and functional basis of sars-cov-2 entry by using human ace2 novel coronavirus infection (covid-19) in humans: a scoping review and meta-analysis covid-19: four fifths of cases are asymptomatic, china figures indicate clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis self-reported olfactory and taste disorders in sars-cov-2 patients: a crosssectional study pulmonary embolism in covid-19 patients: awareness of an increased prevalence pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study anosmia as a prominent symptom of covid-19 infection features of anosmia in covid-19 anosmia in covid-19 patients covid-19 and anosmia: a review based on up-to-date knowledge analysis of epidemiological and clinical features in older patients with corona virus disease 2019. (covid-19) out of wuhan epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019. (covid-19) with gastrointestinal symptoms clinical features of patients infected with 2019 novel coronavirus in wuhan case-fatality rate and characteristics of patients dying in relation to covid-19 in italy clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china functional exhaustion of antiviral lymphocytes in covid-19 patients elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients clinical characteristics of coronavirus disease 2019 in china a single-cell atlas of the peripheral immune response in patients with severe covid-19 antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) antibody responses to sars-cov-2 in patients with covid-19 convergent antibody responses to sars-cov-2 in convalescent individuals clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients chronic inflammation (inflammaging) and its potential contribution to age-associated diseases aging and the immune system: the impact of immunosenescence on viral infection, immunity and vaccine immunogenicity senescence of t lymphocytes: implications for enhancing human immunity cd4 t cell memory derived from young naive cells functions well into old age, but memory generated from aged naive cells functions poorly cellular immune responses to severe acute respiratory syndrome coronavirus (sars-cov) infection in senescent balb/c mice: cd4 + t cells are important in control of sars-cov infection human cd8 + emra t cells display a senescenceassociated secretory phenotype regulated by p38 mapk human peripheral late/exhausted memory b cells express a senescent-associated secretory phenotype and preferentially utilize metabolic signaling pathways considering how biological sex impacts immune responses and covid-19 outcomes gender differences in patients with covid-19: focus on severity and mortality do men have a higher case fatality rate of severe acute respiratory syndrome than women do? sars in singapore -predictors of disease severity the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health sex-based differences in susceptibility to sars-cov infection a multihospital study in wuhan, china: protective effects of non-menopause and female hormones on sars-cov-2 infection. medrxiv impact of sex and gender on covid-19 outcomes in europe the impact of ethnicity on clinical outcomes in covid-19: a systematic review evidence mounts on the disproportionate effect of covid-19 on ethnic minorities covid-19: two thirds of healthcare workers who have died were from ethnic minorities comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis comorbidities in covid-19: outcomes in hypertensive cohort and controversies with renin angiotensin system blockers arterial hypertension and risk of death in patients with covid-19 infection: systematic review and meta-analysis hypertension in patients with coronavirus disease 2019 (covid-19): a pooled analysis effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 controversies of renin-angiotensin system inhibition during the covid-19 pandemic reninangiotensin system inhibitors improve the clinical outcomes of covid-19 patients with hypertension cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) cardiovascular considerations for patients, health care workers, and health systems during the coronavirus disease covid-19, ace2, and the cardiovascular consequences cardiovascular disease and use of reninangiotensin system inhibitors in covid-19 inflammatory response cells during acute respiratory distress syndrome in patients with coronavirus disease 2019 (covid-19) the ace2 expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov-2 pulmonary embolism or pulmonary thrombosis in covid-19? is the recommendation to use high-dose heparin for thromboprophylaxis justified? high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study covid-19 and its implications for thrombosis and anticoagulation cardiovascular complications in covid-19 covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review acute pulmonary embolism in covid-19 patients on ct angiography and relationship to d-dimer levels prevalence and impact of diabetes among people infected with sars-cov-2 diabetes is a risk factor for the progression and prognosis of covid-19 diabetes and infection: assessing the association with glycaemic control in population-based studies prevalence and impact of cardiovascular metabolic diseases on covid-19 in china high glucose-induced replicative senescence: point of no return and effect of telomerase relationship between oxidative stress, er stress, and inflammation in type 2 diabetes: the battle continues are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? covid-19 and diabetes: can dpp4 inhibition play a role? risk of covid-19 for patients with obesity prevalence and prevention of cardiovascular disease and diabetes mellitus obesity and its implications for covid-19 mortality clinical characteristics of 30 medical workers infected with new coronavirus pneumonia weight and prognosis for influenza a(h1n1)pdm09 infection during the pandemic period between 2009 and 2011: a systematic review of observational studies with metaanalysis obesity and infection considerations for obesity, vitamin d, and physical activity amidst the covid-19 pandemic ageing and obesity similarly impair antibody responses obesity is associated with impaired immune response to influenza vaccination in humans the role of adipocytes and adipocyte-like cells in the severity of covid-19 infections cancer patients in sars-cov-2 infection: a nationwide analysis in china risk of covid-19 for patients with cancer controversies about covid-19 and anticancer treatment with immune checkpoint inhibitors pneumonitis in patients treated with anti-programmed death-1/programmed death ligand 1 therapy immune-related adverse events associated with immune checkpoint blockade risk factors for severity and mortality in adult covid-19 inpatients in wuhan preliminary estimates of the prevalence of selected underlying states, conditions among patients with coronavirus disease 2019 worldwide severity and control of asthma in children and adults: the global asthma insights and reality surveys coronavirus disease 2019 and prevalence of chronic liver disease: a meta-analysis pulmonary infectious mortality among patients with end-stage renal disease chronic kidney disease is associated with severe coronavirus disease 2019 (covid-19) infection renal ace2 expression in human kidney disease inflammatory syndrome in chronic kidney disease: pathogenesis and influence on outcomes lymphopenia and autoimmune diseases cryo-em structure of the 2019-ncov spike in the prefusion conformation immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge sars vaccines: where are we? immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates recent advances in the vaccine development against middle east respiratory syndrome-coronavirus duration of antibody responses after severe acute respiratory syndrome landscape of covid-19 candidate vaccines -6 available online at available online at available online at understanding immunosenescence to improve responses to vaccines fluad r -mf59 r -adjuvanted influenza vaccine in older adults efficacy of high-dose versus standard-dose influenza vaccine in older adults efficacy and immunogenicity of high-dose influenza vaccine in older adults by age, comorbidities, and frailty use of convalescent plasma therapy in sars patients in hong kong challenges of convalescent plasma infusion therapy in middle east respiratory coronavirus infection: a single centre experience treatment with convalescent plasma for influenza a (h5n1) infection a serological survey on neutralizing antibody titer of sars convalescent sera the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis effectiveness of convalescent plasma therapy in severe covid-19 patients transfusion-associated circulatory overload after plasma transfusion adverse effects of plasma transfusion incidence and transfusion risk factors for transfusion-associated circulatory overload among medical intensive care unit patients chloroquine is a potent inhibitor of sars coronavirus infection and spread in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial observational study of hydroxychloroquine in hospitalized patients with covid-19 available online at randomised evaluation of covid-19 therapy lopinavirritonavir versus nelfinavir for the initial treatment of hiv infection role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings the first case of 2019 novel coronavirus pneumonia imported into korea from wuhan, china: implication for infection prevention and control measures case of the index patient who caused tertiary transmission of coronavirus disease 2019 in korea: the application of lopinavir/ritonavir for the treatment of covid-19 pneumonia monitored by quantitative rt-pcr a trial of lopinavirritonavir in adults hospitalized with severe covid-19 nuffield department of population health. randomised eval covid-19 pharmacokinetics and hepatotoxicity of lopinavir/ritonavir in noncirrhotic hiv and hepatitis c virus (hcv) co-infected patients drug-induced liver injury associated with antiretroviral therapy that includes hiv-1 protease inhibitors pharmacokinetics and pharmacodynamics of combined use of lopinavir/ritonavir and rosuvastatin in hiv-infected patients drug/drug interaction between lopinavir/ritonavir and rosuvastatin in healthy volunteers protease inhibitor-associated hyperglycaemia management of protease inhibitorassociated hyperlipidemia hyperlipidemia and insulin resistance are induced by protease inhibitors independent of changes in body composition in patients with hiv infection experimental treatment with favipiravir for covid-19: an open-label control study. engineering coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial gilead announces results from phase 3 trial of remdesivir in patients with moderate covid-19 available online at umbilical cord mesenchymal stem cell transplantation in severe and refractory systemic lupus erythematosus survival after mesenchymal stromal cell therapy in steroidrefractory acute graft-versus-host disease: systematic review and metaanalysis transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia extraordinary gurich single-strand rna identified from sars coronavirus contributes an excessive innate immune response delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment effective treatment of severe covid-19 patients with tocilizumab favorable changes of ct findings in a patient with covid-19 pneumonia after treatment with tocilizumab tocilizumab, an anti-il6 receptor antibody, to treat covid-19-related respiratory failure: a case report rapid and severe covid-19 pneumonia with severe acute chest syndrome in a sickle cell patient successfully treated with tocilizumab dissection of the effects of jak and btk inhibitors on the functionality of healthy and malignant lymphocytes covid-19: combining antiviral and anti-inflammatory treatments baricitinib for covid-19: a suitable treatment? th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib baricitinib restrains the immune dysregulation in covid-19 patients. medrxiv available online at effects of ruxolitinib treatment on metabolic and nutritional parameters in patients with myelofibrosis from comfort-i baricitinib induces ldl-c and hdl-c increases in rheumatoid arthritis: a metaanalysis of randomized controlled trials bruton's tyrosine kinase (btk) inhibitors in clinical trials acp-196) in relapsed chronic lymphocytic leukemia the specific bruton tyrosine kinase inhibitor acalabrutinib (acp-196) shows favorable in vitro activity against chronic lymphocytic leukemia b cells with cd20 antibodies ibrutinib treatment improves t cell number and function in cll patients clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury effect of dexamethasone in hospitalized patients with covid-19: preliminary report. medrxiv covid-19: demand for dexamethasone surges as recovery trial publishes preprint august 2020 | volume 11 | article 1991 key: cord-339152-wfakzb6w authors: trovato, maria; sartorius, rossella; d’apice, luciana; manco, roberta; de berardinis, piergiuseppe title: viral emerging diseases: challenges in developing vaccination strategies date: 2020-09-03 journal: front immunol doi: 10.3389/fimmu.2020.02130 sha: doc_id: 339152 cord_uid: wfakzb6w in the last decades, a number of infectious viruses have emerged from wildlife or re-emerged, generating serious threats to the global health and to the economy worldwide. ebola and marburg hemorrhagic fevers, lassa fever, dengue fever, yellow fever, west nile fever, zika, and chikungunya vector-borne diseases, swine flu, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and the recent coronavirus disease 2019 (covid-19) are examples of zoonoses that have spread throughout the globe with such a significant impact on public health that the scientific community has been called for a rapid intervention in preventing and treating emerging infections. vaccination is probably the most effective tool in helping the immune system to activate protective responses against pathogens, reducing morbidity and mortality, as proven by historical records. under health emergency conditions, new and alternative approaches in vaccine design and development are imperative for a rapid and massive vaccination coverage, to manage a disease outbreak and curtail the epidemic spread. this review gives an update on the current vaccination strategies for some of the emerging/re-emerging viruses, and discusses challenges and hurdles to overcome for developing efficacious vaccines against future pathogens. since the start of this century, a certain number of new or neglected pathogens have emerged from wildlife reservoirs and spilt over into human populations, causing severe diseases (1-3). factors such as urbanization, globalization, travels, international commerce, aging, and climate changes have contributed to favor emergence, spread, and transmission of pathogens. contacts among humans and potential zoonotic reservoirs are increasing, the number of travelers and their movements is growing, the aged population are more susceptible to infections, and the geographic distribution of pathogens within a previous endemic zone is changing (4, 5) . during the last decades, the global community faced several outbreaks of emerging and reemerging infectious diseases, with high threats to the health security, biodefense, and economy worldwide (6, 7) . the occurrence of significant disease outbreaks-such as sars (severe acute respiratory syndrome) originating in china in 2002 (8) , the 2009 h1n1 swine flu pandemic from mexico (9) , mers (middle east respiratory syndrome) that occurred in saudi arabia in 2012 (10) , the west african outbreak of ebola virus (ebov) in late 2013 (11) , the zika virus (zikv) outbreak originating in brazil in 2015 (12) , the 2018 health emergence in nigeria caused by lassa virus (13) , and the ongoing coronavirus disease 2019 (covid19) pandemic (14) -has renewed interests in developing strategies to faster prevent, treat, and/or control emerging and re-emerging viruses with high epidemic potential. usually, there is little or no knowledge about identity, epidemiology, and pathogenesis of a new infectious agent appearing for a first time in a certain geographic area (as in case of novel coronaviruses or new influenza variants), as well as the potential to spread out from the zoonotic reservoir, making hard to predict if, where, and when a disease outbreak will occur. the world health organization (who) and the national institutes for allergy and infectious diseases (niaid) published a list of pathogens to be prioritized for research and development, given their epidemic potential. this non-exhaustive list comprises viruses, bacteria, protozoa, and fungi, causing diseases for which efficient countermeasures do not actually exist, or require new therapeutics (15, 16) . as proven by historical records, vaccination has played a pivotal role in reducing morbidity and mortality from devastating infectious diseases, successfully leading to disease eradication (i.e., smallpox), and generally decreasing infectious disease burdens. even in presence of therapeutic options, vaccines are the valuable means to prevent infections and overall represent the much wanted achievement. however, even with worldwide efforts, getting a vaccine to the public takes time, and side effects, dosing issues, and manufacturing problems can all cause delays. thus, we have to use this time with great concern. generally speaking, in case of newly emergent diseases, conventional strategies might raise some issues. the unpredictable identity of largely unknown emerging pathogens, the lack of appropriate experimental animal models, and the time and costs for faster developing, producing, licensing, and globally distributing effective vaccine candidates are some of the major challenges to overcome in case of pandemic threats. hence, new and/or alternative approaches in vaccine design and development are required to rapidly face outbreak situations (17) . this review will discuss the current vaccination strategies for some of the emerging and re-emerging viruses, as well as the approaches that might be suitable in face of global pandemic threats. emerging and re-emerging pathogens represent a constant epidemic threat to humanity not only for the public health consequences but also for the economic, social, and political effects they may globally provoke. therefore, a major public awareness and preparedness would be fundamental in fighting emerging infectious diseases. the terms "emerging and reemerging infectious diseases" mainly refer to two major categories of infectious diseases: newly emergent infections, caused by novel pathogens; and re-emerging infectious diseases, caused by microbes reappearing after previous control, and/or eradication (1). almost 60% of emerging infectious diseases are zoonoses, with the great majority of them originating in wildlife, and the number is constantly increasing. climate changes have been related to the emergence of vector-borne diseases in severe environmental conditions, but this is a most debated issue, as well as the contribution of agricultural practices (18) . in addition, the chances of infectious disease spreading could also include livestock/wildlife animal markets and consumption of those. a study where australia was used as a model of urbanization has proposed a relation among increasing pandemic threats and urbanization: it ascribes the increased threat of pandemic to the high number of major city residents, the exponential intensification of international air traffic, and the commuter mobility network (19) . basically, an epidemic is an event that occurs when there is an increase, often sudden, in the frequency of a disease above what is normally expected in that population, in that area; while pandemic (from: παν = all, and δεµoσ = people) refers to an epidemic that spreads over several countries or continents at the same time, usually affecting a large number of people (20). in the last decades, a certain number of viruses came to light for the first time or reappeared, giving rise to significant epidemics and pandemics (figure 1 and table 1) . epidemic outbreaks of viral diseases were mostly caused by flaviviruses generally transmitted by vectors, including west nile virus (wnv) (21, 22) , zikv (23, 24) , yellow fever virus (yfv) (25, 26) , and dengue virus (denv) (27, 28) . vector-borne diseases, including chikungunya fever caused by chikungunya alphavirus (chikv) (27, 29) , are extremely difficult to eradicate because viruses are maintained in nature by propagation among vectors and hosts, without human-human contact. moreover, dry and hot climate conditions seem to foster mosquitoes to bite humans than animals, increasing the risk of spreading diseases with a devastating impact (30) . today, most areas of the world are endemic for at least one flavivirus, with denv being the most prevalent, and approximately 50-100 million people are infected each year. among viral hemorrhagic fevers, lassa fever (lf) is a rodent-borne acute disease caused by lassa virus (lasv) (31) , endemic in many west african countries, including nigeria that experienced a high mortality rate in the 2018 outbreak (32) . ebola virus disease (evd) and marburg virus disease (mvd) are caused by members of the filoviridae family, ebov (33), and marburg virus (marv) (34), respectively. the 2013-2016 ebola outbreak in west africa was the largest since the virus was first discovered in 1976, with a case fatality rate for zaire ebolavirus of 75% (35) , while the largest recorded mvd outbreak occurred in angola in 2004 (36) . concerning pandemics, flu pandemics were reported three times during the twentieth century; genome analysis of pandemic influenza viruses dated 1918 (h1n1), 1957 (h2n2), and 1968 (h3n2) demonstrated that all viral strains fully or partially originated from non-human reservoirs, and that the ultimate origin of ha (hemagglutinin) genes are from avian influenza viruses (37) , with the 1918 strain likely being the ancestor of the subsequent epidemic variants. hence, the 1918 influenza pandemic has been called the mother of all pandemics (38 nhps, non-human primates. frontiers in immunology | www.frontiersin.org during the first 12 months, after the first reported case (39) . the age of deceased people was below 65 years in almost 80% of cases, a peculiarity compared with the seasonal influenza epidemic. the mortality rates observed in 1968 and 1918 flu were of 0.03% and 1-3% due to h3n2 and h1n1, respectively, and ranging from 2.9 and 9.1% in 2009 (40) . the 2009 h1n1 pandemic has been commonly referred to as swine flu for the swine origin of the virus, first isolated in mexico and united states in april 2009. the viral genome sequencing indicated that the virus contains a combination of genes never reported in swine or humans before. it has been demonstrated that the swine has become a reservoir of h1 viruses with the potential to cause future pandemics (37) . a (h1n1)pdm09 virus monovalent vaccine was produced in late 2009 (41) , but the virus has not been eradicated and it continues to circulate as a seasonal variant, causing hospitalization, and death (42) . in november 2002, a first case of sars was reported in guangdong (china), and after 7 months, the coronavirus (cov) causing the disease, named sars-cov, spread in 37 countries, giving rise to lower respiratory tract infections, with a poor outcome in 10% of cases (43, 44) . middle east respiratory syndrome-cov, the causative agent of mers, was isolated in 2012. the coronavirus has caused isolated mers outbreaks thereafter, becoming endemic in arabian peninsula, with a case fatality rate of 34.4% (43) (44) (45) . on march 11, 2020, who has declared the coronavirus disease 2019 (covid-19) outbreak a global pandemic. the disease is caused by a novel coronavirus, known as sars-cov-2, that shares almost 88% of the genome with that of sars-cov (46, 47) . actually more than 5.9 million (as of august 1, 2020) of people are infected, with an overall case fatality rate of 0.1-15.2% (48) . in case of global public health emergencies, governmental and private organizations, vaccine developers, and regulatory authorities should all massively collaborate in selecting and funding the most suitable vaccine platform and strategy to quickly act and curtail disease outbreaks. at the outset of a disease outbreak, gaps in knowledge of identity, pathogenesis, epidemiology of the new emerging pathogen, time required to study the immune responses correlating with the outcome of the viral infection, and the lack of appropriate preclinical models susceptible to infection for testing a vaccine candidate pose several barriers and impediments to expedite vaccine design and development, and thus to ensure global vaccination coverage in time. in the fight against newly emergent viruses, vaccine design might benefit from a range of platform technologies, including nucleic acid vaccines, viral-vector vaccines, and recombinant protein-based vaccines (likely to be administered with adjuvants) (17, 49) . compared with conventional vaccines, such as live attenuated and inactivated vaccines, molecular-based platforms might offer a more versatile tool against new emergent viruses, allowing a more fast, low-cost, and scalable vaccine manufacturing. essentially, these platforms rely on the use of a system to deliver and present a new antigen (or a synthetic gene) to rapidly target an emergent pathogen. theoretically, once a platform has previously met safety and efficacy requirements to be moved and advanced into the market, a candidate vaccine against a new virus might profit from the same system, production, and purification protocols, only replacing the disease target antigen (or inserted gene), thus streamlining the vaccine discovery. in inactivated vaccine, the virus is rendered uninfectious using chemicals, such as formaldehyde or heat. this technology, conceived in the nineteenth century, is used for few vaccines still in use (i.e., inactivated polio, whole cell pertussis, and hepatitis a) (50) . live attenuated vaccines are obtained by passing the virus through animal or human cells until it picks up mutations that make it unable to cause the disease (i.e., measles, mumps, chickenpox, etc.); the attenuated smallpox was used for the massive vaccination campaign that successfully eradicated the infection (51) , and currently, attenuated influenza viruses are used as vaccines against the seasonal influenza (52). the advantages of live attenuated vaccines are the intrinsic adjuvant properties, the ability to infect cells (figure 2) , and to activate the innate immune response. interestingly, a safe sars-cov-2 inactivated vaccine (picovacc) has been recently described as being able to induce specific neutralizing antibodies (nabs) in experimental animal models (53) , and a phase iii clinical trial (nct04456595) will soon assess efficacy and safety of this candidate in health care professionals ( table 2) . nucleic acid vaccines include either mrna or plasmid dna (pdna) vaccines (figure 2) . two types of mrna vaccines were developed: conventional non-replicating mrna vaccines and self-amplifying vaccines (or viral replicons). the in vitro enzymatic transcription (ivt) of a dna template plasmid, containing the promoter sequence for the dna-dependent rna polymerase, provides a mature mrna molecule, with the open reading frame that encodes the target antigen, the 5 and 3 flanking untranslated regions (utrs), the 5 cap, and the terminal poly(a) tail. self-amplifying rna (sam) vaccines are commonly based on alphavirus genomes, where genes coding for the structural proteins are replaced with that encoding the target antigens, while the rna replication machinery sequences are conserved, allowing intracellular antigen-encoding rna amplification and higher antigen expression levels than the conventional mrna vaccines (17, 54) . once the mrna vaccine is delivered to the host cells and reaches the cytoplasm, it is translated in vivo by the host cellular machinery, providing the corresponding posttranslationally modified antigen (figure 2) , thus mimicking the in vivo natural infection. mrna vaccines activate the innate immune system, triggering host immune sensing receptors, and successively promoting adaptive immune responses (55) . several figure 2 | platforms for vaccine manufacturing: a graphical overview. nucleic acid, viral-vector, protein-based, live attenuated and inactivated vaccines are schematically illustrated. nucleic acid vaccines: conventional non-replicating mrna vaccine, containing the target gene sequence, can be encapsulated into a delivery system to aid its cellular uptake. once released from endosome into the cytosol, it is translated by the host cellular machinery into the target antigen. a pdna carrying a gene target reaches the nucleus to achieve transcription and translation into the cytosol. pdna can be internalized by somatic cells (i.e., myocytes) and then the secreted antigen can be taken up by apcs or naïve b cell, priming immune responses. viral vectored vaccines: defective viral vector, carrying a transgene cassette, can be employed as a system to deliver a transgene and allow the expression of the heterologous antigen within the infected cell. a recombinant replicating viral vector retains the ability to replicate and produce progeny virus particles that can then infect cells, leading to transgene expression and ag processing and presentation. protein-based vaccines: recombinant subunit vaccine or a vlp can be taken up by apcs for mhc presentation and b-cell recognition through bcr. virus vaccines: compared with an inactivated virus, a live attenuated virus retains the ability to replicate and infect cells, mimicking the natural infection. apcs, antigen-presenting cells; mhc, major histocompatibility complex; ag, antigen; pdna, plasmid dna; ep, electroporation; bcr, b-cell receptor; and vlp, virus-like particle. technological innovations have allowed to overcome some of the concerns associated with instability, half-life, inefficient in vivo delivery, and high innate immunogenicity of mrna platform (56) . mrna vaccines do not produce infectious particles and potentially do not integrate into the host genome, reducing safety issues, and no anti-vector immunity is elicited. they can be quickly produced (likely within the time required to get genomic information from the new emergent virus), saving time and cutting costs. thus, the mrna platform offers a promising attractive alternative to conventional vaccines, should a disease outbreak occur. no rna vaccine has been yet licensed for humans, but encouraging results from preclinical and human clinical trials have shown that mrna vaccines are able to induce safe and long-lasting immunity against different infectious viral diseases, including zika (57), influenza (58-61), ebola (61), dengue (62) , and other viral diseases (17) . a sars-cov-2 mrnabased vaccine entered clinical phases just 2 months after the identification of the viral genome sequence (nct04283461), and a phase iii study (nct04470427) will assess its effectiveness to prevent covid-19 (63) (64) (65) . a clinical study (nct04449276) is currently evaluating a similar vaccine in healthy adults ( table 2) . the dna-based strategy, like the mrna-based technology, offers a valuable platform to design and deliver any target of choice, due to safety profile, stability, ease of gene manipulation, and large-scale vaccine manufacturing, in short (66, 67), on cellular uptake and in vivo long-term gene expression, potentially providing advantages over mrna vaccines in terms of protein coding capacity, and amount and extent of antigen production. unlike mrna, pdna needs to cross both plasma and nuclear membranes to enter into a cell target, reach the nucleus, and achieve transcription (figure 2) . advances in pdna delivery devices (i.e., use of gene gun; in vivo electroporation, ep), and delivery systems (i.e., encapsulation in lnps; adsorption to polymers), have greatly enhanced molecular stability, delivery efficiency, uptake, and antigen expression. in addition, the use of optimized pdna formulations and encoding molecular adjuvants, to be administered in prime-boost strategies or simultaneously with other vaccine platforms, has generally improved the low protective immunestimulatory profile of pdna (67) . however, some potential safety concerns should be considered, including long-term persistence upon administration, which could eventually lead to genomic integration events, antibodies against bacteria-derived plasmids that could potentially trigger autoimmune diseases, and unwanted side effects due to encoded and co-delivered molecular adjuvants (17, 67) . even though no dna vaccine has been yet licensed for use in humans (four for veterinary use), this platform has shown great promise for several emerging viral diseases, including ebola and marburg (68), mers (69), west nile (70), dengue (71), chikungunya (72) , and other viral diseases (17) , and more recently for covid-19 (73) . currently, dna-based vaccine candidates, encoding the s protein from sars-cov-2, have moved into clinical phase i/ii development (63, 65, 74) (table 2 ). recombinant viral vector-based platform employs either live replicating often attenuated or non-replicating viruses as vector vaccines (figure 2 ). viral vector vaccines represent the biotechnological evolution of live attenuated and inactivated vaccines: a viral backbone devoid of the replication machinery to be used as a shuttle to express in vivo the chosen target antigen. several viral backbones have been exploited to generate viral-vector vaccines. targeted deletion of replication genes represents the non-empirical way of virus attenuation, allowing the generation of a wide array of viral vectors, engineered by insertion of a transgene cassette. the modified virus ankara (mva) is an attenuated form of the vaccinia virus (vacv), derived from more than 570 passages in chick embryo fibroblasts, a method that empirically modifies the viral genome, without affecting the immunogenicity (75) . it is able to infect multiple cell types but cannot replicate inside the infected cells, ruling out the safety concerns related to the use of live vaccines. one of the drawbacks in the use of a viral vector vaccine is that multiple immunizations lead to the host response against the structural viral proteins, limiting the efficacy of vaccination, as demonstrated in a study based on cellular immune response. to overcome this limitation, the heterologous prime-boost regimen has been introduced in several clinical trials, where two different viral vectors or a pdna prime-viral vector boost were tested (76) . risks of integration into the host genome do potentially exist, as some viral vectors enter to the nucleus of cells to achieve transcription and replication. a major restrain in the production of viral vector vaccines is the time-consuming manufacturing; several attempts to accelerate vaccine production are in development, like selecting cell lines with higher yield or choosing the best promoter for transgene expression to reduce vaccine doses (77) . among the available viral vectors, the adenoviruses are the most used in priming the immune response, being able to induce humoral and cellular responses (78) . a pre-existing anti-vector immune response jeopardized the vaccine response in adenoviral-based clinical trials (79) . to avoid pre-existing immunity, adenoviral vectors of non-human origin or rare serotypes have been used as vaccine platform. the use of chimpanzee adenoviral vectors proved to be safe and effective in clinical trials conducted against ebola (80) and respiratory syncytial virus (rsv) (81) . vesicular stomatitis virus (vsv), a single-stranded negative sense rna virus that naturally infects livestock, represents an attractive safe alternative over other viral vectors due to low risk of pre-existing immunity, lack of dna molecules during replication, and ability of vsv-based vaccines to induce effective humoral responses (82) . for humans, two viral-vector vaccines are available: imojev, a japanese encephalitic virus (jev) vaccine; and dengvaxia, a dengue vaccine (both from sanofi pasteur). both are produced using the chimeric yfv as vector: two of the yfv genes have been replaced by genes encoding the pre-membrane (prm) and the envelope (e) protein of jev or denv, and the chimeric viruses are propagated in cell culture (83, 84) . conversely, several viral vector vaccines have been licensed for veterinary use because of the less stringent regulatory requirements (76) . to face covid-19, an adenovirus type 5 vector expressing sars-cov-2 s protein (ad5-ncov) has been advanced into phase ii trial (nct04341389), while a phase iii study (isrctn89951424) is currently investigating the chimpanzee adenoviral vector (chadox1 ncov-19), expressing the same protein (63, 65, 74) ( table 2) . recombinant protein-based vaccines consist of immunogenic proteins from the target pathogen. once identified, recombinant proteins can be produced on a large scale, in bioreactors, using heterologous expression systems, like bacteria, yeast, plants, insect, or mammalian cell lines, depending on the posttranscriptional pattern of modification required (85) . vaccines based on recombinant proteins represent a safe platform because they do not contain pathogen-derived genetic information, and the manufacturing does not require manipulation of live pathogens. they might represent a platform of choice when a fast response to an epidemic is on demand, as the vaccine production can start once the genome of the new virus has been sequenced, even before the virus isolation. protein-based vaccines can be obtained producing recombinant virus subunits (suvs) that can be administered in combination with adjuvants to improve the host immune response against the recombinant viral antigens (86) . recombinant proteins derived from viral capsid can selfassemble into virus-like particles (vlps), high ordered and repetitive structures devoid of the viral genome. vlps display antigenic epitopes in their original conformation in high copy number, they retain the size and geometrical organization of the original virus (mainly icosahedral or rod shape), preserving the viral immunogenicity due to the ability to crosslink b cell receptor on b cell surface (87) , and to be taken up by antigenpresenting cells (apcs) (88, 89) (figure 2) . several strategies have been proposed to improve dendritic cell (dc) uptake, by expressing targeting molecules such as antibodies directed against endocytic receptors, and to augment immunogenicity, through simultaneous delivery of maturation stimuli, like tlr agonists (90, 91). when not able to self-assemble into a vlp, the selected antigen can be expressed as chimeric protein: several vlp platforms are available for the display of heterologous antigens on the viral coat proteins. recombinant vlps from plant virus, like tobacco mosaic virus (92) , or alpha mosaic virus (93) , are easily produced, competing for speed and cost of production with vlp platform based on mammalian viruses (94) . the most used vlp platform is the hbcag-vlp, the core antigen from hepatitis b virus (hbv) (95) . it is also possible to chemically attach the heterologous antigen to a preformed vlp by using conjugation methods (96) . although this strategy could increase the manufacturing costs, it might be suitable when the expression of recombinant antigens affects the vlp assembly. to table 2) . it is worth mentioning that kim and colleagues designed and developed a sars-cov-2 subunit vaccine within 4 weeks of the identification of sars-cov-2 s protein n-terminal domain s1 sequence. delivery of recombinant subunit vaccines by microneedle array resulted in potent antibody response in mice (97) , and vaccination with a sars-cov-2 spike s1-fc fusion protein induced antibody responses in small animal models and nabs in monkeys (98) . in table 2 are listed the vaccine candidates that currently moved into clinical trials for preventing the viral infectious diseases discussed in the following section. west nile virus includes five lineages; among them, lineage 1 was classified as the most virulent, while lineage 2 is considered more attenuated. however, during a serious outbreak in hungary in 2008, the sequencing of lineage 2 showed some genetic mutations that demonstrated the increased virulence of this strain and its explosion throughout the central europe (99, 100) , causing renewed interest in the development of a vaccine against wnv. 20 years after the epidemic that hit the united states, no wnv vaccine has been yet released for human use, while four vaccine formulations are on the market for veterinary use, three based on the whole inactivated virus (wn innovator, vetera wnv, and prestige wnv), and one on recombinant vaccine expressing wnv prm/e into a canarypox backbone (recombitek equine wnv) (101, 102) . these vaccines completely protect horses from viral infection but require subsequent administrations and several booster doses overtime. for the development of a vaccine for humans, many different platforms were used in preclinical studies, and many of them entered into phase i/ii trials, including hydrogen peroxideinactivated whole virus (hydrovax) vaccine (nct02337868) (103), a recombinant truncated form of wnv e protein (104), recombinant chimeric live attenuated viral vectors, employing yfv (105), or mva (106) delivering wnv prm/e proteins (nct00442169), pdna vaccines encoding prm/e (nct00106769) (70, 107) . all the envelope-based vaccines induced nabs against both wnv lineages 1 and 2, but some candidates are unable to generate long-lasting antibody responses, requiring multiple administrations (103, 108, 109) . thus, further improvements are needed for the development of next-generation vaccines (110) . recently, a wnv replicationdeficient vaccine candidate with a deletion of the non-structural protein ns1 has been shown to protect mice from a highly lethal viral challenge, after a single dose, without adverse effects (111) . during the 2015 outbreak in brazil, an abnormal microcephaly number and other birth defects in newborns were reported (112) . for this reason, vaccination of pregnant and of reproductiveage women became an urgency. shan and colleagues developed a candidate vaccine, using a live attenuated viral strain containing a deletion in the 3 region of the virus genome. this vaccine induced strong and protective antibody response, after a single injection in mice and macaques, and reduced viral rna in placental and fetal tissues in infected mice (113) . the immunized mice also developed a robust t-cell response (114) . although promising, this attenuated virus-based formulation does not meet the safety standards required to be used to vaccinate pregnant women, whose prophylaxis requires a vaccine that fulfill higher safety standards. a number of different replication-deficient viral vectors have been recently developed and are currently under evaluation. immunization of mice with a vaccine based on mva delivering the zikv prm and the e structural proteins (mva-zikv) elicited nabs and potent zikv-specific cd8 + t-cell responses, mainly with an effector memory phenotype (115) . a rhesus adenovirus serotype 52 vector (rhad52), expressing zikv prm and e proteins, induced high titer of zikv-specific antibodies after the first prime, offering complete protection against subcutaneous zikv challenge, in mice (116) , and rhesus monkeys (117) . these adenoviral-based vaccines induced antibodies that were also maternally transmitted (118) . in addition, abbink et al. using the rhesus macaque model demonstrated that a complete anti-zikv immunity can only be achieved through vaccination with a combination of different vaccine platforms (117) . zikv vaccine candidates currently in phase i clinical trials include inactivated and live attenuated vaccines, mrna and pdna vaccines, and recombinant viral-vectored vaccines, mainly targeting the prm and e proteins (119) . a dna-based vaccine encoding the prm signal sequence from jev and zikv e proteins moved into phase ii (nct03110770), showing immunogenicity and safety in humans (120) . a protective and efficacious vaccine against yfv is currently available. to date, the main type of yf vaccine produced on a large scale is based on the live attenuated 17d virus vaccine. this vaccine is obtained after numerous passages of asibi virus strain in mouse and chicken embryo that generate a strain with accumulated mutations in the envelope protein. these mutations affect the virus binding to the host receptor, reducing its neurotropism and vicerotropism, and mosquito transmissibility (121) . because the vaccine is produced in chicken embryo, there are issues related to manufacturing costs and vaccine availability. the interruption of vaccination coverage against yf in endemic countries has caused major outbreaks in africa and south america in 2015 and 2016, which exhausted the 17d vaccine stockpiles leading to the use of an emergency "fractional dose" campaign in the democratic republic of congo (122) . thus, the fluctuating demand for doses during outbreaks makes the accessibility to the vaccine still a problem to be solved. the need for a vaccine against denv has become an urgency only in recent decades. dengue fever is caused by four distinct virus serotypes, denv1-4, able to circulate simultaneously in endemic areas, making extremely difficult the development of a broad protective vaccine. recently, the food and drug administration approved the first dengue vaccine by sanofi-pasteur, named cyd-tdv or dengvaxia (123, 124), a tetravalent live attenuated virus vaccine on yfv backbone, whose release has generated controversy due to evidence that the administration can increase the risk of a more severe form of the illness in people with a pre-existing immunity toward other denv strains (125, 126) . for this reason, the use of dengvaxia is strictly limited, depending on age (between 9 and 16) and serostatus of recipients to vaccinate (exclusively individuals who had a previous denv infection), generating concerns about its costbenefit balance. studies for the development of a safer vaccine are still ongoing, and candidate vaccines include a tetravalent dengue purified inactivated virus vaccine, currently in phase i/ii clinical trial (nct02421367), and two live attenuated tetravalent chimeric tdv (denvax), and tvd 003/005 (tetravax-dv) vaccines, currently in phase iii clinical trials (nct02747927; nct02406729) (127). no vaccine is actually available to prevent chikv infection. among the candidates in ongoing studies, two of them achieved and completed phase i or ii trials: vla1553 and mv-chik vaccines. vla1553 candidate (by valneva) is a live chikv (la réunion isolate lr2006 opy1) attenuated by a partial deletion of the gene encoding the non-structural replicase complex protein. this vaccine induced immunity lasting over 20 months after a single shot immunization (nct03382964). mv-chik vaccine is a live attenuated measles-vectored chikv vaccine that induced chikv-specific nabs and shown to be well tolerated by all the participants (nct02861586) (128) . recently, moderna therapeutics tested a vaccine based on engineered mrna encoding chikv structural polyproteins (mrna-1388) in a phase i clinical trial. as shown in preclinical studies, this formulation induced strong immune responses after one single injection, totally protecting mice from developing the disease (129) . several vaccine platforms have been tested in preclinical animal models and shown to be able to protect animals from marv infection and to induce both humoral and cellular immune responses. these include vlps (137) , dna vaccines (68), recombinant adenoviral vectors (138) , and rvsv (139, 140) . many works have emphasized the use of a multivalent vaccine formulation to achieve protection against different filoviruses. vaccination with a single dose of a trivalent formulation based on rvsv expressing glycoproteins from ebov, sudan ebolavirus (sudv), and the angola strain of marv elicited antibodies specific for the three glycoproteins in non-human primates (nhps) and a balanced t-cell response sufficient to protect against the viral challenges (141) . similarly, vlps delivering a trimeric hybrid glycoprotein from marv, ebov, and sudv fully protected vaccinated animals from marv challenge, inducing specific nabs (142) . using an enhanced dna-based platform encoding the envelope glycoprotein from marv and ebov, shedlock and colleagues showed that a polyvalent-filoviral vaccine candidate, delivered by in vivo ep, elicited in preclinical models robust nabs and cytotoxic t cells, completely protecting animals from the viral challenge, after a single dose administration (68) . actually, a multivalent phase i study (nct02891980) is evaluating safety and immunogenicity of two heterologous and two homologous prime-boost regimens using a mva multi-filo and ad26 zaire ebola (ad26.zebov) vaccines (143) in healthy volunteers, with the aim to analyze the protective response to different filoviruses. coronaviruses are a group of single-stranded rna viruses that have been present in humans for at least 500-800 years and all originated in bats (144, 145) . earlier than 2019, six coronaviruses had been known to cause diseases in humans: hcov-229e, hcov-043, hcov-nl63, hcov-hkn1, sars coronavirus (sars-cov), and mers coronavirus (mers-cov) (146) . in late 2019 and early 2020, a novel coronavirus was discovered to be the cause of a rapidly spreading outbreak of respiratory disease, including potentially fatal pneumonia, in wuhan, china. the virus, provisionally designated 2019-ncov and later given the official name sars-cov-2, owing to its similarity to sars-cov (then named sars-cov-1), was isolated and the viral genome sequenced. sars-cov-2 was characterized as a beta-coronavirus (147) . the disease caused by the virus was officially named coronavirus disease 2019 (covid-19) by who. coronaviruses are capable of adapting quickly to new hosts through the processes of genetic recombination and mutation in vivo. point mutations alone are not sufficient to create a new virus. however, this may occur when the same host is simultaneously infected with two coronavirus strains, enabling recombination of genomic fragments of hundreds or thousands of base pairs long and thus making a new virus (148, 149) . this susceptibility enabled the emergence, in approximately two decades, of three new human coronavirus species with epidemic potential: sars-cov-1, mers-cov, and sars-cov-2. coronaviruses enter cells via binding to a host receptor followed by membrane fusion. the angiotensin-converting enzyme 2 (ace2) was identified as the cell receptor for sars-cov (150) , and recently also for the new sars-cov-2 (151), while mers-cov binds the dipeptidyl peptidase 4 (dpp4) receptor, also known as cd26 (152) . the s protein is used for virus-cell receptor interaction during viral entry (153) . transmission of the virus during the viremic stage of disease is primarily via respiratory secretions (droplets) or direct contact. sars-cov-2 is extremely contagious, with an estimated basic reproduction number (r0) of 2.24-3.58 (154) . in contrast, the r0 for both sars-cov-1 and mers-cov is less than 1 (155). it soon became apparent that infected individuals might be capable of transmitting the virus during the prodromal period (156) . social distancing strategies (quarantine and community containment) represent the only efficacious means of controlling coronavirus spread in the absence of effective drugs or vaccine against the pathogens. of importance, for preventing the spread of the disease caused by contact with patients or contaminated fomites, hygiene measures are also mandatory, such as washing hands with soap and water or with alcohol-based preparations. indeed, coronaviruses are able to survive on various surfaces for few days but can be inactivated by disinfection (157) . finally, because it has been demonstrated that the overlap between human and animal ecosystems have given to coronaviruses the opportunity to cross the species barrier, to prevent future zoonotic diseases, a coordination with veterinary experts as well as stricter laws governing the trade of wild animals would be necessary. humans are extremely exposed to these pathogens because these viruses had not previously circulated in the human population, as testified by the absence of antibodies against coronavirus in healthy people. in addition, the innate immune response has demonstrated to be insufficient in controlling coronavirus infection because decreases in viral load are coincident with the specific antibody response (158, 159) . in this context, vaccines represent a much expected resource. a hopeful premise is represented by the successful containment of coronavirus epidemics in farm animals by vaccines, based on either killed or attenuated virus (160) , and concerning sars-cov-2 by the finding that specific antibodies are detectable in 100% of patients with covid-19, 17-19 days after symptom onset (161) , and that the magnitude of antibody titers positively correlated with viral neutralization potency (162) . after the sars outbreak, several vaccines were formulated based on various strategies, as recombinant s protein-based vaccines, attenuated and whole inactivated vaccines, as well as vectored vaccines. pre-clinical data showed animal protection from challenge with sars-cov-1. however, sterilizing immunity was not always achieved (163) . in few cases, the use of live virus as a vaccine resulted in complication including lung damage, eosinophil infiltration, and liver damage in animal models. moreover, a study of vaccination with inactivated sars-cov-1 in nhps reported enhancement of disease caused by specific epitopes on the s protein [reviewed in (64) ]. another issue is related to the length of a protective immune response. both humoral and cellular responses have been found important for lasting protection. in long-term studies of recovered sars patients, antibody responses waned after approximately 6 years, while t-cell responses persisted, suggesting that the latter is required for long-lasting immunity. concerning mers-cov, the vaccines proposed target the s protein (164) (165) (166) , including mucosal vaccine for intranasal administration (167) . however, cases of enhanced lung diseases were also reported in preclinical models of vaccination in mice (168) . new mers-cov vaccines in development also include live attenuated, protein subunit, and dna vaccines (169, 170) . recently, a small animal model that replicates mers-cov transmission has been developed (170) and will help the preclinical studies. following the alarming data and casualties provoked by covid-19, a strong effort by the research community is going on at the moment, and who has been informed of dozens of vaccines in preparation using different platforms, as mentioned in section "vaccine platforms." some of these candidate vaccines are already in phase i/ii clinical trials, while others have been advanced to phase iii studies (63, 65, 74) (table 2 ). however, it is possible that a sars-cov-2 vaccine will not be available for another 12-18 months. recently, a rhesus macaque model that recapitulates sars-cov-2 infection has been developed to study immunopathogenesis and test vaccine candidates (73, 171) . therapy based on passive administration of anti-coronavirus antibodies, isolated from patient sera, also represents a much wanted option for the treatment of coronavirus diseases (172) , and a global effort is pursued in this direction to treat patients before the achievement of a validated vaccine. in addition, researchers are trying to produce in laboratory specific and protective anti-coronavirus antibodies. in the case of sars outbreak, a monoclonal antibody (mab) with neutralizing activity, being able to block receptor association, was identified and described (173) . moreover, neutralizing mabs have also been produced to fight mers-cov infection. in a collaborative study by us and chinese researchers, mabs targeting the receptor (cd26/dpp4) binding domain of mers-cov spike glycoprotein were reported (174) . japanese researchers have also investigated anti-cd26 mab for mers-cov and have identified the humanized mab ys110 as a promising candidate (175) . finally, in the case of sars-cov-2 outbreak, dutch researchers claimed the identification of a human mab named 47d11 able to block sars-cov-2 infection (176). recently, a mab able to cross-neutralize sars-cov-2 has been identified from memory b cells of a sars-cov-infected individual. the antibody, named s309, engages the s receptor-binding domain, recognizing a highly conserved protein/glycan epitope distinct from the receptor-binding motif (177) . more recently, other potent neutralizing antibodies were isolated by different research institutions (178) (179) (180) . amidst the gamut of high-affinity antibodies with the potential to neutralize human pathogenic viruses, single-domain antibodies, referred to as nanobodies or nbs (15 kda), and nanobody-based human heavy chain antibodies (75 kda) derived from camelids might be harnessed as useful therapeutics for the ongoing covid-19 pandemic (181) . camelid heavy-chainonly antibodies (hcabs) are composed of two heavy chains with a single variable domain (vhh) as the target-binding module. recombinant vhhs, devoid of the effector domains, act as single-domain antibodies and harbor advantageous features over conventional antibodies (higher thermal and chemical stability, higher solubility, smaller size, lower susceptibility to steric hindrances, ease of manufacturing, and simple structure) to have been recently proposed as prospective therapeutic candidates against various infectious pathogens (181). vhhs isolated from a llama subcutaneously immunized with perfusionstabilized sars-cov-1 and mers-cov s proteins have been recently characterized and shown to be able to neutralize s pseudotyped viruses in vitro, interfering with the host cell receptor binding (182) . interestingly, sars-cov-1 s-directed vhh cross-reacted with sars-cov-2 receptor binding domain (rbd) and neutralized sars-cov-2 s pseudoviruses in vitro as a bivalent human igg fc-fusion format, underscoring the potential of vhhs to treat coronavirus diseases (182) . because of the global spread of diseases caused by flaviviruses, understanding the cross-reactivity of anti-viral immunity among these viruses is of crucial importance for predicting the evolution of viral disease outbreaks. recently, the analysis of pbmcs isolated from individuals infected by denv or vaccinated with denv tv005 or yf17d vaccines, and pulsed with a pool of antigens from autologous and heterologous flaviviruses, indicated that both cd4 and cd8 t-cell responses were specific, with little or no cross-reactivity, despite the high level of homology (183) . individuals preexposed to denv infection developed t-cell responses against non-structural zika proteins rather than structural envelope protein, suggesting that previous flaviviral infections biased the t-cell response toward more cross-reactive non-structural epitopes (184) . studies enrolling mothers who gave birth to microcephalic babies after zikv infection, showed serological evidence of a pre-existing anti-dengue response, suggesting that vaccination against denv does not protect against zikv microcephaly (185) . however, cross-reactive antibodies between zikv and denv have been described, mainly targeting the structural dimeric envelope protein (186, 187) . the antigenic sequences are both linear and quaternary, with nabs mainly recognizing the latter. the high-conserved e protein fusion loop induces cross-reactive but weak nabs that can be a marker of worst outcome during subsequent flaviviral infections (188) . a research concerning zikv-specific b-cell responses in three denv-experienced donors showed that 5 months after the infection, the pool of antibodies comprised both poorly nabs derived from pre-existing denv-induced memory b cells, associated with an enhanced zikv infection in vitro, and potent zikv-specific antibodies originated de novo (189, 190) . the possibility that wnv-specific antibodies may drive the infection by other flaviviruses is still controversial, even if cross-reactivity was demonstrated. plasma samples from convalescent human wnv patients were shown to enhance zikv infections by antibody-dependent enhancement (ade) phenomenon (191) ; conversely, mice previously infected with zikv and challenged with wnv showed enhanced protection toward the second infection (192) . the immunological flavivirus cross-reactivity, the ade phenomenon (discussed below), genetic mutations that increase the virulence, potential pre-existing immunity concerns, combined with the necessity to increase cost-effectiveness of marketable products are among the issues that have limited the development of successful vaccines until now. the use of t-cell inducing vaccines or proteins with mutations into conserved envelope fusion-loop epitopes might be useful to overcome the cross-reactivity hurdle (193) . known as ade of viral infection, ade is a phenomenon occurring when antibodies facilitate virus entry into the host cells, driving viral replication and increasing infectivity, with subsequent severe outcomes. among the several stumbling blocks in realizing a safe vaccine, ade is a phenomenon largely underestimated, but that can produce severe adverse effects, rendering vaccinated individuals more predisposed to develop harsh symptoms after infection (194) . the first report of ade dates 1964 (195) . the molecular mechanisms disclosed the involvement of fcγr (196) and complement receptors (197) . when an antiviral antibody (induced by vaccination or viral infection) with no neutralizing or sub-neutralizing activity is produced, it can act like a bridge between the virus and the fcγr expressed on the surface of immune cells, leading to viral uptake (figure 3) , as demonstrated for denv, zikv, wnv, influenza, sars-cov, mers-cov, and ebov (194) . the role of complement receptor has been demonstrated in ebov response: two antibodies directed against epitopes in close proximity bind the c1q, forming an immune complex able to enhance the virus entry into a target cell (198) , whereas in an animal model of mers-cov, c3a and c9 protein level increase was observed after passive immunization (199) . the first licensed vaccine against denv (cyd-tdv-dengvaxia) caused hospitalizations in two large multicenter phase iii trials; after result revision, it has been estimated that in seronegative individuals, it can produce adverse effects (194) , and who recommendations are to vaccinate only seropositive individuals in endemic areas of age older than 9 years. using a mathematical model of denv transmission to formulate hypothesis on vaccine trial results, it was speculated that "seronegative recipients gain transient protective cross-reactive immunity akin to that observed for natural infection, " increasing the risk of severe disease after infection, while vaccination of seropositive subjects results in boosting the immune response, producing a protection comparable with the one obtained in individuals who has had two natural infections (200) . the most severe adverse effect after vaccination was registered when a formalin-inactivated vaccine against rsv produced an increase of severe illness in vaccinated infant (hospitalization: 80% rsv vaccinated vs. 5% vaccinated against parainfluenza) and two deaths (201) . afterward, a role for the th2 response was hypothesized in generating the rsv-mediated ade (202) , and it was demonstrated that the formalin-inactivated virus produced ade in monkeys (203) , suggesting that the carbonyl groups on formaldehyde-inactivated rsv were responsible for the th2 response in mice (204) . moreover, the observation that formalin inactivation produced an alteration of antigens, leading to the production of non-nabs, whose avidity did not mature, and the activation of complement were also reported for a measles vaccine (205) . the low-avidity non-nabs are produced in absence of tlr activation (and affinity maturation), and they figure 3 | antibody-dependent enhancement on dengue infection. antibodies generated from a previous denv infection can recognize but do not neutralize another denv serotype and can lead to antibody-dependent enhancement (ade) of entry of the latter virus into host cells. the pre-existing non-(or sub-) neutralizing antibodies bind denv through the fab domains and mediate viral entry into fcγr-expressing cells. on engagement by the fc domains, the virus-antibody immune complex is internalized by the activating fcγriia within the endosome. co-ligation of fcγriia and lilrb1 (leukocyte immunoglobulin-like receptor-b1) to opsonized denv drives the inhibitory signal cascade via immunoreceptor tyrosine-based inhibition motif (itim) pathway, abrogating the expression of isgs (interferon stimulated genes). ligation of fcγriia to immune complex also increases th2 cytokine production and reduces ifnγ, inhibiting the jak/stat signaling pathway, overall resulting in the suppression of the antiviral response and increase of viral replication. nabs, neutralizing antibodies; and itam, immunoreceptor tyrosine-based activation motif. trigger complement activation (206) , enhancing viral infection. to induce potent nabs, the tlr activation has been obtained using a th1-polarizing adjuvant (207) , in association with the candidate vaccine exposing the epitopes of interest. antibody-dependent enhancement has been reported also in many studies focusing on the development of sars and mers vaccines, demonstrating that vaccination with the whole s glycoprotein can increase the susceptibility to viral infection with a mechanism not linked to the virus receptor expression on the host cells (208) , and especially when antibodies are induced with low titer (209) . while for many flaviviruses the mechanism of ade has been explained through evidences that antibodies developed during a primary infection can enhance entry of a heterologous virus via fc-receptor during a secondary infection, for mers-cov and sars-cov, it has also been proposed that nabs that strongly bind the rbd region of the s surface protein can induce conformational changes that enhance the virus entry via canonical viral-receptor-dependent pathways, mimicking viral receptor binding (210, 211) , and antibodies targeting a specific region of the s protein enhanced the viral infection in a sars model of nhps (212) . the high sequence homology and the similarity in structure shared among sars-cov, mers-cov, and sars-cov2 s glycoproteins raises reasonable concerns about the development of covid-19 vaccines based on the s protein. in potential pandemic settings, the clinical development of vaccines is the main aim. however, apart from technical reasons, the vaccine production might be delayed also for economic considerations and safety issues. other strategies may be based on self-disseminating vaccines and induction of trained immunity. to control zoonosis, the formulation of self-disseminating vaccines acts at the level of animal, insect, or environmental reservoir, to directly interfere within the animal-to-human transmission (213) . they are essentially based on replicating viral vectors engineered to express the disease antigen and to target a certain animal population (214) . global vaccination of animals could be achieved to effectively contain an emerging pathogen within the wildlife reservoir, avoiding its global spread. feasibility concerns, costs, and safety issues should be considered when using this strategy to control reservoirs linked to the emergence of high-risk pathogens. in addition, which animal pathogen will cause a human disease is generally unpredictable. it is interesting to underline that a vaccination of great apes with an engineered specific cmv-based vector has been proposed as a strategy to potentially interrupt (or at least decrease) the zoonotic transmission of ebola virus to humans, being able to protect animals from the lethal viral challenge (213, 215) . trained immunity-based vaccines (tibv) might be formulated to stimulate broader anti-infectious responses compared with conventional vaccines for their capacity to increase innate immunity and enhance adaptive responses (216) . this strategy exploits the ability of innate immune cells (monocytes, macrophages, nk cells) to undergo extensive metabolic and epigenetic reprogramming, following certain vaccinations or infections, and to become primed for a quite long period of time to respond more potently to autologous or heterologous re-infection, mounting the so-called "innate immune memory." triggering of pattern recognition receptors (prrs) by microbial effector stimuli results in increased production of pro-inflammatory cytokines and/or reactive oxygen species, and in enhanced immune responses, regardless the primary stimulation (217) . many infectious stimuli are considered potent activators of trained innate immunity, including β-glucan and chitin (components of fungal cell wall), lps (a component of the cell wall of gram-negative bacteria), and the bacille calmette-guérin (bcg) vaccine (218) . thus, tibv should contain pathogen-associated molecular patterns (pamps) to target prrs and subsequently induce trained immune cells. bcg vaccine, vacv, and live attenuated influenza vaccines, together with immunostimulants, could be ascribed to this category of vaccines (216) . it is worth mentioning that a whole-cell killed bacterial vaccine might have played a role in preventing pneumonia and mortality during the 1918 influenza pandemic (219) . recently, a work by berg and colleagues showed that bcg vaccination is associated with the flattening of the curve in the spread of covid-19, suggesting that bcv vaccine might serve as a protective factor against the disease (220). however, it should also be noted that an enhanced immune response mediated by reprogrammed immune cells could contribute to the development or maintenance of inflammatory, neuroinflammatory, and chronic metabolic disorders (221) . the phenomenon of "trained immunity" occurring in the brain is known as microglial priming. exposure of primed microglial cells to a second stimuli can cause an augmented inflammatory response, leading to neuroinflammation and production of neurotoxic molecules. the hyperglycemia condition that characterizes type 2 diabetes could long term affect the cellular metabolism of monocytes and macrophages, leading to increased cytokine production and subsequent diabetes complications, including atherosclerosis. an augmented activation of innate cells may also result in the induction and maintenance of chronic inflammatory disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or sarcoidosis (221) . the covid-19 pandemic experience, combined with the previous viral disease outbreaks, should give blueprints for rapidly responding to the emergence of high-risk pathogens in the future. it is a common belief that vaccines would be the only means of providing long-term immunity and preventing viral diseases. despite the great progress made in vaccine research, we are still unable to produce successful vaccines in a timely manner. human trials take a long time and given a huge list of vaccine candidates, it is hard to choose the most promising one. while the who proposed a solidarity vaccine trial to test all the candidates in rolling trial until they fail, to increase the chances of succeeding, some vaccine stakeholders are considering extreme alternatives for emergency use: intentionally infect young healthy volunteers at low risk in controlled "human challenge trials" to define which vaccine will work (222) . although these approaches are already used for studying influenza (223) and dengue diseases (224) , it is hard to ethically accept this option without a validated therapy. vaccines go through regulatory pathways before the final approval and licensure. in epidemic or pandemic settings, we need to carefully develop a vaccine, as quickly as possible, that adequately proved to be safe and effective (225) . scientists need to fill the gaps in understanding the epidemiology of novel viruses, to identify potential zoonotic reservoirs or spill-over hosts, and the way of transmission of pathogens. once the pathogen is identified, preclinical models need to be developed to study virus-host interactions and early test vaccine candidates, defining the immune correlates of protection. pathogen-specific epitopes need to be identified to guide structure-based vaccines that will elicit protective antibodies, minimizing the induction of non-or weakly nabs that would promote ade of viral infection (226) . moreover, data sharing and collaboration among academia, government, and companies will be essential to coordinate a strategic approach in face of next pandemic threats (227) . all authors equally contributed to this work and read and approved the final manuscript. this work was supported by prin 2017 "nanotechvax tackling biological barriers to antigen delivery by nanotechnological vaccines" prot. 20173zeccm, and consiglio nazionale delle ricerche (cnr), italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." mt was supported by postdoctoral fellowship from cnr, italy: laboratori congiunti bilaterali internazionali (scienze biomediche), project: "new vaccines against poverty-related and neglected tropical diseases." references 1. nii-trebi ni. emerging and neglected infectious diseases: insights, advances, and challenges infectious disease threats in the twenty-first century: strengthening the global response the epidemic of 2019-novel-coronavirus (2019-ncov) pneumonia and insights for emerging infectious diseases in the future major factors affecting the emergence and re-emergence of infectious diseases climate change and multiple emerging infectious diseases infectious disease and economics: the case for considering multi-sectoral impacts emerging infectious diseases: a proactive approach identification of a novel coronavirus in patients with severe acute respiratory syndrome characterizing the epidemiology of the 2009 influenza a/h1n1 pandemic in mexico middle east respiratory syndrome ebola virus disease 2013-2014 outbreak in west africa: an analysis of the epidemic spread and response first report of autochthonous transmission of zika virus in brazil metagenomic sequencing at the epicenter of the nigeria 2018 lassa fever outbreak the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status world health organization r&d blueprint national institutes for allergy and infectious diseases (nih) niaid emerging infectious diseases/pathogens new vaccine technologies to combat outbreak situations global trends in emerging infectious diseases centers for disease control and prevention (cdc) principles of epidemiology in public health practice. an introduction to applied epidemiology and biostatistics the epidemic of west nile virus in the united states nile in europe: an increasing public health problem zika virus, vectors, reservoirs, amplifying hosts, and their potential to spread worldwide: what we know and what we should investigate urgently microcephaly case fatality rate associated with zika virus infection in brazil: current estimates yellow fever: a reemerging threat yellow fever in the americas: the growing concern about new epidemics. f1000res the emergence of arthropod-borne viral diseases: a global prospective on dengue, chikungunya and zika fevers history, epidemiology and diagnostics of dengue in the american and brazilian contexts: a review atypical chikungunya virus infections: clinical manifestations, mortality and risk factors for severe disease during the 2005-2006 outbreak on réunion growth of cities could boost mosquito-borne diseases lassa fever in west african sub-region: an overview epidemiologic and clinical features of lassa fever outbreak in nigeria transmission of ebola virus disease: an overview forty-five years of marburg virus research the ebola outbreak, 2013-2016: old lessons for new epidemics how severe and prevalent are ebola and marburg viruses? a systematic review and meta-analysis of the case fatality rates and seroprevalence antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans influenza: the mother of all pandemics estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study predictors of fatality in pandemic influenza a (h1n1) virus infection among adults estimates of pandemic influenza vaccine effectiveness in europe, 2009-2010: results of influenza monitoring vaccine effectiveness in europe (i-move) multicentre case-control study centers for disease control and prevention (cdc) 2009 h1n1 pandemic (h1n1pdm09 virus sars and mers: recent insights into emerging coronaviruses a tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses world health organization middle east respiratory syndrome coronavirus (mers-cov) the proximal origin of sars-cov-2 genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding available online at challenges and solutions in the development of vaccines against emerging and neglected infectious diseases history of vaccination history of smallpox and its spread in human populations influenza vaccination strategies: comparing inactivated and live attenuated influenza vaccines rapid development of an inactivated vaccine candidate for sars-cov-2 mrna as a transformative technology for vaccine development to control infectious diseases advances in mrna vaccines for infectious diseases mrna vaccines -a new era in vaccinology modified mrna vaccines protect against zika virus infection nucleoside-modified mrna immunization elicits influenza virus hemagglutinin stalk-specific antibodies preclinical and clinical demonstration of immunogenicity by mrna vaccines against h10n8 and h7n9 influenza viruses self-amplifying mrna vaccines expressing multiple conserved influenza antigens confer protection against homologous and heterosubtypic viral challenge dendrimer-rna nanoparticles generate protective immunity against lethal ebola, h1n1 influenza, and toxoplasma gondii challenges with a single dose a tetravalent alphavirus-vector based dengue vaccine provides effective immunity in an early life mouse model the covid-19 vaccine development landscape sars-cov-2 vaccines: status report covid-19: what do we know so far about a vaccine? molecular mechanisms for enhanced dna vaccine immunogenicity engineering dna vaccines against infectious diseases induction of broad cytotoxic t cells by protective dna vaccination against marburg and ebola a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates a west nile virus dna vaccine utilizing a modified promoter induces neutralizing antibody in younger and older healthy adults in a phase i clinical trial immunogenicity and protective efficacy of a vaxfectin-adjuvanted tetravalent dengue dna vaccine a dna vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates dna vaccine protection against sars-cov-2 in rhesus macaques who draft landscape of covid-19 candidate vaccines modified vaccinia virus ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine viruses as vaccine vectors for infectious diseases and cancer viral vectors as vaccine platforms: from immunogenicity to impact effective induction of high-titer antibodies by viral vector vaccines hiv-1 vaccine-induced immunity in the test-of-concept step study: a casecohort analysis chimpanzee adenovirus vector ebola vaccine chimpanzee adenovirus-and mva-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults novel viral vectors in infectious diseases immunogenicity and safety of a single dose of a live attenuated japanese encephalitis chimeric virus vaccine in vietnam: a single-arm, single-center study efficacy and long-term safety of a dengue vaccine in regions of endemic disease current state and recent advances in biopharmaceutical production in escherichia coli, yeasts and mammalian cells application of recombinant dna techniques in the development of viral vaccines the influence of antigen organization on b cell responsiveness vaccine delivery: a matter of size, geometry, kinetics and molecular patterns hiv-1 gag p17 presented as virus-like particles on the e2 scaffold from geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of ifnγ production by cd4+ t cells hepatitis c vlps delivered to dendritic cells by a tlr2 targeting lipopeptide results in enhanced antibody and cell-mediated responses cd40l-containing virus-like particle as a candidate hiv-1 vaccine targeting dendritic cells malarial epitopes expressed on the surface of recombinant tobacco mosaic virus antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and hiv-1 the production of hemagglutinin-based virus-like particles in plants: a rapid, efficient and safe response to pandemic influenza phase i trial of an alhydrogel adjuvanted hepatitis b core virus-like particle containing epitopes of plasmodium falciparum circumsporozoite protein engineering virus-like particles as vaccine platforms microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development recombinant sars-cov-2 spike s1-fc fusion protein induced high levels of neutralizing responses in nonhuman primates explosive spread of a neuroinvasive lineage 2 west nile virus in central europe genetic determinants of virulence in pathogenic lineage 2 west nile virus strains equine vaccine for west nile virus a west nile virus (wnv) recombinant canarypox virus vaccine elicits wnv-specific neutralizing antibodies and cell-mediated immune responses in the horse an observer blinded, randomized, placebo-controlled, phase i dose escalation trial to evaluate the safety and immunogenicity of an inactivated west nile virus vaccine, hydrovax-001, in healthy adults immunogenicity and protective efficacy of a recombinant subunit west nile virus vaccine in rhesus monkeys preclinical and clinical development of a yfv 17 d-based chimeric vaccine against west nile virus immunogenicity and protective efficacy of recombinant modified vaccinia virus ankara candidate vaccines delivering west nile virus envelope antigens a west nile virus dna vaccine induces neutralizing antibody in healthy adults during a phase 1 clinical trial twenty years of progress toward west nile virus vaccine development a1: recombinant subunit west nile virus vaccine for protection of human subjects. united states patent us 20120141520 advanced oxidation technology for the development of a next-generation inactivated west nile virus vaccine replication-defective west nile virus with ns1 deletion as a new vaccine platform for flavivirus zika virus associated with microcephaly a single-dose live-attenuated vaccine prevents zika virus pregnancy transmission and testis damage a live-attenuated zika virus vaccine candidate induces sterilizing immunity in mouse models a vaccine based on a modified vaccinia virus ankara vector expressing zika virus structural proteins controls zika virus replication in mice construction and evaluation of novel rhesus monkey adenovirus vaccine vectors protective efficacy of multiple vaccine platforms against zika virus challenge in rhesus monkeys preventative vaccines for zika virus outbreak: preliminary evaluation recent advances in zika virus vaccines safety, tolerability, and immunogenicity of two zika virus dna vaccine candidates in healthy adults: randomised, open-label, phase 1 clinical trials comparison of the virulent asibi strain of yellow fever virus with the 17d vaccine strain derived from it live attenuated yellow fever 17d vaccine: a legacy vaccine still controlling outbreaks in modern day development of sanofi pasteur tetravalent dengue vaccine from research to phase iii: preclinical, industrial and clinical development of the sanofi pasteur tetravalent dengue vaccine pathogenesis of dengue: challenges to molecular biology antibody-dependent enhancement of severe dengue disease in humans dengue: status of current and under-development vaccines immunogenicity, safety, and tolerability of the measles-vectored chikungunya virus vaccine mv-chik: a double-blind, randomised, placebo-controlled and active-controlled phase 2 trial recent progress in vaccine development against chikungunya virus effective vaccine for lassa fever vaccine platforms for the prevention of lassa fever vaccines inducing immunity to lassa virus glycoprotein and nucleoprotein protect macaques after a single shot isolation of marburg-like virus from a case of haemorrhagic fever in zaire vaccines against ebola virus a review of phase i trials of ebola virus vaccines: what can we learn from the race to develop novel vaccines viruslike particle vaccination protects nonhuman primates from lethal aerosol exposure with marburgvirus (vlp vaccination protects macaques against aerosol challenges) vector choice determines immunogenicity and potency of genetic vaccines against angola marburg virus in nonhuman primates live attenuated recombinant vaccine protects nonhuman primates against ebola and marburg viruses protection against marburg virus using a recombinant vsv-vaccine depends on t and b cell activation singledose trivalent vesiculovax vaccine protects macaques from lethal ebolavirus and marburgvirus challenge cross-protection conferred by filovirus virus-like particles containing trimeric hybrid glycoprotein stability and suitability for storage and distribution of ad26.zebov/mva-bn r -filo heterologous prime-boost ebola vaccine human coronavirus oc43 3cl protease and the potential of ml188 as a broad-spectrum lead compound: homology modelling and molecular dynamic studies epidemiology, genetic recombination, and pathogenesis of coronaviruses available online at a novel coronavirus from patients with pneumonia in china mers: emergence of a novel human coronavirus molecular pathology of emerging coronavirus infections the spike protein of sars-cova target for vaccine and therapeutic development receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc coronavirus infections and immune responses severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study who scientific and technical advisory group for infectious hazards aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study serology characteristics of sars-cov-2 infection since exposure and post symptom onset efficacy of genogroup 1 based porcine epidemic diarrhea live vaccine against genogroup 2 field strain in japan antibody responses to sars-cov-2 in patients with covid-19 rapid generation of neutralizing antibody responses in covid-19 patients sars vaccines: where are we? from sars to mers, thrusting coronaviruses into the spotlight searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design receptor-binding domain-based subunit vaccines against mers-cov intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus development of middle east respiratory syndrome coronavirus vaccines -advances and challenges middle east respiratory syndrome vaccine candidates: cautious optimism sars-cov-2 infection protects against rechallenge in rhesus macaques the convalescent sera option for containing covid-19 potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody a human monoclonal antibody blocking sars-cov-2 infection cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody potent neutralizing antibodies directed to multiple epitopes on sars-cov-2 spike structural basis for neutralization of sars-cov-2 and sars-cov by a potent therapeutic antibody longitudinal isolation of potent near-germline sars-cov-2-neutralizing antibodies from covid-19 patients nanobodies: prospects of expanding the gamut of neutralizing antibodies against the novel coronavirus, sars-cov-2 structural basis for potent neutralization of betacoronaviruses by singledomain camelid antibodies t cell responses induced by attenuated flavivirus vaccination are specific and show limited cross-reactivity with other flavivirus species two is better than one: evidence for t-cell cross-protection between dengue and zika and implications on vaccine design. front immunol strong cd4 t cell responses to zika virus antigens in a cohort of dengue virus immune mothers of congenital zika virus syndrome infants structural basis of potent zika-dengue virus antibody crossneutralization cross-reactivity, and function of antibodies elicited by zika virus infection cross-reactive immunity among flaviviruses zika virus activates de novo and cross-reactive memory b cell responses in dengue-experienced donors dengue virus sero-cross-reactivity drives antibodydependent enhancement of infection with zika virus enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity zika virus infection confers protection against west nile virus challenge in mice nile virus vaccines -current situation and future directions viral-induced enhanced disease illness enhancement of the infectivity of arboviruses by specific antisera produced in domestic fowls the enhancement of virus infectivity by antibody complement receptor mediates enhanced flavivirus replication in macrophages antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody benefits and risks of the sanofi-pasteur dengue vaccine: modeling optimal deployment respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine priming immunization determines t helper cytokine mrna expression patterns in lungs of mice challenged with respiratory syncytial virus antibodydependent enhancement, a possible mechanism in augmented pulmonary disease of respiratory syncytial virus in the bonnet monkey model a potential molecular mechanism for hypersensitivity caused by formalin-inactivated vaccines a role for nonprotective complement-fixing antibodies with low avidity for measles virus in atypical measles lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease a novel respiratory syncytial virus (rsv) f subunit vaccine adjuvanted with gla-se elicits robust protective th1-type humoral and cellular immunity in rodent models antibodydependent infection of human macrophages by severe acute respiratory syndrome coronavirus antibodydependent sars coronavirus infection is mediated by antibodies against spike proteins molecular mechanism for antibody-dependent enhancement of coronavirus entry unexpected receptor functional mimicry elucidates activation of coronavirus fusion immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates emerging viruses and current strategies for vaccine intervention self-disseminating vaccines for emerging infectious diseases a cytomegalovirus-based vaccine provides long-lasting protection against lethal ebola virus challenge after a single dose trained immunity-based vaccines: a new paradigm for the development of broad-spectrum anti-infectious formulations trained immunity: a program of innate immune memory in health and disease infectious agents as stimuli of trained innate immunity efficacy of whole-cell killed bacterial vaccines in preventing pneumonia and death during the 1918 influenza pandemic mandated bacillus calmette-guérin (bcg) vaccination predicts flattened curves for the spread of covid-19 trained innate immunity not always amicable scores of coronavirus vaccines are in competition -how will scientists choose the best? the future of flu: a review of the human challenge model and systems biology for advancement of influenza vaccinology challenge accepted: human challenge trials for dengue tortoises, hares, and vaccines: a cautionary note for sars-cov-2 vaccine development rational vaccine design in the time of covid-19 a strategic approach to covid-19 vaccine r&d a dictionary of epidemiology the authors declare that the research was conducted in the key: cord-353732-7hjsux4m authors: arabi, yaseen m.; al-enezi, farhan; longuere, kajsa-stina; balkhy, hanan h.; al-sultan, mohamed; al-omari, awad; al-hameed, fahad m.; carson, gail; shindo, nahoko; fowler, robert title: feasibility of a randomized controlled trial to assess treatment of middle east respiratory syndrome coronavirus (mers-cov) infection in saudi arabia: a survey of physicians date: 2016-07-12 journal: bmc anesthesiol doi: 10.1186/s12871-016-0198-x sha: doc_id: 353732 cord_uid: 7hjsux4m background: the middle east respiratory syndrome coronavirus (mers-cov) is an emerging respiratory pathogen with a high mortality rate and no specific treatments available to date. the purpose of this study was to determine the feasibility of conducting a randomized controlled trial (rct) of convalescent plasma therapy for mers-cov-infected patients by using mers-cov-specific convalescent plasma obtained from previously recovered patients. methods: a survey was adapted from validated questionnaire originally aimed to measure network capacities and capabilities within the international severe acute respiratory and emerging infection consortium (isaric). the questionnaire was modified for this study to include 26 items that were divided into three main domains of interest: (1) the ability to care for critically ill mers-cov patients; (2) laboratory capacity to diagnose mers-cov and blood bank ability to prepare convalescent plasma; and (3), research capacity to conduct randomized controlled trials. the questionnaire was emailed to physicians. results: of 582 physicians who were invited to the survey, 327 responded (56.2 %). the professional focus of the majority of respondents was critical care (106/249 (43 %)), pediatrics (59/249, (24 %)) or internal medicine (52/249 (21 %)) but none was blood banking. nearly all respondents (251/263 (95 %)) reported to have access to icu facilities within their institutions. most respondents (219/270 (81 %)) reported that intensivists were the most physician group responsible for treatment decisions about critically ill sari patients. while 125/165 respondents (76 %) reported that they conduct research in icus, and 80/161 (49.7 %) had been involved in the conduct of rcts, including using a placebo comparison (60/161 (37 %)), only 49/226 (21 %) of respondents regularly participated in research networks. conclusions: our survey indicated that in the kingdom of saudi arabia (ksa), icus are the most likely clinical locations for conducting a clinical trial of convalescent plasma therapy for mers-cov, and that most icus have experience with such research designs. electronic supplementary material: the online version of this article (doi:10.1186/s12871-016-0198-x) contains supplementary material, which is available to authorized users. a novel coronavirus was first diagnosed in saudi arabia in june 2012 in a man who presented with pneumonia and acute renal failure [1] . this newly discovered virus leading to severe pneumonia and acute respiratory distress syndrome (ards) was later named middle east respiratory syndrome coronavirus (mers-cov). as of july 3, 2016, there have been 1769 laboratoryconfirmed cases of mers-cov infection, 85 % of which occurred in the kingdom of saudi arabia [2] . the case fatality rate is 35 % and reaches 70 % in critically ill patients [3] with no specific therapy available to date. convalescent plasma that contains mers-cov-specific immunoglobulin and is obtained from previously recovered patients has been suggested as a potential therapy for patients with mers-cov infection. convalescent plasma has been used to treat several other viral infections, including the severe acute respiratory syndrome (sars) sars coronavirus (sars-cov), spanish influenza a (h1n1), avian influenza a (h5n1), and 2009 pandemic influenza a (h1n1 pdm09) [4] [5] [6] [7] [8] [9] [10] . a recent meta-analysis of observational studies of passive immunotherapy in sars and severe influenza suggest reductions in mortality with timely use of convalescent blood products, particularly those with neutralizing antibodies [11] . public health england and the international severe acute respiratory & emerging infection consortium (isaric) have published a decision-support tool for clinicians managing cases of mers-cov infection [12] , which relies heavily upon expert opinion and clinical experience of treating patients with severe respiratory disease caused by other viruses including sars and 2009 pandemic influenza a (h1n1pdm09). the document suggests that current evidence is strongest for testing convalescent plasma (cp) or other therapeutics which contain neutralizing antibodies (such as hyperimmune immunoglobulin) for treatment of serious mers-cov illness. given the scattered distribution of cases in saudi arabia, it is unclear whether the clinical and research infrastructure would be sufficient to support the conduction of a randomized controlled trial (rct) of convalescent plasma therapy. such a study will require laboratory ability to diagnose mers-cov infection and identify antibody titers among previously infected but recovered patients, research infrastructure and ability to lead or participate in an rct, and the presence of personal and community equipoise among healthcare workers and patients to enroll in such a trial. therefore, as part of a collaborative effort among colleagues from the gulf states and eastern mediterranean and with the support of the world health organization and international severe acute respiratory and emerging infection consortium, we undertook a survey to assess feasibility of conducting a clinical trial of convalescent plasma therapy for patients with mers-cov infection in ksa. the study targeted health care specialists in internal medicine, critical care, respiratory disease, infectious disease, hematology, and pediatrics, along with clinical microbiologists, hospital epidemiologists and blood bankers. the survey consisted of voluntary anonymous responses to a web-based questionnaire and did not include actual patient data. a validated questionnaire, previously used by isaric to evaluate and map capacity conduct of clinical trials in response to outbreaks, was used as a basis for the development of this survey. omitting questions that were deemed not relevant to this study, and modifying or adding questions that were more appropriate to the current objectives led to a questionnaire of 26 items, divided into three main domains of interest. first, four questions aimed to evaluate the ability and capacity of caring for critically ill patients within the ksa health system (e.g., "how many beds are capable of caring for mechanically ventilated patients in the icu where patients with severe acute respiratory infection (sari) would usually be treated?"). second, the study sought to explore laboratory capacity to diagnose mers-cov infection and blood bank ability to prepare convalescent plasma through seven questions (e.g., "do you have a mechanism to send specific blood donors (e.g., survivors of mers-cov infection) to the blood bank to make donations?" and "what methodologies are available in the laboratories associated with your site for mers-cov testing?"). the third domain aimed to evaluate research capacity needed to conduct clinical trials and whether or not necessary infrastructure to conduct rcts existed in ksa, through 10 questions specifically related to research (e.g., "do you believe that patients at your hospital would participate in randomized studies in which patients are randomly assigned to one of two or more treatments?"). the questionnaire was populated using survey monkey, was emailed through the snowball methodology, to physicians who are possibly involved in the care of mers-cov patients. snowball sampling is a chain referral sampling where the initial participants are asked to assist in recruiting more subjects by passing on the questionnaire. we initiated contact with 250 participants from 48 hospitals in saudi arabia; those in turn invited others, with a total subjects approached being approximately 582. there were three reminders, 2 weeks apart by email to non-respondents, and the data was collected between march and may 2014. a complete list of the survey questions is included in the online supplement (additional file 1). the institutional review board (irb) of ministry of national guard health affairs (rc14/003/r-29 april 2015) approved this study. the first question of the survey was a question about whether the participant consents to the survey. descriptive statistics (frequencies and percentages) were used to quantify the categorical study variables. of 582 physicians who were invited to the survey, 327 responded (56.2 %) ( table 1 ). the professional focus of the majority of respondents was critical care (106/249 (43 %)), pediatrics (59/249 (24 %)) or internal medicine (52/249 (21 %)) but none was blood banking. nearly all respondents (251/263, 95 %) reported to have access to icu facilities within their institutions and 44 % of these icus had more than 20 beds. most respondents (219/ 270 (81 %)) reported that intensivists were the most physician group responsible for treatment decisions about critically ill sari patients. available reported therapies in the icus included extra-corporal membrane oxygenation (ecmo) (48 %), hemodialysis (91 %) and plasmapheresis (82 %). majority of the respondents (235/250 (94 %)) indicated that blood products used in their hospitals were obtained in the same hospital, only 15/250 (6 %) reported that blood products are obtained from a blood bank at another institution. when asked about the feasibility of having mers-cov infection survivors to donate blood/ plasma, 130/237 (55 %) of the respondents reported this would be feasible (table 2) . a majority (240/248 (97 %)) of the respondents reported to have access to the necessary facilities and capabilities for screening for blood-borne viruses in donated blood within their a majority 146/179 (82 %) of respondents said that they had access to registry data for patients receiving care at their sites, and more than 50 % of respondents reported that their institutions had a research administration infrastructure in place to aid with data-sharing agreements (52 %), research contract (49 %), ethics reviews (74 %), and administrative support for research projects (60 %). the retrospective observational studies include case reports, chart review case series, case-control studies, cohort studies average time for the ethical review process (from submission to approval to enroll patients) was approximately 1 to 3 months. although 22 % of respondents said that the patients at their hospital were definitely not or probably not likely to participate in randomized studies and 39 % said that they were 'maybe' prepared to parttake in such studies, 40 % of respondents said they believed their patients would probably or definitely agree to participate. our survey identifies that many healthcare facilities in ksa have the necessary capacity to participate rcts that enroll severely ill patients with mers-cov infection. the survey highlights a few important points in support of this conclusion. first, most respondents believed that the needed infrastructure was in placesuch as data registries, laboratory capacity, ethics review processes, and monitoring units for rcts. second, respondents seemed to be aware of the required capacity for research in mers-cov. third, although not the most common trial design in saudi arabia, most respondents were aware of rcts and what they entail, and a majority of them believed that their patients would consider participating in rcts. however, many barriers to conducting rcts remain. first, there is low community awareness of research in general, which is needed for any rct to take place. second there may be lengthy processes required to obtain regulatory and ethics approval to initiate clinical trials. this survey suggests that the necessary clinical and laboratory facilities exist to conduct such rcts. the presence of icus with sufficient numbers of beds is a necessary component of conducting rcts on potential therapies for critically ill patients with mers-cov. essential laboratory infrastructure for such studies are also available including blood banking, microbiologic diagnostic testing for pneumonia in general and mers-cov in particular. however, as none of the respondents was a blood bank specialist, it is not possible to explore this in greater detail on the basis of this survey alone. a majority of the respondents (54.9 %) reported that sending individuals who had recovered from mers-cov to donate blood is feasible, which is a positive and necessary step for obtaining convalescent plasma. there are a number of limitations associated with this study. because of the snowball methodology, the number of subjects who were approached is only approximate. response rate was modest, and response rates to individual questions varied. due to the anonymity of replies to this survey it has not been possible to follow up on incomplete responses. there were no responses from blood banks. as with other surveys, it is possible that individuals motivated to conduct research or work in institutions that conduct research were more likely to respond to the survey; creating a bias towards more favorable answers for research infrastructure and culture. the study did not include a face-to-face interview, which would have resulted in higher targeted responses. finally, surveys represent stated responses as opposed to measurement of actual practice. our survey results indicate that the research infrastructure at many acute care facilities in saudi arabia is likely generally sufficient to conduct a rct to investigate the efficacy of convalescent plasma treatment for severely ill patients with mers-cov. next steps in such a research program will need to include an observational study of actual clinical and diagnostic practice in the care of patients with mers-cov in ksa. this would help to inform design of a pilot clinical trial with a goal to demonstrate feasibility and safety of convalescent plasma evaluation, and would need to occur before a true evaluation of efficacy could take place. isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a(h1n1) infection sars: systematic review of treatment effects convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities use of convalescent plasma therapy in sars patients in hong kong successful treatment of avian influenza with convalescent plasma convalescent plasma study group family cluster of middle east respiratory syndrome coronavirus infections not applicable. not applicable. the authors declare that they have no competing interests. not applicable. the institutional review board (irb) of ministry of national guard health affairs (rc14/003/r-29 april 2015) approved this study and the first question of the survey was a question about whether the participant consents for the survey. nahoko shindo is a staff member of the world health organization. the author alone is responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the world health organization. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-327063-ea7a1xfl authors: dhama, kuldeep; patel, shailesh kumar; sharun, khan; pathak, mamta; tiwari, ruchi; yatoo, mohd iqbal; malik, yashpal singh; sah, ranjit; rabaan, ali a.; panwar, parmod kumar; singh, karam pal; michalak, izabela; chaicumpa, wanpen; martinez-pulgarin, dayron f.; bonilla-aldana, d. katterine; rodriguez-morales, alfonso j. title: sars-cov-2 jumping the species barrier: zoonotic lessons from sars, mers and recent advances to combat this pandemic virus date: 2020-08-02 journal: travel med infect dis doi: 10.1016/j.tmaid.2020.101830 sha: doc_id: 327063 cord_uid: ea7a1xfl coronavirus disease 2019 (covid-19), caused by sars-cov-2 (severe acute respiratory syndrome coronavirus-2) of the family coronaviridae, appeared in china in december 2019. this disease was declared as posing public health international emergency by world health organization on january 30, 2020, attained the status of a very high-risk category on february 29, and now having a pandemic status (march 11). covid-19 has presently spread to more than 215 countries/territories while killing nearly 0.62 million humans out of cumulative confirmed infected asymptomatic or symptomatic cases accounting to almost 15 million as of july 22, 2020, within a short period of just a few months. researchers worldwide are pacing with high efforts to counter the spread of this virus and to design effective vaccines and therapeutics/drugs. few of the studies have shown the potential of the animal-human interface and zoonotic links in the origin of sars-cov-2. exploring the possible zoonosis and revealing the factors responsible for its initial transmission from animals to humans will pave ways to design and implement effective preventive and control strategies to counter the covid-19. the present review presents a comprehensive overview of covid-19 and sars-cov-2, with emphasis on the role of animals and their jumping the cross-species barriers, experiences learned from sarsand mers-covs, zoonotic links, and spillover events, transmission to humans and rapid spread, and highlights the new advances in diagnosis, vaccine and therapies, preventive and control measures, one health concept along with recent research developments to counter this pandemic disease. in the 21 st century, we have faced a few deadly disease outbreaks caused by pathogenic viruses such as bird flu caused by avian influenza virus h5n1, swine flu caused by reassorted influenza virus h1n1 pandemic 2009 (h1n1pdm2009), severe acute respiratory syndrome (sars) caused by sars-cov (coronavirus), the middle east respiratory syndrome (mers) caused by mers-cov [1] [2] [3] , ebola [4] , zika [5, 6] , nipah virus infections, and the most recent threat [7] , coronavirus disease 2019 (covid19 ) that has been posed by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) of the family coronaviridae, genus betacoronavirus [8] [9] [10] [11] [12] [13] [14] . the sars-cov-2 virus emerged from the city of wuhan, hubei province, china, during december 2019, was declared as public health international emergency by the world health organization (who) on january 31, 2020. consequently, it was categorized in a high-risk category on february 29, 2020, and gained the pandemic status on march 11, 2020 . the disease emerged from wuhan, china, as its epicentre, which moved later to italy, then the usa, and brazil. subsequently, within a short time interval of six months, it has affected nearly 215 countries/territories and claimed near to 0.62 million human deaths out of cumulative confirmed infected asymptomatic or symptomatic cases accounting to almost 15 million. sars-cov-2 has very adversely affected the usa, brazil, india, russia, south africa, peru, mexico, chile, spain, the united kingdom (uk), iran, pakistan, saudi arabia, italy and other countries. the disease incidences are lower in children than adults but exhibit all symptoms of a disease like adults [15] . the lessons learned from earlier threats of sars, mers and the present covid-19 pandemic situations warrants designing and implementing some modified plans and strategies to combat emerging and zoonotic pathogens that could pose pandemic threats/risks while taking away many human lives [11, [16] [17] [18] [19] [20] [21] [22] . researchers and health agencies across the world are putting high efforts to contain/restrain the spread of this deadly disease. they are pacing to develop potential vaccines and therapeutics/drugs [23, 24] . evidence from the initial outbreak indicates earlier cases had links to huanan wholesale seafood market in china [25] and further isolation of sars-cov-2 from different samples of the area (people, animals, birds, discharges, soil, structures) suggests the involvement of intermediate hosts [26] . recently, a literature of review has pointed out the possible potential role of the animal-human interface, zoonotic links and spillover events towards the origin of sars-cov-2/ covid-19 [11, 20, [27] [28] [29] [30] [31] . in the past couple of decade's animal origin viral diseases, especially bats-linked, have increased many folds in humans with noted cross-species transmissions. although many of the illnesses are linked with bats still information on their ecological behaviour, molecular aspects are limited, which could lead to more viral outbreaks shortly [32] . the ongoing covid-19 pandemic has emphasized the importance of understanding the evolution of natural hosts in response to viral pathogens. in a recent study on ace2 receptors, the gene was found under intense selection pressure in bats and positive selection in other selected mammalian hosts [33] . the sars-cov-2 is also thought to have originated from bats, just like sars-cov and mers-cov. civets and dromedary camels are considered as the intermediate host of sars-and mers-cov, respectively, from where they were transmitted to humans [34] . the understanding of genomic signatures of sars-cov-2 with other covs is must for strategic planning through identifying natural or intermediate hosts. using genomic and protein data in a natural vector method (alignment-free approach), phylogenetic analysis revealed the possible transmission path originates from bats to pangolins to humans [35] . however, the likely source of virus origin and the intermediate host of sars-cov-2 are yet to be identified. initially, when the novel virus emerged in china, a hypothesis was put forward, claiming the recent recombination event as the cause of the sars-cov-2 emergence . nevertheless, the phylogenetic and recombination analysis performed within the subgenus of sarbecovirus demonstrated that the novel virus shows discordant clustering with bat-sars-like coronavirus (ratg13) sequences thus rejecting the possibility of a recent recombination event [36] . previously, it was found that the continuous passaging of mers-cov in non-susceptible cells that express viral receptors led to the accumulation of mutations in the spike protein gene. this paid attention to the potential of coronaviruses like mers-cov to undergo mutations that enhance viral entry into novel animal species, thus resulting in cross-species transmission [37] . the covid-19 outbreak is still associated with several unanswered questions like the possibility of shedding of the virus before the onset of clinical signs, whether the transmission is limited to only through respiratory droplets, the possibility of an intermediate host that is responsible for zoonotic spillover, and the possible transmission characteristics [20, 38] . hitherto studies report that the spillover risk remains high from zoonotic viruses and on the same lines a study from north america proposed a hypothesized conceptual model demonstrating sars-cov-2 spillover from humans to naive wildlife host species through the gastrointestinal route where stool from covid-19 infected patient contaminates water bodies and reaches to wildlife hosts [39] . besides, the pandemic imposed a massive blow on the chinese economy, which is not going to heal soon [40] . instead of the current situation, singapore's prime minister lee hsien loong rightly said that the virus might have started in china. however, it does not respect nationality or race. it does not check your passport before it goes into your body, and anybody can be infected. hence, all suspected people need to be tested and quarantined [41] . further research exploring the sars-cov-2 associated zoonosis and mechanisms accounting for its initial transmission from animals to humans, will lead to sort out the spread of this virus as well as design and develop appropriate prevention and control strategies to counter covid-19. the present comprehensive manuscript presents an overview on covid-19, an emerging sars-cov-2 infectious disease while focusing mainly on the events and circumstantial evidences with regards to this virus jumping the species barriers, sharing a few lessons learned from sars-and mers-covs, zoonotic spillover events (zoonosis), acquiring transmission ability to infect humans, and adopting appropriate preventive and control measures [42] . it also highlights the recent advances in sars-cov-2 diagnosis (see supplement material s1), vaccine, drugs and therapies (see supplement material s2), which could aid to counter and restrain this emerging virus at the face of pandemic situations, as well as prevention and control (see supplement material s3). sars-cov-2 is an enveloped virus measuring approximately 50-200 nm in diameter with a single strand positive-sense rna genome ranging from 26 to 32 kilobases in length [43, 44] . it has club-shaped glycoprotein spikes in the envelope, giving it a crown-like or coronal appearance [25] . the genome sars-cov-2 is comprised of 5′ untranslated region (5′ utr) that includes 5′ leader sequence, open reading frame (orf) 1a/b (replicase genes), spike (s) protein, envelop (e) protein, membrane/matrix (m) protein, and accessory proteins (orf 3,6,7a, 7b, 8 and 9b), nucleoprotein (n), and 3′ untranslated region (3′ utr) in their sequence [45] . it has a 50% genetic identity to mers-cov and 80% to sars-cov [43, 46] . the receptor-binding domain (rbd) of virus spikes helps in binding to cellular receptor angiotensin-converting enzyme 2 (ace-2) [47, 48] . orf and rbd of sars-cov-2 may have a role in elucidating cellular interactions and cross-species transmission mechanisms [49] . receptor binding motifs (rbm) have a role in interaction with human receptors, human to human transmission, and cross-species transmission as gln493 provides favourable interaction, and asn501 shows compatibility with human ace-2 [47] . besides, sars-cov-2 has superior transmission competence in comparison to the sars-cov, leading to a continuously increasing number of confirmed cases [50] . sars-cov-2 has the potential to survive in the environment for several days [51] . though believed to be sensitive to environmental factors and alcohol-based sanitizers, bleach, and chloroform, the sars-cov-2 can survive in wet surroundings for days and in closed air conditions up to 12 hours [26, 52] . survival of sars-cov-2 varies with the nature of the surface (glass, fabric, metal, plastic, or paper), environment, and virus load. it can survive on surfaces for hours to several days. it can survive in aerosols for up to 3 hours and on plastic for up to 72 hours [26, 53] . the initial clinical picture of the covid-19 was pneumonia of unknown origin as the first clinical cases were presented with signs of pneumonia [38] . later it was diagnosed as sars-cov-2 infection that was associated with severe pneumonia. hence, initially named as novel coronavirus pneumonia (ncp) [54] . as the outbreak proceeded, a series of cases were produced, developing a wide range of clinical signs with few remaining asymptomatic being in the early incubation stage of the disease. thus covid-19 is characterized by three major patterns of the clinical course of infection, including mild illness producing upper respiratory signs, non-life-threatening pneumonia, and severe pneumonia with acute respiratory distress syndrome (ards) [55, 56] . initially, mild signs appear for 7-8 days, followed by rapid deterioration and ards. it can be mild to moderate in 80% of affected cases, including pneumonia and non-pneumonia cases. in comparison, 13.8% are severe cases, including dyspnea, respiratory distress, hemoptysis, gastrointestinal infection, liver, central nervous system, and lung damage cases [57, 58] . critical cases account for 6.1% and include respiratory failure, septic shock, and multiple organ failure/dysfunction cases. few cases remain asymptomatic and include cases that can become any of the above during infection [26] . thus, the symptoms can be nonspecific and can range from no symptoms (asymptomatic) to severe pneumonia [26] . in this context, a study concluded that the covid-19 is probably overestimated, as around 2.6 million people succumb to respiratory diseases every year in comparison to approximately 400,000 deaths due to the sars-cov-2 infection [59] . the typical clinical signs of covid-19 are fever, chills, cough, fatigue, and chest distress [60, 61] . fever and cough are considered as the most common symptoms in covid-19 patients [62] , followed by headache, dyspnea, sore throat, hemoptysis, myalgia, diarrhoea, nausea, and vomiting are also observed [61, 62] . some patients have shown rhinorrhea, chest pain [25] , nasal congestion [26] , anorexia, pharyngalgia, and abdominal pain [63] . furthermore, neurological symptoms like anosmia and ageusia are also reported as significant clinical symptoms of covid-19 [64] . the characteristic of covid-19 is attacking the lower respiratory tract and producing signs of upper respiratory distress, including rhinorrhea, sneezing, and sore throat [49] . the clinical presentation of individuals infected with sars-cov-2 revealed upper respiratory tract infection, viremia, viral shedding from the nasopharynx, and stool along with the development of nausea, vomiting or diarrhoea after antiviral treatment [65] . on diagnostic imaging using computed tomography (ct scan) and radiography (x-ray) bilateral pneumonia, ground-glass opacity, multiple mottling, pneumothorax, infiltration, consolidation or bronchoinflation sign has been noted in many cases of covid-19 [25, 49, 66] . previously, sars-cov was found to infect the brainstem heavily [67] . even though fever is considered as the most common symptom associated with covid-19 infection, a large proportion of the patients do not express fever during the initial hospital admission [68] . the different transmission routes of sars-cov-2 infections have not yet been entirely ascertained, and are still under investigation. both direct and indirect pathways of transmission are being explored [69] . similar to sars and mers, sars-cov-2 is predominantly spread via the respiratory route [70] . person to person transmission is the main reason for community and global spread. the initial estimated reproduction number (rovalue) of covid-19 was assessed to be from 0.8 to 2.4 in december 2019, which later has been increased to a mean value of 2.6 (range 2.1-5.1) [71] . human-to-human transmission is by face-to-face contact with a sneeze or cough, or from contact with secretions of infected people [56, 70] . nevertheless, the infectivity of other secretions and excretions are not fully understood and may require further study [45] . aerosol and plastic surfaces can sustain virus for hours to days [53] . travelling of infected people is considered as the main reason for the global spread of covid-19 [20, 56] . although the asymptomatic and mild cases are the major hurdle in the evaluation of the real number of infected people, the genuine data on travellers returning from affected countries or areas may prove crucial in estimating the disease incidence [72] . the possible occurrence of super-spreading events is very high at large gatherings, and suspension of gathering during a pandemic may prove crucial in reducing the overall transmission [73] . close contact with any person within 6 feet of the covid-19 patient or anyone having direct contact with secretions of covid-19 patients [74] may set up the infection. unlike sars-cov, most transmissions in covid-19 are during the prodromal period when the infected individuals produce large quantities of virus in the upper respiratory tract, move/travel, and usually work thus spreading virus before illness develops [56] . the rapid spread of covid-19 among the susceptible population can be due to the wide variation in illness degrees that results in a missed diagnosis. heavy viral load in asymptomatic cases and nosocomial transmission is spreading covid-19 unknowingly [75] . recently, high viral load was detected in the sputum of convalescent patients, pointing out the possibility of prolonged shedding of sars-cov-2 even after recovery. this finding, along with the fact that asymptomatic persons can also act as the potential source of infection, may warrant a reassessment in the transmission dynamics of the covid-19 outbreak [76] . the presence of viral nucleic acids in faeces is an important finding, thereby increasing the possibility of faecal-oral transmission. however, symptoms may or may not be manifested [77] . this was found to be the unique feature of covid-19 and was lacking in the previous sars and mers outbreaks. in a study conducted by the chinese cdc, it was found that the majority of patients (80.9%) infected with covid-19 infection were either asymptomatic or had mild pneumonia [56, 66] . furthermore, all forms of sexual contacts have been reported to pose a significantly high risk of disease transmission through respiratory aerosols and fomites. however, to date, no evidence of sexual transmission is available, and further investigation is required in this direction [78] . disease severity is higher in older individuals, especially males with immunocompromised conditions and comorbidities like diabetes, asthma, or cardiovascular diseases [26, 66] . these are considered to be vulnerable to the sars-cov-2 infection. predisposition increases under risk environments where transmission of the virus from affected persons or contaminated fomites to unaffected ones becomes feasible. it was earlier noted that covs are not common to affect immunocompromised patients like other some viral infections (influenza, rhinovirus, adenoviruses, to name a few). the current pandemic has shown sars-cov-2 to affect more lethally than young patients, mainly destroying the lung tissues [79] . till now, evidence regarding the higher susceptibility of pregnant women in comparison to non-pregnant women lacks in covid-19. also, there is no evidence of vertical transmission (mother to fetus/baby transmission) of covid-19 infection [80] . a case study reporting the birth of a healthy infant by a sars-cov-2 infected woman suggests that mother-to-child transmission is unlikely in the case of covid-19. the study also pointed out that on the delivery day, all the samples tested negative except for sputum, which proved positive [81] . however, as per one most recent report, neonates have been found positive for sars-cov-2, indicating the possibility of vertical transmission from infected mothers to their progeny, thus rendering newborns into a high-risk group owing to their immature immune system [82] . individuals harbouring sars-cov-2 may remain asymptomatic for the incubation period [83] . different from sars-cov and mers-cov infection, the median incubation period of covid-19 was found to be four days [68] . the median period from the development of signs to death was 14 days [84] . the case fatality rate (cfr) of covid-19 was found to be lower than mers and sars [62] . however, current disease dynamics with the involvement of many more countries or areas may change the future mortality rate. the recent analysis suggests that the total fatality rate of covid-19 is calculated at 3.46% [75] . however, italy experienced the worst cfr of more than 9% with older people and males suffering from multiple comorbidities as primary victims [85] . sars-cov-2 has shown characteristics of efficient replication in the upper respiratory tract, causing the less abrupt onset of clinical signs just like the common cold and unlike sars-cov [70] . it can also replicate in the lower respiratory tract as has been noted in cases without pneumonia but having lesions in the lungs on radiological examination [70] . the pathogenesis mechanisms of covid-19 are yet to be fully elucidated. however, both cellular and humoral immune responses against sars-cov-2 or its antigenic structures like spike protein (s) are believed to be of importance [86, 87] with disturbed levels of inflammatory mediators playing a mediating role [66] . following receptor binding with angiotensin-converting enzyme 2 (ace2) through receptor binding motif (rbm) of the receptor-binding domain (rbd) of s1 subunit of the sars-cov-2 spike glycoprotein (s), virus gains entry in host cells [47, 88, 89] . s2 subunit helps in the fusion of viral and hosts cell membranes [47, 90] . sars-cov-2 produces cytopathic effects in respiratory and gastrointestinal surface epithelial cells [91] . these include multinucleated syncytial cells, abnormally enlarged pulmonary cells, infiltration with mononuclear cells, lymphocytes infiltration in pulmonary organs, fibrinous exudation, and hyaline deposition [66] . cytokine storm is believed to be involved in this inflammatory pathophysiology of the covid-19 patients producing lung lesions and systemic symptoms [66] . elevated levels of tnf-α, il1b, ifnγ, ip10, gcsf, mip1a, and mcp1, may have stimulated t-helper-1 (th1) cells leading to this inflammatory cascade [66] . however, levels of anti-inflammatory mediators (il4, il10) were also increased, indicating t-helper-2 (th2) stimulation, which suppresses inflammation, unlike what happens in sars [66] . a study documented that the nucleic acid of sars-cov-2 detected in the faecal samples was as accurate as of that of pharyngeal samples obtained from infected patients. moreover, the patients tested positive for sars-cov-2 in stool showed no gastrointestinal symptoms and had no relation to the severity of lung infections [92] . one of the significant clinical signs of covid-19 patients during the initial presentations was gastrointestinal symptoms. hence, the involvement of git in pathogenesis needs to be explored. the significant laboratory findings include lymphopenia, increased values of erythrocyte sedimentation rate, c-reactive protein, lactate dehydrogenase, and decreased oxygenation index [61] . an increase in proinflammatory cytokine and a decrease in antiinflammatory cytokines have also been noted [66] . viral isolation has been achieved from bronchoalveolar lavage of affected persons; however, in the case of pregnant women, serum, faeces, urine, breast milk, umbilical cord blood, placenta, and amniotic fluid were found to be negative for sars-cov-2 [81] . at the same time, the sputum was tested positive [82] . the presence of abnormal coagulation parameters in patients with severe novel coronavirus pneumonia was associated with poor prognosis. the non-survivor patients had higher levels of d-dimer, and fibrin degradation product (fdp) along with longer activated partial thromboplastin time and prothrombin time compared to survivors at the time of admission [93] . though clinical manifestations, pathological changes, and diagnostic laboratory findings can unravel the disease nature helping in devising therapeutic modalities, however, for epidemiological aspects and future prevention and control, simultaneous tracing of the origin and explaining the spillover events can prove beneficial. the sars-cov-2 has first been reported from the pneumonia patients of the wuhan city in hubei province of china. these patients were involved in trading at a wet animal market in the huanan area. it is believed that sars-cov-2 is introduced from the animal kingdom to human populations during november or december 2019, as revealed from the phylogeny of the genomic sequences from the initially reported cases [94] . the spillover of sars-cov-2 from animals to humans took place at the beginning of december 2019 [56] , and the clinical cases appeared around ending december [46, 95] . genetic analysis showed that this novel virus is closely related to bat covs and is similar but distinct from the sars virus [43] . several evidences based on genome sequences, the homology of the ace2 receptor, and the presence of single intact orf on gene 8 indicate bats as a natural reservoir of these viruses. however, an unknown animal is yet to be unravelled as an intermediate host [43, 46, 47, 49, 56] . initial investigations on animal source origin of sars-cov-2 have inconclusively revealed snakes [27] , pangolins, and turtles [96] . the rapid spread of covid-19 followed the initial animal to human spillover through human-to-human transmission. genetic epidemiology had revealed that the spread from the beginning of december when the first cases were retrospectively traced in wuhan was mainly by a human-to-human transmission and not due to continued spillover [56] . these species cross jumping, spillover, and rapid transmission events are linked to viral characteristics, host diversity, and environmental feasibility. coronaviruses being rna viruses have high mutation rates that, besides creating new strains, enable them to adapt to a wide range of hosts. hence, based on genome sequences, all known human covs have emerged from animal sources [97] . this seventh member of the human cov has also been isolated initially from the pneumonia patients who were having direct or indirect links to the huanan seafood market in wuhan china, wherein other animals were also being sold [98] . these include a 49-year-old lady retailer in this wet animal market, a 61-year-old frequent visitor to this market, and a 32-year-old man [74, 98] . further, isolation of the sars-cov-2 from the environmental samples around this market, including people, animals, soil, discharges, or structures, strengthens the claims of involvement of hosts either as a reservoir or intermediate [26, 52, 99] . recently, a pomeranian dog as a probable intermediate host was identified; however, such reports are yet to be validated, and research is underway to explore the emergence of this infectious disease at the animal-human interface [29, 99] . multiple substitutions were observed in ace2 receptors of a dog [96] . in this context, the pomeranian dog of the infected owner found positive for covid-19 suggest the permissiveness of the species for sars-cov-2 as a result of species jumping [99, 100] . among the fifteen dogs tested from different households with confirmed human covid-19 cases in hong kong sar, two dogs were found to be infected with sars-cov-2. the diagnosis was made using quantitative rt-pcr, serology, and viral genome sequencing. virus isolation was also done from the samples obtained from one dog. the genetic sequences of viruses obtained from the two dogs were identical to the ones that were detected from their human cases indicating human-to-dog transmission [101] . moreover, a study reported that the sars-cov-2 might infect the cats and further transmitted by the infected cat to other cats [102] . one cat was tested positive for sars-cov-2 in france that showed mild respiratory and digestive signs. the cat was tested positive by rt-qpcr on the rectal swab, and serological analysis identified the presence of antibodies against sars-cov-2. genome analysis further confirmed that the sars-cov-2 isolated from the cat belongs to the phylogenetic clade a2a seen in french human indicating humanto-animal transmission [103] . this is not the first time that a domestic cat has been found susceptible to zoonotic coronavirus. during the 2003 sars-cov outbreak, domestic cats were tested positive for sars-cov that were living near sars infected humans [21, 104] . even though experimental evidence indicates the possibility of sars-cov-2 transmission from infected to a susceptible cat close, sars-cov-2 transmission between cats or cat-tohumans are not reported under natural conditions [21] . furthermore, along with dogs and cats, the zoo animals like tigers and lions were also reported to get the sars-cov-2 infection and exhibit clinical signs such as vomiting, diarrhoea, dry cough, breathing difficulty and wheezing [105, 106] . spillover of sars-cov-2 was also reported in mink farms of netherlands, further increasing the concern of transmission to humans. outbreaks of sars-cov-2 were reported in two mink farms holding 12,000 and 7500 animals. the virus is suspected to be introduced by a farmworker having covid-19 [107, 108] . host-pathogen interactions and pathogenesis determine the severity and expression of disease [30, [109] [110] [111] . adaptation over time reduces the severity of infection as happened with hcovs; however, the emergence of novel viruses or strains due to genetic alterations or recombinations can enhance hardness producing novel diseases like covid-19 [111, 112] . evolutionarily, the balance of viral-human interaction and immune response against virus enables adaptation, thereby persistence in a host without severe or symptomatic disease when the aggravated pathogenesis results in mortality. hence, loss of sustainable hosts and transmission to novel hosts becomes inevitable for future sustainability [111, 112] . a pathogen cannot kill all its hosts, and for future sustainability, it adapts to some suitable host or spills over to a new host. sars-cov-2 has been implicated to be originated from animals, and associated with animal linkages, spillover events, cross-species barrier jumping and zoonosis [9, 20, 27, [29] [30] [31] 113] . since the beginning of 2002 till the end of 2019, three coronaviruses viz. sars-cov, mers-cov, and sars-cov-2 have caused havoc in the human population globally and will continue to do so. earlier identified betacoronaviruses (sars-cov and mers-cov) were reported in guangdong province of china in november 2002 and saudi arabia in 2012, respectively [114] . sars-cov-2 is the third zoonotic betacoronaviruses recognized in this century. however, the cfr of the sars-cov-2 is lower to date when compared with sars and mers. it should not be overlooked as many asymptomatic cases may remain undiagnosed due to the unavailability of diagnostic kits in china. with nearly 0.62 million deaths till the preparation of the manuscript, sars-cov-2 is proven to be deadliest as far as the number of deaths is concerned in comparison with sars-cov and mers-cov with 774 and 858 associated deaths, respectively [115, 116] . earlier, covid-19 was linked with the exposure to the huanan seafood market. however, individuals with no history of exposure above were also diagnosed with the illness, further supporting the human to human spread through droplets produced by cough and sneeze [66] . the spread of covid-19 that occurred with a high pace and lack of transparency in reporting the disease by the chinese health ministry and failure in the timely implementation of preventive measures has been considered as the primary contributor as stated earlier in sars [19, 117] . both sars-cov and sars-cov-2 showed prominent similarities in their pathogenesis and epidemics. in both cases, bats were considered as the natural host, and the cold temperature and low humidity in cold, dry winter provided conducive environmental conditions that promoted the survival of the virus in the environment [62] . further, moriyama et al. [118] assessed the significance of the environmental factor on host immune system targeting innate and adaptive both responses in the respiratory tract. zoonotic spillover is the transmission of pathogens to humans from vertebrate animals [119] . at present, these spillovers are of significant concern as in the past, many spillovers in the form of nipah, hendra, ebola, sars, mers, and ongoing covid-19 involving many animal species like pigs, horses, monkeys, camels, civets, among others, were documented. bovine covs have been reported to infect children and thus possess zoonotic potential [9, 13, 120, 121] . spillover is governed by the interaction of viral-specific proteins like s protein and host ace2 receptor [30, 31, 111, 112] . these s proteins have rbd in covs, which contain receptor binding motifs (rbm) that help in specific binding to host ace2 receptors [47, 122] . mutations in amino acid sequences of rbds results in a change in specificity of a receptor, interaction and binding, hence alteration in transmissibility, pathogenicity and cross-species jumping with a predisposition to novel and more severe diseases [110, 123] . in the case of sars-cov-2, rbd of s protein has 10-15 times affinity ace2r [88, 110] . it has furin recognition sequence "rrar" at the s1-s2 cleaving site that represents a functional site for the cellular serine protease tmprss2 thus increasing the efficiency of transmission and contagiousness [88, 90, 110] . in addition to enhanced binding affinity, electrostatic complementarity and hydrophobic interactions are critical to enhancing receptor binding and escaping antibody recognition by the rbd of sars-cov-2, thereby further increasing transmission capability and contagiousness [110] . a detailed investigation regarding the emergence of new coronavirus, host range, and transmissibility is crucial to understand such pandemics shortly. the literature revealed that before the appearance of sars-cov and mers-cov, human coronavirus (hcov) strains like hcov-nl63, hcov-229e, hcov-oc43, and hcov-hku1 were the covs strains producing mild infections in humans. however, their natural ancestral hosts were of animal origin, like bats for hcov-nl63, and hcov-229e and rodents were natural hosts for hcov-oc43 and hku1. these four hcovs were initially of low pathogenicity. to enhance the pathogenicity, they used intermediate hosts such as cattle for hcov-oc43 (natural host was rodent), and alpacas for hcov-229e (bats were natural host) and this way acquired the ability to infect human beings with serious health hazards [124] . added to the involvement of bats and pangolins, the recent reports revealing sars-cov-2 infection in cats, dogs, tigers, lions and minks have raised concerns over this virus affecting multiple animal species, and also points out towards the incidences of reverse zoonosis [31, 102, 106, 108, 125, 126] . the ferrets, cats, and primates are suggested to be good candidates for susceptibility to sars-cov-2 [123, 127] . covid-19 research and surveillance in companion and pet animals, livestock animals, zoo animal species, wildlife animal species as well as their handlers, veterinarians, and owners need to be enhanced during the pandemic, which would help to follow better integrated one health strategies [128] and appropriate preventive and mitigation to counter sars-cov-2 effectively [29, 126, [129] [130] [131] [132] [133] . significance of covid-19 monitoring and implementation of suitable public health measures among workers involved in meat and poultry processing facilities/industries has been emphasized, which would protect them as well as aid in preserving the critical meat and poultry production infrastructure and the meat products [134] . the involvement of intermediate hosts in maintaining and transmitting the virus to susceptible host predisposes humans to novel covs leading to the emergence of new diseases in humans. the currently ongoing sars-cov-2 / covid-19 pandemic has put on hold the entire world [111, 135] . the covs have frequently been associated with animal and human diseases and have a zoonotic interface [97, 111] . usually, one or more types of animal hosts are involved in the transmission cycle of covs to humans [30, 97] . that can be natural host, reservoir host, intermediate host or definitive host [31] . bats have been the natural hosts for human covs of alphacoronavirus (hcov-nl63, hcov-229e) and betacoronavirus (sars-cov, mers-cov, sars-cov-2) genera whereas for betacoronavirus members hcov-oc43 and hcov-hku1, rodents are the natural hosts. genome sequence analysis has revealed bats as a natural host for sars-cov-2 [30, 97] . in natural or reservoir hosts, covs adapts well, however, being unstable rna viruses, they keep multiplying continuously without producing disease thereby enabling persistence or survivability and accumulation of mutations over the time resulting in the emergence of newer and novel strains of viruses [97, 111, 136] . these unique strains or viruses occasionally spill over to other species including animals or humans, adapting to their body systems and hence broaden the biological host range for evolutionary sustainability; however, results in epidemiological widening of disease sphere as well [25, 31] . this transmission and adaptation scenario initiates a host-pathogen response resulting in the novel usually severe diseases that can at times be fatal in initial stages or over extended periods until virus pathogen adapts to host or the host develops sufficient immune defence [137, 138] . it has been reported that almost all hcovs have originated from animals like bats (sars-cov, mers-cov, hcov-nl63, and hcov-229e) and rodents (hcov-oc43 and hku1) [139, 140] . additionally, covs have been reported to infect several species of domestic and wild animals either clinically or subclinically [27, 28, 30, 141] . cattle, horses, camels, swine, dogs, cats, birds, rabbits, rodents, ferrets, mink, bats, snakes, frogs, marmots, hedgehogs, malayan pangolin along with other wild animals may serve as a reservoir host of coronavirus [9, 27, 44, 124, [142] [143] [144] [145] [146] [147] . in the context of sars-cov-2, snakes, pangolins and bats have been suspected as intermediate hosts since the first cases of covid-19 had links to huanan sea food market where different animals, birds, and wild animals were being sold along with seafood items [27, 28, 43, 109, 147, 148] . coronaviruses have been reported to cause salivary, enteric and respiratory infections in laboratory animals (mice, rat, guinea pig, and rabbit) and urinary tract infection, respiratory illness and reproductive disorder in poultry [9, 142] . in bovine, canine, feline and swine covs infections have resulted in diarrhoea, enteritis, respiratory illness, gastro-intestinal affections and nervous symptoms [120, 121, [149] [150] [151] . coronavirus, namely-sw1, has been reported in captive beluga whale using a panviral microarray method [152] . among all the assumptions on animal hosts as the intermediate host, genomic and evolutionary information from pangolins reveals the highest closeness to the sars-cov-2 than any other host covs isolates [153] . the spike protein, the main target of many studies searching for a cure of covid-19, has been found highly similar to sars-cov-2 and, thus, could serve as a surrogate system for further evaluations [153] . bats are the natural reservoir host of many covs. as reported earlier, 7 out of 11 alphacoronaviruses and 4 out of 9 betacoronaviruses as per the international committee on taxonomy of viruses (ictv) classification were solely originated from bats [97, 154] . according to the literature, bats have been regarded as a potential wildlife reservoir whereas civets and dromedary camels as intermediate hosts of sars-cov and mers-cov, respectively [155, 156] . the bat coronavirus, batcov ratg13, has shown higher relatedness to sars-cov-2 at the whole genome level and spike gene in particular [153] . coexistence and frequent recombination between highly diversified and prevalent bat sars-related coronaviruses (sarsr-cov) and coronaviruses may suggest the probable emergence of novel viruses shortly [157, 158] . benvenuto et al. [159] analyzed the whole genome sequences of different covs using fast unconstrained bayesian approximation (fubar) to understand the evolutionary and molecular epidemiology of sars-cov-2. the authors concluded that sars-cov-2 clustered with sequences of bat sars-like covs with a few mutations in nucleocapsid and spike glycoprotein, suggesting its probable transmission from the bats [159] . bats, especially horseshoe bats (rhinolophus spp.), are considered to be the known reservoirs of sars-related covs. since the bat origin, covs have always caused outbreaks in humans, studying the diversity and distribution of coronavirus populations in the bats will help to mitigate future outbreaks in humans and animals [160] . interestingly, bats play a crucial role in all the spillovers mentioned above, indicating their importance in the emergence of new viruses. the reason behind the emergence and broad host range of covs in the past and present might be due to unstable rna-dependent rna polymerase (rdrp), lack of proof-reading ability, high frequency of mutations in the receptor-binding domain of spike gene and genetic recombination [140, 161, 162] . bat covs have high diversity and great potential of spillover in different animal species, as reported earlier in civet cat and dromedary camel, leading to well-known pandemics sars and mers, respectively along with the recent spillover in pigs resulted in swine acute diarrhoea syndrome (sads). however, spillover resulted in the emergence of sads-cov, which showed a 95% genomic identity with bat coronavirus, which led to severe mortality with 24,693 deaths in neonatal piglets [160] . fortunately, it did not excel in the form of the third pandemic, and no human cases were reported till date. the spillover responsible for ongoing covid-19 is still under investigation and a matter of great concern for the researchers all around the globe. based on resampling similarity codon usage (rscu), snakes (bungarus multicinctus and naja atra) were suggested as wildlife reservoirs of sars-cov-2 and reported to be associated with the cross-species transmission [27] and later it was disapproved by other researchers [163, 164] . unfortunately, to date, the intermediate host of the sars-cov-2 is abstruse what results in its escalation in the human population around the globe. in this context, analyzing the interaction between the asn501 site in rbd of spike glycoprotein of sars-cov-2 and the residue at 41 sites of ace2 receptor of different hosts (pangolins, turtle, mouse, dog, cat, hamster and bat) revealed that tyrosine has higher receptor binding affinity than histidine suggesting pangolins and turtle be closer than bats to humans and maybe the probable intermediate hosts of sars-cov-2 [96] . however, this hypothesis was also contradicted by li et al. [28] based on an insertion of the unique peptide (prra) in the sars-cov-2 virus, which was lacking in covs from pangolins. moreover, sars-cov-2 showed higher similarity to the betacov/bat/yunnan/ratg13/2013 compared to the ones that were isolated from the pangolins, thereby denied the direct link of the virus from pangolins. however, further studies are required to confirm the role of pangolins in sars-cov-2 spread to humans. the receptor-binding domain of the spike protein of sars-cov interacts with the host receptor ace2 facilitating its potential of cross-species, as well as human-to-human transmission [47] . similarly, the spike protein of sars-cov-2 was reported to recognize ace2 receptors expressed in fish, amphibians, reptiles, birds, and mammals and has a more robust binding capacity (affinity) in comparison to sars-cov [89] . this suggests their involvement as probable natural and intermediate hosts [161] , which may further help in the selection of animal models for epidemic investigation and preventing its spread [47] . bat origin covs have been found to cross the species barrier that favoured their transmission via recombination/mutations in the rbd. the evidence of a virus outbreak that occurred in chinese pig farms suggests its possible cross-species [160, 165] . also, murine cells were found permissive for sars-cov after substitution of his353 with lys353 in the ace2 receptor of a mouse, which suggests the role of residue changes in the cross-species and human-to-human transmission [166] . mutation in residues at position 479 and 487 of receptor binding motif (rbm) of sars-cov was reported to play a role in civet-to-human and human-to-human transmission, respectively [167, 168] . the covs are more prone to recombination and mutations leading to variable host range, and resemblance of receptors in various hosts results in cross-species jumping [31, 112, 169] . genetic divergence due to these genetic alterations results in the evolution of newer viral strains having altered virulence, tissue tropism, and host range [170, 171] . moreover, the presence of threonine at position 487 was reported to enhance the binding affinity of rbm for the ace2 receptor of civet and humans [172] . however, many sarsrelated coronaviruses (sarsr-cov) have been reported in bats and used ace2 receptors for entry into a host cell, which showed its potential to infect humans directly without any intermediate host [173] . in addition to this, no direct transmission of sarsr-cov is reported from bats to humans to date. however, seropositivity on a serological investigation of individuals without prior exposure to sars-cov residing near bat caves in china revealed likely infection of humans by bat sarsr-cov and related viruses [174] . besides, the interspecies transmission potential of sarsr-covs is due to the orf8 gene [175] . as per reports, the sars-cov emerged via recombination of bat sarsr-covs, was transmitted to farmed civets along with other mammals, and these infected civets spread the virus to market civets. the virus was reported to undergo mutations in infected market civets before its spillover to humans. similarly, the mers-cov circulated for 30 years in camels before the pandemic [97, 176] supporting the hypothesis that after species jumping the exogenous viruses opted for adaptation to the environment and host before spillover to humans [177] . moreover, the possible spillover of other circulating bat sarsr-covs to humans from mammalian hosts soon is highly anticipated. the cross-species jumping and adaptation are determined by the presence of specific receptors on host tissues (like ace 2 receptor for hcov-nl63, sars-cov and sars-cov-2, dipeptidyl peptidase-4 for mers-cov, human aminopeptidase n for hcov-229e, 9-oacetylsialic acids for hcov-oc43, hcov-hku1) which help in binding and entry of the virus into host cells [30, 31] . these receptors are present in various body systems in animals and humans, including respiratory and gastrointestinal systems [30] . reservoir host animals including bats and rodents possess these receptors which are similar to those present in camels, masked palm civets (paguma larvata), or bovines, that act as an intermediate host for different covs [30, 110, 111] . presence of some of these receptors in humans like ace2 or dpp4 makes them vulnerable to cov infection like sars-cov and mers-cov causing sars and mers infections, respectively [31, 168] . the mers-cov spike was found to possess the capacity for adapting to species variation in the host receptor dpp4 [37] . the mechanism expressed by mers-cov in adapting to infect cells of new species might be present in the other coronaviruses. ace2 has also been found as a binding receptor for sars-cov-2 [47] . the species-specific variations in the host receptors limit the interaction with cov spike protein, and this is responsible for the development of the species barrier that prevents spillover infection. snakes, civets, and pangolins are considered as the potential intermediate hosts of covid-19. however, further confirmation is required by tracking the origin of the virus. this is critical for preventing additional exposure to this fatal virus [178] . the probability of the sars-cov-2 spread during incubation and convalescent period has been suggested [76] . as per reports, presence of covs has been observed in respiratory droplets, body fluids and inanimate objects with the ability to remain infectious for nine days on contaminated surfaces resulting in its risk of self-inoculation via mucous membranes of the eyes, mouth or nose [179] [180] [181] . nosocomial, as well as human-to-human transmission, have been reported to occur via virus-laden aerosols, contaminated hands or surfaces, and close community contact with an infected person [66, 182, 183] . the ocular route has been reported in the human-to-human transmission of sars-cov-2, as observed in sars-cov, suggesting the involvement of different ways other than the respiratory tract [184, 185] . later on, the probability of the faecal-oral route for potential transmission of the virus was also suggested [186] . the metatranscriptome sequencing of sars-cov-2 in the bronchoalveolar lavage fluid (balf) of infected individuals resulted in polymorphism in few intra-hosts variants, suggesting the in vivo evolution of the virus thereby affecting its virulence, transmissibility, and infectivity [187] . an overview of coronaviruses jumping the cross-species barriers, zoonotic covs transmitted from bats to animals before spillover to humans, and possible prospects for further transmission to mammalian hosts is depicted in figure 1 . the first two decades (2000-2020) of 21 st century have proven a nightmare for the countries around the globe considering the coronavirus zoonosis, including the ongoing crisis of covid-19 which has involved entire fields of global [188, 189] . the countries affected severely by previous covs were even not evolved entirely from the effects of sars and mers when the covid-19 struck almost the entire world. novel coronavirus sars-cov-2 has shaken all the sectors of the countries irrespective of being developed or underdeveloped including healthcare system, economics, trade, infrastructure, service and production sectors [190, 191] . being a zoonotic disease with still unknown intermediate host, undisclosed features of a novel viral pathogen, unclear modes of transmission and ecological aspects, less explored pathogenesis and substantial morbidity and considerable mortality, the safety of all is a matter of great concern, and thus the involvement of various authorities was sought since the inception of disease [26, 31, 192, 193] . the first time the need for one health concept has risen to a level that authorities in various countries implemented coordinated approaches between medical, veterinary, public health, wildlife, food safety, environmental departments and so on [193] [194] [195] . that involved acquiring suggestions, diagnosis and prevention and treatment measures and their implementation in collaboration. non-medical staff in association with the medical staff was employed for initial screening, quarantine, contact tracing when the expertise of molecular biologists or technicians from various disciplines was used in the laboratory diagnosis. medical staff provided the cure and management of the patients when the public health departments, including public health engineering, municipality, food and supplies ensured sanitation, hygiene, food supply and safety. imposing of lockdown was provided by security personnel's and the transport department facilitated the movement of stranded people. thus, this crises management strategy involved various agencies directly or indirectly. however, as the animal, human and environmental health is linked to one another, the prime and future efforts should primarily focus on all these aspects. in addition to regular hand hygiene, respiratory etiquette, social/physical distancing, use of personnel protective equipment (ppe) and food safety recommendations, one health approach encompasses the role of veterinary, medical and environmental specialists for the prevention and control of current covid-19 crises and investigating the animal origin of covid-19, regulating and limiting the sale and farming of wildlife species for food and taking a one health approach to food systems feeding the world for the prevention of future pandemics [193, 196, 197] . considering the contagiousness of the virus, discouraging the working of affected individuals, public health hygiene strategies, and social distancing has been recommended as preventive measures [26, 198] . food hygiene and safety, as recommended by oie [193] and usda [199] , should be followed. as the viral survivability has been demonstrated on various surfaces [53] hence disinfection by using recommended disinfectants is necessary [200] . environmental hygiene and cleanliness are also essential [200] . interaction with animals and improper utilization of animal products during an outbreak should be avoided [193] . though the one health involves mainly public health, animal health and environmental experts, however, for the successful management of current crises and future prevention and control requires the participation of all concerned sectors having a role in public health measures, identifying clinical cases, diagnosis, contact tracing, proper infection control in various settings, isolation, quarantine, cure and management, public awareness, facilitation of infrastructure and other facilities through local administrations [26, 195] . as the human covid-19 cases are on the rise due to efficient human-to-human transmission, there is a subsequent rise in the natural infections of covid-19 among the companion and wild animal species owing to the spillover. this is mainly because of the specific biological and virological characteristics of coronaviruses that gives them the ability to easily cross-species barriers [130] . even though animal-to-human transmission is not reported in covid-19, 'one health' approach is necessary to control this pandemic virus a schematic illustration of covid-19 clinical signs, modes of transmission, important diagnostic methods, and advances in vaccine development along with salient prevention and control strategies are presented in figure 2 . with the rising number and worldwide spread of covid-19, the need for global efforts rely heavily on the investigations carried out at infection sites to trace different aspects of this novel coronavirus outbreak. one of the critical facets and the earliest research must involve determining the root cause, origin, and source of this emerging infectious disease. shreds of evidence have revealed various cross-species jumping or spillover from animals to humans of these zoonotic coronaviruses. detailed serological investigation of all domestic and wild animals residing in the proximity to humans is of utmost necessity to know and prevent likely spillover of many other bat-related covs in the future. rapid detection of spillovers above will only be possible by the implementation of an effective and robust surveillance system for circulating viruses with high zoonotic potential in animals. besides, detection of a pathogen while crossing the species barrier to start circulation among humans and prevention of human-to-human transmission in early-stage may prove crucial in termination of a probable epidemic or pandemic. application of 'one health' concept involving medical, veterinary, wildlife, public health, and other related professionals may help in infection tracing, exploring risk factors and predisposition, minimizing risk to susceptible ones, and finally devising better prevention and control strategies. in the initial stages of the covid-19 outbreak, the steps taken for implementing stringent control and preventive measures have bought us some time. this time has to be efficiently utilized for developing sars-cov-specific therapeutic drugs and vaccines that can prevent the further spread of this fatal pathogen. for the time being early detection, isolation, and management of covid-19 infected cases and for the immediate future awareness and implementation of the necessary steps of prevention and control to under risk and predisposed groups may help in long term prevention and control goals. however, it is clearly understood that relying exclusively on public health measures will not resolve the threat caused by covid-19. hence efforts to curb this emerging zoonosis at all levels need to be enforced under the one health approach. a systematic review of therapeutic agents for the treatment of the middle east respiratory syndrome coronavirus (mers-cov) mers-cov as an emerging respiratory illness: a review of prevention methods clinical predictors of mortality of middle east respiratory syndrome coronavirus (mers-cov) infection: a cohort study ebola virus disease: an emerging zoonosis with importance for travel medicine preliminary estimation of the basic reproduction number of zika virus infection during colombia epidemic zika and microcephaly in latin america: an emerging threat for pregnant travelers fatal zika virus disease in adults: a critical reappraisal of an under-recognized clinical entity advances in designing and developing vaccines, drugs, and therapies to counter ebola virus swine flu is back again: a review emerging novel coronavirus (2019-ncov)-current scenario, evolutionary perspective based on genome analysis and recent developments advances in developing therapies to combat zika virus: current knowledge and future perspectives nipah virus: epidemiology, pathology, immunobiology and advances in diagnosis, vaccine designing and control strategies -a comprehensive review the 2019 coronavirus: learning curves, lessons, and the weakest link spectrum of covid-19 in children novel coronavirus: a crown jewel of pandemics strategies shift as coronavirus pandemic looms novel coronavirus, poor quarantine, and the risk of pandemic the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? history is repeating itself: probable zoonotic spillover as the cause of the 2019 novel coronavirus epidemic susceptibility of felids to coronaviruses coronavirus: global solutions to prevent a pandemic covid-19, an emerging coronavirus infection: advances and prospects in designing and developing vaccines, immunotherapeutics, and therapeutics coronavirus disease pandemic (covid-19): challenges and a global perspective epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study report of the who-china joint mission on coronavirus disease cross-species transmission of the newly identified coronavirus 2019-ncov evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov-2 covid-19: another infectious disease emerging at the animal-human interface. the new zealand medical journal coronaviruses: a paradigm of new emerging zoonotic diseases. pathogens and disease covid-19: animals, veterinary and zoonotic links bat-borne virus diversity, spillover and emergence exceptional diversity and selection pressure on sars-cov and sars-cov-2 host receptor in bats compared to other mammals covid-19: lessons from sars and mers analysis of the hosts and transmission paths of sars-cov-2 in the covid-19 outbreak full-genome evolutionary analysis of the novel corona virus (2019-ncov) rejects the hypothesis of emergence as a result of a recent recombination event adaptive evolution of mers-cov to species variation in dpp4 outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle spillover of sars-cov-2 into novel wild hosts in north america: a conceptual model for perpetuation of the pathogen economic impacts of wuhan 2019-ncov on china and the world fear can be more harmful than the severe acute respiratory syndrome coronavirus 2 in controlling the corona virus disease 2019 epidemic the arrival of sars-cov-2 in venezuela genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission coronavirus 2019-ncov: a brief perspective from the front line identification of a novel coronavirus causing severe pneumonia in human: a descriptive study receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in wuhan, china, as at 22 the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak zhonghua yu fang yi xue za zhi [chinese journal of preventive medicine interim guidance for environmental cleaning in non-healthcare facilities exposed to sars-cov-2 aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov-2 an update on sars-cov-2/covid-19 with particular reference to its clinical pathology, pathogenesis, immunopathology and mitigation strategies technical advisory group for infectious h. covid-19: what is next for public health? the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak -an update on the status systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov-2 and sars-cov sars-cov-2: fear versus data the first 2019 novel coronavirus case in nepal zhonghua jie he he hu xi za zhi = zhonghua jiehe he huxi zazhi = chinese journal of tuberculosis and respiratory diseases clinical characteristics of 50466 patients with 2019-ncov infection clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan anosmia in a healthcare worker with covid-19 in madrid, spain for the singapore novel coronavirus outbreak research t. epidemiologic features and clinical course of patients infected with sars-cov-2 in clinical features of patients infected with 2019 novel coronavirus in wuhan the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients clinical characteristics of coronavirus disease 2019 in china indirect virus transmission in cluster a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster early phylogenetic estimate of the effective reproduction number of sars-cov-2 evidence of sars-cov-2 infection in returning travelers from wuhan, china secondary attack rate and superspreading events for sars-cov-2 novel coronavirus 2019 (covid-19): emergence and implications for emergency care unique epidemiological and clinical features of the emerging 2019 novel coronavirus pneumonia (covid-19) implicate special control measures transmission of 2019-ncov infection from an asymptomatic contact in germany novel coronavirus infection and gastrointestinal tract sexual health in the sars-cov-2 era coronaviruses and immunosuppressed patients: the facts during the third epidemic novel coronavirus infection and pregnancy lack of vertical transmission of severe acute respiratory syndrome coronavirus 2 working committee on p, neonatal management for the p, control of the novel coronavirus i the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application ct imaging of the 2019 novel coronavirus (2019-ncov) pneumonia case-fatality rate and characteristics of patients dying in relation to covid-19 in italy preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies is covid-19 receiving ade from other coronaviruses? microbes and infection the novel coronavirus 2019 (2019-ncov) uses the sars-coronavirus receptor ace2 and the cellular protease tmprss2 for entry into target cells cryo-em structure of the 2019-ncov spike in the prefusion conformation the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade the novel coronavirus: a bird's eye view fecal specimen diagnosis 2019 novel coronavirus-infected pneumonia abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia genomic epidemiology of betacov outbreak of a novel coronavirus composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 origin and evolution of pathogenic coronaviruses a novel coronavirus from patients with pneumonia in china oie. animal and environmental investigations to identify the zoonotic source of the covid-19 virus. 2020. 100. diseases pisfi. coronavirus disease 2019 update (22): companion animal, dog suspected, request for information infection of dogs with sars-cov-2 coronavirus can infect cats -dogs, not so much first detection and genome sequencing of sars-cov-2 in an infected cat in france virology: sars virus infection of cats and ferrets belgian cat infected by owner. the brussels times a tiger at bronx zoo tests positive for covid-19; the tiger and the zoo's other cats are doing well at this time coronavirus rips through dutch mink farms, triggering culls sars-cov2 infection in farmed mink updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan enhanced receptor binding of sars-cov-2 through networks of hydrogenbonding and hydrophobic interactions zoonotic origins of human coronaviruses jumping species-a mechanism for coronavirus persistence and survival. current opinion in virology emerging coronavirus disease (covid-19), a pandemic public health emergency with animal linkages: current status update. the indian journal of animal sciences mers-cov and now the 2019-novel cov: have we investigated enough about coronaviruses? -a bibliometric analysis middle east respiratory syndrome coronavirus (mers-cov) bats, civets and the emergence of sars responding to global infectious disease outbreaks: lessons from sars on the role of risk perception, communication and management seasonality of respiratory viral infections. annual review of virology pathways to zoonotic spillover genomic characterization and phylogenetic classification of bovine coronaviruses through whole genome sequence analysis biological and genetic characterization of a hemagglutinating coronavirus isolated from a diarrhoeic child receptor recognition mechanisms of coronaviruses: a decade of structural studies discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin novel coronavirus (sars-cov-2) epidemic: a veterinary perspective susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 coronavirus sars-cov-2 (covid-19) and companion animal pets is covid-19 the first pandemic that evolves into a panzootic? importance of the one health approach to study the sars-cov-2 in latin america need for integrated surveillance at human-animal interface for rapid detection & response to emerging coronavirus infections using one health approach the risk of sars-cov-2 transmission to pets and other wild and domestic animals strongly mandates a one-health strategy to control the covid-19 pandemic. one health a critical needs assessment for research in companion animals and livestock following the pandemic of covid-19 in humans. vector borne and zoonotic diseases 2020 mitigating the impacts of covid-19 on the livestock sector guidelines to mitigate the impact of the covid-19 pandemic on livestock production and animal health among workers in meat and poultry processing facilities -19 states cov-2: an emerging coronavirus that causes a global threat viral genetics accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence covid-19 and emerging viral diseases: the journey from animals to humans molecular evolution of human coronavirus genomes epidemiology, genetic recombination, and pathogenesis of coronaviruses bat origin of a new human coronavirus: there and back again coronavirus infection in equines: a review emergence of avian infectious bronchitis virus and its variants need better diagnosis, prevention and control strategies: a global perspective synanthropic rodents as virus reservoirs and transmitters. revista da sociedade brasileira de medicina tropical identification of alpha and beta coronavirus in wildlife species in france: bats, rodents, rabbits, and hedgehogs consensus document on the epidemiology of severe acute respiratory syndrome (sars). geneva: world health organization isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins first respiratory transmitted food borne outbreak? canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex. the veterinary clinics of north america: small animal practice canine enteric coronaviruses: emerging viral pathogens with distinct recombinant spike proteins feline coronaviruses: pathogenesis of feline infectious peritonitis identification of a novel coronavirus from a beluga whale by using a panviral microarray probable pangolin origin of sars-cov-2 associated with the covid-19 discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus evidence for camel-to-human transmission of mers coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china new insights into the mechanisms of rna recombination generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure the 2019-new coronavirus epidemic: evidence for virus evolution fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin structure analysis of the receptor binding of 2019-ncov initial public health response and interim clinical guidance for the 2019 novel coronavirus outbreak -united states evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic china coronavirus: six questions scientists are asking complete genome sequence of bat coronavirus hku2 from chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome structure of sars coronavirus spike receptor-binding domain complexed with receptor structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human the outbreak of sars-cov-2 pneumonia calls for viral vaccines what can animal coronaviruses tell us about emerging human coronaviruses? veterinary record potential host range of multiple sars-like coronaviruses and an improved ace2-fc variant that is potent against both sars-cov-2 and sars-cov-1 receptor and viral determinants of sars-coronavirus adaptation to human ace2 isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor serological evidence of bat sars-related coronavirus infection in humans bat coronaviruses in china mers coronavirus neutralizing antibodies in camels zoonotic spillover and emerging viral diseases time to intensify zoonoses surveillance in brazil outbreak of novel coronavirus (sars-cov-2): first evidences from international scientific literature and pending questions severe acute respiratory syndrome coronavirus on hospital surfaces avian influenza and sialic acid receptors: more than meets the eye? the lancet infectious diseases transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents emerging threats from zoonotic coronaviruses-from sars and mers to 2019-ncov ocular tropism of respiratory viruses. microbiology and molecular biology reviews 2019-ncov transmission through the ocular surface must not be ignored first case of 2019 novel coronavirus in the united states genomic diversity of sars-cov-2 in coronavirus disease 2019 patients emerging zoonoses: a one health challenge covid-19: first severe pandemic of the 21st century coronavirus outbreak will set back india's growth recovery covid-19: a look into the modern age pandemic biosafety and biosecurity approaches to restrain/contain and counter sars-cov-2/covid-19 pandemic: a rapid-review oie. questions and answers on the covid-19 calling for a covid-19 one health research coalition from sars to covid-19: a previously unknown sars-related coronavirus (sars-cov-2) of pandemic potential infecting humans -call for a one health approach ohc. covid-19 and one health 3 steps to help prevent another animal-to-human virus pandemic prevention cfdca. covid-19 and animals usda ensures food safety during covid-19 outbreak disinfectants for use against sars-cov none. key: cord-349907-dwhyx97y authors: noh, ji yeong; yoon, sun-woo; kim, doo-jin; lee, moo-seung; kim, ji-hyung; na, woonsung; song, daesub; jeong, dae gwin; kim, hye kwon title: simultaneous detection of severe acute respiratory syndrome, middle east respiratory syndrome, and related bat coronaviruses by real-time reverse transcription pcr date: 2017-02-20 journal: arch virol doi: 10.1007/s00705-017-3281-9 sha: doc_id: 349907 cord_uid: dwhyx97y since severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) coronaviruses (covs) share similar characteristics with respect to clinical signs, etiology, and transmission, methods for a rapid and accurate differential diagnosis are important. therefore, the aim of this study was to develop a duplex real-time reverse transcription (rt)-pcr method for the simultaneous detection of these viruses. primers and probes that target the conserved spike s2 region of human sars-cov, mers-cov, and their related bat covs were designed. the results of real-time rt-pcr showed specific reactions for each virus with adequate detection limits of 50–100 copies/ml and 5–100 copies/ml using puc57-sars-ps2 (a template for sars-cov) and pgem-mers-s2 (a template for mers-cov), respectively. in addition, this real-time rt-pcr system was able to detect the target viruses sars-like bat cov and mers-cov in bat fecal samples and sputum of mers patients, respectively. therefore, this newly developed real-time rt-pcr method is expected to detect not only sars-cov and mers-cov in humans but also several bat covs that are closely related to these viruses in bats. the recent emergence of human coronaviruses (covs) causing severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) with global spread represents a significant threat to public health. both sars-cov and mers-cov cause severe respiratory diseases and belong to the genus betacoronavirus [1] . phylogenetic analysis has shown that sars-cov belongs to lineage b, which includes sars-like bat covs and some other bat-derived covs, whereas mers-cov belongs to lineage c, which also includes bat-derived covs [1] . sars-cov and mers-cov share similar transmission characteristics. both viruses are thought to have originated from bats, which are the primary reservoir of diverse covs [2] . cross-species transmission of these viruses to palm civets and dromedary camels has increased the chance of their zoonotic transmission to humans [3] . nosocomial transmission is thought to be the main cause of human-tohuman transmission of sars-cov and mers-cov [4] . although there have been no sars cases reported since 2004, mers cases have been reported continuously worldwide [3] . in addition, a recent analysis of the sarslike bat covs that use the ace2 receptor highlighted the need for preparedness for their potential zoonotic transmission [5, 6] . bat covs related to sars-cov and mers-cov have been reported continuously worldwide [7] [8] [9] [10] . given the high similarity between sars-cov and mers-cov with respect to clinical signs, etiology, and transmission, establishment of a simultaneous detection method for these viruses would be useful for their accurate diagnosis and monitoring in humans as well as their reservoir, bats. therefore, in this study, a duplex real-time reverse transcription (rt)-pcr method was developed based on primers and probes that target the conserved spike s2 region of sars-cov, sars-like bat covs, mers-cov, and mers-related bat covs. for the universal detection of sars-cov and sars-like bat covs, consensus primers and probes (fig. 1a) were designed based on the conserved sequences of the spike s2 region by aligning the following reference sequences: human sars-covs sino1 (genbank no. ay485277), tor2 (ay274119), and urbani (ay278741); sars-like bat covs b15-21 (ku528591), rp3 (dq071615), rsshc014 (kc881005), rf1 (dq412042), hku3 (dq084199), and 273 (dq648856). for the detection of mers-cov and possible related bat covs, consensus primers and probes (fig. 1b) were designed based on the conserved sequences of the spike s2 region by aligning the following reference sequences: human mers-covs tha-cu (kt225476), jeddah (kf958702), and kor-nih (kt029139); mers-related bat covs a434 (dq648790), pml-phe1 (kc869678), gx2012 (kj473822), hku5 (ef065512), sc2013 (kj473821), and hku4 (ef065508). the designed primers and probes are shown in table 1 . the partial sequences of the s2 region (nucleotides 3221-3620) of the spike gene of the sars-cov sino1 strain (genbank no. ay485277), including the primer-and probe-binding regions, were synthesized and inserted in the puc57 vector at cosmogenetech co., ltd. (seoul, korea). the nucleotide sequence of the mers-cov s2 domain (amino acids 765-1288, emc strain, genbank no. jx869059) was cloned in the pgem vector by bioneer (daejeon, korea). competent escherichia coli cells (dh5a) were transformed with the recombinant plasmid vectors (puc57-sars-ps2 and pgem-mers-s2), and the amplified plasmid was extracted. these plasmids were then used as templates for the positive control as well as in the detection limit test after calculating their copy numbers. a portion of the s2 region of puc57-sars-ps2 was amplified using primers containing a t7 promoter sequence: sars-tem-f (5'-tcg gta cct aat acg act cac tat agg gaa gag cca cca tga agg tgt ttt tgt gtt taa tgg-3') and sars-tem-r (5'-ttt ttt ttt ttt ttt ttt ttt ttt ttt ttt tta att agt cca gca atg aag cc-3'). the amplified pcr product was used as template for in vitro transcription using an mmessage mmachine t7 kit (ambion, usa) following the manufacturer's manual. the in vitrotranscribed mrna was treated with turbo dnase (2 u/ ll) and purified with lithium chloride precipitation solution (7.5 m lithium chloride, 50 mm edta). the final concentration of sars-ps2-based mrna was measured using a nanodrop 2000 spectrophotometer (thermo scientific, usa). the real-time rt-pcr was performed with reverse transcription at 45°c for 10 min followed by 95°c for 2 min and cycling 35 times at 95°c for 10 s and 60°c for 20 s. thermocycling was performed using a lightcycler 96 system (roche, usa), and positive results were estimated according to analysis of the fluorescent curves originating from each probe within 40 cycles. the purified recombinant plasmids were prepared at a concentration of 1 9 10 8 copies/ml for the templates of sars-cov and mers-cov. each template was diluted tenfold in nuclease-free distilled water (ambion, usa). the diluted plasmids were then used as templates for the real-time rt-pcr to evaluate the detection limits of the primers and probes. the in vitro-transcribed mrna of sars-cov ps2 and rna extracted from mers-cov isolate (kor/knih/002_05_2015) were prepared at a concentration of 1 9 10 4 pg/ll. they were then diluted tenfold with nuclease-free distilled water and used as template for the real-time rt-pcr to evaluate the detection limits of the primers and probes. to evaluate the specificity of the primers and probes, several rna viruses, including dengue virus types 2, 3, and 4 (kbpv-vr-29, -30, and -31, respectively; korea bank for pathogenic viruses, seoul, korea), porcine reproductive and respiratory syndrome virus (prrsv) strain cp401-9, porcine epidemic diarrhea virus (pedv) strain dr13 (green cross veterinary products, yongin, korea), and influenza a virus (h1n1) strain sk14 from our laboratory, were tested using the newly developed realtime rt-pcr protocol. in addition, rna extracted from a mers-cov isolate (kor/knih/002_05_2015), which was kindly provided by the korea center for disease control, was tested using the real-time rt-pcr system. a recombinant plasmid including partial sequences of the s2 region (nucleotides 2231-2630 of the spike gene, merslike bat cov strain hku4 [genbank no. ef065508]) was synthesized (puc57-mers-like-cov-ps2) and used as a template for the real-time rt-pcr. agarose gel electrophoresis was performed to observe the target bands as well as for fluorescent detection. rna was extracted from 47 bat samples (feces, urine, and oral swabs) collected between july 2015 and april 2016 in korea. among them, samples b15-8, b15-40, b15-41, b16-6, and b16-40, and b15-21 were determined to be positive for covs according to consensus primers-based rt-pcr, which can detect diverse covs by targeting conserved sequences of the rna-dependent rna polymerase gene [11] . the positive samples were found to have rna sequences that are closely related to those of alphacoronaviruses or sars-like bat cov [7] . real-time pcr was performed with the extracted rnas and compared to the previous rt-pcr results. compared with the results of upe-based real-time rt-pcr as a reference [12] . detection limits of the real-time rt-pcr the tenfold-diluted plasmids (puc57-sars-ps2 and pgem-mers-s2) were tested by real-time rt-pcr as a single template or as mixed templates. the intensity of the fluorescence (fam and hex) emitted from the hydrolyzed probes was measured every cycle. based on the measured amplification curve, the results were expressed as the quantification cycle value (cq value). the cq values obtained from the diluted plasmids were found to be significantly correlated with the copy numbers of each plasmid, with an r 2 value of 0.9847 and 0.9954 for puc57-sars-ps2 and pgem-mers-s2, respectively (fig. 2) . the negative control (distilled water) did not yield an amplification curve. as shown in table 2 , the detection limits of the real-time rt-pcr for puc57-sars-ps2 and pgem-mers-s2 were both 1 9 10 2 copies/ml in the single-template condition and were 5 9 10 1 and 5 9 10 0 copies/ml, respectively, in the mixed-template condition. the detection limits of the real-time rt-pcr for the in vitro-transcribed mrna of puc57-sars-ps2 and viral rna of mers cov (kor/knih/002_05_2015) were 1 9 10 -3 and 1 9 10 0 ng/ml, respectively, in the single-template condition and were 5 9 10 -3 and 5 9 10 1 ng/ml, respectively, in the mixed-template condition ( table 2) . the specificity of the real-time rt-pcr method developed in this study was evaluated using rnas from several rna viruses, including mers-cov (kor/knih/ 002_05_2015), a recombinant plasmid for the bat cov hku4 strain, and rna from a bat fecal sample containing sars-like bat cov. the designed primers and probes were found to be specific for mers-cov and sars-like bat cov (b15-21) ( table 3) . no positive reactions were found for the bat coronavirus strain hku4, dengue virus, prrsv, pedv, or influenza a virus. in the case of the hku4 strain, although the hex fluorescence signal was not detected, a target band of primers for mers-cov was detected by agarose gel electrophoresis. the extracted rnas from a total of 47 bat samples that were previously tested using consensus primer-based rt-pcr [11] were re-evaluated by the real-time rt-pcr method developed in this study. as shown in table 4 , six fecal samples were positive in the consensus-primer-based rt-pcr, and these were further sequenced and analyzed using blastn (http://www.ncbi.nlm.nih.gov). among the six positive samples, five were found to be closely related to alphacoronaviruses, while one sample (b15-21) was closely related to sars-like bat cov [7] . in the real-time pcr developed in this study, five of the alphacoronaviruspositive samples were negative, but only one sars-like bat cov-positive sample was positive. the rna samples from the sputum of mers patients were tested by the newly developed real-time rt-pcr method, and the results were compared to those obtained with upebased real-time rt-pcr as a reference gold-standard test ( table 5) . among the 20 samples tested, 17 were found to be positive by upe-based real-time rt-pcr. when the single set of primers and probe for mers-cov was used, the newly developed real-time rt-pcr results showed 100% consistency with those of the reference test. however, when mixed sets of primers and probes for sars-cov and mers-cov were used, 85% of the results were consistent with those of the reference test. three falsenegative samples had cq values of 33.9, 33.2, and 36.46, respectively, in the reference test. the aim of this study was to develop a duplex real-time rt-pcr method for the simultaneous detection of sars-cov and mers-cov, which are recently emerged pathogens infecting humans. as these viruses are also closely related to several bat covs (sars-like bat covs and mers-related bat covs), the primers and probes were designed based on the conserved region of the spike s2 domains of sars-cov, mers-cov, and their related bat covs. in a previous study, two regions within the spike protein of sars-cov were identified that showed a high degree of sequence conservation with those of other covs [13] . by applying our new approach, we expected to detect not only sars-cov and mers-cov in humans but also several bat covs that are closely related to these viruses. indeed, the newly developed real-time rt-pcr method could detect sars-cov-and mers-cov-specific sequences when the plasmids puc57-sars-ps2 and pgem-mers-s2 were used as templates. in addition, positive results were obtained when rna extracted from mers-cov (kor/knih/002_05_2015), which was isolated from a patient in korea in 2015 [14] , was used as a template. the new real-time rt-pcr method also showed positive results for rna extracted from a fecal sample containing sars-like bat cov (b15-21) [7] . in addition, no nonspecific amplification was found when using rnas obtained from several rna viruses belonging to the families flaviviridae, arteriviridae, and orthomyxoviridae, and the genus alphacoronavirus. the specific binding of the primers and probes used in the real-time rt-pcr was evaluated to determine their detection limits using serially diluted plasmids (puc57-sars-ps2 and pgem-mers-s2). the detection limits of the real time rt-pcr were 50-100 copies/ml and 5-100 copies/ml for puc57-sars-ps2 and pgem-mers-s2, respectively. as the previous real-time rt-pcrs were able to detect as few as 1000 copies/ml and 291 copies/ml for sars-cov and their related bat covs and mers-cov, respectively single template 1 9 10 2 1 9 10 2 1 9 10 -3 1 9 10 0 mixed template 5 9 10 1 5 9 10 0 5 9 10 -3 5 9 10 1 a mrna obtained by in vitro transcription b rna from mers-cov isolate kor/knih/002_05_2015 a samples that were positive in the rt-pcr (11) were sequenced and analyzed using blastn (http:// www.ncbi.nlm.nih.gov) [12, 15] , the duplex real-time rt-pcr method developed in this study can be applied with an adequate limit of detection. when the real-time rt-pcr was applied to the bat samples, it could specifically detect a sars-like bat cov, and no positive reactions were obtained with the other samples, including alphacoronavirus-positive samples. the real-time rt-pcr method developed in this study could not detect the bat cov hku4 strain, which belongs to the same group as human mers-cov. although these viruses are in the same group, the nucleotide sequences were found to be more variable than those of sars-cov and its related bat covs (fig. 1) . however, as a target band was found in the agarose gel electrophoresis of a pcr product from the hku4 template, the negative result obtained with hku4 in the real-time rt-pcr method developed in this study might have been due to inefficient binding of the probe. the new real-time rt-pcr method was also tested with rnas extracted from the sputum of mers patients in korea. the single set of primers and probe for mers-cov could detect the same positive samples detected in the reference test, showing 100% agreement. however, when the mixed sets of primers and probes for sars-cov and mers-cov were used, the agreement was reduced to 85%. three samples that tested positive in the reference test were not detected with the newly developed method. this may be due to the low amount of viral rna in the sputum samples and the reduced sensitivity due to the duplex approach. in addition to using a standard test for the diagnosis of mers-cov infection in humans, consideration of additional tests would be helpful to avoid missing true positives. collectively, the newly developed real-time rt-pcr method was demonstrated to be applicable for the simultaneous detection of sars-cov and mers-cov, showing an adequate detection limit and specificity. by testing the new method with bat samples as well as human samples, it could be applicable to survey sars-cov, mers-cov, and potentially their related bat covs in bats and human samples. it could successfully detect sars-like cov in bat samples but showed limited detection ability for the bat cov hku4 strain, which is related to mers-cov. however, according to a recent finding of emc-like mers-cov, which was detected in bats of saudi arabia [18] , we assume that the new method can be helpful for screening for mers-cov in bat samples. as novel covs continue to emerge around the world, real-time-pcr-based detection methods have been developed by pioneering researchers [12, [15] [16] [17] . these methods are applicable with good limits of detection and have been validated using field samples. however, simultaneous detection of sars-cov, mers-cov, and their related bat covs would be particularly useful for the detection of these viruses in humans as well as in bats, which are a known reservoir of the viruses. therefore, the duplex real-time rt-pcr approach for the simultaneous detection of sars-cov and mers-cov developed in this study might allow more-convenient and rapid detection of these viruses in clinical samples. in addition, this new method can be used to closely monitor related bat covs in bat populations, which are considered to be a primary reservoir of these viruses. epidemiology, genetic recombination, and pathogenesis of coronaviruses ecology, evolution and classification of bat coronaviruses in the aftermath of sars sars and mers: recent insights into emerging coronaviruses transmission characteristics of mers and sars in the healthcare setting: a comparative study isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor a sars-like cluster of circulating bat coronaviruses shows potential for human emergence detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats close relative of human middle east respiratory syndrome coronavirus in bat mers-related betacoronavirus in vespertilio superans bats isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus identification of a novel coronavirus in bats detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus a real-time pcr assay for bat sars-like coronavirus detection and its application to italian greater horseshoe bat faecal sample surveys realtime reverse transcriptase polymerase chain reaction assays for middle east respiratory syndrome development of a molecular-beacon-based multi-allelic real-time rt-pcr assay for the detection of human coronavirus causing severe acute respiratory syndrome (sars-cov): a general methodology for detecting rapidly mutating viruses middle east respiratory syndrome coronavirus in bats, saudi arabia conflict of interest the authors have declared that no competing interests exist. key: cord-353704-lfndq85x authors: ye, zi-wei; yuan, shuofeng; yuen, kit-san; fung, sin-yee; chan, chi-ping; jin, dong-yan title: zoonotic origins of human coronaviruses date: 2020-03-15 journal: int j biol sci doi: 10.7150/ijbs.45472 sha: doc_id: 353704 cord_uid: lfndq85x mutation and adaptation have driven the co-evolution of coronaviruses (covs) and their hosts, including human beings, for thousands of years. before 2003, two human covs (hcovs) were known to cause mild illness, such as common cold. the outbreaks of severe acute respiratory syndrome (sars) and the middle east respiratory syndrome (mers) have flipped the coin to reveal how devastating and life-threatening an hcov infection could be. the emergence of sars-cov-2 in central china at the end of 2019 has thrusted covs into the spotlight again and surprised us with its high transmissibility but reduced pathogenicity compared to its sister sars-cov. hcov infection is a zoonosis and understanding the zoonotic origins of hcovs would serve us well. most hcovs originated from bats where they are non-pathogenic. the intermediate reservoir hosts of some hcovs are also known. identifying the animal hosts has direct implications in the prevention of human diseases. investigating cov-host interactions in animals might also derive important insight on cov pathogenesis in humans. in this review, we present an overview of the existing knowledge about the seven hcovs, with a focus on the history of their discovery as well as their zoonotic origins and interspecies transmission. importantly, we compare and contrast the different hcovs from a perspective of virus evolution and genome recombination. the current cov disease 2019 (covid-19) epidemic is discussed in this context. in addition, the requirements for successful host switches and the implications of virus evolution on disease severity are also highlighted. coronaviruses (covs) belong to the family coronaviridae, which comprises a group of enveloped, positive-sensed, single-stranded rna viruses [1, 2] . these viruses harbouring the largest genome of 26 to 32 kilobases amongst rna viruses were termed "covs" because of their crown-like morphology under electron microscope [2, 3] . structurally, covs have non-segmented genomes that share a similar organization. approximately two thirds of the genome contain two large overlapping open reading frames (orf1a and orf1b), which are translated into the pp1a and pp1ab replicase polyproteins. the polyproteins are further processed to generate 16 non-structural proteins, designated nsp1~16. the remaining portion of the genome contains orfs for the structural proteins, including spike (s), envelope (e), membrane (m) and nucleoprotein (n). a number of lineage-specific accessory proteins are also encoded by different lineages of covs [2, 4] . based on the difference in protein sequences, covs are classified into four genera (alpha-cov, beta-cov, gamma-cov and delta-cov), among which the beta-cov genera contains most hcovs and is subdivided into four lineages (a, b, c and d) [2, 4, 5] . phylogenetic evidence has shown that bats and rodents serve as the gene source of most alpha-covs and beta-covs, while birds are the main reservoir of gamma-covs and delta-covs [2] . for thousands of years, covs have constantly crossed species barriers and some have emerged as important human pathogens [2, 4, [6] [7] [8] . to date, seven human covs (hcovs) are known. among them hcov-229e and hcov-nl63 are alpha-covs. the other five beta-covs include hcov-oc43, hcov-hku1, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus ivyspring international publisher (mers-cov) and sars-cov-2 [2, 5, 9] . hcov-229e, hcov-oc43, hcov-hku1 and hcov-nl63 usually cause mild symptoms, like common cold and/or diarrhea [10, 11] . in contrast, sars-cov, mers-cov and the newly-identified sars-cov-2 are highly pathogenic, causing severe lower respiratory tract infection in relatively more patients with a higher chance to develop acute respiratory distress syndrome (ards) and extrapulmonary manifestations. the first hcov-229e strain, b814, was isolated from the nasal discharge of patients with common cold in mid-1960s [12] . since then, more knowledge was accumulated through extensive studies on hcov-229e and hcov-oc43, both of which cause self-limiting symptoms [13] . indeed, the concept had been widely accepted that infection with hcovs is generally harmless until the outbreak of sars. the sars outbreak occurred in 2003 is one of the most devastating in current history, infecting over 8,000 people with a crude case fatality of approximately 10% [14, 15] . ten years later, the middle east respiratory syndrome (mers) outbreak resulted in a persistent epidemic in the arabian peninsula with sporadic spreading to the rest of the world [16] [17] [18] . the 2019 novel hcov (2019-ncov), which has subsequently been renamed sars-cov-2, is the causative agent of the ongoing epidemic of coronavirus disease 2019 (covid19) , which has claimed more than 3,120 lives and infected more than 91,000 people as of march 3, 2020 [19] . the alarm has been ringing and the world has to prepare for the coming pandemic of sars-cov-2. all seven hcovs have a zoonotic origin from bats, mice or domestic animals [2, 20] . multiple lines of evidence support an evolutionary origin of all hcovs from bats, where viruses are well adapted and non-pathogenic but show great genetic diversity. the covid-19 epidemic has presented enormous medical, scientific, social and moral challenges to china and the world. tracing the zoonotic origins of hcovs provides a framework to understand the natural history, driving force and restriction factors of species jumping. this might also guide or facilitate the search for the reservoir, intermediate and amplifying animal host(s) of sars-cov-2, with important implications in the prevention of future spillovers. in this review we present an overview of the zoonotic origins, interspecies transmission and pathogenesis of hcovs. particularly, we highlight and discuss the common theme that parental viruses of hcovs are typically non-pathogenic in their natural reservoir hosts but become pathogenic after interspecies transmission to a new host. we also review the trend of hcov evolution in which the increase in transmissibility often comes with the decrease in pathogenicity. the outcome of the ongoing sars-cov-2 outbreak is also discussed in this context. animal covs have been known since late 1930s. before the first isolation of hcov-229e strain b814 from the nasal discharge of patients who had contracted common cold, different covs had been isolated in various infected animals, including turkey, mouse, cow, pig, cat and dog [21] [22] [23] [24] [25] [26] . in the past decades, seven hcovs have been identified. a brief summary of the history of hcov discovery in chronological order (table 1) would be informative and instructive. the first hcov-229e strain was isolated from the respiratory tract of patients with upper respiratory tract infection in the year of 1966 [27] , and was subsequently adapted to grow in wi-38 lung cell lines [28] . patients infected with hcov-229e presented with common cold symptoms, including headache, sneezing, malaise and sore-throat, with fever and cough seen in 10~20% cases [29] . later in 1967, hcov-oc43 was isolated from organ culture and subsequent serial passage in brains of suckling mice [28] . the clinical features of hcov-oc43 infection appear to be similar to those caused by hcov-229e, which are symptomatically indistinguishable from infection with other respiratory tract pathogens such as influenza a viruses and rhinoviruses [28] . both hcov-229e and hcov-oc43 are distributed globally, and they tend to be predominantly transmitted during the season of winter in temperate climate [2] . generally, the incubation time of these two viruses is less than one week, followed by an approximately 2-week illness [28] . according to a human volunteer study, healthy individuals infected with hcov-229e developed mild common cold [30] . only a few immunocompromised patients exhibited severe lower respiratory tract infection. sars, also known as "atypical pneumonia", was the first well documented hcov-caused pandemic in human history and the etiological agent is sars-cov, the third hcov discovered [14, 15] . the first case of sars can be traced back to late 2002 in guangdong province of china. the sars epidemic resulted in 8,096 reported cases with 774 deaths, spreading across many countries and continents. apart from the super-spreaders, it was estimated that each case could give rise to approximately two secondary cases, with an incubation period of 4 to 7 days and the peak of viral load appearing on the 10 th day of illness [14, 15] . patients infected with sars-cov initially present with myalgia, headache, fever, malaise and chills, followed by dyspnea, cough and respiratory distress as late symptoms [14, 15] . lymphopenia, deranged liver function tests, and elevated creatine kinase are common laboratory abnormalities of sars [14, 15] . diffuse alveolar damage, epithelial cell proliferation and an increase of macrophages are also observed in sars patients [31] . approximately 20-30% of patients subsequently require intensive care and mechanical ventilation. in addition to lower respiratory tract, multiple organs including gastrointestinal tract, liver and kidney can also be infected in these severe cases, usually accompanied with a cytokine storm, which might be lethal particularly in immunocompromised patients. the virus was first isolated from the open lung biopsy of a relative of the index patient who travelled to hong kong from guangzhou [14, 15] . since then, tremendous efforts have been dedicated to hcov research. hcov-nl63 was isolated from a 7-month-old child from the netherlands during late 2004. it was initially found to be prevalent in young children, the elderly and immunocompromised patients with respiratory illnesses [32] . presentation of coryza, conjunctivitis, fever, and bronchiolitis is common in the disease caused by hcov-nl63 [33] . another independent study described the isolation of the same virus from a nasal specimen from an 8-month-old boy suffering from pneumonia in the netherlands [34] . although it was identified in netherlands, it is actually distributed globally [2] . it has been estimated that hcov-nl63 accounts for approximately 4.7% of common respiratory diseases, and its peak incidence occurs during early summer, spring and winter [2] . hcov-nl63 is associated with obstructive laryngitis, also known as croup [35] . in the same year, hcov-hku1 was isolated from a 71-year-old man who had been hospitalized with pneumonia and bronchiolitis in hong kong [36] . besides community-acquired pneumonia and bronchiolitis, hcov-hku1 was reported to be associated with acute asthmatic exacerbation [37] . similar to hcov-nl63, hcov-229e and hcov-oc43, hcov-hku1 was found worldwide, causing mild respiratory diseases [37] . all these four communityacquired hcovs have been well adapted to humans and are generally less likely to mutate to cause highly pathogenic diseases, though accidents did occur for unknown reasons as in the rare case of a more virulent subtype of hcov-nl63, which has recently been reported to cause severe lower respiratory tract infection in china [38] . generally, when these hcovs acquire the abilities to transmit efficiently and to maintain themselves continuously within humans, they also become less virulent or pathogenic. mers-cov was first isolated in 2012 from the lung of a 60-year-old patient who developed acute pneumonia and renal failure in saudi arabia [17, 18, 39] . whereas most of the laboratory-confirmed cases originate from the middle east, imported cases with occasional secondary spreads to close contacts have been reported in various european countries and tunisia. another secondary outbreak occurred in south korea in 2015 with 186 confirmed cases. clinical manifestations of mers resemble those of sars, characterized by progressive acute pneumonia [17, 18, 39] . unlike sars, many patients with mers also developed acute renal failure, which is thus far unique for mers among hcov-caused diseases [17, 18, 39] . more than 30% of patients present with gastrointestinal symptoms, such as diarrhea and vomiting [17, 18, 39] . as of february 14, 2020, over 2500 laboratory confirmed cases were reported with a high case fatality of 34.4%, making mers-cov one of the most devastating viruses known to humans. during middle to late december 2019, clusters of pneumonia patients retrospectively known to be associated with sars-cov-2 infection were detected in wuhan, hubei province, china [40] . world health organization declared the ongoing outbreak of lower respiratory tract infection caused by sars-cov-2 a public health emergency of international concern and also named the disease covid-19. as of march 3, 2020, 90,053 cases have been confirmed worldwide, with a crude case fatality of 3.4%. notably, the case fatality in hubei, china is 4.2%, whereas the one outside of it is 1.2%. sars-cov-2 causes severe respiratory infection like sars-cov and mers-cov, presented as fever, cough and dyspnea [40] . diarrhea is also seen in some patients [40] . pneumonia is one of the most severe symptoms and can progress rapidly to acute respiratory distress syndrome. although sars-cov and sars-cov-2 are very similar due to high nucleotide sequence homology of 82%, they cluster into different branches in the phylogenetic tree [5] . sars-cov-2 is apparently less pathogenic but more transmissible compared to sars-cov and mers-cov. asymptomatic subjects infected with sars-cov-2 have been reported and might contribute to its rapid spreading around the world. comparing and contrasting sars-cov-2 with the other six hcovs reveal similarities and differences of great interest. first, the incubation period and the duration of the course of hcov disease are very similar. in this regard, sars-cov-2 follows the general trend of the other six hcovs. second, the severity of symptoms of covid-19 lies between sars-cov and the four community-acquired hcovs (i.e. hcov-229e, hcov-oc43, hcov-hku1 and hcov-nl63). on one hand, sars-cov-2 infection exhibits features that are more commonly seen during infection with community-acquired hcovs, including the presentation of non-specific, mild or even no symptoms. on the other hand, a small subset of severe cases of covid-19 can also be seen as in the case of sars-cov infection, although the ratio is a bit lower. third, the transmission of sars-cov-2 also shows interesting patterns characteristic of both community-acquired hcovs and sars-cov. on one hand, the transmissibility of sars-cov-2 is at least as high as that of community-acquired hcovs. on the other hand, it remains to be verified whether the transmissibility of sars-cov-2 decreases after passages in humans as in the cases of sars-cov and mers-cov. finally, same as the other hcovs [41] , sars-cov-2 can be detected in fecal samples. whether fecal-oral transmission of sars-cov-2 plays an important role as in the case of sars-cov at least under some circumstance remains to be clarified by future studies. it is also of particularly great interest to see whether sars-cov-2 might exhibit seasonality as in the cases of community-acquired hcovs. nevertheless, the features of sars-cov-2 including its transmissibility, pathogenicity and sustainable spreading after passages in humans will be influential on the ultimate fate of the ongoing outbreak of covid-19. all four community-acquired hcovs causing mild symptoms have been well adapted to humans. from another perspective, it might also be true that humans have been well adapted to these four hcovs. in other words, both could be the survivors of ancient hcov pandemics. hcovs that cause severe diseases in humans and humans who developed severe hcov diseases have been eliminated. for this to happen, hcovs have to replicate in humans to sufficient extent to allow the accumulation of adaptive mutations that counteract host restriction factors. in this sense, the longer the sars-cov-2 outbreak persists and the more people that it infects, the greater chance that it will fully adapt to humans. if it adapts well, its transmission in humans would be difficult to stop by quarantine or other infection control measures. for many years, the four community-acquired covs circulate in human populations, triggering common cold in immunocompetent subjects. these viruses do not need an animal reservoir. in contrast, highly pathogenic sars-cov and mers-cov have not adapted to humans well and their transmission within humans cannot be sustained. they need to maintain and propagate in their zoonotic reservoirs and seek the chance to spillover to susceptible human targets, possibly via one or more intermediate and amplifying hosts. sars-cov-2 has features that are similar to both sars-cov/mers-cov and the four community-acquired hcovs. it is highly transmissible like community-acquired hcovs, at least for the time being. however, it is more pathogenic than community-acquired hcovs and less pathogenic than sars-cov or mers-cov. it remains to be seen whether it will adapt fully to humans and circulate within humans without a reservoir or intermediate animal host. before discussing the animal origins of hcovs, it will serve us well to discuss the definitions and characteristics of evolutionary, natural, reservoir, intermediate and amplifying hosts of hcovs. an animal serves as the evolutionary host of an hcov if it harbours a closely related ancestor sharing high homology at the level of nucleotide sequence. the ancestral virus is usually well adapted and nonpathogenic in this host. likewise, a reservoir host harbours hcov continuously and for long term. epidemiological data revealed retrospectively that the index case of sars had a contact history with game animals [42] . subsequent seroprevalence investigations indicated that animal traders had a higher prevalence of anti-sars-cov igg compared with that of the general population [42] . masked palm civets (paguma larvata) and a racoon dog in live animal markets were first identified to carry sars-cov-like viruses that are almost identical to sars-cov [43] . this was indirectly supported by the fact that no further sars was reported after killing all civets in the markets. however, it has been reported that masked palm civets from the wild or farms without exposure to the live animal markets were largely negative for sars-cov [44] , suggesting that masked palm civets might only serve as the intermediate amplifying host but not the natural reservoir of sars-cov. notably, since 80% of the different animals in the markets in guangzhou have anti-sars-cov antibodies [45] , the possibilities that multiple species of small mammals might also serve as intermediate amplifying hosts of sars-cov cannot be excluded. all of these appear to be dead-end hosts of sars-cov. subsequent search for the natural animal host of sars-cov unveiled a closely related bat cov, termed sars-related rhinolophus bat cov hku3 (sarsr-rh-batcov hku3), which exists in chinese horseshoe bats [46] . these bats are positive for anti-sars-cov antibodies and genome sequence of sarsr-rh-batcov hku3 [37, 47] . this and other bat covs share 88-92% nucleotide sequence homology with sars-cov. these studies have laid the foundation for the new concept that bats host emerging human pathogens. several sars-like covs (sl-covs) have also been identified from bats, but none except for one designated wiv1 can be isolated as live virus [7] . human angiotensin converting enzyme 2 (ace2) is known to be the receptor of sars-cov. wiv1 derived from fecal sample of bats was demonstrated to use bat, civet and human ace2 as receptor for cell entry [48] . intriguingly, sera of convalescent sars patients were capable of neutralizing wiv1 [48] . thus far, wiv1 represents the most closely related ancestor of sars-cov in bats [7, 48] , sharing 95% nucleotide sequence homology. albeit the high homology between these two viruses, it is generally believed that wiv1 is not the immediate parental virus of sars-cov and bats are not the immediate reservoir host of sars-cov. phylogenetic analysis clusters mers-cov to the same group as bat cov-hku4 and bat cov-hku5. bat cov-hku4 and mers-cov utilize the same host receptor, dipeptidyl peptidase 4 (dpp4), for virus entry [49] . rna-dependent rna polymerase sequences of mers-cov are phylogenetically closer to counterparts in bat beta-covs identified from europe and africa [50, 51] . up to now, no live mers-cov can be found in wild bats. mers-cov and its closest relative bat cov-hku25 share only 87% nucleotide sequence homology [52, 53] . thus, bats might not be the immediate reservoir host of mers-cov. on the other hand, studies in middle east have shown that dromedary camels are seropositive for mers-cov-specific neutralizing antibodies [54] , same as camels of middle east origin in multiple african countries [55] . live mers-cov identical to the virus found in humans was isolated from the nasal swabs of dromedary camels, further indicating that camels serve as the bona fide reservoir host of mers-cov [56] . it is also noteworthy that generally mild symptoms but massive virus shedding were observed in camels experimentally infected with mers-cov [57] . notably, infected camels shed viruses not only through respiratory route but also through fecal-oral route, which is also the main route for virus shedding from bats. however, questions still remain since many confirmed cases of mers have no contact history with camels prior to symptom onset [58] , plausibly ascribed to human-to-human transmission or unknown transmission routes involving unrecognized animal species that harbour mers-cov. these merit further investigations. sars-cov-2 shares 96.2% nucleotide homology with a bat cov ratg13 isolated from rhinolophus affinis bats [8] . as in the cases of sars-cov and mers-cov, the sequence divergence between sars-cov-2 and ratg13 is too great to assign parental relationship. that is to say, bats might not be the immediate reservoir host(s) of sars-cov-2 unless almost identical bat covs are found in future. presumably, the intermediate animal hosts of sars-cov-2 should be among the wildlife species sold and killed at the huanan seafood wholesale market, with which many of the initial cases of covid-19 were associated, indicative of a probable animal-to-human transmission event [40] . several recent studies based on metagenomic sequencing have suggested that a group of endangered small mammals known as pangolins (manis javanica) could also harbour ancestral beta-covs related to sars-cov-2 [59] . these novel pangolin cov genomes share 85-92% nucleotide sequence homology with sars-cov-2. however, they are equally closely related to ratg13 with about 90% identity at the level of nucleotide sequence. they cluster into two sub-lineages of sars-cov-2-like viruses in the phylogenetic tree, one of which share a more similar receptor binding domain (rbd) with sars-cov-2, with 97.4% amino acid sequence identity [59] . in stark contrast, the rbds of sars-cov-2 and ratg13 are more divergent, albeit a higher degree of sequence homology genome-wide. an earlier study on diseased pangolins also reported the detection of viral contigs from lung samples, which turn out to be similarly related to sars-cov-2 [60] . this study adopted different assembly methods and manual curation to generate a partial genome sequence comprising about 86.3% of the full-length viral genome [60] . we cannot exclude the possibility that pangolin is one of the intermediate animal hosts of sars-cov-2 [59] . however, currently there is no evidence in support of a direct pangolin origin of sars-cov-2 due to the sequence divergence between sars-cov-2 and pangolin sars-cov-2-related beta-covs. in addition, the distance between sars-cov-2 and ratg13 is even shorter than that between sars-cov-2 and pangolin sars-cov-2-related beta-covs. the evolutionary pathway of sars-cov-2 in bats, pangolins and other mammals remains to be established. whereas the highest sequence homology has been found in the rbds between sars-cov-2 and pangolin, sars-cov-2-related beta-covs, sars-cov-2 and ratg13 share the highest genome-wide sequence homology. it is highly speculative that the high degree of similarity between the rbds of pangolin sars-cov-2-related beta-covs and sars-cov-2 is driven by selectivitymediated convergent evolution. a counter-proposal is in favour of a recombination between a pangolin sars-cov-2-related beta-cov and ratg13 in the third wild animal species. as a driving force in evolution, recombination is widespread among beta-covs [61] . the jury is still out on the immediate zoonotic origin of sars-cov-2. besides the highly pathogenic hcovs, the zoonotic origin of hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1 have also been studied [9, [62] [63] [64] . phylogenetic evidence indicated that both hcov-nl63 and hcov-229e might have originated from bat covs [62] [63] [64] , while the parental viruses of hcov-oc43 and hcov-hku1 have been found in rodents [9] . it has been reported that a bat cov termed arcov.2 (appalachian ridge cov) detected in north american tricolored bat displayed close relationship with hcov-nl63 [62, 63] . on the other hand, hcov-229e was genetically related to another bat cov, termed hipposideros/ghanakwam/19/2008, which was detected in ghana [65] , while camelids have also been suspected as its intermediate host [66, 67] . for clarity, the current knowledge on animal origins of known hcovs is summarized in figure 1 and table 2 . phylogenetic analysis has provided evidence for interspecies transmission events of hcovs in the history. when hcov-oc43 crossed species to infect humans from domestic livestock around 1890, a pandemic of respiratory infection was recorded [68] . the interspecies transmission history of hcov-229e is less clear. bat alpha-covs closely related to hcov-229e have been found. between them there is an alpaca alpha-cov. several lines of evidence support the transmission of virus from bats to humans directly. first, humans but not alpacas might have contact with bats in a shared ecological niche. instead, humans have close contact with alpacas. second, hcov-229e-related bat alpha-covs are diverse and non-pathogenic in bats, whereas alpaca alpha-cov caused an outbreak of respiratory disease in infected animals [65] . finally, alpaca alpha-cov has not been found in feral animals. thus, the possibility cannot be excluded that alpacas obtain the hcov-229e-related alpha-cov from humans. in fact, bats are the direct source of human pathogenic viruses including rabies virus, ebola virus, nipah virus and hendra virus [69] . it is therefore not too surprising that bats might transmit hcov-229e to humans directly. alternatively, whereas bat alpha-covs serve as the gene pool of hcov-229e, alpacas and dromedary camels might serve as intermediate hosts that transmit viruses to humans, exactly as in the case of mers-cov [69] . mers-cov serves as an excellent example of interspecies transmission from bats to dromedary camels and from dromedary camels to humans. the evolutionary origin of mers-cov from bats is known at its initial identification and has also been strengthened by subsequent findings [49] [50] [51] . it is obvious that bats provide a rich pool of virus species for interspecies exchange of genetic fragments and interspecies transmission. longevity, densely packed colonies, close social interaction and strong ability to fly are all favourable conditions for bats to be an ideal 'virus spreader'. on the other hand, mers-cov has been introduced to dromedary camels for decades. it is well adapted to these camels that have turned from an intermediate host to a stable and natural reservoir host. mers-cov causes very mild disease and maintains a relatively low mutation rate in these animals. its sporadic transmission to humans is an accident and humans remain a dead-end host of mers-cov as its transmission cannot be sustained. in contrast to the role of camels in the transmission of mers-cov, the role of pangolins, if there is any, in the transmission of sars-cov-2 is different. particularly, pangolin beta-covs are highly pathogenic in pangolins. they might be a dead-end host for sars-cov-2-related beta-covs, similar to civets in the case of sars-cov. several possibilities for interspecies transmission of sars-cov-2 from animals to humans have to be ruled in or ruled out in future studies. first, bats could be the reservoir host of a sars-cov-2-related virus almost identical to sars-cov-2. humans might share the ecological niche with bats through butchering or coal mining. second, pangolins could be one of intermediate amplifying host to which a sars-cov-2-related virus had been newly introduced. humans contract the virus through butchering and consumption of game meat. it is possible that many mammals including domestic animals are susceptible to sars-cov-2. a survey of domestic and wild animals for antibodies is warranted. third, as mentioned above, recombination and adaptation of sars-cov-2 might have occurred in a third species that has contact with both bats and pangolins. the search for the animal origins of sars-cov-2 is still on. apart from different types of the animal hosts, three major factors on the viral side are also important in facilitating covs to cross species barriers [70] . first of all, their relatively high mutation rates in rna replication. in comparison to other single-stranded rna viruses, the estimated mutation rates of covs could be regarded as "moderate" to "high" with an average substitution rate being ~10 -4 substitution per year per site [2] , depending on the phase of cov adaptation to novel hosts. covs have a proof-reading exoribonuclease, deletion of which results in exceedingly high mutability and attenuation or even inviability. interestingly, the nucleotide analogue remdesivir is known to suppress cov replication through inhibition of this exoribonuclease and the rna-dependent rna polymerase [70] . remdesivir is one of most promising anti-sars-cov-2 agents to be tested in clinical trials. nevertheless, mutation rates of covs are about a million times higher than those of their hosts. in addition, mutation rate is often high when covs are not well adapted to the host [71] . compared to sars-cov with a high mutation rate [72] , the mutation rate of sars-cov-2 is apparently lower, suggestive of a higher level of adaptation to humans. presumably, it has already been adapted to another host close to humans. in addition to sars-cov-2, this also applies to mers-cov, which is well adapted to dromedary camels. theoretically, it is unlikely that genetic drift would render vaccines and antivirals against sars-cov-2 ineffective quickly. second, the large rna genome in covs exerts extra plasticity in genome modification for mutations and recombination, thereby increasing the probability for interspecies co-evolution, which is advantageous for the emergence of novel covs when the conditions become appropriate. this is supported by the copious unique open reading frames and protein functions encoded towards the 3â�² end of the genome. third, covs randomly and frequently switch templates during rna replication through a unique "copychoice" mechanism. in a host that serves as the mixing vessel, strand switching occurs frequently during cov rna transcription. highly homologous fulllength and subgenomic rnas could recombine to generate new covs. phylogenetic evidence of natural recombination has been found in both hcov-hku1 and hcov-oc43, as well as animal covs such as bat sl-cov and batcov-hku9 [73] . besides three viral factors stated above, viral interaction with host receptor is another key factor influential on interspecies transmission. herein, recombination of sars-cov is taken as a typical example, which also showed evidence of positive selection during interspecies transmission events [74] . based on the comparative analysis between isolates of human and civet sars-covs, sars-cov is thought to undergo rapid adaptation in different hosts, particularly with mutations at the rbd of the s protein [74] . generally, the rbd in the s protein of a cov interacts with the cellular receptor and is intensely selected by the host antibody response. in sars-cov, the rbd is in the 318 th to 510 th amino acids on the s1 fragment, which binds to the human ace2 as well as its coreceptors for viral entry [75] . the rbd of sars-cov is capable of recognizing the ace2 receptors of various animals, including bat, civet, mouse and raccoon dog, allowing interspecies transmission of the virus. in fact, only 6 amino acid residues were observed to be different from human and civet viral isolates in the rbd and 4 of them locate in the receptor-binding motif for interaction with the ace2 receptor. civet sars-cov has k479n and s487t mutations in its rbd, which might increase the affinity of the interaction of spike protein with human ace2 receptor. in other words, these two amino acid substitutions might be critical to viral adaption to humans [75] . it is noteworthy that sars-cov-2 shares the same cellular receptor with sars-cov [8] . a 30% difference between sars-cov-2 and sars-cov in the s1 unit of the s protein implicates that the binding affinity of its s protein with human ace2 might have altered [5] . indeed, a cryo-em study indicates a 10-to 20-fold higher affinity of this binding than that between human ace2 and sars-cov s protein [76] . it will also be of interest to determine whether any other coreceptor might be required for sars-cov-2 transmission. intriguingly, hcov-nl63 also binds to ace2 but with a different part of s [77] . there exist many other hcov receptors, such as aminopeptidase n for hcov-229e, and 9-o-acetylated sialic acid for hcov-oc43. they might also account for successful adaptation of these covs in humans after interspecies transmission from their animal hosts. in addition to cellular receptors, the outcome of interspecies transmission of hcovs is also governed by other host dependency and restriction factors. the divergence of these host proteins between humans and natural reservoir hosts of hcovs such as bats, dromedary camels and rodents might constitute a barrier to interspecies transmission. hcovs have to usurp host dependency factors and subvert host restriction factors for a successful interspecies transmission. in this regard, molecular determinants in this important area of virus-host interaction remain to be identified and characterized. an unbiased genome-wide screening of host dependency and restriction factors for sars-cov-2 using the state-ofthe-art technology of crispr might be fruitful. the diversity of bat covs provides ample opportunities for the emergence of novel hcovs. in this sense, bat covs serve as the gene pool of hcovs. in addition, rapid mutation and genetic recombination also drive hcov evolution and serve as two important steps in this process [2, 6] . for example, the acquisition or loss of novel proteincoding genes has the potential to drastically modify viral phenotypes. among sars-cov accessory proteins, orf8 has been thought to be important in adaptation to humans, as sars-cov-related bat viruses were isolated but found to encode divergent orf8 proteins [48] . a 29-nucleotide deletion characteristic of sars-covs has been found in strains isolated at the beginning of the human epidemic. this deletion splits orf8 into orf8a and orf8b and is thought to be an adaptive mutation that promotes the switch of hosts [78] . besides, sars-cov has a possible recombination history with lineages of alpha-and gamma-covs, where a large number of smaller recombinant regions were identified in the rnadependent rna polymerase [72] . recombination locations were also identified in the nsp9, most of nsp10, and parts of nsp14 [72] . likewise, it has been shown that the epidemic mers-cov experienced recombination events between different lineages, which occurred in dromedary camels in saudi arabia [79] . besides sars-cov and mers-cov, recombination events have also been observed in other hcovs, in which the hcovs recombine with other animal covs in their non-structural genes. it should also be cautioned that artificial selection can contribute to unintended changes in viral genomes, most likely resulting from relieving viruses from selection pressures exerted, such as by the host immune system. an example of these effects is the loss of a full-length orf4 in the hcov-229e prototype strain, owing to a two-nucleotide deletion [80] . while intact orf4 could be observed in bat and camel viruses related to hcov-229e, the alpaca alpha-cov displays a single nucleotide insertion, resulting in a frameshift [81] . last but not least, the evolution of novel hcovs is also driven by the selection pressure in their reservoir hosts. asymptomatic or only mild symptoms were detected when bats were infected with covs, indicating the mutual adaptation between covs and bats [82] . it appeared that bats are well adapted to covs anatomically and physiologically. for example, defects in the activation of proinflammatory response in bats efficiently reduce the pathology triggered by covs [83] . besides, the natural killer cell activity in bats is suppressed due to upregulation of inhibitory natural killer cell receptor nkg2/cd94 and low expression level of major histocompatibility complex class i molecules [84] . moreover, the high level of reactive oxygen species (ros) generated from high metabolic activity of bats could both suppress cov replication and affects proofreading by exoribonuclease [85] , thus providing the selection pressure for the generation of virus strains highly pathogenic when introduced into a new host. more pathogenic cov strains might also evolve by recombination, leading to the acquisition of novel proteins or protein features for host adaptation [82] . thus, it is not by chance that three novel hcovs have emerged in the past two decades. covs are non-pathogenic or cause mild symptoms in their reservoir hosts such as bats and camels. they replicate robustly without eliciting a strong host immune response. herein lie the secrets of why asymptomatic carriers are seen and what causes the severe cases in human infection. the severe symptoms are mainly due to the hyperactivation of immune response and the cytokine storm wherein the stronger the immune response, the more severe the lung damage. in contrast, in asymptomatic carriers, the immune response has been de-coupled from cov replication. the same strategy of delinking the immune response might have beneficial effects in anti-sars-cov-2 therapy. the interferon response is particularly strong in bats. thus, administration of type i interferon at least in the early phase of sars-cov-2 infection in humans should be beneficial. in addition, nlrp3 inflammasome activation in bats is defective [86] . by this reasoning, inhibition of nlrp3 inflammasome with mcc950 might be useful in the treatment of covid-19. the emergence of sars-cov-2 follows the general theme by which sars-cov and mers-cov arose. whereas a bat beta-cov sharing 95% nucleotide homology with sars-cov has been found, there also exists a bat-cov sharing 96% nucleotide homology with sars-cov-2. whereas civets and other animals in the markets have been found to harbour viruses identical to sars-cov, immediate intermediate hosts for sars-cov-2 have not been identified. pangolin beta-covs strikingly homologous to sars-cov-2 have been found, indicating that pangolins might serve as one of intermediate hosts or pangolin beta-covs could contribute gene fragments to the final version of sars-cov-2. although questions remain, there is no evidence that sars-cov-2 is man-made either deliberately or accidentally. covs have returned to the limelight due to the recent outbreak of sars-cov-2. the study of covs in bats and other animals has drastically changed our perception of the importance of zoonotic origins and animal reservoirs of hcovs in human transmission. pervasive evidence has shown that sars-cov, mers-cov and sars-cov-2 have a bat origin and are transmitted to humans via intermediate hosts. given that sars-cov infection originates from the contact between humans and civets in the markets, closing wet markets and killing civets therein could have effectively ended the sars epidemic. by the same reasoning, pangolins should be removed from wet markets to prevent zoonotic transmission, in view of the discovery of multiple lineages of pangolin beta-covs closely related to sars-cov-2. however, whether and how sars-cov-2 is transmitted to humans through pangolins and other mammals remain to be clarified in future investigations. on the other hand, mers-cov has existed in dromedary camels for a long time. these camels serve as an important tool for transportation as well as a main source of meat, milk, leather and wool products for the local people. they are widely distributed across the middle east and africa. it is therefore impossible to sacrifice all camels for the control of mers, as what was done in wild animal markets in china to prevent the spreading of sars-cov and sars-cov-2. to stop the recurrent outbreaks of mers, a comprehensive approach should be taken to develop effective vaccines against mers-cov for camels, in combination with other infection control measures. as we are not able to eliminate these viruses, new genotypes might emerge to cause outbreaks. a variety of zoonotic covs are circulating in the wild. particularly, bat covs with zoonotic potential are so diverse. there are plenty of opportunities that these zoonotic covs evolve and recombine, resulting in the emergence of new covs that are more transmissible and/or deadly in humans in future. the culture of eating wild animals in some places of china should be abandoned to reduce unnecessary contact between humans and animals. with the ordeals of sars, mers and covid-19, a better preparedness and response plan should be in place. in fact, many viruses have existed in the planet for a very long time. they stay in their own natural reservoirs until there is a chance for spillover. although bats have many features that favours the spreading of viruses, the chance for humans to be in contact with bats and other wildlife species can be minimized if people are educated to stay away from them. continuous surveillance in mammals is necessary for better understanding of the ecology of covs and their natural hosts, which will prove useful in preventing animal-to-human transmission and future outbreaks. to conclude, the most effective way to prevent viral zoonosis is for humans to stay away from the ecological niches of the natural reservoirs of the zoonotic viruses. several pieces in the puzzle of the zoonotic origin of sars-cov-2 are still missing. first, if bats transmit an ancestral virus of sars-cov-2 to pangolins, it will be of interest to see under what circumstances bats and pangolins could share the same ecological niche. second, if bats play a more direct role in human transmission, how humans get into contact with bats should be determined. third, if a third mammal acts as the true intermediate host, how it interacts with the different species including humans, bats and pangolins has to be clarified. finally, since many mammals including domestic animals might be susceptible to sars-cov-2, both surveillance and experimental infection should be conducted. should it be a bat, a pangolin or another mammal, it is expected that sars-cov-2 or its parental viruses that are almost identical will be identified in its natural hosts in future. continued investigations in this area will elucidate the evolutionary pathway of sars-cov-2 in animals, with important implications in the prevention and control of covid-19 in humans. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus genetic recombination, and pathogenesis of coronaviruses the molecular biology of coronaviruses a molecular arms race between host innate antiviral response and emerging human coronaviruses genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan interspecies transmission and emergence of novel viruses: lessons from bats and birds bat origin of human coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat origin origin and evolution of pathogenic coronaviruses studies with human coronaviruses. ii. some properties of strains 229e and oc43 clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia virus isolations from common colds occurring in a residential school new respiratory virus severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia from sars to mers: 10 years of research on highly pathogenic human coronaviruses from sars to mers: evidence and speculation the first disease x is caused by a highly transmissible acute respiratory syndrome coronavirus bat origin of a new human coronavirus: there and back again. sci china life sci cultivation of the virus of infectious bronchitis of chickens in embryonated chicken eggs a murine virus (jhm) causing disseminated encephalomyelitis with extensive destruction of myelin transmissible gastroenteritis in pigs electron microscopy of coronavirus-like particles characteristic of turkey bluecomb disease characterization of a feline infectious peritonitis virus isolate recovery and characterization of a coronavirus from military dogs with diarrhea a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease signs and symptoms in common colds effects of a "new" human respiratory virus in volunteers from sars coronavirus to novel animal and human coronaviruses identification of a new human coronavirus understanding human coronavirus hcov-nl63 a previously undescribed coronavirus associated with respiratory disease in humans croup is associated with the novel coronavirus nl63 characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus hku1 and other coronavirus infections in hong kong discovery of a subgenotype of human coronavirus nl63 associated with severe lower respiratory tract infection in china emergence of the middle east respiratory syndrome coronavirus clinical features of patients infected with 2019 novel coronavirus in wuhan detection of human coronaviruses in children with acute gastroenteritis prevalence of igg antibody to sars-associated coronavirus in animal traders isolation and characterization of viruses related to the sars coronavirus from animals in southern china identification of a novel coronavirus in bats antibodies to sars coronavirus in civets severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe receptor usage of a novel bat lineage c betacoronavirus reveals evolution of middle east respiratory syndrome-related coronavirus spike proteins for human dipeptidyl peptidase 4 binding discovery of novel bat coronaviruses in south china that use the same receptor as middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease isolation of mers coronavirus from a dromedary camel bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (mers-cov) after experimental infection( concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers) identification of 2019-ncov related coronaviruses in malayan pangolins in southern china viral metagenomics revealed sendai virus and coronavirus infection of malayan pangolins (manis javanica) evidence of the recombinant origin of a bat severe acute respiratory syndrome (sars)-like coronavirus and its implications on the direct ancestor of sars coronavirus metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat evidence supporting a zoonotic origin of human coronavirus strain nl63 distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats evidence for an ancestral association of human coronavirus 229e with bats surveillance of bat coronaviruses in kenya identifies relatives of human coronaviruses nl63 and 229e and their recombination history link of a ubiquitous human coronavirus to dromedary camels complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia coronavirus diversity, phylogeny and interspecies jumping why are rna virus mutation rates so damn high? moderate mutation rate in the sars coronavirus genome and its implications molecular evolution of human coronavirus genomes sars-cov and emergent coronaviruses: viral determinants of interspecies transmission recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission cryo-em structure of the 2019-ncov spike in the prefusion conformation human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry molecular evolution of the sars coronavirus during the course of the sars epidemic in china spread, circulation, and evolution of the middle east respiratory syndrome coronavirus human coronavirus 229e encodes a single orf4 protein between the spike and the envelope genes identification and characterization of a novel alpaca respiratory coronavirus most closely related to the human coronavirus 229e bats: important reservoir hosts of emerging viruses comparative analysis of bat genomes provides insight into the evolution of flight and immunity the egyptian rousette genome reveals unexpected features of jumping species-a mechanism for coronavirus persistence and survival dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host we thank pearl chan, hinson cheung, terence zwy and dyj wrote the manuscript with inputs from sy, ksy, syf and cpc. all authors read and approved the final version of the manuscript. the authors have declared that no competing interest exists. key: cord-342756-rgm9ffpk authors: senger, mario roberto; evangelista, tereza cristina santos; dantas, rafael ferreira; santana, marcos vinicius da silva; gonçalves, luiz carlos saramago; de souza neto, lauro ribeiro; ferreira, sabrina baptista; silva-junior, floriano paes title: covid-19: molecular targets, drug repurposing and new avenues for drug discovery date: 2020-10-02 journal: mem inst oswaldo cruz doi: 10.1590/0074-02760200254 sha: doc_id: 342756 cord_uid: rgm9ffpk coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a highly contagious infection that may break the healthcare system of several countries. here, we aimed at presenting a critical view of ongoing drug repurposing efforts for covid-19 as well as discussing opportunities for development of new treatments based on current knowledge of the mechanism of infection and potential targets within. finally, we also discuss patent protection issues, cost effectiveness and scalability of synthetic routes for some of the most studied repurposing candidates since these are key aspects to meet global demand for covid-19 treatment. at the moment, the prevention aimed at reducing transmission in the community is the best alternative, indicating that enhanced public health interventions, including social distancing, use of masks and movement restrictions should be implemented to bring the covid-19 pandemic under control. aggressive isolation measures including travel restriction in china, have successfully led to a progressive reduction of covid-19 cases. (8) kissler and colleagues, simulated the relaxation of the protective measures using a post-pandemic mathematical model. (9) the authors demonstrated that social isolation will be necessary at the best scenario until 2021, and reaffirmed the need and importance of maintaining social isolation. they also suggested that in the absence of such restrictions, the pandemic could last until 2024. thus, in this context, the search for new medicines available to combat this disease is urgent. coronaviruses are members of the family coronaviridae that present crown-like spikes on their surface visualised by electron microscopy. the subfamily coronavirinae contains the four genera alpha, beta, gamma, and deltacoronavirus. coronaviruses infect birds (gamma and deltacoronaviruses) and several mammalian species (mainly alpha and betacoronaviruses), including humans. (10, 11) coronaviruses have been isolated from diverse species, including mammals like bats, rodents, bovines, swine, felines, pangolins, horses and others. (12, 13) human coronavirus was first identified in 1966 by tyrrell and bynoe. (14) nowadays the seven coronaviruses that can infect humans (hcovs) are classified in alpha coronavirus (229e, nl63) or beta coronavirus [oc43, hku1, middle east respiratory syndrome coronavirus (mers-cov), severe acute respiratory syndrome coronavirus (sars-cov) and more recently the sars-cov-2]. 229e, nl63, oc43, and hku1 can cause upper and lower respiratory tract infection in adults and children. after the 2000s, two epidemic covs have arisen in humans: the sars-cov and the mers-cov which were reported in 2003 and 2012, respectively. (15, 16) in 2019, after the first report of novel pneumonia in wuhan, china, the seventh hcov was described. it also causes severe acute respiratory syndrome (therefore called sars-cov-2) and spreads very quickly worldwide . coronavirus are single-stranded positive-sense rna viruses and their genome size is approximately 30 kb, which encodes some important structural proteins. (17) the spike (s) glycoprotein is a well characterised protein that mediates coronavirus entry into host cells via fusion of the viral and cellular membranes through a pre to post fusion conformation transition. (18) the s protein s1-s2 subunits bind to cellular receptors that vary according to the coronavirus species: angiotensin-converting enzyme 2 (ace2) in sars-cov, sars-cov-2 and hcov-nl63; and dipeptidyl peptidase 4 (dpp4) and aminopeptidase n (apn) in mers or others alphacoronaviruses like tgev (porcine transmissible gastroenteritis coronavirus) and porcine respiratory coronavirus (prch). (18, 19, 20) other structural proteins are mandatory to assemble the complete viral particle like nucleocapsid protein (n), membrane protein (m) and the envelope protein (e). furthermore, they can be involved in other processes like morphogenesis, envelope formation, budding or pathogenesis. (17, 21, 22) by genomic sequencing analysis of other coronavirus strains and sars-cov-2, andersen and collaborators demonstrated that sars-cov-2 has mutations resulting in six different amino acids at the receptor-binding domain (rbd) that appears to be optimised for binding to the human receptor ace2. (23) they also showed that the gene encoding the spike protein has an insertion of 12 nucleotides giving it a polybasic (furin) cleavage site at the s1-s2. in this way, the high-affinity of the sars-cov-2 spike protein to the human ace2 is a consequence of natural selection on a human or human-like ace2. they suggest some possibilities to explain that: emergence in an animal host before zoonotic transfer; natural selection in humans following zoonotic transfer; or natural selection during the passage. (23) other researchers did a phylogenetic analysis of 160 genomes of sars-cov-2. (24) they showed 3 important variations in the composition of amino acids that allowed them to classify into different groups. group a has two subclusters that are distinguished by the synonymous mutation t29095c. while b is derived from a by two mutations t8782c and c28144t, type c differs from its parent type b by mutation of g26144t. the a and c types are found more often outside east asia, in europeans and americans. while the b type is the most common type in east asia. (24) in december 2019, covid-19 was initially reported as a new viral pneumonia, due to the clinical characteristics of the large number of cases that emerged in wuhan, china. (25, 26) sars-cov-2 typically causes respiratory sickness, the major clinical characteristics ob-served in infected patients are high fever, dry cough and dyspnea (shortness of breath or difficulty in breathing). minor symptoms include headache, diarrhea, nausea, vomiting, loss of smell and taste. (27) this clinical condition can progress to moderate or severe pneumonia. (28) in this case, first there is an accumulation of macrophages in alveoli, followed by release of cytokines and accumulation of fluids. neutrophils can also be recruited by the immune system leading to the destruction of type i and type ii alveolar epithelial cells causing a collapse of the alveoli function and consequently the acute respiratory distress syndrome (ards). in the severe condition, with an increase of the inflammation, the protein rich fluid from lungs enter in the bloodstream causing the systemic inflammatory syndrome (sirs). (29, 30) these complicating factors can lead to a multi-organ failure and septic shock, causing patient death. furthermore, some pre-existing conditions can enhance the risk to develop the severe form of the disease, including age over 60 years and a history of chronic diseases like chronic lung disease, asthma, heart diseases, immunosuppressed patients, cancer, diabetes or chronic kidney disease. (31, 32, 33, 34, 35) finally, the vast majority of people have mild symptoms or are asymptomatic, which is a big problem because they can also transmit the virus to the non-infected population. (36) drug repositioning, repurposing, reprofiling or retasking is the evaluation of existing drugs for new therapeutic purposes. (37) a candidate drug (investigational or approved) for repurposing efforts already has a known safety and toxicity profile, based on at least successful phase i or phase ii clinical trials. (38) considering the whole process, costs of bringing a repurposed drug to the market have been estimated to be ten times lower and the time is shortened by around a half, compared with a new drug. (38) even though the clinical phase iii and regulatory aspects remain similar for developing a new drug, drug repurposing possesses many advantages over developing a new drug from scratch: the reduced time and financial investment for development, the lower risk of failure and a consolidated pharmaceutical supply chain for production and distribution to the patients that effectively need treatment. (39) emerging or reemerging viruses pose major public health concerns globally. (40) for several pathogenic viruses, considerable efforts have focused on vaccine development and other therapies, like transfusion of convalescent plasma. (41, 42) however, during pandemics infected individuals need urgently to be treated on a large scale. a medicine armamentarium for the covid-19 outbreak is needed immediately and drug repurposing could be one of the best strategies to deal with this pandemic. (43, 44) computational and experimental approaches can be used, alone or combined, to achieve a more holistic point of view and increased chance of success in drug repurposing. in the following topic, we will review sars-cov-2 structure and mechanism of infection in order to discuss molecular targets from the virus or its human host that are being considered for drug repurposing and perhaps future development of new drugs. ongoing drug repurposing efforts will be described in more details later in this article, along with some clinical trials that have been carried out so far for covid-19 treatment. finally, as treatment availability is of utmost importance when dealing with a pandemic, we bring a discussion on patent protection and ease of large-scale production of some of the drugs that are more advanced in clinical studies. sars-cov-2: structure, mechanism of infection and drug targets sars-cov-2 structure -electron microscopy imaging of sars-cov-2 virions indicates that they have a spherical or pleomorphic shape, with diameters ranging from 60 to 140 nm, showing prominent spikes of 9-12 nm in their surfaces that resemble a solar corona, hence the name "coronavirus". (45, 46, 47, 48) sars-cov-2 is an enveloped virus with a single-stranded positive sense (5'-3') rna (+ssrna) (~ 30 kb) containing a 5'cap structure and a 3'-poly-a tail. (49, 50) its genomic rna (grna) has a variable number of open reading frames (orfs) that are predicted to encode 16 non-structural (nsp), 4 structural and several accessory proteins ( fig. 1 ). (26, 51, 52, 53, 54) orf1a and orf1b represent more than 2/3 of the whole length of grna, and encode two polyproteins: pp1a (440-500 kda) and pp1ab (740-810 kda). (53, 55) the polyprotein pp1a is translated from orf1a while pp1ab from orf1a/orf1b using a -1 ribosomal frameshift mechanism that occurs near the 3' end of orf1a which allows continued translation of orf1b. (53) together, pp1a and pp1ab originate all nsps (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) , such as m pro (nsp5) protease and rdrp (nsp12) rna polymerase, which form viral replicase/transcriptase complexes (rtcs), and are encapsulated in double-layered vesicles originated from the endoplasmic reticulum (er). (56, 57, 58) the orfs near the 3' end of the grna encode the structural and accessory proteins of sars-covs. (58) the first ones have a crucial role in the assembly of viral particles and virus invasion. (56, 58) the main structural proteins are named: spike (s), envelope (e), nucleocapsid (n) and membrane (m) proteins. most of them reside on the virion surface (s, e, m proteins) while n proteins are found in the core of the particle bound to grna. (59) s proteins are essential for virus attachment and entry into the host cells, tissue tropism and pathogenesis. (58, 60) e proteins exert several roles in virus infection, such as helping in virus assembly and release from infected cells, creating ion channels in cell membranes and suppressing host stress response. (58, 61, 62) n proteins interact with grna to form the ribonucleoprotein. (56, 62) m proteins have a role in virion assembly and in determining the shape of the envelope. they also bind to all other structural proteins promoting, for instance, the stabilisation of n protein-rna complexes. (56, 63) sars-cov-2 mechanism of infection -at present, the mechanisms that underlie sars-cov-2 infection have not been directly described. nonetheless, they seem to be similar to those proposed for other coronaviruses. (58) in one proposal, virus infection starts with the binding of its s proteins to host receptor ace2, a membrane protein largely expressed in the lung and small intestine cells (fig. 2 ). (44, 59, 64) after attachment, s protein is cleaved by host proteases initiating the fusion of virus and cell membranes that culminates in viral grna release into the cytoplasm. this event is proposed to occur through two distinct ways: via plasma membrane (early pathway) or via endosomes (late pathway). in the early pathway, s protein is cleaved by host plasma membrane proteases (e. g., tmprss2) while in the late pathway by endosomal proteases (e. g., cathepsin l). the route taken by the virus to enter the cell appears to be dependent on the availability of these proteases. (59, 64, 65) once in the cytoplasm, grna is readily translated into viral polyproteins (pp1a/pp1ab), which are cleaved into the individual nsps that compose the rtcs (fig. 2) . these complexes recognise transcriptional regulator sequences in grna and begin to transcribe a series of subgenomic rnas that encode structural and accessory proteins, otherwise the whole grna is replicated. (57, 58, 59) upon translation, s, e and m structural proteins are driven to the er-golgi intermediate compartment (ergic) where s proteins go through post-translational modifications (e. g., proteolysis, n-glycosylation). in parallel, a copy of the grna and n proteins bind in the cytoplasm to form the nucleocapsid and move into the ergic. in this compartment, nucleocapsid and the other (1) virus infection initiates with the binding of virus s proteins to the ace2 cellular receptors. after attachment, the virus may enter the cell through two distinct mechanisms: early and late pathways. (2) in the early pathway the genomic ssrna (grna) is liberated into the cytoplasm after the fusion between viral and cell cytoplasmic membranes, an event triggered by membrane proteases (e. g., tmprss2). (3) the grna is immediately translated into two polyproteins that undergo proteolytic cleavage giving rise to all nonstructural proteins (nsps). (4) the nsps form the replication-transcription complexes (rtcs) where the grna (blue ribbon) is replicated and the subgenomic rnas (red ribbon) are transcribed. (5) the subgenomic rnas are translated into viral structural and accessory proteins in the cytosol. (6) upon translation, e, m and s structural proteins are inserted into er and follow the secretory pathway to the er-golgi intermediate compartment (ergic). (7) meanwhile, a copy of the grna binds to n proteins in the cytoplasm forming the nucleocapsid, which is transported to the ergic. (8) virion assembly in the ergic. (9) the new virion travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. (10) alternatively, in the late pathway, the virus can undergo endocytosis to initiate the infection. (11) the virion membrane merges with the endosome membrane after s protein proteolysis by endosomal proteases (e. g., cathepsin l), allowing the grna to be released into the cytoplasm. from this point on, the cycle follows the same pathway described in 3-9 steps. the red arrows indicate the general replication pathway and the blue arrows indicate the grna movement through the cycle. some viral and host proteins have been explored as potential targets for drug repurposing. some of these drug candidates are shown with their site of action indicated in the cycle. (44, 59, 66, 67) viral proteins are assembled into a virion which travels through the cytoplasm inside a vesicle and leaves the cell by exocytosis. (48, 56, 59) candidate drug targets -in the search for a treatment for covid-19, several viral and host molecular proteins have been explored as potential drug targets. overall, they participate in key events of the virus infection cycle, such as cell entry and replication, as well in host metabolic pathways and immune response. in the following topics we will address in more details some of these targets. drugs under investigation for blocking the main steps of the sars-cov-2 virus replication cycle are indicated in fig. 2 . virus targets -during sars-cov-2 grna translation, two proteases, namely m pro and pl pro , act in concert to cleave and release from pp1a/pp1ab the 16 nsps that compose the rtc. (58) therefore, these proteases are essential for virus replication and represent useful targets for therapeutic intervention. recently, sars-cov-2 m pro and pl pro had their 3d structures published -pdb 6lu7, 2.16 å and pdb 6w9c, 2.70 å, respectivelywhich make them particularly useful for computational structure-based drug design methods. (68) 3-chymotrypsin-like protein (synonyms: coronavirus main protease, m pro , 3cl pro ) of sars-cov-2 is a 33.8 kda homodimeric protein (306 aa) that belongs to the cysteine protease class (possibly from c30 family and pa clan). (68, 69) this enzyme catalyses the hydrolysis of peptide bonds of polyproteins (possibly e.c. 3.4.22.69) at sites whose amino acid sequences generally follow the pattern leu-gln* (ser, ala, gly) (*marks the cleavage site). (70, 71) m pro is part of pp1a/pp1ab polyproteins (nsp5). (57) during polyproteins translation, m pro suffers autolyt-ic cleavage and is released from its polyprotein precursor, reaching a mature state that cleaves pp1a/pp1ab at no less than 11 sites downstream of the nsp4 coding region. (57, 68, 70, 72) each protomer of sars-cov-2 m pro is divided into three domains: chymotrypsin and picornavirus 3c protease-like i and ii domains, composed by antiparallel β-barrel structures, and domain iii which contains five α-helices arranged into an antiparallel globular cluster responsible for protease dimerisation (fig. 3a) . domains ii and iii are connected by a long loop, where lies a cleft that serves as a substrate binding site and where catalysis occurs using the cys 145 -his 41 dyad (fig. 3b ). (68, 70, 73) m pro has been widely explored in drug discovery campaigns using experimental and/or computational approaches. (68, 73, 74, 75, 76, 77) moreover, m pro has no human homologue, which reduces the chances of toxic effects of a given inhibitor. (68) potential sars-cov-2 m pro inhibitors include fda-approved antivirals, such as inhibitors of hiv-1 [e. g., lopinavir (1) /ritonavir (2) ] and hcv [e.g., boceprevir (3)] proteases, as well as antineoplastic [e.g. carmofur (4)] and antibacterial [e.g., doxycycline (5)] drugs. (73, 78, 79, 80, 81, 82) chemical structures for compounds 1-14 are shown in fig. 4 . nsp3 is the largest protein encoded by covs (~200 kda). in sars-covs it has 16 domains which include a papain-like proteolytic enzyme from the cysteine protease class (c16 family and ca clan for sars-cov). (57, 69, 83) pl pro is composed of a catalytic domain, an extended right-handed thumb-palm-finger structure with a cys-his-asp catalytic triad, and a ubiquitin-like domain (ubl) (fig. 5a ). the catalytic cys is located in the thumb subdomain while his and asp in the palm subdomain (fig. 5b ). pl pro catalyses the hydrolysis reaction of peptide bonds of pp1a/pp1ab at three sites (nsp1/nsp2, nsp2/nsp3 and nsp3/nsp4) that share the xlxgg* pattern (*represents the cleavage site). (57, 71, 83, 84) this activity is responsible for nsp3 release from polyproteins. pl pro also recognises and hydrolyses ubiquitin and isg15 from cellular proteins. (83, 84) the deubiquitination and deisgylation activities are proposed to modulate the post-translational modifications of signaling molecules that trigger innate immune response of the host. (85) these functions are pivotal for virus infection justifying the search for sars-covs pl pro inhibitors. (84, 86, 87, 88, 89, 90) putative inhibitors of sars-cov-2 pl pro include fda-approved drugs such as fostamatinib disodium (6) (a tyrosine kinase inhibitor used in the treatment of chronic immune thrombocytopenia) and natural products [e.g., platycodin d (7)]. (89, 91) rna-dependent rna polymerase (rdrp, nsp12) is an essential protein responsible for rna synthesis during viral rna transcription and replication cycles. (89) as described for sars-cov, the rna polymerase activity of sar-cov-2 rdrp (804 aa) also seems to require the binding of nsp7 and nsp8 cofactors to enhance rdrp binding and processivity. (92, 93) the overall rdrp structure has a "right hand" rdrp domain, composed by three subdomains (palm, fingers and thumb), and a nidovirus-unique n-terminal extension domain that forms a nidovirus rdrp-associated nucleotidyltransferase (niran) structure. these domains are connected by an interface domain. additionally, this protein also has a n-terminal β-hairpin (fig. 6a) . the active site of rdrp contains conserved polymerase motifs located in the palm subdomain and its configuration is similar to other rna polymerases. (94) rdrp substrates, rna template/primer and nucleotide triphosphate (ntps), access the catalytic centre through the template and ntp entry paths, respectively, while the product-template hybrid is released through the rna exit path. (92, 94) sars-cov-2 rdrp shares 96% identity in amino acid sequence with sars-cov protein. (93) moreover, the accessory proteins also have a high degree of amino acid sequence identity between these viruses: 98.1 % for nsp7 and 97.5 % for nsp8 -sequences obtained from pdb 7bv2 (2.5 å) and pdb 6nur (3.1 å) entries. (92, 95) therefore, it is reasonable to suggest that sars-cov rdrp inhibitors may also bind to the homologous enzyme from sars-cov-2, as demonstrated for remdesivir (8) (fig. 6b) , an adenosine triphosphate analog. (92, 93, 96) other potential inhibitors include clinically available drugs, such as favipiravir (9), a purine nucleic acid analog used in the treatment of influenza, and ribavirin (10), a synthetic guanosine nucleoside indicated for the treatment of hepatitis c virus (hcv) infection. (97, 98) the crucial role of rdrp and the lack of a host homolog turns this enzyme into a valuable target for anti-covs agents. (93) helicase (nsp13) is a multifunctional protein (predicted to have 596 aa in sars-cov-2) essential for sars-covs rna replication and proliferation. (74, 99) sars-cov helicase belongs to helicase superfamily 1 (sf1) and can unwind double-stranded dnas/rnas (helicase activity) in the 5'-3' direction, an energyconsuming process that is driven by the hydrolysis of nucleosides triphosphate (ntpase activity). (57, 100, 101) its structure contains five domains arranged in a triangular pyramid-shape: the triangular base is composed by two "reca-like" (1a and 2a) and 1b domains whereas the zinc binding domain (zbd), in the n-terminal, and the stalk domain are oriented towards the apex. the zbd and 1b domains are connected by the stalk domain ( fig. 7) . (101) the same domain arrangement is predicted to occur in sars-cov-2 enzyme. (74) sars-cov helicase has been explored as a potential target for drugs. (99, 100, 102) recently, some efforts have also been made to predict inhibitors for sars-cov-2 helicase. (74, 103, 104, 105) they include drugs used in the clinics to treat acquired immunodeficiency syndrome (aids), such as vapreotide (11) (somatostatin analog) and atazanavir (12) (hiv protease inhibitor), as well anti-hcv protease inhibitors [e.g., daclatasvir (13) ] and bismuth salts [e.g., bismuth potassium citrate (14) , a compound used in clinical treatment of gastrointestinal diseases]. (103, 104, 106) sars-covs replication also requires other enzymatic activities including (guanine-n7)-methyltransferase (n7-mtase), 2'-o-methyltransferase (2'-omtase) and exoribonuclease (exon). (57, 107, 108) the n7-mtase domain at the c-terminus of nsp14 is responsible for adding a methyl group in the n7 position of rna 5'-guanosine forming the 5'-cap structure of viral rnas. (108) additionally, nsp14 also has a catalytic domain in its nterminus with 3'-5' exon activity, which participates in rna proofreading mechanism (fig. 8 ). (57) 2'-omtase (nsp16) catalyses the methylation reaction at the ribose 2'-o position of the first and second nucleotide of the mrnas and it is one of the sars-cov-2 proteins whose 3d structure has already been resolved (pdb: 6w61, 2.0 å) (fig. 9 ). (109) both nsp14 and 16 use nsp10 as a cofactor to enhance their 3'-5' exon and 2'-omtase activities, respectively. (110) together, these proteins are responsible for inducing rna modifications that are essential for its stability and translation as well to avoid the activation of host immune response. (57, 107, 109) thus, nsp14 and 16 have been considered as potential targets for anti-sars agents that may affect their functions in a direct or indirect way (e.g., by blocking nsp 10 binding). (108,109,110,111,112 ,113,114,115) some compounds have been regarded as potential inhibitors of sars-cov-2 methyl transferases, such as adenine dinucleoside s-adenosylmethionine analogs [e.g., dinucleoside 13 (15) ], predicted for sars-cov n7-mtase activity, and some clinically available drugs [e.g., raltegravir (16) -a hiv integrase inhibitor], predicted for sars-cov-2 2'-omtase activity. (112, 115) chemical structures for compounds 15-29 are shown in fig. 10 . spike protein (s) is a viral type i transmembrane glycoprotein (~180-200 kda) responsible for covs interaction and invasion of host cells. (59, 64) this protein is synthesised as a monomer and suffers post-translational modifications in the er before becoming a trimeric glycosylated protein. (59, 116) its structure is divided into three main domains: an extracellular, a transmembrane and a short intracellular domain (fig. 11 ). (117) the former protrudes from the surface of the virus particle creating a crown-like halo and contains two functional subunits: s1 (bulbous shape), which binds to cellular receptors (e.g., ace2), and s2 (stalk shape) that promotes the fusion between cell and virus membranes. (59) in turn, s1 subunit has two domains: a n-terminal and a c-terminal domain. the latter serves as a rbd for sars-covs being responsible for ace2 recognition and binding. (59, 64, 117) apart from s1 and s2, sars-cov-2 s protein also has a ganglioside-binding subdomain at the tip of n-terminal domain, which may allow this protein to interact with gangliosides on cells' surface. in theory, this domain could facilitate virus attachment to the cell and facilitate the contact with ace2, being a potential site for drug interference. (118) fig. 9: ribbon representation and vdw surface of the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 2'-o-methyltransferase 3d structure (pdb 6w61, 2.0 å, nsp16, pink) in complex with its nsp10 cofactor (light blue). the zinc and chloride ions are represented as gray and green spheres, respectively. image created using maestro, release 2020-1 (maestro, schrödinger, llc, new york, ny, 2020). covs s proteins require proteolytic priming or cleavage to become fusion competent. (59) this is achieved by host proteases (e.g., tmprss2, cathepsin l, furins) which cleave the s2 subunit of s protein at two main positions: s1/s2 interface and s2'. the latter is located immediately upstream of the fusion peptide (fp), the fusion functional element of s2. contrary to sars-cov, sars-cov-2 s2 subunit also has an additional cleavage site for furin proteases in the s1/s2 region, something that also occurs in mers-cov. (60) it is still unclear its role in sars-cov-2 infection, but it is speculated that the ubiquitous expression of furins may increase cell and tissue tropism of sars-cov-2 in comparison to sars-cov, as well altering its transmissibility and pathogenicity. (60, 117) taken together, host proteases represent potential targets for anti-sars-cov-2 drugs, and thus some of them will be discussed in the following topics. (60, 64, 119) covs e proteins are small integral membrane polypeptides (76 -109 aa) encoded by subgenomic rnas. in sars-cov, they are found in virions, but also in large amounts in the ergic where they participate in virus budding and trafficking. their structures are divided into three main domains: n-terminal, transmembrane (tmd) and c-terminal domain. tmd is able to form pentameric α-helical bundles creating ion conductive pores in membranes. (120, 121, 122) the ion channel (ic) activity of e protein has been proposed to alter ion homeostasis, as well induce inflammatory response, which may lead to pulmonary damage. (123, 124) thus, the use of inhibitors of ic activity may represent a possible therapeutic strategy for covs-related diseases, including covid-19. ( this can be exemplified by the fda-approved drugs gliclazide (17), a sulfonylurea administered to non-insulin-dependent diabetes mellitus patients, and memantine (18) , an n-methyl-d-aspartate receptor antagonist used in the management of alzheimer's disease, which have shown ic inhibitory activity in bacteria expressing sars-cov-2 e proteins. (126) host cell targets -a key step in sars-covs infection is the attachment of s protein to host angiotensinconverting enzyme 2 (ace2), a membrane-bound carboxypeptidase (family: m2, clan: ma). (25, 65, 69) this enzyme catalyses the hydrolysis of angiotensin i and angiotensin ii into angiotensin-(1-9) and angiotensin-(1-7) peptides, respectively. (127) ace2 is expressed in the upper respiratory system, type i and ii alveolar epithelial cells in the lungs, heart, endothelial cells, kidney tubular epithelium and other tissues. (128) its role as a functional receptor of sars-cov-2 s protein in host cells makes this protein a potential drug target to treat covid-19. there are several therapeutic strategies targeting ace2 which include: vaccines based on s protein, exogenous administration of a soluble form of ace2, and administration of small molecules [e.g., arbidol (19) , an anti-influenza drug] or antibodies (e.g., sti-1499, an anti-spike antibody) to block the interaction of ace2 with s protein. (66, 129, 130) additionally, compounds could also have an anti-sars-covs activity by disturbing ace2 glycosylation, as proposed for chloroquine (20) . (131) renin-angiotensin-aldosterone system (raas) inhibitors, such as ace inhibitors [ace-i, e.g., captopril (21) ] and angiotensin receptor blockers [arb, e.g., losartan (22) ], are commonly used in clinics to treat hypertension and cardiovascular/renal diseases. (132, 133) recently, some researchers have debated over the administration of these drugs to patients with known or suspected covid-19. (133, 134, 135, 136) the main reason for this discussion is that raas inhibitors may induce ace2 expression, which, in theory, could increase the severity of the infection. (133, 136) nonetheless, evidence suggests that these drugs may protect patients from lung injury by suppressing angiotensin ii signaling mediated by angiotensin receptor 1 (at1r). (135, 136, 137) further investigation is still required to determine whether ace-i and arb have a beneficial or deleterious role in covid-19 treatment. (134) in order to become fusion-competent, sar-covs s proteins must be cleaved by host proteases. in sars-cov-2, this process appears to be mediated primarily by tmprss2 in the plasma membrane. (65) tmprss2 (transmembrane protease, serine 2) is a transmembrane protein (predicted to have 492 aa) from the serine protease class (family: s1 clan: pa). (69, 138) it is highly expressed in the epithelial cells of the prostate, and relatively less in lungs, colon, liver, kidney, and pancreas. its physiological function is still unclear. (138) tmprss2 has a major role in sars-cov-2 cell entry and replication, and thus represents an interesting therapeutic target since its inhibitors could potentially block virus infection in its initial stages. (65, 138) potential tmprss2 blockers include some serine protease inhibitors [e.g., camostat mesylate (23)], commercially available compounds [e. g., zinc64606047 (24), 3-[[5-(3-fluorophenyl)pyrimidin-2-yl]amino~(n)-(4-methyl phenyl)benzamide]] and natural products [e.g., withanone (25) ]. (65, 139, 140, 141) in the absence of exogenous or membrane-bound proteases (e g. tmprss2) to promote sars-covs infection on cells' surface, the virus can also be internalised via endocytosis, also known as "late pathway". (59) this process comprises several factors that operate in a sequential and partially overlapping fashion. (142) endocytosis initiates with the recruitment of endocytic coat proteins (e. g., clathrins) from the cytosol to gather on the inner leaflet of the plasma membrane. then, protein-coated pits bud into vesicles originated from plasma membranes and fuse with each other or with pre-existing early endosomes. (142, 143) gradually, early endosomes mature to late endosomes which have an acid environment (approx. ph 5.5), activating endosomal cathepsin l cysteine proteases (family: c1, clan: ca). (59, 69, 143) these enzymes are responsible for triggering the fusion activity of s protein and subsequent insertion of viral sars-cov grna into the cytosol, especially in cells with lower expression of tmprss2. (64, 65) some key elements of endocytosis have been explored as potential targets for anti-sars-cov drugs, such as agents that increase endosomal ph (avoiding cathepsin l activation) [e.g., chloroquine (20) and hydroxychloroquine (26) ] and cathepsin l inhibitors [e.g., teicoplanin (27) , a glycopeptide antibiotic]. (49, 131, 144) cytokines are short-lived small size proteins (15-20 kda) that exert an important role in autocrine, paracrine, and endocrine signaling controlling the development and function of several immune and nonimmune cells. (145, 146) they are classified in families based on certain properties of their receptor complexes, such as their structure, specificity and composition. (145) one example is interleukin (il)-6 family which includes several cytokines, such as il-6, that use the common signaling receptor subunit glycoprotein 130 kda (gp130). il-6 signaling requires the binding of il-6 to il-6 receptor (il-6r) which consists of a soluble il-6 binding domain (cd126) and membrane-bound gp130. il-6 acts as the main inducer of fever, inflammation and of hepatic acute phase proteins. therefore, il-6/il-6r antagonists have a therapeutic effect in inflammatory diseases. (145, 146) their use in covid-19 treatment may be beneficial to avoid the "cytokines storm" syndrome that some patients with the severe form of the disease may develop. (147) this deleterious event is caused by an amplified immune response and cytokine release that can damage the organs, including the lungs. (66) tocilizumab and sarilumab are two examples of humanised monoclonal antibodies that act as il-6r antagonists which are currently under clinical trials for covid-19 treatment. (148) other targets -apart from sars-covs infection, some relevant molecular targets of other viral diseases and host metabolism have also been investigated in covid-19 drug discovery, such as viral neuraminidases and the dpp4 cell receptor. neuraminidases (na) or sialidases, are glycoside hydrolases (family: gh34) largely found attached to the envelope of influenza viruses. (149, 150) they catalyse the hydrolysis of glycosidic bonds between sialic acid and adjacent sugar residues (ec 3.2.1.18) in glycoproteins, glycolipids and oligosaccharides. (149) na is mainly responsible for cleaving sialic acid acids from cell receptors and on carbohydrate side chains of nascent virion, facilitating its release from infected cells. (150) its critical role in virus infection and proliferation has been exploited by na inhibitors [e.g., oseltamivir (28) ] which are administered in clinics to combat influenza infections. apparently, this therapeutic strategy was employed at the beginning of co-vid-19 outbreak during the peak of influenza season in china when the etiologic agent of this disease was yet unknown. so far, there is no evidence that na inhibitors may have a role in covid-19 management. (66) dipeptidyl peptidase-4 (ddp4), also known as adenosine deaminase complexing protein 2 or t-cell activation antigen cd26, is a type ii transmembrane glycoprotein of 766 aa largely expressed in many tissues (e.g. lungs and immune cells). (151, 152) ddp4 belongs to serine protease group (family: s9, clan: sc) and catalyses the hydrolysis of n-terminal dipeptide, xaa-yaa-|-zaa-, (preferentially when yaa is a proline, as long as zaa is neither proline or hydroxyproline), from a variety of peptide substrates (ec 3.4.14.5), such as chemokines, neuropeptides, vasoactive peptides and growth factors. (69, 71, 152) therefore, it participates in many physiologic and pathological processes, including glucose/insulin metabolism and immune/inflammatory response. (153) in addition, dpp4 acts as the functional cell receptor of mers-cov s protein, helping virus entry into the cell. theoretically, this role may also be played in sars-cov-2 infection, as predicted by computational analysis. (154) recently, there has been a discussion about the use of ddp4 inhibitors, such as sitagliptin (29) (anti-diabetic drug), in the treatment of covid-19. (152, 153, 155, 156) in principle, these inhibitors could be beneficial for type 2 diabetes patients (or even without diabetes) with covid-19 since it could potentially block virus cell entry/replication, as well suppress inflammatory response. (152, 155) however, there is not enough evidence yet to prove this hypothesis. (152, 153, 156) computational approaches -the sars-cov-2 pandemic is creating a fertile ground for computational approaches to identify viable therapeutic options in the short-term, with the number of published studies growing rapidly. (157, 158) classical target-and ligand-based strategies (e.g., molecular docking and similarity analysis), are being applied to fda-approved drugs and compounds in clinical trials to explore possible interactions with viral proteins. (75, 159, 160) artificial intelligence (ai) methods are also being extensively applied to large molecule databases to find existing drugs that could be repurposed based on previously reported antiviral activities. (103, 161, 162, 163) ai approaches can even explore hidden connections between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. (52, 161, 164) these studies emphasize how important computational methods are in modern drug discovery to analyse the huge amount of biomedical data available. table i summarises a selection of studies that suggested potential drugs for repurposing. below we will describe the main computational methods and results. smith and smith performed a large virtual high-throughput screening to identify drugs, natural products and metabolites that could disrupt the interaction between sars-cov-2 s protein and human ace2 receptor. the authors used a supercomputer called summit to conduct the simulations, which included structural modeling of the s protein:ace2 complex, generation of an ensemble of conformations for the complex and molecular docking. (160) the ensemble generation was a crucial step in this work, allowing the authors to explore different conformations of the complex and identify drugs that could interact with binding sites not easily identified in static conformations. using the gromacs molecular dynamics simulation suite, six different clusters of conformations were generated by restrained temperature replica-exchange molecular dynamics simulation (t-remd) and submitted to docking with autodock vina using the sweet-lead compound collection. (170, 171, 172) the authors explored two strategies, consisting of disrupting the s protein: ace2 complex and preventing its formation by docking on the isolated proteins. from this analysis, four compounds [pemirolast (30) , nitrofurantoin (31), isoniazid pyruvate (32) and eriodictyol (33) ] displayed potential to disrupt the s protein: ace2 interaction, and three natural compounds [cepharanthine (34) , ergoloid (35) and hypericin (36) ] showed potential to block host recognition. (160) chemical structures for compounds 30-49 are shown in fig. 12 . ge and coworkers constructed a virus-related knowledge graph (or network) by combining seven networks with information about target-drug and protein-protein interactions, molecule similarity and sequence similarity of human and viral sources to predict drugs targeting sars-cov-2. (164) well known biological and interaction databases were employed, such as drugbank, chembl, bindingdb, and uniprot . (173, 174, 175, 176) the final knowledge graph was assembled by merging the nodes and edges of the seven networks, where each node represent drugs or targets, while the edges between them are the identified relations, such as similarity (i.e., molecular and sequence similarities) and drug-target or target-target interactions. (164) a graph convolutional network (gcn) was used to learn and extract the hidden information on the nodes and edges of the knowledge-graph, allowing the authors to access novel drug-target and target-target interactions and find molecules that could be repurposed to sars-cov-2. (164) gcn's are powerful neural networks that can access the rich information within the nodes of a graph and return insights about possible relations (e.g., potential drugs and targets interactions), representing a valuable tool in the modern drug discovery scenario, where massive biological data are available in databases such as drugbank and chembl. (177, 178) the final knowledge-graph was then mined to gather an initial list of drugs, which was further refined by extracting previous evidence of antiviral activity from pubmed using a deep learning method called biomedical entity relation extraction (bere) and manual inspection. (179) in a subsequent step, the authors elaborated a final list of drug candidates using transcriptome analysis of ten sars-cov patients. the poly-adp-ribose polymerase 1 (parp1) inhibitor, mefuparib hydrochloride (cvl218) (37) , currently in phase i clinical trials, was identified as a potential drug for repurposing. in vitro and in vivo validation showed promising inhibition and safety profiles. concretely, compared to arbidol (19) , one of the standard treatments for covid-19 in china, cvl218 showed higher antiviral efficacy and higher concentration in lungs of rats. (164) the authors also found that cvl218 significantly inhibited the production of il-6 induced by the cpg oligodeoxynucleotide 1826 (cpg-odn1826) in peripheral blood mononuclear cells (pmbc) indicating it might be an alternative to treat the inflammation caused by sars-cov-2. furthermore, cvl218 showed favorable pharmacokinetics and toxicity profiles in rats and monkey models. (164) this integration of artificial intelligence with wet-lab experiments highlights the importance of in silico methods to mine baricitinib (39) rheumatoid arthritis human ap2-associated protein kinase 1 (aak1) knowledge-graph a pilot trial indicated that baricitinib (39) improved clinical parameters (e.g., cough and dyspnea) in a small group of patients. c (161) toremifene (40) (41) and mercaptopurine (42) ] and angiotensin receptor blockers [irbesartan (43) ]. d (52) a: cepharanthin (34) has been shown to decrease the expression levels of s-protein in mrc-5 cells infected with human coronavirus oc43 (hcov-oc43). (165) flavonoids have been described as inhibitors of m pro and ligands of the s-protein of sars-cov, which could give insights on possible antiviral activities of eriodictyol (33), hypericin (36) and other flavonoids on sars-cov-2. (166, 167) b: a recent study showed that atazanavir (12) inhibited m pro in vitro with greater potency than lopinavir (1). (168) c: despite the small size (12 patients) and open label, non-randomised format, baricitinib (39) showed potential safety when used in combination with lopinavir (1) / ritonavir (2). (169) as large amounts of data from databases and accelerate the discovery of potential drugs for the covid-19 pandemic. beck et al. used a natural language processing (nlp)-based approach to estimate the binding affinity of 3,410 fda-approved drugs against potential targets of sars-cov-2, including 3cl pro , s protein, rdrp, helicase, endornase, 3'-to-5'-exonuclease and 2'-o-ribose methyltransferase. (103) the premise of their approach is analogous to understanding text in different languages, for instance by learning the semantic relationships of words to execute a task, such as predicting the most probable word in a text or the sentiment expressed by it. (180, 181, 182) their model, called molecular transformerdrug target interaction (mt-dti) was trained on 1 billion smiles strings and the fasta sequence of target proteins, which bypass the need for 3d structures (e.g., x-ray) of protein-target complexes. (183) the pre-training approach allows the model to be used for other related tasks without the need to train from scratch, which is especially important when not enough data is available, which is the case for sars-cov-2 inhibitors. in addition, this approach is able to transfer more general features learned by the model, making it extremely flexible to deal with related tasks. (184, 185) therefore, the task consisted of training the model to understand the chemical structure of small compounds and protein targets. the authors found that atazanavir (12), remdesivir (8) and efavirenz (38) are potential inhibitors of m pro , while atazanavir (12) also yielded nanomolar predicted binding affinity for rdrp, helicase, endornase, 3'-to-5'-exonuclease and 2'-o-ribose methyltransferase. (103) it is important to highlight that although these drugs were predicted as nanomolar inhibitors, experimental confirmation is essential to validate the computational analysis. researchers at the uk-based company benevolen-tai (https://www.benevolent.com/) analysed medical data from different sources to create a knowledge graph of biomedical information, showing possible drug-target interactions on its nodes and edges (similar to the previously mentioned approach adopted by ge et al.) to explore new strategies for sars-cov-2. (161, 164) among the drugs identified by mining the hidden information within the graph, there were 378 inhibitors of the p2associated protein kinase 1 (aak1), a regulator of viral endocytosis in at2 alveolar epithelial cells. (161) inhibitors of aak1 could then potentially block viral entry into alveolar cells. however, the authors argued that only one of these drugs, baricitinib (39), a janus kinase inhibitor, has an acceptable safety profile. in addition, the low therapeutic dosing of baricitinib (39) (2mg or 4mg daily) makes it a promising drug for repurposing. (161) baricitinib (39) will be further discussed below. zhou et al. developed a network-based approach to identify potential repurposable drugs based on the interactions between their human targets and coronavirusassociated proteins. (52) the premise is that protein targets that are associated with viral infections are part of a subnetwork of the human protein-protein interaction network. the targets of a repurposable drug should be in or close in proximity to this subnetwork. the authors identified many potential targets, such as poly [adp-ribose] polymerase 1 (parp-1), glycogen synthase kinase 3 (gsk-3), and also biological pathways that could be explored, such as nf-kappa b signaling. (52) the most interesting drugs selected by the authors are from a diverse set, including toremifene (40) (selective estrogen receptor modulator), sirolimus (41) (immunosuppressant), mercaptopurine (42) (antineoplastic) and irbesartan (43) (antihypertensive). an interesting finding was that the selected molecules and targets have reported links to viral infection and some have been used for treatment of coronavirus-related disease, such as emodin (44) in sars-cov, and mercaptopurine (42) and toremifene (40) in sars-cov and mers-cov. (52) in addition to identifying repurposable drugs, possible synergistic combinations between them were also suggested by the network, such as sirolimus (41) and dactinomycin (45) , and mercaptopurine (42) and melatonin (46) . (52) in vitro and in vivo studies -genome and replication kinetics of sars-cov, mers-cov and sars-cov-2 viruses are very similar, suggesting that these diseases could be treated by a single drug that modulates the activity of a common target or mechanism among them. (52, 186) so far, no clinically available antiviral drugs have been developed for any of these three viruses. (187) this fact demonstrates that we did not learn a suitable strategy for treatment from these two prior diseases, and we are not yet prepared to deal with this new coronavirus epidemic, regarding therapeutic options. (188) experimental target validation (e.g., by genetic and proteomic approaches), cellular in vitro and in vivo animal models of sars-cov-2 infection, are pivotal for the discovery of molecular pathways involved in pathogenesis, supporting the discovery of new drugs. (189, 190) sars-cov-2 genome encodes less than 30 proteins that can be explored for generating new avenues for further research. heterologous viral protein expression could be used in a myriad of structural and functional targetbased biochemical studies. (191) these assays do not need to be performed in biosafety level 3 laboratory, democratising the early research on searching for a new drug candidate for covid-19 treatment. (192) genome-wide expression studies, nucleotide sequences, protein structures and associated sequences are available online providing a foundation for preclinical drug discovery and development research against sars-cov-2 (available from: https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/). it is good to point out that many drugs show in vitro effect (e.g., target and/or cellular assays) that may not last in vivo, in animal models, including humans. an ideal animal model of covid-19 should reflect the clinical signs, viral replication and pathology reported in humans. (193) in vivo animal infection experiments with sars-cov-2 require infrastructure of a biosafety level 3 laboratory, restricting scientific research to well-equipped research groups. animal models are indispensable, because they could identify toxic hits or molecules that could enhance covid-19 pathogenicity. table ii lists some compounds that have the potential to be clinically tested and that will be discussed in more details below in this topic. remdesivir (8) , a nucleotide analog pro-drug developed by gilead sciences inc. to fight ebola, acts on viral replication by inhibiting rna polymerase. (96) this molecule was also active against sars and mers viruses. (200, 201, 202) wang et al. demonstrated reduced viral copy numbers in cell culture supernatant of vero e6 cells infected with ncov-2019betacov/wuhan/wiv04/20192, in the presence of varying concentrations of remdesivir (8) . (194) this compound blocked virus infection at lowmicromolar concentration (ec 50 = 0.77 μm) and showed a high selectivity index (> 100). the authors also disclosed that remdesivir (8) inhibited virus infection efficiently in human liver cancer huh-7 cells. chloroquine (cq; 20) and its less toxic derivative, hydroxychloroquine (hcq; 26) , are used to treat malaria and lupus/rheumatoid arthritis, respectively. both drugs have already been described as possible antivirals. (195, 203, 204) cq (20) and derivatives probably act by decreasing acidity in endosomes and lysosomes, intervening on glycosylation of cellular virus receptors and modulating host immunological activity. (118, 205) studies in cultures of vero e6 cells have suggested that hcq (26) can affect the virus sars-cov-2 in both entry and post entry stages at host cells. (194) this molecule presented an ec 50 (196) the authors also performed in vitro physiologically based pharmacokinetic models for both drugs, separately. interestingly, when comparing toxicity in five animal models, mcchesney et al. demonstrated that hcq (26) is two to three times less toxic than cq (20) itself. (207) it is also good to point out that chloroquine (20) has shown in vitro activity against many different viruses, but no significant beneficial effect on animal models. (208) the association ritonavir (2)/lopinavir (1) is used together with other antiretrovirals for the treatment of human immunodeficiency virus since the beginning of the century. (209) ritonavir (2) is a potent cyp3a inhibitor therefore inhibiting the metabolism of lopinavir (1), increasing its plasma levels. both are reported as peptidomimetic molecules that inhibit hiv-1 protease activity in a competitive manner. (210) the m pro plays a major role in homeostasis of viral polyproteins essential for viral function and replication, being considered a validated drug target. (211) this combination may be useful against (194) chloroquine (20) (194, 195, 196) ritonavir (2) + lopinavir (1) lopinavir (1) nd (197) nitazoxanide (47) broad-spectrum antiparasitic and antiviral still needs confirmation 2.1 μm 16.8 (194) ivermectin (48) broad-spectrum antiparasitic still needs confirmation 2 μm nd (198) teicoplanin (27) antibiotic for gram-positive bacteria still needs confirmation 1.7 μm nd (199) sars-cov-2 virus by acting on its main protease, an enzyme essential in processing polyproteins translated from viral rna. (212) choy et al. demonstrated that it inhibits sars-cov-2 replication in vero e6 cells with ec 50 value of 26.6 μm. (197) these results corroborate with the study of de wilde et al. that demonstrated antiviral in vitro effect of lopinavir (1), but not ritonavir (2), against sars-cov, mers-cov, and hcov-229e, with mean ec 50 varying from 6.6 to 17.1 μm. (213) other compounds were also tested against sars-cov-2 in vitro. nitazoxanide (47), a broad-spectrum antiparasitic and antiviral, inhibits a low-micromolar range with an ec 50 of 2.1 μm but has a selective index of only 16.8. (194) ivermectin (48), another broad spectrum antiparasitic agent also demonstrated in vitro anti-sars-cov-2 activity. its ec 50 was determined to be 2 μm when added to vero-hslam cells 2 h post-infection and measuring viral rna at 48h post-infection. (198) teicoplanin (27) , an antibiotic used against gram-positive bacterial infections, was found to be active in vitro against sars-cov-2 with an ec 50 value of 1.7 μm, but in vivo efficacy still needs to be determined. (199) a robust preclinical drug discovery pipeline comprising in vitro, and in vivo models of sars-cov-2 infection is particularly important to identify new antivirals for human covid-19 treatment. this pipeline can munition clinical studies with molecules that have a higher chance to become a drug, decreasing attrition rates. clinical studies -over 1452 clinical trials have been unveiled, until the publication of this manuscript, focusing on covid-19 treatment interventions (available from: https://clinicaltrials.gov/ct2/results?cond=covid-19). with a growing number of patients suffering from acute severe respiratory symptoms and hospital capacities reaching its limit, therapeutic options are urgently essential to avoid human mass deaths. drug repurposing approaches of approved (with safe and effectiveness proven) and unapproved molecules, that were promising in pre-clinical and early stages of clinical studies of sars and mers, are in vogue. with this premise, world health organization (who) organised a simplified dynamic platform comparing the effectiveness of treatment strategies around the globe. this clinical trial design, called solidarity, can shrink by 80% the time of clinical studies, compared with the "gold standard" double-blind, placebo-controlled trials. this strategy could overcome the uncertainty of multiple small trials that do not produce a solid base necessary to establish the relative success of arising probable treatments. on the other hand, this shorter time required reflects the compassionate characteristic of the studies, that cannot rule out placebo effects and patient severe adverse effects, including death. (214) currently, who is focusing on four most promising therapies: remdesivir (8); cq (20) or hcq (26) ; ritonavir (2)/lopinavir (1); ritonavir (2) /lopinavir (1) plus interferon-beta, an immune response modulator. (44) a selection of studies with these drugs, alone or in combination, are summarised in table iii. for other studies, the reader can refer to kupferschmidt and cohen, which summarised several clinical studies on drug repurposing for covid-19 reported so far. (44) it is worth noting that many clinical studies are beginning to be carried out in different parts of the world and their number is increasing rapidly day by day. remdesivir (8), a nucleoside analogue that acts by inhibiting rna synthesis, is already in clinical studies for treatment of covid-19. (96) this prodrug was already investigated in clinical trial testing for ebola virus with no effect but showed efficiency against different types of coronaviruses in cell culture and animal models. (200, 221) from the drugs in the solidarity trial, remdesivir (8) has the best potential to be used in clinics, having a low toxicity profile to humans. (44) this molecule was used compassionately in the first covid-19 patient diagnosed in the united states by intravenous treatment and improved patient clinical condition. (222) grein et al. reported a study in a cohort of patients hospitalised for severe covid-19 who were treated with compassionateuse remdesivir (8) , clinical improvement was observed in 68% of the patients (36 of 53). (223) there are two clinical trials at phase iii, being designed to evaluate the efficacy and safety of parenteral remdesivir (8) in mild/moderate (nct04252664) and severe (nct04257656) hospitalised adults with covid-19. (224) these trials are evaluating intravenous remdesivir (8) at a dose of 200 mg on the first day plus 100 mg once daily, for an additional 9 consecutive days. the randomised, double-blind, placebocontrolled, multicentre trials were recently suspended (nct04252664) or terminated (nct04257656) because no eligible patients for enrollment were found, due pandemic control in china. (215) the study nct04257656 showed that remdesivir (8) was not associated with statistically significant clinical benefits in severe forms of covid-19. however, the authors observed a numerical reduction in time to clinical improvement in those treated earlier, but still requires confirmation. another study, performed by beigel et al. (with the same dosage and treatment period than the previous works cited above) enrolled 1063 patients with lower respiratory tract infection, demonstrated that individuals that were treated with remdesivir (8) had a shorter time to recovery than placebo group (11 days compared with 15 days). (216) even with a small scientific ballast, due to limited information about safety and effectiveness of using remdesivir (8), u.s. food and drug administration (fda) approved its emergency use on severe covid-19. (225) the approval was based on review of the topline data on two studies that are recruiting patients, (nct04280705) and (nct04292899). other clinical studies are being conducted to address the effect of remdesivir (8) on co-vid-19 treatment (available from: https://clinicaltrials. gov/ct2/results?cond=covid-19&term=remdesivir&cn try=&state=&city=&dist=). other phase iii clinical trials evaluated for co-vid-19 treatment included cq (20)/hcq (26) . these studies were endorsed by promising in vitro data that suggested an impairment of viral replication by acting on endosome-mediated viral entry or later phases of viral replication. (194, 226) both molecules have a well-studied toxicity profile being used for more than half-century as antimalarials, but in some cases can induce heart side (8) was not associated with clinical benefits in severe forms. however, an observed numerical reduction in time to clinical improvement in patients treated earlier, but still requires confirmation (215) ; nct04257656 (terminated by epidemic control in china with no eligible patients) remdesivir (8) (216) ; nct04280705 chloroquine (20) multicentre > 100 patients 500 mg per day, for 10 days compilation of various clinical studies that are in course, authors conclude that the dosage used could be sufficient (217) phase iib double-blind, randomised 81 patients 600 mg twice daily for 10 days and 450 mg twice on the first day, once daily for more 4 days higher dosage of chloroquine (20) has toxic effects and increased lethality, with any clinical benefit (218) hydroxychloroquine (26) non-randomised, non-double-blind, non-placebo-controlled 36 patients 600 mg of hydroxychloroquine (26) daily for 10 days hydroxychloroquine (26) significantly reduces viral load despite small sample size (219) ritonavir ( effects. (227) these drugs have already been examined against at least five other viruses with good initial in vitro results, but without significant effects in animal models and humans. (205) clinical trials to measure the effectiveness of cq (20) at 500 mg of chloroquine diphosphate (corresponding to 300 mg of chloroquine (20) base) per day, for ten consecutive days on the treatment of covid-19 were performed. (217) based on results for a very limited number of patients (100) , it was concluded that cq (20) presented apparent efficacy and acceptable safety against cov-id-19 associated pneumonia. borba et al. performed a double-blind, randomised clinical trial designed to assess the safety of cq (20) in two dosages: 600 mg twice daily for 10 days with an initial dose of 450 mg twice daily on the first day, decreasing to once daily for four more days. (218) the study suggests that the higher dosage should not be recommended for critically ill patients with covid-19 because of its potential side effects. it is important to note that both doses studied were above the brazilian recommended dose. a controversial study performed by gautret et al. with a small sample size demonstrated that hcq (26) treatment daily at 600 mg significantly decreases viral load in covid-19 patients and its effect is reinforced by azithromycin (az; 49) combination. (219) until this moment there is clinical robustness to support az (49) therapy in covid-19. (228) mehra et al. performed a registry analysis of 96,032 hospitalised patients regarding the use of cq (20) or hcq (26, combined or not with a macrolide) for treatment of covid-19. (229) the observational study verified that with covid-19 requiring hospitalisation a regimen containing cq (20) or hcq (26) had no evidence of benefit, but instead was associated with an increase in the risk of ventricular arrhythmias and a greater hazard for in-hospital death with covid-19. these findings suggest that these drug regimens should not be used outside of clinical trials and urgent confirmation from randomised clinical trials is needed. after the publication, the paper was retracted, which influenced who to return the studies on cq (20) and hcq (26) . (230) recently, on 17 june 2020, who stopped the hcq (26) arm of the solidarity trials again based on the recovery trials (available from: https://www.recoverytrial.net/). in this study 1542 patients were randomised to hcq (26) and compared with 3132 patients randomised to usual care alone. there was no significant difference in any of the parameters evaluated: the primary endpoint of 28-day mortality, hazard ratio and hospital stay duration or other issues. other study reported that hcq (26) did not prevent covid-19 when used as postexposure prophylaxis within 4 days after moderate or high-risk exposure. (231) robust data from well-designed control randomised clinical trials with large number of patients is still expected for concluding on the efficacy of hydroxychloroquine (26) . ritonavir (2) and lopinavir (1) were developed to target hiv-1 protease and postulated to inhibit the 3-chymotrypsin-like protease of sars and mers, being associated with improved clinical outcomes. (232) the combination has beneficial effects in marmoset monkeys infected with the mers-cov virus. (233) (1)/(2) may have clinical efficacy against sars-cov-2, as seen in the response against sars-cov. (234) in vitro sensitivity and satisfactory response in a preliminary non-randomised clinical trial of sars-cov to (1)/(2) have already been reported, encouraging its testing in sars-cov-2. (235) a total of 199 patients confirmed severe sars-cov-2 infection were randomly designated to be given either (1)/(2) (400 mg/100 mg) twice a day for 14 consecutive days plus standard care or standard care alone. (220) this first trial was not promising, no benefit was observed on clinical improvement, mortality and viral loads on severe covid-19 patients. other clinical trials are being carried out and should decide whether these drugs are useful for covid-19 treatment or not. ritonavir (2)/lopinavir (1) plus interferon-beta, a cytokine involved in inflammatory modulation, is the last bet of the solidarity megatrial. sheahan et al. demonstrated that this combination improves pulmonary function but does not reduce virus replication or severe lung pathology in mice model of mers-cov. (236) this combination also showed promising results in mers-cov infection in a nonhuman primate model. (233) the study (nct04276688) administered ritonavir (2)/lopinavir (1) 400 mg + 100 mg/ml twice daily for 14 days and interferon beta-1b 0.25 mg subcutaneous, every alternate day for 14 days. another study (nct04343768) will perform a randomised trial to verify the effects of interferon beta 1a, compared to interferon beta 1b and the base therapeutic treatment in moderate to severe covid-19. currently, there is a lot of research around the world examining drugs that can be used for the treatment of covid-19. unfortunately, until this moment no therapeutic options are promptly effectively available. most of the studies have a small number of patients with variable dose and/or duration, fact that hinders a comparison. to successfully undertake the covid-19 pandemic with new medicines, synchronised clinical trials with randomised, double-blind, placebo-controlled are still needed. infection prevention by social distancing and supportive medical care, are the only strategies to deal with this disease until this moment. the goal of this session is to consider potential obstacles for effective adoption of a repurposed drug to widespread treatment of covid-19. both patent protection and scalability of manufacturing processes are key aspects to consider while choosing the best therapeutic option to adopt when dealing with a pandemic. a patent can be defined as a government license that confers the owner the exclusivity over a new invention or medication. with the patent, the inventor is able to exclude others from making and selling the invention for a determined period of time. when it comes to medication, the market exclusivity granted by the patent generates enormous economic profit for the patent holder, once the company will have the monopoly over the product for around 20 years. once the patent term finishes, generic companies can start producing and selling the drug, increasing the market competition over the product. a strategy adopted by some companies is to maximise the term of patent of successful products, extending market exclusivity, for instance, increasing economic rewards with the invention. in order to extend patent terms, the companies can apply for new formulations, new routes of administration, new uses of the drug among other strategies. (237, 238) this is very important when considering drug repurposing, once those strategies can make it harder to patent a new method of use for the drug, compromising the legal rights of the new repurposed indication. as a consequence, the expected profit associated with repurposing can be severely affected. (39) remdesivir (8) a good example of maximising the term of patent is the drug remdesivir (8) , which was developed by the american pharmaceutical company gilead sciences, inc. in 2011. nowadays, (8) has shown promising results as a suitable repurposed drug in the treatment of the covid-19 disease. indeed, there are many ongoing clinical trials with this drug in cov-id-19 patients. (239, 240) due to the good results concerning remdesivir (8) , in january 2020, the wuhan institute of virology applied for a patent on gilead's remdesivir (8) for the treatment of coronavirus, in an attempt to protect china's economic and medical interests. however, remdesivir (8) has already 133 patents related to the coronavirus filed by gilead science in 43 countries around the globe since 2011. in fact, the company has a robust patent portfolio that includes structures of remdesivir-related compounds (family 1), the manufacturing method of the drug (family 2), the use of remdesivir (8) in treating coronaviridae infection (family 3), among other patents that claims the use of (8) for the treatment of a series of viral infections (families 3 and 4) (fig. 13) . (241) practically, this means that if the (8) is proved to be efficient for the treatment of covid-19, gilead science is the only pharmaceutical company owing the market exclusivity of the drug, at least until 2031. in fact, the company has already started to produce larger amounts of the drug and they already made improvements to optimise the drug manufacturing timeline. now, the company can produce the drug in 6 months, half the time that they used to need to get the final product. in addition, the company has been donating remdesivir (8) to ongoing clinical trials, a gesture that will not only help the trial patients, but gilead itself. (242) baricitinib (39) -in 2009, a patent assigned to incyte corporation disclosed the preparation of several active compounds as jak inhibitors, including baricitinib (39). in the same year, lilly and incyte made an agreement allowing lilly co. to manufacture and commercialise the medicine worldwide, making it lilly co. the only supplier around the globe. (243) there are two patents that protect the drug, one concerning its synthetic pathway and another disclosing the use of (39) in the treatment of rheumatoid arthritis. both of them will expire in nine years, which could open opportunities for generic drug companies to produce baricitinib (39) . nowadays, (39) has been investigated as a drug that could be used in the treatment of covid 19 patients, since its anti-inflammatory activity could minimise inflammatory compli-cations in covid 19 patients. according to the platform clinicaltrials.gov, there are 14 ongoing clinical trials to evaluate the efficacy and safety of baricitinib (39) in the treatment of covid 19. (244) if the drug succeeds, there will be a need for larger and faster production and distribution of the medicine worldwide. there are two patents disclosing a synthetic method for the preparation of (39). the first one is from 2009 and owned by incyte co. and the latest is from 2016, by lilly co. (245, 246) the main difference between them is how the central pyrazole ring is installed in the molecule. in the patent from 2009, baricitinib (39) is obtained by a convergent synthesis. the synthetic pathway starts with the protection in position 7 of 4-chloro-7h-pyrrolo[2,3d]pyrimidine (50) using 2-(trimethylsilyl)ethoxymethyl chloride (51) , affording intermediate (52) . next step is a suzuki-miyaura reaction, coupling the fused ring system to a 4-pyrazoleboronic acid pinacol ester (53), giving key intermediate (54) . parallelly, a couple of steps starting from 2-(chloromethyl)oxirane (55) lead to 1-boc-3-azetidinone (58) that reacts with diethyl cyanomethylphosphonate (59), affording key intermediate (60) . next, the key intermediates (54) and (60) react in the presence of dbu, giving (61) . then, steps involving hydrolysis of the boc, sulfonation and pyrrolopyrimidine deprotection afford (39) with an overall yield of 21 % (fig. 14) . (246) the synthetic pathway described in the patent from 2016 has only six steps in a linear approach, as a consequence, the product is obtained in a higher overall yield when compared to the synthetic pathway discussed above. intermediate (67) is obtained from azetidine-3-ol (64) in a couple of steps, including a sulfonation, an oxidation and the installment of the cyanomethylene moiety (fig. 15 ). furthermore, it is not necessary to protect any position in this sequence. additionally, the oxidation step can be performed under flow conditions. then, intermediate (67) is reacted with ester (53), affording (68) . a suzuki-miyaura reaction involving 7-boc-4-chloro-7hpyrrolo [2,3-d] pyrimidine (69) is applied, allowing the formation of the bound between the azetidinylpyrazole group and the pyrrolo[2,3-d]pyrimidine system. last step is a hydrolysis of the boc, affording baricitinib (39) with an overall yield of 50 % (fig. 15 ). (245) off-patent drugs -fortunately, a number of drugs that have been tested as possible agents in the covid-19 treatment are off-patent, meaning that generic drug companies already manufacture and commercialise the medicine, making it easier for drug repurposing. (39) the off-patent drugs that have been tested in covid-19 clinical trials include cq (20) , hcq (26), ritonavir (2) and lopinavir (1) . (239, 240) a recent research article published by hill and collaborators estimated the minimum cost of production associated with these drugs, showing that all the treatments under evaluation in current clinical trials are cheap to manufacture. however, list prices can be over 100 times higher than the costs to produce the drug. (247) if any of those drugs become approved for the treatment of covid-19, its demand will increase dramatically. therefore, it is important to consider the challenges associated with scaling up the production to meet the demand. for instance, there are few regulatory approved production facilities, and drug manufacturers rely on low-cost suppliers of raw materials in india and china, which might be scarce during a pandemic. consequently, it would be difficult to ensure short term global availability of the treatment, since the production depends on different countries. (248) those conclusions are very helpful in showing the importance of optimising the way of manufacturing the candidate drugs presented above. for this reason, recent and optimised synthetic pathways found in patents and articles for some ritonavir (2) and lopinavir (1) are discussed below, as cq (20) and hcq (26) have now been abandoned. finally, we will briefly discuss the ease of manufacturing in large scale each of the drugs we had the synthesis reviewed here. ritonavir (2) -the first disclosure of ritonavir (2) is presented in a patent from 1994, assigned to abbott laboratories. even though the patent consists of a range of markush structures, among which is found (2), its synthesis is not presented. (249) the synthetic pathway to obtain (2) was disclosed for the first time in a second-generation patent in 1995, by the same company. (250, 251) some drawbacks related to the synthesis presented by abbott include the employment of expensive condensing agents, as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc) and poor reaction yields on the first steps of the synthetic pathway, making this strategy too expensive and not suitable for scale-up production. considering the above deficiencies, a recent chinese patent concerning the synthesis of ritonavir (2) has been released. the synthetic pathway described in the patent starts with a nucleophilic addition-elimination reaction between the starting material (2-isopropylthiazol-4-yl)-nitro-methylamine (71) and n-[(2,2,2-trichloroethoxy)carbonyl]-l-valine (72), generating intermediate (73) . the intermediate is mixed with p-toluenesulfonyl chloride and triethylamine to activate the acid function, followed by the addition of reagent (74) in a one-pot procedure. the condensation allows the formation of intermediate (75) , which is submitted to acidic conditions for a boc deprotection, followed by another nucleophilic additionelimination reaction with reagent (76), thus obtaining the final product ritonavir (2) (fig. 16) . (252) when comparing both patents, it is possible to highlight important advantages present in the latest one, including the use of cheap and easily available reagents, for instance, the use of ptoluenesulfonyl chloride as an amide condensing agent, the synthetic pathway has a high yield (79% overall), low cost and it is ease to scale-up the production. lopinavir (1) -the first synthetic methods related to the synthesis of lopinavir (1) are present in a patent from 1996 owned by abbott laboratories. (249) (1) has 4 chiral centres, and the synthetic strategies reported by abbott are similar, involving the synthesis of a key intermediate amino alcohol unit, that is then connected to the appropriated side chains. some drawbacks that can be pointed out in the synthesis include the use of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (edc) as a condensing agent and the weak base 1-hydroxybenzotriazole, both expensive reagents, making the synthesis not suitable from the viewpoint of cost and industrial ap-plication. in addition, one of the methods described by abbott includes the synthesis of an acid chloride as an intermediate, which is very unstable, and it can be easily decomposed by humidity, making the synthesis not suitable for industrial production. the most recent patent related to the synthesis of lopinavir (1) is dated 2018 and it is from the chinese company shanghai desano pharmaceuticals co. ltd. (253) the patent is described as technical, with the aim of improving the synthesis of (1) presented in the patent from 1996, by abbott laboratories. the patent describes the synthesis of lopinavir (1) by a one-pot procedure starting with the formation of an acid chloride from (2s)-(1tetrahydropyramid-2-one)-3-methylbutanoic acid (77) followed by addition of a weak base and the reactant (78) to the reaction system. the amine function of (78) allows a nucleophilic addition-elimination reaction to occur, affording (1) after treatment with nahco 3 (fig. 17 ). therefore, it is possible to synthesise lopinavir (1) in high yield (90%) in an optimised method, which involves a few reagents, mild conditions and is performed in a one-pot procedure. synthetic scalability and cost-effectiveness -the synthetic pathways described above show how much improvement has been done concerning the preparation of these drugs in a more cost-effective way. furthermore, when comparing the drugs discussed above, it is worth pointing out which one could be the easiest one to produce when analysing the factors that influence the final cost of a synthetic pathway, such as chiral centres, cost of starting materials, number of steps and overall yield. regarding chiral centres, ritonavir (2) and lopinavir (1) have four chiral centres each, and they are not sold as racemate mixtures. moreover, to synthesise those compounds the chiral centres do not come from natural molecules, but have to be synthetically installed, which makes the chiral starting materials more expensive and enantiomeric excess has to be accessed more carefully at the end of the synthetic pathway. as a consequence, it can take longer and more expensive to obtain the final product. on the other hand, baricitinib (39) does not have any chiral centres, making the synthetic pathway easier and cheaper to perform. concerning the number of steps and overall yield, baricitinib (39) has the highest number of steps and lower overall yield when compared to ritonavir (2) and lopinavir (1) . however, one of the steps in the synthesis of baricitinib (39) can be performed under flow conditions, which is very interesting from the industrial point of view. therefore, when taking into account the most recent synthetic pathways proposed for these drugs, the synthesis of baricitinib (39) is the most cost-effective of the three of them. in conclusion -sars-cov-2 infection is a lifethreatening disease with such a high transmission rate that can surpass even the most well-structured health systems in developed high-income countries. while vaccines are the ideal solution for preventing the spread of infectious diseases like covid-19 their development cycle have intrinsic challenges and safety checking steps that require several months or years to complete their development. a similar situation is found for developing new drugs for covid-19 (as with any other disease), because of the long process involving discovery, validation and safety evaluation of new chemical entities for use in human health. in this scenario, repositioning drugs already in clinical use for treatment of covid-19 is an important shortcut, as we have discussed. data are accumulating about the molecular pathology of covid-19 as well as structural information on the viral and host proteins involved in the infection mechanism. this knowledge is essential for allowing researchers to have new insights on approved or investigational drugs that can be repurposed to treat sars-cov-2 infection. as reviewed here, over 10 targets distributed amongst distinct steps of the sars-cov-2 replication cycle or host cell mediators are currently being investigated. here, we highlighted several up-to-date examples of potential repurposable drug candidates proposed from either computational approaches or experimental trials (or their combination) at different levels of validation and stages of development. noteworthy, ai methods hold a great promise in finding occult links between drugs, human and viral targets to find novel bioactivities and even combinations of drugs. remdesivir (8) , cq (20)/hcq (26) (alone or in association with other drugs) and the association lopinavir (1)/ritonavir (2) have been the focus of most clinical studies. so far, results have been mostly disappointing with these trials, stressing the importance of carefully performed studies in patients to attest that biological activities observed in vitro actually be translated into clinical efficacy and safety. at the moment, even with limited information about safety and effectiveness, remdesivir is the only drug approved by the fda for emergency use on severe covid-19. as discussed here, a major concern with drugs for effectively fighting the pandemic is the drug's industrial production cost and its impact on final treatment cost. gilead, remdesivir's owner company, has recently signed non-exclusive voluntary licensing agreements with five generic pharmaceutical manufacturers to produce remdesivir for distribution in 127 countries, nearly all low-income and lower-middle income countries. hopefully, based on the constantly growing scientific knowledge summarised in this review, other treatment options will be revealed soon enough to help stop the havoc caused by the covid-19 pandemic. coronavirus disease (covid-2019) situation report 198 persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of cov-id-19 aerodynamic analysis of sars-cov-2 in two wuhan hospitals sars-cov-2 in wastewater: potential health risk, but also data source first confirmed detection of sars-cov-2 in untreated wastewater in australia: a proof of concept for the wastewater surveillance of covid-19 in the community fiocruz divulga estudo sobre a presença do novo coronavírus em esgotos sanitários the effect of human mobility and control measures on the covid-19 epidemic in china projecting the transmission dynamics of sars-cov-2 through the postpandemic period genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding coronavirus reverse genetic systems: infectious clones and replicons discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals. emerg microbes infect cultivation of viruses from a high proportion of patients with colds isolation and characterization of viruses related to the sars coronavirus from animals in southern china adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus roles of host gene and non-coding rna expression in virus infection structure, function, and evolution of coronavirus spike proteins the mers-cov receptor dpp4 as a candidate binding target of the sars-cov-2 spike. iscience receptor recognition mechanisms of coronaviruses: a decade of structural studies coronavirus envelope protein: current knowledge analyses of coronavirus assembly interactions with interspecies membrane and nucleocapsid protein chimeras the proximal origin of sars-cov-2 phylogenetic network analysis of sars-cov-2 genomes a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china real-time tracking of self-reported symptoms to predict potential covid-19 clinical, laboratory and imaging features of covid-19: a systematic review and meta-analysis coronavirus infections and immune responses targeting potential drivers of co-vid-19: neutrophil extracellular traps similarity in case fatality rates (cfr) of covid-19/sars-cov-2 in italy and china the importance of hypertension as a risk factor for severe illness and mortality in covid-19 clinical characteristics of fatal and recovered cases of coronavirus disease 2019 in wuhan, china: a retrospective study diabetes is a risk factor for the progression and prognosis of covid-19 older people and covid-19: isolation, risk and ageism asymptomatic carriers of covid-19 as a concern for disease prevention and control: more testing, more follow-up drug repositioning: identifying and developing new uses for existing drugs drug repurposing from the perspective of pharmaceutical companies drug repurposing: progress, challenges and recommendations emerging virus diseases: can we ever expect the unexpected? drug repurposing for new, efficient, broad spectrum antivirals deployment of convalescent plasma for the prevention and treatment of covid-19 therapeutic options for the 2019 novel coronavirus (2019-ncov) race to find covid-19 treatments accelerates coronavirus sars-cov-2 disease covid-19: infection, prevention and clinical advances of the prospective chemical drug therapeutics: a review on coronavirus disease covid-19, epidemiology, prevention, and anticipated therapeutic advances a novel coronavirus from patients with pneumonia in china isolation and rapid sharing of the 2019 novel coronavirus (sars-cov-2) from the first patient diagnosed with covid-19 in australia covid-19 infection: origin, transmission, and characteristics of human coronaviruses targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid-19 emerging coronaviruses: genome structure, replication, and pathogenesis genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan networkbased drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 the architecture of sars-cov-2 transcriptome a sars-cov-2 protein interaction map reveals targets for drug repurposing genomic characterization of a novel sars-cov-2 severe acute respiratory syndrome coronavirus 2 (sars-cov-2): an overview of viral structure and host response functional studies of the coronavirus nonstructural proteins coronaviruses and the associated potential therapeutics for the viral infections coronavirus membrane fusion mechanism offers a potential target for antiviral development the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade the coronavirus e protein: assembly and beyond from sars and mers covs to sars-cov-2: moving toward more biased codon usage in viral structural and nonstructural genes pharmacological therapeutics targeting rna-dependent rna polymerase, proteinase and spike protein: from mechanistic studies to clinical trials for covid-19 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor pharmacologic treatments for coronavirus disease novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment structure of mpro from sars-cov-2 and discovery of its inhibitors merops : the database of proteolytic enzymes, their substrates and inhibitors crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors pharmacophores and biological activities of severe acute respiratory syndrome viral protease inhibitors. expert opin ther pat structural basis for the inhibition of sars-cov-2 main protease by antineoplastic drug carmofur structural elucidation of sars-cov-2 vital proteins: computational methods reveal potential drug candidates against main protease, nsp12 polymerase and nsp13 helicase rapid identification of potential inhibitors of sars-cov-2 main protease by deep docking of 1.3 billion compounds insights into the inhibitory potential of selective phytochemicals against mpro of 2019-ncov: a computer-aided study discovery of potential multi-target-directed ligands by targeting host-specific sars-cov-2 structurally conserved main protease potential inhibitors against 2019-ncov coronavirus m protease from clinically approved medicines gc-376, and calpain inhibitors ii, xii inhibit sars-cov-2 viral replication by targeting the viral main protease glecaprevir and maraviroc are high-affinity inhibitors of sars-cov-2 main protease: possible implication in covid-19 therapy fast identification of possible drug treatment of coronavirus disease-19 (covid-19) through computational drug repurposing study computational insights into tetracyclines as inhibitors against sars-cov-2 mpro via combinatorial molecular simulation calculations nsp3 of coronaviruses: structures and functions of a large multi-domain protein the sars-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds structural basis for the ubiquitin-linkage specificity and deis-gylating activity of sars-cov papain-like protease evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors diarylheptanoids from alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus disulfiram can inhibit mers and sars coronavirus papainlike proteases via different modes analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods potential treatments for covid-19; a narrative literature review repurposing of fda-approved antivirals, antibiotics, anthelmintics, antioxidants, and cell protectives against sars-cov-2 papain-like protease structural basis for inhibition of the rna-dependent rna polymerase from sars-cov-2 by remdesivir remdesivir and sars-cov-2: structural requirements at both nsp12 rdrp and nsp14 exonuclease active-sites structure of the rna-dependent rna polymerase from covid-19 virus structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors remdesivir is a direct-acting antiviral that inhibits rna-dependent rna polymerase from severe acute respiratory syndrome coronavirus 2 with high potency favipiravir: pharmacokinetics and concerns about clinical trials for 2019-ncov infection anti-hcv, nucleotide inhibitors, repurposing against covid-19 differential inhibitory activities and stabilisation of dna aptamers against the sars coronavirus helicase development of chemical inhibitors of the sars coronavirus: viral helicase as a potential target delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp13 upon atp hydrolysis identification of myricetin and scutellarein as novel chemical inhibitors of the sars coronavirus helicase, nsp13 predicting commercially available antiviral drugs that may act on the novel coronavirus (sars-cov-2) through a drug-target interaction deep learning model state-of-the-art tools unveil potent drug targets amongst clinically approved drugs to inhibit helicase in sars-cov-2 shahzad-ul-hussan s. identification of potential inhibitors of three key enzymes of sars-cov2 using computational approach sars-coronavirus-2 nsp13 possesses ntpase and rna helicase activities that can be inhibited by bismuth salts crystal structure and functional analysis of the sars-coronavirus rna cap 2′-o-methyltransferase nsp10/nsp16 complex functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase targeting sars-cov-2: a systematic drug repurposing approach to identify promising inhibitors against 3c-like proteinase and 2′-o-ribose methyltransferase coronavirus nsp10, a critical co-factor for activation of multiple replicative enzymes yeast-based assays for the high-throughput screening of inhibitors of coronavirus rna cap guanine-n7-methyltransferase synthesis of adenine dinucleosides sam analogs as specific inhibitors of sars-cov nsp14 rna cap guanine-n7-methyltransferase singh sk. structure-based virtual screening and molecular dynamics simulation of sars-cov-2 guanine-n7 methyltransferase (nsp14) for identifying antiviral inhibitors against covid-19 virtual screening, adme/t, and binding free energy analysis of anti-viral, anti-protease, and anti-infectious compounds against nsp10/ nsp16 methyltransferase and main protease of sars cov-2 targeting sars-cov-2 non-structural protein 16: a virtual drug repurposing study ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence a unique protease cleavage site predicted in the spike protein of the novel pneumonia coronavirus (2019-ncov) potentially related to viral transmissibility structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov-2 infection tmprss2 and covid-19: serendipity or opportunity for intervention? protein-protein interactions of viroporins in coronaviruses and paramyxoviruses: new targets for antivirals? viruses the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis in-silico approaches to detect inhibitors of the human severe acute respiratory syndrome coronavirus envelope protein ion channel structure and inhibition of the sars coronavirus envelope protein ion channel. baric rs severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2: biology and therapeutic options sars-cov-2 e protein is a potential ion channel that can be inhibited by gliclazide and memantine angiotensin-(1-7) and angiotensin-(1-9): function in cardiac and vascular remodelling covid-19 pandemic, coronaviruses, and diabetes mellitus angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target sti-1499, a potent anti-sars-cov-2 antibody, demonstrates ability to completely inhibit in vitro virus infection in preclinical studies new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? recent research advances in renin-angiotensin-aldosterone system receptors covid-19 and the cardiovascular system reply to: 'interaction between raas inhibitors and ace2 in the context of covid-19' angiotensin receptor blockers as tentative sars-cov-2 therapeutics renin-angiotensin-aldosterone system inhibitors in patients with covid-19 sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? tmprss2: a potential target for treatment of influenza virus and coronavirus infections nafamostat mesylate blocks activation of sars-cov-2: new treatment option for covid-19 computeraided screening for potential tmprss2 inhibitors: a combination of pharmacophore modeling, molecular docking and molecular dynamics simulation approaches withanone and withaferin-a are predicted to interact with transmembrane protease serine 2 (tmprss2) and block entry of sars-cov-2 into cells mechanisms of clathrin-mediated endocytosis the endosomal-lysosomal system: from acidification and cargo sorting to neurodegeneration cathepsin l-selective inhibitors: a potentially promising treatment for covid-19 patients interleukin-6 family cytokines cytokines and cytokine receptors covid-19: consider cytokine storm syndromes and immunosuppression covid-19: consider il-6 receptor antagonist for the therapy of cytokine storm syndrome in sars-cov-2 infected patients the carbohydrate-active enzymes database (cazy) in 2013 influenza virus neuraminidase structure and functions coronavirus infections and type 2 diabetesshared pathways with therapeutic implications dpp4 inhibition: preventing sars-cov-2 infection and/or progression of covid-19? covid-19 and diabetes: can dpp4 inhibition play a role? emerging wuhan (covid-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd26 dipeptidyl peptidase-4 (dpp4) inhibition in covid-19 is dpp4 inhibition a comrade or adversary in cov-id-19 infection boosting the arsenal against covid-19 through computational drug repurposing déjà vu: stimulating open drug discovery for sars-cov-2 repurposing therapeutics for covid-19: supercomputer-based docking to the sars-cov-2 viral spike protein and viral spike protein-human ace2 interface peptide-like and small-molecule inhibitors against covid-19 baricitinib as potential treatment for 2019-ncov acute respiratory disease de novo design of new chemical entities (nces) for sars-cov-2 using artificial intelligence deeppurpose: a deep learning library for drug-target interaction prediction and applications to repurposing and screening a data-driven drug repositioning framework discovered a potential therapeutic agent targeting covid-19 natural bis-benzylisoquinoline alkaloids-tetrandrine, fangchinoline, and cepharanthine, inhibit human coronavirus oc43 infection of mrc-5 human lung cells inhibition of sars-cov 3cl protease by flavonoids small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells atazanavir inhibits sars-cov-2 replication and pro-inflammatory cytokine production baricitinib therapy in covid-19: a pilot study on safety and clinical impact gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading sweetlead: an in silico database of approved drugs, regulated chemicals, and herbal isolates for computer-aided drug discovery drugbank 5.0: a major update to the drugbank database for 2018 chembl: towards direct deposition of bioassay data bindingdb: a web-accessible database of experimentally determined proteinligand binding affinities uniprot: the universal protein knowledgebase graph convolutional neural networks for predicting drug-target interactions graph convolutional networks: a comprehensive review bere: an accurate distantly supervised biomedical entity relation extraction network recent trends in deep learning based natural language processing deep learning in natural language generation from images deep learning self-attention based molecule representation for predicting drug-target interaction inductive transfer learning for molecular activity prediction: next-gen qsar models with molpmofit a survey on deep transfer learning the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? covid-19, sars and mers: are they closely related? overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov drug target validation: hitting the target coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 multiparametric assays for accelerating early drug discovery laboratory safety aspects of sars at biosafety level 2 animal models for sars and mers coronaviruses remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro to zika and destroy: an antimalarial drug protects fetuses from zika infection in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov-2 replication in vitro the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro teicoplanin potently blocks the cell entry of 2019-ncov broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection potential antivirals and antiviral strategies against sars coronavirus infections chloroquine and hydroxychloroquine in covid-19 targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro animal toxicity and pharmacokinetics of hydroxychloroquine sulfate of chloroquine and covid-19. antiviral res hiv protease inhibitors: a review of molecular selectivity and toxicity anti-hiv drugs: 25 compounds approved within 25 years after the discovery of hiv 3clpro inhibitors as a potential therapeutic option for covid-19: available evidence and ongoing clinical trials computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with sars-cov-2 protease against covid-19 screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture treating covid-19 -off-label drug use, compassionate use, and randomized clinical trials during pandemics remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid-19 -preliminary report breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 a randomized, controlled trial of ebola virus disease therapeutics first case of 2019 novel coronavirus in the united states compassionate use of remdesivir for patients with severe covid-19 arguments in favour of remdesivir for treating sars-cov-2 infections fda -food and drug administration remdesivir eua letter of authorization effects of chloroquine on viral infections: an old drug against today's diseases cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature clinical pharmacology perspectives on the antiviral activity of azithromycin and use in covid-19 retraction: cardiovascular disease, drug therapy, and mortality in covid-19 retracted: hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid-19: a multinational registry analysis a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 coronaviruses -drug discovery and therapeutic options treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset molecular dynamic simulations analysis of ritronavir and lopinavir as sars-cov 3clpro inhibitors role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov patent protection strategies data exclusivity, and the development of new drugs. ssrn [internet coronavirus puts drug repurposing on the fast track research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases coronavirus patents: china files for remdesivir patent, but gilead sciences will still come out a winner fierce pharma gilead turbocharges production of covid-19 hopeful remdesivir lilly and incyte announce collaboration for development and commercialization of oral anti-inflammatory and autoimmune therapies search results: baricitinib | covid-19 processes and intermediates for the preparation of {1(ethylsulfonyl)-3 inventors; incyte corporation assignee 2009. azetidine and cyclobutane derivatives as jak inhibitors. united states patent us 2009/0233903 a1 minimum costs to manufacture new treatments for covid-19 dozens of coronavirus drugs are in development -what happens next? inventors; abbott laboratories assignee 1995. process for the preparations of a substituted 2,5-diamino-3-hydroxyhexane discovery of ritonavir, a potent inhibitor of hiv protease with high oral bioavailability and clinical efficacy zhou z, inventors; guizhou yongnuo feite bio pharmacy co ltd assignee 2019. preparation method of ritonavir. chinese patent cn 109369562a inventors; yancheng desano pharmaceutical co ltd assignee 2018. method used for preparing lopinavir using one-pot method. chinese patent cn 108218791a all authors contributed to writing, reviewing and editing the manuscript. mrs, tcse and rfd contributed equally to this manuscript; fps-jr performed the manuscript conceptualisation and final review. the authors declare no conflict of interest concerning this manuscript. key: cord-347128-6lyoz8nn authors: kim, cheorl-ho title: sars-cov-2 evolutionary adaptation toward host entry and recognition of receptor o-acetyl sialylation in virus–host interaction date: 2020-06-26 journal: int j mol sci doi: 10.3390/ijms21124549 sha: doc_id: 347128 cord_uid: 6lyoz8nn the recently emerged sars-cov-2 is the cause of the global health crisis of the coronavirus disease 2019 (covid-19) pandemic. no evidence is yet available for cov infection into hosts upon zoonotic disease outbreak, although the cov epidemy resembles influenza viruses, which use sialic acid (sa). currently, information on sars-cov-2 and its receptors is limited. o-acetylated sas interact with the lectin-like spike glycoprotein of sars cov-2 for the initial attachment of viruses to enter into the host cells. sars-cov-2 hemagglutinin-esterase (he) acts as the classical glycan-binding lectin and receptor-degrading enzyme. most β-covs recognize 9-o-acetyl-sas but switched to recognizing the 4-o-acetyl-sa form during evolution of covs. type i he is specific for the 9-o-ac-sas and type ii he is specific for 4-o-ac-sas. the sa-binding shift proceeds through quasi-synchronous adaptations of the sa-recognition sites of the lectin and esterase domains. the molecular switching of he acquisition of 4-o-acetyl binding from 9-o-acetyl sa binding is caused by protein–carbohydrate interaction (pci) or lectin–carbohydrate interaction (lci). the he gene was transmitted to a β-cov lineage a progenitor by horizontal gene transfer from a 9-o-ac-sa–specific hef, as in influenza virus c/d. he acquisition, and expansion takes place by cross-species transmission over he evolution. this reflects viral evolutionary adaptation to host sa-containing glycans. therefore, cov he receptor switching precedes virus evolution driven by the sa-glycan diversity of the hosts. the pci or lci stereochemistry potentiates the sa–ligand switch by a simple conformational shift of the lectin and esterase domains. therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. a clear example of he gene transfer is found in the bcov he, which prefers 7,9-di-o-ac-sas, which is also known to be a target of the bovine torovirus he. a more exciting case of such a switching event occurs in the murine covs, with the example of the β-cov lineage a type binding with two different subtypes of the typical 9-o-ac-sa (type i) and the exclusive 4-o-ac-sa (type ii) attachment factors. the protein structure data for type ii he also imply the virus switching to binding 4-o acetyl sa from 9-o acetyl sa. principles of the protein–glycan interaction and pci stereochemistry potentiate the sa–ligand switch via simple conformational shifts of the lectin and esterase domains. thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. as expected, structural and non-structural proteins of sars-cov-2 are currently being targeted for viral therapeutic designation and development. however, the modern global society needs sars-cov-2 preventive and therapeutic drugs for infected patients. in this review, the structure and sialobiology of sars-cov-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future. mammal-and avian-infectible covs consist of the broad-ranged subfamily of coronavirinae. the ictv recommended classification is as follows: riboviria (realm)-nidovirales (order)-cornidovirineae (suborder)-coronavirida (family)-orthocoronavirinae-coronavirus genus. the mammal-and avian-infectible covs consist of the broad-ranged subfamily of coronavirinae. the ictv recommended classification is as follows: riboviria (realm)-nidovirales (order)-cornidovirineae (suborder)-coronavirida (family)-orthocoronavirinae-coronavirus genus. the mammal-and avian-infectible covs consist of the broad-ranged subfamily of coronavirinae. the ictv recommended classification is as follows: riboviria (realm)-nidovirales (order)-cornidovirineae (suborder)-coronavirida (family)-orthocoronavirinae-coronavirus genus. the mammal-and avian-infectible covs consist of the broad-ranged subfamily of coronavirinae. the ictv recommended classification is as follows: riboviria (realm)-nidovirales (order)-cornidovirineae (suborder)-coronavirida (family)-orthocoronavirinae-coronavirus genus. the four cov genera [11] are the α-cov, β-cov, γ-cov and δ-cov. the 2019-ncov or sars-cov-2 int. j. mol. sci. 2020, 21, 4549 5 of 34 belong to the β-cov genus and are zoonotic and cause mammalian infection, causing respiratory disease in the human lung. the α-cov and β-cov genus target mammal hosts while the δ-cov and γ-cov genus target avians and certain mammals. the β-cov genus has a, b, c and d lineages. among these, lineage b includes sars-cov and sars-cov-2. lineage c includes mers-cov. the b lineage sars-cov and c lineage mers-cov, which are classified as β-covs, exhibit lethal rates of 10% and 35% in humans, respectively. covs in humans are associated with respiratory infections such as colds with clinical importance, as experienced for the previous outbreak in 2003 of sars-human cov (hcov), hcov-hku1 and hcov-nl63 [12, 13] . human infectious hcov includes seven species, including α-cov (hcov-nl63 and hcov-229e) and β-cov (sars-cov, sars-cov-2, hcov-oc43, hcov-hku1 and mers-cov). cov rna sequences mutate at a high frequency. among the known rna viruses, covs bear the longest genome sizes of 26 to 32 kb length rna. nucleotide sequences of cov ssrna genomes isolated from covid-19 patients in wuhan show a high homology of 89% with the nucleotide sequence of the previously known bat sars-like cov-zxc-21 strain and 89% with the previous sars-cov. the initial wuhan cov isolates belong to the β-cov genus and were therefore termed sars-cov-2 or 2019-ncov [14] . sars-cov-2 infects human respiratory tracts and causes outbreaks of pneumonia. sars-cov-2 is a novel cov and originates from the wuhan district in china. the genome sequence of sars-cov-2 exhibits 79% sequence homology with the sars-cov rna sequence and 50% with the mers-cov sequence [15] . covs are 60-140 nm in size and are enveloped (+) ssrna viruses, which feature an rna genome, directly available to function as mrna and thus result in rapid infection. covs exhibit rna genomes of 28-32 kb, comprised of two large overlapping open reading frames (orfs), which encode the virus replicase (transcriptase) and structural proteins. the sars-cov-2 genome is 29,891 bp in size, which encodes 9860 amino acids. the ssrna are capped and tailed with a 5 -capping structure and 3 -poly a tail at the termini. the genome is the same sense as virus mrna indicating that the viral rna is translated through its own (+) rna to synthesize rna dependent rna polymerase (rdrp; pdb: 6m71). generally, viral families are determined by the genome structure and virion morphologies of an envelope or naked capsid. a virus with a naked capsid has a coat of nucleocapsid protein (n) coating the viral genome. viruses with an envelope have lipid envelopes further surrounding the outmost protein layer. the 2019-ncov (sars-cov-2) contains a spike (s) glycoprotein, e, dimeric he enzyme, a membrane matrix glycoprotein (m), n and rna [16] . the structural proteins are the s, n, m and e proteins, while the non-structural proteins are proteases such as nsp3 and nsp5 and rdrp such as nsp12. among the n, m and s glycoproteins, the s glycoprotein is a fusion protein that recognizes the host receptor and enters the host cells [17] . the s, m and e proteins anchored into the endoplasmic reticulum (er) membrane are trafficked to the endoplasmic reticulum-golgi intermediate compartment (ergic). the rna genome linked with nucleoprotein buds into the ergic to form virus particles. assembled virions transported to the vesicular surface are released to the extracellular milieu via exocytosis. the rna generates the replicase as two polyproteins, pp1a and pp1ab. the replicase-encoded viral proteases generate up to 16 nonstructural proteins (nsps) in the cytosol to produce replicase enzyme and the replicase-transcriptase complex (rtc). these enzymes including rtc synthesize rnas for replication and transcription to generate viral rna genome. cov genomes bear two or three protease genes and the coding enzymes cleave the replicases. together with the replicases, nonstructural proteins, termed nsps, assemble into the rtc complex. nsp1 to nsp16 are known to have multiple enzyme regions. for example, nsp1 degrades cellular mrnas and, consequently blocks protein translation in host cells and innate immune responses. nsp2 recognizes the specific protein called prohibitin. nsp3 is a multi-domain transmembrane (tm) protein with diverse activities. ubiquitin-like 1 and acidic domains bind to n protein and adp-ribose-1 -phosphatase (adrp) activity induces cytokine expression. the papain-like protease (plpro)(pdb:6wx4)/ deubiquitinase domain cleaves virus-produced polyprotein. nsp4 is a tm scaffold protein for double-membrane vesicle structure. nsp5 has a main protease domain which also cleaves virus-produced polyprotein and nsp6 acts as a tm scaffold protein. the nsp7 and nsp8 proteins form the nsp7-nsp8 hexadecameric complex, which functions as an rna polymerase-specific clamp and a primase enzyme. nsp9 is an rna-binding protein that activates exon and 2-o-methyltrnasferase (mtase) enzyme activity. nsp10 binds to nsp16 and nsp14 to form a heterodimeric complex. nsp12 is the rdrp and nsps13 is the rna helicase and 5 triphosphatase. nsps14 is a n7 mtase and 3 -5 exoribonuclease. exon of nsap14 acts as an n7 mtase and attaches the 5 cap to viral rnas. viral exoribonuclease enzyme proofreads the viral rna genome, where nsp15 is a viral endoribonuclease (pdb: 6vww). nsp16 has 2-o-mtase enzyme activity, which shields viral rna from melanoma differentiation associated protein-5 recognition. in rna viruses, the s glycoprotein (pdb: 6vsb) is the biggest protein, heavily glycosylated and its n-terminal domain (ntd) sequence binds to the host receptor to enter the er of host cells. sars-cov-2 s-glycoprotein bears 22 n-glycan sequons in each protomer. therefore, the trimeric s glycoprotein surface is dominated by 66 n-glycans. the s glycoprotein mediates direct and indirect interaction of virus with host cells in the infection cycle. all covs exhibit a surface s glycoprotein, which bears the receptor-binding domain (rbd). the s glycoprotein has a distinct spike structure. when s glycoprotein binds to its host receptor, a host furin-like protease cleaves the s glycoprotein, which liberates the spike fusion peptides, allowing entry of the virus into the host cell [18] . the furin-like protease-generated s1 and s2 exist as a s1/s2 complex, where s1 in a homotrimeric form interacts with the host cell membrane and s2 penetrates the cytosolic area. for sars-cov and mers-cov, the s1 c-terminal domains (ctds) have a dual role in virus entry via attachment and fusion. the s1 ntd binds to carbohydrate receptors because the s1 domains act as the rbd. the ctd of s1 recognizes protein receptors via rbds. in rna viruses, the n protein recognizes the viral rna genome. the n protein (pdb: 6m3m) binds to the rna genome via the ntd and ctd. the n protein tethers to the viral rna and replicase-transcriptase complex (rtc). nsp3 (pdb: 6vxs) of cov blocks the innate immune responses of hosts. after entrance into the host cells, for cov transcription and particle release, rna chaperones such as nonspecific nucleic acid binding proteins potentiate ssrna conformation shifts. representatively, the n protein is known as the rna chaperone protein. for example, glycogen synthase kinase 3 (gsk3) phosphorylates the sars-cov n-protein and thus, gsk3 inhibition contributes to reduced replication activity of sars-cov [19] . in addition, heterogeneous nuclear ribonucleoprotein a1 (hnrnp a1) regulates the preformed mrna splicing in the nucleus and continuous translation. hnrnpa1 interacts with sars-cov n protein to form a replication and transcription complex during ssrna genome biosynthesis [20] . e protein is the most abundant structural protein needed to assemble virus particles in the cytosol. as a tm protein, e protein is the smallest structural protein with a mw of 12 kda. the e protein has an ntd in the extracellular region and a ctd in the cytosolic region. the e protein bears an ectodomain in the ntd and an endodomain in the ctd. it has also an ion channel domain. e protein is present in the cytosolic region of infected cells and only a limited amount is incorporated into the envelope of virions [21] . most e proteins assemble and bud in new virus particles. the m protein contains three tm domains and is an abundant structural protein with a mw of 30 kda. it consists of a small glycosylated ntd in the extracellular region and ctd in the cytoplasmic region. the m protein forms a scaffold for virus assembly in the cytosol via binding to s glycoprotein and n protein [22] . for example, e protein and n protein are co-expressed with m protein to form virus-like particles (vlps) that are released from the cells, as the m and e protein are involved in cov assembly. then, covs bud into the ergic, trafficking by membrane vesicles and transported via the exocytosis-secretory pathway [23, 24] . the dimeric m protein binds to the nucleocapsid.-m protein binds to s glycoprotein for s glycoprotein retention in the ergic/golgi complex. the m-n protein complex keeps the n protein-rna complex stable, for nucleocapsid and the viral assembly.-m and e proteins constitute the virus envelope for successful release of virus-like particles. he hemagglutinates and destroys receptors. as rna viruses, covs bear rdes, which are used in effective attachment to hosts and also reversely in detachment from the hosts. for example, enveloped rna viruses evade the hosts via their rdes. currently, rde-related functional enzymes such as neuraminidase (na) and sa-o-acetyl-esterase are known. sa-o-acetyl-esterase was originally identified in influenza c virus and in nidoviruses (cov and torovirus) as well as in salmon anemia virus (teleost orthomyxovirus). the origin and evolution of cov sa-o-acetylesterases are correlated to other viruses. the fusion event of s glycoprotein and he is specific for hcov attachment to sa-associated receptors in the host [25] . the he has acetylesterase activity [26] . in early sa-related biology, influenza a/b viruses were found to recognize chicken erythrocytes in 1942 [27] . they caused hemagglutination through clumping by virus-borne hemagglutinin. these phenomena were widely found in influenza viruses, paramyxoviruses, newcastle disease (ndv) and mumps virus. sas have various derivatives of more than 50 chemically different structures formed from the basic n-acetylneuraminic acid (neu5ac) on the main ring of pyranose and the glycerol side chain. sa are modified by acetyl-, lactyl-, methyl-and sulfo-groups individually or in multiple combinations [28] . multiple enzymes are involved in the modifications [29] . historically, the first discovered sa was crystallized by gunner blix via a hot mild acid extraction of bovine submaxillary mucin in 1936. it consisted of two acetyl groups. among these, only one acetyl group was attached to nitrogen [30] . blix isolated a 9-o-acetyl sa of the common sa n-acetylneuraminic acid (neu5ac), chemically described as 9-o-acetyl-n-acetylneuraminic acid (neu5,9ac2). neu5,9ac2, neu5ac and neu5gc are naturally occurring sa species in mammals. a common modification is o-acetylation. in fact, o-acetylation of sas is common in organisms. the o-acetyl modification occurs in single positions of c-4, c-7, c-8 and c-9 of sa as well as in combined c-positions to yield neu4,5ac2, neu5,9ac2 and neu5,7,9ac3 sas. neu5ac9nac is a chemical and biologic mimic of neu5,9ac2 in the sa-glycans. the c-7 and/or c-9 o-acetylations are catalyzed by the sa o-acetyltransferase enzyme, cas1 domain containing 1 (casd1) ( figure 6 ). casd1 catalyzes the addition of acetyl groups to the sa c-7 at the late golgi apparatus compartment [31] . thereafter, an enzyme termed "migrase" transfers the additional acetyl-group from c-7 to c-9, although this enzyme has not been identified [29] . casd1 uses acetyl-coenzyme a as a donor substrate and cmp-neu5ac as an acceptor substrate, but with weak activity on cmp-neu5gc. and susceptible to esterase enzymes. sialic acid cleavage of the di-acetylated neu5,7,9ac3 by bacterial nas decreases two-fold, when compared to mono-o-acetylated neu5ac. the o-acetylated glycan modification invites interaction with viruses, antibodies and mammalian lectins [32] . therefore, the sa o-acetylation modification confers specific functions to organisms. for example, in the horse, c4-o-acetyl modification of neu5ac (sa) occupies more than 50% of the total sa content. the c4-o-acetylated neu5ac, neu4,5ac2, inhibits the influenza a2 virus ha. de-acetylation reagents such as naoh or naio4 treatment completely hemagglutinate neu4,5ac2 by elimination of the c4-o-acetyl group [33] . the c4-o-acetyl neu5ac species are found in various sources such as equine erythrocyte gm3, starfish a. rubens and fish [34] [35] [36] [37] [38] . c4-o-acetylated neu5ac facilitates the initial attachment of viruses to target cells. like the influenza c virus, infectious salmon anemia virus (isav), a member of the orthomyxoviridae family, contains he and hef proteins to mediate virus entry and exit. c4-o-ac neu5ac is the major receptor determinant of isav in receptor binding and destruction [38] , while the influenza c virus recognizes c9-o-ac neu5ac. the acetylesterase rde of isav cleaves c4-o-ac via 4-sa-o-acetylesterase with a short turnover time, whereas c9-o-ac neu5ac is cleaved by 9-sa-o-acetylesterase with a long turnover time [34] . the position of sa o-acetylation is linked to functions including substrate differentiation of enzymes such as nas and esterase by c4 o-ac. previous development of o-ac site-selective na inhibitors were based on the conceptual consideration of different o-ac positions . the o-ac of sas is site-specific, as c4 of neu5ac is considered to be a potential position for modification. historically, inhibitors of influenza a and b viruses-sialidases were designed by von-itzstein in 1993 [39] . the ac group-based c4 substitution interacts with amino acid glu-119 present in the active site of sialidase. guanidine-attached c4 of c2-c3 unsaturated sa (neu5ac2en) inhibits activity of sialidases isolated from influenza a virus (singapore/1/57) and b virus (victoria/102/85). the same scenario was applied for sialidase inhibition of the human parainfluenza virus type 3, which has hn and fusion proteins [40] . the c4 of neu5ac2en was substituted by alkyl groups such as the o-ethyl group. for example, zanamivir has a substitution with a 4-guanidino group with an ic50 of 25 μm. thus, sialidase inhibition is important for c4 modification of neu5ac2en. later, oseltamivir with the tradename tamiflu (basel, switzerland) and zanamivir with the tradename relenza (london, uk) were established [41] . these drugs exhibit some adverse side effects that restrict clinical use. the sa 9-o-acetylation in hosts allows hosts to evade influenza a virus hemagglutinin (ha) recognition and some lectins of factor h (fh), cd22/siglec-2 and sialoadhesin/siglec-1. instead, the influenza c virus ha recognizes the hosts. β-elimination and permethylation eliminate the 9-o-acetyl group from sas. chemical modification of the c-9 position of neu5,9ac2 generates a 9-n-acetyl biologically, the sa o-acetylation event confers merits to hosts such as protection from pathogenic invasion and maintenance of systemic self-homeostasis. the o-acetylation event of sas protects the sa-containing glycans from neuraminidase (na)/sialidase action, because o-acetyl-groups inhibit microbial na activity. the chemical structure of the o-acetyl group is quite unstable and susceptible to esterase enzymes. sialic acid cleavage of the di-acetylated neu5,7,9ac3 by bacterial nas decreases two-fold, when compared to mono-o-acetylated neu5ac. the o-acetylated glycan modification invites interaction with viruses, antibodies and mammalian lectins [32] . therefore, the sa o-acetylation modification confers specific functions to organisms. for example, in the horse, c4-o-acetyl modification of neu5ac (sa) occupies more than 50% of the total sa content. the c4-o-acetylated neu5ac, neu4,5ac2, inhibits the influenza a2 virus ha. de-acetylation reagents such as naoh or naio 4 treatment completely hemagglutinate neu4,5ac2 by elimination of the c4-o-acetyl group [33] . the c4-o-acetyl neu5ac species are found in various sources such as equine erythrocyte gm3, starfish a. rubens and fish [34] [35] [36] [37] [38] . c4-o-acetylated neu5ac facilitates the initial attachment of viruses to target cells. like the influenza c virus, infectious salmon anemia virus (isav), a member of the orthomyxoviridae family, contains he and hef proteins to mediate virus entry and exit. c4-o-ac neu5ac is the major receptor determinant of isav in receptor binding and destruction [38] , while the influenza c virus recognizes c9-o-ac neu5ac. the acetylesterase rde of isav cleaves c4-o-ac via 4-sa-o-acetylesterase with a short turnover time, whereas c9-o-ac neu5ac is cleaved by 9-sa-o-acetylesterase with a long turnover time [34] . the position of sa o-acetylation is linked to functions including substrate differentiation of enzymes such as nas and esterase by c4 o-ac. previous development of o-ac site-selective na inhibitors were based on the conceptual consideration of different o-ac positions. the o-ac of sas is site-specific, as c4 of neu5ac is considered to be a potential position for modification. historically, inhibitors of influenza a and b viruses-sialidases were designed by von-itzstein in 1993 [39] . the ac group-based c4 substitution interacts with amino acid glu-119 present in the active site of sialidase. guanidine-attached c4 of c2-c3 unsaturated sa (neu5ac2en) inhibits activity of sialidases isolated from influenza a virus (singapore/1/57) and b virus (victoria/102/85). the same scenario was applied for sialidase inhibition of the human parainfluenza virus type 3, which has hn and fusion proteins [40] . the c4 of neu5ac2en was substituted by alkyl groups such as the o-ethyl group. for example, zanamivir has a substitution with a 4-guanidino group with an ic50 of 25 µm. thus, sialidase inhibition is important for c4 modification of neu5ac2en. later, oseltamivir with the tradename tamiflu (basel, switzerland) and zanamivir with the tradename relenza (london, uk) were established [41] . these drugs exhibit some adverse side effects that restrict clinical use. the sa 9-o-acetylation in hosts allows hosts to evade influenza a virus hemagglutinin (ha) recognition and some lectins of factor h (fh), cd22/siglec-2 and sialoadhesin/siglec-1. instead, the influenza c virus ha recognizes the hosts. β-elimination and permethylation eliminate the 9-o-acetyl group from sas. chemical modification of the c-9 position of neu5,9ac2 generates a 9-n-acetyl analog, 9-acetamido-9-deoxy-n-acetylneuraminic acid (neu5ac9nac), a mimic of neu5,9ac2 with influenza c virus-binding capacity, which is not cleaved by the he [42] . sa o-acetylesterase regulates the presence of 7,9-o-ac and 9-o-ac. sa o-acetylation and deacetylation are involved in development, cancer and immunology. sa o-acetylation alters host lectin bindings such as siglecs [29] . the presence of 9-o-ac can also reduce the activity of nas [43] . sa modifications regulate pathogen binding or pathogen nas. influenza a/b/c/d viruses use sa as their entry receptors. influenza a and b subtypes bind to sas via ha and na to allow endocytosis of the virus and fusion of the viral envelope with endosomes. in contrast, influenza c and d subtypes bear only one coated glycoprotein, termed the he fusion protein (hef). the hef acts as the ha and na. hef recognizes 9-o-acetyl sa for entry into cells, while the esterase domain removes 9-o-acetyl-groups and liberates the virus from mucus and mis-assembled virus aggregates after budding. the 9-o-ac on cells prevents the na activity and ha binding of the influenza a type virus [44] . certain viruses use glycoproteins such as ha, he, s and hef for host receptor binding or destruction. coronaviridae, orthomyxoviridae, paramyxoviridae and adenoviridae utilize sas as binding molecules for attachment and entry. however, only limited human pathogens recognize o-ac sa. influenza a and b viruses bear two spikes of receptor-binding ha and na [45] . hef is indeed an ancient type of sa-o-acetylesterase. in contrast to a/b, the influenza c virus bears one spike with triple functions of hef as a homotrimer [46] . each hef subunit bears two neu5,9ac2-binding sites and binds to the 9-o-acetyl group. in parallel, another modification of o-acetylation is found. indeed, influenza c virus bears sa-o-acetylesterase [47] , which converts 5-n-acetyl-9-o-neuac (neu5,9ac2) to 5-neu5ac. the 9-o-acetyl sa is a unique determinant for the influenza c virus receptor and neu5,9ac2 is crucial for receptor activity, but not neu5gc or neu5ac [48] . neu5,9ac2 is an essential determinant for influenza virus c type-specific host cell tropism. nas cleave the α-ketosidic linkages to the d-gal or galnac. sa-o-acetylesterases cleave different o-acetyl linkages ( figure 5 ). the oh-group of tyr224 and the guanidino group of arg236 interact with the ch 3 co-carbonyl oxygen [49] . hef sa-o-acetylesterase is found in several enveloped (+) ssrna viruses of influenza c virus and also in certain covs and toroviruses [47] . the covs are different from the orthomyxoviruses, which hold a segmented (−) ssrna genome and are instead evolutionary linked to the family coronaviridae, order nidovirales [45] . covs and toroviruses of the coronaviridae family are specific for the o-ac sa receptors. their s and he glycoproteins are similar to influenza c virus hef. covs and all toroviruses bear he gene form class i envelope membrane proteins of about 400 amino acid residues which bear 7 to 12 n-glycosylation sites [50] . he multimer forms enter virions. bovine cov (bcov) and hcov-oc43, similar to influenza c virus, recognize neu5,9ac2 and bear sa-9-o-acetylesterase [8] . cov hes are all o-acetylesterases. the he enzymes found in torovirus, cov and influenza c virus are evolutionarily interspecies-mutated with about 30% homology by heterologous rna recombination [51] and horizontal gene transfer. therefore, viral hes are diverse and widespread over evolution. hes as envelope proteins are found in covs, orthomyxoviruses and toroviruses. coronaviral hes are involved in virus attachment to sa species. he protein in β-covs binds to neu5,9ac2 form sa and agglutinates the red blood cells (rbcs) of rodents [52] . as with sa-o-acetylesterase, he potentiates viral entry with the s protein and spreading via the mucosal glycans. it contains a carbohydrate-recognizing domain (crd) known in lectin. the he glycan-binding domain (gbd) mediates virus attachment to sas on host cells. he is the only ha. this indicates that compared to the s glycoprotein, he is only minor a ha and the s glycoprotein mainly attaches to the cell surface. the he protein of murine hepatitis virus (mhv), an enveloped cov, binds to sa-4-acetylester or sa-9-o-acetylester of the carcinoembryonic antigen cell adhesion molecule 1a (ceacam; known as cd66a) as the key receptor [53] . murine covs hes acquired by horizontal gene transfer, bind to c9-o-ac neu5ac. however, some murine cov hes cannot bind to c4-o-ac neu5ac. the original mouse mhv he binds to c9-o-ac neu5ac, while the mhv s-strain he evolutionarily acquired the ability to bind to c4-o-ac neu5ac [12, 53, 54] . in terms of structure, the c5 n-and c9 o-ac neu5ac-accomodating hydrophobic pocket was shifted to a c5 n-and c4-o-ac neu5ac-accomodating pocket [55] . type i he is specific for the 9-o-acetylated sas (9-o-ac-sas). type ii he is specific for 4-o-ac-sas. the sa-binding shift indicates quasi-synchronous adaptations of the sa-recognition sites of the lectin and esterase domains. type i he monomers of β-cov lineage a have a bimodular enzyme-lectin domain similar to cellular glycan/carbohydrate-modifying proteins. originally, he homologs are found in various viruses including toroviruses and orthomyxoviruses such as the influenza virus c/d and isavirus, as well as the exceptional case of β-cov lineage a among covs. the he gene was transmitted to a β-cov lineage a progenitor via horizontal gene transfer from a 9-o-ac-sia-recognizing hef, as shown in influenza virus c/d. he acquisition and expansion occurred by cross-species transmission over he evolution and this phenomenon reflects viral evolutionary adaptation to host sa-containing glycans. therefore, cov he receptor switching precedes virus evolution driven by sa-containing glycan diversity of hosts. for instance, the bcov he prefers 7,9-di-o-ac-sas, which is also a target of the bovine torovirus he. for a more outstanding case, such a switching event occurred in the murine covs for the β-cov lineage a type switch toward o-ac-sa recognition. in the he specificity of murine covs, two different murine cov subtypes of virus group exist with one subtype possessing the typical 9-o-ac-sa (type i) attachment factor and the other exclusively 4-o-ac-sa (type ii) attachment virus group [56] . the first coronaviral he proteins identified were from the porcine hemagglutinating encephalomyelitis virus (phev), bcov and hcov-oc43, which bear sa-9-o-acetylesterases similar to hef [8] . rat cov (rcov) has sa-4-o-acetylesterases, converting neu4,5ac2 to neu5ac [53, 57, 58] . some murine covs prefer 4-o-ac-sas and others 9-o-ac-sas. hcov-oc43 and bcov prefer α2-6-sa 9-o-acetylation by their sa-o-acetyleseterases. the s glycoproteins of bcov and hcov-oc43 are neu5,9ac2-recognizing lectins and agglutinate murine, rat and chicken erythrocytes due to the enriched 9-o-ac-sa species [52] . bcov and hcov-oc43 adapted to sa receptor determinants of 9-o-ac-sa receptors [59] . for a second receptor, the binding of s glycoprotein to neu5,9ac2 receptor is essential for entry into cells. bcov-infection is prevented by prior treatment of cells with na enzyme or with viral sa-o-acetylesterases, blocking the roles of he and s glycoprotein in sa-dependent entry to host cells. several β-cov genera such as bcov bind to o-acetylated sas and bear an acetylesterase enzyme to act as a host cell rde. certain α-cov and γ-cov are deficient for the comparable acetylesterase enzyme but have a preference to neuac or neugc type sa species. infectious bronchitis virus (ibv) and transmissible gastroenteritis virus are such examples. additionally, both α-cov and γ-cov also include sub-members deficient of any sa-recognizing activity. during evolution, some subtypes of sars-cov and hcov-229e acquired sa-binding capacity. the sa-binding activities of bcov, transmissible gastroenteritis coronavirus (tgev) and ibv are well known [60] . in α-covs such as tgev, ha-activity is attributed to the sa-recognizing activity to α2,3-neugc [61, 62] . the sa-binding site is present on the n-terminal region of the s-glycoprotein of tgev. tgev has two types with enteric and respiratory tropism. the respiratory tgev has the porcine aminopeptidase n (papn)-binding domain and sa-binding domain. nucleotide 655 of the s gene is essential for enteric tropism and the s219a mutation of the s glycoprotein confers the enteric to respiratory tropism shift. in addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the y374-t375insnd shift of the s glycoprotein, causes enhanced enteric tract tropism. tgev interacts with sa species on mucin-like glycoprotein (mgp), a highly glycosylated protein, in an sa-dependent manner, on mucin-secreting goblet cells [6] . mgp sa-binding allows virus entry via the mucus layer to the intestinal enterocytes. different from tgev, the s glycoprotein of porcine cov has no hemagglutination activity due to deletion of the sa-binding site of the s glycoprotein [61] . the loss of sa-binding activity is correlated to the non-enteropathogenicity. sas function as ha-mediated entry determinants for tgev, causing the enteropathogenic outcome of the virus, and sa-recognition activity is also responsible for virus amplification in cells. sa-binding activity-deficient tgev can propagate in cells through papn, known as cd13, as a receptor [62, 63] . the sa-binding activity potentiates infection and is crucial for intestinal infection. in β-cov, he mediates viral attachment to o-ac-sas and its function relies on the combined cbd and rde domains. most β-covs target 9-o-ac-sas (type i), but certain strains switched to alternatively targeting 4-o-ac-sas (type ii). for example, the sa-acetylesterase enzyme in bcovs and hcov-oc43 is known to have hemagglutinizing activities as a type of sa-9-o-acetylesterase [8] . the sa-acetylesterase is the he surface glycoprotein in bcov. the three-dimensional structure of bcov he is similar to other viral esterases [9] . the he gene is found only in the β-cov genus. the acetylesterase of murine covs differs in its substrate binding specificity from that of bcov and hcov-oc43, which is specific for o-acetyl residue release from sa c-9. murine covs prefer to esterize 4-o-acetyl-neuac [64] . the β-cov acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65] . as deduced from the sa acetylesterase of hcov-oc43 [8] , the 9-o-ac-sa species is a receptor binding determinant for erythrocytes and entry into cells [59] . the bcov he protein has dual activity of acetylesterase and ha [9] . bcov widely agglutinates erythrocytes and purified he only agglutinates neu5,9ac2-enriched erythrocytes of rats and mice. bcov and hcov-oc43 can agglutinate chicken erythrocytes, while purified he cannot. in contrast to the he protein, purified s glycoprotein can agglutinate chicken erythrocytes [52] , indicating that the major ha is the s protein which acts as the major sa-binding protein. however, the role of o-ac-sas is not certain to be essential in receptors, and sa-binding activity may be essential only to the he protein, but not to the s glycoprotein [54] . in γ-covs, ibv strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. ibv prefers to recognize α2,3-neuac and the sa functions as a host entry receptor for infection [66] . glycosylation of ibv m41 s1 protein rbd is crucial for interaction with chicken trachea tissue and rbd n-glycosylation confers receptor specificity and enables virus replication. the heavy glycosylated m41 rbd has 10 glycosylation sites. n-glycosylation of ibv determines receptor specificity. however, the host receptor has not yet been found. na treatment reduces the binding of soluble s to kidney and tracheal epithelial cells. the ibv s protein recognizes epithelial cells in a sa-dependent manner. the sa-binding ability of ibv is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67] . the sa-binding site is located on s1 of the ibv s protein, although the ibv-specific protein receptor is not known. in contrast to bcov or hcov-oc43, ibv lacks an rde. sa binding of ibv is likely more essential than in other viruses such as tgev. in torovirus, which belongs to the family coronaviridae, the toroviruses are grouped into the torovirinae subfamily and the torovirus genus. the known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. they mildly infect swine and cattle through the he protein, which is similar to the β-cov he protein [68] . the he protein is a class i membrane glycoprotein which forms homodimers with a mw of 65 kda. the rde protein he reversibly binds to glycans [15] through binding to sas. the acetyl-esterase activity disrupts sa binding. he hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pnpa) [69] . similar to cov, torovirus he is an acetylesterase type, which cleaves the o-acetyl group from the sa c-9 position using neu5,9ac2 and n-acetyl-7(8),9-o-neuac [64] . however, torovirus he exhibits a restricted specificity for the neu5,9ac2 substrate, but not for the neu5,7(8),9ac3 substrate, with a unique sa-binding site generated by a single amino acid difference in porcine thr73 and bovine ser64 for each he [70] . the s glycoprotein sars-cov-2 initiates infection of the host cells. the molecular basis of cov attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-em defining the structure of the cov-oc43 s glycoprotein trimer complexed with a 9-o-acetylated sa [56] . cryo-em structures of the trimeric ectodomain of s glycoprotein were observed using forms complexed with neu5ac, neu5gc, sialyl-lewis x (sle x ), α2,3-sialyl-n-acetyl-lactosamine (α2,3-slacnac) and α2,6-slacnac, respectively. the receptor-binding site is commonly conserved in all cov s glycoproteins, which attach to 9-o-ac-sa species with similar ligand-binding pockets to the cov hes and influenza virus c/d hef glycoproteins, indicating conserved recognizing structures [25] . the s glycoprotein-9-o-acetyl-sa interaction resembles the ligand-binding pockets of cov hes and influenza virus c/d he fusion glycoproteins. hcov-oc43 and bcov recognize 9-o-ac-sa. s glycoproteins engage 9-o-acetyl-sas. the 9-o-acetyl sas are the binding site for hcov-oc43 s glycoprotein and related β-1 cov s glycoproteins, however sa-binding sites on the 9-o-acetyl sialyl receptors of mers-cov s glycoprotein and hcov-oc43 s glycoprotein are different [71] . thus, covs use two different entry and attachment receptors. therefore, s glycoproteins of covs are distinct from influenza virus a has, which bind to the neu5ac species by conserved binding sites. the ligand-binding sites of bcov he enzyme, influenza hef enzyme and cov s glycoprotein have evolved 9-o-ac-sa binding through hydrogen bonding with the 9-o-acetyl carbonyl group and hydrophobic pocket formation with the 9-o-acetyl methyl group [71, 72] . however, influenza ha cannot bind to 9-o-acetyl-sas but can bind to neugcs [73] . the hcov-oc43 s glycoprotein, hcov-hku1 s glycoprotein, bcov s glycoprotein and phev s glycoprotein, therefore, share the ligand-binding specificity of influenza c/d hef enzyme, although they are functionally more similar to influenza virus a/b ha, whereas cov he or influenza virus a/b na have rde activities cov hes are functionally similar to influenza virus c/d hef glycoproteins. in cov, the s glycoprotein recognizes the 9-o-ac-sa sugar, while the he acts as the rde enzyme with sa-o-acetyl-esterase activity to release virions from infected host cells. for example, hcov-oc43 also has a similar he as an rde [71] . in influenza c and d viruses, hef glycoproteins act similarly to the cov he [74] . in influenza a virus, rde na releases virions from host cells. however, mers-cov does not have a similar enzyme and thus mer-cov binding to sa receptors is mediated by energetically reversible interactions of the lipid rafts with increased sa receptors [75] , thus enhancing dipeptidyl peptidase 4 (dpp4) or carcinoembryonic antigen-related cell adhesion molecule 5 (ceacam5) recognition power and viral entry [76] and membrane-associated 78-kda glucose-regulated protein (grp78) [77] . mers-cov s glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. mers-cov s glycoprotein attachment is not observed for 9-o-acetylated or 5-n-glycolyl sas, but is observed for α2,3-sa linkage over α2,6-sa linkages. sa-binding sites of mers-cov s glycoprotein and hcov-oc43 s glycoprotein are not conserved [78] , although they engage α2,3-sas on the avian host cell surface [79] . mers-cov recognizes α2,3-sa and to a lesser extent the α2,6-sas and sulfated sle x for binding preference. thus, s glycoproteins may have independently evolved sa recognition. the acquisition of sa-binding ability of mers-cov s seems to be an evolutionarily recent event, because hku4 s1 and hku5 s1 cannot hemagglutinate human erythrocytes [75] , indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of covs. in conclusion, cov recognition of 9-o-ac-sas for infection is based on a conserved sequence for engagement of sa-related carbohydrate ligands across covs and orthomyxoviruses. cov s spikes recognize diverse surface molecules as the attachment or entry site. animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. specifically, s glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80] . covs recognize multiple host receptors via distinct s domains. the host receptors for β-cov sars-cov includes angiotensin-converting enzyme 2 (ace2). as a lineage c β-cov, the mers-cov s glycoprotein binds to dpp4 [81] [82] [83] . mers-cov s glycoprotein recognizes α2,3-sa over α2,6-sa-bearing receptors. the n-terminal subunits of the s1/s1a/s1b/s1d complex of mers-cov recognize dpp4. mers-cov recognizes ceacam5 as the attachment factor for entry [78] . among the six hcovs, the α-cov hcov-229e s protein recognizes human apn (hapn) [84] . α-cov hcov-nl63 and the lineage b β-cov sars-cov s glycoproteins bind to ace2. meanwhile the protein receptors specific for lineage a β-covs such as hcov-hku1 and hcov-oc43 are not known yet. bcov, hcov-oc43, hcov-hku1 and tgev recognize o-acetyl-sas as attachment molecules. in addition to o-acetyl-sa, hcov-hku1 spikes additionally bind to major histocompatibility complex class i (mhc-i) c as attachment sites [85] . sars-cov uses dendritic cell (dc)-specific intercellular adhesion molecule (icam)-3-grabbing nonintegrin (dc-sign) for attachment [86] . for glycan interaction, hcov-nl63 and mouse hepatitis virus utilize heparan sulfate (hs) proteoglycans as attachment enhancers [87] . in general, ace2, apn, heat shock protein a5 (hspa5), furin, heparan sulfate proteoglycans (hspgs) and o-acetyl-sa are covs-recognizing candidates. structure and role of the host sars-cov receptor ace2 sars-cov-2 needs ace2 for entry. host proteases such as human ace2 help viral entry through removement of a barrier to enter human cells through unknown receptors. human ace2 is known for its role as the sars-cov-2 entry receptor and the sars-cov receptor. the enzyme ace-2 in the renin-angiotensin system (ras) is associated with cov entry into lungs. ace2 mediates sars-2002 entry into host cells via s glycoprotein interaction with the ace2 receptor. the ace2 levels on the plasma membrane correlate with virus infectivity. ace2 expression is present in most tissues such as the lung epithelium. it is highly expressed by respiratory epithelial cells and type i/ii lung alveolar epithelial cells [88] . the host receptor is not linked to the classification of covs. mers-cov, a β-cov, does not recognize the ace2 receptor. in contrast, the α-cov hcov-nl63 recognizes the ace2 receptor. ace2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. it contains 17 amino acid residues as a signal peptide in the n-terminal region, a type i membrane-anchored domain in the c-terminal region, an extracellular n-terminal domain with heavy n-glycans, a n-terminal sars-cov-binding and carboxypeptidase site and a short c-terminal cytoplasmic tail. the ace2 gene is located on chromosome xp22. two ace2 forms are known, a membrane-bound form and a soluble form. ace cleaves angiotensin i (ang i) substrate to ang ii. ang ii recognizes the ang ii receptor type 1 (at1r), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ace2 has the opposite function of ace. ace2 is a close homolog to human ace. ace2 activity on ang ii is about 400-fold higher than that on ang i. ang-1 to ang-7 recognize the g protein-coupled receptor (gpcr) mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-ang ii-signaling. therefore, the ace2-ang-1 to ang-7 axis is a target candidate for cardiovascular diseases. ace2 shows similar binding structures between ncov and sars-cov. the three proteins of ace, ang ii and at1r contribute to progression of lung injury in humans. ace2 removes a single amino acid residue from ang ii to yield the vasodilator, named ang 1-ang 7. ace2 cleaves ang-i to ang 1-ang 9 and ang ii to ang-1 to ang-7. the biggest difference between ace2 and ace is that ace2 has a non-inhibitory property by ace inhibitors. pulmonary ace2 is potentially a candidate target in cov-involved inflammatory pathogenesis. if ace inhibitors and ang ii-at1 blockers are dosed, ace2 expression is increased. however, currently we have no conclusive evidence that the inhibitors help sars-cov or sars-cov-2 entry. rather, sars-cov infection reduces ace2 expression. therefore, sars-cov-2 host tropism is not related to ace2 expression. ace2 levels and ang ii/ang 1-7 levels regulate the pathogenic progression. ace2 expression is upregulated by gene polymorphisms and ace inhibitors or angiotensin ii receptor blockers such as sartans. a disintegrin and metallopeptidase domain (adam) family of zn-metalloproteinases belongs to membrane proteins. the well-known adam17 is a tnf-α-converting enzyme (tace), called the sheddase for tnf-α. other adam sheddase family members include adam9, adam10 and adam12. adam17 mediates ace2 shedding. sars-cov s glycoprotein activates cellular tace and consequently facilitates virus entry. soluble ace2 as the n-terminal carboxypeptidase domain form is derived from the original ace2 form by an adam17 metalloprotease in the membrane [89] . adam17 is indeed an enzyme that can convert membrane type pro-tnf-α to soluble tnf-α, a functional proinflammatory cytokine. therefore, adam17 inhibition indicates an anti-inflammatory response and adam17 inhibitors are promising candidates for tnf-α-induced inflammatory diseases. the short c-terminal domain of ace2 is removed by adam17 and tmprss2. however, tmprss2 cleaves ace2 competitively with the adam17 metalloprotease. sars-s protein-ace2 binding leads to adam17/tnf-α-converting enzyme (tace)-cleavage of ace2, facilitating extracellular ace2 shedding and consequent sars-cov entry into host cells [90, 91] . only tmprss2 cleavage allows sars-cov entry into host cells through endocytosis and fusion. soluble ace2 also recognizes the virus and prevents sars-cov-2 infection. sars-cov-2 infection requires membrane ace2 and tmprss2. the ace2-b0at1 complex binds to the s glycoprotein of sars-cov-2. intestinal membrane ace2 and lung tmprss2-shedded ace2 can act as alternative entry sites for sars-cov-2. sars-cov-2 infects the lungs and intestine via tmprss2-cleaved ace2. if tmprss2 is engaged in sars-cov-2 entry and ace2 downregulation, tmprss2 inhibition would lead to covid-19 prevention. although ace2 is expressed both in type i and type ii lung alveolar epithelial cells, sars-cov and sars-cov-2 target only type ii epithelial cells due to the ace2-tmprss2 interaction. therefore, supplementation of ace2 (soluble ace2) or ang-1 to ang-7 should be a way to reduce sars-cov-2-related symptoms. tmprss2-cleaved ace2 is involved in sars-cov and mers-cov infections. sars-cov-2 uses ace2 for cell entry through tmprss2 priming of the s glycoprotein (figure 7) . infection of the h7n9 influenza and h1n1 influenza a subtype viruses are also mediated by tmprss2-cleaved ace2. this implies that tmprss2 can be targeted as a strategic antiviral therapy [92] . transmembrane protease serine 2, termed tmprss2, a type ii tm ser protease (ttsp), also cleaves ace2. the human tmprss2 gene, located on chromosome 21, comprises androgen receptor elements (ares) in the upstream 5 -flanking region [93] . tmprss2 expression is regulated in an androgen-dependent manner. the tmprss2 gene encodes 492 amino acids. the original form is cleaved into the major membrane form and the minor soluble form. tmprss2 activates protease activated receptor 2 (par-2) and activated par-2 upregulates matrix metalloproteinase-2 (mmp-2) and mmp-9. tmprss2-activated hepatocyte growth factor (hgf) induces c-met receptor signaling. tmprss2 activates sars-cov and mers-cov. the sars-cov s glycoprotein is cleaved by host-borne tmprss2, human airway trypsin-like protease (hat), tm protease, serine 13 (mspl), serine protease desc1 (desc1), furin, factor xa and endosomal cathepsin l/b. sars-cov can enter cells upon cleavage by protease tmprss2 or endosomal cathepsin l/b [90] . virus s protein precursor is cleaved by host proteases. the spikes are cleaved by endosomal cathepsin and by golgi or plasma membrane tmprss2 in the step of assembly or attachment and release. the serine protease inhibitor camostat effectively blocks lethal sars-cov infection to mice. however, serine protease and cathepsin inhibitors are not effective. thus, tmprss2 is suggested to be an acting protease for sars-cov entry into host cells, but not by cathepsin. cis-cleavage liberates sars-cov s glycoprotein fragments into the extracellular supernatant. trans-cleavage activates the sars-cov s glycoprotein on the target cells, potentiating efficient sars-cov s glycoprotein-driven viral fusion. tmprss2-activated sars-cov facilitates enveloped virus entry into cells. tmprss2 is important for sars-cov entry and infection [81, [94] [95] [96] . the fact that sars-and mers-cov infections are potentiated by tmprss2 indicates that tmprss2 is a promising target for therapeutic agents. for example, several ser protease inhibitors such as camostat mesylate inhibit tmprss2-ace2-involved sars-cov-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated tmprss2 inhibition in calu-3 cells greatly reduces sars-cov viral titers and improves survival rate in sars-cov infected mice. a treatment of 10-μm camostat blocks mers-cov entry to african green monkey kidney (vero)-tmprss2 cells and blocks viral rna synthesis in calu-3 cells upon mers-cov infection. aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. another serine protease inhibitor, nafamostat, inhibits mers-cov entry and infection by tmprss2 the fact that sars-and mers-cov infections are potentiated by tmprss2 indicates that tmprss2 is a promising target for therapeutic agents. for example, several ser protease inhibitors such as camostat mesylate inhibit tmprss2-ace2-involved sars-cov-2 entry. camostat, a serine protease inhibitor, aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. another serine protease inhibitor, nafamostat, inhibits mers-cov entry and infection by tmprss2 inhibition [93] . nafamostat mesylate blocks the tmprss2-ace2-involved sars-cov-2 envelope-pm fusion and prevents sars-cov-2 entry [95] . nafamostat mesylate inhibits viral entry and thrombosis in covid-19 patients. similarly, an fda-approved mucolytic cough suppressant, bromhexine hydrochloride (bhh), inhibits tmprss2 (ic50 0.75 µm) and hence blocks infection of cov and influenza virus. mprss2 as a host factor plays a pivotal role in sars-cov and mers-cov infections. fda-approved tmprss2 inhibitors are yet under development. because tmprss2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against cov infection. the ser exopeptidase dpp-4/human cd26 (pdb: 4l72), a type ii tm ectopeptidase, functions as a host cell receptor for mers-cov. the rbd structure was characterized by crystallography approaches of the mers-cov s glycoprotein-dpp4 complex. dpp4 is a single type ii tm glycoprotein with a small cytoplasmic tail in the n-terminal region and is present as a homodimeric form. dpp4 cleaves x-proline dipeptides from the n-terminal region. s glycoprotein recognizes sa species and dpp44 as the attachment and entry receptors, respectively. the mers-cov s1 n-terminal domain attaches to dpp4 as the host receptor [81] . the s2 c-terminal domain of mers-cov anchors to cellular pm to enter. mers-cov s glycoprotein is cleaved at a sequence between the s1 and s2 domains [96] . another cleavage site s2 is present in the s2 domain. mers cov s glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express dpp4. binding capacities are hindered by the sa 9-o-acetyl group or sa 5-n-glycolyl group [75] . entry of host cells needs binding of s glycoproteins to the ceacam receptor, forming s-protein-mediated membrane fusion. the trimeric s glycoprotein bears three s1 receptor heads. the three s1 heads of the virus bind to three receptor molecules on the host cell. cholesterol is indirectly involved in membrane fusion through ceacam engagement into "lipid raft" microdomains, increasing multiple s protein interaction with the receptors and triggering membrane fusion [97] . the enveloped cov, mhv, binds to ceacams on cholesterol-depleted cells in bhk cell cultures. the ntd of s1 recognizes ceacam1. for mers-cov, another ceacam5 isoform is the attachment factor for virus entry [75] . the cov s1 ntd has a similar tertiary structure to human galactose-recognizing galectins. mhv s1 ntd binds murine ceacam1a and bcov s1 ntd binds sugar [98] [99] [100] . ceacam1a is a cell adhesion protein (cam) and its mrna is alternatively spliced. the cryo-em structure of mhv s complexed with ceacam1a was elucidated [101] . thus, hcovs evolutionarily combined the galectin gene of hosts into their s1 glycoprotein gene, while bcov s1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. mhv s1 protein also evolutionarily acquired murine ceacam1a-recognizing activity [102] . therefore, covs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80, 103] . on the host side, to escape the lethal pressure from cov infections, hosts have also evolved to acquire sa-binding proteins such as siglecs to inhibit or activate the innate immune cells. both raft and non-raft ceacams are involved in the virus-cell membrane fusion event. formation of ceacam-associated mhv particles or ceacam-induced mhv fusion is possible by gpi-anchored ceacams through the binding between ceacam and s proteins. however, mhv can bind to both gpi-and tm-anchored ceacams. in addition, soluble ceacams also mediate s glycoprotein-driven fusion [104] . this implies that membrane anchors are not intrinsically necessary. in fact, ceacams are present in different tissue-specific isoforms [105] . nevertheless, gpi-anchored ceacams are more effective for mhv infection than tm-anchored ceacams. soluble ceacam receptors can bind to viral s glycoproteins and induce conformational shifts to acceptable s glycoprotein-involved membrane fusions [106] . for example, soluble ceacam forms interacts with s1 fragments [107] and alters the s1-s2 association stability [108] and s1 oxidation confirmation [109] . s proteins are structurally shifted prior to membrane fusion. for the cross-linking of viruses and cells, integral hydrophobic peptides of the s2 chain are embedded into membranes via membrane hydrophobic cholesterols. mers-cov s glycoprotein also recognizes a 78-kda glucose-regulated protein (grp78) or heat shock 70 kda protein 5 (hspa5), known as binding immunoglobulin protein (bip) or byun1, which is encoded by the hspa5 gene in humans. hsp5a is a er-resident unfolded protein response (upr) protein. stressed cell status such as viral infection increase expression and translocation of hspa5 to the pm to form a membrane protein complex. grp78 modulates mers-cov entry in the presence of the dpp4 as a host cell receptor. additionally, lineage d β-cov and bat cov hku9 (bcov-hku9) also bind to grp78 [76] . a cell surface receptor, grp78, was predicted to be another covid-19 receptor as an s glycoprotein binding site [110] . the prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. grp78 or bip is a chaperone protein located in the er lumen [111] . known er-bound enzymes include activating transcription factor 6 (atf6), inositol-requiring enzyme 1 (ire1) and protein kinase rna (pkr)-like er kinase (perk) [112] . depending on threshold of unfolded protein accumulation, grp78 releases ire1, atf6 and perk, and is activated, resulting in translation inhibition and refolding. stress-overexpressed grp78 can avoid er retention and is translocated to the membrane. grp78 translocated to the cell pm can recognize viruses by its substrate-binding domain (sbd) for virus entry into the cell (figure 8 ). in sequence and structural alignments and protein-protein docking, rbd of the cov spike protein recognizes the grp78 sbdβ as the host cell receptor. the predicted region iii (c391-c525) and region iv (c480-c488) of the s glycoprotein and grp78 are highly potential binding sites. region iv is the grp78 binding-driving force. these nine amino acid residues are being molecularly targeted for the designation and simulation of covid-19-specific drugs. this process is the mechanism underlying the cell surface hspa5 (grp78) exposure and this is exploited to be used for pathogen entry. such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, ebola virus, zika virus and hcovs-as well as fungal rhizopus oryzae [113] [114] [115] [116] . therefore, natural products can inhibit cell-surface hspa5 recognition of the viral s glycoprotein. technology of molecular modeling docking with structural bioinformatics. grp78 or bip is a chaperone protein located in the er lumen [111] . known er-bound enzymes include activating transcription factor 6 (atf6), inositol-requiring enzyme 1 (ire1) and protein kinase rna (pkr)-like er kinase (perk) [112] . depending on threshold of unfolded protein accumulation, grp78 releases ire1, atf6 and perk, and is activated, resulting in translation inhibition and refolding. stressoverexpressed grp78 can avoid er retention and is translocated to the membrane. grp78 translocated to the cell pm can recognize viruses by its substrate-binding domain (sbd) for virus entry into the cell (figure 8) . in sequence and structural alignments and protein-protein docking, rbd of the cov spike protein recognizes the grp78 sbdβ as the host cell receptor. the predicted region iii (c391-c525) and region iv (c480-c488) of the s glycoprotein and grp78 are highly potential binding sites. region iv is the grp78 binding-driving force. these nine amino acid residues are being molecularly targeted for the designation and simulation of covid-19-specific drugs. this process is the mechanism underlying the cell surface hspa5 (grp78) exposure and this is exploited to be used for pathogen entry. such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, ebola virus, zika virus and hcovs-as well as fungal rhizopus oryzae [113] [114] [115] [116] . therefore, natural products can inhibit cell-surface hspa5 recognition of the viral s glycoprotein. among the six hcovs, the α-cov hcov-229e s protein recognizes hapn known as cd13 or membrane alanyl aminopeptidase (ec 3.4.11.2). porcine epidemic diarrhea coronavirus virus (pedv) binds to protein receptor apn of human-and pig neuac species as its co-receptor. apart from hapn, tgev and pedv bind to sa species [117] , although sa recognition by tgev is not essential in the among the six hcovs, the α-cov hcov-229e s protein recognizes hapn known as cd13 or membrane alanyl aminopeptidase (ec 3.4.11.2). porcine epidemic diarrhea coronavirus virus (pedv) binds to protein receptor apn of human-and pig neuac species as its co-receptor. apart from hapn, tgev and pedv bind to sa species [117] , although sa recognition by tgev is not essential in the first step of entry cycle. hcov-229e recognizes hapn known as cd13 for its entry receptor. hapn (pdb: 4fyq) or cd13 (ec 3.4.11.2), which is a zn-dependent metalloprotease, has a mw 150 kda with 967 amino acids. cd13 is a type ii tm protein with a short cytoplasmic domain in the n-terminal region and long extracellular region in the ctd. the ctd has a pentapeptide sequence specific for the zinc-mmps. the apn binding domain is located on the ctd of pedv s1 (amino acid 477-629 residues), while the sa-binding domain is found in the n-terminal region of pedv s1 (amino acid 1-320 residues) [118] . cd13 is also a receptor for hcov-229e, human cytomegalovirus, porcine cov tgev, feline infectious peritonitis virus (fipv), feline enteric virus (fecv) and canine-infectious covs [119] [120] [121] [122] . homodimeric cd13 digests luminal peptides. the hapn-encoding anpep gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. if this gene is defective, leukemia or lymphoma are transformed [123] . porcine and human apn exhibit about 80% protein identity. fipv and fecv are in the same group as hcov-229e and tgev. thus, porcine apn is also an attachment site for pig tgev with an additional second receptor. hcov-229e first binds to cd13 and consequently clusters cd13 in caveolae-associated lipid rafts [120] . for glycan interaction, hcov-nl63 and mhv utilize heparan sulfate proteoglycans (hspgs) as attachment enhancers [87, 124] . viruses recognize hspgs as attachment molecules. in the spike (s) protein-deficient virions, the m protein recognizes hspg. the s proteins generally bind to the viral cellular receptor. however, the m protein also acts as a receptor in the early step of hcov-nl63 infection. the m membrane protein of hcov-nl63 recognizes the attachment site of hspgs. hcov-nl63 m protein binds to hspg for the initial attachment of virus to host cells and thereafter, the m and s proteins cooperate for virus entrance into the host cells [125] . hspgs are glycosaminoglycan (gag)-carrying proteins frequently used as a secondary receptor for viral entry. hspgs are composed of covalent-bonded hs chains as a gag form. the hs gag linkage structure of tetrasaccharide exhibits gluaβ1,3glcnacα1,4galβ1,3galβ1,4xylβ-o-serine. glycosyltransferases involved in hs gag synthesis include glcat-ii (glucuronosyltransferase) and glcnact-ii (n-acetylglucosaminyltransferase ii) for heparan sulfate synthesis (figure 9 ). gag is used as docking sites for virus interaction with the host cell surface. gags contain negatively charged n-and o-sulfated sugars [126] . the biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of hs and chondroitin sulfate gag have been elucidated in caenorhabditis elegans [127] . the negative charges mediate the interaction of gags and their ligands through electrostatic forces. interaction of hspg with ligands potentiates many virus infectious cycles. for examples, adeno-associated virus, human t cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis b and c viruses, kaposi's sarcoma-associated herpesvirus, human papilloma viruses and merkel cell polyoma virus recognize the hspgs [128, 129] . hspgs increase virulence upon interaction with viral factors required for viral attachment and replication. in caenorhabditis elegans [127] . the negative charges mediate the interaction of gags and their ligands through electrostatic forces. interaction of hspg with ligands potentiates many virus infectious cycles. for examples, adeno-associated virus, human t cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis b and c viruses, kaposi's sarcoma-associated herpesvirus, human papilloma viruses and merkel cell polyoma virus recognize the hspgs [128, 129] . hspgs increase virulence upon interaction with viral factors required for viral attachment and replication. sas are predominant surface determinants for pathogen attachment, adherence and entry to host cells. eleven representative vertebrate virus families utilize sas as initial entry receptors or as attachment factors. interaction of virus with sa-containing glycans is complex because virus sa-binding lectins are inherently of very low affinity. viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56] . tm s glycoprotein recognizes oligosaccharide receptors. using cryo-em technology and observed structures of s glycoprotein trimers of cov oc43 complexed with 9-o-acetylated sa, s glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. all cov s proteins show conservation in binding to 9-o-acetyl-sas. mers-cov also recognizes 9-carbon sugar sa species. mers-cov s-1a binds to sa species. for example, saα2,3-over saα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by mers-cov s-1a. binding is hence blocked by sa modification to 5-n-neugc and 7, 9-o-neuac species [73] . for example, impairment of ace2 receptor glycosylation does not influence s-glycoprotein-ace2 interaction, however, sars-cov-2 virus entry into respiratory epithelial host cells was downregulated [133] . changes in ace2 n-glycans do not apparently influence interaction with the sars-cov s glycoprotein, but instead, impair viral s glycoprotein-mediated membrane fusion. the receptor glycan structures decide the entry of some human viruses. changes in ace2 receptor sialylation influences interaction affinity between virus ligands and host receptor. inter-species or individual genetic variations such as drift and mutation may occur in sars-covs. this explains currently emerging differences in cov responses within the same population such as humans. on the other hand, from the aspect of virus ligand, the s glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. virus internalization requires potential glycosylation of viral glycoproteins. among the three viral envelope components, s and m are the major glycoproteins and e is nascent and not glycosylated. the m glycoprotein consists of a short glycosylated ectodomain in the n-terminal region. the s glycoprotein expressed in hemagglutinating encephalomyelitis virus is an ha that recognizes n-acetyl-9-o-neuac as a binding receptor expressed on erythrocyte surfaces [134] . for example, bcovs attach to the surface receptor of n-acetyl-9-o-neuac (9-o-acetylated sas) on host cells. tgev and pedv are currently known as a similar class of such covs. pedv infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for pedv. the s glycoprotein of sars-cov-2 utilizes different glycosylation patterns to recognize its receptors. the glycosylation sites in minimal rbd exhibits similar sites to other covs. the trimeric sars-cov-2 s glycoprotein is also highly glycosylated with 66 n-glycans, but a few o-glycans [135] . glycosylation of s glycoproteins leads to immune evasion. in the mers-cov and the bat-specific cov-hku4, glycosylation is linked to zoonotic infection for fusion-based entry [136] . the emerging cov-pandemic requires therapeutic agents to block the recognition, binding, replication, amplification and propagation of the cov in humans. protease inhibitors, rna synthase inhibitors and s2 inhibitors are potential targets, and several agents are currently being evaluated. efficient therapeutic drugs are the most reliable option for patients. the first attachment step of the viral amplification cycle is initiated on the respiratory cell surfaces, driven by the viral s protein. this is a potential therapeutic target. soon after the sars-cov-2 outbreak initiated, the cov s glycoprotein was demonstrated to recognize ace2 as a binding receptor on human cells. human tmprss2 enzyme influences the cov s glycoprotein activation, to facilitate virus infection. ace2 binding and tmprss2 activation facilitate the covs to attach to human host cells. mouse, nonhuman primate and human cells have been analyzed using single-cell rna new generation sequencing (ngs). for example, for human infection, covs can enter nasal goblet secretory cells, because these cells express the proteins required for sars-cov-2 infection. in the lungs, the proteins are stored in the alveoli like air sacs of type ii pneumocytes. in the intestine, the two proteins are expressed in entero-epithelial cells. ace2 gene expression correlates with the ifn-related genes [137] . ace2 helps lung cells to tolerate cellular damage. therefore, covs may evolutionally take advantage of the defense mechanisms of host cells, hijacking such host-borne proteins. in sars-cov-2, the ace2 receptor is an attachment, entry and infection receptor into the cell, when the s glycoprotein is cleaved by a specific serine protease. sars-cov-2 infection is regulated by glycosylated sars-cov-2 viral particles and glycosylated ace2 in the lung epithelial cells. rbd of the cov s glycoprotein recognizes ace2 [82] . amino acid residues 442, 472, 479, 480 and 487 located on the receptor-binding motif (rbm) of the s glycoprotein rbd recognize human ace2 [138] . trimeric viral s glycoprotein is glycosylated and cleaved by a protease, furin, into two subunits, s1 and s2. subunit s1 is further cleaved into the sa and sb domains and the sb domain recognizes human ace2. the n-glycosylated s2 subunit is involved in virus-ace2 complex formation [139] . therefore, the glycosylated ace2 receptor is a key molecule for virus binding and fusion. plasma sera prepared from infected patients is an alternative medication. the who has suggested this trial since the 2014 ebola epidemic and 2015 mers-cov outbreak. in addition, mab therapy is another option. for example, lca50 mab mimics produced by modification of plasma antibodies isolated from mers-cov patients was valuable [140] . low molecular molecules are being examined for anti-virus activities from alkaloids, glycan derivatives and terpenoids. recently, anti-cov drugs are being approached using molecular modeling, docking and simulation methods. computation-assisted drugs via molecular modeling and docking toward drug targets are applied as anti-viral compounds against covs. they target human ace2, plpro (pdb: 3e9s), the cov main proteinase (pdb: 6y84), 3-chymotrypsin-like (3c-like protease; 3clpro), rdrp, helicase, n7 methyltransferase, human ddp4, rbd, protease cathepsin l, type ii tm ser protease or tmprss2. cov 3clpro (pdb: 6wx4) and the plpro cleave the polyproteins to assemble virus proteins. for newborn rna genomes, rdrp is used as a replicase for the complementary rna strand synthesis, which uses the virus rna template. clq and clq-oh are under investigation worldwide to treat covid-19 ( figure 10 ). clq and its derivative clq-oh block cov replication, amplification and spread in in vitro culture via inhibition of ace2 receptor glycosylation. in hcovs, interaction of the s glycoprotein with gangliosides initially occur as the first entry step during the replication cycle of the virus. clq and clq-oh have been alternative drugs for ra and several autoimmune diseases for 70 years, although they are anti-malaria prophylaxis drugs. clq-oh is an aminoquinoline with less toxicity than clq. clq-oh bears an n-hydroxyethyl side chain, which increases its solubility compared to clq [141] . clq-oh modulates activated immune cells via downregulation of tlr signaling and il-6 production [142] . clinical trials are also under consideration for the efficacy and safety of these drugs. regarding the action mechanism(s), clq and clq-oh-mediated inhibition of ace2 terminal glycosylation was considered. in in vitro vero e6 cells, clq significantly inhibits sars-cov spread by interfering with ace2 function, acting at the entry and post-entry steps of sars-cov-2 replication and infection. the binding affinity of ace2 to s glycoprotein is simulated to be lowered by treatment with clq-oh or clq. clq may modify the binding affinity between ace2 and s glycoprotein by alterations in ace2 glycosylation or modification. clq-oh (ec50 0.72 µm) and clq (ec50, 5.47 µm) inhibit sars-cov-2 [143] . gangliosides initially occur as the first entry step during the replication cycle of the virus. clq and clq-oh have been alternative drugs for ra and several autoimmune diseases for 70 years, although they are anti-malaria prophylaxis drugs. clq-oh is an aminoquinoline with less toxicity than clq. clq-oh bears an n-hydroxyethyl side chain, which increases its solubility compared to clq [141] . clq-oh modulates activated immune cells via downregulation of tlr signaling and il-6 production [142] . clinical trials are also under consideration for the efficacy and safety of these drugs. regarding the action mechanism(s), clq and clq-oh-mediated inhibition of ace2 terminal glycosylation was considered. in in vitro vero e6 cells, clq significantly inhibits sars-cov spread by interfering with ace2 function, acting at the entry and post-entry steps of sars-cov-2 replication and infection. the binding affinity of ace2 to s glycoprotein is simulated to be lowered by treatment with clq-oh or clq. clq may modify the binding affinity between ace2 and s glycoprotein by alterations in ace2 glycosylation or modification. clq-oh (ec50 0.72 μm) and clq (ec50, 5.47 μm) inhibit sars-cov-2 [143] . using computer simulation techniques, clq and clq-oh have been suggested to recognize the enzymatic active site of the udp-glcnac 2-epimerase, known as an essential enzyme in sa biosynthesis [144] , blocking the sialylation of host cells. the mechanism underlying the glycosylation inhibition may support the antiviral properties of clq and clq-oh through interactions of clq or clq-oh with ndp-saccharide mutases or glycosyltransferases [145] . clq was reported to inhibit quinone reductase 2 [146] , known as a catalytic mimetic or structural neighbor of udp-glcnac 2epimerases [147, 148] . if clq or clq-oh inhibits sa synthesis, the inhibitory properties may support the antiviral activity of clq or clq-oh against sars-covs because the sars-cov receptor ace2 using computer simulation techniques, clq and clq-oh have been suggested to recognize the enzymatic active site of the udp-glcnac 2-epimerase, known as an essential enzyme in sa biosynthesis [144] , blocking the sialylation of host cells. the mechanism underlying the glycosylation inhibition may support the antiviral properties of clq and clq-oh through interactions of clq or clq-oh with ndp-saccharide mutases or glycosyltransferases [145] . clq was reported to inhibit quinone reductase 2 [146] , known as a catalytic mimetic or structural neighbor of udp-glcnac 2-epimerases [147, 148] . if clq or clq-oh inhibits sa synthesis, the inhibitory properties may support the antiviral activity of clq or clq-oh against sars-covs because the sars-cov receptor ace2 contains sa species. in fact, clq exhibits in vitro anti-sars-cov-1 activity via defective glycosylation of viral ace2 in vero cells [149] . in addition. the interference of clq or clq-oh with sa synthesis may broadly be applicable as an antiviral because the hcovs or other orthomyxoviruses also utilize sas as entry molecules [150] . however, the detailed mechanisms should be further elucidated. the clq treatment efficacy in covid-19 patients has, however, not been conclusively determined. [151] [152] [153] [154] [155] [156] . in avian cov ibv, structural proteins of the ibv virus are co-localized with pm lipid rafts embedded with the ganglioside gm1. hcov-229e entry is prevented by cholesterol depleted conditions because hcov-229e clusters in caveolae-associated lipid rafts [157] . caveolae of caveolin-1, -2 and -3 are cross-linked [158] and control the molecular distribution between rafts and caveolae in a regulatory mechanism. s protein-cd13 cross-linking occurs via cd13-caveolin-1 sequestering. hcov-229e particles similarly exhibit a longitudinal distribution property. hcov-229e-colocalized caveolin-1 undergoes the next step of virus infection. caveolin-1 knockdown inhibited hcov-229e endocytosis and entry and thus caveolin-1 is essential for hcov-229e infection. tgev also endocytoses by a clathrin-mediated mechanism in mdck cells [159] . other viruses including hcov-oc43 also use an entry receptor sequestered to cross-linked caveolae [160] . in sars-cov, the first entry step to host cells needs ace2 in intact lipid rafts by the s glycoprotein [151] . ace2 is associated with caveolin-1 and gm1 in membrane rafts depending on its cell-type specific localization [161] . raft integrity with cholesterol and ace2 is necessary for sars-cov pseudovirus entry into vero e6 cells and for sars-cov-microdomain-based entry. c-type lectin, cd209 l (l-sign), can also form lipid rafts and acts as a sars-cov receptor [162] . information of the cov entry pathways is important for therapeutic designation of sars-cov-targeting drugs, for example, if agents disrupt lipid-raft localization of the ace2 receptor. clq binds the sas and gangliosides in lipid rafts with a high affinity. therefore, clq or clq-oh prevents the s glycoprotein-ganglioside binding. clq (or clq-oh) binding to sa consequently prevents s glycoprotein binding to host receptors. the n-terminal region of sars-cov-2 s glycoprotein interacts with gangliosides. a ganglioside-binding site (gbs) or ganglioside-binding domain (gbd) is present in the ntd of the s glycoprotein of sars-cov-2. using molecular modeling and simulation technology, clq has been suggested to recognize the sas and gangliosides. human type neu5ac binds to clq and clq-oh. thus, sas are binding targets of clq and clq-oh. clq and clq-oh have two specific recognition sites in the polar sugar residues of ganglioside gm1. the first site is found at the tip of the sugar residues of gm1 with an interaction energy of −47 kj/mol. the clq rings face the galnac residue of gm1, while the second site is in a large region of the sugar-ceramide junction and the sugar residues. several amino acid residues of the s protein ntd, which are phe-135, asn-137 and arg-158, recognize the ganglioside gm1. the s glycoprotein ntd-gm1 complex is suggested to form a trimolecular complex with two molecules of ganglioside gm1 anchored to the ntd of s protein [163] . the ace2-binding rbd is suggested to be a potential gbs located on a differential site of the s glycoprotein ntd. the protein sequence interfacing surface of the ntd is the consensus gbds [164] . the amino acids gly, pro and/or ser residues found in gbd motifs are in the same 111-158 amino acids of the ntd as the ganglioside-attachment interface. the gbd is conserved throughout viral isolates from worldwide covid-19 patients. the gbd potentially increases viral attachment ability to pm lipid rafts and contact between host ace-2 and s protein [165] . the interaction between clq-oh and 9-o-acetyl-neuac is also similar to the 9-o-acetyl-neuac-clq interaction. the clq-oh oh group enhances the interaction of clq with sa via a hydrogen bond [163] . in conditions with clq or clq-oh derivative treatment, the s glycoprotein cannot bind to gangliosides in in silico studies, which are used to uncover the action mechanism. clq and clq-oh prevent the binding of s glycoprotein to gangliosides. the clq-sa complex is formed in a mixed surface and balls by the positioning of the negative charged cooh group of neu5ac and one of the two cationic charges of clq [163] . covs preferentially bind to 9-o-acetyl-neuac [60] , differentiating with other viral properties. as clq interacts with the gm1 sugar part, the n-terminal domain of the s protein loses viral attachment capacity to the cell receptors [166] . the s protein ntd and the clq/clq-oh maintain the same position during gm1 binding, consequently preventing gm1 binding to the s protein and the drug at the same time, because the ntd and the clq/clq-oh simultaneously recognize gm1. asn-167 forms a hydrogen bond with the galnac residue, whereas an aromatic phe-135 stacks to the glc residue of gm1. therefore, the antiviral activities of clq and clq-oh is to block the interaction between the sars-cov-2 s glycoprotein and gangliosides on host cell surfaces. the lipid composition of host cell pm can also be a potential target for preventive and therapeutic drugs against such viruses. the sars-cov-2-caused covid-19 pandemic is a global public health issue in the 21st century. in order to coordinate efforts against and mitigate the public health consequences of the spreading and outbreak of the disease, the international community has been exchanging independent information and knowledge. the scientists and epidemiologists exchange covid-19 information to highlight interdisciplinary approaches. the pandemic outbreak issue has raised interest in the pathology and epidemiology of the disease. the current covid-19 pandemic resulted in establishment of the covid action platform of the world economic forum (wef) to perform evidence-based cutting-edge research and analyze the fast-evolving pandemic. the virus outbreak data network (vodan) of the gofair data alliance also pursues to apply the best remedy against the pandemic infection. in the aspect of basic biology, most β-covs recognize 9-o-acetyl sas, but certain viruses have switched to binding 4-o-acetyl sa. originally, he is found in other viruses such as toroviruses and orthomyxoviruses (influenza c/d and isavirus). the exceptional β-cov lineage a is the only one to bear he among the covs. virus entry and replication inhibitors need to be urgently developed. computation-fused artificial intelligence accelerates therapeutic agent development. the sars-cov-2 genome shows 80% similarity to the previous sars-cov and sars-targeting agents can be commonly treated to the related sars-cov-2 patients. for better understanding of the entry pathway of sars-cov-2, the importance of the carbohydrates including sas on cell and virus surfaces is again emphasized. the author declares no conflicts of interest. severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats coronavirus study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 a pneumonia outbreak associated with a new coronavirus of probable bat origin inhibitory effect of silver nanomaterials on transmissible virus-induced host cell infections binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins novel treatment with neuroprotective and antiviral properties against a neuroinvasive human respiratory virus human and bovine coronaviruses recognize sialic acid-containing receptors like those of influenza c viruses isolated he-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity a sars-cov-2-human protein-protein interaction map reveals drug targets and potential drug-repurposing care of ophthalmological patients during the covid-19 pandemic: a rapid scoping review structure, function, and evolution of the hemagglutinin-esterase proteins of corona-and toroviruses identification of a new human coronavirus genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan genomic characterization, and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a review of sars-cov-2 and the ongoing clinical trials the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses proteolytic activation of the sars-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 coronavirus envelope (e) protein remains at the site of assembly analysis of severe acute respiratory syndrome coronavirus structural proteins in virus-like particle assembly human coronavirus: host-pathogen interaction organization of the er-golgi interface for membrane traffic control structural basis for human coronavirus attachment to sialic acid receptors human coronavirus hku1 spike protein useso-acetylated sialic acid as an attachment receptor determinant and employs hemagglutinin-esterase protein as a receptor-destroying enzyme adsorption of influenza hemagglutinins and virus by red blood cells essentials of glycobiology functions and biosynthesis of o-acetylated sialic acids gunnar blix and his discovery of sialic acids. fascinating molecules in glycobiology 9-o-acetylation of sialic acids is catalyzed by casd1 via a covalent acetyl-enzyme intermediate sialic acids and their role in immune defense post-glycosylation modification of sialic acid, and its role in virus pathogenesis novel di-o-acetylated gm3s from equine erythrocytes, one containing 4,9-di-o-acetyl-n-glycolylneuraminic acid and another containing 4-o-acetyl-n-glycolylneuraminic acid and 6-o-acetyl-d-galactose analysis of monosaccharides, fatty constituents, and rare o-acetylated sialic acids from gonads of the starfish asterias rubens the in-situ distribution of glycoprotein-bound 4-o-acetylated sialic acids in vertebrates infectious salmon anemia virus specifically binds to and hydrolyzes 4-o-acetylated sialic acids rational design of potent sialidase-based inhibitors of influenza virus replication synthesis and evaluation of 4-o-alkylated 2-deoxy-2,3-didehydro-n-acetylneuraminic acid derivatives as inhibitors of human parainfluenza virus type-3 sialidase activity why are oseltamivir and zanamivir effective against the newly emerged influenza a virus (a/h1n1)? a synthetic sialic acid analogue is recognized by influenza c virus as a receptor determinant but is resistant to the receptor-destroying enzyme human neuraminidase isoenzymes show variable activities for 9-o-acetyl-sialoside substrates acetyl modified sialic acid in cells and their effects on influenza viruses the viruses and their replication fields virology the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities the receptor-destroying enzyme of influenza c virus is neuraminate-o-acetylesterase influenza c virus uses 9-o-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus structure, and orientation of expressed bovine coronavirus hemagglutinin-esterase protein comparison of the genome organization of toro-and coronaviruses: evidence for two nonhomologous rna recombination events during berne virus evolution the s protein of bovine coronavirus is a hemagglutinin recognizing 9-o-acetylated sialic acid as a receptor determinant the hemagglutinin-esterase of mouse hepatitis virus strain s is a sialate-4-o-acetylesterase attachment of mouse hepatitis virus to o-acetylated sialic acid is mediated by hemagglutinin-esterase and not by the spike protein expression of hemagglutinin esterase protein from recombinant mouse hepatitis virus enhances neurovirulence coronavirus receptor switch explained from the stereochemistry of protein-carbohydrate interactions and a single mutation the sialate-4-o-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 coronaviridae recombinant viral sialate-o-acetylesterases bovine coronavirus uses n-acetyl-9-o-acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells sialic acid receptors of viruses transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants nidovirus sialate-o-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes comparison of hemagglutinating, receptor-destroying, and acetylesterase activities of avirulent and virulent bovine coronavirus strains sialic acid is a receptor determinant for infection of cells by avian infectious bronchitis virus infection of the tracheal epithelium by infectious bronchitis virus is sialic acid dependent structural basis for ligand and substrate recognition by torovirus hemagglutinin esterases evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a the murine coronavirus hemagglutinin-esterase receptor-binding site: a major shift in ligand specificity through modest changes in architecture receptor binding by a ferret-transmissible h5 avian influenza virus an open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly identified influenza d virus: basis for its broad cell tropism identification of sialic acid-binding function for the middle east respiratory syndrome coronavirus spike glycoprotein carcinoembryonic antigen-related cell adhesion molecule 5 is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus and bat coronavirus hku9 both can utilize grp78 for attachment onto host cells cryo-em structure of infectious bronchitis coronavirus spike protein reveals structural and functional evolution of coronavirus spike proteins binding of avian coronavirus spike proteins to host factors reflects virus tropism and pathogenicity discovery of aptamers targeting receptor-binding domain of the sars-cov-2 spike glycoprotein dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc sars-cov-2 receptor and regulator of the renin-angiotensin system: celebrating the 20th anniversary of the discovery of ace2 functional assessment of cell entry and receptor usage for sars-cov-2 and another lineage b betacoronaviruses the human coronavirus hcov-229e s-protein structure and receptor binding identification of major histocompatibility complex class i c molecule as an attachment factor that facilitates coronavirus hku1 spike-mediated infection dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus human coronavirus nl63 utilizes heparan sulfate proteoglycans for attachment to target cells renin-angiotensin system at the heart of covid-19 pandemic a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase tmprss2 and adam17 cleave ace2 differentially and only proteolysis by tmprss2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein tace antagonists blocking ace2 shedding caused by the spike protein of sars-cov are candidate antiviral compounds tmprss2: a potential target for treatment of influenza virus and coronavirus infections prostate-localized and androgen-regulated expression of the membrane-bound serine protease tmprss2 identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay covid-19 and the cardiovascular system proteolytic processing of middle east respiratory syndrome coronavirus spikes expands virus tropism requirements for ceacams and cholesterol during murine coronavirus cell entry crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry structural and molecular evidence suggesting coronavirus-driven evolution of mouse receptor structure, function, and evolution of coronavirus spike proteins soluble receptor potentiates receptor-independent infection by murine coronavirus carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes receptor-induced conformational changes of murine coronavirus spike protein localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein variations in disparate regions of the murine coronavirus spike protein impact the initiation of membrane fusion quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors identification of two mutation sites in spike and envelope proteins mediating optimal cellular infection of porcine epidemic diarrhea virus from different pathways coupling endoplasmic reticulum stress to the cell death program: role of the er chaperone grp78 grp78: a cell's response to stress covid-19 spike-host cell receptor grp78 binding site prediction ebola virus glycoprotein gp1-host cell-surface hspa5 binding site prediction heat shock protein 70 (hsp70) mediates zika virus entry, replication, and egress from host cells natural products may interfere with sars-cov-2 attachment to the host cell role of porcine aminopeptidase n and sialic acids in porcine coronavirus infections in primary porcine enterocytes identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae cd13 (human aminopeptidase n) mediates human cytomegalovirus infection aminopeptidase n is a major receptor for the entero-pathogenic coronavirus tgev feline aminopeptidase n serves as a receptor for feline, canine, porcine, and human coronaviruses in serogroup i variable o-glycosylation of cd13 (aminopeptidase n) heparan sulfate is a binding molecule but not a receptor for ceacam1-independent infection of murine coronavirus membrane protein of human coronavirus nl63 is responsible for interaction with the adhesion receptor sulfotransferases, and sulfated oligosaccharides caenorhabditis elegans early embryogenesis and vulval morphogenesis require chondroitin biosynthesis a) heparan sulfate glycosaminoglycans are receptors sufficient to mediate the initial binding of adenovirus types 2 and 5 herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry specific asparagine-linked glycosylation sites are critical for dc-signand l-sign-mediated severe acute respiratory syndrome coronavirus entry the tetraspanin cd9 facilitates mers-coronavirus entry by scaffolding host cell receptors and proteases tetraspanins: architects of viral entry and exit platforms inhibition of endoplasmic reticulum-resident glucosidases impairs severe acute respiratory syndrome coronavirus and human coronavirus nl63 spike protein-mediated entry by altering the glycan processing of angiotensin i-converting enzyme 2 recognition of n-acetyl-9-o-acetylneuraminic acid by bovine coronavirus and hemagglutinating encephalomyelitis virus site-specific glycan analysis of the sars-cov-2 spike two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues receptor recognition mechanisms of coronaviruses: a decade of structural studies structure, function, and antigenicity of the sars-cov-2 spike glycoprotein prophylactic efficacy of a human monoclonal antibody against mers-cov in the common marmoset aminoquinolines against coronavirus disease 2019 (covid-19): chloroquine or hydroxychloroquine hydroxychloroquine prescription trends and predictors for excess dosing per recent ophthalmology guidelines in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) new insights into the antiviral effects of chloroquine new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? kinetic mechanism of quinone oxidoreductase 2 and its inhibition by the antimalarial quinolines sialic acids as ligands in recognition phenomena chloroquine is a potent inhibitor of sars coronavirus infection and spread avian influenza and sialic acid receptors: more than meets the eye? lipid rafts are involved in sars-cov entry into vero e6 cells the important role of lipid raft-mediated attachment in the infection of cultured cells by coronavirus infectious bronchitis virus beaudette strain pathogens: raft hijackers role of lipid rafts in virus replication secrets of caveolae-and lipid raft-mediated endocytosis revealed by mammalian viruses human aminopeptidase n is a receptor for human coronavirus 229e aetiology: koch's postulates fulfilled for sars virus the coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment hla class i antigen serves as a receptor for human coronavirus oc43 but not ace, is preferentially localized to the apical surface of polarized kidney cells sign) is a receptor for severe acute respiratory syndrome coronavirus structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov-2 infection deciphering the glycolipid code of alzheimer's and parkinson's amyloid proteins allowed the creation of a universal ganglioside-binding peptide hybrid in silico/in vitro approaches for the identification of functional cholesterol-binding domains in membrane proteins hyperglycemia, hydroxychloroquine, and the covid-19 pandemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-350925-1h6pbfwp authors: da silva, priscilla gomes; nascimento, maria são josé; soares, ruben r.g.; sousa, sofia i.v.; mesquita, joão r. title: airborne spread of infectious sars-cov-2: moving forward using lessons from sars-cov and mers-cov date: 2020-10-08 journal: sci total environ doi: 10.1016/j.scitotenv.2020.142802 sha: doc_id: 350925 cord_uid: 1h6pbfwp background although an increasing body of data reports the detection of sars-cov-2 rna in air, this does not correlate to the presence of infectious viruses, thus not evaluating the risk for airborne covid-19. hence there is a marked knowledge gap that requires urgent attention. therefore, in this systematic review, viability/stability of airborne sars-cov-2, sars-cov and mers-cov viruses is discussed. methods a systematic literature review was performed on pubmed/medline, web of science and scopus to assess the stability and viability of sars-cov, mers-cov and sars-cov-2 on air samples. results and discussion the initial search identified 27 articles. following screening of titles and abstracts and removing duplicates, 11 articles were considered relevant. temperatures ranging from 20 °c to 25 °c and relative humidity ranging from 40% to 50% were reported to have a protective effect on viral viability for airborne sars-cov and mers-cov. as no data is yet available on the conditions influencing viability for airborne sars-cov-2, and given the genetic similarity to sars-cov and mers-cov, one could extrapolate that the same conditions would apply. nonetheless, the effect of these conditions seems to be residual considering the increasing number of cases in the south of usa, brazil and india, where high temperatures and humidities have been observed. conclusion higher temperatures and high relative humidity can have a modest effect on sars-cov-2 viability in the environment, as reported in previous studies to this date. however, these studies are experimental, and do not support the fact that the virus has efficiently spread in the tropical regions of the globe, with other transmission routes such as the contact and droplet ones probably being responsible for the majority of cases reported in these regions, along with other factors such as human mobility patterns and contact rates. further studies are needed to investigate the extent of aerosol transmission of sars-cov-2 as this would have important implications for public health and infection-control policies. hcov-oc43, hcov-nl63, hcov-hku1, severe acute respiratory syndrome virus (sars-cov), middle east respiratory syndrome virus (mers-cov) and the emerging sars-cov-2 (responsible for covid-19) (shereen et al., 2020; ye et al., 2020) . coronaviruses usually infect the cells from the respiratory tract and are responsible for different respiratory diseases that range from mild disease to severe acute respiratory syndromes (rothan and byrareddy, 2020; talbot et al., 2008) . human coronaviruses represent a major problem for human health and impose a tremendous economic burden (keogh-brown and smith, 2008; paules et al., 2020) . these viruses are considered a leading cause of morbidity and mortality in humans worldwide, as seen with the past sars and mers outbreaks (kim et al., 2017; qiu et al., 2018) and the current covid-19 global pandemic (peeri et al., 2020) . globally, as of 10:52am cest, 24 september 2020, there have been 31,664,104 confirmed cases of including 972, 221 deaths, reported to the world health organization (who) (who, 2020). viral respiratory infections are known to be spread by contact (direct or indirect) with secretions expelled by the infected person, or through air via droplets and aerosols (kutter et al., 2018) . contact transmission can happen when a healthy person comes in close contact with an infected person (direct contact) or surfaces (fomites) where viruscontaining droplets expelled by an infected person have been deposited (indirect contact) (morawska and cao, 2020) . transmission of viruses through air can happen via droplets or aerosols generated during coughing, sneezing, talking, singing or breathing (jones and cov-2 is that most studies performed only focused on the detection of viral rna and do not correlate to the infectivity of these viral particles. there is an inherent high technical complexity that also hampers the confirmation of the aerosolized sars-cov-2 infectiousness, requiring viral replication to differentiate viable from non/viable virus and including a number of particular methodological requirements, namely proper specimen selection, collection, transport, and storage that preserve viral infectivity (leland and ginocchio, 2007) . provided recent guidelines recommending that handling of material with high concentrations of viable sars-cov-2, such as when performing virus propagation, should be performed only in laboratories capable of meeting strict containment requirements and practices (biosafety level-3), limiting the number of institutions capable of assessing aerosolized sars-cov-2 viability (blacksell et al., 2020; cdc, 2020) . considering the many structural and genetic similarities between sars-cov, mers-cov, and sars-cov-2 (petrosillo et al., 2020) , and taking into consideration previous studies about sars-cov and mers-cov that point out the potential for airborne transmission of these viruses (eissenberg et al., 2020; kutter et al., 2018; olsen et al., 2003; pyankov et al., 2018; qian and zheng, 2018; ramanathan et al., 2020; tellier et al., 2019; yu et al., 2004; zhao et al., 2011) , the likelihood for airborne transmission of sars-cov-2 is very high (morawska and cao, 2020; tellier et al., 2019) . however, to date, only five studies have provided information on sars-cov-2 viability in air (binder et al., 2020; lednicky et al., 2020; lednicky et al., 2020a; santarpia et al., 2020; van doremalen et al., 2020) . thus, there is a marked knowledge gap that requires urgent attention. an opportunity for advancing research in airborne transmission of sars-cov-2 j o u r n a l p r e -p r o o f journal pre-proof is by comparison to the viability of sars-cov and mers-cov. therefore, in this systematic review, the viability/stability of aerosols containing sars-cov and mers-cov viruses will be discussed to provide information on potential mitigation strategies for sars-cov-2 airborne transmission. the present review includes studies published in the past 18 years (1 january 2002 to 25 september 2020), since the emergence of sars-cov (who, 2002) and mers-cov (who, 2012) , in the following databases: pubmed/medline, web of science and scopus. no language restrictions were imposed during the search, retrieving only one article in chinese. with no prior review articles on this topic, an exhaustive search was made, and published research articles were included. the following search terms were used: -sars‖, -mers‖, -airborne‖, -viability‖, -stability‖, -virus‖, -aerosol‖, -coronavirus‖, and -air sample‖. a total of 27 articles were found with potential interest from the initial search and their titles were screened based on their context of research. from those, 20 articles remained, and their abstracts were appropriately reviewed. after this, exclusions were performed based on the following criteria: i) if the virus studied was sars-cov, mers-cov or sars-cov-2; and ii) if the viability of the virus sampled from air was assessed. using these criteria, 18 articles were excluded and 10 additional relevant articles were found while reading the selected articles, with 1 article being excluded. summarizing, 11 articles were reviewed in detail. figure 1 shows the flowchart with the number of studies identified and included/excluded following the preferred reporting items for systematic reviews and meta-analyses (prisma) statement (moher et al., 2009 ). the databases were independently screened by all authors, and relevant information j o u r n a l p r e -p r o o f was extracted. differences on opinions about whether to include an article or not were solved by consensus between all the authors. the selected articles evaluated concerning the objective of the research, sampling site/methods and main conclusions are compiled in table 1 . viral infectivity is defined as the capacity of the virus to attach and enter the host cell and use its resources to ultimately produce new infectious virions (rodríguez et al., 2009 ). in the case of enveloped viruses such as coronaviruses, viral entry is initiated by the interaction of the viral particle with specific proteins on the cell surface. after initial binding of the receptor, these enveloped viruses fuse their envelope with the host cell membrane to deliver their capsid to the target cell (belouzard et al., 2012) . the capsid also confers protection to the viral genome by preventing its degradation by nucleases and other abiotic stresses. therefore capsid integrity is a critical attribute for the virus to successfully infect a host cell (cliver, 2009 ). among the reviewed literature, only a few papers explored viral viability in air samples (agranovski et al., 2004; binder et al., 2020; booth et al., 2005; kim et al., 2016; lednicky et al., 2020; lednicky et al., 2020a; pyankov et al., 2018; santarpia et al., 2020; van doremalen et al., 2013 van doremalen et al., , 2020 xiao et al., 2004) . remarkably, the majority of the literature focuses exclusively on the detection of viral rna in air samples (cheng et al., aiming at simplifying the determination of viral infectivity, alternative strategies to cell culture and tcid 50 determination have been explored. these strategies resort typically combining rt-pcr with a pre-processing step aiming at deconvoluting viable from non-viable virus particles prior to amplification (goyal and cannon, 2006) . a few examples of these methods are (1) enzymatic pre-treatments (such as ribonuclease) (escudero-abarca et al., 2014; monteiro and santos, 2018; nuanualsuwan and cliver, 2002; rönnqvist et al., 2014) ; (2) pre-treatments with intercalating dyes for detection of damaged capsids (leifels et al., 2015; moreno et al., 2015; parshionikar et al., 2010; randazzo et al., 2018) ; (3) porcine gastric mucin binding (dancho et al., 2012; kingsley et al., 2014) ; (4) antibody binding (ogorzaly et al., 2013) ; and (5) integrated cell-culture pcr assays (blackmer et al., 2000; dunams et al., 2012) . overall, developments in these alternative viral infectivity analytical strategies, largely unexplored in the context of current and past coronavirus pandemics, is of paramount importance to enable not only routine fundamental insights into the effective spread of the virus and its societal impact, as well as enabling effective biosensing strategies for on-site determination of viral infectivity. both these currently unpaved avenues are critical to uncover the true impact of airborne spread of sars-cov-2. lednicky et al., 2020; razzini et al., 2020; santarpia et al., 2020; santarpia et al., 2020a santarpia et al., , 2020b zhou et al., 2020) . previous work with sars-cov showed that viral rna, as well as viable virus, were found in air samples (booth et al., 2005; xiao et al., 2004) . several other studies have reported that sars-cov airborne transmission was the main transmission route in indoor cases studied in hong kong's prince of wales hospital xiao et al., 2017; yu et al., 2005) , health care facilities in canada (booth et al., 2005) and in aircraft (olsen et al., 2003) . these results suggest that both sars-cov and sars-cov-2 can potentially be transmitted by aerosols and cause disease, therefore supporting potential airborne transmission. the presence of mers-cov was also confirmed by rt-pcr of viral cultures of 4 out of 7 air samples from two hospitals in south korea (kim et al., 2016) , and showed to be very stable in aerosol at 20°c and 40% relative humidity (van doremalen et al., 2013) . furthermore, the virus demonstrated relatively high robustness in the airborne form under controlled laboratory conditions (pyankov et al., 2018) , suggesting that mers could also be transmitted by aerosols. although not investigating sars-cov-2 viability, some studies suggested that j o u r n a l p r e -p r o o f airborne transmission might occur (buonanno et al., 2020; cai et al., 2020; hamner et al., 2020; . reported that up to 73% of infected patients reported having had no contact with a person with respiratory symptoms or exposure to relevant contaminated areas, which could be explained by a possible airborne transmission of the virus. ong et al., (2020) also showed that air outlet fans located high on the wall behind the bed of one infected patient were contaminated with sars-cov-2, suggesting that virus-containing aerosols produced by the isolated patient were displaced by airflow and deposited on the vents. moreover, sampling methods and environmental conditions are very important factors to consider when studying viral viability and stability in aerosols because air sampling techniques can also affect the viability of virus recovered from air (tseng and li, 2005; verreault et al., 2008) . problems such as inefficiency at the collection of fine particles, dehydration of viruses during the collection process, damage of the viruses during collection due to impaction forces resulting in the loss of viability of some or all the collected viruses, re-aerosolization leading to the loss of viruses from the collection media, and losses due to viruses being trapped by the inlet or the samplers' wall should be taken into consideration when interpreting the results of experiments involving air sampling. noteworthy, samplers based on technologies such as the water-based condensation are considered more suitable for these studies (pan et al., 2016; yu et al., 2018) . physical characteristics of the environment such as ultraviolet light (uv), temperature, relative humidity, as well as wind currents and ventilation systems, are critical environmental factors that will determine the settling time of airborne particles (alonso et al., 2015) . there are three types of uv light: uva (320-400 nm), uvb (280-320 nm), and uvc (200-280 nm) . uvc is known to be absorbed by rna and dna bases, resulting in the photochemical fusion of two adjacent pyrimidines into covalently linked dimers, which in turn lose the ability to pair with each other (perdiz et al., 2000) . previous studies have shown that uvc is able to inactivate aerosolized coronaviruses (darnell et al., 2004; walker and ko, 2007) , with more recent studies on the subject reporting that simulated sunlight is also able to inactivate airborne sars-cov-2, highlighting the hypothesis that persistence and exposure risk to airborne viruses might vary between indoor and outdoor environments schuit et al., 2020) temperature is another significant factor for virus survival because it can affect the state of viral proteins and the virus genome (price et al., 2019) . temperatures above 60 °c for more than 60 min are thought to be sufficient to inactivate most enveloped viruses, and depending on the presence of any surrounding organic material such as saliva, the virus might be insulated against extreme environmental changes (tang, 2009) . in a study by pan et al. (2019) , it was reported that artificial saliva could better protect infectious viruses from deactivation by preventing viruses of reaching the air-water-interface, possibly due to the complex structure of the mucin component. in a similar study, woo et al. (2012) reported that the inactivation efficiency of droplet and aerosolized viruses under different humidity levels and uv irradiation at a constant intensity were low in artificial saliva, indicating that solids present in it might exhibit a protective effect. the relative humidity is also significant for virus survival and stability because phospholipid-protein complexes in enveloped viruses are usually more likely to denature in the air at medium to high relative humidity. in contrast, the protein coats of nonenveloped viruses denature easier at low relative humidity (sobsey and meschke, 2003) , which explains why most enveloped viruses tend to survive longer at a lower relative j o u r n a l p r e -p r o o f humidity (tang, 2009 ). in addition to that, when faced with high humidity, such as in tropical regions as the amazon rainforest where relative humidity values can get close to 100% during the rainy season, viruses are associated with larger droplets that settle down much faster, which can be a limiting factor to transmission (yang and marr, 2011) . in an attempt to study the effects of temperature and relative humidity on the viability of the sars-cov, a study found that low temperature and low humidity was able to prolong survival of virus on contaminated surfaces (chan et al., 2011) . the same was found to be true for mers-cov. a study reported the stability of mers-cov at 20°c and 40% relative humidity; 30°c and 30% relative humidity; and 30°c and 80% relative humidity, and concluded that mers-cov was more stable at lower temperature and lower humidity conditions (van doremalen et al., 2013) . in another study, two sets of climatic conditions were used in order to establish the inactivation of mers-cov: one represented the common indoor office environment (25°c and 79% relative humidity) and the other represented the climatic conditions of the middle eastern region where the virus outbreak started (38°c and 24% relative humidity) (pyankov et al., 2018) . authors found that the virus had a better survival rate at a lower temperature, with virus decay being higher in hot and dry air. in a recent study, atomic force microscopy was applied to investigate the topographical changes of sars-cov-2 virions exposed to high-temperature treatments, reporting that after the treatment, the virus had much fewer less distinct spikes, their trigonal shape not being able to be resolved, suggesting heat-induced inactivation of sars-cov-2 (kiss et al., 2020). another study reported that the virus was stable at 4 °c in virus transport medium, but sensitive to heat, and that at 22 °c and 65% relative humidity had a negative effect on viral survival on smooth surfaces (chin et al., 2020) . other studies have reported the effects of humidity and temperature on sars-cov-2 j o u r n a l p r e -p r o o f transmission based on meteorological data and statistical analysis (auler et al., 2020; ma et al., 2020; méndez-arriaga, 2020; meo et al., 2020; meyer et al., 2020; sajadi et al., 2020; ward et al., 2020; wu et al., 2020; xie and zhu, 2020; yao et al., 2020) . although all of them have reported a correlation between temperature and relative humidity and the number of new covid-19 cases, there is still some controversy regarding whether one or both variables have a positive, negative or no effect on the number of new cases. the main outcomes of these studies are presented on table s1 (supplementary material). given the genetic and structural similarities between sars-cov, mers-cov and sars-cov-2, one might suggest that higher temperatures and relative humidities could have an impact on the viability of sars-cov-2 in the environment. nonetheless, the effect of these conditions seems to be residual (meyer et al., 2020; wu et al., 2020) considering the increasing number of cases in the south of usa, brazil and india, where high temperatures and humidities have been observed. moreover, it should be noted that although sars-cov and mers-cov can give us an idea of how sars-cov-2 might behave, using sars-cov and mers-cov to predict the behaviour and spread patterns of sars-cov-2 is not advisable, as these three viruses are different, and sars-cov-2 might not necessarily follow the same patterns as the aforementioned viruses and more studies are needed in this subject to determine how these environmental variables might impact the virus transmission. other factors, such as human mobility patterns and contact rates, should also be taken into consideration as contributing factors to the different transmission rates in different countries (badr et al., 2020) . in developing countries such as brazil and india, other transmission routes (e.g., the contact and droplet routes) may account for the increasing number of cases rather than the airborne route, as these countries are very densely populated, have overcrowded accommodations and lack of access to basic services, j o u r n a l p r e -p r o o f therefore enabling the contact and droplet routes of transmission and spread of the virus. these individual and collective factors such as political, social, economic and cultural conditions should be considered when analyzing the spread of the virus in these countries, as they may play a more significant role than temperature and relative humidity (auler et al., 2020) . specific measures such as quarantines and lockdowns can also affect the incidence of the virus, as different countries have different approaches and mitigation strategies to deal with the pandemic. among the reviewed literature, only a few papers explored viral viability in air samples, which is probably due to the difficulty and limitation of many research groups regarding bsl-3 facilities. nonetheless, efforts should be directed towards the development of novel or adapted analytical methods to reliably and systematically determine the infectivity of sars-cov-2 viral particles as this would enable not only routine fundamental insights into the effective spread of the virus and its societal impact, as well as enabling effective biosensing strategies for on-site determination of viral infectivity as previously mentioned. currently, there is still debate about whether or not sars-cov-2 is transmitted through aerosols produced by infected people during talking, singing sneezing, coughing and breathing, and further studies regarding this route of transmission are needed in order to explore the feasibility of a new personal bioaerosol sampler for monitoring of viable airborne sars virus. pc4 facility with hepa filters installed in the pipeline connecting sampler and vacuum pump to prevent the equipment contamination. contaminated air was bubbled through porous medium submerged into liquid and subsequently split into multitude of very small bubbles. the particles are scavenged by these bubbles, and, thus, effectively removed. natural decay of the virus in the collection fluid was around 0.75 and 1.76 log during 2 and 4h of continuous operation, respectively. a much higher decay rate (2.58 log) was observed for the bubbling through viral suspension in sterile water. yes. the device filled with virus maintenance fluid was capable of providing a relatively low level of microbial decay and can be evaluated for monitoring of such microorganisms in the air. w. xiao et al., (2004) sars-cov-1 to assess the risk of aerosol transmission in sars patients admitted to a hospital through testing the air samples. air samples were collected from 7 wards and 1 balcony of the hospital, 3 times a day for 3 continuous days. the bioaerosol sampler type fa-2 was used. rt-pcr was used to amplify the n protein gene of the sars-cov. the residual solutions were inoculated into prepared cell cultures to isolate live virus. the positive samples were then identified by indirect immunofluorescence assay and sequence analysis of the pcr products. air sampling was performed using a high-resolution slitsampler system and samples were tested for the presence of sars-cov by rt-pcr and cell culture isolation. pcr-positive viruses were collected from wet and dry air samples but results of viability assays of the samples for infectivity in vero-e6 cell culture were negative. no. not specified. kim et al., to study the possible contribution of contaminated hospital air and surfaces to mers transmission. a suspension containing virus was prepared and aerosolized aerosolised to the experimental aerosol chamber by a 3-jet collison nebulizer nebuliser at the flow rate of 6 l/min of hepafiltered compressed air over 2 mins time. then the nebulizer nebuliser was switched off. the experiments were performed for two sets of parameters of the air. on completion of sampling at each time interval, the bioaerosol samplers were disconnected and aliquots of collecting liquid were acquired and analyzed analysed by end-point titration in vero e6 cells. to evaluate the stability of sars-cov-2 and sars-cov-1 in aerosols and on various surfaces and estimate their decay rates using a bayesian regression model. laboratory under controlled conditions. aerosols (<5 μm) containing sars-cov-2 (10 5.25 50% tcid 50 per milliliter) or sars-cov-1 (10 6.75-7.00 tcid 50 per milliliter) were generated with the use of a three-jet collison nebulizer nebuliser and fed into a goldberg drum to create an aerosolized aerosolised environment. all samples were quantified by end-point titration on vero e6 cells. sars-cov-2 ncov-wa1-2020 (mn985325.1) and sars-cov-1 tor2 (ay274119.3) were the strains used. sars-cov-2 remained viable in aerosols throughout the duration of the experiment (3 hours), with a reduction in infectious titer from 10 3.5 to 10 2.7 tcid 50 per liter of air. yes. not specified. detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units rapid review: aerosol generating procedures in health care, and covid-19 quantitative assessment of the risk of airborne transmission of sars-cov-2 infection: prospective and retrospective applications indirect virus transmission in cluster of covid-19 cases cdc, 2020. interim laboratory biosafety guidelines for handling and processing specimens associated with coronavirus disease ncird), div. viral dis the effects of temperature and relative humidity on the viability of the sars coronavirus escalating infection control response to the rapidly evolving epidemiology of the coronavirus disease 2019 (covid-19) due to sars-cov-2 in hong kong detection of air and surface contamination by sars-cov-2 in hospital rooms of infected patients stability of sars-cov-2 in different environmental conditions capsid and infectivity in virus detection discrimination between infectious and noninfectious human norovirus using porcine gastric mucin inactivation of the j o u r n a l p r e -p r o o f journal pre-proof coronavirus that induces severe acute respiratory syndrome, sars-cov simultaneous detection of infectious human echoviruses and adenoviruses by an in situ nuclease-resistant molecular beacon-based assay treat covid-19 as though it is airborne: it may be molecular methods used to estimate thermal inactivation of a prototype human norovirus: more heat resistant than previously believed? food microbiol a field indoor air measurement of sars-cov-2 in the patient rooms of the largest hospital in iran food microbiology and food safety research and development aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia role of air distribution in sars transmission during the largest nosocomial outbreak in hong kong evidence for probable aerosol transmission of sars-cov-2 in a poorly ventilated restaurant. medrxiv 1-19 effects of temperature variation and humidity on the death of covid-19 in wuhan the temperature and regional climate effects on communitarian covid-19 contagion in mexico throughout phase 1 effect of heat and humidity on the incidence and mortality in world's top ten hottest and top ten coldest countries that higher temperatures are associated with a marginally lower incidence of covid-19 cases preferred reporting items for systematic reviews and meta-analyses: the prisma statement enzymatic and viability rt-qpcr assays for evaluation of enterovirus, hepatitis a virus and norovirus inactivation: implications for public health risk assessment airborne transmission of sars-cov-2: the world should face the reality it is time to address airborne transmission of covid-19 application of viability pcr to discriminate the infectivity of hepatitis a virus in food samples pretreatment to avoid positive rt-pcr results with inactivated viruses development of a quantitative immunocapture real-time pcr assay for detecting structurally intact adenoviral particles in water transmission of the severe acute respiratory syndrome on aircraft air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient determination of the distribution of infectious viruses in aerosol particles using water-based condensational growth technology and a bacteriophage ms2 model efficient collection of viable virus aerosol through laminar-flow, water-based condensational particle growth use of propidium monoazide in reverse transcriptase pcr to distinguish between infectious and noninfectious enteric viruses in water samples coronavirus infections-more than just the common cold the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? distribution and repair of bipyrimidine photoproducts in solar uv-irradiated mammalian cells. possible role of dewar photoproducts in solar mutagenesis covid-19, sars and mers: are they closely related? association between viral seasonality and meteorological factors survival of aerosolized coronavirus in the ambient air ventilation control for airborne transmission of human exhaled bio-aerosols in buildings the impacts on health, society, and economy of sars and h7n9 outbreaks in china: a case comparison study transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination viability rt-qpcr to distinguish between hev and hav with intact and altered capsids simulated sunlight rapidly inactivates sars-cov-2 on surfaces sars-cov-2 rna detection in the air and on surfaces in the covid-19 ward of a hospital in milan application of pcr-based methods to assess the infectivity of enteric viruses in environmental samples ultraviolet light inactivation of murine norovirus and human norovirus gii: pcr may overestimate the persistence of noroviruses even when combined with pre-pcr treatments the epidemiology and pathogenesis of coronavirus j o u r n a l p r e -p r o o f journal pre-proof disease (covid-19) outbreak temperature, humidity, and latitude analysis to estimate potential spread and seasonality of coronavirus disease 2019 (covid-19) aerosol and surface transmission potential of sars-cov-2 aerosol and surface contamination of sars-cov-2 observed in quarantine and isolation care airborne sars-cov-2 is rapidly inactivated by simulated sunlight covid-19 infection: origin, transmission, and characteristics of human coronaviruses guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings virus survival in the environment with special attention to survival in sewage droplets and other environmental media of fecal or respiratory origin pathogenesis of human coronaviruses other than severe acute respiratory syndrome coronavirus the effect of environmental parameters on the survival of airborne infectious agents recognition of aerosol transmission of infectious agents: a commentary collection efficiencies of aerosol samplers for virus-containing aerosols stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions methods for sampling of airborne viruses effect of ultraviolet germicidal irradiation on viral aerosols humidity is a consistent climatic factor contributing to sars-cov-2 transmission transmission of sars-cov-2: implications for infection prevention precautions [www document airborne transmission of severe acute respiratory syndrome coronavirus-2 to healthcare workers: a narrative review effects of relative humidity and spraying medium on uv decontamination of filters loaded with viral aerosols endonasal instrumentation and aerosolization risk in the era of covid-19: simulation, literature review, and proposed mitigation strategies effects of temperature and humidity on the daily new cases and new deaths of covid-19 in 166 countries role of fomites in sars transmission during the largest hospital outbreak in hong kong association between ambient temperature and covid-19 infection in 122 cities from china dynamics of airborne influenza a viruses indoors and dependence on humidity on airborne transmission and control of sars-cov-2 zoonotic origins of human coronaviruses an j o u r n a l p r e -p r o o f journal pre-proof efficient virus aerosol sampler enabled by adiabatic expansion evidence of airborne transmission of the severe acute respiratory syndrome virus temporal-spatial analysis of severe acute respiratory syndrome among hospital inpatients role of two-way airflow owing to temperature difference in severe acute respiratory syndrome transmission: revisiting the largest nosocomial severe acute respiratory syndrome outbreak in hong kong investigating sars-cov-2 surface and air contamination in an acute healthcare setting during the peak of the covid-19 pandemic in london figure 1 key: cord-352322-tsjwnvkk authors: khamassi khbou, médiha; daaloul jedidi, monia; bouaicha zaafouri, faten; benzarti, m’hammed title: coronaviruses in farm animals: epidemiology and public health implications date: 2020-09-25 journal: vet med sci doi: 10.1002/vms3.359 sha: doc_id: 352322 cord_uid: tsjwnvkk coronaviruses (covs) are documented in a wide range of animal species, including terrestrial and aquatic, domestic and wild. the geographic distribution of animal covs is worldwide and prevalences were reported in several countries across the five continents. the viruses are known to cause mainly gastrointestinal and respiratory diseases with different severity levels. in certain cases, cov infections are responsible of huge economic losses associated or not to highly public health impact. despite being enveloped, covs are relatively resistant pathogens in the environment. coronaviruses are characterized by a high mutation and recombination rate, which makes host jumping and cross‐species transmission easy. in fact, increasing contact between different animal species fosters cross‐species transmission, while agriculture intensification, animal trade and herd management are key drivers at the human‐animal interface. if contacts with wild animals are still limited, humans have much more contact with farm animals, during breeding, transport, slaughter and food process, making covs a persistent threat to both humans and animals. a global network should be established for the surveillance and monitoring of animal covs. covs isolated from carnivores, herbivores and omnivores fosters the interspecies transmission and virus adaptation to new hosts (song et al., 2005) . in fact, cross-species transmission and host jumping are the most important driver for virus emergence (holmes, 2016) and covs specifically are dotted with a high rate of mutation and recombination, making them a continuous threat for humanity. mutations result from errors during viral genome replication by the rna-dependent rna polymerase, which reaches 5.7 × 10 -6 nucleotide substitutions per site per day for the sars-cov and the accumulated mutations at some sites could have important implication on virus properties (vega et al., 2004) . recombination appears to be more important in cov genome than mutation; it consists of gaining genome fragment from another cov, which implies the adaptation to new hosts (worobey & holmes, 1999) . as consequences of such genomic mutation and recombination the transmissible gastroenteritis virus (tgev) of swine and the bovine cov (bcov) likely originated from the closely related canine coronavirus (ccov) (pratelli, 2011) . a deletion that occurred in the s protein of the tgev resulted in the rising of the porcine respiratory cov with a marked changing tropism from the gastrointestinal to the respiratory tract (peiris, 2012) . there is evidence that the feline infectious peritonitis virus (fipv) is originated from the feline enteric cov (fecov) by mutation in the region of the s1/s2 of the spike protein (licitra et al., 2013) . the best opportunity that would allow viruses to jump new species occurs at the moment of feeding, when a predator consumes prey and all the viruses infecting it (french & holmes, 2020) . beside feeding, physical contact between different host species increase host range expansion probability (wang, vlasova, kenney, & saif, 2019) as it was the case for the swine influenza viruses (subtypes h1n1 and h1n2) and the nipah virus that were transmitted from pigs to humans (chua et al., 2002; gray et al., 2007) . new porcine cov emerged in china (guangdong province) in 2017, namely the swine acute diarrhoea syndrome cov (sads-cov) with high sequence identity to bat-cov hku2 (gong et al., 2017) . the high density of pig farms and slaughterhouses in guangdong province associated to the wide distribution of bat species explained the cross-species transmission. indeed, as pork meat is considered as the most commonly consumed meat in non-muslim countries, pigs may be an effective intermediate host for the emergence of novel covs of highly public health concerns (wang, su, bi, wong, & gao, 2018 ). coronaviruses of farm animals including large and small ruminants, dromedaries, horses, pigs and chickens were reviewed; cetacean covs were also considered, as marine mammals are a food source in many countries around the world. the aim of this review is to summarize the epidemiological knowledge about covs of farm animals, to discuss the public health implications and to address the gap of knowledge useful for future research direction. & stokstad, 2017). furthermore, high animal density seems to be the main bcov risk factors (boileau & kapil, 2010) . the bcov is one of the main causative agents of neonatal calf diarrhoea during the first month of life (ammar et al., 2014; brandão et al., 2006) and the most common deathly disease in calves (gomez & weese, 2017) . other studies showed that bcov may be involved in 10 to 30% of neonatal diarrhoea cases (alfieri et al., 2018) . in adult cattle, the wd occurs as epidemics during winter and is characterized by a contagious watery diarrhoea, fever, depression and reduced milk yield (toftaker et al., 2017) . singasa, songserm, lertwatcharasarakul, and arunvipas (2017) reported that milk production might decrease up to 10% for 2 weeks during the bcov infection. vaccines are available for the prevention of bcov infections in neonatal calves and in adult. an inactivated virus vaccine was developed for pregnant cows to enhance passive maternal immunization of calves via colostrum and prevent neonatal diarrhoea (decaro et al., 2009) . another inactivated hemagglutinating antigens-enriched vaccine showed their efficacy to protect cattle against wd (takamura, matsumoto, & shimizu, 2002) . the bcov was also associated, alone or with other pathogens, to respiratory infections such as enzootic pneumonia in calves and shipping fever in young cattle (saif, 2010) . the prevention from respiratory cov in cattle is based on multivalent killed or attenuated-live intra-nasally administrated vaccines (hay et al., 2016; richeson et al., 2019) . a bovine-like cov in water buffalo (bubalus bubalis) was first described in 1985 in bulgaria, using serological methods (muniiappa, mitov, & kharalambiev, 1985) . the sequencing of the isolated buffalo cov from italy, showed sequence homology with the bcov, with some differences justifying its classification as a new variant of bcov and was named as bubaline cov (bucov) (decaro, martella, et al., 2008) . in italy, gastroenteritis disease among water buffalo calves is caused by both bucov and bcov (decaro et al., 2010) . closely related strains to the bcov (displaying 98% sequence homology) were also detected among faecal samples of domestic buffaloes in bangladesh (lau et al., 2016) . infection of water buffalo calves was also reported from three districts in egypt, with an overall molecular prevalence of 26.8% (22/82) in faecal samples (elsadek et al., 2019) ( table 2) . although the bcov is initially a cattle virus, it is occurring also in small ruminants, in which it was neglected for a long time due to its low prevalence and insignificant clinical manifestation (amer et al., 2019) . in australia, sheep were reported in 1978 to excrete cov-like particles that were visualized by electron microscopy examination during a diarrhoea episode (tzipori, smith, makin, & mccaughan, 1978) . the infection of small ruminants by the bcov is rather to occur through close contact with cattle in mixed flocks than as a natural infection (tråvén, carlsson, lundén, & larsson, 1999) . the most recent study in ghana reported anti-bcov antibodies in f i g u r e 1 phylogenetic tree of different representative covs based on the spike (s) protein gene (the strain responsible for the pandemic sars-cov-2 in humans is shown with the bolded character among the betacoronavirus genus). the shown animals are hosts from where the corresponding covs were isolated. the sequences of all animal covs were downloaded from the national center for biotechnology information (ncbi) genbank database (https://www.ncbi.nlm.nih.gov/genba nk/). the tree was constructed using the neighbour-joining method (bootstrap resampling = 1,000 replicates and bootstrap values are indicated as % at branch points) (saitou & nei, 1987) . the evolutionary distances were computed using the maximum composite likelihood method (tamura et al., 2004) and are in the units of the number of base substitutions per site. all positions containing either gaps or missing data were eliminated. there were a total of 2044 positions in the final dataset. evolutionary analyses were made with mega7 (kumar et al., 2016) box (burimuah et al., 2020) (table 2) . nowadays, it is well established that dromedaries are the natural host of the mers-cov and a source for human and other domestic animals infections (kandeil et al., 2019) . since the first case of human infected by the mers-cov was identified in september 2012 in saudi arabia (world health organization, 2019), interest to dromedaries as sources of the virus increased and the isolated strains were shown to be genetically very similar to those isolated from humans (omrani, al-tawfiq, & memish, 2015) . the close contacts between infected dromedaries and humans enhance the continuing zoonotic transmission and may explain why the mers-cov continues to occur in humans (de wit et al., 2016) . moreover, the mers-cov was also detected in sheep, goats and donkeys reared close to camels (kandeil et al., 2019) . transmission from dromedaries to humans occurs by direct contact with mucous and nasal discharge or by consumption of meat or raw milk (gossner et al., 2016; mirkena et al., 2018) . indeed, mers-cov rna was detected using qrt-pcr in whole and skimmed milk collected from milking animals that were kept with camels that have frequent contact with multiple origin camels participating at racing events . the mers-cov detection in dromedaries is difficult as the infection is asymptomatic. however, experimental mers-cov infection of dromedaries caused mild to moderate rhinitis with nasal discharge, tracheitis and bronchitis in addition to the shedding of a large amount of virus from the respiratory tract (haverkamp et al., 2018) . several studies showed that the wild strains of the mers-cov circulate in dromedaries in more than 24 countries across africa and the middle east ( figure 2) , with high seroprevalences rates recorded from egypt (71%) (ali et al., 2017) , nigeria (94%) (reusken, messadi, et al., 2014) dromedaries increases with age, reaching 80%-100% in adults (harrath & abu duhier, 2018) . in experimentally challenged dromedaries, a modified orthopox-based vaccine expressing the mers-cov spike protein conferred mucosal immunity. this vaccine induced a significant reduction of virus shedding, and the conferred protection was associated with neutralizing antibodies production (haagmans et al., 2016) . as a strategy to prevent human contaminations, mass vaccination of camels is discussed (dighe et al., 2019) . following a severe diarrhoea outbreak that induced high morbidity and the equine coronavirus (ecov) was isolated for the first time from a foal in north carolina (usa) (guy, breslin, breuhaus, vivrette, & smith, 2000) , since then, multiple outbreaks were documented in different countries but epidemiological information on ecov still scanty (pusterla, vin, leutenegger, mittel, & divers, 2016) . the ecov was reported from japan, saudi arabia, france, the united kingdom and the united states (table 2 ). phylogenetic analyses showed that ecov isolated from japan and the united states, were genetically close (nemoto et al., 2015) . the morbidity rates of ecov infection ranged from 10% to 83% and the lethality rate is classified from rare to 27% (fielding et al., 2015; oue et al., 2011; oue, morita, kondo, & nemoto, 2013; pusterla et al., 2013) . up to 83% of infected horses remain asymptomatic, while their faeces contain the ecov (pusterla et al., 2013) . the transmission route of the ecov occurs through a faecal-oral route (pusterla et al., 2013) and the disease occurs mainly in winter (nemoto et al., 2014) . the ecov could be detected from diarrheic horses 2 to 5 weeks after the beginning of the infection (pusterla et al., 2016) but also from healthy horses (hemida, elmoslemany, et al., 2017) . the peak of faecal shedding occurs 3 to 4 days after the onset of the disease (bryan et al., 2019) and nasal secretions could be positive to the ecov at this moment, but the epidemiological role of these secretions in transmitting the virus is not known (nemoto et al., 2014) . according to the observations of pusterla, vin, leutenegger, mittel, and divers (2018), draft horses showed higher infection rate than other breeds, this could be explained by the stress induced by working conditions. some attempts of vaccination using the bcov gave promising results but need further investigations (nemoto et al., 2017) . six coronaviruses were isolated from pigs belonging to three genera: (table 1) , beside a re-emerging highly virulent strains of the pedv that were described since 2010 (sun et al., 2012) . the tge is the sole mammalian disease caused by a cov in the world (piñeyro et al., 2018) . domestic animals such as cats and dogs may play a role as host for the tgev (sestak & saif, 2002) . as for most of mammalian covs, the tge occurs during winter after an incubation period varying between 18 hr and 3 days (pensaert, 1976) . the course of the disease is characterized by vomiting and profuse diarrhoea and is marked by high morbidity and mortality rates mainly among piglets (animal health australia, 2016). although pigs of all ages are susceptible to the tgev infection, animals older than 5 weeks display milder clinical symptoms than piglets (piñeyro et al., 2018) . as the tgev shares common epitopes for neutralizing antibodies with prcv, a significant decrease of the tge incidence occurred as a consequence of the cross protection conferred by the prcv (wesley & woods, 1996) . the emergence of the phe was traced back to 1957 in canada, with the occurrence of high several episodes of vomiting, wasting and anorexia followed by neurological signs in pig nurseries (roe & alexander, 1958) . in 1962, the virus was isolated from baby pigs suffering from encephalomyelitis (greig et al., 1962) , then several outbreaks with similar clinical signs were reported in canada and europe and the disease was named vomiting and wasting disease (vwd) (cartwright et al., 1969) . in 1971, the phev was classified as a cov (greig et al., 1971 ) and subsequently the disease was reported the colostral immunity transferred to newborn piglets, which is considered as the best way to prevent from the infection (rho et al., 2011) . the porcine epidemic diarrhoea (ped) was reported for the first time in england in 1971, but the viral aetiology was only proved in 1978 (pensaert & de bouck, 1978) . the pedv spread to other european countries (belgium, france, hungary and czech republic…) (pensaert & martelli, 2016) and to asia where it became enzootic particularly in most prosper pork industry countries like china, south korea and philippines (song & park, 2012) . the economic losses induced by the occurrence of the ped are dramatic since, high mortality rate is associated to the infection, mainly among < 10 days old piglets (antas & woźniakowski, 2019) . mortality reached 100% in newborn and suckling piglets in japan during 1993 and 1994 (sueyoshi et al., 1995) and in thailand during 2007 and 2008 (puranaveja et al., 2009 ). in the united states, the pedv emerged in 2013, where almost 10% of the pig population died in less than 1 year after several outbreaks stevenson et al., 2013) and spread to mexico and canada in 2014 (kochhar, 2014) . sow's sera and piglets sera after suckling (paudel et al., 2014) . in japan, live attenuated virus is used since 1997 (usami, yamaguchi, & kumanomido, 1998) and oral vaccination started in south korea and philippines, since 2004 and 2011 respectively (garcia et al., 2018; park et al., 2018) . as new virulent strains are circulating, trials for more effective vaccine are undergoing using swine-poxvirus-based vaccines designed to express the a epitope of the spike protein (yuan, lin, li, he, & fan, 2017) . the porcine respiratory coronavirus (prcv) is a mutant of the tgev, due to a deletion in spike gene (laude et al., 1993) . it was identified first in belgium in 1984 (pensaert, callebaut, & vergote, 1986) and spread to almost all european countries including france (madec et al., 2004) , denmark (have, 1990 ) and the united kingdom (brown & cartwright, 1986) . the prcv was soon reported from the united states (halbur et al., 2003) and japan (usami et al., 1998) . most of the prcv infections are subclinical or inducing mild symptoms (caswell & williams, 2016) , this could be due to the low proinflammatory cytokines synthesis activation (van reeth, labarque, nauwynck, & pensaert, 1999) . the prcv is still detected upto the 21st day in the lungs of experimentally infected pigs and high titres reached 10 8.3 median tissue culture infectious doses (tcdi50) per gram of lung tissue (jung et al., 2007) . faecal shedding of prcv was also reported from 37% (21/57) of sentinel pigs introduced in prcv-infected herds (costantini et al., 2004) . because of the insignificant clinical course of the prcv infection, neither treatment nor vaccination, is applied. however, countries exporting live pigs need negative status to prcv, which could be obtained only if piglets are pre-weaned early at the 7th day, together with strict all-in, all-out managed barns, rigorous disinfection and cleaning of pig barns and regular seronegative tests in sows (burlatschenko & arsenault, 2015) . the porcine deltacoronavirus ( moreover, in orally pdcov-inoculated calves, a persisting faecal viral rna shedding associated to pdcov-specific serum igg antibody responses were observed (jung et al., 2017) . there is no commercial available vaccine for the prevention from the pdcov infection (zhang, liu, et al., 2019) . active surveillance programme effectively led to the decrease of the pdcov herd-level prevalence below 0.5% in two years in canada (ajayi et al., 2018) . the swine acute diarrhoea syndrome virus (sads-cov) has emerged in pigs in china since august 2016 (zhou, sun, et al., 2019) . this virus, also qualified as porcine entreric alphacoronavirus (peav) or swine enteric coronavirus (secov) (pan et al., 2017) was associated with the occurrence of severe diarrhoea of suckling piglets in guangdong province in 2017 (gong et al., 2017; pan et al., 2017) and 2019 (zhou, li, et al., 2019) , and in fujian province in 2018 . the economic loss induced by the sads-cov was dramatically high, as 24,693 piglets in four farms, died from the infection in guangdong province, where the first human sars epidemic started in 2003 . this virus is antigenically distinct from the pedv, the tgev and the pdcov (yang, yu, & huang, 2020) . nevertheless, the complete genome sequencing of the n and the s genes showed a high nucleotides homology (96%-98%) with those of four bat-cov hku2 (gong et al., 2017) . during the first outbreak in china, the sads-cov infection was controlled by immunizing pregnant sows using inactivated filtrated virus obtained from infected piglets intestines (zhou, sun, et al., 2019b) . as there is an urgent need to develop efficient vaccines to control the sads-cov in pigs, trials on attenuation of a virulent strain via cell culture passage are now on-going . decaa new chimeric virus containing the s protein of the pedv on a tgev backbone was discovered in italy in 2016 and might circulate there from mid-2009 to 2012 according to boniotti et al. (2016) . this recombinant virus was also detected in germany, central eastern europe and slovakia precluding for an old circulation in europe (akimkin et al., 2016; belsham et al., 2016; mandelik et al., 2018) . in 2012, a novel beta-cov was isolated from domestic rabbit and characterized as rabbit coronavirus hku14 (rbcov hku14), causing no clinical manifestation. the virus was detected in 8.1% (11/136) rabbit faecal samples using rt-pcr and phylogenetic analysis showed that the novel rbcov hku14 is most closely related to betacoronavirus 1 species (bcov, ecov, ccov, hcov-oc43) . the authors hypothesized that the virus may have emerged as a result of interspecies transmission between different animal species in chinese markets. three species of sea mammals were shown to be susceptible to covs: harbor seals, white beluga whale and bottlenose dolphins. in 1987, three captive seals (phoca vitulina) at the miami seaquarium expressed acute enteritis associated with dehydration and leucocytosis. the pathological examination showed bronchoalveolar oedema and haemorrhage. fluorescent antibody staining yielded positive results to tgev, fipv and ccov antisera, which precluded to an alpha-cov, but the virus was not yet assigned to the genus (bossart & schwartz, 1990 ). ten years later, a captive beluga whale (delphinapterus leucas), in the united states suffering from a generalized pulmonary disease, died from acute liver failure caused by a cov (mihindukulasuriya, wu, st. leger, nordhausen, & wang, 2008) . phylogenetic analyses revealed that the causative agent was closely related to the infectious bronchitis virus (ibv) of chickens, and the pathogen, named bwcov sw1 was assigned to the gamma-cov genus (de groot et al., 2012) . in bottlenose dolphins (tursiops aduncus), the first report of a avian covs are the main representative of the gamma-cov genus, including the ibv in chickens as the most studied cov, and unique among all other animal covs. similar covs to the ibv were detected and isolated from domestic galliformes: turkey (tcov), guinea fowl (gfcov) and pheasants (phcov) (cavanagh, 2005) , but also from non-galliformes namely: columbiformes, psittaciformes and anseriformes (domanska-blicharz et al., 2014; jonassen et al., 2005) . seven other avian covs are included in the delta-cov genus . only the ibv will be treated in this section. the first isolation of the ibv was made in 1930s in the united states (massachusetts) and it was thought that only one serotype exists, until 1956 exists, until , when jungherr et al. (1957 in 2016, valastro et al. (2016) suggested a method to harmonize the nomenclature of the ibv using a phylogeny-based classification and complete genome sequencing of the s1 gene. today, the ibv is divided to seven genotypes including 35 distinct viral lineages based on the complete s1 gene sequencing xu et al., 2019) . the geographic distribution of the ibv is worldwide (figure 2) and some genotypes are present more in certain regions than in others. indeed, two different lineages gi-21 and gii-1 were considered as specific to europe (fan et al., 2019) , while gi-9, gi-27 and giv-1 were considered specific to north america (lin & chen, 2017 ) and the gi-11 is an exclusively south american lineage, very prevalent in brazil and uruguay (marandino et al., 2019) . the ibv is a highly contagious virus of the upper respiratory tract of chickens, leading to 100% morbidity and a mortality rate ranging from 0% to 82% depending upon several factors such as the age of the birds, their immune status, and the involvement of secondary pathogens (ramakrishnan & kappala, 2019) . the ibv infects chickens of all ages, although young are most susceptible, and may die directly from the ibv infection or from mixed infections mainly caused by escherichia coli, leading to heavy losses to the breeding industry (jackwood, 2012) . in brazil, which is considered as the biggest broiler meat exporter (nääs, mollo neto, canuto, waker, oliveira, & vendrametto, 2015) , the total loss (in us$) induced by the ibv infection were estimated per 1,000 birds to 4,210.8 and to 266.3, in breeders and broiler respectively (colvero et al., 2015) . except the sars-cov, the mers-cov and the most recent sars-cov-2, the other documented human covs are specific to human beings (corman et al., 2018) . it is well established that domestic or wild animals were involved in the three epidemics either as reservoir or as intermediate hosts. the & xu, 2003) . several wild animals, including palm civets (paguma larvata), raccoon dogs (nyctereutes procyonoides) and horseshoe bats (rhinolophus hipposideros) were involved in the sars-cov epidemic during 2003 and were tested positive using virological and or serological tests (guan et al., 2003) . while phylogenetic analyses showed that bats are reservoir for the sars-cov and allowing genetic recombination, civets seem to be an intermediate host, as they were tested negative in their wild free lands (su et al., 2016) . the showed that the mers-cov evolve exclusively in camels while humans act as terminal host (dudas et al., 2018) . the epidemiology of mers remains to date not well understood, as heavily exposed persons to infected camels lead only to seropositivity and any of persons confirmed with the mers-cov reported previous exposure to infected animals (hemida, elmoslemany, et al., 2017) . the main factor that could play a role in the transmission of mers-cov from infected dromedaries to humans, is the consumption of raw camel milk . neither the consumption of meat and organs nor the consumption of urine for medicinal use might cause the infection of humans (adney et al., 2014) and further investigations on experimentally inoculated animals are needed to confirm such definite conclusions. for the current covid-19 epidemic, the bat involvement as advanced by is not excluded, as the sars-cov-2 displayed 96% identity to the whole genome of bat-cov. moreover the sars-cov-2 was closely related to five wild animal covs, including civets and pangolin . experimental studies showed that ferrets and cats are highly susceptible to the sars-cov-2. after an intra-nasal inoculation of 10 5 plaque-forming units (pfu) to multiple animal species including dogs, cats and ferrets, the infectious virus was detected in nasal turbinate, soft palate and tonsils of all inoculated ferrets. the sars-cov-2 rna was also detected in rectal swabs and efficacious droplet transmission was confirmed among exposed cats . the phylogenetic studies associated to amino acid sequence comparison of the ace2 receptor has predicted cow, buffalo, goat, sheep and swine, as well as other wild species as the potential intermediate hosts for the sars-cov-2 (qiu et al., 2020) . these findings highlighted the threat from the interspecies transmission of the sars-cov-2 between multiple animal species, and from animals to human and vice versa. multiple factors are playing an important role in increasing the exposure of humans to covs of infected animals. intensification of livestock production implies increasing animal density in farms and increasing movement of human and vehicles on and off farms, which promotes pathogen transmission and spreading (cutler, fooks, & van der poel, 2010) . the intensively development of swine industry the last decade is considered of high risk for the trigger of severe pandemic viruses for humans (borkenhagen, salman, ma, & gray, 2019) . this was shown particularly with swine influenza viruses, and nipah virus. indeed, swine exposed workers displayed significantly higher titers (in hemagglutination inhibition assay) against swine influenza subtypes h1n1 and h1n2 than nonexposed workers (odds ratio = 54.9 and 95% confidence interval: [13.0-232.6] (gray et al., 2007) . in late 1998 and 1999, several fatal encephalitis cases (n = 105) caused by a paramyxovirus were recorded in malaysia (chua, 2000) and quickly spread to singapore (paton et al., 1999) . respiratory disease and encephalitis were recorded few weeks ago in pigs in the same district and the isolated virus, named nipah virus, was shown to be transmitted from bats to pigs, then to humans (chua et al., 2002) . the consequences of anthropogenic activities leading to deforestation, habitat fragmentation and replacement of natural vegetation by crops definitely modify wildlife biology. these anthropogenic activities foster wildlife migration and create new environments that increase host, vector and pathogen contacts (jones et al., 2013) . indeed, the deforestation was incriminated in the occurrence of the nipah virus outbreak by pushing fruitbats (the natural reservoir of the nipah virus) to pig farms high-density areas and fostered the contact between both species (chua et al., 2002) . it is well established that animals and animal product trade foster pathogens spreading (travis, watson, & tauer, 2011) . a study on livestock trade network assessment in europe, showed that pigs (for fattening and slaughter) and cattle (for fattening) were ranked second as the most heavily moved animal species within europe in 2011, which foster the risk for exotic diseases introduction (hardstaff, häsler, & rushton, 2015) . thousands of wild animals are traded in asian markets every day, such as raccoon dogs, pangolin, masked palm civets, ferret badgers and hedgehogs (www.anima lasia.org). as several domestic and wild animals were shown to harbour covs, handling, touching or eating these animals, could increase the risk for humans to be in contact with covs. rodents have a worldwide distribution and were shown to harbour more than 60 known pathogens infecting both animals and humans (meerburg, singleton, & kijlstra, 2009) , including covs . bats also are considered to be the most abundant and diverse vertebrate after rodents (rodhain, 2015) , with more than 1,300 known species and worldwide geographic distribution (teeling, jones, & rossiter, 2016) . bats are considered as an important reservoir of highly lethal zoonotic viruses and were shown to harbour more covs than any other animal species (hu et al., 2015) . indeed, beside the sars-cov, the sars-cov-2 and the mers-cov, two porcine covs, have a bat-cov origin: the sads-cov and the pedv (banerjee, kulcsar, misra, frieman, & mossman, 2019) . the geographical range of some bat species was estimated to expand by near to 400% in the last four decades as a response to climate change (ancillotto, santini, ranc, maiorano, & russo, 2016) . one could imagine, that this expansion would be favourable to a higher contact likelihood with wild and domestic animals. mixing different animal species would increase covs host jumping probability. the bcov contaminates sheep and goats, when they share the same barns or graze with infected cattle (tråvén et al., 1999) . the mers-cov was also detected from sheep, goats and donkeys kept in close contact with infected dromedaries (reusken et al., 2013) . the ability of covs to mutate and to adapt to a new environment makes them a continuous threat to human lives and to domestic animals, mainly of farm industries. pig covs are well described and documented in part because pork meat is the most widely consumed in several countries around the world. nevertheless, data on several animal covs are scarce from multiple countries, mainly from africa. there is an urgent need to characterize the extent of covs infections among domesticated animals in the world using serological investigations for screening, virological studies as confirmatory and phylogenetic studies as comprehensive of the mutation and recombination phenomena. continuous surveillance programme for covs genetic evolution should be implemented and would serve for at risk areas prediction. to date, only one mammal animal cov is registered in the oie list, namely the tgev and one avian, the ibv. more attention should be given to the other animal covs and a specific surveillance system must be implemented in all the oie member countries to monitor the introduction and the emergence of novel cov strains in new areas. strict regulation is to be implemented for farm animal trade. thus, it is urgent to assess if meat, milk, semen and other animal by-products are at risk for humans, either through manipulation, process or consumption. the accumulated knowledge needs to be compiled, broaden and extensive to predict and prevent further fatal pandemic like the covid-19. no ethical approval was required as this is a review article with no original research data. the authors acknowledge the valuable contribution of professor mohamed hussni omar from cornell university for the english revision of the manuscript. the authors declare that they have no conflict of interest. replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels herd-level prevalence and incidence of porcine epidemic diarrhoea virus (pedv) and porcine deltacoronavirus (pdcov) in swine herds in ontario new chimeric porcine coronavirus in swine feces prevalence of endemic enteropathogens of calves in new zealand dairy farms middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia dairy calf rearing unit and infectious diseases: diarrhea outbreak by bovine coronavirus as a model for the dispersion of pathogenic microorganisms systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt bovine-like coronaviruses in domestic and wild ruminants prevalence of rotavirus (garv) and coronavirus (bcov) associated with neonatal diarrhea in calves in western algeria extraordinary range expansion in a common bat: the potential roles of climate change and urbanisation. the science of nature disease strategy: transmissible gastroenteritis (version 4.0). canberra (australia) current status of porcine epidemic diarrhoea (ped) in european pigs bats and coronaviruses characterization of a novel chimeric swine enteric coronavirus from diseased pigs in central eastern europe in 2016 bovine coronavirus associated syndromes. veterinary clinics of north america: food animal practice molecular and antigenic characterization of bovine coronavirus circulating in argentinean cattle during 1994-2010 porcine epidemic diarrhea virus and discovery of a recombinant swine enteric coronavirus animal influenza virus infections in humans: a commentary acute necrotizing enteritis associated with suspected coronavirus infection in 3 harbor seals (phoca vitulina) investigating the introduction of porcine epidemic diarrhea virus into an ohio swine operation molecular analysis of brazilian strains of bovine coronavirus (bcov) reveals a deletion within the hypervariable region of the s1 subunit of the spike glycoprotein also found in human coronavirus oc43 new porcine coronavirus? veterinary record detection of equine coronavirus in horses in the united kingdom sero-prevalence, cross-species infection and serological determinants of prevalence of bovine coronavirus in cattle, sheep and goats in ghana weaning and segregation vomiting and wasting disease of piglets respiratory system coronaviruses in poultry and other birds potential pathogens in feces from unweaned llamas and alpacas with diarrhea seroprevalence of porcine respiratory coronavirus in selected korean pigs decline of transmissible gastroenteritis virus and its complex evolutionary relationship with porcine respiratory coronavirus in the united states development of an immunochromatographic strip for serological diagnosis of porcine hemagglutinating encephalomyelitis virus isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the united states nipah virus: a recently emergent deadly paramyxovirus anthropogenic deforestation, el niño and the emergence of nipah virus in malaysia assessing the economic burden of avian infectious bronchitis on poultry farms in brazil hosts and sources of endemic human coronaviruses advances in virus research respiratory and fecal shedding of porcine respiratory coronavirus (prcv) in sentinel weaned pigs and sequence of the partial s-gene of the prcv isolates identification of a novel coronavirus possibly associated with acute respiratory syndrome in alpacas (vicugna pacos) in california public health threat of new, reemerging, and neglected zoonoses in the industrialized world coronaviridae. in virus taxonomy: ninth report of the international committee on taxonomy of viruses sars and mers: recent insights into emerging coronaviruses spotlight on avian pathology: infectious bronchitis virus respiratory disease associated with bovine coronavirus infection in cattle herds in southern italy a candidate modified-live bovine coronavirus vaccine: safety and immunogenicity evaluation characterisation of bubaline coronavirus strains associated with gastroenteritis in water buffalo (bubalus bubalis) calves biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (bubalus bubalis) calves a systematic review of mers-cov seroprevalence and rna prevalence in dromedary camels: implications for animal vaccination development of a multiplex rt-pcr for the detection of major diarrhoeal viruses in pig herds in china detection and molecular characterization of infectious bronchitis-like viruses in wild bird populations development of multi-specific humanized llama antibodies blocking sars-cov-2/ace2 interaction with high affinity and avidity. emerging microbes and infections porcine deltacoronavirus in mainland china a transmissible gastroenteritis in pigs mers-cov spillover at the camel-human interface investigation of rota and corona viruses as causative agents for diarrhea in egyptian calves genetic analysis of avian coronavirus infectious bronchitis virus in yellow chickens in southern china over the past decade: revealing the changes of genetic diversity, dominant genotypes, and selection pressure coronaviruses: an overview of their replication and pathogenesis disease associated with equine coronavirus infection and high case fatality rate an ecosystems perspective on virus evolution and emergence characterisation of porcine epidemic diarrhea virus isolates during the 2014-2015 outbreak in the philippines vaccines for porcine epidemic diarrhea virus and other swine coronaviruses viral enteritis in calves. the canadian veterinary journal = la revue veterinaire canadienne a new bat-hku2-like coronavirus in swine novel findings from a beta coronavirus outbreak on an american miniature horse breeding farm in upstate new york. equine veterinary education human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection swine workers and swine influenza virus infections encephalomyelitis of swine caused by a haemagglutinating virus vi. morphology of the virus a hemagglutinating virus producing encephalomyelitis in baby pigs isolation and characterization of viruses related to the sars coronavirus from animals in southern china characterization of a coronavirus isolated from a diarrheic foal an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels pathogenicity of three isolates of porcine respiratory coronavirus in the usa livestock trade networks for guiding animal health surveillance sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia infection with a new porcine respiratory coronavirus in denmark: serologic differentiation from transmissible gastroenteritis virus using monoclonal antibodies experimental infection of dromedaries with middle east respiratory syndrome-coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase associations between exposure to viruses and bovine respiratory disease in australian feedlot cattle first detection of bovine coronavirus in yak (bos grunniens) and a bovine coronavirus genome with a recombinant he gene coronavirus infections in horses in saudi arabia and oman mers coronavirus in dromedary camel herd, saudi arabia dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) evolution of rna viruses detection, sequence analysis, and antibody prevalence of porcine deltacoronavirus in taiwan bat origin of human coronaviruses: emerging and re-emerging pathogens in humans and animals review of infectious bronchitis virus around the world porcine deltacoronavirus prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in south korea analysis of the genome sequence of an alpaca coronavirus molecular identification and characterization of novel coronaviruses infecting graylag geese (anser anser), feral pigeons (columbia livia) and mallards (anas platyrhynchos) zoonosis emergence linked to agricultural intensification and environmental change altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus calves are susceptible to infection with the newly emerged porcine deltacoronavirus, but not with the swine enteric alphacoronavirus, porcine epidemic diarrhea virus immunologic differences in strains of infectious bronchitis virus middle east respiratory syndrome coronavirus infection in non-camelid domestic mammals. emerging microbes and infections excretion and persistence of bovine coronavirus in neonatal calves prevalence of a novel bovine coronavirus strain with a recombinant hemagglutinin/ esterase gene in dairy calves in china canada porcine epidemic diarrhea in canada: an emerging disease case study seroprevalence and risk factors for infection with equine coronavirus in healthy horses in the usa mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets coronavirus: how a large rna viral genome is replicated and transcribed first genome sequences of buffalo coronavirus from water buffaloes in bangladesh isolation and characterization of a novel betacoronavirus subgroup a coronavirus, rabbit coronavirus hku14, from domestic rabbits porcine respiratory coronavirus: molecular features and virus-host interactions detection and phylogenetic analysis of porcine deltacoronavirus in korean swine farms causative agents and epidemiology of diarrhea in korean native calves structure, function, and evolution of coronavirus spike proteins full-length genome sequence of porcine deltacoronavirus strain usa/ia/2014/8734 complete genome sequence of a novel swine acute diarrhea syndrome coronavirus susceptibility of chickens to porcine deltacoronavirus infection mutation in spike protein cleavage site and pathogenesis of feline coronavirus infectious bronchitis virus variants: molecular analysis and pathogenicity investigation bovine coronavirus in neonatal calf diarrhoea in iran role of transportation in spread of porcine epidemic diarrhea virus infection. united states novel genotype of infectious bronchitis virus isolated in china coronaviridae une étude épidémiologique à propos des manifestations d'allure grippale chez le porc en croissance the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china first outbreak with chimeric swine enteric coronavirus (secov) on pig farms in slovakia -lessons to learn genetic and antigenic heterogeneity of infectious bronchitis virus in south america: implications for control programmes porcine deltacoronavirus from the united states neonatal calf diarrhea: propagation, attenuation, and characteristics of a coronavirus-like agent rodent-borne diseases and their risks for public health rodent-borne diseases and their risks for public health vers une identification de nouveaux virus respiratoires bovins identification of a novel coronavirus from a beluga whale by using a panviral microarray camel production systems in ethiopia: a review of literature with notes on mers-cov risk factors first detection of equine coronavirus (ecov) in europe detecting and monitoring porcine hemagglutinating encephalomyelitis virus porcine hemagglutinating encephalomyelitis virus: a review first detection and characterisation of porcine hemagglutinating encephalomyelitis virus in the czech republic demonstration of coronavirus infection in buffaloes brazilian chicken meat production chain: a 10-year overview antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine experimental inoculation of equine coronavirus into japanese draft horses complete genome analysis of equine coronavirus isolated in japan genetic evidence of middle east respiratory syndrome coronavirus (mers-cov) and widespread seroprevalence among camels in kenya middle east respiratory syndrome coronavirus (mers-cov): animal to human interaction. pathogens and global health sars-cov-2 infection in farmed minks, the netherlands isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the nc99 strain epidemic of equine coronavirus at obihiro racecourse, hokkaido, japan in 2012 discovery of a novel swine enteric alphacoronavirus (seacov) in southern china prevalence of respiratory pathogens in diseased, non-vaccinated, routinely medicated veal calves porcine epidemic diarrhea vaccine evaluation using a newly isolated strain from korea dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves outbreak of nipah-virus infection among abattoir workers in singapore evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study coronaviruses transmissible gastroenteritis in swine isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis a new coronavirus-like particle associated with diarrhea in swine porcine epidemic diarrhea: a retrospect from europe and matters of debate experimental inoculation of neonatal piglets with feed naturally contaminated with porcine epidemic diarrhea virus first retrospective studies with etiological confirmation of porcine transmissible gastroenteritis virus infection in argentina the evolutionary processes of canine coronaviruses chinese-like strain of porcine epidemic diarrhea virus prevalence of equine coronavirus in nasal secretions from horses with fever and upper respiratory tract infection emerging outbreaks associated with equine coronavirus in adult horses equine coronavirus: an emerging enteric virus of adult horses. equine veterinary education enteric coronavirus infection in adult horses geographic information system emergence of porcine epidemic diarrhea viruses with the novel s genes in tibetan pigs in the qinghai-tibetan plateau in china predicting the angiotensin converting enzyme 2 (ace2) utilizing capability as the receptor of sars-cov-2 hemagglutinating encephalomyelitis coronavirus infection in pigs isolation of mers coronavirus from dromedary camel avian infectious bronchitis virus. recent advances in animal virology middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. the lancet infectious diseases geographic distribution of mers coronavirus among dromedary camels mers-cov infection of alpaca in a region where mers-cov is endemic emerging strains of porcine epidemic diarrhoea virus (pedv) in mexico detection and genetic analysis of porcine hemagglutinating encephalomyelitis virus in south korea vaccination management of beef cattle: delayed vaccination and endotoxin stacking. veterinary clinics of north america -food animal practice molecular epidemiology of coronavirus in faeces of brazilian calves and peruvian camelid herds chauves-souris et virus : des relations complexes a disease of nursing pigs previously unreported in ontario outbreak of diarrhea among preweaning alpacas (vicugna pacos) in the southern peruvian highland retrospective study, full-length genome characterization and evaluation of viral infectivity and pathogenicity of chimeric porcine deltacoronavirus detected in vietnam different lineage of porcine deltacoronavirus in thailand, vietnam and lao pdr in 2015 bovine respiratory coronavirus. veterinary clinics of north america: food animal practice winter dysentery in adult dairy cattle: detection of coronavirus in the faeces. the veterinary record diseases of swine the neighbor-joining method: a new method for reconstructing phylogenetic trees coronaviruses: a paradigm of new emerging zoonotic diseases. pathogens and disease detection of bovine coronavirus in calf diarrheic samples by indirect antigen capture elisa and rt-pcr evaluation of equine coronavirus fecal shedding among hospitalized horses coronavirus envelope protein: current knowledge trends in emerging viral infections of swine susceptibility of ferrets, cats, dogs, and different domestic animals to sars-coronavirus-2. biorxiv genetic characterization of bovine coronavirus in vietnam molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in central region infectious agents associated with diarrhoea in neonatal foals in central kentucky: a comprehensive molecular study porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp from cattle involved in two natural outbreaks of shipping fever epidemiology, genetic recombination, and pathogenesis of coronaviruses an immunohistochemical investigation of porcine epidemic diarrhoea outbreak of porcine epidemic diarrhea in suckling piglets genetic characterization and phylogenetic analysis of porcine deltacoronavirus (pdcov) in shandong province attenuation of a virulent swine acute diarrhea syndrome coronavirus strain via cell culture passage field study of bovine coronavirus vaccine enriched with hemagglutinating antigen for winter dysentery in dairy cows prospects for inferring very large phylogenies by using the neighbor-joining method epidemic and genetic characterization of porcine epidemic diarrhea virus strains circulating in the regions around hunan phylogeny, genes, and hearing: implications for the evolution of echolocation in bats evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of middle east respiratory syndrome coronavirus among livestock in ethiopia a cohort study of the effect of winter dysentery on herd-level milk production bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk -risk factors and spatial analysis serum antibodies to bovine coronavirus in swedish sheep the spread of pathogens through trade in wildlife. revue scientifique et technique de l'oie confirmed cases of sars-cov-2 in animals in the united states antibody response of pregnant sows to porcine epidemic diarrhea virus live vaccine and maternally-derived antibodies of the piglets s1 gene-based phylogeny of infectious bronchitis virus: an attempt to harmonize virus classification. infection high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector-borne and zoonotic diseases differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity mutational dynamics of the sars coronavirus in cell culture and human populations isolated in 2003 prevalence of enteric pathogens in diarrheic and non-diarrheic samples from pig farms with neonatal diarrhea in the north east of spain genetic variability of the s1 subunit of enteric and respiratory bovine coronavirus isolates detection and genetic characterization of deltacoronavirus in pigs porcine coronavirus hku15 detected in 9 us states discovery, diversity and evolution of novel coronaviruses sampled from rodents in china detection and characterization of new coronavirus in bottlenose dolphin bat-origin coronaviruses expand their host range to pigs emerging and re-emerging coronaviruses in pigs. current opinion in virology coronavirus pathogenesis induction of protective immunity against transmissible gastroenteritis virus after exposure of neonatal pigs to porcine respiratory coronavirus prevalence of coronavirus antibodies in iowa swine discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavi discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in gammacoronavirus coronavirus disease 2019, weekly situation report. who/covid19 middle east respiratory syndrome coronavirus (mers-cov) global summary and assessment risk world organisation of animal health (2020) evolutionary aspects of recombination in rna viruses isolation and characterization of a highly pathogenic strain of porcine enteric alphacoronavirus causing watery diarrhoea and high mortality in newborn piglets the structure and functions of coronavirus genomic 3' and 5' ends swine enteric alphacoronavirus (swine acute diarrhea syndrome coronavirus): an update three years after its discovery bovine coronavirus (bocv) infection in calves with diarrhoea and their dams efficacy and immunogenicity of recombinant swinepox virus expressing the truncated s protein of a novel isolate of porcine epidemic diarrhea virus prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate porcine deltacoronavirus: overview of infection dynamics, diagnostic methods, prevalence and genetic evolution genomic characterization and pathogenicity of porcine deltacoronavirus strain chn-hg-2017 from china prevalence and phylogenetic analysis of porcine diarrhea associated viruses in southern china from the re-emerging of sads-cov infection in pig herds in southern china retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin a pneumonia outbreak associated with a new coronavirus of probable bat origin countrywide survey for mers-coronavirus antibodies in dromedaries and humans in pakistan key: cord-356364-ipi81ce3 authors: ho, bo-lin; cheng, shu-chun; shi, lin; wang, ting-yun; ho, kuan-i; chou, chi-yuan title: critical assessment of the important residues involved in the dimerization and catalysis of mers coronavirus main protease date: 2015-12-14 journal: plos one doi: 10.1371/journal.pone.0144865 sha: doc_id: 356364 cord_uid: ipi81ce3 background: a highly pathogenic human coronavirus (cov), middle east respiratory syndrome coronavirus (mers-cov), has emerged in jeddah and other places in saudi arabia, and has quickly spread to european and asian countries since september 2012. up to the 1(st) october 2015 it has infected at least 1593 people with a global fatality rate of about 35%. studies to understand the virus are necessary and urgent. in the present study, mers-cov main protease (m(pro)) is expressed; the dimerization of the protein and its relationship to catalysis are investigated. methods and results: the crystal structure of mers-cov m(pro) indicates that it shares a similar scaffold to that of other coronaviral m(pro) and consists of chymotrypsin-like domains i and ii and a helical domain iii of five helices. analytical ultracentrifugation analysis demonstrated that mers-cov m(pro) undergoes a monomer to dimer conversion in the presence of a peptide substrate. glu169 is a key residue and plays a dual role in both dimerization and catalysis. the mutagenesis of other residues found on the dimerization interface indicate that dimerization of mers-cov m(pro) is required for its catalytic activity. one mutation, m298r, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme. conclusions: mers-cov m(pro) shows substrate-induced dimerization and potent proteolytic activity. a critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral m(pro) family. a highly pathogenic human coronavirus (cov) 1 , middle east respiratory syndrome coronavirus (mers-cov), emerged in jeddah and other places in saudi arabia in september 2012 and purification, the codons of the thrombin cutting recognition sequence and a ndei cutting site were removed and then inserted the codons of leu-arg-leu-lys-gly-gly into the above vector. the forward primer sequence for site-directed mutagenesis was 5'-catcacagcagcggcct gcgtctgaaaggcggcagcggtttggtgaaaatg-3' and the reverse primer was 5'-catttt caccaaaccgctgccgcctttc agacgcaggccgctgctgtgatg-3'. the reading frame of the final plasmid was confirmed by sequencing. the expression vector was transformed into e. coli bl21 (de3) cells (novagen). cultures were grown in 0.8 liters of lb medium at 37°c for 4 h, induced with 0.4 mm isopropyl-β-d -thiogalactopyranoside, and then incubated overnight at 20°c. after centrifuging at 6,000 x g at 4°c for 15 min, the cell pellets were resuspended in lysis buffer (20 mm tris, ph 8.5, 250 mm nacl, 5% glycerol, 0.2% triton x-100, and 2 mm β-mercaptoethanol) and then lysed by sonication. the crude extract was then centrifuged at 12,000 x g at 4°c for 25 min to remove the insoluble pellet. next the supernatant was incubated with 1-ml ni-nta beads at 4°c for 1 h and then loaded onto an empty column. after allowing the supernatant to flow through, the beads were washed with washing buffer (20 mm tris, ph 8.5, 250 mm nacl, 8 mm imidazole, and 2 mm β-mercaptoethanol). the sars-cov papain-like protease [12] (1 mg in 100 mm phosphate buffer (ph 6.5)) was then added and incubated for 3 h. the sars-cov papain-like protease digestion, which removed the 6 x his tag and leu-arg-leu-lys-gly-gly fragment, resulted in a native protein product with an authentic n-terminus. the digest was allowed to flow through and then loaded onto a s-100 gel-filtration column (ge healthcare) equilibrated with running buffer (20 mm tris, ph 8.5, 100 mm nacl, and 2 mm dithiothreitol). the purity of the fractions collected was analyzed by sds-page and the protein was concentrated to 30 mg/ml by amicon ultra-4 10-kda centrifugal filter (millipore). crystals of the mers-cov m pro were obtained at 295 k by the sitting-drop vapor-diffusion method. the protein solution was set up at 5 mg/ml and the reservoir solution consisted of 0.1 m tris, ph 8.4, 15% (w/v) peg 4000 and 0.2 m sodium acetate. clusters of needle crystals appeared in 2 days and were used for micro-seeding. single cystals of rectangle shape and with dimensions of 0.3-0.5 mm were obtained in less than a week. all crystals were cryoprotected in the reservoir solution with 15% glycerol and were flash-cooled in liquid nitrogen. data collection, structure determination and refinement x-ray diffraction data were collected at 100 k on the spxf beamline 13c1 at the national synchrotron radiation research center, taiwan, roc, using a adsc quantum-315r ccd detector (x-ray wavelength of 0.976 å). the diffraction images were processed and scaled using the hkl-2000 package [23] . the structure was solved by the molecular replacement method by phaser [24] using the structure of sars-cov m pro r298a mutant (pdb entry 4hi3; [25] ) as the search model. manual rebuilding of the structure model was performed using coot [26] . structure refinement was carried out using refmac [27] . the data-processing and refinement has been deposited in the protein data bank (pdb entry 5c3n). the colorimetry-based peptide substrate, tsavlq-para-nitroanilide (tq6-pna) (purity 95-99% by hplc; gl biochem ltd, shanghai, china), was used to measure the proteolytic activity of mers-cov m pro and its mutants throughout the course of the study as described previously [25, 28] . this substrate is cleaved at the gln-pna bond to release free pna, resulting in an increase in absorbance at 405 nm. the absorbance at 405 nm was continuously monitored using a jasco v-550 uv/vis spectrophotometer. the protease activity assay was performed in 10 mm phosphate (ph 7.6) at 30°c. the substrate stock solution was 1600 μm and the working concentrations were from 25 to 1200 μm. in the substrate titration assay, the concentration of mers-cov m pro and its mutants, v4r, t126s, e169a, m298r and t126s/m298r was 0.3, 0.4, 0.7, 1.2, 0.15 and 0.26 μm, respectively, while that of sars-cov m pro was 1.1 μm. steady state enzyme kinetic parameters were obtained by fitting the initial velocity (ν 0 ) data to the michaelis-menten eq (1) where k cat is the catalytic constant, [e] is the enzyme concentration, [s] is the substrate concentration and k m is the michaelis constant of the substrate. the program sigmaplot (systat software, inc., richmond, ca) was used for the data analysis. to assess the cooperativity effect, the kinetic parameters were obtained by fitting the initial velocities to the hill eq (2) where k' is a constant that is related to the dissociation constant and h is the hill constant. auc was performed on a xl-a analytical ultracentrifuge (beckman coulter) using an an-50 ti rotor [11, 12, 25, [28] [29] [30] . the sedimentation velocity experiments were carried out using a double-sector epon charcoal-filled centerpiece at 20°c with a rotor speed of 42,000 rpm. protein solutions of 0.05 to 0.5 mg/ml (330 μl) and reference (370 μl) solutions, both containing d 2 o, were loaded into the centerpiece. the absorbance at 280 nm was monitored in a continuous mode with a time interval of 300 s and a step size of 0.003 cm. multiple scans at different time intervals were then fitted to a continuous c(s) distribution model using the sedfit program [31] . additionally, the results with the various different protein concentrations were globally fitted to a monomer-dimer self-association model using the sedphat program to calculate the dissociation constant (k d ) [32] . to measure the substrate-induced dimerization, the active enzyme centrifugation (aec) [33] was performed. briefly, mers-cov m pro of 15 μl (1 mg/ml) was added into the small well of the band-forming centerpiece before the cell assembled. then 330 μl of peptide substrate at 0, 200 and 400 μm in d 2 o were respectively loaded into the bulk sample sector space. at a rotor speed of 42,000 rpm, the protein solution flowed into the substrate-containing channel and form a protein band. it can be detected by absorbance at 250 nm. during the centrifugation, the sediment protein continuously met and cleaved the substrate, which can be detected by absorbance change at 405 nm. the dataset from the multiple scans at 250 nm at various time intervals were fitted to a continuous c(s) distribution model using the sedfit program [31] , while the first five scans (0-30 min) at 405 nm were used to derive the product concentration and then initial velocity values. the protocol followed that of previous studies [28] with some modifications. apparent dissociation constants and stoichiometry of the enzyme-ligand interactions were measured by a thermal activity monitor 2277 from ta instruments (new castle, de). calorimetric titrations of the peptide substrate tq6-pna (0.5 mm in a 250-μl syringe) and m pro (6 μm in a 4-ml ampoule) were carried out at 25°c in 10 mm phosphate buffer (ph 7.6). the peptides were titrated into the enzyme using a 10-μl aliquot for each injection with a time interval of 20 min. a control experiment in the absence of enzyme was performed in parallel to correct for the dilution of heat. the data obtained was then analyzed by integrating the heat effects normalized against the amount of injected protein using curve-fitting based on a 1:1 binding model. this involved the use of digitam software (ta instruments, new castle, de). as part of the present study, an expression vector was constructed and the bl21 (de3) star (invitrogen) strain of e. coli were used to express mers-cov m pro . unlike sars-cov m pro [25, 28] , the mers-cov m pro with 6 x his-tag retained at the c-terminus cannot be expressed. instead, the bacteria are able to express the m pro when there is a n-terminal 6 x his-tag fusion that can be removed during the purification. however, thrombin digestion leaves two extra residues (gly-ser) at the n-terminus of m pro , resulting in protein with no proteolytic activity (data not shown). therefore we used sars-cov papain-like protease [12, 30, 34] , which is a highly active viral deubiquitinase and does not leave any residues at the n-terminus of m pro . after gel-filtration, the purity of authentic n-terminus m pro was about 99% (s2 fig). the size of the mers-cov m pro was found to be close to 30 kda, while any uncut protein was located at higher molecular weight position. the typical yield was about 10 mg after purification from 0.8 liter of e. coli culture. the structure of the mers-cov m pro was determined at 3.0 å resolution by x-ray crystallography (table 1 and fig 1a) . the crystal packing belonged to space group c222 1 , with unit-cell parameters a = 87.2, b = 94.0, c = 155.1 å and α = β = γ = 90°. the final atomic model containing two m pro molecules in a crystallographic asymmetric unit agrees well with the crystallographic data and the expected values of geometric parameters (table 1 ). there are no residues in the disallowed region of the ramachandran plot, while 81.3% of the residues are in the most favored region. the overall dimeric structure of m pro is similar to that of sars-cov m pro ; although a relative shift of 10°to 30°could be observed for the two domain iii within the dimer (s3a fig) . indeed, the r.m.s. distance between equivalent cα atoms of the domain i+ii of the two structures is 0.9 å, while that between the domain iii of the two structures is 3.1 å. compared with the structures of the ligand-bound complex (pdb entry 4ylu) and c148a mutant (pdb entry 4wme) [14, 15] , the r. m. s. distance is 0.8 and 0.7 å over 540 cα atom pairs, respectively (s3b fig) . this indicates that the dimeric structures show no significant difference; although the present structure is a free enzyme and the other two structures involve enzyme-ligand complexes and higher resolution. besides, there is minor difference between the present structure and bat-cov hku4 m pro (pdb entry 2yna), with 80% sequence identity, as the r. m. s. distance over 539 cα atom pairs is 0.8 å (s3b fig). interestingly, the dimerization interface situation with the mers-cov m pro was found to be different to that of sars-cov m pro where there are four amino acid pairs with intermolecular polar interactions (ser1. . .glu166, arg4. . .glu290, ser123. . .arg298 and ser139. . .gln299). there are only two pairs of intermolecular hydrogen bonds, ser1. . .glu169 and ser142. . .gln299 that are associated with the dimer surface of mers-cov m pro according to the current structure ( fig 1b) . this led us to compare the dimerization and catalytic activity of the two types of m pro . in the present study, in addition to using the wild-type mers-cov m pro , we also mutated several residues at the dimerization interface in order to evaluate their role in dimerization and catalysis of mers-cov m pro (see below). to compare catalysis between the two m pro , tq6-pna, a peptide substrate for sars-cov m pro [25, 28] , was used to measure the proteolytic activity. at first, the dependence of the initial additional allowed region 13.6 generously allowed region 5.2 disallowed region 0 a the numbers in parentheses are for the highest-resolution shell. where i hi is the integrated intensity of a given reflection and hi h i is the mean intensity of multiple corresponding symmetry-related reflections. velocity on enzyme concentration was analyzed and showed a nonlinear upward correlation (fig 2a) . the pattern is similar to that of sars-cov m pro , as the monomeric m pro may not have catalytic activity [28] . however, mers-cov m pro displayed a sigmoid curve for its rate constant pattern at various substrate concentrations (fig 2b, open circles) ; this contrast with sars-cov m pro , which exhibited a classical saturation curve (s4 fig). the results were then fitted to the hill equation (eq 2) in order to evaluate the kinetic parameters ( table 2 ). the k cat (2.33 s -1 ) of mers-cov m pro is close to that of sars-cov m pro (2.11 s -1 ), while the hill constant was 1.8, suggesting a significant degree of positive cooperativity among the m pro protomers. the comparable activity levels of the two m pro in the present study is dissimilar to the results obtained during a recent study in which the activity of the mers-cov m pro was found to be 5-fold lower than that of sars-cov [14] . using different substrates may cause the difference. tomar et al. [14] used a longer peptide substrate with residues present at both p and p' table 2 . site. however, in the present studies we used a peptide substrate that contains only p site residues. besides, they utilized fret substrate and could only be used at low substrate concentrations to prevent the inner-filter effect, while we are able to use higher substrate concentrations to capture the kinetic parameters. the cooperativity phenomenon associated with the mers-cov m pro is similar to that of the sars-cov m pro r298a/l monomer mutants; these were found to show monomer to dimer conversion during catalysis [28] . as a result of the above, we investigated the quaternary structure of the m pro by auc (fig 3) . the cumulative spectra (fig 3a) were analyzed using the continuous c(s) distribution model and the results suggested that mers-cov m pro is a monomer in phosphate buffer (s5 fig) and this contrasts with a distribution of 30% monomer and 70% dimer in the presence of 600 μm tq6-pna ( fig 3b) . we also measured the size distribution of m pro at various tq6-pna concentrations (s6 fig). the results indicated that the sedimentation coefficient of the major species was shifted as the substrate dosage changed (s6b fig). more substrate led to the major species moving close to the dimer position. however, before the centrifugation, the enzyme had been mixed with the substrate and the catalysis began, resulting in a mixture of substrate and product with enzyme. it is unable to confirm that our observation is a substrate-induced or substrate/product-induced dimerization. to solve this, a modified auc technique, aec [33] , was utilized to detect the quaternary structure change in the absence and presence of substrate (fig 4) . here the enzyme solution was put into the small well of band-forming centerpiece and then flowed into the substratecontaining channel when the centrifugation began. during the centrifugation, the protein layer gradually sediment and continuously met peptide substrates. not surprisingly, there was a broad distribution between the monomer and dimer species in the presence of 200 μm peptide substrate, while a major species shifted to the dimer in 400 μm substrate (fig 4b) . it suggests that mers-cov m pro acts as a rapid self-associated and substrate-induced dimerization. using different strategies, tomar et al. [14] confirmed that inhibitor binding can also induce and maintain the dimerization of m pro . on the other hand, we measured the velocity of the product formation during the centrifugation; although the rate (0.017 μm/s) is 10-fold lower than that by the spectrometric assay (fig 4b) . the values were derived from a global fit of the auc data to a monomer-dimer self-association model by sedphat [32] . the experiments for the assay were obtained at protein concentration of 1.5 to 30 μm. c the value was from our previous studies for comparison [28] . doi:10.1371/journal.pone.0144865.t002 to quantitatively characterize the monomer-dimer equilibrium of m pro in the absence and presence of substrate, the auc results at protein concentrations of 1.5, 6 and 15 μm were globally fitted to the monomer-dimer self-association model ( table 2 ). the analysis indicated that the k d value of mers-cov m pro in the absence of substrate was 7.7 μm and this decreased 11-fold in the presence of substrate, which brings it close to the value for sars-cov m pro (1.7 μm; table 2 ). thus it can be concluded that, like the sars-cov m pro r298a mutant [25] , the presence of substrate is able to induce the dimerization of mers-cov m pro . in addition, although the k d values of wild-type sars-cov m pro without or with substrates show no significant difference (table 2) , it was possible to detect substrate-induced dimerization at a protein concentration of 1 μm by aec [33] . previous studies have demonstrated that a conserved residue, glu166, plays a pivotal role in connecting the substrate binding site with the dimerization interface of sars-cov m pro [28] . here the equivalent residue, glu169, was mutated and its influence on mers-cov m pro evaluated ( fig 1b) . not surprisingly, compared to the wild-type enzyme, the activity (k cat ) of e169a showed a 6-fold decrease (fig 2b, open diamonds and table 2 ). furthermore, auc analysis suggested that this enzyme consisted of a single species close to monomeric form even in the presence of substrate (fig 3e) . the k d values of the e169a mutant without substrate was 14 μm, which is 2-fold higher than that of the wild-type and this did not decrease in the presence of substrate ( table 2 ). the results suggest that mutation of glu169 is able to block substrate-induced dimerization and that this results in a decrease in enzyme activity. unexpectedly, as well as the monomer, some octamer (6.3%) with a sedimentation coefficient of 4.9 s was observed in the presence of substrate (fig 3e) . previous studies have suggested that a super-active octamer of sars-cov m pro can be locked by 3d domain swapping [35] . in this study, the presence of the octamer form of the mers-cov m pro e169a mutant in the presence of substrate may explain why this mutant is not totally inactive. taking the above as a whole, the dual role of the conserved glu residue in catalysis and dimerization is consistent for both m pro . our results confirmed that there are fewer intermolecular polar interactions at the dimerization interface of mers-cov m pro and this results in the enzyme being in the monomer form in aqueous buffer; this contrasts with sars-cov m pro , which is mostly in the dimer form in similar circumstances due to the greater number of intermolecular interactions (figs 1b and 3b ). based on the sequence alignment, three residues in the dimerization interface vary in coronaviral m pro sequences (s1 fig). in sars-cov m pro , residues arg4, ser123 and arg298 are different to the equivalent ones in mers-cov m pro , which are val4, thr126 and met298, respectively. based on the above, three single mutants, v4r, t126s and m298r, were generated and their proteolytic activity and dimerization were assessed. unexpectedly, both the v4r and the t126s mutant showed a higher k d for the dimer to monomer and a lower level of activity than wild-type m pro (fig 2b and table 2 ). k d values were decreased by 1.5-fold to 2.4-fold for the two mutants in the presence of substrate, which suggests reduced substrate-induced dimerization (fig 3c and 3d) . based on the current structure, residue val4 showed a hydrophobic contact with another protomer's residue gly141 (fig 1b) . mutation of valine to arginine may lose the contact. furthermore, the side chain of residue val131 of mers-cov m pro is hydrophobic while that of the equivalent residue, cys128 of sars-cov m pro , is hydrophilic (fig 1b) . val131 is close to the arg4 and will disfavor the electrostatic interaction of arg4. . .glu290 in mers-cov m pro . these variance may result in v4r failed to form a stable dimer. for the t126s mutant, after compared with the other two mers-cov m pro structures (pdb entry 4ylu and 4wme), we found that the side chain of thr126 is free of rotation. it is able to make a hydrophobic interaction with the residue tyr121, resulting in a 180°-rotation of the phenol ring, and lead to a hydrogen-bond with the backbone amide of leu144 (fig 1b) . it further results in the side chain of leu144 toward another protomer's ile300 and make a hydrophobic contact for the two protomers. mutation of thr126 to serine will lose this contact and may disfavor the dimerization. by way of contrast, the m298r mutant resulted in a stable dimer form in phosphate buffer (fig 3f) . the k d value for the mutant in the absence and presence of substrate were 1.1 and 0.7 μm, respectively, which are very close to those for sars-cov m pro (table 2) . furthermore, the stable dimer form showed higher proteolytic activity and the mutation transformed the enzymes rate constant pattern at various substrate concentrations into a classical saturation curve (fig 2c, open triangles) . in addition, the k m of the m298r mutant is 5.8-fold lower than that of sars-cov m pro , which suggests a higher substrate binding affinity ( table 2 ). it can be concluded that mutation of residue met298 to arginine within the mers-cov m pro results in the stabilization of the dimer formation, which in turn gives rise to more efficient catalysis. based on our structure, arg298 is able to make a hydrogen bonding interaction with thr126. residue thr126 can be replaced by serine because the t126s/m298r double mutant also shows similar dimerization characteristics ( fig 3g) and saturated catalytic pattern to that of the m298r mutant (fig 2c, open hexagons and table 2 ). however, it can only be achieved in the presence of arg298, not met298. the sigmoid nature of the curve describing the rate constant pattern at various substrate concentrations ( fig 2b) means that it is not possible to obtain a k m value for this enzyme, which would allow us to evaluate the substrate binding affinity of the wild-type mers-cov m pro and its mutants; the exceptions being the m298r single mutant and the t126s/m298r double mutant (fig 2) . to further delineate the binding of substrate to the enzyme, itc was used to measure the k d for the substrate (or substrate/product)-enzyme complex and the binding stoichiometry (n) (fig 5) . during the titration, the enzymatic hydrolysis might produce additional heat, resulting in higher δh. so we can only compared the n and k d of the wild-type m pro with those of e169a and m298r mutants. the three enzymes exhibited similar n (0.89 to 1.06) and k d (14.2 to 20.3 μm) . this suggested that the monomeric and dimeric m pro show quite the same substrate binding affinity. such phenomenon is also found in monomeric and dimeric sars-cov m pro , whose n and k d for the same substrate were 0.97 to 1.05 and 29.9 to 33.8 μm, respectively [28] . with the k m ((k -1 +k cat )/k 1 ), k cat and k d (k -1 /k 1 ), we are able to calculate the k 1 and k -1 , the rate of the association and dissociation of the enzyme-substrate complex. the k 1 and k -1 for the m298r mutant and substrate is 0.049 s -1 μm -1 and 0.98 s -1 , while those for sars-cov m pro is 0.00246 s -1 μm -1 and 0.084 s -1 . the rate constants for m298r mutant showed 20-and 12-fold higher than those for sars-cov m pro . the more rapid association and relatively slower dissociation of enzyme-substrate complex may be used to explain why the catalytic efficiency (k cat /k m ) of the mers-cov m pro m298r mutant is higher than that of sars-cov m pro (table 2) . the crystal structure of authentic n-terminus mers-cov m pro was determined and this was found to involve a dimeric form with less intermolecular polar interactions. biochemical and auc studies indicated that mers-cov m pro shows almost the same proteolytic activity as sars-cov m pro ; although it is a monomer in aqueous buffer and displays substrate-induced dimerization (fig 6) . a conserved residue, glu169, plays an essential role in the substrateinduced dimerization of both mers-cov m pro and sars-cov m pro . moreover, mutation of a residue in the dimerization interface, m298r, was found to result in a more stable dimer form in aqueous buffer that had higher enzyme activity; while other two mutations, v4r and t126s, showed the reverse effect. critical assessment of the residues important to dimerization of and catalysis by mers-cov m pro provides valuable insights into the mechanism that controls the monomer-dimer switch of important and valuable enzyme. isolation of a novel coronavirus from a man with pneumonia in saudi arabia is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? emerging human coronaviruses-disease potential and preparedness in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection hospital outbreak of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study close relative of human middle east respiratory syndrome coronavirus in bat ecology, evolution and classification of bat coronaviruses in the aftermath of sars isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors structural and functional characterization of mers coronavirus papain-like protease structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus ligand-induced dimerization of mers coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals structures of the middle east respiratory syndrome coronavirus 3c-like protease reveal insights into substrate specificity the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs quaternary structure of the severe acute respiratory syndrome (sars) coronavirus main protease mechanism of the maturation process of sars-cov 3cl protease structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus processing of x-ray diffraction data collected in oscillation mode phaser crystallographic software mechanism for controlling the monomerdimer conversion of sars coronavirus main protease features and development of coot refmac5 for the refinement of macromolecular crystal structures mutation of glu-166 blocks the substrate-induced dimerization of sars coronavirus main protease structural and functional characterization of human apolipoprotein e 72-166 peptides in both aqueous and lipid environments differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling on the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus three-dimensional domain swapping as a mechanism to lock the active conformation in a super-active octamer of sars-cov main protease espript: analysis of multiple sequence alignments in post-script this research was supported by grants from ministry of science and technology, taiwan (103-2320-b-010-025 and 104-2320-b-010-034) to cyc. we also thank nymu for its financial conceived and designed the experiments: cyc. performed the experiments: blh scc ls tyw kih. analyzed the data: blh tyw. contributed reagents/materials/analysis tools: scc. wrote the paper: cyc. key: cord-354582-fniymnmf authors: ma, zhiqian; li, zhiwei; dong, linfang; yang, ting; xiao, shuqi title: reverse genetic systems: rational design of coronavirus live attenuated vaccines with immune sequelae date: 2020-06-30 journal: adv virus res doi: 10.1016/bs.aivir.2020.06.003 sha: doc_id: 354582 cord_uid: fniymnmf since the end of 2019, the global covid-19 outbreak has once again made coronaviruses a hot topic. vaccines are hoped to be an effective way to stop the spread of the virus. however, there are no clinically approved vaccines available for coronavirus infections. reverse genetics technology can realize the operation of rna virus genomes at the dna level and provide new ideas and strategies for the development of new vaccines. in this review, we systematically describe the role of reverse genetics technology in studying the effects of coronavirus proteins on viral virulence and innate immunity, cell and tissue tropism and antiviral drug screening. an efficient reverse genetics platform is useful for obtaining the ideal attenuated strain to prepare an attenuated live vaccine. coronaviruses are large, enveloped, positive-sense, single-stranded rna viruses with genome sizes ranging from 26 to 32 kb that are distributed broadly among humans, other mammals, and birds and cause respiratory, enteric, hepatic, and neurologic diseases (coronaviridae study group of the international committee on taxonomy of viruses, 2020; cui et al., 2019) . coronaviridae belongs to the order nidovirales along with three other families (arteriviridae, mesoniviridae, and roniviridae). coronaviridae is further classified into four genera (fig. 1a ) based on phylogenetic analyses and genomic structures, namely, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus . the alphacoronaviruses and betacoronaviruses infect only mammals; gammacoronaviruses and deltacoronaviruses infect birds, but some of them can also infect mammals. the genome of coronaviruses is arranged in the order of the 5 0 untranslated region (5 0 utr), open reading frame 1a/b (orf1ab), spike (s) protein, envelope (e) protein, membrane (m) protein, nucleoprotein (n) protein, 3 0 utr, and the poly (a) tail, with regions encoding accessory proteins, including orf3, 6, 7a, 7b, 8, and 9b, between those encoding structural proteins (fig. 1b) . the large replicase polyproteins pp1a and pp1ab, encoded by the partially overlapping 5 0 -terminal orf1a/b within the 5 0 two-thirds of the genome, are proteolytically cleaved into 16 putative nonstructural proteins (nsps; fig. 1b ). in early december 2019, a cluster of cases of pneumonia caused by a novel coronavirus named sudden acute respiratory syndrome coronavirus 2 (sars-cov-2) resulted in tremendous challenges to china's public health and clinical treatment yan et al., 2020) , and now it has been confirmed in more than 211 other countries and territories, causing a major global public health crisis (day, 2020; jernigan and team, 2020; peeri et al., 2020) . sars-cov-2 belongs to the betacoronavirus genus in the family coronaviridae, and is closely related to severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) (drosten et al., 2003; wassenaar and zou, 2020; zaki et al., 2012) . sars-cov, mers-cov and sars-cov-2 are three highly transmissible and pathogenic viruses that have emerged in humans in the past 2 decades and bats are considered their natural hosts . they mainly infect the lower respiratory tract and cause severe pneumonia (hotez et al., 2020) , sometimes leading to fatal acute lung injury and acute respiratory distress syndrome, resulting in high morbidity and mortality. cytokines and chemokines, which are triggered by the innate immune system during virus infections have long been thought to play an important role in immunity, but dysregulated and excessive immune responses may cause immunopathology (channappanavar and perlman, 2017) . research shows that robust sars-cov replication accompanied by delayed ifn-i signaling promotes the accumulation of pathogenic inflammatory monocytemacrophages, resulting in elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific t cell responses . in addition to sars-cov and mers-cov, sars-cov-2 infection also can induce proinflammatory cytokine responses, including interferon gamma (ifn-γ), tumor necrosis factor alpha (tnfα), interleukin-1 (il-1), il-2, il-6 and il-8 (kadkhoda, 2020) . in patients with severe covid-19, lymphopenia is a common feature, with drastically reduced numbers of cd4 + t cells, cd8 + t cells, b cells and natural killer (nk) cells, as well as a reduced percentage of monocytes, eosinophils and basophils. furthermore, sars-cov-2 may have an antibody-dependent enhancement effect (cao, 2020; xu et al., 2020) . although there have been outbreaks of coronavirus diseases, such as sars and mers, when the novel coronavirus sars-cov-2 outbreak occurred, there were no effective vaccines or drugs available to prevent or cure the disease. clinical development of multiple vaccines and screening of specific antiviral drugs were initiated in parallel with attempts to contain the outbreak. currently, more than 115 candidate vaccines for sars-cov-2 are being developed globally. five advanced candidates have recently moved into clinical development, including mrna-1273 from moderna, ad5-ncov from cansino biologicals, ino-4800 from inovio, lv-smenp-dc and pathogen-specific aapc from shenzhen geno-immune medical institute (thanh le et al., 2020) . it became apparent that coronaviruses can cross the species barrier and cause life-threatening infections in humans ( ji et al., 2020; nishiura et al., 2020) . therefore, further attention needs to be paid to these new coronaviruses. in addition, coronaviruses causing high pathogenicity and high mortality in animals have attracted increasing attention. for example, highly virulent porcine epidemic diarrhea virus (pedv), which appeared in china in 2010 and the united states in 2013, caused large global economic losses to the pig industry due to the lack of effective vaccines and drugs (kong et al., 2019; li et al., 2012; stevenson et al., 2013) . another coronavirus, swine acute diarrhea syndrome coronavirus (sads-cov), which is a novel bat-hku2-like coronavirus, was responsible for a large-scale fatal outbreak of in pigs in china that caused the death of more than 24,000 piglets across four farms (zhou et al., 2018) . obviously, the role of vaccines is an effective way not only to enhance the ability of individuals to fight the virus but also to stop the spread of the virus. to be better prepared for future outbreaks of unknown human coronavirus pathogens, platform technologies to accelerate vaccine development should be employed. reverse genetics systems are highly valuable research tools for rna virus vaccine development, which may be more efficient than conventional approaches with live attenuation through passing (forward genetics) or inactivation (stobart and moore, 2014) . reverse genetics systems are useful tools for studying the modifications of viral genomes and for generating recombinant viruses to better understand their fundamental biology, develop novel vaccine candidates, and test antiviral therapeutics. recently, reverse genetics techniques, including targeted rna recombination, in vitro ligation and bacterial artificial chromosome systems, vaccinia virus vectors and transformation associated recombination (tar) cloning, have been successfully used to manipulate the genome of coronaviruses (fig. 2 ). 2.1 reverse genetic system using targeted rna recombination targeted rna recombination was the first reverse genetic system devised for coronaviruses at a time when it was not clear whether a full-length clone could be successfully constructed (masters and rottier, 2005) . constructing an infectious clone of a coronavirus using targeted rna recombination requires two steps: first, constructing a chimeric coronavirus that carries the mouse hepatitis virus (mhv) s gene and has a strict mouse cell affinity and second, rescuing the targeted recombinant coronavirus on a specific cell dependent on the virus (fig. 3a ). targeted rna recombination was the first reverse genetic system devised for mhv. subsequently, the method was applied to the construction of full-length cdna in other coronaviruses, including pedv and feline infectious peritonitis virus (fipv) (haijema et al., 2003; li et al., 2013) . targeted rna recombination presents clear limitations; for example, it does not operate on parts of the genome that code for replicating enzymes. in vitro ligation uses unique type ii restriction endonucleases (e.g., bgli, sapi, and bsai) that cleave several bases away from their recognition site, allowing for the reassembly of authentic coronavirus genomes from smaller fragments. detailed steps can be found in the published literature (cockrell et al., 2017) . the assembled full-length cdna, containing a t7 rna polymerase promoter at the 5 0 end and a poly (a) tail at the 3 0 end, is transcribed in vitro to generate capped full-length transcripts that are used together with capped n gene transcripts to efficiently rescue infectious virus after the transfection of susceptible cells (fig. 3b ). the first coronavirus full-length infectious cdna clone was also generated for tgev using in vitro ligation (yount et al., 2000) . in addition, sars-cov, mers-cov, pedv and sars-cov-2 were constructed using in vitro ligation (scobey et al., 2013; xie et al., 2020; yount et al., 2003; zaki et al., 2012) . this method can avoid the instability and virulence of viral cdna in bacteria, as it depends on the t7 rna polymerase. red arrows indicate human coronavirus outbreaks and identification events. blue arrows indicate animal coronavirus outbreaks and identification events. green arrows indicate the publication of infectious clones using different reverse genetics methods (almazan et al., 2000; li et al., 2013; thao et al., 2020; yount et al., 2002) . the bacterial artificial chromosome (bac) system allows the assembly of a full-length cdna copy of the viral genome in bac plasmids such as pbelobac11, a low-copy-number plasmid that presents strictly controlled replication, leading to one or two plasmid copies per cell (almazan et al., 2008) . bac plasmids contain a 5 0 cytomegalovirus promoter of the viral genome, allowing for the transcription of the viral genome following the transfection of bac dna into mammalian cells. in addition, coronavirus bacs contain a poly (a) tail, a hepatitis d virus ribozyme, and bovine growth hormone termination and polyadenylation signals to create genomic rna with an authentic 3 0 end (fig. 3c ). the first full-length infectious coronavirus cdna clone was generated for transmissible gastroenteritis virus (tgev) using the bac system. in addition, sars-cov, mers-cov, fipv and pedv have been successfully developed (balint et al., 2012; li et al., 2017 ). the bac system presents several advantages, including the high stability of exogenous sequences, unlimited production of the cdna clone, high efficiency of cdna transfection into mammalian cells, easy manipulation for genes and intracellular expression of the viral rna (fehr, 2020) . recently, transformation associated recombination (tar) cloning has been used to construct infectious clones of coronaviruses, including mhv, mers-cov and sars-cov-2 (https://www.biorxiv.org/content/10. 1101/2020.02.21.959817v1). the overall strategy is as follows (fig. 4a ): viral rna is prepared from several overlapping dna fragments by rt-pcr or chemical synthesis (no virus available). the 5 0 -and 3 0 -terminal dna fragments contain the t7-rna polymerase promoter upstream of the coronavirus at the 5 0 end, a restriction endonuclease cleavage site downstream of the poly(a) sequence, and overlapping sequences with the tar plasmid pvc604. then, all the fragments are used to transform saccharomyces cerevisiae (strain vl6-48n), and the positive colonies are screened for the correct assembly of the yeast artificial chromosome (yac) containing virus genome and purified the yac. the plasmid is linearized using restriction endonuclease and subjected to t7 rna polymerase-based in vitro transcription to generate transcripts. the rna transcripts together with capped n gene transcripts are transfected into bhk cells to obtain the corresponding virions. the tar cloning system can rapidly reassemble at least 14 fragments with remarkable efficacy (usually >90% of the clones are correct). the vaccinia virus cloning vector also can be used to construct infectious coronavirus clones and represents a generic approach to reverse genetics (thiel and siddell, 2005) . vaccinia virus vectors allow large fragments (26-31 kb) of foreign genes to be inserted without affecting virus replication, and vaccinia viruses can easily produce high titer strains. the process of cloning coronavirus cdna in vaccinia virus is as follows (fig. 4b) . first of all, the virus genome is analyzed for useful naturally encoded endonuclease restriction sites that can later be used to ligate cloned cdna inserts and generate a set of plasmid dnas that cover the full-length virus genomic sequence. upstream of the viral 5 0 end, there should be an eagi or a bsp120i restriction endonuclease site to allow insertion of the cdna into the noti site of the vaccinia virus genomic dna by in vitro ligation. downstream of the 3 0 end of the virus genome, a poly(a) tail and unique restriction endonuclease site should be cloned. second, long cdna fragments are assembled by in vitro ligation to obtain a full-length virus cdna fragment. third, the full-length fragment or several fragments are inserted into a vaccinia virus genome by in vitro ligation. finally, the vaccinia virus containing the full-length cdna virus genome is linearized and used as a template for in vitro transcription using bacteriophage t7 rna polymerase and transfected into bhk cells to obtain the corresponding virions. currently, vaccinia virus vectors are successfully applied to reverse genetic manipulation of several coronaviruses, including hcov-229e, ibv, mhv and sars-cov (thiel et al., 2001; thiel and siddell, 2005 ; van den worm et al., 2012). chemical synthesis of the viral genome is widely used for the construction of infectious clones. using a chemical synthesis system, the complete genome of the virus can be synthesized to construct a full-length genomic cdna clone, especially in the absence of a natural template. this method was recently used to construct an infectious clone of sars-like cov (becker et al., 2008) . in general, the ability to quickly and efficiently introduce virtually any type of genetic modification (point mutations, insertions, or deletions) into the coronavirus genome and recover recombinant virus is desirable. crispr/cas9 technology is an efficient platform for manipulating the modification of full-length infectious cloning of coronaviruses, which can generate recombinant viruses within a week shen et al., 2019; wang et al., 2019a) . moreover, a combination of bacterial artificial chromosome and lambda red recombination with the i-scei homing endonuclease method was used to engineer genetic alterations of the mers-cov (fehr, 2020; muth et al., 2017) . importantly, the optimized reverse genetics platform used in this study will simplify the construction of mutant infectious clones and help accelerate the progress in coronavirus research. vaccination is expected to be an efficacious strategy in preventing individuals and animals from suffering from coronavirus infections. virus vaccines must be immunogenic and sufficiently stable, safe, and suitable to induce long-lasting immunity. to date, various kinds of candidate vaccines for coronaviruses have been developed, including live attenuated vaccines, subunit vaccines, dna vaccines, inactivated vaccines, and recombinant vector vaccines. each vaccine type has different advantages and disadvantages. for example, traditional inactivated vaccines can generally induce highly potent immune responses and/or protection, but they may induce the antibody-dependent enhancement effect. in addition, they exit recovery of virulence if incomplete inactivation of viruses, resulting in significant safety concerns . although dna vaccines maintain strong safety, they are generally less immunogenic than are whole-virusbased vaccines, usually requiring the optimization of sequences or immune routes; and adjuvant coordination is also often required to achieve immune effects. among them, live attenuated vaccines are considered highly effective because of their ability to replicate within host cells, inducing high levels of antigenic stimulation and robust long-term immunological memory. however, a major safety concern with live attenuated vaccines is the possibility of reversion to a pathogenic form and oral or aerosol route vaccination that can induce mucosal immune responses should be considered. virus-infected cells react quickly to invading viruses by producing type i interferons (ifn-α/β) and type iii interferons (ifn-λ1/λ2/λ3/λ4) and establish an innate antiviral state, which provides a first line of defense against viral infection. type iii ifns are mainly produced by epithelial cells and play a major role in diseases that cause mucosal immunity, such as pedv and pdcov. however, most coronaviruses commonly suppress interferon levels in infected cells. reverse genetics technology provides a very useful platform in the study of virulence and host innate immune-related mutations and in the rational design of coronavirus live attenuated vaccine candidates. according to current research, the 5 0 utr, several structural proteins, nonstructural proteins and the 3 0 utr of coronaviruses all have more or less influence on virulence and the life cycle of the virus. here, we summarize the coronavirus genes associated with virulence, replication and innate immunity (table 1 ) and potential vaccines based on reverse genetics (table 2) . it is well known that among four coronavirus genera, only alphacoronaviruses and betacoronaviruses produce the nsp1 protein, which is located at the n-terminal of the replicase polyprotein pp1a. in terms of both sequence identity and size, nsp1 of alphacoronaviruses shares low similarity with nsp1 of betacoronaviruses. to date, the crystal structure of nsp1 from several coronaviruses (sars-cov, tgev, and pedv) has been resolved, which is important to understand the role of nsp1 in the pathogenesis of viruses and its interaction with host cell proteins (shen et al., 2018 (shen et al., , 2019 . the nsp1 protein of coronaviruses is a vital virulence factor widely demonstrated in both alphacoronaviruses and betacoronaviruses ( jimenez-guardeno et al., 2015; lokugamage et al., 2015; shen et al., 2018 shen et al., , 2019 zhang et al., 2015) . not only inhibiting the expression of host genes with different mechanisms, but also playing a significant regulatory role in evading the host's natural immune response, nsp1 mainly inhibits the production of ifns (zhang et al., , 2018b . additionally, some studies have shown that abolishing the anti-ifn function of nsp1 can attenuate viruses, which has been demonstrated in mhv and sars-cov ( jimenez-guardeno et al., 2015; lei et al., 2013) . the nsp2 proteins of some coronaviruses are dispensable for viral replication in cell culture, but those viruses lacking nsp2, such as mhv and sars-cov, show attenuated viral growth and rna synthesis (graham et al., 2005) . however, the pathogenicity of these viruses in mice has not been further reported. in addition, amino acids 1-120 of tgev nsp2 are involved in the regulation of inflammation via nf-κb activation (wang et al., 2018b) . the multidomain in nsp3, an essential component of the replication/ transcription complex, is the largest protein encoded by the coronavirus (lei et al., 2018) . in all coronaviruses, highly conserved functional domains within nsp3 are present: papain-like protease domains, ubiquitin-like domains, and adp-ribose-phosphatase domains (lei et al., 2018) . using a recombinant sars-cov strain with reduced nsp3 de-adp-ribosylation activity showed that this mutant strain led to virus attenuation in mice but protected them from an otherwise lethal sars-cov infection and significantly enhanced the innate immune response, indicating that it is an important virulence factor for sars-cov . v787s of nsp3 is thought to induce attenuation of mhv, and the mutant mhv strain elicits protective immunity (mielech et al., 2015) . n1347a of nsp3 is also thought to be associated with the virulence of mhv (fehr et al., 2015) . pedv and tgev (590-1215aa, via nf-κb) nsp3 proteins have been proven to evade the host immune system . in addition, 102 and 61 amino acid substitutions were found in nsp2 and nsp3 of sars-cov-2 compared with sars-cov or sars-like bat cov, respectively, and these differences warrant further investigation . the nsp4 genes of coronaviruses are significant in viral replication and pertain to cellular membrane rearrangements (beachboard et al., 2015) . both h120 and f121 in sars-cov nsp4 play critical roles in viral replication (sakai et al., 2017) . however, whether nsp4 affects the virulence of the virus needs to be further verified by using reverse genetics. the coronavirus nsp5 protease (3clpro; mpro) processes nsp proteins at 11 cleavage sites and is essential for virus replication, making it a high value target for the development of anti-coronavirus therapeutics ( jo et al., 2020; tomar et al., 2015) . it has been reported that coronavirus nsp5 cleaves nf-κb essential modulator and stat2, inhibiting ifn production, which has been verified in fipv, pedv and pdcov (chen et al., 2019; wang et al., 2016; zhu et al., 2017) . mhv nsp5 t26i/d65g, which confers resistance to a broad-spectrum coronavirus 3c-like protease inhibitor, also has a slight effect on the virulence and replication of mhv (deng et al., 2014) . coronavirus nsp6 is associated with autophagy (cottam et al., 2014) . scientists used reverse genetics to verify that deletion of any of regions encoding nsp7, nsp8, nsp9 and nsp10 in mhv was lethal to virus (deming et al., 2006) . furthermore, pedv nsp7 was found to inhibit the ifn-β and irf3 promoter activities , and nsp8 can suppress type iii ifn activities (zhang et al., 2018b) . the 148-amino acid nsp10 subunit contains 2 zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3 0 -5 0 exoribonuclease and 2 0 -o-methyltransferase activities. a series of studies have shown that nsp10 is a major regulator of the sars-cov replication function (bouvet et al., 2014) . the single and double mutations r80a/ e82a-exon(+) in mhv nsp10 disrupt the nsp10-nsp14 interaction, and compared to the wild-type virus exon(+), these mutations rendered the virus 5 and 10 times more sensitive to treatment with the rna mutagen 5-fluorouracil, respectively. the results showed that nsp10 is important for cov replication fidelity and support the hypothesis that nsp10 functions to regulate nsp14-exon activity during viral replication (smith et al., 2015) . coronavirus nsp14 has 3 0 -to-5 0 exoribonuclease (exon) and n7-methyltransferase (n7-mtase) activities. the former is a proofreading function that is required for high-fidelity replication. the inactivation of mhv-cov exon(à) activity by the nsp14 mutations d89a and e91a are relatively stable, even after passaging mhv-exon(à) 250 times, without reversion of the exon(à) mutation site. however, novel amino acid changes within the rna-dependent rna polymerase and nsp14 of mhv-exon(à) p250, indicate that multiple replicase proteins could compensate for the exon function during replication and will likely inform the design of countermeasures for endemic and emerging covs by defining novel common targets for stable virus attenuation or direct inhibition (graepel et al., 2017) . mhv-exon activity is required for resistance to the innate immune response (case et al., 2018) . the inactivation of sars-cov exon activity by the nsp14 mutations d90a and e92a was attenuated in both young and aged disease models compared to virulent wt, and it provided complete protection against lethal challenge in a susceptible, immunosenescent mouse model of the lethal sars-cov infection (graham et al., 2012) . the recombinant tgev virus with a mutation in zinc finger 1 of the exon domain of nsp14 induced weak antiviral responses, including reduced expression of beta interferon (ifn-β), tumor necrosis factor (tnf), and interferon-stimulated genes. therefore, coronavirus nsp14 plays a potential role in regulating innate immune responses (becares et al., 2016) . in addition, pedv nsp14 was found to suppress type i and type iii ifn activities (zhang et al., , 2018b . nsp15 encoded by coronavirus is a nidoviral uridylate-specific endoribonuclease (nendou) that plays an essential role in the life cycle of the virus (zhang et al., 2018a) . mhv with the nsp15 h262a mutation that inactivates the endou activity of nsp15, resulted in greatly attenuated disease in mice and stimulated a protective immune response (deng et al., 2017) . pedv endou activity is also a key virulence factor and coronavirus nsp15 is a target for generating live attenuated vaccine (deng et al., 2019) . nsp16 has 2 0 -o-methyltransferase (mtase) activity and it is conserved across the entire coronavirus family, increasing its appeal as an attenuation target (yong et al., 2019) . several recent works have shown that although disruption of nsp16 activity in mhv (d129a), sars-cov (d130a), mers-cov (d130a) and pedv (kdke to aaaa) rendered an attenuated strain, it induced stronger type i and type iii interferon responses and protected animals from the challenge (hou et al., 2019a; menachery et al., 2017a menachery et al., , 2018 . however, without sufficiently attenuated virulence, the nsp16 mutant could be combined with another attenuating mutation in nsp1, nsp14, or s protein, which will produce a stable, attenuated virus capable of protection from heterologous challenge (hou et al., 2019a; menachery et al., 2018) . the coronavirus s protein, which is a class i fusion protein located at the surface of the virus, includes the receptor binding s1 subunit and the membrane fusion s2 subunit (shang et al., 2020a; walls et al., 2020) . the coronavirus s protein is considered a major target for the development of subunit vaccines and major virulence genes. two chimeric viruses with the reciprocally exchanged s gene were generated; one is a highly virulent strain, and the other is an avirulent strain, showing that the s gene is only one of the necessary determinants for virus virulence (wang et al., 2018a) . another report examined the role of the s gene in pedv pathogenesis with different generations of strains in the same way. although the results showed that the s gene plays a role in virulence, other genes might also important in determining virulence (kao and chang, 2019) . three recombinant ibvs, beaur-m41 (s1), beaur-qx (s1) and beaur-m41 (s), based on the beaur backbone expressing a heterologous s1 or s from m41 or qx conferred incomplete protection against homologous challenge based on ciliary activity and clinical signs. however, the protection efficiency of beaur-m41(s) was higher than that of beaur-m41 (s1) (ellis et al., 2018) . coronavirus virions assemble at the endoplasmic reticulum (er)-golgi intermediate compartment (ergic) and some of them, including sars-cov, tgev, ibv, and pedv, possess conserved motifs of yxxφ and/or kxhxx/kkxx in the cytoplasmic tail of the s protein (hou et al., 2019b; ujike et al., 2016) . a recombinant pedv with deletions or a mutation in the two motifs (yxxφ and/or kxhxx/kkxx) showed that the yxxφ motif triggers endocytosis of s proteins and that the motif kvhvq is involved in the retention of the s proteins in the er-golgi intermediate compartment; the loss of both motifs significantly enhances syncytia formation in vero cells and reduces virulence in pigs (hou et al., 2019b) . the ntd of the s protein can attenuate a highly virulent coronavirus, which was confirmed by a recombinant virus (icpc22a-s1δ197aa) and (ictgev-s1δ224aa), but the virus may lose important epitopes for inducing robust protective immunity, which provides new insights for the development of a new attenuated vaccine (hou et al., 2017; wang et al., 2019a) . in addition, 27 amino acid substitutions were found in the sars-cov s protein compared with sars or sars-like cov, and whether these differences could affect the host tropism and transmission property is worthy of future investigation . the coronavirus e protein is the smallest protein of the major structural proteins and is an integral membrane protein involved in several aspects of the virus life cycle, including assembly, budding, and pathogenesis (schoeman and fielding, 2019) . viruses lacking e protein form infectious virions, but their replication efficiency and virulence are significantly reduced. this seems to indicate that the e protein is an important virulence factor in coronavirus but is not essential for some coronaviruses (castano-rodriguez et al., 2018; fett et al., 2013; jimenez-guardeno et al., 2015; shang et al., 2020b) . mers-cov and tgev lacking the e gene are not successfully rescued, but they can spread from cell to cell via expression of the e protein (almazan et al., 2013; ortego et al., 2002) . coronavirus m proteins are major structural proteins and are required for virion assembly. the s, e and m proteins can form virus-like particles (vlps) with structural integrity . both peptides (m1-31 and m132-161) in the sars-cov m protein are immunogenic (he et al., 2005) , but the m protein of sars-cov has not been investigated for its protective efficacy against sars-cov infection. several studies have shown that the coronavirus m protein also inhibits ifn production (shokri et al., 2019; siu et al., 2009) . the mers-cov m protein inhibits type i ifn production through the inhibition of tbk1-dependent phosphorylation of irf3 (lui et al., 2016) . coronavirus n proteins have been associated with multiple functions in the viral life cycle, including the regulation of viral rna synthesis, the packaging of the viral rna in helical nucleocapsids, and virion assembly through the interaction with the viral m protein (mcbride et al., 2014) . expression of the n protein is also necessary for efficient recovery of the virus from infectious cdna clones. unlike the s protein, the n protein has no ability to elicit neutralizing antibodies, but it may induce specific antibody and cellular immune responses. the n protein plays an important role in viral pathogenesis since balb/c mice immunized with recombinant virus mva-mers-n exhibit stronger t cell responses and anti-n monoclonal antibodies protect mice from lethal infection by mhv (nakanaga et al., 1986; veit et al., 2018) . immunodominant sars-cov-n regions n1 (1-422aa) and n3 (110-422aa) produce specific antigens in balb/c mice and react with the serum of sars patients; hence, they can be used as effective sars dna vaccines (dutta et al., 2008) . the sars-cov n protein is also an ifn antagonist that inhibits ifn synthesis by inhibiting irf-3 and nf-κb, interfering with trim25-mediated rig-i ubiquitination or attenuating ptac-mediated rig-i/mda5 activation (ding et al., 2017; hu et al., 2017) . coronavirus accessory proteins have been generally regarded as dispensable for in vitro viral replication. for example, pedv with the orf3 gene entirely deleted could replicate similarly to wild-type pedv (beall et al., 2016) . likewise, deletions of ns6 from pdcov, orf8a from sars-cov, orf3, orf4 and orf5 from mers-cov and orf3 and orf5 from ibv do not affect replication, and the deletion of these accessory proteins could attenuate viruses, showing that accessory proteins are also related to the virulence of viruses (almazan et al., 2013; castano-rodriguez et al., 2018; menachery et al., 2017b; zhang et al., 2020) . the accessory proteins of coronaviruses are often used as target regions for the insertion of exogenous gene since they are dispensable. notably, some accessory proteins have ion channels and are related to the innate immune response (kint et al., 2016; wong et al., 2018; yue et al., 2018; zeng et al., 2016) . some accessory proteins interact with host cells, potentially helping viruses evade the immune system and increase their virulence (comar et al., 2019; fang et al., 2018) . orf4a, orf4b and orf8b of mers-cov seem to be involved in viral evasion of nf-κb mediated host immune responses (lee et al., 2019) . therefore, the mutant virus lacking some accessory proteins is potentially a safe and promising vaccine candidate to prevent coronavirus infection. to date, viral vectors utilizing modified vaccinia virus ankara, measles virus, rv vectors or adenovirus have been used to express the glycoprotein of coronaviruses, including sars-cov and sars-cov-2, and have been shown to be immunogenic according to in vivo assays (kato et al., 2019; thanh le et al., 2020) . to date, a recombinant novel coronavirus vaccine using an adenovirus type 5 vector that expresses the s protein has entered phase i clinical trials (thanh le et al., 2020) . researchers designed recombinant replication-deficient adenovirus-based vaccines expressing the mers-cov s protein that induced the highest neutralizing antibody titer and the strongest cytokine-induced t cell responses, and these may confer protection against mers-cov infection (kim et al., 2019) . recombinant parainfluenza virus 5 (piv5)-expressing the mers-cov s protein can induce neutralizing antibodies and robust t cells in a human dpp4 knock-in c57bl/6 congenic mouse model (hdpp4 ki) and protect mice from fatal mers-cov infection (li et al., 2020b ). an attenuated parainfluenza virus encoding the full-length s protein of sars-cov was used for the vaccination of african green monkeys, and monkeys with this vaccine were protected from subsequent homologous sars-cov infection (bukreyev et al., 2004) . another novel recombinant influenza a virus (h1n 1pdm09) with mers-cov was generated, and the inactivated chimeric bivalent vaccine induced potent and specific neutralizing antibodies against mers-cov and h1n1pdm09 in balb/c mice (shehata et al., 2019) . the use of reverse genetics to construct recombinant vaccine strains could achieve a single immunization and prevent two diseases. coronaviruses generally exhibit restricted cell and tissue tropism, which is dependent on the s glycoprotein of individual coronavirus strains (millet and whittaker, 2015) . understanding the cell and tissue tropism of a virus is helpful to further research the pathogenesis of the virus and save vaccine production costs. vaccines against ibv, both live attenuated and inactivated, are currently produced in embryonated hen eggs, which is a cumbersome and expensive process, because most ibv strains do not replicate in cultured cells. previous studies have shown that the s2 subunit of the avirulent beaur strain is responsible for its extended cellular tropism for vero cells (bickerton et al., 2018b) . later, recombinant ibvs with the immunogenic s1 subunit were derived from the ibv vaccine strain and the virulent field strain was generated using reverse genetics. the recombinant ibvs are able to replicate in both primary chicken kidney and vero cells, which would enable ibv vaccines to be grown in cell lines rather than expensive embryonated hen eggs (bickerton et al., 2018a) . moreover, recombinant ibv with a mutant s2 0 site (furin s2 0 site) led to neurotropism (cheng et al., 2019) . tgev has both enteric and respiratory tropism. tgev s219a in the s protein was required to confer enteric tropism, and a 6 nt insertion at position 1124 could increase virus stability and virus titers after passage in cell cultures (sanchez et al., 2019) . the mhv amino acid lysine at 194 in the nsp1 protein can promote virus replication in the liver, and this study further confirmed that nsp1 is a betacoronavirus virulence factor (zhang et al., 2015) . a recombinant coronavirus would efficiently express green fluorescent protein (gfp), which is commonly applied as a tool for high-throughput drug screens and neutralizing antibody assays. feline coronavirus (fcov) is one of the most significant coronaviruses. tissue culture-adapted type i fcov often loses pathogenicity, which complicates research on type i fcov-induced feline infectious peritonitis (fip). therefore, researchers established a recombinant reporter c3663 virus carrying the nanoluciferase (nluc) gene. they examined the inhibitory effect of 68 compounds on c3663 replication in fcwf-4 cells and infectivity in a canine-derived cell line by using the reporter c3663 virus. finally, they successfully screened a canine cell line, a72, that permitted fcov replication but with low efficiency and aberrant viral gene expression (terada et al., 2019) . coronaviruses have repeatedly crossed species barriers, and some have emerged as important human pathogens. the best-known examples include sars-cov, mers-cov and sars-cov-2 (chan et al., 2020) , all of which pose a marked threat to human health. in addition, four other coronaviruses are known to infect humans, including hcov-229e, hku-nl63, hcov-oc43 and hcov-hku1, which cause mild illnesses yan et al., 2020) . bats are generally thought to be the natural reservoir of a range of coronaviruses . some bat coronaviruses also have the potential to emerge in human populations (letko et al., 2020) , and these include the sars-like virus shc014-cov in horseshoe bats (menachery et al., 2015) . human coronaviruses likely originated from bats and then jumped into another amplification mammalian host before crossing species barriers to infect humans. in addition to coronaviruses harming human health, the new emergence of animal coronaviruses has been a threat to animal health in the past decade. for example, porcine enteric alphacoronavirus, pedv and sads-cov zaki et al., 2012; zhou et al., 2018) . to date, there are no clinically approved vaccines or antiviral drugs available for these coronavirus infections. an ideal live attenuated vaccine should replicate effectively in the host, not causing diseases, but stimulating enough protective immune responses without reverting to a virulent phenotype. one of the most promising applications of reverse genetics is to realize the development of live attenuated vaccines by mutating or deleting virulence-related genes. to produce the ideal coronavirus vaccine strain through reverse genetics, a deeper knowledge of the coronavirus-host interaction, host immune responses, and pathogen immune evasion strategies is needed. for example, host cell pathways affected during coronavirus infection need to be identified, and these proteins of coronavirus suppressing the host innate immunity and the specific signaling pathway also need to be identified. combining reverse genetics with metagenomics and structural biology can help characterize pre-emergent coronavirus populations, allowing the field to make predictions about which zoonotic coronaviruses are likely to emerge, prepare for future outbreaks, and facilitate the development of therapeutic strategies against coronavirus infection ( johnson et al., 2018) . in addition, effective broad-spectrum antiviral drugs are still needed. hopefully, live attenuated vaccines based on reverse genetics will be available in the near future and will prove to be highly effective tools against coronaviruses. engineering infectious cdnas of coronavirus as bacterial artificial chromosomes engineering a replication-competent, propagationdefective middle east respiratory syndrome coronavirus as a vaccine candidate engineering the largest rna virus genome as an infectious bacterial artificial chromosome severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce doublemembrane vesicles molecular characterization of feline infectious peritonitis virus strain df-2 and studies of the role of orf3abc in viral cell tropism characterization of a pathogenic full-length cdna clone and transmission model for porcine epidemic diarrhea virus strain pc22a mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice recombinant infectious bronchitis viruses expressing heterologous s1 subunits: potential for a new generation of vaccines that replicate in vero cells the s2 subunit of infectious bronchitis virus beaudette is a determinant of cellular tropism coronavirus nsp10, a critical co-factor for activation of multiple replicative enzymes mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars covid-19: immunopathology and its implications for therapy murine hepatitis virus nsp14 exoribonuclease activity is required for resistance to innate immunity role of severe acute respiratory syndrome coronavirus viroporins e, 3a, and 8a in replication and pathogenesis genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology dysregulated type i interferon and inflammatory monocytemacrophage responses cause lethal pneumonia in sars-cov-infected mice feline infectious peritonitis virus nsp5 inhibits type i interferon production by cleaving nemo at multiple sites the s2 subunit of qx-type infectious bronchitis coronavirus spike protein is an essential determinant of neurotropism efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses antagonism of dsrna-induced innate immune pathways by ns4a and ns4b accessory proteins during mers coronavirus infection the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 coronavirus nsp6 restricts autophagosome expansion origin and evolution of pathogenic coronaviruses covid-19: italy confirms 11 deaths as cases spread from north mhv-a59 orf1a replicase protein nsp7-nsp10 processing in replication coronaviruses resistant to a 3c-like protease inhibitor are attenuated for replication and pathogenesis, revealing a low genetic barrier but high fitness cost of resistance coronavirus nonstructural protein 15 mediates evasion of dsrna sensors and limits apoptosis in macrophages coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type i and type iii interferon responses the nucleocapsid proteins of mouse hepatitis virus and severe acute respiratory syndrome coronavirus share the same ifn-β antagonizing mechanism: attenuation of pact-mediated rig-i/mda5 activation identification of a novel coronavirus in patients with severe acute respiratory syndrome search for potential target site of nucleocapsid gene for the design of an epitope-based sars dna vaccine the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded rna-binding subunit unique in the rna virus world recombinant infectious bronchitis viruses expressing chimeric spike glycoproteins induce partial protective immunity against homologous challenge despite limited replication in vivo porcine deltacoronavirus accessory protein ns6 antagonizes interferon beta production by interfering with the binding of rig-i/mda5 to double-stranded rna bacterial artificial chromosome-based lambda red recombination with the i-scei homing endonuclease for genetic alteration of mers-cov the nsp3 macrodomain promotes virulence in mice with coronavirus-induced encephalitis the conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection complete protection against severe acute respiratory syndrome coronavirus-mediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking e protein proofreading-deficient coronaviruses adapt for increased fitness over long-term passage without reversion of exoribonuclease-inactivating mutations the nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease a decade after sars: strategies for controlling emerging coronaviruses switching species tropism: an effective way to manipulate the feline coronavirus genome identification of immunodominant epitopes on the membrane protein of the severe acute respiratory syndrome-associated coronavirus the potential role of th17 immune responses in coronavirus immunopathology and vaccine-induced immune enhancement deletion of a 197-amino-acid region in the n-terminal domain of spike protein attenuates porcine epidemic diarrhea virus in piglets engineering a live attenuated porcine epidemic diarrhea virus vaccine candidate via inactivation of the viral 2'-o-methyltransferase and the endocytosis signal of the spike protein deletion of both the tyrosine-based endocytosis signal and the endoplasmic reticulum retrieval signal in the cytoplasmic tail of spike protein attenuates porcine epidemic diarrhea virus in pigs the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination update: public health response to the coronavirus disease 2019 outbreak-united states cross-species transmission of the newly identified coronavirus 2019-ncov the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis identification of the mechanisms causing reversion to virulence in an attenuated sars-cov for the design of a genetically stable vaccine inhibition of sars-cov 3cl protease by flavonoids viral metagenomics, protein structure, and reverse genetics: key strategies for investigating coronaviruses covid-19: an immunopathological view investigation of the role of the spike protein in reversing the virulence of the highly virulent taiwan porcine epidemic diarrhea virus pintung 52 strains and its attenuated counterpart development of a recombinant replicationdeficient rabies virus-based bivalent-vaccine against mers-cov and rabies virus and its humoral immunogenicity in mice superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length spike protein of middle east respiratory syndrome coronavirus infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b bst2 suppresses porcine epidemic diarrhea virus replication by targeting and degrading virus nucleocapsid protein with selective autophagy severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists middle east respiratory syndrome coronavirus-encoded accessory proteins impair mda5-and tbk1-mediated activation of nf-kappab attenuation of mouse hepatitis virus by deletion of the llrkxgxkg region of nsp1 nsp3 of coronaviruses: structures and functions of a large multi-domain protein functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses new variants of porcine epidemic diarrhea virus manipulation of the porcine epidemic diarrhea virus genome using targeted rna recombination development of the full-length cdna clones of two porcine epidemic diarrhea disease virus isolates with different virulence the epidemic of 2019-novel-coronavirus (2019-ncov) pneumonia and insights for emerging infectious diseases in the future single-dose, intranasal immunization with recombinant parainfluenza virus 5 expressing middle east respiratory syndrome coronavirus (mers-cov) spike protein protects mice from fatal mers-cov infection middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin sars-cov nucleocapsid protein antagonizes ifnβ response by targeting initial step of ifn-β induction pathway, and its c-terminal region is critical for the antagonism middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 evaluation of antibody-dependent enhancement of sars-cov infection in rhesus macaques immunized with an inactivated sars-cov vaccine coronavirus reverse genetics by targeted rna recombination the coronavirus nucleocapsid is a multifunctional protein attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2'-o-methyltransferase activity middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis mers-cov accessory orfs play key role for infection and pathogenesis combination attenuation offers strategy for live attenuated coronavirus vaccines murine coronavirus ubiquitin-like domain is important for papain-like protease stability and viral pathogenesis host cell proteases: critical determinants of coronavirus tropism and pathogenesis a novel coronavirus emerging in china-key questions for impact assessment transgene expression in the genome of middle east respiratory syndrome coronavirus based on a novel reverse genetics system utilizing red-mediated recombination cloning protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice initial cluster of novel coronavirus (2019-ncov) infections in wuhan, china is consistent with substantial human-tohuman transmission generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? rapid manipulation of the porcine epidemic diarrhea virus genome by crispr/cas9 technology twoamino acids change in the nsp4 of sars coronavirus abolishes viral replication minimum determinants of transmissible gastroenteritis virus enteric tropism are located in the n-terminus of spike protein coronavirus envelope protein: current knowledge reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry the outbreak of sars-cov-2 pneumonia calls for viral vaccines a recombinant influenza a/h1n1 carrying a short immunogenic peptide of mers-cov as bivalent vaccine in balb/c mice structural basis for the inhibition of host gene expression by porcine epidemic diarrhea virus nsp1 a conserved region of nonstructural protein 1 from alphacoronaviruses inhibits host gene expression and is critical for viral virulence modulation of the immune response by middle east respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex mutations in coronavirus nonstructural protein 10 decrease virus replication fidelity emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences rna virus reverse genetics and vaccine design epidemic and genetic characterization of porcine epidemic diarrhea virus strains circulating in the regions around hunan establishment of a virulent full-length cdna clone for type i feline coronavirus strain c3663 the covid-19 vaccine development landscape reverse genetics of coronaviruses using vaccinia virus vectors infectious rna transcribed in vitro from a cdna copy of the human coronavirus genome cloned in vaccinia virus ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals the contribution of the cytoplasmic retrieval signal of severe acute respiratory syndrome coronavirus to intracellular accumulation of s proteins and incorporation of s protein into virus-like particles recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens. vaccine 36, 1085. van den worm cd8+ t cells responding to the middle east respiratory syndrome coronavirus nucleocapsid protein delivered by vaccinia virus mva in mice structure, function, and antigenicity of the sars-cov-2 spike glycoprotein porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo mers-cov virus-like particles produced in insect cells induce specific humoural and cellular imminity in rhesus macaques the s gene is necessary but not sufficient for the virulence of porcine epidemic diarrhea virus novel variant strain bj2011c porcine transmissible gastroenteritis virus nonstructural protein 2 contributes to inflammation via nf-kappab activation the n-terminal domain of spike protein is not the enteric tropism determinant for transmissible gastroenteritis virus in piglets porcine transmissible gastroenteritis virus inhibits nf-kappab activity via nonstructural protein 3 to evade host immune system sars-cov-2: rapid classification of betacoronaviruses and identification of traditional chinese medicine as potential origin of zoonotic coronaviruses accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3 genome composition and divergence of the novel coronavirus (2019-ncov) originating in china an infectious cdna clone of sars-cov-2 pathological findings of covid-19 associated with acute respiratory distress syndrome preparation of puerarin chitosan oral nanoparticles by ionic gelation method and its related kinetics recent advances in the vaccine development against middle east respiratory syndrome-coronavirus strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus systematic assembly of a fulllength infectious cdna of mouse hepatitis virus strain a59 sars-coronavirus open reading frame-3a drives multimodal necrotic cell death isolation of a novel coronavirus from a man with pneumonia in saudi arabia bat severe acute respiratory syndrome-like coronavirus wiv1 encodes an extra accessory protein, orfx, involved in modulation of the host immune response the nsp1, nsp13, and m proteins contribute to the hepatotropism of murine coronavirus jhm suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 structural and biochemical characterization of endoribonuclease nsp15 encoded by middle east respiratory syndrome coronavirus type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in irf1 signaling genetic manipulation of porcine deltacoronavirus reveals insights into ns6 and ns7 functions: a novel strategy for vaccine design a pneumonia outbreak associated with a new coronavirus of probable bat origin porcine deltacoronavirus nsp5 antagonizes type i interferon signaling by cleaving stat2 coronavirus non-structural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines